The advancement of cancer immunotherapy has revolutionized cancer treatment. In particular, the success of immune checkpoint blockade, including anti-PD-1/L1 and anti-CTLA-4 in the clinic has changed guidelines for treating multiple solid tumours such as melanoma, non-small-cell-lung cancer (NSCLC), renal cell carcinoma and head and neck squamous cell cancers. Adoptive T cell therapy against haematological malignancies has also achieved remarkable success. Two anti-CD19 CAR T cell products have been approved by Food and Drug Administration (FDA) for the treatment of B cell Acute Lymphoblastic Leukaemia (ALL) and non-Hodgkin lymphoma. However, the efficacy of adoptive CAR T cell therapy against solid tumours has been less impressive. Even though numerous preclinical or clinical trials have been conducted to identify novel neoantigens or to improve CAR T cell anti-tumour efficacy against solid tumours, all attempts have so far not matched the extraordinary clinical responses observed in patients with haematological malignancies. There is an urgent need to 1) understand CAR signalling in T cells, 2) identify the key mechanisms intracellularly and extracellularly limiting CAR T cell function, and 3) to develop strategies to overcome the immunosuppressive tumour microenvironment to improve the efficacy of CAR T cells in the context of solid tumours. Recent studies have highlighted the potential of targeting negative regulators of T cell signalling to enhance the efficacy and extend the utility of CAR T cells to solid tumours. Protein tyrosine phosphatases (PTPs) including PTPN2 and PTPN22 are key regulators of T cell signalling and contribute to the maintenance of immune tolerance. PTPN22 is known as a critical autoimmunity-associated PTP that suppresses T cell receptor (TCR) signalling by dephosphorylating substrates such as the Src family kinase LCK and the Syk family kinase ZAP70 in effector/memory T cells. Loss of function SNPs in PTPN2 have also been associated with the development of autoimmunity. Studies from our group have shown that PTPN2 plays pivotal role in negatively regulating T cell receptor (TCR) signalling by dephosphorylating and inactivating the Src family protein tyrosine kinase LCK to limit T cell proliferation and function. PTPN2 also attenuates cytokine signalling by dephosphorylating JAK-1, JAK-3 and their target substrates STAT-1, -3 and -5 in a cell context-dependent manner. Beyond PTPN22 and PTPN2, early studies established that PTP1B could directly dephosphorylate and inactivate JAK-2 and Tyk-2 to attenuate cytokine-induced JAK/STAT signalling. There is also evidence that PTP1B can dephosphorylate STAT family members. In this thesis, we found that basal STAT-5 Y694 phosphorylation and IL-2/IL-7/IL-15 induced STAT-5 Y694 phosphorylation were enhanced by the deletion of PTP1B in T cells. Since CARs signal via LCK, and cytokines can affect CAR T cell function, differentiation and memory, we postulated that inhibiting PTPN2, PTP1B and PTPN22 individually or combinatorically might bolster the anti-tumour activity of CAR T cells. Work in this thesis characterized the role of PTPN22, PTPN2 and PTP1B in adoptive T cell therapy and demonstrated that the deletion or inhibition of PTPN2 or PTP1B resulted in enhanced T cell signalling and promoted CAR T cell anti-tumour efficacy in vitro and in vivo. In addition, we developed a CRISPR/Cas9 RNP gene editing based method to target PTPs in CAR T cells for translational purposes.

Multiple myeloma (MM) is a haematological malignancy of plasma cells with disease present in the bone marrow. Despite the success of new therapies in extending life expectancy, MM is still considered incurable and requires further investigation into new treatments. In the context of refractory/relapsed MM (RRMM), the combination of the two immunotherapies Elotuzumab (Elo, anti-SLAMF7 mAb) and Lenalidomide (Len, IMiD) has resulted in an 82% objective response rate and 16% complete remission in patients. This success led to FDA approval of Elo plus Len treatment for patients with RRMM and has warranted further investigations into the exact mechanism of action. Elo targets SLAMF7 antigen which is highly expressed on MM tumour cells and activates NK cells for antibody dependent cell-mediated cytotoxicity (ADCC) resulting in MM tumour cell death. Len is an immunomodulatory agent commonly used to treat a wide range of haematological cancers. Len has been shown to activate various cells of the immune system as well as directly suppress tumour progression. This thesis aims to identify how the mechanisms of Len intersect with, and enhance, Elo activated NK cell control of tumours. The work from this thesis revealed that to induce ADCC, Elo required both expression of SLAMF7 on MM target cells and NK cell CD16 engagement. Elo-activated NK cells had increased expression of CD107a and CD69, as well as loss of CD16 expression which was a result of ADAM17 induced cleavage. Elo activation of NK cells also secreted increased levels of cytokines and chemokines associated with recruitment of effector immune cells. In both in-vitro and in-vivo studies, Len significantly enhanced Elo-induced healthy donor NK cell killing of MM tumour cells. In contrast, only 4/12 RRMM patient (responders) NK cells induced increased MM cell killing in the context of Elo plus Len. Important for this increased cytotoxicity was the expression of CD54 on NK cells that allowed differentiation between these responder and non-responder patients. CD54 was also more highly expressed on NK cells activated by Elo plus Len compared to Elo activation alone. However, further phenotypic studies and RNA sequencing did not reveal any further mechanisms in NK cells by which Len enhances Elo activation. Further studies revealed that MM tumour (myeloma) cells specifically upregulated CD54 and CD11a expression in response to Elo plus Len activation of NK cells and this did not occur with Elo treatment alone. This increase in CD54/CD11a expression on myeloma cells was also dependent on the presence of CD14+ monocytes. RNA sequencing also revealed enrichment of cytokine and chemokine signalling/secretion pathways in MM cells specifically in the context of Elo and Len activated NK cells and monocytes. In conclusion, these data indicate that a complex network of direct and indirect mechanisms between NK cells, monocytes and myeloma cells contribute to the Elo plus Len treatment induced response in RRMM patients. Elo alone activates NK cells and monocytes, whereas the Len effect is largely on myeloma cells. Collectively the two treatments led to robust myeloma antibody dependent cellular cytotoxicity (ADCC). There was a strong correlation between increased myeloma cell CD54/CD11a expression and myeloma cell ADCC suggesting that stabilisation of the immune synapse with strong adhesion may be a key factor for this improved myeloma cell killing. Finally, this study revealed that upregulation of MM cell CD54 expression on MM cells may be a useful predictive biomarker to stratify RRMM patients for Elo plus Len therapy.

Mucinous ovarian carcinoma (MOC) is a rare subtype of ovarian cancer (OC) distinct from all other subtypes. MOC represents 3% of OC cases; however, it responds very poorly to conventional chemotherapies contributing to the poor outcome of OC. Moreover, there is a lack of effective therapies for MOC since treatments with platinum-based chemotherapy or PARP-inhibitors are unlikely to be effective. The recent description of the molecular landscape of MOC, including contributions by work in this thesis, allowed the identification of novel therapies that target the most recurrent genetic features of the disease. Nevertheless, there is still the need to develop accurate pre-clinical models in order to test the efficacy of these new therapies. This thesis evaluates the transcription factor ELF3 as a new MOC candidate driver gene. We performed a comprehensive analysis of 210 human mucinous ovarian tumours and identified that ELF3 was mutated in 8.2% of our sample cohort. To assess whether ELF3 acts as a tumour suppressor or oncogene in MOC and how its mutated function is expressed in cancer, we evaluated the ability of MOC cells to proliferate, migrate and invade, by introducing truncating mutations using CRISPR-Cas9 technology. It was found that ELF3 could possibly act as a tumour suppressor in MOC; however, its role might depend on coexisting mutations in the cells. This thesis also describes a novel 3D patient-derived therapeutic approach to pre-test the effectiveness of drugs, as monotherapy or in combination, before exposing patients to ineffective therapies or side effects. A mucinous ovarian tumour 3D platform that enables the correlation of patient genetic data with drug testing was established in the first result chapter of this thesis. MOC organoid development was based on the MOC genetic profile and characteristics as well as optimization that adapted human colorectal and pancreatic organoid culture systems. It was found that Wnt-3a is essential for mucinous ovarian tumour organoid long-term derivation. Moreover, MOC organoids faithfully recapitulate MOC primary tumours at the genomic level and retain histological characteristics of their corresponding primary tumours. The development of MOC organoids and 3D spheroids from cell lines together with the description of the genetic profile of MOC allowed the development and testing of a panel of novel therapies that targets the most frequent mutations found in MOC tumours, including a mutant TP53 activator, a Wnt inhibitor, RAF/EGFR/RAS/CDK4 and CDK6 inhibitors, and two antitumour compounds for HER2-overexpressing tumour cells (APR-246, OMP-18R5, BGB-283, BKM120, PD-0332991, PLX4032, Trastuzumab and Lapatinib). The organoids and spheroids showed different responses to the individual therapies tested, depending on the individual genetic features of the models. Spheroids showed sensitivity to PD-0332991, Trastuzumab, PLX4032 , and BKM-120 . MOC organoids also showed different responses: MOC organoid MOCOR-16 showed sensitivity to APR-246, and resistance to Trastuzumab; however, MOCOR-38 was sensitive to Trastuzumab but resistant to APR-246. This chapter also shows the first attempt of drug synergy scoring using automatic robots to create 3D MOC models. Synergistic combinations were identified for one of the spheroid lines when APR-246 and BKM-120 were combined. Interestingly, the spheroids were resistant to APR-246 in monotherapy. If validated independently, these results could potentially represent a more effective treatment for MOC. Antagonistic interactions were also found, with the strongest antagonism occurring between PLX4032 and BGB-283. The findings of this study substantially increase our understanding of mucinous ovarian tumours together with potential personalized therapeutic targets to improve the survival outcomes for women with this deadly disease.

Massively parallel sequencing (MPS) technology has revolutionised the genomic exploration of human disease. This is especially true in the case of cancer, which is primarily driven by the development of acquired genomic aberrations. The body of work described within this thesis represents a broad yet in-depth array of novel applications of MPS technology in the evaluation of haematological malignancies. This field is currently surging in relevance and clinical utility as the ongoing movement of MPS technology from the research to the routine diagnostic setting continues to facilitate the development of increasingly personalised medicine. High impact contributions have been made in a number of areas encompassing myeloid and lymphoid malignancies as well as haematological malignancies as a collective. Key achievements include: quantifying the risk of incidentally detecting germline variants of potential clinical significance during unpaired MPS testing of cancer samples; definitively proving that ASXL1 NM_015338.5:c.1934dup;p.Gly646Trpfs*12 is a true somatic alteration and developing an accurate and sensitive assay for its detection; exploring the pathogenesis of and mechanisms of resistance to histone deacetylase inhibitors in cutaneous T-cell lymphomas as well as defining the clinical features, outcomes and genomic landscape of transformed marginal zone lymphoma. This thesis represents a diverse portfolio of novel research with a strong translational focus. Despite the wide scope of the individual lines of inquiry described herein there is a common thread that binds the narrative together: the pursuit of innovative yet practical ways of utilising the powerful technology now available to improve the genomic characterisation of haematological malignancies and ultimately the lives of the patients and families they affect.

The tumour suppressor gene TP53 is mutated in over half of all cancers, resulting in mutant p53 protein accumulation and poor patient survival. Decades after the discovery that p53 plays a central role in the human body’s defence against cancer, the development of effective therapeutic strategies to target mutant p53 cancers still remains an unmet need in oncology. Eprenetapopt (also known as APR-246 and PRIMA-1MET) is currently in clinical development as a mutant p53 reactivator. The reported mechanism of action of eprenetapopt is to restore wild-type p53 functionality to mutant p53 in cancer cells, triggering p53-dependent cell cycle arrest and caspase- dependent apoptosis. As a result, clinical investigation of eprenetapopt has focused on TP53-mutated cancers, relying solely on TP53 mutation status for patient selection into clinical trials. However, the mechanism of action of eprenetapopt remains contentious, as evidence demonstrates that eprenetapopt maintains strong potency in cancer cells with no mutant p53. Herein, utilising novel unbiased approaches, including CRISPR perturbation screening, metabolomics, proteomics and pan-cancer cellular-feature profiling, this thesis demonstrates two major innovations regarding the determinants of sensitivity and mechanisms of action of eprenetapopt. First, the expression of SLC7A11, which encodes the functional subunit of the cystine- glutamate antiporter, not TP53-mutation status, is the major determinant of sensitivity to eprenetapopt. Second, eprenetapopt triggers a caspase-independent, iron- dependent form of cell death known as ferroptosis, following glutathione depletion and inhibition of iron-sulfur cluster biogenesis. Together, this thesis provides a roadmap for broadening the clinical utility of eprenetapopt as a ferroptosis inducer in haematological and solid malignancies, beyond TP53-mutant cancers.

