This is a competing renewal application of an ongoing merit review program, focused on the mechanisms of leukemogenesis in acute myeloid leukemia (AML). The program aims to develop innovative new therapeutic approaches for AML by identifying new cellular targets in leukemic cells. This is of high clinical translational significance and relevance, as the outcome for most patients with AML remains very poor. Resistance of leukemia cells continues to be a serious problem and identifying pathways that can be therapeutically targeted is of high importance. Work from our laboratory has provided evidence for the existence of negative feedback regulatory loops in AML cells that are engaged in response to antileukemia drugs and mediate leukemic cell resistance. These include activation of pathways that regulate transcription and mRNA translation of pro- leukemogenic genes, and other regulatory negative feedback loops. In efforts to identify pathways associated with leukemic cell resistance, we discovered that there is constitutive activation of the ULK1 kinase in AML cells and identified novel phosphorylation sites that may mediate autophagy-independent activation of downstream effector pathways. Remarkably, we found evidence for interactions of ULK1 with proteins key in the pathophysiology of AML. These include RUNX1 and other core binding factor proteins and CHAF1b & CHAF1a. These unexpected findings raise the intriguing hypothesis that ULK1 directly regulates transcription factors that are important for leukemogenesis and play key roles in the pathophysiology of AML. The current proposal is a systematic approach to define the mechanisms by which ULK1 is engaged in leukemogenesis. The proposal aims to use such information towards identifying novel cellular targets in AML for the development of new therapeutic approaches. Specific aim 1 will define autophagy independent ULK1 effector pathways and will dissect their roles in leukemogenesis. Studies will be performed to examine the roles of unique ULK1 phosphorylation sites in the activation of downstream effectors, including RUNX1, CBFβ and the Core Binding Factor (CBF) complex. The requirement of ULK1 in the regulation of CHAF1b and CHAF1a in leukemogenesis will be assessed, and the functional role of the ULK1-S6K complex in mRNA translation and ribosome function in AML cells will be defined. Specific Aim 2 will determine the role of ULK1 and effector pathways in antileukemic responses in AML models in vivo. AML models will be established using ULK1 conditional knockout mice and AML patient-derived xenografts. The impact of ULK1, as well as ULK1 effector pathways in leukemogenesis and generation of antileukemic responses by hypomethylating agents and chemotherapeutic agents will be examined. Specific aim 3 will examine the antileukemic properties of novel ULK1 inhibitors on primary leukemic progenitors from AML patients. It will systematically study the effects of ULK1 inhibitors on primary cells from A...