CRISPR/Cas9-Mediated Knockdown Of BRCA1/2 Restores Response To Olaparib In Pancreatic Cancer Cell Lines

Abstract Background: 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 and that did not undergo progression during at least 16 weeks of a prior platinum-based chemotherapy regimen. However, germline BRCA mutation has been described in only 4 to 7% of patients with pancreatic adenocarcinoma. Methods: 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 (RNP) was assembled for each mutation and transfected into two PDAC cell lines (T3M4 and Capan-2) and into a breast cancer cell lines (MCF7) as control. Expected mutations were detected using ddPCR assay. Results: Allelic frequencies of 85% for MCF7, 65% for T3M4 and 20% for Capan-2 were found for both BRCA mutations, proving the transfection efficiency of our CRISPR/Cas9 systems. 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 find that olaparib induces more apoptosis after 72h treatment in BRCA knockdown cells than in wild-type cells. Conclusions: The different CRISPR/Cas9 systems allow the in vitro induction of deleterious BRCA1/2 mutations by knock-in in different pancreatic cancer cell lines and increase their sensitivity to olaparib. This strategy might offer a new insight for the management of patients with pancreatic cancer and open new perspective based on the use of CRISPR/Cas9 strategy in vivo.