Medical Biology - Theses

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The role of venetoclax in the treatment of breast cancer

Muttiah, Christine Kumudhini ( 2023-03)

Breast cancer is the most commonly diagnosed cancer in women. The 5-year overall survival for metastatic breast cancer is approximately 30%. Despite rapid developments of new systemic therapies, cancer progression is almost inevitable. Ongoing research about treatment-resistance in breast cancer and mechanisms to overcome this are crucial to improving the lives of women with metastatic disease. Apoptosis is an important process for eliminating cancer cells. The BCL-2 pro-survival proteins and BH3-only proteins are key regulators of cell death. Cancer cells can evade apoptosis through over-expression of the pro-survival proteins. A new class of drug, the BH3 mimetics, inhibit the pro-survival proteins and have undergone extensive investigation in haematological malignancies. These drugs target BCL-2, BCLxL and MCL-1 and have shown promising activity in pre-clinical models. Venetoclax is a BH3 mimetic that specifically and potently binds to BCL-2. Venetoclax has transformed the treatment landscape for BCL-2- positive chronic lymphocytic leukaemia. It achieved fast-track FDA approval due to its success in the clinic. BCL-2 is overexpressed in approximately 70% of oestrogen receptor-positive metastatic breast cancers and approximately 30% of triple negative breast cancers. The aim of this thesis is to investigate the role of venetoclax in the treatment of BCL-2-positive breast cancer. We aim to achieve this through the design and implementation of three phase 1b clinical trials combining venetoclax with standard systemic therapy in the following clinical settings: (1) oestrogen receptor-positive and BCL-2-positive breast cancer in any line of metastatic treatment, (2) oestrogen receptor and BCL-2- positive breast cancer in the early lines of metastatic treatment (1st – 3rd line), (3) metastatic triple negative breast cancer. These clinical trials will firstly explore the safety and tolerability of venetoclax when combined with standard systemic therapy in metastatic breast cancer. Secondly, they will provide data on efficacy, survival and exploratory endpoints that inform future phase II and phase III studies. Findings from these later phase studies would determine whether venetoclax improves clinical outcomes in breast cancer and identify which patient subset might benefit. It is anticipated that due to the rapidly growing drug development in the area of BH3 mimetics, venetoclax may not be the most clinically relevant BH3 mimetic in the future. Therefore our clinical trial protocols could form a basis for future studies that investigate the next generation of BH3 mimetics or novel therapies targeting pro-survival proteins. The mBEP phase Ib clinical trial treated women with oestrogen receptor-positive and BCL-2-postive breast cancer in any line of metastatic treatment. This is the only study forming my thesis that has reached the primary and secondary endpoints. mBEP showed that the combination of tamoxifen and venetoclax was safe and tolerable. Secondary endpoints have confirmed that there is no adverse survival outcome from the addition of venetoclax to standard of care endocrine therapy.
Interleukin-6 family cytokines contribute to pancreatic cancer pathogenesis and can be targeted therapeutically

van Duijneveldt, Gemma Kate ( 2023-01)

