H3K27me3 loss and METTL3-m6A converge to upregulate SOX11/SMARCA4 and drive H3K27-altered spinal cord glioma stemness and proliferation

Abstract Background H3 K27-altered diffuse midline gliomas (H3-DMGs) are driven by stem-like and proliferative tumor cells resulting from global H3K27me3 loss. Here, we aimed to identify key molecular mechanisms involved in the interplay between epigenetic and epitranscriptomic regulation underlying this process. Methods Patient-derived spinal cord H3-DMG stem-like spheres and serum-induced differentiated cells were used to model tumor plasticity. Multi-omics approaches, including single-cell RNA sequencing, spatial transcriptomics, RNA m6A profiling, CUT&Tag, ATAC-seq, and in vitro and in vivo assays were performed to dissect epigenetic and epitranscriptomic regulation of stemness. Results Differentiation reduced stemness markers, proliferation, H3K27M expression, and global m6A levels, while increasing H3K27me3. METTL3 was significantly downregulated during differentiation and positively correlated with stemness and proliferation in patient tumors. METTL3 promoted tumor stemness and growth by stabilizing SOX11 mRNA through YTHDC2-dependent m6A recognition. In parallel, H3K27me3 loss relieved transcriptional repression at both METTL3 and SOX11 loci, leading to SOX11 overexpression. Functionally, SOX11 interacted with SMARCA4, the core ATPase of the SWI/SNF complex, to enhance chromatin accessibility and activate stemness-associated transcriptional programs marked by H3K27ac and H3K4me3 enrichment. Clinically, METTL3 and SOX11 were upregulated in H3K27M-mutant tumors, and high SOX11 expression correlated with increased proliferation, poor prognosis, and enhanced sensitivity to the SMARCA4 inhibitor BRM014. Conclusions H3K27M-driven epigenetic dysregulation promotes SOX11 overexpression through combined H3K27me3 loss and METTL3-mediated m6A modification. SOX11 sustains tumor stemness and proliferation by recruiting SMARCA4 to remodel chromatin and serves as both a prognostic biomarker and a predictor of response to SMARCA4-targeted therapy in spinal cord H3-DMG.