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. 2024 Oct 24;27(3):668–681. doi: 10.1093/neuonc/noae222

Glutamate dehydrogenase 1-catalytic glutaminolysis feedback activates EGFR/PI3K/AKT pathway and reprograms glioblastoma metabolism

Rui Yang 1,✉, Guanghui Zhang 2, Zhen Meng 3, Li Wang 4, Yanping Li 5, Haibin Li 6, Siyuan Yan 7, Xiaonan Wei 8, Shanshan Wang 9, Hongjuan Cui 10,11,✉
PMCID: PMC11889723  PMID: 39446525

Abstract

Background

Glutamine is an important nutrient for cancer cell growth that provides biological sources for nucleic acid and fatty acid synthesis, but the role of glutaminolysis in signal transduction and glioblastoma (GBM) progression remains little known.

Methods

Knockdown and overexpression cells were obtained to explore the functional roles of glutamate dehydrogenase 1 (GDH1) in cell proliferation, tumor formation, and aerobic glycolysis. RNA-seq, Chromatin immunoprecipitation, luciferase assay, and western blot were performed to verify the regulation of the EGFR-AKT pathway by the GDH1 (also known as GLUD1) and KDM6A. Metabolite-level measurements and Seahorse Assay were performed to assess the functional role of GHD1 in reprogramming glycolysis.

Results

Here, we report that GDH1 catalytic glutaminolysis is essential for GBM cell line proliferation and brain tumorigenesis even in high-glucose conditions. Glutamine is metabolized through glutaminolysis to produce α-ketoglutarate (α-KG). We demonstrate that glutamine in combination with leucine activates mammalian TORC1 by enhancing glutaminolysis and α-KG production. α-KG increases the transcription of PDPK1 by reducing the suppressive histone modification H3K27me3 and then promotes the activation of the PI3K/AKT/mTOR pathway. This transcriptional activation induced by α-KG requires histone demethylase KDM6A, which is a 2-oxoglutarate oxygenase that plays an important role in converting α-KG to succinate. Furthermore, we show that GDH1-catalytic glutaminolysis also increases the expression of HK2 and promotes glycolysis in high-glucose conditions dependent on KDM6A-mediated demethylation of H3K27.

Conclusions

These findings suggest a novel function of glutaminolysis in the regulation of signal transduction and metabolism reprogramming and provide further evidence for the unique role of glutaminolysis in GBM progression.

Keywords: EGFR-AKT pathway, GBM, GDH1, GDH1, KDM6A, metabolism reprogramming

Graphical Abstract

Graphical Abstract.

Graphical Abstract

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Key Points.

  1. GDH1-mediated glutaminolysis is essential for GBM growth even under high-glucose conditions.

  2. GDH1-mediated glutaminolysis feedback activates the EGFR/AKT pathway in a KDM6A-dependent manner.

  3. GDH1-mediated glutaminolysis reprograms glycolysis in GBM.

Importance of the Study.

Our study provides a novel mechanism of crosstalk between metabolic, epigenetic transcription machinery and signal transduction in GBM, that GDH1-catalytic glutaminolysis feedback activates epidermal growth factor receptor/phosphatidylinositol 3 kinase/protein kinase B (EGFR/PI3K/AKT) pathway and reprogram GBM metabolism via KDM6A-dependent demethylation.

Glioblastoma (GBM) is the most aggressive and heterogeneous form of central nervous system tumor that has a notoriously poor prognosis.1 The median overall survival time for newly diagnosed GBM patients is about 14.6 and 6.9 months for recurrence patients, despite novel treatment options such as anti-angiogenic therapy and immunotherapy.2 All GBMs are identified as IDH-wt and adult IDH mutated HGGs are identified as astrocytoma IDH-mutant (WHO 4) according to the 2021 WHO CNS5.1 GBM cells exhibit a unique metabolic profile that differs from normal brain cells for adaption to stressful environments, such as GBM cells have high levels of glucose uptake and metabolism.3,4 Besides glucose, glutamine is another important nutriment for cancer cell growth that provides biological sources for nucleic acid and fatty acid synthesis.5,6 Glutamine metabolism is previously considered to be a supplement to glycolysis under low glucose (LG) in GBM.7 However, the role of glutaminolysis in metabolism reprogramming and GBM progression under sufficient glucose, and the underlying mechanism remain unclear.

Glutamate dehydrogenase 1 (GDH1) is a key glutamate dehydrogenase that catalyzes the deamination of glutamate into α-KG, an intermediate in the TCA cycle that provides energy sources for cancer cells.8 GDH1 is frequently upregulated in human cancers and plays important roles in cancer development, such as leukemia, lung cancer, breast cancer, and glioma.9–12 GDH1 promotes human breast and lung cancer growth by regulating redox homeostasis in a GPx1-dependent manner.13 In glioma, GDH1 maintains cell viability upon glucose deficiency by promoting glutaminolysis. Under LG, GDH1-catalytic glutaminolysis upregulates GLUT1 to promote glucose uptake and glioma cell survival by activating IKKβ and NF-κB signaling.14 Furthermore, EGFR induces the transcription of GDH1 and enhances glutamine metabolism through ELK1 activation in GBM.15 EGFR also directly phosphorylates and activates GDH1 that cooperates with RSK2 to drive CREB activation and lung cancer metastasis.16 GDH1-catalytic glutaminolysis involves dysregulated oncogenic kinase signaling, but the role of glutaminolysis in regulating signal transduction and GBM progression remains little known.

