Epigenetics of TET2 Loss in Myelodysplastic Syndromes

Abstract

Myelodysplastic syndromes (MDS) are a class of myeloid malignancy characterized by peripheral blood cytopenias and impaired hematopoietic differentiation. Our understanding of the molecular basis of MDS has improved enormously in recent years due to clinical research efforts to characterize the spectrum of acquired mutations found in patients. This work has revealed that mutations in TET2 are common lesions in MDS and other myeloid malignancies. TET2 function has only recently been elucidated: TET proteins convert 5’-methylcytosine (mC) first to 5’-hydroxymethylcytosine (hmC), apparently the first step in an active DNA demethylation program that leads to the replacement of 5-mC with unmodified cytosine. My thesis work focuses on a characterization of TET2 loss on DNA methylation, and on how TET2 mutations impact patient response to treatment with hypomethylating agents. We examined DNA methylation in a matched set of TET2-WT and -mutant MDS samples, and found that loss of TET2 results in global hypermethylation. This global increase is due to gains in intragenic methylation, specifically localized to intron-exon boundaries. We then used clonal TF1 cell lines with CRISPR/Cas9-engineered TET2 mutations to examine global DNA hydroxymethylation. Loss of TET2 results in a global loss of 5-hmC. By aligning our methylation data with hydroxymethylation data from TET2-WT cells, we were able to identify direct TET2 targets. Because changes in mC/hmC with loss of TET2 appeared to localize to intron-exon boundaries, we investigated the effect of aberrant methylation on mRNA splicing in our TF1 cell system. TET2 loss resulted in an overall increase in exon skipping, consistent with published data on the effect of methylation on splicing, and hypermethylated regions were enriched for alternate splicing events. These findings suggest that the alterations in hematopoietic differentiation seen in TET2-mutant models are due to shifts in the expression of different mRNA isoforms rather than wholesale changes in gene expression. Our data show that loss of TET2 function results in region-specific gains in DNA methylation, and that these alterations affect mRNA splicing by promoting exon skipping. Finally, we have found that presence of TET2 mutations are positively associated with response to HMA therapy.