Medical Biology - Theses
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Characterization of plasmepsin X as a cross-species antimalarial target
The emergence and spread of drug resistance have hindered the campaign for malaria eradication. The development of new drug targets is critical for our anti-malarial arsenal of interventions. Plasmepsins, which are aspartic proteases expressed by malaria parasites, serve important functions for parasite survival. Among the 10 members of this enzyme family, plasmepsin X (PMX) is essential for P. falciparum growth and has been shown to be involved in the egress of merozoites from infected red blood cells and the invasion of merozoites into red blood cells. Several aspartic protease inhibitors have anti-malarial activity on P. falciparum and are proposed to target PfPMX. The aim of this project was to investigate if these compounds affect P. knowlesi growth and whether PMX is a cross-species target for antimalarial development. This work showed that two aspartic protease inhibitors, 49c and 1SR, caused inhibition of P. knowlesi parasite growth. In further studies, live cell imaging demonstrated that these compounds inhibit P. knowlesi parasite growth by blocking parasite egress. Next, the optimal condition for protease activity was characterised after the expression and purification of a functional recombinant P. knowlesi plasmepsin X (rPkPMX). Using a fluorogenic protease assay, both 49c and 1SR were shown to inhibit the activity of rPkPMX. Furthermore, rPkPMX was able to cleave synthetic substrates, which were based on the predicted cleavage sites of PfSUB1, PfRAP1, PfRh2, TgROP1 and TgMIC6 predicted cleavage sites. By screening a panel of aspartic protease inhibitors, the BACE1 inhibitor, LY2886721, was identified as an inhibitor of rPkPMX activity as well as P. knowlesi and P. falciparum parasite growth. Therefore, PMX can be used as a cross-species target for antimalarial drug development.
Smac-mimetic combination therapies for the treatment of cancer and infectious disease
Acute myeloid leukaemia (AML) is an aggressive disease with a current 5-year survival rate of only ~30%, therefore development of more effective treatments is urgently needed. Overexpression of inhibitor of apoptosis (IAP) proteins, responsible for the regulation of tumour necrosis factor (TNF)-mediated apoptosis, have been correlated with cancer progression and treatment resistance. Natural IAP antagonists exist, termed second mitochondria-derived activator of caspases or Smac, leading to the pharmaceutical development of Smac-mimetics. Smac-mimetics, such as the drug birinapant, are currently in clinical trials for a range of cancers, including AML. Although, birinapant has shown promising anti-cancer effects, its efficacy as a single-agent and in combination with numerous anti-cancer therapies, has been variable and limited. Therefore, overcoming patient resistance to Smac-mimetic therapy and potentiating treatment are still major challenges. Using a library of more than 5,700 bioactive compounds, we examined the emergence of resistance to birinapant in AML. Here I present the identification of multidrug resistance protein 1 (MDR1/ABCB1) inhibitors as a class of clinical drugs that can overcome Smac-mimetic resistance in cancer. Inhibition of MDR1 with 3rd generation specific inhibitors increased intracellular levels of birinapant and sensitised AML and ovarian cancer patient cells to birinapant killing. Using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, I show that MDR1 is a predictor of response to birinapant-based therapies in cancer cells. Moreover, the combined therapy of MDR1 inhibitors and birinapant effectively killed leukaemic stem cells (LSCs) and prolonged survival of AML burdened mice in vivo (Chapter 3). The findings observed in Chapter 3 in AML and ovarian cancer cells, were extended to a chronic hepatitis B virus (HBV) infection model (Chapter 4). Birinapant has previously shown efficacy at eradicating HBV infection, however due to toxicity concerns, in-human clinical trials have ceased. In Chapter 4, I demonstrate that a lower concentration of birinapant can be combined with the MDR1 inhibitor zosuquidar, to potentiate birinapant-mediated HBV infected liver cell death and HBV-DNA clearance in vivo. An investigation into the remaining high throughput screen hit compounds, revealed half were psychotropic and inhibited MDR1 to synergise with birinapant. In Chapter 5, I focus on the serotonin receptor 7 (5-HT7) agonists, LP-44 and LP-12, as well as the psychoactive compounds Fluphenazine dihydrochloride, SDZ 21009, GR-127935 and CGP-71683. My identification that these compounds inhibit MDR1 to synergise with birinapant underlines the dependence cancer cells have on MDR1 to evade Smac-mimetic therapy. This thesis presents a comprehensive analysis of birinapant as a substrate for MDR1 and identifies MDR1 as a biomarker of Smac-mimetic therapy. Furthermore, this study provides evidence towards a novel combination regimen combining birinapant with 3rd generation MDR1 inhibitors to potentiate Smac-mimetic therapy for the treatment of AML, ovarian cancer and HBV. Altogether these findings suggest a therapeutic opportunity for Smac-mimetic plus MDR1 inhibitor combination therapy in cancer and infectious disease.
