USP7 Inhibitors Research Articles

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 β-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.

IKZF1 selectively enhances homologous recombination repair by interacting with CtIP and USP7 in multiple myeloma.

Rationale: In multiple myeloma (MM), the activities of non-homologous end joining (NHEJ) and homologous recombination repair (HR) are increased compared with healthy controls. Whether and how IKZF1 as an enhancer of MM participates in the DNA repair pathway of tumor cells remains elusive.Methods: We used an endonuclease AsiSI-based system and quantitative chromatin immunoprecipitation assay (qChIP) analysis to test whether IKZF1 is involved in DNA repair. Immunopurification and mass spectrometric (MS) analysis were performed in MM1.S cells to elucidate the molecular mechanism that IKZF1 promotes DNA damage repair. The combination effect of lenalidomide or USP7 inhibitor with PARP inhibitor on cell proliferation was evaluated using MM cells in vitro and in vivo.Results: We demonstrate that IKZF1 specifically promotes homologous recombination DNA damage repair in MM cells, which is regulated by its interaction with CtIP and USP7. In this process, USP7 could regulate the stability of IKZF1 through its deubiquitinating activity. The N-terminal zinc finger domains of IKZF1 and the ubiquitin-like domain of USP7 are necessary for their interaction. Furthermore, targeted inhibition IKZF1 or USP7 could sensitize MM cells to PARP inhibitor treatment in vitro and in vivo.Conclusions: Our findings identify USP7 as a deubiquitinating enzyme for IKZF1 and uncover a new function of IKZF1 in DNA damage repair. In translational perspective, the combination inhibition of IKZF1 or USP7 with PARP inhibitor deserves further evaluation in clinical trials for the treatment of MM.

Deubiquitination of FBP1 by USP7 blocks FBP1-DNMT1 interaction and decreases the sensitivity of pancreatic cancer cells to PARP inhibitors.

Poly[ADP‐ribose] polymerase (PARP) inhibitors can block DNA single‐strand damage repair and subsequently increase double‐stranded breaks (DSBs) by reducing the activity of the PARP1 protease and by preventing the PARP1 protein from dissociating from chromatin. Tumors with the BRCA mutation are particularly sensitive to PARP inhibitors. So far, PARP inhibitors (Olaparib) have been used to treat pancreatic cancer patients with BRCA mutation. However, these patients are prone to PARP inhibitor resistance. Our previous studies suggest that fructose‐1,6‐bisphosphatase 1 (FBP1) is responsible for the sensitivity to various anticancer agents, such as gemcitabine or mitogen‐activated protein kinase kinase (MEK) inhibitors. In this study, we demonstrate that FBP1 regulates the sensitivity to PARP inhibitors in pancreatic cancer. Then, we showed that nuclear FBP1 is responsible for this process by interacting with DNA (cytosine‐5)‐methyltransferase 1 (DNMT1) and trapping PARP1 in chromatin. Moreover, we revealed that ubiquitin carboxyl‐terminal hydrolase 7 (USP7) binds to and induces the deubiquitination of FBP1, which prevented FBP1 from translocating to the nucleus. Finally, we demonstrated that USP7 inhibitors enhanced the antitumor effect of PARP inhibitors in an FBP1‐dependent manner. Collectively, our results identify a novel USP7–FBP1–DNMT1 signaling axis in pancreatic cancer, which might indicate that USP7 inhibitors and PARP inhibitors might have more powerful antitumor effects than PARP inhibitors alone in pancreatic cancer patients.

USP7 and VCPFAF1 define the SUMO/Ubiquitin landscape at the DNA replication fork.

SummaryThe AAA+ ATPase VCP regulates the extraction of SUMO and ubiquitin-modified DNA replication factors from chromatin. We have previously described that active DNA synthesis is associated with a SUMO-high/ubiquitin-low environment governed by the deubiquitylase USP7. Here, we unveil a functional cooperation between USP7 and VCP in DNA replication, which is conserved from Caenorhabditis elegans to mammals. The role of VCP in chromatin is defined by its cofactor FAF1, which facilitates the extraction of SUMOylated and ubiquitylated proteins that accumulate after the block of DNA replication in the absence of USP7. The inactivation of USP7 and FAF1 is synthetically lethal both in C. elegans and mammalian cells. In addition, USP7 and VCP inhibitors display synergistic toxicity supporting a functional link between deubiquitylation and extraction of chromatin-bound proteins. Our results suggest that USP7 and VCPFAF1 facilitate DNA replication by controlling the balance of SUMO/Ubiquitin-modified DNA replication factors on chromatin.

