USP7 Inhibitors Research Articles

The Next Step Forward in Ubiquitin-Specific Protease 7 Selective Inhibition

Ubiquitin-specific protease 7 is a validated anticancer target; thus, selective USP7 inhibitors are of great interest. In this issue of Cell Chemical Biology, Lamberto etal. (2017) and Pozhidaeva etal. (2017) report important insights into the structural inhibitor-enzyme interplay, lighting the way toward the development of selective inhibitors.

Characterization of naturally occurring pentacyclic triterpenes as novel inhibitors of deubiquitinating protease USP7 with anticancer activity in vitro.

Deubiquitinating protease USP7 is a promising therapeutic target for cancer treatment, and interest in developing USP7 inhibitors has greatly increased. In the present study, we reported a series of natural pentacyclic triterpenes with USP7 inhibitory activity in vitro. Among them, both the ursane triterpenes and oleanane triterpenes were more active than the lupine triterpenes, whereas ursolic acid was the most potent with IC50 of 7.0±1.5 μmol/L. Molecular docking studies showed that ursolic acid might occupy the ubiquitin binding pocket of USP7, with the 17-carboxyl group and 3-hydroxyl group playing a vital role in the USP7-ursolic acid interaction. Using the cellular thermal shift assay, we demonstrated that ursolic acid interacted with USP7 in RPMI8226 human myeloma cells. Ursolic acid dose-dependently inhibited the proliferation of the myeloma cells with IC50 of 6.56 μmol/L, accompanied by reductions in USP7 substrates such as MDM2, UHRF1 and DNMT1. Overexpression of USP7 partially, but significantly attenuated ursolic acid-induced cell death as well as downregulation of MDM2, UHRF1 and DNMT1. In conclusion, we demonstrate for the first time that pentacyclic triterpenes represent a novel scaffold for developing USP7 inhibitors and that USP7 inhibition contributes to the anti-cancer effect of ursolic acid.

Structure-Guided Development of a Potent and Selective Non-covalent Active-Site Inhibitor of USP7

Deubiquitinating enzymes (DUBs) have garnered significant attention as drug targets in the last 5-10 years. The excitement stems in large part from the powerful ability of DUB inhibitors to promote degradation of oncogenic proteins, especially proteins that are challenging to directly target but which are stabilized by DUB family members. Highly optimized and well-characterized DUB inhibitors have thus become highly sought after tools. Most reported DUB inhibitors, however, are polypharmacological agents possessing weak (micromolar) potency toward their primary target, limiting their utility in target validation and mechanism studies. Due to a lack of high-resolution DUB⋅small-molecule ligand complex structures, no structure-guided optimization efforts have been reported for a mammalian DUB. Here, we report a small-molecule⋅ubiquitin-specific protease (USP) family DUB co-structure and rapid design of potent and selective inhibitors of USP7 guided by the structure. Interestingly, the compounds are non-covalent active-site inhibitors.

USP7-Specific Inhibitors Target and Modify the Enzyme's Active Site via Distinct Chemical Mechanisms

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.

Abstract 1691: USP7 inhibitors impair Foxp3+ T regulatory cell function and promote antitumor immunity against solid tumors

Abstract 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

Ubiquitin Signaling: Systematic Profiling in the Immune Response

Abstract Cell signaling induced by cytokine receptors is regulated by ubiquitin (Ub) modification. Ub forms polymerized chains to encrypt the coding for the alteration of signaling molecules in immune response. Here we report a systematic approach for profiling Ub coding. Ubiquitylation of specific target proteins in stimulated and unstimulated human PBMCs and T Leukemia (Jurkat) cells are profiled by ELISA and Immunoblotting UBiTest and Ubi-Omics methods. The cells are treated with the de-ubiquitylase USP7 inhibitor P50378, proteasome inhibitor bortezomib, or a ubiquitin ligase inhibitor. Poly-ubiquitylated cellular proteins is affinity isolated using Tetra Ub Binding Entities (TUBEs), which bind Ub with high affinity (nmol). TUBEs that are selective for isolated K48, K63 and M1 (linear) poly-ubiquitylated proteins are employed. Then, these polyubiquitylated proteins were digested with Lys-C and immunoprecipitated with UbiSite antibody. Ub-rich peptides were analyzed by LC-MS/MS. This UbiSite-Omics approach affords the advantages of both identifying N-terminal ubiquitylation and distinguishing ubiquitination from modification by Ub-like proteins. These data provide proteome-wide profiling of Ub-omics not achievable by traditional proteomics methods and suggest that the TUBE and UbiSite based technology will reveal new Ub landscapes in immune cells. This novel approach will facilitate the discovery of pharmacological markers for drugs, and enable cell biologists to identify patterns of polyUb proteins.

