Sir Peter MacCallum Department of Oncology - Theses
Now showing items 1-12 of 102
Circulating tumour DNA analysis for personalised care in breast cancer
Phenotypic diversity of breast cancers poses insurmountable challenges in the treatment of this lethal disease. Recent advances in next generation sequencing have led to unprecedented insight into the genomic landscape underlying breast tumours. This has resulted in burgeoning development of targeted treatments and predictive biomarkers, several of which have recently demonstrated clinical activity. However, key challenges hinder optimal application. On the background of extensive molecular heterogeneity, most biomarkers represent minority patient subpopulations, hampering clinical development. Furthermore, considerable genomic evolution of breast tumours impacts accuracy of genomic characterisation that is thus far heavily reliant on the sequencing of non-contemporaneous and invasive tumour tissue biopsies. Finally, stratification to genomically-matched targeted therapies also fails to fulfil the extent of its promise. In many cases relentless tumour growth remains unperturbed, while in others resistance ultimately develops. Crucially, molecular mechanisms underlying resistance remain poorly understood, while follow-on treatment options are often poorly defined. Central to the promise of personalised medicine is the robust and accurate characterisation of the tumour genome. Minimal invasiveness and convenience of circulating tumour DNA (ctDNA) analysis, with ability to detect tumour genomic aberrations from a blood draw, highly recommends this approach. Recent technological advances have paved the way to a range of clinical applications, with evolving potential for ctDNA analysis to address the continuum of challenges posed to precision medicine throughout patient management. Toward this end, extensive clinical development is required, while prevailing technological hurdles need to be addressed. This thesis explores a multi-faceted and rigorous approach towards the integration of ctDNA analysis in the management of breast cancer patients. Firstly, the development and validation of multiple assays (allele-specific and NGS-based) tailored to breast cancers, enabled comprehensive genomic analysis with in-built flexibility to be readily applicable to a variety of clinical scenarios. Subsequent establishment of a prospective ctDNA-based molecular profiling program across a large cohort of metastatic breast cancer (mBC) patients demonstrated feasibility of real-time analysis in the clinical setting across a range of genomic targets of variable abundance. Importantly, integration of longitudinal testing in this program throughout patient management demonstrated capacity for ctDNA analysis to reflect genomic evolution in real-time to optimise precision-guided patient management. Finally, exploratory longitudinal ctDNA analysis for the study of resistance mechanisms to CDK4/6i, constituting a novel class of targeted compounds for breast cancer, highlights established and novel resistance markers. Indeed, this study also serves to demonstrate a workable framework for ctDNA analysis as a highly effective approach for the de novo elucidation of resistance mechanisms to novel targeted agents that is relevant across cancer types.
Automated discovery of interacting genomic events that impact cancer survival by using data mining and machine learning techniques
Rapid advancement in genomic technologies has driven down the cost of sequencing significantly. This efficiency has enabled large-scale cancer genomic studies to be conducted, generating a vast amount of data across different levels of omics variables. However, the tasks to extract new knowledge and information from this enormous volume of data present unique challenges. These analyses often require the application of specialised techniques for data mining, integration and interpretation to provide valuable insights. With the rise of machine learning adoption in recent decades, many advanced computational algorithms based on artificial intelligence techniques have also been proposed to analyse these genomics data. Although some of these applications have led to clinically relevant conclusions, many others are still relying on incomplete prior knowledge, or limited to only a selected number of features. These limitations raise the general question about the broader applicability of machine learning in the field of cancer genomics. This research addresses this question by assessing the application of machine learning techniques in the context of breast cancer genomics data. This assessment includes a comprehensive evaluation of computational methods for predicting cancer driver genes and the development of a novel deep learning approach for identifying breast cancer subtypes. The evaluation result of driver gene prediction algorithms suggests that the selection of the best method to be applied to a dataset will primarily be driven by the objectives of the study and the characteristics of the dataset. All of the evaluated approaches could identify well- studied genes, but not all of them performed as well on smaller datasets, subtype-specific cohorts, and in discovering novel genes. To examine the benefit of a more complex machine learning model, this thesis also presents a novel deep learning approach that integrates multi-omics data for predicting various breast cancer’ biomarkers and molecular subtypes. This method combines a semi-supervised autoencoder for dimensionality reduction, and a supervised multitask learning setup for the classifications. Taking an input of gene expression, somatic point mutation and copy number data, the algorithm predicts the ER-Status, HER2-Status and molecular subtypes of breast cancer samples. Further survival analysis of the outputs from this deep learning approach indicates that the predicted subtypes show a stronger correlation with patient prognosis compared to the original PAM50 label. While the outputs from machine learning algorithms still require further validation, the adoption of these complex computational methods in cancer genomics will become increasingly common. Collectively, the results from this thesis suggest that the machine learning analysis of ‘omics data hold great potential in automating the discovery of clinically- relevant molecular features.
