Mutations in the ATM gene have previously been identified in CLL tumours. In this project, I have demonstrated that their detection would have prognostic value. With a prevalence of 12%, ATM mutations represent the commonest single gene defect to be detected in CLL tumours and they identified a subgroup of CLL patients that had a significant reduction in both treatment free and overall survival. Furthermore, ATM mutations provided prognostic information that was independent of age, clinical stage, the mutation status of the IGVH genes and TP53 mutations. The temporal acquisition of the ATM mutations and their relationship with loss of an ATM allele via a chromosomal 11q deletion provides clues into their mechanism of action. There was only a partial correlation between CLL tumours with mutations in the ATM gene and those with a chromosome 11q deletion. In certain cases, the ATM mutations represented germ-line changes and in others were acquired very early in the disease course raising the possibility that they might contribute to the initial clonal transformation process. However, in some CLL tumours, the ATM mutations had been acquired after the development of the tumour clone during disease progression indicating that there may be a step-wise acquisition of ATM allelic defects during the ontogeny of CLL. The ATM protein is the key coordinator of the cellular response to DNA double strand breaks. In this study, I showed that bi-allelic defects in the ATM gene lead to deficient ATM dependent responses, including the up regulation of p53, following both ionising irradiation and also treatment with the chemotherapeutic drug, Fludarabine. Thus an important mechanism accounting for the poor outcome in CLL patients with ATM mutations is likely to relate to chemo-resistance. Interestingly, there were differential responses to DNA damage with both irradiation and fludarabine amongst the category of tumours with an 11q deletion according to the status of the remaining ATM allele. Therefore, ATM mutations can stratify tumours with a chromosome 11q deletion into two functional subgroups. The identification of CLL tumours with ATM mutations would therefore predict those patients that will have a poor clinical outcome and be both more likely to require early treatment for their disease. Patients whose tumours had bi-allelic ATM defects will be expected to have deficient responses to DNA damaging chemotherapeutic drugs, while those with mono-allelic ATM defects might identify a group in whom the use of DNA damaging agents could provide selective pressure for the emergence of sub-clones that have subsequently acquired bi-allelic ATM defects.

Adenovirus E1A (AdE1A) is a viral oncoprotein that targets many cellular proteins and pathways, mainly those involved in transcriptional regulation. Proteasomes represent the major non-lysosomal mechanism responsible for protein degradation. Following interferon-γ treatment, three proteasome subunits are replaced by immunosubunits LMP2, LMP7 and MECL-1 producing immunoproteasomes. The proteasome and immunoproteasome generate peptide antigens for MHC class I presentation to cytotoxic T-cells. In this study, the effect of AdE1A on human immunoproteasomes as well as MHC class I and class II cell surface expression was examined. It was found that AdE1A interacts with the immunoproteasome subunit MECL-1 through its N-terminal and CR3 regions. AdE1A also down-regulated all three immunosubunit expressions during adenovirus infection, transformation and AdE1A transfection, with the exception of Ad5-transformed cells where immunosubunit expression remained unchanged. Furthermore, MHC class I expression remained unaffected in the same three backgrounds. However, in the Ad12 transformants MHC class I was generally reduced prior to IFNγ treatment but was expressed after. MHC class II surface expression, in contrast, was down-regulated in all cases, except in Ad5 infected cells. Similarly, AdE1A reduced IFNγ-stimulated STAT1 phosphorylation and transcriptional response to IFNγ. And finally, T-cell recognition of target cells was reduced in the presence of AdE1A. In conclusion, AdE1A targets the human immunoproteasome, both through direct binding and down-regulation of expression. It also targets the expression of MHC class I and class II surface expression.

