CA2928464A1 - Biomarker for melk activity and methods of using same - Google Patents

Abstract

The methods of the present invention, relate to the surprising determination that the level of phosphorylation of position 406 (e.g., a serine residue.) of human eukaryotic initiation factor 4B (cIF4B), or a corresponding phosphorylatafale amino acid of an ortholog thereof, serves as a bhiraarker for MELK enzymatic (e.g., kinase) and/or oncogenic activity. The methods of the present invention further ceiate to the surprising determination that the level of phosphorylation of position 3 (e.g., a threonine residue) and/or position 10 (e.g., a serine residue) and/or position 11 (e.g., a threonine residue) of human Historic M3, or a corresponding phosphorylatable amino acid of an ortholog thereof, also serves as a biomarker for MELK enzymatic (e.g., kinase) and/or oncogenic activity.

Description

BIOMARKER FOR MEEK _ACTIVITY AND METHODS OF USING SAME
Cross-Reference to Related Applications This application claims the benefit of U.S..Provisioual .ApplieltiedNot..61.1954,046õ.
filed on 17 March 2014, and 611902,877, filed. im12.November 2013; the 'entire contents of each of said applications .is incorporated herein in its entirety by this reference.
Backlyound of the Invention The protein kinate, maternal embryonic leueine zipper kinase (META), is known to be involved M regulating cell cycle progression, cellular proliferation, apoptosis, and mRNA splicing (Badouel et al. (2006) Cell Cycle 5:883-889 and Badouel et al.
(2010) Exp.
Cell Res. 316:2166-2173), MELK has also been :identified using gene expression profile analyses to be associated with a number of cancers, including breast, lung., bladder, lymphoma, and cervical cancer cells and mammary tumor formation in animal models .15 (Komatsu et al. (2013) Int .1 Oncoi. 42:478-506; Pickard et al (2009) .11reast Cancer Res.
1:R60; ilebbard et (2010) Cancer Res. 70:8863-8873; Lin et (2007) Breast Cancer Re. 9:R17; WO 2004/031413; WO 2007/7013665; and WO 2006/085684). Despite this association, however. functional analyses of MELK-modiated oncogenesis have not been performed to date and the mechanisms of MELK-mediated oncogenesis and, by extension, assays for determining agents useful in regulating such oncogenesis, are not known. This lack of understanding has prevented, the identification of biomarkers that reliably report MELK enzymatic and/or oncogenic activity. While certain MELK-targeting inhibitors of kinase activity are known (sec, for example, Chung et aL (2012) Ofleotarget 3:1629-1640), there is a clear need in the art to identify biomarkers f MELK-mcdiated ontogcnesis in order to provide rapid and effective means for evaluating MELK-targeted ant-caner therapies.
Summary of the Invention The present invention is based, at least in part, on the discovery that the level of phosphorylation of position 406 (e.g., a serine residue) of human .eukaryotic initiation factor 4B (e1E4B) is a reliable biomarker for maternal embryonic leucine zipper .kinase (MELK) activity suitable for use in measuring 'NI-ELK enzymatic activity for preclinical and clinical applications. Similarly, the present invention is based, at least hi part, on the discovery that the level of phosphorylation of position 3 (e.g., a threonine residue) and/or position 0 (e.g., a scrim residue) of human Histone I-13 and/or position 1 I (e.g., a threonine residue) is as a reliable biomarker for MELK activity suitable for use in measuring MELK, enzymatic activity or preclinical and clinical applications.
In one aspect, a method of identifying an agent which inhibits kinase or oncogenie activity of human maternal embryonic ICUCITIC zipper kinase (MELK) or an ortholog thereof, comprising a) contacting a sample comprising i) human MELK or an ortholog thereof and ii) human eukaryotie initiation factor 4B (eIF4B) or an ortholog thereof, with the agent; and b) determining the ability of the agent to inhibit Ser-406 phosphorylation of 0 human elF4B or a corresponding phosphorylatable amino acid in the ortholog of human eIF4B, wherein decreased phosphorylation identifies an agent which inhibits .kinase or oneogenic activity of human ME.I.,1( or the ortholog thereof, is provided. In one embodiment, the inhibition of said Ser-406 phosphorylation of human eIF4B or a correspondintt phosphorylatable amino acid in an ortholog of human el F4B is determined 15 by comparing the amount of Ser-406 phosphorylated human elF4B or a corresponding phosphorylatable amino acid in the ortholog of human elF4B, in the sample relative to a control In another embodiment, the control is the amount of Ser-406 phosphorylatcd human ell:4B or a corresponding phosphorylatable amino acid in the ortholog of human cIF4B in the sample relative to said amount in the absence of the agent or at an earlier 20 timepoint after contact of the sample with the agent. In still another embodiment, the inhibition of said Ser-406 phosphorylation of human eiF4B or a corresponding phosphorylatable amino acid in an ortholog of human eIF4B is determined by comparing the ratio of the amount of Ser-406 phosphorylated human eIF4B, or a corresponding phosphorylatable amino acid in the ortholog of human CIF4B, hi the sample relative to the 25 total amount of human efF4f3 or ortholog thereof, to a control. In yet another embodiment, the control is the ratio of the amount of Ser-406 phosphorylated human eIF4B
or a corresponding phosphotylatable amino acid in the ortholog of human eIF4B in the sample relative to said ratio in the absence of the agent or at an earlier timepoint after contact of the sample with the agent. In another embodiment, the method further comprises determining 30 the amount of a protein translated from an RNA with highly structured 5' VTR, optionally wherein the protein is selected from the group consisting of cellular myeloeytomatosis oncogene (e-Myc),X-Iinked inhibitor of apoptosis protein (XIAP), and.
ornithinc decathoxylase (PDC1). In still another embodiment, the method further comprises a step of determining whether the agent directly binds said human eIF4B or said ortholog thereof, or said human MELK or said ortholog thereof In yet another .embodiment, the sample is selected from the group consisting of in vitro, ex vivo, and in vivo SainpieS, in another embodiment, the sample comprises cells (e.g., cancer cells, such as a cancer selected from the group consisting of any cancer in which -MILK or ell:4B is amplified or ovcrexpressed, any cancer having an activating mutation of MELK or 01174B, and any cancer in which MELK or eIF4B is activated by other kinases). In still another embodiment, the cells are Obtained :from a subject. In yet another embodiment, the sample is selected from the group consisting of tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and. bone marrow. hi another embodiment, the amount of Scr-406 phosphorylated human elF4B or a corresponding phosphory.latable amino acid in the ortholog of human eIF4B is determined by an immunoassay using a reagent which specifically binds with Ser-406 phosphorylated human efF4B or corresponding phosphorylated ortholog of human ciF4B (e.g.., a radioimmunoassay, a Western 'blot assay, a proximity ligation assay, an immunafluoresence assay, an .enzyrne immunoassay, an itnmunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, a dot blot assay, or a slot blot assay). In still another embodiment, the enzyme immunoassay is a sandwich enzyme immmmassay using a capture antibody or fragment thereof which specifically binds with human eIF4B or corresponding ortholog of human eIF4B
and a detection antibody or fragment thereof which specifically binds with Ser-406 phosphotylated human eiF4B or a corresponding phosphoiyiated ortholog of human elf4B.
In yet another embodiment, said human elF4B or ortholog thereof, and/or said human.
MELK or ortholog thereof, comprises a nucleic acid sequence or amino acid sequence set forth in Table In another embodiment, the agent is a small molecule, or an antibody or antigen-binding, fragment thereof. in still another embodiment, the agent decreases the amount of Scr-406 phosphotylated human eIF4B or a corresponding phosphorylatable amino acid in the ortholog of human eiF4B by at least 50%.
In another aspect, a method for assessing the efficacy of an agent for inhibiting kinase activity of 'human .MELK or an orthoiog thereof in a subject, comprising a) detecting in a subject sample at a first point in time, the amount of Sr-4O6 phosphotylated human e1f4B or the amount of a human e1f4B ortholog phosphotylated at a corresponding amino acid of human eiF4B; h) repeating step a) during at one or more subsequent points in time after administration of the agent; and c) comparing the amount of phosphorylated human elF4B or ortholog thereof detected hi step a) with said amount detected in step b), wherein a higher amount of Ser-406 phosphorylated human elF4B or the amount of the human e1F413 ortholog phosphorylated at a corresponding amino acid of human eIF4B in the first point in time relative to at least one subsequent point in time, indicates that the agent inhibits kinase activity of human MELK or the ortholog thereof, is provided.
In one embodiment, the amount of Ser-406 phosphorytated human elY4B or a corresponding phosphorylatable amino acid in the orthotog of human .e.IF4B is detennined by an immunoassay using a reagent which specifically binds with Scr-406 pliosphorylated human ell4B or corresponding phosphorylated ortholog of human elF46 (ag, a radioimmunoassay, a Western blot assay, a.proximity ligation assay, an immunofluoresence assay, an enzyme immunoassay, an immtmoprecipitation assay, a chemilimitnescenec assay, an inunimohistochemical assay, a dot blot assay, or a slot blot assay).
in another embodiment, the enzyme immunoassay is a sandwich enzyme immunoassay using a capture antibody or fragment thereof which specifically binds with 'human eliF4B or corresponding ortholog of human elF4B and a detection antibody or fragment thereof which specifically binds with Ser-406 =phosphorylated human elf4B or a corresponding phosphotylated ortbolog of human elF4B. In still another embodiment, the sample is selected from the group consisting of ex vivo and in vivo samples. In yet another embodiment, the sample comprises cancer cells (e.g., cancer cells selected from the group consisting of any cancer in which MELK or eIF4B is amplified or overexpressed, any cancer having an activating mutation of .MELK or eiF4B, and any cancer in which .MELK oreiF4B is activated by other kinases). In another embodiment, the sample is selected from the group consisting of tissue, .whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, .urine, stool, and bone marrow. in still another embodiment, the sample in step a.) and/or step b) is a.
portion of a single sample obtained from the subject. In yet another embodiment, the sample in step a) and/or step b) is a portion of pooled samples obtained from the subject.. In another embodiment, the subject has undergone treatment for cancer, has completed treatment thr cancer, and/or is in remission from cancer between the first point in time and the subsequent point in time. in still another embodiment, said IlUrnall eiF4B
or ortholog thereof, andior said hunian MIlK or ortholog thereof, comprises a nucleic acid sequence or amino acid sequence set forth in Table 1. In yet another embodiment, the agent is a.
small molecule, or an antibody or antigen-binding fragment diet-cot.. In another embodiment, the agent decreases the amount of Ser-406 phosphorylated human elF4B or a - -corresponding phosphorylatable amino acid in the ortholog of human elF4B by at least 50%, In still another aspect, a method of treating a subject afflicted with cancer comprising administering to the subject an agent that inhibits Ser-406 phosphorylation of human e1f4B or a corresponding phosphorylatable amino acid in an ortholog of human.
elf4B, thereby treating the subject afflicted with the cancer, is provided. In one embodiment, the agent is administered, in a pharmaceutically acceptable formulation. In another embodiment, the agent is a small molecule, or an antibody or antigen-binding fragment thereof. In still another embodiment, the agent directly binds said human .eIF4B
or the ortholog thereof, or said. human 'MILK or the ortholog thereof, in yet another embodiment, the cancer is selected from the group consisting of any cancer in which MELK or elf4B is amplified or overcxpressed, any cancer having an activating mutation of MELK or eIF4B, and any cancer in which MELK or ciF4B is activated by other kinases.
In another embodiment, said .human cIF4B or ortholog thereof, and/or said human MELK.
or ortholog thereof, comprises a nucleic acid sequence or amino acid sequence set forth in Table 1. In still another embodiment, the agent is a small molecule, or an antibody or antigen-binding fragment thereof in yet another embodiment, the agent decreases the amount of Ser-406 phosphorylated human elf4B or a corresponding phosphorylatable amino acid in the ortholog of human eIF4B by at least 50%. In another embodiment, the method further comprises administering one or more additional anti-cancer agents.
In yet another aspect, a method of determining the 1:Unction or activity of human MELK or an ortholog, comprising a) detecting in a sample the amount of Ser-406 phosphorylated human eIF4B or the amount of a human eIF4B ortholog phosphorylated at a. corresponding amino acid of human efF4B; b) repeating step a) in the same sample or a test sample at one or more subsequent points in time after manipulation of the sample and/or manipulation of the same sample or test sample; and c) comparing the amount of phosphorylated human elF4B or ortholog thereof detected in step a) with said amount detected in step b), wherein a modulated of Ser-406 phosphorylated human elf4B
or the amount of the human 0:F4B ortholog phosphorylated at a corresponding amino acid of human elF4B in the first point in time relative to a.t least one subsequent point in time and/or at least one subsequent manipulation of the same sample or test sample, indicates that the function or activity of human MEL or an ortholog thereof is modulated, is provided. In one embodiment, the amount of Ser-406 phosphorylated 'human eI.F413 or a corresponding phosphorylatable amino acid in the ortholog of human e1/413 is determined by an immunoassay using a reagent which specifically binds with Ser-406 phosphorylated human elF413 or corresponding phosphorylated ortholog of human CIF4B (e.g., a radioinununoassay, a Western blot assay, a proximity ligation assay, an inununoiltioresence assay, an enzyme immunoassay, an Unnumoprecipitation assay, a chemiluminescence assay, an immtmohistochemical assay, a dot blot assay, or a slot blot assay).
In another embodiment, the enzyme immunoassay is a sandwich enzyme immunoassay using a capture antibody or fra.gment thereof which specifically binds with human e.I.F4B or corresponding ortholog of human elf4B and a detection antibody or fragment thereof which specifically.
binds with Ser-406 phosphorylated human elFzlB or a corresponding phosphorylated ortholog of human e1F4B. In still another embodiment, the sample is selected from the group consisting of in vitro, ex vim, and in vivo samples. In yet another embodiment, the sample comprises cells or the method =uses a cell-based assay. In another embodiment, the cells are cancer cells selected from the group consisting of any cancer in which MELK or .15 elFzlB is amplified or overexpressed, any cancer having an activating mutation of MELK or elf4B, and any cancer in which MELK or elf4B is activated by other kinases. in still another embodiment, the sample is selected from the group consisting of tissue, whole blood, sertmi., plasma, buccal serape, saliva, cerebrospinal fluid, urine, stool, and bone marrow. in yet another embodiment, the same sample or test sample in step a) and/or step b) is a portion of a single sample obtained from a subject. In another embodiment, the same sample or test sample in step a) and/or step b) is a portion of pooled samples obtained from a subject. In still another embodiment, the subject has undergone treatment for cancer, has completed treatment for cancer, and/or is in remission from cancer between the first point in time and the subsequent point in time, In yet another embodiment, said human elF4B or orthoiog thereof, and/or said human MELK or ortholog thereof, comprises a nucleic acid sequence or amino acid sequence set forth in Table I . In another embodiment, the manipulation of the sample is selected from the group consisting of contacting the sample with a test agent, contacting the sample with an upstream signal of the MELK
signaling pathway, and contacting the sample with a 'NI-ELK inhibitor.. In still another embodiment, the test agent is a small molecule, or an antibody or antigen-binding fragment thereof In yet another embodiment, the test agent decreases the amount of Ser-406 phosphorylated human e1f4B or a corresponding phosphorylatable amino acid in the ortholog of human elF4B by at least 50%.

In another aspect, a method of identifying an agent which inhibits kinase or oncogenic activity of human MELK or an ortholog thereof, comprising: a) contacting a sample comprising i) human MELK or an ortholog thereof and human ilistone 113 or an ortholog thereof, with the agent; and b) determining the ability of the agent to inhibit Thr-3 phosphorylation of human Histone H3 or a corresponding phosphorylatable amino acid in the ortholog of human Histone 1-13; and/or Ser- 1 0 phosphorylation of human Histone H3 or a corresponding phosphorylatable amino acid in the ortholog of human hiStOriC
H3o andfor Thr-I phosphorylation of human Histone H3 or a corresponding phosphotylatable amino acid in the ortholog of human Histone H3, wherein decreased phosphorylation identifies an agent which inhibits kinase or oncogenic activity of human MELK or the ortholog thereof is provided. N one embodiment, the inhibition of said 'Thr-3 phosphorylation and/or Ser-10 phosphorylation and/or Thr 11 phosphorylation of human Histone H3, or a corresponding phosphotylatable amino acid in an orthoiog of human Histone H3, is determined by comparing the amount of Thr-3 phosphorylated human Histone H3 and/or Scr-I0 .15 phosphorylated human Histone H3 and/or Thr-I 1 phosphorylated human .Histone H3, or a corresponding phosphorylatable amino acid in the ortholog of human Histone H3, in the sample relative to a control. In another embodiment. the control is the amount of Thr-3 phosphorylated and/or Ser-1 0 phosphorylated and/or =Thr-1 phosphorylated human Historic H13, or a coiresponding phosphotylatable amino acid in the ortholog of human Histone H3, in the sample relative to said amount in the absence of the agent or at an earlier timepoint after contact of the sample with the agent. in still another embodiment, the inhibition of' said Thr-3 phosphorylation and/or Ser- 10 phosphm-ylation and/or Thr-1 1 phosphorylation of human Histone 1-13. or a cotTesponding phosphorylatable amino acid in an ortholog of human Histone H3, is determined by comparing the ratio of the amount of Thr-3 phosphorylated and/or Ser-1 0 phosphorylated and/or 'Thr-1 1 phosphorylated human Histone 143, or a corresponding phosphorylatable amino acid in the ortholog of human Historic: 143, in the sample relative to the total amount of human Histone H3 or ortholog thereof, to a control. In vet another embodiment, the control is the ratio of the amount of Thr-3 phosphotylated and/or Sec-TO phosphorylated andlor Thr-11 phosphorylated human Histone 113, or a corresponding phosphorylatable amino acid in the ortholog of human Histone 113 in the sample relative to said ratio in the absence of the agent or at an earlier timepoint after contact of the sample with the agent. In another embodiment, the method further comprises determinino the amount of a mitosis-specific protein. In still another embodiment, the method further comprises a step of determining whether the agent directly binds said human Histone 1-1.3 or said ortholog thereof, or said human MELK or said ortholog thereof.
111 yet another embodiment, the sample is selected from the group consisting of in vitro, ex vivo, and in vivo samples. In another embodiment, the sample comprises cells, such as cancer cells (e.g., cells front a cancer selected from the. group consisting of any cancer in.
which MEEK or Histone H3 is amplified or overexpressed, any cancer having an activating mutation of MELK or -Histone H3, and any cancer in which MELK or 'Historic 1-13 is activated by other .kinases). In still another embodiment, the cells are obtained from a subject. In yet another embodiment, the sample is selected from the group consisting of tissue, .whole blood, scrum, plasma, buccal scrape, saliva, cerebrospinal fluid, .urine, stool, and hone marrow. In another embodiment, the amount of Thi-.3 phosphorylated andior Ser-1 0 phosphorylated and/or Thal I phosphorylated human Histone H3, or a corresponding phosphorylatable amino acid in the ortholoa of human Histone H3, is determined by an immunoassay using a reagent which specifically binds with Thr-.15 plaospholated or Ser-10 phosphorylated or Thr-I 1 phosphorylated .human ..Histone R3, or corresponding phosphory fated ortholog of human Histone 113, :in still another embodiment, the immunoassay is a radioimmunoassay, a Western blot assay, a proximity ligation assay, immunofitioresence assay, an enzyme immunoassay, an immunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, a dot blot assay, or a slot blot assay. In yet another embodiment, the enzyme immunoassay .is a sandwich enzyme immunoassay thing a capture antibody or fragment thereof which specifically binds -with.
human Histone H3 or corresponding ortholog of human Histone H3 and a detection antibody or fragment thereof which. specifically binds with Thr-3 phosphorylated or .Ser-1 0 phosphorylated or Thr-I phosphorylated human Histone H3, or a corresponding phosphorylated dialog: of human .Histone H.3. In another embodiment, the human .Histone 1:13 or ortholog thereof, andlor said. human MELK or ortholog thereof, comprises a nucleic acid sequence or amino acid sequence set forth in Table 1. In still another embodiment, the agent is a small molecule, or an antibody or antigen-binding fragment thereof.
In yet another embodiment., the agent decreases the amount of Thr-3 phosphorylated and/or Ser.-1.0 phosphorylated and/of Thr] I phosphorylated human Histone 113, or. a corresponding phosphorylatable amino acid in the ortholog of human Histone H3, by at least 50%.
In still another aspect, a method for assessing the efficacy of an agent thr inhibiting kinase activity of human MELK or an ortholog thereof in a. subject, comprising: a) detecting in a subject sample at a first point in time, the amount of Thr-3 phosphorylated and/or Ser-10 phospbotylated and/or Thr-11 phospbotylated human Historic 1:13, or the amount of a human Histone H3 ortholog phosphorylated at a corresponding amino acid of human Histone H3; b) repeating step a) during at one or more subsequent points in time after administration of the agent; and c) comparing the amount of phosphorylated human Histone H3 or ortholog thereof detected in step a) with said amount detected in step b), wherein a higher amount of Thr-3 phosphorylated. and/or Ser-10 phosphorylated and/or Thr-11 phosphorylated human Histone143, or the amount of the human Histone H3 ortholog phosphorylated at a corresponding amino add of human Histone 143, in the first point in time relative to at least one subsequent point M time, indicates that the agent inhibits kinase activity of human NIELK or the ortholog thereof is provided.
In one embodiment, the amount of Thi-3 phosphotylated and/or Ser-10 phosphorylated ardor Thr-11 phosphorylated human Histonc H3, or a corresponding phosphorylatabic amino acid in the ortholog of human Histone H3, is determined by an immunoassay using a reagent .15 which specifically binds with Thr-3 phosphorylated human Historic H3 or Ser-I0 phosphorylated human Histone H3 or Thr-11 phosphorylated human Histone H3, or corresponding phosphory Wed ortholog of human Histone H3. :In another embodimentõ the immunoassay is a radioinuntinoassay, a "Western blot assay, a proximity ligation assay, an immunofluoresence assay, an enzyme immunoassay, an immunopreeipitation assay, a chemiluminescence assay, an immunohistochenneal assay, a dot blot assay, or a slot blot assay. In still another embodiment, the enzyme immunoassay is a sandwich enzyme immunoassay using a capture antibody m fragment thereof which specifically binds with human Histone 1-13 or corresponding ortholou of human Histone 1-13 and a detection antibody or fragment thereof whiCh specifically binds with Thr-3 phosphorylated or Scr-10 phosphorylated or Thr-1 1 phosphoryiated human Histone H3, or a corresponding phosphor5/lated ortholog of human Histone 113. in yet another embodiment, the sample is selected from the group consisting of ex vivo and in vivo samples. In another embodiment;
the sample comprises cancer cells (e.g., cancer cells selected from the group consisting of any cancer in which MELT( or Histone 113 is amplified or overexpressed, any cancer having an activating mutation of NIELK or Histone H3, and any cancer in which MELK or Histone H3 is activated by other kinases). In still another embodiment, the sample is selected from the group consisting of tissue, whole blood, serum, plasma, buccal serape, saliva, cerebrospinal fluid, urine, stool, and bone marrow. In yet another embodiment, the sample in step a) and/or step b) is a portion of a single sample obtained from the subject. hi another embodiment, the sample in step a) and/or step b) is a portion of pooled samples obtained from the subject. In still another embodiment, the. subject has undergone treatment for cancer, has completed treatment for cancer, ardor is in remission from cancer between the first point in time and the subsequent point in time.. In yet another embodiment, the human Historic 1113 or ortholog thereof, and/or said human MELK or ortholog thereof comprises a nucleic. acid sequence or amino acid sequence set forth in Table 1. In another embodiment, the agent is a small molecule, or an antibody or antigen-binding fragment thereof. In still another embodiment, the agent decreases the amount .of Thr-3 phosphorylated arid/or Ser-1 0 phosphorylated and/or Thr- Ii phosphorylated human Historic H3, or a corresponding phosphorylata.ble amino acid in the ortholog of human Histone H3, by at least 50%.
In yet another aspect, a method of treating a subject afflicted with cancer comprising administering to the subject an agent that inhibits ishr-3 =phosphorylation and/or Ser-10 plaospholation. and/or Thr-11 phosphorylation of human -Histone H3, or a corresponding phosphorylatable amino acid in an ortholog of human Histone H3, thereby treating the subject afflicted with the cancer is provided. In one embodiment, the agent is administered in a pharmaceutically acceptable formulation. In another embodiment, the agent is a small molecule, or an antibody or antigen-binding fragment thereof. In still another embodiment, the agent directly binds said human 'Riskin 1-13 or the ortholog thereof, or said human MELK or the ortholog thereof. In yet another embodiment, the cancer is selected from the group consisting of any cancer in which MEEK or Histone H3 is amplified or oyerexpressed, any cancer having an activating mutation of MELK or Histone I-13, and any cancer in which MELK or 'Historic H3 is activated by other kinases, in another embodiment, the human Histone H3 or dialog; thereof,' and/or said human .MELK
or ortholog, thereof, comprises a nucleic acid sequence or amino acid sequence set forth in Table 1. In still another embodiment, the agent is a small molecule, or an antibody or antigen-binding fragment thereof. In yet another embodiment, the agent decreases the amount of Thr-3 phosphorylated. and/or Ser-10 phosphory kited and/or Thr- ii phosphorylated human Historic. H3, or a corresponding phosnhorylatablc amino acid in the ortholog of human .Histone H3, by at least 50%. In another embodiment, the method further comprises administering one or more additional anti-cancer agents, In another aspect, a method of determining the function or activity of human MELK
or an ortholog, comprising: a) detecting in a sample the amount of Thr-3 phosphorylated and/or Ser-10 phosphorylated and/or Thr-11 phosphorylated human histone 113 or the amount of a human :Histone H3 ortholog phosphorylated at a corresponding amino acid. of human Histone H3; b) repeating step a) in the same sample or a test sample at one or more subsequent points in time after manipulation of the sample and/or manipulation of the same sample or test sample and c) comparing the amount of phosphorylated human Historic H3 or ortholog thereof detected in step a) with said amount detected in step b), wherein a modulated amount of Thr-3 phosphoryiated and/or Send 0 phosphorylated and/or Thr-1 phosphorylated human Histone H3, or the amount of the human Historic:, H3 ortholog phosphorylated at a corresponding amino acid of human Historic H3, in the first point in.
time relative to at least one subsequent point in time and/or at least one subsequent.
manipulation of the same sample or test: sample, indicates that the function or activity of human MELK or an ortholog thereof is modulated is provided. in one embodiment, the amount of Thr-3 phosphorylated and/or Ser-10 phosphorylated and/or Thr-11 phosphorylated human Histone H3, or a corresponding phosphorylatable amino acid in the ortholog of human Historic H3, is determined by an immunoassay using a reagent which.
specifically binds with .thr-3 phosphorylated or Scr-I 0 phosphorylated or 'Br-phosphorylated human Histone 1-13, or corresponding phosphorylated ortholog of human Histone H3. In another embodiment, the immunoassay is a radioimmunoassay, a Western blot assay, a proximity ligation assay, an immunofluoresence assay, an enzyme immunoassay, an immunoprecipitation assay, a ehemilumineseenee assay, an immunohistochemical assay, a dot blot assay, or a slot blot assay. in still another embodiment, the enzyme immunoassay is a sandwich enzyme immunoassay using a capture antibody or fragment thereof which specifically binds with human Histone H3 or corresponding ortholog of human Historic 113 and a detection antibody or fragment thereof which specifically binds with Thr-3 phosphorylated or 5er-10 phosphorated or Thy-1 phosphorylated human Historic H3, or a corresponding phosphorylated ortholog of human Historic H3, in yet another embodiment, the sample is selected from the group consisting of in vitro, ex vivo.. and in vivo samples. In another embodiment, the sample comprises cells or the method uses a cell-based assay, in still another embodiment, the cells are cancer cells selected from the group consisting of any cancer in which MELK or Histone 1713 is amplified or overexpressed, any cancer having an activating mutation of MELK or Histone 1-13, and any cancer in which MELK or Histone1:13 is activated by other kinases.
In yet another embodiment, the sample is selected from the group consisting of tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and.
bone marrow. In another embodiment, the same sample or test sample in step a) and/or step b) is a portion of a single sample obtained from a subject. In still another embodiment: the same sample or test sample in step a) and/or step b) is a portion of pooled samples obtained from a subject. In yet another embodiment, the subject has undergone treatment for cancer, has completed treatment for cancer, andior is in remission from cancer between the first point in time and the subsequent point in time. In another embodiment, the human Histone H3 or ortholog thereof, and/or said human Nil-EL,K or orthoIog thereof, comprises a nucleic acid sequence or amino acid sequence set forth in Table 1. In still another embodiment: the manipulation of the sample is selected from the group consisting of contacting the sample with a test agent, contacting the sample with an upstream signal of the MELK
signaling pathway, and contacting the sample with al4.ELE, inhibitor. In yet another embodiment, .15 the test agent is a small molecule, or an antibody or antigen-binding fragment thereof: In another embodiment, the test agent decreases the amount of Thr-3 phosphoiylated and/or Seral 0 phoaphorylated. and/or Thr-11 phosphorylated human Histone H3, or a corresponding phosphorylatable amino acid in the ortholog of human Histone H3, by at least 50%, It will also be understood that certain embodiments of the present invention can be used with more than one method described herein, according to knowledge available to the skilled artisan.
Brief Description of Figures

