WO2011016869A1 - Methods and compositions for drug discovery - Google Patents

Abstract

This disclosure provides methods and compositions to enable drug discovery, particularly for potential therapeutics for diseases or disorders related to, or correlated with, changes in patterns of histone modification.

Description

METHODS AND COMPOSITIONS FOR DRUG DISCOVERY

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 61/231,745 filed on August 6, 2009.

FIELD OF THE INVENTION

[0002] The present invention relates to methods of screening therapeutic agents including already-approved drug agents, biologies and small molecules against cell lines, primary cells and tissue samples to determine changes in local or global histone modification patterns.

BACKGROUND OF THE INVENTION

[0003] A potential therapeutic target for many diseases are histone proteins, particularly histones exhibiting post-translational modifications (e.g., increased or decreased acetylation or methylation). There is evidence of a relationship between histone modification and patterns of gene expression and chromatin structure; and histone modification may correlate with the prognosis of diseases, such as cancer. There is a need for additional investigation into the nature of the relationship between patterns of histone modifications and disease.

SUMMARY OF THE INVENTION

[0004] In one aspect, the disclosure provides a method of identifying a therapeutic target comprising:

(a) obtaining a sample derived from a subject with a disease or disorder; (b) isolating from said sample one or more partial or whole histones, wherein such isolating is achieved by targeting one or more modified histone residues correlated with said disease or disorder; (c) analyzing the binding partners of said one or more partial or whole histones; and (d) comparing said binding partners to the binding partners of one or more partial or whole histones isolated from a subject who does not exhibit said disease or disorder; and (e) identifying the binding partners that exhibit enhanced or reduced levels of binding to histones derived from a subject with a disease or disorder compared to the level of binding to histones derived from a subject who does not exhibit said disease or disorder. In some embodiments, the one or more modified histone residues is selected from the group consisting of: H3 K9, H3 Kl 8, H4 Kl 2, H3 K4, H3K27, H3K36, H3 and H4 pan-acetylation, and H4 R3. In another embodiment, the modification of said one or more modified histone residues is selected from the group consisting of: H3 K9 acetylation, H3K9 monomethylation, H3K9 dimethylation, H3K9 trimethylation, H3 K18 acetylation, H4 K12 acetylation, H4K20 monomethylation, H4K20 dimethylation, H4K20 trimethylation, H3 K4 monomethylation, H3K4 dimethylation, H3K4 trimethylation, H3K36 monomethylation, H3K36 dimethylation, H3K36 trimethylation, H3K27 monomethylation, H3K27 dimethylation, H3K27 trimethylation, and H4 R3 dimethylation. [0005] In other embodiments, the binding partner is DNA, RNA, or protein. In a preferred embodiment, the binding partner is protein. In yet another embodiment, the biological sample is a human or non-human animal, hi preferred embodiments, the sample is from a human subject. In some embodiments, the sample is a bodily fluid. In some embodiments the sample is selected from the group consisting of: whole blood, peripheral blood, a cell line, lung biopsy, prostate biopsy, colon biopsy, pancreas biopsy, breast biopsy, kidney biopsy, gall bladder biopsy, uterus biopsy, thyroid biopsy, bladder biopsy, and skin biopsy.

[0006] hi some embodiments, the subject exhibits a disease or disorder that is cancer.

[0007] hi some embodiments, the analyzing comprises using a technique selected from the group consisting of mass-spectroscopy, flow cytometry, immunological assay, ChIP, ChIP-Seq, RNA Alternative Splicing, two-dimensional imaging and three-dimensional imaging. In some embodiments, the analyzing comprises using the technique of MALDI-TOF.

[0008] hi a second aspect, this disclosure provides a method of drug screening comprising: (a) obtaining a sample derived from one or more subjects with a disease or disorder; (b) applying a chemical compound library to said sample; and (c) identifying one or more compounds that can reverse or prevent one or more histone modifications present in said sample, hi some embodiments, said one or more histone modifications are selected from the group consisting of: H3 K9 acetylation, H3 Kl 8 acetylation, H3K27 methylation, H3K36 methylation, H4 Kl 2 acetylation, H4K20 methylation, H3 K4 dimethylation, H3 and H4 pan- acetylation, and H4 R3 dimethylation. In some embodiments, said disease or disorder is cancer. In some embodiments, the sample is a cellular live sample, cellular immobilized sample, cell suspension, peripheral blood, bodily fluids containing cells exhibiting patterns of histone modifications, or tissue biopsy, hi some embodiments, said sample is selected from the group consisting of: cells from a tumor bank, live cancer cells, drug-treated live cancer cells, whole blood, peripheral blood, a cell line, lung biopsy, prostate biopsy, colon biopsy, pancreas biopsy, breast biopsy, kidney biopsy, gall bladder biopsy, uterus biopsy, thyroid biopsy, bladder biopsy, and skin biopsy.

