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WO2018175673A1 - Méthodes et compositions de détection et de traitement de cancer - Google Patents

Méthodes et compositions de détection et de traitement de cancer Download PDF

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Publication number
WO2018175673A1
WO2018175673A1 PCT/US2018/023683 US2018023683W WO2018175673A1 WO 2018175673 A1 WO2018175673 A1 WO 2018175673A1 US 2018023683 W US2018023683 W US 2018023683W WO 2018175673 A1 WO2018175673 A1 WO 2018175673A1
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cancer
epicenter
transcription
sox9
composition
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PCT/US2018/023683
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English (en)
Inventor
Elaine Fuchs
Rene Adam
Yeijing GE
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The Rockefeller University
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Publication of WO2018175673A1 publication Critical patent/WO2018175673A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • SCs stem cells
  • Tumorigenesis arises when normal SCs accumulate mutations that cause them to derail, shifting their homeostatic balance to favor tissue growth at the expense of differentiation.
  • differentiation imbalance is transient, cancers are refractory to tissue restoration cues, seemingly hijacking these normal cellular programs to fuel their molecular thirst for uncontrolled growth.
  • mice with gene mutations that enhance hair follicle SC activity heal their wounds faster, but they also exhibit enhanced susceptibility to squamous cell carcinomas (Guasch et al., 2007; Fiance et al., 2014).
  • mice whose skin possesses mutations that impede HFSC activation display reduced efficiency in wound closure, but also an increased resistance to cancers (Schober et al, 2007;
  • HFSCs hair follicle
  • Epd epidermis
  • HFSCs hair follicle
  • EpdSCs epidermis
  • both EpdSCs and HFSCs in the vicinity of the wound site are mobilized toward it, re-epitheiializing the wound bed and restoring the skin barrier (Ito et al ., 2005; Jensen et al, 2009; Levy et al., 2007; Tumbar et al., 2004).
  • Each lineage can also participate in cancer progression when its SCs acquire oncogenic HRAS mutations (Lapouge et al, 2011; White et al, 2011).
  • HFSCs in the bulge niche are distinguished from EpdSCs in the basal epidermal layer by a cohort of transcription factors (TFs) that include SOX9, LHX2, TCF3/4, NFATcl , NFIB and FOXCl (Blanpam et al, 2004; Morns et al., 2004; Tumbar et al, 2004). SOX9 appears to be particularly important in governing the fate of skin SCs.
  • TFs transcription factors
  • SOX9 is the only HFSC TF that remains expressed, albeit at reduced levels, as HFSCs mobilize and re-epithelialize the injured skin (Adam et al, 2015). Once HFSC progenies reach the epidermis and the wound is healed, SOX9 is no longer detected, indicative of a fate switch. Interestingly, SOX9 is also one of the few HFSC TFs that remain expressed in the tumor-initiating (stem) cells of SCCs (Lapouge et al., 2011; Schober and Fuchs, 201 1 ).
  • the present invention describes a composition for treating cancer, wherein the composition comprises a modulator of a transcription epicenter, wherein the epicenter regulates cancer.
  • the modulator comprises an inhibitor of the activity of the transcription epicenter.
  • the modulator comprises an activator of the activity of the transcription epicenter.
  • the modulator modulates the activation of an epicenter regulator, wherein the regulator is selected from the group consisting of polymerases, aceiyltransferases, histone deacetylases, methylases, histone demethylases, transcription factors, coactivators, corepressors, and enhancers.
  • the modulator modulates the activation of one or more transcription factors selected from the group consisting of SOX9, KLF5, GAT A, API (JUN FOS), AP2, TCF, LHX2, NFI, ETS2, and STAT3.
  • the inhibitor is one or more molecules selected from the group consisting of a small interfering RNA (siRNA), a small guide R.NA (gRNA), a microRNA, an antisense nucleic acid, a ribozyme, an expression vector encoding a trans dominant negative mutant, an antibody, a peptide, a chemical compound and a small molecule.
  • siRNA small interfering RNA
  • gRNA small guide R.NA
  • microRNA an antisense nucleic acid
  • a ribozyme an expression vector encoding a trans dominant negative mutant
  • an antibody a peptide
  • a chemical compound a small molecule
  • the activator is one or more compound selected from the group consisting of a chemical compound, a protein, a peptidomimetic, an antibody, a nucleic acid molecule.
  • the composition comprises a modulator wherein the modulator binds a region of DNA, wherein the region of DNA comprising at least one binding transcription factor binding motif.
  • the transcription factor binding motif is selected from the group consisting of a SOX9 binding motif, a KLFS binding motif, and a combination thereof.
  • the cancer is selected from the group consisting of squamous cell carcinoma and human head and neck squamous cell carcinoma.
  • the invention relates to a method for treating cancer in a subject, the method comprising administering to subject in need thereof a modulator of an epicenter, wherein the epicenter is accessible, and wherein the epicenter regulates cancer.
  • the modulator comprises an inhibitor of the activity of the transcnption epicenter.
  • the modulator comprises an activator of the activity of the transcription epicenter.
  • the modulator modulates the activation of an epicenter regulator, wherein the regulator is selected from the group consisting of polymerases, acetyltransferases, histone deacetylases, methylases, hi stone demethylases, transcription factors, coactivators, corepressors, and enhancers.
  • the modulator modulates the activation of one or more transcription factors selected from the group consisting of SOX9, KLF5, ETS, AP-1, GAT A, GRHL, AP2, TCF, and NFL
  • the inhibitor is one or more molecules selected from the group consisting of a small interfering R A (siR A), a small guide R A (gRNA), a microRNA, an antisense nucleic acid, a ribozyme, an expression vector encoding a transdominant negative mutant, an antibody, a peptide, a chemical compound and a small molecule.
  • the activator is one or more compound selected from the group consisting of a chemical compound, a protein, a peptidomimetic, an antibody, a nucleic acid molecule.
  • the modulator binds a region of DNA, wherein the region of DNA comprising at least one binding transcription factor binding motif.
  • the transcription factor binding motif is selected from the group consisting of a SOX9 binding motif, a KLF5 binding motif, and a combination thereof.
  • the cancer is squamous cell carcinoma. In some embodiments, the cancer is human head and neck squamous cell carcinoma.
  • the invention relates to a method of diagnosing cancer in a subject, the method comprising the step of: (a) obtaining a sample from the subject, (b) detecting epicenter activity relative to a comparator control in the sample, and (c) thereby diagnosing the subject with cancer.
  • the sample is a human sample.
  • the sample is skin tissue.
  • the cancer is squamous cell carcinoma.
  • the cancer is head and neck squamous cell carcinoma.
  • the comparator control is at least one selected from the group consisting of: a positive control, a negative control, a historical control, a historical norm, or the level of a reference molecule in the biological sample.
  • the invention relates to a method of identifying a cancer marker in a sample, the method comprising the steps of (a) classifying the transcriptional state of the sample, (b) performing transcriptome analysis to identify modulated transcription factors, and (c) identifying a cancer marker.
  • the sample is a population of cells.
  • the cells are tumor stem cells.
  • the samples are sorted by transcriptional state wherein transcriptional state is characterized as significantly altered open chromatin state relative to a comparator control.
  • transcriptionally sorted samples are statistically evaluated.
  • the sorted samples are mapped to genes.
  • the genes are classified as either induced or suppressed relative to a comparator control.
  • the comparator control is at least one selected from the group consisting of: a positive control, a negative control, a historical control, a historical norm, or the level of a reference molecule in the biological sample.
  • the genes are subjected to pathway enrichment analysis.
  • the genes are screened for key regulatory elements.
  • the regulator ⁇ ' elements are mapped to transcription epicenters.
  • the epicenters are screened for genes corresponding to the regions containing the epicenters.
  • the epicenters are linked to specific transcription factors.
  • the transcription factors are screened in databases.
  • the invention relates to a composition for treating wounds comprising an inhibitor of SOX9. In some embodiments, the invention relates to a method for treating a subject having a wound, the method comprising administering an inhibitor to a subject in need thereof and inhibitor of SOX9.
  • the invention relates to a composition for detecting transcription epicenters comprising a plurality of probes and a plurality of promoters.
  • the plurality of probes comprises two probes.
  • the plurality of probes comprises a fluorescent protein.
  • the fluorescent protein is selected from the group consisting of RFP, iRFP and GFP.
  • the plurality of promotors comprises two promoters.
  • the two promotors are selected from a group consisting of a H2B, SOX9, KLF5, PGK and miR21.
  • the invention also provides a method of screening for a composition that modulates a transcription epicenter, wherein the epicenter regulates cancer.
  • the method comprises: (a) administering an epicenter reporter construct into cell, (b) detecting epicenter activity relative to a comparator control in the cell, and (c) thereby identifying a composition that modulate the epicenter.
  • Figure 1 depicts results of example experiments demonstrating that tumor SCs undergo global changes in chromatin accessibility compared to their normal counterparts.
  • Figure 1 A depicts results from ATAC-seq performed on FACS sorted tumor (SCC-SCs) and normal (HFSCs and EpdSCs) stem cells. Genome-wide ATAC signals are plotted pair-wise on x- and y-axis to calculate their correlation coefficient of determination (R2).
  • Figure IB depicts whole genome ATAC signals in SCC-, Epd- and HF-SCs are z-score-transformed and averaged across 100-bp genomic windows. Hierarchical clustering shows shared open chromatin regions between normal EpdSCs and HFSCs compared to SCC-SCs.
  • Heatmap shows gain ( ellow), loss (blue) or no change (black) of ATAC signals from normal to stress comparison, 'n' indicates number of genomic windows plotted.
  • Figure 1C depicts cumulative density showing genes that are strongly upregulated in tumorigenic (T) versus normal (N) SCs are those that have gained ATAC peaks (log2FC more positive, green curve right shift), whereas those that exhibit marked tumor-associated declines in expression are genes that have lost ATAC peaks (log2FC more negative, red curve left shift).
  • KS one-sided test was performed to compare genes that had gained or lost ATAC peaks relative to all genes.
  • Figure I D depicts SCC-SC, EpdSC and HFSC ATAC tracks of representative genes that are either constitutively active in all conditions, induced in tumor or suppressed in tumor.
  • Figure 2 depicts the results of example experiments illustrating that SCC-SCs express both epidermal and HF lineage markers.
  • Figure 2A depicts de novo motif analysis identifying enriched TF motifs associated with ATAC-peaks that are unique to HFSCs or EpdSCs compared to peaks that are common to both lineages.
  • Figure 2C depicts immunofluorescence results revealing lineage-specificity in SOX9 and KLF5 expression patterns under skin homeostasis.
  • Figure 2D depicts AT AC tracks revealing enhanced chromatin accessibility within the KLF5 and SOX9 regulatory regions that are specific to SCC-SCs and not seen in the homeostatic SCs of the opposite lineage.
  • Figure 2E depicts de novo motif analysis identifying enriched motifs for TFs associated with AT AC peaks that are unique to SCC-SCs compared to those that are shared between SCC-SCs and their normal counterparts.
  • Figure 3 depicts the results of example experiments illustrating that lineage infidelity is a functiono ally obligatory hallmark of tumorigenesis.
  • FIG. 3C depicts the results from genes associated with AT AC peaks in SCC-SCs containing SOX9 or KLF5 motifs (see Methods Details) that were analyzed by MolSigDB (Broad Institute) for their associated molecular pathways. Note preference of KLF5 for proliferation pathway genes and SOX9 for invasion pathway genes.
  • Figure 3D depicts that overexpression (O/E) of KLF5 in vivo was achieved by lentiviral (IN) TRE-KLF5 infection of E9.5 K14-rtTA embryos.
  • FIG. 3F depicts keratinocytes from transgenic K14-rtTA embryos that were infected with ientiviral TRE-SOX9 or TRE- KLF5 (TRE-emply was used as control) and treated with doxy cy dine for three days, and then subjected to Boy den chamber assays. A layer of matrigel is placed on the lower filter. Keratinocytes in the upper chamber are assayed for their ability to respond to stimulatory dermal fibroblasts conditioned media in the lower chamber and degrade matrigel.
  • Figure 4 depicts the results of example experiments illustrating that epithelial wounding transiently inflicts and relies on lineage infidelity for repair.
  • Figure 4B depicis a schematic of inducible KI.F5 and SOX9 knockout by in vivo CRISPR/CAS to analyze
  • LV Ientiviral
  • tamoxifen was administered to pregnant females, and at P3, half of back skin of pups was directly frozen for sectioning and immune-labeling; the other half was treated with EDTA to remove and discard the epidermis. The dermis, including HFs, was then engrafted onto Nude mice.
  • Figure 5 depicts the results of example experiments illustrating that wounded and tumori genie SCs display similar trans criptonies and genome-wide chromatin accessibilities.
  • Figure 5 A and Figure 5B depict that unsupervised hierarchical clustering and principle component analysis (PC A) of transcriptome data reveals similarities between SCC-SCs and Wound (Wd)-SCs relative to homeostatic SCs (HFSCs and EpdSCs).
  • Figure 5C depicts that gene set enrichment analysis (GSEA) reveals striking parallels in transcriptome changes that occur in tumor and wound versus homeostatic SCs. Gene changes in tumor vs. normal are compared against pre-ranked changes in wound vs. normal. Left panel, up-regulated genes; right panel, down-regulated genes.
  • GSEA gene set enrichment analysis
  • Figure 5D depicts ATAC signals of SCC- and Wd-SC plotted on x- and y-axis, respectively, and that their correlation was calculated (coefficient of determination R2).
  • Figure 5E depicts results from genome-wide ATAC signals in Wd-, SCC-, Epd- and HF-SCs that are z-score-transformed and averaged across 100-bp genomic windows.
  • Hierarchical clustering shows divergence between normal (Epd and HF) and stress-experienced (Wd and SCC) SCs, and heatmap shows gain
  • FIG. 5F depicts ATAC tracks for Wd-, SCC-, Epd- and HF-SCs and H3K27Ac ChlPseq track for SCC-SCs which are shown for I LF5 (left) and mir21 (right) genes. Boxed peaks denote epicenters (ECs) cloned for testing enhancer activities with eGFP reporters in vivo (the tested KLF5 EC is the second boxed peak on the right). Grey shaded box indicates predicted TF motifs within ECs.
  • FIG. 5G depicts that epifluorescence shows stress-induced EC driven reporter activity (eGFP) in the tumor and wound but not in normal skin, while all conditions show comparable LV transduction (H2BRFP). At least five biologically independent experiments were performed; shown are representative images.
  • SE super-enhancers
  • Figure 6 A depicts immunofluorescence results revealing pETS2 in tumor and wound but not in normal skin.
  • Figure 6B depicts that forced activation of ETS2 in the skin epithelium is achieved by transducing K14rtTA embryos with lentivirai TRE- T72D-Ets2, and then treating neonatal mice with doxycycline for 4 weeks starting at P0 (Yang et al., 2015).
