USP7 inhibitors suppress tumour neoangiogenesis and promote synergy with immune checkpoint inhibitors by downregulating fibroblast VEGF.

BACKGROUND: Understanding how to modulate the microenvironment of tumors that are resistant to immune checkpoint inhibitors represents a major challenge in oncology.Here we investigate the ability of USP7 inhibitors to reprogram the tumor microenvironment (TME) by inhibiting secretion of vascular endothelial growth factor (VEGF) from fibroblasts. METHODS: To understand the role played by USP7 in the TME, we systematically evaluated the effects of potent, selective USP7 inhibitors on co-cultures comprising components of the TME, using human primary cells. We also evaluated the effects of USP7 inhibition on tumor growth inhibition in syngeneic models when dosed in combination with immune checkpoint inhibitors (ICIs). RESULTS: Abrogation of VEGF secretion from fibroblasts in response to USP7 inhibition resulted in inhibition of tumor neoangiogenesis and increased tumor recruitment of CD8-positive T-lymphocytes, leading to significantly improved sensitivity to immune checkpoint inhibitors. In syngeneic models, treatment with USP7 inhibitors led to striking tumor responses resulting in significantly improved survival. CONCLUSIONS: USP7-mediated reprograming of the TME is not linked to its previously characterized role in modulating MDM2 but does require p53 and UHRF1 in addition to the well-characterized VEGF transcription factor, HIF-1α. This represents a function of USP7 that is unique to fibroblasts, and which is not observed in cancer cells or other components of the TME. Given the potential for USP7 inhibitors to transform "immune desert" tumors into "immune responsive" tumors, this paves the way for a novel therapeutic strategy combining USP7 inhibitors with immune checkpoint inhibitors (ICIs).

Cellular Assays for Dynamic Quantification of Deubiquitinase Activity and Inhibition.

Deubiquitinases (DUBs) are proteolytic enzymes that catalyze the removal of ubiquitin from protein substrates. The critical role of DUBs in regulating protein ubiquitination makes them attractive drug targets in oncology, neurodegenerative disease, and antiviral development. Biochemical assays for quantifying DUB activity have enabled characterization of substrate preferences and discovery of small molecule inhibitors. However, assessing the efficacy of these inhibitors in cellular contexts to support clinical drug development has been limited by a lack of tractable cell-based assays. To address this gap, we developed a two-color flow cytometry-based assay that allows for sensitive quantification of DUB activity and inhibition in living cells. The utility of this system was demonstrated by quantifying the potency of GRL0617 against the viral DUB SARS-CoV-2 PLpro, identifying potential GRL0617 resistance mutations, and performing structure-function analysis of the vOTU domain from the recently emerged Yezo virus. In addition, the system was optimized for cellular DUBs by modifying a GFP-targeting nanobody to recruit USP7 and USP28 to benchmark a panel of reported inhibitors and assess inhibition kinetics. Together, these results demonstrate the utility of these assays for studying DUB biology in a cellular context with potential to aid in inhibitor discovery and development.

YM155 inhibits neuroblastoma growth through degradation of MYCN: A new role as a USP7 inhibitor.

Amplification of the MYCN gene (MNA) is observed in approximately 25 to 35% of neuroblastoma patients, and is a well-recognized marker of tumor aggressiveness and poor outcome. Targeting MYCN is a novel therapy strategy to induce tumor regression. Here, we discovered that a BIRC5/Survivin inhibitor, YM155, specifically inhibits MNA neuroblastoma cell growth in vitro. We found that YM155 promotes MYCN degradation in MNA cells. Further, we found that YM155 inhibits USP7 deubiquitinase activity in vitro, using Ub-aminomethylcoumarin (Ub-AMC) as substrate. Results from in vivo studies further demonstrated that YM155 significantly inhibited the tumor growth in MNA neuroblastoma xenograft model. Our data support a novel mechanism of action of YM155 in inhibition of growth of cancer cells through inducing MYCN degradation by inibition of activity of deubiquitinase like USP7.

Inhibition of USP7 suppresses advanced glycation end-induced cell cycle arrest and senescence of human umbilical vein endothelial cells through ubiquitination of p53.

