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

项目总结 这个项目的首要目标是了解DNA是如何激活非凋亡性细胞死亡的 损害反应(DDR)。作为对基因组侮辱的回应，DDR激活DNA修复和细胞周期停滞 解决损伤，促进细胞存活。或者，在严重损坏的情况下，DDR将被激活 细胞凋亡性死亡这些关键的支持生存和支持死亡的反应都受到P53的调控。中心性 在DDR中使用p53使细胞能够快速而灵活地对不同类型的DNA损伤做出反应。然而，在 P53的缺失，这个模型预测的结果是什么？虽然我们可能会认为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作为回应执行细胞死亡 为了破坏DNA的药物。这表明存在另一条迄今未被描述的途径。 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，在缺乏P53的细胞中，非凋亡性死亡优先被激活。我们的策略是刻画 这一新的DNA损伤诱导的非凋亡性死亡将进行全基因组CRISPR筛查。 全基因组CRISPR筛查通常不能识别死亡调控基因。为了克服这一限制，我们 设计了一种新的计算药物致死率的实验和计算方法 单基因敲除。基于我们筛选的结果，在目标1中，我们将测试ROS和 在缺乏P53的情况下，线粒体通透性转换(MPT)是DNA损伤诱导的死亡所必需的。 我们将使用CRISPR/Cas9介导的基因敲除来比较DNA损伤诱导的MPT和典型的MPT。我们 将使用荧光显微镜监测MPT的激活，并使用电子显微镜来表征线粒体 形态特征。我们的CRISPR筛查还发现转化生长因子-β信号是DNA损伤的负面调节因子- 诱导非凋亡性死亡。在目标2中，我们将确定转化生长因子-β途径组件对 抑制非凋亡性死亡，并确定这一知识在细胞系中的普适性。我们 将把这一探索扩展到体内的小鼠模型，即有和没有功能性p53产生的癌症。我们的 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 预测哪些癌症会对破坏DNA的药物产生反应，以及哪些死亡途径可以成为靶点 以提高治疗效果。

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.