2.5 Figure I shows that .M.T-:1,K interacts with el:F.4B. Flag-MELK was conditionally expressed in .MDA-MB-468 cells. Mitotic lysates were subjected to anti-Flag immunoprecipitation followed by tandem mass spectrometric analysis. The left panel shows the number of peptides recovered from the immunoprecipitates. The right panel shows validation of the interaction between MELK and ell:4B during mitosis.
Note that Flag-MELK is doxycycline-inducible.
Figure 2 shows the results of peptide library screening to identify an optimal substrate motif for MELK. The top panel shows the reSitits ofa apatially arrayed peptide library subjected to la vitro phosphorylation using recombinant fullalength MEEK. Each peptide contains one residue fixed at one of nine positions relative to the centrally fixed phosphoaceeptor (Le., serine or threonine). Reactions were spotted onto the membrane and the spots were exposed to a phosphor storage screen. The bottom pwiel shows a sequence logo generated using quantified and normalized data from the screen. Note that MEEK has a strong selection for al-017nm at the -3 position relative to the plaosphoacceptor site.
Figure 3 shows that :MEEK phosphorylates elF4B at S406 in vitro. Recombinant full-length MEEK or the kinase domain of MEEK was subjected to in vitro kinase assays using immunoprecipitated Flag-elf4B (wild type) or Flag-elF4B (S406A). Robust phosphorylation of eIF4B at S406 was observed in the presence of MEEK. This phosphorylation was abolished when wild type (wt) efF4B was nplaced with a.
mutant e1F4B (S406A).
Figure 4 shows that MEEK does not phosphorylates elE4B at S422 in vitro.
Recombinant full-length or kinase domain of MEEK was subjected to in vitro kinase assay using immunopreeipitated Flag-elf-4B (wild type) or Flag-eif4B (S422,A).
Reactions were analyzed by immunoblotting.
Figure 5 shows that MEEK regulates phosphorylation of el:F.4B at S406 in vivo.

Left panels show MEEK knockdown impairs the phosphorylation of ell:4B at S406.
MDA-MB-468 cells stably expressing. tetracycline-inducible (tet-on) small hairpin MEEK
(shMELK) in the presence or absence of doxycycline were harvested through treatment of nocodazole, and subjected to immunobloning. Right panels show that a MEEK
inhibitor impairs the phosphorylation of efF4B at S406. Mitotic MDA-MB-468 cells were treated.
for 30 min with vehicle or 200 IN OTSSP167, a MELK. inhibitor (Chung et al.
(2012) Oneotorget 3:1629-1.640). Lysates were used for immunoblotting.
Figure 6 shows the results of treating mitotic cells fbr 30 min with inTOR
inhibitors (e.g., Rapamycin and Torin 1) versus treating such cells with MEEK inhibitors (e.g., OTSSP67). The results indicate that MEEK inhibition, but not niTOR inhibition, suppressed the phosphorylation of elf4B at S406.
Figure 7 shows that knocking down 'MEEK or elf4B decreases the protein abundance of MAP, oNlye, and ODC I during mitosis. MDA-MB-468 and MDA-MB-231.
eons stably expressing tet-on shMELK or sh-elF4.13 were treated with doxycycline or vehicle control. Mitotic cells were harvested by nocodazole-induced arrest at prometaphase. Note that the inRNA of MAP, e-Myc, and ODC I have been shown to contain structured 5't3TR and their total levels remain unchanged.

Figure 8 shows that knocking down MELK decreases the translation of lueiferase driven by the 5 tiTR of e-Mye or ODC I during mitosis. .MDA-MB-231 cells stably expressing tet-on sliMELK were triuisfeeted with the indicated bicistronic vector in the presence or absence of doxycycline. Nocodazolc-arrested mitotic cells were harvested two days after transfection and subjected to a lueiferase assay. The ratio of Renilla Ineiferase to firefly lueiferase (RL/FL) was normalized to the value of control vector. Note that the left bar corresponds to =Dox H and the right bar corresponds to Dox (+) for each pair of bars shown in the graph reporting relative RUFL ratios.
Figure 9 shows that MELK phosphorylates Historic H3 at Thr-3, Ser-10 and Thr-in vitro. Recombinant Histone H3 was incubated with or without recombinant MELK
(kinase domain) for 30 min. at 30 'C in the presence of ATP. Reactions were terminated by adding SOS sample buffer. Samples were then subjected to immunoblotting using the indicated antibodies.
Figure 10 shows that knocking down MELK decreases the mitotic =phosphorylation offlistone H.3 at Thr-3. Ser-10 and Thr-11, but not Scr-28. MDA-MB-468 cells stably transduced with let-on shMELK were untreated or treated with doxycycline (200 niv'ml) in order to induce gene silencing. Cells were then treated with nocodazole (200 ngittil) for 2,0 hours. Mitotic cells were harvested by Shake-off and cell lysates were subjected to immunoblotting using the indicated antibodies, Figure 11. shows that knocking down MELK. does not affect the phosphorylation of Aurora kinases, which are known kinases for .Histone H3 at Set- 10. MDA-MB-468 cells stably transdueed with tet-on slaMD.:K were untreated or treated with doxycycline (200 ngfrni) to induce gene silencing. Cells were treated with nocodazole (200 ng/m1) for 20 hours. Mitotic .cells were harvested by Shake-off and cell lysates were subjected to immunoblotting using the indicated antibodies.
Figure 12 shows that a MELK. inhibitor suppresses MELK-induced phosphorylation of Histone 143 at Ser loin vitro. Recombinant Histone H3 was incubated without or with recombinant MELK (kinase domain) for 30 min. at 30 nC. in the absence or presence of OISSP167 000 final). Reactions were terminated by adding SOS
sample buffer. Samples were subjected to immunoblotting using the indicated antibodies.
Figure 13 shows that a MELK inhibition suppresses the mitotic phosphoryiation of Histone H3 at Thr-3, Ser-10 and Thr-11, but not Ser-28, in a concentration-dependent manner. Mitotic cells were harvested through noeodazole-induced cell cycle arrest at prometaphase (200 nglinl nocodazole, 20 hours). Cells were treated with the small chemical inhibitor of MELK. OISSP167, for 30 min, at the indicated concentrations. Cell lysates were then prepared for immunoblotting.
Detailed Description of the invention The methods of the present invention relate to the surprising determination that the level of phosphorylation of position 406 (e.g , a serint residue) of human eukatyotic initiation factor 4B (cIF4B), or a corresponding phosphorylatable amino acid of an ortholog thereof, SMITS as a biornarker for MELK enzymatic (e.g., kinase) and/or oncogenic activity. Specifically, decreased phosphorylation of, for example, Ser-406 of human elF48 (e.g., by directly or indirectly inhibiting MELK-mediated phosphorylation of Ser-406) corresponds with a reduction in MELK enzymatic activity (e.g., kinase activity) and MELK-mediated oncogenic effects. Such a biomarker is particularly advantageous for preelinical and clinical applications because the phosphorylation state of elF4B is directly associated with the MELK oncogene itself Similarly, the methods of the present invention also relate to the surprising, determination that the level of phosphorylation of position 10 (e.g, a sante residue) of human Histone H3, or a corresponding phosphorylatable amino acid of an ortholog thereof, and/or the level of phosphorylation of position Ii (e.g., a threonine residue) of human Hlistone 1:13, or a corresponding phosphorylatable amino acid of an ortholog thereof, serves as a biomarker for MELK enzymatic (e.g., kinase) and/or oncogenic activity. Specifically, decreased phosphorylation of for example.
Thr-3 of human Histone H3 (e.g., by directly or indirectly inhibiting MELK-mediated phosphorylation of Thr-3) andlor 5er-1 0 of human Histone 1-13 (e.g., by directly or indirectly inhibiting MELK-mediated phosphorylation of Ser-10) and/or Thr-1 I
of human HistoricH3 (e.g., by directly or indirectly- inhibiting MELK-mediated phosphorylation of Thr-11 ) corresponds with a reduction in MELK enzymatic activity (e.g., kinase activity) and MELK-mediated oneogenic effects. Such a bioniarker is particularly advantageous for preclinicai and clinical applications because the phosphorylation state of Histone H3 is directly associated, with the MELK oncogene itself, 'in some embodiments. Scr-406 of human elF411, Thr-3 of human Ristone H3, Ser-10 of human Historic R3, and/or Thr-11 of human Historic H3, as well as any corresponding phosphorylatable amino acid of an ortholog thereof, including in any combination thereof, are contemplated for use according to the present invention. In other embodiments, Ser-28 of human elF4B or a corresponding phosphorylatable amino acid of an ortholog thereof is not regulated by MELK
and is not used according to the present invention.
A. MELK, elF411, and Histone 113 'Molecules As used 'herein, "MELK" refers to the MELK member of the protein kinase superfannly and is alternatively known as "pEG3 kinase," "protein kinase 43,"
"protein kinase," and "serineitinvoninc-protein kinasc MELK.÷ At least nine splice variants encoding nine distinct human MELK isoforms exist. Human MELK transcript variant 1 (N.M_014791,3) encodes the long human N1F.1,1. isofonn (NP 55606.1). Him:tan MELK
transcript variant 2 (NM.J01256685.1) lacks an exon in the 3' coding region compared to transcript variant 1., but maintains the reading frame and results in an isoform NP 001243614. that is shorter than isotbrm 1. Human :MELK transcript variant 3 (NM_00.1256687.1) tacks an en in the 5' coding region compared to transcript variant I.
but maintains the reading frame and .results M an isoform NP 0012436!6.i) that is shorter than isoform 1. Human MELK transcript variant 4 (NK.001256688.1) lacks two consecutive exons in the 5 coding region compared to transcript variant 1, but maintains the reading frame and results in an isoform (NP2)01243617.1) that is shorter than isoform 1. Human MELK transcript variant 5 NM 001256689.1) initiates translation at an alternate start codon and lacks an man in the 5' coding -.region compared to transcript variant 1 and thus results in an isoform (NP_001243618.1 ) that is shorter than and has a distinct N-terminus from isolbrin 1. Human MELK transcript variam 6 (NM...001.256690.1) initiates translation at an alternate start codon and.
lacks two consecutive exons in the 5' coding region compared to transcript variant 1, but mantas the reading frame and results in an isoform (NP_001.243619.1) that is shorter than and has a distinct N-terminus from isoform 1. Human :MELK transcript variant 7 (NM 001256691.1) initiates translation at an alternate start codon and lacks two exons in the 5' coding ration compared to transcript variant I, but maintains the reading frame and results in :311 isoform (NP 001243620.1) that is shorter than and has a distinct N-terminus from isoform 1. Human MELK transcript variant 8 (N1VILp01256692.1) lacks three exons in the 5' coding region and initiates translation at a downstream, in-frame start codon compared to tran.script variant I and results in an isoform (NP_001243621.1) that has a shorter N.-terminus than isoform 1. Finally, human MELK. transcript variant 9 (NM...001256693i ) lacks two consecutive exons in the 5' coding region and initiates translation at a downstream, in-frame start codon compared to transcript variant I and results in an isoform (NP_001 243622.1) that has a shorter N-terminus than isoform 1. The protein domains and structural basis for the regulation of MBA( autophospbotylation and activation of kinase activities on target proteins is known (sec, at least Cao et al. (2013) floS One 8:e70031 and Carievari etal. (2013) Biochemistry 52:6380-6387).
Mouse MELK nucleic acid (NM_010790.2) and amino acid (NP 034920.2) sequences are publicly available on the GenBank database maintained by the U.S. National Center for Biotechnology Information, Nucleic acid and polypeptide sequences of MELK
orthologs in species other than mice and humans are also well known and include, for example, chimpanzee MELK (XM_001169038.3, .X11_001169038.1, XM_001168991.3, XP._001168991.1, XM_00 1168745,3, XP_001168745.1, XM._001168775,3, X11_001168775.1, XM. J/03951427.1, XP 003951476.1, XM520578.4, X11_520578.3, XM_001168822.3, .XP_0011688221, XM_003312085.2, .XP_003312133.1 , KM_003951428.1, XP 003951477.1, xm..903312086 2, and XP 003312134.1), monkey MELK po 1 Ii 5(176.2 and X11_001115076.2), dog MELK (XM..903431578.1.
X11_003431626.1, X.M._538730.3, .XP 538730.2, XM_003431579.1., and XP 003431627,1), cow MELK (NM_001111260.1 and NP_001104730.1), rat MELK
(NM 00 1108662.1 and N11.p01102132.1), chicken MELK (NM...001031509.1 and N11_001026680.1), and zebrafish MELK (NM 206888.2 and NP 996771.2).
As used herein, "e1F4B" refers to the eukaryotic translation initiation factor member of the eukatyotic translation initiation factor family and is alternatively known as "Elf-4B" and ¨PRO1843." Human elf4B nucleic acid (NM...001417,4) and amino acid (NP_001408.2) sequences are publicly available on the GenBank database maintained by the U.S. National Center for Biotechnology Information. Nucleic acid and polypeptide sequences of CIF413 orthologs in species other than humans are also well known and include, for example, mouse efF4B (NM_145625.3 and NP 663600.2), chimpanzee elF413 (XM:_003313676.1, XP j003313724.1, XM_001142097.3, and XP_001142097.3), monkey cIF4B (NM._001195808.1 and NP j)01182737.1), dog elF4B (XM_853888.2, X11_858981.2, XM_. 853812.2, and X11_858905.2), cow eIF4B (NM001035028.2 and N11_001030200,1), rat elf4B (NM...001008324.1 and NP_001008325.1), and.
chicken elF413 (2CM_003643408.2 and XP 003643456.2). In addition, "Ser-406" of eIF4B
refers to the amino acid numbering of the human elf4B. Accordingly, a skilled artisan will readily understand that Ser-406 of the human e1F4B polypeptide is conserved across numerous species and that although those specific residues may be referenced herein, the methods of the present invention apply equally well to the corresponding residues (e.g., phosphorylatable amino acids) of isoforms, hornologs, and orthologs in other species corresponding to said Ser-406 of human ciF4B.
As used herein, the term "Historic 113" refers to the 143 member of the Historic family, which comprises proteins used to form the structure of nueleosomes in eukaryotic cells. lEukaryotes have chromatin arranged around proteins in the fOrm of aucleosomes, which are the smallest subunits of chromatin and include approximately 146-147 base pairs of DNA wrapped around an octanier of core historic proteins (two each of H2A.
H2B, 143, and 114). Mammalian cells have three known sequence variants of 11 istoncH3 proteins, denoted 143,1, 113.2 and 113.3, that are highly conserved differing in sequence by only a few amino acids. Post-translational modification of Histone 113 residues are important in many cellular processes and pliosphorylation of serine 10 and/or scrim 28 arc important for cell division and proliferation regulation. Phosphorylated Histone 113 at serine I
0 is a specific biomarker for mitotic cells, similar to other well-known mitosis-specific markers, such as phosphorylated MPM-2, phosphorylated retinoblastoma protein 1 (Rb), phosphorylated cdc2. BubRi, cyclin131, cdc25e, cdkl, cdc27, and the like. Any scrim:, threonine, or tyrosine residue can be phosphorylated. hi some embodiments, other possible phosphorylation sites include threonine 3, threoninc 6, threonine Ii,scrim 31, tyrosine 41, serine 57, threonine 80, and threonine 107.
As used herein, the term 1-listone143" can refer to 113.1, 113.2, or 1-13.3 individually or collectively. These amino acid sequences inciude a methionine as residue number 1 that is cleaved off when the protein is processed. Thus, for example, serine 11 in the Historic 113 amino acid sequences Shown in Table 1 below corresponds to serine (Ser) 10 of the present invention. These three protein variants are encoded by at least fifteen different genes/transcripts. Sequences encoding the Histone 113.1 variant are publicly available as HISTII13A (NM J03529.2; NP_003520.1), HIST] H3B (NM _003537.3; NPL003528.1), HIST1H3C (NM 003531.2; NP J)03522A), HIST1H3D (NNI_003530.3; NP 003521.2), HIS1"1113E (NM 003532.2; NP 003523.1). HIST11-13F(NK_021018.2-, NP 066298.1).
HrsT 1113G (NM 003534.2; NP003525,1), tirsT1113H (NM._9035362; Np003527.1), .111ST11131 (NM 003533.2; NP2103524.1)õ and HIST1113,1 (NM 003535.2; NP
003526.1).
Sequences encoding the Histone 143,2 variant are publicly available as (NM_001005464, 2; N1L001005464.1), H1ST2H3C (N,M.021059.2; NP 066403.2), and H1ST2H3D (NM 001123375.1; NP 0011168471). Sequences encoding the Histone 113.3 variant are publicly available as 113F3A (NM 002107.3; NPJ)02098,1) and H3F3B
(NM 005324.3; NP _005315.1). See U.S. Pat. Publ. 2012/0202843 for additional details.
Moreover, polypeptide sequences for Histone H3 orthologs, as well as nucleic acid sequences that encode such polypeptides, are well-known in many species, and include, for example, Historic H3.1 orthologs in mice (Nii4_013550.4; NP 03857&2), chimpanzee (XN1_527253.4; XP 527253.2). monkey (XM...0010882982; XP_001088298.1), dog (XM_003434195.1; XP 003434243.1), cow (XM.....002697460.1; XP 002697506.1).
rat (XM_001055231.2; XP_001055231.1), and zebrafish (NM 001100173.1;
NP..õ001093643.1). :Histone 113.2 orthologs in mice (NM...178215.1;
NP....835587.1), chimpanzee (XM_524859.4; )<P 524859.2). monkey (XM001084245.2;
X. P...001084245 .1 ), dog (X1V1... 003640147,1; XP _003640195 1) cow (CM.
J102685500.1;
XP_0026855461), rat (NM_001107698.1; NP_001101168.1), chicken (XM_001233027.2;