[0009] hi a third aspect, this disclosure provides a method of screening biological molecules, comprising: (a) obtaining a sample derived from one or more subjects with a disease or disorder; (b) applying one or more biological molecules to said sample; and (c) identifying one or more of said biological molecules that can reverse or prevent one or more histone modifications present in said sample. In some embodiments, said one or more histone modifications are selected from the group consisting of: H3 K9 acetylation, H3 Kl 8 acetylation, H3K27 methylation, H3K36 methylation, H4 Kl 2 acetylation, H4K20 methylation, H3 K4 dimethylation, H3 and H4 pan-acetylation, and H4 R3 dimethylation. hi some embodiments, said disease or disorder is cancer, hi some embodiments, the sample is a cellular live sample, cellular immobilized sample, cell suspension, peripheral blood, bodily fluids containing cells exhibiting patterns of histone modifications, or tissue biopsy, hi some embodiments, said sample is selected from the group consisting of: cells from a tumor bank, live cancer cells, drug-treated live cancer cells, whole blood, peripheral blood, a cell line, lung biopsy, prostate biopsy, colon biopsy, pancreas biopsy, breast biopsy, kidney biopsy, gall bladder biopsy, uterus biopsy, thyroid biopsy, bladder biopsy, and skin biopsy.

[0010] In a third aspect, this disclosure provides a method of drug screening comprising: (a) obtaining a sample derived from one or more subjects with a disease or disorder; (b) applying one or more already- approved drug agents to said sample; and (c) identifying one or more of said already-approved drug agents that can reverse or prevent one or more histone modifications present in said sample. In some embodiments, said one or more histone modifications are selected from the group consisting of: H3 K9 acetylation, H3 Kl 8 acetylation, H3K27 methylation, H3K36 methylation, H4 Kl 2 acetylation, H4K20 methylation, H3 K4 dimethylation, H3 and H4 pan-acetylation, and H4 R3 dimethylation. In some embodiments, said disease or disorder is cancer. In some embodiments, the sample is a cellular live sample, cellular immobilized sample, cell suspension, peripheral blood, bodily fluids containing cells exhibiting patterns of histone modifications, or tissue biopsy. In some embodiments, said sample is selected from the group consisting of: cells from a tumor bank, live cancer cells, drug-treated live cancer cells, whole blood, peripheral blood, a cell line, lung biopsy, prostate biopsy, colon biopsy, pancreas biopsy, breast biopsy, kidney biopsy, gall bladder biopsy, uterus biopsy, thyroid biopsy, bladder biopsy, and skin biopsy..

[0011] In a fifth aspect, this disclosure provides a method of sharing information among health-care workers and/or researchers comprising: (a) obtaining a database of samples from subject populations stratified according to histone modification profiles, wherein said database is provided on a computer-readable medium; and (b) using a computer to access said database. In some embodiments, said database further comprises a feature to permit submission of subject information. In some embodiments, said subject information is selected from the group consisting of: disease status; gender; race; age; geographical location; current drug regimen, drug regimen, genomic information, and medical history.

INCORPORATION BY REFERENCE

[0012] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION

[0013] This disclosure provides methods and compositions to identify a target(s) for therapies for diseases caused by, or correlated with, aberrant histone post-translational modification patterns (e.g., increased or decreased histone acetylation or methylation at one or more specific residues, when compared to a normal subject). In addition, this disclosure provides readout methods of screening for drugs, compounds or biological molecules (i.e., biologies) to treat, prevent, or reverse histone modifications and histone modifications patterns, especially histone modifications correlated with certain cancers (e.g., prostate cancer). In addition, this disclosure provides methods and/or systems of compiling information useful to physicians (or other health-care workers) treating subjects experiencing a disease or disorder related to, or correlated with, aberrant histone modifications and histone modification patterns and to researchers interested in studying, or obtaining, the histone modification profiles of subjects in populations for the purpose of patient sample profiling. Such methods of patient profiling allow researchers to predict patient response to therapeutics, patient prognosis either alone or in combination with various two-dimensional and three-dimensional imaging techniques allowing for the detection of the earliest morphological and/or molecular pathological changes detectable in otherwise normal cells that may exhibit signs of irregular metabolic changes due or related to a variety of biochemical processes or events including, but not limited to, substrates involved in the nucleotide synthesis process, ATP production, and conformational changes in the structure of histone proteins. Such changes may result in a cascade of molecular events affecting control of gene expression locally (i.e., at the promotor sites of genes) or globally across the entire genome. These changes produce in cancer cells a unique phenotype that exhibits a unique pattern or patterns of local or global histone modifications predictive of clinical outcome in a patient.

