WO2011137302A1 - Procédés d'identification de voies de signalisation intracellulaire régulées de manière aberrante dans des cellules cancéreuses - Google Patents

Procédés d'identification de voies de signalisation intracellulaire régulées de manière aberrante dans des cellules cancéreuses Download PDF

Info

Publication number
WO2011137302A1
WO2011137302A1 PCT/US2011/034476 US2011034476W WO2011137302A1 WO 2011137302 A1 WO2011137302 A1 WO 2011137302A1 US 2011034476 W US2011034476 W US 2011034476W WO 2011137302 A1 WO2011137302 A1 WO 2011137302A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
cancer
histone modification
aberrantly
signaling pathway
Prior art date
Application number
PCT/US2011/034476
Other languages
English (en)
Inventor
Andrew S. Chi
Bradley Bernstein
Esther Rheinbay
Original Assignee
The General Hospital Corporation
Trustees Of Boston University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The General Hospital Corporation, Trustees Of Boston University filed Critical The General Hospital Corporation
Publication of WO2011137302A1 publication Critical patent/WO2011137302A1/fr

Links

Classifications

    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/30—Detection of binding sites or motifs
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B5/00—ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding

Definitions

  • Cell processes such as proliferation, differentiation, apoptosis, mobility, and self- renewal are governed by signaling pathways. Understanding signal pathway regulation and mapping which pathways are activated or inactivated in a particular cell, cell type, tissue or organ at which points during development and upon which intra- and extra-cellular cues is an important aim in biomedical research.
  • cancer cells exhibit aberrant growth capabilities and modified differentiation potential when compared to normal cells. These characteristics are often the result of underlying changes in signaling pathways.
  • Certain aspects of the invention relate to the use of chromatin profiling to acquire genome-wide histone modification maps. Such maps may be generated for cancer cells, including cancer stem cells, tumors and cancer cell lines, and their normal cell counterparts, with subsequent identification of aberrantly regulated (e.g. , activated or inactivated) signaling pathways of prognostic and therapeutic significance. Certain aspects of the invention relate to chromatin profiling of cancer cells, including cancer stem cells, tumors and cancer cell lines, to identify new pathways for therapeutic intervention. In certain embodiments, the invention relates to profiling of cancer cells, including cancer stem cells, tumors and cancer cell lines, derived from individual patients for prognostic purposes and to guide therapeutic course of treatment (personalized medicine).
  • chromatin profiling to acquire genome-wide histone modification maps. Such maps may be generated for cancer cells, including cancer stem cells, tumors and cancer cell lines, and their normal cell counterparts, with subsequent identification of aberrantly regulated (e.g. , activated or inactivated) signaling pathways of prognostic and therapeutic significance
  • the methods provided herein can be used to identify aberrantly regulated pathways or processes, with the identity of these pathways or processes providing useful prognostic and therapeutic information.
  • the methods provided herein may comprise the identification of targets for therapeutic intervention to treat a disease, for example a cancer.
  • the method described herein may be applied to specific cancer cell models (including cancer cell lines), for example to gain general insight into a given cancer type, or may be applied to an individual patient' s tumor, for example to tailor cancer treatment (personalized medicine).
  • Certain aspects of the invention relate to the use of ChlP-Seq chromatin profiling technology (Mikkelsen et al., 2007) to localize specific covalent histone modifications genome-wide in cancer cells, including cancer stem cells, tumors and cancer cell lines, and the corresponding normal tissue cells, including stem cells and primary or other normal cell lines, from which the cancer cells were derived.
  • the methods described herein may be applied to any histone modification or chromatin marker, e.g. any of the modifications described in Table 1 and/or that are depicted Figure 1 , and may use chromatin profiling technology other than ChlP-Seq, including microarrays (e.g.
  • ChlP-on-chip also known as ChlP-chip; Bernstein, BE et al. Cell. 2005 Jan 28; 120(2): 169-81).
  • methods are provided that utilize one or more histone modifications selected from the group consisting of: histone 3 lysine 4 trimethylation (H3K4me3), histone 3 lysine 27
  • H3K27me3 histone 3 lysine 36 trimethylation
  • H3K36me3 histone 3 lysine 36 trimethylation
  • the methods described herein comprise comparing the genomic profiles of specific histone modifications in cancer cells , including cancer stem cells, tumors and cancer cell lines, against normal cells (including tissues, stem cells and primary cultured cells) from the same tissue type from which the cancer cells were derived.
  • this comparison may reveal one or more gene(s), including master regulator transcription factors, and pathways that may be considered aberrantly regulated specifically in the malignant (cancer) cells, for example the genes exhibit active chromatin in the cancer cells and inactive chromatin in the normal tissue cells.
  • the aberrantly regulated genes may be used to identify specific intracellular signaling pathways that are aberrantly regulated in the cancer cells.
  • Newly aberrantly regulated (e.g. , active or inactive) intracellular signaling pathways identified by the methods described herein may include, but are not limited to, the PI3K/Akt, Notch, Wnt, Ras/Raf/MAPK, Janus kinase, and integrin/FAK pathways.
  • the identified pathways may represent therapeutic targets which can be exploited for treatment.
  • modulation of the identified pathway can be used to inhibit the growth of the cancer cells, to induce apoptosis in the cancer cells, or to induce differentiation of cancer cells.
  • the methods described herein may be applied to any tumor type for which cells (including stem cells and cell lines) can be derived.
  • the cancer cells are glioblastoma stem cells that are compared against normal neural stem cells.
  • Certain aspects of the invention relate to methods of identifying aberrantly regulated intracellular signaling pathways in a cancer cell, wherein the methods include: (a) generating one or more genome-wide histone modification maps for a cancer cell; (b) comparing the one or more genome-wide histone modification maps for the cancer cell with the one or more genome-wide histone modification maps for the normal cell equivalent and identifying aberrantly active genes or aberrantly repressed (inactive) genes in the cancer cell based on the histone modifications prevalent at the promoter or control region of the gene, wherein aberrantly active genes or aberrantly repressed genes in the cancer cell are those genes that are differentially expressed in the cancer cell as compared to the normal cell, (c) clustering the aberrantly active genes or aberrantly repressed genes identified in (b) and applying network inference methods and motif analysis to build connectivity between these genes; and (d) identifying one or more intracellular signaling pathways that is aberrantly regulated (active or inactive) in the cancer cell based on the clustering and analysis performed in
  • the methods of identifying aberrantly regulated intracellular signaling pathways in a cancer cell further include identifying a normal, non-malignant cell equivalent based on one or more existing genome-wide histone modification maps or based on one or more genome-wide histone modification maps generated for the normal, non- malignant cell.
  • the genome- wide histone modification map of any one of the preceding methods is generated by performing chromatin precipitation of chromatin obtained from a cancer cell and a normal cell equivalent using histone modification-specific antibodies.
  • the cancer cell of any one of the preceding methods is an isolated cell that is optionally isolated from a tumor.
  • the genome-wide histone modification map of any one of the preceding methods is generated by identifying the DNA sequence of the precipitated chromatin and by correlating a chromatin state at a promoter with an active state of the promoter or an inactive state of the promoter. In certain embodiments, this correlation is performed by (a) assigning the active state to DNA sequences that are precipitated by histone modification-specific antibodies that recognize histone modifications that are associated with an active chromatin state but not by histone modification-specific antibodies that recognize histone modifications that are associated with an inactive chromatin state; or (b) by assigning the inactive state to DNA sequences that are precipitated by histone modification-specific antibodies that recognize histone modifications that are associated with an inactive chromatin state.
  • the correlation of a chromatin state at a promoter with an inactive state of the promoter is performed by assigning the inactive state to DNA sequences that i) are precipitated by histone modification-specific antibodies that recognize histone modifications that are associated with an inactive chromatin state, and ii) are precipitated by histone modification-specific antibodies that recognize histone modifications that are associated with an active chromatin state.
  • the correlation of a chromatin state at a promoter with an inactive state of the promoter is performed by assigning the inactive state to DNA sequences that are not precipitated by histone modification-specific antibodies that recognize histone modifications that are associated with an inactive chromatin state and are not precipitated by histone modification-specific antibodies that recognize histone modifications that are associated with an active chromatin state.
  • the histone modification-specific antibodies used in the preceding methods recognize histone modifications that are associated with an active chromatin state and are selected from the group consisting of H3K4 methylation, H3K36 methylation, H3K79 methylation, and histone acetylation.
  • the histone modification-specific antibodies used in the preceding methods recognize histone modifications that are associated with an inactive (chromatin state are selected from the group consisting of H3K27 methylation and H3K9 methylation.
  • the chromatin state as identified at the promoter in any one of the preceding methods is validated by gene expression analysis of the gene that is operated (controlled) by the promoter. In certain embodiments, this validation is performed with DNA microarray or RNA sequencing.
  • genes identified by any one of the preceding methods are clustered for biological/functional annotation.
  • any one of the preceding methods further includes identifying a transcription factor that is aberrantly expressed in a cancer cell, wherein identifying the transcription factor includes: i) identifying one or more DNA transcription factor binding motif that is prevalent at the promoter or control region of the aberrantly active genes and/or aberrantly inactive genes in the cancer cell, and ii) identifying a transcription factor that associates with (binds to) the prevalent DNA binding motif.
  • Certain aspects of the invention relate to methods of identifying a therapeutic course of treatment for a cancer in an individual, wherein the therapeutic course of treatment targets one or more molecules linked to an identified aberrantly regulated intracellular signaling pathway, and wherein the aberrantly regulated intracellular signaling pathway is identified by any one of the preceding methods.
  • methods of treating cancer in an individual include: i) identifying a therapeutic course of treatment for the cancer in the individual according to the preceding method, and ii) treating the individual with one or more agents that modulates the identified aberrantly regulated intracellular signaling pathway in an amount effective to treat the cancer, wherein the one or more agents that modulate the identified aberrantly regulated intracellular signaling pathway inhibit an aberrantly active intracellular signaling pathway and/or activate an aberrantly inactive intracellular signaling pathway.
