WO2013123411A1 - Methods for diagnosing and treating cancer - Google Patents

Abstract

Methods for the diagnosis and treatment of cancer are provided. In certain aspects, overexpression of KDM2A (also referred to as FBXLl 1 or JHDMIA) is associated with a poor prognosis in certain cancers, such as non-small cell lung cancer (NSCLC). Methods of predicting response to KDM2A inhibitor therapies are also provided. Inhibitors of KDM2A may be used in various aspects to treat a cancer.

Description

DESCRIPTION

METHODS FOR DIAGNOSING AND TREATING CANCER

BACKGROUND OF THE INVENTION

[0001] This application claims the benefit of United States Provisional Patent Application No. 61/600,210, filed February 17, 2012, the entirety of which is incorporated herein by reference.

[0002] This invention was made with government support under 1R01CA157919-01 awarded by the National Institutes of Health and RP 1 10183 awarded by Cancer Prevention and Research Institute of Texas. The government has certain rights in the invention.

1. Field of the Invention

[0003] The present invention relates generally to the fields of molecular biology and medicine. More particularly, it concerns methods of diagnosing and treating cancer. 2. Description of Related Art

[0004] Non-small cell lung cancer (NSCLC) is the most commonly occurring type of lung cancer responsible for 157,300 cancer deaths out of 222,520 cases in 2010 in the United States. Although dys-regulation of epigenetic processes, which are defined as heritable changes in gene expression or cellular phenotypes without alterations in DNA sequences, are a major contributor for the development of many cancer types (1, 2), dys-regulated epigenetic enzymes have not been identified in NSCLC. Of epigenetic signals, histone lysine (K) methylation represents an addition of methyl groups to lysine residues in histones (i.e., the major DNA partner proteins) and has emerged as a hallmark associated with epigenetic regulation of gene expression at the genome-wide levels. In recent breakthrough studies, we and others identified many specific histone lysine demethylases that are capable of "enzymatically removing methyl groups" (demethylating) from methylated lysine residues in histones (3-10). Although it has been increasingly evident that several lysine methylati on- modifying enzymes are linked with oncogenic properties to promote cell growth and cell invasion via alterations in epigenetic landscapes (1 1, 12), no histone lysine demethylase with oncogenic function in NSCLC has been described. In search for specific demethylases linked to the development of NSCLC, we identify the histone lysine demethylase KDM2A as a novel anti-cancer target and diagnostic, both prognostic and predictive, biomarker.

SUMMARY OF THE INVENTION

[0005] The present invention overcomes limitations in the prior art by providing new methods for the diagnosis and treatment of cancer. In certain aspects, overexpression of KDM2A (also referred to as FBXL1 1 or JHDM1A) is associated with a poor prognosis in certain cancers, such as non-small cell lung cancer (NSCLC). Inhibitors of KDM2A may be used in various aspects to treat a cancer. In various embodiments, overexpression of KDM2A may indicate a favorable or therapeutic response of a cancer patient, such as a patient with NSCLC, to therapy with a KDM2A inhibitor anti -cancer therapy.

[0006] In some aspects, the present invention relates to a method of providing a prognosis or prediction for a subject determined to have a cancer, comprising: obtaining expression or genomic amplification information of KDM2A (FBXL1 1) in a cancer sample of a subject by testing said sample, and providing a prognosis or prediction for the subject based on the expression information, wherein, as compared with a reference expression level, increased expression of KDM2A indicates a poor survival, a high risk of recurrence, or a favorable response to a KDM2A inhibitor therapy; and decreased expression of KDM2A indicates a favorable survival, a low risk of recurrence, or an unlikely or low response to a KDM2A inhibitor therapy. In some embodiments, the cancer is not prostate cancer. [0007] An aspect of the present invention relates to a method of providing a prognosis or prediction for a subject determined to have a cancer, comprising: a) obtaining a cancer sample from said subject; b) measuring expression or DNA amplification of KDM2A (FBXL1 1) in said sample via an in vitro test; and c) providing a prognosis or prediction for the subject based on the expression or DNA amplification, wherein a gene copy number of greater than about (2.5, 3, 3.5, 4, 4.5, or 5), an expression level of KDM2A of greater than about (1.5, 2, 2.5, or 3) standard deviations above the mean expression level of KDM2A in a healthy tissue sample of the same tissue type as the cancer sample, or an expression level of KDM2A of greater than the maximal expression level of KDM2A in a healthy tissue sample of the same tissue type as the cancer sample indicates a poor survival, a high risk of recurrence, or a favorable or a high response to a KDM2A inhibitor therapy; and wherein a gene copy number of less than about 2.5, an expression level of KDM2A of less than about 2 standard deviations above the mean expression level of KDM2A in a healthy tissue sample of the same tissue type as the cancer sample, and an expression level of KDM2A of less than the maximal expression level of KDM2A in a healthy tissue sample of the same tissue type as the cancer sample indicates a favorable survival, a low risk of recurrence, or an unlikely or a low response to a KDM2A inhibitor therapy. The in vitro test may be selected from the group consisting of quantitative RT-PCR, immunohistochemistry (IHC), DNA-PCR, and fluorescence in situ hybridization (FISH). The method may further comprise administering a KDM2A (FBXL11) inhibitor to said subject if said measuring detects in said sample a gene copy number of greater than about 2.5, an expression level of KDM2A of greater than about 2 standard deviations above the mean expression level of KDM2A in a healthy tissue sample of the same tissue type as the cancer sample, or an expression level of KDM2A of greater than the maximal expression level of KDM2A in a healthy tissue sample of the same tissue type as the cancer sample.

[0008] The cancer may be a lung cancer, ovarian cancer, breast cancer, pancreatic cancer, multiple myeloma, esophageal cancer, or hepatoma. In some embodiments, the cancer is a lung cancer such as, e.g., non-small cell lung cancer (NSCLC). The sample may comprise a blood sample or a human tumor. The subject may be a human. The sample may comprise a cancer tissue from a cancer biopsy or surgically resected tissue. The method may comprises obtaining or receiving said sample. The sample may be paraffin-embedded and/or frozen. [0009] The measuring may comprise R A quantification. The RNA quantification may comprises cDNA microarray, quantitative RT-PCR, in situ hybridization, Northern blotting, or nuclease protection. Said measuring may comprise protein quantification. The protein quantification may comprise immunohistochemistry, an ELISA, a radioimmunoassay (RIA), an immunoradiometric assay, a fluoroimmunoassay, a chemiluminescent assay, a bioluminescent assay, a gel electrophoresis, or a Western blot analysis. Said measuring may comprise DNA gene copy quantification. The DNA gene quantification may comprise comparative genomic hybridization (CGH), DNA-PCR, or fluorescence in situ hybridization (FISH). In some embodiments, providing the prognosis or prediction comprises generating a classifier based on the expression, wherein the classifier is defined as a weighted sum of expression levels of KDM2A. In some embodiments, providing the prognosis or prediction comprises classifying a group of subjects based on the classifier associated with individual subjects in the group with a reference value. The classifier may be generated on a computer. The classifier may be generated by a computer readable medium comprising machine executable instructions suitable for generating a classifier. In some embodiments, the method further comprises reporting said prognosis or prediction. The method may further comprise prescribing or administering an anti-cancer therapy to said subject based on said prediction. The anticancer therapy may be an adjuvant therapy, a prevention therapy, a neoadjuvant therapy, or a metastatic therapy. The cancer may be a stage I cancer, a stage II cancer, a stage III cancer, or a stage IV cancer.

[0010] Another aspect of the present invention relates to a kit comprising a plurality of antigen-binding fragments that bind to KDM2A (FBXL11) or a plurality of primers or probes that bind to transcripts of KDM2A to assess expression levels, wherein said kit is housed in a container. The kit may comprise an immunoassay. In some embodiments, the immunoassay comprises a lateral flow assay.

[0011] Yet another aspect of the present invention relates to a method for treating a cancer in a subject, comprising: a) selecting a subject predicted to have a favorable or high response to a KDM2A inhibitor therapy in accordance with claim 1; and b) administering to the subject a pharmacologically effective dose of an inhibitor of KDM2A (FBXL11). In some embodiments, the subject is a human. The cancer may be a lung cancer, ovarian cancer, breast cancer, pancreatic cancer, or multiple myeloma. In some embodiments, the cancer is a lung cancer such as, e.g., non-small cell lung cancer (NSCLC). The inhibitor may be an antibody. The antibody may be a human or humanized monoclonal antibody. The antibody may be a monovalent antibody or a multivalent antibody. The antibody may be conjugated to a reporter molecule such as, e.g. , a radioligand or a fluorescent label. The antibody may be an anti-KDM2A scFv, F(ab) or F(ab)2. The inhibitor may be an antisense nucleic acid, a shRNA, a siRNA, or a siNA. In some embodiments, the inhibitor is a small molecule. The administration may be systemic, local, regional, parenteral, intravenous, intraperitoneal, via inhalation, oral, or intra-tumoral injection.

[0012] Another aspect of the present invention relates to a composition comprising a KDM2A inhibitor for use in treating cancer in a patient from whom a cancer sample has been tested by an in vitro test and determined to exhibit a gene copy number of greater than about 2.5, an expression level of KDM2A of greater than about 2 standard deviations above the mean expression level of KDM2A in a healthy tissue sample of the same tissue type as the cancer sample, or an expression level of KDM2A of greater than the maximal expression level of KDM2A in a healthy tissue sample of the same tissue type as the cancer sample. The KDM2A inhibitor may comprise an antibody or an siRNA. In some embodiments, the in vitro test is quantitative RT-PCR, immunohistochemistry (IHC), DNA-PCR, or fluorescence in situ hybridization (FISH). [0013] Yet another aspect of the present invention relates to a method for treating a hyperproliferative disease, comprising administering to a subject a pharmacologically effective dose of an inhibitor of KDM2A (FBXL1 1) to a subject. The hyperproliferative disease may be a cancer such as, e.g., a lung cancer, ovarian cancer, breast cancer, pancreatic cancer, or multiple myeloma. In some embodiments, the cancer is a lung cancer, such as, e.g., NSCLC. The inhibitor may be an antibody. The antibody may be a human or humanized monoclonal antibody. The antibody may be a monovalent or a multivalent antibody. The antibody may be conjugated to a reporter molecule such as, e.g., a radioligand or a fluorescent label. The antibody may be an anti-KDM2A scFv, F(ab) or F(ab)2. The targeting compound may be an antisense nucleic acid, a shRNA, a siRNA, or a siNA. The administration may be systemic, local, regional, parenteral, intravenous, intraperitoneal, oral, via inhalation, or intra-tumoral injection.

[0014] The tissue sample may be collected from a subject with a cancer and, optionally, stored or shipped prior to testing. The collection may comprise surgical resection. The sample of tissue may be stored in RNALater™ or flash frozen, such that RNA may be isolated at a later date. RNA may be isolated from the tissue and used to generate labeled probes for a nucleic acid microarray analysis. The RNA may also be used as a template for qRT-PCR in which the expression of a plurality of biomarkers is analyzed. The expression data generated may be used to derive a score which may predict an individual's response to a KDM2A inhibitor as a cancer therapy or predict an individual's survival from cancer, e.g., using the Kaplan-Meier analysis method. The score may be used to predict whether the subject will be a short-term or a long-term cancer survivor.

