WO2014002007A1 - Method of predicting or monitoring response to igf-1r and ir inhibitors using biomarkers - Google Patents

Description

METHOD OF PREDICTING OR MONITORING RESPONSE TO IGF-IR AND IR

INHIBITORS USING BIOMARKERS

FIELD OF THE INVENTION

The present invention relates to a method of monitoring response of a subject having cancer to treatment with a compound which is an Insulin Like Growth Factor- 1 Receptor (IGF-IR) and Insulin Receptor (IR) inhibitor. The method of the present invention comprises detecting gene signature with at least one biomarker selected from the group consisting of Early Growth Response- 1 (EGR-1), Dual Specificity Phosphatase-5 (DUSP-5) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in a sample.

BACKGROUND OF THE INVENTION

IGF-IR signaling controls proliferation, differentiation, growth and cell survival in many tissues [PLoS genetics, 2012, 8(3), el002609, doi: 10.1371/journal.pgen.1002609]. The Insulin-like Growth Factor (IGF) family and the IGF-IR play an important role in cancer. IGF-IR is a receptor tyrosine kinase (RTK) that functions as a heterotetramer and is structurally related to the Insulin Receptor (IR) with 60% homology in its amino acid sequence. Deregulation of the IGF- IR signaling pathway is involved in many malignancies including colorectal, breast, pancreatic, lung, head and neck, prostate, renal, ovarian and endometrial cancer as well as sarcomas. The intricate and complex IGF-IR signaling pathway provides many opportunities for therapeutic intervention and several novel therapeutics aimed at IGF-IR are being developed [Recent Patents on Anti-Cancer Drug Discovery, 2012, 7, 14-30].

Various compounds targeting IGF-IR are under different stages of clinical or preclinical development and belong to the following categories: growth hormone releasing hormone antagonists (e.g. JV-1-38), growth hormone receptor antagonists (e.g. pegvisomant, (Pfizer)), IGF-IR antibodies (e.g. CP-751,871, (Pfizer)), AVE1642 (Sanofi-Aventis)/EM164, IMC-A12 (ImClone LLC), SCH-717454 (Merck), BIIB022 (Biogen Idee), AMG 479 (Amgen), MK- 0646/h7C10(Merck) and IGF-IR tyrosine kinase inhibitors (e.g. BMS-536942 (Bristol-Myers Squibb), BMS-554417 (Bristol-Myers Squibb), NVP-AEW541(Novartis), NVP-ADW742 (Novartis), AG1024 (Calbiochem-EMD Biosciences), OSI-906 (OSI Pharmaceuticals), potent quinolinyl-derived imidazo (l,5-a)pyrazine (PQIP, Insmed ), picropodophyllin (PPP, Axelar AB), Nordihydroguaiaretic acid (INSM-18/NDGA, Insmed) [Recent Patents on Anti-Cancer Drug Discovery, 2009, 4, 54-72]. A-928605(trans-3-[2-(2-Chlorobenzyl)-lH-benzimidazol-5-yl]-l-[4-(4-morpholinyl)cyclohexyl]- lH-pyrazolo[3,4-d]pyrimidin-4-amine) (Abbott) is a dual IGF-1R and IR inhibitor which is efficacious in in vivo models against neuroblastoma, pancreatic cancer and non-small cell lung cancer (NSCLC) [Recent Patents on Anti-Cancer Drug Discovery, 2012, 7, 14-30].

In parallel to development of new therapies targeting IGF-1R, efforts at identifying biomarkers have increased [Recent Patents on Anti-Cancer Drug Discovery, 2012, 7, 14-30]. It is a known fact that certain cancer drugs are effective in some patients, but not in others. This results from genetic variation among tumors/cancers. In fact, variable patient response is particularly pronounced with respect to targeted therapeutics. Therefore, the full potential of targeted therapies cannot be realized without suitable tests for determining which patients will benefit from which drugs. It has been found by researchers that the development of improved diagnostics based on the discovery of biomarkers has the potential to identify those patients most likely to show a clinical response to a given drug. This would significantly reduce the size, length and cost of clinical trials. In fact, the use of biomarkers in the early clinical trials of rationally designed molecularly targeted anticancer agents has the potential to increase patients' benefit from early clinical trials, accelerate the drug development process, exploit the ability to generate information about human cancer and decrease the risk of late and costly drug attrition [Clinical Pharmacology & Therapeutics, 2009, 85, 131-133].

These findings indicate a need for the discovery of clinically useful biomarkers and diagnostic methods based on such biomarkers to assist in the determination of cancer treatment progress and patient outcome during the course of disease.

BRIEF DESCRIPTION OF THE FIGURES

Figure I: Effect of Compound A on down stream targets of Ras/Raf/ERK pathway in HEK- IGF1R cells using Microarray.

Figure II: Effect of Compound A on down stream targets of Ras/Raf/ERK pathway in HEK- IGF1R cells using real time quantitative polymerase chain reaction (RTQPCR).

Figure III: Effect of Compound A on down stream targets of Ras/Raf/ERK pathway in A673 cells using RTQPCR.

Figure IV: Effect of Compound A on down stream targets of Ras/Raf/ERK pathway in Colo- 205 cells using RTQPCR.

Figure V: Effect of Compound A on down stream targets of Ras/Raf/ERK pathway in tumor tissue obtained from Colo205 xenograft model using RTQPCR. SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a method of monitoring response of a subject having cancer to treatment with a compound, which is an IGF-IR (Insulin Like Growth Factor- 1 Receptor) and IR (Insulin Receptor) inhibitor.

In another aspect, the present invention provides a method of monitoring the response of a subject having cancer to the treatment with a compound which is an IGF-IR and IR inhibitor; said method comprising determining the expression level of at least one of the biomarker transcripts selected from the group consisting of Early Growth Response- 1 (EGR-1), Dual Specificity Phosphatase-5 (DUSP-5) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in a sample pre-therapy and post-therapy.

In another aspect, the present invention provides a method of monitoring response of a subject having cancer to treatment with a compound, which is an IGF-IR and IR inhibitor; said method comprising the steps of:

a) determining the expression level of at least one of the biomarker transcripts selected from the group consisting of Early Growth Response- 1 (EGR-1), Dual Specificity Phosphatase-5 (DUSP-5) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in a sample obtained from the subject before administration of the compound;

b) administering to the said subject the compound and obtaining a sample from the subject after the administration;

c) determining the expression level of at least one of the said biomarker transcripts in the sample obtained in step (b);

d) comparing the expression level of at least one of the said biomarker transcripts as determined in step (a) with the expression level determined in step (c); and

e) predicting that the said subject shall respond to the treatment with the compound when the expression level of at least one of the said biomarker transcripts is decreased.

In another aspect, the present invention also relates to a method of testing whether the compound, which is an IGF-IR and IR inhibitor produces a therapeutic response in a subject having cancer.

In another aspect, the present invention relates to a method of determining response of a cancer or a tumor to the treatment with a compound, which is an IGF-IR and IR inhibitor; said method comprises comparing expression level of at least one of the biomarker transcripts selected from the group of EGR-1, DUSP-5 or FOS in a sample before administering the compound with the expression level of the said biomarker transcript in a sample after administering the said compound. Decrease in the expression level of at least one of the said biomarker transcripts after administering the compound which is an IGF-IR and IR inhibitor would help in identifying that the cancer or tumor is responsive to treatment with the compound, which is an IGF-IR and IR inhibitor.

In another aspect, the present invention provides a kit for performing the method of determining response of a cancer or a tumor to the treatment with a compound, which is an IGF-IR and IR inhibitor; wherein the said kit comprising instructions for its use.

Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to be understood that this invention is not limited to particular embodiments. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Definitions

As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly indicates otherwise.

Unless defined otherwise, the technical and scientific terms used herein have the meaning as commonly understood by one of the ordinary skill in the art to which the invention belongs. However, the following terms have the meanings ascribed to them unless specified otherwise. Biomarker: The term "biomarker", as used herein, refers to a molecule or molecular species (such as a protein or gene) used to indicate or measure a biological process. A biological process is a process occurring in living organisms which is regulated by many means, examples include control of gene expression, protein modification or interaction with a protein or substrate molecule. Detection and analysis of a biomarker specific to a disease can aid in the identification, diagnosis, and treatment of the disease, or act as a drug response marker for the disease. For example, the level of a particular protein found in blood may be an indicator of a specific blood-associated disorder. In the present invention the terms "biomarker" or "drug response marker" are used interchangeably as the biomarkers are used to monitor response of subject administered with a compound, which is an IGF-IR and IR inhibitor. Biomarker can be detected at mRNA level (transcript) or at the protein level. For the purpose of the invention, the terms "biomarker" or "biomarker transcript" are used interchangeably.

IGF-IR and IR inhibitor: As used herein the term "IGF-IR and IR inhibitor" refers to small molecules which act as inhibitors of IGF-IR and IR. Inhibiting the IGF-IR and IR leads to inhibition of disorders or diseases related to IGF-IR and IR. In the present invention, the disorder or disease mediated by IGF-IR and IR is cancer. Cancer is an abnormal growth of cells which tend to proliferate in an uncontrolled way. Deregulation of the IGF-IR signaling pathway is involved in many malignancies including colorectal, breast, pancreatic, lung, head and neck, prostate, renal, ovarian and endometrial cancer as well as sarcomas. The IGF-IR and IR inhibitor used in accordance with method of the present invention finds use in the treatment of cancer wherein treating cancer refers to ameliorating, mitigating or delaying the onset of the effects of cancer. Various IGF-IR and IR inhibitors such as BMS-536942 (Bristol-Myers Squibb), BMS- 554417 (Bristol-Myers Squibb), NVP-AEW541 (Novartis), NVP-ADW742 (Novartis), AG1024 (Calbiochem-EMD Biosciences), OSI-906 (OSI Pharmaceuticals), CP-751,871 (Pfizer), AVE1642/EM164 (Sanofi Aventis/ImmunoGen), IMC-A12 (Imclone) and the compound A (as described herein) can be used to treat different types of cancers.

Tumor: The term "tumor" as used herein refers to a group of cells that are cancerous in origin and grow uncontrollably. Tumors from various types of cancers such as colorectal, breast, pancreatic, lung, head and neck, prostate, renal, ovarian are a few examples of tumors that can be treated with compounds which are IGF-IR and IR inhibitors.

RNA: The term RNA (ribonucleic acid) as used herein means a polymer composed of ribonucleotides.

DNA: The term DNA (deoxyribonucleic acid) as used herein means a polymer composed of deoxyribonucleotides.

Oligonucleotide: The term "oligonucleotide" as used herein means a polymer composed of either DNA or RNA, and used as probes to find a complementary sequence of DNA or RNA. Protein or polypeptide: The terms "protein" or "polypeptide" are used interchangeably. They refer to a chain of two or more amino acids, which are linked together with peptide or amide bonds, regardless of post-translational modification (e.g., glycosylation or phosphorylation). Sample: The term "sample" as used herein relates to a material or mixture of materials, typically, although not necessarily, in fluid form, containing one or more components of interest. Samples include, but are not limited to, biological samples obtained from natural biological sources, such as whole blood, cells or tissues. The samples may be derived from a tissue biopsy or another clinical procedure, and may include tumor tissue or cells extracted from subjects including mammals or cancer patients or cells from an in vitro culture. The sample may be in the form of an explant or xenograft. The tissue of the present invention may be surrogate tissue, i.e. any tissue that can be used as a substitute or replacement for tumor tissue in monitoring biological responses. The surrogate tissue may be non-proliferating peripheral mononuclear cells or proliferating cells, such as buccal mucosa tissue cells. For example, the surrogate tissue may be a blood sample obtained from a subject.

Subject: The term "subject" as used herein may refer to any mammal but may be selected either from a human or an experimental animal such as a mouse or a rat. Preferably, the mammal is human. For the purpose of the invention, the terms "subject or subjects" and "patient or patients" may be used interchangeably.