Time-resolved in vivo ubiquitinome profiling by DIA-MS reveals USP7 targets on a proteome-wide scale

Mass spectrometry (MS)-based ubiquitinomics provides system-level understanding of ubiquitin signaling. Here we present a scalable workflow for deep and precise in vivo ubiquitinome profiling, coupling an improved sample preparation protocol with data-independent acquisition (DIA)-MS and neural network-based data processing specifically optimized for ubiquitinomics. Compared to data-dependent acquisition (DDA), our method more than triples identification numbers to 70,000 ubiquitinated peptides in single MS runs, while significantly improving robustness and quantification precision. Upon inhibition of the oncology target USP7, we simultaneously record ubiquitination and consequent changes in abundance of more than 8,000 proteins at high temporal resolution. While ubiquitination of hundreds of proteins increases within minutes of USP7 inhibition, we find that only a small fraction of those are ever degraded, thereby dissecting the scope of USP7 action. Our method enables rapid mode-of-action profiling of candidate drugs targeting DUBs or ubiquitin ligases at high precision and throughput.

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/β‐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.

Crotonylation Sensitizes IAPI-Induced Disruption of Latent HIV by Modulating ncNF-κB Signaling at the Step of p100 Cleavage Into p52

The eradication of infection with human immunodeficiency virus type 1 (HIV) is difficult to achieve, largely due to stable viral reservoirs. While many strategies have been tested, a clinical meaningful success has yet to be achieved, indicating that our understanding of HIV latency is incomplete and strategies to disrupt latency are not yet ideal. In this report, we show that crotonylation, a recently discovered epigenetic modification involved in HIV transcription from latency, also acts to enhance AZD5582-induced noncanonical NF-κB (ncNF-κB) signaling, further augmenting HIV latency reversal via this pathway. This was tested in Jurkat and U1 cell line models of latency, HIV latently infected primary CD4+ T cells and resting CD4+ T cells isolated from ART-suppressed people living with HIV ex vivo . Surprisingly, crotonylation upregulates the levels of the active p52 subunit of the NF-κB transcription factor induced by AZD5582 in Jurkat models of HIV latency. Quantitative mass spectrometry screening and biochemical analysis suggest that the ubiquitination E3 ligase TRIM27 may be involved in p100 cleavage into p52 activated by AZD5582, part of the heterodimer to activate NF-κB for the transcription of HIV. When TRIM27 is depleted, transcription of HIV and AZD5582-induced latency reversal are reduced. Interestingly, both p100 and p52 levels are reduced by TRIM27 siRNA knockdown or by pharmacologic inhibition of TRIM27 deubiquitinase partner protein USP7 during the induction of ncNF-κB signaling. These observations reveal the complexity of HIV transcriptional machinery, particularly of NF-κB factors known to be essential for HIV latency disruption.

TRIP13 modulates protein deubiquitination and accelerates tumor development and progression of B cell malignancies.

Multiple myeloma (MM), a terminally differentiated B cell malignancy, remains difficult to cure. Understanding the molecular mechanisms underlying the progression of MM may identify therapeutic targets and lead to a fundamental shift in treatment of the disease. Deubiquitination, like ubiquitination, is a highly regulated process, implicated in almost every cellular process. Multiple deubiquitinating enzymes (DUBs) have been identified, but their regulation is poorly defined. Here, we determined that TRIP13 increases cellular deubiquitination. Overexpression of TRIP13 in mice and cultured cells resulted in excess cellular deubiquitination by enhancing the association of the DUB USP7 with its substrates. We show that TRIP13 is an oncogenic protein because it accelerates B cell tumor development in transgenic mice. TRIP13-induced resistance to proteasome inhibition can be overcome by a USP7 inhibitor in vitro and in vivo. These findings suggest that TRIP13 expression plays a critical role in B cell lymphoma and MM by regulating deubiquitination of critical oncogenic (NEK2) and tumor suppressor (PTEN, p53) proteins. High TRIP13 identifies a high-risk patient group amenable to adjuvant anti-USP7 therapy.

