Medical Biology - Theses
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ItemThe role of venetoclax in the treatment of breast cancer( 2023-03)Breast cancer is the most commonly diagnosed cancer in women. The 5-year overall survival for metastatic breast cancer is approximately 30%. Despite rapid developments of new systemic therapies, cancer progression is almost inevitable. Ongoing research about treatment-resistance in breast cancer and mechanisms to overcome this are crucial to improving the lives of women with metastatic disease. Apoptosis is an important process for eliminating cancer cells. The BCL-2 pro-survival proteins and BH3-only proteins are key regulators of cell death. Cancer cells can evade apoptosis through over-expression of the pro-survival proteins. A new class of drug, the BH3 mimetics, inhibit the pro-survival proteins and have undergone extensive investigation in haematological malignancies. These drugs target BCL-2, BCLxL and MCL-1 and have shown promising activity in pre-clinical models. Venetoclax is a BH3 mimetic that specifically and potently binds to BCL-2. Venetoclax has transformed the treatment landscape for BCL-2- positive chronic lymphocytic leukaemia. It achieved fast-track FDA approval due to its success in the clinic. BCL-2 is overexpressed in approximately 70% of oestrogen receptor-positive metastatic breast cancers and approximately 30% of triple negative breast cancers. The aim of this thesis is to investigate the role of venetoclax in the treatment of BCL-2-positive breast cancer. We aim to achieve this through the design and implementation of three phase 1b clinical trials combining venetoclax with standard systemic therapy in the following clinical settings: (1) oestrogen receptor-positive and BCL-2-positive breast cancer in any line of metastatic treatment, (2) oestrogen receptor and BCL-2- positive breast cancer in the early lines of metastatic treatment (1st – 3rd line), (3) metastatic triple negative breast cancer. These clinical trials will firstly explore the safety and tolerability of venetoclax when combined with standard systemic therapy in metastatic breast cancer. Secondly, they will provide data on efficacy, survival and exploratory endpoints that inform future phase II and phase III studies. Findings from these later phase studies would determine whether venetoclax improves clinical outcomes in breast cancer and identify which patient subset might benefit. It is anticipated that due to the rapidly growing drug development in the area of BH3 mimetics, venetoclax may not be the most clinically relevant BH3 mimetic in the future. Therefore our clinical trial protocols could form a basis for future studies that investigate the next generation of BH3 mimetics or novel therapies targeting pro-survival proteins. The mBEP phase Ib clinical trial treated women with oestrogen receptor-positive and BCL-2-postive breast cancer in any line of metastatic treatment. This is the only study forming my thesis that has reached the primary and secondary endpoints. mBEP showed that the combination of tamoxifen and venetoclax was safe and tolerable. Secondary endpoints have confirmed that there is no adverse survival outcome from the addition of venetoclax to standard of care endocrine therapy.
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ItemStatistical and Machine Learning models for estimation of missing values in label-free mass spectrometry quantification( 2022)Mass spectrometry (MS) enables high throughput identification and quantification of proteins in complex biological samples and can provide insights into the global function of biological systems, aberrations and disease progression. Label-free quantification is cost effective, suitable for analysis of human samples and can profile proteins from a broad range of abundance. Despite rapid developments in label-free data acquisition workflows, the number of proteins commonly quantified across samples can be limited. This results in missing values in the measurements between samples, which present substantial challenges for downstream data analysis tasks and biomedical discoveries. This thesis provides two solutions for the treatment of missing values in label- free mass spectrometry: (i) imputation of missing values after quantification using Barycenter computation from Optimal Transport discipline in Machine Learning research, and (ii) a deep learning solution for sequence identification transfer between precursor ions across samples at the quantification step. In the two methodological manuscripts arose from this thesis, I demonstrate how these two solutions enhance data completeness in label-free mass spectrometry acquisition, thereby facilitating biomedical discoveries. I then provide a perspective on the future directions of these two works. The tools developed in this work are available on open-source software repositories and are used by the proteomics and bioinformatics community in medical research projects.
