WO2012034076A2 - Etv1 as a diagnostic, prognostic and therapeutic target for gastrointestinal stromal tumors - Google Patents

Etv1 as a diagnostic, prognostic and therapeutic target for gastrointestinal stromal tumors Download PDF

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WO2012034076A2
WO2012034076A2 PCT/US2011/051089 US2011051089W WO2012034076A2 WO 2012034076 A2 WO2012034076 A2 WO 2012034076A2 US 2011051089 W US2011051089 W US 2011051089W WO 2012034076 A2 WO2012034076 A2 WO 2012034076A2
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etvl
expression
etv1
gist
kit
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WO2012034076A3 (en
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Ping Chi
Yu Chen
Charles Sawyers
C. David Allis
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Memorial Sloan Kettering Cancer Center
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Memorial Sloan Kettering Cancer Center
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • Gastrointestinal stromal tumor is the most common human sarcoma.
  • GIST is primarily defined by activating mutations in the KIT or PDGFRA receptor tyrosine kinases.
  • KIT is a protein-tyrosine kinase receptor known to interact with stem cell factor. Binding of stem cell factor to KIT results in receptor dimerization and activation of protein kinase activity. The activated receptor autophosphorylates at tyrosine residues that serve as docking sites for signal transduction molecules contain SH2 domains.
  • the general structure of the KIT protein comprises an extracellular domain, a transmembrane segment, a juxtamembrane domain, and a protein kinase domain that contains an insert of about 80 amino acid residues. Both the active and inactive conformations of KIT have been determined by X-ray crystallography.
  • KIT is highly expressed in interstitial cells of Cajal (ICCs)—the presumed cell of origin for GIST— as well as in hematopoietic stem cells, melanocytes, mast cells and germ cells.
  • ICCs Cajal
  • the present invention encompasses the finding that the ETS family member ETV1 is highly expressed in the subtypes of ICCs sensitive to oncogenic KIT mediated transformation (Kwon, J. G. et al., 2009, Gastroenterology , 136: 630-639; incorporated herein by reference), and is required for their development.
  • the present invention also encompasses the finding that ETVl is universally highly expressed in GISTs.
  • the present invention further encompasses the finging that ETVl is required for growth of imatinib- sensitive and resistant GIST cell lines.
  • the present invention demonstrates, through transcriptome profiling and global analyses of ETVl -binding sites, that ETVl is a master regulator of an ICC- GIST-specific transcription network mainly through enhancer binding.
  • the ETVl transcriptional program is further regulated by activated KIT, which prolongs ETVl protein stability and cooperates with ETVl to promote tumorigenesis.
  • activated KIT which prolongs ETVl protein stability and cooperates with ETVl to promote tumorigenesis.
  • GIST arises from ICCs with high levels of endogenous ETVl expression that, when coupled with an activating KIT mutation, drives an oncogenic ETS transcription program.
  • the present invention therefore provides the insight that GIST differs from other ETO-dependent tumors such as prostate cancer, melanoma, and Ewing sarcoma where genomic translocation or amplification drives aberrant ETS expression (Tomlins, S. A. et ah, 2005, Science, 310: 644-648, incorporated herein by reference; Mertens, F. et ah, 2009, Semin Oncol, 36: 312-323, incorporated herein by reference; Jane-Valbuena, J. et ah, 2010, Cancer Res, 70: 2075-2084, incorporated herein by reference) and represents a novel mechanism of oncogenic transcription factor activation.
  • ETVl expression can be used to enable diagnosis, prognosis, and can be a therapeutic target for gastrointestinal stromal tumors or GISTs.
  • the present invention provides systems and technologies for detecting, characterizing, and/or treating gastrointestinal stromal tumors or GISTs, for example through assessment of level and/or activity of an ETS family member such as ETVl.
  • a method for determining the likelihood of gastrointestinal stromal tissue being cancerous or being gastrointestinal stromal tumor comprising (a) determining a level of expression or activity of ETVl in a gastrointestinal stromal tissue sample obtained from a subject; (b) comparing the determined ETVl level with that of a reference correlated with a predetermined probability of being cancerous or being GIST; and (c) based on the comparing, determining that the tissue sample has an increased or decreased probability, relative to the reference, of being cancerous or being GIST.
  • determining the level of expression or activity involves determining the expression of a nucleic acid.
  • determining the level of expression or activity involves determining the expression of a polypeptide.
  • determining the level of expression activity of ETVl involves determining an ETVl regulated expression signature.
  • an increase in the level of ETVl correlates with the onset of or progression of a GIST phenotype. In other embodiments, a decrease in the level of ETVl correlates with the regression of a GIST phenotype.
  • determining the level of expression comprises performing Northern blotting, Western blotting, protein gel electrophoresis, immunoprecipitation, ELISA, PCR, RT-PCR, differential display, serial analysis of gene expression, array analysis, and/or immunohistochemistry.
  • the step (b) of comparing comprises establishing that the determined level in the subject tissue sample is higher than the reference level, and the reference level correlates with that observed in normal tissue, so that step (c) of determining comprises detemining that the tissue sample has an elevated likelihood of being cancerous or GIST.
  • the step (a) of determining comprises performing an analysis selected from the group consisting of Northern blotting, Western blotting, protein gel electrophoresis, immunoprecipitation, ELISA, PCR, RT-PCR, differential display, serial analysis of gene expression, array analysis, immunohistochemistry, or a combination thereof, so that the ETVl level of expression or activity is determined.
  • the step (a) of determining comprises performing immunohistochemistry on the tissue sample, using an ETVl antibody or antigen binding fragment thereof.
  • the antibody or antigen binding fragment binds to an amino acid sequence or portion thereof as set forth in SEQ ID NO: 1.
  • a method for determining the likelihood of cancerous gastrointestinal stromal tissue responding to therapy comprising: (a) determining a level of expression or activity of ETVl in a cancerous gastrointestinal stromal tissue sample obtained from a subject; (b) comparing the determined ETVl level with that of a reference correlated with a predetermined probability of responding to a therapeutic regimen; and (c) based on the comparing, determining that the subject from which the tissue sample was obtained has an increased or decreased probability, relative to the reference, of responding to the therapeutic regimen.
  • the reference is an average of responses derived from a population of individuals.
  • a methhod for identifying or characterizing an agent that affects GIST phenotype comprising: (a) contacting cultured GIST tissue or cells with an agent to be characterized; and (b) determining that ETVl level of expression or activity is altered when the agent is present as compared with when it is absent.
  • a method of treating GIST by administering to a subject suffering from or susceptible to GIST a therapy that alters ETVl level of expression or activity.
  • the step of administering comprises administering a therapy that modulates expression or activity of an ETVl regulator so that ETVl level of expression or activity is altered.
  • a method of treating GIST in a subject in need thereof comprising administering a therapeutically effective amount of pharmaceutical composition comprising an agent identified by the methods disclosed herein.
  • the composition inhibits EVT1 expression.
  • the composition inhibits EVT1 expression.
  • composition promotes the degradation of ETVl protein.
  • kits for use in detecting ETVl expression comprises an agent capable of detecting the level of ETVl expression or activity, a container comprising the agent for detecting the level of ETVl in a sample, a control, and instructions to provide guidance for carrying out an assay embodied by the kit and for making a determination of ETV1 based upon that assay.
  • the agent is an antibody or ETV1- binding fragment thereof, oligonucleotide probe, oligonucleotide primer, aptamer, polypeptide, peptide, ligand, hormone, lipid, carbohydrate, lectin, or small molecule.
  • the agent is an anti-ETVl antibody or an antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof is attached to a substrate, the substrate is applied to a sample from a patient or to a surface that may contain ETV1, and the surface of the substrate is then processed to assess whether specific binding occurs between the antibody and ETV1 or other component of the sample.
  • the control is a control slide comprising tumor samples for testing reagents in the kit.
  • a method comprising the steps of: (a) monitoring ETV1 level of expression or activity of ETV1 in a subject suffering from GIST; (b) detecting a change in the monitored ETV1 level over time; and (c) initiating or altering therapy administered to the subject after detecting the change.
  • the subject is undergoing a first therapy and the step of initiating or altering comprises ceasing the first therapy, modifying the first therapy, adding a second therapy, or combinations thereof.
  • a method of inhibiting the growth of ICC cancerous cell with aberrant ETV1 expression comprising contacting the cell with an ETV1 inhibitory agent so as to decrease the level or activity of ETV1, thereby inhibiting the growth of the ICC cancerous cell.
  • the ETV1 inhibitory agent is selected from the group consisting of a targeted ribozyme, a targeted antisense oligonucleotide, and a targeted siRNA
  • FIG. 1 depicts an exemplary result illustrating ETVl is universally highly expressed and required for tumour growth and survival in GIST.
  • FIG. 1A depicts an exemplary result illustrating a Venn diagram of GIST-signature genes from three datasets.
  • FIG. IB depicts an exemplary result illustrating expression of ETVl in multiple tumour types from the ExpO dataset. Box, 25-75 percentile; error bar, 10-90 percentile; dots, outliers.
  • FIG. 1C depicts an exemplary result illustrating ETVl and KIT mRNA levels by qRT-PCR of GIST and non-GIST samples, whose details are described in Full Methods.
  • FIG. ID depicts an exemplary result illustrating immunoblotting of selected tumour tissues and cell lines from FIG. 1C.
  • FIG. 1G depicts an exemplary result illustrating ETVl mRNA levels of preimplanted GIST882 cells and explanted xenografts at week 10. Mean+SD.
  • FIG. 2 depicts an exemplary result illustrating ETVl is expressed in the subtypes of ICCs susceptible to oncogenesis and is required for their development.
  • FIG. 2A depicts an exemplary result illustrating a schematic showing localization of ICC-MY (yellow arrowheads), ICC-IM (yellow arrows) and ICC-SMP (white arrowheads) in the large intestine.
  • M mucosa
  • CM circular muscle
  • LM longitudinal muscle. All three ICC subtypes express Kit (red).
  • FIG. 1 depicts an exemplary result illustrating ETVl is expressed in the subtypes of ICCs susceptible to oncogenesis and is required for their development.
  • FIG. 2A depicts an exemplary result illustrating a schematic showing localization of ICC-MY (yellow arrowheads), ICC-IM (yellow arrows) and ICC-SMP (white arrowheads) in the large intestine.
  • M mucosa
  • CM circular
  • FIG. 2B depicts an exemplary result illustrating co-immunofluorescence (divided into two microscopy fields) of Kit (red), Etvl (green) and DAPI (blue) of the large intestine of wild-type mice.
  • FIG. 2C depicts an exemplary result illustrating co-immunofluorescence of Kit (red), Pgp9.5 (green), and DAPI (blue) of the large intestine of Etvl+/+ and Etvl-/- mice.
  • FIG. 2D depicts an exemplary result illustrating representative deconvoluted whole-mount Kit-immunofluorescence images of the large intestine of Etvl+/+ and Etvl-/- mice. A single microscopy field focused to the ICC-MY and ICC-SMP planes are shown. Scale bar, 20 ⁇ .
  • FIG. 3 depicts an exemplary result illustrating ETVl regulates GIST-signature genes predominantly through enhancer binding.
  • FIG. 3A depicts an exemplary result illustrating a ranked list of ETVl regulated genes generated based on the average fold-change by the two ETVl hairpins in two cell lines.
  • FIG. 3B depicts an exemplary result illustrating a heatmap of expression of the 48 genes with average downregulation > 1.7-fold. For each gene, table shows p- value of GIST vs. other tumour types from the ExpO dataset, calculated by OncomineTM (NS: p>0.05), and the presence of ETVl binding sites from ChlP-Seq analysis.
  • FIG. 3 depicts an exemplary result illustrating ETVl regulates GIST-signature genes predominantly through enhancer binding.
  • FIG. 3A depicts an exemplary result illustrating a ranked list of ETVl regulated genes generated based on the average fold-change by the two ETVl hairpins in two cell lines.
  • FIG. 3C depicts an exemplary result illustrating GSEA plots of the shETVl ranked list using three gene sets: GIST signature genes from ExpO dataset, ICC-MY and ICC-DMP signature genes in mouse small intestine. ES, enrichment score; FDR, false discovery rate.
  • FIG. 3D depicts an exemplary result illustrating pie charts of genomic structure and distribution of ETVl ChlP-Seq peaks. TSS, transcription start site; TES, transcription end site.
  • FIG. 3E depicts an exemplary result illustrating representative ChlP-Seq reads in top ETVl target genes.
  • FIG. 3F depicts an exemplary result illustrating the consensus sequence motif identified in the ETVl binding sites by the MEME program.
  • FIG. 3G depicts an exemplary result illustrating pie chart of genes with ETVl binding sites divided into promoter only, enhancer only and both.
  • FIG. 4 depicts an exemplary result illustrating KIT signalling synergizes with ETVl in GIST tumouri genesis by stabilization of ETVl protein.
  • FIG. 4 A depicts an exemplary result illustrating immunoblots of GIST882 cells treated with the imatinib (1 ⁇ ) and PD325901 (100 nM) for the indicated time points.
  • FIG. 4B depicts an exemplary result illustrating immunoblots of GIST882 cells treated for 2 hours with imatinib or PD325901 in combination with cyclohexamide (10 ⁇ g ml) or MG132 (10 ⁇ ).
  • FIG. 4 A depicts an exemplary result illustrating immunoblots of GIST882 cells treated with the imatinib (1 ⁇ ) and PD325901 (100 nM) for the indicated time points.
  • FIG. 4B depicts an exemplary result illustrating immunoblots of GIST882 cells treated for 2 hours with imatinib or PD325901 in
  • FIG. 4C depicts an exemplary result illustrating a Venn diagram of genes downregulated by 1.4-fold by shETVl and by imatinib in GIST882 cells. P-value: Fisher's exact test based on number of expressed genes.
  • FIG. 4D depicts an exemplary result illustrating percent of all genes, imatinib-downregulated genes, shETVl- downregulated genes, and overlapping genes with ETVl enhancer peaks.
  • FIG. 4E depicts an exemplary result illustrating an immunoblot of NIH3T3 cells expressing ETVl and either KITwt or ⁇ 560 two hours after treatment with PD325901, imatinib, or MG132.
  • FIG. 4G depicts an exemplary result illustrating a photograph of 4 representative explanted xenografts at 4 weeks after implanting. Scale bar 1 cm.
  • FIG. 4H depicts an exemplary result illustrating a model of the role of ETVl in ICC maintenance and GIST oncogenesis. Normal level of KIT activation by KIT ligand (red triangle) stabilizes ETVl transcription factor through the MAPK pathway, and results in physiological ETVl transcriptional output critical for ICC development (middle).
  • FIG. 5 depicts an exemplary result illustrating ETVl expression levels from GIST containing microarray expression datasets.
  • FIG. 5A depicts an exemplary result illustrating expression of ETVl (37156_at, Affymetrix U95A-Av2) of individual tumour samples of 9 sarcoma types from Segal dataset.
  • FIG. 5B depicts an exemplary result illustrating expression of ETVl (IMAGE:81320, Stanford spotted platform) of individual tumour samples of 6 sarcoma types from Nielsen dataset.
  • FIG. 5C depicts an exemplary result illustrating expression of ETVl (22191 l_at, Affymetrix HG_U133_Plus_2) of 32 GIST samples from Yamaguchi dataset annotated by location.
  • FIG. 5D depicts an exemplary result illustrating expression of ETV1 (22191 l_at, Affymetrix HG_U133_Plus_2) of 29 GIST samples from Ostrowski dataset annotated by TKI mutation status.
  • FIG. 6 depicts an exemplary result illustrating ETV1 immunofluorescence in four GIST (GIST1-4) samples and a leiomyosarcoma (LMS) control sample.
  • GIST1-4 GIST
  • LMS leiomyosarcoma
  • Paraffin embedded human GIST samples were stained with ETVl-specific primary antibody and Alexa-594 anti- rabbit secondary antibody.
  • Alexa 594 left column; DAPI: middle column; Alexa/DAPI merge: right column.
  • Scale bar 20 um.
  • FIG. 7 depicts an exemplary result illustrating effect of shETVl on ETV1 protein and mRNA levels and cell cycle profile.
  • FIG. 7 A depicts an exemplary result illustrating ETV1 mRNA and protein levels in GIST48, GIST882, and U20S cells 4-days after shRNA-mediated ETV1 knockdown.
  • FIG. 8 depicts an exemplary result illustrating an assessment for ETV1 translocation and amplification in GIST.
  • FIG. 8A-B depicts an exemplary result illustrating representative FISH of the ETV1 locus in two GIST clinicial specimens. Green are two probes in the gene body and downstream of ETV1 and red are two probes upstream of ETV1. There's no evidence of "breakaway" or amplification of ETV1.
  • FIG. 8C depicts an exemplary result illustrating qRT- PCR of same cDNA as in FIG. 1C, which used primers targeting ETV1 exons 6 and 7. THis qRT-PCR used primers targeting exons 2 and 3. UP arrows indicate regions of translocation in prostate cancers. Comparing to FIG. 1C, there is no evidence that early exons before
  • FIG. 9 depicts an exemplary result illustrating expression of ETV1 in the murine small intestine.
  • FIG. 9A depicts an exemplary result illustrating the myenteric ICC network (ICC-MY, yellow arrowheads) is localized adjacent to the myenteric plexus (MP) between the circular (CM) and longitudinal (LM) muscle layers.
  • the deep muscle plexus ICC network (ICC- DMP, white arrowheads) is localized just below the mucosa (M) in the circular muscle layer.
  • FIG. 9B depicts an exemplary result illustrating both ICC subsets express Kit though ICC-DMP stains much more dimly.
  • FIG. 10 depicts an exemplary result illustrating Etvl is required for ICC-MY but not ICC-DMP development of the murine small intestine.
  • FIG. 10A depicts an exemplary result illustrating co-immunofluorescence of Kit (red), Pgp9.5 (green), and DAPI (blue) of the small intestine of Etvl+/+ and Etvl-/- mice showing significant loss of ICC-MY (yellow arrowheads) and preservation of ICC-DMP (white arrowheads).
  • FIG. 10B depicts an exemplary result illustrating whole mount Kit immunofluorescence taken at the ICC-MY plane and ICC-DMP plane, also showing loss of ICC-MYs and preservation of ICC-DMPs. Scale bar 20uM.
  • FIG. 11 depicts an exemplary result illustrating Etvl is required for ICC-IM development of the murine gastric fundus.
  • FIG. 11 A depicts an exemplary schematic showing ICC-IMs present in both the circular and longitudinal muscle layers.
  • FIG. 11B depicts an exemplary result illustrating co-immunofluorescence of Kit (red), Pgp9.5 (green), and DAPI (blue) of the gastric fundus of Etvl+/+ and Etvl-/- mice showing significant loss of ICC-IM.
  • FIG. l lC depicts an exemplary result illustrating whole mount Kit immunofluorescence taken at the circular muscle ICC-IM and longitudinal muscle ICC-IM planes, also showing loss of ICC- IMs. Scale bar 20 uM.
  • FIG. 12 depicts an exemplary result illustrating Etvl is required for ICC-MY development of the murine cecum.
  • FIG. 12 A depicts an exemplary schematic of ICC types of the cecum. Compared to the rest of the large intestine, ICC-IM and ICC-SMP is very sparse (see FIG. 2).
  • FIG. 12B depicts an exemplary result illustrating co-immunofluorescence of Kit (red), Pgp9.5 (green), and DAPI (blue) of the cecum of Etvl+/+ and Etvl-/- mice showing significant loss of ICC-MY.
  • FIG. 12C depicts an exemplary result illustrating whle mount Kit
  • FIG. 13 depicts an exemplary result illustrating quantification of ICC subclasses remaining in Etvl-/- mice expressed as precent of Etvl-/- mice.
  • immunofluorescence images from at least four fields each from two Etvl+/+ and Etvl-/- mice (at least eight fields total) of the stomach, small intestine, cecum, and large intestine were examined.
  • a percentage of ICCs were obtained by dividing the number of KIT-positive immunostaining cells with the number of DAPI-positive nuclei. Plotted is the ratio of percent of ICCs in Etvl-/- mice compared to Etvl+/+ mice (Mean + SD).
  • FIG. 14 depicts an exemplary result illustrating Kit and Anol immunofluorescence in GI tract of Etvl+/+ and Etvl-/- mice.
  • Kit and Anol label all ICC subsets with similar intensity except in ICC-DMP of the small intestine where Kit staining is faint and Anol is stronger (FIG. 14B, white arrows).
  • FIG. 14A depicts an exemplary result illustrating global loss of ICC-MY and ICC-IM in stomach of Etvl-/- mice.
  • FIG. 14B depicts an exemplary result illustrating global loss of ICC-MY and ICC-IM in small intestine of Etvl-/- mice.
  • FIG. 14C depicts an exemplary result illustrating global loss of ICC-MY and ICC-IM in large intestine of Etvl-/- mice.
  • ICC- DMP (FIG. 14B) are preserved in the small intestine
  • ICC-SMP (FIG. 14C) are preserved in the colon (white arrows).
  • Scale bar 20 um.
  • FIG. 15 depicts an exemplary result illustrating whole mont Pgp9.5
  • FIG. 16 depicts an exemplary result illustrating overlap of expressed probes (total 38985) perturbed 1.7-fold by ETVl shRNA in GIST48 and GIST 882 cells.
  • FIG. 16 depicts an exemplary result illustrating overlap of expressed probes (total 38985) perturbed 1.7-fold by ETVl shRNA in GIST48 and GIST 882 cells.
  • FIG. 16C depicts an exemplary result illustrating a Venn diagram of probes downregulated by either ETVlshl or ETVlsh2 in GIST48 (blue) and GIST882 (orange).
  • FIG. 16D depicts an exemplary result illustrating a Venn diagram of probes upregulated by either ETVlshl or ETVlsh2 in GIST28 (green) and GIST882 (red). P- values for significance of overlap were calculated using Fisher Exact Test.
  • FIG. 17 depicts an exemplary result illustrating qRT-PCR validation of top downregulated genes by ETVl knockdown.
  • GIST48 and GIST882 cells were infected with shScr, ETVlshl, and ETVlsh2 lentiviruses in an independent experiment from the expression profiling experiment.
  • RNA was harvested 4 days after lentiviral infection without drug selection and qRT-PCR performed.
  • FIG. 18 depicts an exemplary result illustrating GSEA enrichment of GIST-signature datasets.
  • FIG. 18A depicts an exemplary result illustrating enrichment plots of Segal GIST- signature gene sets on the "shETVl” ranked list.
  • FIG. 18B depicts an exemplary result illustrating enrichment plots of Nielsen GIST-signature gene sets on the "shETVl” ranked list.
  • FIG. 21 depicts an exemplary result illustrating soft agar colony formation assays of NIH3T3 cells transduced with different permutations of ETVl and KIT.
  • NIH3T3 cells transduced with EGFP or ETVl expression virus were subsequently transduced with different combinations of empty vector, wild-type KIT, or activating mutant KIT ( ⁇ 560).
  • 5,000 cells/well of 6-well plate were seeded in triplicate of each transduced line.
  • FIG. 21 A depicts an exemplary result illustrating photographs of representative wells of soft agar colony formation 3 weeks after plating 5,000 cells/well. Scale bar 2mm.
  • Combination therapy refers to those situations in which two or more different pharmaceutical agents are administered in overlapping regimens so that the subject is simultaneously exposed to both agents.
  • ETVl regulator refers to any endogenous intracellular or extracellular substance that modulates the transcription, binding, activity or stability of ETVl.
  • the ETVl regulator may be KIT.
  • Gene expression signature refers to a unique and distinct pattern of expression of a given gene in a particular tissue.
  • a gene expression signature may be correlated with and indicate a particular phenotype, aberrant expression pattern, condition, probability of disease, prognosis, preferred treatment, our liklihood of success of a particular treatment.
  • Overexpression refers to expression of a nucleic acid sequence or protein in a cancer cell at a higher or lower level, respectively, than that level typically observed in a non-tumor cell (i.e., normal control).
  • the level of expression of a nucleic acid or a protein that is overexpressed in the cancer cell is at least 10%, 20%, 40%, 60%, 80%, 100%, 200%, 400%, 500%, 750%, 1,000%, 2,000%, 5,000%, or 10,000% greater in the cancer cell relative to a normal control.
  • the level of overexpression in a cancer cells is at least two-fold or more greater than the relative level of expression in a non-cancer cell.
  • Reference sample may include, but is not limited to, any or all of the following: a cell or cells, a portion of tissue, blood, serum, ascites, urine, saliva, and other body fluids, secretions, or excretions.
  • sample also includes any material derived by processing such a sample. Derived samples may include nucleotide molecules or polypeptides extracted from the sample or obtained by subjecting the sample to techniques such as amplification or reverse transcription of mRNA, etc.
  • Subject refers to any organism upon which embodiments of the invention may be used or administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals ⁇ e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.).
  • a disease, disorder, or condition e.g., GIST
  • GIST a disease, disorder, or condition
  • GIST a disease, disorder, or condition
  • Early-stage GIST is frequently asymptomatic.
  • an individual who is suffering from GIST has GIST, but does not display any symptoms of GIST and/or has not been diagnosed with GIST.
  • an individual who is suffering from GIST is an individual who has upregulated ETV1 expression.
  • Susceptible to An individual who is "susceptible to" a disease, disorder, or condition (e.g. , GIST) is at risk for developing the disease, disorder, or condition.
  • an individual who is susceptible to a disease, disorder, or condition does not display any symptoms of the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition is an individual who displays conditions associated with development of the disease, disorder, or condition (e.g., the individual has elevated levels of ETV1 expression). In some embodiments, a risk of developing a disease, disorder, and/or condition is a population-based risk (e.g., carrier of KIT ex on 9 mutation, carrier of KIT ex on 11 mutation, carrier of KIT exon 13 mutation; etc.).
  • a population-based risk e.g., carrier of KIT ex on 9 mutation, carrier of KIT ex on 11 mutation, carrier of KIT exon 13 mutation; etc.
  • Symptoms are reduced: According to the present invention, "symptoms are reduced” when one or more symptoms of a particular disease, disorder or condition is reduced in magnitude (e.g., intensity, severity, etc.) or frequency. For purposes of clarity, a delay in the onset of a particular symptom is considered one form of reducing the frequency of that symptom. Many GIST patients with smaller tumors have no symptoms. Larger tumors can cause symptoms that are related to the increased mass being accommodated in the abdominal cavity.
  • exemplary symptoms of GIST may include, but are not limited to, digestive discomfort, sensations of abdominal fullness, abdominal pain, visible enlargement of the abdomen, vomiting or diarrhea, bowel obstruction, perforation of the stomach or gut lining, black or tarry stools, vomiting of blood, anemia fatigue, and weight loss. It is not intended that the present invention be limited only to cases where the symptoms are eliminated. The present invention specifically contemplates treatment such that one or more symptoms is/are reduced (and the condition of the subject is thereby "improved"), albeit not completely eliminated.
  • therapeutic regimen refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. It may comprise a treatment or series of treatments designed to achieve a particular effect i.e., reduction or elimination of a detrimental condition or disease such as GIST.
  • the treatment may include administration of one or more compounds either simultaneously, sequentially or at different times, for the same or different amounts of time. Alternatively, or additionally, the treatment may include exposure to radiation, chemotherapeutic agents or surgery.
  • a “treatment regimen” may include genetic methods such as gene therapy, gene ablation or other methods known to reduce expression of a particular gene or translation of a gene-derived mRNA.
  • Therapeutic agent refers to any agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect.
  • therapeutically effective amount refers to an amount of a therapeutic protein (e.g., ETV1 antibody) which confers a therapeutic effect on the treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment.
  • the therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect).
  • the "therapeutically effective amount” refers to an amount of a therapeutic protein or composition effective to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity or frequency of symptoms of the disease.
  • a therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses.
  • a therapeutically effective amount (and/or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration, on combination with other pharmaceutical agents.
  • the specific therapeutically effective amount (and/or unit dose) for any particular patient may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and/or rate of excretion or metabolism of the specific fusion protein employed; the duration of the treatment; and like factors as is well known in the medical arts.
  • treatment refers to any administration of a substance (e.g., provided compositions) that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition (e.g., GIST).
  • a substance e.g., provided compositions
  • Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition.
  • such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.
  • treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.
  • Tumor sample is taken broadly to include cell or tissue samples removed from a tumor, cells (or their progeny) derived from a tumor that may be located elsewhere in the body (e.g., cells in the bloodstream or at a site of metastasis), or any material derived by processing such a sample. Derived tumor samples may include nucleic acids or proteins extracted from the sample or obtained by subjecting the sample to techniques such as amplification or reverse transcription of mRNA, etc.
  • Tumor subclass is the group of tumors that display one or more phenotypic or genotypic characteristics that distinguish members of the group from other tumors.
  • ETS family member ETV1 is highly expressed in the subtypes of ICCs sensitive to oncogenic KIT-mediated transformation (Kwon, J. G. et ah, 2009, Gastroenterology, 136: 630-639), and is required for their development.
  • ETV1 expression can be used as a diagnostic, prognostic or therapeutic target for GIST.
  • GISTs Gastrointestinal stromal tumors
  • GISTs occur in the wall of the bowel and have been proposed to arise from the interstitial cells of Cajal (ICCs). GISTs are the most common mesenchymal neoplasms of the gastrointestinal tract, and can also originate in the mesentery and omentum. The differential diagnosis of these tumors includes desmoid fibromatosis, Schwannoma, leiomyosarcoma, and, in some cases, high grade sarcomas. Accurate diagnosis of GISTs is important, because imatinib mesylate (GLEEVEC® manufactured by Novartis, Switzerland) has been shown to significantly inhibit these tumors.
  • GLEEVEC® imatinib mesylate
  • Embodiments of the present invention are based on the surprising discovery of the oncogenic contributions of ETV1 in ICC cells and GIST. The data described below
  • ETV1 is a superior marker for GIST because it is a master regulator of an ICC-GIST-specific transcription network. Moreover, ETV1 controls this network through stabilizing interactions with KIT.
  • the synergistic interaction of KIT and ETV1 provides a new paradigm for the development of ICC cancers, including GIST. Analysis, qualification or quantification of ETV1 expression, either alone or in combination with analysis of KIT, provides refined diagnostic capabilities.
  • therapies directed toward ETV1 either alone or in combination with therapies directed at KIT or conventional cancer therapies, provide promising new uses and treatments for GIST- afflicted patients.
  • Embodiments of the present invention may be used for the diagnosis or prognosis of GIST, and may further be used in GIST-directed therapies.
  • the invention therefore provides a method comprising providing a GIST tumor sample; detecting expression or activity of a gene encoding a ETV1 polypeptide in the sample; determining whether the tumor sample is marked by elevated ETV1 expression relative to a non-cancerous negative control sample.
  • the method further comprises detecting expression or activity of a gene encoding a KIT polypeptide in the sample and detecting expression or activity of a gene encoding a ETVI polypeptide in the sample.
  • the determining step is based on the results of all detecting steps.
  • the invention provides a method comprising providing a GIST tumor sample; detecting expression or activity of a gene encoding a ETV1 polypeptide in the sample; and determining whether the tumor is marked by normal or reduced ETV1 expression relative to a cancerous positive control sample.
  • the method further comprises detecting expression or activity of a gene encoding a KIT polypeptide in the sample and detecting expression or activity of a gene encoding a ETV1 polypeptide in the sample.
  • the determining step is based on the results of all detecting steps.
  • the tumor sample may be a blood sample, a urine sample, a serum sample, an ascites sample, a saliva sample, a cell, or a portion of tissue.
  • the methods may further comprise providing diagnostic, prognostic, or predictive information based on the determining step. Determining may include diagnosis, probabiltiy or predisposition of GIST, or stratifying the tumor (and thus stratifying a subject having the tumor), e.g., for a clinical trial, or classifying for purposes of determining treatment.
  • the methods may further comprise selecting a treatment based on the detrmining step. Additionally or alternatively, the methods may further comprise determining the specific mutations within the ETV1 markers and the correlation of those mutations with the severity of disease, a desired course of treatment, and the prognosis for recovery.
  • GIST samples may be obtained by methods known to those of skill in the art, including biopsy or post-surgery. GISTs may occur anywhere in the gastrointestinal tract but are most common in the stomach and small bowel (roughly 60% and 30%, resp.), while 10% arise in other parts of the gastrointestinal tract (esophagus, colon, and rectum), and a small percentage are extragastrointestinal, arising in the mesentery, omentum, retroperitoneum, or pelvis.
  • Endoscopic, fine needle and surgical biopsy techniques may be used to retrieve the samples.
  • the main goal for a localized GIST is complete surgical resection with negative margins and preservation of an intact pseudocapsule.
  • personalized treatment regimens may be devised depending on the level of ETV1 expresssion, with higher level of ETV1 indicating a more aggressive treatment regimen.
  • Particular levels of expression may require either gastric wedge resection, partial gastrectomy or total gastrectomy.
  • Embodiments of the invention encompass novel therapies to directly or indirectly (i.e., by affecting an upstream regulator or downstream target) treat elevated or otherwise aberrant (e.g., mutated) ETV1 expression.
  • These treatments may be combined with known surgical or pharmacological treatments for GIST.
  • the methods disclosed herein may be used after surgical intervention to target an affected area.
  • embodiments of the invention may be used simultaneously with or immediately before or after current pharmacological and chemotherapeutic treatments.
  • the tyrosine kinase inhibitor imatinib mesylate may be used in the adjuvant setting following complete gross resection.
  • the kinase inhibitor sunitinib malate (SU 11248 or Sutent, Pfizer, New York, NY) may be used.
  • RADOOl manufactured by Novartis, Switzerland
  • AKT is a survival pathway that is activated by KIT and many other receptors. It is hoped that the simultaneous inhibition of KIT and mTOR using GLEEVEC® and RADOOl will result in increased effectiveness over GLEEVEC® alone.