[0015] The expression of KDM2A may be measured by a variety of techniques that are well known in the art. Quantifying the levels of the messenger RNA (mRNA) of a biomarker or quantifying the DNA copy numbers of its gene may be used to measure the expression of the biomarker. Alternatively, quantifying the levels of the protein product of a biomarker may be to measure the expression or amplification of the biomarker. Additional information regarding the methods discussed below may be found in Ausubel et al. (2003) or Sambrook et al. (1989). One skilled in the art will know which parameters may be manipulated to optimize detection of the mR A or protein of interest. In some embodiments, determination of KDM2A gene copy amplifications may be used as an alternative to measuring KDM2A expression, e.g., to provide a good or poor prognosis to a patient or predict response to a KDM2A inhibitor.

[0016] A nucleic acid microarray may be used to quantify the differential expression of KDM2A. Microarray analysis may be performed using commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GeneChip® technology (Santa Clara, CA) or the Microarray System from Incyte (Fremont, CA). For example, single- stranded nucleic acids (e.g., cDNAs or oligonucleotides) may be plated, or arrayed, on a microchip substrate. The arrayed sequences are then hybridized with specific nucleic acid probes from the cells of interest. Fluorescently labeled cDNA probes may be generated through incorporation of fluorescently labeled deoxynucleotides by reverse transcription of RNA extracted from the cells of interest. Alternatively, the RNA may be amplified by in vitro transcription and labeled with a marker, such as biotin. The labeled probes are then hybridized to the immobilized nucleic acids on the microchip under highly stringent conditions. After stringent washing to remove the non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. The raw fluorescence intensity data in the hybridization files are generally preprocessed with the robust multichip average (RMA) algorithm to generate expression values.

[0017] Quantitative real-time PCR (qRT-PCR) may also be used to measure the differential expression of KDM2A. In qRT-PCR, the RNA template is generally reverse transcribed into cDNA, which is then amplified via a PCR reaction. The amount of PCR product is followed cycle-by-cycle in real time, which allows for determination of the initial concentrations of mRNA. To measure the amount of PCR product, the reaction may be performed in the presence of a fluorescent dye, such as SYBR Green, which binds to double- stranded DNA. The reaction may also be performed with a fluorescent reporter probe that is specific for the DNA being amplified. [0018] A non-limiting example of a fluorescent reporter probe is a TaqMan® probe (Applied Biosystems, Foster City, CA). The fluorescent reporter probe fluoresces when the quencher is removed during the PCR extension cycle. Multiplex qRT-PCR may be performed by using multiple gene-specific reporter probes, each of which contains a different fluorophore. Fluorescence values are recorded during each cycle and represent the amount of product amplified to that point in the amplification reaction. To minimize errors and reduce any sample-to-sample variation, qRT-PCR may be performed using a reference standard. The ideal reference standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment.

[0019] Suitable reference standards include, but are not limited to, mR As for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and β-actin. The level of mRNA in the original sample or the fold change in expression of each biomarker may be determined using calculations well known in the art.

[0020] Immunohistochemical staining may also be used to measure the differential expression of a plurality of biomarkers. This method enables the localization of a protein in the cells of a tissue section by interaction of the protein with a specific antibody. For this, the tissue may be fixed in formaldehyde or another suitable fixative, embedded in wax or plastic, and cut into thin sections (from about 0.1 mm to several mm thick) using a microtome. Alternatively, the tissue may be frozen and cut into thin sections using a cryostat. The sections of tissue may be arrayed onto and affixed to a solid surface (i.e., a tissue microarray). The sections of tissue are incubated with a primary antibody against the antigen of interest, followed by washes to remove the unbound antibodies. The primary antibody may be coupled to a detection system, or the primary antibody may be detected with a secondary antibody that is coupled to a detection system. The detection system may be a fluorophore or it may be an enzyme, such as horseradish peroxidase or alkaline phosphatase, which can convert a substrate into a colorimetric, fluorescent, or chemiluminescent product. The stained tissue sections are generally scanned under a microscope. Because a sample of tissue from a subject with cancer may be heterogeneous, i.e., some cells may be normal and other cells may be cancerous, the percentage of positively stained cells in the tissue may be determined. This measurement, along with a quantification of the intensity of staining, may be used to generate an expression value for the biomarker.

[0021] An enzyme-linked immunosorbent assay, or ELISA, may be used to measure the differential expression of KDM2A. There are many variations of an ELISA assay. All are based on the immobilization of an antigen or antibody on a solid surface, generally a microtiter plate. The original ELISA method comprises preparing a sample containing the biomarker proteins of interest, coating the wells of a microtiter plate with the sample, incubating each well with a primary antibody that recognizes a specific antigen, washing away the unbound antibody, and then detecting the antibody-antigen complexes. The antibody-antibody complexes may be detected directly. For this, the primary antibodies are conjugated to a detection system, such as an enzyme that produces a detectable product. The antibody-antibody complexes may be detected indirectly. For this, the primary antibody is detected by a secondary antibody that is conjugated to a detection system, as described above. The microtiter plate is then scanned and the raw intensity data may be converted into expression values using means known in the art. [0022] An antibody microarray may also be used to measure the differential expression of KDM2A. For this, a plurality of antibodies is arrayed and covalently attached to the surface of the microarray or biochip. A protein extract containing the biomarker proteins of interest is generally labeled with a fluorescent dye.

[0023] The labeled biomarker proteins are incubated with the antibody microarray. After washes to remove the unbound proteins, the microarray is scanned. The raw fluorescent intensity data may be converted into expression values using means known in the art.

[0024] Luminex multiplexing microspheres may also be used to measure the differential expression of KDM2A. These microscopic polystyrene beads are internally color-coded with fluorescent dyes, such that each bead has a unique spectral signature (of which there are up to 100). Beads with the same signature are tagged with a specific oligonucleotide or specific antibody that will bind the target of interest (i.e., biomarker mRNA or protein, respectively). The target, in turn, is also tagged with a fluorescent reporter. Hence, there are two sources of color, one from the bead and the other from the reporter molecule on the target. The beads are then incubated with the sample containing the targets, of which up 100 may be detected in one well. The small size/surface area of the beads and the three dimensional exposure of the beads to the targets allows for nearly solution-phase kinetics during the binding reaction. The captured targets are detected by high-tech fluidics based upon flow cytometry in which lasers excite the internal dyes that identify each bead and also any reporter dye captured during the assay. The data from the acquisition files may be converted into expression values using means known in the art. [0025] In situ hybridization may also be used to measure the differential expression of KDM2A. This method permits the localization of mRNAs of interest in the cells of a tissue section. For this method, the tissue may be frozen, or fixed and embedded, and then cut into thin sections, which are arrayed and affixed on a solid surface. The tissue sections are incubated with a labeled antisense probe that will hybridize with an mRNA of interest. The hybridization and washing steps are generally performed under highly stringent conditions. The probe may be labeled with a fluorophore or a small tag (such as biotin or digoxigenin) that may be detected by another protein or antibody, such that the labeled hybrid may be detected and visualized under a microscope. Multiple mRNAs may be detected simultaneously, provided each antisense probe has a distinguishable label. The hybridized tissue array is generally scanned under a microscope. Because a sample of tissue from a subject with cancer may be heterogeneous, i.e., some cells may be normal and other cells may be cancerous, the percentage of positively stained cells in the tissue may be determined. This measurement, along with a quantification of the intensity of staining, may be used to generate an expression value for each biomarker.

[0026] As used herein, "obtaining a biological sample" or "obtaining a blood sample" refer to receiving a biological or blood sample, e.g., either directly or indirectly. For example, in some embodiments, the biological sample (e.g., comprising cancer tissue surgically resected from a patient or obtained from a cancer biopsy) is directly obtained from a subject at or near the laboratory or location where the biological sample will be analyzed. In other embodiments, the biological sample may be drawn or taken by a third party and then transferred, e.g., to a separate entity or location for analysis. In other embodiments, the sample may be obtained and tested in the same location using a point-of care test. In these embodiments, said obtaining refers to receiving the sample, e.g., from the patient, from a laboratory, from a doctor's office, from the mail, courier, or post office, etc. In some further aspects, the method may further comprise reporting the determination to the subject, a health care payer, an attending clinician, a pharmacist, a pharmacy benefits manager, or any person that the determination may be of interest.

[0027] "Patient response" can be assessed using any endpoint indicating a benefit to the patient, including, without limitation, (1) inhibition, to some extent, of disease progression, including slowing down and complete arrest; (2) reduction in the number of disease episodes and/or symptoms; (3) reduction in lesional size; (4) inhibition (i.e., reduction, slowing down or complete stopping) of disease cell infiltration into adjacent peripheral organs and/or tissues; (5) inhibition (i.e., reduction, slowing down or complete stopping) of disease spread; (6) relief, to some extent, of one or more symptoms associated with the disorder; (7) increase in the length of disease-free presentation following treatment; and/or (8) decreased mortality at a given point of time following treatment.

[0028] "Prognosis" refers to as a prediction of how a patient will progress, and whether there is a chance of recovery. "Cancer prognosis" generally refers to a forecast or prediction of the probable course or outcome of the cancer. As used herein, cancer prognosis includes the forecast or prediction of any one or more of the following: duration of survival of a patient susceptible to or diagnosed with a cancer, duration of recurrence-free survival, duration of progression free survival of a patient susceptible to or diagnosed with a cancer, response rate in a group of patients susceptible to or diagnosed with a cancer, duration of response in a patient or a group of patients susceptible to or diagnosed with a cancer, and/or likelihood of metastasis in a patient susceptible to or diagnosed with a cancer. Prognosis also includes prediction of favorable responses to cancer treatments, such as a conventional cancer therapy or response to a KDM2A inhibitor.

[0029] The invention is based, in part, on the discovery that KDM2A can be a prognostic biomarker or a predictive biomarker in cancers. A prognostic diagnostic biomarker can be used predicts patient outcome independent of treatment. A predictive diagnostic biomarker can be used to predict outcome of treatment with a specific agent or modality. For example Her2 overexpression is a negative (poor) prognostic biomarker for breast cancer, but a positive predictive biomarker for Her-2 targeted therapies, e.g., Her2 overexpression or gene amplification predicts good response to Herceptin.

[0030] By "subject" or "patient" is meant any single subject for which therapy is desired, including humans, cattle, dogs, guinea pigs, rabbits, chickens, and so on. Also intended to be included as a subject are any subjects involved in clinical research trials not showing any clinical sign of disease, or subjects involved in epidemiological studies, or subjects used as controls.

[0031] As used herein, "increased expression" refers to an elevated or increased level of expression in a cancer sample relative to a suitable control (e.g., a non-cancerous tissue or cell sample, or a reference standard), wherein the elevation or increase in the level of gene expression is statistically-significant (p<0.05). Whether an increase in the expression of a gene in a cancer sample relative to a control is statistically significant can be determined using an appropriate t-test (e.g., one-sample t-test, two-sample t-test, Welch's t-test) or other statistical test known to those of skill in the art. Genes that are overexpressed in a cancer can be, for example, genes that are known, or have been previously determined, to be overexpressed in a cancer.

