WO2019106126A1 - Modulateurs mdm2 pour le diagnostic et le traitement du liposarcome - Google Patents

Modulateurs mdm2 pour le diagnostic et le traitement du liposarcome Download PDF

Info

Publication number
WO2019106126A1
WO2019106126A1 PCT/EP2018/083086 EP2018083086W WO2019106126A1 WO 2019106126 A1 WO2019106126 A1 WO 2019106126A1 EP 2018083086 W EP2018083086 W EP 2018083086W WO 2019106126 A1 WO2019106126 A1 WO 2019106126A1
Authority
WO
WIPO (PCT)
Prior art keywords
mdm2
liposarcoma
chromatin
inhibitor
subject
Prior art date
Application number
PCT/EP2018/083086
Other languages
English (en)
Inventor
Laurent LE CAM
Madi CISSE
Lætitia LINARES
Nelly FIRMIN
Romain RISCAL
Sébastien CARRERE
Emilie SCHREPFER
Original Assignee
INSERM (Institut National de la Santé et de la Recherche Médicale)
Université De Montpellier
Institut Régional Du Cancer De Montpellier
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by INSERM (Institut National de la Santé et de la Recherche Médicale), Université De Montpellier, Institut Régional Du Cancer De Montpellier filed Critical INSERM (Institut National de la Santé et de la Recherche Médicale)
Publication of WO2019106126A1 publication Critical patent/WO2019106126A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to methods for the diagnosis of subjects suffering from liposarcoma exhibiting recruitment of MDM2 to chromatin or liposarcoma resistant to inhibitors of p53 and MDM2 interaction.
  • the present invention also relates to methods and pharmaceutical compositions for the treatment of liposarcoma.
  • LPS liposarcoma
  • GIST Gastrointestinal stromal tumors
  • LPS LPS
  • WD-LPS well-differentiated LPS
  • DD-LPS dedifferentiated LPS
  • the frequency of MDM2 amplification is such (almost 100%) that it is currently used for routine diagnosis to distinguish WD/DD-LPS from other sarcoma subtypes that more commonly harbor p53 mutations.
  • This very peculiar genetic profile of WD/DD-LPS raises important questions regarding the strong selective pressure that leads to the systematic gene amplification of MDM2 in LPS.
  • the pro-oncogenic properties of the MDM2 protein have been mainly attributed so far to its well-characterized E3 ubiquitin ligase activity targeting p53 for proteasomal degradation.
  • This MDM2 function has been the rationale for the development of targeted therapies for tumors harboring wild-type p53 that aimed at blocking the interaction between p53 and MDM2 and reactivating p53 tumor suppressor functions.
  • this class of inhibitors which includes Nutlin3a (Nutlin) and its derivatives, efficiently stabilizes p53 in vitro, most clinical trials have shown that they provide a poor, if any, therapeutic benefit for LPS patients.
  • the present invention relates to methods for the diagnosis of subjects suffering from liposarcoma exhibiting recruitment of MDM2 to chromatin or liposarcoma resistant to inhibitors of p53 and MDM2 interaction.
  • the present invention also relates to methods and pharmaceutical compositions for the treatment of liposarcoma.
  • the present invention also relates to methods and pharmaceutical compositions for the treatment of cancer types exhibiting recruitment of MDM2 to chromatin and cancer types exhibiting exacerbated serine and glycine metabolism.
  • MDM2 functions in serine metabolism are exacerbated in liposarcomas and support the notion that the selective pressure for MDM2 amplification in LPS reflects their addiction to serine metabolism.
  • the inventors found that most, if not all, LPS cell lines, as well as fresh LPS patient samples, display increased levels of chromatin-bound MDM2 in comparison to other types of sarcomas.
  • the inventors demonstrate that DD-LPS cells lines are highly dependent on serine metabolism in vitro.
  • LPS cell lines and LPS patient-derived xenograft (PDX) mouse models demonstrate that genetic or pharmacological inhibition of chromatin-bound MDM2, as well as targeting of key genes involved in serine metabolism or transport including PHGDH, PSAT1, PSPH, SLC1A4 and strategies limiting serine availability or intake, represent very efficient therapeutic strategies for LPS.
  • the inventors also provide molecular evidences explaining the poor therapeutic response of LPS patients to Nutlin, and demonstrate that chromatin-bound MDM2 and serine metabolism are critical for LPS development.
  • the inventors also define new therapeutic strategies for LPS, cancer types exhibiting recruitment of MDM2 to chromatin and cancer types exhibiting exacerbated serine and glycine metabolism based on a new class of MDM2 inhibitors that block chromatin-bound MDM2 activities and its effects on serine metabolism.
  • the present invention relates to an inhibitor of the MDM2 recruitment to chromatin or inhibitor of MDM2-mediated control of serine metabolism for use in the treatment of cancer exhibiting recruitment of MDM2 to chromatin.
  • the present invention relates to an inhibitor of the MDM2 recruitment to chromatin or inhibitor of MDM2-mediated control of serine metabolism for use in the treatment of cancer exhibiting exacerbated serine and glycine metabolism.
  • the present invention relates to a MDM2 inhibitor for use in the treatment of liposarcoma in a subject in need thereof.
  • the present invention relates to an inhibitor of MDM2 recruitment to chromatin for use in the treatment of liposarcoma in a subject in need thereof.
  • the present invention relates to an inhibitor of MDM2 recruitment to chromatin by inducing its degradation for use in the treatment of liposarcoma in a subject in need thereof.
  • the present invention relates to an inhibitor of MDM2-mediated control of serine metabolism for use in the treatment of liposarcoma in a subject in need thereof.
  • the term“subject” denotes a mammal.
  • a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with liposarcoma.
  • a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with liposarcoma exhibiting increased levels of chromatin-bound MDM2.
  • the term“subject” refers to any subject (preferably human) afflicted with or susceptible to be afflicted with liposarcoma resistant to inhibitors of targeting p53 and MDM2 interaction. In some embodiments, the term “subject” refers to any subject afflicted with or susceptible to be afflicted with liposarcoma receiving a serine and glycine deprived diet.
  • the term“subject” refers to any subject afflicted with or susceptible to be afflicted with cancer types exhibiting increased recruitment of MDM2 to chromatin, including liposarcoma, ovarian cancers, glioblastoma, breast cancers, melanoma, colorectal cancers, kidney cancers, bone cancer, brain cancer, skin cancer, AML and lung cancer or cancer exhibiting exacerbated serine and glycine metabolism, including advanced melanoma and lung cancer.
  • liposarcoma or“LPS” has its general meaning in the art and refers to soft tissue sarcomas of mesenchymal origin such as revised in the World Health Organisation Classification ICD10 C49.9.
  • the term“liposarcoma” also refers to well-differentiated and de- differentiated liposarcoma (WD- and DD-LPS).
  • the term “liposarcoma” also relates to Malignant mesenchymal neoplasms, a type of soft tissue sarcoma, a group of lipomatous tumors of varying severity ranging from slow-growing to aggressive and metastatic.
  • Liposarcomas are most often located in the lower extremities or retroperitoneum, but they can also occur in the upper extremities, neck, peritoneal cavity, spermatic cord, breast, vulva and axilla.
  • the term “liposarcoma” also relates to Dedifferentiated liposarcoma and Well-differentiated liposarcoma.
  • the term“liposarcoma” refers to liposarcoma exhibiting recruitment of MDM2 to chromatin and MDM2-mediated control of serine metabolism independently of p53.
  • the term“liposarcoma” refers to liposarcoma resistant to inhibitors of p53 and MDM2 protein interaction.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • MDM2 has its general meaning in the art and refers to mouse double minute 2.
  • MDM2 refers also refers to oncoprotein, E3 ubiquitin-protein ligase having the UniProtKB accession number Q00987.
  • MDM2 inhibitor has its general meaning in the art and refers to compounds that selectively blocks or inactivates MDM2.
  • the term“MDM2 inhibitor” according to the invention refers to compounds inhibiting the MDM2 recruitment to chromatin.
  • the term“MDM2 inhibitor” also refers to inhibitors targeting MDM2-mediated control of serine metabolism.
  • the term“MDM2 inhibitor” also refers to compounds inhibiting MDM2- mediated control of serine/glycine and glutathione metabolism.
  • MDM2 inhibitor also refers to compounds inhibiting p53 -independent functions of chromatin-bound MDM2 in serine metabolism.
  • the term“MDM2 inhibitor” also refers to a compound that selectively blocks MDM2 translocation to chromatin.
  • MDM2 inhibitor also refers to a compound that selectively induces MDM2 degradation.
  • MDM2 inhibitor also refers to a compound that selectively blocks the MDM2 recruitment to chromatin, inhibits MDM2 and ATF3/4 (activating transcription factor 3 and 4) protein interaction and then inhibits the expression of genes involved in serine metabolism including phosphoglycerate dehydrogenase ( PHGDH ), phosphoserine aminotransferase ( PSAT1 ), and phosphoserine phosphatase ( PSPH) and solute carrier family 1 member 4 ( slclA4 ).
  • PHGDH phosphoglycerate dehydrogenase
  • PSAT1 phosphoserine aminotransferase
  • PSPH solute carrier family 1 member 4
  • slclA4 solute carrier family 1 member 4
  • Compounds inhibiting MDM2-mediated control of serine metabolism or serine metabolism inhibitors refer to PHGDH inhibitors, PSAT inhibitors, PSPH inhibitors, and SLC1A4 inhibitors.
  • the term“selectively blocks or inactivates” refers to a compound that preferentially binds to and blocks or inactivates MDM2, PHGDH, PSAT, PSPH, and SLC1A4 with a greater affinity and potency, respectively, than other ubiquitin-protein ligases, or related enzymes or related transporters.
  • MDM2 inhibitor also refers to a compound that inhibits MDM2, PHGDH, PSAT, PSPH, and SLC1A4 expression.
  • a MDM2 inhibitor is a small organic molecule, a polypeptide, an aptamer, an antibody, an intra-antibody, an oligonucleotide or a ribozyme.
  • Tests and assays for determining whether a compound is a MDM2 inhibitor are well known by the skilled person in the art such as described in (Qin et ah, 2016; US8329723).
  • MDM2 inhibitors are well-known in the art as illustrated by (Qin et ah, 2016; US8329723).
  • the MDM2 inhibitor is selected from the group consisting of SP141, 6-Methoxy-l-naphthalen-2-yl-9H-P-carboline; SP141 nanoparticles (SP141NP); SPl4l-loaded IgG Fc-conjugated maleimidyl-poly(ethylene glycol)-co-poly(e- caprolactone) (Mal-PEG-PCL) nanoparticles (SPl4lFcNP); 6-Mcthoxy- 1 -quinolin-4-yl-9H-P- carboline; 6-Methoxy- 1 -naphthalen- 1 -yl-9H-P-carboline; 6-Methoxy- 1 -phenanthren-9-yl-9H- b-carboline; 7-Mcthoxy- 1 -phcnanthrcn-9-yl-9H-P-carbolinc; miR-509-5p; shRNA and compounds described in Qin et ah,
  • the present invention relates to the MDM2 inhibitor of the invention in combination with ATF3 inhibitor and/or ATF4 inhibitor for use in the treatment of liposarcoma in a subject in need thereof.
  • the MDM2 inhibitor of the invention is a PHGDH inhibitor.
  • PHGDH has its general meaning in the art and refers to phosphoglycerate dehydrogenase having the UniProtKB accession number 043175, Catalyzes the oxidation of 3 -phosphoglycerate (3PG) to 3-phosphohydroxypyruvate (3P-Pyr), the first step of the serine biosynthesis pathway.
  • Tests and assays for determining whether a compound is a PHGDH inhibitor are well known by the skilled person in the art such as described in (Hamanak et al, 2017; Wang et ah, 2017; Mullarky et al, 2016; Pacold et al, 2016).
  • the PHGDH inhibitors are well-known in the art as illustrated by (Hamanak et al, 2017; Wang et al, 2017; Mullarky et al., 2016; Pacold et al., 2016).
  • the PHGDH inhibitor is selected from the group consisting of CBR-5884; CBR-5807 (Disulfiram); CBR-6936; CBR-9480; Piperazine-l- carbothio amide PHGDH inhibitors such as NCT-503 and NCT-502; PKUMDL-WQ-2101; PKUMDL- WQ-2201 ; PKUMDL-WQ-2202; PKUMDL-WQ-2203; shRNA (Hamanak et al, 2017; Wang et al., 2017; Mullarky et al., 2016; Pacold et al., 2016).
  • the MDM2 inhibitor of the invention is a PSAT inhibitor.
  • PSAT has its general meaning in the art and refers to phosphoserine aminotransferase having the UniProtKB accession number Q9Y617, catalyzes the conversion of 3-phosphohydroxypyruvate (3P-Pyr) to 3 -phosphoserine (3P-Ser).
  • the PSAT inhibitors are well-known in the art as illustrated by (Ye et al., 2012).
  • the PSAT inhibitor is selected from the group consisting of siRNA described in (Ye et al., 2012) and shRNA.
  • the MDM2 inhibitor of the invention is a PSPH inhibitor.
  • PSPH has its general meaning in the art and refers to phosphoserine phosphatase having the UniProtKB accession number P78330, catalyzes the last step in the biosynthesis of serine, the conversion of 3 -phosphoserine (3P-Ser) to serine.
  • PSPH inhibitors are well-known in the art as illustrated by (US 20140087970).
  • the PSPH inhibitor is selected from the group consisting of AP3; shRNA and compounds described in (US 20140087970).
