WO2012071469A2 - Histone demethylase inhibitors and uses thereof for treatment o f cancer - Google Patents

Histone demethylase inhibitors and uses thereof for treatment o f cancer Download PDF

Info

Publication number
WO2012071469A2
WO2012071469A2 PCT/US2011/061954 US2011061954W WO2012071469A2 WO 2012071469 A2 WO2012071469 A2 WO 2012071469A2 US 2011061954 W US2011061954 W US 2011061954W WO 2012071469 A2 WO2012071469 A2 WO 2012071469A2
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
mir
cells
subject
histone
Prior art date
Application number
PCT/US2011/061954
Other languages
French (fr)
Other versions
WO2012071469A3 (en
Inventor
Hui Zhang
Tao Ye
Junmin Quan
Jing Wang
Original Assignee
Nevada Cancer Institute
Shenzhen Graduate School Of Peking University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nevada Cancer Institute, Shenzhen Graduate School Of Peking University filed Critical Nevada Cancer Institute
Publication of WO2012071469A2 publication Critical patent/WO2012071469A2/en
Publication of WO2012071469A3 publication Critical patent/WO2012071469A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
    • C07D295/20—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carbonic acid, or sulfur or nitrogen analogues thereof
    • C07D295/215—Radicals derived from nitrogen analogues of carbonic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/40—Oxygen atoms
    • C07D211/44—Oxygen atoms attached in position 4
    • C07D211/46—Oxygen atoms attached in position 4 having a hydrogen atom as the second substituent in position 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/56—Nitrogen atoms
    • C07D211/58—Nitrogen atoms attached in position 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/60—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D211/62—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals attached in position 4

