Scientists at the Institute of Cancer Research (ICR) in the U.K. have harnessed CRISPR-Cas9 genome-editing technology to uncover why some tumors are resistant to poly(ADP-ribose) polymerase  inhibitors (PARPi), such as olaparib and talazoparib. The studies, headed by Chris Lord, Ph.D., professor of Cancer Genomics at ICR, found that mutations in the PARP1 gene prevent the enzyme from being trapped by the drugs when it binds to DNA, resulting in resistance to PARPi therapy in vitro and animal models in vivo, as well as in an ovarian cancer patient who had developed resistance to olaparib.

The researchers hope that their results will help clinicians select the most appropriate treatments and drug regimens for patients with breast and ovarian cancer. “PARPi are hugely exciting new drugs which are especially effective in women with BRCA mutations—but unfortunately, as with many other treatments, it is common for cancer cells to eventually develop resistance,” comments co-researcher Stephen Pettitt, Ph.D., staff scientist in cancer genomics at the ICR. “Our study has discovered one of the reasons why resistance to PARPi such as olaparib might occur. Testing for the mutations we have identified could offer even more personalized treatment for women with breast and ovarian cancer, by allowing doctors to judge whether and for how long olaparib should be used.”

The ICR team, together with colleagues in the U.K., U.S., and Bulgaria, report on their findings in Nature Communications, in a paper entitled “Genome-Wide and High-Density CRISPR-Cas9 Screens Identify Point Mutations in PARP1 Causing PARP Inhibitor Resistance.”

The PARP enzyme PARP1 acts as DNA damage sensor, which attaches to and coordinates the repair of single- or double-stranded breaks in DNA. Drugs that target PARP1 and PARP2 cause the death of cancer cells that already have defects in genes, such as the tumor suppressors BRCA1 or BRCA2, because this double deficit in repair mechanisms is effectively lethal to the cells. Studies have shown that as well as blocking the catalytic activity of PARP1, most clinical PARPi cause cytotoxicity by trapping PARP1 where it binds to DNA at the sites of DNA damage.

To try and understand the mechanisms of PARPi toxicity in even greater detail, the ICR researchers developed genome-wide, high-density CRISPR-Cas9 “tag-mutate-enrich” mutagenesis screens. The approach involved generating mutations in specific sections of the PARP1 gene, and tagging the mutated proteins so that the effects on cancer cell response to drug therapy could be tracked. Using this method the team could generate and identify near full-length mutant forms of PARP1 that cause resistance to PARPi both in vitro and in tumor-bearing animal models. Mice with PARP1-mutant tumors were more resistant to therapy with talazoparib, whereas the drug delayed tumor growth and increased survival in control animals.

Specific mutations in the DNA-binding region of the PARP1 gene were found to disrupt the ability of the enzyme protein to bind to DNA, and so prevented PARPi from trapping them at the site of DNA damage. Interestingly, even mutations at sites on the PARP1 gene that are not known to be directly involved with DNA binding caused PARPi resistance, suggesting that they may also prevent PARP1 trapping. Further analyses suggested that these mutations may affect amino acids that are involved in hydrogen-bonding interactions that act to bridge the DNA-binding and catalytic domains of the protein, which could ultimately impact on PARP1 trapping. “Our genetic screens also uncovered several clusters of mutations that suggest that regions of PARP1 outside the DNA-binding domain can influence trapping, observations that are consistent with inter-domain interactions being critical for PARP1 binding and activation,” the authors state.

More surprisingly, some cancer cells with mutant BRCA1 were resistant to PARP1 inhibitors, even when the enzyme couldn't carry out DNA repair. The team's analyses suggested that some residual BRCA1 function may be retained in these cells, which is enough to support cell survival even when PARP1 is mutated.  “Our experiments also showed that PARP1 mutation can be tolerated in certain BRCA1 mutant, PARPi-sensitive tumour cells,” the researchers comment. “This suggests that PARP1 trapping still underlies the increased cytotoxicity of PARPi in these tumour cells but that some residual BRCA1 function allows these cells to tolerate PARP1 mutations.…”

In parallel with their screening studies, the researchers analyzed cells from an ovarian cancer patient who had developed olaparib resistance. They identified a specific mutation in the patient's PARP1 gene that meant the enzyme could still be recruited to sites of DNA damage, but didn’t bind well to the DNA, so wasn’t trapped by the drug. “…we also observed a PARP1 mutation that abolished trapping in a patient with de novo resistance to olaparib, suggesting that such mutations can arise in patients and could potentially contribute to resistance.”

The team says that the finding that PARP1 mutations outside of the DNA-binding domains can still influence PARP1 trapping, and so PARPi resistance “…reinforces the importance of trapped PARP1 as a cytotoxic DNA lesion and suggests that PARP1 mutations are also tolerated in cells with a pathogenic BRCA1 mutation where they result in distinct sensitivities to chemotherapeutic drugs compared to other mechanisms of PARPi resistance.”

They further suggest that their “tag-mutate-enrich” approach could, in principle, be used to generate full-length mutants of any gene associated with a particular disease. “This 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 evolution of cancers into drug-resistant forms is a major challenge we face in getting cancer treatments to work,” states Dr. Lord. “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. 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.

Charles Swanton, Ph.D., Cancer Research UK's chief clinician, adds, “This ambitious study using state-of-the-art molecular technologies shows new ways in which tumors become resistant to PARPi, a family of drugs discovered and developed by Cancer Research UK–funded scientists. Importantly, this resistance may influence the success of future treatment options, so increasing our understanding of how resistance occurs means we may be able to design even better therapies and predict how well a patient may respond to future treatment.”

“Studies like this, which build on the development of PARPi as a brand new treatment option for some women with breast cancer, could help take us a step closer to an even more personalized approach to treating the disease,” noted Baroness Delyth Morgan, chief executive at Breast Cancer Now, which partly funded the study. “It is vital that we understand exactly how and when cancer cells begin to adapt to and resist treatment, so that we can remain one step ahead of often elusive cancer cells.”



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