Modeling Chromosome Instability in High-Grade Serous Ovarian Cancer: Minor Instability Permitted by TP53 Loss Is Exacerbated by BRCA1 Mutation

Daniel Bronder, Darawalee Wangsa, Dali Zong, Thomas J. Meyer, Irena Bockaj, René Wardenaar, Paul Minshall, Daniela Hirsch, Kerstin Heselmeyer-Haddad, Anthony Tighe, Louisa Nelson, Diana Spierings, Joanne McGrail, Maggie Cam, André Nussenzweig, Floris Foijer, Thomas Ried, Stephen S. Taylor

High-grade serous ovarian cancer (HGSOC) originates in the fallopian tube and is characterized by ubiquitous TP53 mutations, high levels of chromosome instability (CIN) and resulting aneuploidy. CIN can be driven by various mechanisms, e.g., mitotic chromosome mis-segregation and replication stress. However, the underlying genetic lesions that drive CIN in HGSOC are unknown, in particular mutations in mitotic and DNA replication genes are rare. To determine if known genetic drivers of HGSOC, such as mutations in TP53 and BRCA1 and the overexpression of MYC, are sufficient to induce CIN, we focused on the human, fallopian-tube-derived, non-transformed, hTERT-immortalized, p53-proficient, FNE1 cell line, as a model system. In a bottom-up approach, we sequentially mutagenized the tumor suppressors TP53 (P) and BRCA1 (B), using CRISPR/Cas9-mediated gene editing, and subsequently overexpressed the oncogene MYC (M), establishing single- (P), double- (PB and PM) and triple- mutant (PBM) FNE1-derived subclones. Transcriptomics showed positive enrichment of mitotic spindle, G2/M checkpoint and E2F target genes in P cells, suggesting that p53 loss alone induces profound cell cycle deregulation. Unsupervised hierarchical clustering based on gene expression of 479 cell cycle regulators, from these three gene sets, resulted in both separation of wildtype from mutant cells, as well as separation of the individual mutant subclones. Concurrently, the mutants exhibited both upregulation and downregulation of genes versus wildtype. Shallow-depth whole-genome sequencing of single cells showed aneuploidy in most mutants at varying degrees. In two of three PB double-mutant subclones the emergence of tetraploidy was observed. Thus, we show that HGSOC-characterizing genetic aberrations are sufficient to cause aneuploidy in a fallopian tube derived model system.

PI: Thomas Ried