Abstract 1734: Crispr/Cas9 screening of Brca1 and Brca2 susceptibility genes in breast and ovarian cancer precursor cells can identify phenotypically different mutants with variable penetrance

Abstract

Abstract BRCA1 and BRCA2 mutations are the most significant risk factors for breast and ovarian cancer identified so far. A recent analysis of breast/ovarian cancer risks in approx. 12,000 BRCA1 carriers and 7,000 BRCA2 carriers found that PTVs (protein truncating variants) in the central region of both genes are more associated with ovarian cancer development (Ovarian Cancer Cluster Regions (OCCRs)), while PTVs in the 5’ and 3’ regions are more associated with breast cancer development (Breast Cancer Cluster Regions (BCCRs)). We aimed to identify the functional consequences of different BRCA1/2 mutants by creating various PTVs in BCCR and OCCR regions of both genes in precursor cell types for Basal-like Breast Cancer (BLBC) and High-Grade Serous Ovarian Cancer (HGSOC). To this end, we created partially transformed models of breast and fallopian tube epithelial cells (MCF12A and FT282, respectively) via an overexpression of a hot-spot p53 mutation (R175H), frequently observed in BLBC and HGSOC. We then generated PTVs in MCF12Ap53R175H and FT282p53R175H cells in two regions of BRCA1 (exon 11 (OCCR) and 16 (BCCR)) and two regions of BRCA2 (exon 9 (BCCR) and 13 (OCCR)) using CRISPR/Cas9 system. We targeted the olfactory receptor OR10A4 as a negative control. We clonally derived cell lines and genotyped PTVs via molecular cloning and Sanger sequencing. We established models of MCF12Ap53R175H and FT282p53R175H cells carrying PTVs for almost all regions. Mostly heterozygous PTVs survived; except of BRCA1 BCCR PTVs, which were lethal in FT282p53R175H, while MCF12Ap53R175H cells survived homozygous PTVs in this region. Typically, all PTVs led to diminished growth, although FT282p53R175H with BRCA2 OCCR PTVs showed similar proliferation rate as control. Heterozygous BRCA1/2 mutations were sufficient to induce nonsense mediated decay of BRCA1/2 mRNA transcripts. BRCA1 OCCR PTVs also specifically increased processing of delta11q splice isoform, known to decrease PARP inhibitor and Cisplatin sensitivity in BRCA1 mutant cell lines. Consistently, derived BRCA1/2 mutant clones showed increased Cisplatin resistance. Immunofluorescence and flow cytometry confirmed decreased BRCA1 protein levels upon introduction of BRCA1 PTVs. Our studies demonstrate that different BRCA1/2 mutations have different phenotypic effects in breast and ovarian cancer precursor cells. We found that BRCA1 and BRCA2 BCCR PTVs in FT282p53R175H had more severe effect on survival and proliferation than OCCR PTVs. This suggests that OCCR PTVs may have survival advantage in FT282p53R175H cells, compared to BCCR PTVs. Next, we will perform high-throughput CRISPR/Cas9 screen of hundreds of BRCA1 and BRCA2 PTVs to functionally validate the genetic epidemiology data indicating the different disease risks for different germline mutations in these susceptibility genes. Citation Format: Justyna Kanska, Kruttika Dabke, Zachary Schwartz, Norma Rodriquez-Malave, Nikoo Safi, Simon Gayther. Crispr/Cas9 screening of Brca1 and Brca2 susceptibility genes in breast and ovarian cancer precursor cells can identify phenotypically different mutants with variable penetrance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1734.