Abstract 410: Genome-wide and focused CRISPR screens to study PARP inhibitor resistance mediated by mutations in PARP1
Authors: Pettitt Stephen J. , Krastev Dragomir B. , Brandsma Inger , Dréan Amy , Song Feifei , Harrell Maria I. , Brough Rachel , Frankum Jessica , Lee Jung-Min , Swisher Elizabeth M. , Yusa Kosuke , Ashworth Alan , Lord Christopher J. 
Published: August 2018

Abstract
To investigate mechanisms of PARP inhibitor (PARPi) cytotoxicity, we carried out genome wide CRISPR-Cas9 screens for mutants resistant to the potent PARPi talazoparib (BMN 673) in mouse ES cells and human breast cancer cells. Many talazoparib resistant clones had loss of function Parp1 mutations as expected from to the proposed role of trapped PARP1 in cytotoxicity. However, we also isolated a point mutation affecting a single amino acid in the Parp1 DNA binding domain. This mutant encodes a stable Parp1 protein that cannot bind DNA and does not become trapped in the presence of inhibitors. Thus the CRISPR screen implicated PARP1 DNA binding directly in determination of PARPi cytotoxicity and was extremely informative about the mechanism of action compared to conventional loss-of-function mutagenesis.
We extended this approach by synthesising a high-density focused sgRNA library targeting only PARP1. We developed a reporter cell line that allows us to selectively isolate in-frame mutations that preserve PARP1 protein expression. By deep sequencing mutagenised and appropriately selected cells we identified a series of subtle mutations in PARP1 that result in PARP inhibitor resistance, giving us a detailed insight into structure-function relationships in PARP1. Among these, we found mutants that display trapping despite conferring PARPi resistance, suggesting that PARP trapping is not sufficient for cytotoxicity. Mutations that confer resistance are restricted to DNA binding domains and a network of residues in the WGR and helical domains of PARP1 that may be involved in intramolecular activation of PARP1 upon DNA binding and thus affect trapping.
We also established that PARP1 loss can be tolerated in some BRCA1 mutant breast and ovarian tumour cell lines, despite the well-described synthetic lethality of PARP1 and BRCA1 loss. Further investigation indicated that these cell lines that tolerate PARP1 loss of function mutations have exon 11 BRCA1 mutations, and may retain some function due to a splice variant that skips exon 11. siRNA knockdown of remaining BRCA1 function in SUM149 or COV362 cells with PARP1 mutations elicited the expected synthetic lethality. A PARP1 mutation identified in a PARPi resistant patient is localised to the WGR domain cluster identified in our screens and abolishes PARP1 trapping in a microirradiation assay. PARP1 mutation also caused talazoparib resistance in a SUM149 xenograft model. These results raise the possibility that PARP1 loss may be a clinical mechanism of PARP inhibitor resistance. Importantly, BRCA1 mutant cells with PARP1 mutations had distinct drug sensitivities to other known mechanisms of PARPi resistance - for example PARP1 mutation did not result in cross resistance to cisplatin or camptothecin, unlike BRCA1 reversion or 53BP1 loss. This suggests that studying the mechanism of acquired PARPi resistance may be important to inform subsequent therapy choice.
Citation Format: Stephen J. Pettitt, Dragomir B. Krastev, Inger Brandsma, Amy Dréan, Feifei Song, Maria I. Harrell, Rachel Brough, Jessica Frankum, Jung-Min Lee, Elizabeth M. Swisher, Kosuke Yusa, Alan Ashworth, Christopher J. Lord. Genome-wide and focused CRISPR screens to study PARP inhibitor resistance mediated by mutations in PARP1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 410.