Functional and structural characterisation of the epigenetic regulator, SMCHD1
Structural Maintenance of Chromosomes Hinge Domain-containing protein 1 (SMCHD1) has been established as an epigenetic regulator, with critical roles in X-chromosome inactivation, autosomal gene silencing and genomic imprinting. Recently, variations in SMCHD1 have been associated with two human conditions: facioscapulohumeral muscular dystrophy (FSHD) and Bosma arhinia microphthalmia syndrome (BAMS). There has therefore been a growing interest in unveiling SMCHD1’s atomic structure and the molecular mechanisms underlying its function in both a healthy and diseased state. To provide a better understanding of Smchd1’s molecular structure and function, I successfully expressed and purified the full-length 2007-amino acid mouse Smchd1 protein. Electron microscopy analyses of the Smchd1 dimer revealed an elongated rod-like structure that displays a high conformational flexibility, similar to that of other structural maintenance of chromosomes (SMC) proteins. This flexibility is largely conferred by the intermediate region of the protein that connects Smchd1’s two functional domains: the N-terminal GHKL ATPase and the C-terminal SMC hinge domain. In follow-up studies of the two individual domains, we revealed the first atomic-resolution structure of Smchd1’s hinge domain, providing a novel insight into its DNA-binding and dimerisation modes. Contrary to previously suggested models describing the DNA interaction mode of canonical SMC proteins, I showed that nucleic acids are not threaded through the central pore region of the Smchd1 hinge domain. Subsequent immunofluorescence studies additionally revealed that the hinge domain targets full-length Smchd1 to chromatin, and that a functional hotspot within the hinge is required for chromatin localisation in cells. SMCHD1’s ATPase domain has been of particular interest due to the identification of disease-related variants that are frequently located within this region of the protein. However, the mechanisms by which some of these pathogenic variants affect SMCHD1 function are poorly understood. Using analytical ultracentrifugation, I demonstrated that the wild-type SMCHD1 ATPase undergoes dimerisation, which was reliant on the inclusion of both the UBL domain and the presence of substrate, ATP. Follow-up cellular studies revealed that Smchd1’s catalytic activity, as well as the presence of the newly- identified UBL domain, are both necessary for the localisation of full-length Smchd1 to chromatin. Together, these studies provide an insight into the molecular basis of Smchd1 function and highlight how chromatin binding may be compromised in human disease. Future studies will further investigate the cellular localisation and dimerisation properties of disease-associated SMCHD1 variants, contributing towards our ongoing drug development program aimed at developing therapeutic treatments for FSHD patients.
