It is well known that cancer cells are loaded with excess proteins. To survive this proteotoxic crisis cancer cells rely heavily on the ubiquitin proteasome system (UPS). Valosin containing protein (VCP) or p97 has recently emerged as a central player of UPS. Moreover, p97 inhibitors were shown to possess potent anti-cancer properties. Owing to the essential function of p97, it can be assumed that general p97 inhibition will be equally toxic to surrounding normal tissues. Interestingly, p97 employs diverse sets of cofactors to govern distinct biological pathways. Thus understanding the p97-cofactor interaction in specific pathways may give us the ultimate edge by allowing us to abolish those cancer specific p97-cofactor interactions in our fight against cancer. This thesis explains novel function of two bonafide p97 cofactors SPRTN and FAF1 in DNA replication. Our discovery of a new human syndrome followed by its characterisation that monogenic and biallelic mutations in SPRTN leads to premature aging and juvenile hepatocellular carcinoma, have led us to identify the critical function of SPRTN in DNA replication. Our follow up work further revealed that SPRTN is a novel protease in humans and has the essential function in covalent DNA-protein crosslinks (DPCs) repair. To this end, we have found that SPRTN proteolytically removes CHK1 from chromatin, which is evolutionary conserved and vital for replication fork progression to preserve genomic integrity. Additionally, this thesis also explains a novel mechanism through which FAF1 downregulation in cancer cells might promote genomic instability. Our data supports a model where the p97-^FAF1 complex binds to ubiquitinated CDT1 and degrades it via the proteasome after CDT1 mediated replication initiation, to prevent re-replication and genomic instability. The notion that high replication stress in some cancers can be utilised as an Achilles heel, led to the search for novel factors involved in DNA replication owing to its high therapeutic potential. Replication factors with enzymatic activities are especially appealing due to the relative ease of targeting them by small molecule inhibitors. This thesis thus highlights the great potential of the p97 systems in our battle against cancer.

Colorectal cancer (CRC) is one of the top three most common cancers and among the top four causes of cancer-related deaths worldwide (Torre et al., 2015). CRC patients are well characterised on the transcriptome and proteome level, but proteomics data on representative cell lines as model systems for pre-clinical drug sensitivity studies lag behind. Here, label-free quantitative mass spectrometry was used to characterise the kinomes and full proteomes of 65 CRC cell lines, collectively termed the CRC65 cell line panel. This data was integrated with proteomics data on patient samples, as well as public transcriptome and drug sensitivity datasets, which were reanalysed from raw data in order to unify and streamline the data processing. Protein/mRNA ratios were constant across these datasets, enabling linear prediction of protein abundance from mRNA abundance after appropriate adjustment, which was used for mRNA-guided missing value imputation. An exploration of secondary imputation methods prompted the development of a complementary method for minimum-guided missing value imputation. Combining the proteomics datasets on cell lines and patients led to the discovery of integrated proteomic subtypes of CRC and enabled the identification of representative cell lines for each of them. Modelling publicly available dose-response data generated by four large-scale drug sensitivity studies as a function of kinome/full proteome profiles fuelled the prediction of drug sensitivity for cell lines and patients, allowed the identification of drugs differentially effective between the different integrated proteomic subtypes and revealed MERTK as a predictive biomarker for resistance towards MEK1/2 inhibitors. This predictive role of MERTK was subsequently confirmed using in vitro experiments, while immunohistochemistry of TMAs from 1,074 tumours generated as part of the QUASAR2 clinical trial unveiled that MERTK is also a prognostic biomarker in CRC. This dataset will be made available to the scientific community to facilitate the design of prospective clinical studies.

