PROJECT SUMMARY Ependymoma (EPN) is fatal in 50% of children and has not seen any therapeutic improvements in over 30 years. Standard therapy remains limited to surgery, radiation, and chemotherapy in limited situations. In the past decade our understanding of the molecular biology of EPN has been advanced by molecular analysis of patient samples but these advances have failed to improve outcomes. Genomic studies clearly define multiple subgroups including Posterior fossa A and B as well as supratentorial subgroups. Despite these advances the heterogeneity in EPN remains a problem. To address this problem, we performed single cell RNA sequencing analysis of EPN patient samples and identified an undifferentiated progenitor population that expresses high levels of pluripotent cell markers. In parallel we performed an unbiased epigenome-wide RNAi screen targeting 410 chromatin regulators to identify novel epigenetic regulators critical for EPN cell growth. We identified the Polycomb repressive complex 1 (PRC1) protein BMI1 as a top hit, that when depleted or chemically inhibited severely suppressed EPN cell growth. Interestingly BMI1 is most highly expressed in the undifferentiated population identified by our scRNA-seq and associated with worse outcomes. Importantly the functional significance of BMI1 has not previously been evaluated in EPN. We hypothesize that high expression of BMI1 promotes de-differentiation and self-renewal of the UEC subpopulation, thereby locking the cells in a highly tumorigenic stem-like state. To address our hypothesis, we will first investigate the inhibition of BMI1 function on PFA tumor cell self-renewal and differentiation of aggressive PFA subpopulations in vitro. We will then establish the therapeutic efficacy of targeting BMI1 with novel small molecule inhibitors in EPN in vivo. Completion of this work will establish a direct role for BMI1 in EPN stem cell differentiation and provide justification for use of BMI1 inhibitors in human pediatric clinical trials.