Sir Peter MacCallum Department of Oncology - Theses
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ItemMolecular regulation of adipogenesis in secondary lymphoedema: a common complication of cancer therapies( 2018)Secondary lymphoedema is a common, chronic disease caused by inadequate drainage of interstitial tissue fluid from a limb due to damaged lymphatic vessels. It may develop after radiation therapy or cancer surgery involving lymph node dissection, in particularly for breast cancer, as well as a variety of other conditions. The resulting accumulation of interstitial fluid promotes pathological changes including oedema, expansion of fat and dermal fibrosis, which contribute to extensive chronic tissue swelling, typically in the upper or lower limbs. There is no curative treatment or molecular-based therapy for secondary lymphoedema, and current treatments for this condition have limited efficacy so it is an important unmet clinical need in medicine with an estimated 300,000 patients in Australia. A pharmacological intervention to restrict or reduce expansion of fat tissue and associated tissue swelling would be highly beneficial for patients however, the molecular mechanisms driving the development of fat in secondary lymphoedema are unknown. Here, a surgical mouse tail model of secondary lymphoedema is employed to investigate molecular pathways that drive expansion of fat tissue in this condition, and to explore a potential pharmacological approach for restricting this process. The mouse model of secondary lymphoedema employed exhibited key pathophysiological features of clinical secondary lymphoedema such as tissue swelling, subcutaneous oedema, dermal fibrosis and excess formation of fat. With the expansion of fat occurring during the early phase of lymphoedema, and elevated levels of fat persisting in the chronic phase, this mouse lymphoedema model was well suited for studying molecular mechanisms driving the initial expansion of fat as well as the persistence of excess fat tissue in the chronic setting. Whole-genome microarray analysis was used to study the mouse lymphoedema model which revealed that mRNA for insulin-like growth factor binding protein 5 (IGFBP5), an inhibitor of the insulin-like growth factor 1 receptor (IGF1R) adipogenic signalling pathway, was down-regulated in the model compared to controls. Further analyses by immunohistochemistry revealed the presence of IGF1 and IGF1R on adipocytes in the model and in a clinical sample of secondary lymphoedema, and activated IGF1R in the clinical sample. Moreover, the levels of IGF1 (an activating ligand for IGF1R) associated with adipocytes, were elevated in the mouse lymphoedema model. Thus, the IGF1R signalling axis could be active and promote expansion of fat tissue in secondary lymphoedema. Importantly, pharmacological targeting of this pathway in the mouse lymphoedema model with linsitinib, a small molecule tyrosine kinase inhibitor of IGF1R, led to significant reductions in tissue swelling and fat expansion which was associated with decreases in both the number and size of adipocytes. Furthermore, linsitinib also restricted the degree of dermal fibrosis in the mouse lymphoedema model. The work presented in this thesis demonstrated the role of the IGF1R signalling pathway in promoting adipogenesis in a mouse model of secondary lymphoedema. Hence, pharmacologically targeting IGF1R might be a viable therapeutic approach for restricting expansion of fat and tissue swelling in secondary lymphoedema patients.
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ItemIdentification and characterisation of novel mutant p53 regulators( 2018)The p53 protein is a transcription factor and has been considered as a master tumour suppressor. TP53 gene is frequently mutated in human cancers (more than 50%). Mutation in p53 not only loses its tumour suppressive functions but also gains new oncogenic properties, including increased cell proliferation, higher resistance to therapeutics and enhanced metastasis. These are consistent with the association of mutant p53 cancers with poor clinical prognosis. Importantly, some cancer cells are dependent on mutant p53 expression for survival and proliferation. This opens an exciting therapeutic window for the treatment of multiple cancer types. Despite the prominent roles of mutant p53 in malignant progression, its