Sir Peter MacCallum Department of Oncology - Theses
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Imaging and blood biomarkers: towards personalized medicine in head and neck cancer
Head and neck cancer is the 6th most common malignancy, accounting for approximately 4% of malignancies, and 1 – 2% of cancer-related deaths. Radiation therapy utilises high energy radiation to kill cancer cells. In head and neck cancer, radiotherapy is one of the main treatment modalities, particularly in curative-intent treatments. Despite advancements in imaging and radiation treatment planning and delivery, the prescribed dose and radiation treatment workflow remained unchanged and is largely ‘one size fits all’. Similarly, the survivorship program for patients with head and neck cancer is ‘one size fits all’, often one standard institutional follow up schedule for all patients treated for head and neck cancer, regardless of expected risk of treatment-related late toxicities, patients’ subsequent risk of recurrence and second malignancy. This thesis focuses on the value and efficacy of imaging and blood biomarkers in improving treatment personalisation in patients with head and neck cancer. In chapters 1 and 2, I explored the use of imaging and blood markers in the pre-, during, and post-radiotherapy settings to further improve risk stratification. In chapter 1, I investigated the potential use of readily available and ‘cheap’ blood biomarkers (neutrophil and lymphocyte counts) as predictors of subsequent outcomes in a large cohort of patients with oropharyngeal cancer. In chapter 2, I designed and conducted a prospective observational study to systematically characterize the kinetics of gross tumour volume and apparent diffusion coefficient (ADC) changes observed in magnetic resonance imaging (MRI) and circulating tumour cells (CTCs) counts during radiotherapy in patients with head and neck squamous cell cancer. In the survivorship period (Chapter 3), I evaluated the effectiveness of current surveillance program and investigated the potential use of PET imaging and alternative imaging frequencies to improve the cost-effectiveness of the survivorship program. In this chapter, I found that 70% of disease recurrence occur within 2 years and the probability of a surveillance imaging detecting a recurrence in an asymptomatic patient with no adverse clinical finding is very low. Furthermore, in patients with human papillomavirus (HPV)-related oropharyngeal cancer, achieving a complete response on post-treatment PET imaging has a negative predictive value of any subsequent recurrence of 92%, so the yield of surveillance imaging is very low in this group. Using a partially observed Markov decision model, a potentially effective surveillance program with less frequent imaging was propositioned in this chapter. Finally, in chapter 4, I assessed the potential use of re-irradiation in the era of modern imaging and new radiation treatment techniques including intensity-modulated radiotherapy (IMRT), proton therapy and stereotactic body radiotherapy (SBRT). I showed the value of different imaging modality (dual energy CT and MRI) in target delineation in patients who had previous radiation. In addition, I demonstrated that the local control rate for each treatment technique is similar. Although wide field radiotherapy (IMRT and proton therapy) had improve disease-specific survival, treatment with these techniques are longer (typically 6 to 7 weeks) and had higher toxicity rates than SBRT (delivered over 5 treatments).
Identification and Characterisation of the Cellular and Molecular Changes in Intestinal Metaplasia Associated with Progression to Gastric Cancer
Gastric cancer (GC) patients are mainly asymptomatic and present at an advanced stage with a 5-year survival rate of only 20-30% in most countries. It is crucial to gain greater insight into the process of gastric carcinogenesis in order to develop tools that will allow improved patient stratification and targeted surveillance. The relationship between intestinal metaplasia (IM), a key premalignant lesion in gastric carcinogenesis, and the diffuse type of gastric cancer (DGC) was investigated using data mining of GC patient pathology reports and existing gene expression data. IM and DGC were shown to be associated both histologically and molecularly in a proportion of cases suggesting that IM might be a precursor lesion to a subset of DGC cases. Complete and incomplete IM subtypes, the latter being associated with a greater risk of progression, were characterised at the molecular level using gene expression data acquired from macro-dissected IM tissue. Complete IM was associated with genes highly expressed in the small intestine whereas incomplete IM was associated with genes expressed both in the colon and in GC suggesting it is molecularly closer to a state of malignancy. Using OPAL multiplex immunohistochemistry, the macrophage and T cell landscape in IM was investigated. In addition to traditional “M1-like” (IRF8) and “M2-like” (CD163, CD206) macrophage populations, a novel hybrid subgroup “M1/2” containing macrophages expressing both M1 and M2 markers in different combinations was identified. Overall, complete IM was characterised by M2 macrophages and greater levels of T cell infiltration whereas incomplete IM was characterised by M1/2 macrophages and fewer CD8 and double negative (DN) T cells. Spatial cell analysis showed significantly fewer CD8 and DN T cells in the vicinity of incomplete IM epithelial cells suggesting reduced immune-surveillance may play a key role in progression to dysplasia. IM subtyping is affected by intra-observer and inter-observer variation. To address this, the potential of CD10 and Das1 as biomarkers for subtyping complete and incomplete IM was investigated. Overall CD10 was shown to be an outstanding biomarker for complete IM and Das1 was shown to have potential as an additional risk associated biomarker. GC animal models are associated with long duration and high cost. Optimised protocols for growing and characterising gastrointestinal organoids from gastroscopy biopsies were developed. IM organoid cultures were successfully established and characterised. Additionally a human gastrointestinal organoid biobank was created. In conclusion this thesis offers potential evidence of IM as a precursor lesion to DGC, characterises the molecular differences between complete and incomplete IM and shows key differences in the innate and adaptive immune system between IM subtypes and how these may play a role in progression to GC. It also identifies two biomarkers with potential clinical utility for subtyping IM and describes the methodology for growing IM organoids with future potential as a model for studying gastric carcinogenesis. Finally future studies are required to gain further insight into the cellular and molecular evolution of gastric carcinogenesis which should lead to better management of patients with premalignant lesions and ultimately to the prevention of GC.
