PROJECT SUMMARY/ABSTRACT Acute myeloid leukemia (AML) is a hematological cancer characterized by the quick proliferation and accumulation of immature myeloid cells that are impaired to differentiate into normal blood cells. As one of the deadliest subtypes of leukemia, AML is often driven by chromosomal translocations that fuse the multiple lineage leukemia gene MLL1 to either of the YEATS domain-containing proteins, ENL or AF9. Recent studies have indicated that ENL but not AF9 is essential for the maintenance of MLL-rearranged leukemia cells, making ENL as a valuable target for the development of therapeutics for AML. The ENL YEATS domain serves a critical role in the recognition of histone lysine acetylation in chromatin. By developing small molecules that selectively target the ENL YEATS domain to inhibit its recognition of histone acetylation in chromatin, multiple compounds have been identified that inhibit the growth of MLL-rearranged leukemia cells. Encouraged by this strong preliminary study, the current application is focused on expanding the drug discovery endeavor in targeting ENL for the development of MLL-rearranged leukemia therapeutics by pursuing three short-term specific aims. In the first aim, NanoBRET systems will be developed for ENL and its close paralogue AF9 for the analysis of ENL inhibitors in their cellular permeability, stability, selectivity, and drug residence time in cells. In the second aim, thorough characterization of developed small molecule ENL inhibitors will be conducted and obtained knowledge will be used for the development of novel inhibitors with improved potency and selectivity. In the third aim, a currently booming drug discovery concept, proteolysis targeting chimera (PROTAC), will be applied to the ENL drug discovery effort for the development of ENL-targeting PROTAC molecules. The success of the project will make a number of potential MLL-rearranged leukemia therapeutics available for further preclinical and clinical evaluations.