FIGURE 2 from D-2-HG Inhibits IDH1^mut Glioma Growth via FTO Inhibition and Resultant m6A Hypermethylation

IDH1^mut production of D-2-HG inhibits gliomasphere growth. IDH1^mut inhibitors (C35 = 2 µmol/L; AG881 = 1 µmol/L) enhance growth of IDH1^mutpatient-derived gliomaspheres HK252 (A) and HK211 (B). C, Effect of IDH1^mut forced expression in IDH1^wt gliomaspheres (HK385, HK217, and HK250) on gliomasphere growth relative to vector control [ANOVA, F(1,1056) = 199.0, P ≤ 0.0001; asterisks indicate post hoc Newman–Keuls comparison with vector], showing decreased overall growth in IDH1^mut. D, Octyl-D-2-HG (0.5 mmol/L) and FTO inhibitor MA (100 µmol/L) treatment inhibited growth of IDH1^wt gliomaspheres (HK217 and HK250) [ANOVA, F(2,996) = 80.4, P ≤ 0.0001; asterisks indicate post hoc Newman–Keuls comparison of octyl-D-2-HG or MA with PBS treatment]. Top: IDH1^mut inhibitor (C35 = 2 µmol/L, or AG881 = 1 µmol/L) treatment reduces intracellular D-2-HG content (n = 1) in IDH1^wt gliomaspheres [HK385 (E), HK217 (F), HK250 (G)] with IDH1^mut forced expression. D-2-HG levels in untreated vector gliomaspheres were below detection threshold and were not visible. Bottom: IDH1^wt gliomaspheres with IDH1^mut forced expression exhibited differences in gliomasphere growth [ANOVA, E: F(1,353) = 9.6, P ≤ 0.002, F: F(1,890) = 30.6, P ≤ 0.0001, G: F(1,833) = 73.0, P ≤ 0.0001], and gliomasphere growth. IDH1^mut forced expression spheres could be enhanced with IDH1^mut inhibitor treatment [ANOVA, E: F(2,353) = 1.3, P ≤ 0.3, F: F(1,890) = 13.1, P ≤ 0.0003, G: F(1,833) = 14.8, P ≤ 0.0001]. Asterisks indicate post hoc Newman–Keuls comparisons between IDH1^mut and vector, or between groups indicated by the horizontal bars. H, 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 IDH1^wt (I, J) and hemizygous IDH1^mut/− BT142 (K) gliomaspheres following treatment with octyl-D-2-HG (0.5 mmol/L) or FTO inhibitor (FB23–2 = 3 µmol/L; MA = 100 µmol/L). L, Octyl-D-2-HG (0.5 mmol/L) or FTO inhibitor (FB23–2 = 3 µmol/L) treatment reduced PCNA expression in Western blots of IDH1^wt gliomaspheres. M,IDH1^wt gliomaspheres (HK217 and HK250) exhibiting IDH1^mut forced expression demonstrated reduced PCNA expression via Western blot analysis. Treatment with IDH1^mut inhibitor (AG881 = 1 µmol/L) was able to restore PCNA expression in IDH1^mutgliomaspheres, but not in vector controls. N, IDH1^mut inhibitor (AG881 = 1 µmol/L) treatment enhanced PCNA expression in native IDH1^mut patient-derived gliomaspheres (HK211, HK252). PCNA protein expression was determined relative to GAPDH protein loading controls. *, 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.