The incidence of oesophageal adenocarcinoma (OAC) has risen rapidly over the last four decades and has a high overall mortality rate that has shown only incremental improvements over the same duration. OAC develops from the precursor intestinal metaplasia of the oesophageal mucosa known as Barrett’s oesophagus. However, limited knowledge of the molecular mechanisms driving disease progression makes effective clinical management of OAC challenging. One of the common genetic events associated with the progression from Barrett’s oesophagus to OAC is loss of the tumour suppressor, SMAD4 (mutated in 13% or loss of function in 34% of OAC cases). Herein, this thesis firstly investigated the effect of SMAD4 inactivation in Barrett’s carcinogenesis. Genetic knockdown or knockout of SMAD4 was sufficient to initiate tumourigenesis of dysplastic Barrett’s oesophagus cell line, CP-B, in vivo, establishing SMAD4 loss as a crucial event driving progression to OAC. Further, low coverage whole genome sequencing (LC-WGS) analysis revealed that tumourigenic SMAD4 knockdown/knockout CP-B cell lines exhibited distinctive and consistent copy number alterations (CNAs) compared to non-tumourigenic SMAD4 wild-type parental cells. Amongst the alterations we observed were loss of chromosome arm 14q, while amplified regions include chromosome arms 6q and 12p, consistent with common CNAs found in patient tumours. This high genomic instability, characterized by structural chromosomal rearrangements within the tumours following SMAD4 loss, implicates SMAD4 as a protector of genome integrity in OAC development and progression. Moreover, initial in vitro data shows that SMAD4 knockout in CP-B cell line, results in differential expression of transforming growth factor-beta (TGF-beta) pathway target genes (such as ACTA2, CRYAB, PTK2B, ATF3 and CDC6) and loss of cell cycle arrest in response to TGF-beta1 cytokine compared to SMAD4 wild-type parental cells. Furthermore, SMAD4 knockout negatively regulated transcript expression of the multifunctional tumour suppressor INK4/ARF locus, demonstrating the novel and complex network of SMAD4 tumour suppressive activity. This thesis also focused on deciphering the functional role of growth factor receptor bound protein 7 (GRB7) amplification and overexpression in OAC and its potential targeting. GRB7 gene is positioned within known 17q12 amplicon, together with HER2 gene encoding for human epidermal growth factor receptor 2 (HER2). GRB7 is an adaptor molecule that mediates networking of multiple cell surface receptors with downstream signalling pathways. GRB7 high expression was found to be associated with worse survival in OAC patient cohort. Further, genetic GRB7 knockdown (siRNA) inhibited cell proliferation and clonogenic survival and induced apoptosis in GRB7 amplified and overexpressing OAC cell lines (OE19 and Eso26), without altering proliferation of the cells with normal GRB7 expression. Furthermore, whilst HER2 amplification and overexpression was also observed in OE19 and Eso26 cells, they were not uniquely sensitive to trastuzumab (HER2 inhibitor), with Eso26 cells exhibiting resistance in vitro. Taken together, initial findings raise the possibility that GRB7 may be a better molecular therapeutic target than HER2 in OAC with the 17q12 amplicon. To address this possibility, OE19 and Eso26 cell line xenograft models with inducible expression of shRNA targeting GRB7 were used. Consistent with in vitro findings, HER2 expression did not predict sensitivity to trastuzumab, with Eso26 xenografts remaining refractory to trastuzumab treatment. Of high importance, mimicking GRB7 inhibition with inducible-shRNA significantly inhibited tumour growth in both OE19 and Eso26 xenografts. Thus, this part of the thesis demonstrates the functional role of GRB7 overexpression as an oncogenic driver independent of HER2. In summary, the identification of functional genetic drivers and a deeper understanding of the mechanisms that underlie tumour progression in Barrett’s carcinogenesis will lead to improved strategies for the clinical management of OAC patients. To this end, SMAD4 loss was sufficient for progression from dysplasia to OAC. Tumours driven by SMAD4 loss exhibit distinctive CNAs consistent with OAC and metastatic potential. In addition, GRB7 high expression predicts poor outcome in patients with OAC and as such, GRB7 represents an oncogenic driver that could be used as a therapeutic target. Crucially, this thesis provides in vitro and in vivo preclinical and molecular biology evidence for the potential therapeutic benefit of targeting GRB7 in cancer.