  • PKC412 an inhibitor of protein kinase C
  • PKC412 is less specific than GLEEVEC®, inhibiting PKC, and kinases of KIT, VEGF, PDGF 42.
  • Amgen of Thousand Oaks, CA are developing AMG706 that is thought to have a similar mechanism of action as SU11248.
  • Bristol-Myers Squibb of New York, NY are developing BMS-354825 that is an inhibitor of both KIT and PDGFRA.
  • Pharmaceutical treatment directed towards EDTV1 may be added to such existing treatments. 2. ETV1 expression or activity correlates with GIST
  • ETV1 is a transcription factor belonging to the large ETS (E-twenty six) family and PEA3 subfamily.
  • ETS family members are defined by a highly conserved DNA binding domain, the ETS domain, which is a winged helix-turn-helix structure that binds to DNA sites with a central GGA DNA sequence.
  • Multiple ETS factors have been found to be associated with cancer.
  • Ewing's sarcoma the ERG ETS transcription factor is fused to the EWS gene (Ida, K., et al., 1995, Int. J. Cancer, 63(4):500-4; incorporated herein by reference).
  • the ETV1 protein (SEQ ID NO: 1) and its readily derived mRNA sequences are universally highly expressed in gastroinstestinal stromal tumors (GISTs) and are required for growth of imatinib-sensitive and resistant GIST cell lines.
  • GISTs gastroinstestinal stromal tumors
  • Activated KIT through MEK, prolongs ETV1 protein stability and cooperates with ETV1 to promote tumorigenesis.
  • GIST arises from interstitial cells of Cajal (ICCs) exhibiting high levels of endogenous ETV1 expression that, when coupled with an activating KIT mutation, drives an oncogenic ETS transcriptional program.
  • ETV1 This oncogenic role for ETV1 in GIST differs from classical models of other ETS-dependent tumors such as prostate cancer, melanoma and Ewing sarcoma where genomic translocation or amplification drives aberrant ETS expression and promote tumorigenesis (Tomlins, S.A., et al., 2005, Science, 310(5748):644-8; Mertens, F. et al., 2009, Semin. Oncol., 26, 312-23; Jane-Valbuena, J. et al., 2010, Cancer Res., (70), 2075-84; each of which are incorporated herein by reference).
  • ETV1 is universally highly expressed in all GISTs makes it immediately useful as a candidate diagnostic biomarker, because the current standard of KIT immunreactivity is negative in about 5% of all GISTs (Miettinen, M. et al., 2006, Arch. Pathol. Lab. Med., 130: 1466-78; incorporated herein by reference).
  • the human ETV1 gene comprises approximately 100 kb of Chromosome 7. At least seven different transcript variants of the ETV1 gene have been identified.
  • ETV1 variant 1 (Accession No. NM_004956) is shown in Table 1 as SEQ ID NO: 31. Additional ETV1 variants encompassed by embodiments of the present invention include Accession Nos. NM_001163150, NM_001163151, NM_001163149, NM_001163148, NM_001163147, and NM_001163152 the entire contents of which are incorporated herein by reference.
  • Embodiments of the invention are based on the surprising discovery that ETV1 is highly expressed in all GISTs and at significanly higher levels than any other tumor type.
  • Analysis of ETVl mRNA and protein levels in GIST and other sarcomas in clinical samples has proven that ETVl mRNA and protein are highly and exclusively expressed in all GIST tumor samples and GIST cell lines.
  • RNAi techniques used to reduce EVTl translation have proven that ETVl is required for GIST growth and survival.
  • In situ hybidization analyses of GIST samples and cell lines further indicates that high levels of ETVl expression are
  • EVTl is a master regulator of an ICC-GIST-specific transcription network through mediation of enhancer binding.
  • ETVl as a master regulator facilitates diagnostic uses of ETVl expression and activity levels. It is well known in the art that the gene expression profile of a tumor or subclass of tumor may be associated with different prognoses. Such information may include, but is not limited to, the average life expectancy of a patient, the likelihood that a patient will survive for a given amount of time (e.g., 6 months, 1 year, 5 years, etc.), the likelihood that a patient will be cured of a disease, the likelihood that a patient's disease will respond to a particular therapy (wherein response may be defined in any of a variety of ways). For example, differences in the prognosis of patients with KIT positive and PDGFRA positive GISTs have been described.
  • Methods of the present invention utilize a novel parameter for diagnosis and prognosis of GIST cancers: ETVl expression and/or mutation.
  • a method is provided for determining the liklihood of gastrointestinal stromal tissue being cancerous and/or afflicted by a GIST. The method comprises determining a level of expression or activity of ETVl in a gastrointestinal stromal tissue sample obtained from a subject;
  • the present invention offers the possibility of providing additional diagnostic, prognostic, or predictive information based on modification of existing protocols to include ETV1.
  • the present invention also offers the possibility of analyzing tumor sample archives containing tissue samples that were obtained from patients and stored with information regarding the progress of the patient's disease.
  • tumor sample archives consist of tumor samples embedded in paraffin blocks.
  • These tumor samples can be analyzed for their expression of polypeptides encoded by the marker genes of the present invention, particularly ETV1.
  • immunohistochemistry can be performed using antibodies that bind to the marker genes of the present invention, particularly ETV1.
  • Tumors with elevated, mutated or otherwise aberrant expression may then be identified and correlated with the severity of the tumor, responses to therapy and other avaible clincial information, e.g., age at death, length of survival, etc.
  • ETV1 expression in the samples may be quantitated or semi-quantitated relative to a control.
  • the information derived from these samples may be used to construct a reference database or library.
  • the average expression across a population or subpopulation of normal and afflicted individuals may be determined, thereby creating a reference correlated with a predetermined probability of being cancerous.
  • ETV1 expression analysis comprises identifying whether ETV1 (the gene or gene product) is upregulated or downregulated relative to a reference or baseline.
  • the reference may be derived from average expression across a population of individuals or may be single prior sample derived from the subject.
  • the analyses of samples from single individuals or across a population i.e., large group of individuals provides a library of data sets for use as a reference or baseline. Differential expression relative to the reference can then be determined, which provides a qualitative or quantitative comparative value that is informative about the diagnosis of GIST, long-term prognosis, response to therapy, etc.
  • the differential expression values for ETVl can be cross-referenced with analyses of other oncogenic GIST events (e.g., KIT mutations) to provide a synergistic diagnosis.
  • the differential expression values relative to a reference may also be annotated with medical information about the subject.
  • Determining whether the tissue sample has an increased or decreased probability, relative to the reference, of being cancerous or GIST can be performed using a statistical test to determine statistical significance of any differential expression observed.
  • statistical significance is determined using a parametric statistical test.
  • the parametric statistical test can comprise, for example, a fractional factorial design, analysis of variance (ANOVA), a t- test, least squares, a Pearson correlation, simple linear regression, nonlinear regression, multiple linear regression, or multiple nonlinear regression.
  • the parametric statistical test can comprise a one-way analysis of variance, two-way analysis of variance, or repeated measures analysis of variance.
  • statistical significance is determined using a nonparametric statistical test.
  • Examples include, but are not limited to, a Wilcox on signed- rank test, a Mann- Whitney test, a Kruskal-Wallis test, a Friedman test, a Spearman ranked order correlation coefficient, a Kendall Tau analysis, and a nonparametric regression test.
  • statistical significance is determined at a p-value of less than about 0.05, 0.01, 0.005, 0.001, 0.0005, or 0.0001.
  • the degree of differential expression can also be taken into account.
  • ETVl can be considered as differentially expressed when the fold-change in expression compared to control level is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 2.7, 3.0, 4, 5, 6, 7, 8, 9 or 10-fold different in the sample versus the control.
  • the differential expression takes into account both overexpression and underexpression.
  • a gene or gene product can be considered up or downregulated if the differential expression meets a statistical threshold, a fold- change threshold, or both.
  • the criteria for identifying differential expression can comprise both a p-value of 0.001 and fold change of at least 2.0-fold (up or down).
  • the reference to which differential expression of ETVl is determined is a normal gene.
  • the reference may be a housekeeping gene such as 18S rRNA or GAPDH.
  • differential expression may be determined relative to ETVl expression in noncancerous gastrointestinal ICCs derived from healthy subjects.
  • differential expression may be determined relative to ETVl expression in cancerous ICCs cells or GIST cell lines. Fold changes in ETVl expression may be calculated by the delta-delta Ct method (M.W. Pfaffl, A new mathematical model for relative quantification in real-time RT-PCR, Nucleic Acids Res. 29 (2001) e45) or other methods known to those of skill in the art.
  • the invention provides a method of determining ETVl mRNA expression, polypeptide levels or activity and correlating the results obtained therein with a diagnosis or prognostic assessement of GIST cancer and severity.
  • these methods include a step of detecting expression or activity of a gene encoding a ETVl polypeptide.
  • the gene products are detected in a tumor sample and can be polypeptides or polynucleotides, e.g., mRNA. a. Nucleic acid analysis
  • ETVl detection at the nucleic acid level is also encompassed by embodiments of the present invention.
  • Nucleic acid analyses can be performed on genomic DNA, messenger RNAs, and/or cDNA.
  • nucleic acids are extracted from a biological sample.
  • nucleic acids are analyzed without having been amplified.
  • nucleic acids are amplified using techniques known in the art (such as polymerase chain reaction (PCR)) and amplified nucleic acids are used in subsequent analyses. Multiplex PCR, in which several amplicons (e.g., from different genomic regions) are amplified at once using multiple sets of primer pairs, may be employed, (see generally, Bustin, S.A., 2000 J. Molecular Endocrinology 25: 169-193.)
  • Microarray analysis is one means by which polynucleotides can be used to detect or measure gene expression.
  • Expression of a gene can also be measured by a variety of techniques that make use of a polynucleotide corresponding to part or all of the gene rather than a binding agent for a polypeptide encoded by the gene.
  • Appropriate techniques include, but are not limited to, in situ hybridization, Northern blot, and various nucleic acid amplification techniques such as PCR, rtPCR, quantitative rtPCR, and the ligase chain reaction. Quantitative rtPCR and in situ hybridization technique are discussed in further detail in the examples below.
  • PCR and considerations for primer design are well known in the art and are described, for example, in Newton, et al. (eds.) PCR: Essential data Series, John Wiley & Sons; PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1995; White, et al.. (eds.) PCR Protocols: Current methods and Applications, Methods in Molecular Biology, The Humana Press, Totowa, NJ, 1993. b. Polypeptide analysis
  • a polypeptide may be detected using any of a variety of techniques and binding agents. Any such technique and agent may be used according to the present invention.
  • the binding agent is an antibody that binds specifically to ETV1 or KIT.
  • the invention also encompasses the use of protein arrays, including antibody arrays, for detection of a polypeptide. The use of antibody arrays is described, for example, in Haab et al., "Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions", Genome Biol. 2(2):2001, 2001. Other types of protein arrays are known in the art.
  • antibodies that bind specifically to ETV1 or KIT may be generated by methods well known in the art and described, for example, in Harlow, E, Lane, E, and Harlow, E, (eds.) Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1998. Details and references for the production of antibodies may also be found in U.S. Patent No. 6,008,337.
  • Antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric (e.g.,
  • the invention encompasses the use of "fully human” antibodies produced using the XenoMouseTM technology (AbGenix Corp., Fremont, CA) according to the techniques described in U.S. Patent No. 6,075,181.
  • Polyclonal ETV1 antibodies are commercially available (e.g., rabbit polyclonal antibodies ab81806 from Abeam of Cambridge, MA and PRB-326C from Covance).
  • Antibody detection methods are well known in the art including, but are not limited to, enzyme-linked immunosorbent assays (ELISAs) and Western blots. Some such methods are amenable to being performed in an array format.
  • polypeptides are detected using other specific binding agents known in the art for the detection of polypeptides, such as aptamers (Aptamers, Molecular Diagnosis, Vol. 4, No. 4, 1999), reagents derived from combinatorial libraries for specific detection of proteins in complex mixtures, random peptide affinity reagents, etc.
  • aptamers Adamers, Molecular Diagnosis, Vol. 4, No. 4, 1999
  • reagents derived from combinatorial libraries for specific detection of proteins in complex mixtures random peptide affinity reagents, etc.
  • any appropriate binding agent for detecting a polypeptide may be used in conjunction with the present invention, although antibodies may represent a particularly appropriate modality.
  • a single binding agent e.g., antibody
  • multiple binding agents directed either against the same or against different polypeptides (e.g., ETV1 or KIT) can be used to increase the sensitivity or specificity of the detection technique or to provide more detailed information than that provided by a single binding agent.
  • the invention encompasses the use of a battery of binding agents that bind to polypeptides encoded by the marker genes identified herein.
  • these agents can also be used in conjunction with binding agents against polypeptides encoded by other useful marker genes (e.g., CD34 when used with GISTs).
  • the polypeptides are detected within a tumor sample that has been obtained from a subject, e.g., a tissue sample, cell sample, cell extract, body fluid sample, etc.
  • a subject e.g., a tissue sample, cell sample, cell extract, body fluid sample, etc.
  • the invention encompasses the recognition that polypeptides encoded by EDTV1 at various levels (optionally in conjuction with KIT polypeptides) may be present in serum, enabling their detection through a blood test rather than requiring a biopsy specimen.
  • One of ordinary skill in the art will readily be able to develop appropriate assays for polypeptides encoded by the marker genes described herein and to apply them to the detection of such polypeptides in serum. Similar methods may be applied to other body fluid samples, e.g., ascites, urine, saliva, etc.
  • binding can be detected by adding a detectable label to the binding agent.
  • binding can be detected by using a labeled secondary binding agent that associates specifically with the primary binding agent, e.g., as is well known in the art of antigen/antibody detection.
  • the detectable label may be directly detectable or indirectly detectable, e.g., through combined action with one or more additional members of a signal producing system. Examples of directly detectable labels include radioactive,
  • paramagnetic, fluorescent, light scattering, absorptive and colorimetric labels include chemiluminescent labels, e.g., enzymes that are capable of converting a substrate to a chromogenic product such as alkaline phosphatase, horseradish peroxidase and the like.
  • the complex may be visualized or detected in a variety of ways, with the particular manner of detection being chosen based on the particular detectable label.
  • Representative detection means include, e.g., scintillation counting, autoradiography, measurement of paramagnetism, fluorescence measurement, light absorption measurement, measurement of light scattering and the like.
  • IHC immunohistochemistry
  • ELISA ELISA
  • FACS fluorescence activated cell sorting
  • the detection techniques of the present invention will include a negative control, which can involve applying the test to a control sample (e.g., from a normal non-cancerous tissue) so that the signal obtained thereby can be compared with the signal obtained from the tumor sample being tested.
  • a control sample e.g., from a normal non-cancerous tissue
  • an appropriate negative control can involve performing the test on a portion of the sample with the omission of the primary binding agent.
  • the results of the inventive detection techniques can be presented in any of a variety of formats.
  • the results can be presented in a qualitative fashion.
  • the test report may indicate only whether or not a particular polypeptide marker was detected, perhaps also with an indication of the limits of detection.
  • the results may be presented in a semiquantitative fashion.
  • various ranges may be defined, and the ranges may be assigned a score (e.g., 0 to 3 where 0 means no binding detected and 3 means strong binding detected) that provides a certain degree of quantitative information.
  • a score may reflect various factors, e.g., the number of cells in which the polypeptide is detected, the intensity of the signal (which may indicate the level of expression of the polypeptide), etc.
  • results may be presented in a quantitative fashion, e.g., as a percentage of cells in which the polypeptide is detected, as a protein concentration, etc.
  • the type of output provided by a test will vary depending upon the technical limitations of the test and the biological significance associated with detection of the polypeptide. For example, in the case of one polypeptide marker a purely qualitative output (e.g., whether or not the polypeptide is detected at a certain detection level) provides significant information. In another case a more quantitative output (e.g., a ratio of the level of expression of the polypeptide in the sample being tested versus the normal level) is necessary.
  • Detectable moieties e.g., a purely qualitative output (e.g., whether or not the polypeptide is detected at a certain detection level) provides significant information.
  • a more quantitative output e.g., a ratio of the level of expression of the polypeptide in the sample being tested versus the normal level
  • certain molecules used in accordance with and/or provided by the invention comprise one or more detectable entities or moieties, i.e., such molecules are "labeled" with such entities or moieties.
  • detectable agents include, but are not limited to: various ligands, radionuclides; fluorescent dyes; chemiluminescent agents (such as, for example, acridinum esters, stabilized dioxetanes, and the like); bioluminescent agents; spectrally resolvable inorganic fluorescent semiconductors nanocrystals (i.e., quantum dots); microparticles; metal nanoparticles (e.g., gold, silver, copper, platinum, etc.); nanoclusters; paramagnetic metal ions; enzymes; colorimetric labels (such as, for example, dyes, colloidal gold, and the like); biotin; dioxigenin; haptens; and proteins for which antisera or monoclonal antibodies are available.
  • chemiluminescent agents such as, for example, acridinum esters, stabilized dioxetanes, and the like
  • bioluminescent agents spectrally resolvable inorganic fluorescent semiconductors nanocrystals
  • the detectable moiety is biotin.
  • Biotin can be bound to avidins (such as streptavidin), which are typically conjugated (directly or indirectly) to other moieties (e.g., fluorescent moieties) that are detectable themselves.
  • a detectable moiety is a fluorescent dye.
  • Numerous known fluorescent dyes of a wide variety of chemical structures and physical characteristics are suitable for use in the practice of the present invention.
  • a fluorescent detectable moiety can be stimulated by a laser with the emitted light captured by a detector.
  • the detector can be a charge- coupled device (CCD) or a confocal microscope, which records its intensity.
  • Suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'- dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., carboxytetramethyl- rhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine Red, tetramethylrhodamine (TMR), etc.
  • hydroxycoumarin, aminomethylcoumarin AMCA
  • Oregon Green Dyes e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514., etc.
  • Texas Red Texas Red-X
  • SPECTRUM REDTM SPECTRUM GREENTM
  • cyanine dyes e.g., CY-3TM, CY-5TM, CY-3.5TM, CY- 5.5TM, etc.
  • ALEXA FLUORTM dyes e.g., ALEXA FLUORTM 350, ALEXA FLUORTM 488, ALEXA FLUORTM 532, ALEXA FLUORTM 546, ALEXA FLUORTM 568, ALEXA FLUORTM 594, ALEXA FLUORTM 633, ALEXA FLUORTM 660, ALEXA FLUORTM 680, etc.
  • BODIPYTM dyes e.g., BODIPYTM FL, BODIPYTM R6G, BOD
  • BODIPYTM TR BODIPYTM TR, BODIPYTM 530/550, BODIPYTM 558/568, BODIPYTM 564/570,
  • IRDyes e.g., IRD40, IRD 700, IRD 800, etc.
  • suitable fluorescent dyes and methods for coupling fluorescent dyes to other chemical entities such as proteins and peptides, see, for example, "The Handbook of Fluorescent Probes and Research Products", 9th Ed., Molecular Probes, Inc., Eugene, OR.
  • Favorable properties of fluorescent labeling agents include high molar absorption coefficient, high fluorescence quantum yield, and photostability.
  • labeling fluorophores exhibit absorption and emission wavelengths in the visible (i.e., between 400 and 750 nm) rather than in the ultraviolet range of the spectrum (i.e., lower than 400 nm).
  • a detectable moiety may include more than one chemical entity such as in fluorescent resonance energy transfer (FRET). Resonance transfer results an overall enhancement of the emission intensity. For instance, see Ju et. al. (1995) Proc. Nat'l Acad. Sci. (USA ) 92: 4347, the entire contents of which are herein incorporated by reference.
  • the first fluorescent molecule absorbs light and transfers it through the resonance of excited electrons to the second fluorescent molecule (the “acceptor” fluor).
  • both the donor and acceptor dyes can be linked together and attached to the oligo primer. Methods to link donor and acceptor dyes to a nucleic acid have been described previously, for example, in U.S. Pat. No. 5,945,526 to Lee et al., the entire contents of which are herein incorporated by reference.
  • Donor/acceptor pairs of dyes that can be used include, for example, fluorescein/tetramethylrohdamine, IAEDANS/fluroescein, EDANS/DABCYL, fluorescein/fluorescein, BODIPY FL/BODIPY FL, and Fluorescein/ QSY 7 dye. See, e.g., U.S. Pat. No. 5,945,526 to Lee et al. Many of these dyes also are commercially available, for instance, from Molecular Probes Inc. (Eugene, Oreg.).
  • Suitable donor fluorophores include 6- carboxyfluorescein (FAM), tetrachloro-6-carboxyfluorescein (TET), 2'-chloro-7'-phenyl-l,4- dichloro-6-carboxyfluorescein (VIC), and the like.
  • FAM 6- carboxyfluorescein
  • TET tetrachloro-6-carboxyfluorescein
  • VIC 2'-chloro-7'-phenyl-l,4- dichloro-6-carboxyfluorescein
  • a detectable moiety is an enzyme.
  • suitable enzymes include, but are not limited to, those used in an ELISA, e.g., horseradish peroxidase, beta-galactosidase, lucif erase, alkaline phosphatase, etc..
  • Other examples include beta- glucuronidase, beta-D-glucosidase, urease, glucose oxidase, etc.
  • An enzyme may be conjugated to a molecule using a linker group such as a carbodiimide, a diisocyanate, a glutaraldehyde, and the like.
  • a detectable moiety is a radioactive isotope.
  • a molecule may be isotopically-labeled (i.e., may contain one or more atoms that have been replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature) or an isotope may be attached to the molecule.
  • Non- limiting examples of isotopes that can be incorporated into molecules include isotopes of hydrogen, carbon, fluorine, phosphorous, copper, gallium, yttrium, technetium, indium, iodine, rhenium, thallium, bismuth, astatine, samarium, and lutetium (i.e., 3H, 13C, 14C, 18F, 19F, 32P, 35S, 64Cu, 67Cu, 67Ga, 90Y, 99mTc, l l lln, 1251, 1231, 1291, 1311, 1351, 186Re, 187Re, 201T1, 212Bi, 213Bi, 211At, 153Sm, 177Lu).
  • isotopes of hydrogen, carbon, fluorine, phosphorous, copper, gallium, yttrium, technetium, indium, iodine, rhenium, thallium, bismuth,
  • signal amplification is achieved using labeled dendrimers as the detectable moiety (see, e.g., Physiol Genomics 3:93-99, 2000), the entire contents of which are herein incorporated by reference.
  • Fluorescently labeled dendrimers are available from Genisphere (Montvale, NJ). For example, these may be chemically conjugated to nucleic acid primers by methods known in the art. d. Detection of mutations
  • the invention also encompasses the detection of mutations within ETV1.
  • detection of mutations can be used to classify the severity of mutation and, therefore, the prognosis of the subject in response to coventional or novel treatments disclosed herein.
  • Mutations may include, but are not limited to, deletions, additions, substitutions, and amplification of regions of genomic DNA that include all or part of a gene. Methods for detecting such mutations are well known in the art and include direct sequencing, denaturing HPLC and combinations thereof. (Segal, N. H. et ah, 2003, Am J Pathol, 163: 691- 700, incorporated herein by reference; Visel, A.
  • Mutations may result in overexpression or inappropriate expression of ETV1 (i.e., aberrant expression). Additionally or alternatively mutations may result in an overly activated gene product (e.g., polypeptide).
  • mutation analysis of particular ETV1- relevant genes or proteins may provide further diagnostic or prognostic information. For example, diagnostic KIT mutations have been identified within mutations in ex on 9, 11, 13 or
  • ETV1 mutations may be analyzed by the exemplary methods below. i. Allele-specific amplification
  • gene mutations are detected using an allele-specific amplification assay.
  • This approach is variously referred to as PCR amplification of specific allele (PASA) (Sarkar, et al., 1990 Anal. Biochem. 186:64-68), allele-specific amplification (ASA) (Okayama, et al., 1989 J. Lab. Clin. Med. 114: 105-113), allele-specific PCR (ASPCR) (Wu, et al. 1989 Proc. Natl. Acad. Sci. USA. 86:2757-2760), and amplification-refractory mutation system (ARMS) (Newton, et al., 1989 Nucleic Acids Res. 17:2503-2516).
  • PASA PCR amplification of specific allele
  • ASPCR allele-specific PCR
  • ARMS amplification-refractory mutation system
  • amplification primers may be designed such that they can distinguish between different alleles (e.g., between a wild-type allele and a mutant allele).
  • the presence or absence of amplification product can be used to determine whether a gene mutation is present in a given nucleic acid sample.
  • allele specific primers can be designed such that the presence of amplification product is indicative of a gene mutation.
  • allele specific primers can be designed such that the absence of amplification product is indicative of a gene mutation.
  • two complementary reactions are used.
  • One reaction employs a primer specific for the wild type allele ("wild-type-specific reaction") and the other reaction employs a primer for the mutant allele (“mutant-specific reaction”).
  • the two reactions may employ a common second primer.
  • PCR primers specific for a particular allele e.g., the wild- type allele or mutant allele
  • the mismatch may be located at/near the 3' end of the primer, leading to preferential amplification of the perfectly matched allele. Whether an amplification product can be detected from one or in both reactions indicates the absence or presence of the mutant allele.
  • Detection of an amplification product only from the wild-type-specific reaction indicates presence of the wild-type allele only (e.g., homozygosity of the wild-type allele).
  • Detection of an amplification product in the mutant- specific reaction only indicates presence of the mutant allele only (e.g. homozygosity of the mutant allele).
  • Detection of amplification products from both reactions indicate (e.g., a heterozygote).
  • this approach will be referred to as "allele specific amplification (ASA)."
  • Allele-specific amplification can also be used to detect duplications, insertions, or inversions by using a primer that hybridizes partially across the junction.
  • the extent of junction overlap can be varied to allow specific amplification.
  • Amplification products can be examined by methods known in the art, including by visualizing (e.g., with one or more dyes) bands of nucleic acids that have been migrated (e.g., by electrophoresis) through a gel to separate nucleic acids by size.
  • visualizing e.g., with one or more dyes
  • bands of nucleic acids that have been migrated e.g., by electrophoresis
  • electrophoresis e.g., electrophoresis
  • an allele-specific primer extension (ASPE) approach is used to detect gene mutations.
  • ASPE employs allele-specific primers that can distinguish between alleles (e.g., between a mutant allele and a wild-type allele) in an extension reaction such that an extension product is obtained only in the presence of a particular allele (e.g., mutant allele or wild-type allele).
  • Extension products may be detectable or made detectable, e.g., by employing a labeled deoxynucleotide in the extension reaction. Any of a variety of labels are compatible for use in these methods, including, but not limited to, radioactive labels, fluorescent labels, chemiluminescent labels, enzymatic labels, etc.
  • a nucleotide is labeled with an entity that can then be bound (directly or indirectly) by a detectable label, e.g., a biotin molecule that can be bound by streptavidin-conjugated fluorescent dyes.
  • a detectable label e.g., a biotin molecule that can be bound by streptavidin-conjugated fluorescent dyes.
  • reactions are done in multiplex, e.g., using many allele-specific primers in the same extension reaction.
  • extension products are hybridized to a solid or semi-solid support, such as beads, matrix, gel, among others.
  • the extension products may be tagged with a particular nucleic acid sequence (e.g., included as part of the allele-specific primer) and the solid support may be attached to an "anti-tag" (e.g., a nucleic acid sequence
  • Extension products can be captured and detected on the solid support. For example, beads may be sorted and detected.
  • LUMINEXTM MAP system which can be adapted for cystic fibrosis mutation detection by TM Bioscience and is sold commercially as a universal bead array (TAG-ITTM).
  • a single nucleotide primer extension (SNuPE) assay is used, in which the primer is designed to be extended by only one nucleotide.
  • the identity of the nucleotide just downstream (e.g., 3') of the 3' end of the primer is known and differs in the mutant allele as compared to the wild-type allele.
  • SNuPE can be performed using an extension reaction in which the only one particular kind of deoxynucleotide is labeled (e.g., labeled dATP, labeled dCTP, labeled dGTP, or labeled dTTP).
  • the presence of a detectable extension product can be used as an indication of the identity of the nucleotide at the position of interest (e.g., the position just downstream of the 3' end of the primer), and thus as an indication of the presence or absence of a mutation at that position.
  • SNuPE can be performed as described in U.S. Pat. No. 5,888,819; U.S. Pat. No. 5,846,710; U.S. Pat. No. 6,280,947; U.S. Pat. No. 6,482,595; U.S. Pat. No. 6,503,718; U.S. Pat. No. 6,919,174; ; Piggee, C. et al. Journal of Chromatography A 781 (1997), p.
  • primer extension can be combined with mass spectrometry for accurate and fast detection of the presence or absence of a mutation. See, U.S. Pat. No.
  • Suitable mass spectrometric format includes, but is not limited to, Matrix-Assisted Laser Desorption/Ionization, Time-of- Flight (MALDI-TOF), Electrospray (ES), IR-MALDI, Ion Cyclotron Resonance (ICR), Fourier Transform, and combinations thereof.
  • MALDI-TOF Time-of- Flight
  • ES Electrospray
  • IR-MALDI Ion Cyclotron Resonance
  • ICR Ion Cyclotron Resonance
  • an oligonucleotide ligation assay (“OLA” or “OL”) is used.
  • OLA employs two oligonucleotides that are designed to be capable of hybridizing to abutting sequences of a single strand of a target molecules.
  • one of the oligonucleotides is biotinylated, and the other is detectably labeled, e.g., with a streptavidin-conjugated fluorescent moiety. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate that can be captured and detected.
  • nucleic acids are analyzed by hybridization using one or more oligonucleotide probes specific for a region in the gene corresponding to one or more ETV1 mutations, and under conditions sufficiently stringent to disallow a single nucleotide mismatch.
  • suitable nucleic acid probes can distinguish between a normal gene and a mutant gene containing one or more mutations. For example, suitable nucleic acid probes specifically bind to a normal gene but not to a mutant gene. Alternatively, nucleic acid probes specifically bind to an ETV1 mutant gene containing one or more mutations but not to a normal gene.
  • Probes of the present invention include those that are capable of specifically hybridizing a mutant ETV1 allele containing one or more mutations. Probes of the present invention also include those that are capable of specifically hybridizing a normal allele in a particular region of the ETV1 gene and therefore capable of distinguishing a normal allele from a mutant ETV1 allele. Thus, for example, one of ordinary skill in the art could use probes of the invention to determine whether an individual is homozygous or heterozygous for a particular allele.
  • nucleic acid hybridization techniques are well known in the art. See, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions such that sequences having at least a desired level of complementary will stably hybridize, while those having lower complementary will not. For examples of hybridization conditions and parameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press,
  • Nucleic acid probes may comprise ribonucleic acids and/or deoxyribonucleic acids.
  • provided nucleic acid probes are oligonucleotides (i.e., "oligonucleotide probes").
  • oligonucleotide probes are long enough to bind specifically to a homologous region of the gene, but short enough such that a difference of one nucleotide between the probe and the nucleic acid sample being tested disrupts hybridization.
  • the sizes of oligonucleotide probes vary from approximately 10 to 100 nucleotides.
  • oligonucleotide probes vary from 15 to 90, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to 40, 15 to 35, 15 to 30, 18 to 30, or 18 to 26 nucleotides in length.
  • the optimal length of an oligonucleotide probe may depend on the particular methods and/or conditions in which the oligonucleotide probe may be employed.
  • nucleic acid probes are useful as primers, e.g., for nucleic acid amplification and/or extension reactions.
  • nucleic acid probes are labeled with a detectable moiety as described herein.
  • Another aspect of the invention makes use of therapeutic materials and methods for effecting repression of ETV1. For example, by preventing transcription, repressing mRNA transcript levels, inhibiting translation, or otherwise directly or indirectly interfering with the activity of ETV1, it is possible to inhibit growth of GIST cells without affecting growth of noncancerous cells. Similarly, treatment of overexpressed or otherwise aberrant ETV1 expression in cancerous ICC cells or GIST cells decreases cell cycle progession, increases apoptosis and decreases tumorigenicity. ETV1 expression or activity may be modulated either directly through interaction with the ETV1 gene, mRNA or protein itself, or indirectly through one or more upstream regulator or downstream targets of ETVl. For example, ETVl repression may modulated through KIT or PDGFRA receptor tyrosine kinases.
  • each of the therapies described herein may be directed specifically against ETVl.
  • approaches and therapies can be modified for combination therapies against ETVl and KIT and/or PDGFRA.
  • the present invention also encompasses therapies that interfere with the interaction of ETVl and KIT. a. Antibodies
  • Antibodies are increasingly used for therapeutic treatments of cancer, autoimmunity and infectious diseases. Many of these antibody therapies have shown considerable success, as evidence by the approval of more than 30 monoclonal antibody-based therapies in the past 25 years. Antibodies can be engineered to almost any extracellular or cell surface target. In a cancer treatment context, they may be used to stimulate an immune response against cancerous cells, to block or activate specific cell receptors involved in cell division or apoptosis, or to sensitize a cancer cell to chemotherapy. Antibodies may also be used to deliver radioisotopes or other toxic agents to cancerous cells or tumors.
  • the present invention encompass the use of therapeutic antibodies in the treatment of ETVl -mediated cancers, particularly GISTs.
  • the antibodies may be monoclonal or polyclonal, and may act either directly or indirectly against GIST.
  • inactivating antibodies against the KIT receptor indirectly affect ETVl levels. Inhibition of KIT signaling by such antibodies may result in rapid loss of ETVl protein through destabihzation of the ETVl protein.
  • KIT-MEK signaling stabilizes ETVl protein. Any inactivating or blocking antibodies against KIT are a potential therapy for ETVl -mediated oncogenesis.
  • Anti-ETVl antibodies suitable for the invention include antibodies or fragments of antibodies that bind immunospecifically to any ETVl epitopes.
