A multi-scale analysis of the mutagenicity of cisplatin and its modulation of acquired chemoresistance in high-grade serous ovarian cancer

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PROJECT SUMMARY. High-grade serous ovarian cancer (HGSOC) accounts for up to 80% of ovarian cancer deaths, due to recurrence in over half of patients associated with platinum-based chemoresistance. Intratumor heterogeneity (ITH) is thought to be the main contributor in resistance. ITH permits diverse phenotypic landscapes among cells that may provide certain clones advantages in resisting or tolerating chemotherapy. Cisplatin, routinely used in HGSOC treatment, forms platinum-DNA (Pt-DNA) adducts as its main cytotoxic mechanism. However, Pt-DNA adducts are mutagenic, particularly due to the misincorporation of incorrect nucleotides by DNA polymerases β (polβ) and η (polη) opposite these adducts. Despite evidence that polβ, polη, and the bypass of Pt-DNA adducts play roles in cisplatin resistance, the molecular mechanisms that govern this mutagenic bypass are unclear. In addition, HGSOC is characterized by vast genome instability that causes intratumor genetic diversity and augments the possibilities for the acquisition of resistance. As one of the most mutagenic drugs, cisplatin further increases this genetic diversity and induces unwanted mutations that may elicit the emergence of de novo resistant clones. I hypothesize that polβ and polη execute the mutagenic events of cisplatin, and the mutagenicity of cisplatin modulates the de novo origination of resistant clones that propagate resistance in HGSOC. This proposal seeks to define the molecular mechanisms that enable the mutagenic bypass of Pt-DNA adducts, and in conjunction, determine how the mutagenicity of cisplatin modulates the acquisition of HGSOC resistance during cisplatin treatment. Recent lineage tracing through DNA barcoding technologies have enabled exciting quantitative analyses of subclonal architecture throughout treatment of cancer populations. However, subsequent analyses destroy barcoded cellular samples, limiting further studies. COLBERT enables both lineage tracing and isolation through lineage-specific gene expression of a fluorescent reporter. I will employ COLBERT in an unprecedented longitudinal study of de novo resistance that emerges throughout cisplatin treatment of HGSOC cell line populations. This proposal will perform gene expression and whole genome sequencing analyses of individual de novo resistant lineages throughout treatment to determine key de novo mutations that confer resistance, and understand how resistant lineages came to acquire resistance over time. The 2015 Ovarian Cancer Action meeting described the need for studies that aim to better understand clonal diversity and genome instability contributing to acquired HGSOC resistance. This work will address this by uniquely uniting molecular and systems level approaches to provide characterizations of cisplatin’s mutagenicity. This study will provide a holistic understanding of how cisplatin fails to act solely as a cytotoxic drug and induces undesirable mutagenic events, and how these de novo mutations may originate resistance. This work will provide insights into improving personalized therapies and platinum drug design.