Ubiquitin-specific Protease 7 Inhibition Research Articles

Ubiquitin-specific protease-7 promotes expression of airway mucin MUC5AC via the NF-κB signaling pathway

Chronic obstructive pulmonary disease (COPD) and other respiratory diseases frequently present with airway mucus hypersecretion, which not only affects the patient's quality of life but also poses a constant threat to their life expectancy. Ubiquitin-specific protease 7 (USP7), a deubiquitinating enzyme, affects cell differentiation, tissue growth, and disease development. However, its role in airway mucus hypersecretion induced by COPD remains elusive. In this study, USP7 expression was significantly upregulated in airway epithelial samples from patients with COPD, and USP7 was also overexpressed in mouse lung and human airway epithelial cells in models of airway mucus hypersecretion. Inhibition of USP7 reduced the expression of nuclear factor kappa B (NF-κB), phosphorylated-NF-κB (p-NF-κB), and phosphonated inhibitor of nuclear factor kappa B (p-IκBα), and alleviated the airway mucus hypersecretion in vivo and in vitro. Further research revealed that USP7 stimulated airway mucus hypersecretion through the activation of NF-κB nuclear translocation. In addition, the expression of mucin 5AC (MUC5AC) was suppressed by the NF-κB inhibitor erdosteine. These findings suggest that USP7 stimulates the NF-κB signaling pathway, which promotes airway mucus hypersecretion. This study identifies one of the mechanisms regulating airway mucus secretion and provides a new potential target for its prevention and treatment.

USP7 Deregulation Impairs S Phase Specific DNA Repair after Irradiation in Breast Cancer Cells.

The ubiquitin specific protease 7 (USP7) is a deubiquitinating enzyme with numerous substrates. Aberrant expression of USP7 is associated with tumor progression. This study aims to investigate how a deregulated USP7 expression affects chromosomal instability and prognosis of breast cancer patients in silico and radiosensitivity and DNA repair in breast cancer cells in vitro. The investigations in silico were performed using overall survival and USP7 mRNA expression data of breast cancer patients. The results showed that a high USP7 expression was associated with increased chromosomal instability and decreased overall survival. The in vitro experiments were performed in a luminal and a triple-negative breast cancer cell line. Proliferation, DNA repair, DNA replication stress, and survival after USP7 overexpression or inhibition and irradiation were analyzed. Both, USP7 inhibition and overexpression resulted in decreased cellular survival, distinct radiosensitization and an increased number of residual DNA double-strand breaks in the S phase following irradiation. RAD51 recruitment and base incorporation were decreased after USP7 inhibition plus irradiation and more single-stranded DNA was detected. The results show that deregulation of USP7 activity disrupts DNA repair in the S phase by increasing DNA replication stress and presents USP7 as a promising target to overcome the radioresistance of breast tumors.

Blocking Ubiquitin-Specific Protease 7 Induces Ferroptosis in Gastric Cancer via Targeting Stearoyl-CoA Desaturase.

Gastric cancer (GC) presents a formidable global health challenge, and conventional therapies face efficacy limitations. Ubiquitin-specific protease 7 (USP7) plays pivotal roles in GC development, immune response, and chemo-resistance, making it a promising target. Various USP7 inhibitors have shown selectivity and efficacy in preclinical studies. However, the mechanistic role of USP7 has not been fully elucidated, and currently, no USP7 inhibitors have been approved for clinical use. In this study, DHPO is identified as a potent USP7 inhibitor for GC treatment through in silico screening. DHPO demonstrates significant anti-tumor activity in vitro, inhibiting cell viability and clonogenic ability, and preventing tumor migration and invasion. In vivo studies using orthotopic gastric tumor mouse models validate DHPO's efficacy in suppressing tumor growth and metastasis without significant toxicity. Mechanistically, DHPO inhibition triggers ferroptosis, evidenced by mitochondrial alterations, lipid Reactive Oxygen Species (ROS), Malondialdehyde (MDA) accumulation, and iron overload. Further investigations unveil USP7's regulation of Stearoyl-CoA Desaturase (SCD) through deubiquitination, linking USP7 inhibition to SCD degradation and ferroptosis induction. Overall, this study identifies USP7 as a key player in ferroptosis of GC, elucidates DHPO's inhibitory mechanisms, and highlights its potential for GC treatment by inducing ferroptosis through SCD regulation.

