WO2002012894A9 - Facteur 6 du genre kruppel (klf6), proteine supprimant les tumeurs et diagnostics, therapies et criblages bases sur cette proteine - Google Patents

Facteur 6 du genre kruppel (klf6), proteine supprimant les tumeurs et diagnostics, therapies et criblages bases sur cette proteine

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Publication number
WO2002012894A9
WO2002012894A9 PCT/US2001/025046 US0125046W WO0212894A9 WO 2002012894 A9 WO2002012894 A9 WO 2002012894A9 US 0125046 W US0125046 W US 0125046W WO 0212894 A9 WO0212894 A9 WO 0212894A9
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WO
WIPO (PCT)
Prior art keywords
gene
klfό
cancer
klf6
protein
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PCT/US2001/025046
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English (en)
Other versions
WO2002012894A1 (fr
Inventor
Scott Friedman
Dan Li
Goutham Narla
John Martignetti
Karen Heath
Original Assignee
Sinai School Medicine
Scott Friedman
Dan Li
Goutham Narla
John Martignetti
Karen Heath
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Publication date
Application filed by Sinai School Medicine, Scott Friedman, Dan Li, Goutham Narla, John Martignetti, Karen Heath filed Critical Sinai School Medicine
Priority to EP01963875A priority Critical patent/EP1332362A4/fr
Priority to CA002419064A priority patent/CA2419064A1/fr
Priority to AU2001284790A priority patent/AU2001284790A1/en
Publication of WO2002012894A1 publication Critical patent/WO2002012894A1/fr
Publication of WO2002012894A9 publication Critical patent/WO2002012894A9/fr
Priority to US12/769,595 priority patent/US20110059899A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • C12Q2600/136—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

  • KLF6 KRUPPEL-LIKE FACTOR 6
  • a TUMOR SUPPRESSOR PROTEIN AND DIAGNOSTICS, THERAPEUTICS
  • the present invention relates to identification of tumor suppressor activity of a protein, and to related diagnostic and therapeutic compositions and methods.
  • the discovery of this tumor suppressor activity provides for screening targets as well, particularly screening for compounds that overcome gene inactivation or alteration.
  • P53 is a tumor suppressor gene whose antitumor activity is in part related to its ability to upregulate p21 in normal tissue.
  • the loss of p53 in at least 50% of tumors leads to uncontrolled growth.
  • up to 50% of human tumors have a mechanism of tumorigenesis other than p53 signaling.
  • CCA Carcinoembryonic Antigen
  • AFP Fetoprotein
  • PSA Prostate Specific Antigen
  • the assays using these markers have not, to date, been markedly predictive of the presence of cancer in these individuals, as verified by other clinical diagnoses. The sensitivity and specificity of these assays has been disappointingly low. Time-consuming and labor-intensive clinical assessments (e.g. palpations, x-rays, mammograms, biopsies) have remained the accepted methods for diagnosing cancer.
  • the present invention addresses both therapeutic and diagnostic needs by disclosing the role of KLF6 as a tumor suppressor gene and as a marker for the detection of cancer.
  • KLF6 is a gene encoding a novel zinc finger transcription factor protein. The gene was first cloned and reported as CPBP (Koritshconer, et al., Journal of Biol.
  • KLF6 was subsequently cloned from humans and rats as an immediate-early gene induced in hepatic stellate cells in early liver injury, it is expressed in all mammalian cell types (Ratziu, et al, Proc. Natl. Acad. Sci. USA 1998, 95:9500-9505). KLF6 has been localized to human chromosome lOp (Ratziu, et al, 1998, supra), a region which is deleted in various tumors including neuroblastomas and melanomas. While the expression pattern and chromosomal location of KLF6 were known, the present invention provides the first evidence that KLF6 is inactivated in cancers and acts as a tumor suppressor gene.
  • the present invention relates to the discovery that KLF6 is inactivated in cancers, and plays a role of a tumor suppressor gene.
  • the invention provides a method for detecting inactivation or alteration of a KLF6 gene.
  • the method comprises detecting a mutation of genomic DNA comprising the KLF6 gene, wherein such a mutation results in inactivation or alteration of KLF6.
  • This method is particularly useful for obtaining a diagnosis or specific prognosis of a cancer, particularly neuroblastoma, glioblastoma, melanoma, prostate cancer, breast cancer, ovarian cancer, head and neck cancer, hepatocellular cancer, lung cancer, and colon cancer. Detecting the inactivation or alteration of KLF6 allows the phenotype of a tumor to be determined, or can show that a person is at risk for developing certain tumors.
  • the invention further provides a method for diagnosis or prognosis of a cancer, comprising detecting inactivation or alteration of a KLF6 gene.
  • inactivation or alteration of the KLF6 gene is indicative of the presence of a cancer or a specific prognosis for outcome of treatment of the cancer.
  • detection of inactivation or alteration of KLF6 involves detecting mutation of the KLF-6 gene in a functional domain, such as the activation domain, the DNA binding domain, as well as a putative Casein Kinase II phosphorylation site, or protein kinase C phosphorylation sites.
  • the invention further provides a method for diagnosis or prognosis of a cancer, which method comprises detecting a loss of heterozygosity at the KLF6 locus, wherein said loss of heterozygosity is indicative of the presence of a cancer or a specific prognosis of the cancer.
  • kits for detecting inactivation or alteration of a KLF6 gene comprising a detection assay, e.g., an immunoassay, PCR-based assay, DNA sequencing, Western blotting, detection of gross chromosomal rearrangements, alterations in mRNA levels, detection of non- wild-type splicing patterns of mRNA transcripts or a hybridization assay, for inactivation or alteration of a KLF6 gene.
  • a kit of the invention provides for detection of mutations or deletion of a KLF6 gene.
  • the invention provides a method of treating such hyperplasia, more particularly cancer, in a subject.
  • the method comprises administering an amount of a vector that expresses a gene encoding functional KLF6 effective to express a functional level of KLF6 into cells of the subject. More particularly this expression vector is useful for expressing the KLF6 protein in somatic cell types for human gene therapy.
  • the target cells are tumor cells wherein the KLF6 gene is inactivated.
  • the invention provides a vector, such as a defective virus (particularly a neurotrophic virus) or non- viral vector, that comprises a gene encoding a functional human KLF6 operatively associated with a regulatory sequence that allows expression of the KLF6 gene in human target cells in vivo.
  • This regulatory sequence preferably comprises a promoter that provides for a high level of expression of the KLF6 gene.
  • the invention provides a pharmaceutical composition for treating a cancer, comprising such vector and a pharmaceutically acceptable carrier.
  • Another subject of the invention is a method of preventing or treating mammalian cancer cells lacking endogenous KLF6 protein, or expressing altered forms or levels of endogenous KLF6 protein, which method comprises introducing a KLF6 tumor suppressor gene encoding a KLF6 protein into the mammalian cancer cells, whereby the mammalian cancer cells' neoplastic phenotype is suppressed.
  • the mammalian cancer cell lacks the wild-type KLF6 tumor suppressor gene or has a mutated or methylated KLF6 gene.
  • the mammalian cell may also present a haploinsufficiency for the KLF6 gene.
  • the KLF6 gene is derived from any species, and may more particularly be derived from the same mammalian species as the mammalian cancer cells.
  • the invention contemplates preventing or treating human hyperplasia of cells in a subject by administering an effective amount of a functional KLF6 protein, or analogues thereof, to the subject.
  • KLF6 mRNA is upregulated in hepatocytes after partial hepatectomy. Rats were subjected to either 2/3 partial hepatectomy or sham hepatectomy. Hepatocytes were isolated at the intervals shown and analyzed for KLF6 mRNA by RNAse protection. Expression of S14 ribosomal mRNA was used as a normalization control in the same sample. Data are expressed as KLF6 mRNA relative to hepatocytes from sham hepatectomized animals analyzed in parallel. There is biphasic induction of KLF6 mRNA with peaks at 1 and 12 hours.
  • FIG. 1 A and 2B. P21 induction and decreased cell growth was observed following induction of KLF6 in vivo and in cultured cells.
  • PCNA were observed in transgenic mice.
  • Hepatocytes were isolated from either wild type or transgenic mice at 4 weeks, and analyzed for expression of KLF6, p21 and PCNA by Western blot. In transgenic mice there was a ⁇ 3 fold increase in KLF6 accompanied by a 10-fold increase in p21 and a 70% reduction in PCNA expression. Data from three animals are shown; results were comparable in three separate transgenic lines B.
  • Reduced hepatocyte proliferation was observed in transgenic mice. Proliferation of hepatocytes 24 hours after isolation from transgenic mice was reduced by 70% compared to cells isolated from wild type littermates, as assessed by incorporation of 3 H thymidine.
  • FIG. 3 A, 3B and 3C Transactivation of the p21 promoter by KLF6 does not require p53.
  • Figure 4A, 4B, 4C, 4D and 4E Suppression of transformed phenotypes of glioma by KLF6.
  • KLF6, or the KLF6-DN mutant were seeded in soft agar and colonies were allowed to form. Representative fields from a single experiment are shown. Panel B shows the number of colonies formed from an experiment performed in triplicate. The calculated standard deviations are shown. C. The proliferation of these same cells was determined by counting cell numbers. Results over a five day period are plotted. Error bars represent the standard deviation of a single assay performed in triplicate. Small dashed lines represent the KLF6 transfected cells. Larger dashed lines represent control cells and KLF6-DN cells are depicted as a solid line. All assays were performed at least two times with two different independently created cell lines. Similar results were obtained. D. DBTRG-05MG cells were transiently transfected with KLF6 expression plasmids along with a p21 promoter-luciferase reporter.
  • Results are expressed as fold over control transfected cells with all values being normalized to total protein. Assay were performed in triplicate.
  • Figure 5A, 5B, 5C, 5D and 5E Suppression of c-sis/PDGF- B transformation by KLF6.
  • A. NIH 3T3 cells were co-transfected with the c-sfs/PDGF-BB and either a control plasmid, KLF6 or KLF6-DN and focus forming assays were performed. Cells were fixed with methanol and foci visualized by Giemsa staining.
  • B Data expressed as number of foci with standard errors from triplicate measurements are shown.
  • FIG 6A and 6B Suppression of tumor formation in animals by KLF6.
  • DBTRG-05MG cells transduced with either the control (A) vector or KLF6 (B) were injected subcutaneously into 10 nude mice and total tumor volume per animal was measured over a seven week period.
  • Figure 7A, 7B, 7C, and 7D Loss of heterozygosity analysis at the KLF6 locus in prostate cancer.
  • Loss of heterozygosity (LOH) analysis of eight prostate cancer patients using normal and tumor DNA Microsatellite markers flanking the KLF6 gene are shown.
  • FIG. 8 Prostate cancer-derived point mutations of KLF6 abrogate its growth suppressive activity.
  • PC3 cells were transfected with either the P 169 or X 137 mutant proteins, or wild type human KLF6. DNA synthesis was assayed 24 hours after transfection. DNA synthesis in cells expressing each of the point mutants was greater than in cells transfected with KLF6 or empty vector pCI-neo. Cells transfected with wild type KLF6 proliferated 40% less than the empty vector transfected cells. Average of three independent experiments is shown. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention is based on the identification of KLF6 as a tumor suppressor gene, and on the discovery that this gene was inactivated or altered in cancers.
  • the inventors first showed that KLF6 is rapidly upregulated in hepatocytes following partial hepatectomy in both wild type and p53 null animals.
  • Transgenic mice overexpressing KLF6 driven by a hepatocyte-specif ⁇ c promoter exhibit a runted phenotype, decreased hepatocyte mass, and impaired synthetic function associated with induction of p21 WAF1 /CIP1.
  • Induction of KLF6 in NIH 3T3 cells also induces p21 and provokes growth arrest.
  • KLF6 transactivat.es the p21 promoter and upregulates endogenous p21.
  • the data reveal a unique role for KLF6 in provoking growth arrest in vivo and in vitro by upregulating the cyclin dependent kinase inhibitor p21. This suggests that KLF6 provides an alternative mechamsm for p21 upregulation independent of p53.
  • the inventors then examined human tumors in which the p53 gene was intact, to determine if KLF6 gene was inactivated or if expression of the KLF6 protein was altered.
  • a human glioblastoma cell line (CRL2020) with a known chromosome 1 Op deletion has very low levels of KLF6. Additionally, KLF6 is not expressed in a neuroblastoma cell line, SKNML. These data link KLF6 to tumorigenesis and suggest that KLF6 is a tumor suppressor gene and can act independently of p53.
  • the inventors confirmed that KLF6 expression is attenuated in a variety of glial tumor cell lines. They further showed that expression of KLF6 into these cells inhibits their transformed phenotypes in vitro and reduces their ability to form tumors in mice. Additionally, KLF6 may block transformation by specific lesions found in glial tumors such as PDGFR amplification as evidenced by its ability to block the formation of foci induced by c-sis/PDGF-BB in NIH 3T3 cells.
