Glutamine anaplerosis is required for amino acid biosynthesis in human meningiomas

Abstract

Background

We postulate that meningiomas undergo distinct metabolic reprogramming in tumorigenesis and unraveling their metabolic phenotypes provide new therapeutic insights. Glutamine catabolism is key to the growth and proliferation of tumors. Here, we investigated the metabolomics of freshly resected meningiomas and glutamine metabolism in patient-derived meningioma cells.

Methods

¹H NMR spectroscopy of tumor tissues from meningioma patients was used to differentiate the metabolite profiles of grade-I and grade-II meningiomas. Glutamine metabolism was examined using ¹³C/¹⁵N glutamine tracer, in 5 patient-derived meningioma cells.

Results

Alanine, lactate, glutamate, glutamine, and glycine were predominantly elevated only in grade-II meningiomas by 74%, 76%, 35%, 75%, and 33%, respectively, with alanine and glutamine levels being statistically significant (P ≤ .02). ¹³C/¹⁵N glutamine tracer experiments revealed that both grade-I and -II meningiomas actively metabolize glutamine to generate various key carbon intermediates including alanine and proline that are necessary for the tumor growth. Also, it is shown that glutaminase (GLS1) inhibitor, CB-839 is highly effective in downregulating glutamine metabolism and decreasing proliferation in meningioma cells.

Conclusion

Alanine and glutamine/glutamate are mainly elevated in grade-II meningiomas. Grade-I meningiomas possess relatively higher glutamine metabolism providing carbon/nitrogen for the biosynthesis of key nonessential amino acids. GLS1 inhibitor (CB-839) is very effective in downregulating glutamine metabolic pathways in grade-I meningiomas leading to decreased cellular proliferation.

Key Points.

1. Metabolomics of meningioma tumors revealed that alanine and glutamine/glutamate were predominantly elevated in grade-II meningiomas.

2. Stable isotope-based ¹³C/¹⁵N glutamine flux analysis revealed that grade-I meningiomas possess relatively higher glutamine metabolism.

3. GLS1 inhibitor (CB-839) is highly effective in downregulating glutamine-dependent metabolic pathways.

Importance of the Study.

NMR-based metabolomics of meningiomas revealed that alanine and glutamine were significantly elevated in grade-II meningiomas. Stable isotope-based ¹³C/¹⁵N glutamine flux studies showed that glutamine facilitates the biosynthesis of nonessential amino acids including alanine. GLS1 inhibitor (CB-839) is shown to reduce proliferation significantly in both grade-I and -II meningioma cells. Currently, no chemotherapy is part of the standard of care and targeting glutamine metabolism using GLS1 inhibitors may provide opportunities in the therapeutic intervention of meningiomas.

Meningiomas are the most frequently reported central nervous system (CNS) tumors accounting for 37.0% with an incidence rate of 8.33/100 000.¹ 80% of meningiomas are slow-growing and are benign (WHO grade-I). Whereas, atypical meningiomas (WHO grade-II), accounting for 18.0%, exhibit increased cellular abnormalities, grow at a faster rate than benign meningiomas, and show higher recurrence rate.² Anaplastic meningiomas (WHO grade III) are very rare contributing only up to 2.0% to the total meningiomas.^2,3 High index of recurrence within or after 5 years of the first surgery is common in grade-II meningiomas, and it is associated with lower survival rates. Currently, no chemotherapy is part of the standard of care for meningiomas, leaving surgery followed by radiotherapy as the only available treatment option. Metabolism is the key to many biological processes including cancer.^4–6 Both benign and malignant tumors are known to have altered metabolic profiles. Quantification of metabolite levels along with metabolic flux of essential nutrients provides valuable information that is necessary to derive valuable biological insights. Unraveling metabolic phenotypes of various histological subtypes of meningiomas may identify new targets that can be further exploited for therapeutic intervention.

Earlier magnetic resonance spectroscopy (MRS) studies have reported elevated levels of alanine in meningioma patients without making any quantitative comparison between the histological subtypes.^7–12 Here, we have characterized two most common histological subtypes of meningiomas, by ex vivo ¹H NMR spectroscopy of surgically resected tumors from patients undergoing craniotomy. We have observed elevated levels of many nonessential amino acids such as alanine, glutamine, glutamate, and glycine with alanine and glutamine showing statistical significance (P < .02) in grade-II meningiomas. These results have led us to probe a detailed investigation on amino acid biosynthesis in meningiomas. Glucose and glutamine are the 2 major nutrients present in the circulation which are required for many biochemical synthesis pathways to act as carbon and nitrogen sources, respectively.^13,14 Cellular biosynthesis of alanine by alanine aminotransferase (ALT) requires pyruvate and glutamate as precursors, while the latter acting as a nitrogen donor. Tumor cells produce excess amount of pyruvate through Warburg glycolysis whereas glutamate can be generated from glutamine metabolism via glutaminase (GLS).¹⁴ Aggressive recurrent meningiomas overexpress glutamate transporters which may contribute to elevated levels of glutamate in the tumor tissues.^15–17 Additionally, these cells might have an active glutamine metabolism to supply glutamate as the nitrogen source for alanine biosynthesis. Similarly, biosynthesis of aspartate relies on glutamine-derived glutamate as both carbon and nitrogen source. Serine de novo synthesis also depends on glutamate to donate amino group for glycine synthesis by serine hydroxymethyltransferase (SHMT).¹⁸ Proline is also synthesized from glutamate that is known to play a key role in tumor proliferation in many human cancers through the oncogene c-MYC regulation.^19,20 Also, c-MYC is known to stimulate glutamine catabolism by upregulating GLS.^20,21 Here, we have investigated the metabolomics of human meningiomas, and the role of glutamine metabolism in patient-derived grade-I and grade-II meningioma cells. We also probed the effect of a GLS1 inhibitor (CB-839) on the proliferation and glutamine metabolism in these cells, using ¹³C and ¹⁵N-labeled glutamine as tracers. The results suggest that glutamine catabolism may be a key requirement for the cellular proliferation of human meningiomas with grade-I tumors showing relatively higher glutamine metabolic activity along with increased sensitivity to GLS1 inhibitor.