The combined effect of USP7 inhibitors and PARP inhibitors in hormone-sensitive and castration-resistant prostate cancer cells.

Purpose of the studyReduced levels of the tumor suppressor protein CCDC6 sensitize cancer cells to the treatment with PARP-inhibitors. The turnover of CCDC6 protein is regulated by the de-ubiquitinase USP7, which also controls the androgen receptor (AR) stability. Here, we correlated the expression levels of CCDC6 and USP7 proteins in primary prostate cancers (PC). Moreover, we tested the efficacy of the USP7 inhibitors, in combination with PARP-inhibitors as a novel therapeutic option in advanced prostate cancer.Experimental techniquesPC cells were exposed to USP7 inhibitor, P5091, together with cycloheximide, to investigate the turnover of the USP7 substrates, AR and CCDC6. As outcome of the AR downregulation, transcription targets of AR and its variant V7 were examined by qPCR. As a result of CCDC6 degradation, the induction of PARP inhibitors sensitivity was evaluated by analyzing PC cells viability and foci formation. We scored and correlated CCDC6 and USP7 expression levels in a prostate cancer tissue microarray (TMA).ResultsP5091 accelerated the degradation of AR and V7 isoform affecting PSA, UBE2C, CDC20 transcription and PC cells proliferation. Moreover, P5091 accelerated the degradation of CCDC6 sensitizing the cells to PARP-inhibitors, that acted sinergistically with genotoxic agents. The immunohistochemical analysis of both CCDC6 and USP7 proteins exhibited significant correlation for the intensity of staining (p ≤ 0.05).Data interpretationThus, CCDC6 and USP7 represent predictive markers for the combined treatment of the USP7-inhibitors and PARP-inhibitors in advanced prostate cancer.

Therapeutic inhibition of USP7-PTEN network in chronic lymphocytic leukemia: a strategy to overcome TP53 mutated/deleted clones.

Chronic Lymphocytic Leukemia (CLL) is a lymphoproliferative disorder with either indolent or aggressive clinical course. Current treatment regiments have significantly improved the overall outcomes even if higher risk subgroups - those harboring TP53 mutations or deletions of the short arm of chromosome 17 (del17p) - remain highly challenging. In the present work, we identified USP7, a known de-ubiquitinase with multiple roles in cellular homeostasis, as a potential therapeutic target in CLL. We demonstrated that in primary CLL samples and in CLL cell lines USP7 is: i) over-expressed through a mechanism involving miR-338-3p and miR-181b deregulation; ii) functionally activated by Casein Kinase 2 (CK2), an upstream interactor known to be deregulated in CLL; iii) effectively targeted by the USP7 inhibitor P5091. Treatment of primary CLL samples and cell lines with P5091 induces cell growth arrest and apoptosis, through the restoration of PTEN nuclear pool, both in TP53-wild type and -null environment. Importantly, PTEN acts as the main tumor suppressive mediator along the USP7-PTEN axis in a p53 dispensable manner. In conclusion, we propose USP7 as a new druggable target in CLL.

Usp7 promotes medulloblastoma cell survival and metastasis by activating Shh pathway

The ubiquitin-specific protease Usp7 plays roles in multiple cellular processes through deubiquitinating and stabilizing numerous substrates, including P53, Pten and Gli. Aberrant Usp7 activity has been implicated in many disorders and tumorigenesis, making it as a potential target for therapeutic intervention. Although it is clear that Usp7 is involved in many types of cancer, its role in regulating medulloblastoma (MB) is still unknown. In this study, we show that knockdown of Usp7 inhibits the proliferation and migration of MB cells, while Usp7 overexpression exerts an opposite effect. Furthermore, we establish Usp7 knockout MB cell line using the CRISPR/Cas9 system and further confirm that Usp7 knockout also blocks MB cell proliferation and metastasis. In addition, we reveal that knockdown of Usp7 compromises Shh pathway activity and decrease Gli protein levels, while P53 level and P53 target gene expression have no obvious changes. Finally, we find that Usp7 inhibitors apparently inhibit MB cell viability and migration. Taken together, our findings suggest that Usp7 is important for MB cell proliferation and metastasis by activating Shh pathway, and is a putative therapeutic target for MBs.