Virtual screening of natural products targeting ubiquitin-specific protease 7

Ubiquitin-specific protease 7 (USP7) is a promising prognostic and druggable target for cancer therapy. Inhibition of USP7 can activate the MDM2-P53 signaling pathway, thereby promoting cancer cell apoptosis. This study based on watvina molecular docking of virtual screening method and biological evaluation found the new USP7 inhibitors targeting catalytic active site. Three hits were screened from 3760 natural products and validated as USP7 inhibitors by enzymatic and kinetic assays. The IC50 values of scutellarein (Scu), semethylzeylastera (DML) and salvianolic acid C (SAC) were 3.017, 6.865 and 8.495 μM, respectively. Further, we reported that the hits could downregulate MDM2 and activate p53 signal pathway in HCT116 cells. Molecular dynamics simulation was used to investigate the binding mechanism of USP7 to Scu, the compound with the best performance, which formed stable contact with Val296, Gln297, Phe409, Tyr465 and Tyr514. These interactions are essential for maintaining the biological activity of Scu. Three natural products are suitable as lead compounds for the development of novel USP7 inhibitors, especially anti-colon cancer drugs. Communicated by Ramaswamy H. Sarma

Abstract PR04: USP7 inhibitors prevent triple negative breast cancer metastasis by inducing UHRF1 protein degradation

Abstract Functional inactivation of RB1 occurs frequently in triple negative breast cancer (TNBC), the most aggressive breast cancer subtype with poor clinical prognosis when tumors metastasize. Ubiquitin-like with PHD and RING Finger domains 1 (UHRF1) is a key epigenetic regulator that is controlled by the RB-E2F pathway and is frequently overexpressed in many types of cancers, including TNBC. Using TCGA and METABRIC databases, we found that UHRF1 overexpression is negatively associated with breast cancer patient survival, including TNBC. Genetic ablation of UHRF1 using CRISPR/Cas9 in TNBC cell lines resulted in a robust decrease in tumorigenicity, including decreased cell clonogenicity (soft agar colony assay), migration (scratch wound healing assay), and invasion (Transwell assay) in vitro and decreased tumor growth and metastasis using orthotopic xenograft in vivo models. Despite strong evidence showing UHRF1 as a promising therapeutic target in several cancers, there is a current lack of UHRF1-specific inhibitors. To overcome this limitation, we explored targeting Ubiquitin-Specific Protease 7 (USP7), a deubiquitinase that prevents UHRF1 degradation, as a therapeutic strategy to trigger UHRF1 degradation. Here, we demonstrate that genetic inactivation or pharmacological inhibition of USP7 decreases TNBC tumor growth and metastasis in a UHRF1-degradation dependent manner. Phenocopying the effects of knocking out UHRF1, genetic ablation of USP7 using CRISPR/Cas9 in TNBC cell lines also resulted in significant decreases in cell clonogenicity, migration, and invasion in vitro. Intriguingly, mice bearing USP7 knockout tumors resulted in a far more robust decrease in tumor growth and absolute absence of metastatic disease. We established that many of the effects driven by USP7 ablation were mediated by the targeted degradation of UHRF1 by comparing vector control cell lines with UHRF1 knockout cells treated with the highly selective USP7 inhibitors, XL177A or FT671. In the absence of UHRF1, USP7 inhibitors showed no effects in cell migration and invasion in the three TNBC cell lines used in this study. Of promise, NSG mice bearing MDA-MB-231 tumors were recruited into vehicle or 50 mg/kg FT671 treatment groups and dosed daily via oral gavage for 20 days. For mice treated with FT671, we observed a significant decrease in tumor growth, with decreased UHRF1 levels in tumors, with minimal systemic toxicities. This study suggests that USP7 is a target with potential clinical relevance in suppressing tumor growth and metastasis in TNBC, and possibly other TP53-null cancers. These are promising results that present a novel opportunity for future clinical interventions for TNBC patients. Citation Format: Ahhyun Kim, Claudia Benavente. USP7 inhibitors prevent triple negative breast cancer metastasis by inducing UHRF1 protein degradation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr PR04.