Pancreatic cancer is a malignancy of significant unmet need. There have been negligible improvements in survival outcomes over the past four decades, with poor prognosis driven by late-stage diagnosis, which is in turn driven by multiple factors including a lack of early screening options, non-specific symptoms, and an aggressive tumour phenotype. The inflammatory and fibrotic nature of the disease presents many difficulties as the dense stroma and associated immune exclusion limit therapeutic efficacy, while the complex cellular network is highly heterogenous and drives pro-tumorigenic programmes through a multitude of cell-type and context dependent signalling pathways. The inherent intertumoral and intratumoral heterogeneity, in combination with the intricate and intertwining signalling pathways occurring within the pancreatic tumour microenvironment has complicated the delineation of pathogenic signalling mechanisms and identification of reliable therapeutic targets. The IL-6 family of cytokines and associated downstream mediators JAK and STAT3 have, however, been implicated in several pro-tumorigenic processes that drive tumour initiation and disease progression, though additional work is required to disentangle the mechanistic complexities of these signalling pathways. Clinical patient samples and associated data were analysed with the aim to understand the value of IL 6 family cytokine expression in predicting disease stage or survival outcomes. Though several associations were identified, our data did reflect the variation often observed in the literature between different patient cohorts, highlighting the need to consider multiple features during analysis. Of the IL-6 family cytokines, pancreatic IL-11 was associated with advanced disease stage, while IL-11, LIF, an OSM were individually associated with survival outcomes. Importantly, an additional analysis considering simultaneous expression of multiple IL-6 family cytokines demonstrated a significant relationship between elevated cytokine expression and reduced recurrence-free and overall survival. This represented an opportunity to increase the prognostic value of IL-6 family cytokines in pancreatic cancer through combinatorial approaches rather than individual analysis. Previous work highlighted a role of IL-11 in solid tumours, with IL-11 also being implicated in pancreatic ductal adenocarcinoma (PDAC). We utilised a PDAC mouse model crossed with IL-11R knockout mice to investigate the contribution of IL-11 signalling to disease pathogenesis. Analysis revealed that STAT3 was activated in PDAC tumours, with this reduced in mice lacking IL 11R. However, this reduction in STAT3 activation was not significantly associated with reduced disease progression or alterations to the tumour microenvironment. While the current data suggests that abrogation of IL-11 signalling is not sufficient to drive meaningful biological change, additional analyses are required. There are currently limited therapeutic options available for pancreatic cancer patients. Recently, the use of chemotherapy/immunotherapy in combination with drugs targeting components of the tumour microenvironment have shown promise. In this study, we generated IL-11, IL-11R, and gp130 nanobodies as a potential new therapeutic opportunity targeting these pro-tumorigenic signalling pathways within PDAC tumours. Characterisation of these nanobodies revealed one IL-11 and two gp130 nanobodies that were able to inhibit downstream STAT3 activation and reduce PDAC cell proliferation and migration in vitro. These nanobodies represent a novel approach by which IL-11 or multiple IL-6 family cytokine signalling pathways could be inhibited. In particular, the use of gp130 nanobodies may provide additional therapeutic benefit by simultaneously targeting multiple IL-6 family members, and thereby limiting potential compensatory mechanisms. Overall, this work has contributed to understanding the associations and roles of IL-6 family cytokines in pancreatic cancer, and characterised a novel therapeutic with which to target these pathways. The complexity of the tumour microenvironment poses obstacles in terms of our ability to understand and outline the mechanisms underlying tumorigenesis, but each additional piece of evidence is vital in informing the development of new and innovative therapies that are crucial to improving clinical outcomes for pancreatic cancer patients.
In vivo imaging of plasma cell dynamics in the bone marrow niche

Rimes, Joel Scott ( 2022-12)

Plasma cells are critical effector cells that form part of the adaptive immune system. They produce antibodies that can bind to and flag foreign pathogens for clearance by other components of the immune system. Plasma cells are long-lived immune cells that are preferentially found in bone marrow, where they home to and reside after generation in peripheral immune sites. However, much remains unknown regarding the specifics of their behaviour. The duration plasma cells can survive in the bone marrow, the factors required for their maintenance, the potential existence and composition of a ‘plasma cell niche’, as well as their kinetic behavioural profile are still debated. In this thesis, I explore the role of the bone marrow in the regulation of plasma cell maintenance in the context of autoimmunity and immunisation. To achieve this, I combined in situ imaging techniques in live mice with a novel B6.MRL-Fas/lpr and B6.AIDcre confetti mouse model that allows visualisation of plasma cells in the bone marrow. Using multiphoton in vivo microscopy, plasma cells of the bone marrow were revealed to exist in dense clusters in a highly non-migratory state. These clusters consisted of largely plasma cells of single founder origin and thus, likely generated via plasma cell proliferation in the bone marrow. Additionally, plasma cell clusters appeared to manifest uniquely in bone marrow environments and not in peripheral tissues such as spleen, lymph node, or kidney. Investigation of dysregulated and pathogenic plasma cell biology was performed in murine models of systemic lupus erythematosus (SLE). These systems demonstrated a highly dysregulated phenotype, exhibiting significantly expanded bone marrow plasma cell clusters. Additionally, standard glucocorticoid therapy failed to significantly reduce plasma cell populations in the bone marrow of diseased mice, suggesting the need for identification for new drug targets for the clearance of potentially pathogenic plasma cell clusters. A novel intravital imaging and RNA-sequencing paired analysis was established, termed high-resolution imaging of the direct environment with single cell RNA-sequencing (HIDE-n-seq). This approach suggested that altered proliferation, not survival, drove cluster formation in the bone marrow. Furthermore, we identified potential regulators of plasma cell clustering that could be candidates for design of new therapies for SLE patients.
Computational tools for long-read DNA methylation analysis and benchmarking complex single-cell genomics pipelines

Su, Shian ( 2023-03)

