OTUD6A promotes de novo pyrimidine synthesis by deubiquitinating and stabilizing CAD in cervical cancer.

Carbamoyl-phosphate synthetase II, aspartate transcarbamylase and dihydroorotase (CAD) is a multifunctional, rate-limiting enzyme involved in de novo pyrimidine synthesis. Its activity is tightly regulated, primarily through phosphorylation and allosteric mechanisms. However, the contribution of other post-translational modifications to CAD regulation remains largely unexplored. Here we identify ubiquitination as a novel regulatory mechanism controlling CAD stability. We show that CAD undergoes K29-linked ubiquitination and proteasomal degradation following ubiquitination at lysine residues K1325 and K1411 within its carbamoyl-phosphate synthetase II (CPS II) domain. Inhibiting CAD ubiquitination by mutating these lysine sites promotes its stability, de novo pyrimidine synthesis and tumor growth. Notably, CAD protein expression is elevated in cervical cancer and is associated with poor prognosis. Furthermore, we identify OTU domain-containing protein 6 A (OTUD6A) as a deubiquitylase that directly interacts with the CPS II domain of CAD, leading to its deubiquitylation and stabilization. OTUD6A overexpression enhances de novo pyrimidine synthesis and tumor growth in a CAD-dependent manner. OTUD6A is also upregulated in cervical cancer, positively correlating with CAD protein levels and poor prognosis of cervical cancer patients. Collectively, our results reveal the OTUD6A-CAD axis as a critical regulator of pyrimidine metabolism in cervical cancer, highlighting a potential vulnerability for therapeutic targeting.