USP7 Promotes TGF-β1 Signaling by De-Ubiquitinating Smad2/Smad3 in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a long-term, progressive, and irreversible pulmonary interstitial disease. The activation of Smad family member 2 (Smad2) and Smad3 transcription factors by transforming growth factor β-1 (TGF-β1) is a critical event in the pathogenesis of IPF. However, there is still a lack of understanding regarding the molecular mechanisms governing Smad2 and Smad3 proteins. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a vital role in regulating protein stability within cells. However, its regulation of the TGF-β signaling pathway and its significance in IPF remain undiscovered. This study aims to clarify the function of USP7 in the TGF-β signaling pathway, while simultaneously exploring the specific molecular mechanisms involved. Additionally, this study seeks to evaluate the therapeutic potential of targeted USP7 inhibitors in IPF, thereby providing novel insights for the diagnosis and management of IPF. We first detected the expression of USP7 in lung tissues of mice with Bleomycin (BLM)-induced pulmonary fibrosis and in Beas-2B cells treated with or without TGF-β1 through Western blot analysis. Subsequently, we explored the influence of USP7 on fibrotic processes and the TGF-β1 signaling pathway, utilizing in vitro and in vivo studies. Finally, we assessed the effectiveness of USP7-specific inhibitors in an IPF murine model. In the present study, USP7 was found to de-ubiquitinate Smad2 and Smad3, consequently increasing their stability and promoting the TGF-β1-induced production of profibrotic proteins including α-smooth muscle actin (α-SMA) and fibronectin 1 (FN-1). Inhibition or knockdown of USP7 resulted in decreased levels of Smad2 and Smad3 proteins, leading to reduced expression of FN-1, Collagen Type I Alpha 1 Chain (Col1A1), and α-SMA induced by TGF-β1 in human pulmonary epithelial cells. These findings demonstrate that overexpression of USP7 reduces Smad2/3 ubiquitination, whereas inhibition or knockdown of USP7 enhances their ubiquitination. USP7 is abundantly expressed in IPF lungs. The expressions of USP7, Smad2, and Smad3 were upregulated in bleomycin-induced lung injury. The USP7 inhibitor P22077 reduced the expression of FN-1 and type I collagen as well as Smad2/3 and collagen deposition in lung tissue in a model of pulmonary fibrosis induced by bleomycin. This study demonstrates that USP7 promotes TGF-β1 signaling by stabilizing Smad2 and Smad3. The contribution of USP7 to the progression of IPF indicates it may be a viable treatment target.

The ubiquitin specific protease 7 stabilizes HPV16E7 to promote HPV-mediated carcinogenesis.

Human papillomavirus (HPV) encoded E7 oncoprotein plays an important role in supporting the viral productive cycle and inducing cancer phenotypes. The ability of E7 to exercise these functions, partly, depends upon its steady-state level. HPV manipulates the host de-ubiquitination pathway to maintain the stability of its viral proteins. In this study, we uncovered that HPV interacts with the host ubiquitin specific protease 7 (USP7), a universal de-ubiquitinating enzyme, leading to the stabilization of E7 oncoprotein. We observed that HPV16E7 complexes with USP7 via the E7-CR3 domain, and this E7-USP7 complex exists predominantly in the nucleus. Our results showed that USP7 stabilizes and prolongs the half-life of HPV16E7 by antagonizing ubiquitination and proteasomal degradation. Consistently, when we inhibited USP7 activity using HBX 19818, HPV16E7 protein level was reduced and its turnover was increased. We also provide evidence that HBX 19818-induced USP7 inhibition can halt HPV-mediated carcinogenesis, including cell proliferation, invasion, migration and transformation. These findings indicate that USP7 plays an essential role in stabilizing E7. The specific and potent inhibitory effects of HBX 19818 on HPV-induced carcinogenesis provide a molecular insight, suggesting the potential of targeting USP7 as a new therapeutic approach for the treatment of HPV-associated cancers.

USP7 Inhibition Suppresses Neuroblastoma Growth via Induction of p53-Mediated Apoptosis and EZH2 and N-Myc Downregulation.