  • Backskin was analyzed for pETS2, KLF5 and SOX9. Arrows point to the ectopic KLF5 expression in the HF and SOX9 in the Epd.
  • Figure 6C depicts that immunofluorescence reveals KLF5 and SOX9 co-expression in EpdSCs at the wound edge five days after full-thickness punch wound.
  • Figure 6D depicts that forced activation of LF5 in the skin epithelium is achieved by transducing l 4rtTA embryos with lentiviral TRE-KLF5, and inducing pups with doxycycline for two weeks starting at P0.
  • Backskin was analyzed for KLF5 and SOX9 expression.
  • Figure 6E depicts AT AC peaks at the SOX9 locus of Wd-, SCO, Epd- and HF-SCs contrasting with SOX9 and LHX2 ChJPseq peaks at the SOX9 locus of HFSC-SCs. Boxed areas are magnified (HFSC-EC green, Tumor-EC red). Note silencing of SOX9's homeostatic enhancers and activation of new, tumor-specific enliancers in SCC-SCs.
  • FIG. 6F depicts epi fluorescence results showing that SOX9's homeostatic enhancer drives GFP reporter activity only in HFSCs and not in tumor or wound states, while SOX9's SCOspecific enhancer drives GFP reporter activity only in the SCC and not in wound or normal homeostasis.
  • Figure 3 where low stress ATAC peaks driver reporter expression in both SCC and wound but not normal homeostasis. All reporters have comparable lentiviral infectivity (H2BRFP).
  • Figure 6G depicts that forced activation of SOX9 in the skin epithelium is achieved by transducing TRE-SOX9 embryos with lentiviral rtTA-H2BGFP, and then treating animals with doxyc cline just prior to split-thickness engraftrnent (see Method Details). Skin grafts were then analyzed two weeks later, with half of the graft used for sectioning and immunofluorescence for SOX9, H2BGFP, KLF5 and pETS2, and the other half for EdU pulse and analyses.
  • Figure 7 depicts the results of example experiments illustrating a model for sustained imeage infidelity in diverging tumors from wounds.
  • skin EpdSCs and HFSCs govern their own fates by activating lineage-specific homeostatic epicenters (ECs) for KLFS and SOX9, respectively, in wound-repair, wound- ECs regulated by RAS/MAPK-induced, stress-responsive TFs ETS2 among others, become activated to drive expression of both KLF5 and SOX9 genes, irrespective of SC origin. This leads to transient lineage infidelity, which is resolved when RAS/MAPK and pETS2 activity wanes upon restoration of the skin barrier.
  • ECs lineage-specific homeostatic epicenters
  • Figure 8 depicts the results of example experiments illustrating that attributes of ATAC-seq peaks in tumor and wound SCs confirm their quality and usefulness to identify gene regulatory regions.
  • ENCODE ChlPseq
  • Figure 8B depicts the distribution of AT AC peaks (total numbers listed) over annotated genomic regions shown as pie charts for SCC-, Epd- and HF-SC chromatin. Note consistency with enriched signals over intergenic regulatory regions.
  • Figure 8C depicts that the location of AT AC peak marks within an epicenter (EC) within the super-enhancer of the Cxcll4 gene that is known to be active in HFSCs, and bound by an entire suite of TFs that are flanked by H3K27Ac signals (Adam et al., 2015).
  • the current ATAC dataset for EpdSCs, SCC-SCs and HFSCs were plotted, to illustrate ATAC can be used to identify bona fide gene regulatory regions, in this case active in HFSC and silent in SCC-SC and Epd SC.
  • Figure 9 depicts the results of example experiments illustrating that
  • S2KLF5 is and SOX9 are co-expressed in hyperplastic and benign tumor stages.
  • FIG. 10 depicts the results of example experiments illustrating efficient knockdown of KLF5 in SCC.
  • SCC cells Prior to injections, SCC cells were transduced with a GFP lenti virus (to mark the tumor cells as opposed to stroma) and also a KLF5 or Scramble control shRNA hairpin and puromycin selection marker, administered for 2d to obtain stable integration.
  • Scale bar 50 urn.
  • Two different KLF5 shRNA hairpins were tested and gave consistent results. Three biologically independent experiments were performed; shown are representative images.
  • Figure 11 depicts the results of example experiments illustrating efficient CRISPR/CAS-mediated SOX9 and KLF5 gene ablation in vivo.
  • Amniotic sacs of living Rosa26-Loxp-STOP-Loxp-Cas9-P2A-GFP E9.5 embryos were injected in utero with lend virus (LV) harboring PGK-CreER and a U6- sgRNA against KLF5 or SOX9 or Scramble control.
  • LV lend virus
  • Embryos were removed for immunofluorescence analysis of either sagittal skin sections (Figure 1 1 A, E18.5, KLF5 ablated; Scr) or whole mount ( Figure 1B, E16.5, SOX9 ablated; Scr).
  • KLF5 and GFP are mutually exclusive in the epidermis, reflective of efficient KLF5 knockout in LV -transduced cells.
  • green GFP boxed insets in lower left corners of mainframes
  • P-cadherin (PCAD) and LHX2 served as controls to mark FIFs and were not affected by SOX9 ablation.
  • Scale bar : 50um for sagittal sections, 200 ⁇ for whole mount images. At least two distinct sgRNAs were tested for each gene and both gave consistent results. At least 5 biologically independent experiments were performed; shown are representative images.
  • Figure 12 depicts the results of example experiments illustrating that wound and tumor transcriptomes and chromatin accessibility profiles share similarities distinct from proliferative progenies of stem cells.
  • Figure 12B depicts gene set enrichment analysis (GSEA) compares the transcripts up or downregulated by 53 ⁇ 44X (p ⁇ 0.05) in stressed conditions vs. normal compared against pre-ranked changes in proliferative TAC (transient amplifying cells, derived from HFSCs) vs. homeostatic HFSCs. Comparisons of signatures of TACs and Wd-SCs are shown in the top panels; TACs and SCC-SCs in the bottom panels. Note that these signatures do not significantly overlap, indicating that the similarities seen between Wd-SCs and SCC-SCs go beyond merely similarities in proliferative features.
  • GSEA gene set enrichment analysis
  • Figure 12C depicts that strong AT AC signals are centered throughout the transcription start sites (TSS, left) and CTCF motif sites (right) of the wounded (Wd) SC chromatin. Plots of the genome-wide data illustrate the expected peaks (numbers on X axis in kb; Y axis, average number of sequencing reads at each position).
  • Figure 12D depicts the distribution of AT AC peaks over annotated genomic regions of Wd-SC chromatin, shown as a pie chart, and highlights the enrichment of AT AC signals over intergenic regulator ⁇ ' regions.
  • Figure 13A depicts ATAC peaks at the Seal and Hesl locus
  • FIG. 13B depicts immunofluorescence results revealing the presence of AP I factors JUN and FOS as well as pSTAT3 in tumor and wound. FOS is absent in homeostatic skin and JUN, pSTAT3 are weak.
  • Figure 13C depicts that forced activation of ETS2 in the skin epithelium is achieved by transducing E9.5 KMrtTA embryos in utero with lentivirus harboring TRE-ETS2 (T72D).
  • Figure 14 depicts the results of example experiments illustrating that KLF5 reprograms HF into epidermis and sustained SOX9 pushes wound into tumor.
  • Figure 14A depicts that forced activation of KLF5 in the skin epithelium is achieved by transducing KMrtTA embryos with lentivirai TRE-KLF5, and inducing pups with doxycycline for 2 weeks starting at P0. Back skin was analyzed for Kl 0, LORICRIN, and TENASCIN C.
  • FIG. 14B and Figure 14C depict that forced activation of SOX9 in the skin epithelium was achieved by transducing E9.5 TRESOX9 embryos in utero with lentivirus harboring PGK-rtTA-H2BGFP. Doxycyclme was administered starting at E18.5 and at P3, split- thickness grafts were performed on host Nude mice (see Method Details).
  • Figure 15 is an image demonstrating that epicenter activity in cultured mouse and human SCC lines.
  • Figure 16 is an image demonstrating epicenter activity in huma and neck squamous cell carcinoma models.
  • the present invention provides compositions and methods for detecting and treating cancer.
  • the present invention is partly based upon the observation that human patients suffering from chronic wounds have increased susceptibility to cancers.
  • the present invention is partly based on the observation that mice with gene mutations that enhance hair follicle stem cell activity heal their wounds faster but also exhibit susceptibility to squamous cell carcinoma.
  • mice whose skin possesses mutations that impede hair follicle stem cell activation display reduced efficiency in wound closure, but an increased resistance to cancers.
  • the present invention provides methods for detecting and modulating transcription epicenters and super-enhancer regions in DNA that are specifically accessible in cells transitioning to a cancerous state.
  • the present invention provides compositions and methods for preventing and treating cancer through use of modulators of epicenters and super-enhancer regions.
  • the present invention also provides a composition for detecting accessible promoter regions for the purpose of identifying new epicenters and relevant transcription factors that bind thereto that may be new targets for cancer therapeutics.
  • an element means one element or more than one element.
  • abnormal when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cell s or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, ceils or components thereof that display the "normal” (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.
  • alteration refers to a mutation in a gene in a cell that affects the function, activity, expression (transcription or translation) or conformation of the polypeptide that it encodes.
  • Mutations encompassed by the present invention can be any mutation of a gene in a ceil that results in the enhancement or disruption of the function, activity, expression or conformation of the encoded polypeptide, including the complete absence of expression of the encoded protein and can include, for example, missense and nonsense mutations, insertions, deletions, frameshifts and premature terminations.
  • mutations encompassed by the present invention may alter splicing the mRNA (splice site mutation) or cause a shift in the reading frame (frameshift).
  • a disease or disorder is "alleviated” if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced.
  • "Alleviating" specific cancers and/or their pathology includes degrading a tumor, for example, breaking down the structural integrity or connective tissue of a tumor, such that the tumor size is reduced when compared to the tumor size before treatment, "Alleviating" cancer includes reducing the rate at which the cancer spreads to other organs.
  • amplification refers to the operation by which the number of copies of a target nucleotide sequence present in a sample is multiplied.
  • apper any device including, but not limited to, a hypodermic syringe, a pipette, an iontophoresis device, a patch, and the like, for administering the compositions of the invention to a subject.
  • antibody refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact
  • immunoglobulins derived from natural sources or from recombinant sources and can be immuno-reactive portions of intact immunoglobulins.
  • Antibodies are typically tetramers of immunoglobulin molecules.
  • the antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab) 2 , as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al, 1988, Science 242:423-426).
  • antibody fragment refers to at least one portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody.
  • antibody fragments include, but are not limited to. Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, sdAb (either VL or VH), camelid VHH domains, scFv antibodies, and multi-specific antibodies formed from antibody fragments.
  • scF refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it was derived.
  • an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N- terminal and C -terminal ends of the polypeptide, the scFv may comprise Vi-Iinker-Vn or may comprise Vi-i-linker-VL.
  • antibody light chain refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations, ⁇ and ⁇ light chains refer to the two major antibody light chain isotypes.
  • synthetic antibody an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein.
  • the term should also be construed to mean an antibody which has been generated by the synthesis of a DM A molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or ammo acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
  • an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample.
  • an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific.
  • an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.
  • the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A” and the antibody, will reduce the amount of labeled A bound to the antibody.
  • a particular structure e.g., an antigenic determinant or epitope
  • antigen or "Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologicaily-competent cells, or both.
  • any macromolecule including virtually ail proteins or peptides, can serve as an antigen.
  • antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein.
  • an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a ' " gene" at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a ceil or a biological fluid.
  • anti-tumor effect refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition.
  • An "anti-tumor effect” can also be manifested by the ability of the peptides,
  • polynucleotides, cells and antibodies of the invention in prevention of the occurrence of tumor in the first place.
  • cancer as used herein is defined as disease characterized by the abnormal growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, squamous cell carcinoma, sarcoma and the like.
  • the term “marker” or “biomarker” is meant to include a parameter which is useful according to this invention for determining the presence and/or severity of disease.
  • the level of a marker or biomarker "significantly" differs from the level of the marker or biomarker in a reference sample if the level of the marker in a sample from the patient differs from the level in a sample from the reference subject by an amount greater than the standard error of the assay employed to assess the marker, and preferably at least 10%, and more preferably 25%, 50%, 75%, or 100%.
  • control or reference standard describes a material comprising none, or a normal, low, or high level of one of more of the marker (or biomarker) expression products of one or more the markers (or biomarkers) of the invention, such that the control or reference standard may serve as a comparator against which a sample can be compared.
  • determining the level of marker (or biomarker) expression is meant an assessment of the degree of expression of a marker in a sample at the nucleic acid or protein level, using technology available to the skilled artisan to detect a sufficient portion of any marker expression product.
  • “Differentially increased expression” or “up regulation” refers to biomarker product levels which are at least 10% or more, for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% higher or more, and/or 1.1 fold, 1.2 fold, 1.4 fold, 1.6 fold, 1.8 fold, 2.0 fold higher or more, and any and ail whole or partial increments
  • “Differentially decreased expression” or “down regulation” refers to biomarker product levels which are at least 10% or more, for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% lower or less, and/or 2.0 fold, 1.8 fold, 1.6 fold, 1.4 fold, 1.2 fold, 1.1 fold or less lower, and any and all whole or partial increments therebetween tha a control.
  • a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
  • a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., DNA, cDNA, rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same ammo acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.
  • Effective amount or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result. Such results may include, but are not limited to, the inhibition of virus infection as determined by any means suitable in the art.
  • endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • expression is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
  • an “immunoassay” refers to a biochemical test that measures the presence or concentration of a substance in a sample, such as a biological sample, using the reaction of an antibody to its cognate antigen, for example the specific binding of an antibody to a protein. Both the presence of the antigen or the amount of the antigen present can be measured.
  • inhibitor means to suppress or block an activity or function by at least about ten percent relative to a control value.
  • the activity- is suppressed or blocked by 50% compared to a control value, more preferably by 75%, and even more preferably by 95%.
  • an "instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of a component of the invention in a kit for detecting biomarkers disclosed herein.
  • the instructional material of the kit of the invention can, for example, be affixed to a container which contains the component of the invention or be shipped together with a container which contains the component. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional materia] and the component be used cooperatively by the recipient.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • nucleic acid bases In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosme, “T” refers to thymidine, and “L! refers to uridine.
  • label when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to a probe to generate a "labeled" probe.
  • the label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may cataly ze chemical alteration of a substrate compound or composition that is detectable (e.g., avidin-biotin).
  • primers ca be labeled to detect a PCR product.
  • the "level" of one or more biomarkers means the absolute or relative amount or concentration of the biomarker in the sample.
  • marker expression encompasses the transcription, translation, post-translation modification, and phenotypic manifestation of a gene, including all aspects of the transformation of information encoded in a gene into RNA or protein.
