Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. There are no available therapeutics that slow or halt the progressive loss of dopamine-producing neurons, which underlies the primary clinical symptoms. Currently approved PD drugs can provide symptomatic relief by increasing brain dopamine content or activity; however, the alleviation is temporary, and the effectiveness diminishes with the inevitable progression of neurodegeneration. Discovery and development of disease-modifying neuroprotective therapies has been hampered by insufficient understanding of the root cause of PD-related neurodegeneration. The etiology of PD involves a combination of genetic and environmental factors. Although a single cause has yet to emerge, genetic, cell biological and neuropathological evidence implicates mitochondrial dysfunction and protein aggregation. Postmortem PD brains show pathognomonic Lewy body intraneuronal inclusions composed of aggregated α-synuclein, indicative of failure to degrade misfolded protein. Mutations in the genes that code for α-synuclein, as well as the E3 ubiquitin ligase Parkin, cause rare inherited forms of PD. While many ubiquitin ligases label proteins with ubiquitin chains to mark proteins for degradation by the proteasome, Parkin has been shown to mark dysfunctional mitochondria for degradation by mitophagy. The ubiquitin proteasome system participates in several aspects of the cell’s response to mitochondrial damage, affording numerous therapeutic opportunities to augment mitophagy and potentially stop PD progression. This review examines the role and therapeutic potential of such UPS modulators, exemplified by both ubiquitinating and deubiquitinating enzymes.

TAM receptors (Tyro3, Axl, and Mer) are receptor tyrosine kinases (RTKs) that are expressed by multiple immune cells including NK cells. Although RTKs typically enhance cellular functions, TAM receptor ligation blocks NK‐cell activation. The mechanisms by which RTKs block NK‐cell signaling downstream of activating receptors are unknown. In this report, we demonstrate that TAM receptors attenuate NK cell responses via the activity of E3 ubiquitin ligase Casitas B lineage lymphoma b (Cbl‐b). Specifically, we show that Tyro3, Axl, and Mer phosphorylate Cbl‐b, and Tyro3 ligation activates Cbl‐b by phosphorylating tyrosine residues 133 and 363. Ligation of TAM receptors by their ligand Gas6 suppresses activating receptor‐stimulated NK‐cell functions such as IFN‐γ production and degranulation, in a TAM receptor kinase‐ and Cbl‐b‐dependent manner. Moreover, Gas6 ligation induces the degradation of LAT1, a transmembrane adaptor protein required for NK cell activating receptor signaling, in WT but not in Cbl‐b knock‐out NK cells. Together, these results suggest that TAM receptors may attenuate NK‐cell function by phosphorylating Cbl‐b, which in turn dampens NK‐cell activation signaling by promoting the degradation of LAT1. Our data therefore support a mechanism by which RTKs attenuate, rather than stimulate, signaling pathways via the activation of ubiquitin ligases.

Post-translational modifications (PTMs) by ubiquitin (Ub) are versatile, highly dynamic, and involved in nearly all aspects of eukaryote biological function. The reversibility and heterogeneity of Ub chains attached to protein substrates have complicated their isolation, quantification, and characterization. Strategies have emerged to isolate endogenous ubiquitylated targets, including technologies based on the use of Ub-binding peptides, such as tandem-repeated Ub-binding entities (TUBEs). TUBEs allow the identification and characterization of Ub chains, and novel substrates for deubiquitylases (DUBs) and Ub ligases (E3s). Here we review their impact on purification, analysis of pan or chain-selective polyubiquitylated proteins and underline the biological relevance of this information. Together with peptide aptamers and other Ub affinity-based approaches, TUBEs will contribute to unraveling the secrets of the Ub code.