The Hippo pathway is an important regulator of organ growth during development where it ensures correct scaling of tissues and organs. It is well-conserved in animals and deregulation of the pathway has been found to drive development of a range of cancers. The Hippo pathway was first identified in the fruit fly, Drosophila melanogaster, and has been extensively studied in a number of different tissues. As well as controlling organ growth in Drosophila, the Hippo pathway has been repurposed to control the binary cell fate choice of the R8 photoreceptor cell in the Drosophila eye. The R8 photoreceptor cell is one of eight photoreceptor cell types in the Drosophila eye and is responsible for much of colour vision in Drosophila. There are two main subtypes of R8 cells – around 30% are the 'pale' (p) subtype and express Rhodopsin 5 (Rh5; responsive to blue light); the remaining 70% are the 'yellow' (y) subtype and express Rh6 (responsive to green light). The Hippo pathway acts in a bistable feedback loop to both specify and maintain R8 subtype specification. This bistable feedback loop is composed of the Hippo pathway kinase, Warts, the downstream transcriptional coactivator, Yorkie, and the Pleckstrin-homology domain containing protein, Melted. Yorkie and Melted both promote pR8 cell fate, while Warts represses pR8 cell fate and promotes yR8 cell fate. Although we have begun to study the role of the Hippo pathway in R8 cell fate choices, we still lack a clear understanding how the Hippo pathway functions in these cells. I sought to further our understanding of the Hippo pathway in R8 cells by answering three key questions: (1) Which Hippo pathway proteins control R8 cell fate? (2) What is the subcellular localisation of Hippo pathway components in R8 cells? (3) What are the target genes of the Hippo pathway in each R8 subtype? (1) I used genetic experiments to modulate the expression of Hippo pathway genes and found that Crumbs and Cka promote pR8 cell fate, while the apical spectrin cytoskeleton and the 14-3-3 proteins promote yR8 cell fate. Modulating other Hippo pathway regulators did not obviously alter the ratio of R8 subtypes, suggesting that not all Hippo pathway proteins function in R8 cell fate choices. (2) Using confocal and multiphoton microscopy, I showed that only Warts differed in expression or localisation between R8 subtypes, suggesting that regulation of Warts levels in R8 cells controls the Hippo pathway's role in R8 cell fate. I also showed that the upstream Hippo pathway proteins, Tao, Merlin and alpha-spectrin are relocalised between late pupal and adult photoreceptor cells, hinting that there may be differences in their mechanisms of action between these two stages. (3) Targeted DamID-seq showed that Yorkie and its binding partner Scalloped reside at over a thousand loci in each R8 subtype. Yorkie and Scalloped target genes included known target genes from proliferating Drosophila tissues, as well as genes that are specific to photoreceptor cells. I also investigated the role of a target gene in pR8 cells, ninaB, and found that it promoted pR8 cell fate in male Drosophila retinas. These results provide insight into the role of the Hippo pathway in R8 cells and highlight similarities and differences between the roles of the Hippo pathway in proliferating cells and in R8 cell fate specification.