Chimeric antigen receptor (CAR) T cell therapies have been highly effective and clinically approved for treating haematological malignancies, however trials in solid cancers have shown limited efficacy, likely due in part to the increased complexity of the immunosuppressive tumour microenvironment (TME) in solid cancers. CAR T cells are inhibited by immunosuppressive proteins, cytokines or physical barriers deployed by the tumour to evade and avoid destruction by anti-tumour immunity. One such process involves the accumulation of extracellular adenosine (eADO), in the TME which has potent immunosuppressive effects on T cells and other immune cells. eADO has four known G protein coupled receptors, the A1R, A2AR, A2BR and A3R, of which the A2AR is primarily responsible for suppressing T cell function. Our previous studies highlighted a major impediment to pharmacological blockade of the A2AR which was predicted to be hindered by poor solubility and suboptimal in vivo pharmacokinetic profile [1]. This became apparent when comparing the effectiveness with genetic deletion of A2AR in CAR T cells to pharmacological blockade, in which the CAR T cells generated from A2AR-/- mice elicited comparatively greater efficacy in vivo when combined with anti-PD-1 blockade [1, 2]. This thesis therefore investigated multiple gene editing strategies to modulate adenosine receptor signalling, firstly by overexpressing the alternative signalling A1R or A3R in human or mouse CAR T cells. A1R or A3R have been shown to act by the opposing downstream signalling pathway to A2AR, and thus it is hypothesised that A1R or A3R overexpression can reverse suppression and supercharge CAR T cells in the presence of eADO. Interestingly, A1R or A3R overexpression did not confer protection to suppression by eADO in both mouse and human models, but A1R expression instead enhanced effector and terminal differentiation, activation, and baseline cytokine production of CAR T cells. This however translated to higher expression of exhaustion markers, loss of memory associated gene expression and reduced stem-like memory fraction in the CAR T cell product, ultimately leading to reduced persistence in vivo, and limiting the therapeutic efficacy of this approach. Alternatively, a previous publication from our lab briefly examined short-hairpin RNA (shRNA) mediated silencing of A2AR expression [1]. While shRNA-mediated silencing of the A2AR was able to partially reverse suppression by eADO, much like A1R expression, it also led to effector differentiation, activation, and increased baseline cytokine production. Importantly, while shRNA-mediated silencing of the A2AR also resulted in reduced persistence in vivo, it was able to mediate modest anti-tumour efficacy leading to reduced tumour growth and increased mouse survival. Both overexpression and knockdown approaches are limited by sub-optimal persistence in vivo which limited their overall therapeutic efficacies. Yet these results contradicted our prior observations of CAR T cells derived from A2AR-/- mice and from studies in the Lymphocytic choriomeningitis virus (LCMV) setting, whereby A2AR deletion was linked to increased T cell numbers [1, 3]. Therefore, the final gene-editing approach examined in this thesis utilised CRISPR/Cas9 protocols to achieve full deletion of the A2AR in CAR T cells. CRISPR/Cas9 methodologies are currently being used in clinical trials and therefore deleting the A2AR in CAR T cells using this approach is highly novel and clinically translatable. To reasons unknown, CRISPR/Cas9 mediated deletion of A2AR had minimal effects on CAR T cell memory phenotypes and no adverse effects on engraftment or persistence in vivo. Furthermore, CRISPR/Cas9-mediated deletion of A2AR in CAR T cells led to enhanced therapeutic efficacy in both mouse and human models, thus representing a potent approach to targeting the A2AR. In conclusion, future studies comparing full A2AR deletion to A2AR silencing/ pharmacological blockade or A1R overexpression may be of interest to fully elucidate the mechanisms of adenosine receptor signalling on T cell persistence and memory.

Infection is a near-universal human experience and is responsible for substantial child mortality across the globe, despite impressive reductions in child mortality and morbidity since the twentieth century. Antibiotics and other antimicrobial drugs have transformed our ability to prevent and treat infection. In general, these drugs are so safe, effective and widely available that overuse and inappropriate use are common. This is a cause of real problems in hospitals and the community, with unintended consequences of antimicrobial use including rising antimicrobial resistance. Antimicrobial stewardship (AMS) is aimed at improving the safety and efficacy of prescribing and has received growing attention in recent years. However, evidence to support and improve AMS for Australian children in hospitals is lacking. Australian hospitals are mandated to implement AMS programs and provide access to appropriate national and/or local prescribing guidelines. However, hospitals are under no current obligation to provide appropriately targeted AMS for the children in their care. Prior to mid-2019, national antimicrobial guidelines contained little paediatric and no neonatal advice. Since 2013, the voluntary National Antimicrobial Prescribing Survey (NAPS) has provided national reports on prescribing. However, until now, paediatric-specific data have not been reported. Compared with the literature on adult AMS, research on paediatric AMS is lacking, with few high-quality studies on interventions to improve care. This situation creates challenges for child healthcare providers and paediatric AMS program leaders, and more evidence is required to prioritise and improve care. The overall aims of this thesis are to improve the understanding of current antimicrobial use and stewardship for children in Australian hospitals and determine priorities to improve antimicrobial use now and in the future. This is achieved by analysing antimicrobial prescribing epidemiology and quality using national datasets, including national point prevalence survey and cohort study data. Chapter 1 reviews antimicrobial prescribing to children in hospitals, including in Australia. Chapter 2 presents the first analysis of paediatric antimicrobial prescribing to children in hospitals throughout Australia using NAPS data. Chapter 3 turns to high-risk groups, presenting the first nationwide analysis of prescribing for neonatal sepsis and fungal infections, again using NAPS data. Chapter 4 presents an analysis of antimicrobial prescribing in a contemporary cohort of immunocompromised children with fever and neutropenia, including prescribing quality and outcomes. Chapter 5 presents an interventional study evaluating the implementation of Australian guidelines on antibiotic duration and intravenous-to-oral switch. This is an example of the evidence translation and implementation approach needed for sustainable AMS improvement. Chapter 6 concludes the thesis, discussing the implications of the research and the paediatric AMS horizon in Australia. The analyses reported here reveal unnecessary variations in care and systemic inequities, which have implications for policy and guidelines. Non-metropolitan and non-tertiary hospitals in general provide lower-quality antimicrobial prescribing to children. This is likely to reflect decreased access to high-quality AMS resources, including guidelines and personnel, suggesting the need for systemic improvements. Neonates in Australian hospitals receive highly structured care in terms of antimicrobial choice and indications, but variations in dosing are substantial and undesirable, reflecting the lack of use of national guidelines. Prescribing for febrile neutropenia is highly diverse and often includes empiric aminoglycosides, which this research reveals are associated with real harm, suggesting the need for national guidelines to optimise care. Finally, the standard management of infections in hospitals involves excessive intravenous therapy, which is associated with unnecessarily increased hospital length of stay. As demonstrated, this can be improved with a structured AMS program, which should be available wherever children are treated in hospital. The information generated by this thesis provides new evidence on current antimicrobial prescribing practice and priorities and demonstrates the importance of utilising routinely collected data for the surveillance and improvement of paediatric AMS. Since this body of research began, national guidelines and paediatric-specific resources are now being developed, establishing new benchmarks. Along with continuous surveillance, these must be implemented appropriately to improve care. The research collaborations and networks developed during the production of this thesis will be used to support future surveillance and implementation work, which is needed to address AMS priorities in Australia and support the research and development of paediatric AMS across the globe.

Over the past decade, outcomes for patients with advanced melanoma have been transformed with the introduction of immune checkpoint inhibitors and molecular targeted therapies directed against the MAPK pathway. Prior to the introduction of these treatments, the median overall survival (OS) of patients with melanoma was 9-12 months. In contrast, patients treated with combination anti-CTLA-4 and anti-PD1 immune checkpoint inhibitors (ipilimumab-nivolumab) now exhibit OS rates at the 5-year landmark that exceed 50%. This stunning revolution in systemic therapeutics for melanoma has paved the way for the use of anti-PD1 immunotherapy in various other solid cancer types. The central aim of this thesis is to provide additional knowledge that may improve outcomes for patients with advanced melanoma through the refinement and development of new approaches to the use of immunotherapy. The first chapter addresses the sequencing of systemic treatment in patients with melanoma brain metastases where previously outcomes were dismal with a median OS of 6 months or less. Phase II studies show that the first line combination dabrafenib-trametinib (BRAF-MEKi) or ipilimumab-nivolumab both display intracranial response rates of 50-55%. Notably responses to targeted therapy are relatively shortly lived at approximately 6 months whereas immunotherapy displays potentially durable intracranial activity. Critically, the real-world outcomes and the sequencing of targeted therapy and immunotherapy is currently unknown. This thesis shows that ipilimumab-nivolumab given after progression of BRAF-MEKi has modest intracranial activity with objective response rates of less than 5%. Given this unexpected result, whole transcriptome sequencing of BRAF V600 melanoma brain metastases naive to systemic treatment and those excised after progression of BRAF-MEKi treatment was performed to identify potential mechanisms of resistance to both targeted and immunotherapy. Gene set testing of this cohort showed enrichment of the Innate PD-1 Resistance Signature (IPRES) with upregulation of endothelial and myeloid cell activation genes. Furthermore, macrophage infiltration of the BRAF-MEKi resistant brain metastases was higher than lesions naive to treatment which suggests that this cell type is involved with resistance to immunotherapy particularly in the central nervous system (CNS). The second chapter of this thesis investigates whether the addition of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) can augment adoptive cell transfer (ACT) immunotherapy in combination with BRAF-MEKi. Other laboratories have shown that the addition of BRAF-MEKi with pmel-1 ACT can augment anti-tumour activity against the murine melanoma cell line SM1 in vivo. The addition of BRAF-MEKi not only enhanced anti-tumour activity by blockade of the MAPK pathway, but also exerted immunomodulatory effects including upregulation of melanoma differentiation antigens and antigen presentation pathways. The addition of CDK4/6i might enhance anti-tumour activity of combination BRAF-MEKi with ACT as melanoma exhibits a high rate of molecular aberrations that impact upon the cell cycle pathways and are an emerging therapeutic target. CDK4/6i such as palbociclib might further enhance ACT by inducing cell cycle arrest; moreover, several papers also show immunomodulatory effects such as increased tumour expression of major histocompatibility complex (MHC) class I and inhibition of T regulatory cells in the tumour microenvironment. The data contained herein shows that the CDK4/6 inhibitor, palbociclib did not further enhance expression of MHC Class I on SM1 or YOVAL1.1 melanoma cell lines by itself or in combination with BRAF-MEKi. However, combination BRAF-MEK-CDK4/6 inhibitor with OT-1 adoptive cell transfer leads to robust anti-tumour activity in vivo against YOVAL1.1. The third chapter of the thesis explores whether the addition of denosumab to standard immune checkpoint inhibitor regimens improves outcomes for patients with advanced melanoma. Denosumab is an anti-RANKL antibody and is an approved agent for osteoporosis, prevention of bone metastases and giant cell carcinoma. Recent pre-clinical studies show denosumab can enhance in vivo antitumour activity in pre-clinical models of melanoma, renal cell and prostate carcinoma. Given there are no known overlapping toxicities of denosumab with nivolumab or with the combination ipilimumab-nivolumab, we developed a Phase Ib/II trial of Ipilimumab-Nivolumab-Denosumab or Nivolumab-Denosumab for Unresectable or Metastatic Melanoma (NCT03161756). This multi-centre, investigator-initiated study was opened in December 2017. Available data of the combination of nivolumab-denosumab shows no concerning safety signals. Overall, this thesis sought to identify optimal treatment sequencing strategies for brain metastases, the development of novel immunotherapy strategies involving CDK4/6i with adoptive cell transfer and the safety of combining denosumab with nivolumab and ipilimumab-nivolumab.

Background Neural stem cells are cells that can self-renew and differentiate to give rise to specialized cell types, such as neurons. The appropriate regulation of neural stem cell activities during development guarantees that the correct number of functional neurons are generated to form the adult nervous system. Dysregulation of this process can result in a number of neurological diseases. Too few neural stem cells (and their neuronal progeny) can lead to microcephaly, and too many neural stem cells (and their neuronal progeny) can lead to brain tumour formation. Therefore, understanding the mechanisms regulating neural stem cell activities may shed light on novel therapeutics for neurological diseases. Drosophila neural stem cells, the neuroblasts (NBs), have been an advantageous model for the study of neural stem cell biology during development and in disease contexts. In the developing larval Central Nervous System (CNS), NB activities are maintained by intrinsic regulatory machinery as well as extrinsic cues from glial cells, which form a microenvironment enwrapping NBs. Dysregulation of either mechanism can have severe consequences. Deregulation of NB intrinsic regulators such as a homeobox transcription factor called Prospero (Pros) can result in decreased differentiation and an expansion of NBs, resulting in increased brain size. The disruption of glial niche or glial derived signals can lead to an alteration of NB proliferation rate, thus affecting the number of neurons in the adult CNS. While multiple intrinsic factors regulating NB activities during development have been intensively studied, it remained poorly understood how NB intrinsic metabolic pathways are important for NB proliferation under physiological and disease context. Furthermore, the mechanisms of how NBs respond to extrinsic cues and how the glial niche relay extrinsic signals to control NB activities is still not well understood. Hence, the aim of my study is to expand our knowledge on how intrinsic metabolic adaptations and extrinsic cues regulate the progenitor pool of NBs and the rate at which neuronal progeny is produced. Aims, methods and results: In this work, I have addressed two key questions: 1) what metabolic rewiring events occur as a result of NB tumour formation caused by dedifferentiation? 2) How do glial niche and glial derived signals non-autonomously regulate NB proliferative activities? 1) Metabolic rewiring is known to accompany aberrant proliferation and has recently been shown to drive tumour growth. Utilising the Drosophila NB tumour model caused by deregulation of pros (a homeobox transcription factor), we conducted transcriptomics and metabolomics studies to examine metabolic changes that occur as a result of defective NB differentiation. I found that glutathione metabolism is altered as a result of pros loss-of-function (LOF) tumour formation. It is known that the Pros homologue PROX1 is deregulated in neuroblastoma. Therefore, findings from our work may inform the search of novel metabolite-based targeted therapies for the treatment of neuroblastoma. 2) In the larval CNS, NBs re-enter into cell cycle from a quiescent state in response to extrinsic signals from their microenvironment made up of glial cells. In my study, I have uncovered several new regulators of glia-NB interaction using genetic analyses. I found that lipid metabolism and Hedgehog (Hh) in the glial niche are involved in regulating NB proliferation. Glial Hh is autonomously required for the growth of glial niche, which is key in maintaining NB proliferation during development. Upon glial overgrowth mediated by FGF activation, excessive Hh from the glial niche triggers a delay in NB cell cycle progression. In this glial overgrowth context, Hh function is modulated by two lipid regulators, Fatty acid synthase 1 (Fasn1) and Lipid droplet storage 2 (Lsd-2). I demonstrated that Fasn1 can modulate Hh palmitoylation. However, it remains unclear how Lsd-2 modulates Hh function, despite the observation that Lsd-2 and Hh both localize to the surface of lipid droplets localized to glial cells. I also found that glial derived chitinase like proteins, called imaginal disc growth factors (IDGFs) are important non-autonomous regulators of NB proliferation. I showed that IDGF2 is necessary for NB exit from quiescence and is required to sustain NB proliferation at later larval stages. Collectively, these findings revealed novel mechanism by which Drosophila neural stem cell activities are regulated during development and during tumorigenesis.