Definitions

  • the present invention relates to, inter alia, compounds and compositions that inhibit histone demethylases.
  • the present invention concerns histone demethylase inhibitor compounds and derivatives thereof, pharmaceutical compositions comprising histone demethylase inhibitor compounds, and methods of using these compounds and compositions to modulate histone methylation, inhibit growth and proliferation of cancer cells, and promote differentiation of cancer cells in vitro and in vivo, as well as for treatment of cancer in a subject.
  • the present invention also concerns the use of histone demethylases as a therapeutic, diagnostic, or prognostic biomarker for identifying tumors comprising cells expressing one or more pluripotent or multipotent stem cell markers.
  • the present invention further relates to modulating or inhibiting histone demethylase activity in pluripotent/multipotent cells using inhibitory nucleic acids.
  • the invention additionally encompasses the use of histone demethylase inhibitors that alter the expression or enzyme activity of histone demethylases to modulate the properties of pluripotent embryonic stem/iPS (induced pluripotent stem) cells or the re-reprogramming of iPS cells from somatic cells for stem cell-based therapy.
  • Histone methylation is a major covalent modification of histones that is often regarded as part of the "histone code” that provides the structural and functional characteristics of chromatin to epigenetically define gene expression patterns in a particular cell (Klose, R.J. et al., (2007) Nat. Rev. Mol. Cell Biol. 8: 307-18; Shi, Y. (2007) Nat. Rev. Genet. 8: 829-33). While methylation of histone H3 at lysines 9 (K9) and 27 (K27) suppresses gene expression, the mono-, di-, and tri-methylations of lysine 4 (K4) in histone H3 (H3K4) associate with actively transcribed genes (Klose, R.J.
  • Histone methylation is dynamically controlled by specific histone methyltransferases and demethylases (Agger, K. et al. (2008) Curr. Opin. Genet. Dev. 18: 159-68).
  • the methylations at H3K4 are primarily catalyzed by histone methyltransferase complexes composed of the members of MLL (Mixed Lineage Leukemia) SET-domain methyltransferases, ASH2, WDR5, and RBBP5 (Klose, R.J. et al., (2007) Nat. Rev. Mol. Cell Biol. 8: 307-18; Wysocka, J. et al. (2005) Cell 121 : 859-72).
  • MLL Mated Lineage Leukemia
  • H3K4 The methylations at H3K4 allow the direct binding of proteins that contain a plant homeodomain finger domain (PHD), such as ING2, NURF, and BPTF, which in turn remodel chromatin and promote active gene expression
  • PLD plant homeodomain finger domain
  • ING2 plant homeodomain finger domain
  • BPTF BPTF
  • H3K4 histone demethylases
  • LSD1 also called KDM1, AOF2, or BHC110
  • JAREDl family 1 A-1D
  • FBXL10 FBXL10
  • H3K4 methylation is also regulated by other mechanisms.
  • previous studies indicate that a novel
  • CUL4- and DDB l -containing ubiquitin E3 ligase complex is also required for H3K4 methylation through their interaction with the WD40 repeat proteins WDR5 and RBBP5 (Higa, L. et al. (2006) Nat. Cell Biol. 8: 1277-83).
  • Lysine-specific demethylase 1 belongs to the flavin adenine dinucleotide (FAD)-dependent amine oxidase family, and specifically catalyzes the demethylation of di- and mono-methylated H3K4 through amine oxidation (Shi, Y. (2007) Nat. Rev. Genet. 8: 829-33; Shi, Y. et al., (2004) Cell 119: 941-53; Fomeris, F. et al. (2008) Trends Biochem. Sci. 33: 181- 9).
  • FAD flavin adenine dinucleotide
  • demethylation by LSD1 requires a protonated nitrogen in the methylated histone, precluding it from removing the methyl group from tri -methylated H3K4 (Shi, Y. (2007) Nat. Rev. Genet. 8: 829-33; Shi, Y. et al., (2004) Cell 119: 941-53; Culhane, J.C. et al., (2007) Curr. Opin. Chem. Biol. 11 561 -8).
  • LSD1 is highly conserved among species (Shilatifard, A. (2008) Curr. Opin. Cell Biol. 20: 341-8). Several studies from model organisms such as Drosophila revealed that LSD1 is highly expressed in primordial germ cells of females and mutation of the LSD1 gene in
  • Drosophila leads to sex-specific embryonic lethality or sterility in the remaining female offspring (DiStefano, L. et al., (2007) Curr. Biol. 17: 808-120). In mice, loss of LSD1 causes embryonic lethality (Wang, J. et al. (2009) Nat. Genet. 41: 125-9).
  • LSD1 Because the catalytic domain of LSD1 shares significant sequence and structural similarity with other members of the amine oxidase family, most of current investigation on LSD1 function involve the use of non-selective amine oxidase inhibitors, which were developed against two major isoforms of monoamine oxidases, MAO-A and MAO-B, and act through the irreversible modification of the covalently bound FAD at high concentrations (milimolars) (Shi, Y. et al., (2004) Cell 119: 941-53; Culhane, J.C. et al., (2007) Curr. Opin. Chem. Biol. 11 561-8; Schulte, J.H. et al. (2009) Cancer Res.
  • Cancer stem cells also often called cancer or tumor initiating cells, have been found in a number of cancers and are considered as the origin of various heterogeneous cancer populations due to their pluripotent or multipotent stem cell property (Lapidot, T et al., (1994) Nature 367: 645-8; Singh, S. . et al., (2004) Nature 432: 396-401; Bapat, S.A. et al., (2005) Cancer Res. 65: 3025-9; Maitland, N.J. et al., (2005) BJU Int. 96: 1219-23; Zhong, X. et al. (2010) J. Biol. Chem. 285: 41961-71 ; Yang, X. et al.
  • histone demethylase inhibitors that do not display the in vivo toxicity observed in the use of monoamine oxidase inhibitors.
  • histone demethylase inhibitors would be useful in targeting pluripotent and/or multipotent cancer stem cells, which are known to be resistant to currently available anticancer therapies.
  • the compounds, compositions, and methods of the present invention fulfill this need that was heretofore unrecognized.
  • the present invention relates to histone demethylase inhibitor compounds and compositions comprising histone demethylase inhibitors for treating cancers, such as germ cell tumors e.g., teratomas, embryonal carcinomas, seminomas, choriocarcinomas, tumors of yolk sac, ovarian teratocarcinomas/embryonal carcinomas, and cancer stem cell-like cells that express pluripotent stem cell markers such as Oct4, Sox2 and/or Lin28 and high levels of histone demethylases, such as the LSD1 protein.
  • cancers such as germ cell tumors e.g., teratomas, embryonal carcinomas, seminomas, choriocarcinomas, tumors of yolk sac, ovarian teratocarcinomas/embryonal carcinomas, and cancer stem cell-like cells that express pluripotent stem cell markers such as Oct4, Sox2 and/or Lin28 and high levels of histone demethylases, such as the LSD1 protein.
  • Cancer stem cells or cancer initiating cells that express pluripotent stem cell markers Oct4, Sox2, and/or Lin28 have been identified in many different cancer types, such as, germ cell tumors, ovarian cancers, breast cancers, and lung cancers. These cells are believed to be responsible for the recurrence of cancer and are resistant to currently available anticancer therapies.
  • the compounds disclosed herein are also useful for targeting prostate cancers, breast cancers, gliomas, glioblastomas, bladder cancers, colorectal cancers, lung cancers, skin cancers, leukemias, lymphomas, and neuroblastomas.
  • the present invention also concerns the modulation or inhibition of histone demethylases, such as LSD1, to modulate histone methylation, inhibit the growth, proliferation, and/or survival of cancer cells, as well as to affect the growth and differentiation of embryonic stem cells or the reprogramming of iPS cells from somatic cells that can be applied for stem cell-based therapy.
  • histone demethylase inhibitors of the invention can be used to eliminate embryonic
  • carcinomas/teratomas during stem cell-based therapy which are caused by incomplete differentiation of stem cells or iPS cells.
  • the present invention also embraces the use of histone demethylases, e.g., LSD1 , as therapeutic targets and biomarkers of cancers, such as germ cell tumors or cancers characterized by the presence of pluripotent and/or multipotent stem cell-like cells.
  • the present invention provides a compound represented by structural formula I:
  • Ri is an optionally substituted heterocyclic group
  • R and R' are independently selected from the group consisting of: H, CH3, NO2, S02N(CH3)2, S0 2 N((CH 3 )S0 2 ), COOH, COOCH 3 , CO(N(CH 3 )), alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl, heterocycloalkyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylarninocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, trifluoromethyl, pentafluoroethyl, halogen
  • n and n are independently integers from 1 to 4.
  • T, U, V, and Z are independendy selected from CH, N, and CR;
  • L is selected from NH, CH 2 , O, S, and S0 2 ;
  • X is selected from N or CH
  • Y and Y' are independently selected from O and S;
  • R 3 is an optionally substituted aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group.
  • R ⁇ is an optionally substituted heterocyclic group
  • R 2 , R4, R5, Re, and R7 are selected from the group consisting of: H, CH 3 , N0 2 , S0 2 N(CH 3 ) 2 , S0 2 N((CH 3 )S0 2 ), COOH, COOCH 3 , CO(N(CH 3 )), alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl, heterocycloalkyl, alkoxy, alkylcarbonyloxy,
  • arylcarbonyloxy alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, trifluoromethyl, pentafluoroethyl, halogen, cyano, thio, amido, ether, ester, hydroxyl, hydroxyalkyl, saturated or unsaturated fatty acids, azido, phosphonamido, sulfonamido, lactam, phosphate, phosphonato, phosphinato, amino, acylamino, amidino, imino, guanidino, sulfhydryl, alkylthio, aryl
  • Ri is:
  • 3 ⁇ 4 and R9 are selected from alkyl or aryl, and X, Y and Z are selected from N or C.
  • Ri is:
  • n 0 to 10.
  • R3 may be an optionally substituted aryl or heteroaryl group, an optionally substituted cycloalkyl group, or an optionally substituted heterocycloalkyl group selected from piperazine and piperidine.
  • R3 may be substituted with a group selected from an optionally substituted amidino group and a guanidine group.
  • R3 is:
  • Rio and Rn are selected from alkyl or aryl
  • Y is selected from a substituted aryl or heteroaryl group, an optionally substituted cycloalkyl group, or an optionally substituted heterocycloalkyl group.
  • R3 is:
  • n 0 to 10.
  • the present invention provides a compound represented by the structural formula ⁇ :
  • Ri is an optionally substituted heterocyclic group
  • R 2 is selected from the group consisting of: H, CH 3 , N0 2 , S0 2 N(CH 3 ) 2 , S0 2 N((CH 3 )S0 2 ), COOH, COOCH 3 , CO(N(CH 3 )), alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl, heterocycloalkyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbon
  • the Ri heterocyclic group is selected from piperazine and piperidine. In another embodiment, Ri is substituted with a group selected from an amidino group and a guanidino group.
  • R 3 may be an optionally substituted aryl group, an optionally substituted cycloalkyl group, or an optionally substituted heterocycloalkyl group selected from piperazine and piperidine.
  • R 3 may be substituted with a group selected from an amidino group and a guanidine group.
  • X is a functional group selected from the group consisting of CH2, S, NH, and
  • V is O or S
  • J, K, L, M, Y, Z and Z' are independently N, CR', or CH;
  • Ri comprises a functional group selected from the group consisting of hydrogen, a carboxamide (aminocarbonyl), a carboxamidine (carboximidamide), acyl, an alkylsulfonyl, an arylsulfonyl, guanidine, and an aminosulfonyl, any of which may be optionally substituted;
  • R, R', and R" are independently selected from the group consisting of: H, CH 3 , NO2,
  • R3 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, and
  • heterocycloalkyl group any of which is optionally substituted.
  • X is a functional group selected from the group consisting of CH 2 , S,
  • Y and Z are independently N or CH;
  • Ri comprises a functional group selected from the group consisting of hydrogen, a carboxamide (aminocarbonyl), a carboxamidine (carboximidamide), acyl, an alkylsulfonyl, an arylsulfonyl, guanidine, and an aminosulfonyl, any of which may be optionally substituted;
  • R 2 comprises a functional group selected from the group consisting of hydrogen, alkyl, nitro, sulfonamide, sulfonimide, amide, and carboxyalkyl, any of which is optionally substituted;
  • R3 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, and heterocycloalkyl group, any of which is optionally substituted.
  • the compound of the invention may be represented by the following structural formula (also referred to herein as "CBBIOOI”):
  • the compound of the invention may be represented by the following structural formula (also referred to herein as "CBB1002”):
  • the compound of the invention may also be represented by the following structural formula (also refe).
  • the compound of the invention may be represented by the following structur
  • the compound of the invention may be represented by the following structural
  • the compound of the invention is represented by the following structural formula (also referred to herein as "CBB1006”):
  • CBB 1007 The compound of the invention may also be represented by the following structural formula (also referred to herein as "CBB 1007"):
  • the compound of the invention is represented by the following structural formula (also referred to herein as "CBB1008”):
  • the compound of the invention is represented by the following structural formula (also referred to herein as "CBB1009”):
  • CBBIOIO structural formula
  • the compound of the invention may be represented by the following struc
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the compound as disclosed herein, and a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical composition may further comprise an anticancer agent.
  • Another aspect of the invention provides a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the compounds of the invention.
  • the cancer can be one that is characterized by the presence of pluripotent and/or multipotent cancer cells, such as, for example, embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, ovarian cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian epithelial cancer, gliomas, glioblastoma, lung cancer, skin cancer, leukemia, lymphoma, colorectal cancer, and bladder cancer.
  • pluripotent and/or multipotent cancer cells such as, for example, embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, ovarian cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian epithelial cancer, gliomas, glioblast
  • the invention provides a method of treating breast cancer, ovarian cancer, or prostate cancer in a subject, comprising administering a therapeutically effective amount of the compounds of the invention, wherein the compound modulates one or more histone methylation events in the subject.
  • the invention provides a method of treating breast or ovarian cancer in a subject, comprising administering a therapeutically effective amount of the compounds of the invention, wherein the compound modulates one or more histone methylation events in the subject.
  • the one or more histone methylations events may occur at one or more lysine residues of histone H3 or histone H4, such as, e.g., lysine 4, lysine 9, lysine 27, lysine 36, or lysine 79 of histone H3 or lysine 20 of histone H4.
  • the compounds of the invention may optionally be coadministered with a therapeutically effective amount of an anticancer agent.
  • a method for inhibiting the growth, proliferation, and/or survival of cancer cells and/or promoting differentiation of cancer stem cells comprising contacting the cells with an effective amount of the compounds disclosed herein, which may modulate one or more histone methylation events in the cancer cells, such as, e.g. at lysine 4, lysine 9, lysine 27, lysine 36, or lysine 79 of histone H3 or lysine 20 of histone H4.
  • the cancer cells may comprise cells that are characterized by the expression of LSD1 and may comprise pluripotent and/or multipotent cancer cells.
  • the cancer cells may be derived from a cancer selected from the group consisting of embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian epithelial cancer, gliomas, glioblastoma, lung cancer, skin cancer, colorectal cancer, leukemia, lymphoma and bladder cancer.
  • the cancer cells may be breast, ovarian or prostate cancer cells.
  • a method of modulating one or more histone methylation events in a cell comprising contacting the cell with an effective amount of the compounds of the invention.
  • the one or more histone methylation events may occur at, for example, lysine 4, lysine 9, lysine 27, or lysine 42 of histone H3.
  • the cell may be derived from a cancer characterized by the expression of LSD1.
  • the cell may also be derived from a cancer comprising pluripotent and/or multipotent cancer cells.
  • the cell may also be derived from a cancer selected from the group consisting of embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian epithelial cancer, gliomas, glioblastoma, lung cancer, skin cancer, colorectal cancer, leukemia, lymphoma and bladder cancer.
  • a cancer selected from the group consisting of embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian epithelial cancer, gliomas, glioblastoma, lung cancer, skin cancer, colorectal cancer, leukemia, lymphoma and bladder cancer.
  • a cancer selected from the group consisting of embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, chor
  • Another aspect of the present invention provides a method of detecting or diagnosing cancer in a subject, comprising measuring an effective amount of one or more histone demethylases in a sample from the subject; and comparing the amount to a reference value, wherein an increase or decrease in the amount of the one or more histone demethylases relative to the reference value indicates that the subject has cancer.
  • an increase in one or more histone demethylases relative to the reference value indicates that the subject has cancer.
  • an increase in LSDl relative to the reference value indicates that the subject has cancer.
  • a decrease in one or more histone demethylases relative to the reference value indicates that the subject has cancer.
  • the sample can be whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsies, lymphatic fluid, ascites fluid, interstitial fluid, bone marrow, cerebrospinal fluid (CSF),seminal fluid, saliva, mucous, sputum, sweat, or urine.
  • CSF cerebrospinal fluid
  • the subject can be one who has been previously diagnosed as having cancer, one who has not been previously diagnosed as having cancer, or one who is asymptomatic for cancer.
  • the measuring may comprise detecting the presence or absence of the one or more histone demethylases, quantifying the amount of the one or more histone demethylases, and qualifying the type of the one or more histone demethylases.
  • the reference value can be an index value, a value derived from one or more cancer risk prediction algorithms, a value derived from a subject not suffering from cancer, or a value derived from a subject diagnosed with cancer.
  • the one or more histone demethylases may comprise LSDl . In one embodiment, the one or more histone demethylases are measured by PCR. In another embodiment, the one or more histone demethylases are measured by immunoassay.
  • a method for monitoring the progression of cancer in a subject comprising (a) measuring an effective amount of one or more histone demethylases in a first sample from the subject at a first period of time; (b) measuring an effective amount of one or more histone demethylases in a second sample from the subject at a second period of time; and (c) comparing the amounts of the one or more histone demethylases detected in step (a) to the amount detected in step (b), or to a reference value, wherein an increase in one or more histone demethylases from the measurement in step (a) to the measurement in step (b) and/or relative to the reference value, indicates increased progression of cancer and, wherein a decrease in one or more histone demethylases from the measurement in step (a) to the measurement in step (b) and/or relative to the reference value, indicates regression of cancer.
  • the monitoring can comprise evaluating changes in the risk of developing cancer in the subject.
  • the first sample is taken from the subject prior to being treated for cancer.
  • the second sample may be taken from the subject after being treated for cancer.
  • the monitoring further comprises selecting a treatment regimen for the subject and/or monitoring the effectiveness of a treatment regimen for cancer, wherein the treatment for cancer comprises surgical intervention, administration of anticancer agents, surgical intervention following or preceded by administration of anticancer agents, or taking no further action.
  • the present invention provides a use of compounds of the invention in the manufacture of a medicament for the treatment of cancer.
  • the present invention provides a method for inhibiting the growth, proliferation, and/or survival of cancer cells, and/or promoting differentiation of cancer cells, comprising contacting the cancer cells with an effective amount of compounds of the invention.
  • the present invention provides a method of modulating one or more histone methylation events in a cell, comprising contacting the cell with an effective amount of the compounds of the invention.
  • the present invention provides a method for selecting a subject for treatment with a compound of the invention comprising: (a) measuring the level of one or more histone demethylases in said subject; and (b) comparing the level of the one or more histone demethylases detected in step (a) to a reference value; wherein when the level of one or more histone demethylases in the subject is greater than the reference value, the subject is selected for treatment with compounds of the invention.
  • a kit comprising reagents that detect one or more histone demethylases, a sample derived from a subject having normal control levels, and optionally instructions for using the reagents in the methods described herein.
  • the detection reagents may further comprise one or more antibodies or fragments thereof, one or more aptamers, one or more oligonucleotides, or combinations thereof.
  • Figures 1 A and IB show the crystal structure of LSDl and its interactions with substratelike peptide inhibitors ( Figure 1A) and small molecule inhibitors ( Figure IB).
  • Figure 1A discloses the "H3K4" sequence as SEQ ID NO: 13 and the "H3K4M” sequence as SEQ ID NO: 14.
  • Figure IB shows an illustration of the de novo designed non-peptide chemical scaffold that binds to LSDl with similar mode to that of the H3 4M peptide.
  • the guanidinium groups of the inhibitors form strong hydrogen bonds with the negatively charged residues of LSDl, and the hydrophobic substituents dock into the deep pocket that is close to FAD. Other interactions are also indicated.
  • Figure 1C shows the synthetic scheme of the histone demethylase inhibitors of the invention, using CBBIOOI as an example.
  • Figure ID shows the chemical structures of exemplary histone demethylase compounds of the invention.
  • Figure 2A-G show various analyses of exemplary compounds of the invention on LSD1-dependent demethylation in vitro.
  • Figures 2A and 2B show the results of in vitro LSDl -dependent demethylase assays.
  • Figure 2 A is a gel showing purified recombinant GST-LSDl protein expressed and isolated from the E. coli BL21 strain. Purified recombinant GST-LSDl protein was used for a demethylation assay.
  • Figure 2B shows the results of in vitro demethylase assays, demonstrating that purified LSDl protein can demethylate a histone H3 peptide substrate containing dimethylated K4 to mono-methylated and non-methylated forms in a time-dependent manner.
  • Recombinant LSDl was incubated at 30 °C for 1 hour with the di-methylated H3K4 peptide (H3K4Me2) and various concentrations of CBB1002 ( Figure 2B) or other exemplary compounds ( Figure 2C).
  • the demethylated products, mono-methylated (H3K4Mel) and non-methylated (H3K4meO), were analyzed by mass-spectrometry (MS).
  • Figure 2C shows the MS peak areas integrated and used to calculate IC50 of CBB 1002, 1003, and 1007 at 0, 1, 2, 5, 10, 20, 50, 100 ⁇ , respectively.
  • Figure 2D shows the IC50 of CBBlOOl-1009 in vitro for LSDl using the mass- spectrometry assay as calculated in B and C.
  • Figure 2E shows that exemplary compounds of the invention do not inhibit LSD2 and JARID1A.
  • LSDl, LSD2 and JARID1A demethylation reactions were analyzed using the di- methylated H3K4 substrate peptide in the presence of 0, 20, and 50 ⁇ CBB1003 and 1007 as in Figure 2B. The inhibitory effects were plotted and compared.
  • Figures 2F-G show the inhibitory effect of exemplary compounds of the invention on LSDl demethylase using methylated histone as a substrate.
  • Methylated histones and LSDl were assayed as in Figure 2B with 10 ⁇ CBB 1001-1009 (Figure 2F); or with various concentrations of CBB1002, 1003, and 1007 ( Figure 2G) as indicated.
  • the in vitro inhibitory effects of CBB compounds on the mono-, di-, and tri-methylated histone H3K4, di-methylated H3K9, and histone H3 by LSDl were analyzed by Western-blotting with specific antibodies.
  • Figures 3A -3C show the results of LSDl demethylase assays and inhibition of activity by synthetic LSDl inhibitor compounds in cultured cancer cells.
  • Figure 3 A shows the in vivo effects of LSDl inhibitory compounds of the invention on methylation of histone H3.
  • Pluripotent F9 teratocarcinma cells were treated with 10 ⁇ CBBlOOl-1009 for 24 hours.
  • Total histones were extracted and the levels of methylated H3K4, H3K9, and histone H3 were monitored by Western blotting with specific antibodies, respectively.
  • Figure 3B shows the dose effects of CBBIOOI, 1002, 1003, and 1007 on histone H3 methylation in F9 cells as assayed in Figure 3A.
  • the effects of LSD1 ablation by specific siRNA on histone methylation in F9 cells (left panels).
  • the F9 cells were treated with 50 nM luciferase (Luc) or LSD1 specific siRNAs for 48 hours and the methylation of H3K4 was analyzed.
  • Figure 3C shows the effects of LSD 1 inhibitors of the invention on epigenetic suppressed gene expression.
  • F9 cells were treated with 10 ⁇ CBBlOOl-1009 for 24. hours.
  • the activation of epigenetically suppressed CHRM4 and SCN3A genes were monitored by quantitative RT- PCR using the beta-actin gene as a control.
  • Only CBB1003, 1004, and 1006-1008 can activate the expression of CHRM4 and SCN3A genes by inhibiting LSD1.
  • Figure 3D shows the dose-dependent effects of CBB1003 and 1007 on the activation of the expression of CHRM4 and SCN3A genes in F9 and HeLa cervical carcinoma cells. The effects of LSD1 siRNAs in F9 cells were also included.
  • FIGS 4A-C show LSD1 inhibitors of the invention induce the expression of differentiation genes and are selectively permeable to cells.
  • Figures 4A and 4B show the results of in vivo analysis of LSD1 inhibitors CBB1003- CBB1007 on gene activation.
  • Pluripotent F9 teratocarcinoma cells were cultured with various concentrations of CBB1003 and CBB1007 as indicated for 24 hours.
  • the activation of the expression of SCN3A, CHRM4/M4-ArchR, and differentiation gene FOXA2 was quantified by quantitative real-time RT-PCR.
  • Figure 4B shows the IC5 0 of LSD1 inhibitors with regard to the epigenetic activated gene expression of SCN3A.
  • concentrations of CBB1003 or 1007 in Figure 3B were the same as Fig. 4A, respectively.
  • Figure 4C shows CBB1003 and CBB1007 are permeable to cells, but not CBB1002.
  • CBB1002, CBB1003 and 1007 were incubated with F9 cells for 2 hours, using dimethyl sulfoxide (DMSO) as a control. After extensive washing of treated cells, the compounds were extracted and their presence (arrows) was analyzed by mass-spectrometry using pure compounds as a control.
  • Figures 5A-5H show the results of cancer cells treated with exemplary LSDl inhibitory compounds of the invention.
  • Figures 5A-5I demonstrate that LSDl compounds selectively inhibit the growth of pluripotent embryonic carcinoma, teratocarcinoma and seminoma cells but not non-pluripotent cells.
  • Figure 5A and 5B show the inhibition of growth of F9 cells when treated with LSDl inhibitory compounds.
  • mouse pluripotent F9 teratocarcinoma cells were treated with DMSO and 50 ⁇ LSDl compounds CBBlOOl-3 and 1007 for 30 hours and the cell numbers were counted.
  • CBB1003 and 1007 significandy inhibit the growth of F9 cells but not CBBIOOI and 1002.
  • dose-response of F9 cells to various concentrations of CBB 1003 and 1007 are as indicated for 30 hours.
  • Figures 5C and 5D depict the dose-dependent response of F9 cells to various doses of LSDl compounds, as analyzed by MTT proliferation assays.
  • the percentage of compound-treated cells relative to the control is shown (see data in Figure 5A).
  • Figure 5E shows the inhibition of bromodeoxyuridine (BrdU) incorporation after F9 is treated by CBB 1003 for 24 hours .
  • Figure 5F shows the relative BrdU incorporation after treating F9 cells with 50 nM luciferase (Luc) and LSDl siRNAs for 48 hours.
  • Figures 5G and H show photographs depicting the growth of pluripotent NCCIT mixed embryonic carcinoma/seminoma cancer cells and pluripotent human testicular embryonic carcinoma NTERA-2 cells in the presence of LSDl inhibitor compounds and in non-pluripotent HeLa and 293 cancer cells.
  • Figures 5G and 5H show the growth of pluripotent NCCIT and NTERA-2 cells was inhibited by 50 ⁇ CBB1003 or CBB1007 at 30 hours but non-pluripotent HeLa, 293, and ⁇ 3 ⁇ 3 cells were not inhibited by LSDl inhibitors.
  • Figure 51 shows the growth of pluripotent mouse embryonic stem (ES) cells were inhibited by exemplary compounds of the invention.
  • the mouse ES cells were treated with either DMSO or 50 ⁇ CBB 1003 or CBB 1007 for 30 hours as indicated.
  • Figure 6A-C show inactivation of LSDl blocks the growth of pluripotent F9 and ES cells but not HeLa cells.
  • HeLa and F9 cells were transfected with 50 nM of luciferase (Luc), LSDl or LSD2 specific siRNAs for 48 hours. The cells were examined for growth inhibition and for LSD1 and LSD2 protein levels by blotting with anti-LSDl or LSD2 antibodies
  • Figure 6A shows photographs depicting growth inhibition assays of HeLa and F9 cells treated with LSD1 or LSD2 siRNA as indicated. Loss of LSD1 inhibits the growth of F9 cells but not HeLa cells. Loss of LSD2 has an opposite effects towards F and HeLa cells as compared to that of LSD 1 inhibition.
  • Figure 6B shows the results of Western blotting with anti-LSDl antibodies in HeLa and F cells with or without siRNA treatment as in Figure 6A.
  • Figure 6C shows Western blots depicting LSD1 inhibition or siRNA-based ablation of LSD1 causes the downregulation of Sox2 and Oct4 protein expression.
  • Figure 6D shows high protein levels of LSD1 in pluripotent F9, NCCIT, and NTERA-2 cancer cells that also express Oct4 and Sox2 pluripotent stem cell markers. LSD2 expression was very low in F9 and NCCIT cells.
  • Figures 7A and 7B show photographs showing immunohistological analyses of human testicular normal tissues surrounding seminomas, which were stained with anti-LSDl or Oct4 antibodies.
  • Figures 7A-B indicate elevated LSD1 protein levels in human testicular seminomas that express Oct4.
  • LSD1 and Oct4 proteins are low or non-detectable in normal testis tissue.
  • Figure 7A-7B immunohistological staining of human testicular normal tissues surrounding seminomas were stained with anti-LSDl (top) or Oct4 (bottom) antibodies.
  • three normal human testicular tissues were examined and all displayed low levels of LSD1 and Oct4. One of them is shown (Case D6).
  • Figure 7B six human testicular seminomas were stained with anti-LSDl and Oct4 antibodies. All of them displayed elevated protein levels of LSD1 and Oct4.
  • One of the seminomas was shown (Case C4).
  • Figure 8 shows Western blots showing inhibition of LSD1 demethylase activity by LSD1 inhibitors using methylated histone H3 at K4 (H3K4) isolated in mininucleosomes as a substrate.
  • Figures 9A and 9B show selective inhibition of pluripotent NCCIT cells by LSD1 inhibitor CBB 1007, but not non-pluripotent HeLa and 293 cells.
  • Figure 10 show a set of photographs showing the sensitivity of ovarian adenocarcinoma cells IGROV-1 and ovarian teratocarcinoma cells PA-1 to treatment with LSD1 inhibitor compounds CBB 1007 and 1010.
  • DMSO was used as a control.
  • Figure 11 show a set of photographs comparing the sensitivity of F9 teratoma cells and HeLa cells to RBBP5 siRNA treatment. Loss of RBBP5 altered the methylation of H3K4 and selectively inhibited the growth of pluripotent F9 cells but not non-pluripotent HeLa cells.
  • Figure 12 show a set of photographs depicting inhibition of IGROV-1 and PA-1 cell growth in the presence of RBBP5 siRNA.
  • Figures 13A-E show LSDl inhibitory compounds selectively inhibit the growth of pluripotent ovarian teratocarcinoma PA-1 cells, but not non-pluripotent ovarian carcinoma Hs38.T cells. Active growing PA-1 and Hs38.T cells were treated with indicated concentration of LSDl inhibitory compounds CBB1003 and CBB1007 for 30 hours.
  • Figures 14A-F shows LSDl inhibitory compounds selectively inhibit the growth of a subset of ovarian and breast cancer cells that express at least one of pluripotent or multipotent stem cell protein markers, as shown in Figure 18.
  • Figure 14A shows the growth inhibition of a series of A2780 cells, a human ovarian carcinoma cell, treated at various concentrations of LSDl inhibitors CBB1003 and 1007 for 30 hours.
  • Figure 14B shows the growth inhibition of a series of T47D cells, a human ductal breast epithelial carcinoma cell, treated at various concentrations of LSDl inhibitors for 60 hours.
  • Figure 14C shows the inhibition of cell viability assays after the treatment of various doses of CBB1003 and 1007 in A2780 (top graph) and T47D (lower graph) cells from Figures 14A and 14B, respectively.
  • Figure 14D shows IGROV1, a human ovarian carcinoma cell, was also growth-inhibited by CBB1007 and CBBIOIO as indicated after 30 hours. DMSO was used as a control.
  • Figure 14E shows S OV-3, a human ovarian carcinoma cell, was also growth-inhibited by LSDl inhibitors CBB1003 and CBB1007 as indicated after 30 hours.
  • Figure 14F shows MCF-7, a breast adenocarcinoma cell, was also growth-inhibited by
  • Figuresl5A-D show in vivo analysis of CBB1003 and CBB1007 on LSDl demethylation and gene activation in ovarian and breast cancer cells.
  • Figure 15 A shows the in vivo effect of LSDl compounds on methylation of histone H3 in A2780 cells.
  • A2780 cells were treated with various concentrations of CBB1003 (#3, left) and CBB1007 (#7, right) for 30 hours.
  • Total histones were extracted and the levels of methylated H3K4, H3K9, and histone H3 were monitored by Western blotting with specific antibodies, respectively.
  • Figure 15B shows the in vivo effect of LSDl compounds on methylation of histone H3 in T47D cells.
  • T47D cells were treated with various concentrations of CBB1003 (#3, left) and CBB1007 (#7, right) for 30 hours.
  • Total histones were extracted and the levels of methylated H3K4, H3K9, and histone H3 were monitored by Western blotting with specific antibodies, respectively.
  • Figure 15C shows the treatment with CBB1003 and CBB1007 for 30 hours induced the downregulation of DNMTl protein in F9 teratocarcinoma cells (on left) and ablation of LSDl by siRNA for 48 hours also induced the downregulation of DNMTl protein in F9 teratocarcinoma cells (on right).
  • CUL1 serves as a protein loading control.
  • Figure 15D shows the loss of LSDl in ovarian IGROV1 and A2780 or breast T47D carcinoma cells induced the expression of differentiation genes such as HNF4a and FOXA2.
  • IGROV1, A2780, and T47D cells were transfected with either 50 nM of a control siRNA for luciferase (Luc) or LSDl siRNA and incubated for 60 hours.
  • the expression of HNF4a and FOXA2 mRNAs was quantified by quantitative real-time RT-PCR.
  • Figures 16A-B show inactivation of LSDl inhibited the growth of cancer stem cells A2780, S OV3, and T47D but not non-stem Hs38.T ovarian carcinoma cells.
  • Figure 16A shows A2780, SKOV3 and Hs38.T cells transfected with 50 nM of luciferase (Luc) or LSDl specific siRNAs for 60 hours. The cells were examined for growth inhibition. Only cancer stem cell -like cells A2780 and SKOV3 were growth inhibited after loss of LSDl but not non-stem cell Hs38.T.
  • Figure 16B shows T47D cells transfected with 50 nM of luciferase (Luc) or LSDl specific siRNAs for 60 hours. The cells were examined for growth inhibition. Cancer stem celllike cells T47D were growth inhibited after loss of LSDl.
  • Figure 17A-C shows inactivation of RBBP5 or WDR5 that reduced the methylation of histone H3 at lysine 4 (H3K4) selectively inhibited the growth of pluripotent or multipotent F9, PA-1 , SKOV3, A2780, IGROV1 , and T47D cancer stem cells but not non-stem HeLa cancer cells.
  • Figure 17A and B F9, PA-1 , HeLa, SKOV3, A2780, IGROV1 , and T47D cells were transfected with 50 nM of luciferase (Luc) or LSD1 specific siRNAs. The cells were examined for growth inhibition after 48 hours (F9 and PA-1) or 60 hours (all other cells).
  • FIG. 17C Loss of RBBP5 or WDR5 reduced the tri- and monomethylation of histone H3 at lysine 4 (H3K4) but not methylation at H3K9. Actin is used as a protein loading control.
  • FIG 18 shows LSD 1 -sensitive cells express pluripotent or multipotent stem cell proteins/markers.
  • Pluripotent teratocarcinoma/embryonic carcinomas cells F9, NTERA-2, PA-1 cells, ovarian carcinoma cells SKV03, IGROV1 , A2780, Hs38.T, breast carcinoma cells T47D and MCF7, as well as non-stem cervical carcinoma cell HeLa were lysed and equal proteins were analyzed for their expression of pluripotent or multipotent stem cell proteins Oct4, Sox2, Lin28, Nanog, Sall4, Klf4, and LSD1 and CUL1 (control) by immunoblotting with specific antibodies, respectively.
  • the present invention relates to, inter alia, histone demethylase inhibitor compounds that were rationally designed using the substrates of LSD1 protein as a template. These compounds are highly specific towards histone demethylases such as LSD1 and are fundamentally different from broad spectrum monoamine oxidase inhibitors known in the art, which non-specifically inhibit several members of amine oxidase family proteins as well as histone demethylases.
  • the methods disclosed herein are based on the finding that inhibition of histone demethylases or modulation of histone methylations at lysine 4 (H3K4) can selectively inhibit or alter the growth of pluripotent and/or multipotent cancer cells such as, e.g., pluripotent teratocarcinoma cells, mixed germ tumor cells, seminoma cells, embryonal carcinoma cells, ovarian teratocarcinoma cells, or ovarian or breast cancer stem cell-like cells which display stem cell or progenitor cell properties.
  • pluripotent teratocarcinoma cells e.g., pluripotent teratocarcinoma cells, mixed germ tumor cells, seminoma cells, embryonal carcinoma cells, ovarian teratocarcinoma cells, or ovarian or breast cancer stem cell-like cells which display stem cell or progenitor cell properties.
  • the present invention further relates to methods of treating cancers, such as those characterized by the presence of such pluripotent and/or multipotent cancer cells and methods of inhibiting growth, proliferation, and/or survival of cancer cells, such as pluripotent and/or multipotent cancer cells.
  • the present invention further embraces the use of histone demethylases as therapeutic, diagnostic, or prognostic biomarkers for detecting, diagnosing, or monitoring the progression of cancer by measuring expression levels of one or more histone demethylases in a sample from a subject, as well as kits for measuring expression levels of one or more histone demethylases in a subject.
  • the present invention also encompasses methods of inhibiting histone demethylases to modulate the growth, survival, and differentiation of normal stem cell or related iPS (induced pluripotent stem) cells.
  • references to “a nucleic acid” includes one or more nucleic acids, and/or compositions of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
  • administering should be understood to mean providing a compound of the invention to a subject in need of treatment.
  • aryl means a monocyclic or fused multicyclic aromatic ring assembly containing six to ten ring carbon atoms. A similar term in this context is a “conjugated” ring assembly.
  • aryl can be phenyl, benzyl, or naphthyl.
  • Heteroaryl is as defined for aryl where one or more of the ring members are a heteroatom.
  • a “heteroaryl” ring may include a stable 5-, 6-, or 7-membered monocyclic or 7-, 8-, 9-, 10-, 11- or 12-membered bicyclic aromatic heterocyclic ring which consists of carbon atoms and one or more heteroatoms, e.g., 1 or 1-2 or 1 -3 or 1 -4 or 1 -5 or 1 -6 heteroatoms, or e.g., 1 , 2, 3, 4, 5, or 6 heteroatoms, independently selected from the group consisting of nitrogen, oxygen and sulfur.
  • the nitrogen atom may be substituted or unsubstituted (i.e., N or NR wherein R is H or other substituents, as defined).
  • the nitrogen and sulfur heteroatoms may optionally be oxidized.
  • heteroaryl includes, without limitation, pyrrole, furan, pyridine, indole, indazole, quinoxaline, quinoline, benzofuran, benzopyran, benzothiopyran, benzo[l,3]dioxole, imidazole, benzo-imidazole, pyrimidine, furan, oxazole, isoxazole, triazole, tetrazole, pyrazole, thiene, thiophene, thiazole, isothiazole, pyrazine, pyridazine, pyrimidine, naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, methylenedioxyphenyl, isoquinoline, naphthrydine, purine, deaza
  • the rings In the case of multicyclic aromatic rings, only one of the rings needs to be aromatic (e.g., 2,3-dihydroindole), although all of the rings may be aromatic (e.g., quinoline).
  • the second ring can also be fused or bridged.
  • the aryl or heteroaryl aromatic ring can be substituted at one or more ring positions with such substituents such as, for example, H, CH 3 , N0 2 , S0 2 N(CH 3 )2, S0 2 N((CH 3 )S0 2 ), COOH, COOCH 3) CO(N(CH 3 )), alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyi, heterocyclyl, alkylaryl, heteroaryl, heterocycloalkyl, alkoxy (i.e., methoxy, ethoxy, etc), alkylcarbonyloxy,
  • substituents such as, for example, H, CH 3 , N0 2 , S0 2 N(CH 3 )2, S0 2 N((CH 3 )S0 2 ), COOH, COOCH 3) CO(N(CH 3 )), alkyl, alkenyl, alkynyl, aryl, aralky
  • arylcarbonyloxy alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, trifluoromethyl, pentafluoroethyl, halogen (i.e., chloro, fluoro, bromo, iodo), cyano, thio, amido, ether, ester, hydroxyl, hydroxyalkyl, saturated or unsaturated fatty acids, azido, phosphonamido, sulfonamide lactam, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino
  • Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings, which are not aromatic so as to form a multicyclic system (e.g., tetralin, methylenedioxyphenyl).
  • a “biomarker” in the context of the present invention is a molecular indicator of a specific biological property; a biochemical feature or facet that can be used to measure the progress of disease or the effects of treatment.
  • Biomarker encompasses, without limitation, proteins, nucleic acids, and metabolites, together with their polymorphisms, mutations, variants, modifications, subunits, fragments, protein-ligand complexes, and degradation products, protein- ligand complexes, elements, related metabolites, and other analytes or sample-derived measures. Biomarkers can also include mutated proteins or mutated nucleic acids.
  • a combination of biomarkers, or “profile” can comprise a validated selection of optimal biomarkers.
  • biomarkers as disclosed herein include one or more histone demethylase genes and proteins.
  • Cycloalkyl means a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing the number of ring atoms indicated.
  • C3-10 cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.
  • heterocycloalkyl as used herein to describe compounds of the invention includes, but is not limited to, morpholine, pyrrolidine, piperazine, piperidine, piperidinylone, etc.
  • the cycloalkyl or heterocycloalkyl ring can be substituted at one or more ring positions with such substituents such as, for example, H, CH 3 , N0 2 , S0 2 N(CH 3 ) 2 ,
  • alkylthiocarbonyl trifluoromethyl, pentafluoroethyl, halogen (i.e., chloro, fluoro, bromo, iodo), cyano, thio, amido, ether, ester, hydroxyl, hydroxyalkyl, saturated or unsaturated fatty acids, azido, phosphonamido, sulfonamido, lactam, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, guanidine, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfiny
  • Detect or “detection” refers to identifying the presence, absence or amount of the object to be detected.
  • diagnosis is not limited to a definitive or near definitive determination that an individual has a disease, but also includes determining that an individual has an increased likelihood of having or developing the disease, compared to healthy individuals or to the general population.
  • expression and “expression levels” include but are not limited to one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
  • a “formula,” “algorithm,” or “model” is any mathematical equation, algorithmic, analytical or programmed process, or statistical technique that takes one or more continuous or categorical inputs (herein called “parameters”) and calculates an output value, sometimes referred to as an "index” or “index value.”
  • Parameters continuous or categorical inputs
  • Non-limiting examples of “algorithms” include sums, ratios, and regression operators, such as coefficients or exponents, biomarker value
  • transformations and normalizations including, without limitation, those normalization schemes based on clinical parameters, such as gender, age, smoking status, or ethnicity
  • rules and guidelines including, without limitation, those normalization schemes based on clinical parameters, such as gender, age, smoking status, or ethnicity
  • statistical classification models including, without limitation, those normalization schemes based on clinical parameters, such as gender, age, smoking status, or ethnicity
  • neural networks trained on historical populations.
  • biomarkers of the present invention are linear and non-linear equations and statistical classification analyses to "correlate" the relationship between levels of biomarkers detected in a subject sample and the subject's risk of cancer.
  • histone demethylase is a protein that catalyzes the demethylation of histones.
  • histone demethylases examples include, but are not limited to, LSD1 (also called “KDM1”, “AOF2", “KIAA0601” "FAD-binding protein BRAF35-HDAC complex, 110 kDa subunit” or “BHC110”), LSD2, members of the JARID1 family containing the Jumonji (JmjC) domain, and FBXL10.
  • LSD1 also called “KDM1", “AOF2", “KIAA0601” "FAD-binding protein BRAF35-HDAC complex, 110 kDa subunit” or “BHC110”
  • LSD2 members of the JARID1 family containing the Jumonji (JmjC) domain
  • FBXL10 Jumonji
  • histone demethylases may typically be characterized by a SWIRM domain, a FAD binding motif, and/or an amine oxidase domain or JmjC domain (Klose, R.J. et al., (2007) Nat. Rev
  • histone demethylase inhibitor includes, for example, the compounds disclosed herein, but also encompasses inhibitory nucleic acids, such as antisense nucleic acids and small interfering RNAs (siRNAs).
  • inhibitory nucleic acids such as antisense nucleic acids and small interfering RNAs (siRNAs).
  • siRNAs small interfering RNAs
  • histone demethylase inhibitors are believed to modulate one or more histone methylation events on one or more histones present in a cell, including, e.g., histone HI, histone H2A, histone H2B, histone H3, histone H4, and histone H5.
  • Measurement means assessing the presence, absence, quantity or amount (which can be an effective amount) of either a given substance within a clinical or subject-derived sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise evaluating the values or categorization of a subject's clinical parameters. Measurement or measuring may also involve qualifying the type or identifying the biomarker(s). Measurement of the biomarkers of the invention may be used to diagnose, detect, or identify cancer in a subject, to monitor the progression or prognosis of cancer in a subject, to predict the recurrence of cancer in a subject, or to classify a subject as having a low risk or a high risk of developing cancer or a recurrence of cancer.
  • Modulating or “modulate” in the context of the present invention means increasing, decreasing, or otherwise altering, adjusting, varying, changing, enhancing or inhibiting a biological event.
  • “Modulating one or more histone methylation events” means that methylation of a histone protein is increased or decreased in response to stimuli, i.e., by administration of a histone demethylase inhibitor or loss of components histone methyltransferase complexes, such as MLL, WDR5 or RBBP5 (Klose, R.J. et al., (2007) Nat. Rev. Mol. Cell Biol. 8: 307-18; Shi, Y. (2007) Nat. Rev. Genet. 8: 829-33; Agger, K. et al.
  • Histone methylation may occur at any lysine residue present in a histone protein, such as, but not limited to, lysine 4, lysine 9, lysine 14, lysine 27, lysine 36, and lysine 79 of histone H3.
  • Histone methylation also may occur at any other lysine residue in other histone proteins, such as histone HI, histone H2A, histone H2B, histone H4 (e.g., lysine 20), and histone H5 (comprising subfamily members, such as e.g., H1F, H1H1, H2AF, H2A1 , H2A2, H2BF, H2B1 , H2B2, H3A1 , H3A2, H3A3, H41 , H44).
  • Specific family members of the foregoing histone subfamilies are known in the art and include, without limitation, H1F0, H1FNT, H1FOO, HIFX,
  • HISTIHIA HISTIHIB
  • HISTIHIC HISTIHID
  • HISTIHIE HISTIHIT
  • H2AFB 1 H2AFB2, H2AFB 3, H2AFJ, H2AFV, H2AFX, H2AFY, H2AFY2, H2AFZ, HIST1 H2AA, HIST1H2AB, HIST1H2AC, HIST1H2AD, HIST1H2AE, HIST1H2AG, HIST1H2AI, HIST1H2AJ,
  • HIST1H2AK HIST1 H2AL, HIST1 H2AM, HIST2H2AA3, HIST2H2AC, H2BFM, H2BFO, H2BFS, H2BFWT, HIST1H2BA, HIST1H2BB, HIST1H2BC, HIST1 H2BD, HIST1 H2BE, HIST1 H2BF, HIST1H2BG, HIST1 H2BH, HIST1H2BI, HIST1H2BJ, HIST1H2BK,
  • HIST1H4D HIST1H4E, HIST1H4F, HIST1H4G, HIST1H4H, HIST1H4I, HIST1H4J, HIST1H4K, HIST1H4L, and HIST4H4.
  • “Risk” in the context of the present invention relates to the probability that an event will occur over a specific time period, as in the development or growth or metastasis of cancer, and can mean a subject's "absolute” risk or “relative” risk.
  • Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period.
  • Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed.
  • Odds ratios the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no-conversion.
  • Alternative continuous measures which may be assessed in the context of the present invention include time to development of cancer, or progression to a different stage of cancer, including progression or development of cancer and therapeutic cancer conversion risk reduction ratios.
  • Risk evaluation in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a "normal” condition to an at-risk condition for developing cancer, or from an at-risk condition to cancer, or development of recurrent cancer.
  • Risk evaluation can also comprise prediction of other indices of cancer, either in absolute or relative terms in reference to a previously measured population.
  • the methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to cancer, thus diagnosing and defining the risk spectrum of a category of subjects defined as at risk for developing cancer.
  • the invention can be used to discriminate between normal and at-risk subject cohorts.
  • the present invention may be used so as to discriminate at-risk conditions from cancerous conditions, or cancerous conditions from normal.
  • Such differing use may require different biomarker combinations in individual panel or profile, mathematical algorithm, and/or cut-off points, but be subject to the same aforementioned measurements of accuracy for the intended use.
  • sample in the context of the present invention is a biological sample isolated from a subject and can include, by way of example and not limitation, whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsies, lymphatic fluid, ascites fluid, interstitial fluid (also known as "extracellular fluid” and encompasses the fluid found in spaces between cells, including, inter alia, gingival crevicular fluid), bone marrow, seminal fluid, cerebrospinal fluid (CSF), saliva, mucous, sputum, sweat, urine, or any other secretion, excretion, or other bodily fluids.
  • interstitial fluid also known as "extracellular fluid” and encompasses the fluid found in spaces between cells, including, inter alia, gingival crevicular fluid
  • CSF cerebrospinal fluid
  • saliva mucous, sputum, sweat, urine, or any other secretion, excretion, or other bodily fluids.
  • Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which presents the probability of obtaining a result at least as extreme as a given data point, assuming the data point was the result of chance alone. A result is often considered highly significant at a p-value of 0.05 or less.
  • stem cell refers to an undifferentiated cell that can be induced to proliferate or differentiate.
  • the stem cell is capable of self-maintenance or self-renewal, meaning that with each cell division, one daughter cell will also be a stem cell.
  • Stem cells can be obtained from embryonic, post-natal, juvenile, or adult tissue.
  • Stem cells can be pluripotent or multipotent.
  • pluripotent refers to cells with the potential to make any differentiated cell in the body, but cannot contribute to making the components of the extraembryonic membranes which are derived from the trophoblast.
  • pluripotent stem cells Three types have been confirmed to date: embryonic stem (ES) cells, embryonic germ (EG) cells, and embryonic carcinoma (EC) cells.
  • ES embryonic stem
  • EG embryonic germ
  • EC embryonic carcinoma
  • a cell is “pluripotent” if it expresses the marker genes Oct4, Sox2, and/or Lin28.
  • multipotent is used to describe cells that can only differentiate into a limited number of types.
  • the bone marrow contains multipotent stem cells that give rise to all the cells of the blood but may not be able to differentiate into other cells types.
  • Pluripotent and multipotent cells encompass any cancer stem cell/cancer stem celllike cell or cancer/tumor initiating cells.
  • a “cancer stem cell” in the context of the present invention relates to cells that are considered as the origin of various heterogeneous cancer populations due to their pluripotent or multipotent stem cell properties. Such cells can be of hematogenic in origin or may be present in solid tumors.
  • the term "progenitor cell” refers to an undifferentiated cell derived from a stem cell, and is not itself a stem cell. Some progenitor cells can produce progeny that are capable of differentiating into more than one cell type.
  • a "subject" in the context of the present invention is preferably a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of cancer, such as nude mice.
  • a subject can be male or female.
  • a subject can be one who has been previously diagnosed or identified as having cancer, and optionally has already undergone, or is undergoing, a therapeutic intervention or treatment for the cancer.
  • a subject can also be one who has not been previously diagnosed as having cancer.
  • a subject can be one who exhibits one or more risk factors for cancer, or a subject who does not exhibit risk factors for cancer, or a subject who is
  • a subject can also be one who is suffering from or at risk of developing cancer, or who is suffering from or at risk of developing a recurrence of cancer.
  • a subject can also be one who is suffering from or at risk of developing metastatic cancer.
  • a subject can also be one who has been previously treated for cancer, whether by administration of anticancer agents, radiation therapy, surgery, or any combination of the foregoing.
  • substituted means that any one or more hydrogen atoms on the designated atom is replaced with a selection from the indicated groups, provided that the designated atom's normal valency is not exceeded, and that the substitution results in a stable compound.
  • 2 hydrogen atoms on the atom are replaced.
  • Keto substituents are not present on aromatic moieties.
  • Non-limiting examples of such groups include, without limitation, H, CH3, NO2, S0 2 N(CH 3 ) 2 , S0 2 N((CH 3 )S0 2 ), COOH, COOCH3, CO(N(CH 3 )), alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heterocyclyl, alkylaryl, heteroaryl, heterocycloalkyl, alkoxy (i.e., methoxy, ethoxy, etc), alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbortyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylaminocarbonyl, alkoxycarbonyl, aminocarbonyl
  • the compounds, compositions, biomarkers, kits, and methods of the present invention can be used in the diagnosis, prognosis, monitoring, and treatment of cancer in vivo as well as for the inhibition of growth and/or proliferation of cancer cells in vitro, preferably those
  • pluripotent and or multipotent cancer cells such as, e.g., embryonic carcinomas, teratomas, seminomas, germ cell tumors, choriocarcinomas, yolk sac tumors, ovarian epithelial cancers, but can also be used to detect, diagnose, monitor, or treat a wide variety of other cancers, including but not limited to solid tumors (e.g., tumors of the head and neck, lung, breast, colon, colorectal, prostate (which can be androgen dependent), bladder, rectum, brain, gastric tissue, bone, ovary, thyroid, or endometrium), hematological malignancies (e.g., leukemias, lymphomas, myelomas), carcinomas (e.g.
  • pluripotent and or multipotent cancer cells such as, e.g., embryonic carcinomas, teratomas, seminomas, germ cell tumors, choriocarcinomas, yolk sac tumors, ovarian epithelial
  • Non-limiting examples of these cancers include diffuse large B- cell lymphoma (DLBCL), T-cell lymphomas or leukemias, e.g., cutaneous T-cell lymphoma (CTCL), noncutaneous peripheral T-cell lymphoma, lymphoma associated with human T-cell lymphotrophic virus (HTLV), adult T-cell leukemia/lymphoma (ATLL), as well as acute lymphocytic leukemia, acute nonlymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, myeloma, multiple myeloma, mesothelioma,
  • DLBCL diffuse large B- cell lymphoma
  • CTCL cutaneous T-cell lymphoma
  • HTLV human T-cell lymphotrophic virus
  • ATLL adult T-cell leukemia/lymphoma
  • lung cancer e.g., small cell carcinoma and non-small cell lung carcinoma, including squamous cell carcinoma and adenocarcinoma
  • lung cancer e.g., small cell carcinoma and non-small cell lung carcinoma, including squamous cell carcinoma and adenocarcinoma
  • Therapeutic agents for treating or reducing the risk of cancer include, without limitation of the following, radiation therapy with or without “anticancer agents", such as, but not limited to, an alkylating agent, an antibiotic agent, an antimetabolic agent, a hormonal agent, a plant- derived agent, an anti-angiogenic agent, a differentiation inducing agent, a cell growth arrest inducing agent, an apoptosis inducing agent, a cytotoxic agent, a biologic agent, a gene therapy agent, a retinoid agent, an histone deacetylase inhibitor, an enzyme inhibitor, a cytokine, a chemokine, an antibody, a DNA molecule, an RNA molecule, a small molecule, a peptide, or a peptidomimetic, or any combination thereof.
  • anticancer agents such as, but not limited to, an alkylating agent, an antibiotic agent, an antimetabolic agent, a hormonal agent, a plant- derived agent, an anti-angiogenic agent,
  • treating in its various grammatical forms in relation to the present invention refers to preventing (e.g.,- chemoprevention), curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease state, disease progression, disease causative agent (e.g., bacteria or viruses) or other abnormal condition.
  • treatment may involve alleviating a symptom (i.e., not necessary all symptoms) of a disease or attenuating the progression of a disease.
  • Treatment of cancers refers to partially or totally inhibiting, delaying or preventing the progression of cancer including cancer metastasis; inhibiting the growth, proliferation and/or survival of cancer cells, inhibiting, delaying or preventing the recurrence of cancer including cancer metastasis; or preventing the onset or development of cancer (e.g., chemoprevention) in a subject.
  • the term "therapeutically effective amount" is intended to qualify a desired biological response, such as, e.g., is partial or total inhibition, delay or prevention of the progression of cancer including cancer metastasis; inhibition, delay or prevention of the recurrence of cancer including cancer metastasis; or the prevention of the onset or development of cancer (e.g., chemoprevention) in a subject.
  • the present invention provides a compound of formula V:
  • Ri comprises a functional group selected from the group consisting of a carboxamide (aminocarbonyl), a carboxamidine (carboximidamide), acyl, an alkylsulfonyl, an arylsulfonyl, and an aminosulfonyl, any of which may be optionally substituted;
  • R. 2 comprises a functional group selected from the group consisting of hydrogen, alkyl, nitro, sulfonamide, sulfonimide, amide, and carboxyalkyl, any of which is optionally substituted;
  • R3 comprises one selected from the group consisting of aryl, heteroaryl, cycloalkyl, and heterocycloalkyl group, any of which is optionally substituted.
  • a terminal guanidine or carboxamidine can be substituted, replacing any NH or NH 2 hydrogen with an alkyl, hydroxyl, alkoxy, or sulfur containing functional group, such as sulfhydryl, sulfone, sulfonyl, and the like.
  • R 2 is a functional group selected from the group consisting of hydrogen, methyl, nitro, N,N-dimethylsulfonamide, MeS0 2 NMeS0 2 -, N-methylcarboxamide, and carboxymethyl.
  • R3 in the compound of formula V R3 is selected from the group consisting of:
  • a terminal guanidine or carboxamidine can be substituted, replacing any NH or NH2 hydrogen with an alkyl, hydroxyl, alkoxy, or sulfur containing functional group, such as sulfhydryl, sulfone, sulfonyl, and the like.
  • the compound of formula V is selected from on of compounds 1- 19 below:
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the compound of formula V or any of the disclosed genus, subgenus, or the species disclosed herein, in conjunction with a pharmaceutically acceptable carrier or diluent.
  • Pharmaceutical compositions of the invention can further include an anticancer agent as described herein.
  • the compounds of the present invention may exist in one or more particular geometric, optical, enantiomeric, diastereomeric, epimeric, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; a- and ⁇ -forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and half chair-forms; and
  • the compounds of the invention when used in pharmaceutical or diagnostic applications may be prepared in a racemic mixture or an essentially pure enantiomer form, with an enantiopurity of at least 90% enantiomeric excess (EE), preferably at least 95% EE, more preferably at least 98% EE, and most preferably at least 99% EE.
  • Enantiomeric excess values provide a quantitative measure of the excess of the percentage amount of a major isomer over the percentage amount of a minor isomer which is present therewith, and may be readily determined by suitable methods well-known and established in the art, as for example chiral high pressure liquid chromatography (HPLC), chiral gas chromatography (GC), nuclear magnetic resonance (NMR) using chiral shift reagents, etc.
  • a “pharmaceutical composition” is a formulation containing the compounds of the present invention in a form suitable for administration to a subject.
  • pharmaceutically acceptable refers to those compounds, materials, compositions, carriers, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), and transmucosal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens;
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as
  • ethylenediaminetetraacetic acid ethylenediaminetetraacetic acid
  • buffers such as acetates, citrates or phosphates
  • agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compounds and pharmaceutical compositions of the invention may be administered in therapeutically effective amounts via any of the usual and acceptable modes known in the art, either singly or in combination with one or more therapeutic agents.
  • a therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors involved, as readily determinable within the skill of the art.
  • Suitable therapeutic doses of the compounds of the invention may be in the range of 1 microgram ( ⁇ %) to 1000 milligrams (mg) per kilogram body weight of the recipient per day, and any increment in between, such as, e.g., 1, 2, 3, 5, 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 ⁇ g (1 mg); 2, 3, 5, 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mg.
