Volume 26, Issue 6, Pages 830-841.e3 (December 2017) In Vivo Imaging of Glutamine Metabolism to the Oncometabolite 2-Hydroxyglutarate in IDH1/2 Mutant Tumors  Lucia Salamanca-Cardona, Hardik Shah, Alex J. Poot, Fabian M. Correa, Valentina Di Gialleonardo, Hui Lui, Vesselin Z. Miloushev, Kristin L. Granlund, Sui S. Tee, Justin R. Cross, Craig B. Thompson, Kayvan R. Keshari  Cell Metabolism  Volume 26, Issue 6, Pages 830-841.e3 (December 2017) DOI: 10.1016/j.cmet.2017.10.001 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell Metabolism 2017 26, 830-841.e3DOI: (10.1016/j.cmet.2017.10.001) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Glutamine Flux Is Directed toward Production of 2-HG in IDH1/2 Mutants In Vitro (A) Representative metabolic map of TCA cycle metabolism in mIDH1 chondrosarcoma cells (JJ012) grown with 5 mM [13C5] Gln. The time course analysis shows nearly all of the glutamate and 2-HG pool is labeled in all carbons after 24 hr. The map also shows evidence of reductive carboxylation taking place as observed in the m+5 labeling of isocitrate and citrate, and it extends to aspartate metabolism as well as malate and fumarate, as evidenced by m+3 labeling of these metabolites. The LC-MS method was sensitive enough to detect the second pass of the oxidative reduction of TCA cycle, as shown by the m+3 labeling of citrate and isocitrate. The representative metabolic map for mIDH2 cells (CS1) is shown in Figure S1. (B and C) show labeling of 2-HG from JJ012 and CS1 after 24 hr incubation with various combinations of unlabeled and [13C6 ] Gluc [1-13C] Gln. Cells incubated with [13C6] Gluc show ∼5% m+2 fractional enrichment. (D) JJ012 and CS1 cells were grown in media supplemented with 5 mM glutamine (Gln +) and in glutamine-depleted media (Gln −). JJ012 cells do not produce 2-HG in the absence of glutamine, while CS1 cells are capable of limited 2-HG production in these conditions. Data are representative of mean ± SD of three replicates from three experiments carried independently. Cell Metabolism 2017 26, 830-841.e3DOI: (10.1016/j.cmet.2017.10.001) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Kinetics of Glutamine-Derived Glutamate and 2-HG In Vitro Provide Evidence for the Possible Translation to In Vivo HP-MRI (A–D) Fractional enrichment of glutamate and 2-HG from glutamine in vitro was determined by LC-MS analysis following the time course m+5 labeling of these metabolites from [13C5] Gln. (A) and (B) show absolute glutamate pools (blue) and m+5 labeling obtained from LC-MS over time (red) for JJ012 and CS1 cells, respectively. Glutamate enrichment reached approximately 80% by 3 hr on JJ012 and CS1. (C) and (D) show total 2-HG fractional enrichment with absolute glutamate pools (blue) and m+5 labeling from LC-MS over time (red) for JJ012 and CS1 cells, respectively. At 8 hr, approximately 80% of 2-HG has been labeled in both cell lines. Data are representative of mean ± SD of three replicates from three experiments carried independently. Cell Metabolism 2017 26, 830-841.e3DOI: (10.1016/j.cmet.2017.10.001) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 The Rate of 2-HG Production in mIDH1 Cells Can Be Modulated In Vitro using an mIDH1 Inhibitor Drug (A and B) IC50 plot of JJ012 and CS1 cells, respectively, grown in media supplemented with 5 mM glutamine and increasing concentrations of IDH1 inhibitor (IDHi) AGI-5198. JJ012 cells show sensitivity to the drug, with a maximum inhibition of 2-HG production of approximately 90%. CS1 cells show limited sensitivity to AGI-5198. (C and D) Isotopic enrichment in JJ012 cells incubated with [13C5] Gln without (control) or with (IDHi) 2 μM of IDHi demonstrates the glutamate pool is unchanged by inhibition of mutant IDH1, while the apparent rate of 2-HG production is reduced as a function of total 2-HG pools. Data are representative of mean ± SD of three replicates from three experiments carried independently. Cell Metabolism 2017 26, 830-841.e3DOI: (10.1016/j.cmet.2017.10.001) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 [1-13C] Glutamine Can Be Hyperpolarized with Minimal PG Formation (A) Schematic representation of metabolism of HP [1-13C] Gln into [1-13C] 2-HG in vivo: (i) A sample of [1-13C] Gln dissolved in NaOH, combined with OX063 radical and Gd-Dota, is hyperpolarized at 3.35 T for >1 hr, rapidly dissolved in Tris buffer, and intravenously injected into mice. (ii) HP [1-13C] Gln is converted to HP [1-13C] Glu by glutaminase (GLS). (iii) HP [1-13C] Glu is metabolized to HP [1-13C] 2-oxoglutarate (2-OG) by glutamate dehydrogenase (GDH). (iv) HP [1-13C] 2-OG is converted to HP [1-13C] 2-HG by mutant isocitrate dehydrogenase 1/2 (mIDH1/2). (B) Degradation of glutamine in solution leads to the formation of PG, which we minimized during preparation. HP [1-13C] Gln shows resonance at 175 ppm and PG at 180 ppm following dissolution. (C) The T1 relaxation [1-13C] Gln is 31 ± 3 s at 1 Tesla. (D) Chemical shifts at physiological pH for [1-13C] 2-HG (180.8 ppm), [5-13C] PG (180 ppm), [1-13C] Glu (175.5 ppm), [1-13C] Gln (174.8 ppm), and 13C Urea (163 ppm) measured at 14 Tesla. Cell Metabolism 2017 26, 830-841.e3DOI: (10.1016/j.cmet.2017.10.001) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 The Real-Time Formation of 2-HG Can Be Detected In Vivo using HP [1-13C] Gln as an Imaging Probe (A) Representative heatmap of spectral data from a mouse with a mIDH1 tumor xenograft following injection of [1-13C] Gln. The top picture shows distribution of glutamine and glutamate over the whole body. The middle picture shows accumulation of 2-HG in the tumor region only. The bottom picture shows the 13C urea phantom used for resonance reference. The dotted lines highlight tumor and urea phantom. White line at the bottom is 10 mm for scaling. (B) Selected voxels showing the presence of a second peak approximately 5.5 ppm away from [1-13C] Gln+Glu in tumor regions only. (C) Bar plots representing relative [1-13C] 2-HG signal integration from animals with JJ012 (n = 11) and CS1 (n = 8) tumors. Signal integrations were normalized to 13C urea signals from the same acquisition experiment. (D) Relative [1-13C] 2-HG signal integration from longitudinal study of animals with JJ012 tumors (n = 3∗) at baseline (without treatment) and treated with IDH1 inhibitor. Cell Metabolism 2017 26, 830-841.e3DOI: (10.1016/j.cmet.2017.10.001) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Kinetics of Glutamine Conversion to 2-HG In Vivo Are Fast Enough for HP Imaging (A–C) Tumors were extracted from mice and xenografted with JJ012 and CS1 cells following a 12 s intravenous infusion of [13C5] Gln, and metabolite levels (glutamate and 2-HG) were quantified. (A) Representative 1H-NMR spectra of tissue extracts from JJ012 (top) and CS1 (bottom) tumors used for quantification of total glutamate (Glu) and 2-HG pools. For glutamate quantification, the area of gamma proton signal was used (dark pink area), and for 2-HG, the area of one beta proton was used (light blue area). (B) Bar plots showed concentration of glutamate and 2-HG in tumors as resolved from the 1H-NMR spectra. For both metabolites, JJ012 shows higher levels than CS1. (C) Concentration of [13C5] 2-HG in JJ012 and CS1 tumors 30 min after the end of infusion was determined via LC-MS. Both JJ012 (black dot) and CS1 (gray square) show similar levels of labeling after 30 min. Additionally, we quantified concentrations of [13C5] 2-HG at 1, 5, and 10 min to assess initial rates of labeling in JJ012 tumors. Quantitative data are representative of mean ± SD of three replicates from three experiments carried out independently. Cell Metabolism 2017 26, 830-841.e3DOI: (10.1016/j.cmet.2017.10.001) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 Glucose Carbons Are Not Incorporated into 2-HG in the Time Frame of an HP Injection (A–G) Tumors were extracted from mice and xenografted with JJ012 and CS1 cells following a 12 s intravenous infusion of [13C6] Gluc or [13C5] Gln, and fractional enrichment (for each tracer) of selected metabolites (glyceraldehyde-3-phosphate, isocitrate, and 2-HG) was obtained. (A) and (B) show fractional enrichment of glyceraldehyde-3-phosphate, confirming the metabolism of glucose in the tumor (m+3, middle set). (C) and (D) show enrichment of isocitrate to confirm entry of glucose and glutamine carbons into the TCA cycle (m+2 and m+4, respectively). (E) and (F) show fractional enrichment of 2-HG m+5 in tumors infused with [13C5] Gln, while no fractional enrichment of m+2 is detected in tumors infused with [13C6] Gluc. (G) shows fractional enrichment of 2-HG m+5 from [13C5] Gln with or without mIDH inhibitor. These results show similar inhibition levels detected in imaging experiments. Qualitative data are representative of mean ± SD of three replicates from three experiments carried out independently. Cell Metabolism 2017 26, 830-841.e3DOI: (10.1016/j.cmet.2017.10.001) Copyright © 2017 Elsevier Inc. Terms and Conditions