[0032] As used herein, "decreased expression" refers to a reduced or decreased level of expression in a cancer sample relative to a suitable control (e.g., a non-cancerous tissue, cell sample, cancer sample with low expression, a reference standard), wherein the reduction or decrease in the level of gene expression is statistically-significant (p<0.05). In some embodiments, the reduced or decreased level of gene expression can be a complete absence of gene expression, or an expression level of zero. Whether a decrease in the expression of a gene in a cancer sample relative to a control is statistically significant can be determined using an appropriate t-test (e.g., one-sample t-test, two-sample t-test, Welch's t-test) or other statistical test known to those of skill in the art. Genes that are underexpressed in a cancer can be, for example, genes that are known, or have been previously determined, to be underexpressed in a cancer.

[0033] In a further embodiment, the marker level may be compared to the level of the marker from a control, wherein the control may comprise one or more tumor samples (e.g., colon cancer samples) taken from one or more patients determined as having a good prognosis ("good prognosis" control) or a poor prognosis ("poor prognosis" control), or both.

[0034] The control may comprise data obtained at the same time (e.g., in the same hybridization experiment) as the patient's individual data, or may be a stored value or set of values, e.g. stored on a computer, or on computer-readable media. If the latter is used, new patient data for the selected marker(s), obtained from initial or follow-up samples, can be compared to the stored data for the same marker(s) without the need for additional control experiments.

[0035] A good or bad prognosis may, for example, be assessed in terms of patient survival, likelihood of disease recurrence or disease metastasis (patient survival, disease recurrence and metastasis may for example be assessed in relation to a defined timepoint, e.g. at a given number of years after cancer surgery (e.g. surgery to remove one or more tumors) or after initial diagnosis. In one embodiment, a good or bad prognosis may be assessed in terms of overall survival or disease free survival.

[0036] For example, "good prognosis" may refer to the likelihood that a patient afflicted with cancer will remain disease-free (e.g., cancer-free) or survive despite the presence of the cancer. "Poor prognosis" may be used to mean the likelihood of a relapse or recurrence of the underlying cancer or tumor, metastasis, or death. Cancer patients classified as having a "good prognosis" may remain free of the underlying cancer or tumor or survive despite the presence of cancer or tumor. For example, cancerous cells and/or tumors from a cancer may continue to exist in a patient with a good prognosis, but the patient's immune system may slow or prevent the progression or growth of the cancer, thus allowing the patient to continue to survive. In contrast, "bad prognosis" cancer patients experience disease relapse, tumor recurrence, metastasis, and death. In particular embodiments, the time frame for assessing prognosis and outcome is, for example, less than one year, one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, or more years. In certain aspects, the relevant time for assessing prognosis or disease-free survival time may begin at the time of the surgical removal of the tumor or suppression, mitigation, or inhibition of tumor growth. A "good prognosis" refers to the likelihood that a cancer patient will survive for a period of at least five, such as for a period of at least ten years. In further aspects of the invention, a "poor prognosis" refers to the likelihood that a cancer patient, such as a melanoma patient, will experience disease relapse, tumor recurrence, metastasis, or death within less than ten years, such as less than five years. Time frames for assessing prognosis and outcome provided herein are illustrative and are not intended to be limiting.

[0037] The term "high risk" means the patient is expected to have a distant relapse in a shorter period less than a predetermined value (for example, from a control), for example in less than 5 years, preferably in less than 3 years. The term "low risk" means the patient is expected to have a distant relapse in a longer period greater than a predetermined value, for example, after 5 years, preferably in more than ten years. Time frames for assessing risks provided herein are illustrative and are not intended to be limiting.

[0038] The term "antigen binding fragment" herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments. Multivalent antibodies, such as monospecific bivalent or tetravalent antibodies, are known in the art (e.g., Cuesta et al. Trends Biotechnol. 28(7):355-62, 2010). Antibodies are preferably humanized when administered to a human subject.

[0039] The term "primer," as used herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Primers may be oligonucleotides from ten to twenty and/or thirty base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded and/or single-stranded form, although the single-stranded form is preferred.

[0040] Embodiments discussed in the context of methods and/or compositions of the invention may be employed with respect to any other method or composition described herein. Thus, an embodiment pertaining to one method or composition may be applied to other methods and compositions of the invention as well.

[0041] As used herein the terms "encode" or "encoding" with reference to a nucleic acid are used to make the invention readily understandable by the skilled artisan; however, these terms may be used interchangeably with "comprise" or "comprising" respectively. [0042] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one.

[0043] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more.

[0044] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. [0045] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0047] FIGS. 1A-F: KDM2A is frequently overexpressed in NSCLC cell lines and patients. FIG 1A & FIG. IB, KDM2A mRNA levels were higher in most NSCLC cell lines than in two normal lung cell lines. FIG. 1C & FIG. ID, KDM2A mRNA levels were up- regulated in an NSCLC patient set from UT MDACC (referred as to "UT MDACC set') as compared to 40 normal lung tissues. KDM2A levels were higher in 14% NSCLC patients (N=14/98 patients) as compared to the highest normal value. FIG. IE & FIG. IF, KDM2A protein levels were statistically higher in NSCLC tumors than in normal tissues. High KDM2A levels were present in 40% of tumor samples as compared to 12% of normal tissues

[0048] FIGS. 2A-K: High KDM2A levels correlate with poor patient survival in three independent patient sets. FIG. 2A & FIG. 2B, KDM2A mRNA expression levels are associated with poor survival in the MDACC patient set. FIG. 2C, Clinical characteristics of the UT MDACC patient set (N=98). FIG. 2D & FIG. 2E, In the publicly available NCI Director's Challenge Consortium patient set (444 lung adenocarcinoma samples), high KDM2A mRNA levels are associated with poor survival. FIG. 2F, Clinical characteristics of NSCLC tumors in the NCI Director's Challenge Consortium patient set FIG. 2G & FIG. 2H, In the NCI Director's Challenge Consortium patient set, other histone demethylases, such as KDM4A and J ARID ID, did not correlate with patient survival FIG. 21 & FIG. 2J, KDM2A protein levels measured by IHC correlate with poor overall survival in the NSCLC IHC set (N=79). FIG. 2K, Clinical characteristics of squamous cell carcinoma (SCC) in the NSCLC IHC set.

[0049] FIGS. 3A-C: In the NCI Director's Challenge Consortium patient set, high ALK levels are associated with poor prognosis and are mutually exclusive with high KDM2A levels. (FIG. 3A & FIG. 3B) In the NCI Director's Challenge Consortium patient set, high ALK mRNA levels were associated with poor prognosis of overall survival (FIG. 3B) but not progression free survival (FIG. 3A). FIG. 3C, In the NCI Directors Challenge Consortium patient set, high expression levels of ALK are mutually exclusive with high expression levels of KDM2A.

[0050] FIGS. 4A-F: The KDM2A gene is amplified in KDM2A-overexpressing NSCLC cell lines and in a subset of NSCLC and other cancer patients. A-D) The KDM2A gene is amplified in three KDM2A-overexpressing NSCLC cell lines (HI 792, HI 975 & H23) but not in two cell lines with low KDM2A levels (H460 & H2122). E & F) The KDM2A (FBXL11) gene appears to be significantly amplified in a subset of NSCLC (10-27%), breast cancer (19- 33%), esophageal cancer (43-47%), hepatoma patients (6-10%). [0051] FIGS. 5A-N: KDM2A knockdown in KDM2A-overexpressing NSCLC cell lines but not in cell lines with low KDM2A levels suppresses cell growth and colony formation in vitro. FIGS. 5A-D, siRNA mediated knockdown of KDM2A in H1975 cells suppressed cell proliferation. FIGS. 5E-J, siRNA mediated knockdown of KDM2A in HI 792 and H23 cells inhibited cell proliferation. FIG. 5K & FIG. 5L, siRNA-mediated knockdown of KDM2A in cell lines with low KDM2A levels (H460 and H2122) did not inhibit cell proliferation. FIG. 5M & FIG. 5N, KDM2A knockdown inhibited colony formation of H1975 and H1792 cells.

[0052] FIGS. 6A-F: KDM2A knockdown in KDM2A-overexpressing NSCLC cell lines completely inhibits cell migration and invasion in vitro. FIG. 6A & FIG. 6B, siRNA- mediated knockdown of KDM2A in HI 792 and HI 975 cells remarkably impeded cell invasion. FIGS. 6C - F, siRNA mediated knockdown of KDM2A suppressed cell migration of HI 792 and HI 975 cells but not control cell lines with low KDM2A levels (H460 and H2122).

[0053] FIGS. 7A-D: Ectopic expression of KDM2A in KDM2A-depleted cells rescues defects in cell growth and invasion by KDM2A knockdown. FIG. 7A & FIG. 7B, Ectopic expression of KDM2A but not its catalytic mutant (mKDM2A) rescued defective proliferation of KDM2A-depleted H1792 (FIG. 7A) and H1975 (FIG. 7B) cells. FIG. 7C & FIG. 7D, Ectopic expression of KDM2A but not its catalytic mutant mKDM2A restored defective invasion of KDM2A-depleted H1792 (FIG. 7C) and H1975 (FIG. 7D) cells. For these rescue experiments, KDM2A knockdown efficiency was reduced.

[0054] FIGS. 8A-B: Venn diagrams and heat maps of KDM2A-regulated genes. FIG. 8A, A partial list of genes, which are consistently 1.5 fold up-regulated by KDM2A knockdown, are presented. The order of genes in the list does not necessarily reflect the importance of KDM2A-regulated genes, because it is based simply on the mean of fold changes in expression that were induced by two siKDM2As (siKDM2A-3 and -4) in HI 792 and HI 975 cells. Cells were treated with siControl RNA or two different siKDM2As and harvested 48 h later. The mRNA levels in KDM2A knockdown cells were measured by Affymetrix U133P and compared with those in siControl-treated cells. FIG. 8B, A partial list of genes, which are consistently 2 fold down-regulated by KDM2A knockdown, are presented. [0055] FIGS. 9A-D: KDM2A down-regulates expression of DUSP3 and HDAC3 while indirectly activating the cell cycle gene NEK7 and the cell invasion-associated gene NANOS 1 (FIG. 9A and FIG. 9B). Expression levels of HDAC3, DUSP3, GPR157, TMEM65, TIMM17, NANOS 1 and NEK7 in H1975 (FIG. 9A) and H1792 (FIG. 9B) cells after KDM2A knockdown were analyzed by quantitative RT-PCR. (C and D) KDM2A was localized at HDAC3, DUSP3, GPR157, TMEM65, and TIMM17 genes in H1792 (FIG. 9C) and H1975 (FIG. 9D) cells. Chromatin levels of KDM2A were analyzed by qChlP. Data are presented as the mean ± SEM (error bars).

