DNMT3A mutations mediate the epigenetic reactivation of the leukemogenic factor MEIS1 in acute myeloid leukemia

Abstract

Close to half of de novo acute myeloid leukemia (AML) cases do not exhibit any cytogenetic aberrations. In this regard, distortion of the DNA methylation setting and the presence of mutations in epigenetic modifier genes can also be molecular drivers of the disease. In recent years, somatic missense mutations of the DNA methyltransferase 3A (DNMT3A) have been reported in ~20% of AML patients; however, no obvious critical downstream gene has been identified that could explain the role of DNMT3A in the natural history of AML. Herein, using whole-genome bisulfite sequencing and DNA methylation microarrays, we have identified a key gene undergoing promoter hypomethylation-associated transcriptional reactivation in DNMT3 mutant patients, the leukemogenic HOX cofactor MEIS1. Our results indicate that, in the absence of mixed lineage leukemia fusions, an alternative pathway for engaging an oncogenic MEIS1-dependent transcriptional program can be mediated by DNMT3A mutations.

Figure 1

Complete DNA methylomes of DNMT3A wild-type and mutant AML cell lines. (a) Global DNA methylation levels in the DNMT3A mutant (OCI-AML3, outer circle) and wild-type (AML5, inner circle) cell lines analyzed by whole-genome bisulfite sequencing. Mean DNA methylation levels are displayed for 10 Mb genomic segments and all chromosomes. (b) Genome-wide analysis of DNA methylation levels at the CpG level (upper panel) and absolute number of hypermethylated (>0.66 methylation level; lower panel) CpG dinucleotides. (c) Genome-wide CpG methylation levels (upper panel) and DNA methylation profile exemplified by chromosome 1 (lower panel). (d) DNA methylation levels in DMRs hypomethylated in AML3. (e) Difference in promoter methylation (x axis; OCI-AML3 vs AML5) is associated with differential gene expression (y axis; OCI-AML3 vs AML5). Applied thresholds are indicated by dotted lines (δ DNA methylation >0.2; 1.5-fold change in gene expression). The 292 identified hypomethylated and overexpressed genes are highlighted in purple (upper left quadrant).

Figure 2

DNMT3A mutations in AML are associated with a DNA hypomethylation signature characterized by poor patient survival and MEIS1 induction. (a) Hierarchical clustered DNA methylation levels (green: 0% red: 100%) of 68 AML samples and 28 CpG sites significantly differentially methylated between DNMT3A mutant (red) and wild-type patients (blue). The red boxes indicate samples assigned to the DNMT3A mutant-related hypomethylated cluster. Differential survival analysis (5-year OS) of patients within (red line) or outside (blue line) the identified hypomethylated cluster (n=64, right panel). (b) Hierarchical cluster of the 28 CpG sites related to DNMT3A mutation in 194 primary AML patient samples. The red boxes indicate samples assigned to the DNMT3A mutant-related hypomethylated cluster. Differential survival analysis (5-year OS) of patients within (red line) or outside (blue line) the identified hypomethylated cluster in the independent patient cohort (n=139, right panel).

Figure 3

DNMT3A mutations in AML are associated with a DNA hypomethylation signature characterized by MEIS1 induction. (a) DNA methylation level of the 28 CpG sites among the 12 candidate genes in the OCI-AML3 and AML5 cell lines analyzed by DNA methylation array (upper panel). Relative gene expression levels of the 12 genes related to DNMT3A mutation profiled by quantitative PCR in OCI-AML3 (black) and AML5 (gray) cell lines (lower panel). (b) Protein levels of MEIS1 in OCI-AML3 and AML5 cells analyzed by immunoblotting (top left panel). Quantitative chromatin immunoprecipitation assay to assess DNMT3A occupancy at the MEIS1 studied CpG sites in AML5 cells. Data are presented as fold enrichment±s.e.m. Data of four independent experiments are shown. Significance of Student's t-tests is shown. IgG, immunoglobulin G (top right panel). Standard deviations are indicated by error bars. Bottom panel, association between the DNMT3A mutation-related differentially methylated CpG sites in MEIS1 and transcriptional activity using matched data from 170 primary AML samples (Cancer Genome Atlas Research Network). Mean DNA methylation levels over the five CpG sites of MEIS1 and gene expression levels in DNMT3A mutant patients are shown.