The intermediary metabolite α-KG is known to play crucial roles in various cellular pathways. For example, α-KG activates NRF2 and its target antioxidant genes to promote cellular antioxidant response and development of PA embryos.17α-KG interacts with CanKK2 to activate AMPK and promote anoikis resistance of lung cancer cells.10 α-KG inhibits Wnt-Hippo signaling to restore the gut barrier and attenuate colitis.18 In glioma, α-KG directly interacts with and activates IKKβ and NF-κB signaling to increase compensatory glucose uptake and brain tumor growth.14 Apart from its metabolic functions, α-KG is also the co-substrate for α-KG-dependent dioxygenases that include histone demethylases. The intracellular α-KG regulates different histone demethylation modifications in different cell types, which may be due to the different sensitivity of different demethylases to a-KG/Fe(II).19 Histone methylations are involved in oncogenic kinase signaling, but the mechanism of crosstalk between metabolic, epigenetic transcription machinery and signal transduction in GBM remains unknown.

In this study, we investigated the functional role and regulatory mechanisms of GDH1-catalytic glutaminolysis in GBM cell survival, reprogramming of glucose metabolism, and tumor growth. Our findings demonstrated that GDH1-mediated glutaminolysis was essential for GBM growth even upon glucose sufficiency. Furthermore, GDH1-produced α-KG increased the transcription of PDPK1 to amplify the EGFR-activated AKT/mTOR signal pathway by reducing H3K27me3. In addition, we found that KDM6A, α-KG-dependent demethylases, was required for GDH1 to activate HK2 transcription and aerobic glycolysis. In summary, our findings revealed a novel mechanism by which GDH1-mediated glutaminolysis regulates the EGFR-AKT-mTOR pathway and reprograms glycolysis, providing further insights into the unique role of glutaminolysis in the progression of GBM.

Materials and Methods

Ethics Statement

All animal experiments were approved by the Institute Animal Care and Use Committee at Liaocheng University.

Cells and Cell Culture

Human GBM cell line LN229 was obtained from the American Type Culture Collection. HEK 293 cells were obtained from the National Infrastructure of Cell Line Resource (Beijing, China). All the cell lines were authenticated by short tandem repeat analysis tested for mycoplasma. Cells were maintained in Dulbecco’s modified Eagle’s Medium–high glucose (HG) or LG (Gibco BRL) in 5% CO2 at 37 °C. Primary human GBM cells (GBM02) were obtained as previously reported.20 Primary GBM cells were cultured in NeurobasalTM Medium (Gibco) and supplemented with 2% B27 Neuro Mix (Thermo Fisher Scientific).

Transfection and generation of stable cell lines

GDH1-shRNA, KDM6A-shRNA, and shNT target sites were designed and synthesized at Beijing Genomics Institute (BGI) and cloned into the lentiviral vector pLKO.1. Flag-GDH1 wt and Flag-GDH1 R496S were purchased from YouBo Biotechnology (Changsha) and constructed into the lentiviral vector pCDH-CMV-MCS-EF1. HEK293 cells were transfected using Effectene transfection reagent (QIAGEN) according to the manufacturer’s protocol. Stable transfections were selected with puromycin for 1 week. Primer sequences are listed in Supplementary Table S1.

Cell Viability

Cell viabilities were examined by using CCK-8 (Beyotime). LN229 cells (1 × 103/well) or GBM02 cells (2 × 103/well) were seeded into 96-well plates. Added 20 μL CCK-8 into medium and cultured for 2 hours. The absorbance was measured at a wavelength of 450 nm.

Intracellular Metabolite Measurements

For intracellular metabolite measurements, 2 × 106 GBM cells were collected and homogenized in PBS. Proteins were removed by using a 10 KD Amicon Ultra Centrifugal Filter. The intracellular levels of α-KG (Abcam, ab83431) and succinate (Abcam, ab204718) were determined by using commercial kits following the manufacturer’s instructions.

Western Blot

Cells were collected and lysed by RIPA buffer (Beyotime), and western blot assays were performed as previously described.20 The antibodies were listed in Supplementary Table S2.

RNA Sequence

Total RNA was isolated from 3 independent samples of LN229-shGDH1 or shNT cells by using Trizol (Thermo, Shanghai, China). The library construction, RNA-seq, GO and PANTHER analyses, and GSEA were performed by Consure Biotechnology Co.Ltd. The RNA-seq data was uploaded to the GEO dataset, the GEO accession number is GSE268158.

Chromatin Immunoprecipitation Assay

The Chromatin Immunoprecipitation (ChIP) Assay was performed as previously described.15 The chromatin was immunoprecipitated with the specific antibodies shown in Supplementary Table S2. The ChIP PCR sequences are shown in Supplementary Table S1.