  • the term "antibodies” is intended to include immunoglobulins and fragments thereof which are specifically reactive to the designated protein or peptide, or fragments thereof.
  • Suitable antibodies include, but are not limited to, human antibodies, primatized antibodies, chimeric antibodies, bi-specific antibodies, humanized antibodies, conjugated antibodies (i.e. , antibodies conjugated or fused to other proteins, radiolabels, cytotoxins), Small Modular
  • SMIPsTM ImmunoPharmaceuticals
  • antibodies also includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. , bi-specific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
  • an "antibody fragment” includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; single domain antibodies; diabodies; triabodies; tetrabodies; linear antibodies; single-chain antibody molecules; and multi specific antibodies formed from antibody fragments.
  • antibody fragment also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.
  • antibody fragments include isolated fragments, "Fv” fragments, consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker ("ScFv proteins”), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.
  • Fv fragments
  • ScFv proteins peptide linker
  • Antibodies can be generated using methods well known in the art. For example, protocols for antibody production are described by Harlow and Lane, Antibodies: A Laboratory Manual, (1988). Typically, antibodies can be generated in mouse, rat, guinea pig, hamster, camel, llama, shark, or other appropriate host. Alternatively, antibodies may be made in chickens, producing IgY molecules (Schade et al., (1996) ALTEX 13(5):80-85). In some embodiments, antibodies suitable for the present invention are subhuman primate antibodies. For example, general techniques for raising therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al., international patent publication No.
  • monoclonal antibodies may be prepared using hybridoma methods (Milstein and Cuello, (1983) Nature 305(5934):537-40.). In some embodiments, monoclonal antibodies may also be made by recombinant methods (U.S. Pat. No. 4,166,452).
  • antibodies suitable for the invention may include humanized or human antibodies.
  • Humanized forms of non-human antibodies are chimeric Igs, Ig chains or fragments (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of Abs) that contain minimal sequence derived from non-human Ig.
  • a humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain.
  • Humanization is accomplished by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody (Riechmann et al., Nature 332(6162):323-7, 1988; Verhoeyen et al., Science. 239(4847):1534-6, 1988.).
  • humanized antibodies are chimeric Abs (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent Abs.
  • Humanized antibodies include human Igs (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit, having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • Humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which most if not all of the CDR regions correspond to those of a non-human Ig and most if not all of the FR regions are those of a human Ig consensus sequence.
  • the humanized antibody optimally also comprises at least a portion of an Ig constant region (Fc), typically that of a human Ig (Riechmann et al., Nature 332(6162):323-7, 1988; Verhoeyen et al., Science 239(4847): 1534-6, 1988.).
  • Fc Ig constant region
  • Human antibodies can also be produced using various techniques, including phage display libraries (Hoogenboom et al., Mol Immunol. (1991) 28(9): 1027-37; Marks et al., J Mol Biol. (1991) 222(3):581-97) and the preparation of human monoclonal antibodies (Reisfeld and Sell, 1985, Cancer Surv. 4(l):271-90).
  • phage display libraries Hoogenboom et al., Mol Immunol. (1991) 28(9): 1027-37; Marks et al., J Mol Biol. (1991) 222(3):581-97
  • human monoclonal antibodies Reisfeld and Sell, 1985, Cancer Surv. 4(l):271-90.
  • introducing human Ig genes into transgenic animals in which the endogenous Ig genes have been partially or completely inactivated can be exploited to synthesize human antibodies.
  • human antibody production is observed, which closely resembles that seen in humans in all respects, including
  • Synthetic double-stranded oligodeoxynucleotides act as "decoy" cis elements to block the binding of transcription factors to promoter regions of target genes, resulting in the inhibition of gene transactivation in vitro and in vivo (see, e.g., Bielinska et al., (1990) Science 250:997- 1000). Furthermore, certain studies have described application of decoy strategy as in vivo gene therapy (see, e.g., Morishita et al., (1997) Nature Med. 8:894-899; and Kawamura et al. (1999) Gene Therapy 6:91-7).
  • Embodiments of the present invention may utilize promoter competition whereby nucleic acid sequences encoding ETVl cis-acting elements are introduced in high copy number into cells. These sequenes functions as decoys to competitively bind away ETVl from its natural enhancer sequences, with a corresponding suppression of the ETVl -regulated ICC-GIST- specific transcription network.
  • One embodiment of this approach utilizes oligonucleotides, modified to facilitate entry into cells that compete with the native cellular sequence for binding to ETVl.
  • Transcription factor decoys of the presently claimed invention are recognized and bound by ETVl such it can no longer bind to native response elements and regulate gene expression.
  • Decoys can comprise one or more duplex nucleic acid structures. These structures are recognized by the DNA binding domain of the ETVl.
  • the decoys can comprise one or more sequences of known ETVl binding sites. However, any sequence that has affinity for ETVl is suitable for use as a decoy and is contemplated by the presently claimed invention.
  • Embodiments of the invention include various decoy nucleic acid sequences including duplex, hairpin, and cruciform oligonucleotides containing one or more ETVl binding site, which, in certain aspects of the invention, inhibit cancerous cell growth without adversely or minimally affecting non-cancerous cells.
  • Nucleic acid sequences with at least 70% homology to known sequences may also be recognized and bound by ETVl. Thus a range of sequences may be effectively employed as decoys.
  • a pool of sequences identical or divergent to known ETVl binding sequences may be selected to generate therapeutic treatments of various potency.
  • the decoy sequences are palindromic cis-transcription elements comprising a synthetic single-stranded oligonucleotide composed one or more ETVl binding sites that capable of self-hybridizing to form a duplex or hairpin structure.
  • hairpin oligonucleotide decoys containing a duplex portion with one or more ETVl binding site may be introduced into cells and
  • cruciform ETVl -binding decoys may be constructed through standard genetic manipulations of known sequences. Such cruciform DNA can increase the potency of the transcription factor decoy to inhibit gene transcription. Similar DNA structures are known to be generated during genetic recombination and from palindromic sequences under the effect of supercoiling, indicating a biological role for such structures. c. Dominant negative therapy
  • Embodiments of present invention further encompass dominant negative derivatives of ETVl that are capable of interfering with transcription factor function of native ETVl.
  • Such dominant negative versions of transcription factors are known to those of skill in the art, and readily created by deleting the transcriptional activation domain.
  • U.S. Pat. No. 5,869,040 describes dominant negative forms of the nuclear proto-oncogene E2F transcription factor protein and methods of their use to treat cancer.
  • Various ETVl -derived dominant negative proteins may be derived so long as they maintain sequence-specific DNA binding capabilities.
  • Dominant interfering mutations may be made to either the DNA binding domain or the activation domains.
  • Dominant-negative ETVl mutatnts are known in the art (Shin et al., J Cell Biochem. 2008 Oct 15;105(3):866-74). d. Aptamer therapy
  • Aptamers are macromolecules composed of nucleic acid (e.g., RNA, DNA) that bind tightly to a specific molecular target (e.g., ETVl polypeptide or an epitope thereof).
  • a particular aptamer may be described by a linear nucleotide sequence and is typically about 15-60 nucleotides in length. Without wishing to be bound by any theories, it is contemplated that the chain of nucleotides in an aptamer form intramolecular interactions that fold the molecule into a complex three-dimensional shape, and this three-dimensional shape allows the aptamer to bind tightly to the surface of its target molecule.
  • aptamers may be obtained for a wide array of molecular targets, including proteins and small molecules.
  • aptamers have very high affinities for their targets (e.g., affinities in the picomolar to low nanomolar range for proteins).
  • Aptamers are chemically stable and can be boiled or frozen without loss of activity. Because they are synthetic molecules, they are amenable to a variety of modifications, which can optimize their function for particular applications. For example, aptamers can be modified to dramatically reduce their sensitivity to degradation by enzymes in the blood for use in in vivo applications. In addition, aptamers can be modified to alter their biodistribution or plasma residence time.
  • aptamers that can bind ETV1 or a fragment thereof can be achieved through methods known in the art.
  • aptamers can be selected using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method (Tuerk, C, and Gold, L., Science 249:505-510 (1990)).
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • a large library of nucleic acid molecules e.g., 1015 different molecules
  • the target molecule e.g., an ETV1 protein or an ETV1 epitope.
  • the target molecule is allowed to incubate with the library of nucleotide sequences for a period of time.
  • aptamers with the highest affinity for the target molecule can then be purified away from the target molecule and amplified enzymatically to produce a new library of molecules that is substantially enriched for aptamers that can bind the target molecule.
  • the enriched library can then be used to initiate a new cycle of selection, partitioning, and amplification. After 5-15 cycles of this iterative selection, partitioning and amplification process, the library is reduced to a small number of aptamers that bind tightly to the target molecule.
  • aptamers in the mixture can then be isolated, their nucleotide sequences determined, and their properties with respect to binding affinity and specificity measured and compared. Isolated aptamers can then be further refined to eliminate any nucleotides that do not contribute to target binding and/or aptamer structure, thereby producing aptamers truncated to their core binding domain. See Jayasena, S. D. Clin. Chem. 45: 1628-1650 (1999) for review of aptamer technology; the entire teachings of which are incorporated herein by reference. e. Antisense and interfering RNA therapy
  • Antisense molecules are RNA or single-stranded DNA molecules with nucleotide sequences complementary to a specified mRNA.
  • a laboratory-prepared antisense molecule is injected into cells containing the normal mRNA transcribed by a gene under study, the antisense molecule can base-pair with the mRNA, preventing translation of the mRNA into protein.
  • the resulting double-stranded RNA or RNA/DNA is digested by enzymes that specifically attach to such molecules. Therefore, a depletion of the mRNA occurs, blocking the translation of the gene product so that antisense molecules find uses in medicine to block the production of deleterious proteins.
  • Antisense molecules suitable for inhibiting ETV1 activity can be designed based on the sequences described above and known in the art.
  • the antisense molecules may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
  • these cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells, or tissues.
  • RNA molecules may be modified to increase intracellular stability and half-life.
  • flanking sequences at the 5' and/or 3' ends of the molecule Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2'O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • This concept can be extended by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
  • RNA interference is a mechanism of post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA), which is distinct from the antisense approach described above. dsRNA molecules are believed to direct sequence-specific degradation of mRNA in cells of various lineages after first undergoing processing by an RNase Ill-like enzyme called DICER (Bernstein et al., Nature 409:363, 2001) into smaller dsRNA molecules comprised of two 21 nucleotide strands, each of which has a 5' phosphate group and a 3' hydroxyl, and includes a 19 nucleotide region precisely complementary with the other strand, so that there is a 19 nucleotide duplex region flanked by 2 nucleotide 3' overhangs. RNAi is thus mediated by short interfering RNAs (siRNA), which typically comprise a double-stranded region
  • siRNAs can have a range of lengths, e.g., the double- stranded portion can range from 15-29 nucleotides. It will also be appreciated that the siRNA can have a blunt end or a 3' overhang at either or both ends. If present, such 3' overhang is often from 1 -5 nucleotides in length.
  • siRNA has been shown to downregulate gene expression when transferred into mammalian cells by such methods as transfection, electroporation, or microinjection, or when expressed in cells via any of a variety of plasmid-based approaches.
  • RNA interference using siRNA is reviewed in, e.g., Tuschl, T., Nat. Biotechnol., 20:446-448, May 2002. See also Yu, J., et al., Proc. Natl. Acad. Sci., 99(9), 6047-6052 (2002); Sui, G., et al., Proc. Nail. Acad. Sci., 99(8), 5515-5520 (2002); Paddison, P., et al., Genes and Dev., 16, 948-958 (2002);
  • RNAi in vivo inhibition of specific gene expression by RNAi has been achieved in various organisms including mammals.
  • Song et al., Nature Medicine, 9:347-351 (2003) discloses that intravenous injection of Fas siRNA compounds into laboratory mice with autoimmune hepatitis specifically reduced Fas mRNA levels and expression of Fas protein in mouse liver cells.
  • Several other approaches for delivery of siRNA into animals have also proved to be successful. See e.g., McCaffery et al., Nature, 418:38-39 (2002); Lewis et al., Nature Genetics, 32:107-108 (2002); and Xia et al., Nature Biotech., 20: 1006-1010 (2002).
  • the siRNA may consist of two individual nucleic acid strands or of a single strand with a self-complementary region capable of forming a hairpin (stem-loop) structure.
  • a hairpin stem-loop
  • a number of variations in structure, length, number of mismatches, size of loop, identity of nucleotides in overhangs, etc., are consistent with effective siRNA-triggered gene silencing. While not wishing to be bound by any theory, it is thought that intracellular processing (e.g., by DICER) of a variety of different precursors results in production of siRNA capable of effectively mediating gene silencing.
  • target exons rather than introns, and it may also be particularly desirable to select sequences complementary to regions within the 3' portion of the target transcript. Generally it is preferred to select sequences that contain approximately equimolar ratio of the different nucleotides and to avoid stretches in which a single residue is repeated multiple times.
  • siRNA may thus comprise RNA molecules typically having a double-stranded region approximately 19 nucleotides in length typically with 1-2 nucleotide 3' overhangs on each strand, resulting in a total length of between approximately 21 and 23 nucleotides.
  • siRNA also includes various RNA structures that may be processed in vivo to generate such molecules. Such structures include RNA strands containing two complementary elements that hybridize to one another to form a stem, a loop, and optionally an overhang, preferably a 3' overhang.
  • the stem is approximately 19 bp long, the loop is about 1-20, preferably about 4-10, and more preferably about 6-8 nucleotides long and/or the overhang is typically about 1-20, and preferably about 2-15 nucleotides long.
  • the stem is minimally 19 nucleotides in length and may be up to approximately 29 nucleotides in length. Loops of 4 nucleotides or greater are less likely subject to steric constraints than are shorter loops and therefore may be preferred.
  • the overhang may include a 5' phosphate and a 3' hydroxyl. The overhang may, but need not, comprise a plurality of U residues, e.g., between 1 and 5 U residues.
  • the siRNA compounds suitable for the present invention can be designed based on the ETV1 sequence described herein and can be synthesized using conventional RNA synthesis methods. For example, they can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Various applicable methods for RNA synthesis are disclosed in, e.g., Usman et al., J. Am. Chem. Soc, 109:7845- 7854 (1987) and Scaringe et al., Nucleic Acids Res, 18:5433-5441 (1990). Custom siRNA synthesis services are available from commercial vendors such as Ambion (Austin, Tex., USA), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (Rockford, 111., USA),
  • siRNAs may be comprised entirely of natural RNA nucleotides, or may instead include one or more nucleotide analogs and/or modifications as mentioned above for antisense molecules.
  • the siRNA structure may be stabilized, for example by including nucleotide analogs at one or more free strand ends in order to reduce digestion, e.g., by exonucleases. This may also be accomplished by the inclusion.
  • siRNA molecules may be generated by in vitro transcription of DNA sequences encoding the relevant molecule. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7, T3, or SP6.
  • the siRNA compounds can also be various modified equivalents of the siRNA structures.
  • modified equivalent means a modified form of a particular siRNA compound having the same target-specificity (i.e., recognizing the same mRNA molecules that complement the unmodified particular siRNA compound).
  • a modified equivalent of an unmodified siRNA compound can have modified ribonucleotides, that is, ribonucleotides that contain a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate (or phospodiester linkage).
  • an "unmodified ribonucleotide” has one of the bases adenine, cytosine, guanine, and uracil joined to the ⁇ carbon of beta-D-ribo- furanose.
  • Modified siRNA compounds contain modified backbones or non-natural amino acids
  • internucleoside linkages e.g., modified phosphorous-containing backbones and non- phosphorous backbones such as morpholino backbones; siloxane, sulfide, sulfoxide, sulfone, sulfonate, sulfonamide, and sulfamate backbones; formacetyl and thioformacetyl backbones; alkene-containing backbones; methyleneimino and methylenehydrazino backbones; amide backbones, and the like.
  • modified phosphorous-containing backbones and non- phosphorous backbones such as morpholino backbones
  • siloxane sulfide, sulfoxide, sulfone, sulfonate, sulfonamide, and sulfamate backbones
  • formacetyl and thioformacetyl backbones alkene-containing backbones
  • siRNA may be generated by intracellular transcription of small RNA molecules, which may be followed by intracellular processing events. For example, intracellular transcription is achieved by cloning siRNA templates into RNA polymerase III transcription units, e.g., under control of a U6 or HI promoter. In one approach, sense and antisense strands are transcribed from individual promoters, which may be on the same construct. The promoters may be in opposite orientation so that they drive transcription from a single template, or they may direct synthesis from different templates. In a second approach siRNAs are expressed as stem-loop structures.
  • the siRNAs of the invention may be introduced into cells by any of a variety of methods. For instance, siRNAs or vectors encoding them can be introduced into cells via conventional transformation or transfection techniques. As used herein, the terms
  • transformation and “transfection” are intended to refer to a variety of aft-recognized techniques for introducing foreign nucleic acid (e.g., DNA or RNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, injection, or electroporation.
  • foreign nucleic acid e.g., DNA or RNA
  • inventive vectors that direct in vivo synthesis of siRNA constitutively or inducibly can be introduced into cell lines, cells, or tissues.
  • inventive vectors are gene therapy vectors (e.g., adenoviral vectors, adeno-associated viral vectors, retroviral or lentiviral vectors, or various nonviral gene therapy vectors) appropriate for the delivery of an siRNA-expressing construct to mammalian cells, most preferably human cells.
  • gene therapy vectors e.g., adenoviral vectors, adeno-associated viral vectors, retroviral or lentiviral vectors, or various nonviral gene therapy vectors
  • the present invention includes gene therapy approaches to the treatment of diseases or clinical conditions associated with inflammation in, for example, airway (e.g., airway hyperresponsiveness), digestive, pulmonary or reproductive tract.
  • the invention includes methods of treating a disease or clinical condition associated with elevated ETV1 expression in, for example, GIST by administering siRNA compositions comprising siRNA that targets ETV1.
  • the compositions may be administered parenterally, orally, inhalationally, etc.
  • siRNA compositions reduce the level of the target transcript and its encoded protein by at least 2-fold, preferably at least 4-fold, more preferably at least 10-fold or more.
  • the ability of a candidate siRNA to reduce expression of the target transcript and/or its encoded protein may readily be tested using methods well known in the art including, but not limited to, Northern blots, RT-PCR, microarray analysis in the case of the transcript, and various immunological methods such as Western blot, ELISA, immunofluorescence, etc., in the case of the encoded protein. Efficacy may be tested in appropriate animal models or in human subjects.
  • siRNA compounds may be administered to mammals by various methods through different routes. For example, they can be administered by intravenous injection. See Song et al., Nature Medicine, 9:347-351 (2003). They can also be delivered directly to a particular organ or tissue by any suitable localized administration methods. Several other approaches for delivery of siRNA into animals have also proved to be successful. See e.g., McCaffery et al., Nature, 418:38-39 (2002); Lewis et al., Nature Genetics, 32: 107-108 (2002); and Xia et al., Nature Biotech., 20: 1006-1010 (2002).
  • they may be delivered encapsulated in liposomes, by iontophoresis, or by incorporation into other vehicles such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.
  • siRNA compounds in e.g., small hairpin form (shRNA)
  • shRNA small hairpin form
  • Numerous studies have demonstrated that while double-stranded siRNAs are very effective at mediating RNAi, short, single-stranded, hairpin-shaped RNAs can also mediate RNAi, presumably because they fold into intramolecular duplexes that are processed into double-stranded siRNAs by cellular enzymes.
  • the RNAi caused by the encoded shRNAs can be made stable and heritable. Not only have such techniques been used to "knock down” the expression of specific genes in mammalian cells, but they have now been successfully employed to knock down the expression of exogenously expressed transgenes, as well as endogenous genes in the brain and liver of living mice. See generally Hannon, Nature. 418:244- 251 (2002) and Shi, Trends Genet, 19:9-12 (2003); see also Xia et al., Nature Biotech., 20: 1006- 1010 (2002).
  • siRNA compounds targeted at different sites of the nucleic acids encoding one or more interacting protein members of a protein complex identified in the present invention may also be designed and synthesized according to general guidelines provided herein and generally known to skilled artisans. See e.g., Elbashir, et al. (Nature 411: 494-498 (2001). For example, guidelines have been compiled into "The siRNA User Guide” which is available at the website of The Rockefeller University, New York, NY.
  • Small molecule modulators e.g., inhibitors or activators
  • Small molecule modulators of gene expression are also within the scope of the invention and may be detected by screening libraries of compounds using, for example, cell lines that express a ETV1 polypeptide or a version of a ETV1 polypeptide that has been modified to include a readily detectable moiety. Methods for identifying compounds capable of modulating gene expression are described, for example, in U.S. Patent No. 5,976,793.
  • the invention encompasses compounds that modulate ETV1 activity.
  • Methods of screening for such interacting compounds are well known in the art and depend, to a certain degree, on the particular properties and activities of the polypeptide encoded by the gene. Representative examples of such screening methods may be found, for example, in U.S. Patent No. 5,985,829, U.S. Patent No. 5,726,025, U.S. Patent No. 5,972,621, and U.S. Patent No.
  • the invention encompasses methods of screening for molecules that modulate the activity of a polypeptide encoded by a marker gene, particularly the ETV1 gene. g. Vaccination
  • the invention also encompasses the use of polynucleotide sequences corresponding to ETV1, or portions thereof, as DNA vaccines.
  • DNA vaccines comprise polynucleotide sequences, typically inserted into vectors, that direct the expression of an antigenic polypeptide within the body of the individual being immunized. Details regarding the development of vaccines, including DNA vaccines for various forms of cancer may be found, for example, in Brinckerhoff LH, Thompson LW, Slingluff CL, Melanoma Vaccines, Curr. Opin. Oncol, 12(2): 163-73, 2000 and in Stevenson FK, DNA vaccines against cancer: from genes to therapy, Ann. Oncol., 10(12): 1413-8, 1999 and references cited therein.
  • the polypeptides, or fragments thereof, that are encoded by marker genes may also find use as cancer vaccines. Such vaccines may be used for the prevention and/or treatment of cancer.
  • the invention includes pharmaceutical compositions comprising the inventive antibodies, polynucleotides, small molecules, etc. described above.
  • Pharmaceutical compositions comprising the inventive antibodies, polynucleotides, small molecules, etc. described above.
  • compositions may optionally comprise one or more additional therapeutically-active substances.
  • compositions are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation.
  • Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys.
  • Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • a pharmaceutical composition in accordance with the invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • compositions in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • compositions may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • a pharmaceutically acceptable excipient includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.
  • an excipient is approved for use in humans and for veterinary use.
  • an excipient is approved by United States Food and Drug Administration.
  • an excipient is pharmaceutical grade.
  • an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British
  • compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.
  • Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs.
  • liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example,
  • oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents.
  • compositions are mixed with solubilizing agents such an CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents.
  • Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution.
  • Sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • Fatty acids such as oleic acid can be used in the preparation of injec tables.
  • Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • the rate of drug release can be controlled.
  • biodegradable polymers include poly(orthoesters) and poly( anhydrides).
  • Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
  • compositions for rectal administration are typically suppositories which can be prepared by mixing compositions with suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
  • suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and/or fillers or extenders (e.g. starches, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g. glycerol), disintegrating agents (e.g.
  • the dosage form may comprise buffering agents.
  • solution retarding agents e.g. paraffin
  • absorption accelerators e.g. quaternary ammonium compounds
  • wetting agents e.g. cetyl alcohol and glycerol monostearate
  • absorbents e.g. kaolin and bentonite clay
  • lubricants e.g. talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate
  • the dosage form may comprise buffering agents.
  • Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or
  • embedding compositions which can be used include polymeric substances and waxes.
  • Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • inventive antibodies, polynucleotides, small molecules, etc. described above and/or pharmaceutical compositions thereof may be administered to a subject using any amount and any route of administration effective for treating a disease, disorder, and/or condition (e.g., GIST).
  • a disease, disorder, and/or condition e.g., GIST
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular composition, its mode of administration, its mode of activity, and the like.
  • Compositions in accordance with the invention are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
  • compositions in accordance with the present invention may be administered by any route.
  • pharmaceutical compositions are provided by any route.
  • pharmaceutical compositions are provided by any route.
  • routes including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (e.g. by powders, ointments, creams, gels, lotions, and/or drops), mucosal, nasal, buccal, enteral, vitreal, intratumoral, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; as an oral spray, nasal spray, and/or aerosol, and/or through a portal vein catheter.
  • routes including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (e.g. by powders, ointments, creams, gels, lotions, and/or drops), mucosal, nasal, buc
  • the most appropriate route of administration will depend upon a variety of factors including the nature of the active ingredient, the condition of the patient (e.g., whether the patient is able to tolerate particular routes of administration), etc.
  • the invention encompasses the delivery of the pharmaceutical compositions by any appropriate route taking into consideration likely advances in the sciences of drug delivery. 7. Therapy selection
  • Another aspect of the invention relates to the selection of a treatment regimen based on ETVl over-expression or particular activating mutations. For example, it is known in the art that certain anti-cancer theraperies, particularly chemotherapy and small-molecule
  • the present invention provides methods of identifying patients with aberrant ETVl expression who may benefit from one therapy over another.
  • therapy may be personalized based on over-expression versus mutation, and sub-categorized based on the type of ETVl mutation and/or other abberations with known GIST-related proteins such as KIT or PDGFRA.
  • kits to test for the presence of ETVl in a tumor sample can comprise, for example, an antibody for detection of a polypeptide or a probe for detection of a polynucleotide.
  • the kit can comprise a reference or control sample, instructions for processing samples, performing the test and interpreting the results, buffers and other reagents necessary for performing the test.
  • the kit comprises one or more antibodies (monoclonal or polyclonal) for ETVl. In some embodiments monoclonal antibodies are preferred.
  • the kit comprises a panel of antibodies, e.g., for ETVl and KIT; for ETVl and PDGFRA; or for ETVl, KIT and PDGFRA.
  • the kit comprises pairs of primers or probes for detecting expression of one or more of the marker genes of the invention, e.g., for ETVl and KIT; for ETVl and PDGFRA; or for ETVl, KIT and PDGFRA.
  • the kit comprises a cDNA or oligonucleotide array for detecting expression of one or more of the marker genes of the invention.
  • Example 1 ETV1 as a diagnostic, prognostic and therapeutic target for Gastrointestinal stromal tumours or GIST
  • the present Example describes how the ETS family member ETV1 is highly expressed in the subtypes of ICCs sensitive to oncogenic KIT mediated transformation (Kwon, J. G. et al., 2009, Gastroenterology, 136: 630-639; incorporated herein by reference), and is required for their development.
  • the present Example describes how ETV1 expression can be used as a diagnostic, prognostic or therapeutic target for GIST.
  • the GIST882 cell line obtained from an imatinib naive patient harbours a homozygous ex on 13 KIT mutation (K642E) and is maintained in RPMI supplemented with 15% FBS, lOmM HEPES pH 7.5.
  • the GIST48 cell line, obtained from an imatinib-resistant patient harbour a homozygous exon 11 KIT mutation (V560D) and a secondary heterozygous Exon 17 KIT mutation (D820A), is maintained in Ham's F10 media supplemented with 15% FBS, 0.5% MITO+ Serum Extender (BD Biosciences), and 30 mg/L bovine pituitary extract (BD
  • the U20S osteosarcoma, LNCaP prostate cancer, and NIH-3T3 mouse embryonic fibroblast cells were obtained from ATCC and cultured as recommended.
  • GIST48 and GIST882 cells were established in the Fletcher laboratory (DFCI). All other cells were obtained from ATCC. Etvl-/- mice, with targeted deletion of the ETS domain, was obtained from the Jessell laboratory (Columbia) and CB17-SCID mice was from Taconic. Antibody sources are: ETV1, ANOl, PGP9.5 (Abeam), KIT for WB, P-Tyr703-KIT (Cell Signaling), P-Tyr204-ERK, GAPDH (Santa Cruz), and anti-mouse Kit for IF (clone ACK2, E- Biosciences).
  • mouse anti-phospho-tyr204 ERK for WB (Santa Cruz Biotechnologies), mouse anti- GAPDH for WB (Santa Cruz Biotechnologies), ACK2 rat anti-mouse Kit for IF (E-Biosceinces), rabbit anti-PGP9.5 for IF (Abeam), rabbit anti-ANOl for IF (Abeam).
  • Secondary antibodies were Alexa-488 labelled anti-rabbit antibody and Alexa-594 labelled anti-rat antibody
  • PD0325901 was synthesized in the MSKCC Organic Synthesis Core Facility by O. Ouerfelli and imatinib was a gift from Novartis.
  • GIST samples Five publically deposited database containing expression of GIST samples are as follows: Expression Project for Oncology (ExpO, GSE2109) containing 7 GIST samples among over 2,000 tumours of all types, NIELSEN_SARCOMA (GSE3443) containing 10 GIST samples among 44 sarcoma samples (Nielsen, T. O. et al., 2002, Lancet, 359: 1301-1307;
  • SEGAL_SARCOMA GSE2719 containing 5 GIST samples among 50 sarcoma samples (Segal, N. H. et al., 2003, Am J Pathol, 163: 691-700; incorporated herein by reference), YAMAGUCHI_GIST (GSE8167) with 32 GIST samples (Yamaguchi, U. et al., 2008, J Clin Oncol, 26: 4100-4108; incorporated herein by reference), and OSTROWSKI_GIST (GSE17743) with 29 GIST samples (Ostrowski, J. et al., 2009, BMC Cancer, 9: 413; incorporated herein by reference).
  • ETV1 of a given sample was calculated as the Z-score (i.e., standard deviations from median) of ETV1 probe signal among all probes.
  • Z-score i.e., standard deviations from median
  • the three gene sets, EXPO_GIST_SIG, NIELSEN_GIST_SIG, and SEGAL_GIST_SIG contain 102, 156, and 30 genes respectively (Supplementary Table 1).
  • ICC-MY and ICC-DMP signature genes sets were defined as genes expressed higher in ICC-MY and ICC- DMP compared to muscle by 1.5 Z-score with p ⁇ 0.05 and contain 61 and 81 genes respectively.
  • G1-G8 are GISTs: Gl, G2 and G4 (KIT exonl l mutation), G3 (KIT exon 9 mutation), G5 and G6 (KIT and PDGFRA wild-type), G7 and G8 (PDGFRA D842V mutation); S1-S5 are leiomyosarcoma, Gl leiomyoma, Gl leiomyoma, intra-abdominal desmoid tumour, and Gl schwannoma respectively. FISH of tumor samples
  • BACs one covering ETV1 gene body and one on the 3' side (RP11-138H16, RP11-124L22, CHORI) were labelled with Spectrum Green-d-UTP (Vysis) and two BACs covering 5' upstream region of ETV1 (RP11-703 A4, RP11-115D14) were labelled with
  • tissue samples were grounded in 500 ⁇ Trizol (Invitrogen) using PowerGen homogenizer (Fisher Scientific), followed by addition of 100 ⁇ chloroform. The samples were then centrifuged at 10,000g x 15 minutes. The upper phase was mixed with equal volume of 70% ethanol, and the RNA was further purified using an RNeasy column.
  • CTSL1 F: AGGCGCGTGACTGGTTGAGC (SEQ ID NO: 2), R: TGCATCGCCTTCCACTTGGTCC (SEQ ID NO: 3)
  • DUSP6 F: TGCCGGGCGTTCTACCTGGA (SEQ ID NO: 4), R:
  • ETVl-Exon23 F: AACAGAGATCTGGCTCATGATTCA (SEQ ID NO: 6), R:
  • CTTCTGCAAGCCATGTTTCCTGTA 9 (SEQ ID NO: 7)
  • CTTAAAGCCTTGTGGTGGGAAG 9 (SEQ ID NO: 9)
  • KIT F: GGGATTTTCTCTGCGTTCTG (SEQ ID NO: 10), R:
  • CACGTCAGCCTGCGGTTCTTC SEQ ID NO: 13
  • PROM1 F: GCCACCGCTCTAGATACTGC (SEQ ID NO: 14), R:
  • PTPRE F: TGCACGCGGAGCAGAAGGTG (SEQ ID NO: 16), R:
  • RPL27 F: CATGGGCAAGAAGAAGATCG (SEQ ID NO: 18), R:
  • TIMP3 F: CCAGCGCAAGGGGCTGAACT (SEQ ID NO: 20), R:
  • ETVlshl TRCN0000013923, targeting GTGGGAGTAATCTAAACATTT (SEQ ID NO: 22) in 3' UTR and ETVlsh2:
  • TRCN0000013925 targeting CGACCCAGTGTATGAACACAA (SEQ ID NO: 23 in exon 7) were purchased from Open Biosystems and pLKO.l shScr (targeting
  • CCTAAGGTTAAGTCGCCCTCG (SEQ ID NO: 24) was purchased from Addgene (Sarbassov, D. D., et al., 2005, Science, 307: 1098-1101; incorporated herein by reference).
  • Lentiviruses were generated by co-transfecting the shETVl hairpin constructs with psPax2 and pVSVG (Addgene) into 293FT cells (Invitrogen) using Lipofectamine 2000 (Invitrogen). Viruses were harvested at 48 and 72 hours after transfection and concentrated 100-fold using Lenti-X concentrator (Clontech) and stored at -80 in aliquots.
  • Infection was performed in 12-well dishes in the presence of 7.5 ⁇ g ml polybrene (Sigma) by a 1 hour centrifugation at 500 x g.
  • To determine virus titre in GIST48 and GIST882 cells two steps were taken. First, the relative titre of shScr, ETVlshl, and ETVlsh2 was obtained by counting puromycin resistant colonies after infection of U20S cells, whose growth is insensitive to ETV1 knockdown. Next, the absolute titre of the shScr was calculated by counting puromyicn resistant colonies after infection into GIST48 and GIST882 cells.
  • ETV1 cDNA Open Biosystems
  • EGFP EGFP were cloned into MSCV-puro.
