P19.04.B EXAMINATION OF THE ROLES OF KDM6A AND KDM6B HISTONE DEMETHYLASES IN ISOCITRATE DEHYDROGENASE (IDH) MUTANT GLIOMA

Abstract

Abstract Glioma, comprising 80% percent of all malignant brain tumors, is one of the most aggressive and difficult to treat tumors. Isocitrate dehydrogenase (IDH) is an enzyme that catalyzes the oxidative decarboxylation of isocitrate, producing α-ketoglutarate. Most IDH-mutant gliomas have a heterozygous point mutation in IDH1 that causes an arginine-to-histidine substitution at amino acid 132. Mutant IDH enzyme leads to 2-hydroxyglutarate accumulation in cells, inhibiting DNA and histone demethylases and ultimately generating a hypermethylated epigenetic phenotype. Considering that the mutant IDH enzyme generates a discrete epigenetic profile, we aimed to develop an epigenetic-based therapy approach for IDH mutant glioma. To this end, we performed chemical library screens consisting of inhibitors of important epigenetic enzymes and identified GSK-J4 as a chemical selectively targeting IDH1 mutant cells.GSK-J4 is a well-known inhibitor for histone demethylases KDM6A and KDM6B; therefore, we aimed to phenocopy the effects of the chemical approach with the genetic approach by depleting KDM6A and/or KDM6B using CRISPR/Cas9. We successfully established IDH-mutant primary glioma cell lines deficient in KDM6A and/or KDM6B expression, as revealed by qRT-PCR and Western Blots. Cells with double knockout were markedly slower in growth compared to controls which had received nontargeting gRNA or single gRNAs targeting each enzyme. In the future, we will examine changes in cell cycle and apoptosis pathways upon KDM6A and KDM6B knockouts and test the response of these cells to mutant IDH inhibitors, TMZ, or irradiation. In parallel, genome-wide changes in gene expression and cellular pathways, histone modifications, and chromatin accessibility will be analyzed, via RNA-seq, ChIP-seq, and ATAC-seq techniques, respectively.Even though KDM6A and KDM6B have been shown to have important but sometimes conflicting roles in various tumor types, no study showed their roles in IDH mutant glioma. Therefore, revealing the functional mechanisms of KDM6A and KDM6B in IDH-mutant gliomas will serve as an important step that leads to the development of novel epigenetic-based therapeutic approaches for IDH-mutant gliomas.This project is supported by TUBITAK with the given grant number: 219S882.