Fig. 3
PARP1 mediates PARPi sensitivity in BRCA1 mutant cell lines. a Cas9-expressing BRCA1 mutant SUM149 cells were transduced with PARP1-ZnF or hsvTK (non-targeting control) lentiviral sgRNA particles, selected in puromycin and exposed to the indicated talazoparib concentrations for 7 days. Representative SRB-stained 24-well plate image is shown. b Western blot showing PARP1 expression in talazoparib-resistant SUM149 clones TR1 and TR2. Par, parental SUM149 cells. TR2 has an in-frame PARP1-p.43_45delMFD mutation. c SUM149 clones TR1 and TR2 are highly talazoparib-resistant. Plot shows survival relative to DMSO-exposed cells after exposure to the indicated concentration of talazoparib for 7 days. Mean of five replicates plotted, error bars show SD. d PARP1 mutant SUM149-TR2 xenografts do not respond to talazoparib treatment, whereas PARP1 wild-type SUM149 xenografts do. Top panels show Kaplan–Meier survival curves, with a tumour volume of 1000 mm3 (after which mice were sacrificed) used as a surrogate end point. Lower panels show volume of individual tumours, normalised according to tumour volume on the day of randomisation (indicated by the arrow). e Pools of PARP1 guides cause talazoparib resistance in the BRCA1 mutant ovarian cancer cell line COV362. Cas9-expressing COV362 cells were transduced with the indicated lentiviral guide pool, selected in puromycin and assayed as in c. f Silencing of residual BRCA1 function, or BRCA2, induces synthetic lethality with PARP1 genetic loss in SUM149 cells. B1.S* is a SUM149 derivative with a secondary BRCA1 mutation that confers PARP inhibitor resistance. SRB-stained 24-well plate image is shown, 7 days after siRNA transfection. g Colony forming assay using SUM149-Cas9 parental cells and the talazoparib-resistant daughter clone, TR2. Cells were transfected as in f, plated on 6-well plates and colonies stained and counted 2 weeks later. siRNA targeting BRCA1 or BRCA2 significantly reduces survival in PARP1 mutant cells (TR2) compared to the parental SUM149 line (t-test). h BRCA1 and BRCA2 silencing is also synthetically lethal in COV362 PARP1 mutant clones A4 (p.119_120delKS) and D1 (p.848delY/YK). Colony formation assay as in g, p values for t-test shown. Mean of three replicates plotted, error bars show SD
