A Genetic Screen Using the PiggyBac Transposon in Haploid Cells Identifies Parp1 as a Mediator of Olaparib Toxicity

Abstract

Genetic perturbation screens have the potential to dissect a wide range of cellular phenotypes. Such screens have historically been difficult in diploid mammalian cells. The recent derivation of haploid embryonic stem cells provides an opportunity to cause loss of function mutants with a random mutagen in a mammalian cell with a normal genetic background. We describe an approach to genetic screens that exploits the highly active piggyBac transposon in haploid mammalian cells. As an example of haploid transposon (HTP) screening, we apply this approach to identifying determinants of cancer drug toxicity and resistance. In a screen for 6-thioguanine resistance we recovered components of the DNA mismatch repair pathway, a known requirement for toxicity. In a further screen for resistance to the clinical poly(ADP-ribose) polymerase (PARP) inhibitor olaparib we recovered multiple Parp1 mutants. Our results show that olaparib toxicity to normal cells is mediated predominantly via Parp1, and suggest that the clinical side effects of olaparib may be on target. The transposon mutant libraries are stable and can be readily reused to screen other drugs. The screening protocol described has several advantages over other methods such as RNA interference: it is rapid and low cost, and mutations can be easily reverted to establish causality.

Trapping Poly(ADP-Ribose) Polymerase

“…Other data have also confirmed that wild-type cells are more sensitive to PARP inhibitor cytotoxicity than PARP1 2/2 mouse cells (Horton et al, 2005;Kedar et al, 2012). The effects of PARP1 ablation are not cell type specific, as it was observed in chicken cells (Murai et al, 2012(Murai et al, , 2014a, mouse cells (Horton et al, 2005;Kedar et al, 2012;Pettitt et al, 2013), and human cancer cells Murai et al, 2012Murai et al, , 2014aPettitt et al, 2013;Das et al, 2014). Although the PARP1 loss-of-function mutant cell lines also showed resistance to veliparib, this effect was observed only at very high drug concentrations, consistent with the relatively weak trapping effects of veliparib (Murai et al, 2012).…”

“…A study by Pettitt et al (2013) has shown that either complete loss or reduction of PARP1 expression is associated with acquired resistance to PARP inhibitors. Cells with complete loss of PARP1 expression are ∼100-fold more resistant to olaparib and talazoparib than the parental cell line, while reduction of PARP1 expression by siRNA treatment is associated with partial resistances to the PARP inhibitors (Pettitt et al, 2013).…”

“…Cells with complete loss of PARP1 expression are ∼100-fold more resistant to olaparib and talazoparib than the parental cell line, while reduction of PARP1 expression by siRNA treatment is associated with partial resistances to the PARP inhibitors (Pettitt et al, 2013). Similarly, Singh et al (2013) reported that a common characteristic in several Ewing's sarcoma cell lines that have developed resistance to talazoparib (500-to 1000-foldhigher IC 50 as compared with the parental cell lines) is the loss or reduction of PARP1 expression.…”

“…Depletion of PARP renders cells resistant to both talazoparib and olaparib (Murai et al, 2012(Murai et al, , 2014a. Likewise, cells with PARP1 loss-of-function mutations are 100-fold more resistant to olaparib and talazoparib than wild-type cells, and restoring PARP1 expression causes these cells to revert to wildtype sensitivity (Pettitt et al, 2013). These data indicate that PARP1 protein is the critical mediator of the cytotoxic effect of PARP inhibitors, arguing against the suggestion that these differences in cytotoxicity result from off-target activities.…”

Blueberries inhibit cyclooxygenase-1 and cyclooxygenase-2 activity in human epithelial ovarian cancer

“…However, the majority of medicines used exhibit unwanted cell toxicity (14)(15)(16)(17). Chemotherapy can cause different side effects, such as nausea and vomiting, distress, sexual dysfunction, fatigue and memory loss (18).…”