Minerva Elements Records

Permanent URI for this collection

http://hdl.handle.net/11343/251382

Search Results

Now showing 1 - 10 of 81

Hyper-nutritional cell culture medium distorts the expression of anti-cancer drug targets

Cheng, Tianhong ( 2023-07)

In vitro cell cultures are instrumental in deciphering the mechanisms that underlie cell processes in vivo including differentiation, migration, growth and mechanics, all of which are impacted by both biochemical and biomechanical microenvironments. Evolving technologies provide continuous improvement to cell culture towards more physiologically relevant systems that better emulate in vivo conditions. However, the cell culture media as one of the fundamental elements for in vitro cell culture have been overlooked with incremental rather than the transformational improvements that are required. Nutrient availability in the tumour microenvironment is essential for metabolism, cellular processes and may be a determinant of anti-cancer drug responses. Nevertheless, the overwhelming majority of current in vitro biomedical research is conducted in unphysiological conditions, using conventional cell culture medium developed generations ago with hyper-physiological level of nutrients. In addition, most of the cell culture were in static conditions, lacking the flow/renewal of the in vivo situation to provide appropriate biochemical and mechanical cues and prevent accumulation of metabolic by-products. We therefore developed an improved physiological medium, “Melbourne Medium” (MM) with human plasma-like composition for continuous supply to multi-well cell culture environments using the custom-built RPM2 multiplexed superfusion system. Our focus here is on the imperative to understand the influence of physiological media on cellular behaviours by contrast with conventional hyper-nutritional media. Culture of non-small cell lung cancer (NSCLC) A549 cells in MM slowed proliferation, promoted an epithelial-mesenchymal transition (EMT)-like phenotype with an increased motility and induced paclitaxel resistance. Global proteomic analyses revealed expected differential expression of metabolism-related gene ontologies, but also multiple biological pathways of critical importance to the growth and spread of NSCLC. Notably, significant distortion was identified in the expression of key anti-cancer drug targets including EGFR, STAT3, TGFBI and Smad3, as well as tubulins, specifically betaII and betaIV tubulin isoforms implicated in paclitaxel resistance. The most up- and down- regulated proteins in MM, CNBP and 15-hydroxy prostaglandin dehydrogenase (15-PGDH), respectively, are each associated with lung cancer survival, the former association being discovered in this thesis study. The cell metabolism processes critical to tumour progression were perturbed by hyper-nutritional medium, as indicated by both proteomic and metabolomic analyses. For the first time, by applying the RPM2 multiplexed superfusion of media, we established that the physiological nature of MM could be maintained, while minimising accumulation of both bioactive metabolic by-products and previously unappreciated metabotoxin formation. Together, these studies have demonstrated that hyper-nutritional (conventional) media profoundly distort tumour cell functions, whereas superfusion of MM facilitated a more physiological metabolic environment, associated with improved drug target validation and drug screening.
Development of a CRISPR/Cas9 based RiboTag strategy to identify regulators of melanoma immune interaction

Kretzmer, Freya Maria ( 2023-04)