Synthesis and biological evaluation of thiazole derivatives as novel USP7 inhibitors

Herpesvirus-associated Ubiquitin-Specific Protease (HAUSP, also called USP7) interacts with and stabilizes Mdm2, and represents one of the first examples that deubiquitinases oncogenic proteins. USP7 has been regarded as a potential drug target for cancer therapy. Inhibitors of USP7 have been recently shown to suppress tumor cell growth in vitro and in vivo. Based on leading USP7 inhibitors P5091 and P22077, we designed and synthesized a series of thiazole derivatives. The results of in vitro assays showed that the thiazole compounds exhibited low micromolar inhibition activity against both USP7 enzyme and cancer cell lines. The compounds induced cell death in a p53-dependent and p53-independent manner. Taken together, this study may provide thiazole compounds as a new class of USP7 inhibitors.

The USP7 Inhibitor P5091 Induces Cell Death in Ovarian Cancers with Different P53 Status

Background/Aims: Ovarian cancer is often diagnosed at later stages with poor prognosis. Recent studies have associated the expression of deubiquitylase USP7 with the survival of ovarian cancers. Being a cysteine protease, USP7 could become a target for pharmacological intervention. Therefore, in this study, we assessed the influence of its inhibitor P5091 on ovarian cancer cells. Methods: Ovarian cancer cells were treated with P5091, and cell proliferation was measured with MTT assay; cell morphology was inspected under a phase-contrast microscope; cell cycle and cell death were examined by flow cytometry. To gain mechanistic insights into its effects, immunoblotting was performed to detect USP7, HDM2, p53, p21, apoptosis and autophagy related proteins. Results: P5091 effectively suppressed the growth of ovarian cancer cells, caused cell cycle blockage, and induced necrosis and apoptosis with more severe phenotypes observed in HeyA8 cells with wild-type p53 than in OVCAR-8 cells with mutant p53. P5091 also prompted autophagy, with more efficient p62 degradation in HeyA8. Conclusion: P5091 shows efficacy in suppressing ovarian cancers harbouring wild-type and mutant p53. Its effects seemed to be enhanced by wild-type p53. The potency of this USP7 inhibitor also correlated with autophagy to some extent. Therefore, the pharmacological targeting of USP7 may serve as a potential therapeutic strategy and warrants further investigation.

Inhibition of USP10 Induces Degradation of Oncogenic FLT3: A Novel Approach to Therapy of Leukemia

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. Overall, the survival with current chemotherapy is only 20-40%, declining steadily with advancing age. Approximately 30% of AML patients have mutations that constitutively activate the FLT3 gene. The most common FLT3 mutation results in tandem duplications within the juxtamembrane domain, observed in 20-25% of AML patients (internal tandem duplication, ITD), associated with markedly decreased survival. FLT3 kinase domain inhibitors, including SU11248, SU5416, CEP-701 and PKC412 (midostaurin), have been shown to induce partial, and usually brief, remissions in clinical trials of relapsed AML patients when administered as single agents. In a large trial in newly diagnosed patients, however, midostaurin was shown to increase survival when combined with standard chemotherapy.[1] This study supports the notion that inhibition of FLT3 may be important, at least in patients with mutations in the FLT3 gene. Since drug resistance develops in some patients with newly diagnosed AML and virtually all patients with advanced disease, additional strategies to target FLT3 would be of value.We discovered that the deubiquitinating enzyme (DUB) ubiquitin specific protease 10 (USP10) removes a degradative ubiquitin tag from mutant FLT3 thereby contributing to high levels of the oncogenic protein in AML (Fig 1a). Screening of our preclinical DUB inhibitor library for ability to selectively kill growth factor-independent FLT3-ITD-positive Ba/F3 cells over IL-3-dependent parental Ba/F3 cells identified HBX19818, a reported USP7 inhibitor, as the top hit. The effects are not unique to the Ba/F3 system: when profiled against a panel of 7 leukemia cell lines, HBX19818 conferred a substantial growth suppressive effect only to those expressing the FLT3-ITD oncoprotein (Fig 1b). As an initial assessment of the mechanism of HBX19818 we confirmed that it does promote ubiquitin-mediated degradation of FLT3-ITD (Fig 1c) and that the effect is selective as HBX19818 does not impact protein levels of wt FLT3. HBX19818 is published as an irreversible USP7 inhibitor,[2] however DUBome selectivity profiling data we generated identifies USP10 as the most potently inhibited DUB of the compound (USP10 IC50 = 14 µM). We went on to validate USP10 as the DUB that stabilizes FLT3-ITD using a combination of small molecule and genetic experiments. Notably, HBX19818 binds and inhibits USP10 in cells (data not shown), small hairpin knockdown of USP10 phenocopies the antiproliferative and FLT3 degradation effects of HBX19818 (Figure 1d and data not shown), and a direct interaction between USP10 and FLT3-ITD is observed in co-immunoprecipitation experiments (Fig 1e). Additionally, SAR studies reveal correlation among USP10 IC50, FLT3-ITD degradation and anti-proliferative effects for the HBX19818 chemical series, and we identified a second chemotype that phenocopies its effects. In support of the translational potential of USP10 inhibition for FLT3 mutant AML, we observed that both USP10 inhibitor series synergize with FLT3 kinase inhibitors, suppress growth of mutant FLT3-expressing primary AML cells and primagraft AML cells and, importantly, display the ability to overcome the FLT3 inhibitor-resistant mutant FLT3-ITD-F691L among other FLT3 kinase inhibitor-resistant mutants (Fig. 1f and data not shown).Overall, our data strongly support degradation of mutant FLT3 as an alternative approach to therapeutically target FLT3. This approach, which focuses on targeting USP10, could prove more efficacious than kinase inhibitors by simultaneously blocking both enzymatic and scaffolding functions of FLT3, and blocking compensatory increases in FLT3 protein or resistant point mutations associated with some kinase inhibitors. Importantly, this is the first demonstration of stabilization of an AML mutant driver protein by a DUB enzyme and introduces a novel therapy for FLT3 mutant-positive AML.