[0014] Currently, antibodies exist that are capable of detecting the presence of modified histones in a sample (e.g., cancer biopsy). Modifications in certain histone residues have been correlated with specific cancers such as prostate cancer. Specifically, antibodies exist that can detect H3 K9 acetylation, H3 Kl 8 acetylation, H4 Kl 2 acetylation, H3 K4 dimethylation, and H4 R3 dimethylation, all of which have been correlated with the prognosis of cancers (e.g., prostate cancer). See, e.g., Seligson et al., (2005) Nature 435: 1262-66; Suka et al., (2001) MoI Cell 8:473-79.

[0015] This disclosure provides methods of using such antibodies (or other molecules described herein) or non-antibody-based methods or label-free methods to identify binding partners of modified histone proteins (e.g., H3 K9 acetylation, H3K9 methylated, H3 Kl 8 acetylation, H3K27 methylated, H3K36 methylated, H4 Kl 2 acetylation, H4K20 methylated, H3 K4 dimethylation, and H4 R3 dimethylation). Although in preferred embodiments antibodies to specific modified histone residues are used, this disclosure also encompasses the use of synthetic molecules (e.g., aptamers) designed to specifically bind modified histone proteins. The modified histone proteins may be either whole histones or partial histones. Proteins (or other types of molecules, e.g., DNA, RNA) that preferentially bind a modified histone molecule over an unmodified histone molecule may play an important role in the pathogenesis of disease, e.g., cancer. In some examples, specimens from subjects with cancer (e.g., prostate cancer) may be prepared, and then one or more of the antibodies described herein is used to immunoprecipitate a modified partial or whole histone protein.

Immunoprecipitation techniques are well-known in the art. As a control, antibodies to unmodified histone proteins (e.g., antibodies to H3 or H4) may be applied to control samples from the same subject or to a subject who does not exhibit signs or symptoms of prostate cancer. Other controls known in the art to control for nonspecific binding may also be used. The binding partners (e.g., protein) that specifically bind more strongly to modified histones than to unmodified histones, can then be identified by mass spectrometry platforms, such as electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS), quadrupole TOF- MS, High Definitions MS technologies designed to use with UPLC-MS, MS-MS, MSe, and/or matrix- assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF-MS). These techniques may also provide high-resolution, exact mass, mass precursor, fragment ion data over a wide dynamic range, information on the number of histone precursor variants, histone variants, and the proportion of each one within a sample culminating into a histone mass library/histone ion catalog of the specimen. This can permit high resolution correlation studies to identify the specific variants that can have an impact on disease presence/progression, stage, etc.

[0016] The methods described herein may provide an improved rational approach for selecting therapeutic targets. This process may also be repeated using multiple samples in order to detect binding trends, and to correlate the presence of specific binding partners with the degree (or grade) of cancer. For example, analysis of multiple samples can be used to identify binding partners (e.g., proteins, DNA, or RNA), that preferentially bind to modified histone proteins present in samples from subjects with low-grade tumors. The presence of such binding partners, and the degree of binding, can then be analyzed in samples from subjects with increasing grades of tumors. A binding partner (e.g., protein, DNA, RNA) that exhibits increased binding to modified histones in samples from subjects with higher grades of tumors may play an especially important role in disease pathogenesis or progression. Histone variants with one-carbon methyl group or acetyl group, two-carbon methyl group or acetyl group, three-carbon methyl group or acetyl group or higher carbon number- methyl group or acetyl group may selectively bind to a binding partner (e.g., protein, DNA, RNA) that exhibits decreased binding to modified histones thereby causing a lower percent cell staining yet still detecting an IHC signal from cancer cells in samples from subjects with higher grades of tumors or cancers with an aggressive profile. Such variants may render cancer cells or cancer stem cells or dormant cancer cells to be more prone to reverting to a cellular phenotype that exhibits lower levels of global patterns of histone modifications, which is the hallmark of cancers in patients with highest risk of cancer recurrence. These altered binding patterns of global histone modifications is due to selective carbon moiety binding preferences of certain cancer cells within a population of cancer cells having the innate characteristics to develop into aggressive cancers.

