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

DNA methyltransferase inhibitors (DNMTI) 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 downregulation 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 patients with MDS. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/19/3/451/F1.large.jpg.