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.

摘要： 胰腺导管腺癌（PDA）是肿瘤学中最困难的挑战之一。激活 在95%的PDA病例中发现了K-ras癌基因突变，但目前还没有能够 有效地针对PDA中这种（或任何其他）高流行率改变。另一种策略是针对关键的 PDA细胞依赖的生物过程，但正常细胞可以放弃。这方面的一个例子是进口 外源性半胱氨酸（以胱氨酸的氧化形式）通过胱氨酸/谷氨酸反向转运蛋白称为系统xc−。 在许多癌细胞系中，系统xc−的抑制已被证明可诱导一种特殊形式的非- 凋亡性细胞死亡，称为铁凋亡，其在机制上不同于坏死性凋亡，自噬， parthanatos和其他形式的非凋亡性细胞死亡。它的特点是快速，铁依赖性 脂质ROS的积累导致在不存在DNA切割的情况下膜完整性的丧失。尽管 系统xc−抑制在肿瘤细胞中的显著作用，种系系统xc−敲除小鼠是可行的， 健康的成年人，证明正常细胞通常不需要胱氨酸输入。半胱氨酸是限速蛋白 谷胱甘肽（GSH）合成的前体，GSH是一种非蛋白质三肽，对解毒至关重要。 活性氧（ROS）。谷胱甘肽过氧化物酶4的抑制剂也可诱导铁凋亡 （GPX 4），其使用GSH作为辅因子来解毒脂质过氧化物。然而，谷胱甘肽本身的消耗并没有被 显示出诱导铁凋亡，原因尚不清楚。我们假设半胱氨酸的耗尽是 定性上不同于谷胱甘肽的消耗，并且额外的半胱氨酸衍生代谢物发挥了 在脂质ROS的解毒和铁凋亡的控制中起关键作用。 已鉴定出几种系统xc−抑制剂，它们在体外可有效诱导铁凋亡，包括 erastin、柳氮磺胺吡啶和索拉非尼以及新的抑制剂正在迅速开发。的首要目标 该建议是通过系统xc-抑制来确定铁凋亡诱导的潜在机制， 包括鉴定铁凋亡敏感性的决定因素。我们带来了一系列创新的 工具，包括半胱氨酸和甲硫氨酸碳标记，质谱，化学生物学方法， 可诱导慢病毒shRNA敲减关键代谢酶，系统生物学技术，小动物 成像和使用基因工程小鼠模型的转化治疗。除了2D细胞系 和类器官培养模型，我们还提出了一个复杂的基因工程小鼠的试点数据， 在已建立的K-ras/p53突变胰腺肿瘤中急性缺失系统xc−的模型。这 六等位基因双重组酶小鼠策略为系统xc-1的评估提供了理想的遗传策略。 在PDA中发挥作用，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，我们将评估一个候选机制的途径，使用一种新的组合， 两种重新利用的临床开发的药物，每种药物都具有良好的耐受性。