  • LEO loss of heterozygosity
  • Tumor suppressor genes produce cancer via a two hit mechanism in which a first mutation, such as a point mutation (or a small deletion or insertion) inactivates one allele of the tumor suppressor gene. Often, this first mutation is inherited from generation to generation.
  • a second mutation often a spontaneous somatic mutation such as a deletion which deletes all or part of the chromosome carrying the other copy of the tumor suppressor gene, results in a cell in which both copies of the tumor suppressor gene are inactive. As a consequence of the deletion in the tumor suppressor gene, one allele is lost for any genetic marker located close to the tumor suppressor gene.
  • the tumor tissue loses heterozygosity, becoming homozygous or hemizygous. This loss of heterozygosity provides strong evidence for the existence of a tumor suppressor gene in the lost region.
  • This approach provided a way to find additional mutations in the KLF6 gene that alter the activity of the protein in a variety of cancers, such as prostate colon, breast, ovarian, head and neck cancer, hepatocellular carcinoma, and lung cancer.
  • inactivation or alteration of KLF6 and “inactivation or alteration of KLF6 gene” are used interchangeably to refer to a modification of the genomic sequence of KLF6 that results in impairment of transcription or translation of the gene or of activity of the gene product.
  • deletion of the segment of the chromosome containing KLF6, i.e., deletion of the KLF6 gene results in inactivation or alteration of that allele of the gene.
  • Inactivation or alteration of one allele may reduce the level of expression of KLF6 to below that necessary for proper cellular regulation. No KLF6 expression occurs with inactivation or alteration of both alleles.
  • KLF6 genomic DNA means the KLF6 gene with introns and exons, upstream and downstream regulatory sequences, and flanking markers or satellite sequences. In addition to gross chromosomal deletions or gene deletions, the term
  • Modification of genomic DNA refers to any mutation of the DNA that impairs gene expression or protein activity, or to any polymorphism which may be associated with a cancer.
  • mutations that lead to insertion of a heterologous sequence in the gene, truncation of the gene, or a nonsense mutation, a frameshift mutation, a splice-site mutation, a missense mutation, a translocation, or a methylation can result in inactivation or alteration of the gene.
  • point mutations can impact mRNA stability and translation efficiency, for example by introducing a base that affects secondary structure of the message.
  • Other point mutations can lead to expression of an inactive protein or of a protein with reduced activity. In the latter circumstance, protein expression may be detectable (e.g. , by immunoassay), so only analysis of the KLF6 gene sequence permits identification of an inactivating point mutation. ⁇
  • cancer refers to a malignant condition of uncontrolled cell growth.
  • the mass of cancerous cells is a tumor.
  • the cells are tumor cells.
  • Cancers or malignant tumors are classified according to the type of tissue from which they originate.
  • the broadest division of cancers separates the carcinomas, tumors which arise from epithelial tissues, and the sarcomas, which arise from all other tissues.
  • Epithelium is tissue that covers the internal or external surfaces of the body. Thus, skin, the lining of the mouth, stomach, intestines, bladder and so on are all epithelial tissue.
  • the present invention is directed to any type of hyperplasia, more particularly to any type of cancer, as well as any type of benign tumors, including hyperproliferative disorders. Sporadic as well as familial forms of cancers are encompassed.
  • the skin which consists of a type of epithelium called squamous epithelium, can give rise to squamous cell carcinomas.
  • epithelium a type of epithelium called basal epithelium
  • melanocytes which give rise to melanomas.
  • Adenocarcinoma is a cancer originating in glandular cells. Adenocarcinomas occur in the lungs, from small glands in the bronchi; in the stomach from one of the several types of glands lining it; and in the colon, breast, ovaries, testes, prostate and in other locations. Adenocarcinomas arising from different organs can often be identified by the pathologist microscopically, even when they are removed from a different location where they may have metastasized, such as the liver. Thus, it is common to refer to an adenocarcinoma of the stomach which has metastasized to the liver, or one from the colon metastasized to the lungs. Adenocarcinomas are the most common cell type of cancer, since they include almost all breast cancers, all colon cancers, all prostate cancers, and a fair percentage of lung cancers. The invention more particularly targets solid tumors, including carcinomas.
  • KLF6 is inactivated in these tumors.
  • solid tumors according to the invention include sarcomas and carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell
  • tumors that can be diagnosed (including determination of a diagnosis or prognosis, or determination of a relative risk of developing a tumor) or treated in accordance with the present invention: neuroblastoma, glioblastoma, melanomas, and hyperproliferative disorders.
  • Other preferred examples include prostate cancer, colon carcinoma, lung carcinoma, small cell lung carcinoma, breast cancer, ovarian cancer, hepatocellular carcinoma, and head and neck cancer.
  • Leukemia is also encompassed.
  • aa amino acids
  • bp base pairs
  • cDNA DNA complementary to RNA
  • FISH fluorescent in situ hybridization
  • GFP green fluorescent protein
  • kb(s) kilobase or 1000 bp
  • nt nucleotide
  • oligo oligodeoxyribonucleotide
  • ORF open reading frame
  • PCNA proliferating cell nuclear antigen
  • RFLP restriction fragment length polymorphism
  • SSC 0.15M NaCl/0.015M Na, citrate pH7.6
  • TTR transthyretin promoter
  • UTR untranslated region(s)
  • SNP single nucleotide polymorphism.
  • the term “about” or “approximately” means within an acceptable error for the type of measurement used to obtain a value, e.g. within 20%, preferably within 10%, and more preferably within 5% of a given value or range.
  • the term means within an order of magnitude, and preferably a factor of two, of a value.
  • isolated means that the referenced material is free of components present in the natural environment in which the material is normally found. In particular, isolated biological material is free of cellular components.
  • an isolated nucleic acid includes a PCR product, an isolated mRNA, a cDNA, or a restriction fragment.
  • an isolated nucleic acid is preferably excised from the chromosome in which it may be found, and more preferably is no longer joined to non-regulatory, non-coding regions, or to other genes, located upstream or downstream of the gene contained by the isolated nucleic acid molecule when found in the chromosome.
  • the isolated nucleic acid lacks one or more introns. Isolated nucleic acid molecules can be inserted into plasmids, cosmids, artificial chromosomes, and the like.
  • a recombinant nucleic acid is an isolated nucleic acid.
  • An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein.
  • An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in an organism.
  • An isolated material may be, but need not be, purified.
  • purified refers to material that has been isolated under conditions that reduce or eliminate unrelated materials, i. e. , contaminants.
  • a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell; a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within a cell.
  • a purified tumor cell is preferably substantially free of other normal cells.
  • substantially free is used operationally, in the context of analytical testing of the material.
  • purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.
  • nucleic acid molecule e.g., KLF6, cDNA, gene, etc.
  • normal text e.g. , KLF6 or Kruppel-like factor 6
  • KLF6 Zf9/CPBP
  • Kruppel-like family of transcription factors that is rapidly induced as an immediate early gene in hepatic stellate cells during acute liver injury (Ratziu, et al, Proc. Natl. Acad. Sci. USA 1998,
  • Hepatic regeneration is a complex response requiring coordinated expression of signaling intermediates and both proliferative and antiproliferative signals (Fausto, J. Hepatol. 2000, 32:19-31).
  • Adult hepatocytes are terminally differentiated and mitotically quiescent, yet the normal resting liver retains the ability to regenerate after partial hepatectomy or liver injury associated with parenchymal cell loss (Michalopoulos and DeFrances, Science 1997, 276: 60-66).
  • the KLF6 sequence encodes a 283 amino acid protein with two distinct domains, a 201 aa proline- and serine-rich amino terminal activation domain, and a carboxy terminal 82aa zinc finger C 2 H 2 DNA-binding domain.
  • this DNA binding domain (from aminoacid 202 to aminoacid 283) shows homology to other members of the KLF family.
  • the KLF6 activation domain (from aminoacid 1 to aminoacid 201) is unique and only shares partial homology with another member of the
  • KLF6 has been established as a transcription factor that binds to promoter regions of genes containing a "GC Box" motif (Ratziu, et al, 1998, supra). KLF6 regulates the expression of a placental glycoprotein (Koritschoner, et al. , J. Biol. Chem., 1997, 272:9573-9580), HIV-1 (Suzuki, et al, J. Biochem (Tokyo), 1998, 124:389-385), collagen al (I) (Ratziu et al. , 1998, supra), TGFbl , types I and II TGFb receptors
  • KLF6 or the KLF6 gene we include the published gene sequence (Ratziu, et al, 1998, supra), as well as its homologues, analogues, and derivatives.
  • sequence SEQ ID NO:25 is a KLF6 genomic sequence that is not meant to restrict the present invention but that may be useful to locate specific mutations described below.
  • a “vector” is a recombinant nucleic acid construct, such as plasmid, phage genome, virus genome, cosmid, or artificial chromosome, to which another DNA segment may be attached.
  • the vector may bring about the replication of the attached segment, e.g., in the case of a cloning vector.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i. e. , it is capable of replication under its own control.
  • vector includes both viral and nonviral means for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo.
  • Non- viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA- protein complexes, and biopolymers.
  • Niral vectors include retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, he ⁇ es simplex, Epstein-Barr and adenovirus vectors, as set forth in greater detail below.
  • a vector may also contain one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).
  • a “cassette” refers to a segment of D ⁇ A that can be inserted into a vector at specific restriction sites.
  • the segment of D ⁇ A encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation.
  • a cell has been "transfected” by exogenous or heterologous D ⁇ A when such D ⁇ A has been introduced inside the cell.
  • a cell has been "transformed” by exogenous or heterologous D ⁇ A when the transfected D ⁇ A is expressed and effects a function or phenotype on the cell in which it is expressed.
  • heterologous refers to a combination of elements not naturally occurring.
  • heterologous D ⁇ A refers to D ⁇ A not naturally located in the cell, or in a chromosomal site of the cell.
  • a heterologous expression regulatory element is such an element operatively associated with a different gene than the one it is operatively associated with in nature, such as a CMN promoter operatively associated with a KLF6 coding region.
  • an KLF6 gene is heterologous to vector D ⁇ A in which it is inserted for cloning or expression.
  • nucleic acid molecule refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine;
  • R ⁇ A molecules or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "D ⁇ A molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded D ⁇ A-D ⁇ A, D ⁇ A-R ⁇ A and R ⁇ A-R ⁇ A helices are possible.
  • nucleic acid molecule and in particular D ⁇ A or R ⁇ A molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.
  • this term includes double-stranded D ⁇ A found, inter alia, in linear (e.g. , restriction fragments) or circular D ⁇ A molecules, plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of D ⁇ A ( . e. , the strand having a sequence homologous to the mR ⁇ A).
  • a "recombinant D ⁇ A molecule” is a D ⁇ A molecule that has undergone a molecular biological manipulation.
  • Antisense nucleic acids may be used as probes or used to inhibit expression of KLF6.
  • An "antisense nucleic acid” is a single-stranded nucleic acid molecule which, on hybridizing with complementary bases in an R ⁇ A or D ⁇ A molecule, inhibits the latter's role. If the R ⁇ A is a messenger R ⁇ A transcript, the antisense nucleic acid is a countertranscript or mR ⁇ A-interfering complementary nucleic acid.
  • antisense broadly includes R ⁇ A-R ⁇ A interactions, R ⁇ A-D ⁇ A interactions, ribozymes, and R ⁇ ase-H mediated arrest.
  • Antisense nucleic acid molecules can be encoded by a recombinant gene for expression in a cell (e.g., U.S. Patent ⁇ os. 5,814,500 and 5,811,234), or alternatively they can be prepared synthetically (e.g. , U.S. Patent No. 5,780,607).
  • synthetic oligonucleotides envisioned for this invention include oligonucleotides that contain phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl, or cycloalkl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are those with CH 2 -NH-O-CH 2 , CH 2 - N(CH 3 )-O-CH 2 , CH 2 -O-N(CH 3 )-CH 2 , CH 2 -N(CH 3 )-N(CH 3 )-CH 2 and O-N(CH 3 )-CH 2 -CH 2 backbones (where phosphodiester is O-PO 2 -O-CH 2 ).
  • the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et al, Science, 1991, 254:1497).
  • oligonucleotides may contain substituted sugar moieties comprising one of the following groups at the 2' position: OH, SH, SCH 3 , F, OCN, O(CH 2 ) n NH 2 or O(CH 2 ) n CH 3 where n is from 1 to about 10; C t to C 10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF 3 ; OCF 3 ; O-; S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH 3 ; SO 2 CH 3 ; ONO 2 ;NO 2 ; N 3 ; NH 2 ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted sialyl; a fluorescein moiety; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of
  • Oligonucleotides may also have sugar mimetics such as cyclobutyls or other carbocyclics in place of the pentofuranosyl group.
