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Objectives: One in two patients with stage 4 melanoma will not survive their first year after diagnosis. Although recent treatment advances have improved survival, there is an unmet need for alternative therapeutics. Chimeric antigen receptor (CAR) T cell therapy involves re-directing autologous T lymphocytes against tumour-associated antigens via a receptor which couples an antigen-binding domain to CD3-zeta and costimulatory signalling domains. Melanoma cell adhesion molecule (MCAM) is upregulated in melanoma, promotes invasive cell behaviour and angiogenesis, and predicts poor survival. CAR T cells redirected against this tumour-associated antigen could represent a novel treatment option in late-stage melanoma. Methods: 5 single chain variable fragments (scFvs), derived from MCAM-specific antibody sequences, were cloned into a retroviral second-generation, 4-1BB costimulated CAR construct with CD8 hinge and transmembrane regions. CARs were screened for their ability to activate the JRT3-T3.5 cell line in response to MCAM-expressing cells. Selected CAR constructs were then expressed in primary T cells, and T cell effector functions were assessed in response to target cells. CAR T cells were also cocultured with HUVEC cells in monolayers and networks. Results: M1.BBz and M40.BBz CARs drove modest activation of the JRT3-T3.5 cell line in response to target cells expressing the MCAM ectodomain. These CARs redirected primary T cells effector functions, causing MCAM-dependent pro-inflammatory cytokine release, upregulation of cell surface activation markers and tumour cell lysis. However, the CAR T cells also targeted HUVEC cells, causing significant destruction of capillary-like networks. Conclusions: Whilst this study demonstrates the feasibility of generating MCAM-specific CAR T cells, further work is needed to confirm their anti-tumour efficacy in vivo. Ultimately, the potential for on-target/off-tumour toxicity, particularly towards the vasculature, must be considered. Further engineering has the potential to increase their safety and specificity, but the risk of toxicities may ultimately preclude the safe targeting of MCAM in this context.

Intra-cellular signalling determines how cells process information. Through the integration of diverse chemical and physical stimuli, cells can enact transcription, among other changes to modulate growth, fate and survival. It is through the dysregulation of such processes that many diseases, including cancer originate. For many years, our study of signalling processes has been based on discrete 'pathways', characterised mostly by small-scale studies. However, as more system-wide data becomes available, it is becoming increasingly obvious that intra-cellular signalling is more like a dense and inter-connected network, with intense and functional cross-talk between pathways. In my PhD project, I utilised both new 'omics' data and a plethora of network-based approaches to guide our understanding of various signalling-related disease contexts. Networks provide a convenient framework with which to explicitly control the level of prior-knowledge required to understand complex 'omics' data. Escaping the study bias that has so-far dominated our characterisation of intra-cellular signalling is vital if we are to progress in our understanding of complex cellular behaviours.

Melanoma is an exceedingly aggressive and resistant to treatment form of cancer. Therefore, the identification of novel therapeutic approaches continues to be a crucial task. Understanding the pathophysiological role of potential drug targets for this disease enables the discovery of new therapeutic strategies. The two-pore channel 2, TPC2, is located on late endosomes, lysosomes, and melanosomes. Here, we evaluated the effect of TPC2 knockout (KO) on human metastatic melanoma (CHL-1) and murine primary melanoma (B16) cells. TPC2- depleted melanoma cell lines were created using CRISPR-Cas9 gene editing. CHL-1, an amelanotic metastatic melanoma cell line, exhibited in vitro induced migratory traits after TPC2 depletion. In contrast, B16 TPC2 KO cells displayed a diminished ability to migrate in vitro. TPC2 KO activated genes regulated by YAP/TAZ, which are important regulators of tumorigenesis and metastasis, in CHL-1. TPC2 KO and RNA interference knockdown decreased the expression levels of ORAI1, a component of store-operated Ca2+ entry (SOCE), and PKC-II, a component of the HIPPO pathway that adversely affects YAP/TAZ activity. These findings are explained by a cellular mechanism mediated by ORAI1/ Ca2+/PKC-II. Furthermore, CHL-1 and B16 TPC2 KO proteomic signatures were examined to WT cells. The differential expression of proteins between WT and KO cells enables us to evaluate TPC2's role in various crucial pathways and processes. Accordingly, the bioenergetic phenotype of TPC2-deficient cells was assessed. TPC2 depletion boosted ATP synthesis in CHL-1 and B16 TPC2 KO cells relative to WT cells, but increased lactate generation in B16 cells. TPC2 deficiency enhanced anaerobic respiration in B16 cells but reduced it in CHL-1 cells. OCR/ECAR analysis confirmed TPC2-KO cells' metabolic shift. CHL-1 TPC2 KO cells are more aerobic due to a reduced OCR/ECAR ratio. OCR/ECAR was elevated in B16 KO cells in complete media. B16 KO cells increased oxygen utilisation and lactate production without glucose. In addition, the cytoskeleton and synthesis and release of melanin were altered in TPC2 KO cells. In vivo evaluation of B16 TPC2 KO melanoma cell lines following subcutaneous injection revealed a significantly reduced tumour volume compared to WT cells. However, histopathological examination of mice injected with KO cells revealed that the hepatic and splenic structures were drastically changed. Clinical association is vital to determining correlations between molecular discoveries and cancer patient survival. TPC2 high-expression patients had a shorter overall survival time than low-expression patients. Furthermore, high TPC2 expression is associated with lower survival in BRAF mutant melanoma. In summary, the work presented in this thesis indicates that TPC2 plays vital functions in melanoma's progression, invasion, and metastasis.

For over a century, it has been apparent that tumours behave somewhat like wounds that fail to heal. A number of studies have established several cellular and molecular parallels between wound healing and cancer, and a key one of these is inflammation, with both wounds and tumours triggering an inflammatory response that plays an essential role in the outcome of these tissue lesions. As a consequence, any potential therapy with the capacity to alter the wound/tumour inflammatory response is now considered clinically relevant. In this regard, infection is known to modulate the inflammatory response to both cancer and wound healing. Serendipitous findings extending back to those of Coley in the early 1900s, have shown cancer remissions occurring upon treatment with heat-killed bacteria, suggesting that infection can enhance, or prime, the host immune response to better recognise and eradicate cancers. During tissue repair, there is evidence that some aspects of the wound inflammatory response may be activated by microbial antigens, although, of course, if infection becomes overwhelming, this can lead to chronic, non-healing wounds. Whilst the longer term consequences of infection on cancer and wound healing have been partially explored, rather little is known about the precise cellular and molecular mechanisms by which bacteria-induced inflammation can influence these consequences, for example, how infection alters the behaviour of inflammatory cells and their cellular interactions with the tissue insult. Here I used the translucent larval zebrafish (Danio rerio), which has relatively recently become a powerful model of inflammatory-related human conditions, in combination with high-resolution confocal imaging and a novel bespoke automated cell tracking and cell-cell interaction software, to investigate how inflammatory cells behave and interact with repairing wounds and early cancer cells in sterile versus infection conditions. Using this approach, I showed that infection, delivered either systemically or locally, induced changes in the number and behaviour of inflammatory cells recruited to acute wounds and to pre-neoplastic cells, and these alterations led to a significant delay in wound healing, and to a reduction in the number of pre-neoplastic cells, which correlated with increased numbers of pro-inflammatory (tnfα-positive) macrophages. I expanded these studies into adult zebrafish where I observed that infection-induced inflammation inhibits the growth of established human-like melanomas. Finally, I tested a more therapeutically viable anti-cancer strategy by using a novel particle-based protocell system to selectively load macrophages and neutrophils, via their natural foreign body phagocytic abilities, with immunostimulatory, tumour-suppressing molecules (anti-miR223 or R848). I showed that anti-miR223-loaded protocells or R848-loaded protocells can effectively "reprogramme" macrophages (and neutrophils) towards a more pro-inflammatory, and thus anti-cancer state, in zebrafish (and cultured human macrophages), which led to a significant reduction in both larval pre-neoplastic burden and in the size and number of adult melanomas also.

Introduction: Melanoma is a paradigm of how the treatment landscape can be revolutionised over a decade. The advent of immune checkpoint blockade (ICB) has dramatically improved the outcomes of patients with both early and advanced stage disease. Nevertheless, patients are still dying of their disease, highlighting the urgent need to identify which patients will respond to therapy, in order to improve patient stratification and avoid unnecessary toxicity risk. Thus, precision immuno-oncology approaches are required to improve cancer care. Therapy decisions need to be made based on patient-specific immunological signatures, but clinically validated biomarkers for this purpose are currently lacking. The primary aim of this work was to explore liquid biopsy as a minimally invasive approach to investigate peripheral T cell evolution early on treatment as a potential predictive biomarker of response to immunotherapy in melanoma. Additionally, the impact of patient clinical variables on peripheral T cell and T cell receptor (TCR) repertoire evolution under the selective pressure of ICB in advanced melanoma was investigated. Methods: To assess the peripheral T cell compartment of metastatic melanoma patients undergoing first line anti-PD1 based ICB treatment, peripheral blood samples were taken prior to and early on treatment then analysed by flow cytometry. Phenotypic assessment of peripheral T cells and TCR sequencing data were used to correlate changes in peripheral T cell subset dynamics and TCR repertoire to patient clinical variables before treatment (T0) and after the first cycle of ICB at week 3 (W3). A prospective study was set up to investigate whether changes in peripheral T cells early on adjuvant ICB therapy can predict patient response in stage III melanoma. Results: Flow cytometry revealed expansion of a subset of CD8+ memory immune effector cytotoxic T cells in peripheral blood. At W3 after therapy initiation, expansion of these cells was significantly greater in patients that responded to ICB. An increase of 0.8% in the ratio of this subset of T cells relative to all CD8+ memory cells at W3 was associated with improved survival and separated 6-month responders from non-responders; this was confirmed in a separate validation cohort. Analysis from later W9 treatment time point revealed the presence of the T cell subset, however there was no longer a correlation with response, highlighting the dynamic nature of the immune signature and indicating that these changes occur early and are transient. Evaluation of whether changes in W3 T cell subset expansion were associated with immune related adverse events revealed no correlation between T cell subset expansion and grade of toxicity, suggesting that these cells are not a predictive marker of immunotherapy toxicity in this setting. Notably, expansion of a separate regulatory T cell subset did correlate with grade of toxicity. Investigation of clinical variables associated with the immune signature demonstrated a correlation between the immune effector T cell subset abundance and age at T0 (r=0.40), which reduced following treatment at W3 (r=0.07). However, at W3 two significantly opposing patterns (p=0.03) of TCR repertoire rearrangement were observed in patients who responded to treatment, with patients ≥70 years of age showing an increase in TCR clonality and patients < 70 years of age showing an increase in TCR diversity. Conclusion: This work has identified a peripheral blood early immune signature characterised by turnover of a specific subset of immune effector T cells. The magnitude of immune signature changes following the first cycle of ICB therapy anticipated which patients would go on to respond and ultimately correlated with overall survival. Additionally, a regulatory T cell subset demonstrated a correlation with toxicity grade. Furthermore, investigation of the clinical correlates associated with the immune signature identified a model whereby age does not affect peripheral immune effector T cell subset expansion in response to ICB, but does influence immunotherapy-induced peripheral TCR repertoire evolution. Thus, T cell repertoire analysis should be contextualised by patient age. Finally, the prospective study to assess the feasibility of translating the immune signature findings into the adjuvant setting to identify patients at high risk of relapse and predict response to ICB therapy in stage III melanoma remains undoubtedly clinically relevant.

RAC1 GEFs have been shown to be capable of determining the signalling outputs of RAC1 signalling, specifically P-REX1 has been shown to interact with and facilitate RAC1 signalling through FLII. P-REX1 is capable of enhancing cell migration and this is dependent on FLII and the downstream phosphorylation of MLC2 enhancing cell contractility. P-REX1 shows limited mutations in melanoma, in contrast P-REX2 is highly mutated and thus may have greater clinical significance. Limited data in the literature shows that SNPs and truncations within P-REX2 appear to escape PTEN regulation and assist in oncogenic transformation. Less is known about how P-REX2 may direct RAC1 signalling. I sought to investigate the role of P-REX2 and its truncations in melanoma initiation and progression. Given the known interaction between P-REX1 and FLII, I sought to determine if this interaction persists with the P-REX1 paralogue P-REX2 and elucidate any potentially oncogenic implications. I demonstrate, through over expression studies and within the endogenous oncogenic setting, that PREX-2 interacts with FLII, which has not yet been reported in the current literature. Furthermore, truncations of P-REX2 not only maintain the ability to interact with FLII but also have a greater capacity to induce oncogenic transformation, in comparison to full-length P-REX2. These interactions have multiple, quantifiable effects on the intracellular structure, migration and invasive capacity of melanocytes, ultimately leading to a highly oncogenic phenotype. This thesis presents novel research into the roles and interactions of PREX-2, an under-studied protein with a link to the formation of melanoma. Through detailed characterisation of the molecular interactions and the intracellular and in-vivo effects of PREX-2, the work presented here shines further light onto the molecular mechanisms that underly the formation and progression of melanoma.