  • marker expression includes transcription into messenger RNA (mRNA) and translation into protein, as well as transcription into types of RNA such as transfer RNA (tRNA) and ribosomal RNA
  • microarray and “array” refers broadly to both “DNA microarrays” and “DNA chip(s),” and encompasses all art-recognized solid supports, and all art-recognized methods for affixing nucleic acid molecules thereto or for synthesis of nucleic acids thereon.
  • Preferred arrays typically comprise a plurality of different nucleic acid probes that are coupled to a surface of a substrate in different, known locations.
  • These arrays also described as “microarrays” or colloquially “chips” have been generally described in the art, for example, U.S. Pat. Nos.
  • Arrays may generally be produced using a variety of techniques, such as mechanical synthesis methods or light directed synthesis methods that incorporate a combination of photolithographic methods and solid phase synthesis methods. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. Nos. 5,384,261 , and 6,040,193, which are incorporated herein by reference in their entirety for all purposes.
  • arrays may be nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate.
  • Arrays may be packaged in such a manner as to allow for diagnostic use or can be an all-inclusive device; e.g., U.S. Pat. Nos. 5,856,174 and 5,922,591 incorporated in their entirety by reference for all purposes.
  • Arrays are commercially available from, for example, Affymetrix (Santa Clara, Calif.) and Applied Biosy stems (Foster City, Calif.), and are directed to a variety of purposes, including genotyping, diagnostics, mutation analysis, marker expression, and gene expression monitoring for a variety of eukaryotic and prokaryotie organisms.
  • the number of probes on a solid support may be varied by changing the size of the individual features. In one embodiment, the feature size is 20 by 25 microns square, in other embodiments features may be, for example, 8 by 8, 5 by 5 or 3 by 3 microns square, resulting in about 2,600,000, 6,600,000 or 18,000,000 individual probe features.
  • detection means assessing the presence, absence, quantity or amount (which can be an effective amount) of either a given substance within a clinical or subject-derived sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise evaluating the values or categorization of a subject's clinical parameters.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • nucleotide sequence that encodes a protein or an R A may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an mtron(s).
  • operbiy linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operabiy linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operabiy linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operabiy linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • parenteral administration of an immunogenic composition includes, e.g., subcutaneous (s.c), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.
  • patient refers to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein.
  • the patient, subject or individual is a human.
  • nucleic acid as used herein is defined as a chain of nucleotides.
  • nucleic acids are polymers of nucleotides.
  • nucleic acids and polynucleotides as used herein are interchangeable.
  • nucleic acids are polynucleotides, which can be hvdrolyzed into the monomeric "nucleotides.”
  • the monomelic nucleotides can be hvdrolyzed into nucleosides.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
  • recombinant means i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCRTM, and the like, and by synthetic means.
  • polypeptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of ammo acid residues covalentiy linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified
  • polypeptides derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof
  • the term "providing a prognosis” refers to providing a prediction of the probable course and outcome of disease, including prediction of severity, duration, chances of recovery, etc.
  • the methods ca also be used to devise a suitable therapeutic plan, e.g., by indicating whether or not the condition is still at an early stage or if the condition has advanced to a stage where aggressi v e therapy would be ineffective.
  • a “reference level” of a biomarker means a level of the biomarker that is indicative of a particular disease state, phenotype, or lack thereof, as well as combinations of disease states, phenoty pes, or lack thereof.
  • a “positive" reference level of a biomarker means a level that is indicative of a particular disease state or phenotype.
  • a “negative” reference level of a biomarker means a level that is indicative of a lack of a particular disease state or phenotype.
  • sample or “biological sample” as used herein means a biological material isolated from an individual.
  • the biological sample may contain any biological material suitable for detecting the desired biomarkers, and may comprise cellular and/or non-cellular material obtained from the individual.
  • Standard control value refers to a predetermined amount of a particular protein or nucleic acid that is detectable in a sample.
  • the standard control value is suitable for the use of a method of the present in vention, in order for comparing the amount of a protein or nucleic acid of interest that is present in a sample.
  • An established sample serving as a standard control provides an average amount of the protein or nucleic acid of interest in the sample that is typical for an average, healthy person of reasonably matched background, e.g., gender, age, ethnicity, and medical history.
  • a standard control value may vary depending on the protein or nucleic acid of interest and the nature of the sample.
  • subject refers to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein.
  • patient, subject or individual is a human.
  • terapéutica as used herein means a treatment and/or prophylaxis.
  • a therapeutic effect is obtained by suppression, remission, or eradication of a disease state.
  • a “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear
  • vector includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, poly lysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • ranges throughout this disclosure, v arious aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Description
  • the present invention relates to the identification of transcription epicenters and super-enhancer regions, and using modulators of the activity of transcription epicenters and super-enhancer regions.
  • Epicenters are short regions of DNA that can be bound by proteins, for example transcription factors, to increase the likelihood that transcription of a particular gene or genes can occur.
  • epicenters are regulatory regions of DNA where one or more transcription factors bind.
  • epicenters are shouldered by active histone markers (e.g. H3K27Ac).
  • super- enhancers are broad open chromatin domains marked with active histone markers, comprising one or more epicenters.
  • the present invention provides compositions and methods for detecting and treating cancer.
  • the composition comprises a modulator of transcription epicenter activity or super-enhancer activity.
  • the composition modulates the accessibility of the transcription epicenter or super-enhancer.
  • the transcription epicenter is a short (around one kilobase of nucleotides in length) active subdomain within super- enhancers, which is particularly enriched for transcription factor binding sites and allows for cooperative binding of a cohort of cell type- and state- specific transcription factors.
  • Epicenters are tissue-, lineage- and cell-stage specific, change dynamically depending on the microenvironment and thus drive gene expression in a highly spatio-temporally selective manner.
  • the super-enhancer region is a densely spaced cluster of active enhancers (often larger than 10 kilobases of nucleotides in length) with unusually strong enrichment for the binding of cell-type specific transcription factors and transcriptional coactivators, including Mediator (MEDl ).
  • Super-enhancers are also marked by highly concentrated active histone markers (including H3K27ac), and harbor multiple clusters of epicenters to drive cell type- and state- specific gene expression. Super-enhancers associate with critical cell-identity genes and thus dictate cellular behavior and fate.
  • the composition comprises a modulator of one or more epicenter regulator proteins or other components associated with transcription epicenter or super-enhancer accessibility .
  • the modulator modulates the expression or activity of the one or more epicenter regulator proteins.
  • Exemplar ⁇ ' epicenter regulatory proteins associated with transcription epicenter or super-enhancer accessibility include, but is not limited to, polymerases,
  • the composition modulates the assembly of transcription machinery specific to the assembly of active transcription complexes within epicenters or super- enhancer regions.
  • the composition comprises a modulator of one or more factors that bind to the transcription epicenter or super-enhancer.
  • Exemplary factors include, but are not limited to, transcription factors, coacti vators, corepressors, and enhancers.
  • the modulator modulates the expression or activity of the one or more factors.
  • the modulator modulates the formation of a complex of the one or more factors.
  • the invention provides compositions and methods for modulating epicenters and/or super-enhancer regions.
  • modulating epicenters and/or super-enhancer regions include but is not limited to modulating the accessibility of the transcription epicenter and/or super-enhancer, modulating proteins or other components associated with transcription epicenter or super- enhancer accessibility, desired transcription factors, and the likes.
  • the modulator modulates binding of the one or more factors to the transcription epicenter or super-enhancer.
  • the composition modulates one or more transcription factors identified to be oncogenic. In one embodiment, the composition modulates one or more transcription factors identified to regulate distinct cell phenotypes. In one embodiment, the composition modulates one or more transcription factors identified to regulate cellular communication. In one embodiment, the composition modulates one or more transcription factors identified to regulate cellular movements and adhesion. In one embodiment, the one or more transcription factors include, but are not limited to, SOX9, KLF5, GATA, API (JUN/FOS), AP2, TCF, LHX2, NFI, ETS2, and STATS. In one embodiment, the composition modulates one or more of SOX9 and KLF5.
  • the present invention provides methods for treating and preventing cancer.
  • the method comprises modulating epicenters and super-enhancers that are specifically accessible in cells transitioning to a cancerous state.
  • the method comprises
  • the method comprises modulating epicenters or super-enhancer regions by administering compositions described herein. For example, in one
  • the method comprises administering a modulator of one or more transcription factors, as described herein.
  • the present invention also provides methods for detecting new epicenters and relevant transcription factors that bind thereto that may be new targets for cancer diagnostics and therapeutics.
  • the present invention provides transcription probes indicating the accessibility of certain promoter regions, transcription factors, epicenters, super-enhancers, and/or epicenter regulating proteins.
  • the present invention provides a method of diagnosing cancer in a subject by detecting the activity of a transcription epicenter or super-enhancer.
  • the method comprises detecting the accessibility of the transcription epicenter or super-enhancer.
  • the method comprises detecting the presence or abundance of one or more components associated with accessibility of the transcription epicenter or super-enhancer.
  • the method comprises detecting the presence or abundance of one or more factors that bind to the transcription epicenter or super-enhancer.
  • the method comprises detecting the presence or abundance of one or more transcripts associated with the transcription epicenter or super-enhancer.
  • the invention provides a composition for treating wounds.
  • the composition comprises a modulator epicenter activity such that when administered, wound healing is improved.
  • the composition comprises an activator of epicenter activity and-' or transcription factor activity that promote wound healing, cell proliferation, cell migration, differentiation, remodeling and homeostasis.
  • the composition comprises an inhibitor of epicenter activity and/or transcription factors that prevent wound healing, cell proliferation, cell migration, differentiation, remodeling and homeostasis.
  • the present invention provides methods for treating wounds comprising administering to a subject a composition that modulates epicenter activity.
  • the method comprises administering to a subject in need thereof, a composition that modulated epicenter activity, wherein when the composition is administered, wound healing is improved.
  • the method comprises administering a composition modulates the activity of one or more epicenters.
  • the method comprises administering a composition modulates the activity of one or more transcription factors.
  • the composition provides a modulator (e.g., an inhibitor or activator) of transcription epicenter activity or super-enhancer activity.
  • the invention relates to compositions that modulate chromatin remodeling, thereby modulating the accessibility of epicenters or super-enhancers.
  • the invention relates to modulators of histone modifications (e.g., mono-methylation, di-methylation, tri-methylation, acetylation).
  • the present invention relates to modulators of specific enzymes that modify chromatin (e.g., acetyltransferases, deacetylases, methyltransferases, and kinases).
  • the present invention relates to modulators of ATP -dependent chromatin remodeling (e.g., modulators of remodel ers of chromatin including but not limited to SWI/SNF, ISWI, NuRD/Mi-2/CHD, 1NO80 and SWR1).
  • modulators of ATP -dependent chromatin remodeling e.g., modulators of remodel ers of chromatin including but not limited to SWI/SNF, ISWI, NuRD/Mi-2/CHD, 1NO80 and SWR1).
  • the composition provides a modulator of one or more transcription factors the bind transcription epicenters. In some embodiments, the composition provides a modulator of one or more transcription factors that modulate epicenter activity.
  • the transcription factors include but are not limited to SOX9, KLF5, GAT A, AP I (JUN/FOS), AP2, TCP, LHX2, NFI, ETS2, and STAT3.
  • the invention provides compositions and methods for modulating epicenters and/or super-enhancer regions.
  • modulating epicenters and/or super-enhancer regions include but is not limited to modulating the accessibility of the transcription epicenter and/or super-enhancer, modulating proteins or other components associated with transcription epicenter or super- enhancer accessibility, desired transcription factors, and the likes.
  • inhibitors are used to prevent expression of or activity of one or more transcription epicenters. In some embodiments, the inhibitors are used to prevent binding of proteins to transcription epicenters.
  • the proteins are transcription factors, regulators of transcription factor expression or activity, or regulators of transcription.
  • the inhibitors of transcription factor expression are regulators of transcription and translation of transcription factors, including but are not limited to siRNA, antisense nucleic acids, ribozymes, small molecules, and antagonists.
  • the regul ators of transcription factor activity are enzymes including but not limited to kinases.
  • regulators of transcription include by are not limited to polymerases, acetyltransferases, histone deacetylases, and methylases.
  • siRNA is used to decrease the activity of one or more epicenters.
  • the siRNA is used to decrease the level of one or more of transcription factor (e.g., SOX9, LF5, GATA, API (JUN/FOS), AP2, TCF, LHX2, NFI, ETS2, and STAT3) binding to an epicenter.
  • the siRNA is used to decrease the level of one or more epicenter regulator protein.
  • RNA interference is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA.
  • siRNAs short 21-25 nucleotide small interfering RNAs, or siRNAs, by a nbonuciease known as Dicer.
  • the siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC), unwinding in the process.
  • RISC RNA-induced silencing complex
  • Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA.
  • the bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See, for example, U.S. Patent No.
  • RNA Interference RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DN A Press, Eaglevender, PA (2003); and Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003).
  • Soutschek et al. 2004, Nature 432: 173-178) describe a chemical modification to siRNAs that aids in intravenous systemic delivery.
  • Optimizing siRNAs involves consideration of overall G/C content, C/T content at the termini, Tm and the nucleotide content of the 3' overhang. See, for instance, Schwartz et al, 2003, Cell, 1 15: 199-208 and Khvorova et al, 2003, Cell 115:209-216. Therefore, the present invention also includes metliods of decreasing levels of the desired transcription factor at the protein level using RNAi technology. In doing so, the present invention includes methods of decreasing the activity of one or more epicenters.
  • the invention includes an isolated nucleic acid encoding an inhibitor, wherein an inhibitor such as an siRNA or antisense molecule, inhibits the desired one or more epicenters, one or more transcription factors binding thereto, a derivative thereof, a regulator thereof, or a downstream effector, operably linked to a nucleic acid comprising a promoter/regulatory sequence such that the nucleic acid is preferably capable of directing expression of the protein encoded by the nucleic acid.
  • the invention encompasses expression vectors and methods for the introduction of exogenous DNA into cells with concomitant expression of the exogenous DNA in the cells such as those described, for example, in Sambrook et al.
  • the desired epicenter, one or more transcription factors binding thereto, or a regulator thereof can be inhibited by way of inacti vating and/or sequestering one or more of the epicenters, transcription factors, or a regulator thereof.
  • inhibiting the effects of the epicenter or one or more transcription factors binding thereto can be accomplished by using a transdominant negative mutant.
  • the invention includes a vector comprising an siRNA or antisense polynucleotide.
  • the siRNA or antisense polynucleotide is capable of inhibiting the expression of the one or more transcription factors or other proteins involved in the regulation of the epicenter.
  • the incorporation of a desired polynucleotide into a vector and the choice of vectors is well-known in the art as described in, for example, Sambrook et al, supra, and Ausubel et al., supra, and elsewhere herein.
  • the siR A or antisense polynucleotide can be cloned into a number of types of vectors as described elsewhere herein.
