P01.03 * FUNCTIONAL CHARACTERISATION OF THE ISOCITRATE DEHYDROGENASE 1 (IDH 1) R132H MUTATION IN GLIOMAS

Abstract

INTRODUCTION: Diffuse gliomas are the most prevalent brain tumors in adults and cannot be cured. Amino-acid substitution at position 132 of cytoplasmic enzyme isocitrate dehydrogenase 1 (IDH1) are characteristic for lower grade gliomas and therefrom derived secondary glioblastomas (GBM). The most frequent mutation observed in low grade gliomas is IDH1 c.395G>A (p.R132H). The wild type IDH1 converts isocitrate to α-ketoglutarate while reducing nicotinamide adenine dinucleotide phosphate (NADP). In contrast, IDH1 R132H produces high amounts of 2-hydroxyglutarate (2HG), which leads to a hypermethylation phenotype and is therefore considered to be an oncometabolite. Nevertheless, the accumulation of 2HG cannot explain all the observed phenomena in gliomas. We aimed to investigate other mechanisms by which IDH1 R132H mutation contributes to gliomagenesis. MATERIALS AND METHODS: We have stably transduced three cell lines (a established GBM cell line, a primary patient-derived GBM cell line, and an immortalized astrocyte cell line) with IDH1 c.395G>A cDNA using viral vectors. Transduction success was validated on DNA, RNA and protein levels using Sanger sequencing, allele-specific PCR and Western blots. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to measure Krebs cycle metabolite concentrations. In order to find effects independent of 2HG, we added the oncometabolite to empty vector controls. NADP+/NADPH quantification kits were used to determine the concentrations of this coenzyme. RESULTS: We confirmed the presence of IDH1 c.395G>A;p.R132H mutation in the cells transduced with the mutant variant of IDH1. There was an up to 1700-fold increase of the oncometabolite 2HG in the cells with the mutation compared to an empty vector control. The levels of glutamate significantly decreased in cells with the IDH1 mutation as well as in empty vector controls after 2HG treatment. We found a ∼50% decrease in NADPH concentration in GBM cells with the IDH1 mutation but not in astrocytes. Interestingly, there was a decrease in proliferation of GBM cells with IDH1 R132H. This effect was confirmed in 3D spheroid cell cultures. CONCLUSION: The mechanisms of how IDH1 R132H mutations contribute to gliomagenesis are still far from clear. We found that mutations in IDH1 lead to changes in Krebs cycle metabolite concentrations and energy homeostasis while decreasing proliferation in vitro. We are currently analyzing the effect of IDH1 R132H on the transcriptome level in comparison with IDH1 wt cells treated with 2HG. Based on these profiles we aim to investigate possible genetic mechanisms which are independet of the oncometabolite 2HG. These results will also be presented.