Pre-therapy: As used herein the term "pre-therapy" denotes determining the expression level of at least one of the biomarker transcripts selected from the group of EGR-1, DUSP-5 or FOS in a sample before either contacting the cancer cell from cell culture or from tumor tissue from a subject with a compound which is an inhibitor of IGF-IR and IR, or before administering the compound which is an inhibitor of IGF-IR and IR to a subject.

Post-therapy: As used herein the term "post-therapy" denotes determining the expression level of the biomarker transcript in a sample after either contacting the cancer cell from cell culture or from tumor tissue from a subject with a compound which is an inhibitor of IGF-IR and IR, or after administering the compound which is an inhibitor of IGF-IR and IR to a subject.

Tissue, cells or xenografts extracted from mammals having tumors are capable of use with the methods of the present invention. Where the methods involve in vitro analysis of cells, a number of different cell lines can be used. Examples include, without limitation, HEK-IGF1R, A673 and Colo-205 cell lines.

In an aspect, the present invention provides a method of monitoring the response of a subject having cancer to the treatment with a compound which is an IGF-IR and IR inhibitor; said method comprising determining the expression level of at least one of the biomarker transcripts selected from the group consisting of Early Growth Response- 1 (EGR-1), Dual Specificity Phosphatase-5 (DUSP-5) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in a sample pre-therapy and post-therapy.

In another aspect, the present invention provides a method of monitoring response of a subject having cancer to treatment with a compound, which is an IGF-IR and IR inhibitor; said method comprising the steps of: (a) determining the expression level of at least one of the biomarker transcripts selected from the group consisting of Early Growth Response- 1 (EGR-1), Dual Specificity Phosphatase-5 (DUSP-5) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in a sample obtained from the subject before administration of the compound, which is an IGF-IR and IR inhibitor;

(b) administering to the subject the compound, which is an IGF-IR and IR inhibitor and obtaining a sample from the subject after the said administration;

(c) determining the expression level of at least one of the said biomarker transcripts in the sample obtained in step (b);

(d) comparing the expression level of at least one of the said biomarker transcripts as determined in step (a) with the expression level determined in step (c); and

(e) predicting that said subject shall respond to the treatment with the compound when the expression level of at least one of the said biomarker transcripts is decreased.

According to the present invention, the cancer is selected from the group consisting of: astrocytoma, basal or squamous cell carcinoma, brain cancer, gliobastoma, bladder cancer, breast cancer, colon carcinoma, colorectal cancer, chrondrosarcoma, cervical cancer, adrenal cancer, choriocarcinoma, esophageal cancer, endometrial carcinoma, erythroleukemia, Ewing's sarcoma, gastrointestinal cancer, head and neck cancer, hepatoma, glioma, hepatocellular carcinoma, leukemia, leiomyona, melanoma, non-small cell lung cancer, neural cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, thymona, thyroid cancer, testicular cancer or osteosarcoma.

In an embodiment of the invention, the compound which is an IGF-IR and IR inhibitor is administered in vivo to an experimental animal having a tumor, or to a subject having cancer. In another aspect, the present invention relates to method of determining response of a cancer or a tumor to the treatment with a compound, which is an IGF-IR and IR inhibitor; said method comprises comparing the expression level of at least one of the biomarker transcripts selected from the group of EGR-1, DUSP-5 or FOS in a sample before treatment with the compound with the expression level of the said biomarker in a sample after treatment with the said compound. Decrease in the expression level of at least one of said biomarker transcripts after administering the IGF-IR and IR inhibitor would help in identifying that the tumor is responsive to treatment with the compound, which is an IGF-IR and IR inhibitor. In an embodiment, the method of identifying response of a cancer cell or a tumor to the treatment with a compound, which is an IGF-IR and IR inhibitor comprises contacting the compound to a cancer cell or a tumor tissue from a subject, ex vivo, for example a xenograft or explant.

In another embodiment, the method of identifying response of a tumor comprises contacting the tumor tissue or cancer cells with the compound, which is an IGF-IR and IR inhibitor.

In an aspect of the invention, the level of at least one of the biomarkers selected from the group of EGR-1, DUSP-5 and FOS is measured in a tumor tissue extracted from the subject having cancer wherein the subject is administered with a compound which is an IGF-IR and IR inhibitor.

According to the present invention the cancer cell is from the cancer selected from the group consisting of: astrocytoma, basal or squamous cell carcinoma, brain cancer, gliobastoma, bladder cancer, breast cancer, colon carcinoma, colorectal cancer, chrondrosarcoma, cervical cancer, adrenal cancer, choriocarcinoma, esophageal cancer, endometrial carcinoma, erythroleukemia, Ewing's sarcoma, gastrointestinal cancer, head and neck cancer, hepatoma, glioma, hepatocellular carcinoma, leukemia, leiomyona, melanoma, non-small cell lung cancer, neural cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, thymona, thyroid cancer, testicular cancer or osteosarcoma.

The IGF-IR and IR inhibitor can be contacted with tumor tissue or cancer cells in vivo, in vitro or ex vivo using known methods. In an embodiment, the cancer cells in culture or tumor tissues can be exposed to the compound, which is an IGF-IR and IR inhibitor over a time period ranging from 3 to 12 hours. In another embodiment, the experimental animals can be exposed to plurality of concentrations of the compound, which is an IGF-IR and IR inhibitor. For example, the experimental animals can be treated with the concentrations of the compound, which is an IGF-IR and IR inhibitor ranging from 10 to 400 mg/kg. In an embodiment, prolonged administration of the inhibitor can be achieved by treating the experimental animals continuously on consecutive days.

According to the invention, the compound which is an IGF-IR and IR inhibitor is selected from: a compound of formula I (as described herein), BMS-536924 (Bristol-Myers Squibb), BMS- 554417 (Bristol-Myers Squibb), BVP51004 (Biovitrium), NVP-ADW 742 (Novartis), NVP- AEW541 (Novartis), Tyrphostin AG1024 (tyrosine kinase inhibitor), A-928605 (Abbott), AG1024 (Calbiochem-EMD biosciences), OSI-906 (OSI Pharmaceuticals), quinolinyl-derived imidazo (l,5-a)pyrazine PQIP (OSI Pharmaceuticals), picropodophyllin PPP(AXL-1717) (Karolinska Institute/Biovitrium), CP-751871 (Pfizer), IMC-A12 (Imclone), AMG-479 (Amgen), AMG-655 (Amgen), MK0646/h7C10 (Merck/Pierre Fabre), R1507 (Roche), AVE- 1642/EM164 (Sanofi Aventis/immunoGen), SCH-717454 (19D12) (Schering-Plough/Medrex), BIIB022 (Biogen Idee), a-IR3 (mouse monoclonal antibody), di-diabody (Imclone), INSM- 18/NDGA (Insmed), XL-228/EXEL2280 (Exelixis), ANT-429 (Antrya), ATL-1101 (Antisense Therapeutics), rhIGFBP3 (Insmed) or BMS-754807 (Bristol-Myers Squibb).

In an embodiment the compound which is an IGF-IR and IR inhibitor is a compound of the following formula I

wherein R 1 is halogen; R 2 is C(0)OR 3 and R 3 is H or Q-C3 alkyl; or a pharmaceutically acceptable salt thereof. The compounds of formula I are described in PCT Appln. No. PCT/US 12/34188; which is incorporated herein by reference.

In an embodiment, the compound of formula I is (S)-ethyl 4-(2-carbamoyl -5-chloro-3-(2- (phenoxymethyl) morpholinosulfonyl)-lH-indol-7-ylamino) piperidine-l-carboxylate methane sulfonate (hereinafter referred to as Compound A). In a specific embodiment, the compound is a crystalline form of (S)-ethyl 4-(2-carbamoyl-5-chloro-3-(2-(phenoxymethyl) morpholinosulfonyl)-lH-indol-7-ylamino) piperidine-l-carboxylate methane sulfonate (crystalline form of Compound A) disclosed in PCT Appln. PCT/IB2012/051967 incorporated herein by reference. The measuring of expression level of the biomarker transcripts EGR-1, DUSP-5 and FOS, either before or after administration of a compound which is an IGF-IR and IR inhibitor can be carried out using known methods, for instance, microarray or RTQ-PCR as described in "The Open Pathology Journal, 2008, 2, 96-101" or "The Journal of Investigative Dermatology, 2004, 159- 167". RNA is extracted from mammalian tumor tissue, cancer cells or xenografts that have been treated with a compound which is an IGF-IR and IR inhibitor or with vehicle. The RNA is then converted to cDNA and analyzed by hybridization to a microarray to determine expression level of the said biomarker transcripts. Alternatively, RNA extracted from tumor tissue, cells or xenografts is used for real time quantitative polymerase chain reaction (RTQ-PCR) analysis.

In an embodiment, according to the methods of the present invention, the biomarker EGR- 1 in a sample e.g. cancer cells in culture or a xenograft mouse model, is down regulated when the sample is treated with a compound which is an IGF-IR and IR inhibitor. The length of the EGR- 1 polynucleotide that can be used in the methods of the present invention is 3136 base pair (bp) (accession no. NM_001964.2) and the length of the corresponding protein sequence is 543 amino acids. The DNA sequence (SEQ ID NO: 1) of EGR-1 is:

gcgcagaact tggggagccg ccgccgccat ccgccgccgc agccagcttc cgccgccgca

6 ggaccggccc ctgccccagc ctccgcagcc gcggcgcgtc cacgcccgcc cgcgcccagg

12 gcgagtcggg gtcgccgcct gcacgcttct cagtgttccc cgcgccccgc atgtaacccg

18 gccaggcccc cgcaactgtg tcccctgcag ctccagcccc gggctgcacc cccccgcccc

24 gacaccagct ctccagcctg ctcgtccagg atggccgcgg ccaaggccga gatgcagctg

30 atgtccccgc tgcagatctc tgacccgttc ggatcctttc ctcactcgcc caccatggac

36 aactacccta agctggagga gatgatgctg ctgagcaacg gggctcccca gttcctcggc

42 gccgccgggg ccccagaggg cagcggcagc aacagcagca gcagcagcag cgggggcggt

48 ggaggcggcg ggggcggcag caacagcagc agcagcagca gcaccttcaa ccctcaggcg

54 gacacgggcg agcagcccta cgagcacctg accgcagagt cttttcctga catctctctg

60 aacaacgaga aggtgctggt ggagaccagt taccccagcc aaaccactcg actgcccccc

66 atcacctata ctggccgctt ttccctggag cctgcaccca acagtggcaa caccttgtgg

72 cccgagcccc tcttcagctt ggtcagtggc ctagtgagca tgaccaaccc accggcctcc

78 tcgtcctcag caccatctcc agcggcctcc tccgcctccg cctcccagag cccacccctg

84 agctgcgcag tgccatccaa cgacagcagt cccatttact cagcggcacc caccttcccc

90 acgccgaaca ctgacatttt ccctgagcca caaagccagg ccttcccggg ctcggcaggg

96 acagcgctcc agtacccgcc tcctgcctac cctgccgcca agggtggctt ccaggttccc

102 atgatccccg actacctgtt tccacagcag cagggggatc tgggcctggg caccccagac

108 cagaagccct tccagggcct ggagagccgc acccagcagc cttcgctaac ccctctgtct

114 actattaagg cctttgccac tcagtcgggc tcccaggacc tgaaggccct caataccagc

120 taccagtccc agctcatcaa acccagccgc atgcgcaagt accccaaccg gcccagcaag

126 acgccccccc acgaacgccc ttacgcttgc ccagtggagt cctgtgatcg ccgcttctcc

132 cgctccgacg agctcacccg ccacatccgc atccacacag gccagaagcc cttccagtgc

138 cgcatctgca tgcgcaactt cagccgcagc gaccacctca ccacccacat ccgcacccac

144 acaggcgaaa agcccttcgc ctgcgacatc tgtggaagaa agtttgccag gagcgatgaa

150 cgcaagaggc ataccaagat ccacttgcgg cagaaggaca agaaagcaga caaaagtgtt

156 gtggcctctt cggccacctc ctctctctct tcctacccgt ccccggttgc tacctcttac 1621 ccgtccccgg ttactacctc ttatccatcc ccggccacca cctcataccc atcccctgtg 1681 cccacctcct tctcctctcc cggctcctcg acctacccat cccctgtgca cagtggcttc 1741 ccctccccgt cggtggccac cacgtactcc tctgttcccc ctgctttccc ggcccaggtc 1801 agcagcttcc cttcctcagc tgtcaccaac tccttcagcg cctccacagg gctttcggac 1861 atgacagcaa ccttttctcc caggacaatt gaaatttgct aaagggaaag gggaaagaaa 1921 gggaaaaggg agaaaaagaa acacaagaga cttaaaggac aggaggagga gatggccata 1981 ggagaggagg gttcctctta ggtcagatgg aggttctcag agccaagtcc tccctctcta 2041 ctggagtgga aggtctattg gccaacaatc ctttctgccc acttcccctt ccccaattac 2101 tattcccttt gacttcagct gcctgaaaca gccatgtcca agttcttcac ctctatccaa 2161 agaacttgat ttgcatggat tttggataaa tcatttcagt atcatctcca tcatatgcct

