The deubiquitylase OTUB1 drives gemcitabine resistance in pancreatic cancer by enhancing pyrimidine metabolism through modulating DHODH mRNA stability.

Gemcitabine resistance is a major clinical challenge in pancreatic cancer (PC); therefore, strategies to combat gemcitabine resistance are urgently required. Reprogramming pyrimidine metabolism by oncogenic signaling contributes to cancer progression and confers chemoresistance to many cancers. The current study identified the deubiquitinating enzyme OTUB1 as a promising therapeutic target for combating gemcitabine resistance in PC. OTUB1 was found to be aberrantly expressed in PC and remarkably correlated with poor patient survival. Both in vivo and in vitro, OTUB1 knockdown increased the gemcitabine efficacy of PC cells by inhibiting pyrimidine metabolism. Furthermore, OTUB1 enhanced de novo nucleotide pyrimidine synthesis in PC cells by upregulating dihydroorotate dehydrogenase (DHODH), a critical rate-limiting enzyme for pyrimidine de novo biosynthesis. Mechanistically, OTUB1 suppressed the degradation and polyubiquitination of the RNA-binding protein DEAD-box helicase 3 X-linked (DDX3X), which in turn stabilized DDX3X-mediated DHODH mRNA. OTUB1 interacts with DDX3X, and the binding stabilizes DDX3X through its deubiquitinase activity. In addition, a small-molecule OTUB1 inhibitor combined with gemcitabine treatment could synergistically inhibit tumor growth in high-OTUB1-expressing murine tumoroids. Collectively, OTUB1 could impart gemcitabine resistance by promoting de novo pyrimidine synthesis, and targeted suppression of OTUB1 could be an effective strategy to overcome gemcitabine resistance in PC.