Medicine (RMH) - Theses
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ItemInvestigating the functional biology of chimeric antigen receptor T cells( 2017)Despite the success of autologous chimeric antigen receptor (CAR) T cells to treat patients with refractory B cell acute lymphoblastic leukaemia, (ALL) and lymphoma, there are many aspects of CAR T cell biology that remains unknown. For this reason, this thesis explored whether the recognition of antigen via either antigen receptor (CAR vs endogenous T cell receptor (TCR)) affected the CAR-T cell immune synapse, receptor signalling and tumour target killing kinetics. By addressing this issue we aim to translate this new knowledge to the clinic and broaden the CAR T therapy success to patients with a wider range of cancer subtypes. To explore the above questions, a novel transgenic mouse (designated CAR.OT-I) was developed, in which CD8+ T cells co-expressed the OVA257-specific T cell receptor (TCR) and a second generation CAR with an scFv specific for human HER2. Chapter 3 of this thesis validated the model system and compared CTL activation from CAR.OT-I and OT-I mice. Chapter 4 used time-lapse and confocal microscopy to explore whether the killing kinetics of CAR.OTI CTL was different when stimulated via with OVA257-pulsed (TCR) or HER2-expressing tumour cells (CAR). This thesis showed for the first time, individual CAR.OT-I CTL killed multiple tumour cells (‘serial killing’) and detached faster from dying targets after CAR ligation. Furthermore, in chapter 5, the CAR immune synapse gross molecular structure was described for the first time. This disrupted immune synapse had Lck micro-clusters, poor actin clearance and no peripheral LFA-1 clustering. Finally, phosphoprotein signalling and Ca2+ flux studies revealed faster, stronger signalling initiated via CAR compared to TCR ligation. This observation was also correlated with faster recruitment of cytotoxic granules to the target cell after CAR ligation. Taken together, the chapter 5 data reveals the mechanisms whereby CAR ligation initiates rapid tumour killing and detachment (Chapter 4). Information from this body of work can be used to inform on the next generation of CAR designs and provides a baseline for comparing CAR and TCR killing events.
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ItemCharacterisation of the nuclear pore abnormalities in the intestinal zebrafish mutant, flotte lotte (flo)( 2012)The evolution of eukaryotic cells is defined by the compartmentalisation of the genetic material inside the nucleus, segregated from cytoplasm by a nuclear envelope. This barrier is punctuated by approximately 3000 large multi-protein structures known as nuclear pore complexes, which permit the bidirectional transport of protein and RNA molecules between the nucleus and cytoplasm. This study provided the opportunity to investigate the importance of the nuclear pore protein Elys during vertebrate development. Zebrafish mutants generated by ethylnitrosourea (ENU) mutagenesis provide a powerful tool for dissecting the genetic regulation of developmental processes. Our interest has focused on a panel of ENU generated mutants exhibiting a variety of defects in the formation and differentiation of the intestinal epithelium. One of these mutants, flotte lotte (flo), harbours a premature stop codon in the coding sequence of the nuclear pore component elys (embryonic large molecule derived from yolk sac). Elys is an essential component of the nuclear pore complex, yet surprisingly, its mutation in the flo mutant does not result in a global dysfunction in nuclear pore formation throughout the developing zebrafish embryo. Instead, flo mutants exhibit tissue-specific abnormalities in the development of the intestinal epithelium, liver, pancreas and eye; organs that are highly proliferative from 48hpf. We show that this time-point coincides with the exhaustion of maternally-deposited stocks of elys mRNA from flo embryos. Not surprisingly, we found that the ensuing inability to create new nuclear pore complexes appears to impact most severely on these rapidly proliferating tissues. Using multi-photon microscopy we reconstructed three-dimensional renditions of the endodermal organs in wild-type and flo larvae. Compared to the highly elaborated and polarized intestinal epithelium of wild-type zebrafish, the intestinal epithelium in flo is thin, unfolded and poorly polarised. Moreover, nuclear pore complexes in flo intestinal cells are not embedded in the nuclear envelope but are found in profuse cytoplasmic aggregates. Catastrophic levels of apoptosis accompany the loss of a functional nuclear envelope in intestinal epithelial cells. Thus, flo mutants provide an opportunity to identify signals that commit nuclear pore-deficient cells to an apoptotic fate. We found that the apoptotic response observed in the flo intestine is mediated via a Tp53-independent mechanism. Since Elys function is critical for the integrity of proliferative cells in zebrafish, we investigated whether ELYS is also critical for the proliferation of human cancer cells. We discovered a strong up-regulation of ELYS expression in many cancers when compared to their respective normal tissues. We observed ELYS to be ranked in the top 1% of all up-regulated genes investigated in gene expression studies of colorectal, kidney, liver and breast cancers available in the Oncomine database. The discovery that ELYS is frequently over-expressed in human colorectal cancer suggests that our functional genomics approach to novel cancer gene discovery using zebrafish mutants is valid. Moreover, we propose that targeted approaches to disabling ELYS synthesis or function may activate apoptosis in colorectal cancer cells and provide a useful therapeutic approach in the future.