Materials and Methods

Patients

Thirty-three tumor tissue samples were collected from WHO grade-I (n = 23) and grade-II (n = 10) meningioma patients (Table 1) who were undergoing surgical excision of the tumors at the Houston Methodist Hospital. All surgeries were performed by the neurosurgeon and the co-author (D.S.B.). Informed consent was obtained from each patient following an Institutional Review Board (IRB) protocol approved by Houston Methodist Hospital and Research Institute. The clinical characteristics of study participants are provided in Table 1. Also, a grade-I meningioma patient was recruited (at the Medical University of Vienna, Austria, approved by their IRB) to demonstrate differences in the levels of key metabolites between meningioma tumor and the surrounding normal brain, by in vivo metabolic mapping using 7T magnetic resonance spectroscopic imaging (MRSI).

Table 1.

Patients Characteristics Showing Meningioma Tumor Grade, MIB-1 Index, and Mitotic Activity

Patient	Sex (M/F)	Age (yr)	Meningioma Grade (IHC)	MIB-1 (%)	#Mitoses/ 10 HPFs
1	F	61	Grade I	1	1
2	M	80	Grade I	10	3
3	M	60	Grade I	2	1
4	F	59	Grade I	1	Not identified
5	F	45	Grade I	2-3	1
6	F	73	Grade I	1-4	1
7	F	49	Grade I	5-8	3
8	F	57	Grade I	2-3	1
9	F	65	Grade I	2	Not identified
10	M	40	Grade I	3-4	Not identified
11	F	41	Grade I	2	NA
12	F	36	Grade I	5	3
13	F	67	Grade I	2-3	1
14	F	44	Grade I	5-10	3
15	F	37	Grade I	5	1
16	F	69	Grade I	1-2	Not identified
17	F	45	Grade I	2-4	Inconspicuous
18	M	29	Grade I	3-10	3
19	F	54	Grade I	2	Not identified
20	M	73	Grade I	2	Inconspicuous
21	M	68	Grade I	5	2
22	F	73	Grade I	2-3	1
23	M	73	Grade I	2-3	1
24	F	61	Grade II	8	6
25	F	75	Grade II	12	6
26	F	65	Grade II	5	4
27	F	85	Grade II	NA	2c
28	F	52	Grade II	5-6	5
29a	F	75	Grade II	20	16
30	M	34	Grade II	15	5
31	F	46	Grade II	14	6
32	M	66	Grade II	8	6
33b	F	46	Grade II	14	6

Abbreviations: F, female; HPF, high power field; IHC, immunohistochemistry; M, male; NA, not available.

Grade-I meningioma patients showed lower MIB-1 index in the range 1%-5% (except 4 patients #2, 7, 14, and 18 which showed MIB-1 index in the range of 5%-10%), whereas grade-II patients showed an elevated MIB-1 index of 5%-20%. Low mitotic rate (≤3 mitoses/10 HPFs) was observed in grade-I patients compared to grade-II patients (4-16 mitoses/10 HPFs).

^aSame as patient #25, tumor recurred in this patient and underwent a second surgery 9 months after the initial surgery.

^bSame as patient #31, tumor invaded to the facial region, and the specimen was collected from this region.

^cOne of the grade-II patients (patient #27) had 2 mitoses/10 HPFs, but the patient presented with brain invasion, and hence it was classified as a grade-II meningioma.