Neuroblastoma (NB) is a pediatric malignancy originating from neural crest cells of the sympathetic nervous system that accounts for 15% of all pediatric cancer deaths. Despite advances in treatment, high-risk NB remains difficult to cure, highlighting the need for novel therapeutic approaches. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a critical role in tumor suppression and DNA repair, and USP7 overexpression has been associated with tumor aggressiveness in a variety of tumors, including NB. Therefore, USP7 is a potential therapeutic target for NB. The tumor suppressor p53 is a known target of USP7, and therefore reactivation of the p53 pathway may be an effective therapeutic strategy for NB treatment. We hypothesized that inhibition of USP7 would be effective against NB tumor growth. Using a novel USP7 inhibitor, Almac4, we have demonstrated significant antitumor activity, with significant decreases in both cell proliferation and cell viability in TP53 wild-type NB cell lines. USP7 inhibition in NB cells activated the p53 pathway via USP7 and MDM2 degradation, leading to reduced p53 ubiquitination and increased p53 expression in all sensitive NB cells. In addition, USP7 inhibition led to decreased N-myc protein levels in both MYCN-amplified and -nonamplified NB cell lines, but no correlation was observed between MYCN amplification and treatment response. USP7 inhibition induced apoptosis in all TP53 wild-type NB cell lines. USP7 inhibition also induced EZH2 ubiquitination and degradation. Lastly, the combination of USP7 and MDM2 inhibition showed enhanced efficacy. Our data suggests that USP7 inhibition may be a promising therapeutic strategy for children with high-risk and relapsed NB.

Abstract 6157: USP7 is a promising thersapeutic target for neuroblastoma

Abstract Background: The ubiquitin-proteasome system (UPS) plays an essential role in post-translational modification of proteins to maintain proteostasis. Ubiquitination of target protein is coordinated by different enzymes, which add and link ubiquitin to each other in specific ways that determine the fate of the protein for to be degraded or to be included into protein complex to induce signaling pathways. Ubiquitination is further regulated by deubiquitinases, which remove ubiquitin and rescue target protein from being degraded or diverted from it signaling role. UPS dysregulation has been linked to many human diseases, including cancer. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a critical role in immune response, tumor suppression and DNA repair. USP7 overexpression has been associated with tumor aggressiveness in a variety of tumors, including in neuroblastoma (NB). Therefore, USP7 is a potential therapeutic target for NB. Some new specific USP7 inhibitors are highly potent and selective and have demonstrated significant antitumor activity in preclinical models of adult cancer. We hypothesize that inhibition of USP7 alone or in combination with epigenetic drugs like HDAC inhibitors will be more effective against NB tumor growth. To evaluate the efficacy of USP7 inhibition with specific USP7 inhibitors alone or with combination with epigenetic drugs like HDAC inhibitors (Saha) or chemotherapy against NB tumor growth, NB cell lines were treated with increasing concentrations of USP7 inhibitors alone or in combination with chemotherapy or with HDAC inhibitors. Cell proliferation was measured using continuous live cell imaging and cell viability using AlamarBlue assay. Apoptosis was measured by caspase cleavage using Caspase 3/7 apoptosis assay as well as by PARP cleavage by Western Blotting (WB). Changes in various proteins were measured by WB and co-immunoprecipitation were performed to analyze the ubiquitin status of specific protein targets regulated by USP7. Pharmacologic inhibition of USP7 resulted in significant decreases in both cell proliferation and cell viability in vitro only in p53 wild-type NB cell lines. There was no correlation seen between MYCN amplification status and treatment response. USP7 inhibition induced apoptosis as well as necroptosis with dose-dependent increase. Treatment with USP7 inhibitors led to decrease p53 ubiquitination leading increased of p53 protein expression in all sensitive cell lines tested and decreased MYCN protein level in MYCN-amplified cell lines. EZH2, which is a predominant target of USP7 is significantly downregulated with increasing dose of USP7 inhibitors. In addition, the combination of USP7 inhibitors with chemotherapy commonly used in NB or with combination with HDAC inhibitors showed enhanced efficacy. Our data suggests that USP7 inhibition may be a promising therapeutic strategy for children with high-risk and relapsed neuroblastoma. Citation Format: Christophe Le Clorennec, Karen Lee, Peter Zage. USP7 is a promising thersapeutic target for neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6157.

USP7 Inhibits Osteoclastogenesis via Dual Effects of Attenuating TRAF6/TAK1 Axis and Stimulating STING Signaling.

