Medical Biology - Theses
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ItemIdentifying and characterising novel regulators of TRAIL-induced cell death and cholangitis-like liver injury( 2022)Primary sclerosing cholangitis (PSC) is a progressive, idiopathic cholangiopathy characterised by chronic inflammation of the biliary epithelium and cholestasis. PSC promotes fibrotic scarring of the intrahepatic and extrahepatic bile ducts often leading to premature death due to irreversible liver damage. Chronic persistent inflammation in the biliary tree further predisposes to the development of malignant cholangiocarcinoma (CCA). Tumour necrosis factor (TNF)-Related Apoptosis Inducing Ligand (TRAIL)/TRAIL-receptor-mediated signalling was shown to play a substantial role in the pathogenesis of human sclerosing cholangitis-like disease in mice with TRAIL- mediated apoptosis contributing to the disease. However, the etiology and exact pathogenic mechanisms of TRAIL-dependent PSC, or inflammation-associated cholangiocarcinogenesis are largely unclear. Various genetic and environmental factors have been reported to play role in the pathogenesis of PSC. Recently, mutations in ZFYVE19 gene (protein name: ANCHR) were described as a novel cause of neonatal sclerosing cholangitis and hepatic fibrosis termed ZFYVE19 disease. However, the mechanism by which ZFYVE19/ANCHR is involved in the pathogenesis of sclerosing cholangiopathy in these patients has not been yet explored. In Chapters 3 & 4 of my thesis, I identify and characterise two novel regulators of TRAIL-induced cell death, the Abscission/NoCut Checkpoint Regulator (ANCHR/ZFYVE19) and its interacting protein E3 ligase Mind Bomb 2 (MIB2) and show that the loss of ANCHR or MIB2 sensitises TRAIL-resistant cancer cells to caspase-8- dependent death. Moreover, loss of ANCHR alone sensitises CCA cells to death in vitro. Given that the loss of ZFYVE19/ANCHR in a cohort of patients was associated with PSC and cholestasis, and TRAIL/TRAIL-R-mediated apoptosis has been suggested to play an essential role in a PSC-like disease in mice, I further interrogate the physiological consequences of ANCHR loss in mice, particularly focusing on TRAIL-mediated cell death in the liver. In Chapters 3 & 4 I demonstrate a role for ANCHR in limiting TRAIL-induced cell death in vivo and show that loss of ANCHR in mice sensitises to TRAIL-mediated liver cell death. A significant increase in liver cell death in Zfyve19 knock-out mutant mice is observed compared to the wild-type mice after anti-TRAIL-R2 monoclonal antibody (MD5- 1) injection, with concurrent increase in cholangiocyte cell death, suggesting a role for ANCHR in limiting the TRAIL-mediated cholangitis in mice by limiting TRAIL-induced cell death in the liver. Lastly, in the Chapter 5 of this thesis, I demonstrate, as a proof-of-concept, that ANCHR and MIB2 can be efficiently targeted and degraded using the emerging degradation tag (dTAG) PROteolysis-TArgeting Chimera molecules (PROTACs). Our preliminary results serve as a basis for future research and suggest that anti-apoptotic ANCHR and MIB2 are feasible targets for target-specific protein degradation for development of future TRAIL therapeutics. Overall, this thesis expands our understanding on how TRAIL-signalling is regulated and provides a mechanism for the interplay between ZFYVE19/ANCHR loss and TRAIL- mediated PSC-like liver disease. Furthermore, our studies provide correlative evidence for the relationship between the PSC pathology seen in patients carrying bi-allelic nonsense mutations in the ZFYVE19 gene and an overactive TRAIL signalling or overactive liver sensitivity to endogenous TRAIL.