  • the MDM2 inhibitor of the invention is a SLC1A4 inhibitor.
  • SLC1A4 has its general meaning in the art and refers to Solute Carrier Family 1 Member 4, the neutral amino acid transporter of serine having the UniProtKB accession number P43007.
  • SLC1A4 also refers to ASCT1, the Alanine/S erine/Cysteine/Threonine Transporter 1. Tests and assays for determining whether a compound is a SLC1A4 inhibitor are well known by the skilled person in the art such as described in (Foster et ah, 2017).
  • the SLC1A4 inhibitors are well-known in the art as illustrated by (Foster et al, 2017).
  • the SLC1A4 inhibitor is selected from the group consisting of Phenylglycine analogs such as L-4C1PG; shRNA and compounds described in (Foster et al., 2017).
  • the MDM2 inhibitor and serine metabolism inhibitor of the invention is an aptamer.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996). Then after raising aptamers directed against MDM2, PHGDH, PSAT, PSPH and/or SLC1A4 of the invention as above described, the skilled man in the art can easily select those inhibiting MDM2, PHGDH, PSAT, PSPH and/or SLC1A4.
  • the MDM2 inhibitor and serine metabolism inhibitor of the invention is an antibody (the term including“antibody portion”) directed against MDM2, PHGDH, PSAT, PSPH and SLC1A4.
  • the antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a chimeric antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a light chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody.
  • the portion of the antibody comprises a F(ab')2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a variable domain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises one or more CDR domains of the antibody.
  • antibody includes both naturally occurring and non-naturally occurring antibodies. Specifically, “antibody” includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, “antibody” includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man.
  • Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with antigenic forms of MDM2, PHGDH, PSAT, PSPH and SLC1A4. The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization.
  • suitable intervals e.g., twice-weekly, weekly, twice-monthly or monthly
  • suitable intervals e.g., twice-weekly, weekly, twice-monthly or monthly
  • the animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization.
  • Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides.
  • Other suitable adjuvants are well-known in the field.
  • the animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal administration or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes.
  • the antigen may be provided as synthetic peptides corresponding to antigenic regions of interest in MDM2, PHGDH, PSAT, PSPH and SLC1A4.
  • lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma.
  • cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods, as described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996).
  • cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen.
  • Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation.
  • an antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region designated an F(ab’)2 fragment, retains both of the antigen binding sites of an intact antibody.
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule.
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
  • the Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
  • CDRs complementarity determining regions
  • FRs framework regions
  • CDR1 through CDRS complementarity determining regions
  • compositions and methods that include humanized forms of antibodies.
  • humanized describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules.
  • Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference.
  • the above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 also propose four possible criteria which may use in designing the humanized antibodies.
  • the first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies.
  • the second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected.
  • the third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected.
  • the fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3 A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs.
  • the above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies.
  • One of ordinary skill in the art will be familiar with other methods for antibody humanization.
  • humanized forms of the antibodies some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen.
  • Suitable human immunoglobulin molecules would include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules.
  • a "humanized" antibody retains a similar antigenic specificity as the original antibody.
  • Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference.
  • mice have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies.
  • the animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest.
  • monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans.
  • KAMA human anti-mouse antibody
  • the present invention also provides for F(ab') 2 Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non human sequences.
  • the present invention also includes so-called single chain antibodies.
  • the various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM.
  • IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4.
  • the MDM2 inhibitor and serine metabolism inhibitor of the invention is a Human IgG4.
  • the invention provides a multispecific antibody comprising a first antigen binding site from an antibody of the present invention directed against MDM2, PHGDH, PSAT, PSPH or SLC1A4 and at least one second antigen binding site.
  • the second antigen-binding site binds to ATF3 or ATF4.
  • the second antigen-binding site is used for recruiting a killing mechanism such as, for example, by binding an antigen on a human effector cell as a BiTE (Bispecific T-Cell engager) antibody which is a bispecific scFv2 directed against target antigen and CD3 on T cells described in US7235641, or by binding a cytotoxic agent or a second therapeutic agent.
  • a killing mechanism such as, for example, by binding an antigen on a human effector cell as a BiTE (Bispecific T-Cell engager) antibody which is a bispecific scFv2 directed against target antigen and CD3 on T cells described in US7235641, or by binding a cytotoxic agent or a second therapeutic agent.
  • effector cell refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response.
  • Exemplary immune cells include a cell of a myeloid or lymphoid origin, for instance lymphocytes (such as B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, mast cells and granulocytes, such as neutrophils, eosinophils and basophils.
  • lymphocytes such as B cells and T cells including cytolytic T cells (CTLs)
  • CTLs cytolytic T cells
  • FcRs Fc receptors
  • an effector cell is capable of inducing ADCC, such as a natural killer cell.
  • monocytes, macrophages, which express FcRs are involved in specific killing of target cells and presenting antigens to other components of the immune system.
  • an effector cell may phagocytose a target antigen or target cell.
  • the expression of a particular FcR on an effector cell may be regulated by humoral factors such as cytokines.
  • An effector cell can phagocytose a target antigen or phagocytose or lyse a target cell.
  • Suitable cytotoxic agents and second therapeutic agents are exemplified below, and include toxins (such as radiolabeled peptides), chemotherapeutic agents and prodrugs.
  • the second antigen-binding site binds a tissue-specific antigen, promoting localization of the bispecific antibody to a specific tissue.
  • the second antigen-binding site binds to an antigen located on the same type of cell as the [MDM2, PHGDH, PSAT, PSPH and SEC 1A4] -expressing cell, typically a tumor-associated antigen (TAA), but has a binding specificity different from that of the first antigen-binding site.
  • TAA tumor-associated antigen
  • the second antigen-binding site binds to a different epitope of [MDM2, PHGDH, PSAT, PSPH or SFC1 A4]
  • the second antigen-binding site may alternatively bind an angiogenic factor or other cancer-associated growth factor, such as a vascular endothelial growth factor, a fibroblast growth factor, epidermal growth factor, angiogenin or a receptor of any of these, particularly receptors associated with cancer progression.
  • the second antigen-binding site is from a second antibody or ADC of the invention, such as the antibody of the present invention.
  • Exemplary formats for the multispecific antibody molecules of the invention include, but are not limited to (i) two antibodies cross-linked by chemical heteroconjugation, one with a specificity to [MDM2, PHGDH, PSAT, PSPH or SLC1A4] and another with a specificity to a second antigen; (ii) a single antibody that comprises two different antigen-binding regions; (iii) a single-chain antibody that comprises two different antigen-binding regions, e.g., two scFvs linked in tandem by an extra peptide linker; (iv) a dual- variable-domain antibody (DVD- Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-IgTM) Molecule, In : Antibody Engineering, Springer Berlin Heidelberg (2010)); (v) a chemically- linked bispecific (Fab')2 fragment; (
  • IgG-like molecules with complementary CH3 domains to force heterodimerization is IgG-like molecules with complementary CH3 domains to force heterodimerization.
  • Such molecules can be prepared using known technologies, such as, e.g., those known as Triomab/Quadroma (Trion Pharma/Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche) and electrostatically-matched (Amgen), FUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono), Biclonic (Merus) and DuoBody (Genmab A/S) technologies.
  • the bispecific antibody is obtained or obtainable via a controlled Fab-arm exchange, typically using DuoBody technology.
  • a controlled Fab-arm exchange typically using DuoBody technology.
  • In vitro methods for producing bispecific antibodies by controlled Fab-arm exchange have been described in W02008119353 and WO 2011131746 (both by Genmab A/S).
  • a bispecific antibody is formed by "Fab-arm" or "half- molecule" exchange (swapping of a heavy chain and attached light chain) between two monospecific antibodies, both comprising IgG4-like CH3 regions, upon incubation under reducing conditions.
  • the resulting product is a bispecific antibody having two Fab arms which may comprise different sequences.
  • bispecific antibodies of the present invention are prepared by a method comprising the following steps, wherein at least one of the first and second antibodies is the antibody of the present invention : a) providing a first antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a first CH3 region; b) providing a second antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a second CH3 region; wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions; c) incubating said first antibody together with said second antibody under reducing conditions; and d) obtaining said bispecific antibody, wherein the first antibody is the antibody of the present invention and the second antibody has a different binding specificity, or vice versa.
  • the reducing conditions may, for example, be provided by adding a reducing agent, e.g. selected from 2-mercaptoethylamine, dithiothreitol and tris(2- carboxyethyl)phosphine.
  • Step d) may further comprise restoring the conditions to become non reducing or less reducing, for example by removal of a reducing agent, e.g. by desalting.
  • the sequences of the first and second CH3 regions are different, comprising only a few, fairly conservative, asymmetrical mutations, such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions.
  • the antibody according to the invention is a single domain antibody.
  • the term“single domain antibody” (sdAb) or “VHH” refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called“nanobody®”. According to the invention, sdAb can particularly be llama sdAb.
  • the term“VHH” refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3.
  • CDRs complementarity determining region
  • CDR complementarity determining region
  • VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation.
  • the VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation.
  • VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2.
  • Antigen- specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells.
  • VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations.
  • the high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B-cells in the lymphoid organs of immunized animals.
  • the affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations).
  • VHHs derived from camelid are readily expressed in and purified from the E.
  • VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells.
  • the“Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695).
  • The“Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E.
  • coli see for example US 6,765,087 and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).
  • moulds for example Aspergillus or Trichoderma
  • yeast for example Saccharomyces, Kluyveromyces, Hansenula or Pichia