Ubiquitination is a reversible post-translational modification implicated in cell differentiation, homeostasis, and organ development. Several deubiquitinases (DUBs) decrease protein ubiquitination through the hydrolysis of ubiquitin linkages. However, the role of DUBs in bone resorption and formation is still unclear. In this study, we identified DUB ubiquitin-specific protease 7 (USP7) as a negative regulator of osteoclast formation. USP7 combines with tumor necrosis factor receptor-associated factor 6 (TRAF6) and inhibits its ubiquitination by impairing the Lys63-linked polyubiquitin chain. Such impairment leads to the suppression of receptor activator of NF-κB ligand (RANKL)-mediated nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) activation without affecting TRAF6 stability. USP7 also protects the stimulator of interferon genes (STING) against degradation, inducing interferon-β (IFN-β) expression in osteoclast formation, thereby inhibiting osteoclastogenesis cooperatively with the classical TRAF6 pathway. Furthermore, USP7 inhibition accelerates osteoclast differentiation and bone resorption both in vitro and in vivo. Contrarily, USP7 overexpression impairs osteoclast differentiation and bone resorption in vitro and in vivo. Additionally, in ovariectomy (OVX) mice, USP7 levels are lower than those in sham-operated mice, suggesting that USP7 plays a role in osteoporosis. Altogether, our data reveal the dual effect of USP7-mediated TRAF6 signal transduction and USP7-mediated protein degradation of STING in osteoclast formation.

USP7 Inhibitors in Cancer Immunotherapy: Current Status and Perspective.

Ubiquitin-specific protease 7 (USP7) regulates the stability of a plethora of intracellular proteins involved in the suppression of anti-tumor immune responses and its overexpression is associated with poor survival in many cancers. USP7 impairs the balance of the p53/MDM2 axis resulting in the proteasomal degradation of the p53 tumor suppressor, a process that can be reversed by small-molecule inhibitors of USP7. USP7 was shown to regulate the anti-tumor immune responses in several cases. Its inhibition impedes the function of regulatory T cells, promotes polarization of tumor-associated macrophages, and reduces programmed death-ligand 1 (PD-L1) expression in tumor cells. The efficacy of small-molecule USP7 inhibitors was demonstrated in vivo. The synergistic effect of combining USP7 inhibition with cancer immunotherapy is a promising therapeutic approach, though its clinical efficacy is yet to be proven. In this review, we focus on the recent developments in understanding the intrinsic role of USP7, its interplay with other molecular pathways, and the therapeutic potential of targeting USP7 functions.

Neuroinflammation inhibition by small-molecule targeting USP7 noncatalytic domain for neurodegenerative disease therapy.

Neuroinflammation is a fundamental contributor to progressive neuronal damage, which arouses a heightened interest in neurodegenerative disease therapy. Ubiquitin-specific protease 7 (USP7) has a crucial role in regulating protein stability in multiple biological processes; however, the potential role of USP7 in neurodegenerative progression is poorly understood. Here, we discover the natural small molecule eupalinolide B (EB), which targets USP7 to inhibit microglia activation. Cocrystal structure reveals a previously undisclosed covalent allosteric site, Cys576, in a unique noncatalytic HUBL domain. By selectively modifying Cys576, EB allosterically inhibits USP7 to cause a ubiquitination-dependent degradation of Keap1. Keap1 function loss further results in an Nrf2-dependent transcription activation of anti-neuroinflammation genes in microglia. In vivo, pharmacological USP7 inhibition attenuates microglia activation and resultant neuron injury, thereby notably improving behavioral deficits in dementia and Parkinson’s disease mouse models. Collectively, our findings provide an attractive future direction for neurodegenerative disease therapy by inhibiting microglia-mediated neuroinflammation by targeting USP7.

A Designer Nanoparticle Platform for Controlled Intracellular Delivery of Bioactive Macromolecules: Inhibition of Ubiquitin-Specific Protease 7 in Breast Cancer Cells.