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ItemIdentification of genes that regulate lymphatic endothelial cell migration through siRNA screening( 2013)The lymphatic vasculature is vital for tissue fluid homeostasis. It also enables effective immune surveillance, and aids the transport and absorption of fatty acids. Damaged or faulty lymphatic vessels can lead to an accumulation of lymph fluid, a condition known as lymphoedema. Metastatic cancer can also spread via the lymphatic vessels; expression of the lymphangiogenic growth factors VEGFC and VEGFD is correlated with tumour progression and negatively affects overall patient survival. Given the importance of lymphangiogenesis and vessel remodelling in development, and in pathological conditions such as cancer, it would be of interest to understand the molecules required for new lymphatic vessel growth to occur. One aspect of lymphangiogenesis that can be modelled in vitro is cell migration. The primary approach of this thesis was to utilise RNA interference technology to screen the entire human genome for molecules that regulate lymphatic endothelial cell (LEC) migration. An experimental procedure was established that was compatible with transfection of small interfering RNA (siRNA) in primary LECs, and enabled accurate quantification of LEC migration in a scratch-wound assay. Using this approach, a smallscale pilot screen of genes for 133 protein tyrosine kinases and tyrosine kinase-like molecules was performed. Several promising candidate genes were identified, whose roles in LEC migration were confirmed using siRNA and pharmacological inhibitors. The success of this approach enabled the screening of a genome-wide SMARTpool siRNA library, which was performed in a high-throughput manner with the aid of automation and robotics. A total of 154 genes were validated with medium or high confidence as regulators of LEC migration in vitro. This dataset enabled the collation of maps detailing signalling cascades and processes that may control the migration of this specialised cell type. While a proportion of the genes identified have been previously linked to cell migration or development of the vasculature, many were not known to play a role in cell migration or lymphatic biology. A parallel microarray study investigated the transcriptional response that occurred during LEC migration. This demonstrated that the majority of genes identified by the functional screen as being important for this process are not up-regulated during migration. However, gene ontology enrichment analysis found that genes associated with mitosis and the cell cycle are down-regulated during the early stages of cell migration. This prompted an investigation of the connection between cell cycle regulation and the ability of a cell to migrate effectively. Utilising high-content image analysis, the genome-wide LEC migration screen dataset was reanalysed to identify genes that, upon siRNA-mediated knockdown, led to cell cycle arrest. Importantly, this study showed that LECs are unable to migrate when arrested in S or G2/M phase, and identified a potential role for the Hippo/Warts pathway in ensuring that cells do not migrate during these phases. This thesis has revealed molecular regulators of lymphatic endothelial cell migration through a genome-wide siRNA screen. Identification of novel genes important for the lymphatics will increase the understanding of LEC signalling networks, and has implications for the development of therapeutic agents that could be used in preventing lymphogenous and distant organ tumour metastasis, or treating conditions such as lymphoedema or lymphangioma. Future investigations will define the biochemical signalling events that occur in LEC migration, and explore the significance of these pathways in vivo.
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ItemBiological function of the proteolytic processing of vascular endothelial growth factor-D in cancer( 2012)The Vascular Endothelial Growth Factors are a family of secreted homodimeric glycoproteins and primary regulators of angiogenesis, the growth of new blood vessels, and lymphangiogenesis, the growth of new lymphatic vessels. The mammalian family consists of VEGF, PlGF, VEGF-B, VEGF-C and VEGF-D and they exert biological effects in tissues by binding and activating their cognate receptors, VEGFR-1, VEGFR-2 and VEGFR-3, expressed on endothelial cells. VEGF-C and VEGF-D comprise a subset of the VEGF family as they are initially synthesised as precursor proteins composed of an N- and C-terminal propeptide flanking the central VEGF homology domain, which contains receptor binding sites. Proteolytic processing of these propeptides liberates the mature form consisting of homodimers of the VEGF homology domain, with enhanced affinity for receptors. VEGF-D binds VEGFR-2 and VEGFR-3, and when expressed in tissues promotes blood and lymphatic vessel growth. In animal models of cancer VEGF-D expression induces lymphangiogenesis, increasing lymph node metastasis, and stimulates tumour angiogenesis enhancing tumour growth. VEGF-D can be expressed in a range of prevalent human cancers and this expression correlates with lymph node metastasis and shorter overall patient survival in studies of breast, colorectal and ovarian cancer. Previous analysis of full-length VEGF-D in vivo demonstrated variability in the angiogenesis and lymphangiogenesis induced in tissues, however, processing was not blocked in these model systems so partially and fully processed VEGF-D may have contributed to the effects observed. The activity of full-length VEGF-D, which has not been processed, is not known. In addition, the bioactivity of partially processed VEGF-D and the effect that