Abstract 2799: Unexpected sustained hypoxic glutamine metabolism and therapeutic opportunities
Authors: Le Anne , Hamaker Max , Zhang Haixia , Zimmerman Lisa J. , Dinavahi Ramani , Gao Ping , Liebler Daniel C. , Slebos Robbert J.C. , Moseley Hunter , Higashi Richard M. , Lane Andrew , Fan Teresa W. M. , Dang Chi V. 
Published: June 2012

Abstract
Rationale: As the result of genetic alterations and tumor hypoxia, cancer cells re-program metabolism to meet increased energy demand for enhanced anabolism, cell proliferation, and protection from oxidative damage and cell death signals. Whereas glycolysis and its regulation by the MYC oncogene product (Myc) have been intensively studied, glutaminolysis and anapleurosis, especially under hypoxia, have not been defined.
Objectives: We sought to determine whether glutamine, a key source of nitrogen and anabolic carbon skeleton for mammalian cells, is still utilized under hypoxic condition and how it is regulated by MYC.
Methods: We used NMR and FT-ICR-MS to resolve 13C and 15N labeling patterns of metabolites derived from labeled glutamine or glucose in the P493 human B lymphoma model that bear a tetracycline-repressible MYC, under normoxic and hypoxic conditions. Levels of specific enzymes, which are involved in glucose and glutamine metabolism, were determined by immunoblotting and LC-MRM-MS and compared with the metabolomic profiles.
Measurements and Main Results: Myc regulates almost the entire glycolytic pathway. As expected for hypoxia, many glycolytic enzymes levels and lactate production were elevated, and 13C- glucose catabolism by the TCA cycle was significantly diminished. Myc elevated glutaminase (GLS, which converts glutamine to glutamate and NH4+), the glutamine transporter SLC1A5, and the transaminases GOT1 and GPT2, suggesting that transamination is favored with Myc activation. While GLS-mediated NH4+ release persisted under hypoxia, the Myc-induced transaminases favor the production of α-ketoglutarate without additional NH4+ production. The 13C labeling pattern of Ala is consistent with active mitochondrial GPT2 that transaminates 13C-pyruvate derived from 13C- glucose. Given that mitochondrial function is generally believed to be diminished by hypoxia, glutamine metabolism unexpectedly persisted in hypoxia with continued oxidation of glutamine carbons by the TCA cycle. The key enzymes and main products of 13C labeled glutamine were not decreased by hypoxia, and glutamine contributed to substantial de novo glutathione production in hypoxia. Because glutamine metabolism is sustained in hypoxia, which is pervasive in the tumor microenvironment, we sought to target glutaminase in vivo. We found that small molecule inhibition of glutaminase could diminish lymphoma and pancreatic cancer xenograft growth in vivo.
Conclusions: Our studies reveal a previously unsuspected sustained glutamine metabolism in hypoxia. Glutamine contributes to TCA cycle carbons as well as enhances glutathione synthesis in hypoxia, suggesting that cancer cells reprogram metabolism via both glucose and glutamine to adapt to the tumor microenvironment. Our findings indicate that key nodal points in glutamine metabolism in addition to those in glycolysis could be targeted for therapy.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2799. doi:10.1158/1538-7445.AM2011-2799