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(2020)ThesisActivated PI3K Delta Syndrome 1 (APDS1) is a rare condition caused by heterozygous gain-of-function (GOF) mutation in PIK3CD, which encodes the leukocyte-restricted p110δ catalytic subunit of phosphoinositol 3-kinase (PI3K). PI3K is activated downstream of many T cell receptors and has been implicated in the control of T cell differentiation, activation and proliferation. APDS1 patients have increased PI3K activity leading to several immune manifestations including hepatosplenomegaly, chronic herpes-virus infections, recurrent respiratory tract infections and impaired Ab responses. Here we phenotyped the T cells in a CRISPR/Cas9 mouse model harbouring the ortholog of the common mutation seen in ~80% of patients (Pik3cdE1020K). This revealed the peripheral blood T cells in these mice phenocopy what is observed in the blood of APDS1 patients. Analysis of the spleen revealed increased CD4+ T cell numbers which contributed to the splenomegaly seen in Pik3cd GOF mice. Moreover, T cell activation was deeply disturbed across the lymphoid organs, with a decreased frequency of naïve T cells and a concurrent increase in memory T cells. Mixed bone marrow chimeras revealed the T cell activation was extrinsically driven. Further, chimeras revealed that Pik3cd GOF in myeloid cells was not sufficient to drive this T cells activation. Similarly, we saw few changes in CD4+ T cells activation in vitro when they interacted with Pik3cd GOF APCs during antigen presentation. Thus, it remains unclear what drives this T cell activation. In addition, this thesis examined the effect of Pik3cd GOF on T cell function. In vitro polarising cell cultures of Pik3cd GOF naïve CD4+ and CD8+ T cells revealed altered cytokine expression, indicating Pik3cd GOF alters T cell differentiation. As impaired humoral response is a marked characteristic of these patients, we also analysed Pik3cd GOF Tfh cell function by using an in vivo antigen specific model. We observed similar frequencies of Tfh cells generated, however the Pik3cd GOF Tfh cells showed impaired ability to sustain GCs. Together, the results presented in this thesis reveal that PI3K plays a key role in modulating T cell activation, differentiation and function and provide new insights into the pathophysiology of APDS1 patients.
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(2021)ThesisBacterial superantigens are a key determinant in the incessant war between man and pathogen. Several notable examples include Streptococcal pyrogenic exotoxins (SPEs), Staphylococcus aureus enterotoxin D (SED) and Peptostreptococcus magnus protein L (PpL), all of which are virulence factors that evade our adaptive immune system. However, one in particular known as Staphylococcus aureus protein A (SpA), is a classic example of a potent B cell superantigen that causes numerous pathologies in humans such as septicaemia, endocarditis and toxic shock syndrome. The highlighted feature of SpA is that it exclusively targets VH3 germline derived B cells and immunoglobulins to perturb our immune system’s natural defence mechanisms. The VH3 germline happens to be the most frequently used germline class in our naïve B cell repertoire, and this is what makes this antigen so ‘super’. Humans have exploited this feature for basic applications as its affinity for immunoglobulins provided an ideal purification reagent for recombinantly expressed antibodies. We’ve exploited it once before, but the question is can we exploit it once more? This thesis seeks to answer the question whether naturally occurring interactions between superantigens and antibodies can in fact be manipulated. Using principles of protein engineering and design, I have outlined a rational strategy to which SpA can be engineered such that its affinity and specificity can be shifted towards a non-VH3 germline class. The success of this approach was based on strategically designing a tailored library of SpA domain mutants by targeting key contact residues that govern its binding. Coupling this with phage display technology enabled high throughput selection of superior variants that not only showed exclusive affinity to recombinantly expressed VH1-69 germline antibodies but also the ability to maintain functionality in context to live B cells. Having never been attempted before, the novelty of this work engenders a myriad of potential biotech applications for engineered SpA. More importantly however, the findings from this work proves that superantigen properties can be exploited. During the course of this Ph.D., the COVID19 pandemic quickly emerged as a world health crisis. The desperate search for therapeutic intervention quickly ensued. Success from the previous work on bacterial superantigens begged the question whether the same principles of molecular engineering can be adopted in context to antibody engineering. Here, I showcase how SARS antibodies can be engineered such that their affinity and specificity is shifted towards the COVID19 virus. For this, I adopt the same rationale of targeted mutagenesis using structural insights for tailored library design and phage display technology for high throughput screening. This enabled the discovery of several high affinity antibody candidates that were not only mutationally robust against the emergent strains but also harboured potent neutralising capabilities. This work offers an excellent strategy to rapidly generate highly desirable COVID19 antibodies that have strong applications as anti-viral drug therapies and/or reagents for diagnostic tools. Furthermore, it provides a framework for general antibody discovery techniques that can be used to combat current and future infectious disease outbreaks.
