Sir Peter MacCallum Department of Oncology - Theses
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ItemNo Preview AvailablePrecision Medicine in Breast and Thoracic Oncology( 2023-07)Treatment of breast and thoracic malignancies remains at the forefront of innovative treatment of cancer. However, due to a lack of testing for the underlying molecular drivers of these cancers, many patients who would benefit from Precision Medicine are deprived of more targeted treatments known to have better response rates and favourable side effect profiles. The current practice of germline sequencing of hereditary breast cancer genes which is based on family history and histology, underestimates the frequency of carriers of pathogenic variants by at least 50%, with consequences to the patients and their families. My PhD focused on the offer of universal germline testing and somatic sequencing to prove its utility in clinical management and to offer a model moving forward to address these deficiencies. The MAGIC study, which included 474 patients with newly diagnosed high-grade in situ or invasive breast cancer, detected pathogenic variants in 31 patients (6.5%). 18 out of 31 of those patients would have been missed if they had gone ahead with current testing guidelines, and 14 of those 18 patients' clinical management changed, highlighting the value of universal germline testing as opposed to the current criteria-based offer of testing. In thoracic oncology, obtaining genomic information with tissue biopsies remains technically challenging and fails to appreciate the evolution of thoracic malignancies and tumour heterogeneity. Liquid biopsies offer much promise in addressing these issues in a less invasive way and to detect new therapeutic targets, monitor response and function as a biomarker. My study used the Resolution Bioscience liquid biopsy platform in stage II-III non-small cell lung cancer, which had a detection rate of 49% with 45% concordance compared to the gold standard platform, MSK-IMPACT. Also, a correlation was found between ctDNA shedding with higher volumes of cancer and squamous cell histology but with no relationship with smoking, age, or sex. This work demonstrated the ability to utilise liquid biopsies in clinical practice without the requirement for tissue biopsies in certain clinical situations. HER2 is well known therapeutic target in breast oncology but remains relatively new to thoracic oncology. Currently in Australia, routine testing for HER2 is not done, and in the United States, although recommended, every centre does not perform comprehensive molecular profiling for the detection of HER2. My study at Memorial Sloan Kettering, New York, USA, analysed 2200 consecutive specimens, and 114 patients had HER2 alteration (5.1%). The IHC data was not available for all specimens. The study's primary objective was to demonstrate the superiority of next-generation sequencing (NGS) over IHC (immunohistochemistry). IHC only identified 20/51 (39%) of the HER2 alterations. This study also demonstrated the value of the identification of the exact mutation and the co-mutation profile, as this influences the response to treatment. In conclusion, the offer of universal germline testing of women with newly diagnosed breast cancer identified more carriers with germline pathogenic variants, which led to management change. Liquid biopsies can be utilised in the detection of therapeutic targets and can be utilised for the management of pre surgical patients. NGS was found to be superior to IHC in the detection of HER2 targets in thoracic oncology, and the identification of the exact HER2 mutation and co-mutation profile is invaluable in the effective management of thoracic oncology patients with HER2 alterations.