[0056] FIGS. 10A-F: KDM2A represses DUSP3 expression and demethylates H3K36me2 at the DUPS3 gene promoter. FIG. 10A, Western blot analysis showed that KDM2A knockdown increased DUSP3 protein levels. FIG. 10B and FIG. IOC, DUSP3 mRNA levels in KDM2A-depleted HI 792 (FIG. 10B) and HI 975 (FIG. IOC) cells were repressed by ectopic expression of wild type KDM2A but not its catalytic mutant mKDM2A. FIG. 10D, Schematic representation of the DUSP3 gene. Arrows indicate the PCR-amplified region, and TSS denotes transcription start site. FIG. 10E and FIG. 10F, Chromatin levels of KDM2A, H3K36me2, H3K9me3 and H3 at the DUSP3 gene were compared between control and siKDM2A-treated cells by qChlP. H1792 (FIG. 10E) and H1975 (FIG. 10F) cells were used, and anti-H3 was used as a ChIP control. Data are presented as the mean ± SEM (error bars).

[0057] FIGS. 11A-J: KDM2A-mediated repression of DUSP3 inhibits DUSP3 -catalyzed dephosphorylation of ERKl/2 and JNKl/2 to promote the growth and invasiveness of NSCLC cells. FIG. 11A, KDM2A knockdown decreased phosphorylation levels of ERKl/2 and JNKl/2. H1792 cells treated with siControl or siKDM2As were examined by Western blot analysis. FIG. 11B, KDM2A knockdown did not have any effect on phosphorylation levels of EGFR during serum activation. KDM2A-depleted HI 792 cells and control cells were stimulated with 10% serum for 5, 15 or 30 min after 18 h of serum starvation. Subsequently, protein extracts were examined by Western blot analysis. FIG. 11C, Ectopic expression of DUSP3 decreased phosphorylation levels of ERKl/2 and JNKl/2. H1792 cells transfected with GFP or DUSP3 were examined by Western blot analysis. FIG. 11D, KDM2A knockdown decreased phosphorylation levels of ERKl/2 and JNKl/2 during serum activation. FIG. HE, Double knockdown of KDM2A and DUSP3 restored phosphorylation levels of ERKl/2 and JNKl/2 in KDM2A-depleted cells during serum activation. KDM2A- depleted and DUSP3/KDM2A-depleted H1792 cells were stimulated by serum. FIG. 11F, DUSP3 knockdown rescued the growth defect of KDM2A-depleted H1792 cells. FIG. 11G, DUSP3 knockdown revived the invasive defect of KDM2A-depleted H1792 cells. The siControl-treated cells were used as controls. FIG. 11H & FIG. HI, DUSP3 was localized in cytosol in H1975 (FIG. 11H) and H1792 (FIG. HI) cells. Cytoplasmic and nuclear fractions of HI 975 and HI 792 cells were examined by Western blot analysis. p84 and β-actin were used as a nuclear marker and a loading control, respectively. WCL, whole cell lysates. FIG. 11J, There is an inverse association between DUSP3 and KDM2A mRNA levels in the publicly available NCI Director's Challenge Consortium patient set (444 lung adenocarcinoma samples). [0058] FIGS. 12A-E: HDAC3 directly down-regulates NEK7 and NANOS1. (FIG. 12A and FIG. 12B) HDAC3 was recruited to the NANOS1 and NEK7 (but not GPR157) gene in H1975 (FIG. 12A) and H1792 (FIG. 12B) cells. Chromatin levels of HDAC3 were measured by qChlP. (FIG. 12C) siRNA-mediated HDAC3 knockdown efficacy was analyzed in KDM2A-overexpressing cell lines HI 975 and HI 792 by quantitative RT-PCR. FIGS. 12D- E, HDAC3 knockdown increased expression levels of NANOS1 and NEK7 in HI 975 (FIG. 12D) and H1792 (FIG. 12E) cells. The mRNA levels of individual genes were quantified by qRT-PCR.

[0059] FIGS. 13A-E: KDM2A-mediated repression of HDAC3 contributes to cell growth and invasion. FIGS. 13A-B, HDAC3 mRNA levels were analyzed in KDM2A-depleted HI 975 (FIG. 13A) and HI 792 (FIG. 13B) cells after ectopic expression of GFP, wild type KDM2A, and its catalytic mutant mKDM2A. FIGS. 13C-D, HDAC3 knockdown rescued the growth defect of KDM2A knockdown cells in H1975 (FIG. 13C) and H1792 (FIG. 13D) cells. FIG. 13E, HDAC3 knockdown restored the invasive defect of KDM2A knockdown HI 975 cells. The siControl-treated cells were used as controls. Data are presented as the mean ± SEM (error bars) of three independent experiments.

[0060] FIGS. 14A-E: KDM2A is required for in vivo growth and invasion of NSCLC cells. (FIGS. 14A-C) KDM2A knockdown completely inhibited lung tumor colonization. HI 792 cells treated with siControl RNA or two siRNAs against KDM2A were injected into mice via tail veins, and lung tumor formation was monitored (FIG. 14A). Representative pictures for normal lung and tumor lesion (white arrow) were shown in (FIG. 14B), and lung metastatic lesions were analyzed by H&E staining (FIG. 14C). FIGS. 14D-E, KDM2A knockdown abrogated tumor formation and metastasis of NSCLC cells in an orthotopic lung model. HI 792 cells treated with siControl or siKDM2A-#3 were implanted into the parenchyma of left sided lung, and the contralateral lungs and lymph nodes were monitored for metastasis (FIG. 14D). Representative pictures for normal lung and tumor lesion (black arrows) were shown in (FIG. 14E), and lung lesions were analyzed by H&E staining (FIG. 14E).

[0061] FIGS. 15A-B: KDM2A mRNA levels are up-regulated in subsets of pancreatic (FIG. 15A) and breast (FIG. 15B) tumors, whereas low levels of KDM2A are observed in normal counterpart tissues. Gene expression data were from Oncomine database.

[0062] FIG. 16: Identification of KDM2A-interacting proteins (ΗΡΙγ and SKPlb) by anti-FLAG affinity purification and mass spectrometric analysis.

[0063] FIG. 17: A detailed view (A) and a simplified representation (B) for the regulatory pathways underlying KDM2A-controlled cell growth and invasion. KDM2A up-regulates two phosphorylated MAPKs (phospho-ERK/2 and phospho-JNKl/2) by directly repressing expression of DUSP3. DUPS3 dephosphorylates and inactivates ERK1/2 and JNK1/2 that stimulate cell growth and invasiveness (KDM2A -| DUSP3 -| MAPKs). In addition, KDM2A transcriptionally inhibits HDAC3 expression to up-regulate HDAC3 -repressed genes, such as the cell cycle gene NEK7 and cell invasion-associated gene NANOS1 (KDM2A -| HDAC3 -| NEK7/NANOSl). The down-regulation of DUSP3 and HDAC3 by KDM2A merges to promotes the growth and invasion of NSCLC cells. TRKs, Tyrosine receptor kinases; me, methyl group; p: phosphorylated DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Non-small cell lung cancer (NSCLC) represents approximately 80% of lung cancer that causes more cancer related deaths (157,300 deaths in 2010) in the United States than any other malignancy. However, prognostic biomarkers and anti-cancer targets for NSCLC are rare. Here, we show that the histone lysine demethylase KDM2A (also known as FBXL11/JHDM1), an epigenetic enzyme, is overexpressed in significant portions of non- small cell lung cancer (NSCLC) patients. Specifically, quantitative analysis of mRNA levels indicate that KDM2A is up-regulated in about 14% of NSCLC patients from UTMDACC (N=14/98), and immunohistochemical analysis showed that KDM2A protein levels are increased in up to 40% of another independent group of NSCLC patients. Notably, patient outcome analysis in three independent groups of NSCLC patients demonstrate that NSCLC patients (Stages I, II and III) with high KDM2A levels (at both mRNA and protein levels) are significantly associated with poor overall survival as compared with those with low KDM2A expression, indicating that KDM2A is a novel negative prognostic biomarker for survival of NSCLC patients. Importantly, KDM2A knockdown in three representative KDM2A-overexpressing NSCLC cell lines but not in two cell lines with low KDM2A levels significantly suppresses cell proliferation and markedly inhibits cell migration and invasion in vitro. Because such drastic inhibition by KDM2A knockdown were exclusive to KDM2A- overexpressing NSCLC cell lines, KDM2A is likely an important predictive diagnostic biomarker to predict treatment response to KDM2A directed anti-cancer therapies. Subsequently, these in vitro migration and invasion results were confirmed by mouse xenograft experiments showing that KDM2A knockdown remarkably inhibited lung tumor formation and metastasis of KDM2A-overexpressing NSCLC cells in vivo. Interestingly, the KDM2A gene undergoes amplification in three KDM2A-overexpressing NSCLC cell lines but not in two cell lines with low KDM2A levels and appears to be significantly amplified in NSCLC patients. Furthermore, ectopic expression of KDM2A but not its catalytic mutant (H212A) significantly rescues defective proliferation and invasion of KDM2A-depleted cells and restores expression of KDM2A-regulated genes. These findings provide direct evidence that changes in cellular characteristics and gene expression by KDM2A knockdown are largely dependent on its catalytic activity and therefore define KDM2A as a new druggable anti-cancer target. Mechanistically, the dual-specificity phosphatase DUSP3 gene is defined as a key target of KDM2A, and DUSP3 is found to dephosphorylate ERKl/2 and JNKl/2 in NSCLC cells. KDM2A activates ERKl/2 and JNKl/2 via the transcriptional repression of DUSP3 to promote the growth and invasiveness of NSCLC cells. Additional results indicate that the transcriptional down-regulation of HDAC3 expression by KDM2A antagonizes HDAC3 -mediated repression of cell cycle- and invasiveness-associated gene to promote the proliferation and invasion of NSCLC cells. Finally, our analysis of KDM2A expression profiles in other cancer types suggests that KDM2A is overexpressed in significant subsets of pancreatic and breast cancer patients, opening the possibility that KDM2A may have a similar clinical value in these cancer types. Taken together, our findings identify KDM2A as a novel prognostic biomarker for poor outcome in NSCLC patients as well as a new predictive biomarker and also establish KDM2A as a new anti-cancer target for the treatment of KDM2A-overexpressing NSCLC patients.

