WO2013123411A1 - Procédés de diagnostic et de traitement du cancer - Google Patents

Procédés de diagnostic et de traitement du cancer Download PDF

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Publication number
WO2013123411A1
WO2013123411A1 PCT/US2013/026458 US2013026458W WO2013123411A1 WO 2013123411 A1 WO2013123411 A1 WO 2013123411A1 US 2013026458 W US2013026458 W US 2013026458W WO 2013123411 A1 WO2013123411 A1 WO 2013123411A1
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kdm2a
cancer
sample
expression
nsclc
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PCT/US2013/026458
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English (en)
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Klaus W. Wagner
Min Gyu LEE
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Board Of Regents, The University Of Texas System
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • 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
  • Non-small cell lung cancer 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.
  • 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.
  • 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.
  • KDM2A also referred to as FBXL1 1 or JHDM1A
  • NSCLC non-small cell lung cancer
  • Inhibitors of KDM2A may be used in various aspects to treat a cancer.
  • 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.
  • 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.
  • KDM2A FBXL1 1
  • 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
  • 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.
  • KDM2A FBXL11
  • the cancer may be a lung cancer, ovarian cancer, breast cancer, pancreatic cancer, multiple myeloma, esophageal cancer, or hepatoma.
  • the cancer is a lung cancer such as, e.g., non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • kits 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.
  • the immunoassay comprises a lateral flow assay.
  • 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).
  • the subject is a human.
  • the cancer may be a lung cancer, ovarian cancer, breast cancer, pancreatic cancer, or multiple myeloma.
  • 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.
  • Another aspect of the present invention relates to a composition
  • 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.
  • the in vitro test is quantitative RT-PCR, immunohistochemistry (IHC), DNA-PCR, or fluorescence in situ hybridization (FISH).
  • 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.
  • 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.
  • 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 RNALaterTM 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.
  • KDM2A 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.
  • mRNA messenger RNA
  • 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.
  • 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).
  • Affymetrix GeneChip® technology Santa Clara, CA
  • the Microarray System from Incyte Incyte (Fremont, CA).
  • single- stranded nucleic acids e.g., cDNAs or oligonucleotides
  • 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.
  • 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.
  • RMA robust multichip average
  • Quantitative real-time PCR may also be used to measure the differential expression of KDM2A.
  • 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.
  • 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.
  • 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.
  • Suitable reference standards include, but are not limited to, mR As for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and ⁇ -actin.
  • GPDH glyceraldehyde-3-phosphate-dehydrogenase
  • ⁇ -actin glyceraldehyde-3-phosphate-dehydrogenase
  • 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.
  • 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.
  • 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.
  • 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.
  • An enzyme-linked immunosorbent assay may be used to measure the differential expression of KDM2A.
  • an ELISA assay 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.
  • 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.
  • 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.
  • An antibody microarray may also be used to measure the differential expression of KDM2A.
  • 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.
  • 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.
  • 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 is also tagged with a fluorescent reporter.
  • 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.
  • 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.
  • 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.
  • 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.
  • obtaining a biological sample or “obtaining a blood sample” refer to receiving a biological or blood sample, e.g., either directly or indirectly.
  • the biological sample e.g., comprising cancer tissue surgically resected from a patient or obtained from a cancer biopsy
  • the biological sample is directly obtained from a subject at or near the laboratory or location where the biological sample will be analyzed.
  • 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.
  • the sample may be obtained and tested in the same location using a point-of care test.
  • 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.
  • 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.
  • 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.
  • cancer 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • a suitable control e.g., a non-cancerous tissue, cell sample, cancer sample with low expression, a reference standard
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a good or bad prognosis may be assessed in terms of overall survival or disease free survival.
  • 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.
  • 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.
  • "bad prognosis” cancer patients experience disease relapse, tumor recurrence, metastasis, and death.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • primer 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.
  • 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.
  • 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.
  • 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
  • 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. 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.
  • FIG. 2K Clinical characteristics of squamous cell carcinoma (SCC) in the NSCLC IHC set.
  • 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.
  • 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%).
  • 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
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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 ⁇ -actin were used as a nuclear marker and a loading control, respectively.
  • WCL whole cell lysates.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • FIG. 16 Identification of KDM2A-interacting proteins ( ⁇ and SKPlb) by anti-FLAG affinity purification and mass spectrometric analysis.
  • 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 -
  • Non-small cell lung cancer 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.
  • prognostic biomarkers and anti-cancer targets for NSCLC are rare.
  • the histone lysine demethylase KDM2A also known as FBXL11/JHDM1
  • FBXL11/JHDM1 an epigenetic enzyme
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • KDM2A 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.
  • 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.
  • KDM2A overexpression activates the downstream effectors (i.e., ERK1/2 and J K1/2) via epigenetic repression of DUSP3.
  • 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).