Abstract Tumors employ diverse strategies to suppress and evade the immune system’s ability to recognize and destroy tumor cells. Immune-suppressive Foxp3+ regulatory T cells (Tregs) in the tumor microenvironment correlate with poor prognosis in solid tumors. Therefore, selective depletion of tumor-associated Tregs or impairment of Treg function is considered an attractive cancer immunotherapy approach. The pro-tumorigenic deubiquitylase (DUB) USP7 is also essential for Treg functions. USP7 controls Treg function largely by regulating post-translational modification of Foxp3 and TIP60. Deletion of USP7 in Tregs results in impairment of Treg functions and autoimmunity in mice. Progenra has developed potent, selective covalent irreversible USP7 inhibitors that impair Treg functions ex vivo and in vivo. Most importantly, USP7 inhibitors exhibit robust antitumor activity against several syngeneic solid tumor models in immunocompetent mice. In addition, Progenra’s USP7 inhibitors enhance the efficacy of anti-PD1 antibody, anti-CTLA4 antibody, and cancer vaccines. These studies strongly suggest that USP7 inhibitors alone or in combination regimens can improve the efficacy and expand the scope of cancer immunotherapy. Citation Format: Suresh Kumar, Jian Wu, Feng Wang, Liqing Wang, Lee Chen, Ivan Sokirniy, Hui Wang, David Sterner, Charles Grove, Brigid Cunnion, Joseph Weinstock, Michael Mattern, Wayne Hancock. Covalent irreversible usp7 inhibitors for cancer immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B202.

Abstract Cancer cells evade destruction by co-opting checkpoint signaling in the immune system. Therapeutics aimed at preventing this, such as anti-PD1 and anti-CTLA4 antibodies, promote cytotoxic T lymphocyte (CTL)-mediated antitumor immune responses against advanced solid tumors. Harnessing the antitumor activity of CTLs, and that of their innate counterpart natural killer (NK) cells, is a promising approach. Activation of CTLs and NK cells is tightly regulated. CTLs require costimulatory signals, such as CD28, for robust activation. NK cell activation can be kept in check by a variety of inhibitory receptors, such as those of the TAM tyrosine kinase receptor family. Ultimately, these signaling events are orchestrated by the ubiquitin signaling system. One ubiquitin ligase in particular, Cbl-b (Casitas B-lineage lymphoma proto-oncogene b), is a negative regulator of both CTL and NK cell activation. In T cells, Cbl-b attenuates TCR signaling by negatively regulating several downstream signaling components, enforcing the requirement for costimulation. In NK cells, Cbl-b regulates TAM receptor internalization at the plasma membrane via ubiquitylation, and this process is important in enabling inhibitory signaling via the TAM receptors. Accordingly, mice deficient in active Cbl-b exhibit hyper-responsive immunity and reject a variety of implanted metastatic and nonmetastatic tumors. Outgrowth of spontaneous tumors in these mice is also significantly delayed. This tumor resistance is mediated by activated CD8+ T cells and NK cells. Thus, Cbl-b is a promising target for developing small-molecule cancer immunotherapy agents. Using proprietary HTS technologies, Progenra has discovered novel and selective Cbl-b inhibitors that decrease ubiquitylation of TAM receptor family members (Tyro3). These inhibitors also promote CD3-mediated activation of T cells in a CD28-independent manner, markedly increasing proliferation, IL-2 secretion, and TNFα production in T cells. Additionally, these Cbl-b inhibitors increase NK cell activation (as measured by IFNγ and degranulation) in ex vivo assays. ADME/DMPK as well as in vivo efficacy data will be discussed. These molecules are expected to synergize with other approved immunotherapy agents. Citation Format: Christopher Riling, Ivan Sokirniy, Brigid Cunnion, Emily Todd, Michael Mattern, Jian Wu, Taku Kambayashi, Suresh Kumar. Small-molecule Cbl-b inhibitors as novel intracellular checkpoint inhibitors for cancer immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A206.

