Medical Biology - Theses
Permanent URI for this collection
Search Results
1 - 6 of 6
-
ItemNo Preview AvailableIdentifying vulnerabilities in treatment resistant lung adenocarcinoma( 2024-01)With close to 15,000 new diagnoses in 2022, lung cancer is the 5th most diagnosed form of cancer and the leading cause of cancer-related death in Australia. Whilst clinical advancements in cancer treatment has improved survival outcomes for patients, the heterogeneity of the disease remains a clinical challenge. Particularly, mutations in KRAS are common, and historically, KRAS-mutant lung cancer has been difficult to treat with impaired responses to classical therapeutic strategies, such as chemotherapy and radiotherapy. With approximately 16-17% of all lung cancer cases harbouring mutations in KRAS, understanding the molecular and cellular mechanisms that govern tumourigenesis and progression are key to developing new treatment strategies to improve patient survival. Recently, the development of targeted therapies and immunotherapy have improved outcomes for KRAS-mutant lung adenocarcinoma (LUAD) patients. However, the stratification of tumour suppressor genes co-mutated with KRAS has highlighted distinct patterns of therapeutic response. The three most common tumour suppressor genes found co-mutated with KRAS are TP53, KEAP1 and STK11 (also known as Lkb1). Whilst loss-of-function mutations in either of these 3 tumour suppressor genes display aggressive forms of the disease, mutations in either KEAP1 and/or STK11 display impaired response to chemotherapy. Immune checkpoint blockers (ICBs) that target the PD-1/PD-L1 axis form a class of immunotherapy aimed to unleash the cytotoxic killing ability of CD8 effector T cells against tumours, are currently first-line treatment. However, patients who harbour mutations in both KRAS and STK11 (KL-mutant LUAD) or KEAP1 (KK-mutant LUAD) fail to respond to ICB. Critically, KK-mutant LUAD are also recalcitrant to newer KRAS inhibitors, such as Sotorasib (KRASG12C inhibitor). Therefore, a greater understanding of the tumour-intrinsic characteristics that govern underlying cancer processes for KL and KK mutant LUAD are required to improve therapeutic outcomes. Thus, the overarching goal of this body of work was to identify novel vulnerabilities distinct to these subtypes of KRAS-mutant LUAD and develop new personalised treatment modalities for patients. To investigate the impaired immunotherapeutic response of KL-mutant LUAD, the development and validation of top gene candidates from a whole-genome CRISPR/Cas9 screen was performed in a genotype specific manner. The use of pre-clinical genetically engineered mouse models (GEMMs) was also leveraged to replicate the disease in vivo. Impaired CD8 T cell function is a defining characteristic of KL-mutant LUAD. To replicate this feature of disease, I developed an unbiased in vitro CRISPR/Cas9 whole-genome knockout (KO) screening platform whereby murine KL-mutant lung tumour cells are co-cultured with cytotoxic CD8 OT-I T cells to mimic tumour-immune interactions in vivo. This strategy successfully identified components of the MHC-I antigen presentation (Tap2 and B2m) and Jak/Stat signalling (Jak1, Jak2, Stat1, Stat2, Ifngr1 and Ifngr2) pathways, readily reported to confer resistance to immunotherapy, validating my screening approach. Importantly, this platform also identified known immunotherapeutic candidates (Ptpn2 and Serpinb9) that sensitised KL-mutant tumour cells to killing by cytotoxic CD8 T cells. Excitingly, Kdm2b an epigenetic regulator of the variant polycomb repressive complex 1 (PRC1.1) was identified as a novel gene candidate that sensitised KL-mutant tumour cells to CD8 T cell killing. Furthermore, through in vitro co-culture assays and in vivo growth assays, Kdm2b inhibition sensitised the growth and proliferation of KL-mutant tumour cells following immune pressure. Critically, the KO of Kdm2b synergised with anti-PD-1 treatment in vivo, highlighting a novel immunotherapeutic strategy for the treatment of KL-mutant LUAD in the clinic. To investigate characteristics unique to KK-mutant LUAD, as a means to design personalised treatment approaches, an in depth investigation of commonly utilised models of Keap1-deficiency was undertaken. These studies focused on interrogating the cellular origin of KK-mutant LUAD. Integrative transcriptomic analysis through RNA-sequencing (RNA-seq) technologies identified the upregulation of ciliated cell markers in lung tumours that arose in multiple Keap1-deficient GEMMs. Whilst the use of cell specific adeno-Cre viruses did not define a putative cell-of-origin for Keap1-mutant