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ItemThe molecular control of lymphocyte fate timers( 2022)Activation of lymphocytes induces proliferation and clonal expansion of antigen specific cells. During this response numerous fate and differentiation decisions occur resulting in a robust and heterogeneous immune cell population. Recent studies support a quantitative model of the lymphocyte response where signals regulate three independent timed cellular modules, the time to divide (Division), the time in which cells are ‘licensed’ for successive divisions before returning to quiescence (Division Destiny) and the time to die (Death). Division destiny time was shown to be inherited through generations and regulated by the production and loss of the oncoprotein Myc with cell division stopping when levels fall below a threshold. These results provided an example mechanism for timed fate control, linking regulator protein expression levels to timed outcomes. In this thesis, timed regulatory mechanisms for the analogous division and death timers were investigated, and the limiting molecular components described. Using quantitative cell-based assays for B cell proliferation, we found the rate of D-Cyclin production and loss is linked to activation signal strength and correlates with division times. D-cyclin levels must reach a threshold level for cells to enter the first division and this constraint of expression level also applies for subsequent division rounds. Inhibitors of D-Cyclin/CDK catalytic activity slowed division entry and subsequent division rates, while the forced over-expression of Cyclin D3 increased B cell division rates, thus indicating D-Cyclin protein level and activity regulate and govern division times. To study the death timer, we analysed CpG stimulated B cells as they die over a precise, predictable period. Induction of pro- and anti-apoptotic molecule expression was observed to rise and fall in B cells prior to, and during, the death phase. A timer model based on stochastic differences in molecular expression, coupled to death if the protein ensemble fell below a critical value was developed. To test this model, we used data from Bax/Bak KO single cell FACS expression, that do not die, to predict death frequency in wild type cells. Weighting the combined effect of BCL-2, MCL1, BCL-xL and BIM, predicted death and identified the collective ‘threshold’ required for B cell survival, supporting the timer model. Together the findings presented here support conceptual models that can link quantitative molecular changes to the control of multiple cell fates in response to complex signal combinations and further support the hypothesis of independent timed fate outcomes for division and death controlled by the levels of key regulating proteins over time.
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ItemTherapeutic targeting of mutant TP53 in human cancers( 2021)TP53 is the most frequently mutated gene in human cancers (~50%), and these mutations sustain tumour development through three different, albeit not mutually exclusive effects: loss of wt TP53 functions (i.e. mutant TP53 is unable to transactivate wt TP53 target genes thereby resulting in the loss of tumour suppression function); dominant negative effects (i.e. mutant TP53 inhibits wt TP53 to exert its tumour suppression function through the formation of wt TP53/mutant TP53 mixed tetramers ); gain of function that allow mutant TP53 to exert neomorphic functions through protein-protein interactions that wt TP53 does not engage in. While loss of wt TP53 function is widely accepted to be critical for tumorigenesis, the importance of the role of the gain of function effects of mutant TP53 are still under debate. Here, by removal of mutant TP53 in human cancer cell lines as well as in patient derived colon cancer organoids, which were driven by mutant TP53, we reveal that the removal of mutant TP53 did not impair the survival or proliferation of tumour cells both in vivo or in vitro, nor did it render the tumour cells sensitive to chemotherapeutic drugs. This demonstrates that the gain of function effects of mutant TP53 are not essential to sustain tumour expansion and survival. Therefore, drugs that could abolish mutant TP53 expression in tumour cells would not be expected to have significant therapeutic benefits as anti-cancer therapy. An alternative therapeutic strategy for mutant TP53 cancers would be to restore wt tumour suppressor functions. APR-246 is a small molecule drug that was reported to restore wt TP53 function to tumour cells expressing mutant TP53. However, there is also evidence that APR-246 may be able to kill tumour cells independent of mutant TP53. Here, by generating isogenic background tumour cell lines with different TP53 states, we reveal that APR-246 can kill tumour cells irrespective of the TP53 status. We also discovered that APR-246 can exert its killing effects by orchestrating different forms of programmed cell death depending on the type of tumour cell. In mouse Eu-Myc lymphoma cells, low doses of APR-246 induced intrinsic apoptosis while high doses of APR-246 resulted in cell lysis even when BAX and BAK, the essential effectors of apoptosis, were absent. Our work also showed that necroptosis and ferroptosis can also contribute to APR-246 induced killing of malignant cells, again dependent on the type of tumour cells. In summary, APR-246 can kill tumour cells by inducing different forms of programmed cell death that were all independent of mutant TP53. These findings have important implications for the clinical application of APR-246 in cancer patients.
