The long-term goal of this proposal is to define the contribution of altered epigenetic patterns and genome organization to the pathogenesis of acute myeloid leukemia. Acute myeloid leukemia (AML) is a devastating cancer that is initiated by somatic mutations in hematopoietic stem/progenitor cells. AML cells are also characterized by DNA methylation changes and altered gene expression patterns, but the relationships between AML mutations, DNA methylation, and transcriptional activity in AML are poorly understood. We have performed comprehensive epigenetic analysis to investigate the regulatory mechanisms that control expression of the HOX gene loci in AML cells, which encode transcription factors that maintain normal hematopoietic stem cell identity and promote self-renewal in AML. These studies have identified specific long-range three- dimensional (3D) genome interactions at the HOXA locus that are increased in AML vs. normal hematopoietic stem cells. Further analysis has showed that the loci involved in these interactions have AML-specific epigenetic changes suggesting they may be enhancers. We have extended these studies by performing a genome-wide analysis of DNA methylation and 3D genome architecture in primary AML samples. This demonstrated that AMLs with canonical mutations in either IDH1 or IDH2 have focal hypermethylation at enhancers that form direct interactions with genes relevant for AML pathogenesis, including MYC and ETV6. Based on these findings, we hypothesize that epigenetic changes at specific regulatory enhancers in AML cells can cause the dysregulation of genes that contribute to AML pathogenesis. Here we propose to test this hypothesis by performing detailed, mechanistic studies of enhancers and gene regulation in primary AML samples and AML cell line models. In Aim 1, we will use capture-HiC to perform in-depth studies of the HOXA locus in primary AML samples and AML cell lines that will define the relationships between AML mutations, enhancer interactions, and HOXA gene expression. We will then use massively parallel reporter assays, CRISPR/Cas9 mediated genome editing, and functional studies in vitro and in vivo to identify the specific enhancers and epigenetic pathways that regulate expression of HOXA genes. In Aim 2, we will use in situ HiC to define the 3D genome organization of primary AML samples with mutations in IDH1 and IDH2 that have focal DNA hypermethylation at enhancers. We will integrate these data with DNA methylation, chromatin profiling, and gene expression to determine how DNA methylation influences enhancer-promoter interactions and gene regulation in AML cells. Together, these studies will provide mechanistic insights into HOX gene regulation that may guide therapeutic approaches that target the HOX self-renewal pathway in AML cells, and determine the extent to which DNA methylation contributes to the leukemia phenotype by altering the function of regulatory enhancers.