CDK12 and CDK13 cooperatively regulate RNA polymerase II elongation and alternative polyadenylation of mRNA
Transcription driven by RNA Polymerase II (POLII) is a multi-step process that is strictly regulated by Cyclin dependent kinases (CDKs) at multiple checkpoints. Compared to the regulation of transcription initiation and pause release, the roles of CDKs in regulating transcription elongation remain poorly defined. To investigate the individual and shared roles of CDK12 and CDK13 in transcription regulation, a set of cell lines containing analog-sensitive variants of CDK12 and CDK13 were constructed using CRISPR-Cas9 homology-directed repair (HDR) technology. Multiple Next-Generation Sequencing (NGS) based assays including RNA-Seq, ChIP-Seq, PRO-Seq, TT-Seq were utilised to provide a comprehensive characterisation of the consequences of acute inhibition of CDK12, CDK13 or both kinases using these analog sensitive CDK12 and CDK13 cell lines. Selective inhibition of CDK12 or CDK13 led to various molecular responses including selective changes in gene expression, alternations in polyadenylation site usage as well as mild reduction in POLII elongation rate and processivity. In contrast, dual inhibition of CDK12 and CDK13 caused dramatic changes in transcription genome-wide, including the induction of widespread alternative polyadenylation events, reduction in POLII elongation rates and processivity, substantial changes in gene expression, as well as the near complete loss of POLII Ser2 phosphorylation. These observations illustrated that both CDK12 and CDK13 are regulators of POLII transcription elongation. Furthermore, the substantial differences between selective and simultaneous inhibition of CDK12 and CDK13 revealed the redundant and individual roles of CDK12 and CDK13 in maintaining global transcription elongation. To identify substrates of CDK12 and CDK13 that might be responsible for the phenotypes caused by CDK12 and CDK13 inhibition, phospho-proteomic analysis was performed to identify putative CDK12 and CDK13 substrates. The analysis revealed that CDK12 and CDK13 shared multiple substrates and functional redundancy between CDK12 and CDK13 in phosphorylating these substrates was identified. In order to identify the putative substrates that were responsible for the transcriptional changes upon CDK12 and CDK13 inhibition, a novel siRNA screen method “mini-bulk” CEL-Seq2 siRNA screen was developed and utilised. The screen revealed that SF3B1 and SRRM2 could be the potential substrates of CDK12 and CDK13 that were partially responsible for the transcriptional phenotype caused by the dual inhibition of CDK12 and CDK13, as depletion of SF3B1 and SRRM2 led to similar differential gene expression and alternative polyadenylation profiles as CDK12 and CDK13 inhibition. Finally, as the phospho-proteomic analysis also revealed that CDK12 and CDK13 might regulate phosphorylation of multiple translation regulators, the effect of CDK12 and CDK13 inhibition on protein translation was also investigated. Both nascent protein labelling as well as polysome profiling revealed that CDK12 and CDK13 function was required to maintain global translation. In conclusion, this thesis explored the role of CDK12 and CDK13 in POLII driven transcription and protein translation. CDK12 and CDK13 were shown to cooperatively regulate POLII transcription elongation processivity and alternative polyadenylation, potentially through regulating POLII Ser2 phosphorylation and the phosphorylation of other CDK12 and CDK13 substrates.