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ItemMechanism of Action of Two Small Molecule Necroptosis Inhibitors( 2021)Necroptosis is a form of programmed cell death that is controlled by a defined set of protein effectors, despite displaying the morphological characteristics of unregulated lytic cell death (necrosis). Recently, there has been increasing interest in this type of cell death, ignited by studies demonstrating that necroptosis is involved in the pathophysiology of various diseases – including inflammatory conditions, degenerative conditions, infectious diseases and cancers – and further kindled by the progression of small molecule necroptosis inhibitors into clinical trials. The best studied form of necroptosis is driven by the tumour necrosis factor (TNF) signalling pathway, which is initiated by TNF binding to its cell surface receptor TNFR1. Importantly, TNF-induced necroptosis is regulated by three key proteins: the kinases RIPK1 and RIPK3, and the pseudokinase MLKL, which acts as the cell death executioner. To identify novel inhibitors of necroptotic cell death, several small molecule screens were performed at WEHI. This PhD thesis details the investigation into the mechanism of action of two small molecule necroptosis inhibitors identified from these screens. I employed a suite of chemical biology, biochemistry and cell biology approaches to deduce the cellular targets of these small molecules and investigate their anti-necroptotic activity. Chapter 2 examines the identification and mechanism of action of Compound 2, a more potent necroptosis inhibitor than its parent compound, Compound 1, which was identified from a small molecule screen against MLKL. I determined that Compound 2 targets all three necroptotic effector proteins – MLKL, RIPK1 and RIPK3 – in vitro and in cells, to potently block necroptosis in human and murine cells at nanomolar concentrations. Moreover, this study highlights that necroptosis can be potently inhibited by targeting multiple effectors, suggesting that targeting multiple proteins in the pathway may be an ideal strategy for inhibiting necroptosis in a therapeutic context. Chapter 3 explores the cellular activity and cellular targets of ABT-869, an inhibitor of necroptosis identified from a phenotypic screen using a cell line expressing a constitutively active MLKL mutant. I determined that ABT-869 blocks necroptotic cell death by targeting RIPK1 and possibly RIPK3, although whether ABT-869 targets RIPK3 directly or indirectly, as a result of RIPK1 inhibition, remains to be elucidated. Furthermore, this study raises some interesting questions regarding the involvement of RIPK1 downstream of MLKL activation, which could contribute to an improved understanding of how necroptosis is regulated at the molecular level. Together, these two novel inhibitors of necroptosis identified from small molecule screens were found to block necroptotic cell death by targeting known components of the TNF-induced necroptosis pathway. This research provides insight into how small molecules can modulate necroptotic signalling by interacting with key necroptotic proteins to inhibit cell death.
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ItemThe role of RIPK3 ubiquitylation and MLKL signalling during cell death and autophagy( 2021)Receptor Interacting Serine/Threonine Kinase-3 (RIPK3) is essential for necroptosis, an inflammatory form of programmed cell death pathway implicated in innate immunity, kidney ischemia reperfusion injury, and systemic inflammatory response syndrome. In the classical model, cells committed to necroptosis phosphorylate RIPK1, which in turn drives RIPK3 phosphorylation and oligomerisation. Active RIPK3 oligomers subsequently phosphorylate mixed lineage kinase domain-like protein (MLKL) pseudokinase which induces its translocation to the plasma membrane. The necroptosis pathway culminates in MLKL perforating the plasma membrane as a prelude to cellular rupture and release of inflammatory cytokines and damage-associated molecular patterns to the extracellular milieu. In addition to being a pro-necroptotic kinase, RIPK3 is also capable of triggering apoptosis when its kinase activity is restrained. Moreover, numerous death-independent roles of RIPK3 have been described in the context of inflammation such as arthritis, viral infection, or colitis whereby RIPK3 