Diabetes mellitus is a n arising public health concern, and diabetic foot is one of the most common complications of diabetes. Current management for diabetic foot cannot reach optimal remission. In this study, we aim to explore the mechanism underlying the pathogenesis of diabetic foot and provide novel strategies for the treatment of diabetic foot. A total of 10 normal skin tissues and 20 diabetic foot ulcer specimens are collected. Cell proliferation is determined by CCK-8 assay. Cell cycle is determined by flow cytometry, and cell senescence is evaluated by ß-galactosidase staining. Co-immunoprecipitation assay is used to explore the interaction between USP7 and p53. Advanced glycation end products (AGEs) are used to establish diabetic cell model, and streptozotocin (STZ) is used to establish diabetic rat model. Our results showed that USP7 expression is increased in diabetic foot ulcer and in human umbilical vein endothelial cells (HUVECs) after treatment with AGEs. Inhibition of USP7 can reduce cell cycle arrest and cell senescence in HUVECs. Moreover, USP7 can interact with p53 and promote its expression through mediating its deubiquitination. Knockdown of p53 can reverse USP7-mediated cell cycle arrest and cell senescence in HUVECs. In diabetic rats, HBX 41108, the specific inhibitor of USP7, can significantly accelerate wound healing. Our study reveals that the inhibition of USP7 can suppress AGEs-induced cell cycle arrest and cell senescence of HUVECs through promoting p53 ubiquitination. USP7 is a potential target for the treatment of diabetic foot ulcers.

A patent review of ubiquitin-specific protease 7 (USP7) inhibitors (2014-present).

INTRODUCTION: Ubiquitin-specific protease 7 (USP7) plays a critical role in multiple signaling pathways, and many recent studies have proved its association with many diseases. The USP7-murine double minute 2-p53 pathway and the relationship between USP7 and the important immune protein PD-L1 in cancer progression and metastasis have been clarified. Recently, USP7 has emerged as a promising and potent therapeutic target for cancer and has attracted both academic and industrial attention. AREAS COVERED: This review focuses on the structure, activity, and applications of USP7 inhibitors in cancer therapy. It also focuses on patents reported since 2014. EXPERT OPINION: Recently, USP7 has attracted considerable attention owing to its physiological and pathophysiological roles in cancer progression, and few studies have focused on the development of USP7 inhibitors. Compared with micromolar first-generation USP7 inhibitors, second-generation USP7 inhibitors exhibit higher potency (at nanomolar level for both USP7 and cell inhibitory activities), higher selectivity, and better pharmacokinetic properties, and they largely broaden the range of candidates for further clinical tests. However, there is still a need for a more precise description of compounds with receptors, the structural diversity of these compounds, and screening methods.

Virtual Screening Inhibitors of Ubiquitin-specific Protease 7 Combining Pharmacophore Modeling and Molecular Docking.

Ubiquitin-specific protease 7 (USP7) is one of the most extensively studied deubiquitinases. USP7 exhibits a high expression signature in various malignant tumors, suggesting that it is a marker of tumor prognosis and a potential drug target for anti-tumor therapy. In this study, virtual screening based on pharmacophore model and biological evaluation have been applied for the discovery of novel USP7 inhibitors targeting the catalytic active site. The TS-4 was screened from 215,480 small molecules and was found to have USP7 inhibitory activity. Preliminary in vitro studies disclosed its antiproliferative activity on human colon cancer cell lines (HCT-116 and RKO), compared with normal colon cell line (CCD841CoN). Molecular dynamics (MD) simulation revealed the combine mechanism between USP7 with the TS-4. The TS-4 formed stable interactions with Asp295, Phe409 and Tyr514, which were critical to enhance its biological activity. This compound will serve as a promising hit compound for facilitating the further design of novel USP7 inhibitors.

Characterizing (un)binding mechanism of USP7 inhibitors to unravel the cause of enhanced binding potencies at allosteric checkpoint.

The ability to predict the intricate mechanistic behavior of ligands and associated structural determinants during protein-ligand (un)binding is of great practical importance in drug discovery. Ubiquitin specific protease-7 (USP7) is a newly emerging attractive cancer therapeutic target with bound allosteric inhibitors. However, none of the inhibitors have reached clinical trials, allowing opportunities to examine every aspect of allosteric modulation. The crystallographic insights reveal that these inhibitors have common properties such as chemical scaffolds, binding site and interaction fingerprinting. However, they still possess a broader range of binding potencies, ranging from 22 nM to 1,300 nM. Hence, it becomes more critical to decipher the structural determinants guiding the enhanced binding potency of the inhibitors. In this regard, we elucidated the atomic-level insights from both interacting partners, that is, protein-ligand perspective, and established the structure-activity link between USP7 inhibitors by using classical and advanced molecular dynamics simulations combined with linear interaction energy and molecular mechanics-Poisson Boltzmann surface area. We revealed the inhibitor potency differences by examining the contributions of chemical moieties and USP7 residues, the involvement of water-mediated interactions, and the thermodynamic landscape alterations. Additionally, the dissociation profiles aided in the establishment of a correlation between experimental potencies and structural determinants. Our study demonstrates the critical role of blocking loop 1 in allosteric inhibition and enhanced binding affinity. Comprehensively, our findings provide a constructive expansion of experimental outcomes and show the basis for varying binding potency using in-silico approaches. We expect this atomistic approach to be useful for effective drug design.