2221 gaccccttgc tcccttcaat gctagaaaat cgagttggca aaatggggtt tgggcccctc 2281 agagccctgc cctgcaccct tgtacagtgt ctgtgccatg gatttcgttt ttcttggggt 2341 actcttgatg tgaagataat ttgcatattc tattgtatta tttggagtta ggtcctcact

2401 tgggggaaaa aaaaaaaaga aaagccaagc aaaccaatgg tgatcctcta ttttgtgatg 2461 atgctgtgac aataagtttg aacctttttt tttgaaacag cagtcccagt attctcagag

2521 catgtgtcag agtgttgttc cgttaacctt tttgtaaata ctgcttgacc gtactctcac

2581 atgtggcaaa atatggtttg gtttttcttt tttttttttt ttgaaagtgt tttttcttcg

2641 tccttttggt ttaaaaagtt tcacgtcttg gtgccttttg tgtgatgcgc cttgctgatg

2701 gcttgacatg tgcaattgtg agggacatgc tcacctctag ccttaagggg ggcagggagt 2761 gatgatttgg gggaggcttt gggagcaaaa taaggaagag ggctgagctg agcttcggtt 2821 ctccagaatg taagaaaaca aaatctaaaa caaaatctga actctcaaaa gtctattttt 2881 ttaactgaaa atgtaaattt ataaatatat tcaggagttg gaatgttgta gttacctact

2941 gagtaggcgg cgatttttgt atgttatgaa catgcagttc attattttgt ggttctattt

3001 tactttgtac ttgtgtttgc ttaaacaaag tgactgtttg gcttataaac acattgaatg

3061 cgctttattg cccatgggat atgtggtgta tatccttcca aaaaattaaa acgaaaataa 3121 agtagctgcg attggg

This EGR-1 polynucleotide encodes the following amino acid sequence: EGR-1 Polypeptide (SEQ ID NO: 2):

Met Ala Ala Ala Lys Ala Glu Met Gin Leu Met Ser Pro Leu Gin He

1 5 10 15

Ser Asp Pro Phe Gly Ser Phe Pro His Ser Pro Thr Met Asp Asn Tyr

20 25 30

Pro Lys Leu Glu Glu Met Met Leu Leu Ser Asn Gly Ala Pro Gin Phe

35 40 45

Leu Gly Ala Ala Gly Ala Pro Glu Gly Ser Gly Ser Asn Ser Ser Ser

50 55 60

Ser Ser Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Asn Ser Ser

65 70 75 80

Ser Ser Ser Ser Thr Phe Asn Pro Gin Ala Asp Thr Gly Glu Gin Pro

85 90 95

Tyr Glu His Leu Thr Ala Glu Ser Phe Pro Asp He Ser Leu Asn Asn

100 105 110 Glu Lys Val Leu Val Glu Thr Ser Tyr Pro Ser Gin Thr Thr Arg Leu

115 120 125

Pro Pro lie Thr Tyr Thr Gly Arg Phe Ser Leu Glu Pro Ala Pro Asn

130 135 140

Ser Gly Asn Thr Leu Trp Pro Glu Pro Leu Phe Ser Leu Val Ser Gly 145 150 155 160

Leu Val Ser Met Thr Asn Pro Pro Ala Ser Ser Ser Ser Ala Pro Ser

165 170 175

Pro Ala Ala Ser Ser Ala Ser Ala Ser Gin Ser Pro Pro Leu Ser Cys

180 185 190

Ala Val Pro Ser Asn Asp Ser Ser Pro He Tyr Ser Ala Ala Pro Thr

195 200 205

Phe Pro Thr Pro Asn Thr Asp lie Phe Pro Glu Pro Gin Ser Gin Ala

210 215 220

Phe Pro Gly Ser Ala Gly Thr Ala Leu Gin Tyr Pro Pro Pro Ala Tyr 225 230 235 240

Pro Ala Ala Lys Gly Gly Phe Gin Val Pro Met He Pro Asp Tyr Leu

245 250 255

Phe Pro Gin Gin Gin Gly Asp Leu Gly Leu Gly Thr Pro Asp Gin Lys

260 265 270

Pro Phe Gin Gly Leu Glu Ser Arg Thr Gin Gin Pro Ser Leu Thr Pro

275 280 285

Leu Ser Thr He Lys Ala Phe Ala Thr Gin Ser Gly Ser Gin Asp Leu

290 295 300

Lys Ala Leu Asn Thr Ser Tyr Gin Ser Gin Leu He Lys Pro Ser Arg 305 310 315 320

Met Arg Lys Tyr Pro Asn Arg Pro Ser Lys Thr Pro Pro His Glu Arg

325 330 335

Pro Tyr Ala Cys Pro Val Glu Ser Cys Asp Arg Arg Phe Ser Arg Ser

340 345 350

Asp Glu Leu Thr Arg His He Arg He His Thr Gly Gin Lys Pro Phe

355 360 365

Gin Cys Arg He Cys Met Arg Asn Phe Ser Arg Ser Asp His Leu Thr 370 375 380 Thr His He Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp He

385 390 395 400

Cys Gly Arg Lys Phe Ala Arg Ser Asp Glu Arg Lys Arg His Thr Lys

405 410 415

He His Leu Arg Gin Lys Asp Lys Lys Ala Asp Lys Ser Val Val Ala

420 425 430

Ser Ser Ala Thr Ser Ser Leu Ser Ser Tyr Pro Ser Pro Val Ala Thr

435 440 445

Ser Tyr Pro Ser Pro Val Thr Thr Ser Tyr Pro Ser Pro Ala Thr Thr

450 455 460

Ser Tyr Pro Ser Pro Val Pro Thr Ser Phe Ser Ser Pro Gly Ser Ser

465 470 475 480

Thr Tyr Pro Ser Pro Val His Ser Gly Phe Pro Ser Pro Ser Val Ala

485 490 495

Thr Thr Tyr Ser Ser Val Pro Pro Ala Phe Pro Ala Gin Val Ser Ser

500 505 510

Phe Pro Ser Ser Ala Val Thr Asn Ser Phe Ser Ala Ser Thr Gly Leu

515 520 525

Ser Asp Met Thr Ala Thr Phe Ser Pro Arg Thr He Glu He Cys

530 535 540

In another embodiment, according to the methods of the present invention, the biomarker DUSP-

5 in a sample e.g. cancer cells in culture or a xenograft mouse model, is down regulated when the sample is treated with a compound which is an IGF-IR and IR inhibitor. The length of the

DUSP-5 polynucleotide that can be used in the methods of the present invention is 2545 bp

(accession no. NM_004419.3) and the length of the corresponding protein sequence is 384 amino acids. The DNA sequence (SEQ ID NO: 3) of DUSP-5 is:

1 actcattcac ataaaacgct gcgcggccgg cggaatcccc ggcttctagg gcggcgagcg

61 gccgggctgg ctatcgagcg agcggggcgg gaacgcggag ttgcgccgcc gctcgggcgc

121 cgggctccgt cgcggccgca gccccgcggg tcgccctccc gtgcctcgcc cgcggacacc

181 ctggccgtgg acaccctggc cgtgggcacc cgcggggcgc gcggcgcggg gccgctggcc

241 ggcggcggcg gcggcatgaa ggtcacgtcg ctcgacgggc gccagctgcg caagatgctc

301 cgcaaggagg cggcggcgcg ctgcgtggtg ctcgactgcc ggccctatct ggccttcgct

361 gcctcgaacg tgcgcggctc gctcaacgtc aacctcaact cggtggtgct gcggcgggcc

421 cggggcggcg cggtgtcggc gcgctacgtg ctgcccgacg aggcggcgcg cgcgcggctc

481 ctgcaggagg gcggcggcgg cgtcgcggcc gtggtggtgc tggaccaggg cagccgccac

541 tggcagaagc tgcgagagga gagcgccgcg cgtgtcgtcc tcacctcgct actcgcttgc

601 ctacccgccg gcccgcgggt ctacttcctc aaagggggat atgagacttt ctactcggaa 661 tatcctgagt gttgcgtgga tgtaaaaccc atttcacaag agaagattga gagtgagaga 721 gccctcatca gccagtgtgg aaaaccagtg gtaaatgtca gctacaggcc agcttatgac 781 cagggtggcc cagttgaaat ccttcccttc ctctaccttg gaagtgccta ccatgcatcc 841 aagtgcgagt tcctcgccaa cctgcacatc acagccctgc tgaatgtctc ccgacggacc 901 tccgaggcct gcgcgaccca cctacactac aaatggatcc ctgtggaaga cagccacacg 961 gctgacatta gctcccactt tcaagaagca atagacttca ttgactgtgt cagggaaaag 1021 ggaggcaagg tcctggtcca ctgtgaggct gggatctccc gttcacccac catctgcatg 1081 gcttacctta tgaagaccaa gcagttccgc ctgaaggagg ccttcgatta catcaagcag 1141 aggaggagca tggtctcgcc caactttggc ttcatgggcc agctcctgca gtacgaatct 1201 gagatcctgc cctccacgcc caacccccag cctccctcct gccaagggga ggcagcaggc 1261 tcttcactga taggccattt gcagacactg agccctgaca tgcagggtgc ctactgcaca 1321 ttccctgcct cggtgctggc accggtgcct acccactcaa cagtctcaga gctcagcaga 1381 agccctgtgg caacggccac atcctgctaa aactgggatg gaggaatcgg cccagcccca 1441 agagcaactg tgatttttgt ttttaagact catggacatt tcatacctgt gcaatactga 1501 agacctcatt ctgtcatgct gccccagtga gatagtgagt ggtcaccagg cttgcaaatg 1561 aacttcagac ggacctcagg gtaggttctc gggactgaag gaaggccaag ccattacggg 1621 agcacagcat gtgctgacta ctgtacttcc agacccctgc cctcttggga ctgcccagtc 1681 cttgcacctc agagttcgcc ttttcatttc aagcataagg caataaatac ctgcagcaac 1741 gtgggagaaa gaagttgctg gaccaggaga aaaggcagtt atgaagccaa ttcattttga 1801 aggaagcaca atttccacct tattttttga actttggcag tttcaatgtc tgtctctgtt 1861 gcttcggggc ataagctgat caccgtctag ttgggaaagt aaccctacag ggtttgtagg 1921 gacatgatca gcatcctgat ttgaaccctg aaatgttgtg tagacaccct cttgggtcca 1981 atgaggtagt tggttgaagt agcaagatgt tggcttttct ggattttttt tgccatgggt 2041 tcttcactga ccttggactt tggcatgatt cttagtcata cttgaacttg tctcattcca 2101 cctcttctca gagcaactct tcctttggga aaagagttct tcagatcata gaccaaaaaa 2161 gtcatacctt cgaggtggta gcagtagatt ccaggaggag aagggtactt gctaggtatc 2221 ctgggtcagt ggcggtgcaa actggtttcc tcagctgcct gtccttctgt gtgcttatgt 2281 ctcttgtgac aattgttttc ctccctgccc ctggaggttg tcttcaagct gtggacttct 2341 gggatttgca gattttgcaa cgtggtacta cttttttttt ctttttgtct gttagttatt