  • the MDM2 inhibitor and serine metabolism inhibitor of the invention is an MDM2, PHGDH, PSAT, PSPH and SLC1A4 expression inhibitor.
  • a gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA.
  • Gene products also include messenger RNAs, which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins (e.g., MDM2, PHGDH, PSAT, PSPH and SLC1A4) modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation, myristilation, and glycosylation.
  • An“inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene.
  • Expression inhibitors for use in the present invention may be based on antisense oligonucleotide constructs.
  • Anti-sense oligonucleotides including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of proteins, and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding MDM2, PHGDH, PSAT, PSPH and SLC1A4 can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion.
  • Small inhibitory RNAs and short hairpin RNAs (shRNAs) can also function as expression inhibitors for use in the present invention.
  • Gene expression can be reduced by contacting the subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that MDM2, PHGDH, PSAT, PSPH and SLC1A4 expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al.
  • Ribozymes can also function as expression inhibitors for use in the present invention.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of mRNA sequences are thereby useful within the scope of the present invention.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
  • antisense oligonucleotides and ribozymes useful as inhibitors of expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3’ ends of the molecule, or the use of phosphorothioate or 2’-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
  • Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing MDM2, PHGDH, PSAT, PSPH and SLC1A4.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno- associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno- associated virus
  • SV40-type viruses polyoma viruses
  • Epstein-Barr viruses Epstein-Barr viruses
  • papilloma viruses herpes virus
  • Non-cytopathic viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest.
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.
  • Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle).
  • retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • Standard protocols for producing replication-deficient retroviruses including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles
  • KRIEGLER A Laboratory Manual
  • MURRY Method of Recombinant retroviruses by the packaging cell line
  • Methods in Molecular Biology vol.7, Humana Press, Inc., Cliffton, N.J., 1991.
  • adeno-viruses and adeno-associated viruses are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • the adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hematopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions.
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also function in an extrachromosomal fashion.
  • Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g., SANBROOK et al, "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
  • inhibitors according to the invention as described above are administered to the subject in a therapeutically effective amount.
  • a “therapeutically effective amount” of the inhibitor of the present invention as above described is meant a sufficient amount of the inhibitor for treating cancer at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the inhibitors and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific inhibitor employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific inhibitor employed; the duration of the treatment; drugs used in combination or coincidential with the specific inhibitor employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the inhibitor of the present invention for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the inhibitor of the present invention, preferably from 1 mg to about 100 mg of the inhibitor of the present invention.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the compound according to the invention may be used in a concentration between 0.01 mM and 20 mM, particularly, the compound of the invention may be used in a concentration of 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 20.0 mM.
  • the compound of the present invention is administered to the subject in the form of a pharmaceutical composition.
  • the compound of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • “Pharmaceutically” or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the active principle in the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the compound of the present invention can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine,
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized agent of the present inventions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions the typical methods of preparation are vacuum-drying and freeze drying techniques which yield a powder of the compound of the present invention plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • the preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • the MDM2 inhibitor of the invention is administered in combination with an ATF3 inhibitor and/or an ATF4 inhibitor.
  • the compound of the present invention is used as neo-adjuvant in the treatment of liposarcoma.
  • the compound of the present invention is used in combination with strategies aiming at limiting serine and glycine availability, including specific diet such as serine and glycine deprived diet, in the treatment of liposarcoma.
  • the compound of the present invention is administered sequentially or concomitantly with one or more therapeutic active agent such as chemotherapeutic or radiotherapeutic agents.
  • the compound of the present invention is administered with a chemotherapeutic agent.
  • chemotherapeutic agent refers to chemical compounds that are effective in inhibiting tumor growth.
  • examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolo melamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,
  • calicheamicin especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Intl. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrol
  • paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-l 1 ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and phannaceutically acceptable salts, acids or derivatives of any of the above.
  • the compound of the present invention is administered with a targeted cancer therapy.
  • Targeted cancer therapies are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules ("molecular targets") that are involved in the growth, progression, and spread of cancer.
  • Targeted cancer therapies are sometimes called “molecularly targeted drugs", “molecularly targeted therapies”, “precision medicines”, or similar names.
  • the targeted therapy consists of administering the subject with a tyrosine kinase inhibitor as defined above.
  • compound of the present invention is administered with an immunotherapeutic agent.
  • immunotherapeutic agent refers to a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the body's immune response against cancer cells and/or that decreases the side effects of other anticancer therapies. Immunotherapy is thus a therapy that directly or indirectly stimulates or enhances the immune system's responses to cancer cells and/or lessens the side effects that may have been caused by other anti-cancer agents. Immunotherapy is also referred to in the art as immunologic therapy, biological therapy biological response modifier therapy and biotherapy.
  • immunotherapeutic agents examples include, but are not limited to, cytokines, cancer vaccines, monoclonal antibodies and non-cytokine adjuvants.
  • the immunotherapeutic treatment may consist of administering the subject with an amount of immune cells (T cells, NK, cells, dendritic cells, B cells).
  • Immunotherapeutic agents can be non-specific, i.e. boost the immune system generally so that the human body becomes more effective in fighting the growth and/or spread of cancer cells, or they can be specific, i.e. targeted to the cancer cells themselves immunotherapy regimens may combine the use of non-specific and specific immunotherapeutic agents.
  • Non-specific immunotherapeutic agents are substances that stimulate or indirectly improve the immune system.
  • Non-specific immunotherapeutic agents have been used alone as a main therapy for the treatment of cancer, as well as in addition to a main therapy, in which case the non-specific immunotherapeutic agent functions as an adjuvant to enhance the effectiveness of other therapies (e.g. cancer vaccines).
  • Non-specific immunotherapeutic agents can also function in this latter context to reduce the side effects of other therapies, for example, bone marrow suppression induced by certain chemotherapeutic agents.
  • Non-specific immunotherapeutic agents can act on key immune system cells and cause secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agents can themselves comprise cytokines.
  • Non- specific immunotherapeutic agents are generally classified as cytokines or non-cytokine adjuvants.
  • cytokines have found application in the treatment of cancer either as general non-specific immunotherapies designed to boost the immune system, or as adjuvants provided with other therapies.
  • Suitable cytokines include, but are not limited to, interferons, interleukins and colony- stimulating factors.
  • Interferons (IFNs) contemplated by the present invention include the common types of IFNs, IFN-alpha (IFN-a), IFN-beta (IFN-b) and IFN- gamma (IFN-g).
  • IFNs can act directly on cancer cells, for example, by slowing their growth, promoting their development into cells with more normal behaviour and/or increasing their production of antigens thus making the cancer cells easier for the immune system to recognise and destroy.
  • IFNs can also act indirectly on cancer cells, for example, by slowing down angiogenesis, boosting the immune system and/or stimulating natural killer (NK) cells, T cells and macrophages.
  • Recombinant IFN-alpha is available commercially as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation).
  • Interleukins contemplated by the present invention include IL-2, IL-4, IL-l l and IL-12. Examples of commercially available recombinant interleukins include Proleukin® (IL-2; Chiron Corporation) and Neumega® (IL- 12; Wyeth Pharmaceuticals). Zymogenetics, Inc.
  • Colony-stimulating factors contemplated by the present invention include granulocyte colony stimulating factor (G-CSL or filgrastim), granulocyte-macrophage colony stimulating factor (GM-CSL or sargramostim) and erythropoietin (epoetin alfa, darbepoietin). Treatment with one or more growth factors can help to stimulate the generation of new blood cells in subjects undergoing traditional chemotherapy.
  • CSLs can be helpful in decreasing the side effects associated with chemotherapy and can allow for higher doses of chemotherapeutic agents to be used.
  • Various-recombinant colony stimulating factors are available commercially, for example, Neupogen® (G-CSL; Amgen), Neulasta (pelfilgrastim; Amgen), Leukine (GM-CSL; Berlex), Procrit (erythropoietin; Ortho Biotech), Epogen (erythropoietin; Amgen), Arnesp (erytropoietin).
  • immunotherapeutic agents can be active, i.e. stimulate the body's own immune response, or they can be passive, i.e.
  • Passive specific immunotherapy typically involves the use of one or more monoclonal antibodies that are specific for a particular antigen found on the surface of a cancer cell or that are specific for a particular cell growth factor.
  • Monoclonal antibodies may be used in the treatment of cancer in a number of ways, for example, to enhance a subject’s immune response to a specific type of cancer, to interfere with the growth of cancer cells by targeting specific cell growth factors, such as those involved in angiogenesis, or by enhancing the delivery of other anticancer agents to cancer cells when linked or conjugated to agents such as chemotherapeutic agents, radioactive particles or toxins.
  • Monoclonal antibodies currently used as cancer immunotherapeutic agents that are suitable for inclusion in the combinations of the present invention include, but are not limited to, rituximab (Rituxan®), trastuzumab (Herceptin®), ibritumomab tiuxetan (Zevalin®), tositumomab (Bexxar®), cetuximab (C-225, Erbitux®), bevacizumab (Avastin®), gemtuzumab ozogamicin (Mylotarg®), alemtuzumab (Campath®), and BL22.
  • Other examples include anti-CTLA4 antibodies (e.g.
  • antibodies include B cell depleting antibodies.
  • Typical B cell depleting antibodies include but are not limited to anti-CD20 monoclonal antibodies [e.g.
  • the immunotherapeutic treatment may consist of allografting, in particular, allograft with hematopoietic stem cell HSC.
  • the immunotherapeutic treatment may also consist in an adoptive immunotherapy as described by Nicholas P. Restifo, Mark E.
  • the subject In adoptive immunotherapy, the subject’s circulating lymphocytes, NK cells, are isolated amplified in vitro and readministered to the subject.
  • the activated lymphocytes or NK cells are most preferably be the subject’s own cells that were earlier isolated from a blood or tumor sample and activated (or“expanded”) in vitro.