Biological therapeutics represent an increasing and critical component of newly approved drugs; however, the inability to deliver biologics intracellularly in a controlled manner remains a major limitation. We have developed a semi-synthetic, tunable phage-like particle (PLP) platform derived from bacteriophage λ. The shell surface can be decorated with small-molecule, biological and synthetic moieties, alone or in combination and in defined ratios. Here, we demonstrate that the platform can be used to deliver biological macromolecules intracellularly and in a controlled manner. Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme that has been widely recognized as an ideal target for the treatment of a variety of cancers. Recently, UbV.7.2, a novel biologic derived from the ubiquitin scaffold, was developed for inhibition of USP7, but issues remain in achieving efficient and controlled intracellular delivery of the biologic. We have shown that decoration of PLPs with trastuzumab (Trz), a HER2-targeted therapeutic used in the treatment of various cancers, results in specific targeting and uptake of Trz-PLPs into HER2-overexpressing breast cancer cells. By simultaneously decorating PLPs with Trz and UbV.7.2, we now show that these particles are also internalized by HER2-positive cells, thus providing a means for intracellular delivery of the biologic in a controlled fashion. Internalized particles retain USP7 inhibition activity of UbV.7.2 and alter the metabolic and proteomic landscapes of these cells. This study demonstrates that the λ "designer nanoparticles" represent a powerful system for the intracellular delivery of biologics in a defined dose.

USP7 sustains an active epigenetic program via stabilizing MLL2 and WDR5 in diffuse large B-cell lymphoma.

Activated B-cell-like (ABC)-diffuse large B-cell lymphoma (ABC-DLBCL) is a common subtype of non-Hodgkin's lymphoma with poor prognosis. The survival of ABC-DLBCL relies on constitutive activation of BCR signaling, but the underlying molecular mechanism is not fully addressed. By mining The Cancer Genome Atlas database, we found that the expression of ubiquitin-specific protease 7 (USP7) is significantly elevated in three cancer types including DLBCL. Interestingly, unlike germinal center B-cell-like (GCB)-DLBCL, ABC-DLBCL shows upregulated expression of USP7. Inhibiting the enzymatic activity of USP7 (P22077) has a drasticeffect on ABC-DLBCL, but not GCB-DLBCL cells. Compared to GCB-DLBCL, ABC-DLBCL cells show transcriptional upregulation of multiple components of BCR-signaling. USP7 inhibition significantly reduces the expression of upregulated components of BCR signaling. Mechanistically, USP7 inhibition greatly reduces the methylation of histone 3 on lysine 4 (H3K4me2), which is an epigenetic marker for active enhancers. USP7 inhibition greatly reduces the protein level of WDR5 and MLL2, key components of lysine-specific methyltransferase complex (complex of proteins associated with Set1 [COMPASS]). In ABC-DLBCL cells, USP7 stabilizes WDR5 and MLL2. In patients, the expression of USP7 is significantly associated with components of BCR signaling (LYN, SYK, BTK, PLCG2, PRKCB, MALT1, BCL10, and CARD11) and targets of BCR signaling (MYC and IRF4). In summary, we demonstrated an essential role of USP7 in ABC-DLBCL by organizing an oncogenic epigenetic program via stabilization of WDR5 and MLL2. Targeting USP7 might be a novel and efficient approach to treat patients with ABC-DLBCL and it might be better than targeting individual components such as BTK in BCR signaling.

P22077 enhances the antitumor efficacy of Cisplatin and its mechanism

Activation of DNA damage repair pathways in tumor cells may reduce the treatment efficacy of platinum-based chemotherapeutic agents. Ubiquitin-specific protease 7 (USP7) is one of the deubiquitinating enzymes that can remove the ubiquitin from target proteins and protect substrate proteins from degradation. Although ubiquitin-specific protease 7(USP7) is highly expressed in cervical cancer tissues and plays an important role in DNA damage repair, the role of USP7 inhibition in the antitumor efficacy of cisplatin remains unknown. This study explored the effects and mechanisms of a USP7 inhibitor P22077 on the anti-cervical cancer efficacy of cisplatin. In in vitro studies, P22077 and cisplatin both significantly reduced HeLa cell proliferation and colony formation, and the combination produced preferable effects. In in vivo xenograft tumor model, P22077 and cisplatin both demonstrated significant antitumor efficacy. The drug combination produced greater antitumor activity than the individual drug alone. Cisplatin evoked DNA damage repair-related molecules and P22077 tended to prevent this change. The drug combination produced higher cell death rate than the individual drug alone. Collectively, These results suggest that the USP7 inhibitor P22077 alone has significant antitumor efficacy and also can enhance the antitumor effects of cisplatin. The USP7 inhibitor P22077 combined with cisplatin may be an effective treatment strategy for cervical cancer.