  • the aberrantly regulated intracellular signaling pathway identified by any one of the preceding methods is selected from the group consisting of: PI3K/Akt, Notch, Wnt, Ras/Raf/MAPK, Janus kinase, and integrin/FAK.
  • the cancer cell of any one of the preceding methods is a cancer stem cell.
  • the preceding methods relate to a cancer stem cell that is a glioblastoma-derived stem cell (GSC) and optionally relate to a normal cell equivalent that is a human embryonic stem cell-derived neural stem cell (NCS) or a normal human astrocyte (NHA).
  • GSC glioblastoma-derived stem cell
  • NCS human embryonic stem cell-derived neural stem cell
  • NHA normal human astrocyte
  • the preceding methods relate to a cancer stem cell that is a glioblastoma-derived stem cell (GSC) and a transcription factor that is LEF1.
  • GSC glioblastoma-derived stem cell
  • the DNA transcription factor binding motif is selected from the group consisting of 5' CTTTGAT 3' (SEQ ID NO: 1); CTTTGA (SEQ ID NO: 2), CTTTGT (SEQ ID NO: 3), CTTTGATC (SEQ ID NO: 4), ATCAATCA (SEQ ID NO: 5); TCAAAG (SEQ ID NO: 6), MCTTTGWWSNY ((SEQ ID NO: 7), NWTCAAAGNN (SEQ ID NO: 8) and reverse complements of the foregoing, and/or (b) the aberrant regulated gene is selected from the group consisting of: HOXC4, HOXA3, SHOX2, EN1, HEY1, SALL3, OLIG2, ASCL1, SIM2, TFAP2A, ISL2, NR5A2, PRRX1,
  • Certain aspects of the invention relate to methods of identifying the nearest cell equivalent of a cell, wherein the methods include: (a) obtaining a histone modification profile (signature) for a first cell and obtaining a histone modification profile (signature) for at least two other cells, (b) comparing the histone modification profile (signature) of the first cell with the histone modification profile (signature) of the at least two other cells, (c) identifying a set genes that exhibit a high histone modification profile (signature) variability across the cells compared in (b), (d) determining that another cell is the nearest cell equivalent of the first cell if the number of genes of the other cell identified in (c) that exhibit a similar or essentially identical histone modification profile (signature) to the first cell is highest among the at least two other cells.
  • methods of identifying the nearest cell equivalent of a cell are provided, wherein the first cell is a cancer stem cell and the at least two other cells are two or more non-cancer cells.
  • methods of identifying the nearest cell equivalent of a cell wherein obtaining a histone modification profile (signature) includes performing chromatin precipitation of chromatin obtained from the first cell using histone modification-specific antibodies.
  • methods of identifying the nearest cell equivalent of a cell wherein obtaining a histone modification profile (signature) for the two or more other cells includes querying a public database.
  • methods of identifying the nearest cell equivalent of a cell wherein the first cell and the two or more other cells are hierarchically clustered according to the genes identified in (c) of the foregoing method of identifying the nearest cell equivalent of a cell are provided.
  • Certain aspects of the invention relate to methods for selectively killing a cancer cell, wherein the methods include contacting a mixture of cells with one or more agents that modulate the aberrantly regulated intracellular signaling pathway(s) in an amount effective to kill the cancer cell, wherein the aberrantly regulated intracellular signaling pathway(s) are identified according to the methods described herein, and wherein the one or more agents that modulate the identified aberrantly regulated intracellular signaling pathway(s) inhibit an aberrantly active intracellular signaling pathway and/or activate an aberrantly inactive intracellular signaling pathway.
  • the methods for selectively killing a cancer cell include contacting a mixture of cells with a small molecule or a siRNA.
  • the methods for selectively killing a cancer cell include contacting a glioblastoma stem cell and the identified aberrantly active intracellular signaling pathway(s) are Wnt signaling pathway and/or the Notch signaling pathway.
  • methods for selectively killing a cancer cell wherein the aberrantly regulated intracellular signaling pathway(s) is an aberrantly active Wnt signaling pathway and wherein the Wnt inhibitor: i) stabilizes the beta-catenin destruction complex, ii) inhibits GSK3 alpha/beta, Hi) interferes with the association of beta- catenin and LEF1 or iv) suppresses beta-catenin expression.
  • the preceding methods for selectively killing a cancer cell include a Wnt inhibitor that is XAV939, BIO, cercosporin or indomethacin.
  • methods for selectively killing a cancer cell wherein the aberrantly regulated intracellular signaling pathway(s) is an aberrantly active Notch signaling pathway and wherein the Notch inhibitor is DAPT.
  • methods for selectively killing a cancer cell wherein the cell is contacted in vivo.
  • methods for selectively killing a cancer cell are provided, wherein the methods are part of a post surgical treatment regimen in an individual having undergone surgery to remove a cancer.
  • Certain aspects of the invention relate to methods for selectively killing a cancer stem cell, wherein the methods include identifying aberrantly regulated intracellular signaling pathway(s) in a cancer stem cell according to the methods described herein, and contacting a mixture of cells with a modulator of the aberrantly regulated intracellular signaling pathway(s) in an amount effective to kill the cancer stem cell.
  • the methods for selectively killing a cancer cell include contacting a mixture of cells with a small molecule or a siRNA.
  • the methods for selectively killing a cancer cell include contacting a glioblastoma stem cell and the identified aberrantly active intracellular signaling pathway(s) are Wnt signaling pathway and/or the Notch signaling pathway.
  • methods for selectively killing a cancer cell wherein the aberrantly regulated intracellular signaling pathway(s) is an aberrantly active Wnt signaling pathway and wherein the Wnt inhibitor: i) stabilizes the beta-catenin destruction complex, ii) inhibits GSK3 alpha/beta, Hi) interferes with the association of beta- catenin and LEF1 or iv) suppresses beta-catenin expression.
  • the preceding methods for selectively killing a cancer cell include a Wnt inhibitor that is XAV939, BIO, cercosporin or indomethacin.
  • methods for selectively killing a cancer cell wherein the aberrantly regulated intracellular signaling pathway(s) is an aberrantly active Notch signaling pathway and wherein the Notch inhibitor is DAPT.
  • methods for selectively killing a cancer cell wherein the cell is contacted in vivo.
  • methods for selectively killing a cancer cell are provided, wherein the methods are part of a post surgical treatment regimen in an individual having undergone surgery to remove a cancer.
  • Certain aspects of the invention relate to methods of identifying aberrantly regulated intracellular signaling pathways in a cancer cell as described herein, wherein the methods further comprise validating the identified aberrantly regulated intracellular signaling pathway in the cancer cell that affects cancer cell growth and/or survival, wherein validating comprises contacting the cancer cell with one or more modulator of the aberrantly regulated signaling pathway and measuring cancer cell growth and/or survival in the presence and absence of the one or more modulator, wherein when cancer cell growth and/or survival is less in the presence of the modulator when compared to cancer cell growth and/or survival in the absence of the one or more modulator, the aberrantly regulated intracellular signaling pathway affects cancer cell growth and/or survival, optionally wherein the cancer cell is contacted in vitro.
  • Certain aspects of the invention relate to methods of diagnosing or confirming a diagnosis that a cancer cell exhibits an aberrantly regulated intracellular signaling pathway, the methods include: a) obtaining the cell, b) performing any one of the methods for identifying aberrantly regulated intracellular signaling pathways in a cancer cell as described herein, and c) diagnosing based on the results obtained in (b) if the cell exhibits an aberrantly regulated intracellular signaling pathway.
  • the methods of diagnosing or confirming a diagnosis that a cancer cell exhibits an aberrantly regulated intracellular signaling pathway are provided, wherein the cancer cell is a glioblastoma-derived stem cell.
  • methods of diagnosing or confirming a diagnosis that a cancer cell exhibits an aberrantly regulated intracellular signaling pathway are provided, wherein the aberrantly regulated intracellular signaling pathway is an aberrantly active Wnt signaling pathway or an aberrantly active Notch signaling pathway.
  • Certain aspects of the invention relate to methods of diagnosing cancer or confirming a cancer diagnosis, the methods include: (a) obtaining a cell, (b) performing any one of the methods for identifying aberrantly regulated intracellular signaling pathways in a cancer cell as described herein, wherein when the cell obtained in (a) exhibits an aberrantly regulated intracellular signaling pathway the cell is a cancer cell, and c) diagnosing cancer or confirming a cancer diagnosis based on whether the cell is a cancer cell.
  • the methods of diagnosing cancer or confirming a cancer diagnosis are provided, wherein the cancer cell is a glioblastoma-derived stem cell.
  • the aberrantly regulated intracellular signaling pathway is an aberrantly active Wnt signaling pathway or an aberrantly active Notch signaling pathway.
  • Certain aspects of the invention relate to methods of diagnosing cancer or confirming a cancer diagnosis, the methods include: (a) obtaining a cell, (b) performing any one of the methods for identifying an aberrantly regulated transcription factor in a cancer cell as described herein, wherein when the cell obtained in (a) exhibits an aberrantly expressed transcription factor the cell is a cancer cell, and c) diagnosing cancer or confirming a cancer diagnosis based on whether the cell is a cancer cell.
  • the methods of diagnosing cancer or confirming a cancer diagnosis are provided, wherein the aberrantly expressed transcription factor is a homeobox transcription factor, is a helix-loop-helix motif family transcription factor, or is associated with development, morphogenesis or the regulation of a cellular process.
  • the aberrantly expressed transcription factor is a homeobox transcription factor, is a helix-loop-helix motif family transcription factor, or is associated with development, morphogenesis or the regulation of a cellular process.
  • the aberrantly expressed transcription factor is LEF 1.
  • Certain aspects of the invention relate to methods of identifying the cancer type and/or origin of a cancer cell, the methods include: (a) obtaining a cancer cell from a tumor, (b) performing any one of the methods for identifying aberrantly regulated intracellular signaling pathways in a cancer cell as described herein, and/or performing any one of the methods to of identify the nearest cell equivalent of a cell, (c) comparing the histone modification profile (signature) obtained in (b) to either the histone modification profile (signature) obtained from a cancer cell of known cancer type and/or origin from a subject or comparing the histone modification profile (signature) obtained in (b) with the histone modification profile (signature) of one or more cancer cell of known cancer type and/or origin from a public database, and (d) identifying the origin and/or cancer type of the cancer cell from a tumor obtained in (a), wherein when the cancer cell from a tumor exhibits a histone modification profile (signature) that is closely similar or identical to the histone modification profile (signature)
  • the methods of identifying the cancer type and/or origin of a cancer cell are provided, wherein the cancer cell is a glioblastoma-derived stem cell.