Characterisation of the Plasmodium aspartyl proteases DNA-damage inducible protein 1 (DDI1) and Plasmepsin VII (PMVII)
Plasmodium falciparum resistance to artemisinin-(ART) based combination therapies (ACTs) and other antimalarials poses a major threat to malaria control and elimination. Current efforts are aimed towards identifying potent antimalarials which inhibit multiple stages of the parasite lifecycle or discovering novel drug targets which may help overcome ART-resistance. This work aimed to characterise two aspartyl proteases of P. falciparum which may hold promise as antimalarial targets. One strategy recently proposed to overcome ART-resistance is the synergistic use of a parasite-selective proteasome inhibitor to sensitise ART-resistant parasites to artemisinin. Therefore, development of an inhibitor targeting a parasite-specific protein involved in the P. falciparum ubiquitin-proteasome system (UPS) could yield a combination therapy to tackle ART-resistance. DNA-damage inducible protein 1 (DDI1) is a previously uncharacterised essential aspartyl protease in P. falciparum. Recent studies have shown that the catalytic domain of human DDI2 upregulates the UPS in mammalian cells. In other organisms, DDI1 plays a role in shuttling proteins to the proteasome for degradation via its ubiquitin-like domain. We hypothesise PfDDI1 is an active aspartyl protease and plays a role in the parasite’s UPS. To investigate the role of DDI1 in the UPS and parasite survival, we identified a DDI1 orthologue in P. falciparum and characterised this using several strategies. We utilised CRISPR-Cas9 to knock out, tag and inducibly knock down DDI1 across the asexual lifecycle of P. falciparum, and study the effect of this on parasites. Expression of recombinant DDI1 proteins provided insight into the protease activity and substrate repertoire of PfDDI1. Together these studies provide insight into the domain architecture, essentiality and function of PfDDI1 and clues into its potential as an antimalarial target. Development of an antimalarial to block parasite transmission between humans and mosquitos is also a viable strategy to reduce malaria burden. In this study, we also explore a potential transmission-blocking target, Plasmepsin VII (PMVII) and create tools to enable further study of this aspartyl protease in sexually reproductive gametocytes. These tools are vital to determine the function and substrate repertoire of PMVII and elucidate its potential as an antimalarial target.
Investigation of mammary gland development and resident macrophages by 3D and intravital imaging
The mammary gland is a fascinating organ that develops after birth and is capable of remodelling through multiple rounds of reproduction. The behaviour of mammary epithelial cells and how these interact dynamically with their environment are poorly understood. Cell morphology and arrangement can be addressed by three-dimensional (3D) confocal imaging to provide large-scale, subcellular resolution views of tissue architecture. Further insight can be gained from intravital imaging that allows direct observation of cell behaviour in vivo, but this has rarely been implemented for the normal mammary gland. Mammary ducts are embedded in adipose tissue, making in vivo imaging of mammary ducts extremely challenging. Chapter 3 provides a detailed protocol for an intravital imaging method that was adapted and optimised for the mouse mammary gland. This technique enables high-resolution, 3D intravital imaging of the mammary gland for up to twelve hours. The skin flap surgical technique was modified to expose the entire inguinal mammary gland, allowing rare accessible epithelial structures to be identified. Additional fine microdissection of connective tissue maximised the resolution of imaging. Significant measures were taken to achieve as near to physiological conditions as possible, including creating a sealed environment over the exposed tissue. Strategies used for image analysis are then discussed, including image stabilisation, cell tracking and 3D visualisation. This technique advances our ability to observe mammary cell behaviour in vivo and will enable future investigation of rare events that are spatially and temporally regulated, such as stem cell behaviour, tumour initiation and microenvironment interactions. Mammary gland morphogenesis occurs by migration of terminal end buds through the mammary fat pad. Terminal end buds are large, club-like structures comprising a cap layer and a multi-layered body that give rise to bilayered ducts. Epithelial progenitors within terminal end buds generate mature cells of ducts but how these behave and cooperate to generate the bilayer is not well understood. Chapter 4 describes the lineage-specific behaviours of terminal end bud progenitors as observed by intravital microscopy. Cap cell migration into the body was recorded at high resolution in vivo for the first time. High-dimensional image quantification of cap cell behaviour showed that most cap cells that migrate into the body die rapidly but a small proportion survive long term. Progenitors for the luminal lineages were observed to have contrasting behaviours, with hormone-sensing progenitors being highly migratory. Single cell transcriptomic analysis of terminal end buds is described, providing possible molecular drivers of the distinctive behaviour of hormone-sensing progenitors. This work provides an unprecedented view of mammary stem cell behaviour, making an important contribution to our understanding of how cellular behaviour drives organogenesis. Chapter 5 describes a previously uncharacterised population of resident intra-epithelial macrophages that were revealed by 3D confocal imaging. These cells, termed mammary ductal macrophages, are regularly positioned over the entire mammary gland at all stages of development. They do not migrate but monitor the epithelium by dendrite movement, allowing them to rapidly sense and respond to epithelial damage. Ductal macrophages proliferate in pregnancy to maintain their density on the epithelium in lactation. During involution following weaning, they rapidly phagocytose dying alveolar cells to facilitate remodelling. Breast tumour-associated macrophages are pro-tumorigenic and strongly resemble ductal macrophages, not stromal macrophages. Macrophages are emerging as important targets for breast cancer treatment, therefore, better understanding of parallels between DM function in healthy and perturbed tissue may enable development of improved cancer therapies. Finally, in Chapter 6, the presented results are summarised and their context within the field, wider implications and possible future directions are discussed. Overall, this thesis presents original research that advances our technical ability to address questions of cell dynamics in the mammary gland, provides important insights into mammary stem cell behaviour during morphogenesis, and characterises a novel tissue-resident macrophage population, finding a key role for these in mammary gland remodelling.