The ubiquitin-specific protease 7 (USP7) has emerged as an attractive oncology/immune-oncology target owing to its critical roles in several cancer-related signaling pathways as well as its essential role in maintaining functions of Foxp3+ T-regulatory cells (Tregs), the key players in tumor immune evasion. Progenra has developed a series of compounds that inhibit purified USP7 selectively and attenuate USP7 activity in cells and in vivo; these inhibitors exert antitumor activity directly and also facilitate immune-mediated antitumor activity by suppressing Treg functions. However, the precise mechanism of action of these compounds remains unclear. In this study, using a combination of NMR spectroscopy, mass spectrometry, and single amino-acid substitution approaches, we have now demonstrated that our USP7 inhibitors specifically target the catalytic pocket of USP7 and modify its active site cysteine (Cys223) by forming a covalent adduct. Consistent with the covalent binding mechanism, pharmacokinetic studies revealed long-lasting, irreversible USP7 inhibition after a short pulse treatment with inhibitor, accompanied by changes in the level and ubiquitylation of various pharmacodynamic markers, including the Treg lineage-specific transcription factor Foxp3. Detailed knowledge of the mechanism of USP7 inhibition will permit the rational design of improved inhibitors as a new class of anticancer agent. Citation Format: Feng Wang, Jian Wu, Liqing Wang, Ivan Sokirniy, Hui Wang, Lee Chen, Brigid Cunnion, David Sterner, Charles Grove, Thomas Bregnard, Joseph Weinstock, Michael Mattern, Irina Bezsonova, Wayne W. Hancock, Suresh Kumar. Characterization of selective active-site targeted covalent inhibitors of usp7 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B193.

Tumor microenvironment (TME) plays critical role in suppressing the immune system’s ability to recognize and destroy cancer cells. Although T cell checkpoint blockers such as anti-PD1 antibodies unleash anti-tumor immune response against a variety of tumors, the complex immunosuppressive tumor milieu necessitates development of additional therapeutic agents to potentiate active drugs and expand the realm of the revolutionary cancer immunotherapy. The presence of highly immunosuppressive Foxp3+ T-regulatory cells (Tregs) in the TME has been correlated with poor prognosis. Thus, depletion of Tregs or impairment of Treg function is considered an attractive therapeutic approach. USP7, a deubiquitylase (DUB) implicated as a critical node in several cancer signaling pathways has emerged as an essential factor in maintaining Treg functions. Treg specific deletion of USP7 impairs Treg function leading to lethal autoimmunity. Using the UbiProTM discovery platform, Progenra has identified selective covalent USP7 inhibitors that were subjected to lead optimization and preclinical evaluation. Potent and selective candidate USP7 inhibitors have been shown to impair Treg functions ex vivo and in vivo as well as exhibit powerful anti-tumor activity against syngeneic lung tumor models in immunocompetent mice. Most importantly, Progenra’s USP7 inhibitors enhances the efficacy of anti-PD1 antibody and cancer vaccines. Along with the already established direct anti-tumor activities of USP7 inhibitors, these studies provide a strong rationale for combining USP7 inhibitors to suppress Treg functions and improve the efficacy of currently approved cancer immunotherapy agents. Citation Format: Suresh Kumar, Jian Wu, Liqing Wang, Feng Wang, Ivan Sokirniy, Hui Wang, David Sterner, Charles Grove, Joseph Weinstock, Michael Mattern, Wayne Hancock. USP7 inhibitors impair Foxp3+ T regulatory cell function and promote antitumor immunity against solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1691. doi:10.1158/1538-7445.AM2017-1691

Abstract The ubiquitin-specific protease 7 (USP7) has emerged as an attractive therapeutic target owing to its critical roles in several cancer signaling pathways as well as its essential role in maintaining Foxp3+ T-regulatory cell (Treg) functions. Pharmacological inhibition of USP7 using Progenra’s small molecule inhibitors resulted in direct anti-tumor activity as well as the unleashing of anti-tumor immunity by suppressing Treg functions. However, the precise mechanism of action these compounds was not well characterized. Using a combination of NMR spectroscopy, mass spectrometry, and single amino acid substitution approaches, we have now demonstrated that our USP7 inhibitors specifically target the catalytic cleft of USP7 and covalently modify its active site cysteine (Cys223). Pharmacokinetic studies revealed sustained irreversible USP7 inhibition after short term inhibitor treatment as well as subsequent changes in the level and ubiquitylation of various pharmacodynamic markers, including the Treg lineage specific transcription factor Foxp3. Detailed knowledge of the mechanism of USP7 inhibition has allowed us to incorporate rational design strategies into the lead optimization to obtain improved molecules that will serve as the basis of a new class of anti-cancer immunotherapy agents. Citation Format: Feng Wang, Jian Wu, Liqing Wang, Ivan Sokirniy, Hui Wang, Charles Grove, Phuong Nguyen, Thomas Bregnard, Joseph Weinstock, Michael Mattern, Wayne Hancock, Irina Bezsonova, Suresh Kumar. Characterization of selective covalent inhibitors of USP7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5336. doi:10.1158/1538-7445.AM2017-5336

