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:
- 金额:$ 3.24万
- 依托单位:
- 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:
- 关键词:ApoptosisApoptoticBiological AssayCRISPR screenCRISPR/Cas technologyCancer ModelCancer PatientCell Cycle ArrestCell DeathCell Death InductionCell LineCell SurvivalCellsCessation of lifeClustered Regularly Interspaced Short Palindromic RepeatsCommunitiesComputer AnalysisComputing MethodologiesDNA DamageDNA RepairDataDeath RateEnvironmentEvolutionExcisionFluorescence MicroscopyFluorescent DyesG1 ArrestGene DeletionGenesGenetic ScreeningGenomeGenomicsGenotypeGoalsGrowthInduction of ApoptosisKnock-outKnowledgeLinkMalignant NeoplasmsMalignant neoplasm of lungMeasurementMeasuresMediatingMitochondriaMonitorMorphologyMutateNecrosisOutcomeOutputPathway interactionsPermeabilityPharmaceutical PreparationsPhenotypePhosphorylationPhosphotransferasesPopulationRegulator GenesResearchSignal TransductionTP53 geneTechniquesTestingTherapeuticTransforming Growth Factor betaTransforming Growth Factor beta ReceptorsTransmission Electron MicroscopyTreatment EfficacyWorkanalysis pipelinecancer cellcancer therapycancer typechemotherapydesignfitnessflexibilitygenome-wideimprovedin vivoinsightknockout genemouse modelnovelpredictive modelingresponsewhole 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.
项目摘要
该项目的总体目标是了解非凋亡细胞死亡如何被DNA激活
伤害响应(DDR)。为了应对基因组侮辱,DDR激活DNA修复和细胞周期停滞
解决损害并促进细胞存活。或者,在严重损坏的情况下,DDR将激活
凋亡细胞死亡。这些关键的亲阳性和亲死亡反应都受p53的调节。中心性
DDR中p53的p53允许细胞快速而灵活地响应不同类型的DNA损伤。但是,在
缺乏p53,该模型预测了什么结果?虽然我们可能期望p53去除废除
细胞周期停滞和细胞凋亡,许多p53突变的癌症仍然能够响应细胞死亡
减轻DNA的药物。这表明存在另外一个未描述的途径
将DDR与细胞死亡联系起来。我们发现DNA损伤还能够诱导非凋亡细胞死亡。
此外,在缺乏p53的细胞中,非凋亡死亡更有可能激活。我们表征的策略
这种新型的DNA损伤引起的非凋亡死亡是进行全基因组CRISPR屏幕。
全基因组CRISPR筛选通常不会识别死亡调节基因。为了克服这一限制,我们
设计了一种新的实验和计算方法,用于计算每种药物诱导的死亡率
单基因淘汰赛。根据屏幕的结果,在AIM 1中,我们将测试ROS和ROS的假设
在没有p53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介导的敲除将DNA损伤引起的MPT与规范MPT进行比较。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。我们的CRISPR屏幕还确定TGF-β信号传导是DNA损伤的负调节剂
诱发非凋亡死亡。在AIM 2中,我们将确定TGF-β途径成分有助于
抑制非凋亡死亡,并确定这些知识跨细胞系的普遍性。我们
将把这种探索扩展到具有和不具有功能性p53的癌症的体内小鼠模型。我们的
DNA损伤引起的非凋亡死亡的表征将提高我们对p53-的理解
突变的癌症对化学治疗剂反应。最终,我们希望这项工作将提高我们的能力
预测哪些癌症将对破坏DNA的药物有反应,以及可以针对哪些死亡途径
提高治疗效率。
项目成果
期刊论文数量(2)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Megan Elizabeth Honeywell其他文献
Megan Elizabeth Honeywell的其他文献
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{{ truncateString('Megan Elizabeth Honeywell', 18)}}的其他基金
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 - 财政年份:2022
- 资助金额:
$ 3.24万 - 项目类别:
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