The appropriate regulation of gene expression programs is essential for normal cellular function. In cancer, mutations derail normal developmental gene expression programs resulting in a malignant epigenetic state. Traditionally, therapies have attempted to kill cancer cells by targeting rapid proliferation or by directly disrupting the mutated protein. More recent therapies, such as BET inhibitors, attempt to directly target the oncogenic epigenetic state. All of these therapeutic approaches are frequently hampered by the emergence of drug resistance. In many cases, resistance is acquired through genetic changes. However, there is increasing evidence that drug resistance can also arise through non-genetic/epigenetic mechanisms. This has major implications for cancer treatment, as the processes that drive genetic and non-genetic resistance are completely different and epigenetic changes, unlike genetic changes, are potentially reversible. Our laboratory previously generated a model of non-genetic resistance to BET inhibitors in acute myeloid leukaemia (AML). Resistance coincided with the acquisition of a less differentiated stem cell like phenotype and was stable upon drug withdrawal. To discover targets that can overcome the resistant epigenetic state, I performed a focused CRISPR-Cas9 screen, which identified the enhancer regulator, LSD1, as key to maintaining the resistance phenotype. Treatment with an LSD1 inhibitor overcame resistance through time-dependent reprogramming of the resistant cells. This epigenetic reprogramming resulted in both differentiation and the formation of new enhancers around critical BET inhibitor target genes. Through functional genomics experiments, I demonstrated that re-sensitization of the resistant cells was driven by the new enhancer formation, rather than differentiation. Mechanistically, the enhancer remodeling is caused by upregulation of Irf8, which together with the pioneer factor, Pu.1, initiates the formation of new enhancers around BET inhibitor target genes, leading to a restored BET inhibitor transcriptional response. Motivated by the poor clinical responses and rapid acquisition of resistance to BET inhibitors in AML, I also sought to develop an assay that could identify new therapeutic targets that may be more effective. Transcription factors (TFs) are ideal targets, however they are difficult to disrupt directly. To circumvent this, I developed an assay that can identify what cofactors (which are often druggable) are required to drive the transcriptional activity of a given TF. This assay works by combining the Gal4 transactivation system with CRISPR-Cas9 screening. Preliminary screens using the transcription factors, VP64 and MYB, were able to identify previously validated cofactors for each TF. VP64 was dependent on MED25 and MYB was dependent on P300. A number of other potential specific cofactors were identified and will be validated in future work. This assay not only provides the potential to identify novel therapeutic targets, but will also provide insight into the poorly characterized interface between TFs and cofactors.

While adoptive cell transfer (ACT) therapy using chimeric antigen receptor (CAR) T cells can be effective in the treatment of haematological B cell malignancies, the treatment of solid tumours has been challenging. Limiting factors such as low levels of CAR T cell activity and poor infiltration into solid tumours, antigen heterogeneity and immunosuppressive microenvironments are playing important roles in solid tumour resistance to CAR T cell therapy. Therefore, a better understanding of these limiting factors is necessary for overcoming these challenges and addressing tumour resistance to immunotherapy. Multiple studies have investigated different strategies to increase the efficacy of CAR T cell therapy in solid tumours including the modification of CAR T cell structure and using a combination of checkpoint inhibitors. Recently a study from our laboratory demonstrated the eradication of established large tumours including E0771-Her2 breast cancer, 24JK-Her2 sarcoma, and MC38-Her2 colon carcinoma using adoptive cell transfer incorporating vaccination (ACTIV) therapy. In ACTIV therapy, tumour-bearing mice were preconditioned with whole-body irradiation and then treated with dual specific CAR T cells and vaccinia virus VV-gp100 in addition to IL-2 administration. The dual specific CAR T cell possessed a CAR specific for Her2 together with a TCR specific for the premelanosome protein, Pmel. Pmel serves as a strong immunogen that is incorporated in a vaccinia virus, VV-gp100, in ACTIV therapy and facilitates dual-specific (CARaMEL) T cell activation, proliferation and infiltration. Despite the significant results of ACTIV therapy in the elimination of the above tumours, we identified a relatively resistant tumour, AT3-Her2 breast cancer tumour. Since an understanding of mechanisms of tumour resistance is essential for potential extension of ACTIV therapy to a broader range of tumours, we used E0771-Her2 and AT3-Her2 tumours as comparative tumour models for studying limitations in effective ACTIV therapy and proposing potential approaches to overcome those limitations. In our study E0771-Her2 and AT3-Her2 tumours were representative of sensitivity and resistance to ACTIV therapy respectively. We identified the relative resistance of AT3-Her2 tumours to CARaMEL T cell cytotoxicity and poor T cell infiltration into tumours as two main limiting factors in effective ACTIV therapy of AT3-Her2 tumours. We used two approaches to address these challenges. Firstly, we used the combination of oncolytic vaccinia virus VV-dd with ACTIV therapy and showed a significant improvement in therapeutic efficacy of ACTIV therapy. In addition, we showed that oncolytic VV-dd can increase apoptosis in AT3-Her2 tumours. Analysis of T cell proliferation and distribution showed a higher T cell infiltration using ACTIV+VV-dd therapy. In another approach to address the relative resistance of AT3-Her2 tumours to CARaMEL T cell killing, we used an IAP antagonist, SMAC-mimetic drug, named AZD5582 in combination with ACTIV therapy. Our results showed a significantly higher level of CARaMEL T cell cytotoxicity using a combination of the CARaMEL T cells with AZD5582 in vitro. In addition, inhibition of AT3-Her2 tumour growth increased remarkably using AZD5582 in combination with ACTIV therapy. Further analysis showed that this therapy could also increase T cell infiltration into AT3-Her2 tumours and induce apoptosis in tumour cells. Therefore, both strategies demonstrated the promising potential for increasing the therapeutic efficacy of ACTIV therapy and its extension to a broader range of solid tumours.