XP_001233028.1), and zebra:fish (XM...002662732.1; XP 002662778.1). Simiiariy Histone 113.3 orthologs in mice (XM...892026.4; XP 897119.3), monkey (Xg..001085836.2;
XP_001085836.1), cow (NM 001099370.1.; NP_001092840.1), rat (NM 053985.2:
NP 46437.!), chicken (NM 205296.1; NP 990627.1.). and zebratish (M4_200003.1;
NP 956297.!). are well-known. Antibodies for the detection of phosphorylated histone, such as phosphorylated Historic 113 at Thr-3, Ser-10, Thr-I 1, and other phosphmlatahle residues of Histone 113, as well as methods for making such antibodies are known in the art. in addition, for example, Seri 0" of Histone 113 refers to the amino acid numbering of the human 'Historic 113, Accordingly, a skilled artisan will readily understand that Ser-10 of the human Histone 113 polypeptide is conserved across numerous species and that although those specific residues may be referenced herein, the methods of the present invention apply equally well to the corresponding residues (e.g., phosphorylatablc amino acids) of isoforms, homologs, and orthologs in other species corresponding to said Ser-I 0 of human Histone H3, The same applies to Thr-3 and Thr-1 I.
Representative MELK,eIF4B, and Histone 113 orthologs are provided herein (e.g., at least at Table I and the Examples) as follows:
Table Human MELK (isoibrin 1) cDNA Saporta (NM 0.14791.3) 1 atqaaatt 4t.gt4A4cn t,e.'tc,atat tatgaatt4c at4a4.a.ctat tqggatag4t:
61 ggctttgcaa aggtcaaact tgcctgccat atccttactg gagagatggt agctataaaa 121 atcatggata aaaacacact agggagtgat ttgccccgga tcaaaacgga gattgaggcc 181 ttgaagaacc tgagacatca gcatatatgt caactctaccatgtgctaga qadagccsac 241 aaaatattca tggttcttga gtactgccct ggaggagagc tgtttgacta tataatttcc.
301 caggatcgcc tgtcagaaga ggagacccgg gttgtcttcc gtcagatagt atctgctgtt 3E1 gcttatgtgc acagccaggg ctatgctcac agggacctca agccagaaaa tttgctgttt 421 gatgaatatc atat aaa gctgattgac tttggtctct gtgcaaaacc caagggtaac 401 aaggattacc atctacagac atgctgtggg agtctggctt atgcagcacc tgagttaata 541 caaggcaaat catatcttgg atcagaggca gatgtttgga gcatgggcat actgttatat 601 gttcttatgt gtggatttct accatttgat gatgataatg taatggcttt atacaagaag 661 attatgagag gaaaatatga tgttcccaag tggctctctc ccagtagcat tctgcttctt i0 721 caacaaatgc tgcaggtgga cccaaagaaa cggatttcta tgaaaaatct attgaaccat 781 ccctqqatca tgoaagatta caactatcct gttgagtggc aaagcaagaa tccttttatt 841 cacotcgatg atgattgcgt aacagaactt. tctgtacatc acagaaacaa caggoaaaca 801 atggaggatt taatttcact gtggcagtat gatcacctca cggctaccta tcttctgctt 961 ctagccaaga aggctcgggg aaaaccagtt cgtttaaggc tttcttcttt ctcctgtgga 1021 caagccagtg ctaccccatt cacagacatc aagtcaaata attggagtct ggaagatgtg 1081 accgtaagtg ataaaaatta tgtggcggga ttaatagact atgattggtg tgaagatgat 1141 ttatcaacag gtgctgctac tccccgaaca tcacagttta ccaagtactg gacagaatca 1201 aatgqggtgg aatctaaatc attaactcca gccttatgca gaacacctgc aaataaatta 1261 aagaacaaag aaaatgtata tactcctaag tctgctgtaa agaatgaaga gtactttatg 1321 tttcctgagc caaagactcc agttaataag aaccagcata agagagaaat actcactacg 1381 ccaaatcgtt acactacacc ctcaaaagct agaaaccagt gcctgaaaga aactccaatt 1441 aaaataccag taaattcaac aggaacagac aagttaatga caggtgtcat tagccctgag 1501 aggcggtgcc gctcagtgga attggatctc aaccaagcac atatggagga gactccaaaa 1561 agaaagggag ccaaagtgtt tgggagcctt gaaagggggt tggataaggt tatcactgtg 1621 ctcaccagga gcaaaaggaa gggttctgcc agagacgggc ccagaagact aaagcttcac 1681 tataacgtga ctacaactag attagtgaat ccagatcaac tgttgaatga aataatgtct 1741 attcttccaa agaagcatgt tgactstgta caaaagggtt atacactgaa gtgtcaaaca 1801 cagtcagatt ttgggaaagt gacaatgcaa tttgaattag aagtgtgcca gcttcaaaaa 1861 cccgatgtgg tgggtatcag gaggcagcgg cttaagggcg atgcctgggt ttacaaaaga 1921 ttagtggaag acatcctatc tagctgcaag dtataa Human MELK(jsoform. 1) Araino Acid. Sequence (NP _55606.1) 1 mkdydellky yelhatigtg gfakyklach iltgemvaik imdkntlgsd Iprikteiea 51 ikhlrhqhic glyhvletan kifmvleycp ggelfdylis qdrisaeetr vvft.givsav 121 ayvhaggyah rdlkpanlIf davhklklid fglcakpkgm kdyhlgtccg slavaapeli 181 qgkaylgaea dvwamgilly vlmcgflpfd ddnvmalykk imrgkydvpk wispasilii 241 qgmlgvdpkk rismknlinh pwimgdynyp vawqsknpfi hldddcvtel avhhrnnrqt 301 mediialwqy dhltatylli lakkargkpv r1r1saf5cg gasatpftdi kannwsledv 361 tasdknyvag lidydwcedd Istgaatprt sqftkywtes hgvesksltp alcrtpankl.
421 knkanyytpk savkneayfm fpapktpvnk nqbkrailtt pnryttpaka rcqclketpi 481 kipvhstgtd klmtgvispa rrcrsveldl nqahmeetpk rkgakvfgsl ergldkvitv 541 Itrakrkgsa rdgprriklh ynytttrivn pdglinaims iipkkhvdfv qkgvtlkcgt 601 qsdfgkvtraq feievcqlqk pdvvgirrqr lkgdawvykr Ivediisack v Human MELK (isoform 2) cDNA. Sequence (NM_001256685.1.) 1 atgaaagatt atgatgaact tctcaaatat tatgaattac atgaaactat tgggacaggt 61 ggctttgcaa aggtcaaact tgcctgccat atccttactg gagagatggt agctataaaa 121 atcatggata aaaacacact agggagtgat ttgccccgga tcaaaacgga gattgaggcc 181 ttgaagaacc tgagacatca gcatatatgt caactctacc atgtgctaga gacagccaac 241 aaaatattca tggttcttga gtactgccct ggaggagagc tqtttgacta tataatttcc 301 caggatcgcc tgtcagaaga ggagacccgg gttgtcttcc gtcagatagt atctgctgtt 361 gcttatgtgc acagccaggg ctatgctcac agggacctca agccagaaaa tttgctgttt 421 gatgaatatc ataaattaaa gctgattgac tttggtctct gtgcaaaacc caagggtaac 461 aaggattacc atctacagac atgctgtggg agtctggctt atgcagcacc tgagttaata 541 caaggcaaat catatcttgg atcagaggca gatgtttgga gcatqggcat actgttatat 601 gttcttatgt gtggatttct accatttgat gatgataatg taatggcttt atacaagaag 661 attatgagag gaaaatatga tgttcocaag tggctctctc ccagtagcat tctgcttctt 721 caacaaatgc tgcaggtgga cccaaagaaa cggatttcta tgaaaaatct attgaaccat 761 ccctggatca tgcaagatta caactatcct gttgagtggc aaagcaagaa tccttttatt 841 cacctcgatg atgattgcgt aacagaactt tctgtacatc acagaaacaa caggcaaaca 901 atggaggatt taatttcact gtggcagtat gatcacctca cggctaccta tcttctgctt 961 ctagccaaga aggctcgggg aaaaccagtt cgtttaaggc tttcttcttt ctcctgtgga 1021 caagccagtg ctaccccatt cacagacatc aagtttacca agtactggac agaatcaaat 1081 ggggtggaat ctaaatcatt aactccagcc ttatgcagaa cacctgcaaa taaattaaag 1141 aacaaagaaa atgtatatac toctaagtot gctgtaaaga atgaagagta ctttatgttt 1201 cctgagccaa agactccagt taataagaac cagcataaga gagaaatact cactacgcca 1261 aatcgttaca ctacaccctc aaaagctaga aaccagtgcc tgaaagaaac tccaattaaa 1321 ataccagtaa attcaacagg aacagacaag ttaatgacag gtgtcattag ccctgagagg 1381 cggtgccgct cagtggaatt ggatctcaac caagcacata tggaggagac tccaaaaaga 1441 aagggagcca aagtgtttgg gagccttgaa agggggttgg ataaggttat cactgtgctc 1501 accaggagca aaaggaaggg ttctgccaga gacgggccca gaagactaaa gcttcactat 1561 aacgtgacta caactagatt agtgaatcca gatcaactgt tgaatgaaat aatgtctatt 1621 cttccaaaga agcatqttga ctttgtacaa aaggqttata cactgaaqtg tcaaacacag 1681 tcagattttg ggaaagtgac aatgcaattt gaattagaag tgtgccagct tcaaaaaccc 1741 gatgtggtgg gtatcaggag gcagcggctt aagggcgatg cctgggttta caaaagatta 1801 gtggaagaca tcctatctag ctgcaaggta taa Human MELK (isoform..2).AmMe=AcidScipetice (NP 00.12436.14.1) 1 mkdydaa.lky yelllatigtg gfakvklaCil. iltgamvaik imdkatigsd lp.tiktedea 61 IknIthgnic calynv-letan kifmvlaycp ggalfdylis qdriaaaetr. vvfrqivsav 121 ayvhsclgyah rdIkpenlIf dayhkIklid fglcakpkgn kdyhlgtccg alayaapeli 181 qgkayigsea dvwsmgilly vimcgflpfd ddnvmalykk imrgkydvpk wispasilli 241 qqmiqvdpkk rismkhlinh pwimgdynyp vemgeknpfl hidddcvtel svhhrnnrqt 301 madIialwqv dh1taty1.11 lakkargkpv rIrinsfscg qasatpftdi kftkywtesn 361 gvesksitpa Icrtpankik nkenvvtpks avkneeyfmf pepktpvnkn qhkreilttp 421 ntvttpskar nqciketpik ipvnatgtdk Intgviaper rcraveldin qahmeetpkt 481 kgakvfgsle rgidkvitvi trskrkgsar dgprrlklhv nvtttrIvnp dgllneimsi 541 Ipkkhvdfvg kgytikcqtg sdfgkvtmqf elevcqlqkp dvvgirrqrl kgdawvykri 601 vedilasckv Human MELT( (isoform 3) cliNA. Sequence (1 M I) 1 atgaaagatt atgatgaact tctcaaatat tatgaattac atgaaactat tgggacaggt 61 ggctttgcaa aggtcaaact tgcctgccat atccttactg gagagatggt agctataaaa 121 atcatggata aaaacacact agggagtgat ttgccccgga tcaaaacgga gattgaggcc 181 ttgaagaacc tgagacatca gcatatatgt caactctacc atgtgctaga gacagccaac 241 aaaatattca tggttcttga ggaaaatttg ctgtttgatg aatatcataa attaaagctg 301 attgactttg gtctctgtgc aaaacccaag ggtaacaagg attaccatct acagacatgc 361 tgtgggagtc tggcttatgc agcacctgag ttaatacaag gcaaatcata tcttggatca 421 gaggcagatg tttggagcat gggcatactg ttatasgttc ttatgtgtgg atttctacca 461 tttgatgatg ataatgtaat ggctttatac aagaagatta tgagaggaaa atatgatgtt 541 cccaagtggc tctctcccag tagcattctg cttcttcaac aaatgctgca ggtggaccca 601 aagaaacgga tttctatgaa aaatctattg aaccatccct ggatcatgca agattacaac 661 tatcctgttg agtggcaaag caagaatcct tttattcacc tcgatgatqa ttgcqtaaca 721 gaactttctg tacatcacaq aaacaacagg caaacaatgg aggatttaat ttcactgtqg 751 cagtatgatc acctcacggc tacctatctt ctgcttctag ccaagaaggc tcggggaaaa 841 ccagttcgtt taaggctttc ttct.ttctcc tgtggacaag ccagtgctac cccattcaca 401 gacatcaagt caaataattg gagtctggaa gatgtgaccg caagtgataa aaattatgtg 961 gcgggattaa tagactatga ttggtgtgaa gatgatttat caacaggtgc tgctactccc 1021 cgaacatcac agtttaccaa gtactqgaca gaatcaaatg gggtgqaatc taaatcatta 1081 actccagcct tatgcagaac acctgcaaat aaattaaaga acaaagaaaa tgtatatact 1141 cctaagtctg ctqtaaagaa tgaagagtac tttatgtttc ctgagccaaa gactccagtt 1201 aataagaacc agcataagag agaaatactc actacgccaa atcgttacac tacaccctca 1261 aaagctagaa accagtgcct gaaagaaact ccaattaaaa taccagtaaa ttcaacagga 1321 acagacaagt taatgacagg tgtcattagc cctgagaggc ggtgccgctc agtggaattg 1381 gatctcaacc aagcacatat ggaggagact ccaaaaagaa agggagccaa agtgtttggg 1441 agccttqaaa ggqggttgga taaggttatc actgtgctca ccaggagcaa aaggaaggqt 1501 tctgccagag acgggcccag aagactaaag cttcactata acgtgactac aactagatta 1561 gtgaatccag atcaactgtt gaatgaaata atgtctattc ttccaaagaa gcatgttgac 1621 tttgtacaaa agggttatac actgaagtgt caaacacagt cagattttgg gaaagtgaca 101 atgcaatttg aattagaagt gtgccagctt caaaaacccg asgtggtggg tatcaggagg 1741 cagcggctta agggcgatgc ctgggtttac aaaagattag tggaagacat cctatctagc 1801 tgcaaggtat aa Human Mf..,1,1( (1solorm 3) Amino Acid. Sequence (NP _00124361.6.1) 1 mkdvdelikv yelhetigtg gfakvklach iltgemvaik imdkntigsd lpriktelea 61 IknIrhqhic givhvletan kifmvieenl ifdeyhkiki idfgicakpk gnkdyhIcitc 121 cgslayaape liqgksvIgs eadviesmgil lyv1mcgflp fdddnvmaly kkimrgkydv 181 pkwlspssil 11qqm1qvdp kkrismknll shpwimgdyn ypvewq.aknp fihldddcvt 241 elsvhhrnnr gtmedlisiw gydhltatyl illakkargk pvr1r1ssfs cggasatpft 301 diksnnwsle dvtasdknyv aglidydwce ddIstgaatp rtsgftkpft esngvesksi 361 tnalcrtnan kiknkesvvt nksavkneey fmfnepktpv nksqhkrei1 ttnnryttps 421 karnqclket pi.kipvnstg tdklmtgvis perrcrsvel dingahmeet pkrkgakvfg ak 02928464 2016-04-21 481 siergldkvi tvItrskrkg sardgprrik ihynvtttrl vnpdglInei mslipkkhvd 541 fvqkgytIkc qtqsdfgkvt mgfelevcgl qkpdv-vgirr cirlkgdavvy krIvedilss 601 ckv .5 Human MEM (isoform 4).:eD.NA Sequcmce. (NM 001256688.1) atgaeagatt atgatgaact tctcaaatat tatgaattac atgaaactat tgggacaggt 61 ggctttgcaa aggtcaaact tgcctgccat atccttactg gagagatggt agctataaaa 121 atcatggata aaaacacact agggagtgat ttgccccgga tcaaaacgga gattgaggcc 131 ttgaagaacc tgagacatca gcatatatgt caactctacc atgtgctaga gacagccaac 241 aaaatattca tggttcttga gggtaacaag gattaccatc tacagacatg ctgtgggagt 301 ctggcttatg cagcacctga gttaatacaa ggcaaatcat ascttggasc agaggcagat 361 gtttggagca tgggcatact gttatatgtt cttatgtgtg gatttctacc atttgatgat 421 gataatgtaa tggctttata caagaagatt atgagaggaa aatatgatgt tcccaagtgg 481 ctctctccca gtagcattct gcttcttcaa caaatgctgc aggtggaccc aaagaaacgg 541 atttctatga aaaatctatt gaaccatccc tggatcatgc aagattacaa ctatcctgtt 601 gagtggcaaa gcaagaatcc ttttattcac ctcgatgatg attgcgtaac agaactttct 661 gtacatcaca gaaacaacag gcaaacaatg gaggatttaa tttcactgtg gcagtatgat 721 caccLcacgg ctacctatxt tc:.:gcttcta gccitageagg c:.:cgginaaa accagttcgt 751 ttaaggcttt cttctttctc ctgtggacaa gccagtgcta ccccattcac agacatcaag 841 tcaaataatt ggagtctgga agatgtgacc gcaagtgata aaaattatgt ggcgggatta 901 atagactatg attggtgtga agatgattta tcaacaggtg ctgctactcc ccgaacatca 961 cagtttacca agtactggac agaatcaaat ggggtggaat ctaaatcatt aactccagcc 1021 ttatgcagaa cacctgcaaa taaattaaag aacaaagaaa atgtatatac tcctaagtct 1051 gctgtaaaga atgaagagta ctttatgttt cctgagccaa agactccagt taataagaac 1141 cagcataaga gagaaatact cactacgcca aatcgttaca ctacaccctc aaaagctaga 1201 aaccagtgcc tgaaagaaac tccaattaaa ataccagtaa attcaacagg aacagacaag 1261 ttaatgacag gtgtcattag ccctgagagg cggtgccgct cagtggaatt ggatctcaac 1321 caagcacata tggaggagac tccaaaaaga aagggagcca aagtgtttgg gagccttgaa 131 agggggttgg ataaggttat cactgtgctc accaggagca aaaggaaggg ttctgccaga 1441 gacgggccca gaagactaaa gcttcactat aacgtgacta caactagatt agtgaatcca 1501 gatcaactgt tgaatgaaat aatgtotatt cttccaaaga agcatgttga ctttgtacaa 1561 aagggttata. cactgaagtg tcaaacacag tcagastttg ggaaagtgac aatgcaattt 1621 gaattagaag tgtgccagct tcaaaaaccc gatgtggtgg gtatcaggag gcagcggctt 161 aagggcgatg cctgggttta caaaagatta gtggaagaca tcctatctag ctgcaaggta 1741 taa Human MELK (isofarm 4) Amino Acid Sequence (NP 00124361.7,1) 1 mkdydeilky yelhetigtg gfakvklach iitgemvaik imdkntigsd ipriktelea 61 iknirhghic. glyhvietan kifmvlegnk dynlqtccge layaapelig gksvigsead 121 vwsmgiilyv lmcgflpfdd dnvmaiykki mrgkydvpkw Ispssiillq qmigvdpkkr 181 ismknlInhp wimgdyhypv awgsknpfih idddcvtels vhhrnnrgtm edlisiwgyd 241 hitatylill akkargkpvr Irlssfscgg asatpftdik snnwsledvt asdknyvagl 301 idydwceddl stgaatprts gftkywtesn avesksitpa Icrtpanklk nkenvytpks 361 avkneeytmf pepktpvnkn qhkrelittp nryttpskar ngclketpik ipvnstgtdk ak 02928464 2016-04-21 421 Imtgvisper rcrsveldln qahmeetpkr kgakvfgsle rgldkvitvl trskrkgaar 46.1 dgprr1k1hy nvtttrIvnp dg1Ineimai 1pkkhvdfvq kgytIkcgtq sdfgkvtmqf 541 elevcgIgkp dvvgirrqrI kgdawvykri vedilssckv .5 Human MEM (isoform 5).eD.NA Sequence4NM.....001256689.1) atgatgaact tctcaaatat tatgaattac atgaaactat tgggacagag tgatttgc.cc 61 cggatcaaaa cggagattgaggccttgaag aacctgagac atcagcatat atgtcaaetc 121 taccatgtgc tagagacagc caacaaaata ttcatggttc ttgagtactg ccctggagga 131 gagctgtttg actatataat ttcccaggat cgcctgtcag aagaggagac ccgggttgtc 241 ttccgtcaga tagtatctgc tgttgcttat gtgcacagcc agggctatgc tcacagggac 301 ctcaagccag aaaatttgct gtttgatgaa tatcasaaat taaagctgat tgactttggt 361 ctctgtgcaa aacccaaggg taacaaggat taccatctac agacatgctg tgggagtctg 421 gcttatgcag cacctgagtt aatacaaggc aaatcatatc ttggatcaga ggcagatgtt 481 tggagcatgg gcatactgtt atatgttctt atgtgtggat ttctaccatt tgatgatgat 15 541 aatqtaatgg ctttatacaa gaagattatq agaggaaaat atgatgttcc caagtggctc 601 tctcccagta gcattctgct tcttcaacaa atgctgcagg tggacccaaa gaaacggatt 661 tctatgaaaa atctattgaa ccatccctgg atcatgcaag attacaacta tcctgttgag 721 tggcaaagca agaatccttt tattcacctc gatgatgatt gcgtaacage actttctgta 761 catcacagaa acaacaggca aacaatggag gasttaattt cactgtggca gtatgatcac 20 841 ctcacggcta cctatcttct gcttctagcc aagaaggctc ggggaaaacc agttcgttta 901 aggctttctt ctttctcctq tggacaagcc agtgctaccc cattcacaga catcaagtca .961 aataattgga gtctggaaga tgtgaccgca agtgataaaa attatgtggc gggattaata 1021 gactatqatt ggtgtgaaga tgatttatca acaggtgctg ctactccccg aacatcacag 1.051 tttaccaagt actggacaga atcaaatggg gtggaatcta aatcastaac tccagcctta tgcagaacac ctgcaaataa attaaagaac aaagaaaatg tatatactcc taagtctgct 1201 gtaaagaatg aagagtactt tatgtttcct gagccaaaga ctccagttaa taagaaccag 1261 cataagagag aaatactcac tacgccaaat cgttacacta caccctcaaa agctagaaac 1321 cagtgcctga aaqaaactcc aattaaaata ccagtaaatt caacaggaac agacaagtta 1351 atgacaggtg tcattagccc tgagaggcgg tgccgctcag tggaattgga tctcaaccaa gcacatatgg aggagactcc aaaaaqaaag ggagccaaag tgtttqggag ccttgaaaqg 1501 gggttggata aggttatcac tgtgctcacc aggagcaaaa ggaagggttc tgccagagec 1561 gggcccagaa. gactaaagct tcactataac gtgactacaa ctagattagt gaatccagat 1621 ceactgttga atgaaataat gtctattctt ccaaagaagc atgttgactt tgtacaaaag 1.6E.j1 ggttatacac tgaagtgtca aacacagtca gattttggga aagtgacaat gcaatttgaa ttagaagtgt gccagcttca aaaacccgat gtggtgggta tcaggaggca gcggcttaag 1801 ggcgatgcct gggtttacaa aagattagtg gaagacatcc tatctagctg caaggtataa Human MELK. (isoform. 5) Amino Acid Sequence (NP 001243618.1) 1 mmnfsnimnv mkliggsdlp riktelealk nIrlaghicql vhvletanki nmvieycpgg 40 61 elfdyiisqd rlseeetrvv frgivsavay vhsggyahrd Ikpenilfde yhkIklidfg 121 Icakpkgnkd yhIgtccgs1 ayaapeliqg ksylgseadv wsmgillyvi mcgflpfddd 151 nvmalykkim rgkydvpkwl spssilliqg mlqvdpkkri smknlinhpw imqdynypve 241 wqsknpfihl dddcvtelsv hhrnnrgtme dlislwqydh Itatvillla kkargkpvrl.
301 rissfacgqa satpetdiks nnwsladvta sdknvvagli dvdwceddls tgaatprtsq 361 ftkywtesng vesksitpal crtpankikn kenvytpkaa vkneeyfmfp epktpvnknq 421 hkrei1ttpn ryttpakarn qc1ketpiki pvnstgtdk1 mtgvi:sperr crsveIdIng 481 ahmeetpkrk gakvfgsler gldkvitylt rskrkgsard gprriklhyn vtttrivnpd 541 glineirasil pkkhvdfvqk gvtlkcqtqs dfgkvtalgfe loycgiqkpd vvgirrgylk 601 gdawvykrIv adilssckv Hainan MELK Osamu 6) eDNA Sequence (NK.001256690.1) 1 atgatgaact tctcaaatat tatgaattac atgaaactat tgggacagta ctgccctgga 61 ggagagctgt ttgactatat aatttcccag gatcgcctgt cagaagagga gacccgggtt 0 121 gtcttccgtc agatagtatc tgctgttgct tatgtgcaca gccagggcta tgctcacagg 181 gacctcaagc cagaaaattt gctgtttgas gaatascata aattaaagct gattgacttt 241 ggtctctgtg caaaacccaa gggtaacaag gattaccatc tacagacatg ctgtgggagt 301 ctggcttatg cagcacctga gttaatacaa ggcaaatcat atcttggatc agaggcagat 361 gtttggagca tgqgcatact gttatatgtt cttatgtgtg gatttctacc atttgatgat 421 gataatgtaa tggctttata caagaagatt atgagaggaa aatatgatqt tcccaagtgg 481 ctctctccca gtagcattct gcttcttcaa caaatgctgc aggtgqaccc aaagaaacqg 541 atttctatga aaaatctatt gaaccatccc tggatcatgc aagattacaa ctatcctgtt 601 gagtggcaaa gcaagaatcc ttttattcac ctcgatgatg attgcgtaac agaactttct 661 gtacatcaca gaaacaacag gcaaacaatg gaggatttaa tttcactgtg gcagtatgat 721 cacctcacgg ctacctatct tctgcttcta gccaagaagg ctcggggaaa accagttcgt 781 ttaaggcttt cttctttctc ctgtgqacaa gccagtgcta ccccattcac agacatcaag 841 tcaaataatt ggagtctgga agatgtgacc gcaagtgata aaaattatgt ggcgggatta 901 atagactatg attggtgtga agatgattta tcaacaggtg ctgctactcc ccgaacatca 961 cagtttacca agtactggac agaatcaaat ggggtggaat ctaaatcatt aactccagcc 1021 ttatgcagaa cacctgcaaa taaattaaag aacaaagaaa atgtatatac tcctaagtct 1061 gctgtaaaga atgaagagta ctttatgttt cctgagccaa agactccagt taataagaac 1141 cagcataaga gagaaatact cactacgcca aatcgttaca ctacaccctc aaaagctaga 1201 aaccagtgcc tgaaagaaac tccaattaaa ataccagtaa attcaacagg aacagacaag 1261 ttaatgacag gtgtcattag ccctgagagg cggtgccgct cagtggaatt ggatctcaac 1321 caagcacata tggaggagac tccaaaaaga aagggagcca aagtgtttgg gagccttgaa 1381 agggggttgg ataaggttat cactgtgctc accaggagca aaaggaaggg ttctgccaga 1441 gacgggccca. gaagactaaa gcttcactas aacgtgacta caactagast agtgaatcca 1501 gatcaactgt tgaatgaaat aatgtctatt cttccaaaga agcatgttga ctttgtacaa 1561 aagggttata cactgaagtg tcaaacacag tcagattttg ggaaagtgac aatgcaattt 1621 gaattagaag tgtgccagct tcaaaaaccc gatgtggtgg gtatcaggag gcagcggctt 1481 aagggcgatg cctgggttta caaaagatta gtggaagaca tcctatctag ctgcaaggta 1741 taa Flu man MELK (isoform 6) Amino Acid Sequence (NP 001243619.1) 1 mmnfsnimny mkilgwycpg gelfdyiiag drIseeetrv vfrgivaava yvhaggyahr 61 dIkpenIlfd eynklklidf glcakpkgnk dyhlgtccgs layaapeliq gksylgsead 121 vwsmgillyv Imcgflpfdd dnvmalykki mrgkydvpkw ispssilliq gmiqvdpkkr 131 ismkniinhp wimgdynypv swgsknnfin Idddcvtals vhhtnnrgtm edlisiwqvd 241 hitaty1111 akkargkpvr irissfscgq asatpftdik snniesledvt azdknyvagi 301 idydwceddi stgaatprts qftkywtesn gvesksltpa icrtpankik nkenvytpks 861 avkneeyfmf pepktpvnkn qhkrenttp nryttpskar nvIketpik ipvnstgtdk 421 Imtgvisper rcrsveldin gahmeetpkr kgakvfgzie rgidkvitvi trskrkgsar 431 dgprrIkihv nvrttrIvnp dqll4th. Ipkkilvdfvg kgytIkccitg sdfgkvtmqf 541 elevcgigkp dvvgirrgrl kgdawvykri vedilssckv Human MEEK (isoform 7) eDNA Sequence (NM...001256691.1) 1 atgatgaact tctcaaatat tatgaattac atgaaactat tgggacagag tgatttgccc 61 cggatcaaaa cggagattga ggccttgaag aacctgagac atcagcatat atgtcaactc 0 121 taccatgtgc tagagacagc caacaaaata ttcatggttc ttgaggaaaa tttgctgttt 181 gatgaatatc ataaattaaa gctgattgac tttggtctct gtgcaaaacc caagggtaac 241 aaggattacc atctacagac atgctgtggg agtctggctt atgcagcacc tgagttaata 301 caaggcaaat catatcttgg atcagaggca gatgtttgga gcatgggcat actgttatat 361 gttcttatgt gtggatttct accatttgat gatgataatg taatggcttt atacaagaag 421 attatgagag qaaaatatga tgttcccaag tggctctctc ccagtagcat tctgcttctt 481 caacaaatgc tgcaggtgga cccaaagaaa cggatttcta tgaaaaatct attgaaccat 541 ccctggatca tgcaagatta caactatcct gttgagtggc aaagcaagaa tccttttatt 401 cacctcgatg atgattgcgt aacagaactt tctgtacatc acagaaacaa caggcaaaca 661 atggaggatt taatttcact gtggcagtat gatcacctca cggctaccta tcttctgctt 721 ctagccaaga aggctcgggg aaaaccagtt cgtttaaggc tttcttcttt ctcctgtgga 781 caagccagtg ctaccccatt cacagacatc aagtcaaata attggagtct gqaagatgtg 841 accgcaagtg ataaaaatta tgtggcggga ttaatagact atgattggtg tgaagatgat 901 ttatcaacag gtgctgctac tccccgaaca tcacagttta ccaagtactg gacagaatca 961 aatggggtgg aatctaaatc attaactcca gccttatgca gaacacctgc aaatatta 1021 aagaacaaag aaaatgtata tactcctaag tctgctgtaa agaatgaaga gtactttatg 106'1 tttcctgagc caaagactcc agttaataag aaccagcata agagagaaat actcactacg 1141 ccaaatcgtt acactacacc ctcaaaagct agaaaccagt gcctgaaaga aactccaatt 1201 aaaataccag taaattcaac aggaacagac aagttaatga caggtgtcat tagccctgag 1261 aggcggtgcc gctcagtgga attggatctc aaccaagcac atatggagga gactccaaaa 1321 agaaagggag ccaaagtgtt tgggagcctt gaaagggggt tggataaggt tatcactgtg 1361 ctcaccagga gcaaaaggaa gggttotgcc agagacgggc ccagaagact aaagcttcac 1441 tataacgtga. ctacaactag attagtgaas ccagascaac tgttgaatga aataatgtct 1501 attcttccaa agaagcatgt tgactttgta caaaagggtt atacactgaa gtgtcaaaca 1561 cagtcagatt ttgggaaagt gacaatgcaa tttgaattag aagtgtgcca gcttcaaaaa 1621 cccgatgtgg tgggtatcag gaggcagcgg cttaagggcg atgcctgggt ttacaaaaga 1481 ttagtggaag acatcctatc tagctgcaag gtataa Human MELK. Osamu 7) Amino Acid Sequence (NP 001243620.1) 1 mmnfanimnv mkliggsdlp riktelealk nlrhghicql vhvletanki nmvieenllf 61 deyhkiklid fgicakpkgn kdyhlgtccg slayaapeli qgksyigaea dvwamgilly 121 vlmcgflpfd ddnvmalykk imrgkydvpk wispssiill ggmiqvdpkk rismkniinh 181 pwimgdynyp vewgzknpfi hidddcvtel svhhrnnrgt medlisiwgy dhltatylli 241 lakkargknv rirlssfscg gasatpftdi ksnnwsledv tasdknyva* lidydwcedd 301 istgaatprt agftkvwtes ngvesksltp alcrtpankl knkenvytpk savkneeyfm 361 fpepktpvnk nOkreiltt pnryttpska rnciciketpi kipvnatgtd kimtgviape 421 rrcrsweldl nqahmeetpk rkgakvfgal ergldkvitv Ittskrkgaa rdgprtlklh 481 ynvtttrIvn pdglIneims ilpkkhvdfv gkgytikcgt gsdfgkvtmg felevcgigk 541 pdvvgirrqr lkgdawvykr ivedlissck v Hunian MELK (isoform 8) cDNA Sequence (NM:001256692.1) 1 atggttcttg aggaaaattt gctgtttgat gaatatcata aattaaagct gattgacttt 61 ggtctctgtg caaaacccaa gggtaacaag gattaccatc tacagacatg ctgtgggagt 121 ctggcttatg cagcacctga gttaatacaa ggcaaatcat atcttggatc agaggcagat 161 gtttggagca tgggcatact gttatatgtt cttatgtgtg gatttctacc atttgatgat 241 gataatgtaa. tggctttata caagaagatt atgagaggaa aatatgatgt tcccaagtgg 301 ctctctccca gtagcattct gcttcttcaa caaatgctgc aggtggaccc aaagaaacgg 361 atttctatga aaaatctatt gaaccatccc tggatcatgc aagattacaa ctatcctgtt 421 gagtggcaaa gcaagaatcc ttttattcac ctcgatgatg attgcgtaac agaactttct 451 gtacatcaca qaaacaacag gcaaacaatg gaggatttaa tttcactgtq gcagtatgat 541 cacctcacgg ctacctatct tctgcttcta gccaagaagq ctcggggaaa accagttcgt 601 ttaaggcttt cttctttctc ctgtggacaa gccagtgcta ccccattcac agacatcaag 661 tcaaataatt ggagtctgga agatgtgacc gcaagtgata aaaattatgt ggcgggatta 721 atagactatg attggtgtga agatgattta tcaacaggtg ctgctactcc ccgaacatca 781 cagtttacca agtactggac agaatcaaat ggggtggaat ctaaatcatt aactccagcc 841 ttatgcagaa cacctgcaaa taaattaaag aacaaagaaa atgtatatac tcctaagtct .901 gctgtaaaga atgaagagta ctttatgttt cctgagccaa agactccagt taataagaac 961 cagcataaga gagaaatact cactacgcca aatcgttaca ctacaccctc aaaagctaga 1021 aaccagtgcc. tgaaagaaac tccaattaaa ataccagtaa attcaacagg aacagacaag 101 ttaatgacag gtgtcattag ccctgagagg cggtgccgct cagtggaatt ggatctcaac 1141 caagcacata tggaggagac tccaaaaaga aagggagcca aagtgtttgg gagccttgaa 1201 agggggttgg ataaggttat cactgtgctc accaggagca aaaggaaggg ttctgccaga 1261 gacgggccca gaagactaaa gcttcactat aacgtgacta caactagatt agtgaatcca 1321 gatcaactgt tgaatgaaat aatgtctatt cttccaaaga agcatgttga ctttgtacaa 1381 aagggttata cactgaagtg tcaaacacag tcagattttg ggaaagtgac aatgcaattt 1441 gaattagaag tgtgccagct tcaaaaaccc gatgtggtgg gtatcaggag gcagcggctt 1501 aagggcgatg cctgggttta caaaagatta gtggaagaca tcctatctag ctgcaaggta 1661 taa Human MELK. (isofoim 8) Amino Acid Sequence (NP_O.0124362 ) 1 mvleenilfd exyhkIllidf glcakpkgak dvhlgtccgs layaapellq gksylgsead 61 vwsmgillyv Imcgflpfdd dnvmalykki mrgkvdvpkw ispssillig gmiclvdpkkr 121 ismkniinhp wimgdynypv essigsknpfih idddcvteis vhhrnnrgtm edIisiwcivd 161 hltaty1111 akkargkpvr 1r1sfscgq asatpftdik snniesledvt asdknyvagl 241 idydwceddl stgaatprts qftkywtean gveaksltpa Icrtpanklk nkenvytpka 301 avkneeyfmf pepktpvnkn ghkreilttp nryttpskar ngclketpik ipvnstgtdk 361 imtgvisper rcrsveldln gahmeetpkr kgakvfqsle rgldkvitvl trskrkgsar 421 dgprriklhy nvtttrivnp dglineimsi ipkkhvdfvg, kgytikcqtq sdfgkvtmcif 461 elevcglqkp dvvg1rrqr1 kgdavvykr1 vedilsckv Human MELK (isoform 9) cDNA. Sequence (NM_001256693.1.) 1 atgggcetac tgttatatgt tcttatgtgt ggatttctag catttgatga tgataatgta 61 atggctttat agaagaagat tatgagagga aaatatgatg ttcgcaagtg gctctctccc 121 agtagcattc tgcttcttca acaaatgctg caggtggacc caaagaaacg gatttctatg 181 aaaaatctat tgaaccatcc ctggatcatg caagattaca actatcctgt tgagtggcaa 241 agcaagaatc cttttattca cctcgatgat gattgcgtaa cagaactttc tgtacatcac 301 agaaacaaca ggcaaagaat ggaggattta atttcactgt ggcagtatga tcacctcacg 361 gctacctatc ttctgcttct agccaagaag gctcggggaa aacgagttcg tttaaggctt 421 tcttctttct cgtgtggace agccagtgct acggcattca cagacatcaa gtgaaataat 451 tggagtctgg aagatgtgac cgcaagtgat aaaaattatg tggggggatt aatagagtat 541 gattggtgtg aagatgattt atcaacaggt gctgctactc cccgaacatc acagtttacc 601 aagtactgga gagaatcaaa tggggtggaa tctaaatgat taagtccagg cttatgcaga 661 acagctgcaa ataaattaaa gaacaaagaa aatgtatata ctcctaagtc tgctgtaaag 721 aatgaagagt agtttatgtt tcctgaggca aagactcgag ttaataagaa ccagcataag 15 761 agagaaatac tgactacgcc aaatggttac actacacgct caaaaggtag aaaccagtgc 841 ctgaaagaaa ctccaattaa aataccagta aattcaacag gaacagacaa gttaatgaca 901 ggtgtgatta gccctgagag gcggtgccgc tgagtggaat tggatctcea gcaagcagat 961 atggaggaga ctccaaaaag aaagggaggc aaagtgtttg ggagcgttga aagggggttg 1021 gataaggtta tcactgtggt caccaggagc aaaaggaagg gttctgcceg agacggggcc agaagactaa agcttcacta taacgtgact acaactagat tagtgaatcc agatcaactg 1141 ttgaatgaaa taatgtctat tcttgcaaag aaggatgttg actttgtaca aaagggttat 1201 agactgaagt gtcaaacaca gtcagatttt gggaaagtga caatgcaatt tgaattagaa 1261 gtgtgccagc ttcaaaaacc ggatgtggtg ggtatcagga ggcagcggct taagggggat 1321 gcctgggttt acaaaagatt agtggaagac atcctatcta gctggaaggt ataa Human MELK (isoform 9) Amino Acid Sequence (NP 001243622,1) 1 mgilivvImc gfipfdddnv malykkimrg kvdvpkwlep ssI1.1i.qqm1 qvdpkkriam 61 kniinhpwim gdynypvewq sknpfillIdd dgvtalavhh tnnrcItmedl islwqydhlt 121 atyaillakk argkpvriri asfscggasa tpftdikenn wsledvtazd knyvaglidy 181 dwcaddIstg aatprtsqft kywtesngva sksltpalgr tpanklknke nvytpksavk 241 neeyfmfpep ktpvnknqhk reilttpnry ttpakarnqg Iketpikipv natgtdklmt 301 gvisperrcr sveldlnqah meetpkrkga kvfgslergl dkvitvItrs krkgsardgp 361 velklhyrIvt ttrivnpdql Ineimsilpk Ithvdfvqkgy tIkcqtcodf gtvtmifele 421 vcgicikpdvv girrqr1kgd awvykrIvediicv Mouse NI-ELK CDNA Sequence (NM_010790.2) 1 atgaaagatt atgacgaagt cctcaaatac tatgaactat atgaaacgat tgggacaggt 61 gggtttgcaa aggtcaaact ggcctgccat gtcctcactg gagagatggt agctataaaa 121 atcetggata agaatgcggt agggagtgat ttgccgcgag tcaaaactga gatcgatgcg 181 gtgaagagtc tgagacatca gcacatatgt caggtctacg atgtgctgga gacaaagaac 241 aaaatattga tggttctgga gtactgtgca ggaggagagc tgtttgacta cataatgtcg 301 caggatcgcc tgtcggaaga ggagacccgg gtcgtcttcc gtcagatact gtctgcagtt 361 gggtatgtgc agaggcaggg gtatgccgac agggacctca aacgagaaaa tttattattt 421 gatgaaaatc ataagctaaa gctgattgac tttggtcttt gtgcaaaacc gaagggcaac 461 aaggactacg atctggagac gtgctgtggg agccttgctt atgcagctcc tgaactaata 541 caagggaagt cgtaccttgg attagaggca gatgtttgga .gcatqggc.a:t octc.t4tat 601 gtgctcatgt gtggatttct accatttgat gatgataatg tpatggcttt gtacaagaag 661 ataatgagag ggaaatacga agttcctaag tggctctctc ccagtagcat tqtgcttctc.
721 cagcagatgt tgcaggtgga cccaaagaaa cggatttcta tgagaaatct cctgaaccat 731 ccctgggtca tgcaagatta cagctgtccc gtggagtggc aaagcaagac tcctttgact 841 cacctcgatg aggattgcgt gacagagctt tctgtacatc accgcagcag caggcagaca 901 atggaggatt taatttcgtc gtggcagtac gatcacctca cagccaccta ccttctgctt 961 ctagccaaga aggcccgggg gaagccggct cgtctacagc tcctgtcctt ctcttgtgga 1021 accgccagca ccactccaaa gtcaaagaat ctgagcctgg aagatatgag cacaagtgat iO 1081 gataactgtg tggctggatt gatagactat gaattgtgtg aagataaatt attagctccc 1141 aagacgccac aggttaccaa acacttggca gaatcaaatc acgcagcatc taaatcacca 1201 gcgocagggg tacgcagagc agtggcaaat aaattaatgg acaaagaaaa tgtgtgcact 1261 cccaagtctt ctgtgaagaz tgaagagcag tttgtatttt ctgagccgaa gattccagtt 1321 agtaagaacc agtataagag agaaataccc gcctcaccaa cccgtttccc aacacctgca 136'1 aaagctagag cccagtgcct gagagaagcc ccggttagaa caccagggaa ttccgcagga 1441 gcagacacac taacgacagg tgtcattagc cccgagagga ggtgccgttc aatggacgtg 1501 gatctcaacc aggcacacat ggaggatacc ccgaaaaaga aaggaaccaa tgtgtttggg 1561 agccttgaga gaggactgga taaggttctc actgcgctca caaggaacaa gaagaagggc 1621 tctgccagag atggaccaag aaagcgaaag ctgcactaca atgtgactac aactcgcctg 1681 gtgaaccccg accagctcct gagcgaaatc atggctattc ttccaaagaa gaacgtggac 1741 ttcgtacaga. aaggttacac tctaaagtgt caaacgcagt ctgattttgg caaagtgaca 1801 atgcagtttg aactggaagt gtgccagctg cagagacctg acgtggtagg catccggaga 1861 cagcggctga agggtgatgc ctgggtttac aagagattag tggaagatat cttgtctggc 1921 tgcaagatgt ga Mouse MELK Amino Acid Sequence (NP...034920.2) 1 mkdydellky yelyetigtg gfakvklach vItgemvaik imdknalgsd Iprvkteida 61 1kslrhghic qiyhvletkn kifmvieycp ggelidyiis qdrIseeetr vyfrgilsav 121 ayvhsqgyah rdlkpenllf denhklkiid fgicakpkgn kdyhlqtccg 51avaapeli 131 ggksylgsea dvwsmgilly vlmcgflpfd ddnvmalykk imrgkyevpk wlepssilil.
241 qqmiqvdpkk riemrnilch pwvmgdyscp vewqsktplt hidedcvtel avhhrs3Nt 301 medlisawgy dhItatyllI lakkargkpa rigllefscg tasttpkekn 1s1edmstad 361 dncvaglidy eicedkilap ktpqvtkhia esnhaasksp apgyrrayan klmdkenvct.
421 pkssvkneeq fyfsepkipv sknqykreip asptrfptpa karagclrea pvrtpgnsag 481 adtittgvis perrcrsmdv cangahmedt pkkkgtnvfg siergidkvi taltrnkkkg 541 sardgprkrk Ihynvtttrl vnpdglisei maiipkknvd fvqkgytIkc qtqsdfgkvt 601 mgfelevcgl qrpdyvgarr grlkgdawyy krlyedilsg ckm Human ciF413 cDNA Sequence (NNL)0141.7.4) 1 atggcggcct cagcaaaaaa gaagaataag aaggggaaga ctatctccct aacagacttt 61 ctggctgagg atgggggtac tggtggagga agcacctatg tttccaaacc agtcagctgg 121 gctgatgaaa cggatgacct ggaaggagat gtttcgacca cttggcacag taacgatgac 181 gatgtgtata. gggcgcctcc aattgaccgt tccatccttc ccactgctcc acgggctgct 241 cgggaaccca atatcgaccg gagccgtctt cccaaatcgc caccctacac tgcttttcta 301 ggaaacctac cctatgatgt tacagaagag tcaattaagg aattctttcg aggattaaat - 30 .-361 atcagtgcag tgcgtttacc acgtgaadcc agcaatCcag .agaggttqaa aggttttggt 421 tatgctgaat ttgaggacct ggattgcctg ctcagtgccc tgagtctaaa tgasgagtct 481 ctaggtaaca ggagaattcg agtggacgtt gctgatcaag cacaggataa. agacagggat 541 gatcgttctt ttggccgtga tagaaatcgg gattctgaca aaacagatac agactggagg 601 gctcgtcctg ctacagacag ctttgatgac tacccaccta gaagaggtga tgatagcttt 661 ggagacaagt atcgagatcg ttatgattca gaccggtatc gggatgggta tcgggatggg 721 tatcgggatg gcccacgccg ggaratggat cgatatggtg gccgggatcg ctatgatgac 781 cgaggcagca gagactatga tagaggctat gattcgcgga taggcagtgg cagaagagca 841 tttggcagtg ggtatcgcag ggatgatgac tacagaggag gcggggaccg ctatgaagac 901 cgatatgaca gacgggatga tcggtcgtgg agctcgagag atgattactc tcgggatgat 961 tataggcgtg atgatagagg tccccoccaa agacccaaac tgaatctaaa gcctcggagt 1021 actoctaagg aagatgattc ctotgctagt acctcocagt coactcgagc tgcttctatc 1061 tttggagggg caaagcctgt tgacacagct gctagagaaa gagaagtaga agaacggcta 1141 cagaaggaac aagagaagtt gcagcgtcag ctggatgagc caaaactaga acgacggcct 1201 cgggagagac acccaagctg gcgaagtgaa gaaactcagg aacgggaacg grcgaggaca 1261 ggaagtgagt catcacaaac tgggacctcc accacatcta gcagaaatgc acgaaggaga 1321 gagagtgaga agtctctaga aaatgaaaca ctcaataagg aggaagattg cctcc 1381 acttctaaac ctcccaaacc tgatcagccc ctaaagqtaa tgccagcccc tccaccaaag 1441 gagaatgctt gggtgaagcg aagttctaac cctcctgctc gatctcagag ctcagacaca 1501 gagcagcagt cccctacaag tggtggggga aaagtagctc cagctcaacc atctgaggaa 1561 ggaccaggaa ggaaagarga aaaraaagta gatgggatga atgccccaaa aggccaaact 1621 gggaactcta gccgtggtcc aggagacgga gggaacagag accactggaa ggagtcagat 101 aggaaagatg gcaaaaagga tcaagactcc agatctgcac ctgagccaaa gaaacctgag 1741 gaaaatccag cttccaagtt cagttctgca agcaagtatg ctgctctctc tgttgatggt 1801 gaagatgaaa atgagggaga agattatgcc gaatag Human ciF4B Amino Acid Sequence (NP 001408.2) 1 maasakkknk kgktisitdf laedggtggg styvskpvsw adetddlegd vsttwnsndd 61 dvyrappidr siiptapraa repnidrszl pksppytafl gnlpydvtee sikeffrgin 121 isavriprep snperlkgfg yaefedldsi 1sa1sinees lgnrrirvdv adqaqdkdrd 181 drsfgrdrnr dsdktdtdwr arpatdsfdd ypprrgddsf gdkyrdxyds dryrdgyrdg 241 yrdgprrdmd ryggrdrydd rgsrdydrgy dsrigsgrra fgsgvrrddd vrgggdrved 301 ryarrddrsw nsrddynrdd yrrddrgppg rpkInIkprs tpkeddssan tsgstraasi 361 fggakpvdta arareveeri gkegekigrg Idapklerrp rerhpswrse etgerersrt 421 gsessgtgts ttssrnarrr esekslenet Inkeedchsp tskppkpdgp lkvmpapppk 481 enawvkrnsn pparsgssdt eggsptsggg kvapagaasee gpgrkdenkv dgmnapkgqt 541 gnssrgpgdg gnrdhwkesd rkdgkkdgds rsapepkkpe enpankfssa skyaalsvdg 601 edenegedya e Mouse e1F4B cDNA Sequence (NM 1456253) 1 atggcggcct cagcaaaaaa gaagaataag aaggggaaga ccatctccct aacggacttt 61 ctagctgagg atggaggaac tggtggagga agcacctatg tccccaaacc agtcagctgg 121 gctgatgaaa cagacgatct ggaaagagat atgtcaacaa cttagcata* taacgatgat 161 gacgtgtaca gggcgcctcc aattgaccgt tccatccttc ccactgctcc acgggctgct 241 cgggaaccca atattgaccg gagccgtctt cdcaagtcgc cAccdtacAc tgctttcdta.
801 gggaatctgc cctatgatgt gacagaagac=t:ccattaagg..att*.xtttag .aggattaaat 361 atcagcgctg tacgcttacc acgggaaccc agdaatccag.acaggttgaa. aggtttcggc.
421 tacgcagaat ttgaggacct ggattctctg ctcagtgctc tgagtctcaa tgaagagtct 481 ctaggtaaca ggagaattcg tgtggatgtt gctgatcaag cacaggataa agacagggat 541 gaccgttctt ttggtcgaga tagaaatcgg gattctgaca aaacagacac agactggagg 601 gcccgtccca ccacagacag ttttgatgac tacccaccta gaagaggcga tgatagcttt 661 ggagacaagt atcgagatcg ttacgattca gaccggtatc gggatgggta tagggacgga 721 tatcgcgacg gcccacgcag agacatggac cgctatgggg gccgggatcg ctatgatgac iO 781 cgaggdagca gagactatga ccgaggctat gactccagga taggcagtgg cagaagggca 841 tttggaagtg ggteccggag agatgatgac tacagaggag gtggggaccg ctatgaagec 901 dgctatgada gacgggatga tcggtcgtgg agdtccaggg atgactactc tdgggatgat 861 tataggcgtg atgacagagg tcccccccag agacccagac tgaacctcaa gcctcgaagc 1021 gctcctaagg aggatgacgc ctccgccagc acctcccagt ccagccgggc agcctccatc 106'1 tttggagggg cgaagcctgt tgacacagct gctagggaaa gagaagtaga ggagcggcta 1141 cagaaggagd aggagaagct gdagcgtcag ctggatgagc daaaactaga ccgdcggccc 1201 cgggagagac acccaagctg gcgaagtgaa gaaactcagg aaagagaacg gtcaaggaca 1261 ggaaqtgagt catcgcaqac tggqgcctca gccacatctg gcagaaatac acgaaqgaga 1321 gagagtgaga agtctctaga aaatgaaacc ctcaataaag aagaagactg tcactctcca 1381 acctctaagc ctcctaaacc tgaccagcct ctaaaggtaa tgccagcccc tccaccaaag 1441 gagaatgcgt gggtgaagcg aagctctaac cctcctgccc gatctcagag ctcagacaca 1501 gagcagccgt cccctacaag tggtggaggg aaagtagctg cagtccagcc ccctgaggaa 1561 ggaccatcaa gaaaagatgg aaataaagtg gatgtggtgg gtgccacaca aggccaagct 1621 ggaagctgca gccgtggtcc cggggatgga gggagcagag accactggaa ggacttggat 1651 aggaaggatg gcaaaaaaga tcaagactcc agatctgcgc ctgagccaaa gaaacctgag 1741 gagaacccag cctctaagtt cagctctgca agcaagtacg ctgctctgtc tgtggatggc 1801 gaggatgagg atgagggcga cgactgcact gagtag Mouse elF4B Amino Acid Sequence (NP 663600.2) 1 maasakkknk kgktisitdf laedggtggg styvpkpvaw adetddlegd vsttwhandd 61 dvyrappidr sidptapraa repnidrari pkeppytafi gnlpydvted sikdffrgin 121 isavrlprep wapdrakgfg yaefedldsi laale1seea Igarrirvdv adgaldkdrd 181 drsfgrdrnt dsdktdtdwr arpttdsfdd ypprtgddsf gdkyrdryda drytdgyrdg 241 yrdgprrdmd ryggrdrydd rgsrdydrgy dsrigsgrra fgsgyrrddd yrgggdry'ad 301 rydrrddrsw asrddvardd yrrddrgppq rprinikprs apkeddasaa taqsaraasi 361 fggakpvdta arereveeri gkegekigrq IdepkIdrrp zerhpswzse etgerersrt 421 gsessqtgas atagrnttrr eseksienet inkeedchap tskppkpdqp Ikvmpapppk 451 enawykrsen pparagasdt eqpsptsggg kvaavqppee gpsrkdgnkv dvvgatqgqa 541 gscstgpgdg gardhwkdid rkdgkkdqds raapepkkpe enpaskfssa skyaalsvdg 601 ededegddct e Monkey elf4.13 cDNA Sequence (NM_001195808 I) 1 ctctcccaac atggcggcct cagcaaaaaa daagaataag aadgggaaga ctatCtCcCt 61 aacagacttt ctggctgagg atgggggtac tggtggagga agcacctatg tttccaaacc 121 agtcagctgg gctgatgaaa cggatgadct glaaggagat .gttteaacas 4tgg.c.aag.=
181 taatgacgac gatgtgtaca gggdgcctd.r4=aattgadgt tdatccttp ccactqctcc.
241 acgggctgct cgggaaccca atatcgaccg gagccgtztt cccaaatcgp caccctacac 301 tgcttttcta gggaacctac cctatgatgt gacagaagaa tcaattaagg aattctttag 361 aggattaaat atcagtgcag tgcgtttacc acgtgaaccc agcaatccag agaggttgaa 421 aggttttggt tatgctgaat ttgaggacct ggattccctg ctcagtgccc tgagtctcaa 431 tgaagagtct ctaggtaaca ggagaattcg agtggacgtt gctgatcaag cacaggataa 641 agacagggat gatcgttctt ttggccgtga tagaaatcgg gattctgaca aaacagatac .601 agactggagg gctcgtcctg ctacagacag ctttgatgac tacccaccta gaagaggtga iO 661 tgatagcttt ggagacaagt atcgagatcg ttatgattca gaccggtatc gggatgggta 721 tcgggatggc ccacgccggg atatggatcg atatggtggc cgggatcgct atgatgacog 781 aggoagcaga gactatgata gaggctatga ttcccggata ggcagtggoa gaagagcatt 841 tggcagtggg tatcgcaggg atgatgacta cagaggaggc ggggaccgat atgaagaccg 901 atacgacaga cgggatgatc ggtcgtggag ctccagagat gattactctc gggatgatta 961 taggcgcgat gacagaggtc cccctcaaag acccaaactg aatctaaagc ctcggagtac 1021 tcctaaggaa gatgattcct ctgntagtac ctcccagtcc agtcgagctg cttctatctt 1081 tggaggggca aagcctgttg acacagctgc tagagaaaga gaagtagaag aacggctaca 1141 gaaggaacaa qagaagttgc agcqtcagct ggatgaqcca asactagasc gacggcctcg 1201 ggagagacac ccaagctggc gaagtgaaga aactcaggaa cgggaacggt cgaggacagg 1261 aagtgagtca tcacagactg ggacctccgc cacatctggc agaaawcac gaaggagaga 1321 gagtgagaag tctctagaaa atgaaacact caataaggag gaagattgtc actctccaac 1361 ttctaaacct cccaaacctg atcagcccct aaaggtaatg ccagcccctc caccaaagga 1441 gaatgcttgg gtgaagcgaa gttctaacc tccagctcga tctcagagct cagacacaga 1501 gcagcaatcc cctacaagtg gtgggggaaa agtagctcca gctcaaccat ctgaggaagg 1561 accagcaagg aaagatgaaa ataaagtaga tgggatgaat gtcccaaaag gccaaactgg 1621 gacctctagc cgtggaccag gagacggagg gaacaaagac cactggaagg agtcagatag 1681 gaaagasggc aasaaggatc aagacsccag atctgcacct gagccsaaga aacctgagga 1741 aaatccagct tcgaagttca gttctgcaag caagtatgct gctctctctg ttgatggtga 1801 agatgaaaac gagggagaag attatgccga atagacctct acatcctgtg ctttctccsa 1861 gtttctctcc accctggaac attcmagagc aaatcaaaac ctctatccag acaagacaaa 1921 ataaaactca ccatctcctg aagacctttc ttaccttttt ttaaaaacaa aaaatgaaat.
1981 tattttgcat gctgctgcag cctttaaagt attaaagtaa ctggagaatc gccaatatag 2041 ccagagagaa agggactaca gctttttaga ggaagagttg tggtgtgtta Monkey eIF4B Amino Acid Sequence (NP_)011.82737.1) 1 maasakkknk kgktisltdf laedggtggg styvskpvsw adetddlegd vsttwhsndd 61 dvyrappidr silptapraa repnidrsrl pksppytafl gnipvdvtee sikeffrgin 121 isavrlprep snperikgfg yaefedidsI Isalsinees ignrrirvdv adqagdkdrd 131 drsfgrdrnr dsdktdtdwr arpatdsfdd ypprrgddsf. gdkyrdryds dryrdgyrdg 241 prrdmdrygg rdryddrgsr dvdrgydsri gsgrrafgsg vrrdddyrgg gdrvadrydr 301 rddrswssrd dysrddyrrd drgppqrpk1 nikpzetpke ddssastsgs sraasifgga 361 kpvdtaarer eyeerigkeg ekigrgIdep klerrprerh pswrseetge rersrtgses 421 sqtgtsatsg rnarrzesek slenetinke edchsptskp pkpdqpikvm papppkenaw 461 vkrssnppar scpsdteqqs ptsgggkvap acIpseegpar kdenkvdgmn vpkgqtgtss 541 rgpgdggnkd hwkesdrkdg kkdqdsrsap epkkpeenpa skfsaaskya alsvdgeden .601 egedyae Cow cl.F4B eDNA Sequence (NM_0010350282) 1 atggcggcct cagcgaaaaa gaagaataag aaggggaaga ctatctcnct aacagacttt 61 ctggctgagg atggagggac tggtggaggc agcacctatg tccccaaacc agtcagctgg 121 gctgatgaaa cagacgatct ggaaggggar gtttcaacca crtggcatag taatgatgat 181 gatgtgtatc gggcacdtcc aattgaccgt tccatdctgc cdactgctdc acgggctgct 241 cgggaadcca atatcgaccg gagccgtctt cccaaatctc caccctacac tgcttttcta iO 301 gggaacctgc cctatgatgt gadagaagad tccattaagg aattctttag aggattaaat 361 atcagtgcag tgogtttado gcgtgaaccc agdaatcctg agaggttaaa aggttttggt 421 tatgcagagt ttgaggacct ggattccttg ctcagtqcct tgagcctcaa cgaagagtct 461 ctaggtaaca ggagaattcg agtggacgtt gctgatcaag cacaggataa agacagggat 541 gatcgttctt ttggccgaga tagaaatcgt gattctgaca aaacagatac agactggagg 601 gccdgtcctg ctgcagacag ctttgatgac tacccgccca gaaggggtga tgatagctrt 661 ggagacaagt atdgagatcg ttacgattca gacagatatc gtgatgggta tcgggacagt 721 taccgtgatg gcccacgccg ggacatggat cgatacgggg gccgagatcg ctatgatgac 781 cgaggtggca gagactatga cagaggctac gattccagga taggcagtgg cagaagagca 841 ttcggtagcg ggtaccggag ggatgatgac tacagaggag gcggggaccg ctatgaagac 901 agatacgaca gacgagatga ccggtcctgg agttccagag atgattactc tcgggatgat 961 tacaggcggg atgatagagg tccccctcaa agacccaaac tgaacdtaaa gcctcggagt 1021 actcctaagg aagatgattc ctccgctagc acctcdcagt cdagtcgtgc agcctctatc 101 tttggagggg caaagcctgt tgacacagct gctagagaac gagaagtaga agagcggcta 1141 cagaaggaac aggagaaact gcagcgtcag ctggatgagc caaaactaga acgacggcct 1201 cgggagagac acccaagctg gcgaagtgaa gaaactcagg aacgggaacg atcgaggaca 1261 ggaagtgagt catcacagac tgggacctca gccacatctg gcagaaatgc aagaagaaga 1321 gagagtgaga agsctttaga aaatgaaacd cccaataaag aggaagactg tcagtctcca 1381 acttctaagc ctcccaaacd tgaacagcct ctaaaggtaa tgccagcccc tccaccaaag 1441 gagaatgctt gggtgaagcg aagttctaac cctcctgctc gatctcagag ctcagacaca 1501 gagcagcagt cccctacaag tggtdgaggg aaagtagttc cagctcaact atctgadgaa 1561 ggatcagcaa ggaaagatga aaataaagta gatggggtga gtgccccaaa aggccaaagt 1621 gggagctcca gccgtggtcc gggagatggg gggaacaaag accactggaa ggaggcagac 1681 aggaaagatg gcaaaaagga tcacgactcc agatctgcac ctgagccaaa gaaagctgaa 1741 gaaaatccag ccscgaagtt cagatctgca agcaagtacg ctactctcgc cattgacggt 1801 gaagatgaaa atgagggaga ttacaccgaa tag COW eiFAIB Amino Acid Sequcnce (NP 001030200.i) 1 maasakkknk kgktisitdf laedggtggg styvpkpvsw adetddlegd vettwhsndd 61 dvyrappidr siiptapraa repnidrsrl pksppytafl gnipvdvted sikeffrgin 121 isavrIprep snperikgfg yaefed1ds1 Isalsinees ignrrirvdv adqagdkdrd 181 drafgrdrnr dsdktdtdwr azpaadeedd ypprzgddsf gdkyrdryds dryzdgyrds 241 yrdgprrdmd ryggrdrydd rggrdydrgv dsrigsgrra fgsgyrrddd yrgggdryed 301 rydrrddrsw ssrddysrdd yz/ddrgppq rpkinIkprs tpkeddssas tsqssraasi 361 fggakpvdta arereveeri gkegeklqrq idepkierrp rerhpswrse etgerersrt 421 gseseqtgts atagrnarrl esekslenet pnkeedcqap tekppkpegp Ikvmpapppk 01 enawykresn pparsgasdt eggspteggg kvvpaqlsee gaarkdenkv dgvaapkgqs 541 gsssrgpgdg gnkdhwkaad rkdgkkdhds rsapepkkae enpaskfrsa skyatiaidg 601 adenegdyte Rat ofF4B cDNA Sequence (NM 001008324, D.
1 atggcggcct cagcaaaaaa gaagaataag: aaggggaaga ccat.ctccct aacagacttt 61 ctagctgagg atgggggaac tggtggagga agcacctatg tccccaaacc agtcagctgg 121 gctgatgaaa cagacgatct ggaaggagat gtgtcaacaa cttggcatag taacgatgac 151 gatgtgtaca gggcacctcc tattgaccgt tccatccttc ccactgctcc acgggctgct 241 cgggaaccca. atattgatcg gagccgtctt cccaagtcac caccctacac tgctttccta 301 gggaatctgc cctatgatgt gacagaagac tctattaagg atttctttag aggattaaat 361 atcagcgctg tacgcttgcc gcgtgagccc agcaatccag acaggttgaa aggttttggc 421 tatgccgaat ttgaggatct ggattctctg ctcagtgctc tgagtctcaa tgaagagtct 15 451 ctaggtaaca ggagaattcg ggtggatgtt gctgatcaag cacaggataa agacagggat 541 gaccgttctt ttggtcgaga tagaaatcgg gattctgaca agacagacac agactggagg 601 gcccgtcctg ccacagacag ctttgatgac tacccaccta gacgaggtga tgacagcttc 661 ggagecaagt atcgagatcg ttacgagtca gaccggtatc gggatgggta tagggecgga 721 tatcgggacg gcccacgcag agacatggac cgctatgggg gccgggatcg ctatgatgac 20 781 cgaggcagca gagactatga ccgaggctat gactccagga taggcagtgg cagaagagca 841 tttggaagtg ggtaccggag ggatgacgac tacagaggag gtggggaccg ctatgaagat .901 cgctatgaca gacgggacga tcggtcatgg agctccaggg acgattactc tcgggacgat 961 tacaggcgtg atgacagagg tcccccccaa agacccaaac tgaatctaaa gcctcggagt 1021 actcctaaag aagatgattc ctctgctagc acctcccagt ccagccgagc ggcttctatc 25 1081 tttggagggg cgaagcctgt tgacacagct gctagagaaa gagaagtaga ggagcggcta 1141 cagaaggagc aggagaagct gcagcgtcag ctggatgagc caaaactaga ccgccggccc 1201 cgggagagac acccaagttg gcgaagtgaa gaaactcagg aaagagaacg gtcgaggaca 1261 ggaagtgagt catcgcagac tgggacctca gccacatctg gcagaaatac acgaaggaga 1321 gagagtgaga agtctctaga aaatgaaacc ctcaataaag aagaagactg tcactctcca 30 1381 acctctaagc ctcctaaacc tgaccagcct ctaaaggtaa tgccagcccc tccaccaaag 1441 gagaatgcgt gggtgaagcg aagctotaac cctcctgctc gatctoagag ctcagacaca 1501 gagcagccgt cccctacaag tggtggaggg aaagttgctc cagctcagcc ctctgaggaa 1561 ggaccatcaa ggaaagatga aactaaagtg gatggggtga gcaccaccaa aggccagact 1621 ggacactcca gccgtggtcc tggggatgga gggagcagag accactggaa ggagttggat 35 1651 aggaaggacg gcaaaaaaga tcaagactcc agatctgcac ctgagccaaa gaaatctgag 1741 gagaaccgag cctctaagtt cagttctgca agcaagtacg ctgctctgtc tgtggacggt 1801 gaggatgagg atgagggaga cgactgcact gagtag Rat e1F4)3 Amino Acid Sequence (NP 3)01.008325.1) maasakkknk kgktialtdf laedggtggg styvpkpvsw adetddlegd vsttwhsndd 61 dvyrappidr siiptapraa repnidrsrl pksppytafl gnipydvted sikdffrg1n 121 isavrlprep snpdrikgfg yaefedidsi Isalslnees Ignrrirvdv adqaqdkdrd 131 drsf*rdrnr dsdktdtdwr arpatdsfdd ypnrrgddsf. gdkyrdryes dryrd*yrdg 241 yrdgprrdmd ryggrdrydd rgsrdydrgv darigsgrra fgsgyrrddd yrgggdryed - 35 .-301 rydrrddrsw ssrddysrdd yrrddrgppq rpkInlkprs tpkeddssas tsgssraasi 861 fggakpvdta arerevaeri qkageklqrg Idepkldrrp rerhpswrse etgerersrt 421 gsessgtgts atsgrntrrr esekalenet Inkeedchap tskppkpdcrp ikvmpapppk 4S1 enawvkrsen pparecp3dt ecip3ptsggg kvapagpsee gp3rkdetkv dgvt-stkgqt 541 ghssrgpgdg gardhwkaid rkdgkkdqds raapepkkse enraakfssa akyaalsvdg 601 adadegddct e Human Historic HI I Amino Acid Sequence ('W 003520.1) 1 martkqtark stggkaprkg latkaarksa patggykkph ryrpgtvair eirrygkate 61 Ilirklpfqr Ivreiagdft tarfgssav malgeaceay Ivgifedthl c,eihakrvti 121 mpkdiglerr irgera Mouse Histone H3.1 Amino Acid Sequence (NP 038578.2):
1 martkqtark stggkaprkg latkaarksa patggvickph ryrpgtvair eirrycikete 61 Ilirklpfqr Ivreiagdfk tdirfqssav maigeacsay 1vg1iedtn1 caihakrvti 121 mpkdiglarr irgers Human Histone H3,2 Amino Acid Sequence (NP...00l 005464.4 1 martkqtark stggkaprkg latkaarksa patggvkkph ryrpgtvair eirryqkste 61 Ilirkipfqr lvreiagdfk tdIrfqssav malgeaseay Ivglfedtn1 caihakrvti 121 mpkdiglarr irgera Mouse Histone 1112 Amino Acid Sequence(NP=835587,1):
martkqtark stggkaprkq latkaarkta paragvkkpb tyrpgtvair .eirrygksta 61 11.irkipfqr ivrelazidfk tdIrfq5sav. malgeaseay Ivgifedtni calhakrvti.
121 mpkdiglarr irgera Human Histone .H3.3 Amino Acid Sequence (NP 002098.1):
1 martkqtark stggkaprkq latkaarkza pstggvkkph ryrpgtvalr eirryqkste 61 Ilirkipfgr ivreiacidfk tdirfgsaai galqeaseay Ivgifedtni caihakrvti 121 mpkdiglarr irgera Mouse Histone 113.3 Amino Acid Sequence (NP 032237,1):
1 martkgtark stggkaprkg latkaarksa patggvkkph ryrpgtvair eirryqkate 61 llirklpfeir lvreiagdfk td1rfgaaai galgeas:eay 1vg1fedtn1 caihakryti 121 mpkdiglarr irgers Nucleic acid and protein molecules (e.g., those of .MELK, elf4B,, and orthologs thereof across species) that differ due to degeneracy of the genetic code or due to encoding or having "non-essential", "conservative", "stereoisomers", or "unconventional" amino acids that do .not appreciably alter the enzymatic (e.g., kinase) and/or eiF4B
Ser-406-regulatory ability of MELK are included within the scope of the invention, A
"conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Stereoisomers (e.g., D-ainino acids) of the twenty conventional amino acids, unnatural amino acids such as alpha,alpha-disubstituted amino acids, N-alkyl amino acids; lactic acid, and other .unconventional amino acids may also he suitable components for poiypeptides described herein. There is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequences that can code for the protein, as defined by the genetic code (shown below).
Likewise, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.
GENETIC CODE
Alan ine (Ala, A) GCA, SCC, GCS, GOT
Ar gi nine (Arg, R) AGA, AOG, CGA, (XX:, OGG, cGT
Asparacarie (Asn, N) MC, MT
Aspartic acid (Asp, D) GAO, GAT
Cysteine (Cy, C) TGOõ TGT
Glutamic acid (Giu, E) GM, GAG
Glutamine (Gin, Q) CAA, CAG
Glycine (Giv, G) GGA, GGC, GGG, GGT
Histidine (His, H) CAC, CAT
Isoleucine (Ile, 1) ATA, ATC, ATT
Leucine (Leu, L) CTA, CTC, CTG, OTT, TTA, TTG
Lysine (Lys, K) AAA, MG
Methionine (Met, M) ATG
Phenyialanine (Phe, F) TTC, Terr Proline (Pro, P) CCA, CCO, CCG, COT
Serine (Ser, S) AGO, ACT, TCA, TOO, TOG, TCT
Threonine (Thr, T) ACA, ACC, ACS, ACT
Tryptophan (Trp, TGG
Tyrosine (Tyr, Y) TAO, TAT
Vali /le (Val, V) GTA, GPO, GTG, GTT
Termination signal (end) TM, TAG, TGA
An important and well known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (for example, illustrated above).
Therefore, a number of different nucleotide sequences may code for a given amino acid sequence.
Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others) Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such .methylations do not affect the coding relationship between the trinucleotid.c codon and the corresponding amino acid. In addition, a skilled artisan will understand how to mutate nucleotides of a specific codon so as to specifically alter an.
encoded amino acid based on the relevant codon chart. Additional desired .nucleic acid and/or amino acid modifications can be engineered using site-directed .mutagenesis and PC:R.-mediated mutagenesis techniques.
The "nucleic acid" can take any of a number of forms (e.g.. DNA, naRNA, cDNA) that encode a biomarker described herein. For example, such biomarker nucleic acid_ molecules include 'DNA (e.g, genornie 'DNA and cDNA) comprising the entire Of a partial .15 sequence of a desired gene or the complement or hybridizing fragment of such a sequence.
The biomarker nucleic acid molecules also include RNA comprising the entire or a partial.
sequence of a desired one or the complement of such a sequence, wherein all thymidine residues are replaced with arichne residues. A "transcribed polynueleotide" is a polynucleotide (e.g., an RNA, a eDNA, or an analog of one of an RNA or .cDNA) which is complementary to or homologous with all or a portion of a mature RNA made by transcription of a biomarker of the present invention, at least in part, and normal post-transcriptional processing 4..g.õ splicing), if any, of the transcript, and reverse transcription of the transcript.
The terms "homology" or "identity," as used interchangeably herein, refer to sequence similarity between two polynueleotide sequences or between two polypeptide sequences, with identity being a more strict comparison. The phrases "percent identity or homology" and "% identity or homology" refer to the percentage of sequence similarity found. in a comparison of two or more polynueleotide sequences or two or more poiypeptide sequences. Two or more sequences can be anywhere from 0-100% similar, or any integer value there between. Identity or similarity can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of similarity or identity between polynucleotide sequences is a function of the number of identical or matching nucleotides.
at positions shared by the polynucleotide sequences. A degree of identity of polypeptide sequences is a function of the .number of identical amino acids at positions shared by the polypeptide sequences. .A degree of homology or similarity of polypeptide sequences is a function of the number of amino acids at positions shared by the polypeptide sequences.
The term "substantial homology" refers to homology plat. least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more (e.g., about 96%, 963%, 97%, 97.5%, 98%, 98.5%, 99%, 993%, 99.6%, 99.7%, )9.8% 99.9% or mom). In one embodiment, biomarker nucleic acid molecules encode a protein or portion thereof which includes an amino acid sequence which is sufficiently homologous to an amino acid sequence described herein such that the protein or portion thereof maintains, fOr example, the ability to phosphotylate elF4Bõ to phosphorylate Histone H3, and/or the ability to be phosphorylated by MEM.
The comparison a sequences and determination of 'percent homology between two .15 sequences can be .accomplished using a .mathematical algorithm. The alignment can be performed using the Clustal Method., Multiple alignment parameters include CAP
Penalty ::40, Gap Length Penalty 10, For DNA alignments, the pairwise alignment parameters can be Htuple,--,2, Gap penaitv5. Window,---4, and Diagonal saved4. For .protein alignments, the pairwise alignment parameters can be Ktuple,----1, Gap penaltr3, Window,----5, and Diagonals Saved,----5. Similarly, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch J. Ma BIoL
(48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG
software package (available online), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. in yet another embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available online), using a NWSgapdna.C.MP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid. or nucleotide sequences is determined -using the algorithm of E. Meyers and W.
Miller (CAMS, 4:11.-17 (1989)) which has been incorporated into the ALIGN program (version 2.0) (available online), using a .PAM1.20 weight residue table, a gap length penalty of 12 and a gap penalty of 4, Methods for the production of nucleic acids (e.g. , MELK, eIF4B, and/or mRNA.s translated from nucleic acids having structured 5 regions are known in the art and include standard hybridization, PCR, and/or synthetic .nucleic acid techniques. The .nucleic acid so amplified can be cloned into an appropriate vector and characterized. by DNA
sequence analysis.
A "biomarker protein" is a protein encoded by or corresponding to a biomarker of the present invention. The terms "protein" and "polypeptide" are used interchangeably herein. In one embodiment, the. protein is at least 50%, 60%, 70%, 80%, 90%, and 95% or mom (e.g, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%., 99.1%, 99.1%, 99.3%, 99,4%, 99.5%, 99.6%, 991%, 99.8%, 99,9% or more) homologous to the entire amino acid sequence of a MELK andfor eiF4B and/or Histone H3 protein described herein. In addition, biologically active portions of .MELK and/or eIF4B and/or Historic.
H3 proteins described herein are included which have at least 50%, 6%, 70%, 80%, 9%, and 95% or more (e.g., 95.5%, 96%, 96_5%, 97%, 97.5%, 98%, 983%, 99%, 991%, 992%, 99.3%, 99.4%, 993%, 99.6%, 99.7%, 99.8%, 99.9% or more) homology to a fragment of a MELK
and/or e1F4B andlor Histone H3 protein described herein, e.g., a domain or motif, and that is capable of phosphorylating elF4B, phosphorylating Histone H3, or beine phosphorylated by MELK. Typically, biologically active portions (peptides, e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40. 50, 100, 150, 200, 250, 300, 350) 400, 450, or more amino acids in length) comprise a domain or motif, e.g., a MELK
kinase domain., eI.F4B domain encompassing an amino acid residue phosphorylatable by MELK
such as a MELK.-mediated phosphorylation substrate motif having an ar6nine a.t