The Interaction of NEK2 with USP7 Causes Resistance to Proteasome Inhibitor in Multiple Myeloma

Background: Drug resistance is the major reason for treatment failure in multiple myeloma (MM). Our previous work has demonstrated that high expression of chromosomal instability (CIN) genes results in increased cell survival and drug resistance and is associated with poor outcome in MM and other cancers. NEK2 was the most significant CIN gene to induce drug resistance and poor outcome in MM. Though we have demonstrated that NEK2 induces drug resistance through activation of efflux drug pumps, the detail mechanisms remain unknown.Materials and Methods: Multiple MM cell lines, xenograft MM NOD-Rag1nullmice with both subcutaneous and by tail vein injection, and primary MM samples were included in this study to test NEK2 inhibitors and gene expression profiles (GEP). The tandem affinity purification - mass spectrometry (TAP-MS) and co-immunoprecipitation (Co-IP) were applied for the identification of NEK2 interacting partners. The dual-luciferase reporter assay and GEPs were performed to determine whether the interaction of NEK2 with USP7 activates the canonical NF-kB signaling pathway. Chromatin immunoprecipitation (ChIP)-qPCR and western blots were carried out to determine the direct target genes of the axis NEK2/USP7/NF-kB.Results: We demonstrate that NEK2 is stabilized by the de-ubiquitinating enzyme USP7, leading to accumulation of NEK2 within the cancer cells. Accumulation of NEK2 activates the canonical NF-kB through the AKT/IKK/IKB-a/p65 axis and, using this pathway as framework, we uncovered a novel bona fide NF-kB target, heparanase, a protein that is known to promote bone destruction in MM patients. A NF-kB or USP7 inhibitor could overcome NEK2-induced bortezomib resistance. The NEK2 inhibitor, INH1, showed great therapeutic effects on MM in vitro and in vivo . To corroborate our findings, we subcutaneously injected NEK2-OE ARP1 MM cells into NOD.Cg-Rag1 mice and targeted NEK2 with the small molecule drugs INH1 or P5091 (USP7 inhibitor) alone or in combination with bortezomib. As expected bortezomib had very little effect in reducing tumor burden since NEK2-OE cells are highly resistant to this drug. Inhibition of NEK2 with P5091 or INH1 alone had a strong anti-tumor effect and a synergistic effect was observed when combined with bortezomib.Conclusion: Our data present a novel mechanism for the interaction of NEK2 with USP7 driving drug resistance in MM. We conclude that (1) USP7 in MM cells leads to the de-ubiquitination and escape of proteasomal degradation of NEK2, leading to its accumulation in cancer cells. (2) Accumulated NEK2 binds and phosphorylates PP1a, suppressing its phosphatase activity thus promoting AKT overactivation. (3) AKT then phosphorylates the IKKa/b complex, triggering the canonical NF-kB cascade which phosphorylates and targets IKBa for proteasomal degradation, releasing the sequestered p65 to be phosphorylated at S536 and translocation to the nucleus where it drives bortezomib resistance by activating its target genes. Our data also show that INH1 and P5091 can deplete NEK2 protein in vitro and in vivo and suppress NEK2 downstream targets resulting in overcoming bortezomib resistance. Our findings should facilitate novel targeted approaches to the therapy and prevention of myeloma progression and relapse. DisclosuresNo relevant conflicts of interest to declare.