Cancer is the leading cause of disease burden in Australia. Targeted therapy utilises oncogenic mutations in tumours by modulating cancer-promoting proteins and signalling pathways. While these drugs deliver promising initial responses, cancers invariably develop resistance resulting in the recurrence of the cancer. A common theme of resistance mechanisms is the ability of cancer to hijack alternative signalling pathways to evade therapy. The identification and evaluation of these relatively understudied pathways could facilitate the development of novel therapies to overcome resistance development. One such understudied subset of signalling molecules, the WNT-binding RTKs, are at the interface of two protein families frequently dysregulated in cancer – receptor tyrosine kinases (RTKs) and WNTs. RTKs are targets of many drugs used clinically for cancer treatment, often through the inhibition of their kinase activity or their interactions with activating ligands. WNTs are key signalling morphogens in cancer but the complexity of their signalling has made them difficult drug targets. RYK is a WNT-binding RTK pivotal for embryonic development but with unusual biochemical properties that have meant it is understudied in a cancer context. This Thesis sought to uncover a role for RYK in cancer and evaluate its potential as a therapeutic target. This Thesis revealed the overexpression of RYK mRNA in lung squamous cell carcinoma and glioblastoma samples before identifying human tumour cell lines that readily express RYK mRNA and might be reliant on RYK signalling for its tumourigenic nature. Genetic perturbation of RYK through siRNA knockdown and CRISPR/Cas9 inactivation facilitated the discovery of human tumour cells from four different tumour types that were dependent on RYK for their viability. To assess the clinical expression of RYK protein, a novel chicken anti-RYKEC antiserum was generated, validated and utilised to uncover an upregulation of RYK in a subset of human NSCLC tumours. This antiserum also identified RYK expression in tumours from breast cancer patients and found an inverse correlation between RYK expression and the tumour grade of breast cancers. The establishment of a novel bioassay in pre-osteoblast cells dependent on WNT3A/RYK signalling is described. This assay is used to optimise the expression and purification of two RYK/WNT signalling inhibitors and confirm their RYK/WNT-inhibitory activity. One of these inhibitors, a neutralising anti-RYK antibody – RWD1 – then demonstrated an anti-cancer effect by inhibiting the viability of the RYK-dependent human tumour cells in vitro. RWD1 also reduced the growth of A549 NSCLC tumour xenografts, demonstrating the anti-cancer effect of targeting RYK signalling in vivo. RNA-sequencing analysis revealed an upregulation of epithelial to mesenchymal transition (EMT) genes in human tumour cells upon RWD1 treatment, implying RYK signalling supresses EMT. CRISPR/Cas9-mediated inactivation of RYK increased the metastatic potential of A549 tumour xenografts in vivo. This Thesis uncovered a dual role for RYK signalling in a cancer setting. RYK was found to promote the viability and growth of human tumour cells while also suppressing EMT and metastatic growth. The findings of these parallel pro- and anti-tumour functions of RYK signalling, suggest that RYK signalling could be exploited to deliver a therapeutic effect in cancer.