Chemicals and Reagents

Methanol, chloroform, GLS1 inhibitor (CB-839), chloroform-d (CDCl₃), [U-¹³C]glutamine, [α-¹⁵N]glutamine, [¹⁵N-amide]glutamine, and [3-¹³C]alanine were purchased from Millipore Sigma (St. Louis, MO, USA). D₂O, DCl, and NaOD were purchased from Cambridge isotope laboratories (Tewksbury, MA, USA). 3-(trimethylsilyl)-1-propane sulfonic acid-d₆ sodium salt (DSS-d₆) solution was purchased from Chenomx Inc. (Edmonton, Canada). Calcein-AM and Ethidium Homodimer were obtained from Biotium (Fremont, CA, USA), and Hoechst 33342 was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Cell Lines

Grade-I meningioma cell lines (GAR, SAM, and JEN) were gifts from Dr. Randy L. Jensen (University of Utah, Salt Lake City, UT).^22,23 Two grade-II meningioma cell lines (MNG2-66 and MNG2-77) were generated from surgically resected tissue specimens from atypical meningioma patients. Tumor tissue was washed with cold PBS (phosphate-buffered saline) and manually minced using a scalpel. The cells were cultured in high glucose (25 mM) DMEM supplemented with 20% fetal bovine serum (FBS), 2.0 mM glutamine, and 1.0 mM pyruvate or ABM astrocyte basal media (Lonza, Walkersville, MD, USA). MNG2-66 cells grew well in ABM whereas MNG2-77 grew in DMEM. Tumor majority was observed after 2-3 passages, and these cells were authenticated for the presence of meningioma features against the parent surgical tissue specimens (University of Arizona Genetics Core, Tucson, AZ, USA), and the details have been provided in the Supplementary information (hematoxylin and eosin [H&E] staining images: Supplementary Figure S1; the allele reports: Supplementary Figures S2 and S3). Further, patient-derived tumor cells and corresponding grade-specific tumor tissues showed similar metabolite profiles (Supplementary Figures S4 and S5).

[U-¹³C]Glutamine, [α-¹⁵N]Glutamine, [¹⁵N-Amide]Glutamine, or [3-¹³C]Alanine Metabolism Studies

Both grade-I and grade-II meningioma cells were grown in T-75 flasks (n = 4) as described above at 37°C under humidified air with 5% CO₂

. When cells reached confluency, they were treated with 4.0 mM [U-¹³C]glutamine or [α-¹⁵N]glutamine or [¹⁵N-amide]glutamine or 2.0 mM [3-¹³C]alanine and incubated for 24 h. Cells were harvested in 50% methanol, snap-frozen in liquid N₂, and lysates were stored at −80°C until further analysis by gas chromatography-mass spectrometry (GC-MS) for mass isotopomer distribution (MID) of various metabolites.

Effect of GLS1 Inhibitor on the Proliferation of Meningioma Cells

Grade-I cells (JEN, SAM, and GAR) and grade-II cells (MNG2-77) were plated in 96-well plates at 1500-3000 cells per well in 0.2 mL DMEM containing either 11.0 mM or 25.0 mM d-glucose. Another grade-II cell line (MNG2-66) was grown in ABM media. 24 hours after seeding, the respective media was replaced with fresh media containing the GLS1 inhibitor, CB-839 at various concentrations (0.1, 1.0, and 5.0 µM) and incubated for 24, 48, 72, and 144 (only for grade-II cells) hours.²⁴ Cell viability was evaluated using the dyes Calcein-AM (1 µM), Ethidium Homodimer (2.5 µM), and Hoechst 33342 (5 µg/mL). Dyes were mixed together at the specified concentrations, diluted in complete medium, and added to each well. Cells were incubated with dyes for 20 min at 37°C in humidified atmosphere of air:CO₂ (95:5, v/v). After incubation, images were acquired using the epifluorescence microscope Axio Observer 7 (Zeiss, Oberkochen, Germany). 8-20 images per each 4 replicate wells per treatment group were obtained. ImageJ (FIJI, version 1.8_172) was used for image processing and counting the cells, raw cell counts of each treated group were normalized to the cell numbers in the DMSO treated group (vehicle) at each time-point, and the normalized data were plotted on Prism 9.0 (GraphPad).

To test the effect of CB-839 on glutamine metabolism in grade-II cells that had higher MIB-1 index, we used MNG2-77 cells, primed the media with 5.0 µM CB-839 (test, n = 4-8) or with DMSO and were incubated for 48 h at 37°C under humidified air with 5% CO₂. Finally, cells were treated with 4.0 mM [U-¹³C]glutamine or [α-¹⁵N]glutamine in the presence of CB-839 or DMSO for 24 h. The cells were harvested in 50% methanol, snap-frozen in liquid N₂, and lysates were stored at −80°C until further analysis by GC-MS.

Methanol-Chloroform Extraction of Tumor Tissues

Metabolites were extracted from pre-weighed frozen tumor tissues (~50 mg) from each patient following the methanol:chloroform extraction method as described in our recent publication.²⁵ The aqueous-methanol layers were dried in a CentriVap vacuum concentrator (Labconco, Kansas City, MO, USA), and the residue was reconstituted in 180 µL D₂O containing 1.0 mM DSS-d₆ (internal standard). The pH of the final solution was adjusted to 7.4.