Luciferase Assay

The PDPK1 wild or mutate promoter were constructed into pGL3-Bacic. LN229 cells stably expressing shNT or shGDH1 were transfected with promoter reporters or pGL3-basic plasmids. The different cells were lysed by the Renilla-Lumi buffer after 24 hours. The dual luciferase activities were tested according to manufacturers’ instructions (Promega, E1910).

Metabolite-Level Measurements

For metabolite-level measurements by LC-MS, LN229 cells were treated with R162 for 24 hours or 72 hours. Samples (1 × 107 cells/sample) were collected and homogenized by ice-cold methnol/water. The samples were vortexed and sonicated, and then centrifugated at 12 000 rpm for 10 minutes. The supernatant was used for LC-MS/MS (ABSciex QTRAP6500+) analysis. The metabolites in energetic metabolism were measured and analyzed by Metware Biotechnology Inc.

Seahorse Assay

For seahorse assay, GBM cells were treated with R162 for 72 hours and seeded into XF-96 well plates and then cultured for 6 hours. Cells were maintained in seahorse DMEM with 2 µM glutamine in a non-CO2 incubator at 37 °C for 60 minutes. The glycolysis, glycolytic reserve, glycolytic capacity, and non-glycolytic acidification were determined by the XF-96p analyzer (Seahorse Bioscience).

Animal Study and Immunohistochemistry

Four-week-old female BALB/c nude mice were obtained from the Beijing Laboratory Animal Research Center and housed in the SPF room. For animal studies, 5 × 105 LN229 cells (shNT, shGDH1, shGDH1/rGDH1 wt, shGDH1/rGDH1 R496S, Empty vector, GDH1, and GDH1/shKDM6A) were intracranially injected into nude mice (10/group). Three animals of each group were euthanized 14 days after GBM cell injection, the other animals were monitored for survival. IHC and H&E Staining were performed to evaluate tumor formation and phenotype as previously described.20 Animal experiments were performed in compliance with the guidelines of the Institute for Laboratory Animal Research, Liaocheng University.

Statistical Analysis

The statistical analyses were performed by using GraphPad Prism version 7.0 (GraphPad Software). The significance of quantitative data was assessed by 2-tailed unpaired student’s t-tests. All the experiments were repeated at least 3 independent times. P < .05 was considered statistically significant.

Results

1. GDH1-Mediated Glutaminolysis is Essential for GBM Growth Even Under High-Glucose Condition

To evaluate the critical role of glutamine metabolism in cell growth, we knocked down GDH1 that is a key enzyme of glutaminolysis by using short hairpin RNA in GBM cells (Figure 1A). Consistent with previous studies, GDH1 depletion remarkably inhibited the viability of GBM cells under low-glucose, but not high-glucose conditions in a short time (Figure 1B). However, GDH1 silencing also greatly reduced cell proliferation in high-glucose conditions with the extension of time, especially over 72 hours (Figure 1C). We further examined whether GDH1 depletion reduced cell growth dependent on its catalytic ability of glutaminolysis. Knockdown of GDH1 significantly decreased intracellular α-KG levels in GBM cells under HG (Supplementary Figure S1A). We supplemented GDH1-silence GBM cells with methyl-α-KG, which restored α-KG levels and partially recovered the viability of GBM cells under HG (Supplementary Figure S1B and S1C). We next reconstituted wild-type GDH1 (rGDH1 wt) and GDH1 496 arginine mutant (rGDH1 R496S) that was defined as 443 arginine in the previous sequence,21 which lacks the dehydrogenase activity into GDH1 depletion cells (Figure 1D and Supplementary Figure S1D). We found that reconstituted expression of GDH1 wt but not GDH1 R496S restored intracellular α-KG levels and rescued cell viability (Figure 1E and Supplementary S1E). In intracranial tumor models, GDH1 depletion inhibited brain tumorigenesis, and this effect was rescued by rGDH1 wt but not rGDH1 R496S expression (Figure 1F). All these results demonstrated that GDH1-mediated glutaminolysis was essential for GBM growth even under high-glucose conditions, suggesting that glutaminolysis was not only a supplement to glycolysis under LG.

Figure 1.

Figure 1.

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GDH1 promotes GBM cell survival and tumor growth dependent on its dehydrogenase activity. (A-C) LN229 or GBM02 cells stably expressing shNT or shGDH1. GDH1 expression was examined (A). Cells were treated with high glucose (HG) (25 mM) or low glucose (LG) (5 mM) for the indicated time. Cell viability was determined by CCK-8 assay (B). Cells were cultured under HG conditions for the indicated time. Cell viability was determined (C). (D-F) LN229 or GBM02 cells stably expressing shNT or shGDH1 were reconstituted with Flag-rGDH1 wt or Flag-rGDH1 R496S and then cultured under HG conditions. GDH1 and Flag expression were examined (D). Intracellular a-KG levels were examined (E). Orthotopic tumor growth abilities and survival rates of mice (n = 6) were determined (F). Data represent the mean ± SD (*P < .05; **P < .01).