Deubiquitinase USP7 Regulates Erythroid Development Via Deubiquinating GATA1

Erythropoiesis is the process which produces mature red blood cells from haematopoietic stem cell. Ubiquitination/deubiquititation plays important roles in many cellular processes. Previous studies have documented the roles of ubiquitination and several ubiquitinases in erythropoiesis. In contrast, the roles of deubiquitiation/deubiquitinases in erythropoiesis remain unexplored. In this study, we found that the expression of deubiquitinase USP7 was significantly upregulated during erythropoiesis. We then employed shRNA mediated knockdown approach to explore the role USP7, we found that USP7 knockdown significantly delayed terminal erythroid differentiation, inhibited the hemoglobin expression and cell proliferation, and induced apoptosis. To explore the underlying mechanisms for the observed phenotypic changes, we compared the expression of several key erythroid transcription factors including GATA1, KLF1, LSD1, SOX1 and IKAROS between control and USP7-knockdown cells by western blot analysis. We found that only the protein level of GATA1 but not other transcription factors was significantly decreased following USP7 knockdown. Moreover, GATA1 mRNA level was not affected by USP7 knockdown. The effect of USP7 on the stability of GATA1 protein was further demonstrated by the findings that inhibition of USP7 by USP7 inhibitors also resulted in decreased GATA1 protein levels and that knockdown/inhibition of USP7 shorten the GATA1 half-life and increased the ubiquitination of GATA1. Furthermore, we performed Co-IP, pull down and in vitro ubiquitization experiment which documented that USP7 directly binds to and deubiquinates GATA1. Our findings demonstrate that GATA1 is the substrate of USP7 and that USP7 stabilizes GATA1 by deubiquinating GATA1. To examine whether the impaired erythropoiesis following USP7 knockdown is due to decreased GATA1, we ectopically expressed GATA1 in USP7 knockdown cells and showed that the delayed differentiation was rescued by GATA1 overexpression. To demonstrate the role of USP7 in vivo, we established a stress erythropoiesis mouse model by phlebotomy and then treated the mice by intraperitoneal injection of USP7 inhibitor. We found USP7 inhibitor delayed the recovery of anemia. Collectively, our findings not only identified previously unknown roles of USP7 in erythropoiesis but also identified a novel regulatory mechanism for GATA1 expression.AcknowledgmentsThis work was supported in part by grants 81270576, 81470362 and 81530005 from Natural Science Foundation of China, by NIH grants DK100810 and DK26263, and by postgraduate innovation project of Central South University of China 2016zzts165.The authors declare no conflict of interest. DisclosuresNo relevant conflicts of interest to declare.

USP7 promotes hepatoblastoma progression through activation of PI3K/AKT signaling pathway.

Hepatoblastoma (HB) is an embryonic solid tumor and the most common primary malignant liver tumor in children. HB usually occurs in infants and children. Although treatment diversity is increasing, some patients still have very poor prognosis. Many studies have investigated USP7 inhibitors for tumors. Using database information, we found that USP7 is highly expressed in HB. Lentivirus-mediated USP7 knockdown and overexpression was performed in HB cell lines HepG2 and Huh6. CCK8 and transwell assays were used to determine cell viability and metastasis. Flow cytometry was used to study cell cycle and apoptosis. Levels of proteins were detected using western blots. Downregulation of USP7 resulted in significant decrease in cell proliferation, clonal formation, and cell migration and invasion. With overexpression of USP7, cellular malignant behavior increased. Cell cycle assays showed that USP7 knockdown inhibited G1 to S phase transition in the cell cycle. Upregulation of USP7 promoted the transition. Animal experiments showed USP7 facilitated tumor growth in vivo. Western blots indicated that USP7 may affect HB tumorigenesis through the PI3K/AKT signaling pathway. Furthermore, USP7 inhibitor P5091 inhibited HB development and PI3K/AKT pathway. USP7 upregulation contributed to HB genesis and development through the PI3K/AKT signaling pathway. USP7 could be a potential target for future HB treatment.

The Role of PARP Inhibitors in the Treatment of Prostate Cancer: Recent Advances in Clinical Trials.

Poly (adenosine diphosphate-ribose) polymerase inhibitors (PARPis) belong to a class of targeted drugs developed for the treatment of homologous recombination repair (HRR)-defective tumors. Preclinical and limited clinical data suggest that PARP inhibition is effective against prostate cancer (PC) in patients with HRR-deficient tumors and that PARPis can improve the mortality rate of PC in patients with BRCA1/2 mutations through a synthetic lethality. Olaparib has been approved by the FDA for advanced ovarian and breast cancer with BRCA mutations, and as a maintenance therapy for ovarian cancer after platinum chemotherapy. PARPis are also a new and emerging clinical treatment for metastatic castration-resistant prostate cancer (mCRPC). Although PARPis have shown great efficacy, their widespread use is restricted by various factors, including drug resistance and the limited population who benefit from treatment. It is necessary to study the combination of PARPis and other therapeutic agents such as anti-hormone drugs, USP7 inhibitors, BET inhibitors, and immunotherapy. This article reviews the mechanism of PARP inhibition in the treatment of PC, the progress of clinical research, the mechanisms of drug resistance, and the strategies of combination treatments.

Restraining CDK1-cyclin B activation: PP2A on the cUSP(7).

