Targeting glutamine metabolism disables Warburg physiology by inhibiting proximal glycolysis and Krebs cycle rewiring

Glutamine plays a critical role in multiple metabolic reactions that support tumor anabolic processes. In spite of this, targeted inhibition of glutaminase, which converts exogenous glutamine into glutamate has met with limited clinical success. We hypothesized that broadly inhibiting glutamine metabolism would more effectively shut down tumor growth. To this end we employed a novel prodrug of the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) in a number of syngeneic mouse models of cancer including MC38 (derived from colon cancer), 3LL (derived from lung cancer) and B16 (derived from melanoma). To better understand the anti-tumor mechanisms, we used LC-MS-based metabolomics to profile metabolic flux in these different tumor models with [U-13C]-glucose/glutamine as tracer. We observed that when compared to 3LL and B16, MC38 was highly sensitive to anti-glutamine treatment. As expected, tumor growth inhibition was correlated with inhibition of (the glutamine requiring) purine nucleotide synthesis. Surprisingly however, treatment with the glutamine antagonist markedly inhibited “proximal” glycolytic reactions as determined by inhibition of the generation of glucose-6-P and fructose 1,6-bisphosphate. This inhibition correlated with a decrease in FDG-PET. Likewise, we observed a marked decrease in one carbon metabolism as measured by serine. Strikingly, glutamine antagonism eliminated the glucose-derived succinate. Instead, a dramatic rewiring of the Krebs cycle was identified, which showed an alternative source of succinate was derived from the GABA shunt. In contrast, not only were these pathways upregulated in the relatively resistant B16 and 3LL tumors, glutamine antagonism only minimally affected these pathways. Furthermore, glutamine antagonism led to a marked decrease in kynurenine. Kynurenine is the result of tryptophan metabolism by IDO and is a potent immunosuppressive metabolite and promoter of metastasis. Decreased kynurenine levels were not the result of inhibition of IDO activity by the glutamine antagonist but rather due to a decrease in IDO expression. Interestingly, the hierarchy of susceptibility to glutamine metabolism and the inhibition of these specific pathways (proximal glycolysis, one carbon metabolism and Krebs cycle) correlated with the hierarchy of susceptibility to anti-PD-1 suggesting that tumor specific metabolism might contribute to resistance to immunotherapy. Overall these results accentuate the importance of glutamine requiring metabolic pathways independent of the role of exogenous glutamine uptake. Further, our results highlight the lack of plasticity of highly coordinated tumor metabolic programming; targeting glutamine metabolism inhibited proximal glycolysis, one carbon metabolism and Krebs cycle. In doing so, we identify novel susceptibilities to exploit for inhibiting tumor growth. Citation Format: Liang Zhao, Matthew Arwood, Min-Hee Oh, Wei Xu, Im-Hong Sun, Im-Meng Sun, Chirag Patel, Robert Leone, Jesse Alt, Rana Rais, Barbara Slusher, Jonathan D. Powell. Targeting glutamine metabolism disables Warburg physiology by inhibiting proximal glycolysis and Krebs cycle rewiring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4376.