HGG-18. Manipulation of the RNA modification m6A to induce tumor-specific antigens in high-grade glioma

Abstract In tumors with limited actionable genomic mutations, products of RNA dysregulation offer potential targets for immunotherapy. RNA modifications regulate gene expression and RNA dysregulation in cancer. N6-methyladenosine (m6A) is the most abundant mRNA modification, and deposition is primarily dependent on the methyltransferase-like 3 (METTL3)-containing writer complex. m6A placement is reversible and detected by reader proteins that dictate the fate of marked mRNA. m6A contributes to isoform diversity by influencing RNA processing such as alternative splicing, isoform stability, and alternative polyadenylation. In high-grade glioma (HGG), RNA modifications are largely unexplored. We hypothesize that tumor-intrinsic RNA modifications can be leveraged to generate tumor-specific antigens targetable by immunotherapy. Analysis of RNA sequencing data from the Children’s Brain Tumor Network revealed greater RNA isoform variation in H3K27M diffuse midline glioma (DMG) tumors compared to histone wild-type and H3G34R HGGs (p 0.05). Among a panel of 65 genes that modulate RNA modifications, 50 showed significant expression differences between DMG and normal brain tissues (p 0.05). To overcome limitations of traditional short-read RNA sequencing, we performed Oxford Nanopore Technologies long-read RNA sequencing on 22 distinct patient-derived HGG cell lines (12 H3K27M, 7 H3 wild-type, and 3 H3G34R) to examine full-length mRNA structure. We next sought to influence antigen expression through manipulation of m6A using METTL3 inhibitor STC-15. Liquid chromatography/mass spectrometry confirmed global reduction of m6A by 81.7% (p 0.05), with preserved METTL3 protein expression by western blot. Direct long-read RNA sequencing of 2 separate DMG cell lines revealed enrichment of m6A across the gene body and at the 3’ end of mRNA that was depleted after STC-15 treatment. IRIS-long computational analysis was performed to identify isoform-specific tumor antigens on STC-15-treated cells to identify antigens induced by METTL3 inhibition. This study establishes a foundational strategy for future epitranscriptome-based therapeutic approaches.