DNA methylation epimutations in leukemia

About This Grant

Project Summary/Abstract Acute myeloid leukemia (AML) is a lethal hematologic malignancy caused by mutations in hematopoietic stem cells. Normal karyotype AML (NK-AML) accounts for ~50% of cases and presents unique challenges due to its clinical and genetic heterogeneity. Prior research has shown that NK-AML develops from pre-existing clonal bone marrow diseases, including clonal hematopoiesis (CH) and myelodysplastic syndromes (MDS). In some CH and MDS patients, transformation to AML occurs in the absence of new mutations, indicating that epigenetic factors play a role in AML development. Recent studies by our lab using long-read DNA sequencing investigated DNA methylation as a potential source of novel contributing factors to AML development. These studies discovered thousands of discrete blocks with Allele-Specific DNA methylation (ASM) in primary AML samples that are sequence-independent and distinct from imprinted genes. These allele-specific “epimutations” are remarkably stable and are conserved in patient-matched samples obtained at presentation and relapse. Epimutations were also identified in remission samples from patients with genetic evidence of clonal disease and in single-cell clones expanded from normal hematopoietic stem cells. Furthermore, recurrent epimutations are associated with differential chromatin accessibility at genes that regulate stem cell differentiation and self-renewal, including GATA2 and genes in the HOXB cluster. Based on this evidence, we hypothesize that these allele-specific DNA methylation epimutations are clonal events that arise in normal cells and can disrupt gene regulation and be selected for during leukemogenesis. In this proposal we will investigate the role of DNA methylation epimutations in NK-AML using primary human AML samples, normal hematopoietic stem cells, and model systems via 3 aims. Aim 1 will define the landscape of recurrent epimutations in NK-AML using long-read native DNA sequencing. We will assess epimutation frequency and stability and explore their functional impact via RNA-seq, histone modification profiling, and Fiber-seq chromatin accessibility profiling. Aim 2 will use high resolution chromatin structure analysis and targeted epigenome editing to investigate the mechanisms of allele-specific gene regulation at the GATA2 locus. This aim will then test the functional consequences of allele-specific GATA2 activity by generating human AML cells with allele-specific silencing of GATA2 and engrafting them in humanized mice. These critical experiments will directly test whether epigenetic allele-specific expression of GATA2 confers a selective advantage for human AML cells in vivo. Finally, we will develop a novel targeted sequencing assay capable of detecting both mutations and epimutations and determine whether this approach improves the identification of persistent AML-related clones predictive of relapse in NK-AML patients in remission after chemotherapy. Together these studies will define the functional role of DNA methylation epimutations in AML and provide a novel molecular tool for monitoring AML patients and predicting relapse risk.