  • at least one module in each promoter functions to position the start site for RNA synthesis.
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing ceils from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be earned on a separate piece of DNA and used in a co-transfection procedure.
  • Both selectable markers and reporter genes may be flanked with appropriate regulator ⁇ - sequences to enable expression in the host cells.
  • Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neomycin resistance and the like.
  • an antisense nucleic acid sequence which is expressed by a plasmid vector is used to inhibit a desired epicenter or one or more transcription factors binding thereto.
  • the antisense expressing vector is used to transfect a mammalian cell or the mammal itself, thereby causing reduced epicenter activity, endogenous expression of the one or more transcription factors or epicenter regulator proteins.
  • Antisense molecules and their use for inhibiting gene expression are well known in the art (see, e.g., Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press).
  • Antisense nucleic acids are DNA or RNA molecules that are complementary, as that term is defined elsewhere herein, to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize to the corresponding mRNA, forming a double- stranded molecule thereby inhibiting the translation of genes.
  • antisense molecules may be provided to the cell via genetic expression using DNA encoding the antisense molecule as taught by Inoue, 1993, U.S. Patent No. 5,190,931.
  • antisense molecules of the invention may be made synthetically and then provided to the ceil.
  • Antisense oligomers of between about 10 to about 30, and more preferably about 15 nucleotides, are preferred, since they are easily synthesized and introduced into a iarget cell.
  • Synthetic antisense molecules contemplated by the invention include oligonucleotide derivatives known in the art which have improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243),
  • compositions and methods for the synthesis and expression of antisense nucleic acids are as described elsewhere herein.
  • Ribozymes and their use for inhibiting gene expression are also well known in the art (see. e.g., Cech et al., 1992, J. Biol. Chem. 267: 17479-17482; Hampel et al., 1989, Biochemistry 28:4929-4933; Eckstein et al., Internationa] Publication No, WO 92/07065; Altaian et al , U.S. Patent No. 5, 168,053). Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases.
  • RNA molecules can be engineered to recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, 1988, J. Amer. Med. Assn. 260:3030).
  • ech 1988, J. Amer. Med. Assn. 260:3030.
  • a major advantage of this approach is the fact that ribozymes are sequence-specific.
  • Tetrahymena-type ribozymes recognize sequences which are four bases in length, while hammerhead-type ribozymes recognize base sequences 11-18 bases in length. The longer the sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species.
  • hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating specific mRNA species, and 18-base recognition sequences are preferable to shorter recognition sequences which may occur randomly within various unrelated mRNA molecules.
  • a ribozyme is used to inhibit a desired epicenter, one or more regulators of epicenter activity, or one or more transcription factors binding thereto.
  • Ribozymes useful for inhibiting the expression of a target molecule may be designed by incorporating target sequences into the basic ribozyme structure which are complementary, for example, to the mRNA sequence of the collagen modifying enzyme of the present invention.
  • Ribozymes targeting a desired collagen modifying enzyme may be synthesized using commercially available reagents (Applied Bios stems. Inc., Foster City, CA) or they may be genetically expressed from DNA encoding them.
  • a small molecule agonist may be obtained using standard methods known to the skilled artisan. Such methods include chemical organic synthesis or biological means. Biological means include purification from a biological source, recombinant synthesis and in vitro translation systems, using methods well known in the art.
  • Combinatorial libraries of moieculariy diverse chemical compounds potentially useful in treating a variety of diseases and conditions are well known in the art as are method of making the libraries.
  • the method may use a variety of techniques well- known to the skilled artisan including solid phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, tagging techniques, and generating unbiased molecular landscapes for lead discovery vs. biased structures for lead development.
  • an activated core molecule is condensed with a number of building blocks, resulting in a combinatorial library of covalently linked, core-building block ensembles.
  • the shape and rigidity of the core determines the orientation of the building blocks in sh ape space.
  • the libraries can be biased by changing the core, linkage, or building blocks to target a characterized biological structure (“focused libraries") or synthesized with less structural bias using flexible cores.
  • the small molecule is able to inhibit one or more epicenters or transcription factor binding domains. In one embodiment, the small molecule induces the expression of the intracellular enzyme procollagen-lysine, 2- oxoglutarate 5-dioxygenase 2 (PLOD2). In one embodiment, the small molecule is minoxidil or a salt or chemical analog thereof.
  • one or more epicenters, one or more epicenter regulator proteins, or one or more transcription factors binding thereto can be inhibited by way of inactivating and/or sequestering the epicenter, regulator of epicenter activity, or one or more transcription factors binding thereto.
  • inhibiting the effects of an epicenter, regulator of epicenter activity, or one or more transcription factors binding thereto can be accomplished by using a transdominant negative mutant.
  • an antibody specific for the epicenter, regulator, or one or more transcription factor otherwise known as an antagonist to the epicenter, regulator, or one or more transcription factor may be used.
  • the antagonist is a protein and/or compound having the desirable property of interacting with a binding partner of the epicenter, regulator, or one or more transcription factor, and thereby competing with the corresponding protein.
  • the antagonist is a protein and/or compound having the desirable property of interacting with the epicenter, regular, or one or more transcription factor and thereby sequestering the epicenter, regulator, or one or more transcription factor.
  • any antibody that can recognize and bind to an antigen of interest is useful in the present invention.
  • Methods of making and using antibodies are well known in the art.
  • polyclonal antibodies useful in the present invention are generated by immunizing rabbits according to standard immunological techniques well-known in the art (see, e.g. , Harlow et al., 1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, NY).
  • Such techniques include immunizing an animal with a chimeric protein comprising a portion of another protein such as a maltose binding protein or glutathione (GSH) tag polypeptide portion, and/or a moiety such that the antigenic protein of interest is rendered immunogenic (e.g.
  • GSH glutathione
  • chimeric proteins are produced by cloning the appropriate nucleic acids encoding the marker protein into a plasmid vector suitable for this purpose, such as but not limited to, pMAL-2 or pCMX.
  • the invention should not be construed as being limited solely to methods and compositions including these antibodies or to these portions of the antigens. Rather, the invention should be construed to include other antibodies, as that term is defined elsewhere herein, to antigens, or portions thereof.
  • the present invention should be construed to encompass antibodies, inter alia, bind to the specific antigens of interest, and they are able to bind the antigen present on Western blots, in solution in enzyme linked immunoassays, in fluorescence activated cells sorting (FACS) assays, in magnetic-activated cell sorting (MACS) assays, and in immunofluorescence microscopy of a cell transiently transfected with a nucleic acid encoding at least a portion of the antigenic protein, for example.
  • FACS fluorescence activated cells sorting
  • MCS magnetic-activated cell sorting
  • the antibody can specifically bind with any portion of the antigen and the full- length protein can be used to generate antibodies specific therefor.
  • the present invention is not limited to using the full-length protein as an immunogen. Rather, the present invention includes using an immunogenic portion of the protein to produce an antibody that specifically binds with a specific antigen. That is, the invention includes immunizing an animal using an immunogenic portion, or antigenic determinant, of the antigen.
  • polyclonal antibodies The generation of polyclonal antibodies is accomplished by inoculating the desired animal with the antigen and isolating antibodies which specifically bind the antigen therefrom using standard antibody production methods such as those described in, for example, Harlow et al. (1988, in: Antibodies, A Laborator ' Manual, Cold Spring Harbor, NY).
  • Monoclonal antibodies directed against full length or peptide fragments of a protein or peptide may be prepared using any well-known monoclonal antibody- preparation procedures, such as those described, for example, in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, NY) and in Tuszynski et al. (1988, Blood, 72: 109-115). Q uantities of the desired peptide may also be synthesized using chemical synthesis technology. Alternatively, DNA encoding the desired peptide may be cloned and expressed from an appropriate promoter sequence in cells suitable for the generation of large quantities of peptide. Monoclonal antibodies directed against the peptide are generated from mice immunized with the peptide using standard procedures as referenced herein.
  • Nucleic acid encoding the monoclonal antibody obtained using the procedures described herein may be cloned and sequenced using technology which is available in the art, and is described, for example, in Wright et al. (1992, Critical Rev. Immunol. 12: 125-168), and the references cited therein. Further, the antibody of the invention may be "humanized' " using the technology described in, for example, Wright et al, and in the references cited therein, and in Gu et al. (1997, Thrombosis and
  • Hematocyst 77:755-759 Hematocyst 77:755-759), and other methods of humanizing antibodies well-known in the art or to be developed.
  • the present invention also includes the use of humanized antibodies specifically reactive with epitopes of an antigen of interest.
  • the humanized antibodies of the invention have a human framework and have one or more complementarity determining regions (CDRs) from an antibody, typically a mouse antibody, specifically reactive with an antigen of interest.
  • CDRs complementarity determining regions
  • the antibody used in the invention is humanized, the antibody may be generated as described in Queen, et al. (U.S. Patent No. 6, 180,370), Wright et ai., (supra) and in the references cited therein, or in Gu et ai. (1997, Thrombosis and Hematocyst 77(4):755-759).
  • the method disclosed in Queen et al. is directed in part toward designing humanized immunoglobulins that are produced by expressing recombinant DNA segments encoding the heavy and light chain
  • complementarity determining regions from a donor immunoglobulin capable of binding to a desired antigen, such as an epitope on an antigen of interest, attached to DNA segments encoding acceptor human framework regions.
  • a desired antigen such as an epitope on an antigen of interest
  • the invention in the Queen patent has applicability toward the design of substantially any humanized immunoglobulin.
  • the expression control sequences can be eukar otic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells or the expression control sequences can be prokaryotic promoter systems in vectors capable of transforming or transfecting prokaryotic host cells.
  • the host is maintained under conditions suitable for high level expression of the introduced nucl eotide sequences and as desired the collection and purification of the humanized light chains, heavy chains, light/heavy chain dimers or intact antibodies, binding fragments or other immunoglobulin forms may follow (Beychok, Ceils of Immunoglobulin Synthesis, Academic Press, New York, (1979), which is incorporated herein by reference).
  • the invention also includes functional equivalents of the antibodies described herein.
  • Functional equivalents have binding characteristics comparable to those of the antibodies, and include, for example, hybridized and single chain antibodies, as well as fragments thereof. Methods of producing such functional equivalents are disclosed in PCT Application WO 93/21319 and PCT Application WO 89/09622.
  • Functional equivalents include polypeptides with amino acid sequences substantially the same as the amino acid sequence of the variabl e or hypervariable regions of the antibodies.
  • " ⁇ S ubstantially the same" amino acid sequence is defined herein as a sequence with at least 70%, preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least 99% homology to another amino acid sequence (or any integer in between 70 and 99), as determined by the FASTA search method in accordance with Pearson and Lipman, 1988 Proc. Nat'l. Acad. Sci. USA 85: 2444-2448.
  • Chimeric or other hybrid antibodies have constant regions derived substantially or exclusi vely from human antibody constant regions and variable regions derived substantially or exclusively from the sequence of the variable region of a monoclonal antibody from each stable hybridoma.
  • Single chain antibodies or Fv fragments are polypeptides that consist of the v ariable region of the heavy chain of the antibody linked to the variable region of the light chain, with or without an interconnecting linker.
  • the Fv comprises an antibody combining site.
  • Functional equivalents of the antibodies of the invention further include fragments of antibodies that have the same, or substantially the same, binding
  • Such fragments may contain one or both Fab fragments or the F(ab')2 fragment.
  • the antibody fragments contain all six
  • complement determining regions of the whole antibody although fragments containing fewer than all of such regions, such as three, four or five complement determining regions, are also functional.
  • the functional equivalents are members of the IgG immunoglobulin class and subclasses thereof, but may be or may combine with any one of the following immunoglobulin classes: IgM, IgA, IgD, or IgE, and subclasses thereof.
  • Heavy chains of various subclasses, such as the IgG subclasses, are responsible for different effector functions and thus, by choosing the desired heavy chain constant region, hybrid antibodies with desired effector function are produced.
  • Exemplary constant regions are gamma 1 (IgG 1), gamma 2 (IgG2), gamma 3 (IgG3), and gamma 4 (IgG4).
  • the light chain constant region can be of the kappa or lambda type.
  • the immunoglobulins of the present invention can be monovalent, divalent or polyvalent.
  • Monovalent immunoglobulins are dimers (HL) formed of a hybrid heavy chain associated through disulfide bridges with a hybrid light chain.
  • Divalent immunoglobulins are dimers (HL) formed of a hybrid heavy chain associated through disulfide bridges with a hybrid light chain.
  • immunoglobulins are tetramers (H2L2) formed of two dimers associated through at least one disulfide bridge.
  • the present invention includes compositions and methods of treating wounds in a subject. In various embodiments, the present invention includes compositions and methods of diagnosing and treating cancer. Examples of wounds include skin wounds, skin ulcers, skin punctures, incision wounds, lacerations, avulsions, and the like.
  • the composition for treating a wound comprises an activator of one or more epicenters or one or more transcription factor binding thereto. In one embodiment, the activator of the invention increases epicenter activity. In one embodiment, the activator of the invention increases the amount of transcription factor polypeptide, the amount of transcription factor mRNA, the amount of transcription factor activity, or a combination thereof.
  • the transcription factor is SOX9, KLF (for example KLF5), GAT A, API (JUN/FQS), AP2, TCF, LHX2, Ni l. ETS2, and STAT3.
  • transcription factors encompasses the increase in transcription factor expression, including transcription, translation, or both.
  • an increase in the accessibility or activity of the epicenter or level of one or more transcription factors includes an increase in epicenter or transcription factor activity.
  • increasing the epicenter activity or level or activity of the transcription factor includes, but is not limited to, increasing the amount of transcription factor polypeptide, increasing transcription, translation, or both, of a nucleic acid encoding transcription factor; and it also includes increasing any activity of a transcription factor polypeptide as well. It additionally includes transcriptional activity localized to the epicenter or regulated by the activity of the epicenter.
  • the present invention relates to the prevention and treatment of a disease (e.g., cancer) or disorder by administration of a transcription factor polypeptide, a recombinant transcription factor polypeptide, an active transcription factor polypeptide fragment, an activator of transcription factor expression or activity, or activator of epicenter activity.
  • a disease e.g., cancer
  • a recombinant transcription factor polypeptide e.g., an active transcription factor polypeptide fragment
  • an activator of transcription factor expression or activity e.g., epicenter activity.
  • an increase in the activity of the epicenter or level of epicenter regulator protein or transcription factor binding thereto encompasses the increase of regulator protein or transcription factor protein expression. Additionally, the skilled artisan would appreciate, that an increase in the level of regulator protein or transcription factor includes an increase in epicenter or transcription factor activity. Thus, increasing the level or activity of regulator protein or transcription factor includes, but is not limited to, increasing transcription, translation, or both, of a nucleic acid encoding regulator protein or transcription factor; and it also includes increasing any activity of one or more regulator proteins or transcription factors as well. Activation of transcription factors can be assessed using a wide variety of methods, including those disclosed herein, as well as methods well-known in the art or to be developed in the future.