2401 tctccagggg aaaaggcaat aattttctaa gacccgtgtg aatgtgaaga aaagcagtat 2461 gttactggtt gttgttgttg ttcttgtttt ttatagtgta aaataaaaat agtaaaagga

2521 gaaaagcaaa aaaaaaaaaa aaaaa

This DUSP-5 polynucleotide encodes the following amino acid sequence: DUSP-5 Polypeptide (SEQ ID NO: 4):

Met Lys Val Thr Ser Leu Asp Gly Arg Gin Leu Arg Lys Met Leu Arg

1 5 10 15

Lys Glu Ala Ala Ala Arg Cys Val Val Leu Asp Cys Arg Pro Tyr Leu

20 25 30

Ala Phe Ala Ala Ser Asn Val Arg Gly Ser Leu Asn Val Asn Leu Asn

35 40 45

Ser Val Val Leu Arg Arg Ala Arg Gly Gly Ala Val Ser Ala Arg Tyr

50 55 60

Val Leu Pro Asp Glu Ala Ala Arg Ala Arg Leu Leu Gin Glu Gly Gly 65 70 75 80 Gly Gly Val Ala Ala Val Val Val Leu Asp Gin Gly Ser Arg His Trp

85 90 95

Gin Lys Leu Arg Glu Glu Ser Ala Ala Arg Val Val Leu Thr Ser Leu

100 105 110

Leu Ala Cys Leu Pro Ala Gly Pro Arg Val Tyr Phe Leu Lys Gly Gly

115 120 125

Tyr Glu Thr Phe Tyr Ser Glu Tyr Pro Glu Cys Cys Val Asp Val Lys

130 135 140

Pro lie Ser Gin Glu Lys He Glu Ser Glu Arg Ala Leu He Ser Gin 145 150 155 160

Cys Gly Lys Pro Val Val Asn Val Ser Tyr Arg Pro Ala Tyr Asp Gin

165 170 175

Gly Gly Pro Val Glu lie Leu Pro Phe Leu Tyr Leu Gly Ser Ala Tyr

180 185 190

His Ala Ser Lys Cys Glu Phe Leu Ala Asn Leu His lie Thr Ala Leu

195 200 205

Leu Asn Val Ser Arg Arg Thr Ser Glu Ala Cys Ala Thr His Leu His

210 215 220

Tyr Lys Trp He Pro Val Glu Asp Ser His Thr Ala Asp He Ser Ser 225 230 235 240

His Phe Gin Glu Ala He Asp Phe He Asp Cys Val Arg Glu Lys Gly

245 250 255

Gly Lys Val Leu Val His Cys Glu Ala Gly He Ser Arg Ser Pro Thr

260 265 270

He Cys Met Ala Tyr Leu Met Lys Thr Lys Gin Phe Arg Leu Lys Glu

275 280 285

Ala Phe Asp Tyr He Lys Gin Arg Arg Ser Met Val Ser Pro Asn Phe

290 295 300

Gly Phe Met Gly Gin Leu Leu Gin Tyr Glu Ser Glu He Leu Pro Ser 305 310 315 320

Thr Pro Asn Pro Gin Pro Pro Ser Cys Gin Gly Glu Ala Ala Gly Ser

325 330 335

Ser Leu He Gly His Leu Gin Thr Leu Ser Pro Asp Met Gin Gly Ala

340 345 350 Tyr Cys Thr Phe Pro Ala Ser Val Leu Ala Pro Val Pro Thr His Ser

355 360 365

Thr Val Ser Glu Leu Ser Arg Ser Pro Val Ala Thr Ala Thr Ser Cys

370 375 380

In yet another embodiment, according to the methods of the present invention, the biomarker FOS in a sample e.g. cancer cells in culture or a xenograft mouse model, is down regulated when the sample is treated with a compound which is an IGF-IR and IR inhibitor. The length of the FOS polynucleotide that can be used in the methods of the present invention is 2158 bp (accession no. NM_005252.3) and the length of the corresponding protein sequence is 380 amino acids. The DNA sequence (SEQ ID NO: 5) of FOS is:

1 attcataaaa cgcttgttat aaaagcagtg gctgcggcgc ctcgtactcc aaccgcatct

61 gcagcgagca tctgagaagc caagactgag ccggcggccg cggcgcagcg aacgagcagt

121 gaccgtgctc ctacccagct ctgctccaca gcgcccacct gtctccgccc ctcggcccct

181 cgcccggctt tgcctaaccg ccacgatgat gttctcgggc ttcaacgcag actacgaggc

241 gtcatcctcc cgctgcagca gcgcgtcccc ggccggggat agcctctctt actaccactc

301 acccgcagac tccttctcca gcatgggctc gcctgtcaac gcgcaggact tctgcacgga

361 cctggccgtc tccagtgcca acttcattcc cacggtcact gccatctcga ccagtccgga

421 cctgcagtgg ctggtgcagc ccgccctcgt ctcctccgtg gccccatcgc agaccagagc

481 ccctcaccct ttcggagtcc ccgccccctc cgctggggct tactccaggg ctggcgttgt

541 gaagaccatg acaggaggcc gagcgcagag cattggcagg aggggcaagg tggaacagtt

601 atctccagaa gaagaagaga aaaggagaat ccgaagggaa aggaataaga tggctgcagc

661 caaatgccgc aaccggagga gggagctgac tgatacactc caagcggaga cagaccaact

721 agaagatgag aagtctgctt tgcagaccga gattgccaac ctgctgaagg agaaggaaaa

781 actagagttc atcctggcag ctcaccgacc tgcctgcaag atccctgatg acctgggctt

841 cccagaagag atgtctgtgg cttcccttga tctgactggg ggcctgccag aggttgccac

901 cccggagtct gaggaggcct tcaccctgcc tctcctcaat gaccctgagc ccaagccctc

961 agtggaacct gtcaagagca tcagcagcat ggagctgaag accgagccct ttgatgactt

1021 cctgttccca gcatcatcca ggcccagtgg ctctgagaca gcccgctccg tgccagacat

1081 ggacctatct gggtccttct atgcagcaga ctgggagcct ctgcacagtg gctccctggg

1141 gatggggccc atggccacag agctggagcc cctgtgcact ccggtggtca cctgtactcc

1201 cagctgcact gcttacacgt cttccttcgt cttcacctac cccgaggctg actccttccc

1261 cagctgtgca gctgcccacc gcaagggcag cagcagcaat gagccttcct ctgactcgct

1321 cagctcaccc acgctgctgg ccctgtgagg gggcagggaa ggggaggcag ccggcaccca

1381 caagtgccac tgcccgagct ggtgcattac agagaggaga aacacatctt ccctagaggg

1441 ttcctgtaga cctagggagg accttatctg tgcgtgaaac acaccaggct gtgggcctca

1501 aggacttgaa agcatccatg tgtggactca agtccttacc tcttccggag atgtagcaaa

1561 acgcatggag tgtgtattgt tcccagtgac acttcagaga gctggtagtt agtagcatgt

1621 tgagccaggc ctgggtctgt gtctcttttc tctttctcct tagtcttctc atagcattaa

1681 ctaatctatt gggttcatta ttggaattaa cctggtgctg gatattttca aattgtatct

1741 agtgcagctg attttaacaa taactactgt gttcctggca atagtgtgtt ctgattagaa

1801 atgaccaata ttatactaag aaaagatacg actttatttt ctggtagata gaaataaata

1861 gctatatcca tgtactgtag tttttcttca acatcaatgt tcattgtaat gttactgatc

1921 atgcattgtt gaggtggtct gaatgttctg acattaacag ttttccatga aaacgtttta

1981 ttgtgttttt aatttattta ttaagatgga ttctcagata tttatatttt tattttattt 2041 ttttctacct tgaggtcttt tgacatgtgg aaagtgaatt tgaatgaaaa atttaagcat 2101 tgtttgctta ttgttccaag acattgtcaa taaaagcatt taagttgaat gcgaccaa

This FOS polynucleotide encodes the following amino acid sequence: FOS Polypeptide (SEQ ID NO: 6):

Met Met Phe Ser Gly Phe Asn Ala Asp Tyr Glu Ala Ser Ser Ser Arg

1 5 10 15

Cys Ser Ser Ala Ser Pro Ala Gly Asp Ser Leu Ser Tyr Tyr His Ser

20 25 30

Pro Ala Asp Ser Phe Ser Ser Met Gly Ser Pro Val Asn Ala Gin Asp

35 40 45

Phe Cys Thr Asp Leu Ala Val Ser Ser Ala Asn Phe lie Pro Thr Val

50 55 60

Thr Ala He Ser Thr Ser Pro Asp Leu Gin Trp Leu Val Gin Pro Ala 65 70 75 80

Leu Val Ser Ser Val Ala Pro Ser Gin Thr Arg Ala Pro His Pro Phe

85 90 95

Gly Val Pro Ala Pro Ser Ala Gly Ala Tyr Ser Arg Ala Gly Val Val

100 105 110

Lys Thr Met Thr Gly Gly Arg Ala Gin Ser He Gly Arg Arg Gly Lys

115 120 125

Val Glu Gin Leu Ser Pro Glu Glu Glu Glu Lys Arg Arg He Arg Arg

130 135 140

Glu Arg Asn Lys Met Ala Ala Ala Lys Cys Arg Asn Arg Arg Arg Glu 145 150 155 160

Leu Thr Asp Thr Leu Gin Ala Glu Thr Asp Gin Leu Glu Asp Glu Lys

165 170 175

Ser Ala Leu Gin Thr Glu He Ala Asn Leu Leu Lys Glu Lys Glu Lys

180 185 190

Leu Glu Phe He Leu Ala Ala His Arg Pro Ala Cys Lys He Pro Asp

195 200 205

Asp Leu Gly Phe Pro Glu Glu Met Ser Val Ala Ser Leu Asp Leu Thr

210 215 220

Gly Gly Leu Pro Glu Val Ala Thr Pro Glu Ser Glu Glu Ala Phe Thr 225 230 235 240 Leu Pro Leu Leu Asn Asp Pro Glu Pro Lys Pro Ser Val Glu Pro Val

245 250 255

Lys Ser He Ser Ser Met Glu Leu Lys Thr Glu Pro Phe Asp Asp Phe

260 265 270

Leu Phe Pro Ala Ser Ser Arg Pro Ser Gly Ser Glu Thr Ala Arg Ser

275 280 285

Val Pro Asp Met Asp Leu Ser Gly Ser Phe Tyr Ala Ala Asp Trp Glu

290 295 300

Pro Leu His Ser Gly Ser Leu Gly Met Gly Pro Met Ala Thr Glu Leu

305 310 315 320

Glu Pro Leu Cys Thr Pro Val Val Thr Cys Thr Pro Ser Cys Thr Ala

325 330 335

Tyr Thr Ser Ser Phe Val Phe Thr Tyr Pro Glu Ala Asp Ser Phe Pro

340 345 350

Ser Cys Ala Ala Ala His Arg Lys Gly Ser Ser Ser Asn Glu Pro Ser

355 360 365

Ser Asp Ser Leu Ser Ser Pro Thr Leu Leu Ala Leu

370 375 380

The present invention also relates to a method for testing whether a compound which is an IGF- IR and IR inhibitor produces a therapeutic response in a subject having cancer; said method comprising the steps of:

(a) determining the expression level of at least one of the biomarker transcripts selected from the group consisting of Early Growth Response-1 (EGR-1), Dual Specificity Phosphatase-5 (DUSP-5) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in a sample obtained from the said subject;

(b) administering to the subject, the compound, which is an IGF-1R and IR inhibitor and obtaining sample from the subject after the administration;

(c) determining the expression level of at least one of the said biomarker transcripts in the sample obtained in step (b);

(d) comparing the expression level of at least one of the said biomarker transcripts as determined in step (a) with the expression level determined in step (c);

(e) predicting that said subject shall respond to the treatment with the compound when the expression level of at least one of the said biomarker transcripts is decreased. In another aspect, the present invention provides a method of determining response of a cancer or a tumor to the treatment with a compound, which is an IGF-IR and IR inhibitor; said method comprising comparing expression level of at least one of the biomarker transcripts selected from the group of Early Growth Response- 1 (EGR-1), Dual Specificity Phosphatase-5 (DUSP-5) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in a sample before treatment with said compound with the expression level of the said biomarker transcript in a sample after treatment with the said compound.