-3 amino acid residue positions relative to serine/threonine (e.g., Ser406 or Ser422 of human eIF4B), or Historic .H3 domain encompassing an amino acid residue phosphorylatable by MELK
such as a human Histone H3 region comprising Sex TO. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the activities described herein.
Methods for the production of proteins (e.g., MELK and/or e1F4B and/or =Histone H3) are known in the art and include e.g , the expression of the protein in appropriate cells starting from a cDNA or the production by subsequent addition of amino acids to a starting amino acid (see Current 'Protocols, 'John Wiley & Sons, Inc,, New York), Furthermore, methods for the production of protein fragments are known in the art and include the cleavage of the protein with appropriate proteases or the generation of nucleic acid - 40 .-fragments encoding the protein fragments and subsequent expression of the fragments in appropriate cells. Methods for the production of mutated proteins, e.g., by exchanging and/or deleting one or more amino acids, are known in the art.
B. Diagnostic Methods Methods are provided for identifying agents, such as small molecules and antibodies, which inhibit oncogenic and/or kinase activity of human --MELK or an ortholog thereof; comprising: a) contacting a sample comprising i.) human MELK or an ortholog thereof and ii) human eukaryotic initiation factor 4B (eIF4B) or an ortholog thereof, with the agent; and b) determining the ability of the agent to inhibit Ser-406 phosphorylation of human e1F4B or a corresponding phosphorylatable amino acid in the ortholog of human e1f4B, wherein decreased phosphoi-ylation identifies an agent which inhibits .kina.se or oneogenic activity of human MELK or the ortholog thereof. Similarly, methods are provided for identifying agents, such as small molecules and antibodies, which inhibit oncogenic andlor kinase activity of human MELK or an ortholog thereof, comprising: a) contacting a sample comprising i) human MELK or an ortholog thereof and ii) human Histone H3 or an ortholog thereof with the agent; and b) .detertnining the ability attic agent to inhibit 'flu-3 phosphorylation and/or Ser-1.0 phosphorylation and/or Thr-11 phosphoration of human Histone 113, or a corresponding phosphotylatable amino acid in the ortholog of human HistoneH.3, wherein decreased phosphorylation identifies an agent which inhibits .kinase or ortcogenic activity of 'human MELK or the ortholog thereof These methods are also referred to herein as drug screening assays and typically include the step of screening a candidate/test compound or agent for the ability to interact with (e.g., bind to) a MELK and/or cIF4B and/or Historic .H3 protein, to modulate the phosphorylation of elf4B by MELK, to :modulate the interaction of a phosphorylatable residue of elF4B with a.
MELK-mediated intracellular signalinn target, to modulate the phosphorylation of Histone 1113 by MELK., and/or to modulate the interaction of a phosphorylatable residue of Histone H3 with a MELK-mediated intracellular signaling target. Test compounds or agents which have one or more of these a.bilittes can be used as drugs to treat disorders characterized by aberrant, abnormal, andlor unwanted MELK and/or e1f4B andior Histone11.3 nucleic acid expression aid/or protein activity, such as cancer. Candidate/test compounds include, kir example, small organic and. inorganic molecules (e.g., molecules Obtained frçm.
combinatorial and natural product libraries). Similarly, antibody agents that, for example bind to .NIELK in a manner that modulates phosphorylation of a residue by MELK, modulates efF4B activity normally driven by N1ELK-mediated phosphorylation, and/or modulates HistoncH3 activity normally driven by MELK-mediated phosphorylation, can be useful agents. The skilled artisan can also readily make other modulatory agents, such.
as aptamersõ antisense RNA, siRNA, that are capable of interacting with M.E.LK
nucleic acids and/or proteins to affect MELK-mediated phosphorylation of eIF4B or Histone 1-13 (see, at least Chung et al, (2012) 3:1629-1640; WO 2013/109388; WO
2012/016082; WO
20131045539; each of which is incorporated herein in its entirety by this reference.
The term "sample," "tissue sample," "subject sample," "subject cell or .tissue sample" or "specimen" each refer to a collection of similar cells obtained from a tissue of a subject or subject either as in vitro (e.g., cultured), ex vivo, or in vivo (e.g., isolated primary cells) samples. The source of the tissue sample may be solid tissue as from a =fresh, frozen and/or preserved organ, tissue sample, biopsy, or aspirate, blood or any blood constituents;
bodily fluids such as whole blood, serum, plasma, buccal serape, saliva, cerebrospinal fluid, urine, stool, and bone marrow, amniotic fluid, peritoneal fluid or interstitial fluid; or cells from any time in gestation or development of the subject. The tissue sample may contain compounds that are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics or the like, The sample may further comprise cancer cells, such as ovarian, lung, breast, and multiple myeloma cancer cells or any cancer in which MEEK and/or eiF4B and/or Histone :113 is amplified or overexpressedõ
has an activating mutation, or is activated by other .kinases.
The terms "subject" and "patient" are used interchangeably. A.s used herein, the terms "subject" and "subjects" refer to an animal, e.g., a mammal including a non-primate (e.g., a cow, pig, horse, donkey, goat, camel, cat, dog, guinea pig, rat, mouse, Sheep) and a.
primate (e.g., a .monkey, such as a cynornolgous monkey, gorilla, chimpanzee and a.
human).
The term "inhibit" refers to a statistically significant decrease in a metric of interest, such as the reduction of .Thr-3 phosphotyiated and/or Ser-10-phosphotylated and/or Thr-11-phosphotylated Historic H3, Ser-406-phosphorylated e1F4B, .NIELK enzymatic.
activity (e.g., kinase activity), cancer progression, and the like. Such statistically significant dectvase can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more relative to a control. For example, a test compound administered and analyzed according to the methods described herein can comprise a bona fide inhibitor of -NIELK
enzymatic activity (e.g., kinase activity) by decreasing Ser-406-phosphorylated efF413 amounts, Thr-3 phosphorylated Histone H3 amounts, Ser-1 0-phosphorylated Historic H3 amounts, and/or Thr-11 -phosphorylated Histone H3 amounts by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more relative to that of no MELK ligand administration or over a given amount of time. In one embodiment, the term "MELK inhibitor" is a substance, such as a small molecule, antibody, antisense nucleic acid, small interfering nucleic acid, which interferes with the phosphorylation of human cIF4B at Ser-406 or at a corresponding pliosphoiylation site in an el.F4B ortholog thereof the phosphorylation of human Histone 1-13 at Thr-3 or at a corresponding phosphorylation site in a Histone 113 ortholog thereof, the phosphorylation of human Histone H3 at Ser- 10 or at a corresponding phosphorylation site in a Histone H3 ortholog thereof, and/or the phosphorylation of human Histone H3 at Thr-11 or at a corresponding phosphorylation site in a Histone H3 ortholog thereof Exemplary MELK inhibitors are well known in the art, such as OTSSP167, siomyein A.
thiostrepton, and anti-MELK antibodies are disclosed, for example, in Chung a a/. pm) oncotarget 3:1629-1640; WO 2013/045539; WO 201.3/109388; and WO 2012/01.6082; each of which is incorporated in its entirety herein by this reference.
The term "altered amount" of a biomarker or 'altered level" of a biomarker refers to increased or decreased expression, modification, and/or activity of a biomarker of the Present invention, at least in part in a sample as compared to that in a control sample.
The amount of a biomarker in a subject: is "significantly" hi her or lower than the normal amount of a biomarker, if the amount of the biomarker is greater or less, respectively, than the normal level by an amount greater than the standard error of the assay employed to assess amount, or at least two, three, four, five, ten or more times that amount.
Alternatively, the amount of the biomarker in the subject can be considered "significantly"
higher or lower than the normal amount if the amount is at least about two, at least about three, at least about thur, or at least about five times, higher or lower, respectively, than the.
normal amount of the biomarker (e.g., in a control sample or the average expression level of the biomarkers of the present invention ia several control samples).
"Likely to," as used herein, refers to an increased probability, that an item, object, thinly, or person will occur such as at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more (or any range inclusive).
Thus, in one embodiment, an agent that is likely to inhibit MELK-mediated phosphorylation of - 43 .-efF413 has an increased probability of inhibiting Ser-406 phosphorylation of human efF4B
or a corresponding phosphorylatable amino acid in an ortholog of human eIF4B.
In another embodiment, an agent that is likely to inhibit MELK-mediated phosphorylation of Histone H3 has an increased probability of inhibiting Thr-3 phosphorylation, Ser40 phosphorylation, and/or Thr-11 phosphorylation of human Histone H3, or a corresponding phosphorylatable amino acid in an ertheing of human Historic H3, Test compounds of the present invention, at least in part, can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including:
biological libraries; spatially addressable parallel solid phase or solution phase libraries;
synthetic library methods rewiring deconvolution; the 'one-bead one-compound library method; and synthetic library methods using affinity chromatography selection.
The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide Oli,g,0111CT or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Dm 12:145).
Examples of .methods for the synthesis of molecular libraries can be found in the art, .for example in; DeWitt etal. (1993) Proc. Nail. Acad. Sc). USA. 90;6909; Erb et at (1994) Proc. Natl. /lead Set USA 91;11422; Zuckermann et al. (1994)J. .Med.
Chem.
37:2678; Cho et (1993) Science 261:1303; Carrell (1994) Angew. Chem. hit.
Ed Engl. 33:2059; Carell ei al. (1994) Angew. Chem. Int Ed. Engl. 33:2061; and in Gallop et (1994)J Med. Chem. 37:1233.
Libraries of compounds may be presented in solution (e.g., Houghten (1992) ilioiechnique,s 13:412-421); or on beads (Lain (1991) Nature 354:82-84), chips (Fodor (1993)Nature 364:555-556), bacteria (Ladner USP 5,223,409), spores (Ladner USP
'409), plasmids (Call et al. (1992) Proc 'lead Sc) USA 89:1865-1869) or on Phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249404-406); (Cwirla ci at (1990) Proc. Nall. Acad. Sc). 87:6378-6382); (Felici (1991) Blat 222:301-310);
(Ladner supra.).
In one embodiment, the inhibition of Ser-406 phosphorylation of human elF4B or a corresponding phosphorylatable amino acid in an onholog of human elF4B is determined by comparing the amount of Ser-406 phosphorylated human elf4B or a corresponding phosphorylatable amino acid in the ortholog of human elf4B, in the sample relative to a control. The control can be the amount of amount of Ser-406 phosphorylated human elF4B
or a corresponding phosphorylatable amino acid in the orthelog of human c1F4B
in the sample relative to said amount n the absence of the agent or at an earlier timepoint after contact of the sample with the agent, The phosphorylation level of eIF4B is generally determined by measuring the amount of phosphorylatcd elF4B protein and, optionally, of unphosplaorylated eTF4Bõ and normalizing the amount of phosphotylated protein to the total protein in the sample being analyzed. The calculated response phosphorylation level in the presence of the test compound and the basal or background phosphorylation 1CVCIS (e.g., in the absence of the test compound or at a earlier timepoint after test compound administration) are thus not affected by differences in the absolute quantity of the indicator protein at a given time, The discriminatory time point, or predetermined time after administering the test compoimd to cells, can be selected to achieve a calibrated statistically significant difference between Ser-406 phosphorylation levels in the sample relative to controls. The difference may be maximal at the predetermined time but that is .not required and depends on other parameters of the teL in addition, whereas the calculation of ratios as described 'herein is beneficial in providing useful comparative numbers, calculation of absolute differences between phosphorylated elF4B levels upon administration of test compounds relative to controls., and between test subjects and control subjects, could also be employed and would be effective.
In some embodiments, the methods described above can further comprise' determining the amount of determining the amount of a protein translated from an rtiRNA
with hiuhly structured 5' untransiated region (5'UTR), optionally wherein the protein is selected from the group consisting of cellular .myelocytomatosis oncogene (c-Myc), X-linked inhibitor of apoptosis protein .(XIAP), and ornithine decarboxylase (0D-C.1), lt is known that elF48 stimulates the hehease activity of eIF4A few unwinding the secondary structure of 515TR, of mRN.A and that eif4.13 is important for the translation of mRNA with structured 5' UTR (Dmitriev ei al. (2003) Mol. Cell &oL 23:8925-8933 and Shahbazian at (2010) lila Cell Blot 30 1.478-1485). The skilled artisan is well aware of mRNA with structured 5' iTTR encoding oneogenic proteins, such as c-Mye, X1AP (X-linked inhibitor of apoptosis protein), ODC (omithine decarboxylase), -VEGF, H1F-lalpha, and the like (see, at least Bert et al (2006) RNA 12:1074-1083).
=Phosphotylation is a biochemical reaction in -whiCh a phosphate Ltroup is added to Ser., Thr or Ti residues of a protein and is catalyzed by protein kinase enzymes.
Phosphorylation normally modifies the functions of target proteins, often causing - 45 .-activation, As pan of the cells homeostatic mechanisms, phosphotylation is only a transient process that is reversed by other enzyme called phosphatases.
Therefore, protein phosphorylation levels change over time and can be evaluated, in a number of well-known manners, including, for example, by immunological approaches. For example, the amount of Ser-406 phosphorylated human elF4B or a corresponding phosphorylata.ble amino acid in an ortholog of human elF4B is determined by an immunoassay using a reagent which specifically binds with Ser-406 phosphorylated human clE4B or corresponding phosphorylated ortholog of human elF4B, Such an immunoassay comprise a number of well known forms, including, without limitation, a radioimmunoassay, a Western blot assay, an immunofluorescence assay, an enzyme immunoassay, an immunopreeipitation assay, a chemil uminescence assay, an immunohistoellemical assay, a dot blot assay, or a slot blot assay. General techniques to be used in performing the various immunoassays noted above and other variations of the techniques, such as in situ proximity ligation assay (PLA), fluorescence polarization immunoassay (FP1A), fluorescence immunoassay (11A), enzyme immunoassay (HA), nephelometric inhibition immunoassay (NIA), enzyme linked immunosorbent assay (ELBA), and radioimmunoassay (RIA),.ELISA, etc. alone or in combination or alternatively with NMR, MALD1-TOF, LC-MS/MS, are known to those of ordinary skill in the art.
In one embodiment, the enzyme immunoassay is a sandwich enzyme immunoassay.
using a capture antibody or .fragment thereofwhich specifically binds with human eIF4B or corresponding ortholog of human elf4B and a detection antibody or fragment thereof which specifically hinds with Ser-406 phosphorylated human efF4B or a corresponding phosphorylated ortholog of human elF413., Such an enzyme immunoassay is particularly advantageous because identifying differences in protein levels between related k Mase family members or isoforms taia,en the .relatively high homology between kinases among themselves and also among their phosphorylated forms.
Immunological reagents for identifying ellF413 in both phosphorylated and non-phosphorylated forms, as well as for detecting MELK, are well 'known in the art and can be generated using standard techniques, such as by inoculating 'host animals with appropriate elF413 phosphor-peptides. Such anti-MI:1,1c anti-elf4B, andfor anti-phospho-elF413 antibody reagents (e.g., monoclonal antibody) can be used to isolate andior determine the amount of the respective proteins such as in a cellular lysate. Such reagents can also be used to monitor protein levels in a cell or tissue, e.g., white blood cells or 'lymphocytes, as part of a clinical testing procedure, e.g., in order to monitor an optimal dosage of an inhibitory agent. Detection can be facilitated by coupling (e.g., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive .materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphata.so, p-galactosidase, or acetyleholinesterase;
examples of suitable prosthetic group complexes include streptavidintiotin and avidinfbiotin; examples of suitable fluorescent materials include umbelliferoneõ fluorescein, :fluorescein isothiocyanate, .thodamine, dichlorotriazinylamine -fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent .material includes luininol;
examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include I 251 131l, -.35s or 'HI.
The screeninti assays described above can further be adapted to identify candidate/test compounds which modulate (e.g., stimulate or inhibit) the interaction (and most likely MELK and elF4.13 oncogenie activity as well) between an cIF4B
protein and a target e1f4B protein with which the el.F4B protein normally interacts or modulates to verify that MELK-mediated enzymatic activity has been reduced in accordance with the reduced amounts of phosphorylated elF4B kvct, Exiimples of such target molecules or substrates include certain protein encoded by mRNA with structured 5'1JTR, such as those described further herein.
As described .above for the identification of eIF4B phosphorylation, Thr-3 phosphoryl.ation, Ser-I0 phosphorylation, and/of Thr-11 phosphoryl.ation of human Histone H3, or a corresponding phosphorylatable amino acid in an ortholog of human Histone 113, and modulation (e.g., inhibition) thereof, can similarly be determined.
In another embodiment, the invention provides assays for screening candidatettest compounds which interact with (e.g., bind to) MELK and/or elF4B and/or Historic 1113 protein. "Binding compound" shall refer to a binding composition, such as a small molecule, an antibody, a peptide, a peptide or non-peptide ligandõ a protein, an.
oligonticleotideõ an oligonueleotide analog, such as a peptide nucleic acid, a lectinõ or any other molecular entity that is capable of specifically binding to a target protein or molecule or stable complex formation with an analyte of interest, such as a complex of proteins.
"Binding moiety" means any molecule to which molecular tags can be directly or indirectly attached that is capable of specifically binding, to an analyte. Binding moieties include, but are not limited to, antibodies, antibody binding compositions, peptides, proteins, nucleic acids and organic molecules having a molecular weight of up to about 1000 &lions and containing atoms selected from the group consisting a hydrogen, fluoride, carbon, oxygen, nitrogen, sulfur and phosphorus, Typically, the assays are cell-based assays.
The cell, for example, can be of mammalian origin expressing MEEK and/or el.F4B and/or Histone H3, e.g., a cancer cell.
In other embodiments, the assays are cell-freeassaywhich include the steps of combining a MELK and/or eIF4B and/or Histone H3 protein or a.biologically active portion thereof; and a candidate/test compound, e.g., under conditions which allow for interaction of (e.g., binding of) the candidate/test compound to the MEEK
and/or el .F413 and/or Histone 1.13 protein or biologically active portion thereof to form a complex, and detecting the formation of a complex, in which the ability of the candidate compound to interact with (e.g., bind to) the MEEK and/or eIF4B andlor Histone H3 polypeptide or biologically active fragment thereof is indicated by the presence of the candidate compound .15 in the complex. Formation of complexes between the MEEK and/or ell/'4B
and/or Histone protein and the candidate compound can be quantitated, for example, using standard immunoassays. Such analyses would identify test compounds as MELK and/or elF4B