  • Certain aspects of the invention relate to methods of diagnosing or confirming the diagnosis of a secondary tumor (metastasis), the methods include: (a) obtaining a first cell from a subject, (b) performing any one of the methods for identifying aberrantly regulated intracellular signaling pathways in a cancer cell as described herein, and/or performing any one of the methods to of identify the nearest cell equivalent of a cell, c) comparing the histone modification profile (signature) obtained in (b) to a histone modification profile (signature) of one or more cancer cell obtained from a subject or comparing the histone modification profile (signature) obtained in (b) with the histone modification profile
  • (signature) of one or more cancer cell from a public database and d) diagnosing that the first cell is obtained from a secondary tumor when the first cell: i) exhibits an aberrantly regulated intracellular signaling pathway, and ii) exhibits a histone modification profile (signature) that indicates that the first cell is located at a site that normally encompasses cancer cells with a histone modification profile (signature) that is different from the first cell, and/or e) diagnosing the origin of the first cell, wherein when the first cell exhibits a histone modification profile (signature) that is closely similar or identical to the histone modification profile (signature) of a cancer cell of a specific origin, then the first cell is diagnosed to be of that origin.
  • Certain aspects of the invention relate to methods of diagnosing or confirming the diagnosis of a primary tumor or a secondary tumor, the methods include: a) obtaining a cancer cell from a tumor, b) performing any one of the methods for identifying aberrantly regulated intracellular signaling pathways in a cancer cell as described herein, and/or performing any one of the methods to of identify the nearest cell equivalent of a cell, c) comparing the histone modification profile (signature) obtained in (b) to either the histone modification profile (signature) obtained for a normal cell equivalent from the site of the tumor or comparing the histone modification profile (signature) obtained in (b) with results from a public database for a normal cell from the site of the tumor, and d) diagnosing if the cancer cell is from a primary tumor or a secondary tumor, wherein when the histone modification profile (signature) obtained for the cancer cell and the normal cell in (c) indicates that they are nearest cell equivalents the tumor is a primary tumor, and wherein when the histone modification profile (signature)
  • methods of diagnosing or confirming the diagnosis of a primary tumor or a secondary tumor wherein the cancer cell is a cancer stem cell, optionally a glioblastoma-derived stem cell.
  • Certain aspects of the invention relate to identifying the effects of environmental conditions on a histone modification profile (signature) of a cell, the methods include: (a) obtaining a cell which has been exposed to a particular environmental condition or exposing a cell to a particular environmental condition, (b) performing on the cell obtained in (a) any one of the methods for identifying aberrantly regulated intracellular signaling pathways in a cell as described herein to obtain a histone modification profile of the cell, (c) comparing the histone modification profile (signatures) of the cell to a histone modification profile
  • the cell of (a) is a cancer cell.
  • aspects of the invention relate to comparing the effects of environmental conditions on histone modification profiles (signatures) of cells obtained from or exposed to different environmental conditions
  • the methods include: (a) obtaining a first cell which has been exposed to a particular environmental condition or exposing a first cell to a particular environmental condition, (b) obtaining a second cell which has been exposed to an environmental condition different from that of the first cell or exposing a second cell to an environmental condition different from that of the first cell, (c) performing on each of the first and second cell any one of the methods for identifying aberrantly regulated intracellular signaling pathways in a cancer cell as described herein to obtain a histone modification profile (signature), (d) comparing the histone modification profile (signatures) obtained for the first cell to the histone modification profile (signatures) obtained for the second cell, or comparing a histone modification profile (signatures) obtained for the first cell and the histone modification profile (signatures) for the second cell to a histone modification profile (signature) obtained from a public database
  • a histone modification profile is obtained for each of two or more (multiple) cells obtained from or exposed to two or more different environmental conditions.
  • the first cell of (a) and/or second cell of (b) is a cancer cell.
  • Figure 1 depicts various histone modifications on histone H3.
  • Figure 2 A depicts bar graphs showing signal density for H3K4me3 and H3K27me3 at the OLIG2 promoter in various GBM cancer stem cells, providing a comparison of promoter chromatin state of glioma marker OLIG2 in normal and malignant neural cells;
  • Figure 2B depicts a bar graph showing OLIG2 gene expression concordant with chromatin state;
  • Figure 2C shows relative enrichment of factors with certain functional annotation: two glioblastoma stem cell lines are enriched for homeo-box related transcription factor and development terms.
  • Figure 3 depicts a bar graph showing percent active developmental transcription factors.
  • Figure 4 depicts heat maps (A and B) showing genes over-expressed in GBM tumor tissue.
  • Figure 5 provides an overview of determining the circuitry of GBM stem cell transcription factors.
  • Figure 6 provides an overview of conducting a motif search in the promoters of GBM stem cell transcription factors.
  • Figure 7 depicts an example of a LEF1 binding site consensus motif, chromatin state in normal and GBM stem cells and box plots showing LEF1 expression in primary tumors and normal brain.
  • FIG 8 provides an overview of the Wnt pathway.
  • Figure 9A shows overrepresentation of the LEF1 motif in glioma transcription factors
  • Figure 9B shows a heatmap of Wnt pathway genes that are differentially expressed in GBM compared to normal brain (significantly different genes only);
  • Figure 9C depicts a bar graph showing decrease of relative expression of glioma transcription factor in normal and malignant stem cells after inhibition with Wnt pathway inhibitors;
  • Figure 9D photographs of cells (neurospheres) showing cell death upon treatment with Wnt pathway inhibitors. Cells were assayed after exposure to inhibitor for 3d.
  • Figure 10 provides an overview of determining and validating the glioma transcription factor circuitry.
  • Figure 11A shows a heatmap comparing epigenomic (chromatin) profiles of various cell types, revealing that the chromatin profile of epitope H3K4 of the serum-grown cells is more closely related to differentiated primary normal astrocytes than to their counterpart cancer stem cells.
  • Figure 11B depicts a graph showing a subset of epigenomic profiles of epitopes H3K4 and H3K27 from Figure 11 A.
  • transcription factors OLIG2 and ASCL1 previously identified as aberrantly active in glioma stem cells, are shown to be repressed through H3K27me3 when glioma stem cells are grown in serum.
  • Figure 12 depicts a graph showing fold induction of the transcription factor, AXIN2, in ASCL1 -transformed astrocytes versus control cells, suggesting that ASCL1 functions upstream of the Wnt pathway to regulate AXIN2 expression.
  • Certain aspects of the invention relate to the use of chromatin profiling to acquire genome-wide histone modification maps. For example, these maps may be generated for cancer cells (including cancer stem cells, tumor cells and cancer cell lines) and their normal counterparts.
  • methods are provided for identification of aberrantly regulated (including activated and inactivated) signaling pathways that utilize histone modification maps.
  • the identification of aberrantly regulated signaling pathways in certain cells, for example cancer cells may be of prognostic and therapeutic significance and may, for example, aid the identification of a treatment regimen for a disease, such as cancer.
  • methods are provided that use chromatin profiling of cancer cells to identify new pathways for therapeutic intervention.
  • methods are provided for profiling cancer cells derived from individual patients for prognostic purposes and/or to guide therapeutic course of treatment (e.g., in the practice of personalized medicine).
  • Chromatin profiles may be used to determine the patterns of covalent histone modifications genome-wide, for example in cancer stem cells or other cancer cells. Any method for interrogating histone modification patterns can be used in connection with the invention.
  • the invention relates to the use of ChlP-Seq chromatin profiling technology (Mikkelsen et al., 2007), although other methods known in the art for chromatin profiling may be used, such as ChlP-chip, as may other nucleic acid quantification methods for use with histone modifications.
  • the methods described herein may be applied to any histone modification or chromatin marker. In specific embodiments, histone
  • H3K4me3 histone 3 lysine 4 trimethylation
  • H3K27me3 histone 3 lysine 27 trimethylation
  • H3K36me3 histone 3 lysine 36 trimethylation
  • the chromatin profiles that may be obtained for example from cancer cells may be compared to non-malignant cells that represent the most similar normal counterpart of the cancer cells. This can be done through direct analysis of the non-malignant cells in parallel to the cancer profiling.
  • a classifier provided herein may be used that integrates the cancer profiles with chromatin profiling data for normal human tissues that are in the public domain. The classifier may then be used to identify the most similar normal cell types for use in the comparative analysis.
  • genes including master regulator transcription factors, and pathways that are considered aberrantly regulated specifically in the cancer cells.
  • the genes may be identified by comparing the chromatin profiles for the cancer cells and the normal counterparts. For example, by comparing the chromatin signatures, patterns suggestive of specific gene activity in the tumors may be identified. These methods may be particularly applicable in cases of transcription factor genes, as chromatin is a much more robust readout of the state of such genes than RNA profiling.
  • methods are provided using the information about aberrantly regulated genes in a combination of network inference and direct interaction analysis through motif analysis to identify specific intracellular signaling pathways that are aberrantly regulated in the cancer cells.
  • Newly identifiable aberrantly regulated intracellular signaling pathways include, but are not limited to, the PI3K/Akt, Notch, Wnt, Ras/Raf/MAPK, Janus kinase, and integrin/FAK pathways.
  • the identified aberrantly regulated pathways represent targets that can be exploited for treatment of a disease.
  • the methods described herein may be applied to any tumor type for which cells can be derived.
  • the tumor is a glioblastoma.
  • Histone modifications are a component of chromatin, which packages the DNA and influence how genes within the DNA function (e.g. , whether the respective gene is active or inactive).
  • Specific types of histone modifications at the start of a gene are associated with different gene functional states such as active, or inactive states.
  • “Inactive" states as used herein include (temporarily) repressed, silent, or permanently silenced states. Some histone modifications tend to be found at genes that are important for embryonic development or preferentially silenced in cancer.
  • methods are provided applying a chromatin profiling, such as a ChlP-Seq assay, to identify the locations of specific histone modifications across the entire genome. Such data may then be used to determine the regulation state of many, most of, or all of the genes in the cell.
  • Histones undergo posttranslational modifications which alter their interaction with DNA and nuclear proteins and influence, for example gene regulation, DNA repair and chromosome condensation.