Metabolic reprogramming is one of eight hallmarks of cancer; altered lipid metabolism such as enhanced fatty acid (FA) synthesis and uptake support the membrane biogenesis and ATP requirements for melanoma cell growth and division. As yet, few druggable oncoproteins directly responsible for altered metabolism in cancer cells have been identified. To thrive, however, cancer cells must avoid the reduction in cell growth signalling and generation of toxic lipid peroxides that ordinarily accompany excess free FA. Here, we have uncovered the frequent up- regulation and amplification in melanoma of the enzyme Diacylglycerol O- acyltransferase 1 (DGAT1). DGAT1 catalyses triacylglyceride synthesis, the final step in lipid droplet formation, which allow cancer cells to tolerate excess FA. Using zebrafish transgenesis, we found forced Dgat1 expression in melanocytes accelerated melanoma development driven by oncogenic BRAF or NRAS. Strikingly, in p53 mutant zebrafish melanocytes forced Dgat1 expression alone induced melanoma formation. Utilising both in vitro and in vivo models we found that the pro-oncogenic activity of DGAT1 was mediated through the stimulation of mTOR- S6K, signalling melanoma cell growth and protecting melanoma cells from cell death induced by oxidative stress and lipotoxicity. Inhibition of DGAT1 leads to a decrease in mTOR signalling through S6K and increased production of toxic metabolites leading to a loss of mitochondrial membrane potential and an increase in reactive oxygen species, ultimately leading to melanoma cell death. Together, our data identifies DGAT1 as a bona fide oncogene that stimulates cell growth and suppresses oxidative stress while enabling fatty acid accumulation and thus, a putative therapeutic target in melanoma.

The aim of the work undertaken in this thesis was to explore the potential of synthetic peptides to enhance anti-cancer photodynamic therapy (PDT) treatment by improving targeting to tumour tissue. Chapter 1 provides an introduction to the challenges associated in the treatment of cancer, the use of peptides as therapeutics or targeting agents and the potential / limitations of current PDT. Chapter 2 discusses the methods used in each of the results chapters 3-6. The first results chapter (Chapter 3) describes how ferrocene can be used to quench ROS generation from a Rose Bengal sensetiser when both were separated by a short integrin targeting RGD containing peptide. The addition of trypsin was shown to cleave the quencher and enhance RB mediated ROS generation. In chapter 4, the sensetiser-peptide conjugate RB-C(KLAKLAK)2 was prepared, and its PDT efficacy determined in melanoma cells and melanoma tumours in mice. The results showed that the PDT treatment using RB-C(KLAKLAK)2 was significantly more efficacious than RB mediated PDT or the treatment of RB-C(KLAKLAK)2 alone. This was attributed to the peptide sensitizing the cell to ROS generation. This work was extended in Chapter 5 where the contribution of both the sensetiser and peptide to PDT mediated efficacy was studied by preparing the methylene blue - peptide analogue MB-C(KLAKLAK)2 and the shorter peptide analogue RB-CKLAKLAK. The results demonstrated that the choice of sensetiser does impact PDT efficacy but the peptide length had only a minor effect. The final results chapter, Chapter 6 describes the preparation of biotin labelled αvβ6 integrin targeting peptide A20FMDV2 and its conjugation to the surface of avidin coated liposomes. Improved uptake of the A20FMDV2 targeting liposomes was observed in αvβ6 integrin expressing BxPC-03 cells when compared to Panc-01 cells that do not over express this protein. Collectively, the results described in this thesis demonstrate the role peptides can play to enhance anti-cancer PDT efficacy or improve the delivery of drug delivery vehicles such as liposomes.

The introduction of immune checkpoint blockade (ICB) therapy for metastatic melanoma (MM) has dramatically improved survival in this patient group. However, only some patients experience durable clinical response, and many will develop immune-related adverse events (irAEs). The identification of predictive biomarkers which can be assessed in a biopsy-free manner may facilitate early detection of non-responders and optimal targeting of treatment. There is emerging evidence to show that the circulating monocyte population has a prognostic and predictive role in the context of ICB, but this has yet to be fully characterised. Circulating-cell free DNA (cfDNA) is derived predominantly from leukocytes, but in patients with cancer may comprise circulating tumour DNA (ctDNA). Assessment of epigenetic cfDNA profiles may provide tumour profiling and reflect immune responses as a 'liquid biopsy'. Although epigenetic tumour profiles have diagnostic and prognostic utility across multiple cancer types, epigenetic cfDNA profiles in MM and following ICB are not yet characterised. In this thesis I dissect both circulating myeloid and epigenetic cfDNA profiles in MM patients. Firstly, I explore MM-associated monocyte transcriptomic profiles with bulk RNA sequencing (RNA-seq), characterising on-treatment modulation and association with clinical response. I then use scRNA-seq to explore the transcriptional heterogeneity of the peripheral monocyte compartment, and describe monocyte subset-wise responses to ICB. Lastly, I characterise epigenetic cfDNA profiles in this cohort, revealing distinct 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) cfDNA profiles in MM and following ICB. In this thesis I present novel findings regarding peripheral myeloid and epigenetic cfDNA responses to ICB and demonstrate that these profiles may have potential diagnostic and predictive clinical utility.

Witnessing a decade since the Physics Nobel Prize awarding for the ground breaking studies on the 'two-dimensional material graphene', graphene oxide (GO) stands as a promising candidate under the spotlight of biomedical research. Amongst the most questioned applications lies the possibility of its future use for intravenous (IV) treatment, imaging, diagnostic or theranostic purposes. Numerous studies are highlighting the trade-off between the physicochemical properties of GO materials and their in vivo safety, biodistribution and performances. However, little is known about the in vivo interaction of GO with biofluids. To date, no study investigated the interactions of GO with proteins upon their IV administration. This is a major gap, as it has been shown that the adsorption of blood proteins on the surface of nanoparticles can modulate their in vivo effects and fate. Our group and others have found that GO flakes are able to travel from blood to bladder, without causing renal impairment. In this thesis, we show that renal excretion could be explored to recover protein-coated GO flakes after their IV administration. We developed a robust method to simultaneously recover GO and remove free proteins from urine. Our results suggest that the proteomic content of mouse urine is enriched upon GO excretion. Further on, inspired by the proteomic studies in our laboratory showing that in vivo protein-coated nanoparticles can be explored as a disease biomarker resource, we questioned the possibility of using our method for scavenging disease-related proteins. Using B16-F10 melanoma tumour bearing mice, we provided evidence that the excretion of GO boosts the levels of disease-relevant molecules into the urine samples. In summary, this project reveals a new method for the in vivo GO-protein interaction studies and disease biomarker scavenging.

Cutaneous melanoma arises due to the uncontrolled proliferation of melanocytes. It is the deadliest form of skin cancer and its incidence, particularly in younger adults, is rapidly rising worldwide. Over the past decade, multiple large-scale sequencing studies have uncovered the molecular landscape of cutaneous melanoma. In particular, an improved understanding of the melanoma genome and regulation of the immune system have led to the development of effective targeted and immunotherapies that have revolutionised the treatment landscape. Nevertheless, only a subset of patients demonstrate durable responses to these therapies and identifying those patients most likely to benefit remains an important unmet need. Melanoma is an aggressive malignancy that often metastasises beyond its primary site. It has a particular propensity to metastasise to the brain and the mechanisms underlying this devastating complication remain incompletely understood. Melanoma risk stratification has traditionally relied on the examination of clinical and pathological features, however it has become clear that traditional staging may fall short in accurately assessing an individual patients' risk. There is therefore a need for robust prognostic biomarkers capable of identifying truly high-risk patients, or patients with biologically indolent tumours, which could be used as an adjunct to conventional clinicopathologic assessments, and would afford a unique insight into the underlying tumour cell biology. In this thesis, I explore the mutational landscape of cutaneous melanoma focussing on four key clinical cohorts; adolescent cutaneous melanoma, patients with brain metastases, widespread lethal metastatic disease and patients with high-risk primary melanoma. I use a range of high-throughput sequencing technologies to identify the tumour-specific somatic alterations characterising these cohorts and apply detailed multi-site phylogenetic analyses to uncover some of the key evolutionary changes. In the final chapter, I apply deep RNA sequencing to primary melanomas embedded within a prospective phase III clinical trial, to define and validate a gene expression signature identifying primary melanoma patients at higher risk of adverse outcomes. Together, these studies demonstrate how detailed analyses of molecular sequencing can uncover novel biological insights. It is hoped that further prospective molecular analyses coupled with high-quality experimental validation will pave the way towards novel therapeutic strategies for these patient cohorts.

ShcD/RaLP is the least characterised member of the Shc adaptor family of proteins. Shc adaptor proteins share the modular structural domains known as phosphotyrosine binding (PTB), Src homology 2 (SH2), and one or two collagen homology (CH1/CH2) domains. These domains are involved in binding activated receptor tyrosine kinases and co-ordinating the activation of various signalling pathways. ShcD is highly expressed in melanomas where high expression levels correlate with increasing vertical growth phase and invasive properties. Since ShcD is a signalling adaptor, its role in migration and invasion is likely to depend on the proteins with which it forms complexes. Previous studies in this lab have shown that the signalling scaffolding protein Gab1 interacts with the SH2 domain of ShcD. Interestingly a possible phosphorylation site has been identified by mass spectrometry within the SH2 domain of ShcD which could regulate binding to Gab1. I have made a S551D phosphomimic mutation in the SH2 domain of ShcD and shown that this inhibits interaction with Gab1 and other tyrosine phosphorylated proteins by GST pull-downs. This suggests that phosphorylation of the SH2 domain by a Ser/Thr kinases may negatively regulate ShcD-Gab1 induced signalling. Future studies will examine the effect of the mutation on ShcD mediated migration and identifying the biological function of kinases that phosphorylate the SH2 domain of ShcD.

Myeloid Derived Suppressor Cells (MDSCs) are a heterogeneous immune population found within the tumour microenvironment (TME). We sought to explore the potential role of microenvironment components on the suppressive behaviour, development and maintenance of MDSCs, focusing mainly on the role of the SIRPα-CD47 signalling axis. In an orthotopic melanoma model, we observed an increase in Ly6C-expressing myeloid cells (indicating monocytic-myeloid derived suppressor cells (M-MDSCs) and monocytic dendritic cells (moDC)) as tumours developed. These cells were suppressive, able to block T CD8+ cell proliferation. To model the effect of tumour-derived factors on M-MDSCs and moDCs development and function, we developed a culture system using hematopoietic stem cells cultured with GM-CSF and melanoma tumour condition media (TCM). Similar to the in vivo setting, exposure to TCM skewed the myeloid compartment towards an M-MDSC and moDC phenotype (based on Ly6C expression) that potently suppressed CD8+ T cell proliferation to a greater extent than GM-CSF induced MDSCs. Further characterisation of the TME by single-cell RNA sequencing and flow cytometry revealed specific expression of the signal-regulatory protein alpha (SIRPα) in M-MDSC and moDC cells fraction and elevated expression of its cognate ligand, CD47 by other immune cells. Thus, we investigated the impact of CD47-SIRPα interaction on MDSC function. Engagement of SIRPα on moDCs and M-MDSCs by recombinant CD47 in vitro induced intracellular signalling via SHP2, and inhibited the phagocytic capability of these cells. Moreover, persistent activation of this programme translated to an increase in their suppressive phenotype quantified by elevated expression of immune checkpoint molecules, inhibitory factors and reactive oxygen species. Knowing this axis promoted a pro-tumour, suppressive phenotype, we then investigated the consequence of its disruption on tumour growth in vivo. Neutralization of SIRPα on moDCs and M-MDSCs in established tumours resulted in a significant decrease in growth, which was driven by a reprogramming of moDCs and M-MDSCs. Disruption of the CD47-SIRPα axis was sufficient to rescue their phagocytic capability, which in turn enhanced their ability to process and present antigen to tumour infiltrating T cells. These functional changes were accompanied by metabolic adaptations. In summary, we report the CD47-SIRPα axis functions as a mechanism used to support moDC and M-MDSC suppressive function in the TME, and its disruption in early tumour-infiltrating monocyte progenitors shows potential to restore anti-tumour features of myeloid cells and in turn promote the T cell mediated anti-tumour immune response.