USP7 inhibition induces apoptosis in glioblastoma by enhancing ubiquitination of ARF4

BackgroundGlioblastomas (GBMs) are grade IV central nervous system tumors characterized by a poor prognosis and a short median overall survival. Effective induction of GBM cell death is difficult because the GBM cell population is genetically unstable, resistant to chemotherapy and highly angiogenic. In recent studies, ubiquitin-specific protease 7 (USP7) is shown to scavenge ubiquitin from oncogenic protein substrates, so effective inhibition of USP7 may be a potential key treatment for GBM.MethodsImmunohistochemistry and western blotting were used to detect the expression of USP7 in GBM tissues. In vitro apoptosis assay of USP7 inhibition was performed by western blotting, immunofluorescence, and flow cytometry. Anti-apoptotic substrates of USP7 were defined by Co-IP and TMT proteomics. Western blotting and IP were used to verify the relationship between USP7 and its substrate. In an in vivo experiment using an intracranial xenograft model in nude mice was constructed to assess the therapeutic effect of target USP7.ResultsImmunohistochemistry and western blotting confirmed that USP7 was significantly upregulated in glioblastoma samples. In in vitro experiments, inhibition of USP7 in GBM induced significant apoptosis. Co-IP and TMT proteomics identified a key anti-apoptotic substrate of USP7, ADP-ribosylation factor 4 (ARF4). Western blotting and IP confirmed that USP7 interacted directly with ARF4 and catalyzed the removal of the K48-linked polyubiquitinated chain that binded to ARF4. In addition, in vivo experiments revealed that USP7 inhibition significantly suppressed tumor growth and promoted the expression of apoptotic genes.ConclusionsTargeted inhibition of USP7 enhances the ubiquitination of ARF4 and ultimately mediates the apoptosis of GBM cells. In a clinical sense, P5091 as a novel specific inhibitor of USP7 may be an effective approach for the treatment of GBM.

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.

Abstract 1338: Discovery of novel, potent USP7 inhibitors that upregulate p53 leading to anti-proliferative effects in cancer cells

Abstract Introduction: Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that regulates several proteins involved in cell cycle, DNA repair, genomic stability, and epigenetics and has been implicated in cancer progression. A key substrate of USP7 is MDM2, the oncogenic E3 ubiquitin ligase that promotes degradation of the tumor suppressor p53. USP7-mediated stabilization of MDM2 leads to the degradation of p53, preventing cell cycle arrest and the induction of apoptosis and promoting tumor cell growth. In addition to the MDM2-p53 pathway, USP7 regulates a number of other substrates involved in cancer, including PIM2 kinase, MYCN, the DNA methyltransferase DNMT1, and the PTEN tumor suppressor. Consistent with its function promoting oncogenic signaling pathways, genetic and pharmacological inhibition of USP7 has been shown to inhibit growth of a number of tumor cell lines in vitro and in vivo, and this anti-tumor activity is significantly enriched among cells expressing wild type p53. Thus, inhibition of USP7 is a promising therapeutic strategy, especially in cancers carrying wild type p53 that can be reactivated by suppression of MDM2. Results: We have discovered a new class of potent and selective USP7 inhibitors. These compounds bind to USP7 and prevent deubiquitinase activity in biochemical activity assays with picomolar potency. Our compounds induce accumulation of p53 and exhibit IC50s below 50 nM in cell viability assays in Multiple Myeloma and Acute Myeloid Leukemia (AML) cell lines. In AML cell lines, our USP7 inhibitors strongly synergize with the approved Bcl-2 inhibitor venetoclax. Unlike MDM2 antagonists currently undergoing clinical trials in AML, these USP7 inhibitors do not lead to a significant increase in MDM2 levels, and our compounds result in a more modest induction of p53, both of which could provide significant safety benefits. We have demonstrated differential sensitivity to MDM2 antagonists and our USP7 inhibitors in select cell lines and show that this effect is dependent upon p53 induction. Our compounds are orally bioavailable with a desirable PK profile in mice and induce p53 in tumor cells in CDX mouse models of Multiple Myeloma. Conclusions: We have identified novel, potent, orally bioavailable USP7 inhibitors that lead to p53 accumulation and cytotoxicity in cancer cells. We demonstrate mechanistic differences between USP7 and MDM2-p53 antagonists, which may lead to a safety advantage. We observe strong synergy between USP7 inhibitors and an approved treatment. Our lead compounds have favorable drug-like properties, promising mouse PK profiles, and can induce p53 accumulation in mouse CDX tumor models. These data demonstrate the therapeutic potential of our USP7 inhibitors and reveal specific opportunities for their use in the treatment of Multiple Myeloma and AML. Citation Format: Alan Futran, Tao Lu, Katherine Amberg-Johnson, Xiaoxiao Yang, Saidi He, Jeffrey Bell, Sarah Boyce, Markus Dahlgren, Karen Dingley, Liping Fang, Heidi Koldsoe, Zef Konst, Fang-Yu Lin, Robert Pelleltier, Heng Qian, Mats Svensson, Michael Trzoss, Jie Xu, Shuping Xu, Zhaowu Xu, Engin Yapici, Yan Zhang, Li Xing, Takao Suzuki, Xianhai Huang, Jiayi Xu, Hamish Wright, Kristian Jensen, Wayne Tang, Tao Guo, Karen Akinsanya, David Madge. Discovery of novel, potent USP7 inhibitors that upregulate p53 leading to anti-proliferative effects in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1338.