As an epigenetic enzyme, KDM2A is overexpressed in a significant portion of patients, i.e., up to 40% of NSCLC patients at the protein level. In addition, high KDM2A levels are associated with poor prognosis of NSCLC patients. These findings clearly define the clinical importance of KDM2A overexpression. Because KDM2A regulates a list of genes, such as DUSP3 and HDAC3, in NSCLC cells by likely altering the epigenetic landscape of methylation status at histone H3 lysine 36, KDM2A-targeted therapy would be categorized into a novel class of anti-cancer epigenetic approaches. This epigenetic targeting strategy is promising because other types of epigenetic strategies, such as targeting DNA methylation, have been successful in the clinic and have been approved by the FDA for certain types of cancer (e.g. leukemia). Importantly, knockdown of KDM2A drastically inhibits cell growth and invasion of the model NSCLC cells in vitro and in vivo, and these inhibitory effects are specific to KDM2A-overexpressing cells. Therefore, KDM2A expression can be a promising diagnostic biomarker for predicting response of anti-KDM2A therapies. EGFR overexpression and mutations occur in 50% and about 10% of NSCLC patients, respectively. A number of FDA-approved strategies involving small-molecule inhibitors (e.g., gefitinib and erlotinib) and large molecules (e.g. cetuximab) were developed to target EGFR for NSCLC patients. However, most of NSCLC patients, who receive targeted therapies, eventually show drug resistance (13, 14). For this reason, the identification of new drug targets may provide a better therapeutic strategy for NSCLC patients. It has been known that genetic aberrations of EGFR and KRAS in NSCLC frequently up-regulate activities of their downstream effectors, such as ERK1/2 and JNK1/2 (13, 14). Importantly, our findings indicate that KDM2A overexpression activates the downstream effectors (i.e., ERK1/2 and J K1/2) via epigenetic repression of DUSP3. Thus, these findings may not only justify KDM2A-targeted therapy for KDM2A-overexpressing NSCLC patients but also offer new strategy to target the undruggable RAS (KDM2A ultimately activates ERK1/2 and JNK1/2 downstream of RAS). Notably, expression status of KDM2A is mutually exclusive with that of the kinase ALK. Therefore, KDM2A-targeted therapy may provide a unique, distinct and first-in-class strategy to treat cancer patients and will also enable combination therapies (e.g., combinations of KDM2A inhibitors, EGFR targeting molecules and classic chemotherapy) that should not be cross-resistant. [0064] In summary, data provided herein identifies KDM2A as a druggable anti-cancer target in a prevalent cancer population with few therapy options and establishes KDM2A as a novel prognostic biomarker for poor outcome in NSCLC patients and a predictive biomarker for response to KDM2A directed anti-cancer therapies. These findings provide support for the development of KDM2A inhibitors. The mutual exclusiveness of expression status between KDM2A and the kinase ALK makes KDM2A a strongly attractive anti-cancer drug target.

I. EXAMPLES

[0065] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1

Materials and Methods

[0066] Cell culture reagents were purchased from Gibco (Invitrogen/Gibco, Carlsbad, CA, USA); all other chemicals were from Sigma Aldrich (St Louis, MO, USA). NSCLC and adjacent tissue mRNA samples were kind gifts from Dr. Xifeng Wu (UTMDACC). Formalin fixed paraffin embedded samples for IHC were obtained from Biomax and Imgenex and processed as described in the manufacturers' instructions. All NSCLC cell lines were purchased from ATCC (Rockville, MD, USA) or kind gifts from Dr. Heymach (UTMDACC). The KDM2A specific antibodies were purchased from Novus (NB 100-74602 for WB) and Abgent (API 043c for IHC). Microarray hybridization and data analysis

[0067] Total cellular RNA were prepared, and labeled cRNAs were hybridized to oligonucleotide microarrays consisting of 54,613 gene probes (U133P GeneChip; Affymetrix). Probe set 208988_at was used to examine mRNA expression of KDM2A.

[0068] For analysis of genes regulated by KDM2A target, two cell lines (HI 792 and H1975) were treated with two different siKDM2A (siKDM2A-3 and -4), and harvested 48 h later. Expression levels of KDM2A knockdown cells were measured by Affymetrix U133P and compared with those of siControl-treated cells.

[0069] The NCI Director's Challenge Consortium data set represents an Affymetrix U133A microarray data set of tumor samples from early stages of 444 lung NSCLC adenocarcinoma patients with health outcomes. Patient samples for this group were collected at the University of Michigan, H. Lee Moffitt Cancer Center, Memorial Sloan-Kettering Cancer Center, Dana-Farber Cancer Institute, and Ontario Cancer Institute (15).

Quantitative RT-PCR

[0070] For quantitative RT-PCR, 103 NSCLC and 40 normal lung samples were collected, and verified by histological examination. NSCLC tumors include adenocarcinoma, squamous cell carcinoma to large cell carcinoma and ranged from clinical stages I, II to IIA. mRNA was isolated using the RNeasy kit (QIAGEN). Single-stranded cDNA was synthesized using the iScript™ cDNA Synthesis Kit (Bio-rad). Quantitative PCR was performed in triplicates using SYBR-green, ABI reagents and ABI real time PCR equipment (StepOnePlus). Gene-specific primers for KDM2A and actin (control gene) were designed for both cDNA and DNA evaluations. Fold mRNA overexpression or DNA amplification was calculated according to relative quantification protocols. Immunohistochemistry (IHC)

[0071] The avidin-biotin immunoperoxidase method was performed on de-paraffinized zinc formalin-fixed, paraffin-embedded sections, which were purchased from Biomax and Imgenex. Slides placed in citrate buffer were heated with a microwave for 20 min prior to the application of the anti-KDM2A antibody for 1 h at room temperature. This work was performed in collaboration with Dr. Mien-Chie Hung and Dr. Yong-Kun Wei.

Statistical analysis for patient survival

[0072] KDM2A high and low expression was correlated with recurrence and overall survival using the Kaplan-Meier method. Significance was tested by the log-rank (Mantel- Cox) method unless indicated, and p values less than 0.05 were considered statistically significant. GraphPad Prism software was used for all statistical analysis.

Analysis of gene copy number

[0073] Real-time quantitative PCR was performed using ABI real time PCR equipment (StepOnePlus) and ABI TaqMan copy number assays detecting emitted fluorescence as FAM is released from the probe during amplification. For each test sample, triplicate wells were set up for KDM2A, Cyclin Dl and a reference control gene RNAseP, which is on a different chromosome.

Cell proliferation

[0074] Cell viability was assessed by the MTS assay (Promega). Cells were plated at a density of 1500 to 2000 cells per well in 96 well plates, treated with control siRNA or siKDM2A at a final concentration of 20 -100 nM. siRNA design and transfection protocol

[0075] siRNAs against KDM2A were purchased from Dharmacon and Sigma. The selected sequences were: siKDM2A-3 5' AA-caaggagagugugguguuu-dTdT 3' and siKDM2A- 4 5' AA-uuacgaagccucacacuau-dTdT 3'. siRNAs were transfected using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) at the following concentrations: 2.5 x 10⁴ cells/ml in a six-well plate format with a final volume of 2 ml, final siRNA concentration of 20 nM and 5 μΐ LF per well. Following 72 to 96h incubation, cells were harvested for mRNA and protein analysis. siR A duplexes against Luciferase GL3 R A (5' AA- CTTACGCTGAGTACTTCGA-dTdT 3') or an FITC-conjugated siRNA from Dharmacon were used as controls.

Migration and invasion assays [0076] To examine in vitro migratory abilities of NSCLC cells, cell migration assay was carried out using 24-well plates with membrane filters inserts with pore sizes of 8 um. In brief, 1.0 x 10⁵ cells were seeded on the insert membrane in 500 μΐ serum free medium in 24- well plates. 24h later, cells that moved to the other side of the membrane were stained and counted. For the cell invasion assay, the Boyden chamber assay with a modification was performed. Cells were seeded on the matrigel-coated membrane in the inserts, and cells that invaded the matrigel and migrated to the other side of the membrane were stained and counted.

Expression constructs and transfection protocol

[0077] We generated cDNA constructs encoding KDM2A and its catalytic mutant mKDM2A using standard cloning methodology. Eukaryotic expression plasmids (2 μg) were transfected into 3 x 10⁵ cells in a six-well plate using 5 μΐ of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

Quantitative ChIP assay

[0078] ChIP assay was performed as previously described (16). DNA was purified from chromatin fragments immunoprecipitated by antibodies and methyl marks, amplified by quantitative PCR using specific primer sets for individual genes, normalized to input, and calculated as % of input. Relative occupancy indicates the fold change in % of input over the control (e.g., anti-KDM2A /IgG).

Mice and xenograft studies [0079] Female athymic nu/nu mice (Charles River Laboratory) were used. Mice were injected via tail veins or into lung with HI 792 cells (1 x 10⁶) that were ex vivo treated with control siRNA or two siKDM2As for 72h. For each group, 3-6 mice were used as follows: siControl (n=4), siKDM2A-3 (n=3) and siKDM2A-4 (n=3) for tail vein injection; siControl (n=6) and siKDM2A-3 (n=6) for lung orthotopic injection. Four to thirteen weeks later, mice were sacrificed and the lungs were examined for lung metastasis, which were confirmed by histology.

Affinity purification

[0080] Affinity chromatography purification using anti-FLAG resin was carried out as previously described (17). In brief, KDM2A-containing complex was isolated from 100- 200 mg of nuclear extracts isolated from a 293 cell line expressing FLAG-tagged KDM2A using anti-Flag M2 affinity resin. KDM2A-interacting proteins were silver-stained and identified by liquid chromatography-tandem mass spectroscopic analysis.

EXAMPLE 2

The histone lysine demethylase KDM2A is a novel anti-cancer drug target for non-small cell lung cancer (NSCLC) and a novel prognostic and predictive biomarker for NSCLC patients

The histone lysine demethylase KDM2A is overexpressed in 14-40% of NSCLC patient tumors.

[0081] In an effort to identify histone lysine demethylases that may be dys-regulated or overexpressed in subsets of NSCLC, we carried out bioinformatic analysis of mRNA expression profiles of all known histone lysine demethylases in 54 NSCLC cell lines. Of the examined histone lysine demethylases, KDM2A mRNA expression clearly displayed the largest standard deviation, indicating that KDM2A may be highly dys-regulated in NSCLC (Table 1). In addition, KDM2A mRNA levels of most NSCLC cell lines were higher than those of normal bronchial epithelial cells (FIGS. 1A-B). These results led us to choose KDM2A as the top candidate for subsequent expression analysis in primary NSCLC patient samples. [0082] As shown in FIGS. 1A-B, KDM2A is frequently overexpressed in NSCLC cell lines and patients. FIG. 1A & FIG. IB, KDM2A mRNA levels were higher in most NSCLC cell lines than in two normal lung cell lines. Profiling of KDM2A mRNA expression in two normal lung cell lines and 54 NSCLC cell lines (FIG. 1A) and comparison of of KDM2A mRNA levels between two normal lung cell lines and 54 NSCLC cell lines (FIG. IB). Affymetrix U133P, probe set 208988_at. [0083] As shown in FIGS. 1 C- D, KDM2A mRNA levels were up-regulated in an NSCLC patient set from UT MDACC (referred as to "UT MDACC set") as compared to 40 normal lung tissues. (FIG. 1C) KDM2A expression levels were compared between 40 pairs of cancer (Black bars) and normal (Gray bars) tissues. (FIG. ID) KDM2A levels were higher in 14% NSCLC patients (N= 14/98 patients) as compared to the highest normal value (500). KDM2A mRNA expression was evaluated by quantitative RT-PCR. The lowest PCR value of KDM2A mRNA levels was set at "1".

[0084] As shown in FIGS. 1E-F, KDM2A protein levels were statistically higher in tumors than in normal tissues. High KDM2A levels were present in 40% of tumor samples as compared to 12% of normal tissues (FIG. IE). KDM2A levels in 159 NSCLC and 32 normal lung tissue samples were measured by immunohistochemistry (IHC). Representative pictures of two normal and tumor samples are shown in (FIG. IF).

