Fig. 2

Exogenous supplementation of selected anaplerotic substrates mitigates the toxicity of enolase inhibition. a Schematic representing different cellular metabolites that converge to replenish the TCA cycle carbon atoms. Exogenously supplemented anaplerotic substrates (alanine, acetate, aspartate, fatty acids, lactate, methylpyruvate, pyruvate, oxaloacetate, and oxovalerate) are indicated in blue, while cataplerotic substrates are indicated in red. b Dose response curves of ENO1-deleted, ENO1-rescued and ENO1 wild type cells to POMHEX in pyruvate free DMEM (N=12) and DMEM exogenously supplemented with 5 mM pyruvate (N=6) and 2.5 mM methyl pyruvate (N=4). Cells were seeded in 96-well plates in pyruvate free DMEM or DMEM supplemented with the anaplerotic substrates and treated with serial dilutions of POMHEX. Crystal violet staining was performed to measure the terminal cell density and assess the effect of POMHEX and the degree of rescue of POMHEX toxicity by exogenous supplementation of anaplerotic substrates. Cell density is expressed relative to the untreated controls. A shift in IC50 (blue horizontal arrows) indicates alleviation of POMHEX toxicity by addition of exogenous anaplerotic substrates. IC50 of POMHEX for each cell line in different medium condition is indicated. (see Supplemental Figure S4 and S5 for a panel of anaplerotic substrates)