Hippo pathway target genes and transcription factors in organ growth control
The Hippo pathway, first discovered in the fruit fly Drosophila melanogaster, is an evolutionarily conserved regulator of organ growth. The Hippo pathway controls organ growth by regulating the nuclear activity of the transcriptional coactivator Yorkie (Yki). Yki binds to sequence-specific transcription factors to regulate target gene expression, including the TEAD/TEF family transcription factor Scalloped (Sd). It was recently discovered that the Hippo pathway controls the growth of organs in Drosophila by balancing transcriptional activation mediated by Yki and Sd, and transcriptional repression mediated by Tondu-domain- containing Growth Inhibitor (Tgi) and Sd. If this fine balance is disrupted and Yki is hyperactivated, organs can grow beyond their normal size. Importantly, deregulation of Hippo signalling drives tumorigenesis in a variety of human cancers. The high degree of conservation of the Hippo pathway throughout the animal kingdom makes the fruit fly a powerful model organism to understand how this pathway regulates tissue growth during development and cancer. The Hippo pathway regulates the expression of genes involved in cell proliferation, survival, stemness, differentiation, and migration. However, we lack a clear understanding of the full transcriptional program that the Hippo pathway regulates in growing tissues beyond a small number of well-studied target genes. In this thesis, I aimed to deepen our understanding of the transcriptional program that the Hippo pathway controls in the growing Drosophila eye. To do this, I addressed three key questions: 1) What are the target genes of Yki, Sd and Tgi? 2) What gene expression changes occur as a result of hyperactive Yki? 3) What additional transcriptional regulators control Hippo pathway target gene expression and eye growth? 1) Using targeted DamID-seq, I identified target genes of Yki, Sd and Tgi in growing Drosophila eye discs. I found a very high degree of overlapping target genes between Yki, Sd, and Tgi, indicating that Sd is the key transcription factor that mediates binding of both Yki and Tgi to their target gene loci. Additionally, I found a strong enrichment of the AP-1 binding motif in Yki, Sd, and Tgi target genes, suggesting that these transcription factors co-regulate many target genes. 2) I performed comprehensive analyses of Yki, Sd, and Tgi target genes, and the changes in gene expression and chromatin accessibility caused by Yki hyperactivation. This highlighted key biological processes and signalling pathways that the Hippo pathway could regulate during eye growth, including: MAPK pathway, glutathione S-transferases (GSTs), cuticle development, extracellular matrix (ECM), and retinal determination and differentiation. 3) Using targeted DamID-seq, I found that the Drosophila AP-1 transcription factor Jra/Jun shares 71% of its target genes with Yki and/or Sd. However, using genetic studies, I showed that AP-1 transcription factors do not regulate physiological Drosophila eye growth, and are not essential regulators of Hippo pathway target gene expression. The results presented in this thesis provide new insights into the target genes and transcriptional program that is regulated by the Hippo pathway during Drosophila eye growth. They have identified previously unexplored biological processes that the Hippo pathway might regulate in the eye (e.g. cuticle development), and reinforced known roles for the Hippo pathway (e.g. cell fate specification). My findings also extend our understanding of the functional links between AP-1 and the Hippo pathway in growing organs. Importantly, the data generated in this thesis will provide a rich resource for researchers interested in studying the direct target genes of both Hippo signalling and AP-1 transcription factors.
Novel biomarkers for melanoma immunotherapy
Traditionally, metastatic melanoma had a dismal prognosis, but the recent advent of immune checkpoint inhibitors (ICI) has extended survival from months to years for some patients. There is an urgent need to identify prognostic and predictive biomarkers for melanoma patients treated with ICI, given that only a minority of patients respond, coupled with the potential treatment related toxicities. This thesis aimed to investigate clinical factors, functional PET imaging and tumour immune profiling as candidate biomarkers for ICI in patients with melanoma. Firstly, Chapter 3 focused on baseline performance status as a biomarker for outcome following anti-PD-1. The hypothesis was that unlike cytotoxic chemotherapy, baseline performance status was not correlated with outcome following ICI, owing to its distinct mechanism of action. However, in the cohort of 91 patients treated with anti-PD-1 at Peter MacCallum Cancer Centre, poor performance status was correlated with poor survival and low response rate to anti-PD-1. Furthermore, patients with poor performance status were more likely to be hospitalised and more likely to die in hospital. Patient characteristics and blood parameters were further examined in Chapter 4, but specific to a cohort of patients with melanoma brain metastases. Melanoma commonly metastasise to the central nervous system and this is associated with extremely poor survival. Recently, combination ICI has resulted in intracranial responses and durable survival. Most of the existing literature in biomarkers in melanoma brain metastases also predates the introduction of ICI, therefore investigation of biomarkers in patients with melanoma brain metastases treated with ICI is needed. A post-hoc analysis of patients with melanoma brain metastases as part of the phase II Anti-PD1 Brain Collaboration study was performed to identify possible predictors of clinical outcome or toxicity. In this study, patients were randomised to receive either nivolumab monotherapy or nivolumab in combination with ipilimumab. High C-reactive protein, a marker of systemic inflammation, was correlated with poor survival. Treatment with combination ICI, hypernatraemia and increased body mass index were associated with higher likelihood of severe toxicity at 120 days, whereas CRP was not associated with higher toxicity. The thesis then went on to examine the role of FDG PET functional imaging as a source of biomarkers for outcome following ICI in Chapter 5. Baseline pre-treatment tumoural FDG-PET avidity (measured by SUVmax or metabolic tumour volume) as well as FDG-avidity in the immune system (measured by spleen to liver ratio) were assessed in relation to survival outcomes. Interestingly, tumoural PET avidity was not correlated with survival, whereas high spleen to liver ratio was correlated with poor survival after ipilimumab. This was subsequently validated in a combined cohort of patients from two