Targeting cysteine import to induce ferroptotic cell death in pancreatic cancer

摘要： 胰腺导管腺癌(PDA)是肿瘤学面临的最大挑战之一。正在激活 K-ras癌基因突变在95%的PDA病例中被发现，但目前还没有能够 有效地瞄准这种(或任何其他)PDA的高发改变。另一种战略是瞄准危急情况 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导入 外源半胱氨酸(以半胱氨酸的氧化形式)通过被称为系统XC−的半胱氨酸/谷氨酸逆向转运蛋白。 在许多癌细胞系中，XC−系统的抑制已被证明诱导了一种特殊形式的非 凋亡性细胞死亡，称为铁性下垂，在机制上不同于坏死性下垂，自噬， 死亡论和其他形式的非凋亡性细胞死亡。它的特点是速度快，铁依赖 在没有DNA裂解的情况下，脂质ROS的积累会导致膜完整性的丧失。尽管 XC−系统对肿瘤细胞、生殖系XC−基因敲除小鼠的显著抑制作用 成年后身体健康，证明正常细胞通常不需要胱氨酸进口。半胱氨酸是限速药 谷胱甘肽(GSH)是合成谷胱甘肽的前体，谷胱甘肽是一种非蛋白质三肽，对人体的解毒至关重要 活性氧(ROS)。谷胱甘肽过氧化物酶4抑制剂也可引起铁下垂 (Gpx4)，使用GSH作为辅助因子来解毒过氧化脂质。然而，谷胱甘肽本身的耗尽并没有 被证明会导致铁性下垂，原因尚不清楚。我们假设半胱氨酸的耗尽是 与谷胱甘肽的耗竭和额外的半胱氨酸−衍生代谢物在 在脂类ROS的解毒和铁性下垂的控制中的关键作用。 XC−系统的几种抑制剂已经被确定在体外有效地诱导铁下垂，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。的首要目标是 这个建议是为了确定通过抑制系统XC−诱导铁性下垂的潜在机制。 包括对铁性下垂敏感性决定因素的鉴定。我们带来了一系列创新的 工具，包括半胱氨酸和蛋氨酸碳标记、质谱学、化学生物学方法、 可诱导慢病毒shRNA击倒关键代谢酶，系统生物学技术，小动物 利用基因工程小鼠模型进行成像和转化治疗。除了2D细胞系之外 和器官培养模型，我们还提供了一个复杂的基因工程小鼠的试验数据 在已建立的K-−/P53突变的胰腺肿瘤中实现系统XC ras的急性缺失的模型。这 六等位基因双重组小鼠策略为系统XC−的评价提供了一种理想的遗传策略 在掌上电脑中的功能，并作为基因−定义的原始细胞的来源，以促进我们建议的 机理研究。最后，我们将使用一种新的组合来评估候选的机械路径 两种用途改变的临床−开发的药物，每一种都单独耐受性良好。

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.