New Prognostic and Predictive Markers in Cancer Progression

Biomarkers are of critical medical importance for oncologists, allowing them to predict and detect disease and to determine the best course of action for cancer patient care. Prognostic markers are used to evaluate a patient’s outcome and cancer recurrence probability after initial interventions such as surgery or drug treatments and, hence, to select follow-up and further treatment strategies. On the other hand, predictive markers are increasingly being used to evaluate the probability of benefit from clinical intervention(s), driving personalized medicine. Evolving technologies and the increasing availability of “multiomics” data are leading to the selection of numerous potential biomarkers, based on DNA, RNA, miRNA, protein, and metabolic alterations within cancer cells or tumor microenvironment, that may be combined with clinical and pathological data to greatly improve the prediction of both cancer progression and therapeutic treatment responses. However, in recent years, few biomarkers have progressed from discovery to become validated tools to be used in clinical practice. This Special Issue comprises eight review articles and five original studies on novel potential prognostic and predictive markers for different cancer types

Defining the mechanism of action in a novel class of anti-PD-1 antibodies

Background. Despite the relative success of anti-PD-1 antibodies over the years in cancer immunotherapy, the mechanism of action beyond blockade of the ligand binding site remains inadequately defined. Given that anti-PD-1 therapy is effective in only a fraction of cancer patients, it is imperative to elucidate the mechanisms imparted by anti-PD-1 antibodies and how they are involved in restoring activity to functionally exhausted PD-1+ memory T cells. Hypothesis. The two hypotheses investigated are: 1) The novel non-blocking anti-PD-1 antibody discovered in our lab differs in its mechanism of action from the traditional blocking PD-1 antibodies, 2) Anti-PD-1 mediated downregulation contributes to the relief of T cell exhaustion and occurs through an antibody-Fc interaction with FcγR expressing cells. Objectives. The research plans are: 1) to develop an experimental strategy to evaluate the functional activity in primary, memory T cells responding to the anti-PD-1 therapies utilizing coinhibitory conditions with PD-L1 and 2) to identify the cell populations and associated determinants for anti-PD-1 mediated downregulation on primary, memory T cells. Experimental strategy. 1) We used PBMCs isolated from chronically infected HIV+ donors with high expression of PD-1 to measure the activation of key mediators of the T cell activation pathway. We were able to evaluate the restoration of phosphoprotein activity and calcium signaling to these functionally exhausted primary T cells at early timepoints when stimulated by CD3/CD28/PD-L1 and treated with anti-PD-1. 2) We isolated different fractions of the PBMC composition to determine if any effector cells were involved in PD-1 downregulation. Furthermore, we sought to establish a contact-based dependency test to determine which receptor was necessary for the anti-PD-1 downregulation effect. Results. 1) We demonstrated that both blocking and non-blocking antibodies were able to restore T cell signaling but the non-blocking antibody restored significantly more Akt signaling and the combination of the two anti-PD-1 antibodies enhanced the restoration of Ca2+ signaling dramatically. 2) Anti-PD-1 mediated downregulation was observed only in the presence of monocytes and CD64 was found to be the FcγR with the highest impact for this effect. Conclusions. 1) The non-blocking PD-1 antibody was shown to be effective in restoring T cell functionality and appeared to preferentially restore signaling through the CD28 associated pathway while the blocking PD-1 antibody exerted its effect over the TCR pathway. 2) The requirement for CD64 expressing monocytes was shown for the antibody-mediated downregulation of PD-1 on memory T cells. -- Contexte: Malgré le succès relatif des anticorps anti-PD-1 au fil des années dans l'immunothérapie anticancéreuse, le mécanisme d'action au-delà du blocage du site de liaison du ligand reste mal défini. Étant donné que le traitement anti-PD-1 n'est efficace que chez une fraction des patients cancéreux, il est impératif d'élucider les mécanismes conférés par les anticorps anti-PD-1 et comment ils sont impliqués dans la restauration de l'activité de la mémoire PD-1+ fonctionnellement épuisée T cellules. Hypothèse: Les deux hypothèsesétudiées sont: 1) Le nouvel anticorps anti-PD-1 non bloquant découvert dans notre laboratoire diffère dans son mécanisme d'action des anticorps bloquants traditionnels PD-1, 2) La régulation négative médiée par anti-PD-1 contribue à la soulagement de l'épuisement des cellules T et se produit par le biais d'une interaction anticorps-Fc avec les cellules exprimant FcγR. Objectifs: Les plans de recherche sont les suivants: 1) développer une stratégie expérimentale pour évaluer l'activité fonctionnelle des lymphocytes T mémoires primaires répondant aux thérapies anti-PD-1 utilisant des conditions co-inhibitrices avec PD-L1 et 2) pour identifier les populations cellulaireset les déterminants associés pour la régulation négative à médiation anti- PD-1 sur les lymphocytes T primaires à mémoire. Stratégie expérimentale: 1) Nous avons utilisé des PBMC isolées de donneurs VIH + infectés de manière chronique avec une forte expression de PD-1 pour mesurer l'activation de médiateurs clés de la voie d'activation des lymphocytes T. Nous avons pu évaluer la restauration de l'activité des phosphoprotéines et la signalisation du calcium vers ces lymphocytes T primaires fonctionnellement épuisés à des moments précoces lorsqu'ils sont stimulés par CD3 / CD28 / PD-L1 et traités avec de l'anti-PD-1. 2) Nous avons isolé différentes fractions de la composition de PBMC pour déterminer si des cellules effectrices étaient impliquées dans la régulation négative de PD-1. En outre, nous avons cherché à établir un test de dépendance basé sur le contact pour déterminer quel récepteur était nécessaire pour l'effet de régulation négative anti-PD-1. Résultats: 1) Nous avons démontré que les anticorps bloquants et non bloquants étaient capables de restaurer la signalisation des lymphocytes T, mais l'anticorps non bloquant a restauré significativement plus de signalisation Akt et la combinaison des deux anticorps anti- PD-1 a considérablement amélioré la restauration de la signalisation Ca 2+. 2) une régulation négative à médiation anti-PD-1 a été observée uniquement en présence de monocytes et CD64 s'est avéré être le FcyR avec l'impact le plus élevé pour cet effet. Conclusion: 1) L'anticorps PD-1 non bloquant s'est avéré efficace pour restaurer la fonctionnalité des lymphocytes T et semblait restaurer préférentiellement la signalisation via la voie associée au CD28 tandis que l'anticorps bloquant PD-1 exerçait son effet sur la voie TCR. 2) L'exigence de monocytes exprimant CD64 a été montrée pour la régulation à la baisse médiée par les anticorps de PD-1 sur les cellules T mémoire

