Abstract
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.
Highlights
Inhibitors of poly-ADP-ribose polymerase 1 (PARPi) are highly effective in killing cells deficient in homologous recombination (HR); PARPi have been clinically utilized to successfully treat BRCA2-mutant tumors
In order to gain a broader understanding of factors governing PARPi response, we performed a series of genome-wide CRISPR knockout and transcriptional activation screens in an isogenic pair of wildtype and BRCA2knockout HeLa cell lines
HeLa cells infected with the Brunello library were divided into PARPi (5 μM olaparib)- or vehicle (DMSO)- treated arms, and after 4 days surviving cells were harvested for single-guide RNAs (sgRNAs) sequencing and bioinformatic analysis using the MAGeCK (Model-based Analysis of Genome-wide CRISPR/ Cas[9] Knockout) algorithm[11] (Fig. 1a)
Summary
Inhibitors of poly-ADP-ribose polymerase 1 (PARPi) are highly effective in killing cells deficient in homologous recombination (HR); PARPi have been clinically utilized to successfully treat BRCA2-mutant tumors. In order to better understand the mechanisms regulating cellular sensitivity and resistance to PARP inhibitors, we designed complementary genome-wide CRISPR screens in a pair of parental wildtype and BRCA2-knockout (BRCA2KO) HeLa cell lines. This approach allowed us to investigate which specific genetic changes lead to PARPi sensitivity in inherently resistant cells (parental) or to resistance in intrinsically sensitive cells (BRCA2KO) in an otherwise isogenic background. We show that the mechanism of resistance caused by KAT5 depletion involves 53BP1-mediated regulation of end resection at DSBs. In contrast, loss of HUWE1 restores HR in BRCA2-deficient cells by increasing RAD51 levels
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