Mammographic screening has led to an increased detection of in situ and invasive breast carcinoma. However, lesions with uncertain malignant potential (B3) are also frequently detected with routine mammographic screening. They are recognised as B3 due to their unpredictable but significant association with malignancy either on subsequent excision (i.e. upgrade) or later development of cancer. Atypical ductal hyperplasia (ADH) has long been thought of as a direct precursor of only low-grade (LG) neoplasia pathway, whereas breast papillary lesions (PL) are thought to be a risk factor for breast carcinoma. However, we have a limited understanding of how breast cancer progresses from these early lesions. Additionally, other B3 lesions are often misdiagnosed as ADH due to the subjectivity of pathologists’ criteria, therefore, unnecessary surgical excision is often recommended. Conversely, 10-15% of ADH patients subsequently develop cancer despite being surgically excised. Currently, there is no biomarker for accurate risk prediction of later cancer or upgrade for any of these B3 lesions. The first chapter of this thesis describes a novel method developed to utilise low-input DNA for low-coverage whole genome sequencing (LCWGS) for copy number (CN) profiling, which enabled us to study more ADH cases than previously possible. The second study showed that ADH can be not only a direct precursor of the LG- breast cancer pathway, but also could progress to any grades of ER+ cancer, including high grade (HG), ER- and ERBB2 amplified cancer. To our knowledge, this multipotent nature of ADH with the inclusion of specific initiating CN events for LG and HG pathways was never shown previously. This study also identified a possible progression biomarker based on seven CN loci for individual risk prediction as well as for upgrade. The third study showed in detail that breast PL could be a direct precursor of any grade of breast carcinoma. No previous study has combined the mutational landscape and CN profile to identify the early clonal expansion for these lesions. It was shown in this study that in the absence of PIK3CA mutation, there were three specific CN aberrations (16q loss/1q gain or 11q loss) suggested as cancer-associated, which could be informative in the clinical setting for accurate risk prediction for both upgrades and later development of cancer. Collectively, this thesis aimed at understanding breast cancer progression and has been able to suggest a revised breast cancer progression model including ADH and PL as precursors of both LG and HG breast cancer pathways. This knowledge could be used in the clinical setting in future for personalising management of the patients diagnosed with these B3 lesions in core biopsies. If validated in independent cohorts these potential biomarkers will be developing into a diagnostic assay, which may help to reduce unnecessary surgeries for patients by identifying the group of patients at “low-risk” for developing cancer.

High-grade serous ovarian cancer (HGSC) is common, with poor prognosis. Limited therapeutic options are available, and the development of new therapies is of high priority. The RNA Polymerase I (Pol I) transcription inhibitor CX-5461 has shown efficacy in both chemotherapy-sensitive and -resistant ovarian cancer through its ability to activate the DNA damage checkpoint. Here, we combine a genome-wide RNAi screening approach with a focussed drug screen to identify potential targets whose inhibition can enhance the efficacy of CX-5461. We demonstrate that CX-5461 combined with knockdown of homologous recombination DNA repair genes shows cooperative cell proliferation inhibition in several HGSC cell lines. We also demonstrate combinatorial efficacy between CX-5461 and topoisomerase 1 (TOP1) depletion or the TOP1 poison Topotecan. The combination induces cell death, cell cycle arrest and senescence even after drug withdrawal. The mechanism of their cooperativity relies on a cell cycle-independent, nucleolar DNA damage response (DDR) associated with topological stress at the ribosomal DNA and is independent of the ability to inhibit PoI I transcription or induce global replication stress. Despite dose-limiting toxicities hampering the broad use of Topotecan in the clinic, combined treatment with CX-5461 and low-dose Topotecan exhibits striking therapeutic efficacy in vivo, thus providing evidence for a novel strategy to treat HGSC.