Ubiquitin specific protease 7 (USP7), the most widely studied among the nearly 100 deubiquitinating enzymes, supports cancer by positively affecting tumor growth and negatively affecting the patient's immune response to tumors. Great interest exists, therefore, in developing USP7 inhibitors for clinical evaluation. While the proteasome inhibitor field has enjoyed clinical success, very few clinically appropriate effectors of deubiquitinating (protease) or ubiquitinating (ligase) enzymes have appeared. The ubiquitin protease/ligase field is moving from the initial discovery of potent, selective modulators with cell proof of concept and in vivo activity to the optimization of these molecules to impart drug-like properties or the discovery of new inhibitor scaffolds by improved screening or rational design. This Perspective focuses on the current status of USP7 inhibitors from various organizations active in developing these compounds for the clinic and suggests undertakings that are both achievable and necessary to lead to successful clinical outcomes for USP7 inhibitors in cancer treatment.

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.

Abstract Studies have been performed to demonstrate that effectors of enzymes of the ubiquitin proteasome pathway associated with T cell activation are able to impair tumor survival by abrogating the immune evasion mechanism. Immune evasion is a collective term for the various strategies employed by tumors to suppress the host immune system's ability to recognize and destroy cancer cells. Significant advances in the understanding of the mechanisms of cancer immune evasion have led to the successful development of biologic immunotherapies that showed clinical efficacy against hematological as well as solid tumors. These include monoclonal antibodies, allogeneic hematopoietic stem cell transplantation, vaccination, cytokines, T cell checkpoint blockade and adoptive transfer of engineered T cells. Owing to the multifaceted nature of cancer immune evasion, however, several different approaches will likely be necessary for effective therapeutic management. We have adopted a dual targeting approach focused on the ubiquitin proteasome pathway to develop novel small molecule cancer immunotherapies. Using the UbiProTM discovery platform, we have identified inhibitors of a deubiquitylase (DUB) that is highly expressed in regulatory T cells (Tregs) and plays a critical role in promoting Treg functions. These inhibitors are shown to suppress Treg functions and promote T-effector cell function ex vivo and in vivo. In addition, we have identified inhibitors of an E3 ubiquitin ligase that negatively regulates T-effector cell function. These inhibitors are shown to activate T-cells and NK cell functions in vitro. A combination of such inhibitors should provide powerful anti-cancer immunotherapy. Citation Format: Suresh Kumar, Liqing Wang, Jian Wu, Feng Wang, Saket Agarwal, Matthew Kodrasov, Wayne Hancock, Michael Mattern. Targeting the ubiquitin signaling system for cancer immunotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B007.

