Therapies that target various aspects of cancer cell metabolism are currently being developed and primarily focused on glucose metabolism (2
). The dependence of cancer cells on glutamine for various processes is well documented (2
) and has also been a target of interest for therapy; however, clinical trials have yielded little success due to a lack of efficacy or undesirable side effects of glutamine analogs (2
). Here, we explore an inhibitor of glutamine metabolism, BPTES, which allosterically inhibits glutaminase (GLS, but not GLS2) and is not a glutamine analog (19
). Specifically, we studied the dependency of cancer cells with IDH1 mutation on glutaminase activity for maintenance of α-KG homeostasis.
The discovery of IDH1 mutations (6
) identified a metabolic genetic alteration present in a large fraction of gliomas and cytogenetically normal AML (6
). Genetically, a clustering of heterozygous mutations in IDH1 at a single residue indicates a gain-of-function mutation which is supported by the gain of a new enzymatic activity by mutant IDH1. Rather than converting isocitrate to α-KG, mutant IDH1 consumes α-KG and produces 2-HG. Studies have demonstrated that glutamine serves as a cellular source of α-KG consumed by mutant IDH1 (9
). It is not currently understood whether inhibiting mutant IDH1 and reducing 2-HG production would be therapeutically useful, because a role for mutant IDH1 or 2-HG in tumor maintenance has not been established. Thus, we sought to slow mutant IDH1 cell growth by inhibiting glutaminase.
Here we demonstrate that BPTES inhibits glutaminase activity and consequently lowers glutamate and α-KG levels in mutant and WT IDH1 cells, but only growth of mutant IDH1 cells is preferentially slowed in response to BPTES treatment. Intriguingly, BPTES treatment was associated with elevated glycolytic intermediates, which may reflect a compensatory increase in glycolysis to produce α-KG and maintain homeostasis. The notion that glutaminase inhibition perturbs α-KG homeostasis and causes growth inhibition is supported by rescue experiments using a membrane permeable α-KG precursor. Further, inhibition of enzymes which convert glutamate to α-KG showed selectivity for mutant IDH1 cells but only under glucose deprived conditions. Mutant IDH1 cells, however, were not more susceptible to glutamine deprivation than WT IDH1 cells, and withdrawing glutamine altogether will eventually slow the growth of any cell which requires glutamine as an essential nutrient. This result raises the possibility that inhibition of glutaminase may have a different therapeutic result on IDH1 mutant cells compared to inhibition of glutamine uptake.
Our metabolomic studies revealed a number of interesting findings. Even though BPTES treatment lowered glutamate and α-KG levels, 2-HG levels were not significantly decreased. Accordingly, if 2-HG competes with α-KG for binding sites on α-KG-dependent enzymes occupancy of these sites with α-KG would fall with BPTES treatment. While the effect would likely not be significant in wild-type cells, where α-KG presumably fills most sites, it could contribute to impaired cell growth upon BPTES treatment of cells expressing mutant IDH1. Second, BPTES treatment decreased subsequent TCA cycle intermediates, succinate and malate. Additionally, levels of glycolytic intermediates were increased demonstrating that metabolism is perturbed even far from the TCA cycle. We hypothesize that glycolytic intermediates are increased because of increased glycolytic flux to compensate for lowered α-KG levels; however, we have not ruled out the possibility that intermediates build up due to decreased glycolytic flux. One intriguing observation is the diametrically opposite changes in citrate levels between treated WT and R132H IDH1 cells. Currently, the reasons for these differences are not fully understood, and the dissection of these mechanisms is beyond the scope of this paper. Nonetheless, differences in intermediary metabolism between WT and mutant IDH1 cells are sufficient to provoke a gain of sensitivity to glutaminase inhibition in cells with mutant IDH1.
While reduction of glutaminase activity is significant in mutant IDH1 cells, the effects of BPTES on growth are modest (about 20% growth reduction). The modest growth reduction is not particularly surprising, since D54 cells have a genetic background that can tolerate significant siRNA-mediated reduction of glutaminase level. However, the simple overexpression of mutant IDH1 alters intermediary metabolism sufficiently to sensitize mutant IDH1 cells to glutaminase inhibition. We speculate that glioma cells with a naturally occurring IDH1 mutation would demonstrate increased susceptibility to glutaminase inhibition; however, no such cell lines exist at present.
Additionally, cellular metabolism is incredibly dynamic and appears to compensate for changes in intermediary metabolism, such as increased glycolysis, upon BPTES treatment. As a result, we propose that glutaminase inhibition will not be effective as a single arm therapy but will be a part of a more complex strategy that may involve simultaneous inhibition of glycolysis.
Like all treatments, there could be potential disadvantages to this therapeutic strategy since glioblastoma cells engineered with mutant IDH1 or IDH2 could have a pseudohypoxic phenotype (18
). Previous studies documented that exogenous α-KG could reactivate prolyl hydroxylase, decrease HIF-1α levels, and inhibit cell growth resulting from pseudohypoxia elicited by mutations in SDH and FH (20
). As such, reducing α-KG by glutaminase inhibition could hypothetically enhance pseudohypoxia and favor tumor growth.
Our study demonstrates that glutaminase could be a potential therapeutic target in mutant IDH1 cancer cells. However, further work is needed to investigate the metabolic consequences and biochemical specificity for mutant IDH1 cells in response to glutaminase inhibition. An understanding of these effects will be useful in developing combination therapies to augment the effects of glutaminase inhibition and provide insights into how IDH1 mutation and 2-HG production affect cellular physiology.