Development of Cancer Immunotherapeutics Targeting Complement Regulatory Protein CD55

CD55 is one of the complement regulatory/inhibitory proteins and is over-expressed on a wide range of solid tumours. CD55 is also known to be deposited within tumour stroma and is secreted in an active soluble form, mediated by matrix metalloproteinase-7. The complement cascade forms part of the innate immune system and culminates in cell lysis of targeted cells. As a complement regulatory protein, the primary function of CD55 is to accelerate the decay of complement components preventing formation of the membrane attack complex. CD55 is also known to be a ligand for the T cell early activation antigen CD97, and their interaction has been shown to inhibit the proliferation of activated T cells. This project aimed to develop anti-tumour immunotherapeutics aimed at exploiting CD55 as a tumour associated antigen. Initial strategies were to develop monoclonal antibodies, specific to identified epitopes from within the CD55 protein sequence, capable of binding, and neutralising CD55s decay accelerating activity. Developed antibodies would also have the potential to induce antibody dependent cell cytotoxicity, thus blocking CD55 protection of tumours and mediating an active anti-tumour response. Antibodies were raised specific to CD55 derived linear peptides, which have been used for the assessment of CD55 expression in breast tumour sections. Monoclonal antibodies failed to recognise natively expressed protein on viable tumour cells and alternate strategies were developed. An effective immunotherapy for the treatment of cancer would engage both cellular and humoral mediated responses for effective clearance of target cells. In order to achieve this, a DNA vaccine incorporating a human IgG Fc tail was developed expressing the active sites of CD55, containing HLA-A*201 restricted heteroclitic epitopes. The vaccines were used to immunise HLA-A*201 HHDII transgenic mice and CD55 specific responses were assessed. One of the vaccines analysed, elicited CD55 specific antibodies capable of recognising tumour cells in vitro and also generated epitope specific CD8+ T cell mediated lysis of epitope bearing cells. The frequency of CD55 specific T cells was obtained via antigen specific IFN gamma release ELISPOT assays and the cytokine profile of responses generated was assessed via luminex analysis. In conclusion, CD55 remains a viable target for immunotherapies aimed at CD55 bearing tumours. DNA vaccines encoding modified epitopes are capable or raising cellular and humoral responses to this antigen and further studies should be completed in order to determine anti-cancer effects in tumour bearing models