either promotes or dampens the secretion of pro-inflammatory cytokines. Understanding the molecular regulation of RIPK3 will thereby facilitate the ongoing pre-clinical development of RIPK3 inhibitors. Like most proteins, post-translational modification (PTM) is a critical fine tuner of RIPK3 activities. Ubiquitylation, in particular, has recently garnered attention in the cell death field as loss of this PTM may result in hyperactive RIPK3 which consequently accelerates death and inflammation. However, the post-translational control of RIPK3 signalling is not fully understood. Using mass-spectrometry, I identified a novel ubiquitylation site on murine RIPK3 on lysine 469 (K469). Complementation of RIPK3-deficient cells with a RIPK3-K469R mutant demonstrated that the decoration of RIPK3 K469 by ubiquitin limits both RIPK3-mediated caspase-8 activation and apoptotic killing, in addition to RIPK3 autophosphorylation and MLKL-mediated necroptosis. Unexpectedly, the overall ubiquitylation of mutant RIPK3-K469R was enhanced, which largely resulted from additional RIPK3 ubiquitylation upstream on lysine 359 (K359). Loss of RIPK3-K359 ubiquitylation reduced RIPK3-K469R hyper-ubiquitylation and also RIPK3-K469R killing. Collectively, I therefore propose that ubiquitylation of RIPK3 on K469 functions to prevent RIPK3 hyper-ubiquitylation on alternate lysine residues, which otherwise promote RIPK3 oligomerisation and consequent cell death signalling. I further investigated the consequence of abolishing RIPK3 K469 ubiquitylation by generating Ripk3K469R/K469R mice. In agreement with in vitro findings, primary fibroblasts with mutant RIPK3-K469R enhanced apoptosis, and in vivo studies demonstrate that RIPK3-K469 ubiquitylation contributes to pathogen clearance. Specifically, when Ripk3K469R/K469R mice were challenged with Salmonella enterica serovar Typhimurium, bacterial loads in the spleen and liver were significantly increased relative to wildtype control animals. The increased bacterial burden in the mutant mice was consistent with reduced IFNg produced in the serum, while the elevated MCP-1 cytokine upon infection might be indicative of heightened immune infiltrates. Although necroptosis signalling clearly triggers cell death, how it might impact other cellular responses remains unclear. Therefore, to further delineate the functional outcomes of necroptotic activity I examined how its signalling impacts autophagy. The autophagy pathway is triggered when cells are deprived of nutrients. Although regarded as a pro-survival pathway which acts to recycle and remove damaged organelles, studies have recognised that autophagic pathways can impact cell death processes. In apoptosis, for instance, autophagy acts to limit pro-inflammatory IFN-b secretion, thus decreasing apoptotic immunogenicity. Nonetheless, little is known about the status of autophagy during necroptosis. I demonstrate through various genetic, imaging, and pharmacological approaches that active MLKL translocates to autophagic membranes during necroptosis. However, contrary to previous findings which reported the activation of autophagy upon necroptotic activity based on increased lipidated LC3B, a commonly used marker of autophagy induction, I challenged this conclusion by demonstrating that the accumulation of active LC3B during necroptosis is a consequence of reduced autophagic flux. Therefore, unlike apoptosis which proceeds in tandem with autophagy, the induction of necroptosis negates autophagy in an MLKL-dependent manner. While the function of MLKL-mediated autophagy inhibition warrants further investigation, I propose that attenuating autophagy during necroptosis contributes to the immunogenicity of this cell death modality by limiting the ability of the cell to clear damaged organelles and immunogenic molecules. Overall, my research has helped in outlining how a key necroptotic molecule RIPK3 is regulated post-translationally and how this is relevant in the context of microbial defence. I have also defined novel functional roles for necroptosis signalling in the regulation of autophagic responses. Understanding the molecular regulation of necroptosis signalling and how this cell death pathway is linked to other cellular responses, such as autophagy, is important for the accurate design of new therapeutics to target these pathways in pathological settings.