Nek2 Stabilization By Usp7 Leads to Activation of NF-Kb in Multiple Myeloma

NIMA (Never In Mitosis Gene A)-Related Kinase 2 (Nek2), a centrosomal Serine/Threonine kinase, is a key player in numerous malignancies. Overexpression of Nek2 has been related to many cancers including Multiple Myeloma (MM). In MM, Nek2 is one of the chromosomal instability genes associated with drug resistance and disease relapse. However, very little is known about the mechanisms that lead to these Nek2-driven disparities. Here, we show that the Ubiquitin Specific Peptidase 7 (USP7) stabilizes Nek2 leading to activation of NF-kb pathway.Using gene expression profile (GEP) data from patients and cell lines we discovered that Nek2 overexpression leads to increases of several targets of the NF-kb pathway. We, thus, hypothesize that Nek2 is activating NF-kb. To address this, we overexpressed Nek2 and tested the classic canonical NF-kb hallmarks proteins by western blotting. Nek2 overexpression led to an increase in phosphorylation of IKK, activator of NF-kb, and to decrease levels of IKb-alpha, a negative regulator of the pathway. Nek2 overexpression also increased nuclear and phosphorylated p65 on residue S536, known as active transcriptional site. To further confirm that Nek2 is activating canonical NF-kb luciferase assay was performed. The luciferase reporter is driven by a p65 promoter and in cells overexpressing Nek2 luciferase levels were increased.To characterize Nek2 interacting partners a tandem affinity purification/mass spectrometry (TAP/MS) approach was performed. We found that Nek2 binds to Usp7, a deubiquitinase overexpressed in numerous cancers. This led to hypothesize that that Nek2, a known target of the ubiquitin proteasome system, is being stabilized by the Usp7 contributing to its overexpression and the increased activation of the NF-kb pathway. To test our hypothesis, we treated different cancer cell lines with the commercially available Usp7 inhibitor, P5091, or silenced the protein using shRNA. In both case, we found a reduction in Nek2 protein level. Additionally, we overexpressed Usp7 and Nek2 increased confirming that Usp7 stabilizes Nek2. To further show that Usp7 stabilizes Nek2 by de-ubiquitination, we overexpressed Usp7 and analyzed Nek2 ubiquitination after immunoprecipitation. When Usp7 was overexpressed no ubiquitination of Nek2 was detected.Finally, by using GEP data from MM patients, we found that individuals who overexpressed Nek2 along with an active NF-kb signature have worst event free survival as well as overall survival, indicating Nek2 overexpression leading to increased NF-kb signature has clinical significance. DisclosuresNo relevant conflicts of interest to declare.

Abstract B042: Potent and selective small molecule USP7 inhibitors for cancer immunotherapy

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.

CDDO-Me reveals USP7 as a novel target in ovarian cancer cells.