Abstract Immunotherapy using biological agents such as anti-PD1 antibody has revolutionized cancer treatment. Nevertheless, patient response has been highly variable, with many patients showing no response. Although the precise mechanism of resistance is unknown, the complex immunosuppressive tumor milieu necessitates development of additional therapeutic agents to potentiate active drugs and expand the scope of the revolutionary cancer immunotherapy. Regulatory T cells (Tregs) present in tumors dampen the anti-tumor activities of effector T cells and are correlated with poor prognosis. Therefore, depletion of Tregs or impairment of Treg function is considered an attractive therapeutic approach. USP7, a deubiquitylase (DUB) implicated as a critical node in several cancer signaling pathways, is essential for maintaining Treg function, and Treg specific deletion of USP7 results in impaired Treg functions and lethal autoimmunity. Progenra has identified selective small molecule USP7 inhibitors that were subsequently lead optimized and evaluated in ADME/PK studies. Potent, selective and irreversible USP7 inhibitors have been shown to impair Treg functions and exhibit powerful anti-tumor activity against syngeneic lung tumor models in immunocompetent mice. In addition, Progenra's USP7 inhibitors synergize with anti-PD1 antibody and cancer vaccines. These studies suggest that USP7 inhibitors alone or in combination can improve the efficacy and expand the scope of cancer immunotherapy Citation Format: Suresh Kumar, Jian Wu, Liqing Wang, Jack Wang, Ivan Sokirniy, Hui Wang, Glen Fegley, Joseph Weinstock, Michael Mattern, Wayne W. Hancock. Potent and selective small molecule USP7 inhibitors for cancer immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B042.

Abstract Immune evasion is a hallmark feature of tumors as they employ various strategies to suppress the immune system's ability to recognize and destroy cancer cells. T cell checkpoint inhibitors such as anti-PD1 and anti-CTLA-4 antibodies spearhead the immune response against a variety of tumors. Numerous studies suggest that the complex immunosuppressive milieux require the development of additional therapeutic agents to potentiate active drugs and thereby broaden the utility and increase the therapeutic indices of revolutionary immune-oncology treatment modalities. The presence of immunosuppressive regulatory T cells (Tregs) in tumors, keeping tumoricidal Teffector cells in check, signals poor prognosis. Thus, depletion of Tregs or impairment of Treg function is an attractive therapeutic approach for cancer. USP7, a deubiquitylase enzyme implicated as a critical node in several cancer signaling pathways, has recently emerged as an essential factor in maintaining Treg functions. Progenra identified small molecule USP7 inhibitors and employed them to show that Treg specific inhibition of USP7 results in impairment of Treg function leading to immune activation, commensurate with the ablation of Foxp3, a transcription factor that is a target of USP7 and is essential to Treg activation. This USP7 inhibitor class was subsequently lead optimized, and selected USP7 inhibitors were evaluated in ADME/PK studies and shown to impair Treg functions and to exhibit powerful anti-tumor activity against syngeneic lung tumor models in immunocompetent mice. In addition, Progenra's USP7 inhibitors significantly augmented the antitumor activity of anti-PD1 antibody, CAR T cell therapy, and cancer vaccines. These studies provide a strong rationale for the use of USP7 inhibitors in combination therapy protocols to improve the efficacy of currently approved cancer immunotherapy agents. Citation Format: Suresh Kumar, Jian Wu, Liquing Wang, Feng Wang, Matthew P. Kodrasov, Saket Agarwal, Ivan Sokirniy, Thomas Yeckley, Joseph Weinstock, Michael R. Mattern, Wayne W. Hancock. Small molecule T-reg inhibitors for cancer immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 559.

The approval by the FDA and clinical use of immune checkpoint inhibitors, such as anti-PD1 and anti-CTLA4 antibodies, that activate cytotoxic T lymphocyte (CTL) mediated anti-tumor immune responses against advanced solid tumors has resulted in a paradigm shift in cancer therapy. Antitumor activity of CTLs and NK cells is tightly regulated by intracellular signaling events orchestrated by the ubiquitin signaling system. In particular, Cbl-b (Casitas B-lineage lymphoma proto-oncogene b), a RING finger E3-ubiquitin ligase primarily expressed in immune cells, acts as an intracellular checkpoint and a master negative regulator of both CTLs and NK cells. Accordingly, Cbl-b-/- mice have been observed to reject a variety of implanted metastatic and non-metastatic tumors and to delay significantly the outgrowth of spontaneous tumors; these observed antitumor effects were seen to be mediated by activated CD8+ T cells and NK cells. Based on the overwhelming evidence supporting the role of Cbl-b in immune suppression, this ubiquitin conjugating enzyme is considered a novel target for developing small molecule cancer immunotherapy agents. Using proprietary high throughput screening technologies, Progenra identified novel, selective Cbl-b inhibitors that were shown to decrease ubiquitination of substrates such as TAM receptor kinases (Tyro3) and to activate T cells (as judged by increased IL-2 production) and NK cells (as judged by increased IFNγ production and degranulation) in ex vivo functional assays. ADME/DMPK evaluation and in vivo efficacy of selected Cbl-b inhibitors will be discussed in relation to the therapeutic utility of this class of small molecule agent, which is expected to potentiate the anticancer effects of approved immunotherapy agents. Citation Format: Saket Agarwal, Jian Wu, Chris Riling, Matthew Kodrasov, Joseph Weinstock, Ivan Sokirniy, Michael Mattern, Taku Kambayashi, Suresh Kumar. Cbl-b inhibitors as novel intra-cellular checkpoint inhibitors for cancer immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2228.

