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2011/096210 Al (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date Λ 1 1 August 2011 (11 .08.2011) 2011/096210 Al (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12N 15/09 (2006.01) A61P 35/00 (2006.01) kind of national protection available): AE, AG, AL, AM, A61K 31/713 (2006.01) C12Q 1/68 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, (21) Number: International Application DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, PCT/JP201 1/000582 HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (22) International Filing Date: KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, 2 February 201 1 (02.02.201 1) ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, (25) Filing Language: English SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, (26) Publication Language: English TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: (84) Designated States (unless otherwise indicated, for every 61/301,020 3 February 2010 (03.02.2010) US kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, (71) Applicant (for all designated States except US): ON- ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, COTHERAPY SCIENCE, INC. [JP/JP]; 2-1, Sakado 3- TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, chome, Takatsu-ku, Kawasaki-shi, Kanagawa, 2130012 EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (JP). LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, (72) Inventors; and GW, ML, MR, NE, SN, TD, TG). (75) Inventors/Applicants (for US only): HAMAMOTO, Ryuji [JP/JP]; c/o THE UNIVERSITY OF TOKYO, 3-1, Declarations under Rule 4.17: Hongo 7-chome, Bunkyo-ku, Tokyo, 1138654 (JP). — as to applicant's entitlement to apply for and be granted NAKAMURA, Yusuke [JP/JP]; c/o THE UNIVERSITY a patent (Rule 4.1 7(H)) OF TOKYO, 3-1, Hongo 7-chome, Bunkyo-ku, Tokyo, 1138654 (JP). TSUNODA, Takuya [JP/JP]; c/o ON- — as to the applicant's entitlement to claim the priority of COTHERAPY SCIENCE, INC., 2-1, Sakado 3-chome, the earlier application (Rule 4.17(Hi)) Takatsu-ku, Kawasaki-shi, Kanagawa, 2 130012 (JP). Published: (74) Agents: SHIMIZU, Hatsushi et al; Kantetsu Tsukuba — with international search report (Art. 21(3)) Bldg. 6F, 1-1-1, Oroshi-machi, Tsuchiura-shi, Ibaraki, 3000847 (JP). — with sequence listing part of description (Rule 5.2(a)) (54) Title: PRMT1 AND PRMT6 FOR TARGET GENES OF CANCER THERAPY AND DIAGNOSIS (57) Abstract: Objective methods for diagnosing a predisposition to developing cancer, particularly bladder cancer, gastric can © cer, colorectal cancer, breast cancer, esophageal cancer, lung cancer, lymphoma, pancreatic cancer and testicular cancer, are de scribed herein. In one embodiment, the diagnostic method involves determining an expression level of PRMT1 or PRMT6 gene. The present invention further provides methods of screening for therapeutic agents useful in the treatment of PRMT1 or PRMT6 associated diseases, such as a cancer, e.g., bladder cancer, diffuse-type gastric cancer, breast cancer, esophageal cancer, NSCLC, SCLC, lymphoma, pancreatic cancer, testicular cancer, cervical cancer, osteosarcoma, prostate cancer and CML. The present in- vention further provides methods of inhibiting the cell growth and treating or alleviating symptoms of PRMT1 or PRMT6 associ- ¾ ated diseases. The present invention also features products, including double-stranded molecules and vectors encoding thereof as well as to compositions containing them. Description Title of Invention: PRMTl AND PRMT6 FOR TARGET GENES OF CANCER THERAPY AND DIAGNOSIS Technical Field [0001] The present invention relates to methods of detecting and diagnosing a predisposition to developing cancer, particularly bladder cancer, diffuse-type gastric cancer, breast cancer, esophageal cancer, NSCLC, SCLC, lymphoma, pancreatic cancer, testicular cancer, cervical cancer, osteosarcoma, prostate cancer and CML. The present invention also relates to methods of screening for a candidate substance for treating and preventing cancer with over-expression of PRMTl or PRMT6, particularly bladder cancer, diffuse-type gastric cancer, breast cancer, esophageal cancer, NSCLC, SCLC, lymphoma, pancreatic cancer, testicular cancer, cervical cancer, osteosarcoma, prostate cancer and CML. Moreover, the present invention relates to a double-stranded molecule which reduces PRMT6 gene expression and uses thereof. [0002] Priority The present application claims priority to US Serial No. 61/301,020, filed February 3, 2010, the disclosures of which are incorporated herein by reference in their en tireties. Background Art [0003] SMYD3, a