PROJECT SUMMARY & ABSTRACT Enhancer of Zeste Homolog 2 (EZH2), the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), induces trimethylation of histone H3 lysine 27 (H3K27me3) for repressing the target gene expression. Numerous studies have reported that EZH2 promotes oncogenesis in a range of human cancers. In particular, independent studies have demonstrated the expression of EZH2 to be essential for tumorigenicity of acute leukemias with the Mixed Lineage Leukemia gene rearrangement (MLL-r), which accounts for approximately 60–80% of infantile and ~10% of adult acute lymphoblastic leukemia (ALL) cases, as well as ~50% of infantile and ~7% of adult acute myeloid leukemia (AML) cases. Leukemia patients with MLL-r generally exhibit very poor prognosis in the clinic, demanding new treatment strategies. However, increasing evidence including ours now support that EZH2’s oncogenic functions go beyond PRC2 and its enzymatic function for H3K27me3 deposition, and has the new, PRC2-independent activity (non-canonical) to sustain oncogenesis. The latter non-canonical function of EZH2 partly explains why the current existing enzymatic inhibitors of EZH2 have rather limited antitumor effect. In this project, we aim to employ the Proteolysis Targeting Chimera (PROTAC) technology to develop novel pharmacological ‘degraders’ for depleting EZH2 functions in cancer as a new and more effective therapeutic agent. We have generated highly promising preliminary results showing that, compared to the EZH2 enzymatic inhibitor, our lead E3 ligase-based EZH2-targeting PROTAC (aka EZH2 degrader) efficiently induced depletion of EZH2, thereby suppressing both canonical (PRC2-dependent) and non-canonical (PRC2- independent) activities of EZH2 in tumor. The EZH2 degrader also displays a much stronger potency in killing the aggressive MLL-r AML cells in vitro. Additionally, preliminary characterization of this lead compound revealed an excellent drug-like potential in mice. In this project, I will further (i) determine the effect and potency of our EZH2 degrader in suppressing tumor growth in vivo by using the genetically engineered mouse model (GEMM) and human patient-derived xenograft (PDX) models of MLL-r leukemias (Aim 1) and (ii) define its molecular effects in the MLL-r leukemia cells, focusing on both canonical and non-canonical functions of EZH2, by using the integrated genomic profiling technologies (Aim 2). Results from this project will reveal a promising preclinical strategy for the treatment of human cancers showing EZH2 dependency. Recent FDA approval of compounds targeting epigenetic proteins makes a strong argument.