USP25 promotes pathological HIF-1-driven metabolic reprogramming and is a therapeutic target in pancreatic cancer

Deubiquitylating enzymes (DUBs) play an essential role in targeted protein degradation and represent an emerging therapeutic paradigm in cancer. However, their therapeutic potential in pancreatic ductal adenocarcinoma (PDAC) has not been explored. Here, we developed a DUB discovery pipeline, combining activity-based proteomics with a loss-of-function genetic screen in patient-derived PDAC organoids and murine genetic models. This approach identified USP25 as a master regulator of PDAC growth and maintenance. Genetic and pharmacological USP25 inhibition resulted in potent growth impairment in PDAC organoids, while normal pancreatic organoids were insensitive, and caused dramatic regression of patient-derived xenografts. Mechanistically, USP25 deubiquitinated and stabilised the HIF-1a transcription factor. PDAC is characterised by a severely hypoxic microenvironment, and USP25 depletion abrogated HIF-1a transcriptional activity and impaired glycolysis, inducing PDAC cell death in the tumor hypoxic core. Thus, the USP25/HIF-1a axis is an essential mechanism of metabolic reprogramming and survival in PDAC, which can be therapeutically exploited. Murine PDAC organoids were derived from primary PDAC tumors from KPCY mice (KPCY mice: Pdx1-Cre; LSL-KrasG12D; Trp53flox/flox; Rosa26-LSL-YFP). Organoids were grown as 3D cultures, in Matrix gel with defined stem cell medium. Organoids were transduced with lentiviruses containing shRNAs targeting either Usp25 or the YFP reporter as a control. Post-transduction, organoids recovered for 72hrs and selected with puromycin for 30 hours prior to analysis.