  • Human wild-type KIT in MSCV-IRES-EGFP vector was obtained from Gary Gilliland and generation of ⁇ 560 mutation was performed using QuikChange II XL site-directed mutagenesis kit
  • ETV1 expression cells were selected with puromycin (1 ⁇ g/ml).
  • KIT expression GFP FACS demonstrated >90 infection and therefore, cells were not further enriched.
  • Photometric Coolsnap HQ camera Images were taken with at 20X (N.A. 0.75) or 60X (N.A. 1.4) objectives. Monochrome images taken with DAPI, FITC, and Texas Red filter sets were pseudo- colored blue, green, and red respectively and merged using Image J (http://rsbweb.nih.gov/ij/). The exposure, threshold, and maximum were identical between Etvl-/- and Etvl+/+ images and the gamma was 1.0.
  • mouse GI tissue was fixed in ice-cold acetone for >1 hours. They were then rehydrated in PBS, stretched, and the mucosa of these GI tissues was blunt dissected away. Samples were then blocked in 5% goat serum, incubated with ACK2 rat- anti-KIT antibody or PGP9.5 antibody, followed by secondary antibody. Images were taken with at 20X (N.A. 0.75) or 60X (N.A. 1.4) objectives with Z-step of ⁇ through the muscle layer. The Z-stack of images was deconvoluted using Autoquant X2 software (Media Cybernetics). Movies were generated from deconvolued stacks using ImageJ software.
  • GIST882 cells were treated with vehicle or ⁇ imatinib for 8 hours in triplicate and profiled as above.
  • GSEA determines whether predefined sets of genes (e.g., genes within a Gene Ontology category) are disproportionally overrepresented in the top or the bottom of the list instead of randomly across the list (Subramanian, A. et al., 2005, Proc Natl Acad Sci U S A, 102, 15545-15550; incorporated herein by reference).
  • predefined sets of genes e.g., genes within a Gene Ontology category
  • ETV1 knockdown we used the ranked list of genes generated above from the most upregulated by ETV1 knockdown to most downregulated by ETV1 knockdown. For genes with multiple probes by the Illumina HT-12 array, median signal was used.
  • ChlP-Seq was performed as previously described (Goldberg, A. D. et al., 2010, Cell, 140: 678-691 ; incorporated herein by reference). Briefly, 108 GIST48 cells were crosslinked for 15-minutes in 1 % paraformaldehyde, washed, and lysed. Chromatin was sheared using Bioruptor (Diagenode) to fragments of -150 bp and was incubated with anti-rabbit IgG dynabeads (Invitrogen) pre-conjugated with anti-ETVl antibody, washed, and eluted. The eluted chromatin was reverse-cross-linked, and DNA was column purified.
  • the purified ChIP DNA was blunt- ended, ligated to Solexa adaptors, amplified with 18-cycles of PCR, and sequenced on Solexa Genome Analyzer.
  • F GCCCGCTGTTGCAGCTTGTT (SEQ ID NO: 25), R:
  • GAPDH promoter TCCCAAAGTCCTCCTGTTTCA (SEQ ID NO: 29), R:
  • CAGCAGGACACTAGGGAGTCAA SEQ ID NO: 30
  • GIST-signature genes from three datasets containing both GIST and non- GIST malignancies met the following two criteria: 1) q ⁇ 0.05, and 2) a Z-score expression difference >1.5 between GIST and non-GIST tumours. Expression profiling of GIST cell lines with different shRNA conditions was performed in duplicate on Illumina Human HT-12 array. GSEA was performed using MSigDB C2, MSigDB C4, and the GIST and ICC signature gene sets.
  • ETV1 peak was assigned to all genes if it was in the promoter regions of multiple genes.
  • An enhancer peak within a gene body was assigned to the host gene, but otherwise was assigned to the nearest gene.
  • Nucleotide sequences of all ETV1 peaks were retrieved from the reference human genome and subjected to motif analysis by the program MEME (Bailey, T. L., et al., 2006, Nucleic Acids Res, 34: W369-373; incorporated herein by reference).
  • ETV1 was of immediate interest since ETS family transcription factors are well established oncogenes in Ewing sarcoma, melanoma, and prostate cancer (Tomlins, S. A. et al., 2005, Science, 310: 644- 648; Mertens, F. et al., 2009, Semin Oncol, 36: 312-323; Jane-Valbuena, J. et al., 2010, Cancer Res, 70: 2075-2084; each of which is incorporated herein by reference).
  • ETV1 mRNA and protein levels of ETV1 in GIST and other sarcomas in clinical samples GIST cell lines (imatinib-resistant GIST48 and imatinib- sensitive GIST882), the U20S osteosarcoma cell line, and the LNCaP prostate cancer cell line known to overexpress ETV1 due to translocation (Tomlins, S. A. et al., 2007, Nature, 448: 595-599; incorporated herein by reference) (FIG 1C, D).
  • ETV1 mRNA and protein were highly and exclusively expressed in all GISTs and GIST cell lines, and in LNCaP cells.
  • CM inner circular muscle
  • M mucosa
  • LM outer longitudinal muscle
  • ICC-MY myenteric ICCs
  • ICC-IM intramuscular ICCs
  • ICC-SMP submucosal ICCs
  • FIG. 9A ICC-DMPs
  • ETVl peaks ETVl peaks
  • GGAA ETS core consensus motif
  • FIG. 3F Integrative analyses of the ETVl ChlP-Seq data with the transcriptomes from shRNA- mediated ETVl suppression in GIST cells showed that 38 of the top 48 shETVl downregulated genes contain ETVl peaks (FIG. 3B, E, FIG. 19).
  • ETV1 is universally highly expressed in all GISTs makes it immediately useful as a candidate diagnostic biomarker, since the current standard of KIT immunoreactivity is negative in about 5% of all GISTs (Miettinen, M. & Lasota, 2006, Arch Pathol Lab Med, 130: 1466-1478; incorporated herein by reference). While transcription factors has classically been considered “undruggable”, reports of successful inhibition of the NOTCH transcription factor complex and AR activity by blocking coactivator binding have challenged this paradigm
  • ETV1 inhibitors may yield novel therapeutic agents for imatinib-resistant GIST.
  • the invention includes embodiments in which more than one, or all of the group members are presenting, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitation, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.
  • compositions of the invention e.g., any HCV genotype/subtype, any HCV antibody, any epitope, any pharmaceutical composition, any method of administration, any therapeutic application, etc.
  • any particular embodiment of the compositions of the invention can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

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Abstract

Uses of ETVl for diagnostic, prognostic or therapeutic purposes with gastrointestinal stromal tumors (GISTs).

Description

ETV1 AS A DIAGNOSTIC, PROGNOSTIC AND THERAPEUTIC TARGET FOR GASTROINTESTINAL STROMAL TUMORS
Priority Claim
[0001] The present application claims priority to U.S. Provisional Patent Application No. 61/403,016 that was filed on September 9, 2010. The entire contents of this priority application are incorporated herein by reference.
Government Support
[0002] The inventions described herein were made with Government Support under Contract Nos. K08CA140946 (YC), 5F32CA130372 (PC), CA47179 (CRA, RGM), CA148260 (RGM) awarded by the National Cancer Institute (NCI) and Contract No. R21MH087840 (DZ) awarded by the National Institute of Mental Health (NIMH). The Government has certain rights in the invention.
[0003] The inventions described herein were also supported in part by the NCI- ASCO Cancer Foundation Clinical Investigator Team Leadership Supplemental Award (RGM), ASCO YIA (PC), Doris Duke (CLS), Charles H Revson (YC), the Charles A. Dana (YC) Foundations, ACS MRSG CCE-106841 (CRA), P01CA47179 (CRA, RGM), Life Raft Group (CRA), GIST Cancer Research Fund (CRA), Shuman Family Fund for GIST Research (CRA, RGM), Cycle for Survival (RGM) and Startup Funds from Albert Einstein College of Medicine (DZ).
Background
[0004] Gastrointestinal stromal tumor (GIST) is the most common human sarcoma.
According to current practices, GIST is primarily defined by activating mutations in the KIT or PDGFRA receptor tyrosine kinases. (Heinrich, M. C. et ah, 2003, Science, 299: 708-710, incorporated herein by reference; Hirota, S. et al, 1998, Science, 279: 577-580; incorporated herein by reference.) KIT is a protein-tyrosine kinase receptor known to interact with stem cell factor. Binding of stem cell factor to KIT results in receptor dimerization and activation of protein kinase activity. The activated receptor autophosphorylates at tyrosine residues that serve as docking sites for signal transduction molecules contain SH2 domains. Activated KIT fuctions to further activate several known signal transduction pathways, including phosphatidylinositol 3- kinase and Ras/mitogen-activated protein kinases.
[0005] The general structure of the KIT protein comprises an extracellular domain, a transmembrane segment, a juxtamembrane domain, and a protein kinase domain that contains an insert of about 80 amino acid residues. Both the active and inactive conformations of KIT have been determined by X-ray crystallography.
KIT is highly expressed in interstitial cells of Cajal (ICCs)— the presumed cell of origin for GIST— as well as in hematopoietic stem cells, melanocytes, mast cells and germ cells. (Hirota, S. et al, 1998, Science, 279: 577-580, incorporated herein by reference; Kindblom, L. G., et.al., 1998, Am J Pathol, 152: 1259-1269, incorporated herein by reference.) Yet, families harbouring germline activating KIT mutations and mice with knock- in KIT mutations almost exclusively develop ICC hyperplasia and GIST. (Antonescu, C. R., 2006, Semin Diagn Pathol, 23: 63-69, incorporated herein by reference; Nakai, N. et al., 2008, J Pathol, 214: 302-311, incorporated herein by reference; Rubin, B. P. et al., 2005, Cancer Res, 65: 6631-6639, incorporated herein by reference; Sommer, G. et al., 2003, Proc Natl Acad Sci U S A, 100: 6706-6711, incorporated herein by reference.)
Summary
[0006] The present invention encompasses the finding that the ETS family member ETV1 is highly expressed in the subtypes of ICCs sensitive to oncogenic KIT mediated transformation (Kwon, J. G. et al., 2009, Gastroenterology , 136: 630-639; incorporated herein by reference), and is required for their development. The present invention also encompasses the finding that ETVl is universally highly expressed in GISTs. The present invention further encompasses the finging that ETVl is required for growth of imatinib- sensitive and resistant GIST cell lines.
[0007] Among other things, the present invention demonstrates, through transcriptome profiling and global analyses of ETVl -binding sites, that ETVl is a master regulator of an ICC- GIST-specific transcription network mainly through enhancer binding. The ETVl transcriptional program is further regulated by activated KIT, which prolongs ETVl protein stability and cooperates with ETVl to promote tumorigenesis. Without wishing to be bound by any particular theory, it is proposed that GIST arises from ICCs with high levels of endogenous ETVl expression that, when coupled with an activating KIT mutation, drives an oncogenic ETS transcription program. The present invention therefore provides the insight that GIST differs from other ETO-dependent tumors such as prostate cancer, melanoma, and Ewing sarcoma where genomic translocation or amplification drives aberrant ETS expression (Tomlins, S. A. et ah, 2005, Science, 310: 644-648, incorporated herein by reference; Mertens, F. et ah, 2009, Semin Oncol, 36: 312-323, incorporated herein by reference; Jane-Valbuena, J. et ah, 2010, Cancer Res, 70: 2075-2084, incorporated herein by reference) and represents a novel mechanism of oncogenic transcription factor activation. According to the present invention, ETVl expression can be used to enable diagnosis, prognosis, and can be a therapeutic target for gastrointestinal stromal tumors or GISTs. The present invention provides systems and technologies for detecting, characterizing, and/or treating gastrointestinal stromal tumors or GISTs, for example through assessment of level and/or activity of an ETS family member such as ETVl.
[0008] In a first aspect, there is provided a method for determining the likelihood of gastrointestinal stromal tissue being cancerous or being gastrointestinal stromal tumor (GIST), comprising (a) determining a level of expression or activity of ETVl in a gastrointestinal stromal tissue sample obtained from a subject; (b) comparing the determined ETVl level with that of a reference correlated with a predetermined probability of being cancerous or being GIST; and (c) based on the comparing, determining that the tissue sample has an increased or decreased probability, relative to the reference, of being cancerous or being GIST. In particular embodiments, determining the level of expression or activity involves determining the expression of a nucleic acid. In other embodiments, determining the level of expression or activity involves determining the expression of a polypeptide. In yet other embodiments, determining the level of expression activity of ETVl involves determining an ETVl regulated expression signature.
[0009] In certain embodiments, an increase in the level of ETVl correlates with the onset of or progression of a GIST phenotype. In other embodiments, a decrease in the level of ETVl correlates with the regression of a GIST phenotype.
[0010] In additional embodiments, determining the level of expression comprises performing Northern blotting, Western blotting, protein gel electrophoresis, immunoprecipitation, ELISA, PCR, RT-PCR, differential display, serial analysis of gene expression, array analysis, and/or immunohistochemistry.
[0011] In a related embodiment, the step (b) of comparing comprises establishing that the determined level in the subject tissue sample is higher than the reference level, and the reference level correlates with that observed in normal tissue, so that step (c) of determining comprises detemining that the tissue sample has an elevated likelihood of being cancerous or GIST.
[0012] In another related embodiment, the step (a) of determining comprises performing an analysis selected from the group consisting of Northern blotting, Western blotting, protein gel electrophoresis, immunoprecipitation, ELISA, PCR, RT-PCR, differential display, serial analysis of gene expression, array analysis, immunohistochemistry, or a combination thereof, so that the ETVl level of expression or activity is determined. In additional embodiments, the step (a) of determining comprises performing immunohistochemistry on the tissue sample, using an ETVl antibody or antigen binding fragment thereof. In a particular embodiment, the antibody or antigen binding fragment binds to an amino acid sequence or portion thereof as set forth in SEQ ID NO: 1. [0013] In a second aspect, there is provided a method for determining the likelihood of cancerous gastrointestinal stromal tissue responding to therapy, comprising: (a) determining a level of expression or activity of ETVl in a cancerous gastrointestinal stromal tissue sample obtained from a subject; (b) comparing the determined ETVl level with that of a reference correlated with a predetermined probability of responding to a therapeutic regimen; and (c) based on the comparing, determining that the subject from which the tissue sample was obtained has an increased or decreased probability, relative to the reference, of responding to the therapeutic regimen. In a particular embodiment, the reference is an average of responses derived from a population of individuals.
[0014] In a third aspect, there is provided a methhod for identifying or characterizing an agent that affects GIST phenotype comprising: (a) contacting cultured GIST tissue or cells with an agent to be characterized; and (b) determining that ETVl level of expression or activity is altered when the agent is present as compared with when it is absent.
[0015] In a fourth aspect, there is provided a method of treating GIST by administering to a subject suffering from or susceptible to GIST a therapy that alters ETVl level of expression or activity. In a particular embodiments, the step of administering comprises administering a therapy that modulates expression or activity of an ETVl regulator so that ETVl level of expression or activity is altered.
[0016] In a fifth aspect, there is provided a method of treating GIST in a subject in need thereof comprising administering a therapeutically effective amount of pharmaceutical composition comprising an agent identified by the methods disclosed herein. In certain embodiments, the composition inhibits EVT1 expression. In other embodiments, the
composition promotes the degradation of ETVl protein.
[0017] In a sixth aspect, there is provided a kit for use in detecting ETVl expression. The kit comprises an agent capable of detecting the level of ETVl expression or activity, a container comprising the agent for detecting the level of ETVl in a sample, a control, and instructions to provide guidance for carrying out an assay embodied by the kit and for making a determination of ETV1 based upon that assay. In particular embodiments, the agent is an antibody or ETV1- binding fragment thereof, oligonucleotide probe, oligonucleotide primer, aptamer, polypeptide, peptide, ligand, hormone, lipid, carbohydrate, lectin, or small molecule. In certain embodiments, the agent is an anti-ETVl antibody or an antigen-binding fragment thereof. In certain embodiments, the antibody or antigen-binding fragment thereof is attached to a substrate, the substrate is applied to a sample from a patient or to a surface that may contain ETV1, and the surface of the substrate is then processed to assess whether specific binding occurs between the antibody and ETV1 or other component of the sample. In still another embodiment, the control is a control slide comprising tumor samples for testing reagents in the kit.
[0018] In a seventh aspect, there is provided a method comprising the steps of: (a) monitoring ETV1 level of expression or activity of ETV1 in a subject suffering from GIST; (b) detecting a change in the monitored ETV1 level over time; and (c) initiating or altering therapy administered to the subject after detecting the change. In a particular embodiment, the subject is undergoing a first therapy and the step of initiating or altering comprises ceasing the first therapy, modifying the first therapy, adding a second therapy, or combinations thereof.
[0019] In an eighth aspect, there is provided a method of inhibiting the growth of ICC cancerous cell with aberrant ETV1 expression comprising contacting the cell with an ETV1 inhibitory agent so as to decrease the level or activity of ETV1, thereby inhibiting the growth of the ICC cancerous cell. In particular embodiments, the ETV1 inhibitory agent is selected from the group consisting of a targeted ribozyme, a targeted antisense oligonucleotide, and a targeted siRNA
Description of the Drawing
[0020] The Figures described below, that together make up the Drawing, are for illustration purposes only, not for limitation. [0021] FIG. 1 depicts an exemplary result illustrating ETVl is universally highly expressed and required for tumour growth and survival in GIST. FIG. 1A depicts an exemplary result illustrating a Venn diagram of GIST-signature genes from three datasets. FIG. IB depicts an exemplary result illustrating expression of ETVl in multiple tumour types from the ExpO dataset. Box, 25-75 percentile; error bar, 10-90 percentile; dots, outliers. FIG. 1C depicts an exemplary result illustrating ETVl and KIT mRNA levels by qRT-PCR of GIST and non-GIST samples, whose details are described in Full Methods. Mean+SD, n=3. FIG. ID depicts an exemplary result illustrating immunoblotting of selected tumour tissues and cell lines from FIG. 1C. FIG. IE depicts an exemplary result illustrating growth curves of GIST and U20S cells after shRNA-mediated ETVl suppression compared to control. Mean+SEM, n=3. FIG. IF depicts an exemplary result illustrating tumour volume over time in SCID mice implanted with GIST882 cells after shRNA-mediated ETVl suppression compared to scrambled shRNA controls. Mean+SEM, * p<0.05; n=7, 10, 8 for scrambled, ETVlshl, and ETVlsh2 respectively. FIG. 1G depicts an exemplary result illustrating ETVl mRNA levels of preimplanted GIST882 cells and explanted xenografts at week 10. Mean+SD.
[0022] FIG. 2 depicts an exemplary result illustrating ETVl is expressed in the subtypes of ICCs susceptible to oncogenesis and is required for their development. FIG. 2A depicts an exemplary result illustrating a schematic showing localization of ICC-MY (yellow arrowheads), ICC-IM (yellow arrows) and ICC-SMP (white arrowheads) in the large intestine. M: mucosa, CM: circular muscle, LM: longitudinal muscle. All three ICC subtypes express Kit (red). FIG. 2B depicts an exemplary result illustrating co-immunofluorescence (divided into two microscopy fields) of Kit (red), Etvl (green) and DAPI (blue) of the large intestine of wild-type mice. FIG. 2C depicts an exemplary result illustrating co-immunofluorescence of Kit (red), Pgp9.5 (green), and DAPI (blue) of the large intestine of Etvl+/+ and Etvl-/- mice. FIG. 2D depicts an exemplary result illustrating representative deconvoluted whole-mount Kit-immunofluorescence images of the large intestine of Etvl+/+ and Etvl-/- mice. A single microscopy field focused to the ICC-MY and ICC-SMP planes are shown. Scale bar, 20 μιη.
[0023] FIG. 3 depicts an exemplary result illustrating ETVl regulates GIST-signature genes predominantly through enhancer binding. FIG. 3A depicts an exemplary result illustrating a ranked list of ETVl regulated genes generated based on the average fold-change by the two ETVl hairpins in two cell lines. FIG. 3B depicts an exemplary result illustrating a heatmap of expression of the 48 genes with average downregulation > 1.7-fold. For each gene, table shows p- value of GIST vs. other tumour types from the ExpO dataset, calculated by Oncomine™ (NS: p>0.05), and the presence of ETVl binding sites from ChlP-Seq analysis. FIG. 3C depicts an exemplary result illustrating GSEA plots of the shETVl ranked list using three gene sets: GIST signature genes from ExpO dataset, ICC-MY and ICC-DMP signature genes in mouse small intestine. ES, enrichment score; FDR, false discovery rate. FIG. 3D depicts an exemplary result illustrating pie charts of genomic structure and distribution of ETVl ChlP-Seq peaks. TSS, transcription start site; TES, transcription end site. FIG. 3E depicts an exemplary result illustrating representative ChlP-Seq reads in top ETVl target genes. FIG. 3F depicts an exemplary result illustrating the consensus sequence motif identified in the ETVl binding sites by the MEME program. FIG. 3G depicts an exemplary result illustrating pie chart of genes with ETVl binding sites divided into promoter only, enhancer only and both. FIG. 3H depicts an exemplary result illustrating plot of percent of all genes, genes averagely downregulated 1.4-fold by shETVl (n=410), and genes averagely upregulated 1.4-fold by shETVl (n=380) with promoter only, enhancer only and both promoter and enhancer ETVl binding. Fold enrichment over all genes is shown above the plots.
[0024] FIG. 4 depicts an exemplary result illustrating KIT signalling synergizes with ETVl in GIST tumouri genesis by stabilization of ETVl protein. FIG. 4 A depicts an exemplary result illustrating immunoblots of GIST882 cells treated with the imatinib (1 μΜ) and PD325901 (100 nM) for the indicated time points. FIG. 4B depicts an exemplary result illustrating immunoblots of GIST882 cells treated for 2 hours with imatinib or PD325901 in combination with cyclohexamide (10 μg ml) or MG132 (10 μΜ). FIG. 4C depicts an exemplary result illustrating a Venn diagram of genes downregulated by 1.4-fold by shETVl and by imatinib in GIST882 cells. P-value: Fisher's exact test based on number of expressed genes. FIG. 4D depicts an exemplary result illustrating percent of all genes, imatinib-downregulated genes, shETVl- downregulated genes, and overlapping genes with ETVl enhancer peaks. FIG. 4E depicts an exemplary result illustrating an immunoblot of NIH3T3 cells expressing ETVl and either KITwt or ΚΙΤΔ560 two hours after treatment with PD325901, imatinib, or MG132. FIG. 4F depicts an exemplary result illustrating growth of xenografts of engineered NIH3T3 cells stabilizing the indicated genes (n=12, Mean +SEM). FIG. 4G depicts an exemplary result illustrating a photograph of 4 representative explanted xenografts at 4 weeks after implanting. Scale bar 1 cm. FIG. 4H depicts an exemplary result illustrating a model of the role of ETVl in ICC maintenance and GIST oncogenesis. Normal level of KIT activation by KIT ligand (red triangle) stabilizes ETVl transcription factor through the MAPK pathway, and results in physiological ETVl transcriptional output critical for ICC development (middle). In the absence of ETVl, there is decreased ICC development, which phenocopies genetic loss of KIT signalling (left). Activating mutation of KIT (e.g. ΚΙΤΔ560) leads to constitutive activation of the KIT-MAPK signalling pathway, increased stabilization and augmented ETVl transcriptional output that promotes tumourigenesis (right).
[0025] FIG. 5 depicts an exemplary result illustrating ETVl expression levels from GIST containing microarray expression datasets. FIG. 5A depicts an exemplary result illustrating expression of ETVl (37156_at, Affymetrix U95A-Av2) of individual tumour samples of 9 sarcoma types from Segal dataset. FIG. 5B depicts an exemplary result illustrating expression of ETVl (IMAGE:81320, Stanford spotted platform) of individual tumour samples of 6 sarcoma types from Nielsen dataset. FIG. 5C depicts an exemplary result illustrating expression of ETVl (22191 l_at, Affymetrix HG_U133_Plus_2) of 32 GIST samples from Yamaguchi dataset annotated by location. FIG. 5D depicts an exemplary result illustrating expression of ETV1 (22191 l_at, Affymetrix HG_U133_Plus_2) of 29 GIST samples from Ostrowski dataset annotated by TKI mutation status.
[0026] FIG. 6 depicts an exemplary result illustrating ETV1 immunofluorescence in four GIST (GIST1-4) samples and a leiomyosarcoma (LMS) control sample. Paraffin embedded human GIST samples were stained with ETVl-specific primary antibody and Alexa-594 anti- rabbit secondary antibody. Alexa 594: left column; DAPI: middle column; Alexa/DAPI merge: right column. Scale bar = 20 um.
[0027] FIG. 7 depicts an exemplary result illustrating effect of shETVl on ETV1 protein and mRNA levels and cell cycle profile. FIG. 7 A depicts an exemplary result illustrating ETV1 mRNA and protein levels in GIST48, GIST882, and U20S cells 4-days after shRNA-mediated ETV1 knockdown. FIG. 7B depicts an exemplary result illustrating cell-cycle analysis of GIST48 and GIST882 cells 5 days after infection with shETVl viruses containing scrambled or the ETVl-specific shRNAs. Cells were infected at MOI=5, fixed 5 days after infection and stained with propidium iodide.
[0028] FIG. 8 depicts an exemplary result illustrating an assessment for ETV1 translocation and amplification in GIST. FIG. 8A-B depicts an exemplary result illustrating representative FISH of the ETV1 locus in two GIST clinicial specimens. Green are two probes in the gene body and downstream of ETV1 and red are two probes upstream of ETV1. There's no evidence of "breakaway" or amplification of ETV1. FIG. 8C depicts an exemplary result illustrating qRT- PCR of same cDNA as in FIG. 1C, which used primers targeting ETV1 exons 6 and 7. THis qRT-PCR used primers targeting exons 2 and 3. UP arrows indicate regions of translocation in prostate cancers. Comparing to FIG. 1C, there is no evidence that early exons before
translocation hotspots are significantly under-expressed compared to exons 6 and 7. Mean + SD, n=3. [0029] FIG. 9 depicts an exemplary result illustrating expression of ETV1 in the murine small intestine. FIG. 9A depicts an exemplary result illustrating the myenteric ICC network (ICC-MY, yellow arrowheads) is localized adjacent to the myenteric plexus (MP) between the circular (CM) and longitudinal (LM) muscle layers. The deep muscle plexus ICC network (ICC- DMP, white arrowheads) is localized just below the mucosa (M) in the circular muscle layer. FIG. 9B depicts an exemplary result illustrating both ICC subsets express Kit though ICC-DMP stains much more dimly. Co-immunofluorescence of Kit (red), Etvl (green), and DAPI (blue) shows that the ICC-MY, but not ICC-DMP express Etvl. FIG. 9C depicts an exemplary result illustrating Kit and Etvl mRNA expression level from expression profiles ICC-MY (n=3), ICC- DMP (n=3), and muscle (n=2) isolated from mouse small intestine (GSE7809, Mean + SD).
[0030] FIG. 10 depicts an exemplary result illustrating Etvl is required for ICC-MY but not ICC-DMP development of the murine small intestine. FIG. 10A depicts an exemplary result illustrating co-immunofluorescence of Kit (red), Pgp9.5 (green), and DAPI (blue) of the small intestine of Etvl+/+ and Etvl-/- mice showing significant loss of ICC-MY (yellow arrowheads) and preservation of ICC-DMP (white arrowheads). FIG. 10B depicts an exemplary result illustrating whole mount Kit immunofluorescence taken at the ICC-MY plane and ICC-DMP plane, also showing loss of ICC-MYs and preservation of ICC-DMPs. Scale bar 20uM.
[0031] FIG. 11 depicts an exemplary result illustrating Etvl is required for ICC-IM development of the murine gastric fundus. FIG. 11 A depicts an exemplary schematic showing ICC-IMs present in both the circular and longitudinal muscle layers. FIG. 11B depicts an exemplary result illustrating co-immunofluorescence of Kit (red), Pgp9.5 (green), and DAPI (blue) of the gastric fundus of Etvl+/+ and Etvl-/- mice showing significant loss of ICC-IM. FIG. l lC depicts an exemplary result illustrating whole mount Kit immunofluorescence taken at the circular muscle ICC-IM and longitudinal muscle ICC-IM planes, also showing loss of ICC- IMs. Scale bar 20 uM. [0032] FIG. 12 depicts an exemplary result illustrating Etvl is required for ICC-MY development of the murine cecum. FIG. 12 A depicts an exemplary schematic of ICC types of the cecum. Compared to the rest of the large intestine, ICC-IM and ICC-SMP is very sparse (see FIG. 2). FIG. 12B depicts an exemplary result illustrating co-immunofluorescence of Kit (red), Pgp9.5 (green), and DAPI (blue) of the cecum of Etvl+/+ and Etvl-/- mice showing significant loss of ICC-MY. FIG. 12C depicts an exemplary result illustrating whle mount Kit
immunofluorescence taken at the ICC-MY plane also showing loss of ICC-MY. Scale bar 20 uM.
[0033] FIG. 13 depicts an exemplary result illustrating quantification of ICC subclasses remaining in Etvl-/- mice expressed as precent of Etvl-/- mice. To quantify ICC subsets, immunofluorescence images from at least four fields each from two Etvl+/+ and Etvl-/- mice (at least eight fields total) of the stomach, small intestine, cecum, and large intestine were examined. A percentage of ICCs were obtained by dividing the number of KIT-positive immunostaining cells with the number of DAPI-positive nuclei. Plotted is the ratio of percent of ICCs in Etvl-/- mice compared to Etvl+/+ mice (Mean + SD).
[0034] FIG. 14 depicts an exemplary result illustrating Kit and Anol immunofluorescence in GI tract of Etvl+/+ and Etvl-/- mice. Kit and Anol label all ICC subsets with similar intensity except in ICC-DMP of the small intestine where Kit staining is faint and Anol is stronger (FIG. 14B, white arrows). FIG. 14A depicts an exemplary result illustrating global loss of ICC-MY and ICC-IM in stomach of Etvl-/- mice. FIG. 14B depicts an exemplary result illustrating global loss of ICC-MY and ICC-IM in small intestine of Etvl-/- mice. FIG. 14C depicts an exemplary result illustrating global loss of ICC-MY and ICC-IM in large intestine of Etvl-/- mice. ICC- DMP (FIG. 14B) are preserved in the small intestine, and ICC-SMP (FIG. 14C) are preserved in the colon (white arrows). Scale bar, 20 um.
[0035] FIG. 15 depicts an exemplary result illustrating whole mont Pgp9.5
immunofluorescence of small and large intestine to examine the neuronal myenteric plexus in Etvl+/+ and Etvl-/- mice. Whole mount Z-stack of Pgp9.6 staining of the small and large intestine was performed and an exemplary Z-section of the myenteric plexus is shown. Scale bar 20 uM.
[0036] FIG. 16 depicts an exemplary result illustrating overlap of expressed probes (total 38985) perturbed 1.7-fold by ETVl shRNA in GIST48 and GIST 882 cells. FIG. 16A and FIG. 16B depict an exemplary result illustrating a Venn diagram of probes downregulated by ETVshl (blue), downregulated by ETVlsh2 (orange), upregulated by ETVlshl (red) and upregulated by ETVlsh2 (green), all 1.7-fold compared to scrambled shRNA. FIG. 16C depicts an exemplary result illustrating a Venn diagram of probes downregulated by either ETVlshl or ETVlsh2 in GIST48 (blue) and GIST882 (orange). FIG. 16D depicts an exemplary result illustrating a Venn diagram of probes upregulated by either ETVlshl or ETVlsh2 in GIST28 (green) and GIST882 (red). P- values for significance of overlap were calculated using Fisher Exact Test.
[0037] FIG. 17 depicts an exemplary result illustrating qRT-PCR validation of top downregulated genes by ETVl knockdown. GIST48 and GIST882 cells were infected with shScr, ETVlshl, and ETVlsh2 lentiviruses in an independent experiment from the expression profiling experiment. RNA was harvested 4 days after lentiviral infection without drug selection and qRT-PCR performed. Data represents 2-AACt using RPL27 as housekeeping gene control and shScr as normalization sample. Mean + SD, n=3.
[0038] FIG. 18 depicts an exemplary result illustrating GSEA enrichment of GIST-signature datasets. FIG. 18A depicts an exemplary result illustrating enrichment plots of Segal GIST- signature gene sets on the "shETVl" ranked list. FIG. 18B depicts an exemplary result illustrating enrichment plots of Nielsen GIST-signature gene sets on the "shETVl" ranked list.
[0039] FIG. 19 depicts an exemplary result illustrating qPCR ChIP validation of ETVl binding to DUSP6 and GPR20 locus in GIST48 and GIST882 cells (Mean + SD, n=3). [0040] FIG. 20 depicts an exemplary result illustrating mRNA level of ETVl by qRT-PCT in GIST882 cells after imatinib treatment for the indicated time points. Samples normalized to endogenous transcript (Mean + SD, n=3).
[0041] FIG. 21 depicts an exemplary result illustrating soft agar colony formation assays of NIH3T3 cells transduced with different permutations of ETVl and KIT. NIH3T3 cells transduced with EGFP or ETVl expression virus were subsequently transduced with different combinations of empty vector, wild-type KIT, or activating mutant KIT (Δ560). 5,000 cells/well of 6-well plate were seeded in triplicate of each transduced line. FIG. 21 A depicts an exemplary result illustrating photographs of representative wells of soft agar colony formation 3 weeks after plating 5,000 cells/well. Scale bar 2mm. FIG. 21B depicts an exemplary result illustrating colongy quantification (Mean + SD, n=3).
Definitions
[0042] Combination therapy: The term "combination therapy", as used herein, refers to those situations in which two or more different pharmaceutical agents are administered in overlapping regimens so that the subject is simultaneously exposed to both agents.
