Inhibition of glutaminase preferentially slows growth of glioma cells with mutant IDH1

Abstract

Mutation at the R132 residue of isocitrate dehydrogenase 1 (IDH1), frequently found in gliomas and acute myelogenous leukemia, creates a neoenzyme that produces 2-hydroxyglutarate (2-HG) from α-ketoglutarate (α-KG). We sought to therapeutically exploit this neoreaction in mutant IDH1 cells that require α-KG derived from glutamine. Glutamine is converted to glutamate by glutaminase and further metabolized to α-KG. Therefore, we inhibited glutaminase with siRNA or the small molecule inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and found slowed growth of glioblastoma cells expressing mutant IDH1 compared with those expressing wild-type IDH1. Growth suppression of mutant IDH1 cells by BPTES was rescued by adding exogenous α-KG. BPTES inhibited glutaminase activity, lowered glutamate and α-KG levels, and increased glycolytic intermediates while leaving total 2-HG levels unaffected. The ability to selectively slow growth in cells with IDH1 mutations by inhibiting glutaminase suggests a unique reprogramming of intermediary metabolism and a potential therapeutic strategy.

Figure 1

Pathway showing production of 2-HG from glutamine and inhibitor targets.

Figure 2

Validation of Tet-Inducible, Stable D54 Glioblastoma Lines. A. Western blot showing doxycyline-induced expression of 6X-His-Tag-IDH1. B. 2-hydroxyglutarate LC/MS retention peaks. C. 2-HG and α-KG levels measured by LC/MS.

Figure 3

Mutant IDH1 cells depend on glutaminase for cell growth and glutaminase inhibition is negated by dimethyl-α-ketoglutarate. A. Anti-glutaminase siRNA (siGLS) slows growth of mutant IDH1 cells. Western blot shows decreased levels of glutaminase in response to siGLS. B. Effects of BPTES in the absence or presence of 1mM dimethyl-α-ketoglutarate were measured. B shows one representative experiment of three with similar trends and the average and SEM of four replicates at each concentration. *corresponds to p-value ≤ 0.05. For A, p-value is for siGLS compared to siCont. Cell number was normalized to day 0. For B, the p-value was for D54 + R132H compared to D54 and D54 + WT IDH1. Fold growth represents the ratio of alamarBlue fluorescence units of treated cells to vehicle treated cells.

Figure 4

Metabolic changes result from overexpression of mutant IDH1 and 48 hrs of treatment with 10 µM BPTES. Glutaminase activity (A), and glutamate, α-KG, and 2-HG levels (B) were measured in WT and mutant IDH1 cells. C. Levels of other metabolites measured using LC/MS in response to BPTES treatment. For B and C, * represents a p-value ≤0.05. The p-value is for either D54 WT IDH1 DMSO versus 10 µM BPTES or D54 R132H IDH1 DMSO versus 10 µM BPTES.