In an embodiment, the IGF-IR and IR inhibitor used in the method of determining response is the compound of Formula I.

In another embodiment, the IGF-IR and IR inhibitor used in the method of determining response is compound A.

In an embodiment, the biomarker transcript used in the method of determining response is Early Growth Response- 1 (EGR-1).

In another embodiment, the biomarker transcript used in the method of determining response is Dual Specificity Phosphatase-5 (DUSP-5).

In yet another embodiment, the biomarker transcript used in the method of determining response is FBJ murine osteosarcoma viral oncogene homolog (FOS).

In another embodiment, the present invention provides a kit for performing the method of the present invention, comprising:

(a) an IGF-IR and IR inhibitor;

(b) means for measuring expression level of at least one of the biomarker transcripts selected from the group of Early Growth Response-1 (EGR-1), Dual Specificity Phosphatase-5 (DUSP-5) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in a sample; ;

(c) instructions for use of the kit.

In an embodiment, in the kit of the present invention, the means for measuring the expression level of the biomarker transcripts can be microarray or RTQ-PCR. In an embodiment, expression level of the biomarker transcripts are measured as described herein, particularly, in reference to the examples but not limited thereto.

In an aspect of the invention, the sample used in a method of the present invention, is a tumor tissue extracted from the subject having cancer. The tumor tissue is from a tumor selected from the group consisting of astrocytoma, basal or squamous cell carcinoma, brain cancer, gliobastoma, bladder cancer, breast cancer, colon carcinoma, colorectal cancer, chrondrosarcoma, cervical cancer, adrenal cancer, choriocarcinoma, esophageal cancer, endometrial carcinoma, erythroleukemia, Ewing's sarcoma, gastrointestinal cancer, head and neck cancer, hepatoma, glioma, hepatocellular carcinoma, leukemia, leiomyona, melanoma, non- small cell lung cancer, neural cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, thymona, thyroid cancer, testicular cancer or osteosarcoma.

In another aspect of the invention, the sample is a surrogate tissue. The surrogate tissue can be a blood sample of the subject having cancer which comprises serum or plasma.

The methods of the present invention have been established by biological assays which are described in detail in subsequent examples. These examples are herein provided for the purpose of illustration only and are not intended to limit the scope of the invention.

EXAMPLES

Example 1

Evaluation of Compound A in HEK-IGF1R cells using Microarray.

HEK-IGF1R cells (Human Embryonic Kidney cells over expressed for IGF1R obtained from Merck) were treated with Compound A (20mM stock was prepared in DMSO; final concentration in the assay was 1 μΜ) for 3, 6 and 12 hours. The cells treated with the compound A are referred to as "treated cells" and the cells not treated with the Compound A are referred to as "untreated cells". Treated and untreated cells were either stimulated with ^g/mL of IGF1 or were left unstimulated for another half an hour duration. RNA was isolated and subjected to microarray using alumina platform. Data was obtained after image quantification and analyzed using Genespring (GX 10.0. Agilent Technologies, USA). After normalization differentially expressed genes were filtered for significant difference from their respective time point control (p-value <0.05) using ANOVA with Tukey's multiple comparison test. Represented relative gene expression values were calculated [NT (normalized test)/ NC (normalized control)] and represented on log₂ scale. Values for the stimulated control (SC) group are expressed as fold change over the unstimulated control. Efficacy of cells treated with Compound A is expressed as fold change over the stimulated control. Data is expressed as mean + SEM, n=2. A change below and above +1 is considered significant in either direction.

RNA isolation

Cancer cells that were treated with Compound A under in vitro conditions were washed in nuclease free water and homogenized in 800μΕ lysis buffer with 8μί β-mercaptoethanol (Nucleopore RNASure Mini kit, Qiagen GmbH, Germany). The tubes were then centrifuged at 10,000g for 1 minute. This supernatant was used to obtain total RNA. The final elution was carried out in 32uL nuclease free water. The quality and quantity of the RNA was determined using a UV- visible spectrophotometer (NanoDrop ND-1000 spectrophotometer, NanoDrop products, USA). The 260/280 & the 260/230 ratios for all the samples used in the study were within 1.8 - 2.1, indicating highly pure total RNA species. The RNA samples were stored at -80°C until further use.

Gene expression profiling by oligonucleotide microarray

Microarray was performed using the Illumina Sentrix BeadChip® HumanHT-12 Expression BeadChip containing approximately 48,000 probes.

Result:

The results obtained are depicted in Figure I. HEK-IGFIR cells not treated with the Compound A (Stimulated Control) showed over expression of EGR1, FOS and DUSP5 at mRNA level after stimulation with IGF1. HEK-IGFIR cells treated with the Compound A showed significant inhibition (down regulation) of transcript level of the biomarkers EGR1, FOS and DUSP5 at mRNA level at all the time points i.e. at 3, 6 and 12 hours.

Example 2

Evaluation of Compound A in HEK-IGFIR cells using RTQPCR assay.

HEK-IGFIR cells were treated with the following compounds: (i) 0.1 and 1 μΜ of Compound A (stock of 20mM was prepared in DMSO); (ii) 0.1 and 1 μΜ of OSI-906 (Linsitinib, Active Biochem catalogue no. A- 1058) (stock of 20mM was prepared in DMSO) and (iii) 0.1 and 1 μΜ of BMS-754807 (Active Biochem, catalogue no. A-1013) (stock of 20mM was prepared in DMSO) for 3, 6 and 12 hours. OSI-906 and BMS-754807 are IGF-1R tyrosine kinase inhibitors. Cells not treated with the said compounds are referred to as untreated cells. Treated and untreated cells were either stimulated with ^g/mL of IGFl or were left unstimulated for another half an hour duration. Stimulated untreated cells are vehicle (DMSO) control cells. RNA was isolated according to procedure provided in Example 1 and utilized for RTQPCR assay.

Gene expression was calculated by the 2^"ΔΔα method as described in METHODS, 2001, 25, 402- 408 and then expressed on log₂ scale. A change in 2-fold or more is considered significant in either direction. Values for the stimulated control group are expressed as fold change over the unstimulated control. Efficacy of said compounds is expressed as fold change over the stimulated control. Data is expressed as mean + SEM, n=3 per group.

RTQ-PCR

Three biomarker transcripts (EGR-1, DUSP-5 and FOS) from IGF1R signaling pathway were analyzed by RTQ-PCR to measure the molecular response of the Compound A, OST906 and BMS-754807 in HEK-IGFIR cells. The primer sequences used in the RTQ-PCR for the said transcripts are listed below:

EGR-1 Forward Primer acctcatacc catcccctgt 20 (SEQ ID NO: 7) Reverse Primer tgtcctggga gaaaaggttg 20 (SEQ ID NO: 8)

DUSP-5 Forward Primer gctcccactt tcaagaagca 20 (SEQ ID NO: 9) Reverse Primer aggtaagcca tgcagatggt 20 (SEQ ID NO: 10)

FOS Forward Primer cgtgccagac atggacctat 20 (SEQ ID NO: 11)

Reverse Primer gtgaagagaa ggaagacg 18 (SEQ ID NO: 12)

GAPDH Forward Primer tgtgtccgtc gtggatctga 20 (SEQ ID NO: 13)

Reverse Primer cctgcttcac caccttcttg a 21 (SEQ ID NO: 14) cDNA synthesized from total RNA isolated was used as the template. All the PCR reactions were performed using the QuantiFast SYBR Green PCR Kit (Qiagen GmbH, InVitrogen Corporation, USA). Each PCR reaction contained IX master mix, 1 μL· of the diluted cDNA, and 250 nM of forward and reverse primers designed to yield 80 to 125-bp amplicons. Following the initial 3 minutes enzyme activation at 95°C a PCR reaction was carried out through 40 cycles (95°C for 10 seconds and 60°C for 30 seconds). Reactions were carried out on an Eppendorf Realplex 4 (Eppendorf AG, Hamburg, Germany). Experiments were performed in duplicate for each data point and glyceraldehydes 3-phosphate dehydrogenase (GAPDH) gene (housekeeping gene) was used as endogenous reference control. Each data point was normalized with its corresponding reference control. Results are expressed as mean + 2 standard error based on Log₂ transformation of normalized RT-QPCR values of the assayed genes. The fold change in expression of each gene was calculated using the ΔΔ Ct method as described in Livak et al (METHODS, 2001, 25, 402-408). The cycling threshold (C_t) value for each of the transcript was calculated after normalization of the data using data for housekeeping gene and the fold difference in the expression level of the transcripts was calculated. Data is represented graphically as the relative fold change (log₂ scale) of transcript levels as compared to cells treated with vehicle (DMSO) control.

Result:

The results obtained are depicted in Figure II. Analysis of mRNA level of biomarker transcripts EGRl, FOS and DUSP5 in HEK-IGFIR cells not treated with Compound A, OSI-906 and BMS- 754807 showed over expression of the said transcripts in comparison to unstimulated control. HEK-IGFIR cells treated with Compound A showed significant inhibition (down regulation) of transcript level of EGRl, FOS and DUSP5 at mRNA level at all the time points i.e. at 3, 6 and 12 hours. Efficacy (down regulation of biomarker transcripts) of Compound A is comparable to the known tyrosine kinase inhibitors OSI-906 and BMS-754807.

Example 3

Evaluation of Compound A in A673 cells using RTQPCR assay.

A673 cells (rhabdomyosarcoma cells; ATCC no. CRL-1598) were treated with the following compounds: (i) 0.1 and 1 μΜ of Compound A (20mM stock was prepared in DMSO); (ii) 0.1 and 1 μΜ of OSI-906 (Linsitinib, Active Biochem catalogue no. A- 1058) (20mM stock was prepared in DMSO) and (iii) 0.1 and 1 μΜ of BMS-754807 (Active Biochem, catalogue no. A- 1013) (20mM stock was prepared in DMSO) for 3, 6 and 12 hours. OSI-906 and BMS-754807 are IGF-1R tyrosine kinase inhibitors. Cells not treated with the said compounds are referred to as untreated cells. Treated and untreated cells were either stimulated with ^g/mL of IGF1 or were left unstimulated for another half an hour duration. Stimulated untreated cells are vehicle (DMSO) control cells. RNA was isolated according to procedure provided in Example 1 and utilized for RTQPCR assay.

Gene expression was calculated by the 2^"ΔΔα method (METHODS, 2001, 25, 402-408) and then expressed on log₂ scale. A change in 2-fold or more is considered significant in either direction. Values for the stimulated control group are expressed as fold change over the unstimulated control and for the groups treated with the said compounds are expressed as fold change over the stimulated control.

Result:

The results obtained are depicted in Figure III. Analysis of mRNA level of transcripts EGR1, FOS and DUSP5 in A673 cells not treated with Compound A, OSI-906 and BMS-754807 showed over expression of the said transcripts in comparison to unstimulated control. A673 cells treated with Compound A showed significant inhibition (down regulation) of biomarker transcript level of EGR1, FOS and DUSP5 at mRNA level at all the time points i.e. at 3, 6 and 12 hours. Efficacy (down regulation of biomarker transcripts) of Compound A is comparable to the known tyrosine kinase inhibitors OSI-906 and BMS-754807.

Example 4

Evaluation of Compound A in Colo-205 cells using RTQPCR assay.

Colo-205 cells (colorectal adenocarcinoma cells; ATCC number: CCL-222) were treated with the following compounds: (i) 0.1 and 1 μΜ of Compound A (20mM stock was prepared in DMSO); (ii) 0.1 and 1 μΜ of OSI-906 (Linsitinib, Active Biochem catalogue no. A-1058) (20mM stock was prepared in DMSO) and (iii) 0.1 and 1 μΜ of BMS-754807 (Active Biochem, catalogue no. A-1013) (20mM stock was prepared in DMSO) for 3, 6 and 12 hours. OSI-906 and BMS-754807 are IGF-1R tyrosine kinase inhibitors. Cells not treated with the said compounds are referred to as untreated cells. Treated and untreated cells were either stimulated with ^g/mL of IGF1 or were left unstimulated for another half an hour duration. Stimulated untreated cells are vehicle (DMSO) control cells. RNA was isolated according to procedure provided in Example 1 and utilized for RTQPCR assay.