Chemokine-mediated control of immunity to tumours and infectious pathogens

The ability of immune cells to migrate to distinct niches and peripheral sites is critical for their appropriate differentiation and for execution of their effector functions. This migration is facilitated to a large degree by the expression of chemokine receptors, which allow for migration in a spatiotemporally-controlled manner. The work presented in this thesis addresses two distinct issues regarding how regulation of immune cell migration affects development of anti-tumour immunity and infectious immunity. In the first part of this thesis, a novel role for the atypical chemokine receptor ACKR4 in controlling anti-tumour immune responses was identified. As a scavenging receptor, ACKR4 regulates the bioavailability of the CCR7 ligands, CCL19 and CCL21, and the CCR9 ligand, CCL25. These ligands have previously been shown to be critical for many aspects of immune homeostasis, as well as contributing to tumour cell growth and metastasis. However, the contribution of ACKR4 in regulating tumour-specific responses has been unclear. Using multiple orthotopic, transgenic and chemically-induced models of cancer, loss of ACKR4 resulted in inhibited tumour growth. In the absence of ACKR4, enhanced CCL21 levels were associated with enhanced tumour infiltration of IFNy+ CD8+ T cells. The reduced tumour growth seen was dependent on the enhanced CD8+ response, with depletion of CD8+ T cells restoring growth of Ackr4–/– tumours to wildtype levels. The enhanced CD8+ T cell response was not a result of altered priming in draining lymph nodes, although there was increased intratumoural proliferation of CD8+ T cells. Furthermore, ACKR4-deficient tumours showed increased retention of CD103+ DCs, with these cells previously being shown to be critical for effective recruitment of CD8+ T cells to tumours. Moreover, intratumoural administration of CCL21 into wildtype tumours also enhanced the accumulation of DCs, suggesting a direct role for the scavenging ability of ACKR4. These data support the notion that ACKR4, through its regulation of CCL21 bioavailability, controls DC migration in tumours thus regulating the development of anti-tumour immune responses. Furthermore, multiple immunotherapies show increased efficacy in the absence of ACKR4, suggesting ACKR4 may be useful as a potential novel target for immunotherapy. In the second part of this thesis, the role of CCR2 on memory CD4+ T cells was explored. Relatively little is understood about the generation, maintenance and effector functions of memory CD4+ T cells, despite correlations with improved disease outcomes. Furthermore, how these cells migrate to inflammatory sites is still largely unknown. In this project, CCR2 was identified as being enriched on antigen-specific memory CD4+ T cells in response to infection with the extracellular bacteria Streptococcus pneumoniae and infection with influenza A virus. Competitive co-transfer of wildtype and CCR2- deficient TCR-transgenic CD4+ T cells showed enhanced contraction of Ccr2–/– cells, suggesting a cell-intrinsic role for CCR2 in CD4+ T cell maintenance. CCR2-deficient effector cells were unaffected in their ability to secrete cytokines or enter into effector sites. Moreover, despite being numerically reduced at memory timepoints compared with CCR2-sufficent cells, they were equally capable of expanding upon secondary challenge. These data highlight CCR2 as an important regulator of CD4+ T cell memory maintenance. Taken together, this project has furthered our understanding of the complexity of cell migration in dictating immune responses. The identification of CCR2 as a mediator of memory CD4+ T cell generation may allow further investigation into how these cells are induced and maintained. In ACKR4, a novel level of post-transcriptional regulation of intratumoural DC trafficking has been identified, with this having the potential to be a tractable target for therapeutic manipulation in malignant disease.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 201

