Proteasome-associated DUB inhibition by VLX1570 targets UCHL5/USP14 and enhances cisplatin efficacy in neuroblastoma

Neuroblastoma is the most common extracranial solid tumor in children, and high-risk disease still carries an unfavorable prognosis despite intensive multimodality therapy. Deubiquitinating enzymes (DUBs) associated with the 19S proteasome, such as ubiquitin C-terminal hydrolase L5 (UCHL5) and ubiquitin-specific protease 14 (USP14), are key regulators of protein homeostasis and have attracted interest as potential therapeutic targets. In this study, we examined UCHL5 and USP14 expression in clinical neuroblastoma specimens compared with normal peripheral nerve tissue by immunohistochemistry, and investigated the effects of pharmacologic inhibition of proteasome-associated DUB activity using the small-molecule inhibitor VLX1570 in a panel of human neuroblastoma cell lines by assessing cell viability, cell-cycle distribution, apoptosis, clonogenic growth, and markers of proteasome stress, endoplasmic reticulum stress, and autophagy, as well as in a xenograft mouse model. Cisplatin remains a cornerstone of induction and consolidation chemotherapy for high-risk neuroblastoma, and resistance or intolerance to cisplatin-based regimens represents a major barrier to cure; therefore, we specifically evaluated whether VLX1570 could potentiate cisplatin-induced cytotoxicity in vitro and in vivo. UCHL5 and USP14 were markedly upregulated in neuroblastoma tissues relative to normal peripheral nerve, and VLX1570 treatment significantly reduced neuroblastoma cell growth, induced G2/M arrest and caspase-dependent apoptosis, promoted accumulation of polyubiquitinated proteins, activated unfolded protein response signaling, and modulated autophagy-related proteins. Notably, combining VLX1570 with cisplatin produced greater suppression of cell viability and clonogenic survival, and enhanced apoptotic signaling, than either treatment alone in vitro. Consistently, VLX1570 significantly suppressed tumor growth in xenograft models, while co-treatment with cisplatin further delayed tumor progression and reduced proliferative indices without overt systemic toxicity. Taken together, these data demonstrate that UCHL5 and USP14 are overexpressed in neuroblastoma and that pharmacologic inhibition of proteasome-associated DUB activity with VLX1570 exerts antitumor effects and enhances the efficacy of cisplatin, supporting further evaluation of DUB inhibitor-cisplatin combination strategies in neuroblastoma.