Targeting cysteine import to induce ferroptotic cell death in pancreatic cancer

靶向半胱氨酸输入诱导胰腺癌铁死亡细胞

• 批准号: 10088424
• 负责人: Kenneth P Olive
• 金额: $ 38.51万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-08 至 2022-03-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10088424
• 关键词: Acute; Address; Adult; Alleles; Amino Acids; Apoptosis; Autophagocytosis; BAY 54-9085; Biological Process; Biology; Cancer Etiology; Cancer cell line; Carbon; Caspase; Catabolic Process; Cell Culture Techniques; Cell Death; Cell Death Process; Cell Line; Cell Nucleus; Cell division; Cells; Chemicals; Clinic; Clinical; Complex; Cultured Cells; Cysteine; Cystine; DNA; Dangerousness; Data; Development; Disulfides; Drug Metabolic Detoxication; Enzymes; Evaluation; Event; Exhibits; FDA approved; Generations; Genetically Engineered Mouse; Glutamates; Glutathione; Goals; High Prevalence; Human; Hypersensitivity; In Vitro; Individual; Iron; KRAS2 gene; Kidney; Knockout Mice; Label; Lipid Peroxidation; Lipid Peroxides; Lipids; Malignant Neoplasms; Malignant neoplasm of pancreas; Mass Spectrum Analysis; Mediator of activation protein; Membrane; Metabolic; Metabolism; Methionine; Modeling; Molecular; Morphologic artifacts; Mus; Mutation; Necrosis; Normal Cell; Oncogenic; Oncology; Organoids; Oxides; Pancreas; Pancreatic Ductal Adenocarcinoma; Pathologic Processes; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Phospholipids; Play; Process; Proliferating; Property; Reaction; Reactive Oxygen Species; Regimen; Resistance; Role; Source; Sulfasalazine; Supporting Cell; System; Systems Biology; TP53 gene; Techniques; Therapeutic; Therapeutic Uses; Toxic effect; United States; Ursidae Family; animal imaging; anti-cancer therapeutic; antiporter; buthionine; chemotherapy; clinical translation; cyclophilin D; erastin; experimental study; genetic approach; genotoxicity; glutathione peroxidase; in vivo; inhibitor/antagonist; innovation; knock-down; macromolecule; mitochondrial metabolism; mortality; mouse model; mutant; neoplastic cell; next generation; novel; novel strategies; pancreatic ductal adenocarcinoma cell; pancreatic neoplasm; programs; recombinase; screening; small hairpin RNA; therapeutic target; tool; transcription factor; tumor

Abstract: Pancreatic ductal adenocarcinoma (PDA) is one of the most difficult challenges in oncology. Activating mutations in the K-ras oncogene are found in 95% of PDA cases, but agents are not yet available that can effectively target this (or any other) high prevalence alteration in PDA. An alternative strategy is to target critical biological processes that PDA cells depend on but normal cells can forego. An example of this is the import of exogenous cysteine (in the oxidized form of cystine) via the cystine/glutamate antiporter called System xc−. The inhibition of System xc− in many cancer cell lines has been shown to induce a peculiar form of non- apoptotic cell death, called ferroptosis, which is mechanistically distinct from necroptosis, autophagy, parthanatos, and other forms of non-apoptotic cell death. It is characterized by the rapid, iron-dependent accumulation of lipid ROS leading to loss of membrane integrity in the absence of DNA cleavage. Despite the dramatic effects of System xc− inhibition in tumor cells, germline System xc− knockout mice are viable and healthy as adults, proving that normal cells do not usually require cystine import. Cysteine is the rate-limiting precursor for the synthesis of glutathione (GSH), a non-protein tripeptide that is critical for the detoxification of reactive oxygen species (ROS). Ferroptosis can also be induced by inhibitors of glutathione peroxidase 4 (GPX4) which detoxifies lipid peroxides using GSH as a co-factor. Yet depletion of GSH itself has not been shown to induce ferroptosis, for reasons that are unclear. We hypothesize that depletion of cysteine is qualitatively distinct from the depletion of glutathione and that additional cysteine−derived metabolites play a critical role in the detoxification of lipid ROS and control of ferroptosis. Several inhibitors of System xc− have been identified that effectively induce ferroptosis in vitro, including erastin, sulfasalazine, and sorafenib, and new inhibitors are rapidly being developed. The overarching goal of this proposal is to determine the underlying mechanisms of ferroptosis induction through System xc− inhibition, including the identification of determinants of ferroptosis sensitivity. We bring to bear a range of innovative tools, including cysteine and methionine carbon labeling, mass spectrometry, chemical biology approaches, inducible lentiviral shRNA knockdown of key metabolic enzymes, systems biology techniques, small animal imaging, and translational therapeutics using genetically engineered mouse models. In addition to 2D cell line and organoid culture models, we also present pilot data from a sophisticated genetically engineered mouse model that enables the acute deletion of System xc− in established K-ras/p53 mutant pancreatic tumors. This six-allele dual recombinase mouse strategy provides an ideal genetic strategy for the evaluation of System xc− function in PDA and serves as a source for genetically−defined primary cells to facilitate our proposed mechanism studies. Finally, we will evaluate a candidate mechanistic pathway using a novel combination of two repurposed, clinically−developed agents, each of which is individually well-tolerated.