[0017] A similar approach can be used to monitor the treatment of subjects suffering from diseases such as cancer (e.g., prostate cancer). For example, a subject undergoing a chemotherapeutic regimen may provide tissue specimen over time. Global histone modification patterns can be analyzed in order to monitor the subject's response to treatment. Moreover, the presence of a binding partner (that binds a specific modified histone) described herein can also be monitored over time, in order to monitor a subject's response to treatment. In some cases, it may be easier to monitor the binding partner or assess the recurrence risk of cancer in a patient or a patient's response to treatment by actually determining global patterns of binding moieties and through performing either a negative or positive carbon number quantitation as the earliest sign of cancer recurrence in patients. The label-free or antibody-based methods of detecting global levels of carbon-moieties in cancer cells linked to specific histone marks or histone variants can then be used to analyze the over-riding ability of certain cancer cells by detecting their unique combination and patterns of one-carbon methyl group or acetyl group or multi-carbon methyl group or acetyl group and to develop a single-molecule (i.e., single carbon molecule) assay predictive of a patient's response to therapeutics or prognosis of cancer or other diseases. Such prediction of therapeutic response or prognosis of disease is achieved by detecting and analyzing the underlying cellular metabolic alterations of a cancer cell or other diseased cell and comparing and comparing this "molecular metabolic signature" to the "molecular metabolic signature" obtained from a normal cell or cells or a cancer cell or cells less prone to become aggressive during the onset of cancer or progression of cancer and during periods where therapeutics are administered to patients.

[0018] Another way to monitor drug action during discovery and development can employ using radiolabeled carbon to track the metabolic histone products through secreted bodily fluids over time. Specific live cells, cell-lines, xenograft models, specific genetically engineered cells or epigentically-modified cells (global histone modifications in particular), stem cells, cancer stem cells, pluripotent stem cells,

hematopoietic stem cells could be drug treated and assayed for various levels of such histone variants to determine specific epigenetic histone and histone variant signatures. Disease specific cell-lines with varying characteristics including cancer cell-lines naturally exhibiting or engineered to exhibit one or more of such histones, histone precursor variants or histone variants may be implanted into preclinical animal models treated with different drugs, immunotherapies, biologies, chemotherapeutics, radiation, hormone therapeutics, etc. to assess the mechanism of action of such drugs and to extrapolate information as to potential toxicity, efficacy of such drugs and postulate potential methods of individual therapies or combination therapies, adjuvant and new-adjuvant therapies. Individually or as in a panel such library of histone proteins, histone precursor variants and histone variants pertaining to different diseases may be screened by high throughput techniques using Mass Spectroscopy label-free detection methods or antibody-based methodologies including multi-color flow cytometry to provide cellular or system biology information linking such histone proteins, histone precursor variants, histone variants to the onset, progression of disease and survival probabilities of patients, as well as providing context-based, predictive histone biomarker profile that may be linked to susceptibility to a disease and vulnerability to particular therapy or combination of therapies. Using advanced two-dimensional or three-dimensional imaging tools global levels of radiolabeled carbon moieties (present as molecular binding partners to histone marks and/ or histone variants) can be assessed, and as integral components of histone marks and/ or histone variants, can be assayed to assess the prognosis of a cancer patient or response of a cancer patient to administered therapeutics or in selection of said therapeutics.

[0019] Such histone marks, histone precursor variants and histone variants are used to develop therapeutics that mechanistically cause hyper expression of such modifications and hyper activation of enzymes or substrates causing such modifications within cells or conversely result in a reduction in the quantities of such histone modifications or reduction of enzymatic activities of the enzymes or global levels of substrates for these enzymes involved in such post translational modifications linked to the clinical outcome of a cancer patient.

[0020] Such histone marks, histone precursor variants or histone variants linked to a particular known or unknown disease states or in case of cancer may be enriched by immobilization in primary tumor tissue specimens, FFPE-tissue or freshly resected tumors or other biologically relevant specimens and subsequently probed with primary and labeled secondary antibodies to generate a chemiluminescent epigenetic signature that can be used to quantify the exact or relative amount of these proteins. Instrumentations capable of separating proteins based on isoelectric focusing (IEF) may be used in conjunction with application of ultraviolet light onto the surface of the specimens containing cells or tissue or other physiologically relevant specimens in order to activate various chemical coatings to form covalent bonds with the histone proteins, histone precursor variants, histone variants and thereby provide for a nano-immunoassay protein analysis. Such characterization is used to study mechanism of signaling pathways modulated by varying levels of histone marks, histone precursor variants, histone variants to gain better clinical understanding of the role of such post translational histone modifications may have on the onset, progression of disease or alterations in the survival probability in patients treated with various drugs or therapeutic regimens.