Abstract 277: Discovery of promising antineoplastic drugs against the USP7 deubiquitinating enzyme: A pharmacophore-based FDA-approved and investigational drugs repurposing study

Abstract The deubiquitinating enzyme ubiquitin-specific protease 7 (USP7) is a novel protein target for various hematological and solid-organ malignancies [1]. Its inhibition is thus considered a highly promising strategy for the development of new antineoplastic drugs. In this study, we applied an integrated approach with a major focus on pharmacophore modeling and screening combined with molecular dynamic simulations and free energy-based calculations to identify promising drugs as USP7-small molecule inhibitors. We started this drug repurposing study with the development of structure-based complex pharmacophore model hypotheses using the crystal structure of USP7. This pharmacophore modeling strategy was found to be most successful for the identification of promising molecules in drug discovery as we have recently reported [2]. We used three pharmacophore hypotheses to screen 6654 FDA-approved and investigational drugs as derived from the NPC's NIH library. Our pharmacophore screening led to the identification of 100 drugs that have well matched several chemical features of our hypotheses based on fitness scoring. All drugs were analyzed using 10-ns MD simulations and MM/GBSA system free energy. The 100 selected molecules encompassed over 35 different classes of drugs, among which antibiotics (13 identified), antivirals (6 identified) and anti-inflammatory drugs (5 identified) were the most represented. It is important to highlight that we found 4 antineoplastic drugs, suggesting potential interactions with the USP7 enzyme. Considering the important role of USP7 in cancer development and viral infections [3], we decided to further analyze all the discovered anticancer and antiviral drugs using 100-ns MD simulations. Additionally, we included the ten most stable molecules based on their system free energy (MM/GBSA scores lower than -69.0 kcal/mol) and all discovered anti-inflammatory drugs. In total, we ran 100-ns MD simulations for 24 molecules and a known USP7 inhibitor. Depending on this analysis, certain drugs will be tested in vitro to assess their level of USP7 inhibition. Our results thus far demonstrate the potential role of selected approved/investigational drugs as promising drugs that target USP7. These findings may open the route for efficient identification and characterization of promising USP7 inhibitors that could advance to clinical studies.

Suppression of USP7 induces BCR-ABL degradation and chronic myelogenous leukemia cell apoptosis

Chronic myelogenous leukemia (CML) is a clonal malignancy of hematopoietic stem cells featured with the fusion protein kinase BCR-ABL. To elicit the mechanism underlying BCR-ABL stability, we perform a screen against a panel of deubiquitinating enzymes (DUBs) and find that the ubiquitin-specific protease 7 (USP7) drastically stabilizes the BCR-ABL fusion protein. Further studies show that USP7 interacts with BCR-ABL and blocks its polyubiquitination and degradation. Moreover, USP7 knockdown triggers BCR-ABL degradation and suppresses its downstream signaling transduction. In line with this finding, genetic or chemical inhibition of USP7 leads to BCR-ABL protein degradation, suppresses BCR/ABL signaling, and induces CML cell apoptosis. Furthermore, we find the antimalarial artesunate (ART) significantly inhibits USP7/BCR-ABL interaction, thereby promoting BCR-ABL degradation and inducing CML cell death. This study thus identifies USP7 as a putative Dub of BCR-ABL and provides a rationale in targeting USP7/BCR-ABL for the treatment of CML.