and/or Histone H3 ligands.
To perform the above drug screening assays, it ..can be desirable to immobilize either MELK and/or elF4B and/or Histone H3 or its target molecule to facilitate separation of complexes frotn uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Interaction (e.g., binding of) of MELK
and/or 61'4B and/or Histone 113 to a target molecule, in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants.
Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
In one embodiment, a fusion polypeptide can be provided which adds a domain that allows the polypeptide to be bound to a matrix. For example, Outathione-S-transferase-MELK, Histone H3, and/or -cIF4B fusion polypeptides can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St, Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., 35 S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of MELK-, Histone H3-, andfor el F4B-binding polypeptide found in the bead fraction quantitated from the gel using standard eleetrophoretic techniques.
Other techniques for immobilizing poi-peptides on matrices en also be used in the exemplary drug screening assays of the invention. For example, MELK and/or efF4B
andfor Histone H3 or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Bionnylated MELK and/or e1F4B and/or Histone H3 molecules can be prepared from biotin-NHS (N-bydroxy-succinimide)nsing techniques well known in the aft (e.g., biotinylation kit, Pierce Chemicals, 'Rockford, Ill), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chenricall). Alternatively, antibodies reactive with .MELK. and/or a:4B and/or Fiistone H3, but which do not interfere with binding of the polypeptide to its target molecule can be derivatized to the wells of the plate.: and MELK
and/or elF4B and/or Historic .113 trapped in the wells by antibody conjugation. As .15 described above, preparations of a MELK- and/or cIF4B-binding polypeptide and a candidate compound are incubated in the :MELK- and/or ellF4B- and/or Histone presentinu; wells of the plate, and the amount of complex trapped in the well can be quantitated. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodeteetion of complexes using antibodies reactive with the MELIA( and/or eIF4B andlor Histone H3 target molecule, or which are reactive with MELK and/or ciF4B and/or Histonc H3 polypeptid.e and compete with the target molecule; as well as enzyme-linked. assays which rely on detecting an enzymatic activity associated with the target molecule.
In another aspect, a method for assessing the efficacy a an agent for inhibiting kinase activity of human MELK or an ortholog thereof in a subject, comprising:
a) detecting in a subject sample at a first point in time, the amount of Scr-406 phosphorylated human eIF4B or the amount of a human eIF4B ortholog phosphorylated at a corresponding amino acid of human eiF4B; b) repeating step a) during at one or more subsequ.ent points in time after administration of the agent; and e) comparing the amount of phosphorylated human elF413 or ortholog thereof detected in step a) with said amount detected in step b), wherein a hi her amount of Ser-406 phosphorylated human el.F4B or the amount of the human e1f4B ortholog phosphorylated at a corresponding amino acid of human el.F4B in the first point in time relative to at least one subsequent point in time, indicates that the agent inhibits kinase activity of MELK or the ortholog thereof, is provided.
.Sirimilarly, a method for assessing the efficacy of an agent for inhibiting kinase activity of human MELK.
or an ortholleg thereof in a subject, .comprising: a.) detecting in a subject sample at a first point in time, the amount of Thr-3 phosphorylated. Ser-I TO phosphorylated.
and/or Thr-4 phosphorylated human Histone H3, or the amount of a human Historic -113 ortholog phosphorylated at a corresponding amino acid of human Historic 113; h) repeating step a) during at one or more stibsequent points in time after administration of the agent: and e) comparing the amount of phosphorylated human -Historic 1-13 or ortholog thereof detected in step a) with said amount detected in step b), wherein a higher amount of Thr-3 I 0 phosphorylated, Ser- I 0 phosphorylated, and/or Thr-1 phosphorylated human Histone H3, or the amount of the human Histone H.3 ortholog: phosphorylated at a corresponding amino acid of human Histone H3 in the first point in time relative to at least one subsequent point in time, indicates that the agent inhibits kinase activity of M-ELK or the ortholog thereof, is provided.
As used herein, "time course" shall refer to the amount of time between an initial event and a subsequent event. For example, with respect to a subjeetts cancer 'progression, time cow-se may relate to a subject's disease and may be measured by gauging simifleant events in the course of the disease, wherein the first event may be diagnosis and the subsequent event may he proliferation, metastasis, etc.
Once binding is confirmed, additional assays, such as kinase assays to determine inhibition of phosphotylation effects, can be pertbrined according to -well-known methods in the art. For example, assays for determining TvIELK kinase activity are well known in the art (see, for example, the publications described herein and incorporated by reference in their enfirety). Briefly., MELK can be incubated with a suitable substrate in a buffer allowing phosphorylation of elT4B or Histone H3. Phosphorylation of the substrate can be detected -using a labeled phosphate group, such as the use of the radioactive label. 3:P
present as the ATP source in the buffer. Alternatively, antibodies specific for the phosphotylated products of elF4B catalytic activity can be used to detect activity. As will be apparent to those of ordinary skill in the art, the assays are easily amenable to high through-put technologies using robotics and automated processes,.
Alternatively, the MELT( kinase activity can be assayed using a synthetic substrate, such as a peptide library.
MELT( activity can also be assayed by detecting downstream targets of the .kinase such as those described herein.

