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The immune response to a given cancer can profoundly influence a tumour's trajectory and response to treatment, but the ability to analyse this component of the microenvironment is still limited. To this end, a number of immune marker gene signatures have been reported which were designed to enable the profiling of the immune system from transcriptomics data from tissue and blood samples. Our initial analyses of these resources suggested that these existing signatures had a number of serious deficiencies. In this study, a co-expression based approach led to the development of a new set of immune cell marker gene signatures (ImSig). ImSig supports the quantitative and qualitative assessment of eight immune cell types in expression data generated from either blood or tissue. The utility of ImSig was validated across a wide variety of clinical datasets and compared to published signatures. Evidence is provided for the superiority of ImSig and the utility of network analysis for data deconvolution, demonstrating the ability to monitor changes in immune cell abundance and activation state. ImSig was also used to examine immune infiltration in the context of cancer classification and treatment. Patient-matched ER+ breast cancer samples before and after treatment with letrozole were analysed. Significant elevation of infiltration of macrophages and T cells on treatment was observed in responders but not in non-responders, potentially revealing a biomarker for response. ImSig was also used to study the immune infiltrate in 12 cancer types. By computing the relative abundance of immune cells in these samples, their relationship to survival was investigated. It was interesting to observe that half of the cancers showed trends towards poor prognosis with increased infiltration of immune cells. ImSig alongside the network-based framework can also be used for a more explorative analysis such as to identify biomarkers and activation or differentiation states of immune cells. Melanoma is a highly immunogenic cancer and has shown tremendous success with immune checkpoint inhibitors in a subset of patients. In chapter-6, the molecular subgrouping of melanoma was explored using a network-based approach. Despite the plethora of evidence suggesting various aspects of the immune system to contribute towards the response to immunotherapy in melanoma, there has been little to no effort to consider this heterogeneity while developing molecular subgroups. The use of ImSig was therefore explored for the stratification of melanoma patients into immuno-subgroups. The subgrouping methodology divided the tumours into four groups with different immune profiles. Interestingly, these groupings showed prognostic significance, reiterating the need to consider the heterogeneity of immune cells in future studies. On identifying the most dominant phenotypes that contribute towards prognosis of these patients and in comparison to the published subgroupings of melanoma, we argue that the subgroup of samples enriched in keratin genes are not clinically meaningful. ImSig and the associated analysis framework described in this work, support the retrospective analysis of tissue derived transcriptomics data enabling better characterisation of immune infiltrate associated with disease, and in so doing, provide a resource useful for prognosis and potentially in guiding treatment.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents a curative treatment option for AML. Alloreactive donor T cells can produce the graft-versus-leukemia (GVL) effect which represents a major therapeutic benefit of allo-HSCT. However, evading from allogeneic immune surveillance, potentially through the downregulation of human leukocyte antigen class II (HLA-II) antigen presentation machinery, can contribute to AML relapse. Lysine-specific demethylase 1 (LSD1) is an emerging epigenetic therapeutic target in AML. The present study aims to explore whether LSD1 inhibition can enhance AML immunogenicity to promote T-cell mediated immune response. The immunological effects of a clinical-stage LSD1 inhibitor bomedemstat (IMG-7289) were examined in both human and murine AML cell models in vitro. The results demonstrated that bomedemstat treatment significantly enhanced the expression of class II transactivator (CIITA). It subsequently led to the upregulation of HLA-DR in certain human AML cell lines when stimulated by IFN-γ. Bomedemstat also markedly upregulated the expression of CD86 in all human AML cell lines tested. The study also demonstrated that bomedemstat treatment significantly increased the production of pro-inflammatory cytokines IL-12 and CXCL-10. In murine AML models, bomedemstat concurrently upregulated the expression of major histocompatibility complex class II (MHC-II) and CD86 in H9M-transformed cells without IFN-γ stimulation. This effect was not observed in MN1-transformed cells. Bomedemstat-treated H9M cells were subsequently shown to induce antigen-dependent T cell activation. Functional assays revealed that bomedemstat treatment sensitized H9M cells to antigen-dependent immune killing effect mediated by CD4+ T cells. In conclusion, the current study demonstrates both phenotypically and functionally that LSD1 inhibition by bomedemstat treatment can enhance AML immunogenicity. This is evident by the increased antigen presentation, co-stimulation and production of inflammatory cytokines. These findings suggest that LSD1 inhibition may be a relevant strategy to pursue as a maintenance therapy after allo-HSCT. / Thesis / Master of Science in Medical Sciences (MSMS) / Acute myeloid leukemia (AML) is an aggressive blood cancer characterized by the rapid growth of abnormal blood cells in the bone marrow. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an important treatment for AML that involves taking blood stem cells from a healthy donor and transplanting them into an AML patient. Meanwhile, immune cells such as T cells from the donor can help destroy leukemia cells. However, AML frequently reappears after allo-HSCT and new therapies are needed to prevent the disease from coming back. The present study investigates whether blocking a protein called lysine-specific demethylase 1 (LSD1) can increase T cells’ ability to identify and kill cancer cells. The results demonstrate that treatment with an LSD1 blocker called bomedemstat can enhance the recognition of AML cells by T cells, thereby enhancing their immune response. These findings suggest that blocking LSD1 is a promising approach to enhance the effectiveness of allo-HSCT.

