CRISPR/Cas9-mediated knock-in of BRCA1/2 mutations restores response to olaparib in pancreatic cancer cell lines

Abstract

Pancreatic cancer is one of the most aggressive diseases with a very poor outcome. Olaparib, a PARP inhibitor, as maintenance therapy showed benefits in patients with metastatic pancreatic adenocarcinoma bearing germline BRCA1/2 mutations. However, germline BRCA mutation has been described in only 4–7% of patients with pancreatic adenocarcinoma. A CRISPR/Cas9-mediated system was used to knock-in the c.763G > T p.(Glu255*) and c.2133C > A p.(Cys711*) mutations in cell lines to obtain truncated BRCA1 and BRCA2 proteins, respectively. A CRISPR/Cas9 ribonucleoprotein complex was assembled for each mutation and transfected into two pancreatic cell lines (T3M4 and Capan-2) and into a breast cancer cell lines (MCF7) as control. BRCA protein levels were significantly decreased in all BRCA-depleted cells (P < 0.05), proving the transfection efficiency of our CRISPR/Cas9 systems. As expected, the calculated olaparib IC50 were significantly reduced for all cell lines harbored BRCA1 or BRCA2 mutations compared to wild-type BRCA1/2 cells (P < 0.01). Furthermore, we observed a higher induction of apoptosis after 72 h olaparib treatment in BRCA-depleted cells than in wild-type cells. This strategy might offer new insights into the management of patients with pancreatic cancer and open up new perspectives based on the in vivo use of CRISPR/Cas9 strategy.