  • the compound of the present invention is administered with a radio therapeutic agent.
  • radiotherapeutic agent as used herein, is intended to refer to any radiotherapeutic agent known to one of skill in the art to be effective to treat or ameliorate cancer, without limitation.
  • the radiotherapeutic agent can be an agent such as those administered in brachytherapy or radionuclide therapy.
  • Such methods can optionally further comprise the administration of one or more additional cancer therapies, such as, but not limited to, chemotherapies, and/or another radiotherapy.
  • said additional active compounds may be contained in the same composition or administrated separately.
  • the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of liposarcoma in a subject in need thereof.
  • kits comprising the compound of the invention. Kits containing the compound of the invention find use in therapeutic methods.
  • the present invention relates to a method for diagnosing liposarcoma exhibiting recruitment of MDM2 to chromatin in a subject in need thereof, comprising the step of determining the level of serine and/or glycine in a biological sample obtained from said subject.
  • the present invention relates to a method for diagnosing liposarcoma exhibiting recruitment of MDM2 to chromatin in a subject in need thereof, comprising the step of determining the level of chromatin-bound MDM2 in a biological sample obtained from said subject.
  • the present invention relates to a method for diagnosing liposarcoma exhibiting recruitment of MDM2 to chromatin in a subject in need thereof, comprising the step of determining the level of serine and/or glycine and chromatin-bound MDM2 in a biological sample obtained from said subject.
  • the present invention further comprises the steps of comparing the level of serine/glycine and/or of chromatin-bound MDM2 in a biological sample obtained from said subject with a predetermined reference value and concluding that the subject have liposarcoma exhibiting recruitment of MDM2 to chromatin when the level of serine/glycine and/or of chromatin-bound MDM2 is higher than the predetermined reference value or concluding that the subject don’t have liposarcoma exhibiting recruitment of MDM2 to chromatin when the level of serine/glycine and/or chromatin-bound MDM2 is lower than the predetermined reference value.
  • the present invention also relates to a method for diagnosing liposarcoma exhibiting recruitment of MDM2 to chromatin in a subject in need thereof, comprising the steps of: i) determining the level of serine/glycine and/or chromatin-bound MDM2 in a biological sample obtained from said subject, ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the subject have liposarcoma exhibiting recruitment of MDM2 to chromatin when the level determined at step i) is higher than the predetermined reference value or concluding that the subject don’t have liposarcoma exhibiting recruitment of MDM2 to chromatin when the level determined at step i) is lower than the predetermined reference value.
  • the present invention relates to a method for diagnosing liposarcoma resistant to inhibitors of p53 and MDM2 interaction in a subject in need thereof, comprising the step of determining the level of serine and/or glycine in a biological sample obtained from said subject.
  • the present invention relates to a method for diagnosing liposarcoma resistant to inhibitors of p53 and MDM2 interaction in a subject in need thereof, comprising the step of determining the level of chromatin-bound MDM2 in a biological sample obtained from said subject.
  • the present invention relates to a method for diagnosing liposarcoma resistant to inhibitors of p53 and MDM2 interaction in a subject in need thereof, comprising the step of determining the level of serine/glycine and/or glycine and chromatin- bound MDM2 in a biological sample obtained from said subject.
  • the present invention further comprises the steps of comparing the level of serine/glycine and/or chromatin-bound MDM2 with a predetermined reference value and concluding that the subject have liposarcoma resistant to inhibitors of p53 and MDM2 interaction when the level of serine/glycine and/or of chromatin-bound MDM2 is higher than the predetermined reference value or concluding that the subject don’t have liposarcoma resistant to inhibitors of p53 and MDM2 interaction when the level of serine/glycine and/or chromatin-bound MDM2 is lower than the predetermined reference value.
  • the present invention also relates to a method for diagnosing liposarcoma resistant to inhibitors of p53 and MDM2 interaction in a subject in need thereof, comprising the steps of: i) determining the level of serine/glycine and/or chromatin-bound MDM2 in a biological sample obtained from said subject, ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the subject have liposarcoma resistant to inhibitors of p53 and MDM2 interaction when the level determined at step i) is higher than the predetermined reference value or concluding that the subject don’t have liposarcoma resistant to inhibitors of p53 and MDM2 interaction when the level determined at step i) is lower than the predetermined reference value.
  • the term“biological sample” refers to any biological sample derived from the subject such as liposarcoma sample, biopsy sample, biopsy sample obtained during surgery, blood sample, plasma sample, or serum sample. Said biological sample is obtained
  • inhibitor of p53 and MDM2 interaction has its general meaning in the art and refers to compounds inhibiting the MDM2-p53 protein-protein interaction and the action of MDM2 toward p53, compounds targeting the MDM2-p53 interaction, and compounds targeting the p53 pathway.
  • the inhibitors of p53 and MDM2 interaction are well-known in the art as illustrated by (Khoo et al, 2014; Li and Lozano, 2013; Saiki et ah, 2015; Rew and Sunk, 2014).
  • the inhibitors of p53 and MDM2 interaction is selected from the group consisting of nutlin and nutlin analogs; Nutlin 3 a; RG7112 (also known as RO5045337); RG7388; Ro-2443; RO5503781; MI-773; MI-219, MI-713, MI-888; DS-3032b; Benzodiazepinediones (including TDP521252); Sulphonamides (including NSC279287); Chromenotriazolopyrimidine compounds; morpholinone and piperidinones (AM 8553 and AMG 232); piperidines; Terphenyls; Chalcones ; Pyrazoles; imidazoles; Imidazole- indoles ; Isoindolinone ; Pyrrolidinone (including PXN822) ; Piperidines; spirooxindoles; Naturally derived prenylated xanthones; SAH-8; sMT
  • the“reference value” refers to a threshold value or a cut-off value.
  • the setting of a single “reference value” thus allows discrimination between a liposarcoma exhibiting recruitment of MDM2 to chromatin and liposarcoma not exhibiting recruitment of MDM2 to chromatin or a liposarcoma resistant to inhibitors of p53 and MDM2 interaction and liposarcoma which is not resistant to inhibitors of p53 and MDM2 interaction with respect to the cancer relapse and overall survival (OS) for a subject.
  • a “threshold value” or “cut off value” can be determined experimentally, empirically, or theoretically.
  • a threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art.
  • the threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative).
  • the optimal sensitivity and specificity can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
  • ROC Receiver Operating Characteristic
  • the person skilled in the art may compare the level (obtained according to the method of the invention) with a defined threshold value.
  • the threshold value is derived from the level (or ratio, or score) determined in a biological sample derived from one or more subjects having liposarcoma exhibiting recruitment of MDM2 to chromatin or liposarcoma resistant to inhibitors of p53 and MDM2 interaction. Furthermore, retrospective measurement of the level (or ratio, or scores) in properly banked historical subject samples may be used in establishing these threshold values.
  • Predetermined reference values used for comparison may comprise “cut-off’ or “threshold” values that may be determined as described herein.
  • Each reference (“cut-off’) value for the level of serine/glycine and/or chromatin-bound MDM2 of interest may be predetermined by carrying out a method comprising the steps of
  • step e providing, for each sample provided at step a), information relating to the liposarcoma exhibiting recruitment of MDM2 to chromatin or liposarcoma resistant to inhibitors of p53 and MDM2 interaction or the actual clinical outcome for the corresponding cancer subject (i.e. the duration of the event-free survival (EFS), metastasis- free survival (MFS) or the overall survival (OS) or both);
  • EFS event-free survival
  • MFS metastasis- free survival
  • OS overall survival
  • the levels of serine/glycine and/or of chromatin-bound MDM2 have been assessed for 100 cancer samples of 100 subjects.
  • the 100 samples are ranked according to their level.
  • Sample 1 has the best level and sample 100 has the worst level.
  • a first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples.
  • the next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100.
  • Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated.
  • the reference value is selected such as the discrimination based on the criterion of the minimum p value is the strongest. In other terms, the level corresponding to the boundary between both subsets for which the p value is minimum is considered as the reference value. It should be noted that the reference value is not necessarily the median value of the levels of serine/glycine and/or chromatin-bound MDM2.
  • the reference value (cut-off value) may be used in the present method to discriminate cancer samples and therefore the corresponding subjects.
  • Kaplan-Meier curves of percentage of survival as a function of time are commonly to measure the fraction of subjects living for a certain amount of time after treatment and are well known by the man skilled in the art.
  • the reference value may correspond to the level of serine/glycine and/or of chromatin-bound MDM2 determined in a sample associated with liposarcoma exhibiting recruitment of MDM2 to chromatin or liposarcoma resistant to inhibitors of p53 and MDM2 interaction.
  • a higher or equal level of serine/glycine and/or of chromatin- bound MDM2 than the reference value is indicative of a subject having liposarcoma exhibiting recruitment of MDM2 to chromatin or liposarcoma resistant to inhibitors of p53 and MDM2 interaction
  • lower level of serine/glycine and/or of chromatin-bound MDM2 than the reference value is indicative of a subject having liposarcoma not exhibiting recruitment of MDM2 to chromatin or liposarcoma not resistant to inhibitors of p53 and MDM2 interaction.
  • the reference value may correspond to the level of serine/glycine and/or of chromatin-bound MDM2 determined in a sample associated with liposarcoma not exhibiting recruitment of MDM2 to chromatin or liposarcoma not resistant to inhibitors of p53 and MDM2 interaction.
  • higher level of serine/glycine and/or of chromatin-bound MDM2 than the reference value is indicative of a subject having liposarcoma exhibiting recruitment of MDM2 to chromatin or liposarcoma resistant to inhibitors of p53 and MDM2 interaction
  • a lower or equal level of serine/glycine and/or of chromatin-bound MDM2 than the reference value is indicative of a subject having liposarcoma not exhibiting recruitment of MDM2 to chromatin or liposarcoma not resistant to inhibitors of p53 and MDM2 interaction.
  • a score which is a composite of the level of serine/glycine and/or of chromatin-bound MDM2 may also be determined and compared to a reference value wherein a difference between said score and said reference value is indicative of a subject having a liposarcoma exhibiting or not recruitment of MDM2 to chromatin or a liposarcoma resistant or not to inhibitors of p53 and MDM2 interaction.
  • the score may be generated by a computer program.
  • Analyzing the level of serine/glycine and/or of chromatin-bound MDM2 may be assessed by any of a wide variety of well-known methods for detecting serine/glycine and/or MDM2 recruitment to chromatin.
  • the level of serine and/or glycine is assessed by mass spectrometry (MS) chromatogram, liquid chromatography-mass spectrometry (LC-MS) such as described in Riscal et al., 2016.
  • the level of chromatin-bound MDM2 is assessed by extracting liposarcoma cell, and measuring level of chromatin-associated MDM2 by immunoblot analyses, quantitative immunoblotting, western blot, immunoprecipitation assays, Quantitative chromatin immunoprecipitation (qChIP) assays such as described in Riscal et al., 2016.
  • the level of chromatin-bound MDM2 is assessed by measuring the level of MDM2 and ATF3/4 protein interaction. In some embodiment, the level of chromatin-bound MDM2 is assessed by Proximity Ligation Assay (PLA).
  • PLA Proximity Ligation Assay
  • analyzing the level of serine/glycine and/or of chromatin-bound MDM2 may also be assessed by assessing bio markers of serine/glycine metabolism such as by measuring expression level of enzymes involved in serine/glycine metabolism such as PHGDH (phosphoglycerate dehydrogenase), PSAT (phosphoserine aminotransferase, PSAT1), PSPH (phosphoserine phosphatase) and SLC1A4 (Solute Carrier Family 1 Member 4 inhibitor).