histone lysine methyltransferase, stimulates proliferation of cells and plays an important role in human carcinogenesis through its methyltransferase activity [PTL1, NPLs 1-5]. Arginine methyltransferases have also been characterized as tran scriptional regulators, similar to lysine methyltransferases. In mammalian cells, protein arginine methyltransferases (PRMTs) have been classified into type I (PRMTl, 3, 4, 6 and 8) and type II (PRMT5, 7 and FBXOl 1), depending on their specific catalytic activity [NPLs 6, 7]. Type I PRMT activity is defined by the formation of asymmetric omega-N G, NG-dimethylarginine residues, whereas type II activity is defined by the formation of symmetric omega- NG, NG-dimethylarginine residues [NPLs 8, 9]. Despite a large body of information for the prominent role of PRMTs in transcriptional regulation, their physiological function and involvement in human disease is still not well understood. [0004] PRMTl (NM_001536.3, NM_198318.2, NM_198319.2) (for example SEQ ID NO: 1 encoded by SEQ ID NO: 2) is known to possess type I activity and catalyze methylation of the third arginine of histone H4 [NPLs 10, 11]. PRMTl was originally identified as an interacting protein for both the BTG1 and BTG2 proteins, as well as the interferon-alpha/beta receptor [NPLs 12, 13]. PRMTl has served as the pro- totypical PRMT because it was the first eukaryotic PRMT to be cloned and has been shown to act as a coactivator of nuclear receptor-mediated gene transcription together with p300/CBP, a histone acethyltransferase, and PRMT4/CARM 1 (coactivator- associated arginine methyltransferase 1) [NPLs 11, 14]. PRMT6 (NM_018137.2) (SEQ ID NO: 3 encoded by SEQ ID NO: 4) is also a type I enzyme and is the major protein arginine methyltransferase responsible for the methylation of the second arginine of histone H3 [NPLs 15, 16]. PRMT6 was shown to antagonize the MLL-complex-dependent methylation of the Lys-4 residue [NPL 16]. The biological role of PRMT6 is not clarified, but it has been suggested that its activity may affect gene regulation primarily through modifying protein-nucleic acid interactions. PRMT6 localizes exclusively in the nucleus, and methylates glycine- and arginine-rich (GAR) sequences in proteins [NPL 17]. Although PRMTl and PRMT6 share substrates, some PRMT6-specific cellular targets do not contain the GAR consensus sequence, including high mobility group proteins (HMGAla and HMGAlb) [NPL 18], DNA polymerase beta [NPL 19] and HIV-1 trans-activator of transcription (Tat) protein [NPL 20]. Citation List Patent Literature [0005] [PTL1] WO2005/07 1102 Non-Patent Literature [0006] [NPL 1] Hamamoto R et al. Nat Cell Biol 2004;6:73 1-40 [NPL 2] Hamamoto R et al. Cancer Sci 2006;97: 113-8 [NPL 3] Kunizaki M et al. Cancer Res 2007;67:10759-65 [NPL 4] Silva FP et al. Oncogene 2008;27:2686-92 [NPL 5] Tsuge M et al. Nat Genet 2005;37: 1104-7 [NPL 6] Bedford MT et al., Mol Cell 2009;33:1-13 [NPL 7] Pahlich S et al. Biochim Biophys Acta 2006;1764:1890-903 [NPL 8] Gary JD et al. Prog Nucleic Acid Res Mol Biol 1998;61:65-131 [NPL 9] Scott HS et al. Genomics 1998;48:330-40 [NPL 10] Huang S et al. Genes Dev 2005;19:1885-93 [NPL 11] Strahl BD et al. Curr Biol 2001;1 1:996-1000 [NPL 12] Lin WJ et al. J Biol Chem 1996;271:15034-44 [NPL 13] Abramovich C et al., EMBO J 1997;16:260-6 [NPL 14] Koh SS et al. J Biol Chem 2001;276:1089-98. [NPL 15] Guccione E et al. Nature 2007;449:933-7 [NPL 16] Hyllus D et al. Genes Dev 2007;21:3369-80 [NPL 17] Frankel A et al. J Biol Chem 2002;277:3537-43 [NPL 18] Sgarra R et al. J Biol Chem 2006;281:3764-72 [NPL 19] El-Andaloussi N et al. Mol Cell 2006;22:51-62 Summary of Invention [0007] In order to investigate possible roles of PRMTs in human carcinogenesis, the ex pression profiles of all human PRMTs in clinical tissues were examined to identify a methyltransf erase that can contribute to human carcinogenesis. We found that ex pression levels of PRMTl and PRMT6 were significantly up-regulated in various types of cancer, compared with the levels in corresponding normal (non-cancer) tissues. We identified two Type I PRMTs (PRMTl and PRMT6) overexpressed in various types of human cancer. Since the genes are scarcely expressed in adult normal organs, PRMTl and 6 are appropriate and promising molecular targets for novel therapeutic approaches with minimal adverse effect. Functionally, knockdown of endogenous PRMTl or 6 by siRNA in cancer cell lines results in drastic suppression of cancer cell growth, demon strating the essential role of these genes in maintaining viability of cancer cells.
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