Reprogramming of metabolic pathways is crucial to satisfy the bioenergetic and biosynthetic demands and maintain the redox status of rapidly proliferating cancer cells. In tumors, the tricarboxylic acid (TCA) cycle generates biosynthetic intermediates and must be replenished (anaplerosis), mainly from pyruvate and glutamine. We recently described a novel enolase inhibitor, HEX, and its pro-drug POMHEX. Since glycolysis inhibition would deprive the cell of a key source of pyruvate, we hypothesized that enolase inhibitors might inhibit anaplerosis and synergize with other inhibitors of anaplerosis, such as the glutaminase inhibitor, CB-839. We analyzed polar metabolites in sensitive (ENO1-deleted) and resistant (ENO1-WT) glioma cells treated with enolase and glutaminase inhibitors. We investigated whether sensitivity to enolase inhibitors could be attenuated by exogenous anaplerotic metabolites. We also determined the synergy between enolase inhibitors and the glutaminase inhibitor CB-839 in glioma cells in vitro and in vivo in both intracranial and subcutaneous tumor models. Metabolomic profiling of ENO1-deleted glioma cells treated with the enolase inhibitor revealed a profound decrease in the TCA cycle metabolites with the toxicity reversible upon exogenous supplementation of supraphysiological levels of anaplerotic substrates, including pyruvate. ENO1-deleted cells also exhibited selective sensitivity to the glutaminase inhibitor CB-839, in a manner rescuable by supplementation of anaplerotic substrates or plasma-like media PlasmaxTM. In vitro, the interaction of these two drugs yielded a strong synergistic interaction but the antineoplastic effects of CB-839 as a single agent in ENO1-deleted xenograft tumors in vivo were modest in both intracranial orthotopic tumors, where the limited efficacy could be attributed to the blood-brain barrier (BBB), and subcutaneous xenografts, where BBB penetration is not an issue. This contrasts with the enolase inhibitor HEX, which, despite its negative charge, achieved antineoplastic effects in both intracranial and subcutaneous tumors. Together, these data suggest that at least for ENO1-deleted gliomas, tumors in vivo—unlike cells in culture—show limited dependence on glutaminolysis and instead primarily depend on glycolysis for anaplerosis. Our findings reinforce the previously reported metabolic idiosyncrasies of in vitro culture and suggest that cell culture media nutrient composition more faithful to the in vivo environment will more accurately predict in vivo efficacy of metabolism targeting drugs.

中文翻译：

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回补功能受损是神经胶质瘤糖酵解抑制剂毒性的主要原因

代谢途径的重新编程对于满足生物能量和生物合成需求以及维持快速增殖的癌细胞的氧化还原状态至关重要。在肿瘤中，三羧酸 (TCA) 循环产生生物合成中间体，必须补充（厌食），主要来自丙酮酸和谷氨酰胺。我们最近描述了一种新型烯醇化酶抑制剂 HEX 及其前药 POMHEX。由于糖酵解抑制会剥夺细胞的关键丙酮酸来源，我们假设烯醇化酶抑制剂可能会抑制回补并与其他回补抑制剂（如谷氨酰胺酶抑制剂 CB-839）协同作用。我们分析了用烯醇化酶和谷氨酰胺酶抑制剂处理的敏感（ENO1 缺失）和耐药（ENO1-WT）神经胶质瘤细胞中的极性代谢物。我们研究了是否可以通过外源性回补代谢物减弱对烯醇化酶抑制剂的敏感性。我们还在体外和体内颅内和皮下肿瘤模型中确定了烯醇化酶抑制剂和谷氨酰胺酶抑制剂 CB-839 在神经胶质瘤细胞中的协同作用。用烯醇酶抑制剂处理的 ENO1 缺失神经胶质瘤细胞的代谢组学分析显示，TCA 循环代谢物显着减少，在外源补充超生理水平的回补底物（包括丙酮酸）后毒性可逆。ENO1 缺失的细胞还表现出对谷氨酰胺酶抑制剂 CB-839 的选择性敏感性，其方式可通过补充回补底物或血浆样培养基 PlasmaxTM 来挽救。体外，这两种药物的相互作用产生了很强的协同作用，但 CB-839 作为单一药物在体内 ENO1 缺失的异种移植肿瘤中的抗肿瘤作用在两种颅内原位肿瘤中都是适度的，其中有限的疗效可归因于血液-脑屏障 (BBB) 和皮下异种移植物，其中 BBB 渗透不是问题。这与烯醇化酶抑制剂 HEX 形成对比，后者尽管带有负电荷，但在颅内和皮下肿瘤中均具有抗肿瘤作用。总之，这些数据表明，至少对于 ENO1 缺失的神经胶质瘤，体内肿瘤——与培养的细胞不同——显示出对谷氨酰胺分解的有限依赖性，而是主要依赖于糖酵解来回补。