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Objective:
Glutamine metabolism is altered in tumors with comparable importance to glucose metabolism. A subset of cancers demonstrate glutamine “addiction.” CB-839 is an inhibitor of the first step in glutamine catabolism which involves the enzyme glutaminase (GLS). The use of CB-839 in combination with taxol chemotherapy was furthered to phase II clinical trials in breast cancer without limiting toxicity. However, efficacy was highly variable. In preclinical models, efficacy of glutaminase inhibition is directly correlated to degree of glutamine catabolism. Glutamine metabolism is complex, involving both redox pathways in the cytosol and anaplerotic pathways in the mitochondria. Kinetic analyses have been performed on positron emission tomography (PET) metabolite analogs, specifically glutamine analogs (2S,4R)-4-[18F]fluoroglutamine ([18F]4F-Gln) and [5-11C] glutamine, as well as glutamate analog (4S)-4-(3-[18F]fluoropropyl)-L-glutamate ([18F]4F-FSPG). These kinetic analyses explore a hypothesis that glutamate arising from mitochondrial glutamine metabolism is stored in mitochondrial pools as a buffer to mediate oxidative stress from proliferation or chemotherapies that induce oxidative stress like paclitaxel. We propose that analyzing the subcellular distributions of [18F]4F-Gln, [18F]4F-FSPG, 13C glutamine and 13C glutamate both in vitro and in vivo will confirm this mitochondrial glutamate pool hypothesis, and thus aid in clarifying the mechanism glutamine addiction in a subset of patients with breast cancer.

Materials and Methods:
To further assess glutamine metabolism compartmentation, we are developing and testing an expansion of the modeling approach used for nonmetabolized [18F]4F-Gln by performing kinetic analysis of administered [5-11C] glutamine and downstream metabolites ([5-11C] glutamate and [11C] CO2). This analysis will guide the design and interpretation of experiments utilizing a preclinical model of breast cancer used by our group to model [18F]4F-Gln PET tracer data. Subcellular isolation protocols are being employed to compare cytosolic and mitochondrial 13C and PET glutamine and glutamate pools, with and without CB-839, in vitro, with validation experiments in flank xenograft tumors in vivo.

Results:
[5-11C] glutamine kinetic modeling has been conducted to further support a two-compartment model of glutamine metabolism. The isolation method has been validated by both western blot analysis and oxygraph experiments of the subcellular fractions to assess both separation efficiency and vitality of the isolated mitochondria.

Conclusion:
A predictive molecular imaging biomarker with the ability to distinguish which subset of patients with breast cancer may benefit from glutaminase inhibition could both improve the efficacy of clinical trials and decrease the opportunity cost of deploying glutaminase inhibitors in patients. A more mechanistic understanding of glutamine addiction may aid in the development of molecular imaging protocols of glutamine metabolism. We have developed a preclinical model to characterize this mechanism in breast cancer.