Abstract A61: Deleterious consequences of BRCA1 or BRCA2 mutations via CRISPR-Cas9 in ancestrally distinct triple-negative breast cancer cell lines

Abstract

Abstract There is a critical need to identify additional viable treatment options for triple negative breast cancer (TNBC) that is more likely to develop in African American (AA) women who have a lower incidence of BCa, in general, but have a higher mortality from all invasive BCa subtypes compared to Caucasian (CA) women. The ground-breaking genomic editing CRISPR-Cas9 technology was used to better understand cancer driving mutations amongst two different genetic backgrounds. Using this tool, potentially deleterious mutations in the BRCA1 or BRCA2 DNA repair genes were introduced in triple negative breast cancer (TNBC) lines from African or Caucasian ancestries. The dependence of the tumor protein p53 (TP53) activity due to the R248Q mutation was also be examined by reverting that mutation to wild-type. As a positive control, we also targeted the “187delAG” mutation in the BRCA1 gene. This mutation is a two base-pair deletion (AG), causing a frameshift mutation resulting in a stop codon and premature truncation leading functional loss of BRCA1. Following successful introduction of this variant in two TNBC cell lines (AA: HCC70 & CA: HCC1143), two other variants identified previously by next generation sequencing of 32 high risk patients (young African American women with breast cancer) were also tested. To introduce the variants, guide RNA (gRNA) sequences were designed using the online CRISPR design tool (http://crispr.mit.edu). Three optimal gRNAs were chosen to limit off-target effects, mismatches close to the PAM site and minimize the distance to the target site. To ensure homologous recombination repair, template donor sequences (100bp) were designed with the target variant as well as a silent mutation site to introduce either an Hpy8I or SsiI cut site. Cells were transfected via Lipofectamine Messenger Max with the donor sequence, mRNA for the Cas9 protein and gRNA from the GeneArt CRISPR T7 Strings system. The most efficient gRNA was chosen for further optimization. Finally, the R248Q TP53 mutation in both cell lines was reverted to wild-type. The introduction of these mutations were tested for the effects on cell survival in response to ionizing radiation and bleomycin to determine the functional consequences. Future studies will introduce variants of unknown significance for functional testing to better understand potential drug targets in precision medicine. Citation Format: Marcus Spears, Luisel Ricks-Santi, John Tyson McDonald. Deleterious consequences of BRCA1 or BRCA2 mutations via CRISPR-Cas9 in ancestrally distinct triple-negative breast cancer cell lines. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr A61.