Medical Biology - Theses

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http://hdl.handle.net/11343/239

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Investigating molecular interactions in necroptosis and MLKL-mediated cell death

Jacobsen, Annette Vivi ( 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.
Mechanism of PINK1 activation by autophosphorylation

Gan, Zhong Yan ( 2023)

PINK1 is a protein kinase that functions with the E3 ubiquitin (Ub) ligase Parkin to trigger the autophagic elimination of damaged mitochondria, a process known as mitophagy. Loss-of-function mutations in PINK1 or Parkin result in the development of early onset Parkinson’s disease (EOPD). PINK1 is normally rapidly degraded by the proteasome to restrain mitophagy under basal conditions. However, in response to mitochondrial damage, PINK1 is stabilised on the mitochondrial surface where it activates by autophosphorylation. Active PINK1 then phosphorylates Ub and the Ub-like domain of Parkin to initiate mitophagy. Previous structural analysis of phosphorylated Pediculus humanus corporis (Ph, body louse) PINK1 in complex with Ub revealed how PINK1 engages and phosphorylates Ub, but how PINK1 initially activates by autophosphorylation remained unknown. The work in this thesis has elucidated the mechanism of PINK1 activation using a combination of structural, biochemical and cell biological approaches. In Chapter 3, a crystal structure of unphosphorylated PhPINK1 was determined, revealing the conformational state of PINK1 prior to activation. Comparison with the previous Ub-bound structure of PhPINK1 suggested that large scale conformation changes occur during PINK1 activation. Chapter 4 builds upon the findings of Chapter 3 and describes the events that occur during PINK1 activation. Unexpected oligomerisation of PhPINK1 enabled the use of cryo-electron microscopy (cryo-EM) to solve the structure of PhPINK1 in a symmetric dimerised state trapped in the process of trans-autophosphorylation at Ser202. Dimerisation and autophosphorylation of PINK1 could be validated in vitro using PhPINK1, and in cells using human PINK1. Furthermore, a structure of phosphorylated PhPINK1 revealed conformational changes that occur following Ser202 autophosphorylation, resulting in the formation of the Ub binding site that enables PINK1 to phosphorylate Ub. Chapter 5 explores the potential for oxidative regulation of PINK1 activity via a specific Cys residue in the P-loop of PINK1. Together, this thesis resolves the mechanism of PINK1 activation by autophosphorylation and implicates mitochondrial ROS in the regulation of PINK1 activity. The resolved mechanism further explains the pathological basis of a subset of EOPD patient mutations in PINK1 and reveal new potential therapeutic strategies to target and activate PINK1.
Identifying and characterising novel regulators of TRAIL-induced cell death and cholangitis-like liver injury

Gabrielyan, Anna ( 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.
Interrogating the cells-of-origin of BRCA mutant cancers to identify therapeutic targets for cancer prevention

Joyce, Rachel ( 2022)

It is currently estimated that approximately one woman dies every minute of breast cancer across the globe. While the greatest risk factor for developing breast or ovarian cancer is merely having female reproductive organs, the cumulative life-time risk of developing breast and ovarian cancer for women who carry pathogenic mutations in their BRCA genes is significantly higher than non-carriers. Men who harbour mutations in their BRCA genes are also at increased risk of developing breast cancer within their lifetime. Currently there are no clinically approved strategies for breast or ovarian cancer prevention in BRCA mutation carriers beyond highly invasive and irreversible surgical procedures such as prophylactic mastectomy and bilateral salpingo-oophorectomy. Targeted therapeutic strategies for cancer prevention in BRCA mutation carriers are thus a sought-after alternative. Significant headway has been made by our research group and others in identifying RANK-ligand inhibition as a putative chemoprevention strategy for the onset of breast cancer in female BRCA1 mutation carriers; subsequently, a phase 3 international clinical trial BRCA-P (ClinicalTrials.gov Identifier: NCT04711109) is currently recruiting female BRCA1 mutation carriers to assess the efficacy of RANK-ligand inhibition in preventing breast cancer development using the FDA-approved drug denosumab. A portion of this thesis describes the functional and biological consequences of denosumab treatment on the putative cell-of-origin of BRCA1 mutant breast cancer, the RANK+ luminal progenitor, from patients enrolled in the Melbourne Health BRCA-D pre-operative window study; these patients received denosumab treatments prior to undergoing prophylactic mastectomies. This work indicated that BRCA1 mutation carriers who received 1 denosumab injection per month for 3 months had significantly reduced numbers of RANK+ luminal progenitors in their breast epithelium, and these cells also displayed decreased colony forming activity ex vivo, compared to cells from untreated BRCA1 mutation carriers. This thesis also seeks to shed light on the biological mechanisms driving ovarian cancer development in BRCA1 mutation carriers, and describes novel subsets of BRCA1 mutant fallopian tube secretory cells that are putative cancer cells-of-origin. To date, there have been no prospective studies or chemoprevention trials for breast cancer development in BRCA2 mutation carriers. As such, there is a pressing need for the identification of novel therapeutic pathways for breast cancer prevention in these patients; this thesis makes several promising developments in this effort. Using preneoplastic breast tissue samples from BRCA2 mutation carriers and wildtype patients, luminal cells, including a subset of ERBB3lo luminal progenitors and mature luminal cells, were found to be expanded in breast tissue epithelium of BRCA2 mutation carriers. ERBB3lo luminal progenitors from preneoplastic BRCA2mut/+ patients were found to have increased colony forming activity ex vivo, and exhibited upregulation of genes involved in mTORC1 signalling, protein synthesis and proteostasis. Indeed, a functional protein synthesis assay revealed increased protein translation in preneoplastic luminal cells from BRCA2 mutation carriers compared to wildtype patients ex vivo. A genetically engineered mouse model of BRCA2 mutant breast cancer was used to faithfully recapitulate the preneoplastic phenotype of luminal epithelium identified in BRCA2 mutation carriers, and showed a significant delay of BRCA2mut/+ mammary tumourigenesis upon short-term treatment with an mTORC1 inhibitor in vivo. In summary, the findings detailed in this thesis describe several developments in our understanding of the mechanisms of breast and ovarian cancer development in BRCA mutation carriers, and uncover mTORC1 inhibition as a putative strategy to delay or prevent the onset of breast cancer in BRCA2 mutation carriers. Cumulatively this work provides important insights of clinical significance for women harbouring mutations in their BRCA genes.
Intestinal Microfold Cells Orchestrate Microbe- Immune Interactions

