Sir Peter MacCallum Department of Oncology - Theses
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ItemProfiling the immune and genomic landscape of anal squamous cell carcinoma and establishing preclinical models to explore new therapeutic options( 2019)Anal SCC is a rare disease that has increased significantly in both incidence and mortality over the last fourty years. Definitive chemoradiotherapy is the primary modality of treatment, offering a 5-year overall survival rate of 65%. For patients with locally persistent or recurrent disease, salvage surgery is an option with a 5-year overall survival of 50%. However, for those patients with un-resectable locoregional or metastatic disease, there are limited treatment options, and patients face a dismal outcome. Progress in identifying new treatment options for patients with anal cancer has been hampered by a deficiency in understanding the underpinnings of the disease and a lack of appropriate preclinical models. This thesis has focussed on addressing both of these deficiencies in addition to assessing the success of salvage surgery at a quaternary centre in Australia. Firstly, an attempt has been made to further our understanding of the biology of Anal SCC. This was undertaken by exploring the immune and genomic landscape of ASCC, to identify potential prognostic and therapeutic biomarkers. This has provided insight into the prognostic power of assessing the CD8+ immune infiltrate in Anal SCC. It has also identified PI3K aberrations as a frequent genomic event that may serve as a future therapeutic target. Secondly, it has led to the establishment of both human and mouse preclinical models of this disease. This includes the world’s first panel of human anal SCC cells lines and a syngeneic mouse model. Both of these pre-clinical models have been validated and characterised, with features closely resembling the human disease. These models can now act as a platform to further explore and facilitate investigation into potential new therapeutic options in this disease.
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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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ItemEffectiveness and cost-effectiveness of programs for BRCA pathogenic variant carrier cancer risk management( 2019)Background Women who inherit a germline pathogenic variant in the BRCA1 or BRCA2 genes have a significantly elevated lifetime risk of breast and ovarian cancer. Women who are aware of their BRCA carrier status can mitigate their increased risk by undergoing intensive breast cancer screening from a young age for early detection, and risk-reducing surgery for prevention of breast and/or ovarian cancer. The effectiveness of these interventions is dependent on BRCA carriers taking up these risk management strategies at an appropriate time, considering factors such as their age, personal preferences, and life stage. The most effective approach to ensuring carriers adhere to risk management recommendations is unknown. This project evaluates the lifetime health outcomes and cost-effectiveness of long-term clinical management of BRCA carriers in the context of structured clinical programs, using real-world data. Methods This thesis describes the development and outcomes of a discrete-time state-based microsimulation model. The model, named miBRovaCare, simulates the gene-specific natural histories for breast and ovarian cancer in BRCA carriers. Cost-effectiveness and cost-utility analyses were performed to evaluate the lifetime outcomes of different approaches to clinical management of carriers from the perspective of the Australian public healthcare system. The comparator for the base case analysis was the natural history (no cancer risk management). The interventions included: (i) a structured familial cancer service with a multidisciplinary high-risk clinic, and (ii) a formal annual carrier review program. For the intervention arms, BRCA carriers could undergo annual breast imaging, risk-reducing bilateral or contralateral mastectomy, and risk-reducing bilateral salpingo-oophorectomy. Uptake of and adherence to these strategies (patient behaviour) was based on an analysis of 983 BRCA carriers seen through a clinic in Melbourne, Australia. Additional model inputs were obtained from a local hospital database, the literature, government reports, and expert opinion. Costs and health outcomes were discounted by 5%. Results Long-term management of BRCA carriers through a familial cancer service is likely to be cost-effective, with or without an annual review program. A familial cancer service was the preferred strategy if the willingness-to-pay was at least $29,000 per quality-adjusted life-year (QALY) for BRCA1 carriers and $57,000 per QALY for BRCA2 carriers. Inclusion of an annual review program for BRCA1 carriers had a 75% probability of being cost-effective at a willingness-to-pay threshold of $50,000 per QALY. For BRCA2 carriers, a familial cancer service with or without an annual review program had only a 37% probability of being cost-effective at a $50,000 per QALY willingness-to-pay threshold. Discounting of health outcomes had, by far, the greatest impact on cost-effectiveness outcomes. Conclusions This thesis describes a novel microsimulation model for optimising clinical management of BRCA carriers. BRCA carriers are likely to benefit from access to structured clinical programs and regular review, due to fewer cancer diagnoses, improved life expectancy and an increase in QALYs. Genetic testing for hereditary breast and ovarian cancer predisposition syndromes is steadily expanding, and may even be available at a population-based level in the near future. Maximising adherence to evidence-based risk management guidelines along with access to appropriate follow-up services will therefore be increasingly important. The model developed for this thesis can enable faster evaluation of emerging risk management strategies and behavioural interventions, and can be easily adapted to alternative settings and healthcare systems.