[0021] In another embodiment, the role of such histone protein modifications including all forms of post translational modifications of his tones for determination of the risk of recurrence of cancer and prediction of response to various therapeutics including chemotherapy may be determined by a quantitative and comparative method whereby such histone proteins, histone precursor variants, histone variants are measured in individual cells enriched prior or during application of flow cytometry and compared with other cells within the enriched population as well as the population of non-enriched cells and in parallel conduct a differential signal subtraction where individual patient specimens with similar histone proteins, histone precursor variants, histone variants are clustered together as we have done previously with the demonstration of the histone code in retrospectively collected FFPE-tissue specimens in patients with cancer and conversely cluster the patients that have substantially different patterns of histone protein, histone precursor variant, histone variants in another cluster. This method provides for super stratification of patients that not only allows for classic separation of patient population into distinct groups but also within each group sub-stratifies them with high degree of resolution based on specific, topologically and etiologically relevant cellular localization of the histone proteins, histone precursor variants, histone variants epigenetic signatures that may be exhibited in the nucleus of normal cells and cancer cells chronologically and in sequence or possibly in patterns similar to global cellular patterns of histone modifications previously observed. Such ability to super stratify patients into distinct groups and sub-groups with unique epigenetic molecular signatures addressing the inherent heterogeneity of the disease, including cancer, provides with accurate therapeutic targets for therapeutic intervention and ultimate practice of highly individualized personal medicine.

[0022] In another embodiment using AQUA™ technology for MS quantification, histone protein biomarkers, histone precursor variants, histone variants are treated with synthetic tryptic peptide that corresponds to a peptide of interest and as such one stable isotope-labeled amino acid incorporated in order to form a histone protein library including histone precursor variants, histone variants. This library can then be used to study the mechanism of action of various signal transduction pathways and to better understand the role of individual gene or groups of genes that act as initial switches to turn on or turn off specific epigenetic changes that in turn further regulate the role of other components of cellular machinery including the putative role of RNAi, miRNAs, siRNA and other silencing and regulating species of RNAs that effect changes in the non-coding regions of the DNA that in turn control the specific modifications on the histone tails. We hereby claim that histone precursor variants or histone variants or combination of histone proteins, histone precursor variants, histone variants may form permanent or intermediary species of histone macromolecules capable of impacting the chromatin structure along the entire human genome and either in the direction of stabilization of chromosomal structure and integrity in the healthy, normal cells during normal cellular functions or alternatively in an adverse way impacting the chromatin structure and contributing to the chromosomal instability and aberrations in key regions of the DNA and during this uncontrolled process and inadvertently turn genes off or on haphazardly and in varying degrees and speed and thereby render normal cells unable to stop their uncontrolled proliferation which is a process that can possibly be reversed by controlling the epigenetic master switches turned back on through a sequential and chronological additions of specific amino acid residues to the histone tails and only in a predetermined fashion and epigenetically programmed sequence of events that they were once committed to within the progenitor stem cells, pluripotent stem cells, hematopoietic stem cells, normal somatic cells and in close proximity to the influx of normal extracellular matrix effects regulating the normal exchange of metabolic ingredients and reversible cellular and intracellular metabolic processes.

[0023] In another embodiment, each histone protein, histone precursor variant, histone variant identified by various quantitative methods mentioned may be assayed against the presence or absence of specific kinases in cell-lines devoid of the other kinases or in as many as permutations thereof desired by researcher to assess the potential role of each histone protein, histone precursor variant, histone variant in turning on and off genetic switches or alternatively conduct such experiments in a high throughput fashion and assay for the potential therapeutic efficacy or toxicity of thousands of compounds in a catalog of biologies or library of immunotherapeutic or chemotherapeutic agents and look for cellular modulations in the direction of increasing global cellular patterns of histone modifications, i.e. global cellular patterns of hyper acetylations of histone proteins. [0024] In another embodiment, such histone proteins, histone precursor variants, histone variants may be assayed in tumor tissue specimens or other physiologically relevant specimens to determine possible correlations with specific mutations present in patient samples. These mutations can be germ-line mutations or somatic mutations. For example, histone variants may be correlated with BRAF and KRAS mutations in patients with melanoma and colon cancers to better understand the role of inhibitors of Aurora, CSFlR, JAK2 kinases and the efficacy of such therapies for various inflammatory diseases and many other types of cancers.

[0025] Specific types of cancers or malignant tumors, either primary or secondary, for which drugs, or drug targets, can be identified using the methods described herein, or which can be treated or monitored using the methods described herein include breast cancer, skin cancer, bone cancer, prostate cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, gall bladder, pancreas, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor, gallstones, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms, intestinal ganglioneuromas, hyperplastic corneal nerve tumor, marfanoid habitus tumor, WiIm' s tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignant melanomas, epidermoid carcinomas, and other carcinomas and sarcomas.

