FIGURE 1 from D-2-HG Inhibits <i>IDH1<sup>mut</sup></i> Glioma Growth via FTO Inhibition and Resultant m6A Hypermethylation
Authors: Pianka Sean T. , Li Tie , Prins Terry J. , Eldred Blaine S.C. , Kevan Bryan M. , Liang Haowen , Zapanta Rinonos Serendipity , Kornblum Harley I. , Nathanson David A. , Pellegrini Matteo , Liau Linda M. , Nghiemphu Phioanh Leia , Cloughesy Timothy F. , Lai Albert 
Published: March 2024

<p>IDH1<sup>mut</sup> production of D-2-HG induces RNA m6A hypermethylation in gliomas. <b>A,</b><i>IDH1<sup>mut</sup></i> patient gliomas manifested RNA m6A hypermethylation, <i>IDH1<sup>mut</sup></i> (<i>n</i> = 12) versus <i>IDH1<sup>wt</sup></i> (<i>n</i> = 12; <i>P</i> ≤ 0.04). <b>B,</b><i>IDH1<sup>mut</sup></i> gliomaspheres exhibited increased m6A content, <i>IDH1<sup>mut</sup></i> (<i>n</i> = 4) versus <i>IDH1<sup>wt</sup></i> (<i>n</i> = 7; <i>P</i> ≤ 0.04). In NHA lines, U87 cells, and <i>IDH1<sup>wt</sup></i> gliomaspheres, <i>IDH1<sup>mut</sup></i> forced expression elevates intracellular D-2-HG content (<i>n</i> = 1; <b>C</b>) and induce m6A enrichment [ANOVA, <i>F</i>(1,27) = 115.0, <i>P</i> ≤ 0.0001; asterisks indicate <i>post hoc</i> Newman–Keuls comparison with vector for each cell type] (<b>D</b>). <b>E,</b> Octyl-D-2-HG treatment (0.5 mmol/L) of <i>IDH1<sup>wt</sup></i> gliomaspheres increased m6A content compared with PBS treatment controls (<i>n</i> = 6; paired <i>t</i> test; <i>P</i> ≤ 0.01). <b>F,</b> D-2-HG content in HK385, U87, and NHA cells following treatment with IDH1<sup>mut</sup> inhibitors (C35 = 2 µmol/L; AG881 = 1 µmol/L), quantified using D-2-HG enzymatic assay. <b>G,</b> Effect of IDH1<sup>mut</sup> inhibitor on m6A content in HK385, U87, and NHA cells with <i>IDH1<sup>mut</sup></i> forced expression [ANOVA, IDH1<sup>mut</sup> inhibitor treatment: <i>F</i>(8,32) = 12.9, <i>P</i> ≤ 0.0001; vector vs. <i>IDH1<sup>mut:</sup> F</i>(1,32) = 79.22, <i>P</i> ≤ 0.0001; inhibitor and <i>IDH1</i> status interaction: <i>F</i>(8,32) = 7.3, <i>P</i> ≤ 0.0001; asterisks indicate <i>post hoc</i> Newman–Keuls comparison with vector or between the groups as indicated by horizontal bars]. Pooled data from native <i>IDH1<sup>mut</sup></i> gliomaspheres (HK252, HK211) demonstrating a reduction in D-2-HG (one-tailed paired <i>t</i> test; <i>P</i> ≤ 0.04; <b>H</b>) and m6A content (paired <i>t</i> test; <i>P</i> ≤ 0.003; <b>I</b>) following IDH1<sup>mut</sup> inhibitor AG881 treatment (1.0 µmol/L) in comparison with DMSO control. <b>J,</b> Volcano plot showing the RADAR log<sub>2</sub> fold change in m6A expression against −log<sub>10</sub> converted <i>P</i> values between <i>IDH1<sup>mut</sup></i> and <i>IDH1<sup>wt</sup></i> U87, NHA, and gliomasphere cells. Transcript sites with an absolute value log<sub>2</sub> fold change of less than 0.5 are excluded (void region in between vertical, dashed red lines). The horizontal, dashed red line indicates the demarcation of RADAR <i>P</i> values <0.01. <b>K,</b> Venn diagram showing the total number of genes fitting TCGA criteria (TCGA <i>P</i> value <1.0 × 10<sup>−6</sup>; absolute value log<sub>2</sub> fold change in RNA expression of 1.0 or greater between <i>IDH1<sup>mut</sup></i> and <i>IDH1<sup>wt</sup></i> patients) and RADAR criteria (<i>P <</i> 0.01) for all transcripts. Note that the total RADAR gene number is slightly larger here than the totals in <b>J</b>, because this list includes all <i>P</i> < 0.01 transcripts (including those with low magnitude fold changes). <b>L,</b> Supervised hierarchical clustering heat map of intersectional TCGA and RADAR data between U87, NHA, and gliomasphere cell lines that conformed to m6A and RNA expression conventions (i.e., an increase in RADAR m6A expression corresponded to a decrease in TCGA RNA expression). Heat map colors represent sample (columns) natural log converted normalized RADAR m6A values as a difference from the transcript (rows) average across genotype. A transcript of interest, <i>ATF5</i>, is identified. *, <i>P</i> ≤ 0.05; **, <i>P</i> ≤ 0.01; ***, <i>P</i> ≤ 0.001; and ****, <i>P</i> ≤ 0.0001 compared with relevant controls. Unless otherwise stated, <i>P</i> values indicate unpaired Student <i>t</i> test comparisons with the control, or between two groups as indicated by the horizontal line.</p>