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

基本信息

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
项目类别：
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

项目摘要

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）是肿瘤学中最困难的挑战之一。 95% 的 PDA 病例中均发现 K-ras 癌基因突变，但目前尚无药物可以治愈有效地针对 PDA 中的这种（或任何其他）高患病率变化另一种策略是针对关键的变化。 PDA 细胞依赖但正常细胞可以放弃的生物过程就是一个例子。外源半胱氨酸（胱氨酸的氧化形式）通过称为系统 xc− 的胱氨酸/谷氨酸逆向转运蛋白。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- 细胞凋亡，称为铁死亡，其机制与坏死性凋亡、自噬、 parthanatos 和其他形式的非凋亡细胞死亡其特点是快速、铁依赖性。尽管没有 DNA 裂解，脂质 ROS 的积累仍会导致膜完整性丧失。 System xc− 抑制对肿瘤细胞产生显着影响，种系 System xc− 敲除小鼠可存活并与成人一样健康，证明正常细胞通常不需要半胱氨酸输入。合成谷胱甘肽 (GSH) 的前体，谷胱甘肽是一种非蛋白质三肽，对于解毒至关重要谷胱甘肽过氧化物酶 4 抑制剂也可诱导活性氧 (ROS)。 (GPX4) 使用 GSH 作为辅助因子来解毒脂质过氧化物，但 GSH 本身尚未被耗尽。研究表明，半胱氨酸的消耗会导致铁死亡，但原因尚不清楚。与谷胱甘肽的消耗在性质上不同，并且额外的半胱氨酸衍生的代谢物起着在脂质ROS的解毒和铁死亡的控制中发挥关键作用。已鉴定出几种可在体外有效诱导铁死亡的系统 xc− 抑制剂，包括 erastin、柳氮磺吡啶和索拉非尼以及新的抑制剂正在迅速开发。该提案是为了确定通过系统 xc− 抑制诱导铁死亡的潜在机制，包括确定铁死亡敏感性的决定因素。我们带来了一系列创新。工具，包括半胱氨酸和蛋氨酸碳标记、质谱、化学生物学方法，关键代谢酶的诱导型慢病毒 shRNA 敲低、系统生物学技术、小动物除了 2D 细胞系之外，还使用基因工程小鼠模型进行成像和转化治疗。和类器官培养模型，我们还提供了来自复杂基因工程小鼠的试验数据该模型能够在已建立的 K-ras/p53 突变胰腺肿瘤中急性删除系统 xc−。六等位基因双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 中发挥功能，并作为基因定义的原代细胞的来源，以促进我们提出的最后，我们将使用一种新颖的组合来评估候选机制途径。两种经过重新调整用途的临床开发药物，每种药物均具有良好的耐受性。