RUNX1, also known as CBFa, is a master regulator of haematopoiesis. In Acute Myeloid Leukaemia (AML) it is frequently disrupted by translocations to different epigenetic regulators, resulting in the expression of core-binding factor fusion proteins. We compared the chromatin landscape of t(8;21) and t(3;21) AML which express RUNX1-ETO and RUNX1-EVI1, respectively. We found that the diverse clinical outcomes of patients with these two forms of AML are reflected in fundamental differences in gene expression and chromatin landscape. Despite both fusion proteins sharing a RUNT DNA binding domain, we show that RUNX1-EVI-1 targets a more immature stem cell-related gene expression program of genes as compared to RUNX1-ETO. Despite the differences in the epigenomic landscape of t(3;21) and t(8;21) leukaemia, knockdown of either core-binding factor fusion protein activates a common myeloid differentiation program involving up regulation of C/EBPa. By blocking C/EBPa DNA binding through a dominant negative partner, we showed that this factor is required for the downstream effects of RUNX1-EVI-1 knockdown. Even in the continued presence of RUNX1-EVI-1, ectopic expression of C/EBPa. is sufficient to initiate myeloid differentiation in t(3;21) cells. Overall, this suggests that deregulation of C/EBPa is a common pathway in the development of both t(8;21) and t(3;21) AML.

Prostate epithelial cells are exquisitely sensitive to Nuclear Receptor (NR) ligands. These compounds exert anti-proliferative effect over non-malignant cells RWPE-1 while malignant PC-3 cells retain their proliferative ability. The Nuclear Receptor Co-Repressor 1 (NCOR1) complexes with Histone Deacetylases (HDACs) to repress the action of unliganded NRs, hence, inhibiting their transcriptional and phenotypical effects. NCOR1 was found to be over-expressed in PC-3 cells when compared to non-malignant RWPE-1 cells. Chemical inhibition of NCOR1, via the HDAC inhibitor SAHA, or NCOR1 knock-down, via shRNA, restored PC-3 cells sensitivity to NR agonists with exception of Vitamin D and Thyroid Hormone T3. NCOR1-knock down led also to a re-expression of basally repressed genes, as measured via Microfluidic Gene-Card analysis (Q-RTPCRm). CDKN1A was de-repressed by the knock down and its activation via VDR was modeled with a systems biology approach to identify the mechanistic events behind CDKN1A transcriptional regulation via miRNAs. Differential equation models revealed a time-sensitive activation, VDR-dependent, of the miRNA-miR106b that leads to a steep degradation of CDKN1A mRNA levels within the first 30 minutes. These results support the idea of a corrupted regulatory network that squelches NR activity in prostate epithelial cells.

A shortlist of candidate tumour endothelial markers was generated by Microarray comparison of differential gene expression between multiple patient-matched colorectal cancer and normal colon samples. This list was narrowed through a process of literature review, real-time quantitative polymerase chain reaction and immunohistochemistry. Through siRNA knockdown and analysis in in vitro models of angiogenesis, it has been demonstrated that a decrease in an novel target’s expression significantly decreases cellular migration, communication and chemotaxis, without adversely affecting cell viability or proliferation in HUVEC. Vaccination with a murine Fc fusion protein in combination with Freund’s adjuvant stimulated a specific immune response to this self-antigen, by breaking immune tolerance. The resulting increase in specific IgG1 antibody titers, indicative of Th2 T-cell response, resulted in a significant reduction in physiological angiogenesis in the subcutaneous sponge assay, and a significant decrease in colorectal tumour growth in a murine subcutaneous CT26 tumour model. The gene of interest represents a novel tumour endothelial marker in colorectal cancer. A hypothesised mechanism for the observed effects is an inhibition of endothelial calcium influx, leading to decreased angiogenic potential in tumour endothelial cells.