separate European centres. High spleen to liver ratio was correlated with low albumin in a multivariate analysis, thus suggesting a possible association with systemic inflammation. Early on-treatment PET (EOT PET) were assessed in a small subset of 16 patients, and several challenges were identified that may limit the use of FDG PET in this early juncture as a biomarker for outcome after ICI. In-depth characterisation of tumoural immune landscape is crucial to improving the understanding of melanoma immuno-biology, with potential implications for biomarker development. Chapter 6 aimed to compare the immune profile of UV related skin cancers (melanoma, cutaneous squamous cell carcinoma and Merkel cell carcinoma) using orthogonal methods of bulk RNA-sequencing and multi-spectral immunohistochemistry. The three skin cancers showed distinct immune landscapes, with melanoma having a significantly higher intratumoural T cell infiltrate compared to Merkel cell carcinoma, whereas PD-L1 density was highly variable across three skin cancers. Transcriptomic analyses of melanoma samples with high PD-L1 density were associated with upregulation of genes related to leucocyte proliferation, migration and adaptive immune responses, in contrast to MCC samples with high PD-L1 density, where such a signature was not observed. Lastly, an in-depth case study of six patients highlighted how multi-factorial biomarkers such as clinical factors, functional PET imaging, baseline blood parameters, and multi-spectral immunohistochemistry can be applied together. In conclusion, this thesis evaluated multi-factorial biomarkers including clinical, functional imaging and tumoural immune profiling biomarkers. These studies add to the evolving literature on biomarkers associated with ICI treatment. It is envisaged that with time, these complementary methods of understanding the patient and tumoural immune environment can aid rational selection of immune based therapies for patients with advanced melanoma.
Examining the effects of BRAF, MEK and CDK4/6 inhibition on anti-tumor immunity in BRAFV600 melanoma
The recent advent of targeted and immune-based therapies has revolutionized the treatment of melanoma, and transformed outcomes for patients with metastatic disease. However, the mechanisms underpinning the clinical efficacy of these approaches are still being elucidated. The majority of patients develop resistance to the current standard-of-care targeted therapy, dual BRAF and MEK inhibition (BRAFi+MEKi), prompting evaluation of a new combination incorporating a CDK4/6 inhibitor. Based on promising preclinical data, combined BRAF, MEK and CDK4/6 inhibition (triple therapy) has recently entered clinical trials for the treatment of BRAFV600 melanoma. Interestingly, while BRAFi+MEKi therapy was initially developed on the basis of potent tumor-intrinsic effects, it was later discovered to have significant immune-potentiating activity. Recent studies have also identified immune-related impacts of CDK4/6 inhibition, though these are less well defined and appear to be both immune-potentiating and immune-inhibitory. BRAFV600 melanoma patients are also eligible for immunotherapies, and hence the immunomodulatory activity of these targeted inhibitors makes first-line treatment decisions complex. The aim of this thesis was to examine the immunomodulatory effects of BRAF, MEK and CDK4/6 inhibition, with an ultimate goal of providing critical information to aid in the clinical management of BRAFV600 melanoma patients. Examining mechanisms of the immunomodulatory effects of targeted therapies requires preclinical mouse models of melanoma that are both immunogenic, and harbor the oncogenic drivers targeted by the therapies being evaluated. To address this, we developed a novel immunogenic BrafV600ECdkn2a-/-Pten-/- melanoma mouse model, called YOVAL1.1. YOVAL1.1 tumors are transplantable in immunocompetent mice and amenable to standard-of-care melanoma therapies, including BRAFi+MEKi and immune checkpoint blockade. This, coupled with the Cdkn2a status, which infers some sensitivity to CDK4/6 inhibitors, makes this an ideal preclinical model to evaluate the immunomodulatory effects of the triple therapy. Using this model, we demonstrated that triple therapy promotes durable tumor control through tumor-intrinsic mechanisms, while promoting immunogenic cell death and T cell infiltration. However, despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade. Flow cytometric and single cell RNA-seq analyses of tumor infiltrating immune populations revealed that triple therapy markedly depleted pro-inflammatory macrophages and cross priming CD103+ dendritic cells, the absence of which correlated with poor overall survival and clinical responses to immune checkpoint blockade in melanoma patients. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T cell responses ex vivo. Hence, while combined BRAF, MEK and CDK4/6 inhibition demonstrated favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact on anti-tumor immunity. Several recent studies have reported immune-potentiating effects of CDK4/6 inhibition, and subsequent synergy with immune checkpoint blockade. However, T cells are the primary target of these immunotherapies, and an understanding of the direct effects of CDK4/6 inhibition on this cellular subset was lacking. In this thesis, using integrated epigenomic, transcriptomic and single cell CITE-seq analyses, we identified a novel role for CDK4/6 in regulating T cell fate. Specifically, we demonstrated that CDK4/6 inhibition promoted the phenotypic and functional acquisition of T cell memory. Genome-wide CRISPR/Cas9 screening and phospho-proteomics revealed that memory formation in response to CDK4/6 inhibition was cell intrinsic and required RB. Pre-conditioning human CAR T cells with a CDK4/6 inhibitor enhanced their persistence and tumor control, and clinical treatment with a CDK4/6 inhibitor promoted expansion of memory T cells in a melanoma patient, priming a response to immune checkpoint blockade. Collectively these findings highlight the multi-faceted immunomodulatory activity of BRAF, MEK and CDK4/6 inhibition. The addition of a CDK4/6 inhibitor to dual BRAFi+MEKi led to the depletion of intratumoral myeloid subsets that may be critical for supporting a therapeutically beneficial T cell response. In contrast, as an individual therapy, CDK4/6 inhibition promoted effector and memory T cell activity, suggesting that, with optimal scheduling to prevent myeloid depletion, CDK4/6 inhibitors may be used to enhance and prolong BRAFi/MEKi-induced anti-tumor T cell immunity. Defining the mechanisms that underpin the clinical efficacy of these available therapies is a critical step forward in optimising novel combination and scheduling approaches to combat melanoma and improve patient outcomes.