  • a desired dose may preferably be presented as two, three, four, five, six, or more sub-doses administered at appropriate intervals throughout the day.
  • sub-doses may be administered in unit dosage forms, for example, containing from 1 ⁇ g to 1000 mg of active ingredient per unit dosage form. Alternatively, if the condition of the recipient so requires, the doses may be administered as a continuous infusion.
  • the mode of administration and dosage forms will of course affect the therapeutic amounts of the compounds which are desirable and efficacious for the given treatment application.
  • orally administered dosages typically are at least twice, e.g., 2-10 times, the dosage levels used in parenteral administration methods, for the same active ingredient.
  • dosage levels for delta receptor binding compounds of the invention may be on the order of 5-200 mg 70 kg body weight/day.
  • typical active agent dose levels are on the order of 10-100 mg per tablet.
  • the compounds of the present invention may be administered per se as well as in the form of pharmaceutically acceptable esters, salts, and ethers, as well as other physiologically functional derivatives of such compounds.
  • Compounds of the invention may be amorphous or polymorphic.
  • crystal polymorphs means crystal structures in which a compound (or a salt or solvate thereof) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystal forms usually have different X-ray diffraction patterns, infrared spectral, melting points, density hardness, crystal shape, optical and electrical properties, stability and solubility.
  • crystal lattice forms include, but are not limited to, cubic, isometric, tetragonal, orthorhombic, hexagonal, trigonal, triclinic, and monoclinic. Recrystallization solvent, rate of crystallization, storage temperature, and other factors may cause one crystal form to dominate. Crystal polymorphs of the compounds can be prepared by crystallization under different conditions.
  • the compounds of the present invention can exist in either hydrated or unhydrated (the anhydrous) form or as solvates with other solvent molecules.
  • “Solvate” means solvent addition forms that contain either stoichiometric or non stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water the solvate formed is a hydrate; and if the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one or more molecules of water with one molecule of the substance in which the water retains its molecular state as 3 ⁇ 40. Non-limiting examples of hydrates include monohydrates, dihydrates, etc. Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc.
  • Examples of pharmaceutically acceptable acid addition salts include those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; as well as organic acids such as acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, 3-(4-hydroxybenzoyl)benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,
  • benzenesulfonic acid 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4- toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy- 2-ene-l -carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, p-toluenesulfonic acid, and salicylic acid and the like.
  • Examples of a pharmaceutically acceptable base addition salts include those formed when an acidic proton present in the parent compound is replaced by a metal ion, such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferable salts are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins.
  • organic bases include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diemylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, tromethamine, N-methylglucamine, polyamine resins, and the like.
  • organic bases are isopropylamine, diethylamine, ethanolamine, trimemylamine, dicyclohexylamine, choline, and caffeine.
  • Compounds of the invention can be administered as pharmaceutical compositions by any conventional route, in particular enterally, e.g., orally, e.g., in the form of tablets or capsules, or parenterally, e.g., in the form of injectable solutions or suspensions, topically, e.g., in the form of lotions, gels, ointments or creams, or in a nasal or suppository form or in inhaled forms.
  • compositions comprising a compound of the present invention in free form or in a pharmaceutically acceptable salt form in association with at least one pharmaceutically acceptable carrier or diluent can be manufactured in a conventional manner by mixing, granulating or coating methods.
  • oral compositions can be tablets or gelatin capsules comprising the active ingredient together with a pharmaceutically acceptable carrier, including any one or a combination of the following components: a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners.
  • diluents
  • compositions can be aqueous isotonic solutions or suspensions, and suppositories can be prepared from fatty emulsions or suspensions.
  • the compositions can be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they can also contain other therapeutically valuable substances.
  • Suitable formulations for transdermal applications include an effective amount of a compound of the present invention with a carrier.
  • a carrier can include absorbable
  • transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.
  • Matrix transdermal formulations can also be used.
  • Suitable formulations for topical application, e.g., to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels well-known in the art. Such can contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.
  • the active compounds can be prepared with pharmaceutically acceptable carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No.
  • Compounds of the invention can be administered in therapeutically effective amounts in combination with one or more anticancer agents as defined herein. For example, synergistic effects can occur with other substances used in the treatment of cancers. Where the compounds of the invention are administered in conjunction with other therapies, dosages of the coadministered compounds will of course vary depending on the type of co-drug employed, on the specific drug employed, on the condition being treated and so forth.
  • the terms “combination treatment”, “combination therapy”, “combined treatment” or “combinatorial treatment”, used interchangeably, refer to a treatment of an individual with at least two different therapeutic agents.
  • the terms “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of
  • pharmaceutical combination means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients.
  • a "fixed combination” means that the active ingredients, e.g. a compound as disclosed herein and an anticancer agent, are both administered to a patient simultaneously in the form of a single entity or dosage.
  • a "non-fixed combination” means that the active ingredients, e.g. a compound as disclosed herein and anticancer agent, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the 2 compounds in the body of the patient.
  • cocktail therapy e.g. the administration of 3 or more active ingredients.
  • Anticancer agents for treating or reducing the risk of cancer include, without limitation of the following, radiation therapy, compounds such as, but not limited to, an alkylating agent, an antibiotic agent, an antimetabolic agent, a hormonal agent, a plant-derived agent, an anti- angiogenic agent, a differentiation inducing agent, a cell growth arrest inducing agent, an apoptosis inducing agent, a cytotoxic agent, a biologic agent, a gene therapy agent, a retinoid agent, an histone deacetylase inhibitor, a tyrosine kinase inhibitor, an enzyme inhibitor, a cytokine, a chemokine, an antibody, a DNA molecule, an RNA molecule, a small molecule, a peptide, or a peptidomimetic, or any combination thereof.
  • radiation therapy compounds such as, but not limited to, an alkylating agent, an antibiotic agent, an antimetabolic agent, a hormonal agent, a plant-derived agent, an
  • anti-cancer agents encompass those described herein, as well as inhibitory nucleic acids, including any pharmaceutically acceptable salts or hydrates of such agents, or any free acids, free bases, or other free forms of such agents.
  • alkylating agents include, but are not limited to, bischloroethylamines (nitrogen mustards, e.g., Chlorambucil, Cyclophosphamide, Ifosfamide, MechJorethamine, Melphalan, uracil mustard), aziridines (e.g., Thiotepa), alkyl alkone sulfonates (e.g., Busulfan), nitrosoureas (e.g., Carmustine, Lomustine, Streptozocin), non-classic alkylating agents
  • antibiotic agents include, without limitation, anthracyclines (e.g., phenyl)-2-aminoethracyclines
  • Doxorubicin, Daunorubicin, Epirubicin, Idarubicin, and Anthracenedione Mitomycin C, Bleomycin, Dactinomycin, and Plicatomycin.
  • antimetabolic agents include, but are not limited to, Fluorouracil (5-FU),
  • Floxuridine (5-FUdR), Methotrexate, Leucovorin, Hydroxyurea, Azacytidine, Flavopiridol, Thioguanine (6-TG), Mercaptopurine (6-MP), Cytarabine, Pentostatin, Fludarabine Phosphate, Cladribine (2-CDA), Asparaginase, Gemcitabine, and Pemetrexed.
  • hormonal agents include, but are not limited to, synthetic estrogens (e.g., Diethylstibestrol), antiestrogens (e.g., Tamoxifen, Toremifene, Fluoxymesterol, and Raloxifene), antiandrogens (e.g., Bicalutamide, Nilutamide, and Flutamide), aromatase inhibitors (e.g., Aminoglutethimide, Anastrozole, and Tetrazole), luteinizing hormone release hormone (LHRH) analogues, Ketoconazole, Goserelin Acetate, Leuprolide, Megestrol Acetate, Prednisone, and Mifepristone.
  • synthetic estrogens e.g., Diethylstibestrol
  • antiestrogens e.g., Tamoxifen, Toremifene, Fluoxymesterol, and Raloxifene
  • antiandrogens e.g., Bicalutamide, Ni
  • plant-derived agents include, but are not limited to, vinca alkaloids (e.g., vinca alkaloids).
  • Vincristine, Vinblastine, Vindesine, Vinzolidine, and Vinorelbine e.g., Etoposide (VP- 16) and Tenyposide (VM-26)
  • taxanes e.g., Paclitaxel and Docetaxel
  • biologic agents include, but are not limited to, immunomodulating proteins such as cytokines (such as interleukin-2 (IL-2, Aldesleukin), Epoietin- a; EPO), granulocyte- CSF (G-CSF; Filgrastin), and granulocyte, macrophage-CSF (GM-CSF; Sargramostim and interferons, e.g., interferon-a, interferon- ⁇ (fibroblast interferon) and interferon- ⁇ (lymphocyte interferon)), bacillus Calmette-Guerin, levamisole, and octreotide, monoclonal antibodies against tumor antigens (such as Herceptin (trastuzumab), Rituxan (rituximab), Myelotarg (gemtuzumab ozogamicin) and Campath (alemtuzumab), endostatin, tumor suppressor genes (e.g., DPC4, NF- 1, NF-2
  • retinoid agents include all natural, recombinant, and synthetic derivatives or mimetics of vitamin A, for example, retinyl palmitate, retinoyl-beta-glucuronide (vitamin Al beta-glucuronide), retinyl phosphate (vitamin Al phosphate), retinyl esters, 4-oxoretinol, 4- oxoretinaldehyde, 3-dehydroretinol (vitamin A2), 11-cis -retinal (11-cis-retinaldehyde, 11-cis or neo b vitamin Al aldehyde), 5,6-epoxyretinol (5,6-epoxy vitamin Al alcohol), anhydroretinol (anhydro vitamin Al) and 4-ketoretinol (4-keto- vitamin Al alcohol), all-trans retinoic acid (ATRA; Tretinoin; vitamin A acid; 3,7-dimethyl-9-(2,6,6,-
  • lipid formulations of all-trans retinoic acid e.g., ATRA-IV
  • 9-cis retinoic acid (9-cis-RA; Alitretinoin; Panretin ⁇ ; LGD1057)
  • Fenretinide N-(4-hydroxyphenyl)retinamide; 4-HPR
  • Acitretin (Ro 10-1670), Tazarotene (ethyl 6-[2-(4,4- dimethylthiochroman-6-yl)-ethynyl] nicotinate), Tocoretinate (9-c
  • histone deacetylase inhibitors include, without limitation, MS-275, depsipeptide, Cl-994, Apicidin, A-161906, Scriptaid, PXD-101, CHAP, LAQ-824, Butyric acid, depudecin, oxamflatin, trichostatin A, trichostatin C, suberoylanilide hydroxamic acid (SAHA), m-Carboxycinnamic acid bishydroxamide (CBHA), Pyroxamide; Salicylbishydroxamic acid; Suberoyl bishydroxamic acid (SBHA); Azelaic bishydroxamic acid (ABHA); Azelaic-1- hydroxamate-9-anilide (AAHA); 6-(3-Chlorophenylureido) carpoic hydroxamic acid (3C1- UCHA); MW2796; MW2996, trapoxin A, sodium butyrate, isovalerate, valerate, 4- phenyl
  • tyrosine kinase inhibitors include, e.g., DMPQ (5,7-dimethoxy-3-(4- pyridinyl)quinoline dihydrochloride), Aminogenistein (4'-amino-6-hydroxyflavone), Erbstatin analog (2,5-dihydroxymethylcinnamate, methyl 2,5-dihydroxycinnamate), Imatinib (GleevecTM' GlivecTM; STI-571 ; 4-[(4-methyl-l-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2- yrimidinyl] amino] -phenyl ]benzamide methanesulfonate), LFM-A13 (2-Cyano-N-(2,5- dibromophenyl)-3-hydroxy-2-butenamide), PD153035 (ZM 252868; 4-[(3-bromophenyl)
  • Erlotinib and derivatives e.g., Tarceva®; NSC 718781, CP- 358774, OSI-774, R1415; N-(3-emynylphenyl)-6,7-bis(2-memoxyethoxy)-4-qwnazolinamine.
  • the invention encompasses the use of inhibitory nucleic acids.
  • Inhibitory nucleic acids may be designed based on the sequences of one or more histone demethylases as defined herein, either in whole or in part (i.e., sequences of conserved domains), or may be designed based on identification of biomarkers that indicate cancer status and progression of disease in a subject.
  • the invention features histone demethylase inhibitory nucleic acid molecules.
  • Histone demethylase inhibitory nucleic acid molecules are essentially oligomers or oligonucleotides that may be employed as single-stranded or double-stranded nucleic acid molecule to decrease or ablate histone demethylase expression.
  • the histone demethylase inhibitory nucleic acid molecule is a double- stranded RNA used for RNA interference (RNAi)-mediated knock-down of histone demethylase gene expression.
  • a double-stranded RNA (dsRNA) molecule is made that includes between eight and twenty-five (e.g., 8, 10, 12, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25) consecutive nucleotides.
  • the dsRNA can be two complementary strands of RNA that have duplexed, or a single RNA strand that has self -duplexed (small hairpin (sh)RNA).
  • dsRNAs are about 21 or 22 base pairs, but may be shorter or longer (up to about 29 nucleotides) if desired.
  • Double stranded RNA can be made using standard techniques (e.g., chemical synthesis or in vitro transcription). Kits are available, for example, from Ambion (Austin, Tex.) and Epicentre (Madison, Wis.). Methods for expressing dsRNA in mammalian cells are described in Brummelkamp et al., (2002) Science 296: 550-553; Paddison et al., (2002) Genes & Devel. 16:948-958. Paul et al., (2002) Nature Biotechnol.
  • An inhibitory nucleic acid molecule that "corresponds" to one or more histone demethylase genes comprises at least a fragment of the double-stranded gene, such that each strand of the double-stranded inhibitory nucleic acid molecule is capable of binding to the complementary strand of a target histone demethylase gene.
  • the inhibitory nucleic acid molecule need not have perfect correspondence to the reference histone demethylase sequence.
  • a siRNA has at least about 85%, 90%, 95%, 96%, 97%, 98%, or even 99% sequence identity with the target nucleic acid. For example, a 19 base pair duplex having a 1 - 2 base pair mismatch is considered useful in the methods of the invention.
  • the nucleotide sequence of the inhibitory nucleic acid molecule exhibits 1, 2, 3, 4, 5 or more mismatches.
  • the inhibitory nucleic acid molecules provided by the invention are not limited to siRNAs, but include any nucleic acid molecule sufficient to decrease the expression of a histone demethylase nucleic acid molecule or polypeptide.
  • Each of the DNA sequences provided herein may be used, for example, in the discovery and development of therapeutic antisense nucleic acid molecule to decrease the expression of one or more histone demethylases.
  • the invention further provides catalytic RNA molecules or ribozymes. Such catalytic
  • RNA molecules can be used to inhibit expression of a histone demethylase nucleic acid molecule in vivo.
  • the inclusion of ribozyme sequences within an antisense RNA confers RNA-cleaving activity upon the molecule, thereby increasing the activity of the constructs.
  • the design and use of target RNA-specific ribozymes is described in Haseloff et al., (1998) Nature 334: 585-591 and U:S. Patent Application Publication No. 20030003469, each of which is incorporated by reference.
  • the catalytic nucleic acid molecule is formed in a hammerhead or hairpin motif.
  • the inhibitory nucleic acid molecules of the invention may be administered systemically in dosages between about 1 and 100 mg/kg (e.g., 1, 5, 10, 20, 25, 50, 75, and 100 mg/kg). The dosage may range from between about 25 and 500 mg/m 2 /day.
  • a human subject having cancer can receive a dosage between about 50 and 300 mg/m 2 /day (e.g., 50, 75, 100, 125, 150, 175, 200, 250, 275, and 300).
  • the amounts of the inhibitory nucleic acid molecules administered to the subject will depend, of course, on whether it is administered alone or in combination with another anticancer agent, such as the histone demethylase inhibitor compounds disclosed herein.
  • inhibitory nucleic acid molecule is based on 2' -modified oligonucleotides containing oligodeoxynucleotide gaps with some or all internucleotide linkages modified to phosphorothioates for nuclease resistance.
  • the presence of methylphosphonate modifications increases the affinity of the oligonucleotide for its target RNA and thus reduces the IC50.
  • This modification also increases the nuclease resistance of the modified oligonucleotide. It is understood that the methods and reagents of the present invention may be used in conjunction with any technologies known to those skilled in the art that may be developed to enhance the stability or efficacy of an inhibitory nucleic acid molecule.
  • Inhibitory nucleic acid molecules include oligomers containing modified backbones or non-natural internucleoside linkages. Oligomers having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are also considered to be oligomers.
  • Oligomers that have modified oligonucleotide backbones include, for example,
  • phosphorothioates chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl- phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, thionophosphoramidates,
  • Oligomers having modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • Oligomers may also contain one or more substituted sugar moieties. Such modifications include 2'-0-methyl and 2'-methoxyethoxy modifications. Another desirable modification is 2'- dimethylaminooxyethoxy, 2'-aminopropoxy and 2'-fluoro. Similar modifications may also be made at other positions on an oligonucleotide or oligomer, particularly the 3' position of the sugar on the 3' terminal nucleotide. Oligomers may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;
  • the present invention provides a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the compound of formula V or any of the disclosed genus, subgenus, or species disclosed herein.
  • the cancer comprises pluripotent and/or multipotent cancer cells expressing LSDl.
  • the cancer comprises cells expressing at least one pluripotent or multipotent stem cell protein marker (Lapidot, T et al., (1994) Nature 367: 645-8; Singh, S.K. et al., (2004) Nature 432: 396-401 ; Bapat, S.A. et al., (2005) Cancer Res. 65: 3025- 9; Maidand, N.J.
  • the at least one pluripotent stem cell protein marker is selected from the group consisting of Oct4, Sox2, Lin28, Nanog, Klf4, Sall4, Lin28B, cMyc, nMyc,
  • the cancer cell can express any two of these pluripotent stem cell protein markers, such as Oct4 and Sox2, Oct4 and Lin28, or Sox2 and Lin28. In some embodiments, the cancer cell may express any three of these pluripotent stem cell protein markers, such as Oct4, Sox, 2, and Lin28. In some such embodiments, the cancer cells can express the three cancer stem cell protein markers Wnt/beta-catenin, Notch, and Hedgehog, or three cancer stem cell markers can be CD133 + , CD44 + , and CD24 " . In some embodiments, the cancer cell can express and four, five, six, seven, eight, nine, ten, up to all of the pluripotent or multipotent stem cell protein markers described herein. In some embodiments, the presence of any known or discovered pluripotent or multipotent stem cell protein can be used as a marker indicating treatment with compounds of the present invention.
  • the cancer to be treated can be at least one selected from the group consisting of: embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian cancer, endometrial cancer, cervical cancer, retinoblastoma, kidney cancer, liver cancer, gastric cancer, brain cancer, medulloblastoma, medulloepithelioma, glioma, glioblastoma, lung cancer, bronchial cancer, mesothelioma, skin cancer, colon and rectal cancer, bladder cancer, pancreatic cancer, lip and oral cancer, laryngeal and pharyngeal cancer, melanoma, pituitary cancer, penile cancer, parathyroid cancer, thyroid cancer, pheochromocytoma and paraganglioma, thymoma and thymic Carcinoma, leukemia, lympho
  • the cancer comprises breast cancer. In some such embodiments, the cancer comprises ovarian cancer.
  • Any cancer treatment regimen, in accordance with methods of the invention, can include administering a therapeutically effective amount of an anticancer agent in conjunction with compounds of the invention.
  • the present invention provides for the use of a compound of formula V or any of the disclosed genus, subgenus, or species disclosed herein, in the manufacture of a medicament for the treatment of cancer.
  • the cancer comprises pluripotent and/or multipotent cancer cells expressing LSD1.
  • the cancer comprises cells expressing at least one pluripotent or multipotent stem cell protein marker.
  • the at least one pluripotent stem cell protein marker is selected from the group consisting of Oct4, Sox2, Lin28, Nanog, Klf4, and Sall4, Lin28B, cMyc, nMyc, LMyc, Wnt3a, miR-291-3p, miR-294, miR-295, miR-290/371 cluster, miR-302 cluster, miR-363 cluster, miR-520 cluster, miR-92b, miR148/152, miR-124, miR-615, miR-708, miR-9, Klfl, Klf2, Klf5, Esrrb, Esrrg, Soxl, Sox3, Soxl5, Soxl8, Smad7, Nr5al, Nr5a2,
  • the cancer comprises at least one cancer selected from the group consisting of: embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian cancer, endometrial cancer, cervical cancer, retinoblastoma, kidney cancer, liver cancer, gastric cancer, brain cancer, medulloblastoma, medulloepithelioma, glioma, glioblastoma, lung cancer, bronchial cancer, mesothelioma, skin cancer, colon and rectal cancer, bladder cancer, pancreatic cancer, lip and oral cancer, laryngeal and pharyngeal cancer, melanoma, pituitary cancer, penile cancer, parathyroid cancer, thyroid cancer, pheochromocytoma and paraganglioma, thymoma and thymic Carcinoma, leukemia, lymphoma
  • the medicament further comprises a therapeutically effective amount of an anticancer agent as described herein.
  • the present invention provides a method for inhibiting the growth, proliferation, and/or survival of cancer cells, comprising contacting the cancer cells with an effective amount of the compound of formula V or any of the disclosed genus, subgenus, or species disclosed herein.
  • methods for inhibiting the growth, proliferation, survival of cancer cells, and/or promoting the differentiation of cancer cells can further include contacting the cancer cells with an anticancer agent.
  • the cancer cells comprise pluripotent and/or multipotent cancer cells expressing LSD1.
  • the cancer cells comprise cells expressing at least one pluripotent or multipotent stem cell protein marker.
  • the at least one pluripotent stem cell protein marker is selected from the group consisting of Oct4, Sox2, Lin28, Nanog, Klf4, and Sall4, Lin28B, cMyc, nMyc, LMyc, Wnt3a, miR-291-3p, miR-294, miR-295, miR-290/371 cluster, miR-302 cluster, miR-363 cluster, miR-520 cluster, miR-92b, miR148/152, miR-124, miR-615, miR-708, nuR-9, Klfl, KIf2, Klf5, Esrrb, Esrrg, Soxl, Sox3, Soxl5, Soxl8, Smad7, Nr5al, Nr5a2, Wnt beta-catenin, Met, Notch, Hedgehog, Sonic Hedgehog, CD133, CD44, CD44 + /CD247ESA +
  • the cancer cells comprise at least one cancer selected from the group consisting of: embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian cancer, endometrial cancer, cervical cancer, retinoblastoma, kidney cancer, liver cancer, gastric cancer, brain cancer, medulloblastoma, medulloepithelioma, glioma, glioblastoma, lung cancer, bronchial cancer, mesothelioma, skin cancer, colon and rectal cancer, bladder cancer, pancreatic cancer, lip and oral cancer, laryngeal and pharyngeal cancer, melanoma, pituitary cancer, penile cancer, parathyroid cancer, thyroid cancer,
  • the at least one cancer comprises breast cancer. In some such embodiments, the at least one cancer comprises ovarian cancer.
  • methods for inhibiting the growth, proliferation, survival of cancer cells, and/or promoting the differentiation of cancer cells can be conducted in vivo. In some embodiments, methods for inhibiting the growth, proliferation, survival of cancer cells, and/or promoting the differentiation of cancer cells conducted in vitro.
  • the present invention provides a method of modulating one or more histone methylation events in a cell, comprising contacting the cell with an effective amount of the compound of formula V or any of the disclosed genus, subgenus, or species disclosed herein.
  • the one or more histone methylation events occur at lysine 4, lysine 9, lysine 27, lysine 36, lysine 79 of histone H3 or lysine 20 of histone H4.
  • the cell in methods of modulating one or more histone methylation events in a cell, is derived from a cancer expressing LSD1.
  • the cell in methods of modulating one or more histone methylation events in a cell, is derived from a cancer comprising pluripotent and/or multipotent cancer cells.
  • the cell in methods of modulating one or more histone methylation events in a cell, is derived from a cancer expressing at least one pluripotent stem cell protein marker.
  • the at least one pluripotent stem cell protein marker is selected from the group consisting of Oct4, Sox2, Lin28, Nanog, Klf4, and Sall4, Lin28B, cMyc, nMyc, LMyc, Wnt3a, miR-291-3p, miR-294, miR-295, miR-290/371 cluster, miR-302 cluster, nuR-363 cluster, miR- 520 cluster, miR-92b, miR148/152, miR-124, miR-615, miR-708, miR-9, Klfl, Klf2, Klf5, Esrrb, Esrrg, So l, Sox3, Sox 15, Sox 18, Smad7, Nr5al, Nr5a2, Wn ⁇ eta-catenin, Met, Notch, Hedgehog, Sonic Hedgehog, CD133, CD44, CD44 + /CD247ESA ⁇ CD347CD
  • the cell in methods of modulating one or more histone methylation events in a cell, is derived from a cancer selected from the group consisting of: embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian cancer, endometrial cancer, cervical cancer, retinoblastoma, kidney cancer, liver cancer, gastric cancer, brain cancer, medulloblastoma, medulloepithelioma, glioma, glioblastoma, lung cancer, bronchial cancer, mesothelioma, skin cancer, colon and rectal cancer, bladder cancer, pancreatic cancer, lip and oral cancer, laryngeal and pharyngeal cancer, melanoma, pituitary cancer, penile cancer, parathyroid cancer, thyroid cancer, pheochromocytoma and paraganglioma, thymo
  • the present invention provides a method for monitoring the progression of cancer in a subject, comprising:
  • the monitoring comprises evaluating changes in the risk of developing cancer in the subject.
  • the subject comprises one who has previously been treated for cancer, one who has not been previously treated for cancer, or one who has not been previously diagnosed with cancer.
  • the sample in methods for monitoring the progression of cancer in a subject, is whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsies, lymphatic fluid, ascites fluid, interstitial fluid, bone marrow, cerebrospinal fluid (CSF), seminal fluid, saliva, mucous, sputum, sweat, or urine.
  • CSF cerebrospinal fluid
  • the first sample is taken from the subject prior to being treated for cancer.
  • a second sample can be taken from the subject after being treated for cancer, allowing for comparison with the first sample, with an increase or decrease in one or more histone demethylases being an indication of progression or regression of cancer, respectively.
  • an increase in LSD1 indicates progression of cancer and the need for an alternate treatment.
  • a decrease in LSD1 indicates regression of cancer and that the treatment regimen is effective.
  • the method further comprises selecting a treatment regimen for the subject and/or monitoring the effectiveness of a treatment regimen for cancer.
  • the treatment for cancer comprises surgical intervention, administration of anticancer agents, surgical intervention following or preceded by administration of anticancer agents, or taking no further action.
  • the reference value comprises an index value, a value derived from one or more cancer risk prediction algorithms, a value derived from a subject not having cancer, or a value derived from a subject diagnosed with cancer.
  • the measuring comprises detecting the presence or absence of the one or more histone demethylases, quantifying the amount of the one or more histone demethylases, and qualifying the type of the one or more histone demethylases.
  • the one or more histone demethylases comprises LSD1.
  • the one or more histone demethylases are measured by PCR.
  • the one or more histone demethylases are measured by immunoassay.
  • the cancer in methods for monitoring the progression of cancer in a subject, is characterized by the presence of pluripotent and/or multipotent cells.
  • the cancer in methods for monitoring the progression of cancer in a subject, is selected from the group consisting of: embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian cancer, endometrial cancer, cervical cancer, retinoblastoma, kidney cancer, liver cancer, gastric cancer, brain cancer, medulloblastoma, medulloepithelioma, glioma, glioblastoma, lung cancer, bronchial cancer, mesothelioma, skin cancer, colon and rectal cancer, bladder cancer, pancreatic cancer, lip and oral cancer, laryngeal and pharyngeal cancer, melanoma, pituitary cancer, penile cancer, parathyroid cancer, thyroid cancer,
  • the cancer is breast cancer. In some such embodiments, the cancer is ovarian cancer.
  • the present invention provides a method for selecting a subject for treatment with a compound of formula V, or any genus, subgenus, the species disclosed herein, the method comprising:(a) measuring the level of one or more histone demethylases in said subject; and(b) comparing the level of said one or more histone demethylases detected in step (a) to a reference value; wherein when the level of one or more histone demethylases in the subject is greater than the reference value, the subject is selected for treatment with the compounds of the invention.
  • the one or more histone demethylases comprises LSD1.
  • the method further includes measuring the level of at least one pluripotent stem cell protein marker and comparing the measured level of at least one pluripotent stem cell protein marker to a reference value for each marker, wherein when the level of at least one pluriponent stem cell protein marker is greater than its reference value, treatment with the compound of formula V or any of the disclosed genus, subgenus, or species disclosed herein is indicated.
  • the at least one pluripotent stem cell protein marker is selected from the group consisting of Oct4, Sox2, Lin28, Nanog, Klf4, and Sall4, Lin28B, cMyc, nMyc, LMyc, Wnt3a, miR-291-3p, miR-294, miR-295, miR-290/371 cluster, miR-302 cluster, miR-363 cluster, miR-520 cluster, miR-92b, miR 148/152, miR-124, miR-615, miR-708, nuR-9, Klfl, Klf2, lf5, Esrrb, Esrrg, Soxl, Sox3, Soxl5, Soxl8, Smad7, Nr5al, Nr5a2, Wnt beta-catenin, Met, Notch, Hedgehog, Sonic Hedgehog, CD133, CD44, CD44 + /CD247ESA + , CD34 + /CD38 ⁇ CD90, CD133, CD44, CD44 + /CD247
  • the present invention provides methods of treating cancer by administering a therapeutically effective amount of the histone demethylase inhibitor compounds disclosed herein.
  • "Treating" cancer can result in a reduction in size of a tumor.
  • a reduction in size of a tumor may also be referred to as “tumor regression".
  • tumor size is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor size is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75% or greater.
  • Size of a tumor may be measured by any reproducible means of measurement. The size of a tumor may be measured as a diameter of the tumor.
  • Treating cancer can result in a reduction in tumor volume.
  • tumor volume is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor volume is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75% or greater.
  • Tumor volume may be measured by any reproducible means of measurement.
  • Treating cancer may result in a decrease in number of tumors.
  • tumor number is reduced by 5% or greater relative to number prior to treatment; more preferably, tumor number is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%.
  • Number of tumors may be measured by any reproducible means of measurement.
  • the number of tumors may be measured by counting tumors visible to the naked eye or at a specified magnification.
  • the specified magnification is 2X, 3X, 4X, 5X, 10X, or 50X.
  • Treating cancer can result in a decrease in number of metastatic lesions in other tissues or organs distant from the primary tumor site.
  • the number of metastatic lesions is reduced by 5% or greater relative to number prior to treatment; more preferably, the number of metastatic lesions is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%.
  • the number of metastatic lesions may be measured by any reproducible means of measurement.
  • the number of metastatic lesions may be measured by counting metastatic lesions visible to the naked eye or at a specified magnification.
  • the specified magnification is 2X, 3X, 4X, 5X, 10X, or 50X.
  • Treating cancer can result in an increase in average survival time of a population of treated subjects in comparison to a population receiving carrier alone.
  • the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days.
  • An increase in average survival time of a population may be measured by any reproducible means.
  • An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound.
  • An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.
  • Treating cancer can result in an increase in average survival time of a population of treated subjects in comparison to a population of untreated subjects.
  • the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days.
  • An increase in average survival time of a population may be measured by any reproducible means.
  • An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound.
  • An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.
  • Treating cancer can result in increase in average survival time of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present invention, or a pharmaceutically acceptable salt thereof.
  • the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days.
  • An increase in average survival time of a population may be measured by any reproducible means.
  • An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound.
  • An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.
  • Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving carrier alone. Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present invention, or a pharmaceutically acceptable salt thereof.
  • the mortality rate is decreased by more than 2%; more preferably, by more than 5%; more preferably, by more than 10%; and most preferably, by more than 25%.
  • a decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means.
  • a decrease in the mortality rate of a population may be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with an active compound.
  • a decrease in the mortality rate of a population may also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with an active compound.
  • Treating cancer can result in a decrease in tumor growth rate.
  • tumor growth rate is reduced by at least 5% relative to number prior to treatment; more preferably, tumor growth rate is reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%.
  • Tumor growth rate may be measured by any reproducible means of measurement. Tumor growth rate can be measured according to a change in tumor diameter per unit time.
  • Treating cancer can result in a decrease in tumor regrowth.
  • tumor regrowth is less than 5%; more preferably, tumor regrowth is less than 10%; more preferably, less than 20%; more preferably, less than 30%; more preferably, less than 40%; more preferably, less than 50%; even more preferably, less than 50%; and most preferably, less than 75%.
  • Tumor regrowth may be measured by any reproducible means of measurement. Tumor regrowth is measured, for example, by measuring an increase in the diameter of a tumor after a prior tumor shrinkage that followed treatment. A decrease in tumor regrowth is indicated by failure of tumors to reoccur after treatment has stopped.
  • Treating cancer can result in a reduction in the rate of cancer cell proliferation.
  • the rate of cellular proliferation is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%.
  • the rate of cellular proliferation may be measured by any reproducible means of measurement.
  • the rate of cellular proliferation is measured, for example, by measuring the number of dividing cells in a tissue sample per unit time.
  • Treating cancer can result in a reduction in the proportion of proliferating cells.
  • the proportion of proliferating cells is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%.
  • the proportion of proliferating cells may be measured by any reproducible means of measurement.
  • the proportion of proliferating cells is measured, for example, by quantifying the number of dividing cells relative to the number of nondividing cells in a tissue sample. T he proportion of proliferating cells can be equivalent to the mitotic index.
  • Treating cancer can result in a decrease in the number or proportion of cells having an abnormal appearance or morphology.
  • the number of cells having an abnormal morphology is reduced by at least 5% relative to its size prior to treatment; more preferably, reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%.
  • An abnormal cellular appearance or morphology may be measured by any reproducible means of measurement.
  • An abnormal cellular morphology can be measured by microscopy, e.g., using an inverted tissue culture microscope.
  • An abnormal cellular morphology can take the form of nuclear pleiomorphism.
  • Also provided herein are methods for inhibiting the growth, proliferation, and/or survival of cancer cells comprising contacting the cells with an effective amount of a histone demethylase inhibitor as defined herein.
  • Cell growth, proliferation, and/or survival can be measured by any method known to those skilled in the art, such as BrdU incorporation, 3 H- thymidine incorporation, 5-ethynyl-2'-deoxyuridine incorporation, LIVE/DEAD
  • kits such as PhiPhiLux (Oncolmmunin), Caspase 3 Activity Assay and Homogeneous Caspases Assay (Roche Applied Science), Caspase-Glo (Promega), Apo-ONE Homogeneous Caspase 3/7 assay (Promega), CaspACE colorimetric and fluorometric Assay Systems (Promega), EnzChek Caspase-3 Assay Kit (Invitrogen), Image-iT LIVE Green Caspase- 3 and -7 Detection Kit (Invitrogen), Active Caspase-3 Detection
  • Methods of measuring and/or assessing cell growth, proliferation, and survival can also encompass measurement of the reaction between a histone and histone demethylase protein can be accomplished by any means known in the art. These include, without limitation Western blotting, measuring formation of formaldehyde, mass spectrometry, and measuring formation of peroxide.
  • One method comprises monitoring the interaction between a histone demethylase protein and CoREST, a protein that is believed to interact with histone demethylases such as LSD1.
  • This method may comprise contacting a histone demethylase protein and a CoREST protein in the presence of one or more histone demethylase inhibitors; and determining the level of interaction between the histone demethylase and CoREST, wherein a different level of interaction between the histone demethylase and CoREST in the presence of the histone demethylase inhibitor relative to the absence of the inhibitor indicates that the inhibitor modulates the interaction between the histone demethylase protein and CoREST, thereby causing the inhibition of growth, proliferation, and/or survival of cancer cells.
  • the method may further comprise at least one other component of a histone demethylase transcription complex, such as, for example, HDACl, HDAC2, BHC80 and HDC80 (Gocke, C. B. and Yu, H. (2008) PLoS One 22: e3255; Roizman, B. (2011) J. Virol. 85: 7474-82; Shi, Y. J. et al. (2005) Mol. Cell 19: 857- 64; Lee, M. G. et al. (2005) Nature 437: 432-5).
  • the method may also comprise determining the effect of the inhibitor on a biological activity of the histone demethylase.
  • a method may further comprise contacting a histone demethylase and CoREST with a histone demethylase inhibitor and determining the biological activity of the histone demethylase, wherein a different activity of the histone demethylase in the presence of the inhibitor relative to the absence of the inhibitor indicates that the inhibitor modulates the biological activity of a histone demethylase, thereby causing the inhibition of growth, proliferation, and/or survival of cancer cells.
  • the methods disclosed herein may comprise at least a portion of a protein of interest, e.g., a histone demethylase protein, CoREST protein, or other component of a histone demethylase transcription complex, fused direcdy or indirectiy to another moiety or label, e.g., a fluorophore or radioactive label or another peptide that may be useful in identifying, quantitating, isolating or purifying the reagent.
  • a protein of interest e.g., a histone demethylase protein, CoREST protein, or other component of a histone demethylase transcription complex, fused direcdy or indirectiy to another moiety or label, e.g., a fluorophore or radioactive label or another peptide that may be useful in identifying, quantitating, isolating or purifying the reagent.
  • a reporter gene may encode, e.g., a promoter and/or enhancer, thereof, operably linked to a reporter gene and (ii) contacting the cell or cell lysate with a histone demethylase inhibitor and (iii) determining the level of expression of the reporter gene, wherein a higher level of expression of the reporter gene in the presence of the inhibitor relative to the absence of the inhibitor indicates that the inhibitor increases the level of expression of the histone demethylase gene, whereas a lower level of expression of the reporter gene in the presence of the inhibitor relative to the absence of the inhibitor indicates that the inhibitor decreases the level of expression of the histone demethylase gene.
  • a reporter gene may encode, e.g., a promoter and/or enhancer, thereof, operably linked to a reporter gene and (ii) contacting the cell or cell lysate with a histone demethylase inhibitor and (iii) determining the level of expression of the reporter gene, wherein a higher level of expression of the
  • Assays for inhibiting the growth, proliferation, survival of cancer cells, and/or promotion of differentiation of cancer cells may further comprise testing the effect of the histone demethylase inhibitor on the demethylase activity in a cell.
  • an inhibitor may be contacted with or administered into a cell and the level of expression of one or more genes whose expression is regulated by methylation may be measured.
  • the level of protein e.g., a histone demethylase protein or protein associated with a histone demethylase, may be measured.
  • the present invention provides a method of detecting or diagnosing cancer in a subject, comprising:
  • an increase in one or more histone demethylases relative to the reference value indicates that the subject has cancer.
  • an increase in LSD1 relative to the reference value indicates that the subject has cancer.
  • a decrease in one or more histone demethylases relative to the reference value indicates that the subject has cancer.
  • the sample in methods of detecting or diagnosing cancer in a subject, is whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsies, lymphatic fluid, ascites fluid, interstitial fluid, bone marrow, cerebrospinal fluid (CSF), seminal fluid, saliva, mucous, sputum, sweat, or urine.
  • CSF cerebrospinal fluid
  • the subject comprises one who has been previously diagnosed as having cancer, one who has not been previously diagnosed as having cancer, or one who is asymptomatic for cancer.
  • the measuring comprises detecting the presence or absence of the one or more histone demethylases, quantifying the amount of the one or more histone demethylases, and qualifying the type of the one or more histone demethylases.
  • the reference value comprises an index value, a value derived from one or more cancer risk prediction algorithms, a value derived from a subject not suffering from cancer, or a value derived from a subject diagnosed with cancer.
  • the one or more histone demethylases comprises LSD1.
  • the one or more histone demethylases are measured by PCR.
  • the one or more histone demethylases are measured by immunoassay.
  • the cancer in methods of detecting or diagnosing cancer in a subject, is characterized by the presence of pluripotent and/or multipotent cells.
  • the method further comprising measuring the level of at least one pluripotent or multipotent stem cell protein marker.
  • the at least one pluripotent stem cell protein marker is selected from the group consisting of Oct4, Sox2, Lin28, Nanog, Klf4, and Sall4, Lin28B, cMyc, nMyc, LMyc, Wnt3a, miR-291-3p, miR- 294, miR-295, miR-290/371 cluster, miR-302 cluster, miR-363 cluster, miR-520 cluster, miR- 92b, miR148/152, miR-124, miR-615, miR-708, miR-9, Klfl, Klf2, Klf5, Esrrb, Esrrg, Soxl , Sox3, Soxl5, Soxl8, Smad7, Nr5al , Nr5a2, Wnt/beta-catenin, Met, Notch, Hedgehog, Sonic Hedgehog, CD133, CD44, CD447CD247ESA + , CD347CD
  • the cancer in methods of detecting or diagnosing cancer in a subject, is selected from the group consisting of: embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian cancer, endometrial cancer, cervical cancer, retinoblastoma, kidney cancer, liver cancer, gastric cancer, brain cancer, medulloblastoma, medulloepithelioma, glioma,
  • glioblastoma lung cancer, bronchial cancer, mesothelioma, skin cancer, colon and rectal cancer, bladder cancer, pancreatic cancer, lip and oral cancer, laryngeal and pharyngeal cancer, melanoma, pituitary cancer, penile cancer, parathyroid cancer, thyroid cancer, pheochromocytoma and paraganglioma, thymoma and thymic Carcinoma, leukemia, lymphoma, plasma cell neoplasms, myeloproliferative disorders, islet cell tumor, small intestine cancer, transitional cell cancer, pleuropulmonary blastoma, gestational trophoblastic cancer, esophageal cancer, central nervous system cancer, head and neck cancer, endocrine cancer, cardiovascular cancer, rhabdomyosarcoma, soft tissue carcinomas, carcinomas of bone, cartilage, fat, vascular, neural, and hematopoietic tissues and AIDS-related cancer
  • biomarkers disclosed herein can be detected in the practice of the present invention.
  • at least one (1), at least two (2), at least three (3), at least four (4) or more biomarkers e.g., histone demethylases as described herein
  • the histone demethylases include, e.g., LSDl, LSD2, JARTD family members, and FBXLIO, but are not limited to these examples.
  • the invention also includes cancer candidate genes that are inhibited by, modulated by or otherwise affected by the action of one or more histone demethylases, which are useful as therapeutic targets.
  • biomarkers can be measured to aid cancer diagnosis or prognosis.
  • the methods for detection of the biomarkers can be used to monitor responses in a subject to cancer treatment.
  • the methods for detecting biomarkers can be used to assay for and to identify compounds that modulate expression of these biomarkers in vivo or in vitro, which may be useful in preventing or treating cancer in subjects. Differentiation of non-cancer and cancer status may be by the detection of one or more of the biomarkers disclosed herein.
  • exemplary biomarkers that may independently discriminate between cancer statuses include detection or measurement of one or more histone demethylases. Combinations of biomarkers are also useful in the methods of the invention for the determination of cancer and cancer status.
  • Methods for identifying a candidate compound for treating cancer may comprise, for example, contacting one or more of the biomarkers of the invention with a test compound; and determining whether the test compound interacts with the biomarker, wherein a compound that interacts with the biomarker is identified as a candidate compound for treating cancer.
  • Compounds suitable for therapeutic testing may be screened initially by identifying compounds which interact with one or more biomarkers listed in identified herein.
  • screening might include recombinantly expressing a biomarker of this invention, purifying the biomarker, and affixing the biomarker to a substrate.
  • Test compounds can then be contacted with the substrate, typically in aqueous conditions, and interactions between the test compound and the biomarker are measured, for example, by measuring elution rates as a function of salt concentration.
  • Certain proteins may recognize and cleave one or more biomarkers of this invention, in which case the proteins may be detected by monitoring the digestion of one or more biomarkers in a standard assay, e.g., by gel electrophoresis of the proteins.
  • the ability of a test compound to inhibit the activity of one or more of the biomarkers of this invention may be measured.
  • the techniques used to measure the activity of a particular biomarker will vary depending on the function and properties of the biomarker. For example, an enzymatic activity of a biomarker may be assayed provided that an appropriate substrate is available and provided that the concentration of the substrate or the appearance of the reaction product is readily measurable.
  • the ability of potentially therapeutic test compounds to inhibit or enhance the activity of a given biomarker may be determined by measuring the rates of catalysis in the presence or absence of the test compounds.
  • the ability of a test compound to interfere with a non-enzymatic (e.g., structural) function or activity of one of the biomarkers of this invention may also be measured.
  • the self-assembly of a multi-protein complex which includes one of the biomarkers of this invention may be monitored by spectroscopy in the presence or absence of a test compound.
  • the biomarker is a non-enzymatic enhancer of transcription
  • test compounds which interfere with the ability of the biomarker to enhance transcription may be identified by measuring the levels of biomarker-dependent transcription in vivo or in vitro in the presence and absence of the test compound.
  • Test compounds capable of modulating the activity of any of the biomarkers of this invention may be administered to subjects who are suffering from or are at risk of developing cancer.
  • the administration of a test compound which increases the activity of a particular biomarker may decrease the risk of cancer in a subject if the activity of the particular biomarker in vivo prevents the accumulation of proteins for cancer.
  • screening a test compound includes obtaining samples from test subjects before and after exposure to a test compound.
  • the levels in the samples of one or more of the biomarkers of this invention may be measured and analyzed to determine whether the levels of the biomarkers change after exposure to a test compound.
  • the samples may be analyzed by any appropriate means known to one of skill in the art.
  • the levels of one or more of the biomarkers of this invention may be measured directly by Western blot using radio- or fluorescently-labeled antibodies which specifically bind to the biomarkers.
  • changes in the levels of mRNA encoding the one or more biomarkers may be measured and correlated with the administration of a given test compound to a subject.
  • the changes in the level of expression of one or more of the biomarkers may be measured using in vitro methods and materials.
  • human tissue cultured cells which express, or are capable of expressing, one or more of the biomarkers of this invention may be contacted with test compounds.
  • Subjects who have been treated with test compounds will be routinely examined for any physiological effects which may result from the treatment.
  • the test compounds will be evaluated for their ability to decrease disease likelihood in a subject.
  • test compounds will be screened for their ability to slow or stop the progression of the disease.
  • Methods of identifying therapeutic targets for cancer generally comprise comparing an expression profile of a cancer cell with an expression profile of one a reference cell, wherein the comparison is capable of classifying proteins or transcripts in the profile as being associated with cancer invasion.
  • Reference cells may be normal cells (cells that are not cancer cells) or cancer cells a different stage from the cancer cells being compared to.
  • the reference cells may be primary cultured cells, fresh blood cells, established cell lines or other cells determined to be appropriate to one of skill in the art.
  • Transcripts and proteins associated with cancer invasion include cells that differentiate between the states of cancer and between normal and cancer cell lines.
  • the transcripts and proteins may also differentiate between different types of cancer.
  • the proteins may be secreted proteins, such that they are easily detectable from a blood sample.
  • the subjects may be subjects who have been determined to have a high risk of cancer based on their family history, a previous treatment, subjects with physical symptoms known to be associated with cancer, subjects identified through screening assays (e.g., routine cancer screening) or other techniques. Other subjects include subjects who have cancer and the test is being used to determine the effectiveness of therapy or treatment they are receiving. Also, subjects could include healthy people who are having a test as part of a routine examination, or to establish baseline levels of the biomarkers. Samples may be collected from subjects who had been diagnosed with cancer and received treatment to eliminate the cancer, or who are in remission.
  • the risk of cancer can be detected by measuring an "effective amount" of the biomarkers of the present invention in a sample (e.g., a subject derived sample), and comparing the effective amounts to reference values, often utilizing mathematical algorithms or formulae in order to combine information from results of multiple individual biomarkers into a single measurement.
  • Subjects identified as having an increased risk of cancer can optionally be selected to receive treatment regimens or therapeutic interventions, such as administration of compounds such as "anticancer agents" as defined herein, or implementation of surgical interventions (which may follow or precede administration of therapeutic agents or other therapies), biological therapies (“biotherapies”), or radiological therapies to prevent or delay the onset or recurrence of cancer or metastasis of cancer.
  • the present invention may be used to make continuous or categorical measurements of the risk of conversion to cancer, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at-risk for developing cancer.
  • the methods of the present invention can be used to discriminate between normal and at-risk subject cohorts.
  • the present invention may be used so as to discriminate at-risk from cancerous, or cancerous from normal. Such differing use may require different biomarker combinations in individual panel or profile, mathematical algorithm, and/or cut-off points, but be subject to the same aforementioned measurements of accuracy for the intended use.
  • Identifying the at-risk subject enables the selection and initiation of various therapeutic interventions or treatment regimens in order to delay, reduce, or prevent that subject's conversion to cancer.
  • Levels of an effective amount of biomarker proteins, nucleic acids, polymorphisms, metabolites, or other analytes also allows for the course of treatment of cancer to be monitored.
  • a biological sample can be provided from a subject undergoing treatment regimens, e.g., therapeutic treatments, for cancer.
  • treatment regimens can include, but are not limited to, surgical intervention, radiological therapies, and treatment with therapeutic agents used in subjects diagnosed or identified with cancer.
  • biological samples are obtained from the subject at various time points before, during, or after treatment.
  • determining the cancer status by comparison of a subject's biomarker profile to a reference biomarker profile can be repeated more than once, wherein the subject's biomarker profile can be obtained from a separate sample taken each time the method is repeated. Samples may be taken from the subject at defined time intervals, such as, e.g., 24 hours, 48 hours, 72 hours, or any suitable time interval as would be performed by those skilled in the art.
  • Differences in the genetic makeup of subjects can result in differences in their relative abilities to metabolize various drugs, which may modulate the symptoms or risk factors of cancer.
  • Subjects that have cancer, or at risk for developing cancer can vary in age, ethnicity, and other parameters. Accordingly, use of the biomarkers disclosed herein, both alone and together in combination with known genetic factors for drug metabolism, allow for a pre-determined level of predictability that a putative therapeutic or prophylactic to be tested in a selected subject will be suitable for treating or preventing cancer in the subject.
  • a test sample from the subject can also be exposed to a therapeutic agent or a drug, and the level of one or more of biomarker proteins, nucleic acids, polymorphisms, metabolites or other analytes can be determined.
  • the level of one or more biomarkers can be compared to sample derived from the subject before and after subject management for cancer, e.g., treatment or exposure to a therapeutic agent or a drug, or can be compared to samples derived from one or more subjects who have shown improvements in cancer risk factors as a result of such treatment or exposure.
  • Nucleic acids may be obtained from the samples in many ways known to one of skill in the art, for example, extraction methods, including e.g., solvent extraction, affinity purification and centrifugation. Selective precipitation can also purify nucleic acids. Chromatography methods may also be utilized including, gel filtration, ion exchange, selective adsorption, or affinity binding.
  • the nucleic acids may be, for example, RNA, DNA or may be synthesized into cDNA.
  • the nucleic acids may be detected using microarray techniques that are well known in the art, for example, Affymetrix arrays followed by multidimensional scaling techniques. See R. Ekins, R. and Chu, F.W. (1999) Trends Biotechnol. 17: 217-218; D. D.
  • the sample can be prepared to enhance detectability of the biomarkers.
  • a blood serum sample from the subject can be preferably fractionated by, e.g., Cibacron blue agarose chromatography and single stranded DNA affinity chromatography, anion exchange chromatography, affinity
  • chromatography e.g., with antibodies
  • the method of fractionation depends on the type of detection method used. Any method that enriches for the protein of interest can be used.
  • preparation involves fractionation of the sample and collection of fractions determined to contain the biomarkers.
  • Methods of pre-fractionation include, for example, size exclusion chromatography, ion exchange chromatography, heparin chromatography, affinity
  • a sample can be pre-fractionated by removing proteins that are present in a high quantity or that may interfere with the detection of biomarkers in a sample.
  • serum albumin is present in a high quantity and may obscure the analysis of biomarkers.
  • a blood serum sample can be pre- fractionated by removing serum albumin.
  • Serum albumin can be removed using a substrate that comprises adsorbents that specifically bind serum albumin.
  • a column which comprises, e.g., Cibacron blue agarose (which has a high affinity for serum albumin) or antiserum albumin antibodies can be used.
  • a sample can be pre-fractionated by isolating proteins that have a specific characteristic, e.g. are glycosylated.
  • a blood serum sample can be fractionated by passing the sample over a lectin chromatography column (which has a high affinity for sugars). Glycosylated proteins will bind to the lectin column and non-glycosylated proteins will pass through the flow through. Glycosylated proteins are then eluted from the lectin column with an eluant containing a sugar, e.g., N-acetyl-glucosamine and are available for further analysis.
  • a sugar e.g., N-acetyl-glucosamine
  • affinity columns which are suitable for pre-fractionating blood serum samples.
  • An example of a type of affinity chromatography available to pre-fractionate a sample is a single stranded DNA spin column. These columns bind proteins which are basic or positively charged. Bound proteins are then eluted from the column using eluants containing denaturants or high pH.
  • a sample can be fractionated using a sequential extraction protocol. In sequential extraction, a sample is exposed to a series of adsorbents to extract different types of biomarkers from a sample.
  • a sample is applied to a first adsorbent to extract certain proteins, and an eluant containing non-adsorbent proteins (i.e., proteins that did not bind to the first adsorbent) is collected. Then, the fraction is exposed to a second adsorbent. This further extracts various proteins from the fraction. This second fraction is then exposed to a third adsorbent, and so on.
  • Any suitable materials and methods can be used to perform sequential extraction of a sample. For example, a series of spin columns comprising different adsorbents can be used. In another example, multi-well plates comprising different adsorbents at its bottom can be used.
  • sequential extraction can be performed on a probe adapted for use in a gas phase ion spectrometer, wherein the probe surface comprises adsorbents for binding biomarkers.
  • the sample is applied to a first adsorbent on the probe, which is subsequendy washed with an eluant. Biomarkers that do not bind to the first adsorbent are removed with an eluant. The biomarkers that are in the fraction can be applied to a second adsorbent on the probe, and so forth.
  • the advantage of performing sequential extraction on a gas phase ion spectrometer probe is that biomarkers that bind to various adsorbents at every stage of the sequential extraction protocol can be analyzed directly using a gas phase ion spectrometer.
  • biomarkers in a sample can be separated by. high- resolution electrophoresis, e.g., one or two-dimensional gel electrophoresis.
  • a fraction containing a biomarker can be isolated and further analyzed by gas phase ion spectrometry.
  • two- dimensional gel electrophoresis is used to generate two-dimensional array of spots, including one or more biomarkers. See, e.g., Jungblut and Thiede, (1997) Mass Spectr. Rev. 16: 145-162.
  • the two-dimensional gel electrophoresis can be performed using methods known in the art. See, e.g., Academic Press, Science, etc.
  • biomarkers in a sample are separated by, e.g., isoelectric focusing, during which biomarkers in a sample are separated in a pH gradient until they reach a spot where their net charge is zero (i.e., isoelectric point).
  • This first separation step results in one-dimensional array of biomarkers.
  • the biomarkers in one- dimensional array is further separated using a technique generally distinct from that used in the first separation step.
  • biomarkers separated by isoelectric focusing are further separated using a polyacrylamide gel, such as polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE).
  • SDS-PAGE gel allows further separation based on molecular mass of biomarkers.
  • two-dimensional gel electrophoresis can separate chemically different biomarkers in the molecular mass range from 1000-200,000 Da within complex mixtures.
  • Biomarkers in the two-dimensional array can be detected using any suitable methods known in the art.
  • biomarkers in a gel can be labeled or stained (e.g., Coomassie Blue or silver staining). If gel electrophoresis generates spots that correspond to the molecular weight of one or more biomarkers of the invention, the spot can be excised and further analyzed by, for example, gas phase ion spectrometry, mass spectrometry, or high performance liquid chromatography.
  • the gel containing biomarkers can be transferred to an inert membrane by applying an electric field. Then a spot on the membrane that approximately corresponds to the molecular weight of a biomarker can be analyzed by e.g., gas phase ion spectrometry, mass spectrometry, or HPLC.
