Influence of targeted knockout of the BRCA1 gene on the pharmacologic profile of the mouse breast cancer cell line EMT6 in vitro and in vivo

Around 10% of breast cancer cases are attributed to genetic disorders like mutations in BRCA-1/2 genes. Targeted therapy of BRCA-deficient cancers has been achieved using poly(ADP-ribose) polymerase (PARP) inhibitors, which block BRCA-independent DNA repair. To study the effect of this common mutation on sensitivity towards innovative therapies in more detail, we knocked out the BRCA1 gene in the murine EMT6 breast cancer cell line. Subsequently, we analyzed the sensitivity towards PARP as well as checkpoint (CP) inhibitors in the mutated as well as the parental line in vitro and in vivo. The EMT6/BRCA1 -/- cell line was created by a CRISPR/CAS9 based removal of Exon2 of the BRCA1 gene. The homozygous clone of the modified cell line was used for drug testing in comparison to the wildtype (wt) cell line. PARP inhibitors Niraparib, Rucaparib, Talazoparib and Olaparib were investigated in EMT6/BRCA1 wt as well as EMT6/BRCA1 -/- in vitro in 2D as well as 3D cell culture using different cell concentrations. The latter showed higher sensitivity towards Talazoparib and Olaparib in 2D assays: The respective IC 50 values were 2-4 times lower in the EMT6/BRCA1 -/- as compared to EMT6/BRCA1 wt . In 3D assays Niraparib, Rucaparib and Talazoparib showed pronounced activity in the EMT6/BRCA1 -/- : IC 50 values depicted a 2-3fold difference between the two investigated lines. To further characterize the two lines, tumor growth and sensitivity towards PARP as well as CP inhibitors was determined in vivo. Tumor growth behavior of subcutaneously implanted breast cancer cells was similar in both lines: mean doubling times were 1.9 (± 0.38) days for EMT6/BRCA1 -/- and 1.62 (± 0.37) days for EMT6/BRCA1 wt . Both lines showed a distinct sensitivity profile towards CP inhibitors: anti-CTLA4 was more active in EMT6/BRCA1 -/- : optimal T/C (test/control) value of 22% (KO line) was accompanied by 34.9% in the parental line. In contrast, anti-PD1 treatment led to optimal T/C values of 30.9% in the wt line and 54.8% in the KO line. The evaluation of Niraparib as well as Olaparib in mono-and combined therapy with anti-CTLA4 and anti-PD1 in vivo is currently under investigation. Weekly cytokine analysis in the serum of tumor bearing mice will elucidate possible interactions between PARP and CP inhibitors and give guidance to optimal combination and schedules. We expect that further evaluation of PARP inhibitors in combination with different immune modulatory interventions in our mouse breast cancer model will increase the clinical utility of this strategy for the treatment of patients with BRCA1 mutated cancers. In general, the possibility to compare a mutated vs a parental line in vivo will help to identify novel promising combination as well as patient stratification strategies for immuno-oncology. Citation Format: Anya Avrutskaya, Cordula Tschuch, William Durham, Gerhard Kelter, Astrid Jensen, Armin Maier, Aidan Synnott, Anne-Marie Zuurmond, Julia Schuler. Influence of targeted knockout of the BRCA1 gene on the pharmacologic profile of the mouse breast cancer cell line EMT6 in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3101.