Pharmacological inhibition of USP7 suppresses growth and metastasis of melanoma cells in vitro and in vivo.

Melanoma is a highly aggressive type of skin cancer. The development of diverse resistance mechanisms and severe adverse effects significantly limit the efficiency of current therapeutic approaches. Identification of the new therapeutic targets involved in the pathogenesis will benefit the development of novel therapeutic strategies. The deubiquitinase ubiquitin-specific protease-7, a potential target for cancer treatment, is deregulated in types of cancer, but its role in melanoma is still unclear. We investigated the role and the inhibitor P22077 of ubiquitin-specific protease-7 in melanoma treatment. We found that ubiquitin-specific protease-7 was overexpressed and correlated with poor prognosis in melanoma. Further, pharmacological inhibition of ubiquitin-specific protease-7 by P22077 can effectively inhibit proliferation, and induce cell cycle arrest and apoptosis via ROS accumulation-induced DNA damage in melanoma cells. Inhibition of ubiquitin-specific protease-7 by P22077 also inhibits melanoma tumour growth in vivo. Moreover, inhibition of ubiquitin-specific protease-7 prevented migration and invasion of melanoma cells in vitro and in vivo by decreasing the Wnt/ß-catenin signalling pathway. Taken together, our study revealed that ubiquitin-specific protease-7 acted as an oncogene involved in melanoma cell proliferation and metastasis. Therefore, ubiquitin-specific protease-7 may serve as potential candidates for the treatment of melanoma.

Recent advances on the intervention sites targeting USP7-MDM2-p53 in cancer therapy.

The ubiquitin-specific protease 7 (USP7)-murine double minute 2 (MDM2)-p53 network plays an important role in the regulation of p53, a tumor suppressor which plays critical roles in regulating cell growth, proliferation, cell cycle progression, apoptosis and immune response. The overexpression of USP7 and MDM2 in human cancers contributes to cancer initiation and progression, and their inhibition reactivates p53 signalings and causes cell cycle arrest and apoptosis. Herein, the current state of pharmacological characterization, potential applications in cancer treatment and mechanism of action of small molecules used to target and inhibit MDM2 and USP7 proteins are highlighted, along with the outcomes in clinical and preclinical settings. Moreover, challenges and advantages of these strategies, as well as perspectives in USP7-MDM2-p53 field are analyzed in detail. The investigation and application of MDM2 and USP7 inhibitors will deepen our understanding of the function of USP7-MDM2-p53 network, and feed in the development of effective and safe cancer therapies where USP7-MDM2-p53 network is implicated.

Prognostic role of USP7 expression in cancer patients: A systematic review and meta-analysis.

BACKGROUND: Numerous studies have examined the prognostic value of ubiquitin-specific protease 7 (USP7) in cancer, but the results remain controversial. Differences in assessment assays (mRNA/protein) used could be a potential confounding factor. Thus, we extracted studies that measured the protein expression and performed a meta-analysis to assess the prognostic role of USP7 expression in cancer and to identify clinicopathological features associated with USP7 expression. METHODS: PubMed, Scopus, Web of Science Core Collection, Wiley Online Library, and Google Scholar were searched from inception to July 2020. Pooled hazard ratios were calculated to evaluate the association between USP7 expression and overall survival (OS). In addition, pooled odds ratios were calculated to identify clinicopathological features associated with USP7 expression. RESULTS: Eight studies in China were included in our meta-analysis, which had a total of 1192 patients and assessed five types of cancer. The pooled results revealed that a high expression of USP7 was associated with poor OS, especially in epithelial ovarian cancer (EOC). Moreover, USP7 expression was increased in patients with tumour-node-metastasis (TNM) stages III-IV, poor pathological grade, and positive lymph node metastasis. For patients with EOC, a high USP7 expression positively correlated with lymph node metastasis. CONCLUSION: A high USP7 expression may promote cancer progression and predict unfavourable prognosis of cancer patients, especially those with EOC. Our findings suggest that USP7 inhibitors might be promising therapeutics for cancer patients with such characteristics.

