Molecular targeting of the deubiquitinase USP14 to circumvent cisplatin resistance in ovarian carcinoma and identification of novel inhibitors

Aim: This study aims to investigate the biological role of the proteasome-associated deubiquitinase ubiquitinspecific protease 14 (USP14) in ovarian carcinoma drug resistance and to identify novel USP14 inhibitors (USP14i) for further preclinical development. Methods: USP14 expression was evaluated in clinical samples from 134 ovarian carcinoma patients and in a broad panel of human ovarian carcinoma cell lines. Functional studies, including gain-and loss-of-function assays, migration and invasion, and apoptosis induction assays, were conducted using cisplatin-sensitive IGROV-1 cells and their cisplatin-resistant derivative IGROV-1/Pt1. A library of 1,056 small molecules was screened using an optimized hydrolysis assay. Docking and molecular dynamics simulations were employed to predict binding modes of candidate inhibitors within the USP14 domain. Results: In clinical specimens, USP14 mRNA expression was associated with tumor grade. Exogenous overexpression of USP14 enhanced the survival of cisplatin-resistant IGROV-1/Pt1 cells, but not parental IGROV-1 cells, upon cisplatin exposure. USP14 knockdown by small interfering RNAs in resistant cells reduced aggressive features and restored cisplatin sensitivity, whereas no sensitization was observed in IGROV-1 cells. Medium-throughput screening identified five candidate molecules, among which ARN12502 showed the strongest inhibitory activity against USP14. ARN12502 exhibited an IC50 of 18.4 mu M, and molecular dynamics simulations confirmed stable binding in two distinct modes. In proteasome sensor-expressing cells, ARN12502 displayed proteasomeinhibitory activity. Conclusion: USP14 contributes to the aggressiveness of ovarian carcinoma, particularly to the cisplatin-resistant phenotype, and represents a relevant promising druggable target. ARN12502 serves as a starting point for chemical optimization toward the development of more potent USP14i.