抽象的： 胰腺导管腺癌（PDA）是肿瘤学最困难的挑战之一。激活 在95％的PDA病例中发现了K-RAS癌基因中的突变，但尚未可用的代理可以 有效地针对PDA中的（或任何其他）高患病率改变。另一种策略是针对关键 PDA细胞依赖但正常细胞可以放弃的生物学过程。一个例子是导入 外源性半胱氨酸（以氧化形式为胱氨酸），通过胱氨酸/谷氨酸抗植物剂，称为系统XC-。 许多癌细胞系中系统XC-的抑制作用已显示出诱导的一种非 - 凋亡细胞死亡，称为铁凋亡，在机械上与坏死性，自噬， Parthanatos和其他形式的非凋亡细胞死亡。它的特征是快速，铁依赖性 在没有DNA裂解的情况下，脂质ROS的积累导致膜完整性丧失。尽管有 系统XC-抑制在肿瘤细胞中的戏剧性作用，种系系统XC-基因敲除小鼠是可行的，并且 作为成年人健康，证明正常细胞通常不需要胱氨酸进口。半胱氨酸是限制速率 谷胱甘肽（GSH）合成的前体，这是一种非蛋白三肽，对排毒至关重要 活性氧（ROS）。谷胱甘肽过氧化物酶4的抑制剂也可以诱导铁凋亡 （GPX4）使用GSH作为副因素来解毒脂质过氧化物。然而，GSH本身的耗尽并不是 出于不清楚的原因，证明会诱导铁铁作用。我们假设半胱氨酸的消耗是 定性上与谷胱甘肽的耗尽不同，而额外的半胱氨酸代谢产物发挥作用 在脂质ROS的排毒和控制铁氧化作用中的关键作用。 已经确定了系统XC-的几种抑制剂 Erastin，sulfasalazine和Sorafenib以及新的抑制剂正在迅速发展。总体目标 该建议是通过系统XC-抑制作用来确定铁凋亡诱导的潜在机制， 包括鉴定确定铁凋亡敏感性。我们带来一系列创新的 工具，包括半胱氨酸和甲二碳标记，质谱法，化学生物学方法， 关键代谢酶，系统生物学技术，小动物的诱导式慢病毒shRNA敲低 成像和使用一般工程的小鼠模型进行翻译治疗。除了2D细胞系 和器官培养模型，我们还提供了来自精致工程鼠标的飞行员数据 在已建立的K-RAS/p53突变体胰腺肿瘤中急性缺失的模型。这 六位基因双重重组酶小鼠策略为评估系统XC-提供了理想的遗传策略 在PDA中的功能，并作为遗传定义的原代细胞的来源，以促进我们提出的 机理研究。最后，我们将使用新颖的组合来评估候选机械途径 两个重新利用的临床发达的药物，每种药物都具有良好的耐受性。