Deubiquitylating Enzyme USP-7, a Novel Therapeutic Target in Multiple Myeloma.

Ubiquitin-Proteasome Signaling (UPS) pathway is mediated via a large number of components, suggesting many potential sites of pharmacological intervention. Therapeutic targeting of UPS is exemplified by the recent FDA approval of dipeptidyl boronic acid bortezomib first-in-class proteasome inhibitor for the treatment of relapsed/refractory, relapsed, and newly diagnosed multiple myeloma (MM). As with other agents, however, dose-limiting toxicities and the development of resistance limit its long-term utility. Recent efforts have been devoted to discovery and development of small molecule inhibitors of the major components of UPS, such as E1-conjugating enzyme inhibitors, E3 ubiquitin ligase inhibitors (e.g inhibitors of TRAF6, SCF-p27, POSH, MDM2-P53) or Deubiquitylating enzyme (DUB) inhibitor. Therapeutic strategies using these small molecule inhibitors will allow for specific targeting of the UPS and therefore may be less likely than proteasome inhibitors to trigger off-target activities and associated toxicities. In this context, Ubiquitin Specific Protease-7 (USP-7), a deubiquitylating enzyme, is of particular interest since it regulates activity of key biological signaling pathways such as p53, the transcription factor FOXO-4, Cdk inhibitor p27, and PI3K/Akt. In the present study, we examined the anti-tumor activity of a novel USP-7 inhibitor P5091 (Progenra, Inc., Malvern, PA) in MM cells using both in vitro and in vivo model systems.

We first confirmed the functional specificity of P5091 using different experimental strategies: 1) Cell-free based experiments using a novel reporter (Ub-PLA₂)-based assay showed a potent, specific and selective deubiquitylating activity of P5091 against USP-7 (EC₅₀ = 5.6 mM) versus other DUBs and proteases (EC₅₀ 25 to > 50 mM); 2) Treatment of MM.1S MM cells with P5091 blocked USP-7 deubiquitylating activity, without altering proteasome activity; and 3) treatment of HCT-116 (wild type) cells with P5091 markedly decreased viability; conversely, knockout of USP-7 in HCT-116 conferred resistance to P5091 (P < 0.001; n=2). Taken together, these data demonstrate that P5091 targets USP-7. We next examined the effects of P5091 in MM cells. Protein expression analysis showed that USP-7 is highly expressed in MM cells. Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, U266, KMS-12BM, and INA-6) and primary patient cells for 72h significantly decreased their viability (IC₅₀ range 5-10 mM) (P < 0.001; n=3) without affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index for P5091. P5091-induced apoptosis was confirmed in MM.1S and RPMI-8226 cells, as evidenced by marked increase in Annexin V+ and PI- cell population (P < 0.001, n=3). Importantly, transfection of USP-7 siRNA, but not scrambled (genome control) siRNA, inhibits growth of MM.1S cells, similar to USP-7 inhibitor P5091 (P < 0.001; n=2). In addition, p5091 triggered apoptosis in MM cells even in the presence of MM bone marrow stromal cells. Mechanistic studies further showed that P5091-triggered apoptosis in MM cells is associated with 1) activation of caspase-8, caspase-9, caspase-3, and PARP; 2) activation of Noxa, PUMA, and Bid; 3) downregulation of MM cell growth and survival proteins: Akt, Bcl_xl, phospho-S6 ribosomal protein, and c-Myc; 4) inhibition of migration of MM cells and angiogenesis; and 5) suppression of USP-7 specific substrate HDM2 with concurrent increased expression of CDK inhibitor p21. Importantly, blockade of HDM2 and p21 using siRNA strategy significantly abrogates P5091-induced MM.1S cell death (P value <0.001; n=2). We next examined the in vivo efficacy of p5091 using human plasmacytoma xenograft mouse model. Treatment of tumor-bearing mice with P5091 (10 mg/kg IV, twice a week for three weeks), but not vehicle alone, significantly (P < 0.001) inhibits MM tumor growth and prolongs survival of these mice (P < 0.001; 13.6 days prologation of survival in mice receiving P5091 vs. vehicle-treated mice). The concentrations of P5091 were well tolerated by mice, without significant weight loss.

These preclinical studies will allow for the development of next generation UPS-based therapies and provide the rationale for novel therapeutics targeting USP7 to improve patient outcome in MM.

Chauhan:Progenra, Inc: Consultancy. Nicholson:Progenra, Inc: Employment.