Epigenetic repurposing by CBFA2T3-GLIS2 reveals a druggable DNA methylation axis in high-risk pediatric AML vulnerability in pediatric AML

The CBFA2T3-GLIS2 (C/G) fusion defines an infant-restricted, clinically devastating subtype of pediatric AML with relapse rates exceeding 90%. While C/G is known to remodel the enhancer landscape, the epigenetic mechanisms that sustain leukemic identity and enforce apoptotic resistance remain undefined.To resolve this, we performed integrative multi-omic epigenetic profiling across primary patient samples, C/G⁺ AML lines, and a developmentally faithful cord blood CD34⁺ HSPC model. Genome-wide DNAm mapping (meEM-seq), chromatin profiling (CUT&RUN, ATAC-seq), and transcriptomics uncovered a promoter-biased hypermethylation program unique to C/G⁺ AML, with ~50% of altered CpGs localized to promoters (Δbeta≥0.2 vs NBM). Despite unaltered global 5mC levels, C/G bound and transcriptionally activated DNMT3B, which imposed focal promoter hypermethylation specifically at transcriptionally active, enhancer-connected loci. Strikingly, these hypermethylated promoters did not silence gene expression; instead, they stabilized expression of CRE-linked genes enriched for apoptotic regulation, EMT, KRAS signaling, and heme metabolism-revealing that C/G converts DNA methylation into a stabilizing rather than repressive epigenetic signal.CRISPR-mediated DNMT3B knockout validated this mechanism: DNMT3B loss reduced locus-specific 5mC and downregulated C/G-dependent targets but induced compensatory DNMT1 and UHRF1 upregulation, forming a maintenance shunt that preserved DNAm at apoptotic effector loci and sustained an elevated apoptotic threshold. Targeted dCas9-TET1 demethylation of the pro-apoptotic gene PMAIP1 (NOXA) restored transcriptional inducibility and apoptotic signaling, directly confirming that DNAm constrains activation of death pathways. In vivo, DNMT3B-deficient xenografts regained Venetoclax sensitivity and extended survival, phenocopying DNMT inhibition and validating the therapeutic relevance of this epigenetic axis. Collectively, these findings establish C/G as a developmental epigenetic architect that repurposes DNA methylation to stabilize oncogenic transcriptional circuits and maintain apoptosis resistance. By defining the C/G-DNMT3B-DNMT1/UHRF1 axis and its enhancer-linked promoter hypermethylation signature, this work identifies aberrant DNAm as a structural and druggable vulnerability, providing the mechanistic rationale for integrating DNMT inhibition with BCL-2 blockade in high-risk pediatric AML.