The past decade has seen many advances in the field of cancer immunotherapy, with increased research efforts into optimising adoptive cell therapies. This has been particularly successful in the setting of metastatic melanoma, where both TIL and TCR T-cell therapies have had promising clinical trial results. However, researchers are yet to fully elucidate the reactivity profile of TIL products, and best identify and characterise the tumour-reactive portion of TIL. In the work for this PhD thesis, several different models were explored to best identify the tumour-reactive population within final melanoma TIL products. Through optimisation, a model was chosen where final TIL was co-cultured with autologous patient-derived tumour cell lines and CD137 was selected as a marker of tumour-reactivity to isolate and characterise the tumour-reactive TCRs. Through this approach, several TCRs were identified and a few were chosen for validation, which indicated they were HLA-A*0201-restricted, melanoma-reactive TCRs. In the second part of the project, pre-clinical validation of gp100-reactive TCRs isolated from a single patient TIL product was conducted in a series of flow-cytometry based assays. Through using different model systems to validate and compare the TCRs, an optimal candidate gp100 TCR was identified. This TCR was then directly compared to a control TCR originally isolated from a transgenic mouse model and previously used in gp100 TCR T-cell therapy in the clinic. Comparison with the TIL-derived gp100 TCR showed the two TCRs were comparable with regards to tumour-reactivity in the CD8+ T-cell population, however the TIL-derived TCR was considerably less cross-reactive than the control TCR. Collectively, the work in this PhD thesis demonstrates the safety benefit that TIL-derived TCRs can confer, while proposing an optimised model of identifying and pre-clinically validating tumour-reactive TCRs from TIL products.

Melanoma is the most aggressive skin cancer, traditionally known to be extremely resistant to common chemotherapeutics, and difficult to treat once metastasised. The prognosis of malignant melanoma is strongly dependent on early detection and disease stage. Recently a number of promising anti-melanoma therapies have been developed, such as anti-CTLA4 and anti-PD-1 antibodies, which enhance tumour T-cell activity, and selective BRAF (DBF; dabrafenib) and MEK (TRA; trametinib) inhibitors, that block tumour growth in patients with active BRAF mutations. Although these therapies offer significant improvement in treating melanoma patients, they are associated with high toxicity and drug resistance. Nano-medicine offers unique advantages in treating metastatic melanoma. Nanoparticles (NPs) enhance therapeutic efficacy, providing controlled release and prolonged drug circulation time. Active-targeting enables NP accumulation in tumours using targeting-moieties designed to recognise tumour-associated molecular markers. Super-paramagnetic (SP) iron oxide nanoparticles (SPIONs) have been selected and synthesised in-house as a drug delivery platform.3 These nano-carriers can encapsulate anti-melanoma drugs, allowing the NPs to function as drug delivery systems. Additionally, SP iron oxide cores possess properties for magnetic resonance imaging (MRI) and magnetic hyperthermia therapy (MHT). SPIONs have been fully characterised, including entrapment and drug-release in a controlled environment. A α-melanocyte-stimulating hormone (α-MSH) was incorporated into SPIONs to improve tumour-targeting and demonstrated preferentially increased MC1-receptor mediated-endocytosis in melanoma cell lines. Increased cytotoxicity was demonstrated in vitro using drug-loaded NPs, showing dose-dependent anti-proliferative effects in human and murine melanoma cell lines, using DBF or TRA and a synergistic combination. Drug-loaded NPs were efficacious in drug-sensitive and -resistant melanoma cells lines. In vivo, syngeneic studies with DBF, compared tumour growth with free- and NP-bound drug: mice were treated with DBF daily via oral gavage (31.5 mg/kg) or NP-bound DBF twice weekly via intravenous injection (2.5 mg/kg). Both treatments significantly reduced tumour volume, demonstrating the potential for NPs to decrease systemic toxicity and improve therapeutic efficacy. SPIONs loaded with DBF and TRA, showed an increased in vitro toxicity in human and murine melanoma cells compared to the drug alone. Subsequently, NP-bound DBF significantly reduced tumour volume in syngeneic GEMM5555 tumour-bearing mouse models in vivo, acting as a drug-delivery platform and improving therapeutic efficacy whilst decreasing systemic toxicity commonly associated with conventional chemotherapies. Anti-melanoma SPIONs can represent a lead therapeutic for the treatment of unresectable/recurrent tumours in both patients with locoregional and distant melanoma metastasis.

IFNγ is a key component of our immune system, regulating a plethora of biological processes, including anti-tumour immunity. Its role in tumour immunology is however contradictory, exerting anti-tumour effects early in tumour development but ultimately promoting tumour immune evasion. Its involvement in immunotherapy resistance is also extremely complex and is potentially mediated through multiple cellular processes. In this study, we aimed to generate further insights into the role of sustained IFNγ signalling in adaptation and acquired resistance to therapy in melanoma. To this end, the effects of chronic IFNγ treatment on gene expression of melanoma cell lines were assessed by RNA sequencing. PARP14 was identified as an IFNγ-target gene that is consistently upregulated in melanoma cells under the influence of IFNγ found in the tumour microenvironment as a part of an anti-tumour immune response. RNAseq in melanoma cells manipulated for PARP14 expression suggested that its expression potentially results in tumour progression and therapy resistance in multiple ways. We further show that PARP14 manipulation in melanoma cells affects their responsiveness to IFNγ, impacting their immunogenicity through the deregulation of antigen processing and presentation components. Next, we identified a PARP14-related gene signature, which is associated with immunotherapy resistance and poor prognosis in melanoma. We also showed that Parp14 silencing promotes tumour T cell infiltration and synergizes with immune checkpoint blockade (ICB) in a syngeneic mouse model, providing a strong rationale for PARP14 targeting in combination with ICB. IFNγ signalling has also been linked to resistance to conventional genotoxic therapy. RNA sequencing revealed upregulation of oxidative stress response, as well as DNA damage and repair pathways, in IFNγ-treated cells. The clinical relevance of these findings is highlighted by the fact that high expression of DNA damage response signature correlates with poor prognosis and relapse in melanoma patients. We showed that chronic IFNγ exposure renders melanoma cells resistant to the DNA damaging effects of genotoxic drugs, as well as to DNA damage-induced cell death. We identified PARP14 as an important mediator of the IFNγ- driven resistance to the DNA damaging effects of genotoxic drugs. It has been reported that IFNγ-treated cells are more metastatic when implanted into syngeneic mouse models. The contribution of PARP14 in cell migration and metastasis in melanoma was also assessed. PARP14 expression is elevated in metastatic melanomas compared to the primary tumour site. RNA sequencing analysis revealed that PARP14 silencing in melanoma cells results in the upregulation of cell adhesion genes and downregulation of EMT-related gene signatures. We showed that IFNγ preconditioning enhances the migration and invasion potential of melanoma cells and that this effect is mediated by PARP14.

Bromodomain and extra-terminal (BETs) proteins function as epigenetic readers by docking onto acetylated lysine residues (Kac) on histone tails via tandem bromodomains (BRDs), and recruiting protein binding partners via an extra-terminal recruitment domain (ET). Targeting BET BRDs is efficacious in different malignancy models; however, the underlying mechanisms remain largely unknown in solid tumours. Together with concerns over toxicity, this calls for a greater understanding of BET protein biology and alternative targeting approaches. p53 influences the phenotype observed in haematopoietic cells following BET BRD inhibition, so it was investigated whether p53 has a similar regulatory role in melanoma. Here, I show that melanoma cells undergo senescence or apoptosis following BET BRD inhibition, and that p53 mutation status potentially correlates with this cell fate decision. Furthermore, BET inhibition up-regulates autophagic flux in melanoma and prostate cancer cells. However, the effect of concomitant autophagy inhibition on cellular proliferation is heterogeneous. Finally, ET domain inhibition leads to autophagy- dependent senescence in melanoma, which provides proof-of-principle that both Kac- dependent and independent targeting of BETs yield a loss of cell viability.

Immune checkpoint inhibition using monoclonal antibodies targeting cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed cell death receptor-1 (PD-1) to enhance cytotoxic T-lymphocyte response against tumours has been used in the management of several malignancies including metastatic melanoma. Although preliminary studies in patients with untreated metastatic melanoma demonstrated significantly increased progression-free survival, there is an unmet need for clinical decision-making tools that can be used for monitoring the efficacy of these costly agents, their potential side effects and the identification of patients with the most suitable immunological profile for this treatment approach. The development of non-invasive imaging methods and imaging biomarkers of response to immune checkpoint inhibitors could therefore provide important diagnostic and prognostic information. This project builds on existing technologies in Cambridge to develop structural and functional imaging approaches for monitoring tumour response to immune checkpoint inhibitors. Magnetic resonance imaging (MRI), radiolabelling of neutrophils for single-photon emission computed tomography (SPECT) imaging and intravital imaging are explored as part of a co-clinical study on metastatic melanoma patients and syngeneic mouse models of cancer immunotherapy.

Malignant melanoma is a highly aggressive and often lethal disease. In 2012, patient outcomes were poor after resection of high-risk stage II and III melanoma, and vascular endothelial growth factor (VEGF) has been implicated in melanoma progression. The phase III AVAST-M clinical trial (recruiting between 2007-2012) randomized patients with high risk melanoma to 1 year of adjuvant bevacizumab (anti-VEGF antibody) or observation. Patients treated with bevacizumab experienced superior disease-free survival (DFS), and BRAFV600E mutation was unexpectedly predictive of benefit. The aim in this project was to investigate this novel association between BRAFV600E and VEGF, based on the hypothesis that BRAFV600E melanomas are dependent on VEGF and more sensitive to its inhibition. VEGF and CD31 expression were quantified in tissue microarrays (TMAs) and whole mount sections of AVAST-M melanomas. Although no differences were identified between BRAFV600E and wildtype (WT) tumours from TMAs, there was a trend towards higher VEGF and CD31 expression in BRAFV600E whole mounts. In three independent transcriptomic datasets including NanoString analysis of melanomas from the AVAST-M clinical trial, VEGF itself was not differentially expressed, but all identified receptor kinase ROR2 as overexpressed in BRAFV600E melanomas. Differential ROR2 expression was confirmed in a BRAFV600E/WT isogenic melanoma model. ROR2 expression decreased with pharmacological MEK inhibition, supporting regulation by MAPK signalling. In vitro, ROR2 expression associated with invasive and proliferative phenotypes, with novel evidence that ROR2 regulates VEGF secretion in BRAFV600E mutant melanoma cells. The putative ROR2 ligand Wnt5a was also identified as regulated by MAPK signalling, and MEK inhibition of BRAFV600E mutant ROR2-overexpressing cells was shown to suppress Wnt5a expression and VEGF secretion. An exploratory transcriptomic analysis of bevacizumab treated BRAFV600E/WT isogenic melanoma xenografts identified gene expression changes in the tumour and stroma, that were mutually exclusive between BRAFV600E and WT tumours, both basally and after bevacizumab treatment, and included upregulation of ROR2 in the tumours and Wnt5a in the stroma of BRAFV600E xenografts. Finally, as a predictive biomarker in AVAST-M patient samples, patients with melanomas containing high ROR2 expression trended towards a prolonged DFS after treatment with bevacizumab. In summary, this work identifies Wnt5a-ROR2 signalling as upregulated in BRAF mutant melanomas, contributing to increased VEGF secretion and potentially also to bevacizumab response.

The somatic mutations found in melanomas reflect the biological processes that govern tumour development. They also help shape how tumours evolve and escape immune regulation. Studying these changes can therefore help to refine our understanding of melanoma progression with the added potential to offer new perspectives for disease management. Most prior melanoma sequencing studies have focused on advanced disease. Thus, somatic alterations that influence the behaviour of early-stage tumours have not been fully explored. Consequently, in this thesis I study a collection of 524 primary melanomas on which extensive clinical data have been collected for almost two decades. I describe the mutational landscape of these tumours including driver genes, new recurrent variants, mutually exclusive genetic interactions and copy number alterations. I discuss and associate these features with aspects of tumour pathology, sun exposure, immunogenicity and patient outcomes. To identify genes required for melanoma survival, I intersect my genomic analysis with a dataset of CRISPR-Cas9 dropout screens and discover a melanoma-associated genetic vulnerability mediated by Interferon Regulatory Factor 4 (IRF4). I then begin to experimentally validate and explore the biological pathway by which IRF4 may function in the context of melanoma. Checkpoint inhibitors have revolutionised melanoma care, yet only a minority of patients respond to these treatments and our comprehension of the mechanisms governing PD-L1 expression on melanoma cells is still limited. In the second part of this thesis, I examine the regulation of the key checkpoint receptor PD-L1, which is often upregulated in melanoma to facilitate tumour escape. To improve our understanding of the processes controlling PD-L1 expression, and how the PD-1/PD-L1 axis can be targeted to overcome immune evasion, I employ a genome-wide CRISPR-Cas9 screening approach. I identify genes which elicit downregulation of PD-L1 when disrupted in melanoma cells, capturing several central processes including basal transcription, N-linked glycosylation and intracellular transport. A second extensive screen in eight cancer cell lines of melanoma, bladder and lung cancer origin validate these findings and link novel candidate genes, including Sphingolipid Transporter 1 (SPNS1), to the control of PD-L1 cell surface expression. Additional work is required to further validate and understand the regulation of PD-L1 through SPNS1, as well as its contribution to immune surveillance. In summary, I present the first comprehensive evaluation into the somatic alteration landscape of primary melanomas, gaining insight into the molecular architecture of these tumours. Additionally, I introduce novel genes and processes regulating PD-L1 gene transcription, processing and presentation on the cell surface. Collectively, these results improve our understanding of the genetic processes that govern primary melanomas, as well as providing valuable insights into the mechanism of PD-L1 regulation.