,50 -.Ser-406-phosphorylated elF4B can be analyzed according to any of the methods and using any of the samples described herein (e.g., single subject samples or pooled subject samples). Candidate compounds which produce a statistically significant change in phosphory1ated-c1F4B-dependent responses (e.g., inhibition of human e1F4B
phosphorylation at Ser-406 or a corresponding phosphorylatable amino acid residue in an elf4B ortholog thereof) can be identifitxl. Such statistically significant changes can be measured according to a number of criteria and/or relative to a number of controls. For example, significant modulation of phosphorylation of Ser-406 can be assessed if the output under analysis is inhibited by 1,1-, 1.2-, 1.3-, 1õ4-, 1.5-, L6-, 1.7-, 1.8-, 1.9-, 2.0-, 2,1-, 2.2-, 23-, 2,4-, 25-,26-, 17-, 2.8-, 2.9-, 3.0-, 3.1-, 3.2-, 33-, 3,4-, 3,5-, 36-37-, 3,8-, 3.9-, , 9.0- 9.5-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, 20-fold or more different (including any range inclusive), relative to a control, In one embodiment, between the .first point in time and the subsequent point M time, the subject has undergone treatment for .15 cancer, has .completed treatment for cancer, aid/or is .in remission from cancer.
As described above for the identification and/or analysis of elf4B
phosphotylation, Thr-3 phosphotylated, Ser-10 phosphory kited. and/or .Thr-11 phosphorylated.
Historic H3, or a corresponding phospborylata.ble amino acid in an ortholog of human Histone f13, and.
modulation (e.g., inhibition) thereof, can similarly be identified andlor analyzed.
The temi "cancer" refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell, As .used herein, the term "cancer" includes premalignant as well as malignant cancers. Cancers include, but are .not limited to. B cell cancer, e.g., multiple myeloma.. Waidenstrom's macroglobulinemiaõ the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and. immunocytie amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or .eentral. nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical caner, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, ostcosarcomaõ
chondrosarcoma, cancer of hematological tissues, and the like. Also included are any cancers in which the gene encoding MELK and/or elF4B and/or Histone 113 is amplified or overexpressed, or has an activating mutation, or the .MELK and/or el.F4B and/or Historic H3 is hyper-activated by other kinases. In some embodiments, ovarian cancers, including serous eystadenocarcinoma, head and .neck cancers, including non-small cell lung cancer (NSCLC), squamous cell carcinoma, pancreatic cancer, colon cancer, =prostate cancer, and/or gliomas can be preferred.
"Treat," "treatment," and other forms of this word refer to the administration of an agent that inhibits the ability of I) .NIELK to phosphoryiatc c1F48 and/or Historic H3 and/or 2) the ability of elf4B or Histone .113 to be phosphorylated by MELK, to cause a cancer to be ameliorated, to extend the expected survival time of the subject and/or time to progression of a cancer or the like, "Responsiveness," to "respond" to treatment, and other forms.olthis..verh, as.
used.
herein., refer to the reaction of a subject to treatment with an agent capable of inhibiting the ability of 1) MELK to phosphorylate clF4B or Historic 113 and/or 2) the ability of elF4B or Histone H3 to be phosphorylated by MELK. As an exampte, a subject responds to treatment of the subject or cell thereof with an agent if the assayed condition is .modulated by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more relative to that of no administration of the agent or over a given amount of time, C. Treatment Methods MELK and/or elf4B and/or Historic H3 inhibitors described herein can be osed to treat cancer. In one embodiment, a method of treating a subject afflicted with cancer comprising administering to the subject an agent that inhibits Ser-406 phosphorylation of human el.F4B or a corresponding phosphorylatable amino acid in an orthoiog of human el.F4B, for example an agent that specifically modulates Ser-406 phosphotylation, thereby treating .the subject afflicted with .the cancer, in another embodiment, such MELK and/or elf4B and/or Histone H3 inhibitors can also be used to determine the efficacy, toxicity, or side effects of treatment with such an agent. These methods of treatment generally include the steps of administering modulators in a pharmaceutical composition, as described further below, to a subject in need of such treatment, e.g., a subject with cancer or at risk for developing cancer.