regulation remains partially understood. The major goal of this study is to gain novel insight into the regulation of mutant p53. Our approach has been to identify novel regulators of mutant p53 by utilising whole-genome RNAi discovery screens. These include protein-coding siRNA, microRNA (miRNA) and long noncoding RNA (lncRNA) screens. We have developed a high content assay for these screens. The libraries were subjected to multi-tiered screens on mutant p53-expressing cell lines, including MDA-MB-468, JH-EsoAd1, MDA-MB-231 and AU565. Of 18,120 protein-coding siRNAs, the screen yielded 50 potential mutant p53 candidate regulators. Six of these were conserved between MDA-MB-468 and JH-EsoAd1, and 44 were cell-line specific. Primary lncRNA screen yielded 154 hits, in which 39 and 44 were validated in MDA-MB- 468 and JH-EsoAd1 cell lines, respectively. The miRNA screen yielded a total of 211 mimics and 46 inhibitor hits. Of these, the top 20 miRNA mimics were pursued further. Three potential candidates were identified to down-regulate mutant p53 expression. One of these is miR-504-5p, which has been previously identified to target and down-regulate wild-type p53. Further investigation of these miRNAs led to the identification of MAP1B as a potential regulator of mutant p53. Our result demonstrated that knockdown of MAP1B sensitises mutant p53-expressing cells. The relevance of MAP1B expression to overall survival (OS) of gastric cancer patients (which harbour high TP53 mutation incidence, up to 77 %) was evaluated. Consistently, high MAP1B mRNA expression correlates with poor overall patient survival. Reciprocally, overall survival is improved in patients with low MAP1B mRNA expression. These results suggest a potential role for MAP1B in the oncogenicity of mutant p53 cancer. The mechanism by which MAP1B affects mutant p53 is yet to be elucidated. Overall, this thesis demonstrates the utility of RNAi screen in the exploration of mutant p53 regulation. This study has identified a list of potential regulators that were not previously shown to regulate mutant p53. Future study of these potential novel mutant p53 regulators will help to dissect the regulatory network of mutant p53 and potentially inform on novel therapeutic approaches.
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ItemInvestigating the nucleolar stress response and targets that modulate p53 activation( 2018)The nucleolus is a multifunctional organelle known as the central hub for ribosome biogenesis. Now, it is widely accepted that nucleolus also serves non-canonical role as a stress sensor, responding to numerous internal and environmental stressors. This response is termed the nucleolar stress response (NSR), which can be p53 dependent/independent. One of the cellular insults that activates NSR is perturbation of ribosome biogenesis. In the p53-dependent response, ribosome-free ribosomal proteins L5 and L11 (RPL5 and RPL11) bind to Murine double minute 2 (MDM2), stabilising the p53 protein, leading to cell arrest, apoptosis and/or senescene. However, the exact mechanism by which this process becomes activated remains unclear, and much detail is lacking. In human diseases, ribosomal protein (RP) mutations were frequently observed in Diamond Blackfan Anaemia (DBA) patients, with 20-25% cases of RPS19 mutation. Numerous bodies of evidence suggest that the NSR is the key mechanism of DBA pathogenesis. As such, we hypothesised that a functional genome-wide loss of function (RNAi) screen, performed under the condition of nucleolar stress (due to RPS19 depletion), would identify key proteins that regulate the NSR. Moreover, the candidates identified from this screen would provide new avenues for the treatment of diseases that are caused by abnormal ribosome biogenesis (e.g. ribosomopathies such as DBA and cancer). Prior to the commencement of this study, a genome-wide RNAi screen using Dharmacon human siGENOME SMARTpool siRNAs (4 siRNA duplexes that target the expression of a single gene), was carried out to identify candidates that, when depleted, modulate the p53-mediated NSR caused by RPS19 knockdown. Approximately 400 top candidates that either enhancing or reducing p53 protein expression were selected for secondary screening using the Dharmacon siGENOME deconvoluted SMARTpool siRNA library, assaying each of the individual siRNAs which constituted