Predominant immune cells in colorectal cancer (CRC) are CD4+ T-lymphocytes. The hypotheses are that (i) the CD4+ component in CRC is made up of different functional subtypes; T-helper and T-regulatory cells (TREG), (ii) CD4+ cells in CRC are heterogeneous in their expression of homing markers, and (iii) the presence of homing markers can identify cells with a typical functional response. Peripheral blood lymphocytes were obtained by ficoll separation from 38 patients, with matched tumour (TIL) and lymph node lymphocytes extracted using mechanical disaggregation from 35 and 26 patients respectively. The presence of CD4+CD25+ cells as a putative marker for TREG was assessed. CD4+ cells were stained with a panel of 18 homing markers to identify up-regulation in TIL from CRC. Based on homing markers identified, lymphocyte isolates were separated using Flow Assisted Cell Sorting or MACS separation, and the functional ability of these subpopulations assessed using ELISA. TIL from CRC display up-regulation of homing factors CXCR6, CCR5, and CCR6 (p=0.0001, 0.01782 and 0.5346 respectively). Stimulated CD4+CXCR6+TIL produced interferon-gamma,involved in anti-tumour responses. We have confirmed the presence of a population of inhibitory FoxP3+ and CD4+CD25+ TREG. The techniques validated could be used to monitor the effect of future vaccine trials.

Identification of the role of T cells and their interaction with other cell types remains a major challenge to our understanding of the pathogenesis of rheumatoid arthritis. In this study we have investigated the regulation of the response of T cells infiltrating the rheumatoid joint to IL-6. Furthermore we have investigated the level of T cell activation in the early stages of rheumatoid arthritis. Interleukin-6 is an important regulator of T cell differentiation and survival. It exerts its biological function by either directly binding to the complete IL-6 receptor consisting of CD126 CD130 or via transsignaling, when sIL6R-IL6 complexes bind to CD130. This study addresses the expression and regulation of these receptor components on the T cells infiltrating the rheumatoid joint. While compared to blood T cells, CD126 expression was found at low levels on synovial fluid and tissue T cells, expression of CD130 on synovial tissue T cells was comparable to that of blood T cells, with lower levels in synovial fluid T cells, both at protein and mRNA level. When exposed to sIL6R-IL6 complexes, tissue derived T cells responded with a higher level of STAT3 phosphorylation compared to cells incubated with IL-6, suggestive of transsignaling. High CD130 expression was demonstrable in T cells in the perivascular cuff area. Among a range of cytokines tested, IL-6 reduced CD126 and CD130 expression while IL-10, which is expressed at high levels in the perivascular infiltrate, induced expression of CD130. Taken together these data suggest that the inflammatory microenvironment maintains responsiveness to IL-6 transsignalling by cytokine driven CD130 expression on CD4 positive T cells. To address the question whether the role of T cells changes during the course of progression of RA, we analysed the expression of T cells activation markers on synovial fluid and peripheral blood T cells from patients at the very early stage of disease (within 3 months of disease onset) compared to patients with established or self resolving arthritis. Expression of CD69, CD71 and HLA-DR was upregulated on synovial fluid T cells compared to peripheral blood but there were no differences between the different groups of patients. Furthermore, we quantified the proportion of T cells expressing the invariant TCR Vα24Jα18 in synovial fluid and blood of the same groups of patients. We found a lower frequency of iNKT cells in the synovial fluid of very early arthritis patients compared to other patients. While this is a preliminary result, it suggests that there may be a role for these cells in the regulation of disease susceptibility.

The tumour suppressor, p53, is a vital DNA damage response protein and its means of transcriptional regulation are vast. hnRNPUL-1 is a multifunctional protein and previous studies have implicated it in the modulation of p53 transcriptional activity, although this has been rather poorly understood. Results presented here demonstrate that hnRNPUL-1 represses p53 transcriptional activity and negatively regulates p21 levels. This is consistent with the depletion of hnRNPUL-1 leading to an increase in cells arrested in G1/S. Together these results confirm that hnRNPUL-1 acts as a p53 co-repressor with specific cellular targets. Mutations in p53 and other DNA damage response proteins not only often contribute to the onset of tumourigenesis but can also be the cause of drug resistance during treatment. The development of a novel chemotherapeutic agent, Alchemix (ALX), was based on the requirement for effective treatment in the face of resistance mechanisms. Little has been known about the mechanism of action of ALX up to now. Findings here demonstrate that ALX treatment primarily induces an ATR-dependent DNA damage response that occurs specifically in cycling cells and culminates in cell death via mitotic catastrophe. Results also show that the response elicited by ALX requires TOP2α and TOP2β, as well as its alkylating ability, but does not involve ATM, p53, or components of the MMR and FA pathway. Therefore, ALX has the potential to treat tumours that have developed resistance to conventional chemotherapeutic drugs.