Oesophageal adenocarcinoma is an aggressive malignancy and is associated with extremely poor rates of survival. Its only known precursor is Barrett’s oesophagus, which is a metaplasia that occurs in the lower oesophagus in response to gastro-oesophageal reflux. Despite a number of recent advances, such as the introduction of neoadjuvant therapy and minimally invasive techniques, there has only been a modest improvement in overall survival over the last few decades. One factor that has contributed to this is the limited availability of well- validated, clinically relevant models for research purposes. Therefore, the aim of this thesis, was to develop novel preclinical models for the study of Barrett’s carcinogenesis. Central to this thesis was the optimisation of the patient-derived tumour xenograft model. This is a model in which immunodeficient mice are used as hosts in order to culture pieces of human tumour. Once established, the model provides a perpetual source of tumour tissue. Unfortunately, they have been difficult to establish in oesophageal adenocarcinoma using conventional techniques. Through the use of a novel intramuscular transplantation technique, the efficiency of the model has been greatly improved. Validation has also confirmed that the majority of derived xenografts are representative of the original patient tumour. Importantly, however, validation also confirmed that a subset had undergone lymphomagenic transformation and were no longer representative of the original tumour. Following on from the success of the intramuscular xenograft technique, the model was subsequently used as a source of tissue for the generation of much needed cell lines. Using small pieces of xenograft tissue, rather than single cell suspensions, a robust technique was established for the generation of cell lines. In doing so, a cell line was established that had both metastatic and non-metastatic clones, making it a valuable tool for cancer research. Finally, the intramuscular transplantation technique was also used to successfully culture both normal human oesophageal tissue and metaplastic tissue. Validation of this model also confirmed that the cultured tissue recapitulated the human disease process. In addition to the development and validation of multiple novel models for the study of Barrett’s carcinogenesis, this thesis also begins to explore how these models can be used to investigate clinically significant aspects of disease biology

Multiple myeloma (MM) is a malignant plasma cell disorder that is incurable with currently available therapy. The disease is genetically heterogeneous, with many recurrently mutated genes only seen in small numbers of patients and multiple clones present in each patient. This has limited potential approaches for designing widely applicable genetically targeted therapies. rDNA transcription is consistently dysregulated in cancer, mediated through both oncogenic and tumour-suppressive pathways. RNA polymerase I (Pol I) transcriptional hyperactivity is observed in many cancers, with this dysregulation shown to provoke a survival checkpoint in haematological tumour cells. With the hypothesis that the therapeutic targeting of Pol I transcription may prove an effective strategy across a variety of malignant settings, our laboratory co-developed CX-5461; a highly selective small molecule Pol I-mediated transcription inhibitor, now in phase 1 clinical trials in relapsed / refractory malignancies. We have previously demonstrated that single-agent treatment with CX-5461 provides a significant survival benefit in murine models of B-cell lymphoma and acute myeloid leukaemia. However, despite this improvement, drug resistance and relapse eventually occur, indicating combination drug therapy is essential for long term disease control and implementation in the clinic. This thesis examines combination drug strategies in MM, centred on the therapeutic inhibition of Pol I transcription of ribosomal genes, with the aim of accelerating the clinical use of CX-5461 for MM. A boutique, high-throughput screen in human myeloma cell lines (HMCLs) of CX-5461 in combination with drugs having known clinical or promising preclinical efficacy in MM revealed that CX-5461 increases anti-proliferative effects when combined with a range of other agents, encompassing various targets. The histone deacetylase inhibitor panobinostat and the proteasome inhibitor (PI) carfilzomib demonstrated the most impressive synergy in vitro, both representing drug classes that are actively used to treat patients with MM. In vivo testing demonstrated that the combination of CX-5461 with panobinostat increases survival compared with the single agents in both the Vκ*MYC murine model of MM and in C57BL-KaLwRij mice transplanted with 5T33 myeloma cells. Prolonged combination dosing in the Vκ*MYC model did not cause haematological toxicity beyond that seen with single agents. Investigating the molecular synergistic response to CX-5461 in combination with panobinostat indicated multiple potential mechanisms of synergy, including down-regulation of MYC and enhancement of the DDR elicited by CX-5461 alone. To extend the translation of CX-5461 and its combination with panobinostat into the clinic for MM, where resistance to front-line PI treatment frequently develops, we investigated the synergistic relationship of CX-5461 with each of these drug classes. In addition to the screen finding that CX-5461 synergised with each of panobinostat and carfilzomib, we showed the triplet was synergistic in vitro beyond the individual combinations. Moreover, modelling clinical PI resistance, we generated a cell line that is resistant to the front-line PI bortezomib, and demonstrated that CX-5461 retains its impressive efficacy in this setting, both in vitro and in vivo, using the 5T33-C57BL6/KaLwRij model. Taken together, the results described in this thesis will advance subsequent clinical trials utilising both CX-5461 and its combination with panobinostat in the treatment of relapsed multiple myeloma.