  • a biomarker can be modified before analysis to improve its resolution or to determine its identity.
  • the biomarkers may be subject to proteolytic digestion before analysis. Any protease can be used. Proteases, such as trypsin, that are likely to cleave the biomarkers into a discrete number of fragments are particularly useful. The fragments that result from digestion may function as a fingerprint for the biomarkers, thereby enabling their indirect detection. This is particularly useful where there are biomarkers with similar molecular masses that might be confused for the biomarker in question. Also, proteolytic fragmentation is useful for high molecular weight biomarkers because smaller biomarkers are more easily resolved by mass spectrometry. In another example, biomarkers can be modified to improve detection resolution.
  • neuraminidase can be used to remove terminal sialic acid residues from glycoproteins to improve binding to an anionic adsorbent (e.g., cationic exchange ProteinChip® arrays) and to improve detection resolution.
  • the biomarkers can be modified by the attachment of a tag of particular molecular weight that specifically binds to molecular biomarkers, further distinguishing them.
  • the identity of the biomarkers can be further determined by matching the physical and chemical characteristics of the modified biomarkers in a protein database (e.g., SwissProt).
  • any suitable method such as those described herein as well as other methods known in the art, can be used to measure a biomarker or biomarkers in a sample.
  • the actual measurement of levels or amounts of the biomarkers can be determined using any method known in the art. These methods include, without limitation, mass spectrometry (e.g., laser desorption/ionization mass spectrometry), fluorescence (e.g. sandwich immunoassay), surface plasmon resonance, ellipsometry and atomic force microscopy.
  • Methods may further include, by one or more of microarrays, PCR methods, mass spectrometry (including, for example, and without limitation, ESI-MS, ESI-MS/MS, ESI- MS/(MS)n, matrix-assisted laser desorption ionization time-of -flight mass spectrometry
  • mass spectrometry including, for example, and without limitation, ESI-MS, ESI-MS/MS, ESI- MS/(MS)n, matrix-assisted laser desorption ionization time-of -flight mass spectrometry
  • MALDI-TOF-MS surface-enhanced laser desorption/ionization time-of-flight mass spectrometry
  • SELDI-TOF-MS surface-enhanced laser desorption/ionization time-of-flight mass spectrometry
  • DIOS desorption/ionization on silicon
  • SIMS secondary ion mass spectrometry
  • Q-TOF quadrupole time-of-flight
  • APCI-MS atmospheric pressure chemical ionization mass spectrometry
  • APCI-MS/MS APCI-(MS)n
  • APPI-MS atmospheric pressure photoionization mass spectrometry
  • APPI-MS APPI-MS/MS
  • APPI-(MS)n quadrupole mass spectrometry
  • FTMS Fourier transform mass spectrometry
  • PCR assays may be done, for example, in a multi-well plate formats or in chips, such as the BioTrove OPEN ARRAY Chips (BioTrove, Wobum, MA).
  • sequences within the sequence database entries corresponding to biomarkers of the present invention can be used to construct probes for detecting biomarker RNA sequences in, e.g., Northern blot hybridization analyses or methods which specifically, and, preferably, quantitatively amplify specific nucleic acid sequences.
  • the sequences can be used to construct primers for specifically amplifying the biomarker sequences in, e.g., amplification-based detection methods such as reverse-transcription based polymerase chain reaction (RT-PCR), e.g., quantitative real-time RT-PCR.
  • amplification-based detection methods such as reverse-transcription based polymerase chain reaction (RT-PCR), e.g., quantitative real-time RT-PCR.
  • sequence comparisons in test and reference populations can be made by comparing relative amounts of the examined DNA sequences in subject and reference cell populations.
  • the term “specifically (or selectively) hybridizes" when referring to a nucleic acid refers to a binding reaction that is determinative of the presence of the nucleic acid in a heterogeneous population of nucleic acids.
  • the specified nucleic acid probe may bind or hybridize to a particular nucleic acid of interest at least two times the background and do not substantially bind or hybridize in a significant amount to other nucleic acids present in the sample.
  • Levels of biomarkers can also be determined at the protein level, e.g., by measuring the levels of peptides encoded by the gene products described herein, or activities thereof. Such methods are well known in the art and include, e.g., immunoassays based on antibodies to proteins encoded by the genes, aptamers or molecular imprints. Any biological material can be used for the detection quantification of the protein or its activity. Alternatively, a suitable method can be selected to determine the activity of proteins encoded by the biomarker genes according to the activity of each protein analyzed.
  • the antibody may be monoclonal, polyclonal, chimeric, or a fragment of the foregoing, as discussed in detail herein, and the step of detecting the reaction product may be carried out with any suitable immunoassay.
  • the sample from the subject is typically a biological fluid as described above, and may be the same sample of biological fluid used to conduct the method described above.
  • the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • polyclonal antibodies raised to a biomarker from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with that biomarker and not with other proteins, except for polymorphic variants and alleles of the biomarker. This selection may be achieved by subtracting out antibodies that cross-react with the biomarker molecules from other species.
  • Immunoassays carried out in accordance with the present invention may be homogeneous assays or heterogeneous assays.
  • the immunological reaction usually involves the specific antibody (e.g., anti-biomarker protein antibody), a labeled analyte, and the sample of interest.
  • the signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte.
  • Both the immunological reaction and detection of the extent thereof can be carried out in a homogeneous solution.
  • Immunochemical labels which may be employed include free radicals, radioisotopes, fluorescent dyes, enzymes,
  • the reagents are usually the sample, the antibody, and means for producing a detectable signal.
  • Samples as described above may be used.
  • the antibody can be immobilized on a support, such as a bead (such as protein A and protein G agarose beads), plate or slide, and contacted with the specimen suspected of containing the antigen in a liquid phase.
  • the support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal.
  • the signal is related to the presence of the analyte in the sample.
  • Means for producing a detectable signal include the use of detectable labels.
  • Exemplary detectable labels include magnetic beads (e.g., DYNABEADSTM), fluorescent dyes, enzymes (e.g., horse radish peroxide,
  • radiolabels e.g., S, I, I
  • fluorescent labels e.g., fluorescein, Alexa, green fluorescent protein, rhodamine
  • colorimetric labels such as colloidal gold or colored glass or plastic beads in accordance with known techniques.
  • the biomarker in the sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound biomarker-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the biomarker is incubated simultaneously with the mixture.
  • an indirect assay wherein, for example, a second, labeled antibody is used to detect bound biomarker-specific antibody
  • a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the biomarker is incubated simultaneously with the mixture.
  • an antibody which binds to that site can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step. The presence of the detectable label on the solid support indicates the presence of the antigen in the test sample.
  • Methods for measuring the amount or the presence of antibody-marker complexes include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry).
  • Optical methods include microscopy (both confocal and non-confocal), imaging methods and non-imaging methods.
  • Electrochemical methods include voltametry and amperometry methods. Radio frequency methods include multipolar resonance spectroscopy. Examples of suitable immunoassays include, but are not limited to immunoblotting (e.g., Western blotting, slot blot assay), immunoprecipitation, immunofluorescence methods, chemiluminescence methods,
  • ECL electrochemiluminescence
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • antibodies that specifically bind to a biomarker can be prepared using any suitable methods known in the art. See, e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies: A Laboratory Manual (1988); Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and Kohler & Milstein, Nature 256:495-497 (1975).
  • Such techniques include, but are not limited to, antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al., Science 246: 1275-1281 (1989); Ward et al., Nature 341 :544-546 (1989)).
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • a sample obtained from a subject can be contacted with the antibody that specifically binds the biomarker.
  • the antibody can be fixed to a solid support to facilitate washing and subsequent isolation of the complex, prior to contacting the antibody with a sample.
  • solid supports include glass or plastic in the form of, e.g., a microliter plate, a slide, or wells formed from materials such as latex or polystyrene, a stick, a bead (including magnetic beads), or a microbead such as protein A or protein G agarose.
  • Antibodies can also be attached to a probe substrate or ProteinChip® array. The sample can be diluted with a suitable eluant before contacting the sample to the antibody.
  • Immunoassays can be used to determine presence or absence of a biomarker in a sample as well as the quantity of a biomarker in a sample.
  • the amount of an antibody-marker complex can be determined by comparing to a standard.
  • a standard can be, e.g., a known compound or another protein known to be present in a sample.
  • the test amount of biomarker need not be measured in absolute units, as long as the unit of measurement can be compared to a control.
  • biomarkers can be measured to aid cancer diagnosis or prognosis.
  • the methods for detection of the biomarkers can be used to monitor responses in a subject to cancer treatment.
  • the methods for detecting biomarkers can be used to assay for and to identify compounds that modulate expression of these biomarkers in vivo or in vitro.
  • the biomarkers are used to differentiate between the different stages of tumor progression, thus aiding in determining appropriate treatment and extent of metastasis of the tumor.
  • Pre-translational modified forms include allelic variants, slice variants and RNA editing forms.
  • Post-translationally modified forms include forms resulting from proteolytic cleavage (e.g., fragments of a parent protein), glycosylation, phosphorylation, lipidation, oxidation, methylation, cystinylation, sulphonation and acetylation.
  • Antibodies can also be useful for detecting post-translational modifications of biomarker proteins, polypeptides, mutations, and polymorphisms, such as tyrosine phosphorylation, threonine phosphorylation, serine phosphorylation, glycosylation (e.g., O-GlcNAc). Such antibodies specifically detect the phosphorylated amino acids in a protein or proteins of interest, and can be used in
  • proteins including a specific protein and all modified forms of it is referred to herein as a "protein cluster.”
  • the collection of all modified forms of a specific protein, excluding the specific protein, itself, is referred to herein as a "modified protein cluster.”
  • Modified forms of any biomarker of this invention also may be used, themselves, as biomarkers. In certain cases the modified forms may exhibit better discriminatory power in diagnosis than the specific forms set forth herein.
  • Modified forms of a biomarker including any of the biomarkers as described herein can be initially detected by any methodology that can detect and distinguish the modified from the biomarker.
  • biomarker proteins, polypeptides, mutations, and polymorphisms known to have enzymatic activity the activities can be determined in vitro using enzyme assays known in the art.
  • enzyme assays include, without limitation, kinase assays, phosphatase assays, and reductase assays, among many others.
  • Modulation of the kinetics of enzyme activities can be determined by measuring the rate constant KM using known algorithms, such as the Hill plot, Michaelis- Menten equation, linear regression plots such as Lineweaver-Burk analysis, and Scatchard plot.
  • changes in the activity of a biomarker protein may be measured according to these and other methods known in the art.
  • biomarker protein and nucleic acid metabolites can be measured.
  • the term "metabolite” includes any chemical or biochemical product of a metabolic process, such as any compound produced by the processing, cleavage or consumption of a biological molecule (e.g., a protein, nucleic acid, carbohydrate, or lipid).
  • Metabolites can be detected in a variety of ways known to one of skill in the art, including the refractive index spectroscopy (RI), ultra-violet spectroscopy (UV), fluorescence analysis, radiochemical analysis, near-infrared spectroscopy (near-IR), nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS), mass spectrometry, pyrolysis mass spectrometry, nephelometry, dispersive Raman spectroscopy, gas chromatography combined with mass spectrometry, liquid chromatography (including high- performance liquid chromatography (HPLC)), which may be combined with mass spectrometry, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) combined with mass spectrometry, ion spray spectroscopy combined with mass spectrometry, capillary
  • RI refractive index spectroscopy
  • UV ultra-violet spectroscopy
  • fluorescence analysis radiochemical analysis
  • radiochemical analysis
  • circulating calcium ions can be detected in a sample using fluorescent dyes such as the Fluo series, Fura-2A, Rhod-2, among others.
  • fluorescent dyes such as the Fluo series, Fura-2A, Rhod-2, among others.
  • Other biomarker metabolites can be similarly detected using reagents that specifically designed or tailored to detect such metabolites.
  • Levels of an effective amount of biomarker proteins, nucleic acids, polymorphisms, metabolites, or other analytes can then be determined and compared to a reference value, e.g. a control subject or population whose cancer status is known, or an index value or baseline value.
  • the reference sample or index value or baseline value may be taken or derived from one or more subjects who have been exposed to the treatment, or may be taken or derived from one or more subjects who are at low risk of developing cancer, or may be taken or derived from subjects who have shown improvements in cancer risk factors as a result of exposure to treatment.
  • the reference sample or index value or baseline value may be taken or derived from one or more subjects who have not been exposed to the treatment.
  • samples may be collected from subjects who have received initial treatment for cancer and subsequent treatment for cancer to monitor the progress of the treatment.
  • a reference value can also comprise a value derived from risk prediction algorithms or computed indices from population studies such as those disclosed herein.
  • the biomarkers of the present invention can thus be used to generate a reference biomarker profile of those subjects who do not have cancer, and would not be expected to develop cancer.
  • the biomarkers disclosed herein can also be used to generate a "subject biomarker profile" taken from subjects who have cancer.
  • the subject biomarker profiles can be compared to a reference biomarker profile to diagnose or identify subjects at risk for developing cancer, to monitor the progression of disease, as well as the rate of progression of disease, and to monitor the effectiveness of cancer treatment modalities or subject management.
  • the reference and subject biomarker profiles of the present invention can be contained in a machine-readable medium, such as but not limited to, analog or digital tapes like those readable by a VCR, CD- ROM, DVD-ROM, USB flash media, among others.
  • Such machine-readable media can also contain additional test results, such as, without limitation, measurements of clinical parameters and traditional laboratory risk factors.
  • the machine-readable media can also comprise subject information such as medical history and any relevant family history.
  • the machine-readable media can also contain information relating to other cancer risk algorithms and computed indices such as those described herein.
  • Detection and correlation of biomarkers can be analyzed using any suitable means, including arrays.
  • Nucleic acid arrays may be analyzed using software, for example, Applied Maths, GenExploreTM, 2-way cluster analysis, principal component analysis, discriminant analysis, self-organizing maps; BioDiscovery, Inc., Los Angeles, California (ImaGeneTM, special image processing and data extraction software, powered by MatLab®; GeneSight: hierarchical clustering, artificial neural network (SOM), principal component analysis, time series;
  • Detection and correlation of biomarkers can be analyzed using any suitable means.
  • data generated, for example, by desorption is analyzed with the use of a programmable digital computer.
  • the computer program generally contains a readable medium that stores codes. Certain code can be devoted to memory that includes the location of each feature on a probe, the identity of the adsorbent at that feature and the elution conditions used to wash the adsorbent.
  • the computer also contains code that receives as input, data on the strength of the signal at various molecular masses received from a particular addressable location on the probe. This data can indicate the number of biomarkers detected, including the strength of the signal generated by each biomarker.
  • Data analysis can include the steps of determining signal strength (e.g., height of peaks) of a marker detected and removing "outliers" (data deviating from a predetermined statistical distribution).
  • the observed peaks can be normalized, a process whereby the height of each peak relative to some reference is calculated.
  • a reference can be background noise generated by instrument and chemicals (e.g., energy absorbing molecule) which is set as zero in the scale.
  • the signal strength detected for each biomarker can be displayed in the form of relative intensities in the scale desired (e.g., 100).
  • a standard e.g., a serum protein
  • a standard may be admitted with the sample so that a peak from the standard can be used as a reference to calculate relative intensities of the signals observed for each marker or other biomarkers detected.
  • the computer can transform the resulting data into various formats for displaying.
  • spectrum view or retentate map a standard spectral view can be displayed, wherein the view depicts the quantity of marker reaching the detector at each particular molecular weight.
  • peak map a standard spectral view
  • peak map only the peak height and mass information are retained from the spectrum view, yielding a cleaner image and enabling biomarkers with nearly identical molecular weights to be more easily seen.
  • gel view each mass from the peak view can be converted into a grayscale image based on the height of each peak, resulting in an appearance similar to bands on electrophoretic gels.
  • 3-D overlays In yet another format, referred to as "3-D overlays,” several spectra can be overlaid to study subtle changes in relative peak heights.
  • difference map view two or more spectra can be compared, conveniently highlighting unique biomarkers and biomarkers which are up- or down-regulated between samples. Biomarker profiles (spectra) from any two samples may be compared visually.
  • Spotfire Scatter Plot can be used, wherein biomarkers that are detected are plotted as a dot in a plot, wherein one axis of the plot represents the apparent molecular of the biomarkers detected and another axis represents the signal intensity of biomarkers detected.
  • biomarkers that are detected and the amount of biomarkers present in the sample can be saved in a computer readable medium. This data can then be compared to a control or reference biomarker profile or reference value (e.g., a profile or quantity of biomarkers detected in control, e.g., subjects in whom cancer is undetectable).
  • a control or reference biomarker profile or reference value e.g., a profile or quantity of biomarkers detected in control, e.g., subjects in whom cancer is undetectable.
  • the software can comprise code that converts signal from the mass spectrometer into computer readable form.
  • the software also can include code that applies an algorithm to the analysis of the signal to determine whether the signal represents a "peak" in the signal corresponding to a marker of this invention, or other useful biomarkers.
  • the software also can include code that executes an algorithm that compares signal from a test sample to a typical signal characteristic of "normal” and “cancerous” and determines the closeness of fit between the two signals.
  • the software also can include code indicating which the test sample is closest to, thereby providing a probable diagnosis.
  • multiple biomarkers are measured.
  • the use of multiple biomarkers increases the predictive value of the test and provides greater utility in diagnosis, toxicology, subject stratification and subject monitoring.
  • the process called "Pattern recognition" detects the patterns formed by multiple biomarkers greatly improves the sensitivity and specificity of clinical proteomics for predictive medicine. Subtle variations in data from clinical samples indicate that certain patterns of protein expression can predict phenotypes such as the presence or absence of a certain disease, a particular stage of cancer progression, or a positive or adverse response to drug treatments.
  • Ciphergen's ProteinChip® system employs an analog-to-digital converter (ADC) to accomplish this.
  • ADC analog-to-digital converter
  • the ADC integrates detector output at regularly spaced time intervals into time-dependent bins. The time intervals typically are one to four nanoseconds long.
  • the time-of- flight spectrum ultimately analyzed typically does not represent the signal from a single pulse of ionizing energy against a sample, but rather the sum of signals from a number of pulses. This reduces noise and increases dynamic range. This time-of- flight data is then subject to data processing.
  • data processing typically includes TOF-to- M/Z transformation, baseline subtraction, high frequency noise filtering.
  • TOF-to-M/Z transformation involves the application of an algorithm that transforms times-of-flight into mass-to-charge ratio (MZZ).
  • MZZ mass-to-charge ratio
  • the signals are converted from the time domain to the mass domain. That is, each time-of-flight is converted into mass-to-charge ratio, or MZZ.
  • Calibration can be done internally or externally.
  • the sample analyzed contains one or more analytes of known M/Z. Signal peaks at times-of- flight representing these massed analytes are assigned the known M/Z. Based on these assigned M/Z ratios, parameters are calculated for a mathematical function that converts times-of-flight to M/Z.
  • a function that converts times-of-flight to M/Z is applied to a time-of-flight spectrum without the use of internal calibrants.
  • Baseline subtraction improves data quantification by eliminating artificial, reproducible instrument offsets that perturb the spectrum. It involves calculating a spectrum baseline using an algorithm that incorporates parameters such as peak width, and then subtracting the baseline from the mass spectrum. High frequency noise signals are eliminated by the application of a smoothing function.
  • a typical smoothing function applies a moving average function to each time- dependent bin.
  • the moving average filter is a variable width digital filter in which the bandwidth of the filter varies as a function of, e.g., peak bandwidth, generally becoming broader with increased time-of-flight. See, e.g., International Patent Application Publication No. WO 00/70648.
  • Peak Analysis generally involves the identification of peaks in the spectrum that represent signal from an analyte. Peak selection can, of course, be done by eye. Software is available as part of Ciphergen's ProteinChip® software that can automate the detection of peaks. In general, this software functions by identifying signals having a signal-to-noise ratio above a selected threshold and labeling the mass of the peak at the centroid of the peak signal. In one useful application many spectra are compared to identify identical peaks present in some selected percentage of the mass spectra. One version of this software clusters all peaks appearing in the various spectra within a defined mass range, and assigns a mass (M/Z) to all the peaks that are near the mid-point of the mass (M/Z) cluster.
  • M/Z mass
  • Peak data from one or more spectra can be subject to further analysis by, for example, creating a spreadsheet in which each row represents a particular mass spectrum, each column represents a peak in the spectra defined by mass, and each cell includes the intensity of the peak in that particular spectrum.
  • Various statistical or pattern recognition approaches can applied to the data.
  • the spectra that are generated in embodiments of the invention can be classified using a pattern recognition process that uses a classification model.
  • data derived from the spectra e.g., mass spectra or time-of-flight spectra
  • samples such as "known samples”
  • a "known sample” is a sample that is pre-classified (e.g., cancer or not cancer).
  • Data derived from the spectra e.g., mass spectra or time-of-flight spectra
  • a "known sample” is a sample that is pre-classified.
  • the data that are derived from the spectra and are used to form the classification model can be referred to as a "training data set”.
  • the classification model can recognize patterns in data derived from spectra generated using unknown samples.
  • the classification model can then be used to classify the unknown samples into classes. This can be useful, for example, in predicting whether or not a particular biological sample is associated with a certain biological condition (e.g., diseased vs. non diseased).
  • the training data set that is used to form the classification model may comprise raw data or pre-processed data.
  • raw data can be obtained directly from time-of- flight spectra or mass spectra, and then may be optionally "pre-processed” in any suitable manner.
  • signals above a predetermined signal-to-noise ratio can be selected so that a subset of peaks in a spectrum is selected, rather than selecting all peaks in a spectrum.
  • a predetermined number of peak "clusters" at a common value e.g., a particular time-of-flight value or mass-to-charge ratio value
  • a peak at a given mass-to-charge ratio is in less than 50% of the mass spectra in a group of mass spectra, and then the peak at that mass-to-charge ratio can be omitted from the training data set.
  • Pre-processing steps such as these can be used to reduce the amount of data that is used to train the classification model.
  • Classification models can be formed using any suitable statistical classification (or "learning") method that attempts to segregate bodies of data into classes based on objective parameters present in the data.
  • Classification methods may be either supervised or unsupervised. Examples of supervised and unsupervised classification processes are described in Jain, "Statistical Pattern Recognition: A Review", IEEE Transactions on Pattern Analysis and Machine Intelligence, Vol. 22, No. 1, January 2000, which is herein incorporated by reference in its entirety.
  • supervised classification training data containing examples of known categories are presented to a learning mechanism, which learns one more sets of relationships that define each of the known classes. New data may then be applied to the learning mechanism, which then classifies the new data using the learned relationships. Examples of supervised
  • classification processes include linear regression processes (e.g., multiple linear regression (MLR), partial least squares (PLS) regression and principal components regression (PCR)), binary decision trees (e.g., recursive partitioning processes such as CART - classification and regression trees), artificial neural networks such as back propagation networks, discriminant analyses (e.g., Bayesian classifier or Fischer analysis), logistic classifiers, and support vector classifiers (support vector machines).
  • MLR multiple linear regression
  • PLS partial least squares
  • PCR principal components regression
  • binary decision trees e.g., recursive partitioning processes such as CART - classification and regression trees
  • artificial neural networks such as back propagation networks
  • discriminant analyses e.g., Bayesian classifier or Fischer analysis
  • logistic classifiers logistic classifiers
  • support vector classifiers support vector machines.
  • a preferred supervised classification method is a recursive partitioning process.
  • Recursive partitioning processes use recursive partitioning trees to classify spectra derived from unknown samples. Further details about recursive partitioning processes are provided in U.S. Patent Application Publication No. 20020138208.
  • the classification models that are created can be formed using unsupervised learning methods. Unsupervised classification attempts to learn classifications based on similarities in the training data set, without pre classifying the spectra from which the training data set was derived.
  • Unsupervised learning methods include cluster analyses.
  • a cluster analysis attempts to divide the data into "clusters" or groups that ideally should have members that are very similar to each other, and very dissimilar to members of other clusters. Similarity is then measured using some distance metric, which measures the distance between data items, and clusters together data items that are closer to each other.
  • Clustering techniques include the MacQueen's K-means algorithm and the ohonen's Self-Organizing Map algorithm.
  • the peak intensity data of samples from subjects e.g., cancer subjects, and healthy controls are used as a "discovery set.”
  • This data were combined and randomly divided into a training set and a test set to construct and test multivariate predictive models using a non-linear version of Unified Maximum Separability Analysis ("USMA") classifiers. Details of USMA classifiers are described in U.S. Patent Application Publication No. 20030055615.
  • the invention provides methods for aiding a cancer diagnosis using one or more biomarkers, i.e., one or more histone demethylases as specified herein. These biomarkers can be used alone, in combination with other biomarkers in any set, or with entirely different biomarkers in aiding cancer diagnosis.
  • the biomarkers are differentially present in samples of a cancer subject and a normal subject in whom cancer is undetectable. For example, some of the biomarkers are expressed at an elevated level and/or are present at a higher frequency in cancer subjects than in normal subjects, while some of the biomarkers are expressed at a decreased level and or are present at a lower frequency in cancer subjects than in normal subjects. Therefore, detection of one or more of these biomarkers in a person would provide useful information regarding the probability that the person may have cancer.
  • the data from the sample may be fed directly from the detection means into a computer containing the diagnostic algorithm.
  • the data obtained can be fed manually, or via an automated means, into a separate computer that contains the diagnostic algorithm.
  • embodiments of the invention include methods involving correlating the detection of the biomarker or biomarkers with a probable diagnosis of cancer.
  • the correlation may take into account the amount of the biomarker or biomarkers in the sample compared to a control amount of the biomarker or biomarkers (up or down regulation of the biomarker or biomarkers) (e.g., in normal subjects in whom cancer is undetectable).
  • the correlation may take into account the presence or absence of the biomarkers in a test sample and the frequency of detection of the same biomarkers in a control.
  • the correlation may take into account both of such factors to facilitate determination of whether a subject has cancer or not.
  • the correlation may take into account the amount of the biomarker or biomarkers in the sample compared to a control amount of the biomarker or biomarkers (up or down regulation of the biomarker or biomarkers) (e.g., in normal subjects or in non-cancer subjects such as where cancer is undetectable).
  • a control can be, e.g., the average or median amount of biomarker present in comparable samples of normal subjects in normal subjects or in non-cancer subjects such as where cancer is undetectable.
  • the control amount is measured under the same or substantially similar experimental conditions as in measuring the test amount.
  • the correlation may take into account the presence or absence of the biomarkers in a test sample and the frequency of detection of the same biomarkers in a control.
  • the correlation may take into account both of such factors to facilitate determination of cancer status.
  • the methods further comprise managing or modifying clinical treatment of a subject based on the status of the cancer. For example, if the result of the methods of the present invention is inconclusive or there is reason that confirmation of status is necessary, the physician may order more tests (e.g., CT scans, PET scans, MRI scans, PET-CT scans, X-rays, biopsies, blood tests (LFTs, LDH).
  • tests e.g., CT scans, PET scans, MRI scans, PET-CT scans, X-rays, biopsies, blood tests (LFTs, LDH).
  • the physician may schedule the subject for treatment.
  • the subject may receive therapeutic treatments (such as administration of anticancer agents, either in lieu of, or in addition to, surgery. No further action may be warranted.
  • a maintenance therapy or no further management may be necessary.
  • Anticancer agents may include, one or more of an alkylating agent, an antibiotic agent, an antimetabolic agent, a hormonal agent, a plant-derived agent, an anti-angiogenic agent, a differentiation inducing agent, a cell growth arrest inducing agent, an apoptosis inducing agent, a cytotoxic agent, a biologic agent, a gene therapy agent, a retinoid agent, an histone deacetylase inhibitor, an enzyme inhibitor, a cytokine, a chemokine, an antibody, a DNA molecule, an RNA molecule, a small molecule, a peptide, or a peptidomimetic, or any combination thereof, but are not limited to these examples.
  • the invention also provides for such methods where the biomarkers (or specific combination of biomarkers) are measured again after clinical treatment of a subject.
  • the methods are used to monitor the status of the cancer, e.g., response to cancer treatment, remission of the disease or progression of the disease.
  • the methods can be repeated after each treatment the subject receives, allowing the physician to follow the effectiveness of the course of treatment. If the results show that the treatment is not effective, the course of treatment can be altered accordingly.
  • a diagnosis based on the presence or absence in a test subject of any the biomarkers of this invention is preferably communicated to the subject as soon as possible after the diagnosis is obtained.
  • the diagnosis may be communicated to the subject by the subject's treating physician.
  • the diagnosis may be sent to a test subject by email or communicated to the subject by phone.
  • a computer may be used to communicate the diagnosis by email or phone.
  • the message containing results of a diagnostic test may be generated and delivered automatically to the subject using a combination of computer hardware and software which will be familiar to artisans skilled in telecommunications.
  • One example of a healthcare- oriented communications system is described in U.S. Patent No. 6,283,761 ; however, the present invention is not limited to methods which utilize this particular communications system.
  • all or some of the method steps including the assaying of samples, diagnosing of diseases, and communicating of assay results or diagnoses, may be used.
  • Methods of the invention for determining the cancer status of a subject include for example, obtaining a biomarker profile from a sample taken from the subject; and comparing the subject's biomarker profile to a reference biomarker profile obtained from a reference population, wherein the comparison is capable of classifying the subject as belonging to or not belonging to the reference population; wherein the subject's biomarker profile and the reference biomarker profile comprise one or more biomarkers as described herein.
  • the method may further comprise repeating the method at least once, wherein the subject's biomarker profile is obtained from a separate sample taken each time the method is repeated.
  • Samples from the subject may be taken at any time, for example, the samples may be taken 24 hours apart or any other time determined useful.
  • the reference biomarker profile can be obtained from a population comprising a single subject, at least two subjects, at least 20 subjects or more. The number of subjects will depend, in part, on the number of available subjects, and the power of the statistical analysis necessary.
  • a dataset can be analyzed by multiple classification algorithms. Some classification algorithms provide discrete rules for classification; others provide probability estimates of a certain outcome (class). In the latter case, the decision (diagnosis) is made based on the class with the highest probability. For example, consider the three-class problem: healthy, benign, and cancer. Suppose that a classification algorithm (e.g. nearest neighbor) is constructed and applied to sample A, and the probability of the sample being healthy is 0, benign is 33%, and cancer is 67%. Sample A would be diagnosed as being cancer. This approach, however, does not take into account any "fuzziness" in the diagnosis, e.g., that there was a certain probability that the sample was benign.
  • a classification algorithm e.g. nearest neighbor
  • sample B which has a probability of 0 of being healthy or benign and a probability of 1 of being cancer.
  • Other classification algorithms and formulae include, but are not limited to, Principal Component Analysis (PCA), cross-correlation, factor rotation, Logistic Regression (LogReg), Linear
  • LDA Discriminant Analysis
  • ELDA Eigengene Linear Discriminant Analysis
  • SVM Support Vector Machines
  • RF Random Forest
  • RPART Recursive Partitioning Tree
  • SC Shrunken Centroids
  • Boosting Decision Trees, Neural Networks, Bayesian Networks, Support Vector Machines, Leave-One-Out (LOO), 10-Fold cross-validation (10-Fold CV), and Hidden Markov Models, among others.
  • the present invention provides a kit comprising reagents that detect one or more histone demethylases, a sample derived from a subject having normal control levels, and optionally instructions for using the reagents in the methods disclosed herein.
  • the kit can include detection reagents further including one or more antibodies or fragments thereof, one or more aptamers, one or more oligonucleotides, or combinations thereof.
  • kits for qualifying cancer status and/or detecting or diagnosing cancer wherein the kits can be used to detect the biomarkers of the present invention.
  • the kits can be used to detect any one or more of the biomarkers described herein, which biomarkers are differentially present in samples of cancer subjects and normal subjects.
  • the kits of the invention have many applications.
  • the kits can be used in any one of the methods of the invention described herein,. such as, inter alia, to differentiate if a subject has cancer or has a negative diagnosis, thus aiding a cancer diagnosis.
  • the kits can be used to identify compounds that modulate expression of one or more of the biomarkers by using in vitro or in vivo animal models for cancer.
  • kits of the present invention include a biomarker-detection reagent, e.g., nucleic acids that specifically identify one or more biomarker nucleic acids by having homologous nucleic acid sequences, such as oligonucleotide sequences or aptamers, complementary to a portion of the biomarker nucleic acids or antibodies to proteins encoded by the biomarker nucleic acids packaged together.
  • the oligonucleotides can be fragments of the biomarker genes.
  • the oligonucleotides may be single stranded or double stranded.
  • the oligonucleotides can be 200, 150, 100, 50, 25, 10 or less nucleotides in length.
  • the kit may contain in separate containers a nucleic acid or antibody (either already bound to a solid matrix or packaged separately with reagents for binding them to the matrix), control formulations (positive and/or negative), and/or a detectable label such as fluorescein, green fluorescent protein, rhodamine, cyanine dyes, Alexa dyes, luciferase, radiolabels, among others.
  • Instructions e.g., written, tape, VCR, CD-ROM, etc.
  • the assay may for example be in the form of a Northern hybridization or a sandwich ELISA as known in the art.
  • biomarker detection reagents can be immobilized on a solid matrix such as a porous strip to form at least one biomarker detection site.
  • the measurement or detection region of the porous strip may include a plurality of sites containing a nucleic acid.
  • a test strip may also contain sites for negative and/or positive controls. Alternatively, control sites can be located on a separate strip from the test strip.
  • the different detection sites may contain different amounts of immobilized nucleic acids, e.g., a higher amount in the first detection site and lesser amounts in subsequent sites.
  • the number of sites displaying a detectable signal provides a quantitative indication of the amount of biomarkers present in the sample.
  • the detection sites may be configured in any suitably detectable shape and are typically in the shape of a bar or dot spanning the width of a test strip.
  • the kit contains a nucleic acid substrate array comprising one or more nucleic acid sequences, e.g., primers for nucleic acid amplification.
  • the nucleic acids on the array specifically identify one or more nucleic acid sequences represented by the biomarkers of the present invention.
  • the expression of 2, 3, or all 4 of the sequences represented by the biomarkers described herein can be identified by virtue of binding to the array.
  • the substrate array can be on, e.g., a solid substrate, e.g., a "chip" as described in U.S. Patent No. 5,744,305.
  • the substrate array can be a solution array, e.g., xMAP (Luminex, Austin, TX), Cyvera (Illumina, San Diego, CA), CellCard (Vitra Bioscience,
  • the kit may also contain reagents, and/or enzymes for amplifying or isolating sample DNA.
  • the kits may include reagents for real-time PCR, for example, TaqMan probes and or primers, and enzymes.
  • a kit comprises: (a) a substrate comprising an adsorbent thereon, wherein the adsorbent retains or is otherwise suitable for binding a biomarker, and (b) instructions to detect the biomarker or biomarkers by contacting a sample with the adsorbent and detecting the biomarker or biomarkers retained by the adsorbent.
  • the kit may comprise an eluant (as an alternative or in combination with instructions) or instructions for making an eluant, wherein the combination of the adsorbent and the eluant allows detection of the biomarkers using gas phase ion spectrometry.
  • Such kits can be prepared from the materials described above, and the previous discussion of these materials (e.g., probe substrates, adsorbents, washing solutions, etc.) is fully applicable to this section and will not be repeated.
  • the kit may comprise a first substrate comprising an adsorbent thereon (e.g., a particle functionalized with an adsorbent) and a second substrate onto which the first substrate can be positioned to form a probe, which may be removed and inserted into machine, such as, e.g., a gas phase ion spectrometer.
  • the kit may comprise a single substrate, which is in the form of a probe with adsorbents on the substrate that can be removed and inserted into a machine.
  • the kit may further comprise a pre- fractionation spin column (e.g., Cibacron blue agarose column, anti-HSA agarose column, K-30 size exclusion column, Q-anion exchange spin column, single stranded DNA column, lectin column, etc.).
  • a kit comprises (a) an antibody that specifically binds to a biomarker; and (b) a detection reagent.
  • An antibody may be, for example, an antibody directed against the gene products of a histone demethylase gene.
  • the kit may further comprise pre-fractionation spin columns.
  • the kit may further comprise instructions for suitable operation parameters in the form of a label or a separate insert.
  • the kit may further comprise a standard or control information so that the test sample can be compared with the control information standard to determine if the test amount of a biomarker detected in a sample is a diagnostic amount consistent with a diagnosis of cancer.
  • the docking template structure of LSD1 was derived from the crystal structure of LSD1 bound to the substrate-like peptide (PDB code: 2vld) (Forneris, F. et al., (2007) J. Biol. Chem. 282: 20070-4). The peptide and water molecules were removed from the crystal structure and the polar hydrogen atoms were added to the amino acid residues before docking. The active site of LSD1 was used in the design of chemical molecules. Docking was performed using the latest version of AutoDock 4.0 (Morris, G.M. et al. (1998) J. Comput. Chem. 19: 1639-1662). The illustrated structures were made by Pymol (DeLano, W.L. (2002) The PvMOL Molecular Graphics System, DeLano Scientific, Palo Alto, CA, USA).
  • Pluripotent mouse teratocarcinoma F9 human mediastinal mixed germ NCCIT cells (an intermediate cell between seminoma and embryonic carcinoma), and human testicular embryonic carcinoma NTERA-2 cells were obtained from American Type Culture Collection.
  • NCCIT cells were grown in RPMI-1640 medium.
  • F9 cells were grown on petri dishes coated with 0.1 % gelatin.
  • Histone H3 peptides with dimethylated lysine 4 were purchased from AnaSpec.
  • reaction mixture 0.5 uL of the reaction mixture were equally mixed with 0.5 uL MALDI matrix (2mg/mL a-cyano-4-hydroxycinamic acid with 0.1% trifluoroacetic acid) and spotted onto the Opti-TOFTM 384 Well Insert (Applied Biosystems) to allow solvent evaporation and pepudes/matrix co-crystallization.
  • MALDI matrix 2mg/mL a-cyano-4-hydroxycinamic acid with 0.1% trifluoroacetic acid
  • Opti-TOFTM 384 Well Insert Applied Biosystems
  • AAGGAAAGCUAGAAGAAAA (SEQ ID NO:7), which matches 100% for both human and mouse LSDl genes, and was designed and synthesized by Dharmacon.
  • Testicular carcinoma tissue microarray slides (with 5 micron thickness tissues) were obtained from U.S. Biomax, Inc. Slides were baked and deparaffinized according to standard protocol (Shim, E.H. et al. (2003) Cancer Res. 63: 1583-8). Antigen retrieval was carried out in DivaDecloaker at 90°C for 30 minutes (for LSDl staining) or 30 seconds at 125°C, 30 seconds (for Oct4 staining) using the Decloaking Chamber (Biocare Medical). Slides were immersed in 3% H2O2 for 5 minutes to inactivate the endogenous peroxidase. Nonspecific binding sites were blocked using Background Sniper for 15 minutes.
  • Rabbit monoclonal antibody against LSDl (C69G12, Cell Signaling) was diluted 1 :800 in Renoir Red Diluent, and the mouse monoclonal antibody for Oct4 (Biocare Medical) was pre- diluted by the supplier in DaVinci Green Diluent. Slides were incubated with primary antibodies at 4°C overnight, washed and then incubated with Rabbit-Probe or Mouse-Probe MACH3 HRP- polymer detection system according to the supplier's instructions.
  • the positively charged residues (Arginine 2 and Arginine 8) of the peptide establish favorable electrostatic interactions with a cluster of negatively charged residues on LSDl surface that involve Aspartic acid (Asp) 375, Glutamic acid (Glu) 379, Asp 553, Asp 555, Asp 556, Asp 557, and Glu 559 (Figure 1A).
  • the funnel channel that provides access to FAD is blocked by the peptide inhibitor.
  • a non-peptide chemical scaffold that binds to LSDl with similar non- covalent binding mode to that of the peptide inhibitor was designed de novo (Figure IB).
  • Example 2 LSD1 compounds inhibit LSD1 demethylase activity in vitro
  • a recombinant fusion protein of glutathione-S- transferase and human LSD1 was expressed in bacteria, affinity purified, and examined for its ability to demethylate a synthetic histone H3 amino terminal substrate peptide that contains the di-methylated K4 in vitro ( Figure 2).
  • the LSD1 protein displayed a dose- and time-dependent demethylase activity, yielding the intermediate mono-methylated and final unmethylated H3K4 peptides (Shi, Y. et al. (2004) Cell 119: 941-53), which were separated, resolved, and semi- quantified by MALDI TOF-TOF mass -spectrometry (Figure 2B).
  • Example 2 LSDl compounds inhibit LSDl demethylase activity in vivo
  • mice F9 embryonic carcinoma/teratoma cells were treated with synthetic LSDl compounds and then monitored for the accumulation of di- methylated H3K4 by Western blotting with anti-di-methylated H3K4 antibodies. While compounds CBB IOOI, CBB 1002, CBB 1004, CBB 1005, and CBB1009 did not have noticeable effects on di-methylated H3K4 at high concentrations (250 ⁇ , Figure 3A-B), compounds CBB 1003 and CBB 1007 at low concentrations led to the significant increase of di-methylated H3 4 after treatment ( Figure 3A-B).
  • LSD1 inactivation A hallmark of LSD1 inactivation is that the resultant increase of di-methylated H3K4 can lead to the activation of epigenetically suppressed genes such as M4-ArchR and SCN3A in cultured cells (Shi, Y. et al., (2004) Cell 119: 941-53).
  • epigenetically suppressed genes such as M4-ArchR and SCN3A in cultured cells (Shi, Y. et al., (2004) Cell 119: 941-53).
  • LSD1 inhibitory compounds To investigate whether LSD1 inhibitory compounds can induce epigenetically suppressed gene expression, the activation of M4-ArchR and SCN3A genes was monitored by quantitative RT-PCR after addition of LSD1 compounds to the cells (Figure 3C-D).
  • Example 3 LSD1 inhibitors selectively inhibit the growth of pluripotent embryonic carcinoma, teratoma, and seminoma cells
  • LSD1 is highly conserved among high eukaryotes and null mutation of LSD1 genes in the mouse causes embryonic lethality, suggesting LSD1 is essential for development.
  • the physiological function of LSD1 is quite a puzzle since it can only demethylate di- and mono- methylated but not tri-methylated H3K4, while the members of JARID1 family (1 A-1D) that contain the Jumonji C (JmjC) domain can demethylate tri-, di, and mono-methylated H4K43.
  • loss of LSD1 expression by siRNA-mediated ablation in many cancer or normal cells usually did not cause inhibition of cell growth (Shi, Y. et al., (2004) Cell 119: 941- 53; Wang, Y.
  • CBB1006 and CBB1008 correlate with their ability to induce di-methylation of H3K4 and activation of epigenetically suppressed gene expression ( Figures 3, 4, and 5A-5E), suggesting that the growth inhibition is a consequence of specific LSD1 inhibition by these compounds in F9 cells.
  • LSD1 inhibitory compounds are highly selective towards pluripotent mouse F9 embryonic carcinoma cells but not other types of cancer or normal cells.
  • the mouse F9 cells express the pluripotent stem cell markers Oct4 and Sox2 and retain stem cell-like properties, such as rapid spherical growth and the ability to differentiate into less pluripotent cells under cell culture conditions (Strickland, S. et al., (1980) Cell 21: 347-55; Cheng, L. et al. (2007) J. Pathol. 211 : 1-9). Inoculation of F9 cells into immunodeficient mice can induce teratomas which are capable of differentiating into a wide range of tissue types (Strickland, S. et al., (1980) Cell 21 : 347-55). Unlike F9 cells, cancer cell lines such as HeLa, 293, and immortal ⁇ 3 ⁇ 3 cells do not express pluripotent stem cell markers Oct4 and Sox2 and are incapable of differentiation and hence are considered non-stem cell lineage (Figure 6D).
  • human pluripotent mediastinal mixed germ NCCIT cells, an intermediate cell between seminoma and embryonic carcinoma, and pluripotent human testicular embryonic carcinoma NTERA-2 cells were examined. It was found that the growth of both human NCCIT and NTERA-2 cells are also highly sensitive to LSD1 inhibitors ( Figure 5G and 5H, and Figure 9A), similar to that of mouse teratoma F9 cells ( Figure 5A-5E). These studies indicate that embryonic carcinomas, teratomas, seminomas, possibly other stem cell- derived cells of pluripotent germ cell tumors (Cheng, L. et al. (2007) J.
  • Pathol. 211 : 1-9) are most sensitive to LSDl inhibitors, suggesting that LSDl plays an essential function for these pluripotent cancer cells. Similar results were seen with the ovarian adenocarcinoma cell line IGROVl and ovarian teratocarcinoma cell line PA-1 ( Figure 10). Both IGROVl and PA-1 express pluripotent stem cell markers Oct4 and Sox2, and high levels of LSDl ( Figure 18).
  • IGROVl is a human epithelial ovarian cancer cell line originally derived from a stage ⁇ ovarian adenocarcinoma patient with an endometrioid, serous clear and undifferentiated tumor.
  • Figure 11 shows that F9 cells are sensitive to RBBP5 siRNA, but HeLa cells are not.
  • RBBP5 is an essential component of MLL histone methyltransferase complex that methylates histone H3 at lysine 4 (K4), while non-pluripotent cancer HeLa cells are not very sensitive to RBBP5 siRNA.
  • Loss of RBBP5 reduces histone H3 at lysine 4 tri-, di-, and mono-methylations (Figure 17C), while inhibition of LSDl leads to an increased di-, and mono-methylation in histone H3 at lysine 4 (H3K4)( Figures 3A-B, 8, and 15A-C).
  • Figure 11 demonstrates that pluripotent cancer stem cells are very sensitive to changes in histone H3 methylation status, while non-pluripotent cancer cells are not.
  • the growth of ovarian cancer stem cell-like cancer cell IGROVl and ovarian teratoma cell PA-1 is also very sensitive to the changes of histone methylation at lysine 4 (K4) caused by loss of RBBP5 using siRNA-mediated ablation (Figure 12).
  • LSDl compounds specifically target at LSDl and modulate histone methylation at H3K4 in vivo and loss or inhibition of LSDl blocks the growth of pluripotent teratoma, embryonic carcinoma, and seminoma cells, but without obvious effects on the proliferation of other non-pluripotent cancer or normal cells. Similar growth-inhibitory effects after modulation of H3K4 methylation by loss of RBBP5 were also observed. This selectivity of LSDl inhibitors and alteration of H3K4 methylation towards pluripotent cancer cells suggests that these LSDl inhibitory compounds may provide a novel therapeutic tool to specifically inhibit stem cell-like cancers without affecting the growth of other cells.
  • pluripotent F9, NCCIT, and NTERA-2 cancer cells are selectively sensitive to LSDl inhibition or inactivation
  • levels of LSDl in these cells were examined and compared with that of other non-pluripotent cancer cells such as HeLa and 293.
  • the pluripotent F9, NCCIT, and NTERA-2 cells express high levels of LSDl protein, while LSDl is much lower in HeLa and 293 cells ( Figure 6D).
  • the high levels of LSDl in F9, NCCIT, and NTERA-2 cells are correlated with the expression of pluripotent stem cell markers Oct4 and Sox2 ( Figure 6D), which are not present in HeLa or 293 cells.
  • histone H3 and di-methylated histone H4K4 are similar to histone H3 and di-methylated histone H4K4 by their specific antibodies.
  • histone H3 and dimethylated H3K4 antibodies recognize additional protein bands that migrate slower than histone H3, suggesting that histone H3 is likely further modified.
  • Elevated levels of LSDl may render the pluripotent cancer cells more dependent on LSDl, which may underlie the selective sensitivity of these cancer stem cells towards the inactivation of LSDl by specific LSDl inhibitors or siRNAs.
  • LSDl protein levels in human testicular seminomas is consistent with the observation that pluripotent F9, NCCrr, and NTERA-2 cancer cells also contain high levels of LSDl, Oct4, and Sox2 protein and that these cells are highly sensitive to LSDl inhibitors.
  • LSDl can serve as a biomarker for teratomas, seminomas, as well as other undifferentiated germ cell tumors or cancers with stem cell properties, and LSDl inhibitors may be used to selectively treat these cancers in vivo without affecting other normal cells.
  • Example 5 Ovarian and Breast Cancer Cells Selectively Sensitive to LSDl Inhibitors
  • LSDl inhibitors selectively inhibited the proliferation of pluripotent cancer cells including teratocarcinoma, embryonic carcinoma, or seminoma cells that express stem cell markers Oct4 and Sox2, while displaying minimum growth inhibitory effects on non-pluripotent cancer or normal somatic cells.
  • pluripotent cancer cells including teratocarcinoma, embryonic carcinoma, or seminoma cells that express stem cell markers Oct4 and Sox2, while displaying minimum growth inhibitory effects on non-pluripotent cancer or normal somatic cells.
  • ovarian and breast cancer cells that are selectively sensitive to LSDl inhibitors are identified ( Figures 10 and 14A-F). These cancer cells express pluripotent or multipotent stem cell proteins, such as Sox2, Lin28, KLF4, and/or Oct4 ( Figure 18), and are potential cancer stem cell-like cells.
  • MCF-7 human breast adenocarcinoma, SKOV-3 human ovarian carcinoma, and T47D human breast ductal carcinoma cells were obtained from American Type Culture Collection (ATCC).
  • IGROV1 human ovarian carcinoma cells were from National Cancer Institute and A2780 human ovarian carcinoma cells were from Sigma-Aldrich. They were maintained in Dulbecco's Modified Eagle medium or RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics.
  • F9, NCCIT, and HeLa cells were cultured as described previously (Wang et al. Novel Histone Demethylase LSD1 Inhibitors Selectively Target Cancer Cells with Pluripotent Stem Cell Properties.
  • Anti-Lin28, DNMT1, Nanog, Sall4, histone H3, tri-, di-, and monomethylated histone H3 at lysines 4 (H3K4) and 9 (H3K9), and Klf4 antibodies were from Abcom.
  • Anti-Sox2, Oct4, and CUL1 antibodies were described previously (Wang et al., 2011 supra).
  • CUL4-DDB 1 ubiquitin ligase interacts with multiple WD40-repeat proteins and regulates histone methylation. Nat Cell Biol, 8:1277-1283, (2006)).
  • total RNAs were extracted from A2780 or T47D cells or other cells using TRizol Reagent
  • RNA was used in each real time-PCR (RT-PCR) reaction using the respective primers for the target mouse genes (Shi et al. Histone
  • SCN3A forward 5'.- C ACTACTTCCTACTTC AATGGCA-3 ' (SEQ ID NO:l) and reverse 5'- A A ACAGCG ATAAG A AGGCCC AG-3 ' (SEQ ID NO:15); 2) CHRM4., forward 5'- TC AC ACCTGTC A ATGGCAGC-3 ' (SEQ ID NO: 3) and reverse 5'- GCC AGTAGCCCTTGATGATG-3 ' (SEQ ID NO:4); 3) beta-actin, forward 5'- TCC AGCCTTCCTTCTTGGGTATG-3 ' (SEQ ID NO:5) and reverse 5'-
  • RBBP5 siRNA GAGCCGAGAUGGUCAUAAAUU (SEQ ID NO: 10); WDR5 siRNA: CAGAGGATAACCTTGTTTA (SEQ ID NO:l l); and Luciferase (Luc):
  • CGTACGCGGAATACTTCGA SEQ ID NO: 12
  • the siRNAs were designed and synthesized by Dharmacon.
  • LSD1 inhibitory compounds CBB1003 and CBB1007 can specifically inhibit the growth of pluripotent cancer cells such as teratocarcinoma, embryonic carcinoma, and seminoma cells but with minimum effects towards non-pluripotent cancer cells or normal somatic cells such as HeLa, 293, and ⁇ 3 ⁇ 3 (see also Wang et al. 2011 i «pra)( Figures 5 and 9). LSD1 inhibitors also had minimum effects towards the growth of ovarian carcinoma cells such as Hs38.T cells that are non-cancer stem cells ( Figure 13)(Gao et al.
  • PA-1 a human ovarian teratocarcinoma cell line
  • CBB1007 Figure 13B
  • CBIOIO Figure 13C
  • This cell line expresses pluripotent stem cell proteins Oct4, Sox2, and Lin28, as shown in Figure 18, Lane 5.
  • Ovarian and breast cancer cells that are non-teratoma, embryonic carcinoma, or seminoma in origin were also screened.
  • the A2780 cell a human ovarian carcinoma that was reported to express pluripotent stem cell proteins Sox2 and Lin28 was tested with CBB1003 and CBB1007 at various concentrations and was growth-inhibited by LSDl inhibitors (Zhong et al. Identification of microRNAs regulating reprogramming factor LIN28 in embryonic stem cells and cancer cells. / Biol Chem, 285:41961-41971 (2010).), with the results shown in Figure 14A and depicted graphically in the top graph of Figure 14C.
  • IGROV1 and SKOV-3 two additional human ovarian carcinoma that also have been indicated to express Oct4, Sox2, and/or Lin28 (Peng et al.
  • Pluripotency factors Lin28 and Oct4 identify a sub-population of stem cell-like cells in ovarian cancer.
  • FEBS Lett, 527: 10-4 (2002) were also sensitive to LSDl inhibitors of the invention as shown in Figures 14D and 14E). Notably, it was found all these cells expressing one or more pluripotent stem cell markers showed excellent sensitivity towards LSDl inhibitors CBB1003 and 1007 (Fig. 14). In addition to CBB1003 and CBB1007, IGROV1 cells also displayed excellent sensitivity towards a new derivative of LSDl inhibitor, CBBIOIO ( Figure 14D).
  • T47D a human ductal breast epithelial carcinoma cell line expressing Lin28 ( Zhong et al. supra), was found to be highly sensitive towards LSDl inhibitory compounds CBB1003 and CBB1007 (Fig. 14B and 14C).
  • MCF-7 a breast adenocarcinoma cell line obtained from ATCC that expresses pluripotent stem cell protein Sox2 ( Figure 18), also showed significant sensitivity to LSDl inhibitors (Fig. 14F).
  • LSDl compounds are active in cancer cells to inhibit LSDl demethylation activity
  • LSDl is a demethylase specific for mono- and di-methylated H3K4 and loss of LSDl in vivo causes the accumulation of these methylated forms of H3K4 (Shi et al. Histone demethylation mediated by the nuclear amine oxidase homolog LSDl. Cell, 119:941-953 (2004), Schulte et al. Lysine-specific demethylase 1 is strongly expressed in poorly differentiated neuroblastoma: implications for therapy. Cancer Res, 69:2065-2071 (2009), Wang et al. LSDl is a subunit of the NuRD complex and targets the metastasis programs in breast cancer. Cell, 138:660-72 (2009)).
  • LSDl can demethylate non-histone proteins such as DNMT1 to stabilize its protein stability and regulate global DNA methylation in mouse ES cells (Wang et al. The lysine demethylase LSDl (KDM1) is required for maintenance of global DNA methylation. Nat Genet;41 : 125-9 (2009)). Loss of LSDl leads to the destabilization of DNMT1 in mouse ES cells (Wang et al. 2009 supra). It was found that inhibition of LSDl by CBB1003 and 1007 can also induce DNMT1 destabilization and downregulation (Figure 15C), indicating that the effect of LSDl inhibitors are specific for LSD demethylation activity in vivo.
  • RNA-interference-based ablation was used in these cancer cells to determine the effect of loss of LSDl .
  • loss of LSDl by treatment of cells with specific LSDl siRNAs led to the growth inhibition of A2780, IGROVl, SKVO-3, and T47D (Fig. 16A and 16B).
  • the loss of LSDl by siRNA in non-stem Hs38 cells did not have much effect on its growth (Fig. 16A), consistent with the effect of LSDl inhibitor on this cell line, and suggesting that LSDl in this non-stem cell like ovarian cancer cell is not essential.
  • the non-sensitive HeLa or Hs38.T cells do not express any of the pluripotent stem cell proteins.
  • A2780, IGROV1, and T47D, as well as SKOV-3 and MCF-7 may retain some properties of pluripotent or multipotent stem cells, such as the transcriptional circuitry of pluripotent stem cells, thereby conferring their sensitivity towards LSD1 inhibitors.
  • This Example indicates that maintenance of proper levels of H3K4 methylation in these cells is needed for these cells to proliferate and survive, and any changes in histone methylation may adversely affect the ability of these cells to maintain stem cell-like properties for proliferation, survival, or differentiation.
  • This Example further indicates that methylation of histone H3 at lysine 4 serves as a good target for these cancer stem cell-like cells.