Gene expression was calculated by the 2^"ΔΔα method (METHODS, 2001, 25, 402-408) and then expressed on log₂ scale. A change in 2-fold or more is considered significant in either direction. Values for the stimulated control group are expressed as fold change over the unstimulated control and for the groups treated with the said compounds are expressed as fold change over the stimulated control.

Result:

The results obtained are depicted in Figure IV. Analysis of mRNA level of transcripts EGR1, FOS and DUSP5 in Colo-205 cells not treated with Compound A, OSI-906 and BMS-754807 showed over expression of the said transcripts in comparison to unstimulated control. Colo-205 cells treated with Compound A showed significant inhibition (down regulation) of transcript level of EGR1, FOS and DUSP5 at mRNA level at all the time points i.e. at 3, 6 and 12 hours. Efficacy (down regulation of biomarker transcripts) of Compound A is comparable to the known tyrosine kinase inhibitors OSI-906 and BMS-754807.

In vivo experiment

Animals used in the experiments were housed and cared for, in accordance with the Guidelines in force published by CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals), Tamil Nadu, India. Procedures using laboratory animals were approved by the IAEC (Institutional Animal Ethics Committee) of Piramal Enterprises Limited (Formerly Piramal Healthcare Limited), Goregaon (East), Mumbai, India.

Example 5

Evaluation of Compound A in tumor tissue obtained from Colo-205 xenograft model using RTQPCR assay.

Colo-205 cells were harvested and re-suspended in saline at 6 x 10⁶ cells per 0.2 mL per mouse volume and injected to severe combined immunodeficient (SCID) mice on right flank. Animals were observed for the tumors till the tumors size attained a diameter of -150-200 mm mm and were randomized into control and treatment group. Treatment group received various doses of Compound A (10, 30, 100, 200, 400 mg/kg) or 50 mg/kg OSI-906 (Linsitinib, Active Biochem catalogue no. A-1058) in water by oral administration route for 3 days. On day 3 after 2 hours of treatment for the day, treated and untreated animals were either stimulated with

of IGF1 or were left unstimulated for another half an hour duration. After sacrificing the animals, the tumors were stored and RNA was isolated and subjected to RTQPCR assay.

Gene expression was calculated by the 2^"ΔΔα method (METHODS, 2001, 25, 402-408) and then expressed on log₂ scale. A change in 2-fold or more is considered significant in either direction. Values for the stimulated control group are expressed as fold change over the unstimulated control and for the groups treated with the said compounds are expressed as fold change over the stimulated control.

Result:

Results obtained are depicted in Figure V. Analysis of mRNA level of biomarker transcripts EGR1, FOS and DUSP5 in tumors isolated from animals not treated with Compound A or OSI-906 showed over expression of said biomarker transcripts in comparison to unstimulated control. The expression of the transcripts EGRl, FOS and DUSP5 in tumors of animals treated with Compound A or OST906 are as follows:

(i) Expression of FOS: No inhibition of FOS was observed in tumors isolated from animals treated with 10 mg/kg of the Compound A. Significant inhibition was observed in tumors isolated from animals treated with 30, 100, 200 and 400 mg/kg of the Compound A. The inhibition at these concentrations is comparable to inhibition shown by 50 mg/kg of OST906 in the experimental animals.

(ii) Expression of EGRl: Significant inhibition of EGRl was observed in tumors isolated from animals treated with higher concentrations of the Compound A. OSI- 906 showed higher inhibition of EGRl at the dose of 50 mg/kg in the experimental animals.

(iii) Expression of DUSP-5: Significant inhibition of EGRl was observed in tumors isolated from animals treated with all concentrations of the Compound A. The inhibition was comparable to inhibition shown by 50 mg/kg of OSI-906 in the experimental animals.

SEQUENCE LISTING

SEQ ID NO: 1

1 gcgcagaact tggggagccg ccgccgccat ccgccgccgc agccagcttc cgccgccgca 61 ggaccggccc ctgccccagc ctccgcagcc gcggcgcgtc cacgcccgcc cgcgcccagg 121 gcgagtcggg gtcgccgcct gcacgcttct cagtgttccc cgcgccccgc atgtaacccg 181 gccaggcccc cgcaactgtg tcccctgcag ctccagcccc gggctgcacc cccccgcccc 241 gacaccagct ctccagcctg ctcgtccagg atggccgcgg ccaaggccga gatgcagctg 301 atgtccccgc tgcagatctc tgacccgttc ggatcctttc ctcactcgcc caccatggac 361 aactacccta agctggagga gatgatgctg ctgagcaacg gggctcccca gttcctcggc 421 gccgccgggg ccccagaggg cagcggcagc aacagcagca gcagcagcag cgggggcggt 481 ggaggcggcg ggggcggcag caacagcagc agcagcagca gcaccttcaa ccctcaggcg 541 gacacgggcg agcagcccta cgagcacctg accgcagagt cttttcctga catctctctg 601 aacaacgaga aggtgctggt ggagaccagt taccccagcc aaaccactcg actgcccccc 661 atcacctata ctggccgctt ttccctggag cctgcaccca acagtggcaa caccttgtgg 721 cccgagcccc tcttcagctt ggtcagtggc ctagtgagca tgaccaaccc accggcctcc 781 tcgtcctcag caccatctcc agcggcctcc tccgcctccg cctcccagag cccacccctg 841 agctgcgcag tgccatccaa cgacagcagt cccatttact cagcggcacc caccttcccc 901 acgccgaaca ctgacatttt ccctgagcca caaagccagg ccttcccggg ctcggcaggg 961 acagcgctcc agtacccgcc tcctgcctac cctgccgcca agggtggctt ccaggttccc 1021 atgatccccg actacctgtt tccacagcag cagggggatc tgggcctggg caccccagac 1081 cagaagccct tccagggcct ggagagccgc acccagcagc cttcgctaac ccctctgtct 1141 actattaagg cctttgccac tcagtcgggc tcccaggacc tgaaggccct caataccagc 1201 taccagtccc agctcatcaa acccagccgc atgcgcaagt accccaaccg gcccagcaag 1261 acgccccccc acgaacgccc ttacgcttgc ccagtggagt cctgtgatcg ccgcttctcc 1321 cgctccgacg agctcacccg ccacatccgc atccacacag gccagaagcc cttccagtgc 1381 cgcatctgca tgcgcaactt cagccgcagc gaccacctca ccacccacat ccgcacccac 1441 acaggcgaaa agcccttcgc ctgcgacatc tgtggaagaa agtttgccag gagcgatgaa 1501 cgcaagaggc ataccaagat ccacttgcgg cagaaggaca agaaagcaga caaaagtgtt 1561 gtggcctctt cggccacctc ctctctctct tcctacccgt ccccggttgc tacctcttac

1621 ccgtccccgg ttactacctc ttatccatcc ccggccacca cctcataccc atcccctgtg 1681 cccacctcct tctcctctcc cggctcctcg acctacccat cccctgtgca cagtggcttc 1741 ccctccccgt cggtggccac cacgtactcc tctgttcccc ctgctttccc ggcccaggtc 1801 agcagcttcc cttcctcagc tgtcaccaac tccttcagcg cctccacagg gctttcggac 1861 atgacagcaa ccttttctcc caggacaatt gaaatttgct aaagggaaag gggaaagaaa 1921 gggaaaaggg agaaaaagaa acacaagaga cttaaaggac aggaggagga gatggccata 1981 ggagaggagg gttcctctta ggtcagatgg aggttctcag agccaagtcc tccctctcta 2041 ctggagtgga aggtctattg gccaacaatc ctttctgccc acttcccctt ccccaattac

2101 tattcccttt gacttcagct gcctgaaaca gccatgtcca agttcttcac ctctatccaa

2161 agaacttgat ttgcatggat tttggataaa tcatttcagt atcatctcca tcatatgcct

2221 gaccccttgc tcccttcaat gctagaaaat cgagttggca aaatggggtt tgggcccctc 2281 agagccctgc cctgcaccct tgtacagtgt ctgtgccatg gatttcgttt ttcttggggt

2341 actcttgatg tgaagataat ttgcatattc tattgtatta tttggagtta ggtcctcact

2401 tgggggaaaa aaaaaaaaga aaagccaagc aaaccaatgg tgatcctcta ttttgtgatg 2461 atgctgtgac aataagtttg aacctttttt tttgaaacag cagtcccagt attctcagag

2521 catgtgtcag agtgttgttc cgttaacctt tttgtaaata ctgcttgacc gtactctcac

2581 atgtggcaaa atatggtttg gtttttcttt tttttttttt ttgaaagtgt tttttcttcg

2641 tccttttggt ttaaaaagtt tcacgtcttg gtgccttttg tgtgatgcgc cttgctgatg

2701 gcttgacatg tgcaattgtg agggacatgc tcacctctag ccttaagggg ggcagggagt 2761 gatgatttgg gggaggcttt gggagcaaaa taaggaagag ggctgagctg agcttcggtt 2821 ctccagaatg taagaaaaca aaatctaaaa caaaatctga actctcaaaa gtctattttt 2881 ttaactgaaa atgtaaattt ataaatatat tcaggagttg gaatgttgta gttacctact 2941 gagtaggcgg cgatttttgt atgttatgaa catgcagttc attattttgt ggttctattt 3001 tactttgtac ttgtgtttgc ttaaacaaag tgactgtttg gcttataaac acattgaatg 3061 cgctttattg cccatgggat atgtggtgta tatccttcca aaaaattaaa acgaaaataa 3121 agtagctgcg attggg

SEQ ID NO: 2

Met Ala Ala Ala Lys Ala Glu Met Gin Leu Met Ser Pro Leu Gin He

1 5 10 15

Ser Asp Pro Phe Gly Ser Phe Pro His Ser Pro Thr Met Asp Asn Tyr

20 25 30

Pro Lys Leu Glu Glu Met Met Leu Leu Ser Asn Gly Ala Pro Gin Phe

35 40 45

Leu Gly Ala Ala Gly Ala Pro Glu Gly Ser Gly Ser Asn Ser Ser Ser

50 55 60

Ser Ser Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Asn Ser Ser 65 70 75 80

Ser Ser Ser Ser Thr Phe Asn Pro Gin Ala Asp Thr Gly Glu Gin Pro

85 90 95

Tyr Glu His Leu Thr Ala Glu Ser Phe Pro Asp He Ser Leu Asn Asn

100 105 110

Glu Lys Val Leu Val Glu Thr Ser Tyr Pro Ser Gin Thr Thr Arg Leu

115 120 125

Pro Pro He Thr Tyr Thr Gly Arg Phe Ser Leu Glu Pro Ala Pro Asn

130 135 140

Ser Gly Asn Thr Leu Trp Pro Glu Pro Leu Phe Ser Leu Val Ser Gly 145 150 155 160

Leu Val Ser Met Thr Asn Pro Pro Ala Ser Ser Ser Ser Ala Pro Ser

165 170 175

Pro Ala Ala Ser Ser Ala Ser Ala Ser Gin Ser Pro Pro Leu Ser Cys

180 185 190

Ala Val Pro Ser Asn Asp Ser Ser Pro He Tyr Ser Ala Ala Pro Thr

195 200 205

Phe Pro Thr Pro Asn Thr Asp He Phe Pro Glu Pro Gin Ser Gin Ala 210 215 220 Phe Pro Gly Ser Ala Gly Thr Ala Leu Gin Tyr Pro Pro Pro Ala Tyr 225 230 235 240