  • Nucleotide units having nucleosides other than adenosine, cytidine, guanosine, thymidine and uridine may be used, such as inosine.
  • a “gene” is used herein to refer to a portion of a DNA molecule that includes a polypeptide coding sequence operatively associated with expression control sequences.
  • a gene includes both transcribed and untranscribed regions.
  • the transcribed region may include introns, which are spliced out of the mRNA, and 5'- and 3'-untranslated (UTR) sequences along with protein coding sequences.
  • a gene can be a genomic or partial genomic sequence, in that it contains one or more introns.
  • the term gene may refer to a cDNA molecule (i.e., the coding sequence lacking introns).
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence are determined by a translation start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
  • "Expression control sequences” e.g., transcriptional and translational control sequences, are regulatory sequences that flank a coding sequence, such as promoters, enhancers, suppressors, terminators, and the like, and that provide for the expression of a coding sequence in a host cell. In eukaryotic cells, a polyadenylation signal and transcription termination sequence will usually be located 3 ' to the coding sequence.
  • a ribosome binding site is an expression control sequence.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced (if it contains introns) and translated into the protein encoded by the coding sequence.
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al, 1989). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a T m of
  • Moderate stringency hybridization conditions correspond to a higher T m , e.g., 40% formamide, with 5x or 6x SCC.
  • High stringency hybridization conditions correspond to the highest T m , e.g., 50% formamide, 5x or 6x SCC.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the IX degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T m for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher T m ) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating
  • a minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; preferably at least about 15 nucleotides; and more preferably the length is at least about 20 nucleotides.
  • standard hybridization conditions refers to a T m of 55 °C, and utilizes conditions as set forth above.
  • the T m is 60 °C; in a more preferred embodiment, the T m is 65 °C.
  • high stringency refers to hybridization and/or washing conditions at 68°C in 0.2XSSC, at 42°C in 50% formamide,
  • oligonucleotide refers to a nucleic acid, generally of at least 10, preferably at least 15, and more preferably at least 20 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest. Oligonucleotides can be labeled, e.g. , with 32 P-nucleotides or nucleotides to which a ligand molecule, such as biotin, has been covalently conjugated.
  • a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid.
  • oligonucleotides (one or both of which may be labeled) can be used as PCR or sequencing primers, either for cloning full length or a fragment of KLF6, or to detect the presence of nucleic acids encoding KLF 6.
  • an oligonucleotide of the invention can form a triple helix with a KLF 6 DNA molecule.
  • a library of oligonucleotides arranged on a solid support can be used to detect various KLF6 polymo ⁇ hisms of interest.
  • oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.
  • homologous in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a "common evolutionary origin,” including proteins from superfamilies (e.g., the immunoglobulm superfamily) and homologous proteins from different species (e.g. , myosin light chain, etc.) (Reeck et al. , Cell, 1987, 50:667). Such proteins (and their encoding genes) have sequence homology, as reflected by their high degree of sequence similarity.
  • sequence similarity in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al, supra).
  • sequence similarity when modified with an adverb such as “highly, " may refer to sequence similarity and may or may not relate to a common evolutionary origin.
  • two DNA sequences are "substantially homologous" or “substantially similar” when at least about 30%, and preferably at least about 50%, of the nucleotides match over the defined length of the DNA sequences.
  • Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks or from commercial sources (BLAST, DNA Strider, DNA Star, FASTA, etc.) using standard or default parameters, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g. , Sambrook et al, 1989; DNA Cloning, Nols. I & IT, supra; Nucleic Acid Hybridization, supra.
  • two amino acid sequences are "substantially homologous" or “substantially similar” when greater than about 80%, preferably greater than about 90%, of the amino acids are identical, or greater than about 85%, preferably greater than about 95%, are similar (functionally identical).
  • the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any of the sequence alignment programs described above.
  • a gene encoding KLF6, whether genomic D ⁇ A or cD ⁇ A, can be isolated from any source, particularly from a human cD ⁇ A or genomic library. Methods for obtaining KLF 6 gene are well known in the art, as described above (see, e.g., Sambrook et al, 1989).
  • the D ⁇ A may be obtained by standard procedures known in the art from cloned D ⁇ A (e.g.
  • a D ⁇ A "library” preferably is obtained from a cDNA library prepared from tissues with high level expression of the protein (e.g., a hepatocyte cell library, since these cells evidence high levels of expression of KLF 6), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell (see, e.g. , Sambrook et al , 1989; DNA cloning, supra).
  • Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will not contain intron sequences.
  • the gene should be molecularly cloned into a suitable vector for propagation of the gene.
  • Identification of the specific DNA fragment containing the desired KLF 6 gene may be accomplished in a number of ways. For example, a portion of a KLF6 gene exemplified infra can be purified and labeled to prepare a labeled probe, and the generated DNA may be screened by nucleic acid hybridization to the labeled probe (Benton and Davis, Science, 1977, 196:180; GrunsteinandHogness, Proc. Natl. Acad. Sci. USA, 1975, 72:3961). Those DNA fragments with substantial homology to the probe, such as an allelic variant from another individual, will hybridize. In a specific embodiment, highest stringency hybridization conditions are used to identify a homologous KLF6 gene.
  • the gene encodes a protein product having the isoelectric, electrophoretic, amino acid composition, partial or complete amino acid sequence, antibody binding activity, or ligand binding profile of KLF6 protein as disclosed herein.
  • the presence of the gene may be detected by assays based on the physical, chemical, immunological, or functional properties of its expressed product.
  • the present invention also relates to cloning vectors containing genes encoding analogs and derivatives of KLF6 of the invention, that have the same or homologous functional activity as KLF6. Furthermore, although the present invention relates to human KLF6, in certain embodiments, such as for gene therapy, non-human variants of KLF6 are contemplated.
  • the derivative or analog is functionally active, i. e. , capable of exhibiting one or more functional activities associated with a full-length, wild- type KLF6 of the invention.
  • Such functions include p53 -independent upregulation of p21 , and inhibition of cell growth.
  • KLF6 are contemplated, as are fusion proteins that contain functional domains (discussed below).
  • KLF6 derivatives can be made by altering encoding nucleic acid sequences by substitutions, additions or deletions that provide for functionally equivalent molecules.
  • derivatives are made that have enhanced or increased functional activity relative to native KLF6.
  • chimeric derivatives with a greater self-aggregation potential e.g., prepared by inco ⁇ orating an additional or stronger aggregation sequence, may have increased functional activity.
  • KLF6 derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a KLF 6 protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a conservative amino acid substitution.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity and, if present, charge, which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations will not be expected to affect apparent molecular weight as determined by polyacrylamide gel electrophoresis, or isoelectric point. Particularly preferred substitutions are:
  • KLF6 derivatives and analogs of the invention can be produced by various methods known in the art. The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro. In the production of the gene encoding a derivative or analog of KLF6, care should be taken to ensure that the modified gene remains within the same translational reading frame as the KLF 6 gene, uninterrupted by translational stop signals, in the gene region where the desired activity is encoded.
  • the -ZE ⁇ ' -encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, C, etal, 3. Biol. Chem., 1978, 253:6551; Zoller and Smith, DNA, 1984, 3:479- 488; Oliphant etal, Gene, 1986, 44:177; Hutchinson etal, Proc.
  • the identified and isolated gene can then be inserted into an appropriate cloning vector.
  • vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Examples of vectors include, but are not limited to, E. coli, bacteriophages such as lambda derivatives, or plasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX vectors, pmal-c, pFLAG, etc.
  • the insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini.
  • the ends of the DNA molecules may be enzymatically modified.
  • any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.
  • Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.
  • the cloned gene is contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, e.g. , E. coli, and facile purification for subsequent insertion into an appropriate expression cell line, if such is desired.
  • a shuttle vector which is a vector that can replicate in more than one type of organism, can be prepared for replication in both E. coli and S ⁇ cch ⁇ romyces cerevisi ⁇ e by linking sequences from an E. coli plasmid with sequences from the yeast 2 ⁇ plasmid.
  • the nucleotide sequence coding for KLF6, including derivatives or analogs thereof, or a functionally active chimeric protein thereof (herein collectively KLF6”), can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence in vivo.
  • an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence in vivo.
  • the nucleic acid encoding KLF6 of the invention is operationally associated with a promoter in an expression vector of the invention. Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences.
  • An expression vector also preferably includes a replication origin.
  • an KLF6 polypeptide of the invention can be prepared using well-known techniques in peptide synthesis, including solid phase synthesis (using, e.g. , BOC of FMOC chemistry), or peptide condensation techniques.
  • polypeptide and protein may be used interchangeably to refer to the gene product (or corresponding synthetic product) of a KLF6 gene.
  • protein may also refer specifically to the polypeptide as expressed in cells.
  • a peptide is generally a fragment of a polypeptide, e.g., of about six or more amino acid residues.
  • the necessary transcriptional and translational signals can be provided on a recombinant expression vector, or they may be supplied by the native gene encoding KLF6 and/or its flanking regions.
  • control sequences whether a promoter or enhancer, or both, permit high level expression of KLF6 in the target cell, particularly for gene therapy (described infra).
  • Potential host- vector systems include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, adeno-associated virus, he ⁇ es virus, etc.); insect cell systems infected with virus (e.g.
  • baculovirus a virus containing yeast vectors
  • bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA.
  • the expression elements of vectors vary in their strengths and specificities. Depending on the host- vector system utilized, any one of a number of suitable transcription and translation elements may be used.
  • a recombinant KLF6 protein of the invention, or functional fragment, derivative, chimeric construct, or analog thereof, may be expressed chromosomally, after integration of the coding sequence by recombination. In this regard, any of a number of amplification systems may be used to achieve high levels of stable gene expression (See Sambrook et al, 1989).
  • any of the methods previously described for the insertion of DNA fragments into a cloning vector may be used to construct expression vectors containing a gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination).
  • Expression of KLF6 protein may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression.
  • the promoter permits high level expression in a mammalian, and more preferably human, host cell.
  • viral promoters some of which are listed below, often permit high level expression. Other such promoters are known.
  • Promoters which may be used to control KLF6 gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U.S.PatentNos.5,168,062 and 5,385,839), the SV40 early promoter region (Benoist and Chambon, Nature, 1981, 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al, Cell, 1980, 22:787- 797), the he ⁇ es thymidine kinase promoter (Wagner et al. , Proc. Natl. Acad. Sci. USA, 1981, 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster etal, Nature,
  • prokaryotic expression vectors such as the ⁇ -lactamase promoter (Nilla- Kamaroff, et al, Proc. ⁇ afi. Acad. Sci. USA, 1978, 75:3727-3731), or the tac promoter (DeBoer, et al, Proc. ⁇ atl. Acad. Sci. USA, 1983, 80:21-25); see also "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK
  • elastase I gene control region which is active in pancreatic acinar cells (Swift etal, Cell, 1984, 38:639-646; Ornitz etal, Cold Spring Harbor Symp. Quant. Biol, 1986, 50:399-409; MacDonald, Hepatology, 1987, 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, Nature, 1985, 315:115-122), immunoglobulm gene control region which is active in lymphoid cells (Grosschedl et al. , Cell,
  • alpha 1-antitrypsingene control region which is active in the liver (Kelsey et al, Genes and Devel., 1987, 1:161-171), beta- globin gene control region which is active in myeloid cells (Mogram et al, Nature, 1985, 315:338-340; Kollias et al, Cell, 1986, 46:89-94), myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readheadet /., Cell, 1987, 48:703-712), myosin light chain-2 gene control region which is active in skeletal muscle (Sani, Nature, 1985, 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al, Science, 1986, 234:1372-1378).
  • promoters useful for practice of this invention are ubiquitous promoters (e.g., HPRT, vimentin, actin, tubulin), intermediate filament promoters (e.g., desmin, neurofilaments, keratin, GFAP), therapeutic gene promoters (e.g., MDR type, CFTR, factor NIII), tissue-specific promoters (e.g., actin promoter in smooth muscle cells), promoters which are preferentially activated in dividing cells, promoters which respond to a stimulus (e.g., steroid hormone receptor, retinoic acid receptor), tetracycline-regulated transcriptional modulators, retroviral LTR, metallothionein, Ela, and MLP promoters. Tetracycline-regulated transcriptional modulators are described in
  • Promoters specific for expression in cells of the CNS can be used to drive expression of KLF6, e.g., for gene therapy of neuroblastoma or another CNS tumor.
  • KLF6 e.g., for gene therapy of neuroblastoma or another CNS tumor.