[0043] ETVl regulator'. The term "ETVl regulator", as used herein, refers to any endogenous intracellular or extracellular substance that modulates the transcription, binding, activity or stability of ETVl. For example, in particular embodiments, the ETVl regulator may be KIT.
[0044] Gene expression signature: The term "gene expression signature" or "expression signature", as used herein, refers to a unique and distinct pattern of expression of a given gene in a particular tissue. A gene expression signature may be correlated with and indicate a particular phenotype, aberrant expression pattern, condition, probability of disease, prognosis, preferred treatment, our liklihood of success of a particular treatment. [0045] Overexpression: The terms "overexpressed" or "underexpressed", or "over- expression" or "under-expression", or "upregulated" or "downregulated", as used herein, refer to expression of a nucleic acid sequence or protein in a cancer cell at a higher or lower level, respectively, than that level typically observed in a non-tumor cell (i.e., normal control). In certain embodiments, the level of expression of a nucleic acid or a protein that is overexpressed in the cancer cell is at least 10%, 20%, 40%, 60%, 80%, 100%, 200%, 400%, 500%, 750%, 1,000%, 2,000%, 5,000%, or 10,000% greater in the cancer cell relative to a normal control. In other embodiments, the level of overexpression in a cancer cells is at least two-fold or more greater than the relative level of expression in a non-cancer cell.
[0046] Reference sample: As used herein, a reference sample may include, but is not limited to, any or all of the following: a cell or cells, a portion of tissue, blood, serum, ascites, urine, saliva, and other body fluids, secretions, or excretions. The term "sample" also includes any material derived by processing such a sample. Derived samples may include nucleotide molecules or polypeptides extracted from the sample or obtained by subjecting the sample to techniques such as amplification or reverse transcription of mRNA, etc.
[0047] Subject: As used herein, the term "subject" or "patient" refers to any organism upon which embodiments of the invention may be used or administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals {e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.).
[0048] Suffering from'. An individual who is "suffering from" a disease, disorder, or condition (e.g., GIST) has been diagnosed with and/or exhibits one or more symptoms of the disease, disorder, or condition. Early-stage GIST is frequently asymptomatic. In some embodiments, an individual who is suffering from GIST has GIST, but does not display any symptoms of GIST and/or has not been diagnosed with GIST. In some embodiments, an individual who is suffering from GIST is an individual who has upregulated ETV1 expression. [0049] Susceptible to: An individual who is "susceptible to" a disease, disorder, or condition (e.g. , GIST) is at risk for developing the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition does not display any symptoms of the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition is an individual who displays conditions associated with development of the disease, disorder, or condition (e.g., the individual has elevated levels of ETV1 expression). In some embodiments, a risk of developing a disease, disorder, and/or condition is a population-based risk (e.g., carrier of KIT ex on 9 mutation, carrier of KIT ex on 11 mutation, carrier of KIT exon 13 mutation; etc.).
[0050] Symptoms are reduced: According to the present invention, "symptoms are reduced" when one or more symptoms of a particular disease, disorder or condition is reduced in magnitude (e.g., intensity, severity, etc.) or frequency. For purposes of clarity, a delay in the onset of a particular symptom is considered one form of reducing the frequency of that symptom. Many GIST patients with smaller tumors have no symptoms. Larger tumors can cause symptoms that are related to the increased mass being accommodated in the abdominal cavity. To give but a few examples, exemplary symptoms of GIST may include, but are not limited to, digestive discomfort, sensations of abdominal fullness, abdominal pain, visible enlargement of the abdomen, vomiting or diarrhea, bowel obstruction, perforation of the stomach or gut lining, black or tarry stools, vomiting of blood, anemia fatigue, and weight loss. It is not intended that the present invention be limited only to cases where the symptoms are eliminated. The present invention specifically contemplates treatment such that one or more symptoms is/are reduced (and the condition of the subject is thereby "improved"), albeit not completely eliminated.
[0051] Therapeutic regimen: As used herein, the term "therapeutic regimen" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. It may comprise a treatment or series of treatments designed to achieve a particular effect i.e., reduction or elimination of a detrimental condition or disease such as GIST. The treatment may include administration of one or more compounds either simultaneously, sequentially or at different times, for the same or different amounts of time. Alternatively, or additionally, the treatment may include exposure to radiation, chemotherapeutic agents or surgery. In addition, a "treatment regimen" may include genetic methods such as gene therapy, gene ablation or other methods known to reduce expression of a particular gene or translation of a gene-derived mRNA.
[0052] Therapeutic agent'. As used herein, the phrase "therapeutic agent" refers to any agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect.
[0053] Therapeutically effective amount'. As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic protein (e.g., ETV1 antibody) which confers a therapeutic effect on the treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect). In particular, the "therapeutically effective amount" refers to an amount of a therapeutic protein or composition effective to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity or frequency of symptoms of the disease. A therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses. For any particular therapeutic protein, a therapeutically effective amount (and/or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration, on combination with other pharmaceutical agents. Also, the specific therapeutically effective amount (and/or unit dose) for any particular patient may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and/or rate of excretion or metabolism of the specific fusion protein employed; the duration of the treatment; and like factors as is well known in the medical arts.
[0054] Treatment'. As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a substance (e.g., provided compositions) that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition (e.g., GIST). Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.
[0055] Tumor sample: The term "tumor sample" as used herein is taken broadly to include cell or tissue samples removed from a tumor, cells (or their progeny) derived from a tumor that may be located elsewhere in the body (e.g., cells in the bloodstream or at a site of metastasis), or any material derived by processing such a sample. Derived tumor samples may include nucleic acids or proteins extracted from the sample or obtained by subjecting the sample to techniques such as amplification or reverse transcription of mRNA, etc.
[0056] Tumor subclass: A tumor subclass is the group of tumors that display one or more phenotypic or genotypic characteristics that distinguish members of the group from other tumors. Detailed Description of Certain Embodiments
[0057] Among other things, the present disclosure demonstrates that the ETS family member ETV1 is highly expressed in the subtypes of ICCs sensitive to oncogenic KIT-mediated transformation (Kwon, J. G. et ah, 2009, Gastroenterology, 136: 630-639), and is required for their development. According to the present invention, ETV1 expression can be used as a diagnostic, prognostic or therapeutic target for GIST.
1. Gastrointestinal stromal tumors (GISTs)
[0058] GISTs occur in the wall of the bowel and have been proposed to arise from the interstitial cells of Cajal (ICCs). GISTs are the most common mesenchymal neoplasms of the gastrointestinal tract, and can also originate in the mesentery and omentum. The differential diagnosis of these tumors includes desmoid fibromatosis, Schwannoma, leiomyosarcoma, and, in some cases, high grade sarcomas. Accurate diagnosis of GISTs is important, because imatinib mesylate (GLEEVEC® manufactured by Novartis, Switzerland) has been shown to significantly inhibit these tumors.
[0059] Currently, the diagnosis of GISTs relies heavily on expression of the KIT marker. Recommendations in the literature emphasize a diffuse, strong KIT immunoreactivity for the diagnosis of GIST. CD34 immunostaining can also aid in the diagnosis, but a subset of cases is immunonegative while many other types of sarcomas are immunoreactive for this marker. In the vast majority of GISTs, high levels of KIT expression are accompanied by a KIT mutation in exon 9, 11, 13 or 17. Recently, a subset of GISTs have been found to have PDGFRA mutations rather than KIT mutations. Patients with GISTs containing mutations in PDGFRA belong to the PDGFRA positive subclass and may still benefit from imatinib therapy. However, this subclass of tumors often fail to react with antibodies against KIT and hence may remain undiagnosed as GISTs. Furthermore, identification of PDGFRA positive mutant GISTs currently requires molecular analysis, a laborious process that is not ideal for application in a routine clinical setting. In addition, some GISTs with KIT mutations may have low KIT expression by immunohistochemistry yet will still respond to imatinib therapy.
[0060] Therefore, there is need in the art for alternative and superior GIST markers that are capable of providing diagnosis and classification of GIST, as well as providing prognistic information on the efficacy of GIST treatments. Such markers would also be targets for GIST therapies.
[0061] Embodiments of the present invention are based on the surprising discovery of the oncogenic contributions of ETV1 in ICC cells and GIST. The data described below
demonstratest that ETV1 is a superior marker for GIST because it is a master regulator of an ICC-GIST-specific transcription network. Moreover, ETV1 controls this network through stabilizing interactions with KIT. The synergistic interaction of KIT and ETV1 provides a new paradigm for the development of ICC cancers, including GIST. Analysis, qualification or quantification of ETV1 expression, either alone or in combination with analysis of KIT, provides refined diagnostic capabilities. Likewise, therapies directed toward ETV1, either alone or in combination with therapies directed at KIT or conventional cancer therapies, provide promising new uses and treatments for GIST- afflicted patients.
[0062] Embodiments of the present invention may be used for the diagnosis or prognosis of GIST, and may further be used in GIST-directed therapies. According to one aspect, the invention therefore provides a method comprising providing a GIST tumor sample; detecting expression or activity of a gene encoding a ETV1 polypeptide in the sample; determining whether the tumor sample is marked by elevated ETV1 expression relative to a non-cancerous negative control sample. In certain embodiments, the method further comprises detecting expression or activity of a gene encoding a KIT polypeptide in the sample and detecting expression or activity of a gene encoding a ETVI polypeptide in the sample. According to such embodiments, the determining step is based on the results of all detecting steps. [0063] In another aspect, the invention provides a method comprising providing a GIST tumor sample; detecting expression or activity of a gene encoding a ETV1 polypeptide in the sample; and determining whether the tumor is marked by normal or reduced ETV1 expression relative to a cancerous positive control sample. In certain embodiments, the method further comprises detecting expression or activity of a gene encoding a KIT polypeptide in the sample and detecting expression or activity of a gene encoding a ETV1 polypeptide in the sample. According to such embodiments, the determining step is based on the results of all detecting steps.
[0064] In any of the above methods, the tumor sample may be a blood sample, a urine sample, a serum sample, an ascites sample, a saliva sample, a cell, or a portion of tissue. As described in greater detail below, the methods may further comprise providing diagnostic, prognostic, or predictive information based on the determining step. Determining may include diagnosis, probabiltiy or predisposition of GIST, or stratifying the tumor (and thus stratifying a subject having the tumor), e.g., for a clinical trial, or classifying for purposes of determining treatment. In certain embodiments, the methods may further comprise selecting a treatment based on the detrmining step. Additionally or alternatively, the methods may further comprise determining the specific mutations within the ETV1 markers and the correlation of those mutations with the severity of disease, a desired course of treatment, and the prognosis for recovery.
[0065] GIST samples may be obtained by methods known to those of skill in the art, including biopsy or post-surgery. GISTs may occur anywhere in the gastrointestinal tract but are most common in the stomach and small bowel (roughly 60% and 30%, resp.), while 10% arise in other parts of the gastrointestinal tract (esophagus, colon, and rectum), and a small percentage are extragastrointestinal, arising in the mesentery, omentum, retroperitoneum, or pelvis.
Endoscopic, fine needle and surgical biopsy techniques may be used to retrieve the samples. [0066] In regard to current treatment, the main goal for a localized GIST is complete surgical resection with negative margins and preservation of an intact pseudocapsule. However, in accordance with embodiments of the invention personalized treatment regimens may be devised depending on the level of ETV1 expresssion, with higher level of ETV1 indicating a more aggressive treatment regimen. Particular levels of expression may require either gastric wedge resection, partial gastrectomy or total gastrectomy.
[0067] Embodiments of the invention encompass novel therapies to directly or indirectly (i.e., by affecting an upstream regulator or downstream target) treat elevated or otherwise aberrant (e.g., mutated) ETV1 expression. These treatments may be combined with known surgical or pharmacological treatments for GIST. For example, the methods disclosed herein may be used after surgical intervention to target an affected area. Alternatively or additionally, embodiments of the invention may be used simultaneously with or immediately before or after current pharmacological and chemotherapeutic treatments. For example, the tyrosine kinase inhibitor imatinib mesylate may be used in the adjuvant setting following complete gross resection. Alternatively, the kinase inhibitor sunitinib malate (SU 11248 or Sutent, Pfizer, New York, NY) may be used. RADOOl (manufactured by Novartis, Switzerland) is currently in Phase I clinical trials. RADOOl inhibits mTOR, a downstream target in the AKT pathway. AKT is a survival pathway that is activated by KIT and many other receptors. It is hoped that the simultaneous inhibition of KIT and mTOR using GLEEVEC® and RADOOl will result in increased effectiveness over GLEEVEC® alone. Novartis have also begun Phase I and II clinical trials with PKC412, an inhibitor of protein kinase C (PKC). PKC412 is less specific than GLEEVEC®, inhibiting PKC, and kinases of KIT, VEGF, PDGF 42. Amgen of Thousand Oaks, CA are developing AMG706 that is thought to have a similar mechanism of action as SU11248. Bristol-Myers Squibb of New York, NY are developing BMS-354825 that is an inhibitor of both KIT and PDGFRA. Pharmaceutical treatment directed towards EDTV1 may be added to such existing treatments. 2. ETV1 expression or activity correlates with GIST
[0068] ETV1 is a transcription factor belonging to the large ETS (E-twenty six) family and PEA3 subfamily. ETS family members are defined by a highly conserved DNA binding domain, the ETS domain, which is a winged helix-turn-helix structure that binds to DNA sites with a central GGA DNA sequence. Multiple ETS factors have been found to be associated with cancer. In a condition called Ewing's sarcoma, the ERG ETS transcription factor is fused to the EWS gene (Ida, K., et al., 1995, Int. J. Cancer, 63(4):500-4; incorporated herein by reference). The fusion of TEL to the JAK2 protein results in early pre-B acute lymphoid leukemia (Peeters, P., et al., 1997, Blood, 90(7):2535-40; incorporated herein by reference) and ERG and ETV1 are ETS family members which are over-expressed, and whose gene fusions are found, in prostate cancer (Tomlins, S.A., et al., 2005, Science, 310(5748):644-8; incorporated herein by reference).
[0069] As described herein, the ETV1 protein (SEQ ID NO: 1) and its readily derived mRNA sequences are universally highly expressed in gastroinstestinal stromal tumors (GISTs) and are required for growth of imatinib-sensitive and resistant GIST cell lines. Activated KIT, through MEK, prolongs ETV1 protein stability and cooperates with ETV1 to promote tumorigenesis. GIST arises from interstitial cells of Cajal (ICCs) exhibiting high levels of endogenous ETV1 expression that, when coupled with an activating KIT mutation, drives an oncogenic ETS transcriptional program. This oncogenic role for ETV1 in GIST differs from classical models of other ETS-dependent tumors such as prostate cancer, melanoma and Ewing sarcoma where genomic translocation or amplification drives aberrant ETS expression and promote tumorigenesis (Tomlins, S.A., et al., 2005, Science, 310(5748):644-8; Mertens, F. et al., 2009, Semin. Oncol., 26, 312-23; Jane-Valbuena, J. et al., 2010, Cancer Res., (70), 2075-84; each of which are incorporated herein by reference). The fact that ETV1 is universally highly expressed in all GISTs makes it immediately useful as a candidate diagnostic biomarker, because the current standard of KIT immunreactivity is negative in about 5% of all GISTs (Miettinen, M. et al., 2006, Arch. Pathol. Lab. Med., 130: 1466-78; incorporated herein by reference).
[0070] The human ETV1 gene comprises approximately 100 kb of Chromosome 7. At least seven different transcript variants of the ETV1 gene have been identified. ETV1 variant 1 (Accession No. NM_004956) is shown in Table 1 as SEQ ID NO: 31. Additional ETV1 variants encompassed by embodiments of the present invention include Accession Nos. NM_001163150, NM_001163151, NM_001163149, NM_001163148, NM_001163147, and NM_001163152 the entire contents of which are incorporated herein by reference.
Table 1: Human ETV1
Protein Sequence (GeneBank: AAD29877.1): (SEQ ID NO: 1)
MDGFYDQQVPYMVTNSQRGRNCNEKPTNVRKRKFINRDLAHDSEELFQDLSQLQETWLAEAQVP DNDEQFVPDYQAESLAFHGLPLKIKKEPHSPCSEISSACSQEQPFKFSYGEKCLYNVSAYDQKP QVGMRPSNPPTPSSTPVSPLHHASPNSTHTPKPDRAFPAHLPPSQSIPDSSYPMDHRFRRQLSE PCNSFPPLPTMPREGRPMYQRQMSEPNIPFPPQGFKQEYHDPVYEHNTMVGSAASQSFPPPLMI KQEPRDFAYDSEVPSCHSIYMRQEGFLAHPSRTEGCMFEKGPRQFYDDTCVVPEKFDGDIKQEP GMYREGPTYQRRGSLQLWQFLVALLDDPSNSHFIAWTGRGMEFKLIEPEEVARRWGIQKNRPAM NYDKLSRSLRYYYEKGIMQKVAGERYVYKFVCDPEALFSMAFPDNQRPLLKTDMERHINEEDTV PLSHFDESMAYMPEGGCCNPHPYNEGYVY
DNA Sequence (GeneBank: NM_004956): (SEQ ID NO: 31)
AAGGAAACCAGCCCAATAGTGGACCTAACACCCCAATAATGGGAACTGGCGACTCGAAACACAC GGTTTCAGTATCCCCAGGCGCCTTAACAATAAGCAGCGGGTTACAGAGGAAATCGAGTCTCTCC GTGGCGCTTCCAGGCTGAGGACCCAGCGCCTACCGGCCGAGCACCCCCTAGGGCAGAGGGGCGA GGGAGACCCGCGGGAAGGGGCAGCAAGCCGCCTGATTGACAGCCCGAAATCTGATCTTGTGAAA GAGACGCGGAGCCAATGGGAGGCAGCGATCCATCAGTTTGGATTGGAGGCCCCTGGAGGAGAAG TAGAGGAAAAACCACCGAATTGAGCTCTGTCAGAGGCGCTTTCGGCTTCCAAGGGGGAAGTGCT GGGCTATAATTAATGTTTTTATTAAATTTGGAGGGAAGTTTTTGCAGCCTTTCGCCTAGCGTGG CCTTCAGTCCCGTTAGGTGCAAAGCAAGTCTCGTTGATCGCCATTGCTAGTTTTGCACACGTTT GCGAATCAGAGCTGCCCGGGGTACACCGACCGCGCAGGGAAACATCGAGAGTGTAAATAAATAC ATCGCCTCTTGTTCGGATTTTTGCTACTACCGAAAATATGTAAATTGTGAACTCTGTTGGCTCT CTTTGGATCCAGGTTGATAGAAGTCCAGATCCTGAGGAAATCTCCAGCTAAATGCTCAAAATAT AAAATACTGAGCTGAGATTTGCGAAGAGCAGCAGCATGGATGGATTTTATGACCAGCAAGTGCC TTACATGGTCACCAATAGTCAGCGTGGGAGAAATTGTAACGAGAAACCAACAAATGTCAGGAAA AGAAAATTCATTAACAGAGATCTGGCTCATGATTCAGAAGAACTCTTTCAAGATCTAAGTCAAT TACAGGAAACATGGCTTGCAGAAGCTCAGGTACCTGACAATGATGAGCAGTTTGTACCAGACTA TCAGGCTGAAAGTTTGGCTTTTCATGGCCTGCCACTGAAAATCAAGAAAGAACCCCACAGTCCA TGTTCAGAAATCAGCTCTGCCTGCAGTCAAGAACAGCCCTTTAAATTCAGCTATGGAGAAAAGT GCCTGTACAATGTCAGTGCCTATGATCAGAAGCCACAAGTGGGAATGAGGCCCTCCAACCCCCC CACACCATCCAGCACGCCAGTGTCCCCACTGCATCATGCATCTCCAAACTCAACTCATACACCG AAACCTGACCGGGCCTTCCCAGCTCACCTCCCTCCATCGCAGTCCATACCAGATAGCAGCTACC CCATGGACCACAGATTTCGCCGCCAGCTTTCTGAACCCTGTAACTCCTTTCCTCCTTTGCCGAC GATGCCAAGGGAAGGACGTCCTATGTACCAACGCCAGATGTCTGAGCCAAACATCCCCTTCCCA CCACAAGGCTTTAAGCAGGAGTACCACGACCCAGTGTATGAACACAACACCATGGTTGGCAGTG CGGCCAGCCAAAGCTTTCCCCCTCCTCTGATGATTAAACAGGAACCCAGAGATTTTGCATATGA CTCAGAAGTGCCTAGCTGCCACTCCATTTATATGAGGCAAGAAGGCTTCCTGGCTCATCCCAGC AGAACAGAAGGCTGTATGTTTGAAAAGGGCCCCAGGCAGTTTTATGATGACACCTGTGTTGTCC CAGAAAAATTCGATGGAGACATCAAACAAGAGCCAGGAATGTATCGGGAAGGACCCACATACCA ACGGCGAGGATCACTTCAGCTCTGGCAGTTTTTGGTAGCTCTTCTGGATGACCCTTCAAATTCT CATTTTATTGCCTGGACTGGTCGAGGCATGGAATTTAAACTGATTGAGCCTGAAGAGGTGGCCC GACGTTGGGGCATTCAGAAAAACAGGCCAGCTATGAACTATGATAAACTTAGCCGTTCACTCCG CTATTACTATGAGAAAGGAATTATGCAAAAGGTGGCTGGAGAGAGATATGTCTACAAGTTTGTG TGTGATCCAGAAGCCCTTTTCTCCATGGCCTTTCCAGATAATCAGCGTCCACTGCTGAAGACAG ACATGGAACGTCACATCAACGAGGAGGACACAGTGCCTCTTTCTCACTTTGATGAGAGCATGGC CTACATGCCGGAAGGGGGCTGCTGCAACCCCCACCCCTACAACGAAGGCTACGTGTATTAACAC AAGTGACAGTCAAGCAGGGCGTTTTTGCGCTTTTCCTTTTTTCTGCAAGATACAGAGAATTGCT GAATCTTTGTTTTATTTCTGTTGTTTGTATTTTATTTTTAAATAATAATACACAAAAAGGGGCT TTTCCTGTTGCATTATTCTATGGTCTGCCATGGACTGTGCACTTTATTTGAGGGTGGGTGGGAG TAATCTAAACATTTATTCTGTGTAACAGGAAGCTAATGGGTGAATGGGCAGAGGGATTTGGGGA TTACTTTTTACTTAGGCTTGGGATGGGGTCCTACAAGTTTTGAGTATGATGAAACTATATCATG TCTGTTTGATTTCATAACAACATAAGATAATGTTTATTTTATCGGGGTATCTATGGTACAGTTA ATTTCACGTTGTGTAAATATCCACTTGGAGACTATTTGCCTTGGGCATTTTCCCCTGTCATTTA TGAGTCTCTGCAGGTGTACAAAAAAACCCCAATCTACTGTAAATGGCAGTTTAATTGTTAGAAA TGACTGTTTTTGCACCACTTGTAAAAAGGTATTTAGCGATTGCATTTGCTGTTTGTTGTTTTAT TTTGCTTTATATATGACTTGCAGAGGATAACCATAAAATGGGTAATTCTCTCTGAAGTTGAATA ATCACCATGACTGTAAATGAGGGGCACAATTTTGGACTCTGGCGCCAAACTGAGTCATAGGCCA GTAGCATTACGTGTATCTGGTGCCACCTTGCTGTTTAGATACAAATCATACCGTCTTTTAAATA TTTTGAAGCCCATTTCAGTTAAATAATGACATGTCATGGTCCTTTGGAATCTTCATTTAAATGT TAAATCTGGAATCAAAATGAAGCAAAAAATATCTGTCTCCTTTTCACTTTCTTCAGTACATAAA TACATTATTTAATCAATAAGAATTAACTGTACTAAATCATGTATTATGCTGTTCTAGTTACAGC AAACACTCTTTAAGAAAAATATCCAATACACTAAATAGGTACTATAGTAATTTTTAGACATGGT ACCCATTGATATGCATTTAAACCTTTTACTGCTGTGTTATGTTGATAACATATATAAATATTAG ATAATGCTAATGCTTCTGCTGCTGTCTTTTCTGTAATATTCTCTTTCATGCTGAATTTACTATG ACCATTTATAAGCAGTGCAGTTAACTACAGATAGCATTTCAGGACAAAATAGATGACTCAAACC ATTTATTGCTTAAAAAATAGCTTACGCCATGCTATGCTATAAGCAGCTTTTATGCACATTGACA AATGAAGAGTAAGCTTCAGCTTGCTAAAGGAAACTGTGGAACCTTTTGTAACTTTTGGTGATAT GGAAAATTATTTACAAACCGTCAAAGAATATGAGGAAGTTGCTGTATGACATAGTGCTGGCACT GATATTATCCATCATCTCTTTTTGGACACTTCTGTAAATGTGATTGGATTGTTTGAAAGAAGAT TTAAAGTTTCAAAGTTTTTTGTTCTGTTTTTGCTTTGCATTTGGAGAAAATATTGAAAGCAGGG TATGTTGTTTCATTCACCTTGAAAAAACCATGAGTAAATGGGGATATAGAATCTCTGAATAGCT CGCTAAAAGATTCAAGCAAGGGACATGAATTTTGTTCCATCTATCAATAATATCCAGAAGAACA ACTTTTTTAAAGAGTCTATAGCAAAAAGCAAAAAAAAAAAAAAATTCTAAACACAAAGTCAAAA TAAACCTATTGTAAAAGCATTTCGTGATGAGCATGAAAAAGATTGTTTAAAGATGATCCCCCCA GCTACCCATTTTCCAAAACTACACAGATCACAGCTCATTTCTCTAAGTGGAGCAGTTATCAAGA AACCCAAACACCAAAATTGCTACTCTTCACATTTAATCCTACAAAAAGTACTCCAATTTCAAAA TATGTATGTAACCTGCGATTTCAATGATTGTTGTTCATATACATCATGTATTATTTTGGCCCAT TTTGGGCCTAAAAAAGAAAACTATGCCTTAAAAATCAGAACCTTTTCTCCCCACTATGCTTATG TGGCCATCTACAGCACTTAGAATAAAAACAGATGTTAAAATATTCAGTGAAAGTTTTATTGGAA AAAGGAATTGAGATATATAATTGAGATTTGGTGAAATTGAAGGAGAAAATTTAAGTGAGTCTTT AAAATATATTCTGAATGAAAACTGTATTGAGGATTCATTTTTGTTCCTTTTTTTTCTTTTTCTC TTTTCTCCTTTTTCTTCTTTTTAATAGTCTAGTTTTAGTCAGTCAGTGAGGAAGAATTGGGCCA TGCTAACGTTATCACAAGAGAACAATGGCAGAAATGGTATTAGTTATATAATATTTAAGGACAA ACTATATGTTTTGCTGTTTTAACGTAGTGACTCACTGAACTAAATACATAATTGACCAACATTA AGTGTATTTCCAATACAGAAGGGTTGAAAATATTACATTATAAACTCTTTTGAAAAATGTATCT AAAATTTTTTAAGTTCTGTTTTGATTCCACTTTTTGGTTGAGTTTTTATGTTTTTGTTTTCAGG TAGATTAATAAATCTGGCAGCTGATTTCTGCAAGATTCTTGTGTTTTGAATTTCTCATTGAATT GGCTACTCAAACATAGAAATCATTTGTTAATGATGTAATGTCTTCTCTCAGCTTTTATCTTCAC TGCTGTTTGCTGTCTCTTGATGATGACATGTTAATACCCAATAGATTAATTGCAACAAACACTT ATACTCAAATAACTAAGTAAAAATAATTTTTCTTGTTATGTCCATGAAAAGTGCTTCAGAATAA AAATCCACAAGACTGACAGTGCAGAACATTTTTCTCAAATCATGGGCGGATCTTGGAGGTCTAG TTTCCCGTAGATGCTGTAACCAATTACCACAACTTCAGTAATTTACACAAATTTATCTTATAGT TCTGGAGGCAGAAGTTCAAAAGAAGCCTTAAGAGACTAAAACCAAGATGTCCTTAGGTCTGGTT CCTTCTGGAGGCTCCAGGGGAGATTCTTCCAGCTTTCACTTCTAGAGTCTGCTGACATTCCTTG GCTCCTGGCTACATCACTTCAATCTCTGCTTCCATGGTCACATACTCTTCTACTATAGTCAAAT TTCCTTCCTGCCTCTTATAAGGATGCTTGTGATTACATTTAGGGGATGCTCAGATAATCCAGGA CAATCTCTCCATCTCAAGATCCTTAACTTAATGACGTGTGCCAAGTCCCTTTGGCTAGATAATT ATTCATAGGTCCCAGGGATTAGGACATGGATGTAAGGGGTGAGGGCAGGGCTGTTATTCAGAAC ACCGCACGGAGGAGGAAGACTGTGTAGCAAAGACTCTAATTGATTTACTCAGGAACAGTGGAGT TCTGCTGAGGGATCTAGGATTTGAAAGTACTAGAGTTTGCTTTTATTTACCACTGAGATATTTT CCCCTTATTCTGCATAAATAATTTTGAAAACTTTCTATATTAAATTTCAACTATTCCACTAAAA TGTCTGGTAATCACATCAAGCCTTTAGATTATTCAAATCCTTCCCCAGCCCCCAGGAAAACACT AAGTCATGAAACAGAAAAACAGAAGGTATGATAATAATAGTAATAACAGTTAAATCAGTGGTCT AATCCAGATTTTATTTTTTAATACATTTCTTTTGGTGTTAATATGGGTTACTATGTGATCTTAT CATTTGCTAGTGATTATTACTTATTAGGTAAGAACAATGTGTAAAATATGTCTATTACTCAAAA GAACAATTGCAAAATGAGTCAACTTATCTTTATATAACCAGGAAAGAAATATATTGCCAGAAGC TACAGAATTTTGCCAGATGATAGGGATTTCTAAAATGAGCCACTTTGTCTATCATGCAGCCTTT TCAGAGCTTGTAATGAGAAAACATTACAGAGGAGAAGGTCATTTGGATGTTTGTTACTTGGAAT CCTAGAAAACAAAAACTAAAATTTAAAAATAAGAAGTGAGTAAGCTATTTTCCATTTGCGATTT GGTATGGAGAAGAGAGGAAATAGAATTATTAAAAAAATACAAATTGGGTAAAAGTGATGGTGGA AAAAATATAAAGAAGGCAAATGTACATATTAAGCAATTCTACTAAGAATTGGAAAAATCAAGTT TCAAAAAGATGGTAATAGTTGGGCATGATACTAGAAAATTTCACCCAGTTTATTCAGAGCTCAA CTAGTACTTTTAGGACTTCTTTTTTTATATACATGAGACTCACTTTGACATACTTAAAAAAAAA ACAGTTTATGGAAAGTACAGTTTAAGAGGAGAATTTGATTAGACTAAGTGGATATCTTTATAGA AATATTAATGATTTCAGAATTTTCAGTTACAAGTGTATATACCGTGGCTATTGTTTATGGATTC ATATGTAAGGTAGGGTCTTTTTTGCATATAGACTCCAGTATTAGTTACTTTCATTCTAAAATTA TATTTATGCTTCTATGGGGAAGAAAATTTTTAATTCACTTGGTTGTATTAAAATTATACTTACG GTTTGAGAAAACATGCTATGAAAATCATGATTATAGCAAATTAAATATGCTCAAAATTTAAATC TAAAATAAAAGCCCAGAAACTGAAAAAAAAAAAAAAAAAA
[0071] Embodiments of the invention are based on the surprising discovery that ETV1 is highly expressed in all GISTs and at significanly higher levels than any other tumor type. Analysis of ETVl mRNA and protein levels in GIST and other sarcomas in clinical samples has proven that ETVl mRNA and protein are highly and exclusively expressed in all GIST tumor samples and GIST cell lines. Moreover, RNAi techniques used to reduce EVTl translation have proven that ETVl is required for GIST growth and survival. In situ hybidization analyses of GIST samples and cell lines further indicates that high levels of ETVl expression are
consistently observed in ICC hyperplasia in the absence of obvious genomic alterations.
Transcriptome profiling and global analyses of ETVl binding sites suggest that EVTl is a master regulator of an ICC-GIST-specific transcription network through mediation of enhancer binding.
3. Diagnostics and methods of use thereof
[0072] The function of ETVl as a master regulator facilitates diagnostic uses of ETVl expression and activity levels. It is well known in the art that the gene expression profile of a tumor or subclass of tumor may be associated with different prognoses. Such information may include, but is not limited to, the average life expectancy of a patient, the likelihood that a patient will survive for a given amount of time (e.g., 6 months, 1 year, 5 years, etc.), the likelihood that a patient will be cured of a disease, the likelihood that a patient's disease will respond to a particular therapy (wherein response may be defined in any of a variety of ways). For example, differences in the prognosis of patients with KIT positive and PDGFRA positive GISTs have been described.
[0073] Methods of the present invention utilize a novel parameter for diagnosis and prognosis of GIST cancers: ETVl expression and/or mutation. In a particular embodiment, a method is provided for determining the liklihood of gastrointestinal stromal tissue being cancerous and/or afflicted by a GIST. The method comprises determining a level of expression or activity of ETVl in a gastrointestinal stromal tissue sample obtained from a subject;
comparing the determined ETVl level with that of a reference correlated with predetermined probablity of being cancerous and/or afflicted by a GIST; and based on the comparing, determining that the tissue sample has an increased or decreased probability, relative to the reference, of being cancerous and/or afflicted by a GIST. Moreover, the present invention offers the possibility of providing additional diagnostic, prognostic, or predictive information based on modification of existing protocols to include ETV1.