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ItemCharacterization of new regulators in TNFR1-mediated death signalling( 2020)Tumor necrosis factor (TNF) is a master inflammatory cytokine that can, depending on the circumstances, promote survival and proliferation or induce cell death. Anti-TNF drugs have proven strikingly successful in treating inflammatory diseases such as rheumatoid arthritis (RA), psoriasis and inflammatory bowel disease (IBD) but it is still unclear exactly why. For a long time, it was thought that they work solely by preventing TNF induced transcription of other inflammatory cytokines, but more recently it has been proposed that one of their major anti-inflammatory functions is to prevent TNF induced death. Therefore, understanding the mechanism by which TNF induced death is regulated may enable the conceptualization of newer or improved approaches in treating a variety of inflammation-associated pathologies. Binding of TNF to its receptor TNFR1 leads to the formation of two distinct signalling complexes. While most previous studies have focused on the membrane-bound, transcription-activating complex (complex-1), the composition and post-translational modifications of the cytosolic, caspase-8-containing, death-inducing complex (complex-2) remain far less well defined. To analyse TNFR1 complex-2 composition at endogenous levels, we decided to generate FLAG tagged caspase-8 knock-in mouse strains. The reagents for the FLAG tag enable very efficient and specific purification and identification of a FLAG tagged protein and its partners. After some preliminary tests and trials, I decided to use a 3x FLAG tag which has been reported to be 20–200 times more sensitive than other FLAG tags in immunoprecipitation and detection assays. Before generating the mouse strains, in Chapter 3 I performed extensive in vitro comparison of N-terminally or C-terminally 3x FLAG-tagged caspase-8 using a doxycycline (Dox)-inducible stably integrated lentiviral system. The results suggested that when expressed above endogenous levels, the expression and killing activity of caspase-8 was unaffected by a 3x FLAG tag. Interestingly, when expressed at physiological levels, C-terminally 3x FLAG tagged caspase-8 appeared to be equivalent to untagged caspase-8 and marginally more efficient in mediating TNF-induced death and complex-2 formation compared to N-terminally 3x FLAG tagged caspase-8. In addition, I immunoprecipitated TNFR1 complex-2 from cells expressing endogenous levels of 3x FLAG tagged caspase-8 and performed a mass spectrometry (MS) analysis. According to this analysis, Tankyrase-1 (TNKS1/PARP5a/ ARTD5), a member of the poly ADP-ribose polymerase (PARP) superfamily, appears to be a novel interactor of complex-2. Based on our in vitro data, we generated N-terminally or C-terminally 3x FLAG tagged caspase-8 knock-in mice using CRIPSR/Cas9 technology and these mice were characterized in Chapter 4. Homozygous N-terminally or C-terminally 3x FLAG tagged caspase-8 knock-in mice were viable, fertile and developed normally, indicating that N-terminally or C-terminally 3x FLAG tagged caspase-8 were expressed and active in vivo, at least to heterozygous caspase-8 levels. As expected, the expression of N-terminal or C-terminal 3x FLAG tagged caspase-8 was detectable in tissue and cells from knock-in mice by Western blot and immunofluorescence stain using an anti-FLAG M2 antibody. The 3x FLAG tagged caspase-8 displayed similar tissue distribution and comparable expression levels as endogenous caspase-8. The cell death assay suggested that the primary cells and transformed cells from 3x FLAG tagged caspase-8 knock-in mice responded similarly as wild-type cells to apoptotic and necroptotic stimulations. Moreover, by performing anti-FLAG immunoprecipitation, I successfully purified endogenous TNFR1 complex-2 from knock-in mice derived cells. These data indicated that 3x FLAG tagged caspase-8 knock-in mouse strains are useful tools to study caspase-8 and caspase-8-containing protein complexes at physiological levels. In Chapter 5, I characterized tankyrases-mediated poly(ADP-ribosyl)ation (PARsylation) as a novel checkpoint that limits TNF-induced cytotoxicity. Using primary cells from the 3x FLAG tagged caspase-8 knock-in mice described in Chapter 4, I found that the enzyme tankyrase-1 (TNKS1/TNKS/PARP5a/ARTD5), which was identified by mass spectrometry in Chapter 3, is recruited to the endogenous TNFR1 complex-2. Western blot data indicates that tankyrase-2 (TNKS2/PARP5b/ARTD6) may also be recruited. Tankyrases are poly ADP-ribose polymerases and belong to an ancient group of enzymes that post-translationally modify proteins with ADP-ribose. I found that during TNF signalling, complex-2 becomes poly(ADP-ribosyl)ated (PARsylated) in a tankyrases-dependent manner. Furthermore, tankyrases-specific inhibitors sensitized cells to TNF-induced cell death, which correlated with increased levels of complex-2. This suggested that normally tankyrases help limit TNF induced death. Mechanistically, I showed that tankyrases may modulate the stability of complex-2 by recruiting the E3 ubiquitin ligase RNF146, that in turn promotes ubiquitylation and degradation of complex-2. Moreover, inactivation of tankyrases dramatically increased the killing of the clinical Smac-mimetic (SM) birinapant in a primary acute myeloid leukemia (AML) model. Taken together, this thesis describes 3x FLAG tagged caspase-8 knock-in mice as new tools to study caspase-8 and caspase-8-containing protein complexes at physiological levels. Furthermore, this study identifies tankyrases-mediated PARsylation as a novel checkpoint in TNF signalling that expands our understanding of how TNF induced death is regulated and provides a rationale to use tankyrases inhibitors for cancer therapy.
