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 to improve outcomes in myeloid malignancies. However, 5-azaC treatment causes gene dysregulation inconsistent with DNA hypomethylation changes, suggesting alternative mechanisms of action by 5-azaC. As a ribonucleoside analogue, 5-azaC is more readily incorporated into nascent RNA. 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 effect of 5-azaC causes transcriptional repression, disrupting genes involved in cell cycle regulation and DNA repair. Mechanistically, 5-azaC impairs two specific m5C-mediated transcriptional regulatory pathways. First, depletion of m5C in chromatin-associated RNA (caRNA) disrupts the MBD6-mediated H2AK119ub deubiquitination. In parallel, this also impairs SRSF2 recruitment and the downstream H3K27ac deposition by p300. Indeed, 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. Furthermore, we performed a leukemia cell line screen and identified that TET2 and IKZF1 depletion can sensitize 5-azaC treatment, consistent with the observed RNA-dependent cytotoxicity of 5-azaC in leukemic cells. In summary, our findings highlight the transcription repression by 5-azaC through depleting caRNA m5C, providing additional insights into the mechanism of action for 5-azaC, the prediction of its efficacy, and future directions for therapy developments based on 5-azaC. HIGHLIGHTO_LIRNA-dependent effects of 5-azaC are sufficient to drive leukemia cell cytotoxicity through transcriptional repression. C_LIO_LI5-azaC-induced caRNA m5C depletion impairs MBD6 binding and H2AK119ub deubiquitination. C_LIO_LI5-azaC-induced caRNA m5C depletion disrupts SRSF2 chromatin-binding, impeding p300 recruitment and H3K27ac deposition. C_LIO_LITET2 or IKZF1 depletion synergizes leukemia sensitivity to 5-azaC C_LI