Medicine (St Vincent's) - Theses
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ItemImproving epigenetic therapies for haematological malignancies( 2016)The work presented here is the product of several years of clinical trial and laboratory endeavour focused on the theme of understanding and improving currently available epigenetic therapies for blood cancers, and understanding fundamental aspects of clinical trial design. Several classes of epigenetically active drugs with proven clinical usefulness have received regulatory approval for the therapy of haematological malignancies in the last 5 years. At the same time, advances in high throughput sequencing technologies have provided landmark discoveries that reinforce the hypothesis that epigenetic mechanisms play a central role in the neoplastic process, as well as giving us new opportunities to develop targeted therapies. Some of the most exciting new targeted biological therapies arising from these discoveries such as inhibitors of histone methyl-transferases, the BET bromodomain inhibitors and the isocitrate dehydrogenase inhibitors are only now in the earliest phase of clinical development with initial signals that many of these agents will be both tolerable and active therapies for blood and solid tumours. In the context of the field at the start of 2010, only two classes of agent were available in clinical trials and for early investigator-initiated development. Even today, only these classes of epigenetic agents have marketing approval. As a consequence, histone deacetylase inhibitors and DNA methyl-transferase inhibitors are the focus of this thesis. The thesis presents investigator-initiated trials of agents in these two drug classes as well as the theory associated with the design of the trials. The literature review in Chapter 1 establishes the foundation for the clinical trial work. Here, the mechanism of action of the histone deacetylase inhibitors and the DNA-demethylating agents is reviewed. Activity of these agents in industry-sponsored clinical trials is presented as well as key toxicity data that informs the hypotheses and design of the clinical research presented in the thesis. The theories underlying the combination therapies that form the backbone of the thesis are discussed. Eltrombopag, a thrombopoietin agonist, is given particular prominence for its potential as a supportive care agent. The clinical trials that represent the majority of the thesis were designed to explore the critical questions of improving the tolerability of the DNA-demethylating agent azacitidine, and developing novel combination therapies for HDAC inhibitors. As experimental design is a critical question, a discussion on clinical trial design is presented in Chapter 2, which closes with a relevant case study. Several clinical trials were developed during the conduct of this higher degree from concept through to funding and clinical recruitment. Three, presented in Chapters 3, 4, and 7 have completed accrual and a further trial (Chapter 5) has been interrupted due to administrative challenges and is currently being reworked. The protocol for each study was in itself an extensive body of novel work contributing to the thesis. These are included in the appendices but summarised within the body of the thesis. Chapter 3 presents finalised and novel data analysed in depth of a trial of the combination of eltrombopag, a thrombopoietin-receptor agonist and azacitidine. The purpose of the study was to develop a supportive care therapy for patients with myelodysplastic syndromes, a disease that causes low platelets (thrombocytopenia), and has a risk of transforming to acute myeloid leukaemia, an almost invariably fatal event. Thrombopoietin mimetic agents have a somewhat complicated relationship with myeloid malignancies in the minds of researchers and while the receptor (mpl) and the role of the mpl receptor in haematopoietic stem cell survival and fate has been the subject of the research for at least 20 years, many questions about their theoretical safety remain open. There is considerable debate over the theoretical safety of mpl in agonists in patients with myeloproliferative diseases. Hence, attention is given to the rationale for the choice of eltrombopag for myelodysplastic syndrome in Chapter 1; also, the established body of preclinical data is presented to provide a context. This particular trial, “AzaE”, has been presented in international fora and the data has provided a foundation for a 170-centre, 43-country international randomised study that, if positive, may change clinical practice for patients with myelodysplastic syndrome and thrombocytopenia. Hence Chapter 3 is a central part of the novel data in this thesis. Based on the hypothesis that HDAC inhibition would up-regulate CD20 on the surface of malignant B-cells, a collaborating colleague, Mark Bishton, developed a phase 1 clinical trial of the combination of the radio labelled rituximab and the HDACi panobinostat. Dr Bishton and Prof Seymour wrote the trial protocol. As Principal Investigator of the study, I set