  • regulator protein or one or more transcription factor can be readily assessed using methods that assess the level of a nucleic acid encoding regulator protein or transcription factor (e.g., mRNA) and/or the level of regulator protein or transcription factor polypeptide in a biological sample obtained from a subject.
  • a nucleic acid encoding regulator protein or transcription factor e.g., mRNA
  • regulator protein or transcription factor polypeptide in a biological sample obtained from a subject.
  • An epicenter, regulator protein or transcription factor activator can include, but should not be construed as being limited to, a chemical compound, a protein, a peptidomimetic, an antibody, a nucleic acid molecule.
  • an epicenter activator, regulator protein activator, or transcription factor activator encompasses a chemical compound, a protein, a peptidomimetic, an antibody, and/or a nucleic acid molecule that increases the level, enzymatic activity, or the like of epicenter, regulator protein, or transcription factor, in some embodiments, the enzymatic activity is post-translational modification (e.g., phosphorylation, acetylation, SUMOylation, ubiquityiation).
  • an epicenter, regulator protein, or transcription factor activator encompasses a chemically modified compound, and derivatives, as is well known to one of skill in the chemical arts.
  • an increase in the level of transcription factor or regulator protein encompasses the increase in regulator protein or transcription factor expression, including transcription, translation, or both.
  • an increase in the level of transcription factor includes an increase in regulator protein or transcription factor activity (e.g., enzymatic activity, receptor binding activity, etc.).
  • increasing the level or activity of regulator protein or transcription factor includes, but is not limited to, increasing the amount of regulator protein or transcription factor polypeptide, increasing transcription, translation, or both, of a nucleic acid encoding regulator protein or transcription factor; and it also includes increasing any activity of a regulator protein or transcription factor polypeptide as well.
  • the epicenter, regulator protein, and one or more transcription factor activator compositions and methods of the invention can selectively acti vate epicenter, regulator protein, or transcription factor.
  • the present invention relates to disease treatment (i.e., cancer treatment) by administration of a regulator protein or transcription factor polypeptide, a recombinant regulator protein or transcription factor polypeptide, an active regulator protein or transcription factor polypeptide fragment, or an activator of regulator protein or transcription factor expression or activity.
  • an epicenter activator, regulator protein activator, or transcription factor activator includes such activators as discovered in the future, as can be identified by well-known criteria in the art of pharmacology, such as the physiological results of activation of epicenters, regulator proteins, or transcription factors as described in detail herein and/or as known in the art. Therefore, the present invention is not limited in any way to any particular epicenter, regulator protein, or transcription factor activator as exemplified or disclosed herein; rather, the invention encompasses those activators that would be understood by the routineer to be useful as are known in the art and as are discovered in the future.
  • an epicenter activator, regulator protein activator, or transcription factor activator are well known to those of ordinary skill in the art, including, but not limited to, obtaining an activator from a naturally occurring source.
  • an epicenter, regulator protein, or transcription factor activator can be synthesized chemically.
  • the routineer would appreciate, based upon the teachings provided herein, that an epicenter, regulator protein, or transcription factor activator can be obtained from a recombinant organism.
  • Compositions and methods for chemically synthesizing epicenter, regulator protein, or transcription factor activators and for obtaining them from natural sources are well known in the art and are described in the art.
  • an activator can be administered as a small molecule chemical, a protein, a nucleic acid construct encoding a protein, or combinations thereof.
  • Numerous vectors and other compositions and methods are well known for administering a protein or a nucleic acid construct encoding a protein to cells or tissues. Therefore, the invention includes a method of administering a protein or a nucleic acid encoding a protein that is an activator of an epicenter, regulator protein, or one or more transcription factor.
  • antisense is described as a form of inhibiting a regul ator of epicenter, regulator protein or transcription factor in order to increase the amount or activity of epicenter, regulator protein, or transcription factor.
  • Antisense oligonucleotides are DNA or RNA molecules that are complementary to some portion of a mRNA molecule.
  • antisense oligonucleotides When present in a cell, antisense oligonucleotides hybridize to an existing mRNA molecule and inhibit translation into a gene product. Inhibiting the expression of a gene using an antisense oligonucleotide is well known in the art (Marcus-Sekura, 1988, Anal. Biochem. 172:289), as are methods of expressing an antisense oligonucleotide in a cell (Inoue, U.S. Pat. No. 5,190,931).
  • the methods of the invention include the use of antisense oligonucleotide to diminish the amount of a molecule that causes a decrease in the amount or activity or epicenter, regulator protein, or transcription factor, thereby increasing the amount or activity of epicenter, regulator protein, or transcription factor.
  • Contemplated in the present invention are antisense oligonucleotides that are synthesized and provided to the cell by way of methods well known to those of ordinary skill in the art.
  • an antisense oligonucleotide can be synthesized to be between about 10 and about 100, more preferably between about 15 and about 50 nucleotides long.
  • the synthesis of nucleic acid molecules is well known in the art, as is the synthesis of modified antisense
  • oligonucleotides to improve biological activity in comparison to unmodified antisense oligonucleotides (Tullis, 1991, U. S. Pat. No. 5,023,243).
  • the expression of a gene may be inhibited by the hybridization of an antisense molecule to a promoter or other regulatory element of a gene, thereby affecting the transcription of the gene.
  • Methods for the identification of a promoter or other regulatory element that interacts with a gene of interest are well known in the art, and include such methods as the yeast two hybrid system (Battel and Fields, eds.. In: The Yeast Two Hybrid System, Oxford University Press, Gary, N.C.).
  • ribozyme for inhibiting gene expression is well known to those of skill in the art (see, e.g., Cech et a!,, 1992, J. Biol. Chem. 267: 17479; Hampel et al., 1989, Biochemistry 28: 4929: Altman et al, U.S. Pat. No. 5,168,053). Ribozymes are catalytic RNA molecules with the ability to cleave other single-stranded RNA molecules.
  • Ribozymes are known to be sequence specific, and can therefore be modified to recognize a specific nucleotide sequence (Cech, 1988, J. Amer. Med, Assn. 260:3030), allowing the selective cleavage of specific mRNA molecules. Given the nucleotide sequence of the molecule, one of ordinary skill in the art could synthesize an antisense oligonucleotide or ribozyme without undue experimentation, provided with the disclosure and references incorporated herein.
  • a regulator protein or transcription factor polypeptide, a recombinant regulator protein or transcription factor polypeptide, or an active regulator protein or transcription factor polypeptide fragment can be administered singly or in any combination thereof. Further, a regulator protein or transcription factor polypeptide, a recombinant regulator protein or transcription factor polypeptide, or an active regulator protein or transcription factor polypeptide fragment can be administered singly or in any combination thereof in a temporal sense, in that they may be administered simultaneously, before, and/or after each other.
  • a regulator protein or transcription factor polypeptide, a recombinant regulator protein or transcription factor polypeptide, or an active regulator protein or transcription factor polypeptide fragment can be used to prevent or treat a disease or disorder such as cancer, and that an activator can be used alone or in any combination with another regulator protein or transcription factor polypeptide, recombinant regulator protein or transcription factor polypeptide, active regulator protein or transcription factor polypeptide fragment, or regulator protein or transcription factor activator to effect a therapeutic result.
  • a polypeptide, a recombinant polypeptide, an active polypeptide fragment, or activator as a preventative measure against a disease or disorder such as cancer.
  • methods of increasing the level or activity of an epicenter, regulator protein, or transcription factor encompass a wide plethora of techniques for increasing not only epicenter, regulator protein, or transcription factor activity, but also for increasing expression of a nucleic acid encoding regulator protein or transcription factor.
  • the present invention encompasses a method of preventing a wide variety of diseases where increased expression and/or activity of regulator protein or transcription factor mediates, treats or prevents the disease. Further, the invention encompasses treatment or prevention of such diseases discovered in the future.
  • the invention encompasses administration of a polypeptide, a recombinant polypeptide, an active polypeptide fragment, or an activator to practice the methods of the invention; the skilled artisan would understand, based on the disclosure provided herein, how to formulate and administer the appropriate polypeptide, recombinant polypeptide, active polypeptide fragment, or activator to a subject.
  • the present invention is not limited to any particular method of administration or treatment regimen.
  • the term "pharmaceutically-acceptable carrier” means a chemical composition with which an appropriate polypeptide, recombinant poly peptide, active polypeptide fragment, or activator, may be combined and which, following the combination, ca be used to administer the appropriate polypeptide, recombinant polypeptide, active polypeptide fragment, or activator to a subject.
  • the invention relates to probes used to detect epicenters, for example cancer-related epicenters.
  • the probes are transcription reporter constructs comprising promoters of genes determined to be regulated by transcription factors associated with epicenters of interest.
  • the construct comprises promoters or genes that are involved in signaling pathways of interest.
  • the pathway s of interest include lineage infidelity, cell differentiation, stem cell plasticity in different microenvironments, epithelial-mesenchymal transition, cancer, and wound healing.
  • the probes comprise an internal H2BRFP control and eGFP tagged to a promoter of a transcription factor of interest (e.g., SOX9, KLF5, GATA, API (JIJN/FOS), AP2, TCF, LHX2, NFI, ETS2, and STAT3, either individually or as a cohort).
  • a transcription factor of interest e.g., SOX9, KLF5, GATA, API (JIJN/FOS), AP2, TCF, LHX2, NFI, ETS2, and STAT3, either individually or as a cohort.
  • the reporter is a fluorescent protein.
  • the reporter is a iuciferase reporter.
  • the reporter is a CRE recombinase.
  • the reporter is a CAS9 enzyme.
  • the invention provides methods of treating or preventing cancer.
  • the invention provides methods of treating cancer comprise modulating epicenters or super-enhancers determined to be differentially regulated in cancer.
  • the method comprises modulating epicenter activity.
  • the invention provides methods for modulating the assembly of transcriptional machinery specific to one or more epicenters.
  • the method comprises administering to a subject in need thereof, one or more compositions of the invention described herein.
  • the invention provides methods for detecting or diagnosing cancer in a subject or biological sample obtained from a subject.
  • the method comprises detecting the accessibility of the
  • the method comprises detecting the presence or abundance of one or more components associated with accessibility of the transcription epicenter or super-enhancer. In one embodiment, the method comprises detecting the presence or abundance of one or more factors that bind to the transcription epicenter or super-enhancer. In one embodiment, the method comprises detecting the presence or abundance of one or more transcripts associated with transcription epicenter or super-enhancer.
  • One aspect of the invention provides a method of treating cancer in an individual, the method comprising administering to the individual an effective cancer- inhibiting amount of an inhibitor of epicenter activity.
  • the invention further provides a method of inhibiting cancer in an individual in need thereof, the method comprising administering to the individual an effective cancer-inhibiting amount of any one of the compositions described herein.
  • the disclosed compounds can be used to prevent, abate, minimize, control, and/or lessen cancer in humans and animals.
  • the disclosed compounds can also be used to slow the rate of primary cancerous growth.
  • the disclosed compounds when administered to a subject in need of treatment can be used to stop the spread of cancer cells.
  • the compounds disclosed herein can be administered as part of a combination therapy with one or more drugs or other pharmaceutical agents.
  • the decrease in cancer and reduction in primary cancerous growth afforded by the disclosed compounds allows for a more effective and efficient use of any pharmaceutical or drug therapy being used to treat the patient.
  • control of cancer by the disclosed compound affords the subject a greater ability to concentrate the disease in one location.
  • the invention provides methods for preventing cancer or other cancerous cells as well as to reduce the rate of tumor growth.
  • the methods comprise administering an effective amount of one or more of the disclosed compounds to a subject diagnosed with a cancer or cancerous cells or to a subject having cancer or cancerous cells.
  • cancers that can be treated by the disclosed methods and compositions: Acute Lymphoblastic; Acute Myeloid
  • Gallbladder Cancer Gastric (Stomach) Cancer; Gastrointestinal Carcinoid Tumor;
  • Gastrointestinal Stromal Tumor GIST
  • Germ Cell Tumor Extracranial; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma; Glioma, Childhood Brain Stem; Glioma, Childhood Cerebral Astrocytoma; Glioma, Childhood Visual Pathway and Hypothalamic; Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer; Histiocytosis, Langerhans Cell; Hodgkin Lymphoma; Hypopharyngeal Cancer; Hypothalamic and Visual Pathway Glioma;
  • Intraocular Melanoma Islet Cell Tumors; Kidney (Renal Cell) Cancer; Langerhans Ceil Histiocytosis; Laryngeal Cancer; Leukemia, Acute Lymphoblastic; Leukemia, Acute Myeloid; Leukemia, Chronic Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer; Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphoma, AIDS-Related; Lymphoma, Burkitt; Lymphoma, Cutaneous T-Cell ; Lymphoma, Hodgkin; Lymphoma, Non-Hodgkin; Lymphoma,
  • Macrogiobulinemia Waldenstrom; Malignant Fibrous Histiocytoma of Bone and Osteosarcoma; Medulloblastoma; Melanoma; Melanoma, intraocular (Eye); Merkel Cell Carcinoma; Mesothelioma; Metastatic Squamous Neck Cancer with Occult Primary; Mouth Cancer; Multiple Endocrine Neoplasia Syndrome, (Childhood); Multiple Myeloma/Plasma Cell Neoplasm; Mycosis; Fungoides;
  • Sarcoma Kaposi; Sarcoma, Soft Tissue; Sarcoma, Uterine; Sezary Syndrome; Skin Cancer (Nonmeianoma); Skin Cancer (Melanoma); Skin Carcinoma, Merkel Ceil; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma; Squamous Ceil Carcinoma, Head and Neck Squamous Ceil Carcinoma, Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric) Cancer; Supratentorial Primitive
  • T-Celi Lymphoma Cutaneous; Testicular Cancer; Throat Cancer; Thymoma and Thymic Carcinoma; Thyroid Cancer; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Urethral Cancer; Uterine Cancer, Endometrial; Uterine Sarcoma; Vaginal Cancer; Vulvar Cancer; Waldenstrom Macroglobulinemia; and Wilms Tumor.
  • Chemotherapeutic agents include cytotoxic agents (e.g., 5-fluorouracil, cispiatin, carboplatin, methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucine phosphate sodium, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cispiatin, interferon alfa-2a recombinant, paclitaxel, teniposide, and streptozoci), cytotoxic alkylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethyl esul
  • topoisomerase II inhibitors e.g., mitoxantron, amonafide, m- AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene HCL, daunorubicin, deoxydoxorubicin, menogaril, ⁇ , ⁇ -dibenzyl daunomycin, oxanthrazole, rubidazone, VM- 26 and VP- 16
  • synthetics e.g., hydroxyurea, procarbazine, ⁇ , ⁇ '-DDD, dacarbazine, CCNU, BCNU, cis-diamminedichloroplatimun, mitoxantrone, CBDCA, levamisole, hexamethy imelamine, all-trans retinoic acid, gliadei and porfimer sodium).
