DNA Hydroxymethylation Profiling Reveals that WT1 Mutations Result in Loss of TET2 Function in Acute Myeloid Leukemia

Abstract

Summary Somatic mutations in IDH1/2 and TET2 result in impaired TET2 mediated conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). The observation that WT1 inactivating mutations anti-correlate with TET2/IDH1/2 mutations in AML led us to hypothesize that WT1 mutations may impact TET2 function. WT1 mutant acute myeloid leukemia (AML) patients have reduced 5-hmC levels similar to TET2/IDH1/2-mutant AML. These mutations are characterized by convergent, site-specific alterations in DNA hydroxymethylation, which drive differential gene expression more than alterations in DNA promoter methylation. WT1 overexpression increases global levels of 5-hmC, and WT1 silencing reduced 5-hmC levels. WT1 physically interacts with TET2 and TET3, and WT1 loss of function results in a similar hematopoietic differentiation phenotype as observed with TET2 deficiency. These data provide a novel role for WT1 in regulating DNA hydroxymethylation and suggest that TET2 IDH1/2, and WT1 mutations define a novel AML subtype defined by dysregulated DNA hydroxymethylation.

Mutant IDH : A Targetable Driver of Leukemic Phenotypes Linking Metabolism, Epigenetics and Transcriptional Regulation

“…Compared to normal bone marrow controls and AML patients without IDH1m and IDH2m, these groups featured dominant cytosine hypermethylation patterns [25]. The hypermethylation signature was confirmed in studies of AML patients using Illlumina arrays [1] as well as enhanced reduced representation bisulfite sequencing (ERRBS) [26,27]. Mouse models expressing IDH1m recapitulated the hypermethylation signatures.…”

“…IDH1 and IDH2 mutations were mutually exclusive, and their associated cytosine hypermethylation and hydroxymethylation signatures were highly overlapping [11,25,26], suggesting a common downstream mechanism of action in AML. Likewise, differentially expressed genes in IDH1m and IDH2m patients compared with normal bone marrow controls also showed a significant overlap of gene sets involved in cell cycle, DNA repair processes as well as hematopoiesis [25] suggesting a role in leukemia pathogenesis.…”

“…AML patients with TET2 mutations tended to cluster with IDH1 and IDH2 mutant cases in DNA methylation profiling suggesting common targets of aberrant cytosine hypermethylation [25]. Furthermore, TET2m AMLs exhibited a dominance of regional 5hmC loss and aberrant gene expression signatures compared with normal bone marrow controls that highly overlapped with those of IDH1 and IDH2 mutant cases [25,26]. Given that TET2 is dependent on α-KG, and that 2-HG produced by IDH1m and IDH2m enzymes is structurally similar to α-KG [17], it was theorized that 2-HG might competitively inhibit TET2.…”

“…5hmC has been identified as a mark within intergenic regions, including distal regulatory elements (enhancers, CTCF binding sites, DNase hypersensitivity sites), near transcription factor bindings sites, as well as within genic regions [29][30][31][32][33][34][35][36]. Rampal et al [26] reported the first high-resolution genome-wide mapping of 5hmC patterns in AML patients. Genome-wide 5hmC profiling in IDHm AML patient samples revealed a dominant pattern of focal and regional 5hmC loss compared with nonmutated AMLs, although there was also a smaller subset of focal regions with increased 5hmC compared with normal bone marrow controls.…”

“…Further evaluation of the mutational landscape in AML revealed that mutations in the gene encoding the transcription factor WT1 are also almost mutually exclusive with mutations in IDH1, IDH2 and anticorrelated with mutations in TET2 [11,25,26,41]. DNA methylation profiling in WT1 mutant (WT1m) AML patient samples revealed a cytosine methylation pattern that featured hypermethylation compared with normal bone marrow controls.…”