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ItemUnderstanding retinal diseases with genotypic and transcriptomic data analysis( 2021)The retina is light-sensitive eye tissue responsible for vision, but little is known about the genetic regulation of retinal gene expression. Investigating key drivers of gene regulation in the retina in healthy and diseased individuals remains a fundamental challenge in macular degeneration research, especially given the difficulty of accessing human retinal tissue. Deciphering the effects of genetic variation on retinal gene expression will underpin the development of novel treatment avenues for otherwise untreatable diseases causing blindness. A method to investigate these further focuses on the effects of genetic variants on gene expression levels derived from transcriptomic data. This type of ‘omics analysis, known as expression quantitative trait (eQTL) analysis integrates genotype and gene-expression data. The genotyping data for this thesis was generated in collaboration with scientists from the TIGEM, Italy, who first assembled the retinal transcriptome. We aimed to identify the genetic variants that modulate gene expression using a cohort of 41 individual donors of healthy retinal tissue. We performed retinal eQTL analysis using this independent cohort and compared our results with recently published retinal eQTL studies. After observing a weak eQTL signal potentially due to the small sample size, we explored potential strategies to mitigate the multiple testing burden so as to improve statistical power. To this end, we performed eQTL power analyses and limited both the set of variants and genes under consideration by thresholding on allele frequency and gene transcriptional abundance as well as disease relevance. Further, eQTL analysis was used to interpret the genetics of Macular Telangiectasia II, a blinding retinal degenerative disease. This included genome-wide and targeted interrogation of the signals from the largest genome-wide association study to date for this disease.
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ItemIdentifying Novel Strategies to Enhance the Anti-cancer Activity of Venetoclax by Manipulating NOXA Expression( 2021)Apoptosis is a form of programmed cell death. The intrinsic pathway of apoptosis is governed by the BCL2 family proteins. Targeting BCL2 proteins by small molecules that mimicking the BH3-only proteins to induce apoptosis has proven to be a successful strategy for cancer therapy. Venetoclax, a specific inhibitor of BCL2, has exhibited remarkable efficacy in treating cancers that rely on BCL2 for survival. However, the activity of venetoclax is often limited in other cancers whose survival relies on MCL1, another BCL2 family member. Selective MCL1 inhibitors have been developed and are currently being evaluated in clinical trials. However, the clinical development of these agents has been hampered by toxicity, especially cardiac toxicity. Potentially, another strategy to target MCL1 is by modulating NOXA, a BH3-only protein that selectively binds to MCL1 and mediates its degradation. I hypothesised that increased NOXA expression would prime cancer cells to venetoclax killing and that this would reduce their co-dependence on MCL1. In order to identify new targets to modulate NOXA expression, I generated and validated cell lines that report on NOXA transcription and then carried out CRISPR-Cas9 genetic screens in those NOXA reporter cell lines. In CRISPR-Cas9 loss-of-function screens focused on epigenetic regulators, I found several genes whose mutation or loss modulated NOXA expression, including CTBP1, CHTOP, ZMYM3, SPEN, HSPA1A, KEAP1, FOXA1, HDAC3 and SAP30. Some of these factors have been targeted for cancer therapies, for example KEAP1 and HDAC3, while the others have not yet been recognized for their therapeutic possibilities. Subject to their validation, my results have identified interesting novel mechanisms of NOXA regulation, thus providing the rationale basis for the development of new anti-cancer agents. In CRISPR-Cas9 tiling screens that focused on the NOXA promoter region, five cis-regulatory elements were identified that contributed to regulation of NOXA expression. Among them, a hypermethylated element on the NOXA promoter was found to be important for repressing NOXA expression across diverse cell lines derived from blood cancers. Disrupting this region led to NOXA induction. Potentially, the findings could provide a rational basis of combining hypomethylating agents with venetoclax in a range of haematological malignancies. In summary, several potential NOXA regulating proteins and DNA elements were discovered by the CRISPR-Cas9 screening approaches. Once validated, these findings should provide new insights into NOXA regulation.