[0085] Table 1: Expression analysis of histone demethylases using whole-genome Affymetrix data of 54 non-small cell lung cancer (NSCLC) cell lines. Results shows that the histone lysine demethylase KDM2A displayed the largest normalized standard deviation (STD). Normalized STD indicates STD normalized to the mean. MAX and ΜΓΝ represent the maximum and minimum values that are normalized to the mean.

Table 1: Expression analysis of histone demethylases using whole-genome Affymetrix data

[0086] For analysis of NSCLC patient samples, we first isolated mRNA from tumor samples of 98 NSCLC patients in early stages (Stage I, II and III) from the UT MD Anderson Cancer Center (referred as to "UT MDACC set"). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of KDM2A mRNA levels demonstrated that KDM2A mRNA levels were increased in 14% (N=14/98) of NSCLC patients as compared to the highest normal value (500). Consistent with cell line data, KDM2A mRNA levels in NSCLC patients were significantly higher than those in 40 adjacent normal tissues (FIGS. 1C-D).

[0087] To confirm whether KDM2A is also overexpressed at the protein level in NSCLC samples, we evaluated KDM2A protein expression in 159 early-stage NSCLC patient samples and 32 normal lung tissues (from Biomax company) by immunohistochemical (IHC) analysis. This analysis showed that KDM2A protein levels were increased in up to 40% of NSCLC patients as compared to KDM2A-low tumor samples and most normal tissues (FIGS. 1E-F). These results establish KDM2A overexpression as one common molecular event in NSCLC and indicate that KDM2A possesses a typical characteristic of oncogene, i.e., overexpression in cancer.

KDM2A is a novel negative prognostic biomarker for NSCLC patient survival and is mutually exclusive with ALK.

[0088] To assess whether KDM2A is a prognostic biomarker, we carried out overall survival rate analysis using the Kaplan-Meier method. Analysis of the UT MDACC patient set demonstrated that high levels of KDM2A mRNA were significantly associated with poor patient survival (p= 0.0048) independent of age and gender (FIGS. 2A-C). The median survival time for KDM2A-overexpressing patients was 14.9 months, whereas that for KDM2A-low patients was 56.1 months. [0089] To further verify the results from the UT MDACC patient set, we analyzed survival of a second independent NSCLC patient group from the NCI Director's Challenge Consortium set that represents tumor samples from 444 lung NSCLC adenocarcinoma patients with health outcomes (15). For this analysis, we compared patients with high KDM2A mRNA expression levels (i.e., more than two standard deviations above the mean) with the other patients. Consistent with results of the UT MDACC patient set, survival analysis demonstrated that patients with high KDM2A levels had significantly higher recurrence rates and shorter overall survival times than the rest of patients (FIGS. 2D-F). In contrast, high levels of other histone demethylases KDM4A and JARIDld were not associated with shorter survival rates (FIGS. 2G-H).

[0090] Because the above survival analyses of the two patient sets were based on mRNA levels of KDM2A, we also assessed patient survival rates on the basis of KDM2A protein levels that were defined by immunohistochemical analysis. Specifically, we analyzed 79 NSCLC patients with adenocarcinoma, squamous cell carcinoma, and large cell carcinoma [US patients (N=21) and Korean patients (N=58)]. Consistent with the above results, patients with high KDM2A levels (N=37) had a worse clinical outcome (p=0.017) (FIG. 21). Interestingly, survival analysis of patients (N=45) with only squamous cell carcinoma (a subtype of NSCLC) also showed that high KDM2A levels correlated with poor clinical outcome (p=0.026) (FIGS. 2J-K).

[0091] As shown in FIGS. 2A-B, KDM2A mRNA expression levels are associated with poor survival in the MDACC patient set. FIG. 2A, KDM2A mRNA levels in patients (N=98, Pathology mix of all NSCLC) who were diagnosed between 11/9/1994 to 6/9/1999. Follow- up time, 43.1 month (2.3-180.7); Median survival time (MST), 45.6 mo. FIG. 2B, High KDM2A mRNA levels were associated with poor survival in the MDACC patient set. The cut-off value (line) is 500, which represents the highest normal value among 40 normal lung tissues. The lowest PCR value of KDM2A mRNA levels examined was set at "1". [0092] FIG. 2C shows clinical characteristics of UT MDACC NSCLC patient set (N=98). NO, No regional lymph nodes; Nl, Metastasis in hilar lymph nodes; N2, Metastasis in ipsilateral mediastinal lymph nodes. There is no significant difference in gender, age, smoking status, stage and NSCLC histology between patients with high KDM2A and those with low KDM2A. [0093] As shown in FIGS. 2D-E, in a publicly available NCI Director's Challenge Consortium patient set (444 lung adenocarcinoma samples), high KDM2A mRNA levels are associated with poor survival (FIG. 2D, progression free survival; FIG. 2E, overall survival). KDM2A mRNA levels were measured by Affymetrix U133A microarray gene expression analysis. Pathology: NSCLC adenocarcinoma; Clinical stages: I, II and III. Statistical significance was tested by the Gehan-Breslow-Wilcoxon method. FIG. 2F shows clinical characteristics of NSCLC tumors in the NCI Director's Challenge Consortium patient set. FIGS. 2G-H, In the NCI Director's Challenge Consortium patient set, other histone demethylases, such as KDM4A (FIG. 2G) and J ARID ID (FIG. 2H), did not correlate with patient survival, although high and low levels were separated by four different criteria (> 2 STD, > 1 STD, Mean, and < -1 STD). FIGS. 2I-J show KDM2A protein levels measured by IHC correlate with poor overall survival in the NSCLC IHC set (N=79). The survival curve in I represents all types of NSCLC, while that in J does only squamous cell carcinoma (SCC). NSCLC patients from Biomax (N=21) and Imgenex (N=58, from Korea); Clinical stages: Stage I (N=37), II (N=23) and III (N=20); Pathology: SCC (N=45), adenocarcinoma (N=16), bronchioloalveolar carcinoma (N=9) and large cell carcinoma (N=7). FIG. 2K shows clinical characteristics of squamous cell carcinoma (SCC) patients in the NSCLC IHC set. NO: No regional lymph nodes; Nl : Metastasis in hilar lymph nodes; N2: Metastasis in ipsilateral mediastinal lymph nodes.

[0094] In addition, we also investigated whether high KDM2A levels are mutually exclusive with anaplastic lymphoma kinase (ALK) using the NCI Director's Challenge Consortium patient set. ALK has been known to act as an oncogene by forming a fusion protein with the echinoderm microtubule-associated protein-like 4 (EML4) (18). Although high ALK levels were associated with overall poor prognosis (but not progression free survival), KDM2A and ALK were mutually exclusive with high KDM2A levels (FIGS. 3A- C). [0095] Together, these results from three independent survival analyses clearly indicated that NSCLC patients (Stage I, II and III) with high KDM2A levels at the mRNA and protein levels have poor prognosis for recurrence and overall survival, indicating that KDM2A is a novel prognostic biomarker for a subset of NSCLC patient population.

KDM2A gene is amplified in three representative NSCLC cell lines with high KDM2A levels and appears to be frequently amplified in a subset of NSCLC (10-27%).

[0096] To understand how KDM2A is overexpressed in NSCLC, we investigated whether the KDM2A gene is directly amplified in the three KDM2A-overexpressing NSCLC cell lines (H1792, H1975 and H23). The cell lines were chosen on the basis of similar NSCLS histology, gender and KRAS mutation status (FIG. 4A). The KDM2A mRNA levels of all five selected cell lines were assessed by quantitative RT-PCR and Western blotting (FIGS. 4B-C). Our analysis revealed that KDM2A gene amplification occurred in all three KDM2A- overexpressing NSCLC cell lines but not in two control cell lines with low KDM2A expression, uncovering gene amplification as a mechanism underlying KDM2A overexpression (FIG. 4D). In addition, analysis of a publicly available database showed that KDM2A appeared to be significantly amplified in a subset of NSCLC (10-27%) patients (FIG. 4E). The KDM2A gene is in very close proximity to the Cyclin Dl gene on chromosome l lql3.3, which is amplified in approximately 5% of lung cancer patients. As shown in FIG. 4D, KDM2A amplification did not always coincide with Cyclin Dl gene amplification, indicating that amplification of the two neighboring genes is likely independent. Given the fact that many classic oncogenes often undergo gene amplification, KDM2A amplification further supports its likely oncogenic property.

[0097] As shown in FIGS. 4A-D, the KDM2A gene is amplified in KDM2A- overexpressing NSCLC cell lines and in a subsets of NSCLC and other cancer patients. The KDM2A gene is amplified in three KDM2A-overexpressing NSCLC cell lines (HI 792, HI 975 & H23) but not in two cell lines with low KDM2A levels (H460 & H2122). FIG. 4A, Characteristics of NSCLC cell lines with high KDM2A levels (HI 975, H23 and HI 792) or low KDM2A levels (H460 and H2122). KDM2A mRNA levels, histology, gender and mutations status were summarized. Relative KDM2A mRNA levels were measured by quantitative RT-PCR. Adeno, Adenocarcinoma; LC, Large cell; BA, Bronchoalveolar. FIGS. 4B-C, KDM2A expression levels were assessed by quantitative RT-PCR (FIG. 4B) and Western Blot (FIG. 4C). FIG. 4D, Amplification of KDM2A and CyclinDl genes. Gene copy numbers of KDM2A and Cyclin D 1 genes were quantified. Note that normal copy number is 2.

The inhibitory effects of KDM2A knockdown on cellular growth and invasive phenotypes are exclusive to KDM2A-overexpressing NSCLC cell lines. [0098] Because the oncogenes are often important for cell growth, migration and invasion, we sought to determine the role of KDM2A in these cellular properties. We depleted KDM2A mRNA in the three KDM2A-overexpressing NSCLC cell lines (HI 792, HI 975 and H23) and two cell lines with low KDM2A levels (H460 and H2122) using RNA interference (siRNAs). KDM2A knockdown significantly inhibited cell proliferation of HI 792, HI 975 and H23 in vitro (FIGS. 5A-J), whereas it did not affect that of H460 and H2122 (FIGS. 5K- L). Consistent with its effect on cell growth, KDM2A knockdown suppressed colony formation ability of H1975 and H1792 cells (FIGS.5M-N). In addition, KDM2A knockdown remarkably inhibited cell migration and invasion of the three KDM2A-overexpressing NSCLC cell lines, but not the two cell lines with low KDM2A levels (FIGS. 6A-F). Such inhibition of cell growth, migration and invasion by KDM2A knockdown occurred exclusively in KDM2A-overexpressing NSCLC cell lines. Dependency of the growth and invasiveness of NSCLC cells on KDM2A is consistent with the oncogene addiction model, in which the growth and survival of cancer cells can often be dependent on a single oncogene. Together, KDM2A is likely to be a useful predictive biomarker for patient treatment selection for KDM2A-directed therapies.