  • Antiproliferative agents are compounds that decrease the proliferation of cells.
  • Antiproliferative agents include alkylating agents, antimetabolites, enzymes, biological response modifiers, miscellaneous agents, hormones and antagonists, androgen inhibitors (e.g., fiutamide and ieuproiide acetate), anti estrogens (e.g., tamoxifen citrate and analogs thereof, toremifene, droloxifene and raloxifene), Additional examples of specific antiproliferative agents include, but are not limited to levamisole, gallium nitrate, granisetron, sargramostim strontium-89 chloride, filgrastim, pilocarpine, dexrazoxane, and ondansetron.
  • the inhibitors of the invention can be administered alone or in combination with other anti-cancer agents, including cytotoxic/antineoplastic agents and anti-angiogenic agents.
  • Cytotoxic/anti-neoplastic agents are defined as agents which attack and kill cancer cells.
  • Some cytotoxic/anti-neoplastic agents are alkylating agents, which alkylate the genetic material in tumor cells, e.g., cis-platin, cyclophosphamide, nitrogen mustard, trimethyiene thiophosphoramide, carmustine, busuifaii, chlorambucil, belustine, uracil mustard, chlomaphazm, and dacabazine.
  • cytotoxic/ anti -neoplastic agents are antimetabolites for tumor cells, e.g., cytosine arabinoside, fluorouracil, methotrexate, mercaptopuirine, azathioprime, and procarbazine.
  • Other cytotoxic/anti- iieopiastic agents are antibiotics, e.g., doxorubicin, bleomycin, dactinomycin, daunorubicin, mithramycin, mitomycin, mytomycin C, and daunomycin.
  • doxorubicin e.g., doxorubicin, bleomycin, dactinomycin, daunorubicin, mithramycin, mitomycin, mytomycin C, and daunomycin.
  • mitotic inhibitors (vinca alkaloids).
  • cytotoxic/anti-neoplastic agents include taxol and its derivatives, L-asparaginase, anti-tumor antibodies, dacarbazme, azacytidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone, and vmdesine.
  • Anti-angiogenic agents are well known to those of skill in the art. Suitable anti-angiogenic agents for use in the methods and compositions of the present disclosure include anti-VEGF antibodies, including humanized and chimeric antibodies, anti-VEGF aptamers and antisense oligonucleotides. Other known inhibitors of angiogenesis include angiostatin, endostatin, interferons, inter! eukin 1 (including alpha and beta) interleukin 12, retinoic acid, and tissue inhibitors of metalloproteinase-1 and -2. ( ⁇ -1 and -2). Small molecules, including topoisomerases such as razoxane, a topoisomerase II inhibitor with anti-angiogenic activity, can also be used.
  • anii-cancer agents that can be used in combination with the disclosed compounds include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretaraine; ambomycin; anietantrone acetate; aminoglutethimide; amsacrine; anastrozoie; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropinmine; busulfan: cactinomycin; calusterone; caraceniide; carbetimer; carbopiatin; carmustine; carubicin hydrochloride; carzelesm; cede
  • spirogermanium hydrochloride spiromustine: spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride;
  • temoporfin teniposide; teroxirone; testoiactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate giucuronate; triptorelin; tubuiozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate;
  • vmdesine vindesme sulfate; vinepidme sulfate; vinglycinate sulfate; vinieurosine sulfate; vinorelbine tartrate; vmrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;
  • anti-cancer drugs include, but are not limited to: 20-epi-l,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin:
  • acylfulvene acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;
  • anagre!ide anastrozole; andrographolide; angiogenesis inhibitors; antagonist D;
  • antagonist G antagonist G; antarelix; anti-dorsalizmg morphogenetic protein- 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL- PTBA; arginine deaminase; asulacnne; atamestane; atrimustine; axinastatin 1 ; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol;
  • batimastat BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisazindinylspermme; bisnafide; bistratene A; bizelesm; breflate; bropirimine: budotiiane; buthionme sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canary pox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole;
  • CaRest M3 CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline
  • sulfonamide cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; cranibescidin 816; crisnatoi; cryptophycin 8; ciyptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycm; cytarabme ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone;
  • dexifosfamide dexrazoxane
  • dexverapamil diaziquone
  • didemnin B didox
  • diethylnorspermine dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; camrabine;
  • fenretinide filgrastim; finasteride; flavopiridol ; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formes tane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idraniantone; iimofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor- 1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane;
  • iododoxorubicin ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin
  • B itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; lemamycin; lenograstim; lentinan sulfaie; Ieptolsiatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; ievamisole; liarozole; linear poly amine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine;
  • losoxantrone lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat: masoprocol; maspin; matrilysin inhibitors; matrix metalioproteinase inhibitors; menogaril; merbarone; meterelin;
  • nartograstim nedaplatin
  • nemorubicin nedaplatin
  • neridronic acid neutral endopeptida.se
  • iiilutaniide nisamycin; nitric oxide modulators; nitroxide antioxidant; nitruilyn; 06- benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin;
  • oxaunomycin paclitaxel; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;
  • perflubron perfosfarnide
  • perillyl alcohol phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; piacetm A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis- acridone; prostaglandin J2; proteasome inhibitors; protein A-hased immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins;
  • pyrazoloacridine pyridoxylated hemoglobin polyoxy ethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesvl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RH retinamide; rogletimide; rohitukme; romurtide; roquinimex; rubiginone Bl ; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1 ; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofuran;
  • sobuzoxane sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongi statin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipi amide; stromelysin inhibitors; suifmosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic giycosaminogiycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thalibiastine;
  • thymopoietin receptor agonist thymotrinan
  • thyroid stimulating hormone tin eth l etiopurpurin
  • tirapazamine titanocene bichloride
  • topsentin toremifene
  • totipotent stem cell factor translation inhibitors
  • tretinoin triacetyluridine
  • triciribine trimetrexate
  • the anticancer drug is 5-fluoro uracil, taxol, or leucovorin.
  • the present invention envisions treating a disease, for example, cancer and the like, in a mammal by the administration of therapeutic agent.
  • Administration of the therapeutic agent in accordance with the present invention may be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitio ers.
  • the administration of the agents of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration is
  • the amount administered will vary depending on various factors including, but not limited to, the composition chosen, the particular disease, the weight, the physical condition, and the age of the mammal, and whether prevention or treatment is to be achieved. Such factors can be readily determined by the clinician employing animal models or other test systems which are well known to the art
  • One or more suitable unit dosage forms having the therapeutic agent(s) of the invention which, as discussed below, may optionally be formulated for sustained release (for example using microencapsulation, see WO 94/07529, and U.S. Pat. No. 4,962,091 the disclosures of which are incorporated by reference herein), can be administered by a variety of routes including parenteral, including by intravenous and intramuscular routes, as well as by direct injection into the diseased tissue.
  • the therapeutic agent or modified cell may be directly injected into the tumor.
  • the formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to pharmacy. Such methods may include the step of bringing into association the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired deliver ⁇ ' system.
  • the therapeutic agents of the invention are prepared for administration, they are preferably combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form.
  • a pharmaceutically acceptable carrier diluent or excipient to form a pharmaceutical formulation, or unit dosage form.
  • the total active ingredients in such formulations include from 0.1 to 99.9% by weight of the formulation.
  • a ''pharmaceutically acceptable is a carrier, diluent, excipient, and/or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
  • the active ingredient for administration may be present as a powder or as granules; as a solution, a suspension or an emulsion.
  • compositions containing the therapeutic agents of the invention can be prepared by procedures known in the art using well known and readily available ingredients.
  • the therapeutic agents of the invention can also be formulated as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous or intravenous routes.
  • the pharmaceutical formulations of the therapeutic agents of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension.
  • the therapeutic agent may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small volume infusion containers or in multi-dose containers with an added preservative.
  • the active ingredients may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyopliilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen -free water, before use.
  • the unit content of active ingredient or ingredients contained in an individual aerosol dose of each dosage form need not in itself constitute an effective amount for treating the particular indication or disease since the necessary effective amount can be reached by administration of a plurality of dosage units. Moreover, the effective amount may be achieved using less than the dose in the dosage form, either individually, or in a series of administrations.
  • the pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are well-known in the art.
  • pharmaceutically acceptable carriers such as phosphate buffered saline solutions pH 7.0-8.0.
  • the agents of this invention can be formulated and administered to treat a variety of disease states by any means that produces contact of the active ingredient with the agent's site of action in the body of the organism. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
  • water, suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions.
  • Solutions for parenteral administration contain the active ingredient, suitable stabilizing agents and, if necessary, buffer substances.
  • Antioxidizing agents such as sodium bisulfate, sodium sulfite or ascorbic acid, either alone or combined, are suitable stabilizing agents.
  • parenteral solutions can contain preservatives such as benzalkonium chloride, methyl- or propyl -paraben and chlorobutanol.
  • Suitable pharmaceutical carriers are described in Remington's
  • the active ingredients of the invention may be formulated to be suspended in a pharmaceutically acceptable composition suitable for use in mammals and in particular, in humans.
  • a pharmaceutically acceptable composition suitable for use in mammals and in particular, in humans.
  • Such formulations include the use of adjuvants such as muramyl dipeptide derivatives (MDP) or analogs that are described in U. S. Patent Nos. 4,082,735; 4,082,736; 4,101,536; 4,185,089; 4,235,771 ; and 4,406,890,
  • MDP muramyl dipeptide derivatives
  • Other adjuvants, which are useful include alum (Pierce Chemical Co.), lipid A, trehalose dimycolate and
  • DDA dimethyldioctadecylammonium bromide
  • IL-12 IL-12
  • Other components may include a polyoxypropyiene-polyoxy ethylene block polymer
  • control release preparations can include appropriate macromolecules, for example polymers, polyesters, poly amino acids, polyvinyl, pyrolidone, ethylenevinylacetate, methyl cellulose, carboxymethyl cellulose or protamine sulfate.
  • the agent can be incorporated into particles of polymeric materials such as polyesters, poly amino acids, hydrogels, poly (lactic acid) or
  • these agents can also be used to trap the compound in microcapsules.
  • the pharmaceutical composition of the present invention may be delivered via various routes and to various sites in a mammal body to achieve a particular effect (see, e.g., Rosenfeld et al., 1991 ; Rosenfeld et al, 1991a; Jaffe et al., supra; Berkner, supra).
  • Rosenfeld et al., 1991 Rosenfeld et al, 1991a
  • Jaffe et al., supra Berkner, supra.
  • Local or systemic delivery' can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, peritoneal, subcutaneous, intradermal, as well as topical administration.
  • each dosage unit e.g., a teaspoonful, tablet, solution, or suppository
  • each dosage unit e.g., a teaspoonful, tablet, solution, or suppository
  • the term ''unit dosage form refers to physically discrete units suitable as unitar ' dosages for human and mammal subjects, each unit containing a predetermined quantity of the compositions of the present invention, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle, where appropriate.
  • the specificati ons for the unit dosage forms of the present invention depend on the particular effect to be achieved and the particular
  • the invention provides methods of detecting or diagnosing cancer, the method comprising identifying the presence of an epicenter in a cancer in a patient.
  • the presence of an epicenter is determined using a method such as ATAC-seq in biological samples collected from an individual.
  • the method relates to evaluating expression levels or activity of one or more transcription factors relative to a comparator control .
  • the one or more transcription factors is determined using trans criptonie analysis of a DNA sample collected from a biological sample determined to have one or more epicenters or super-enhancers or transposase accessible regions.
  • the DNA sample is collected, isolated, and purified using methods known to one skilled in the art.
  • the transcriptome analysis includes using ATAC-seq data identifying regions of DNA, and using HOMER software (Heinz et al, 2010) in addition to the MolSigDB database (Mootha et al. , 2003) to search for and identify genes with binding motifs of transcription factors determined to be enriched in epicenters or super- enhancers differentially regulated in a cancer sample relative to a comparator control.
  • the invention contemplates the identification of differentially accessible epicenters, transcription factors binding thereto, and genes regulated thereby, in order to identify markers differentially expressed between normal, and cancer subjects.
  • the invention further contemplates using methods known to those skilled in the art to detect and to measure the level of differentially expressed marker of expression products, such as RNA and protein, to measure the level of one or more differentially expressed marker expression products, for example transcription factor or targeted gene expression level or expression products.
  • Methods of detecting or measuring gene expression may utilize methods that focus on cellular components (cellular examination), or methods that focus on examining extracellular components (fluid examination). Because gene expression involves the ordered production of a number of different molecules, a cellular or fluid examination may be used to detect or measure a variety of molecules including RNA, protein, and a number of molecules that may be modified as a result of the protein's function.
  • Typical diagnostic methods focusing on nucleic acids include amplification techniques such as PCR and RT-PCR (including quantitative variants), and hybridization techniques such as in situ hybridization, microarrays, blots, and others.
  • Typical diagnostic methods focusing on proteins include binding techniques such as ELISA,
  • the genes identified as being differentially expressed may be assessed in a variety of nucleic acid detection assays to detect or quantify the expression level of a gene or multiple genes in a given sample.
  • nucleic acid detection assays For example, traditional Northern blotting, nuclease protection, RT-PCR, microarray, and differential display methods may be used for detecting gene expression levels.
  • Methods for assaying for mRNA include Northern blots, slot blots, dot blots, and hybridization to an ordered array of oligonucleotides. Any method for specifically and quantitatively measuring a specific protein or mRNA or DN A product can be used. However, methods and assays are most efficiently designed with array or chip hybridization-based methods for detecting the expression of a large number of genes. Any hybridization assay format may be used, including solution-based and solid support-based assay formats.
  • the protein products of the genes identified herein can also be assayed to determine the amount of expression.
  • Methods for assaying for a protein include Western blot, immunoprecipitation, and radioimmunoassay.
  • the proteins analyzed may be localized intracellularly (most commonly an application of immunohistochemistry) or extracellularly (most commonly an application of immunoassays such as ELISA).
  • Biological samples may be of any biological tissue or fluid. Frequently the sample will be a "clinical sample" which is a sample derived from a patient.
  • Controls groups may either be normal or samples from known stages of cancer. As described below, comparison of the expression patterns of the sample to be tested with those of the controls can be used to diagnose between normal and cancer subjects. In some instances, the control groups are only for the purposes of establishing initial cutoffs for the assays of the invention. Therefore, in some instances, the systems and methods of the invention can diagnose between normal and cancer subjects without the need to compare with a control group.