CLUSTER HOMOLOG OF IMMUNOGLOBULIN-LIKE RECEPTOR GENES IN CHICKEN IMMUNE RESPONSES

This dissertation explores the identity and role of immunoglobulin-like (Ig-like) receptors in chickens, with focus on their implications in disease and disease progression. These receptors, wisely expressed across immune cells, interact with the major histocompatibility complex (MHC) class I molecules to modulate immune responses in mammals. Due to the insufficient representation of chicken Ig-like receptors in online databases, this study systematically annotates the chicken Cluster Homolog of Immunoglobulin-like Receptors (CHIR) genes using advanced bioinformatic techniques, aligning with the release of the 7th edition of the chicken genome assembly that comprises builds for a broiler and layer chicken. The analysis identifies over 150 CHIR genes, refining functional classifications of activatory (CHIRA), inhibitory (CHIRB), bifunctional (CHIRAB), and CHIR-like (CHIRL) genes through InterProScan, phylogeny and motif searches. Variations in CHIR gene counts across different chicken lines (broiler, N = 124, layer, N = 70) suggest links to selective breeding demands, emphasizing their importance in poultry health and production. Phylogenetically, CHIRs show close relationships with other poultry Ig-like receptors, and structural comparisons indicate analogous roles to Ig-like receptors in the human and rat. As an outcome of the analysis, CHIR genes were renamed with the Chicken Genome Nomenclature Consortium from “chicken homolog of Ig-like receptors” to “cluster homolog of Ig-like receptors”. Reanalyzing next-generation sequencing data reveals CHIR genes are expressed across all tissues of a UCD001 line, with generally higher expression in blood-containing organs. Examination of CHIR gene single nucleotide polymorphisms across various in inbred lines (UCD001, UCD003, Line 0, Line 6, Line 7, Line 15, Line N, Line P, Line C, and Line W) indicates an overall variant rarity and slightly more occurrence in CHIRB genes. Over 1,000 protein-encoding variants are associated with differential resistance and susceptibility to Marek’s disease (P \u3c 0.05). Two in vitro approaches assessed the roles of CHIR molecules in modulating immune responses or targeting pathogens. Re-examination of RNA-sequencing data of MHC-I types B2 and B19 macrophages, temporally stimulated with interferon-gamma, revealed dynamic and opposite CHIR expression trends, with B2s showing an increase and B19s displaying a decrease until returning to basal levels at 24 to 48 hours. These findings suggest nuanced and distinct regulatory patterns of CHIRs in different haplotypes during immune responses. Additionally, CHIR sequences were aligned for the design of small interfering RNA molecules targeting the CHIRB functional group on macrophages retrieved from birds of congenic (UCD331 and UCD335) and mixed (WVU1952) backgrounds. CHIRB silencing was observed to enhance cellular nitrate release and impact H2O2, particularly in specific MHC-I haplotypes and in different genetic backbones, in avian influenza virus infection. While this dissertation enhances our understanding of chicken Ig-like receptors and cellular involvement, it also acknowledges certain limitations, such as variations in gene annotations. Nevertheless, CHIRs merit a sizeable acknowledgment as pivotal contributors to the immune response, particularly in their intricate interactions with the MHC. Future studies integrating this understanding into breeding plans or other interventions becomes a strategic imperative for optimizing poultry health and immunity, ensuring wellbeing, and in turn, a more resilient and sustainable food supply

The anti-cancer potential of polyphenols in the treatment of leukaemia.

