Degradation of D-2-hydroxyglutarate in the presence of isocitrate dehydrogenase mutations

Abstract

D-2-Hydroxyglutarate (D-2-HG) is regarded as an oncometabolite. It is found at elevated levels in certain malignancies such as acute myeloid leukaemia and glioma. It is produced by a mutated isocitrate dehydrogenase IDH1/2, a low-affinity/high-capacity enzyme. Its degradation, in contrast, is catalysed by the high-affinity/low-capacity enzyme D-2-hydroxyglutarate dehydrogenase (D2HDH). So far, it has not been proven experimentally that the accumulation of D-2-HG in IDH mutant cells is the result of its insufficient degradation by D2HDH. Therefore, we developed an LC-MS/MS-based enzyme activity assay that measures the temporal drop in substrate and compared this to the expression of D2HDH protein as measured by Western blot. Our data clearly indicate, that the maximum D-2-HG degradation rate by D2HDH is reached in vivo, as v_max is low in comparison to production of D-2-HG by mutant IDH1/2. The latter seems to be limited only by substrate availability. Further, incubation of IDH wild type cells for up to 48 hours with 5 mM D-2-HG did not result in a significant increase in either D2HDH protein abundance or enzyme activity.

Figure 1

Representative chromatograms of 2-HG and the internal standard 2-HG-d₃. (a) Standard sample (conc[iS] = 10 µM, conc[2-HG] = 14.1 µM), and (b) biological sample (cell culture supernatant of HCT116 IDH2R172K, conc[iS] = 20 µM, conc[2-HG] = 14.1 µM). iS = internal standard.

Figure 2

The D2HDH assay was optimized by testing different assay conditions using cell homogenates from MCF7 cells. In all plots, 2-HG concentration normalized to protein content per aliquot was set 100% at t = 0 min. (a) Frozen cell pellets yield lower enzymatic rates than freshly prepared cell pellets (n = 5 for both). (b) Impact of different redox equivalents: PMS shows a significantly higher degradation rate than FAD and INT (ANOVA: p = 2.04 × 10⁻¹⁵). (c) pH 7.0, pH 7.6, and pH 8.0 were compared to establish optimal pH conditions for the D2HDH activity assay. D-2-HG degradation rate was highest at pH 7.6 (ANOVA: p = 3.01 × 10⁻⁴), reflecting mitochondrial pH.

Figure 3

Enzyme activity and protein abundance of D2HDH was tested in three different cell lines. (a) Using MCF7 cells, a K_m of 26.4 µM (standard error 1.65; n = 3) was determined for D2HDH. (b) Comparison of three different cell lines shows D2HDH activity to be lower in HT1080 than MCF7 and C7H2 cells. (c) Relative protein abundance for D2HDH (normalized to MCF7) is different for the cell lines tested but does not reflect D2HDH enzyme activity. (n = 3, ANOVA p: 0.0007, TukeyHSD: HT1080 vs. C7H2 p = 8.20 × 10⁻⁶; MCF7 vs. C7H2 p = 9.23 × 10⁻⁴, MCF7 vs. HT1080 p = 2.51 × 10⁻⁴). For Western blot data see also Supplementary Fig. S4.

Figure 4

Enzyme activity of D2HDH in MCF7 does not change as a function of D-2-HG concentration and duration of treatment: (a) 24 h (ANOVA p = 0.7789) or (b) 48 h (ANOVA p = 0.0694) (n = 2 for each time point). D2HDH protein abundance in MCF7 after 24 h (c) and 48 h (d) of D-2-HG treatment, (n = 3) from Western blot. No significant difference in protein abundance was observed, ANOVA: p = 0.50478 (c) and p = 0.06892 (d), respectively.

Figure 5

Comparing degradation rates for D-2-HG. (a) Degradation rates of D-2-HG by D2HDH in MCF7 and different HCT116 cells are shown at v_max of MCF7 (n = 2–3). (b) Degradation rates were recalculated correcting for differences in D2HDH protein abundance. (c) D2HDH abundance by Western blot in cells of the HCT116 panel and in MCF7 cells were normalized to expression in parental HCT116. Expression differences are significant between parental HCT116 and IDH2-R140Q (TukeyHSD p = 0.0324) and for MCF7 against all HCT116 panel cell lines (n = 3–7, see Supplementary Table S4).

Figure 6

Comparison of D-2-HG formation by mutated IDH to D-2-HG degradation by D2HDH in cell-based enzyme assays in (a) HCT116 IDH1-R132H cells and (b) HCT116 IDH2-R172K cells (n = 2): Degradation capacity is found low and is not further increased at higher substrate concentrations. In this assay degradation is already at v_max with D-2-HG spike added on endogenous D-2-HG resulting in c(2-HG) > 200 µM. In contrast, formation of D-2-HG still increases upon increasing the concentration of α-ketoglutarate from 1 mM to 10 mM, proving a high D-2-HG production capacity. Again, endogenous D-2-HG raises the starting level in the 2-HG production assay. (c) Reaction velocity as a function of αKG-concentrations normalized to total protein for the HCT116 cell panel (wild type IDH and different IDH1/2 mutations).