Reduced methylation of PFKFB3 in cancer cells shunts glucose towards the pentose phosphate pathway

Haem oxygenase (HO)-1/carbon monoxide (CO) protects cancer cells from oxidative stress, but the gas-responsive signalling mechanisms remain unknown. Here we show using metabolomics that CO-sensitive methylation of PFKFB3, an enzyme producing fructose 2,6-bisphosphate (F-2,6-BP), serves as a switch to activate phosphofructokinase-1, a rate-limiting glycolytic enzyme. In human leukaemia U937 cells, PFKFB3 is asymmetrically di-methylated at R131 and R134 through modification by protein arginine methyltransferase 1. HO-1 induction or CO results in reduced methylation of PFKFB3 in varied cancer cells to suppress F-2,6-BP, shifting glucose utilization from glycolysis toward the pentose phosphate pathway. Loss of PFKFB3 methylation depends on the inhibitory effects of CO on haem-containing cystathionine beta-synthase (CBS). CBS modulates remethylation metabolism, and increases NADPH to supply reduced glutathione, protecting cells from oxidative stress and anti-cancer reagents. Once the methylation of PFKFB3 is reduced, the protein undergoes polyubiquitination and is degraded in the proteasome. These results suggest that the CO/ CBS-dependent regulation of PFKFB3 methylation determines directional glucose utilization to ensure resistance against oxidative stress for cancer cell survival.