[0074] The present invention also offers the possibility of analyzing tumor sample archives containing tissue samples that were obtained from patients and stored with information regarding the progress of the patient's disease. In general such archives consist of tumor samples embedded in paraffin blocks. These tumor samples can be analyzed for their expression of polypeptides encoded by the marker genes of the present invention, particularly ETV1. For example, immunohistochemistry can be performed using antibodies that bind to the
polypeptides. Tumors with elevated, mutated or otherwise aberrant expression may then be identified and correlated with the severity of the tumor, responses to therapy and other avaible clincial information, e.g., age at death, length of survival, etc. ETV1 expression in the samples may be quantitated or semi-quantitated relative to a control. The information derived from these samples may be used to construct a reference database or library. In particular, the average expression across a population or subpopulation of normal and afflicted individuals may be determined, thereby creating a reference correlated with a predetermined probability of being cancerous. Once suitable prognostic or predictive correlations are identified, a patient's likely outcome can be predicted based on the level and type of ETV1 expression in his or her tumor.
[0075] In some embodiments, ETV1 expression analysis comprises identifying whether ETV1 (the gene or gene product) is upregulated or downregulated relative to a reference or baseline. The reference may be derived from average expression across a population of individuals or may be single prior sample derived from the subject. The analyses of samples from single individuals or across a population (i.e., large group of individuals) provides a library of data sets for use as a reference or baseline. Differential expression relative to the reference can then be determined, which provides a qualitative or quantitative comparative value that is informative about the diagnosis of GIST, long-term prognosis, response to therapy, etc. The differential expression values for ETVl can be cross-referenced with analyses of other oncogenic GIST events (e.g., KIT mutations) to provide a synergistic diagnosis. The differential expression values relative to a reference may also be annotated with medical information about the subject.
[0076] Determining whether the tissue sample has an increased or decreased probability, relative to the reference, of being cancerous or GIST can be performed using a statistical test to determine statistical significance of any differential expression observed. In some embodiments, statistical significance is determined using a parametric statistical test. The parametric statistical test can comprise, for example, a fractional factorial design, analysis of variance (ANOVA), a t- test, least squares, a Pearson correlation, simple linear regression, nonlinear regression, multiple linear regression, or multiple nonlinear regression. Alternatively, the parametric statistical test can comprise a one-way analysis of variance, two-way analysis of variance, or repeated measures analysis of variance. In other embodiments, statistical significance is determined using a nonparametric statistical test. Examples include, but are not limited to, a Wilcox on signed- rank test, a Mann- Whitney test, a Kruskal-Wallis test, a Friedman test, a Spearman ranked order correlation coefficient, a Kendall Tau analysis, and a nonparametric regression test. In some embodiments, statistical significance is determined at a p-value of less than about 0.05, 0.01, 0.005, 0.001, 0.0005, or 0.0001.
[0077] The degree of differential expression can also be taken into account. For example, ETVl can be considered as differentially expressed when the fold-change in expression compared to control level is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 2.7, 3.0, 4, 5, 6, 7, 8, 9 or 10-fold different in the sample versus the control. The differential expression takes into account both overexpression and underexpression. A gene or gene product can be considered up or downregulated if the differential expression meets a statistical threshold, a fold- change threshold, or both. For example, the criteria for identifying differential expression can comprise both a p-value of 0.001 and fold change of at least 2.0-fold (up or down).
[0078] In some embodiments, the reference to which differential expression of ETVl is determined is a normal gene. For example, the reference may be a housekeeping gene such as 18S rRNA or GAPDH. In alternative embodiments, differential expression may be determined relative to ETVl expression in noncancerous gastrointestinal ICCs derived from healthy subjects. In other embodiments, differential expression may be determined relative to ETVl expression in cancerous ICCs cells or GIST cell lines. Fold changes in ETVl expression may be calculated by the delta-delta Ct method (M.W. Pfaffl, A new mathematical model for relative quantification in real-time RT-PCR, Nucleic Acids Res. 29 (2001) e45) or other methods known to those of skill in the art.
[0079] In one aspect, the invention provides a method of determining ETVl mRNA expression, polypeptide levels or activity and correlating the results obtained therein with a diagnosis or prognostic assessement of GIST cancer and severity. Generally, these methods include a step of detecting expression or activity of a gene encoding a ETVl polypeptide. In certain embodiments it may prove advantageous to combine detection of the ETVl marker with detection of the KIT and/or PDGFRA markers. The gene products are detected in a tumor sample and can be polypeptides or polynucleotides, e.g., mRNA. a. Nucleic acid analysis
[0080] ETVl detection at the nucleic acid level is also encompassed by embodiments of the present invention. Nucleic acid analyses can be performed on genomic DNA, messenger RNAs, and/or cDNA. In many embodiments, nucleic acids are extracted from a biological sample. In some embodiments, nucleic acids are analyzed without having been amplified. In some embodiments, nucleic acids are amplified using techniques known in the art (such as polymerase chain reaction (PCR)) and amplified nucleic acids are used in subsequent analyses. Multiplex PCR, in which several amplicons (e.g., from different genomic regions) are amplified at once using multiple sets of primer pairs, may be employed, (see generally, Bustin, S.A., 2000 J. Molecular Endocrinology 25: 169-193.)
[0081] Microarray analysis is one means by which polynucleotides can be used to detect or measure gene expression. Expression of a gene can also be measured by a variety of techniques that make use of a polynucleotide corresponding to part or all of the gene rather than a binding agent for a polypeptide encoded by the gene. Appropriate techniques include, but are not limited to, in situ hybridization, Northern blot, and various nucleic acid amplification techniques such as PCR, rtPCR, quantitative rtPCR, and the ligase chain reaction. Quantitative rtPCR and in situ hybridization technique are discussed in further detail in the examples below. PCR and considerations for primer design are well known in the art and are described, for example, in Newton, et al. (eds.) PCR: Essential data Series, John Wiley & Sons; PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1995; White, et al.. (eds.) PCR Protocols: Current methods and Applications, Methods in Molecular Biology, The Humana Press, Totowa, NJ, 1993. b. Polypeptide analysis
[0082] As is well known in the art, a polypeptide may be detected using any of a variety of techniques and binding agents. Any such technique and agent may be used according to the present invention. In certain preferred embodiments, the binding agent is an antibody that binds specifically to ETV1 or KIT. The invention also encompasses the use of protein arrays, including antibody arrays, for detection of a polypeptide. The use of antibody arrays is described, for example, in Haab et al., "Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions", Genome Biol. 2(2):2001, 2001. Other types of protein arrays are known in the art. In general, antibodies that bind specifically to ETV1 or KIT may be generated by methods well known in the art and described, for example, in Harlow, E, Lane, E, and Harlow, E, (eds.) Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1998. Details and references for the production of antibodies may also be found in U.S. Patent No. 6,008,337. Antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric (e.g.,
"humanized"), single chain antibodies, Fab fragments, antibodies generated using phage display technology, etc. The invention encompasses the use of "fully human" antibodies produced using the XenoMouse™ technology (AbGenix Corp., Fremont, CA) according to the techniques described in U.S. Patent No. 6,075,181. Polyclonal ETV1 antibodies are commercially available (e.g., rabbit polyclonal antibodies ab81806 from Abeam of Cambridge, MA and PRB-326C from Covance). Antibody detection methods are well known in the art including, but are not limited to, enzyme-linked immunosorbent assays (ELISAs) and Western blots. Some such methods are amenable to being performed in an array format.
[0083] In addition, in certain embodiments of the invention the polypeptides are detected using other specific binding agents known in the art for the detection of polypeptides, such as aptamers (Aptamers, Molecular Diagnosis, Vol. 4, No. 4, 1999), reagents derived from combinatorial libraries for specific detection of proteins in complex mixtures, random peptide affinity reagents, etc. In general, any appropriate binding agent for detecting a polypeptide may be used in conjunction with the present invention, although antibodies may represent a particularly appropriate modality.
[0084] In certain embodiments of the inventive methods, a single binding agent (e.g., antibody) is used whereas in other embodiments of the invention multiple binding agents, directed either against the same or against different polypeptides (e.g., ETV1 or KIT) can be used to increase the sensitivity or specificity of the detection technique or to provide more detailed information than that provided by a single binding agent. Thus the invention encompasses the use of a battery of binding agents that bind to polypeptides encoded by the marker genes identified herein. Of course these agents can also be used in conjunction with binding agents against polypeptides encoded by other useful marker genes (e.g., CD34 when used with GISTs).
[0085] In general, the polypeptides are detected within a tumor sample that has been obtained from a subject, e.g., a tissue sample, cell sample, cell extract, body fluid sample, etc. The invention encompasses the recognition that polypeptides encoded by EDTV1 at various levels (optionally in conjuction with KIT polypeptides) may be present in serum, enabling their detection through a blood test rather than requiring a biopsy specimen. One of ordinary skill in the art will readily be able to develop appropriate assays for polypeptides encoded by the marker genes described herein and to apply them to the detection of such polypeptides in serum. Similar methods may be applied to other body fluid samples, e.g., ascites, urine, saliva, etc.
[0086] In certain embodiments, binding can be detected by adding a detectable label to the binding agent. In other embodiments, binding can be detected by using a labeled secondary binding agent that associates specifically with the primary binding agent, e.g., as is well known in the art of antigen/antibody detection. The detectable label may be directly detectable or indirectly detectable, e.g., through combined action with one or more additional members of a signal producing system. Examples of directly detectable labels include radioactive,
paramagnetic, fluorescent, light scattering, absorptive and colorimetric labels. Indirectly detectable labels include chemiluminescent labels, e.g., enzymes that are capable of converting a substrate to a chromogenic product such as alkaline phosphatase, horseradish peroxidase and the like.
[0087] Once a labeled binding agent has bound a polypeptide marker, the complex may be visualized or detected in a variety of ways, with the particular manner of detection being chosen based on the particular detectable label. Representative detection means include, e.g., scintillation counting, autoradiography, measurement of paramagnetism, fluorescence measurement, light absorption measurement, measurement of light scattering and the like.
Depending upon the nature of the sample, appropriate detection techniques include, but are not limited to, immunohistochemistry (IHC), radioimmunoassay, ELISA, immunoblotting and fluorescence activated cell sorting (FACS). In the case where the polypeptide is to be detected in a tissue sample, e.g., a biopsy sample, IHC is a particularly appropriate detection technique.
[0088] In certain embodiments, the detection techniques of the present invention will include a negative control, which can involve applying the test to a control sample (e.g., from a normal non-cancerous tissue) so that the signal obtained thereby can be compared with the signal obtained from the tumor sample being tested. In tests in which a secondary binding agent is used to detect a primary binding agent that binds to the polypeptide of interest, an appropriate negative control can involve performing the test on a portion of the sample with the omission of the primary binding agent.
[0089] In general, the results of the inventive detection techniques can be presented in any of a variety of formats. The results can be presented in a qualitative fashion. For example, the test report may indicate only whether or not a particular polypeptide marker was detected, perhaps also with an indication of the limits of detection. The results may be presented in a semiquantitative fashion. For example, various ranges may be defined, and the ranges may be assigned a score (e.g., 0 to 3 where 0 means no binding detected and 3 means strong binding detected) that provides a certain degree of quantitative information. Such a score may reflect various factors, e.g., the number of cells in which the polypeptide is detected, the intensity of the signal (which may indicate the level of expression of the polypeptide), etc. The results may be presented in a quantitative fashion, e.g., as a percentage of cells in which the polypeptide is detected, as a protein concentration, etc. As will be appreciated by one of ordinary skill in the art, the type of output provided by a test will vary depending upon the technical limitations of the test and the biological significance associated with detection of the polypeptide. For example, in the case of one polypeptide marker a purely qualitative output (e.g., whether or not the polypeptide is detected at a certain detection level) provides significant information. In another case a more quantitative output (e.g., a ratio of the level of expression of the polypeptide in the sample being tested versus the normal level) is necessary. c. Detectable moieties
[0090] As noted above, in certain embodiments, certain molecules (e.g., nucleic acid probes, antibodies, etc.) used in accordance with and/or provided by the invention comprise one or more detectable entities or moieties, i.e., such molecules are "labeled" with such entities or moieties.
[0091] Any of a wide variety of detectable agents can be used in the practice of the present invention. Suitable detectable agents include, but are not limited to: various ligands, radionuclides; fluorescent dyes; chemiluminescent agents (such as, for example, acridinum esters, stabilized dioxetanes, and the like); bioluminescent agents; spectrally resolvable inorganic fluorescent semiconductors nanocrystals (i.e., quantum dots); microparticles; metal nanoparticles (e.g., gold, silver, copper, platinum, etc.); nanoclusters; paramagnetic metal ions; enzymes; colorimetric labels (such as, for example, dyes, colloidal gold, and the like); biotin; dioxigenin; haptens; and proteins for which antisera or monoclonal antibodies are available.
[0092] In some embodiments, the detectable moiety is biotin. Biotin can be bound to avidins (such as streptavidin), which are typically conjugated (directly or indirectly) to other moieties (e.g., fluorescent moieties) that are detectable themselves.
[0093] Below are described some non-limiting examples of other detectable moieties. i. Fluorescent dyes
[0094] In certain embodiments, a detectable moiety is a fluorescent dye. Numerous known fluorescent dyes of a wide variety of chemical structures and physical characteristics are suitable for use in the practice of the present invention. A fluorescent detectable moiety can be stimulated by a laser with the emitted light captured by a detector. The detector can be a charge- coupled device (CCD) or a confocal microscope, which records its intensity.
[0095] Suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'- dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., carboxytetramethyl- rhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine Red, tetramethylrhodamine (TMR), etc.), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin,
hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon Green Dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514., etc.), Texas Red, Texas Red-X, SPECTRUM REDTM, SPECTRUM GREENTM, cyanine dyes (e.g., CY-3TM, CY-5TM, CY-3.5TM, CY- 5.5TM, etc.), ALEXA FLUORTM dyes (e.g., ALEXA FLUORTM 350, ALEXA FLUORTM 488, ALEXA FLUORTM 532, ALEXA FLUORTM 546, ALEXA FLUORTM 568, ALEXA FLUORTM 594, ALEXA FLUORTM 633, ALEXA FLUORTM 660, ALEXA FLUORTM 680, etc.), BODIPYTM dyes (e.g., BODIPYTM FL, BODIPYTM R6G, BODIPYTM TMR,
BODIPYTM TR, BODIPYTM 530/550, BODIPYTM 558/568, BODIPYTM 564/570,
BODIPYTM 576/589, BODIPYTM 581/591, BODIPYTM 630/650, BODIPYTM 650/665, etc.), IRDyes (e.g., IRD40, IRD 700, IRD 800, etc.), and the like. For more examples of suitable fluorescent dyes and methods for coupling fluorescent dyes to other chemical entities such as proteins and peptides, see, for example, "The Handbook of Fluorescent Probes and Research Products", 9th Ed., Molecular Probes, Inc., Eugene, OR. Favorable properties of fluorescent labeling agents include high molar absorption coefficient, high fluorescence quantum yield, and photostability. In some embodiments, labeling fluorophores exhibit absorption and emission wavelengths in the visible (i.e., between 400 and 750 nm) rather than in the ultraviolet range of the spectrum (i.e., lower than 400 nm). [0096] A detectable moiety may include more than one chemical entity such as in fluorescent resonance energy transfer (FRET). Resonance transfer results an overall enhancement of the emission intensity. For instance, see Ju et. al. (1995) Proc. Nat'l Acad. Sci. (USA ) 92: 4347, the entire contents of which are herein incorporated by reference. To achieve resonance energy transfer, the first fluorescent molecule (the "donor" fluor) absorbs light and transfers it through the resonance of excited electrons to the second fluorescent molecule (the "acceptor" fluor). In one approach, both the donor and acceptor dyes can be linked together and attached to the oligo primer. Methods to link donor and acceptor dyes to a nucleic acid have been described previously, for example, in U.S. Pat. No. 5,945,526 to Lee et al., the entire contents of which are herein incorporated by reference. Donor/acceptor pairs of dyes that can be used include, for example, fluorescein/tetramethylrohdamine, IAEDANS/fluroescein, EDANS/DABCYL, fluorescein/fluorescein, BODIPY FL/BODIPY FL, and Fluorescein/ QSY 7 dye. See, e.g., U.S. Pat. No. 5,945,526 to Lee et al. Many of these dyes also are commercially available, for instance, from Molecular Probes Inc. (Eugene, Oreg.). Suitable donor fluorophores include 6- carboxyfluorescein (FAM), tetrachloro-6-carboxyfluorescein (TET), 2'-chloro-7'-phenyl-l,4- dichloro-6-carboxyfluorescein (VIC), and the like. ii. Enzymes
[0097] In certain embodiments, a detectable moiety is an enzyme. Examples of suitable enzymes include, but are not limited to, those used in an ELISA, e.g., horseradish peroxidase, beta-galactosidase, lucif erase, alkaline phosphatase, etc.. Other examples include beta- glucuronidase, beta-D-glucosidase, urease, glucose oxidase, etc. An enzyme may be conjugated to a molecule using a linker group such as a carbodiimide, a diisocyanate, a glutaraldehyde, and the like.
Hi. Radioactive isotopes [0098] In certain embodiments, a detectable moiety is a radioactive isotope. For example, a molecule may be isotopically-labeled (i.e., may contain one or more atoms that have been replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature) or an isotope may be attached to the molecule. Non- limiting examples of isotopes that can be incorporated into molecules include isotopes of hydrogen, carbon, fluorine, phosphorous, copper, gallium, yttrium, technetium, indium, iodine, rhenium, thallium, bismuth, astatine, samarium, and lutetium (i.e., 3H, 13C, 14C, 18F, 19F, 32P, 35S, 64Cu, 67Cu, 67Ga, 90Y, 99mTc, l l lln, 1251, 1231, 1291, 1311, 1351, 186Re, 187Re, 201T1, 212Bi, 213Bi, 211At, 153Sm, 177Lu). iii. Labeled dendrimers
[0099] In some embodiments, signal amplification is achieved using labeled dendrimers as the detectable moiety (see, e.g., Physiol Genomics 3:93-99, 2000), the entire contents of which are herein incorporated by reference. Fluorescently labeled dendrimers are available from Genisphere (Montvale, NJ). For example, these may be chemically conjugated to nucleic acid primers by methods known in the art. d. Detection of mutations
[00100] The invention also encompasses the detection of mutations within ETV1. In certain embodiments of the invention, detection of mutations can be used to classify the severity of mutation and, therefore, the prognosis of the subject in response to coventional or novel treatments disclosed herein. Mutations may include, but are not limited to, deletions, additions, substitutions, and amplification of regions of genomic DNA that include all or part of a gene. Methods for detecting such mutations are well known in the art and include direct sequencing, denaturing HPLC and combinations thereof. (Segal, N. H. et ah, 2003, Am J Pathol, 163: 691- 700, incorporated herein by reference; Visel, A. et al., 2009, Nature, 457: 854-858, incorporated herein by reference.) Mutations may result in overexpression or inappropriate expression of ETV1 (i.e., aberrant expression). Additionally or alternatively mutations may result in an overly activated gene product (e.g., polypeptide). In addition, mutation analysis of particular ETV1- relevant genes or proteins may provide further diagnostic or prognostic information. For example, diagnostic KIT mutations have been identified within mutations in ex on 9, 11, 13 or
17 11 '12. (Jane-Valbuena, J. et al., 2010, Cancer Res, 70: 2075-2084, incorporated herein by reference; Nielsen, T. O. et al., 2002, Lancet, 359: 1301-1307, incorporated herein by reference.)
[00101] ETV1 mutations may be analyzed by the exemplary methods below. i. Allele-specific amplification
[00102] In some embodiments, gene mutations are detected using an allele-specific amplification assay. This approach is variously referred to as PCR amplification of specific allele (PASA) (Sarkar, et al., 1990 Anal. Biochem. 186:64-68), allele-specific amplification (ASA) (Okayama, et al., 1989 J. Lab. Clin. Med. 114: 105-113), allele-specific PCR (ASPCR) (Wu, et al. 1989 Proc. Natl. Acad. Sci. USA. 86:2757-2760), and amplification-refractory mutation system (ARMS) (Newton, et al., 1989 Nucleic Acids Res. 17:2503-2516). The entire contents of each of these references is incorporated herein by reference. This method is applicable for single base substitutions as well as micro deletions/insertions.
[00103] For example, for PCR-based amplification methods, amplification primers may be designed such that they can distinguish between different alleles (e.g., between a wild-type allele and a mutant allele). Thus, the presence or absence of amplification product can be used to determine whether a gene mutation is present in a given nucleic acid sample. In some embodiments, allele specific primers can be designed such that the presence of amplification product is indicative of a gene mutation. In some embodiments, allele specific primers can be designed such that the absence of amplification product is indicative of a gene mutation. [00104] In some embodiments, two complementary reactions are used. One reaction employs a primer specific for the wild type allele ("wild-type-specific reaction") and the other reaction employs a primer for the mutant allele ("mutant-specific reaction"). The two reactions may employ a common second primer. PCR primers specific for a particular allele (e.g., the wild- type allele or mutant allele) generally perfectly match one allelic variant of the target, but are mismatched to other allelic variant (e.g., the mutant allele or wild-type allele). The mismatch may be located at/near the 3' end of the primer, leading to preferential amplification of the perfectly matched allele. Whether an amplification product can be detected from one or in both reactions indicates the absence or presence of the mutant allele. Detection of an amplification product only from the wild-type-specific reaction indicates presence of the wild-type allele only (e.g., homozygosity of the wild-type allele). Detection of an amplification product in the mutant- specific reaction only indicates presence of the mutant allele only (e.g. homozygosity of the mutant allele). Detection of amplification products from both reactions indicate (e.g., a heterozygote). As used herein, this approach will be referred to as "allele specific amplification (ASA)."
[00105] Allele-specific amplification can also be used to detect duplications, insertions, or inversions by using a primer that hybridizes partially across the junction. The extent of junction overlap can be varied to allow specific amplification.
[00106] Amplification products can be examined by methods known in the art, including by visualizing (e.g., with one or more dyes) bands of nucleic acids that have been migrated (e.g., by electrophoresis) through a gel to separate nucleic acids by size. ii. Allele-specific primer extension
[00107] In some embodiments, an allele-specific primer extension (ASPE) approach is used to detect gene mutations. ASPE employs allele-specific primers that can distinguish between alleles (e.g., between a mutant allele and a wild-type allele) in an extension reaction such that an extension product is obtained only in the presence of a particular allele (e.g., mutant allele or wild-type allele). Extension products may be detectable or made detectable, e.g., by employing a labeled deoxynucleotide in the extension reaction. Any of a variety of labels are compatible for use in these methods, including, but not limited to, radioactive labels, fluorescent labels, chemiluminescent labels, enzymatic labels, etc. In some embodiments, a nucleotide is labeled with an entity that can then be bound (directly or indirectly) by a detectable label, e.g., a biotin molecule that can be bound by streptavidin-conjugated fluorescent dyes. In some embodiments, reactions are done in multiplex, e.g., using many allele-specific primers in the same extension reaction.
[00108] In some embodiments, extension products are hybridized to a solid or semi-solid support, such as beads, matrix, gel, among others. For example, the extension products may be tagged with a particular nucleic acid sequence (e.g., included as part of the allele-specific primer) and the solid support may be attached to an "anti-tag" (e.g., a nucleic acid sequence
complementary to the tag in the extension product). Extension products can be captured and detected on the solid support. For example, beads may be sorted and detected. One such system that can be employed in this manner is the LUMINEXTM MAP system, which can be adapted for cystic fibrosis mutation detection by TM Bioscience and is sold commercially as a universal bead array (TAG-ITTM).
[00109] Additional ASPE methods and reagents are described in, e.g., U.S. Patent Publication No. 2008/0138803 Al, the entire contents of which are herein incorporated by reference.
Hi. Single nucleotide primer extension
[00110] In some embodiments, a single nucleotide primer extension (SNuPE) assay is used, in which the primer is designed to be extended by only one nucleotide. In such methods, the identity of the nucleotide just downstream (e.g., 3') of the 3' end of the primer is known and differs in the mutant allele as compared to the wild-type allele. SNuPE can be performed using an extension reaction in which the only one particular kind of deoxynucleotide is labeled (e.g., labeled dATP, labeled dCTP, labeled dGTP, or labeled dTTP). Thus, the presence of a detectable extension product can be used as an indication of the identity of the nucleotide at the position of interest (e.g., the position just downstream of the 3' end of the primer), and thus as an indication of the presence or absence of a mutation at that position. SNuPE can be performed as described in U.S. Pat. No. 5,888,819; U.S. Pat. No. 5,846,710; U.S. Pat. No. 6,280,947; U.S. Pat. No. 6,482,595; U.S. Pat. No. 6,503,718; U.S. Pat. No. 6,919,174; ; Piggee, C. et al. Journal of Chromatography A 781 (1997), p. 367-375 ("Capillary Electrophoresis for the Detection of Known Point Mutations by Single-Nucleotide Primer Extension and Laser-Induced Fluorescence Detection"); Hoogendoom, B. et al., Human Genetics (1999) 104:89-93, ("Genotyping Single Nucleotide Polymorphism by Primer Extension and High Performance Liquid
Chromatography")), the entire contents of each of which are herein incorporated by reference.
[00111] In some embodiments, primer extension can be combined with mass spectrometry for accurate and fast detection of the presence or absence of a mutation. See, U.S. Pat. No.
5,885,775 to Haff et al. (analysis of single nucleotide polymorphism analysis by mass spectrometry); U.S. Pat. No. 7,501,251 to Koster (DNA diagnosis based on mass spectrometry); the teachings of both of which are incorporated herein by reference. Suitable mass spectrometric format includes, but is not limited to, Matrix-Assisted Laser Desorption/Ionization, Time-of- Flight (MALDI-TOF), Electrospray (ES), IR-MALDI, Ion Cyclotron Resonance (ICR), Fourier Transform, and combinations thereof. iv. Oligonucleotide ligation assay
[00112] In some embodiments, an oligonucleotide ligation assay ("OLA" or "OL") is used. OLA employs two oligonucleotides that are designed to be capable of hybridizing to abutting sequences of a single strand of a target molecules. Typically, one of the oligonucleotides is biotinylated, and the other is detectably labeled, e.g., with a streptavidin-conjugated fluorescent moiety. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate that can be captured and detected. See e.g., Nickerson et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:8923-8927, Landegren, U. et al. (1988) Science 241: 1077-1080 and U.S. Pat. No. 4,998,617, the entire contents of which are herein incorporated by reference in their entirety. v. Hybridization approach
[00113] In some embodiments, nucleic acids are analyzed by hybridization using one or more oligonucleotide probes specific for a region in the gene corresponding to one or more ETV1 mutations, and under conditions sufficiently stringent to disallow a single nucleotide mismatch. In certain embodiments, suitable nucleic acid probes can distinguish between a normal gene and a mutant gene containing one or more mutations. For example, suitable nucleic acid probes specifically bind to a normal gene but not to a mutant gene. Alternatively, nucleic acid probes specifically bind to an ETV1 mutant gene containing one or more mutations but not to a normal gene. Probes of the present invention include those that are capable of specifically hybridizing a mutant ETV1 allele containing one or more mutations. Probes of the present invention also include those that are capable of specifically hybridizing a normal allele in a particular region of the ETV1 gene and therefore capable of distinguishing a normal allele from a mutant ETV1 allele. Thus, for example, one of ordinary skill in the art could use probes of the invention to determine whether an individual is homozygous or heterozygous for a particular allele.
[00114] Nucleic acid hybridization techniques are well known in the art. See, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions such that sequences having at least a desired level of complementary will stably hybridize, while those having lower complementary will not. For examples of hybridization conditions and parameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press,
Plain view, N.Y.; Ausubel, F. M. et al. 1994, Current Protocols in Molecular Biology. John Wiley
& Sons, Secaucus, N.J.
[00115] Nucleic acid probes may comprise ribonucleic acids and/or deoxyribonucleic acids. In some embodiments, provided nucleic acid probes are oligonucleotides (i.e., "oligonucleotide probes"). Generally, oligonucleotide probes are long enough to bind specifically to a homologous region of the gene, but short enough such that a difference of one nucleotide between the probe and the nucleic acid sample being tested disrupts hybridization. Typically, the sizes of oligonucleotide probes vary from approximately 10 to 100 nucleotides. In some embodiments, oligonucleotide probes vary from 15 to 90, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to 40, 15 to 35, 15 to 30, 18 to 30, or 18 to 26 nucleotides in length. As appreciated by those of ordinary skill in the art, the optimal length of an oligonucleotide probe may depend on the particular methods and/or conditions in which the oligonucleotide probe may be employed.
[00116] In some embodiments, nucleic acid probes are useful as primers, e.g., for nucleic acid amplification and/or extension reactions. In some embodiments, nucleic acid probes are labeled with a detectable moiety as described herein.
4. Therapeutic methods
[00117] Another aspect of the invention makes use of therapeutic materials and methods for effecting repression of ETV1. For example, by preventing transcription, repressing mRNA transcript levels, inhibiting translation, or otherwise directly or indirectly interfering with the activity of ETV1, it is possible to inhibit growth of GIST cells without affecting growth of noncancerous cells. Similarly, treatment of overexpressed or otherwise aberrant ETV1 expression in cancerous ICC cells or GIST cells decreases cell cycle progession, increases apoptosis and decreases tumorigenicity. ETV1 expression or activity may be modulated either directly through interaction with the ETV1 gene, mRNA or protein itself, or indirectly through one or more upstream regulator or downstream targets of ETVl. For example, ETVl repression may modulated through KIT or PDGFRA receptor tyrosine kinases.
[00118] Each of the therapies described herein may be directed specifically against ETVl. Alternatively, those of skill in the art will appreciate that the approaches and therapies can be modified for combination therapies against ETVl and KIT and/or PDGFRA. The present invention also encompasses therapies that interfere with the interaction of ETVl and KIT. a. Antibodies
[00119] Antibodies are increasingly used for therapeutic treatments of cancer, autoimmunity and infectious diseases. Many of these antibody therapies have shown considerable success, as evidence by the approval of more than 30 monoclonal antibody-based therapies in the past 25 years. Antibodies can be engineered to almost any extracellular or cell surface target. In a cancer treatment context, they may be used to stimulate an immune response against cancerous cells, to block or activate specific cell receptors involved in cell division or apoptosis, or to sensitize a cancer cell to chemotherapy. Antibodies may also be used to deliver radioisotopes or other toxic agents to cancerous cells or tumors.
[00120] The present invention encompass the use of therapeutic antibodies in the treatment of ETVl -mediated cancers, particularly GISTs. The antibodies may be monoclonal or polyclonal, and may act either directly or indirectly against GIST. In a particular embodiment, inactivating antibodies against the KIT receptor indirectly affect ETVl levels. Inhibition of KIT signaling by such antibodies may result in rapid loss of ETVl protein through destabihzation of the ETVl protein. As described in detail below, KIT-MEK signaling stabilizes ETVl protein. Any inactivating or blocking antibodies against KIT are a potential therapy for ETVl -mediated oncogenesis.
[00121] Anti-ETVl antibodies suitable for the invention include antibodies or fragments of antibodies that bind immunospecifically to any ETVl epitopes. As used herein, the term "antibodies" is intended to include immunoglobulins and fragments thereof which are specifically reactive to the designated protein or peptide, or fragments thereof. Suitable antibodies include, but are not limited to, human antibodies, primatized antibodies, chimeric antibodies, bi-specific antibodies, humanized antibodies, conjugated antibodies (i.e. , antibodies conjugated or fused to other proteins, radiolabels, cytotoxins), Small Modular
ImmunoPharmaceuticals ("SMIPs™ ), and antibody fragments. As used herein, the term "antibodies" also includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. , bi-specific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
[00122] As used herein, an "antibody fragment" includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; single domain antibodies; diabodies; triabodies; tetrabodies; linear antibodies; single-chain antibody molecules; and multi specific antibodies formed from antibody fragments. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. For example, antibody fragments include isolated fragments, "Fv" fragments, consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker ("ScFv proteins"), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.
[00123] Antibodies can be generated using methods well known in the art. For example, protocols for antibody production are described by Harlow and Lane, Antibodies: A Laboratory Manual, (1988). Typically, antibodies can be generated in mouse, rat, guinea pig, hamster, camel, llama, shark, or other appropriate host. Alternatively, antibodies may be made in chickens, producing IgY molecules (Schade et al., (1996) ALTEX 13(5):80-85). In some embodiments, antibodies suitable for the present invention are subhuman primate antibodies. For example, general techniques for raising therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al., international patent publication No. WO 91/11465 (1991), and in Losman et al., Int. J. Cancer 46: 310 (1990). In some embodiments, monoclonal antibodies may be prepared using hybridoma methods (Milstein and Cuello, (1983) Nature 305(5934):537-40.). In some embodiments, monoclonal antibodies may also be made by recombinant methods (U.S. Pat. No. 4,166,452).
[00124] In some embodiments, antibodies suitable for the invention may include humanized or human antibodies. Humanized forms of non-human antibodies are chimeric Igs, Ig chains or fragments (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of Abs) that contain minimal sequence derived from non-human Ig. Generally, a humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization is accomplished by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody (Riechmann et al., Nature 332(6162):323-7, 1988; Verhoeyen et al., Science. 239(4847):1534-6, 1988.). Such
"humanized" antibodies are chimeric Abs (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In some embodiments, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent Abs. Humanized antibodies include human Igs (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit, having the desired specificity, affinity and capacity. In some instances,
corresponding non-human residues replace Fv framework residues of the human Ig. Humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which most if not all of the CDR regions correspond to those of a non-human Ig and most if not all of the FR regions are those of a human Ig consensus sequence. The humanized antibody optimally also comprises at least a portion of an Ig constant region (Fc), typically that of a human Ig (Riechmann et al., Nature 332(6162):323-7, 1988; Verhoeyen et al., Science 239(4847): 1534-6, 1988.).
