Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma

Abstract

L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, 13C–glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.

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Figure 1: Gln starvation reduces GBM cell proliferation.
Figure 2: The effects of Glu secretion and GLS inhibition on GBM cell growth and metabolism.
Figure 3: GS sustains cell growth during Gln starvation.
Figure 4: GS activity regulates cell growth and purine availability under Gln starvation.
Figure 5: Gln metabolism in differentiated and GBM stem-like (GSC) primary human GBM cells.
Figure 6: Gln metabolism in GBM patients and primary orthotopic xenografts.
Figure 7: Glutamine supply for GBM tumours with low GS expression.
Figure 8: Astrocytes provide GBM cells with Gln.

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Acknowledgements

This study has been supported by Cancer Research UK. S.T. is a recipient of an AIRC/Marie Curie International Fellowship for Cancer Research. The human and animal metabolomic studies were supported by The Norwegian Cancer Society, The Norwegian Research Council, Helse Vest, Haukeland University Hospital and the K.G-Jebsen Foundation. We acknowledge A. Golebiewska, V. Baus-Talko, N. Van Den Broek, G. MacKay, C. Nixon and E. MacKenzie for excellent technical assistance and A. King for excellent editorial work.

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Contributions

S.T. conceived the study, designed and performed most experiments, interpreted the data, and wrote the manuscript, A.O. performed the experiments in orthotopic xenograft models, S.U.A. and A.J.C. provided the differentiated and stem-like primary glioblastoma cells, L.Z. supervised the analysis of LC-MS samples, O.K. performed the MRI analysis, F.F. processed the orthotopic and clinical GBM samples, H.M. provided the tissue microarray, A.K.H. designed and provided the iRFP and iRFP–GS constructs, A.Weinstock, A.Wagner and E.R. generated and employed the metabolic modelling, S.C.B. and S.L.L. provided the primary astrocytes, M.L.-J., S.H.M. and P.Ø.S. provided the surgical specimens from the patients, S.P.N. and R.B. conceived and supervised the experiments in orthotopic models and human patients, and E.G. conceived and supervised the study, interpreted the data, and revised the manuscript.

Corresponding author

Correspondence to Eyal Gottlieb.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 5 Glutamine starvation reduces GBM cell proliferation.

(a) Representative microscopic fields of cells incubated for 6 days with or without Gln. (b) Cell cycle distribution of cells incubated for 3 days with or without Gln. Mean ± S.E.M. n = 3 independent experiments. p value refers to a two-tailed t test for unpaired samples. Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.

Supplementary Figure 6 Exchange rates of metabolites in GBM cell lines.

Cells were incubated for 24 h +/− U-13C5 Gln, secretion rates (positive bars) and consumption rates (negative bars) of the indicated metabolites are shown. The sum of all isotopologues is reported for clarity. Alanine (Ala) exchange rates are magnified in the inset. Mean ± S.E.M. n = 3 independent experiments.

Supplementary Figure 7 Effects of Gln availability and Glutaminase inhibition on GBM cell lines growth and redox state.

(a) Correlation between Gln consumption and growth inhibition caused by Gln starvation is shown in six cell lines. Mean ± S.E.M. n = 3 independent experiments. (b) Citrate isotopologues distribution of 13C5-Gln derived atoms. 13C6-Citrate was not detected. Mean ± S.E.M. n = 3 independent experiments. (c) Oxidized (GSSG) to reduced (GSH) glutathione ratio. LN18 cells were incubated for 24 h +/− Gln and supplemented with Glu, α-ketoglutarate dimethylester (dm-αKG), sulfasalazine (SSZ), or cystine, as indicated. GSSG/GSH ratio was assessed by HPLC-MS, and shown as % of untreated control. Data derive from one experiment performed once. Raw data of independent repeats are provided in the statistics source data Supplementary Table 5. (di) Cells were incubated with 0, 2.5, 5, 10, 15, 30 μM BPTES for 72 h, in the absence (solid line) or presence (dotted line) of 4 mM dm-αKG and counted. In all conditions cells were exposed to 0.3%DMSO. Mean ± S.E.M. n = 3 independent experiments.

Supplementary Figure 8 GS expression and its effects on colony formation capacity, and glucosamine biosynthetic pathway in GBM cells.