The development of immunotherapy approaches has revolutionised the field of melanoma treatment by prolonging the survival of patients. Nevertheless, a significant proportion of patients either do not respond to immunotherapy or relapse. To understand the mechanisms of the melanoma immune crosstalk resulting in resistance to immunotherapy, we aim to dissect pathways and gene expression signatures in melanoma to identify new potential therapeutic targets or prognostic biomarkers. As a highly heterogeneous tissue melanoma lesions not only contain transformed melanocytes, but also cells of the tumour microenvironment. Therefore, most of sequencing approaches rely on mechanical processes in order to dissect tissues and isolate the target cells to study melanoma cell specific expression patterns. But the extensive processing can introduce stress related artifacts in the analysis. In order to find an alternative approach which avoids the extensive processing but still gives access to tumour cell-specific transcripts, a murine RiboTag melanoma model was generated. An adapted CRISPR/Cas9 approach was used to endogenously fuse the ribosomal protein L8 (RPL8) with a triple HA peptide tag. The incorporation of the tagged RPL8 into translating polyribosomes enabled the isolation ribosome-associated mRNAs by immunoprecipitation. We used RiboTag melanoma cells in a C57BL/6 mouse model for immune checkpoint blockade (ICB) therapy and found the expression of Cnot9, a subunit of the CCR4-NOT complex, significantly downregulated in ICB-treated melanomas. In previous exome sequencing studies analysing cutaneous melanoma CNOT9 has been described to harbour a hotspot mutation, but the consequences for progression or successes of therapy approaches remained unknown. To investigate the impact of these mutations on melanoma-immune interactions, we performed a Gene Set Enrichment Analysis (GSEA) of a cutaneous melanoma dataset and observed an enrichment of immune activation and antigen presentation signatures in tumours bearing CNOT9 mutations. A knockdown of CNOT9 in human MZ7 melanoma cells induced similar expression patterns, suggesting that this immune-activated status of CNOT9 mutated melanoma may be due to a partially impaired functionality. To further evaluate possible functional effects of the mutation, the protein structure of CNOT9 was analysed by modelling and mutations were localised. This structural study revealed that the S87P and P131L point mutations occur in the concave surface of CNOT9, which is described to be the binding pocket of Roquin and CNOT4. In literature, both proteins are described to be involved in translational regulation of immune response-associated mRNA. To further investigate the functional effects of the mutations and potential impact on interactions with binding partners, melanoma cells expressing CNOT9 variants bearing either the S87P or P131L point mutation were established. The use of RiboTag in combination with an experimental model for ICB therapy facilitated a comprehensive characterisation of melanoma-specific gene expression patterns during therapy, revealing a downregulated expression of Cnot9 in inflamed melanomas. Melanoma-associated mutations in CNOT9 as well as a CNOT9 knockdown induced inflammatory response pathways, indicating an involvement of CNOT9 in immune regulatory processes in melanoma. Thus, our work can contribute to a better understanding of the interactions between melanoma and the immune system, which in turn offers the opportunity of improving the tailored treatment of malignant melanoma.
Interrogating the cells-of-origin of BRCA mutant cancers to identify therapeutic targets for cancer prevention

Joyce, Rachel ( 2022)

It is currently estimated that approximately one woman dies every minute of breast cancer across the globe. While the greatest risk factor for developing breast or ovarian cancer is merely having female reproductive organs, the cumulative life-time risk of developing breast and ovarian cancer for women who carry pathogenic mutations in their BRCA genes is significantly higher than non-carriers. Men who harbour mutations in their BRCA genes are also at increased risk of developing breast cancer within their lifetime. Currently there are no clinically approved strategies for breast or ovarian cancer prevention in BRCA mutation carriers beyond highly invasive and irreversible surgical procedures such as prophylactic mastectomy and bilateral salpingo-oophorectomy. Targeted therapeutic strategies for cancer prevention in BRCA mutation carriers are thus a sought-after alternative. Significant headway has been made by our research group and others in identifying RANK-ligand inhibition as a putative chemoprevention strategy for the onset of breast cancer in female BRCA1 mutation carriers; subsequently, a phase 3 international clinical trial BRCA-P (ClinicalTrials.gov Identifier: NCT04711109) is currently recruiting female BRCA1 mutation carriers to assess the efficacy of RANK-ligand inhibition in preventing breast cancer development using the FDA-approved drug denosumab. A portion of this thesis describes the functional and biological consequences of denosumab treatment on the putative cell-of-origin of BRCA1 mutant breast cancer, the RANK+ luminal progenitor, from patients enrolled in the Melbourne Health BRCA-D pre-operative window study; these patients received denosumab treatments prior to undergoing prophylactic mastectomies. This work indicated that BRCA1 mutation carriers who received 1 denosumab injection per month for 3 months had significantly reduced numbers of RANK+ luminal progenitors in their breast epithelium, and these cells also displayed decreased colony forming activity ex vivo, compared to cells from untreated BRCA1 mutation carriers. This thesis also seeks to shed light on the biological mechanisms driving ovarian cancer development in BRCA1 mutation carriers, and describes novel subsets of BRCA1 mutant fallopian tube secretory cells that are putative cancer cells-of-origin. To date, there have been no prospective studies or chemoprevention trials for breast cancer development in BRCA2 mutation carriers. As such, there is a pressing need for the identification of novel therapeutic pathways for breast cancer prevention in these patients; this thesis makes several promising developments in this effort. Using preneoplastic breast tissue samples from BRCA2 mutation carriers and wildtype patients, luminal cells, including a subset of ERBB3lo luminal progenitors and mature luminal cells, were found to be expanded in breast tissue epithelium of BRCA2 mutation carriers. ERBB3lo luminal progenitors from preneoplastic BRCA2mut/+ patients were found to have increased colony forming activity ex vivo, and exhibited upregulation of genes involved in mTORC1 signalling, protein synthesis and proteostasis. Indeed, a functional protein synthesis assay revealed increased protein translation in preneoplastic luminal cells from BRCA2 mutation carriers compared to wildtype patients ex vivo. A genetically engineered mouse model of BRCA2 mutant breast cancer was used to faithfully recapitulate the preneoplastic phenotype of luminal epithelium identified in BRCA2 mutation carriers, and showed a significant delay of BRCA2mut/+ mammary tumourigenesis upon short-term treatment with an mTORC1 inhibitor in vivo. In summary, the findings detailed in this thesis describe several developments in our understanding of the mechanisms of breast and ovarian cancer development in BRCA mutation carriers, and uncover mTORC1 inhibition as a putative strategy to delay or prevent the onset of breast cancer in BRCA2 mutation carriers. Cumulatively this work provides important insights of clinical significance for women harbouring mutations in their BRCA genes.
Development of new methods for accurate estimation of tumour heterogeneity