The multistep process of cancer metastasis is only partly understood and the complexity of the metastatic cascade is the major obstacle for the development of efficient treatment strategies. This complexity is especially acute in plastic cancers such as melanoma, which switch between different migratory strategies depending on intrinsic signalling pathways and external cues. By taking advantage of the tractable zebrafish, which allows visualisation of tumour cell migration and invasion throughout the living animal with unprecedented spatial and temporal resolution, we developed zebrafish transplantation models that revealed novel aspects of cancer metastasis. Using the developed in vivo models, we discovered that WDR5 and RbBP5, members of the methyltransferase complex that controls the tri-methylation of H3K4 (associated with an open chromatin formation) are critical for tumour cell migration through confined spaces. Moreover, we identified TIAM1, as a potential tumour suppressor in colorectal cancer and revealed that this function is attributed to the nuclear fraction of TIAM1. Finally, we discovered that invading melanoma cells migrated along the outer surface of the blood vasculature, resembling extravascular migratory metastasis (EVMM), an alternative form of metastatic cells spread. EVMM represents an underexplored mode of metastatic cell spread distinct from intravascular dissemination with angiotropic melanoma cells disseminating along the vascular surface in a pericyte location. The extravascular migration of melanoma cells resembles the embryonic migration of melanoblasts and is attributed to the aberrant regulation of neural crest developmental genes. We developed in vitro co- culture models and a high-throughput screening platform to explore this mode of metastasis. The identification of melanoma cell lines that performed EVMM and those that did not suggested that EVMM might be specific to melanoma cell lines derived from lung metastasis. Importantly, we could show the NF-kB signalling pathway is central to this phenomenon. Activation of this pathway promoted the spreading along vascular surfaces whereas its inhibition promoted the initial association with the endothelium. Furthermore, we showed that paracrine factors, secreted by EVMM-performing cells, guide this migratory phenotype. This adds important mechanistic detail to a poorly understood, but potentially lethal, mode of melanoma cell dissemination.

In patients with early-stage cancer, ctDNA detection rates can be low due to the presence of few or no copies of any individual mutation in each sample. Sensitivity may be increased by collecting larger plasma volumes, but this is not feasible in practice. Although cancers typically have thousands of mutations in their genome, previous analyses measured only individual or up to 32 tumour-specific mutations in plasma. Here, we demonstrate that sensitivity can be greatly enhanced for any given input DNA mass by analysing a large number of mutations via sequencing. We sequenced in plasma 10^2-10^4 mutated loci per patient, using custom capture panels, whole exome (WES) or whole genome sequencing (WGS). We developed a method for INtegration of VAriant Reads (INVAR) that aggregates reads carrying tumour mutations across multiple mutant loci, and assigns confidence to error-suppressed reads based on mutation context, fragment length and tumour representation. This workflow combines a number of concepts in a novel approach in order to quantify ctDNA with maximal sensitivity. We applied INVAR to plasma sequencing data from 45 patients with stage II-IV melanoma and 26 healthy individuals. ctDNA was detected to 1 mutant molecule per million, and tumour volumes of ~1cm^3. We show that this algorithm is applicable across targeted and untargeted sequencing methods. In patients with stage II-III melanoma who relapsed after resection, ctDNA was detected within 6 months post-surgery and prior to relapse in 50% of cases, compared to 16% in a similar cohort analysed with digital PCR. In addition, INVAR may enhance detection of ctDNA in samples with limited input or sequencing coverage by aggregating signal across a large number of mutations. Using low-depth WGS (0.6x), ctDNA was detected to 1 mutant per 10,000 molecules. Given that 60 genome copies of cfDNA may be obtained from 1 drop of blood (50-75μL), we suggest that INVAR may enable cancer monitoring from limited samples volumes. As tumour sequencing becomes more widespread allowing identification of a large number of mutations per patient, this method has potential to quantify ctDNA with enhanced sensitivity, and to enable routine cancer monitoring using low-depth sequencing, potentially from low-volume blood samples that might be self-collected.

As our understanding of the cancer genome has progressed, traditional chemotherapeutic agents are being replaced, in part, by targeted therapies. Development of these therapies is driven by our understanding of genetic vulnerabilities harboured by tumours. The aim of this project is to identify novel synthetic lethal interactions within melanoma. Synthetic lethality describes a relationship between two genes, where loss of either of the pair is compatible with cellular viability, but simultaneous loss of both genes induces cell death. This approach can therefore exploit somatically acquired mutations to specifically kill tumour cells and to define new therapeutic targets. Abstract In order to do this, we selected a panel of putative synthetic lethal interactions and built a bespoke multiplex CRISPR-Cas9 library to interrogate 1192 gene-pair interactions. We deployed this library on a panel of melanoma cell lines and a retinal pigment epithelial cell line as a normal comparator. We went on to develop a bioinformatic pipeline with which to analyse the paired screen output and using this pipeline we have identified a number of novel synthetic lethal relationships. Using low throughput assays we validated a number of the interactions found by our screen. Finally, using human tumour expression data, we identified a candidate pair with potential therapeutic relevance. Using external datasets and isogenic knockout models we have further validated this relationship and performed mechanistic experiments to explore the role of these genes within the cell and generate a hypothesis to explain why loss of these two genes results in cell death.

Skin cancers are the most common human malignancies, with rising incidence despite public health warnings into the dangers of excessive UV exposure. Skin cancers are divided in two major types: (i) keratinocyte or non-melanoma skin cancers (NMSC), including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), and (ii) melanoma. Melanoma represents less than 5% of all skin cancers, however it has the highest mortality rates. Several factors are known to induce melanoma progression, such as the overexpression of serine protease inhibitors (serpins). Vaspin (also known as visceral adipose tissue-derived serpin; serpinA12) is the most recently identified serpin. Vaspin is expressed in the skin epidermis, its expression is dysregulated in psoriatic skin, but its potential role in melanoma has not been explored. In the current work, I investigated the expression and biological role of vaspin in melanoma. I observed that vaspin is overexpressed in melanoma cells compared to benign nevi or melanocytes. Additionally, I found that vaspin does not regulate proliferation, survival or migration, however it induces cell invasion. I further showed that vaspin-induced invasion is dependent on matrix metalloproteinases (MMPs). Cutaneous SCC (cSCC) is the second most common skin cancer, comprising 20% of NMSC. cSCC arise from the aberrant proliferation of keratinocytes, although this process is also dysregulated in other skin diseases. Wingless-Type MMTV Integration Site Family (Wnt)/β-catenin signalling is upregulated in many cancers and is important for keratinocyte proliferation. Gene expression analysis has indicated that Wnt/β-catenin signalling is altered in cSCC. In the second part of this work, I investigated the Wnt/β-catenin signalling pathway as a possible driver of cSCC development. I demonstrated that β-catenin is expressed and active in human cSCC cell lines. However, stimulation with recombinant Wnt member 3 (Wnt3a) did not increase the levels of β-catenin in cSCC cells. Knockdown of β-catenin led to reduction in cSCC proliferation, without affecting cell viability and surprisingly, without affecting the classical β-catenin target genes. Two pharmacological inhibitors of the β-catenin interaction with TCF or LEF transcription factors, BC21 and PRI-724, respectively, reduced cSCC cell viability in a dose-dependent manner. Surprisingly, this effect was preserved in cells, in which β-catenin was knocked down, indicating that the observed reduction of cell viability by the compounds was independent of β-catenin. Subsequently, I found that BC21 and PRI-724 are reducing cell viability by altering the expression of cell cycle regulators, thereby inducing cell cycle arrest. In summary, I describe for the first time the biological effect of vaspin in the context of melanoma. Additionally, I describe the role of β-catenin signalling in cSCC cells and the effect of BC21 and PRI-724 inhibitors.

Dacarbazine (Dac) is a chemotherapeutic for melanoma. Its poor solubility, short half-life, and side effects limit its therapeutic use. Nanoparticles used as drug delivery systems (DDS) improve drug pharmacological properties, target site concentration, and stability. This study aimed to improve the therapeutic efficiency and decrease the systemic toxicity of Dacarbazine using nanostructured lipid carriers (NLC) for topical administration. Different NLC compositions were synthesised using a laboratory-based high sheer dispersion (HSD) method, and applying varying processes parameters. The preparations were optimized to achieve the desired NLC characteristics (average size 155±2 nm, polydispersity index (PDI) 0.1±0.05 and zeta potential -43.7±0.6) assessed using hydrodynamic light scattering (DLS), zetasizer and transmission electron microscopy (TEM). The optimized NLC (NLC/PI) were then used to encapsulate Dacarbazine. The resultant NLC/PI-Dac was characterized (average size 190±10 nm, PDI 0.2±0.03 and zeta potential -43.5 ± 1.2) and crystallinity determined using X-Ray Diffraction (XRD). The pharmacological properties of NLC/PI-Dac were then investigated and the percentage of encapsulation efficiency and drug loading capacity were determined to be 98.5±0.2% and 23.4±0.2%, respectively. The in vitro toxicity of NLC/PI-Dac was assessed using a melanoma cell line (A375). The in vitro drug release profile of NLC-Dac showed a biphasic pattern; 50% released over 2 hours and remainder within 30 hours. No toxicity was measured for the different NLC/PI concentrations after 24, 48 and 72 hours treatment, but there was an increase in toxicity of Dacarbazine after NLC/PI encapsulation (NLC/PI-Dac) at all concentrations and all-time points. NLC/PI and NLC/PI-Dac were stable in size, PDI and zeta potential when stored at 4°C for 3 months and the developed process was transferable to industrial-scale synthesis using the Micro Jet Reactor technique. NLCs were shown to be a suitable drug delivery system for Dacarbazine, achieving a desirable drug loading, encapsulation efficiency and drug release profile.

Developing personalized cancer therapies based on cancer genomics methodologies forms the basis for future cancer therapeutics. A genomics platform was developed based on canine cancer to produce a proof-of-concept for personalized genomics led therapeutic choices but also developing personalized therapeutics for canine cancer patients themselves. The platform identified the genetic state of a canine cancer patient within two drugable pathways; p53 and HSP90/IRF1. The former gene was wild-type p53 thus directing the use of p53 activating molecules. The latter mutations in both HSP90 and IRF1 suggested an investigation into HSP90 and interferon signalling molecules as drug leads. Drugs that target both of these pathways were subsequently used to measure drug effects in cell line models but also to identify novel biomarkers of drug responses. My study focused on the effect of the HSP90-inhibitor Ganetespib had on its client proteins, particularly IRF-1. Briefly my results indicated the following:(i) Ganetespib downregulated IRF-1 protein levels in A375 cell lines and this attenuation was not mediated by either MDM2 or CHIP (E3 ligase). IFNγ- induced IRF-1 was also observed to be downregulated when Ganetespib was used in combination therapy.(ii) Insitu proximity ligation assay showed induced HSC70 upregulation upon HSP90 inhibition by Ganetespib and HSC70/MDM2 complexes were seen to be stabilized compared to the usage of MDM2/p53 inhibitor-nutlin. I hypothesize that MDM2/HSC70 complex might chaperon IRF-1 into lysosome for degradation via chaperon mediated autophagy pathway. (iii) My results also indicate that Ganetespib can downregulate IFN γ- induced PDL-1 expression in melanoma cell lines. Pre-sensitizing the cells with Ganetespib prior to the addition of IFNγ could attenuate PDL-1 to basal levels. (iv) My results also showed that the downregulation of PDL-1 by Ganetespib is an IRF-1 dependent mechanism. Therefore, my results suggest that HSP90 represents an important emerging target for cancer therapy because its inactivation results in the simultaneous blockade of multiple signalling pathways and can also sensitize tumor cells to other anticancer agents. Targeting HSP90 could also help to disrupt PD1/PDL- 1 interaction and activate immune system to recognise tumor cells. I conclude that HSP90 and IRF-1 play a critical role in types II interferon pathways and these findings establish a novel basis for the design of future Ganetespib-based combinatorial approaches to improve patient outcomes in this disease. These approaches finally demonstrate that cancer genomics can stratify choice of cancer drugs used on patients but also provide evidence that cancer patient samples can be used for the specific stratification of cancer drug choice based on cancer genomics data.