LUAD, RNA-seq analysis revealed the upregulation of the Sonic hedgehog (Shh) pathway, a critical pathway implicated in ciliogenesis and cilia formation within the lung, was restricted to Keap1-mutant tumours. With multiple Shh pathway inhibitors being trialled in the clinic, these results suggest a much-needed, novel therapeutic strategy for the treatment of KK-mutant LUAD. Taken together, my work highlights novel treatment strategies within KL- and KK-mutant LUAD, providing robust investigative studies on the utility of pre-clinical models for KRAS-mutant LUAD. I have utilised sophisticated pre-clinical GEMMs to replicate the human disease in a living organism and interrogate underlying characteristics that define these subtypes of KRAS-mutant LUAD, with future results from these studies guiding new treatment approaches in the clinic. Moreover, my PhD studies have established a CRISPR screening platform that may serve as the foundation of future work to investigate and interrogate novel immunotherapeutic targets in other non-responsive subtypes of lung cancer, and other cancer types.
-
ItemInvestigating molecular interactions in necroptosis and MLKL-mediated cell death( 2022)Necroptosis is a lytic, caspase-independent form of regulated cell death that involves the swelling of cells and organelles, the breakdown of cellular membranes, and the release of damage associated molecular patterns and other cellular contents. From an evolutionary perspective, necroptosis is thought to have arisen as a defence against infection, particularly in the case of intracellular bacteria and viruses. Additionally, there is a growing body of evidence demonstrating that dysregulated necroptosis contributes to the pathology of a broad range of noninfectious diseases, including those involving neurodegeneration, inflammation, autoimmunity, and ischaemic tissue death, as well as having a role in the progression of some types of cancer. As such, having a clear understanding of the molecular processes involved in necroptosis is critical for both increasing our understanding of its role disease and determining the most effective strategies for pharmacological manipulation of the pathway. Although necroptosis can be activated by a range of intracellular and extracellular receptors, necroptotic signalling through tumour necrosis factor receptor 1 (TNFR1) is the most well-studied, as it has relevance to a broad range of disease states. For necroptosis to proceed through TNFR1, there are two critical signalling events that need to occur. Firstly, receptor interacting protein kinase 1 (RIPK1) needs to disengage from the membrane-bound TNFR1 receptor complex to form a death-promoting cytoplasmic complex which, importantly, includes caspase 8 (CASP8). Secondly, proteolytic activity of CASP8 needs to be restricted to allow RIPK1 to associate with the related kinase RIPK3, through their RIP homotypic interaction motifs. This association enables RIPK3-mediated phosphorylation and activation of the mixed lineage kinase domain-like (MLKL) pseudokinase, enabling MLKL to oligomerise and translocate to cell membranes, where it can initiate cell death. However, although this core signalling axis is well defined, the peripheral signalling events that control necroptotic cell death are not well established, particularly relating to events that occur at the level, or downstream, of MLKL activation. This thesis uses two different strategies to increase our understanding of factors that influence MLKL activation and subsequent cell death. Firstly, in Chapter 3, I developed a method for generating reconstitutable MLKL-/- human cell lines using CRISPR-Cas9 delivered by integrase-deficient lentivirus, which enabled me to test the functional consequences of mutations to MLKL without the complicating influence of endogenous MLKL. The results of these reconstitution studies, which are documented in Chapter 4, helped identify several novel residues that play an important role in MLKL function, including some that have previously been found to be mutated in human cancer. In Chapter 5, I explored the mechanism of action of AMG-47a, a small molecule identified in a phenotypic screen focussed on drugs that inhibit necroptosis downstream of MLKL activation. The key findings of this investigation include confirming RIPK1 and RIPK3 as the relevant targets of AMG-47a during TNFR1-dependent necroptosis and uncovering a potential role for RIPK1 downstream of MLKL activation. Together, my research has contributed to our understanding of how MLKL activation is controlled and expanded our knowledge of the molecular processes downstream of MLKL activation.