  • the promoters for the human tyrosine hydroxylase gene (Kim et al, Nucl. Acids Res., 1998, 26:1793-800), human dopamine beta-hydroxylase (DBH) gene (Zellmer et al, J. of
  • Vectors are introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (liposome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wuetal, J. Biol. Chem., 1992,267:963-967; Wuand Wu, J. Biol. Chem., 1988, 263:14621-14624; Canadian Patent Application No. 2,012,311).
  • the present invention provides for evaluating cancer in a subject or patient based on detecting whether KLF6 has been inactivated. This evaluation can provide either a diagnosis or a prognosis of cancer, or both. The present invention also provides for determining a relative risk of developing a cancer.
  • diagnosis in any grammatical form refers to the identification of a particular disease condition in a subject or patient. As the skilled physician knows, almost any diagnosis is based on a multiple of deteiminants, including symptomology, histology, and other criteria, which together form a diagnosis. Thus, when used herein, diagnosis according to the invention is one component or determinant of the final diagnosis.
  • prognosis or prognosis in any grammatical form refers to prediction of a disease outcome, e.g., whether the subject suffering from the disease is likely to improve or regress.
  • relative risk means the probability of the specified outcome in a given individual.
  • cancer any form of cancer, e.g., as discussed above, can be evaluated using the diagnostic methods of the invention.
  • the cancer is neuroblastoma, glioblastoma, melanomas, prostate cancer, colon carcinoma, lung carcinoma, small cell lung carcinoma, breast cancer, ovarian cancer, hepatocellular carcinoma, and head and neck cancer.
  • Leukemia is also encompassed.
  • kits contain, at least, a detection assay for inactivation or alteration of KLF 6, or for detecting sequence changes in the gene.
  • the absence, alteration or reduction of level of the KLF6 protein is detected by assessing the level of regulation of p21, or of a gene that is up- regulated or down-regulated by the induction of a functional KLF6, such as a gene selected from Tables 1 and 2:
  • level of regulation more particularly refers to the level of activation or inhibition of transcription of such down-stream genes. This may be assessed either by nucleic acid based assays, or protein based assays.
  • the assays described below for direct determination of KLF6 mRNA or KLF6 protein can be easily adapted to the determination of the level of regulation of these genes.
  • the assays employ microarray technology, whichpermits simultaneous detection of numerous expressed genes and provides an expression profile.
  • a normal cell and a cell to be tested are transfected with a reporter gene operatively linked to all or part of the promoter of any of these effector genes. The level of expression of the reporter gene is determined in the test cell in comparison with the normal control cell.
  • Reporter gene assays of the invention may use one or more of the commonly used detection techniques involving isotopic, colorimetric, flourimetric, or luminescent enzyme substrates and immuno-assay based procedures with isotopic, colorimetric, or chemiluminescent end points.
  • the assays of the invention include, but are not limited to, using the reporter genes for the following proteins: CAT (chloramphenicol acetyltransferase) GAL
  • ⁇ -galactosidase ⁇ -galactosidase
  • LUC luciferase
  • GFP green fluorescent protein
  • hGH human growth hormone
  • SEAP secreted form of the human placental alkaline phosphatase
  • Nucleic acid assays for inactivation or alteration of KLF6 are either based on detection of mutations or modifications in the KLF6 gene, or on detection of an altered form or decreased level of mRNA that encodes the KLF6 protein.
  • the nucleic acid (DNA or mRNA) to be assayed may be obtained from any cell source, depending on the assay being applied. Tumor cells may particularly be a useful source for these assays.
  • Non-limiting examples of cell sources available in clinical practice include without limitation blood cells, buccal cells, cervicovaginal cells, epithelial cells from urine, fetal cells, or any cells present in tissue obtained by biopsy.
  • Cells may also be obtained from body fluids, including without limitation blood, plasma, serum, lymph, milk, cerebrospinal fluid, saliva, sweat, urine, feces, and tissue exudates.
  • DNA or mRNA is extracted from the cell source or body fluid using any of the numerous methods that are standard in the art. It will be understood that the particular method used to extract the nucleic acid will depend on the nature of the source. Generally, the minimum amount of DNA to be extracted for use in the present invention is about 25 pg (corresponding to about 5 cell equivalents of a genome size of 3 x 10 9 base pairs).
  • Mutations of the KLF6 genomic DNA include an insertion in the gene, deletion of the gene, truncation of the gene (e.g., due to a nonsense, missense, or frameshift mutation), or disregulation of gene expression (e.g., due to a frameshift mutation or a splice-site mutation), as well as translocation or methylation. Examples of such mutations are set forth in Tables 3 a and 3b below, a well as in Example 7 infra. Table 3 a: Missense mutations
  • KLF6 is heterozygously or homozygously deleted from chromosome 10. Identification of gene deletion is readily accomplished using nucleic acid probes, PCR analysis, or direct sequencing. Identification of a frameshift mutation is readily accomplished using enzymes and analyzing the patterns of the cleaved products. Determination of polymo ⁇ hic positions is achieved by any means known in the art, including but not limited to direct sequencing, hybridization with allele-specific oligonucleotides, allele-specific PCR, ligase- PCR, HOT cleavage, denaturing gradient gel electrophoresis (DGGE), single-stranded conformational polymo ⁇ hism (SSCP), or Mass Spectrometry (MS).
  • direct sequencing hybridization with allele-specific oligonucleotides, allele-specific PCR, ligase- PCR, HOT cleavage, denaturing gradient gel electrophoresis (DGGE), single-stranded conformational polymo ⁇ hism (SSCP), or Mass
  • Denaturing high performance liquid chromatography may also be a convenient qualitative technique to screen for the presence of mutations or polymo ⁇ hims.
  • DHPLC is a highly sensitive PCR-based technique for nucleotide variant detection which relies on the principle of heteroduplex analysis by ion-pair reverse-phase liquid chromatography under partially denaturing conditions (Liu et al., Nuc Acids Res. 1998, 26:1396-400; O'Donovan et al. Genomics. 1998, 52:44-9).
  • Direct sequencing may be accomplished by any method, including without limitation, by enzymatic sequencing, using the Sanger method; by chemical sequencing, using the Maxam-Gilbert method; mass spectrometry sequencing; and sequencing using a chip-based technology (see, e.g., Little et al, Genet. Anal., 1996, 6:151).
  • DNA from a subject is first subjected to amplification by polymerase chain reaction (PCR) using specific amplification primers.
  • PCR polymerase chain reaction
  • Gene expression, or lack of gene expression, can be directly evaluated by detecting KLF6 mRNA.
  • Methods for detecting mRNA include Northern blotting and reverse transcriptase (RT)-PCR. These methods can be used to determine whether or not expression occurs, and whether a truncated (or oversized) message is expressed. All of these factors can help establish inactivation or alteration of KLF6.
  • Loss of heterozygosity (LOH) primers allow rapid and cost-efficient screening of a test sample, e.g. a tumor sample to assess for KLF6 copy number, e.g., if one copy/allele has been deleted.
  • Loss of heterozygosity (LOH) analysis is a powerful method for isolating, detecting, and confirming cancer susceptibility genes. It detects whether or not two copies of a gene are present in a tumor sample. In the case of tumor suppressor genes, both copies of the gene need to be functionally inactivated. LOH can be used to analyze if one copy has been deleted or lost.
  • a loss of heterozygosity (LOH) at the KLF6 locus can be associated with a cancer, for example prostate cancer, colon cancer, breast, ovarian cancer, hepatocellular carcinoma, small cell lung cancer, or head and neck cancer.
  • This allelic imbalance can be readily assessed by analyzing microsatellite markers or SNPs (single nucleotide polymo ⁇ hisms), or by means of any other standard method well known by one skilled in the art. Analysis of microsatellite markers is currently a preferred technique. For that pu ⁇ ose primer sequences are designed to flank the KLF 6 gene. Preferred pairs of primers useful for detecting these microsatellite markers are part of the present invention:
  • Antisense 5'-TTT CCA GCC CAC TGT CTT CTT GAC-3' (SEQ. ID NO. 2)
  • Antisense 5'-GAT GTG TTT GGC TCA GGG A-3' (SEQ. ID NO. 6)
  • the allelic imbalance may be determined by investigating
  • SNPs are sequence variations between individuals that may be identified by standard methods well-known to one skilled in the art. Examples of ⁇ -ZFo ' -related SNPs identified from a screening of 50 healthy unrelated individuals are described hereafter. The position of these SNPs is given here with regard to the named exons. A minus sign "-" means
  • Exon 1 (these are present within the promoter region and may thus play a role in transcriptional efficiencies): position -80 C > T position -4 O A
  • SNPs is given with regard to the first nucleotide of SEQ ID No. 25.
  • the S P at position 3023 as above described is of particular importance. This variant is present in 26 out of 142 patients with prostate cancer from families with an inherited predisposition to prostate cancer. It is only present in 8 out of 103 control individuals. This S ⁇ P thus represents a significant predictor of cancer risk (p ⁇ 0.009). This S ⁇ P is all the more interesting in that it can be easily determined in blood samples. It can be detected prior to development of a cancer, in particular prostate cancer. This substitution of nucleotide 3023 (Gtg -> Atg) was also found in head and neck cancer, as well as ovarian, breast, and lung cancer.
  • a nucleic acid assay kit of the invention will comprise a nucleic acid that specifically hybridizes under stringent conditions to a KLF6 gene, or KLF6 mRNA and an assay detector, e.g. , a label.
  • an assay detector e.g. , a label.
  • the kit is an amplification (such as PCR)-based kit
  • a primer pair will be included; in this case, the detector may simply be a reagent such as ethidium bromide to quantify amplified DNA.
  • a nucleic acid based kit of the invention includes primer pairs for PCR analysis of a KLF6 mutations or SNPs, more particularly at a functional domain, such as the activation domain, the DNA binding domain, as well as a putative Casein Kinase II phosphorylation site, or protein kinase C phosphorylation sites.
  • a functional domain such as the activation domain, the DNA binding domain, as well as a putative Casein Kinase II phosphorylation site, or protein kinase C phosphorylation sites.
  • Optional components include buffer or buffer reagents, nucleotides, and instructions for use of the kit. If possible, a positive control for this assay is also included.
  • microarrays for identifying loss of heterozygosity or point mutations
  • the present invention makes use of microarrays for identifying loss of heterozygosity at the KLF6 locus, by detecting microsatellite markers.
  • oligonucleotide probes that may be selected within the above- described three pairs of oligonucleotides are attached on a solid support.
  • Other oligonucleotides e.g. oligonucleotides for example, allowing the detection of additional microsatellite markers, may be advantageously used, preferably in combination with at least one oligonucleotide selected within the above three pairs of oligonucleotides.
  • Microarrays may be designed so that the same set of identical oligonucleotides is attached to at least two selected discrete regions of the array, so that one can easily compare a normal sample, contacted with one of said selected regions of the array, against a test sample, contacted with another of said selected regions. These arrays avoid the mixture of normal sample and test sample, using microfluidic conduits.
  • the microarray techniques developed by Nanogen, Inc may be of particular interest in that respect.
  • the invention makes use of microarrays for identifying mutations in the KLF6 gene, more particularly SNPs.
  • Affymetrix may be particularly useful in that request.
  • all types of microarrays also called “gene chips” or “DNA chips” may be adapted, either for the detection of microsatellite markers or for the identification of mutations.
  • microarrays are well known in the art (see for example the following: U. S . Pat Nos. 6,045,996; 6,040,138; 6,027,880;6,020,135; 5,968,740; 5,959,098; 5,945,334; 5,885,837; 5,874,219; 5,861,242; 5,843,655; 5,837,832; 5,677,195 and 5,593,839).
  • the solid support on which oligonucleotides are attached may be made from glass, silicon, plastic (e.g., polypropylene, nylon), polyacrylamide, nitrocellulose, or other materials.
  • One method for attaching the nucleic acids to a surface is by printing on glass plates, as is described generally by Schena et al. , Science 1995, 270:467-470. This method is especially useful for preparing microarrays of cDNA. See also DeRisi et al, Nature Genetics 1996, 14:457-460, ; Shalon et al. , Genome Res. 1996, 6:639-645; and Schena et al , Proc. Natl. Acad.
  • microarrays Another method of making microarrays is by use of an inkjet printing process to bind genes or oligonucleotides directly on a solid phase, as described, e.g., in U.S. Patent No. 5,965,352.
  • Other methods for making microarrays e.g., by masking (Maskos and Southern,
  • any type of array for example, dot blots on a nylon hybridization membrane (see Sambrook et al, Molecular Cloning A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989) could be used, although, as will be recognized by those of skill in the art, very small arrays will be preferred because hybridization volumes will be smaller.