2. GDH1-Mediated Glutaminolysis is a Critical Amplifier of EGFR-Activated AKT/mTOR Signal Pathway

To better determine the molecular mechanism of GDH1-catalytic glutaminolysis action, we employed microarray gene expression of LN229 cells with or without GDH1 knockdown. We identified 623 GDH1-responsive genes (≥ ± 1.4 fold), with 224 responsive genes enriched in signal transduction (Figure 2A and Supplementary Figure S2A). Further pathway enrichment assay suggested a general role of GDH1 in transcriptional control of the PI3K-AKT pathway (Supplementary Figure S2B). Therefore, we detected whether GDH1 regulates PI3K-AKT signaling. The results have shown that the phosphorylation levels of AKT, mTOR, and p70S6K were remarkably reduced after GDH1 depletion (Figure 2B). Overexpression of GDH1 increased the phosphorylation levels of AKT, mTOR, and p70S6K (Figure 2C). Our previous study has shown that EGFR activation increases transcription of GDH1 and subsequently promotes glutaminolysis.15 Here, we found that GDH1 did not affect the activation of EGFR (Figure 2B and C). Previous studies have demonstrated that EGFR activates the PI3K-AKT pathway through a phosphorylation cascade reaction. We hypothesized that GDH1 might promote EGFR-mediated activation of the PI3K-AKT pathway. Indeed, in response to EGF stimulation, the levels of AKT, mTOR, and p70S6K phosphorylation, but not EGFR and MEK1 were significantly reduced in GDH1-depletion cells compared to that in control cells (Figure 2D). In addition, the expression of GDH1 was increased after EGF treatment in control cells but not in GDH1 depletion cells (Supplementary Figure S3A).

Figure 2.

Figure 2.

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GDH1 promotes EGFR-mediated PI3K/AKT/mTOR signal pathway. (A) RNA-seq were performed in LN229 cells expressing shNT and shGDH1. The statistics of genes in pathway iterm were shown. (B) LN229 or GBM02 cells stably expressing shNT or shGDH1 were cultured under HG conditions. Immunoblotting of indicated proteins were shown. (C) LN229 or GBM02 cells stably expressing GDH1 were cultured under HG conditions. Immunoblotting of indicated proteins were shown. (D) LN229 or GBM02 cells stably expressing shNT or shGDH1 were treated with EGF (50 ng/mL) for the indicated time. Immunoblotting of indicated proteins were shown. (E) LN229 or GBM02 were cultured with or without glutamine and then treated with or without EGF. Immunoblotting of indicated proteins were shown.

It has been shown that the PI3K-AKT pathway increases the expression of cell cycle genes, such as CCNB1, CCND1, and CCNE.22 In line with this, GSEA of our microarray data showed that GDH1 silence led to lower expression of a large amount of cell cycle genes including those critical for G1/S progression (Supplementary Figure S2C). These results were confirmed by qRT-PCR and WB assay, which revealed that GDH1 depletion significantly reduced the expression of CCND1 and CCNE1 (Supplementary Figure S2D and S2E). Furthermore, we detected whether glutamine was essential for the activation of the EGFR-AKT-mTOR pathway. Indeed, in response to EGF stimulation, the levels of AKT, mTOR, and p70S6K phosphorylation were remarkably increased in high glutamine conditions (Figure 2E). All these results suggested that GDH1-mediated glutaminolysis might be a critical amplifier of EGFR activated AKT/mTOR signal pathway.

3. GDH1-Produced α-KG Activates AKT/mTOR Pathway Through Regulating Histone Demethylation

To determine the critical role of GDH1-catalytic glutaminolysis in the AKT/mTOR signal pathway, we constructed GDH1 silence GBM cells with reconstituted expression of GDH1 wt or GDH1 R496S (Figure 1D). In response to EGF stimulation, the reduced phosphorylated levels of AKT, mTOR, and p70S6K induced by GDH1 depletion were remarkably recovered by GDH1 restoration but not GDH1 R496S (Figure 3A). Previous studies have demonstrated that α-KG, the metabolite of glutamine, could regulate histone demethylation in mammals. Here, we examined whether GDH1-catalytic glutaminolysis promotes the EGFR-mediated PI3K-AKT pathway by αKG-dependent demethylases in GBM. GBM cells cultured in a glutamine-free medium presented increases in tri-methylation and decreases in mono-methylation on H3K4 and H3K27, while H3K36 and H3K79 methylations remained unchanged, and H3K9 methylations only changed in GBM02 cells but not LN229 cells (Figure 3B).

Figure 3.

Figure 3.