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ItemIdentification of candidate genes predisposing to familial colorectal cancer by germline whole exome sequencing( 2019)Abstract Colorectal cancer (CRC) is the third most common cause of cancer-related death worldwide. Approximately 5% of CRC is attributable to an inherited high-penetrance pathogenic variant in a known CRC-predisposing gene, often associated with a family history of CRC, a young onset of CRC and/or colonic polyposis. Identification of an inherited genetic variant predisposing to CRC in an individual with CRC enables predictive testing in blood relatives, and implementation of risk reduction strategies for individuals with the causative variant within the family. In individuals with familial or young onset CRCs/colonic polyposis in whom a causative germline variant in a known CRC-predisposing gene has not been identified, it is hypothesised that novel CRC-predisposing genes harbouring rare high risk germline variants remain to be discovered. Germline whole exome sequencing (WES) offered a novel and comprehensive approach to identifying potential pathogenic variants in candidate CRC-predisposing genes. This thesis used WES of germline DNA in individuals with unexplained CRC and a high a priori risk of an inherited predisposition to CRC, to prioritise candidate CRC-predisposing genes that were subsequently re-sequenced in a validation cohort of young-onset CRC cases. A discovery cohort was recruited comprising 54 individuals who met one or more of the following criteria: CRC and a family history of CRC (n=29), a diagnosis of CRC before 40 years of age (n=20), or a diagnosis of 20 or more bowel polyps (n=10). No known germline predisposition to CRC had been identified in these individuals through clinical testing. Rare (minor allele frequency of <0.5%) loss-of-function (nonsense, frameshift or essential splice site) variants or missense variants predicted to be deleterious using a Combined Annotation Dependent Depletion C Score of >10 were prioritised for analysis. Three individuals with pathogenic variants in known CRC-predisposing genes were identified and excluded from further analyses. In the remaining 51 individuals, four separate analysis approaches were used to prioritise candidate CRC-predisposing genes containing prioritised variants: 1) genes with variants in multiple families, 2) genes with a shared variant in multiple family members, 3) genes within known CRC pathways, and 4) genes intolerant of loss-of-function variants. Forty-five candidate CRC-predisposing genes were prioritised using these analyses. Segregation analysis of potentially pathogenic variants within a family (n=1) and loss-of-heterozygosity studies in tumour tissue (n=4) were performed but hampered by lack of availability of samples. Re-sequencing of the 45 candidate CRC-predisposing genes in a validation cohort of 333 individuals with unexplained young onset CRC identified seven genes (WNT10A, NEDD4, MS4A15, TNFRSF10B, LRRK2, INPP5E, UFL1) with a loss-of-function variant in 1 or more individuals. None of the seven genes harboured LoF variants in >1% of the validation cohort, indicating that a novel gene likely accounts for very few individuals with unexplained CRC and a high a priori risk of an inherited predisposition to CRC, pointing to a very high degree of genetic heterogeneity in such cases. Future studies will require very large numbers of cases and controls to confirm the association of these genes with CRC predisposition.