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ItemDevelopment of an Editable Approach to the Study Parasite-Erythroid Interactions( 2020)Malaria remains responsible for an enormous health burden worldwide; considerable research effort is being devoted to finding ways to combat the disease and its transmission. Malaria is caused by Plasmodium species, and P. falciparum causes the most serious disease. The blood stage of the P. falciparum remains critically important to understand for development of treatments and vaccines. To initiate invasion, the P. falciparum merozoite recognises specific proteins on the host red cell membrane, known as invasion receptors. In order to study parasite–host interactions, laboratory adapted P. falciparum strains that invade mature human red cells have been used. Gene modification methods are well established for P. falciparum; however, genetic manipulation of the red cell has not been extensively applied because erythrocytes are not nucleated. The in vitro cultivation of erythroid cell lines facilitates both the scalable production of host cells to support P. falciparum invasion and editing of nucleated precursors that can be genetically modified in a precise manner. In this project, two erythroid cell lines – the Human Umbilical cord blood Derived Erythroid Progenitors (HUDEP-2) and the Bristol Erythroid Line- Adult (BEL-A), both of which can differentiate to more mature forms in vitro – were studied as possible host models. A FACS antibody panel, based on the stage-specific profile of HUDEP-2 and BEL-A cells, provided the means to analyse host invasion receptors as well as erythroid maturation markers. Band 3 is a red cell membrane protein with an uncertain role in merozoite invasion. A gene knockout was constructed in expansion stage BEL-A cells using the lentiviral CRISPR/Cas9 system, targeting band 3 which may be involved in merozoite invasion of human erythrocytes. Single-cell-derived clones were isolated and preliminary validation using PCR and flow cytometry was performed to verify disruption of band 3. Completion of work to validate and functionally characterise the band 3 knockout, and experiments to assess effects on invasion, were curtailed by COVID-19 stay-at-home orders issued to Melbourne between March and July 2020. In summary, a genetically editable in vitro erythroid model was defined to study the function of host invasion receptors for P. falciparum merozoite invasion. Clonal band 3- deficient BEL-A cells were generated, thus paving the way for studying their role as invasion receptors.
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ItemCharacterization of plasmepsin X as a cross-species antimalarial target( 2019)The emergence and spread of drug resistance have hindered the campaign for malaria eradication. The development of new drug targets is critical for our anti-malarial arsenal of interventions. Plasmepsins, which are aspartic proteases expressed by malaria parasites, serve important functions for parasite survival. Among the 10 members of this enzyme family, plasmepsin X (PMX) is essential for P. falciparum growth and has been shown to be involved in the egress of merozoites from infected red blood cells and the invasion of merozoites into red blood cells. Several aspartic protease inhibitors have anti-malarial activity on P. falciparum and are proposed to target PfPMX. The aim of this project was to investigate if these compounds affect P. knowlesi growth and whether PMX is a cross-species target for antimalarial development. This work showed that two aspartic protease inhibitors, 49c and 1SR, caused inhibition of P. knowlesi parasite growth. In further studies, live cell imaging demonstrated that these compounds inhibit P. knowlesi parasite growth by blocking parasite egress. Next, the optimal condition for protease activity was characterised after the expression and purification of a functional recombinant P. knowlesi plasmepsin X (rPkPMX). Using a fluorogenic protease assay, both 49c and 1SR were shown to inhibit the activity of rPkPMX. Furthermore, rPkPMX was able to cleave synthetic substrates, which were based on the predicted cleavage sites of PfSUB1, PfRAP1, PfRh2, TgROP1 and TgMIC6 predicted cleavage sites. By screening a panel of aspartic protease inhibitors, the BACE1 inhibitor, LY2886721, was identified as an inhibitor of rPkPMX activity as well as P. knowlesi and P. falciparum parasite growth. Therefore, PMX can be used as a cross-species target for antimalarial drug development.