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GDH1 promotes EGFR-mediated PI3K/AKT/mTOR pathway by regulating histone demethylation. (A) LN229 or GBM02 cells stably expressing shNT or shGDH1 were reconstituted with Flag-rGDH1 wt or Flag-rGDH1 R496S and then treated with EGF. Immunoblotting of indicated proteins were shown. (B) LN229 or GBM02 cells were cultured with or without glutamine for 2 days. Immunoblotting of indicated proteins were shown. (C) Simplified schematic of the reaction mechanism of α-KG-dependent dioxygenases. (D and E) LN229 or GBM02 cells stably expressing shNT or shGDH1 were reconstituted with Flag-rGDH1 wt or Flag-rGDH1 R496S. Analysis of the α-KG/succinate ratio was shown (D). Data represent the mean ± SD (**P < .01). Immunoblotting of indicated proteins were shown (E). (F) LN229 or GBM02 cells stably expressing shNT or shGDH1 were cultured with supplementation as indicated (dimethyl-α-ketoglutarate, 0.5 mM) and then treated with EGF (50 ng/mL). Immunoblotting of indicated proteins were shown.

The 2-oxoglutarate oxygenase couples with α-KG to give succinate and carbon dioxide, which is accompanied by histone demethylations (Figure 3C). Indeed, GDH1 depletion significantly reduced the α-KG/succinate ratio in GBM cells suggesting the decreased conversion of α-KG to succinate, which were recovered by GDH1 restoration but not GDH1 R496S (Figure 3D). In line with this result, increased tri-methylation and decreased mono-methylation of H3K4 and H3K27, but not H3K9, H3K36, and H3K79 methylation, was seen in GDH1 silenced cells; effects rescued by GDH1 restoration, but not GDH1 R496S (Figure 3E and Supplementary Figure S3B). Moreover, DM-α-KG reversed the decreases in phosphorylated levels of AKT, mTOR, and p70S6K observed in GDH1 depletion cells that responded to EGF stimulation (Figure 3F), confirming that these effects induced by attenuation in glutamine-dependent α-KG.

4. GDH1 Activates the Transcription of PDPK1 to Amplify EGFR-Mediated PI3K-AKT Pathway by Modifying H3K27me3

To further clarify the underlying molecular mechanism of GDH1-mediated activation of the EGFR-AKT pathway, we detected some key regulators of the PI3K-AKT pathway. The results have shown that the expression of PDPK1 was significantly reduced in GDH1 silencing cells and increased in GDH1 overexpression cells, whereas the expressions of PI3Kp110α, PI3Kp850α, and PTEN were not changed (Figure 4A and B and Supplementary Figure S4A-E). Next, we examined whether GDH1 promoted PDPK1 expression by activating its transcription. We generated a series of luciferase reporter constructs containing PDPK1 promoter, then investigated the effect of GDH1 silencing on the luciferase activity of these constructs. GDH1 depletion significantly reduced the luciferase activity of PDPK1 promoter/reporter constructs that contained the region from positions –476 to 0 but not -934 to –513 (Figure 4C).

Figure 4.

Figure 4.

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GDH1 activates the transcription of PDPK1 and PI3K/AKT/mTOR pathway by modifying H3K27me3. (A) Immunoblotting of indicated proteins in LN229 or GBM02 cells stably expressing shNT or shGDH1. (B) Immunoblotting of indicated proteins in LN229 or GBM02 cells stably expressing empty vector or GDH1. (C) LN229 cells stably expressing shNT or shGDH1 were transfected with PDPK1-Luc. The relative luciferase activities were normalized to those of the cells expressing shNT and to Renilla controls. (D) ChIP qRT-PCR assays were performed with H3K27me3 antibody and amplified with primers targeting sites in LN229 or GBM02 cells stably expressing shNT or shGDH1. The relative ChIP DNA levels were normalized to input DNA. (E) LN229 or GBM02 cells stably expressing empty vector or GDH1 were cultured with supplementation as indicated (PDPK1 inhibitor NSC156529) and then treated with EGF. Immunoblotting of indicated proteins were shown. (F and G) LN229 cells stably expressing e GDH1 were treated with or without NSC156529. Cell growth curves were examined (F). Intracellular a-KG levels were determined (G). (C, D, and F) Data represent the mean ± SD (**P < .01).

Furthermore, the knockdown of GDH1 significantly increased the suppressive histone modification H3K27me3 that enriched in PDPK1 promoter (Figure 4D). Overexpression of GDH1 significantly amplified EGFR-mediated phosphorylation of AKT, mTOR, and p70S6K, which were abolished by the inhibitor of PDPK1 (Figure 4E). In addition, inhibition of PDPK1 attenuated the increased cell proliferation induced by GDH1 overexpression, whereas did not affect the product of α-KG (Figure 4F and G). All these results demonstrated that GDH1 activated the transcription of PDPK1 by reducing H3K27me3 enrichment at the PDPK1 promoter, and then amplified the EGFR-mediated PI3K-AKT pathway.