[00125] Human antibodies can also be produced using various techniques, including phage display libraries (Hoogenboom et al., Mol Immunol. (1991) 28(9): 1027-37; Marks et al., J Mol Biol. (1991) 222(3):581-97) and the preparation of human monoclonal antibodies (Reisfeld and Sell, 1985, Cancer Surv. 4(l):271-90). Similarly, introducing human Ig genes into transgenic animals in which the endogenous Ig genes have been partially or completely inactivated can be exploited to synthesize human antibodies. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene
rearrangement, assembly, and antibody repertoire (Fishwild et al., Nat Biotechnol. 1996;
14(7):845-51; Lonberg et al., Nature 1994;368(6474):856-9; Lonberg and Huszar, Int. Rev. Immunol. 1995;13(l):65-93; Marks et al., Biotechnology (NY) 1992; 10(7):779-83). b. Binding site oligonucleotides
[00126] Synthetic double-stranded oligodeoxynucleotides act as "decoy" cis elements to block the binding of transcription factors to promoter regions of target genes, resulting in the inhibition of gene transactivation in vitro and in vivo (see, e.g., Bielinska et al., (1990) Science 250:997- 1000). Furthermore, certain studies have described application of decoy strategy as in vivo gene therapy (see, e.g., Morishita et al., (1997) Nature Med. 8:894-899; and Kawamura et al. (1999) Gene Therapy 6:91-7).
[00127] Embodiments of the present invention may utilize promoter competition whereby nucleic acid sequences encoding ETVl cis-acting elements are introduced in high copy number into cells. These sequenes functions as decoys to competitively bind away ETVl from its natural enhancer sequences, with a corresponding suppression of the ETVl -regulated ICC-GIST- specific transcription network.
[00128] One embodiment of this approach utilizes oligonucleotides, modified to facilitate entry into cells that compete with the native cellular sequence for binding to ETVl.
Transcription factor decoys of the presently claimed invention are recognized and bound by ETVl such it can no longer bind to native response elements and regulate gene expression. Decoys can comprise one or more duplex nucleic acid structures. These structures are recognized by the DNA binding domain of the ETVl. The decoys can comprise one or more sequences of known ETVl binding sites. However, any sequence that has affinity for ETVl is suitable for use as a decoy and is contemplated by the presently claimed invention.
[00129] Embodiments of the invention include various decoy nucleic acid sequences including duplex, hairpin, and cruciform oligonucleotides containing one or more ETVl binding site, which, in certain aspects of the invention, inhibit cancerous cell growth without adversely or minimally affecting non-cancerous cells. Nucleic acid sequences with at least 70% homology to known sequences may also be recognized and bound by ETVl. Thus a range of sequences may be effectively employed as decoys. A pool of sequences identical or divergent to known ETVl binding sequences may be selected to generate therapeutic treatments of various potency. In particular embodiments, the decoy sequences are palindromic cis-transcription elements comprising a synthetic single-stranded oligonucleotide composed one or more ETVl binding sites that capable of self-hybridizing to form a duplex or hairpin structure.
[00130] A similar approach can be used with hairpin oligonucleotide decoys containing a duplex portion with one or more ETVl binding site may be introduced into cells and
successfully function as decoys to alter gene expression. Those of skill in the art will also appreciate that cruciform ETVl -binding decoys may be constructed through standard genetic manipulations of known sequences. Such cruciform DNA can increase the potency of the transcription factor decoy to inhibit gene transcription. Similar DNA structures are known to be generated during genetic recombination and from palindromic sequences under the effect of supercoiling, indicating a biological role for such structures. c. Dominant negative therapy
[00131] Embodiments of present invention further encompass dominant negative derivatives of ETVl that are capable of interfering with transcription factor function of native ETVl. Such dominant negative versions of transcription factors are known to those of skill in the art, and readily created by deleting the transcriptional activation domain. U.S. Pat. No. 5,869,040, for example, describes dominant negative forms of the nuclear proto-oncogene E2F transcription factor protein and methods of their use to treat cancer. Various ETVl -derived dominant negative proteins may be derived so long as they maintain sequence-specific DNA binding capabilities. Dominant interfering mutations may be made to either the DNA binding domain or the activation domains. Dominant-negative ETVl mutatnts are known in the art (Shin et al., J Cell Biochem. 2008 Oct 15;105(3):866-74). d. Aptamer therapy
[00132] Aptamers are macromolecules composed of nucleic acid (e.g., RNA, DNA) that bind tightly to a specific molecular target (e.g., ETVl polypeptide or an epitope thereof). A particular aptamer may be described by a linear nucleotide sequence and is typically about 15-60 nucleotides in length. Without wishing to be bound by any theories, it is contemplated that the chain of nucleotides in an aptamer form intramolecular interactions that fold the molecule into a complex three-dimensional shape, and this three-dimensional shape allows the aptamer to bind tightly to the surface of its target molecule. Given the extraordinary diversity of molecular shapes that exist within the universe of all possible nucleotide sequences, aptamers may be obtained for a wide array of molecular targets, including proteins and small molecules. In addition to high specificity, aptamers have very high affinities for their targets (e.g., affinities in the picomolar to low nanomolar range for proteins). Aptamers are chemically stable and can be boiled or frozen without loss of activity. Because they are synthetic molecules, they are amenable to a variety of modifications, which can optimize their function for particular applications. For example, aptamers can be modified to dramatically reduce their sensitivity to degradation by enzymes in the blood for use in in vivo applications. In addition, aptamers can be modified to alter their biodistribution or plasma residence time.
[00133] Selection of aptamers that can bind ETV1 or a fragment thereof can be achieved through methods known in the art. For example, aptamers can be selected using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method (Tuerk, C, and Gold, L., Science 249:505-510 (1990)). In the SELEX method, a large library of nucleic acid molecules (e.g., 1015 different molecules) is produced and/or screened with the target molecule (e.g., an ETV1 protein or an ETV1 epitope). The target molecule is allowed to incubate with the library of nucleotide sequences for a period of time. Several methods, known in the art, can then be used to physically isolate the aptamer target molecules from the unbound molecules in the mixture, which can be discarded. The aptamers with the highest affinity for the target molecule can then be purified away from the target molecule and amplified enzymatically to produce a new library of molecules that is substantially enriched for aptamers that can bind the target molecule. The enriched library can then be used to initiate a new cycle of selection, partitioning, and amplification. After 5-15 cycles of this iterative selection, partitioning and amplification process, the library is reduced to a small number of aptamers that bind tightly to the target molecule. Individual molecules in the mixture can then be isolated, their nucleotide sequences determined, and their properties with respect to binding affinity and specificity measured and compared. Isolated aptamers can then be further refined to eliminate any nucleotides that do not contribute to target binding and/or aptamer structure, thereby producing aptamers truncated to their core binding domain. See Jayasena, S. D. Clin. Chem. 45: 1628-1650 (1999) for review of aptamer technology; the entire teachings of which are incorporated herein by reference. e. Antisense and interfering RNA therapy
[00134] Antisense molecules are RNA or single-stranded DNA molecules with nucleotide sequences complementary to a specified mRNA. When a laboratory-prepared antisense molecule is injected into cells containing the normal mRNA transcribed by a gene under study, the antisense molecule can base-pair with the mRNA, preventing translation of the mRNA into protein. The resulting double-stranded RNA or RNA/DNA is digested by enzymes that specifically attach to such molecules. Therefore, a depletion of the mRNA occurs, blocking the translation of the gene product so that antisense molecules find uses in medicine to block the production of deleterious proteins. Methods of producing and utilizing antisense RNA are well known to those of ordinary skill in the art (see, for example, C. Lichtenstein and W. Nellen (Editors), Antisense Technology: A Practical Approach, Oxford University Press (December, 1997); S. Agrawal and S. T. Crooke, Antisense Research and Application (Handbook of Experimental Pharmacology, Volume 131), Springer Verlag (April, 1998); I. Gibson, Antisense and Ribozyme Methodology: Laboratory Companion, Chapman & Hall (June, 1997); J. N. M. Mol and A. R. Van Der Krol, Antisense Nucleic Acids and Proteins, Marcel Dekker; B. Weiss, Antisense Oligonodeoxynucleotides and Antisense RNA Novel Pharmacological and
Therapeutic Agents, CRC Press (June, 1997); Stanley et al., Antisense Research and
Applications, CRC Press (June, 1993); C. A. Stein and A. M. Krieg, Applied Antisense Oligonucleotide Technology (April, 1998)).
[00135] Antisense molecules suitable for inhibiting ETV1 activity can be designed based on the sequences described above and known in the art. The antisense molecules may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells, or tissues.
[00136] RNA molecules may be modified to increase intracellular stability and half-life.
Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2'O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept can be extended by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
[00137] RNA interference (RNAi) is a mechanism of post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA), which is distinct from the antisense approach described above. dsRNA molecules are believed to direct sequence-specific degradation of mRNA in cells of various lineages after first undergoing processing by an RNase Ill-like enzyme called DICER (Bernstein et al., Nature 409:363, 2001) into smaller dsRNA molecules comprised of two 21 nucleotide strands, each of which has a 5' phosphate group and a 3' hydroxyl, and includes a 19 nucleotide region precisely complementary with the other strand, so that there is a 19 nucleotide duplex region flanked by 2 nucleotide 3' overhangs. RNAi is thus mediated by short interfering RNAs (siRNA), which typically comprise a double-stranded region
approximately 19 nucleotides in length typically with 1-2 nucleotide 3' overhangs on each strand, resulting in a total length typically of between approximately 21 and 23 nucleotides.
[00138] It will also be appreciated that siRNAs can have a range of lengths, e.g., the double- stranded portion can range from 15-29 nucleotides. It will also be appreciated that the siRNA can have a blunt end or a 3' overhang at either or both ends. If present, such 3' overhang is often from 1 -5 nucleotides in length.
[00139] siRNA has been shown to downregulate gene expression when transferred into mammalian cells by such methods as transfection, electroporation, or microinjection, or when expressed in cells via any of a variety of plasmid-based approaches. RNA interference using siRNA is reviewed in, e.g., Tuschl, T., Nat. Biotechnol., 20:446-448, May 2002. See also Yu, J., et al., Proc. Natl. Acad. Sci., 99(9), 6047-6052 (2002); Sui, G., et al., Proc. Nail. Acad. Sci., 99(8), 5515-5520 (2002); Paddison, P., et al., Genes and Dev., 16, 948-958 (2002);
Brummelkamp, T. et al., Science, 296, 550-553 (2002); Miyagashi, M. and Taira, K., Nat.
Biotech., 20, 497-500 (2002); Paul, C., et al., Nat. Biotech., 20, 505-508 (2002).
[00140] Indeed, in vivo inhibition of specific gene expression by RNAi has been achieved in various organisms including mammals. For example, Song et al., Nature Medicine, 9:347-351 (2003) discloses that intravenous injection of Fas siRNA compounds into laboratory mice with autoimmune hepatitis specifically reduced Fas mRNA levels and expression of Fas protein in mouse liver cells. Several other approaches for delivery of siRNA into animals have also proved to be successful. See e.g., McCaffery et al., Nature, 418:38-39 (2002); Lewis et al., Nature Genetics, 32:107-108 (2002); and Xia et al., Nature Biotech., 20: 1006-1010 (2002).
[00141] As described in these and other references, the siRNA may consist of two individual nucleic acid strands or of a single strand with a self-complementary region capable of forming a hairpin (stem-loop) structure. A number of variations in structure, length, number of mismatches, size of loop, identity of nucleotides in overhangs, etc., are consistent with effective siRNA-triggered gene silencing. While not wishing to be bound by any theory, it is thought that intracellular processing (e.g., by DICER) of a variety of different precursors results in production of siRNA capable of effectively mediating gene silencing. Generally it is desirable to target exons rather than introns, and it may also be particularly desirable to select sequences complementary to regions within the 3' portion of the target transcript. Generally it is preferred to select sequences that contain approximately equimolar ratio of the different nucleotides and to avoid stretches in which a single residue is repeated multiple times.
[00142] siRNA may thus comprise RNA molecules typically having a double-stranded region approximately 19 nucleotides in length typically with 1-2 nucleotide 3' overhangs on each strand, resulting in a total length of between approximately 21 and 23 nucleotides. As used herein, siRNA also includes various RNA structures that may be processed in vivo to generate such molecules. Such structures include RNA strands containing two complementary elements that hybridize to one another to form a stem, a loop, and optionally an overhang, preferably a 3' overhang. Typically, the stem is approximately 19 bp long, the loop is about 1-20, preferably about 4-10, and more preferably about 6-8 nucleotides long and/or the overhang is typically about 1-20, and preferably about 2-15 nucleotides long. In certain embodiments of the invention the stem is minimally 19 nucleotides in length and may be up to approximately 29 nucleotides in length. Loops of 4 nucleotides or greater are less likely subject to steric constraints than are shorter loops and therefore may be preferred. The overhang may include a 5' phosphate and a 3' hydroxyl. The overhang may, but need not, comprise a plurality of U residues, e.g., between 1 and 5 U residues.
[00143] The siRNA compounds suitable for the present invention can be designed based on the ETV1 sequence described herein and can be synthesized using conventional RNA synthesis methods. For example, they can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Various applicable methods for RNA synthesis are disclosed in, e.g., Usman et al., J. Am. Chem. Soc, 109:7845- 7854 (1987) and Scaringe et al., Nucleic Acids Res, 18:5433-5441 (1990). Custom siRNA synthesis services are available from commercial vendors such as Ambion (Austin, Tex., USA), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (Rockford, 111., USA),
ChemGenes (Ashland, Mass., USA), Proligo (Hamburg, Germany), and Cruachem (Glasgow, UK). [00144] Inventive siRNAs may be comprised entirely of natural RNA nucleotides, or may instead include one or more nucleotide analogs and/or modifications as mentioned above for antisense molecules. The siRNA structure may be stabilized, for example by including nucleotide analogs at one or more free strand ends in order to reduce digestion, e.g., by exonucleases. This may also be accomplished by the inclusion. Alternatively, siRNA molecules may be generated by in vitro transcription of DNA sequences encoding the relevant molecule. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7, T3, or SP6.
[00145] The siRNA compounds can also be various modified equivalents of the siRNA structures. As used herein, "modified equivalent" means a modified form of a particular siRNA compound having the same target-specificity (i.e., recognizing the same mRNA molecules that complement the unmodified particular siRNA compound). Thus, a modified equivalent of an unmodified siRNA compound can have modified ribonucleotides, that is, ribonucleotides that contain a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate (or phospodiester linkage). As is known in the art, an "unmodified ribonucleotide" has one of the bases adenine, cytosine, guanine, and uracil joined to the Γ carbon of beta-D-ribo- furanose.
[00146] Modified siRNA compounds contain modified backbones or non-natural
internucleoside linkages, e.g., modified phosphorous-containing backbones and non- phosphorous backbones such as morpholino backbones; siloxane, sulfide, sulfoxide, sulfone, sulfonate, sulfonamide, and sulfamate backbones; formacetyl and thioformacetyl backbones; alkene-containing backbones; methyleneimino and methylenehydrazino backbones; amide backbones, and the like.
[00147] siRNA may be generated by intracellular transcription of small RNA molecules, which may be followed by intracellular processing events. For example, intracellular transcription is achieved by cloning siRNA templates into RNA polymerase III transcription units, e.g., under control of a U6 or HI promoter. In one approach, sense and antisense strands are transcribed from individual promoters, which may be on the same construct. The promoters may be in opposite orientation so that they drive transcription from a single template, or they may direct synthesis from different templates. In a second approach siRNAs are expressed as stem-loop structures. The siRNAs of the invention may be introduced into cells by any of a variety of methods. For instance, siRNAs or vectors encoding them can be introduced into cells via conventional transformation or transfection techniques. As used herein, the terms
"transformation" and "transfection" are intended to refer to a variety of aft-recognized techniques for introducing foreign nucleic acid (e.g., DNA or RNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, injection, or electroporation.
[00148] Vectors that direct in vivo synthesis of siRNA constitutively or inducibly can be introduced into cell lines, cells, or tissues. In certain preferred embodiments of the invention, inventive vectors are gene therapy vectors (e.g., adenoviral vectors, adeno-associated viral vectors, retroviral or lentiviral vectors, or various nonviral gene therapy vectors) appropriate for the delivery of an siRNA-expressing construct to mammalian cells, most preferably human cells. Thus the present invention includes gene therapy approaches to the treatment of diseases or clinical conditions associated with inflammation in, for example, airway (e.g., airway hyperresponsiveness), digestive, pulmonary or reproductive tract.
[00149] The invention includes methods of treating a disease or clinical condition associated with elevated ETV1 expression in, for example, GIST by administering siRNA compositions comprising siRNA that targets ETV1. The compositions may be administered parenterally, orally, inhalationally, etc.
[00150] Typically, siRNA compositions reduce the level of the target transcript and its encoded protein by at least 2-fold, preferably at least 4-fold, more preferably at least 10-fold or more. The ability of a candidate siRNA to reduce expression of the target transcript and/or its encoded protein may readily be tested using methods well known in the art including, but not limited to, Northern blots, RT-PCR, microarray analysis in the case of the transcript, and various immunological methods such as Western blot, ELISA, immunofluorescence, etc., in the case of the encoded protein. Efficacy may be tested in appropriate animal models or in human subjects.
[00151] siRNA compounds may be administered to mammals by various methods through different routes. For example, they can be administered by intravenous injection. See Song et al., Nature Medicine, 9:347-351 (2003). They can also be delivered directly to a particular organ or tissue by any suitable localized administration methods. Several other approaches for delivery of siRNA into animals have also proved to be successful. See e.g., McCaffery et al., Nature, 418:38-39 (2002); Lewis et al., Nature Genetics, 32: 107-108 (2002); and Xia et al., Nature Biotech., 20: 1006-1010 (2002). Alternatively, they may be delivered encapsulated in liposomes, by iontophoresis, or by incorporation into other vehicles such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.
[00152] In addition, they may also be delivered by a gene therapy approach, e.g., using a DNA vector from which siRNA compounds in, e.g., small hairpin form (shRNA), can be transcribed directly. Numerous studies have demonstrated that while double-stranded siRNAs are very effective at mediating RNAi, short, single-stranded, hairpin-shaped RNAs can also mediate RNAi, presumably because they fold into intramolecular duplexes that are processed into double-stranded siRNAs by cellular enzymes. Sui et al., Proc Natl Acad Sci USA, 99:5515- 5520 (2002); Yu et al., Proc Natl Acad Sci USA, 99:6047-6052 (2002); and Paul et al., Nature Biotech., 20:505-508 (2002)). This discovery has significant and far-reaching implications, since the production of such shRNAs can be readily achieved in vivo by transfecting cells or tissues with DNA vectors bearing short inverted repeats separated by a small number of (e.g., 3 to 9) nucleotides that direct the transcription of such small hairpin RNAs. Additionally, if mechanisms are included to direct the integration of the transcription cassette into the host cell genome, or to ensure the stability of the transcription vector, the RNAi caused by the encoded shRNAs, can be made stable and heritable. Not only have such techniques been used to "knock down" the expression of specific genes in mammalian cells, but they have now been successfully employed to knock down the expression of exogenously expressed transgenes, as well as endogenous genes in the brain and liver of living mice. See generally Hannon, Nature. 418:244- 251 (2002) and Shi, Trends Genet, 19:9-12 (2003); see also Xia et al., Nature Biotech., 20: 1006- 1010 (2002).
[00153] Additional siRNA compounds targeted at different sites of the nucleic acids encoding one or more interacting protein members of a protein complex identified in the present invention may also be designed and synthesized according to general guidelines provided herein and generally known to skilled artisans. See e.g., Elbashir, et al. (Nature 411: 494-498 (2001). For example, guidelines have been compiled into "The siRNA User Guide" which is available at the website of The Rockefeller University, New York, NY.
/. Small molecule therapy
[00154] Small molecule modulators (e.g., inhibitors or activators) of gene expression are also within the scope of the invention and may be detected by screening libraries of compounds using, for example, cell lines that express a ETV1 polypeptide or a version of a ETV1 polypeptide that has been modified to include a readily detectable moiety. Methods for identifying compounds capable of modulating gene expression are described, for example, in U.S. Patent No. 5,976,793.
[00155] More generally, the invention encompasses compounds that modulate ETV1 activity. Methods of screening for such interacting compounds are well known in the art and depend, to a certain degree, on the particular properties and activities of the polypeptide encoded by the gene. Representative examples of such screening methods may be found, for example, in U.S. Patent No. 5,985,829, U.S. Patent No. 5,726,025, U.S. Patent No. 5,972,621, and U.S. Patent No.
6,015,692. The skilled practitioner will readily be able to modify and adapt these methods as appropriate for a given polypeptide. Thus the invention encompasses methods of screening for molecules that modulate the activity of a polypeptide encoded by a marker gene, particularly the ETV1 gene. g. Vaccination
[00156] The invention also encompasses the use of polynucleotide sequences corresponding to ETV1, or portions thereof, as DNA vaccines. Such vaccines comprise polynucleotide sequences, typically inserted into vectors, that direct the expression of an antigenic polypeptide within the body of the individual being immunized. Details regarding the development of vaccines, including DNA vaccines for various forms of cancer may be found, for example, in Brinckerhoff LH, Thompson LW, Slingluff CL, Melanoma Vaccines, Curr. Opin. Oncol, 12(2): 163-73, 2000 and in Stevenson FK, DNA vaccines against cancer: from genes to therapy, Ann. Oncol., 10(12): 1413-8, 1999 and references cited therein. The polypeptides, or fragments thereof, that are encoded by marker genes may also find use as cancer vaccines. Such vaccines may be used for the prevention and/or treatment of cancer.
5. Pharmaceutical compositions
[00157] The invention includes pharmaceutical compositions comprising the inventive antibodies, polynucleotides, small molecules, etc. described above. Pharmaceutical
compositions may comprise therapeutically active ingredient in accordance with the invention (e.g., ETV1 or variants thereof, and/or ETV1 inhibitors), together with one or more
pharmaceutically acceptable excipients. Such pharmaceutical compositions may optionally comprise one or more additional therapeutically-active substances.
[00158] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys.
[00159] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.
[00160] A pharmaceutical composition in accordance with the invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
[00161] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.
[00162] Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of
Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excipients used in formulating
pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.
[00163] In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British
Pharmacopoeia, and/or the International Pharmacopoeia.
[00164] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.
[00165] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents. In certain embodiments for parenteral administration, compositions are mixed with solubilizing agents such an CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
[00166] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injec tables.
[00167] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
[00168] In order to prolong the effect of an active ingredient, it is often desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly( anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
[00169] Compositions for rectal administration are typically suppositories which can be prepared by mixing compositions with suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
[00170] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and/or fillers or extenders (e.g. starches, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g. glycerol), disintegrating agents (e.g. agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarding agents (e.g. paraffin), absorption accelerators (e.g. quaternary ammonium compounds), wetting agents (e.g. cetyl alcohol and glycerol monostearate), absorbents (e.g. kaolin and bentonite clay), and lubricants (e.g. talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents.
[00171] Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or
preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
[00172] General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).
6. Administration
[00173] The inventive antibodies, polynucleotides, small molecules, etc. described above and/or pharmaceutical compositions thereof may be administered to a subject using any amount and any route of administration effective for treating a disease, disorder, and/or condition (e.g., GIST). The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular composition, its mode of administration, its mode of activity, and the like. Compositions in accordance with the invention are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
[00174] Pharmaceutical compositions in accordance with the present invention may be administered by any route. In some embodiments, pharmaceutical compositions are
administered by one or more of a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (e.g. by powders, ointments, creams, gels, lotions, and/or drops), mucosal, nasal, buccal, enteral, vitreal, intratumoral, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; as an oral spray, nasal spray, and/or aerosol, and/or through a portal vein catheter.
[00175] In general the most appropriate route of administration will depend upon a variety of factors including the nature of the active ingredient, the condition of the patient (e.g., whether the patient is able to tolerate particular routes of administration), etc. The invention encompasses the delivery of the pharmaceutical compositions by any appropriate route taking into consideration likely advances in the sciences of drug delivery. 7. Therapy selection
[00176] Another aspect of the invention relates to the selection of a treatment regimen based on ETVl over-expression or particular activating mutations. For example, it is known in the art that certain anti-cancer theraperies, particularly chemotherapy and small-molecule
pharmaceutical therapies, produce superior result in cancer cells that are positive for a particular mutant allele or over-/under-expression of a particular protein or enzyme. The present invention provides methods of identifying patients with aberrant ETVl expression who may benefit from one therapy over another. For example, therapy may be personalized based on over-expression versus mutation, and sub-categorized based on the type of ETVl mutation and/or other abberations with known GIST-related proteins such as KIT or PDGFRA.
8. Kits
[00177] Another aspect of the invention provides a kit to test for the presence of ETVl in a tumor sample. The kit can comprise, for example, an antibody for detection of a polypeptide or a probe for detection of a polynucleotide. In addition, the kit can comprise a reference or control sample, instructions for processing samples, performing the test and interpreting the results, buffers and other reagents necessary for performing the test. In one embodiment the kit comprises one or more antibodies (monoclonal or polyclonal) for ETVl. In some embodiments monoclonal antibodies are preferred. In other preferred embodiments of the invention, the kit comprises a panel of antibodies, e.g., for ETVl and KIT; for ETVl and PDGFRA; or for ETVl, KIT and PDGFRA. In certain embodiments of the invention the kit comprises pairs of primers or probes for detecting expression of one or more of the marker genes of the invention, e.g., for ETVl and KIT; for ETVl and PDGFRA; or for ETVl, KIT and PDGFRA. In certain embodiments of the invention the kit comprises a cDNA or oligonucleotide array for detecting expression of one or more of the marker genes of the invention. [00178] It will be appreciated that any of the therapeutic applications discussed above may be used either alone or in combination with one another, or in combination with conventional GIST therapies such as surgery, chemotherapy, radiation or dietary modifications.
Examples
Example 1: ETV1 as a diagnostic, prognostic and therapeutic target for Gastrointestinal stromal tumours or GIST
[00179] The present Example describes how the ETS family member ETV1 is highly expressed in the subtypes of ICCs sensitive to oncogenic KIT mediated transformation (Kwon, J. G. et al., 2009, Gastroenterology, 136: 630-639; incorporated herein by reference), and is required for their development.
[00180] Among other things, the present Example describes how ETV1 expression can be used as a diagnostic, prognostic or therapeutic target for GIST.
Materials and Methods
Cell lines, Antibodies, and Reagents
[00181] The GIST882 cell line obtained from an imatinib naive patient, harbours a homozygous ex on 13 KIT mutation (K642E) and is maintained in RPMI supplemented with 15% FBS, lOmM HEPES pH 7.5. The GIST48 cell line, obtained from an imatinib-resistant patient, harbour a homozygous exon 11 KIT mutation (V560D) and a secondary heterozygous Exon 17 KIT mutation (D820A), is maintained in Ham's F10 media supplemented with 15% FBS, 0.5% MITO+ Serum Extender (BD Biosciences), and 30 mg/L bovine pituitary extract (BD
Biosciences)( Bauer, S., et al., 2006, Cancer Res, 66: 9153-9161; incorporated herein by reference). The U20S osteosarcoma, LNCaP prostate cancer, and NIH-3T3 mouse embryonic fibroblast cells were obtained from ATCC and cultured as recommended.
[00182] GIST48 and GIST882 cells were established in the Fletcher laboratory (DFCI). All other cells were obtained from ATCC. Etvl-/- mice, with targeted deletion of the ETS domain, was obtained from the Jessell laboratory (Columbia) and CB17-SCID mice was from Taconic. Antibody sources are: ETV1, ANOl, PGP9.5 (Abeam), KIT for WB, P-Tyr703-KIT (Cell Signaling), P-Tyr204-ERK, GAPDH (Santa Cruz), and anti-mouse Kit for IF (clone ACK2, E- Biosciences).
[00183] The following antibodies are used: rabbit anti-ETVl WB, IHC, and ChIP (Abeam), mouse anti-KIT for WB (Cell Signaling), rabbit anti-Phospho-tyr703 KIT for WB (Cell
Signalling), mouse anti-phospho-tyr204 ERK for WB (Santa Cruz Biotechnologies), mouse anti- GAPDH for WB (Santa Cruz Biotechnologies), ACK2 rat anti-mouse Kit for IF (E-Biosceinces), rabbit anti-PGP9.5 for IF (Abeam), rabbit anti-ANOl for IF (Abeam). Secondary antibodies were Alexa-488 labelled anti-rabbit antibody and Alexa-594 labelled anti-rat antibody
(Invitrogen). PD0325901 was synthesized in the MSKCC Organic Synthesis Core Facility by O. Ouerfelli and imatinib was a gift from Novartis.
Expression Data Mining
[00184] Five publically deposited database containing expression of GIST samples are as follows: Expression Project for Oncology (ExpO, GSE2109) containing 7 GIST samples among over 2,000 tumours of all types, NIELSEN_SARCOMA (GSE3443) containing 10 GIST samples among 44 sarcoma samples (Nielsen, T. O. et al., 2002, Lancet, 359: 1301-1307;
incorporated herein by reference), SEGAL_SARCOMA (GSE2719) containing 5 GIST samples among 50 sarcoma samples (Segal, N. H. et al., 2003, Am J Pathol, 163: 691-700; incorporated herein by reference), YAMAGUCHI_GIST (GSE8167) with 32 GIST samples (Yamaguchi, U. et al., 2008, J Clin Oncol, 26: 4100-4108; incorporated herein by reference), and OSTROWSKI_GIST (GSE17743) with 29 GIST samples (Ostrowski, J. et al., 2009, BMC Cancer, 9: 413; incorporated herein by reference). Normalized expression of ETV1 of a given sample was calculated as the Z-score (i.e., standard deviations from median) of ETV1 probe signal among all probes. GIST-signature gene sets derived from three datasets (ExpO, Nielsen, and Segal) containing both GIST and non-GIST malignancies included genes that met the following two criteria: 1) multiple-hypothesis testing significance of q<0.05, and 2) a mean expression that is 1.5 Z-score higher in GIST than non-GIST tumours. The three gene sets, EXPO_GIST_SIG, NIELSEN_GIST_SIG, and SEGAL_GIST_SIG contain 102, 156, and 30 genes respectively (Supplementary Table 1).
[00185] For mouse ICC gene expression, we analyzed an expression dataset of FACS sorted ICC-MY, ICC-DMP, and smooth muscle cells from the mouse small intestine (GSE7809)( Chen, H. et al., 2007, Physiol Genomics, 31 : 492-509; incorporated herein by reference). ICC-MY and ICC-DMP signature genes sets were defined as genes expressed higher in ICC-MY and ICC- DMP compared to muscle by 1.5 Z-score with p<0.05 and contain 61 and 81 genes respectively.
Tumor Samples
[00186] Clinical samples from patients with GIST and other sarcomas were obtained according to MSKCC IRB protocol and frozen and paraffin embedded tissue samples were banked. All GIST and non-GIST tumours have been pathologically reviewed and confirmed by a sarcoma expert (CRA). All GIST tumours used have been sequenced for KIT and PDGFA mutations. For tumours in FIG. 1, G1-G8 are GISTs: Gl, G2 and G4 (KIT exonl l mutation), G3 (KIT exon 9 mutation), G5 and G6 (KIT and PDGFRA wild-type), G7 and G8 (PDGFRA D842V mutation); S1-S5 are leiomyosarcoma, Gl leiomyoma, Gl leiomyoma, intra-abdominal desmoid tumour, and Gl schwannoma respectively. FISH of tumor samples
[00187] Two BACs, one covering ETV1 gene body and one on the 3' side (RP11-138H16, RP11-124L22, CHORI) were labelled with Spectrum Green-d-UTP (Vysis) and two BACs covering 5' upstream region of ETV1 (RP11-703 A4, RP11-115D14) were labelled with
Spectrum Orange-d-UTP. Paraffin-embedded slides were de- waxed with xylene (Fisher), and then heated in 1 OmM sodium citrate (PH 6) by microwaving for 5 to 10 minutes in a tender cooker (Nordic Ware). Slides were co-denatured with the probes for 6 minutes at 75°C using HYbrite (Vysis), and kept in a dark, moist chamber at 37°C for 16 hours. Slides were then washed with 50% formamide (Fisher)/2XSSC at 45°C according to standard procedure. Nuclei were stained with DAPI, and slides were mounted with Vectashield (Vector). Results were viewed under a Zeiss microscope, and images were captured with an ISIS camera.
RNA isolation and qRT-PCR
[00188] For tissue culture cells, RNA was isolated using RNeasy (Qiagen). For frozen samples of clinical specimens and for explanted xenografts, tissue samples were grounded in 500 μΐ Trizol (Invitrogen) using PowerGen homogenizer (Fisher Scientific), followed by addition of 100 μΐ chloroform. The samples were then centrifuged at 10,000g x 15 minutes. The upper phase was mixed with equal volume of 70% ethanol, and the RNA was further purified using an RNeasy column.