  • the H3 and H4 histones have long tails protruding from the nucleosome which can be covalently modified, for example by methylation, acetylation, phosphorylation, ubiquitination, sumoylation, citrullination and ADP-ribosylation (as depicted in Figure 1).
  • the core of the histones H2A and H2B can also be modified.
  • Histone modifications may include:
  • Histone HI for example HI (phospho SI + T3), HI (phospho S35), HI (acetyl K63);
  • H2A asymmetric di methyl R3
  • H2A symmetric di methyl R3
  • H2A acetyl K5
  • H2A mono methyl R17
  • H2A symmetric di methyl R77
  • H2A Hydroxy P26
  • H2A mono methyl K125
  • H2A tri methyl K125
  • H2A tri methyl K127
  • H2A tri methyl K127
  • H2A phospho S129
  • Histone H2B Modifications for Histone H2B, for example H2B (acetyl K5), H2B (di methyl K5), H2B (Hydroxy P10), H2B (di methyl K43);
  • H3 Modifications for Histone H3, for example H3 (mono methyl R2), H3 (citrulline 2 + 8 + 17), H3 (mono methyl K4), H3 (di methyl K4), H3 (tri methyl K4), H3 (di+tri methyl K4), H3 (acetyl K9), H3 (acetyl K9, phospho S10), H3 (mono methyl K9), H3 (di methyl K9), H3 (tri methyl K9), H3 (phospho S10), H3 (asymmetric di methyl R17), H3 (acetyl K18), H3 (acetyl K27), H3 (di methyl K27), H3 (tri methyl K27), H3 (mono methyl K27,tri methyl K27 + K4), H3 (mono methyl K36), H3 (tri methyl K36), H3 (Hydroxy P38), H3 (mono methyl K79), H3 (di methyl K
  • Histone modifications that are particularly useful in the methods described herein are modifications that are associated with a particular chromatin state, e.g. at a gene promoter.
  • Histone modifications that are associated with an open or active chromatin state include, for example, mono-methylation of either H3K4, H3K9, H3K27, and H3K79; di-methylation of H3K79; tri-methylation of H3K4; and acetylation H3K9 and H3K14.
  • Histone modifications that are associated with a closed or inactive chromatin state include, for example, tri- methylation of H3K9 or H3K27.
  • Antibodies specific for each of the histone modifications described herein are commercially available, for example, they may be obtained from Abeam, Cambridge, MA. Most of the commercially available antibodies are suitable for the chromatin precipitation assays described herein.
  • Chromatin immunoprecipitation- massively parallel DNA sequencing is used to analyze a set of DNA-associated proteins. It can be used to precisely map global DNA binding sites for any protein of interest, e.g. transcription factor, restriction enzyme, or other chromatin associated proteins, on a genome scale. Chromatin immunoprecipitation may also be combined with microarray "ChlP-on-chip," which requires a hybridization array. ChlP-on-chip may introduce some bias, as an array is restricted to a fixed number of probes. Sequencing, by contrast, is thought to have less bias, although a certain sequencing bias for different sequencing technologies may occur.
  • DNA modifications Determining how proteins interact with DNA to regulate gene expression enhances comprehension of many biological processes and disease states.
  • the epigenetic information obtained from an understanding of protein-DNA interactions is complementary to genotype and expression analysis.
  • Specific DNA sites that are in direct physical interaction with transcription factors and other proteins, such as histones, may be isolated by ChIP, which produces a library of target DNA sites bound by a protein in vivo.
  • Massively parallel sequence analyses may be used in conjunction with whole-genome sequence databases to analyze the interaction pattern of a protein of interest (e.g. transcription factors, polymerases or transcriptional machinery) with DNA (Johnson et al. (2007) Science 316: 1497-1502), or to analyze the pattern of an epigenetic chromatin modification of interest (e.g. histone modifications or DNA modifications).
  • ChIP may be used to selectively enrich for DNA sequences bound by a particular protein (e.g. transcription factor or histone) in living cells by cross-linking DNA-protein complexes and using an antibody that is specific against a protein of interest.
  • a particular protein e.g. transcription factor or histone
  • Specific ChIP protocols are well known in the art. After precipitation of chromatin oligonucleotide adapters may be added to the small stretches of DNA that are bound to the protein of interest to enable massively parallel sequencing. After size selection, the resulting ChlP-DNA fragments are sequenced simultaneously using a genome sequencer. A single sequencing run can scan for genome- wide associations with high resolution. ChlP-on-chip may require large sets of tiling arrays (of overlapping probes designed to densely represent a genomic region of interest) and may provide a lower resolution.
  • Massively parallel sequencing is known in the art and many sequencing methods may be used. Some technologies may use cluster amplification of adapter-ligated ChIP DNA fragments on a solid flow cell substrate. The resulting high density array of template clusters on the flow cell surface may then be submitted to sequencing-by-synthesis in parallel using for example fluorescently labeled reversible terminator nucleotides. Templates are sequenced base-by-base during each read. The resulting data may be analyzed using data collection and analysis software that aligns sample sequences to a known genomic sequence.
  • Sensitivity of this technology may depend on factors such as the depth of the sequencing run (the number of mapped sequence tags), the size of the genome and the distribution of the target factor.
  • the precision of the ChlP-Seq assay is not limited by the spacing of predetermined probes. By integrating a large number of short reads, highly precise binding site localization may be obtained. ChlP-Seq data can be used to locate the binding site within few tens of base pairs of the actual protein binding site, and tag densities at the binding sites may allow quantification and comparison of binding affinities of a protein to different DNA sites.
  • ChlP-Seq data is obtained from cancer cells
  • the data may require additional data processing and analysis since cancer cells may have mutated and abnormal DNA with copy number changes.
  • analytical steps during the generation and processing of chromatin modification maps must compensate for DNA copy number changes, including genomic amplifications or deletions.
  • ChlP-Seq data generated from a cancer cell may be used to identify the locations of histone
  • Chromatin state at promoters can be validated by gene expression analysis of the gene that is operated (controlled) by the promoter.
  • the validation is performed with DNA microarray.
  • a DNA microarray consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides of a specific DNA sequence, known as probes. This can be a short fragment of a gene or other DNA element that are used to hybridize a cDNA sample (called target) under high-stringency conditions.
  • Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target.
  • the probes are attached to a solid surface (glass, plastic or a silicon chip) by a covalent bond to a chemical matrix (e.g. via epoxy- silane, amino-silane, lysine, polyacrylamide or others), representing what is commonly known as a "gene chip”, “genome chip”, “DNA chip” or “gene array”, e.g. Affymetrix, Santa Clara, CA) or a microscopic bead (e.g. polystyrene beads, Illumina, San Diego, CA).
  • DNA microarrays may for example be used to measure changes in expression levels.
  • the process of measuring gene expression via cDNA e.g. RNA after reverse transcription
  • expression profiling is called "expression analysis” or "expression profiling.”
  • the validation of chromatin state at promoters is performed by RNA sequencing.
  • RNA sequencing high-throughput sequencing technology is used to profile the entity of messenger RNA molecules (the "transcriptome"). Such data could be used to validate inferences made from chromatin data with regard to the expression of a given protein coding or non-coding gene or a set of protein coding or non-coding genes within a pathway.
  • RNA sequencing refers to the use of high-throughput sequencing technologies to sequence cDNA (the sequence libraries may be created extracting mRNA using its poly(A) tail according to known methodologies, which is added to the mRNA molecule post- transcriptionally and thus splicing has taken place) in order to get information about the RNA content of a sample. RNA sequencing may also provide information, for example, on how different alleles of a gene are expressed, can detect post-transcriptional mutations and can identify gene fusions.
  • the sequencing chemistry may involve a variety of methods known in the art such as pyrosequencing (454 Sequencing, Branford, CT), polymerase-based sequence-by-synthesis (Illumina, San Diego, CA), or ligation-based sequencing (SOLiDTM, Applied Biosystems, Foster City, CA).
  • the amplification approach may involve a variety of methods known in the art such as emulsion PCR or bridge amplification, producing reads ranging from approximately 30bp to 250bp.
  • RNA sequencing may be combined with other techniques such as Sanger sequencing providing larger sequences obtained from the same sample. These larger reads may be used as a "skeleton” or a "template” to help assemble reads in difficult regions (e.g. regions with repetitive sequences).
  • One approach is to align the millions of reads to a "reference genome”.
  • Tools for aligning genomic reads to a reference genome are known in the art.
  • gene expression analysis may be used to associate the active state of a gene with the state as it is determined by the data obtained from the ChlP-Seq assay.
  • Gene expression analysis is, however, a less sensitive method for determining the active state of genes that are active at very low levels, such as, for example, genes important for development.
  • Gene expression analysis may be combined with ChlP-Seq assays to verify the active state of the associated genes on a global scale. Determination of abnormally regulated genes and/or pathways: Described herein are methods comprising the use of ChlP-Seq and optionally comprising the use of gene expression analysis to determine one or more genes and/or one or more signaling pathways that are aberrantly regulated (e.g.
  • cancer cells such as cancer stem cells, tumors and cell lines
  • a normal cell equivalent such as a normal cell of the same type or tissue.
  • abnormally regulated genes can be revealed.
  • a set of (one or more) genes may exhibit active histone modifications in cancer cells but exhibits silencing histone modifications in the normal cell equivalent.
  • Genes that are normally silenced are considered genes that are "aberrantly active" in the cancer cells.
  • Genes that are normally active are normally active (genes that are active in the normal cell equivalent of a cancer cell) but are inactive (e.g. repressed or silent) in the cancer cell are considered genes that are "aberrantly repressed" in the cancer cells.
  • genetic analyses may be used to identify common biological properties and/or similar biological functions. Such analyses may reference databases (such as public databases) that compile known functions of genes (i.e. make available functional connotations for specific genes or gene products). These connotations may be broad bio-functional categories, such as
  • transcription factor activity a dominant common function of a set of genes can be "transcription factor activity.”
  • Certain transcription factors can play critical roles in cell, organ and tissue development and their activity can drive a stem cell, e.g. to self -renew, to differentiate into a specific cell type, to apoptose, or to become a tumorigenic cancer stem cell.
  • This cellular reprogramming is accomplished by transcription factors through interactions with DNA, which may lead to activating or silencing genes controlled by the transcription factor. Since genes encoding transcription factors may also be controlled by transcription factors, activation of such genes by the first transcription factor may lead to subsequent activation of genes that are not controlled by the first transcription factor but are controlled by the activated second transcription factor.
