Supplementary MaterialsSupplementary Figures. carrying heterogeneous tumours, AZD-1775 delivered a greater therapeutic benefit than olaparib treatment. This suggests that despite the restoration of some or gene function in revertant tumour cells, vulnerabilities still exist that could be therapeutically exploited. Introduction Heterozygous germ-line mutations in the or tumour suppressor genes strongly predispose to cancers of the breast, ovary, pancreas, and prostate (1,2). BRCA1 and BRCA2 are involved in homologous recombination (HR), a process used to repair DNA double-strand breaks (DSBs), and other DNA lesions that impair replication forks (3C6). Extensive preclinical and clinical Isotretinoin kinase inhibitor data has established that loss of or function is associated with sensitivity to small molecule PARP inhibitors (7). Recently, the PARP inhibitor (PARPi) Lynparza (olaparib/AZD2281 C KuDOS/AstraZeneca) was approved for the treatment of platinum-responsive, or mutant high-grade serous ovarian carcinomas (HGSOC) (8). Despite a number of profound and sustained anti-tumour responses in patients treated with PARP inhibitors, drug resistance limits the overall effectiveness of these agents (9C12). A number of mechanisms of PARP inhibitor resistance have been identified, including upregulation of PgP drug transporters, loss of 53BP1 Isotretinoin kinase inhibitor or REV7 function, or secondary revertant mutations within the or genes themselves (13,14). These secondary gene mutations restore or open reading frames and encode proteins that have partial function (13C16). In some mutant patients, initial clinical responses to PARPi are seen, followed by the emergence of profoundly PARPi resistant lesions (13). The gradual emergence of PARPi resistance during treatment has led to the hypothesis that PARPi treatment might provide a Darwinian selective pressure effect, where a secondary mutant clone has a selective advantage over non-secondary mutant tumour clones, once PARPi treatment is applied (7,17). Although this hypothesis has not MAPT as yet been tested, if such a Darwinian process did exist, a secondary mutant clone might be expected to gradually dominate a tumour cell population over the course of PARPi therapy. To date, only one approach for targeting tumour cell clones with secondary mutations has been proposed, namely the use of thiopurines (18). The wide application of thiopurines in the treatment of cancer has been limited by safety concerns (18), suggesting that additional therapeutic approaches for targeting secondary mutant tumour cells might also be required. We set out to assess, both and gene mutations are selected for by PARPi treatment in a Darwinian fashion. To do this, we used CRISPR-Cas9 mediated gene targeting in or mutant tumour cells to generate daughter clones with secondary mutations. By mixing these secondary Isotretinoin kinase inhibitor mutant clones with parental tumour cells in co-cultures or tumour xenografts, we established that PARPi treatment can select for secondary mutant clones in a Darwinian fashion. Using these same systems, we also found that exposure to a clinical WEE1 kinase inhibitor (AZD-1775) minimized the selection of secondary mutant tumour cells, targeting parental and secondary mutant cells to a similar extent, whilst having minimal effects on non-tumour cells. Materials and Methods Cell lines CAPAN1 and SUM149 cells were obtained from American Type Tissue Collection. DLD1-or primers described below. PCR products were subcloned using TOPO TA Cloning Kit, with pCR2.1-TOPO (Invitrogen). Sanger sequencing confirmed secondary or mutations from 20 subcloned colony sequences per cell line. gRNA (in pMA-T vector): gRNA contains the mutation Isotretinoin kinase inhibitor specific to the CAPAN1 cell line. Primers for PCR amplification were: Isotretinoin kinase inhibitor co-culture drug exposure assays Cells were plated in a fixed starting ratio of secondary mutant:parental cells in either 24 well or 6 well plates, or T75 flasks and exposed to either olaparib, talazoparib, or AZD-1775 for 14.