the original SMARTpool siRNA screened in the primary screen. The 400 candidates were then categorised into 3 categories: high confidence (3/4 or 4/4 duplex hits- if the candidate had 3 or more individual siRNAs that demonstrate the same phenotype as what was observed in the primary screen), medium confidence (2/4 duplex hits) and low confidence (1/4 duplex hits). High and medium confidence candidates identified from the screens included RPL5, RPL11, p53, HEAT Repeat Containing 3 (HEATR3), Cirrhosis autosomal recessive 1A (CIRH1A), Retinoid X receptor alpha (RXRA), Insulin like growth factor 1 receptor (IGF1R), Nucleophosmin (NPM) and Phosphatase and tensin homolog (PTEN). As part of these thesis studies, the high and medium confidence candidates identified in the screen were further validated using an in vitro candidate-based approach. The knockdown of candidates in A549 human lung cancer cells was first confirmed at the mRNA level, followed by measuring the resulting p53 expression via immunofluorescence and western blot. This was further complemented by examining how the cell cycle was affected, using flow cytometry. Apart from NPM, all the candidates were confirmed preventing p53 expression induced by RPS19 knockdown, demonstrating their important role underlying NSR. One of these candidates, HEATR3, was further evaluated to garner some insights into its normal role within cells, and how it influences the NSR. Due to its novelty and previous literature demonstrating its role in ribosome 60S subunit biogenesis in yeast (Syo1), HEATR3 was further investigated to determine its specific role in the p53-mediated NSR and its normal cell functioning. Due to lack of robust tools (e.g. specific antibody) to study the endogenous function of HEATR3, a cell line stably expressing myc-tagged HEATR3 (A549-MT-HEATR3) was generated and used in most of the studies in this thesis. We aimed to investigate the subcellular localisation of HEATR3 by nuclear-cytoplasmic fractionation and immunofluorescence analysis. With these assays, the endogenous HEATR3 and MT-HEATR3 were found to be located in the nucleus. Furthermore, HEATR3 depletion prevented p53 activation/stabilisation after challenging cells with multiple RP depletion (knockdown of RPS6, RPS14 and RPL26). In contrast, HEATR3 depletion was unable to prevent the NSR induced by certain drugs/chemicals, including Doxorubicin, Camptothecin, Etoposide, CX-5461 and MG-132. In particular, HEATR3 depletion only rescued the p53 response with RP depletion/Leptomycin B treatment, suggesting specificity of HEATR3 involvement with these specific stresses. Overexpressed HEATR3 (MT-HEATR3) binds to overexpressed RPL5 and RPL11 (FLAG-tagged RPL5 an FLAG-tagged RPL11) evidenced in co-immunoprecipitation (CoIP) analysis, while its binding to endogenous RPL5 is specific, but not endogenous RPL11. We aimed to determine other binding partners of MT-HEATR3 using mass spectrometry; data analysis was prohibited due to sample variability. Furthermore, we sought to determine whether HEATR3 depletion would alter the localisation of total RPL5/RPL11 (endogenous proteins), however, due to non-specificity of the antibodies, interpretation of the data was prevented. In addition, subcellular fractionation analysis suggested that HEATR3 depletion might not alter abundance of ribosome-free endogenous RPL5 and RPL11. While MT-HEATR3 may bind to 5S rRNA (statistically not significant), our ‘typical’ and ‘high salt’ polysome profiling analysis showed that HEATR3 is required for 60S ribosome subunit biogenesis and translation, which HEATR3 depletion reduced 60S ribosome subunit abundance and polysomes abundance. Particularly, HEATR3 knockdown alter translation of certain subsets of mRNA (decrease in some RPs mRNA translation and increase of Bcl-XL mRNA translation). Overall, studies in this thesis have demonstrated the strength of functional genome-wide RNAi screens in identifying novel modifier genes of the p53-mediated NSR. We have also demonstrated the importance of HEATR3 in p53-mediated NSR and its normal function in ribosome biogenesis for the first time, and have begun to characterise the HEATR3 protein. The findings in this thesis will facilitate further studies in elucidating the impact of HEATR3 in human diseases and its potential role as a therapeutic target in diseases where the NSR is activated.