The genetic causes of the majority of hereditary breast cancer families remain unresolved (BRCAx families) and lack of this information compromises primary and secondary cancer prevention for the affected women and their family members. Despite intensive efforts, no major new breast cancer predisposition genes with equivalent impact have been discovered since the identification of BRCA1 and BRCA2 in the 1990s. Research from our laboratory provided direct demonstration that the remaining hereditary causes of breast cancer are not due to a few BRCA1-like genes (high penetrance and high carrier frequency) but instead by numerous moderate penetrance genes, each accounting for only a small fraction of families. This finding highlighted the need for very large case-control studies as the only way of resolving the missing breast cancer heritability. In this thesis I aimed to carry out such a study. Using existing germline whole exome and targeted sequencing data, 162 candidate genes were selected for validation in ~8,000 unrelated BRCAx cases and cancer-free controls. Combined with the existing targeted sequencing data of ~4,000 cases and controls, this analysis represents one of the largest targeted sequencing studies of its kind. A significant association with breast cancer predisposition was confirmed for known breast cancer genes PALB2, CHEK2 and ATM. Among the 162 candidate genes, nearly twice as many had more LoF variants in the cases than the controls, compared to those with more variants in the controls than the cases, demonstrating a high enrichment for genuine breast cancer predisposition genes. Most novel candidate genes appeared to convey only low to moderate risks, with a total of 35 genes identified that had an OR>2. Despite the very large sample size, the number of carriers of LoF variants for any candidate gene was still small, further demonstrating the extreme genetic heterogeneity of BRCAx families, and that case-control data is still insufficient on its own to claim the discovery of a specific new breast cancer gene. Nevertheless, this large-scale case-control data will be a valuable guide for future validation. Many of the candidate genes such as NTHL1, WRN, BAP1, PARP2 and CDK9 play essential roles in DNA damage repair, consistent with the function of all the established breast cancer genes. An intriguing exception is CTH which is involved in the trans-sulfuration pathway, and if confirmed, would be the first moderate penetrance breast cancer predisposition not involved in DNA repair. This study also re-evaluated some contentious genes and provided strong evidence to reject RINT1 and RECQL as breast cancer predisposition genes. On the other hand, by combining both case-control data with tumour sequencing data, RAD51C was identified as a triple-negative breast cancer predisposition gene. This study has shown that because of the high genetic heterogeneity of BRCAx families, future validation studies will require either an extremely large sample size (>10,000 subjects), and/or the inclusion of approaches providing independent evidence, such as tumour sequencing to identify bi-allelic inactivation and specific mutational signatures.

The immune response is strongly associated with outcome in CRC (stages I-III). Cytotoxic CD8+ T-cells are the most important subset of immune cells positively associated with outcome, in most solid malignancies and especially CRC. However, in the advanced stage of CRC, this is not always the case. Stage-IV CRC metastasises (mCRC) commonly to the liver, which this thesis addresses. The gold standard for treatment of colorectal liver metastasis (CRLM) is surgical liver resection. Indeed, improvements in surgical techniques have greatly improved the 5-year survival of these patients; however, up to 60% of patients still recur following surgical liver resection. Understanding the progression of mCRC in the context of the immune response is the main focus of this thesis. To investigate the immune response at the primary site of CRC, a unique retrospective cohort of de novo or synchronous mCRC patients (n=109) was explored. Included in this cohort were patients that had microsatellite unstable tumours (MSI) (n=12), which in the metastatic setting have a reduced overall survival (OS) and have been found to respond to checkpoint blockade inhibition (CBI). I analysed the primary tumours of these patients in the context of tumour infiltrating lymphocytes (TILs), immune escape mechanisms and oncogenic potential of these primary tumours, where the patients had synchronous metastatic disease. Despite high frequencies of cytotoxic CD8+ T-cells in some tumours, there was no association with OS, indicating the tumour had surpassed immune control. Expression of PD-L1 >1% on tumour cells was independently correlated with OS in multivariate analysis suggesting that tumour cells have the ability to progress in part at least by evading the immune response. To evaluate the immune response at the metastatic site in the liver, a prospective cohort of CRLM patients was recruited (n=11) to examine the immune context in these tumours. Patients undergoing