探討磷酸甘油酸脫氫酶為藥物標靶於神經母細胞瘤之治療與機制研究

Abstract

神經母細胞瘤是原發自交感神經系統或腎上腺且好發於兒童時期的常見的惡性腫瘤之一。在本研究中，我們透過分析了神經母細胞瘤患者的基因表現得知磷酸甘油酸脫氫酶（Phosphoglycerate dehydrogenase, PHGDH）與高危險神經母細胞瘤中惡性指標基因MYCN具有高度相關性且發揮關鍵作用。PHGDH是絲胺酸生物合成中關鍵酵素，在合成下游甘胺酸合成代謝中是不可或缺的角色。近年來的研究顯示，癌症中重組代謝體是一種有效且新穎之治療策略。因此，我們好奇已用來治療白血病以及在不同癌症研究指出擁有抗腫瘤能力之化療藥物高三尖杉酯碱 (Homoharringtonine, HHT)是否能有效治療高危險神經母細胞瘤。首先，我們利用分子對接模型(Docking)預測出HHT的連結位以及透過等溫滴定微量熱法 (Isothermal Titration Calorimetry, ITC) 發現PHGDH能夠和HHT結合且比本身受質NAD+有較強的結合力。也透過競爭實驗發現HHT可能會和NAD+競爭同一結合位。進一步利用X-ray結晶學試圖解析以及分析PHGDH和 HHT結合位。此外，我們也透過不同種細胞實驗來研究此抑制劑的作用和分子機制。在細胞實驗中發現，HHT使細胞以及粒線體的氧化壓力產物 (Reactive oxidative species，ROS) 上升而促使細胞凋亡。透過代謝體實驗結果顯示， HHT也能使得細胞中三羧酸循環 (Tricarboxylic acid cycle, TCA cycle) 表現量上升以及絲胺酸/甘胺酸合成路徑表現量下降。綜合以上實驗結果顯示， HHT會透過調節細胞代謝體含量以及氧化壓力產物來達到治療神經母細胞瘤的效果。最後，透過動物實驗得知，此抑制劑之藥效作用可成功的抑制腫瘤生長的能力，並且能夠顯著提升存活能力。因此，我們的研究顯示HHT可能為潛在治療神經母細胞瘤病患的治療藥物。

Neuroblastoma is the most common extracranial childhood tumor of the sympathetic nervous system. To date, only few somatic mutations have been found in neuroblastoma, and tremendous efforts are now being made to identify druggable targets for effective treatment of high-risk disease. A recent study has revealed a non-oncogene dependency of phosphoglycerate dehydrogenase (PHGDH) in MYCN-amplified neuroblastoma. By analyzing independent datasets, we demonstrated that high expression of PHGDH was associated with poor even-free and overall survival in patients with neuroblastoma. We used two neuroblastoma cell lines to demonstrate that PHGDH knockdown significantly reduced proliferation and viability, whereas PHGDH overexpression conferred growth advantage. Homoharringtonine (HHT) is a Chinese traditional medicine that is currently approved for the treatment of chronic myeloid leukemia, and may have broad anticancer effects on various cancer types. Using molecular docking, we first demonstrated that HHT had a high binding affinity to PHGDH. We then performed isothermal titration calorimetry (ITC) assay to confirm the ability of HHT to inhibit the enzymatic activity of PHGDH by competing with its substrate NAD+, and used X-ray crystallography to further ascertain their binding. Treatment of four neuroblastoma cell lines with HHT significantly reduced proliferation and increased apoptosis at nanomolar concentrations. Overexpression of PHGDH conferred resistance to HHT in these neuroblastoma cells, suggesting the drug–target relationship. Given an essential role of PHGDH in cellular and mitochondrial metabolism, we determined whether these pathways are perturbed by HHT treatment. Metabolite analysis revealed that treatment of two neuroblastoma lines with HHT led to a decrease of glycine, but not serine, accompanied with an increase of metabolites of tricarboxylic acid (TCA) cycle. We also observed an increase of cellular and mitochondrial reactive oxygen species (ROS) after HHT treatment in these cells. We then examined the in vivo efficacy of HHT in the neuroblastoma xenograft model, demonstrating that HHT treatment led to reduced tumor growth and improved mouse survival with no apparent toxicity to organs including liver, spleen, and kidney. Taken together, these findings suggest that reprogramming serine/glycine metabolism with a new PHGDH inhibitor might be a powerful therapeutic strategy for different types of neuroblastoma.