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ItemThe control of melanoma by the Hippo pathway( 2018)Melanoma is an aggressive cancer with extremely unfavourable prognosis. Two main types of melanoma include cutaneous melanoma (CM) accounting for around 95% and uveal melanoma (UM) around 5%. In Australia, melanoma is in the top five most commonly diagnosed cancers, estimated to contribute to over 10% of all new cancer diagnoses in 2017 (Cancer Australia, 2018.). While the overall death rate caused by all cancer is decreasing, the mortality of melanoma has increased in recent years (Howlader et al., 2012; AIHW, 2017). Patients diagnosed only with primary melanoma have relatively high survival rates, whereas when patients are diagnosed with metastatic melanoma, the survival rate is very low (Gershenwald et al., 2017). Currently, the mechanisms that drive melanoma progression and metastasis remain poorly understood; but better therapies are definitely required. BRAF mutations are most common in melanoma, occurring in around 50% of this disease (Akbani et al., 2015), which provides a possibility for targeted therapy. Indeed, the United States Food and Drug Administration (USFDA) has approved BRAF inhibitors (BRAFi) and MEK inhibitors (MEKi) as the standard treatment for metastatic melanoma patients harbouring BRAF mutations. However, drug resistance occurs in the majority of these patients within two years of treatment (Long et al., 2016). Therefore there is an urgent need to understand the mechanism of BRAFi and MEKi resistance, and find new therapeutic strategies for melanoma. One gene that has been linked to BRAFi resistance is the YAP, which is the key downstream effector of a pathway called the Hippo pathway. The Hippo pathway is an important regulator of organ growth in development. Deregulation of the Hippo pathway stimulates the activity of the YAP oncoprotein, which can cause several human cancers (Zanconato, Cordenonsi and Piccolo, 2016). However, the impacts of YAP deregulation in melanoma are not thoroughly understood. In this project, the roles of YAP in melanoma were examined. Firstly, the impacts of knockdown, overexpression, and activation of YAP on anchorage-independent growth of melanoma cells were assessed using soft agar assays. The results showed that either YAP activation or overexpression promotes colony formation, whilst YAP knockdown reduces this, suggesting potential influences of YAP on melanoma tumorigenesis. Secondly, the effects of YAP in melanoma invasion and metastasis were investigated. Melanoma cells stably expressing an active YAP mutant (YAP-5SA) have a greater invasive ability, as determined with transwell invasion assays. A spontaneous murine metastasis model was used to investigate the impact of YAP on metastasis. The results demonstrated that YAP-5SA promotes metastasis to multiple organs such as the lung and the liver; YAP-5SA enhances vascularity and necrosis of primary melanoma. Thirdly, mechanisms responsible for YAP-induced invasion were explored. Four potential target genes of YAP, derived from RNA-sequencing data, were found crucial, as well as the key YAP transcription factor partners, TEAD1-4. Finally, a lipid-lowering drug called simvastatin was found to kill melanoma cells and inhibits YAP activity in vitro. A post-translational modification, geranylgeranylation, was found to be essential in the statin-induced melanoma cell death and YAP inactivation; RhoA and other geranylgeranylated proteins might be important in these phenotypes. To conclude, this study explored the role of YAP in melanoma metastatic progression, and identified crucial transcription factors and target genes that mediate YAP-induced impacts on melanoma invasion. Additionally, inhibition of YAP and its mechanism in melanoma cells was preliminarily assessed using simvastatin. Understanding the molecular mechanism of melanoma metastasis and inhibition may help us establish more effective therapies for this disease.
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ItemImmune markers to predict response to neoadjuvant chemoradiotherapy and identifying methods to incorporate immunotherapy in locally advanced rectal cancer( 2018)The standard of care for locally advanced rectal cancer (T3-4 +/- N+) is neoadjuvant chemoradiotherapy (CRT) followed by total mesorectal excision (TME). However, this has been challenged recently with increasing interest and trials assessing the efficacy and safety of avoiding TME. This concept is known as the “watch and wait” strategy, if patients were deemed to have clinical complete response (cCR). The current limitation is the risk of local tumour regrowth rate between 20-30%, as cCR is not equivalent to pathological complete response (pCR). Therefore, this thesis describes the development of a novel immune cytotoxic assay for measuring patient-matched cytotoxic T cell-mediated killing of rectal cancer organoids. Subsequently a proof of principle prospective observational study was conducted, showing in those patients with pCR, their cytotoxic T cell-mediated killing were highest when compared to non-pCR. Furthermore, this was a stark difference without overlapping of 95% confidence interval when compared to the partial and non-responding T cell-mediated killing of rectal cancer organoids. At the other end of the spectrum, those that had failed to achieve any response to neoadjuvant therapy will not have any other therapeutic option left to increase their tumour response rate. Promising emerging therapies employing immunotherapy by check-point inhibition and/or targeted-vaccine are now highly relevant, especially inhighly immunogenic colorectal cancer such as microsatellite instability high subset due to high somatic mutation. A similar tumour microenvironment has been documented after induction radiotherapy, with success of check-point inhibition shown only in mouse models. Using the immune cytotoxic assay, this thesis demonstrates the increased in patient-matched T cell-mediated killing of rectal cancer organoids in the presence of check-point inhibition. This opens another avenue to explore the utility of immunotherapy using a T cell-organoid model, with the potential for investigating and identifying novel markers to immune resistance.