-
ItemGenome in 3D – regulation of lymphocyte development by genome architecture( 2020)The functionally and phenotypically diverse cell populations that make up the immune system arise from the expression of a select part of a genome at a given time. The mechanisms governing such differential transcription are still, surprisingly, not fully understood. Only recently has the three-dimensional organisation of chromatin in the interphase nucleus been acknowledged to play a crucial role in modulating transcription. For instance, distal cis-regulatory elements like enhancers can form long-range chromatin loops with promoters to drive transcription, and these chromatin loops are in turn harboured in topologically associating domains (TADs), shielded from interference by outside elements. These three-dimensional structures can be lineage-specific and their roles during cellular differentiation are beginning to be uncovered. The chromosome conformation, or genome architecture, in B- and T-lymphocytes, captured previously as Hi-C data in the lab, has provided immense information about lineage-specific DNA interactions that might be critical during differentiation. Based on this resource, the work herein aimed to develop an approach to identify, characterise and functionally dissect any novel and critical regulatory elements. Using this strategy, I have identified several putative T- and B-cell specific elements and subsequently adopted the CRISPR/Cas9 platform in generating large deletions as to dissect these elements. The approach has identified and confirmed the enhancers of T cell-specific transcription factors Bcl11b and Gata3. Upon closer inspection an uncharacterised long non-coding RNA (lncRNA) Gm13218 was uncovered to associate with the enhancer of Gata3. Given the recent recognition of lncRNAs as important regulator of the 3D genome, I have retrieved the full-length sequence and characterised its expression pattern. It was found that expression of Gm13218 is highly correlated with that of Gata3 during early T cell development in thymus as well as T helper 2 (TH2) cell differentiation. Knockdown and overexpression of Gm13218 transcripts, CRISPR-mediated silencing, activation, demethylation of the locus as well as interference of transcription elongation suggest that Gm13218 may be involved in the establishment, but not the maintenance of Gata3 expression. By utilising Hi-C, RNA-seq, cell division and cell cycle indicators, the spatiotemporal dynamics of genome architecture during B cell activation and terminal differentiation into antibody-secreting cells was examined. It was revealed that genome organisation exhibit two discrete waves of restructuring – the first occurs just prior to the first cell division, with the resulting genome architecture being inherited through the subsequent rapid clonal expansion for many days until the second wave of restructuring upon differentiation into plasmablast. In addition, the first restructuring event was shown to precede the first DNA replication phase, suggesting that genome reorganisation is independent of, and well partitioned from, DNA synthesis and mitosis. In contrast, transcription underwent very early burst and was altered throughout the entire differentiation process. Further analysis suggests that transcription is intricately intermingled with genome organisation in a reciprocal fashion. Overall, the work in this thesis has revealed a number of important findings regarding how the 3D genome controls the development and function of the immune system.
