Sir Peter MacCallum Department of Oncology - Theses
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ItemCRISPR/Cas9 Engineering of Next Generation Armoured CAR T Cells( 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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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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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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ItemThe role of USP9X in Low-Grade Serous Ovarian Cancer( 2023)Low-grade serous ovarian carcinoma (LGSOC) is a rare histotype of epithelial ovarian cancer (EOC), and accounts for approximately 3-5% of diagnosed EOC cases. LGSOC is characterised by wildtype TP53 expression, frequent aberrance in the RAS/RAF signalling pathway, and relative genomic stability which in part explains LGSOC resistance to current standard-of-care platinum-based chemotherapeutics. Current chemotherapy strategies for LGSOC have predominantly been driven by that of the far more common high-grade serous subtype. Optimal cytoreductive surgery is challenging, given that the majority of LGSOC diagnoses are at a late stage where the cancer has metastasised from the primary site. Recently, alternative therapy strategies including targeted therapy of the RAS/RAF pathway have shown efficacy against tumours, but further characterisation into potential novel drivers of this disease is required to expand the treatment repertoire for patients suffering from this disease. Previous sequencing studies elucidated USP9X as one such potential driver of LGSOC. USP9X is a deubiquitinase involved in protein turnover. The gene has been implicated as both oncogenic and tumour-suppressive depending on the cancer type being investigated. In the context of LGSOC, little is known as to the role that this gene has in disease development. This thesis evaluated 121 LGSOC cases, 71 sequenced via targeted sequencing, 49 through whole exome sequencing, and 1 by whole genome sequencing. Sequencing results identified USP9X mutations at a frequency of 14%, and as the most frequently mutated non-RAS/RAF gene in the assessed cohort. Interrogation into the allelic status of these mutations revealed more than half of the mutations were inactivating, suggesting a tumour-suppressive function; USP9X was elucidated to follow a classical two-hit tumour suppressor model. Gene knockdown and knockout experiments on LGSOC cell lines highlighted a potential perturbance to clonogenic survival, but not to migration and proliferation. Mass spectrometry analysis on USP9Xnull LGSOC cell lines identified the molecular chaperone BAG3 as a likely direct substrate of USP9X, and the deubiquitinase as a potential regulator of the mTORC signalling pathway. Assessment of the global proteomic perturbations as a result of USP9X downregulation suggested the downstream consequences of USP9X suppression are likely to be decreased cell adhesion, and potentially increased cell migration and invasion.
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ItemNo Preview AvailableDeveloping novel methods of infection surveillance in haematology-oncology patients and implications for health policy( 2021)Healthcare-associated and opportunistic infections are a leading cause of mortality, morbidity, and increased healthcare costs in haematology-oncology patients. Surveillance is recognised as the cornerstone of infection prevention to guide clinical decision making and to monitor quality improvement. The utility of current case ascertainment methods is poorly delineated in patients with underlying malignancy. Administrative data comprise a standardised ontology to classify disease with the potential to support large scale infection surveillance programmes; however, it is unclear if administrative data can support surveillance activities in high-risk settings. The overall aims of this thesis were to: (i) present and argue the case for novel case ascertainment methods; (ii) develop and apply a methodology using administrative data to identify infection in haematology-oncology patients, and determine the classification performance and healthcare funding implications of these data in line with current health policy; and (iii) establish and evaluate a hospital-wide linked dataset integrating multiple data sources, together with administrative data, to achieve maximal performance for automated surveillance in patients with haematological malignancies. Methods include a systematic review to describe the scope of existing surveillance methods among haematology-oncology units, analysis of continuous statewide surveillance datasets and hospital-level administrative data extracts, performance evaluation of administrative data to classify infection and simulation of pay-for-performance funding methodology in a cancer casemix, and development of a hospital-wide linked dataset to identify discrete data combination yielding highest performance for automated infection surveillance. The thesis findings demonstrate significant heterogeneity in existing infection monitoring methods among haematology-oncology patients. Estimates of the burden of disease in a predefined haematology-oncology population relative to a statewide cohort were determined, together with longitudinal trends in incidence over time. Administrative data show to be a feasible alternative to current surveillance data to enable standardised comparison of intra- and interhospital infection epidemiology in patients with underlying malignancy, however, at the expense of poor-to-moderate classification performance associated with significant shortfalls in hospital remuneration. Linkage of administrative data with microbiology, histopathology, and antimicrobial-dispensing data according to specific data combinations demonstrated improvements in classification performance for discrete opportunistic and healthcare-associated infections in patients with haematological malignancy. It was demonstrated that although administrative data enable standardised comparison of infection epidemiology, these data are an unreliable proxy for infection surveillance in Australian haematology-oncology units. Refinements to current pay-for-performance funding specifications are necessary before administrative data can reliably be used as quality improvement measures in a cancer casemix. This thesis posits data linkage as an efficient means to optimise the utility of administrative data, together with hospital-level datasets, to support an automated surveillance strategy in haematology-oncology patients. Future research agenda are outlined regarding the evaluation of electronic medical record data and other codified nomenclature to support electronic surveillance and quality improvement monitoring.