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ItemInvestigating the molecular characteristics of TILs and the role of tissue-resident memory CD8+ T cells in response to immune checkpoint inhibition in breast cancer( 2023-04)The presence of high amounts of tumor-infiltrating lymphocytes (TILs) are considered a robust prognostic biomarker and are associated with improved patient survival outcomes, particularly in triple-negative (TN) and HER2 overexpressing breast cancer (BC) subtypes [1]. Although TILs in BC vastly contain T cells [2], the qualitative properties of tumor-infiltrating T cells and their associations with BC patient prognosis remain poorly understood. We hypothesized that T cells infiltrating breast tumors contain heterogeneous subsets and exhibit qualitative differences specifically in BC subtypes with higher levels of TILs. To explore this in detail, in Chapter 3 we investigated the immune composition of TILs and the qualitative properties of BC-associated T cells including the differentiation of memory T cells in patient primary and metastatic tumors. We demonstrated that primary TNBC and HER2+ BC tumors contained significantly greater frequencies of T cells compared to other BC subtypes and metastatic lesions. Specifically, high TIL BC tumors contained a greater accumulation of CD8+ T cells with a unique feature of tissue-resident memory (TRM) phenotype. These CD8+ TRM cells expressed increased levels of immune checkpoint molecules compared with circulating T cell (TCIRC) counterparts. Moreover, bulk RNA sequencing of FACS-sorted CD8+ T cell sub-populations revealed the distinct transcriptional program of CD8+ TRM cells that were previously described in infection models. Furthermore, unbiased single-cell RNA sequencing (scRNA-seq) of over 6000 CD3+ T cells from primary TNBC patient tumors revealed the diverse TIL sub-clusters and validated the transcriptional profile of CD8+ TRM cells. In Chapter 4, we investigated the mechanistic development of resident CD8+ T cells and their functional association in response to immune checkpoint inhibition in murine models of TNBC. To this end, we tracked the differentiation of CD8+ TRM-like cells in mammary tumors and showed that the intratumoral resident CD8+ T cells transcriptionally resembled CD8+ TRM cells in TNBC patients. Furthermore, phenotypic, and transcriptional profiling revealed two distinct intratumoral CD8+ resident sub-populations: one more enriched in markers of terminal exhaustion (TEX-like) and the other exhibited a bona-fide resident memory phenotype (TRM-like). Notably, we showed that therapeutic blockade of both PD-1 and CTLA-4 checkpoint receptors was most effective in controlling BC tumor growth and resulted in greater accumulation of CD8+ TRM-like cells in the local mammary tissue. Furthermore, CD8+ TRM-like cells produced increased levels of IFN-g and TNF cytokines following re-stimulation with autologous cancer cells and mediated direct lysis of TNBC tumor cells. Finally, we showed that the CD8+ TRM-like cells in the local tumor-free mammary tissue following treatment with anti-PD-1 and anti-CTLA-4 therapy conferred strong local tissue protection from secondary TNBC tumor challenge. Strikingly, a CD8+ TRM gene signature extracted from tumor-free tissue following inhibition of PD-1 and anti-CTLA-4 receptors demonstrated an improved clinical outcome in TNBC patients treated with PD1/PDL-1 checkpoint inhibitors. Overall, this work demonstrates that CD8+ TRM cells play a vital role in BC anti-tumor immunity and are a crucial determinant for favorable clinical outcomes in TNBC, useful in identifying patient prognosis and predicting therapy responses to PD1/PDL-1 immune checkpoint inhibitors.
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ItemUncovering epigenetic regulators of bivalent MHC class I genes in cancer( 2023-06)Recent breakthroughs in cancer immunotherapy have transformed the management of many malignancies and renewed interest in the molecular understanding of tumour antigen presentation. However, despite the success of these therapies, resistance remains a significant challenge for many patients. One prominent resistance mechanism involves the disruption of major histocompatibility complex class I (MHC-I) antigen presentation, which has been demonstrated to occur through inactivating mutations or transcriptional silencing in the MHC-I antigen presentation pathway, with the latter presenting as a potentially reversible and, therefore, targetable mechanism of resistance. In this thesis, I investigate the epigenetic mechanisms underlying MHC-I resistance in cancer. Firstly, I elucidate the significance of the polycomb repressive complex 2 (PRC2) in maintaining transcriptional repression of MHC-I, which is conserved across different species, including humans, mice, and Tasmanian devils. The silencing of MHC-I by PRC2 in cancer cells facilitates the evasion of T-cell killing. However, I demonstrate that this can be overcome through genetic or pharmacological depletion of PRC2. By conducting ChIP-sequencing, I identify that silenced MHC-I genes in cancer cells exhibit bivalent modifications, specifically repressive H3K27me3 and activating H3K4me3 marks, which is a developmental process seen in embryonic stem cells and maintained during neural progenitor differentiation. Collectively, these findings reveal how cancer cells can co-opt an evolutionarily conserved, lineage-specific function of PRC2 to silence MHC-I antigen presentation and evade immune surveillance. Driven by the observation that bivalency is often dysregulated in cancer, I set out to identify the regulators of bivalent chromatin. Building upon the previous observation that MHC-I is bivalently modified, I leveraged this characteristic as a