Cao, Wang ( 2022)

Microbiota–immune cell interactions play a vital role in defenses against potentially harmful external organisms such as viruses and bacteria, and environmental agents including food. Microfold (M) cells are specialized cells within the epithelium of the intestines that sample the gut contents and pass them to the local guardians – a complex array of immune cells. Once harmful invaders are detected by immune cells, they swing into action to fight the infection. M cells provide the pivotal link between the gut lumen and the immune cell network, positioned to rapidly orchestrate appropriate immune responses. Exactly how M cells orchestrate these events, however, is not clear. Despite their critical function, to date few specific tools have existed to study intestinal M cells, the molecular mechanisms that regulate their generation, or how they drive mucosal immunity. To overcome this gap, we have generated novel gene modified mouse strains to allow us to visualize M cells and tease apart their behavior. I discovered that M cells are present along the entire intestinal tract, and not just localized to the Peyer’s patch as previously thought. Analysis of gut epithelial cells at different sites along the gut using single cell RNA sequencing revealed tissue-specific heterogeneity allowing us to define distinct gene signatures for M cells based on their location. These molecular blueprints identify distinct maturation programs that reflect local environmental cues shaped by the ingested material and the microbiota. Collectively, these features shape the delicate network of immune cells and show how the body can regulate gut region-specific diseases.
Exploring the ubiquitin proteasomal system in Toxoplasma gondii

Khurana, Sachin ( 2022)

The phylum Apicomplexa comprises a group of unicellular eukaryotic organisms including Plasmodium spp., Toxoplasma gondii and Cryptosporidium spp. Infection with Toxoplasma gondii causes severe congenital birth defects and disease in immunocompromised individuals and persists for life. Efforts to develop effective vaccines have been unsuccessful and emergence of drug resistance has reduced the utility of current antiparasitic compounds, highlighting the need for new therapeutics. My PhD focussed on two areas: Firstly, understanding degradative ubiquitination in apicomplexan parasites as a step towards developing first in class anti-apicomplexan PROTACS. PROTACS are heterobifunctional molecules which on one end bind an E3 ubiquitin ligase and force it to bind with a neo-substrate which is then degraded via the proteasome. This technique has shown great promise in developing new therapeutic approaches to treat disease like cancer but has never been tested against any apicomplexan parasite. The absence of conventionally used E3 ligases for PROTAC development became a challenge which I decided to overcome during my thesis. I developed a new system to screen for degradative parasite E3 ubiquitin ligases that can be used to enhance current therapies. The second part of my PhD focused on the role of ubiquitination during differentiation of Toxoplasma gondii into latent stages. We performed a whole genome CRISPR screen to identify genes required for differentiation into latent forms. The screen identified several genes involved in protein ubiquitination. These encompassed a complete multi protein E3 ubiquitin ligase complex that regulates differentiation from acute to latent forms and was the orthologue of the GID/CTLH E3 ligase complex which has been characterised in yeast and humans. Genetic removal of this complex resulted in markedly reduced cyst formation in vitro even in conditions favouring differentiation and reduced burden in the brain in vivo. Furthermore, we have identified potential substrates of this E3 complex and the likely mechanism by which it regulates differentiation. This work highlights the importance of ubiquitination during stage transition into latent forms and highlights the fascinating biological processes that ubiquitination regulates.
Understanding how malaria-induced T-bet expression impacts the development of protective immunity to infection