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ItemThe molecular dissection of host manipulation by Toxoplasma gondii bradyzoites( 2020)Toxoplasma gondii is an obligate intracellular parasite, which chronically infects one-third of the world’s population. Toxoplasma infection severely affects immune-compromised individuals, resulting in birth defects, blindness, or brain encephalitis. Individuals often become infected through the consumption of contaminated food and water sources. Toxoplasma has two life stages during the lytic life cycle, the fast disease-causing tachyzoites and the chronic bradyzoites. Following host cell invasion, Toxoplasma tachyzoites extensively manipulate their host cell by exporting a distinct repertoire of effector proteins across the newly-established parasitophorous vacuole. This process interferes with the host's transcriptional program and is thought to enable parasite persistence and dissemination in spite of the host’s immune response. Eventually, Toxoplasma forms bradyzoite cysts in the visceral organs, which are a reservoir for disease reactivation. In the current scientific literature, the disparity in our knowledge between tachyzoite- and bradyzoite-host interactions is large. Almost nothing is known on how this chronic-stage of infection persists post-cyst formation and what role host manipulation plays in latency. Therefore, in this thesis I aim to understand whether Toxoplasma bradyzoites modulate the host transcriptional response for their survival and, if so, what role could dense granule protein export have in this process. I explore the host transcriptional profile of bradyzoite containing cells using RNA sequencing to question what role host manipulation plays in latency. I show that bradyzoite-containing host cells have a unique transcriptional landscape when compared to tachyzoite infection, and, by pairing this technique with protein export deficient parasites, I show that many of these changes are dependent parasite protein export. Next, I investigate whether the known tachyzoite effector proteins have a function in chronic infection. IST, an inhibitor of host IFN-gamma signalling, was identified as the only known tachyzoite effector to be expressed, synthesised, and exported in bradyzoites, suggesting a role for this effector protein in the chronic stage. Furthermore, I demonstrate that effector proteins are critical in protecting bradyzoite infected host cells from undergoing cell death upon IFN-gamma-mediated cell death, purposing three models that enable cyst persistence. This thesis explores bradyzoite-host interactions to interrogate the possible mechanism behind Toxoplasma’s lifelong infections.
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ItemControl of the Intrinsic Pathway of Apoptosis( 2019)Apoptosis is a cellular process of programmed cell death. The intrinsic pathway of apoptosis is triggered by mitochondrial outer membrane permeabilization, a point of no return that coincides with the release of cytochrome c into the cytosol where it activates the main effectors of cellular destruction: the caspases. The mitochondrial pathway that is centered on MOMP is tightly regulated by BCL2 family proteins, which includes some members that promote apoptosis and others that inhibit it. The interplay between these proteins with opposing roles determines whether a cell will die or survive. In a healthy cell, pro-survival BCL2 proteins inhibit the effector proteins BAX and BAK. BH3-only proteins are activated in response to cellular stress and promote apoptosis by neutralizing pro-survival proteins. Targeting BCL2 proteins to provoke apoptotic cell death has proven to be a successful strategy for cancer therapy with the BCL2-selective drug venetoclax exhibiting remarkable efficacy in treating cancers that rely on BCL2 for their survival. MCL1, a protein related to BCL2, is likewise critical for the survival of many cancer cells, making it another attractive anti-cancer drug target. Selective MCL1 inhibitors have been developed and are currently being evaluated in clinical trials to establish their safety and efficacy. Safety is a particular concern for MCL1 inhibitors because MCL1 is also essential for the survival of many cells in critical organs and tissues throughout the body. It remains to be seen if a sufficient therapeutic window will exist when MCL1 is targeted systemically. An alternative and potentially safer strategy to modulate MCL1 survival function would be to target pathways that regulate its activity in particular contexts. In Chapter 3 and 4, I focus on one such mechanism of MCL1 regulation: its turnover by the ubiquitin proteasome system. My work in Chapter 3 elucidated details of how MCL1 protein turnover is regulated by BH3-only protein NOXA. Using CRISPR-Cas9 screen, I discovered that the mitochondrial