DHODH is a synthetic lethal target in PIK3CA-mutant colorectal cancer.

Cancer cells acquire distinct metabolic and signaling dependencies driven by oncogenic mutations. Defining these mutation-specific liabilities can uncover therapeutic opportunities. Here, we identify dihydroorotate dehydrogenase (DHODH) as a selective metabolic dependency in PIK3CA-mutant colorectal cancer (CRC). DHODH sustains WDR77 O-GlcNAcylation and protein stability by promoting the generation of uridine diphosphate (UDP)-N-acetylglucosamine (UDP-GlcNAc), thereby maintaining PI3K-AKT signaling. Genetic or pharmacological inhibition of DHODH reduces WDR77 protein abundance, decreases phosphorylated AKT, and impairs cancer cell self-renewal and tumor initiation. Uridine supplementation restores WDR77 and AKT signaling, whereas O-GlcNAc transferase (OGT) depletion abrogates this rescue, establishing a UDP-dependent mechanism linking pyrimidine metabolism to signaling maintenance. Treatment with the DHODH inhibitor HL6 recapitulates the genetic phenotypes and suppresses tumor growth in xenograft, orthotopic, and patient-derived CRC models. This study demonstrates that the metabolic regulation of protein stability represents a critical mechanism underlying oncogene-specific dependencies in CRC.