Pietrzak, Halina Mary ( 2022)

Malaria is a globally significant parasitic disease infecting millions of people annually. Clinical immunity to infection takes years of frequent exposure to develop and only partially protects the host against clinical symptoms, with individuals in endemic areas often developing chronic, asymptomatic infections. These observations suggest defects in the generation, maintenance, or effector capacity of immune memory induced in response to infection. Antibody responses are a critical component of clinical immunity to malaria. Recent work from our group demonstrated that inflammatory pathways contributing to the development of clinical malaria episodes play a negative role in the induction of humoral immunity. IFN-g produced in response to acute malaria infection was found to upregulate the expression of transcription factor T-bet in T follicular helper cells (Tfh), the key T cell subset required to provide help to B cells for the induction of protective antibody responses to infection. T-bet expression in Tfh cells impairs their normal differentiation and compromises downstream humoral responses to acute infection. The contribution of T-bet expression to the development of Tfh memory cells in malaria is unknown. To investigate this, the Tfh memory cell compartment was examined using PBMC samples from human P. vivax patients, and a murine model of severe malaria infection. Together, these analyses involving flow cytometry, adoptive transfer, and RNA-sequencing approaches revealed that the T-bet influences the composition and development of the Tfh memory cell compartment in malaria. Specifically, the main results from this investigation revealed that T-bet expression in CD4+ T cells impairs the development of Tfh central memory (TfhCM) cells which are an important compartment that support and bolster long-lived memory responses. This data provides evidence that malaria-induced inflammation negatively impacts the development of memory populations required for an efficient response to malaria, thus restraining a potent immune response to re-infection.
Discovery of antimalarials with novel mechanisms of action

Bailey, Brodie ( 2022)

Today, despite an annual investment of $3 billion from governments, NGOs and pharmaceutical companies, malaria remains one of the most devastating parasitic diseases in human history. Of great concern is the emergence of resistance to all currently available antimalarial treatments, particularly the front-line artemisinin combination therapies. Therefore, an urgent need has arisen towards the development of antimalarials with novel mechanism of action to combat the rise of resistance. To discover new antimalarial chemotypes a high-throughput screen of the Janssen Jumpstarter library against asexual stage P. falciparum was undertaken and uncovered the novel 2-(N-phenyl carboxamide) triazolopyrimidine scaffold. This thesis describes the optimisation and mechanistic characterisation of the 2-(N-phenyl carboxamide) triazolopyrimidine antimalarial class. In Chapter 2, the optimisation and phenotypic characterisation of the triazolopyrimidine series was conducted. In the optimisation process, the structure activity relationship was defined and delivered analogues with EC50 values below 100 nM against the asexual stage parasite. Phenotypic characterisation determined this class exhibited a slow to moderate rate of kill and arrested asexual stage development at the trophozoite stage. Equipotent activity against P. falciparum multi-drug resistant field strains and moderately reduced activity in P. knowlesi indicated a distinct mechanism of action to clinically relevant antimalarials and a conserved target, albeit with potential species differentiation. In Chapter 3, the mechanism of action of triazolopyrimidine series was explored using chemoproteomic and chemogenomic techniques. Chemical probes were synthesised for use in affinity pulldown and fluorescent imaging. However, these probes were unable to clearly identify the molecular target of the chemical series. Resistance selection was undertaken and whole genome sequencing of resistant clones identified amplifications and non-synonymous point mutations in a putative mitochondrial carrier protein (PF3D7_0407500) of unknown function. Metabolomic analysis of drug treated parasites indicated disruptions in pyrimidine metabolism and depletion of pantothenate metabolites. The unique metabolic signature reveals a potential multifaceted and indirect effect on the function of the electron transport chain and CoA pathways of the mitochondria. Finally, Chapter 4 describes research towards the validation of the putative mitochondrial carrier protein (PF3D7_0407500) as the molecular target of the triazolopyrimidine series. Genetic validation of the target is described in which we attempt to introduce the resistance conferring mutations into wild-type parasites. Another component of the validation focuses on the characterisation of the indirect effect of the triazolopyrimidine series on the function of the mitochondria. Pantothenate uptake experiments show that a frontrunner triazolopyrimidine analogue blocks the flux of this metabolite into the parasite. To definitively confirm the function of the carrier protein, recombinant expression systems were trialed unsuccessfully to enable biochemical substrate screening and structural studies. Together this research describes the exploration of a novel antimalarial class with a distinct mechanism of action. Future research will aim to genetically validate the carrier protein and further mechanistically characterise its involvement in mitochondria function and development of the malaria parasite. Using these compounds as tools, we have uncovered a novel carrier protein that has a unique function that could represent a novel druggable target for future antimalarial development.
Chimeric Antigen Receptor T cell therapy in Diffuse Midline Glioma

