GENE-38. IDH1MUT INDUCES N6-METHYLADENOSINE (m6A) RNA HYPERMETHYLATION VIA D-2-HG IN GLIOMA

Abstract

Abstract INTRODUCTION IDH1 MUT confers slower tumor growth rates and improved prognoses for glioma patients, but the precise mechanisms underlying these clinically relevant benefits remain largely unknown. IDH1MUT gliomas produce high levels of D-2-HG, a putative oncometabolite that inhibits a range of α-ketoglutarate-dependent dioxygenases. D-2-HG-mediated inhibition of TET-family proteins involved in DNA demethylation is known to be a key driver leading to the oncogenic DNA hypermethylation phenotype (known as G-CIMP) seen in IDH1MUT gliomas. Recent evidence indicates that D-2-HG also functionally inhibits FTO and AlkBH5, two dioxygenases responsible for demethylation of RNA N6-methyladenosine (m6A) sites. This study sought to determine if D-2-HG mediates an increase in m6A content in IDH1MUT gliomas. METHODS Total RNA was isolated from patient tumor samples, patient-derived gliomaspheres, human embryonic kidney cell (HEK293T), normal human astrocyte (NHA), and U87 glioma model systems expressing either IDH1MUT or IDH1WT. Relative abundance of m6A modifications was determined both quantitatively and qualitatively using colorimetric ELISA-like assays and m6A-antibody dot blots, respectively. RESULTS Quantification of m6A abundance in IDH1MUT patient tumor samples and patient-derived gliomaspheres revealed an increase in m6A content compared to IDH1WT samples. Forced expression of IDH1MUT in HEK293T, NHA, and U87 cells increased intracellular D-2-HG content and global m6A abundance in purified RNA. Additionally, D-2-HG treatment of IDH1WT cell lines increased m6A abundance, including in IDH1WT gliomaspheres. Conversely, inhibition of D-2-HG generation in IDH1MUT cell lines decreased m6A abundance. Data will also be presented suggesting that increased m6A abundance is associated with decreased cellular proliferation. CONCLUSIONS These results indicate that increased intracellular D-2-HG arising in the context of IDH1MUT mediates increases in RNA m6A methylation in glioma. The association with m6A hypermethylation and reduced growth suggests that RNA methylation provides a novel therapeutic target.