readout and conducted whole genome CRISPR/Cas9 screens to pinpoint key regulators involved. I uncover specific roles of the PRC2.1 and PRC1.1 sub-complexes in maintaining silencing of bivalent gene expression. Unexpectedly, I make the intriguing discovery that genetic depletion or pharmacological inhibition of Menin, traditionally known as a co-activator and a component of the KMT2A/B H3K4me3 methyltransferase complexes, phenocopies the effects of polycomb disruption. This results in the derepression of bivalent genes in cancer and human pluripotent and embryonic stem cells, findings which challenge the existing paradigm whereby disruption of the KMT2A/B and polycomb complexes is expected to have opposing effects on bivalent gene regulation. Furthermore, my research reveals an essential role of KMT2A/B in MHC-I gene expression following Menin inhibition and, therefore, highlights the existence of Menin-independent and Menin-dependent functions of KMT2A/B. Finally, I demonstrate that targeting the Menin-KMT2A interaction leads to the release and redistribution of KMT2A from active genes to bivalent genes, which creates a permissive chromatin environment that facilitates gene activation. My research has uncovered previously unknown roles for specific components of the KMT2A/B and polycomb complexes in regulating bivalency. Moreover, these findings have significant implications for cancer therapy. By identifying strategies to overcome transcriptional MHC-I repression, my work provides a compelling rationale for utilising inhibitors targeting PRC2 and Menin in the treatment of difficult-to-treat malignancies. Additionally, these insights offer potential avenues for developing novel therapeutic approaches to effectively treat cancers characterised by low MHC-I expression.
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ItemDevelopment and Implementation of Robotic Colorectal Cancer Surgery( 2023-03)Robotic colorectal surgery is increasingly being used around the world, but its status in Australia has not been explored. Whilst retrospective studies and a few trials have demonstrated its feasibility and safety, there is limited research on the potential advantages of using a robotic platform for complex oncologic procedures, where the penetration of minimally invasive surgery remains anecdotal. In addition, current evidence on the costs of robotic colorectal surgery compared to conventional laparoscopic surgery is limited and potentially outdated. This thesis addresses these gaps by examining the adoption of robotic colorectal surgery in Australia, revealing a dramatic increase in its use, particularly in the private sector. It also confirms that the implementation of robotic surgery for complex cancer work, such as complete mesocolic excision for right-sided colon cancer and beyond total mesorectal excision surgery for advanced or recurrent pelvic malignancies, is feasible. This expands our limited understanding of how minimally invasive techniques can be applied to navigate complex oncological scenarios, providing valuable insights into the technical aspects involved. The thesis also sheds light on the costs associated with robotic colorectal surgery compared to a conventional laparoscopic approach. Whilst it highlights the increased total costs, it acknowledges the limitations of the current data in this evolving field. Ultimately, this work provides baseline data to inform future economic evaluations, which will be required to support the wider adoption of robotic colorectal surgery in the public sector.
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ItemNo Preview AvailableTracking Disease In Breast Cancer Using Circulating Tumour DNA( 2023-03)Plasma circulating tumour DNA (ctDNA) correlates with disease status in breast cancer and can provide more comprehensive genomic information than the heterogeneous nature of a single biopsy site. In metastatic breast cancer it allows the study of underlying cancer driving mutations, characterization of treatment resistance and can potentially guide future treatment adaptation and selection to improve patient survival. In early breast cancer, it has also been explored as a potential minimal residual disease (MRD) biomarker with the promise that it can significantly predict disease relapse and aid in prognostic stratification. This work has utilised ctDNA as a minimally invasive strategy to study molecular information in patients undergoing novel combination and targeted therapies in breast cancer. Using different sequencing technologies, this work has shown the feasibility and ability of ctDNA to predict and profile the molecular subtypes of breast cancer that would respond to different treatments. Additionally, serial analysis of ctDNA has been able to accurately monitor disease during therapy. When breast cancer patients progress on treatment, the molecular changes captured by ctDNA were studied to characterize genomic resistance mechanisms associated with the applied therapies, providing insights into future strategies to circumvent these changes. Nevertheless, the reliance on genetic information alone has limited the sensitivity and specificity of ctDNA as a prognostic biomarker in cancer of low volume disease. This thesis has therefore explored new methodologies to interrogate the epigenetic profile of ctDNA in breast cancer. When coupled with ctDNA genetic information, a breast cancer ctDNA classifier was derived and tested for its sensitivity and specificity for disease detection in patients with oligometastatic disease. As a result, this work provides a platform for future research to refine the use of combined genomic and epigenomic ctDNA test as a sensitive and specific disease monitoring strategy in high risk early and metastatic breast cancer.