  • transcription factors may be active and critical to a cell's function, generally they are present in only low amounts in the cell. Therefore, detecting changes in their activity is difficult using standard methods of detection and analysis, such as those that employ microarrays to detect and/or quantify gene expression.
  • the methods provided herein, analyzing changes in histone modifications allow the detection and determination of developmentally important transcription factors that are abnormally active or inactive in cancer cells. Identifying and determining abnormally regulated (e.g. , active, or inactive) transcription factors in cancer cells may not be accomplishable using established methods other than those provided herein.
  • Cancer stem cells may be present in some or all human tumors and are likely the subpopulation of tumor cells responsible for growth of the tumor, although they may represent only a small minority of the total cellular mass of the tumor. Certain tumors, for example malignant gliomas, are capable of recurring and/or progressing following conventional surgical and radiation therapy, suggesting the presence of cancer stem cells.
  • the term "stem cell” is known in the art to mean a cell (i) that is capable of generating one or more kinds of progeny with reduced proliferative or developmental potential; (ii) that has extensive proliferative capacity; and (Hi) that is capable of self-renewal or self-maintenance.
  • cancer stem cells are defined as cells that can undergo self-renewal as well as abnormal proliferation and differentiation to form a tumor. Functional features of tumor stem cells are that they are tumorigenic; they can give rise to additional tumorigenic cells by self-renewal; and they can give rise to non-tumorigenic tumor cells.
  • tumorigenic refers to a cell derived from a tumor that is capable of forming a tumor, particularly an isolated cell or isolated cell population that is capable of forming a tumor. Tumorigenicity may be tested, for example by dissociating and transplanting an isolated cell or isolated cell population into a suitable animal model, such as an immuno-compromised mouse.
  • a "tumorigenic” cell refers to a cell derived from a tumor or other tissue that when dissociated, transplanted into a suitable animal model and tested, is capable of forming a tumor.
  • non-tumorigenic cell refers to a cell derived from a tumor or other tissue that when dissociated, transplanted and tested under identical conditions does not form a tumor in an animal model.
  • the cell population may be derived from a single cloned tumor cell (i.e., a clonal cell line established in vitro from a tumor cell) or a cell population substantially enriched in tumor stem cells.
  • tumor stem cells may proliferate extensively and may give rise to additional tumor stem cells as well as to other tumor cells that lack tumorigenic potential.
  • An additional trait of tumor stem cells can be their resistance to therapeutics, such as chemotherapy, which may ultimately prove fatal.
  • the developmental origin of tumor stem cells can vary among different types of cancers. Tumor stem cells may arise for example as a result of genetic damage that deregulates normal mechanisms of proliferation and differentiation of stem cells.
  • Cancer stem cell isolation Described herein are methods for isolating cell populations enriched in cancer stem cells and isolated cell populations substantially enriched in cancer stem cells that are tumorigenic.
  • Primary cancers can be isolated, plated and grown under culture conditions that promote separation of cell types into subpopulations.
  • Populations of cancer stem cell-enriched cultures can be substantially freed of non-tumorigenic cells, for example by means of subcloning.
  • a general method of isolating a cell population substantially enriched in tumorigenic stem cells may comprise at least one or more of the following steps: (a) mincing a tissue sample from a tumor, with optional enzymatic treatment (e.g., using trypsin) to obtain a single cell suspension using standard methods known in the art; (b) plating and culturing the resulting sample in vitro under conditions suitable to culture the cancer cells; (c) passaging cells by dissociating potential cell aggregates (e.g.
  • tissue culture plates used for culturing may be coated with a cell-adhesive layer (e.g. coated with a solution containing laminin and poly-L-omithine, fibronectin, vitronectin, gelatin, or other suitable mixture) and cells may be cultured under conditions that promote attachment and/or aggregation of cells.
  • a cell-adhesive layer e.g. coated with a solution containing laminin and poly-L-omithine, fibronectin, vitronectin, gelatin, or other suitable mixture
  • cells may be cultured under conditions that promote attachment and/or aggregation of cells.
  • Culture conditions may also be suitable to promote cell migration of specific subpopulations of cells, e.g. from cell aggregates onto the substrate (migratory cell population that migrate away from the tumor mass).
  • the choice of cell-adhesive layer will vary depending upon the type of tumor and may also be selected according to the particular characteristics of the migratory cell population. For example, some populations of migratory cells isolated from aggressive brain tumors are highly enriched in cancer stem cells.
  • Such "neurogenic" cancer stem cells have the capacity or propensity to differentiate into one or more cell types of the nervous system or a nervous tissue, including both neuronal cell types and glial cell types. These cancer stem cells can undergo self-renewal as well as abnormal proliferation and differentiation to cells expressing markers of neuronal and/or glial cells, and may form a tumor of the CNS.
  • the tumor-derived stem cells may be propagated, expanded and passaged in vitro (at least, for example, 5, 10, 15, 20, 25, 30, 35, 40 or more passages) using standard culture conditions.
  • standard culture conditions refer to culture conditions suitable for the maintenance and propagation of stem cells without components added to stimulate these cells to differentiate along a particular lineage, for example the neural lineage. Standard culture conditions for cultivating stem cells, including methods for generating clonal cultures have been developed and are known in the art.
  • Isolated cancer stem cells may be analyzed for specific cellular markers, for example cellular markers of multipotency.
  • the markers may be detected using immunohistochemical detection methods, e.g. employing antibodies that are specific for certain stem-cell markers or lineage markers (e.g. neuronal and glial lineage).
  • Suitable stem cell and lineage markers and techniques for their detection are known in the art and are described in detail, for example, in Scheffler et al., Proc. Natl. Acad. Sci. 102(26):9353-9358, 2005.
  • lineage markers specific for any tissue of origin of a tumor, and other tumor markers may be detected by immunocytochemistry or, e.g. , by flow cytometry using suitable antibodies using techniques well known in the art.
  • the tumorigenicity of an isolated cancer stem cell can be confirmed, for example, by demonstrating tumor growth in a suitable host animal.
  • the host animal can be a model organism such as nematode, fruit fly, zebrafish; preferably a laboratory mammal such as a mouse (nude mouse, SCID mouse, NOD/SCID mouse, Beige/SCID mouse, FOX/SCID mouse), rat, rabbit, or primate.
  • Severely immunodeficient NOD-SCID mice are particularly suitable animal recipients of transplanted human cancer stem cells.
  • Single-cell suspensions or suspensions with a few aggregates of cells, such as 20,000 cells; ideally less than 100; preferably less than 10 cells) are prepared from the isolated cancer stem cells and
  • Suitable routes may include parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intracerebral, or intraocular injections, for example.
  • the cells may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • An in vivo assay may be useful for initial verification of the tumorigenicity of a tumor-derived stem cell line. Once tumorigenicity is established, an animal model can be used for a wide array of biological and molecular assays to characterize the tumorigenic stem cells and the tumors that arise therefrom.
  • tumors from which tissue samples containing tumor stem cells may be isolated and/or enriched for, and to which the diagnostic methods described herein may be applied include sarcomas and carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, mesothelioma, Ewing's tumor,
  • lymphangioendotheliosarcoma synovioma, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, astrocytic tumors (e.g.
  • oligodendroglial tumors and mixed gliomas e.g. , oligodendroglioma, anaplastic oligodendroglioma, oligoastrocytoma, anaplastic oligoastrocytoma
  • ependymal tumors e.g.
  • ependymoma anaplastic ependymoma, myxopapillary ependymoma, subependymoma), choroid plexus tumors, neuroepithelial tumors of uncertain origin (astroblastoma, chordoid glioma, gliomatosis cerebri), neuronal and mixed-neuronal-glial tumors (e.g.
  • ganglioglioma and gangliocytoma desmoplastic infantile astrocytoma and ganglioglioma, dysembryoplastic neuroepithelial tumor, central neurocytoma, cerebellar liponeurocytoma, paraganglioglioma), pineal parenchymal tumors, embryonal tumors (medulloepithelioma, ependymoblastoma, medulloblastoma, primitive neuroectodemmal tumor, atypical teratoid/rhabdoid tumor), peripheral neuroblastic tumors, tumors of cranial and peripheral nerves (e.g.
  • schwannoma neurinofibroma, perineurioma, malignant peripheral nerve sheath tumor
  • meningeal tumors e.g. , meningeomas, mesenchymal, non-meningothelial tumors, haemangiopericytomas, melanocytic lesions
  • germ cell tumors tumors of the sellar region (e.g. , craniopharyngioma, granular cell tumor of the neurohypophysis), hemangioblastoma, melanoma, and
  • glioblastoma cancer stem cells from human glioblastoma tumors. These cells are thought to be the most malignant (tumorigenic) cells of the tumor and are thought to be responsible for repopulating the tumor after intervention/treatment and for the resistance of the tumor to therapy.
  • Nearest cell equivalent Described herein are methods for determining the "nearest cell equivalent" of a cell of interest, that is a cell that is more closely related to the cell of interest than any other cell tested.
  • a cell of interest may be a cancer cell, such as a cancer stem cell, a tumor cell or a cell line.
  • methods for determining the nearest cell equivalent of a cell of interest are provided using histone modification profiles. Other cell characteristics may also be used to determine the nearest cell equivalent of a cell of interest. These characteristics may be used alone or in combination with histone modification profiles to determine the nearest cell equivalent of a cell of interest.
  • Such characteristics include, but are not limited to: i) location, such as a "nearest neighbor" cell, which is physically located (found) in close vicinity to the location from which the cell of interest has been isolated; ii) expression of lineage or differentiation markers; Hi) expression of cellular receptors that are not lineage or differentiation markers; iv) global gene expression profiles (e.g. determined by DNA microarray); v) global chromatin profiles; and vi) phenotypical characteristics: doubling rate, capacity for cell division, nutrient dependency, invasiveness (migration potential), ability to form colonies (colony formation assays), presence/absence of check point control responses.
  • the "nearest cell equivalent" of a cell of interest is the “nearest normal cell” of a cancer cell, or the “nearest normal stem cell” of a cancer stem cell.
  • the "nearest normal” (stem) cell equivalent of a cancer (stem) cell is a normal (non-cancer) (stem) cell that is more closely related to the cancer (stem) cell than any other (stem) cells tested.
  • methods for determining the nearest normal (stem) cell equivalent of a cancer (stem) cell are provided using histone modification profiles for determining the nearest normal (stem) cell equivalent.
  • Each cell type has a unique histone modification profile. For each histone
  • each cell type has a different set of genes that are marked by that particular histone modification. These unique histone modification profiles can thus be viewed as its chromatin "signature".