5. KDM6A is Required for GDH1 to Amplify EGFR-Mediated PI3K-AKT Pathway Activation and Brain Tumor Growth

KDM6A was reported as a α-KG-dependent demethylase that catalyzes the demethylation of H3K27me3.23 Treated GBM cells with GSK-J4, an inhibitor of H3K27me3-specific JmjC-family histone demethylases (Figure 5A), induced a dose-dependent increase in H3K27me3 concomitant with decrease in H3K27me1 (Figure 5B), which was consistent with effect when GBM cells were cultured in the absence or presence of glutamine (Figure 3B). To determine whether KDM6A was a key factor for GDH1 amplified EGFR-mediated AKT pathway activation, we generated stable KDM6A-knockdown cells, in which the total levels of H3K27me3 were significantly increased (Supplementary Figure S5A). Furthermore, the knockdown of KDM6A significantly decreased KDM6A levels at PDPK1 promoter (Supplementary Figure S5B). In KDM6A silencing cells, overexpression of GDH1 failed to increase the expression of PDPK1 and phosphorylated levels of AKT, mTOR, and p70S6K (Figure 5C). In line with these results, the knockdown of KDM6A markedly increased H3K27me3 levels at PDPK1 promoter, and overexpression of GDH1 failed to rescue the effect (Figure 5D).

Figure 5.

Figure 5.

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KDM6A is required for GDH1 to amplify EGFR-mediated PI3K-AKT pathway activation. (A) Simplified schematic of the reaction mechanism of α-KG-dependent dioxygenases that catalyze the demethylation of H3K27me3. (B) LN229 or GBM02 cells were treated with GSK-J4 for the indicated concentration. H3K27me3 and H3K27me1 levels were examined. (C) LN229 or GBM02 cells stably expressing shNT or shKDM6A were transfected with GDH1. Immunoblotting of indicated proteins were shown. (D) ChIP qRT-PCR assays were performed with H3K27me3 antibody and amplified with PDPK1 promoter in LN229 or GBM02 cells stably expressing shNT or shGDH1 were transfected with or without GDH1. The relative ChIP DNA levels were normalized to input DNA. (E) LN229 or GBM02 cells stably expressing shNT or shKDM6A were supplemented with or without glutamine for 24 hours and then treated with EGF for 30 minutes. Immunoblotting of indicated proteins were shown. (F and G) LN229 cells stably expressing empty vector or GDH1 were treated with or without GSK-J4. Intracellular a-KG levels were determined (F). Cell growth curves were examined (G). (D, F, and G) Data represent the mean ± SD (**P < .01).

Furthermore, we detected whether KDM6A was essential for the amplification of EGFR/AKT/mTOR pathway glutamine induced by glutamine. Indeed, the expression of PDPK1 was inhibited in KDM6A silencing cells. Besides this, in response to EGF stimulation, the levels of AKT, mTOR, and p70S6K phosphorylation were not changed under high glutamine conditions (Figure 5E). Then we inhibited KDM6A in stable GDH1 overexpression cells by using KDM6A shRNA or inhibitor GSK-J4, the results showed that inhibition of KDM6A did not affect GDH1-catalytic glutaminolysis (Figure 5F and Supplementary Figure S5C). However, inhibition of KDM6A could partly suppress cell proliferation induced by GDH1 overexpression (Figure 5G and Supplementary Figure S5D). In intracranial tumor models, overexpression of GDH1 significantly promoted the growth of brain tumors and decreased the survival time of mice, and these effects were reversed by KDM6A knockdown (Supplementary Figure S6A). In addition, overexpression of GDH1 increased p-AKT, p-mTOR, and p-p70S6K in the tumors, which were reversed by KDM6A knockdown (Supplementary Figure S6B). All these results demonstrated that KDM6A was required for GDH1 to amplify EGFR-mediated PI3K-AKT pathway activation.

6. GDH1-Produced α-KG Remodel Glycolysis Through Regulating HK2 in a KDM6A-Dependent Manner

Previous studies have shown that GDH1 can promote glucose uptake and tumor cell survival under LG conditions.14 However, the potential role of GDH1 in glucose metabolism reprogramming under HG and the underlying mechanism remains unclear. Treated GBM cells with R162 for 24 hours, a specific inhibitor of GDH1, induced profound alterations in the glutamine metabolism and TCA cycle, but not glycolysis under HG conditions. With the extension of time, inhibition of GDH1 also significantly suppressed glycolysis, including a dramatic decrease in almost all glycolysis intermediates after 72 hours (Figure 6A). Moreover, treatment with GDH1 inhibitor R162 for 72 hours markedly reduced the abilities of glycolysis in GBM cells (Figure 6B and C). Molecularly, treatment with R162 induced a time-dependent increase in H3K27me3 concomitant with the decrease in HK2 (Figure 6D).

Figure 6.

Figure 6.

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GDH1-catalytic glutaminolysis remodels glycolysis through regulating HK2 in a KDM6A-dependent manner. (A) Mass spectrometry analyses of metabolites in energetic metabolism in LN229 cells treated with DMSO or GDH1 inhibitor R162 (20 μM) for the indicated time. A heat map representing significantly different metabolites was shown. (B and C) LN229 or GBM02 cells were treated with DMSO or R162 for 72 hours. The glycolytic rate of the indicated GBM cells was examined by ECAR assay. (D) LN229 or GBM02 cells were treated with R162 for the indicated time. Expression of H3K27me3 and HK2 were determined. (E and F) LN229 or GBM02 cells stably expressing shNT or shGDH1 were reconstituted with Flag-rGDH1 wt or Flag-rGDH1 R496S. HK2 expression was examined (E). ChIP qRT-PCR assays were performed with H3K27me3 antibody and amplified with primers targeting site (F). (G and H) LN229 or GBM02 cells stably expressing shNT or shKDM6A were transfected with GDH1. HK2 expression was examined (E). ChIP qRT-PCR assays were performed with H3K27me3 antibody and amplified with primers targeting site (F). (B, C, F, and G) Data represent the mean ± SD (**P < .01).

