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A Novel Small Molecule Inhibitor Of Deubiquitylating Enzyme USP14 and UCHL5 Induces Apoptosis In Myeloma Cells and Overcomes Bortezomib Resistance

ASH 2013 Annual Meeting Abstract 1923 (Poster Presentation)

Session:652. Myeloma: Pathophysiology and Pre-Clinical Studies, excluding Therapy: Poster I
Time:Saturday, December 7, 2013, 5:30 PM-7:30 PM
Location:Hall G (Ernest N. Morial Convention Center)

Ze Tian, Ph.D1, Padraig D'Arcy, Ph.D2, Xin Wang, Ph.D3, Arghya Ray4, Yu-Tzu Tai, PhD5, Yiguo Hu, PhD6, Ruben Carrasco, MD, PhD7, Deepika Das, Ph.D4, Paul Richardson, MD8, Stig Linder, MD, PhD9, Dharminder Chauhan, PhD10 and Kenneth C. Anderson, MD7

Affiliations: 1Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, 22Department of Oncology and Pathology, Karolinska Institute, Stockholm, Sweden, 3Department of Oncology and Pathology, Karolinska Institute, Karolinska Institute, Stockholm, Sweden, 4Medical Oncology, Dana Farber Cancer Institute, Boston, 5LeBow Institute for Myeloma Therapeutics and the Jerome Lipper Multiple Myeloma Center, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, 6Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, 7Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, 8Dana-Farber Cancer Institute, Boston, MA, 9Karolinska Institute, Stockholm, Sweden, 10LeBow Institute for Myeloma Therapeutics and Jerome Lipper Center for Multiple Myeloma Research, Harvard Medical School, Dana-Farber Cancer Institute, Boston, MA

IntroductionProteasome inhibitors have demonstrated that targeting ubiquitin proteasome pathway (UPS) is an effective therapy in multiple myeloma (MM). More recent studies have focused on targeting enzymes modulating protein ubiquitin conjugation/deconjugation upstream of the proteasome rather than the proteasome itself, with the goal of producing more specific, potent, and less toxic therapies targeting UPS. Ubiquitylation is a dynamic reversible process coordinated by many enzymes: ubiquitin ligases attach ubiquitin to proteins allowing for their degradation, whereas deubiquitylating enzymes (DUBs) deconjugate ubiquitin from target proteins, thereby preventing their proteasome-mediated degradation. Importantly, many human diseases are linked to dysfunction of ubiquitin ligases and/or DUBs, suggesting that inhibitors of ubiquitylating or DUBs represent a potential therapeutic strategy.In mammalian cells, three DUBs are associated with the proteasome: USP14, UCHL5/Uch37, and Rpn11. In the present study, we examined the expression of USP14 and UCHL5 in MM by western blot and Immunohistochemistry (IHC).

Results Our results show that DUBs USP14 and UCHL5 are more highly expressed in primary MM patient tumor cells and MM cell lines than in normal plasma cells and peripheral blood mononuclear cells (PBMCs). Additionally, USP14 and UCHL5 siRNA knockdown significantly decrease MM cell viability (p < 0.001) in a CellTiter Glo assay. A novel 19S regulatory particle inhibitor b-AP15 selectively blocks deubiquitylating activity of USP14 and UCHL5 without inhibiting activity of other DUBs, proteases and proteasome; Importantly, b-AP15 decreases cell viability in MM cell lines as well as patient MM cells, without markedly affecting PBMCs from normal healthy donors. Moreover, b-AP15 inhibits proliferation of MM cells even in the presence of bone marrow stroma cells and overcomes bortezomib-resistance. Mechanistic studies show that b-AP15 triggers MM cell arrest via downregulation of CDC25C, CDC2 and cyclinB1, followed by caspase-dependent apoptosis through activation of intrinsic and extrinsic apoptotic pathways. b-AP15, like bortezomib, induces ER stress evidenced by the upregulation of ER stress-related proteins p-IRE-alpha, and p-eIF2. In vivostudies using subcutaneous and disseminated human MM xenograft models show that b-AP15 is well tolerated, inhibits tumor growth, and prolongs survival (p < 0.001). In concert with our in vitro study, IHC analysis of tumor tissues showed inhibition of proliferation, induction of apoptosis, and accumulation of ubiquitinated proteins, assessed by staining with ki67, caspase-3, and UB-k48 antibodies, respectively. Finally, combining b-AP15 with SAHA, lenalidomide, or dexamethasone induces synergistic anti-MM activity.

Conclusion Our preclinical data showing efficacy of b-AP15 in MM disease models validates targeting DUBs upstream of the proteasome in the ubiquitin proteasomal cascade to overcome proteasome inhibitor resistance, and provides the framework for clinical evaluation of USP14/UCHL5 inhibitors to improve patient outcome in MM.

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