WO 2014/209957 Al 31 December 2014 (31.12.2014) P O P C T
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(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 WO 2014/209957 Al 31 December 2014 (31.12.2014) P O P C T (51) International Patent Classification: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, A61K 31/282 (2006.01) A61P 35/00 (2006.01) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, A61K 31/28 (2006.01) HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, (21) International Application Number: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, PCT/US20 14/043802 OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (22) International Filing Date: SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, 24 June 2014 (24.06.2014) TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (25) Filing Language: English (84) Designated States (unless otherwise indicated, for every (26) Publication Language: English kind of regional protection available): ARIPO (BW, GH, (30) Priority Data: GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, 61/838,560 24 June 2013 (24.06.2013) US UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, (71) Applicant: DUKE UNIVERSITY [US/US]; 2812 Erwin EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, Road, Suite 306, Durham, NC 27705 (US). MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, (72) Inventors: THIELE, Dennis, J.; 2705 Creek Ridge Lane, KM, ML, MR, NE, SN, TD, TG). Chaple Hill, NC 275 14 (US). OHRVK, Helena; Duke University, 2812 Erwin Road, Suite 306, Durham, NC Published: 27705 (US). — with international search report (Art. 21(3)) (74) Agent: REYNOLDS, Anne, M.; Michael Best & Friedrich — before the expiration of the time limit for amending the LLP, 100 East Wisconsin Avenue, Site 3300, Milwaukee, claims and to be republished in the event of receipt of WI 53202-4108 (US). amendments (Rule 48.2(h)) (81) Designated States (unless otherwise indicated, for every — with sequence listing part of description (Rule 5.2(a)) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (54) Title: CANCER TREATMENT or other p ti - rug Cytoplasm © FIGURE 1 (57) Abstract: Described herein are methods of inhibiting the proliferation of cancer cells and methods of treating cancer, as well as o methods of predicting responsiveness of subjects to certain cancer treatments and methods of identifying subjects as candidates for o certain cancer treatments. CANCER TREATMENT CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to United States Provisional Patent Application No. 61/838,560, filed on June 24, 2013, the entire contents of which are hereby incorporated by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under R01 DK074192 awarded by the National Institutes of Health (National Institute of Diabetes and Digestive and Kidney Diseases). The United States government has certain rights in the invention. BACKGROUND [0003] Cisplatin is a platinum-based anti-cancer drug that is highly effective and currently in broad clinical use for the treatment of a number of cancers, including head, neck, ovarian, breast, pancreatic, testicular, melanoma, bladder, lung, sarcoma, squamous cell carcinoma, small cell lung cancer as well as others. Cisplatin and related platinum-based chemotherapeutic agents such as oxaliplatin, carboplatin, satraplatin, picoplatin and the like, are typically administered at high concentrations to enhance efficacy, but can have significant side-effects such as ototoxicity and nephrotoxicity. Furthermore, many cancer cells may develop resistance to platinum-based chemotherapeutics. Some forms of resistance may be intrinsic, wherein the cells or patients may be inherently resistant for reasons that are not well understood. SUMMARY [0004] In one aspect, the disclosure provides a method of reducing the proliferation of a cancer cell, comprising contacting the cancer cell with a platinum-based chemotherapeutic and a cysteine protease inhibitor, such as a cathepsin inhibitor (e.g., a Cathespin L inhibitor). [0005] In another aspect, the disclosure provides a method of treating cancer in a subject in need of treatment, comprising administering to the subject a platinum-based chemotherapeutic and a cysteine protease inhibitor, such as a cathepsin inhibitor (e.g., a Cathespin L inhibitor), in amounts effective to treat the cancer. [0006] In another aspect, the disclosure provides a method of predicting responsiveness of a subject having cancer to treatment with a platinum-based chemotherapeutic agent, comprising: providing a nucleic acid-containing sample obtained from the