Decitabine and azacitidine exert multifaceted effects beyond DNA hypomethylation in myeloid neoplasms

Decitabine (DAC) and azacitidine (AZA) are widely recognized as hypomethylating agents for treating myeloid neoplasms; however, recent studies show that their actions extend beyond DNA hypomethylation. DAC acts primarily through incorporation into DNA, forming DNMT1-DNA protein crosslinks (DNMT1-DPCs). Proteolytic processing of these adducts depletes DNMT1 and causes DNA hypomethylation, which can reactivate repressed gene programs, promote myeloid differentiation, and induce innate immune signaling through endogenous retrovirus reactivation. Conversely, the persistence of unresolved DNMT1-DPCs causes replication stress and mitotic perturbation, thereby contributing to direct cytotoxicity. SUMOylation initiates DNMT1-DPC resolution, which involves ubiquitylation by RNF4 and TOPORS, followed by clearance through the proteasome and SPRTN, indicating that the balance between DPC formation and clearance shapes DAC sensitivity. In contrast, AZA exerts prominent RNA-dependent effects in addition to its DNA-mediated effects. Following incorporation into mRNA, AZA induces ribosome stalling and collisions, activating the GCN2-dependent integrated stress response and promoting cytotoxicity. Together with emerging evidence for cell-extrinsic effects, these findings highlight that DAC and AZA are azanucleosides that exert multifaceted effects beyond canonical DNA hypomethylation.