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  • Mitochondrial depolarization induced by DHODH inhibition Fig


    Mitochondrial depolarization induced by DHODH inhibition (Figure 5A) may also contribute to the selective effects of DHODH inhibitors on KRAS mutant cells. If so, the effect may arise more from pro-apoptotic effects of mitochondrial depolarization (Lemasters et al., 1998) than from impaired oxidative phosphorylation. Oncogenic KRAS mutations have been associated with decreased, rather than increased, oxidative phosphorylation: expression of mutant KRAS leads to an increased dependence on anabolic glucose metabolism (Weinberg et al., 2010, Ying et al., 2012), while ablation of mutant KRAS leads to an increased dependence on oxidative phosphorylation (Viale et al., 2014). However, to the extent that KRAS mutant Bikinin exhibit enhanced flux through de novo pyrimidine biosynthesis, they may also exhibit a greater dependence on electron transport through DHODH to maintain consistently hyperpolarized mitochondrial membrane potentials and a pro-survival phenotype. A precedent for an association between hyperpolarized mitochondrial potentials and a pro-survival phenotype can be found in the example of CD8+ memory T cells, for which surplus capacity for oxidative phosphorylation enhances cellular survival (van der Windt et al., 2012). Measurements of metabolite levels in the current study are also consistent with the idea that KRAS mutant cells are differentially dependent on mitochondrial electron transport via DHODH, which may contribute to differential effects of DHODH inhibition. In KRAS mutant cells, brequinar treatment is associated with energy stress (Figure S7). However, brequinar does not induce similar energy stress in WT cells. Taken together, these data support the hypothesis that KRAS mutant cells are differentially dependent on flux through DHODH for survival, due in part to its role in primary energy metabolism. One possibility is that in KRAS mutant cells, increased electron transport via DHODH partially compensates for decreased electron transport via aerobic glucose metabolism. In addition to a role for DHODH inhibition in primary energy metabolism, the observation of decreased steady-state levels of glutamine and glutamate and increased isotopic exchange into glutamate (a proxy for metabolic flux) upon treatment with a DHODH inhibitor (Figures 5C, 6C, 7G, S5, and S7) suggests a link between DHODH activity and glutamine metabolism, which was previously demonstrated to be crucial for the growth and survival of KRAS mutant cancer cells (Son et al., 2013, Weinberg et al., 2010). These observations, as well as the increased steady-state level of aspartate observed upon DHODH inhibition, are consistent with increased conversion of glutamine and oxaloacetate to αKG and aspartate via GOT1. Therefore, the decreased steady-state levels of glutamine appear not to be caused by a lack of available glutamine as a metabolic substrate, but rather by increased consumption of glutamine. Since the decrease in glutamine levels appears to be associated with increased glutaminolysis rather than a decreased ability to accumulate glutamine, it might be hypothesized that this decrease in glutamine levels does not contribute to impaired growth and survival in KRAS mutant cells. However, the observation that uridine rescues both the effect of brequinar on glutamate flux and its cellular phenotypic effect suggests that the metabolic effects of DHODH do contribute to its broader biological effects. In addition, the results are consistent with synergism between GLS1 inhibition and DHODH inhibition both at the level of regulating glutamine levels and (at least for some GLS1 inhibitors) at the level of growth and survival. One hypothesis that accounts for the current observations is that DHODH activity helps to mediate the balance between two distinct glutaminolysis-dependent redox metabolic pathways that have been implicated in growth/survival in KRAS mutant cells: generation of mitochondrial reactive oxygen species (ROS) (Weinberg et al., 2010) and flux through GOT1 enabling generation of NADPH and maintenance of reduced glutathione (Son et al., 2013, Yun et al., 2015).