Metastasis is the lethal aspect of cancer for most patients. Remodelling of lymphatic vessels associated with a tumour is a key initial step in metastasis because it facilitates the entry of cancer cells into the lymphatic vasculature and their spread to lymph nodes and distant organs. Although it is clear that vascular endothelial growth factors (VEGFs), such as VEGFC and VEGFD, are key drivers of lymphatic remodelling in cancer, the means by which many signalling pathways in endothelial cells are co-ordinately regulated to drive growth and remodelling of lymphatics in cancer is not understood. In this thesis, I seek to understand the broader molecular mechanisms that control cancer metastasis through the analysis of microRNAs which act to co-ordinately regulate signalling pathways involved in complex biological responses, such as lymphatic remodelling, in health and disease. Here, using high-throughput small RNA sequencing, I found that a specific microRNA, miR-132, is up-regulated in expression in lymphatic endothelial cells (LECs) in response to stimulation with VEGFC and VEGFD. Interestingly, inhibiting the effects of miR-132 in LECs in vitro blocked proliferation and tube formation of these cells induced by VEGFC and VEGFD - LEC proliferation and tube formation are key steps in lymphatic remodelling. Moreover, I demonstrated that miR-132 is expressed in the lymphatic vessels of a subset of human breast tumours which were previously found to express high levels of VEGFD. In order to dissect the complexity of molecular regulation by miR-132 in lymphatic biology, my collaborators and I identified miR-132 target mRNAs in LECs, using high-throughput sequencing after RNA-protein cross-linking and immunoprecipitation of the Argonaute protein (Argonaute HITS-CLIP), which led us to define the miR-132-mRNA interactome in LECs. We found that this microRNA in LECs is involved in the control of many different molecular pathways mainly involved in cell proliferation and regulation of the extracellular matrix and cell-cell junctions. It is logical that miR-132 regulates such pathways given they are involved in the processes of LEC proliferation and tube formation, which I showed are dependent on miR-132 in my in vitro studies. Finally, I demonstrated that inhibiting the effects of miR-132 in a mouse ear model of lymphangiogenesis, using an antagomiR inhibitor of miR-132 coupled to cholesterol, blocked the complex remodelling of lymphatic vessels stimulated by VEGFC, in vivo. It was noteworthy that all aspects of lymphatic remodelling induced by VEGFC were restricted by inhibition of miR-132, including the enlargement, branching and sprouting of lymphatic vessels. Thus the inhibitory effect of targeting this microRNA on lymphangiogenesis and lymphatic remodelling can be considered comprehensive. The research described in this thesis identified miR-132 as a critical regulator of lymphangiogenesis and lymphatic remodelling, and delineated molecular mechanisms by which this microRNA influences these important biological processes. This work also identified new molecular pathways which are involved in modifying the lymphatic vasculature in response to key lymphangiogenic growth factors. In-so-doing, these studies identified potential therapeutic targets for drugs designed to block the growth and remodelling of tumour lymphatics, and thereby restrict the metastatic spread of cancer.

Global initiatives applying next generation sequencing to thousands of primary and metastatic tumour samples have detailed the genomic landscape of breast cancer. These unique insights have transformed our understanding of the biological processes underlying these tumours. Despite this, the clinical relevance of these findings remains poorly understood. This is because these datasets are frequently heterogeneous, include patients that may not have received contemporary treatments, often have limited clinical annotation, and commonly lack long-term follow-up for patient survival outcomes. In order to better assess the clinical relevance of genomic driver alterations in breast cancer, this research project performed genomic sequencing using primary tumour samples from patients enrolled in two pivotal phase 3 clinical trials – the Breast International Group 1-98 (BIG 1-98) study and the Suppression of Ovarian Function Trial (SOFT). This provided a well-defined population with hormone receptor-positive, human epidermal growth factor receptor (HER) 2-negative early breast cancer in both postmenopausal and premenopausal populations, as well as long-term survival outcome data collection. Crucially, this allowed for further investigation of late distant recurrence, a unique feature of hormone receptor-positive, HER2-negative breast cancers, as well as the ability to focus on distinct high-risk subpopulations, such as breast cancer arising in young women. In the postmenopausal BIG 1-98 study, comprehensive panel sequencing was performed using 538 tumour samples from patients with hormone receptor-positive, HER2-negative early breast cancer. PIK3CA mutations were the most common driver (49%) and were associated with a reduction in the risk of distant recurrence. Contrastingly, TP53 mutations, amplifications on 11q13 and 8p11, and increasing number of driver alterations were associated with an increase in the risk of distant recurrence. Additionally, PIK3CA mutations were predictive of a greater magnitude of benefit from letrozole compared with tamoxifen. Using the same dataset, we further investigated the association of oncogenic drivers with late distant recurrence (5 years or greater from the time of randomisation). PIK3CA mutations were associated with reduced risk of late distant recurrence, whereas amplifications on 8p11 and BRCA2 mutations were associated with increased risk of late distant recurrence. In the premenopausal SOFT study, comprehensive panel sequencing was performed using 1258 tumour samples from the entire study cohort, and whole exome sequencing was performed using 82 tumour samples from very young patients with high-risk, hormone receptor-positive, HER2-negative early breast cancers. Subgroups of tumours with distinct copy number-amplifications had a higher risk of distant recurrence than tumours that were amplification-devoid, validating previously reported data. Importantly, the study further defined unique molecular characteristics that were enriched in very young women and may explain their poor prognosis. This includes subgroups of tumours with homologous recombination deficiency, a high-risk PIK3CA mutated subgroup, amplification-enriched subsets, and a low estrogen receptor expressing subtype with immune infiltration. Taken together, these results highlight the clinical relevance of genomic sequencing of hormone receptor-positive, HER2-negative early breast cancers. They provide a new prognostic framework using genomic stratification, and highlight molecular targets that should be prioritised for future clinical trial research.

Malignant mesothelioma is an aggressive cancer of the mesothelium for which asbestos-exposure is the predominant etiological factor. The standard treatment regime for mesothelioma is systemic chemotherapy, which exhibits poor efficacy with a median post-diagnosis survival of 12 months. This highlights the need for alternate therapies, such as targeted therapies, to improve patient outcomes. Genomic and cell biological analyses of mesotheliomas indicate that deficiency of key tumour suppressor genes including BAP1 or members of the Hippo tumour suppressor pathway such as NF2, are frequent genetic aberrations in this cancer. Inhibition of cancer-promoting events that occur downstream of these genetic aberrations represents a potential mechanism by which mesothelioma patient outcomes can be improved. Originally identified in Drosophila, the Hippo pathway is highly conserved in mammals and regulates the activity of the oncoproteins and transcriptional coactivators YAP and TAZ and their nuclear interaction partners, the TEAD family of transcription factors. YAP/TAZ-TEAD hyperactivation is an oncogenic driver in Hippo pathway deregulated mesotheliomas, indicating that inhibition of these proteins could represent a new treatment strategy for this disease. An emerging class of small molecule inhibitors of YAP/TAZ-TEAD target the recently identified post-translational modification of TEAD palmitoylation. One such compound, VT107, has shown promising pre-clinical activity in in vitro and in vivo models of Hippo pathway deregulated mesothelioma. Whilst VT107 is a promising therapeutic, the current understanding of its molecular impact is incomplete. Moreover, further identification of therapeutic targets in BAP1 or NF2 deficient mesothelioma cells might enable development of additional targeted therapies to treat candidate tumours. To address these considerations, I aimed to answer three key questions: 1) Which genes are potential therapeutic targets for treatment of NF2 or BAP1 mutant mesothelioma?; 2) How does VT10- 7 modulate the behaviour and transcription regulatory activity of YAP/TAZ-TEAD?; and 3) How does VT107 inhibit the proliferation of Hippo pathway mutant mesothelioma cells? 1) To identify candidate therapeutic targets for treatment of NF2 or BAP1 deficient mesothelioma, I conducted a genome-wide synthetic lethality screen. Due to technical limitations with BAP1 deficient cells, I conducted the screen with only NF2 deficient cells. While I identified a mild synthetic lethal interaction between NF2 and CREB3, I concluded that the screen was unsuccessful in identifying robust synthetic lethal interaction partners of NF2. 2) I assessed the molecular impact of VT107 in a Hippo pathway mutant mesothelioma cell line (NCI-H2052) using single molecule tracking of YAP and TEAD1. With this advanced live microscopy approach, I observed that VT107 increased the nuclear mobility, and reduced the DNA residence-time, of both YAP and TEAD1. Thus, I provided a high resolution molecular understanding of how VT107 influences the ability of YAP and TEAD1 to regulate transcription. 3) I conducted a genome-wide CRISPR screen to investigate the mechanism of action of VT107 in NCI-H2052 cells. I identified that loss of SOCS3, VGLL4, BTAF1 and DR1, among other genes, increased cellular resistance to VT107. I also found that loss of several genes, including TCEB2, could confer VT107 sensitivity. These findings provide unbiased insights into the mechanism by which YAP/TAZ-TEAD inhibition modulates the proliferation of mesothelioma cells. Furthermore, I identified potential drivers of resistance to VT107- information which might guide its optimal application in the clinic for the treatment of Hippo pathway mutant mesothelioma.

Cytotoxic secretory granules (CGs) are unique and specialised lysosomal organelles found specifically in cytotoxic lymphocytes: Natural Killer (NK) cells and CD8+ T cells or Cytotoxic T-Lymphocytes (CTLs). CGs precisely store and deliver perforin and granzymes; molecules that synergistically induce the controlled death of infected and neoplastic cells. The precise molecular mechanisms that govern CG formation remain poorly understood and this work aimed to explore the potential role of the MiT/TFE family of proteins in transcriptionally regulating this process. Three members of the MiT/TFE family (MITF, TFEB and TFE3) are largely recognised as the master regulators of lysosome biogenesis and as the main drivers in the formation of organelles with a lysosomal nature. Under this hypothesis the role of the microphthalmia associated transcription factor (MITF) was studied. CTLs expressing mutant MITF showed a reduced ability to expand and kill target cells in vitro in response to antigen-specific stimulation. However, it was determined that the defect was not intrinsic to the lymphocytes but associated to antigen presenting cells required for CTLs activation. Having ruled out MITF in the formation of CGs, the role of transcription factor EB (TFEB) and transcription factor E3 (TFE3) was explored next. By studying the subcellular trafficking of these two proteins during mouse CTL activation, this work described that TFEB, but not TFE3, translocated from the cytosol into the nucleus. Despite the nuclear TFEB presence, RNA-sequencing of CTLs did not conclusively showed upregulation of canonical TFEB target genes. To gain further understanding on the role of TFEB in CTLs, the lysosomal storage disorder (LSD) Niemann-Pick type C1 (NP-C1) was implemented as a model. This devastating disease, caused by mutations in the lysosomal cholesterol transporter, NPC1 proved to be suitable for this purpose, given that it was shown here that TFEB is retained in the cytosol of CTLs in a mouse model of the disease and this was accompanied by a significant reduction of their cytotoxic function. The implementation of various microscopy techniques led to the discovery that NPC1-defficient CTLs accumulate aberrant autophagic bodies that fuse, or embed, lipid-dense CGs. These observations were corroborated in CTLs derived from a cohort NP-C1 patients. Finally, it was established that reconstituting nuclear TFEB was sufficient to restore autophagic dysregulation, but it was not linked to cytotoxic improvement. Contrastingly, pharmacological depletion of aberrantly accumulated lipids within CGs of NPC1-defficient CTLs, completely restored their cytotoxic function. The work presented here shows that the mechanism behind it is linked to lipid inactivation of the pore forming protein, perforin, and the consequent inability of NPC1-defficient CTLs to kill target cells. Overall, the evidence presented here suggests that TFEB plays a critical role in CTL activation which might be unrelated to its canonical lysosomal biogenesis transcriptional regulation. Moreover, while studying this transcription factor in CTLs in the context of the LSD, NP-C1, a number of phenotypical abnormalities were discovered, including accumulation of autophagic bodies and reduced cytotoxicity. Importantly, the impairment in the autophagic flux in NPC1-defficient CTLs was linked to limited TFEB nuclear translocation while the diminished capacity to kill target cells was associated with lipid accumulation within CGs.

Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene are among the most frequently occurring somatic mutations in acute myeloid leukaemia (AML), a disease that presents with devastating prognosis. FLT3 is primarily expressed on hematopoietic progenitor cells, and during early haematopoiesis coordinates a ligand-dependent signalling cascade that regulates the proliferation and maturation of the progenitor pool. FLT3 internal tandem duplication (FLT3-ITD), the most common type of FLT3 mutation, promotes constitutive FLT3 kinase activity and hyperactivation of downstream signalling pathways including STAT5, PI3K/mTOR and MAPK. Though our understanding of the molecular pathways downstream of mutant FLT3 have greatly improved in the modern genomic sequencing era, the repertoire of molecular signalling events induced by mutant-FLT3 to drive leukaemogenesis has not been fully characterised. In this thesis, a murine model of MLL-rearranged AML harbouring inducible FLT3-ITD expression was developed and used, along with human AML cell lines, to demonstrate that FLT3-ITD promotes serine uptake and serine biosynthesis via transcriptional regulation of neutral amino acid transporters (SLC1A4 and SLC1A5) and genes in the de novo serine biosynthesis pathway (PHGDH and PSAT1). Mechanistically, dysregulation of serine metabolism in FLT3-ITD-driven AML is dependent on the mTORC1-ATF4 axis, which drives RNA-Pol II occupancy at PHGDH, PSAT1, SLC1A4 and SLC1A5. Genetic or pharmacological inhibition of the de novo serine biosynthesis pathway, in vitro and in vivo, selectively inhibited the proliferation of FLT3-ITD-driven AML cells. Pharmacological inhibition of the de novo serine biosynthesis pathway using WQ-2101, an inhibitor of PHGDH, the first rate-limiting enzyme of the de novo serine biosynthesis pathway, sensitises FLT3-ITD-driven AML cells, including primary patient samples, to the standard of care chemotherapy agent cytarabine via exacerbation of DNA damage. Given that transcriptional activation of de novo serine biosynthesis and serine uptake was mediated by mTORC1 (a master regulator of biomass production and cellular metabolism), and that therapeutic responses in vitro and in vivo to mTORC1 inhibitors are poor, a small molecule compound screen utilising 181 epigenetic inhibitory compounds to determine novel synthetic lethal interactions between epigenetic regulators and mTORC1 inhibition was performed. This analysis revealed that inhibition of lysine-specific methyltransferase SETD8, the only known enzyme that catalyses H4K20 monomethylation (H4K20me1), synergised with mTORC1 inhibition. Transcriptional profiling suggested dual SETD8/mTORC1 inhibition preferentially suppressed mTORC1 target genes mediating amino acid biosynthesis and transamination (including de novo serine biosynthesis) to a greater extent than either single agent SETD8 or mTORC1 inhibition alone. Importantly, these observations were independent of global transcriptional repression induced by impaired cell viability or suppression of global transcription. Thus, this preliminary work suggests mTORC1 and/or its target genes and pathways may be dependent on SETD8 and/or H4K20me1 or, alternatively, mTORC1 functionally regulates SETD8. Collectively, the results presented herein provide novel insights into FLT3-ITD-induced metabolic reprogramming events in AML and identify a targetable metabolic dependency in this poor prognosis subtype of disease. In addition, these results provide the preliminary basis of a SETD8/mTORC1 synthetic dependency that can be exploited in FLT3-ITD-driven AML.

Multiple myeloma (MM) is an incurable plasma cell neoplasm. The incorporation of immunomodulatory imide drugs (IMiDs) – thalidomide, lenalidomide and pomalidomide - into clinical practice has improved survival and quality of life of MM patients. However, most patients develop therapeutic resistance to IMiDs, representing a significant clinical problem. IMiDs exert anti-MM effects by engaging Cereblon (CRBN), a ligand of the E3 ligase complex CUL4-DDB1-RBX1. By binding CRBN, IMiDs promote ubiquitination and proteasomal degradation of important MM cell transcription factors, IKZF1 (Ikaros) and IKZF3 (Aiolos). Degradation of such neosubstrates has also been shown to augment an anti-cancer immune response by activation of cytotoxic effector cells against MM plasma cells. Finally, it has been demonstrated that IMiDs modulate events occurring within the bone marrow microenvironment with negative impact on the MM-sustaining niche. Despite these recent advances in the characterisation of IMiD mechanism of action, the precise molecular events underpinning the anti-MM activity of IMiDs remain incompletely understood. Further insight is required to improve clinical responses to IMiDs and overcome therapy resistance in MM. In this work, genome-wide approaches such as RNA-sequencing and CRISPR-based functional genomics screening were employed to dissect the molecular mechanisms of IMiD-sensitivity and -resistance in models of acquired resistance to lenalidomide. RNA-sequencing revealed a strong interferon-like transcriptional response and increased transcription of genes downstream the MAPK pathway, together with downregulation of MYC-responsive genes, following IMiD treatment of IMiD-sensitive MM cells. Conversely, IMiD-resistant cells displayed reduced transcriptional changes following IMiD exposure as a likely consequence of CRBN downregulation and subsequent attenuation of neosubstrate degradation. Genome-wide CRISPR screening demonstrated that loss of CRBN and other genes regulating protein degradation, such as subunits of the COP9 signalosome, but also loss of genes that are not implicated in protein turnover, such as NCOR1 and EDC4, is sufficient to cause resistance to IMiDs in IMiD-sensitive cells. CRIPSR screening also demonstrated that resensitisation of IMiD-resistant cells to IMiDs can occur through loss of ATXN7, TOP2B and other genes implicated in several distinct biological processes, such as glucose metabolism, cell cycle progression and RNA processing. Dexrazoxane, a pan-TOP2 inhibitor and TOP2B-selective degrader, was discovered to possess anti-proliferative properties and combinatorial activity with lenalidomide in certain MM cell lines. To develop a deeper understanding of the biology of CRBN and IMiDs, proximity labelling with BioID2 was employed to characterise the interactome of CRBN in the presence and absence of an IMiD. Co-treatment of BioID2-CRBN-expressing OPM2 cells with IMiDs and/or the proteasome inhibitor bortezomib, followed by liquid chromatography mass spectrometry proteomics, allowed identification of known CRBN interactors (COP9 signalosome subunits) and neosubstrates (IKZF1, IKZF3 and CK1alpha). This approach enabled further characterisation of CRBN-associated proteome and identification of new CRBN and CRBN-IMiD interacting partners, such as MEF2C, DVL1, DVL2, MYH9, PRRC2C and EDC4. This data led to the hypothesis that CRBN may be implicated in several novel biological processes, such as RNA processing and protein translation regulation. Additionally, some of the newly identified interacting partners may modulate the MM niche in the bone marrow and may help understand the occurrence of side effects to IMiD therapy. This work has further defined transcriptional signatures of IMiD treatment and of acquired IMiD-resistance. TOP2B was found to be a potential druggable vulnerability of MM cells with acquired IMiD-resistance. The unbiased study of the CRBN-interacting proteome has helped broaden the knowledge on its biological activities both in presence and absence of an IMiD. Collectively, this thesis provides new and important insight into IMiD mechanism of action and further expands knowledge of IMiD biology.

Intra and inter-tumour heterogeneity poses a challenge for associating molecular and immunohistochemical markers with clinical outcomes. Sequencing technologies has enabled detailed assessment of tumour heterogeneity, facilitating the genomic characterisation of tumours. Whilst such technologies have revealed mutational landscapes and have identified key driver alterations for tumorigenesis, pan-cancer clonal evolution reconstructions are lacking. In order to bridge this gap, I used discrete-time branching models to derive biological insights into tumour progression and reconstructed the clonal evolution in 1,800 patients, successfully linking mutations with growth patterns of disease progression. I first modified a discrete time-branching process to account for individual clonal subpopulations and derived analytical solutions for expectation and variance of both clonal and tumour expansions. Additionally, I derived the expected time for any given clone to successfully expand as \hat{\tau}, and with the use of these analytical solutions, I showed the likely driver and clonal compositions of the tumours and their phylogenies. Secondly, I generated a database of results from four different versions of time-branching process models that covered multiple parameters. Here I identified how an increase in diversity arises by both increased mutation rate and reduced fitness. I further corroborated that total number of drug resistant cells is directly proportional to lineage extinction probability (\delta) and tumour size as shown in previous studies. I also showed that this effect can be extrapolated to other types of functional passenger mutations involved in cancer-specific mortality. Moreover, I showed how commonly used sequencing cut-offs limit the accurate inference of tumour’s average selective advantage and driver mutation rate. Thirdly, I identified that a minimum distance metric can provide accurate fits of simulated cancer cell fractions to real patient tumour data. This metric showed at least 80% accuracy to identify the initial parameters of s and u and at least 40% accuracy to recover the correct evolutionary trajectory. Fourthly, I applied this fitting procedure to reconstruct the evolutionary trajectories of 1,800 tumours from different cancer sequencing studies. The best fits derived suggests that the most likely parameters for the evolution of solid tumours are high driver mutation rates and weak driver effects of fitness. Fifthly, using The Cancer Genome Atlas cohort, I identified an association between predicted degree of clonality and survival, and found branched topologies are common in malignancies with adverse prognosis. In the TRACERx non-small-cell lung cancer cohort, I identified that clonal reconstructions agreed with previously reported phylogenies. Additionally, using data from the Breast International Group 1-98, I identified the role of tumour fitness in determining clinical outcome, and the evolutionary dynamics of TP53 and PIK3CA mutations conducive to distant metastasis. Finally, using data from a metastatic melanoma patient collected through the CASCADE melanoma study, I was able to propose a pattern of dissemination from the primary to metastatic sites in the liver and brain based on the phylogenies recovered from my data-fitting procedure. This study demonstrates the power of the discrete-time branching process in reconstructing tumour evolution, and its potential to uncover insights in the dynamics of tumour growth that are missed by current methods.

Phenotypic diversity of breast cancers poses insurmountable challenges in the treatment of this lethal disease. Recent advances in next generation sequencing have led to unprecedented insight into the genomic landscape underlying breast tumours. This has resulted in burgeoning development of targeted treatments and predictive biomarkers, several of which have recently demonstrated clinical activity. However, key challenges hinder optimal application. On the background of extensive molecular heterogeneity, most biomarkers represent minority patient subpopulations, hampering clinical development. Furthermore, considerable genomic evolution of breast tumours impacts accuracy of genomic characterisation that is thus far heavily reliant on the sequencing of non-contemporaneous and invasive tumour tissue biopsies. Finally, stratification to genomically-matched targeted therapies also fails to fulfil the extent of its promise. In many cases relentless tumour growth remains unperturbed, while in others resistance ultimately develops. Crucially, molecular mechanisms underlying resistance remain poorly understood, while follow-on treatment options are often poorly defined. Central to the promise of personalised medicine is the robust and accurate characterisation of the tumour genome. Minimal invasiveness and convenience of circulating tumour DNA (ctDNA) analysis, with ability to detect tumour genomic aberrations from a blood draw, highly recommends this approach. Recent technological advances have paved the way to a range of clinical applications, with evolving potential for ctDNA analysis to address the continuum of challenges posed to precision medicine throughout patient management. Toward this end, extensive clinical development is required, while prevailing technological hurdles need to be addressed. This thesis explores a multi-faceted and rigorous approach towards the integration of ctDNA analysis in the management of breast cancer patients. Firstly, the development and validation of multiple assays (allele-specific and NGS-based) tailored to breast cancers, enabled comprehensive genomic analysis with in-built flexibility to be readily applicable to a variety of clinical scenarios. Subsequent establishment of a prospective ctDNA-based molecular profiling program across a large cohort of metastatic breast cancer (mBC) patients demonstrated feasibility of real-time analysis in the clinical setting across a range of genomic targets of variable abundance. Importantly, integration of longitudinal testing in this program throughout patient management demonstrated capacity for ctDNA analysis to reflect genomic evolution in real-time to optimise precision-guided patient management. Finally, exploratory longitudinal ctDNA analysis for the study of resistance mechanisms to CDK4/6i, constituting a novel class of targeted compounds for breast cancer, highlights established and novel resistance markers. Indeed, this study also serves to demonstrate a workable framework for ctDNA analysis as a highly effective approach for the de novo elucidation of resistance mechanisms to novel targeted agents that is relevant across cancer types.

Rapid advancement in genomic technologies has driven down the cost of sequencing significantly. This efficiency has enabled large-scale cancer genomic studies to be conducted, generating a vast amount of data across different levels of omics variables. However, the tasks to extract new knowledge and information from this enormous volume of data present unique challenges. These analyses often require the application of specialised techniques for data mining, integration and interpretation to provide valuable insights. With the rise of machine learning adoption in recent decades, many advanced computational algorithms based on artificial intelligence techniques have also been proposed to analyse these genomics data. Although some of these applications have led to clinically relevant conclusions, many others are still relying on incomplete prior knowledge, or limited to only a selected number of features. These limitations raise the general question about the broader applicability of machine learning in the field of cancer genomics. This research addresses this question by assessing the application of machine learning techniques in the context of breast cancer genomics data. This assessment includes a comprehensive evaluation of computational methods for predicting cancer driver genes and the development of a novel deep learning approach for identifying breast cancer subtypes. The evaluation result of driver gene prediction algorithms suggests that the selection of the best method to be applied to a dataset will primarily be driven by the objectives of the study and the characteristics of the dataset. All of the evaluated approaches could identify well- studied genes, but not all of them performed as well on smaller datasets, subtype-specific cohorts, and in discovering novel genes. To examine the benefit of a more complex machine learning model, this thesis also presents a novel deep learning approach that integrates multi-omics data for predicting various breast cancer’ biomarkers and molecular subtypes. This method combines a semi-supervised autoencoder for dimensionality reduction, and a supervised multitask learning setup for the classifications. Taking an input of gene expression, somatic point mutation and copy number data, the algorithm predicts the ER-Status, HER2-Status and molecular subtypes of breast cancer samples. Further survival analysis of the outputs from this deep learning approach indicates that the predicted subtypes show a stronger correlation with patient prognosis compared to the original PAM50 label. While the outputs from machine learning algorithms still require further validation, the adoption of these complex computational methods in cancer genomics will become increasingly common. Collectively, the results from this thesis suggest that the machine learning analysis of ‘omics data hold great potential in automating the discovery of clinically- relevant molecular features.