up the study, moving it from protocol to execution. I oversaw the conduct of the study as the principal investigator and reviewed and approved the clinical data. The combination was toxic and the lessons learned are cogent and argue against further developing this sort of combination therapy. This study is presented in Chapter 4. In order to address the moderate activity of the HDACi inhibitor romidepsin and to broaden its clinical indications, a phase I/II study was designed in collaboration with academic partners from Yale University. The strategy behind the design of that study, the protocol itself, and initial data from six patients that have been recruited is presented in Chapter 5. That study has been halted due to regulatory challenges in the USA and Yale University that interrupted the research without warning. The trial strategy is discussed and preliminary results are presented. HDACi are highly active in cancers of the immune system, where the symptomatic and physical manifestations are to a considerable extent driven by cytokine changes which serve a both disease epiphenomena and drivers of the malignancy itself. In Chapter 6, I investigate the effect of the HDACi on human monocyte derived dendritic cells, identifying a mechanism by which these agents are likely shift the T-cell populations, altering patient symptoms and potentially inducing clinical responses. The findings support the contention that HDACi are immunosuppressive and do not make suitable partner drugs for treatments that benefit from an intact antigen presentation apparatus. Histone deacetylase inhibitors and DNA demethylating agents are often conceptually explained as driving a single cellular mechanism. However, the effects within the cell are pleiotropic and there are very few studies that provide data on predictors for response, especially so for the HDAC inhibitors. Therefore, a part of this work was to design a clinical trial that enables future scientific studies on predictors of response to HDAC inhibitors. Several academic questions were faced in developing the approach to this study. Chapter 7 presents the approach to the design of the clinical study, and presents the clinical features of the patients who have been recruited and key response data. An overview of planned experiments is presented. Having concerned itself with the design of investigator-initiated clinical trials of novel combinations and presented successes and failures of trial design and execution as well as original laboratory work that may inform combinations, the thesis concludes with a discussion on future directions for the projects arising from this work and epigenetic agents in general.
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ItemUnderstanding and manipulating epigenetic deregulations in osteosarcoma( 2016)Osteosarcoma (OS) is the most common cancer of bone and the 5th leading cause of cancer related death in young adults. Current 5-year survival rates have plateaued at ~70% for patients with localised disease. Those with disseminated disease have an ~20% 5-year survival. An improved understanding of the molecular genetics of OS translating into the identification of effective therapeutic targets may yield new approaches to improve outcomes for OS patients. To this end, I applied previously described murine models that replicate human OS to identify and understand dysregulated microRNAs (miRNA) and epigenetic modulators in OS. miRNA and epigenetic modulator expression patterns were profiled in murine primary osteoblasts, osteoblast cultures and primary OS cell cultures (from primary and paired metastatic locations) isolated from two genetically engineered murine models of OS. The differentially expressed miRNA were further assessed by a cross species comparison to human osteoblasts and osteosarcoma cultures. This led to the identification of miR-155-5p, miR-148a-3p and miR 335-5p as deregulated miRNA in OS. Additionally, miR-155-5p suppression or miR-148a-3p overexpression potently reduced proliferation and induced apoptosis in OS cells, yet strikingly, did not impact normal osteoblasts. To define how these miRNAs regulated OS cell fate, I used an integrated computational approach to identify putative candidate targets and then correlated these with the cell biological impact. While I could not resolve the mechanism through which miR-148a-3p or miR-335-5p impact OS, I identified that miR-155-5p overexpression suppressed its target Ripk1 (receptor (TNFRSF)-interacting serine-threonine kinase 1) expression, and miR-155-5p inhibition elevated Ripk1 levels. Ripk1 is directly involved in apoptosis/necroptosis. In OS cells, siRNA or small molecule inhibition against Ripk1 prevented cell death induced by the sequestration of miR-155-5p. Collectively I have shown that miR-148a-3p and miR-155-5p are species-conserved deregulated miRNA in OS. Modulation of these miRNA was specifically toxic to tumour cells but not to normal osteoblasts, raising the possibility that these may be tractable targets for miRNA based therapies for OS. Additionally, I investigated the