  • PHGDH phosphoglycerate dehydrogenase
  • PSAT phosphoserine aminotransferase
  • PSPH phosphoserine phosphatase
  • SLC1A4 Solute Carrier Family 1 Member 4 inhibitor
  • Analyzing the bio marker expression level may be assessed by any of a wide variety of well-known methods for detecting expression of a transcribed nucleic acid or translated protein.
  • the biomarker expression level is assessed by analyzing the expression of the protein translated from said gene. Said analysis can be assessed using an antibody (e.g., a radio-labeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative (e.g., an antibody conjugate with a substrate or with the protein or ligand of a protein of a protein/ligand pair (e.g., biotin-streptavidin)), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically to the protein translated from the gene encoding for the biomarker.
  • an antibody e.g., a radio-labeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody
  • an antibody derivative e.g., an antibody conjugate with a substrate or with the protein or ligand of a protein
  • Methods for measuring the expression level of a biomarker in a sample may be assessed by any of a wide variety of well-known methods from one of skill in the art for detecting expression of a protein including, but not limited to, direct methods like mass spectrometry- based quantification methods, protein microarray methods, enzyme immunoassay (EIA), radioimmunoassay (RIA), Immunohistochemistry (IHC), Western blot analysis, ELISA, Luminex, ELISPOT and enzyme linked immunoabsorbant assay and undirect methods based on detecting expression of corresponding messenger ribonucleic acids (mRNAs).
  • the mRNA expression profile may be determined by any technology known by a man skilled in the art.
  • each mRNA expression level may be measured using any technology known by a man skilled in the art, including nucleic microarrays, quantitative Polymerase Chain Reaction (qPCR), next generation sequencing and hybridization with a labelled probe.
  • the binding partner may be an antibody that may be polyclonal or monoclonal, preferably monoclonal (e.g., a isotope-label, element-label, radio-labeled, chromophore- labeled, fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative (e.g., an antibody conjugate with a substrate or with the protein or ligand of a protein of a protein/ligand pair (e.g., biotin-streptavidin)), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically to the protein translated from the gene encoding for the biomarker of the invention.
  • the binding partner may be an aptamer.
  • binding partners of the invention such as antibodies or aptamers, may be labelled with a detectable molecule or substance, such as an isotope, an element, a fluorescent molecule, a radioactive molecule or any others labels known in the art.
  • a detectable molecule or substance such as an isotope, an element, a fluorescent molecule, a radioactive molecule or any others labels known in the art.
  • Labels are known in the art that generally provide (either directly or indirectly) a signal.
  • the term "labelled", with regard to the antibody is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as an isotope, an element, a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance.
  • a detectable substance such as an isotope, an element, a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • Indocyanine Indocyanine
  • radioactive molecules include but are not limited to radioactive atom for scintigraphic studies such as 1123, 1124, Inl l l, Rel86, Rel88, specific isotopes include but are not limited to 13C, 15N, 1261, 79Br, 8lBr.
  • the afore mentioned assays generally involve the binding of the binding partner (ie. antibody or aptamer) to a solid support.
  • Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidene fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, silicon wafers.
  • substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidene fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, silicon wa
  • an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies which recognize said biomarker. A sample containing or suspected of containing said biomarker is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labelled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art such as Singulex, Quanterix, MSD, Bioscale, Cytof.
  • an Enzyme-linked immunospot (ELISpot) method may be used.
  • the sample is transferred to a plate which has been coated with the desired anti biomarker capture antibodies.
  • Revelation is carried out with biotinylated secondary Abs and standard colorimetric or fluorimetric detection methods such as streptavidin-alkaline phosphatase and NBT-BCIP and the spots counted.
  • the bead may be a cytometric bead for use in flow cytometry.
  • Such beads may for example correspond to BDTM Cytometric Beads commercialized by BD Biosciences (San Jose, California).
  • cytometric beads may be suitable for preparing a multiplexed bead assay.
  • a multiplexed bead assay such as, for example, the BD(TM) Cytometric Bead Array, is a series of spectrally discrete beads that can be used to capture and quantify soluble antigens.
  • beads are labelled with one or more spectrally distinct fluorescent dyes, and detection is carried out using a multiplicity of photodetectors, one for each distinct dye to be detected.
  • a number of methods of making and using sets of distinguishable beads have been described in the literature. These include beads distinguishable by size, wherein each size bead is coated with a different target-specific antibody (see e.g. Fulwyler and McHugh, 1990, Methods in Cell Biology 33:613-629), beads with two or more fluorescent dyes at varying concentrations, wherein the beads are identified by the levels of fluorescence dyes (see e.g. European Patent No.
  • beads distinguishably labelled with two different dyes, wherein the beads are identified by separately measuring the fluorescence intensity of each of the dyes (see e.g. U.S. patent Nos. 4,499,052 and 4,717,655).
  • Both one-dimensional and two-dimensional arrays for the simultaneous analysis of multiple antigens by flow cytometry are available commercially. Examples of one-dimensional arrays of singly dyed beads distinguishable by the level of fluorescence intensity include the BD(TM) Cytometric Bead Array (CBA) (BD Biosciences, San Jose, Calif.) and Cyto-Plex(TM) Flow Cytometry microspheres (Duke Scientific, Palo Alto, Calif.).
  • An example of a two-dimensional array of beads distinguishable by a combination of fluorescence intensity (five levels) and size (two sizes) is the QuantumPlex(TM) microspheres (Bangs Laboratories, Fisher, Ind.).
  • An example of a two- dimensional array of doubly-dyed beads distinguishable by the levels of fluorescence of each of the two dyes is described in Fulton et al. (1997, Clinical Chemistry 43(9): 1749-1756).
  • the beads may be labelled with any fluorescent compound known in the art such as e.g. FITC (FL1), PE (FL2), fluorophores for use in the blue laser (e.g.
  • bead is a magnetic bead for use in magnetic separation. Magnetic beads are known to those of skill in the art. Typically, the magnetic bead is preferably made of a magnetic material selected from the group consisting of metals (e.g. ferrum, cobalt and nickel), an alloy thereof and an oxide thereof. In another particular embodiment, bead is bead that is dyed and magnetized.
  • metals e.g. ferrum, cobalt and nickel
  • bead is bead that is dyed and magnetized.
  • protein microarray methods may be used.
  • at least one antibody or aptamer directed against the biomarker is immobilized or grafted to an array(s), a solid or semi- so lid surface(s).
  • a sample containing or suspected of containing the biomarker is then labelled with at least one isotope or one element or one fluorophore or one colorimetric tag that are not naturally contained in the tested sample.
  • the array is then washed and dried.
  • quantifying said biomarker may be achieved using any appropriate microarray scanner like fluorescence scanner, colorimetric scanner, SIMS (secondary ions mass spectrometry) scanner, maldi scanner, electromagnetic scanner or any technique allowing to quantify said labels.
  • the antibody or aptamer grafted on the array is labelled.
  • reverse phase arrays may be used.
  • at least one sample is immobilized or grafted to an array(s), a solid or semi- so lid surface(s).
  • An antibody or aptamer against the suspected biomarker is then labelled with at least one isotope or one element or one fluorophore or one colorimetric tag that are not naturally contained in the tested sample. After a period of incubation of said antibody or aptamer with the array sufficient to allow the formation of antibody-antigen complexes, the array is then washed and dried.
  • detecting quantifying and counting by D-SIMS said biomarker containing said isotope or group of isotopes, and a reference natural element, and then calculating the isotopic ratio between the biomarker and the reference natural element may be achieved using any appropriate microarray scanner like fluorescence scanner, colorimetric scanner, SIMS (secondary ions mass spectrometry) scanner, maldi scanner, electromagnetic scanner or any technique allowing to quantify said labels.
  • any appropriate microarray scanner like fluorescence scanner, colorimetric scanner, SIMS (secondary ions mass spectrometry) scanner, maldi scanner, electromagnetic scanner or any technique allowing to quantify said labels.
  • said direct analysis can also be assessed by mass Spectrometry.
  • Mass spectrometry-based quantification methods may be performed using either labelled or unlabelled approaches (DeSouza and Siu, 2012). Mass spectrometry-based quantification methods may be performed using chemical labeling, metabolic labelingor proteolytic labeling. Mass spectrometry-based quantification methods may be performed using mass spectrometry label free quantification, LTQ Orbitrap Velos, LTQ-MS/MS, a quantification based on extracted ion chromatogram EIC (progenesis LC-MS, Liquid chromatography-mass spectrometry) and then profile alignment to determine differential expression of the biomarker.
  • the biomarker expression level is assessed by analyzing the expression of mRNA transcript or mRNA precursors, such as nascent RNA, of biomarker gene. Said analysis can be assessed by preparing mRNA/cDNA from cells in a sample from a subject, and hybridizing the mRNA/cDNA with a reference polynucleotide. The prepared mRNA/cDNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses, such as quantitative PCR (TaqMan), and probes arrays such as GeneChip(TM) DNA Arrays ( AFF YMETRIX) .
  • mRNA transcript or mRNA precursors such as nascent RNA
  • the analysis of the expression level of mRNA transcribed from the gene encoding for biomarkers involves the process of nucleic acid amplification, e. g., by RT- PCR (the experimental embodiment set forth in U. S. Patent No. 4,683, 202), ligase chain reaction (Barany, 1991), self sustained sequence replication (Guatelli et al, 1990), transcriptional amplification system (Kwoh et al., 1989), Q-Beta Replicase (Lizardi et al., 1988), rolling circle replication (U. S. Patent No. 5,854, 033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art.
  • RT- PCR the experimental embodiment set forth in U. S. Patent No. 4,683, 202
  • ligase chain reaction Barany, 1991
  • self sustained sequence replication (Guatelli et al, 1990)
  • transcriptional amplification system Kwoh e
  • amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between.
  • amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.
  • the present invention relates to MDM2 inhibitor of the invention for use in the treatment of liposarcoma exhibiting recruitment of MDM2 to chromatin in a subject in need thereof, wherein the subject was being classified as having liposarcoma exhibiting recruitment of MDM2 to chromatin by performing the method according to the invention.
  • the present invention relates to MDM2 inhibitor of the invention for use in the treatment of liposarcoma resistant to inhibitors of p53 and MDM2 interaction in a subject in need thereof, wherein the subject was being classified as having liposarcoma resistant to inhibitors of p53 and MDM2 interaction by performing the method according to the invention.
  • the method of the invention allows to define a subgroup of subjects who will be responder or non responder to MDM2 inhibitor of the invention.
  • the present invention relates to the MDM2 inhibitor of the invention for use in the treatment of liposarcoma exhibiting recruitment of MDM2 to chromatin in a subject in need thereof.
  • a further aspect of the invention relates to a method for treating liposarcoma exhibiting recruitment of MDM2 to chromatin in a subject in need thereof comprising the steps of:
  • the method of the invention allows to define a subgroup of subjects who will be responder or non responder to inhibitors of p53 and MDM2 interaction.
  • the present invention relates to the MDM2 inhibitor of the invention for use in the treatment of liposarcoma resistant to inhibitors of p53 and MDM2 interaction in a subject in need thereof
