Medical Biology - Theses
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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.