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PARP Inhibitor Resistance Gene Identified Using CRISPR Screen

NEW YORK (GenomeWeb) – Resistance to PARP inhibitor drugs can arise from mutations affecting the enzyme-coding gene PARP1, according to research from a team based in the UK, Bulgaria, and the US.

As they reported yesterday in Nature Communications, the researchers used CRISPR-Cas9-based mutagenesis — applying the gene editing technology in a so-called "tag-mutate-enrich" strategy — to uncover and characterize alterations prompting resistance to the PARP inhibitor talazoparib. Among them were PARP1 mutations linked to PARP inhibitor resistance in vitro in mouse or human cell lines and in subsequent in vivo analysis of talazoparib-treated, PARP1-mutant mice.

The team also tracked down a PARP1 mutation in an ovarian cancer patient with resistance to the PARP inhibitor olaparib, demonstrating the potential importance of PARP1 resistance mutations in the clinic.

"Studies like this can tell us how and why drug resistance occurs, and give us new ways of predicting the likely response to new-style targeted drugs," senior author Chris Lord, a cancer genomics researcher at the Institute of Cancer Research, said in a statement. "We hope our research will help doctors use the best drug right from the outset, respond quickly to early signs of resistance, and work out the best ways to combine treatments to overcome drug resistance."

Using a set of lentiviral single guide RNAs, the researchers targeted more than 19,100 genes in mouse embryonic stem cells exposed to talazoparib for several days. When treatment-resistant cell colonies did arise, they expanded the colonies and tracked down the CRISPR/Cas9 mutations contributing to resistance using PCR and Sanger sequencing.

From a collection of two-dozen talazoparib-resistant mouse embryonic stem cell clones, the team focused in on resistance-related mutations in PARP1, which turned up in nine of the clones. That led to a series of follow-up experiments — including green fluorescent protein-tagging, CRISPR-Cas9 gene editing, and Ion Torrent PGM sequencing analyses of PARP1 in the HeLa human cell line — to get a clearer look at the types of PARP1 mutations that can confer talazoparib resistance.

"[T]he tagging of genes with C-terminal GFP coding sequences … followed by the targeted mutagenesis of these genes via CRISPR-Cas9 mediated mutagenesis allows, in principle, full-length mutants of any gene of interest associated with a selectable  phenotype to be identified," the authors explained, noting that the same strategy "could be employed in the analysis of other resistance mutations observed in patients being treated with targeted therapies in order to annotate likely drivers and passengers of resistance."

The analyses suggested mutations in the DNA-binding zinc finger domain of the PARP1 gene contribute to cell growth in the presence of the PARP inhibiting drug, for example. But mutations outside this region were also linked to resistance, the researchers reported, including other mutations that affect PARP1 trapping by PARP inhibitors at DNA damage sites.

When the team screened and assessed BRCA1 mutation-containing breast and ovarian cancer cell lines, meanwhile, it identified PARP inhibitor resistance involving PARP1 in these cells as well, despite the synthetic lethality that has been documented for BRCA1 and PARP1 double knockouts. 

Further, the PARP1/BRCA1 double mutants appeared to show distinct drug sensitivity patterns, the authors reported, noting that "knowledge of the mechanism of resistance in patients that relapse could inform the best subsequent treatment."