Glioma is the most common primary adult brain cancer, affecting over 20,000 Americans each year. Though past molecular characterizations defined glioma subtypes exhibiting different disease trajectories and treatment responses, there remains a paucity of personalized therapeutic approaches. The goal of this study is to delineate and exploit a putative mechanism of decreased malignancy governed by RNA hypermethylation. Gliomas exhibiting characteristic mutations in isocitrate dehydrogenase 1 (IDHMUT) grow significantly slower than IDHWT tumors. This confers a clear clinical advantage. IDHMUT disrupts normal IDH1 enzymatic activity, leading to the production of an oncometabolite called D-2-hydroxyglutarate (D2HG). D2HG directly inhibits ?- ketoglutarate-dependent dioxygenases involved in a range of cellular functions, including demethylation of DNA and RNA. D2HG-mediated inhibition of TET-family DNA demethylases leads to hypermethylated DNA profiles in IDHMUT gliomas, with the degree of methylation being positively associated with improved survival. FTO and AlkBH5 are the only two known RNA demethylases, and both are sensitive to D2HG-mediated inhibition. However, RNA hypermethylation has not been previously investigated in glioma. This proposal aims to perform the first `epitranscriptomic' profiling of IDHMUT and IDHWT gliomas, evaluating mRNA methylation in patient tumor samples and patient-derived glioma stem cells. RNA methylation occurs most frequently in the form of N6-methyladenosine (m6A), and is linked to a number of post-transcriptional processes including mRNA stability, intracellular localization, and translation. I will employ a recently developed methodology known as m6A RNA immunoprecipitation sequencing (MeRIP-Seq) to study m6A abundance and localization with transcript-level resolution. MeRIP-Seq is a modified version of RNA-Seq, and I will utilize both techniques to identify m6A-enriched transcripts and quantify overall transcript abundances using these respective approaches. This will allow me to investigate whether IDHMUT gliomas exhibit a reproducible set of m6A- enriched transcripts, and evaluate the effect of m6A enrichment on transcript stability. I will also assess m6A profiles in patient tumors as a biomarker of improved outcomes. To elucidate whether m6A hypermethylation is a key factor in reducing cell proliferation as seen in the clinically favored IDHMUT subtype, I will inhibit FTO in IDHWT glioma cells and determine if inhibition recapitulates the IDHMUT m6A hypermethylation phenotype, decreases tumor proliferation, and increases overall survival. Taken together, this work will provide insight into the mechanism of m6A-mediated gene regulation and malignancy in glioma, while evaluating the clinical efficacy of FTO inhibition as a novel therapeutic approach.
Glioma is a devastating form of brain cancer that remains incurable in nearly all patients. I propose that RNA methylation plays an important role in determining molecular phenotypes associated with malignancy and proliferative potential. This work will yield mechanistic insights into RNA methylation in glioma, and examine whether methylation regulators are efficacious treatment targets.