Cancer is the leading cause of death in Canada and one of the leading causes of death worldwide. Conventional cancer therapies such as chemotherapy often include severe side effects that can decrease the quality of life of patients undergoing treatment. Immunotherapy is designed to harness the host immune response and enhance its ability to seek out and kill cancer cells. Immunotherapy has gained traction in the past decade due to its improved safety and efficacy over conventional cancer therapies. However, there is room for improvement as most patients fail to respond to immunotherapy. The work described in this dissertation involves the development of therapeutic combination platforms that are designed to improve upon immunotherapy outcomes. Murine tumor models were used to develop a better understanding of biological processes associated with therapeutic efficacy. These findings can be used for the development of therapeutic strategies that can further improve the efficacy of cancer immunotherapy. / Cancer immunotherapy has demonstrated immense promise in the past decade. Immune checkpoint therapy has shown unprecedented responses in many cancers; however most patients fail to respond to checkpoint therapy. This highlights the need to develop a better understanding of factors in the tumor microenvironment that can influence therapeutic outcomes. In this body of work, we have utilized oncolytic viruses (OVs) to enhance immunogenicity in the tumor and study the cellular mechanisms that enable a therapeutic response. We utilize a combination of OVs and low dose chemotherapy to further sensitize murine models of mismatch repair-deficient colorectal cancer to checkpoint therapy. Using a Clariom S transcriptome assay we found that the combination induced gene signatures associated with the recruitment and activation of myeloid subsets. When we assessed tumor infiltrates, we found that the combination induced the chemoattraction of several myeloid subsets, including type I conventional DCs (cDC1s) which are known for their role in antigen presentation. Using Batf3-/- mice, we demonstrated that the therapeutic efficacy of our combination platform was dependent on the presence of cDC1s. In this dissertation, we also studied the role of OV-induced type I IFN (IFNI) in enabling or suppressing antitumor immunity. We found that OVs induced the upregulation of PD-L1 in an IFN-I-dependent manner in cancer cells and circulating immune cells. Inhibition of IFN-I signaling using an anti-IFNAR monoclonal antibody partially prevented OV-induced upregulation of PD-L1. Furthermore, the combination of OV and v | P a g e IFNAR blockade enhanced the effector functions of tumor-specific T cells and led to better tumor control compared to OV monotherapy. Altogether, these findings demonstrate that OVs can be an effective agent for enhancing immunogenicity in the tumor and promoting the infiltration of inflammatory myeloid subsets. By combining OVs with checkpoint or IFNAR inhibitors, we prevent the onset of immunosuppression and enable a favorable therapeutic response. / Thesis / Doctor of Philosophy (Medical Science)

Tumours are highly heterogeneous collections of tissues characterised by a repertoire of heavily mutated and rapidly proliferating cells. Evading immune destruction is a fundamental hallmark of cancer, and elucidating the contextual basis of tumour-infiltrating leukocytes is pivotal for improving immunotherapy initiatives. However, progress in this domain is hindered by an incomplete characterisation of the regulatory mechanisms involved in cancer immunity. Addressing this challenge, this thesis is formulated around a fundamental line of inquiry: how do we quantitatively describe the immune system with respect to tumour heterogeneity? Describing the molecular interactions between cancer cells and the immune system is a fundamental goal of cancer immunology. The first part of this thesis describes a three-stage association study to address this challenge in pancreatic ductal adenocarcinoma (PDAC). Firstly, network-based approaches are used to characterise PDAC on the basis of transcription factor regulators of an oncogenic KRAS signature. Next, gene expression tools are used to resolve the leukocyte subset mixing proportions, stromal contamination, immune checkpoint expression and immune pathway dysregulation from the data. Finally, partial correlations are used to characterise immune features in terms of KRAS master regulator activity. The results are compared across two independent cohorts for consistency. Moving beyond associations, the second part of the dissertation introduces a causal modelling approach to infer directed interactions between signaling pathway activity and immune agency. This is achieved by anchoring the analysis on somatic genomic changes. In particular, copy number profiles, transcriptomic data, image data and a protein-protein interaction network are integrated using graphical modelling approaches to infer directed relationships. Generated models are compared between independent cohorts and orthogonal datasets to evaluate consistency. Finally, proposed mechanisms are cross-referenced against literature examples to test for legitimacy. In summary, this dissertation provides methodological contributions, at the levels of associative and causal inference, for inferring the contextual basis for tumour-specific immune agency.

