PROJECT SUMMARY Our proposal investigates a mechanism underlying innate immune dysfunction in acute myeloid leukemia (AML), the leading cause of leukemia-related deaths in the U.S. The innate immune system naturally defends against malignancy, however AML evades immunosurveillance to drive disease progression. Natural killer (NK) cells are the primarily innate lymphoid cell (ILC) responsible for anti-tumor immune surveillance, and reduced NK cell function both in de novo AML and in the post-transplant setting is correlated with poor outcomes. We have recently discovered that AML patients carry a fundamental defect in NK cell development leading to specific depletion of a sub-population of NK cells with critical roles in coordinating innate and adaptive immune responses, as well as mature NK cell development and function. We have shown that NK cells develop from a common innate lymphoid cell precursor (ILCP), which generates a series of NK developmental intermediates (NKDIs) leading to mature, cytotoxic NK cells. ILCPs also give rise to the other members of the ILC family, a diverse group of non-cytotoxic, cytokine-producing “helper” ILCs that are known to be pro-tumorigenic. Our preliminary studies show that AML disrupts the NK lineage, shifting production towards helper ILCs. As these populations all stem from the ILCP, this suggests AML is acting on ILCPs to alter lineage fate specification. Lineage specification occurs through carefully controlled activities of transcription factors that modify the epigenomic landscape generating stable cell type-specific gene expression patterns. Our preliminary studies have uncovered an aberrant, helper ILC-like DNA methylation signature in NKDIs isolated from AML patients and following leukemic cell co-culture. One key transcription factor is the aryl hydrocarbon receptor (AHR), which we have found shifts the helper ILC/NK ratio in the presence of AHR ligands ectopic produced by AML cells. We propose a strategy where the combination of AHR inhibition and hypomethylating agents (HMAs) guides development to restore NK cell differentiation from the ILCP. In this proposal, we will determine how AML drives this fate decision and promotes the generation of helper ILCs by performing detailed epigenetic and functional analyses of ILCPs isolated from normal donors and AML patients, including investigation in an immunocompetent murine AML model. We will investigate functional and epigenetic poising of lineage fate including the role of AHR. Secondly, we will determine the relationship of the NK cell defect in AML patients to epigenetic programming and disease progression, and directly test the impact of HMAs on ILCP and NKDI development. We will also determine the preclinical efficacy of combining both HMA and a novel AHR inhibitor to restore normal NK cell epigenetic programming and enhance NK cell generation to improve outcomes in preclinical models of AML. Maintaining functionally mature NK cells and supporting im...