To further determine whether GDH1 remodeled aerobic glycolysis through its catalytic glutaminolysis, we next detected the expression of HK2 in GDH1 silencing cells with reconstituted rGDH1 wt or rGDH1 R496S. The results revealed that GDH1 downregulation reduced the levels of HK2, which were rescued by reconstituted expression of GDH1 wt but bot GDH1 R496S (Figure 6E). Knockdown of GDH1 significantly increased the suppressive histone modification H3K27me3 enriched in HK2 promoter, and the effect was abolished by reconstituted expression of GDH1 wt but bot GDH1 R496S (Figure 6F). Moreover, in KDM6A silencing cells, overexpression of GDH1 failed to increase the expression of HK2 (Figure 6G). KDM6A downregulation markedly increased H3K27me3 levels at HK2 promoter, and overexpression of GDH1 failed to rescue the effect (Figure 6H). All these results suggested that GDH1-catalytic glutaminolysis remodels glycolysis by regulating HK2 in a KDM6A-dependent manner.

Discussion

Glutamine is a major material source of energy and biomass synthesis, which is necessary to maintain rapid tumor growth, metastasis, and redox homeostasis.24,25 The conversion of glutamate to α-KG is catalyzed by glutamate dehydrogenases (GDH1 and GDH2), which are frequently upregulated in multiple cancers.26 GDH1-catalytic glutaminolysis is involved in the regulation of epithelial-mesenchymal transition, redox homeostasis, and anoikis resistance, which are critical for cancer development.13,27 However, previous studies of the functional role of GDH1 in glioma were focused on glucose deficiency conditions. For example, GDH1 is activated upon glucose deficiency and maintains glioma cell survival by upregulating GLUT1 through activating IKKβ and NF-κB signaling.14 Here, we verified that GDH1 depletion did not affect the viability of GBM cells under high-glucose conditions in a short time, that was consistent with previous studies. However, GDH1 silencing also greatly reduced cell proliferation under high glucose for a long time. Moreover, we demonstrated that GDH1 promoted cell proliferation and brain tumor growth dependent on its dehydrogenase activity, suggesting that GDH1-catalytic glutaminolysis was not only a supplement to glycolysis and essential for GBM growth.

Glutamine-derived metabolites are also involved in promoting cellular cascade reactions in cancer, such as hypoxia response, immune response, and chromatin reorganization,28,29 but the role of Glutamine intermediates in signal transduction remains unclear. In this study, RNA-seq results revealed that GDH1-responsive genes were enriched in signal transduction, especially in transcriptional control of the PI3K/AKT pathway. Our previous study has demonstrated that GDH1 is a downstream target gene of EGFR/ELK1 pathway.15 Meanwhile, the AKT/mTOR/p70S6K pathway is an important downstream signaling of EGFR, that is critical for cancer development.30 Here, we found that GDH1 depletion were remarkably inhibited EGFR-activated AKT/mTOR/p70S6K pathway, that was recovered by GDH1 restoration but not GDH1 R496S that lacks dehydrogenase activity. Furthermore, GBM cells cultured in glutamine-free medium presented reductions in EGFR-induced AKT/mTOR/p70S6K activation, compared with those cultured under high glutamine. Our results reveal a novel mechanism that GDH1-mediated glutaminolysis is a critical amplifier of EGFR-activated AKT/mTOR signal pathway.

As an intermediate of glutamine metabolism, α-KG enters into the TCA cycle and provides energy sources for cancer cells.31 Besides a metabolic intermediate, α-KG is also the co-substrate for α-KG-dependent histone demethylases.19 The intracellular α-KG regulates different histone demethylation modifications in different cell types. α-KG decreases H3K9me3 and H3K27me3 in embryonic stem cells to maintain pluripotency.23 Reduced α-KG alters H3K27me3 levels and enriches H3K27me3 at Col3A1 and PLK1 promoter region to support pro-fibrotic fibroblast phenotype in lung fibrosis.32 In this study, we found that reduced GDH1-catalytic glutaminolysis increased H3K4me3 and H3K27me3 levels and specially enriched H3K27me3 association with PDPK1 promoter region. These results suggested that GDH1 activates the transcription of PDPK1 to amplify the EGFR-mediated PI3K-AKT pathway by modifying H3K27me3. Previous studies have demonstrated that EGFR stimulates Ras proteins through dimerization, leading to a phosphorylation cascade that activates the PI3K/AKT pathway.33 Our findings provide a supplementary mechanism of EGFR activates PI3K/AKT pathway, that EGFR activates PI3K/AKT/mTOR pathway through glutaminolysis-mediated epigenetic transcriptional control of PDPK1. As IDH1R132 mutation disrupts isocitric acid production in mitochondria toward α-KG and H3K27M mutation leads to a global reduction of H3K27me3, glutaminolysis-mediated H3K27me3 changes may be a characteristic feature of GBM.