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ItemNo Preview AvailableUridine 34 tRNA modification and its involvement if prostate cancer( 2023-05)Prostate cancer (PCa) is predicted to become the deadliest neoplasia in men in Australia by 2044. This poses a challenge to the research community to find novel therapeutic strategies to treat the most aggressive forms of PCa. At the cellular level, many tumour types, including PCa, show an increase in protein synthesis to sustain their high metabolic demand and growth rates. Transfer RNAs (tRNAs) are short, heavily modified RNA molecules that play a central role in protein synthesis, decoding the messenger RNA (mRNA) through direct base pairing between the tRNA anticodons and the mRNA codons. A subset of tRNAs with a uridine in position 34 (U34) in the anticodon needs to be modified to mcm5s2U34 to base pair efficiently with the corresponding A-ending mRNA codons. Synthesis of mcm5s2U34 is a multistep process in which the Elongator complex (ELP1-6), ALKBH8 and CTU1/2 act in a sequential manner. Silencing of the Elongator complex has been reported to diminish the translational rates of mRNAs enriched in A-ending codons in different cancer types, including melanoma, intestine tumour and breast cancer. Our group has previously shown that ELP3, the catalytic subunit of the Elongator complex, is over- expressed in PCa. In this thesis we present compelling evidence of the relevance of the mcm5s2U34 tRNA modification pathway, highlighting the effect on cellular metabolism and protein synthesis. We studied the role of EPL3 in the PCa cell lines DU 145, LNCaP, BM67 and untransformed prostate epithelial cell line PNT1A. In PCa cell lines, lack of ELP3 strongly reduces the proliferative rate, the reactive oxygen species (ROS) detoxifying potential and induces metabolic rearrangement. On the contrary, in the benign cell line PNT1A, ELP3 depletion does not induce the same effects. We investigated the role of ELP3 by doing protein mass-spectrometry in DU 145 ELP3-depleted cell lines, which revealed impairment of many cellular pathways, such as DNA replication and repair, tRNA and RNA metabolism, metabolic pathways and protein synthesis. Also in DU 145, polysome profiling experiments showed a striking downregulation of the global level of translation upon ELP3 depletion. To ascertain the role of ELP3 in the translation of A-ending codons, we developed two reporter gene assays, one based on flow cytometry, the other one based on confocal microscopy. Both revealed that upon ELP3 depletion the reporter gene enriched in A-ending codons is translated 50% less efficiently than the reporter gene enriched in synonymous G-ending codons. Furthermore, the bioinformatic analysis of the codon usage in the proteomic datasets, clearly shows that proteins enriched in A-ending codons are downregulated in ELP3 KO cell lines. Altogether, our results suggest that the lack of U34 modification greatly reduces the translation of mRNAs enriched in A-ending codons, thereby impacting protein synthesis in a codon-specific manner. In turn, this results in the adaptation of PCa cells to the lack of mcm5s2U34 tRNA modification, leading to global downregulation of protein synthesis, mediated by impairment of key signalling pathways. Furthermore, our data points out that inhibition of the mcm5s2U34 tRNA modification pathway seems to affect only neoplastic cells, making it an interesting pathway to be explored in the design of novel therapeutic strategies.
