5-Azacytidine Transiently Restores Dysregulated Erythroid Differentiation Gene Expression in TET2-Deficient Erythroleukemia Cells.

DNA methyltransferase inhibitors (DNMTIs) like 5-Azacytidine (5-Aza) are the only disease-modifying drugs approved for the treatment of higher-risk myelodysplastic syndromes (MDS), however less than 50% of patients respond, and there are no predictors of response with clinical utility. Somatic mutations in the DNA methylation regulating gene tet-methylcytosine dioxygenase 2 (TET2) are associated with response to DNMTIs, however the mechanisms responsible for this association remain unknown. Using bisulfite padlock probes, mRNA sequencing, and hydroxymethylcytosine pull-down sequencing at several time points throughout 5-Aza treatment we show that TET2 loss particularly influences DNA methylation (5mC) and hydroxymethylation (5hmC) patterns at erythroid gene enhancers and is associated with down-regulation of erythroid gene expression in the human erythroleukemia cell line TF-1. 5-Aza disproportionately induces expression of these down regulated genes in TET2KO cells and this effect is related to dynamic 5mC changes at erythroid gene enhancers after 5-Aza exposure. We identified differences in remethylation kinetics after 5-Aza exposure for several types of genomic regulatory elements, with distal enhancers exhibiting longer-lasting 5mC changes than other regions. This work highlights the role of 5mC and 5hmC dynamics at distal enhancers in regulating the expression of differentiation-associated gene signatures, and sheds light on how 5-Aza may be more effective in patients harboring TET2 mutations. Implications: TET2 loss in erythroleukemia cells induces hypermethylation and impaired expression of erythroid differentiation genes which can be specifically counteracted by 5-Azacytidine, providing a potential mechanism for the increased efficacy of 5-Aza in TET2-mutant MDS patients.