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

## TL;DR

This study shows how different thymidylate synthase inhibitors affect DNA uracilation and mutagenesis in colon cancer cells.

## Contribution

The study reveals drug-specific genomic uracil patterns and their link to APOBEC3 activation and mutagenesis in cancer cells.

## Key findings

- Genomic uracilation patterns differ between raltitrexed and 5FdUR treatments.
- High-dose 5FdUR increases C-to-T mutations linked to APOBEC3 activity in DNA repair-deficient cells.
- Mutagenic responses correlate with reduced cytotoxicity in treated cancer cells.

## Abstract

Distinct genomic uracil patterns and APOBEC3 activation differentiate cellular responses to TS inhibitors, connecting uracil metabolism to mutagenesis and drug tolerance in colon cancer cells.

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.