[0099] siRNA-mediated knockdown of KDM2A in HI 975 cells suppressed cell proliferation are shown in FIGS. 5A-D. Efficacy of siRNA knockdown was assessed by quantitative RT-PCR (FIG. 5A) and Western blot (FIG. 5B) results are shown. Cell growth assay was performed using MTT assay (FIG. 5C). The same number of cells were seeded, and images were taken 72h after siRNA treatment (FIG. 5D). FIGS. 5E-J show siRNA- mediated knockdown of KDM2A in HI 792 and H23 cells inhibited cell proliferation. Efficacy of siRNA knockdown was assessed by quantitative RT-PCR (FIG. 5E) and Western blot (FIG. 5F). Cell growth assay was performed using MTS assay (FIG. 5G & FIG. 5F). The same number of cells were seeded, and images were taken 72h after siRNA treatment (FIGS. 5I-J). As shown in FIGS. 5K-L, siRNA-mediated knockdown of KDM2A in cell lines with low KDM2A levels (H460 and H2122) did not inhibit cell proliferation. Efficacy of siRNA knockdown (Bottom panels) was assessed by Western blot. KDM2A knockdown suppressed colony formation of HI 975 (FIG. 5M) and HI 792 (FIG. 5N) cells.

[00100] siRNA-mediated knockdown of KDM2A in H1792 (FIG. 6A) and H1975 (FIG. 6B) cells remarkably impeded cell invasion. Cells were treated with siRNA for 96 hours and were incubated for cell invasion for 18h. As shown in FIGS. 6C-F, siRNA mediated knockdown of KDM2A suppressed cell migration of H1792 and H1975 cells but not control cell lines with low KDM2A levels (H460 and H2122). siRNA-mediated knockdown of KDM2A in HI 792 (FIG. 6C) and HI 975 (FIG. 6D) cells markedly suppressed cell migration. Cells were treated with siRNA for 96 hours and were incubated for cell migration for 18h. As shown in FIGS. 6E-F, siRNA-mediated knockdown of KDM2A in control cell lines with low KDM2A levels (H460 and H2122) did not inhibit cell migration. H2122 cells had a poor migration ability. Cells were treated with siRNA for 96 hours and were incubated for cell migration for 18h. The regulation of cell proliferation and invasion by KDM2A are dependent largely on the enzymatic activity of KDM2A, which may be a promising anti-cancer target.

[00101] KDM2A is an epigenetic enzyme that removes methyl groups from dimethylated lysine 36 at histone H3 (H3K36me2) (9). To assess whether KDM2A-driven phenotypes, such as cell proliferation, migration and invasion, are dependent on the demethylase activity, we transfected KDM2A-depleted cells with expression plasmids encoding wild type KDM2A or its catalytically inactive mutant mKDM2A (H212A). Interestingly, these rescue experiments showed that wild-type KDM2A, but not its mutant, significantly restored defective proliferation and invasion by KDM2A knockdown (FIGS. 7A-D). These results indicate that the drastic inhibitory effect of KDM2A knockdown on cell proliferation and invasion are dependent largely on its catalytic activity, establishing KDM2A as an attractive new druggable anti-cancer target.

KDM2A directly down-regulates HDAC3 and DUPS3 expression while indirectly up- regulating expression of the cell cycle-regulatory gene NEK7 and the invasion-associated gene NANOSl.

[00102] To understand the mechanisms underlying KDM2A-mediated regulation of cell proliferation and invasion, HI 792 and HI 975 cells were treated with two different siRNAs against KDM2A (siKDM2A-3 or -4). Then, the whole genome mRNA levels were compared between KDM2A-depleted cells and control siRNA-treated cells. This analysis revealed a list of genes that were consistently up- or down- regulated by both siRNAs against KDM2A in both cell lines (FIGS. 8A-B). We confirmed the microarray results by individual analyzing several highly regulated genes by quantitative RT-PCR (FIGS. 9A-B). We selected DUSP3, HDAC3 NEK7, and NANOSl because of their functional implications in cancer development (see also below). For example, NEK7 regulates cell cycle progression (19) while NANOS l promotes cell invasion (20). Notably, KDM2A knockdown up-regulated up to 9 fold DUSP3 expression (FIGS. 9A-B). We additionally included GPR157, TMEM65 and TIMM17 genes as controls. Quantitative chromatin immunoprecipitation (qChIP) assay (a method measuring chromatin levels of chromatin-associated proteins or chromatin marks) showed that HDAC3, GPR157, TMEM65 and TIMM17, which were up-regulated by KDM2A knockdown, were KDM2A target genes (FIGS. 9C-D). In contrast, NANOSl and NEK7 genes, which were down-regulated by KDM2A knockdown, were not directly targeted by KDM2A (FIGS. 9C- D).

KDM2A activates the MAPKs (ERK1/2 and JNK1/2) by transcriptionally repressing the MAPK phosphatase DUSP3 gene via H3K36me2 demethylation.

[00103] The above results indicate that DUSP3 is a KDM2A target gene and is highly up- regulated by KDM2A knockdown. This prompted us to investigate the role of DUSP3 in mediating the cellular function of KDM2A. In line with its effect on mRNA levels of DUSP3, KDM2A depletion also increased the protein levels of DUSP3 (FIG. 10A). To further assure that DUSP3 expression is regulated by KDM2A, we examined whether ectopic expression of KDM2A represses DUSP3 levels in KDM2A knockdown cells. Results showed that ectopic expression of wild-type KDM2A but not its catalytic mutant mKDM2A significantly suppressed DUSP3 expression, confirming a specific transcriptional regulation of DUPS3 by KDM2A and its enzymatic activity (FIGS. lOB-C). KDM2A has been implicated in gene repression at the transcriptional levels (21, 22), because it erases the gene activation mark H3K36me2 (23). In support with KDM2A's role as a transcriptional co- repressor, quantitative ChIP results showed that KDM2A depletion up-regulated specifically H3K36me2 occupancy at the promoter of the DUSP3 gene in H1792 and H1975 cells (FIGS. 10D-F). These results indicate that KDM2A-mediated repression oiDUSP3 may result from KDM2A-catalyzed demethylation of H3K36me2 at the DUSP3 gene.

[00104] DUSP3 has been known to inhibit the activity of EGFR in the lung cancer cell line H1299 and the activities of ERK1/2 and ΓΝΚ1/2 in HeLa cells by dephosphorylating these kinases (24-27). ERK1/2 and ΓΝΚ1/2 are MAPKs that play an key role in regulating cell signaling processes, including cell growth and invasiveness (28-30). We assessed whether increases in DUSP3 by KDM2A knockdown affect phosphorylation levels of EGFR, ERK1/2 and ΓΝΚ1/2 as well as the MAPK p38 and the serine-threonine protein kinase AKT1. Western blot results demonstrated that increased levels in DUSP3 levels by KDM2A depletion remarkably down-regulated phospho-ERKl/2 and phospho-JNKl/2 but did not affect phosphorylation levels of EGFR, p38, and AKT (FIGS. 11A-B). Consistently, phospho-ERKl/2 and phospho-JNKl/2 levels were drastically reduced by ectopic expression of DUSP3 (FIG. 11C). [00105] To assess whether increases in DUSP3 by KDM2A knockdown decreases phosphorylation levels of ERK1/2 and JNK1/2 during MAPK signaling, we first serum- starved KDM2A-depeleted H1792 cells and then monitored phospho-ERKl/2, phospho- J K1/2 and phospho-p38 between control cells and KDM2A knockdown cells after serum treatment. Increases in DUSP3 levels delayed phosphorylation of ERKl/2 and JNKl/2 but did not change phopsho-EGFRs (FIG. 11B & FIG. 11D). To ascertain whether phospho- ERKl/2 and phospho-J Kl/2 are dephosphorylated in a DUSP3-dependent manner, we depleted DUSP3 in KDM2A knockdown cells and serum-starved these double knockdown cells, followed by serum treatment. As shown in FIG. HE, DUSP3 knockdown recovered phospho-ERKl/2 and to a lesser extent phospho- JNKl/2. These results indicate that KDM2A activates ERKl/2 and JNKl/2 by transcriptionally repressing DUSP3. KDM2A promotes the growth and invasiveness of NSCLC cells in a DUSP3-dependent manner, and low DUSP3 levels are significantly associated with high KMD2A levels.

[00106] To test whether KDM2A regulates cellular growth and invasiveness in a DUSP3- dependent manner, we examined the effect of DUSP3 knockdown on the growth and invasiveness of KDM2A-depleted cells. The double knockdown experiments showed that DUSP3 depletion significantly rescued defective growth of KDM2A-depleted cells while markedly restoring their invasiveness (FIGS. 11F-G). Because it has been reported that the subcellular localization of DUSP3 is cell type-dependent (31), we determined whether DUSP3 is localized in cytosol in NSCLC cells. Our results showed that DUSP3 is a cytosolic protein, indicating that DUSP3-catalyzed dephosphorylation of ERKl/2 and JNKl/2 takes place exclusively in cytosol (FIGS. 11H-I). Next, we assessed whether KDM2A levels are inversely associated with DUSP3 levels. Analysis of the publicly available microarray dataset from the NCI Director's Challenge Consortium showed that DUSP3 levels inversely correlated with KDM2A levels (FIG. 11J). These results suggest that the transcriptional repression of DUSP3 levels by KDM2A plays a critical role in the growth and invasiveness of KDM2A-overexpressing NSCLC cells.

KDM2A-mediated repression of HDAC3 contributes to cellular growth and invasiveness, and HDAC3 directly represses NEK7 and NANOS1.

[00107] The invasiveness-associated gene NANOS1 and the cell cycle-regulatory gene NEK7 were up-regulated by KDM2A, although they were not direct target genes of KDM2A (FIG. 9). This led us to hypothesize that these genes may be down-regulated by a transcriptional co-repressor encoded by a KDM2A-repressed gene. Of the KDM2A-repressed genes, HDAC3 is a well-known transcriptional co-repressor and an epigenomic modifier (32- 34). We tested the possibility that HDAC3 may directly down-regulate expression levels of the NANOS1 and NEK7 genes. In line with our hypothesis, qChIP experiments with anti- HDAC3 antibodies revealed that HDAC3 was recruited to the NANOS1 and NEK7 gene but not the GPR157 gene (FIGS. 12A-B). In addition, we determined the effect of HDAC3 knockdown on expression levels of NANOS1 and NEK7. As shown in FIGS. 12C-E, HDAC3 depletion by two independent siRNAs increased expression levels of the NANOS1 and NEK7 genes. These results indicate that KDM2A increases expression of the HDAC3 target genes NANOS1 and NEK7 by directly repressing HDAC3 expression at the transcriptional level.

[00108] To ascertain that KDM2A represses HDAC3 expression, we examined whether ectopic expression of KDM2A in KDM2A knockdown cells represses HDAC3 levels. Results showed that ectopic expression of wild-type KDM2A but not its catalytic mutant mKDM2A significantly inhibited HDAC3 expression, indicating a specific transcriptional regulation of HDAC3 by KDM2A and its demethylase activity (FIGS. 13A-B).