Already-Approved Drug, Small Molecule or Biologies Screening

[0026] This disclosure provides methods of screening of already approved drug agents, small molecule compounds (e.g., new chemical entities or already-known small molecule chemical entities) or biological molecules (or any combination of the foregoing) capable of reversing or preventing a specific histone modification pattern, which can either improve a cancer patient's clinical outcome or contribute to rapid progression and recurrence of cancer. In some cases, a cell line derived from a subject exhibiting a disease or disorder (or live cells from a tumor bank implanted in a live animal) e.g., cancer, prostate cancer, etc, is used to screen for an already-approved drug agent, biologic or small molecule compound (or any combination of the foregoing) capable of reversing a specific histone modification. By "already-approved drug agent" is meant any drug agent approved for marketing and use by a regulatory agency charged with such approval, e.g., the U.S. Food and Drug Administration, the European Medicines Agency, the Japanese Ministry of Health, Labor and Welfare, and the like. For example, a cultured cell line or live primary cells may exhibit one or more of the following histone modifications: H3 K9 acetylation, H3K36 acetylation, H3K9 methylation, H3 Kl 8 acetylation, H4 Kl 2 acetylation, H4K20 acetylation, H3K27 methylation, H3 K4 dimethylation, and H4 R3 dimethylation. Following the administration of an already-approved drug agent, biologic or small molecule compound (or any combination of the foregoing) to said live primary cells or cultured cell line, the level of histone modification and unique carbon moiety profile can be assessed by one or more techniques described herein, e.g., immunohistochemistry, Maldi-TOF, radiolabeled carbon, single carbon molecule detection using HPLC, GC/MS, GC/MS/MS, etc.

[0027] Alternatively, the activity of a promoter at or near one of the histone modifications described herein may be assessed. Promoter activity can be quantified by measuring a property of the reporter polypeptide (e.g., enzymatic activity or fluorescence), reporter polypeptide expression (e.g., by an ELISA assay), or reporter mRNA expression (e.g., by a fluorescent hybridization technique). Suitable reporter polypeptides include, e.g., firefly luciferase, Renilla luciferase, fluorescent proteins (e.g., enhanced green fluorescent protein), β-galactosidase, β lactamase, and horseradish peroxidase. A positive result in any of the assays described herein, such as a significantly higher level of activity for a test agent than for a control agent, may be interpreted as a preliminary indication that a test agent is capable of affecting histone modification.

[0028] Various types of tissues and live primary cells, and cell lines can be used for the drug screening assays described herein. In some cases, the cell lines are derived from a subject with any of the cancers described herein (e.g., prostate cancer). In some cases, tissue biopsies are used for drug screenin. In some embodiments, the tissue is selected from the group consisting of: whole blood, peripheral blood, a cell line, lung biopsy, prostate biopsy, colon biopsy, pancreas biopsy, breast biopsy, kidney biopsy, gall bladder biopsy, uterus biopsy, thyroid biopsy, bladder biopsy, and skin biopsy.

[0029] The conditions for the assays may vary and depend upon the nature of the assay protocol being utilized and the cells and agent being employed. For such assays, the cell culture period prior to an endpoint assay may vary from at least about 3 days to at least about 40 days, e.g., 5, 6, 9, 10, 12, 14, 20, 21, 25, 26, 27, 30, 32, 34, 36, 38, or other period from at least about 3 days to at least about 40 days. Additionally, in most cases the time for the test agent incubation ranges from at least about 30 minutes to about 40 days, e.g., 1 hour, 2 hours, 12 hours, 18 hours, 1 day, 3 days, 5 days, 7 days, 14 days, 21 days, 25 days, 30 days, 34 days, or any other period from at least about 30 minutes to at least about 40 days.

[0030] In some embodiments, the agent to be tested is an siRNA, including, but not limited to, a double stranded RNA that comprises about 19 base pairs of a target gene sequence and is capable of inhibiting target gene expression of RNA interference. See, e.g., Scherr et al., (2007), Cell Cycle, 6(4):444-449. In some embodiments, the siRNAs to be assayed include, but are not limited to, whole-genome siRNA libraries, as described in, e.g., Miyagishi et al., (2003), Oligonucleotides, 13(5):325-333; and Huesken et al., (2005), Nat. BiotechnoL, 8:995-1001. Suitable whole genome siRNA libraries, e.g., arrayed siRNA libraries that are commercially available include, the "Human Whole Genome siRNA Set V4.0" from Qiagen (Valencia, CA); the "Human siGENOME siRNA Library - Genome" from Dharmacon, Inc. (Lafayette, CO); and the Silencer® Human Genome siRNA Library from Ambion (Austin, TX). Methods and reagents for introducing siRNAs include, but are not limited to, commercial reagents such as Lipofectamine™ RNAiMAX

(Invitrogen, Carlsbad, CA), TransMessenger Transfection Reagent (Qiagen, Valencia, CA), or Dharma FECT^® (Dharmacon, Lafayette, CO). See, e.g., Krausz (2007), MoI. Biosyst., 3(4):232-240. In some embodiments, a viral RNAi library is used as described in, e.g., Root et al., (2006), Nat. Methods, 3(9):715- 719.