liver resection were included in the study, and freshly isolated lymphocytes from the tumour; normal liver and peripheral blood were analysed by flow cytometry. These tumours were found to have a reduced infiltration of cytotoxic CD8+ T-cells and increased infiltration of CD4+ T-cells, including T-regulatory cells that are known to suppress immune responses. This immune milieu in these tumours alludes to a reduced cytotoxic and increased immunosuppressive environment. To investigate the functional capacity of these immune cells, a novel immune cytotoxic assay was developed. This assay involved co-culturing patient-derived tumouroids with expanded autologous TILs to assess dynamic interaction of function of these cells. TILs expanded from these tumours were able to kill matched tumouroids, further indicating that when removed from the immunosuppressive TME these cells have functional ability to kill tumouroids. To assess if these TILs respond to CBI, addition of anti-PD-1 antibody was included in the co-culture assays where no improvement in killing was observed. Further investigation of other immune cell subsets in CRLM tumours was undertaken to gain an insight into other immune cell populations that may contribute to the tumour microenvironment (TME). One population of unconventional T-cells abundant in the liver are mucosal-associated invariant T-cells (MAIT cells). These cells play a role in bacterial infections and bridge a gap between innate and adaptive immune responses, and their role in tumour immunity is less defined. These cells are of interest in the context of the TME as they have cytotoxic capability and rapid produce cytokine. When assessing MAIT cell presence in CRLM (n=25) by flow cytometry, MAIT cell frequency was reduced in the tumour compared to surrounding normal liver. The phenotype of MAIT cells in the tissue was phenotypically distinct compared to the periphery, with high expression of PD-1 and CD69, both markers of activation. To assess the potential of MAIT cells, peripheral MAIT cells were isolated from healthy donors and co-cultured with patient-derived tumouroids in an unstimulated and stimulated state. MAIT cells in both states were able to kill patient-derived tumouroids. This is the first documentation of MAIT cell killing with patient-derived material. Despite PD-1 expression on these cells, addition of anti-PD-1 antibody did not enhance this killing. Even though at reduced frequency in the tumour, MAIT cells are activated and may contribute to the tumour microenvironment (TME) in CRLM. Patients with advanced-stage CRC have a reduced survival compared to earlier stage CRC patients. In the context of the immune response, it is evident that these tumours have evaded the immune response to progress to metastasis. The work of this thesis highlights that at the primary tumour site of patients with de novo mCRC, despite high frequencies of cytotoxic CD8+ T-cells, there is an inability at controlling tumour progression. This has likely arisen through immune evasive mechanisms. Therefore there should be a focus on improving the immunogenicity of these tumours to again be recognised by the immune cells. Secondly, at the metastatic site, the TME is immunosuppressed and reinvigorating the function of cytotoxic immune cells present may restore improved immune responses. Importantly, understanding the immune biology of these tumours will provide greater guidance to improve potential immune therapies for these patients into the future.

Mutations in the tumour suppressor gene, TP53 occur in more than 50% of human cancers. Mutant p53 proteins not only lose their tumour suppressive capacities, but also gain oncogenic functions, broadly referred to as gain of function (GOF). Cancer cells frequently accumulate mutant p53 and may become addicted to this protein for their survival. During development, mutant p53, like its wild-type counterpart, is inherently labile, however in cancer cells mutant p53 frequently accumulates. In part, this is due to the interrupted auto-regulatory loop with MDM2. However, MDM2 alone cannot explain the stability of mutant p53 in many cancer contexts. We therefore argued that additional factors are responsible for its stability in cancer cells. In order to identify the major players in the regulation of mutant p53, we performed a high-throughput RNAi screen through which we evaluated 18,120 genes for their effects on mutant p53 levels in two different mutant p53 expressing cell lines. Based on network analyses, pathway analyses and extensive literature mining, we selected 37 candidate genes to be validated through p53 immunoblotting. From the validated genes, we have selected one candidate for further studies. The results described in this thesis highlight a previously unknown mode of mutant p53 regulation in cancer cells. Future studies will explore in greater depth the functional consequences of this new interaction with mutant p53. Excitingly, these studies expose new vulnerabilities for therapeutic intervention and these opportunities for targeting aggressive cancers with mutant p53 will also be the focus of ongoing research.