No description available.

In the last decade cancer immunotherapy has taken huge strides forward from bench to bedside and being approved as drugs. Cancer immunotherapy harnesses the power of patient’s own immune system to fight cancer. Approaches are diverse and include antibodies, therapeutic vaccines, adoptively transferred T-cells, immune checkpoint inhibitors, oncolytic viruses and immune cell activators such as toll-like receptor (TLR) agonists. Excellent clinical responses have been observed for certain cancers with checkpoint antibodies and chimeric antigen receptor (CAR)-engineered T-cells. It is however becoming evident that strategies need to be combined for broader effective treatment responses because cancers evolve to escape immune recognition. A conditionally replication-competent oncolytic adenovirus (Ad5PTDf35-[Δ24]) was engineered to secrete Helicobacter pylori Neutrophil Activating Protein (HP-NAP, a TLR-2 agonist) to combine viral oncolysis and immune stimulation. Treatment with Ad5PTDf35-[Δ24-sNAP] improved survival of mice bearing human neuroendocrine tumors (BON). Expression of HP-NAP in the tumor microenvironment promoted neutrophil infiltration, proinflammatory cytokine secretion and increased necrosis. We further studied the ability of HP-NAP to activate dendritic cells (DCs) a key player in priming T-cell responses. HP-NAP phenotypically matured and activated DCs to secrete the T-helper type-1 (Th-1) polarizing cytokine IL-12. HP-NAP-matured DCs were functional; able to migrate to draining lymph nodes and prime antigen-specific T-cell proliferation. CAR T-cells were engineered to secrete HP-NAP upon T-cell activation. Secreted HP-NAP was able to mature DCs, leading to a reciprocal effect on the CAR T-cells with improved cytotoxicity in vitro. Semliki Forest virus (SFV), an oncolytic virus with natural neuro-tropism was tagged with central nervous system (CNS)-specific microRNA target sequences for miR124, miR125 and miR134 to selectively attenuate virus replication in healthy CNS cells. Systemic infection of mice with the SFV4miRT did not cause encephalitis, while it retained its ability to replicate in tumor cells and cure a big proportion of mice bearing syngeneic neuroblastoma and gliomas. Therapeutic efficacy of SFV4miRT inversely correlated with type-I antiviral interferon response (IFN-β) mounted by tumor cells. In summary, combining immunotherapeutic strategies with HP-NAP is a promising approach to combat cancers and SFV4miRT is an excellent candidate for treatment of neuroblastomas and gliomas.