Pro Ala Ala Lys Gly Gly Phe Gin Val Pro Met He Pro Asp Tyr Leu

245 250 255

Phe Pro Gin Gin Gin Gly Asp Leu Gly Leu Gly Thr Pro Asp Gin Lys

260 265 270

Pro Phe Gin Gly Leu Glu Ser Arg Thr Gin Gin Pro Ser Leu Thr Pro

275 280 285

Leu Ser Thr He Lys Ala Phe Ala Thr Gin Ser Gly Ser Gin Asp Leu

290 295 300

Lys Ala Leu Asn Thr Ser Tyr Gin Ser Gin Leu He Lys Pro Ser Arg 305 310 315 320

Met Arg Lys Tyr Pro Asn Arg Pro Ser Lys Thr Pro Pro His Glu Arg

325 330 335

Pro Tyr Ala Cys Pro Val Glu Ser Cys Asp Arg Arg Phe Ser Arg Ser

340 345 350

Asp Glu Leu Thr Arg His He Arg He His Thr Gly Gin Lys Pro Phe

355 360 365

Gin Cys Arg He Cys Met Arg Asn Phe Ser Arg Ser Asp His Leu Thr

370 375 380

Thr His He Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp He 385 390 395 400

Cys Gly Arg Lys Phe Ala Arg Ser Asp Glu Arg Lys Arg His Thr Lys

405 410 415

He His Leu Arg Gin Lys Asp Lys Lys Ala Asp Lys Ser Val Val Ala

420 425 430

Ser Ser Ala Thr Ser Ser Leu Ser Ser Tyr Pro Ser Pro Val Ala Thr

435 440 445

Ser Tyr Pro Ser Pro Val Thr Thr Ser Tyr Pro Ser Pro Ala Thr Thr

450 455 460

Ser Tyr Pro Ser Pro Val Pro Thr Ser Phe Ser Ser Pro Gly Ser Ser 465 470 475 480

Thr Tyr Pro Ser Pro Val His Ser Gly Phe Pro Ser Pro Ser Val Ala

485 490 495 Thr Thr Tyr Ser Ser Val Pro Pro Ala Phe Pro Ala Gin Val Ser Ser

500 505 510

Phe Pro Ser Ser Ala Val Thr Asn Ser Phe Ser Ala Ser Thr Gly Leu

515 520 525

Ser Asp Met Thr Ala Thr Phe Ser Pro Arg Thr He Glu lie Cys

530 535 540

SEQ ID NO: 3 actcattcac ataaaacgct gcgcggccgg cggaatcccc ggcttctagg gcggcgagcg gccgggctgg ctatcgagcg agcggggcgg gaacgcggag ttgcgccgcc gctcgggcgc cgggctccgt cgcggccgca gccccgcggg tcgccctccc gtgcctcgcc cgcggacacc ctggccgtgg acaccctggc cgtgggcacc cgcggggcgc gcggcgcggg gccgctggcc ggcggcggcg gcggcatgaa ggtcacgtcg ctcgacgggc gccagctgcg caagatgctc cgcaaggagg cggcggcgcg ctgcgtggtg ctcgactgcc ggccctatct ggccttcgct gcctcgaacg tgcgcggctc gctcaacgtc aacctcaact cggtggtgct gcggcgggcc cggggcggcg cggtgtcggc gcgctacgtg ctgcccgacg aggcggcgcg cgcgcggctc ctgcaggagg gcggcggcgg cgtcgcggcc gtggtggtgc tggaccaggg cagccgccac tggcagaagc tgcgagagga gagcgccgcg cgtgtcgtcc tcacctcgct actcgcttgc ctacccgccg gcccgcgggt ctacttcctc aaagggggat atgagacttt ctactcggaa tatcctgagt gttgcgtgga tgtaaaaccc atttcacaag agaagattga gagtgagaga gccctcatca gccagtgtgg aaaaccagtg gtaaatgtca gctacaggcc agcttatgac cagggtggcc cagttgaaat ccttcccttc ctctaccttg gaagtgccta ccatgcatcc aagtgcgagt tcctcgccaa cctgcacatc acagccctgc tgaatgtctc ccgacggacc tccgaggcct gcgcgaccca cctacactac aaatggatcc ctgtggaaga cagccacacg gctgacatta gctcccactt tcaagaagca atagacttca ttgactgtgt cagggaaaag ggaggcaagg tcctggtcca ctgtgaggct gggatctccc gttcacccac catctgcatg gcttacctta tgaagaccaa gcagttccgc ctgaaggagg ccttcgatta catcaagcag aggaggagca tggtctcgcc caactttggc ttcatgggcc agctcctgca gtacgaatct gagatcctgc cctccacgcc caacccccag cctccctcct gccaagggga ggcagcaggc tcttcactga taggccattt gcagacactg agccctgaca tgcagggtgc ctactgcaca ttccctgcct cggtgctggc accggtgcct acccactcaa cagtctcaga gctcagcaga agccctgtgg caacggccac atcctgctaa aactgggatg gaggaatcgg cccagcccca agagcaactg tgatttttgt ttttaagact catggacatt tcatacctgt gcaatactga agacctcatt ctgtcatgct gccccagtga gatagtgagt ggtcaccagg cttgcaaatg aacttcagac ggacctcagg gtaggttctc gggactgaag gaaggccaag ccattacggg agcacagcat gtgctgacta ctgtacttcc agacccctgc cctcttggga ctgcccagtc cttgcacctc agagttcgcc ttttcatttc aagcataagg caataaatac ctgcagcaac gtgggagaaa gaagttgctg gaccaggaga aaaggcagtt atgaagccaa ttcattttga aggaagcaca atttccacct tattttttga actttggcag tttcaatgtc tgtctctgtt gcttcggggc ataagctgat caccgtctag ttgggaaagt aaccctacag ggtttgtagg gacatgatca gcatcctgat ttgaaccctg aaatgttgtg tagacaccct cttgggtcca atgaggtagt tggttgaagt agcaagatgt tggcttttct ggattttttt tgccatgggt tcttcactga ccttggactt tggcatgatt cttagtcata cttgaacttg tctcattcca cctcttctca gagcaactct tcctttggga aaagagttct tcagatcata gaccaaaaaa gtcatacctt cgaggtggta gcagtagatt ccaggaggag aagggtactt gctaggtatc ctgggtcagt ggcggtgcaa actggtttcc tcagctgcct gtccttctgt gtgcttatgt 2281 ctcttgtgac aattgttttc ctccctgccc ctggaggttg tcttcaagct gtggacttct 2341 gggatttgca gattttgcaa cgtggtacta cttttttttt ctttttgtct gttagttatt 2401 tctccagggg aaaaggcaat aattttctaa gacccgtgtg aatgtgaaga aaagcagtat 2461 gttactggtt gttgttgttg ttcttgtttt ttatagtgta aaataaaaat agtaaaagga 2521 gaaaagcaaa aaaaaaaaaa aaaaa

SEQ ID NO: 4

Met Lys Val Thr Ser Leu Asp Gly Arg Gin Leu Arg Lys Met Leu Arg

1 5 10 15

Lys Glu Ala Ala Ala Arg Cys Val Val Leu Asp Cys Arg Pro Tyr Leu

20 25 30

Ala Phe Ala Ala Ser Asn Val Arg Gly Ser Leu Asn Val Asn Leu Asn

35 40 45

Ser Val Val Leu Arg Arg Ala Arg Gly Gly Ala Val Ser Ala Arg Tyr

50 55 60

Val Leu Pro Asp Glu Ala Ala Arg Ala Arg Leu Leu Gin Glu Gly Gly 65 70 75 80

Gly Gly Val Ala Ala Val Val Val Leu Asp Gin Gly Ser Arg His Trp

85 90 95

Gin Lys Leu Arg Glu Glu Ser Ala Ala Arg Val Val Leu Thr Ser Leu

100 105 110

Leu Ala Cys Leu Pro Ala Gly Pro Arg Val Tyr Phe Leu Lys Gly Gly

115 120 125

Tyr Glu Thr Phe Tyr Ser Glu Tyr Pro Glu Cys Cys Val Asp Val Lys

130 135 140

Pro lie Ser Gin Glu Lys He Glu Ser Glu Arg Ala Leu He Ser Gin 145 150 155 160

Cys Gly Lys Pro Val Val Asn Val Ser Tyr Arg Pro Ala Tyr Asp Gin

165 170 175

Gly Gly Pro Val Glu lie Leu Pro Phe Leu Tyr Leu Gly Ser Ala Tyr

180 185 190

His Ala Ser Lys Cys Glu Phe Leu Ala Asn Leu His lie Thr Ala Leu

195 200 205

Leu Asn Val Ser Arg Arg Thr Ser Glu Ala Cys Ala Thr His Leu His 210 215 220 Tyr Lys Trp He Pro Val Glu Asp Ser His Thr Ala Asp lie Ser Ser

225 230 235 240

His Phe Gin Glu Ala lie Asp Phe lie Asp Cys Val Arg Glu Lys Gly

245 250 255

Gly Lys Val Leu Val His Cys Glu Ala Gly He Ser Arg Ser Pro Thr

260 265 270

He Cys Met Ala Tyr Leu Met Lys Thr Lys Gin Phe Arg Leu Lys Glu

275 280 285

Ala Phe Asp Tyr He Lys Gin Arg Arg Ser Met Val Ser Pro Asn Phe

290 295 300

Gly Phe Met Gly Gin Leu Leu Gin Tyr Glu Ser Glu He Leu Pro Ser 305 310 315 320

Thr Pro Asn Pro Gin Pro Pro Ser Cys Gin Gly Glu Ala Ala Gly Ser

325 330 335

Ser Leu He Gly His Leu Gin Thr Leu Ser Pro Asp Met Gin Gly Ala

340 345 350

Tyr Cys Thr Phe Pro Ala Ser Val Leu Ala Pro Val Pro Thr His Ser

355 360 365

Thr Val Ser Glu Leu Ser Arg Ser Pro Val Ala Thr Ala Thr Ser Cys

370 375 380

SEQ ID NO: 5

1 attcataaaa cgcttgttat aaaagcagtg gctgcggcgc ctcgtactcc aaccgcatct 61 gcagcgagca tctgagaagc caagactgag ccggcggccg cggcgcagcg aacgagcagt 121 gaccgtgctc ctacccagct ctgctccaca gcgcccacct gtctccgccc ctcggcccct 181 cgcccggctt tgcctaaccg ccacgatgat gttctcgggc ttcaacgcag actacgaggc 241 gtcatcctcc cgctgcagca gcgcgtcccc ggccggggat agcctctctt actaccactc 301 acccgcagac tccttctcca gcatgggctc gcctgtcaac gcgcaggact tctgcacgga 361 cctggccgtc tccagtgcca acttcattcc cacggtcact gccatctcga ccagtccgga 421 cctgcagtgg ctggtgcagc ccgccctcgt ctcctccgtg gccccatcgc agaccagagc 481 ccctcaccct ttcggagtcc ccgccccctc cgctggggct tactccaggg ctggcgttgt 541 gaagaccatg acaggaggcc gagcgcagag cattggcagg aggggcaagg tggaacagtt 601 atctccagaa gaagaagaga aaaggagaat ccgaagggaa aggaataaga tggctgcagc 661 caaatgccgc aaccggagga gggagctgac tgatacactc caagcggaga cagaccaact 721 agaagatgag aagtctgctt tgcagaccga gattgccaac ctgctgaagg agaaggaaaa 781 actagagttc atcctggcag ctcaccgacc tgcctgcaag atccctgatg acctgggctt 841 cccagaagag atgtctgtgg cttcccttga tctgactggg ggcctgccag aggttgccac 901 cccggagtct gaggaggcct tcaccctgcc tctcctcaat gaccctgagc ccaagccctc 961 agtggaacct gtcaagagca tcagcagcat ggagctgaag accgagccct ttgatgactt 1021 cctgttccca gcatcatcca ggcccagtgg ctctgagaca gcccgctccg tgccagacat 1081 ggacctatct gggtccttct atgcagcaga ctgggagcct ctgcacagtg gctccctggg 1141 gatggggccc atggccacag agctggagcc cctgtgcact ccggtggtca cctgtactcc 1201 cagctgcact gcttacacgt cttccttcgt cttcacctac cccgaggctg actccttccc 1261 cagctgtgca gctgcccacc gcaagggcag cagcagcaat gagccttcct ctgactcgct 1321 cagctcaccc acgctgctgg ccctgtgagg gggcagggaa ggggaggcag ccggcaccca 1381 caagtgccac tgcccgagct ggtgcattac agagaggaga aacacatctt ccctagaggg 1441 ttcctgtaga cctagggagg accttatctg tgcgtgaaac acaccaggct gtgggcctca 1501 aggacttgaa agcatccatg tgtggactca agtccttacc tcttccggag atgtagcaaa 1561 acgcatggag tgtgtattgt tcccagtgac acttcagaga gctggtagtt agtagcatgt 1621 tgagccaggc ctgggtctgt gtctcttttc tctttctcct tagtcttctc atagcattaa