Morin Acts as a USP7 Inhibitor to Hold Back the Migration of Rheumatoid Arthritis Fibroblast-Like Synoviocytes in a "Prickle1-mTORC2" Dependent Manner.

INTRODUCTION: The aim of this study is to investigate the effect and detailed mechanisms of morin, an anti-arthritis compound widely distributed in foods of plant origin, on the pathological migration of fibroblast-like synoviocytes (FLS). METHODS AND RESULTS: The migration of FLS collected from arthritis rats and MH7A cells is induced by platelet-derived growth factor, and an arthritis model in rats is established by Freund's complete adjuvant. The results show that morin remarkably restrains FLS migration but slightly affects FLS apoptosis and proliferation. Moreover, in the progression of FLS migration, focal adhesion (FA) turnover is inhibited by morin via lowering the activation of Paxillin and focal adhesion kinase (FAK) and internalization of integrin ß1. Morin disrupts the formation of mTOR complex 2 (mTORC2) and the activation of AKT (S473) and PKCα (S657), and MHY1485 reverses morin-limited FLS migration. Of note, the protein stability of Prickle1, a binding factor of Rictor, is reduced by morin, and MG132 but not Baf A1 shows a repressive effect. Finally, the target protein is identified as ubiquitin-specific protease 7 (USP7) but not USP9X. USP7 overexpressing plasmid weakens morin-affected protein and ubiquitination of Prickle1, and mechanisms are confirmed in vivo by using an overexpressing plasmid and inhibitor. CONCLUSION: Morin restricts FLS migration and arthritis by intervening in "USP7-Prickle1-mTORC2" signaling and FA turnover.

USP7 limits CDK1 activity throughout the cell cycle.

Chemical inhibitors of the deubiquitinase USP7 are currently being developed as anticancer agents based on their capacity to stabilize P53. Regardless of this activity, USP7 inhibitors also generate DNA damage in a p53-independent manner. However, the mechanism of this genotoxicity and its contribution to the anticancer effects of USP7 inhibitors are still under debate. Here we show that, surprisingly, even if USP7 inhibitors stop DNA replication, they also induce a widespread activation of CDK1 throughout the cell cycle, which leads to DNA damage and is toxic for mammalian cells. In addition, USP7 interacts with the phosphatase PP2A and supports its active localization in the cytoplasm. Accordingly, inhibition of USP7 or PP2A triggers very similar changes of the phosphoproteome, including a widespread increase in the phosphorylation of CDK1 targets. Importantly, the toxicity of USP7 inhibitors is alleviated by lowering CDK1 activity or by chemical activation of PP2A. Our work reveals that USP7 limits CDK1 activity at all cell cycle stages, providing a novel mechanism that explains the toxicity of USP7 inhibitors through untimely activation of CDK1.

Design, synthesis, biological evaluation and structure-activity relationship study of quinazolin-4(3H)-one derivatives as novel USP7 inhibitors.

Recent research has indicated that the abnormal expression of the deubiquitinase USP7 induces tumorigenesis via multiple cell pathways, and in particular, the p53-MDM2-USP7 pathway is well understood. USP7 is emerging as a promising target for cancer therapy. However, there are limited reports on USP7 inhibitors. Here we report design, synthesis and biological evaluation of novel quinazolin-4(3H)-one derivatives as potent USP7 inhibitors. Our results indicated that the compounds C9 and C19 exhibited the greatest potency against the USP7 catalytic domain, with IC50 values of 4.86 µM and 1.537 µM, respectively. Ub-AMC assays further confirmed IC50 values of 5.048 µM for C9 and 0.595 µM for C19. MTT assays indicated that gastric cancer MGC-803 cells were more sensitive to these compounds than BGC-823 cells. Flow cytometry analysis revealed that C9 restricted cancer cell growth at the G0/G1 and S phases and inhibited the proliferation and clone formation of MGC-803 cells. Further biochemical experiments indicated that C9 decreased the MDM2 protein level and increased the levels of the tumour suppressors p53 and p21 in a dose-dependent manner. Docking studies predicted that solvent exposure of the side chains of C9 and C19 would uniquely form hydrogen bonds with Met407 of USP7. Additionally, C9 exhibited a remarkable anticancer effect in a zebrafish gastric cancer MGC-803 cell model. Our results demonstrated that quinazolin-4(3H)-one derivatives were suitable as leads for the development of novel USP7 inhibitors and especially for anti-gastric cancer drugs.