Transdifferentiation of cancer stem cells in brain tumours: Lessons from Drosophila neural stem cells
Background: In human brain cancers, glioblastoma stem cells (GSCs) originate from neoplastic transformation of neural stem cells (NSCs) or dedifferentiation of other neural cells. Similar to normal NSCs, GSCs possess stem-cell properties to self-renew and differentiate into multiple neural lineages. However, GSCs are more plastic than normal NSCs, as they can transdifferentiate into other cell types. At the present, we do not fully understand the cellular step-wise conversion from GSCs into other distinct cell lineages, and the molecular mechanism responsible for this event. Drosophila NSCs, called neuroblasts (NBs), also asymmetrically divide to renew themselves, and generate neurons or glia that make up the adult central nervous system. Disrupting either asymmetric cell division or neuronal maintenance allows differentiated cells to dedifferentiate into ectopic NBs, which then continue to proliferate and form tumours. Methods and aims: We utilised several in vivo brain tumour models in Drosophila to study how GSCs function in brain cancers and how they undergo transdifferentiation. In this thesis, I induced loss-of-function of transcription factors Prospero or Nerfin-1 in NB lineages to generate dedifferentiation-driven tumours, and found a class of cells which exhibited glial cell identity. I sought to investigate the behaviours and characteristics of these ectopic glia to elucidate some aspects about GSC transdifferentiation by answering three questions: (1) What are the cell numbers and cell types within the pros- and nerfin-1- tumours. (2) Do ectopic glia arise by transdifferentiation of NBs in the tumours? (3) Which transcription factors and signal transduction pathways drive the formation of ectopic glia, and are ectopic glia required for tumour growth? Results: (1) I found that the expansion of ectopic glia population is correlated with overall tumour growth. Ectopic glia exhibit glial identity and their formation is not dependent on the location of tumours in the central nervous system. (2) By performing live-cell imaging of pros- tumours and molecular marker analysis, I found a subset of NBs switch to glial cell fate. (3) I performed genetic experiments to manipulate the transcription factors dichaete, tailless and glial cells missing (gcm) in pros- tumour and found that they are required for ectopic glial formation and tumour growth. Their target gene reversed polarity (repo) regulates the formation of ectopic glia, which in turn, promote the tumour growth. I showed that Notch promotes tumour growth independently of its effect on ectopic glial formation, as Notch regulates the tumour growth in the absence of ectopic glia. I also showed that FGF signalling pathway promotes tumour growth by regulating ectopic glia formation, as it does not affect the tumour growth in the absence of ectopic glia. Hippo pathway also plays a role in promoting the formation of ectopic glia and tumour growth. Our study of pros- and nerfin-1- tumour models in the context of transdifferentiation may extend our understanding of the biology of NBs and may shed light on GSC behaviours upon their transdifferentiation into different cell types. We can use the underlying mechanisms of these phenotypes to gain a better understanding of the transdifferentiation events at the molecular and cellular levels. As most genes and signalling pathways examined in this study are also found in human brain cancers, this study will enhance the knowledge of how cell fate changes can influence the tumour malignancy.