Developing new high-throughput assaying techniques necessitates the development of novel bioinformatics software that can not only extract insight from newly generated data types, but also evaluate the efficacy of newly developed tools. To this end, I created the NanoMethViz package to enhance the exploratory data analysis of DNA methylation data obtained from ONT long-read sequencing through the provision of data management and visualisation tools. The application of this software to female mouse placenta and neural stem cell samples enabled the study of methylation patterns in the context of X-inactivation. Additionally, the proliferation of single-cell analysis techniques and the need for comprehensive pipeline-level benchmarking led me to create the CellBench package, which can automatically execute complete combinations of methods to fully characterise the performance of single-cell analysis pipelines. This package establishes a benchmarking framework for combinations of methods that promotes modular code without duplication, resulting in readable, reproducible, and extensible pipeline benchmarking code. Both packages are open source and available through the R/Bioconductor repository, providing useful support for researchers who are working with these emerging and quickly advancing genomic technologies.
The Function and Druggability of Cryptosporidium parvum Aspartyl Proteases

Wiradiputri, Kharizta ( 2023-05)

Cryptosporidium is an apicomplexan parasite that causes cryptosporidiosis, a severe and life-threatening diarrhoea. Cryptosporidium is widely known as the second leading cause of diarrhoeal disease and deaths in children worldwide after rotavirus. Despite the massive impact on human health, there is no effective drug or vaccine to combat Cryptosporidium. The only FDA-approved drug is nitazoxanide, but it is not effective in children and people with compromised immune systems (eg. AIDS), who are the most vulnerable towards Cryptosporidium infection. Anthroponotic Cryptosporidium hominis (C. hominis) and zoonotic Cryptosporidium parvum (C. parvum) cause most human infections. Cryptosporidium parasites trigger disease by causing tissue damage, which is the result of parasite lytic life cycle. Throughout the life cycle, Cryptosporidium parasites rely on secretory proteins to invade and interact with the host cells, and these are commonly proteolytically matured by proteases. Aspartyl proteases are the best studied in apicomplexan species and have been shown to have interesting and important roles. In Chapter 1, I review the literature around the role of secretory protein maturation by proteases, in particular aspartyl proteases, and their potential as therapeutic targets. The most advanced in this respect is drugs targeting aspartyl proteases in the malaria-causing parasite, Plasmodium falciparum (P. falciparum). Almost nothing is understood about the function or druggability of Cryptosporidium aspartyl proteases. In this thesis, I explore both areas. I focus on two aspartyl proteases out of the five, as these are proved easiest to generate reagents to. In Chapter 3, I demonstrate that C. parvum aspartyl protease 1 (CpASP1) likely acts as a maturase of a secretory protein, rhoptry protein 4 (CpROP4). We show that CpASP1 is likely essential for the parasite survival and furthermore, can be potentially blocked by two inhibitors designed to target aspartyl proteases of P. falciparum. Strikingly, both compounds effectively block parasite growth in vitro and exhibit anti-cryptosporidial activity in vivo, indicating CpASP1 as a promising candidate for drug development. In Chapter 4, I characterized a second C. parvum aspartyl protease, CpASP5. We have discovered parasite adhesin, C. parvum glycoprotein 40 (CpGP40) as a likely substrate of CpASP5, and characterized its cleavage site. Interestingly, the loss of CpASP5 is likely lethal for the parasite, suggesting its requirement for progression of parasite life cycle. Together, this is the first study to reveal the function and druggability of Cryptosporidium aspartyl protease to treat cryptosporidiosis. Aspartyl proteases in apicomplexan parasites mature effector proteins that are exported by the parasites into the host. I am interested in the interaction between Cryptosporidium and its host, understanding what secretory proteins are involved. In Chapter 5, through biotin-based proximity labelling (PL), I identified candidate proteins that are likely to be exported by C. parvum into the host and are potentially the substrates of CpASP1 or CpASP5.
Moving towards personalised therapeutics for bowel cancer using patient-derived tumour organoids

Tan, Tao ( 2023-03)