¹H NMR Experiments

¹H NMR spectra of above solutions were collected on a Bruker 600 MHz spectrometer (¹H frequency) equipped with a cryogenically cooled ¹H/¹³C detection probe (Bruker Biospin, Billerica, MA, USA). Nuclear Overhauser Effect Spectroscopy (NOESY) pulse sequence with water pre-saturation was used for collecting the ¹H NMR data. The acquisition and processing parameters are described in our recent publication.²⁵ We have quantified water-soluble metabolites in the tumor extracts by analyzing the ¹H NMR data and their assignments were validated using chemical shifts reported in the literature.^25–27 The concentrations (µmol/g of wet weight of tissue) of the metabolites were determined as described previously.²⁵ In vivo 7T MRSI metabolite mapping experimental details are reported earlier.²⁸

Preparation of Cell Extracts for GC-MS Analysis

For ¹³C isotopomer analysis of intracellular metabolites by GC-MS, snap-frozen cell lysates were thawed and centrifuged to remove the precipitated proteins. 50 nmols of sodium 2-oxobutyrate (internal standard) was added to the lysates and dried samples were derivatized by trimethylsilylation. 3 µL of the derivatized solution was injected onto an Agilent 6970 gas chromatograph equipped with a fused silica capillary GC column coupled with an Agilent 5973 mass selective detector.²⁹ The measured MID of carbon isotopomers of various metabolites were corrected for natural abundance of ¹³C isotopomer (1.1%).

Western Blot Analysis

The meningioma cells grown in dishes were lysed in RIPA buffer with protease inhibitor, and proteins were separated using SDS-PAGE. Proteins from gel were transferred to PVDF (polyvinylidene fluoride) membrane using standard Towbin buffer. Blots were blocked with 5% nonfat dried milk in PBS containing 0.1% Tween-20, incubated with primary antibodies of GLS1, c-MYC, pyrroline-5-carboxylate synthase (P5CS), and pyrroline-5-carboxylate reductase 1 (PYCR1) and secondary antibodies at room temperature. Washed blots were incubated with SuperSignal West Femto solution and exposed to X-ray films.

Statistical Analysis

The means and standard deviations of metabolites quantified were compared between grade-I and grade-II meningiomas using Student’s t test. Metabolites showing P-value ≤.05 were considered statistically significant.

Results

Aqueous Metabolite Profiles of Grade-I and Grade-II Meningiomas

Aqueous extracts of surgically resected grade-I and grade-II meningioma tumor tissues were analyzed by ¹H NMR spectroscopy. Figure 1 shows representative brain MRI images of grade-I and grade-II meningioma patients along with H&E-stained images of resected tumor tissues from respective patients (A), ¹H NMR spectra of tumor extracts of grade-I and grade-II meningiomas (B), and bar charts showing the concentrations of key metabolites (C). We identified the following metabolites in the tumor extracts: leucine, isoleucine, valine, lactate, alanine, acetate, glutamate, succinate, glutamine, aspartate, creatine (Cr)/phosphocreatine (PCr), phosphocholine (PC), glycerophosphocholine (GPC), myo-inositol (mI), scyllo-inositol (sI), taurine (Tau), hypotaurine (H-Tau), glycine, phosphoethanolamine (PE), d-mannitol, and glucose. d-mannitol is an exogenous compound infused in patients during the craniotomy to reduce cerebral edema and the intracranial pressure.³⁰ From Figure 1B and C, it is evident that lactate (76%), alanine (74%), glycine (33%), glutamate (35%), glutamine (75%), and succinate (69%) were highly elevated in grade-II tumors while taurine (21%) and aspartate (16%) were decreased when compared to the grade-I tumors. The levels of glycolytic metabolites, such as lactate, alanine, and glycine were elevated only in grade-II meningiomas with alanine being significant (P ≤ .02). Additionally, the tricarboxylic acid (TCA) cycle intermediates—glutamate, glutamine, and succinate levels were also elevated in grade-II meningiomas with glutamine being significant (P ≤ .02). PC and GPC were quantified together due to their signal overlap, and we observed a statistically significant increase (68%) in the levels of (PC + GPC) in grade-II meningiomas (Figure 1B and C). This observation is in agreement with previous MRS reports showing elevated levels of total choline in gliomas.^7–12 Concentrations of all the detected metabolites are given in Supplementary Table S1. In order to illustrate the metabolic differences between the tumor and surrounding normal brain, we have shown the 7T in vivo MRSI metabolite mapping from a grade-I meningioma patient (Supplementary Figure S6). Also, to further demonstrate differences in the steady-state levels of various key metabolites between meningiomas and normal surrounding brain, we used ex vivo ¹H NMR data from a non-tumor tissue (collected from a brain tumor patient) and compared with both grades of meningiomas (Supplementary Figure S7).

Fig. 1.

(A) Post-contrast T1w MR images of grade-I and grade-II meningiomas and respective H&E staining of the resected tumors. (B) ¹H NMR spectra of tumor extracts from grade-I and grade-II meningiomas (*solvent impurity, isopropanol; d-mannitol is infused in patients during the craniotomy to reduce cerebral edema and the intracranial pressure). (C) Metabolite concentrations of grade-I and grade-II meningiomas. Abbreviations: GPC, glycerophosphocholine; H&E, hematoxylin and eosin; mI, myo-inositol; PC, phosphocholine; PE, phosphoethanolamine.