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ItemDefining signalling pathways that control the response of endothelium to cancer therapy( 2018)Targeting non-transformed stromal components of the tumour microenvironment (TME) has become clinically attractive in treating cancer over the last few decades. On this basis, vascular endothelial growth factor A (VEGFA) inhibitors which suppress blood vessel sprouting (angiogenesis) by blocking VEGFA signalling have been developed and integrated into modern cancer treatment regimens. However, tumour response to VEGFA inhibitors is highly complex and variable. In addition to cancer cells, the TME is composed of various stromal cell types that play an important role in modifying the tumour response by, for example, supporting the development of resistance to VEGFA blockade. Moreover, the indiscriminate large-scale application of VEGFA inhibitors (with or without chemotherapeutic agents) in clinical oncology, resulting in overall modest patient benefit and the inevitable occurrence of resistance, has underscored a pressing need for rational use of these expensive agents. To address the challenges in deciphering the role of each TME component and thus the mechanisms of resistance, this Thesis focused on the main cellular target of VEGFA inhibitors — human microvascular blood endothelial cells (ECs). To identify molecular modifiers of the EC response to VEGFA inhibitors (in this Thesis bevacizumab, a humanised anti-VEGFA neutralising monoclonal antibody, was used), a pooled genetic screening platform was developed. This involved a three-dimensional microcarrier-based culture system, CRISPR–Cas9-driven genetic loss-of-function (LOF) and VEGFA-dependent serum-free culture conditions for applying selective pressure. A pooled kinome-wide CRISPR–Cas9-based screen identified 18 candidate genes that upon LOF were significantly enriched or depleted in the bevacizumab versus control treatment arm. Candidate evaluation using small interfering RNA (siRNA) validated ACTR2, BRD2, BRD3, BRD4, TAOK1 and TRRAP LOF as mediators of EC resistance to bevacizumab; TLK1 and TLK2 LOF as sensitisers of ECs to bevacizumab. Further analysis of the most significant validated candidate genes BRD2, BRD3 and BRD4 (encoding members of the bromodomain and extraterminal domain (BET) family of proteins) using the BET bromodomain inhibitors (BETi) JQ1 and I-BET762 reproduced the effect of siRNA-mediated knockdown of BRD2, BRD3, or BRD4 on the EC response to bevacizumab. Markedly, a survival- and/or proliferation-inhibiting effect of BETi was observed regardless of the presence of bevacizumab. However, this inhibitory effect was unexpectedly attenuated when cells were co-treated with bevacizumab under VEGFA-dependent culture conditions. These results collectively indicated an interaction between BETi and bevacizumab. Investigation of the mechanistic basis for such interaction using RNA sequencing suggested a role for epigenetic regulation of chromosomal activity in modifying the EC response to co-treatment with BETi and bevacizumab. With development and application of a minimally biased and systematic screening approach, this Thesis identified and validated novel molecular modifiers of the EC response to bevacizumab. A previously unreported interaction between BET protein activity and VEGFA signalling in the context of bevacizumab treatment in ECs was revealed. Importantly, these observations will prompt further investigation of the role of epigenetic regulation in vascular biology, tumour angiogenesis and response to cancer therapy. These findings could facilitate clinical development of predictive and/or response biomarkers and strategies to overcome therapeutic resistance, ultimately enabling the rational use of VEGFA inhibitors.
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ItemLaminin-511: a key regulator of breast cancer metastasis( 2018)Cancer progression is invariably associated with changes in the stromal microenvironment, most notably, the extracellular matrix (ECM) composition. Laminins (LM) are a family of large trimeric ECM proteins comprised of at least 16 isoforms that differ by their α, β and γ chain combination. Their expression and function in normal development and in pathologies are temporally regulated in a tissue specific manner and dictated in part by their interactions with specific cell surface integrin receptors. Work from our laboratory indicates that the more recently discovered LM-511 (α5β1γ1 trimer, formerly LM-10) is the most relevant isoform contributing to breast cancer metastasis. Unlike LM-111 (α1β1γ1 trimer, formerly LM-1) and LM-332 (α3β3γ2 trimer, formerly LM-5), LM-511 expression is often sustained or increased in advanced tumours and metastases (Chia et al., 2007). Consistent with this