Effects of thymidylate synthase inhibitors differ in genomic uracilation and mutagenic potential

Genomic uracil and its respective repair play key roles in colorectal, gastric, and other solid tumor therapies targeting thymidylate biosynthesis. Previously, we established that treating HCT116 colon cancer cell lines with either raltitrexed (RTX) or 5-fluoro-2'-deoxyuridine (5FdUR), two potent inhibitors of thymidylate synthase, results in characteristic genomic uracil patterns. Here, we focus on drug-specific differences in the genomic uracil profiles and their associations with altered cytotoxicity and drug-induced mutagenesis. We demonstrated that biased uracilation preferentially affects functionally related genes in a drug-specific manner, highlighting the biological significance of genomic uracilation. Mutational analysis further revealed a significant increase in the frequency of C-to-T somatic transitions, selectively in response to high-dose 5FdUR treatment, in DNA repair-deficient cells. The mutational spectra and the clustered nature of these transitions suggested the involvement of APOBEC3 DNA cytidine deaminases, several of which were induced under these conditions. Notably, this mutagenic response coincides with decreased cytotoxicity compared with the low-dose 5FdUR or any efficient doses of RTX, providing insights that may be relevant for personalized cancer therapy.