GluCEST MRI: A Biomarker for Glutamine Metabolism in Cancer
Rong Zhou1, Puneet Bagga1, Kavindra Nath1, David Mankoff1, Hari Hariharan1, and Ravinder Reddy1

1Radiology, University of Pennsylvania, Philadelphia, PA, United States

Synopsis

We presented the very first evaluation of glutamate chemical exchange saturation transfer (GluCEST) MRI to detect pharmacodynamic effect of small molecule drugs that target cancer glutaminolysis pathway. Conversion of glutamine to glutamate is a rate limiting step along this pathway. Inhibition of this conversion leads to reduction of cellular glutamate concentration that can be detected by GluCEST in vivo and confirmed by ex vivo high resolution 1H MRS of tumor homogenates.

Introduction

Metabolic analyses have revealed that cancer orchestrates the use of glucose (via glycolysis or Warburg effect) and glutamine to meet the demand of energy and building blocks necessary for survival and proliferation.1, 2 In contrast to normal cells, many cancers extensively use the amino acid glutamine via glutaminolysis pathway.4, 5 Mitochondrial enzyme glutaminase (GLS), which converts glutamine to glutamate (Glu) is the first and rate-limiting step and inhibition of GLS leads to over 2 fold change of tumor Glu concentration. Emerging therapeutics targeting cancer GLS are currently in clinical trials of cancers including the triple negative cancers, which have few therapeutic options but are highly dependent on glutamine for survival and growth. Non-invasive and sensitive method that can assess the pharmacodynamics (PD) of this new category of drugs is needed to facilitate their development and clinical implementation. Based on our prior development of chemical exchange saturation transfer (CEST) MRI to map the brain Glu concentration, here we propose to examine the utility of GluCEST 6 for monitoring the change of tumor Glu level in response to GLS inhibition.

Methods

All the experiments were performed under an approved IACUC protocol of the University of Pennsylvania. Human triple negative breast cancer cells (HCC1806) are inoculated subcutaneously in the flank of athymic nu/nu mice and xenografts were grown to about 500 mm3 before assigned to CB-839 or Vehicle (VHE) group. A selective, potent and reversible GLS inhibitor, CB-839,7 was obtained from Calithera via material transfer agreement. CB-839, dissolved in a vehicle (25% (w/v) hydroxypropyl-β-cyclodextrin in 10 mmol/L citrate, pH 2) was administered orally (200 mg/kg) twice daily, while control mice received the same volume of vehicle solution. A total of four doses are administered. Each mouse underwent the GluCEST MRI before treatment and 4 hours after the last dose. All MRI studies are performed using custom-built slot-tube resonator (15 mm dia) tuned to 1H on a 9.4T horizontal bore spectrometer. GluCEST MRI was performed using a custom-programmed RF spoiled gradient echo readout pulse sequence in 10 mm slices with a frequency selective continuous wave saturation preparation pulse. CEST images were collected using a 1 second saturation pulse at peak B1 of 250 Hz for the frequencies ± 2.5-3.5 ppm from water resonance with a step size of 0.25 ppm. The B0-corrected images at 3 ppm (M+3ppm) and -3 ppm (M−3ppm) were used for computing the percent GluCEST asymmetry value, which is equal to 100×[(M−3ppm − M+3ppm)/M−3ppm]. Regions of interest were manually segmented from T2-weighted images.

