Abstract 447: The IDH1 mutation in human glioblastoma and its effects on epigenetic modification and cell fate selection
Authors: Garrett Matthew C. , Mottahedeh Jack , Sperry Jantzen , Eskin Ascia , Coppola Giovanni , Shih Ya-shin , Lai Albert , Chou Arthur , Liau Linda , Prins Rob , Cloughesy Timothy , Wu Hong , Nelson Stanley , Kornblum Harley 
Published: May 2015

Abstract
Introduction: In GBM IDH mutations are associated with relatively prolonged survival compared to other subtypes. The mutations in both IDH1 result in the abnormal production of 2-hydroxyglutarate (2-HG). Although the subject of intense study, the function of 2-HG in GBM is not entirely understood. The investigation of IDH mutant tumors has been hampered by the lack of appropriate models. We have now utilized the neurosphere culture method to generate cells from 7 different tumors bearing IDH1 mutations. This study tests the validity of these lines. We utilize both these primary cultures and mouse neurospheres to test the hypotheses that mutant IDH1 is pro-proliferative and influences cell fate.
Methods: We successfully propagated and obtained microarray expression data on 7 neurosphere lines bearing IDH1 mutations and 61 IDH1 wildtype cell lines for comparison. We additionally performed methylation sequencing on 3 IDH1 mutant and 3 IDH1 wildtype lines to identify sets of genes that are consistently methylated and down-regulated in these IDH1mutant cell lines. We treated 6 IDH1 mutant lines with a small molecule IDH1 mutant inhibitor that prevents 2-HG formation to to determine effects on proliferation and whether down-regulated genes were rescued by the inhibition of 2-HG formation. Finally we treated fetal murine neural progenitors with exogenous 2-HG to determine the effects on differentiation status as well as on gene expression.
Results: Gene expression studies of the 7 IDH1 mutant neurosphere lines revealed 103 genes that were 4-fold down-regulated in IDH1mutant tumors. Of those 103 genes, 32 were also found to be methylated and down-regulated in IDH1mutant brain tumor samples found in public databases. Methylation sequencing revealed that 31 of these 32 genes were heavily methylated in our three IDH1mutant cell lines, while only 12 of these genes were methylated in the IDH1WT cell lines. Treatment of IDH1mutant neurosphere cultures with a small molecule inhibitor resulted in diminished growth capacity. Microarray analysis demonstrated that only one of the downregulated genes was was consistently rescued, Inhibitor of DNA binding 2 (ID2). Application of exogenous 2-HG to murine neurosphere cultures promototed the appearance of oligodendrocytes and neurons, and also induced downregulation of ID2 and upregulation of Olig 2.
Discussion: Our data suggest that there is likely a conserved set of “key gateway genes” that are frequently down-regulated in these IDH1mutant tumors and that patient-derived glioma cell lines maintain these epigenetic changes. Inhibition of 2-HG formation may be an effective therapy evidenced by the fact that tumor cells grow slower without 2-HG production. However inhibition of 2-HG production did not appear to reverse the majority of epigenetic changes in gene expression. The Id2 gene is consistently rescued by 2-HG inhibition and may be an important target for future tumor therapy.
Citation Format: Matthew C. Garrett, Jack Mottahedeh, Jantzen Sperry, Ascia Eskin, Giovanni Coppola, Ya-shin Shih, Albert Lai, Arthur Chou, Linda Liau, Rob Prins, Timothy Cloughesy, Hong Wu, Stanley Nelson, Harley Kornblum. The IDH1 mutation in human glioblastoma and its effects on epigenetic modification and cell fate selection. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 447. doi:10.1158/1538-7445.AM2014-447