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Figure S1: Characterisation of CAPAN1-B2S* Figure S2: Characterisation of SUM149-B1S* Figure S3: Sensitivity of ddPCR assay. Figure S4: Olaparib and talazoparib select for secondary mutant tumour cells. Figure S5: DLD1 tumour cells have a fitness advantage over DLD1.BRCA2-/- cells in vitro. Figure S6: Olaparib has little efficacy in mixed CAPAN-1 xenografts. Figure S7: Exome sequencing of CAPAN-1.B2.S* shows retention of TP53 mutations. Figure S8: Exome sequencing of SUM149.B1.S* show retention of TP53 mutation. Figure S9: BRCA-proficient and -deficient cells exhibit sensitivity to additional DNA damaging agents. Figure S10: AZD-1775 causes an active S phase reduction in both CAPAN1 and CAPAN-1.B2.S* cells.

ddPCR probe and primer design

Although PARP inhibitors (PARPi) target homologous recombination defective tumours, drug resistance frequently emerges, often via poorly understood mechanisms. Here, using genome-wide and high-density CRISPR-Cas9 “tag-mutate-enrich” mutagenesis screens, we identify close to full-length mutant forms of PARP1 that cause in vitro and in vivo PARPi resistance. Mutations both within and outside of the PARP1 DNA-binding zinc-finger domains cause PARPi resistance and alter PARP1 trapping, as does a PARP1 mutation found in a clinical case of PARPi resistance. This reinforces the importance of trapped PARP1 as a cytotoxic DNA lesion and suggests that PARP1 intramolecular interactions might influence PARPi-mediated cytotoxicity. 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 (BRCA1 reversion, 53BP1, REV7 (MAD2L2) mutation), suggesting that the underlying mechanism of PARPi resistance that emerges could influence the success of subsequent therapies., The mechanisms of PARP inhibitor (PARPi) resistance are poorly understood. Here the authors employ a CRISPR mutagenesis approach to identify PARP1 mutants causing PARPi resistance and find that PARP1 mutations are tolerated in BRCA1 mutated cells, suggesting alternative resistance mechanisms.

Although PARP inhibitors target BRCA1- or BRCA2-mutant tumor cells, drug resistance is a problem. PARP inhibitor resistance is sometimes associated with the presence of secondary or “revertant” mutations in BRCA1 or BRCA2. Whether secondary mutant tumor cells are selected for in a Darwinian fashion by treatment is unclear. Furthermore, how PARP inhibitor resistance might be therapeutically targeted is also poorly understood. Using CRISPR mutagenesis, we generated isogenic tumor cell models with secondary BRCA1 or BRCA2 mutations. Using these in heterogeneous in vitro culture or in vivo xenograft experiments in which the clonal composition of tumor cell populations in response to therapy was monitored, we established that PARP inhibitor or platinum salt exposure selects for secondary mutant clones in a Darwinian fashion, with the periodicity of PARP inhibitor administration and the pretreatment frequency of secondary mutant tumor cells influencing the eventual clonal composition of the tumor cell population. In xenograft studies, the presence of secondary mutant cells in tumors impaired the therapeutic effect of a clinical PARP inhibitor. However, we found that both PARP inhibitor–sensitive and PARP inhibitor–resistant BRCA2 mutant tumor cells were sensitive to AZD-1775, a WEE1 kinase inhibitor. In mice carrying heterogeneous tumors, AZD-1775 delivered a greater therapeutic benefit than olaparib treatment. This suggests that despite the restoration of some BRCA1 or BRCA2 gene function in “revertant” tumor cells, vulnerabilities still exist that could be therapeutically exploited. Mol Cancer Ther; 16(9); 2022–34. ©2017 AACR.

In 2014, olaparib (Lynparza) became the first PARP (Poly(ADP-ribose) polymerase) inhibitor to be approved for the treatment of cancer. When used as single agents, PARP inhibitors can selectively target tumour cells with BRCA1 or BRCA2 tumour suppressor gene mutations through synthetic lethality. However, PARP inhibition also shows considerable promise when used together with other therapeutic agents. Here, we summarise both the pre-clinical and clinical evidence for the utility of such combinations and discuss the future prospects and challenges for PARP inhibitor combinatorial therapies.

Although PARP inhibitors target BRCA1 or BRCA2 mutant tumour cells, drug resistance is a problem. PARP inhibitor resistance is sometimes associated with the presence of secondary or “revertant” mutations in BRCA1 or BRCA2. Whether secondary mutant tumour cells are selected for in a Darwinian fashion by treatment is unclear. Furthermore, how PARP inhibitor resistance might be therapeutically targeted is also poorly understood. Using CRISPR-mutagenesis, we generated isogenic tumour cell models with secondary BRCA1 or BRCA2 mutations. Using these in heterogeneous in vitro culture or in vivo xenograft experiments where the clonal composition of tumour cell populations in response to therapy was monitored, we established that PARP inhibitor or platinum salt exposure selects for secondary mutant clones in a Darwinian fashion, with the periodicity of PARP inhibitor administration and the pre-treatment frequency of secondary mutant tumour cells influencing the eventual clonal composition of the tumour cell population. In xenograft studies the presence of secondary mutant cells in tumours impaired the therapeutic effect of a clinical PARP inhibitor. However, we found that both PARP inhibitor sensitive and PARP inhibitor resistant BRCA2 mutant tumour cells were sensitive to AZD-1775, a WEE1 kinase inhibitor. In mice carrying heterogeneous tumours, AZD-1775 delivered a greater therapeutic benefit than olaparib treatment. This suggests that despite the restoration of some BRCA1 or BRCA2 gene function in “revertant” tumour cells, vulnerabilities still exist that could be therapeutically exploited.