The thermodynamic state of aerobic glycolytic flux plays a critical role in stabilizing aerobic glycolytic flux of cancer cells

Inhibition of aerobic glycolysis (AG) is a strategy for cancer treatment, but its clinical translation has not been successful, suggesting that the mechanism underlying the inhibition of AG is not completely understood. We introduce thermodynamic state of AG (TSAG) into AG flux control, which refers the actual change of Gibbs free energy (ΔG) of reactions along the pathway. By using PKM2 knockdown and knockout models, we found that TSAG is resistant to PKM2 perturbation and it can effectively counteract the inhibitory effect of PKM2 perturbation on AG flux. Moreover, we deciphered the interrelationship between TSAG, AG rate, intermediate concentrations, and PK activity, revealed that TSAG is a system property of AG independent of cancer cells’ pathophysiological features and it plays important roles in stabilizing AG flux responding to perturbation. We propose that both inhibition of glycolytic enzyme and disruption of TSAG are required for effective AG inhibition. ### Competing Interest Statement The authors have declared no competing interest. * 3-PG : 3-phosphoglycerate 2-PG : 2-phosphoglycerate AIM : allosteric inhibitor mixture AG : Aerobic glycolysis Ala : Alanine F6P : fructose 6-phosphate FBP : fructose 1,6-bisphosphate FBS : fetal bovine serum G6P : glucose 6-phosphate G6PDH : glucose-6-phosphate dehydrogenase GA3P : glyceraldehyde 3-phosphate GAPDH : glyceraldehyde 3-phosphate dehydrogenase ΔG : Gibbs free energy Glc : glucose HK : hexokinase LDH : lactate dehydrogenase MTS : methanethiosulfonate NC : negative control PEP : phosphoenolpyruvate PES : phenazine ethosulfate PFK : phosphofructokinase PGI : phosphohexose isomerase PGK : phosphoglycerate kinase Phe : phenylalanine PK : pyruvate kinase Pro : proline Pyr : pyruvate Q : reaction quotient Ser : serine TPI : triose phosphate isomerase Trp : tryptophan [x], the concentration of x : e.g., [PK] refers the concentration of PK TSAG : thermodynamic state of AG UPLC : ultra performance liquid chromatography Val : valine