[00189] For qRT-PCR, RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (ABI) and PCR was run using Power SYBR master mix (ABI) on a Realplex machine (Eppendorf). Expression was normalized to the ribosomal protein RPL27 (de Jonge, H. J. et al., 2007, PLoS One, 2: e898; incorporated herein by reference). Primers were designed using primer blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast) except when cited and purchased from Operon. The following primers pairs were used:
CTSL1: F: AGGCGCGTGACTGGTTGAGC (SEQ ID NO: 2), R: TGCATCGCCTTCCACTTGGTCC (SEQ ID NO: 3)
DUSP6: F: TGCCGGGCGTTCTACCTGGA (SEQ ID NO: 4), R:
GGCGAGCTGCTGCTACACGA (SEQ ID NO: 5)
ETVl-Exon23: F: AACAGAGATCTGGCTCATGATTCA (SEQ ID NO: 6), R:
CTTCTGCAAGCCATGTTTCCTGTA9 (SEQ ID NO: 7)
ETVl-Exon67: F: CTACCCCATGGACCACAGATTT (SEQ ID NO: 8), R:
CTTAAAGCCTTGTGGTGGGAAG9 (SEQ ID NO: 9)
KIT: F: GGGATTTTCTCTGCGTTCTG (SEQ ID NO: 10), R:
GATGGATGGATGGTGGAGAC (SEQ ID NO: 11)
PLAT: F: CACTGGGCCTGGGCAAACATA (SEQ ID NO: 12), R:
CACGTCAGCCTGCGGTTCTTC (SEQ ID NO: 13)
PROM1: F: GCCACCGCTCTAGATACTGC (SEQ ID NO: 14), R:
GCTTTTCCTATGCCAAACCA (SEQ ID NO: 15)
PTPRE: F: TGCACGCGGAGCAGAAGGTG (SEQ ID NO: 16), R:
GCCGTGCATGGTCTGCAGGT (SEQ ID NO: 17)
RPL27: F: CATGGGCAAGAAGAAGATCG (SEQ ID NO: 18), R:
TCCAAGGGGATATCCACAGA (SEQ ID NO: 19)
TIMP3: F: CCAGCGCAAGGGGCTGAACT (SEQ ID NO: 20), R:
TAGCCGCCCTTCTGCCGGAT (SEQ ID NO: 21)
Lentiviral knockdown
[00190] Two pLKO.l constructs against ETVl (ETVlshl : TRCN0000013923, targeting GTGGGAGTAATCTAAACATTT (SEQ ID NO: 22) in 3' UTR and ETVlsh2:
TRCN0000013925, targeting CGACCCAGTGTATGAACACAA (SEQ ID NO: 23 in exon 7) were purchased from Open Biosystems and pLKO.l shScr (targeting
CCTAAGGTTAAGTCGCCCTCG (SEQ ID NO: 24) was purchased from Addgene (Sarbassov, D. D., et al., 2005, Science, 307: 1098-1101; incorporated herein by reference). Lentiviruses were generated by co-transfecting the shETVl hairpin constructs with psPax2 and pVSVG (Addgene) into 293FT cells (Invitrogen) using Lipofectamine 2000 (Invitrogen). Viruses were harvested at 48 and 72 hours after transfection and concentrated 100-fold using Lenti-X concentrator (Clontech) and stored at -80 in aliquots. Infection was performed in 12-well dishes in the presence of 7.5 μg ml polybrene (Sigma) by a 1 hour centrifugation at 500 x g. To determine virus titre in GIST48 and GIST882 cells, two steps were taken. First, the relative titre of shScr, ETVlshl, and ETVlsh2 was obtained by counting puromycin resistant colonies after infection of U20S cells, whose growth is insensitive to ETV1 knockdown. Next, the absolute titre of the shScr was calculated by counting puromyicn resistant colonies after infection into GIST48 and GIST882 cells.
Stable gene expression
[00191] ETV1 cDNA (Open Biosystems) and EGFP were cloned into MSCV-puro. Human wild-type KIT in MSCV-IRES-EGFP vector was obtained from Gary Gilliland and generation of Δ560 mutation was performed using QuikChange II XL site-directed mutagenesis kit
(Stratagene). Retrovirus was produced in 293FT cells by standard methods using ecotropic packaging vector and infected into NIH-3T3 cells at MOI=5. For ETV1 expression, cells were selected with puromycin (1 μg/ml). For KIT expression, GFP FACS demonstrated >90 infection and therefore, cells were not further enriched.
Cell growth and cell cycle analysis after shRNA-mediated knockdown
[00192] For GIST48 and GIST882 cells, cells were plated at 2 x 105 cells per well in a 24- well plate on day 0 and infected with pLKO.l hairpin viruses (MOI=5) on day 1. Cells were not further puromycin selected. Triplicate wells were counted every three days until day 13. For U20S cells, cells were plated at 1 x 105 cells per well of 24-well plate on day 0 and infected at MOI=5 in 24-well plates on Day 1. On day 3, they were split into 96-well plates (2,500/well). Viability was assessed using Cell Titer Glo (Promega). For cell cycle analysis, cells were trypsinized on day 4 after infection, fixed in 70% ethanol and incubated with 20 μg ml propidium iodide (Sigma) and 0.2 mg/ml RNAse A (Invitrogen) for 2 hours prior to FACS analysis on a FACSCalibur™ (BD Biosciences). Fluorescence intensity of Gl and G2 peaks were normalized between samples and cell cycle analysis was performed using FlowJo software.
Xenograft Growth
[00193] For GIST882, cells were infected at MOI=l on day 0 and briefly selected with 5 μg/ml puromycin between days 2-4. On day 4, 5x106 cells resuspended in 100 μΐ of 1: 1 mix of growth media and Matrigel™ (BD Biosciences) were subcutaneously injected into CB17-SCID mice (Taconic). Tumour sizes were measured weekly staring 6 weeks after xenografting. After 10 weeks, xenografts were explanted and RNA isolated for qRT-PCR analysis. For NIH-3T3 cells stably expressing combinations of vector, ETV1, KIT, and ΚΙΤΔ560, 1x106 cells were implanted as above and tumour sizes were measured starting 2 weeks after xenografting.
Anchorage independent growth
[00194] Transduced NIH-3T3 cells were suspended in soft agar with 20% calf-serum at 5,000 cells per well in a6-well plate in triplicate. After 3 weeks, each well was stained with 0.5% crystal violet, imaged and counted using GelCount (Oxford Optronix). Immunofluorescence
[00195] For ETVl immunofluorescence of paraffin sections of tumour samples, heat- mediated antigen retrieval was performed followed by staining with ETVl antibody at 2.5 μg ml and Alexa-594 labeled anti -rabbit secondary antibody.
[00196] For immunofluorescence of cryostat sections of the mouse GI tract, mouse stomach, small intestine, cecum, and large intestine were dissected and fixed in 4% paraformaldehyde for 2 hours followed by an overnight incubation in 30% sucrose. They were then embedded in OCT, flash frozen, and cut into 5 μπι sections using a Cryostat. Etvl-/- and Etvl+/+ littermate controls were embedded onto the same block to ensure identical processing. Tissue sections were blocked overnight using 5% goat serum, incubated with primary antibodies at 4° C overnight, and secondary antibody for 2 hours at room temperature. Slides were mounted using Prolong® Gold (Invitrogen) and images were taken on a Nikon Eclipse TE2000-E microscope using a
Photometric Coolsnap HQ camera. Images were taken with at 20X (N.A. 0.75) or 60X (N.A. 1.4) objectives. Monochrome images taken with DAPI, FITC, and Texas Red filter sets were pseudo- colored blue, green, and red respectively and merged using Image J (http://rsbweb.nih.gov/ij/). The exposure, threshold, and maximum were identical between Etvl-/- and Etvl+/+ images and the gamma was 1.0.
[00197] For whole-mount staining, mouse GI tissue was fixed in ice-cold acetone for >1 hours. They were then rehydrated in PBS, stretched, and the mucosa of these GI tissues was blunt dissected away. Samples were then blocked in 5% goat serum, incubated with ACK2 rat- anti-KIT antibody or PGP9.5 antibody, followed by secondary antibody. Images were taken with at 20X (N.A. 0.75) or 60X (N.A. 1.4) objectives with Z-step of Ιμπι through the muscle layer. The Z-stack of images was deconvoluted using Autoquant X2 software (Media Cybernetics). Movies were generated from deconvolued stacks using ImageJ software.
[00198] To quantify ICC subsets, immunofluorescence images from at least four fields each from two Etvl+/+ and Etvl-/- mice (at least eight fields total) of the stomach, small intestine, cecum, and large intestine were examined. A percentage of ICCs were obtained by dividing the total number of KIT-positive immunostaining cells with the number of DAPI-positive nuclei. The results are shown as percentage of residual KIT-positive immunostaining in Etvl-/- mice compared to Etvl+/+ controls.
Expression Profiling
[00199] For profiling of ETVl knockdown, GIST48 and GIST882 cells plated in 12-well plates were infected in triplicates with shScr, and ETVlshl and ETVlsh2 viruses at MOI=5. Four days after infection, RNAs were isolated from duplicate samples and protein lysates were obtained from the third for confirmatory ETVl Western Blot. The duplicate samples were expression profiled using an Illumina Human HT-12 array. Raw data was imported into
Genespring 10, quartile normalized and log 2 transformed. The log transformed expression value of each probe was averaged across duplicates. For each probe, the average change by ETVl knockdown is calculated as:
((ETV 1 shl GisT48-ShScrGisT48)+(ETV 1 sh2GisT48-ShScrGisT48)+(ETV 1 sh 1 GIST882- ShScrGisT882)+(ETVlsh2GisT882-ShScrGisT882))/4
[00200] We next generated a ranked list of genes based on the averaged log2 transformed fold change. The top forty-eight genes showed an averaged decrease of >1.7-fold with ETVl knockdown and shown in FIG. 2. We defined a "shETVl DN" gene set as the 410 genes with average decrease >1.4-fold and a "shETVl UP" gene set as the 380 genes with average increase of > 1.4-fold.
[00201] For profiling of imatinib treatment, GIST882 cells were treated with vehicle or ΙμΜ imatinib for 8 hours in triplicate and profiled as above. Gene set enrichment analysis
[00202] Given a ranked list of genes, GSEA determines whether predefined sets of genes (e.g., genes within a Gene Ontology category) are disproportionally overrepresented in the top or the bottom of the list instead of randomly across the list (Subramanian, A. et al., 2005, Proc Natl Acad Sci U S A, 102, 15545-15550; incorporated herein by reference). To assess the phenotypic association with ETV1 knockdown, we used the ranked list of genes generated above from the most upregulated by ETV1 knockdown to most downregulated by ETV1 knockdown. For genes with multiple probes by the Illumina HT-12 array, median signal was used. We used gene sets from the Molecular Signatures Database C2v2.5 with 1892 curated gene sets of chemical and genetic perturbations and canonical pathways and C5v2.5 with 1454 Gene Ontology gene sets. In addition, we added five custom gene sets including the three GIST signature gene sets
EXPO_GIST_SIG, NIELSEN_GIST_SIG, and SEGAL_GIST_SIG and two ICC signature gene sets ICC_MY_SIG, and ICC_DMP_SIG defined above.
ChlP-Seq
[00203] ChlP-Seq was performed as previously described (Goldberg, A. D. et al., 2010, Cell, 140: 678-691 ; incorporated herein by reference). Briefly, 108 GIST48 cells were crosslinked for 15-minutes in 1 % paraformaldehyde, washed, and lysed. Chromatin was sheared using Bioruptor (Diagenode) to fragments of -150 bp and was incubated with anti-rabbit IgG dynabeads (Invitrogen) pre-conjugated with anti-ETVl antibody, washed, and eluted. The eluted chromatin was reverse-cross-linked, and DNA was column purified. The purified ChIP DNA was blunt- ended, ligated to Solexa adaptors, amplified with 18-cycles of PCR, and sequenced on Solexa Genome Analyzer. We performed independent qPCR validation of separate ChIP samples using the following primers:
DUSP6 Promoter. F: GCCCGCTGTTGCAGCTTGTT (SEQ ID NO: 25), R:
GCCGGCTGGAACAGGTTGTG (SEQ ID NO: 26) GPR20 Enhancer. F: CCCTCCCAGGCTCTCCCCAC (SEQ ID NO: 27), R:
TCCGGGCCTGCTCTCTGTCC (SEQ ID NO: 28)
GAPDH promoter. F: TCCCAAAGTCCTCCTGTTTCA (SEQ ID NO: 29), R:
CAGCAGGACACTAGGGAGTCAA (SEQ ID NO: 30)
[00204] All reads (~36-bp) were aligned to the human genome (hgl8) using the ELAND alignment software within the Illumina Analysis Pipeline. Unique reads mapped to a single best- matching location with no more than two mismatches were kept and used to generate genome- wide distribution of ETVl -binding and for peak identification. Redundant reads were considered once. The software MACS (Zhang, Y. et al., 2008, Genome Biol, 9: R137; incorporated herein by reference) was applied to the ChlP-Seq data with sequencing data from input DNA as control for identifying genomic regions enriched with ETVl ChIP signals (i.e., peaks). For each peak, we subsequently calculated the number of reads (extending 200-nt in 3 '-direction) covering each nucleotide position and then defined the maximal count as peak height.
Results and Discussion
[00205] All mined datasets were downloaded Gene Expression Omnibus (GSE2109, GSE7809, GSE2719, GSE3443, GSE8167, GSE17743) and were analyzed by Oncomine™ or using Genespring 10. GIST-signature genes from three datasets containing both GIST and non- GIST malignancies met the following two criteria: 1) q<0.05, and 2) a Z-score expression difference >1.5 between GIST and non-GIST tumours. Expression profiling of GIST cell lines with different shRNA conditions was performed in duplicate on Illumina Human HT-12 array. GSEA was performed using MSigDB C2, MSigDB C4, and the GIST and ICC signature gene sets. For ChlP-Seq, sheared chromatin enriched by ETVl IP was sequenced on Solexa Genome Analyzer, aligned using ELAND alignment software. Peaks were identified by MACS using input DNA as control using a FDR <1 . [00206] Using FDR < 1% (as defined by MACS) and a height of > 15 reads, we defined 14,741 peaks. These peaks were classified as promoter peaks (within +/- 2kb of transcription start sites), enhancer peaks (from -50 kb of transcription start sites to + 5kb of transcription end sites, but excluding promoter regions), or otherwise intergenic peaks based on their genomic distances to known Refseq gene annotation. To associate an ETV1 peak with a specific gene, we used the following protocol. A promoter peak was assigned to all genes if it was in the promoter regions of multiple genes. An enhancer peak within a gene body was assigned to the host gene, but otherwise was assigned to the nearest gene. Nucleotide sequences of all ETV1 peaks were retrieved from the reference human genome and subjected to motif analysis by the program MEME (Bailey, T. L., et al., 2006, Nucleic Acids Res, 34: W369-373; incorporated herein by reference).
[00207] To correlate gene expression with ETV1 binding sites, we first limited the analysis the genes with peaks to those that were also represented on the mRNA microarray (Illumina HT- 12). Overlapping analysis of genes whose expression was perturbed by ETV1 knockdown and those with ETV1 binding peaks was performed using Excel macros and significance was calculated using Fisher's exact test.
[00208] Reasoning that transcription factors are likely to play critical roles in defining the cellular context, we utilized three expression datasets (Nielsen, T. O. et al., 2002, Lancet, 359: 1301-1307; Segal, N. H. et al., 2003, Am J Pathol, 163: 691-700; each of which is incorporated herein by reference) to search for GIST specific genes that might provide new molecular insights. We identified an eleven-gene signature exclusively associated with GIST in all three datasets that included the ETS family transcription factor ETV1 (FIG. 1A). Examination of individual tumour samples revealed that ETV1 is highly expressed in all GISTs and at significantly higher levels than any other tumour type (FIG. IB, FIG. 5). ETV1 was of immediate interest since ETS family transcription factors are well established oncogenes in Ewing sarcoma, melanoma, and prostate cancer (Tomlins, S. A. et al., 2005, Science, 310: 644- 648; Mertens, F. et al., 2009, Semin Oncol, 36: 312-323; Jane-Valbuena, J. et al., 2010, Cancer Res, 70: 2075-2084; each of which is incorporated herein by reference).
[00209] Next, we assessed mRNA and protein levels of ETV1 in GIST and other sarcomas in clinical samples, GIST cell lines (imatinib-resistant GIST48 and imatinib- sensitive GIST882), the U20S osteosarcoma cell line, and the LNCaP prostate cancer cell line known to overexpress ETV1 due to translocation (Tomlins, S. A. et al., 2007, Nature, 448: 595-599; incorporated herein by reference) (FIG 1C, D). ETV1 mRNA and protein were highly and exclusively expressed in all GISTs and GIST cell lines, and in LNCaP cells. As expected, KIT mRNA and protein were highly expressed in all GIST tumours and GIST cell lines, but not in other sarcomas or non-GIST cell lines, and phospho-KIT was only seen in GIST samples with activating KIT mutations. Four additional GIST samples amenable to immunohistochemical analysis all showed strong nuclear ETV1 staining whereas a leiomyosarcoma control sample did not (FIG. 6). These data show that ETV1 is universally highly expressed in all GISTs both at transcript and protein levels.
[00210] To explore the requirement of ETV1 in GIST pathogenesis, we performed RNAi experiments using two ETV1 -specific hairpins validated for both protein and mRNA suppression (FIG. 7A). Infection with either hairpin resulted in growth inhibition of both GIST cell lines, but did not affect the growth of U20S cells. Consistent with the level of ETV1 knockdown, ETVlsh2 was more growth suppressive than ETVlshl in both GIST cell lines (FIG. IE). Cell cycle analysis showed that ETV1 knockdown resulted in both decreased cell cycle progression and increased apoptosis (FIG. 7B). ETV1 knockdown also impaired the tumourigenicity of GIST882 cells in SCID mouse xenografts, and those tumours that did grow had escaped
ETV1 suppression (FIG. IF). Collectively, these observations indicate that ETV1 is required for GIST growth and survival.
[00211] Next, we addressed the mode of high ETV1 expression in GIST. FISH on 4 GIST samples and 2 GIST cell lines showed no evidence of amplification or "breakaway" between the 3' and 5' ends of ETV1 locus. qRT-PCR showed no evidence of differential ex on expression, which is expected with ETV1 translocation (FIG. 8). Furthermore, no focal ETV1 amplification was found in 40 GIST tumours and 6 GIST cell lines in a recent 250K SNP array study
(Beroukhim, R. et al., 2010, Nature, 463: 899-905; incorporated herein by reference). The fact that high levels of ETV1 expression are consistently observed in the absence of obvious genomic alterations raises the possibility that the ICCs that give rise to GIST may endogenously express ETV1.
[00212] The musculature of the GI tract is organized into two principal layers— the inner circular muscle (CM) layer beneath the mucosa (M) and the outer longitudinal muscle (LM) layer (Ward, S. M., et al., 2001, Am J Physiol Gastrointest Liver Physiol, 281: G602-611;
incorporated herein by reference). In the large intestine, myenteric ICCs (ICC-MY) form a network between the CM and LM layers surrounding the neuronal myenteric plexus, intramuscular ICCs (ICC-IM) are singly dispersed in the CM, and submucosal ICCs (ICC-SMP) form network surrounding the submucosal plexus (FIG. 2A). In the small intestine, ICC-IMs and ICC-SMPs are absent and ICC-DMPs form a network around the deep muscular plexus in the CM close to the mucosa (FIG. 9A). All four ICC subtypes are identified by positive membrane expression of Kit (Ward, S. M., et al., 2001, Am J Physiol Gastrointest Liver Physiol, 281:
G602-611; incorporated herein by reference) (FIG. 2B and FIG. 9B). In the large intestine, ICC- MYs and ICC-IMs but not ICC-SMPs stain with nuclear Etvl (FIG. 2B). In the small intestine, ICC-MYs but not ICC-DMPs stain with nuclear Etvl (FIG. 9B). This finding is further supported by our analysis of a published ICC expression dataset from mouse small intestine (Chen, H. et al., 2007, Physiol Genomics, 31: 492-509; incorporated herein by reference) showing that Etvl is only highly expressed in ICC-MYs (FIG. 9C). Notably, in the KitA558 mutant mice only ICC-MY and ICC-IM develop hyperplasia while ICC-SMP and ICC-DMP do not (Kwon, J. G. et al., 2009, Gastroenterology, 136: 630-639; incorporated herein by reference). These data suggest that ETV1 is a lineage- specific transcription factor for the ICCs that give rise to GIST.
[00213] We therefore asked if Etvl is required for the normal development of ICCs by examining the GI tracts of Etvl-/- mice (Arber, S., et al., 2000, Cell, 101: 485-498; incorporated herein by reference). Cross section and reconstructed whole-mount images from Etvl-/- mice showed significant loss of Kit-positive ICC-IMs and ICC-MYs in the large intestine (FIG. 2C-D, FIG. 13), small intestine, stomach, and cecum (FIGS. 10-13). In contrast, ICC-DMPs and ICC- SMPs in the small and large intestine respectively were preserved, consistent with absent Etvl expression in these ICC subtypes. These results were confirmed with a second ICC marker Anol (Gomez-Pinilla, P. J. et al., 2009, Am J Physiol Gastrointest Liver Physiol, 296: G1370-1381; incorporated herein by reference) (FIG. 14). Immunostaining with the neuronal marker PGP9.5 confirmed the integrity of the myenteric plexus in Etvl-/- mice (FIG. 2C, FIGS. 10-12, FIG. 15). Collectively, these data indicate that Etvl is selectively required for development of ICC-MY and ICC-IM and, by implication, a lineage-specific survival factor for the ICC-GIST lineage.
[00214] To identify ETV1 target genes in GIST, we analyzed the effect of shRNA-mediated ETV1 suppression on the transcriptomes of GIST48 and GIST882 cells. The overlap of genes perturbed by both ETV1 -specific hairpins and across both cell lines was highly statistically significant, suggesting that ETV1 regulates a core set of genes in GIST (FIG. 16). To minimize cell line-specific and off-target effects, we generated a ranked gene list based on the average change in gene expression induced by the two ETV1 -specific hairpins in both GIST cell lines (FIG. 3A, B). We independently confirmed downregulation of 5 of these genes using real-time RT-PCR (FIG. 17). Among the 48 genes suppressed >1.7-fold by ETV1 knockdown, 32 were expressed at higher levels in human GIST samples relative to other tumour types in the ExpO expression dataset (FIG. 3B). We performed gene set enrichment analysis (GSEA)(
Subramanian, A. et al., 2005, Proc Natl Acad Sci U S A, 102, 15545-15550; incorporated herein by reference) of the "shETVl" ranked list using >3,000 gene sets in the Molecular Signature Database along with 5 custom gene sets defined by GIST-signature genes from the Segal, Nielsen, and ExpO datasets and by ICC-MY- and ICC-DMP-signature genes. All three GIST sets along with the ICC-MY set were among the most negatively enriched gene sets while the ICC-DMP set was not (FIG. 3C, FIG. 18). These data suggest that ETVl is a master regulator of a transcriptional program conserved in ICC-IM/MYs and GISTs.
[00215] To define the direct transcriptional targets of ETVl in GIST, we performed genome- wide analyses of ETVl -binding sites using ChlP-Seq in GIST48 cells. We identified 14,741 ETVl -binding sites (ETVl peaks) which are enriched in promoter regions (FIG. 3D). Motif analysis of the peaks identified the ETS core consensus motif, GGAA, in -90% of peaks (FIG. 3F). Integrative analyses of the ETVl ChlP-Seq data with the transcriptomes from shRNA- mediated ETVl suppression in GIST cells showed that 38 of the top 48 shETVl downregulated genes contain ETVl peaks (FIG. 3B, E, FIG. 19). Analysis of genes with 1.4-fold change by shETVl knockdown revealed that both shETVl upregulated and shETVl downregulated genes are enriched for ETVl peaks. Furthermore, enhancer binding and in particular enhancer and promoter binding is highly predicative of transcriptional activation by ETVl (FIG. 3H). Since enhancers are in general cell-lineage specific (Heintzman, N. D. et al., 2009, Nature, 459: 108- 112; Visel, A. et al., 2009, Nature, 457: 854-858; each of which is incorporated herein by reference), our data suggest that these ICC-GIST-lineage specific genes are likely directly regulated by ETVl binding to their enhancer regulatory elements.
[00216] The dual requirement of KIT and ETVl in normal ICC development and GIST survival raise the possibility that KIT and ETVl cooperate in GIST oncogenesis. Inhibition of KIT signalling by imatinib in imatinib-sensitive GIST882 cells resulted in rapid loss of ETVl protein, without significant effect on ETVl mRNA levels (FIG. 4A, B, FIG. 20). Similar results were observed with the MEK inhibitor PD325901. This loss of ETVl protein was faster than the natural degradation rate, as revealed by cyclohexamide experiments to inhibit protein synthesis, and was rescued from proteosomal degradation by MG132 (FIG. 4B). Therefore, KIT-MEK signalling stabilizes ETVl protein. Consistent with this KIT-MEK-ETV1 signalling pathway model, the overlap between genes transcriptionally altered by imatinib treatment (KIT-regulated) and by ETVl knockdown in GIST882 cells is highly significant (FIG. 4C). Furthermore, these ETVl transcriptional targets preferentially contain ETVl enhancer peaks (FIG. 4D), indicating that KIT signalling influences the ETVl transcriptional output of the tissue and lineage-specific genes in GIST.
[00217] Having established a signalling pathway from KIT to ETVl, we explored their potential cooperativity in tumourigenesis by expressing ETVl, wild-type KIT, KIT harbouring a common GIST mutation (ΚΙΤΔ560) and control vectors in combination in NIH3T3 cells. KIT- dependent stabilization of ETVl protein was recapitulated in this system (FIG. 4E). In anchorage independent colony formation assays, ETVl significantly increased the number and size of colonies in ΚΙΤΔ560 expressing cells but was insufficient to confer anchorage-independent growth on its own (FIG. 21). Furthermore, ΚΙΤΔ560 and ETVl strongly cooperated in conferring tumourigenic growth in SCID mice (FIG. 4F, G).
[00218] Taken together, these findings establish an oncogenic role for ETVl in GIST that differs from classical models of ETS-driven malignancies where structural alterations (e.g., TMPRSS2-ETV1 translocation in prostate cancer, ETVl amplification in melanoma) lead to aberrant expression and promote tumourigenesis (Tomlins, S. A. et al., 2005, Science, 310: 644- 648; Jane-Valbuena, J. et al., 2010, Cancer Res, 70: 2075-2084; each of which is incorporated herein by reference). Rather, ETVl expression in GIST is inherited from ICC-MY/IM cells, where ETVl is also a survival factor. We further established that KIT activity, through MEK, stabilizes ETVl, providing a mechanism for KIT-ETV1 cooperativity (FIG. 4H). These observations provide an explanation for why patients and mice with germline activating KIT mutations develop neoplasia in only the ICC-MY/IM lineage. While the mechanism of ETV1- mediated oncogenesis in GIST differs from other ETS-driven cancers, we anticipate that the ETVl -dependent transcriptional program defined here may serve as a valuable resource for further understanding of other ETV1- and other ETS-driven transcriptional programs in various cellular contexts such as prostate cancer.
[00219] The fact that ETV1 is universally highly expressed in all GISTs makes it immediately useful as a candidate diagnostic biomarker, since the current standard of KIT immunoreactivity is negative in about 5% of all GISTs (Miettinen, M. & Lasota, 2006, Arch Pathol Lab Med, 130: 1466-1478; incorporated herein by reference). While transcription factors has classically been considered "undruggable", reports of successful inhibition of the NOTCH transcription factor complex and AR activity by blocking coactivator binding have challenged this paradigm
(Andersen, R. J. et al.,2010, Cancer Cell, 17: 535-546; Moellering, R. E. et al., 2009, Nature, 462: 182-188; each of which is incorporated herein by reference). ETV1 inhibitors may yield novel therapeutic agents for imatinib-resistant GIST.
Equivalents and Scope
[00220] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.
[00221] In the claims articles such as "a", "an" and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Thus, for example, reference to "an antibody" includes a plurality of such antibodies, and reference to "the cell" includes reference to one or more cells known to those skilled in the art, and so forth. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are presenting, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitation, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for anyone of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.
[00222] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. It is noted that the term "comprising" is intended to be open and permits the inclusion of additional elements or steps.
[00223] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understand of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the state ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. [00224] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any HCV genotype/subtype, any HCV antibody, any epitope, any pharmaceutical composition, any method of administration, any therapeutic application, etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.
[00225] The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.
Other Embodiments
[00226] Those of ordinary skill in the art will readily appreciate that the foregoing represents merely certain preferred embodiments of the invention. Various changes and modifications to the procedures and compositions described above can be made without departing from the spirit or scope of the present invention, as set forth in the following claims.

Claims

Claims We claim:
1. A method for determining the likelihood of gastrointestinal stromal tissue being cancerous or gastrointestinal stromal tumor (GIST), comprising:
(a) determining a level of expression or activity of ETV1 in a gastrointestinal stromal tissue sample obtained from a subject;
(b) comparing the determined ETV1 level with that of a reference correlated with a predetermined probability of being cancerous or GIST; and
(c) based on the comparing, determining that the tissue sample has an increased or decreased probability, relative to the reference, of being cancerous or GIST.
2. The method of claim 1 , wherein determining the level of expression or activity involves determining the expression of a nucleic acid.
3. The method of claim 1, wherein determining the level of expression or activity involves determining the expression of a polypeptide.
4. The method of claim 1 , wherein determining the level of expression or activity of ETV1 involves determining an ETV1 regulated expression signature.
5. The method of claim 1, wherein the step (b) of comparing comprises establishing that the determined level in the tissue sample is higher than the reference level, and the reference level correlates with that observed in normal tissue, so that step (c) of determining comprises detemining that the tissue sample has an elevated likelihood of being cancerous or GIST.
6. The method of claim 1 , wherein the step (a) of determining comprises performing an analysis selected from the group consisting of Northern blotting, Western blotting, protein gel electrophoresis, immunoprecipitation, ELISA, PCR, RT-PCR, differential display, serial analysis of gene expression, array analysis, immunohistochemistry, and combinations thereof, so that the ETV1 level of expression or activity is determined.
7. The method of claim 6, wherein the step (a) of determining comprises performing immunohistochemistry on the tissue sample, using an ETV1 antibody or antigen binding fragment thereof.
8. The method of claim 7, wherein the ETV1 antibody or antigen binding fragment binds to an amino acid sequence as set forth in SEQ ID NO: 1.
9. A method for determining the likelihood of cancerous gastrointestinal stromal tissue responding to therapy, comprising:
(a) determining a level of expression or activity of ETV1 in a cancerous
gastrointestinal stromal tissue sample obtained from a subject;
(b) comparing the determined ETV1 level with that of a reference correlated with a predetermined probability of responding to a therapeutic regimen; and
(c) based on the comparing, determining that the subject from which the tissue sample was obtained has an increased or decreased probability, relative to the reference, of responding to the therapeutic regimen.
10. The method of claim 9, wherein the reference is an average of responses derived from a population of individuals.
11. A method for identifying or characterizing an agent that affects GIST phenotype comprising:
(a) contacting cultured GIST tissue or cells with an agent to be characterized; and
(b) determining that ETVl level of expression or activity is altered when the agent is present as compared with when it is absent.
12. The method in claim 11, wherein an increase in the level of ETVl correlates with the onset of or progression of a GIST phenotype.
13. The method in claim 11, wherein a decrease in the level of ETVl correlates with the regression of a GIST phenotype.
14. The method of claim 11, wherein the step (b) of determining comprises performing an analysis selected from the group consisting of Northern blotting, Western blotting, protein gel electrophoresis, immunoprecipitation, ELISA, PCR, RT-PCR, differential display, serial analysis of gene expression, array analysis, immunohistochemistry, and combinations thereof, so that the ETVl level of expression or activity is determined.
15. A method of treating GIST by administering to a subject suffering from or susceptible to GIST a therapy that alters ETVl level of expression or activity.
16. The method of claim 15, wherein the step of administering comprises
administering a therapy that modulates expression or activity of an ETVl regulator so that ETVl level of expression or activity is altered.
17. A method of treating GIST in a subject in need thereof comprising administering a therapeutically effective amount of pharmaceutical composition comprising an agent identified by the method of claim 11.
18. The method of claim 17, wherein the pharmaceutical composition inhibits EVTl expression.
19. The method of claim 17, wherein the pharmaceutical composition promotes the degradation of ETV1 protein.
20. A kit for use in detecting ETV1 expression comprising:
(a) an agent capable of detecting the level of ETV1 expression or activity;
(b) a container comprising the agent for detecting the level of ETV1 expression or activity in a sample;
(c) a control; and
(d) instructions to provide guidance for carrying out an assay embodied by the kit and for making a determination of ETV1 based upon that assay.
21. The kit of claim 20, wherein the agent is an ETV1 antibody or ETVl-binding fragment thereof, oligonucleotide probe, oligonucleotide primer, aptamer, polypeptide, peptide, ligand, hormone, lipid, carbohydrate, lectin, or small molecule.
22. The kit of claim 21, wherein the agent is an ETV1 antibody or ETVl-binding fragment thereof.
23. The kit of claim 21, wherein the ETVl antibody or ETVl -binding fragment thereof is attached to a substrate, which substrate is applied to a sample from a patient or to a surface that may contain ETVl and the surface of the substrate is then processed to assess whether specific binding occurs between the antibody or fragment thereof and ETVl in the sample.
24. The kit of claim 21, wherein the control is a control slide comprising tumor samples.
25. A method comprising steps of:
(a) monitoring the level of ETVl expression or activity in a subject suffering from GIST;
(b) detecting a change in the monitored level of ETVl expression or activity over time; and
(c) initiating or altering therapy administered to the subject after detecting the change.
26. The method of claim 25, wherein the subject is undergoing a first therapy and the step of initiating or altering comprises ceasing the first therapy, modifying the first therapy, adding a second therapy, or combinations thereof.
27. A method of inhibiting the growth of ICC cancerous cell with aberrant ETVl expression or activity comprising contacting the cell with an ETVl inhibitory agent so as to decrease the expression or activity of ETVl, thereby inhibiting the growth of the ICC cancerous cell.
28. The method of claim 27, wherein the ETVl inhibitory agent is a targeted antisense oligonucleotide or a targeted siRNA.
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CN112082976A (en) * 2019-06-14 2020-12-15 天津方得生物科技有限公司 In-vitro drug sensitivity detection method based on drug probe and tissue slice

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WO2013036755A1 (en) * 2011-09-09 2013-03-14 Sloan-Kettering Institute For Cancer Research Etv1 antibodies and uses thereof
CN112082976A (en) * 2019-06-14 2020-12-15 天津方得生物科技有限公司 In-vitro drug sensitivity detection method based on drug probe and tissue slice

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