Abstract 417: Identification of novel inhibitors of the deubiquitinase USP14 for targeting cisplatin-resistant ovarian carcinoma

Abstract Deubiquitinases (DUBs) including the proteasome-associated USP14 have emerged as drug targets in cancer and may contribute to drug resistance. Since DUBs are deregulated in ovarian carcinoma, the disease management may benefit from novel inhibitor development. The aim of this study was to identify novel USP14 inhibitors (USP14i) to test in preclinical ovarian cancer models. Cellular pharmacology approaches were employed to examine USP14 role in cisplatin-sensitive and resistant cells. An assay for compound screening, previously developed by Lee and colleagues (PMID: 23788557), was optimized using Ub-Rho110-G. Molecular docking was used to evaluate the best ligand interaction of putative inhibitors. In 134 specimens representative of the frequency occurrence of the histological ovarian carcinoma subtypes, USP14 mRNA levels were associated with tumor grade. Exogenous over-expression of USP14 increased survival upon cisplatin exposure in cisplatin-resistant IGROV-1/Pt1 cells, but not in cisplatin-sensitive IGROV-1 cells, supporting a role for this DUB in the cisplatin-resistant phenotype. Knockdown of USP14 in IGROV-1/Pt1 cells by small-interfering RNA transfection impaired cell aggressive features such as proliferative, migratory and invasive abilities; sensitization to cisplatin was evident in IGROV-1/Pt1 but not in IGROV-1 cells, suggesting that USP14 acts on resistance-relevant substrates. A diversity set of 1056 small molecules from a library of ca. 11, 000 compounds was screened. Setting a relatively low threshold of inhibition of USP14 activity, 5 initial hits were identified. The most interesting compound, referred to as Cpd1, displayed an IC50 of 18.4 μM. As Cpd1 represents a novel chemical class of USP14i, a docking protocol targeting the USP domain of USP14 was employed to identify its potential binding modes (BMs). Molecular dynamics simulations were then performed using the best docking results to assess the stability of the identified BMs and evaluate the interactions between Cpd1 and USP14. The simulations revealed excellent stability for two BMs and provided insights into the interaction mechanism. In cells expressing a proteasome sensor vector, Cpd1 displayed proteasome inhibiting activity, also evident in intact cells by active-site direct labeling with HA Ub-vinyl sulfone. The identified hit represents a new chemical class of USP14i and is a starting point for future chemical optimization aiming at identifying more potent compounds. In conclusion, USP14 appears to be a relevant and druggable target in ovarian carcinoma. Citation Format: Cristina Corno, Debora Russo, Francesco Pignotti, Francesca De Giorgi, Ilaria Penna, Francesco Saccoliti, Matteo Costantino, Luca Mirra, Pietro Pettinari, Nunzio Perta, Chiara M. Ciniselli, Pietro Pratesi, Rita Scarpelli, Fabio Bertozzi, Paolo Verderio, Giovanni L. Beretta, Daniele Di Marino, Tiziano Bandiera, Paola Perego. Identification of novel inhibitors of the deubiquitinase USP14 for targeting cisplatin-resistant ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 417.