Abstract The conjugation and deconjugation of ubiquitin controls the stability, localization and activity of many cellular proteins. Deconjugation of ubiquitin is performed by a family of ~80 deubiquitylating enzymes (DUBs) including USP7 which specifically removes ubiquitin from HDM2, Foxp3, claspin and other therapeutically relevant substrates. Importantly, USP7 is overexpressed in human multiple myeloma cells and the expression level of USP7 inversely correlates with prognosis in multiple myeloma patients. Previously, we reported the discovery and characterization of P0005091 a USP7 inhibitor which exhibited anti-tumor activity in in vitro and in vivo models of multiple myeloma. Additionally, we reported that P0005091 inhibits USP47 a closely related DUB which is an emerging oncology target in its own right. Furthermore, a structural analog of P0005091, P0022077 has recently been reported by another research group to inhibit neuroblastoma growth in an orthotopic mouse model suggesting that in addition to hematological tumors, solid tumors may be treatable via inhibition of USP7. To obtain additional starting points for a USP7 or a USP7/USP47 specific drug discovery program we recently performed a new screening campaign. By utilizing a biophysical assay platform we were able to exclude compounds that bind to the active site of USP7 thus increasing the possibility of identifying allosteric modifiers of USP7 activity. Hits from this screen are currently the subject of hit to lead optimization and will be evaluated in biophysical and biochemical assays as well as in vitro and in vivo models of hematological malignancies. Data will be presented describing these results. Citation Format: Benjamin Nicholson, Matthew P. Kodrasov, James P. LaRocque, Michael J. Eddins, Suresh Kumar, Dharminder Chauhan, Kenneth C. Anderson, Michael R. Mattern. Inhibition of USP7 for the treatment of multiple myeloma and other malignancies. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A21.

Abstract Ubiquitylation and deubiquitylation control the stability, localization and activity of many cellular proteins. Deubiquitylation is performed by a family of ∼80 enzymes (DUBs) and many of these DUBs have been implicated in a wide range of diseases including cancer. In particular, USP2 removes ubiquitin selectively from Cyclin D1, Cyclin A1, Aurora A, MDM2 and FAS which are all implicated in oncogenic processes. Importantly, USP2 is overexpressed in prostate and ovarian cancers and inhibition of USP2 has been shown to sensitize prostate carcinoma cells to apoptosis. Therefore USP2 is an attractive therapeutic target. Progenra's unique UbiProTM discovery platform was utilized to screen ∼200K member diversity based library of small molecules for identifying novel USP2 inhibitors. Hits from the screen were subjected to orthogonal selectivity assays to identify USP2 specific inhibitors. Here we report discovery and hit to lead optimization of novel USP2 inhibitors showing cellular efficacy and anti-cancer activities against relevant cancer cell models. The most promising compounds are being further developed as novel anti-cancer agents. Citation Format: Suresh Kumar, James P. LaRocque, Jeffrey G. Marblestone, Jian Wu, Joseph Weinstock, David E. Sterner, Michael R. Mattern. Discovery and development of novel USP2 inhibitors for cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5433. doi:10.1158/1538-7445.AM2015-5433