Abstract

A compound of formula I: or a pharmaceutically acceptable salt, Ri is an optionally substituted heterocyclic group; R and R' are independently H, CH3, NO2, SO2N(CH3)2, SO2N((CH3)SO2), COOH, COOCH3, CO(N(CH3)), alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl, heterocycloalkyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, trifluoromethyl, pentafluoroethyl, halogen, cyano, thio, amido, ether, ester, hydroxyl, hydroxyalkyl, saturated or unsaturated fatty acids, azido, phosphonamido, sulfonamido, lactam, phosphate, phosphonato, phosphinato, amino, acylamino, amidino, imino, guanidino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, cyano, or azido; m and n are independently integers from 1 to 4; T, U, V, and Z are independently CH, N, or CR; L is NH, CH2, O, S, or SO2; X is N or CH; Y and Y' are independently O or S; and R3 is an optionally substituted aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group.

Description

HISTONE DEMETHYLASE INHIBITORS AND USES THEREOF FOR
TREATMENT OF CANCER
STATEMENT OF RELATED APPLICATION
This application claims priority under 35 U.S.C. 119(e) to U.S. Provisional Application
Number 61/416,566, filed November 23, 2010, and is incorporate herein by reference in its entirety.
GOVERNMENT INTEREST STATEMENT
This invention was made in whole or in part with government support under grant number NIH CA098955 awarded by the National Institutes of Health. The government may have certain rights in the invention.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 17, 2011, is named 08939313.txt and is 3,883 bytes in size.
FIELD OF THE INVENTION
The present invention relates to, inter alia, compounds and compositions that inhibit histone demethylases. In particular, the present invention concerns histone demethylase inhibitor compounds and derivatives thereof, pharmaceutical compositions comprising histone demethylase inhibitor compounds, and methods of using these compounds and compositions to modulate histone methylation, inhibit growth and proliferation of cancer cells, and promote differentiation of cancer cells in vitro and in vivo, as well as for treatment of cancer in a subject. The present invention also concerns the use of histone demethylases as a therapeutic, diagnostic, or prognostic biomarker for identifying tumors comprising cells expressing one or more pluripotent or multipotent stem cell markers. The present invention further relates to modulating or inhibiting histone demethylase activity in pluripotent/multipotent cells using inhibitory nucleic acids. The invention additionally encompasses the use of histone demethylase inhibitors that alter the expression or enzyme activity of histone demethylases to modulate the properties of pluripotent embryonic stem/iPS (induced pluripotent stem) cells or the re-reprogramming of iPS cells from somatic cells for stem cell-based therapy.
BACKGROUND OF THE INVENTION
Histone methylation is a major covalent modification of histones that is often regarded as part of the "histone code" that provides the structural and functional characteristics of chromatin to epigenetically define gene expression patterns in a particular cell (Klose, R.J. et al., (2007) Nat. Rev. Mol. Cell Biol. 8: 307-18; Shi, Y. (2007) Nat. Rev. Genet. 8: 829-33). While methylation of histone H3 at lysines 9 (K9) and 27 (K27) suppresses gene expression, the mono-, di-, and tri-methylations of lysine 4 (K4) in histone H3 (H3K4) associate with actively transcribed genes (Klose, R.J. et al., (2007) Nat. Rev. Mol. Cell Biol. 8: 307-18; Shi, Y. (2007) Nat. Rev. Genet. 8: 829-33; Agger, K. et al. (2008) Curr. Opin. Genet. Dev. 18: 159-68).
Histone methylation is dynamically controlled by specific histone methyltransferases and demethylases (Agger, K. et al. (2008) Curr. Opin. Genet. Dev. 18: 159-68). In particular, the methylations at H3K4 are primarily catalyzed by histone methyltransferase complexes composed of the members of MLL (Mixed Lineage Leukemia) SET-domain methyltransferases, ASH2, WDR5, and RBBP5 (Klose, R.J. et al., (2007) Nat. Rev. Mol. Cell Biol. 8: 307-18; Wysocka, J. et al. (2005) Cell 121 : 859-72). The methylations at H3K4 allow the direct binding of proteins that contain a plant homeodomain finger domain (PHD), such as ING2, NURF, and BPTF, which in turn remodel chromatin and promote active gene expression (Klose, R.J. et al., (2007) Nat. Rev. Mol. Cell Biol. 8: 307-18; Shi, Y. (2007) Nat. Rev. Genet. 8: 829-33; Li, H. et al., (2006) Nature 442: 91-95; Wysocka, J. et al., (2006) Nature 442: 86-90). The methyl groups in H3K4 are removed by histone demethylases LSD1 (also called KDM1, AOF2, or BHC110) and the members of JAREDl family (1 A-1D), as well as FBXL10, leading to transcriptional inactivation (Shi, Y. (2007) Nat. Rev. Genet. 8: 829-33; Agger, K. et al. (2008) Curr. Opin.
Genet. Dev. 18: 159-68). In addition to methylases and demethylases, H3K4 methylation is also regulated by other mechanisms. For example, previous studies indicate that a novel
CUL4- and DDB l -containing ubiquitin E3 ligase complex is also required for H3K4 methylation through their interaction with the WD40 repeat proteins WDR5 and RBBP5 (Higa, L. et al. (2006) Nat. Cell Biol. 8: 1277-83).
Lysine-specific demethylase 1 (LSD1) belongs to the flavin adenine dinucleotide (FAD)-dependent amine oxidase family, and specifically catalyzes the demethylation of di- and mono-methylated H3K4 through amine oxidation (Shi, Y. (2007) Nat. Rev. Genet. 8: 829-33; Shi, Y. et al., (2004) Cell 119: 941-53; Fomeris, F. et al. (2008) Trends Biochem. Sci. 33: 181- 9). In contrast to the JARID1 family of Jumonji C (JmjC) domain-containing demethylases that can remove the methyl group from tri-, di-, and mono-methylated H3K42, demethylation by LSD1 requires a protonated nitrogen in the methylated histone, precluding it from removing the methyl group from tri -methylated H3K4 (Shi, Y. (2007) Nat. Rev. Genet. 8: 829-33; Shi, Y. et al., (2004) Cell 119: 941-53; Culhane, J.C. et al., (2007) Curr. Opin. Chem. Biol. 11 561 -8).
LSD1 is highly conserved among species (Shilatifard, A. (2008) Curr. Opin. Cell Biol. 20: 341-8). Several studies from model organisms such as Drosophila revealed that LSD1 is highly expressed in primordial germ cells of females and mutation of the LSD1 gene in
Drosophila leads to sex-specific embryonic lethality or sterility in the remaining female offspring (DiStefano, L. et al., (2007) Curr. Biol. 17: 808-120). In mice, loss of LSD1 causes embryonic lethality (Wang, J. et al. (2009) Nat. Genet. 41: 125-9). Because the catalytic domain of LSD1 shares significant sequence and structural similarity with other members of the amine oxidase family, most of current investigation on LSD1 function involve the use of non-selective amine oxidase inhibitors, which were developed against two major isoforms of monoamine oxidases, MAO-A and MAO-B, and act through the irreversible modification of the covalently bound FAD at high concentrations (milimolars) (Shi, Y. et al., (2004) Cell 119: 941-53; Culhane, J.C. et al., (2007) Curr. Opin. Chem. Biol. 11 561-8; Schulte, J.H. et al. (2009) Cancer Res. 69: 2065-71 ; Stavropoulos, P. et al., (2007) Expert Opin. Ther. Targets 19: 809-20; Szewczuk, L.M. et al., (2007) Biochemistry 46: 6892-902; Yang, M. et al. (2007) Biochemistry 46: 8058-65; Yang, M. et al, (2007) Nat. Struct. Mol. Biol. 14: 535-9; Wang, Y. et al., (2009) Cell 138: 600-72).
However, these monoamine oxidase inhibitors induce substantial toxicity in vivo by interfering with the activities of many other FAD-dependent amine oxidases.
Cancer stem cells, also often called cancer or tumor initiating cells, have been found in a number of cancers and are considered as the origin of various heterogeneous cancer populations due to their pluripotent or multipotent stem cell property (Lapidot, T et al., (1994) Nature 367: 645-8; Singh, S. . et al., (2004) Nature 432: 396-401; Bapat, S.A. et al., (2005) Cancer Res. 65: 3025-9; Maitland, N.J. et al., (2005) BJU Int. 96: 1219-23; Zhong, X. et al. (2010) J. Biol. Chem. 285: 41961-71 ; Yang, X. et al. (2010) Cancer Res.70: 9463-72; Peng, S. et al. (2008) Oncogene 29: 2153-9; Viswanathan, S. R. et al. (2009) Nat. Genet. 41 : 843-848; Marquardt, J. U. et al. (2010) J. Hepatology 53: 568-77; Kitamura, H. et al. (2009) Lung Cancer 66: 275-81 ; Levy, C. et al. (2006) TRENDS in Mol. Medicine 12: 406-14; Tysnes, B. B. (2010) Neoplasia 12: 506-15; Holmberg, J. et al. (2011) PLoS One 6: el8454; Mishra, L. et al. (2009) Hepatology 49: 318-29). Development of therapeutic drugs that target cancer stem cells is an unmet medical demand since cancer stem cells appear to be more resistant to conventional chemotherapy or radiotherapy and often act as the source for recurring drug resistant cancers after treatment (Lapidot, T et al., (1994) Nature 367: 645-8; Maitland, N.J. et al., (2005) BJU Int. 96: 1219-23; Bapat, S.A. (2010) Reproduction 140: 33-41). It is well established that pluripotent stem cells have distinct patterns of histone methylation and other epigenetic modifications for their maintenance and self renewal (Mikkelsen, T.S. et al., (2007) Nature 448: 553-60); and reprogramming of somatic cells into the induced pluripotent stem (iPS) cells by expression of pluripotent stem cell genes Oct4, Sox2, and Lin28 is associated with dramatic rearrangement of histone methylation (Takahashi, K. et al., (2007) Cell 131: 861-72; Mikkelsen, T.S. et al., (2008) Nature 454: 49-55; Yu, J. et al. (2007) Science 318: 1917-20; Ho, R. et al. (2011) J. Cell Physiol. 226: 868-78; Mallanna, S. and
Rizzino, A. (2010) Dev. Biol. 344: 16-25; Patel, M. and Yang, S. (2010) Stem Cell Review 6: 367-80). However, the exact role of each histone methylase and demethylase in defining the specific transcriptional program in pluripotent stem cells, cancer stem cells, and somatic cells remains unclear.
Therefore, there remains a need in the art to develop specific inhibitors of histone demethylase inhibitors that do not display the in vivo toxicity observed in the use of monoamine oxidase inhibitors. Such histone demethylase inhibitors would be useful in targeting pluripotent and/or multipotent cancer stem cells, which are known to be resistant to currently available anticancer therapies. The compounds, compositions, and methods of the present invention fulfill this need that was heretofore unrecognized.
SUMMARY OF THE INVENTION
The present invention relates to histone demethylase inhibitor compounds and compositions comprising histone demethylase inhibitors for treating cancers, such as germ cell tumors e.g., teratomas, embryonal carcinomas, seminomas, choriocarcinomas, tumors of yolk sac, ovarian teratocarcinomas/embryonal carcinomas, and cancer stem cell-like cells that express pluripotent stem cell markers such as Oct4, Sox2 and/or Lin28 and high levels of histone demethylases, such as the LSD1 protein. Cancer stem cells or cancer initiating cells that express pluripotent stem cell markers Oct4, Sox2, and/or Lin28 have been identified in many different cancer types, such as, germ cell tumors, ovarian cancers, breast cancers, and lung cancers. These cells are believed to be responsible for the recurrence of cancer and are resistant to currently available anticancer therapies. In addition, the compounds disclosed herein are also useful for targeting prostate cancers, breast cancers, gliomas, glioblastomas, bladder cancers, colorectal cancers, lung cancers, skin cancers, leukemias, lymphomas, and neuroblastomas. The present invention also concerns the modulation or inhibition of histone demethylases, such as LSD1, to modulate histone methylation, inhibit the growth, proliferation, and/or survival of cancer cells, as well as to affect the growth and differentiation of embryonic stem cells or the reprogramming of iPS cells from somatic cells that can be applied for stem cell-based therapy. The histone demethylase inhibitors of the invention can be used to eliminate embryonic
carcinomas/teratomas during stem cell-based therapy, which are caused by incomplete differentiation of stem cells or iPS cells. In addition, the present invention also embraces the use of histone demethylases, e.g., LSD1 , as therapeutic targets and biomarkers of cancers, such as germ cell tumors or cancers characterized by the presence of pluripotent and/or multipotent stem cell-like cells.
Accordingly, in one aspect, the present invention provides a compound represented by structural formula I:
Figure imgf000007_0001
or a pharmaceutically acceptable salt thereof, wherein:
Ri is an optionally substituted heterocyclic group;
R and R' are independently selected from the group consisting of: H, CH3, NO2, S02N(CH3)2, S02N((CH3)S02), COOH, COOCH3, CO(N(CH3)), alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl, heterocycloalkyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylarninocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, trifluoromethyl, pentafluoroethyl, halogen, cyano, thio, amido, ether, ester, hydroxyl, hydroxyalkyl, saturated or unsaturated fatty acids, azido, phosphonamido, sulfonamide, lactam, phosphate, phosphonato, phosphinato, amino, acylamino, amidino, imino, guanidino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, cyano, and azido;
m and n are independently integers from 1 to 4;
T, U, V, and Z are independendy selected from CH, N, and CR;
L is selected from NH, CH2, O, S, and S02;
X is selected from N or CH;
Y and Y' are independently selected from O and S;
and R3 is an optionally substituted aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group.
In other aspects, the sented by formula Π:
Figure imgf000008_0001
or a pharmaceutically acceptable salt thereof, wherein: R\ is an optionally substituted heterocyclic group; R2, R4, R5, Re, and R7 are selected from the group consisting of: H, CH3, N02, S02N(CH3)2, S02N((CH3)S02), COOH, COOCH3, CO(N(CH3)), alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl, heterocycloalkyl, alkoxy, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, trifluoromethyl, pentafluoroethyl, halogen, cyano, thio, amido, ether, ester, hydroxyl, hydroxyalkyl, saturated or unsaturated fatty acids, azido, phosphonamido, sulfonamido, lactam, phosphate, phosphonato, phosphinato, amino, acylamino, amidino, imino, guanidino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, cyano, and azido; X is selected from N or CH, and R3 is an optionally substituted aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group. In some embodiments, the Ri heterocyclic group of formula I or Π is selected from piperazine and piperidine. In another embodiment, Ri is substituted with a group selected from an amidino group and a guanidino group.
In one embodiment, Ri is:
Figure imgf000009_0001
wherein ¾ and R9 are selected from alkyl or aryl, and X, Y and Z are selected from N or C.
In one embodiment, Ri is:
Figure imgf000009_0002
wherein n = 0 to 10.
R3 may be an optionally substituted aryl or heteroaryl group, an optionally substituted cycloalkyl group, or an optionally substituted heterocycloalkyl group selected from piperazine and piperidine. R3 may be substituted with a group selected from an optionally substituted amidino group and a guanidine group.
In one embodiment, R3 is:
Figure imgf000009_0003
wherein Rio and Rn are selected from alkyl or aryl, and Y is selected from a substituted aryl or heteroaryl group, an optionally substituted cycloalkyl group, or an optionally substituted heterocycloalkyl group.
In one embodiment, R3 is:
Figure imgf000009_0004
wherein n = 0 to 10. In some embodiments, the present invention provides a compound represented by the structural formula ΙΠ:
Figure imgf000010_0001
or a pharmaceutically acceptable salt thereof, wherein: Ri is an optionally substituted heterocyclic group; R2 is selected from the group consisting of: H, CH3, N02, S02N(CH3)2, S02N((CH3)S02), COOH, COOCH3, CO(N(CH3)), alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl, heterocycloalkyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, trifluoromethyl, pentafluoroethyl, halogen, cyano, thio, amido, ether, ester, hydroxyl, hydroxyalkyl, saturated or unsaturated fatty acids, azido, phosphonamido, sulfonamido, lactam, phosphate, phosphonato, phosphinato, amino, acylamino, amidino, imino, guanidino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfmyl, sulfonate, sulfamoyl, sulfonamido, nitro, cyano, and azido; and R3 is an optionally substituted aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group.
In one embodiment, the Ri heterocyclic group is selected from piperazine and piperidine. In another embodiment, Ri is substituted with a group selected from an amidino group and a guanidino group.
In one embodiment, Ri is:
Figure imgf000010_0002
wherein n = 0 to 10.
R3 may be an optionally substituted aryl group, an optionally substituted cycloalkyl group, or an optionally substituted heterocycloalkyl group selected from piperazine and piperidine. R3 may be substituted with a group selected from an amidino group and a guanidine group. In one embodiment, R3 is:
Figure imgf000011_0001
wherein n = 0 to 10.
In some embodim of formula IV:
Figure imgf000011_0002
or pharmaceutically acceptable salt thereof;
wherein X is a functional group selected from the group consisting of CH2, S, NH, and
S02;
V is O or S;
J, K, L, M, Y, Z and Z' are independently N, CR', or CH;
Ri comprises a functional group selected from the group consisting of hydrogen, a carboxamide (aminocarbonyl), a carboxamidine (carboximidamide), acyl, an alkylsulfonyl, an arylsulfonyl, guanidine, and an aminosulfonyl, any of which may be optionally substituted;
R, R', and R" are independently selected from the group consisting of: H, CH3, NO2,
S02N(CH3)2, S02N((CH3)S02), COOH, COOCH3, CO(N(CH3)), alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl, heterocycloalkyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, trifluoromethyl, pentafluoroethyl, halogen, cyano, thio, amido, ether, ester, hydroxyl, hydroxyalkyl, saturated or unsaturated fatty acids, azido, phosphonamido, sulfonamido, lactam, phosphate, phosphonato, phosphinato, amino, acylamino, amidino, imino, guanidino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfmyl, sulfonate, sulfamoyl, sulfonamido, nitro, cyano, and azido; m, n, and p are independently integers from 1 to 4;
R3 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, and
heterocycloalkyl group, any of which is optionally substituted.
In some aspects, the formula V:
Figure imgf000012_0001
or pharmaceutically acceptable salt thereof;
wherein X is a functional group selected from the group consisting of CH2, S,
NH, and S02;
Y and Z are independently N or CH;
Ri comprises a functional group selected from the group consisting of hydrogen, a carboxamide (aminocarbonyl), a carboxamidine (carboximidamide), acyl, an alkylsulfonyl, an arylsulfonyl, guanidine, and an aminosulfonyl, any of which may be optionally substituted;
R2 comprises a functional group selected from the group consisting of hydrogen, alkyl, nitro, sulfonamide, sulfonimide, amide, and carboxyalkyl, any of which is optionally substituted; and
R3 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, and heterocycloalkyl group, any of which is optionally substituted.
In one embodiment, the compound of the invention may be represented by the following structural formula (also referred to herein as "CBBIOOI"):
Figure imgf000012_0002
In another embodiment, the compound of the invention may be represented by the following structural formula (also referred to herein as "CBB1002"):
Figure imgf000013_0001
The compound of the invention may also be represented by the following structural formula (also refe
Figure imgf000013_0002
In another embodiment, the compound of the invention may be represented by the following structur
Figure imgf000013_0003
In another embodiment, the compound of the invention may be represented by the following structural
Figure imgf000013_0004
In other embodiments, the compound of the invention is represented by the following structural formula (also referred to herein as "CBB1006"):
Figure imgf000014_0001
The compound of the invention may also be represented by the following structural formula (also referred to herein as "CBB 1007"):
Figure imgf000014_0002
In another embodiment, the compound of the invention is represented by the following structural formula (also referred to herein as "CBB1008"):
Figure imgf000014_0003
In another embodiment, the compound of the invention is represented by the following structural formula (also referred to herein as "CBB1009"):
Figure imgf000014_0004
The compound of the invention may also be represented by the following structural formula (also referred to herein as "CBBIOIO"):
Figure imgf000015_0001
Figure imgf000015_0002
In other embodiments, the compound of the invention may be represented by the following struc
Figure imgf000015_0003
In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the compound as disclosed herein, and a pharmaceutically acceptable carrier or diluent. The pharmaceutical composition may further comprise an anticancer agent.
Another aspect of the invention provides a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the compounds of the invention. The cancer can be one that is characterized by the presence of pluripotent and/or multipotent cancer cells, such as, for example, embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, ovarian cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian epithelial cancer, gliomas, glioblastoma, lung cancer, skin cancer, leukemia, lymphoma, colorectal cancer, and bladder cancer. In another aspect, the invention provides a method of treating breast cancer, ovarian cancer, or prostate cancer in a subject, comprising administering a therapeutically effective amount of the compounds of the invention, wherein the compound modulates one or more histone methylation events in the subject.
In another aspect, the invention provides a method of treating breast or ovarian cancer in a subject, comprising administering a therapeutically effective amount of the compounds of the invention, wherein the compound modulates one or more histone methylation events in the subject. The one or more histone methylations events may occur at one or more lysine residues of histone H3 or histone H4, such as, e.g., lysine 4, lysine 9, lysine 27, lysine 36, or lysine 79 of histone H3 or lysine 20 of histone H4. The compounds of the invention may optionally be coadministered with a therapeutically effective amount of an anticancer agent.
In another aspect, a method for inhibiting the growth, proliferation, and/or survival of cancer cells and/or promoting differentiation of cancer stem cells is provided, comprising contacting the cells with an effective amount of the compounds disclosed herein, which may modulate one or more histone methylation events in the cancer cells, such as, e.g. at lysine 4, lysine 9, lysine 27, lysine 36, or lysine 79 of histone H3 or lysine 20 of histone H4. The cancer cells may comprise cells that are characterized by the expression of LSD1 and may comprise pluripotent and/or multipotent cancer cells. The cancer cells may be derived from a cancer selected from the group consisting of embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian epithelial cancer, gliomas, glioblastoma, lung cancer, skin cancer, colorectal cancer, leukemia, lymphoma and bladder cancer. Preferably, the cancer cells may be breast, ovarian or prostate cancer cells.
In another aspect, a method of modulating one or more histone methylation events in a cell is provided, comprising contacting the cell with an effective amount of the compounds of the invention. The one or more histone methylation events may occur at, for example, lysine 4, lysine 9, lysine 27, or lysine 42 of histone H3. The cell may be derived from a cancer characterized by the expression of LSD1. The cell may also be derived from a cancer comprising pluripotent and/or multipotent cancer cells. The cell may also be derived from a cancer selected from the group consisting of embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian epithelial cancer, gliomas, glioblastoma, lung cancer, skin cancer, colorectal cancer, leukemia, lymphoma and bladder cancer. Preferably the cell is a breast, ovarian, or prostate cancer cell.
Another aspect of the present invention provides a method of detecting or diagnosing cancer in a subject, comprising measuring an effective amount of one or more histone demethylases in a sample from the subject; and comparing the amount to a reference value, wherein an increase or decrease in the amount of the one or more histone demethylases relative to the reference value indicates that the subject has cancer. In some embodiments, an increase in one or more histone demethylases relative to the reference value indicates that the subject has cancer. In some such embodiments, an increase in LSDl relative to the reference value indicates that the subject has cancer. In some embodiments, a decrease in one or more histone demethylases relative to the reference value indicates that the subject has cancer.
The sample can be whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsies, lymphatic fluid, ascites fluid, interstitial fluid, bone marrow, cerebrospinal fluid (CSF),seminal fluid, saliva, mucous, sputum, sweat, or urine.
The subject can be one who has been previously diagnosed as having cancer, one who has not been previously diagnosed as having cancer, or one who is asymptomatic for cancer.
The measuring may comprise detecting the presence or absence of the one or more histone demethylases, quantifying the amount of the one or more histone demethylases, and qualifying the type of the one or more histone demethylases.
The reference value can be an index value, a value derived from one or more cancer risk prediction algorithms, a value derived from a subject not suffering from cancer, or a value derived from a subject diagnosed with cancer.
The one or more histone demethylases may comprise LSDl . In one embodiment, the one or more histone demethylases are measured by PCR. In another embodiment, the one or more histone demethylases are measured by immunoassay.
In another aspect, a method for monitoring the progression of cancer in a subject, is provided, comprising (a) measuring an effective amount of one or more histone demethylases in a first sample from the subject at a first period of time; (b) measuring an effective amount of one or more histone demethylases in a second sample from the subject at a second period of time; and (c) comparing the amounts of the one or more histone demethylases detected in step (a) to the amount detected in step (b), or to a reference value, wherein an increase in one or more histone demethylases from the measurement in step (a) to the measurement in step (b) and/or relative to the reference value, indicates increased progression of cancer and, wherein a decrease in one or more histone demethylases from the measurement in step (a) to the measurement in step (b) and/or relative to the reference value, indicates regression of cancer. The monitoring can comprise evaluating changes in the risk of developing cancer in the subject. In one embodiment, the first sample is taken from the subject prior to being treated for cancer. The second sample may be taken from the subject after being treated for cancer. In other embodiments, the monitoring further comprises selecting a treatment regimen for the subject and/or monitoring the effectiveness of a treatment regimen for cancer, wherein the treatment for cancer comprises surgical intervention, administration of anticancer agents, surgical intervention following or preceded by administration of anticancer agents, or taking no further action.
In another aspect, the present invention provides a use of compounds of the invention in the manufacture of a medicament for the treatment of cancer.
In another aspect, the present invention provides a method for inhibiting the growth, proliferation, and/or survival of cancer cells, and/or promoting differentiation of cancer cells, comprising contacting the cancer cells with an effective amount of compounds of the invention.
In another aspect, the present invention provides a method of modulating one or more histone methylation events in a cell, comprising contacting the cell with an effective amount of the compounds of the invention.
In another aspect, the present invention provides a method for selecting a subject for treatment with a compound of the invention comprising: (a) measuring the level of one or more histone demethylases in said subject; and (b) comparing the level of the one or more histone demethylases detected in step (a) to a reference value; wherein when the level of one or more histone demethylases in the subject is greater than the reference value, the subject is selected for treatment with compounds of the invention.
In another aspect of the present invention, a kit is provided, comprising reagents that detect one or more histone demethylases, a sample derived from a subject having normal control levels, and optionally instructions for using the reagents in the methods described herein. The detection reagents may further comprise one or more antibodies or fragments thereof, one or more aptamers, one or more oligonucleotides, or combinations thereof. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In cases of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
The following Detailed Description, given by way of example, but not intended to limit the invention to specific embodiments described, may be understood in conjunction with the accompanying Figures, incorporated herein by reference, in which:
Figures 1 A and IB show the crystal structure of LSDl and its interactions with substratelike peptide inhibitors (Figure 1A) and small molecule inhibitors (Figure IB).
In Figure 1 A, the crystal structure of LSDl was used as a template to design the binding of a substrate-like peptide H3K4M, in which the lysine 4 in histone H3 was replaced with a methionine, which binds to LSDl with high binding affinity (AT; = 0.05 μΜ). Figure 1A discloses the "H3K4" sequence as SEQ ID NO: 13 and the "H3K4M" sequence as SEQ ID NO: 14.
Figure IB shows an illustration of the de novo designed non-peptide chemical scaffold that binds to LSDl with similar mode to that of the H3 4M peptide. The guanidinium groups of the inhibitors form strong hydrogen bonds with the negatively charged residues of LSDl, and the hydrophobic substituents dock into the deep pocket that is close to FAD. Other interactions are also indicated.
Figure 1C shows the synthetic scheme of the histone demethylase inhibitors of the invention, using CBBIOOI as an example.
Figure ID shows the chemical structures of exemplary histone demethylase compounds of the invention. Figure 2A-G show various analyses of exemplary compounds of the invention on LSD1- dependent demethylation in vitro.
Figures 2A and 2B show the results of in vitro LSDl -dependent demethylase assays.
Figure 2 A is a gel showing purified recombinant GST-LSDl protein expressed and isolated from the E. coli BL21 strain. Purified recombinant GST-LSDl protein was used for a demethylation assay.
Figure 2B shows the results of in vitro demethylase assays, demonstrating that purified LSDl protein can demethylate a histone H3 peptide substrate containing dimethylated K4 to mono-methylated and non-methylated forms in a time-dependent manner. Recombinant LSDl was incubated at 30 °C for 1 hour with the di-methylated H3K4 peptide (H3K4Me2) and various concentrations of CBB1002 (Figure 2B) or other exemplary compounds (Figure 2C). The demethylated products, mono-methylated (H3K4Mel) and non-methylated (H3K4meO), were analyzed by mass-spectrometry (MS).
Figure 2C shows the MS peak areas integrated and used to calculate IC50 of CBB 1002, 1003, and 1007 at 0, 1, 2, 5, 10, 20, 50, 100 μΜ, respectively.
Figure 2D shows the IC50 of CBBlOOl-1009 in vitro for LSDl using the mass- spectrometry assay as calculated in B and C.
Figure 2E shows that exemplary compounds of the invention do not inhibit LSD2 and JARID1A. LSDl, LSD2 and JARID1A demethylation reactions were analyzed using the di- methylated H3K4 substrate peptide in the presence of 0, 20, and 50 μΜ CBB1003 and 1007 as in Figure 2B. The inhibitory effects were plotted and compared.
Figures 2F-G show the inhibitory effect of exemplary compounds of the invention on LSDl demethylase using methylated histone as a substrate. Methylated histones and LSDl were assayed as in Figure 2B with 10 μΜ CBB 1001-1009 (Figure 2F); or with various concentrations of CBB1002, 1003, and 1007 (Figure 2G) as indicated. The in vitro inhibitory effects of CBB compounds on the mono-, di-, and tri-methylated histone H3K4, di-methylated H3K9, and histone H3 by LSDl were analyzed by Western-blotting with specific antibodies.
Figures 3A -3C show the results of LSDl demethylase assays and inhibition of activity by synthetic LSDl inhibitor compounds in cultured cancer cells.
Figure 3 A shows the in vivo effects of LSDl inhibitory compounds of the invention on methylation of histone H3. Pluripotent F9 teratocarcinma cells were treated with 10 μΜ CBBlOOl-1009 for 24 hours. Total histones were extracted and the levels of methylated H3K4, H3K9, and histone H3 were monitored by Western blotting with specific antibodies, respectively. Only CBB1003, 1004, and 1006-1008 had significant effects on the mono- and di- methylated H3K4 by inhibiting LSD1. .
Figure 3B shows the dose effects of CBBIOOI, 1002, 1003, and 1007 on histone H3 methylation in F9 cells as assayed in Figure 3A. The effects of LSD1 ablation by specific siRNA on histone methylation in F9 cells (left panels). The F9 cells were treated with 50 nM luciferase (Luc) or LSD1 specific siRNAs for 48 hours and the methylation of H3K4 was analyzed.
Figure 3C shows the effects of LSD 1 inhibitors of the invention on epigenetic suppressed gene expression. F9 cells were treated with 10 μΜ CBBlOOl-1009 for 24. hours. The activation of epigenetically suppressed CHRM4 and SCN3A genes were monitored by quantitative RT- PCR using the beta-actin gene as a control. Only CBB1003, 1004, and 1006-1008 can activate the expression of CHRM4 and SCN3A genes by inhibiting LSD1.
Figure 3D shows the dose-dependent effects of CBB1003 and 1007 on the activation of the expression of CHRM4 and SCN3A genes in F9 and HeLa cervical carcinoma cells. The effects of LSD1 siRNAs in F9 cells were also included.
Figures 4A-C show LSD1 inhibitors of the invention induce the expression of differentiation genes and are selectively permeable to cells.
Figures 4A and 4B show the results of in vivo analysis of LSD1 inhibitors CBB1003- CBB1007 on gene activation. Pluripotent F9 teratocarcinoma cells were cultured with various concentrations of CBB1003 and CBB1007 as indicated for 24 hours. The activation of the expression of SCN3A, CHRM4/M4-ArchR, and differentiation gene FOXA2 was quantified by quantitative real-time RT-PCR.
Figure 4B shows the IC50 of LSD1 inhibitors with regard to the epigenetic activated gene expression of SCN3A. The concentrations of CBB1003 or 1007 in Figure 3B were the same as Fig. 4A, respectively.
Figure 4C shows CBB1003 and CBB1007 are permeable to cells, but not CBB1002. CBB1002, CBB1003 and 1007 were incubated with F9 cells for 2 hours, using dimethyl sulfoxide (DMSO) as a control. After extensive washing of treated cells, the compounds were extracted and their presence (arrows) was analyzed by mass-spectrometry using pure compounds as a control. Figures 5A-5H show the results of cancer cells treated with exemplary LSDl inhibitory compounds of the invention. Figures 5A-5I demonstrate that LSDl compounds selectively inhibit the growth of pluripotent embryonic carcinoma, teratocarcinoma and seminoma cells but not non-pluripotent cells.
Figure 5A and 5B show the inhibition of growth of F9 cells when treated with LSDl inhibitory compounds. In Figure 5A, mouse pluripotent F9 teratocarcinoma cells were treated with DMSO and 50 μΜ LSDl compounds CBBlOOl-3 and 1007 for 30 hours and the cell numbers were counted. CBB1003 and 1007 significandy inhibit the growth of F9 cells but not CBBIOOI and 1002. In Figure 5B, dose-response of F9 cells to various concentrations of CBB 1003 and 1007 are as indicated for 30 hours.
Figures 5C and 5D depict the dose-dependent response of F9 cells to various doses of LSDl compounds, as analyzed by MTT proliferation assays. In Figure 5C, the percentage of compound-treated cells relative to the control is shown (see data in Figure 5A). In Figure 5D, the percentage of CBB 1003- and 1007 -treated cells relative to control cells (DMSO treated), assayed by the MTT proliferation assay using indicated concentrations for 30 hours.
Figure 5E shows the inhibition of bromodeoxyuridine (BrdU) incorporation after F9 is treated by CBB 1003 for 24 hours .
Figure 5F shows the relative BrdU incorporation after treating F9 cells with 50 nM luciferase (Luc) and LSDl siRNAs for 48 hours.
Figures 5G and H show photographs depicting the growth of pluripotent NCCIT mixed embryonic carcinoma/seminoma cancer cells and pluripotent human testicular embryonic carcinoma NTERA-2 cells in the presence of LSDl inhibitor compounds and in non-pluripotent HeLa and 293 cancer cells. Figures 5G and 5H show the growth of pluripotent NCCIT and NTERA-2 cells was inhibited by 50 μΜ CBB1003 or CBB1007 at 30 hours but non-pluripotent HeLa, 293, and ΝΓΗ3Τ3 cells were not inhibited by LSDl inhibitors.
Figure 51 shows the growth of pluripotent mouse embryonic stem (ES) cells were inhibited by exemplary compounds of the invention. The mouse ES cells were treated with either DMSO or 50 μΜ CBB 1003 or CBB 1007 for 30 hours as indicated.
Figure 6A-C show inactivation of LSDl blocks the growth of pluripotent F9 and ES cells but not HeLa cells. HeLa and F9 cells were transfected with 50 nM of luciferase (Luc), LSDl or LSD2 specific siRNAs for 48 hours. The cells were examined for growth inhibition and for LSD1 and LSD2 protein levels by blotting with anti-LSDl or LSD2 antibodies
Figure 6A shows photographs depicting growth inhibition assays of HeLa and F9 cells treated with LSD1 or LSD2 siRNA as indicated. Loss of LSD1 inhibits the growth of F9 cells but not HeLa cells. Loss of LSD2 has an opposite effects towards F and HeLa cells as compared to that of LSD 1 inhibition.
Figure 6B shows the results of Western blotting with anti-LSDl antibodies in HeLa and F cells with or without siRNA treatment as in Figure 6A.
Figure 6C shows Western blots depicting LSD1 inhibition or siRNA-based ablation of LSD1 causes the downregulation of Sox2 and Oct4 protein expression.
Figure 6D shows high protein levels of LSD1 in pluripotent F9, NCCIT, and NTERA-2 cancer cells that also express Oct4 and Sox2 pluripotent stem cell markers. LSD2 expression was very low in F9 and NCCIT cells.
Figures 7A and 7B show photographs showing immunohistological analyses of human testicular normal tissues surrounding seminomas, which were stained with anti-LSDl or Oct4 antibodies. Figures 7A-B indicate elevated LSD1 protein levels in human testicular seminomas that express Oct4. In Figure 7A, LSD1 and Oct4 proteins are low or non-detectable in normal testis tissue. In Figure 7A-7B, immunohistological staining of human testicular normal tissues surrounding seminomas were stained with anti-LSDl (top) or Oct4 (bottom) antibodies. In Figure 7A, three normal human testicular tissues were examined and all displayed low levels of LSD1 and Oct4. One of them is shown (Case D6). In Figure 7B, six human testicular seminomas were stained with anti-LSDl and Oct4 antibodies. All of them displayed elevated protein levels of LSD1 and Oct4. One of the seminomas was shown (Case C4).
Figure 8 shows Western blots showing inhibition of LSD1 demethylase activity by LSD1 inhibitors using methylated histone H3 at K4 (H3K4) isolated in mininucleosomes as a substrate.
Figures 9A and 9B show selective inhibition of pluripotent NCCIT cells by LSD1 inhibitor CBB 1007, but not non-pluripotent HeLa and 293 cells.
Figure 10 show a set of photographs showing the sensitivity of ovarian adenocarcinoma cells IGROV-1 and ovarian teratocarcinoma cells PA-1 to treatment with LSD1 inhibitor compounds CBB 1007 and 1010. DMSO was used as a control. Figure 11 show a set of photographs comparing the sensitivity of F9 teratoma cells and HeLa cells to RBBP5 siRNA treatment. Loss of RBBP5 altered the methylation of H3K4 and selectively inhibited the growth of pluripotent F9 cells but not non-pluripotent HeLa cells.
Figure 12 show a set of photographs depicting inhibition of IGROV-1 and PA-1 cell growth in the presence of RBBP5 siRNA.
Figures 13A-E show LSDl inhibitory compounds selectively inhibit the growth of pluripotent ovarian teratocarcinoma PA-1 cells, but not non-pluripotent ovarian carcinoma Hs38.T cells. Active growing PA-1 and Hs38.T cells were treated with indicated concentration of LSDl inhibitory compounds CBB1003 and CBB1007 for 30 hours.
Figures 14A-F shows LSDl inhibitory compounds selectively inhibit the growth of a subset of ovarian and breast cancer cells that express at least one of pluripotent or multipotent stem cell protein markers, as shown in Figure 18.
Figure 14A shows the growth inhibition of a series of A2780 cells, a human ovarian carcinoma cell, treated at various concentrations of LSDl inhibitors CBB1003 and 1007 for 30 hours.
Figure 14B shows the growth inhibition of a series of T47D cells, a human ductal breast epithelial carcinoma cell, treated at various concentrations of LSDl inhibitors for 60 hours.
Figure 14C shows the inhibition of cell viability assays after the treatment of various doses of CBB1003 and 1007 in A2780 (top graph) and T47D (lower graph) cells from Figures 14A and 14B, respectively.
Figure 14D shows IGROV1, a human ovarian carcinoma cell, was also growth-inhibited by CBB1007 and CBBIOIO as indicated after 30 hours. DMSO was used as a control.
Figure 14E shows S OV-3, a human ovarian carcinoma cell, was also growth-inhibited by LSDl inhibitors CBB1003 and CBB1007 as indicated after 30 hours.
Figure 14F shows MCF-7, a breast adenocarcinoma cell, was also growth-inhibited by
LSDl inhibitors after 30 hours.
Figuresl5A-D show in vivo analysis of CBB1003 and CBB1007 on LSDl demethylation and gene activation in ovarian and breast cancer cells.
Figure 15 A shows the in vivo effect of LSDl compounds on methylation of histone H3 in A2780 cells. A2780 cells were treated with various concentrations of CBB1003 (#3, left) and CBB1007 (#7, right) for 30 hours. Total histones were extracted and the levels of methylated H3K4, H3K9, and histone H3 were monitored by Western blotting with specific antibodies, respectively.
Figure 15B shows the in vivo effect of LSDl compounds on methylation of histone H3 in T47D cells. T47D cells were treated with various concentrations of CBB1003 (#3, left) and CBB1007 (#7, right) for 30 hours. Total histones were extracted and the levels of methylated H3K4, H3K9, and histone H3 were monitored by Western blotting with specific antibodies, respectively.
Figure 15C shows the treatment with CBB1003 and CBB1007 for 30 hours induced the downregulation of DNMTl protein in F9 teratocarcinoma cells (on left) and ablation of LSDl by siRNA for 48 hours also induced the downregulation of DNMTl protein in F9 teratocarcinoma cells (on right). CUL1 serves as a protein loading control.
Figure 15D shows the loss of LSDl in ovarian IGROV1 and A2780 or breast T47D carcinoma cells induced the expression of differentiation genes such as HNF4a and FOXA2. IGROV1, A2780, and T47D cells were transfected with either 50 nM of a control siRNA for luciferase (Luc) or LSDl siRNA and incubated for 60 hours. The expression of HNF4a and FOXA2 mRNAs was quantified by quantitative real-time RT-PCR. The induction of epigenetically suppressed genes, such as SCN3A and CHRM4, were used as basic gene levels, after the loss of LSDl by siRNA.
Figures 16A-B show inactivation of LSDl inhibited the growth of cancer stem cells A2780, S OV3, and T47D but not non-stem Hs38.T ovarian carcinoma cells.
Figure 16A shows A2780, SKOV3 and Hs38.T cells transfected with 50 nM of luciferase (Luc) or LSDl specific siRNAs for 60 hours. The cells were examined for growth inhibition. Only cancer stem cell -like cells A2780 and SKOV3 were growth inhibited after loss of LSDl but not non-stem cell Hs38.T.
Figure 16B shows T47D cells transfected with 50 nM of luciferase (Luc) or LSDl specific siRNAs for 60 hours. The cells were examined for growth inhibition. Cancer stem celllike cells T47D were growth inhibited after loss of LSDl.
Figure 17A-C shows inactivation of RBBP5 or WDR5 that reduced the methylation of histone H3 at lysine 4 (H3K4) selectively inhibited the growth of pluripotent or multipotent F9, PA-1 , SKOV3, A2780, IGROV1 , and T47D cancer stem cells but not non-stem HeLa cancer cells. Figure 17A and B: F9, PA-1 , HeLa, SKOV3, A2780, IGROV1 , and T47D cells were transfected with 50 nM of luciferase (Luc) or LSD1 specific siRNAs. The cells were examined for growth inhibition after 48 hours (F9 and PA-1) or 60 hours (all other cells).
Figure 17C. Loss of RBBP5 or WDR5 reduced the tri- and monomethylation of histone H3 at lysine 4 (H3K4) but not methylation at H3K9. Actin is used as a protein loading control.
Figure 18 shows LSD 1 -sensitive cells express pluripotent or multipotent stem cell proteins/markers. Pluripotent teratocarcinoma/embryonic carcinomas cells F9, NTERA-2, PA-1 cells, ovarian carcinoma cells SKV03, IGROV1 , A2780, Hs38.T, breast carcinoma cells T47D and MCF7, as well as non-stem cervical carcinoma cell HeLa were lysed and equal proteins were analyzed for their expression of pluripotent or multipotent stem cell proteins Oct4, Sox2, Lin28, Nanog, Sall4, Klf4, and LSD1 and CUL1 (control) by immunoblotting with specific antibodies, respectively.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to, inter alia, histone demethylase inhibitor compounds that were rationally designed using the substrates of LSD1 protein as a template. These compounds are highly specific towards histone demethylases such as LSD1 and are fundamentally different from broad spectrum monoamine oxidase inhibitors known in the art, which non-specifically inhibit several members of amine oxidase family proteins as well as histone demethylases. In addition, the methods disclosed herein are based on the finding that inhibition of histone demethylases or modulation of histone methylations at lysine 4 (H3K4) can selectively inhibit or alter the growth of pluripotent and/or multipotent cancer cells such as, e.g., pluripotent teratocarcinoma cells, mixed germ tumor cells, seminoma cells, embryonal carcinoma cells, ovarian teratocarcinoma cells, or ovarian or breast cancer stem cell-like cells which display stem cell or progenitor cell properties. Thus, the present invention further relates to methods of treating cancers, such as those characterized by the presence of such pluripotent and/or multipotent cancer cells and methods of inhibiting growth, proliferation, and/or survival of cancer cells, such as pluripotent and/or multipotent cancer cells. The present invention further embraces the use of histone demethylases as therapeutic, diagnostic, or prognostic biomarkers for detecting, diagnosing, or monitoring the progression of cancer by measuring expression levels of one or more histone demethylases in a sample from a subject, as well as kits for measuring expression levels of one or more histone demethylases in a subject. In addition, the present invention also encompasses methods of inhibiting histone demethylases to modulate the growth, survival, and differentiation of normal stem cell or related iPS (induced pluripotent stem) cells.
Definitions
As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "a nucleic acid" includes one or more nucleic acids, and/or compositions of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
The terms "administration" and or "administering" a compound should be understood to mean providing a compound of the invention to a subject in need of treatment.
As used herein, "aryl" means a monocyclic or fused multicyclic aromatic ring assembly containing six to ten ring carbon atoms. A similar term in this context is a "conjugated" ring assembly. For example, aryl can be phenyl, benzyl, or naphthyl. "Heteroaryl" is as defined for aryl where one or more of the ring members are a heteroatom. A "heteroaryl" ring may include a stable 5-, 6-, or 7-membered monocyclic or 7-, 8-, 9-, 10-, 11- or 12-membered bicyclic aromatic heterocyclic ring which consists of carbon atoms and one or more heteroatoms, e.g., 1 or 1-2 or 1 -3 or 1 -4 or 1 -5 or 1 -6 heteroatoms, or e.g., 1 , 2, 3, 4, 5, or 6 heteroatoms, independently selected from the group consisting of nitrogen, oxygen and sulfur. The nitrogen atom may be substituted or unsubstituted (i.e., N or NR wherein R is H or other substituents, as defined). The nitrogen and sulfur heteroatoms may optionally be oxidized. It is to be noted that total number of S and O atoms in the aromatic heterocycle is not more than 1. For example, heteroaryl includes, without limitation, pyrrole, furan, pyridine, indole, indazole, quinoxaline, quinoline, benzofuran, benzopyran, benzothiopyran, benzo[l,3]dioxole, imidazole, benzo-imidazole, pyrimidine, furan, oxazole, isoxazole, triazole, tetrazole, pyrazole, thiene, thiophene, thiazole, isothiazole, pyrazine, pyridazine, pyrimidine, naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, methylenedioxyphenyl, isoquinoline, naphthrydine, purine, deazapurine, indolizine and the like. In the case of multicyclic aromatic rings, only one of the rings needs to be aromatic (e.g., 2,3-dihydroindole), although all of the rings may be aromatic (e.g., quinoline). The second ring can also be fused or bridged.
The aryl or heteroaryl aromatic ring can be substituted at one or more ring positions with such substituents such as, for example, H, CH3, N02, S02N(CH3)2, S02N((CH3)S02), COOH, COOCH3) CO(N(CH3)), alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyi, heterocyclyl, alkylaryl, heteroaryl, heterocycloalkyl, alkoxy (i.e., methoxy, ethoxy, etc), alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, trifluoromethyl, pentafluoroethyl, halogen (i.e., chloro, fluoro, bromo, iodo), cyano, thio, amido, ether, ester, hydroxyl, hydroxyalkyl, saturated or unsaturated fatty acids, azido, phosphonamido, sulfonamide lactam, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, guanidine, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamide, nitro, cyano, azido, etc. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings, which are not aromatic so as to form a multicyclic system (e.g., tetralin, methylenedioxyphenyl).
A "biomarker" in the context of the present invention is a molecular indicator of a specific biological property; a biochemical feature or facet that can be used to measure the progress of disease or the effects of treatment. "Biomarker" encompasses, without limitation, proteins, nucleic acids, and metabolites, together with their polymorphisms, mutations, variants, modifications, subunits, fragments, protein-ligand complexes, and degradation products, protein- ligand complexes, elements, related metabolites, and other analytes or sample-derived measures. Biomarkers can also include mutated proteins or mutated nucleic acids. A combination of biomarkers, or "profile" can comprise a validated selection of optimal biomarkers. Specifically, "biomarkers" as disclosed herein include one or more histone demethylase genes and proteins.
As used herein, "contacting a cell" refers to a condition in which a compound or other composition of matter is in direct contact with a cell, or is close enough to induce a desired biological effect in a cell. "Cycloalkyl" means a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing the number of ring atoms indicated. For example, C3-10 cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.
"Heterocycloalkyl" means cycloalkyl, as defined in this application, provided that one or more of the ring carbons indicated, are replaced by a moiety that can include, without limitation, -0-, - N=, - R-, -C(O) -, -S-, -S(O) - or -S(0)2-, wherein R can be hydrogen,
Figure imgf000029_0001
or a nitrogen protecting group. For example, heterocycloalkyl as used herein to describe compounds of the invention includes, but is not limited to, morpholine, pyrrolidine, piperazine, piperidine, piperidinylone, etc. The cycloalkyl or heterocycloalkyl ring can be substituted at one or more ring positions with such substituents such as, for example, H, CH3, N02, S02N(CH3)2,
S02N((CH3)S02), COOH, COOCH3, CO(N(CH3)), alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heterocyclyl, alkylaryl, heteroaryl, heterocycloalkyl, alkoxy (i.e., methoxy, ethoxy, etc), alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl,
alkylthiocarbonyl, trifluoromethyl, pentafluoroethyl, halogen (i.e., chloro, fluoro, bromo, iodo), cyano, thio, amido, ether, ester, hydroxyl, hydroxyalkyl, saturated or unsaturated fatty acids, azido, phosphonamido, sulfonamido, lactam, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, guanidine, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamide, nitro, cyano, azido, etc.
"Detect" or "detection" refers to identifying the presence, absence or amount of the object to be detected.
As used herein, the term "diagnosis" or "diagnose" is not limited to a definitive or near definitive determination that an individual has a disease, but also includes determining that an individual has an increased likelihood of having or developing the disease, compared to healthy individuals or to the general population.
As used herein, "expression" and "expression levels" include but are not limited to one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
A "formula," "algorithm," or "model" is any mathematical equation, algorithmic, analytical or programmed process, or statistical technique that takes one or more continuous or categorical inputs (herein called "parameters") and calculates an output value, sometimes referred to as an "index" or "index value." Non-limiting examples of "algorithms" include sums, ratios, and regression operators, such as coefficients or exponents, biomarker value
transformations and normalizations (including, without limitation, those normalization schemes based on clinical parameters, such as gender, age, smoking status, or ethnicity), rules and guidelines, statistical classification models, and neural networks trained on historical populations. Of particular use in combining the biomarkers of the present invention are linear and non-linear equations and statistical classification analyses to "correlate" the relationship between levels of biomarkers detected in a subject sample and the subject's risk of cancer.
A "histone demethylase" is a protein that catalyzes the demethylation of histones.
Examples of histone demethylases known in the art include, but are not limited to, LSD1 (also called "KDM1", "AOF2", "KIAA0601" "FAD-binding protein BRAF35-HDAC complex, 110 kDa subunit" or "BHC110"), LSD2, members of the JARID1 family containing the Jumonji (JmjC) domain, and FBXL10. In eukaryotes, histone demethylases may typically be characterized by a SWIRM domain, a FAD binding motif, and/or an amine oxidase domain or JmjC domain (Klose, R.J. et al., (2007) Nat. Rev. Mol. Cell Biol. 8: 307-18; Shi, Y. (2007) Nat. Rev. Genet. 8: 829-33; Agger, K. et al. (2008) Curr. Opin. Genet. Dev. 18: 159-68). The presence of these domains can be determined using tools available in the art including GenBank, BLAST and the Conserved Domain Search Program available from the National Center for Biotechnology Information (NCBI).
A "histone demethylase inhibitor" includes, for example, the compounds disclosed herein, but also encompasses inhibitory nucleic acids, such as antisense nucleic acids and small interfering RNAs (siRNAs). Without wishing to be bound by theory, histone demethylase inhibitors are believed to modulate one or more histone methylation events on one or more histones present in a cell, including, e.g., histone HI, histone H2A, histone H2B, histone H3, histone H4, and histone H5. "Measuring" or "measurement" means assessing the presence, absence, quantity or amount (which can be an effective amount) of either a given substance within a clinical or subject-derived sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise evaluating the values or categorization of a subject's clinical parameters. Measurement or measuring may also involve qualifying the type or identifying the biomarker(s). Measurement of the biomarkers of the invention may be used to diagnose, detect, or identify cancer in a subject, to monitor the progression or prognosis of cancer in a subject, to predict the recurrence of cancer in a subject, or to classify a subject as having a low risk or a high risk of developing cancer or a recurrence of cancer.
"Modulating" or "modulate" in the context of the present invention means increasing, decreasing, or otherwise altering, adjusting, varying, changing, enhancing or inhibiting a biological event. "Modulating one or more histone methylation events" means that methylation of a histone protein is increased or decreased in response to stimuli, i.e., by administration of a histone demethylase inhibitor or loss of components histone methyltransferase complexes, such as MLL, WDR5 or RBBP5 (Klose, R.J. et al., (2007) Nat. Rev. Mol. Cell Biol. 8: 307-18; Shi, Y. (2007) Nat. Rev. Genet. 8: 829-33; Agger, K. et al. (2008) Curr. Opin. Genet. Dev. 18: 159- 68). Histone methylation may occur at any lysine residue present in a histone protein, such as, but not limited to, lysine 4, lysine 9, lysine 14, lysine 27, lysine 36, and lysine 79 of histone H3. Histone methylation also may occur at any other lysine residue in other histone proteins, such as histone HI, histone H2A, histone H2B, histone H4 (e.g., lysine 20), and histone H5 (comprising subfamily members, such as e.g., H1F, H1H1, H2AF, H2A1 , H2A2, H2BF, H2B1 , H2B2, H3A1 , H3A2, H3A3, H41 , H44). Specific family members of the foregoing histone subfamilies are known in the art and include, without limitation, H1F0, H1FNT, H1FOO, HIFX,
HISTIHIA, HISTIHIB, HISTIHIC, HISTIHID, HISTIHIE, HISTIHIT, H2AFB 1, H2AFB2, H2AFB 3, H2AFJ, H2AFV, H2AFX, H2AFY, H2AFY2, H2AFZ, HIST1 H2AA, HIST1H2AB, HIST1H2AC, HIST1H2AD, HIST1H2AE, HIST1H2AG, HIST1H2AI, HIST1H2AJ,
HIST1H2AK, HIST1 H2AL, HIST1 H2AM, HIST2H2AA3, HIST2H2AC, H2BFM, H2BFO, H2BFS, H2BFWT, HIST1H2BA, HIST1H2BB, HIST1H2BC, HIST1 H2BD, HIST1 H2BE, HIST1 H2BF, HIST1H2BG, HIST1 H2BH, HIST1H2BI, HIST1H2BJ, HIST1H2BK,
HIST1H2BL, HIST1 H2BM, HIST1H2BN, HIST1 H2BO, HIST2H2BE, HIST1H3A,
HIST1H3B, HIST1H3C, HIST1H3D, HIST1HE3, HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, HIST1HEJ, HIST2H3C, HIST3H3, HIST1H4A, HIST1H4B, HIST1H4C,
HIST1H4D, HIST1H4E, HIST1H4F, HIST1H4G, HIST1H4H, HIST1H4I, HIST1H4J, HIST1H4K, HIST1H4L, and HIST4H4.
"Risk" in the context of the present invention relates to the probability that an event will occur over a specific time period, as in the development or growth or metastasis of cancer, and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no-conversion. Alternative continuous measures which may be assessed in the context of the present invention include time to development of cancer, or progression to a different stage of cancer, including progression or development of cancer and therapeutic cancer conversion risk reduction ratios.
"Risk evaluation," or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a "normal" condition to an at-risk condition for developing cancer, or from an at-risk condition to cancer, or development of recurrent cancer. Risk evaluation can also comprise prediction of other indices of cancer, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to cancer, thus diagnosing and defining the risk spectrum of a category of subjects defined as at risk for developing cancer. In the categorical scenario, the invention can be used to discriminate between normal and at-risk subject cohorts. In other embodiments, the present invention may be used so as to discriminate at-risk conditions from cancerous conditions, or cancerous conditions from normal. Such differing use may require different biomarker combinations in individual panel or profile, mathematical algorithm, and/or cut-off points, but be subject to the same aforementioned measurements of accuracy for the intended use.
A "sample" in the context of the present invention is a biological sample isolated from a subject and can include, by way of example and not limitation, whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsies, lymphatic fluid, ascites fluid, interstitial fluid (also known as "extracellular fluid" and encompasses the fluid found in spaces between cells, including, inter alia, gingival crevicular fluid), bone marrow, seminal fluid, cerebrospinal fluid (CSF), saliva, mucous, sputum, sweat, urine, or any other secretion, excretion, or other bodily fluids.
By "statistically significant", it is meant that the alteration is greater than what might be expected to happen by chance alone (which could be a "false positive"). Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which presents the probability of obtaining a result at least as extreme as a given data point, assuming the data point was the result of chance alone. A result is often considered highly significant at a p-value of 0.05 or less.
As used herein, the term "stem cell" refers to an undifferentiated cell that can be induced to proliferate or differentiate. The stem cell is capable of self-maintenance or self-renewal, meaning that with each cell division, one daughter cell will also be a stem cell. Stem cells can be obtained from embryonic, post-natal, juvenile, or adult tissue. Stem cells can be pluripotent or multipotent. As used herein, the term "pluripotent" refers to cells with the potential to make any differentiated cell in the body, but cannot contribute to making the components of the extraembryonic membranes which are derived from the trophoblast. Three types of pluripotent stem cells have been confirmed to date: embryonic stem (ES) cells, embryonic germ (EG) cells, and embryonic carcinoma (EC) cells. Typically a cell is "pluripotent" if it expresses the marker genes Oct4, Sox2, and/or Lin28. The term "multipotent" is used to describe cells that can only differentiate into a limited number of types. For example, the bone marrow contains multipotent stem cells that give rise to all the cells of the blood but may not be able to differentiate into other cells types. Pluripotent and multipotent cells encompass any cancer stem cell/cancer stem celllike cell or cancer/tumor initiating cells. A "cancer stem cell" in the context of the present invention relates to cells that are considered as the origin of various heterogeneous cancer populations due to their pluripotent or multipotent stem cell properties. Such cells can be of hematogenic in origin or may be present in solid tumors. The term "progenitor cell" refers to an undifferentiated cell derived from a stem cell, and is not itself a stem cell. Some progenitor cells can produce progeny that are capable of differentiating into more than one cell type.
A "subject" in the context of the present invention is preferably a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of cancer, such as nude mice. A subject can be male or female. A subject can be one who has been previously diagnosed or identified as having cancer, and optionally has already undergone, or is undergoing, a therapeutic intervention or treatment for the cancer. Alternatively, a subject can also be one who has not been previously diagnosed as having cancer. For example, a subject can be one who exhibits one or more risk factors for cancer, or a subject who does not exhibit risk factors for cancer, or a subject who is
asymptomatic for cancer. A subject can also be one who is suffering from or at risk of developing cancer, or who is suffering from or at risk of developing a recurrence of cancer. A subject can also be one who is suffering from or at risk of developing metastatic cancer. A subject can also be one who has been previously treated for cancer, whether by administration of anticancer agents, radiation therapy, surgery, or any combination of the foregoing.
The term "substituted", as used herein, means that any one or more hydrogen atoms on the designated atom is replaced with a selection from the indicated groups, provided that the designated atom's normal valency is not exceeded, and that the substitution results in a stable compound. When a substituent is keto (i.e., =0), then 2 hydrogen atoms on the atom are replaced. Keto substituents are not present on aromatic moieties. Ring double bonds, as used herein, are double bonds that are formed between two adjacent ring atoms (e.g., C=C, C=N or N=N). Non-limiting examples of such groups include, without limitation, H, CH3, NO2, S02N(CH3)2, S02N((CH3)S02), COOH, COOCH3, CO(N(CH3)), alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heterocyclyl, alkylaryl, heteroaryl, heterocycloalkyl, alkoxy (i.e., methoxy, ethoxy, etc), alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbortyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, trifluoromethyl, pentafluoroethyl, halogen (i.e., chloro, fluoro, bromo, iodo), cyano, thio, amido, ether, ester, hydroxyl, hydroxyalkyl, saturated or unsaturated fatty acids, azido, phosphonamido, sulfonamide-, lactam, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, guanidino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonate sulfamoyl, sulfonamido, nitro, cyano, azido, etc.
The compounds, compositions, biomarkers, kits, and methods of the present invention can be used in the diagnosis, prognosis, monitoring, and treatment of cancer in vivo as well as for the inhibition of growth and/or proliferation of cancer cells in vitro, preferably those
characterized by the presence of pluripotent and or multipotent cancer cells, such as, e.g., embryonic carcinomas, teratomas, seminomas, germ cell tumors, choriocarcinomas, yolk sac tumors, ovarian epithelial cancers, but can also be used to detect, diagnose, monitor, or treat a wide variety of other cancers, including but not limited to solid tumors (e.g., tumors of the head and neck, lung, breast, colon, colorectal, prostate (which can be androgen dependent), bladder, rectum, brain, gastric tissue, bone, ovary, thyroid, or endometrium), hematological malignancies (e.g., leukemias, lymphomas, myelomas), carcinomas (e.g. bladder carcinoma, renal carcinoma, breast carcinoma, colorectal carcinoma) (Saigusa, S. et al., (2010) Ann. Surg. Oncol. 16(12): 3488-3498, or neuroblastoma. Non-limiting examples of these cancers include diffuse large B- cell lymphoma (DLBCL), T-cell lymphomas or leukemias, e.g., cutaneous T-cell lymphoma (CTCL), noncutaneous peripheral T-cell lymphoma, lymphoma associated with human T-cell lymphotrophic virus (HTLV), adult T-cell leukemia/lymphoma (ATLL), as well as acute lymphocytic leukemia, acute nonlymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, myeloma, multiple myeloma, mesothelioma, childhood solid tumors, glioblastoma (Field, M. et al., (2010) Clin. Neurosurg. 57: 151-159), meduUoblastoma, neuroblastoma, retinoblastoma, glioma (Du, Z. et al., (2009) Glia 57(7): 724-33), Wilms' tumor, bone cancer and soft-tissue sarcomas, common solid tumors of adults such as head and neck cancers (e.g., oral, laryngeal and esophageal), genitourinary cancers (e.g., prostate, including androgen-dependent and independent prostate, bladder, renal, uterine, ovarian, testicular, rectal and colon)
(Sotomayor, P. et al., (2009) Prostate 69(4): 401-410; Mowla, S.J. et al., (2010) Methods of Cancer Diagnosis, Therapy and Prognosis 6(4): 211-226), lung cancer (e.g., small cell carcinoma and non-small cell lung carcinoma, including squamous cell carcinoma and adenocarcinoma) (Chen, Y.C. et al., (2008) PLoS ONE 3(7): e2637; Karoubi, G. et al., (2009) Interact.
Cardiovasc. Thorac. Surg. 8: 383-397), breast cancer (Ezeh, U.I. et al., (2005) Cancer 104(10): 2255-65), pancreatic cancer, skin cancers (Katona, T.M. et al., (2007) Appl. Immunohistochem. Mol. Morphol. 15: 359-262), stomach cancer, brain cancer, liver cancer, adrenal cancer, kidney cancer, thyroid cancer, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, metastatic skin carcinoma, medullary carcinoma, osteosarcoma, Ewing's sarcoma, veticulum cell sarcoma, and Kaposi's sarcoma. Also included are pediatric forms of any of the cancers described herein.
Therapeutic agents for treating or reducing the risk of cancer include, without limitation of the following, radiation therapy with or without "anticancer agents", such as, but not limited to, an alkylating agent, an antibiotic agent, an antimetabolic agent, a hormonal agent, a plant- derived agent, an anti-angiogenic agent, a differentiation inducing agent, a cell growth arrest inducing agent, an apoptosis inducing agent, a cytotoxic agent, a biologic agent, a gene therapy agent, a retinoid agent, an histone deacetylase inhibitor, an enzyme inhibitor, a cytokine, a chemokine, an antibody, a DNA molecule, an RNA molecule, a small molecule, a peptide, or a peptidomimetic, or any combination thereof.
The term "treating" in its various grammatical forms in relation to the present invention refers to preventing (e.g.,- chemoprevention), curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease state, disease progression, disease causative agent (e.g., bacteria or viruses) or other abnormal condition. For example, treatment may involve alleviating a symptom (i.e., not necessary all symptoms) of a disease or attenuating the progression of a disease.
Treatment of cancers, as used herein, refers to partially or totally inhibiting, delaying or preventing the progression of cancer including cancer metastasis; inhibiting the growth, proliferation and/or survival of cancer cells, inhibiting, delaying or preventing the recurrence of cancer including cancer metastasis; or preventing the onset or development of cancer (e.g., chemoprevention) in a subject.
As used herein, the term "therapeutically effective amount" is intended to qualify a desired biological response, such as, e.g., is partial or total inhibition, delay or prevention of the progression of cancer including cancer metastasis; inhibition, delay or prevention of the recurrence of cancer including cancer metastasis; or the prevention of the onset or development of cancer (e.g., chemoprevention) in a subject.
In some embodiments, the present invention provides a compound of formula V:
Figure imgf000037_0001
or pharmaceutically acceptable salt thereof;
wherein Ri comprises a functional group selected from the group consisting of a carboxamide (aminocarbonyl), a carboxamidine (carboximidamide), acyl, an alkylsulfonyl, an arylsulfonyl, and an aminosulfonyl, any of which may be optionally substituted;
R.2 comprises a functional group selected from the group consisting of hydrogen, alkyl, nitro, sulfonamide, sulfonimide, amide, and carboxyalkyl, any of which is optionally substituted; and
R3 comprises one selected from the group consisting of aryl, heteroaryl, cycloalkyl, and heterocycloalkyl group, any of which is optionally substituted.
In some such embodiments, in the compound of formula V Ri is the carboxamidine: - (CN=H)-NH2. In some such embodiments, a terminal guanidine or carboxamidine can be substituted, replacing any NH or NH2 hydrogen with an alkyl, hydroxyl, alkoxy, or sulfur containing functional group, such as sulfhydryl, sulfone, sulfonyl, and the like.
In some such embodiments, in the compound of formula V R2 is a functional group selected from the group consisting of hydrogen, methyl, nitro, N,N-dimethylsulfonamide, MeS02NMeS02-, N-methylcarboxamide, and carboxymethyl.
In some such embodiments, in the compound of formula V R3 is selected from the group consisting of:
Figure imgf000037_0002
NH , > and H In some such embodiments, a terminal guanidine or carboxamidine can be substituted, replacing any NH or NH2 hydrogen with an alkyl, hydroxyl, alkoxy, or sulfur containing functional group, such as sulfhydryl, sulfone, sulfonyl, and the like.
In some embodiments, the compound of formula V is selected from on of compounds 1- 19 below:
Figure imgf000038_0001
Figure imgf000039_0001