work, our laboratory showed that LM-511 promotes pro-metastatic responses in vitro including cell adhesion, migration, invasion and protease (MMP-9) expression and this correlated with high expression of LM-binding integrin β4 receptor. To provide more direct evidence for the role of LM-511 and integrin β4 in breast cancer metastasis, here we have used a gene knockdown approach to suppress their expression in a syngeneic mouse model of spontaneous breast cancer metastasis. We demonstrate for the first time that suppression of tumour LM511 or its integrin α6β4 receptor significantly reduces circulating tumour cells and metastasis, particularly to bone, the major site of metastasis for breast cancer. When repeated in an experimental model of metastasis, bone metastasis was significantly reduced by the down-regulation of LMα5 or integrin β4 expression. Collectively, our results identify a role for LM-511 and integrin β4receptor in both early and late stage breast cancer metastasis. We present evidence that LM-511 and integrin β4 regulate metastasis by modulating epithelial-mesenchymal transition (EMT) and that this pathway is enhanced via MMP-9 proteolytic cleavage of LM-511. Protein analysis and immunofluorescence in vitro and immunohistochemistry in vivo demonstrated that when LMα5 or integrin β4 is down-regulated, the expression of mesenchymal markers N-cadherin, vimentin, SNAI1/2, TWIST and ZEB1/2 is reduced. Studies have reported that interactions between tumour cells and surrounding ECM can influence the acquisition of drug resistance (Senthebane et al., 2017). Herein, we show that when LM-511 or β4 is suppressed tumour cells become more sensitive to chemotherapeutic agents. Furthermore, treating cells with Lebein-1 (disintegrin) blocks LM/integrin interactions and further enhances their sensitivity to current therapies. In summary, we show that interfering with LM-511 production or integrin β4 receptor expression significantly reduces triple negative (TN) breast cancer metastasis to bone and soft tissues and enhances their sensitivity to current breast cancer therapies.
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ItemDefining the translational landscape of MYC-driven cancer cells in response to therapeutic targeting of the ribosome( 2018)Recent studies by our group and others have demonstrated that oncogene- driven hyper-activation of ribosome synthesis and activity is a vulnerability that can be targeted for cancer treatment. Specifically, combined inhibition of ribosome synthesis using the Pol I inhibitor CX-5461 and mTORC1-dependent mRNA translation using everolimus (EV) synergistically improved survival benefit of a MYC-driven mouse model of B-lymphoma [1]. Despite this promising outcome, a few questions remain: 1) what are the molecular mechanisms that trigger the pro-death pathways in these lymphoma cells in response to CX-5461-everolimus co-treatment (CX-5461+EV); 2) what mechanisms confer resistance to this combination therapy; 3) how can we improve the efficacy of this ribosome targeting therapy. Since both inhibitors target the ribosome, this thesis applies poly(ribo)some profiling analysis as the principal technique to address these research questions. mRNA translation is an important step in gene expression and can be examined in a genome-wide and high-throughput manner using polysome profiling analysis. Firstly, multiple aspects of this method were optimised to enable robust characterisation of the cellular translational landscape in this MYC-driven model of B-lymphoma (Chapter 3). Polysome profiling analysis was then utilised to evaluate changes in the translatome of CX-5461+EV- treated MYC-driven B-lymphoma cells in vivo (Chapter 4). The analysis revealed that the synergistic improvement in therapeutic efficacy in response to CX-5461+EV treatment is due to selective reduction in the translation of mRNAs encoding component of translational apparatus, which is associated with decreased translation efficiency of metabolism-related mRNAs. We have also demonstrated that the resistance to CX-5461+EV treatment was not due to the lack of on target activity of the drugs. Rather, it is due to a translation-driven activation of a metabolism-dependent, pro-survival response via the cAMP-EPAC1/2-Rap1 pathway. Importantly, the inhibition of this pro-survival pathway by the anti-diabetic drug metformin, which inhibits energy metabolism, re-sensitise the CX-5461+EV-resistant cells to CX- 5461+EV in vitro and in vivo. Together, this thesis highlights the role of translational changes in drug response and resistance development in the context of MYC-driven cancer. Furthermore, this work also identifies the potential of combined therapeutic targeting of the ribosome and metabolism for the treatment of MYC-driven haematological cancers.
