Abstract A36: A transposon-based genetic screen in haploid mouse embryonic stem cells identifies Parp1 as a major mediator of olaparib toxicity

Abstract

Abstract Haploid embryonic stem cells have recently been isolated from activated mouse oocytes. Due to the ease of random gene disruption in haploid cells, these have great promise for forward genetic screens. We have used the piggyBac transposon, a highly active insertional mutagen, to generate stable libraries of mutants in these cells. These libraries comprise at least 200,000 mutants. We have exposed these libraries to several drugs to generate resistant clones. Mapping the transposon integration sites in these clones can identify genes required for toxicity of the drugs to normal cells. In a proof of principle screen we isolated clones resistant to 6-thioguanine that had insertions in components of the DNA mismatch repair pathway, which is known to be required for toxicity of this purine analogue. In a screen for mutants resistant to the poly-(ADP-ribose) polymerase (PARP) 1/2 inhibitor olaparib, we identified two mutants with different insertions in Parp1 that were more than 100-fold resistant to olaparib than wild type cells. These mutants lacked Parp1 protein expression and radiation-induced poly-(ADP-ribose) formation and were also resistant to other PARP inhibitors. Removal of the transposon by re-expressing transposase in the cells resulted in reversion of the phenotype. Knockdown of PARP1 by siRNA in human cell lines also led to olaparib resistance. The finding that PARP1 itself is required for toxicity of olaparib supports the hypothesis that PARP1 is a component of the toxic lesion. There were several other resistant mutants isolated in the screen, but no other genes had multiple resistant clones with insertions. Removal of the transposon did not revert the phenotype in these clones, suggesting that they arose from background mutations in cell culture. Interestingly many of these clones also lacked Parp1 protein expression, suggesting that inactivation of Parp1 may also be the mechanism of resistance. Therefore Parp1 may be the major determinant of olaparib toxicity to wild type cells. Conditional gene targeting technology is well-established in diploid embryonic stem cells. By combining targeted mutation in a cancer gene with random transposon mutagenesis, it may also be possible to use this screening system to identify synthetic lethal interactions. We have successfully used gene targeting to modify these cells (at the Hprt locus) and are developing strategies to monitor the loss of mutants from a pooled culture by high throughput sequencing of transposon integration sites. Citation Format: Stephen J. Pettitt, Farah L. Rehman, Ilirjana Bajrami, Helen Pemberton, Rachel Brough, Iwanka Kozarewa, Christopher J. Lord, Alan Ashworth. A transposon-based genetic screen in haploid mouse embryonic stem cells identifies Parp1 as a major mediator of olaparib toxicity. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A36.