Glutamine Carbons Contribute to Amino Acid Biosynthesis in Meningiomas

To determine the level of glutamine dependency in grade-I and -II tumors, we performed GC-MS-based [U-¹³C]glutamine flux measurements using patient-derived grade-I and grade-II meningioma cells. GC-MS MID analysis of cells treated with [U-¹³C]glutamine showed the presence of active glutamine catabolism (Figure 2). Glutamine is used as a carbon source for TCA cycle intermediates in both grades evidenced by the detection of ¹³C labeled isotopomers of citrate, glutamate, fumarate, malate, and aspartate including the glycolytic metabolites—pyruvate, lactate, and alanine (Figure 2A and C). Notably, the presence of M+3 alanine isotopomer clearly shows that glutamine is the carbon source for alanine biosynthesis in addition to the glucose-derived pyruvate. Additionally, glutamine carbons contributed to the biosynthesis of proline (via glutamine-derived glutamate) which are detected through M+5 proline (¹³C labeling of 43.24 ± 0.39%, 33.11 ± 0.67% in MNG2-66 and MNG2-77, respectively; 20.0 ± 1.14%, 17.05 ± 1.67%, 19.90 ± 0.57% for GAR, JEN, and SAM, respectively). Lower levels of M+5 proline in grade-I are significant because proline synthesis is shown to be tightly regulated by c-MYC gene which was shown earlier to be absent or relatively weak in grade-I meningiomas.²¹ However, our current results do not provide any direct link between c-MYC and proline biosynthesis (Supplementary Figure S8). Next, the presence of both M+4 and M+5 citrate isotopomers indicates that all these cells possess both canonical TCA cycle oxidation and reductive carboxylation pathways of glutamine metabolism (Figure 2). The reductive carboxylation of glutamine produces M+5 citrate (3.30 ± 0.39% and 10.02 ± 0.24% in MNG2-66 and MNG2-77, respectively; 4.25 ± 0.75%, 3.82 ± 0.44%, and 5.94 ± 1.03% in GAR, JEN, and SAM, respectively) through the addition of an unlabeled carbon by isocitrate dehydrogenase (IDH) acting in the reverse direction (green curved arrow, Figure 2B), whereas canonical TCA cycle oxidation of glutamine loses a ¹³C carbon as ¹³CO₂ and thus produces M+4 citrate (34.49 ± 0.99% and 36.05 ± 0.59% in MNG2-66 and MNG2-77, respectively; 38.27 ± 1.15%, 37.59 ± 1.20%, and 38.78 ± 0.62% in GAR, JEN, and SAM, respectively) (blue arrow, Figure 2B). Citrate leaves TCA cycle and cleaved by ATP-citrate lyase (ACLY) in the cytosol to produce M+2 acetyl-CoA which acts as precursor for lipid de novo synthesis. The ratio of (M+5/M+4) citrate gives a measure of the relative flux of reductive carboxylation to the oxidation of glutamine via the canonical route.³¹ The (M+5/M+4) ratio of citrate in MNG2-66 and MNG2-77 was 9.58 ± 1.20% and 27.81 ± 1.06%, respectively, and this ratio in GAR, JEN, and SAM was 9.21 ± 1.63%, 9.95 ± 1.33%, and 6.57 ± 0.59%, respectively; suggesting MNG2-77 has ~3-4 times higher reductive carboxylation flux than the other 4 meningioma cells (Figure 2A and C).

Fig. 2.

(A) ¹³C MID of the metabolites during [U-¹³C]glutamine metabolism in grade-II meningioma cells. (B) Schema showing the [U-¹³C]glutamine metabolism via canonical TCA cycle and reductive carboxylation pathways. (C) ¹³C MID of various metabolites during [U-¹³C]glutamine metabolism in grade-I meningioma cells. Abbreviations: MID, mass isotopomer distribution; TCA, tricarboxylic acid.

Glutamine Acts as Nitrogen Donor for De Novo Amino Acid Biosynthesis in Meningiomas

