FTO inhibition attenuates gliomasphere growth. A, Increase in U87 RNA m6A content relative to control (0 µmol/L) following the application of FTO inhibitors FB23-2 (left) or MA (right) at various doses. Dose-dependent inhibition of U87 cell viability by FTO inhibitors FB23-2 (B) and MA (C) evaluated by MTT assay. D, Time course of m6A enrichment in U87 cells treated with the FTO inhibitor FB23-2 (3 µmol/L, red) or DMSO (blue). One representative dot blot comparison between FB23-2–treated or DMSO-treated U87 cells is shown below the graph. Growth inhibition following FTO inhibitor treatment (FB23-2 = 3 µmol/L; MA = 100 µmol/L) in IDH1wt HK217 (E) and HK250 (F) gliomaspheres. G, Treatment of both vector control and IDH1mut forced expressing U87 cells with MA results in decreased viability (ANOVA, F(3,16) = 271.4, P ≤ 0.0001; asterisks indicate post hoc Newman–Keuls comparisons at each dose between IDH1mut and vector). H, Treatment of HK385 gliomaspheres with the FTO inhibitor FB23-2 (3 µmol/L) attenuates sphere growth in vector gliomaspheres but not those exhibiting IDH1mut forced expression [ANOVA, treatment: F(1,937) = 51.1, P ≤ 0.0001; vector vs. IDH1mut: F(1,937) = 29.4, P ≤ 0.0001; interaction: F(1,937) = 31.3, P ≤ 0.0001; asterisks indicate post hoc Newman–Keuls comparison with DMSO or between groups as indicated by horizontal bars]. I, IDH1mut inhibitor (C35 = 2 µmol/L; or AG881 = 1 µmol/L) treatment enhanced growth in IDH1mut forced expression HK252 gliomaspheres, thereby restoring sensitivity to FB23-2 (3 µmol/L) [ANOVA, IDHmut inhibitor treatment: F(2,1133) = 10.9, P ≤ 0.0001; FTO inhibitor treatment: F(1,1133) = 73.7, P ≤ 0.0001; interaction: F(2,1133) = 9.5, P ≤ 0.0001; asterisks indicate post hoc Newman–Keuls comparison with DMSO or between groups as indicated by the horizontal bars]. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; and ****, P ≤ 0.0001 compared with relevant controls. Unless otherwise stated, P values indicate unpaired Student t test comparisons with the control or between two groups as indicated by the horizontal line.
Funding
U.S. Department of Defense (DOD)
HHS | NIH | National Cancer Institute (NCI)
UC | UCLA | Jonsson Comprehensive Cancer Center (JCCC)
HHS | NIH | National Institute of General Medical Sciences (NIGMS)
ARTICLE ABSTRACT
IDH1mut gliomas produce high levels of D-2-hydroxyglutarate (D-2-HG), an oncometabolite capable of inhibiting α-ketoglutarate–dependent dioxygenases critical to a range of cellular functions involved in gliomagenesis. IDH1mut gliomas also exhibit slower growth rates and improved treatment sensitivity compared with their IDH1wt counterparts. This study explores the mechanism driving apparent reduced growth in IDH1mut gliomas. Specifically, we investigated the relationship between IDH1mut and the RNA N6-methyladenosine (m6A) demethylases FTO and ALKBH5, and their potential for therapeutic targeting. We investigated the role of D-2-HG and m6A in tumor proliferation/viability using glioma patient tumor samples, patient-derived gliomaspheres, and U87 cells, as well as with mouse intracranial IDH1wt gliomasphere xenografts. Methylation RNA immunoprecipitation sequencing (MeRIP-seq) RNA sequencing was used to identify m6A-enriched transcripts in IDH1mut glioma. We show that IDH1mut production of D-2-HG is capable of reducing glioma cell growth via inhibition of the m6A epitranscriptomic regulator, FTO, with resultant m6A hypermethylation of a set of mRNA transcripts. On the basis of unbiased MeRIP-seq epitranscriptomic profiling, we identify ATF5 as a hypermethylated, downregulated transcript that potentially contributes to increased apoptosis. We further demonstrate how targeting this pathway genetically and pharmacologically reduces the proliferative potential of malignant IDH1wt gliomas, both in vitro and in vivo. Our work provides evidence that selective inhibition of the m6A epitranscriptomic regulator FTO attenuates growth in IDH1wt glioma, recapitulating the clinically favorable growth phenotype seen in the IDH1mut subtype.
We show that IDH1mut-generated D-2-HG can reduce glioma growth via inhibition of the m6A demethylase, FTO. FTO inhibition represents a potential therapeutic target for IDH1wt gliomas and possibly in conjunction with IDH1mut inhibitors for the treatment of IDH1mut glioma. Future studies are necessary to demonstrate the role of ATF5 downregulation in the indolent phenotype of IDH1mut gliomas, as well as to identify other involved gene transcripts deregulated by m6A hypermethylation.