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ItemCharacterising the molecular heterogeneity of neuroendocrine prostate cancer( 2023-04)Prostate cancer is primarily made up of androgen receptor-driven adenocarcinoma, but some patients may progress to a more aggressive and lethal neuroendocrine prostate cancer (NEPC) phenotype lacking AR expression. NEPC is highly heterogeneous, posing a challenge in detection and treatment. While mutations such as RB1, PTEN, and TP53 have been identified as genomic alterations in NEPC progression, they do not fully explain the heterogeneity and varied response to treatment. Understanding the molecular drivers behind the heterogeneity and therapy resistance of NEPC is vital in improving detection and treatment. Therefore, the main goal of the thesis is to characterise the transcriptional heterogeneity of tumour cells of NEPCs and the cells of the tumour microenvironment (TME), to understand the biology of these tumours and determine whether there are any consistent therapeutic targets. I hypothesise that by characterising the different levels of tumour heterogeneity in NEPCs, new insights into how to treat these tumours can be detected. To do so, I focus on three main questions 1) What is the level of intra-tumour heterogeneity of NEPC pathologies? 2) Are there common and/or distinct transcriptional profiles among NEPC pathologies? 3) How does the tumour microenvironment (TME) vary across different pathologies in NEPC? To extensively analyse the transcriptional heterogeneity of NEPC, single-cell RNA sequencing technology was used on a novel cohort of nine patient-derived xenografts (PDX) models that recapitulate the pathological and clinical heterogeneity of NEPC. I analysed scRNA-seq data from 18,632 cells from 9 patient-derived xenografts (PDX) models of NEPC to detect transcriptional heterogeneity between sub-populations. PDXs are a powerful tool to analyse the heterogeneity of NEPC due to their ability to retain the genomic and phenotypic features of the original tumour in vivo. The opportunity to study rare types of NEPC using PDX models is particularly valuable, as it allows for a more comprehensive understanding of the disease. Additionally, the feasibility of using PDX models for NEPC research is enhanced by the availability of fresh tissue with more viable cells for single-cell experiments. This makes PDX models an attractive option for studying the disease's biology and potential treatment options in a more comprehensive and detailed way. The thesis is divided into three distinct studies. The first study comprehensively characterises the degree of intra-tumour heterogeneity in each NEPC sample. This is achieved through a rigorous quality control process and standardisation of a single-cell RNA sequencing pipeline, which accurately assesses the levels of heterogeneity. Analysis revealed the presence of 3-8 subpopulations in each sample. The degree of heterogeneity changed depending on the pathology; small cell NEPC showed more heterogeneity than any other pathology. One key finding is that at least one chemo-resistant cluster was detected in each pathology, and those clusters showed a unique transcriptional profile. These findings suggest that the intra-transcriptional heterogeneity of NEPC could be a critical factor in identifying potential therapeutic targets. The second section of the thesis aims to examine the inter-patient heterogeneity of NEPC. The analysis of NEPC cells revealed that certain pathologies exhibit a similar transcriptional profile, while others show a closer resemblance to adenocarcinoma. Although these pathologies share the expression of neuroendocrine markers, there is evident heterogeneity in the underlying biology driving each phenotype. Both small and large cell neuroendocrine pathologies exhibit enriched LEF1, YAP, Notch and NMYC signalling pathways and hallmarks of aggressiveness, which are associated with poor prognosis for patients. Notably, the NED cell populations revealed a distinct profile of enriched markers and pathways, such as TNFA via NFKB and KRAS signalling. Unexpected cell co-expression of both neuroendocrine and adenocarcinoma markers was observed in NED pathologies. The co-expression analysis has uncovered this unexpected cell plasticity in NEDs, exposing that they retain adenocarcinoma features and exhibit a transcriptional profile similar to adenocarcinoma. The data indicate the presence of two molecular profiles of NEPC; it is important to consider these molecular profiles in developing personalised therapeutic strategies, as they can potentially improve patient outcomes and response to treatment. The third section of the thesis is dedicated to the investigation of the TME of NEPC. The study focused on murine non-cancerous cells extracted from tumours collected from PDXs. The analysis revealed the presence of heterogeneous subpopulations of fibroblasts, including the novel antigen-presenting CAF, as well as the inflammatory and myofibroblasts CAF populations. Based on their gene expression profile, these cells may represent a dynamic state of cancer-associated fibroblasts (CAFs), whose role is likely to be influenced by cell-to-cell interactions within the TME. Notably, a significantly increased proportion of myofibroblasts (myCAFs) and inflammatory CAFs (iCAFs) were observed in the small and large cell pathologies. This suggests that the TME may play a crucial role in the progression and aggressiveness of NEPC and highlights the importance of considering the TME when developing therapeutic strategies for these patients. The detection of transcriptional sub-populations in NEPC has provided insights into the underlying molecular mechanisms that make finding effective treatments for these tumours so challenging. These sub-populations exhibit unique gene expression profiles, resulting in a high degree of heterogeneity within NEPC. This heterogeneity is likely a key contributor to the failure of standard prostate cancer treatments in NEPC, as the different sub-populations may respond differently to various therapies. Furthermore, these sub-populations may play a role in the resistance to existing treatments, making them potential targets for the development of new therapeutics. Overall, the characterisation of the transcriptional heterogeneity of NEPC represents a significant step towards developing more personalised and effective treatments for this aggressive cancer.