  • Cells that are closely related have signatures that are more similar to each other. Described herein are methods for determining the cell types that are most closely related, comparing an unknown or diseased cell type to a library (or reference dataset) of chromatin signatures. For example, chromatin promoter states of cancer (stem) cells generated using the methods described herein may be compared against the chromatin promoter states of normal cell types that are in the public domain.
  • the promoter signal for the normal cell types in public databases may optionally be normalized.
  • a set of "most informative genes" may be generated in all cell types by selecting genes that show the highest variability across cell types. Cell types may then be clustered based on signal of the selected genes, generating a hierarchical map. Promoter signals for the set of most informative genes obtained from a cell of interest (e.g. a cancer cell) may then be used to identify the most closely related cell types in the hierarchical map of normal tissues. In certain embodiments, methods are provided that use the most related normal cell types (nearest cell equivalents) identified as described herein in chromatin state comparison with the cancer stem cells, to determine aberrantly regulated signaling pathways.
  • a cell of interest e.g. a cancer cell
  • Network inference methods and motif analysis can be applied.
  • Connectivity is the relationship between individual genes in a cell-specific or general regulatory network.
  • a “network inference method”, as used herein, is a method to identify regulatory interactions between genes as components of regulatory pathways or networks. This includes, but is not limited to, identification of protein-protein interaction pathways, identifications of DNA binding sites in the regulatory region of one gene that can be bound by a protein that specifically binds to this site (“binding motif identification”), or co- expression analysis (for example by evaluation of DNA microarray data).
  • Bining motif identification identification of DNA binding sites in the regulatory region of one gene that can be bound by a protein that specifically binds to this site
  • Bining motif identification identifications of DNA binding sites in the regulatory region of one gene that can be bound by a protein that specifically binds to this site
  • Bining motif identification identifications of DNA binding sites in the regulatory region of one gene that can be bound by a protein that specifically binds to this site
  • Bind motif identification identifications of DNA binding sites in the regulatory
  • the methods described herein may be used in diagnosing cancer or confirming a diagnosis of cancer. Changes in histone modification profiles, as described herein, can deregulate mechanisms such as transcriptional control which may lead to the inappropriate silencing or activation of cancer-associated genes. These changes may be inheritable at the cellular level and may contribute to the clonal expansion of cancer cells. Changes in histone modification profiles leading to aberrantly regulated intracellular signaling pathways that can be indicative of cancer cells can be exploited in the clinic as biomarkers for cancer detection, diagnosis and prognosis. For example, the methods described herein may be used in diagnosing or confirming a diagnosis that a cancer cell exhibits an aberrantly regulated intracellular signaling pathway.
  • Such methods include: a) obtaining the cell, such as during surgery or from a biopsy, b) performing any one of the methods for identifying aberrantly regulated intracellular signaling pathways in a cancer cell as described herein, and c) diagnosing based on the results obtained in (b) if the cell exhibits an aberrantly regulated intracellular signaling pathway.
  • the methods described herein may be used in diagnosing or confirming a cancer diagnosis.
  • Such methods include: (a) obtaining a cell, (b) performing any one of the methods for identifying an aberrantly regulated transcription factor in a cancer cell as described herein, wherein when the cell obtained in (a) exhibits an aberrantly expressed transcription factor the cell is a cancer cell, and c) diagnosing cancer or confirming a cancer diagnosis based on whether the cell is a cancer cell.
  • the methods described herein also facilitate diagnosis of the cancer type of the cancer and the origin of the cancer based on analysis of histone modification profiles and nearest cell equivalent analysis as described herein.
  • Such methods include: (a) obtaining a cancer cell from a tumor, (b) performing any one of the methods for identifying aberrantly regulated intracellular signaling pathways in a cancer cell as described herein, and/or performing any one of the methods to of identify the nearest cell equivalent of a cell, (c) comparing the histone modification profile (signature) obtained in (b) to either the histone modification profile (signature) obtained from a cancer cell of known cancer type and/or origin from a subject or comparing the histone modification profile (signature) obtained in (b) with the histone modification profile (signature) of one or more cancer cell of known cancer type and/or origin from a public database, and (d) identifying the origin and/or cancer type of the cancer cell from a tumor obtained in (a), wherein when the cancer cell from a tumor exhibits a histone modification profile (sig
  • the methods described herein further facilitate diagnosis of a secondary tumor (metastasis) based on analysis of histone modification profiles and nearest cell equivalent analysis as described herein.
  • Such methods include: (a) obtaining a first cell from a subject, (b) performing any one of the methods for identifying aberrantly regulated intracellular signaling pathways in a cancer cell as described herein, and/or performing any one of the methods to of identify the nearest cell equivalent of a cell, c) comparing the histone modification profile (signature) obtained in (b) to a histone modification profile (signature) of one or more cancer cell obtained from a subject or comparing the histone modification profile (signature) obtained in (b) with the histone modification profile (signature) of one or more cancer cell from a public database, and d) diagnosing that the first cell is obtained from a secondary tumor when the first cell: i) exhibits an aberrantly regulated intracellular signaling pathway, and ii) exhibits a histone modification profile (signature) that indicates that the first cell is
  • These methods may also be employed to distinguish between a primary tumor and a secondary tumor. For example, by: a) obtaining a cancer cell from a tumor, b) performing any one of the methods for identifying aberrantly regulated intracellular signaling pathways in a cancer cell as described herein, and/or performing any one of the methods to of identify the nearest cell equivalent of a cell, and c) comparing the histone modification profile (signature) obtained in (b) to either the histone modification profile (signature) obtained for a normal cell equivalent from the site of the tumor or comparing the histone modification profile (signature) obtained in (b) with results from a public database for a normal cell from the site of the tumor, it can be determined that the cancer cell is from a primary tumor or a secondary tumor, wherein when the histone modification profile (signature) obtained for the cancer cell and the normal cell in (c) indicates that they are nearest cell equivalents the tumor is a primary tumor, and wherein when the histone modification profile (signature) obtained for the cancer cell and the normal cell
  • a tumor from which a cancer cell is obtained is from a primary tumor or a secondary tumor, such as a metastasis.
  • these methods can also be employed to identify the effects of environmental conditions on a histone modification profile (signature) of a cell, the methods include: (a) obtaining a cell which has been exposed to a particular environmental condition or exposing a cell to a particular environmental condition, (b) performing on the cell obtained in (a) any one of the methods for identifying aberrantly regulated intracellular signaling pathways in a cell as described herein to obtain a histone modification profile of the cell, (c) comparing the histone modification profile (signatures) of the cell to a histone modification profile
  • the cell of (a) is a cancer cell.
  • these methods can be employed to compare the effects of environmental conditions on histone modification profiles (signatures) of cells obtained from or exposed to different environmental conditions, the methods include: (a) obtaining a first cell which has been exposed to a particular environmental condition or exposing a first cell to a particular environmental condition, (b) obtaining a second cell which has been exposed to an environmental condition different from that of the first cell or exposing a second cell to an environmental condition different from that of the first cell, (c) performing on each of the first and second cell any one of the methods for identifying aberrantly regulated intracellular signaling pathways in a cancer cell as described herein to obtain a histone modification profile (signature), (d) comparing the histone modification profile (signatures) obtained for the first cell to the histone modification profile (signatures) obtained for the second cell, or comparing a histone modification profile (signatures) obtained for the first cell and the histone modification profile (signatures) for the second cell to a histone modification profile (signature) obtained from a public
  • the condition is exposure to
  • the condition is exposure to radiation.
  • cancer stem cells such as glioblastoma-derived stem cells.
  • Aberrantly regulated intracellular signaling pathway include those described herein, such as aberrantly active Wnt signaling pathway or an aberrantly active Notch signaling pathway.
  • Diagnosis, treatment and prognosis of most human tumors of the central nervous system is presently based almost exclusively on histopathological criteria such as cytological appearance, necrosis, and tumor cell or endothelial cell proliferation.
  • histopathological criteria such as cytological appearance, necrosis, and tumor cell or endothelial cell proliferation.
  • Glioblastoma the most common type of primary brain tumor in adults, is fatal despite currently available multimodal treatments such as surgery, radiation, and
  • cancer stem cells possess characteristics of adult organ stem cells such as self -renewal and multi-lineage differentiation. These cells may be referred to as cancer stem cells or CSCs.
  • CSCs cancer stem cells
  • GBM is among several cancers where CSCs have been isolated that have been associated with highly tumorigenic phenotypes. These cells may exhibit the CD133 cell surface antigen, which is also expressed on normal neural and hematopoietic stem cells.
  • CD133-positive GBM cancer stem cells have been shown to display enhanced resistance to ionizing radiation due to their increased activation of the DNA damage checkpoint (Bao et al. Nature 2006;444:756-60). Additionally, the CD133-positive human GBM cancer stem cells have been shown to mediate enhanced resistance to a variety of chemotherapeutic agents (Liu et al. Mol. Cancer 2006;5:67). These observations suggest that the cancer stem cell subpopulation of GBM cells, resistant to radio-chemotherapy and chemotherapy and more likely to survive those modalities, may be the source of cancer recurrence, and hence novel therapies will need to be developed that target GBM cancer stem cells.
  • GSCs glioblastoma-derived stem cells
  • Glioblastoma stem cells were isolated and propagated as previously described (Wakimoto, Kesari et al. 2009). Briefly, surgical specimens of GBM were collected at Massachusetts General Hospital with approval by the Institutional Review Board. Mechanically minced tissues were digested with 0.1% Trypsin and 10 U/mL of DNasel at 37C for 45 min. After washes, tissues were triturated and passed through a 100-um cell strainer.