[0031] Optionally, the test agents to be screened are small molecules. The test molecules may be individual small molecules of choice or in some cases, the small molecule test agents to be screened come from a combinatorial library, i.e., a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks." For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. Indeed, theoretically, the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds. See, e.g., Gallop et al., (1994), J. Med. Chem., 37(9), 1233-1251. Preparation and screening of combinatorial chemical libraries are known in the art. Combinatorial chemical libraries include, but are not limited to: diversomers such as hydantoins, benzodiazepines, and dipeptides, as described in, e.g., Hobbs et al., (1993), Proc. Natl. Acad. Sci. U.S.A., 90:6909-6913; analogous organic syntheses of small compound libraries, as described in Chen et al., (1994), J. Amer. Chem. Soc, 116:2661- 2662; Oligocarbamates, as described in Cho, et al., (1993), Science, 261:1303-1305; peptidyl phosphonates, as described in Campbell et al., (1994), J. Org. Chem., 59: 658-660; and small organic molecule libraries containing, e.g., thiazolidinones and metathiazanones (U.S. Pat. No. 5,549,974), pyrrolidines (U.S. Pat. Nos. 5,525,735 and 5,519,134), benzodiazepines (U.S. Pat. No. 5,288,514).

[0032] Numerous combinatorial libraries are commercially available from, e.g., ComGenex (Princeton, NJ); Asinex (Moscow, Russia); Tripos, Inc. (St. Louis, MO); ChemStar, Ltd. (Moscow, Russia); 3D

Pharmaceuticals (Exton, PA); and Martek Biosciences (Columbia, MD).

[0033] Promising test compounds may be screened in secondary screens for toxicity or effectiveness. In some cases, a promising test agent may be tested along with a second compound, particularly a compound with a known therapeutic effect, in order to measure synergism between the two compounds. For example test agents to be screened may be used in combination with a known histone modification agent, e.g, HDAC inhibitor, SAHA, tricostatin A, etc., prodrugs, drug analogs or a chemotherapeutic agent.

[0034] An "antibody" as the term is used herein, includes reference to any molecule, whether naturally- occurring, artificially induced, or recombinant, which has specific immunoreactive activity. Generally, though not necessarily, an antibody is a protein that includes two molecules, each molecule having two different polypeptides, the shorter of which functions as the light chains of the antibody and the longer of which polypeptides function as the heavy chains of the antibody. Normally, as used herein, an antibody will include at least one variable region from a heavy or light chain. Additionally, the antibody may comprise combinations of variable regions. The combination may include more than one variable region of a light chain or of a heavy chain. The antibody may also include variable regions from one or more light chains in combination with variable regions of one or more heavy chains. An antibody can be an immunoglobulin molecule obtained by in vitro or in vivo generation of the humoral response, and includes both polyclonal and monoclonal antibodies. Furthermore, the present invention includes antigen binding fragments of the antibodies described herein, such as Fab, Fab', F(ab)₂, and Fv fragments, fragments comprised of one or more CDRs, single-chain antibodies (e.g., single chain Fv fragments (ScFv)), disulfide stabilized (dsFv) Fv fragments, heteroconjugate antibodies (e.g., bispecific antibodies), pFv fragments, heavy chain monomers or dimers, light chain monomers or dimers, and dimers consisting of one heavy chain and one light chain, all of which are encompassed by the terms "antibody" or "antibody structure." Such antibody fragments may be produced by chemical methods, e.g., by cleaving an intact antibody with a protease, such as pepsin or papain, or via recombinant DNA techniques, e.g., by using host cells transformed with truncated heavy and/or light chain genes. Synthetic methods of generating such fragments are also contemplated. Heavy and light chain monomers may similarly be produced by treating an intact antibody with a reducing agent, such as dithiothreitol or beta-mercaptoethanol, or by using host cells transformed with DNA encoding either the desired heavy chain or light chain or both. An antibody immunologically reactive with a particular antigen can be generated in vivo or by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors.

Information sharing/portals

[0035] This disclosure provides a method of sharing information among health-care workers and/or researchers comprising: (a) obtaining a database of samples from subject populations stratified according to histone modification profiles, wherein said database is provided on a computer-readable medium; and (b) using a computer to access said database. In some embodiments, said database further comprises a feature to permit submission of subject information. In some embodiments, said subject information is selected from the group consisting of: disease status; gender; race; age; geographical location; current drug regimen, drug regimen, genomic information, and medical history. Especially useful information that can be included in the database is the grade of tumor from the subject, which can be correlated with global histone modification patterns, as well as with binding partners of specific modified histones. The computerized database can be used to track tissue samples from various subjects. Such tissue samples may be annotated with some or all of the information described herein.