The CD40 ligand (CD40L) molecule is central to innate and adaptive immunity. CD40L expression is very tightly regulated whereas its CD40 receptor is constitutively expressed by many different cell types. CD40L is expressed transiently on helper T cells (Th) only after activation by specific immune recognition molecules carried by professional antigen presenting cells, in particular, dendritic cells (DC). CD40L subsequently binds to CD40 on DC to enable full Th activation. CD40 ligated DC produce interleukin-12 (IL-12) and contribute both to the development of IFNγ-secreting natural killer cells, a vital component of innate immunity, and of IFNγ-secreting type 1 Th (Th1) cells. CD40 ligated DC also contribute to the development of IL-4- and IL-10-secreting Th2 cells. CD40L on Th cells also binds CD40 on macrophages to enhance their cytotoxic functions. CD40L-expressing Th cells provide the ‘help’ pivotally required to activate other components of adaptive immunity responsible both for clearing invading pathogens and generating the memory cells required to prevent re-infection. Th-supplied CD40L binds (i) B cell CD40 to switch production of antibodies to more potent effector molecules that have higher avidity for antigen, and (ii) DC CD40 to prime then expand antigen-specific cytotoxic T lymphocytes (CTL). Activated NK cells and CTL are required both to eradicate malignant cells and cells infected with viruses or other intracellular pathogens. Genetic CD40L deficiency causes the very rare HyperIgM Syndrome Type 1 (HIGM1), which is realistically modelled by genetically engineered CD40L-deficient mice. Neither CD40L-deficient patients nor mice make effective antibodies or mount cellular immune responses that would defend them against intracellular pathogens such as parasites. Consequently, the only potentially curative therapy is allogeneic stem cell transplantation or CD40L gene replacement. Here, we used a retroviral vector, which constitutively expressed CD40L, to genetically modify CD40L-deficient bone marrow cells, which were used to reconstitute partially the immunity of CD40L-deficient mice. The crucial importance of tight regulation of CD40L expression was revealed when these mice later developed lethal thymic T cell malignancy. Growing tumours escape immune vigilance by genetic alterations that reduce their sensitivity to IFNγ. Using murine tumour models, we incorporated transgenic CD40L expression in therapeutic tumour vaccines to show that CD40L gene transfer augmented the immunogenicity of the host’s tumour thus reducing its tumorigenicity. We translated this finding clinically to safety and immunogenicity testing of a transgenic CD40L- and IL-2- expressing leukaemia vaccine. Finally, the common viral respiratory pathogen, respiratory syncytial virus (RSV) mainly infects young infants and the elderly to cause potentially lethal pneumonia. Both groups have reduced cellular and humoral immunity, which predisposes them to re-infection with RSV. Using a murine model, we showed first that simultaneous adenoviral expression of CD40L augmented primary RSV-specific Th1 responses that were associated with accelerated pulmonary viral clearance. Second, we showed that expression of CD40L in RSV-F and RSV-G subunit DNA vaccines elevated antibody and cellular immune responses to RSV challenge four and eight months after the initial immunisation. These results demonstrate the potent ability of CD40L gene transfer to solve the absolute immune deficiency caused by genetic lesions of CD40L. However, physiological regulation of the transgene is required to prevent serious adverse consequences. In contrast, no adverse effects were observed after transgenic CD40L expression was used to overcome relative immune deficiencies imposed by malignancy and RSV infection. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1298200 / Thesis (Ph.D.) - University of Adelaide, School of Medicine, 2007

abstract: The immune system plays a dual role during neoplastic progression. It can suppress tumor growth by eliminating cancer cells, and also promote neoplastic expansion by either selecting for tumor cells that are fitter to survive in an immunocompetent host or by establishing the right conditions within the tumor microenvironment. First, I present a model to study the dynamics of subclonal evolution of cancer. I model selection through time as an epistatic process. That is, the fitness change in a given cell is not simply additive, but depends on previous mutations. Simulation studies indicate that tumors are composed of myriads of small subclones at the time of diagnosis. Because some of these rare subclones harbor pre-existing treatment-resistant mutations, they present a major challenge to precision medicine. Second, I study the question of self and non-self discrimination by the immune system, which is fundamental in the field in cancer immunology. By performing a quantitative analysis of the biochemical properties of thousands of MHC class I peptides, I find that hydrophobicity of T cell receptors contact residues is a hallmark of immunogenic epitopes. Based on these findings, I further develop a computational model to predict immunogenic epitopes which facilitate the development of T cell vaccines against pathogen and tumor antigens. Lastly, I study the effect of early detection in the context of Ebola. I develope a simple mathematical model calibrated to the transmission dynamics of Ebola virus in West Africa. My findings suggest that a strategy that focuses on early diagnosis of high-risk individuals, caregivers, and health-care workers at the pre-symptomatic stage, when combined with public health measures to improve the speed and efficacy of isolation of infectious individuals, can lead to rapid reductions in Ebola transmission. / Dissertation/Thesis / Doctoral Dissertation Applied Mathematics 2016