To test whether glutamine donates nitrogen for the amino acid biosynthesis in grade-I and grade-II meningiomas, we treated meningioma cells with [α-¹⁵N]glutamine and measured M+1 (¹⁵N) mass isotopomers of various amino acids. Both grade-II meningioma cells showed ¹⁵N labeling in the amino acids—alanine, aspartate, serine, and glycine. MNG2-77 showed relatively higher levels of ¹⁵N-labeling in these amino acids (Figure 3A). ¹⁵N-labeling in alanine (3.98 ± 0.65% in MNG2-66 and 12.68 ± 6.42% in MNG2-77) is an evidence for the transfer of amine nitrogen from glutamine-derived glutamate catalyzed by ALT. Higher level of ¹⁵N-labeling was detected in aspartate (20.43 ± 1.08% in MNG2-66 and 44.14 ± 0.66% in MNG2-77) suggesting higher aspartate aminotransferase (AST) activity in these cells. Thus, the current study clearly demonstrated that glutamine-derived glutamate acts as a nitrogen source in the biosynthesis of nonessential amino acids alanine and aspartate in human meningiomas (Figure 3B). We also detected a small ¹⁵N-labeling in serine, glycine, leucine, and isoleucine, suggesting that glutamine also donates nitrogen to the synthesis of these amino acids. Similar pattern of ¹⁵N transfer from glutamate to various amino acid pools was detected in grade-I meningioma cells (Figure 3C). All the 3 grade-I meningioma cells showed much higher ¹⁵N-labeling in alanine (38.14 ± 1.84%, 37.91 ± 5.10%, 40.19 ± 1.0% in GAR, SAM, and JEN, respectively), aspartate (47.55 ± 3.17%, 52.90 ± 3.38%, 52.73 ± 0.18% in GAR, SAM, and JEN, respectively), serine (18.0 ± 1.06%, 15.15 ± 2.83%, 20.62 ± 0.68% in GAR, SAM, and JEN, respectively), and glycine (14.87 ± 0.67%, 11.75 ± 1.46%, 16.73 ± 0.25% in GAR, SAM, and JEN, respectively). Relatively higher levels of ¹⁵N-labeling seen in various amino acids in GAR, SAM, and JEN cells indicates higher glutamine dependency of grade-I cells. This observation has been supported by higher GLS1 expression in grade-I meningiomas (Supplementary Figure S8).

Fig. 3.

(A) ¹⁵N MID of the amino acids during [α-¹⁵N]glutamine metabolism in grade-II meningioma cells. (B) Schema illustrating [α-¹⁵N]glutamine acting as a nitrogen donor for the synthesis of nonessential amino acids. (C) ¹⁵N MID of various metabolites during [α-¹⁵N]glutamine metabolism in grade-I meningioma cells. Abbreviation: MID, mass isotopomer distribution.

c-MYC-Dependent Proline Synthesis

c-MYC is known to enhance glutamine metabolism in human prostate cancer cells leading to the elevated enzymatic activities of GLS, P5CS, and PYCR1 (involved in proline synthesis).²⁰ Therefore, we measured the levels of c-MYC, and the 2 enzymes involved in the proline de novo synthesis to determine if there is any correlation between c-MYC and proline biosynthesis in meningiomas.^20,32 c-MYC is highly expressed in all the 3 grade-I tumors, and very little expression was detected in the grade-II tumors (Supplementary Figure S8). However, PYCR1 (the rate-limiting enzyme in proline synthesis) was highly expressed in all the 5 meningioma cells (Supplementary Figure S8).

Effect of GLS Inhibitor on the Proliferation of Meningioma Cells

After demonstrating higher glutamine dependence by grade-I cells, next, we wanted to determine whether targeting the key enzyme (GLS1) can alter the glutamine dependence of these cells and affect their proliferation. Grade-I meningioma cells showed a higher dose-dependent decrease (75%, relative to the vehicle) in the cell numbers after 48- and 72-h treatment with CB-839 compared to grade-II cells (Figure 4A). When treated with CB-839 for 144 h, the grade-II cells MNG2-66 and MNG2-77 showed decrease in cell numbers by 32% and 52% respectively (Figure 4B). Due to longer doubling times (3-4 days), grade-II cells were treated with CB-839 for up to 144 h. We also tested the cytostatic effectiveness of CB-839 in meningioma cells by measuring the cell proliferation after 72 h when the inhibitor treatment was stopped. We detected an increase in the proliferation in the range of 10%-38% in all the 5 cell lines (Supplementary Figure S9).

Fig. 4.

Graphs showing the effect of CB-839 on proliferation of (A) grade-I and (B) grade-II meningioma cells along with representative fluorescence micrographs of vehicle (DMSO) and CB-839 (5.0 µM, 72 h/144 h) treatments (blue stain: all nuclei; red stain: dead cells).

Next, we wanted to determine the effect of CB-839 on glutamine metabolism in grade-II cells (MNG2-77) that had higher MIB-1 index (Supplementary Figure S10). This particular grade-II cell line showed relatively higher GLS1 activity compared to the other grade-II cell line (MNG2-66). Figure 5A depicts the uptake of [U-¹³C]glutamine and its differential conversion to [U-¹³C]glutamate in MNG2-77 cells in the presence and the absence of CB-839. The MIDs of various [U-¹³C]glutamine-derived TCA cycle metabolites (citrate, aspartate, malate, and proline) are shown in Figure 5A. We observed statistically significant (P < .0001 for all) reduction in the levels of M+5 of glutamate (54.05%), proline (63.60%), and citrate (59.70%) and also in M+4 of malate (78.73%), aspartate (77.34%), and citrate (75.50%) (P < 1 × 10⁻⁹ for all) in MNG2-77 cells treated with CB-839 with respect to the vehicle treatment. We detected a 60% decrease in the relative flux through reductive carboxylation of glutamine via M+5 citrate in MNG2-77 cells treated with CB-839 compared to the vehicle treatment (Figure 5A). We also investigated the effect of CB-839 on the biosynthesis of nonessential amino acids in MNG2-77 cells treated with [α-¹⁵N]glutamine. The relative levels of M+1 glutamate directly derived from [α-¹⁵N]glutamine as well as M+1 labeled amino acids (alanine, aspartate, serine, and glycine) in the presence and absence of CB-839 are shown in Figure 5B. Cells treated with CB-839 showed a statistically significant reduction in the levels of M+1 of glutamate (52.53%), alanine (84.70%), aspartate (56.40%), serine (76.06%), and glycine (42.07%) when compared to the vehicle-treated group (P < 1 × 10⁻⁶ for all, except glycine, P = .168). Glutamine is the major source of cellular nitrogen for the de novo synthesis of various amino acids, and CB-839 treatment effectively has reduced glutamine metabolic flux through the inhibition of GLS1 activity (Figure 5B; Supplementary Figure S10) and hence the cellular proliferation of meningioma cells.