Proteomics of broad deubiquitylase inhibition unmasks redundant enzyme function to reveal substrates and assess enzyme specificity.

Deubiquitylating enzymes (DUBs) counteract ubiquitylation to control stability or activity of substrates. Identification of DUB substrates is challenging because multiple DUBs can act on the same substrate, thwarting genetic approaches. Here, we circumvent redundancy by chemically inhibiting multiple DUBs simultaneously in Xenopus egg extract. We used quantitative mass spectrometry to identify proteins whose ubiquitylation or stability is altered by broad DUB inhibition, and confirmed their DUB-dependent regulation with human orthologs, demonstrating evolutionary conservation. We next extended this method to profile DUB specificity. By adding recombinant DUBs to extract where DUB activity was broadly inhibited, but ubiquitylation and degradation were active at physiological rates, we profiled the ability of DUBs to rescue degradation of these substrates. We found that USP7 has a unique ability to broadly antagonize degradation. Together, we present an approach to identify DUB substrates and characterize DUB specificity that overcomes challenges posed by DUB redundancy.

Proteomics-Based Identification of DUB Substrates Using Selective Inhibitors.

Deubiquitinating enzymes (DUBs) catalyze the removal of ubiquitin, thereby reversing the activity of E3 ubiquitin ligases and are central to the control of protein abundance and function. Despite the growing interest in DUBs as therapeutic targets, cellular functions for DUBs remain largely unknown and technical challenges often preclude the identification of DUB substrates in a comprehensive manner. Here, we demonstrate that treatment with potent DUB inhibitors coupled to mass spectrometry-based proteomics can identify DUB substrates at a proteome-wide scale. We applied this approach to USP7, a DUB widely investigated as a therapeutic target and identified many known substrates and additional targets. We demonstrate that USP7 substrates are enriched for DNA repair enzymes and E3 ubiquitin ligases. This work provides not only a comprehensive annotation of USP7 substrates, but a general protocol widely applicable to other DUBs, which is critical for translational development of DUB targeted agents.

An Integrated in silico Approach and in†vitro Study for the Discovery of Small-Molecule USP7 Inhibitors as Potential Cancer Therapies.

The ubiquitin-specific protease 7 (USP7) is a highly promising well-validated target for a variety of malignancies. USP7 is critical in regulating the tumor suppressor p53 along with numerous epigenetic modifiers and transcription factors. Previous studies showed that USP7 inhibitors led to increased levels of p53 and anti-proliferative effects in hematological and solid tumor cell lines. Thus, this study aimed to identify potent and safe USP7 hit inhibitors as potential anti-cancer therapeutics via an integrated computational approach that combines pharmacophore modeling, molecular docking, molecular dynamics (MD) simulations and post-MD free energy calculations. In this study, the crystal structure of USP7 has been extensively investigated using a combination of three different chemical pharmacophore modeling approaches. We then screened ∼220.000 drug-like small molecule library and the hit ligands predicted to be nontoxic were evaluated further. The identified hits from each pharmacophore modeling study were further examined by 1-ns short MD simulations and MM/GBSA free energy analysis. In total, we ran 1 ns MD simulations for 1137 selected on small compounds. Based on their average MM/GBSA scores, 18 ligands were selected for 50 ns MD simulations along with one highly potent USP7 inhibitor used as a positive control. The in vitro enzymatic inhibition assay testing of our lead 18 molecules confirmed that 7 of these molecules were successful in USP7 inhibition. Screening results showed that within the used screening approaches, the most successful one was structure-based pharmacophore modeling with the success rate of 75 %. The identification of potent and safe USP7 small molecules as potential inhibitors is a step closer to finding appropriate effective therapies for cancer. Our lead ligands can be used as a scaffold for further structural optimization and development, enabling further research in this promising field.

Inhibition of ubiquitin-specific protease 7 sensitizes acute myeloid leukemia to chemotherapy.