Coming of age with Li-Fraumeni syndrome: perspectives of young people and health professionals
This thesis is situated in the discourse of risk that defines our technology-driven modern society, wherein one’s health is constructed as a personal and moral responsibility. A key contributor to the individualisation of risk in modern medicine is genomic technology. As genomics becomes progressively normalised in mainstream society, individuals of younger and younger ages are seeking to learn of their genetic risk of disease, including cancer. Young people occupy a formative and transitional life stage with complex processes of human development, making them a unique population for which genetic services are currently ill-equipped to serve appropriately. This research contributes to a new field of genetic counselling research that aims to explore and meet the distinctive developmental needs of young people with genetic disease. This thesis comprises of three inter-related studies that explore the psychosocial implications of living with a devastating, early onset inherited cancer condition, Li- Fraumeni syndrome (LFS), from the perspectives of young people and health professionals in Australasia. Informed by a pragmatic-critical realist stance, this thesis uses a mixed-method approach divided into a qualitative and quantitative phase. The qualitative phase consists of two studies, the first is a systematic review and thematic synthesis of 39 studies investigating how young people experience inherited disease with similar implications to LFS. The second and principal study of this research is an interpretive description of how young people experience LFS in their daily lives. To develop findings, I conducted interviews with 30 adolescents and young adults (aged 15- 39 years) with, or at 50% risk of, a pathogenic germline mutation in TP53 from across Australia, and used reflexive thematic analysis. The quantitative phase consists of a survey of 43 Australasian health professionals who care for young people with LFS to document their current practices and how they meet the developmental needs of this population. The first key finding is that experiences of cancer, either familial or personal, strongly influence how young people make sense and meaning of LFS, how they consider genetic testing, and their perceptions of cancer risk and mortality. The nature of LFS in terms of its high penetrance, early onset, and varied phenotype meant young people’s experiences were unique in oncology and genetic settings and require in-depth exploration during genetic counselling. The second key finding was that young people’s growing autonomy from family complicated the process of genetic testing, which was recognised by health professionals who worked to foster their autonomous decision- making. Genetic testing and whole-body cancer risk management represented instrumental actions of control for young people to mitigate their cancer risk from LFS, a perspective shared and promoted by health professionals. Intensive risk management and risk-reducing surgery, however, was emotionally and logistically burdensome for some young people, constructing the body as a material object be scrutinised in great detail by health professionals and blurring boundaries between self and body. The third key finding was that young people felt strongly about preventing the passage of their LFS- causing genetic variant to the next generation, reporting that pre - implantation genetic diagnosis was their preferred method for having a ‘healthy’ biological family. Few had reached a life stage, however, where they appreciated the ethical, financial and logistical burdens of this technology, and many deferred reproductive decision-making until they were ‘ready’. This research argues that young people with inherited disease have unique psychosocial and clinical needs that are directly tied to the formative developmental tasks of their life stage. They require specialist youth-friendly counselling that considers and appreciates their developmental needs both during genetic testing and beyond. Youth-friendly genetic counselling must therefore be longitudinal, incorporating psychosocial check-ups as a key clinical interaction. Further, youth-friendly models of care promote the distinct value of genetic counsellors in managing rare and complex inherited conditions. As the mainstreaming of genetic services begins to outsource tasks of genetic education and pre-test counselling, the genetic counselling profession must make better use of their psychosocial counselling skills to serve high-needs populations. In this context, young people stand out as a key focus. Finally, this research argues that attending genetic counselling exposes young people to a discourse of risk bounded by an ethos of responsibility. Undergoing genetic testing, subjecting their bodies to intensive surveillance and risk-reducing surgery, and having a family with reproductive technology all stand out as the ‘right thing’ to do when living with genetic risk of disease. Yet, each presented complex psychosocial implications for young people that were intrinsically linked to the broader effects of our modern risk society defined by Beck (1992) and Giddens (1991). Young people’s decision-making and preferences during this developmentally labile life stage therefore need to be interpreted in relation to normative societal pressures that dictate expected behaviour around risk.
