Thymidylate synthase inhibitory drugs induce p53-dependent pathways differently

Thymidylate synthase (TS) is a key enzyme in thymidylate biosynthesis and an established target of chemotherapeutics such as 5-fluoro-2-deoxyuridine (5FdUR) and raltitrexed (RTX). Inhibition of TS disrupts the dUTP:dTTP balance, leading to uracil misincorporation, futile base excision repair cycles, DNA strand breaks, and ultimately cell death. Beyond its catalytic role, TS also binds RNA, autoregulating its own translation and interacting with transcripts such as p53 and c-myc, thereby linking TS activity to broader post-transcriptional regulatory networks. These interactions, together with regulation by miRNAs and lncRNAs, suggest that TS inhibition may provoke cellular responses extending beyond DNA metabolism. Non-coding RNAs, including miRNAs, snoRNAs, snRNAs, and lncRNAs, may play critical roles in shaping these outcomes. To dissect these mechanisms, we investigated the transcriptomic effects of TS inhibition in mismatch repair-deficient, UNG-inhibited HCT116 colon cancer cells treated with 5FdUR or RTX. Both drugs induced DNA damage responses and S-phase arrest, yet displayed distinct transcriptional signatures. Moreover, TS-RNA immunoprecipitation sequencing revealed direct RNA-binding targets of TS, highlighting its contribution as a post-transcriptional regulator. Our findings underscore the multifaceted impact of TS inhibition, linking enzymatic disruption to RNA-level regulation and revealing drug-specific differences in cellular responses.