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ItemImproving Ovarian Cancer Care Along The Disease Trajectory: From Prevention to Disease Management( 2019)The overarching objective of this thesis was to investigate key clinical care questions along the ovarian cancer disease trajectory and formulate strategies to improve care provision. This thesis focused on three time points on the trajectory: i) cancer prevention; ii) tolerability of experimental treatment at disease recurrence; and iii) development of malignant bowel obstruction. For women at high risk of developing ovarian cancer, such as those with a germline BRCA1 or BRCA2 mutation (gBRCA1/2mut), risk-reducing salpingo-oophorectomy (RRSO) is the most effective preventative method and recommended by peak bodies. RRSO involves complete resection of the ovaries and fallopian tubes up to the insertion into the cornua of the uterus. The resection of the fallopian tubes is necessary as it is known that BRCA1- and BRCA2-associated gynaecologic cancers appear to originate in the fimbrial end of the fallopian tubes rather than the ovaries, even though they have been typically labelled as “serous ovarian cancer”. Previous research had raised concerns that the quality of RRSO surgery and associated pathology evaluation performed in Australasian women had been suboptimal prior to 2008. I therefore undertook an investigation of the quality of contemporary RRSO and compared it with data from the historical research, examining the clinical predictors of RRSO quality. Across 78 institutions in Australia and New Zealand, a total of 164 study participants had RRSO performed and the quality of RRSO, in particularly the related pathology examination, had improved significantly compared to the previous report. The proportions of women who received adequate surgery and pathology evaluation were 99% and 66% respectively, compared to 91% and 23%, respectively (P<0.001) in the previous study. Implementable clinical strategies to potentially further improve quality were identified and included i) to direct RRSO referral to a gynaecologic oncologist (rather than general gynaecologist or general surgeon) and ii) to ensure the reporting pathologist is aware that the intent of surgery is risk-reduction in a high-risk woman, in order to prompt adequate processing and review of the RRSO specimens for occult cancer and pre-malignant lesions. In women with gBRCA1/2mut who undergo RRSO, whether hysterectomy should be performed at the time of RRSO to prevent uterine cancer was controversial. I therefore investigated the incidence of uterine cancer for Australasian gBRCA1/2mut carriers. Of the 828 eligible women identified from a prospective multi-institutional follow up study (kConFab), there were five cases of uterine cancer. Compared to the expected incidence in the Australian population, the standardised incidence ratio (SIR) was 2.45 (95%CI: 0.80-5.72; P=0.11). All five cases were of endometrioid adenocarcinoma histology and importantly, none had serous histology. Of those cases, three had a history of breast cancer and exposure to tamoxifen, a known risk factor for uterine cancer. Therefore, this finding did not justify the need for routine hysterectomy at the time of RRSO to reduce uterine cancer incidence in Australasian gBRCA1/2mut carriers. Disease progression in women with advanced ovarian cancer typically involves the omentum and peritoneum which causes abdominal symptoms. The goal of systemic treatment is to improve symptoms and delay further disease progression. Therefore, it is crucial that we consider the burden of treatment-related toxicities particularly in evaluation of novel drugs. Phase I clinical trials typically would include patients with different solid tumour types and are primarily intended to assess the safety and tolerability of investigational agents. Using the National Cancer Institute Phase I database, I performed a retrospective analysis of the adverse events (AEs) reported in women with gynaecological cancers compared to patients with other cancer types. Patients