  • the present invention relates to the MDM2 inhibitor of the invention for use in the treatment of liposarcoma resistant to inhibitors of p53 and MDM2 interaction in a subject in need thereof, wherein said MDM2 inhibitor is SP141.
  • the present invention relates to the MDM2 inhibitor of the invention for use in the treatment of liposarcoma resistant to inhibitors of p53 and MDM2 interaction like nutlin in a subject in need thereof, wherein said MDM2 inhibitor is SP141.
  • a further aspect of the invention relates to a method for treating liposarcoma in a subject in need thereof comprising the steps of:
  • a further aspect of the invention relates to a method for treating liposarcoma resistant to inhibitors of p53 and MDM2 interaction in a subject in need thereof comprising the steps of: a) determining whether a subject afflicted with liposarcoma will be a responder or a non-responder to inhibitors of p53 and MDM2 interaction by performing the method according to the invention, and
  • the present invention relates to the MDM2 inhibitor of the invention for use in preventing liposarcoma resistance to inhibitors of p53 and MDM2 interaction in a subject in need thereof.
  • the present invention relates to the MDM2 inhibitor of the invention for use in enhancing the potency of inhibitors of p53 and MDM2 interaction administered to a subject afflicted with liposarcoma in need thereof.
  • a further aspect of the invention relates to a method of treating a liposarcoma exhibiting recruitment of MDM2 to chromatin in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the MDM2 inhibitor of the invention.
  • a further aspect of the invention relates to a method of treating a liposarcoma resistant to inhibitors of p53 and MDM2 interaction in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the MDM2 inhibitor of the invention.
  • a further aspect of the present invention relates to a method of preventing resistance to an inhibitor of p53 and MDM2 interaction in a subject afflicted with liposarcoma comprising administering to the subject a therapeutically effective amount of the MDM2 inhibitor of the invention.
  • a further aspect of the present invention relates to a method for enhancing the potency of inhibitors of p53 and MDM2 interaction administered to a subject afflicted with liposarcoma, the method comprising administering to the subject a pharmaceutically effective amount of the MDM2 inhibitor of the invention.
  • a further aspect of the invention relates to a method of treating a subject afflicted with liposarcoma and providing a diagnosis of liposarcoma exhibiting recruitment of MDM2 to chromatin or liposarcoma resistant to inhibitors of p53 and MDM2 interaction, comprising the steps of:
  • the present invention relates to a method of screening a candidate compound for use as a drug for the treatment of liposarcoma in a subject in need thereof, wherein the method comprises the steps of:
  • MDM2, PHGDH, PSAT, PSPH and/or SLC1A4 expressing cell providing a cell, tissue sample or organism expressing MDM2, PHGDH, PSAT, PSPH and/or SLC1A4, liposarcoma-derived cell lines, xenograft (liposarcoma-derived cell lines) mouse models and xenograft (PDX) mouse models, providing a candidate compound such as a small organic molecule, an oligonucleotide, a polypeptide, an aptamer, antibody or an intra-antibody,
  • measuring MDM2, PHGDH, PSAT, PSPH and/or SLC1A4 activities involves determining the Ki of the-MDM2, PHGDH, PSAT, PSPH and/or SLC1A4 cloned and transfected in a stable manner into a CHO cell line or liposarcoma-derived cell lines, measuring the level of chromatin-bound MDM2, measuring the level of serine and/or glycine and measuring serine metabolism in the present or absence of the candidate compound.
  • Tests and assays for screening and determining whether a candidate compound is a MDM2 inhibitor, inhibitor of the MDM2 recruitment to chromatin, PHGDH inhibitor, PSAT inhibitor, PSPH inhibitor and/or SLC1A4 inhibitor are well known in the art (Riscal et al, 2016; Qin et al, 2016; Wang et al, 2017; Hamanak et al., 2017; Mullarky et al, 2016; Pacold et al, 2016; Ye et al., 2012; US 20140087970; Foster et al., 2017).
  • In vitro and in vivo assays may be used to assess the potency and selectivity of the candidate compounds to inhibit MDM2, PHGDH, PSAT, PSPH and/or SLC1A4 activity.
  • Activities of the candidate compounds, their ability to bind MDM2, PHGDH, PSAT, PSPH and/or SLC1A4 and their ability to inhibit MDM2, PHGDH, PSAT, PSPH and/or SLC1A4 activity may be tested using isolated cancer cell or CHO cell line cloned and transfected in a stable manner with the human MDM2, PHGDH, PS AT l, PSPH and/or SLC1A4 cDNAs.
  • Activities of the candidate compounds and their ability to bind to MDM2, PHGDH, PSAT, PSPH and/or SLC1A4 proteins may be assessed by the determination of the Ki of the MDM2, PHGDH, PSAT, PSPH and/or SLC1A4 cloned and transfected in a stable manner into a CHO cell line or liposarcoma-derived cell lines, measuring the level of chromatin-bound MDM2, measuring the level of serine and/or glycine and measuring serine metabolism in the present or absence of the candidate compound.
  • FIGURES
  • Figure 1 Increased recruitment of MDM2 to chromatin upon Nutlin treatment explains its low clinical efficiency in LPS.
  • Figure 2 A subset of Human liposarcomas display increased expression of genes involved in serine metabolism. RT-qPCR analysis of MDM2, PHGDH, PSAT1, PSPH and SLC1A4 mRNA levels in human samples of WD/DD-LPS and in leimyo sarcomas, or in normal abdominal adipose tissue.
  • C A p53 KO clone of IB115 generated by CRISPR/Cas9 technology was transduced with lentivirus expressing control- (CTRL) or PHGDH - shRNAs and then subcutaneously implanted in nude mice that were fed with chow or a serine/gly cine-deprived diet (-SG) of equivalent calorific value and equal total amino acid content, as indicated. Tumor volume was measured at the indicated time points.
  • C Quantitative immunoblot analysis of PHGDH and ACTIN (Act, loading control) protein levels in whole cell extracts prepared from the same tumor samples than in B).
  • p53 null IB115 cells generated using the CRISPR/Cas9 technology were transduced with lentiviruses encoding control- (CTRL) or MDM2 - shRNAs and then subcutaneously implanted in nude mice that were fed with control chow or a serine/glycine deprived diet (-SG). Tumor volume was measured at the indicated time points.
  • Tumor volume was measured at the indicated time points.
  • FIG. 6 MDM2 inhibitor, a new therapy for Liposarcoma.
  • Table 1 MDM2 inhibitor, a new therapy for Tumor with chromatin bound MDM2.
  • MDM2 localization in a panel of cancer cell lines following biochemical fractionation.
  • the inventors have shown that a fraction of MDM2 localizes to chromatin independently of p53, ranging from 20% to 80% of the total amount of MDM2 (Riscal et al., 2016).
  • levels of chromatin-bound MDM2 can be used as a bio marker to predict the efficacy of drugs that modulate MDM2-p53 interaction.
  • agents that reduce the levels of chromatin-bound MDM2 represent new therapies for cancers that display high levels of MDM2, including liposarcomas.
  • pLKOT Puro sh MDM2 Sigma- Aldrich mission clone #3377)
  • pMKO.l puro sh/ 53 a gift from W. Hahn, Addgene clones #10671 and #10672
  • pLKO.l puro sh PHGDH Sigma-Aldrich mission clone #28532
  • Lentivirus allowing stable expression of shRNA targeting the firefly luciferase gene was used as a control shRNA.
  • Lentiviral particles were produced as previously reported in packaging HEK293 cells (Hatchi et al, 2011). Transduced liposarcoma cells were selected with puromycin (2 pg/ml, invitrogen) or hygromycin (50pg/ml) during 48h.
  • mice Bilateral subcutaneous injections of 5.10 6 cells were performed on 8-week-old CD-l- Foxnlnu mice (Charles River). One week before injection, mice were fed with control diet (Amino Acid diet, TD99366, Harlan) or the same diet lacking serine and glycine (Harlan). The diets had equal calorific value (3,9 kCal/g) and equal amount of total amino acids (l79.6g/Kg). Total food uptake was controlled to be identical in all experimental groups. Mice were housed in a pathogen free barrier facility in accordance with the regional ethic committee for animal warfare (n°CEEA-LR-l2067).
  • cell culture reagents were purchased from Gibco (Invitrogen). Cells were kept at 37 °C in a humidified 5% C02 incubator. Sarcoma cells were maintained in DMEM Glutamax supplemented with 10% fetal bovine serum (FBS) (Hyclone, Thermo Scientific). For amino acid starvation experiments, cells were cultured in DMEM lacking serine and glycine (-SG medium, Biological Industries). SP141 was added in the culture medium at a final concentration of ImM.
  • FBS fetal bovine serum
  • cells were transduced overnight with lentiviral particles encoding firefly luciferase (control) or shRNAs targeting MDM2 or PHGDH, selected with puromycin (2 pg/ml) for 72h and then seeded in triplicates in 6-well plates (3.10 5 cells per well) in regular DMEM. 24 hrs later, cells were washed with PBS and then cultured in complete or - SG medium. Cell numbers were determined by manual counting after trypan-blue exclusion every 24h during 4 days or using a real time cell analysis system - xCEFFigence (Roche). The Cell index is the quantitative measure of cell number.
  • Total mRNAs were isolated from sarcoma cells or human samples using TriZol Reagent (Invitrogen). cDNAs were synthesized from 500ng of total RNA using SuperscriptTM III Reverse Transcriptase (Invitrogen). Real-time quantitative PCRs were performed on a FightCycler 480 SW 1.5 apparatus (Roche) with Platinum Taq DNA polymerase (Invitrogen) and a SYBR Green mix containing 3 mM MgCl2 and 30 mM of each dNTP using the following amplification procedure: 45 cycles of 95°C for 4 s, 60°C for 10 s, and 72°C for 15 s. The relative mRNA copy number was calculated using Ct value and was normalized with TBP or b2- microglobulin transcripts.
  • SRB sulforhodamine B
  • Serine and glycine are 2 nonessential amino acids involved in several essential anabolic pathways and in the control of the redox status of the cell.
  • the intracellular levels of serine and glycine are controlled by two main pathways: de novo synthesis from the glycolytic intermediate 3-phosphoglycerate, and by uptake of extracellular serine and glycine from the external medium.
  • the inventors found that chromatin-bound MDM2 directly controls at the transcrpitional level 3 genes that encode key enzymes of de novo serine synthesis (PHGDH, PSAT and PSPH) as well as neutral amino-acid transporters that can uptake serine and glycine (SLC1A4).
  • LPS tumor samples display high levels of the 3 enzymes responsible for de novo serine synthesis (PHGDH, PSAT and PSPH) and also of the serine carrier SLC1A4 ( Figure 2).
  • MDM2 is a master regulator of Serine metabolism in LPS development Consistent with MDM2 functions in serine metabolism, the inventors found that MDM2 inhibition by shRNA impacts on LPS cell growth, and this effect was potentialized when these cells were cultured in a serine/gly cine-deprived medium ( Figure 4A). The inventors also confirmed the importance of MDM2-mediated control of serine/gly cine metabolism on LPS growth in vivo. LPS cells stably expressing control or MDM2 shRNAs were subcutaneously injected in nude mice that were fed with a normal diet or a serine/gly cine deprived diet of equivalent calorific value and equal content in total amino acids.
  • SP141 a drug that promotes MDM2 auto-ubiquitination and its proteasomal degradation.
  • SP 141 induces cell cycle arrest and cell death selectively in LPS cancer cell lines in vitro and inhibits tumor growth in vivo, but has little or no effect on other sarcoma cells. Strikingly, decreased biodisponibility of serine and glycine, obtained with a serine and glycine deprived diet, further decreased the tumorigenic potential of SP 141 -treated LPS cells ( Figure 5A and C).
  • the inventors determined the IC50 of SP141 on 12 LPS cell lines and 5 sarcomas cell lines and show that SP141 is selectively efficient in LPS, as compared to other sarcoma subtypes (Figure 6A).
  • the inventors show that the effect of SP141 is rescued upon overexpression of a MDM2 iso form that is preferentially localized to chromatin (MDM2-deltaAD), in vitro and in vivo, showing that the effect observed with the SP141 is due to MDM2 transcriptional activity on serine metabolism (Figure 6B and C).
  • the inventors determined the IC50 of SP141 on cell lines that are p53WT and resistant to Nutlin-3A. They have shown that all those cell lines are sensible to SP141. (Table 1).
  • the inventors assume that the very specific cytogenetic signature of LPS, characterized by the constant amplification of the MDM2 gene, illustrates the importance of MDM2- associated metabolic functions in LPS pathogenesis. The inventors expect that these results will lead to new therapeutic strategies targeting MDM2 and/or serine metabolism in advanced LPS. The inventors expect these new strategies to be more efficient that current chemotherapies based on doxorubicin. The inventors expect these new strategies can be applicable to all cancer type with chromatin bound MDM2.

