Cellular glutamine pool size change in response to glutaminase inhibition detected by [¹⁸F](2S,4R)4-fluoroglutamine PET

Objectives: Glutamine addiction has been observed in a number of aggressive cancers. Reliance on glutamine has been exploited for therapeutic gain with the development of targeted inhibitors of glutaminase, the enzyme that converts glutamine to glutamate in the first step of the glutaminolysis pathway. The glutaminase inhibitor CB-839 (Calithera, Inc.) has shown antitumor efficacy in a triple-negative breast cancer (TNBC) cell lines with inherently elevated levels of glutaminase, but not receptor-positive cell lines with inherently low glutaminase activity. CB-839 has advanced into early clinical trials with promising results in TNBC patients. Currently, there is no clinical biomarker available to predict therapeutic efficacy and measure pharmacodynamic effect of glutamine-targeted cancer therapy. The radiotracer [¹⁸F](2S,4R)4-fluoroglutamine ([¹⁸F]4F-Gln) shares similar transport properties with native glutamine, but is minimally metabolized. As such [¹⁸F]4F-Gln is an ideal radiotracer to infer glutamine pool size through estimates of distribution volume (DV). In this study, we studied differences in DV at baseline and after glutaminase inhibition in two mouse xenografts of human breast cancer with inherently different levels of glutaminase activity using dynamic PET imaging. We also tested whether changes in tumor-to-blood ratios (T:B) could serve as a simple proxy for DV changes.

Methods: TNBC (HCC-1806, high inherent glutaminase activity) and receptor-positive (MCF-7, low inherent glutaminase activity) xenografts were established in athymic nu/nu mice. PET studies were performed on a dedicated small animal scanner (A-PET) at baseline and after treatment with CB-839 or a vehicle solution. Dynamic PET imaging was obtained for one hour after injection of [¹⁸F]4F-Gln (300-350 μCi) via the tail vein. Images were analyzed with AMIDE data analysis software. Kinetic analysis was performed with PMOD. T:B ratios were calculated at 30-45 minutes and 45-60 minutes.

Results: Logan plot analysis revealed late linearity, consistent with largely reversible transport. In a two-compartment model with irreversible trapping, k₃ was low (less than 0.01 in most cases), also consistent with largely reversible transport. There was strong correlation between DV as estimated by the Logan plot and single-compartment model (r>0.95), but not with a two-compartment model. A strong correlation was also seen between T:B ratios and DV estimates by Logan plot and a single-compartment model (r>0.9), but not between T:B ratios and a two-compartment model. For the remainder of this analysis, the two-compartment model was not considered. MCF-7 tumors demonstrated over 60% larger DVs and T:B ratios pre-treatment compared to TNBC tumors. Upon glutaminase inhibition, CB-839-treated TNBC tumors demonstrated a greater than 30% mean increase in DV and T:B ratio, not seen in the MCF-7 tumors nor the vehicle treated TNBC tumors.

Conclusion: As a minimally metabolized radiotracer, [¹⁸F]4F-Gln is a marker of glutamine pool size and can infer changes in tumor glutaminolysis with glutaminase inhibition through estimation of DV and T:B ratios. These findings indicate promise for [¹⁸F]4F-Gln as a biomarker to assess glutaminase-directed therapy. Research Support: This study is supported by funding from Susan G. Komen Foundation Grant SAC140060 (DM), Department of Energy Grant DE-SE0012476 (DM and RHM), R21-CA-198563 (RZ) and R01CA211337 (DM and RZ).