Hollizeck, Sebastian ( 2022)

It is now understood that intra-tumor heterogeneity is one of the leading determinants of therapeutic resistance and treatment failure and one of the main reasons for poor overall survival in cancer patients. However, the possibility to study this phenomenon is so far underexplored as the acquisition of multi region data sets from the different tumour sites can be ethically challenging. With circulating tumour DNA (ctDNA) used as a proxy for tumour biopsies, it is possible to analyse a snapshot of the unified heterogeneity in each patient, but there is still an unmet need for new methods to optimize the analysis of these large-scale, high-dimensional data to derive new treatment targets. The contributions of this work include the development of multiple new methods, which show that the analysis of bulk sequencing from tumour tissue and ctDNA has unrealised potential for both diagnostic and research questions. This thesis presents three distinct but related projects, which explore the analysis of tumour heterogeneity at different levels and depths, focusing on method development. First, we developed a workflow to improve the detection of somatic variants present at very low allele frequencies. When multiple samples, separated in time or space, from the same patient were available, we were able to substantially improve the detection threshold of variants. These low abundance variants are invaluable in a clinical setting, where they can indicate an arising resistance mechanism or relapse of disease. With the improved sensitivity of our method, the treatment of patients can be adjusted earlier and more accurately. We then used our new analysis workflows to explore evolutionary trajectories and resistance pathways of five lung cancer patients enrolled in the CASCADE autopsy program. In addition to analysis of somatic variants, we used copy number analysis and structural variants to contrast and compare each sample within a patient to generate phylogenies to visualise the evolutionary distances and a pseudo time scale to assess the timing of mutations. Clear genomic determinants of treatment resistance were identified for three of the five cases with non-small cell lung cancer and the diversity of these genomic mechanisms profoundly highlighted the true extent of inter-patient heterogeneity. This work included the identification of a novel genomic resistance mechanism to the drug selpercatinib, a small molecule inhibitor of REK kinase. Among the remaining two patients, treatment resistance was mediated by transformation of their disease from non-small cell lung cancer to a small cell lung cancer histological phenotype. These two cases showed distinct evolutionary trajectories compared to the other non-small cell lung cancer cases, with similarity in their nuclear and mitochondrial phylogenies, but no clear genetic determinant for the small cell transformation, highlighting the additional importance of non-genomic mechanisms which can drive resistance in this disease. Finally, we developed a method, called MisMatchFinder, to monitor tumour heterogeneity and evolution over time through ctDNA. We tailored the method to be fully tumour agnostic and enabled it to be easily applied in the clinical setting by using low-coverage whole genome sequencing. The method uses highly specialised filtering steps to enrich the tumour signal and eliminate the background noise from normal cell-free DNA and sequencing errors in these data. We showed that the method could accurately detect specific cancer-related signatures at low tumour purity and tumour burden in simulated and patient data for melanoma and breast cancer. In summary, with this work we contributed multiple new methods to study, measure and understand genetic tumour heterogeneity. This understanding is crucial for the continuous optimisation of cancer management and the development of new and effective treatments for patients.
Characterization and prevention of stunning, a cytotoxic T lymphocyte inactivating program that impairs adoptive T cell therapy against cancer