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ItemNovel approaches to harness the anti-tumour activity of natural killer cells( 2022)Despite revolutionary advances in cancer treatment with immunotherapy, durable clinical benefit is limited to a subset of patients. Current forms of immunotherapy, including checkpoint inhibition and chimeric antigen receptor T cells, are primarily restricted to targeting cytotoxic CD8+ T cells of the adaptive immune system. Natural killer (NK) cells are the cytotoxic effector cells of the innate immune response and are emerging as a promising and alternative target for cancer immunotherapy, however, comprehensive functional genetic studies examining anti-tumour NK cell activity are limited. Using genome-scale clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein 9 screening technology, we sought to comprehensively identify and validate tumour genes that potently influence sensitivity to primary NK cells. We first demonstrate that B16-F10 mouse melanoma cells lacking genes encoding proteins involved in interferon-gamma (IFN-gamma) signaling and antigen processing/presentation are highly sensitive to killing by NK cells, a process dependent on the absence of major histocompatibility complex class I expression. As checkpoint inhibition-resistant melanoma patients present with loss-of-function mutations within these pathways, our findings highlight intratumoural NK cells as a potent strategy to limit and/or overcome resistance to CD8+ T cell attack during conventional checkpoint inhibition immunotherapy. We additionally identify Rnf31 as a novel negative regulator of tumour cell sensitivity to NK cell killing. Rnf31 encodes for HOIP, which has an established role in driving tumour necrosis factor (TNF)-mediated gene induction and inhibition of TNF-induced cell death in certain cell types. HOIP-deficient melanoma cells not only exhibited increased sensitivity to NK cells, but also to antigen-specific CD8+ T cells. We surprisingly demonstrate that HOIP protects tumour cells from apoptosis induced by combined IFN-gamma and TNF, rather than TNF alone. We provide valuable mechanistic insight into the transcription-dependent form of apoptosis induced by combined IFN-gamma and TNF limited by HOIP, pharmacological validation using a HOIP inhibitor, and in vivo validation using HOIP-deficient B16-F10 ovalbumin-expressing tumours that exhibit enhanced CD8+ T cell-mediated control. Inhibition of tumour HOIP activity may therefore enhance NK and CD8+ T cell anti-tumour responses through unlocking the apoptotic potential of combined IFN-gamma and TNF secreted by these immune cells, representing a potential new combinatorial target for current and emerging immunotherapies. Collectively, we impartially identify tumour-specific genes that powerfully modulate tumour cell sensitivity to primary NK cells. We provide validation of established yet clinically relevant tumour genes associated with evasion from CD8+ T cells that mediate resistance to checkpoint inhibition immunotherapy, and additionally validate Rnf31 as a novel tumour regulator of not only sensitivity to NK cells, but also CD8+ T cells. We lastly present appropriate methodology and molecular tools that may facilitate analogous screening within NK cell lines to identify targetable regulators of tumour cell-induced IFN-gamma production by NK cells. Taken together, we advocate that NK cells may play an important role in combating resistance to checkpoint inhibition therapy in melanoma and identify HOIP as a potential combinatorial tumour target that may enhance future NK cell-based immunotherapy endeavours through secreted IFN-gamma and TNF, which may form the basis of a future immunotherapeutic strategy.
