Cytotoxicity caused by 5-methylcytosine depletion on chromatin-associated RNA by 5-azacytosine in leukemia [CUT&Tag]

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. Some patients benefited from 5-azaC treatment did not display notable DNA demethylation. These observations suggest alternative mechanisms of action by 5-azaC. As a ribonuleoside analogue, 5-azaC is more readily incorporated into nascent RNA. Here, we demonstrate that RNA 5-methycytosine (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 process by 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 removal. 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 leukemia cell line screen and identifies that TET2 and IKZF1 depletion can sensitize 5-azaC treatment, consistent with the observed RNA-dependent cytotoxicity of 5-azaC in leukemia cells. In summary, our findings highlight the transcription regulation by 5-azaC through depleting caRNA m5C, providing insights into the mechanism of action for 5-azaC, the prediction of its efficacy, and future directions for therapy developments based on 5-azaC.