outcome of targeting deregulate epigenetic modulators to achieve therapeutic efficacy in OS. The serine/threonine p21-activating kinases (Pak) are dysregulated in multiple cancers; however, their role in osteosarcoma (OS) is yet unknown. Using primary and metastatic OS tumour propagated cultures from murine models of OS I identified Pak1 and Pak2 as deregulated in OS. While reduced Pak1 expression in murine OS was not found conserved in human OS, the increased expression of Pak2, consistently existed across human and mouse OS. By inhibiting Pak2 gene expression levels using siRNA or pharmacological inhibition using 1,1′-Disulfanediyldinaphthalen-2-ol (IPA-3) and FRAX486, I demonstrated Pak2 inhibition to induce apoptosis in fibroblastic and osteoblastic murine OS cells in addition to primary human OS xenograft derived OS cells. The effects of Pak2 reduction or inhibition on cell proliferation and viability were proportional to the magnitude of Pak2 overexpression. Pak2 inhibition in normal osteoblasts, which have low basal Pak2 expression, did not induce changes in cell viability or proliferation. These findings demonstrate the efficacy of targeting Pak2 activity as a potential therapeutic strategy in OS. Finally, I investigated the effects of osteoblast restricted deletion of the oncogenic miR-17-92 cluster in delaying the onset of OS in Osx-Cre+ p53fl/fl Rbfl/fl murine models. While within my timeline I was unable to resolve the effects of conditional deletion of miR-17-92 cluster on OS onset, my parallel investigation of this deletion on skeletal and haematopoietic development in Osx-Cre+ R26eYFPki/+ miR17+/+, Osx-Cre+ R26eYFPki/+ miR17fl/+ and Osx-Cre+ R26eYFPki/+ miR17fl/fl models demonstrated unaltered parameters.
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ItemThe contribution of genetic variations in the region of the parathyroid hormone-like hormone, PTHLH, gene to breast cancer susceptibility( 2016)Aims: • Analyse the evidence for PTHLH and PTHrP, its protein product, playing a role in breast cancer and update the empirical definition of the gene. • Describe the 3-Dimensional structure of the PTHLH region and determine its system of regulatory interactions, including remote regulatory elements affecting PTHLH. • Integrate existing tools in addition to the novel perspectives generated above to enable a comprehensive annotation and analysis of genetic variants identified through molecular epidemiological techniques including Genome-Wide Association Studies (GWAS) and somatic DNA sequencing of tumour tissue to derive putative molecular mechanisms for the region’s involvement in breast cancer susceptibility. Methodology: • Review published studies and databases, integrating findings from diverse sources. • Acquire and analyse DNA and RNA sequencing, regulatory, expression, algorithm-inferred, and proximity-ligation data from multiple public data sources including ENCODE, ROADMAP, dbGAP, COSMIC and other to update the definition of PTHLH, and advance concepts of structure and regulatory function in the region. • Acquire and analyse primary GWAS data, performing imputation with multiple algorithms and references, and annotating associated variants with a suite of tools. • Use genome browsers including UCSC, WUSTL, and Golden Helix SVS, and their associated databases and tools, to analyse and visually integrate findings. Results: • PTHrP has multiple discretely functional segments active throughout the cell. It likely plays a bivalent and context-dependent role in cancer biology. Analysis of somatic variation in cancer suggests PTHrP may have a tumourigenic role within the nucleus. • PTHLH sits within a 1.3Mb TAD featuring multiple sub-structures that are integrated with the region’s regulatory function. There are activated chromatin hubs (ACHs) at protein-coding genes with evidence of extensive interaction between them. This is facilitated by the TAD’s structure, collocating them at the neck of the TAD. • The ACHs at MRPS35, KLHL42, and CCDC91 each monopolise a subordinate regulatory sub-net with a hierarchical structure. They each appear to act as important remote regulatory elements that integrate regulatory signals generated within their respective sub-nets, transferring them to PTHLH, and other genes, via ACH-ACH interactions. • There appear to be multiple discrete GWAS breast cancer association signals in the PTHLH region. Annotation of the associated variants suggests three particular regulatory elements may be its key drivers. In the context of the regulatory concepts developed in this thesis, the variants may affect a particular regulatory signal at multiple points in its assembly. PTHLH is the likely downstream target of this signal. • There are multiple poorly-describe coding, and non-coding, genes in the region that are also potential actors in breast cancer and should be investigated. Conclusion: • The PTHLH region is likely involved in the pathogenesis of breast cancer through the modification of PTHLH expression. There are likely to be other mechanisms in parallel that are yet to be fully described.