-
ItemUnderstanding the molecular mechanisms of AML development and treatment using phosphoproteomics( 2020)AML (Acute Myeloid Leukemia) is a rapidly progressing cancer of the blood and bone marrow where the accumulation of abnormal myeloid cells crowds out healthy blood cells. Despite the improvements in understanding the biology of AML, these advancements are not reflected in the trajectory of the survival rate. One reason for the poor translation of research results into novel therapies, is the genetic and epigenetic heterogeneity of the disease, which hampers the development of reliable AML models. Mouse models of AML are designed to mimic human disease by expressing known oncogenes in the hematopoietic system of mice. Although the oncogene-specific disease pathology appears to reflect what is observed in human patients, the translation of novel treatments into the clinic has often been unsuccessful. Expression of the hematopoietic transcription factor EVI1 has been identified as a marker for poor prognosis in human AML patients. In this thesis, I aimed to develop a mouse model, that recapitulates a particularly aggressive and treatment-resistant form of AML by overexpressing EVI1 together with the fusion-oncogene MLL-AF9. Although I successfully generated EVI-expressing AMLs, the presence of the transcription factor did not affect disease features such as latency, disease pathology, morphology and immunophenotype of leukemic cells. However, when cultured in vitro, leukemic cells expressing EVI1 were more resistant to Smac mimetic combination treatments and the chemotherapeutic agent Ara-C, therefore reflecting the treatment resistance observed in human patients. Although some targeted treatments are now available, chemotherapy remains the standard of care therapy for AML. Therefore, to improve disease outcome, novel treatments are desperately needed. The IAPs (Inhibitor of Apoptosis Proteins) have been identified as an attractive therapeutic target in a number of cancers including AML. Smac mimetics, a class of drugs that specifically inhibit IAPs, have been found to selectively induce cell death in leukemic cells when combined with an inhibitor of the MAP kinase p38 both in vivo and in vitro. To understand the molecular mechanisms behind this synergistic killing, I studied the phosphoproteome of treated cells using mass spectrometry and revealed that the PI3K/Akt/mTOR survival pathway was activated following Smac mimetic treatment. Upon p38 inhibition, this survival signaling did not occur. Furthermore, CSF1R was identified as a potential regulator of the PI3K/Akt/mTOR pathway. In support of this finding, the combination of a Smac mimetic and CSF1R inhibitor, resulted in synergistic cell killing in vitro. To study proteins, gene tagging provides a valuable tool for a range of applications including real-time monitoring of target protein activities, modifications of proteins and protein-protein interactions. The CRISPR/Cas9 technology is a powerful tool for modifying any DNA of interest and enables the tagging of endogenous genes. However, the targeted insertion of foreign DNA into cell lines is challenging. I aimed to generate endogenously FLAG-tagged proteins in cell lines by targeting components of the TNF pathway using the CRISPR-Cas9 knock-in technology. Although ssDNA containing the FLAG sequence was inserted into the genome of mouse dermal fibroblasts (MDFs) at the correct site, next generation sequencing revealed that this process was both inefficient and error-prone and resulted in the introduction of mutations. Nonetheless, I was able to enrich for tagged RIPK1 following FLAG pull-down, which was detectable via both immunohistochemistry and mass spectrometry.
-
ItemNovel genes and mechanisms in monogenic autoinflammatory disorders( 2018)Monogenic autoinflammatory disorders are a heterogeneous group of rare conditions characterised by innate immune dysregulation. Patients often present early in life with recurrent fevers and features of systemic inflammation without high titres of autoantibodies or self-reactive T cells. Since the introduction of whole exome sequencing in the diagnostic evaluation of patients with suspected monogenic autoinflammatory disorders, the number of genetically defined conditions has greatly increased as has the phenotypic diversity. This study aimed to optimise methods of validating the pathogenicity of previously undescribed variants in vitro and to establish a national registry for patients with suspected or confirmed monogenic autoinflammatory disorders. Two variants in inflammasome forming proteins were evaluated. An inflammasome is a large multiprotein complex that forms in response to danger or pathogens. It serves as a platform for caspase-1 activation, resulting in cleavage of pro-IL-1 beta and pro-IL-18 to their active forms, as well as inflammatory cell death, pyroptosis. The first variant investigated was found through Sanger sequencing of MEFV in a family with a dominantly inherited suppurative dermatological condition. The novel variant p.Glu244Lys pyrin segregated with disease and was associated with increased inflammasome activation in vitro. This residue was shown using immunoprecipitation to be important for the binding of the regulatory proteins 14-3-3 and the substitution to lysine