Identify new regulators of TNFR1-induced necroptotic pathway
Acute Myeloid Leukaemia (AML) is a vastly heterogeneous blood disorder with a poor prognosis for patients older than 65. Our group has been focused on developing new treatments for AML to replace the standard intensive chemotherapy. Previous data showed that the SMAC-mimetic birinapant in combination with the caspase inhibitor IDN could kill different types of AML both in vitro and in vivo through activation of necroptosis cell death pathway. However, over 50% of the patient samples tested in study showed resistance to necroptosis. This project aims to determine the molecular mechanisms that mediate necroptosis resistance in AML and identify new regulators of necroptotic pathway. The results obtained in this study will expand the knowledge of necroptosis signalling in leukaemia and will contribute to the optimal clinical use of birinapant/IDN drug combination. This project contains 2 parts; (1) We will use human AML cell lines that are resistant to necroptosis to determine the molecular changes involved in cell death resistance. (2) We will use CRISPR/Cas9 knock out screen in human AML cell lines that are sensitive to necroptosis, trying to identify new regulators of TNFR1-induced necroptotic pathway. Together these experimental approaches will allow a better understanding of the regulation of TNF-necroptosis signalling in AML. By overexpression wild-type RIPK3 in the KG-1 cell line, we successfully sensitised KG- 1 cells to necroptosis, which indicates that the KG-1 endogenous RIPK3 is dysfunctional. By cDNA sequence of KG-1 endogenous RIPK3, we detected several mutant base pairs, which may lead to the dysfunction, but this result needs further prove by genome sequence, which is undergoing. By CRISPR knock out screen, we found several targets that may lead to the necroptotic resistant, and MAGE3 is the most research-worthy one. Knockout MAGEB3 in the MV4;11 cell line led to the downregulation of RIPK3 and the necroptotic resistance. However, this result could not be repeated on the U937 cell line, and the mechanism of how MAGEB3 regulates RIPK3 is still unclear. Further research will be done on MAGEB3 to have a better understanding of the role of MAGEB3 in the necroptotic pathway. Together, all these results gave a better understanding of the necroptotic pathway and may contribute to the treatment of AML.
Intracellular competition regulates B lymphocyte differentiation
The production of antibodies, with their potential to recognise unique targets and prevent repeat infections, is an important aspect of immune health. In order to generate free antibodies, the cells responsible, B cells, must undergo a differentiation step to transform from lymphoblast to antibody secreting cell (ASC). This differentiation step prevents further antibody modifications and hence the timing for optimal immunity requires a delicate balance between expanding useful clones and providing early protection. How differentiation is controlled to achieve this balance for an effective immune response is of great interest. In this study, the progression from naive B cells to ASC was investigated in the context of an emerging model for competing cell fates. By this model, alternate cell fates, such as division, death and differentiation, are pursued independently in individual cells but are in competition such that events which occur earlier prevent those that require more time from being observed. Evaluation and testing of this model requires careful measurement of distributions of times to fates which is only possible with single cell fate tracking. Here I have developed and applied methods for live cell imaging and analysis for assessing and evaluating cell fate changes over time. Using these methods, several modes of regulating differentiation times were revealed. Low levels of stimulation through CD40 produced a greater proportion of antibody secreting cells per generation as division is slowed and more time is allowed for differentiation, consistent with competing cell fates. A second mechanism was found where increasing division numbers directly reduced the amount of time required for cells to differentiate, without modulating division times, ensuring the natural development of ASC during the ongoing immune response. A direct method of uncensoring was explored where cell cycle inhibitors were used to prevent division, with the hypothesis that more cells would go on to differentiate in the absence of competition. Various inhibitors were assessed for their suitability to this task, and a panel of compounds were found to be suitable for uncensoring underlying differentiation and cell death times. Findings from this study are consistent with the model of independent and competing cell fates, and significantly advance our understanding of how antibody responses are controlled and can be modelled at the cell population level.
