Medical Biology - Theses
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ItemInterrogating the cells-of-origin of BRCA mutant cancers to identify therapeutic targets for cancer prevention( 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.
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ItemNo Preview AvailableCharacterisation of the receptor tyrosine pseudokinases, EphB6 and EphA10( 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.
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ItemIdentification of synthetic lethal interactions with the KRAS oncogene for targeted cancer treatment( 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.