The Warburg effect has recently reemerged as an exciting anticancer target. However, until relatively recently, the only inhibitors available were unspecific agents like 2-deoxyglucose (2-DG) and 3-bromopyruvate, which have effects apart from glycolysis inhibition that could play important roles in inhibiting growth. While more recent screens have identified blockers of specific glycolytic enzymes, questions about the role of glycolysis inhibition in their effects on proliferation–when those have even been reported–remain. Taken together with the fact that knockouts or knockdowns do not necessarily provide evidence that enzymatic inhibition of a target will produce therapeutic effects, the evidence that targeting glycolysis could be an effective antiproliferative strategy remains weak. We suspected that examining the effects of a panel of structurally and mechanistically diverse glycolysis-inhibiting compounds rather than focusing on a single compound would be informative. Any one compound that inhibits glycolysis could reduce proliferation via off-target effects. However, if inhibiting glycolysis is indeed a good way to prevent cell growth, then if we identify a sufficient number of glycolysis inhibitors and all are antiproliferative, it could provide strong support for the idea that effects on glycolysis are responsible for effects on proliferation. In contrast, if even one effective glycolysis inhibitor were to fail to block proliferation, it would falsify the mechanism. We screened the NCI’s Mechanistic Set III using a novel multiread assay based on an intramolecular FRET sensor for ATP expressed stably in K562 cells, which can maintain ATP levels via glycolysis. We identified 7 inhibitors of oxidative phosphorylation-dependent ATP production and 10 glycolysis inhibitors. We drew upon the NCI's measurements of the compound set's effects on proliferation in K562 cells, and found that the inhibitors of oxidative phosphorylation-dependent ATP production were no more effective at inhibiting growth than the overall compound collection. Some had no effect on growth, suggesting that this is pathway is not a valid antiproliferative target in K562 cells. By contrast, all of the glycolysis inhibitors were in the top third of most effective antiproliferative compounds. The chances of obtaining 10 compounds as effective at blocking proliferation from the library solely by chance, estimated using the binomial distribution, are less than one in 500,000. This provides strong support for the idea that inhibiting glycolysis is likely an effective means of blocking proliferation of K562 cells, and points to a strategy that can be used to provide small-molecule support for pursuing specific glycolytic enzymes or other cellular targets for cancer therapies.

Citation Format: Ziyan Zhao, Adam Zweifach. Glycolysis inhibitors are antiproliferative in leukemic cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B020.