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(2020)ThesisThe accelerated progress in genomic technologies enables a systematic molecular reclassification of cancers, and can guide therapeutic opportunities. The clinical translation of genomic insights has lagged behind the pace of scientific discovery. This work analyses practical approaches to using genomic output to inform patient care. The Molecular Screening and Therapeutics program enabled patients with advanced cancer to access molecular tumour profiling in the Australian setting. Molecular results were reviewed by a molecular tumour board to determine the therapeutic relevance of results, and to guide therapeutic options through the program, or via other clinical trials/ or compassionate access schema. Enrolment was agnostic to tumour histology, with a focus on rare and less common cancers, where histology specific trials are limited. Our findings underscore the importance of trials using genomic selection as a vehicle for accessing novel therapies for these populations. The histology-agnostic approach required innovative approaches to trial endpoints to enable comparisons between widely different cancer types, as well as translational correlates to assist in interpretation of clinical benefit. We developed workflows to triage incidentally identified germline variants of clinical importance. This was necessary because of increasing uncertainty regarding traditional clinical parameters to guide prioritisation of cases for follow up, given this was not the primary purpose of the tumour profiling. A separate analysis of the therapeutic implications of germline variation showed that this is likely to be an increasingly important factor in coming years. Lastly, clinicians’ experiences with navigating and communicating genomic profiling results was explored, given their key role in translating genomic results into treatment selection. Most oncologists felt responsible for this, but often wanted assistance. Oncologists reported gene and variant interpretation, and access to the recommended therapies as the main barriers to treatment. This PhD contributes to the development of precision oncology through practical approaches to the clinical translation of genomic output. Future endeavours should prioritise the evaluation of impact on patient outcomes, the implications of germline findings, and further innovation in trials design to enable greater access to, and faster progress in the development of new cancer treatments.
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(2021)ThesisActivated PI3K Delta Syndrome (APDS) is a class of immunodeficiencies caused by mutations that dysregulate phosphoinositide-3-kinase (PI3K) signalling in lymphocytes. Gain-of-function (GOF) mutations in the PIK3CD gene, encoding the lymphocyte-restricted p110δ catalytic subunit, and loss-of-function (LOF) mutations the PIK3R1 gene, encoding the p85 regulatory subunits, both result in manifestation of APDS (APDS1 and APDS2, respectively). PI3K is activated downstream of the B cell receptor and has been implicated in B cell development and function. APDS patients have increased PI3K activity leading to several immune manifestations, including recurrent respiratory tract infections and impaired antibody responses. Paradoxically, a proportion of APDS patients also present with antibody-mediated autoimmune manifestations. To determine how dysregulated PI3K affects B cell biology, we generated two novel mouse models using CRISPR/Cas9-mediated gene editing to introduce the ortholog of the common mutation seen in ~80% of APDS1 patients (Pik3cdE1020K), or to target a splice site that is commonly mutated in APDS2 patients (Pik3r1E11SpD). Analysis of Pik3cdE1020K mice revealed defects in B cell development both in the bone marrow and the periphery, mirroring what is observed in APDS1 patients. In contrast, Pik3r1E11SpD mice had a largely unaltered peripheral B cell compartment. However, in vitro stimulation of follicular B cells from Pik3r1E11SpD mice revealed impaired isotype switching to IgG subclasses, comparable to that observed in Pik3cdE1020K B cells, and consistent with antibody defects seen in APDS1 and APDS2 patients. To further understand the autoimmune features in APDS1 patients, we utilised a mouse model of B cell self-reactivity. Pik3cdE1020K mice were crossed with the SWHEL transgenic mouse model. Using mice that express low-affinity HEL3X as a membrane self-antigen, mixed bone marrow chimeras were generated, whereby Pik3cdE1020K SWHEL B cells developed in the presence of self-antigen. Strikingly, these B cells failed to undergo anergy and instead expanded in the periphery, differentiating into self-reactive plasmablasts, secreting high titres of IgM autoantibody, and seeding germinal centres. Together, these findings highlight the importance of regulated PI3K signalling in B cell development and function and suggest a novel mechanism in which dysregulated PI3K signalling switches a tolerogenic signal into an immunogenic signal. Thus, this thesis provides further insights into the pathophysiology of APDS patients.