The DNA damage response (DDR) is a highly organised process, which involves the spatial and temporal orchestrated removal and recruitment of proteins, achieved by the use of dynamic posttranslational modifications (PTMs). Ubiquitination has emerged as one of the major PTMs in the DDR after DNA double strand breaks. Intensive studies have been performed to understand the regulation and impact of ubiquitination in the DDR. Regulation of important DDR players such as 53BP1, BRCA1 or RAD51 strictly depend on the signaling cascade initiated by the two main ubiquitin E3 ligases RNF8 and RNF168 at the site of DNA damage. Recently, it has emerged that the AAA ATPase p97 (VCP/Cdc48) plays a crucial role in the ubiquitin axis of the DDR. p97 interacts with a vast set of cofactors that target p97 to specific substrates and assist it in its function as a molecular segregase. However, the exact role of p97 and its cofactors in the response to ionising radiation induced DSBs and ubiquitination is still poorly understood. In this thesis, three p97 cofactors and their role in the DSB-triggered ubiquitination have been studied. The results show that the so-far poorly characterised UBE4A and UBE4B, two E3/E4 ubiquitin enzymes and p97 associated proteins (cofactors), play critical roles in the regulation of ubiquitination at the sites of DNA damage and absence of either affects both K48- and K63-linked ubiquitin chains. Furthermore, another cofactor of p97, the deubiquitinating enzyme Ataxin-3, affects the regulation of RNF8 and RNF168 at the sites of DNA damage. All three proteins, therefore, help to fine-tune the DDR and the recruitment of critical DDR proteins including BRCA1 or 53BP1. Together, the results of this thesis discover and characterise three new players to the ubiquitination axis at the site of DNA damage. Moreover, Ataxin-3 appears to be an interesting druggable target for two-hit radiotherapy for cancer treatment, namely, the inhibition of Ataxin-3 deubiquitinating activity in combination with ionising radiation.

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Myeloma remains largely incurable and there is an unmet need for new therapies. B-cell maturation antigen (BCMA) is an attractive therapeutic target due to its restricted expression on normal and malignant plasma cells(PC). A natural ligand for BCMA is a proliferating ligand (APRIL) which also naturally binds another antigen expressed on myeloma cells: Transmembrane activator and CAML interactor (TACI). APRIL is an attractive binder as it is a compact, self-protein which binds both BCMA and TACI with high affinity. BCMA and TACI expression was quantified on tumour cells from a large number of patients to explore the clinical utility of targeting these antigens. Tumour BCMA expression was found in all patients, persisted with relapse and extramedullary spread and low levels was associated with improved patient outcome. However BCMA expression was variable, often low and TACI was co-expressed on tumour from 78% of patients. Recognising that dual antigen targeting of BCMA and TACI may increase target antigens on tumour and reduce the risk of antigen negative disease escape, an APRIL based chimeric antigen receptor(ACAR) was optimised and in vitro activity against BCMA and TACI targets demonstrated. Target kill was seen at low levels of antigen and low effector to target ratios and further, potent ACAR efficacy was demonstrated in vivo in an intramedullary murine myeloma model. A targeted mutagenesis strategy was also used to generate a BCMA specific APRIL mutant. When APRIL mutant was used in a CAR(ACAR-mut), specificity was confirmed for BCMA targets, however there was no significant T cell expansion against primary tumour. In a second strategy, APRIL based bispecific molecules were engineered consisting of a CD3 specific single chain variable fragment linked to an APRILWT(APRILiTE). Preliminary studies using unpurified and unquantified protein in co-cultures of target and activated T cells, demonstrated cytolysis of BCMA expressing cells and primary myeloma cells using APRILiTE. Therefore, APRIL based therapeutic strategies provide a novel and promising approach to immunotherapy in myeloma.

Firefly luciferase is ubiquitously used as a genetic reporter for the non-invasive bioluminescence imaging of small animal models. This widespread use of Firefly luciferase in vivo has been facilitated by genetic engineering producing mutants which are extremely stable at physiological conditions. In addition, the red-shifting of bioluminescence has resulted in the enhanced penetration of light emission through biological tissue. However, the use of bioluminescence in vivo is still largely limited to the tracking of single events within a model. This is due to the differential attenuation of light < 600nm, making the spectral unmixing of bioluminescent signals extremely challenging. Consequently, there is a real need to move bioluminescence into the near-infrared for dual-colour imaging. As we seem to have reached the limits of mutational based red-shifting, research has more recently focused upon chemical modification of the D-luciferin substrate. But any modification of the DLuciferin substrate is inevitably going to require subsequent mutagenesis of Firefly luciferase to optimise the light emitting reaction. The first part of this project describes the development and validation of a high throughput screening platform for bioluminescent proteins, to advance the identification of mutants with enhanced characteristics. Focus then turns to the use of genetically engineered Firefly luciferase colour mutants for in vivo bioluminescence imaging. Small animal tumour models, representing increasing tissue depth, were engrafted with Firefly luciferase colour mutants to explore the feasibility of dual colour imaging and establish the true benefit of red-shifting bioluminescence. Finally, bioluminescence in the near infra-red is used for dual bioluminescence imaging, tracking two tumour populations in a B-cell lymphoma mouse model through the spectral unmixing of Firefly luciferase colour mutants with the novel substrate infra-luciferin.