Prostate cancer is the most common male cancer in the UK and USA, with a 1/13 chance of diagnosis and a 1/30 lifetime risk of death from the disease. Current treatment options include radiotherapy, surgery and hormone therapy, however 1/3 patients escape from all therapies and novel therapies are urgently required for this patient group. The University of Birmingham gene therapy group constructed two oncolytic adenovirus vectors, CRAd-NTR and vNR6, both of which contained the E1B-55K deletion and expressed the transgene nitroreductase for combined oncolytic virotherapy and enzyme/prodrug gene therapy. The latter of these two vectors, vNR6, expressing nitroreductase from the pIX virus promoter demonstrated the greatest cytotoxicity at low virus concentrations however also showed some lytic activity to non-transformed human fibroblasts. Our collaborators at the Institut Català d'Oncologia designed a panel of oncolytic adenovirus vectors with the E1A CR2 24 deletion and the E1A promoter replaced by an insulated E2F-1 promoter. The latest two in this series of vectors, termed ICOVIR-5 and ICOVIR-7, provide potential oncolytic backbones for the introduction of the therapeutic transgene nitroreductase. The aim of this thesis was therefore to ‘arm’ the ICOVIR based vectors with nitroreductase for combined oncolytic virotherapy and enzyme/prodrug therapy. At the beginning of this study no reports were published with either ICOVIR-5 or ICOVIR-7 based vectors. It was therefore first decided to construct both vectors expressing the marker transgene eGFP. These vectors were characterised in terms of cytotoxicity, transgene expression, DNA replication and E1A expression. Furthermore, these vectors were compared to the vNR3, an E1B-55K deleted virus similar to vNR6, but the eGFP ORF replacing that of pIX. The ICOVIR-7 based vectors were identified as being the most tumour selective vectors and demonstrated no cytotoxicity to non-transformed human fibroblasts, and were therefore chosen for the introduction of the therapeutic transgenes. The new ICOVIR-7 based vectors were constructed to express either wildtype, double mutant or triple mutant nitroreductase. Double and triple mutant nitroreductase are two previously characterised mutant nitroreductases, which show enhanced catalytic activity for the prodrug CB1954. The new nitroreductase expressing ICOVIR-7 vectors were characterised in terms of virus mediated cytotoxicity, tumour selectivity, E1A and NTR expression and cytotoxicity with the prodrug CB1954. One vector, expressing double mutant nitroreductase, showed the highest tumour selectivity and greatest combined cytotoxicity with the prodrug CB1954. Furthermore, this vector showed greater tumour selectivity and combined cytotoxicity than the E1B-55K deleted vector vNR6.

Epstein-Barr virus (EBV) is strongly associated with rare but aggressive lymphoproliferative diseases of NK and T cell origin. The finding of clonal and episomal forms of the virus in tumour cells from these clinically diverse diseases indicates involvement of EBV at an early stage of lymphomagenesis. However, many fundamental questions about EBV’s contribution to pathogenesis remain unanswered. In vivo analyses herein found that infection of tonsillar (but not peripheral blood) T cells occasionally occurred in primary and persistent infection, whilst infection of NK cells was a rare event. By contrast, a high EBV load was found in peripheral blood NK cells from 3 adult patients with EBV-associated haemophagocytic-lymphohistiocytosis, a disease previously associated with CD8+ T cell infection in children. Complementary studies examined EBV latent gene expression in EBV+ T/NK malignancies. Notwithstanding an apparent absence of latent membrane protein 2A and 2B (LMP2A/B), these tumour cells were recognised and killed by LMP2-specific cytotoxic T lymphocytes. This paradox was resolved by identifying a novel LMP2 mRNA, initiated from within the terminal repeat (TR) region of the viral genome and containing downstream epitope-encoding exons. Expression of LMP2-TR in T/NK cell lines and primary tissue implicates this truncated viral protein in T/NK lymphomagenesis.