Activation of non-apoptotic cell death by the DNA damage response

DNA 损伤反应激活非凋亡细胞死亡

• 批准号: 10388929
• 负责人: Megan Elizabeth Honeywell
• 金额: $ 3.15万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10388929
• 关键词: Apoptosis; Apoptotic; Biological Assay; CRISPR screen; CRISPR/Cas technology; Cancer Model; Cancer Patient; Cell Cycle Arrest; Cell Death; Cell Line; Cell Survival; Cells; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Computer Analysis; Computing Methodologies; Conflict (Psychology); DNA Damage; DNA Repair; Data; Death Rate; Environment; Evolution; Excision; Fluorescence Microscopy; Fluorescent Dyes; G1 Arrest; Gene Deletion; Genes; Genetic Screening; Genome; Genomics; Genotype; Goals; Growth; Knock-out; Knowledge; Link; Malignant Neoplasms; Malignant neoplasm of lung; Measurement; Measures; Mediating; Mitochondria; Monitor; Morphology; Mutate; Necrosis; Outcome; Output; Pathway interactions; Permeability; Pharmaceutical Preparations; Phenotype; Phosphotransferases; Population; Regulator Genes; Research; Signal Transduction; TP53 gene; Techniques; Testing; Transforming Growth Factor beta; Transforming Growth Factor beta Receptors; Transmission Electron Microscopy; Treatment Efficacy; Work; analysis pipeline; base; cancer cell; cancer therapy; cancer type; chemotherapy; design; fitness; flexibility; genome-wide; improved; in vivo; insight; knockout gene; mouse model; novel; predictive modeling; response; whole genome

PROJECT SUMMARY The overarching goal of this project is to understand how non-apoptotic cell death is activated by the DNA damage response (DDR). In response to genomic insult, the DDR activates DNA repair and cell cycle arrest to resolve the damage and promote cell survival. Alternatively, in cases of severe damage, the DDR will activate apoptotic cell death. These critical pro-survival and pro-death responses are all regulated by p53. The centrality of p53 in the DDR allows cells to quickly and flexibly respond to different types of DNA damage. However, in the absence of p53, what outcome is predicted by this model? While we might expect that p53 removal abrogates both cell cycle arrest and apoptosis, many p53-mutated cancers are still able to execute cell death in response to DNA-damaging drugs. This suggests the presence of an additional and heretofore undescribed pathway linking the DDR to cell death. We found that DNA damage is also capable of inducing non-apoptotic cell death. Furthermore, non-apoptotic death is preferentially activated in cells that lack p53. Our strategy for characterizing this novel DNA damage-induced non-apoptotic death was to perform a whole-genome CRISPR screen. Genome-wide CRISPR screens do not typically identify death regulatory genes. To overcome this limitation, we devised a new experimental and computational method for calculating the drug-induced death rate of each single-gene knockout. Based on the results of our screen, in Aim 1 we will test the hypothesis that ROS and mitochondrial permeability transition (MPT) are required for DNA damage-induced death in the absence of p53. We will use CRISPR/Cas9 mediated knockout to compare DNA damage-induced MPT to canonical MPT. We will monitor activation of MPT using fluorescence microscopy, and use TEM to characterize mitochondrial morphologies. Our CRISPR screen also identified TGF-β signaling as a negative regulator of DNA damage- induced non-apoptotic death. In Aim 2, we will identify TGF-β pathway components that contribute to the suppression of non-apoptotic death, and determine the generalizability of this knowledge across cell lines. We will extend this exploration to an in vivo mouse model of cancers generated with and without functional p53. Our characterization of DNA damage-induced non-apoptotic death will improve our understanding of how p53- mutated cancers respond to chemotherapeutics. Ultimately, we hope that this work will improve our ability to predict which cancers will respond to DNA-damaging drugs, as well as which death pathways can be targeted to enhance treatment efficacy.

项目摘要 这个项目的首要目标是了解非凋亡性细胞死亡是如何被DNA激活的。 损伤响应（DDR）。作为对基因组损伤的反应，DDR激活DNA修复和细胞周期停滞， 解决损伤并促进细胞存活。或者，在严重损坏的情况下，DDR将启动 细胞凋亡这些关键的促生存和促死亡反应都是由p53调节的。的中心地位 DDR中的p53蛋白的表达使细胞能够快速灵活地对不同类型的DNA损伤做出反应。但在 如果没有p53，这个模型预测的结果是什么？虽然我们可能认为p53的去除会废除 细胞周期停滞和细胞凋亡，许多p53突变的癌症仍然能够执行细胞死亡， 破坏DNA的药物这表明存在一个额外的和迄今为止未描述的途径 将DDR与细胞死亡联系起来我们发现，DNA损伤也能够诱导非凋亡性细胞死亡。 此外，非凋亡性死亡在缺乏p53的细胞中优先被激活。我们的特征化策略 这种新的DNA损伤诱导的非凋亡性死亡是为了进行全基因组CRISPR筛选。 全基因组CRISPR筛选通常不能识别死亡调控基因。为了克服这一局限性，我们 设计了一种新的实验和计算方法来计算每种药物引起的死亡率， 单基因敲除基于我们的筛选结果，在目标1中，我们将检验ROS和 线粒体通透性转换（MPT）是在p53不存在下DNA损伤诱导的死亡所必需的。 我们将使用CRISPR/Cas9介导的敲除来比较DNA损伤诱导的MPT与经典MPT。我们 将使用荧光显微镜监测MPT的激活，并使用TEM表征线粒体 形态学我们的CRISPR筛选还将TGF-β信号转导确定为DNA损伤的负调节因子- 诱导非凋亡性死亡。在目标2中，我们将确定TGF-β通路的组成部分，有助于 抑制非凋亡性死亡，并确定这种知识在细胞系中的普遍性。我们 将这种探索扩展到有和没有功能性p53产生的癌症的体内小鼠模型。我们 DNA损伤诱导的非凋亡性死亡的特征将提高我们对p53- 突变的癌症对化疗药物有反应。最终，我们希望这项工作将提高我们的能力， 预测哪些癌症会对DNA损伤药物产生反应，以及哪些死亡途径可以被靶向 以提高治疗效果。