Deciphering the Role of Reductive Stress in Non Small Cell Lung Cancer

解读还原应激在非小细胞肺癌中的作用

• 批准号: 10540372
• 负责人: Liron Bar-Peled
• 金额: $ 37.66万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-13 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10540372
• 关键词: Antioxidants; Applications Grants; Aspartate; Biochemical; Biologic Characteristic; Biological Assay; Biological Markers; Biology; CRISPR screen; Cancer Model; Cancer Patient; Cancer cell line; Cell Proliferation; Cells; Chemicals; Companions; Complex; Cysteine; Dependence; Drug Metabolic Detoxication; Electron Transport; Environment; Enzymes; Equilibrium; Foundations; Functional disorder; Gene Deletion; Generations; Genes; Genetic; Genetic Transcription; Genomics; Goals; Growth; Homeostasis; Impairment; In Vitro; Lead; Maintenance; Malignant Neoplasms; Malignant neoplasm of lung; Measures; Mediating; Metabolic; Metabolic Pathway; Metabolism; Methods; Mitochondria; Modeling; Modification; Mutation; Non-Small-Cell Lung Carcinoma; Oncogenic; Oxidation-Reduction; Oxidative Stress; Pathway interactions; Patients; Pharmacology; Play; Point Mutation; Production; Proliferating; Proteins; Proteomics; Reactive Oxygen Species; Regulation; Research; Respiration; Role; Series; Signal Transduction; Signal Transduction Pathway; Signaling Protein; Stress; Supplementation; Technology; Testing; Therapeutic; Tumor Suppressor Proteins; Work; anti-cancer therapeutic; biomarker identification; cancer cell; cancer therapy; cancer type; cell growth; cohort; efficacy evaluation; efficacy testing; experimental study; functional genomics; genome-wide; in vivo; insight; lung cancer cell; metabolomics; mitochondrial metabolism; mutant; next generation; novel; novel therapeutic intervention; pharmacologic; potential biomarker; protein function; reconstitution; response; small molecule inhibitor; synergism; therapeutic development; therapeutic evaluation; transcription factor; transcriptomics; translational therapeutics; tumor growth

Project Summary Control of the redox homeostasis is essential to cancer cell proliferation and requires the delicate maintenance of oxidative and reductive metabolic pathways. This equilibrium is controlled by signal transduction pathways and imbalances lead to redox stress that potently blocks cancer growth. Much work has focused on the role of oxidative stress in cancer proliferation, however, the converse– reductive stress and its impact on malignant cells is poorly understood. We have studied the role of redox control in cancer in the context of modification of proteinaceous cysteines by reactive oxygen species and the NRF2 transcription factor pathway. NRF2 functions as the master regulator of the cellular antioxidant response and promotes the expression of key metabolic and detoxification genes to generate a reductive environment and negate oxidative stress. NRF2 is activated in many cancers including ~30% of non small cell lung cancers (NSCLC) through mutation of its negative regulator KEAP1. While NRF2 has been extensively studied in KEAP1-mutant NSCLCs, we wondered what role this pathway plays in the proliferation of NSCLC cell lines which are wildtype (WT) for KEAP1. To this end, we pharmacologically activated NRF2 in 50+ NSCLC cell lines (WT for KEAP1) and measured their proliferation. Unexpectedly, we find that in ~16% of NSCLC cell lines, NRF2 activation results in a severe block in proliferation. A genome wide CRISPR screen identifies that genes involved in mitochondrial metabolism, mitochondrial fusion and the electron transport chain (ETC) are major sensitizers to NRF2 activation when lost and can function as companion biomarkers for NRF2-sensitivity. In line with the generation of reductive stress following NRF2 activation, key cysteines on enzymes involved in mitochondrial metabolism and mitochondrial fusion are reduced as determined by chemical proteomic platforms. To explain these surprising biological characteristics we propose the following hypothesis: NRF2 activation in a subset of NSCLC cell lines promotes an overly reductive environment that decreases the activity of key enzymes in mitochondrial metabolism and mitochondrial respiration and fusion. The inactivation of these pathways synergize to block cell growth. In this grant application, we build on our research surrounding NRF2 sensitization and mechanistically characterize the role of reductive stress in NSCLC proliferation. In this grant application, we will comprehensively define KEAP1-dependence by identifying NRF2 regulation of mitochondrial metabolism/fusion at the protein, cellular and organismal levels. The research proposed herein, takes full advantage of a series of recently conceived methods: chemical proteomics, genome-wide CRISPR screens and untargeted metabolomics, which have previously been deployed in isolation, to be used in an integrated manner to effectively dissect how protein reduction underlies protein malfunction and KEAP1-dependence. These studies will not only provide a comprehensive understanding of NRF2/KEAP1 biology but may also lay the foundation for developing translational therapeutics to benefit lung cancer patients with deregulated NRF2 signaling.

项目摘要 氧化还原稳态的控制对于癌细胞增殖是必不可少的，并且需要精细的维持。 氧化和还原代谢途径。这种平衡是由信号转导途径控制的 失衡导致氧化还原压力，从而有效地阻止癌症生长。许多工作都集中在 氧化应激在癌细胞增殖中的作用，然而，匡威还原应激及其对恶性肿瘤的影响， 对细胞了解甚少。我们已经研究了氧化还原控制在癌症中的作用， 蛋白质半胱氨酸通过活性氧和NRF 2转录因子途径。NRF 2功能 作为细胞抗氧化反应的主要调节剂，并促进关键代谢和 解毒基因，以产生还原环境和否定氧化应激。NRF 2在许多情况下被激活。 癌症，包括~30%的非小细胞肺癌（NSCLC），通过其负调节因子突变 KEAP 1.虽然NRF 2在KEAP 1突变型NSCLC中已被广泛研究，但我们想知道NRF 2在KEAP 1突变型NSCLC中的作用， 在KEAP 1的野生型（WT）NSCLC细胞系的增殖中起作用。为此我们 在50+ NSCLC细胞系（KEAP 1的WT）中，NRF 2活化并测量其增殖。 出乎意料的是，我们发现在~16%的NSCLC细胞系中，NRF 2激活导致增殖严重阻滞。 全基因组CRISPR筛选鉴定出参与线粒体代谢、线粒体融合 和电子传递链（ETC）是NRF 2活化的主要敏化剂， NRF 2敏感性的伴随生物标志物。与NRF 2后还原应激的产生一致 激活后，参与线粒体代谢和线粒体融合的酶上的关键半胱氨酸减少 如通过化学蛋白质组学平台所确定的。为了解释这些令人惊讶的生物特征， 我提出了以下假设：NRF 2在NSCLC细胞系亚群中的激活促进了过度还原性的 降低线粒体代谢和线粒体内关键酶活性的环境 呼吸和融合这些途径的失活协同作用以阻断细胞生长。在这份资助申请中， 我们建立在我们围绕NRF 2致敏的研究基础上，并从机制上描述了还原性NRF的作用。 在NSCLC增殖中的应激。在本资助申请中，我们将全面定义KEAP 1依赖性， 在蛋白质、细胞和生物体水平鉴定NRF 2对线粒体代谢/融合的调节。 本文提出的研究，充分利用了一系列最近设想的方法：化学 蛋白质组学，全基因组CRISPR筛选和非靶向代谢组学，这些以前一直是 单独部署，以综合方式使用，以有效剖析蛋白质减少如何构成 蛋白质故障和KEAP 1依赖。这些研究不仅提供了一个全面的 了解NRF 2/KEAP 1生物学，但也可能为开发转化疗法奠定基础 使NRF 2信号失调的肺癌患者受益。