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 IDH1R132H (loss of wild-type allele), heterozygous IDH1R132H 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 IDH1R132H biology and the underlying mechanism of the glutamate effect. METHODS RNA sequencing data of IDH1R132H-heterozygous and IDH1R132H-hemizygous glioma cells cultured under two-dimensional (2D) and 3D conditions were subjected to unsupervised hierarchal clustering and gene set enrichment analysis. IDH1R132H-heterozygous and IDH1R132H-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 IDH1R132H-heterozygous cells. RESULTS In contrast to IDH1R132H-heterozygous cells, 3D-cultured but not 2D-cultured IDH1R132H-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 IDH1R132H-heterozygous cells, GLUD2 expression was required for glutamate, but not AGI-5198, stimulation. CONCLUSION 3D culture is more relevant to IDH1R132H glioma biology. The importance of redox homeostasis in IDH1R132H glioma suggests that metabolic pathway(s) can be explored for therapeutic targeting, whereas IDH1R132H inhibitors may have counterproductive consequences in patient treatment.

中文翻译：

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细胞外谷氨酸和IDH1R132H抑制剂通过增强氧化还原电位来促进神经胶质瘤的生长。

目的异体柠檬酸脱氢酶1（IDH1）基因的体细胞突变（主要用组氨酸替代Arg132）与患者更好的生存率相关，但神经胶质瘤的复发和进展几乎不可避免，从而导致发病率和死亡率成比例的增加。我们以前的研究表明，与半合IDH1R132H（野生型等位基因缺失）相反，杂合IDH1R132H本质上具有抑制神经胶质瘤的作用，但其对三维（3D）生长的抑制作用却被细胞外谷氨酸所抵消，并降低了当量。这项研究试图了解3D培养在IDH1R132H生物学中的重要性以及谷氨酸作用的潜在机制。方法对在二维（2D）和3D条件下培养的IDH1R132H-杂合和IDH1R132H-半合胶质瘤细胞的RNA测序数据进行无监督分层聚类和基因集富集分析。在皮下和颅内移植中比较了IDH1R132H-杂合和IDH1R132H-杂合的肿瘤生长。针对谷氨酸脱氢酶2基因（GLUD2）表达的短发夹RNA被用来确定谷氨酸和突变IDH1抑制剂AGI-5198对IDH1R132H-杂合细胞中氧化还原电位的影响。结果与IDH1R132H-杂合细胞相比，3D培养而非2D培养的IDH1R132H-杂合细胞聚集了更多的恶性神经胶质瘤，具有胶质母细胞瘤间质特征，并表现出侵袭性的肿瘤生长。尽管细胞外谷氨酸和AGI-5198均可刺激IDH1R132H杂合细胞3D生长的氧化还原电位，但谷氨酸刺激需要GLUD2表达，而AGI-5198则不需要。结论3D培养与IDH1R132H胶质瘤生物学更相关。氧化还原稳态在IDH1R132H胶质瘤中的重要性表明，可以探索代谢途径进行靶向治疗，而IDH1R132H抑制剂在患者治疗中可能产生适得其反的后果。