PROJECT SUMMARY/ABSTRACT Kinase inhibitor therapy has made a minimal impact on the clinical treatment of patients with Acute Myeloid Leukemia (AML). We have shown that inhibition of the epigenetic regulator lysine-specific demethylase 1 (LSD1) augments the efficacy of kinase inhibition in AML, including drugs targeting FLT3, cKIT and JAK2. This occurs via the repression of the MYC super enhancer (MYC-SE), leading to a loss of MYC gene expression and consequently decreased expression of a pro-proliferative gene expression program. However, we lack a complete mechanistic understating of how kinase plus LSD1 inhibition produces this effect, or whether the drug combination has important effects that are independent from the MYC-SE. Our long-term objective is to establish the efficacy of kinase plus LSD1 inhibition in AML, translating this concept into new effective treatment for patients with AML. The overall objective of this proposal is to: 1) define the mechanistic basis for drug responses to FLT3 plus LSD1 inhibition in FLT3-mutant AML and 2) evaluate the potential of dual MEK and LSD1 inhibition in NRAS-mutant AML. Our central hypothesis is that the suppression of MYC-target genes is an essential mechanism of kinase plus LSD1 inhibition-mediated cell death. In Aim 1, we will investigate three possible mechanisms for FLT3 plus LSD1 inhibition-induced suppression of MYC target genes: 1) Via inactivation of the MYC-SE leading to decreased MYC gene expression resulting in a loss of MYC-target gene expression, 2) Via inhibition of LSD1-dependent activation of MYC-target genes and 3) through inhibition of signaling pathways down-stream of activated FLT3, resulting in a loss of MYC binding to the promoters of target genes. We will also perform correlative studies investigating these mechanisms in AML patients enrolled in the FRIDA trial, receiving FLT3 plus LSD1-inhbitor therapy. In Aim 2, we will evaluate the efficacy and mechanism of action of MEK plus LSD1 inhibitor therapy in NRAS-mutant AML, using an integrated evaluation of chromatin and signaling pathway dynamics. We will also employ multiple mouse models of NRAS-mutant AML including a patient-derived xenograft model. At the completion of these studies, our expected outcomes are to 1) have identified how FLT3 plus LSD1 inhibition drives cell death in FLT3-mutant AML and 2) establish the preclinical efficacy of dual MEK plus LSD1 inhibition in NRAS-mutant AML. These studies will provide key pre-clinical rationale for expanding the indications for kinase plus LSD1 inhibitor to a larger proportion of patients with AML.