  • nucleic acid hybridization and wash conditions are chosen so that the attached oligonucleotides "specifically bind” or “specifically hybridize” to at least a portion of the KLF6 gene present in the tested sample , i.e., the probe hybridizes, duplexes or binds to the KLF6 locus with a complementary nucleic acid sequence but does not hybridize to a site with a non-complementary nucleic acid sequence.
  • one polynucleotide sequence is considered complementary to another when, if the shorter of the polynucleotides is less than or equal to 25 bases, there are no mismatches using standard base-pairing rules or, if the shorter of the polynucleotides is longer than 25 bases, there is no more than a 5% mismatch.
  • the polynucleotides are perfectly complementary (no mismatches). It can easily be demonstrated that specific hybridization conditions result in specific hybridization by carrying out a hybridization assay including negative controls (see, e.g., Shalon et al, supra, and Chee et al, Science 1996, 274:610-614).
  • Optimal hybridization conditions will depend on the length (e.g. , oligomer versus polynucleotide greater than 200 bases) and type (e.g., RNA, DNA, PNA) of labeled probe and immobilized polynucleotide or oligonucleotide.
  • length e.g. , oligomer versus polynucleotide greater than 200 bases
  • type e.g., RNA, DNA, PNA
  • General parameters for specific (i.e., stringent) hybridization conditions for nucleic acids are described in Sambrook et al. , supra, and in Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, 1987.
  • typical hybridization conditions are hybridization in 5 x SSC plus 0.2% SDS at 65 °C.
  • a variety of methods are available for detection and analysis of the hybridization events.
  • detection and analysis are carried out fluorimetrically, colorimetrically or by autoradiography.
  • emitted radiation such as fluorescent radiation or a particle emission
  • information may be obtained about the hybridization events.
  • the fluorescence emissions at each site of transcript array can, preferably be detected by scanning confocal laser microscopy.
  • a separate scan, using the appropriate excitation line, is carried out for each of the two fluorophores used.
  • a laser can be used that allows simultaneous specimen illumination at wavelengths specific to the two fluorophores and emissions from the two fluorophores can be analyzed simultaneously (see Shalon et al. Genome Res. 1996, 6:639-695).
  • Signals are recorded and, in a preferred embodiment, analyzed by computer, e.g., using a 12 bit analog to digital board.
  • the scanned image is despeckled using a graphic program (e.g., Hijaak Graphics Suite) and then analyzed using an image gridding program that creates a spreadsheet of the average hybridization at each wavelength at each site. If necessary, an experimentally determined correction for "cross talk" (or overlap) between the channels for the two fluors may be made. For any particular hybridization site on the transcript array, a ratio of the emission of the two flourophonres can be calculated.
  • a perturbation in addition to identifying a perturbation as positive or negative, it is advantageous to determine the magnitude of the perturbation. This can be carried out, as noted above, by calculating the ratio of the emission of the two fluorophores used for differential labeling, or by analogous methods that will be readily apparent to those of skill in the art.
  • KLF6 Protein Based Assays
  • this assay may be the most informative, since KLF6 mRNA levels may appear high, but a mutation in the sequence may make the mRNA less effective for translation, resulting in reduction or elimination of protein expression.
  • KLF6 is detected by immunoassay.
  • immunoassay For example, as exemplified infra, Western blotting permits detection of the presence or absence of KLF6.
  • Other immunoassay formats e.g., as discussed above in connection with LE ⁇ ' -specific antibodies, can also be used in place of Western blotting.
  • a biochemical assay can be used to detect expression of KLF6, e.g., by the presence or absence of a band by polyacrylamide gel electrophoresis; by the presence or absence of a chromatographic peak by any of the various methods of high performance liquid chromatography, including reverse phase, ion exchange, and gel permeation; by the presence or absence of KLF6 in analytical capillary electrophoresis chromatography, or any other quantitative or qualitative biochemical technique known in the art.
  • tissue that expresses KLF6 may be used, in particular when searching protein isoforms associated with an increased risk of cancer.
  • the tissue is a biopsy tissue obtained from a subj ect.
  • the tumor cells should be purified from other tissue to ensure that contaminating KLF6 from normal cells is not detected.
  • Antibodies that are capable of binding to KLF6 are then contacted with samples of the tissue under conditions that permit antibody binding to determine the presence or absence of KLF6.
  • antibodies that distinguish polymo ⁇ hic variants of KLF 6 can be used.
  • the antibodies may be polyclonal or monoclonal, preferably monoclonal.
  • Measurement of specific antibody binding to cells may be accomplished by any known method, e.g., quantitative flow cytometry, or enzyme-linked or fluorescence-linked immunoassay.
  • the presence or absence of a particular mutation, and its allelic distribution i.e., homozygosity vs. heterozygosity is determined by comparing the values obtained from a patient with norms established from populations of patients having known polymo ⁇ hic patterns.
  • kits of the invention provides a KLF6 detector, e.g., a detectable antibody (which may be directly labeled or which may be detected with a secondary labeled reagent).
  • a KLF6 detector e.g., a detectable antibody (which may be directly labeled or which may be detected with a secondary labeled reagent).
  • anti-tumor gene therapy refers to a gene therapy targeted to cells of a tumor, t ' .e., cancer, which causes tumor necrosis, apoptosis, growth regulation, i.e., regression or suppression of the tumor.
  • anti-tumor gene therapy refers to administration or delivery of a gene encoding KLF6, either alone or in combination with other genes effective for treating tumors.
  • anti-tumor gene therapies of the prior art include, but are by no means limited to, introduction of a suicide gene; introduction of an apoptosis gene; introduction of a tumor suppresser gene; and introduction of an oncogene antagonist gene.
  • anti-tumor genes such as KLF6
  • KLF6 anti-tumor genes
  • immunostimulatory genes to enhance recruitment and activation of immune effector cells.
  • a viral, such as adenovirus, vector is used (see, e.g., PCT Publication No. WO 95/14101), the presence of adeno viral antigens could also provide an adjuvant effect to overall enhanced immune responsiveness.
  • Gene therapy refers to transfer of a gene encoding an effector molecule into cells, in this case of the tumor.
  • Gene therapy vectors include, but are not limited to, viral vectors (including retroviruses and DNA viruses), naked DNA vectors, and DNA-transfection agent admixtures.
  • a therapeutically effective amount of the vectors are delivered in a pharmaceutically acceptable carrier.
  • the phrase "therapeutically effective amount” is used herein to mean an amount sufficient to reduce by at least about 15 percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably prevent, a clinically significant deficit in the activity, function and response of the host, e.g., tumor progression, metastasises, or progression to the next stage of cancer.
  • a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host, e.g., to induce remission, reduce tumor size or burden, or both, or increase the time from treatment until relapse.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • the term "pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
  • Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E. W. Martin. Such methods, including routes of administration and dose, are well known in the art. These are discussed in greater detail in a section directed to "Gene Therapy Vectors" below, as well as in the references disclosed therein.
  • Gene therapy in accordance with the invention can be used to treat any cancer, but particularly tumors where KLF 6 is inactivated in the tumor cells of the cancer.
  • this does not necessarily have to be the case: increasing the level of expression of KLF6 beyond endogenous levels is expected to slow cell growth even further.
  • assays that detect expression e.g., Northern assays
  • translation e.g., immunoassays
  • KLF6 may not differentiate a defective gene product from wild-type, thus delivery of the wild-type gene may be useful even if it appears that the cell expresses KLF6.
  • KLF6 gene therapy of a tumor can be combined with other anti- tumor therapies, including but by no means limited to suicide gene therapy, anti-oncogene or tumor suppressor gene therapy, administration of tumor growth inhibitors, administration of angiogenesis inhibitors, immune therapies with an immunologically active polypeptide (including immunostimulation, e.g. , in which the active polypeptide is a cytokine, lymphokine, or chemokine, and vaccination, in which the active polypeptide is a tumor specific or tumor associated antigen), and conventional cancer therapies (chemotherapy and radiation therapy).
  • immunologically active polypeptide including immunostimulation, e.g. , in which the active polypeptide is a cytokine, lymphokine, or chemokine
  • vaccination in which the active polypeptide is a tumor specific or tumor associated antigen
  • conventional cancer therapies chemotherapy and radiation therapy
  • Suicide gene therapies Introduction of genes that encode enzymes capable of conferring to tumor cells sensitivity to chemotherapeutic agents (suicide gene) has proven to be an effective anti-tumor gene therapy.
  • a representative example of such a suicide gene is thymidine kinase of he ⁇ es simplex virus. Additional examples are thymidine kinase of varicella zoster virus and the bacterial gene cytosine deaminase which can convert 5-fluorocytosine to the highly toxic compound 5-fluorouracil.
  • the prodrug useful in the methods of the present invention is any that can be converted to a toxic product, i. e. , toxic to tumor cells.
  • the prodrug is converted to a toxic product by the gene product of the therapeutic nucleic acid sequence in the vector useful in the method of the present invention.
  • Representative examples of such a prodrug is ganciclovir, which is converted in vivo to a toxic compound by HSV-tk.
  • pro-drugs include acyclovir, FIAU [l-(2-deoxy-2-fluoro- ⁇ -D-arabinofuranosyl)-5-iodouracill, 6- methoxypurine arabino-side for VZV-tk, and 5-fluorocytosine for cytosine deambinase.
  • Anti-oncogene and tumor suppressor gene therapies Tumor initiation and progression in many cancer types are linked to mutations in oncogenes (e.g., ras, myc) and tumor suppresser genes (e.g., retinoblastoma protein, p53).
  • oncogenes e.g., ras, myc
  • tumor suppresser genes e.g., retinoblastoma protein, p53.
  • a number of approaches are being pursued using anti-oncogene molecules including monoclonal antibodies, single chain antibody vectors, antisense oligonucleotide constructs, ribozymes and immunogenic peptides (Chen, Mol. Med. Today, 1997, 3:160-167; Spitz, et al, Anticancer Res., 1996, 16:3415-3422; Indolfi et al, Nat.
  • a vector is any means for the transfer of a nucleic acid according to the invention into a host cell.
  • Preferred vectors for transient expression are viral vectors, such as retroviruses, he ⁇ es viruses, adenoviruses and adeno-associated viruses.
  • viral vectors such as retroviruses, he ⁇ es viruses, adenoviruses and adeno-associated viruses.
  • a gene encoding a functional KLF6 protein or polypeptide domain fragment thereof can be introduced in vivo, ex vivo, or in vitro using a viral vector or through direct introduction of DNA.
  • Expression in targeted tissues can be effected by targeting the transgenic vector to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue-specific promoter, or both. Targeted gene delivery is described in PCT Publication No.
  • Viral vectors are DNA-based vectors and retro viral vectors such a lenti virus (Park etal, Nat. Genet. 2000, 24(l):49-52). Methods for constructing and using viral vectors are known in the art (see, e.g., Miller and Rosman, BioTechniques, 1992, 7:980-990).
  • the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell.
  • the genome of the replication defective viral vectors which are used within the scope of the present invention lack at least one region which is necessary for the replication of the virus in the infected cell.
  • These regions can either be eliminated (in whole or in part), be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution (by other sequences, in particular by the inserted nucleic acid), partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents. Preferably, the replication defective virus retains the sequences of its genome which are necessary for encapsidating the viral particles. Defective retrovirus vectors may be preferred.
  • DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to he ⁇ es simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV) (Kay et al, at. Genet. 2000, 24(3):257-61), and the like.
  • HSV he ⁇ es simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted.
  • vectors examples include, but are not limited to, a defective he ⁇ es virus 1 (HSV1) vector (Kaplitt et al. , Molec. Cell. Neurosci., 1991, 2:320-330), defective he ⁇ es virus vector lacking a glyco-protein L gene (Patent Publication RD 371005 A), or other defective he ⁇ es virus vectors (PCT Publication Nos. WO 94/21807 and WO 92/05263); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet etal. (J. Clin.
  • a defective he ⁇ es simplex virus has been shown to be effective for delivery of genes, particularly to cells of the CNS (see, e.g., Belloni et al, Human Gene Therapy, 1996, 7:2015-24).
  • Recombinant defective adenoviruses have been used for transferring foreign genes into cells, particularly for gene therapy of tumors (as noted above), and for delivery of therapeutic genes to cells of the central nervous system.
  • PCT Publication Nos.WO 94/08026 and WO 94/08026 describe recombinant adenovirus vectors for the transfer of foreign genes into the central nervous system (CNS) .
  • Other examples of gene delivery to the CNS include the following : French Publication No.
  • FR2717824 discloses adenoviruses containing DNA from glial derived neutrophilic factors, which infected nerve cells very efficiently; various publications describe adenoviral vectors that express glial maturation factor (FR2717497), brain derived neurotropic factor (FR2717496) and acidic fibroblast growth factor (FR2717495); PCT Publication No. WO 95/26409 describes adenoviruses containing the DNA sequence for basic fibroblast growth factor to infect cells directly or via implants to treat neurological disorders; PCT Publication No. WO 96/00790 describes adenoviruses containing DNA encoding superoxide dismutase (SOD) to treat neurodegenerative diseases and excessive SOD expression; and PCT Publication No. WO 96/01902 describes adenoviruses expressing nitric oxide synthase for gene therapy where angiogenesis is required for treating disorders of the CNS.