Abstract

L'invention concerne des méthodes pour le diagnostic de sujets souffrant d'un liposarcome caractérisé par le recrutement de MDM2 sur la chromatine ou résistant aux inhibiteurs de l'interaction p53 et MDM2 et des méthodes pour le traitement du liposarcome. Les inventeurs montrent que les fonctions métaboliques commandées par MDM2 sont exacerbées dans le liposarcome et que la pression sélective pour l'amplification de MDM2 dans LPS reflète probablement leur dépendance vis-à-vis du métabolisme de la sérine. À l'aide d'une collection de lignées cellulaires dérivées de patients LPS et de modèles murins de xénogreffes (PDX), les inventeurs démontrent que le ciblage génétique ou pharmacologique de MDM2 liée à la chromatine, ou du métabolisme de la sérine tel que le ciblage de PHGDH, PSAT, PSPH, SLC1A4 et de la sérine, représente une stratégie thérapeutique efficace pour LPS. Les inventeurs apportent également des preuves moléculaires expliquant la faible réponse thérapeutique des patients LPS à la nutline démontrant que le développement de LPS dépend du recrutement de MDM2 sur la chromatine et du métabolisme de la sérine indépendamment de p53. Les inventeurs définissent ainsi de nouvelles stratégies thérapeutiques pour LPS sur la base d'une nouvelle classe d'inhibiteurs de MDM2 ciblant le recrutement de MDM2 sur la chromatine et le métabolisme de la sérine. Par conséquent, l'invention concerne une méthode pour diagnostiquer les sujets atteints d'un liposarcome caractérisé par le recrutement de MDM2 sur la chromatine ou résistant aux inhibiteurs de l'interaction de p53 et MDM2; et un inhibiteur du recrutement de MDM2 sur la chromatine ou un inhibiteur de la régulation du métabolisme de la sérine médiée par MDM2 destiné à être utilisé dans le traitement dudit liposarcome, des types de cancers caractérisés par un recrutement de MDM2 sur la chromatine et des types de cancer caractérisés par un métabolisme exacerbé de la sérine et de la glycine.
PCT/EP2018/083086 2017-12-01 2018-11-30 Modulateurs mdm2 pour le diagnostic et le traitement du liposarcome WO2019106126A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP17306681 2017-12-01
EP17306681.2 2017-12-01

Publications (1)

Publication Number Publication Date
WO2019106126A1 true WO2019106126A1 (fr) 2019-06-06

Family

ID=60673425

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2018/083086 WO2019106126A1 (fr) 2017-12-01 2018-11-30 Modulateurs mdm2 pour le diagnostic et le traitement du liposarcome

Country Status (1)

Country Link
WO (1) WO2019106126A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021206105A1 (fr) * 2020-04-07 2021-10-14 テオリアサイエンス株式会社 Inhibiteur de sécrétion de vésicule extracellulaire pour inhiber la sécrétion de vésicule extracellulaire, et son utilisation
WO2023012343A1 (fr) 2021-08-06 2023-02-09 Institut Du Cancer De Montpellier Procédés de traitement du cancer

Citations (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0126450A2 (fr) 1983-05-19 1984-11-28 Ioannis Dr. Tripatzis Particule et méthode de détection d'antigènes et/ou anticorps utilisant cette particule
US4499052A (en) 1982-08-30 1985-02-12 Becton, Dickinson And Company Apparatus for distinguishing multiple subpopulations of cells
US4717655A (en) 1982-08-30 1988-01-05 Becton, Dickinson And Company Method and apparatus for distinguishing multiple subpopulations of cells
WO1990007861A1 (fr) 1988-12-28 1990-07-26 Protein Design Labs, Inc. IMMUNOGLOBULINES CHIMERIQUES SPECIFIQUES CONTRE LA PROTEINE TAC p55 DU RECEPTEUR D'IL-2
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5229275A (en) 1990-04-26 1993-07-20 Akzo N.V. In-vitro method for producing antigen-specific human monoclonal antibodies
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
US5567610A (en) 1986-09-04 1996-10-22 Bioinvent International Ab Method of producing human monoclonal antibodies and kit therefor
US5573905A (en) 1992-03-30 1996-11-12 The Scripps Research Institute Encoded combinatorial chemical libraries
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5591669A (en) 1988-12-05 1997-01-07 Genpharm International, Inc. Transgenic mice depleted in a mature lymphocytic cell-type
US5598369A (en) 1994-06-28 1997-01-28 Advanced Micro Devices, Inc. Flash EEPROM array with floating substrate erase operation
US5800988A (en) 1992-08-21 1998-09-01 Vrije Universiteit Brussel Immunoglobulins devoid of light chains
US5854033A (en) 1995-11-21 1998-12-29 Yale University Rolling circle replication reporter systems
US5859205A (en) 1989-12-21 1999-01-12 Celltech Limited Humanised antibodies
WO1999032619A1 (fr) 1997-12-23 1999-07-01 The Carnegie Institution Of Washington Inhibition genetique par de l'arn double brin
US5981732A (en) 1998-12-04 1999-11-09 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-13 expression
US6046321A (en) 1999-04-09 2000-04-04 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-i1 expression
US6107091A (en) 1998-12-03 2000-08-22 Isis Pharmaceuticals Inc. Antisense inhibition of G-alpha-16 expression
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO2001036646A1 (fr) 1999-11-19 2001-05-25 Cancer Research Ventures Limited Inhibition d"expression genique a l"aide d"arn bicatenaire
WO2001068836A2 (fr) 2000-03-16 2001-09-20 Genetica, Inc. Procedes et compositions d'interference d'arn
US6365354B1 (en) 2000-07-31 2002-04-02 Isis Pharmaceuticals, Inc. Antisense modulation of lysophospholipase I expression
US6410323B1 (en) 1999-08-31 2002-06-25 Isis Pharmaceuticals, Inc. Antisense modulation of human Rho family gene expression
US6566135B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of caspase 6 expression
US6566131B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of Smad6 expression
US6573099B2 (en) 1998-03-20 2003-06-03 Benitec Australia, Ltd. Genetic constructs for delaying or repressing the expression of a target gene
US6765087B1 (en) 1992-08-21 2004-07-20 Vrije Universiteit Brussel Immunoglobulins devoid of light chains
US6838254B1 (en) 1993-04-29 2005-01-04 Conopco, Inc. Production of antibodies or (functionalized) fragments thereof derived from heavy chain immunoglobulins of camelidae
US7109304B2 (en) 2003-07-31 2006-09-19 Immunomedics, Inc. Humanized anti-CD19 antibodies
US7235641B2 (en) 2003-12-22 2007-06-26 Micromet Ag Bispecific antibodies
WO2008119353A1 (fr) 2007-03-29 2008-10-09 Genmab A/S Anticorps bispécifiques et procédés de production de ceux-ci
WO2011131746A2 (fr) 2010-04-20 2011-10-27 Genmab A/S Protéines contenant des anticorps fc hétérodimères et leurs procédés de production
US8329723B2 (en) 2009-04-24 2012-12-11 John K Buolamwini 1-aryl- or 1-heteroaryl-pyrido[B]indoles and uses thereof in treating cancers
US20140087970A1 (en) 2011-03-30 2014-03-27 Whitehead Institute For Biomedical Research Serine biosynthesis pathway inhibition for treatment of cancer

Patent Citations (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4499052A (en) 1982-08-30 1985-02-12 Becton, Dickinson And Company Apparatus for distinguishing multiple subpopulations of cells
US4717655A (en) 1982-08-30 1988-01-05 Becton, Dickinson And Company Method and apparatus for distinguishing multiple subpopulations of cells
EP0126450A2 (fr) 1983-05-19 1984-11-28 Ioannis Dr. Tripatzis Particule et méthode de détection d'antigènes et/ou anticorps utilisant cette particule
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5567610A (en) 1986-09-04 1996-10-22 Bioinvent International Ab Method of producing human monoclonal antibodies and kit therefor
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
US5591669A (en) 1988-12-05 1997-01-07 Genpharm International, Inc. Transgenic mice depleted in a mature lymphocytic cell-type
US5693762A (en) 1988-12-28 1997-12-02 Protein Design Labs, Inc. Humanized immunoglobulins
WO1990007861A1 (fr) 1988-12-28 1990-07-26 Protein Design Labs, Inc. IMMUNOGLOBULINES CHIMERIQUES SPECIFIQUES CONTRE LA PROTEINE TAC p55 DU RECEPTEUR D'IL-2
US5693761A (en) 1988-12-28 1997-12-02 Protein Design Labs, Inc. Polynucleotides encoding improved humanized immunoglobulins
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5859205A (en) 1989-12-21 1999-01-12 Celltech Limited Humanised antibodies
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5229275A (en) 1990-04-26 1993-07-20 Akzo N.V. In-vitro method for producing antigen-specific human monoclonal antibodies
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
US5573905A (en) 1992-03-30 1996-11-12 The Scripps Research Institute Encoded combinatorial chemical libraries
US5800988A (en) 1992-08-21 1998-09-01 Vrije Universiteit Brussel Immunoglobulins devoid of light chains
US5874541A (en) 1992-08-21 1999-02-23 Vrije Universiteit Immunoglobulins devoid of light chains
US6015695A (en) 1992-08-21 2000-01-18 Vrije Universiteit Brussel Immunoglobulins devoid of light chains
US6765087B1 (en) 1992-08-21 2004-07-20 Vrije Universiteit Brussel Immunoglobulins devoid of light chains
US6838254B1 (en) 1993-04-29 2005-01-04 Conopco, Inc. Production of antibodies or (functionalized) fragments thereof derived from heavy chain immunoglobulins of camelidae
US5598369A (en) 1994-06-28 1997-01-28 Advanced Micro Devices, Inc. Flash EEPROM array with floating substrate erase operation
US5854033A (en) 1995-11-21 1998-12-29 Yale University Rolling circle replication reporter systems
WO1999032619A1 (fr) 1997-12-23 1999-07-01 The Carnegie Institution Of Washington Inhibition genetique par de l'arn double brin
US6506559B1 (en) 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
US6573099B2 (en) 1998-03-20 2003-06-03 Benitec Australia, Ltd. Genetic constructs for delaying or repressing the expression of a target gene
US6107091A (en) 1998-12-03 2000-08-22 Isis Pharmaceuticals Inc. Antisense inhibition of G-alpha-16 expression
US5981732A (en) 1998-12-04 1999-11-09 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-13 expression
US6046321A (en) 1999-04-09 2000-04-04 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-i1 expression
US6410323B1 (en) 1999-08-31 2002-06-25 Isis Pharmaceuticals, Inc. Antisense modulation of human Rho family gene expression
WO2001036646A1 (fr) 1999-11-19 2001-05-25 Cancer Research Ventures Limited Inhibition d"expression genique a l"aide d"arn bicatenaire
WO2001068836A2 (fr) 2000-03-16 2001-09-20 Genetica, Inc. Procedes et compositions d'interference d'arn
US6365354B1 (en) 2000-07-31 2002-04-02 Isis Pharmaceuticals, Inc. Antisense modulation of lysophospholipase I expression
US6566135B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of caspase 6 expression
US6566131B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of Smad6 expression
US7109304B2 (en) 2003-07-31 2006-09-19 Immunomedics, Inc. Humanized anti-CD19 antibodies
US7235641B2 (en) 2003-12-22 2007-06-26 Micromet Ag Bispecific antibodies
WO2008119353A1 (fr) 2007-03-29 2008-10-09 Genmab A/S Anticorps bispécifiques et procédés de production de ceux-ci
US8329723B2 (en) 2009-04-24 2012-12-11 John K Buolamwini 1-aryl- or 1-heteroaryl-pyrido[B]indoles and uses thereof in treating cancers
WO2011131746A2 (fr) 2010-04-20 2011-10-27 Genmab A/S Protéines contenant des anticorps fc hétérodimères et leurs procédés de production
US20140087970A1 (en) 2011-03-30 2014-03-27 Whitehead Institute For Biomedical Research Serine biosynthesis pathway inhibition for treatment of cancer

