CBFA2T3-GLIS2 establishes transcriptionally permissive enhancer hypermethylation associated with venetoclax response in pediatric AML

CBFA2T3-GLIS2 (C/G) defines a high-risk pediatric AML subtype in which de novo enhancer rewiring sustains an aggressive leukemic transcriptional state. The early epigenomic mechanisms reinforcing this program and linking it to leukemic survival remain incompletely defined. Here, we identify enhancer-proximal hypermethylation as a noncanonical, transcription-permissive feature of C/G AML. Using primary C/G-positive AML samples and a developmentally relevant C/G-transformed cord blood hematopoietic stem/progenitor cell model, we integrated DNA methylation, chromatin state, fusion occupancy, and gene expression across early and leukemia-stage contexts. Early C/G-associated hypermethylated regions localized near H3K27ac/H3K4me1-marked active or primed cis-regulatory elements, were enriched for ERG/ETS-like motifs, and showed related architecture in leukemia-stage contexts. DNMT3B was prominently increased in C/G contexts, and its loss selectively altered methylation-associated gene expression without measurably changing global 5mC. Genetic DNMT3B loss increased venetoclax sensitivity in vitro, while pharmacologic DNMT3B inhibition with Nanaomycin-A similarly enhanced venetoclax response. During venetoclax treatment, DNMT3B-deficient xenografts showed reduced disease burden and prolonged survival. To assess whether this interaction extended to clinically used hypomethylating agents, azacitidine or decitabine plus venetoclax induced apoptosis-related transcriptional programs and mitochondrial depolarization. These findings identify hypermethylated active cis-regulatory elements as C/G-associated states linked to transcriptional dysregulation and venetoclax response.