Investigating drug response and resistance to IDH1R132H inhibition and hypomethylating agents in AML
Mutations in epigenetic regulators frequently occur in AML. Furthermore, the epigenetic landscape is typically dysregulated in AML. Targeted epigenetic therapeutics that directly inhibit mutant epigenetic oncogenic drivers, such as AG120 inhibition of IDH1R132H, have shown clinical success for the treatment of IDH1-mutant AML. Other epigenetic therapies target wildtype epigenetic regulators, such as DNMT1 inhibition by hypomethylating agents, with the aim of reprogramming the transcriptional networks driving malignant progression. Despite some patients achieving clinical remission, many AML patients do not respond to epigenetic therapies or relapse post-treatment. Furthermore, the contribution of mutant epigenetic regulators to leukaemogenesis is relatively poorly understood. A clinically and pathophysiologically relevant murine model of IDH1R132H driven AML co-expressing oncogenic DNMT3A and Nras was utilised to investigate the cellular and molecular consequences of IDH1R132H expression in leukaemia initiation and progression. Results from this thesis suggest that IDH1R132H governs distinct leukaemic properties within the different AML cell-types; IDH1R132H drove the expression of genes that underpin HSC-like self-renewal and proliferation in leukaemic progenitor-like cells, whilst IDH1R132H most profoundly impaired the differentiation capacity of leukaemic immature neutrophils. Furthermore, this thesis demonstrated that AG120 was a highly effective and on-target IDH1R132H inhibitor for the treatment of IDH1R132H driven in vivo AML, and induced neutrophil-skewed differentiation with distinct kinetics throughout the hierarchy of leukaemic cells. Mechanisms of resistance to the hypomethylating agents AZA and GDAC were investigated utilising a genome-wide CRISPR/Cas9 screen in vitro. Members of the pyrimidine salvage pathway were implicated in the resistance to hypomethylating agents. Loss of DCK and UCK2 conferred resistance to GDAC and AZA, respectively, whilst SLC29A1 loss mediated resistance to both hypomethylating agents in both in vitro and in vivo models of haematological malignancies. This thesis demonstrated that DCK and UCK2 loss remained sensitive to AZA and GDAC, whilst cells with SLC29A1 loss were sensitive to inhibition of DHODH. This research provided comprehensive insight of the impact of IDH1R132H on distinct leukaemic cell subsets, transcriptional mechanisms underlying leukaemogenesis, and the transcriptional perturbations that may contribute to AG120 resistance. Furthermore, the results of this thesis suggest that AZA and GDAC treatment are potential therapeutic avenues for AML resistance driven by DCK or UCK2 depletion or loss, respectively. Moreover, the results described herein suggest that targeting the de novo pyrimidine synthesis pathway, through DHODH inhibition, is a therapeutic strategy to overcome resistance to hypomethylating agents mediated by the depletion of pyrimidine salvage pathway enzymes.
Deciphering tumour heterogeneity in acute myeloid leukaemia at the single cell level
The advent of next-generation sequencing (NGS) has allowed researchers to appreciate the enormous heterogeneity that exists between cells within a single tumour. This intratumour heterogeneity leads to diverse phenotypic outcomes, resulting in functionally distinct subpopulations of cancer cells. This functional heterogeneity fuels tumour evolution and therapeutic resistance and is thus a major barrier to producing cures in cancer. Acute myeloid leukaemia (AML) is an aggressive and heterogeneous malignancy with a high relapse rate. The prevailing paradigm to explain relapse in AML posits that genetic heterogeneity leads to pre-existing or acquired mutations that render certain cells refractory to therapy, resulting in the outgrowth of a resistant clone. Large-scale sequencing studies aimed at cataloguing genetic heterogeneity in AML have revealed several important observations. Firstly, AML has one of the lowest mutational burdens of any cancer. Secondly, a significant proportion of clinical relapse events cannot be attributed to an underlying genetic change. These important findings raise the possibility that mutations alone are insufficient to fully explain therapeutic resistance in AML. Indeed, we are now beginning to appreciate that both tumour evolution and clinical relapse can be driven by non-genetic processes. However, characterising the full extent of non-genetic heterogeneity and its relative contribution to both the evolutionary trajectory of the disease and therapeutic resistance requires innovative single cell methodologies. Single-cell RNA sequencing (scRNA-seq) has been instrumental in revealing the phenotypic heterogeneity of rare subpopulations of cells within a complex tumour. However, it is difficult to infer clonal relationships from scRNA-seq alone and this has hampered our ability to understand how individual malignant cells evolve over time. To overcome some of these challenges, we present a lentiviral method of tagging cells with unique heritable barcodes that are stably transcribed into RNA molecules in cells and therefore highly detected in microfluidic scRNA-seq workflows. This strategy, termed Single-cell Profiling and LINeage TRacking with expressed barcodes (SPLINTR), offers the ability to match the gene expression programmes of individual cells to their clonal lineage, in order to establish how initial transcriptional differences amongst heterogeneous malignant cells can shape thier future clonal behaviour during cancer progression. We apply our SPLINTR barcoding system to an in vivo model of clonal competition in order to determine the early transcriptional signatures that are associated with future clonal dominance in AML. We discover that clonal dominance is largely an intrinsic property amongst genetically identical clones. However, we find the deterministic nature of dominance is altered by the presence of other distinct competing mutational clones. Furthermore, SPLINTR enabled us to retrospectively identify a novel set of differentially expressed genes contained within certain clones prior to transplantation, which distinguished them from losing clones and was associated with their future dominance during disease progression. Finally, we find that resistance occurs to BET inhibitor therapy in the clinic in the absence of a clear genetic event. scRNA-seq of paired baseline and relapse AML patient bone marrow samples revealed than non-genetic resistance originates from either a population of pre-existing cells that phenotypically resemble LSCs, or through transcriptional adaptation as a result of therapeutic pressure. We then use SPLINTR coupled with scRNA-seq to interrogate our previously published in vitro model of non-genetic resistance to BET bromodomain inhibition. This provided further evidence that Lamarckian evolution in the form of gradual transcriptional adaptation drives non-genetic resistance. Future work aims to unravel the epigenetic states that mediate non-genetic transcriptional adaptation in a broader therapeutic context in AML. Collectively, the research presented in this thesis demonstrates the importance of applying novel single cell technologies to investigations of cellular diversity in cancer and highlights the underappreciated role of non-genetic heterogeneity in driving both disease evolution and therapeutic resistance in AML. These studies provide the molecular tools and rationale to further define the mechanisms by which non-genetic heterogeneity shapes cellular behaviour in cancer.
