Targeting O-GlcNAcylated METTL3 impedes MDS/AML progression via diminishing SRSF1 m⁶A modification

N6-methyladenosine (m⁶A) modification, primarily regulated by methyltransferase-like protein 3 (METTL3), plays a pivotal role in RNA metabolism and leukemogenesis. However, the post-translational mechanisms governing METTL3 stability and function remain incompletely understood. Given the widespread occurrence of O-GlcNAcylation on nuclear and cytosolic proteins, we hypothesized that METTL3 might undergo O-GlcNAcylation, thereby influencing its stability and oncogenic function in myeloid malignancies. In this study, we found that METTL3 is O-GlcNAcylated in both myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), and its expression positively correlates with O-GlcNAcylation levels. Functional assays demonstrated that O-GlcNAcylation enhances METTL3 protein stability and promotes leukemic cell survival. Mechanistically, O-GlcNAcylated METTL3 stabilizes mRNA of serine- and arginine-rich splicing factor 1 (SRSF1), leading to increased expression of the anti-apoptotic protein MCL-1. This, in turn, suppresses apoptosis and supports MDS/AML cell viability. Targeting the O-GlcNAcylated form of METTL3 using a competitive peptide significantly inhibited MDS/AML progression in preclinical models. In conclusion, our findings reveal a novel O-GlcNAcylation-dependent mechanism that regulates METTL3 stability and oncogenic activity through the m⁶A-SRSF1-MCL-1 axis, highlighting a potential therapeutic strategy for MDS and AML.