  • Cells were plated in EF medium composed of Neurobasal medium (Invitrogen, Carlsbad, CA) supplemented with 3 mmol/L L-glutamine (Mediatech, Manassas, VA), IX B27 supplement (Invitrogen), 0.5X N2 supplement (Invitrogen), 2 ⁇ g/mL heparin (Sigma, Ronkonkoma, NY), 20 ng/mL recombinant human EGF (R & D systems, Minneapolis, MN), 20 ng/mL recombinant human FGF2 (R & D systems), and 0.5X penicillin G/streptomycin sulfate.
  • Neurobasal medium Invitrogen, Carlsbad, CA
  • L-glutamine Mediatech, Manassas, VA
  • IX B27 supplement Invitrogen
  • 0.5X N2 supplement Invitrogen
  • 2 ⁇ g/mL heparin Sigma, Ronkonkoma, NY
  • NS medium composed of Neurobasal supplemented with B27, N2, 3 mmol/L glutamine, penicillin/streptomycin, 20 ng/mL recombinant human EOF and 20 ng/mL recombinant human FGF2 on polyornithine- coated and laminin-coated plates. Cells were passaged by manual trituration (Shin,
  • NHA Normal human astrocytes
  • ChlP-Seq Assay ChIP assays were carried out on cultures of approximately 10M
  • Immunoprecipitations were performed using antibody against K4me3 (Millipore 07473), K27me3 (Millipore 07449) or K36me3 (Abeam 9050). ChIP DNA samples were used to prepare sequencing libraries, which were then sequenced on the Illumina Genome Analyzer by standard procedures and read alignment and density map generation were performed as previously described (Mikkelsen, Ku et al. 2007).
  • ChlP-Seq Data Processing To account for copy-number variations in the genomes of the cancer cell lines, we used a non-enriched whole cell extract ChlP-Seq dataset to normalize ChlP-Seq signal from H3K4me3 and H3K27me3 experiments. We assume the non-enriched whole cell extract sequencing reads will be proportional to the quantity (or copies) of a given locus in the genome. First, H3K4me3 and H3K27me3 mean density was calculated in promoter regions (-500bp to 2kb relative to transcription start site) for human RefSeq genes (hgl8; (Ku, Koche et al. 2008)) and normalized by whole-cell extract signal. Because coverage for whole-cell extract sequencing is sparse due to the generally low number of reads, we used a lOOkb region surrounding each promoter for signal/background calculation.
  • ChlP-Seq peaks at promoters were defined as present or absent as follows: for each ChIP sequencing output, we reassigned reads to random locations in the genome and generated the distribution of mean density of 2.5kb regions normalized by lOOkb of surrounding WCE background. Based on this distribution, we chose an empirical p- value cutoff threshold for classifying a positive signal at a promoter.
  • Gene Expression Analysis of Cultured Cells The chromatin state of promoters were correlated to gene expression using microarray analysis. Total RNA was isolated from cells using Trizol Reagent (Invitrogen) and purified using the RNeasy Kit (Qiagen, Valencia, CA). Gene expression data were acquired with Affymetrix Human Genome U133 2.0 ArraysTM and normalized using the Genepattern expression data analysis package with RMA and default parameters (Reich, Lief eld et al. 2006).
  • GSC chromatin promoter states generated using the above method were compared against the chromatin promoter states of normal cell types that we have previously characterized and/or that are in the public domain.
  • the promoter signal for the normal cell types in this database was standardized in that 2.5kb region.
  • we generated a set of most informative genes in all cell types by selecting genes that showed the highest variability across cell types. Cell types were clustered based on signal of the selected genes, generating a hierarchical map pf pairwise correlation.
  • GSC cell promoter signal for the same set of genes was used to identify the most closely related cell types in the hierarchical map of normal tissues. These related normal cell types are then used in the chromatin state comparison with the cancer stem cells.
  • the small molecule inhibitors used were XAV939 (Cayman Chemical, Ann Arbor, MI), which stabilizes the beta-catenin destruction complex (Huang, Mishina et al. 2009), and BIO (Cayman Chemical), a
  • GSK3 alpha/beta inhibitor which activates Wnt target gene expression.
  • DAPT Calbiochem, San Diego, CA
  • Notch pathway target genes were included in the aberrantly active GSC TF list and predicted to have LEFl targets.
  • GSC cultures were dissociated and plated into 6 well plates. After 24 hours, XAV939 (1 ⁇ ), BIO (1 ⁇ ), DAPT (1 ⁇ ), both XAV939 and DAPT (at ⁇ each) or no inhibitor were added and the cells were incubated for 48 hours.
  • PCR primer pairs were designed to amplify designated regions of the cDNA that spanned intron/exon borders in order to avoid amplifying genomic DNA using Primer3 (fokker.wi.mit.edu/primer3/input.htm).
  • Real-time PCR assays were carried out on an ABI 7500 detection system using the Quantitect SYBR green PCR mix (Qiagen).
  • Cytotoxicity Assays for small molecule inhibitors of Wnt and Notch activity Cells were dissociated and plated into 96 well plates. After 24 hours, the following inhibitors were added at various concentrations: XAV939, DAPT, both XAV939 and DAPT, cercosporin (Sigma), which interferes with the association of beta-catenin and LEF1 (Lepourcelet, Chen et al. 2004) and indomethacin (Sigma), which suppresses beta-catenin expression (Goessling, North et al. 2009). After 5 days of incubation with inhibitor, cells were assayed using the CellTiter 96 Aqueous MTS-based cell viability assay (Promega, Madison, WI) according the manufacturer's specifications. RESULTS
  • GBM-stem cells are the most malignant cells of the tumor and are thought to be responsible for repopulating the tumor after treatment and for the tumor's resistance to therapy.
  • Histone modifications in GBM-stem cells were studied on a global scale using ChlP- Seq assays and ChlP-Seq data processing (as described in Methods). Histone modifications are a component of chromatin, which packages the DNA and influence how genes within the DNA function (whether they are active or silent). Specific types of histone modifications at the start of a gene are associated with different gene functional states such as active, silent, or permanently silenced. Some histone modifications tend to be found at genes that are important for embryonic development or preferentially activated in cancer ( Figure 2 A-C). Using ChlP-Seq assays, locations of specific histone modifications across the entire DNA were identified and the activation state of every gene in the cell was determined.
  • ChlP-Seq of cancer cells requires additional data processing and analysis since cancer cells have mutated and abnormal DNA. For example, analytical steps during the generation and processing of chromatin modification maps must compensate for DNA copy number changes, including genomic amplifications or deletions.
  • ChlP-Seq data generated from the GBM cancer stem cells was used to determine the activation state of every gene in each cell line.
  • the locations of histone modifications that are associated with gene activation, silencing, and embryonic development were identified.
  • gene expression analysis the active state of a gene can be associated with that inferred from the ChlP-Seq assay.
  • Gene expression analysis is, however, a less sensitive method for determining the active state of genes that are active at very low levels, such as genes important for development. This step was taken to validate on a global scale the results obtained from the ChlP-Seq assay, i.e. that gene expression analysis data was consistent with the active state of the associated genes ( Figures 3 and 4 A, B).
  • Each cell type has a unique histone modification profile. For each histone
  • each cell type has a different set of genes that are marked by that particular histone modification. These unique histone modification profiles can thus be viewed as its chromatin "signature".
  • Cells that are closely related have signatures that are more similar to each other.
  • a library or reference dataset
  • Comparing the GBM chromatin signature to the reference dataset reveals the GBM cancer stem cells to be most closely related to neural cell types and other cancer cells.
  • abnormally activated or silenced genes can be revealed.
  • a set of genes that had active histone modifications in GBM cancer stem cells but had silenced histone modifications in their normal neural stem cell counterparts was identified. Since these genes are normally silenced, they were considered to be abnormally active genes in the cancer stem cells.
  • CTTTGA_V$LEF1_Q2 CTTTGT_V$LEF1_Q2 (motifs that match TRANSFAC motif V$LEF1_Q2); Uniprobe motifs (mouse): CTTTGATC (primary, SEQ ID NO: 4),
  • the Wnt signaling pathway is important during development of multiple organs, including the brain.
  • the results obtained indicated that the Wnt signaling pathway, through its final common transcription factor LEFl, may be controlling the activity of the set of identified abnormally active genes, and that the Wnt signaling pathway itself was abnormally active in the GBM stem cells ( Figure 9B).
  • Figure 9B By investigating the activity of genes in the Wnt signaling pathway in human GBM tumors and normal brains using the methods described herein, it was determined that the Wnt signaling pathway is active in human GBM tumors relative to normal brain tissue.
  • glioblastoma-derived cancer stem cells were grown in the presence and absence of fetal calf serum (FCS) (as described above in the Methods section). Cells grown in FCS are less tumorigenic than their counterpart cells grown in the absence of FCS, and cells grown in FCS do not exhibit stem-cell like properties (Wakimoto, Kesari et al. 2009).
  • Figure 11A shows a heatmap comparing chromatin profiles (epitope H3K4me3) of various cell types, revealing that the chromatin profile of epitope H3K4me3 of the serum- grown cells is more closely related to differentiated primary normal astrocytes than to its counterpart profile of cancer stem cells.
  • FIG. 1 IB Data presented in Figure 1 IB shows that in the GBM8 cell line, transcription factors OLIG2 and ASCLl, previously identified as aberrantly active in glioma stem cells, are repressed through H3K27me3 when glioma stem cells are grown in serum. This data indicates that epigenomic profiling can be used to identify aberrant programs associated with different environmental exposures (e.g., small molecules, drugs, chemicals such as pesticides, etc.).
  • environmental exposures e.g., small molecules, drugs, chemicals such as pesticides, etc.
  • Transcription factors may regulate gene activity in the Wnt signaling pathway
  • FIG. 12 shows fold induction of the Wnt signaling pathway gene, AXIN2, suggesting that transcription factors act upstream of the Wnt pathway.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Biophysics (AREA)
  • Evolutionary Biology (AREA)
  • Medical Informatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Theoretical Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Physiology (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des outils techniques et analytiques pour (i) profiler la chromatine de l'ensemble du génome dans des cellules cancéreuses; (ii) identifier dans quelles mesures le paysage de la chromatine diffère des cellules homologues non malignes; et (iii) utiliser ces informations afin de découvrir des voies de régulation aberrantes qui entraînent des tumeurs chez l'être humain. L'invention concerne également des méthodes permettant d'identifier des processus ou des voies régulés de manière aberrante, grâce à l'identité de ces voies ou processus, ce qui permet d'obtenir un pronostic utile, des informations thérapeutiques utiles et des cibles pour une intervention thérapeutique afin de traiter une maladie, par exemple, un cancer. Les procédés peuvent être appliqués à des modèles de cellules cancéreuses spécifiques (y compris des cellules souches cancéreuses, des cellules tumorales, des lignées cellulaires cancéreuses, ainsi que leurs homologues normales), afin de découvrir un type de cancer donné, ou ils peuvent être appliqués à une tumeur d'un patient précis, par exemple afin d'élaborer un traitement anti-cancer sur-mesure (médicament personnalisé).
PCT/US2011/034476 2010-04-29 2011-04-29 Procédés d'identification de voies de signalisation intracellulaire régulées de manière aberrante dans des cellules cancéreuses WO2011137302A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US34375110P 2010-04-29 2010-04-29
US61/343,751 2010-04-29

