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Title: Identifying and overcoming mechanisms of resistance to Wee1 kinase inhibitor (AZD1775) in high grade serous ovarian cancer
Author: Gomez, Miriam Kathleen
ISNI:       0000 0004 8509 1430
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2019
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High grade serous ovarian cancer (HGSOC) comprises 75-80% of all ovarian cancer cases and is characterised by p53 mutation and genetic heterogeneity. There are two major molecular categories: homologous recombination repair (HR) pathway proficient and deficient. Most cases are treated with a combination of surgery and chemotherapy. Patients with HR deficient cancers respond well initially, but the majority experience relapse after first line chemotherapy due to drug resistance. As p53 is mutated in virtually all HGSOC cases, these cancers rely heavily on the G2/M checkpoint for DNA damage repair and survival. The G2/M checkpoint is controlled by the phosphorylation status of Cdk1. Wee1 kinase responds to DNA damage by phosphorylating Cdk1 and causing G2 arrest, giving time for DNA repair before entry into mitosis. We and others have hypothesised that combining a DNA damaging agent with Wee1 kinase inhibition would be particularly effective in HGSOC. AZD1775 is a small molecule inhibitor of Wee1 kinase that forces cells into mitosis without the repair of damaged DNA. The overall aim of the thesis was to characterise mechanisms of resistance to AZD1775 in cultured HGSOC cells and identify ways to overcome resistance that could be ready to deploy as the inhibitor moved into the clinic. It was hypothesised that resistance to AZD1775 could occur in a number of different ways through changes in key DNA damage response and cell cycle control pathways. To address the additional hypothesis that mechanisms of resistance to AZD1775 could be different in HR-deficient and proficient HGSOC, cell lines from each category were used to generate resistant clones for mechanistic studies. Given the increased use of the PARP inhibitor olaparib as a therapy for HGSOC, olaparib resistant clones were also isolated from the same HR-deficient and proficient HGSOC cell lines with the supplementary aim of investigating the effectiveness of AZD1775 therapy in olaparib resistant HGSOC. I first investigated the effect of AZD1775 as a single agent and in combination with the DNA damaging agent cisplatin and olaparib in a panel of twelve HGSOC cell lines in cell growth and viability assays. AZD1775 enhanced the sensitivity to cisplatin and olaparib in three cell lines from the panel, two of which were HR pathway deficient. To study mechanisms of resistance to Wee1 inhibition, two cell lines, ES-2 and OVCAR8, were chosen representing HR proficient and deficient HGSOC to isolate clones resistant to AZD1775, AZD1775 in combination with cisplatin and olaparib alone. Compared to the parental lines, AZD1775 resistant clones retained normal cell cycle profiles in the presence of AZD1775. No evidence was found for mutation in the coding exons of the Wee1 kinase gene in resistant clones. Western blot analysis revealed reduced Cdk1 expression in many of the resistant clones, while others showed increased Wee1 kinase expression. RNA sequencing based comparison of AZD1775 resistant clones with the parental ES-2 cell line showed an upregulation of TGFβ signalling that fed into the cell cycle control pathway via multiple cell cycle inhibitors. An alternative potential mechanism of resistance in some ES-2 and OVCAR8 clones involved upregulation of the TNF and NF-κB signalling pathway leading to evasion of apoptosis. An attempt to overcome AZD1775 resistance by blocking TGFβ or TNFNF- κB signalling pathways was successful for one resistant ES-2 clone using a TGFβR1 inhibitor. Western blotting of olaparib resistant clones isolated from ES-2 and OVCAR8 showed known changes responsible for olaparib resistance, including reduced expression of 53BP1 and down regulation of Poly(ADP-ribose)glycohydrolase. All but one of the olaparib resistant clones tested remained sensitive to AZD1775. In conclusion, mechanisms of resistance to two important inhibitors for the improved treatment of HGSOC have been identified and ways to overcome resistance in the clinic have been suggested.
Supervisor: Melton, David ; Gourley, Charlie Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: ovarian cancer ; HGSOC ; drug resistance ; p53 ; Wee1 kinase ; AZD1775 ; olaparib ; Wee1 kinase inhibition ; AZD1775 resistant clones