Identifying Plasmodium vivax encoded proteins that may prevent host cell death during liver development
Malaria is a major global health problem and a leading cause of death worldwide. The mechanism behind some parts of the parasite life cycle are still obscure, especially the liver stage which is essential for parasite development and maturation. It is likely that the parasite prevents the host hepatocyte from undergoing cell death during invasion. This is especially relevant for Plasmodium vivax as the hypnozoite can lay dormant in a liver cell for months, even years. We hypothesise that P. vivax encodes proteins to inhibit host cell death in liver. We used the computer algorithm I-TASSER to identify several P. vivax proteins which were predicted to have similar structures to human proteins involved in cell death. We expressed these P. vivax proteins in mammalian cells and performed functional tests to investigate their potential roles experimentally. Identification of P. vivax proteins that influence host cell death would improve our understanding of how P. vivax can survive for prolonged periods in the host cell during liver stage and may accelerate the development of new drugs for malaria liver stage, which is necessary for the ultimate goal of eliminating malaria.
Low birthweight and infant growth among children in Papua New Guinea - effect of malaria and other infectious diseases during childhood
Globally, young children continue to die or fail to thrive from treatable and preventable causes including low birthweight (LBW), childhood undernutrition (wasting, stunting, underweight) and infectious diseases. Reducing the burden of these are an essential pillar of global child health and survival targets. These global trends are reflected in Papua New Guinea (PNG), a resource constrained setting that continues to observe high rates of illness and death in young children with LBW, childhood undernutrition and infectious diseases (especially malaria, pneumonia and diarrhoea) continuing to be the leading causes. Improving child health and survival in PNG requires evidence about the risk factors for LBW, sub-optimal growth and infectious diseases, as well as the inter-relatedness of risk factors, that can inform policies and identify appropriate interventions and strategies. This thesis aims to address critical knowledge gaps in this area and provide insights to inform interventions and strategies aimed at reducing low birthweight, childhood undernutrition and malaria in young children in PNG and globally. LBW is caused by a multitude of factors which are often inter-related and with that, it is often difficult to distinguish between independently direct and indirect effects. Moreover, current interventions targeted at preventing LBW generally assume a single dominant cause overlooking the inter-relatedness of risk factors and the possibility of factors exerting joint effects on LBW. These are difficult to establish with widely used standard statistical methods and have therefore been rarely investigated. Using structural equation modelling, we showed intermittent preventive treatment of malaria during pregnancy with sulphadoxine-pyrimethamine (SP) plus azithromycin (AZ) to be independently directly associated with reduced probability of LBW. Unexpectedly, anaemia at enrolment was also directly associated with reduced probability of LBW. Maternal undernutrition at enrolment was independently directly associated with increased probability of LBW. No significant indirect associations between risk factors and LBW were established. After birth, children in lowlands PNG experience high rates of malaria, pneumonia and diarrhoea alongside undernutrition. Infections and undernutrition are believed to have a bi-directional relationship and whilst the effect of undernutrition on risks of illness and death is well known, little is known about the effects of malaria, pneumonia and diarrhoea on growth faltering, particularly among PNG children. By using multivariable regression and distributed lag models for data analysis, we observed malaria pneumonia and diarrhoea to have a differential impact on child growth. The effect of acute malaria on child growth was observed to be long-term while pneumonia and diarrhoea had short-term effects lasting up to 3 months. Of these three diseases, malaria was once ranked the top leading infectious cause of childhood morbidities and mortality in PNG, especially in lowlands PNG. The nationwide scale-up of malaria control interventions significantly reduced overall malaria transmission between 2008 and 2014 but a detailed understanding of the impact of this changing transmission on the epidemiology and risk profile of malaria infections and disease due to the two main species in young children was lacking. By analysing three consecutive longitudinal child cohorts (1-5-year-old children) conducted over the period of improved control (2013), we observed a differential impact of improved control on P. falciparum and P. vivax. Additionally, we showed that with declining malaria transmission, burden of malaria infections and illness were highly spatially localised to areas that had the highest burden prior to scale-up, highlighting potential hotspots of transmission. Collectively, the findings from this thesis provide important insights for improving child health in PNG and globally.