- c7 -The term "administering" is intended to include routes of administration which allow the agent to perform its intended function of inhibiting the ability of MELK. to phosphorylate elF4B, the ability of elF4B to be phosphorylated by MELK, the ability of MELK to phosphorylate Histone H3, and/or the ability of Histone H3 to be phosphorylated by MELK. Examples of routes of administration which can be used include injection (subcutaneous, intravenous, parenterally, intraperitorteally, inirathecal, etc.), oral, inhalation, and transdennal, The injection can be bolus injections or can be continuous infusion. Depending on the route of administration, the agent can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally affect its ability to perform its intended function. The agent may be administered alone, or in conjunction with a pharmaceutically acceptable carrier. The agent also may be administered as a prodrug, which is converted to its active form in vivo.
The term "effective amount" of all went inhibiting the ability of MEEK to phosphorylate eIF4B and/or the ability of elF4B to be phosphorylated by MELK
is that .15 amount necessary or sufficient to inhibit the ability of MELK to phosphorylate etF4B
and/or the ability of elf4B to be phosphorylated by MELK in the subject or population of subjects as measured, for example, by the levels of Ser-406-phosphotylated human efF4B
or a corresponding phosphorylatable residue in an el.FLIB ortholq, thereof according to the methods described above. The same analysis applies to inhibiting the ability of MELK to phosphorylate Historic 11:3 and/or the ability of Histone 1:13 to be phosphorylated by MELK.
The effective amount can vary depending on such factors as the type of therapeutic agent(s) employed, the size of the subject, or the severity of the disorder.
It will be appreciated that individual dosages may be varied depending upon the requirements of the subject in the judgment of the attending clinician, the severity of the condition being treated and the particular compound being employed. In determining the therapeutically effective amount or dose, a number of additional factors may be considered by the attending clinician, including, but not limited to: the pharmacodynanne characteristics of the particular agent and its mode and route of administration; the desired time course of treatment; the species of mammal; its size, age, and general health; the specific disease involved; the degree of or involvement or the severity of the disease; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the kind of concurrent treatment; and other relevant circumstances.

-Treatment can he initiated with smaller dosages that are less than the effective dose of the compound. Thereafter, in one embodiment, the dosage should be increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired.
The effectiveness of any particular agent to treat cancers can be monitored by comparing two or more samples obtained .from subjects undergoing cancer treatment. In general, a first sample is Obtained from the subject prior to beginning therapy and one or more samples during treatment. ln such a use, a baseline of expression of cells from subjects with cancer prior to therapy is determined and .then changes in .the baseline state of expression of cells from subjects with cancer is monitored during the course of therapy.
Alternatively, two or more successive samples obtained during treatment can be used without the need of a pre-treatment baseline sample. insuch a use, the first sample obtained from the subject is used as a baseline for determining whether the expression of cells from subjects with metabolic disorders is increasing or decreasing.
.MEL.K and/or .c.I.F43 and/or Historic 143 inhibitors can be administered in.
pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of the inhibitor formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. For example, formulations can be adapted for (I) oral adminisnation, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin, buccal, or sublingual surfaces; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) nasal/aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound, based on well-known methods in the pharmaceutical arts.
The phrase "pharmaceutically acceptable" is employed herein to refer to those agents, materials, compositions, and/or dosage thrills 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.
The phrase "pharmaceutically-acceptable carrier" as used herein means a phannaceuticaily-acceptable material, composition or vehicle, such as a liquid or solid -filler, diluent, excipientõ solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium .carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and. soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, .mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) *ink acid;
(16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21.) other .non-toxic compatible substances employed in pharmaceutical formulations.
The term 'pharmaceutically-acceptable salts" refers to the relatively non-toxic, inorganic and organic acid addition salts of the agents that reduce the phosphorylation levels of .PKG=lota and/or activity encompassed by the invention, These salts can be prepared in situ during the final isolation and purification of the agents, or by separately reacting a purified agents agent in its free base loyal with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, Imitate, benzoate, lactate, phosphate, tosylate, citrate, malcate, fumarate, SUCCitlatO, tartrate, napthylatc, :rnesylate, glucoheptonate, lactobionateõ and lattrylsulphonate salts and the like (See, for example, Berge el al. (1977) 'Pharmaceutical Salts", 1.
Pharm. Sa 66:1-19).
In addition, the methods described herein can further comprise treating subjects with MELK and/or eiF4B and/or .1'h-stone H3 inhibitors in addition to administering one or more additional anti-cancer agents and/or use samples from subjects exposed to such anti-cancer agents. Anti-cancer agents are well known to the skilled artisan and include, without limitation, chemotherapy and radiation, as well as immunotherapyõ hormone therapy, and gene therapy using nucleic acid molecules and/or proteins that are linked to the initiation, progression, and/or pathology of a tumor or cancer.
Chemotherapy includes the administration of a chemotherapeutic .ai:ient. Such a chemotherapeutic agent may be, but is not limited to, those selected from among the following groups of compounds: platinum compounds, cytotoxic antibiotics, antimetabolities, anti-initotic agents, alkylating agents, arsenic compounds, DNA
topoisomcrase inhibitors, taxanes, nucleoside analogues, plant alkaloids, and toxins; and synthetic derivatives thereof. Exemplary compounds include, but are not limited to, alkylating agents: eisplatin, treosulfaa and trofosfamide; plant alkaloids:
vinblastine, paclitaxel, doeetaxol, DNA topoisomerase inhibitors: teniposidc, crisnatol, and mitomycin;
anti-folates: methotrexate, .mycophenolie acid, and hydroxyurea; pvrimidine analogs: 5-fluorouracil, doxilluridineõ and cytosine ara.binoside, wino analogs:
mercaptopurine and thioquanine; DNA antimetabolites: 2-deoxy-5-.fluorouridine: aphidieolin glyeinate, and pyrazoloimidazole; and antitnitotic agents'. haliehondrin, colehicine, and rhizoxin.
Compositions comprising one or more chemotherapeutic agents (e.g., FLAG, CHOP) may also be used. FLAG comprises .fludarabine, cytosine arabinoside (Ara-C) and (i-CSF.
CHOP comprises cyclophosphamidc, vincristine, doxorubicin, and prednisone. In another embodiment, PARP (e.g., PARP- I and/or PARP-2) inhibitors are used and such inhibitors are well known in the an (e.g., Olaparibõ,\BT-888, 851-201, BGP-1.5 (N-Gene Research Laboratories, Inc.); INO-1001 (1notek Pharmaceuticals Inc.); P134 (Soriano et at.. 2001;
Pacher et al., 2002b); 3-aminobenzamide .(Trevip,en.); 4-amino-1,8-naplithalimid.e;
(Trevigen); 6(5111)-placnanthridinone (Trevigen); benzamide (U.S. Pat. Re, 36,397); and NU1.025 (Bowman ei al.). in still another embodiment, the chemotherapeutic agents are platinum compounds, such as cisplatin, .earboplatin, oxaliplatin, nedaplatin, and iproplatin, Other antineoplastic platinum coordination compounds are well known in the art, can he modified. according to well-known methods in the. art, and include the compounds disclosed in U.S. Pat. Nos. 4,996,337,4,9469954, 5,09.1,521, 5,434,256, 5,527,905, and 5,633,243, all of which are incorporated herein by reference. The thregoing examples of chemotherapeutic agents are illustrative, and are not intended, to be limiting.
Radiation therapy can also comprise an additional anti-cancer agent... The radiation used in radiation therapy can be ionizing radiation, Radiation therapy can also be gamma rays. X-rays, or proton beams, Examples of radiation therapy include, 'but are not limited to, external-beam radiation therapy, interstitial implantation of radioisotopes (1-125, Pd-1.03, lr-192), intravenous administration of radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal -32P radiation therapy, and/or total abdominal and pelvic radiation therapy. For a general overview of radiation therapy, see Hellman, Chapter 16:
Principles of Cancer Management: Radiation Therapy, 6th edition, 2001, DeVita et al., eds,, J. B. Lippencott Company, Philadelphia, The radiation therapy can be administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source. The radiation treatment can also be administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body dose to cancer cells or a tumor mass. Also encompassed is the use of photodynamie therapy comprising the administration of photosensitizers, such as hematoporphyrin and its derivatives, Vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A; and DMHA.
Additional anti-cancer agents include immunotherapy, hormone therapy, and gene therapy. Such therapies include, but are not limited to, the use of antisense polynucleotides, .15 ribozymes, RNA interference molecules, triple helix polynucleotides and the like, where the nucleotide sequence of such compounds are Mated to the nucleotide sequences of DNA
and/or RNA of genes that are linked to the initiation, progression, and/or pathology of a tumor or cancer. For example, oncogenes, growth factor genes, growth factor receptor genes, cell cycle genes. DNA repair genes, and others, may be targeted in such therapies.
immunotherapy may comprise, for example, use of cancer vaccines and/or sensitized antigen presenting cells. Immuncitherapy can also involve derepression of immtmoinhibitory pathways, such as by targeting PD-1,1, PD-1, CTLA-4, and the like. The immunotberapy can involve passive immunity for short-term protection of a host, achieved by the administration of an antibody directed against a cancer antigen or disease antigen (e.g., administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen). Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines.
Hormonal therapeutic treatments can comprise, ibr example, hormonal agonists, hormonal antagonists (e.g flutamide, bicaintamide, tamoxi fen, raloxifene, leuprolide acetate (LUPRON), LII-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glueoeorticoids, mineralocorticoids, estrogen, testosterone, progestins), vitamin A derivatives (e.g., all-trans retinoie acid (ATRA));

- -vitamin D3 analogs; antigestagens (e.g., mifepristone, onapristone), or antiandrogens cyprotcrone acetate).
In one embodiment, anti-cancer therapy used for cancers whose phenotype is determined by the methods of the invention can comprise one or more types of therapies described herein including, but not limited to, chemotherapeutic agents, immunotherapeutics, anti-angiogenic agents, cytokines, hormones, antibodies, polynueleotid.es, radiation and photod.ynamic therapeutic agents. For example, combination therapies can comprise one or more chemotherapeutic agents and radiation, one or more chemotherapeutic agents and immunotherapy, or one Or more chemotherapeutic agents, radiation and chemotherapy, Exemplification This invention is further illustrated by the following examples, which should not be construed as limiting.
Example Materials and Methods For Examples 2-3 a. Plasmids Human el F4B was cloned from the reverse transcription products of total RNA
extracted from human mammary epithelial cells (HMECO, using the primers (forward:
ATGCCGCCCTCAGCAAAAAAG; reverse: CTATTCGGCATAATCTTCTC), The 1,8 kb PCR product was then used as template for ;Amplifying Flag-tagged or HA-tagged eIF4B
with restriction sites. The constructs (pWzi-Flag-elf4B, pfrex-elF4B-HA) were yen fled by sequencing. Site-directed mutagenesis of elF4B was performed using Quickehange XL
(Stratagene), and all mutant constructs were confirmed by sequencing.
To generate pLKO-wt-on shRNA targeting human elF46, synthesized oligonucleotides were annealed and ligated with digested pLKO vector. The sequences tbr saambie, sh-e1F4B-1, sh-elf4B-2 are GICiGACICTIGAAAGTACTAT, GGACCAGGAAGGAAAGATGAA, and GCGGAGAAACACC11GAICTT:
respectively.
b, Retroviral and lentivirai gene delivery Retrovimses were generated by transfecting HEK293T cells with retroviral plasmids and packaging DNA. Generally, 1.6 ug pWz1 DNA, 1.2 tig pC0-VSVG and 1.2 pg pCG-gapipol, 12 ul lipid of Metafeetene Pro (13iontex) were used. DNA and lipid were diluted in 300 pi PBS respectively and mixed, .After 15 minutes (min.) of incubation, they were added to one 6-cm dish that was seeded with 3 million FIEK293T cells one day earlier. Vitali supernatiult was collected 48 hours (h) and 72 hours post-transfeetion. The supernatant was filtered through 0.45 pm membrane, and was added to target cells in the presence of 8 item] polybrene (Millipore). Lentiviruses were generated with a similar approach with the exception of HEK293T cells that were transfected with 2 pg PLKO
DNA, 1.5 mg pCMV-dR8.91, and 0,5 g p.MD2-VSVG. Cells were selected with antibiotics starting 72 h after initial infection. Purornycin and blasticidin were used at the final concentration s of 1.5 Wail and 4 pginil respectively.

C. immunoblotting For treatment with nocodazole, cells were refreshed with medium containing nocodazole (200 ngind). Twenty hours after treatment, floating mitotic cells were harvested by gemal shake-off For drug treatment, cells were seeded in multi-well plate, in the presence of OTSSP167 (ChemExpress, HY15512; 10 mM stock made in DMSO).
Cells were harvested and lysed with RITA buffer (25 mM Tris, pH 7.4, 150 mM
NaCI, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodeeYlsulfate) supplemented with protease inhibitors cocktail (Roche) and phosphatasc inhibitors cocktail (Thermo Scientific). Cleared lysates were analyzed for protein concentration using a BCA
kit (Thermo Scientific). Equal amount of protein (10-20 pg) was resolved on SDS-PAGE, and was subsequendõ, tiansferred onto a nitrocellulose or polyvinylidene difluoride membrane (Amersham). The membrane was blocked with 5% non-fat milk and was then ineu.bated with primary antibodies overnight at 4 C. After washing, the membrane was incubated with fluorophore-eorijugated secondary antibodies for 1 h at room temperature.
The membrane was then washed and scanned with an Odyssey Infrared scanner (Li-Car Bioseiences).
The folio-wing antibodies were used for immunobiotting or immunoprecipitation, MELK (Epitomics, 2916), p-elF4B (S406) (Cell Signaling, 8151), p-efF4B (S422) (Cell Signaling, 3591), elf4B (Cell Signaling, 3592) c-Mye (Cell Signaling, 5605), XIAP (( ell Signaling, 2045), p-Akt (S473) (Cell Signaling. 4060), p-MAPK (T202/Y204) (Cell Signaling, 4370), cleaved PARP (Asp214) (Cell Signaling, 9541), Aurora A (Cell Signaling, 4718), Aurora B (Cell Signaling, 3094), p-Aurora A (T288)/Aurora B
(T232)1Aurora C(T198) (Cell Signaling, 2914), p-Histone H3 (13) (Cell Signaling, 13576), p-Histone H3 (S10) (Cell Signaling, 3377), p-Histone H3 (11 1 )(Cell Signaling, 9767), p-Histone H3 (S28) (Cell Signaling, 9713), ODC (Sigma, 01136), Vineulin (Sigma, V9131, alpha-tubulin (Sigma, T9016), anti-HA magnetic beads (Pierce, 88836), anti-Flag magnetic beads (Sigma, M8823). Secondary antibodies used were Mesa Fluor 680 goat anti-rabbit 1.gCi .(Invitrogen, .A-21109) and 'IRDye800-conjugated anti-mouse IgG
(Rockland).
d. in vitro kinase assay Flag-tagged elF4B or Flag-elF4B (S406A) was transfected into HEK293T cells (4 ng DNA for cells in one 60 mm dish). Thirty-six hours aftettransf&tiOnõ.ceils Were lySed with IP buffer (100 niM Naa, 50 niM. Iris, pH 7.5,0.5% NP-40, 0.5% SOdit1111 d.coxycholateõ supplemented with proteaselphosphata.se inhibitor cocktail).
Lysates were cleared via incubating with anti-mouseIgG conjugated to magnetic beads (4'C, 30 rain), and then immunoprecipitated with anti-Flag M2 magnetic heads (Sigma) (4'C, 120 min).
The beads with bound antigens were washed 5 times with IP buffer. Beads during the last wash were aliquoted into 1.5 ml mierocentrifuge tubes. After removal of JP
buffer, the beads were washed once with lx kinase buti-Cr without ATP (5 nilvi iris. pH
7.5, 5 inM -13 glyeerophosphate, 2 miVI. dithiothreitol, 0.1 niN1 Na3VO4, 10 .niM. MgC12;
Cell Signaling).
After the wash, 40 pi lx .kinase buffer with 200 DIM .ATP was added to each tube, followed by 5 W. buffer without or with 500 ng recombinant MELK. The reaction was incubated at 30*C for 30 min, and terminated by adding 40 Id 2x SDS sample buffer. The samples were then boiled and subjected to immunobiotting. kinase assays with 'Historic H3 were performed as above, except that recombinant Histon 113.1 (New England BioLabs, M2503S) was used (50 ng per reaction), c. Positional scanning peptide library screen Active full-length human 111.ELK was purified from insect cells. The positional scanning peptide library screen was performed as described in Turk et aL
(2006).Mt.
Protoca 1:375. Briefly, a set of 180 (or 198) biotin-conjugated peptides with the following sequence, Y-A-X-X-X-X-X-SiT-X-X-X-X-A,G-K-K-biotin, was used. In the sequence, str means an .equimolar mixture of Ser and Thr. For each peptide, one of the nine X positions represents one of the twenty total amino acids. Peptides were arrayed in a 384-well plates in buffer containing 50 .triN1 FI.EPES, pH 7,5, 20 inNI MgCl.
0.02 mglini BSA, 0.01% Br ii 35, 5 .niN4. DTTõ 0.5 DIM EGTAõ and .active MELK and y432P1-ATP was added to wells (final [peptide] = 50 p.M, and [ATP = 100 tM, 0,025 it,Ci/1.i1 in each well).
After incubating for 2 h at 300C, aliquots of the reactions were spotted onto a streptavidin membrane. The membrane was quenched, washed extensively, dried, and exposed to a phosphor storage screen.
Example 2: Phosphorylation Status of elf4B is a Biomarker of MELK Enzymatic and Oncogenic Activity To seek a potential molecular mechanism underlying the importance of MELK for cancer (such as basal-like breast cancer (BBC)), multiple experimental approaches, .includinR irnmunoprecipitation-tandem mass spectrometry, and pbospho-peptide .mapping, were explored. When Flag-tagged MELK. was immunopreeipitated in mitotic cell lysates and subsequently subject to mass spectrometry analysis, it was found that a translation.
initiation factor; elF4B, had a strong association with MELK duria4. mitosis (Figure 1).
Using positional phospho-peptide .mapping, an optimal substrate motif for MELK
was identified having a strong selection for arginine at the -3 position relative to serinefthreonine (Figure 2).
5 There are two regions flanking the residues of human el:174B (i.e., at Ser406 and Ser422), which contain the MEEK phosphorylation .motif (Figures 3 and 4). To test whether MELK is capable of phosphorylating human cl:P1B at these two sites, in vitro kinase assays were performed using purified recombinant MELK with immunopreeipitated it was found. that Ser406, but not Ser422,. of human elf4B was readily phosphotylated by full-length MELK or the kinase domain of M.E.L.K, and that the observed.
phosphorylation was abolished when the twine at 406 of human .eIF4B was mutated to alanine or other manipulations of .MELK (Figures 3-5). These results are specifically dependent upon MELK, as inhibition of mitotic cells with inToR inhibitors, such as rapamycin and torus, do not produce the same results (Figure 6; van Ciorp et al (2009) Oncogene 28;95-106). These data indicate that Mai( is a kinase that phosphorylates human elF4B at S406 and that efF4B orthologs in other species are similarly phosphotylated due to the highly conserved sequence and structural composition of the e1F413 polypeptide region harboring .the phosphorN;,lation site (Table 2).

Table 2 elF 4B S406 Human S406 RERHPSWRSE
Chimpanzee S406 RERHPSWRSE
Monkey S402 RERHPSWRSE
Cattle S406 REM-WSW/6E
Dog S406 RERHPSWRSE
Mouse S406 RERUIPSWRSE
Rat S406 RERUIPSWRSE
Zebra fish S403 RERHPSWRSE
Fission yeast S315 RERSTSRKPS
It is known that eIF4B stimulates the holicase activity of ellF4A for unwinding the second.ary structure of 5F1JTR of mRNA (Dmitriev etal. (2003) Afol. Cell Biol.
23:8925-8933 and Shahbazian et at (2010) .A:161. Cell Biol. 10 1478-1485). Many of these mRNAs encode oncogenic proteins, such as c-Myc, MAP (X-linked inhibitor of apoptosis protein) and ODC. (ornithine decarboxylase). It was determined herein that down-regulation of MELK reduced phosphorylation of elf4B (p-cIF4B) at S406 in MDA-MB-468 cells during mitosis, which also resulted in markedly reduced levels of c-Myc, MAP and ODC1 (Figures 7 and 8). Thus, MELK-mediated 5406 phosphorylation of efF4B during mitosis, is functionally important for the optimal translation of inRNAs with highly structured 51UTR, many of which are known to be oncogenic, such as c-I\4ye, MAP, and ODC1.
Together, these data indicate that MELK is a novel kinase that regulates elF4B
during mitosis and thereby mediates the translation of mRN As that harbor structured 5'41TR and are important thr the survival and proliferation of cancer cells. Thus, the level of elF4B
phosphorylation mediated by MELK is a target engagement biomarker for MELK
oncogenic activity useful for pnclinical and clinical applications.
Example 3: Phosphorylation Status of Histone 113 is a Biomarker of MELK
Enzymatic and Oneogenic Activity Since MELK. protein abidance is higheSt during mitotis, a. cell cycle phase when Histon H3 is heavily phosphorylated, a link between MELK and 'Hist= H3 phosphorylation was suggested. In fact, the region flanking the residues of human Histone H3 at Thr-11 contains the optimal MELK phosphorylation motif described in Example 2 above.

SUBSTITUTE SHEET (RULE 26) To test whether MELK is capable of phosphorylating human Histone H3 at threonine 3 (Thr3), scrim 10 (Seri()) and threonine 1.1 (Thri ), in vitro kinase assays were performed using a purified recombinant kinase domain of human MELK and human Histone H3. It was found that Thr3, Seri , and Thri] of human Histone H3 were readily phosphorylatcd by the kinase domain of MELK (Figure 9).
Down-regulation of MELK reduced phosphorylation of Histone H3 at Thr3. Ser10 and Thri 1 in MDA-MB-468 cells during mitosis, but did not affect the phosphorYlation of Aurora kinases A, B, or C. which are known kinases that phosphorylate Histone H3 at Seri 0 (Figures 10 and 11). Similarly, inhibition of MEEK using the small chemical MEEK
inhibitor, OTSSP167, in MDA-MB-468 cells teduced phosphorylation of Historic 113 at Thr3iSer10/Thrli (Figure .12). An OTSSP167 concentration-dependent reduction in phosphorylation of Histone H3 at Thr3, Serl 0 and Thri], but not Ser28, was also observed (Figure 13).
These data indicate that :MELK is a kirtasq :that phosphorylates human Histone .1-13 at least at Thr3, Seri 0 and Thri], but not Ser28, and that Histone .H.3 orthologs in other species are similarly phosphorylated due to the highly conserved sequence and structural composition of the :Histone 113 polypeptido region harboring the phosphorylation site (Table 3).
Table 3 Historic 113 Human T3/S101T11 MART KQTARKSTGGKA
Chimpanzee T3/S101111 MA RTKQTARKSICiGKA
Monkey T3/S101-.11 MA RTKQTARKSTGGKA
Cattle 10fT I 1 MARTKQTARKSTGGKA
Dog T3/Slorr 1 MARTKQTARKSTGUICk Mouse: TIS 101'11 141 AR TKQT ARKS TO G KA
Rat 1'3/SI0ST 11 MARTKQTARKSIGGKA
Zebrafish T3/S10./T1 I MARTKQTARKSTGOKA
Fission yeast 13 /SlOiT I I MARTKQTARKSTGCIKA
Together, these data indicate that MELK is a novel kinase that regulates Histone H3 phosphorylation during mitosis and is therefore potentially important fbr the proliferation of cells (e.g.., cancer cells). Thus, the level of :Histone 113 phosphorylation mediated by MEEK is a target engagement biomarker for MELK oncogenic activity useful for preclinieal and clinical applications.

SUBSTITUTE SHEET (RULE 26) Incorporation by Reference The contents of all references, patent applications, patents, and published patent applications, as well as the Figures and the Sequence Listing, cited throughout this application are hereby incorporated by reference.
Equivalents Those skilled in the art will recognize, or be able to ascertain using no morc...than routine experiffientation, many equivalents to the specific embodiments of Ow invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (114)

What is chinned:

1. A method of identifying an agent which inhibits kinase or oncogenic activity of human maternal embryonic leucine zipper kinase (MELK) or an ortholog thereof comprising:
a) contacting a sample comprising i) human MELK or an ortholog thereof and ii) human eukaryotic initiation factor 4B (eIf4B) or an ortholog thereof, with the agent; and b) determining the ability of the agent to inhibit Ser-406 phosphorylation of human eIf4B or a corresponding phosphorylatable amino acid in the ortholog of human eIF4B, wherein decreased phosphorylation identifies an agent which inhibits kinase or oncogenic activity of human MELK or the ortholog thereof.

2. The method of claim 1, wherein the inhibition of said Ser-406 phosphorylation of human eIF4B or a corresponding phosphorylatable amino acid in an ortholog of human eIf4B is determined by comparing the amount of Ser-406 phosphorylated human eIf4B or a corresponding phosphorylatable amino acid in the ortholog of human eIf4B, in the sample relative to a control.