Prostate cancer (PCa) is one of the most diagnosed tumours in Australia, pertaining to more than 30% of all cancer detection (AIHW 2020). Most of these diagnoses are correlated to men with early, localised stages of the disease that are easily treated with radical proctectomy surgery and/or radiation. One third of these patients will relapse with their disease progressing to the either the recurrent localised stage or the metastatic stage. These advanced cases will have to undergo Androgen Deprivation Therapy (ADT), as hormone dependence is a key driver of prostate tumorigenesis. ADT will help increase patient survival by halting cancer progression, while minimising symptoms. Although this type of therapy can initially alleviate tumour burden, patients will inevitably be subjected to cancer recurrence where their tumours continue to progress despite castrate levels of androgens. An observed second-order effect of ADT is that it creates a therapeutic selection pressure within PCa, leading to the proliferation and establishment of cells that are resistant to androgen ablation. These hormone-refractory cells lead to the development of Castration Resistant Prostate Cancer (CRPC), a stage of the disease that ultimately leads to death. Despite the development of second-generation ADT or chemotherapeutic agents, no effective therapies have been developed yet to combat this lethal stage of the disease. Therefore, there is a need for therapies that can target pathways and mechanisms that are not related to androgen signalling. MYC overexpression is one of the most frequently characterised genomic aberrations within men with CRPC. MYC mRNA overexpression is found in 30% of men with localised disease and in 85% of men with CRPC. Although MYC seems like a prime therapeutic target to combat PCa, previous attempts at developing MYC inhibitors have failed due to major off-target effects making these therapies dangerous. The overexpression of MYC is tightly correlated with increased ribosome biogenesis because MYC-driven tumours have an inherent need for protein synthesis to sustain infinite cell proliferation. The rate limiting step of ribosome biogenesis is the transcription of a ribosome RNA precursor by RNA polymerase I within the nucleolus. MYC orchestrates the over-transcription of this precursor by tightly interacting with Pol I associated components. PIM kinases and the PI3K pathways also tightly interact with MYC in order to increase its stability and prevent its degradation. It can be inferred from this that MYC-driven tumours are highly proliferative, lack any type of cell cycle regulation, and are constitutively producing proteins to sustain their uncontrolled growth. Administration of CX-5461, an RNA Pol I inhibitor, was followed by potent anti-proliferative activity and cell cycle arrest within various in vitro and in vivo models of prostate cancer. Strikingly, the combination of Pol I inhibition along with CX-6258, a PIM kinase inhibitor, was characterised by potent reduction in invasive lesions within two mouse models of prostate cancer. Although both drugs combined seemed to have potential additive effect, little is understood about the mechanism of action of these two inhibitors together. Therefore, one of the main aims of this thesis is to understand the efficacy of this combination. Both drugs will be tested on different subtypes of human prostate cancer in the explant settings in order to correlate patient-drug response with specific genomic or pathological profiles. A further goal for this project will be to utilize mass spectrometry to characterise the main phosphorylation events that occur as a result of the administration of these inhibitors, both alone and in combination. This will further the understanding of the mechanistic action of both drugs combined. Due to the limited efficacy of CX-5461 as a single agent, PMR-116, a novel Pol I inhibitor, will also be tested within different models of prostate cancer to evaluate as a potential anti-cancer drug. The final aim of this thesis will be to combine CX-5461 with targeted 177Lu-PSMA-617 therapy, as the latter has recently been shown to potentiate radiation-induced DNA damage. Through the various aims of this thesis, we hope to find and characterise the efficacy of RNA polymerase I therapies on prostate cancer both as single agents or combined therapies.

The phosphoinositide 3-kinase (PI3K) pathway is one of the most commonly activated pathways in a variety of cancers and it has recently been highlighted as one of the primary modulators of cell metabolism. This has opened promises and challenges for the development of therapeutic strategies to target metabolism in cancer cells harbouring mutations in the PI3K pathway. Through an inducible “exon switch” approach, our laboratory has previously generated mice ubiquitously expressing a mutation in Pik3ca, the gene coding for the subunit p110alpha of PI3K. By using this mouse model (UbCreERPik3caH1047R) our laboratory has shown that mutations in the PI3K pathway lead to dramatic severe defects in glucose homeostasis resulting in hypoglycaemia and hypoinsulinemia. In this thesis the causes responsible for the metabolic dysfunction observed in these mutant mice are investigated. This thesis provides evidence that mutations in the PI3K pathway lead to increased glucose uptake by the tissues, inhibition of hepatic gluconeogenesis and inhibition of insulin release from the pancreas. Previous studies performed on the UbCreERPik3caH1047R mouse model have also shown increased body weight and organomegaly. This thesis demonstrates that the increase in body weight, resulting from the activation of the Pik3caH1047R mutation was not associated with adiposity. On the contrary, mutations in the PI3K pathway determine loss of body fat and increased lipolysis in the adipose tissue of mice, whilst the tissue growth is associated to hypertrophy or hyperplasia. Furthermore, oncogenic activation of the Pik3caH1047R mutation in vivo leads to alteration of the respiratory exchange rate and energy expenditure of the mice and stimulates browning of the adipose tissue. This thesis also shows that activation of the PI3K pathway alters the expression of genes and proteins involved in metabolic pathways, and that these alterations are organ-specific, therefore opening promises for customising treatments to individual patients.

Head and neck cancer is the 6th most common malignancy, accounting for approximately 4% of malignancies, and 1 – 2% of cancer-related deaths. Radiation therapy utilises high energy radiation to kill cancer cells. In head and neck cancer, radiotherapy is one of the main treatment modalities, particularly in curative-intent treatments. Despite advancements in imaging and radiation treatment planning and delivery, the prescribed dose and radiation treatment workflow remained unchanged and is largely ‘one size fits all’. Similarly, the survivorship program for patients with head and neck cancer is ‘one size fits all’, often one standard institutional follow up schedule for all patients treated for head and neck cancer, regardless of expected risk of treatment-related late toxicities, patients’ subsequent risk of recurrence and second malignancy. This thesis focuses on the value and efficacy of imaging and blood biomarkers in improving treatment personalisation in patients with head and neck cancer. In chapters 1 and 2, I explored the use of imaging and blood markers in the pre-, during, and post-radiotherapy settings to further improve risk stratification. In chapter 1, I investigated the potential use of readily available and ‘cheap’ blood biomarkers (neutrophil and lymphocyte counts) as predictors of subsequent outcomes in a large cohort of patients with oropharyngeal cancer. In chapter 2, I designed and conducted a prospective observational study to systematically characterize the kinetics of gross tumour volume and apparent diffusion coefficient (ADC) changes observed in magnetic resonance imaging (MRI) and circulating tumour cells (CTCs) counts during radiotherapy in patients with head and neck squamous cell cancer. In the survivorship period (Chapter 3), I evaluated the effectiveness of current surveillance program and investigated the potential use of PET imaging and alternative imaging frequencies to improve the cost-effectiveness of the survivorship program. In this chapter, I found that 70% of disease recurrence occur within 2 years and the probability of a surveillance imaging detecting a recurrence in an asymptomatic patient with no adverse clinical finding is very low. Furthermore, in patients with human papillomavirus (HPV)-related oropharyngeal cancer, achieving a complete response on post-treatment PET imaging has a negative predictive value of any subsequent recurrence of 92%, so the yield of surveillance imaging is very low in this group. Using a partially observed Markov decision model, a potentially effective surveillance program with less frequent imaging was propositioned in this chapter. Finally, in chapter 4, I assessed the potential use of re-irradiation in the era of modern imaging and new radiation treatment techniques including intensity-modulated radiotherapy (IMRT), proton therapy and stereotactic body radiotherapy (SBRT). I showed the value of different imaging modality (dual energy CT and MRI) in target delineation in patients who had previous radiation. In addition, I demonstrated that the local control rate for each treatment technique is similar. Although wide field radiotherapy (IMRT and proton therapy) had improve disease-specific survival, treatment with these techniques are longer (typically 6 to 7 weeks) and had higher toxicity rates than SBRT (delivered over 5 treatments).

Gastric cancer (GC) patients are mainly asymptomatic and present at an advanced stage with a 5-year survival rate of only 20-30% in most countries. It is crucial to gain greater insight into the process of gastric carcinogenesis in order to develop tools that will allow improved patient stratification and targeted surveillance. The relationship between intestinal metaplasia (IM), a key premalignant lesion in gastric carcinogenesis, and the diffuse type of gastric cancer (DGC) was investigated using data mining of GC patient pathology reports and existing gene expression data. IM and DGC were shown to be associated both histologically and molecularly in a proportion of cases suggesting that IM might be a precursor lesion to a subset of DGC cases. Complete and incomplete IM subtypes, the latter being associated with a greater risk of progression, were characterised at the molecular level using gene expression data acquired from macro-dissected IM tissue. Complete IM was associated with genes highly expressed in the small intestine whereas incomplete IM was associated with genes expressed both in the colon and in GC suggesting it is molecularly closer to a state of malignancy. Using OPAL multiplex immunohistochemistry, the macrophage and T cell landscape in IM was investigated. In addition to traditional “M1-like” (IRF8) and “M2-like” (CD163, CD206) macrophage populations, a novel hybrid subgroup “M1/2” containing macrophages expressing both M1 and M2 markers in different combinations was identified. Overall, complete IM was characterised by M2 macrophages and greater levels of T cell infiltration whereas incomplete IM was characterised by M1/2 macrophages and fewer CD8 and double negative (DN) T cells. Spatial cell analysis showed significantly fewer CD8 and DN T cells in the vicinity of incomplete IM epithelial cells suggesting reduced immune-surveillance may play a key role in progression to dysplasia. IM subtyping is affected by intra-observer and inter-observer variation. To address this, the potential of CD10 and Das1 as biomarkers for subtyping complete and incomplete IM was investigated. Overall CD10 was shown to be an outstanding biomarker for complete IM and Das1 was shown to have potential as an additional risk associated biomarker. GC animal models are associated with long duration and high cost. Optimised protocols for growing and characterising gastrointestinal organoids from gastroscopy biopsies were developed. IM organoid cultures were successfully established and characterised. Additionally a human gastrointestinal organoid biobank was created. In conclusion this thesis offers potential evidence of IM as a precursor lesion to DGC, characterises the molecular differences between complete and incomplete IM and shows key differences in the innate and adaptive immune system between IM subtypes and how these may play a role in progression to GC. It also identifies two biomarkers with potential clinical utility for subtyping IM and describes the methodology for growing IM organoids with future potential as a model for studying gastric carcinogenesis. Finally future studies are required to gain further insight into the cellular and molecular evolution of gastric carcinogenesis which should lead to better management of patients with premalignant lesions and ultimately to the prevention of GC.

Transcription driven by RNA Polymerase II (POLII) is a multi-step process that is strictly regulated by Cyclin dependent kinases (CDKs) at multiple checkpoints. Compared to the regulation of transcription initiation and pause release, the roles of CDKs in regulating transcription elongation remain poorly defined. To investigate the individual and shared roles of CDK12 and CDK13 in transcription regulation, a set of cell lines containing analog-sensitive variants of CDK12 and CDK13 were constructed using CRISPR-Cas9 homology-directed repair (HDR) technology. Multiple Next-Generation Sequencing (NGS) based assays including RNA-Seq, ChIP-Seq, PRO-Seq, TT-Seq were utilised to provide a comprehensive characterisation of the consequences of acute inhibition of CDK12, CDK13 or both kinases using these analog sensitive CDK12 and CDK13 cell lines. Selective inhibition of CDK12 or CDK13 led to various molecular responses including selective changes in gene expression, alternations in polyadenylation site usage as well as mild reduction in POLII elongation rate and processivity. In contrast, dual inhibition of CDK12 and CDK13 caused dramatic changes in transcription genome-wide, including the induction of widespread alternative polyadenylation events, reduction in POLII elongation rates and processivity, substantial changes in gene expression, as well as the near complete loss of POLII Ser2 phosphorylation. These observations illustrated that both CDK12 and CDK13 are regulators of POLII transcription elongation. Furthermore, the substantial differences between selective and simultaneous inhibition of CDK12 and CDK13 revealed the redundant and individual roles of CDK12 and CDK13 in maintaining global transcription elongation. To identify substrates of CDK12 and CDK13 that might be responsible for the phenotypes caused by CDK12 and CDK13 inhibition, phospho-proteomic analysis was performed to identify putative CDK12 and CDK13 substrates. The analysis revealed that CDK12 and CDK13 shared multiple substrates and functional redundancy between CDK12 and CDK13 in phosphorylating these substrates was identified. In order to identify the putative substrates that were responsible for the transcriptional changes upon CDK12 and CDK13 inhibition, a novel siRNA screen method “mini-bulk” CEL-Seq2 siRNA screen was developed and utilised. The screen revealed that SF3B1 and SRRM2 could be the potential substrates of CDK12 and CDK13 that were partially responsible for the transcriptional phenotype caused by the dual inhibition of CDK12 and CDK13, as depletion of SF3B1 and SRRM2 led to similar differential gene expression and alternative polyadenylation profiles as CDK12 and CDK13 inhibition. Finally, as the phospho-proteomic analysis also revealed that CDK12 and CDK13 might regulate phosphorylation of multiple translation regulators, the effect of CDK12 and CDK13 inhibition on protein translation was also investigated. Both nascent protein labelling as well as polysome profiling revealed that CDK12 and CDK13 function was required to maintain global translation. In conclusion, this thesis explored the role of CDK12 and CDK13 in POLII driven transcription and protein translation. CDK12 and CDK13 were shown to cooperatively regulate POLII transcription elongation processivity and alternative polyadenylation, potentially through regulating POLII Ser2 phosphorylation and the phosphorylation of other CDK12 and CDK13 substrates.