Fig. 5.

Effect of CB-839 on (A) [U-¹³C]glutamine and (B) [α-¹⁵N]glutamine metabolism in MNG2-77. The inhibitor caused a drastic reduction in the levels of glutamine-derived glutamate along with other key metabolites.

Ammonia Recycling Is Active in Meningioma Cells

It is known that ammonia is generated during glutamine metabolism and is accumulated in the tumor cells. Recently, it has been shown that ammonia generated from glutamine catabolism is recycled into amino acid biosynthesis in various human cancers to support cancer growth.³³ We wanted to test if meningiomas have a similar ammonia recycling mechanism using [¹⁵N-amide]glutamine. In both grade-I and grade-II meningioma cells, ¹⁵NH₃ released by GLS gets recycled into [α-¹⁵N]glutamate by combining with α-KG via glutamine dehydrogenase (GDH) which we measured through the detection of M+1 [α-¹⁵N]glutamate (4.3 ± 1.0%) (Supplementary Figure S11). These findings are in agreement with a recent report in human breast cancer cells.³³ Surprisingly, we also detected M+2 [¹⁵N₂] glutamine (2.9 ± 0.3%) which can only be produced from M+1 [α-¹⁵N]glutamate recondensing with ¹⁵NH₃, via glutamine synthase (GS).

Alanine Contributes to TCA Cycle Flux in Grade-II Meningiomas

Since we observed a significantly higher level of alanine in grade-II meningiomas, we wanted to investigate whether the role of alanine is to act as an alternative nutrient. To test this, we treated the grade-II tumor cells with [3-¹³C]alanine. Metabolic flux analysis showed that [3-¹³C]alanine contributed to the synthesis of [3-¹³C]pyruvate through the presence of M+1 pyruvate (7.17 ± 0.99% in MNG2-66 and 4.45 ± 0.47% in MNG2-77) which further entered the TCA cycle via [2-¹³C]acetyl-CoA to produce M+1 citrate (8.92 ± 0.46% in MNG2-66 and 7.33 ± 0.51% in MNG2-77), glutamate (6.01 ± 0.44% in MNG2-66 and 1.45 ± 0.06% in MNG2-77), and malate (4.55 ± 0.73% in MNG2-66 and 3.17 ± 0.59% in MNG2-77) (Supplementary Figure S12). Interestingly, we detected very little ¹³C labeling in the lactate (2.07 ± 0.28% in MNG2-66 and 2.89 ± 0.28% in MNG2-77) which suggested that alanine carbons may be preferentially used in mitochondrial oxidative metabolism in the TCA cycle and not affecting glycolytic fluxes and cytosol NAD⁺/NADH ratio. Similar observation was made by Sousa et al where they showed endogenously produced alanine by pancreatic stellate cells (PSCs) cells are used up by pancreatic cancer cells as alternative carbon fuel.³⁴

Discussion

Although earlier MRS studies reported elevated levels of alanine in meningiomas, there are no comparative studies showing differential levels of alanine between grade-I and grade-II meningiomas.^7–12 Here, we quantified the levels of alanine along with other key metabolites in surgically resected tumor tissues obtained from grade-I and grade-II meningioma patients. One of the main observations from this analysis is that alanine (74%), glutamate (35%), glutamine (75%), and (PC + GPC) (68%) were highly elevated in grade-II tumors (Figure 1C). Further, we illustrated in a grade-I meningioma patient using in vivo 7T MRSI metabolic imaging that clearly shows the differences in the levels of various key metabolites (ie, alanine, glutamine, glutamate, glycine, choline) between the tumor and surrounding brain (Supplementary Figure S6). This in vivo metabolic MRSI data fully supports our ex vivo metabolomics data derived from resected tumor tissues from patients.