Improving outcomes for patients with colorectal cancer (CRC) requires better use of current therapy options based on molecular biomarkers and predictive cancer models and the discovery of new therapeutic opportunities. Recently, patient-derived tumour organoids (PDTOs) have emerged as a promising tool for personalisation of cancer treatment. Nonetheless, multiple challenges remain to be overcome to streamline PDTO drug testing and translate PDTO assays into clinical practise. In this thesis, we aimed to advance PDTO use for personalisation of CRC treatment, by (i) developing PDTO-based assays to identify the most effective standard-of-care treatment for each patient, and (ii) validating new synergistic drug combinations identified through CRC cell line-based drug combination screens. In addition, we (iii) leveraged genome-wide CRISPR-Cas9 knock-out screens in CRC cell lines to identify novel biomarkers for cetuximab resistance. Using a newly developed suspension culture method, CRC PDTOs could be established with a high success rate (81.1%) and rapidly expanded for drug testing, whilst recapitulating the main genomic and histopathological features of original tumours. Assay results generated using our CRC PDTO platform were robust and reproducible, and classification of PDTO drug sensitivity data achieved 84.6% accuracy for predicting treatment responses of patients with metastatic disease. We successfully used our CRC PDTO platform to validate synergistic drug combinations and respective biomarkers identified in a large-scale CRC cell line-based drug repurposing screen, demonstrating PDTO complementarity with CRC cell line and animal models. In addition, a whole-genome CRISPR-Cas9 knock-out screen revealed novel negative regulators of cetuximab resistance in RAS WT CRC cells. In conclusion, our findings provide a pathway for the development of PDTO-based diagnostic applications for CRC and highlight new candidate drug combinations and cetuximab response biomarkers for CRC, warranting further dedicated follow-up studies. The CRC PDTO biobank established as a result of this work will provide an invaluable resource for future discovery and translational research.
A Quantitative Analysis of Natural Killer Cell Homeostasis, Competition, and Collaboration

Hennessy, Robert John ( 2022-12)

Contemporary Immunology views Natural Killer (NK) cells as critical facilitators of immune protection in various pathological settings. Still, this has not always been the case; a somewhat challenging history of NK cell research has delayed full scientific appreciation of their importance and modus operandi, which rendered NK cells a mysterious and misunderstood immune cell subset for several decades. In more recent years, NK cells are receiving a resurgence in clinical attention owing to characterisation of their potent anti-tumour and immunomodulatory properties; however, as modern Immunology remains in the aftermath of an uncertain era for NK cells, harnessing this revolutionary therapeutic potential has proven difficult. NK cells are key inducers of early inflammation and systemic immune activation, as well as expert decision makers in the destruction of harmful cells versus protection of healthy tissue. As may be expected, catastrophic consequences can occur to a host if these processes are not properly regulated. There is growing appreciation in the research community regarding the sheer complexity and redundancy in regulatory processes that maintain NK cell homeostasis and functions, as well as the plethora of cytokines and cell-cell interactions that govern this regulated behaviour. As a means of dissecting these complex processes, we have applied a reductionist approach to study how various individual signals are integrated into the internal machinery of an NK cell to produce different outcomes. To this end, we applied quantitative methods previously established in adaptive T and B lymphocytes to delineate and quantify parameters relating to survival and proliferation. In this work, we uncovered that stimulatory proliferative signals from the cytokines IL-15, IL-18, and IL-12 are offset by enhanced propensity for NK cell death, which limits the overall efficiency of their expansion during stimulation. These responses were largely dependent on direct interactions between NK cells via Fas and FasL, which induce fratricidal killing of each other. These competitive relationships between fellow NK cells were heavily dependent on the type and dose of cytokine present. Further, our investigation of NK cell interactions led us to identify that NK cells also facilitate advantageous interactions with other NK cells in more homeostatic contexts, which were dependent on IL-15. We discovered that these homotypic collaborative interactions are the result of complex interactions and bidirectional signalling events between SLAM family receptors 2B4 and CD48, which together facilitate IL-15 responsiveness and education events, thereby enhancing NK cell fitness and function, respectively. This work offers valuable insights to improve in vitro culture protocols in the clinical cultivation of NK cells for immunotherapies, such as Adoptive Cell Therapy, as well as indicating broader and nuanced roles of immune and target cell interactions in the stimulation and regulation of NK cell fitness, function, and homeostasis.
The Parasite Genetic and Host Immunological Determinants of Immune Escape in Plasmodium falciparum Malaria

Naung, Myo ( 2022-12)

