FIGURE 3 from D-2-HG Inhibits IDH1^mut 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

Role of m6A demethylases FTO and ALKBH5 in mediating m6A hypermethylation and growth inhibition. A, Western blot images of FTO, ALKBH5 and α-tubulin protein content following DARTS assay in U87 cells. B, Normalized densities of Western blots in A, showing increased protected protein density in FTO lysates treated with D-2-HG over ALKBH5. The effect of FTO versus ALKBH5 lysates on protected protein density was evaluated via ANOVA [F(1,30) = 27.2, P ≤ 0.0001; asterisks indicate post hoc Newman–Keuls comparison within treatment of D-2HG, or between groups as indicated by the horizontal bars]. The densities of FTO and ALKBH5 DARTS products were normalized with parallel FTO and ALKBH5 Western blot densities of mock DARTS products (no protease added). C, Western blot analysis showing downregulation of FTO and ALKBH5 following shRNA knockdown in U87. D, Greater effect of FTO than ALKBH5 knockdown (shRNA) on increasing m6A content in U87. Treatment with shFTO and shALKBH5 was similar to shFTO only treatment. E, MTT assays demonstrating that shRNA treatment (shFTO, or shFTO and shALKBH5) reduces the viability of U87 cell lines [ANOVA, F(3,8) = 39.1, P ≤ 0.0001]. Treatment effect was heterogenous regarding FTO and ALKBH5, with shFTO demonstrating an effect on cell viability and shALKBH5 having no effect in comparison with a scrambled sequence (NSC; post hoc Newman–Keuls comparisons, indicated by the asterisks and brackets). Western blots showing downregulation of FTO and ALKBH5 following shRNA knockdown in HK217 (F) and HK250 (G) gliomaspheres. FTO knockdown (shRNA) has a greater effect on m6A content increases than ALKBH5 in HK217 (H) and HK250 (I) gliomaspheres. FTO knockdown (shRNA) is more effective at reducing sphere growth than ALKBH5 in HK217 (J) and HK250 (K) gliomaspheres. Western blots showing successful overexpression of FTO^wt and inactivated FTO^mut control in HK217 (L) and HK250 (M) gliomaspheres. FTO^wt overexpression increases gliomasphere growth in comparison with inactivated FTO^mut control in HK217 (N, P ≤ 0.0006) and HK250 (O, P ≤ 0.002) gliomaspheres. *, 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)

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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.

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Keywords

CNS Cancers Gliomas, Glioblastomas Oncogenes & Tumor Suppressors Epigenetics Cell Death And Senescence Effectors of apoptosis Genome Biology Comparative transcriptomics Gene Regulation Posttranscriptional and translational control Metabolism Metabolic pathways

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FIGURE 3 from D-2-HG Inhibits IDH1mut Glioma Growth via FTO Inhibition and Resultant m6A Hypermethylation