Abstract
BackgroundReprogramming of metabolic pathways is crucial to satisfy the bioenergetic and biosynthetic demands and maintain the redox status of rapidly proliferating cancer cells. In tumors, the tricarboxylic acid (TCA) cycle generates biosynthetic intermediates and must be replenished (anaplerosis), mainly from pyruvate and glutamine. We recently described a novel enolase inhibitor, HEX, and its pro-drug POMHEX. Since glycolysis inhibition would deprive the cell of a key source of pyruvate, we hypothesized that enolase inhibitors might inhibit anaplerosis and synergize with other inhibitors of anaplerosis, such as the glutaminase inhibitor, CB-839.MethodsWe analyzed polar metabolites in sensitive (ENO1-deleted) and resistant (ENO1-WT) glioma cells treated with enolase and glutaminase inhibitors. We investigated whether sensitivity to enolase inhibitors could be attenuated by exogenous anaplerotic metabolites. We also determined the synergy between enolase inhibitors and the glutaminase inhibitor CB-839 in glioma cells in vitro and in vivo in both intracranial and subcutaneous tumor models.ResultsMetabolomic profiling of ENO1-deleted glioma cells treated with the enolase inhibitor revealed a profound decrease in the TCA cycle metabolites with the toxicity reversible upon exogenous supplementation of supraphysiological levels of anaplerotic substrates, including pyruvate. ENO1-deleted cells also exhibited selective sensitivity to the glutaminase inhibitor CB-839, in a manner rescuable by supplementation of anaplerotic substrates or plasma-like media PlasmaxTM. In vitro, the interaction of these two drugs yielded a strong synergistic interaction but the antineoplastic effects of CB-839 as a single agent in ENO1-deleted xenograft tumors in vivo were modest in both intracranial orthotopic tumors, where the limited efficacy could be attributed to the blood-brain barrier (BBB), and subcutaneous xenografts, where BBB penetration is not an issue. This contrasts with the enolase inhibitor HEX, which, despite its negative charge, achieved antineoplastic effects in both intracranial and subcutaneous tumors.ConclusionTogether, these data suggest that at least for ENO1-deleted gliomas, tumors in vivo—unlike cells in culture—show limited dependence on glutaminolysis and instead primarily depend on glycolysis for anaplerosis. Our findings reinforce the previously reported metabolic idiosyncrasies of in vitro culture and suggest that cell culture media nutrient composition more faithful to the in vivo environment will more accurately predict in vivo efficacy of metabolism targeting drugs.
Highlights
Reprogramming of metabolic pathways is crucial to satisfy the bioenergetic and biosynthetic demands and maintain the redox status of rapidly proliferating cancer cells
Enolase inhibition represses oxidative phosphorylation, depletes tricarboxylic acid (TCA) cycle metabolites, and induces bioenergetic collapse We previously demonstrated that glioma cells with ENO1 passenger deletions are selectively susceptible to inhibition of ENO1’s redundant paralog ENO2 through the collateral lethality paradigm [8]
We found that glioma cell lines that have either homozygous (D423) or heterozygous (D502, U343) deletions of ENO1 are selectively more sensitive to the enolase inhibitor POMHEX than are ENO1 rescued (D423 ENO1) and wildtype cells (LN319) (Fig. 1 a-c)
Summary
Reprogramming of metabolic pathways is crucial to satisfy the bioenergetic and biosynthetic demands and maintain the redox status of rapidly proliferating cancer cells. We conceived the framework of collateral lethality, whereby incidental loss of passenger metabolic genes—genes that are lost along with driver tumor suppressor genes—can be exploited therapeutically by targeting the redundant isoforms of the passenger genes [7, 8] Using this concept of collateral lethality, we validated that in a subset of gliomas with 1p36 deletions, passenger deletion of the glycolytic gene ENO1 selectively renders cancer cells sensitive to inhibition of the redundant isoform ENO2 [8]. To exploit this therapeutic opportunity in this subset of tumors, we developed a specific inhibitor of enolase, HEX and a pro-drug thereof, POMHEX. Metabolomic and biochemical data strongly indicated both specific and dose-dependent inhibition of glycolysis by these enolase inhibitors [9]
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