  • SOD superoxide dismutase
  • Adenoviruses can be genetically modified to reduce the levels of viral gene transcription and expression, including adenoviruses defective in the El and E4 regions (PCT Publication No. WO 96/22378) and adenoviruses with an inactivated El region but also with altered genomic organization reducing the number of viable viral particles produced if recombination occurs with the host genome (PCT Publication No. WO 96/13596).
  • PCT Publication No. WO 96/10088 describes defective adenoviruses with an inactivated IVa2 gene.
  • PCT Publication No. WO 95/02697 describes an adenovirus defective in regions El and E2, E4, or Ll-L5.
  • plasmovirus Combination virus
  • a gene can be introduced using a combined virus, also termed plasmovirus (Genopoietic, France) vector system.
  • Plasmovirus systems permit one cycle of infectious virus formation in infected host cells.
  • a complementing gene(s) for defective viral genome sequences and the defective viral sequences are both provided to target cells in vivo or in vitro.
  • the primary infected cells produce infectious, defective virus.
  • plasmovirus technology amplifies gene delivery in vitro and, particularly, in vivo.
  • Non-viral vectors can be introduced in vivo by lipofection, as naked DNA, or with other transfection facilitating agents (peptides, polymers, etc.).
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et. al, Proc. Natl. Acad. Sci. USA, 1987, 84:7413-7417; Feigner and Ringold, Science, 1989, 337:387-388; see Mackey et al, Proc. Natl. Acad. Sci. USA, 1988, 85:8027-8031; Ulmer et al, Science, 1993, 259:1745-1748).
  • lipid compounds and compositions for transfer of nucleic acids are described in PCT Publication Nos. WO 95/18863 and WO 96/17823, and in U.S. Patent No. 5,459,127.
  • Lipids may be chemically coupled to other molecules for the pu ⁇ ose of targeting (see Mackey et. al, supra).
  • Targeted peptides e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • a nucleic acid in vivo, is also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., PCT Publication No. WO 95/21931), peptides derived from DNA binding proteins (e.g. , PCT Publication No. WO 96/25508), or a cationic polymer (e.g. , PCT Publication No. WO 95/21931).
  • a cationic oligopeptide e.g., PCT Publication No. WO 95/21931
  • peptides derived from DNA binding proteins e.g. , PCT Publication No. WO 96/25508
  • a cationic polymer e.g. , PCT Publication No. WO 95/21931
  • naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, e.g., Wu et al, J. Biol. Chem., 1992, 267:963-967; Wu and Wu, J. Biol. Chem., 1988, 263:14621- 14624; Canadian Patent Application No. 2,012,311; Williams etal, Proc. Natl. Acad. Sci.
  • Gene correction in vitro may also be achieved by various techniques, including chimeraplasty (Tagalakis etal, J. Biol. Chem., 2001 , 276(16): 13226-30; Ken et al, Semin. Liver Dis 1999, 19(1):93-104). This technique is based on the observation that oligonucleotides containing complementary RNA/DNA hybrid regions are more active than duplex DNA in homologous pairing reactions in vitro.
  • the chimeric molecules are designed with a homologous targeting sequence comprised of a DNA region flanked by blocks of 2'-O-methyl RNA residues (the chimeric strand), its complementary all-DNA strand, thymidine hai ⁇ in caps, a single-strand break, and a double-stranded clamp region.
  • the oligonucleotide can align in perfect register with a genomic target except for the designed single base pair mismatch, which is recognized and corrected by harnessing the cell's endogenous DNA repair system.
  • transposon technology successfully reported for the nonhomologous insertion of foreign genes into genomes of adult mammals using naked DNA (Yart etal, Nat. Genet 2000, 251(1):35-41).
  • Linear DNA concatamers provide another approach for achieving expression of a transgene in vivo (Chen etal, Mol. Ther.2001, 3(3):403-10).
  • the present invention further provides a method for preventing or treating hype ⁇ lasia of cells in a subject, in need of such treatment, wherein an effective amount of a functional KLF6 protein is administered to the subject.
  • the hype ⁇ lasia may be a cancer as described above.
  • the invention thus encompasses pharmaceutical compositions comprising a KLF6 protein as an active ingredient, with a pharmaceutically acceptable carrier.
  • KLF6 analogues KLF6 analogues.
  • the invention contemplates using analogues, derivatives or mimetics of the KLF6 protein as the active ingredient.
  • active ingredient is designed so that it may not be cleaved by proteolytic enzymes, such as enzymes of the digestive tract.
  • a KLF6 protein may be modified by combining it to a translocation peptide sequence.
  • Peptide sequences have been identified that mediate membrane transport, and accordingly provide for delivery of polypeptides to the cytoplasm.
  • such peptides can be derived from the antennapedia homeodomain helix 3 to generate membrane transport vectors, such as penetratin (PCT Publication WO 00/29427; see also Fischer et al, J. Pept. Res. 2000, 55:163-72; DeRossi et al, Trends in Cell Biol. 1998, 8:84-7; Brugidou et al, Biochem. Biophys. Res. Comm. 1995, 214:685-93).
  • PCT Publication WO 00/29427 see also Fischer et al, J. Pept. Res. 2000, 55:163-72; DeRossi et al, Trends in Cell Biol. 1998, 8:84-7; Brugidou et al,
  • Protein transduction domains which include the antennapedia domain and the HIV TAT domain (see Vives et al. , J. Biol. Chem. 1997, 272 : 16010- 17), posses a characteristic positive charge, which led to the development of cationic 12-mer peptides that can be used to transfer therapeutic proteins and DNA into cells (Mi etal, Mol. Therapy 2000, 2:339-47).
  • Therapeutic polypeptides can be generated by creating fusion proteins or polypeptide conjugates combining a translocation peptide sequence with a therapeutically functional sequence. For example, p21 WAF1 -derived peptides linked to a translocation peptide inhibited ovarian tumor cell line growth (Bonfantiet /., Cancer Res. 1997, 57:1442-1446). These constructs yield more stable drug-like polypeptides able to penetrate cells and effect a therapeutic outcome. These constructs can also form the basis for rational drug design approaches. In addition, complexes containing tetrameric streptavidin, e.g., including a biotinylated protein, translocate into the cytoplasmic efficiently with preservation of protein function.
  • tetrameric streptavidin e.g., including a biotinylated protein
  • a preferred such construct employs a Protein A-streptavidin fusion protein, which can bind a targeting antibody and the active protein, which can be biotinylated (see, e.g., U.S. Patent No. 5,328,985; Sano and Cantor, Bio/Technology 1991, 9:1378-81; Ohno et al, Biochem. Mol. Med. 1996, 58:227-33; Yu et al, DNA and Cell Biol. 2000, 19:383-8).
  • the concentration or amount of the active ingredient depends on the desired dosage and administration regimen, as discussed below. Suitable dose ranges may include from about 1 mg/kg to about 100 mg/kg of body weight per day.
  • compositions may also include other biologically active compounds.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • a composition comprising "A” (where "A” is a single protein, DNA molecule, vector, recombinant host cell, etc.) is substantially free of “B” (where “B” comprises one or more contaminating proteins, DNA molecules, vectors, etc.) when at least about 75% by weight of the proteins, DNA, vectors (depending on the category of species to which A and B belong) in the composition is "A".
  • "A” comprises at least about 90% by weight of the A+B species in the composition, most preferably at least about 99% by weight. It is also preferred that a composition, which is substantially free of contamination, contain only a single molecular weight species having the activity or characteristic of the species of interest.
  • the pharmaceutical composition of the invention can be introduced parenterally, transmucosally, e.g., orally (per os), nasally, or rectally, or transdermally.
  • Parental routes include intravenous, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration.
  • administration is targeted to the cancer tissue.
  • the active ingredient can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss: New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.317-327; see generally ibid.). To reduce its systemic side effects, this may be a preferred method for introducing the agent.
  • the therapeutic compound can be delivered in a controlled release system.
  • a polypeptide may be administered using intravenous infusion with a continuous pump, in a polymer matrix such as poly-lactic/glutamic acid (PLGA), a pellet containing a mixture of cholesterol and the active ingredient (SilasticRTM; Dow Corning, Midland, MI; see U.S. Patent No. 5,554,601) implanted subcutaneously, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.
  • a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng.
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press: Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley: New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al, J. Neurosurg. 71:105 (1989)).
  • the present invention provides for further enhancement of the anti-tumor effect by including additional anti-tumor treatments with the anti-tumor gene.
  • additional anti-tumor treatments with the anti-tumor gene for example, the present invention contemplates further combinations with tumor growth inhibitors, anti- angiogenesis treatment, tumor antigen and whole tumor vaccines, chemotherapeutic agents, radiation, and surgery (tumor resection).
  • Tumor growth inhibitors are used herein to refer to a protein that inhibits tumor growth, such as but not limited to interferon (TFN)- ⁇ , tumor necrosis factor (TNF)- ⁇ , TNF- ⁇ , and similar cytokines.
  • a tumor growth inhibitor can be an antagonist of a tumor growth factor.
  • Such antagonists include, but are not limited to, antagonists of tumor growth factor (TGF)- ⁇ and IL-10.
  • TGF tumor growth factor
  • the present invention contemplates administration of tumor growth inhibitor proteins systemically, or alternatively by gene therapy. In a specific gene therapy embodiment, the gene therapy vector is administered directly to the tumor.
  • Anti-angiogenic factors Tumor angiogenesis is an integral part of tumor progression and a variety of therapies targeted to inhibit angiogenesis are under development as cancer therapies. Anti-angiogenesis molecules vary from anti-angiogenic proteins to small molecules that block growth factor receptor mediated effects. Anti-angiogenesis therapies primarily reverse the growth/apoptosis balance of the tumor and induce dormancy. Once the administration of these therapies is halted, angiogenesis can resume and tumor growth progresses.
  • An "anti-angiogenic factor” is a molecule that inhibits angiogenesis, particularly by blocking endothelial cell migration.
  • Such factors include fragments of angiogenic proteins that are inhibitory (such as the ATF of urokinase), angiogenesis inhibitory factors, such as angiostation (O'Reilly et al, Cell, 1994, 79:315-328) and endostatin; tissue inhibition of metalloproteinase (Johnson et al, J. Cell. PhysioL, 1994, 160:194-202); soluble receptors of angiogenic factors, such as the urokinase receptor or FGF/NEGF receptor (Wilhem et al, FEBS Letters, 1994, 337:131-134); molecules which block endothelial cell growth factor receptors (O'Reilly et.
  • inhibitory such as the ATF of urokinase
  • angiogenesis inhibitory factors such as angiostation (O'Reilly et al, Cell, 1994, 79:315-328) and endostatin
  • tissue inhibition of metalloproteinase Johnson et al, J
  • an anti-angiogenic factor for use in the invention is a protein or polypeptide, which may be encoded by a gene transfected into tumors using vectors of the invention.
  • the vectors of the invention can be used to deliver a gene encoding an anti-angiogenic protein into a tumor in accordance with the invention.
  • Immune activation administration of various immunostimulatory molecules (cytokines, lymphokines, and chemokines, for example), such as CM-CSF and IL-2, can stimulate any immune response in conjunction with the tumor suppressor activity of KLF6.
  • immunostimulatory molecules cytokines, lymphokines, and chemokines, for example
  • the immunostimulatory molecules can be delivered as proteins, e.g., by intravenous injection, or as therapeutic expression vectors, for expression in the host.
  • TAA tumor associated antigens
  • Chemotherapeutic agents are effective in inhibiting tumor growth and metastasis
  • the vectors and methods of the present invention are advantageously used with other treatment modalities, including without limitation surgery, radiation, chemotherapy, and other gene therapies.
  • the vectors of the invention can be administered in combination with nitric oxide inhibitors, which have vasoconstrictive activity and reduce blood flow to the tumor.
  • a vector of the invention can be administered with a chemotherapeutic such as, though not limited to, taxol, taxotere and other taxoids (e.g., as disclosed in U.S. Patent Nos. 4,857,653; 4,814,470; 4,924,011, 5,290,957; 5,292,921; 5,438,072; 5,587,493; European Patent No. EP 253 738; and PCT Publication Nos.
  • chemotherapeutic such as, though not limited to, taxol, taxotere and other taxoids (e.g., as disclosed in U.S. Patent Nos. 4,857,653; 4,814,470; 4,924,011, 5,290,957; 5,292,921; 5,438,072; 5,587,493; European Patent No. EP 253 738; and PCT Publication Nos.