enrolled in the 150 phase I trials identified were divided into three groups to allow comparison; i) females with gynaecological cancers (n=685); ii) females with non-gynaecological cancers (n=1698); and iii) males with cancers (n=1886). Of those females with gynaecological cancers, the majority had ovarian cancer (n=527, 77%) followed by cervical cancer (n=76, 11%) and uterine cancer (n=72, 11%). Overall, females with gynaecological cancers were reported to have a significantly greater number of AEs during treatment (mean, 17.1 vs 14.7 vs 13.5, P<0.001), despite being similar at baseline (mean 7.0 vs 7.4 vs 7.0, P=0.09). In terms of AE severity, the main difference was a higher prevalence of grade 2 AEs reported in women with gynaecological cancers (mean 4.6 vs 3.9 vs 3.5). The five most prevalent AEs in women with gynaecological cancers were nausea (n=617, 90%), fatigue (n=587, 86%), anaemia (n=381, 56%), anorexia (n=357, 52%) and vomiting (n=355, 52%). These findings highlighted the need to improve the management for low-grade AEs in particular abdominal-related AEs for women with gynaecological cancers being treated on clinical trials. The inclusion of specific supportive care protocols/strategies into clinical trial protocols will better address symptom burden and improve quality of life. Malignant bowel obstruction (MBO) is a common complication for women with recurrent ovarian cancer that causes protracted and debilitating symptoms. Recognising variation in clinical practice and the unmet need for evidence-based treatment, I conducted a literature review to summarise current treatment strategies for MBO in women with advanced gynaecological cancers from a multidisciplinary perspective. A pilot interprofessional MBO program was developed by the MBO working group (which I co-led with Dr Stephanie Lheureux) and implemented in June 2016 at Princess Margaret Cancer Centre to support women who had, or were at risk, of developing MBO. The integrated model of care consisted of: i) standardised MBO symptom triage tools; ii) establishment of MBO multidisciplinary case conferences; iii) consensus on MBO care algorithms for in-patients and out-patients; iv) development of patient education materials for MBO; and v) prompt access to allied health professionals and supported advanced care planning. To assess the impact of the interprofessional MBO program, I reviewed all consecutive patients presenting with MBO from April 2014 to March 2018 (i.e. before and after the implementation of the program) and compared their outcomes. Of the 169 patients included, the majority (n=124, 73%) had recurrent ovarian cancer. There were 106 patients admitted prior to implementation of the MBO program (baseline group) whilst 63 patients were managed under the MBO program (MBO program group). Overall, the MBO program group had a significantly shorter average accumulated hospital length of stay (LOSsum) by 9 days (13 vs 22 days, adjusted P=0.006). Furthermore, their median overall survival post MBO diagnosis was approximately 5 months longer compared to the baseline group (243 vs 99 days, P=0.002). This retrospective, single institution study suggests a beneficial impact towards improving the complex care of women with advanced gynaecological cancers who developed MBO. Following on from this retrospective study, I developed a prospective MBO study incorporating patient reported outcomes to validate these findings, which is currently recruiting (MAMBO study, N=61/150 NCT03260647). In conclusion, the body of academic work carried out in this thesis has addressed known clinical gaps in ovarian cancer care throughout the disease trajectory and generated specific care recommendations to guide risk-reducing surgery management, to improve symptom burden whilst undergoing cancer treatment, and to improve management of malignant bowel obstruction. Broadly, this research will help clinicians, peak bodies and health funders implement evidence based care and institutional and national policies to facilitate better care provision for patients with ovarian cancer.