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ItemDiscovering the molecular basis of CRISPR3-Cas13b for precise silencing of tumour transcripts( 2023-03)Cancers are caused by the accumulation of genomic mutations and gene expression dysregulations. In many patients, standard therapies such as chemotherapy often result in severe toxicity due to off-target effects that takes place in non-tumour tissues. Additionally, certain genetic aberrations confer resistance to these standard therapies and mediate cancer relapse. Recently, advanced sequencing technologies led to a better understanding of tumour genomics, which opened up a new era of precision and personalized medicine. These insights from tumour genomics enabled the identification of several hundreds of aberrant oncogenes called tumour drivers that confer clonal expansions and malignancy, which are considered high-priority targets of precision oncology. Currently, there are two main precision medicine approaches to target tumour drivers. The first is adoptive cell transfer involving in vitro engineering patients’ immune cells to recognize tumour antigens and lyse tumour cells, but this approach is restricted to tumour antigens on the cell. The second is the use of small inhibitory molecules and antibodies to target tumour drivers at a protein level. However, the drug development process using conventional pipelines is time-consuming, limiting the number of cancer drugs that are available to target newly identified tumour drivers in a personalized manner. These limitations highlight the need to develop a new design-flexible precision medicine approach to target tumour drivers in a personalized manner. The recent breakthroughs in the field of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) offer a promising opportunity to precisely target tumour driver genes. Theoretically, the most extensively studied CRISPR-Cas9 can cleave any oncogene and induces its loss of function. Undoubtedly, it has been a powerful tool to study cancer functional genomics in basic research with immense potentials as modular cancer therapeutics. Nevertheless, safety concerns due to off-target effects and irreversible chromosomal instabilities largely constrain the therapeutic applications of classical CRISPR-Cas9. On the other hand, the discovery of a novel RNA-guided RNA targeting CRISPR-Cas13 may address some of the limitations of Cas9 to target tumour drivers at the RNA level. In the introduction of this thesis (Chapter 1), I discuss the molecular basis of various CRISPR effectors with special focus on RNA-targeting enzymes. I also highlight how recent scientific discoveries in CRISPR filed translate into various tumour precision medicine applications. Finally, I chart progress in the CRISPR-Cas13 field and discuss its advantages and limitations as a programmable tool to target tumour drivers in a personalized manner. In my Ph.D. research project, we hypothesized that the CRISPR-PspCas13b nucleoprotein complex may be re-engineered to silence tumour driver transcripts with single-nucleotide accuracy. However, the poor understanding of the molecular principles governing PspCas13b target recognition and cleavage limits its utility and development as programmable cancer therapeutics. Thus, this research focuses on revealing the molecular mechanisms of PspCas13b in order to reprogram this ribonuclease to target tumour transcripts that are currently ‘undruggable’. Excitingly, our Single-Base Tiled crRNA screens (SiBTil), unbiased computational analysis, and comprehensive spacer-target mutagenesis revealed key determinants of PspCas13b activity. De novo design of crRNAs harbouring base-paired or mismatched guanosine bases at key spacer positions greatly enhances the silencing efficacy of otherwise inefficient crRNAs, expanding the targeting spectrum of this enzyme. We also reveal the interface between mismatch tolerance and intolerance, which unlocks an unexpected single-base precision targeting capability of this RNA nuclease. Notably, our de novo design principles enable potent and selective silencing of various gene fusion transcripts and their downstream oncogenic networks, without off-targeting of non-translocated variants that share extensive sequence homology. We demonstrate that PspCas13b targeting the breakpoint of fusion transcripts enables efficient suppression of ancestral and single-nucleotide mutants (e.g., BCR-ABL1 T315I) that often drive clinical cancer relapse. Our transcriptomic and proteomic analyses suggest PspCas13b is highly specific and has no global off-targeting or collateral activity against endogenous transcripts and proteins. Collectively, my Ph.D. research provides new design principles for PspCas13b programming to specifically recognise and degrade any ‘undruggable’ fusion oncogenic transcript, thus providing a new conceptual framework for personalized oncology.