Scheffler, Christina Maria ( 2022)

Adoptive T cell therapy (ACT) is a promising immunotherapeutic approach to fight cancer by utilising cytotoxic T lymphocytes (CTL), which specifically target and eradicate tumour cells. However, one major limitation of this therapy is the ability of tumours to interfere with the CTL through immune escape mechanisms. In a mouse model of B-cell lymphoma and ACT, we investigate the mechanisms underlying this failure. Our laboratory previously found that tumour antigen-specific CTL fail to eradicate lymphoma cells once the tumour burden reaches a certain threshold. In this case a major proportion of the CTL is lost while those remaining lose their effector function, a process we call “stunning”. Our tumour model enables us to investigate the underlying mechanisms of stunning by comparing characteristics of CTL and tumour cells in a small tumour setting, where CTL successfully eradicate tumour cells with a large tumour setting, where stunning occurs. In Chapter 3, we describe findings that suggest that the number of tumour cells presenting a CTL’s specific antigen rather than the total tumour burden determines ACT outcome in our murine lymphoma model. Once the number of antigen+ tumour target cells reaches a critical threshold, all CTL are rendered dysfunctional. We therefore hypothesize that a large heterogenous tumour may be more efficiently killed by using a number of CTL specific for several different antigens simultaneously, where each antigen is only expressed on a subpopulation of tumour cells to prevent CTL dysfunction due to high target cell density. We further showed that increasing numbers of antigen+ tumour cells at the time of adoptive transfer induce the rapid expansion of an Interleukin (IL)-18-receptor-alpha (IL18Ra) and Neuropilin-1 (NRP-1) double positive CTL population, with reduced cytotoxicity. The higher the number of antigen+ tumour cells encountered by the CTL upon transfer, the lower the cytotoxicity, cytokine production and T cell receptor (TCR) expression of this double positive CTL population and the higher its proliferation and expression of exhaustion associated molecules. Once the number of antigen+ tumour cells reaches a certain threshold, all CTL become IL18Ra+NRP-1+ double positive and totally lose their effector functions. We therefore propose that stunning is a T cell inactivation mechanism by which high numbers of target cells induce the rapid expansion of an inactive population of adoptively transferred CTL which blunts anti-tumour immunity. As CTL become rapidly inactivated and show IL18Ra and NRP-1 expression early after the adoptive transfer, we suggest that the frequency of IL18Ra+NRP-1+ CTL can be used as an early diagnostic marker of whether the therapy is going to be successful for an individual patient. In Chapter 4, we aimed to investigate which CTL intrinsic pathways are involved in poor CTL persistence upon stunning. We were able to show that CTL were rapidly deleted followed by efficient expansion and found that the pro-apoptotic protein Bcl-2-interacting mediator of cell death (Bim) as well as the death receptor Fas are involved in the process. KO of both molecules improved CTL persistence but did not improve tumour killing. We propose that KO of Fas and Bim in combination with T cell modifications that enhance effector function and prevent CTL dysfunction may be utilized to optimise ACT outcomes. In summary, this thesis contributes to the understanding of mechanisms that may obstruct successful ACT and offers possible ways of how to improve the therapy outcome by preventing CTL deletion as well as dysfunction. It further describes IL18Ra and NRP-1 as early markers of dysfunctional adoptively transferred CTL, which could potentially be used to predict therapy outcome.
Characterisation of the receptor tyrosine pseudokinases, EphB6 and EphA10

Liang, Lung-Yu ( 2022)