(a) Cells were incubated for 24h +/− Gln and GLUL (GS), GLS, and MYC mRNA relative expression was assessed by qPCR. Actin expression was used for normalization. Data derive from one experiment performed once. Raw data of independent repeats are provided in the statistics source data Supplementary Table 5. (b) SF188 and U251 cells stably expressing a non-targeting control shRNA (shNTC) and two sequences targeting GS (shGS-1 and shGS-2) were cultured for 12–17 days +/− Gln in medium supplemented with glutamate (4 mM), and ammonia (0.8 mM) as indicated. The quantification of colonies surface area was obtained as described in the Methods section. Mean ± S.E.M., n = 4 independent experiments. (c) iRFP4 and iRFP-GS5 expressing LN18 cells were incubated +/− Gln for 24 h in medium supplemented with 0.8 mM 15NH4+. The intracellular isotopologues distribution of UDP N-acetylglucosamine is shown as % of control (value obtained for the metabolites in iRFP4 cells + Gln). Data derive from one experiment performed twice. Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.

Supplementary Figure 9 Heterogeneous GS expression in human GBM biopsies.

The whole bioptic tissues of three patients included in the tissue microarray were stained for GS, and Hematoxilin and Eosin [H&E], as indicated. Low magnifications of whole biopsies are reported in panels (a,b,h,i,n,o). For each biopsy, tissue regions framed in blue and green are magnified in dg,jm,qt. Biopsy 1 shows necrotic tissue [nec] surrounded by a solid tumour core [TC] with uniformly low GS staining (d,f). (d,e) Invasive tumour areas [TI] show infiltrative cells with heterogeneous immunoreactivity for GS. (c) Normal astrocytes [black arrows] in the adjacent normal brain [Br] were positive for GS, while neurons [white arrows] were GS-negative. Biopsy 2 shows distinct areas with low/absent (k) and high (j) immunoreactivity for GS. GS-negative inflammatory tissue [i] surrounding a necrotic region [nec] is evident in panels h,i,km. Biopsy 3 shows distinct areas with low/absent (q) and high (r) immunoreactivity for GS. (p) GS-positive reactive astrocytes [arrows] with extensive cellular processes forming a network of GS positive fibers surrounding GS-negative tumour cells.

Supplementary Figure 10 Glucose and glutamine metabolic fates in GBM patients, and orthotopic mouse models.

(a) Serum levels of 13C6 and 13C0 glucose in three GBM patients injected with 13C6 glucose before surgical intervention for tumour removal. The dashed line represents the time at which glucose infusion starts. Black arrow points at the time of surgical resection. Values are reported as % of the basal values obtained before infusion. (b) 13C Lactate and (c) 13C Glutamate enrichment in serum at time of tumour resection, in tumour tissue, and in adjacent edematous tissue, of seven (1-7) GBM patients injected with 13C6-glucose. The % of lactate or glutamate incorporating one or more 13C carbons over the total amount of lactate or glutamate detected is reported; na: not available, nd: not detectable. Values were corrected for the natural abundance of 13C. (d,e) Isotopologue distribution of metabolites obtained in the liver of mice orthotopically xenografted with human P3 GBM and injected with 13C6 Glucose (d) or 13C5-Gln (e). The values are mean ± S.E.M. n = 3 mice. (f,g) Isotopologues of Gln (f) and glucose (g) found in the serum of mice orthotopically xenografted with the human GS-positive T16 GBM, and infused for 4h into the carotid artery with 13C5-Gln. Mean ± S.E.M. n = 3 mice. (hj) Isotopologue distributions of Gln, Glu and α-kg in T16 tumours, contralateral brain, and liver tissues of mice infused as in (f,g). Mean ± S.E.M. n = 3 mice. (k) Astrocytes were incubated for 24 hours +/− Gln, and asparagine consumption is reported. Data derive from one experiment performed twice. Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.

Supplementary Figure 11 A toy network used for metabolic modelling.

A toy network (a) and its optimal flux distribution (b).

Supplementary Figure 12 Unprocessed scans of western blots accompanied by size markers.

Red dotted lines delineate the region presented in the respective Figures as indicated. Images were obtained using a Licor Odyssey scanner and acquired using Image Studio 2.0.

Supplementary Table 1 Formulation of DMEM and Serum-like Modified Eagles Medium (SMEM).
Supplementary Table 2 Exchange rates of metabolites in GBM cell lines.
Supplementary Table 3 Representation of SMEM growth medium in the metabolic model Recon1.
Supplementary Table 4 Weighted costs of growth limiting metabolites.

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Tardito, S., Oudin, A., Ahmed, S. et al. Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma. Nat Cell Biol 17, 1556–1568 (2015). https://doi.org/10.1038/ncb3272

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