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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：
10559522
负责人：
Megan Elizabeth Honeywell
金额：
$ 3.24万
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/10559522
关键词：
Apoptosis Apoptotic Biological Assay CRISPR screen CRISPR/Cas technology Cancer Model Cancer Patient Cell Cycle Arrest Cell Death Cell Death Induction Cell Line Cell Survival Cells Cessation of life Clustered Regularly Interspaced Short Palindromic Repeats Communities Computer Analysis Computing Methodologies 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 Induction of Apoptosis 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 Phosphorylation Phosphotransferases Population Regulator Genes Research Signal Transduction TP53 gene Techniques Testing Therapeutic Transforming Growth Factor beta Transforming Growth Factor beta Receptors Transmission Electron Microscopy Treatment Efficacy Work analysis pipeline 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.

项目总结