The Hippo pathway, first discovered in the fruit fly Drosophila melanogaster, is an evolutionarily conserved regulator of organ growth. The Hippo pathway controls organ growth by regulating the nuclear activity of the transcriptional coactivator Yorkie (Yki). Yki binds to sequence-specific transcription factors to regulate target gene expression, including the TEAD/TEF family transcription factor Scalloped (Sd). It was recently discovered that the Hippo pathway controls the growth of organs in Drosophila by balancing transcriptional activation mediated by Yki and Sd, and transcriptional repression mediated by Tondu-domain- containing Growth Inhibitor (Tgi) and Sd. If this fine balance is disrupted and Yki is hyperactivated, organs can grow beyond their normal size. Importantly, deregulation of Hippo signalling drives tumorigenesis in a variety of human cancers. The high degree of conservation of the Hippo pathway throughout the animal kingdom makes the fruit fly a powerful model organism to understand how this pathway regulates tissue growth during development and cancer. The Hippo pathway regulates the expression of genes involved in cell proliferation, survival, stemness, differentiation, and migration. However, we lack a clear understanding of the full transcriptional program that the Hippo pathway regulates in growing tissues beyond a small number of well-studied target genes. In this thesis, I aimed to deepen our understanding of the transcriptional program that the Hippo pathway controls in the growing Drosophila eye. To do this, I addressed three key questions: 1) What are the target genes of Yki, Sd and Tgi? 2) What gene expression changes occur as a result of hyperactive Yki? 3) What additional transcriptional regulators control Hippo pathway target gene expression and eye growth? 1) Using targeted DamID-seq, I identified target genes of Yki, Sd and Tgi in growing Drosophila eye discs. I found a very high degree of overlapping target genes between Yki, Sd, and Tgi, indicating that Sd is the key transcription factor that mediates binding of both Yki and Tgi to their target gene loci. Additionally, I found a strong enrichment of the AP-1 binding motif in Yki, Sd, and Tgi target genes, suggesting that these transcription factors co-regulate many target genes. 2) I performed comprehensive analyses of Yki, Sd, and Tgi target genes, and the changes in gene expression and chromatin accessibility caused by Yki hyperactivation. This highlighted key biological processes and signalling pathways that the Hippo pathway could regulate during eye growth, including: MAPK pathway, glutathione S-transferases (GSTs), cuticle development, extracellular matrix (ECM), and retinal determination and differentiation. 3) Using targeted DamID-seq, I found that the Drosophila AP-1 transcription factor Jra/Jun shares 71% of its target genes with Yki and/or Sd. However, using genetic studies, I showed that AP-1 transcription factors do not regulate physiological Drosophila eye growth, and are not essential regulators of Hippo pathway target gene expression. The results presented in this thesis provide new insights into the target genes and transcriptional program that is regulated by the Hippo pathway during Drosophila eye growth. They have identified previously unexplored biological processes that the Hippo pathway might regulate in the eye (e.g. cuticle development), and reinforced known roles for the Hippo pathway (e.g. cell fate specification). My findings also extend our understanding of the functional links between AP-1 and the Hippo pathway in growing organs. Importantly, the data generated in this thesis will provide a rich resource for researchers interested in studying the direct target genes of both Hippo signalling and AP-1 transcription factors.

Traditionally, metastatic melanoma had a dismal prognosis, but the recent advent of immune checkpoint inhibitors (ICI) has extended survival from months to years for some patients. There is an urgent need to identify prognostic and predictive biomarkers for melanoma patients treated with ICI, given that only a minority of patients respond, coupled with the potential treatment related toxicities. This thesis aimed to investigate clinical factors, functional PET imaging and tumour immune profiling as candidate biomarkers for ICI in patients with melanoma. Firstly, Chapter 3 focused on baseline performance status as a biomarker for outcome following anti-PD-1. The hypothesis was that unlike cytotoxic chemotherapy, baseline performance status was not correlated with outcome following ICI, owing to its distinct mechanism of action. However, in the cohort of 91 patients treated with anti-PD-1 at Peter MacCallum Cancer Centre, poor performance status was correlated with poor survival and low response rate to anti-PD-1. Furthermore, patients with poor performance status were more likely to be hospitalised and more likely to die in hospital. Patient characteristics and blood parameters were further examined in Chapter 4, but specific to a cohort of patients with melanoma brain metastases. Melanoma commonly metastasise to the central nervous system and this is associated with extremely poor survival. Recently, combination ICI has resulted in intracranial responses and durable survival. Most of the existing literature in biomarkers in melanoma brain metastases also predates the introduction of ICI, therefore investigation of biomarkers in patients with melanoma brain metastases treated with ICI is needed. A post-hoc analysis of patients with melanoma brain metastases as part of the phase II Anti-PD1 Brain Collaboration study was performed to identify possible predictors of clinical outcome or toxicity. In this study, patients were randomised to receive either nivolumab monotherapy or nivolumab in combination with ipilimumab. High C-reactive protein, a marker of systemic inflammation, was correlated with poor survival. Treatment with combination ICI, hypernatraemia and increased body mass index were associated with higher likelihood of severe toxicity at 120 days, whereas CRP was not associated with higher toxicity. The thesis then went on to examine the role of FDG PET functional imaging as a source of biomarkers for outcome following ICI in Chapter 5. Baseline pre-treatment tumoural FDG-PET avidity (measured by SUVmax or metabolic tumour volume) as well as FDG-avidity in the immune system (measured by spleen to liver ratio) were assessed in relation to survival outcomes. Interestingly, tumoural PET avidity was not correlated with survival, whereas high spleen to liver ratio was correlated with poor survival after ipilimumab. This was subsequently validated in a combined cohort of patients from two separate European centres. High spleen to liver ratio was correlated with low albumin in a multivariate analysis, thus suggesting a possible association with systemic inflammation. Early on-treatment PET (EOT PET) were assessed in a small subset of 16 patients, and several challenges were identified that may limit the use of FDG PET in this early juncture as a biomarker for outcome after ICI. In-depth characterisation of tumoural immune landscape is crucial to improving the understanding of melanoma immuno-biology, with potential implications for biomarker development. Chapter 6 aimed to compare the immune profile of UV related skin cancers (melanoma, cutaneous squamous cell carcinoma and Merkel cell carcinoma) using orthogonal methods of bulk RNA-sequencing and multi-spectral immunohistochemistry. The three skin cancers showed distinct immune landscapes, with melanoma having a significantly higher intratumoural T cell infiltrate compared to Merkel cell carcinoma, whereas PD-L1 density was highly variable across three skin cancers. Transcriptomic analyses of melanoma samples with high PD-L1 density were associated with upregulation of genes related to leucocyte proliferation, migration and adaptive immune responses, in contrast to MCC samples with high PD-L1 density, where such a signature was not observed. Lastly, an in-depth case study of six patients highlighted how multi-factorial biomarkers such as clinical factors, functional PET imaging, baseline blood parameters, and multi-spectral immunohistochemistry can be applied together. In conclusion, this thesis evaluated multi-factorial biomarkers including clinical, functional imaging and tumoural immune profiling biomarkers. These studies add to the evolving literature on biomarkers associated with ICI treatment. It is envisaged that with time, these complementary methods of understanding the patient and tumoural immune environment can aid rational selection of immune based therapies for patients with advanced melanoma.

The recent advent of targeted and immune-based therapies has revolutionized the treatment of melanoma, and transformed outcomes for patients with metastatic disease. However, the mechanisms underpinning the clinical efficacy of these approaches are still being elucidated. The majority of patients develop resistance to the current standard-of-care targeted therapy, dual BRAF and MEK inhibition (BRAFi+MEKi), prompting evaluation of a new combination incorporating a CDK4/6 inhibitor. Based on promising preclinical data, combined BRAF, MEK and CDK4/6 inhibition (triple therapy) has recently entered clinical trials for the treatment of BRAFV600 melanoma. Interestingly, while BRAFi+MEKi therapy was initially developed on the basis of potent tumor-intrinsic effects, it was later discovered to have significant immune-potentiating activity. Recent studies have also identified immune-related impacts of CDK4/6 inhibition, though these are less well defined and appear to be both immune-potentiating and immune-inhibitory. BRAFV600 melanoma patients are also eligible for immunotherapies, and hence the immunomodulatory activity of these targeted inhibitors makes first-line treatment decisions complex. The aim of this thesis was to examine the immunomodulatory effects of BRAF, MEK and CDK4/6 inhibition, with an ultimate goal of providing critical information to aid in the clinical management of BRAFV600 melanoma patients. Examining mechanisms of the immunomodulatory effects of targeted therapies requires preclinical mouse models of melanoma that are both immunogenic, and harbor the oncogenic drivers targeted by the therapies being evaluated. To address this, we developed a novel immunogenic BrafV600ECdkn2a-/-Pten-/- melanoma mouse model, called YOVAL1.1. YOVAL1.1 tumors are transplantable in immunocompetent mice and amenable to standard-of-care melanoma therapies, including BRAFi+MEKi and immune checkpoint blockade. This, coupled with the Cdkn2a status, which infers some sensitivity to CDK4/6 inhibitors, makes this an ideal preclinical model to evaluate the immunomodulatory effects of the triple therapy. Using this model, we demonstrated that triple therapy promotes durable tumor control through tumor-intrinsic mechanisms, while promoting immunogenic cell death and T cell infiltration. However, despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade. Flow cytometric and single cell RNA-seq analyses of tumor infiltrating immune populations revealed that triple therapy markedly depleted pro-inflammatory macrophages and cross priming CD103+ dendritic cells, the absence of which correlated with poor overall survival and clinical responses to immune checkpoint blockade in melanoma patients. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T cell responses ex vivo. Hence, while combined BRAF, MEK and CDK4/6 inhibition demonstrated favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact on anti-tumor immunity. Several recent studies have reported immune-potentiating effects of CDK4/6 inhibition, and subsequent synergy with immune checkpoint blockade. However, T cells are the primary target of these immunotherapies, and an understanding of the direct effects of CDK4/6 inhibition on this cellular subset was lacking. In this thesis, using integrated epigenomic, transcriptomic and single cell CITE-seq analyses, we identified a novel role for CDK4/6 in regulating T cell fate. Specifically, we demonstrated that CDK4/6 inhibition promoted the phenotypic and functional acquisition of T cell memory. Genome-wide CRISPR/Cas9 screening and phospho-proteomics revealed that memory formation in response to CDK4/6 inhibition was cell intrinsic and required RB. Pre-conditioning human CAR T cells with a CDK4/6 inhibitor enhanced their persistence and tumor control, and clinical treatment with a CDK4/6 inhibitor promoted expansion of memory T cells in a melanoma patient, priming a response to immune checkpoint blockade. Collectively these findings highlight the multi-faceted immunomodulatory activity of BRAF, MEK and CDK4/6 inhibition. The addition of a CDK4/6 inhibitor to dual BRAFi+MEKi led to the depletion of intratumoral myeloid subsets that may be critical for supporting a therapeutically beneficial T cell response. In contrast, as an individual therapy, CDK4/6 inhibition promoted effector and memory T cell activity, suggesting that, with optimal scheduling to prevent myeloid depletion, CDK4/6 inhibitors may be used to enhance and prolong BRAFi/MEKi-induced anti-tumor T cell immunity. Defining the mechanisms that underpin the clinical efficacy of these available therapies is a critical step forward in optimising novel combination and scheduling approaches to combat melanoma and improve patient outcomes.

Background: In human brain cancers, glioblastoma stem cells (GSCs) originate from neoplastic transformation of neural stem cells (NSCs) or dedifferentiation of other neural cells. Similar to normal NSCs, GSCs possess stem-cell properties to self-renew and differentiate into multiple neural lineages. However, GSCs are more plastic than normal NSCs, as they can transdifferentiate into other cell types. At the present, we do not fully understand the cellular step-wise conversion from GSCs into other distinct cell lineages, and the molecular mechanism responsible for this event. Drosophila NSCs, called neuroblasts (NBs), also asymmetrically divide to renew themselves, and generate neurons or glia that make up the adult central nervous system. Disrupting either asymmetric cell division or neuronal maintenance allows differentiated cells to dedifferentiate into ectopic NBs, which then continue to proliferate and form tumours. Methods and aims: We utilised several in vivo brain tumour models in Drosophila to study how GSCs function in brain cancers and how they undergo transdifferentiation. In this thesis, I induced loss-of-function of transcription factors Prospero or Nerfin-1 in NB lineages to generate dedifferentiation-driven tumours, and found a class of cells which exhibited glial cell identity. I sought to investigate the behaviours and characteristics of these ectopic glia to elucidate some aspects about GSC transdifferentiation by answering three questions: (1) What are the cell numbers and cell types within the pros- and nerfin-1- tumours. (2) Do ectopic glia arise by transdifferentiation of NBs in the tumours? (3) Which transcription factors and signal transduction pathways drive the formation of ectopic glia, and are ectopic glia required for tumour growth? Results: (1) I found that the expansion of ectopic glia population is correlated with overall tumour growth. Ectopic glia exhibit glial identity and their formation is not dependent on the location of tumours in the central nervous system. (2) By performing live-cell imaging of pros- tumours and molecular marker analysis, I found a subset of NBs switch to glial cell fate. (3) I performed genetic experiments to manipulate the transcription factors dichaete, tailless and glial cells missing (gcm) in pros- tumour and found that they are required for ectopic glial formation and tumour growth. Their target gene reversed polarity (repo) regulates the formation of ectopic glia, which in turn, promote the tumour growth. I showed that Notch promotes tumour growth independently of its effect on ectopic glial formation, as Notch regulates the tumour growth in the absence of ectopic glia. I also showed that FGF signalling pathway promotes tumour growth by regulating ectopic glia formation, as it does not affect the tumour growth in the absence of ectopic glia. Hippo pathway also plays a role in promoting the formation of ectopic glia and tumour growth. Our study of pros- and nerfin-1- tumour models in the context of transdifferentiation may extend our understanding of the biology of NBs and may shed light on GSC behaviours upon their transdifferentiation into different cell types. We can use the underlying mechanisms of these phenotypes to gain a better understanding of the transdifferentiation events at the molecular and cellular levels. As most genes and signalling pathways examined in this study are also found in human brain cancers, this study will enhance the knowledge of how cell fate changes can influence the tumour malignancy.