  • the present invention relates to the identification of biomarkers associated with cancer, and/or cancer related conditions. Accordingly, the present invention features methods for identifying subjects who are at risk of developing cancer and/or cancer related conditions, including those subjects who are asymptomatic or only exhibit nonspecific indicators of cancer and/or cancer related conditions by detection of the biomarkers disclosed herein. These biomarkers are also useful for monitoring subjects undergoing treatments and therapies for cancer and/or cancer conditions, and for selecting or modifying therapies and treatments that would be efficacious in subjects having cancer and/or cancer conditions, wherein selection and use of such treatments and therapies slow the progression of cancer and/or cancer conditions, or prevent their onset.
  • the invention provides improved diagnosis and prognosis of cancer and/or cancer conditions.
  • the risk of developing cancer and/or cancer conditions can be assessed by measuring one or more of the biomarkers described herein, and comparing the measured values to reference or index values. Such a comparison can be undertaken with mathematical algorithms or formula in order to combine information from results of multiple individual biomarkers and other parameters into a single measurement or index.
  • Subjects identified as having an increased risk of cancer and-'or cancer conditions can optionally be selected to receive treatment regimens, such as administration of prophylactic or therapeutic compounds or combination with other anti-cancer agents, including cytotoxic/antineoplastic agents and anti -angiogenic agents to prevent, treat or delay the onset of cancer and/or cancer conditions.
  • Identifying a subject before they develop cancer and/or cancer related conditions enables the selection and initiation of various therapeutic interventions or treatment regimens in order to delay, reduce or prevent that subject's conversion to a disease state.
  • Monitoring the levels of at least one biomarker also allows for the course of treatment of cancer and/or cancer conditions to be monitored.
  • a sample can be provided from a subject undergoing treatment regimens or therapeutic interventions, e.g., drug treatments, for cancer and/or cancer conditions.
  • Such treatment regimens or therapeutic interventions can include dietary modification, dietary supplementation, surgical intervention, administration of pharmaceuticals, anti-cancer agents, including cytotoxic/antineoplastic agents and and -angiogenic agents, and treatment with
  • Samples can be obtained from the subject at various time points before, during, or after treatment.
  • the biomarkers of the present invention can thus be used to generate a biomarker profile or signature of subjects: (i) who do not have and are not expected to develop cancer and/or cancer related conditions and/or (ii) who have or expected to develop cancer and/or cancer related conditions.
  • the biomarker profile of a subject can be compared to a predetermined or reference biomarker profile to diagnose or identify subjects at risk for developing cancer and/or cancer related conditions, to monitor the progression of disease, as well as the rate of progression of disease, and to monitor the effectiveness of cancer and/or cancer rel ated condition treatments.
  • Data concerning the biomarkers of the present invention can also be combined or correlated with other data or test results, such as, without limitation, measurements of clinical parameters or other algorithms for cancer and-'or cancer related conditions.
  • the machine-readable media can also comprise subject information such as medical history and any relevant family history.
  • the present invention also provides methods for identifying agents for treating cancer and/or cancer related conditions that are appropriate or otherwise customized for a specific subject.
  • a test sample from a subject, exposed to a therapeutic agent or a drug can be taken and the level of one or more biomarkers can be determined.
  • the level of one or more biomarkers can be compared to a sample derived from the subject before and after treatment, or ca be compared to samples derived from one or more subjects who have shown improvements in risk factors as a result of such treatment or exposure.
  • the invention is a method of diagnosing cancer. In one embodiment, the method includes distinguishing between normal and cancer subjects.
  • methods are disclosed herein that may be of use to determine whether a subject has a cancer.
  • these methods may utilize a biological sample (such as urine, saliva, blood, serum, amniotic fluid, or tears), for the detection of one or more markers of the invention in the sample.
  • a biological sample such as urine, saliva, blood, serum, amniotic fluid, or tears
  • the invention provides a biomarker for the detection of cancer from non-cancer.
  • the biomarker for the detection of cancer from non-cancer includes but is not limited to SOX9, KLF5, GAT A, API (JUN/FOS), AP2, TCF, LHX2, NFI, ETS2, and STATS.
  • the method comprises detecting one or more markers in a biological sample of the subject.
  • the level of one or more of markers of the invention in the biological sample of the subject is compared with the level of a corresponding biomarker in a comparator.
  • comparators include, but are not limited to, a negative control, a positive control, an expected normal background value of the subject, a historical normal background value of the subject, an expected normal background value of a population that the subject is a member of, or a historical normal background value of a population that the subject is a member of.
  • the in vention is a method of monitoring the progression of cancer in a subject by assessing the level of one or more of the markers of the invention in a biological sample of the subject.
  • the subject is a human subject, and may be of any race, sex and age.
  • Information obtained from the methods of the invention described herein can be used alone, or in combination with other information (e.g., disease status, disease history, vital signs, blood chemistry, etc.) from the subject or from the biological sample obtained from the subject.
  • information e.g., disease status, disease history, vital signs, blood chemistry, etc.
  • the level of one or more markers of the invention is determined to be increased when the level of one or more of the markers of the invention is increased by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, or by at least 100%, when compared to with a comparator control.
  • a biological sample from a subject is assessed for the level of one or more of the markers of the invention in the biological sample obtained from the patient.
  • the level of one or more of the markers of the invention in the biological sample can be determined by assessing the amount of polypeptide of one or more of the biomarkers of the invention in the biological sample, the amount of mRNA of one or more of the biomarkers of the invention in the biological sample, the amount of enzymatic activity of one or more of the biornarkers of the invention in the biological sample, or a combination thereof.
  • the invention includes detecting an extracellular mRNA in a biological sample, wherei the extracellular mRNA is detected in a cell-free fluid phase portion of the biological sample.
  • Biomarkers generally can be measured and detected through a variety of assays, methods and detection systems known to one of skill in the art.
  • Various methods include but are not limited to refractive index spectroscopy (RI), ultra-violet spectroscopy (UV), fluorescence analysis, electrochemical analysis, radiochemical analysis, near- infrared spectroscopy (near-IR), infrared (IR) spectroscopy, nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS), mass spectrometry, pyroiysis mass spectrometry, nephelometry, dispersive Raman spectroscopy, gas chromatography, liquid chromatography, gas chromatography combined with mass spectrometry, liquid chromatography combined with mass spectrometry, matrix- assisted laser desorption ionization -time of flight (MALDI-TOF) combined with mass spectrometry, ion spray spectroscopy combined with mass spectrometry, capillary electrophoresis, colori
  • biomarkers can be measured using the above-mentioned detection methods, or other methods known to the skilled artisan.
  • Other biomarkers can be similarly detected using reagents that are specifically designed or tailored to detect them.
  • biomarkers and their measurements can be combined in the compositions and methods of the present invention.
  • the protein form of the biomarkers is measured.
  • the nucleic acid form of the biomarkers is measured.
  • the nucleic acid form is mRNA.
  • measurements of protein biomarkers are used in conj unction with measurements of nucleic acid biomarkers.
  • Methods for detecting mRNA such as RT-PCR, real time PGR, branch
  • sequences in sequence database entries or sequences disclosed herein can be used to construct probes for detecting biomarker RNA sequences in, e.g. , Northern blot hybridization analyses or methods which specifically, and, preferably, quantitatively amplify specific nucleic acid sequences.
  • sequences in sequence database entries or sequences disclosed herein can be used to construct primers for specifically amplifying the biomarker sequences in, e.g., amplification-based detection methods such as reverse-transcription based polymerase chain reaction (RT-PCR).
  • RT-PCR reverse-transcription based polymerase chain reaction
  • RNA can also be measured using, for example, other target amplification methods (e.g., TMA, SDA, NASBA), signal amplification methods (e.g., bDNA), nuclease protection assays, in situ hybridization and the like.
  • target amplification methods e.g., TMA, SDA, NASBA
  • signal amplification methods e.g., bDNA
  • nuclease protection assays e.g., in situ hybridization and the like.
  • the concentration of the biomarker in a sample may be determined by any suitable assay.
  • a suitable assay may include one or more of the following methods, an enzyme assay, an immunoassay, mass spectrometry, chromatography, electrophoresis or an antibody microarray, or any combination thereof
  • the system and methods of the invention may include any method known in the art to detect a biomarker in a sample.
  • the invention described herein also relates to methods for a multiplex analysis platform.
  • the method comprises an analytical method for multiplexing analytical measurements of markers.
  • the present invention is a method for treating a wound.
  • method comprising administering to a subject a composition as described herein, that when administered to said subject improves wound healing.
  • the method comprises administering to a subject a modulator of epicenter activity, wherein the modulator activates transcription factor activity wherein the transcription factor activity improves wound healing.
  • the method comprises administering to subject a modulator of epicenter activity, wherein the modulator inhibits transcription factor activity, wherein the inhibition of transcription factor activity improves wound healing.
  • the modulated transcription factors include but are not limited to SOX9, KLF5, GATA, API (JUN/FOS), AP2, TCF, LHX2, NFI, ETS2, and STATS.
  • Example 1 Stem Cell Lineage Infidelity at the Crossroads of Wound-Repair and Cancer
  • Tissue stem cells govern tissue regeneration and wound-repair. Tumors often hijack these normal cellular programs and exploit them for malignancy. Here, such a phenomenon was identified in skin, where stem cells of the epidermis and hair follicle remain faithfully restricted to fueling their own tissue during homeostasis. They lose lineage fidelity during tumorigenesis. Moreover, breakdown of stem cell lineage confinement - granting privileges associated with both fates - is not only a hallmark, but also obligatory for malignancy . Intriguingly, it was found that lineage plasticity is also critical in wound-repair, where it functions transiently to redirect fates.
  • FACE Fluorescence Activated Cell Sorting
  • Purification of adult populations was performed using P60 second telogen WT mice. Purification of tumor populations was performed using transplants from TGFbRII-deficient; HRasG12V background (SCCs). Purification of wound population was performed using SGX9CreER; R26YFP mice, treated with Tamoxifen at P53, wounded at P60 (partial thickness wound using a Dremel head gently scraping against backskin), and collected at P67.
  • the backskin was first scraped from dermal side to remove fat; for tumor isolation, tumor was first chopped and the placed in collagenase (Sigma, 0.25% in HBSS) for 1 hour at 37°C and spin down 300 g 4°C; for wound isolation, the wound center was dissected and EDTA (50 mM) treated for 30min at 37°C after which the epidermis is peeled off. The remaining epidermal side (adult skin) or cell mixture (tumor) or epidermis (wound) was then transferred to trypsin (Gibco, 0.25% in PBS) at 37°C for 10 minutes.
  • Single-cell suspensions were obtained by scraping the skin from epidermal side (adult skin) or pipetting (tumor or wound) gently. The cells were then filtered with 70 ⁇ , ⁇ followed by 40 ⁇ strainers, and pelleted at 300 g 4°C.
  • EdU was administrated through intraperitoneal injection at 100 ⁇ (5 nig/ml) per 20 g mouse weight for 1 hour before euthanasia. Cells were first stained with Live/Dead Blue (Life Tech, 1 ; 100), and then fixed, permeabilized, stained with anti- GFP (1 : 1,000), followed by EdU Click-iT reactions and Alexa Fluor (IntAo) staining.
  • Tumor grafts formed from previously characterized aggressive SCCs (Yang et al, GFP tagged) were gated as GFP+IntA6+IntB4+ for SCCSCs. Whether cultured, or injected directly into recipient mice, these basal SCC cells have been previously shown to have tumor-initiating properties characteristic of cancer stem cells (Schober et al, Yang et al). The findings presented herein corroborate this behavior as shown by the tumor-initiating assays in figures throughout the manuscript. Wounded SCs were gated as YFP+TntA6+ for repairing cells that are HFSCs originated. The following antibodies were used:
  • CD34_eFluor660 (1 : 100, eBioscience 50-0341-80), lntA6 PE (1 : 100, BD Bioscience 551129), IntB4 Alexa647 (1 : 1,000, eBioscience 14-0291-81), Scal _PerCP-Cy5.5
  • ATAC-seq libraries were made from freshly FACS -sorted SCs, with two biologically independent replicates for ceil population. Library preparation and analy sis was performed as described (Buenrostro et al, 2013). Briefly, Freshly FACS-sorted cells (20K-100K) are subject to tagmentation reaction with 2-10 ul TnD transposase, cleaned up and PGR amplified with 10-15 cycles. Library concentration and quality was confirmed with D1000 Tape Station prior to sequencing.
  • R26YFP female mice were subjected to full-thickness 7 mm punch wound, and 7 days post- wounding, single ceil resuspensions were obtained by FACS as Lineage-
  • RNA+Integrina6+ (HF-derived) and Lineage-YFP-Integrina6+ ( Epd-den ved).
  • Cells were lysed with TrizolLS (Invitrogen) and total RNA was isolated with the Direct-zol RNA MiniPrep kit (Zymo Research) and submitted to the Genomics Resources Core Facility of the Weill Cornell Medical College for quality control (determined using Agilent 2100 Bioanalyzer, with all samples passing the quality threshold of RNA integrity numbers (RIN 8). Library construction was performed using IlluminaTruSeq Stranded mRNA Sample Prep Kit, and sequencing was performed on an Illumina HiSeq2000 sequencing machine. For RNA quantitative PCR, complementary DNAs were generated from !
  • Samples are then incubated with primary antibody overnight at 4°C, washed in PBS, secondary antibody 1 hour at room temperature (sections) or overnight at 4°C, washed in PBS, and mounted in ProLong Gold with DAP1 (Life Tech).
  • the following primary antibodies and dilutions were used: KLF-5 (goat, 1 :50, R&D), SOX9 (rabbit, 1 :300, E.Fuchs), P-Cadherin (goat, 1 :400, R&D), LHX2 (rabbit, 1 :2000, E.
  • GFP (chicken, 1 :2000, Abeam), JUN (rabbit, 1 : 1000, Cell Signaling), FOS (rabbit, 1 : 100, Abeam), pSTAT3 (rabbit, 1 : 1000, Ceil Signaling), pETS2 (rabbit, 1 :200, Thermo Fisher Sci), K5 (rabbit, 1 :500, E. Fuchs), 10 (rabbit, 1 : 1 ,000, Covance), CD 104 (rat, eBioscience, 1 : 1,000), Secondary antibodies were conjugated to Alexa488, 546, 647 (1 : 1,000, Life Technologies A- 11006).
  • Images were captured on a Zeiss Axioplan2 using a Plan- Apochromat 20x/0.8 air objective. For whole mount imaging, z stacks of 20-40 planes (0.25 mm) were acquired. Images were processed using ImageJ and Adobe Photoshop CSS.
  • Human HNSCC cell line A431 cells were LV-transduced with EC reporter in culture, FACS sorted with internal control color H2BRFP, and grafted (50 ) onto nude backskin and harvest in 3 weeks for primary tumors, or tail vein injected (1M) into nude and harvest in 2 months for lung mets.
  • CRISPR/CAS Knockouts in Vivo were LV-transduced with EC reporter in culture, FACS sorted with internal control color H2BRFP, and grafted (50 ) onto nude backskin and harvest in 3 weeks for primary tumors, or tail vein injected (1M) into nude and harvest in 2 months for lung mets.
