PROJECT SUMMARY Glioblastoma (GBM) is the most common malignant primary brain tumor in adults. Despite progress in understanding the genetic and epigenetic underpinnings, GBM remains an incurable disease with a median overall survival of approximately 15 months. Therefore, novel therapies are required to combat this disease. Immunotherapy treatments are effective in some cancers but have not shown promise in GBM therapy. Research Gap: One of the likely reasons for this is that we do not fully understand the epigenetic pathways controlling tumors and the tumor microenvironment. Our preliminary data identified a novel epigenetic regulator, polycomb group ring finger 1 (PCGF1), of the disialoganglioside GD2 CAR T cell antigen, which has been investigated in pediatric cancers but not GBM. To identify the epigenetic pathways controlling the expression of tumor antigens in GBM, we performed a pooled CRISPR screen for targets that control GD2 and identified PCGF1 as a novel regulator of GD2 levels. We discovered that PCGF1 knockdown (KD) selectively increases GD2 levels in GBM cells and not in untransformed cells, suggesting a mechanism that links PCGF1 and GD2 in rapidly proliferating GBM cells. Importantly, PCGF1 regulation of GD2 is functional since PCGF1 KD sensitizes GBM cells to GD2-CAR T cell killing in vitro and in vivo. Mice orthotopically bearing GBM cells with PCGF1 KD and GD2-CAR T cells survive longer than those with Control KD and GD2-CAR T-cells. We have found that standard-of-care treatment that includes temozolomide (TMZ) increases PCGF1 levels. Consistent with this, we discovered that PCGF1 levels are higher in PDX samples from tumors from recurrent GBM patients receiving TMZ/radiation than treatment naïve newly diagnosed GBM tumors. These findings suggest that the levels of PCGF1 vary with different treatments in GBM and can be used to stratify patients who will receive GD2- CAR T cell therapy within clinical trials. However, to do this we need to determ