Results and Discussion

By utilizing a slot-tube RF resonator in which tumor is relatively isolated from the breathing motion, reliable B0 and B1 homogeneity maps were obtained in our studies (Fig 1A-B). Regions which had B0 variation beyond ±0.3 ppm or relative B1 ¬≠below 0.8 or above 1.2 were excluded for analyses. Pre- and post-treatment GluCEST map from a mouse in CB-839 group is shown in Fig 1C, revealing an overall decrease of GluCEST value with heterogeneity across the tumor after treatment (bottom vs. upper panel). Our data show a marked reduction of GluCEST value in 3 out of 4 CB-839 treated mice while there is minimal change in all VEH treated mice (Fig 2). Compared to average 0.32% increase of GluCEST in VEH group (n=4), GluCEST value decreases 3.5% in CB-839 group (n=4), consistent with extract data (below) since about 1.7% GluCEST change per millimolar Glu was calibrated in phantom and brain.6,8 Using high resolution 1H MRS of perchloric acid extraction of the tumors, we confirmed that tumor Glu level in CB-839 treated mice was reduced over 2 mM compared to VEH treated ones, meanwhile glutamine was increased and alanine (Ala) unchanged (Fig 3). Taking together, these data corroborate a reduction of tumor GluCEST value with a decrease in Glu concentrations by 1H MRS, strongly supporting the concept of GluCEST as a marker of GLS inhibition. The presence of Ala, which also exhibits GluCEST effect, could contribute to baseline GluCEST value. It is noted that cellular Glu and Ala pool are linked via the alanine transferase (ALT) reaction (Fig 4), hence upon GLS inhibition, Ala concentration is also expected to decrease as Glu, leading to a serendipitous increase of GluCEST sensitivity. Tumor intracellular pH (pHi) also contributes to GluCEST value, however a pHi drop following the treatment is expected to cause an underestimation of the change in GluCEST value and we will examine it further. In summary, our preliminary results presented here suggest GluCEST is a promising PD marker for a potent glutaminase inhibitor.

Note

RZ and PB are equal contribution.

Acknowledgements

We thank Calithera and Dr. Susan Demo for providing the reagent and helpful discussions, and technical support from SAIF (small animal imaging facility) of Radiology Dept. This study is supported by the National Center for Research Resources and the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health through Grant P41-EB015893.

References

1. Le A, Lane AN, Hamaker M, Bose S, Gouw A, Barbi J, et al. Glucose-independent glutamine metabolism via TCA cycling for proliferation and survival in B cells. Cell Metab 2012, 15(1): 110-121.

2. DeBerardinis RJ, Mancuso A, Daikhin E, Nissim I, Yudkoff M, Wehrli S, et al. Beyond aerobic glycolysis: Transformed cells can engage in glutamine metabolism that exceeds the requirement for protein and nucleotide synthesis. Proceedings of the National Academy of Sciences 2007, 104(49): 19345-19350.

3. Parlati F. CB-839, a selective glutaminase inhibitor, synergizes with signaling pathway inhibitors to produce an anti-tumor effect in cell lines and tumor xenografts. American Association of Cancer Research Annual Meeting 2015 2015; Philadelphia; 2015.

4. Dang CV. Rethinking the Warburg effect with Myc micromanaging glutamine metabolism. Cancer Res 2010, 70(3): 859-862.

5. Filipp FV, Ratnikov B, De Ingeniis J, Smith JW, Osterman AL, Scott DA. Glutamine-fueled mitochondrial metabolism is decoupled from glycolysis in melanoma. Pigment Cell & Melanoma Research 2012, 25(6): 732-739.

6. Cai K, Haris M, Singh A, Kogan F, Greenberg JH, Hariharan H, et al. Magnetic resonance imaging of glutamate. Nat Med 2012, 18(2): 302-306.

7. Gross MI, Demo SD, Dennison JB, Chen L, Chernov-Rogan T, Goyal B, et al. Antitumor Activity of the Glutaminase Inhibitor CB-839 in Triple-Negative Breast Cancer. Mol Cancer Ther 2014, 13(4): 890-901.

8. Haris M, Nath K, Cai K, Singh A, Crescenzi R, Kogan F, et al. Imaging of glutamate neurotransmitter alterations in Alzheimer's disease. NMR Biomed 2013, 26(4): 386-391.

Figures

Fig 1 B0 (A), B1 (B) maps of tumor ROI and average value from pre- and post-scan. GluCEST map overlaid on T2W image of tumor (C). The tumor is marked by dotted white line. The colored ROI includes region that can be corrected for B0 and B1 (see text).

Fig 2 GluCEST values from individual mice in CB-839 and VEH group (mouse ID are shown on x-axis).

Fig 3 Left panels: 1H MRS (cropped) of PCA extracts of tumors treated by VEH or CB-839 (4 doses over 2 days). Right: average concentration of Gln, Glu and Ala in VEH or CB treated tumors. Gln or gln = glutamine; succ = succinate; Ala = alanine.

Fig 4 Tumor cellular Glu and Ala are linked by alanine transferase (ALT). GLS inhibition leads to reduced TCA cycle products and Ala via decreased Glu (adapted from ref 3).



Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
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