  • sgRNAs CR1SPR guide RNAs
  • LV-CreER driven CAS9 activation was induced by administrating a single dose of tamoxifen (200 ⁇ at 20 mg/mL per 40 g body weight) by oral gavage at El 8.5, as intraperitoneal injection of tamoxifen at doses sufficient to induce recombination frequently led to aborted litters.
  • plentiCRISPRv2 vectors Guides against KLF5 or SOX9 were selected as described above and the most efficient guides were then subcloned into plentiCRISPRv2 vectors (Sanjana et al, 2014). sgRNAs against Scrambled sequences were used as controls and done side-by-side with sgRNAs against target sites throughout the experiments to rule out phenotypic changes due to nonspecific targeting. plentiCRJSPRv2 vector carrying targeting sgRNA was then packaged into LV and used to transduce SCC-SCs cells, which were then selected with puromycin (3 ⁇ ig/ml) for 2 days, and then transplanted into the backs of imniunosuppressed Nude mice. Quantitative PCR was performed prior to transplantation to validate the effecti v eness of the gene knockout. At least two small guides for each gene were tested and results were consistent. The small guide sequences used are found in Table 2.
  • SCC-SCs were transduced with pLentiV2 Crispr guides against Scramble, KLF5 or SOX9, respectively, briefly selected with puromycin for 2 days, and then transplanted onto immunocompromised nude back skin via intradermal injection as previously described (Ge et al., 2016). Animals were monitored every 3 days for a month. Tumor size was measured using a digital caliper, and tumor volume was calculated using the formula (p(length x width)2)/6. GraphPad Prism software was used to generate the tumor growth curves and to calculate the P value by two-way ANOVA with repeated measurement test.
  • split- and Full-thickness grafts were performed as previously described (Nowak et al. , 2008; Rhee et al., 2006). Briefly, for full-thickness graft, head skins from early postnatal (P3) mice were dissected from the scramble guide (LV-CreER-sgScr) and guide against gene of interest (LV-CreER-sgGene) transduced R26-LSL-Cas9-P2A-GFP animals and placed onto the backs of anesthetized female nu/nu (Nude) recipient mice. Grafts were secured by sterile gauze and cloth bandages, which were removed after healmg (14 days).
  • grafts For split-thickness grafts, head skin from P3 mice was placed dermis- side up in 50 ttiM EDTA in PBS for 1 hour at 37°C. The epidermis was removed as a single sheet from hair follicle/dermis. Dermis was then grafted in the same manner as full thickness grafts. In all cases, paired sgScr and sgGene partial-thickness grafts of comparable size were grafted side-by-side, with full-thickness grafts as control on the same nude recipient.
  • mice SCC-SC cell lines isolated from malignant SCCs were generated previously in the Fuchs' laboratory (Yang et a!., 2015).
  • Mouse keratmocytes were isolated and cultured as previously described (Blanpain et al., 2004: Guasch et al, 2007).
  • SCC-SC cells were cultured in E intermediate calcium medium (contains 300 ⁇ calcium); mouse keratmocytes were cultured in E low calcium medium (contains 50 ⁇ calcium).
  • E intermediate calcium medium contains 300 ⁇ calcium
  • E low calcium medium contains 50 ⁇ calcium
  • keratmocytes are cultured in regular culture media until close to confluent, and starved in serum-free P media containing 0.3 mM calcium overnight.
  • cells are treated with mitomycin C (10 g/ml) for 2 hours at 37°C to arrest cell proliferation.
  • Coat insert 24 mm Corning Transwell polycarbonate membrane cell culture inserts with 8.0 ⁇ pore
  • fibronectin on bottom side of, and matrigel on top side of the membrane.
  • Incubate 24 hours trypsinize cells from both sides of membrane, add media slowly and remove top or bottom ceils carefully, resuspend in collection tubes and count cells from both sides.
  • plentiCRISPRv2 vector carrying targeting sgRNA was packaged into lentivirus and used to transduce SCC-SCs cells, which were then selected with puromycin (3 .ug/'nil) for 2 days, and transplanted onto immunosuppressant mice back. Real-time PCR were performed prior to transplanting to validate gene knockout by effective sgRNA.
  • HNSCC human head and neck squamous cell carcinoma
  • SCC9, SCC15, SCC25 lentiviruses harboring the desired epicenter reporter and a ubiquitously active control PGK-H2BRFP gene.
  • RFP+ transduced cells were purified by FACS, expanded, and then grafted (5 OK) onto Nude mouse backskin.
  • Raw sequenced reads were aligned to the Mouse reference genome (Version mmlO from UCSC) using STAR (Version 2.4.2) aligner. Aligned reads were quantified against the reference annotation (mmlO from UCSC) to obtain FPKM
  • Pathway analysis using GSEA Gene Set Enrichment Analysis Software
  • GSEA Gene Set Enrichment Analysis Software
  • Software from Broad institute was used to identify functions of differentially expressed genes. Genes were ranked by the t-statistic value obtained from comparisons and the pre- ranked version of the tool was used to identify significantly enriched biological pathways Pathways enriched with FDR ⁇ 0.25 were considered to be significant.
  • 50-bp paired-end reads were aligned to 10 mm using bowtie with the parameters -X 2000 and -m 1. Duplicates were removed using Picard. Peaks were called using MACS2 (2, 1 .1.20160309) (Zhang et al., 2008) with the parameter -keep-dup all.
  • CTCF sites were derived from CTCF Peaks called in E14.5 C57BL/6 Limb embryo from ENCODE (Accession: ENCFF001YA ) and converted to mmlO coordinates using liftOver.
  • Z-score transformed wiggle files were created using the average ATAC signal per base-pair over the genome, excluding chromM and chroniY for each sample. Scores were then averaged over 100-bp non-overlapping windows. To filter out regions of background signal it required that the windows average greater than 1 in replicates. Significance was assayed between groups using a t-test (p- value ⁇ 0.05). Filtered data was then clustered using Cluster 3.0 (Eisen et al, 1998) and visualized using Java Treeview (Saldanha, 2004). Significantly altered windows were then associated with genes and subjected to pathway enrichment using GREAT (McLean et al., 20 0).
  • ATAC-seq peaks for tumor and epidermis were associated with genes using GREAT and quantified as peaks per gene.
  • Log?.FC (Tumor/Normal) of gene expression was presented as a cumulati ve distribution for a) genes demonstrating an increase in number of ATAC peak associations in tumor relative epidermis (gain of at least 6 peaks, green), loss of ATAC peaks (loss of at least 1 peak, red), and all genes (black).
  • ATAC-seq identified not only pan SC marker genes that were invariably expressed regardless of malignancy states (e.g., miR-205, Krtl 4), but also many genes that were either induced (e.g. miR-21, Gsta4, K17) or suppressed (e.g. Isnil, Ackr4) specifically in tumor SCs.
  • HFSC-specific ATAC peaks precisely captured previously annotated regulator ' regions from ChlP-seq where the full suite of HFSC identity TFs is bound and shouldered by active histone marker H3K27Ac ( Figure 8C) (Folgueras et al , 2013; Kadaja et al, 2014; Keyes et al, 2013; Lien et al, 2014), so called 'epicenters' (ECs). Numerous of such ECs sit within H3K27Ac marked, broad open chromatin domains known as 'super-enhancers' (Whyte et al, 2013), notable as regulators of stem cell key identity genes in the HFs (Adam et al, 2015).
  • the SCC-SC- unique ATAC peaks contained TF motifs for both EpdSCs (KLF5, GRHL, AP2) and HFSCs (SOX9, TCF, NFI) (Figure 2E).
  • the regulator ' regions for both KLF5 and SOX9 displayed a considerably broadened open-chromatin state not seen in the opposite SC lineage ( Figure 2C).
  • EpdSCs that ectopically expressed SOX9 showed only a modest increase in proliferation relative to control skin (Figure 3D), but instead displayed a robust ability to penetrate raatngel and migrate to the bottom well in a Boyden chamber assay (Figure 3F), These findings functionally corroborate in silico analysis of chromatin and transcriptional landscapes of SCC-SCs, and support a more prominent role for KLF5 in SC proliferation and an enhanced role for SOX9 in invasion.
  • CRISPR/CAS was used to specifically ablate SOX9 and KLF5 individually in the skin epithelium in vivo. This was accomplished by engineering lentiviral (LV) vectors harboring CRISPR small guide (sg) RNAs and PGK- CreER. LVs were then delivered in utero into the amniotic sacs of living E9.5 embryos harboring a Lox-Stop-Lox-Cas9-P2A-eGFP cassette knocked into the ubiquitously active Rosa26 locus (Piatt et al., 2014) ( Figure 4B).
  • LV lentiviral
  • the LV is transduced within a day exclusively into the single layer of unspecified epidermal progenitors, and thereafter is stably propagated to the developing epidermis and HF progeny (Beronja et al ,, 2010).
  • the pregnant females harboring the transduced embryos were treated with tamoxifen to induce Cre recombinase and permanently activate CAS 9 and eGFP expression in transduced clones.
  • split-thickness grafting was conducted. This method is analogous to partial-thickness wounding in that it challenges targeted HFSCs to re-epithelialize the skin denuded of its epidermis ( Figure 4B).
  • AT AC -marked-EC s regulatory elements within super- enhancers that contain AT AC peaks with densely clustered TF motifs
  • AT AC -marked-EC s regulatory elements within super- enhancers that contain AT AC peaks with densely clustered TF motifs
  • ETS2 was particularly interesting given that it is phosphoryiated at T72 and activated by ERK1/2, which is transiently elevated during wound-repair and consti utively activated in HRASG12V induced tumorigenesis (Yang et al, 1996). Moreover, when engineered to be constitutively active (T72D mutation) in skin epidermis, ETS2 functions as an oncogenic driver in SCCs (Yang et al, 2015).
  • the mutant, ETS2 was therefore used in vivo to address whether stress- responsive TFs cause EpdSC genes activation in HFSCs. Indeed, in addition to inducing the cohort of stress responsive TFs (Figure 13C), KLF5 was activated in HFSCs ( Figure 6B), similar to the ectopic KLF5 induction that had been seen in wound-experienced HFSCs (see Figure 4A).
  • SOX9 would be induced in EpdSCs under stress.
  • the SOX9 locus also acquired stress-specific ATAC peaks unique to the wound and tumor (Figiire 13D).
  • SOX9 was also ectopically induced in EpdSCs upon ETS2 (T72D) expression ( Figure 6B).
  • SOX9 was again ectopically induced ( Figure 6C).
  • KLF5 antagonizes SOX9, as posited previously based upon expression patterns and the presence of KI.F5 motifs in the SOX9 promoter (Bell et al, 2013; McConnell et al., 201 1 ; Nandan et al., 2014). Consistent with this hypothesis, when KMrtTA embryos were transduced with a doxyeycime-inducible KLF5 LV, and then induced KLF5 in adult HFs, SOX9 expression waned (Figure 6D), along with HFSC marker TENASCIN C, while EpdSC markers KI O and LORICRIN were activated ( Figure 14 A).
  • tumor-EC low-stress wound-induced regulator
  • inducible epithelial CRJSPR/CAS targeting and in utero lentiviral CreER and guide RNA delivery enabled us to expose hitherto unrecognized roles for KLF5 and SOX9 in wound and SCC as elaborated upon below.
  • KLF5 is an Epidermal Lineage Factor Required for Wound-repair and SCC
  • KLF5 skin functions (Ohnishi et al., 2000; Shindo et al, 2002; Sur et al., 2002). It was discovered that KLF5 is not only spatially associated with but also functionally essential for EpdSC fate. KLFS is induced early in HFSCs upon epidermal injury, and by virtue of its ability to suppress SOX9 and reprogram HFSCs to EpdSCs, KLF5 plays an integral role in the fate switch, enabling HFSCs to participate in epidermal re-epitheiialization during wound-repair.
  • EpdSCs in response to injury.
  • mobilized EpdSCs at the wound edge adopted certain features of HFSCs before resolving back to EpdSC.
  • mobilized HFSCs adopted features of EpdSCs before losing their own identity as they re-epithelialized denuded epidermis and restored the skin barrier.
  • a sensitive method, "assay for transposase accessible chromatin with high throughput sequencing" (ATAC-seq) (Buenrostro et al., 2013) was exploited a, to systemically interrogate the open chromatin landscape of purified tumorigenic SCs and compare them to those found in normal EpdSCs and HFSCs first during homeostatic and then under wound conditions.
  • Such comparative analy sis led to a surprising phenotype where two otherwise confined skin lineages - EpdSCs and HFSCs - are breached and manifested simultaneously in the tumor SCs. Intriguingly, it was found that this feature also occurs during a wound-response, but it is transient and resolves itself upon wound- closure.
  • CRISPR/CAS was used to ablate genes encoding two distinguishing lineage TFs, SOX9 (HFSCs) and LF5 (EpdSCs). It was demonstrated that in tumongenesis, co-expression of SOX9 and KLF5 is not only a hallmark of SCC, but also a requirement for malignant transformation. Lineage infidelity also appears to be crucial for wound-repair, as even though co-expression of SOX9 and KLF5 is transient, without either one, wounded SCs fail to contribute effectively to the healing process.
  • Example 2 Epicenter activity in multiple squamous cell carcinoma models
  • Figure 15 is an image demonstrating that epicenter activity in cultured mouse and human SCC lines. Lentiviral epicenter reporters were stably transduced into
  • SCC line mouse skin SCC, human skin SCC, and human head and neck SCCs.
  • Candidate test EC's (KlfSEC and miR21 EC) are compared against negative control EC (Ctrl EC) in each experiment. In each cases, significant EC reporter activity induction was observed in SCCs (** p ⁇ 0.01 , *** pO.001, paired student's test). Five biologically independent experiments were performed.
  • Figure 16 is an image demonstrating epicenter activity in human head and neck squamous cell carcinoma models.
  • Human head and neck squamous carcinoma (SCC9) cells were iransduced with lentiviral reporters for either stress-specific epicenter Klf5-EC or control EC (Ctrl-EC), xenografted onto immunosuppressant mice. Tumors were dissected 6 weeks post graft, and examined for GFP expression. RFP expression served as internal controls for lentiviral reporter transduction. Specific GFP expression was only observed in lfSEC but not in CtrlEC. Three independent results were conducted.

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Abstract

La présente invention concerne des compositions et des méthodes de diagnostic, de traitement de cancer et de prévention contre ce dernier chez un individu. L'invention concerne également des méthodes de criblage d'une composition qui module un épicentre souhaité.
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US20110269691A1 (en) * 2008-08-15 2011-11-03 Hearing Vincent J Compositions and Methods for Treating Pigmentary Conditions and Melanoma

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US20100028364A1 (en) * 2006-10-27 2010-02-04 The University Of Western Ontario Inhibition of sox9 function in the treatment of proteoglycan-associated pathophysiological conditions
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