Abstract Human malaria remains a major global public health problem with an estimated 241 million clinical cases and 627,000 deaths in 2020, expected to increase in future years. Highly effective vaccines are urgently needed to progress the control and elimination of the disease. There are dozens of candidates in development, however only one vaccine (RTS, S) targeting the most virulent human malaria parasite, Plasmodium falciparum, has reached Phase 4 implementation trials with 50% efficacy that is short-lived and strain specific. As WHO has outlined a goal for malaria vaccines with a 75% efficacy against clinical malaria in all malaria-endemic countries by 2030, novel approaches are needed to increase efficacy. The limited efficacy of malaria vaccines to date has been in part attributed to the extreme diversity of parasite antigens being developed as ‘subunit’ vaccines, with only one or two randomly selected allelic variants as the basis for inducing immune responses. Antigen diversity has evolved as a means for malaria parasites to evade host immune responses - a process known as an immune escape. Pinpointing specific antigen polymorphisms that drive immune escape would help to prioritise antigens and alleles for inclusion in vaccine formulations. In my Ph.D. project, I investigated the hypothesis that specific polymorphisms in leading P. falciparum vaccine candidates are associated with immune escape. To test this hypothesis, I first analysed the publicly available MalariaGEN genome sequence data to catalogue the global genetic diversity of the genes encoding 25 leading P. falciparum vaccine candidate antigens. Predicted regions of immune selection were identified on both the linear gene sequence and the 3-dimensional protein structure. We then focused on two cohorts of children from malaria endemic regions of PNG conducted during moderate and high transmission periods. We analysed samples from 758 children, conducting multiplexed high-throughput assays on serum samples to measure IgG responses against 27 antigens, and targeted amplicon sequencing of 38 parasite antigen genes in sequentially collected samples from each child to measure the rate of allelic turnover for each antigen. The analysis identified critical immune escape genes and their specific polymorphisms that contribute to immune escape. The relationship between measures of genetic diversity and immune selection in the global data, and the antibody response in the children identifies antigens driving immune escape and those where diversity did not appear to contribute to immune escape. This research provides a vital framework for the prioritization of vaccine candidate antigens and a ‘serotype classification system’ to identify immune escape polymorphisms and for evaluating strain specific efficacy during vaccine trials.
Benchmarking long-read RNA-seq analysis methods

Dong, Xueyi ( 2022-12)

Application of Oxford Nanopore Technologies' long-read sequencing platform to transcriptomic analysis is increasing in popularity. To explore how much additional benefit it brings over short-read RNA-seq, as well as provide guidance in the choice of suitable long-read RNA-seq data analysis methods, I first created a long-read RNA-seq analysis pipeline using a mix of long-read specific preprocessing tools together with established short-read methods for differential expression analysis. Using this pipeline, I analyzed two nanopore long-read RNA-seq datasets and compared the results to short-read experiments with equivalent samples. The results showed that performing differential gene expression analysis using this pipeline can yield comparable results to those obtained using short-read data. Secondly, I designed a comprehensive long-read benchmarking experiment with ground-truth provided by sequin spike-ins and an in silico mixture design. Samples were deeply sequenced using both short-read and long-read technologies. By analyzing this dataset, I found that StringTie2 and bambu outperformed other tools from the 6 isoform detection tools tested, DESeq2, edgeR, and limma-voom were best amongst the 5 differential transcript expression tools tested, and there was no clear front-runner for performing differential transcript usage analysis between the 5 tools compared. Finally, I used the best-in-class methods identified in the benchmark study to detect aberrant minor class RNA splicing due to the knockdown of the RNPC3 gene in A549 cell line samples. Differentially expressed and differentially used transcripts detected on the long-read RNA-seq data were highly correlated with annotated minor intron-containing genes. I also visualized the long- and short-read coverage of the five top genes with differential transcript usage, which further highlighted the strength of long-reads in detecting and visualizing transcript splicing. The datasets generated and benchmarking results presented in this thesis provide useful resources and insights for long-read RNA-seq analysis to the research community.
Deconvolving gene expression changes associated with time and cell division following B cell activation

Tarasova, Ilariya ( 2022-12)

B cells, a type of lymphocyte, play a critical role in the adaptive immune system. They have a sophisticated decision-making mechanism of differentiation into multiple effector cell types. When stimulated, B cells divide and change levels of key regulatory molecules that alter and guide the differentiation path of each individual cell. However, the mechanisms governing this process and how cells transit to new cell types are still poorly understood at the molecular level. On the one hand, the timing of the division burst occurring during the proliferation phase of the B cells response is controlled by protein Myc, whose expression is shown to be dependent on time after activation. On the other hand, several studies have documented a connection between B cell differentiation, including isotype switching, and cell division progression. Thus, the question of globally assessing which gene changes can be affected by time or division arose. Therefore, the aim of the current study focused on quantifying the effect of time and division on the whole transcriptome. Here, we examine the transcription profiles of B cells in different conditions and investigate changes between two fundamental B cell activation programs: proliferation only and proliferation with further differentiation. As a result, we developed a method for quantifying time and division effects on the B cell transcriptome and highlighted the overall behaviour of genes. We also provided a high-level visualisation of the influence of these two variables on each gene transcript. Further investigation of the data revealed clustered and shared expression trends over time and division progression. Moreover, we interrogated common regulatory features and identified potential key regulators of underlying transcriptional programs in an unbiased manner. Together the findings presented here support the hypothesis of a time-dependent proliferation program and division-dependent differentiation. Overall, the statistical approach and analysis described in the current work could be applied to any cells that can evolve or transform into other cell types over time and during division progression.

Medical Biology - Theses

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