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Cells respond to the induction of DNA damage with activation of the DNA damage response (DDR), a complex signalling network which orchestrates cell cycle arrest and DNA repair in order to maintain genomic stability and cell viability. Activation of these signalling pathways enables cancer cells to survive DNA damaging chemo- or radiotherapy and contributes to the development of therapy resistance. Therefore, components of the DDR have become attractive targets for chemo- or radiosensitisation. Furthermore, cancer cells frequently exhibit defects in certain DDR components and may, as a consequence, become highly dependent on remaining DDR pathways to survive DNA damage. Two apical mediators of the DDR are the serine/threonine protein kinases ATM and ATR. ATM is frequently mutated in non-small cell lung cancer (NSCLC), and defects in ATM may render the tumour cells dependent on ATR signalling for survival. In this study, we characterised the functional consequences of ATM mutations in NSCLC cell lines and established an immunohistochemistry-based assay to identify patients with loss of ATM expression. As a single agent, pharmacological ATR inhibition (ATRi) was selectively cytotoxic for cells deficient in both ATM and p53. Furthermore, ATRi in combination with either ATM or PARP inhibition selectively killed tumour cells with mutant p53. We show that following ATR inhibition, ATM and p53 perform critical cell cycle checkpoint functions, independently of each other. Our results suggest that while retained function in any of these pathways is sufficient to maintain cell viability, functional loss of ATM, ATR and p53 results in premature mitotic entry, chromosome fragmentation and mitotic catastrophe. We conclude that in NSCLC the functional status of both ATM and p53 determines the cellular response to ATR inhibition, and propose that a combination of ATR inhibition with ATM or PARP inhibition may have broad utility for the treatment of p53-mutated NSCLC.

Defects within the DNA damage response (DDR) pathways are common in human malignancies. This is especially true in high-grade serous ovarian cancer (HGSOC) where defects within the Homologous Recombination (HR) pathway may be present in up to 50% of tumours. Oncolytic adenovirus is a potential novel therapy for human malignancies. These viruses infect malignant cells and multiply selectively within them causing cell death and release of mature virions. Here, I have investigated the role of the DDR in determining the efficacy of the E1A-CR2 deleted adenovirus type 5 (Ad5) vector, dl922-947, in ovarian cancer. I show that infection with dl922-947 stimulates a robust DDR within the host cell, which the virus manipulates in order to ensure optimal viral replication. In a panel of HGSOC cell lines, the extent of overreplication of genomic DNA and the degree of genomic damage following infection with dl922-947 was shown to correlate closely with viral efficacy. Functional HR, however, promoted viral DNA replication and augmented overall anti-cancer efficacy. Mechanistically, both BRCA2 and RAD51 localised to viral replication centres within the infected cell nucleus. RAD51 co-localisation was also demonstrated in cells with defective HR and occurred independently of BRCA2. In addition, a direct interaction was identified between RAD51 and adenovirus E2 DNA binding protein. Using functional assays of HR competence, I show that Ad5 infection does not alter cellular ability to repair DNA double-strand break damage via HR. These data suggest that oncolytic adenoviral therapy may be most clinically relevant in tumours with intact HR function. Using a high-throughput siRNA DNA repair screen, potential novel targets have been identified that can increase the efficacy of dl922-947 (for example: NONO) and also result in increased resistance (RPA). These results highlight the complex interplay between adenovirus and host cell. Further understanding of these pathways is vital to increase efficacy, develop biomarkers and improve patient selection into clinical trials for these therapies.

Thesis (Ph.D.)--Flinders University, School of Medicine, Dept. of Gastroenterology. / Typescript bound. Includes bibliographical references: (leaves 215-233) Also available in an electronic version via the Web.