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ItemIdentifying heterogenous mechanisms of drug resistance in high grade serous ovarian cancer( 2023-05)Ovarian, fallopian tube and primary peritoneal cancer is the sixth most common cause of cancer death for women in Australia. A diagnosis of ovarian cancer is life-altering with a five-year survival of only 45.7%. While clinical factors such as disease stage are important for prognosis and response to therapy, the underlying genomic, transcriptomic and immune characteristics of the tumour are also key determinants of outcome. High grade serous ovarian cancer (HGSC) is the most common epithelial ovarian cancer subtype, and is characterised by frequent germline, somatic and epigenetic aberrations which result in homologous recombination DNA repair (HR) deficiency. Aneuploidy and frequent somatic copy number aberrations (SCNA) are common features of the genomic landscape of HGSC. Despite often initially responding to therapy, acquired resistance is extremely common, arising through a variety of genomic, transcriptomic, adaptive and microenvironmental mechanisms. Owing to the scarcity of biopsies or surgery in relapsed disease, the frequency and distribution of these acquired resistance mechanisms is only partially known. In particular, there is a paucity of information about resistance mechanisms in late-stage disease following the multiple lines of therapy that a typical patient with HGSC receives. This thesis addressed these important gaps in knowledge by examining resistance mechanisms in a cohort of women with HR deficient HGSC who underwent a research autopsy, providing unique insight into the architecture of end stage disease. Importantly, resistance mechanisms were frequently subclonal, often with more than one mechanism detected within a single metastatic site. Evidence of convergent evolution was also observed, including of reversion mutations and BRCA1-specific mechanisms of HR restoration. An increased frequency of whole genome duplication, an important event in cancer evolution, was noted in the end-stage samples compared to what is known in primary tumours. Following this observation, the transcriptomic differences between tumours with and without WGD were assessed. Examination of the transcriptomic differences in whole genome duplication revealed downregulation of CIITA and other MHC-II genes, highlighting a potential means of immune escape and a potential therapeutic vulnerability. This thesis also examined mechanisms of resistance specifically occurring following receipt of poly-ADP ribose polymerase (PARP) inhibitor therapy, and the natural history and evolution of reversion mutations in HR deficient HGSC, showing that they are in fact not frequently present outside of a progressing or resistant cohort. The findings of this thesis add to the current knowledge of resistance to therapy, demonstrate the need for accessible genomic testing in the clinical management of HGSC and indicate that further exploration of resistance should focus on transcriptomics, epigenetics, and the tumour microenvironment.
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ItemElucidating epigenetic mechanisms of cancer immune evasion( 2023-03)Cancer immunotherapies have revolutionised the management of a wide range of haematological and solid organ malignancies, due to their potential to induce durable remissions in a proportion of responders. However, primary or acquired resistance remains problematic for the majority of patients, and typically arises from tumour-intrinsic properties that reduce immunogenicity or extrinsic factors promoting an immunosuppressive tumour microenvironment. Effective tumour antigen presentation via major histocompatibility proteins (MHC-I and/or MHC-II) to immune effector cells is a critical component of the adaptive anti-cancer response and genetic disruption of the MHC-I and/or MHC-II antigen presentation pathways, either through inactivating mutations or transcriptional silencing, is a well-recognised cause of resistance to both pharmacological and cellular immunotherapies. In this thesis, I explore epigenetic mechanisms of MHC-I and MHC-II repression in cancer and identify an evolutionarily conserved role for Polycomb repressive complex 2 (PRC2) in MHC-I silencing. I also discover two key mechanisms of MHC-II regulation in acute myeloid leukaemia and melanoma: transcriptional repression of MHC-II pathway genes by the C-terminal binding protein (CtBP) co-repressor complex, and post-translational regulation of CIITA, the master regulator of MHC-II expression, by the FBXO11-containing E3 ubiquitin ligase complex. Targeting of these repressive pathways efficiently upregulates cell surface MHC expression and augments in vitro and in vivo adaptive immune responses. These findings provide valuable biological insights into mechanisms of cancer immune evasion and establish the scientific rationale for further pre-clinical and translational studies of these novel therapeutic strategies to overcome immunotherapy resistance via restoration of tumour antigen presentation.