Melanoma is the most lethal type of skin cancer with over 132,000 cases occurring globally each year and continually rising incidence. BRAFV600E inhibitors have led to clinically significant improvements in outcomes for melanoma patients, yet many patients with metastatic melanoma rapidly succumb to the disease due to eventual chemoresistance or insensitivity to the drug. Thus, it is critical to identify new therapies that can act alone, or be combined with available treatments for enhanced efficacy and/or to overcome drug resistance. Evidence from human melanoma indicates that the melanocyte lineage is critical for melanoma survival and contributes to therapeutic resistance. MITF is a highly conserved “master melanocyte transcription factor” with a complex role in melanoma. Our lab has previously developed a temperature sensitive BRAFV600E mitfavc7 zebrafish melanoma model carrying a human oncogene and mitfavc7 splice site mutation that enables the conditional control of its endogenous activity by changes to water temperature. As part of my PhD project, I characterized and compared two new models developed since then: a very aggressive BRAFV600E mitfavc7p53M214K melanoma model with three driving mutations and a slower developing BRAF-independent mitfavc7p53M214K. I showed that the MITF activity is crucial for melanocyte survival in both models and that both mutated BRAF and p53 deficiency are oncogenic with low levels of MITF, and result in fish nevi and melanoma resembling the pathology of human disease. Both models are also relevant to a low-MITF subclass of human melanomas that emerged from a recent classification by The Cancer Genome Atlas Network. In addition, I established that, similarly to the BRAFV600Emitfavc7, complete inhibition of MITF activity leads to rapid tumour regression, but once its activity is restored the melanomas recur at the same site as the original tumour. I used histopathology studies and melanocyte lineage transgenes to identify and visualize subpopulations of cells remaining at the site of regression in these new zebrafish melanoma models. I hypothesised that these are the cells of origin for tumour recurrence (melanoma stem or progenitor cells), showed that some of them express a cancer stem cell marker aldehyde dehydrogenase, and attempted to target these subpopulations using 5-nitrofurans (a prodrug NFN1, shown previously by our lab to target ALDHhigh subpopulations in context of melanoma) in fish after melanoma regression. Finally, I also developed and described a new primary zebrafish melanoma cell line that I derived from one of these zebrafish tumours. This study is still in progress, but the cell line will be a useful tool for further investigation of these proposed melanoma progenitor cells in vitro, with potential applications for lineage tracing and transplantations.

Malignant melanoma is one of the most aggressive types of malignancies in humans resulting in 50,000 deaths worldwide annually. Malignant melanoma is highly immunogenic and it is now well established that the host immune system can detect and kill melanoma. In this thesis, our work has focused on establishing zebrafish as a viable model system to study CD4+ T cell mediated immune responses against melanoma as well as an in-vivo model system compatible for testing multiple immunotherapy strategies. We have created BAC transgenic zebrafish with vibrantly labeled CD4+ cells allowing us to scrutinize the development and specialization of zebrafish CD4+ leukocytes in-vivo. We demonstrate the utility of this zebrafish resource for interrogating the complex behavior of immune cells at cellular resolution by the imaging of intimate contacts between CD4+ T cells and mononuclear phagocytes. We reveal that sub-specialization of CD4+ T cells in to TH1, TH2 and Treg cells is conserved in zebrafish and most importantly, as in mammals, we show that zebrafish CD4+ T cells will infiltrate melanoma tumours and obtain a phenotype consistent with a type 2 immune microenvironment. We have built a Tamoxifen inducible CreERT2 system, which can be used to specifically induce the expression of selected immune-regulatory molecules by the zebrafish melanocytes. We report that forced expression of IFNγ in the tumour microenvironment using this system has resulted in enhanced tumour regressing in our autochthonous zebrafish melanoma model. However, new tumour nodules were also observed to develop in the vicinity of regressing nodules which are unpigmented and potentially hypo-immunogenic. In parallel, we have observed that forced secretion of the ectopic domain of zebrafish PDL1 homologue suppresses tumour formation but again late escapee tumours were unpigmented. RNA sequencing of the resistant tumours (IFNγ or PDL1 expressing) could reveal unique but also shared signatures for progressing tumours, which can be used to predict immunotherapy responses in human patients.

There have been dramatic advances in melanoma therapy in the last 10 years, yet there is still a demand for effective and affordable therapies. To identify novel therapeutic pathways a transcriptome analysis was performed on zebrafish melanoma models representing the different stages of melanoma progression. Transcriptomic differences between pre-malignant and malignant conditions highlighted lipid metabolism as a potential mediator of progression. A mass spectrometry analysis confirmed multiple changes in lipid composition between wild type fish, pre-malignant and advanced melanoma models. To better investigate metabolism a positron emission tomography (PET) technique was developed in zebrafish. Tumours in the zebrafish were successfully scanned with FDG used to detect human tumours. A novel tracer of unconjugated FA was then developed and, consistent with inferences from the transcriptome and mass spectrometry, was shown to be incorporated into tumours. Demonstrating the feasibility of PET in zebrafish now opens the way to systematic use of this organism in tracer development with potential time-saving and cost benefits. One of the most significantly up-regulated genes exclusive to the malignant state encodes lipoprotein lipase (LPL). LPL is involved in the release and uptake of FA from circulating triglyceride. LPL was found to increase the rate of tumour appearance and tumour growth in a zebrafish tumour assay. LPL was expressed in human tumours and expression correlated with progression. Melanoma cell lines expressed LPL and knocking-down LPL resulted in reduced cell numbers. The effect was most dramatic in WM852 cells. A novel role for LPL in autophagy was identified. WM852 cells treated with LPL siRNA showed a stabilisation of p62/SQSTM and induction of LC3B II. Electron microscopy revealed large autolysosomal vacuoles in the cytoplasm. Additionally many cells showed damaged mitochondria with absent cristae. The dependency of cells on LPL seemed to be modified by the co-expression of fatty acid synthase (FASN) required for de novo FA synthesis, as the magnitude of the effect of LPL-knockdown was dependent on the levels of FASN expressed in melanoma cell lines. Moreover, combining LPL and FASN inhibitors synergised to kill cells previously less sensitive to LPL inhibitor. FASN and LPL co-inhibition could provide a unique combinatorial therapeutic strategy.

The typical response of cancer patients to treatment is only temporary, and is often followed by relapse. The failure of various therapeutic strategies is commonly attributed to the emergence of drug resistance. The response patterns for patients under such treatments indicate that complex dynamics regulate the response of the tumour to the therapy. The environment in which the tumour lives (the stroma) is known to be a modulator of multiple mechanisms that lead to drug resistance and seems to be a likely candidate for explaining some of this complexity. Understanding the role of stromal cells in the promotion of drug resistance is critical for the design of optimal treatment strategies, and for the development of novel therapies that selectively target both the tumour and the stroma. In this thesis we design two novel mathematical models that describe cancer growth within its environment and the evolution of drug resistance within spatially complex and temporally dynamic tumours. A compartment model captures clinically observed dynamics and allows direct comparison with experimental data, facilitating model parametrisation and the understanding of inter-tumour heterogeneity. An individual cell-based model highlights the key role of local interactions, determining heterogeneity at the tissue scale, that will eventually determine treatment outcome. A non-spatial approximation of this second model allows us to find analytic guidelines for the design of effective therapy. These tools allow the simulation of a range of treatment strategies (including combination of different drugs and variation of schedule) as well as the investigation of therapy response based on patient- or organ-specic parameters. The work developed in this dissertation is based on the paradigmatic biology of melanoma and non-small cell lung cancer. Its results are therefore applicable to a variety of cancer treatments that target similar processes, and whose therapeutic failure can be attributed to environment-mediated drug resistance.

Cutaneous melanoma is a cancer of melanocytes, the pigment-producing cells in our skin. It is one of the most aggressive human malignancies, constituting only about 2% of all dermatological cancers but being responsible for over 75% of all deaths from skin cancer. It has recently become a major public health problem, as it is now the fifth most common cancer in the United Kingdom after its incidence more than quadrupled in the last three decades. For these reasons, understanding the biological processes that are involved in its development is of great importance for devising novel treatments and for the management of patients in the clinic. The study of the genetic factors that influence melanoma risk can uncover mechanisms that are relevant in the transition from a benign melanocyte to a malignant melanoma. Approximately 10% of all melanoma cases are familial, and about half of these familial cases can be explained by pathogenetic variants in genes such as cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase 4 (CDK4), breast cancer 2 (BRCA2), BRCA1-associated protein-1 (BAP1) and in the promoter of the telomerase reverse transcriptase (TERT). However, about 50% of all familial melanoma cannot be explained by mutations in known genes. In this dissertation, I detail the methodology I followed in an effort to uncover additional high-penetrance melanoma susceptibility genes. I analysed exome and genome sequence data from a total of 184 individuals that belong to 105 melanoma-prone families from the United Kingdom, The Netherlands and Australia that did not have any pathogenetic variants in known susceptibility genes. I applied different gene prioritisation strategies and developed novel software tools in order to devise a list of plausible melanoma susceptibility candidate genes; these analyses suggested that genes regulating telomere function could be influencing melanoma risk. After performing functional experimental analyses, our research team was able to determine that carriers of rare variants in the protection of telomeres (POT1) gene, a member of the shelterin complex that safeguards telomere integrity, are at high risk for developing melanoma. We successfully described the mechanism by which this happens, showing that the variants identified either disrupt POT1 mRNA splicing or abolish the ability of POT1 to bind to telomeres, and lead to increased telomere length in carriers when compared to melanoma cases with wild-type POT1. The main finding of the work described in this dissertation is the identification of telomere dysfunction as an important contributor to the risk of developing melanoma, and possibly other cancers. Our analyses suggest that POT1 is the second most commonly mutated high-penetrance melanoma susceptibility gene reported thus far, and moreover, that rare variants in this gene constitute the first hereditary mechanism for telomere lengthening in humans.

Melanoma is one of the most common cancers in the world. For primary melanoma, early diagnosis and surgical excision are effective treatments but, despite the new targeted therapies and immunotherapies, there is still a need for more effective treatment options to improve overall survival for patients with metastatic melanoma. Chemotherapy with genotoxic agents remains the main approach for most cancers, but DNA repair pathways in cancer cells reduce their effectiveness. So disruption of key DNA repair pathways, such as nucleotide excision repair (NER), could be an effective option to combine with chemotherapy for melanoma. The structure-specific endonuclease ERCC1-XPF, which heterodimerises through the C-terminal helix-hairpin-helix (HhH)2 domains of both proteins, is essential for NER. The aim of my project was to determine the mechanism involved in regulating the level of the ERCC1-XPF heterodimer with a view to disrupting NER activity. The project started by determining the ERCC1 and XPF response in six melanoma cell lines to the chemotherapeutic cisplatin at the mRNA and protein levels. Although the mRNA and protein levels of both ERCC1 and XPF increased, there was variable consistency between the cell lines, raising the possibility that post translational modification may play an important role in the regulation of ERCC1- XPF activity. We chose to focus on ubiquitination, because it can affect a protein’s activity at both expression and activation levels and several examples of ubiquitinated DNA repair proteins were known. In the pilot study we found that ERCC1 was accumulated after proteasome inhibitor treatment and decreased by treatment with a translation inhibitor in two melanoma cell lines, suggesting that ERCC1 may be ubiquitinated. By cotransfecting His-tagged ubiquitin and non-tagged ERCC1 constructs into melanoma cells and performing an ubiquitin assay, we found that ERCC1 was degraded by the proteasome system through polyubiquitination or multiple monoubiquitination. To determine the nature of the ubiquitination type, we mutated each of the seven Lys residues on ubiquitin and carried out additional assays with ubiquitin single and combination mutants, and discovered that Lys33 was most likely involved in the proteasome dependent degradation of ERCC1. By immunoprecipitation with an antibody to linear ubiquitin from melanoma cell extracts containing a ubiquitin construct with all seven Lys residues mutated to Arg, we found that the N-Met of ubiquitin was also most likely involved in ERCC1 ubiquitination. To determine which Lys of ERCC1 is used by ubiquitin, we did another series of in vivo ubiquitin assays with full length and truncated ERCC1 constructs and found that the key amino acid is most likely within the C-terminal XPF binding domain of ERCC1. By cotransfecting the full length ERCC1 and ERCC1 truncation constructs together with full length XPF, we showed that the ubiquitination of ERCC1 was not an artefact resulting from overexpression of ERCC1 alone and that the stability of XPF was dependent on the overexpression and stability of ERCC1. We then made single lysine and lysine combination mutants in the XPF binding domain of ERCC1 and found that none of the lysines were essential for ubiquitination of ERCC1, indicating that a non- lysine amino acid might be used for ubiquitination. However, using a transfection-based NER assay in ERCC1-deficient cells, we found that ubiquitination of Lys 295 could be involved in regulating the DNA repair activity of ERCC1-XPF. The in vivo ubiquitin assay result after cotransfection of ERCC1 and XPF, which showed that XPF was dependent on the presence of ERCC1 for stability, but not vice versa, was inconsistent with previous published data suggesting that heterodimerization was essential for the stability of both proteins. Instead we hypothesised that homodimerization of ERCC1 might be another mechanism to keep ERCC1 stable and obtained evidence for this at the overexpression level by immunoprecipitation following cotransfection of Myc-tagged ERCC1 and Flag-tagged ERCC1 or ERCC1 truncations, which was supported at the endogenous expression level by size exclusion chromatography on melanoma cell extracts to identify ERCC1 in different molecular weight fractions. In the previous in vivo ubiquitin assay, we found that levels of transfected full length ERCC1 and XPF were dramatically reduced by cotransfection with the Flag-tagged ERCC1 (220-297) construct that just contains the XPF binding domain of ERCC1. This led to another hypothesis, that the ERCC1 (220-297) peptide can decrease endogenous levels of ERCC1 and XPF and so be a potential drug in combination with cisplatin chemotherapy. This hypothesis was verified in stable transgenic cell lines expressing ERCC1 (220-297) which showed reduced levels of ERCC1 and XPF and of NER and increased sensitivity to cisplatin and UV irradiation. Based on the above results and supporting bioinformatics analysis we have made the following conclusions: ERCC1 is regulated by the ubiquitin-proteasome degradation pathway through linkages most likely involving Lys33 and N-Met; the XPF binding domain is most likely the key domain for ERCC1 ubiquitination; XPF stability is dependent on the presence of ERCC1 and seems affected by ERCC1 ubiquitination; ERCC1 seems to be ubiquitinated in a non-conventional lysine-independent manner and ubiquitination of Lys 295 might be involved in the regulation of the DNA repair activity of ERCC1- XPF; homodimerization is most likely a novel mechanism to keep ERCC1 stable; the ERCC1 (220-297) peptide can destabilise both ERCC1 and XPF and could be a potential drug in combination with genotoxic therapies.