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ItemInvestigating the role of Mucosal-Associated Invariant T (MAIT) cells in cancer( 2021)The success of immunotherapy in patients has highlighted the importance of the anti-tumour role of the immune system. The function of conventional T cells within the tumour microenvironment (TME) have been intensively studied, while the role of mucosal-associated invariant T (MAIT) cells is yet to be determined. MAIT cells are abundant in humans and enriched in mucosal tissues, such as the colon and lung, and have been found within primary and metastatic tumours. Upon activation, MAIT cells exert rapid effector functions and can secrete both the anti-tumour cytokines (IFNg and TNF) and pro-tumour cytokines (IL-17 and IL-22). MAIT cells also produce granzyme B and perforin, suggesting they are capable of killing target cells. Although direct evidence of MAIT cells precise function in cancer is limited, some studies show that increased numbers of MAIT cells within tumours are correlated with a good prognosis, whilst other studies have indicated MAIT cells are associated with a poorer prognosis. These divergent results have made it difficult to interpret whether MAIT cells have an anti-tumour or pro-tumour role. Therefore, this thesis investigated the role of MAIT cells in cancer and the potential for MAIT cells to be exploited for adoptive cellular therapy. The first results chapter of this thesis explores the anti-tumour role of MAIT cells in both murine and ex vivo human models. It was observed that at steady-state, MAIT cells negatively regulate NK cell maturation and anti-tumour activity. Conversely, activating MAIT cells through either pulsing of tumour targets or intranasal administration of free MAIT cell antigen, led to robust protection against the development of lung metastases. Upon further investigation, it was discovered that activated MAIT cells enhance NK cell maturation and anti-tumour activity in an IFNg-dependent manner. This chapter proposes the existence of a MAIT-NK cell axis that can control NK cell mediated anti-tumour efficacy. The second results chapter aims to further improve the anti-tumour efficacy of activated MAIT cells, by combining this therapy with additional immunotherapies. The additional immunotherapies tested in combination with MAIT cell activation were selected on the basis of their ability to activate MAIT cells and/or NK cells. Notably, additional therapies that increased both MAIT cell and NK cell activity were most promising. This chapter also found that intravenous administration of MAIT cell antigen led to systemic expansion of MAIT cells and an increase in MAIT cells within the tumour tissue, broadening the application of activating MAIT cells in the clinic. The third results chapter aims to investigate the potential of MAIT cells in the context of Chimeric antigen receptor (CAR) T cell therapy. CAR T cell therapy is currently ineffective in solid tumours, due to the immunosuppressive TME, antigen heterogeneity, poor trafficking to solid tumours and decreased persistence. Furthermore, this therapy requires autologous generation of CAR cells in order to avoid graft versus host disease (GVHD). Excitingly, MAIT cells represent an allogeneic source of CAR cells as they are not restricted to conventional MHC. Chapter 5 demonstrates that MAIT cells are able to be efficiently transduced with CAR and upon target recognition CAR MAIT cells produce cytokines and directly kill tumour cells. Collectively, this data illustrates the potential anti-tumour activity of MAIT cells through a MAIT-NK cell axis. Furthermore, this thesis demonstrates the potential for MAIT cells to be used in adoptive cellular therapy, namely as CAR MAIT cells.
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ItemInvestigating mechanisms for Elotuzumab and Lenalidomide therapy in Multiple Myeloma( 2021)Multiple myeloma (MM) is a haematological malignancy of plasma cells with disease present in the bone marrow. Despite the success of new therapies in extending life expectancy, MM is still considered incurable and requires further investigation into new treatments. In the context of refractory/relapsed MM (RRMM), the combination of the two immunotherapies Elotuzumab (Elo, anti-SLAMF7 mAb) and Lenalidomide (Len, IMiD) has resulted in an 82% objective response rate and 16% complete remission in patients. This success led to FDA approval of Elo plus Len treatment for patients with RRMM and has warranted further investigations into the exact mechanism of action. Elo targets SLAMF7 antigen which is highly expressed on MM tumour cells and activates NK cells for antibody dependent cell-mediated cytotoxicity (ADCC) resulting in MM tumour cell death. Len is an immunomodulatory agent commonly used to treat a wide range of haematological cancers. Len has been shown to activate various cells of the immune system as well as directly suppress tumour progression. This thesis aims to identify how the mechanisms of Len intersect with, and enhance, Elo activated NK cell control of tumours. The work from this thesis revealed that to induce ADCC, Elo required both expression of SLAMF7 on MM target cells and NK cell CD16 engagement. Elo-activated NK cells had increased expression of CD107a and CD69, as well as loss of CD16 expression which was a result of ADAM17 induced cleavage. Elo activation of NK cells also secreted increased levels of cytokines and chemokines associated with recruitment of effector immune cells. In both in-vitro and in-vivo studies, Len significantly enhanced Elo-induced healthy donor NK cell killing of MM tumour cells. In contrast, only 4/12 RRMM patient (responders) NK cells induced increased MM cell killing in the context of Elo plus Len. Important for this increased cytotoxicity was the expression of CD54 on NK cells that allowed differentiation between these responder and non-responder patients. CD54 was also more highly expressed on NK cells activated by Elo plus Len compared to Elo activation alone. However, further phenotypic studies and RNA sequencing did not reveal any further mechanisms in NK cells by which Len enhances Elo activation. Further studies revealed that MM tumour (myeloma) cells specifically upregulated CD54 and CD11a expression in response to Elo plus Len activation of NK cells and this did not occur with Elo treatment alone. This increase in CD54/CD11a expression on myeloma cells was also dependent on the presence of CD14+ monocytes. RNA sequencing also revealed enrichment of cytokine and chemokine signalling/secretion pathways in MM cells specifically in the context of Elo and Len activated NK cells and monocytes. In conclusion, these data indicate that a complex network of direct and indirect mechanisms between NK cells, monocytes and myeloma cells contribute to the Elo plus Len treatment induced response in RRMM patients. Elo alone activates NK cells and monocytes, whereas the Len effect is largely on myeloma cells. Collectively the two treatments led to robust myeloma antibody dependent cellular cytotoxicity (ADCC). There was a strong correlation between increased myeloma cell CD54/CD11a expression and myeloma cell ADCC suggesting that stabilisation of the immune synapse with strong adhesion may be a key factor for this improved myeloma cell killing. Finally, this study revealed that upregulation of MM cell CD54 expression on MM cells may be a useful predictive biomarker to stratify RRMM patients for Elo plus Len therapy.