Developing new therapies to treat advanced prostate cancer
Prostate cancer (PCa) is one of the most diagnosed tumours in Australia, pertaining to more than 30% of all cancer detection (AIHW 2020). Most of these diagnoses are correlated to men with early, localised stages of the disease that are easily treated with radical proctectomy surgery and/or radiation. One third of these patients will relapse with their disease progressing to the either the recurrent localised stage or the metastatic stage. These advanced cases will have to undergo Androgen Deprivation Therapy (ADT), as hormone dependence is a key driver of prostate tumorigenesis. ADT will help increase patient survival by halting cancer progression, while minimising symptoms. Although this type of therapy can initially alleviate tumour burden, patients will inevitably be subjected to cancer recurrence where their tumours continue to progress despite castrate levels of androgens. An observed second-order effect of ADT is that it creates a therapeutic selection pressure within PCa, leading to the proliferation and establishment of cells that are resistant to androgen ablation. These hormone-refractory cells lead to the development of Castration Resistant Prostate Cancer (CRPC), a stage of the disease that ultimately leads to death. Despite the development of second-generation ADT or chemotherapeutic agents, no effective therapies have been developed yet to combat this lethal stage of the disease. Therefore, there is a need for therapies that can target pathways and mechanisms that are not related to androgen signalling. MYC overexpression is one of the most frequently characterised genomic aberrations within men with CRPC. MYC mRNA overexpression is found in 30% of men with localised disease and in 85% of men with CRPC. Although MYC seems like a prime therapeutic target to combat PCa, previous attempts at developing MYC inhibitors have failed due to major off-target effects making these therapies dangerous. The overexpression of MYC is tightly correlated with increased ribosome biogenesis because MYC-driven tumours have an inherent need for protein synthesis to sustain infinite cell proliferation. The rate limiting step of ribosome biogenesis is the transcription of a ribosome RNA precursor by RNA polymerase I within the nucleolus. MYC orchestrates the over-transcription of this precursor by tightly interacting with Pol I associated components. PIM kinases and the PI3K pathways also tightly interact with MYC in order to increase its stability and prevent its degradation. It can be inferred from this that MYC-driven tumours are highly proliferative, lack any type of cell cycle regulation, and are constitutively producing proteins to sustain their uncontrolled growth. Administration of CX-5461, an RNA Pol I inhibitor, was followed by potent anti-proliferative activity and cell cycle arrest within various in vitro and in vivo models of prostate cancer. Strikingly, the combination of Pol I inhibition along with CX-6258, a PIM kinase inhibitor, was characterised by potent reduction in invasive lesions within two mouse models of prostate cancer. Although both drugs combined seemed to have potential additive effect, little is understood about the mechanism of action of these two inhibitors together. Therefore, one of the main aims of this thesis is to understand the efficacy of this combination. Both drugs will be tested on different subtypes of human prostate cancer in the explant settings in order to correlate patient-drug response with specific genomic or pathological profiles. A further goal for this project will be to utilize mass spectrometry to characterise the main phosphorylation events that occur as a result of the administration of these inhibitors, both alone and in combination. This will further the understanding of the mechanistic action of both drugs combined. Due to the limited efficacy of CX-5461 as a single agent, PMR-116, a novel Pol I inhibitor, will also be tested within different models of prostate cancer to evaluate as a potential anti-cancer drug. The final aim of this thesis will be to combine CX-5461 with targeted 177Lu-PSMA-617 therapy, as the latter has recently been shown to potentiate radiation-induced DNA damage. Through the various aims of this thesis, we hope to find and characterise the efficacy of RNA polymerase I therapies on prostate cancer both as single agents or combined therapies.
Investigating the metabolic consequences resulting from the oncogenic activation of the PI3K-AKT pathway
The phosphoinositide 3-kinase (PI3K) pathway is one of the most commonly activated pathways in a variety of cancers and it has recently been highlighted as one of the primary modulators of cell metabolism. This has opened promises and challenges for the development of therapeutic strategies to target metabolism in cancer cells harbouring mutations in the PI3K pathway. Through an inducible “exon switch” approach, our laboratory has previously generated mice ubiquitously expressing a mutation in Pik3ca, the gene coding for the subunit p110alpha of PI3K. By using this mouse model (UbCreERPik3caH1047R) our laboratory has shown that mutations in the PI3K pathway lead to dramatic severe defects in glucose homeostasis resulting in hypoglycaemia and hypoinsulinemia. In this thesis the causes responsible for the metabolic dysfunction observed in these mutant mice are investigated. This thesis provides evidence that mutations in the PI3K pathway lead to increased glucose uptake by the tissues, inhibition of hepatic gluconeogenesis and inhibition of insulin release from the pancreas. Previous studies performed on the UbCreERPik3caH1047R mouse model have also shown increased body weight and organomegaly. This thesis demonstrates that the increase in body weight, resulting from the activation of the Pik3caH1047R mutation was not associated with adiposity. On the contrary, mutations in the PI3K pathway determine loss of body fat and increased lipolysis in the adipose tissue of mice, whilst the tissue growth is associated to hypertrophy or hyperplasia. Furthermore, oncogenic activation of the Pik3caH1047R mutation in vivo leads to alteration of the respiratory exchange rate and energy expenditure of the mice and stimulates browning of the adipose tissue. This thesis also shows that activation of the PI3K pathway alters the expression of genes and proteins involved in metabolic pathways, and that these alterations are organ-specific, therefore opening promises for customising treatments to individual patients.
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.