MITF is the master melanocyte transcription factor and has a complex role in melanoma. Both gain- and loss-of function mutations in MITF have been identified in melanoma, although its’ role in melanoma development and the effects of targeting MITF are unknown. Using a temperature-sensitive mitf zebrafish mutant I show that low levels of MITF are oncogenic with BRAFV600E in melanoma progression. By pathology and MITF target gene expression, BRAFV600Emitfavc7 tumours are distinct from BRAFV600Ep53M214K tumours, and represent two melanoma subtypes. Melanomagenesis can also be driven independently of BRAFV600E, in a transgenic zebrafish with mutations in mitf and p53, representing a new melanoma model. Abrogating MITF activity in BRAFV600Emitfavc7 melanoma leads to regression of the tumour, characterised by macrophage infiltration and increased apoptosis. This result confirms the dependence on MITF activity in BRAFV600Emitfavc7 melanomas and highlights the role of MITF as a therapeutic target for melanoma. Exome and transcriptome sequencing has been carried out to gain insight into the expression and genomic mutational landscape that is driven by these melanoma transgenic models and results in these genotype-phenotype specific subtypes observed.

The cancers of the skin, melanoma and the keratinocyte cancers, basal cell andsquamous cell carcinomas (BCC and SCC), are among the most common cancersin white populations. While ultraviolet radiation (UVR) is their principal cause,links with non-UVR-related factors have also been noted. Ultimately, theinteraction of these elements results in malignancy however, understanding oftheir specific contributions remains incomplete. This thesis reports findings fromsix studies aiming to investigate gaps in current knowledge of the role of UVR andnon-UVR-related risk factors on skin cancer. The papers are groupedaccording to the aspects of skin cancer epidemiology and aetiology they address. The first two papers address the descriptive epidemiology of melanoma inEngland, a country with low ambient solar UVR. They arise from ecologicalstudies using national melanoma registration data and document rising trends inmelanoma incidence by anatomic site (Paper 1), and by region of residence andsocio-economic deprivation (Paper 2). Their findings were consistent with thesuggestion that increases in recreational UVR exposure are driving rises inmelanoma rates. These results emphasise both the need to closely monitor UVRexposure and melanoma trends and the importance of public health campaigns. The second group of three papers considers the assessment of associations ofnutritional factors with keratinocyte cancer. Two studies use data from aprospective cohort to evaluate the relationship between dietary intake (Paper 3)and blood concentrations (Paper 4) of omega-3 and omega-6 polyunsaturatedfatty acids (PUFA) in relation to BCC and SCC risk. Associations with both PUFAtypes were observed. In addition, Paper 5, a three-way correlational assessment,demonstrated that questionnaire and blood circulating levels of omega-3 PUFAwere highly correlated with measures of skin bioavailability. Collectively, thesestudies give evidence for associations of these nutrients with skin cancer and forthe utility of both intake and biomarker measures for assessing the relationships. The final paper explores the relationship between a widely cited non-UVR riskfactor, namely scars and cancers of the skin. It reports a systematic review of allpublished observational studies quantifying this association. While innumerablecase reports were found, quantitative analyses were rare. The review identifieda major gap in the literature where knowledge of scar malignancies is notevidence-based, but rather founded mainly on cumulative anecdotal reporting. Taken together, this body of published work highlights the largely unrecognisedcomplexity of the aetiology of cancers of the skin. Future research must bebroad in scope in order to advance understanding of the interaction betweenUVR and other risk factors and to provide a base for health messages aimed atreducing the burden of these malignancies.

One key to understanding how cells integrate and how they respond to diverse stimuli in order to direct a transcriptional response is knowing how a transcription factor may be directed to an appropriate subset of its target genes. One mechanism with which this may be achieved is by modulation of the transcription factor’s post-translational modification status. The microphthalmia-associated transcription factor (MITF) is the master regulator of the melanocyte lineage, and it is also a lineage addiction gene in melanoma. Low or high levels of MITF expression induce a reversible cell cycle arrest. Invasive behaviour is characteristic of low MITF expression; differentiation a product of high MITF activity; and moderate levels of MITF expression promote proliferation. A major, unaddressed problem is how DNA binding by MITF may be differentially directed such that it regulates either a proliferation-associated or a differentiation-associated gene expression programme appropriate to the cellular microenvironment. This thesis explores the function and regulation of the signalling pathways controlling novel post-translational modifications of MITF. One such modification, in the DNA binding domain of MITF, defines a key switch that controls MITF’s DNA binding affinity and specificity. Moreover, a novel set of MITF target genes are revealed that extend its control beyond pigmentation and cell cycle regulation to implicate MITF as an overall regulator of cell behaviour in the melanocyte lineage.

Melanoma is the most aggressive skin cancer with high recurrence and low survival rate. In addition to genetic mechanisms, resistance also arises from phenotypic heterogeneity in which a proportion of cells, the so-called melanoma stem or initiating cells, survive therapy. Due to a lack of reliable markers, however, there is still debate about the existence of these cells in melanoma. Consistent with phenotypic heterogeneity, previous observations in our laboratory have demonstrated that cells in melanoma can reversibly segregate in vivo into different subpopulations with different properties, such as differentiation or increased invasive capacity (potentially attributed to the existence of de-differentiated stem-like cells). To characterise these cells, a dual reporter lentiviral system was engineered, expressing fluorescent proteins under cell stage/phenotype-specific promoters. The promoters for the transcription factors POU3F2 (Brn2) (to mark de-differentiated cells) and the microphthalmia-associated transcription factor (MITF) (to mark proliferating and differentiated cells) were chosen. Lentivirally-transduced cells were used to screen a library of kinase inhibitors for their potential to affect promoter activity in vitro. The RhoA/ROCK pathway, known to contribute to invasion and metastases, was identified to play a role in Brn2 promoter activity and exhibited differential effects on both the MITF and Brn2 promoters in 501mel and SKmel28 cell lines. Through investigation of other signalling pathways involved in melanoma metastasis, we also identified the co-activator Mastermind-like 1 (MAML1), previously reported to act in the Notch pathway, as an activator of the Brn2 promoter via the transcription factor TCF3, and the MITF promoter through the lymphoid-enhancer binding factor 1 (LEF1). The effects of MAML1 on Brn2 and MITF promoter activity were potentiated by β-catenin. These findings provide new opportunities for the identification of therapeutic targets to prevent metastases formation in melanoma.

Advanced melanoma poses a major therapeutic challenge, and despite the development of recent promising therapeutic agents, resistance to treatment remains a problem. Until recently, despite low response rates, alkylating agents dacarbazine and temozolomide (TMZ) were the standard of care for the treatment of advanced melanoma. The cytotoxic effects of these agents relies upon the formation of alkylated base lesions such as O6-methylguanine (O6MeG), which is repaired by a protein implicated in TMZ resistance called O6-methylguanine-DNA methyltransferase (MGMT). Failure to resolve such alkylated bases results in DNA replication-associated double-strand breaks (DSBs). The type 1 insulin-like growth factor receptor (IGF-1R) mediates a number of characteristics common to cancers, including proliferation and survival, and evidence suggests it may also contribute to resistance to many anticancer agents. The aims of this study were to test whether melanoma cells could be sensitized to TMZ by small molecule IGF1R inhibitors, and to explore the mechanism of any chemosensitization. This study found an association between basal IGF1R phosphorylation and in vitro TMZ resistance in seven MGMT-proficient melanoma cell lines, suggesting that IGF1R activation may be linked with TMZ resistance. Furthermore, IGF1R inhibition caused dose-dependent sensitization of melanoma cells to TMZ, regardless of BRAF mutation status. This reduction in cell survival was not accompanied by an increase in apoptosis, but rather Chk2 activation and an accumulation of cells in the G2/M phase of the cell cycle, suggesting a possible effect of IGF1R inhibition on DNA repair. IGF1R depletion was found to increase MGMT protein levels and activity, but this effect was not seen in IGF1R inhibited cells. In addition, IGF1R inhibition was not epistatic with MGMT inhibition, and IGF1R inhibition reduced survival of TMZ treated MGMT-null cells. This suggested that TMZ sensitization by IGF1R inhibition was independent of MGMT. IGF1R inhibition did however cause an increase in the accumulation of TMZ-induced RPA foci, and delay in resolution of RAD51 foci. Together with the finding that IGF1R inhibition reduced survival in PARP inhibited melanoma cells, these results suggested that IGF1R inhibition influenced DSB repair by homologous recombination. Finally, the combination of IGF1R inhibition with TMZ was tested in a mouse model and was found to be tolerable. TMZ or IGF-1R inhibitor alone caused minor reduction in melanoma xenograft growth rates (rate reduction by 13% and 25% respectively), while combination treatment caused supra-additive growth delay (72%) that was significantly different from other treatment groups (p<0.05). The findings of this study suggest IGF1R inhibition as a possible option in overcoming alkylating drug resistance in melanoma.

The changes in splicing of MDM4 in human melanomagenesis are critical to p53 activity and represent potential therapeutic targets. Genomic characteristics of melanoma and precursor lesions, such as copy number variation and gene expression, have been investigated in detail. However, there has never been a detailed survey of splicing changes that occur during melanomagenesis. Splicing changes can be a very early event in melanoma tumor progression, with characteristic changes in the p53 pathway already in place in early nevi. MDM4 is upregulated in a strong majority of melanoma cases and has been described as a “key therapeutic target in cutaneous melanoma”. Identifying splicing changes in human tissue specimens will provide therapeutic targets during initiation, dysplasia, and progression toward metastatic melanoma.Splicing of MDM4 has been characterized in melanomas with increased MDM4 isoform expression over canonical MDM4-FL. However, these studies have utilized new or existing data obtained by next-generation sequencing (NGS) methods, typically Illumina sequencing, relying on short reads. A difficulty associated with NGS analysis is the loss of “connectivity” data in which full transcripts are inferred from the presence of splice junction reads. To address this problem, long-read, third generation sequencing, was utilized to read the entire length of transcripts, intact. Oxford Nanopore flow cell sequencing allowed for quantification and identification of the MDM4 transcripts, both alternative and canonical, present in melanoma specimens. RT-PCR and Nanopore sequencing provided a direct view into the isoform landscape of melanoma specimens while RT-qPCR quantified the expression of major MDM4 isoforms represented in the samples. This study identifies and quantifies MDM4 isoforms present in malignant melanoma tumor samples and provides evidence of a novel hybrid MDM4-A & MDM4-S isoform (MDM4-A/S).