[00109] The pathologic role of HDAC3 for cancer development might be tissue-dependent. In colorectal tumors, HDAC3 appeared to be up-regulated and to repress the tumor suppressor p21 (35). In contrast, recent studies demonstrated that HDAC3 knockout mice developed hepatoma (36, 37) and that HDAC3 was shown to potentiate apoptosis by down- regulating the proto-oncogene c-Jun (32). To directly address the role of HDAC3 in KDM2A-mediated regulation of cell growth and invasiveness, we depleted HDAC3 in KDM2A knockdown cells using siHDAC3. Consistent with the role of HDAC3 in tumor suppression, HDAC3 knockdown significantly restored the proliferation and invasiveness of KDM2A-depleted cells (FIGS. 13C-E). These results indicate that the transcriptional repression of HDAC3 by KDM2A contributes to the growth and invasiveness of KDM2A- overexpressing NSCLC cells.

The in vivo mouse xenograft models confirm the oncogenic effects of KDM2A on the growth and invasiveness of NSCLC cells.

[00110] To demonstrate the oncogenic role of KDM2A in NSCLC tumorigenesis in vivo, we carried out in vivo mouse xenograft experiments in which KDM2A-overexpressing H1792 cells were treated with control siRNA or siRNA against KDM2A ex vivo and injected into mice via their tail veins. In sharp contrast to the development of multiple lung metastases of control siRNA-treated cells within 3-4 months, the formation of lung metastases was completely abolished by KDM2A knockdown mediated by two independent siRNAs against KDM2A (FIGS. 14A-B). Lung tumors were confirmed by H&E staining (FIG. 14C). Moreover, we further sought to confirm the oncogenic role of KDM2A in a lung orthotopic NSCLC model. We implanted siRNA-treated HI 792 NSCLC cells into the mouse lung. KDM2A knockdown drastically suppressed tumor formation of cancer cells in lungs and completely abrogated their metastasis to contralateral lung and mediastinal lymph node (LN) (FIGS. 14D-E). These tumors were verified by H&E staining (FIG. 14E). These results indicate that KDM2A may play a critical role in tumorigenesis and metastasis of NSCLC in vivo.

KDM2A is overexpressed and appears to be amplified in other types of cancer.

[00111] Using a publicly available mRNA microarray data from the Oncomine database, we found that in addition to NSCLC, KDM2A was overexpressed in significant portions of pancreatic and breast tumors (FIG. 15). Although the functional significance of KDM2A overexpression in these cancer types is currently unknown, these results strongly suggest that KDM2A may act as a common oncogene in several tumor types and be used as an anti-cancer drug target to treat these types of tumors as well. Consistent with this, analysis of the publicly available database demonstrated that KDM2A appeared to be frequently amplified a subset of breast cancer (19-33%), esophageal cancer (43-47%) and hepatoma (6-10%) patients (FIG. 4E).

Mass spectrometric analysis identifies novel KDM2A-interacting proteins.

[00112] In an effort to identify new KDM2A-interacting proteins, we generated an HEK293 stable cell line that expresses high levels of FLAG-tagged KDM2A. The KDM2A- associated proteins were eluted from anti-FLAG affinity resins that were incubated with nuclear extracts (FIG. 16). A mass spectrometric analysis of individual bands led to the identification of novel KDM2A partner proteins, including Skpl and ΗΡ Ιγ, which will enable to design and screen potential inhibitors against protein-protein interactions.

[00113] In conclusion, we identify the histone demethylase KDM2A as a novel oncogenic promoter for NSCLC. Specifically, our results indicate that KDM2A is frequently overexpressed and gene-amplified in NSCLC cell lines and patient samples and that RNAi- mediated knockdown of KDM2A remarkably inhibited cell proliferation, migration and invasion of NSCLC cells in vitro and in vivo. Notably, the tumor-promoting properties of KDM2A are largely dependent on its demethylase activity. Our mechanistic studies demonstrated that KDM2A is a transcriptional co-repressor of both HDAC3 and the MAPK phosphatase DUSP3 in NSCLC cells. HDAC3 represses cell cycle- and invasion-associated genes while DUSP3 down-regulates activities of two major types of MAPKs (i.e., ERK1/2 and to a lesser extent JNK1/2) by dephosphorylating them. Importantly, these two novel pathways— the repression of a histone deacetylase and the activation of DUSP3 -repressed MAPKs by KDM2A— appear to be in parallel but converge on cell proliferation and invasion (FIG. 17).

* * *

[00114] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims

A method of providing a prognosis or prediction for a subject determined to have a cancer, comprising: a) obtaining a cancer sample from said subject; b) measuring expression or DNA amplification of KDM2A (FBXL1 1) in said sample via an in vitro test; and c) providing a prognosis or prediction for the subject based on the expression or DNA amplification, wherein a gene copy number of greater than about 2.5, an expression level of KDM2A of greater than about 2 standard deviations above the mean expression level of KDM2A in a healthy tissue sample of the same tissue type as the cancer sample, or an expression level of KDM2A of greater than the maximal expression level of KDM2A in a healthy tissue sample of the same tissue type as the cancer sample indicates a poor survival, a high risk of recurrence, or a favorable or a high response to a KDM2A inhibitor therapy; and wherein a gene copy number of less than about 2.5, an expression level of KDM2A of less than about 2 standard deviations above the mean expression level of KDM2A in a healthy tissue sample of the same tissue type as the cancer sample, and an expression level of KDM2A of less than the maximal expression level of KDM2A in a healthy tissue sample of the same tissue type as the cancer sample indicates a favorable survival, a low risk of recurrence, or an unlikely or a low response to a KDM2A inhibitor therapy.

The method of claim 1, wherein said in vitro test is selected from the group consisting of quantitative RT-PCR, immunohistochemistry (IHC), DNA-PCR, or fluorescence in situ hybridization (FISH).

The method of claim 1, wherein the method further comprises administering a KDM2A (FBXL11) inhibitor to said subject if said measuring detects in said sample a gene copy number of greater than about 2.5, an expression level of KDM2A of greater than about 2 standard deviations above the mean expression level of KDM2A in a healthy tissue sample of the same tissue type as the cancer sample, or an expression level of KDM2A of greater than the maximal expression level of KDM2A in a healthy tissue sample of the same tissue type as the cancer sample.

4. The method of claim 1 , wherein the cancer is a lung cancer, ovarian cancer, breast cancer, pancreatic cancer, multiple myeloma, esophageal cancer, or hepatoma.

5. The method of claim 4, wherein the cancer is a lung cancer.

6. The method of claim 4, wherein the lung cancer is non-small cell lung cancer (NSCLC).

7. The method of claim 1, wherein the sample comprises a blood sample.

8. The method of claim 1, wherein the sample comprises a human tumor.

9. The method of claim 1, wherein the subject is a human.

10. The method of claim 1, wherein the sample comprises a cancer tissue from a cancer biopsy or surgically resected tissue.

11. The method of claim 1, wherein the method comprises obtaining or receiving said sample.

12. The method of claim 11, wherein said sample is paraffin-embedded.

13. The method of claim 11, wherein said sample is frozen.

14. The method of claim 1, wherein said measuring comprises RNA quantification.

15. The method of claim 14, wherein the RNA quantification comprises cDNA microarray, quantitative RT-PCR, in situ hybridization, Northern blotting, or nuclease protection.

16. The method of claim 1, wherein said measuring comprises protein quantification.

17. The method of claim 16, wherein said protein quantification comprises immunohistochemistry, an ELISA, a radioimmunoassay (RIA), an immunoradiometric assay, a fluoroimmunoassay, a chemiluminescent assay, a bioluminescent assay, a gel electrophoresis, or a Western blot analysis.

18. The method of claim 1, wherein said measuring comprises DNA gene copy quantification.

19. The method of claim 18, wherein said DNA gene quantification comprises comparative genomic hybridization (CGH), DNA-PCR, or fluorescence in situ hybridization (FISH).

20. The method of claim 1, wherein providing the prognosis or prediction comprises generating a classifier based on the expression, wherein the classifier is defined as a weighted sum of expression levels of KDM2A.

21. The method of claim 20, wherein providing the prognosis or prediction comprises classifying a group of subjects based on the classifier associated with individual subjects in the group with a reference value.

22. The method of claim 20, wherein the classifier is generated on a computer.

23. The method of claim 20, wherein the classifier is generated by a computer readable medium comprising machine executable instructions suitable for generating a classifier.

24. The method of claim 1, further comprising reporting said prognosis or prediction.

25. The method of claim 1, further comprising prescribing or administering an anti-cancer therapy to said subject based on said prediction

26. The method of claim 25, wherein the anticancer therapy is an adjuvant therapy, a prevention therapy, a neoadjuvant therapy, or a metastatic therapy.

27. The method of claim 1, wherein the cancer is a stage I cancer.

28. The method of claim 1, wherein the cancer is a stage II cancer.

29. The method of claim 1, wherein the cancer is a stage III cancer.

30. The method of claim 1, wherein the cancer is a stage IV cancer.

31. A kit comprising a plurality of antigen-binding fragments that bind to KDM2A (FBXL1 1) or a plurality of primers or probes that bind to transcripts of KDM2A to assess expression levels, wherein said kit is housed in a container.

32. The kit of claim 31, wherein the kit comprises an immunoassay.

33. The kit of claim 32, wherein the immunoassay comprises a lateral flow assay.

34. A method for treating a cancer in a subject, comprising: a) selecting a subject predicted to have a favorable or high response to a KDM2A inhibitor therapy in accordance with claim 1 ; and b) administering to the subject a pharmacologically effective dose of an inhibitor of KDM2A (FBXL1 1).

35. The method of claim 34, wherein the subject is a human.

36. The method of claim 34, wherein the cancer is a lung cancer, ovarian cancer, breast cancer, pancreatic cancer, or multiple myeloma.

37. The method of claim 36, wherein the cancer is a lung cancer.

38. The method of claim 37, wherein the lung cancer is non-small cell lung cancer (NSCLC).

39. The method of claim 36, wherein the inhibitor is an antibody.

40. The method of claim 39, wherein the antibody is a human or humanized monoclonal antibody.

41. The method of claim 39, wherein the antibody is a monovalent antibody.

42. The method of claim 39, wherein the antibody is a multivalent antibody.

43. The method of claim 39, wherein the antibody is conjugated to a reporter molecule.

44. The method of claim 43, wherein the reporter molecule is a radioligand or a fluorescent label.

45. The method of claim 44, wherein the antibody is an anti-KDM2A scFv, F(ab) or F(ab)2.

46. The method of claim 34, wherein the inhibitor is an antisense nucleic acid, a shRNA, a siRNA, or a siNA.

47. The method of claim 34, wherein the administration is systemic, local, regional, parenteral, intravenous, intraperitoneal, via inhalation, oral, or intra-tumoral injection.

48. A composition comprising a KDM2A inhibitor for use in treating cancer in a patient from whom a cancer sample has been tested by an in vitro test and determined to exhibit a gene copy number of greater than about 2.5, an expression level of KDM2A of greater than about 2 standard deviations above the mean expression level of KDM2A in a healthy tissue sample of the same tissue type as the cancer sample, or an expression level of KDM2A of greater than the maximal expression level of KDM2A in a healthy tissue sample of the same tissue type as the cancer sample.

49. The composition of claim 48, wherein the KDM2A inhibitor comprises an antibody or an siRNA.

50. The composition of claim 48, wherein the in vitro test is quantitative RT-PCR, immunohistochemistry (IHC), DNA-PCR, or fluorescence in situ hybridization (FISH).