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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要该项目的首要目标是了解 DNA 如何激活非凋亡细胞死亡损伤反应 (DDR) 响应基因组损伤，DDR 激活 DNA 修复和细胞周期停滞。或者，在严重损伤的情况下，DDR 将激活。这些关键的促生存和促死亡反应均由 p53 调节。 DDR 中的 p53 使细胞能够快速灵活地应对不同类型的 DNA 损伤。如果没有 p53，该模型会预测什么结果？虽然我们可能期望 p53 去除会消除？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作为反应这表明存在一种额外的、迄今为止未被描述的途径。我们发现 DNA 损伤也能够诱导非凋亡细胞死亡。此外，我们的表征策略是在缺乏 p53 的细胞中优先激活非凋亡性死亡。这种新颖的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 筛选通常无法识别死亡调控基因。为了克服这一限制，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根据我们的筛选结果，在目标 1 中，我们将检验 ROS 和在 p53 缺失的情况下，DNA 损伤诱导的死亡需要线粒体通透性转变 (MPT)。我们将使用 CRISPR/Cas9 介导的基因敲除来比较 DNA 损伤诱导的 MPT 与典型的 MPT。将使用荧光显微镜监测 MPT 的激活，并使用 TEM 表征线粒体我们的 CRISPR 筛选还鉴定出 TGF-β 信号传导是 DNA 损伤的负调节因子。在目标 2 中，我们将鉴定有助于诱导非凋亡性死亡的 TGF-β 途径成分。抑制非凋亡性死亡，并确定该知识在细胞系中的普遍性。将把这一探索扩展到使用和不使用功能性 p53 产生的体内癌症小鼠模型。 DNA 损伤诱导的非凋亡性死亡的表征将提高我们对 p53- 最终，我们希望这项工作能够提高我们治疗突变癌症的能力。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作为目标以提高治疗效果。