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ItemDefining functional drivers of oesophageal tumourigenesis( 2019)The incidence of oesophageal adenocarcinoma (OAC) has risen rapidly over the last four decades and has a high overall mortality rate that has shown only incremental improvements over the same duration. OAC develops from the precursor intestinal metaplasia of the oesophageal mucosa known as Barrett’s oesophagus. However, limited knowledge of the molecular mechanisms driving disease progression makes effective clinical management of OAC challenging. One of the common genetic events associated with the progression from Barrett’s oesophagus to OAC is loss of the tumour suppressor, SMAD4 (mutated in 13% or loss of function in 34% of OAC cases). Herein, this thesis firstly investigated the effect of SMAD4 inactivation in Barrett’s carcinogenesis. Genetic knockdown or knockout of SMAD4 was sufficient to initiate tumourigenesis of dysplastic Barrett’s oesophagus cell line, CP-B, in vivo, establishing SMAD4 loss as a crucial event driving progression to OAC. Further, low coverage whole genome sequencing (LC-WGS) analysis revealed that tumourigenic SMAD4 knockdown/knockout CP-B cell lines exhibited distinctive and consistent copy number alterations (CNAs) compared to non-tumourigenic SMAD4 wild-type parental cells. Amongst the alterations we observed were loss of chromosome arm 14q, while amplified regions include chromosome arms 6q and 12p, consistent with common CNAs found in patient tumours. This high genomic instability, characterized by structural chromosomal rearrangements within the tumours following SMAD4 loss, implicates SMAD4 as a protector of genome integrity in OAC development and progression. Moreover, initial in vitro data shows that SMAD4 knockout in CP-B cell line, results in differential expression of transforming growth factor-beta (TGF-beta) pathway target genes (such as ACTA2, CRYAB, PTK2B, ATF3 and CDC6) and loss of cell cycle arrest in response to TGF-beta1 cytokine compared to SMAD4 wild-type parental cells. Furthermore, SMAD4 knockout negatively regulated transcript expression of the multifunctional tumour suppressor INK4/ARF locus, demonstrating the novel and complex network of SMAD4 tumour suppressive activity. This thesis also focused on deciphering the functional role of growth factor receptor bound protein 7 (GRB7) amplification and overexpression in OAC and its potential targeting. GRB7 gene is positioned within known 17q12 amplicon, together with HER2 gene encoding for human epidermal growth factor receptor 2 (HER2). GRB7 is an adaptor molecule that mediates networking of multiple cell surface receptors with downstream signalling pathways. GRB7 high expression was found to be associated with worse survival in OAC patient cohort. Further, genetic GRB7 knockdown (siRNA) inhibited cell proliferation and clonogenic survival and induced apoptosis in GRB7 amplified and overexpressing OAC cell lines (OE19 and Eso26), without altering proliferation of the cells with normal GRB7 expression. Furthermore, whilst HER2 amplification and overexpression was also observed in OE19 and Eso26 cells, they were not uniquely sensitive to trastuzumab (HER2 inhibitor), with Eso26 cells exhibiting resistance in vitro. Taken together, initial findings raise the possibility that GRB7 may be a better molecular therapeutic target than HER2 in OAC with the 17q12 amplicon. To address this possibility, OE19 and Eso26 cell line xenograft models with inducible expression of shRNA targeting GRB7 were used. Consistent with in vitro findings, HER2 expression did not predict sensitivity to trastuzumab, with Eso26 xenografts remaining refractory to trastuzumab treatment. Of high importance, mimicking GRB7 inhibition with inducible-shRNA significantly inhibited tumour growth in both OE19 and Eso26 xenografts. Thus, this part of the thesis demonstrates the functional role of GRB7 overexpression as an oncogenic driver independent of HER2. In summary, the identification of functional genetic drivers and a deeper understanding of the mechanisms that underlie tumour progression in Barrett’s carcinogenesis will lead to improved strategies for the clinical management of OAC patients. To this end, SMAD4 loss was sufficient for progression from dysplasia to OAC. Tumours driven by SMAD4 loss exhibit distinctive CNAs consistent with OAC and metastatic potential. In addition, GRB7 high expression predicts poor outcome in patients with OAC and as such, GRB7 represents an oncogenic driver that could be used as a therapeutic target. Crucially, this thesis provides in vitro and in vivo preclinical and molecular biology evidence for the potential therapeutic benefit of targeting GRB7 in cancer.