KDM6A, an iron and α-KG dependent H3K27me3 demethylase, is upregulated and correlated with poor prognosis in GBM.20 As GDH1-catalytic glutaminolysis regulated the transcription of PDPK1 dependent on α-KG-mediated demethylation of H3K27me3, we hypothesized that KDM6A might be a critical factor of GDH1-catalytic glutaminolysis in regulation of EGFR-activated PI3K/AKT/mTOR pathway. Indeed, we found that KDM6A was required for GDH1-catalytic glutaminolysis to amplify EGFR-mediated PI3K-AKT pathway activation. There are close links between different metabolic pathways, for example, glutamine metabolism promotes glucose uptake upon glucose deficiency in GBM.34 Of note, our results revealed that prolonged inhibition of GDH1-catalytic glutaminolysis significantly reduced the glycolysis abilities of GBM cells cultured in HG by decreasing HK2 expression. In addition, KDM6A-modified histone demethylation is involved in hypoxic reprogramming in cancer metabolism.35 KDM6A promotes aerobic glycolysis by removing suppressive H3K27me3 of HK2 and PKM2 in GBM cells.20 In this study, we found that GDH1-produced α-KG remodels glycolysis by regulating HK2 in a KDM6A-dependent manner. Moreover, activation of the PI3K/AKT pathway is identified as an initiator of glycolysis in GBM cells.36 Therefore, besides directly activates the transcription of HK2, GDH1-catalytic glutaminolysis may promote glycolysis via EGFR-activated PI3K/AKT/mTOR pathway. Benefit from “Warburg effect” that tumor cells prefer aerobic glycolysis and cannot metabolize ketones, the ketogenic diet has gained popularity.37 As glutamine metabolism is a supplement to glycolysis in GBM, a ketogenic diet in combination with GDH1 inhibitors and PI3K inhibitors appears to have great potential in the treatment of GBM.

In summary, our study demonstrates that GDH1-catalytic glutaminolysis feedback activates EGFR/PI3K/AKT pathway and reprograms glioblastoma metabolism via KDM6A-dependent demethylation, provides a novel mechanism of crosstalk between metabolic, epigenetic transcription machinery and signal transduction in GBM progression.

Supplementary material

Supplementary material is available online at Neuro-Oncology (https://academic.oup.com/neuro-oncology).

noae222_suppl_Supplementary_Materials
noae222_suppl_supplementary_materials.zip (2.7MB, zip)

Contributor Information

Rui Yang, Biomedical Laboratory, School of Medicine, Liaocheng University, Liaocheng, China.

Guanghui Zhang, Medical College, Henan University of Chinese Medicine, Zhengzhou, China.

Zhen Meng, Biomedical Laboratory, School of Medicine, Liaocheng University, Liaocheng, China.

Li Wang, Biomedical Laboratory, School of Medicine, Liaocheng University, Liaocheng, China.

Yanping Li, Precision Medicine Laboratory for Chronic Non-communicable Diseases of Shandong Province, Institute of Precision Medicine, Jining Medical University, Jining, China.

Haibin Li, Precision Medicine Laboratory for Chronic Non-communicable Diseases of Shandong Province, Institute of Precision Medicine, Jining Medical University, Jining, China.

Siyuan Yan, Precision Medicine Laboratory for Chronic Non-communicable Diseases of Shandong Province, Institute of Precision Medicine, Jining Medical University, Jining, China.

Xiaonan Wei, Precision Medicine Laboratory for Chronic Non-communicable Diseases of Shandong Province, Institute of Precision Medicine, Jining Medical University, Jining, China.

Shanshan Wang, Precision Medicine Laboratory for Chronic Non-communicable Diseases of Shandong Province, Institute of Precision Medicine, Jining Medical University, Jining, China.

Hongjuan Cui, Jinfeng Laboratory, Chongqing, China; Medical Research Institute, State Key Laboratory of Resources Insects, Southwest University, Chongqing, China.

Funding

This work was supported by National Nature Science Foundation of China (No. 82002639, 82472859 to R.Y.), Shandong Provincial Natural Science Foundation, China (No. ZR2020QH235, ZR2024MH151 to R.Y.) and Project of Shandong Province Higher Educational Youth Innovation Science and Technology Program (No. 2023KJ263 to S.Y.).

Conflict of interest statement

The authors have declared no conflict of interest.

Authorship statement

R.Y. contributed to the design of the work, performance of experiments, interpretation of data, and writing the paper. H.C. contributed to design of the work and editing the paper. G.Z., Z.M., Y.L., H.L., S.Y. contributed to performance of experiments. L.W., X.W. and S.W. contributed to interpretation of data.

Data availability

The data generated in this study are available within the article and its supplementary data files.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

noae222_suppl_Supplementary_Materials
noae222_suppl_supplementary_materials.zip (2.7MB, zip)

Data Availability Statement

The data generated in this study are available within the article and its supplementary data files.

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