FIGURE 2 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 inhibits gliomasphere growth. IDH1<sup>mut</sup> inhibitors (C35 = 2 µmol/L; AG881 = 1 µmol/L) enhance growth of <i>IDH1<sup>mut</sup></i><b></b>patient-derived gliomaspheres HK252 (<b>A</b>) and HK211 (<b>B</b>). <b>C,</b> Effect of <i>IDH1<sup>mut</sup></i> forced expression in <i>IDH1<sup>wt</sup></i> gliomaspheres (HK385, HK217, and HK250) on gliomasphere growth relative to vector control [ANOVA, <i>F</i>(1,1056) = 199.0, <i>P</i> ≤ 0.0001; asterisks indicate <i>post hoc</i> Newman–Keuls comparison with vector], showing decreased overall growth in <i>IDH1<sup>mut</sup></i>. <b>D,</b> Octyl-D-2-HG (0.5 mmol/L) and FTO inhibitor MA (100 µmol/L) treatment inhibited growth of <i>IDH1<sup>wt</sup></i> gliomaspheres (HK217 and HK250) [ANOVA, <i>F</i>(2,996) = 80.4, <i>P</i> ≤ 0.0001; asterisks indicate <i>post hoc</i> Newman–Keuls comparison of octyl-D-2-HG or MA with PBS treatment]. Top: IDH1<sup>mut</sup> inhibitor (C35 = 2 µmol/L, or AG881 = 1 µmol/L) treatment reduces intracellular D-2-HG content (<i>n</i> = 1) in <i>IDH1<sup>wt</sup></i> gliomaspheres [HK385 (<b>E</b>), HK217 (<b>F</b>), HK250 (<b>G</b>)] with <i>IDH1<sup>mut</sup></i> forced expression. D-2-HG levels in untreated vector gliomaspheres were below detection threshold and were not visible. Bottom: <i>IDH1<sup>wt</sup></i> gliomaspheres with <i>IDH1<sup>mut</sup></i> forced expression exhibited differences in gliomasphere growth [ANOVA, E: <i>F</i>(1,353) = 9.6, <i>P</i> ≤ 0.002, F: <i>F</i>(1,890) = 30.6, <i>P</i> ≤ 0.0001, G: <i>F</i>(1,833) = 73.0, <i>P</i> ≤ 0.0001], and gliomasphere growth. <i>IDH1<sup>mut</sup></i> forced expression spheres could be enhanced with IDH1<sup>mut</sup> inhibitor treatment [ANOVA, E: <i>F</i>(2,353) = 1.3, <i>P</i> ≤ 0.3, F: <i>F</i>(1,890) = 13.1, <i>P</i> ≤ 0.0003, G: <i>F</i>(1,833) = 14.8, <i>P</i> ≤ 0.0001]. Asterisks indicate <i>post hoc</i> Newman–Keuls comparisons between <i>IDH1<sup>mut</sup></i> and vector, or between groups indicated by the horizontal bars. <b>H,</b> Treatment with octyl-D-2-HG (1.0 mmol/L) or FTO inhibitors (FB23–2 = 3 µmol/L; MA = 100 µmol/L) appeared to induce apoptosis (green fluorescence), but not necrosis (red fluorescence), in U87 cells using an Abcam Apoptosis/Necrosis Assay Kit (10X magnification). Quantification of increased apoptotic Caspase 3/7 activity (detected with Promega Caspase-Glo 3/7 Assay kit) in <i>IDH1<sup>wt</sup></i> (<b>I</b>, <b>J</b>) and hemizygous <i>IDH1<sup>mut/</sup></i><sup>−</sup> BT142 (<b>K</b>) gliomaspheres following treatment with octyl-D-2-HG (0.5 mmol/L) or FTO inhibitor (FB23–2 = 3 µmol/L; MA = 100 µmol/L). <b>L,</b> Octyl-D-2-HG (0.5 mmol/L) or FTO inhibitor (FB23–2 = 3 µmol/L) treatment reduced PCNA expression in Western blots of <i>IDH1<sup>wt</sup></i> gliomaspheres. <b>M,</b><i>IDH1<sup>wt</sup></i> gliomaspheres (HK217 and HK250) exhibiting <i>IDH1<sup>mut</sup></i> forced expression demonstrated reduced PCNA expression via Western blot analysis. Treatment with IDH1<sup>mut</sup> inhibitor (AG881 = 1 µmol/L) was able to restore PCNA expression in <i>IDH1<sup>mut</sup></i><b></b>gliomaspheres, but not in vector controls. <b>N,</b> IDH1<sup>mut</sup> inhibitor (AG881 = 1 µmol/L) treatment enhanced PCNA expression in native <i>IDH1<sup>mut</sup></i> patient-derived gliomaspheres (HK211, HK252). PCNA protein expression was determined relative to GAPDH protein loading controls. *, <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>