RNA exonuclease REXO4 resolves m6A-marked R-loops and suppresses anti-tumor immunity.

Transcription blockage frequently occurs in tumor cells, and aberrant R-loop formation during this process drives genome instability. However, the regulation of R-loop homeostasis and its contribution to tumorigenesis remain to be investigated. Here, we report that the RNA exonuclease REXO4 resolves R-loops by 3'-5' exonucleolytic cleavage of the RNA strand within RNA-DNA hybrids. Accessible RNA ends, generated by endonucleases, are required for this process, and N6-methyladenosine (m6A) modification on RNA moieties promotes REXO4 localization and R-loop removal in human cells. REXO4 ablation-induced DNA damage stimulates an interferon response and tumor immune infiltration, suppressing mouse squamous cell carcinoma (SCC) progression. Importantly, inhibition of REXO4 potentiates the anti-tumor efficacy of PD-1 blockade against SCC by recruiting and activating CD8+ T cells. Thus, our study provides mechanistic insight into how m6A couples with an exonuclease in R-loop clearance and genome maintenance and uncovers a druggable epitranscriptional machinery that constrains the innate immune response and enables SCC immune evasion.