Resistance of acute myeloid leukemia (AML) to therapeutic agents is frequent. Consequently, the mechanisms leading to this resistance must be understood and addressed. In this paper, we demonstrate that inhibition of deubiquitinylase USP7 significantly reduces cell proliferation in vitro and in vivo, blocks DNA replication progression and increases cell death in AML. Transcriptomic dataset analyses reveal that a USP7 gene signature is highly enriched in cells from AML patients at relapse, as well as in residual blasts from patient-derived xenograft (PDX) models treated with clinically relevant doses of cytarabine, which indicates a relationship between USP7 expression and resistance to therapy. Accordingly, single-cell analysis of AML patient samples at relapse versus at diagnosis showed that a gene signature of the pre-existing subpopulation responsible for relapse is enriched in transcriptomes of patients with a high USP7 level. Furthermore, we found that USP7 interacts and modulates CHK1 protein levels and functions in AML. Finally, we demonstrated that USP7 inhibition acts in synergy with cytarabine to kill AML cell lines and primary cells of patients with high USP7 levels. Altogether, these data demonstrate that USP7 is both a marker of resistance to chemotherapy and a potential therapeutic target in overcoming resistance to treatment.

The emerging nature of Ubiquitin-specific protease 7 (USP7): a new target in cancer therapy.

Human ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme that removes the ubiquitin (Ub) protein and spares substrates from degradation. Given its regulation of proteins involved in several cellular processes, abnormal expression and activity of USP7 are associated with several types of disease, including cancer. In this review, we summarize the developments in our understanding of USP7 over the past 5 years, focusing on its role in related cancers. Furthermore, we discuss clinical studies of USP7, including in vivo and pharmacological studies, as well as the development of USP7 inhibitors. A comprehensive understanding of USP7 will expand our knowledge of the structure and function of USP7-mediated signaling and shed light on drug discovery for different diseases in which USP7 is implicated.

Leveraging on Active Site Similarities; Identification of Potential Inhibitors of Zinc-Finger and UFSP domain Protein (ZUFSP).

BACKGROUND: ZUFSP (Zinc-finger and UFSP domain protein) is a novel representative member of the recently characterized seventh class of deubiquitinating enzymes (DUBs). Due to the roles DUBs play in genetic instability, they have become a major drug target in cancer and neurodegenerative diseases. ZUFSP, being a DUB enzyme has also been implicated in genetic stability. However, no lead compound has been developed to target ZUFSP. OBJECTIVE/METHODS: Therefore, in this study, we used a combined drug repurposing, virtual screening and per-Residue Energy Decomposition (PRED) to identify ZUFSP inhibitors with therapeutic potential. 3-bromo-6-{[4-hydroxy-1-3(3-phenylbutanoyl)piperidin-4-yl]methyl}-4H,5H,6H,7H-thieno[2,3- C]pyridine-7-one (BHPTP) which is an inhibitor of USP7 was repurposed to target ZUFSP. The rationale behind this is based on the similarity of the active between USP7 and ZUFSP. RESULTS: PRED of the binding between BHPTP and ZUFSP revealed Cys223, Arg408, Met410, Asn460, and Tyr465 as the crucial residues responsible for this interaction. The pharmacophoric moieties of BHPTP responsible for this binding along with other physiochemical properties were used as a filter to retrieve potential ligands. 799 compounds were retrieved, ZINC083241427, ZINC063648749, and ZINC063648753 were selected due to the binding energy they exhibited. Cheminformatics analysis revealed that the compounds possess high membrane permeability, however, BHPTP had a low membrane permeability. Furthermore, the compounds are drug like, having obeyed Lipinski's rule of five. CONCLUSION: Taken together, findings from this study put ZINC083241427, ZINC063648749, and ZINC063648753 as potential ZUFSP inhibitor, however, more experimental validation is required to unravel the mechanism of actions of these compounds.

Combination of Inhibitors of USP7 and PLK1 has a Strong Synergism against Paclitaxel Resistance.

USP7 is a promising target for the development of cancer treatments because of its high expression and the critical functions of its substrates in carcinogenesis of several different carcinomas. Here, we demonstrated the effectiveness of targeting USP7 in advanced malignant cells showing high levels of USP7, especially in taxane-resistant cancer. USP7 knockdown effectively induced cell death in several cancer cells of lung, prostate, and cervix. Depletion of USP7 induced multiple spindle pole formation in mitosis, and, consequently, resulted in mitotic catastrophe. When USP7 was blocked in the paclitaxel-resistant lung cancer NCI-H460TXR cells, which has resistance to mitotic catastrophe, NCI-H460TXR cells underwent apoptosis effectively. Furthermore, combination treatment with the mitotic kinase PLK1 inhibitor volasertib and the USP7 inhibitor P22077 showed a strong synergism through down-regulation of MDR1/ABCB1 in paclitaxel-resistant lung cancer. Therefore, we suggest USP7 is a promising target for cancer therapy, and combination therapy with inhibitors of PLK1 and USP7 may be valuable for treating paclitaxel-resistant cancers, because of their strong synergism.