Targeting the USP5-HSPB1 axis overcomes ferroptosis resistance in intrahepatic cholangiocarcinoma.

Ferroptosis evasion contributes to therapeutic resistance in intrahepatic cholangiocarcinoma (ICC), yet the ubiquitin-dependent mechanisms governing this process remain poorly defined. Here, we identify ubiquitin-specific protease 5 (USP5) as a clinically relevant ferroptosis suppressor in ICC. USP5 was frequently upregulated in human ICC specimens, and elevated USP5 expression was associated with adverse clinicopathological features and poor outcomes. Mechanistically, USP5 directly interacted with heat shock protein beta-1 (HSPB1) and stabilizes HSPB1 by deubiquitination, thereby attenuating lipid peroxidation-driven ferroptosis. USP5 depletion destabilizes HSPB1, sensitizes ICC cells to ferroptosis, and limits tumor growth, whereas USP5 overexpression promotes ferroptosis resistance and tumor progression. Structure-guided virtual screening and functional validation identified hellebrigenol (Hel) as a candidate USP5 inhibitor that increases HSPB1 ubiquitination and proteasome-dependent turnover, inducing ferroptosis and suppressing ICC progression. Notably, Hel potentiated gemcitabine efficacy, resulting in pronounced tumor inhibition and improved survival. Collectively, these findings establish the USP5-HSPB1 axis as a ferroptosis checkpoint in ICC and suggest pharmacologic USP5 inhibition as a strategy to improve chemotherapy response.