USP7 inhibitors are gaining momentum as a therapeutic strategy to stabilize p53 through their ability to induce MDM2 degradation. However, these inhibitors come with an unexpected p53-independent toxicity, via an unknown mechanism. In this issue of The EMBO Journal, Galarreta et al report how inhibition of USP7 leads to re-distribution of PP2A from cytoplasm to nucleus and an increase of deleterious CDK1-dependent phosphorylation throughout the cell cycle, revealing a new regulatory mechanism for the progression of S-phase cells toward mitosis to maintain genomic integrity.

The USP7-TRIM27 axis mediates non-canonical PRC1.1 function and is a druggable target in leukemia

SummaryIn an attempt to unravel functionality of the non-canonical PRC1.1 Polycomb complex in human leukemogenesis, we show that USP7 and TRIM27 are integral components of PRC1.1. USP7 interactome analyses show that PRC1.1 is the predominant Polycomb complex co-precipitating with USP7. USP7 inhibition results in PRC1.1 disassembly and loss of chromatin binding, coinciding with reduced H2AK119ub and H3K27ac levels and diminished gene transcription of active PRC1.1-controlled loci, whereas H2AK119ub marks are also lost at PRC1 loci. TRIM27 and USP7 are reciprocally required for incorporation into PRC1.1, and TRIM27 knockdown partially rescues USP7 inhibitor sensitivity. USP7 inhibitors effectively impair proliferation in AML cells in vitro, also independent of the USP7-MDM2-TP53 axis, and MLL-AF9-induced leukemia is delayed in vivo in human leukemia xenografts. We propose a model where USP7 counteracts TRIM27 E3 ligase activity, thereby maintaining PRC1.1 integrity and function. Moreover, USP7 inhibition may be a promising new strategy to treat AML patients.

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.

Integrative network analysis reveals USP7 haploinsufficiency inhibits E-protein activity in pediatric T-lineage acute lymphoblastic leukemia (T-ALL)

USP7, which encodes a deubiquitylating enzyme, is among the most frequently mutated genes in pediatric T-ALL, with somatic heterozygous loss-of-function mutations (haploinsufficiency) predominantly affecting the subgroup that has aberrant TAL1 oncogene activation. Network analysis of > 200 T-ALL transcriptomes linked USP7 haploinsufficiency with decreased activities of E-proteins. E-proteins are also negatively regulated by TAL1, leading to concerted down-regulation of E-protein target genes involved in T-cell development. In T-ALL cell lines, we showed the physical interaction of USP7 with E-proteins and TAL1 by mass spectrometry and ChIP-seq. Haploinsufficient but not complete CRISPR knock-out of USP7 showed accelerated cell growth and validated transcriptional down-regulation of E-protein targets. Our study unveiled the synergistic effect of USP7 haploinsufficiency with aberrant TAL1 activation on T-ALL, implicating USP7 as a haploinsufficient tumor suppressor in T-ALL. Our findings caution against a universal oncogene designation for USP7 while emphasizing the dosage-dependent consequences of USP7 inhibitors currently under development as potential cancer therapeutics.

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 4.86μM and 1.537μM, respectively. Ub-AMC assays further confirmed IC50 values of 5.048μM for C9 and 0.595μM for C19. MTT assays indicated that gastric cancer MGC-803cells were more sensitive to these compounds than BGC-823cells. 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-803cells. 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-803cell 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.

Ubiquitin specific protease 7 maintains pluripotency of mouse embryonic stem cells through stabilization of β-catenin.

Embryonic stem cells (ESCs), which are derived from the undifferentiated inner cell mass of the embryo, can differentiate every cell type of the body regarding their pluripotency. Therefore, human or mouse ESCs can be used as an unlimited cell source for numerous researches or therapeutical approaches. However, pluripotency maintenance of ESCs during in vitro culture is challenging because of their endless differentiation capacity. In the current study, the effect of USP7 on pluripotency maintenance of mouse ESCs (mESCs) has been investigated with the help of cell viability assay, morphological analysis, alkaline phosphatase (ALP) staining, qPCR analysis, and Western Blotting. 600 nM P5091 application, which showed no significant toxicity in mESCs, increased the total ubiquitinated protein amount as a proof of the accomplishment of proper USP7 inhibition. Morphological analysis and ALP activity evaluation indicated that dual inhibition of GSK3 and MEK together with leukemia inhibitory factor (LIF) treatment protects the pluripotency in presence of active USP7 enzyme. Yet, inactivation of USP7 reduced the ALP activity and altered the cell morphology in each treatment group. This morphological change and decreased ALP activity refer to differentiated mESCs. These findings were supported by gene expression and protein analysis. Gene expressions and protein amounts of pluripotency related Oct4, Nanog, c-Myc, Sox2 and Klf4 transcription factors decreased significantly after USP7 inhibition. Together with this observation, a remarkable reduction in β-Catenin expression was also noticed. It was also observed that USP7 inactivation shortens the half-live of β-Catenin and GSK3β proteins. This study demonstrates that USP7 activation is crucial for proper pluripotency maintenance, which is provided through β-Catenin stabilization.