Exploiting DNA repair defects in high-grade serous ovarian carcinoma
High-Grade Serous Ovarian Carcinoma (HGSOC) is the most common subtype of ovarian cancer, and is the leading cause of ovarian cancer death. This subtype is molecularly characterised by frequent loss of Homologous Recombination (HR) DNA repair, making it susceptible to Poly-ADP ribose polymerase (PARP) inhibitor treatment. Despite the efficacy of PARP inhibitors (PARPi) in treating HGSOC, disease recurrence is common. A poor understanding of PARPi toxicity and resistance mechanisms has limited improvements in overall survival of patients. This project utilised HGSOC Patient-Derived Xenograft (PDX) and in vitro models to determine underlying mechanisms of PARPi sensitivity and resistance. Characterisation of the HGSOC PDX cohort led to the discovery of 2 models with HR gene secondary mutations, thereby explaining the poor PARPi responses of these tumours. Loss of the Classical Non-Homologous End-Joining (C-NHEJ) DNA repair pathway was also investigated but was not supported as a mechanism of resistance in PDX, or in a genome-wide PARPi-resistance CRISPR screen using a BRCA2-mutant cell line. The CRISPR screen revealed PARP1 mutations as the top hit, but no C-NHEJ mutation hits. CRISPR screens have also been optimised for a BRCA1-mutant cell line and a unique BRCA1 methylated cell line, to enable future validation in different HRD contexts. HR gene methylation analysis in the HGSOC PDX models revealed distinct BRCA1 promoter methylation profiles, subsequently found to represent “homozygous” and “heterozygous” methylation states. Our group demonstrated that all copies of BRCA1 must be methylated for gene silencing and PARPi response, and that methylation can be lost under treatment pressure in patients. I then investigated this mechanism in two HGSOC PDX models with RAD51C promoter methylation. RAD51C promoter methylation was found to have homogeneous or heterogeneous patterns, and both caused gene silencing and PARPi response in the PDX, that could be lost under treatment pressure. One third of patient samples tested appear to have a heterogeneous RAD51C methylation profile and, like the PDX, some responded to chemotherapy. To assess whether global methylation profiles influenced HR gene methylation stability, I carried out genome-wide methylation array analysis of PDX models and clinical samples. Analysis of cyclically PARPi re-treated RAD51C methylated tumours from one PDX model demonstrated increasing global losses in methylation with each treatment cycle. Through characterisation of the HGSOC PDX cohort and in vitro CRISPR screens, I have uncovered both well-established mechanisms of resistance (e.g. secondary mutations), and less studied mechanisms of sensitivity and resistance, such as loss of RAD51C methylation and PARP1 loss. These models provide a platform for further study of these PARPi resistance mechanisms, including potential therapeutics to prevent or overcome their development in the clinic.