Figure imgf000040_0001
In some embodiments, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the compound of formula V or any of the disclosed genus, subgenus, or the species disclosed herein, in conjunction with a pharmaceutically acceptable carrier or diluent. Pharmaceutical compositions of the invention can further include an anticancer agent as described herein.
The compounds of the present invention may exist in one or more particular geometric, optical, enantiomeric, diastereomeric, epimeric, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; a- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and half chair-forms; and
combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms").
The compounds of the invention when used in pharmaceutical or diagnostic applications may be prepared in a racemic mixture or an essentially pure enantiomer form, with an enantiopurity of at least 90% enantiomeric excess (EE), preferably at least 95% EE, more preferably at least 98% EE, and most preferably at least 99% EE. Enantiomeric excess values provide a quantitative measure of the excess of the percentage amount of a major isomer over the percentage amount of a minor isomer which is present therewith, and may be readily determined by suitable methods well-known and established in the art, as for example chiral high pressure liquid chromatography (HPLC), chiral gas chromatography (GC), nuclear magnetic resonance (NMR) using chiral shift reagents, etc.
A "pharmaceutical composition" is a formulation containing the compounds of the present invention in a form suitable for administration to a subject. As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, carriers, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
In general, compounds and pharmaceutical compositions of the invention may be administered in therapeutically effective amounts via any of the usual and acceptable modes known in the art, either singly or in combination with one or more therapeutic agents. A therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors involved, as readily determinable within the skill of the art. Suitable therapeutic doses of the compounds of the invention may be in the range of 1 microgram (μ%) to 1000 milligrams (mg) per kilogram body weight of the recipient per day, and any increment in between, such as, e.g., 1, 2, 3, 5, 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μg (1 mg); 2, 3, 5, 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mg. A desired dose may preferably be presented as two, three, four, five, six, or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example, containing from 1 μg to 1000 mg of active ingredient per unit dosage form. Alternatively, if the condition of the recipient so requires, the doses may be administered as a continuous infusion. The mode of administration and dosage forms will of course affect the therapeutic amounts of the compounds which are desirable and efficacious for the given treatment application.
For example, orally administered dosages typically are at least twice, e.g., 2-10 times, the dosage levels used in parenteral administration methods, for the same active ingredient. In oral administration, dosage levels for delta receptor binding compounds of the invention may be on the order of 5-200 mg 70 kg body weight/day. In tablet dosage forms, typical active agent dose levels are on the order of 10-100 mg per tablet.
The compounds of the present invention may be administered per se as well as in the form of pharmaceutically acceptable esters, salts, and ethers, as well as other physiologically functional derivatives of such compounds. Compounds of the invention may be amorphous or polymorphic. The term "crystal polymorphs", ""polymorphs" or "crystal forms" means crystal structures in which a compound (or a salt or solvate thereof) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystal forms usually have different X-ray diffraction patterns, infrared spectral, melting points, density hardness, crystal shape, optical and electrical properties, stability and solubility. Examples of crystal lattice forms include, but are not limited to, cubic, isometric, tetragonal, orthorhombic, hexagonal, trigonal, triclinic, and monoclinic. Recrystallization solvent, rate of crystallization, storage temperature, and other factors may cause one crystal form to dominate. Crystal polymorphs of the compounds can be prepared by crystallization under different conditions.
Additionally, the compounds of the present invention, for example, the salts of the compounds, can exist in either hydrated or unhydrated (the anhydrous) form or as solvates with other solvent molecules. "Solvate" means solvent addition forms that contain either stoichiometric or non stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water the solvate formed is a hydrate; and if the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one or more molecules of water with one molecule of the substance in which the water retains its molecular state as ¾0. Non-limiting examples of hydrates include monohydrates, dihydrates, etc. Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc.
Examples of pharmaceutically acceptable acid addition salts include those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; as well as organic acids such as acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, 3-(4-hydroxybenzoyl)benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,
benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4- toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy- 2-ene-l -carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, p-toluenesulfonic acid, and salicylic acid and the like. Examples of a pharmaceutically acceptable base addition salts include those formed when an acidic proton present in the parent compound is replaced by a metal ion, such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferable salts are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins. Examples of organic bases include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diemylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, tromethamine, N-methylglucamine, polyamine resins, and the like.
Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimemylamine, dicyclohexylamine, choline, and caffeine.
Compounds of the invention can be administered as pharmaceutical compositions by any conventional route, in particular enterally, e.g., orally, e.g., in the form of tablets or capsules, or parenterally, e.g., in the form of injectable solutions or suspensions, topically, e.g., in the form of lotions, gels, ointments or creams, or in a nasal or suppository form or in inhaled forms.
Pharmaceutical compositions comprising a compound of the present invention in free form or in a pharmaceutically acceptable salt form in association with at least one pharmaceutically acceptable carrier or diluent can be manufactured in a conventional manner by mixing, granulating or coating methods.
For example, oral compositions can be tablets or gelatin capsules comprising the active ingredient together with a pharmaceutically acceptable carrier, including any one or a combination of the following components: a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners. Injectable compositions can be aqueous isotonic solutions or suspensions, and suppositories can be prepared from fatty emulsions or suspensions. The compositions can be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they can also contain other therapeutically valuable substances.
Suitable formulations for transdermal applications include an effective amount of a compound of the present invention with a carrier. A carrier can include absorbable
pharmacologically acceptable solvents to assist passage through the skin of the host. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin. Matrix transdermal formulations can also be used. Suitable formulations for topical application, e.g., to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels well-known in the art. Such can contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.
The active compounds can be prepared with pharmaceutically acceptable carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No.
4,522,81 1.
Techniques for formulation and administration of the disclosed compounds of the invention can be found in Remington: the Science and Practice of Pharmacy, 19th edition, Mack Publishing Co., Easton, Pa. (1995).
Compounds of the invention can be administered in therapeutically effective amounts in combination with one or more anticancer agents as defined herein. For example, synergistic effects can occur with other substances used in the treatment of cancers. Where the compounds of the invention are administered in conjunction with other therapies, dosages of the coadministered compounds will of course vary depending on the type of co-drug employed, on the specific drug employed, on the condition being treated and so forth.
As used herein, the terms "combination treatment", "combination therapy", "combined treatment" or "combinatorial treatment", used interchangeably, refer to a treatment of an individual with at least two different therapeutic agents. The terms "co-administration" or "combined administration" or the like as utilized herein are meant to encompass administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of
administration or at the same time. The term "pharmaceutical combination" means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. A "fixed combination" means that the active ingredients, e.g. a compound as disclosed herein and an anticancer agent, are both administered to a patient simultaneously in the form of a single entity or dosage. A "non-fixed combination" means that the active ingredients, e.g. a compound as disclosed herein and anticancer agent, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the 2 compounds in the body of the patient. The latter also applies to cocktail therapy, e.g. the administration of 3 or more active ingredients.
Anticancer agents for treating or reducing the risk of cancer include, without limitation of the following, radiation therapy, compounds such as, but not limited to, an alkylating agent, an antibiotic agent, an antimetabolic agent, a hormonal agent, a plant-derived agent, an anti- angiogenic agent, a differentiation inducing agent, a cell growth arrest inducing agent, an apoptosis inducing agent, a cytotoxic agent, a biologic agent, a gene therapy agent, a retinoid agent, an histone deacetylase inhibitor, a tyrosine kinase inhibitor, an enzyme inhibitor, a cytokine, a chemokine, an antibody, a DNA molecule, an RNA molecule, a small molecule, a peptide, or a peptidomimetic, or any combination thereof. The "anti-cancer agents" of the invention encompass those described herein, as well as inhibitory nucleic acids, including any pharmaceutically acceptable salts or hydrates of such agents, or any free acids, free bases, or other free forms of such agents. Examples of alkylating agents include, but are not limited to, bischloroethylamines (nitrogen mustards, e.g., Chlorambucil, Cyclophosphamide, Ifosfamide, MechJorethamine, Melphalan, uracil mustard), aziridines (e.g., Thiotepa), alkyl alkone sulfonates (e.g., Busulfan), nitrosoureas (e.g., Carmustine, Lomustine, Streptozocin), non-classic alkylating agents
(Altretamine, Dacarbazine, and Procarbazine), platinum compounds (Carboplatin, oxaliplatin, and Cisplatin).
Examples of antibiotic agents include, without limitation, anthracyclines (e.g.,
Doxorubicin, Daunorubicin, Epirubicin, Idarubicin, and Anthracenedione), Mitomycin C, Bleomycin, Dactinomycin, and Plicatomycin.
Examples of antimetabolic agents include, but are not limited to, Fluorouracil (5-FU),
Floxuridine (5-FUdR), Methotrexate, Leucovorin, Hydroxyurea, Azacytidine, Flavopiridol, Thioguanine (6-TG), Mercaptopurine (6-MP), Cytarabine, Pentostatin, Fludarabine Phosphate, Cladribine (2-CDA), Asparaginase, Gemcitabine, and Pemetrexed.
Examples of hormonal agents include, but are not limited to, synthetic estrogens (e.g., Diethylstibestrol), antiestrogens (e.g., Tamoxifen, Toremifene, Fluoxymesterol, and Raloxifene), antiandrogens (e.g., Bicalutamide, Nilutamide, and Flutamide), aromatase inhibitors (e.g., Aminoglutethimide, Anastrozole, and Tetrazole), luteinizing hormone release hormone (LHRH) analogues, Ketoconazole, Goserelin Acetate, Leuprolide, Megestrol Acetate, Prednisone, and Mifepristone.
Examples of plant-derived agents include, but are not limited to, vinca alkaloids (e.g.,
Vincristine, Vinblastine, Vindesine, Vinzolidine, and Vinorelbine), podophyllotoxins (e.g., Etoposide (VP- 16) and Tenyposide (VM-26)), and taxanes (e.g., Paclitaxel and Docetaxel).
Examples of biologic agents include, but are not limited to, immunomodulating proteins such as cytokines (such as interleukin-2 (IL-2, Aldesleukin), Epoietin- a; EPO), granulocyte- CSF (G-CSF; Filgrastin), and granulocyte, macrophage-CSF (GM-CSF; Sargramostim and interferons, e.g., interferon-a, interferon-β (fibroblast interferon) and interferon-γ (lymphocyte interferon)), bacillus Calmette-Guerin, levamisole, and octreotide, monoclonal antibodies against tumor antigens (such as Herceptin (trastuzumab), Rituxan (rituximab), Myelotarg (gemtuzumab ozogamicin) and Campath (alemtuzumab), endostatin, tumor suppressor genes (e.g., DPC4, NF- 1, NF-2, RB, p53, WTl, BRCA1, and BRCA2), and cancer vaccines (e.g., tumor associated antigens such as gangliosides (GM2), prostate specific antigen (PSA), a-fetoprotein (AFP), carcinoembryonic antigen (CEA) (produced by colon cancers and other adenocarcinomas, e.g., breast, lung, gastric, and pancreatic cancers), melanoma-associated antigens (MART-1, gaplOO, MAGE 1,3 tyrosinase), papillomavirus E6 and E7 fragments, whole cells or portions/lysates of autologous tumor cells and allogeneic tumor cells.
Examples of retinoid agents include all natural, recombinant, and synthetic derivatives or mimetics of vitamin A, for example, retinyl palmitate, retinoyl-beta-glucuronide (vitamin Al beta-glucuronide), retinyl phosphate (vitamin Al phosphate), retinyl esters, 4-oxoretinol, 4- oxoretinaldehyde, 3-dehydroretinol (vitamin A2), 11-cis -retinal (11-cis-retinaldehyde, 11-cis or neo b vitamin Al aldehyde), 5,6-epoxyretinol (5,6-epoxy vitamin Al alcohol), anhydroretinol (anhydro vitamin Al) and 4-ketoretinol (4-keto- vitamin Al alcohol), all-trans retinoic acid (ATRA; Tretinoin; vitamin A acid; 3,7-dimethyl-9-(2,6,6,-trimethyl-l-cyclohenen-l-yl)-2,4,6,8- nonatetraenoic acid [CAS No. 302-79-4]), lipid formulations of all-trans retinoic acid (e.g., ATRA-IV), 9-cis retinoic acid (9-cis-RA; Alitretinoin; Panretin©; LGD1057), (e)-4-[2-(5,6,7,8- tetrahydro-2-naphthalenyl)-l-propenyl]-benzoic acid, 3-methyl-(E)-4-[2-(5,6,7,8-tetrahydro-2- naphthalenyl)-l-propenyl]-benzoic acid, Fenretinide (N-(4-hydroxyphenyl)retinamide; 4-HPR), Etretinate (2,4,6,8-nonatetraenoic acid), Acitretin (Ro 10-1670), Tazarotene (ethyl 6-[2-(4,4- dimethylthiochroman-6-yl)-ethynyl] nicotinate), Tocoretinate (9-cis-tretinoin tocoferil), Adapalene (6-[3-(l-adamantyl)-4-methoxyphenyl]-2-naphthoic acid), Motretinide
(trimethylmethoxyphenyl-N-ethyl retinamide), retinaldehyde, CD437 (also called 6-[3-(l- adamantyl)-4-hydroxphenyl]-2-naphthalene carboxylic acid and AHPN), CD2325, ST1926 ([E- 3-(4'-hydroxy-3'-adamantylbiphenyl-4-yl)acrylic acid), ST1878 (methyl 2-[3-[2-[3-(2-methoxy- l,l-dimethyl-2-oxoethoxy)pheno-xy]ethoxy]phenoxy]isobutyrate), ST2307, ST1898, ST2306, ST2474, MM11453, MM002 (3-Cl-AHPC), MX2870-1, MX3350-1, MX84, and MX90-1, docosahexanoic acid (DHA), phytanic acid, methoprene acid, LG100268 (LG268), LG100324, LGD1057, SRI 1203, SRI 1217, SRI 1234, SRI 1236, SRI 1246, AGN194204, derivatives of 9- cis-RA such as 3-methyl TTNEB and related agents, e.g., Targretin®; Bexarotene; LGD1069; and 4-[l-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl) ethenyl] benzoic acid.
Examples of histone deacetylase inhibitors include, without limitation, MS-275, depsipeptide, Cl-994, Apicidin, A-161906, Scriptaid, PXD-101, CHAP, LAQ-824, Butyric acid, depudecin, oxamflatin, trichostatin A, trichostatin C, suberoylanilide hydroxamic acid (SAHA), m-Carboxycinnamic acid bishydroxamide (CBHA), Pyroxamide; Salicylbishydroxamic acid; Suberoyl bishydroxamic acid (SBHA); Azelaic bishydroxamic acid (ABHA); Azelaic-1- hydroxamate-9-anilide (AAHA); 6-(3-Chlorophenylureido) carpoic hydroxamic acid (3C1- UCHA); MW2796; MW2996, trapoxin A, sodium butyrate, isovalerate, valerate, 4- phenylbutyrate, phenylbutyrate, butyramide, isobutyramide, phenylacetate, 3-bromopropionate, tributyrin, valproic acid, valproate, and Pivanex.
Examples of tyrosine kinase inhibitors include, e.g., DMPQ (5,7-dimethoxy-3-(4- pyridinyl)quinoline dihydrochloride), Aminogenistein (4'-amino-6-hydroxyflavone), Erbstatin analog (2,5-dihydroxymethylcinnamate, methyl 2,5-dihydroxycinnamate), Imatinib (Gleevec™' Glivec™; STI-571 ; 4-[(4-methyl-l-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2- yrimidinyl] amino] -phenyl ]benzamide methanesulfonate), LFM-A13 (2-Cyano-N-(2,5- dibromophenyl)-3-hydroxy-2-butenamide), PD153035 (ZM 252868; 4-[(3-bromophenyl)amino]- 6,7-dimethoxyquinazoline hydrochloride), Piceatannol (trans-3,3',4,5'-tetrahydroxystilbene, 4- [(1 E)-2-(3,5-dihydroxyphenyl)ethenyl]-l ,2-benzenediol), PP1 (4-amino-5-(4-methylphenyl)-7- (t-butyl)pyrazolo[3,4-d]pyrimidine), PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo
[3,4,d]pyrimidine), Pertuzumab (Omnitarg™; rhuMAb2C4), SU4312 (3-[[4-(dimethylamino) phenyl]methylene]-l ,3-dihydro-2H-indol-2-one), SU6656 (2,3-dihydro-N,N-dimethyl-2-oxo-3- [(4,5,6,7-tetrahydro-lH-indol-2-yl)methylene]-lH-indole-5-sulfonamide), Bevacizumab (Avastin®; rhuMAb VEGF), Semaxanib (SU5416), SU6668, and ZD6126. included are inhibitors of EGFR, e.g., Cetuximab (Erbitux; IMC-C225; MoAb C225) and Gefitinib
(IRESSA™; ZD1839; ZD1839; 4-(3-chloro-4-fluoroanilino)-7-methoxy-6-(3-morpholino propoxy)quinazoline), ZD6474 (AZD6474), , and EMD-72000 (Matuzumab), Panitumab (ABX- EGF; MoAb ABX-EGF), ICR-62 (MoAb ICR-62), CI-1033 (PD183805; N-[-4-[(3-Chloro-4- fluorophenyl)ammo]-7-[3-(4-mo holmyl)prorx)xy]-6-quinazolinyl]-2-propenamide), Lapatinib (GW572016), AEE788 (pyrrolo-pyrimidine), EKB-569, and EXEL 7647/EXEL 09999
(EXELIS). Also included are Erlotinib and derivatives, e.g., Tarceva®; NSC 718781, CP- 358774, OSI-774, R1415; N-(3-emynylphenyl)-6,7-bis(2-memoxyethoxy)-4-qwnazolinamine.
Inhibitory Nucleic Acids
The invention encompasses the use of inhibitory nucleic acids. Inhibitory nucleic acids may be designed based on the sequences of one or more histone demethylases as defined herein, either in whole or in part (i.e., sequences of conserved domains), or may be designed based on identification of biomarkers that indicate cancer status and progression of disease in a subject. In certain preferred examples, the invention features histone demethylase inhibitory nucleic acid molecules. Histone demethylase inhibitory nucleic acid molecules are essentially oligomers or oligonucleotides that may be employed as single-stranded or double-stranded nucleic acid molecule to decrease or ablate histone demethylase expression.
In one approach, the histone demethylase inhibitory nucleic acid molecule is a double- stranded RNA used for RNA interference (RNAi)-mediated knock-down of histone demethylase gene expression. In one embodiment, a double-stranded RNA (dsRNA) molecule is made that includes between eight and twenty-five (e.g., 8, 10, 12, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25) consecutive nucleotides. The dsRNA can be two complementary strands of RNA that have duplexed, or a single RNA strand that has self -duplexed (small hairpin (sh)RNA). Typically, dsRNAs are about 21 or 22 base pairs, but may be shorter or longer (up to about 29 nucleotides) if desired. Double stranded RNA can be made using standard techniques (e.g., chemical synthesis or in vitro transcription). Kits are available, for example, from Ambion (Austin, Tex.) and Epicentre (Madison, Wis.). Methods for expressing dsRNA in mammalian cells are described in Brummelkamp et al., (2002) Science 296: 550-553; Paddison et al., (2002) Genes & Devel. 16:948-958. Paul et al., (2002) Nature Biotechnol. 20: 505-508; Sui et al., (2002) Proc. Nad. Acad. Sci. USA 99: 5515-5520; Yu et al., (2002) Proc. Natl. Acad. Sci. USA 99: 6047- 6052; Miyagishi et al., (2002) Nature Biotechnol. 20: 497-500; and Lee et al., (2002) Nature Biotechnol. 20: 500-505, each of which is hereby incorporated by reference.
An inhibitory nucleic acid molecule that "corresponds" to one or more histone demethylase genes comprises at least a fragment of the double-stranded gene, such that each strand of the double-stranded inhibitory nucleic acid molecule is capable of binding to the complementary strand of a target histone demethylase gene. The inhibitory nucleic acid molecule need not have perfect correspondence to the reference histone demethylase sequence. In one embodiment, a siRNA has at least about 85%, 90%, 95%, 96%, 97%, 98%, or even 99% sequence identity with the target nucleic acid. For example, a 19 base pair duplex having a 1 - 2 base pair mismatch is considered useful in the methods of the invention. In other embodiments, the nucleotide sequence of the inhibitory nucleic acid molecule exhibits 1, 2, 3, 4, 5 or more mismatches. The inhibitory nucleic acid molecules provided by the invention are not limited to siRNAs, but include any nucleic acid molecule sufficient to decrease the expression of a histone demethylase nucleic acid molecule or polypeptide. Each of the DNA sequences provided herein may be used, for example, in the discovery and development of therapeutic antisense nucleic acid molecule to decrease the expression of one or more histone demethylases.
The invention further provides catalytic RNA molecules or ribozymes. Such catalytic
RNA molecules can be used to inhibit expression of a histone demethylase nucleic acid molecule in vivo. The inclusion of ribozyme sequences within an antisense RNA confers RNA-cleaving activity upon the molecule, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al., (1998) Nature 334: 585-591 and U:S. Patent Application Publication No. 20030003469, each of which is incorporated by reference. In various embodiments of this invention, the catalytic nucleic acid molecule is formed in a hammerhead or hairpin motif. Examples of such hammerhead motifs are described by Rossi et al., AIDS Research and Human Retroviruses, 8:183, 1992. Example of hairpin motifs are described by Hampel et al., "RNA Catalyst for Cleaving Specific RNA Sequences," filed Sep. 20, 1989, which is a continuation-in-part of U.S. Patent Application Ser. No.
07/247,100 filed Sep. 20, 1988, Hampel and Tritz, (1989) Biochemistry 28: 4929, and Hampel et al., (1990) Nucl. Acids Res. 18: 299. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule. After a subject is diagnosed as having cancer, or at risk for recurrence of cancer, a method of treatment is selected.
The inhibitory nucleic acid molecules of the invention may be administered systemically in dosages between about 1 and 100 mg/kg (e.g., 1, 5, 10, 20, 25, 50, 75, and 100 mg/kg). The dosage may range from between about 25 and 500 mg/m2/day. A human subject having cancer can receive a dosage between about 50 and 300 mg/m2/day (e.g., 50, 75, 100, 125, 150, 175, 200, 250, 275, and 300). The amounts of the inhibitory nucleic acid molecules administered to the subject will depend, of course, on whether it is administered alone or in combination with another anticancer agent, such as the histone demethylase inhibitor compounds disclosed herein. One type of inhibitory nucleic acid molecule is based on 2' -modified oligonucleotides containing oligodeoxynucleotide gaps with some or all internucleotide linkages modified to phosphorothioates for nuclease resistance. The presence of methylphosphonate modifications increases the affinity of the oligonucleotide for its target RNA and thus reduces the IC50. This modification also increases the nuclease resistance of the modified oligonucleotide. It is understood that the methods and reagents of the present invention may be used in conjunction with any technologies known to those skilled in the art that may be developed to enhance the stability or efficacy of an inhibitory nucleic acid molecule.
Inhibitory nucleic acid molecules include oligomers containing modified backbones or non-natural internucleoside linkages. Oligomers having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are also considered to be oligomers.
Oligomers that have modified oligonucleotide backbones include, for example,
phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl- phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. Various salts, mixed salts and free acid forms are also included. Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301 ; 5,023,243; 5,177, 196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131 ; 5,399,676; 5,405,939; 5,453,496; 5,455,233;
5,466,677; 5,476,925; 5,519,126; 5,536,821 ; 5,541,306; 5,550,111; 5,563,253; 5,571,799;
5,587,361; and 5,625,050, each of which is herein incorporated by reference
Oligomers having modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Representative United States patents that teach the preparation of the above oligonucleotides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;
5,489,677; 5,541 ,307; 5,561 ,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;
5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.
Oligomers may also contain one or more substituted sugar moieties. Such modifications include 2'-0-methyl and 2'-methoxyethoxy modifications. Another desirable modification is 2'- dimethylaminooxyethoxy, 2'-aminopropoxy and 2'-fluoro. Similar modifications may also be made at other positions on an oligonucleotide or oligomer, particularly the 3' position of the sugar on the 3' terminal nucleotide. Oligomers may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;
5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;
5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is herein incorporated by reference in its entirety. In other oligomers, both the sugar and the intemucleoside linkage, i.e., the backbone, are replaced with novel groups. The nucleotide units are maintained for hybridization with a histone demethylase nucleic acid molecule. Methods for making and using these oligomers are described, for example, in "Peptide Nucleic Acids (PNA): Protocols and Applications" Ed. P. E. Nielsen, Horizon Press, Norfolk, United Kingdom, 1999. Representative United States patents that teach the preparation of PNAs include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, each of which are herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.
In some embodiments, the present invention provides a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the compound of formula V or any of the disclosed genus, subgenus, or species disclosed herein. In some such embodiments, the cancer comprises pluripotent and/or multipotent cancer cells expressing LSDl. In some such embodiments, the cancer comprises cells expressing at least one pluripotent or multipotent stem cell protein marker (Lapidot, T et al., (1994) Nature 367: 645-8; Singh, S.K. et al., (2004) Nature 432: 396-401 ; Bapat, S.A. et al., (2005) Cancer Res. 65: 3025- 9; Maidand, N.J. et al., (2005) BJU Int. 96: 1219-23; Zhong, X. et al. (2010) J. Biol. Chem. 285: 41961-71 ; Yang, X. et al. (2010) Cancer Res.70: 9463-72; Peng, S. et al. (2008) Oncogene 29: 2153-9; Viswanathan, S. R. et al. (2009) Nat. Genet. 41: 843-848; Marquardt, J. U. et al. (2010) J. Hepatology 53: 568-77; Kitamura, H. et al. (2009) Lung Cancer 66: 275-81 ; Levy, C. et al. (2006) TRENDS in Mol. Medicine 12: 406-14; Tysnes, B. B. (2010) Neoplasia 12: 506-15; Holmberg, J. et al. (2011) PLoS One 6: el8454; Mishra, L. et al. (2009) Hepatology 49: 318-29; Takahashi, K. et al., (2007) Cell 131: 861-72; Mikkelsen, T.S. et al., (2008) Nature 454: 49-55; Yu, J. et al. (2007) Science 318: 1917-20; Ho, R. et al. (2011 ) J. Cell Physiol. 226: 868-78; Mallanna, S. and Rizzino, A. (2010) Dev. Biol. 344: 16-25; Patel, M. and Yang, S. (2010) Stem Cell Review 6: 367-80).
In some embodiments, the at least one pluripotent stem cell protein marker is selected from the group consisting of Oct4, Sox2, Lin28, Nanog, Klf4, Sall4, Lin28B, cMyc, nMyc,
LMyc, Wnt3a, miR-291-3p, miR-294, miR-295, miR-290/371 cluster, miR-302 cluster, miR-363 cluster, miR-520 cluster, miR-92b, miR148/152, miR-124, miR-615, miR-708, niiR-9, Klfl, Klf2, lf5, Esrrb, Esrrg, Soxl, Sox3, Soxl5, Soxl8, Smad7, Nr5al, Nr5a2, Wnt/beta-catenin, Met, Notch, Hedgehog, Sonic Hedgehog, CD133, CD44, CD44+/CD247ESA+, CD34+/CD38", CD90, CD347CD387CD19+, CD34/CD38+/CD123+, CD117, CD20, integrinalpha2betahigh, EpCam, miR-137, miR-301, miR-32, miR-22, miR-135b, miR-204, miR205, miR-lOa, miR196b, miR-448, miR-7-1, miR-128a, miR-196a-l , miR-361, Rexl, CRIPTO/TDGF, Cx43, IGF1, SSEA3, SSEA4, TRA-1-60, TRA-1-81, ZFP42, FOXD3, TERT, Musashi-1, BMI-1, nestin, Ink4a/ARF, Pten", ALDH^, ABCG2, CXCR4, and MTTF.
In some embodiments, the cancer cell can express any two of these pluripotent stem cell protein markers, such as Oct4 and Sox2, Oct4 and Lin28, or Sox2 and Lin28. In some embodiments, the cancer cell may express any three of these pluripotent stem cell protein markers, such as Oct4, Sox, 2, and Lin28. In some such embodiments, the cancer cells can express the three cancer stem cell protein markers Wnt/beta-catenin, Notch, and Hedgehog, or three cancer stem cell markers can be CD133+, CD44+, and CD24". In some embodiments, the cancer cell can express and four, five, six, seven, eight, nine, ten, up to all of the pluripotent or multipotent stem cell protein markers described herein. In some embodiments, the presence of any known or discovered pluripotent or multipotent stem cell protein can be used as a marker indicating treatment with compounds of the present invention.
In some embodiments, the cancer to be treated can be at least one selected from the group consisting of: embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian cancer, endometrial cancer, cervical cancer, retinoblastoma, kidney cancer, liver cancer, gastric cancer, brain cancer, medulloblastoma, medulloepithelioma, glioma, glioblastoma, lung cancer, bronchial cancer, mesothelioma, skin cancer, colon and rectal cancer, bladder cancer, pancreatic cancer, lip and oral cancer, laryngeal and pharyngeal cancer, melanoma, pituitary cancer, penile cancer, parathyroid cancer, thyroid cancer, pheochromocytoma and paraganglioma, thymoma and thymic Carcinoma, leukemia, lymphoma, plasma cell neoplasms, myeloproliferative disorders, islet cell tumor, small intestine cancer, transitional cell cancer, pleuropulmonary blastoma, gestational trophoblastic cancer, esophageal cancer, central nervous system cancer, head and neck cancer, endocrine cancer, cardiovascular cancer, rhabdomyosarcoma, soft tissue carcinomas, carcinomas of bone, cartilage, fat, vascular, neural, and hematopoietic tissues and ADDS-related cancers. In some such embodiments, the cancer comprises breast cancer. In some such embodiments, the cancer comprises ovarian cancer. Any cancer treatment regimen, in accordance with methods of the invention, can include administering a therapeutically effective amount of an anticancer agent in conjunction with compounds of the invention.
In some embodiments, the present invention provides for the use of a compound of formula V or any of the disclosed genus, subgenus, or species disclosed herein, in the manufacture of a medicament for the treatment of cancer. In some such embodiments the cancer comprises pluripotent and/or multipotent cancer cells expressing LSD1. In some such embodiments, the cancer comprises cells expressing at least one pluripotent or multipotent stem cell protein marker. In some embodiments, the at least one pluripotent stem cell protein marker is selected from the group consisting of Oct4, Sox2, Lin28, Nanog, Klf4, and Sall4, Lin28B, cMyc, nMyc, LMyc, Wnt3a, miR-291-3p, miR-294, miR-295, miR-290/371 cluster, miR-302 cluster, miR-363 cluster, miR-520 cluster, miR-92b, miR148/152, miR-124, miR-615, miR-708, miR-9, Klfl, Klf2, Klf5, Esrrb, Esrrg, Soxl, Sox3, Soxl5, Soxl8, Smad7, Nr5al, Nr5a2,
Wnt/beta-catenin, Met, Notch, Hedgehog, Sonic Hedgehog, CD133, CD44, CD44+/CD247ESA+, CD34VCD38", CD90, CD34+/CD387CD19\ CD34/CD38+/CD123+, CD117, CD20, integrinalpJ^beta11'811, EpCam, miR-137, miR-301, miR-32, miR-22, miR-135b, miR-204, miR205, miR-lOa, miR196b, miR-448, nuR-7-1, miR-128a, miR-196a-l, miR-361, Rexl, CRIPTO/TDGF, Cx43, IGF1, SSEA3, SSEA4, TRA-1-60, TRA-1-81, ZFP42, FOXD3, TERT, Musashi-1 , BMI-1 , nestin, Ink4a/ARF, Pten", ALDHhigh, ABCG2, CXCR4, and MITF.
In some such embodiments, the cancer comprises at least one cancer selected from the group consisting of: embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian cancer, endometrial cancer, cervical cancer, retinoblastoma, kidney cancer, liver cancer, gastric cancer, brain cancer, medulloblastoma, medulloepithelioma, glioma, glioblastoma, lung cancer, bronchial cancer, mesothelioma, skin cancer, colon and rectal cancer, bladder cancer, pancreatic cancer, lip and oral cancer, laryngeal and pharyngeal cancer, melanoma, pituitary cancer, penile cancer, parathyroid cancer, thyroid cancer, pheochromocytoma and paraganglioma, thymoma and thymic Carcinoma, leukemia, lymphoma, plasma cell neoplasms, myeloproliferative disorders, islet cell tumor, small intestine cancer, transitional cell cancer, pleuropulmonary blastoma, gestational trophoblastic cancer, esophageal cancer, central nervous system cancer, head and neck cancer, endocrine cancer, cardiovascular cancer, rhabdomyosarcoma, soft tissue carcinomas, carcinomas of bone, cartilage, fat, vascular, neural, and hematopoietic tissues and AEDS-related cancers. In some such embodiments, the cancer comprises breast cancer. In some such embodiments, the cancer comprises ovarian cancer.
In some embodiments, the medicament further comprises a therapeutically effective amount of an anticancer agent as described herein.
In some embodiments, the present invention provides a method for inhibiting the growth, proliferation, and/or survival of cancer cells, comprising contacting the cancer cells with an effective amount of the compound of formula V or any of the disclosed genus, subgenus, or species disclosed herein.
In some embodiments, methods for inhibiting the growth, proliferation, survival of cancer cells, and/or promoting the differentiation of cancer cells can further include contacting the cancer cells with an anticancer agent. In some embodiments, in methods for inhibiting the growth, proliferation, survival of cancer cells, and/or promoting the differentiation of cancer cells the cancer cells comprise pluripotent and/or multipotent cancer cells expressing LSD1.
In some embodiments, in methods for inhibiting the growth, proliferation, survival of cancer cells, and/or promoting the differentiation of cancer cells the cancer cells comprise cells expressing at least one pluripotent or multipotent stem cell protein marker.
In some embodiments, in methods for inhibiting the growth, proliferation, survival of cancer cells, and/or promoting the differentiation of cancer cells the at least one pluripotent stem cell protein marker is selected from the group consisting of Oct4, Sox2, Lin28, Nanog, Klf4, and Sall4, Lin28B, cMyc, nMyc, LMyc, Wnt3a, miR-291-3p, miR-294, miR-295, miR-290/371 cluster, miR-302 cluster, miR-363 cluster, miR-520 cluster, miR-92b, miR148/152, miR-124, miR-615, miR-708, nuR-9, Klfl, KIf2, Klf5, Esrrb, Esrrg, Soxl, Sox3, Soxl5, Soxl8, Smad7, Nr5al, Nr5a2, Wnt beta-catenin, Met, Notch, Hedgehog, Sonic Hedgehog, CD133, CD44, CD44+/CD247ESA+, CD347CD38", CD90, CD34+/CD387CD19+, CD34/CD38+/CD123+, CD117, CD20, integrinalpha2betahigh, EpCam, miR-137, miR-301, miR-32, miR-22, miR-135b, miR-204, miR205, miR-lOa, miR196b, miR-448, nuR-7-1 , miR-128a, miR-196a-l, miR-361, Rexl, CRIPTO/TDGF, Cx43, IGF1, SSEA3, SSEA4, TRA-1-60, TRA-1-81, ZFP42, FOXD3, TERT, Musashi-1, BMI-1, nestin, Ink4a ARF, Pten", ALDHhigh, ABCG2, CXCR4, and MITF.
In some embodiments, in methods for inhibiting the growth, proliferation, survival of cancer cells, and/or promoting the differentiation of cancer cells the cancer cells comprise at least one cancer selected from the group consisting of: embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian cancer, endometrial cancer, cervical cancer, retinoblastoma, kidney cancer, liver cancer, gastric cancer, brain cancer, medulloblastoma, medulloepithelioma, glioma, glioblastoma, lung cancer, bronchial cancer, mesothelioma, skin cancer, colon and rectal cancer, bladder cancer, pancreatic cancer, lip and oral cancer, laryngeal and pharyngeal cancer, melanoma, pituitary cancer, penile cancer, parathyroid cancer, thyroid cancer,
pheochromocytoma and paraganglioma, thymoma and thymic Carcinoma, leukemia, lymphoma, plasma cell neoplasms, myeloproliferative disorders, islet cell tumor, small intestine cancer, transitional cell cancer, pleuropulmonary blastoma, gestational trophoblastic cancer, esophageal cancer, central nervous system cancer, head and neck cancer, endocrine cancer, cardiovascular cancer, rhabdomyosarcoma, soft tissue carcinomas, carcinomas of bone, cartilage, fat, vascular, neural, and hematopoietic tissues and AIDS-related cancers. In some such embodiments, the at least one cancer comprises breast cancer. In some such embodiments, the at least one cancer comprises ovarian cancer.
In some embodiments, methods for inhibiting the growth, proliferation, survival of cancer cells, and/or promoting the differentiation of cancer cells can be conducted in vivo. In some embodiments, methods for inhibiting the growth, proliferation, survival of cancer cells, and/or promoting the differentiation of cancer cells conducted in vitro.
In some embodiments, the present invention provides a method of modulating one or more histone methylation events in a cell, comprising contacting the cell with an effective amount of the compound of formula V or any of the disclosed genus, subgenus, or species disclosed herein.
In some embodiments, in methods of modulating one or more histone methylation events in a cell, the one or more histone methylation events occur at lysine 4, lysine 9, lysine 27, lysine 36, lysine 79 of histone H3 or lysine 20 of histone H4.
In some embodiments, in methods of modulating one or more histone methylation events in a cell, the cell is derived from a cancer expressing LSD1.
In some embodiments, in methods of modulating one or more histone methylation events in a cell, the cell is derived from a cancer comprising pluripotent and/or multipotent cancer cells.
In some embodiments, in methods of modulating one or more histone methylation events in a cell, the cell is derived from a cancer expressing at least one pluripotent stem cell protein marker.
In some embodiments, in methods of modulating one or more histone methylation events in a cell, the at least one pluripotent stem cell protein marker is selected from the group consisting of Oct4, Sox2, Lin28, Nanog, Klf4, and Sall4, Lin28B, cMyc, nMyc, LMyc, Wnt3a, miR-291-3p, miR-294, miR-295, miR-290/371 cluster, miR-302 cluster, nuR-363 cluster, miR- 520 cluster, miR-92b, miR148/152, miR-124, miR-615, miR-708, miR-9, Klfl, Klf2, Klf5, Esrrb, Esrrg, So l, Sox3, Sox 15, Sox 18, Smad7, Nr5al, Nr5a2, Wn^eta-catenin, Met, Notch, Hedgehog, Sonic Hedgehog, CD133, CD44, CD44+/CD247ESA\ CD347CD38", CD90, CD34+/CD387CD19\ CD34/CD38+/CD123+, CD117, CD20, integrinalpha2betahigh, EpCam, miR-137, miR-301, nuR-32, miR-22, miR-135b, miR-204, miR205, miR-lOa, miR196b, miR- 448, miR-7-1, miR-128a, miR-196a-l, miR-361, Rexl , CRTPTO/TDGF, Cx43, IGF1 , SSEA3, SSEA4, TRA-1-60, TRA-1 -81 , ZFP42, FOXD3, TERT, Musashi-1 , BMI-1 , nestin, Ink4a/ARF, Pten , ALDH1"811, ABCG2, CXCR4, and MITF.
In some embodiments, in methods of modulating one or more histone methylation events in a cell, the cell is derived from a cancer selected from the group consisting of: embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian cancer, endometrial cancer, cervical cancer, retinoblastoma, kidney cancer, liver cancer, gastric cancer, brain cancer, medulloblastoma, medulloepithelioma, glioma, glioblastoma, lung cancer, bronchial cancer, mesothelioma, skin cancer, colon and rectal cancer, bladder cancer, pancreatic cancer, lip and oral cancer, laryngeal and pharyngeal cancer, melanoma, pituitary cancer, penile cancer, parathyroid cancer, thyroid cancer, pheochromocytoma and paraganglioma, thymoma and thymic Carcinoma, leukemia, lymphoma, plasma cell neoplasms, myeloproliferative disorders, islet cell tumor, small intestine cancer, transitional cell cancer, pleuropulmonary blastoma, gestational trophoblastic cancer, esophageal cancer, central nervous system cancer, head and neck cancer, endocrine cancer, cardiovascular cancer, rhabdomyosarcoma, soft tissue carcinomas, carcinomas of bone, cartilage, fat, vascular, neural, and hematopoietic tissues and AIDS-related cancers. In some such embodiments, the cancer comprises breast cancer. In some such embodiments, the cancer comprises ovarian cancer.
In some embodiments, the present invention provides a method for monitoring the progression of cancer in a subject, comprising:
(a) measuring an effective amount of one or more histone demethylases in a first sample from the subject at a first period of time;
(b) measuring an effective amount of one or more histone demethylases in a second sample from the subject at a second period of time; and
(c) comparing the amounts of the one or more histone demethylases detected in step (a) to the amount detected in step (b), or to a reference value, wherein an increase in one or more histone demethylases from the measurement in step (a) to the measurement in step (b) and/or relative to the reference value, indicates increased progression of cancer and, wherein a decrease in one or more histone demethylases from the measurement in step (a) to the measurement in step (b) and/or relative to the reference value, indicates regression of cancer. In some embodiments, in methods for monitoring the progression of cancer in a subject, the monitoring comprises evaluating changes in the risk of developing cancer in the subject.
In some embodiments, in methods for monitoring the progression of cancer in a subject, the subject comprises one who has previously been treated for cancer, one who has not been previously treated for cancer, or one who has not been previously diagnosed with cancer.
In some embodiments, in methods for monitoring the progression of cancer in a subject, the sample is whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsies, lymphatic fluid, ascites fluid, interstitial fluid, bone marrow, cerebrospinal fluid (CSF), seminal fluid, saliva, mucous, sputum, sweat, or urine.
In some embodiments, in methods for monitoring the progression of cancer in a subject, the first sample is taken from the subject prior to being treated for cancer.
In addition a second sample can be taken from the subject after being treated for cancer, allowing for comparison with the first sample, with an increase or decrease in one or more histone demethylases being an indication of progression or regression of cancer, respectively. For example, an increase in LSD1 indicates progression of cancer and the need for an alternate treatment. Likewise, a decrease in LSD1 indicates regression of cancer and that the treatment regimen is effective.
In some embodiments, in methods for monitoring the progression of cancer in a subject, the method further comprises selecting a treatment regimen for the subject and/or monitoring the effectiveness of a treatment regimen for cancer.
In some embodiments, in methods for monitoring the progression of cancer in a subject, the treatment for cancer comprises surgical intervention, administration of anticancer agents, surgical intervention following or preceded by administration of anticancer agents, or taking no further action.
In some embodiments, in methods for monitoring the progression of cancer in a subject, the reference value comprises an index value, a value derived from one or more cancer risk prediction algorithms, a value derived from a subject not having cancer, or a value derived from a subject diagnosed with cancer.
In some embodiments, in methods for monitoring the progression of cancer in a subject, the measuring comprises detecting the presence or absence of the one or more histone demethylases, quantifying the amount of the one or more histone demethylases, and qualifying the type of the one or more histone demethylases.
In some embodiments, in methods for monitoring the progression of cancer in a subject, the one or more histone demethylases comprises LSD1.
In some embodiments, in methods for monitoring the progression of cancer in a subject, the one or more histone demethylases are measured by PCR.
In some embodiments, in methods for monitoring the progression of cancer in a subject, the one or more histone demethylases are measured by immunoassay.
In some embodiments, in methods for monitoring the progression of cancer in a subject, the cancer is characterized by the presence of pluripotent and/or multipotent cells.
In some embodiments, in methods for monitoring the progression of cancer in a subject, the cancer is selected from the group consisting of: embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian cancer, endometrial cancer, cervical cancer, retinoblastoma, kidney cancer, liver cancer, gastric cancer, brain cancer, medulloblastoma, medulloepithelioma, glioma, glioblastoma, lung cancer, bronchial cancer, mesothelioma, skin cancer, colon and rectal cancer, bladder cancer, pancreatic cancer, lip and oral cancer, laryngeal and pharyngeal cancer, melanoma, pituitary cancer, penile cancer, parathyroid cancer, thyroid cancer,
pheochromocytoma and paraganglioma, thymoma and thymic Carcinoma, leukemia, lymphoma, plasma cell neoplasms, myeloproliferative disorders, islet cell tumor, small intestine cancer, transitional cell cancer, pleuropulmonary blastoma, gestational trophoblastic cancer, esophageal cancer, central nervous system cancer, head and neck cancer, endocrine cancer, cardiovascular cancer, rhabdomyosarcoma, soft tissue carcinomas, carcinomas of bone, cartilage, fat, vascular, neural, and hematopoietic tissues and AIDS-related cancers. In some such embodiments, the cancer is breast cancer. In some such embodiments, the cancer is ovarian cancer.
In some embodiments, the present invention provides a method for selecting a subject for treatment with a compound of formula V, or any genus, subgenus, the species disclosed herein, the method comprising:(a) measuring the level of one or more histone demethylases in said subject; and(b) comparing the level of said one or more histone demethylases detected in step (a) to a reference value; wherein when the level of one or more histone demethylases in the subject is greater than the reference value, the subject is selected for treatment with the compounds of the invention.
In some embodiments, in methods for selecting a subject with cancer the one or more histone demethylases comprises LSD1.
In some embodiments, in methods for selecting a subject with cancer the method further includes measuring the level of at least one pluripotent stem cell protein marker and comparing the measured level of at least one pluripotent stem cell protein marker to a reference value for each marker, wherein when the level of at least one pluriponent stem cell protein marker is greater than its reference value, treatment with the compound of formula V or any of the disclosed genus, subgenus, or species disclosed herein is indicated.
In some embodiments, in methods for selecting a subject with cancer the at least one pluripotent stem cell protein marker is selected from the group consisting of Oct4, Sox2, Lin28, Nanog, Klf4, and Sall4, Lin28B, cMyc, nMyc, LMyc, Wnt3a, miR-291-3p, miR-294, miR-295, miR-290/371 cluster, miR-302 cluster, miR-363 cluster, miR-520 cluster, miR-92b, miR 148/152, miR-124, miR-615, miR-708, nuR-9, Klfl, Klf2, lf5, Esrrb, Esrrg, Soxl, Sox3, Soxl5, Soxl8, Smad7, Nr5al, Nr5a2, Wnt beta-catenin, Met, Notch, Hedgehog, Sonic Hedgehog, CD133, CD44, CD44+/CD247ESA+, CD34+/CD38\ CD90, CD34+/CD387CD19+,
CD34/CD38+/CD123+, CD117, CD20, integrinalpha2betahigh, EpCam, miR-137, miR-301, miR- 32, miR-22, miR-135b, miR-204, miR205, miR-lOa, miR196b, miR-448, miR-7-1, miR-128a, miR-196a-l, miR-361, Rexl, CRIPTO/TDGF, Cx43, IGFl, SSEA3, SSEA4, TRA-1-60, TRA-1- 81, ZFP42, FOXD3, TERT, Musashi-1, BMI-1, nestin, Ink4a/ARF, Pten", ALDHMgl1, ABCG2, CXCR4, and MITF.
The present invention provides methods of treating cancer by administering a therapeutically effective amount of the histone demethylase inhibitor compounds disclosed herein. "Treating" cancer can result in a reduction in size of a tumor. A reduction in size of a tumor may also be referred to as "tumor regression". Preferably, after treatment, tumor size is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor size is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75% or greater. Size of a tumor may be measured by any reproducible means of measurement. The size of a tumor may be measured as a diameter of the tumor.
Treating cancer can result in a reduction in tumor volume. Preferably, after treatment, tumor volume is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor volume is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75% or greater. Tumor volume may be measured by any reproducible means of measurement.
Treating cancer may result in a decrease in number of tumors. Preferably, after treatment, tumor number is reduced by 5% or greater relative to number prior to treatment; more preferably, tumor number is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%. Number of tumors may be measured by any reproducible means of measurement. The number of tumors may be measured by counting tumors visible to the naked eye or at a specified magnification. Preferably, the specified magnification is 2X, 3X, 4X, 5X, 10X, or 50X.
Treating cancer can result in a decrease in number of metastatic lesions in other tissues or organs distant from the primary tumor site. Preferably, after treatment, the number of metastatic lesions is reduced by 5% or greater relative to number prior to treatment; more preferably, the number of metastatic lesions is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%. The number of metastatic lesions may be measured by any reproducible means of measurement. The number of metastatic lesions may be measured by counting metastatic lesions visible to the naked eye or at a specified magnification. Preferably, the specified magnification is 2X, 3X, 4X, 5X, 10X, or 50X.
Treating cancer can result in an increase in average survival time of a population of treated subjects in comparison to a population receiving carrier alone. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.
Treating cancer can result in an increase in average survival time of a population of treated subjects in comparison to a population of untreated subjects. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.
Treating cancer can result in increase in average survival time of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present invention, or a pharmaceutically acceptable salt thereof. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.
Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving carrier alone. Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present invention, or a pharmaceutically acceptable salt thereof. Preferably, the mortality rate is decreased by more than 2%; more preferably, by more than 5%; more preferably, by more than 10%; and most preferably, by more than 25%. A decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means. A decrease in the mortality rate of a population may be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with an active compound. A decrease in the mortality rate of a population may also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with an active compound.
Treating cancer can result in a decrease in tumor growth rate. Preferably, after treatment, tumor growth rate is reduced by at least 5% relative to number prior to treatment; more preferably, tumor growth rate is reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. Tumor growth rate may be measured by any reproducible means of measurement. Tumor growth rate can be measured according to a change in tumor diameter per unit time.
Treating cancer can result in a decrease in tumor regrowth. Preferably, after treatment, tumor regrowth is less than 5%; more preferably, tumor regrowth is less than 10%; more preferably, less than 20%; more preferably, less than 30%; more preferably, less than 40%; more preferably, less than 50%; even more preferably, less than 50%; and most preferably, less than 75%. Tumor regrowth may be measured by any reproducible means of measurement. Tumor regrowth is measured, for example, by measuring an increase in the diameter of a tumor after a prior tumor shrinkage that followed treatment. A decrease in tumor regrowth is indicated by failure of tumors to reoccur after treatment has stopped.
Treating cancer can result in a reduction in the rate of cancer cell proliferation.
Preferably, after treatment, the rate of cellular proliferation is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The rate of cellular proliferation may be measured by any reproducible means of measurement. The rate of cellular proliferation is measured, for example, by measuring the number of dividing cells in a tissue sample per unit time.
Treating cancer can result in a reduction in the proportion of proliferating cells.
Preferably, after treatment, the proportion of proliferating cells is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The proportion of proliferating cells may be measured by any reproducible means of measurement. Preferably, the proportion of proliferating cells is measured, for example, by quantifying the number of dividing cells relative to the number of nondividing cells in a tissue sample. T he proportion of proliferating cells can be equivalent to the mitotic index.
Treating cancer can result in a decrease in the number or proportion of cells having an abnormal appearance or morphology. Preferably, after treatment, the number of cells having an abnormal morphology is reduced by at least 5% relative to its size prior to treatment; more preferably, reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. An abnormal cellular appearance or morphology may be measured by any reproducible means of measurement. An abnormal cellular morphology can be measured by microscopy, e.g., using an inverted tissue culture microscope. An abnormal cellular morphology can take the form of nuclear pleiomorphism.
Also provided herein are methods for inhibiting the growth, proliferation, and/or survival of cancer cells, comprising contacting the cells with an effective amount of a histone demethylase inhibitor as defined herein. Cell growth, proliferation, and/or survival can be measured by any method known to those skilled in the art, such as BrdU incorporation, 3H- thymidine incorporation, 5-ethynyl-2'-deoxyuridine incorporation, LIVE/DEAD
viability/cytotoxicity kits (Invitrogen), staining with nuclear dyes such as Hoechst, DAPI, MTT, propidium iodide, calcein AM, ethidium homodimer-1 , and trypan blue, cell counting (manual or with the use of kits such as CYQUANT and Click-iT (Invitrogen), measurement of caspase activity by the use of kits such as PhiPhiLux (Oncolmmunin), Caspase 3 Activity Assay and Homogeneous Caspases Assay (Roche Applied Science), Caspase-Glo (Promega), Apo-ONE Homogeneous Caspase 3/7 assay (Promega), CaspACE colorimetric and fluorometric Assay Systems (Promega), EnzChek Caspase-3 Assay Kit (Invitrogen), Image-iT LIVE Green Caspase- 3 and -7 Detection Kit (Invitrogen), Active Caspase-3 Detection Kit (Stratagene), TUNEL and DNA fragmentation assays and kits like Apoptotic DNA Ladder Kit (Roche Applied Science), Cellular DNA Fragmentation ELISA (Roche Applied Science), In Situ Cell Death Detection Kit (Roche Applied Science), DeadEnd TUNEL Systems (Promega), TUNEL Apoptosis Detection Kit (Upstate), Annexin V assays, anti-poly (ADP Ribose) Polymerase (PARP) cleavage, and measurement of mitochondrial membrane potentials, etc. Methods of measuring and/or assessing cell growth, proliferation, and survival can also encompass measurement of the reaction between a histone and histone demethylase protein can be accomplished by any means known in the art. These include, without limitation Western blotting, measuring formation of formaldehyde, mass spectrometry, and measuring formation of peroxide.
One method comprises monitoring the interaction between a histone demethylase protein and CoREST, a protein that is believed to interact with histone demethylases such as LSD1. This method may comprise contacting a histone demethylase protein and a CoREST protein in the presence of one or more histone demethylase inhibitors; and determining the level of interaction between the histone demethylase and CoREST, wherein a different level of interaction between the histone demethylase and CoREST in the presence of the histone demethylase inhibitor relative to the absence of the inhibitor indicates that the inhibitor modulates the interaction between the histone demethylase protein and CoREST, thereby causing the inhibition of growth, proliferation, and/or survival of cancer cells. The method may further comprise at least one other component of a histone demethylase transcription complex, such as, for example, HDACl, HDAC2, BHC80 and HDC80 (Gocke, C. B. and Yu, H. (2008) PLoS One 22: e3255; Roizman, B. (2011) J. Virol. 85: 7474-82; Shi, Y. J. et al. (2005) Mol. Cell 19: 857- 64; Lee, M. G. et al. (2005) Nature 437: 432-5). The method may also comprise determining the effect of the inhibitor on a biological activity of the histone demethylase. For example, a method may further comprise contacting a histone demethylase and CoREST with a histone demethylase inhibitor and determining the biological activity of the histone demethylase, wherein a different activity of the histone demethylase in the presence of the inhibitor relative to the absence of the inhibitor indicates that the inhibitor modulates the biological activity of a histone demethylase, thereby causing the inhibition of growth, proliferation, and/or survival of cancer cells. The methods disclosed herein may comprise at least a portion of a protein of interest, e.g., a histone demethylase protein, CoREST protein, or other component of a histone demethylase transcription complex, fused direcdy or indirectiy to another moiety or label, e.g., a fluorophore or radioactive label or another peptide that may be useful in identifying, quantitating, isolating or purifying the reagent.
Other methods for measuring inhibition of cell growth, proliferation, survival of cancer cells, and/or promotion of differentiation of cancer cells include methods using a reporter gene and a histone demethylase or protein associated with histone demethylase. Such a method may comprise (i) providing a cell or cell lysate comprising an histone demethylase gene or portion, e.g., promoter and/or enhancer, thereof, operably linked to a reporter gene and (ii) contacting the cell or cell lysate with a histone demethylase inhibitor and (iii) determining the level of expression of the reporter gene, wherein a higher level of expression of the reporter gene in the presence of the inhibitor relative to the absence of the inhibitor indicates that the inhibitor increases the level of expression of the histone demethylase gene, whereas a lower level of expression of the reporter gene in the presence of the inhibitor relative to the absence of the inhibitor indicates that the inhibitor decreases the level of expression of the histone demethylase gene. A reporter gene may encode, e.g., firefly luciferase, chloramphenicol acetyltransferase, β- galactosidase, green fluorescent protein, or alkaline phosphatase.
Assays for inhibiting the growth, proliferation, survival of cancer cells, and/or promotion of differentiation of cancer cells may further comprise testing the effect of the histone demethylase inhibitor on the demethylase activity in a cell. For example, an inhibitor may be contacted with or administered into a cell and the level of expression of one or more genes whose expression is regulated by methylation may be measured. Alternatively, or in addition, the level of protein, e.g., a histone demethylase protein or protein associated with a histone demethylase, may be measured.
One skilled in the art may refer to general reference texts for detailed descriptions of known techniques discussed herein or equivalent techniques. These texts include Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al., Molecular Cloning, A Laboratory Manual (3.sup.rd edition), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2000); Coligan et al., Current Protocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., Current Protocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., The Pharmacological Basis of Therapeutics (1975), Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 18.sup.th edition (1990). These texts can, of course, also be referred to in making or using an aspect of the invention
Biomarkers, Kits, and Diagnostic/Prognostic Methods
In some embodiments, the present invention provides a method of detecting or diagnosing cancer in a subject, comprising:
(a) measuring an effective amount of one or more histone demethylases in a sample from the subject; and
(b) comparing the amount to a reference value, wherein an increase or decrease in the amount of the one or more histone demethylases relative to the reference value indicates that the subject has cancer.
In some embodiments, an increase in one or more histone demethylases relative to the reference value indicates that the subject has cancer. For example, in some embodiments, an increase in LSD1 relative to the reference value indicates that the subject has cancer.
In other embodiments, a decrease in one or more histone demethylases relative to the reference value indicates that the subject has cancer.
In some embodiments, in methods of detecting or diagnosing cancer in a subject, the sample is whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsies, lymphatic fluid, ascites fluid, interstitial fluid, bone marrow, cerebrospinal fluid (CSF), seminal fluid, saliva, mucous, sputum, sweat, or urine.
In some embodiments, in methods of detecting or diagnosing cancer in a subject, the subject comprises one who has been previously diagnosed as having cancer, one who has not been previously diagnosed as having cancer, or one who is asymptomatic for cancer.
In some embodiments, in methods of detecting or diagnosing cancer in a subject, the measuring comprises detecting the presence or absence of the one or more histone demethylases, quantifying the amount of the one or more histone demethylases, and qualifying the type of the one or more histone demethylases.
In some embodiments, in methods of detecting or diagnosing cancer in a subject, the reference value comprises an index value, a value derived from one or more cancer risk prediction algorithms, a value derived from a subject not suffering from cancer, or a value derived from a subject diagnosed with cancer. In some embodiments, in methods of detecting or diagnosing cancer in a subject, the one or more histone demethylases comprises LSD1.
In some embodiments, in methods of detecting or diagnosing cancer in a subject, the one or more histone demethylases are measured by PCR.
In some embodiments, in methods of detecting or diagnosing cancer in a subject, the one or more histone demethylases are measured by immunoassay.
In some embodiments, in methods of detecting or diagnosing cancer in a subject, the cancer is characterized by the presence of pluripotent and/or multipotent cells.
In some embodiments, in methods of detecting or diagnosing cancer in a subject, the method further comprising measuring the level of at least one pluripotent or multipotent stem cell protein marker.
In some embodiments, in methods of detecting or diagnosing cancer in a subject, the at least one pluripotent stem cell protein marker is selected from the group consisting of Oct4, Sox2, Lin28, Nanog, Klf4, and Sall4, Lin28B, cMyc, nMyc, LMyc, Wnt3a, miR-291-3p, miR- 294, miR-295, miR-290/371 cluster, miR-302 cluster, miR-363 cluster, miR-520 cluster, miR- 92b, miR148/152, miR-124, miR-615, miR-708, miR-9, Klfl, Klf2, Klf5, Esrrb, Esrrg, Soxl , Sox3, Soxl5, Soxl8, Smad7, Nr5al , Nr5a2, Wnt/beta-catenin, Met, Notch, Hedgehog, Sonic Hedgehog, CD133, CD44, CD447CD247ESA+, CD347CD38", CD90, CD34+/CD387CD19\ CD34/CD38+/CD123+, CD117, CD20, integrinalpha2betahigh, EpCam, miR-137, miR-301, miR- 32, miR-22, miR-135b, miR-204, miR205, miR-lOa, miR196b, miR-448, miR-7-1, miR-128a, miR-196a-l, miR-361, Rexl, CRIPTO/TDGF, Cx43, IGF1, SSEA3, SSEA4, TRA-1-60, TRA-1- 81, ZFP42, FOXD3, TERT, Musashi-1, BMI-1, nestin, Ink4a/ARF, Pten", ALDH^, ABCG2, CXCR4, and MITF.
In some embodiments, in methods of detecting or diagnosing cancer in a subject,the cancer is selected from the group consisting of: embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian cancer, endometrial cancer, cervical cancer, retinoblastoma, kidney cancer, liver cancer, gastric cancer, brain cancer, medulloblastoma, medulloepithelioma, glioma,
glioblastoma, lung cancer, bronchial cancer, mesothelioma, skin cancer, colon and rectal cancer, bladder cancer, pancreatic cancer, lip and oral cancer, laryngeal and pharyngeal cancer, melanoma, pituitary cancer, penile cancer, parathyroid cancer, thyroid cancer, pheochromocytoma and paraganglioma, thymoma and thymic Carcinoma, leukemia, lymphoma, plasma cell neoplasms, myeloproliferative disorders, islet cell tumor, small intestine cancer, transitional cell cancer, pleuropulmonary blastoma, gestational trophoblastic cancer, esophageal cancer, central nervous system cancer, head and neck cancer, endocrine cancer, cardiovascular cancer, rhabdomyosarcoma, soft tissue carcinomas, carcinomas of bone, cartilage, fat, vascular, neural, and hematopoietic tissues and AIDS-related cancers. In some such embodiments, the cancer is breast cancer. In some such embodiments, the cancer is ovarian cancer.
One or more biomarkers disclosed herein can be detected in the practice of the present invention. For example, at least one (1), at least two (2), at least three (3), at least four (4) or more biomarkers (e.g., histone demethylases as described herein) can be detected and used for diagnosis and/or prognosis of cancer according to the methods of the invention disclosed herein. The histone demethylases include, e.g., LSDl, LSD2, JARTD family members, and FBXLIO, but are not limited to these examples. The invention also includes cancer candidate genes that are inhibited by, modulated by or otherwise affected by the action of one or more histone demethylases, which are useful as therapeutic targets.
The methods for detecting these biomarkers in a sample have many applications. For example, one or more biomarkers can be measured to aid cancer diagnosis or prognosis. In another example, the methods for detection of the biomarkers can be used to monitor responses in a subject to cancer treatment. In another example, the methods for detecting biomarkers can be used to assay for and to identify compounds that modulate expression of these biomarkers in vivo or in vitro, which may be useful in preventing or treating cancer in subjects. Differentiation of non-cancer and cancer status may be by the detection of one or more of the biomarkers disclosed herein. For example, exemplary biomarkers that may independently discriminate between cancer statuses include detection or measurement of one or more histone demethylases. Combinations of biomarkers are also useful in the methods of the invention for the determination of cancer and cancer status.
Methods for identifying a candidate compound for treating cancer may comprise, for example, contacting one or more of the biomarkers of the invention with a test compound; and determining whether the test compound interacts with the biomarker, wherein a compound that interacts with the biomarker is identified as a candidate compound for treating cancer.
Compounds suitable for therapeutic testing may be screened initially by identifying compounds which interact with one or more biomarkers listed in identified herein. By way of example, screening might include recombinantly expressing a biomarker of this invention, purifying the biomarker, and affixing the biomarker to a substrate. Test compounds can then be contacted with the substrate, typically in aqueous conditions, and interactions between the test compound and the biomarker are measured, for example, by measuring elution rates as a function of salt concentration. Certain proteins may recognize and cleave one or more biomarkers of this invention, in which case the proteins may be detected by monitoring the digestion of one or more biomarkers in a standard assay, e.g., by gel electrophoresis of the proteins.
The ability of a test compound to inhibit the activity of one or more of the biomarkers of this invention may be measured. The techniques used to measure the activity of a particular biomarker will vary depending on the function and properties of the biomarker. For example, an enzymatic activity of a biomarker may be assayed provided that an appropriate substrate is available and provided that the concentration of the substrate or the appearance of the reaction product is readily measurable. The ability of potentially therapeutic test compounds to inhibit or enhance the activity of a given biomarker may be determined by measuring the rates of catalysis in the presence or absence of the test compounds. The ability of a test compound to interfere with a non-enzymatic (e.g., structural) function or activity of one of the biomarkers of this invention may also be measured. For example, the self-assembly of a multi-protein complex which includes one of the biomarkers of this invention may be monitored by spectroscopy in the presence or absence of a test compound. Alternatively, if the biomarker is a non-enzymatic enhancer of transcription, test compounds which interfere with the ability of the biomarker to enhance transcription may be identified by measuring the levels of biomarker-dependent transcription in vivo or in vitro in the presence and absence of the test compound.
Test compounds capable of modulating the activity of any of the biomarkers of this invention may be administered to subjects who are suffering from or are at risk of developing cancer. For example, the administration of a test compound which increases the activity of a particular biomarker may decrease the risk of cancer in a subject if the activity of the particular biomarker in vivo prevents the accumulation of proteins for cancer. Conversely, the
administration of a test compound which decreases the activity of a particular biomarker may decrease the risk of cancer in a subject if the increased activity of the biomarker is responsible, at least in part, for the onset of cancer. At the clinical level, screening a test compound includes obtaining samples from test subjects before and after exposure to a test compound. The levels in the samples of one or more of the biomarkers of this invention may be measured and analyzed to determine whether the levels of the biomarkers change after exposure to a test compound. The samples may be analyzed by any appropriate means known to one of skill in the art. For example, the levels of one or more of the biomarkers of this invention may be measured directly by Western blot using radio- or fluorescently-labeled antibodies which specifically bind to the biomarkers.
Alternatively, changes in the levels of mRNA encoding the one or more biomarkers may be measured and correlated with the administration of a given test compound to a subject. In a further embodiment, the changes in the level of expression of one or more of the biomarkers may be measured using in vitro methods and materials. For example, human tissue cultured cells which express, or are capable of expressing, one or more of the biomarkers of this invention may be contacted with test compounds. Subjects who have been treated with test compounds will be routinely examined for any physiological effects which may result from the treatment. In particular, the test compounds will be evaluated for their ability to decrease disease likelihood in a subject. Alternatively, if the test compounds are administered to subjects who have previously been diagnosed with cancer, test compounds will be screened for their ability to slow or stop the progression of the disease.
Methods of identifying therapeutic targets for cancer generally comprise comparing an expression profile of a cancer cell with an expression profile of one a reference cell, wherein the comparison is capable of classifying proteins or transcripts in the profile as being associated with cancer invasion. Reference cells may be normal cells (cells that are not cancer cells) or cancer cells a different stage from the cancer cells being compared to. The reference cells may be primary cultured cells, fresh blood cells, established cell lines or other cells determined to be appropriate to one of skill in the art. Transcripts and proteins associated with cancer invasion include cells that differentiate between the states of cancer and between normal and cancer cell lines. The transcripts and proteins may also differentiate between different types of cancer. The proteins may be secreted proteins, such that they are easily detectable from a blood sample.
The subjects may be subjects who have been determined to have a high risk of cancer based on their family history, a previous treatment, subjects with physical symptoms known to be associated with cancer, subjects identified through screening assays (e.g., routine cancer screening) or other techniques. Other subjects include subjects who have cancer and the test is being used to determine the effectiveness of therapy or treatment they are receiving. Also, subjects could include healthy people who are having a test as part of a routine examination, or to establish baseline levels of the biomarkers. Samples may be collected from subjects who had been diagnosed with cancer and received treatment to eliminate the cancer, or who are in remission.
The risk of cancer can be detected by measuring an "effective amount" of the biomarkers of the present invention in a sample (e.g., a subject derived sample), and comparing the effective amounts to reference values, often utilizing mathematical algorithms or formulae in order to combine information from results of multiple individual biomarkers into a single measurement. Subjects identified as having an increased risk of cancer can optionally be selected to receive treatment regimens or therapeutic interventions, such as administration of compounds such as "anticancer agents" as defined herein, or implementation of surgical interventions (which may follow or precede administration of therapeutic agents or other therapies), biological therapies ("biotherapies"), or radiological therapies to prevent or delay the onset or recurrence of cancer or metastasis of cancer.
The present invention may be used to make continuous or categorical measurements of the risk of conversion to cancer, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at-risk for developing cancer. In the categorical scenario, the methods of the present invention can be used to discriminate between normal and at-risk subject cohorts. In other embodiments, the present invention may be used so as to discriminate at-risk from cancerous, or cancerous from normal. Such differing use may require different biomarker combinations in individual panel or profile, mathematical algorithm, and/or cut-off points, but be subject to the same aforementioned measurements of accuracy for the intended use.
Identifying the at-risk subject enables the selection and initiation of various therapeutic interventions or treatment regimens in order to delay, reduce, or prevent that subject's conversion to cancer. Levels of an effective amount of biomarker proteins, nucleic acids, polymorphisms, metabolites, or other analytes also allows for the course of treatment of cancer to be monitored. In this method, a biological sample can be provided from a subject undergoing treatment regimens, e.g., therapeutic treatments, for cancer. Such treatment regimens can include, but are not limited to, surgical intervention, radiological therapies, and treatment with therapeutic agents used in subjects diagnosed or identified with cancer. If desired, biological samples are obtained from the subject at various time points before, during, or after treatment. For example, determining the cancer status by comparison of a subject's biomarker profile to a reference biomarker profile can be repeated more than once, wherein the subject's biomarker profile can be obtained from a separate sample taken each time the method is repeated. Samples may be taken from the subject at defined time intervals, such as, e.g., 24 hours, 48 hours, 72 hours, or any suitable time interval as would be performed by those skilled in the art.
Differences in the genetic makeup of subjects can result in differences in their relative abilities to metabolize various drugs, which may modulate the symptoms or risk factors of cancer. Subjects that have cancer, or at risk for developing cancer can vary in age, ethnicity, and other parameters. Accordingly, use of the biomarkers disclosed herein, both alone and together in combination with known genetic factors for drug metabolism, allow for a pre-determined level of predictability that a putative therapeutic or prophylactic to be tested in a selected subject will be suitable for treating or preventing cancer in the subject.
To identify therapeutic agents or drugs that are appropriate for a specific subject, a test sample from the subject can also be exposed to a therapeutic agent or a drug, and the level of one or more of biomarker proteins, nucleic acids, polymorphisms, metabolites or other analytes can be determined. The level of one or more biomarkers can be compared to sample derived from the subject before and after subject management for cancer, e.g., treatment or exposure to a therapeutic agent or a drug, or can be compared to samples derived from one or more subjects who have shown improvements in cancer risk factors as a result of such treatment or exposure.
Nucleic acids may be obtained from the samples in many ways known to one of skill in the art, for example, extraction methods, including e.g., solvent extraction, affinity purification and centrifugation. Selective precipitation can also purify nucleic acids. Chromatography methods may also be utilized including, gel filtration, ion exchange, selective adsorption, or affinity binding. The nucleic acids may be, for example, RNA, DNA or may be synthesized into cDNA. The nucleic acids may be detected using microarray techniques that are well known in the art, for example, Affymetrix arrays followed by multidimensional scaling techniques. See R. Ekins, R. and Chu, F.W. (1999) Trends Biotechnol. 17: 217-218; D. D. Shoemaker, et al., (2001) Nature 409(6822): 922-927 and U.S. Patent No. 5,750,015. If desired, the sample can be prepared to enhance detectability of the biomarkers. For example, to increase the detectability of protein biomarkers, a blood serum sample from the subject can be preferably fractionated by, e.g., Cibacron blue agarose chromatography and single stranded DNA affinity chromatography, anion exchange chromatography, affinity
chromatography (e.g., with antibodies) and the like. The method of fractionation depends on the type of detection method used. Any method that enriches for the protein of interest can be used. Typically, preparation involves fractionation of the sample and collection of fractions determined to contain the biomarkers. Methods of pre-fractionation include, for example, size exclusion chromatography, ion exchange chromatography, heparin chromatography, affinity
chromatography, sequential extraction, gel electrophoresis and liquid chromatography. The analytes also may be modified prior to detection. A sample can be pre-fractionated by removing proteins that are present in a high quantity or that may interfere with the detection of biomarkers in a sample. For example, in a blood serum sample, serum albumin is present in a high quantity and may obscure the analysis of biomarkers. Thus, a blood serum sample can be pre- fractionated by removing serum albumin. Serum albumin can be removed using a substrate that comprises adsorbents that specifically bind serum albumin. For example, a column which comprises, e.g., Cibacron blue agarose (which has a high affinity for serum albumin) or antiserum albumin antibodies can be used.
In yet another technique, a sample can be pre-fractionated by isolating proteins that have a specific characteristic, e.g. are glycosylated. For example, a blood serum sample can be fractionated by passing the sample over a lectin chromatography column (which has a high affinity for sugars). Glycosylated proteins will bind to the lectin column and non-glycosylated proteins will pass through the flow through. Glycosylated proteins are then eluted from the lectin column with an eluant containing a sugar, e.g., N-acetyl-glucosamine and are available for further analysis.
Many types of affinity columns exist which are suitable for pre-fractionating blood serum samples. An example of a type of affinity chromatography available to pre-fractionate a sample is a single stranded DNA spin column. These columns bind proteins which are basic or positively charged. Bound proteins are then eluted from the column using eluants containing denaturants or high pH. In yet another embodiment, a sample can be fractionated using a sequential extraction protocol. In sequential extraction, a sample is exposed to a series of adsorbents to extract different types of biomarkers from a sample. For example, a sample is applied to a first adsorbent to extract certain proteins, and an eluant containing non-adsorbent proteins (i.e., proteins that did not bind to the first adsorbent) is collected. Then, the fraction is exposed to a second adsorbent. This further extracts various proteins from the fraction. This second fraction is then exposed to a third adsorbent, and so on. Any suitable materials and methods can be used to perform sequential extraction of a sample. For example, a series of spin columns comprising different adsorbents can be used. In another example, multi-well plates comprising different adsorbents at its bottom can be used. In another example, sequential extraction can be performed on a probe adapted for use in a gas phase ion spectrometer, wherein the probe surface comprises adsorbents for binding biomarkers. In this embodiment, the sample is applied to a first adsorbent on the probe, which is subsequendy washed with an eluant. Biomarkers that do not bind to the first adsorbent are removed with an eluant. The biomarkers that are in the fraction can be applied to a second adsorbent on the probe, and so forth. The advantage of performing sequential extraction on a gas phase ion spectrometer probe is that biomarkers that bind to various adsorbents at every stage of the sequential extraction protocol can be analyzed directly using a gas phase ion spectrometer.
In yet another embodiment, biomarkers in a sample can be separated by. high- resolution electrophoresis, e.g., one or two-dimensional gel electrophoresis. A fraction containing a biomarker can be isolated and further analyzed by gas phase ion spectrometry. Preferably, two- dimensional gel electrophoresis is used to generate two-dimensional array of spots, including one or more biomarkers. See, e.g., Jungblut and Thiede, (1997) Mass Spectr. Rev. 16: 145-162. The two-dimensional gel electrophoresis can be performed using methods known in the art. See, e.g., Deutscher ed., Methods in Enzymology vol. 182. Typically, biomarkers in a sample are separated by, e.g., isoelectric focusing, during which biomarkers in a sample are separated in a pH gradient until they reach a spot where their net charge is zero (i.e., isoelectric point). This first separation step results in one-dimensional array of biomarkers. The biomarkers in one- dimensional array is further separated using a technique generally distinct from that used in the first separation step. For example, in the second dimension, biomarkers separated by isoelectric focusing are further separated using a polyacrylamide gel, such as polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). SDS-PAGE gel allows further separation based on molecular mass of biomarkers. Typically, two-dimensional gel electrophoresis can separate chemically different biomarkers in the molecular mass range from 1000-200,000 Da within complex mixtures.
Biomarkers in the two-dimensional array can be detected using any suitable methods known in the art. For example, biomarkers in a gel can be labeled or stained (e.g., Coomassie Blue or silver staining). If gel electrophoresis generates spots that correspond to the molecular weight of one or more biomarkers of the invention, the spot can be excised and further analyzed by, for example, gas phase ion spectrometry, mass spectrometry, or high performance liquid chromatography. Alternatively, the gel containing biomarkers can be transferred to an inert membrane by applying an electric field. Then a spot on the membrane that approximately corresponds to the molecular weight of a biomarker can be analyzed by e.g., gas phase ion spectrometry, mass spectrometry, or HPLC.
Optionally, a biomarker can be modified before analysis to improve its resolution or to determine its identity. For example, the biomarkers may be subject to proteolytic digestion before analysis. Any protease can be used. Proteases, such as trypsin, that are likely to cleave the biomarkers into a discrete number of fragments are particularly useful. The fragments that result from digestion may function as a fingerprint for the biomarkers, thereby enabling their indirect detection. This is particularly useful where there are biomarkers with similar molecular masses that might be confused for the biomarker in question. Also, proteolytic fragmentation is useful for high molecular weight biomarkers because smaller biomarkers are more easily resolved by mass spectrometry. In another example, biomarkers can be modified to improve detection resolution. For instance, neuraminidase can be used to remove terminal sialic acid residues from glycoproteins to improve binding to an anionic adsorbent (e.g., cationic exchange ProteinChip® arrays) and to improve detection resolution. In another example, the biomarkers can be modified by the attachment of a tag of particular molecular weight that specifically binds to molecular biomarkers, further distinguishing them. Optionally, after detecting such modified biomarkers, the identity of the biomarkers can be further determined by matching the physical and chemical characteristics of the modified biomarkers in a protein database (e.g., SwissProt).
Once captured on a substrate, e.g., biochip or antibody, any suitable method, such as those described herein as well as other methods known in the art, can be used to measure a biomarker or biomarkers in a sample. The actual measurement of levels or amounts of the biomarkers can be determined using any method known in the art. These methods include, without limitation, mass spectrometry (e.g., laser desorption/ionization mass spectrometry), fluorescence (e.g. sandwich immunoassay), surface plasmon resonance, ellipsometry and atomic force microscopy. Methods may further include, by one or more of microarrays, PCR methods, mass spectrometry (including, for example, and without limitation, ESI-MS, ESI-MS/MS, ESI- MS/(MS)n, matrix-assisted laser desorption ionization time-of -flight mass spectrometry
(MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), desorption/ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS)n, atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS/MS, and APPI-(MS)n, quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS), and ion trap mass
spectrometry), nucleic acid chips, Northern blot hybridization, TMA, SDA, NASBA, PCR, real time PCR, reverse transcriptase PCR, real time reverse transcriptase PCR, in situ PCR, chromatographic separation coupled with mass spectrometry, protein capture using immobilized antibodies or by traditional immunoassays. See for example, U.S. Patent Nos. 5,723,591 ;