Deubiquitinating enzyme USP7 has been involved in the pathogenesis and progression of several cancers. Targeting USP7 is becoming an attractive strategy for cancer therapy. In this study, we identified synthetic triterpenoid C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid (CDDO-Me) as a novel inhibitor of USP7 but not of other cysteine proteases such as cathepsin B and cathepsin D. CDDO-Me inhibits USP7 activity via a mechanism that is independent of the presence of α, β-unsaturated ketones. Molecular docking studies showed that CDDO-Me fits well in the ubiquitin carboxyl terminus-binding pocket on USP7. Given that CDDO-Me is known to be effective against ovarian cancer cells, we speculated that CDDO-Me may target USP7 in ovarian cancer cells. We demonstrated that ovarian cancer cells have higher USP7 expression than their normal counterparts. Knockdown of USP7 inhibits the proliferation of ovarian cancer cells both in vitro and in vivo. Using the cellular thermal shift assay and the drug affinity responsive target stability assay, we further demonstrated that CDDO-Me directly binds to USP7 in cells, which leads to the decrease of its substrates such as MDM2, MDMX and UHRF1. CDDO-Me suppresses ovarian cancer tumor growth in an xenograft model. In conclusion, we demonstrate that USP7 is a novel target of ovarian cancer cells; targeting USP7 may contribute to the anti-cancer effect of CDDO-Me. The development of novel USP7 selective compounds based on the CDDO-Me-scaffold warrants further investigation.

Ubiquitin-specific Protease-7 Inhibition Impairs Tip60-dependent Foxp3 + T-regulatory Cell Function and Promotes Antitumor Immunity

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.

A SUMO and ubiquitin code coordinates protein traffic at replication factories.

Post-translational modifications regulate each step of DNA replication to ensure the faithful transmission of genetic information. In this context, we recently showed that deubiquitination of SUMO2/3 and SUMOylated proteins by USP7 helps to create a SUMO-rich and ubiquitin-low environment around replisomes that is necessary to maintain the activity of replication forks and for new origin firing. We propose that a two-flag system mediates the collective concentration of factors at sites of DNA replication, whereby SUMO and Ubiquitinated-SUMO would constitute "stay" or "go" signals respectively for replisome and accessory factors. We here discuss the findings that led to this model, which have implications for the potential use of USP7 inhibitors as anticancer agents.

Inhibition OF USP7 Induces Selective Cancer Cell Death in Chronic Lymphocytic Leukemia

Inhibition OF USP7 Induces Selective Cancer Cell Death in Chronic Lymphocytic Leukemia

Abstract 559: Small molecule T-reg inhibitors for cancer immunotherapy

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.

USP7 inhibitors, downregulating CCDC6, sensitize lung neuroendocrine cancer cells to PARP-inhibitor drugs

ObjectivesCCDC6 gene product is a tumor-suppressor pro-apoptotic protein, substrate of ATM, involved in DNA damage response and repair. Altered levels of CCDC6 expression are dependent on post-translational modifications, being the de-ubiquitinating enzyme USP7 responsible of the fine tuning of the CCDC6 stability. Thus, our aim was to investigate CCDC6 and USP7 expression levels in Lung-Neuroendocrine Tumors (L-NETs) to verify if they correlate and may be exploited as novel predictive therapeutic markers. Materials and methodsTumor tissues from 29 L-NET patients were investigated on tissue microarrays. CCDC6 levels were scored and correlated with immunoreactivity for USP7. Next generation sequencing (NGS) of a homogenous group of Large Cell Neuroendocrine Carcinoma (LCNEC) (N=8) was performed by Ion AmpliSeq NGS platform and the Ion AmpliSeq Cancer Hotspot Panel v2. The inhibition of USP7, using P5091, was assayed in vitro to accelerate CCDC6 turnover in order to sensitize the neuroendocrine cancer cells to PARP-inhibitors, alone or in association with cisplatinum. ResultsThe immunostaining of 29 primary L-NETs showed that the intensity of CCDC6 staining correlated with the levels of USP7 expression (p≤0.05). The NGS analysis of 8 LCNEC revealed mutations in the hot spot regions of the p53 gene (in 6 out of 8). Moreover, gene polymorphisms were identified in the druggable STK11, MET and ALK genes. High intensity of p53 immunostaining was reported in the 6 tissues carrying the TP53 mutations. The inhibition of USP7 by P5091 accelerated the degradation of CCDC6 versus control in cycloheximide treated L-NET cells in vitro and sensitized the cells to PARP-inhibitors alone and in combination with cisplatinum. ConclusionOur data suggest that CCDC6 and USP7 have a predictive value for the clinical usage of USP7 inhibitors in combination with the PARP-inhibitors in L-NET in addition to standard therapy.