PROJECT SUMMARY/ABSTRACT MLL-rearranged (MLL-r) leukemias account for 5-10% of human acute leukemia and is associated with poor prognosis. The unmet clinical needs and the lack of an effective targeted therapy to the MLL-r leukemias emphasize the need for novel regimens. Recent cancer epigenetics studies discovered a central role for the histone H3 lysine 79 (H3K79) methyltransferase DOT1L in MLL-r leukemogenesis. Important clinical responses have been noted with DOT1L inhibitor treatment as a single agent, however, it is expected that combination treatments will be necessary. Our preliminary studies based on a DOT1L-inhibitor sensitization screen in MLL-r leukemia have identified suppression of BAZ2A significantly increases the anti-leukemic activity of the DOT1L inhibitor. The objective of this application is to determine the critical epigenetic mechanisms that mediate the availability of SIRT1 to suppress oncogene expression in MLL-r leukemia. Our central hypothesis is that BAZ2A, a chromatin remodeling protein of rDNA loci, mediates redistribution of SIRT1 for histone deacetylation and silencing of MLL-r/DOT1L-driven oncogene. We will dissect the BAZ2A/SIRT1 chromatin targeting mechanisms (Aim 1), investigate the efficacy of DOT1L and BAZ2A combination therapies (Aim 2), and validate a novel saturation CRISPR protein scan technology for de novo discovery of the functional elements in DOT1L and BAZ2A (Aim 3). This study is innovative because (1) it introduces a novel concept of simultaneously targeting multiple components of an epigenetic network to efficiently suppress the cancer programs, and (2) it establishes a brand new genetic screen approach for a sub-protein level functional domain discovery. The impact of this research will be of significance because (1) it immediately provides novel therapeutic opportunities against the difficult-to-treat MLL-r leukemias, and (2) it will help identify novel functional elements in epigenetic regulators for future pharmaceutical targeting.