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.

Blockade of Deubiquitylating Enzyme USP7 in Plasmacytoid Dendritic Cells Stimulates Anti-Myeloma Immunity

Introduction Proteasome inhibitors (PIs) are a backbone standard of care in the treatment of relapsed/refractory and newly diagnosed multiple myeloma (MM). Although PIs represent a major advance, not all MM responds, and relapse develops due to the development of drug-resistance. Besides the 26S proteasome, the ubiquitin proteasome pathway harbors many other sites for therapeutic intervention, with the potential to overcome PI-resistance. We showed that inhibition of deubiquitylating enzyme USP7, upstream of the 20S proteasome, induces cell death even in MM cells resistant to PI therapies. To date, however, the effect of USP7 inhibition on the immunosuppressive and tumor-promoting MM-host bone marrow (BM) accessory cells such as plasmacytoid dendritic cells (pDC) remains unclear. Here, we utilized our co-culture models of pDCs, T cells, and NK cells with autologous patient MM cells to examine whether USP7 inhibition also stimulates anti-MM immunity. Methods MM cell lines were validated by fingerprinting, and MM patient BM/PB samples (n = 9) were obtained after informed consent. We used minimally cytotoxic concentrations of a novel USP7 inhibitor XL177A (0.1-0.5 µM). Activation/maturation assays MM-pDCs were treated with XL177A (0.2 uM; 24h), followed by evaluation of CD86/HLA-DR markers by FACS. USP7-HA-UbVME Labeling: pDC representative Cal-1 cells were treated with DMSO or XL177A for 6h; proteins were labeled with ubiquitin (Ub) active site probe Ub-vinyl methyl ester (HAUbVME) for 30 mins, followed by immunoblot analysis using anti-USP7 antibodies. CTL/NK activity assays MM CD8+ T- or NK-cells cells were cultured with autologous pDCs (1:10 pDC:T/NK ratio) with XL177A for 3 days; after washing to remove drug, cells were cultured with pre-stained (CellTrace Violet) target MM cells (10:1 E/T ratio; T/NK:MM) for 24h, followed by FACS quantification of viable MM cells. Statistical parameters were calculated using GraphPad Prism. Results 1) Treatment of MM cell lines and patient MM cells with XL177A for 24h significantly decreases their viability (IC50 range 0.6 uM to 5 uM) (mean ± SD; p < 0.05; n=4). 2) Blockade of USP7 by XL177A activates MM patient BM-pDCs, evidenced by an increase in pDC maturation markers CD86 and HLA-DR. As a negative control, treatment of pDCs with XL177B, an inactive (R) enantiomer of XL177A, had no effect on CD86 or HLA-DR expression on pDCs. 3) XL177A specificity against USP7 DUB activity was assessed using competition assays between XL177A and Ub-active site labeling probe HAUbVME: treatment of pDC-Cal-1 cells with XL177A blocked the labeling HAUbME probe, confirming its specificity. Similar results were noted using another USP7 inhibitor P5091. 4) Functional studies showed that XL177A stimulates MM-specific CD8+ CTL activity, evidenced by a decrease in the viability of patient MM cells (p = 0.0004; n= 9). 5) XL177A triggered pDC-induced NK cell-mediated MM-specific cytolytic activity (p<0.05). Finally, 6) XL177A decreased Treg (CD4+/CD25+/Foxp3+) populations in MM. Conclusion Targeting USP7 DUB enzyme restores both innate and adaptive immune responses in MM. These findings, coupled with our prior findings showing that inhibition of USP7 overcomes PI-resistance in MM cells, suggest that targeting USP7 is a novel therapeutic strategy to overcome both PI-resistance and immune suppression in MM. Disclosures Buhrlage: Adenoid Cystic Carcinoma Foundation: Other: Scientific Advisory Board. Chauhan:consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.; Oncopeptide AB: Consultancy. Anderson:Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.