Summary The temporal and spatial distribution of signaling proteins is dynamically regulated by post-translational modifications (PTMs). PTMs such as phosphorylation, ubiquitination, or lipid modification dictate protein activities and access to substrates, thereby cellular outcomes. The precise control of signaling pathways is critical to normal hematopoiesis and aberrant signaling leads to malignant transformation of hematopoietic stem and progenitor cells (HSPCs). This application is based on our novel finding that FLT3 (FMS-like tyrosine kinase 3) is palmitoylated and disrupting palmitoylation of oncogenic FLT3 mutants changes their subcellular localization, rewires downstream signaling, and promotes leukemic progression. Internal tandem duplication within FLT3 (FLT3-ITD) is one of the most frequent mutations in acute myeloid leukemia (AML) and correlates with poor prognosis. While wildtype FLT3 receptor tyrosine kinase is activated at the plasma membrane to transduce PI3K/AKT and RAS/MAPK signaling, FLT3-ITD resides in the endoplasmic reticulum (ER) and triggers constitutive STAT5 phosphorylation. Mechanisms underlying this aberrant FLT3-ITD subcellular localization or its impact on leukemogenesis remain poorly understood. We discovered that FLT3-ITD is S-palmitoylated by the ZDHHC6 acyltransferase. Disruption of palmitoylation redirects FLT3-ITD to the plasma membrane and rewires its downstream signaling by activating AKT and ERK pathways in addition to STAT5. Consequently, abrogation of FLT3-ITD palmitoylation via ZDHHC6 depletion promotes FLT3-ITD surface expression, signaling, and increased leukemic progression in xenotransplanted mouse models. Furthermore, we demonstrate that FLT3 proteins are palmitoylated in primary human AML cells. Stabilization of FLT3-ITD palmitoylation by pharmacological inhibition of depalmitoylation synergizes with FLT3 tyrosine kinase inhibitor (TKI) gilteritinib in abrogating the growth of primary FLT3-ITD+ AML cells. The central goal of this grant is to define the molecular basis underlying the regulation of oncogenic FLT3 signaling by palmitoylation and explore its physiological and functional significance in myeloid malignancies. We propose to define roles of ZDHHC6 in FLT3-ITD palmitoylation in vivo in mouse models of myeloproliferative neoplasm (MPN) and AML. We will also identify depalmitoylase(s) for FLT3-ITD that modify FLT3-ITD localization, and activity using combinatorial approaches of targeted and unbiased chemical biology, molecular biology, and genetics. Moreover, we will explore the therapeutic potential of targeting FLT3-ITD depalmitoylation in primary human FLT3-ITD+ AMLs. We will investigate if inhibition of FLT3-ITD depalmitoylase enhances responses to TKI using primary human AML cells as well as patient-derived xenotransplant (PDX) models. These findings provide novel insights into lipid- dependent compartmentalization of FLT3-ITD signaling and suggest targeting depalmitoylation as a new th...