The master-regulators of an epithelial-mesenchymal transition (MR-EMT) have a pivotal role in the regulation of carcinoma development, promoting transformation and generating a migratory and invasive phenotype. Within epithelial cells, the ZEB proteins are co-regulated, jointly repressed by the miR-200 family of microRNAs. However, here it is demonstrated that the expression and regulation of the MR-EMT in malignant melanoma cell lines appears to be fundamentally different, with a hierarchical organisation identified. ZEB2 and SNAIL2 were found to be expressed in melanocytes, whilst ZEB1 and TWIST1 expression was acquired by a sub-set of malignant melanoma cell lines. Melanoma-initiating mutations within B-RAF and NRAS were shown to reversibly promote expression of ZEB1 and TWIST1 at the expense of ZEB2 and SNAIL2. Additionally, ZEB2 and SNAIL2 were identified up-stream of ZEB1 and TWIST1 within the MAPK signalling cascade, with ZEB2 functioning as a repressor of ZEB1. Furthermore, ZEB2 and SNAIL2 were found to positively regulate expression of MITF, a marker of melanocyte differentiation. In contrast, ZEB1 repressed expression of MITF and was the primary transcriptional repressor of E-cadherin, an adhesion molecule vital for the interaction between differentiated melanocytes and keratinocytes. Previously, within epithelial cell lines, all the MR-EMT have been identified as transcriptional repressors of E-cadherin. However, ZEB2 and SNAIL2 were co-expressed with E-cadherin within melanocytes and melanoma cell lines and, along with TWIST1, were not able to independently induce E-cadherin re-activation following repression. Surprisingly, ZEB2 became a repressor of E-cadherin in conjunction with ZEB1. Finally, E-cadherin expression was also shown to be controlled in a ZEB1-dependent manner by the transcriptional co-repressor BRG1, the ATPase subunit of the SWI/SNF chromatin remodelling complex, and by the presence of DNA methylation at the E-cadherin promoter. Indeed, DNA methylation was identified as a possible factor controlling the success rate of metastatic colonisation in melanoma cells, allowing for the dynamic re-expression of E-cadherin at the secondary site. These data demonstrate that in malignant melanoma the expression and regulation of the MREMT is fundamentally different to that of epithelial tumours, with the MR-EMT structured hierarchically, with opposing regulatory functions.

Reactivation of master regulators of epithelial to mesenchymal transition (MR-EMT) represents the molecular basis for tumour cell plasticity, malignant transformation and metastases. However, the current evidence on the specific role of MR-EMT in melanomagenesis has not been fully addressed. The purpose of this investigation was to assess the expression and regulation of these factors in malignant melanoma and to evaluate their prognostic and clinical significance. In vitro experiments indicated that a switch in MR-EMT protein expression ZEB2/SNAI2 to ZEB1/TWIST1 is RAS-RAF-MAPK signalling dependent. In addition, evidence supported a MR-EMT interactome, in which transcriptional repression of ZEB2 by Fra-1 resulted in upregulation of ZEB1, independently of miR-200 family. Further in vitro and immunohistochemical (IHC-P) analyses showed that E-cadherin and VDR protein levels were significantly reduced by the presence of ZEB1 in melanoma cells and archive tissues. Motility assays demonstrated that ZEB1 but not ZEB2 enhances cell migration. IHC-P analyses of ZEB2/SNAI2 (n=142/28) showed a statistically significant gradient of stronger staining at superficial sites compared to the deep sites in a select cohort of independent and matched melanoma tumour samples. In contrast, ZEB1 (n=142) and TWIST1 (n=133) showed higher staining in deep sites of primary melanomas and metastases. Trend analyses showed a significant MR-EMT switch in this progression series from high levels of ZEB2/SNAI2 in naevi towards high ZEB1/TWIST1 expression in melanomas. In primary melanomas these factors were also significant in Kaplan Meier survival curves and after two step cluster analysis the combined profile of ZEB1[superscript high]/TWIST1[superscript high]/ZEB2[superscript low] predicted the worse prognosis (P=0.001). Multivariate Cox regression analyses of IHC-P staining indicated that only the gain of ZEB1 (P<0.002, n=98) and superficial TWIST1 (P=0.012) were associated with poor metastasis-free survival and independent of breslow depth. In conclusion, the reversible switch between ZEB1/TWIST1 and ZEB2/SNAI2 is controlled by RAS-RAF-MAPK pathway activity and constitutes an independent factor of poor prognosis in patients with malignant melanoma.

The most frequent mutation identified in both benign nevi and malignant melanoma is the constitutively activating V600E substitution of BRAF. However, how additional mutations co-operate with BRAFV600E to promote subtypes of melanoma in animals is only beginning to be understood. In this thesis, I generate and analyze zebrafish BRAFV600E melanoma models and also develop the first animal model for BRAFV600E nevus recurrence. In my first data chapter, I develop a unique animal model of nevus recurrence. In people it is not uncommon for a nevus to recur following removal, even when no pigmented nevus cells remain. The biology of how and why this happens is unknown. By partial amputation of the nevus in the zebrafish tail fin, we described both nevus regrowth, as well as nevi that do not regrow. Utilising melanin as a lineage tracer, I was able to show that recurrent nevi are repopulated from an unpigmented precursor population. This suggested that BRAFV600E nevi are supported by an undifferentiated stem cell population that is recruited to regenerate and pigment the nevus after removal. In my second data chapter, I use genetics to develop BRAFV600E zebrafish models of melanoma. In collaboration with Dr. James Lister and Professor Jeroen den Hertog, I describe three differing models of zebrafish melanoma. All three models show progression to melanoma, and in collaboration with Dr. Marie Mathers I establish that while BRAFV600E is present in all three models, co-operating mutations affect melanoma pathology. In my third data chapter, I develop tools to study the molecular differences in the BRAFV600E melanoma models. I described the optimisation of a broad range of antibodies, raised against human peptides due to the lack of reliable antibodies in the zebrafish field. I use punch core biopsies of both zebrafish and human tumours, and whole sagittal sections of juvenile zebrafish, to show specific staining throughout many organs of the developing fish. I then use some of these antibodies to analyse molecular pathways in the melanoma models.

Malignant melanoma results in 132,000 cases worldwide each year with an incidence rate that is increasing faster than for any other skin cancer. In the UK, cutaneous melanoma is the sixth most commonly diagnosed cancer and the second most common in young people aged 15-34 (excluding non-melanoma skin cancer). Furthermore, while less common than NMSC, malignant melanoma accounts for 4% of skin cancer cases and 74% of skin cancer-related deaths. Although early surgical removal of primary tumours is an effective treatment, patients that develop metastatic melanoma have a very poor prognosis (5 year survival rate is only 5%). Elevated expression of a number of DNA repair genes has been reported in primary melanomas that subsequently metastasised when compared to non-recurrent primary tumours. In addition, patients who do not respond to chemotherapy have elevated expression of DNA repair genes. One chemotherapeutic that is effective against a range of other cancers, but not melanoma is cisplatin. Elevated levels of the DNA repair protein ERCC1, which is needed to remove cisplatin-induced DNA damage, has been found to be an indicator of poor prognosis in ovarian and lung cancer. To test our hypothesis that elevated ERCC1 levels account for an increased resistance to cisplatin in melanoma, a xenograft experiment was performed. Our results show that ERCC1 proficient melanoma xenografts initially responded to cisplatin treatment however resistance soon followed. Tumours deficient in ERCC1 however could be cured after only two treatments of cisplatin, indicating a novel method to overcome chemoresistance in metastatic melanoma. The aim of the project was to identify novel compounds to improve therapy of melanoma. To achieve this, in collaboration with Dr Patton we performed a cell culture screen to identify compounds which display specificity against melanoma cell lines. In addition, we sought to identify compounds which would overcome cisplatin resistance. We identified a series of nitrofuran compounds which are potent against melanoma and neuroblastoma cell lines and enhanced the toxicity of cisplatin through an ERCC1 independent pathway. In addition, we showed that melanin pigmentation is protective against nitrofuran toxicity. We have proposed the structure specific endonuclease, ERCC1-XPF, as a drug target to overcome chemoresistance. We collaborated with Professor Walkinshaw to perform an in silico screen for protein-protein interaction inhibitors to disrupt the obligate dimerization between ERCC1 and XPF. In addition we directly inhibited the endonuclease activity by developing XPF endonuclease domain inhibitors and utilised a range of biochemical, molecular biology and cell culture assays to validate ERCC1-XPF inhibitors. Furthermore, we developed an in vitro endonuclease assay for ERCC1-XPF, FEN1 and DNase1 and utilised these to demonstrate compound specificity of our validated ERCC1-XPF inhibitors. In collaboration with MRC Technology we utilised the ERCC1-XPF endonuclease assay to perform a high throughput screen. We characterised hit compounds to demonstrate physical binding and in vitro specificity for ERCC1-XPF. In conclusion, we have discovered new compounds which may prove beneficial for the treatment of malignant melanoma.

Examining surface shape appearance by touching and observing a lesion from different points of view is a part of the clinical process for skin lesion diagnosis. Motivated by this, we hypothesise that surface shape embodies important information that serves to represent lesion identity and status. A new sensor, Dense Stereo Imaging System (DSIS) allows us to capture 1:1 aligned 3D surface data and 2D colour images simultaneously. This thesis investigates whether the extra surface shape appearance information, represented by features derived from the captured 3D data benefits skin lesion analysis, particularly on the tasks of segmentation and classification. In order to validate the contribution of 3D data to lesion identification, we compare the segmentations resulting from various combinations of images cues (e.g., colour, depth and texture) embedded in a region-based level set segmentation method. The experiments indicate that depth is complementary to colour. Adding the 3D information reduces the error rate from 7:8% to 6:6%. For the purpose of evaluating the segmentation results, we propose a novel ground truth estimation approach that incorporates a prior pattern analysis of a set of manual segmentations. The experiments on both synthetic and real data show that this method performs favourably compared to the state of the art approach STAPLE [1] on ground truth estimation. Finally, we explore the usefulness of 3D information to non-melanoma lesion diagnosis by tests on both human and computer based classifications of five lesion types. The results provide evidence for the benefit of the additional 3D information, i.e., adding the 3D-based features gives a significantly improved classification rate of 80:7% compared to only using colour features (75:3%). The three main contributions of the thesis are improved methods for lesion segmentation, non-melanoma lesion classification and lesion boundary ground-truth estimation.

Interleukin-2 activates lymphocytes to become highly cytotoxic for a wide range of tumour cell types in vitro (Iymphokine activated killer or LAK cells), and in animal models. However, only limited therapeutic benefit was observed in clinical trials of LAK cell therapy. This project aimed to investigate the molecular and cellular interactions involved in the production and effector functions of LAK cells, to identify factor(s) which might be responsible for the poor clinical responses observed in LAK cell therapy. Tumour cell lines were heterogeneous in their response to killing by cytokines (TNFα, LT, IFNγ and IL-1β), and purified monocytes or lymphocytes, but were consistently highly sensitive to LAK cell cytotoxicity. Autologous monocytes and lymphocytes were not killed by LAK cells, in contrast to human umbilical vein endothelial cells and fibroblasts. Supernatants from LAK cells were considerably less cytotoxic than the effector cells, and physical separation of effector and target cells resulted in inhibition of killing. Lymphocyte and LAK cell cytotoxicity was associated predominantly with the CD8+ (cytotoxic T-cell) lymphocyte sub-population, and was significantly inhibited by anti-TNFα and anti-LT, demonstrating that these cytokines were the primary effector molecules in this system. LAK cells and A375 melanoma cells showed procoagulant activity, predominantly via the tissue factor pathway, and LAK cells also possessed surface factor V. In addition, A375 cells were highly fibrinolytic. Tumour cell killing by LAK cells was inhibited by plasma, and further experiments determined that polymerised fibrin, but not fibrin monomer was responsible. From these results it was suggested that culture of small numbers of cells from tumour biopsies, and the determination of their sensitivity to cytotoxic drugs, cytokines and effector cells may lead to more effective treatment protocols for immunotherapy of individual tumours. In order to enhance the efficacy of immunotherapy, further in vivo research is required to elucidate the interactions between immune effector cells and the coagulation/fibrinolytic systems.