Background: Leukaemia is a complex disease affecting all blood cell lineages. It affects millions of people worldwide each year and mortality rates are high, despite considerable improvements in treatment. Thus, new therapies for leukaemia are urgently needed to improve leukaemia patients' health and survival. Since polyphenols exert pro-apoptotic effects in solid tumours, our study investigated the effects of polyphenols in haematological malignancies.Methods: The effects of eight polyphenols (quercetin, chrysin, apigenin, emodin, aloe-emodin, rhein, cis-stilbene and trans-stilbene) was studied on cellular proliferation, the induction of apoptosis and cell cycle progression in four lymphoid (JURKAT, MOLT-3, CCRF-CEM and U937) and four myeloid (HL-60, THP-1, K562 and KG-la) leukaemia cells lines, together with normal haematopoietic control cells (CD34+ HSC and CD133+ HSC) from cord blood. Further to this, an investigation was made of the effects of the most promising polyphenols used in combination with nine standard chemotherapeutic agents (etoposide, doxorubicin, cyclophosphamide, chlorambucil, cisplatin, methotrexate, 6-mercaptopurine, 5-fluorouracil and imatinib). For this polyphenol and chemotherapy combination work four leukaemia cells lines were used: the two most sensitive (JURKAT and CCRFM-CEM) and two most resistant (KGla and THP-1) to polyphenol treatment. Subsequently, an investigation was undertaken to identify potential mechanisms of action of these polyphenol when used alone and in combination with chemotherapeutics. The extrinsic and intrinsic apoptotic pathways were investigated together with effects on glutathione levels and DNA damage.Results: Emodin, quercetin, and cis-stilbene were the most effective polyphenols at decreasing cell viability and inducing apoptosis. Lymphoid cell lines were normally more sensitive to polyphenol treatment compared to myeloid cell lines; however those myeloid (KG-la and K562) cell lines which were most polyphenol resistant; were however affected by emodin and quercetin at micromolar treatment doses. Non-tumour cells were less sensitive to all polyphenols compared to the leukaemia cells. Mechanistically, most polyphenols alone depleted glutathione (GSH) levels associated with a direct activation in caspase 8 and/or caspase 9 in leukaemia cell lines at 24 h. Polyphenols also had differential capacities to induce DNA damage in the leukaemia cell lines. Polyphenols acted synergistically in lymphoid cell lines and differently in myeloid cell lines producing either synergistic, additive, competitive antagonistic or antagonistic effects; when they were combined with toposiomerase inhibitor agents (etoposide and doxorubicin) and alkylating agents (cyclophosphamide and chlorambucil, cisplatin). In contrast, they worked antagonistically with anti-metabolites agents (methotrexate and 6-mercaptopurine) in both lymphoid and myeloid leukaemia cell lines. Mechanistically the synergistic induction of apoptosis observed following the combination of polyphenols withchemotherapeutic agents was caused by the direct activation of intrinsic or /and extrinsic apoptotic pathway through the up-regulation of caspase 8 or caspase 9 within the lymphoid and myeloid leukaemia cell line. Furthermore, it has been shown the synergistic effects observed when polyphenols and chemotherapy agents were combined was correlated with down regulation of GSH levels and an induction of DNA damage which drove apoptosis. Alternatively where there was an antagonist effect there was an up-regulation of GSH levels, a reduction in DNA damage and the level of apoptosis.Conclusions: These findings demonstrate that polyphenols induce apoptosis and arrest cell cycle in leukaemia cell lines which could translate to anti-cancer activities in leukaemia, although the effects were dependant on polyphenol type and origin of the cell line investigated. Importantly, the differential sensitivity of emodin, quercetin, and cis-stilbene between leukaemia and normal cells suggests that polyphenols are potential therapeutic agents for leukaemia. Furthermore, this study concluded that the efficacy of standard chemotherapeutic agents were differentially modulated by polyphenols, producing either synergistic, additive or competitive antagonistic/antagonistic effects, which was dependent on type of polyphenol, chemotherapy agent and cell line. Interestingly the study showed that synergistic or antagonistic effects observed following the combination treatments were strongly dependent on the modulation of glutathione levels in association with the formation of H2AX nuclear foci and DNA damage in leukaemia cell lines