5,801,155 and 6,084,102 and Higuchi, 1992 and 1993. PCR assays may be done, for example, in a multi-well plate formats or in chips, such as the BioTrove OPEN ARRAY Chips (BioTrove, Wobum, MA).
For example, sequences within the sequence database entries corresponding to biomarkers of the present invention can be used to construct probes for detecting biomarker RNA sequences in, e.g., Northern blot hybridization analyses or methods which specifically, and, preferably, quantitatively amplify specific nucleic acid sequences. As another example, the sequences can be used to construct primers for specifically amplifying the biomarker sequences in, e.g., amplification-based detection methods such as reverse-transcription based polymerase chain reaction (RT-PCR), e.g., quantitative real-time RT-PCR. When alterations in gene expression are associated with gene amplification, deletion, polymorphisms, and mutations, sequence comparisons in test and reference populations can be made by comparing relative amounts of the examined DNA sequences in subject and reference cell populations. The term "specifically (or selectively) hybridizes" when referring to a nucleic acid, refers to a binding reaction that is determinative of the presence of the nucleic acid in a heterogeneous population of nucleic acids. Thus, under designated assay conditions, the specified nucleic acid probe (including inhibitory nucleic acids as defined herein) may bind or hybridize to a particular nucleic acid of interest at least two times the background and do not substantially bind or hybridize in a significant amount to other nucleic acids present in the sample.
Levels of biomarkers can also be determined at the protein level, e.g., by measuring the levels of peptides encoded by the gene products described herein, or activities thereof. Such methods are well known in the art and include, e.g., immunoassays based on antibodies to proteins encoded by the genes, aptamers or molecular imprints. Any biological material can be used for the detection quantification of the protein or its activity. Alternatively, a suitable method can be selected to determine the activity of proteins encoded by the biomarker genes according to the activity of each protein analyzed. The antibody may be monoclonal, polyclonal, chimeric, or a fragment of the foregoing, as discussed in detail herein, and the step of detecting the reaction product may be carried out with any suitable immunoassay. The sample from the subject is typically a biological fluid as described above, and may be the same sample of biological fluid used to conduct the method described above.
The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to a biomarker from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with that biomarker and not with other proteins, except for polymorphic variants and alleles of the biomarker. This selection may be achieved by subtracting out antibodies that cross-react with the biomarker molecules from other species.
Immunoassays carried out in accordance with the present invention may be homogeneous assays or heterogeneous assays. In a homogeneous assay the immunological reaction usually involves the specific antibody (e.g., anti-biomarker protein antibody), a labeled analyte, and the sample of interest. The signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte. Both the immunological reaction and detection of the extent thereof can be carried out in a homogeneous solution. Immunochemical labels which may be employed include free radicals, radioisotopes, fluorescent dyes, enzymes,
bacteriophages, or coenzymes.
In a heterogeneous assay approach, the reagents are usually the sample, the antibody, and means for producing a detectable signal. Samples as described above may be used. The antibody can be immobilized on a support, such as a bead (such as protein A and protein G agarose beads), plate or slide, and contacted with the specimen suspected of containing the antigen in a liquid phase. The support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal. The signal is related to the presence of the analyte in the sample. Means for producing a detectable signal include the use of detectable labels. Exemplary detectable labels include magnetic beads (e.g., DYNABEADS™), fluorescent dyes, enzymes (e.g., horse radish peroxide,
35 125 131 alkaline phosphatase and others commonly used in an ELISA), radiolabels (e.g., S, I, I), and fluorescent labels (e.g., fluorescein, Alexa, green fluorescent protein, rhodamine) and colorimetric labels such as colloidal gold or colored glass or plastic beads in accordance with known techniques.
Alternatively, the biomarker in the sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound biomarker-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the biomarker is incubated simultaneously with the mixture. For example, if the antigen to be detected contains a second binding site, an antibody which binds to that site can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step. The presence of the detectable label on the solid support indicates the presence of the antigen in the test sample. Methods for measuring the amount or the presence of antibody-marker complexes include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry). Optical methods include microscopy (both confocal and non-confocal), imaging methods and non-imaging methods. Electrochemical methods include voltametry and amperometry methods. Radio frequency methods include multipolar resonance spectroscopy. Examples of suitable immunoassays include, but are not limited to immunoblotting (e.g., Western blotting, slot blot assay), immunoprecipitation, immunofluorescence methods, chemiluminescence methods,
electrochemiluminescence (ECL) or enzyme-linked immunoassays, e.g., enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).
Those skilled in the art will be familiar with numerous specific immunoassay formats and variations thereof which may be useful for carrying out the method disclosed herein. See generally E. Maggio, Enzyme-Immunoassay, (1980) (CRC Press, Inc., Boca Raton, Fla.); see also U.S. Patent Nos. 4,727,022; 4,659,678; 4,376,110; 4,275,149; 4,233,402; and 4,230,767. These methods are also described in, e.g., Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991); and Harlow & Lane, supra. All of these are incorporated by reference herein.
Using the purified biomarkers or their nucleic acid sequences, antibodies that specifically bind to a biomarker can be prepared using any suitable methods known in the art. See, e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies: A Laboratory Manual (1988); Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and Kohler & Milstein, Nature 256:495-497 (1975). Such techniques include, but are not limited to, antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al., Science 246: 1275-1281 (1989); Ward et al., Nature 341 :544-546 (1989)). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
Generally, a sample obtained from a subject can be contacted with the antibody that specifically binds the biomarker. Optionally, the antibody can be fixed to a solid support to facilitate washing and subsequent isolation of the complex, prior to contacting the antibody with a sample. Examples of solid supports include glass or plastic in the form of, e.g., a microliter plate, a slide, or wells formed from materials such as latex or polystyrene, a stick, a bead (including magnetic beads), or a microbead such as protein A or protein G agarose. Antibodies can also be attached to a probe substrate or ProteinChip® array. The sample can be diluted with a suitable eluant before contacting the sample to the antibody.
Immunoassays can be used to determine presence or absence of a biomarker in a sample as well as the quantity of a biomarker in a sample. The amount of an antibody-marker complex can be determined by comparing to a standard. A standard can be, e.g., a known compound or another protein known to be present in a sample. As noted above, the test amount of biomarker need not be measured in absolute units, as long as the unit of measurement can be compared to a control.
The methods for detecting these biomarkers in a sample have many applications. For example, one or more biomarkers can be measured to aid cancer diagnosis or prognosis. In another example, the methods for detection of the biomarkers can be used to monitor responses in a subject to cancer treatment. In another example, the methods for detecting biomarkers can be used to assay for and to identify compounds that modulate expression of these biomarkers in vivo or in vitro. In a preferred example, the biomarkers are used to differentiate between the different stages of tumor progression, thus aiding in determining appropriate treatment and extent of metastasis of the tumor.
Proteins frequently exist in a sample in a plurality of different forms characterized by a detectably different mass. These forms can result from either, or both, of pre- and post- translational modification. Pre-translational modified forms include allelic variants, slice variants and RNA editing forms. Post-translationally modified forms include forms resulting from proteolytic cleavage (e.g., fragments of a parent protein), glycosylation, phosphorylation, lipidation, oxidation, methylation, cystinylation, sulphonation and acetylation. Antibodies can also be useful for detecting post-translational modifications of biomarker proteins, polypeptides, mutations, and polymorphisms, such as tyrosine phosphorylation, threonine phosphorylation, serine phosphorylation, glycosylation (e.g., O-GlcNAc). Such antibodies specifically detect the phosphorylated amino acids in a protein or proteins of interest, and can be used in
immunoblotting, immunofluorescence, and ELISA assays described herein. These antibodies are well-known to those skilled in the art, and commercially available. Post-translational modifications can also be determined using metastable ions in reflector matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) (Wirth, U. et al. (2002) Proteomics 2(10): 1445-51). The collection of proteins including a specific protein and all modified forms of it is referred to herein as a "protein cluster." The collection of all modified forms of a specific protein, excluding the specific protein, itself, is referred to herein as a "modified protein cluster." Modified forms of any biomarker of this invention also may be used, themselves, as biomarkers. In certain cases the modified forms may exhibit better discriminatory power in diagnosis than the specific forms set forth herein. Modified forms of a biomarker including any of the biomarkers as described herein can be initially detected by any methodology that can detect and distinguish the modified from the biomarker.
For biomarker proteins, polypeptides, mutations, and polymorphisms known to have enzymatic activity, the activities can be determined in vitro using enzyme assays known in the art. Such assays include, without limitation, kinase assays, phosphatase assays, and reductase assays, among many others. Modulation of the kinetics of enzyme activities can be determined by measuring the rate constant KM using known algorithms, such as the Hill plot, Michaelis- Menten equation, linear regression plots such as Lineweaver-Burk analysis, and Scatchard plot. In some instances, changes in the activity of a biomarker protein (which may occur as a result of disease progression) may be measured according to these and other methods known in the art.
Alternatively, biomarker protein and nucleic acid metabolites can be measured. The term "metabolite" includes any chemical or biochemical product of a metabolic process, such as any compound produced by the processing, cleavage or consumption of a biological molecule (e.g., a protein, nucleic acid, carbohydrate, or lipid). Metabolites can be detected in a variety of ways known to one of skill in the art, including the refractive index spectroscopy (RI), ultra-violet spectroscopy (UV), fluorescence analysis, radiochemical analysis, near-infrared spectroscopy (near-IR), nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS), mass spectrometry, pyrolysis mass spectrometry, nephelometry, dispersive Raman spectroscopy, gas chromatography combined with mass spectrometry, liquid chromatography (including high- performance liquid chromatography (HPLC)), which may be combined with mass spectrometry, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) combined with mass spectrometry, ion spray spectroscopy combined with mass spectrometry, capillary
electrophoresis, ion mobility spectrometry, surface-enhanced laser desorption/ionization (SELDI), optical methods, electrochemical methods, atomic force microscopy, radiofrequency methods, surface Plasmon resonance, ellipsometry, NMR and IR detection. (See, International Application Publication Nos. WO 04/056456 and WO 04/088309, each of which are hereby incorporated by reference in their entireties). In this regard, other biomarker analytes can be measured using the above-mentioned detection methods, or other methods known to the skilled artisan. For example, circulating calcium ions (Ca2+) can be detected in a sample using fluorescent dyes such as the Fluo series, Fura-2A, Rhod-2, among others. Other biomarker metabolites can be similarly detected using reagents that specifically designed or tailored to detect such metabolites.
Levels of an effective amount of biomarker proteins, nucleic acids, polymorphisms, metabolites, or other analytes can then be determined and compared to a reference value, e.g. a control subject or population whose cancer status is known, or an index value or baseline value. The reference sample or index value or baseline value may be taken or derived from one or more subjects who have been exposed to the treatment, or may be taken or derived from one or more subjects who are at low risk of developing cancer, or may be taken or derived from subjects who have shown improvements in cancer risk factors as a result of exposure to treatment.
Alternatively, the reference sample or index value or baseline value may be taken or derived from one or more subjects who have not been exposed to the treatment. For example, samples may be collected from subjects who have received initial treatment for cancer and subsequent treatment for cancer to monitor the progress of the treatment. A reference value can also comprise a value derived from risk prediction algorithms or computed indices from population studies such as those disclosed herein.
The biomarkers of the present invention can thus be used to generate a reference biomarker profile of those subjects who do not have cancer, and would not be expected to develop cancer. The biomarkers disclosed herein can also be used to generate a "subject biomarker profile" taken from subjects who have cancer. The subject biomarker profiles can be compared to a reference biomarker profile to diagnose or identify subjects at risk for developing cancer, to monitor the progression of disease, as well as the rate of progression of disease, and to monitor the effectiveness of cancer treatment modalities or subject management. The reference and subject biomarker profiles of the present invention can be contained in a machine-readable medium, such as but not limited to, analog or digital tapes like those readable by a VCR, CD- ROM, DVD-ROM, USB flash media, among others. Such machine-readable media can also contain additional test results, such as, without limitation, measurements of clinical parameters and traditional laboratory risk factors. Alternatively or additionally, the machine-readable media can also comprise subject information such as medical history and any relevant family history. The machine-readable media can also contain information relating to other cancer risk algorithms and computed indices such as those described herein.
Detection and correlation of biomarkers can be analyzed using any suitable means, including arrays. Nucleic acid arrays may be analyzed using software, for example, Applied Maths, GenExplore™, 2-way cluster analysis, principal component analysis, discriminant analysis, self-organizing maps; BioDiscovery, Inc., Los Angeles, California (ImaGene™, special image processing and data extraction software, powered by MatLab®; GeneSight: hierarchical clustering, artificial neural network (SOM), principal component analysis, time series;
AutoGene™; CloneTracker™); GeneData AG (Basel, Switzerland); Molecular Pattern
Recognition web site at MIT's Whitehead Genome Center; Rosetta Inpharmatics, Kirkland, Washington. Resolver™ Expression Data Analysis System; Scanalytics, Inc., Fairfax, VA. Its MicroArray Suite enables researchers to acquire, visualize, process, and analyze gene expression microarray data; TIGR (The Institute for Genome Research) offers software tools for array analysis. For example, see also Eisen and Brown, (1999) Methods Enzymol. 303: 179-205.
Detection and correlation of biomarkers can be analyzed using any suitable means. In one embodiment, data generated, for example, by desorption is analyzed with the use of a programmable digital computer. The computer program generally contains a readable medium that stores codes. Certain code can be devoted to memory that includes the location of each feature on a probe, the identity of the adsorbent at that feature and the elution conditions used to wash the adsorbent. The computer also contains code that receives as input, data on the strength of the signal at various molecular masses received from a particular addressable location on the probe. This data can indicate the number of biomarkers detected, including the strength of the signal generated by each biomarker.
Data analysis can include the steps of determining signal strength (e.g., height of peaks) of a marker detected and removing "outliers" (data deviating from a predetermined statistical distribution). The observed peaks can be normalized, a process whereby the height of each peak relative to some reference is calculated. For example, a reference can be background noise generated by instrument and chemicals (e.g., energy absorbing molecule) which is set as zero in the scale. The signal strength detected for each biomarker can be displayed in the form of relative intensities in the scale desired (e.g., 100). Alternatively, a standard (e.g., a serum protein) may be admitted with the sample so that a peak from the standard can be used as a reference to calculate relative intensities of the signals observed for each marker or other biomarkers detected.
The computer can transform the resulting data into various formats for displaying. In one format, referred to as "spectrum view or retentate map," a standard spectral view can be displayed, wherein the view depicts the quantity of marker reaching the detector at each particular molecular weight. In another format, referred to as "peak map," only the peak height and mass information are retained from the spectrum view, yielding a cleaner image and enabling biomarkers with nearly identical molecular weights to be more easily seen. In yet another format, referred to as "gel view," each mass from the peak view can be converted into a grayscale image based on the height of each peak, resulting in an appearance similar to bands on electrophoretic gels. In yet another format, referred to as "3-D overlays," several spectra can be overlaid to study subtle changes in relative peak heights. In yet another format, referred to as "difference map view," two or more spectra can be compared, conveniently highlighting unique biomarkers and biomarkers which are up- or down-regulated between samples. Biomarker profiles (spectra) from any two samples may be compared visually. In yet another format, Spotfire Scatter Plot can be used, wherein biomarkers that are detected are plotted as a dot in a plot, wherein one axis of the plot represents the apparent molecular of the biomarkers detected and another axis represents the signal intensity of biomarkers detected. For each sample, biomarkers that are detected and the amount of biomarkers present in the sample can be saved in a computer readable medium. This data can then be compared to a control or reference biomarker profile or reference value (e.g., a profile or quantity of biomarkers detected in control, e.g., subjects in whom cancer is undetectable).
When the sample is measured and data is generated, the data is then analyzed by a computer software program. Generally, the software can comprise code that converts signal from the mass spectrometer into computer readable form. The software also can include code that applies an algorithm to the analysis of the signal to determine whether the signal represents a "peak" in the signal corresponding to a marker of this invention, or other useful biomarkers. The software also can include code that executes an algorithm that compares signal from a test sample to a typical signal characteristic of "normal" and "cancerous" and determines the closeness of fit between the two signals. The software also can include code indicating which the test sample is closest to, thereby providing a probable diagnosis.
In preferred methods of the present invention, multiple biomarkers are measured. The use of multiple biomarkers increases the predictive value of the test and provides greater utility in diagnosis, toxicology, subject stratification and subject monitoring. The process called "Pattern recognition" detects the patterns formed by multiple biomarkers greatly improves the sensitivity and specificity of clinical proteomics for predictive medicine. Subtle variations in data from clinical samples indicate that certain patterns of protein expression can predict phenotypes such as the presence or absence of a certain disease, a particular stage of cancer progression, or a positive or adverse response to drug treatments.
Data generation in mass spectrometry begins with the detection of ions by an ion detector as described above. Ions that strike the detector generate an electric potential that is digitized by a high speed time-array recording device that digitally captures the analog signal. Ciphergen's ProteinChip® system employs an analog-to-digital converter (ADC) to accomplish this. The ADC integrates detector output at regularly spaced time intervals into time-dependent bins. The time intervals typically are one to four nanoseconds long. Furthermore, the time-of- flight spectrum ultimately analyzed typically does not represent the signal from a single pulse of ionizing energy against a sample, but rather the sum of signals from a number of pulses. This reduces noise and increases dynamic range. This time-of- flight data is then subject to data processing. In Ciphergen's ProteinChip® software, data processing typically includes TOF-to- M/Z transformation, baseline subtraction, high frequency noise filtering.
TOF-to-M/Z transformation involves the application of an algorithm that transforms times-of-flight into mass-to-charge ratio (MZZ). In this step, the signals are converted from the time domain to the mass domain. That is, each time-of-flight is converted into mass-to-charge ratio, or MZZ. Calibration can be done internally or externally. In internal calibration, the sample analyzed contains one or more analytes of known M/Z. Signal peaks at times-of- flight representing these massed analytes are assigned the known M/Z. Based on these assigned M/Z ratios, parameters are calculated for a mathematical function that converts times-of-flight to M/Z. In external calibration, a function that converts times-of-flight to M/Z, such as one created by prior internal calibration, is applied to a time-of-flight spectrum without the use of internal calibrants. Baseline subtraction improves data quantification by eliminating artificial, reproducible instrument offsets that perturb the spectrum. It involves calculating a spectrum baseline using an algorithm that incorporates parameters such as peak width, and then subtracting the baseline from the mass spectrum. High frequency noise signals are eliminated by the application of a smoothing function. A typical smoothing function applies a moving average function to each time- dependent bin. In an improved version, the moving average filter is a variable width digital filter in which the bandwidth of the filter varies as a function of, e.g., peak bandwidth, generally becoming broader with increased time-of-flight. See, e.g., International Patent Application Publication No. WO 00/70648.
Analysis generally involves the identification of peaks in the spectrum that represent signal from an analyte. Peak selection can, of course, be done by eye. Software is available as part of Ciphergen's ProteinChip® software that can automate the detection of peaks. In general, this software functions by identifying signals having a signal-to-noise ratio above a selected threshold and labeling the mass of the peak at the centroid of the peak signal. In one useful application many spectra are compared to identify identical peaks present in some selected percentage of the mass spectra. One version of this software clusters all peaks appearing in the various spectra within a defined mass range, and assigns a mass (M/Z) to all the peaks that are near the mid-point of the mass (M/Z) cluster.
Peak data from one or more spectra can be subject to further analysis by, for example, creating a spreadsheet in which each row represents a particular mass spectrum, each column represents a peak in the spectra defined by mass, and each cell includes the intensity of the peak in that particular spectrum. Various statistical or pattern recognition approaches can applied to the data.
The spectra that are generated in embodiments of the invention can be classified using a pattern recognition process that uses a classification model. In some embodiments, data derived from the spectra (e.g., mass spectra or time-of-flight spectra) that are generated using samples such as "known samples" can then be used to "train" a classification model. A "known sample" is a sample that is pre-classified (e.g., cancer or not cancer). Data derived from the spectra (e.g., mass spectra or time-of-flight spectra) that are generated using samples such as "known samples" can then be used to "train" a classification model. A "known sample" is a sample that is pre-classified. The data that are derived from the spectra and are used to form the classification model can be referred to as a "training data set". Once trained, the classification model can recognize patterns in data derived from spectra generated using unknown samples. The classification model can then be used to classify the unknown samples into classes. This can be useful, for example, in predicting whether or not a particular biological sample is associated with a certain biological condition (e.g., diseased vs. non diseased).
The training data set that is used to form the classification model may comprise raw data or pre-processed data. In some embodiments, raw data can be obtained directly from time-of- flight spectra or mass spectra, and then may be optionally "pre-processed" in any suitable manner. For example, signals above a predetermined signal-to-noise ratio can be selected so that a subset of peaks in a spectrum is selected, rather than selecting all peaks in a spectrum. In another example, a predetermined number of peak "clusters" at a common value (e.g., a particular time-of-flight value or mass-to-charge ratio value) can be used to select peaks.
Illustratively, if a peak at a given mass-to-charge ratio is in less than 50% of the mass spectra in a group of mass spectra, and then the peak at that mass-to-charge ratio can be omitted from the training data set. Pre-processing steps such as these can be used to reduce the amount of data that is used to train the classification model.
Classification models can be formed using any suitable statistical classification (or "learning") method that attempts to segregate bodies of data into classes based on objective parameters present in the data. Classification methods may be either supervised or unsupervised. Examples of supervised and unsupervised classification processes are described in Jain, "Statistical Pattern Recognition: A Review", IEEE Transactions on Pattern Analysis and Machine Intelligence, Vol. 22, No. 1, January 2000, which is herein incorporated by reference in its entirety. In supervised classification, training data containing examples of known categories are presented to a learning mechanism, which learns one more sets of relationships that define each of the known classes. New data may then be applied to the learning mechanism, which then classifies the new data using the learned relationships. Examples of supervised
classification processes include linear regression processes (e.g., multiple linear regression (MLR), partial least squares (PLS) regression and principal components regression (PCR)), binary decision trees (e.g., recursive partitioning processes such as CART - classification and regression trees), artificial neural networks such as back propagation networks, discriminant analyses (e.g., Bayesian classifier or Fischer analysis), logistic classifiers, and support vector classifiers (support vector machines). A preferred supervised classification method is a recursive partitioning process.
Recursive partitioning processes use recursive partitioning trees to classify spectra derived from unknown samples. Further details about recursive partitioning processes are provided in U.S. Patent Application Publication No. 20020138208. In other embodiments, the classification models that are created can be formed using unsupervised learning methods. Unsupervised classification attempts to learn classifications based on similarities in the training data set, without pre classifying the spectra from which the training data set was derived.
Unsupervised learning methods include cluster analyses. A cluster analysis attempts to divide the data into "clusters" or groups that ideally should have members that are very similar to each other, and very dissimilar to members of other clusters. Similarity is then measured using some distance metric, which measures the distance between data items, and clusters together data items that are closer to each other. Clustering techniques include the MacQueen's K-means algorithm and the ohonen's Self-Organizing Map algorithm.
Learning algorithms asserted for use in classifying biological information are described in, for example, International Application Publication No. WO 01/31580 and U.S. Patent Application Publication Nos. 20020193950, 20030004402, and 20030055615.
More specifically, to obtain the biomarkers the peak intensity data of samples from subjects, e.g., cancer subjects, and healthy controls are used as a "discovery set." This data were combined and randomly divided into a training set and a test set to construct and test multivariate predictive models using a non-linear version of Unified Maximum Separability Analysis ("USMA") classifiers. Details of USMA classifiers are described in U.S. Patent Application Publication No. 20030055615. The invention provides methods for aiding a cancer diagnosis using one or more biomarkers, i.e., one or more histone demethylases as specified herein. These biomarkers can be used alone, in combination with other biomarkers in any set, or with entirely different biomarkers in aiding cancer diagnosis. The biomarkers are differentially present in samples of a cancer subject and a normal subject in whom cancer is undetectable. For example, some of the biomarkers are expressed at an elevated level and/or are present at a higher frequency in cancer subjects than in normal subjects, while some of the biomarkers are expressed at a decreased level and or are present at a lower frequency in cancer subjects than in normal subjects. Therefore, detection of one or more of these biomarkers in a person would provide useful information regarding the probability that the person may have cancer.
In any of the methods disclosed herein, the data from the sample may be fed directly from the detection means into a computer containing the diagnostic algorithm. Alternatively, the data obtained can be fed manually, or via an automated means, into a separate computer that contains the diagnostic algorithm. Accordingly, embodiments of the invention include methods involving correlating the detection of the biomarker or biomarkers with a probable diagnosis of cancer. The correlation may take into account the amount of the biomarker or biomarkers in the sample compared to a control amount of the biomarker or biomarkers (up or down regulation of the biomarker or biomarkers) (e.g., in normal subjects in whom cancer is undetectable). The correlation may take into account the presence or absence of the biomarkers in a test sample and the frequency of detection of the same biomarkers in a control. The correlation may take into account both of such factors to facilitate determination of whether a subject has cancer or not.
The correlation may take into account the amount of the biomarker or biomarkers in the sample compared to a control amount of the biomarker or biomarkers (up or down regulation of the biomarker or biomarkers) (e.g., in normal subjects or in non-cancer subjects such as where cancer is undetectable). A control can be, e.g., the average or median amount of biomarker present in comparable samples of normal subjects in normal subjects or in non-cancer subjects such as where cancer is undetectable. The control amount is measured under the same or substantially similar experimental conditions as in measuring the test amount. The correlation may take into account the presence or absence of the biomarkers in a test sample and the frequency of detection of the same biomarkers in a control. The correlation may take into account both of such factors to facilitate determination of cancer status.
In certain embodiments of the methods of qualifying cancer status, the methods further comprise managing or modifying clinical treatment of a subject based on the status of the cancer. For example, if the result of the methods of the present invention is inconclusive or there is reason that confirmation of status is necessary, the physician may order more tests (e.g., CT scans, PET scans, MRI scans, PET-CT scans, X-rays, biopsies, blood tests (LFTs, LDH).
Alternatively, if the status indicates that treatment is appropriate, the physician may schedule the subject for treatment. In other instances, the subject may receive therapeutic treatments (such as administration of anticancer agents, either in lieu of, or in addition to, surgery. No further action may be warranted. Furthermore, if the results show that treatment has been successful, a maintenance therapy or no further management may be necessary. Anticancer agents may include, one or more of an alkylating agent, an antibiotic agent, an antimetabolic agent, a hormonal agent, a plant-derived agent, an anti-angiogenic agent, a differentiation inducing agent, a cell growth arrest inducing agent, an apoptosis inducing agent, a cytotoxic agent, a biologic agent, a gene therapy agent, a retinoid agent, an histone deacetylase inhibitor, an enzyme inhibitor, a cytokine, a chemokine, an antibody, a DNA molecule, an RNA molecule, a small molecule, a peptide, or a peptidomimetic, or any combination thereof, but are not limited to these examples.
The invention also provides for such methods where the biomarkers (or specific combination of biomarkers) are measured again after clinical treatment of a subject. In these cases, the methods are used to monitor the status of the cancer, e.g., response to cancer treatment, remission of the disease or progression of the disease. The methods can be repeated after each treatment the subject receives, allowing the physician to follow the effectiveness of the course of treatment. If the results show that the treatment is not effective, the course of treatment can be altered accordingly.
A diagnosis based on the presence or absence in a test subject of any the biomarkers of this invention is preferably communicated to the subject as soon as possible after the diagnosis is obtained. The diagnosis may be communicated to the subject by the subject's treating physician. Alternatively, the diagnosis may be sent to a test subject by email or communicated to the subject by phone. A computer may be used to communicate the diagnosis by email or phone. In certain embodiments, the message containing results of a diagnostic test may be generated and delivered automatically to the subject using a combination of computer hardware and software which will be familiar to artisans skilled in telecommunications. One example of a healthcare- oriented communications system is described in U.S. Patent No. 6,283,761 ; however, the present invention is not limited to methods which utilize this particular communications system. In certain embodiments of the methods of the invention, all or some of the method steps, including the assaying of samples, diagnosing of diseases, and communicating of assay results or diagnoses, may be used.
Methods of the invention for determining the cancer status of a subject, include for example, obtaining a biomarker profile from a sample taken from the subject; and comparing the subject's biomarker profile to a reference biomarker profile obtained from a reference population, wherein the comparison is capable of classifying the subject as belonging to or not belonging to the reference population; wherein the subject's biomarker profile and the reference biomarker profile comprise one or more biomarkers as described herein.
The method may further comprise repeating the method at least once, wherein the subject's biomarker profile is obtained from a separate sample taken each time the method is repeated. Samples from the subject may be taken at any time, for example, the samples may be taken 24 hours apart or any other time determined useful.
Such comparisons of the biomarker profiles can determine cancer status in the subject with an accuracy of at least about 60%, 70%, 80%, 90%, 95%, and approaching 100%. The reference biomarker profile can be obtained from a population comprising a single subject, at least two subjects, at least 20 subjects or more. The number of subjects will depend, in part, on the number of available subjects, and the power of the statistical analysis necessary.
A dataset can be analyzed by multiple classification algorithms. Some classification algorithms provide discrete rules for classification; others provide probability estimates of a certain outcome (class). In the latter case, the decision (diagnosis) is made based on the class with the highest probability. For example, consider the three-class problem: healthy, benign, and cancer. Suppose that a classification algorithm (e.g. nearest neighbor) is constructed and applied to sample A, and the probability of the sample being healthy is 0, benign is 33%, and cancer is 67%. Sample A would be diagnosed as being cancer. This approach, however, does not take into account any "fuzziness" in the diagnosis, e.g., that there was a certain probability that the sample was benign. Therefore, the diagnosis would be the same as for sample B, which has a probability of 0 of being healthy or benign and a probability of 1 of being cancer. Other classification algorithms and formulae include, but are not limited to, Principal Component Analysis (PCA), cross-correlation, factor rotation, Logistic Regression (LogReg), Linear
Discriminant Analysis (LDA), Eigengene Linear Discriminant Analysis (ELDA), Support Vector Machines (SVM), Random Forest (RF), Recursive Partitioning Tree (RPART), as well as other related decision tree classification techniques, Shrunken Centroids (SC), StepAIC, Kth-Nearest Neighbor, Boosting, Decision Trees, Neural Networks, Bayesian Networks, Support Vector Machines, Leave-One-Out (LOO), 10-Fold cross-validation (10-Fold CV), and Hidden Markov Models, among others. In some embodiments, the present invention provides a kit comprising reagents that detect one or more histone demethylases, a sample derived from a subject having normal control levels, and optionally instructions for using the reagents in the methods disclosed herein. The kit can include detection reagents further including one or more antibodies or fragments thereof, one or more aptamers, one or more oligonucleotides, or combinations thereof.
The invention provides kits for qualifying cancer status and/or detecting or diagnosing cancer, wherein the kits can be used to detect the biomarkers of the present invention. For example, the kits can be used to detect any one or more of the biomarkers described herein, which biomarkers are differentially present in samples of cancer subjects and normal subjects. The kits of the invention have many applications. For example, the kits can be used in any one of the methods of the invention described herein,. such as, inter alia, to differentiate if a subject has cancer or has a negative diagnosis, thus aiding a cancer diagnosis. In another example, the kits can be used to identify compounds that modulate expression of one or more of the biomarkers by using in vitro or in vivo animal models for cancer.
Generally, kits of the present invention include a biomarker-detection reagent, e.g., nucleic acids that specifically identify one or more biomarker nucleic acids by having homologous nucleic acid sequences, such as oligonucleotide sequences or aptamers, complementary to a portion of the biomarker nucleic acids or antibodies to proteins encoded by the biomarker nucleic acids packaged together. The oligonucleotides can be fragments of the biomarker genes. The oligonucleotides may be single stranded or double stranded. For example the oligonucleotides can be 200, 150, 100, 50, 25, 10 or less nucleotides in length. The kit may contain in separate containers a nucleic acid or antibody (either already bound to a solid matrix or packaged separately with reagents for binding them to the matrix), control formulations (positive and/or negative), and/or a detectable label such as fluorescein, green fluorescent protein, rhodamine, cyanine dyes, Alexa dyes, luciferase, radiolabels, among others. Instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying out the assay and for correlation to cancer status may be included in the kit. The assay may for example be in the form of a Northern hybridization or a sandwich ELISA as known in the art.
For example, biomarker detection reagents can be immobilized on a solid matrix such as a porous strip to form at least one biomarker detection site. The measurement or detection region of the porous strip may include a plurality of sites containing a nucleic acid. A test strip may also contain sites for negative and/or positive controls. Alternatively, control sites can be located on a separate strip from the test strip. Optionally, the different detection sites may contain different amounts of immobilized nucleic acids, e.g., a higher amount in the first detection site and lesser amounts in subsequent sites. Upon the addition of test sample, the number of sites displaying a detectable signal provides a quantitative indication of the amount of biomarkers present in the sample. The detection sites may be configured in any suitably detectable shape and are typically in the shape of a bar or dot spanning the width of a test strip.
Alternatively, the kit contains a nucleic acid substrate array comprising one or more nucleic acid sequences, e.g., primers for nucleic acid amplification. The nucleic acids on the array specifically identify one or more nucleic acid sequences represented by the biomarkers of the present invention. In various embodiments, the expression of 2, 3, or all 4 of the sequences represented by the biomarkers described herein can be identified by virtue of binding to the array. The substrate array can be on, e.g., a solid substrate, e.g., a "chip" as described in U.S. Patent No. 5,744,305. Alternatively, the substrate array can be a solution array, e.g., xMAP (Luminex, Austin, TX), Cyvera (Illumina, San Diego, CA), CellCard (Vitra Bioscience,
Mountain View, CA) and Quantum Dots' Mosaic (Invitrogen, Carlsbad, CA). The kit may also contain reagents, and/or enzymes for amplifying or isolating sample DNA. The kits may include reagents for real-time PCR, for example, TaqMan probes and or primers, and enzymes.
In one embodiment, a kit comprises: (a) a substrate comprising an adsorbent thereon, wherein the adsorbent retains or is otherwise suitable for binding a biomarker, and (b) instructions to detect the biomarker or biomarkers by contacting a sample with the adsorbent and detecting the biomarker or biomarkers retained by the adsorbent. In some embodiments, the kit may comprise an eluant (as an alternative or in combination with instructions) or instructions for making an eluant, wherein the combination of the adsorbent and the eluant allows detection of the biomarkers using gas phase ion spectrometry. Such kits can be prepared from the materials described above, and the previous discussion of these materials (e.g., probe substrates, adsorbents, washing solutions, etc.) is fully applicable to this section and will not be repeated.
In another embodiment, the kit may comprise a first substrate comprising an adsorbent thereon (e.g., a particle functionalized with an adsorbent) and a second substrate onto which the first substrate can be positioned to form a probe, which may be removed and inserted into machine, such as, e.g., a gas phase ion spectrometer. In other embodiments, the kit may comprise a single substrate, which is in the form of a probe with adsorbents on the substrate that can be removed and inserted into a machine. In yet another embodiment, the kit may further comprise a pre- fractionation spin column (e.g., Cibacron blue agarose column, anti-HSA agarose column, K-30 size exclusion column, Q-anion exchange spin column, single stranded DNA column, lectin column, etc.). In another embodiment, a kit comprises (a) an antibody that specifically binds to a biomarker; and (b) a detection reagent. An antibody may be, for example, an antibody directed against the gene products of a histone demethylase gene.
Optionally, the kit may further comprise pre-fractionation spin columns. In some embodiments, the kit may further comprise instructions for suitable operation parameters in the form of a label or a separate insert. Optionally, the kit may further comprise a standard or control information so that the test sample can be compared with the control information standard to determine if the test amount of a biomarker detected in a sample is a diagnostic amount consistent with a diagnosis of cancer.
EXAMPLES
Materials and Methods
Molecular modeling: The docking template structure of LSD1 was derived from the crystal structure of LSD1 bound to the substrate-like peptide (PDB code: 2vld) (Forneris, F. et al., (2007) J. Biol. Chem. 282: 20070-4). The peptide and water molecules were removed from the crystal structure and the polar hydrogen atoms were added to the amino acid residues before docking. The active site of LSD1 was used in the design of chemical molecules. Docking was performed using the latest version of AutoDock 4.0 (Morris, G.M. et al. (1998) J. Comput. Chem. 19: 1639-1662). The illustrated structures were made by Pymol (DeLano, W.L. (2002) The PvMOL Molecular Graphics System, DeLano Scientific, Palo Alto, CA, USA).
Chemical synthesis: The final products are described below and summarized in scheme 1 (Figure 1C). Carboxylic acid 6 (0.06 g, 0.14 mmol) and amine 12 (0.08 g, 0.15mmol) were dissolved in DMF (2 mL) at 0°C, EDCI (0.08 g, 0.4 mmol) and HOBT (0.05 g, 0.4 mmol) were added. The reaction mixture was stirred at room temperature for 20 hours and then diluted with ethyl acetate (150 mL). The organic phase was washed with saturated sodium bicarbonate (20 mL) and brine (20 mL), dried over sodium sulfate (anhydrous) and concentrated in vacuo. The residue was purified by silica gel chromatography, using methanol:dichloromethane (1:15) as eluant, to give the desired coupling compound (0.11 g, 86 %). Ή NMR (300 MHz, CDC13) δ: 1.38 (s, 18H), 1.48 (s, 18H), 1.54 (s, 9H), 2.51 (t, 4H, / = 4.2 Hz), 3.41-3.92 (m, 14H), 7.28-7.41 (m, 4H), 7.51 (d, 2H, J = 8.2 Hz), 7.91 (d, 2H, J = 7.8Hz) ppm. The above product (0.09 g, 0.09 mmol) was treated with trifluroacetic acid:dichloromethane (2 mL, v:v = 1 :1) at room temperature for 3 hours. After the reaction mixture was concentrated in vacuo, it was dissolved in ethyl acetate (50 mL), and washed with saturated sodium bicarbonate (20 mL) and brine (20 mL). The organic phase was dried over sodium sulfate (anhydrous), filtered and concentrated in vacuo to provide the target molecule CBBIOOI (0.04 g, 90%). *H NMR (300 MHz, MeOD) δ: 3.31-3.92 (m, 16H), 4.43 (br, s, 2H), 7.43-7.62 (m, 4H), 7.69 (d, 2H, J = 8.4Hz), 7.91 (d, 2H, J = 8.4 Hz) ppm; 13C NMR (125 MHz, MeOD) δ: 170.5, 169.4, 166.3, 157.0, 140.5, 136.2, 132.9, 129.7,129.5, 129.3, 128.3, 128.1, 127.5, 126.8, 59.6, 50.3, 42.7, 42.5, 42.0 ppm. HRESIMS for C25H33N8O2 [M + H]+ calculated: 477.2726, found: 477.2699.
Cells, peptides, antibodies and recombinant proteins: Pluripotent mouse teratocarcinoma F9, human mediastinal mixed germ NCCIT cells (an intermediate cell between seminoma and embryonic carcinoma), and human testicular embryonic carcinoma NTERA-2 cells were obtained from American Type Culture Collection. Human cervical carcinoma HeLa, human embryonic kidney carcinoma 293, mouse NIH3T3, F9, and NTERA-2 cells were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and antibiotics. NCCIT cells were grown in RPMI-1640 medium. F9 cells were grown on petri dishes coated with 0.1 % gelatin. Histone H3 peptides with dimethylated lysine 4 were purchased from AnaSpec. Rabbit anti-dimethylated 4 of histone H3 (ab32356) and anti-histone H3 (abl791 ) or LSD1 (abl7721) were obtained from Abeam. Sox2 and Oct4 antibodies were from Bethyl Laboratories and Santa Cruz Biotechnology, respectively. Anti-CULl and actin antibodies were described previously (Zheng, J. et al. (2002) Mol. Cell 10: 1519-26). Human LSD1 full length cDNA was obtained from Open Biosystems, cloned into pGEX-KG vector, and expressed as glutathione-S-transferase-LSDl fusion protein in E. coli and affinity purified with glutathione Sepharose-4B beads (GE Healthcare). Mininucleosomes were isolated from HeLa cells according to established procedures (Higa, L.A. et al. (2006) Nat. Cell Biol. 8: 1277-83).
In vitro demethylation assays: Methylated H3K4 peptides or isolated histories were incubated with GST-LSDl protein at 30°C for 1 hour according to previously described methods (Shi, Y. et al. (2004) Cell 119: 941-53). A typical 20 uL reaction contained 4 pg GST-LSDl, 3 μg histones or l ug peptides, LSD1 inhibitors. The reaction products were analyzed by mass spectrometry for peptide substrates and western blotting for histone substrates. For mass spectrometry analysis, 0.5 uL of the reaction mixture were equally mixed with 0.5 uL MALDI matrix (2mg/mL a-cyano-4-hydroxycinamic acid with 0.1% trifluoroacetic acid) and spotted onto the Opti-TOFTM 384 Well Insert (Applied Biosystems) to allow solvent evaporation and pepudes/matrix co-crystallization. The peptides were analyzed by 4800 Plus MALDI TOF/TOF tandem time-of-flight analyzer (Applied Biosystems).
In vivo demethylation and growth assays, quantitative RT-PCR, and siRNA: Actively growing F9 cells were treated with LSDl inhibitors for 30 hours. Cell lysates were prepared and subjected to western blotting for dimethylated H3K4 and histone H3 by specific antibodies. Cell growth, bromodeoxyuridine (BrdU) incorporation, MTT proliferation assays, and siRNA- transfection were described previously (Higa, L.A. et al., (2006) Nat. Cell Biol. 8: 1277-83; Higa, L.A. et al., (2003) Nat. Cell Biol. 5: 1008-15). For quantitative RT-PCR, total RNA was extracted from F9 cells using Trizol Reagent (Invitrogen) and 1 ug total RNA was used in each real time-PCR (RT-PCR) reaction using the respective primers for the target mouse genes (Shi, Y. et al. (2004) Cell 119: 941-53): 1) SCN3A: forward 5'-cactacttcctacttcaatggca-3' (SEQ ID NO:l) and reverse 5'-cagcgataagaaggcccag-3' (SEQ ID NO:2) ; 2) M4-AchR., forward 5'- tcacacctgtcaatggcagc-3' (SEQ ID NO:3) and reverse 5'-gccagtagcccttgatgatg-3' (SEQ ID NO:4); 3) β-actin, forward 5'-tccagccttccttcttgggtatg-3' (SEQ ID NO:5) and reverse 5'- gaaggtggacagtgaggccaggat-3' (SEQ ID NO:6). LSDl siRNA sequence is
AAGGAAAGCUAGAAGAAAA (SEQ ID NO:7), which matches 100% for both human and mouse LSDl genes, and was designed and synthesized by Dharmacon.
Immunohistochemistry: Testicular carcinoma tissue microarray slides (with 5 micron thickness tissues) were obtained from U.S. Biomax, Inc. Slides were baked and deparaffinized according to standard protocol (Shim, E.H. et al. (2003) Cancer Res. 63: 1583-8). Antigen retrieval was carried out in DivaDecloaker at 90°C for 30 minutes (for LSDl staining) or 30 seconds at 125°C, 30 seconds (for Oct4 staining) using the Decloaking Chamber (Biocare Medical). Slides were immersed in 3% H2O2 for 5 minutes to inactivate the endogenous peroxidase. Nonspecific binding sites were blocked using Background Sniper for 15 minutes. Rabbit monoclonal antibody against LSDl (C69G12, Cell Signaling) was diluted 1 :800 in Renoir Red Diluent, and the mouse monoclonal antibody for Oct4 (Biocare Medical) was pre- diluted by the supplier in DaVinci Green Diluent. Slides were incubated with primary antibodies at 4°C overnight, washed and then incubated with Rabbit-Probe or Mouse-Probe MACH3 HRP- polymer detection system according to the supplier's instructions. Slides are developed with 3,3'-diaminobenzidine substrate using the ImmPACT DAB Peroxidase Substrate Kit (Vector Laboratories) for 1-5 minutes, counterstained with hematoxylin, and then mounted with Cytoseal XYL (Richard-Allan Scientific). Unless otherwise specified, all reagents were obtained from Biocare Medical.
Example 1: Synthesis of LSDl Inhibitor Compounds
To understand the physiological function of LSDl in histone methylation and cell growth, compounds that specifically inhibit LSDl demethylase activity were developed. The crystal structure of LSDl in complex with CoREST and a substrate-like peptide inhibitor was used as a template to design specific LSDl inhibitors (Yang, M. et al., (2007) Nat. Struct. Mol. Biol. 14: 535-9; Yang, M. et al., (2006) Mol. Cell 23: 377-87; Chen, Y. et al. (2006) Proc. Natl. Acad. Sci. 103: 13956-61). The substrate-like peptide is derived from the 21 -amino acid residues at amino terminus of histone H3 peptide in which lysine 4 (K4) is replaced by methionine (M4, see, e.g., Figure 1 A, H3K4M), which binds to LSDl with high binding affinity (Ki = 0.05 uM) (Forneris, F. et al., (2007) J. Biol. Chem. 282: 20070-4). The positively charged residues (Arginine 2 and Arginine 8) of the peptide establish favorable electrostatic interactions with a cluster of negatively charged residues on LSDl surface that involve Aspartic acid (Asp) 375, Glutamic acid (Glu) 379, Asp 553, Asp 555, Asp 556, Asp 557, and Glu 559 (Figure 1A). The funnel channel that provides access to FAD is blocked by the peptide inhibitor. Based on the structural features of LSDl active site, especially the highly acidic properties of the surface around the active site, a non-peptide chemical scaffold that binds to LSDl with similar non- covalent binding mode to that of the peptide inhibitor was designed de novo (Figure IB). The guanidinium groups of the small molecules form strong hydrogen bonds with the negatively charged residues of LSDl, and the hydrophobic substituents dock into the deep pocket that is close to FAD. To identify good LSDl inhibitory compounds, a small compound library composed of total 9 small molecules based on the original design was synthesized (Figure IBID). These compounds were designated as CBB1001-CBB1009 (Figure ID). One specific feature of CBB1003 and CBB1007 is that the nitro group of CBB1003 and the methyl ester group of CBB1007 (Figure ID) can form a specific hydrogen bonding interaction with His564 (Figure IB), which improve the inhibition of LSD1. These studies indicate that the designed non-peptide small molecules could act as highly potent and specific LSD1 inhibitors.
The synthesis of the individual LSD1 inhibitor employed conventional solution chemistry, which is shown in Scheme 1 using CBBIOOI as the example (Figure 1C). Mono- methyl isophthalate was converted into the corresponding bromide 3 in 51% yield by a two-step sequence including selective reduction of the acid group in 2 to give a primary alcohol followed by treatment with N-bromosuccinimide and triphenylphosphane. Condensation of bromide 3 with l-[N,N'-bis(tert-butoxycarbonyl)amidino]piperazine (4) in the presence of triethylamine afforded the key intermediate 5, which was hydrolyzed to give the corresponding acid 6. After intermediate 6 was obtained, intermediate 11 was constructed.
Treatment of methyl 4-cyano-benzoate with lithium bis(trimethylsilyl)amide at 0°C was followed by protection of the resulting amidine with di-tert-butyldicarbonate produced ester 8. Saponification of the methyl ester in 8 afforded the corresponding acid which was then condensed with 2-(trimethylsilyl)ethyl piperazine-l-carboxylate to produce fragment 11 with 83% yield. The 2-(trimethylsilyl)ethyl carbamate protecting group in 11 was removed by the action of tetra-n-butylammonium fluoride and the resulting free amine (12) was condensed with acid 6 to provide the corresponding coupling product. This was then treated with trifluoroacetic acid to effect a global deprotection to furnish CBBIOOI in 77% yield. The preparation of additional 8 LSD1 inhibitory compounds (CBB1002-CBB1009) was performed according to synthetic procedures identical to that for the preparation of CBBIOOI except modifications at the R position (Figure IB-ID).
Example 2: LSD1 compounds inhibit LSD1 demethylase activity in vitro
To test the potency of the compounds, a recombinant fusion protein of glutathione-S- transferase and human LSD1 was expressed in bacteria, affinity purified, and examined for its ability to demethylate a synthetic histone H3 amino terminal substrate peptide that contains the di-methylated K4 in vitro (Figure 2). The LSD1 protein displayed a dose- and time-dependent demethylase activity, yielding the intermediate mono-methylated and final unmethylated H3K4 peptides (Shi, Y. et al. (2004) Cell 119: 941-53), which were separated, resolved, and semi- quantified by MALDI TOF-TOF mass -spectrometry (Figure 2B). Under these conditions, while all compounds (CBB1001-CBB1009) exhibited inhibitory effects towards LSD1 demethylase (Figure 2D), CBB 1007 displayed the best inhibitory effect, with the concentration for half inhibition at about 5 uM (Figure 2C and D). All other compounds (CBB1001-CBB 1006, and CBB 1008) displayed half inhibition at about 10-26 uM inhibitor concentrations (Figure 2D). To ensure that LSDl inhibitors can inhibit the activity of LSDl demethylase using the full length histone H3 in its native form, histone H3 was fractionated and isolated in mininucleosomal form from the nuclear fraction of HeLa cells. Isolated histone H3 was then used as a substrate for LSDl demethylation in vitro. It was found that LSDl can demethylate the dimethylated histone H3 and that the LSDl compounds exhibited similar inhibitory effects towards LSDl demethylation activity as with the di-methylated H3K4 peptide (Figure 2F and 2G).
These studies indicate that, unlike the inhibitors of MAO-A and MAO-B, which form a covalent bond with the monoamine oxidases, new LSDl inhibitory compounds can be developed that can non-covalently interact with LSDl to potently inhibit LSDl demethylase activity in vitro.
Example 2: LSDl compounds inhibit LSDl demethylase activity in vivo
Previous studies suggest that LSDl can demethylate di-methylated H3K4 in cultured cells and loss of LSDl causes the accumulation of di-methylated H3K4 (Shi, Y. et al., (2004) Cell 1 19: 941-53; Schulte, J.H. et al. (2009) Cancer Res. 69: 2065-71 ; Wang, Y. et al., (2009) Cell 138: 660-72). To investigate whether new LSDl inhibitors can enter cells and block the demethylase activity of LSDl in vivo, mouse F9 embryonic carcinoma/teratoma cells were treated with synthetic LSDl compounds and then monitored for the accumulation of di- methylated H3K4 by Western blotting with anti-di-methylated H3K4 antibodies. While compounds CBB IOOI, CBB 1002, CBB 1004, CBB 1005, and CBB1009 did not have noticeable effects on di-methylated H3K4 at high concentrations (250 μΜ, Figure 3A-B), compounds CBB 1003 and CBB 1007 at low concentrations led to the significant increase of di-methylated H3 4 after treatment (Figure 3A-B). Among the compounds, compound CBB 1007 exhibited inhibitory activity at about 5 uM, while compound CBB1003 displayed an inhibitory effect at 10 μΜ (Figure 3A-B). The remaining compounds (CBB1006 and CBB1008) behaved similarly but at higher concentrations (5-25 uM). Thus, although all synthetic compounds are active inhibitors for LSDl in vitro, only some of them inhibited LSDl activity in cultured cells, suggesting that inhibition of LSDl H3K4 demethylase activity in vivo is dependent on specific modifications of the scaffold of LSDl compounds. A hallmark of LSD1 inactivation is that the resultant increase of di-methylated H3K4 can lead to the activation of epigenetically suppressed genes such as M4-ArchR and SCN3A in cultured cells (Shi, Y. et al., (2004) Cell 119: 941-53). To investigate whether LSD1 inhibitory compounds can induce epigenetically suppressed gene expression, the activation of M4-ArchR and SCN3A genes was monitored by quantitative RT-PCR after addition of LSD1 compounds to the cells (Figure 3C-D). Consistent with H3 4 di-methylation, while compounds CBBIOOI, CBB1002, CBB1004, CBB1005, and CBB1009 could not activate the expression of these genes (Figure 3C-D), treatment of cells with compounds CBB1003 and CBB1007 led to the activation of M4-ArchR and SCN3 A gene expression (Figure 3C-D and 4A-B). Using this assay, activation of M4-ArchR and SCN3A gene expression was detected at about 500 nM for compound CBB1007 and 1 uM for compound CBB1003, while other compounds (CBB1006 and CBB1008) displayed intermediate inhibitory effects at about 5-20 uM (Figure 3B). These results revealed that while all compounds can inactivate the demethylation activity of LSD1 in vitro, compounds CBB1003 and CBB1007 are the most active compounds to inhibit LSD1 activity in vivo, promoting di-methylation of H3K4 and consequent induction of epigenetically suppressed gene expression.
Example 3: LSD1 inhibitors selectively inhibit the growth of pluripotent embryonic carcinoma, teratoma, and seminoma cells
LSD1 is highly conserved among high eukaryotes and null mutation of LSD1 genes in the mouse causes embryonic lethality, suggesting LSD1 is essential for development. However, the physiological function of LSD1 is quite a puzzle since it can only demethylate di- and mono- methylated but not tri-methylated H3K4, while the members of JARID1 family (1 A-1D) that contain the Jumonji C (JmjC) domain can demethylate tri-, di, and mono-methylated H4K43. In previous reports, loss of LSD1 expression by siRNA-mediated ablation in many cancer or normal cells usually did not cause inhibition of cell growth (Shi, Y. et al., (2004) Cell 119: 941- 53; Wang, Y. et al., (2009) Cell 138: 660-72) presumably due to the presence of other types of H3K4 demethylases (JARIDIA-ID and FBX10) in the same cell that may functionally substitute for LSD1 to demethylate H3K4. After treatment of LSD1 compounds CBB1003 and CBB1007, growth of mouse embryonic carcinoma/teratoma F9 cells was significantly inhibited (Figure 5A- E), as analyzed by cell viability of treated cells and MTT proliferation assays. This growth inhibition of F9 cells is dependent on the dose of compounds CBB1003 or CBB1007, starting at 1-5 uM (Figure 5A-5E), with compound CBB1007 displayed a better inhibitory effect at lower concentrations, and increased in the presence of higher concentrations of the inhibitors.
However, compounds CBBIOOI, CBB1002, CBB1004, CBB1005, and CBB1009 did not exhibit significant inhibitory effects in the proliferation of F9 cells (Figure 5 A), suggesting that the scaffold of LSD1 compounds did not have a general toxicity towards F9 cells. The growth inhibitory effects of compounds CBB1003, CBB1007, and other remaining compounds
(CBB1006 and CBB1008) correlate with their ability to induce di-methylation of H3K4 and activation of epigenetically suppressed gene expression (Figures 3, 4, and 5A-5E), suggesting that the growth inhibition is a consequence of specific LSD1 inhibition by these compounds in F9 cells.
A screen of various cultured cell lines revealed that, consistent with previous reports (Shi, Y. et al., (2004) Cell 119: 941-53; Wang, Y. et al., (2009) Cell 138: 660-72), many cultured cancer or established somatic cell lines, such as human cervical carcinoma HeLa and embryonic kidney carcinoma 293, or immortalized mouse ΝΓΗ 3T3 (Figure 5G and 5H, and Figure 9B), did not display any significant growth inhibition after the treatment of cells with compounds
CBB1003 or CBB1007, even at high concentrations (100-200 uM). These studies suggest that LSD1 inhibitory compounds are highly selective towards pluripotent mouse F9 embryonic carcinoma cells but not other types of cancer or normal cells.
The mouse F9 cells express the pluripotent stem cell markers Oct4 and Sox2 and retain stem cell-like properties, such as rapid spherical growth and the ability to differentiate into less pluripotent cells under cell culture conditions (Strickland, S. et al., (1980) Cell 21: 347-55; Cheng, L. et al. (2007) J. Pathol. 211 : 1-9). Inoculation of F9 cells into immunodeficient mice can induce teratomas which are capable of differentiating into a wide range of tissue types (Strickland, S. et al., (1980) Cell 21 : 347-55). Unlike F9 cells, cancer cell lines such as HeLa, 293, and immortal ΝΓΗ3Τ3 cells do not express pluripotent stem cell markers Oct4 and Sox2 and are incapable of differentiation and hence are considered non-stem cell lineage (Figure 6D).
To further investigate whether human teratoma cells or similar pluripotent germ cell tumors are also sensitive to LSD1 inhibitors, human pluripotent mediastinal mixed germ NCCIT cells, an intermediate cell between seminoma and embryonic carcinoma, and pluripotent human testicular embryonic carcinoma NTERA-2 cells were examined. It was found that the growth of both human NCCIT and NTERA-2 cells are also highly sensitive to LSD1 inhibitors (Figure 5G and 5H, and Figure 9A), similar to that of mouse teratoma F9 cells (Figure 5A-5E). These studies indicate that embryonic carcinomas, teratomas, seminomas, possibly other stem cell- derived cells of pluripotent germ cell tumors (Cheng, L. et al. (2007) J. Pathol. 211 : 1-9), are most sensitive to LSDl inhibitors, suggesting that LSDl plays an essential function for these pluripotent cancer cells. Similar results were seen with the ovarian adenocarcinoma cell line IGROVl and ovarian teratocarcinoma cell line PA-1 (Figure 10). Both IGROVl and PA-1 express pluripotent stem cell markers Oct4 and Sox2, and high levels of LSDl (Figure 18). IGROVl is a human epithelial ovarian cancer cell line originally derived from a stage ΙΠ ovarian adenocarcinoma patient with an endometrioid, serous clear and undifferentiated tumor. These cells were reported to express pluripotent stem cell markers Oct4, Sox2, and Lin28 (Peng, S. et al., (2010) Oncogene 29: 2153-2159)(Figure 18). These cells were treated with dimethyl sulfoxide (DMSO, control), 50 mM CBB1007, or CBBIOIO for 48 hours. Figure 10 shows that, like NCCIT and NTERA-2 cells (Figure 5G-H, and 9A), IGROVl and PA-1 cells are also sensitive to CBB1007 and 1010.
To further examine the critical role of LSDl in pluripotent tumor cells and to confirm the specificity of LSDl inhibitors, the effect of specific ablation of LSDl expression by siRNA- mediated gene silencing in F9 and HeLa cells was also analyzed. After ablating the expression of LSDl with specific siRNA targeted at an mRNA region of LSDl that is completely conserved between mouse and human LSDl genes at the nucleotide levels, only F9 cells displayed the significant growth inhibition and bromodeoxyuridine (BrdU) incorporation while LDS 1 deficiency in HeLa did not cause any reduction in cell growth (Figure 5F, 6A and 6B).
Figure 11 shows that F9 cells are sensitive to RBBP5 siRNA, but HeLa cells are not. RBBP5 is an essential component of MLL histone methyltransferase complex that methylates histone H3 at lysine 4 (K4), while non-pluripotent cancer HeLa cells are not very sensitive to RBBP5 siRNA. Loss of RBBP5 reduces histone H3 at lysine 4 tri-, di-, and mono-methylations (Figure 17C), while inhibition of LSDl leads to an increased di-, and mono-methylation in histone H3 at lysine 4 (H3K4)(Figures 3A-B, 8, and 15A-C). Figure 11 demonstrates that pluripotent cancer stem cells are very sensitive to changes in histone H3 methylation status, while non-pluripotent cancer cells are not. Similarly, the growth of ovarian cancer stem cell-like cancer cell IGROVl and ovarian teratoma cell PA-1 is also very sensitive to the changes of histone methylation at lysine 4 (K4) caused by loss of RBBP5 using siRNA-mediated ablation (Figure 12).
These observations confirm that LSDl compounds specifically target at LSDl and modulate histone methylation at H3K4 in vivo and loss or inhibition of LSDl blocks the growth of pluripotent teratoma, embryonic carcinoma, and seminoma cells, but without obvious effects on the proliferation of other non-pluripotent cancer or normal cells. Similar growth-inhibitory effects after modulation of H3K4 methylation by loss of RBBP5 were also observed. This selectivity of LSDl inhibitors and alteration of H3K4 methylation towards pluripotent cancer cells suggests that these LSDl inhibitory compounds may provide a novel therapeutic tool to specifically inhibit stem cell-like cancers without affecting the growth of other cells.
Example 4: LSDl expression is highly elevated in pluripotent cancer cells
To determine the mechanism by which pluripotent F9, NCCIT, and NTERA-2 cancer cells are selectively sensitive to LSDl inhibition or inactivation, levels of LSDl in these cells were examined and compared with that of other non-pluripotent cancer cells such as HeLa and 293. The pluripotent F9, NCCIT, and NTERA-2 cells express high levels of LSDl protein, while LSDl is much lower in HeLa and 293 cells (Figure 6D). The high levels of LSDl in F9, NCCIT, and NTERA-2 cells are correlated with the expression of pluripotent stem cell markers Oct4 and Sox2 (Figure 6D), which are not present in HeLa or 293 cells. In addition, comparison of histone H3 and di-methylated histone H4K4 by their specific antibodies revealed that histone H3 and dimethylated H3K4 antibodies recognize additional protein bands that migrate slower than histone H3, suggesting that histone H3 is likely further modified. Elevated levels of LSDl may render the pluripotent cancer cells more dependent on LSDl, which may underlie the selective sensitivity of these cancer stem cells towards the inactivation of LSDl by specific LSDl inhibitors or siRNAs.
To determine how the inactivation of LSDl can prevent the pluripotent embryonic carcinoma, teratoma, and seminoma cell from proliferating, inactivation of LSDl and its effect on the expression of Oct4 and Sox2, two most critical stem cell proteins that maintain stem cell property and their self-renewal, was examined (Cheng, L. et al. (2007) J. Pathol. 211 : 1-9; Yamanaka, S. (2009) Cell 137: 13-17). Inactivation of LSDl by treatment of LSDl inhibitors or siRNA caused the significant downregulation of Sox2 and Oct4 expression (Figure 6C), while the expression of other proteins, such as CUL1, were unaltered. Thus, by regulating the stem- cell specific expression of Sox2 and Oct4, as well as possibly other essential stem cell genes, LSDl plays a unique and essential role in the proliferation of many stem cell-like pluripotent cancer cells.
While these studies revealed that LSDl protein levels are highly elevated in established embryonic carcinoma, teratoma, and seminoma cell lines, it was unknown whether LSDl would serve as a good protein biomarker for human teratomas, seminomas, and other pluripotent germ cell tumors in situ (Jones, T.D. et al. (2004) Clin. Cancer Res. 10: 8544-7; Jones, T.D. et al. (2004) Am. J. Surg. Pathol. 28: 935-940). To this end, human seminoma tumor tissue microarrays were analyzed. While normal human testicular tissues surrounding the tumors contain almost un-detectable or very low levels of LSDl (Figure 7 A), immunostaining of tumor microarrays revealed that 6 out 6 human testicular seminomas (100%) displayed high levels of LSDl protein (Figure 7B). These seminomas are likely to be pluripotent, since they are also positive on the expression of Oct4 (Figure 7B) (Jones, T.D. et al. (2004) Am. J. Surg. Pathol. 28: 935-940), a well-known pluripotent stem cell marker. The marked elevation of LSDl protein levels in human testicular seminomas is consistent with the observation that pluripotent F9, NCCrr, and NTERA-2 cancer cells also contain high levels of LSDl, Oct4, and Sox2 protein and that these cells are highly sensitive to LSDl inhibitors. The data suggest that LSDl can serve as a biomarker for teratomas, seminomas, as well as other undifferentiated germ cell tumors or cancers with stem cell properties, and LSDl inhibitors may be used to selectively treat these cancers in vivo without affecting other normal cells.
Example 5: Ovarian and Breast Cancer Cells Selectively Sensitive to LSDl Inhibitors
The previous Examples indicated that LSDl inhibitors selectively inhibited the proliferation of pluripotent cancer cells including teratocarcinoma, embryonic carcinoma, or seminoma cells that express stem cell markers Oct4 and Sox2, while displaying minimum growth inhibitory effects on non-pluripotent cancer or normal somatic cells. In this Example, several ovarian and breast cancer cells that are selectively sensitive to LSDl inhibitors are identified (Figures 10 and 14A-F). These cancer cells express pluripotent or multipotent stem cell proteins, such as Sox2, Lin28, KLF4, and/or Oct4 (Figure 18), and are potential cancer stem cell-like cells. Consistently, ablation of LSDl expression by siRNA also showed that these ovarian and breast cancer-stem cell-like cells are highly sensitive towards LSD1 ablation but not other cancer cells that do not express these stem cell markers (Figures 16A and 16B). Treatment with LSD1 inhibitors or siRNAs led to accumulation of mono- and dimethylated H3K4 and induced genes for differentiation (Figures 15A-D). Loss of H3K4 methylation by specific siRNAs for RBBP5 and WDR5, components of MLL methyltransferase that methylate H3K4, also selectively inhibited the growth of both pluripotent cancer stem cells and the ovarian and breast cancer stem cell-like cells (Figures 12, 17A-C). Finally, this Example shows that LSD1 and histone H3K4 methylation are important for many cancer stem cell-like cells that express pluripotent stem cell proteins.
The PA-1 human ovarian teratocarcinoma, Hs38.T human ovarian adenocarcinoma,
MCF-7 human breast adenocarcinoma, SKOV-3 human ovarian carcinoma, and T47D human breast ductal carcinoma cells were obtained from American Type Culture Collection (ATCC). IGROV1 human ovarian carcinoma cells were from National Cancer Institute and A2780 human ovarian carcinoma cells were from Sigma-Aldrich. They were maintained in Dulbecco's Modified Eagle medium or RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics. F9, NCCIT, and HeLa cells were cultured as described previously (Wang et al. Novel Histone Demethylase LSD1 Inhibitors Selectively Target Cancer Cells with Pluripotent Stem Cell Properties. Cancer Research, on the worldwide web at cancerres.aacrjournals.org: October 5, 2011). They were tested every three months and were authenticated for expression of known protein markers, such as Oct4, Sox2, p53, and pi 6, and morphology by Western blotting and microscopy within last three months.
Anti-Lin28, DNMT1, Nanog, Sall4, histone H3, tri-, di-, and monomethylated histone H3 at lysines 4 (H3K4) and 9 (H3K9), and Klf4 antibodies were from Abcom. Anti-Sox2, Oct4, and CUL1 antibodies were described previously (Wang et al., 2011 supra).
Actively growing A2780, IGROV1, T47D, Hs38.T, PA-1, SKOV-3, and other cells were treated with LSD1 inhibitors for 24-30 hours. Cell lysates were prepared and subjected to Western blotting for detecting methylated H3K4 and histone H3 by specific antibodies. Cell growth, MTT proliferation assays, and siRNA-transfection were described previously (Higa et al. Radiation-mediated proteolysis of CDT1 by CUL4-ROC1 and CSN complexes constitutes a new checkpoint. Nat Cell Biol, 5:1008-1015 (2003); Higa et al. CUL4-DDB 1 ubiquitin ligase interacts with multiple WD40-repeat proteins and regulates histone methylation. Nat Cell Biol, 8:1277-1283, (2006)). For quantitative RT-PCR and Real-time quantitative RT-PCR, total RNAs were extracted from A2780 or T47D cells or other cells using TRizol Reagent
(Invitrogen) and 1 μg (microgram) total RNA was used in each real time-PCR (RT-PCR) reaction using the respective primers for the target mouse genes (Shi et al. Histone
demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell, 119:941-53,
(2004).): 1) SCN3A: forward 5'.- C ACTACTTCCTACTTC AATGGCA-3 ' (SEQ ID NO:l) and reverse 5'- A A ACAGCG ATAAG A AGGCCC AG-3 ' (SEQ ID NO:15); 2) CHRM4., forward 5'- TC AC ACCTGTC A ATGGCAGC-3 ' (SEQ ID NO: 3) and reverse 5'- GCC AGTAGCCCTTGATGATG-3 ' (SEQ ID NO:4); 3) beta-actin, forward 5'- TCC AGCCTTCCTTCTTGGGTATG-3 ' (SEQ ID NO:5) and reverse 5'-
GAAGGTGGACAGTGAGGCCAGGAT -3' (SEQ ID NO:6) 4) FOXA2: Forward primer 5'- CC ATCCG ACTGGAGC AGCTA-3 ' (SEQ ID NO: 8), reverse primer 5- GCTC ATGT ATGTGTTC ATGCC ATTC-3 ' (SEQ ID NO:9). Real-Time RT-PCR was conducted using SYBR premix kit (Applied Biosystems) and 7500 Fast Real-time PCR system (Applied Biosystems)(Wang et al. 2011 supra). LSD1 siRNA sequence is
AAGGAAAGCUAGAAGAAAA (SEQ ID NO:7), which matches 100% for both human and mouse LSD1 genes, RBBP5 siRNA: GAGCCGAGAUGGUCAUAAAUU (SEQ ID NO: 10); WDR5 siRNA: CAGAGGATAACCTTGTTTA (SEQ ID NO:l l); and Luciferase (Luc):
CGTACGCGGAATACTTCGA (SEQ ID NO: 12). The siRNAs were designed and synthesized by Dharmacon.
As indicated in the Examples above, LSD1 inhibitory compounds CBB1003 and CBB1007 can specifically inhibit the growth of pluripotent cancer cells such as teratocarcinoma, embryonic carcinoma, and seminoma cells but with minimum effects towards non-pluripotent cancer cells or normal somatic cells such as HeLa, 293, and ΝΓΗ3Τ3 (see also Wang et al. 2011 i«pra)(Figures 5 and 9). LSD1 inhibitors also had minimum effects towards the growth of ovarian carcinoma cells such as Hs38.T cells that are non-cancer stem cells (Figure 13)(Gao et al. Maintenance of near-diploid karyotype of PA-1 human ovarian teratocarcinoma cells due to death of polyploid cells by chromosome fragmentation/pulverization. Int J Mol Med, 4:291-294 (1999)), as shown in Figure 13E, relative to a control that does not contain any LSD1 inhibitors shown in Figure 13D. Since pluripotent teratoma, embryonic carcinoma, and seminoma cells that are sensitive to LSD1 inhibitors express pluripotent stem cell markers Oct4, Sox2, and Lin28, ovarian and breast cancer cells that were indicated to express one of these pluripotent stem cell proteins were specifically tested. First, PA-1, a human ovarian teratocarcinoma cell line, was tested and was found to be very sensitive to LSDl inhibitors CBB1007 (Figure 13B) and CBIOIO (Figure 13C), relative to a control shown in Figure 13A. This cell line expresses pluripotent stem cell proteins Oct4, Sox2, and Lin28, as shown in Figure 18, Lane 5. Ovarian and breast cancer cells that are non-teratoma, embryonic carcinoma, or seminoma in origin were also screened. The A2780 cell, a human ovarian carcinoma that was reported to express pluripotent stem cell proteins Sox2 and Lin28 was tested with CBB1003 and CBB1007 at various concentrations and was growth-inhibited by LSDl inhibitors (Zhong et al. Identification of microRNAs regulating reprogramming factor LIN28 in embryonic stem cells and cancer cells. / Biol Chem, 285:41961-41971 (2010).), with the results shown in Figure 14A and depicted graphically in the top graph of Figure 14C. In addition, IGROV1 and SKOV-3, two additional human ovarian carcinoma that also have been indicated to express Oct4, Sox2, and/or Lin28 (Peng et al. Pluripotency factors Lin28 and Oct4 identify a sub-population of stem cell-like cells in ovarian cancer. Oncogene, 29:2153-9 (2008); Gan et al. Telomere maintenance in telomerase- positive human ovarian SKOV-3 cells cannot be retarded by complete inhibition of telomerase. FEBS Lett, 527: 10-4 (2002)), according to the micro-array based studies of NCI-60 cancer cell panel, were also sensitive to LSDl inhibitors of the invention as shown in Figures 14D and 14E). Notably, it was found all these cells expressing one or more pluripotent stem cell markers showed excellent sensitivity towards LSDl inhibitors CBB1003 and 1007 (Fig. 14). In addition to CBB1003 and CBB1007, IGROV1 cells also displayed excellent sensitivity towards a new derivative of LSDl inhibitor, CBBIOIO (Figure 14D).
In addition to ovarian cancer cells, T47D, a human ductal breast epithelial carcinoma cell line expressing Lin28 ( Zhong et al. supra), was found to be highly sensitive towards LSDl inhibitory compounds CBB1003 and CBB1007 (Fig. 14B and 14C). MCF-7, a breast adenocarcinoma cell line obtained from ATCC that expresses pluripotent stem cell protein Sox2 (Figure 18), also showed significant sensitivity to LSDl inhibitors (Fig. 14F).
LSDl compounds are active in cancer cells to inhibit LSDl demethylation activity
LSDl is a demethylase specific for mono- and di-methylated H3K4 and loss of LSDl in vivo causes the accumulation of these methylated forms of H3K4 (Shi et al. Histone demethylation mediated by the nuclear amine oxidase homolog LSDl. Cell, 119:941-953 (2004), Schulte et al. Lysine-specific demethylase 1 is strongly expressed in poorly differentiated neuroblastoma: implications for therapy. Cancer Res, 69:2065-2071 (2009), Wang et al. LSDl is a subunit of the NuRD complex and targets the metastasis programs in breast cancer. Cell, 138:660-72 (2009)). To determine the in vivo effects of LSDl inhibitors in A2780 ovarian and T47D breast cancer cells, these cells were treated with CBB1003 and CBB1007 and then monitored the accumulation of methylated H3K4. Notably, CBB1003 and CBB1007 both caused a significant increase of mono- and di-methylated H3K4 after treatment but had very little effects on tri-methylated H3K4 and tri- and dimethylated H3K9 (Fig. 15A and 15B), suggesting that they are specific for LSDl but not other histone demethylases.
It has been shown that LSDl can demethylate non-histone proteins such as DNMT1 to stabilize its protein stability and regulate global DNA methylation in mouse ES cells (Wang et al. The lysine demethylase LSDl (KDM1) is required for maintenance of global DNA methylation. Nat Genet;41 : 125-9 (2009)). Loss of LSDl leads to the destabilization of DNMT1 in mouse ES cells (Wang et al. 2009 supra). It was found that inhibition of LSDl by CBB1003 and 1007 can also induce DNMT1 destabilization and downregulation (Figure 15C), indicating that the effect of LSDl inhibitors are specific for LSD demethylation activity in vivo.
It has been indicated that loss of LSDl in pluripotent teratocarcinoma cells can cause the activation of epigenetically suppressed genes and differentiation genes, such as FOXA2, as a consequence of increased levels of H3K4 methylation (Wang et al. 2011 supra)(Figure 4A). To investigate whether loss of LSDl can also induce the expression of differentiation genes in LSDl -sensitive ovarian and breast cancer cells, the expression of two differentiation genes, HNF4a and FOXA2, was monitored after ablation of LSDl by siRNA. Loss of LSDl dramatically induced the expression of HNF4a and FOXA2 in A2780, T47D, and IGROV1 cells by the real-time quantitative RT-PCR analysis (Fig. 15D), suggesting that inhibition of LSDl can induce A2780, IGROV1, and T47D cells to activate the expression of genes for
differentiation.
RNA-interference analysis
To confirm the specific growth-sensitivity of the ovarian and breast cancer cells to LSDl inhibitors, RNA-interference-based ablation was used in these cancer cells to determine the effect of loss of LSDl . Consistent with findings using the LSDl inhibitors, loss of LSDl by treatment of cells with specific LSDl siRNAs led to the growth inhibition of A2780, IGROVl, SKVO-3, and T47D (Fig. 16A and 16B). The loss of LSDl by siRNA in non-stem Hs38 cells did not have much effect on its growth (Fig. 16A), consistent with the effect of LSDl inhibitor on this cell line, and suggesting that LSDl in this non-stem cell like ovarian cancer cell is not essential.
Sensitivity towards the loss of H3K4 methylation
Since LSDl is a specific demethylase that normally removes di- and monomethylated H3K4, inhibition of LSDl activity in vivo leads the alteration of H3K4 methylation (Fig. 15A- 15B) (Shi et al. Histone demethylation mediated by the nuclear amine oxidase homolog LSDl. Cell 2004;119:941-53). To determine whether methylation of H3K4 is critical to pluripotent or multipotent cancer stem cells, RBBP5 and WDR5 were ablated, these being important components of MLL-WDR5-RBBP5 methyltransferase complex that specifically methylates H3K4 (Klose et al. Regulation of histone methylation by demethylimination and demethylation. Nat Rev Mol Cell Biol, 8:307-18 (2007)). While loss of RBBP5 or WDR5 led to the
downregulation of tri- and monomethylation of H3K4 (Fig. 17C), RBBP5 or WDR5 deficiencies caused the growth inhibition in pluripotent F9 teratocarcinoma cells and ovarian IGROVl, PA-1, and A2780 and breast T47D cancer stem cell-like cells (Figures 11, 12, and 17) . However, ablation of RBBP5 by siRNA did not have significant effects on the growth of non-stem cells such as HeLa or Hs38.T cells (Figure 16A and 17A). These effects are similar to that of loss or inhibition of LSDl, suggesting that the homeostasis regulation of H3K4 methylation is essential for pluripotent cancer stem cells or cancer cells with stem cell properties. Expression of pluripotent stem cell proteins in ovarian and breast cancer cells
To determine whether the sensitivity of ovarian and breast cancer cells towards LSDl inhibitors is correlated with the expression of pluripotent stem cell proteins, the expression of each pluripotent stem cell proteins Oct4, Sox2, Lin28, Nanog, Sall4, and Klf4 in the ovarian and breast cancer cells and teratoma, embryonic carcinoma, seminoma, and non-pluripotent cancer cells such as HeLa or Hs38.T cells was analyzed by immunoblotting analysis (Figure 18). This Example revealed that F9, NCCIT, and PA-1 teratoma cells express Oct4, Sox2, and Lin28, A2780, IGROV1, and T47D express Sox2 and Lin28, SKOV-3 and MCF-7 express Sox2. The non-sensitive HeLa or Hs38.T cells do not express any of the pluripotent stem cell proteins. These studies suggest that A2780, IGROV1, and T47D, as well as SKOV-3 and MCF-7, may retain some properties of pluripotent or multipotent stem cells, such as the transcriptional circuitry of pluripotent stem cells, thereby conferring their sensitivity towards LSD1 inhibitors.
This Example indicates that maintenance of proper levels of H3K4 methylation in these cells is needed for these cells to proliferate and survive, and any changes in histone methylation may adversely affect the ability of these cells to maintain stem cell-like properties for proliferation, survival, or differentiation. This Example further indicates that methylation of histone H3 at lysine 4 serves as a good target for these cancer stem cell-like cells.
Each of the applications and patents cited in this text, as well as each document or reference cited in each of the applications and patents (including during the prosecution of each issued patent; "application cited documents"), and each of the U.S. and foreign applications or patents corresponding to and/or claiming priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference. More generally, documents or references are cited in this text, either in a Reference List before the claims, or in the text itself; and, each of these documents or references ("herein-cited references"), as well as each document or reference cited in each of the herein-cited references (including any manufacturer's specifications, instructions, etc.), is hereby expressly incorporated herein by reference. Documents incorporated by reference into this text may be employed in the practice of the invention.
Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.
Ill

