Abstract P16: CBFA2T3-GLIS2 fusion leads to a distinct DNA methylation enhancer landscape in pediatric acute myeloid leukemia
Authors: Akhilesh Kaushal, Pritam Biswas, Jason Farrar, Samrat Roy Choudhury
Journal: Blood Cancer Discovery (2024)

Abstract Pediatric acute myeloid leukemia (pAML) patients with CBFA2T3-GLIS2 fusions (C/G) have one of the worst clinical outcomes among all pAML subtypes, with poor responses to induction therapy, a high rate of relapse (around 90%), and 5-year survival rates of less than 20%. Although C/G is known to disrupt the balance of ERG-GATA1 transcription factors and de novo super-enhancer (SE) activities, the specific mechanisms by which this fusion protein rearranges DNA methylation (DNAm) to alter enhancer-transcriptional programs are still unknown. In this study, we analyzed the expression of C/G-specific SE-associated genes (n=829, Thirant et al., 2017) in C/G pAML (n=40) compared to normal bone marrow samples (n=71) and other major fusion subgroups of pAML (n=1476) using a published RNA sequencing dataset (Smith et al., 2020). The C/G-SE-associated genes with significantly different (log2FC, ≥±1) expression levels in patient specimens were analyzed for altered DNAm using the Infinium Methylation EPIC v2.0 BeadChip array in C/G+ M-07e cells, compared to CD34+ hematopoietic stem progenitor cells. We observed two groups of SE-associated genes that were differentially upregulated (n=27) or downregulated (n=15) in primary C/G-pAML samples. The C/G-SE-associated genes include BMP2, MED12L, GATA2, and ERG, which have previously been shown to play critical roles in the disease. The differentially expressed genes were primarily enriched for the TGF-β signaling, allograft rejection, and IL6-JAK-STAT3 signaling pathways as predicted by the hallmark MSigDB. The majority of the differentially methylated CpGs (mC) were hypermethylated (~82%) at the promoter region, while the ratio of hypermethylated to hypomethylated mC was nearly equal at the body (0.9) and intergenic regions (0.85). Of the 27 differentially upregulated C/G-SE-associated genes, 18 were hypermethylated, and 3 were hypomethylated at the promoters. Conversely, out of the 15 differentially downregulated C/G-SE-associated genes, 7 were hypermethylated, and 2 were hypomethylated at the promoters. To further investigate the propensity of hypermethylation at these promoters, we evaluated the expression of DNA methyltransferases (DNMT) and found that the C/G binds to the proximal promoters of DNMT1 and DNMT3B, which is consistent with the overexpression of these genes in primary C/G pAML samples. Finally, we generated a CRISPR knockout of DNMT3B in M-07e cells, which led to a decrease in the expression of DNMT3B target genes, including 2 C/G-SE-associated genes (TIMP3, TPSAB1) and reduced cell viability. Our findings demonstrate that the C/G fusion upregulates DNMT1 and DNMT3B to establish a hypermethylated landscape supporting leukemic enhancer-transcriptional programs. Future studies will provide deeper insights into the role of these methylated enhancers in maintaining leukemic phenotype, which could be targeted by combining inhibitors of DNA-methyltransferases and histone acetyltransferases. Citation Format: Akhilesh Kaushal, Pritam Biswas, Jason Farrar, Samrat Roy Choudhury. CBFA2T3-GLIS2 fusion leads to a distinct DNA methylation enhancer landscape in pediatric acute myeloid leukemia [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P16.