E3 ligase MARCH5, the E2 conjugating enzyme UBE2K and the mitochondrial outer membrane protein MTCH2 co-operate to mark MCL1 for degradation by the proteasome. I also demonstrated that this pathway is constitutively active in cells where NOXA is abundantly expressed and showed that manipulating NOXA expression in those cells impacts on MCL1 survival function. Having successfully demonstrated the power of CRISPR-Cas9 screen in Chapter 3, I undertook further screens in Chapter 4 to identify proteins, such as deubiquininating enzymes (DUBs), that might serve to enhance MCL1 protein stability. I did not identify any strong hits from these screens, possibly because multiple DUBs act redundantly on MCL1. Consistent with this hypothesis, only mild impacts on MCL1 protein stability were observed upon deleting DUBs previously reported to act on MCL1. Finally, in Chapter 5, I investigated how BH3 mimetics mimic the activity of BH3-only proteins to induce apoptosis. I studied how selective BH3 mimetic compounds perturb interactions throughout the BCL2 protein network beyond their direct protein targets. I showed that these second order impacts are crucial for effective killing. Apoptosis induced by the BCL2 selective inhibitor venetoclax, for example, typically also involves inhibition of MCL1. The impact on MCL1 in this context occurs as a consequence of displacing BH3-only proteins normally bound to BCL2.
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ItemDefining programs of cell death that can be harnessed to impact on outcomes of chronic viral infection( 2019)Pathogens causing chronic infections have successfully evolved mechanisms to subvert host immunity. Excessive and inappropriate inflammation together with attrition of repeatedly overstimulated high affinity T cells leads to abrogated immunity and persistence of pathogens such as HIV and HBV. T cell exhaustion has been touted as a prelude to T cell deletion during these infections, however, studies indicate that high affinity T cell clones are deleted at the onset of infection. The T cells that remain have lower affinity for pathogen epitopes and hence their response is weaker and more easily antagonised by inhibitory networks, including T-regulatory (Treg) cells. The killing of immune effector cells during chronic overwhelming infections is juxtaposed to the pathogen’s attempts to promote survival of infected target cells. Keeping infected cells alive is imperative for the maintenance of a microbial replicative niche. In this body of work, I dissected the role of host cell molecules and how they contribute to the death and survival of immune and infected cells. Necroptosis did not contribute to the loss of highly functional virus-specific CD8+ T cells during the course of infection. In contrast, when I interfered with death receptor signalling there was a modest rescue of functional CD8+ T cells. This gain in immune function, however, did not translate to improved viral control. The same mechanism I used to promote the survival of T cells made infected target cells refractory to death receptor mediated killing and therefore, offset any gain in immune function. Whilst examining the role of necroptosis in chronic infection, I made the discovery that the necroptotic inducer molecule, RIPK3, has additional non-necroptotic roles. Ripk3-/- mice cleared LCMV with enhanced kinetics compared to wild-type mice and mice that lacked the necroptotic executioner MLKL. I found that in the absence of RIPK3, chronically infected mice had impaired IFNβ responses. Excessive and prolonged IFNβ production is known to impair immunity. This may partially explain why mice lacking RIPK3 had enhanced numbers of granzyme B expressing T cells and controlled infection better than WT animals. The host-viral dynamics that favour displacement of highly functional cells with poorly activated cells makes the immune system highly vulnerable to inhibition through the activity of Treg cells. I next investigated the role of Treg cells in immune dysfunction during chronic infections and I was particularly interested in the cell death and cell survival pathways that contributed to the turnover and accumulation of these cells. I utilised mice with a Treg-specific deletion of Casp8. These mice had twice as many Treg cells as wild-type mice at steady state. Surprisingly, when these mice were infected with chronic LCMV, only 25% of the animals survived to 145 days post infection. Moribund animals succumbed to overt T cell activation and autoimmunity due to a precipitous drop in Treg cell numbers. Survivors, intriguingly, eliminated LCMV in most organs consistent with a massive gain in immune function. The death of the Treg cells was due to necroptosis. When I ablated the necroptotic pathway, through the deletion of Mlkl, I completely prevented the loss of