Structural and functional characterisation of the molecular assembly of two pseudokinase scaffolds
The human pseudokinase SgK269, and its structurally related homologue SgK223, are oncogenic interacting scaffolds that promote the assembly of specific tyrosine kinase signalling pathways. SgK223 and SgK269, as well as the recently discovered PEAK3, belong to the PEAK family of protein pseudokinases. They are large, multidomain proteins that are comprised of an N-terminal region of unknown structure and function, a large unstructured PEST region containing tyrosine phosphorylation sites and a C-terminal domain comprised of a pseudokinase domain flanked by regulatory helices. SgK223 and SgK269 have been shown to localise to focal adhesions and their overexpression leads to increased cell migration and changes in cell morphology, hallmarks of cancerous cells. Recent studies from our lab and others have provided structural insight into the C-terminal domain and flanking alpha helices of SgK223 and SgK269. These structures highlighted a conserved mechanism of dimerisation that drives homo- and hetero-association of SgK223 and SgK269 and plays an important role in cell migration. Additionally, SgK223 and SgK269 were demonstrated to undergo homo- and hetero-oligomerisation through their pseudokinase domains. In contrast to the C-terminal domain, little is known about the function of the N-terminal domains of SgK223 and SgK269, although there is sequence conservation between them. In this study, we have begun characterising the N-terminal domains of SgK223 and SgK269 using biophysical and biochemical techniques, initially demonstrating that these domains are monomeric and appear to have no defined secondary structure. To further investigate SgK223 and SgK269 homo- and hetero-association we carried out single site alanine mutagenesis to determine the energetic hotspots at the dimerisation interface of SgK269. Furthermore, we carried out mutagenesis within the N-lobe of SgK223 and SgK269, to investigate the role of this interface in homo- and hetero-oligomerisation. Additionally, we characterised the PEAK family interactions with the critical interacting signalling adaptor protein, CrkII, using biophysical assays and X-ray crystallography. We found that each member of the PEAK family has a proline-rich motif within their PEST linker that interacts with CrkII N-SH3 domain with ~1-3 uM affinity. The crystal structure of the CrkII N-SH3 domain bound to the SgK269 proline-rich motif demonstrated the critical consensus residues for the PEAK family interaction with CrkII. To further investigate the role of SgK223 and SgK269 homo- and hetero-association in cells, these studies were complemented with localisation microscopy techniques. Utilising mutants of SgK223 and SgK269 that can no longer dimerise or oligomerise, we investigated the importance of SgK223 and SgK269 associations for their localisation and thus, role in signalling. Insights into the scaffolding functions of SgK223 and SgK269 will inform how they contribute to the assembly of signalling pathways and hence their role in cancer.
Establishing pre-clinical models for advanced colorectal cancer
Colorectal cancer (CRC) is the third most common cancer worldwide, and one of the most common cancers in Australia. When detected early, there are multiple treatment options for CRC; however, patients often relapse and ultimately succumb to metastatic disease. 5-Fluorouracil (5-FU)-based chemotherapy is a common first-line treatment for CRC. However, chemotherapy response rates remain low, often due to the development of resistance, which is one of the main limitations in the management of the disease. Our understanding of the progression of CRC, and the development of new therapeutics for CRC, has been facilitated in part by animal models. Unfortunately, many murine models of CRC are adenomas, with few patient-derived models, or models of metastatic disease available. As a result, the opportunity to improve our understanding of the pathogenesis of advanced disease, or test the efficacy of novel therapeutics for advanced disease, is limited. The overarching aim of this PhD thesis was to generate new models of CRC to facilitate the study of this devastating disease. A biobank of 16 CRC patient-derived xenografts (PDXs) was successfully established in immunocompromised mice. These PDX lines recapitulated the histopathological, molecular and genetic features of the original patient tumours. These PDXs represent new pre-clinical tool that will allow for testing the efficacy of potential new therapeutics. A selection of four PDX lines underwent serial 5-FU treatment to generate a library of resistant PDXs, and their matched non-resistant chemonaive controls. The 5-FU resistant PDX tumours underwent an upregulation of the IL-11R/STAT3/Bcl-2 pathway in response to 5-FU. This suggests that IL-11 signalling is elevated in response to 5-FU to promote tumour cell survival. Thus, targeting the IL-11/IL-11R signalling may be a promising strategy to overcome chemoresistance. Finally, four different genetically-engineered mouse models (GEMMs) were established to generate a reproducible model of metastatic CRC. It was found that mutations in Apc (or dysregulation of Wnt signalling) restricted to the colonic epithelium lead to the formation of adenomas, as did the addition of mutations in Tp53. The combination of Apc mutations with Tp53 and Kras mutations lead to an earlier tumour onset, but did not result in metastasis, contrary to previous reports in the literature. It was found however, that mutations in Tp53 and Kras in the stem cell compartment, combined with dysregulation of Wnt signalling, lead to potential metastasis to the liver. However, this did not occur in 100% of the animals, and is thus not amenable to therapeutic studies. Future studies will incorporate alterations to TGFb signalling, in an effort to increase the reproducibility of metastasis. These studies highlight the lack of our understanding of the drivers required for tumour cell metastatic potential. Taken together, the research described in this thesis has led to the generation of a number of new animal models of CRC, that may be of use to future studies of the pathogenesis and treatment of this disease.