Foxp3+ T-regulatory (Treg) cells are known to suppress protective host immune responses to a wide variety of solid tumors, but their therapeutic targeting is largely restricted to their transient depletion or "secondary" modulation, e.g. using anti-CTLA-4 monoclonal antibody. Our ongoing studies of the post-translational modifications that regulate Foxp3 demonstrated that the histone/protein acetyltransferase, Tip60, plays a dominant role in promoting acetylation, dimerization and function in Treg cells. We now show that the ubiquitin-specific protease, Usp7, controls Treg function largely by stabilizing the expression and promoting the multimerization of Tip60 and Foxp3. Genetic or pharmacologic targeting of Usp7 impairs Foxp3+ Treg suppressive functions, while conventional T cell responses remain intact. As a result, pharmacologic inhibitors of Usp7 can limit tumor growth in immunocompetent mice, and promote the efficacy of antitumor vaccines and immune checkpoint therapy with anti-PD1 monoclonal antibody in murine models. Hence, pharmacologic therapy with Usp7 inhibitors may have an important role in future cancer immunotherapy.

USP7 is a deubiquitinating enzyme that plays a pivotal role in multiple oncogenic pathways and therefore is a desirable target for new anti-cancer therapies. However, the lack of structural information about the USP7-inhibitor interactions has been a critical gap in the development of potent inhibitors. USP7 is unique among USPs in that its active site is catalytically incompetent, and is postulated to rearrange into a productive conformation only upon binding to ubiquitin. Surprisingly, we found that ubiquitin alone does not induce an active conformation in solution. Using a combination of nuclear magnetic resonance, mass spectrometry, computational modeling, and cell-based assays, we found that DUB inhibitors P22077 and P50429 covalently modify the catalytic cysteine of USP7 and induce a conformational switch in the enzyme associated with active site rearrangement. This work represents the first experimental insights into USP7 activation and inhibition and provides a structural basis for rational development of potent anti-cancer therapeutics.

Accumulation of Foxp3+ T-regulatory (Treg) cells in the tumor microenvironment is associated with tumor immune evasion and poor patient outcome in the case of many solid tumors. Current therapeutic strategies for blocking Treg functions are not Treg-specific, and display only modest and transient efficacy. Recent studies revealed that ubiquitin-specific protease 7 (USP7) is essential for Treg functions by stabilizing expression of Tip60 and Foxp3, which together are central to the development and maintenance of the Treg cell lineage. Pharmacological inhibition of USP7 is therefore a promising strategy for suppressing Treg functions and promoting anti-tumor immunity. Previously, we reported the P5091 series of small molecule USP7 inhibitors and demonstrated their direct anti-tumor activity in vivo using xenograft models. However, the precise mechanism of action of these compounds was not well defined. In this study, we report the development and characterization of P217564, a second-generation USP7 inhibitor with improved potency and selectivity. P217564 selectively targets the catalytic cleft of USP7 and modifies its active site cysteine (C223) by forming a covalent adduct. Irreversible inhibition of USP7 results in durable downstream biological responses in cells, including down-regulation of Tip60 and consequent impairment of Treg suppressive function. In addition, we demonstrate that both USP7 and various USP7 substrates are subjected to Lys48-mediated ubiquitin modification, consistent with increased proteasomal degradation of these proteins because of USP7 inhibition.

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.