Treg cells and the fatal immune pathology in Treg conditional caspase-8 deficient mice. I found that differential expression of RIPK3 and MLKL in Treg cells made them highly susceptible to necroptosis during chronic infection compared to Tconv cells. This was also the case for human Tregs and I was able to preferentially kill these cells, over Tconv cells, in vitro by driving necroptosis with a clinical stage caspase-8 antagonist called emricasan. Necroptosis is a lytic form of cell death that promotes inflammation and it has been implicated in chronic liver disease. I initially investigated if necroptosis in the liver contributed to the control of chronic LCMV, HBV or the malaria parasite Plasmodium berghei. Ablation of necroptosis had no impact on liver-pathogen dynamics and no impact on general liver function and architecture. In many cell types caspase-8 inhibits death receptor induced necroptosis. So, I reasoned that this molecule must be inhibiting induction of necroptosis in the liver of infected animals. I examined this by infecting mice that had a conditional loss of caspase-8 within hepatocytes. Despite abundant, infection driven, death ligands I observed no necroptosis in the liver. Even drug induced ablation of NF-ĸb survival signalling, downstream of TNF, failed to promote liver necroptosis in the aforementioned scenarios. The liver’s inability to undergo necroptosis was confirmed in mice with a human chimeric liver. I showed this refractoriness was due to liver repression of RIPK3 in humans and mice. The work conducted in this thesis provides important insights into the cell death pathways that are engaged in diverse cell types during chronic viral infections and I provide evidence that antagonising them therapeutically may lead to better clinical outcomes.
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ItemInhibitor of APoptosis proteins (IAPs) and SHARPIN regulate the immune response in the skin to limit inflammation and maintain homeostasis( 2018)The skin is a remarkable organ, a barricade between our vulnerable insides and a constantly changing environment full of physical, chemical, and biological aggressors. Maintenance of barrier integrity, immune surveillance, and rapid response are fundamental, and this multifaceted protection is orchestrated by the epithelial barrier and immune cells. Acute and chronic inflammatory skin diseases can arise due to abnormal over-reactions to the changing environment. A number of these diseases have been associated with genetic aberrations of the TNF super family and innate receptors signalling. My PhD studies have focused on the role of particular E3 ligases in regulating inflammatory signalling in skin homeostasis and inflammation. Inhibitor of APoptosis proteins (IAPs) and the Linear Ubiquitin-chain Assembly Complex (LUBAC) are E3 ubiquitin ligases that play crucial roles in innate immunity by regulating cell death and survival pathways from the TNF and pattern recognition receptor families. Genetic or pharmacological disruption of the IAPs or LUBAC member SHARPIN induce dermatological phenotypes with interesting parallels to a variety of human skin diseases. To investigate the contribution of immune cells to the Sharpincpdm cutaneous phenotype I utilised the transgenic Diphtheria Toxin Receptor (DTR) system to specifically ablate particular immune cell subsets in-vivo. I have found that Langerhans cells play a pivotal role in the cell death mediated skin disease that arises in Sharpin mutant mice, placing them as a potential cellular source of pathogenic TNF in the Sharpincpdm skin, and highlighting a T-cell independent role for Langerhans cells in driving skin inflammation. Epidermal specific genetic deletion of the cellular IAPs (cIAPs) resulted in early post-natal lethality due to widespread dermatoses. Pharmacological loss of cIAP1, cIAP2 and XIAP by subcutaneous injection of an IAP antagonist drug (smac-mimetic; SM) into mice induced a Toxic Epidermal Necrolysis (TEN) like local inflammatory lesion characterised by keratinocyte cell death, immune cell infiltration, and increased production of pro-inflammatory cytokines. Both the genetic and pharmacological phenotypes can be ameliorated by the loss of a single allele of RIPK1. I have conducted a screen injecting SM into a panel of knock-out and mutant mouse strains in order dissect the complex set of interactions initiated by injection of SM and leading to the TEN like lesional response. I found that disruption of IAPs leads to a breakdown in immune tolerance to commensal microorganisms, which can then initiate inflammatory responses in the skin. A full response to SM depends on interactions between innate immune signalling pathways, immune cells, and the microbiota, nicely highlighting the multifaceted processes involved in skin inflammation and cell death.