Project Summary/Abstract Somatic FMS-like tyrosine kinase 3 (FLT3) mutations are associated with a poor prognosis and increased rates of relapse in acute myeloid leukemia (AML). There is evolving evidence describing genetic-dependent mechanisms of resistance in FLT3-mutant AML following targeted therapy with FLT3 inhibitors, including the phenylalanine 691 to leucine (F691L) mutation that has been reported in patients treated with current generation of FLT3 inhibitors. While AML cells containing this mutation experience inhibition of kinase activity and canonical FLT3 signaling following exposure to FLT3i treatment, our preliminary data revealed that FLT3-F691L AML cells continued propagating leukemia both in vitro and in vivo. The perplexing finding that kinase activity canonical FLT3 signaling is suppressed, despite ongoing survival of the AML cells, suggested kinase independent signaling resulting from F691L substitution in FLT3. The long-term goal of this project is understanding the mechanism of resistance underlying the FLT3-F691L mutation, which we believe is not fully explained by gatekeeper functionality solely. The specific aims for this project are: (i) determine the effects of kinase-dependent and -independent signaling in FLT3-ITD and FLT3- F691L AML cells on differentiation, survival, and gene expression, (ii) identify the pathways and molecules mediating kinase-independent signaling in FLT3-F691L AML, and (iii) examine novel therapeutic vulnerabilities for FLT3-F691L AML and evaluate prioritized pharmacologic combination therapy to overcome resistance. The impact of this grant application is highly significant, as it will have translational implications, filling an unmet need for relapsed/refractory FLT3 mutant AML. If this mechanism of resistance holds true, this could inform drug development and combinatorial therapies for various pediatric cancer types in the long-term.