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

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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.