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ItemRepurposing the Hippo pathway for cell fate specification( 2019)The Hippo pathway is an important regulator of organ growth during development where it ensures correct scaling of tissues and organs. It is well-conserved in animals and deregulation of the pathway has been found to drive development of a range of cancers. The Hippo pathway was first identified in the fruit fly, Drosophila melanogaster, and has been extensively studied in a number of different tissues. As well as controlling organ growth in Drosophila, the Hippo pathway has been repurposed to control the binary cell fate choice of the R8 photoreceptor cell in the Drosophila eye. The R8 photoreceptor cell is one of eight photoreceptor cell types in the Drosophila eye and is responsible for much of colour vision in Drosophila. There are two main subtypes of R8 cells – around 30% are the 'pale' (p) subtype and express Rhodopsin 5 (Rh5; responsive to blue light); the remaining 70% are the 'yellow' (y) subtype and express Rh6 (responsive to green light). The Hippo pathway acts in a bistable feedback loop to both specify and maintain R8 subtype specification. This bistable feedback loop is composed of the Hippo pathway kinase, Warts, the downstream transcriptional coactivator, Yorkie, and the Pleckstrin-homology domain containing protein, Melted. Yorkie and Melted both promote pR8 cell fate, while Warts represses pR8 cell fate and promotes yR8 cell fate. Although we have begun to study the role of the Hippo pathway in R8 cell fate choices, we still lack a clear understanding how the Hippo pathway functions in these cells. I sought to further our understanding of the Hippo pathway in R8 cells by answering three key questions: (1) Which Hippo pathway proteins control R8 cell fate? (2) What is the subcellular localisation of Hippo pathway components in R8 cells? (3) What are the target genes of the Hippo pathway in each R8 subtype? (1) I used genetic experiments to modulate the expression of Hippo pathway genes and found that Crumbs and Cka promote pR8 cell fate, while the apical spectrin cytoskeleton and the 14-3-3 proteins promote yR8 cell fate. Modulating other Hippo pathway regulators did not obviously alter the ratio of R8 subtypes, suggesting that not all Hippo pathway proteins function in R8 cell fate choices. (2) Using confocal and multiphoton microscopy, I showed that only Warts differed in expression or localisation between R8 subtypes, suggesting that regulation of Warts levels in R8 cells controls the Hippo pathway's role in R8 cell fate. I also showed that the upstream Hippo pathway proteins, Tao, Merlin and alpha-spectrin are relocalised between late pupal and adult photoreceptor cells, hinting that there may be differences in their mechanisms of action between these two stages. (3) Targeted DamID-seq showed that Yorkie and its binding partner Scalloped reside at over a thousand loci in each R8 subtype. Yorkie and Scalloped target genes included known target genes from proliferating Drosophila tissues, as well as genes that are specific to photoreceptor cells. I also investigated the role of a target gene in pR8 cells, ninaB, and found that it promoted pR8 cell fate in male Drosophila retinas. These results provide insight into the role of the Hippo pathway in R8 cells and highlight similarities and differences between the roles of the Hippo pathway in proliferating cells and in R8 cell fate specification.
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ItemUnderstanding and manipulating epigenetics in cancer( 2019)The appropriate regulation of gene expression programs is essential for normal cellular function. In cancer, mutations derail normal developmental gene expression programs resulting in a malignant epigenetic state. Traditionally, therapies have attempted to kill cancer cells by targeting rapid proliferation or by directly disrupting the mutated protein. More recent therapies, such as BET inhibitors, attempt to directly target the oncogenic epigenetic state. All of these therapeutic approaches are frequently hampered by the emergence of drug resistance. In many cases, resistance is acquired through genetic changes. However, there is increasing evidence that drug resistance can also arise through non-genetic/epigenetic mechanisms. This has major implications for cancer treatment, as the processes that drive genetic and non-genetic resistance are completely different and epigenetic changes, unlike genetic changes, are potentially reversible. Our laboratory previously generated a model of non-genetic resistance to BET inhibitors in acute myeloid leukaemia (AML). Resistance coincided with the acquisition of a less differentiated stem cell like phenotype and was stable upon drug withdrawal. To discover targets that can overcome the resistant epigenetic state, I performed a focused CRISPR-Cas9 screen, which identified the enhancer regulator, LSD1, as key to maintaining the resistance phenotype. Treatment with an LSD1 inhibitor overcame resistance through time-dependent reprogramming of the resistant cells. This epigenetic reprogramming resulted in both differentiation and the formation of new enhancers around critical BET inhibitor