  • WO 91/17976 WO 93/00928, WO 93/00929, and WO 96/01815), or other chemotherapeutics, such as cis-platin (and other platinum intercalating compounds), etoposide and etoposide phosphate, bleomycin, mitomycin C, CCNU, doxorubicin, daunorubicin, idarubicin, ifosfamide, and the like.
  • cis-platin platinum intercalating compounds
  • etoposide and etoposide phosphate bleomycin, mitomycin C, CCNU, doxorubicin, daunorubicin, idarubicin, ifosfamide, and the like.
  • KLF 6 the role of KLF 6 in cancer provides for development of screening assays, particularly for high- throughput screening of molecules that agonize or antagonize the activity of KLF6.
  • indicator cells that are specially engineered to indicate the activity of KLF6, particularly the inhibition of cell growth, can serve as targets to identify either a KLF6 inducer or replacement.
  • KLF6 substitutes i.e., compounds that inhibit cell growth in the absence of KLF 6.
  • candidate compound i.e., compounds that inhibit cell growth in the absence of KLF 6.
  • KLF6 antagonists i. e. , molecules that inhibit KLF6 activity and prevent inhibition of cell growth.
  • the present invention contemplates methods for identifying specific substitutes and antagonists of KLF6 activity using various screening assays known in the art. Any screening technique known in the art can be used to screen for KLF6 agonists or antagonists.
  • the present invention contemplates screens for synthetic small molecules as well as screens for natural molecules that agonize or antagonize the activity of KLF 6 in vivo.
  • natural products libraries can be screened using assays of the invention for molecules that agonize or antagonize KLF6 activity.
  • a green fluorescent protein expression assay permits evaluation of KLF6 activity.
  • GFP can be modified to produce proteins that remain functional but have different fluorescent properties, including different excitation and emission spectra (U.S. Patent No. 5,625,048 and PCT Publication No. WO 98/06737); an enzyme recognition site (PCT Publication No. WO 96/23898); increased intensity compared to the parent proteins (PCT Publication No. WO 97/11094); higher levels of expression in mammalian cells (PCT Publication No. WO 97/26633); twenty times greater fluorescence intensity than wild-type GFP (PCT Publication No.
  • reporter genes include luciferase, ⁇ -galactosidase ( ⁇ -gal or lac-Z), chloramphenicol transferase (CAT), horseradish peroxidase, and alkaline phosphatase.
  • CAT chloramphenicol transferase
  • horseradish peroxidase and alkaline phosphatase.
  • expression of almost any protein can be detected using a specific antibody.
  • Reporter gene expression can be tied to expression or activation of any component of cell signaling downstream of KLF6.
  • a GFP-expression vector containing the p21 gene can be used to evaluate KLF6 activity.
  • candidate compounds can be tested for their ability to induce the expression of p21, as determined by the levels of GFP, as well as inhibiting cell proliferation.
  • This example describes the effects of KLF6 on overall and hepatocyte specific cell growth and proliferation in transgenic mice.
  • Overexpression of KLF6 in hepatocytes results in an antiproliferative effect in mice, displaying reductions in size, liver weight and hepatocyte proliferation.
  • the effects on cell proliferation suggests that KLF6 is an important factor in cell growth and cell cycling.
  • TTR hepatocyte-specific transthyretin
  • RNAse protection assay RNAse protection assay was carried out as previous described (Maher and McGuire, J. Clin. Invest. 1990, 86:1641-1648). Partial hepatectomy. Two-thirds partial hepatectomy or sham laparotomy was performed under ether anesthesia as described previously (Albrecht et al, Hepatology 1997, 25:557-563).
  • KLF6 expression in hepatocytes Hepatocyte expression of KLF6 mRNA was examined in rats following partial hepatectomy using RNAse protection ( Figure 1). This revealed a biphasic pattern of KLF6 induction, with peak levels of expression occurring at one hour and twelve hours. This data suggested a regulatory role for KLF6 in hepatic regeneration. Effects ofKLF6 expression in transgenic mice.
  • TTR transthyretin promoter
  • AST serum aspartate aminotransferase
  • ALT alanine aminotransferase
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase.
  • the transgenic mice had no distortion of liver architecture, although the length of cell plates between portal triads was reduced greatly from the wild-type liver to the transgenic liver.
  • PCNA proliferating cell nuclear antigen
  • expression of proliferating cell nuclear antigen (PCNA) in the hepatocytes of 4 week-old transgenic pups was markedly diminished, indicating that the transgenic mice had reduced hepatocyte proliferation.
  • the transgenic liver showed no increase in cellular apoptosis as assessed by TUNEL.
  • transgenic mice yielded approximately 50% fewer hepatocytes than their non-transgenic littermates following cell isolation using standard methods (Bissell, and Guzelian, Ann. NY Acad. Sci., 1980, 349:85-98).
  • EXAMPLE 2 KLF6 Induced Expression of p21, a Cell-Cycle
  • KLF6 plays an important role in the cell cycle. Additionally, the similarity of the KLF6 transgenic mice to the p21 hepatocyte-specific transgenic mice (Wu, et al, Genes Dev. 1996, 10:245-260) suggested that KLF6 may regulate cell growth and proliferation through the p21 signaling pathway.
  • vTet-KLF ⁇ was constructed by inserting rat KLF6 cDNA into AccI/EcoRV sites of pTet-splice (GIBCO BRL).
  • GBCO BRL pTet-splice
  • a tetracycline-regulated cell line expressing KLF6 was established as described by co-transfecting pTet-KLF6 and pBpuro into the cell line containing pTet-tTAk (Shockett, et al, Proc. Natl. Acad. Sci.
  • DNA synthesis was determined by assaying inco ⁇ oration of 3 H-thymidine inco ⁇ oration, as previously described (Friedman, et al, J. Biol. Chem.1989, 264:10756-10762).
  • P53 null mice were generated as described (Donehower, et al, Nature 1992, 356:215-21), and were a kind gift of Dr. Jeffrey Albrect, U. of Minnesota.
  • KLF 6 induces p21 expression in NIH3T3 cells.
  • an NIH 3T3 cell line was generated in which KLF6 expression was regulated by a tetracycline-responsive promoter. Upon withdrawal of tetracycline, induction of KLF6 led to increased ⁇ 21. This increase in p21 was associated with a marked antiproliferative effect and reduced PCNA expression when compared to the control cell line expressing the tet transactivator and empty expression vector.
  • KLF6 induces p21 independent ofp53.
  • KLF 6 transactivated the p21 promoter in a p53 null cell line (Hep 3B) ( Figure 3 A). Transactivation of the p21 promoter by KLF6 was unaffected by a mutation of the p53 consensus site ( Figure 3B). Furthermore, KLF6 upregulated endogenous p21 following transient transfection in a p53 null cell line (Hep 3B) * ( Figure 3C). The interaction of KLF 6 occurs through binding to GC box motifs within the p21 promoter.
  • KLF6 andp21 show similar patterns of expression post-hepatectomy inp53 null mice.
  • Previous studies have documented that the induction of p21 following partial hepatectomy occurred in a p53-independent manner (Albrecht, et al, Hepatology, 1997, 25:557-563). Therefore, we examined whether the pattern of KLF6 induction following hepatectomy was preserved in p53 null mice, which could explain why p21 is still upregulated in the absence of p53 in this setting.
  • Northern blot analysis of mRNA extracted from the liver remnants we determined that KLF6 was upregulated in an almost identical manner to p21.
  • KLF6 induces p21 in a p53 independent manner, and that the effect of KLF 6 on cell proliferation is mediated through p21.
  • up to 50% of all tumors do not have mutations in p53, suggesting that KLF6 may be involved to a significant level in tumorigenesis.
  • Some tumors and tumor derived cell lines have chromosomal deletions at lOp, the chromosomal location of KLF6. This suggests that inactivation or alteration of KLF6 may result in p53 independent tumorigenesis.
  • Levels of MRNA levels' of mRNA were detected by Northern Blot. DNA Sequencing. The sequencing of cDNA and genomic DNA were done by ABI automated sequencer at the University of Utah DNA sequencing facility on a recharge basis.. Western blots. Levels of protein were detected by Western blot.
  • Cell lines The Memorial Sloan Kettering neuroblastoma cell lines SK-N-ML (a kind gift of Dr. Andrew Chan, Ruttenberg Cancer Center, Mount Sinai School of Medicine), and the human glioblastoma cell line CRL2020 (ATCC Tissue type collection) were analyzed. Control cell lines included Hep 3B, HSC-T6 cells (Bayer AG), and NIH 3T3 fibroblasts.
  • KLF6 mutation in a glioblastoma cell line The human glioblastoma cell line CRL2020 has a known chromosomal lOp deletion. KLF6 has been localized to lOp.
  • KLF6 contains a consistent mutation at a putative Casein Kinase II phosphorylation site (Ser 27 ⁇ Pro 27 ).
  • Casein Kinase II is a highly conserved and ubiquitously expressed enzyme that can modify a range of substrates including transcription factors. In doing so, Casein Kinase II may modulate the transcriptional activity and DNA binding affinity of these substrates (see Ouyang, et al, J. Biol. Chem. 1998, 273:23019-25, and references therein), thus affecting the cell cycle and/or cell growth.
  • KLF6 inactivation in a neuroblastoma cell line MRNA levels of KLF6 were examined in the neuroblastoma cell line SKNML by Northern blot and RT-PCR. KLF6 mRNA is not expressed in this cancerous cell line.
  • KLF6 inactivation in primary tumors The sequence of KLF6 was examined in primary tumors. We have identified mutations in a primary prostate cancer within the open reading frame of KLF6. One of the mutations involves a putative acetylation site. Additionally, several breast tumors have a normal KLF6 sequence.
  • inactivation of KLF 6, whether by gene deletion, mutation, or suppression of expression associates with cancer, and particularly with glioblastoma, neuroblastoma, and prostate cancer.
  • diagnosis and prognosis of cancer, especially neuroblastoma can be based on the level or absence of KLF6 expression, or on the activity of KLF6.
  • the consistent mutation at the putative Casein Kinase II phosphorylation site in the human glioblastoma cell line could provide a useful marker when phenotyping or diagnosing tumors, as could the mutation at the putative acetylation site.
  • the inactivation of KLF6 in tumor cell lines indicates a method for the selection of compounds which overcome the inactivation of KLF6, and lead to the suppression of cell proliferation. Methods and kits for such selection techniques are proposed.
  • KLF6 is also indicated as a therapeutic tumor suppressor gene.
  • Vectors for expression of KLF 6 will be useful in tumor cells in which KLF 6 has been inactivated.
  • overexpression of KLF 6 in other tumor cells is expected to slow or inhibit cell growth.
  • EXAMPLE 4 In vitro analysis of KLF6 expression in glial tumor cell lines
  • KLF 6 levels were determined using total cell extracts derived from a panel of glial tumor cell lines, with normal brain and an astrocyte line (DITNC1) as controls for non-transformed counte ⁇ arts.
  • DITNC1 is a cell line derived from new born rat brain and that retains the characteristics consistent with the phenotype of type 1 astrocytes. Parallel blots were probed with an antibody to PI3-K (p85) to ensure equal loading.
  • Stable transfection in glioblastoma cell line For generation of DBTRG-05MG stable lines, 5 mg of cDNAs in the retroviral vector, pBabepuro, were co-transfected into HEK 293T cells along with 5 mg of a helper virus vector, pCL-ampho. Retroviral particles were collected and similar number of virions were used to infect DBTRG-05MG cells in the presence of 4 mg/ml of polybrene. Infected cells were then subjected to selection in 1 mg/ml of puromycin. For in vivo tumorigenicity assays 5 x 106 of marker selected mass cultures were injected subcutaneously into nude mice and tumor size was monitored for up to 7 weeks.
  • DBTRG-05MG glioblastoma cells transduced with a control plasmid, KLF6, or the KLF6-DN mutant were seeded in soft agar and colonies were allowed to form.
  • KLF6 expression was found to be either absent or extremely low when compared to the astrocyte or fibroblast lines. This implies that KLF6 expression may be lost or attenuated in these tumor cells.
  • Subsequent sequencing of the KLF6 gene did not reveal any intragenic mutations in any of the cell lines or in a panel of 15 primary glial tumors examined.
  • the fact that all cell lines examined in this study display detectable KLF6 transcript in Northern analysis indicating that at least one allele was still intact.
  • KLF6 was introduced through retroviral-mediated gene transfer which would allow for the selection of cells stably expressing this gene. Indeed, Western blot analysis revealed that these cells express KLF ⁇ at levels below that of those found in the DI TNCl astrocytes ⁇ Additionally, a mutant form of KLF6-DN which has the majority of the transactivation domain deleted as a control, was expressed as well. This protein was undetectable, most likely due to the loss of major immunogenic epitopes recognized by the polyclonal antibody.