Wang, Shiqi ( 2022)

Diffuse Midline Glioma-Pons, also known as Diffuse Intrinsic Pontine Glioma (DMG-P or DIPG) is a rare but devastating brain tumour of childhood. The only proven treatment is palliative radiotherapy that extends survival, but does not provide cure. Chimeric Antigen Receptor (CAR) T cell therapy is a promising form of adoptive cell therapy, that re-engineers patient-derived T cells to express a hybrid receptor specific to a tumour-specific antigen of choice. CAR T therapy directed against the CD19 antigen has been extraordinarily successful in various haematological malignancies, and its tumour-specific nature makes this therapy very appealing to apply to DMG-P. This thesis explores the generation and functional testing of a novel CAR T cell for DMG-P, specifically directed against the H3.3K27M peptide, presented in HLA-A*02. I demonstrated that this CAR T cell is functional when the target is amply present using various cell line models. However, my data revealed that this target is not present at an endogenous level on multiple DMG-P cell lines. This suggests that HLA-A*02/H3.3K27M may not be the ideal immunotherapy target, in contrast to previously published data. This thesis also investigates the use of an anti-HER2 CAR, a previously well-established CAR, in DMG-P, demonstrating that there is in vitro and in vivo functionality of this CAR in this rare tumour. Employing this CAR in partnership with other CARs in DMG-P may become important for preventing antigen escape in future. I also identified new immunotherapy targets using a combination of flow cytometry analysis, cell surface mass spectrometry and RNA-sequencing analysis. I generated novel lists of potential new targets for CAR T cell therapy that will provide many opportunities to identify novel therapeutics for paediatric brain cancer in future. Further studies will be required to validate and explore these targets. Results from this thesis illustrate the importance of validation of immunotherapeutic targets. Whilst the H3.3K27M mutation is an important driver of disease, results suggest that the H3.3K27M peptide is not the ideal target for CAR T cell therapy due to the protein expression level, resulting in a limited CAR T cell response. Further studies may be required to either investigate methods to augment CAR function, or to increase expression of the H3.3K27M peptide in HLA-A*02. In addition, ongoing work into generation of new targets for paediatric brain cancer remains crucial, given the paucity of known tumour-associated antigens in this field.
A quantitative analysis of the PD-1 immune checkpoint in T cell proliferation

Biemond, Melissa ( 2022)

Upon activation, T cells undergo a controlled division burst to form a pool of antigen-specific effector cells. Previous work using quantitative T cell assays has demonstrated that parameters including entry into division, subsequent division rate, cell survival, and the number of times the cells divide before returning to quiescence (termed division destiny) determine the size and duration of the division burst. These key variables are independently controlled by the type and strength of T cell receptor (TCR), co-stimulatory and cytokine signals received during activation. Marchingo et al. (2014) established that co-stimulatory inputs linearly sum to determine division destiny and consequently the overall size of the response. I investigated how inhibitory signals integrate into this linear addition model of multiple co-stimulatory signals. Co-inhibitory receptors, such as programmed death receptor 1 (PD-1) are expressed on T cells after activation and are known to inhibit T cell responses. I focused on PD-1 as a quintessential inhibitory signal that has been well established as a cell intrinsic inhibitory signal in T cells. However, the precise mechanisms by which PD-1 modulates T cell proliferative responses are not yet fully understood. Thus, I applied a quantitative approach to investigate the role of PD-1 signalling in CD8+ T cell proliferative responses. I developed a quantitative dendritic cell-T cell co-culture assay for controlled delivery of co-stimulatory and inhibitory signals, including PD-1, to T cells in vitro. Using this system, I discovered that PD-1 signalling reduced proliferation of naive T cells by specifically decreasing division destiny, with no effect on cell survival. Moreover, I uncovered a novel IL-2-independent pathway for PD-1-mediated inhibition, yet IL-2 signalling also amplifies the inhibitory effect of PD-1. I go on to show that PD-1 reduces division destiny independent of TCR, CD28 signals and 4-1BB signals. From these findings I propose that PD-1 integrates with the calculus of multiple co-stimulatory signals by linearly subtracting from division destiny to influence the overall magnitude of the proliferative response.

Medical Biology - Theses

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