Publications (1)

Publication Number Publication Date
WO2011137302A1 true WO2011137302A1 (fr) 2011-11-03

Family

ID=44861922

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/034476 WO2011137302A1 (fr) 2010-04-29 2011-04-29 Procédés d'identification de voies de signalisation intracellulaire régulées de manière aberrante dans des cellules cancéreuses

Country Status (1)

Country Link
WO (1) WO2011137302A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016054270A1 (fr) * 2014-09-30 2016-04-07 Brown University Analyse de réseau génétique basée sur la diffusion de chaleur
WO2017142484A1 (fr) * 2016-02-16 2017-08-24 Agency For Science, Technology And Research Profilage épigénomique révélant le paysage de promoteur somatique d'adénocarcinome gastrique primaire
CN110168646A (zh) * 2016-11-17 2019-08-23 南特生物科学公司 预测抗癌途径的验证
CN112562785A (zh) * 2020-12-10 2021-03-26 哈尔滨医科大学附属第一医院 基于atac测序数据筛选子宫内膜癌关键基因的方法及应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040093331A1 (en) * 2002-09-20 2004-05-13 Board Of Regents, University Of Texas System Computer program products, systems and methods for information discovery and relational analyses
WO2008069906A2 (fr) * 2006-11-14 2008-06-12 The Regents Of The University Of California Expression numérisée de l'analyse génétique
US20080261820A1 (en) * 2005-08-01 2008-10-23 Mount Sinai School Of Medicine Of New York University Methods to Analyze Biological Networks
WO2008130516A1 (fr) * 2007-04-11 2008-10-30 Manel Esteller Marqueurs biologiques épigénétiques pour la détection précoce, l'efficacité thérapeutique, et le suivi de rechutes d'un cancer
US20090170715A1 (en) * 2006-03-31 2009-07-02 Glinsky Gennadi V Prognostic and diagnostic method for cancer therapy

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040093331A1 (en) * 2002-09-20 2004-05-13 Board Of Regents, University Of Texas System Computer program products, systems and methods for information discovery and relational analyses
US20080261820A1 (en) * 2005-08-01 2008-10-23 Mount Sinai School Of Medicine Of New York University Methods to Analyze Biological Networks
US20090170715A1 (en) * 2006-03-31 2009-07-02 Glinsky Gennadi V Prognostic and diagnostic method for cancer therapy
WO2008069906A2 (fr) * 2006-11-14 2008-06-12 The Regents Of The University Of California Expression numérisée de l'analyse génétique
WO2008130516A1 (fr) * 2007-04-11 2008-10-30 Manel Esteller Marqueurs biologiques épigénétiques pour la détection précoce, l'efficacité thérapeutique, et le suivi de rechutes d'un cancer

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ESTELLER: "Aberrant DNA Methylation as a Cancer-Inducing Mechanism.", ANNUAL REVIEW OF PHARMACOLOGY AND TOXICOLOGY, vol. 45, February 2005 (2005-02-01), pages 629 - 656, Retrieved from the Internet <URL:http://www.annualreviews.org/doUabs/10.1146/annurev.pharmtox.45.120403.095832> [retrieved on 20110722] *
MULERO-NAVARRO ET AL.: "Epigenetic biomarkers for human cancer: The time is now.", CRITICAL REVIEWS IN ONCOLOGY/HEMATOLOGY, vol. 68, no. ISS. 1, October 2008 (2008-10-01), pages 1 - 11, Retrieved from the Internet <URL:http://www.sciencedirect.com/science/article/pii/S104084280800053X> [retrieved on 20110722] *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016054270A1 (fr) * 2014-09-30 2016-04-07 Brown University Analyse de réseau génétique basée sur la diffusion de chaleur
WO2017142484A1 (fr) * 2016-02-16 2017-08-24 Agency For Science, Technology And Research Profilage épigénomique révélant le paysage de promoteur somatique d'adénocarcinome gastrique primaire
CN109073659A (zh) * 2016-02-16 2018-12-21 新加坡科技研究局 表观基因组分析揭示了原发性胃腺癌的体细胞启动子局面
CN109073659B (zh) * 2016-02-16 2022-05-17 新加坡科技研究局 表观基因组分析揭示了原发性胃腺癌的体细胞启动子局面
CN110168646A (zh) * 2016-11-17 2019-08-23 南特生物科学公司 预测抗癌途径的验证
CN110168646B (zh) * 2016-11-17 2023-12-19 南特生物科学公司 预测抗癌途径的验证
CN112562785A (zh) * 2020-12-10 2021-03-26 哈尔滨医科大学附属第一医院 基于atac测序数据筛选子宫内膜癌关键基因的方法及应用

Similar Documents

Publication Publication Date Title
Chung et al. The role of GRHL2 and epigenetic remodeling in epithelial–mesenchymal plasticity in ovarian cancer cells
Hlubek et al. Heterogeneous expression of Wnt/β‐catenin target genes within colorectal cancer
Marziali et al. Metabolic/proteomic signature defines two glioblastoma subtypes with different clinical outcome
US7890267B2 (en) Prognostic and diagnostic method for cancer therapy
Alsaqer et al. Inhibition of LSD1 epigenetically attenuates oral cancer growth and metastasis
Tahira et al. Long noncoding intronic RNAs are differentially expressed in primary and metastatic pancreatic cancer
Berthier-Vergnes et al. Gene expression profiles of human melanoma cells with different invasive potential reveal TSPAN8 as a novel mediator of invasion
Nakamura et al. Gene expression profile of metastatic human pancreatic cancer cells depends on the organ microenvironment
Zhang et al. Isoform level expression profiles provide better cancer signatures than gene level expression profiles
Christensen et al. Defining new criteria for selection of cell-based intestinal models using publicly available databases
Yu et al. Down‐regulation of MTHFD2 inhibits NSCLC progression by suppressing cycle‐related genes
US20090098538A1 (en) Prognostic and diagnostic method for disease therapy
CA2660857A1 (fr) Procede de pronostic et diagnostic pour la therapie d&#39;une maladie
Pan et al. MicroRNA-7 targets T-Box 2 to inhibit epithelial-mesenchymal transition and invasiveness in glioblastoma multiforme
Yan et al. Upregulation of DLX2 confers a poor prognosis in glioblastoma patients by inducing a proliferative phenotype
Wei et al. Integrative analysis of biomarkers through machine learning identifies stemness features in colorectal cancer
WO2011137302A1 (fr) Procédés d&#39;identification de voies de signalisation intracellulaire régulées de manière aberrante dans des cellules cancéreuses
Lu et al. Integrated RNA sequencing and single-cell mass cytometry reveal a novel role of LncRNA HOXA-AS2 in tumorigenesis and stemness of hepatocellular carcinoma
Li et al. Identification of hub genes and small molecule drugs associated with acquired resistance to gefitinib in non-small cell lung cancer
Maruschke et al. Gene expression analysis in clear cell renal cell carcinoma using gene set enrichment analysis for biostatistical management
Wang et al. Integrative multi-omics and drug–response characterization of patient-derived prostate cancer primary cells
Yang et al. PLCG2 can exist in eccDNA and contribute to the metastasis of non-small cell lung cancer by regulating mitochondrial respiration
Li et al. RPP30, a transcriptional regulator, is a potential pathogenic factor in glioblastoma
Huo et al. CYP27B1 downregulation: a new molecular mechanism regulating EZH2 in ovarian cancer tumorigenicity
Yang et al. m6A regulator-mediated methylation modification patterns and tumor microenvironment cell-infiltration characterization in head and neck cancer

Legal Events

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

Ref document number: 11775606

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11775606

Country of ref document: EP

Kind code of ref document: A1