Abstract 3737: DNA-methylation is tightly linked with super-enhancer marks to upregulate ERG in ETO2-GLIS2 positive leukemia
Authors: Choudhury Samrat Roy , Bird Jordan T. , Byrum Stephanie , Farrar Jason E. 
Published: June 2022

Infants and young children (&gt; 3 yrs.) harboring ETO2-GLIS2 have the worst outcomes amongst all pediatric acute myeloid leukemia (AML) subtypes, with poor responses to induction therapy, high incidence of relapse, and dismal 5-year survival rates. Previous studies demonstrated that the ETO2-GLIS2 fusion leads to imbalance in the expression of ETS-related transcription factor ERG and GATA1 with neo-super enhancer (SE) activities in a single oncogenic hit (Thirant et al. 2017). ERG was found to co-localize with ETO2-GLIS2 fusion at 90% (372/419) of H3K27ac marked SE-regions and was strongly upregulated (9.44-fold). Herein, we aimed to investigate cooperativity of DNA methylation and chromatin marks on ERG overexpression in this AML subtype. We analyzed transcriptomic data from pediatric ETO2-GLIS2 AML patients (n=40) from dbGAP (phs000465.v19. p8) in comparison to normal bone marrow (NBM) (n=71) samples, confirming upregulation (median log FC: 2.4-fold) in ERG expression in the ETO2-GLIS2 subgroup compared to NBM. Next, we performed an unbiased genome-wide methylation array using Infinium MethylationEPIC 850K-arrays (Illumina). Differences in mean methylation (β-value) at individual CpG sites between NBM and AML samples were considered significant at differences greater than ±10%. We observed reduced methylation at 72% (5/7) CpGs at the ERG-promoter (1500bp upstream of transcription start site) in ETO2-GLIS2 subgroup compared to NBM. Overall median methylation across the promoter was reduced by 12% in the AML subgroup. In comparison, 22 differentially methylated CpGs were observed in the body, of which methylation increased at 68% (15/22) of the CpGs, with median methylation similarly increased by 15% in the AML patients. This represents a classic example of methylation-expression relation, having a promoter demethylation and body-hypermethylation, linked to upregulation of the gene-expression. Next, we investigated the overlaps between CpGs and the distribution of DNAse clusters and H3K27ac marks based on ENCODE data on myeloid leukemia cell line K562. We observed frequent overlaps between demethylated CpGs and DNAse clusters, while H3K27ac broad peaks were coincident with both demethylated CpGs at promoter or hypermethylated CpGs at body. Based on our findings, we thus conclude that DNA-methylation both at the ERG promoter and body is tightly linked to the overlapping chromatin marks of the gene. DNA-methylation might aid in opening of adjacent chromatin on ERG to possibly facilitate SE enrichment and over-expression the gene. Future studies will investigate targeted alterations of DNA-methylation on the gene to test the impact on ERG expression and leukemic growth.

Citation Format: Samrat Roy Choudhury, Jordan T. Bird, Stephanie Byrum, Jason E. Farrar. DNA-methylation is tightly linked with super-enhancer marks to upregulate ERG in ETO2-GLIS2 positive leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3737.