Erythropoietin-producing human hepatocellular (Eph) receptors are the largest receptor tyrosine kinase family, comprising 14 members. Like other receptor tyrosine kinases, Eph receptors consist of extracellular domains capable of ligand binding, a single transmembrane domain, and intracellular components including a tyrosine kinase domain. The cognate ligands of Eph receptors, called ephrins, are also membrane-tethered. Upon cell-cell contact, ligation of ephrins in trans results in dimerisation, oligomerisation and clustering of Eph receptors, a mechanism by which the intracellular tyrosine kinase domain autophosphorylates. The autophosphorylated Eph receptors then transmit signals to downstream effector proteins via their recruitment and phosphorylation. Typical signal outputs arising from clustered Eph receptors include cell adhesion or repulsion, depending on the cellular context. Therefore, Eph receptor/ephrin signalling is critical in embryonic development. Pathologically, deregulated Eph receptors resulting from point mutations and aberrant expression have been linked to many types of malignancies in adults. However, owing to the complexity of the activation mechanisms of Eph receptors, no therapeutics targeting Eph receptors are clinically available to date. Interestingly, two Eph receptor members, EphA10 and EphB6, are categorised as pseudokinases, as they harbour a kinase-like domain devoid of essential residues for kinase activity. Studies have suggested an oncogenic role for EphA10, and EphB6 has been proposed as a potential metastasis suppressor. Nonetheless, how these two receptor tyrosine pseudokinases exert their functions at a protein level remained largely unknown. The specific outstanding questions include: (1) How do the pseudokinase domains of EphA10 and EphB6 function? (2) Do EphA10 and EphB6 have cognate ephrin ligands, and can EphA10 and EphB6 oligomerise at the plasma membrane upon ligating to ephrins? (3) What are the signalling outputs and consequences upon binding to ephrins? This thesis aims to address these outstanding questions, with a primary focus on characterising EphB6. By applying biophysical, biochemical, structural and mass spectrometry approaches, I characterised the intracellular regions of EphB6 and EphA10, and present these studies in this thesis. The intracellular regions of EphB6 and EphA10 exhibited high conformational plasticity in solution. While the pseudokinase domains of EphB6 and EphA10 lack kinase activity, they both retained ATP binding ability, raising the possibility that they can be modulated by conventional small molecule kinase inhibitors. Furthermore, upon phosphorylation by its kinase-active cousin, EphB4, the phosphorylated EphB6 intracellular region was able to bind various Src homology 2 (SH2) domains. This suggests that, once phosphorylated, the EphB6 pseudokinase can act as a signalling hub, by recruiting adaptor and signalling proteins. To elucidate the functions of full-length EphB6, a co-culture system containing EphB6-expressing and ephrinB1-expressing cells was first established. I then employed live cell imaging, which revealed that ephrinB1 is a cognate ligand of EphB6, and is able to induce EphB6 clustering at the plasma membrane. By applying proximity-labelling techniques coupled with mass spectrometry, unique proteins enriched within clustered EphB6 were identified, implying clustered EphB6 is signalling competent and can drive cytoplasmic signal transduction. Phenotypically, clustering of EphB6 appeared to promote formation of tubules interconnecting EphB6 expressing and ephrinB1 expressing cells. By Cryo-electron tomography, our preliminary data suggested that these tubular structures consist of an unprecedented double membrane morphology, raising the prospect that clustered EphB6 may mediate a novel mode of cell-cell communication. Collectively, this thesis presents functional characterisation of EphB6 and EphA10, laying the foundation for future exploration of these two receptor tyrosine pseudokinases.
Cancerous dramaturgy: using biology as a dramaturgical template in writing for performance

Stubbings, Diane ( 2021)

This practice-based research investigates the concept of a biological dramaturgy, and is structured as a dissertation (60%) and accompanying creative works (40%). Working with cancer as a dramaturgical template, the research proposes that a creative writing practice which models itself on the biological processes which drive cancer will foster a dramaturgy that is essentially cancerous in nature. This proposition is tested through three creative experiments which use cancer biology (experiments A and C) and evolutionary-developmental biology (experiment B) as illustrative systems for the generation of writing for performance. The thesis employs a critical framework which synthesises Critical Literary Geography and Systems Biology to allow for a phenomenological account of the creative process. This synthesis enables the articulation of a textual system, one which encapsulates the dwelling within and shaping of imaginative spaces that come through the act of writing, as well as the notion of a dynamic creative system that is generated by concurrent environmental, structural (text-driven) and organisational (author-driven) forces. Through this combined practical and theoretical inquiry – and building on discourse concerning the relationship between form and content in the science play, as well as dramaturgical theories and practices that accentuate process, intertextuality, the organic and the viral – the thesis concludes that the deployment of cancerous processes has the potential to seed and nurture new performance texts that are cancerous in nature. Further, this approach to dramatic composition can be applied to biological processes more broadly, the results of the experiments revealing how a biological organism’s ‘evolution from within’ might be modelled in the dramaturgy of a performance text. The research establishes that working towards a biological dramaturgy requires the nurturing of an embodied sense of the relevant biological processes and a biological sensibility, such that the balance between the environmental, structural and organisational elements of the work might best be negotiated and the author-God resisted. It is also suggested that biologically driven dramaturgies might potentially facilitate a reconfiguration that pushes dramatic form beyond the postdramatic. The practice outcomes of the thesis are demonstrated by three performance texts: Blood & Shadow, Variation for Three Voices on a Letter to Nature and Self Portrait / In Cross-Sections / With Bird.
Antimicrobials in hospitalised and high-risk children: understanding and improving use

