Sir Peter MacCallum Department of Oncology - Theses

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    The role of lysine acetyl transferases and epigenetic regulators in T-cell mediated anti-tumour immune-responses
    Pijpers, Lizzy Maria Gertruda ( 2023-09)
    Adoptive cell therapy using chimeric antigen receptor (CAR) T cells has demonstrated remarkable success in treating haematological malignancies, resulting in the FDA approval of six CAR T cell products for the treatment of B cell Acute Lymphoblastic Leukaemia, multiple myeloma and non-Hodgkin lymphoma. However, CAR T cells have shown limited efficacy against solid tumours in the clinic, which is primarily attributed to the immunosuppressive nature of the tumour microenvironment and antigen heterogeneity of solid tumours. Hence, strategies to overcome these challenges are necessary for CAR T cells to be effective in solid malignancies. Improved clinical outcomes have been associated with CAR T cells exhibiting memory T cell characteristics. While the transcriptional control of memory differentiation is extensively studied, our understanding of the epigenetic mechanisms underlying these gene expression changes have yet to be fully understood. In this thesis, primary human and mouse (CAR) T cells were utilized to study the epigenetic landscape of CD8+ T cell activation and differentiation. A485, a novel histone acetyltransferase inhibitor of P300 and CBP was discovered in a small molecule compound screen to regulate memory T cell differentiation. Treatment of T cells with A485 resulted in memory phenotype differentiation with the corresponding transcriptional changes: upregulation of T cell memory associated genes and downregulation of T cell effector associated genes. In vivo data across various mice tumour models, collectively demonstrated the potent anti-tumour effect of pre-treating (CAR) T cells with A485 prior to adoptive transfer. Given that the P300 and CBP histone acetyltransferase inhibitor A485 was found to regulate memory T cell differentiation, the role of histone acetylation in T cell activation and differentiation was investigated. Histone acetylation was found to be rapidly enhanced upon T cell activation. In addition, P300 and CBP mediated histone acetylation was found to be critical for memory T cell differentiation in the time prior to first T cell division at 24 to 48 hours post activation. H3K27ac ChIP data suggest that P300 and CBP might preferentially acetylate effector-associated super enhancers at 2 hours post T cell activation, thus leading to memory T cell differentiation upon A485 inhibition. Although, P300 and CBP are often described as an entity, this thesis, proposes potential independent roles, with CBP involved in regulating memory T cell differentiation. Overall, the results presented in this thesis improve our understanding of the role of histone acetylation in T cell activation and differentiation.
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    Metabolic crosstalk in the tumour microenvironment
    Vaidyanathan, Srimayee ( 2023-08)
    Metabolic reprogramming is a hallmark of cancer that underpins several tumourigenic processes, including uncontrolled proliferation and therapy resistance. There is growing evidence that non-cancer cell types within the tumour microenvironment (TME) may also undergo metabolic reprogramming, thereby supporting cellular processes that facilitate tumour growth. Cancer-associated fibroblasts (CAFs) are a class of non-cancer cell that are activated in response to diverse cues within the tumour niche. Notably, a subclass of transforming growth factor beta (TGFb)-induced CAFs known as myofibroblast-like CAFs (myoCAFs) play a critical role in producing and remodelling collagenous extracellular matrix (ECM). The resultant dysregulation of ECM architecture is a key feature of solid tumours that promotes pro- oncogenic signalling and metastasis. Although ECM production is known to be a metabolically demanding process, the specific metabolic pathways that underpin myoCAF function are poorly understood. In this thesis, we have used an in vitro myoCAF model to interrogate the metabolic dependencies associated with CAF ECM-remodelling function. We determined that CAFs exhibit upregulated cholesterol biosynthesis relative to normal fibroblasts (NFs), and that this supports augmented endoplasmic reticulum (ER) mass and secretory function. ER function was also underpinned by increased activity of the unfolded protein response (UPR) effector, X-box binding protein 1 (XBP1). Importantly, loss of XBP1 and inhibition of cholesterol biosynthesis both resulted in a loss of CAF ECM-remodelling function, suggesting that cholesterol- dependent regulation of the ER is a targetable metabolic vulnerability in CAFs. CAFs arise within a metabolically challenging environment where many nutrients are limiting. T o examine the effect of nutrient restriction on CAF function, we cultured cells in physiologically-relevant Plasmax media depleted of methionine. We determined that CAFs are more tolerant of methionine deprivation than NFs. Moreover, it was found that CAFs exhibit increased deposition of the ECM component fibrobnectin upon methionine restriction. We demonstrated that CAFs are uniquely dependent on activating transcription factor 4 (ATF4); a key effector of the integrated stress response (ISR) that mediates the response to amino acid deprivation; despite not expressing it at high levels. In addition, we determined that ATF4 is more likely to occupy accessible, promoter-associated regions in CAFs compared with NFs. Finally, we found that ATF4 directly regulates expression of Niban1, which has been shown to promote cellular adaptation to ER stress. Together, these findings highlight the differential roles of ATF4-mediated adaptive metabolic signalling in NFs and CAFs, suggesting that ATF4 is necessary for CAF activation and function. Tumour-associated ECM is produced not just by myoCAFs, but also by malignant cells themselves. We thus interrogated the role of the oncogenic transcriptional coactivator and master metabolic regulator Yes-associated protein (YAP) in ECM production by transformed breast epithelial cells. It was found that YAP transcriptionally regulates ECM-associated pathways through both TEA domain transcription factor (TEAD)-dependent and TEAD- independent mechanisms. Importantly , we found that Y AP regulates glycosaminoglycan (GAG) biosynthesis in an evolutionarily conserved manner. Overall, in this thesis we have identified multiple mechanisms by which myoCAFs co-opt metabolic pathways in order to sustain ECM-remodelling function. In addition, we have identified a novel mechanism by which YAP-hyperactive malignant cells may modify tumour- associated ECM. Thus, this work has identified key metabolic and transcriptional regulators of ECM remodelling, thus advancing our understanding of the cellular pathways that drive tumour progression at the microenvironmental level.
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    CRISPR/Cas9 Engineering of Next Generation Armoured CAR T Cells
    Chen, Amanda Xi Ying ( 2023-08)
    Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable successes in the treatment of certain B cell haematological malignancies; however, its efficacy in the solid tumour setting remains limited. This is attributed to a number of factors, including immunosuppression in the tumour microenvironment (TME), T cell exhaustion, tumour antigen heterogeneity and limited trafficking of CAR T cells into the tumour. A promising strategy to overcome these limitations is by engineering, or “armouring”, CAR T cells to express factors that have the potential to enhance therapeutic efficacy, such as proinflammatory cytokines, chemokines, transcription factors, cell surface molecules and synthetic constructs. This strategy has been assessed in the context of various forms of adoptive cellular therapy (ACT), including tumour-infiltrating lymphocyte (TIL) therapy, as well as transgenic TCR and CAR T cell therapy. Preclinical studies exploring a vast range of armouring genes have demonstrated promising results, with armoured T cell products mediating enhanced anti-tumour efficacy through the capacity to modulate the TME, engage host anti-immune responses and directly enhance T cell function. However, a key limitation of this approach is the potential toxicities caused by peripheral expression of the armouring gene by the engineered T cells, highlighted by the life-threatening side effects observed in an early clinical trial assessing this approach with IL-12-engineered TILs. While strategies have been developed to limit transgene expression to the tumour, such as the synthetic NFAT promoter system, none thus far have successfully demonstrated the ability to prevent clinical toxicities. The advent of CRISPR/Cas9 gene editing, including recent advancements in the engineering of primary T cells, has introduced unique opportunities to enhance the design of armoured T cells. In particular, CRISPR-mediated homology-directed repair (CRISPR-HDR) enables the precise modification of specified genomic sites, including the introduction of a complementary DNA (cDNA) sequence into a target gene locus. Hence, in the context of armoured T cells, CRISPR-HDR can be used to insert armouring genes into a specific endogenous site, creating the possibility to leverage endogenous gene regulatory mechanisms to drive transgene expression. By targeting the armouring gene into the site of an endogenous gene that is exclusively upregulated in intratumoural CAR T cells, this should lead to tumour-restricted expression of the armouring gene. Additionally, integration of the armouring gene can concurrently disrupt expression of the endogenous target gene. Thus, by selecting a target gene that is not only specifically expressed in CAR T cells within the tumour, but also regulates inhibitory T cell functions, this would enable knock-in of a transgene that can enhance therapeutic efficacy while simultaneously knocking out an endogenous inhibitory gene. Therefore, this study assessed the overarching hypothesis that that engineering armoured T cells in accordance with this “knock-in, knock-out” approach would lead to enhanced anti-tumour efficacy while maintaining a favourable safety profile. To explore this hypothesis, a CRISPR-HDR protocol was first developed to engineer primary murine and human transgenic TCR and CAR T cells at high editing efficiencies. This protocol was subsequently applied to generate armoured transgenic TCR and CAR T cells expressing various proinflammatory cytokines under the control of endogenous tumour-specific promoters, and assessed for therapeutic efficacy, mechanism and safety