Abstract PR006: CBFA2T3–GLIS2 fusion reprograms enhancer-linked DNA methylation to enforce apoptotic resistance and defines an epigenetic vulnerability in pediatric AML

Abstract The CBFA2T3–GLIS2 (C/G) fusion defines an exceptionally aggressive, epigenetically driven subtype of pediatric AML (pAML) confined to infants (<3 years). Arising from a pericentric inversion of chromosome 16, it is the most frequent lesion in acute megakaryoblastic leukemia (AMKL; ∼30%) and also occurs in non-AMKL subtypes, indicating lineage-independent pathogenicity. Clinically, C/G+ leukemia shows relapse rates > 90% and < 15% five-year survival, making it among the most treatment-refractory pediatric cancers. Lacking cooperating mutations, C/G acts as a singular oncogenic driver that reprograms the enhancer landscape, yet how it sustains epigenetic programs enforcing leukemic identity and chemoresistance remains unknown. We integrated multi-omic and functional epigenetic profiling across patient samples, C/G+ AML lines, and a developmentally faithful cord-blood CD34+ HSPC co-culture model. DNA methylation (DNAm) was mapped by meEM-seq, chromatin states by CUT&RUN and ATAC-seq, and transcription by RNA-seq. Functional studies included DNMT3B knockout, gRNA-resistant rescue, and inducible dCas9–TET1 demethylation, complemented by promoter-capture Hi-C and C/G+ xenograft validation. C/G+ pAML exhibited a promoter-biased hypermethylation signature (Δβ ≥ 0.2 vs NBM), with ∼50% of altered CpGs at promoters. The fusion bound the DNMT3B promoter, inducing its transcription without global 5mC gain but driving focal hypermethylation at active promoters linked via long-range enhancer loops. These hypermethylated promoters paradoxically stabilized expression of CRE-residing genes (BMP2, MED12, ITGB2) enriched for EMT, KRAS, heme-metabolism, and apoptotic pathways. In the fetal C/G–UCB-HSPC model, these signatures were faithfully recapitulated, confirming that the fusion alone reprograms the fetal hematopoietic epigenome. Motif analysis identified E2F2 and E2F4 as transcription factors stabilizing methylated yet transcriptionally active CREs. DNMT3B loss reduced locus-specific 5mC and target-gene expression but upregulated DNMT1 and UHRF1, suggesting a compensatory maintenance shunt that preserves DNAm at apoptotic loci and raises the apoptotic threshold. Targeted demethylation of PMAIP1 (NOXA) restored its transcription and apoptosis, confirming that DNAm stabilizes but constrains inducibility of pro-apoptotic genes. In vivo, DNMT3B-deficient xenografts regained Venetoclax sensitivity and extended survival, phenocopying DNMT inhibition (DNMTi). This study identifies C/G as an epigenetic architect converting DNAm from a repressive to a stabilizing signal, locking leukemic enhancers into an active yet apoptosis-resistant state. By defining the C/G–DNMT3B–DNMT1/UHRF1 axis and transcription-factor-mediated maintenance of methylated CREs, we reveal a unified mechanism linking developmental origin, transcriptional persistence, and therapy resistance in pAML. These findings position aberrant DNAm as a druggable epigenetic vulnerability and provide the mechanistic rationale for DNMTi + BCL-2 blockade in high-risk, treatment-refractory pAML. Citation Format: Samrat Roy Choudhury, Arundhati Chavan, Rhonda E. Ries, Giselle Almeida Gonzalez, Soheil Meshinchi, Jason Farrar. CBFA2T3–GLIS2 fusion reprograms enhancer-linked DNA methylation to enforce apoptotic resistance and defines an epigenetic vulnerability in pediatric AML [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr PR006.