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ItemThe evolutionary history of genes and transcriptional networks reveals fundamental properties of cancer associated with the breakdown of multicellularity( 2018)All biological systems follow the rules and constraints imposed during their evolution. Current-day gene phenotypes such as gene expression, gene essentiality, gene function and protein localization are linked with the time of evolutionary emergence of genes. In cancer, tumours rely on cellular processes that date back to unicellular ancestors (e.g., cell replication, glycolysis), while dysregulating key pathways linked to the emergence of multicellularity, suggesting that the transition from unicellularity to multicellularity left vulnerabilities in cells that act as guiding principles during cancer development. Therefore, in this thesis I integrate genomics, systems biology and evolutionary biology to investigate fundamental principles of tumourigenesis related to the evolutionary history of genes using gene expression and somatic mutation information across multiple tumour types. First, I coupled the evolutionary age of genes and cellular processes with their expression levels in tumour and normal samples, and found that tumours consistently activate genes from unicellular ancestors while switching off genes related to multicellularity. These consistent patterns were supported by a mutual exclusivity between the activity of genes and transcriptional networks of unicellular and multicellular ancestors, which promoted convergent evolution towards a state of loss of multicellularity. Second, I investigated how somatic mutations disrupted gene regulatory networks. Genes that emerged together with early metazoans were enriched in point mutations and copy- number alterations, indicating that gene innovations that took place at the onset of multicellularity play a fundamental role in cancer development. Importantly, the uncoupling of regulatory networks of unicellular and multicellular ancestors was mostly due to point mutations in gene regulators linking these networks. On the other hand, copy-number aberrations were directly involved in the activation and inactivation of unicellular and multicellular genes, suggesting point mutations and copy-number aberrations play complementary roles in the loss of regulation between unicellular and multicellular transcriptional networks in cancer. Third, I focused on novel transcriptional associations formed during tumourigenesis using gene co-expression module analysis. Significant levels of rewiring between unicellular and multicellular genes were found across tumours. This rewiring was mostly driven by gene amplifications, which promoted the formation of tumour-specific modules composed of novel transcriptional associations between unicellular and multicellular genes, once more linking the genes and regulatory associations evolved at the onset of multicellularity to cancer development. The findings of this work reveal fundamental principles driving cancer development associated with genes and transcriptional networks evolved during the transition from unicellularity to multicellularity. I propose a model whereby activation of programs that date back to unicellular ancestors and the deactivation of multicellular programs is driven by an inherent mutual exclusivity of these genes together with the breakage of regulation between unicellular and multicellular genes by point mutations, whereas the formation of novel transcriptional associations between these genes in tumours is driven by copy-number changes. Finally, I identify potential novel drivers based on their key role in uncoupling unicellular and multicellular transcriptional networks across tumours and suggest novel treatment strategies derived from this evolutionary approach. The results presented in this thesis contribute to our understanding of how past evolutionary events led to vulnerabilities in transcriptional networks that influence cancer development, and highlight the benefits of the integration of evolutionary concepts with genomics and network biology to identify fundamental principles of cancer.
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ItemHow information technology improves the quality and efficiency of medical care and research( 2018)In 2007, the concept of rapid learning healthcare was proposed in the United States of America health system in a response to increasing healthcare costs. Its aim was to accelerate knowledge discovery through a systematic approach to integrating electronic medical records design with analysis infrastructure to rapidly and continuously assess health system performance. The delivery of healthcare is becoming increasingly performed and documented within the electronic domain, and large databases of healthcare-related information being created as a by-product. This has led to an unprecedented level of access to detailed and structured clinical data that could be used to accelerate research. In a rapid learning healthcare system, the high level of integration from electronic record to policy would ensure that each patient and each click of the mouse would drive innovation. The attraction of rapid learning was not to supplant the traditional clinical trial paradigm, but to augment its effectiveness with accelerated analysis of real-world outcomes. The rapid learning concept relied heavily on electronic medical records, administrative systems and disease registries as data sources to power analyses. Electronic health record penetrance in Australia has lagged that achieved in the USA, primarily because of financial assistance provided as part of the HITECH act in the USA. However, one exception is seen in oncology, where radiotherapy is exclusively prescribed electronically. Additionally, there has been a significant shift toward electronic chemotherapy prescribing due to the clinical risk associated with manual systems. Perhaps in oncology there is an opportunity to replicate the successes of data-driven health research achieved elsewhere. The objective of the work contained in this thesis is to develop practical methods to expand and discover the infrastructure required to implement rapid learning health care in the Australian oncology context. Ultimately, the aim is to increase the quality and efficiency of medical care and research by harnessing novel information technology (IT) methods. In addition to leveraging existing secondary databases for health services research and creating high impact linkages with state-level cancer registries, advanced IT methods could also be used to automate manual data extraction tasks in a timely and cost-effective fashion. The integration of these methods into routine clinical practice has enormous implications for tracking patient care quality, and accelerating research by utilising all data by-products of health care.