Potent and selective inhibitors of dihydroorotate dehydrogenase (DHODH), a critical enzyme in the de novo synthesis of pyrimidine ribonucleotides, are being tested in clinical trials for autoimmune disorders, viral infections, and various cancers. A limitation of these DHODH inhibitors (DHODHi) is their immunosuppressive activity, which may dampen antitumor immune responses, highlighting the need for strategies that improve their therapeutic index in oncology. In this study, we explored methods to protect activated T cells from DHODH blockade and sought to identify tumor types that are particularly vulnerable to these compounds. We found that supplementing culture media with cytidine, similar to uridine, can rescue T cells from the effects of DHODH inhibition. Next, we examined cancer cells for alterations in pyrimidine biosynthesis enzyme expression and discovered that cytidine deaminase (CDA)—the enzyme responsible for converting cytidine into uridine—is expressed at low levels in a substantial fraction of cancer cell lines. CDA expression was consistently low in neuroblastoma samples and in cell lines derived from neuroblastoma and small cell lung carcinoma. These findings suggested that, under DHODHi treatment, excess extracellular cytidine could selectively impair the growth of cancer cells with low CDA expression. This hypothesis was supported by experiments showing that cytidine supplementation rapidly reduced viability in low-CDA-expressing cells cultured with fetal bovine serum, although the effect was less pronounced in human serum. Notably, CDA is present both intracellularly and in human plasma, which may influence cytidine metabolism in vivo. Using recombinant CDA, human serum, pharmacologic CDA inhibition, and T cell/cancer co-culture models, our results indicate that the therapeutic window of DHODHi could be expanded by targeting patients with low-CDA-expressing tumors while modulating extracellular cytidine levels or the cytidine-to-uridine ratio. Taken together, this proof-of-principle work supports further investigation into strategies for depleting extracellular CDA as a means to enhance the efficacy and selectivity of DHODH inhibitors in cancer therapy.