3. The method of claim 2, wherein the control is the amount of Ser-406 phosphorylated human eIf4B or a corresponding phosphorylatable amino acid in the ortholog al human eIf4B in the sample relative to said amount in the absence of the agent or at an earlier timepoint after contact of the sample with the agent.

4. The method of claim 1, wherein the inhibition of said Ser-406 phosphorylation of human eIf4B or a corresponding phosphorylatable amino acid in an orthoiog of human eIf4B is determined by comparing the ratio of the amount of Ser-406 phosphorylated human eIf4B, or a corresponding phosphorylatable amino acid in the ortholog of human eIF4B, in the sample relative to the total amount of human EIF4B or ortholog thereof, to a control.

5. The method of claim 4, wherein the control is the ratio of the amount of Ser-406 phosphorylated human eIf4B or a corresponding phosphorylatable amino acid in the ortholog of human eIf4B in the sample relative to said ratio in the absence of the agent or at an earlier timepoint after contact of the sample with the agent.

6, The method of any one of claims 1-5, further comprising determining the amount of a protein translated from an RNA with highly structured 5'UTR optionally wherein the protein is selected from the group consisting of cellular myelocytomatosis oncogene (c-Myc), X-linked inhibitor of apoptosis protein (XIAP), and ornithine decarboxylase (ODCl).

7. The method of claim 1, further comprising a step of determining whether the agent directly binds said human eIF4B or said ortholog thereof, or said human MELK
or said ortholog thereof.

8. The method of claim 1, wherein the sample is selected from the group consisting of in vitro, ex vivo, and in vivo samples.

9. The method of claim 1, wherein the sample comprises cells.

10. The method of claim 9, wherein the cells are cancer cells.

11. The method of claim 10, wherein the cancer is selected from the group consisting of any cancer in which MELK or eIF4B is amplified or overexpressed, any cancer having an activating mutation of MELK or eIF4B, and any cancer in which MELK or eIF4B is activated by other kinases.

12. The method of claim 9, wherein the cells are obtained from a subject.

13. The method of claim 1, wherein the sample is selected from the group consisting of tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow.

14. The method of any one of claims 1-5, wherein the amount of Ser-406 phosphorylated human eIF4B or a corresponding phosphorylatable amino acid in the ortholog of human eIF4B is determined by an immunoassay using a reagent which specifically binds to Ser-406 phosphorylated human eIF4B or corresponding phosphorylated ortholog of human eIF4B.

15, The method of claim 14, wherein the immunoassay is a radioimmunoassay, a Western blot assay, a proximity ligation assay, an immunofluoresence assay, an enzyme immunoassay, an immunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, a dot blot assay, or a slot blot assay.

16. The method of claim 15, wherein the enzyme immunoassay is a sandwich enzyme immunoassay using a capture antibody or fragment thereof which specifically binds with human eIF4B or corresponding ortholog of human eIF4B and a detection antibody or fragment thereof which specifically binds with Ser-406 phosphorylated human eIF4B or a corresponding phosphorylated ortholog of human eIF4B.

17. The method of any one of claims 1-5, wherein said human eIF4B or ortholog thereof, and/or said human MELK or ortholog thereof, comprises a nucleic acid sequence or amino acid sequence set forth in Table 1.

18. The method of any one of claims 1-5, wherein the agent is a small molecule, or an antibody or antigen-binding fragment thereof.

19. The method of any one of claims 1-5, wherein the agent decreases the amount of Ser-406 phosphorylated human eIF4B or a corresponding phosphorylatable amino acid in the ortholog of human eIF4B by at least 50%.

20. A method for assessing the efficacy of an agent for inhibiting kinase activity of human MELK or an ortholog thereof in a subject, comprising:
a) detecting in a subject sample at a first point in time, the amount of Ser-phosphorylated human eIF4B or the amount of a human eIF4B ortholog phosphorylated at a corresponding amino acid of human eIF4B;
b) repeating step a) during at one or more subsequent points in time after administration of the agent; and c) comparing the amount of phosphorylated human eIf4B or ortholog thereof detected in step a) with said amount detected in step b), wherein a higher amount of Ser-406 phosphorylated human eIF4B or the amount of the human eIF4B ortholog phosphorylated at a corresponding amino acid of human eIF4B
in the first point in time relative to at least one subsequent point in time, indicates that the agent inhibits kinase activity of human MELK or the ortholog thereof.

2 . The method of claim 20, wherein the amount of Ser-406 phosphorylated human eIF4B or a corresponding phosphorylatable amino acid in the ortholog of human eIF4B is determined by an immunoassay using a reagent which specifically binds with Ser-phosphorylated human eIF4B or corresponding phosphorylated ortholog of human eIF4B.

22. The method of claim 21, wherein the immunoassay is a radioimmunoassay, a Western blot assay, a proximity ligation assay, an immunofluoresence assay, an enzyme immunoassay, an immunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, a dot blot assay, or a slot blot assay.

23. The method of claim 22, wherein the enzyme immunoassay is a sandwich enzyme immunoassay using a capture antibody or fragment thereof which specifically binds with human eIF4B or corresponding ortholog of human eIF4B and a detection antibody or fragment thereof which specifically binds with Ser-406 phosphorylated human eIF4B or a corresponding phosphorylated ortholog of human eIF4B.

24. The method of claim 20, wherein the sample is selected from the group consisting of ex vivo and in vivo samples.

25. The method of claim 20, wherein the sample comprises cancer cells.

26. The method of claim 25, wherein the cancer cells are cells selected from the group consisting of any cancer in which MELK or eIF4B is amplified or overexpressed, any cancer having an activating mutation of MELK or eIF4B, and any cancer in which MELK
or eIF4B is activated by other kinases.

27. The method of claim 20, wherein the sample is selected from the group consisting of tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow.

28. The method of claim 20, wherein the sample in step a) and/or step b) is a portion of a single sample obtained from the subject.

29. The method of claim 20, wherein the sample in step a) and/or step b) is a portion of pooled samples obtained from the subject.

30. The method of claim 20, wherein between the first point in time and the subsequent point in time, the subject has undergone treatment for cancer, has completed treatment for cancer, and/or is in remission from cancer.

31. The method of claim 20, wherein said human eIF4B or ortholog thereof, and/or said human MELK or ortholog thereof comprises a nucleic acid sequence or amino acid sequence set forth in Table 1.

32. The method of claim 20, wherein the agent is a small molecule, or an antibody or antigen-binding fragment thereof.

33. The method of claim 20, wherein the agent decreases the amount of Ser-phosphorylated human eIF4B or a corresponding phosphorylatable amino acid in the ortholog of human eIF4B by at least 50%.

34. A method of treating a subject afflicted with cancer comprising administering to the subject an agent that inhibits Ser-406 phosphorylation of human eIF4B or a corresponding phosphorylatable amino acid in an ortholog of human eIF4B, thereby treating the subject afflicted with the cancer.

35. The method of claim 34, wherein the agent is administered in a pharmaceutically acceptable formulation.

36. The method of claim 34, wherein the agent is a small molecule, or an antibody or antigen-binding fragment thereof.

37. The method of claim 34, wherein the agent directly binds said human eIF4B or the ortholog thereof, or said human MELK or the ortholog thereof.

38. The method of claim 34, wherein the cancer is selected from the group consisting of any cancer in which MELK or eIF4B is amplified or overexpressed, any cancer having an activating mutation of MELK or eIF4B, and any cancer in which MELK or eIF4B is activated by other kinases.

39. The method of claim 34, wherein said human eIF4B or ortholog thereof, and/or said human MELK or ortholog thereof, comprises a nucleic acid sequence or amino acid sequence set forth in Table 1.

40. The method of claim 34, wherein the agent is a small molecule, or an antibody or antigen-binding fragment thereof.

41. The method of claim 34, wherein the agent decreases the amount of Ser-phosphorylated human eIF4B or a corresponding phosphorylatable amino acid in the ortholog of human eIF4B by at least 50%.

42. The method of claim 34, further comprising administering one or more additional anti-cancer agents.

43. A method of determining the function or activity of human MELK or an ortholog, comprising:
a) detecting in a sample the amount of Ser-406 phosphorylated human eIF4B or the amount of a human eIF4B ortholog phosphorylated at a corresponding amino acid of human eIF4B;
b) repeating step a) in the same sample or a test sample at one or more subsequent points in time after manipulation of the sample and/or manipulation of the same sample or test sample; and c) comparing the amount of phosphorylated human eIF4B or ortholog thereof detected in step a) with said amount detected in step b), wherein a modulated of Ser-406 phosphorylated human eIF4B or the amount of the human eIF4B ortholog phosphorylated at a corresponding amino acid of human eIF4B in the first point in time relative to at least one subsequent point in time and/or at least one subsequent manipulation of the same sample or test sample, indicates that the function or activity of human MEL or an ortholog thereof is modulated.

44. The method of claim 43, wherein the amount of Ser-406 phosphorylated human eIF4B or a corresponding phosphorylatable amino acid in the ortholog of human eIF4B is determined by an immunoassay using a reagent which specifically binds with Ser-phosphorylated human eIF4B or corresponding phosphorylated ortholog of human eIF4B.

45. The method of claim 44, wherein the immunoassay is a radioimmunoassay, a Western blot assay, a proximity ligation assay, an immunofluoresence assay, an enzyme immunoassay, an immunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, a dot blot assay, or a slot blot assay.

46. The method of claim 45, wherein the enzyme immunoassay is a sandwich enzyme immunoassay using a capture antibody or fragment thereof which specifically binds with human elf4B or corresponding ortholog of human elF4B and a detection antibody or fragment thereof which specifically binds with Ser-406 phosphorylated human elF4B or a corresponding phosphorylated ortholog of human elF4B.

47. The method of claim 43, wherein the sample is selected from the group consisting of in vitro, ex vivo, and in vivo samples.

48. The method of claim 43, wherein the sample comprises cells or the method uses a cell-based assay.

49. The method of claim 48, wherein the cells are cancer cells selected from the group consisting of any cancer in which MELK or eIF4B is amplified or overexpressed, any cancer having an activating mutation of MELK or elF4B, and any cancer in which MELK.
or elF4B is activated by other kinases,

50. The method of claim 43, wherein the sample is selected from the group consisting of tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow,

51, The method of claim 43, wherein the same sample or test sample in step a) and/or step b) is a portion of a single sample obtained from a subject.

52. The method of claim 43, wherein the same sample or test sample in step a) and/or step b) is a portion of pooled samples obtained from a subject,

53. The method of claim 43, wherein between the first point in time and the subsequent point in time, the subject has undergone treatment for cancer, has completed treatment for cancer, and/or is in remission from cancer.

54. The method of claim 43, wherein said human e1F4B or ortholog thereof', and/or said human MELK or ortholog thereof, comprises a nucleic acid sequence or amino acid sequence set forth in Table 1.

55. The method of claim 43, wherein the manipulation of the sample is selected from the group consisting of contacting the sample with a test agent, contacting the sample with an upstream signal of the MELK signaling pathway, and contacting the sample with a MELK inhibitor.

56. The method of claim 55, wherein the test agent is a small molecule, or an antibody or antigen-binding fragment thereof

57, The method of claim 55 or 56, wherein the test agent decreases the amount of Ser-406 phosphorylated human elF4B or a corresponding phosphorylatable amino acid in the ortholog of human elF4B by at least 50%.

58. A method of identifying an agent which inhibits kinase or oncogenic activity of human MELK or an ortholog thereof, comprising:
a) contacting a sample comprising i) human MELK or an ortholog thereof and ii) human Histone H3 or an ortholog thereof, with the agent; and b) determining the ability of the agent to inhibit Thr-3 phosphorylation of human Histone H3 or a corresponding phosphorylatable amino acid in the ortholog of human Histone H3; and/or Ser-10 phosphorylation of human Histone H3 or a corresponding phosphorylatable amino acid in the ortholog of human Histone H3; and/or Thr-11 phosphorylation of human Histone H3 or a corresponding phosphorylatable amino acid in the ortholog of human Histone H3, wherein decreased phosphorylation identifies an agent which inhibits kinase or oncogenie activity of human MELK or the ortholog thereof

59. The method of claim 58, wherein the inhibition of said Thr-3 phosphorylated and/or Ser-10 phosphorylation and/or Thr-11 phosphorylation of human Histone H3, or a corresponding phosphorylatable amino acid in an ortholog of human Histone H3, is determined by comparing the amount of Thr-3 phosphorylated human Histone H3 and/or Ser-10 phosphorylated human Histone H3 and/or Thr-11 phosphorylated human Histone H3, or a corresponding phosphorylatable amino acid in the ortholog of human Histone H3, in the sample relative to a control.

60. The method of claim 59, wherein the control is the amount of Thr-3 phosphorylated and/or Ser-10 phosphorylated and/or Thr-11 phosphorylated human Histone H3, or a corresponding phosphorylatable amino acid in the ortholog of human Histone H3, in the sample relative to said amount in the absence of the agent or at an earlier timepoint after contact of the sample with the agent.

61. The method of claim 58, wherein the inhibition of said Thr-3 phosphorylation and/or Ser-10 phosphorylation and/or Thr-11 phosphorylation of human Histone H3, or a corresponding phosphorylatable amino acid in an ortholog of human Histone H3 is determined by comparing the ratio of the amount of Thr-3 phosphorylated and/or Ser-10 phosphorylated and/or Thr-11 phosphorylated human Histone H3, or a corresponding phosphorylatable amino acid in the ortholog of human Histone H3, in the sample relative to the total amount of human Histone H3 or ortholog thereof to a control.

62. The method of claim 61, wherein the control is the ratio of the amount of Thr-3 phosphorylated and/or Ser- 10 phosphorylated and/or Thr-11 phosphorylated human Histone H3, or a corresponding phosphorylatable amino acid in the ortholog of human Histone H3 in the sample relative to said ratio in the absence of the agent or at an earlier timepoint after contact of the sample with the agent.

63. The method of any one of claims 58-62, further comprising determining the amount of a mitosis-specific protein.

64. The method of claim 58, further comprising a step of determining whether the agent directly binds said human Histone H3 or said ortholog thereof, or said human MELK or said ortholog thereof.

65. The method of claim 58, wherein the sample is selected from the group consisting of in vitro, ex vivo, and in vivo samples.

66. The method of claim 58, wherein the sample comprises cells.

67. The method of claim 66, wherein the cells are cancer cells.

68. The method of claim 67, wherein the cancer is selected from the group consisting of any cancer in which MELK or Histone H3 is amplified or overexpressed, any cancer having an activating mutation of MELK or Histone H3, and any cancer in which MELK or Histone H3 is activated by other kinases

69. The method of claim 66, wherein the cells are obtained from a subject.

70. The method of claim 58, wherein the sample is selected from the group consisting of tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow.

71. The method of any one of claims 58-62, wherein the amount of Thr-3 phosphorylated and/or Ser-10 phosphorylated and/or Thr-11 phosphorylated human Histone H3, or a corresponding phosphorylatable amino acid in the orthology of human Historic H3, is determined by an immunoassay using a reagent winch specifically binds with Thr-3 phosphorylated and/or Ser-10 phosphorylated or Thr-11 phosphorylated human Histone H3 or Corresponding phosphorylated orthology of human Histone H3.

72. The method of claim 71, wherein the immunoassay is a radioimmunoassay, a Western blot assay, a proximity ligation assay, an immunofluoresence assay, an enzyme immunoassay, an immunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, a dot blot assay, or a slot blot assay.

73. The method of claim 72, wherein the enzyme immunoassay is a sandwich enzyme immunoassay using a capture antibody or fragment thereof which specifically binds with human Histone H3 or corresponding orthology of human Histone H3 and a detection antibody or fragment thereof which specifically binds with Thr-3 phosphorylated and/or Ser-10 phosphorylated or Thr-11 phosphorylated human Histone H3, or a corresponding phosphorylated orthology of human Histone H3,

74. The method of any one of claims 58-62, wherein said human Histone H3 or orthology thereof and/or said human MELK or ortholog thereof, comprises a nucleic acid.
sequence or amino acid sequence set forth in Table 1.

75. The method of any one of claims 58-62, wherein the agent is a small molecule, or an antibody or antigen-binding fragment thereof.

76. The method of any one of Claims 58-62,wherein the agent decreases the amount of Thr-3 phosphorylated and/or Ser-I0 phosphorylated and/or Thr-11 phosphorylated human Histone H3, or a corresponding phosphorylatable amino acid in the ortholog of human Histone H3 by at least 50%.

77. A method for assessing the efficacy of an agent for inhibiting kinase activity of human MELK or an ortholog thereof in a subject, comprising:
a) detecting in a subject sample at a first point in time, the amount of Thr-3 phosphorylated and/or Ser-10 phosphorylated and/or Thr-11 phosphorylated human Histone H3, or the amount of a human Histone H3 ortholog phosphorylated at a corresponding amino acid of human Histone b) repeating step a) during at one or more subsequent points in time after administration of the agent; and c) comparing the amount of phosphorylated human Histone H3 or ortholog thereof detected in step a) with said amount detected in step b), wherein a higher amount of Thr-3 phosphorylated and/or Ser-10 phosphorylated and/or Thr-11 phosphorylated human Histone H3, or the amount of the human Histone H3 ortholog phosphorylated at a corresponding amino acid of human Histone H3, in the first point in time relative to at least one subsequent point in time, indicates that the agent inhibits kinase activity of human MELK or the ortholog thereof

78. The method of claim 77, wherein the amount of Thr-3 phosphorylated and/or Ser-10 phosphorylated and/or Thr-11 phosphorylated human Histone H3 or a corresponding phosphorylatable amino acid in the ortholog of human Histone H3, is determined by an immunoassay using a reagent which specifically binds with Thr-3 phosphorylated and/or Ser-10 phosphorylated human Histone H3 or Thr-11 phosphorylated human Histone H3, or corresponding phosphorylated ortholog of human Histone H3.

79. The method of claim 78, wherein the immunoassay is a radioimmunoassay, a Western blot assay, a proximity ligation assay, an immunofluoresence assay, an enzyme immunoassay, an immunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, a dot blot assay, or a slot blot assay.

80. The method of claim 79, wherein the enzyme immunoassay is a sandwich enzyme immunoassay using a capture antibody or fragment thereof which specifically binds with human Histone H3 or corresponding orthoIog of human Histone H3 and a detection antibody or fragment thereof which specifically binds with Thr-3 phosphorylated and/or Ser-10 phosphorylated or Thr-11 phosphorylated human Histone H3, or a corresponding phosphorylated ortholog of human Histone H3.

81. The method of claim 77, wherein the sample is selected from the group consisting of at vivo and in vivo samples.

82. The method of claim 77, wherein the sample comprises cancer cells.

83. The method of claim 82, wherein the cancer cells are cells selected from the group consisting of any cancer in which MELK or Histone H3 is amplified or overexpressed, any cancer having an activating mutation MELK or Histone H3, and any cancer in which MELK or Histone H3 is activated by other kinases.

84. The method of claim 77, wherein the sample is selected from the group consisting of tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow.

85. The method of claim 77, wherein the sample in step a) and/or step h) is a portion of a single sample obtained from the subject.

86. The method of claim 77, wherein the sample in step a) and/or step b) is a portion of pooled samples obtained from the subject.

81 The method of claim 77 wherein between the first point in time and the subsequent point in time, the subject has undergone treatment for cancer, has completed treatment for cancer: and/or is in remission from cancer.

88. The method of claim 77, wherein said human Histone H3 or ortholog thereof, and/or said human MELK or ortholog thereof, comprises a nucleic acid sequence or amino acid sequence set forth in Table 1.

89. The method of claim 77, wherein the agent is a small molecule, or an antibody or antigen-binding fragment thereof,

90. The method of claim 77, wherein the agent decreases the amount of Thr-3 phosphorylated and/or Ser-10 phosphorylated and/or Thr-11 phosphorylated human Histone H3, or a corresponding phosphorylatable amino acid in the ortholog of human Histone H3, by at least 50%.

91. A method of treating a subject afflicted with cancer comprising administering to the subject an agent that inhibits Thr-3 phosphorylation and/or Ser-10 phosphorylation and/or Thr-11. phosphorylation of human Histone113, or a corresponding phosphorylatable amino acid in an ortholog of human Histone H3, thereby treating the subject afflicted with the cancer,

92. The method of claim 91, wherein the agent is administered in a pharmaceutically acceptable formulation.

93. The method of claim 91, wherein the vent is a small molecule,or an antibody or antigen-binding fragment thereof

94. The method of claim 91, wherein the.agent directly binds said -human .Histone 113 or the ortholog thereof, or said human MELK or the ortholog thereof.

95. The method of claim 91, wherein the cancer is selected from the group consisting of any cancer in which MELK or Histone H3 is amplified or overexpressed, any cancer having an activating mutation of MELK or Histone H3, and any cancer in which MELK or Histone H3 is activated by other kinases

96. The method of claim 91, wherein said human Histone 143 or ortholog thereof, and/or said human MELK or ortholog thereof, comprises a nucleic acid sequence or amino acid sequence set forth in Table 1.

97. The method of claim 91, wherein the agent is a small molecule, or an antibody or antigen-binding fragment thereof.

98. The method of claim 91, wherein the agent decreases the amount of Thr-3 phosphorylated and/or Ser- 10 phosphorylated and/or Thr-11 phosphorylated human Histone H3, or a corresponding phosphorylatable amino acid in the ortholog of human Histone H3, by at least 50%.

99. The method of claim 91, further comprising administering one or more additional anti-cancer agents.

100. A method of determining the function or activity of human MELK or an ortholog, comprising:
a) detecting in a sample the amount of Thr-3 phosphorylated and/or Ser-10 phosphorylated and/or Thr-11 phosphorylated human histone H,3 or the amount of a human Histone H3 ortholog phosphorylated at a corresponding amino acid of human Histone H3;
b) repeating step a) in the same sample or a test sample at one or more subsequent points in time after manipulation of the sample and/or manipulation of the same sample or test sample; and c) comparing the amount of phosphorylated human Histone H3 or ortholog thereof detected in step a) with said amount detected in step b), wherein a modulated amount of Thr-3 phosphorylated and/or Ser-10 phosphorylated and/or Thr-11 phosphorylated human Histone H3, or the amount of the human Histone H3 ortholog phosphorylated at a corresponding amino acid of human Histone H3, in the first point in time relative to at least one subsequent point in time and/or at least one subsequent manipulation of the same sample or test sample, indicates that the function or activity of human MELK or an ortholog thereof is modulated.

101, The method of claim 100, wherein the amount of Thr-3 phosphorylated and/or Ser-phosphorylated and/or Thr-11 phosphorylated human Histone H3, or a corresponding phosphorylatable amino acid in the ortholog of human Historic H3, is determined by an immunoassay using a reagent which specifically binds with Thr-3 phosphorylated and/or Ser-10 phosphorylated or Thr-11 phosphorylated human Histone H3, or corresponding phosphorylated ortholog of human Histone H3.

102. The method of claim 101, wherein the immunoassay is a radioimmunoassay, a Western blot assay, a proximity ligation assay, an immunofluoresence assay, an enzyme immunoassay, an immunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, a dot blot assay, or a slot blot assay.

103. The method of claim 102, Wherein the enzyme immunoassay is a sandwich enzyme immunoassay using a capture antibody or fragment thereof which specifically binds with human Histone H3 or corresponding ortholog of human Histone H3 and a detection antibody or fragment thereof which specifically binds with Thr-3 phosphorylated and/or Set-10 phosphorylated or Thr-11 phosphorylated human Histone H3, or a corresponding phosphorylated ortholog of human Histone H3.

104. The method of claim 100, Wherein the sample is selected from the group consisting of in vitro, ex vivo, and in vivo samples.

105. The method of claim 100, wherein the sample comprises cells or the method uses a cell-based assay.

106, The method of claim 105, wherein the cells are cancer cells selected from the group consisting of any cancer in which MELK or Histone H3 is amplified or overexpressed, any cancer having an activating mutation of MELK or Histone H3, and any cancer in which MELK or Histone H3 is activated by other kinases.

107. The method of claim 100, wherein the sample is selected from the group consisting of tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow.

108. The method of claim 100, wherein the same sample or test sample in step a) and/or step h is a portion of a single sample obtained from a subject.

109. The method of claim 100, wherein the same sample or test sample in step a) and/or step b) is a portion of pooled samples obtained from a subject.

110. The method of claim 100, wherein between the first point in time and the subsequent point in time, the subject has undergone treatment for cancer, has completed.
treatment for cancer, and/or is in remission from cancer.

111 . The method of claim 100, wherein said human Histone H3 or ortholog thereof and/or said human MELK or ortholog thereof, comprises a nucleic acid sequence or amino acid sequence set forth in Table 1,

112. The method of claim 100, wherein the manipulation of the, sample is selected from the group consisting of contacting the sample with a test agent, contacting the sample with an upstream signal of the MELK signaling pathway, and contacting the sample with a MELK inhibitor.

113: The method of claim 112, wherein the test agent is a small molecule, or an antibody or antigen-binding fragment thereof.

114, The method of claim 112 or 113, wherein the test agent decreases the amount of Thr-3 phosphorylated and/or Ser-I0 phosphorylated andfor Thr-11 phosphorylated human Histone H3, or a corresponding phosphorylatable amino acid in the ortholog of human Histone H3, by at least 50%.