This thesis is situated in the discourse of risk that defines our technology-driven modern society, wherein one’s health is constructed as a personal and moral responsibility. A key contributor to the individualisation of risk in modern medicine is genomic technology. As genomics becomes progressively normalised in mainstream society, individuals of younger and younger ages are seeking to learn of their genetic risk of disease, including cancer. Young people occupy a formative and transitional life stage with complex processes of human development, making them a unique population for which genetic services are currently ill-equipped to serve appropriately. This research contributes to a new field of genetic counselling research that aims to explore and meet the distinctive developmental needs of young people with genetic disease. This thesis comprises of three inter-related studies that explore the psychosocial implications of living with a devastating, early onset inherited cancer condition, Li- Fraumeni syndrome (LFS), from the perspectives of young people and health professionals in Australasia. Informed by a pragmatic-critical realist stance, this thesis uses a mixed-method approach divided into a qualitative and quantitative phase. The qualitative phase consists of two studies, the first is a systematic review and thematic synthesis of 39 studies investigating how young people experience inherited disease with similar implications to LFS. The second and principal study of this research is an interpretive description of how young people experience LFS in their daily lives. To develop findings, I conducted interviews with 30 adolescents and young adults (aged 15- 39 years) with, or at 50% risk of, a pathogenic germline mutation in TP53 from across Australia, and used reflexive thematic analysis. The quantitative phase consists of a survey of 43 Australasian health professionals who care for young people with LFS to document their current practices and how they meet the developmental needs of this population. The first key finding is that experiences of cancer, either familial or personal, strongly influence how young people make sense and meaning of LFS, how they consider genetic testing, and their perceptions of cancer risk and mortality. The nature of LFS in terms of its high penetrance, early onset, and varied phenotype meant young people’s experiences were unique in oncology and genetic settings and require in-depth exploration during genetic counselling. The second key finding was that young people’s growing autonomy from family complicated the process of genetic testing, which was recognised by health professionals who worked to foster their autonomous decision- making. Genetic testing and whole-body cancer risk management represented instrumental actions of control for young people to mitigate their cancer risk from LFS, a perspective shared and promoted by health professionals. Intensive risk management and risk-reducing surgery, however, was emotionally and logistically burdensome for some young people, constructing the body as a material object be scrutinised in great detail by health professionals and blurring boundaries between self and body. The third key finding was that young people felt strongly about preventing the passage of their LFS- causing genetic variant to the next generation, reporting that pre - implantation genetic diagnosis was their preferred method for having a ‘healthy’ biological family. Few had reached a life stage, however, where they appreciated the ethical, financial and logistical burdens of this technology, and many deferred reproductive decision-making until they were ‘ready’. This research argues that young people with inherited disease have unique psychosocial and clinical needs that are directly tied to the formative developmental tasks of their life stage. They require specialist youth-friendly counselling that considers and appreciates their developmental needs both during genetic testing and beyond. Youth-friendly genetic counselling must therefore be longitudinal, incorporating psychosocial check-ups as a key clinical interaction. Further, youth-friendly models of care promote the distinct value of genetic counsellors in managing rare and complex inherited conditions. As the mainstreaming of genetic services begins to outsource tasks of genetic education and pre-test counselling, the genetic counselling profession must make better use of their psychosocial counselling skills to serve high-needs populations. In this context, young people stand out as a key focus. Finally, this research argues that attending genetic counselling exposes young people to a discourse of risk bounded by an ethos of responsibility. Undergoing genetic testing, subjecting their bodies to intensive surveillance and risk-reducing surgery, and having a family with reproductive technology all stand out as the ‘right thing’ to do when living with genetic risk of disease. Yet, each presented complex psychosocial implications for young people that were intrinsically linked to the broader effects of our modern risk society defined by Beck (1992) and Giddens (1991). Young people’s decision-making and preferences during this developmentally labile life stage therefore need to be interpreted in relation to normative societal pressures that dictate expected behaviour around risk.

Mutations in epigenetic regulators frequently occur in AML. Furthermore, the epigenetic landscape is typically dysregulated in AML. Targeted epigenetic therapeutics that directly inhibit mutant epigenetic oncogenic drivers, such as AG120 inhibition of IDH1R132H, have shown clinical success for the treatment of IDH1-mutant AML. Other epigenetic therapies target wildtype epigenetic regulators, such as DNMT1 inhibition by hypomethylating agents, with the aim of reprogramming the transcriptional networks driving malignant progression. Despite some patients achieving clinical remission, many AML patients do not respond to epigenetic therapies or relapse post-treatment. Furthermore, the contribution of mutant epigenetic regulators to leukaemogenesis is relatively poorly understood. A clinically and pathophysiologically relevant murine model of IDH1R132H driven AML co-expressing oncogenic DNMT3A and Nras was utilised to investigate the cellular and molecular consequences of IDH1R132H expression in leukaemia initiation and progression. Results from this thesis suggest that IDH1R132H governs distinct leukaemic properties within the different AML cell-types; IDH1R132H drove the expression of genes that underpin HSC-like self-renewal and proliferation in leukaemic progenitor-like cells, whilst IDH1R132H most profoundly impaired the differentiation capacity of leukaemic immature neutrophils. Furthermore, this thesis demonstrated that AG120 was a highly effective and on-target IDH1R132H inhibitor for the treatment of IDH1R132H driven in vivo AML, and induced neutrophil-skewed differentiation with distinct kinetics throughout the hierarchy of leukaemic cells. Mechanisms of resistance to the hypomethylating agents AZA and GDAC were investigated utilising a genome-wide CRISPR/Cas9 screen in vitro. Members of the pyrimidine salvage pathway were implicated in the resistance to hypomethylating agents. Loss of DCK and UCK2 conferred resistance to GDAC and AZA, respectively, whilst SLC29A1 loss mediated resistance to both hypomethylating agents in both in vitro and in vivo models of haematological malignancies. This thesis demonstrated that DCK and UCK2 loss remained sensitive to AZA and GDAC, whilst cells with SLC29A1 loss were sensitive to inhibition of DHODH. This research provided comprehensive insight of the impact of IDH1R132H on distinct leukaemic cell subsets, transcriptional mechanisms underlying leukaemogenesis, and the transcriptional perturbations that may contribute to AG120 resistance. Furthermore, the results of this thesis suggest that AZA and GDAC treatment are potential therapeutic avenues for AML resistance driven by DCK or UCK2 depletion or loss, respectively. Moreover, the results described herein suggest that targeting the de novo pyrimidine synthesis pathway, through DHODH inhibition, is a therapeutic strategy to overcome resistance to hypomethylating agents mediated by the depletion of pyrimidine salvage pathway enzymes.

The advent of next-generation sequencing (NGS) has allowed researchers to appreciate the enormous heterogeneity that exists between cells within a single tumour. This intratumour heterogeneity leads to diverse phenotypic outcomes, resulting in functionally distinct subpopulations of cancer cells. This functional heterogeneity fuels tumour evolution and therapeutic resistance and is thus a major barrier to producing cures in cancer. Acute myeloid leukaemia (AML) is an aggressive and heterogeneous malignancy with a high relapse rate. The prevailing paradigm to explain relapse in AML posits that genetic heterogeneity leads to pre-existing or acquired mutations that render certain cells refractory to therapy, resulting in the outgrowth of a resistant clone. Large-scale sequencing studies aimed at cataloguing genetic heterogeneity in AML have revealed several important observations. Firstly, AML has one of the lowest mutational burdens of any cancer. Secondly, a significant proportion of clinical relapse events cannot be attributed to an underlying genetic change. These important findings raise the possibility that mutations alone are insufficient to fully explain therapeutic resistance in AML. Indeed, we are now beginning to appreciate that both tumour evolution and clinical relapse can be driven by non-genetic processes. However, characterising the full extent of non-genetic heterogeneity and its relative contribution to both the evolutionary trajectory of the disease and therapeutic resistance requires innovative single cell methodologies. Single-cell RNA sequencing (scRNA-seq) has been instrumental in revealing the phenotypic heterogeneity of rare subpopulations of cells within a complex tumour. However, it is difficult to infer clonal relationships from scRNA-seq alone and this has hampered our ability to understand how individual malignant cells evolve over time. To overcome some of these challenges, we present a lentiviral method of tagging cells with unique heritable barcodes that are stably transcribed into RNA molecules in cells and therefore highly detected in microfluidic scRNA-seq workflows. This strategy, termed Single-cell Profiling and LINeage TRacking with expressed barcodes (SPLINTR), offers the ability to match the gene expression programmes of individual cells to their clonal lineage, in order to establish how initial transcriptional differences amongst heterogeneous malignant cells can shape thier future clonal behaviour during cancer progression. We apply our SPLINTR barcoding system to an in vivo model of clonal competition in order to determine the early transcriptional signatures that are associated with future clonal dominance in AML. We discover that clonal dominance is largely an intrinsic property amongst genetically identical clones. However, we find the deterministic nature of dominance is altered by the presence of other distinct competing mutational clones. Furthermore, SPLINTR enabled us to retrospectively identify a novel set of differentially expressed genes contained within certain clones prior to transplantation, which distinguished them from losing clones and was associated with their future dominance during disease progression. Finally, we find that resistance occurs to BET inhibitor therapy in the clinic in the absence of a clear genetic event. scRNA-seq of paired baseline and relapse AML patient bone marrow samples revealed than non-genetic resistance originates from either a population of pre-existing cells that phenotypically resemble LSCs, or through transcriptional adaptation as a result of therapeutic pressure. We then use SPLINTR coupled with scRNA-seq to interrogate our previously published in vitro model of non-genetic resistance to BET bromodomain inhibition. This provided further evidence that Lamarckian evolution in the form of gradual transcriptional adaptation drives non-genetic resistance. Future work aims to unravel the epigenetic states that mediate non-genetic transcriptional adaptation in a broader therapeutic context in AML. Collectively, the research presented in this thesis demonstrates the importance of applying novel single cell technologies to investigations of cellular diversity in cancer and highlights the underappreciated role of non-genetic heterogeneity in driving both disease evolution and therapeutic resistance in AML. These studies provide the molecular tools and rationale to further define the mechanisms by which non-genetic heterogeneity shapes cellular behaviour in cancer.

Adoptive cell therapy using chimeric antigen receptor (CAR) T cells has shown remarkable efficacy in the treatment of haematological malignancies, with complete remission rates of 90% reported in early clinical trials. Following this success, two CD19 targeting CAR T cell products have been approved in Australia for the treatment of Acute Lymphoblastic Leukaemia (ALL) and non-Hodgkin lymphoma. However, acquired resistance to this therapy through CD19 antigen loss is an emerging problem. Moreover, such promising results have not been recapitulated against solid tumours. This is thought to be due in part to poor CAR T cell trafficking and infiltration, the immunosuppressive tumour microenvironment and antigen heterogeneity in solid tumours, as well as intrinsic and acquired tumour resistance. Therefore, it is necessary to uncover mechanisms of resistance to CAR T cell therapy in both the haematological and solid tumour settings, in order to improve patient outcomes. In this thesis, we sought to uncover the tumour-intrinsic mechanisms of resistance to CAR T cell therapy. Whole-genome loss-of-function CRISPR-Cas9 screening has recently emerged as a powerful tool to screen all protein-coding genes in the genome for conferring resistance to various immune and drug pressures. Herein, we utilised a genome-wide mouse pooled-sgRNA library to investigate genes that protect MC38 mouse adenocarcinoma cells from CAR T cell killing. Here, we identified that loss of cytokine response pathways, in particular, TNF and IFN-y signalling were critical for the effector function of CAR T cells. Additionally, we report for the first time that loss of the transcriptional co-binding partner Cbfb protects tumour cells against T cell killing. Investigation in to the mechanisms of Cbfb-mediated resistance uncovered that loss of tumour cell Cbfb protected tumour cells against T cell-derived TNF. Importantly, we report that mice bearing Cbfb-deficient MC38 tumours did not respond to anti-PD-1 in vivo, and analysis of clinical trial data suggests that in melanoma cohorts, low expression of CBFB or the transcriptional co-binding partner RUNX1 was correlated with a poorer prognosis following immune checkpoint blockade or adoptive cell therapy. Next, we sought to design a rational combination therapy to overcome some of the challenges associated with CAR T cell therapy against solid tumours and enhance CAR T cell therapy in this context. Based on data from the CRISPR screen which highlighted the role of TNF in CAR T cell-mediated cytotoxicity, we sought to enhance TNF-mediated killing using a small-molecule smac-mimetic. We demonstrated that antagonism of the Inhibitor of Apoptosis Proteins with the smac-mimetic birinapant, significantly enhanced CAR T cell killing in a TNF-dependent manner. Using a syngeneic HER2+ self-antigen model, we report that birinapant significantly enhanced CAR T cell-mediated tumour clearance in a combination therapy approach, and we demonstrated enhanced human CAR T cell killing in patient biopsy-derived tumouroids. Critically, we report that birinapant significantly enhanced TNF-mediated “bystander killing”, which occurred in the absence of target antigen expression, which may be a strategy to overcome the challenge of tumour antigen heterogeneity associated with solid tumours. Finally, we sought to uncover mechanisms by which B cells lose CD19 antigen expression following CAR T cell attack. By utilising a second genome-wide CRISPR screening approach, we applied a human pooled sgRNA library to the CD19+ B-ALL cell line MHH-CALL4, and sorted 3 times the cells with the lowest 20% CD19 surface expression by flow cytometry, in order to uncover genes or pathways of interest that are implicated in down-regulation of CD19 surface expression. Along with several known factors, such as CD81 trafficking and N-linked glycosylation, we also identified several novel processes including histone methylation, aryl hydrocarbon receptor processing and E3 ubiquitin ligase activity, which will be the focus of future investigations. In summary, insights gained from the work in this thesis elucidate some of the resistance pathways utilised by tumour cells in order to evade immune pressure from CAR T cells, and highlight the cytotoxic potential of TNF in CAR T cell therapy. In addition, we herein present a novel strategy to enhance CAR T cell efficacy in solid tumours, and demonstrate the potential for combination therapy approaches to overcome the challenges associated with CAR T cell therapy against solid tumours. Taken together, this work significantly addresses several of the existing limitation of CAR T cell therapy in both B cell leukaemias and against solid malignancies, and the novel combination therapy we describe is poised to be rapidly translated in to the clinic.

