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Exploiting DNA repair defects in high-grade serous ovarian carcinoma
Authors
Ksenija Nesic
Publication date
1 January 2020
Publisher
Abstract
© 2020 Ksenija NesicHigh-Grade Serous Ovarian Carcinoma (HGSOC) is the most common subtype of ovarian cancer, and is the leading cause of ovarian cancer death. This subtype is molecularly characterised by frequent loss of Homologous Recombination (HR) DNA repair, making it susceptible to Poly-ADP ribose polymerase (PARP) inhibitor treatment. Despite the efficacy of PARP inhibitors (PARPi) in treating HGSOC, disease recurrence is common. A poor understanding of PARPi toxicity and resistance mechanisms has limited improvements in overall survival of patients. This project utilised HGSOC Patient-Derived Xenograft (PDX) and in vitro models to determine underlying mechanisms of PARPi sensitivity and resistance. Characterisation of the HGSOC PDX cohort led to the discovery of 2 models with HR gene secondary mutations, thereby explaining the poor PARPi responses of these tumours. Loss of the Classical Non-Homologous End-Joining (C-NHEJ) DNA repair pathway was also investigated but was not supported as a mechanism of resistance in PDX, or in a genome-wide PARPi-resistance CRISPR screen using a BRCA2-mutant cell line. The CRISPR screen revealed PARP1 mutations as the top hit, but no C-NHEJ mutation hits. CRISPR screens have also been optimised for a BRCA1-mutant cell line and a unique BRCA1 methylated cell line, to enable future validation in different HRD contexts. HR gene methylation analysis in the HGSOC PDX models revealed distinct BRCA1 promoter methylation profiles, subsequently found to represent “homozygous” and “heterozygous” methylation states. Our group demonstrated that all copies of BRCA1 must be methylated for gene silencing and PARPi response, and that methylation can be lost under treatment pressure in patients. I then investigated this mechanism in two HGSOC PDX models with RAD51C promoter methylation. RAD51C promoter methylation was found to have homogeneous or heterogeneous patterns, and both caused gene silencing and PARPi response in the PDX, that could be lost under treatment pressure. One third of patient samples tested appear to have a heterogeneous RAD51C methylation profile and, like the PDX, some responded to chemotherapy. To assess whether global methylation profiles influenced HR gene methylation stability, I carried out genome-wide methylation array analysis of PDX models and clinical samples. Analysis of cyclically PARPi re-treated RAD51C methylated tumours from one PDX model demonstrated increasing global losses in methylation with each treatment cycle. Through characterisation of the HGSOC PDX cohort and in vitro CRISPR screens, I have uncovered both well-established mechanisms of resistance (e.g. secondary mutations), and less studied mechanisms of sensitivity and resistance, such as loss of RAD51C methylation and PARP1 loss. These models provide a platform for further study of these PARPi resistance mechanisms, including potential therapeutics to prevent or overcome their development in the clinic
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Last time updated on 03/09/2020