Identification of regulators of poly-ADP-ribose polymerase inhibitor response through complementary CRISPR knockout and activation screens

Inhibitors of poly-ADP-ribose polymerase 1 (PARPi) are highly effective in killing cells deficient in homologous recombination (HR); thus, PARPi have been clinically utilized to successfully treat BRCA2-mutant tumors. However, positive response to PARPi is not universal, even among patients with HR-deficiency. Here, we present the results of genome-wide CRISPR knockout and activation screens which reveal genetic determinants of PARPi response in wildtype or BRCA2-knockout cells. Strikingly, we report that depletion of the ubiquitin ligase HUWE1, or the histone acetyltransferase KAT5, top hits from our screens, robustly reverses the PARPi sensitivity caused by BRCA2-deficiency. We identify distinct mechanisms of resistance, in which HUWE1 loss increases RAD51 levels to partially restore HR, whereas KAT5 depletion rewires double strand break repair by promoting 53BP1 binding to double-strand breaks. Our work provides a comprehensive set of putative biomarkers that advance understanding of PARPi response, and identifies novel pathways of PARPi resistance in BRCA2-deficient cells.


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Replication
The three CRISPR screens were performed in duplicate. Western blot experiments, DNA fiber assays, immunofluorescence and comet assays were performed at least twice (with the exception of Supplementary Fig. 9b and 11, which were only done once). All results were reproducible. The experiment in Supplementary Fig. 9b was only performed once because it is a negative result and it addresses an issue not directly linked to the theme of the manuscript and is thus inconsequential. The experiment in Supplementary Fig. 11 was only done once because of time constraints during the revision. The drug sensitivity assays were replicated independently for the number of times indicated in the figure legends. All attempts at replication were successful.

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