Alanine is synthesized from reductive amination of pyruvate by the enzyme ALT with glutamate acting as a nitrogen donor (Figure 3B). The weaker GLS1 expression (Supplementary Figure S8) together with elevated levels of glutamine and glutamate transporters seen in grade-II meningiomas may lead to the accumulation of glutamine and glutamate in the grade-II tumors.^15–17 The elevated levels of glutamate may be playing a key role in the upregulation of alanine de novo synthesis in grade-II meningiomas.³⁵ Recently, it has been demonstrated that stroma associated with PSCs are essential for the metabolism in pancreatic ductal adenocarcinoma (PDAC) which secrete nonessential amino acids, in particular, alanine which was taken up by the PDAC cells as an alternative nutrient, acting as a significant carbon source for the TCA cycle activity.³⁴ Here, we showed that the meningioma cells utilize [3-¹³C]alanine and generate the key TCA cycle intermediates (Supplementary Figure S12). Thus, the elevated levels of alanine found in meningiomas may be used as an additional fuel to support the tumor growth. Interestingly, alanine did not contribute much to the lactate synthesis through pyruvate. These results suggest that meningiomas utilize alanine to fuel mitochondrial metabolism without influencing the glycolytic flux. Alanine de novo synthesis depends on the precursors, pyruvate and glutamate, derived from metabolism of both glucose and glutamine and also glutamate derived from other nutrients. This further signifies the role of acetate and β-hydroxybutyrate as potential alternative energy sources in meningiomas.

Glycine is synthesized from serine by SHMT and many cancer cells have upregulated glycine synthesis pathway,³⁶ which is essential for the biosynthesis of porphyrins and glutathione. Our data showing the elevated levels of glycine (by 33%) in grade-II meningiomas may be due to an increased glycine demand by the tumors for their growth and proliferation.

Glutamine is a major nutrient and nitrogen donor for proliferating cells. Our ¹³C glutamine tracer metabolism data clearly showed that meningiomas are actively uptaking glutamine which is further catabolized to generate key carbon intermediates for anabolic reactions. Recently, it was shown that c-MYC expression was shown to be higher in grade-II compared to grade-I meningiomas.²¹ c-MYC plays a direct role in enhancing GLS expression and proline biosynthesis in cancer cells.²⁰ Our data showing increased proline biosynthesis in both grade-I and -II meningioma cells irrespective of weaker c-MYC expression in grade-II cells may be due to the elevated levels of glutamate as seen in grade-II tumors (Figure 1C) and may not reflect any relationship between c-MYC and proline biosynthesis in atypical meningiomas as reported earlier.²¹ Though the exact role of increased proline synthesis in cancer cell metabolism is not fully clear, it may be possible that generation of NAD⁺ during the conversion of glutamine-derived glutamate to pyrroline-5-carboxylate (P5C) by P5CS, and subsequent conversion of P5C to proline by PYCR1 in the final rate-limiting step of the proline synthesis may allow the TCA cycle to function uninterrupted during oxygen limiting conditions.³⁷

Active glutaminolysis generates ammonia which could be recycled through GDH and GS to maintain glutamate levels up for the synthesis of proline and aspartate. This is indicative of a sophisticated glutamine-driven ammonia recycling mechanism to further augment nitrogen supply for amino acid synthesis in meningiomas.

Overall, blocking glutamine metabolism using GLS1 inhibitor (CB-839) significantly decreased cell proliferation in both grades (I and II) of meningioma cells. A relatively higher glutamine metabolism seen in grade-I meningiomas led to an enhanced sensitivity to CB-839 treatment. Decreased glutamine utilization in the presence of GLS1 inhibitor was also observed in triple-negative breast cancer cells, soft-tissue sarcoma, and brain tumor stem cells.^24,38,39 GLS1 inhibition may play a significant role in the development of targeted therapy for meningiomas, in particular in grade-I tumors, as we have demonstrated its ability to decrease glutamine uptake and proliferation. In conclusion, further detailed in vivo metabolic studies in patients using both MRS and isotopically ¹³C/¹⁵N-labeled glutamine would facilitate the validation of metabolic markers and strategies for the development of targeted therapies using GLS1 inhibitors for the diagnosis and treatment of human meningiomas.

Funding

This study was supported by The Donna and Kenneth R. Peak Foundation, The Kenneth R. Peak Brain and Pituitary Tumor Treatment Center at Houston Methodist Hospital, The Houston Methodist Foundation, The Taub Foundation, The Pauline Sterne Wolff Foundation, The Veralan Foundation, The Marilee A. and Gary M. Schwarz Foundation, The John S. Dunn Foundation, Contributions in honor of Will McKone (D.S.B.), and the Austrian Science Fund (FWF) grants (W.B.).

Conflict of Interest statement. The authors declare no conflict of interest.

Authorship statement. Experimental design: K.P., O.B.I., and D.S.B. Acquisition of data: O.B.I., F.H.C., S.H., S.L., K.P., G.H., W.B., and G.W. Analysis and interpretation of data: O.B.I., K.P., F.H.C., S.H., S.A.H., R.M.B., G.H., W.B., and G.W. Writing, reviewing, and editing: O.B.I., K.P., F.H.C., S.H., M.A.S., R.M.B., D.S.B., G.H., and W.B.