Extracellular glutamate and IDH1R132H inhibitor promote glioma growth by boosting redox potential

Abstract

Purpose: Somatic mutations of the isocitrate dehydrogenase 1 (IDH1) gene, mostly substituting Arg132 with histidine, are associated with better patient survival, but glioma recurrence and progression are nearly inevitable, resulting in disproportionate morbidity and mortality. Our previous studies demonstrated that in contrast to hemizygous IDH1^R132H (loss of wild-type allele), heterozygous IDH1^R132H is intrinsically glioma suppressive but its suppression of three-dimensional (3D) growth is negated by extracellular glutamate and reducing equivalent. This study sought to understand the importance of 3D culture in IDH1^R132H biology and the underlying mechanism of the glutamate effect.

Methods: RNA sequencing data of IDH1^R132H-heterozygous and IDH1^R132H-hemizygous glioma cells cultured under two-dimensional (2D) and 3D conditions were subjected to unsupervised hierarchal clustering and gene set enrichment analysis. IDH1^R132H-heterozygous and IDH1^R132H-hemizygous tumor growth were compared in subcutaneous and intracranial transplantations. Short-hairpin RNA against glutamate dehydrogenase 2 gene (GLUD2) expression was employed to determine the effects of glutamate and the mutant IDH1 inhibitor AGI-5198 on redox potential in IDH1^R132H-heterozygous cells.

Results: In contrast to IDH1^R132H-heterozygous cells, 3D-cultured but not 2D-cultured IDH1^R132H-hemizygous cells were clustered with more malignant gliomas, possessed the glioblastoma mesenchymal signature, and exhibited aggressive tumor growth. Although both extracellular glutamate and AGI-5198 stimulated redox potential for 3D growth of IDH1^R132H-heterozygous cells, GLUD2 expression was required for glutamate, but not AGI-5198, stimulation.

Conclusion: 3D culture is more relevant to IDH1^R132H glioma biology. The importance of redox homeostasis in IDH1^R132H glioma suggests that metabolic pathway(s) can be explored for therapeutic targeting, whereas IDH1^R132H inhibitors may have counterproductive consequences in patient treatment.

Keywords: 3D culture; AGI-5198; Glioma; IDH1 mutation; Redox.

Fig. 1

A distinct gene expression profile from 3D-cultured IDH1^R132H-heterozygous cells. a Unsupervised hierarchical clustering of RNA-sequencing data from 2D- and 3D-cultured IDH1^R132H-heterozygous (het) and IDH1^R132H-hemizygous (hem) BT142 cells revealing a unique cluster of 3D-cultured IDH1^R132H-heterozygous cells distinct from the rest. Each column represents a single sample. Top-20 upregulated (red) and downregulated (blue) genes are indicated. b Differential enrichment of KEGG gene sets between 3D and 2D cultures. The top-10 gene sets based on the most significant false-discovery rates (FDR) and nominal p-values were plotted with the normalized enrichment scores (NES) and the log10-transformed nominal p-values and FDR. Orange dashed lines indicate the cutoff for FDR and nominal p-values at 0.05, and asterisks indicate the gene sets shared between 3D and 2D cultures (see Supplementary Figure 1). c, d Oxidative phosphorylation gene set enrichment plot (c) and hierarchical clustering (d) of 3D-cultured IDH1^R132H glioma cells

Fig. 2

3D culture distinguishes IDH1^R132H-heterozygous cells from IDH1^R132H-hemizygous cells. Unsupervised hierarchical clustering was performed by incorporating the above RNA-sequencing result with the TCGA-LGG data set consisting of IDH-mutant and 1p/19q codeleted, IDH-mutant (without codeletion), and IDH-wildtype gliomas. Enlarged views underscore that 3D culture distinguishes IDH1^R132H-heterozygous (het) cells and IDH1^R132H-hemizygous (hem) in the clustering.

Fig. 3

Loss of IDH1^R132H heterozygosity drives glioma progression. a Enrichment of glioblastoma mesenchymal gene set in 3D-, but not 2D-, cultured IDH1^R132H-hemizygous BT142 cells. b Hierarchical clustering of 3D-cultured cells using the Verhaak_GBM_Mesenchymal gene set. c Histological examination of intracranial tumor growth derived from IDH1^R132H-hemizygous and IDH1^R132H-heterozygous BT142. Micrographs of hematoxylin‑eosin (H‑E) and immunohistochemical staining are presented with 20× objectives and specified antibodies. Scale bar: 200 μm

Fig. 4

Glutamate depends on GLUD2 to boost redox homeostasis of IDH1^R132H-heterozygous cells for 3D growth. Doxycycline (+Dox) induction of GLUD2 shRNA markedly reduced GLUD2 expression at mRNA (a) and protein (b) levels in IDH1^R132H-heterozygous BT142 cells. c GLUD2 shRNA (Dox) obliterated glutamate (+Glu) but not N-acetyl cysteine (+NAC) stimulation of 3D growth. Results are plotted in log2 ratios of treated over untreated (+/−). Similarly, GLUD2 shRNA blocked glutamate effects to increase GSH/GSSG (d) and NADPH/NADP⁺ (e) ratios (n=6) in reference to untreated cells (Ctr). One-way ANOVA was used in reference to the untreated

Fig. 5

AGI-5198 promotes 3D growth of IDH1^R132H-heterozygous cells by boosting redox potential independent of the glutamate/GLUD2 pathway. a AGI-5198 treatment increased 3D growth of IDH1^R132H-heterozygous BT142 irrespective of doxycycline induction. RLU, relative luciferase units. b A diagram depicts how extracellular glutamate (Glu) and AGI-5198 independently negate IDH1^R132H tumor-suppressive activity by enhancing redox homeostasis in glioma. AGI-5198 induced intracerebral tumor growth, as indicated by bioluminescent imaging of live animals (c) and autopsied brains (d), of IDH1^R132H-heterozygous BT142 in the presence of doxycycline induction. The starting time of both treatments is indicated by arrows. AGI-5198 treatment resulted in increased GSH/GSSG (e) and NADPH/NADP⁺ (f) ratios independent of doxycycline induction