cleavage of the N- or C-terminal propeptides has on the biology of the molecule has not been defined. Further, it is not known if proteolytic processing is absolutely required for VEGF-D to promote tumour growth and spread. With the aim of determining the effects of the proteolytic processing of VEGF-D on its biological function and action in cancer, a form of VEGF-D was created with mutations at known cleavage sites, effectively blocking processing of both propeptides. Further, to explore the effects of partial processing and the role of the propeptides, additional mutants were generated in which only the N- or only the C-terminal propeptide were deleted and the cleavage site of the remaining propeptide was mutated to block processing. The research described in this thesis demonstrates that each processing mutant displays unique receptor binding and activation profiles, and activity in in vitro assays. In addition, processing of the N- and C-terminal propeptides is a critical determinant of the interactions of VEGF-D with neuropilins and heparin-containing proteoglycans. Tumour xenograft experiments showed that completely blocking VEGF-D processing significantly reduced tumoural angiogenesis and lymphangiogenesis, and abolishes the contribution of VEGF-D to tumour growth and spread. Further, VEGF-D processing mutants lacking the N- or the C-terminal propeptide displayed distinct effects on tumoural angiogenesis, lymphangiogenesis and tumour growth, however, did not promote metastasis to lymph nodes. This data demonstrates that proteolytic processing of VEGF-D is essential for this protein to enhance angiogenesis and lymphangiogenesis in cancer, and provides a possible strategy by which therapeutics can be targeted to block processing in tumours expressing VEGF-D, in order to restrict the growth and spread of cancer.
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ItemThe U12-dependent spliceosome is essential for regulating gene expression during zebrafish development( 2010)Removal of introns from pre-mRNA is an essential step in generating mature mRNA. The majority of introns are removed by the major, or U2-type, spliceosome, while the minor, or U12-type, spliceosome catalyses the removal of a small set of introns with characteristic features that are highly conserved in metazoans and plants. A novel zebrafish mutant, caliban (cal), with specific U12-type splicing defects, was used to study the biochemistry of the U12-type spliceosome and the role of U12-type introns in the regulation of gene expression. Greater than 99% of vertebrate introns are U2-type introns. However, a second class of U12-type introns exists that is defined by highly conserved consensus splice sites. U12-type introns represent about 0.5% of all introns in vertebrate genomes and usually occur alone alongside U2-type introns in pre-mRNA. U12-type introns are removed by a separate spliceosome that shares many components with the U2-type spliceosome, but contains several unique small nuclear RNAs (snRNAs) and spliceosomal proteins. Several lines of evidence suggest a function for U12-type splicing in the regulation of gene expression. These include the high evolutionary conservation of U12-type introns, their enrichment in certain functional gene groups and the demonstration that their excision can be rate-limiting in the generation of mature mRNAs. Despite these observations, little is known about the role and possible regulatory function of U12-type splicing in vivo. cal is a zebrafish development mutant with abnormalities in the intestinal epithelium, which is poorly polarised and unfolded compared to wildtype (wt). Mutant embryos also show a reduction in size of the liver and the pancreas and display a morphologically abnormal lens in the context of a smaller eye. The genetic lesion in cal was mapped to rnpc3, encoding the zebrafish orthologue of the human U11/U12 snRNP 65KDa protein, a specific component of the U12-type spliceosome. It was shown that cal embryos specifically retain U12-type introns compared to wildtype. Biochemical analyses of U12-type spliceosomal small nuclear ribonucleoproteins (snRNPs) demonstrate abnormal formation of the U11/U12 di-snRNP, which represents the first step in U12-type spliceosome assembly. However, it was also demonstrated that larger spliceosomal particles form and accumulate in cal embryos in the absence of Rnpc3/65K, suggesting a potential novel role for Rnpc3/65K in U12-type spliceosome disassembly and recycling. Whole transcriptome analysis of cal and wt embryos by microarrays and RNA sequencing demonstrated retention of U12-type introns on a global scale as well as a set of about 700 differentially expressed genes between cal and wt at two different developmental time points. Further analysis of gene expression data has led to the emergence of a model in which cal embryos are sustained through the first 48-72hpf by maternally deposited rnpc3 mRNA and Rnpc3/65K protein. After this time, defective U12-type splicing induces a cell cycle arrest in the endoderm-derived tissues of the liver, pancreas and intestine, which are highly proliferative between 72 and 108hpf. These results are leading to further studies with a focus on the role of U12-type splicing in human cancer. It was found that several prominent human tumour suppressor genes such as PTEN, LKB1 and PROX1, contain U12-type introns, and we propose a model by which an intermediate reduction of U12-type splicing efficiency can be tumourigenic by reducing the activity of particular tumour suppressor genes in a dose-dependent fashion. To test this hypothesis, conditional Rnpc3 knockout mouse models are currently being generated on a range of different colorectal cancer-susceptible backgrounds.