Melanoma is a highly aggressive tumour with a poor prognosis for patients with advanced disease because it is resistant to current therapies. Therefore, the development of novel strategies for melanoma treatment is important. The characterization of the molecular mechanisms underlying melanoma proliferation, progression, and survival could help the development of novel targeted melanoma treatments. The MAPK and PI3K pathways both play important roles in melanoma progression. In the MAPK pathway, DUSP6, which acts as a phosphatase to negatively control the activation of ERK1/2, is involved in the development of human cancers. The MAPK pathway also regulates expression of the DNA repair gene ERCC1 following EGF treatment. ERCC1 is essential for nucleotide excision repair, which is one of the major systems for removal of cisplatin induced DNA lesions. The aims of this project were: 1, to investigate the molecular changes in our immortal mouse melanocyte cell lines that were needed for them to form tumours in a xenograft model; 2, to investigate whether the MAPK pathway regulates ERCC1 following cisplatin treatment and protects melanoma cells from death. Through comparison of the RAS/RAF/MEK/ERK (MAPK) and the PI3K/AKT (AKT) signalling pathways between our immortal mouse melanocyte cell lines and their tumour derivatives in our xenograft model, we identified a molecularly distinct subtype of mouse melanoma characterized by reduced ERK and AKT activity and increased expression of DUSP6. Functional analyses employing ectopic overexpression indicated that increased expression of DUSP6 enhanced anchorage independent growth ability and invasive ability in our mouse melanocytes, suggesting that increased DUSP6 expression may contribute to melanoma formation in the xenograft assay. We also demonstrated that higher expression of p-ERK suppressed invasion, but not anchorage independent growth, in our subtype of mouse melanoma by enforced expression of constitutively active MEK1 and MEK2. In addition, the role of DUSP6 in classical human melanoma was investigated in this Genetic changes in melanoma progression study. Inhibition of anchorage independent growth and invasion were observed after exogenous expression of DUSP6 in human melanoma cells. This suggested that DUSP6 played different roles in classic human melanoma than in our distinct subtype of mouse melanoma. Our study also investigated the phosphorylation level of ERK1/2 and the mRNA and protein level of ERCC1 and its partner XPF in the human melanoma cell line following cisplatin treatment. Significant increases in expression of p-ERK, ERCC1 and XPF were found in cisplatin treated cells. Moreover, a MEK inhibitor inhibited ERCC1 induction by cisplatin, but did not significantly affect XPF induction. This suggested that the MAPK pathway was involved in regulation of ERCC1 but not XPF. Furthermore, the DUSP6 level decreased after cisplatin treatment and overexpression of DUSP6 inhibited ERCC1 and XPF induction and reduced resistance to cisplatin. DUSP6 seems to play a crucial role in resistance of melanoma to cisplatin. In addition, a novel larger ERCC1 transcript was identified in human cell lines and was found to be upregulated by cisplatin. The ratio of larger ERCC1 transcript relative to the normal ERCC1 transcript increased following cisplatin treatment. The functions of this larger ERCC1 transcript in cisplatin resistance deserve further study.

Cutaneous melanoma is associated with significant morbidity and mortality representing the most significant cutaneous malignancy. As it is known that early diagnosis and treatment are the most efficient approaches to cure cutaneous melanoma, an improved understanding of the molecular pathogenesis of melanoma and exploration of more reliable molecular biomarkers are particularly essential. Two different types of molecular biomarker for melanoma have been investigated in this thesis. microRNAs (miRNAs) are single-stranded RNA molecules of 20-23 nucleotides in length that are found in both animal and plant cells. miRNAs are involved in the RNA interference (RNAi) machinery to regulate gene expression posttranscriptionally. miRNAs have important roles in cancer: by controlling the expression level of their target genes they can affect cell signalling pathways and have been shown to have both prognostic and therapeutic potential. Importantly for melanoma research, reproducible miRNA expression profiles from formalin-fixed paraffin-embedded (FFPE) tissues can be obtained that are comparable to those from fresh-frozen samples. The aims of the miRNA project were: first, to identify a melanoma-specific miRNA expression profile; secondly, to investigate roles of some of the melanoma-specific miRNAs identified in melanomagenesis. Using miRNA microarray on FFPE samples, I obtained a melanoma-specific miRNA expression profile. 9 of these differentially expressed miRNAs between benign naevi and melanomas (7 downregulated, 2 upregulated in malignancies) were verified by qRT-PCR and the functions of four of these miRNAs were studied. Ectopic overexpression of miR- 200c and miR-205 in A375 melanoma cells inhibited colony forming ability in methylcellulose, an in vitro surrogate assay for tumourigenicity. Moreover, elevation of miR-200c resulted in increased expression levels of E-cadherin through negative regulation of the zinc finger E-box-binding homeobox 2 (ZEB2) gene. Ectopic overexpression of miR-211 in A375 melanoma cells repressed both colony formation in methylcellulose and migratory ability in matrigel, an in vitro surrogate assay for invasiveness. These findings indicate that miR-200c, miR-205 and miR-211 act as tumour suppressors in melanomagenesis. The second biomarker investigated, mutated BRAF, has been seen in 50-70% of spontaneous cutaneous melanoma. The commonest mutation in melanoma is a glutamic acid for valine substitution at position 600 (V600E). Oncogenic BRAF controls many aspects of melanoma cell biology. The aim of this part of the work was: firstly, to study BRAF V600E mutation status in our melanoma tissue microarray (TMA) panel; secondly, to correlate this mutation to various clinicopathological features and evaluate its prognostic value through statistical analyses. BRAF V600E mutations were seen in 20% of the primary and 69% of the metastatic melanomas, respectively. More BRAF V600E mutations were seen in males relative to females. The mutation was also related to cell pigmentation, but not to age, ulceration or solar elastosis. Melanoma patients with the BRAF V600E mutation relapse earlier than patients without this mutation. However, no significant association between the BRAF V600E mutation and overall survival and melanoma specific survival was found.

Nucleotide excision repair (NER) is one of the major repair systems for removal of DNA lesions. The NER pathway has evolved mainly to repair UV-induced DNA damage and is also active against a broad range of endogenously generated oxidative lesions. Defects in NER result in the human inherited disorder xeroderma pigmentosum (XP), which is characterised by UV hypersensitivity and a 1000-fold increased risk of skin cancer. ERCC1 is essential for the NER pathway where it acts in a complex with the XPF protein to make the incision 5' to the DNA lesion. The normal 1.1kb Ercc1 transcript is expressed in all tissues. Our group has discovered a second larger 1.5 kb transcript, which initiates from an alternative promoter, and is the most abundant Ercc1 transcript in mouse skin. The aims of this project were: 1, To investigate the role of ERCC1 and of the 1.5kb skin specific Ercc1 transcript in protecting the skin against UV-induced DNA damage. 2, To study the importance of ERCC1 in melanoma skin cancer and investigate ERCC1as a possible target for therapy against melanoma. Using a panel of Ercc1 wild-type and deficient cells, we established a quantitative western blotting system to study the expression of ERCC1 in a range of mouse tissues and mouse and human cell types. Although the skin-specific Ercc1 transcript was found to be present at much higher levels in the skin of albino compared to pigmented mouse strains, this did not result in an elevated level of ERCC1 protein. We were also unable to demonstrate that UV-irradiation, or other stress-inducing treatments resulted in increased levels of ERCC1 protein in cultured mouse keratinocytes. We investigated the DNA methylation status of the normal Ercc1 promoter and that of two potential upstream promoter regions that were candidates for the source of the 1.5kb skin-specific Ercc1 transcript. We found no evidence that they were the source and, instead, used 5' RACE analysis to locate the skin-specific promoter to a polymorphic region 500bp upstream of the normal initiation site. In albino strains this region contains a SINE element, which we hypothesize could be involved in the production of the skin-specific Ercc1 transcript. We also investigated the protein level of ERCC1 and other DNA repair proteins, including XPF, MSH2, MSH6 and MLH1 in human melanoma cells and ovarian tumour cells. Significantly elevated protein levels of ERCC1 and XPF, as well as the mismatch repair protein MLH1 were found in melanoma cells. This could possibly contribute to the higher resistance to chemotherapy in melanoma, although the melanoma cell lines we tested did not show increased resistance to UV and cisplatin compared to the ovarian cancer cells tested. When Ercc1 proficient mouse melanoma cells were xenografted into nude mice the xenografts grew rapidly. Cisplatin treatment caused an initial shrinkage of the tumours, but re-growth rapidly followed. Cells re-isolated into culture from cisplatin treated xenografts had significantly higher levels of ERCC1 protein than either input cells, or cells re-isolated from untreated xenografts. An isogenic Ercc1 deficient derivative of the Ercc1 proficient mouse melanoma cell line grew as rapidly as the parent line in vitro, but grew much more slowly as xenografts. In addition, the xenografts shrank completely following cisplatin treatment and did not recover. This suggests that ERCC1 could be a drug target for melanoma therapy.

This thesis is concerned with developing and implementing a method for modelling and projecting cancer incidence data. The burden of cancer is an increasing problem for society and therefore, the ability to analyse and predict trends in large scale populations is vital. These predictions based on incidence and mortality data collected by cancer registries, can be used for estimation of current and future rates, which is helpful for public health planning. A large body of work already exists on the use of various modelling strategies, methods and fitting techniques. A multilevel method of preparing the data is proposed, fitted to historical data using regression modelling, to predict future rates of incidence for a given population. The proposed model starts with a model for the total incidence of the population, with each successive level stratifying the data into progressively more specific groupings, based on age. Each grouping is partitioned into subgroups, and each subgroup is expressed as a proportion of the parent group. Models are fitted to each of the proportional age-groups, and a combination of these models produces a model that predicts incidence for a specific age. A simple, efficient implementation of the modelling procedure is described, including key algorithms and measures of performance. The method is applied to data from populations that have very different melanoma incidence (the USA and Australia). The proportional structure reveals that the proportional age trends present in both populations are remarkably similar, indicating that there are links between causative factors in both populations. The method is applied fully to data from a variety of populations, and compared with results from existing models. The method is shown to be able to produce results that are reliable and stable, and are generally significantly more accurate than those of other models.

Immunotherapy offers a promising approach to cancer treatment by harnessing a patient’s own immune system to fight malignant cells. However, the clinical application of immunotherapy has been hindered by the immunosuppressive tumor microenvironment generated by cancer cells as a mechanism to impede immune function and evade immune detection. Clinically used immunotherapies, such as immune checkpoint inhibitors and adoptive cell therapy, aim to overcome the immunosuppressive tumor microenvironment by blocking key regulatory pathways and exogenously activating immune cells. While effective against some cancers, these therapies are still limited by systemic toxicity, poor delivery kinetics, and continuous tumor adaptation that leads to immune escape. Herein, we propose the synthesis of nanoscale branching DNA architectures, known as dendrons, to (1) encode and deliver a DNA sequence, termed G3YSD, capable of activating the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway; and (2) deliver epigenetic modifiers to reprogram immunosuppressive cues in tumor cells. This solution exploits the modularity, programmability, and ease of control over DNA synthesis to generate architectures that exhibit improved delivery kinetics and favorable presentation of cargo to enhance immunomodulatory effects. Our proposed solution directly targets immunosuppressive mechanisms in tumor cells to sensitize them to immune attack and make them more easily recognized by the immune system. Delivery of G3YSD-encoding dendrons to murine B16 melanoma significantly increased the expression of major histocompatibility complex I (MHC I) and programmed cell death-ligand 1 (PD-L1) surface-bound receptors, which are critical for immune signaling pathways. The chemical conjugation of romidepsin, a histone deacetylase inhibitor, to G3YSD-encoding dendrons resulted in more than a 2-fold increase in MHC I expression compared to unconjugated G3YSD sequences and free romidepsin, indicating that the spatial arrangement and presentation of romidepsin has a synergistic impact on cGAS-STING signaling. In addition, pretreatment of B16 melanoma cells with zebularine, a DNA methyltransferase inhibitor, followed by G3YSD-encoding dendrons significantly increased levels of cytotoxic T lymphocyte-mediated lysis in a physiologically relevant co-culture. Developing novel architectures capable of interacting with tumor cells to remodel and overcome immunosuppressive cues will lead to significant advances in the field of immunotherapeutic design and cancer treatment.

The contents of this article are the author’s original unpublished findings. Background: Each year, approximately 6 million Americans are treated for skin cancer, resulting in a total annual medical expense of $8.9 billion 1. The two most common types of skin cancer are basal cell carcinoma (BCC) followed by squamous cell carcinoma (SCC)2. The metastatic potential of these cancers is minimal; however, their treatment may involve severe scarring and disfigurement2. In contrast, melanoma is an aggressive skin malignancy that grows rapidly and can metastasize in a quick manner2. Risk factors of these skin cancers include UV exposure, fair skin complexions, and family history2. To prevent skin cancer, emphasis is placed on sun protection; however, this approach is suboptimal within the population. To combat this, alternatives such as niacinamide, a form of vitamin B3, may be used to reduce the risk of skin malignancies. Objectives: To evaluate the mechanisms and effects of oral niacinamide in the risk reduction of skin cancers among individuals with a history of sun exposure. Methods: A review was conducted by gathering randomized controlled trials and systematic reviews utilizing databases such as ScienceDirect, PubMed, Wiley Online Library, and SpringerLink. Research articles included in this paper have been published within the last eleven years and are inclusive of all races. Results: Niacinamide was associated with a significant reduction in the occurrence of BCCs and SCCs. However, concerning melanoma, its preventive effects were solely investigated through in vitro and in vivo studies. The lack of clinical trials makes it challenging to evaluate its effectiveness for melanoma. Conclusion: The research presented found compelling evidence supporting the potential of niacinamide in reducing the incidence of nonmelanoma skin cancers, particularly basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs). Such results yield to the significance of larger clinical trials aimed at replicating the results obtained from the ONTRAC trial regarding SCCs and BCCs. Despite promising outcomes against nonmelanoma skin cancers, the evidence regarding niacinamide's efficacy in preventing melanoma remains inconclusive. While in vitro and in vivo studies suggest positive effects on melanoma cells, the lack of robust clinical trials specifically evaluating niacinamide's impact on melanoma prevention necessitates further investigation.