  • expression status of KDM2A is mutually exclusive with that of the kinase ALK.
  • 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.
  • combination therapies e.g., combinations of KDM2A inhibitors, EGFR targeting molecules and classic chemotherapy
  • 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.
  • 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).
  • RNA samples 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.
  • KDM2A target 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.
  • 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).
  • 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 iScriptTM 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
  • 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.
  • 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.
  • 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 10 4 cells/ml in a six-well plate format with a final volume of 2 ml, final siRNA concentration of 20 nM and 5 ⁇ LF per well.
  • siR A duplexes against Luciferase GL3 R A (5' AA- CTTACGCTGAGTACTTCGA-dTdT 3') or an FITC-conjugated siRNA from Dharmacon were used as controls.
  • 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).
  • 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.
  • 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.
  • KDM2A mRNA expression clearly displayed the largest standard deviation, indicating that KDM2A may be highly dys-regulated in NSCLC (Table 1).
  • KDM2A mRNA levels of most NSCLC cell lines were higher than those of normal bronchial epithelial cells (FIGS. 1A-B).
  • 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.
  • FIG. 1A 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).
  • 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.
  • UT MDACC set UT MDACC set
  • KDM2A expression levels were compared between 40 pairs of cancer (Black bars) and normal (Gray bars) tissues.
  • KDM2A mRNA expression was evaluated by quantitative RT-PCR. The lowest PCR value of KDM2A mRNA levels was set at "1".
  • 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).
  • 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 ⁇ represent the maximum and minimum values that are normalized to the mean.
  • Table 1 Expression analysis of histone demethylases using whole-genome Affymetrix data
  • qRT-PCR Quantitative reverse transcription polymerase chain reaction
  • KDM2A protein expression was also overexpressed at the protein level in NSCLC samples.
  • IHC immunohistochemical
  • KDM2A is a novel negative prognostic biomarker for NSCLC patient survival and is mutually exclusive with ALK.
  • KDM2A is a prognostic biomarker
  • 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).
  • FIGS. 2A-B KDM2A mRNA expression levels are associated with poor survival in the MDACC patient set.
  • MST Median survival time
  • 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".
  • NO No regional lymph nodes
  • Nl Metastasis in hilar lymph nodes
  • N2 Metastasis in ipsilateral mediastinal lymph nodes.
  • FIGS. 2D-E show that 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).
  • 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. 2G-H histone demethylases
  • the survival curve in I represents all types of NSCLC, while that in J does only squamous cell carcinoma (SCC).
  • 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.
  • ALK anaplastic lymphoma kinase
  • 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%).
  • KDM2A is overexpressed in NSCLC
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • KDM2A knockdown significantly inhibited cell proliferation of HI 792, HI 975 and H23 in vitro (FIGS.
  • KDM2A knockdown suppressed colony formation ability of H1975 and H1792 cells (FIGS.5M-N).
  • 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.
  • KDM2A 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.
  • 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.
  • 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.
  • 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.
  • siRNA-mediated knockdown of KDM2A in control cell lines with low KDM2A levels 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.
  • KDM2A is an epigenetic enzyme that removes methyl groups from dimethylated lysine 36 at histone H3 (H3K36me2) (9).
  • H3K36me2 histone H3
  • 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.
  • 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).
  • DUSP3, HDAC3 NEK7, and NANOSl because of their functional implications in cancer development (see also below).
  • NEK7 regulates cell cycle progression (19) while NANOS l promotes cell invasion (20).
  • KDM2A knockdown up-regulated up to 9 fold DUSP3 expression (FIGS. 9A-B).
  • 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).
  • qChIP Quantitative chromatin immunoprecipitation
  • KDM2A activates the MAPKs (ERK1/2 and JNK1/2) by transcriptionally repressing the MAPK phosphatase DUSP3 gene via H3K36me2 demethylation.
  • 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.
  • KDM2A-mediated repression oiDUSP3 may result from KDM2A-catalyzed demethylation of H3K36me2 at the DUSP3 gene.
  • DUSP3 has been known to inhibit the activity of EGFR in the lung cancer cell line H1299 and the activities of ERK1/2 and ⁇ 1/2 in HeLa cells by dephosphorylating these kinases (24-27).
  • ERK1/2 and ⁇ 1/2 are MAPKs that play an key role in regulating cell signaling processes, including cell growth and invasiveness (28-30).
  • KDM2A knockdown affect phosphorylation levels of EGFR, ERK1/2 and ⁇ 1/2 as well as the MAPK p38 and the serine-threonine protein kinase AKT1.
  • DUSP3 is a cytosolic protein, indicating that DUSP3-catalyzed dephosphorylation of ERKl/2 and JNKl/2 takes place exclusively in cytosol (FIGS. 11H-I).
  • 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).
  • KDM2A-mediated repression of HDAC3 contributes to cellular growth and invasiveness, and HDAC3 directly represses NEK7 and NANOS1.
  • 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.
  • 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.
  • HDAC3 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.
  • HDAC3 knockdown significantly restored the proliferation and invasiveness of KDM2A-depleted cells (FIGS. 13C-E).
  • the in vivo mouse xenograft models confirm the oncogenic effects of KDM2A on the growth and invasiveness of NSCLC cells.
  • 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.
  • 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.
  • KDM2A 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.
  • MAPKs i.e., ERK1/2 and to a lesser extent JNK1/2
  • 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.
  • UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development. Nature 449:731-734. 7. Xiang, Y., Zhu, Z., Han, G., Lin, H., Xu, L., and Chen, CD. 2007. JMJD3 is a histone H3K27 demethylase. Cell Res 17:850-857.
  • KDM2A represses transcription of centromeric satellite repeats and maintains the heterochromatic state. Cell Cycle 7:3539-3547.
  • Extracellular regulated kinases (ERK) 1 and ERK2 are authentic substrates for the dual-specificity protein-tyrosine phosphatase VHR. A novel role in down-regulating the ERK pathway. J Biol Chem 274:13271-13280. 25. Todd, J.L., Rigas, J.D., Rafty, L.A., and Denu, J.M. 2002. Dual-specificity protein tyrosine phosphatase VHR down-regulates c-Jun N-terminal kinase (JNK). Oncogene 21 :2573-2583.
  • Vaccinia Hl-related phosphatase is a phosphatase of ErbB receptors and is down-regulated in non-small cell lung cancer. J Biol Chem 286:10177-10184. 27. Rahmouni, S., Cerignoli, F., Alonso, A., Tsutji, T., Henkens, R., Zhu, C, Louis-dit-
  • Cervix carcinoma is associated with an up- regulation and nuclear localization of the dual-specificity protein phosphatase VHR.
  • HDAC3 Histone deacetylase 3
  • HDAC3 Histone deacetylase 3

