HGG-20. PRMT5 promotes the formation and growth of pediatric high-grade glioma by maintaining tumor stem cell populations

Abstract

BACKGROUND: Pediatric high-grade gliomas (PHGG) are aggressive, undifferentiated CNS tumors comprising two broad subtypes: diffuse midline glioma with H3K27M mutations (DMG) and cortical high-grade glioma (H3K27-wild-type (wt) PHGG). During normal development, PRMT5 promotes stem cell self-renewal through methylation of arginine residues in histone tails. We hypothesized that PRMT5 controls self-renewal essential to the proliferation of PHGG tumor initiating cells (TICs). METHODS: We identified PRMT5 as potentially oncogenic in PHGG through a screen of 4,139 shRNAs targeting 406 genes with epigenetic activity. To elucidate PRMT5’s activity, we used lentiviral shRNA delivery to knock down (KD) PRMT5 expression in four DMG and one H3K27-wt PHGG cell lines. We performed in vitro growth, cell cycle, apoptosis, limiting dilution and bulk RNA-Seq assays to determine the phenotypic effects of PRMT5 KD. To identify PRMT5’s gene targets, we performed cleavage under targets & release using nuclease (CUT&RUN) followed by qPCR and are currently performing CUT&RUN-Seq. We orthotopically implanted PRMT5 KD PHGG cells into mice and tracked survival, tumor growth and tumor histological characteristics. RESULTS: In vitro, PRMT5 KD reduced cell growth (p<0.001), slowed cell cycle progression and increased apoptosis. PRMT KD also slowed neurosphere formation, demonstrating reduced self-renewal (p<7E-9). Geneset expression analysis showed PRMT5 KD reduced expression of self-renewal genes and increased expression of differentiation genes (FDR<0.0001). In vivo, PRMT5 KD reduced tumor growth, as monitored by bioluminescence and MRI, and aggressiveness, based on Ki-67 staining (p<0.05), leading to increased survival (p<0.001). CUT&RUN-qPCR results showed PRMT5 KD led to decreased expression and H3K4me3 promoter occupancy at PAX3, and decreased expression and increased H3K27me3 occupancy at S100A6. PAX3 and S100A6 are oncogenes that preserve TIC self-renewal. CONCLUSION. In vitro experiments show that PRMT5 KD epigenetically reduces TIC self-renewal. In vitro and in vivo, PRMT5 KD reduced PHGG tumor cell growth and aggressiveness.