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(2021)ThesisProstate cancer is a leading cause of cancer-related death globally and is characterised by significant intra- and inter-patient genomic heterogeneity. This heterogeneity manifests as variable disease presentations ranging from indolent cancers for which no treatment is needed to aggressive disease that impacts quality of life and survival. Additional biomarkers that predict clinical course and refine therapeutic decision-making to avoid over- and under-treatment and improve outcomes in men with aggressive disease are urgently needed. Several promising genomic biomarkers have emerged. This thesis firstly summarises the landscape of prostate cancer genomics and its clinical utility in prostate cancer. The second chapter characterises the germline in men with advanced prostate cancer treated at an Australian tertiary centre to identify potential factors that could guide referrals for germline assessment. The third chapter reviews the interactions between two important and therapeutically targetable pathways, androgen receptor and phosphoinositide 3-kinase, in prostate cancer and assesses their therapeutic implications. The fourth chapter of this thesis examines the real-world potential for whole genome sequencing and optical mapping data to inform treatment selection. Finally, cell-free DNA as a predictor of response to novel treatments is explored in Chapter 5. Overall this work assesses the potential for DNA sequencing in prostate cancer to impact patient outcomes.
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(2021)ThesisFusion genes are known to be key diagnostic, prognostic and therapeutic targets in blood cancers. However in sarcoma, the fusion gene landscape has yet to be fully elucidated due to the heterogeneity of fusion gene partners and rarity of the samples. Current fusion gene diagnostics are only able to detect presence of the fusion and are unable to identify novel fusion gene partners or splice isoforms. Therefore to expand on the knowledge of fusion genes in sarcoma, my study aims to sequence RNA from archived sarcoma patient samples by targeting a panel of 255 genes known to be involved in fusion. Through targeted sequencing, accurate fusion gene detection can be achieved along with information on novel fusion gene partners or splice junctions. Finally gene expression analysis was performed to gain insight into the transcriptome of sarcoma and identify the relationship of fusion genes to clinical prognostic markers. In this study, RNA was sourced from formalin fixed paraffin embedded (FFPE) samples, which is often degraded and chemically modified. However based on quality assessment, fusion gene detection could still be carried out in libraries as small as 1 million reads. Fusion genes were found to be identified in 44% (43/98) of patient samples with 38 of these fusions consistent with those reported in literature. Novel fusion isoforms were also found to contain different breakpoints but retain the same protein domains as the recurrent fusion isoform. Through gene expression analysis of fusion positive and negative Ewing’s sarcoma, it was found that 52% of differentially expressed protein coding isoforms contained fewer regulatory elements compared to the principal isoform of the gene. This suggests that presence of the fusion in Ewing’s sarcoma may be related to eased expression of certain genes. To identify any other links fusion genes may have to gene expression, levels of prognostic immune markers were compared to fusion type. While fusion type did not appear to be associated with expression of these immune genes, varying expression levels between samples may potentially act as a predictor for immunotherapy success.
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(2021)ThesisCDK4/6 inhibitors in combination with endocrine therapy have recently become standard-of-care for treatment of advanced/metastatic ER+ breast cancer. Despite initial improvements in patient responses, resistance to combination endocrine–CDK4/6 inhibitor therapy is rapidly emerging. While there have been intense efforts to understand resistance, the mechanisms identified so far from cancer model systems have generally not been reproduced in clinical samples. We hypothesise that this is because most prior models have been of CDK4/6 inhibitor resistance alone, rather than recapitulating the clinical situation of advanced/metastatic disease which is heavily pre-treated with endocrine therapy. To understand the complexities of resistance we adopted multiple approaches: generating a cell line resistant to palbociclib alone and characterising the mechanisms of resistance, generating and comparing cell lines resistant to multiple lines of therapy, and a whole-genome CRISPR/Cas9 screen to identify drivers of resistance to endocrine–CDK4/6 inhibitor treatment. We first generated and characterised a cell line model of palbociclib resistance. Resistance to palbociclib occurred via altered cell cycle kinetics. Moreover, CDK2 inhibition in combination with palbociclib enhanced growth inhibition and promoted the induction of senescence in resistant cells. To complement this model, we developed four unique long-term in vitro models (>16 months) of combination endocrine therapy and palbociclib resistance. Using single-cell RNA sequencing, we identified transcriptional changes within these cells along the trajectory to endocrine–CDK4/6 inhibitor resistance. Finally, using a genome-wide screening approach we discovered knockout of the MAP kinase signalling gene MKK7 mediated resistance to combined endocrine–CDK4/6 inhibition in vitro. Following in vitro validation, we demonstrated MKK7 loss in the metastatic setting using in silico datasets. Overall, we found that novel pathways are altered in resistance to combination therapy compared to cells that are resistant to single arm palbociclib. This includes several pathways that could be targeted therapeutically in drug resistant disease. With the unique laboratory models of endocrine–CDK4/6 inhibitor resistance that were generated as part of this thesis, we are now placed to further develop targeting of these pathways, with the ultimate aim of improving clinical outcomes for patients with this rapidly emerging form of drug resistant breast cancer.