Abstract

La présente invention concerne des procédés de diagnostic et de traitement du cancer. Dans certains aspects, la surexpression de KDM2A (également appelée FBXLl 1 ou JHDMIA) est associée à un pronostic médiocre dans certains cancers, tels que le cancer du poumon non à petites cellules (NSCLC). La présente invention concerne également des procédés de prédiction de la réponse à des thérapies inhibitrices de KDM2A. Des inhibiteurs de KDM2A peuvent être utilisés dans divers aspects pour traiter un cancer.
PCT/US2013/026458 2012-02-17 2013-02-15 Procédés de diagnostic et de traitement du cancer WO2013123411A1 (fr)

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CN111004848A (zh) * 2019-12-11 2020-04-14 中国人民解放军陆军军医大学第一附属医院 Fbxl6作为靶点在制备抗肿瘤药物中的应用
CN111562379A (zh) * 2020-05-25 2020-08-21 郑州大学第一附属医院 一组用于早期食管癌筛查的多指标联合检测胶体金试纸条

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US10189787B2 (en) 2012-10-02 2019-01-29 Gilead Sciences, Inc. Inhibitors of histone demethylases
US10221139B2 (en) 2012-10-02 2019-03-05 Gilead Sciences, Inc. Inhibitors of histone demethylases
US9221801B2 (en) 2013-02-27 2015-12-29 Epitherapeutics Aps Inhibitors of histone demethylases
US9650339B2 (en) 2013-02-27 2017-05-16 Gilead Sciences, Inc. Inhibitors of histone demethylases
US9802941B2 (en) 2014-08-27 2017-10-31 Gilead Sciences, Inc. Compounds and methods for inhibiting histone demethylases
CN107406414A (zh) * 2015-01-09 2017-11-28 基因泰克公司 作为用于治疗癌症的组蛋白脱甲基酶kdm2b的抑制剂的(哌啶‑3‑基)(萘‑2‑基)甲酮衍生物及其相关化合物
CN107406414B (zh) * 2015-01-09 2022-04-19 基因泰克公司 作为用于治疗癌症的组蛋白脱甲基酶kdm2b的抑制剂的(哌啶-3-基)(萘-2-基)甲酮衍生物
CN111004848A (zh) * 2019-12-11 2020-04-14 中国人民解放军陆军军医大学第一附属医院 Fbxl6作为靶点在制备抗肿瘤药物中的应用
CN111004848B (zh) * 2019-12-11 2022-09-23 中国人民解放军陆军军医大学第一附属医院 Fbxl6作为靶点在制备抗肿瘤药物中的应用
CN111562379A (zh) * 2020-05-25 2020-08-21 郑州大学第一附属医院 一组用于早期食管癌筛查的多指标联合检测胶体金试纸条
CN111562379B (zh) * 2020-05-25 2020-12-01 郑州大学第一附属医院 一组用于早期食管癌筛查的多指标联合检测胶体金试纸条

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