Glioma-derived mutations in IDH1 dominantly inhibit IDH1 catalytic activity and induce HIF-1alpha

Abstract

Heterozygous mutations in the gene encoding isocitrate dehydrogenase-1 (IDH1) occur in certain human brain tumors, but their mechanistic role in tumor development is unknown. We have shown that tumor-derived IDH1 mutations impair the enzyme's affinity for its substrate and dominantly inhibit wild-type IDH1 activity through the formation of catalytically inactive heterodimers. Forced expression of mutant IDH1 in cultured cells reduces formation of the enzyme product, alpha-ketoglutarate (alpha-KG), and increases the levels of hypoxia-inducible factor subunit HIF-1alpha, a transcription factor that facilitates tumor growth when oxygen is low and whose stability is regulated by alpha-KG. The rise in HIF-1alpha levels was reversible by an alpha-KG derivative. HIF-1alpha levels were higher in human gliomas harboring an IDH1 mutation than in tumors without a mutation. Thus, IDH1 appears to function as a tumor suppressor that, when mutationally inactivated, contributes to tumorigenesis in part through induction of the HIF-1 pathway.

Fig. 1

Tumor-derived IDH1 mutants have reduced catalytic activity because of impaired isocitrate binding. (A) Structural modeling predicts that mutation of R132 in IDH1 would weaken hydrogen bonding of the enzyme to ICT. Shown is a view of the catalytic active site of human IDH1 bound with NADP⁺ (omitted for clarity), ICT (green), and Ca²⁺. The residues interacting with ICT from the adjacent subunit are labeled with an apostrophe. Hydrogen-bonding interactions are indicated with dashed lines. Simulated H132 mutation (cyan) is superimposed on R132. (B) Tumor-derived IDH1 mutants have reduced catalytic activity in vitro. Left, FLAG-tagged wild-type and mutant IDH1 were expressed in HEK293T cells, purified by immunoprecipitation and eluted by FLAG peptide; right, HIS-tagged wild-type and mutant IDH1 were expressed in E. coli and purified by nickel resin. Specific IDH1 activities for all proteins were measured in the presence of NADP⁺ (10 µM) and ICT (30 µM), with the presence of 2 mM Mn²⁺. Shown are mean values of triplicate experiments ±SD. (C) Kinetic parameters of wild-type and mutant IDH1. Shown are mean values of duplicate experiments ±SD.

Fig. 2

The R132H mutation dominantly inhibits IDH1 activity and reduces cellular levels of α-KG. (A) The WT:R132H heterodimer of IDH1 has low specific activity. The specific activities of WT:WT, R132H:R132H, and WT:R132H dimers were measured under conditions of NADP⁺ (10 µM), ICT (30 µM), and 2 mM MnCl₂. Activities were normalized by protein levels, and wild-type activity was arbitrarily set as 100%. Shown are mean values of triplicate experiments ±SD. (B) A close-up view showing the conformational differences between the IDH1-NADP⁺ and IDH1-NADP⁺-ICT complexes at the active site. The enzyme adopts a quasi-open conformation in the IDH1-NADP⁺ complex (cyan) and a closed conformation in the IDH1-NADP⁺-ICT complex (yellow). The bound ICT and the side chains of several residues in IDH1 involved in ICT binding are shown. (C) The WT:R132H heterodimer loses cooperative binding to ICT. The activities of the WT:WT and WT:R132H enzymes were assayed with increasing concentrations of ICT in the presence of 100 µM NADP⁺ and 2 mM Mn²⁺. Shown are mean values of duplicate assays ±SD. The inset is an expanded view showing the IDH1 activities at lower isocitrate concentrations. (D) Cellular α-KG levels decrease with increasing IDH1^R132H expression. The upper panel is a Western blot showing expression levels of the transfected IDH1^R132H mutant in U-87MG cells. The α-KG level in cells transfected with empty vector was set as 100%, and this value was used to calculate the relative α-KG level in cells transfected with different amounts of IDH1^R132H mutant. Shown are mean values of triplicate assays ±SD.

Fig. 3

α-KG mediates the HIF-1α induction in cells with a decreased IDH1 activity (A) IDH1 knockdown elevates HIF-1α levels in U-87MG glioblastoma cells. IDH1 and HIF-1α protein levels were determined by Western blotting from stable U-87MG cells transduced with empty retrovirus or retrovirus expressing different shRNAs silencing IDH1. (B) Ectopic expression of the IDH1^R132H mutant elevates HIF-1α levels in U-87MG and HEK293T cells. The IDH1^R132H mutant was overexpressed in U-87MG or HEK293T cells, and protein levels were detected by Western blot. CoCl₂-treated cells (a mimetic of hypoxia) and cells overexpressing wild-type IDH1 were also included as controls. (C) A cell-permeable α-KG derivative blocks HIF-1α induction in cells expressing IDH1^R132H. The U-87MG cells were transfected with IDH1^R132H, and different concentrations of octyl-α-KG ester were added to each transfected cell for 4 hours. HIF-1α protein levels were assayed by Western blot.

Fig. 4

IDH1 activity affects the levels of HIF-1α and HIF-1α target genes in gliomas and cultured cells. (A) Overexpression of the IDH1^R132H mutant in U-87MG cells stimulates expression of HIF-1α target genes (Glut1, VEGF, and PGK1) as assayed by QPCR. Shown are mean values of triplicate assays ±SD. (B) Inhibition of IDH1 by oxalomalate activates HIF-1α target genes. U-87MG cells were either untreated (control), treated with 5 mM oxalomalate, an IDH1 inhibitor, or treated with CoCl₂, a hypoxia mimetic. HIF-1α target gene mRNAs were determined. Shown are mean values of triplicate assays ±SD. (C) Immunohistochemistry of HIF-1α was carried out in 12 human gliomas with wild-type IDH1 and 8 gliomas of similar grade harboring a mutated IDH1 allele. Shown are side-by-side comparisons of four gliomas representing different types or grades. Scale bar, 40 µM. Five fields (~173 µm² each) were randomly selected from each sample for quantification of HIF-1α-positive staining area. Statistical analysis was performed using seven IDH1 wild-type and seven IDH1-mutated gliomas.