resulted in the auto-activation of pyrin. The second novel variant was found in two unrelated children with autoinflammation and macrophage activation syndrome. Although different genetic sequencing techniques were used, both children were found to harbour heterozygous p.Trp655Cys NLRC4. In vitro modelling revealed that this variant caused a caspase-1-dependent increase in IL-1 beta and IL-18 release with priming alone. Through the evaluation of the potential mechanisms of auto-activation, a previously unknown leucine rich repeat interface was revealed to exist between two NLRC4 monomers in the oligomeric state. Furthermore, an additional distinct interface was shown to exist between p.Trp655Cys NLRC4 and residues of the adjacent leucine rich repeat domain. Finally, the Australian Autoinflammatory Diseases Registry was established with fifteen tertiary hospitals across six Australian states currently involved. Thirty-seven patients with suspected autoinflammatory disorders have been recruited to date along with seventy-seven family members. The first twenty participants in whom no pathogenic mutation had been detected using National Association of Testing Authorities approved diagnostic testing underwent whole exome sequencing alongside their biological parents to determine variants that may be causing disease. The results of this analysis are presented here, including the identification of a novel variant in SHARPIN, encoding a component of the linear ubiquitin chain assembly complex involved in both the NF-kB and NLRP3 inflammasome pathways. This is the subject of ongoing investigation. The work described in this thesis has led to the first ethically approved national Australian registry for patients with monogenic autoinflammatory disorders. Furthermore, the in vitro validations of several AIDs within this thesis provide exquisite examples of some of the techniques that can be utilised in the future evaluation of variants of interest generated through the recruitment and sequencing of patients through the Australian Autoinflammatory Diseases Registry.
-
ItemA CRISPR/Cas9-based investigation of inflammasomes in infectious disease and autoinflammation( 2017)Inflammasomes are a family of innate immune signalling platforms that are activated in response to tissue damage or infection. Inflammasome stimulation results in activation of the inflammatory protease caspase-1, which induces a lytic cell death program known as pyroptosis, and maturation and release of the pro-inflammatory cytokines Interleukin-1β (IL-1β) and IL-18. The potent inflammatory cascade triggered through activation of the inflammasomes is protective against many bacterial pathogens that either invade host cells or produce toxins that deregulate key homeostatic mechanisms within innate immune cells such as monocytes and macrophages. De-regulation of inflammasome signalling, such as gain-of-function mutations in inflammasome components, can result in autoinflammatory pathology. In order to investigate the function and regulation of inflammasomes, Clustered, Regularly Interspersed, Short, Palindromic Repeats (CRISPR)/Cas9 gene editing technology has been utilised to delete various inflammasome components from human myeloid cell lines or from mice. The alternative inflammatory caspases, caspase-11 in mice and caspases-4 and -5 in humans are activated directly by cytoplasmic lipopolysaccharide (LPS), a key component of the cell wall of gram-negative bacteria. These caspases are able to induce pyroptosis independently of caspase-1, but are only able to trigger IL-1β and IL-18 release in a caspase-1-dependent manner. In this thesis, the roles of caspase-4 and caspase-5 in the response to cytoplasmic lipopolysaccharide (LPS) and invasive gram-negative bacteria have been investigated in a human monocytic cell line. While both caspases responded to infection with live gram-negative bacteria, free LPS that was transfected into the cytoplasm activated only caspase-4. This suggests that caspases-4 and -5 may be activated by distinct stimuli or through different mechanisms. This work also interrogates the role of the inflammasome-forming receptor pyrin, in both autoinflammatory disease and the anti-bacterial immune response. A serine to arginine mutation in pyrin at amino acid position 242 results in a newly described autoinflammatory condition known as Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis (PAAND). A monocytic cell line expressing the S242R mutant of pyrin has been created and it was demonstrated that this mutation results in spontaneous inflammasome activity. Under homeostatic conditions, serine 242 is phosphorylated and interacts with the 14-3-3 family of adapter proteins to keep pyrin inactive. Deletion of specific 14-3-3 isoforms also resulted in spontaneous production of mature IL-1β. Finally, the expression of pyrin in various myeloid compartments and its role in in vivo models of bacterial infection have been investigated using a pyrin-deficient mouse line. Two isoforms of pyrin were detected that were differentially expressed among myeloid populations. Additionally, no role for the pyrin inflammasome was observed in a Dextran Sodium Sulfate (DSS)-induced colitis model, or Citrobacter rodentium, Salmonella Typhimurium or Mycobacterium tuberculosis infection models.