IDH1 Mutation Induces Reprogramming of Pyruvate Metabolism

Abstract

Mutant isocitrate dehydrogenase 1 (IDH1) catalyzes the production of 2-hydroxyglutarate but also elicits additional metabolic changes. Levels of both glutamate and pyruvate dehydrogenase (PDH) activity have been shown to be affected in U87 glioblastoma cells or normal human astrocyte (NHA) cells expressing mutant IDH1, as compared with cells expressing wild-type IDH1. In this study, we show how these phenomena are linked through the effects of IDH1 mutation, which also reprograms pyruvate metabolism. Reduced PDH activity in U87 glioblastoma and NHA IDH1 mutant cells was associated with relative increases in PDH inhibitory phosphorylation, expression of pyruvate dehydrogenase kinase-3, and levels of hypoxia inducible factor-1α. PDH activity was monitored in these cells by hyperpolarized (13)C-magnetic resonance spectroscopy ((13)C-MRS), which revealed a reduction in metabolism of hyperpolarized 2-(13)C-pyruvate to 5-(13)C-glutamate, relative to cells expressing wild-type IDH1. (13)C-MRS also revealed a reduction in glucose flux to glutamate in IDH1 mutant cells. Notably, pharmacological activation of PDH by cell exposure to dichloroacetate (DCA) increased production of hyperpolarized 5-(13)C-glutamate in IDH1 mutant cells. Furthermore, DCA treatment also abrogated the clonogenic advantage conferred by IDH1 mutation. Using patient-derived mutant IDH1 neurosphere models, we showed that PDH activity was essential for cell proliferation. Taken together, our results established that the IDH1 mutation induces an MRS-detectable reprogramming of pyruvate metabolism, which is essential for cell proliferation and clonogenicity, with immediate therapeutic implications.

Figure 1. PDH activity is down-regulated in IDH1 mutant cells as a result of PDK-mediated phosphorylation

PDH activity (A), PDH phosphorylation at Ser293 and Ser300 (B) and PDK1-3 mRNA levels in U87IDHwt and U87IDHmut cells (C).

Figure 2. 2-HG-mediated increase in HIF-1α and PDK3 levels result in reduced PDH activity

Western blots and quantification for PDK3 (A,B) and HIF-1α (C,D) in U87 and NHA models. PDH activity in IDH1 wild-type cells treated with 2-HG in U87 (E) and NHA (F) models. Western blots and quantification for PDK3 (G,H) and HIF-1α (I,J) in U87 and NHA IDH1 wild-type cells treated with 2-HG.

Figure 3. Hyperpolarized ¹³C-MRS can detect reduced PDH activity in IDH1 mutant cells

Dynamic ¹³C-MRS array showing metabolism of hyperpolarized 2-¹³C-pyruvate in U87 (A) and NHA (B) models; insets illustrate the sum of all spectra within dotted region of the array. Hyperpolarized 5-¹³C-glutamate production in mutant and wild-type U87 (C) and NHA (D) models. 2-¹³C-PYR: 2-¹³C-pyruvate, 2-¹³C-PYR Hydrate: 2-¹³C-pyruvate hydrate, 1-¹³C-PYR: 1-¹³C-pyruvate, 1-¹³C-PYR Hydrate: 1-¹³C-pyruvate hydrate, 2-¹³C-LAC: 2-¹³C-lactate, 5-¹³C-GLU: 5-¹³C-glutamate.

Figure 4. Glucose and glutamine flux to glutamate is reduced in U87 IDH1 mutant cells

Schematic representation of ¹³C-labeling of glutamate and 2-HG derived from 1-¹³C-glucose and 3-¹³C-glutamine (A). Build-up of 3-¹³C-glutamate (3-¹³C-GLU) and 3-¹³C-2-HG following perfusion of U87IDHwt and U87IDHmut cells with 3-¹³C-glutamine (3-¹³C-GLN) with quantification of 3-¹³C-glutamate production shown in the inset (B). Build-up of 3-¹³C-lactate (3-¹³C-LAC), 4-¹³C-glutamate (4-¹³C-GLU) and a combined signal of 4-¹³C-glutamate and 4-¹³C-2-HG in U87IDHwt and U87IDHmut cells with quantification of the build-up of glutamate in right inset. Left inset illustrates data from cell extracts resolving 2-HG and glutamate (C).

Figure 5. Glutamate production from glucose is reduced in both U87 and NHA IDH1 mutant cells

Quantification of glutamate produced from 1-¹³C-glucose and 3-¹³C-glutamine (¹³C-GLU) and total glutamate levels (¹H-GLU) in U87 (A) and NHA (B) models. Quantification of 2-HG produced from 1-¹³C-glucose and 3-¹³C-glutamine (¹³C-2-HG) and total 2-HG levels (¹H-2-HG) in both models (C).

Figure 6. DCA inhibits clonogenicity of IDH1 mutant cells

Effect of DCA on PDH activity in U87 (A) and NHA (B) models. Effect of DCA on hyperpolarized 5-¹³C-glutamate production in U87 (C) and NHA (D) models. Effect of DCA on clonogenicity in U87 (E) and NHA (F) models.

Figure 7. DCA inhibits proliferation of patient-derived mutant IDH1 models

Effect of DCA on PDH activity (A), glutamate production (B) and cell proliferation (C) in BT54 and BT142 models.