Sir Peter MacCallum Department of Oncology - Theses
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Molecular and functional analysis of Mucinous Ovarian Carcinoma
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.
Applications of massively parallel sequencing technology in the evaluation of haematological malignancies
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 determinants of sensitivity and mechanism of action of eprenetapopt (APR-246)
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.
Understanding the role of adenosine receptor signalling in chimeric antigen receptor (CAR) T cell therapy in solid cancer
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 . 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 . 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.
Antimicrobials in hospitalised and high-risk children: understanding and improving use
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.
Immunotherapy for Advanced Melanoma
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.
Mechanisms regulating Drosophila neural stem cell proliferation
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.
The control of lymphatic vascular remodelling in cancer by microRNAs
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.
Clinical implications of genomic driver alterations in hormone receptor-positive, human epidermal growth factor receptor (HER) 2-negative early breast 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.
Targeting the Hippo pathway in malignant mesothelioma
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.
The role of the MiT/TFE family of transcription factors in cytotoxic secretory granule formation in cytotoxic Tlymphocytes and the Niemann-Pick type C1 disease as a model
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.
Investigating oncogenic FMS-like tyrosine kinase 3 (FLT3)-induced metabolic reprogramming in acute myeloid leukaemia
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.