Cancer is an evolutionary disease, reliant on genetic diversity and sculpted by selective forces from the immune microenvironment. Here, I use genomics data to decipher the tumor’s evolutionary trajectory and corresponding shifts in the immune contexture to elucidate the events governing tumor immunogenicity and the immune evasive mechanisms evolved by the tumor. To better understand the mutational processes contributing to intratumor heterogeneity in individual tumors, a method to quantify the activity of mutational processes in a single tumor sample was developed and applied to temporally dissected mutations. The clinical relevance of intratumor heterogeneity was examined in the context of immune recognition and modulation. Increased clonal neoantigen burden and minimal neoantigen intratumor heterogeneity were found to associate with improved patient outcome, both in the treatment-naïve and immunotherapy-treated setting. The identification of T-cells recognizing clonal neoantigens further supported the clinical importance of targeting neoantigens present in every cancer cell. Mechanisms of immune evasion were considered through the development of a method to identify loss-of-heterozygosity at the HLA locus, overcoming the challenges posed by the polymorphic nature of the locus. HLA loss-of-heterozygosity was found to be a frequent subclonal event in NSCLC, under strong selective pressure and associated with increased subclonal neoantigen burden. Finally, the immune microenvironment was examined through multi-region RNAseq, permitting the quantification of immune infiltration and allowing for the identification of heterogeneously immune infiltrated tumors. Supporting the interplay between genetic events and the immune contexture, a relationship between the genomic features of the tumor and immune infiltration was observed, with HLA loss-of-heterozygosity specifically identified as occurring within a highly active immune microenvironment. This thesis shows how an improved understanding of the relationship between the tumor and the immune system can illuminate features dictating immune recognition and evasion and how that knowledge may inform the development and implementation of successful immunotherapy.

Peripheral nerves are one of the few adult tissues which can regenerate following injury, and macrophages have many important roles in this multicellular process. Following nerve injury, regrowing axons must traverse tissue termed the ‘nerve bridge’, which forms spontaneously between the nerve stumps (proximal and distal), to reconnect with their original tissue target. Previously, we found that hypoxic macrophages in the bridge induce the formation of a polarised vasculature which dedifferentiated Schwann cells subsequently use as a scaffold to migrate along, taking axons with them into the distal stump. Here, macrophages together with Schwann cells function to clear debris and remodel the environment to facilitate axonal regeneration, demonstrating distinct functions for macrophages within discrete areas of the regenerating nerve. This thesis aims to characterise nerve macrophage populations and to determine whether there is a specific Schwann cell chemoattractant within the bridge. Resident nerve macrophages displayed a distinct gene expression pattern compared to other resident macrophage populations. Moreover, we found two distinct nerve resident populations which can be distinguished by CX3CR1 expression and physiological location. In the injured nerve, the origin and phenotype of macrophage populations is currently unknown. We determined that the majority of macrophages in the bridge and distal stump are monocyte-derived. In further characterisation of bridge macrophages, we have identified an intrinsic ability to differentially sense hypoxia. Here we show data to support a novel role for hypoxic bridge macrophages in promoting Schwann cell migration. Using in vitro chemotaxis assays, we found that hypoxic macrophages are able to induce Schwann cell migration and an unbiased screen identified the chemoattractant factor as CCL3. CCL3 is able to induce Schwann cell migration in chemotactic assays, and knockdown studies showed that CCL3 is the primary chemoattractant secreted by hypoxic macrophages. We also present preliminary in vivo experiments investigating the role of CCL3 following injury, as well as in models of nerve repair. A Schwann cell chemoattractant factor has wide therapeutic implications in peripheral nerve injury, as well as potential uses in the treatment of aberrant nerve growth and tumour spread.