1681 ctaatctatt gggttcatta ttggaattaa cctggtgctg gatattttca aattgtatct

1741 agtgcagctg attttaacaa taactactgt gttcctggca atagtgtgtt ctgattagaa 1801 atgaccaata ttatactaag aaaagatacg actttatttt ctggtagata gaaataaata 1861 gctatatcca tgtactgtag tttttcttca acatcaatgt tcattgtaat gttactgatc

1921 atgcattgtt gaggtggtct gaatgttctg acattaacag ttttccatga aaacgtttta

1981 ttgtgttttt aatttattta ttaagatgga ttctcagata tttatatttt tattttattt

2041 ttttctacct tgaggtcttt tgacatgtgg aaagtgaatt tgaatgaaaa atttaagcat 2101 tgtttgctta ttgttccaag acattgtcaa taaaagcatt taagttgaat gcgaccaa

SEQ ID NO: 6

Met Met Phe Ser Gly Phe Asn Ala Asp Tyr Glu Ala Ser Ser Ser Arg

1 5 10 15

Cys Ser Ser Ala Ser Pro Ala Gly Asp Ser Leu Ser Tyr Tyr His Ser

20 25 30

Pro Ala Asp Ser Phe Ser Ser Met Gly Ser Pro Val Asn Ala Gin Asp

35 40 45

Phe Cys Thr Asp Leu Ala Val Ser Ser Ala Asn Phe lie Pro Thr Val

50 55 60

Thr Ala He Ser Thr Ser Pro Asp Leu Gin Trp Leu Val Gin Pro Ala

65 70 75 80

Leu Val Ser Ser Val Ala Pro Ser Gin Thr Arg Ala Pro His Pro Phe

85 90 95

Gly Val Pro Ala Pro Ser Ala Gly Ala Tyr Ser Arg Ala Gly Val Val

100 105 110

Lys Thr Met Thr Gly Gly Arg Ala Gin Ser He Gly Arg Arg Gly Lys

115 120 125

Val Glu Gin Leu Ser Pro Glu Glu Glu Glu Lys Arg Arg He Arg Arg

130 135 140

Glu Arg Asn Lys Met Ala Ala Ala Lys Cys Arg Asn Arg Arg Arg Glu 145 150 155 160 Leu Thr Asp Thr Leu Gin Ala Glu Thr Asp Gin Leu Glu Asp Glu Lys

165 170 175

Ser Ala Leu Gin Thr Glu lie Ala Asn Leu Leu Lys Glu Lys Glu Lys

180 185 190

Leu Glu Phe He Leu Ala Ala His Arg Pro Ala Cys Lys He Pro Asp

195 200 205

Asp Leu Gly Phe Pro Glu Glu Met Ser Val Ala Ser Leu Asp Leu Thr

210 215 220

Gly Gly Leu Pro Glu Val Ala Thr Pro Glu Ser Glu Glu Ala Phe Thr 225 230 235 240

Leu Pro Leu Leu Asn Asp Pro Glu Pro Lys Pro Ser Val Glu Pro Val

245 250 255

Lys Ser He Ser Ser Met Glu Leu Lys Thr Glu Pro Phe Asp Asp Phe

260 265 270

Leu Phe Pro Ala Ser Ser Arg Pro Ser Gly Ser Glu Thr Ala Arg Ser

275 280 285

Val Pro Asp Met Asp Leu Ser Gly Ser Phe Tyr Ala Ala Asp Trp Glu

290 295 300

Pro Leu His Ser Gly Ser Leu Gly Met Gly Pro Met Ala Thr Glu Leu 305 310 315 320

Glu Pro Leu Cys Thr Pro Val Val Thr Cys Thr Pro Ser Cys Thr Ala

325 330 335

Tyr Thr Ser Ser Phe Val Phe Thr Tyr Pro Glu Ala Asp Ser Phe Pro

340 345 350

Ser Cys Ala Ala Ala His Arg Lys Gly Ser Ser Ser Asn Glu Pro Ser

355 360 365

Ser Asp Ser Leu Ser Ser Pro Thr Leu Leu Ala Leu

370 375 380

SEQ ID NO: 7 acctcatacc catcccctgt SEQ ID NO: 8 tgtcctggga gaaaaggttg SEQIDNO: 9 gctcccactt tcaagaagca

SEQIDNO: 10 aggtaagcca tgcagatggt SEQIDNO: 11 cgtgccagac atggacctat SEQIDNO: 12 gtgaagagaa ggaagacg SEQIDNO: 13 tgtgtccgtc gtggatctga SEQIDNO: 14 cctgcttcac caccttcttg a

Claims

WE CLAIM:

1. A method of monitoring response of a subject having cancer to treatment with a compound which is an Insulin Like Growth Factor- 1 Receptor (IGF-1R) and Insulin receptor (IR) inhibitor, said method comprising the steps of:

(a) determining the expression level of at least one of the biomarker transcripts selected from the group consisting of Early Growth Response- 1 (EGR-1), Dual Specificity Phosphatase-5 (DUSP-5) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in a sample obtained from the subject before administration of the said compound;

(b) administering to the said subject the compound and obtaining a sample from the said subject after the administration;

(c) determining the expression level of at least one of the said biomarker transcripts in the sample obtained in step (b);

(d) comparing the expression level of at least one of the said biomarker transcripts as determined in step (a) with the expression level determined in step (c); and

(e) predicting that the subject shall respond to the treatment with the compound when the expression level of at least one of said biomarker transcripts is decreased.

2. The method according to claim 1, wherein the cancer is selected from the group consisting of: astrocytoma, basal or squamous cell carcinoma, brain cancer, gliobastoma, bladder cancer, breast cancer, colon carcinoma, colorectal cancer, chrondrosarcoma, cervical cancer, adrenal cancer, choriocarcinoma, esophageal cancer, endometrial carcinoma, erythroleukemia, Ewing's sarcoma, gastrointestinal cancer, head and neck cancer, hepatoma, glioma, hepatocellular carcinoma, leukemia, leiomyona, melanoma, non-small cell lung cancer, neural cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, thymona, thyroid cancer, testicular cancer or osteosarcoma.

3. The method according to claim 1, wherein the compound which is an IGF-1R and IR inhibitor is selected from BMS-536924, BMS-554417, BUP51004, NVP-ADW 742, NVP-AEW541, Tyrphostin AG1024, A-928605, AG1024, OSI-906, quinolinyl-derived imidazo (l,5-a)pyrazine PQIP, picropodophyllin PPP(AXL-1717), AG-1024, CP-751871, IMC-A12, AMG-479, AMG-655, MK0646/h7C10, R1507, AVE- 1642, SCH-717454, BIIB022, a-IR3, di-diabody, INSM-18/NDGA, XL-228, ANT-429, ATL-1101, rhIGFBP3, BMS-754807 compound of formula I or a pharmaceutically acceptable salt thereof;

I

wherein R¹ is halogen; R² is C(0)OR³ and R³ is H or Q-C3 alkyl.

4. The method according to claim 3, wherein the compound of formula I is (S)-ethyl 4- (2- carbamoyl -5-chloro-3-(2-(phenoxymethyl) morpholinosulfonyl)-lH-indol-7- ylamino)piperidine-l-carboxylate methane sulfonate (Compound A).

5. The method according to claim 4, wherein the said Compound A is a crystalline form of (S)-ethyl 4-(2-carbamoyl -5-chloro-3-(2-(phenoxymethyl) morpholinosulfonyl)-lH- indol-7-ylamino) piperidine-l-carboxylate methane sulfonate.

6. The method according to claim 1, wherein the biomarker Early Growth Response- 1 (EGR-1) is a polypeptide encoded by a polynucleotide having a sequence of SEQ ID NO: 1.

7. The method according to claim 1, wherein the biomarker Dual Specificity Phosphatase-5 (DUSP-5) is a polypeptide encoded by a polynucleotide having a sequence of SEQ ID NO: 3.

8. The method according to claim 1, wherein the biomarker FBJ murine osteosarcoma viral oncogene homolog (FOS) is a polypeptide encoded by a polynucleotide having a sequence of SEQ ID NO: 5.

9. A method of determining response of a cancer or a tumor to the treatment with a compound, which is an IGF-IR and IR inhibitor; said method comprising comparing expression level of at least one of the biomarker transcripts selected from the group of Early Growth Response- 1 (EGR-1), Dual Specificity Phosphatase-5 (DUSP-5) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in a sample before treatment with the said compound; with the expression level of the said biomarker transcript in a sample after treatment with the said compound.

10. The method according to claim 9, wherein the compound which is an IGF-IR and IR inhibitor is selected from BMS-536924, BMS-554417, BUP51004, NVP-ADW 742, NVP-AEW541, Tyrphostin AG1024, A-928605, AG1024, OSI-906, quinolinyl-derived imidazo (l,5-a)pyrazine PQIP, picropodophyllin PPP(AXL-1717), AG-1024, CP-751871, IMC-A12, AMG-479, AMG-655, MK0646/h7C10, R1507, AVE-1642, SCH-717454, BIIB022, a-IR3, di-diabody, INSM-18/NDGA, XL-228, ANT-429, ATL-1101, rhIGFBP3, BMS-754807 or a compound of formula I or a pharmaceutically acceptable salt thereof;

I

wherein R¹ is halogen; R² is C(0)OR³ and R³ is H or Q Q alkyl.

11. The method according to claim 10, wherein the compound of formula I is (S)-ethyl 4-(2- carbamoyl -5-chloro-3-(2-(phenoxymethyl) morpholinosulfonyl)-lH-indol-7- ylamino)piperidine-l-carboxylatemethane sulfonate (Compound A).

12. The method according to claim 11, wherein the said compound A is a crystalline form of (S)-ethyl 4-(2-carbamoyl -5-chloro-3-(2-(phenoxymethyl) morpholinosulfonyl)-lH- indol-7-ylamino) piperidine-l-carboxylatemethane sulfonate.

13. The method according to claim 9, wherein the sample is a tumor tissue extracted from the subject having cancer.

14. The method according to claim 13, wherein the tumor tissue is from a tumor selected from: astrocytoma, basal or squamous cell carcinoma, brain cancer, gliobastoma, bladder cancer, breast cancer, colon carcinoma, colorectal cancer, chrondrosarcoma, cervical cancer, adrenal cancer, choriocarcinoma, esophageal cancer, endometrial carcinoma, erythroleukemia, Ewing's sarcoma, gastrointestinal cancer, head and neck cancer, hepatoma, glioma, hepatocellular carcinoma, leukemia, leiomyona, melanoma, non-small cell lung cancer, neural cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, thymona, thyroid cancer, testicular cancer or osteosarcoma.

15. The method according to claim 9, wherein the sample is a surrogate tissue.

16. The method according to claim 15, wherein the surrogate tissue is a blood sample of the subject having cancer.

17. The method according to claim 16, wherein the blood sample comprises serum or plasma.

18. A kit for performing the method as claimed in claim 1 comprising:

(a) an IGF-1R and IR inhibitor;

(b) means for measuring expression level of at least one of the biomarker transcripts selected from the group of Early Growth Response- 1 (EGR-1), Dual Specificity Phosphatase-5 (DUSP-5) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in a sample;

(c) instructions for use of the kit.

19. A kit of claim 18, wherein the means for measuring the expression level of the biomarker transcripts is microarray or RTQ-PCR.