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ItemGM-CSF regulation in inflammatory arthritis( 2019)Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease of the joints, affecting 0.5% to 1% of global population. Current targeted therapies antagonize the debilitating effects of key inflammatory mediators and immune cells. However, few patients achieve complete remission, prompting novel therapeutic approach. Granulocyte/Macrophage-Colony Stimulating Factor (GM-CSF) was first identified as a haemopoietic growth factor but is now recognised as a proinflammatory cytokine in a number of autoimmune inflammatory diseases, including RA and Multiple Sclerosis (MS). GM-CSF promotes destructive joint inflammation by priming pro-inflammatory phenotypes of myeloid cells, such as neutrophils, monocytes and macrophages. Accordingly, therapies targeting GM-CSF or its receptor are currently under clinical evaluation in RA and MS and seem promising. However, much remains to be explored how GM-CSF is dynamically regulated during arthritis, especially in autoantibody-mediated inflammation as seen in seropositive RA patients. This work aims to characterize the cellular source of GM-CSF during antibody-induced arthritis and evaluate the cell-intrinsic negative regulation of GM-CSF signalling in myeloid cells and arthritis. I utilized the autoantibody-driven, immune complex-mediated serum transfer induced arthritis (STIA) murine model, which mimics the effector phase of seropositive RA patients. Using the novel GM-CSF dual reporter mice, joint-infiltrating Natural Killer (NK) cells were found to be main GM-CSF-producing cells during STIA. By using NK-deficient (Mcl1fl/fl:Ncr1-Cre) mice, NK-depleted (anti-NK1.1 antibody-treated) mice or specifically deleting GM-CSF production by NK cells (Csf2fl/fl:Ncr1-Cre), I was able to show the importance of NK cells and their GM-CSF-producing function in maintaining arthritis (Chapter 3). GM-CSF on myeloid cells induced the Cytokine Inducible SH2-containing (CIS) protein, a member of the suppressor of cytokine signalling (SOCS) protein family. Using Cish-/- mice, I showed that CIS negatively regulated GM-CSF signalling post activation, which is evident in intracellular signalling pathways, effector cell functions and in antibody-induced arthritis (Chapter 4). Taken together, this study provides new insights into the pathogenesis of antibody-driven, GM-CSF-mediated autoimmune inflammation and provide a rationale towards designing novel anti-inflammatory agents such as NK modulator or CIS mimetics for RA.
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ItemCharacterisation of the Plasmodium aspartyl proteases DNA-damage inducible protein 1 (DDI1) and Plasmepsin VII (PMVII)( 2019)Plasmodium falciparum resistance to artemisinin-(ART) based combination therapies (ACTs) and other antimalarials poses a major threat to malaria control and elimination. Current efforts are aimed towards identifying potent antimalarials which inhibit multiple stages of the parasite lifecycle or discovering novel drug targets which may help overcome ART-resistance. This work aimed to characterise two aspartyl proteases of P. falciparum which may hold promise as antimalarial targets. One strategy recently proposed to overcome ART-resistance is the synergistic use of a parasite-selective proteasome inhibitor to sensitise ART-resistant parasites to artemisinin. Therefore, development of an inhibitor targeting a parasite-specific protein involved in the P. falciparum ubiquitin-proteasome system (UPS) could yield a combination therapy to tackle ART-resistance. DNA-damage inducible protein 1 (DDI1) is a previously uncharacterised essential aspartyl protease in P. falciparum. Recent studies have shown that the catalytic domain of human DDI2 upregulates the UPS in mammalian cells. In other organisms, DDI1 plays a role in shuttling proteins to the proteasome for degradation via its ubiquitin-like domain. We hypothesise PfDDI1 is an active aspartyl protease and plays a role in the parasite’s UPS. To investigate the role of DDI1 in the UPS and parasite survival, we identified a DDI1 orthologue in P. falciparum and characterised this using several strategies. We utilised CRISPR-Cas9 to knock out, tag and inducibly knock down DDI1 across the asexual lifecycle of P. falciparum, and study the effect of this on parasites. Expression of recombinant DDI1 proteins provided insight into the protease activity and substrate repertoire of PfDDI1. Together these studies provide insight into the domain architecture, essentiality and function of PfDDI1 and clues into its potential as an antimalarial target. Development of an antimalarial to block parasite transmission between humans and mosquitos is also a viable strategy to reduce malaria burden. In this study, we also explore a potential transmission-blocking target, Plasmepsin VII (PMVII) and create tools to enable further study of this aspartyl protease in sexually reproductive gametocytes. These tools are vital to determine the function and substrate repertoire of PMVII and elucidate its potential as an antimalarial target.