Anal SCC is a rare disease that has increased significantly in both incidence and mortality over the last fourty years. Definitive chemoradiotherapy is the primary modality of treatment, offering a 5-year overall survival rate of 65%. For patients with locally persistent or recurrent disease, salvage surgery is an option with a 5-year overall survival of 50%. However, for those patients with un-resectable locoregional or metastatic disease, there are limited treatment options, and patients face a dismal outcome. Progress in identifying new treatment options for patients with anal cancer has been hampered by a deficiency in understanding the underpinnings of the disease and a lack of appropriate preclinical models. This thesis has focussed on addressing both of these deficiencies in addition to assessing the success of salvage surgery at a quaternary centre in Australia. Firstly, an attempt has been made to further our understanding of the biology of Anal SCC. This was undertaken by exploring the immune and genomic landscape of ASCC, to identify potential prognostic and therapeutic biomarkers. This has provided insight into the prognostic power of assessing the CD8+ immune infiltrate in Anal SCC. It has also identified PI3K aberrations as a frequent genomic event that may serve as a future therapeutic target. Secondly, it has led to the establishment of both human and mouse preclinical models of this disease. This includes the world’s first panel of human anal SCC cells lines and a syngeneic mouse model. Both of these pre-clinical models have been validated and characterised, with features closely resembling the human disease. These models can now act as a platform to further explore and facilitate investigation into potential new therapeutic options in this disease.

Oncogene-induced senescence (OIS) is a critical anti-tumour mechanism by arresting cell proliferation following oncogenic activation in normal cells. Cell cycle withdrawal along with senescence-associated secretory phenotype, deregulated metabolism and macromolecular changes are the hallmarks of cellular senescence. The PI3K/AKT/mTORC1 pathway is a key signalling pathway for homeostatic control of cell growth, proliferation, and survival. Molecular aberrations in the PI3K/AKT/mTORC1 signalling pathway have been identified in one third of solid tumours and more than 40 cancer types. Paradoxically, PI3K/AKT/mTORC1 hyperactivation causes a senescence-like phenotype in non-transformed cells. While most studies have focused on RAS/MAPK-induced senescence (RIS), little is known about the mechanisms regulating AKT-induced senescence (AIS) and its potential tumour-suppressive function. We hypothesise that understanding the molecular requirements for AIS and determining the essential regulators involved in AIS will identify therapeutic vulnerabilities for treatment of PI3K/AKT/mTORC1-driven cancer. To characterise the molecular landscape of AIS, we performed the RNA-seq and metabolomics analysis in BJ-TERT human skin fibroblasts overexpressing AKT1 or HRASG12V in Chapter 3. Compared to normal proliferating cells, AIS and RIS cells exhibited distinct metabolomes and transcriptomes, supporting metabolic and transcriptional reprogramming in OIS. The common molecular signatures in both AIS and RIS cells well represent the hallmarks of senescence and the molecular signatures that are unique for AIS or RIS are associated with the distinct molecular mechanisms underpinning these two types of OIS. The similar metabolic profile presented by the AIS and RIS cells with enhanced glycolysis, TCA cycle and oxidative phosphorylation to maximise energy production indicates a highly active metabolic status in OIS. To gain more insight of metabolic dependency of AIS, we utilised Cystathionine beta-Synthase (CBS), one of the top candidates in an RNAi screen we recently performed to identify key regulators of AIS and characterised its role in AIS maintenance in chapter 4. CBS acts as a hub for coordinating the transsulfuration and transmethylation pathways, regulating the biosynthesis of hydrogen sulfide (H2S) and glutathione (GSH) and modulating mitochondrial functions and cellular bioenergetics. We demonstrated that 1) the upregulation of transsulfuration pathway activity is an AIS-specific antioxidant response. 2) the metabolic reprogramming potentially couples to epigenetic alterations to maintain AIS. 3) CBS depletion releases oxidative stress through suppression of mitochondrial oxidative phosphorylation and causes AIS escape. The finding of loss of CBS resulting in the release of the senescence brake engaged by AKT hyperactivation promoted us to further evaluate the potential tumour suppressor role of CBS in gastric cancer. We provided in vitro and in vivo evidence to support that CBS depletion synergises with PI3K pathway activation to promote gastric cancer initiation and restoration of CBS inhibited gastric tumour growth via H2S. These findings provided an example of harnessing metabolic vulnerabilities therapeutically for treating cancer. Overall, our study provides novel mechanistic insights into AIS maintenance and more importantly, reveals the essential role of the metabolic reprogramming in AIS maintenance and escape. Further investigation of the molecular mechanisms underpinning PI3K/AKT/mTORC1-driven senescence and tumorigenesis will facilitate developing appropriate strategies to improve human health in aging, aging-related diseases, and cancer.

Radiation therapy (RT) has evolved over more than a century into a well-established, highly sophisticated and major cancer treatment modality today. A paradigm shift that has occurred within the last two decades is the growing understanding that RT can induce host immune responses that contribute to tumour control, beyond direct radiation-induced cytotoxicity. Contemporaneously, the advent of modern cancer immunotherapy such as immune checkpoint inhibitors has revolutionised the field of oncology and highlighted the potential of harnessing the immune system to suppress and eradicate tumours. Inevitably, resistance to cancer immunotherapy has also brought into focus immunological barriers that preclude cancer immunity. In this context, an increasing body of pre-clinical and clinical studies substantiate the use of RT as a unique candidate to complement cancer immunotherapy in non-overlapping mechanisms to overcome such barriers. However, instruction on the optimal integration of RT and cancer immunotherapy is scarce. For the radiation oncologist, an outstanding gap in knowledge is how radiation dose-fractionation influences the immunomodulatory effects of RT and its synergy with cancer immunotherapy. In this PhD project, mouse models of solid cancer were used to systematically interrogate this question by employing a series of rationally selected radiation dose-fractionation regimens to dissect the immunological impact of dose per fraction (DPF) from that of total dose, as represented by biological effective dose (BED). In orthotopic AT3-OVA mammary carcinomas, radiation-induced CD8+ T cell responses were found to be regulated by radiation DPF, rather than BED. By contrast, radiation-induced natural killer (NK) cell responses in the same tumours were independent of radiation DPF but required a sufficient BED. Mechanistic investigations examining the cellular and transcriptional changes in AT3-OVA tumours evoked by radiation demonstrated that the differential regulation of anti-tumour immune responses by radiation DPF and BED was not primarily dictated by differences in tumour cell-intrinsic immunogenicity, but rather by the effector and suppressor dynamics in the tumour immune microenvironment, of which regulatory T cells played a central role. Furthermore, cross-examination of subcutaneously implanted MC38 colon carcinomas and publicly available transcriptomic data of human cancers pre- and post-RT suggested that radiation-induced immune responses are also significantly shaped by the tumour type. Lastly, the impact of radiation dose-fractionation on the anti-tumour activity of immune checkpoint inhibitors targeting the adaptive and innate immune arms was examined in AT3-OVA tumours, confirming the corollary that RT and immune checkpoint inhibitors do not universally synergise, but require selection of radiation regimens and checkpoint targets that are predicated on biological rationale. Overall, this PhD project represents a comprehensive side-by-side pre-clinical study of the effects of radiation dose-fractionation on host anti-tumour immune responses. Results presented herein contribute towards a clearer understanding of this complex and clinically urgent question. More broadly, insights from this project will help guide the refashioning of RT into an exciting key adjunct in the immuno-oncology era.

Secondary lymphoedema is a common, chronic disease caused by inadequate drainage of interstitial tissue fluid from a limb due to damaged lymphatic vessels. It may develop after radiation therapy or cancer surgery involving lymph node dissection, in particularly for breast cancer, as well as a variety of other conditions. The resulting accumulation of interstitial fluid promotes pathological changes including oedema, expansion of fat and dermal fibrosis, which contribute to extensive chronic tissue swelling, typically in the upper or lower limbs. There is no curative treatment or molecular-based therapy for secondary lymphoedema, and current treatments for this condition have limited efficacy so it is an important unmet clinical need in medicine with an estimated 300,000 patients in Australia. A pharmacological intervention to restrict or reduce expansion of fat tissue and associated tissue swelling would be highly beneficial for patients however, the molecular mechanisms driving the development of fat in secondary lymphoedema are unknown. Here, a surgical mouse tail model of secondary lymphoedema is employed to investigate molecular pathways that drive expansion of fat tissue in this condition, and to explore a potential pharmacological approach for restricting this process. The mouse model of secondary lymphoedema employed exhibited key pathophysiological features of clinical secondary lymphoedema such as tissue swelling, subcutaneous oedema, dermal fibrosis and excess formation of fat. With the expansion of fat occurring during the early phase of lymphoedema, and elevated levels of fat persisting in the chronic phase, this mouse lymphoedema model was well suited for studying molecular mechanisms driving the initial expansion of fat as well as the persistence of excess fat tissue in the chronic setting. Whole-genome microarray analysis was used to study the mouse lymphoedema model which revealed that mRNA for insulin-like growth factor binding protein 5 (IGFBP5), an inhibitor of the insulin-like growth factor 1 receptor (IGF1R) adipogenic signalling pathway, was down-regulated in the model compared to controls. Further analyses by immunohistochemistry revealed the presence of IGF1 and IGF1R on adipocytes in the model and in a clinical sample of secondary lymphoedema, and activated IGF1R in the clinical sample. Moreover, the levels of IGF1 (an activating ligand for IGF1R) associated with adipocytes, were elevated in the mouse lymphoedema model. Thus, the IGF1R signalling axis could be active and promote expansion of fat tissue in secondary lymphoedema. Importantly, pharmacological targeting of this pathway in the mouse lymphoedema model with linsitinib, a small molecule tyrosine kinase inhibitor of IGF1R, led to significant reductions in tissue swelling and fat expansion which was associated with decreases in both the number and size of adipocytes. Furthermore, linsitinib also restricted the degree of dermal fibrosis in the mouse lymphoedema model. The work presented in this thesis demonstrated the role of the IGF1R signalling pathway in promoting adipogenesis in a mouse model of secondary lymphoedema. Hence, pharmacologically targeting IGF1R might be a viable therapeutic approach for restricting expansion of fat and tissue swelling in secondary lymphoedema patients.

Precision oncology refers to the use of sophisticated assays to tailor therapy to an individual patient. The feasibility of implementing such a program in a comprehensive cancer centre in Australia is unknown. The utility of precision oncology also depends on understanding how genomic profiles may evolve over time and differ between tumours in the same patient. A precision oncology program called SEGMENT was designed and implemented in a single centre. The program was popular, recruiting well over the study period. Timely acquisition of samples for sequencing was suboptimal from external pathology providers, and proved increasingly expensive during the study period. Delivery of results in a manner where they could be utilized by the patient was challenging in cases where patients were referred late in their natural history. A custom hybrid capture panel worked reliably. A total of 300 patients were recruited to the study, of which 288 had at least one sample received. Accounting for attrition, 214 patients or 71% went onto the main study. The spectrum of mutations and copy number alterations found in this study was similar to published cohorts. There were few differences between primary and metastatic lesions on average. Paired primary and metastatic samples however displayed discordance for both copy number and mutations. This was the case for actionable alterations in ESR1, ERBB2 and very rarely PIK3CA. Approximately 50% of patients had an actionable alteration. Of these 14% of the overall cohort received a therapy matched to their genomic profile. Five patients received matched therapy off trial and 26 received matched trial therapy. Three were zero responses in the off-trial group, and a response rate of 27% in the matched trial therapy group. To explore genomic heterogeneity in greater resolution, 4 patients with advanced breast cancer underwent rapid autopsies to collect large numbers of metastatic samples. Whole exome sequencing was performed on multiple lesions per patient which allowed inference of the subclonal structure. All patients displayed a monophyletic architecture, with truncal driver alterations giving rise to subclones with differing genomic profiles. One patient with a long disease free interval from primary to metastasis showed the acquisition of a new driver in the metastatic lesion. Driver alterations appeared to shape subsequent evolution.