Functional and Molecular Consequences of the Dnmt3aR882H Mutation in Acute Myeloid Leukaemia

The de novo DNA Methyltransferase 3A (DNMT3A) gene is one of the most commonly mutated gene in acute myeloid leukaemia with a normal karyotype (AML-NK). Approximately two thirds of mutations are heterozygous and affect codon R882, located within the methytransferase domain. Also, we and others have shown that these mutations occur in a significant proportion of haematologically normal people and behave as leukaemia-initiating changes.

DNMT3A mediates the transfer of a methyl group from s-adenosylmethionine (SAM) to the 5' carbon of cytosine residues at CpG dinucleotides. The DNMT3A protein domains bind to unmethylated H3K4, a repressive histone mark, as well as the H3K36Me3 mark associated with active transcription. DNA methylation is linked to histone modifications and DNMT3A binds to class I and II histone deacetylases (HDAC) and histone methyltransferases (HMT), such as SETDB1 and G9a, as well components of Polycomb repressive complex 2 (PRC2).

DNMT3A^R882H has been reported to exert a dominant-negative effect on wild type (WT) DNMT3A, with a significant reduction of its enzymatic activity. In AML samples, the R882 mutation correlates with global hypomethylation, particularly at CpG islands, shores and promoters, although some promoter hypermethylation has also been reported. However, the molecular mechanism, through which DNMT3A^R882H alters methylation to drives leukaemia, remains unknown.

In order to understand the molecular and functional consequences of the DNMT3A^R882H mutation in leukaemogenesis we developed a mouse model (Dnmt3a^floxR882H/+), whereby targeting of the Dnmt3a allele does not inactivate the native locus. This allows conditional expression of the mutant protein within haematopoietic stem and progenitor cells that have developed in the presence of normal levels of Dnmt3a. Dnmt3a^floxR882H/+ mice were crossed with mice carrying an interferon-inducible Cre recombinase allele (Mx1-Cre). Upon Cre induction with pIpC the endogenous Dnmt3a exon 23 (last exon), was efficiently replaced by the human exon 23 carrying the R882H mutation. This was confirmed at both the DNA and the RNA level. In order to investigate the molecular effects of Dnmt3a^R882H, we analysed pre-leukaemic haematopoietic cells (in mice 5-6 weeks post Cre-induction). We observed that Dnmt3a^R882H/+ mutant bone marrow (BM) cells had markedly enhanced self-renewal potential as manifested by increased serial replating in cytokine supplemented methylcellulose media in comparison to WT mice. We next examined the relative haemopoietic reconstitution efficiency of Dnmt3a^R882H/+ BM cells in a competitive setting. Dnmt3a^R882H/+ BM (CD45.2) cells were mixed with WT competitor BM (CD45.1) cells at a ratio of 1:2 and transplanted into lethally irradiated recipient mice (CD45.1/45.2). The analysis of blood chimerism in recipients indicated an increased contribution from Dnmt3a^R882H/+ in comparison to WT cells, indicating the enhanced ability of Dnmt3a^R882H/+ BM cells to engraft and reconstitute recipient mice. Currently, we are addressing the role of Dnmt3a^R882H in the spontaneous leukaemia development alone as well as in rational combinations with other mutations present in human AML.

Additionally, to elucidate the molecular mechanism underlying the striking haematopoietic phenotypes ofDnmt3a^R882H/+ we have performed analyses of DNA methylation and hydroxymethylation patterns in the haematopoietic progenitor compartment (lineage negative cell population) of Dnmt3a^R882H/+ and WT mice, using reduced representation oxidative bisulfite sequencing (RRoxBS) in the pre-leukaemic setting. Moreover, due to cross-talk between Dnmt3a mediated DNA methylation and other chromatin states, we have analysed Dnmt3a^R882H-associated changes in histone tail modifications, using ChIP-Seq. As the DNA and histone modifications associated with Dnmt3a^R882H are likely to lead to alter global gene expression, we also performed RNAseq analysis in Dnmt3a^R882H/+ and WT mice.

Currently, we are conducting an integrated analysis, coupling DNA and histone modifications to aberrant gene expression and Dnmt3a^R882H-dependent phenotypes. We predict that this work will provide a detailed understanding of the molecular and cellular effects of DNMT3A-R882H and in so doing reveal rational therapeutic approaches for patients with this common and aggressive subtype of AML.