Neddylation inhibition sensitizes gastric cancer to 5-fluorouracil by targeting the post-translational stability of the metabolic enzyme dihydropyrimidine dehydrogenase.

The therapeutic efficacy of 5-fluorouracil (5-FU), a cornerstone of gastric cancer chemotherapy, is predominantly limited by its catabolic inactivation in tumors. Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme responsible for 5-FU inactivation, and its tumor-specific overexpression constitutes a primary mechanism of 5-FU resistance. Here, we report a novel strategy to increase the sensitivity of 5-FU by targeting the post-translational regulation of DPD. We demonstrate that the neural precursor cell expressed, developmentally downregulated 8-activating enzyme (NAE) inhibitor MLN4924 significantly enhances the antitumor activity of 5-FU in both cellular and animal models of gastric cancer without augmenting systemic toxicity. Mechanistically, MLN4924 treatment inhibits the neddylation of DPD, which is dependent on the NAE1/ubiquitin-conjugating enzyme 12 axis. This inhibition triggers the ubiquitination and subsequent proteasomal degradation of DPD, thereby reducing intracellular 5-FU catabolism and augmenting its cytotoxic effects. Our findings identify neddylation as a previously unrecognized regulatory mechanism governing DPD protein stability and activity. This work identifies the neddylation-DPD axis as a novel therapeutic target and provides a strong rationale for combining NAE inhibition with 5-FU-based chemotherapy in gastric cancer. SIGNIFICANCE STATEMENT: This study establishes neddylation as a previously unrecognized regulatory mechanism that stabilizes the drug-metabolizing enzyme dihydropyrimidine dehydrogenase to drive 5-fluorouracil resistance in gastric cancer. It further unveils that inhibiting neddylation with MLN4924 selectively depletes tumor dihydropyrimidine dehydrogenase, enhancing chemotherapy efficacy without increasing toxicity, thereby proposing a targeted strategy to overcome chemoresistance.