in syngeneic murine models and human xenograft models of ACT. T cells engineered to express TNF, IFN-gamma, IL-2 and IL-12 in a tumour-restricted manner mediated significantly enhanced anti-tumour efficacy; however, their therapeutic effect was largely dependent on the endogenous promoter used. The PD-1 promoter was found to support potent transgene expression in the tumour, which was required for the therapeutic efficacy of TNF, IFN-gamma and IL-2. However, its low level of peripheral expression led to toxicities when used to drive IL-12 expression. Instead, the NR4A2 promoter, which supported highly tumour-restricted transgene expression, was capable of regulating IL-12 production with minimal toxicities while mediating a robust therapeutic response. Hence, these data demonstrate that this CRISPR-HDR strategy is highly customisable, enabling the use of different endogenous promoters to achieve the optimal expression pattern for specific transgenes. In summary, the current study details the development of a novel CRISPR-HDR approach for engineering next generation armoured T cells for ACT, and presents promising preclinical data demonstrating the efficacy, safety, feasibility and translational potential of this approach.
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    Personalising Lung Radiation Therapy — Using Advanced Radiation Planning to Spare Functional Lung
    Bucknell, Nicholas Wentworth ( 2023-07)
    Radiation therapy plays an essential role in the curative treatment of lung cancer. The treatment of lung cancer with thoracic radiation using both fractionated and stereotactic techniques is associated with early and late toxicities, which may impact a patient’s quality of life during and after treatment. Most toxicity is caused by excess dose to organs within close proximity to the tumour. The nondiseased lung, the heart and the oesophagus are organs that limit the dose that can be safely delivered to the tumour. Through improved treatment planning and delivery precision, significant advances have been made in improving outcomes in early-stage lung cancer. The CHISEL trial demonstrated that dose escalation using stereotactic ablative body radiation therapy (SABR) can improve local control and overall survival compared with conventional radiation therapy. SABR minimises radiation dose to surrounding organs by ensuring high conformity to the tumour and using inhomogeneous dose prescription. The impact of SABR on pulmonary function is not well characterised. Regarding advanced disease, despite improvements in treatment planning and treatment delivery, dose-escalation trials, such as RTOG 0617, have consistently demonstrated worse outcomes in the high-dose arm. These outcomes have primarily been attributed to the increased radiation dose delivered to the lung, the heart, and the oesophagus. Radiation-induced lung disease (RILD) is a spectrum of acute and late toxicity, which results in progressive pulmonary function decline. Advanced imaging and treatment using functional lung avoidance (FLA) may reduce the incidence of RILD. In this thesis, I investigate the toxicities of thoracic radiation therapy, analyse the existing literature on FLA and describe the development of an FLA planning technique. Using this technique, I developed a protocol and completed a prospective clinical trial to evaluate the feasibility of integrating the technique into the clinic. Taking the learnings from the trial, I refined the FLA planning technique and improved workflow while maintaining plan quality. Lastly, I investigated the extent of pulmonary function decline after SABR. In this thesis, I find that adapting radiation plans to functional lung is feasible. Most patients experience significant reductions in dose to functional lung when FLA plans are compared with standard plans that do not integrate the functional regions of the lung. To assist with further development of this field, new methods to image and identify functional lung are required and further assessment of FLA using newer forms of planning technology is warranted.
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    Precision Medicine in Breast and Thoracic Oncology
    De Silva, Dilanka ( 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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    Investigating the molecular characteristics of TILs and the role of tissue-resident memory CD8+ T cells in response to immune checkpoint inhibition in breast cancer
    Virassamy, Balaji ( 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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    Uncovering epigenetic regulators of bivalent MHC class I genes in cancer
    Sparbier, Christina ( 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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    Tracking Disease In Breast Cancer Using Circulating Tumour DNA
    Lo, Louisa Lisa ( 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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    Uridine 34 tRNA modification and its involvement if prostate cancer
    Timpone, Clelia ( 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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    Characterising the molecular heterogeneity of neuroendocrine prostate cancer
    Quezada-Urban, Rosalia ( 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.