Suppression of transcription-replication conflicts by sequence-coordinated actions of TRDMT1 and MutLα

TRDMT1 is an RNA methyltransferase that catalyzes 5-methylcytosine (m5C) formation in R-loops to promote transcription-coupled homologous recombination (TC-HR). Although TRDMT1 inhibition selectively kills BRCA1-deficient cancer cells, broader cancer dependencies on TRDMT1 remain unclear. Here, a TRDMT1 inhibitor (TRDMT1i) sensitivity screen across a large panel of cancer cell lines identifies loss of MLH1 or PMS2, two components of the MutLα mismatch repair (MMR) complex frequently inactivated in tumors, as key determinants of TRDMT1 dependency. In contrast, MutLβ and MutSα/β are dispensable for TRDMT1i resistance, revealing a unique MMR-independent function of MutLα. Mechanistically, TRDMT1 and MutLα independently recognize DNA-RNA hybrids and cooperatively suppress co-transcriptional R-loops genome-wide in undamaged cells, with m5C directing pathway choice. Furthermore, MutLα suppresses R-loops through its ATPase and endonuclease activities and through recruitment of EXO1. Combined loss of TRDMT1 and MLH1 causes extensive R-loop accumulation and transcription replication conflicts (TRCs), impairing replication fork progression, inducing DNA damage, and driving apoptosis-mediated synthetic lethality. Importantly, TRDMT1i suppresses growth of MLH1-deficient tumors by inducing TRCs in vivo, suggesting a potential therapeutic strategy for targeting MutLα-deficient tumors. These studies not only expand our understanding of cancer dependency on TRDMT1, but also identify a promising strategy to exploit TRCs in cancer therapy.