Non-Patent Citations (32)

* Cited by examiner, † Cited by third party
Title
"Agnew Chem Intl.", vol. 33, 1994, pages: 183 - 186
"Cell Biology, Biochemistry and Immunology", 1996, ACADEMIC PRESS
"UniProtKB", Database accession no. 043175
"UniProtKB", Database accession no. P43007
"UniProtKB", Database accession no. P78330
"UniProtKB", Database accession no. Q00987
"UniProtKB", Database accession no. Q9Y617
AMBROSINI G ET AL: "Mouse double minute antagonist Nutlin-3a enhances chemotherapy-induced apoptosis in cancer cells with mutant p53 by activating E2F1", ONCOGENE, NATURE PUBLISHING GROUP UK, LONDON, vol. 26, no. 24, 1 May 2007 (2007-05-01), pages 3473 - 3481, XP002554554, ISSN: 0950-9232, DOI: 10.1038/SJ.ONC.1210136 *
BILL KATE LYNN J ET AL: "SAR405838: A Novel and Potent Inhibitor of the MDM2:p53 Axis for the Treatment of Dedifferentiated Liposarcoma", CLINICAL CANCER RESEARCH, vol. 22, no. 5, March 2016 (2016-03-01), pages 1150 - 1160, XP055474400 *
CHRISTOPH R. M?LLER ET AL: "Potential for treatment of liposarcomas with the MDM2 antagonist Nutlin-3A", INTERNATIONAL JOURNAL OF CANCER, vol. 121, no. 1, 1 July 2007 (2007-07-01), pages 199 - 205, XP055158062, ISSN: 0020-7136, DOI: 10.1002/ijc.22643 *
CLARK, W. R.: "The Experimental Foundations of Modern Immunology", 1986, WILEY & SONS, INC.
DE BENEDETTI ET AL., J IMMUNOL, vol. 166, 2001, pages 4334 - 4340
EPRATUZUMAB, LEONARD ET AL., CLINICAL CANCER RESEARCH (Z004, vol. 10, pages 53Z7 - 5334
FULTON ET AL., CLINICAL CHEMISTRY, vol. 43, no. 9, 1997, pages 1749 - 1756
FULWYLER AND MCHUGH, METHODS IN CELL BIOLOGY, vol. 33, 1990, pages 613 - 629
GU LUBING ET AL: "Discovery of Dual Inhibitors of MDM2 and XIAP for Cancer Treatment", CANCER CELL, CELL PRESS, US, vol. 30, no. 4, 22 September 2016 (2016-09-22), pages 623 - 636, XP029762967, ISSN: 1535-6108, DOI: 10.1016/J.CCELL.2016.08.015 *
KRIEGLER: "A Laboratory Manual", 1990, W.H. FREEMAN C.O.
MAGDALENA WIENKEN ET AL: "Mdm2 as a chromatin modifier", JOURNAL OF MOLECULAR CELL BIOLOGY, 9 November 2016 (2016-11-09), XP055473664, ISSN: 1674-2788, DOI: 10.1093/jmcb/mjw046 *
MAGDALENA WIENKEN ET AL: "Mdm2 as a chromatin modifier", JOURNAL OF MOLECULAR CELL BIOLOGY, 9 November 2016 (2016-11-09), XP055500972, ISSN: 1674-2788, DOI: 10.1093/jmcb/mjw046 *
MARKUS P. GHADIMI ET AL: "Pleomorphic liposarcoma : Clinical observations and molecular variables", CANCER., vol. 117, no. 23, 19 May 2011 (2011-05-19), US, pages 5359 - 5369, XP055500786, ISSN: 0008-543X, DOI: 10.1002/cncr.26195 *
MURRY: "Methods in Molecular Biology", vol. 7, 1991, HUMANA PRESS, INC.
NATURE, vol. 256, 1975, pages 495
OU Y ET AL: "p53 Protein-mediated regulation of phosphoglyceratedehydrogenase (PHGDH) is crucial for the apoptotic response upon serine starvation", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 290, no. 1, 2 January 2015 (2015-01-02), pages 457 - 466, XP009507539, ISSN: 0021-9258, DOI: 10.1074/JBC.M114.616359 *
RISCAL ET AL., MOL CELL, 2016
RISCAL R. ET AL.: "Chromatin-bound MDM2 regulates serine metabolism and redox homeostasis independently of p53", MOL. CELL., vol. 62, no. 6, 16 June 2016 (2016-06-16), pages 890 - 902, XP029613076, DOI: doi:10.1016/j.molcel.2016.04.033
RISCAL ROMAIN ET AL: "Chromatin-Bound MDM2 Regulates Serine Metabolism and Redox Homeostasis Independently of p53", MOLECULAR CELL, ELSEVIER, AMSTERDAM, NL, vol. 62, no. 6, 2 June 2016 (2016-06-02), pages 890 - 902, XP029613076, ISSN: 1097-2765, DOI: 10.1016/J.MOLCEL.2016.04.033 *
ROITT, I.: "Essential Immunology", 1991, BLACKWELL SCIENTIFIC PUBLICATIONS
SANBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SUZUKI ET AL., EUROP J OF IMMUNOL, vol. 22, no. 8, 1992, pages 1989 - 1993
WIENKEN MAGDALENA ET AL: "MDM2 Associates with Polycomb Repressor Complex 2 and Enhances Stemness-Promoting Chromatin Modifications Independent of p53", MOLECULAR CELL, vol. 61, no. 1, 2016, pages 68 - 83, XP029381664, ISSN: 1097-2765, DOI: 10.1016/J.MOLCEL.2015.12.008 *
WU ET AL., MOL. BIOL., vol. 294, 1999, pages 151
WU ET AL.: "Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-IgTM) Molecule", ANTIBODY ENGINEERING, SPRINGER BERLIN HEIDELBERG, 2010

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021206105A1 (fr) * 2020-04-07 2021-10-14 テオリアサイエンス株式会社 Inhibiteur de sécrétion de vésicule extracellulaire pour inhiber la sécrétion de vésicule extracellulaire, et son utilisation
JP7442251B2 (ja) 2020-04-07 2024-03-04 テオリアサイエンス株式会社 細胞外小胞の分泌を抑制する細胞外小胞分泌抑制剤、およびその用途
WO2023012343A1 (fr) 2021-08-06 2023-02-09 Institut Du Cancer De Montpellier Procédés de traitement du cancer

Similar Documents

Publication Publication Date Title
US11513122B2 (en) Predicting response to PD-1 axis inhibitors
US20230183346A1 (en) Antibodies having specificity for btla and uses thereof
AU2017315459A1 (en) Methods of treating fibroblast growth factor 19-mediated cancers and tumors
WO2018185516A1 (fr) Méthodes et compositions pharmaceutiques destinées à traiter une toxicité cardiovasculaire induite par une thérapie anticancéreuse
US20210239702A1 (en) Methods for predicting responsiveness of lung cancer patients to her2-targeting therapies
US20160024216A1 (en) Use of egfr biomarkers for the treatment of gastric cancer with anti-egfr agents
WO2019106126A1 (fr) Modulateurs mdm2 pour le diagnostic et le traitement du liposarcome
JP2022519649A (ja) がんの診断および治療方法
EP3898675A1 (fr) Utilisation d'anticorps de liaison à il-1 bêta dans le traitement ou la prévention du syndrome myélodysplasique
WO2019211370A1 (fr) Méthodes et compositions pharmaceutiques pour le traitement du cancer
WO2019234221A1 (fr) Procédés de stratification et de traitement d'un patient souffrant de leucémie lymphoïde chronique
WO2018189403A1 (fr) Méthodes et compositions pharmaceutiques destinées au traitement du cancer
US20210353750A1 (en) Methods of Treating Cancer
US20210047696A1 (en) Methods and pharmaceutical compositions for treating cancer
WO2017129763A1 (fr) Méthodes et compositions pharmaceutiques pour le traitement du cancer de l'estomac à cellules en bague à chaton
US20210164984A1 (en) Methods for predicting outcome and treatment of patients suffering from prostate cancer or breast cancer
US20210355222A1 (en) Methods of Treating Cancer
US20220290151A1 (en) Use of müllerian inhibiting substance inhibitors for treating cancer
WO2019121872A1 (fr) Procédés de diagnostic et de traitement du cancer du foie
US20230250173A1 (en) Biomarkers for pd-1 axis binding antagonist therapy
US20230266332A1 (en) Methods and compositions for preventing and treating a cancer
US20230218608A1 (en) New strategy for treating pancreatic cancer
WO2023041744A1 (fr) Inhibiteurs de bet pour le traitement du cancer à déficit en bap1
US20210290633A1 (en) Combination for treating cancer
CN114728070A (zh) 用于癌症疗法的复合生物标记物

Legal Events

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

Ref document number: 18807647

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18807647

Country of ref document: EP

Kind code of ref document: A1