target genes. Through functional genomics experiments, I demonstrated that re-sensitization of the resistant cells was driven by the new enhancer formation, rather than differentiation. Mechanistically, the enhancer remodeling is caused by upregulation of Irf8, which together with the pioneer factor, Pu.1, initiates the formation of new enhancers around BET inhibitor target genes, leading to a restored BET inhibitor transcriptional response. Motivated by the poor clinical responses and rapid acquisition of resistance to BET inhibitors in AML, I also sought to develop an assay that could identify new therapeutic targets that may be more effective. Transcription factors (TFs) are ideal targets, however they are difficult to disrupt directly. To circumvent this, I developed an assay that can identify what cofactors (which are often druggable) are required to drive the transcriptional activity of a given TF. This assay works by combining the Gal4 transactivation system with CRISPR-Cas9 screening. Preliminary screens using the transcription factors, VP64 and MYB, were able to identify previously validated cofactors for each TF. VP64 was dependent on MED25 and MYB was dependent on P300. A number of other potential specific cofactors were identified and will be validated in future work. This assay not only provides the potential to identify novel therapeutic targets, but will also provide insight into the poorly characterized interface between TFs and cofactors.
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ItemOvercoming Tumour Resistance to Adoptive Immunotherapy by Enhancing CTL function( 2019)While adoptive cell transfer (ACT) therapy using chimeric antigen receptor (CAR) T cells can be effective in the treatment of haematological B cell malignancies, the treatment of solid tumours has been challenging. Limiting factors such as low levels of CAR T cell activity and poor infiltration into solid tumours, antigen heterogeneity and immunosuppressive microenvironments are playing important roles in solid tumour resistance to CAR T cell therapy. Therefore, a better understanding of these limiting factors is necessary for overcoming these challenges and addressing tumour resistance to immunotherapy. Multiple studies have investigated different strategies to increase the efficacy of CAR T cell therapy in solid tumours including the modification of CAR T cell structure and using a combination of checkpoint inhibitors. Recently a study from our laboratory demonstrated the eradication of established large tumours including E0771-Her2 breast cancer, 24JK-Her2 sarcoma, and MC38-Her2 colon carcinoma using adoptive cell transfer incorporating vaccination (ACTIV) therapy. In ACTIV therapy, tumour-bearing mice were preconditioned with whole-body irradiation and then treated with dual specific CAR T cells and vaccinia virus VV-gp100 in addition to IL-2 administration. The dual specific CAR T cell possessed a CAR specific for Her2 together with a TCR specific for the premelanosome protein, Pmel. Pmel serves as a strong immunogen that is incorporated in a vaccinia virus, VV-gp100, in ACTIV therapy and facilitates dual-specific (CARaMEL) T cell activation, proliferation and infiltration. Despite the significant results of ACTIV therapy in the elimination of the above tumours, we identified a relatively resistant tumour, AT3-Her2 breast cancer tumour. Since an understanding of mechanisms of tumour resistance is essential for potential extension of ACTIV therapy to a broader range of tumours, we used E0771-Her2 and AT3-Her2 tumours as comparative tumour models for studying limitations in effective ACTIV therapy and proposing potential approaches to overcome those limitations. In our study E0771-Her2 and AT3-Her2 tumours were representative of sensitivity and resistance to ACTIV therapy respectively. We identified the relative resistance of AT3-Her2 tumours to CARaMEL T cell cytotoxicity and poor T cell infiltration into tumours as two main limiting factors in effective ACTIV therapy of AT3-Her2 tumours. We used two approaches to address these challenges. Firstly, we used the combination of oncolytic vaccinia virus VV-dd with ACTIV therapy and showed a significant improvement in therapeutic efficacy of ACTIV therapy. In addition, we showed that oncolytic VV-dd can increase apoptosis in AT3-Her2 tumours. Analysis of T cell proliferation and distribution showed a higher T cell infiltration using ACTIV+VV-dd therapy. In another approach to address the relative resistance of AT3-Her2 tumours to CARaMEL T cell killing, we used an IAP antagonist, SMAC-mimetic drug, named AZD5582 in combination with ACTIV therapy. Our results showed a significantly higher level of CARaMEL T cell cytotoxicity using a combination of the CARaMEL T cells with AZD5582 in vitro. In addition, inhibition of AT3-Her2 tumour growth increased remarkably using AZD5582 in combination with ACTIV therapy. Further analysis showed that this therapy could also increase T cell infiltration into AT3-Her2 tumours and induce apoptosis in tumour cells. Therefore, both strategies demonstrated the promising potential for increasing the therapeutic efficacy of ACTIV therapy and its extension to a broader range of solid tumours.
