LAUSR

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.