Ubiquitin-specific Protease 7 Inhibitors Research Articles

USP7 inhibitor P22077 inhibits neuroblastoma growth via inducing p53-mediated apoptosis

Neuroblastoma (NB) is a common pediatric cancer and contributes to more than 15% of all pediatric cancer-related deaths. Unlike adult tumors, recurrent somatic mutations in NB, such as tumor protein 53 (p53) mutations, occur with relative paucity. In addition, p53 downstream function is intact in NB cells with wild-type p53, suggesting that reactivation of p53 may be a viable therapeutic strategy for NB treatment. Herein, we report that the ubiquitin-specific protease 7 (USP7) inhibitor, P22077, potently induces apoptosis in NB cells with an intact USP7-HDM2-p53 axis but not in NB cells with mutant p53 or without human homolog of MDM2 (HDM2) expression. In this study, we found that P22077 stabilized p53 by inducing HDM2 protein degradation in NB cells. P22077 also significantly augmented the cytotoxic effects of doxorubicin (Dox) and etoposide (VP-16) in NB cells with an intact USP7-HDM2-p53 axis. Moreover, P22077 was found to be able to sensitize chemoresistant LA-N-6 NB cells to chemotherapy. In an in vivo orthotopic NB mouse model, P22077 significantly inhibited the xenograft growth of three NB cell lines. Database analysis of NB patients shows that high expression of USP7 significantly predicts poor outcomes. Together, our data strongly suggest that targeting USP7 is a novel concept in the treatment of NB. USP7-specific inhibitors like P22077 may serve not only as a stand-alone therapy but also as an effective adjunct to current chemotherapeutic regimens for treating NB with an intact USP7-HDM2-p53 axis.

Conformational stabilization of ubiquitin yields potent and selective inhibitors of USP7

Protein conformation and function are often inextricably linked, such that the states a protein adopts define its enzymatic activity or its affinity for various partners. Here we combine computational design with macromolecular display to isolate functional conformations of ubiquitin that tightly bind the catalytic core of the oncogenic ubiquitin-specific protease 7 (USP7) deubiquitinase. Structural and biochemical characterization of these ubiquitin variants suggest that remodeled backbone conformations and core packing poise these molecules for stronger interactions, leading to potent and specific inhibition of enzymatic activity. A ubiquitin variant expressed in human tumor cell lines binds and inhibits endogenous USP7, thereby enhancing Mdm2 proteasomal turnover and stabilizing p53. In sum, we have developed an approach to rationally target macromolecular libraries toward the remodeling of protein conformation, shown that engineering of ubiquitin conformation can greatly increase its interaction with deubiquitinases and developed powerful tools to probe the cellular role of USP7.

A Small Molecule Inhibitor of Ubiquitin-Specific Protease-7 Induces Apoptosis in Multiple Myeloma Cells and Overcomes Bortezomib Resistance

Bortezomib therapy has proven successful for the treatment of relapsed/refractory, relapsed, and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we show that P5091 is an inhibitor of deubiquitylating enzyme USP7, which induces apoptosis in MM cells resistant to conventional and bortezomib therapies. Biochemical and genetic studies show that blockade of HDM2 and p21 abrogates P5091-induced cytotoxicity. In animal tumor model studies, P5091 is well tolerated, inhibits tumor growth, and prolongs survival. Combining P5091 with lenalidomide, HDAC inhibitor SAHA, or dexamethasone triggers synergistic anti-MM activity. Our preclinical study therefore supports clinical evaluation of USP7 inhibitor, alone or in combination, as a potential MM therapy.

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

Abstract 610 Background and Rationale: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. Results: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, Bclxl, 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. Conclusions: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. Disclosures:Chauhan:Progenra, Inc: Consultancy. Nicholson:Progenra, Inc: Employment.