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ItemIdentify new regulators of TNFR1-induced necroptotic pathway( 2019)Acute Myeloid Leukaemia (AML) is a vastly heterogeneous blood disorder with a poor prognosis for patients older than 65. Our group has been focused on developing new treatments for AML to replace the standard intensive chemotherapy. Previous data showed that the SMAC-mimetic birinapant in combination with the caspase inhibitor IDN could kill different types of AML both in vitro and in vivo through activation of necroptosis cell death pathway. However, over 50% of the patient samples tested in study showed resistance to necroptosis. This project aims to determine the molecular mechanisms that mediate necroptosis resistance in AML and identify new regulators of necroptotic pathway. The results obtained in this study will expand the knowledge of necroptosis signalling in leukaemia and will contribute to the optimal clinical use of birinapant/IDN drug combination. This project contains 2 parts; (1) We will use human AML cell lines that are resistant to necroptosis to determine the molecular changes involved in cell death resistance. (2) We will use CRISPR/Cas9 knock out screen in human AML cell lines that are sensitive to necroptosis, trying to identify new regulators of TNFR1-induced necroptotic pathway. Together these experimental approaches will allow a better understanding of the regulation of TNF-necroptosis signalling in AML. By overexpression wild-type RIPK3 in the KG-1 cell line, we successfully sensitised KG- 1 cells to necroptosis, which indicates that the KG-1 endogenous RIPK3 is dysfunctional. By cDNA sequence of KG-1 endogenous RIPK3, we detected several mutant base pairs, which may lead to the dysfunction, but this result needs further prove by genome sequence, which is undergoing. By CRISPR knock out screen, we found several targets that may lead to the necroptotic resistant, and MAGE3 is the most research-worthy one. Knockout MAGEB3 in the MV4;11 cell line led to the downregulation of RIPK3 and the necroptotic resistance. However, this result could not be repeated on the U937 cell line, and the mechanism of how MAGEB3 regulates RIPK3 is still unclear. Further research will be done on MAGEB3 to have a better understanding of the role of MAGEB3 in the necroptotic pathway. Together, all these results gave a better understanding of the necroptotic pathway and may contribute to the treatment of AML.
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ItemIdentifying Plasmodium vivax encoded proteins that may prevent host cell death during liver development( 2019)Malaria is a major global health problem and a leading cause of death worldwide. The mechanism behind some parts of the parasite life cycle are still obscure, especially the liver stage which is essential for parasite development and maturation. It is likely that the parasite prevents the host hepatocyte from undergoing cell death during invasion. This is especially relevant for Plasmodium vivax as the hypnozoite can lay dormant in a liver cell for months, even years. We hypothesise that P. vivax encodes proteins to inhibit host cell death in liver. We used the computer algorithm I-TASSER to identify several P. vivax proteins which were predicted to have similar structures to human proteins involved in cell death. We expressed these P. vivax proteins in mammalian cells and performed functional tests to investigate their potential roles experimentally. Identification of P. vivax proteins that influence host cell death would improve our understanding of how P. vivax can survive for prolonged periods in the host cell during liver stage and may accelerate the development of new drugs for malaria liver stage, which is necessary for the ultimate goal of eliminating malaria.