McMullan, Brendan Joseph ( 2021)

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.
Identification of synthetic lethal interactions with the KRAS oncogene for targeted cancer treatment

Morgan, Kimberly Jane ( 2021)

Cancer is a major public health issue globally, ranking as the second most common cause of death. Molecularly targeted therapies, focused on exploiting tumour cell dependency on certain oncogenic driver mutations for growth and survival, have greatly improved patient outcomes. However, despite these advances, some of the most frequent oncogenic mutations in cancer, such as those found in KRAS, are extremely challenging to target directly. One promising strategy to expand the range of actionable targets for cancer drug development is the exploitation of synthetic lethal interactions. Synthetic lethality is the term used to describe the death of cells in response to the co-existing disruption of two genes, neither of which is lethal alone. In this setting, targeting a gene that is synthetic lethal with a cancer-relevant mutation has the potential to induce the death of vulnerable cancer cells while leaving healthy cells unaffected. With this background in mind, my lab participated in a focused ENU mutagenesis screen in zebrafish with the aim of identifying genes that are essential for high rates of cell proliferation during endodermal organ development but not required by quiescent tissues. This yielded mutants that exhibited either ‘cell death’ or ‘growth arrest’ phenotypes in the liver, intestine and pancreas. I investigated two of the underlying mutant genes, ahctf1 and rnpc3, for their capacity to engage in synthetic lethal interactions with the kras oncogene. In Chapter 3, I investigated the impact of ahctf1 heterozygosity on the growth and survival of KrasG12V-expressing hepatocytes in a zebrafish model of hepatocellular carcinoma (HCC), TO(krasG12V). ahctf1 encodes Elys, a multifunctional nucleoporin with essential roles in nuclear pore assembly and mitosis. I found that ahctf1 heterozygosity impairs nuclear pore formation, mitotic spindle assembly and chromosome segregation, leading to DNA damage and activation of Tp53-dependent and Tp53-independent cell death pathways which reduced tumour burden. Importantly, ahctf1 heterozygosity did not impact normal liver development, advancing ELYS as an attractive target for cancer therapy with a viable therapeutic window. In Chapter 4, I examined if rnpc3 heterozygosity also reduced tumour burden in the TO(krasG12V) model. rnpc3 encodes 65K, a unique protein component of the U12-dependent spliceosome, a specialised splicing machinery required for the correct splicing of a very small percentage (3.7%) of genes. In hepatocytes expressing krasG12V, rnpc3 heterozygosity reduced the number of cells in S phase of the cell cycle and increased cell death, together reducing tumour burden, without affecting normal tissue. In Chapter 5, I demonstrated that the zebrafish model of HCC is a powerful platform for testing novel therapeutics. I evaluated the efficacy of PRMT5 and KAT6A/B inhibitors early in their development, and showed that they were effective in reducing tumour growth and worthy of future investigation. In conclusion, my studies revealed two promising new targets for cancer treatment. I also demonstrated that the zebrafish HCC model is highly amenable to pharmacological inhibition and provides a valuable system for the pre-clinical examination of drug treatments in vivo.
The control of lymphatic vascular remodelling in cancer by microRNAs

Arcucci, Valeria ( 2021)

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.

Minerva Elements Records

Permanent URI for this collection

Filters

Date

Author

Subject

Type

Settings

Sort By

Results per page

Statistics

Citations

Search Results