PROJECT SUMMARY/ABSTRACT Background: Fms-Like Tyrosine Kinase 3 (FLT3) is the most commonly mutated gene in acute myeloid leukemia (AML) and mutations in FLT3 are associated with high relapse rates. FLT3 tyrosine kinase inhibitors (TKIs) are clinically active in FLT3-mutant AML but duration of response is limited by the development of resistance. Mutations in NRAS and other activation of the RAS pathway are major mechanisms of resistance to FLT3 inhibitors and other AML targeted therapies. Rationale: We have identified suppression of the Mediator/RNA Pol II pathway augments FLT3 TKI-induced apoptosis in the setting of RAS activation. The overall goals of this project are: (1) to prioritize therapeutic targets in the Mediator/RNA Pol II pathway; and (2) to determine the essential Mediator/RNA Pol II-dependent transcriptional targets relevant to FLT3 TKI resistance. Methods: We will identify essential components of the RNA Pol II pathway needed for TKI resistance in AML and evaluate the efficacy of existing RNA Pol II pathway inhibitors in MAPK-activated AML cells. We will determine RNA Pol II-dependent transcriptional changes induced by MAPK activation and FLT3 inhibition in pre-clinical models and patient samples. We will functionally validate the essential downstream transcriptional targets critical to RAS-mediated FLT3 TKI resistance in AML. Expected Results: These studies will rigorously evaluate RNA Pol II as a novel treatment strategy in AML with aberrant RAS/MAPK signaling. If successful, this strategy can be quickly translated to clinical trials in AML and this general approach may also prove efficacious in other RAS-mutant cancers.