  • GFAP Glial Fibrillary Acidic Protein
  • oncogene products have been shown to be activated during glial tumor progression. These include the increased expression/activation of receptors for epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) (T.P. Fleming et al, Cancer Res. 15, 4550 (1992)).
  • EGF epidermal growth factor
  • PDGF platelet-derived growth factor
  • KLF ⁇ expression was able to drastically block transformation by PDGF-BB by greater than 60%.
  • the KLF6-DN mutant failed to produce any detectable inhibitory effect under similar experimental conditions.
  • both KLF ⁇ or control vector produced similar number of marker-selected colonies suggesting that the inhibition observed was not due to a non-specific killing of transfected cells.
  • EXAMPLE 5 Suppression of glioblastoma tumorigenicity in vivo
  • mice When the mice are grouped according to tumor volume, there is an almost exact inverse correlation between control and KLF ⁇ expressing tumors, with 60% of mice injected with control cells falling within the largest volume and 60% of those injected with KLF ⁇ transduced cells in the smallest. Only one mouse injected with KLF ⁇ expressing cells possessed tumors with a volume greater than 3.0 cm 3 . Thus, the expression ofKLF ⁇ into glioblastoma cells leads to partial reversion of their tumorigenic phenotypes both in vitro and in vivo.
  • KLF ⁇ expression is attenuated in a variety of glial tumor cell lines. Expression ofKLF ⁇ into these cells inhibits their transformed phenotypes in vitro and reduces their ability to form tumors in mice. Additionally, KLF ⁇ may block transformation by specific lesions found in glial tumors such as PDGFR amplification as evidenced by its ability to block the formation of foci induced by c-sis/PDGF-BB in NIH 3T3 cells.
  • Embryonic stem cells heterozygous for KLF ⁇ were generated using standard methods as described previously (See for example, Torres, 1998, Cur. Top. Dev. Biol. 36: 99-114).
  • Cell proliferation assay Cell proliferation was determined by cell counting and inco ⁇ oration of 3 H-thymidine, as previously described (Friedman, et al, J. Biol. Chem.1989, 264:10756-10762).
  • KLF ⁇ expression In embryonic stem cells generated by homologous recombination using standard methods, loss of a single allele ofKLF ⁇ leads to increased cell proliferation as assessed by cell counting and inco ⁇ oration of tritiated thymidine to measure DNA synthesis.
  • KLF ⁇ may be used to slow proliferation in non-malignant cells.
  • benign conditions where it is desirable to decrease cell proliferation.
  • benign conditions are hype ⁇ roliferative disorders, such as scar or keloid formation, benign neoplasms such as adenomas, and other hype ⁇ roliferative conditions where cell turnover is increased, such as psoriasis.
  • EXAMPLE 7 Loss of heterozygosity at the KFL6 locus in prostate cancer
  • the genomic structure of the KLF ⁇ gene consists of 4 exons (Genebank accession no. AF001461). PCR amplifications were carried out in a 50 ml volume with 10 ng of genomic DNA; 20 mM of each primer; 10 mM dNTP's; 10 mM Tris, pH 8.8, 50 mM KC1; 1.5 mM MgCl 2 and 0.8 U of AmpliTaq GOLD DNA polymerase (Perkin Elmer). The following amplification conditions were used: 95°C 10 mins for 1 cycle, 95°C 1 min, 55°C 1 min, 72°C 1 min for 44 cycles and finally, 72°C for 10 mins for 1 cycle.
  • Table 8 Oligonucleotide sequences and amplimer sizes for the four KLF ⁇ exons.
  • DHPLC denaturing high performance liquid chromatography
  • Solution B is according to the manufacturer (Transgenomic Inc), 0.1M Triethylammonium acetate (TEAA), 25% acetonitrile.
  • Mutation screening of exon 2 was performed by DNA sequencing.
  • a 50 ml PCR product was amplifed as described above.
  • PCR products were purified using the Qiagen PCR purification kit and sequenced bidirectionally using ABI Bigdye terminator sequencing (Perkin Elmer) on the ABI 3700 DNA Sequencer. Data was analyzed using ABI Sequencing Analysis 3.3 (Perkin Elmer) and Sequencher 3.11 (Gene Codes Co ⁇ oration) software.
  • Microsatellite markers for loss of heterozygosity (LOH) analysis For these analyses, three novel microsatellite markers, D10SXBL1, D10SXBL2, D10SXBL4, were generated, closely flanking the KLF ⁇ gene.
  • Tandem repeats were sought (Bensen, Nuc Acids Res 27:573-580, (1999)) in the 706,257 bp bacterial artificial chromosome DNA sequence NT_024115 containing the KLF ⁇ gene sequence and PCR primers were designed.
  • the sequence, allele size range, and heterozygosity scores for these markers are shown in Table 11. Scores were determined for these novel markers by amplifying each marker from genomic DNA isolated from over 100 healthy Caucasian individuals.
  • three publically available markers from the Marshfield Medical Research Foundation http ://research.marshfieldclinic.org/genetics/) which also flank the KLF ⁇ gene locus, albeit at greater distances, are also shown.
  • Table 11 Oligonucleotide sequences for the six microsatellite markers flanking the KLF ⁇ locus.
  • Sense primers are 5' end-labelled with a fluorescent dye [6-Fam (blue), Tet (green) and Hex (yellow)] detectable by an ABI 377 DNA Sequencer using filter set C.
  • Htz Heterozygosity index.
  • Transactivation assays were performed using 3 ⁇ g DNA containing 1.5 ⁇ gp21 promoter constructs and 1.5 ⁇ gpCI-neo-ZEFo ' orpCI-neo-p53 (giftfromDr. T. Ouchi) was transfected in 3x10 5 PC3 cells plated in 6-well dishes using Lipofectamine 2000 reagent (Life Technologies) . 10 ng TK promoter-Renilla Luciferase construct (Promega) was used to normalize each transfection. Cells were treated and data analyzed using the Promega dual-luciferase Kit. The wild type and mutant p21 constructs are described in Ouchi, et al, Proc Natl Acad Sci USA., 1998, 95:2302-2306.
  • the inventors examined a wide variety of primary prostate tumor samples from well differentiated adenocarcinomas (Gleason 1+1) to poorly differentiated adenocarcinomas (Gleason 5+5) for specific evidence of loss of heterozygosity (LOH) of KLF ⁇ .
  • Microsatellite markers flanking the KLF ⁇ gene were analyzed in nine paired specimens. In total, 6 of the 9 samples (67%) displayed LOH across the KLF ⁇ locus ( Figures 7A, 7B, 7C and 7D).
  • the inventors designed two new microsatellite markers BLl and BL2, which flank the KLF ⁇ gene by approximately 10 kb.
  • patient 9 had loss of only the BLl and BL2 microsatellite markers suggesting that the minimal region of loss on chromosome lOp in prostate cancer is a 100 kb region encompassing the KLF ⁇ gene.
  • the coding region and intron / exon boundaries of the KLF ⁇ gene were then sequenced using genomic DNA extracted from these patient derived tumors.
  • Knudson's "two-hit hypothesis" Knudson, Proc Natl Acad Sci USA, 1971, 68: 820-3
  • all six tumor samples demonstrating LOH possessed mutations in the KLF ⁇ gene, suggesting two inactivating events at the same genetic locus. Sequencing of normal tissue from these six patients did not reveal mutations, confirming that the mutations were somatic.
  • genomic DNA extracted from paired tumor samples from an additional 12 patients with primary prostate cancer were also amplified and sequenced.
  • 16 out of 30 tumor samples (55%) were found to have KLF ⁇ mutations restricted to tumor DNA (Table 12).
  • a total of 15 mutations were identified, with the majority occurring within the activation domain (Table 12).
  • These mutations resulted in nonconservative amino acid changes and the introduction of a premature stop codon. None of these mutations were present in the patients normal tissue or in germline DNA from 50 unaffected, unrelated control individuals.
  • all predicted substituted amino acids, except for the A123D mutation which was functionally characterized occurred within strictly conserved positions within the mouse KLF ⁇ sequence. No common mutations were identified among any of the patients.
  • Table 12 Prostate tumor sample mutations.
  • Luciferase activity assays Luciferase activity assays. Luciferase activity was assayed 24 hours after cotransfection of 293T with either theR64,D123,X137,P169 mutant proteins, or wild type human KLF ⁇ and a p21 promoter reporter cDNA containing a mutated p53 binding site. A 10-fold increase in promoter activity was detected following expression of human KLF ⁇ (p value ⁇ 0.0001). Three of the four tumor derived mutants transactivated the p21 promoter construct ( p value ⁇ 0.05) but to a significantly lower level than wild type KLF ⁇ (p value ⁇ 0.0001) relative to all four of the tumor-derived mutants).
  • wild type KLF ⁇ transactivated the p21 promoter 12 fold (p ⁇ 0.0001).
  • Three of the four tumor derived mutants also transactivated the p21 promoter reporter construct but to significantly lower levels than seen with wild type KLF ⁇ (p value ⁇ 0.0001).
  • the X137 mutant failed to transactivate the p21 promoter reporter construct suggesting a complete loss of transcriptional activity.
  • some of the tumor derived mutants transactivated the p21 promoter luciferase construct, none of these mutants significantly upregulated the endogenous p21 gene. Consistent with their inability to upregulate endogenous p21 , none of the four tumor derived mutants were able to significantly suppress the growth of PC3 cells (Figure 8).
  • the C265 Y mutation occurs in the last zinc finger. This motif shows homology to structures that have been studied to atomic resolution by X- ray crystallography (Kim et al, Nat Struct Biol 1966 3: 940-5). The introduction of a tyrosine at this residue would prevent zinc binding to this motif and hence affect XEEo ' -DNA interactions.
  • the L217S mutation affects a residue conserved across 20 zinc finger containing domains, both in sequence and secondary characteristics.
  • mutation D273G is predicted to occur at and thus destabilize the N-cap position of the putative 273-281 alpha helix and the interaction between the hydrophobic leucine at position 277. Further helical destabilization may arise from the lack of electrostatic interactions between the aspartate' s negatively charged amino side group and the helical dipole positive end.
  • KLF ⁇ mutations may represent a p53-independent pathway in cancer development. In agreement with this, our results identify a p53 -independent pathway for the control of cell proliferation.
  • KLF ⁇ Tumor-associated mutations in KLF ⁇ have been identified predominantly in the activation domain, a distribution which is consistent with the relative sizes of the activation and DNA binding domains (Calculation was done using the Chi-squared contingency test). Given its ubiquitous expression, its ability to suppress growth and reverse the malignant potential of human cancer cells including glioblastoma, KLF ⁇ may have a general role in the development or progression of other human cancers, particularly those associated with loss of heterozygosity at chromosome 10pl5. The below table summarizes the identification of loss of hererozygosity in a number of tumors.
  • NIH3T3 cells were transfected with a plasmid expressing the c-sis PDGV- ' SQ gene, which would functionally mimic the PDGFR overexpression that is seen in these tumors.
  • Cells were either co-transfected with a control vector, a KLF ⁇ expression plasmid, or the KLF6—DN mutant. Cells were fixed with methanol and foci were visualized by Giemsa staining.
  • KLF ⁇ expression was shown to drastically block transformation by PDGF-BB by greater than 60%.
  • the KLF ⁇ — ON mutant failed to produce any detectable inhibitory effect under similar experimental conditions.
  • both KLF ⁇ or control vector produced similar number of marker-selected colonies suggesting that the inhibition observed was not due to a non-specific killing of transfected cells.
  • Similar experiment was also performed with an oncogenic H-Ras allele, H-Ras Arg 12 and KLF ⁇ was also able to block focus formation under similar experimental conditions.
  • Sprouty 1 is an inhibitor of angiogenesis-related growth factor signaling, which is a major component of tumor invasion and growth (Impagnatiello etal, J Cell Biol, 2001, 152(5): 1087-98).
  • the sprouty 1 gene promoter can be activated by KLF ⁇ and the KLF ⁇ protein can bind to a sequence within the sprouty 1 promoter.
  • growth suppression by KLF ⁇ may occur in part by the activation of sproutyl, which in turn deactivates growth factor pathways.

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Abstract

L'invention porte sur l'identification de l'activité de suppression de tumeurs d'une protéine, la KLF6 (KLF6), et sur des compositions et méthodes diagnostiques et thérapeutiques associées. La découverte de cette activité fournit également des cibles de criblage, notamment de criblage de composés qui contrent l'inactivation ou l'altération du gène.
PCT/US2001/025046 2000-08-09 2001-08-09 Facteur 6 du genre kruppel (klf6), proteine supprimant les tumeurs et diagnostics, therapies et criblages bases sur cette proteine WO2002012894A1 (fr)

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