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FIGURE 6 from D-2-HG Inhibits IDH1mut Glioma Growth via FTO Inhibition and Resultant m6A Hypermethylation

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posted on 2024-03-22, 14:21 authored by Sean T. Pianka, Tie Li, Terry J. Prins, Blaine S.C. Eldred, Bryan M. Kevan, Haowen Liang, Serendipity Zapanta Rinonos, Harley I. Kornblum, David A. Nathanson, Matteo Pellegrini, Linda M. Liau, Phioanh Leia Nghiemphu, Timothy F. Cloughesy, Albert Lai

ATF5 is a downstream mediator of the IDH1mut → D-2-HG ⊣ FTO axis leading to gliomasphere growth reduction. A, m6A called peaks of ATF5 mRNA from MeRIP-seq RADAR data aligned to the genome (GRCh38). Inset graph shows m6A enrichment in IDH1mut (salmon) versus IDH1wt (teal) glioma samples for a select coding (exon) region, indicated by the dashed box. IP indicates immunoprecipitated mRNA fraction containing m6A-enriched RNA. INPUT mRNA fraction indicates total mRNA. B, Increased m6A-enriched ATF5 mRNA content in patient IDH1mut gliomas (n = 13) detected by m6A immunoprecipitation qRT-PCR, compared with IDH1wt gliomas (n = 10) (P ≤ 0.05). C, Decreased ATF5 mRNA m6A content detected by ATF5 qRT-PCR in the same IDH1mut gliomas (P ≤ 0.008). D, Pooled data from IDHwt gliomaspheres (HK217 and HK250) showing induction of ATF5 mRNA m6A enrichment following IDH1mut forced expression and its subsequent attenuation following treatment with IDH1mut inhibitor AG881 (1 µmol/L). E, Pooled data from IDHwt gliomaspheres (HK217 and HK250) showing a reduction of ATF5 mRNA expression following IDH1mut forced expression and ATF5 mRNA expression rescue following AG881 (1 µmol/L) treatment. F, ATF5 protein levels are decreased in gliomaspheres following IDH1mut forced expression and are rescued following treatment with IDH1mut inhibitor AG881 (1 µmol/L). ATF5 protein expression was determined relative to α-tubulin protein loading controls. G, Octyl-D-2-HG (0.5 mmol/L) or FTO inhibitor FB23-2 (3 µmol/L) treatment reduced ATF5 protein expression in IDH1wt gliomaspheres. H,IDH1wt gliomaspheres (HK250) transfected with piLenti-siRNA-GFP, expressing shRNA for either NSC or two regions (A, B) of ATF5, show reduced ATF5 expression via Western blot analysis (left) and reduced sphere growth (right). I, Schematic representation of the IDH1mut ⊣ FTO axis resulting in m6A enrichment and subsequent degradation of ATF5 mRNA transcripts leading to increased apoptosis. (P ≤ 0.005). *, 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)

History

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.