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e-Posters

35P - Increased sensitivity to olaparib by BRCA1/2 knockdown using a CRISPR/Cas9-mediated knock-in method in pancreatic cancer cell lines.

Date

06 Oct 2021

Session

e-Posters

Presenters

Andréa Witz

Citation

Annals of Oncology (2021) 32 (suppl_6): S1345-S1371. 10.1016/annonc/annonc740

Authors

A. Witz1, J. Dardare2, M. Husson3, A. Francois4, J. Merlin4, P. Gilson1, A. Harlé1

Author affiliations

  • 1 Université de Lorraine CNRS UMR 7039 CRAN, Service de Biopathologie, Institut de Cancérologie de Lorraine, 54519 - Vandoeuvre les Nancy/FR
  • 2 Université de Lorraine CNRS UMR 7039 CRAN, Service de Biopathologie, Institut de Cancérologie de Lorraine, Vandoeuvre les Nancy/FR
  • 3 Institut de Cancérologie de Lorraine - Alexis Vautrin, 54519 - Vandoeuvre-les-Nancy/FR
  • 4 Institut de Cancérologie de Lorraine - Alexis Vautrin, 54519 - Vandoeuvre les Nancy/FR
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Abstract 35P

Background

Pancreatic cancer is one of the most incurable disease. About 80% of pancreatic adenocarcinoma are locally advanced at diagnosis or metastatic. Therefore, only 20% of patients undergo surgery and the majority have local relapse or metastasis. Chemotherapy regimens are used in adjuvant therapy with gemcitabine or FOLFIRINOX protocol, the standard of care for metastatic pancreatic cancer. Olaparib, a PARP inhibitor, is used as maintenance treatment in patient with metastatic pancreatic adenocarcinoma bearing a germline BRCA1/2 mutation. However, germline BRCA mutations have been described in less than 10% of patients with pancreatic adenocarcinoma. The objective of our study was to determine whether inducing a BRCA1/2 mutation in PDAC cells may restore sensitivity to olaparib.

Methods

We developed a CRISPR/Cas9-mediated knock-in technique to induce deleterious BRCA1 or BRCA2 mutations in pancreatic cancer cell lines. The c.763G>T (p.Glu255Ter) and c.2133C>A (p.Cys711Ter) mutations were selected to obtain truncated and non-functional BRCA1 and BRCA2 proteins respectively. A CRISPR/Cas9 ribonucleoprotein (RNP) was assembled for each mutation and transfected in two PDAC cell lines (T3M4 and Capan-2) and in a breast cancer cell lines (MCF7) as control.

Results

Expected mutations were detected using ddPCR and NGS assays, proving the effectiveness of our CRISPR/Cas9 systems. Allelic frequencies were 90%, 65% and 20% for MCF7, T3M4 and Capan-2 cell-lines respectively. No off-target effects were found in the limit of the sequenced gene-panel. Calculated olaparib IC50 were significantly lower for all cell lines harboring a BRCA1 or a BRCA2 mutations compared to wild type BRCA1/2 cells (p<0.01).

Conclusions

We designed and validated a CRISPR-Cas9 system to induce in vitro deleterious BRCA1/2 genes mutations by knock-in in pancreatic cancer cell-lines and increase their sensitivity to olaparib. This strategy might offer an attractive therapeutic alternative for the management of patients with pancreatic cancer. Further investigations are needed to resolve CRISPR addressing issues in in vivo models and investigate both yield and toxicity.

Legal entity responsible for the study

Institut de Cancérologie de Lorraine.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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