[0036] In some cases, the database includes a program useful for analyzing the data obtained from various subject samples. For example, the program may be used to evaluate trends of tumor grades. The program may be used to evaluate the types of cancers experienced by subjects in particular geographic locations. The database may be used to correlate information about subjects with wide range of cancers, particularly any cancer described herein. Healthcare workers or researchers may also add to the database information, not only concerning, global histone modifications of a subject but also add information regarding any of the binding partners desribed herein, preferably binding partners that preferentially bind modified whole or partial histones over non-modified histones.

[0037] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of identifying a therapeutic target, comprising:

(a) obtaining a sample derived from a subject with a disease or disorder;

(b) isolating from said sample one or more partial or whole histones, wherein such isolating is achieved by targeting one or more modified histone residues correlated with said disease or disorder;

(c) analyzing the binding partners of said one or more partial or whole histones;

(d) comparing said binding partners to the binding partners of one or more partial or whole histones isolated from a subject who does not exhibit said disease or disorder; and

(e) identifying the binding partners that exhibit enhanced or reduced levels of binding to histones derived from a subject with a disease or disorder compared to the level of binding to histones derived from a subject who does not exhibit said disease or disorder.

2. The method of claim 1, wherein said one or more modified histone residues is selected from the group consisting of: H3 K9, H3 K18, H3K27, H3K36, H4 K12, H4K20, H3 K4, and H4 R3.

3. The method of claim 1, wherein the modification of said one or more modified histone residues is selected from the group consisting of: H3 K9 acetylation, H3K9 methylation, H3K27 methylation, H3K36 acetylation, H3 Kl 8 acetylation, H4 Kl 2 acetylation, H4K20 acetylation, H3 K4 dimethylation, and H4 R3 dimethylation.

4. The method of claim 1, wherein said binding partner is DNA, RNA, or protein.

5. The method of claim 1, wherein said binding partner is protein.

6. The method in claim 1 , wherein said biological sample is a human or non-human animal.

7. The method of claim 1, wherein said biological sample is a bodily fluid.

8. The method of claim 1, wherein said sample is selected from the group consisting of: whole blood, peripheral blood, a cell line, lung biopsy, prostate biopsy, colon biopsy, pancreas biopsy, breast biopsy, kidney biopsy, gall bladder biopsy, uterus biopsy, thyroid biopsy, bladder biopsy, and skin biopsy.

9. The method of claim 1, wherein said disease or disorder is cancer.

10. The method of claim 1 , wherein said analyzing comprises using a technique selected from the group selected from: mass-spectroscopy, flow cytometry, immunological assay, radiolabeled carbon moieties, HPLC, GC, GC/MS, and GC/MS/MS.

11. A method of screening one or more small molecule compounds, comprising:

(a) obtaining a sample derived from one or more subjects with a disease or disorder;

(b) applying a chemical compound library to said sample; and

(c) identifying one or more compounds that can reverse or prevent one or more histone modifications present in said sample.

12. The method of claim 11, wherein said one or more histone modifications are selected from the group consisting of: H3 K9 acetylation, H3K9 methylation, H3K27 methylation, H3K36 acetylation, H3 Kl 8 acetylation, H4 K12 acetylation, H4K20 acetylation, H3 K4 dimethylation, and H4 R3 dimethylation.

13. The method of claim 11, wherein said disease or disorder is cancer.

14. The method of claim 11, wherein said sample is a cell line, cellular sample, primary cell or tissue biopsy.

15. The method of claim 11, wherein said sample is selected from the group consisting of: whole blood, peripheral blood, a cell line, lung biopsy, prostate biopsy, colon biopsy, pancreas biopsy, breast biopsy, kidney biopsy, gall bladder biopsy, uterus biopsy, thyroid biopsy, bladder biopsy, and skin biopsy.

16. A method of sharing information among health-care workers and/or researchers comprising:

(a) obtaining a database of samples from subject populations stratified according to histone modification profiles, wherein said database is provided on a computer-readable medium; and

(b) using a computer to access said database.

17. The method of claim 16, wherein said database further comprises a feature to permit submission of subject information.

18. The method of claim 17, wherein said subject information is selected from the group consisting of: disease status; gender; race; age; geographical location; current drug regimen, drug regimen, genomic information, and medical history.

19. A method of screening of a biological molecule, comprising:

(a) obtaining a sample derived from one or more subjects with a disease or disorder;

(b) applying one or more biological molecules to said sample; and (c) identifying one or more of said biological molecules that can reverse or prevent one or more histone modifications present in said sample.

20. A method of screening already-approved drugs, comprising:

(a) obtaining a sample derived from one or more subjects with a disease or disorder;

(b) applying one or more already-approved drugs to said sample; and

(c) identifying one or more of said drugs that can reverse or prevent one or more histone modifications present in said sample.