Claims

CLAIMS We claim:
1. A compound of
Figure imgf000114_0001
or pharmaceutically acceptable salt thereof;
wherein X is a functional group selected from the group consisting of CH2, S,
NH, and S02;
Y and Z are independently N or CH;
Ri comprises a functional group selected from the group consisting of hydrogen, a carboxamide (aminocarbonyl), a carboxamidine (carboximidamide), acyl, an alkylsulfonyl, an arylsulfonyl, guanidine, and an aminosulfonyl, any of which may be optionally substituted;
R.2 comprises a functional group selected from the group consisting of hydrogen, alkyl, nitro, sulfonamide, sulfonimide, amide, and carboxyalkyl, any of which is optionally substituted; and
R3 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, and heterocycloalkyl group, any of which is optionally substituted.
2. The compound of claim 1, wherein Ri is the carboxamidine: -(CN=H)-NH2.
3. The compound of claim 1, wherein R2 is a functional group selected from the group consisting of hydrogen, methyl, nitro, Ν,Ν-dimethylsulfonamide, MeS02NMeS02-, N- methylcarboxamide, and carboxymethyl.
4. The compound of claim 1, wherein R3 is selected from the group consisting of:
Figure imgf000115_0001
5. The compound of claim 1, wherein said compound comprises one selected from the group consisting of:
Figure imgf000115_0002
Figure imgf000116_0001
Figure imgf000116_0002
Figure imgf000116_0003
114
Figure imgf000117_0001
7. The pharmaceutical composition of claim 6, further comprising an anticancer agent.
8. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the compound of claim 1.
9. The method of claim 8, wherein said cancer comprises pluripotent and/or multipotent cancer cells expressing LSD1.
10. The method of claim 8, wherein said cancer comprises cells expressing at least one pluripotent or multipotent stem cell protein marker.
11. The method of claim 10, where said at least one pluripotent stem cell protein marker is selected from the group consisting of Oct4, Sox2, Lin28, Nanog, Klf4, Sall4, Lin28B, cMyc, nMyc, LMyc, Wnt3a, miR-291-3p, miR-294, miR-295, miR-290/371 cluster, miR-302 cluster, miR-363 cluster, miR-520 cluster, miR-92b, miR148/152, miR-124, miR-615, miR-708, miR-9, Klfl, Klf2, Klf5, Esrrb, Esrrg, Soxl , Sox3, Soxl5, Soxl8, Smad7, Nr5al, Nr5a2, Wnt/beta- catenin, Met, Notch, Hedgehog, Sonic Hedgehog, CD133, CD44, CD44+/CD247ESA+, CD34+/CD38", CD90, CD34+/CD387CD19+, CD34/CD38+/CD123+, CD117, CD20, integrinalpha2betahigh, EpCam, miR-137, miR-301, miR-32, miR-22, miR-135b, miR-204, miR205, miR-lOa, miR196b, miR-448, miR-7-1, miR-128a, miR-196a-l, miR-361, Rexl, CRIPTO/TDGF, Cx43, IGF1, SSEA3, SSEA4, TRA-1-60, TRA-1-81, ZFP42, FOXD3, TERT, Musashi-1, BMI-1, nestin, Ink4a/ARF, Pten , ALDH"6*1, ABCG2, CXCR4, and MITF.
12. The method of claim 8, wherein said cancer comprises at least one selected from the group consisting of: embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian cancer, endometrial cancer, cervical cancer, retinoblastoma, kidney cancer, liver cancer, gastric cancer, brain cancer, medulloblastoma, medulloepithelioma, glioma, glioblastoma, lung cancer, bronchial cancer, mesothelioma, skin cancer, colon and rectal cancer, bladder cancer, pancreatic cancer, lip and oral cancer, laryngeal and pharyngeal cancer, melanoma, pituitary cancer, penile cancer, parathyroid cancer, thyroid cancer, pheochromocytoma and paraganglioma, thymoma and thymic carcinoma, leukemia, lymphoma, plasma cell neoplasms, myeloproliferative disorders, islet cell tumor, small intestine cancer, transitional cell cancer, pleuropulmonary blastoma, gestational trophoblastic cancer, esophageal cancer, central nervous system cancer, head and neck cancer, endocrine cancer, cardiovascular cancer, rhabdomyosarcoma, soft tissue carcinomas, carcinomas of bone, cartilage, fat, vascular, neural, and hematopoietic tissues and AIDS-related cancers.
13. The method of claim 12, wherein said cancer comprises breast cancer.
14. The method of claim 12, wherein said cancer comprises ovarian cancer.
15. The method of claim 8, further comprising administering a therapeutically effective amount of an anticancer agent.
16. Use of a compound according to claim 1 in the manufacture of a medicament for the treatment of cancer.
17. The use of claim 16, wherein said cancer comprises pluripotent and/or multipotent cancer cells expressing LSD1.
18. The use of claim 16, wherein said cancer comprises cells expressing at least one pluripotent or multipotent stem cell protein marker.
19. The use of claim 18, where said at least one pluripotent stem cell protein marker is selected from the group consisting of Oct4, Sox2, Lin28, Nanog, Klf4, and Sall4, Lin28B, cMyc, nMyc, LMyc, Wnt3a, miR-291-3p, miR-294, miR-295, miR-290/371 cluster, miR-302 cluster, miR-363 cluster, miR-520 cluster, miR-92b, miR148/152, miR-124, miR-615, miR-708, miR-9, Klfl, Klf2, Klf5, Esrrb, Esrrg, Soxl, Sox3, Soxl5, Soxl8, Smad7, Nr5al, Nr5a2, Wnt/beta- catenin, Met, Notch, Hedgehog, Sonic Hedgehog, CD133, CD44, CD447CD247ESA+, CD34VCD38", CD90, CD347CD387CD19+, CD34/CD38+/CD123+, CD117, CD20, integrinalpha2betahigh, EpCam, miR-137, miR-301, miR-32, miR-22, miR-135b, miR-204, miR205, miR-lOa, miR196b, miR-448, miR-7-1, miR-128a, miR-196a-l, miR-361, Rexl, CRIPTO/TDGF, Cx43, IGFl, SSEA3, SSEA4, TRA-1-60, TRA-1-81 , ZFP42, FOXD3, TERT, Musashi-1, BMI-1, nestin, Ink4a/ARF, Pten , ALDH1"611, ABCG2, CXCR4, and MITF.
20. The use of claim 16, wherein said cancer comprises at least one selected from the group consisting of: embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian cancer, endometrial cancer, cervical cancer, retinoblastoma, kidney cancer, liver cancer, gastric cancer, brain cancer, medulloblastoma, medulloepithelioma, glioma, glioblastoma, lung cancer, bronchial cancer, mesothelioma, skin cancer, colon and rectal cancer, bladder cancer, pancreatic cancer, lip and oral cancer, laryngeal and pharyngeal cancer, melanoma, pituitary cancer, penile cancer, parathyroid cancer, thyroid cancer, pheochromocytoma and paraganglioma, thymoma and thymic Carcinoma, leukemia, lymphoma, plasma cell neoplasms, myeloproliferative disorders, islet cell tumor, small intestine cancer, transitional cell cancer, pleuropulmonary blastoma, gestational trophoblastic cancer, esophageal cancer, central nervous system cancer, head and neck cancer, endocrine cancer, cardiovascular cancer, rhabdomyosarcoma, soft tissue carcinomas, carcinomas of bone, cartilage, fat, vascular, neural, and hematopoietic tissues and AIDS-related cancers.
21. The use of claim 20, wherein said cancer comprises breast cancer.
22. The use of claim 20, wherein said cancer comprises ovarian cancer.
23. The use of claim 16, wherein said medicament further comprises a therapeutically effective amount of an anticancer agent.
24. A method for inhibiting the growth, proliferation, and/or survival of cancer cells, comprising contacting the cancer cells with an effective amount of the compound of claim 1.
25. The method of claim 24, further comprising contacting said cancer cells with an anticancer agent.
26. The method of claim 24, wherein said cancer cells comprise pluripotent and/or multipotent cancer cells expressing LSD1.
27. The method of claim 24, wherein said cancer cells comprise cells expressing at least one pluripotent or multipotent stem cell protein marker.
28. The method of claim 27, where said at least one pluripotent stem cell protein marker is selected from the group consisting of Oct4, Sox2, Lin28, Nanog, Klf4, and Sall4, Lin28B, cMyc, nMyc, LMyc, Wnt3a, miR-291-3p, miR-294, miR-295, miR-290/371 cluster, miR-302 cluster, miR-363 cluster, miR-520 cluster, miR-92b, miR148/152, miR-124, miR-615, miR-708, miR-9, Klfl, Klf2, Klf5, Esrrb, Esrrg, Soxl , Sox3, Soxl5, Soxl8, Smad7, Nr5al, Nr5a2, Wnt/beta- catenin, Met, Notch, Hedgehog, Sonic Hedgehog, CD133, CD44, CD44+/CD247ESA+, CD34VCD38", CD90, CD347CD387CD19\ CD34/CD38+/CD123+, CD117, CD20, integrinalpha2betahigh, EpCam, miR-137, miR-301, miR-32, miR-22, miR-135b, miR-204, miR205, miR-lOa, miR196b, miR-448, miR-7-1, miR-128a, miR-196a-l, miR-361, Rexl,
CRIPTO/TDGF, Cx43, IGF1, SSEA3, SSEA4, TRA-1-60, TRA-1-81, ZFP42, FOXD3, TERT, Musashi-1, BMI-1, nestin, Ink4a/ARF, Pten', ALDH^811, ABCG2, CXCR4, and MITF.
29. The method of claim 24, wherein said cancer cells comprise at least one cancer selected from the group consisting of: embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian cancer, endometrial cancer, cervical cancer, retinoblastoma, kidney cancer, liver cancer, gastric cancer, brain cancer, medulloblastoma, medulloepithelioma, glioma, glioblastoma, lung cancer, bronchial cancer, mesothelioma, skin cancer, colon and rectal cancer, bladder cancer, pancreatic cancer, lip and oral cancer, laryngeal and pharyngeal cancer, melanoma, pituitary cancer, penile cancer, parathyroid cancer, thyroid cancer, pheochromocytoma and paraganglioma, thymoma and thymic Carcinoma, leukemia, lymphoma, plasma cell neoplasms, myeloproliferative disorders, islet cell tumor, small intestine cancer, transitional cell cancer, pleuropulmonary blastoma, gestational trophoblastic cancer, esophageal cancer, central nervous system cancer, head and neck cancer, endocrine cancer, cardiovascular cancer, rhabdomyosarcoma, soft tissue carcinomas, carcinomas of bone, cartilage, fat, vascular, neural, and hematopoietic tissues and AIDS-related cancers.
30. The method of claim 29, wherein said at least one cancer comprises breast cancer.
31. The method of claim 29, wherein said at least one cancer comprises ovarian cancer.
32. The method of claim 24, wherein said method is conducted in vivo.
33. The method of claim 24, wherein said method is conducted in vitro.
34. A method of modulating one or more histone methylation events in a cell, comprising contacting the cell with an effective amount of the compound of claim 1.
35. The method of claim 34, wherein the one or more histone methylation events occur at lysine 4, lysine 9, lysine 27, lysine 36, lysine 79 of histone H3 or lysine 20 of histone H4.
36. The method of claim 34, wherein the one or more histone methylation events occur at lysine 4 of histone H3.
37. The method of claim 34, wherein the cell is derived from a cancer expressing LSD1.
38. The method of claim 34, wherein the cell is derived from a cancer comprising pluripotent and/or multipotent cancer cells.
39. The method of claim 34, wherein said cell is derived from a cancer expressing at least one pluripotent stem cell protein marker.
40. The method of claim 39, wherein said at least one pluripotent stem cell protein marker is selected from the group consisting of Oct4, Sox2, Lin28, Nanog, Klf4, and Sall4, Lin28B, cMyc, nMyc, LMyc, Wnt3a, miR-291-3p, miR-294, miR-295, miR-290/371 cluster, miR-302 cluster, miR-363 cluster, miR-520 cluster, miR-92b, miR148/152, miR-124, miR-615, miR-708, miR-9, Klfl, Klf2, Klf5, Esrrb, Esrrg, Soxl , Sox3, SoxlS, Soxl8, Smad7, Nr5al, Nr5a2, Wnt/beta- catenin, Met, Notch, Hedgehog, Sonic Hedgehog, CD133, CD44, CD44+/CD247ESA+, CD34VCD38", CD90, CD34+/CD387CD19\ CD34/CD38+/CD123+, CD117, CD20, integrinalpha2betahigh, EpCam, miR-137, miR-301, miR-32, miR-22, miR-135b, miR-204, miR205, miR-lOa, miR196b, miR-448, miR-7-1, miR-128a, miR-196a-l, miR-361, Rexl, CRIPTO/TDGF, Cx43, IGFl, SSEA3, SSEA4, TRA-1-60, TRA-1 -81 , ZFP42, FOXD3, TERT, Musashi-1, BMI-1, nestin, Ink4a/ARF, Pten", ALDH^811, ABCG2, CXCR4, and MITF.
41. The method of claim 34, wherein the cell is derived from a cancer selected from the group consisting of: embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian cancer, endometrial cancer, cervical cancer, retinoblastoma, kidney cancer, liver cancer, gastric cancer, brain cancer, medulloblastoma, medulloepithelioma, glioma, glioblastoma, lung cancer, bronchial cancer, mesothelioma, skin cancer, colon and rectal cancer, bladder cancer, pancreatic cancer, lip and oral cancer, laryngeal and pharyngeal cancer, melanoma, pituitary cancer, penile cancer, parathyroid cancer, thyroid cancer, pheochromocytoma and paraganglioma, thymoma and thymic Carcinoma, leukemia, lymphoma, plasma cell neoplasms, myeloproliferative disorders, islet cell tumor, small intestine cancer, transitional cell cancer, pleuropulmonary blastoma, gestational trophoblastic cancer, esophageal cancer, central nervous system cancer, head and neck cancer, endocrine cancer, cardiovascular cancer, rhabdomyosarcoma, soft tissue carcinomas, carcinomas of bone, cartilage, fat, vascular, neural, and hematopoietic tissues and AIDS-related cancers.
42. The method of claim 41, wherein said cancer comprises breast cancer.
43. The method of claim 41, wherein said cancer comprises ovarian cancer.
44. A method of detecting or diagnosing cancer in a subject, comprising:
(a) measuring one or more histone demethylases in a sample from the subject; and (b) comparing the amount to a reference value, wherein an increase or decrease in the amount of the one or more histone demethylases relative to the reference value indicates that the subject has cancer.
45. The method of claim 44, wherein the sample is whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsies, lymphatic fluid, ascites fluid, interstitial fluid, bone marrow, cerebrospinal fluid (CSF), seminal fluid, saliva, mucous, sputum, sweat, or urine.
46. The method of claim 44, wherein the subject comprises one who has been previously diagnosed as having cancer, one who has not been previously diagnosed as having cancer, or one who is asymptomatic for cancer.
47. The method of claim 44, wherein the measuring comprises detecting the presence or absence of the one or more histone demethylases, quantifying the amount of the one or more histone demethylases, and qualifying the type of the one or more histone demethylases.
48. The method of claim 44, wherein the reference value comprises an index value, a value derived from one or more cancer risk prediction algorithms, a value derived from a subject not suffering from cancer, or a value derived from a subject diagnosed with cancer.
49. The method of claim 44, wherein the one or more histone demethylases comprises LSD1 and an increase in LSD1 relative to the reference value indicates that the subject has cancer.
50. The method of claim 44, wherein the one or more histone demethylases are measured by PCR.
51. The method of claim 44, wherein the one or more histone demethylases are measured by immunoassay.
52. The method of claim 44, wherein the cancer is characterized by the presence of pluripotent and/or multipotent cells.
53. The method of claim 44, further comprising measuring the level of at least one plunpotent or multipotent stem cell protein marker.
54. The method of claim 53, wherein said at least one plunpotent stem cell protein marker is selected from the group consisting of Oct4, Sox2, Lin28, Nanog, Klf4, and Sall4, Lin28B, cMyc, nMyc, LMyc, Wnt3a, miR-291-3p, miR-294, miR-295, miR-290/371 cluster, miR-302 cluster, miR-363 cluster, miR-520 cluster, miR-92b, miR148/152, miR-124, miR-615, miR-708, miR-9, Klfl, Klf2, Klf5, Esrrb, Esrrg, Soxl , Sox3, Soxl5, Soxl8, Smad7, Nr5al, Nr5a2, Wnt/beta- catenin, Met, Notch, Hedgehog, Sonic Hedgehog, CD133, CD44, CD44+/CD247ESA\
CD347CD38", CD90, CD34+/CD387CD19+, CD34/CD38+/CD123+, CD117, CD20, integrinalpha2betahigh, EpCam, miR-137, miR-301, miR-32, miR-22, miR-135b, miR-204, miR205, miR-lOa, miR196b, miR-448, nuR-7-1, miR-128a, miR-196a-l, miR-361, Rexl, CRIPTO/TDGF, Cx43, IGF1, SSEA3, SSEA4, TRA-1-60, TRA-1-81 , ZFP42, FOXD3, TERT, Musashi-1, BMI-1, nestin, Ink4a ARF, Pten , ALDHhigh, ABCG2, CXCR4, and MITF.
55. The method of claim 44, wherein the cancer is selected from the group consisting of:
embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian cancer, endometrial cancer, cervical cancer, retinoblastoma, kidney, cancer, liver cancer, gastric cancer, brain cancer,
medulloblastoma, medulloepithelioma, glioma, glioblastoma, lung cancer, bronchial cancer, mesothelioma, skin cancer, colon and rectal cancer, bladder cancer, pancreatic cancer, lip and oral cancer, laryngeal and pharyngeal cancer, melanoma, pituitary cancer, penile cancer, parathyroid cancer, thyroid cancer, pheochromocytoma and paraganglioma, thymoma and thymic Carcinoma, leukemia, lymphoma, plasma cell neoplasms, myeloproliferative disorders, islet cell tumor, small intestine cancer, transitional cell cancer, pleuropulmonary blastoma, gestational trophoblastic cancer, esophageal cancer, central nervous system cancer, head and neck cancer, endocrine cancer, cardiovascular cancer, rhabdomyosarcoma, soft tissue carcinomas, carcinomas of bone, cartilage, fat, vascular, neural, and hematopoietic tissues and ADDS-related cancers.
56. The method of claim 55, wherein said cancer is breast cancer.
57. The method of claim 55, wherein said cancer is ovarian cancer.
58. A method for monitoring the progression of cancer in a subject, comprising:
(a) measuring one or more histone demethylases in a first sample from the subject at a first period of time;
(b) measuring one or more histone demethylases in a second sample from the subject at a second period of time; and
(c) comparing the amounts of the one or more histone demethylases detected in step (a) to the amount detected in step (b), or to a reference value, wherein an increase in one or more histone demethylases from the measurement in step (a) to the measurement in step (b) and/or relative to the reference value, indicates increased progression of cancer and, wherein a decrease in one or more histone demethylases from the measurement in step (a) to the measurement in step (b) and/or relative to the reference value, indicates regression of cancer.
59. The method of claim 58, wherein the monitoring comprises evaluating changes in the risk of developing cancer in the subject.
60. The method of claim 58, wherein the subject comprises one who has previously been treated for cancer, one who has not been previously treated for cancer, or one who has not been previously diagnosed with cancer.
61. The method of claim 58, wherein the sample is whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsies, lymphatic fluid, ascites fluid, interstitial fluid, bone marrow, cerebrospinal fluid (CSF), seminal fluid, saliva, mucous, sputum, sweat, or urine.
62. The method of claim 58, wherein the first sample is taken from the subject prior to being treated for cancer.
63. The method of claim 58, wherein the second sample is taken from the subject after being treated for cancer.
64. The method of claim 58, wherein the monitoring further comprises selecting a treatment regimen for the subject and/or monitoring the effectiveness of a treatment regimen for cancer.
65. The method of claim 64, wherein the treatment for cancer comprises surgical intervention, administration of anticancer agents, surgical intervention following or preceded by
administration of anticancer agents, or taking no further action.
66. The method of claim 58, wherein the reference value comprises an index value, a value derived from one or more cancer risk prediction algorithms, a value derived from a subject not having cancer, or a value derived from a subject diagnosed with cancer.
67. The method of claim 58, wherein the measuring comprises detecting the presence or absence of the one or more histone demethylases, quantifying the amount of the one or more histone demethylases, and qualifying the type of the one or more histone demethylases.
68. The method of claim 58, wherein the one or more histone demethylases comprises LSD1.
69. The method of claim 58, wherein the one or more histone demethylases are measured by PCR.
70. The method of claim 58, wherein the one or more histone demethylases are measured by immunoassay.
71. The method of claim 58, wherein the cancer is characterized by the presence of pluripotent and/or multipotent cells.
72. The method of claim 58, wherein said cancer is selected from the group consisting of: embryonic carcinoma, teratoma, seminoma, germ cell tumors, prostate cancer, breast cancer, neuroblastoma, choriocarcinoma, yolk sac tumors, ovarian cancer, endometrial cancer, cervical cancer, retinoblastoma, kidney cancer, liver cancer, gastric cancer, brain cancer,
medulloblastoma, medulloepithelioma, glioma, glioblastoma, lung cancer, bronchial cancer, mesothelioma, skin cancer, colon and rectal cancer, bladder cancer, pancreatic cancer, lip and oral cancer, laryngeal and pharyngeal cancer, melanoma, pituitary cancer, penile cancer, parathyroid cancer, thyroid cancer, pheochromocytoma and paraganglioma, thymoma and thymic Carcinoma, leukemia, lymphoma, plasma cell neoplasms, myeloproliferative disorders, islet cell tumor, small intestine cancer, transitional cell cancer, pleuropulmonary blastoma, gestational trophoblastic cancer, esophageal cancer, central nervous system cancer, head and neck cancer, endocrine cancer, cardiovascular cancer, rhabdomyosarcoma, soft tissue carcinomas, carcinomas of bone, cartilage, fat, vascular, neural, and hematopoietic tissues and AIDS-related cancers.
73. The method of claim 58, wherein said cancer is breast cancer.
74. The method of claim 58, wherein said cancer is ovarian cancer.
75. A kit comprising reagents that detect one or more histone demethylases and optionally instructions for using the reagents in the method of claim 44 or claim 58.
76. The kit of claim 75, wherein the detection reagents further comprise one or more antibodies or fragments thereof, one or more aptamers, one or more oligonucleotides, or combinations thereof.
77. A method for selecting a subject for treatment with a compound according to claim 1 comprising:
(a) measuring one or more histone demethylases in said subject; and
(b) comparing the level of said one or more histone demethylases detected in step (a) to a reference value; wherein when the level of one or more histone demethylases in said subject is greater than the reference value, the subject is selected for treatment with the compound according to claim 1.
78. The method of claim 77, wherein said one or more histone demethylases comprises LSD1.
79. The method of claim 77, further comprising measuring the level of at least one pluripotent stem cell protein marker and comparing the measured level of at least one pluripotent stem cell protein marker to a reference value for each marker, wherein when the level of at least one pluriponent stem cell protein marker is greater than its reference value, treatment with the compound according to claim 1 is indicated.
80. The method of claim 79, wherein said at least one pluripotent stem cell protein marker is selected from the group consisting of Oct4, Sox2, Lin28, Nanog, Klf4, and Sall4, Lin28B, cMyc, nMyc, LMyc, Wnt3a, miR-291-3p, miR-294, miR-295, miR-290/371 cluster, miR-302 cluster, miR-363 cluster, miR-520 cluster, miR-92b, miR148/152, miR-124, miR-615, miR-708, miR-9, Κ1Π, Klf2, Klf5, Esrrb, Esrrg, Soxl, Sox3, Soxl5, Soxl8, Smad7, Nr5al, Nr5a2, Wnt/beta- catenin, Met, Notch, Hedgehog, Sonic Hedgehog, CD133, CD44, CD44+/CD247ESA+, CD34VCD38", CD90, CD347CD387CD19\ CD34/CD38+/CD123+, CD117, CD20, integrinalpha2betahigh, EpCam, miR-137, miR-301, miR-32, miR-22, miR-135b, miR-204, miR205, miR-lOa, miR196b, miR-448, miR-7-1, miR-128a, miR-196a-l, miR-361, Rexl, CRIPTO/TDGF, Cx43, IGFl, SSEA3, SSEA4, TRA-1-60, TRA-1-81 , ZFP42, FOXD3, TERT, Musashi-1, BMI-1, nestin, Ink4a ARF, Pten", ALDH^8*1, ABCG2, CXCR4, and MITF.
81. A compound of formula I:
Figure imgf000129_0001
a pharmaceutically acceptable salt thereof, wherein:
Ri is an optionally substituted heterocyclic group; R and R' are independently selected from the group consisting of: H, CH3, ΝΟ2,
S02N(CH3)2, S02N((CH3)S02), COOH, COOCH3, CO(N(CH3)), alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl, heterocycloalkyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyi, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, trifluoromethyl, pentafluoroethyl, halogen, cyano, thio, amido, ether, ester, hydroxyl, hydroxyalkyl, saturated or unsaturated fatty acids, azido, phosphonamido, sulfonamido, lactam, phosphate, phosphonato, phosphinato, amino, acylamino, amidino, imino, guanidino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, cyano, and azido;
m and n are independently integers from 1 to 4;
T, U, V, and Z are indendently selected from CH, N, and CR;
L is selected from NH, CH2, O, S, and S02;
X is selected from N or CH;
Y and Y' is selected from O and S; and
R3 is an optionally substituted aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group.
82. A compound of for
Figure imgf000130_0001
n or a pharmaceutically acceptable salt thereof, wherein: Ri is an optionally substituted heterocyclic group; R2, R4, R5, R6, and R7 are selected from the group consisting of: H, CH3, N02) S02N(CH3)2, S02N((CH3)S02), COOH, COOCH3, CO(N(CH3)), alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl, heterocycloalkyl, alkoxy, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyi, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, trifluoromethyl, pentafluoroethyl, halogen, cyano, thio, amido, ether, ester, hydroxyl, hydroxyalkyl, saturated or unsaturated fatty acids, azido, phosphonamido, sulfonamido, lactam, phosphate, phosphonato, phosphinato, amino, acylamino, amidino, imino, guanidino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, cyano, and azido; X is selected from N or CH, and R3 is an optionally substituted aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group.
A compound of formula III:
Figure imgf000131_0001
or a pharmaceutically acceptable salt thereof, wherein: Ri is an optionally substituted heterocyclic group; R2 is selected from the group consisting of: H, CH3, N02, S02N(CH3)2, S02N((CH3)S02), COOH, COOCH3, CO(N(CH3)), alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl, heterocycloalkyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, trifluoromethyl, pentafluoroethyl, halogen, cyano, thio, amido, ether, ester, hydroxyl, hydroxyalkyl, saturated or unsaturated fatty acids, azido, phosphonamido, sulfonamido, lactam, phosphate, phosphonato, phosphinato, amino, acylamino, amidino, imino, guanidino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, cyano, and azido; and R3 is an optionally substituted aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group.
A compound of formula IV:
Figure imgf000132_0001
or pharmaceutically acceptable salt thereof;
wherein X is a functional group selected from the group consisting of CH2, S, NH, and S02;
V is O or S;
J, K, L, M, Y, Z and Z' are independently N, CR', or CH;
Ri comprises a functional group selected from the group consisting of hydrogen, a carboxamide (aminocarbonyl), a carboxamidine (carboximidamide), acyl, an alkylsulfonyl, an arylsulfonyl, guanidine, and an aminosulfonyl, any of which may be optionally substituted;
R, R', and R" are independently selected from the group consisting of: H, CH3, N02, S02N(CH3)2, S02N((CH3)S02), COOH, COOCH3, CO(N(CH3)), alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, heteroaryl, heterocycloalkyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, trifluoromethyl, pentafluoroethyl, halogen, cyano, thio, amido, ether, ester, hydroxyl, hydroxyalkyl, saturated or unsaturated fatty acids, azido, phosphonamido, sulfonamido, lactam, phosphate, phosphonato, phosphinato, amino, acylamino, amidino, imino, guanidino, sulfhydryl, alkylfhio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamido, nitro, cyano, and azido;
m, n, and p are independently integers from 1 to 4;
R3 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, and
heterocycloalkyl group, any of which is optionally substituted.
PCT/US2011/061954 2010-11-23 2011-11-22 Histone demethylase inhibitors and uses thereof for treatment o f cancer WO2012071469A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US41656610P 2010-11-23 2010-11-23
US61/416,566 2010-11-23

Publications (2)

Publication Number Publication Date
WO2012071469A2 true WO2012071469A2 (en) 2012-05-31
WO2012071469A3 WO2012071469A3 (en) 2012-07-26

Family

ID=46146406

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/061954 WO2012071469A2 (en) 2010-11-23 2011-11-22 Histone demethylase inhibitors and uses thereof for treatment o f cancer

Country Status (1)

Country Link
WO (1) WO2012071469A2 (en)

Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013127011A1 (en) 2012-02-27 2013-09-06 British Columbia Cancer Agency Branch Reprogramming effector protein interactions to correct epigenetic defects in cancer
CN103898206A (en) * 2013-05-13 2014-07-02 北京希普生国际生物医学研究院 Gene detection kit for prognostic evaluation of lung cancer, as well as preparation method and applications thereof
WO2014131777A1 (en) * 2013-02-27 2014-09-04 Epitherapeutics Aps Inhibitors of histone demethylases
CN104262246A (en) * 2014-08-29 2015-01-07 西安交通大学 5-phenylnicotinamide Bcr-Abl inhibitors as well as preparation method and application thereof
CN104262238A (en) * 2014-08-29 2015-01-07 西安交通大学 Aromatic heterocyclic biphenyl Bcr-Abl inhibitors as well as preparation method and application thereof
WO2016112284A1 (en) * 2015-01-09 2016-07-14 Genentech, Inc. (piperidin-3-yl)(naphthalen-2-yl)methanone derivatives and related compounds as inhibitors of the histone demethylase kdm2b for the treatment of cancer
US9527835B2 (en) 2014-02-13 2016-12-27 Incyte Corporation Cyclopropylamines as LSD1 inhibitors
CN104262246B (en) * 2014-08-29 2017-01-04 西安交通大学 A kind of 5-phenyl nicotinamide Bcr-Abl inhibitor and its preparation method and application
US9670210B2 (en) 2014-02-13 2017-06-06 Incyte Corporation Cyclopropylamines as LSD1 inhibitors
WO2017099829A1 (en) 2015-12-11 2017-06-15 The General Hospital Corporation Compositions and methods for treating drug-tolerant glioblastoma
US9695167B2 (en) 2014-07-10 2017-07-04 Incyte Corporation Substituted triazolo[1,5-a]pyridines and triazolo[1,5-a]pyrazines as LSD1 inhibitors
US9695180B2 (en) 2014-07-10 2017-07-04 Incyte Corporation Substituted imidazo[1,2-a]pyrazines as LSD1 inhibitors
US9695168B2 (en) 2014-07-10 2017-07-04 Incyte Corporation Substituted imidazo[1,5-α]pyridines and imidazo[1,5-α]pyrazines as LSD1 inhibitors
WO2017114497A1 (en) 2015-12-30 2017-07-06 Novartis Ag Immune effector cell therapies with enhanced efficacy
US9758523B2 (en) 2014-07-10 2017-09-12 Incyte Corporation Triazolopyridines and triazolopyrazines as LSD1 inhibitors
CN107174584A (en) * 2016-03-12 2017-09-19 福建金乐医药科技有限公司 Application of the compound containing piperazine structure in LSD1 inhibitor is prepared
WO2017158136A1 (en) 2016-03-16 2017-09-21 Oryzon Genomics, S.A. Methods to determine kdm1a target engagement and chemoprobes useful therefor
US9790195B2 (en) 2013-08-06 2017-10-17 Imago Biosciences, Inc. KDM1A inhibitors for the treatment of disease
US9802941B2 (en) 2014-08-27 2017-10-31 Gilead Sciences, Inc. Compounds and methods for inhibiting histone demethylases
US9944647B2 (en) 2015-04-03 2018-04-17 Incyte Corporation Heterocyclic compounds as LSD1 inhibitors
WO2018083189A1 (en) * 2016-11-03 2018-05-11 Oryzon Genomics, S.A. Biomarkers for determining responsiveness to lsd1 inhibitors
US9981922B2 (en) 2015-02-12 2018-05-29 Imago Biosciences, Inc. KDM1A inhibitors for the treatment of disease
US9994546B2 (en) 2014-02-13 2018-06-12 Incyte Corporation Cyclopropylamines as LSD1 inhibitors
WO2018106984A1 (en) 2016-12-09 2018-06-14 Constellation Pharmaceuticals, Inc. Markers for personalized cancer treatment with lsd1 inhibitors
CN108957004A (en) * 2018-07-09 2018-12-07 东南大学 Detect application of the reagent of H3K9me2 and H3K36me3 expression quantity in preparation gastric cancer prognosis evaluation reagent kit
US10166221B2 (en) 2016-04-22 2019-01-01 Incyte Corporation Formulations of an LSD1 inhibitor
US10189787B2 (en) 2012-10-02 2019-01-29 Gilead Sciences, Inc. Inhibitors of histone demethylases
WO2019025588A1 (en) 2017-08-03 2019-02-07 Oryzon Genomics, S.A. Methods of treating behavior alterations
US10300051B2 (en) 2014-02-13 2019-05-28 Incyte Corporation Cyclopropylamines as LSD1 inhibitors
US10329255B2 (en) 2015-08-12 2019-06-25 Incyte Corporation Salts of an LSD1 inhibitor
CN110257320A (en) * 2019-06-11 2019-09-20 华夏源（上海）细胞基因工程股份有限公司 The induction of clinical grade iPS a kind of and screening technique
WO2020188089A1 (en) 2019-03-20 2020-09-24 Oryzon Genomics, S.A. Methods of treating attention deficit hyperactivity disorder using kdm1a inhibitors such as the compound vafidemstat
WO2020188090A1 (en) 2019-03-20 2020-09-24 Oryzon Genomics, S.A. Methods of treating borderline personality disorder
WO2021004610A1 (en) 2019-07-05 2021-01-14 Oryzon Genomics, S.A. Biomarkers and methods for personalized treatment of small cell lung cancer using kdm1a inhibitors
US10968200B2 (en) 2018-08-31 2021-04-06 Incyte Corporation Salts of an LSD1 inhibitor and processes for preparing the same
US11390590B2 (en) 2016-08-16 2022-07-19 Imago Biosciences, Inc. Methods and processes for the preparation of KDM1A inhibitors
US11578059B2 (en) 2018-05-11 2023-02-14 Imago Biosciences. Inc. KDM1A inhibitors for the treatment of disease

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5670505A (en) * 1993-11-29 1997-09-23 Fujisawa Pharmaceutical Co., Ltd. Piperazine derivatives
US20060292607A1 (en) * 2005-06-08 2006-12-28 Vanderbilt University Analysis of tissue samples surrounding malignancies
US20070208082A1 (en) * 2005-08-10 2007-09-06 John Hopkins University Polyamines useful as anti-parasitic and anti-cancer therapeutics and as lysine-specific demethylase inhibitors
US20070274912A1 (en) * 2003-09-16 2007-11-29 The Rocefeller University Histone Modifications as Binary Switches Controlling Gene Expression
US20090170796A1 (en) * 2004-12-16 2009-07-02 Yang Shi Histone Demethylation Mediated By The Nuclear Amine Oxidase Homolog LSD1

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5670505A (en) * 1993-11-29 1997-09-23 Fujisawa Pharmaceutical Co., Ltd. Piperazine derivatives
US20070274912A1 (en) * 2003-09-16 2007-11-29 The Rocefeller University Histone Modifications as Binary Switches Controlling Gene Expression
US20090170796A1 (en) * 2004-12-16 2009-07-02 Yang Shi Histone Demethylation Mediated By The Nuclear Amine Oxidase Homolog LSD1
US20060292607A1 (en) * 2005-06-08 2006-12-28 Vanderbilt University Analysis of tissue samples surrounding malignancies
US20070208082A1 (en) * 2005-08-10 2007-09-06 John Hopkins University Polyamines useful as anti-parasitic and anti-cancer therapeutics and as lysine-specific demethylase inhibitors

Cited By (75)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013127011A1 (en) 2012-02-27 2013-09-06 British Columbia Cancer Agency Branch Reprogramming effector protein interactions to correct epigenetic defects in cancer
US9552457B2 (en) 2012-02-27 2017-01-24 British Columbia Cancer Agency Branch Reprogramming effector protein interactions to correct epigenetic defects in cancer
PH12017501306A1 (en) * 2012-10-02 2019-02-27 Epitherapeutics Aps Inhibitors of histone demethylases
US10221139B2 (en) 2012-10-02 2019-03-05 Gilead Sciences, Inc. Inhibitors of histone demethylases
US10189787B2 (en) 2012-10-02 2019-01-29 Gilead Sciences, Inc. Inhibitors of histone demethylases
CN105263906A (en) * 2013-02-27 2016-01-20 Epi医疗公司 Inhibitors of histone demethylases
US9221801B2 (en) 2013-02-27 2015-12-29 Epitherapeutics Aps Inhibitors of histone demethylases
WO2014131777A1 (en) * 2013-02-27 2014-09-04 Epitherapeutics Aps Inhibitors of histone demethylases
US9650339B2 (en) 2013-02-27 2017-05-16 Gilead Sciences, Inc. Inhibitors of histone demethylases
EA030451B1 (en) * 2013-02-27 2018-08-31 Эпитерапьютикс Апс Inhibitors of histone demethylases
AU2014222756B2 (en) * 2013-02-27 2018-07-26 Gilead Sciences, Inc. Inhibitors of histone demethylases
CN103898206A (en) * 2013-05-13 2014-07-02 北京希普生国际生物医学研究院 Gene detection kit for prognostic evaluation of lung cancer, as well as preparation method and applications thereof
US10370346B2 (en) 2013-08-06 2019-08-06 Imago Biosciences, Inc. KDM1A inhibitors for the treatment of disease
US11655226B2 (en) 2013-08-06 2023-05-23 Imago Biosciences, Inc. KDM1A inhibitors for the treatment of disease
US10882835B2 (en) 2013-08-06 2021-01-05 Imago Biosciences, Inc. KDM1A inhibitors for the treatment of disease
US9790195B2 (en) 2013-08-06 2017-10-17 Imago Biosciences, Inc. KDM1A inhibitors for the treatment of disease
US10174030B2 (en) 2014-02-13 2019-01-08 Incyte Corporation Cyclopropylamines as LSD1 inhibitors
US10717737B2 (en) 2014-02-13 2020-07-21 Incyte Corporation Cyclopropylamines as LSD1 inhibitors
US9527835B2 (en) 2014-02-13 2016-12-27 Incyte Corporation Cyclopropylamines as LSD1 inhibitors
US9670210B2 (en) 2014-02-13 2017-06-06 Incyte Corporation Cyclopropylamines as LSD1 inhibitors
US11155532B2 (en) 2014-02-13 2021-10-26 Incyte Corporation Cyclopropylamines as LSD1 inhibitors
US11247992B2 (en) 2014-02-13 2022-02-15 Incyte Corporation Cyclopropylamines as LSD1 inhibitors
US9994546B2 (en) 2014-02-13 2018-06-12 Incyte Corporation Cyclopropylamines as LSD1 inhibitors
US10300051B2 (en) 2014-02-13 2019-05-28 Incyte Corporation Cyclopropylamines as LSD1 inhibitors
US10513493B2 (en) 2014-02-13 2019-12-24 Incyte Corporation Cyclopropylamines as LSD1 inhibitors
US10676457B2 (en) 2014-02-13 2020-06-09 Incyte Corporation Cyclopropylamines as LSD1 inhibitors
US9695167B2 (en) 2014-07-10 2017-07-04 Incyte Corporation Substituted triazolo[1,5-a]pyridines and triazolo[1,5-a]pyrazines as LSD1 inhibitors
US10640503B2 (en) 2014-07-10 2020-05-05 Incyte Corporation Imidazopyridines and imidazopyrazines as LSD1 inhibitors
US10556908B2 (en) 2014-07-10 2020-02-11 Incyte Corporation Substituted imidazo[1,2-a]pyrazines as LSD1 inhibitors
US9695168B2 (en) 2014-07-10 2017-07-04 Incyte Corporation Substituted imidazo[1,5-α]pyridines and imidazo[1,5-α]pyrazines as LSD1 inhibitors
US10047086B2 (en) 2014-07-10 2018-08-14 Incyte Corporation Imidazopyridines and imidazopyrazines as LSD1 inhibitors
US9758523B2 (en) 2014-07-10 2017-09-12 Incyte Corporation Triazolopyridines and triazolopyrazines as LSD1 inhibitors
US10112950B2 (en) 2014-07-10 2018-10-30 Incyte Corporation Substituted imidazo[1,2-a]pyrazines as LSD1 inhibitors
US10125133B2 (en) 2014-07-10 2018-11-13 Incyte Corporation Substituted [1,2,4]triazolo[1,5-a]pyridines and substituted [1,2,4]triazolo[1,5-a]pyrazines as LSD1 inhibitors
US10138249B2 (en) 2014-07-10 2018-11-27 Incyte Corporation Triazolopyridines and triazolopyrazines as LSD1 inhibitors
US10968221B2 (en) 2014-07-10 2021-04-06 Incyte Corporation Substituted [1,2,4]triazolo[1,5-a]pyrazines as LSD1 inhibitors
US9695180B2 (en) 2014-07-10 2017-07-04 Incyte Corporation Substituted imidazo[1,2-a]pyrazines as LSD1 inhibitors
US9802941B2 (en) 2014-08-27 2017-10-31 Gilead Sciences, Inc. Compounds and methods for inhibiting histone demethylases
CN104262246B (en) * 2014-08-29 2017-01-04 西安交通大学 A kind of 5-phenyl nicotinamide Bcr-Abl inhibitor and its preparation method and application
CN104262238A (en) * 2014-08-29 2015-01-07 西安交通大学 Aromatic heterocyclic biphenyl Bcr-Abl inhibitors as well as preparation method and application thereof
CN104262238B (en) * 2014-08-29 2016-08-03 西安交通大学 A kind of heteroaromatic biphenyl class Bcr-Abl inhibitor and its preparation method and application
CN104262246A (en) * 2014-08-29 2015-01-07 西安交通大学 5-phenylnicotinamide Bcr-Abl inhibitors as well as preparation method and application thereof
WO2016112284A1 (en) * 2015-01-09 2016-07-14 Genentech, Inc. (piperidin-3-yl)(naphthalen-2-yl)methanone derivatives and related compounds as inhibitors of the histone demethylase kdm2b for the treatment of cancer
US10280149B2 (en) 2015-01-09 2019-05-07 Genentech, Inc. Therapeutic compounds and uses thereof
US11773068B2 (en) 2015-02-12 2023-10-03 Imago Biosciences, Inc. KDM1A inhibitors for the treatment of disease
US9981922B2 (en) 2015-02-12 2018-05-29 Imago Biosciences, Inc. KDM1A inhibitors for the treatment of disease
US11230534B2 (en) 2015-02-12 2022-01-25 Imago Biosciences, Inc. KDM1A inhibitors for the treatment of disease
US10519118B2 (en) 2015-02-12 2019-12-31 Imago Biosciences, Inc. KDM1A inhibitors for the treatment of disease
US10800779B2 (en) 2015-04-03 2020-10-13 Incyte Corporation Heterocyclic compounds as LSD1 inhibitors
US9944647B2 (en) 2015-04-03 2018-04-17 Incyte Corporation Heterocyclic compounds as LSD1 inhibitors
US11401272B2 (en) 2015-04-03 2022-08-02 Incyte Corporation Heterocyclic compounds as LSD1 inhibitors
US10723700B2 (en) 2015-08-12 2020-07-28 Incyte Corporation Salts of an LSD1 inhibitor
US10329255B2 (en) 2015-08-12 2019-06-25 Incyte Corporation Salts of an LSD1 inhibitor
US11498900B2 (en) 2015-08-12 2022-11-15 Incyte Corporation Salts of an LSD1 inhibitor
EP3386594A4 (en) * 2015-12-11 2019-08-21 The General Hospital Corporation Compositions and methods for treating drug-tolerant glioblastoma
WO2017099829A1 (en) 2015-12-11 2017-06-15 The General Hospital Corporation Compositions and methods for treating drug-tolerant glioblastoma
EP4219689A2 (en) 2015-12-30 2023-08-02 Novartis AG Immune effector cell therapies with enhanced efficacy
WO2017114497A1 (en) 2015-12-30 2017-07-06 Novartis Ag Immune effector cell therapies with enhanced efficacy
CN107174584A (en) * 2016-03-12 2017-09-19 福建金乐医药科技有限公司 Application of the compound containing piperazine structure in LSD1 inhibitor is prepared
WO2017158136A1 (en) 2016-03-16 2017-09-21 Oryzon Genomics, S.A. Methods to determine kdm1a target engagement and chemoprobes useful therefor
US10166221B2 (en) 2016-04-22 2019-01-01 Incyte Corporation Formulations of an LSD1 inhibitor
US11390590B2 (en) 2016-08-16 2022-07-19 Imago Biosciences, Inc. Methods and processes for the preparation of KDM1A inhibitors
WO2018083189A1 (en) * 2016-11-03 2018-05-11 Oryzon Genomics, S.A. Biomarkers for determining responsiveness to lsd1 inhibitors
WO2018106984A1 (en) 2016-12-09 2018-06-14 Constellation Pharmaceuticals, Inc. Markers for personalized cancer treatment with lsd1 inhibitors
WO2019025588A1 (en) 2017-08-03 2019-02-07 Oryzon Genomics, S.A. Methods of treating behavior alterations
US11578059B2 (en) 2018-05-11 2023-02-14 Imago Biosciences. Inc. KDM1A inhibitors for the treatment of disease
US11932629B2 (en) 2018-05-11 2024-03-19 Imago Biosciences, Inc. KDM1A inhibitors for the treatment of disease
CN108957004B (en) * 2018-07-09 2021-10-19 东南大学 Application of reagent for detecting expression levels of H3K9me2 and H3K36me3 in preparation of gastric cancer prognosis evaluation kit
CN108957004A (en) * 2018-07-09 2018-12-07 东南大学 Detect application of the reagent of H3K9me2 and H3K36me3 expression quantity in preparation gastric cancer prognosis evaluation reagent kit
US10968200B2 (en) 2018-08-31 2021-04-06 Incyte Corporation Salts of an LSD1 inhibitor and processes for preparing the same
US11512064B2 (en) 2018-08-31 2022-11-29 Incyte Corporation Salts of an LSD1 inhibitor and processes for preparing the same
WO2020188090A1 (en) 2019-03-20 2020-09-24 Oryzon Genomics, S.A. Methods of treating borderline personality disorder
WO2020188089A1 (en) 2019-03-20 2020-09-24 Oryzon Genomics, S.A. Methods of treating attention deficit hyperactivity disorder using kdm1a inhibitors such as the compound vafidemstat
CN110257320A (en) * 2019-06-11 2019-09-20 华夏源（上海）细胞基因工程股份有限公司 The induction of clinical grade iPS a kind of and screening technique
WO2021004610A1 (en) 2019-07-05 2021-01-14 Oryzon Genomics, S.A. Biomarkers and methods for personalized treatment of small cell lung cancer using kdm1a inhibitors

Also Published As

Publication number Publication date
WO2012071469A3 (en) 2012-07-26

Similar Documents

Publication Publication Date Title
WO2012071469A2 (en) Histone demethylase inhibitors and uses thereof for treatment o f cancer
Wang et al. BRAF‑activated long non‑coding RNA contributes to cell proliferation and activates autophagy in papillary thyroid carcinoma
Jiao et al. Elevated expression level of long noncoding RNA MALAT-1 facilitates cell growth, migration and invasion in pancreatic cancer
Yi et al. High expression of fibronectin is associated with poor prognosis, cell proliferation and malignancy via the NF-κB/p53-apoptosis signaling pathway in colorectal cancer
Wen et al. Evodiamine, a novel inhibitor of the Wnt pathway, inhibits the self-renewal of gastric cancer stem cells
Xie et al. Targeting HPV16 E6-p300 interaction reactivates p53 and inhibits the tumorigenicity of HPV-positive head and neck squamous cell carcinoma
Lai et al. Long noncoding RNA MIAT promotes non-small cell lung cancer proliferation and metastasis through MMP9 activation
Cheng et al. Expression of HAUSP in gliomas correlates with disease progression and survival of patients
Murphy et al. SIX2 and CITED1, markers of nephronic progenitor self-renewal, remain active in primitive elements of Wilms' tumor
Liang et al. Nicotinamide N-methyltransferase promotes epithelial-mesenchymal transition in gastric cancer cells by activating transforming growth factor-β1 expression
Sun et al. Overexpression of pyruvate dehydrogenase E1α subunit inhibits Warburg effect and induces cell apoptosis through mitochondria-mediated pathway in hepatocellular carcinoma
Min et al. The effects of fucodian on senescence are controlled by the p16INK4a-pRb and p14Arf-p53 pathways in hepatocellular carcinoma and hepatic cell lines
US20140179618A1 (en) Novel cancer therapies and methods
Hao et al. UBE2T promotes proliferation and regulates PI3K/Akt signaling in renal cell carcinoma
Jing et al. Decreased expression of TROAP suppresses cellular proliferation, migration and invasion in gastric cancer
Zeng et al. Knockdown of NUPR1 inhibits the growth of U266 and RPMI8226 multiple myeloma cell lines via activating PTEN and caspase activation‑dependent apoptosis
Yu et al. Role of HDAC1 in the progression of gastric cancer and the correlation with lncRNAs
Yang et al. Knockdown of RHOC by shRNA suppresses invasion and migration of cholangiocellular carcinoma cells via inhibition of MMP2, MMP3, MMP9 and epithelial-mesenchymal transition
Wang et al. Oct4 promotes cancer cell proliferation and migration and leads to poor prognosis associated with the survivin/STAT3 pathway in hepatocellular carcinoma
Zeng et al. High expression of TRIM29 (ATDC) contributes to poor prognosis and tumor metastasis by inducing epithelial‑mesenchymal transition in osteosarcoma
Wu et al. Silencing of ASPP2 promotes the proliferation, migration and invasion of triple-negative breast cancer cells via the PI3K/AKT pathway
Li et al. MicroRNA‑103 modulates tumor progression by targeting KLF7 in non‑small cell lung cancer
Lian et al. A novel lncRNA, LL22NC03-N64E9. 1, represses KLF2 transcription through binding with EZH2 in colorectal cancer
Lu et al. CDK5 suppresses the metastasis of gastric cancer cells by interacting with and regulating PP2A
Furusawa et al. Identification of genes and genetic networks associated with BAG3‑dependent cell proliferation and cell survival in human cervical cancer HeLa cells

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11843024

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11843024

Country of ref document: EP

Kind code of ref document: A2