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ItemUnderstanding the role of adenosine receptor signalling in chimeric antigen receptor (CAR) T cell therapy in solid cancer( 2021)Chimeric antigen receptor (CAR) T cell therapies have been highly effective and clinically approved for treating haematological malignancies, however trials in solid cancers have shown limited efficacy, likely due in part to the increased complexity of the immunosuppressive tumour microenvironment (TME) in solid cancers. CAR T cells are inhibited by immunosuppressive proteins, cytokines or physical barriers deployed by the tumour to evade and avoid destruction by anti-tumour immunity. One such process involves the accumulation of extracellular adenosine (eADO), in the TME which has potent immunosuppressive effects on T cells and other immune cells. eADO has four known G protein coupled receptors, the A1R, A2AR, A2BR and A3R, of which the A2AR is primarily responsible for suppressing T cell function. Our previous studies highlighted a major impediment to pharmacological blockade of the A2AR which was predicted to be hindered by poor solubility and suboptimal in vivo pharmacokinetic profile [1]. This became apparent when comparing the effectiveness with genetic deletion of A2AR in CAR T cells to pharmacological blockade, in which the CAR T cells generated from A2AR-/- mice elicited comparatively greater efficacy in vivo when combined with anti-PD-1 blockade [1, 2]. This thesis therefore investigated multiple gene editing strategies to modulate adenosine receptor signalling, firstly by overexpressing the alternative signalling A1R or A3R in human or mouse CAR T cells. A1R or A3R have been shown to act by the opposing downstream signalling pathway to A2AR, and thus it is hypothesised that A1R or A3R overexpression can reverse suppression and supercharge CAR T cells in the presence of eADO. Interestingly, A1R or A3R overexpression did not confer protection to suppression by eADO in both mouse and human models, but A1R expression instead enhanced effector and terminal differentiation, activation, and baseline cytokine production of CAR T cells. This however translated to higher expression of exhaustion markers, loss of memory associated gene expression and reduced stem-like memory fraction in the CAR T cell product, ultimately leading to reduced persistence in vivo, and limiting the therapeutic efficacy of this approach. Alternatively, a previous publication from our lab briefly examined short-hairpin RNA (shRNA) mediated silencing of A2AR expression [1]. While shRNA-mediated silencing of the A2AR was able to partially reverse suppression by eADO, much like A1R expression, it also led to effector differentiation, activation, and increased baseline cytokine production. Importantly, while shRNA-mediated silencing of the A2AR also resulted in reduced persistence in vivo, it was able to mediate modest anti-tumour efficacy leading to reduced tumour growth and increased mouse survival. Both overexpression and knockdown approaches are limited by sub-optimal persistence in vivo which limited their overall therapeutic efficacies. Yet these results contradicted our prior observations of CAR T cells derived from A2AR-/- mice and from studies in the Lymphocytic choriomeningitis virus (LCMV) setting, whereby A2AR deletion was linked to increased T cell numbers [1, 3]. Therefore, the final gene-editing approach examined in this thesis utilised CRISPR/Cas9 protocols to achieve full deletion of the A2AR in CAR T cells. CRISPR/Cas9 methodologies are currently being used in clinical trials and therefore deleting the A2AR in CAR T cells using this approach is highly novel and clinically translatable. To reasons unknown, CRISPR/Cas9 mediated deletion of A2AR had minimal effects on CAR T cell memory phenotypes and no adverse effects on engraftment or persistence in vivo. Furthermore, CRISPR/Cas9-mediated deletion of A2AR in CAR T cells led to enhanced therapeutic efficacy in both mouse and human models, thus representing a potent approach to targeting the A2AR. In conclusion, future studies comparing full A2AR deletion to A2AR silencing/ pharmacological blockade or A1R overexpression may be of interest to fully elucidate the mechanisms of adenosine receptor signalling on T cell persistence and memory.