5-Azacytosine induces cytotoxicity via 5-methylcytosine depletion on chromatin-associated RNA in leukemia.

5-Azacytidine (5-azaC) is a DNA hypomethylating agent clinically used in myeloid malignancies. As a ribonucleoside analogue, 5-azaC incorporates more readily into nascent RNA than DNA. Here, we demonstrate that RNA 5-methylcytosine (m5C) depletion by 5-azaC treatment, particularly at early time points, is sufficient to induce leukemia cell death. In contrast to its DNA demethylation function, the RNA-dependent effects of 5-azaC lead to transcriptional repression, disrupting genes involved in cell-cycle regulation and DNA repair. Mechanistically, depletion of m5C by 5-azaC in chromatin-associated RNA (caRNA) disrupts the MBD6-mediated H2AK119ub deubiquitination. This also impairs SRSF2 recruitment and the downstream H3K27ac deposition by p300. Consistently, loss of the caRNA methyltransferase NSUN2 caused prolonged cell cycle, defective DNA repair, and shifted hematopoietic lineage commitment toward erythropoiesis, mirroring the effects of 5-azaC treatment. Our findings highlight the transcriptional repression by 5-azaC through depleting caRNA m5C, providing additional insights into the mechanism of action for 5-azaC.