subject; and detecting a Ctrl nucleotide sequence selected from the group consisting of SEQ ID NO:l and SEQ ID O:2; wherein the presence of SEQ ID NO: 1 indicates that the subject is a responder to treatment with a platinum-based chemotherapeutic in the absence of a cysteine protease inhibitor, such as a cathepsin inhibitor (e.g., a Cathespin L inhibitor), and wherein the presence of SEQ ID NO:2 indicates that the subject is a non-responder to treatment with a platinum-based chemotherapeutic in the absence of a cysteine protease inhibitor, such as a cathepsin inhibitor (e.g., a Cathespin L inhibitor). [0007] In another aspect, the disclosure provides a method of treating cancer in a subject in need of treatment, comprising: providing a nucleic acid-containing sample obtained from the subject; detecting a Ctrl nucleotide sequence selected from the group consisting of SEQ ID NO:l and SEQ ID NO:2; and if the detecting step detects the presence of SEQ ID NO:2, the method further comprises administering to the subject a therapeutically effective amount of a platinum-based chemotherapeutic and a cysteine protease inhibitor, such as a cathepsin inhibitor (e.g., a Cathespin L inhibitor). [0008] In another aspect, the disclosure provides a method of predicting responsiveness of a subject having cancer to copper chelation therapy, comprising: providing a nucleic acid-containing sample obtained from the subject; and detecting a Ctrl nucleotide sequence selected from the group consisting of SEQ ID NO:l and SEQ ID O:2; wherein the presence of SEQ ID NO: 1 indicates that the subject is a responder to copper chelation therapy, and wherein the presence of SEQ ID NO:2 indicates that the subject is a non-responder to copper chelation therapy. [0009] Other aspects and embodiments are encompassed by the disclosure and will become apparent in light of the following description and drawings. BRIEF DESCRIPTION OF THE DRAWINGS [0010] FIG. 1 depicts a model for the cleavage of the Ctrl ecto-domain. The Ctrl copper/cisplatin transporter is shown in dark gray, with dotted lines pointing to the cisplatin- binding methionine residues. Cisplatin (or other platinum-based chemotherapeutic) is shown as gray balls. The Ctr2 protein, which forms a complex with Ctrl, is shown in light gray. Cathepsin L, a protease which cleaves off the cisplatin-binding ecto-domain of Ctrl, is shown in medium gray. [0011] FIG. 2 is an immunoblot of protein extracts from mouse embryonic fibroblasts (MEFs) from wild-type (Ctr2+ +), heterozygous (Ctr2+ ) and knock out (Ctr2 _) cells, with an anti-Ctrl antibody and an anti-tubulin antibody. [0012] FIG. 3 illustrates two graphs of platinum (left) and copper (white) levels (in ng/mg protein) from wild-type MEFs (Ctr2+ +, white bars) and in knock-out MEFs (Ctr2 _ , gray bars). [0013] FIG. 4 is an immunoblot of protein extracts from wild-type MEFs (lanes 1-4) and Cathepsin L_ MEFs (lanes 5-8) with an anti-Ctrl antibody and an anti-tubulin antibody. Lanes 1 and 2 are duplicate samples from untreated wild type MEFs. Lanes 3 and 4 are duplicate samples from wild-type MEFs treated with the Cathepsin L inhibitor E64d. Lanes 5 and 6 are duplicate samples from untreated Cathepsin L_ MEFs. Lanes 7 and 8 are duplicate samples from Cathepsin L_ MEFs treated with the Cathepsin L inhibitor E64d. [0014] FIG. 5 shows an immunoblot of protein extracts from wild-type MEFs (Ctrl) and from MEFs expressing Ctrl with a Pro25Ala mutation (Ctrl A), with an anti-Ctrl antibody and an anti-actin antibody. Each of the above were either untreated (lanes 1 and 2), or treated with the Cathepsin L inhibitor Z-FY(tBu)-DMK (lanes 3 and 4). F = full-length, T = truncated. [0015] FIG. 6 is a graph showing platinum accumulation in Ctrl knockout MEFs (Ctrl 7 ), wild-type MEFs (Ctrl), or MEFs expressing Ctrl with a Pro25Ala mutation (Ctrl A). Each of the above were either untreated (1, 2 and 3), or treated with the Cathepsin L inhibitor Z- FY(tBu)-DMK (4, 5 and 6). DETAILED DESCRIPTION [0016] The present disclosure is generally directed to methods of treating cancer and to methods of reducing the proliferation of cancer cells. The disclosure may also provide methods of predicting the responsiveness of subjects to certain cancer treatments; for example, the methods may involve identification of subjects as candidates for various cancer treatments. [0017] A major uptake mechanism for the import of cisplatin in both yeast and mammalian cell is the Ctrl copper importer (Ishida et al. (2002) Proc. Natl. Acad. Sci., USA 99: 14298-14302; Pope et al. (2012) Curr. Top. Membr. 69: 97-112; Kuo et al.