Abstract 1400: The function of Brca1 and Brca2 mutations in breast and fallopian tube epithelial cells varies by location

Abstract

Abstract The identification of BRCA1 and BRCA2 mutations has greatly improved risk prediction and prevention strategies for ovarian and breast cancer. However, understanding the variation in cancer risks attributed to different BRCA1 and BRCA2 mutations has proved challenging, because these mutations are functionally recessive at the cellular level. Here, we aimed to identify the functional consequences of different BRCA1 and BRCA2 mutants by creating hundreads of truncating mutations throughout the coding sequence of each gene using a CRISPR/Cas9 screen in precursor cell types for Basal-like Breast Cancer (BLBC) and High-Grade Serous Ovarian Cancer (HGSOC). First, we created partially transformed models of breast and fallopian tube epithelial cells (MCF10A and FT282, respectively) via an overexpression of a hot-spot p53 mutation (R175H). TP53 is frequently mutated in BLBC and HGSOC. Next, we ectopically expressed S. pyogenes Cas9 enabling cells to be amenable to CRISPR/Cas9 screens. We then generated truncating mutations in MCF10Ap53 and FT282p53 cells in two regions of BRCA1 (exon 11 and 16) and two regions of BRCA2 (exon 9 and 13) using CRISPR/Cas9 technology. These regions were selected because they are associated with different risks of ovarian and breast cancer in patients. We also performed knockout of the olfactory receptor gene OR10A4 as a negative control. Modified cells were evaluated for a range of phenotypes including proliferation, anchorage-independent growth, chemoresistance, genomic instability and aneuploidy. MCF10Ap53 and FT282p53 cells engineered to carry BRCA1 or BRCA2 truncating mutations exhibit differences in proliferation, drug sensitivity, DNA damage repair capability and aneuploidy, compared to parental cells and cells carrying the OR10A4 mutation. We also detected phenotypic differences between different BRCA1 and BRCA2 mutants, consistently with the differences in breast and ovarian cancer risks associated with these mutation locations. To functionally study the broad spectrum of BRCA1 and BRCA2 mutants in a single assay, we designed barcoded libraries of lentiviral sgRNA plasmids to generate mutations spanning the entire coding sequences of BRCA1 and BRCA2 genes using CRISPR/Cas9 screen. Each plasmid in these libraries contains two sgRNAs targeting one PAM site and a unique barcode, which allows a robust and unbiased quantification of cells enriched for a given phenotype. In summary, these sgRNA libraries will map the functional differences between approximately 500 mutations spanning BRCA1 and 650 mutations spanning BRCA2 gene. Performing CRISPR/Cas9 screens both in MCF10Ap53 and FT282p53 cells will allow us to relate these functional differences to genetic epidemiological observations demonstrating the different breast and ovarian cancer risks for each gene depending on mutation location. Citation Format: Justyna E. Kanska, Kruttika Dabke, Zachary Schwartz, Norma I. Rodriquez-Malave, Simon Knott, Simon Gayther. The function of Brca1 and Brca2 mutations in breast and fallopian tube epithelial cells varies by location [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 1400.