Enhancing CAR T cell therapy
Adoptive cell therapy using chimeric antigen receptor (CAR) T cells has shown remarkable efficacy in the treatment of haematological malignancies, with complete remission rates of 90% reported in early clinical trials. Following this success, two CD19 targeting CAR T cell products have been approved in Australia for the treatment of Acute Lymphoblastic Leukaemia (ALL) and non-Hodgkin lymphoma. However, acquired resistance to this therapy through CD19 antigen loss is an emerging problem. Moreover, such promising results have not been recapitulated against solid tumours. This is thought to be due in part to poor CAR T cell trafficking and infiltration, the immunosuppressive tumour microenvironment and antigen heterogeneity in solid tumours, as well as intrinsic and acquired tumour resistance. Therefore, it is necessary to uncover mechanisms of resistance to CAR T cell therapy in both the haematological and solid tumour settings, in order to improve patient outcomes. In this thesis, we sought to uncover the tumour-intrinsic mechanisms of resistance to CAR T cell therapy. Whole-genome loss-of-function CRISPR-Cas9 screening has recently emerged as a powerful tool to screen all protein-coding genes in the genome for conferring resistance to various immune and drug pressures. Herein, we utilised a genome-wide mouse pooled-sgRNA library to investigate genes that protect MC38 mouse adenocarcinoma cells from CAR T cell killing. Here, we identified that loss of cytokine response pathways, in particular, TNF and IFN-y signalling were critical for the effector function of CAR T cells. Additionally, we report for the first time that loss of the transcriptional co-binding partner Cbfb protects tumour cells against T cell killing. Investigation in to the mechanisms of Cbfb-mediated resistance uncovered that loss of tumour cell Cbfb protected tumour cells against T cell-derived TNF. Importantly, we report that mice bearing Cbfb-deficient MC38 tumours did not respond to anti-PD-1 in vivo, and analysis of clinical trial data suggests that in melanoma cohorts, low expression of CBFB or the transcriptional co-binding partner RUNX1 was correlated with a poorer prognosis following immune checkpoint blockade or adoptive cell therapy. Next, we sought to design a rational combination therapy to overcome some of the challenges associated with CAR T cell therapy against solid tumours and enhance CAR T cell therapy in this context. Based on data from the CRISPR screen which highlighted the role of TNF in CAR T cell-mediated cytotoxicity, we sought to enhance TNF-mediated killing using a small-molecule smac-mimetic. We demonstrated that antagonism of the Inhibitor of Apoptosis Proteins with the smac-mimetic birinapant, significantly enhanced CAR T cell killing in a TNF-dependent manner. Using a syngeneic HER2+ self-antigen model, we report that birinapant significantly enhanced CAR T cell-mediated tumour clearance in a combination therapy approach, and we demonstrated enhanced human CAR T cell killing in patient biopsy-derived tumouroids. Critically, we report that birinapant significantly enhanced TNF-mediated “bystander killing”, which occurred in the absence of target antigen expression, which may be a strategy to overcome the challenge of tumour antigen heterogeneity associated with solid tumours. Finally, we sought to uncover mechanisms by which B cells lose CD19 antigen expression following CAR T cell attack. By utilising a second genome-wide CRISPR screening approach, we applied a human pooled sgRNA library to the CD19+ B-ALL cell line MHH-CALL4, and sorted 3 times the cells with the lowest 20% CD19 surface expression by flow cytometry, in order to uncover genes or pathways of interest that are implicated in down-regulation of CD19 surface expression. Along with several known factors, such as CD81 trafficking and N-linked glycosylation, we also identified several novel processes including histone methylation, aryl hydrocarbon receptor processing and E3 ubiquitin ligase activity, which will be the focus of future investigations. In summary, insights gained from the work in this thesis elucidate some of the resistance pathways utilised by tumour cells in order to evade immune pressure from CAR T cells, and highlight the cytotoxic potential of TNF in CAR T cell therapy. In addition, we herein present a novel strategy to enhance CAR T cell efficacy in solid tumours, and demonstrate the potential for combination therapy approaches to overcome the challenges associated with CAR T cell therapy against solid tumours. Taken together, this work significantly addresses several of the existing limitation of CAR T cell therapy in both B cell leukaemias and against solid malignancies, and the novel combination therapy we describe is poised to be rapidly translated in to the clinic.
Profiling the immune and genomic landscape of anal squamous cell carcinoma and establishing preclinical models to explore new therapeutic options
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.