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ItemUncovering a role for RYK, a WNT-binding receptor tyrosine kinase, in cancer( 2019)Cancer is the leading cause of disease burden in Australia. Targeted therapy utilises oncogenic mutations in tumours by modulating cancer-promoting proteins and signalling pathways. While these drugs deliver promising initial responses, cancers invariably develop resistance resulting in the recurrence of the cancer. A common theme of resistance mechanisms is the ability of cancer to hijack alternative signalling pathways to evade therapy. The identification and evaluation of these relatively understudied pathways could facilitate the development of novel therapies to overcome resistance development. One such understudied subset of signalling molecules, the WNT-binding RTKs, are at the interface of two protein families frequently dysregulated in cancer – receptor tyrosine kinases (RTKs) and WNTs. RTKs are targets of many drugs used clinically for cancer treatment, often through the inhibition of their kinase activity or their interactions with activating ligands. WNTs are key signalling morphogens in cancer but the complexity of their signalling has made them difficult drug targets. RYK is a WNT-binding RTK pivotal for embryonic development but with unusual biochemical properties that have meant it is understudied in a cancer context. This Thesis sought to uncover a role for RYK in cancer and evaluate its potential as a therapeutic target. This Thesis revealed the overexpression of RYK mRNA in lung squamous cell carcinoma and glioblastoma samples before identifying human tumour cell lines that readily express RYK mRNA and might be reliant on RYK signalling for its tumourigenic nature. Genetic perturbation of RYK through siRNA knockdown and CRISPR/Cas9 inactivation facilitated the discovery of human tumour cells from four different tumour types that were dependent on RYK for their viability. To assess the clinical expression of RYK protein, a novel chicken anti-RYKEC antiserum was generated, validated and utilised to uncover an upregulation of RYK in a subset of human NSCLC tumours. This antiserum also identified RYK expression in tumours from breast cancer patients and found an inverse correlation between RYK expression and the tumour grade of breast cancers. The establishment of a novel bioassay in pre-osteoblast cells dependent on WNT3A/RYK signalling is described. This assay is used to optimise the expression and purification of two RYK/WNT signalling inhibitors and confirm their RYK/WNT-inhibitory activity. One of these inhibitors, a neutralising anti-RYK antibody – RWD1 – then demonstrated an anti-cancer effect by inhibiting the viability of the RYK-dependent human tumour cells in vitro. RWD1 also reduced the growth of A549 NSCLC tumour xenografts, demonstrating the anti-cancer effect of targeting RYK signalling in vivo. RNA-sequencing analysis revealed an upregulation of epithelial to mesenchymal transition (EMT) genes in human tumour cells upon RWD1 treatment, implying RYK signalling supresses EMT. CRISPR/Cas9-mediated inactivation of RYK increased the metastatic potential of A549 tumour xenografts in vivo. This Thesis uncovered a dual role for RYK signalling in a cancer setting. RYK was found to promote the viability and growth of human tumour cells while also suppressing EMT and metastatic growth. The findings of these parallel pro- and anti-tumour functions of RYK signalling, suggest that RYK signalling could be exploited to deliver a therapeutic effect in cancer.