Abstract 5473: Identification of compensatory pathway for glutamate production upon glutaminase 1 inhibition

Abstract Rationale and objectives: A current clinical trial is testing a drug inhibitor for glutaminase 1 (GLS1), the enzyme responsible for glutamine's conversion into glutamate, for cancer therapy. However, the results indicated that monotherapy is not ideal. While metabolic profiling of tumors after treatment with a GLS1 inhibitor revealed numerous upregulated pathways which could explain the resistance, the specific pathways involved were not identified. In this study, using stable isotope-resolved metabolomics, we aim to detail the specific mechanism by which cancer cells are capable of circumventing GLS1 inhibition and compensating for glutamate production. Measurement and Methods: Patient-derived pancreatic tumors were implanted through orthotopic model in the pancreas of mice. After using our innovative delivery method through nanoparticle encapsulation of a selective GLS1 inhibitor, BPTES, we then injected 13C5, 15N2-glutamine via intraperitoneal administration, 3 times, 15 minutes apart. Tumors were harvested 2 hours post first glutamine injection. Metabolites were then extracted from tumors, and analyzed using Agilent 6520 Q-TOF mass spectrometer and 1H-NMR. Metabolite intensities were later normalized to protein concentration following analysis. Results: Our results showed that total glutamate levels were lower in BPTES-NP treated tumors as compared to vehicle control ones. Interestingly, we found an increase in (m+5) labeled glutamate (mass of the parent ions (m) and 5 more mass units due to 13C415N1-glutamate or 13C5-glutamate labelling) in BPTES-NP treated tumors as compared to the vehicle control tumors. Moreover, we found that (m+5) glutamate is a product of (m+7) glutamine being converted to (m+5) alpha-ketoglutaramate (KGM) via glutamine-pyruvate transaminase and further on into alpha-ketoglutarate (aKG) by omega-amidase, which can finally produce the identified (m+5) glutamate through glutamate dehydrogenase. Further analysis using 1H-NMR detailed a significant increase in overall KGM intermediate intensity in treatment groups compared to the control groups, confirming the upregulation of compensatory pathway (glutamine-KGM- aKG-glutamate) to produce glutamate upon treatment of GLS1 inhibitor. Conclusion: These results explain the reasons behind the limited clinical outcomes for single therapy with a GLS1 inhibitor, and provide potential therapeutic targets: glutamine-pyruvate transaminase, for combination treatments with GLS1 inhibitors to prevent the compensation for glutamate production amid GLS1 inhibition. Citation Format: Karim B. Nabi, Jimmy Kirsch, Tu Nguyen, Addison Quinones, Jessica Tan, Felipe Camelo, Marjorie Antonio, Jin Jung, Anne Le. Identification of compensatory pathway for glutamate production upon glutaminase 1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5473.