INTRODUCTION Approximately 9% of all new malignant diagnoses in the UK are due to haematological malignancies. The acute and chronic leukaemias constitute 2.5 % of all cancers and leukaemia is the 12th most common cancer registered in the UK. Approximately 7 000 people are diagnosed with the disease and more than 4 300 people die from leukaemia in the UK each year. As such, they have an important impact on the health of the public and represent a significant cost to the health care budget. AIMS AND OBJECTIVES The research presented in this thesis firstly aimed to quantify the incidence of and mortality from the acute and chronic leukaemias in the UK, and to define their associations with gender, age, socioeconomic class, calendar time, and geographic region of residence. A further aim was to determine whether the use of non-steroidal anti-inflammatory drugs (NSAIDs) had a protective effect on the incidence of and mortality from these leukaemias, as has been shown to be the case for a number of other cancers. Finally, the impact of alcohol consumption on leukaemia incidence and mortality was investigated. A surprising result from the incidence and mortality studies was that survival in AML, but not other leukaemias, was worse with increasing socioeconomic deprivation. This generated an additional hypothesis surrounding potential class bias in bone marrow transplantation in these patients, a new area that was also investigated, in addition to the original aims and objectives of the research. METHODS Both general practice and hospital data were used to conduct these population-based studies. 'The Health Improvement Network' (THIN) general practice dataset was used to conduct the cohort studies of incidence and mortality, as well the case-control studies investigating non-steroidal anti-inflammatory drug use and alcohol consumption, as potential risk factors for leukaemia. Hospital Episode Statistics (HES) data were used to investigate the additional hypothesis generated by results of the incidence and mortality studies, which showed that mortality in AML patients worsens with increasing socioeconomic deprivation. RESULTS A total of 4162 cases of leukaemia were identified, 2314 (56%) of whom were male. The overall incidence of leukaemia is 11.25 per 100 000 person-years and is independent of socioeconomic class. Median survival from leukaemia is 6.58 years and mortality increases with increasing age at diagnosis. The prognosis in AML is dismal and worsens with increasing socioeconomic deprivation, a phenomenon not seen in other leukaemias. Bone marrow transplantation declines with increasing socioeconomic deprivation (p for trend <0.01). Patients with AML in the most deprived socioeconomic quintile are 40% less likely to have a bone marrow transplantation than those in the most advantaged socioeconomic class (OR 0.60, p<0.01, 95% C.I. 0.49 - 0.73), even after adjusting for gender, age at diagnosis, year of bone marrow transplantation and co-morbidity. The risk of leukaemia overall appears to increase marginally with increased use of NSAIDs prior to diagnosis. This is not seen when individual leukaemia subtypes are examined, however, except perhaps in CLL where patients who had received 2-5 prescriptions/year were 29% more likely to be diagnosed with CLL than those who had not had any NSAID prescriptions (O.R. 1.29, p=0.05, 95% C.I. 1.00 - 1.67). There is no statistically significant association between exposure to NSAIDs prior to leukaemia diagnosis, and survival. There is no statistically significant association between alcohol consumptionand risk of developing leukaemia overall, nor with any of the leukaemia subtypes studied here. Alcohol consumption is associated with a lower risk of death in leukaemia overall (HR 0.83, p=0.04, 95% C.I. 0.69 - 0.99), as well as in All (HR 0.14, p<0.01, 95% C.I. 0.04 - 0.44) and CLL (HR 0.71, p=0.02, 95% C.1. 0.53 - 0.96), when compared to those who had not consumed any alcohol.

Cancer cells can exist in a reversible state of dormancy (G0 phase of the cell cycle). Relapse of acute myeloid leukaemia (AML) is likely due to dormant cells escape frontline treatment. Dormant AML cells have been identified in the bone marrow (BM) endosteal region which is characterised by an excess of TGFβ1 and a shortage of nutrients. As the first step in this project, we developed an in vitro model of AML cell dormancy by exploiting these features. Following a preliminary investigation of four AML cell lines, the CD34+CD38- line TF1-a was selected for in-depth investigation. TF1-a showed significant inhibition of proliferation, with features of dormancy and stemness, in response to 72 hours of TGFB1+mTOR inhibitor treatment (mTOR pathway inhibition mimics major effects of nutrient scarcity) without affecting cell viability or inducing differentiation of these cells. Secondly, whole human genome gene expression profiling using Affymetrix microarray strips (HuGene2.1ST) was conducted to molecularly characterise the dormancy-induced TF1-a cells. As a result, we identified 240 genes which were significantly up-regulated by at least twofold, including genes involved in adhesion, stemness, chemoresistance, tumor suppression and genes involved in canonical cell cycle regulation. The most upregulated gene was osteopontin (17.1fold). Immunocytochemistry of BM biopsies from AML patients confirmed high levels of osteopontin in the cytoplasm of blasts near the paratrabecular BM. Osteopontin and other genes identified in this model, including well-characterised genes (e.g. CD44, CD47, CD123, ABCC3 and CDKN2B) as well as little-known ones (e.g. ITGB3, BTG2 and PTPRU), are potential therapeutic targets in AML. AML cells which are identified in the bone marrow (BM) endosteal region are likely to survive chemotherapy for several reasons including the low concentration of the drug delivered to this poorly perfused area of the BM. We hypothesised that these cells might induce dormancy features that help them escaping chemotherapy. Moreover, little is known regarding the molecular changes in those AML cells surviving genotoxic stress. The third aim of this study was to investigate the AML cells surviving genotoxic stress. Therefore, we developed and characterised an in vitro model of prolonged sub-lethal genotoxicity in AML cells by utilising the TF1-a cells treated with etoposide for 6 days. TF1-a cells survived this conditioning with significant inhibition of proliferation and limited damage and apoptosis. The molecular signature of these cells was characterized using GEP of the whole human genome and was compared to that of the dormancy-induced TF1-a cells in an aim to identify genes commonly up-regulated in both scenarios that might act as possible drivers of dormancy in AML cells residing in a sub-lethal genotoxic environment. In this context, 31 genes were significantly commonly upregulated in both scenarios including ITGB3, SLFN5, C15orf26 and GRAP2 which are candidates for in-depth investigation in AML. To sum up, in this study we developed and molecularly characterised in vitro models of dormancy and sub-lethal genotoxicity in AML cells with stemness characteristics through novel approaches that took bone marrow microenvironmental features into consideration. These features likely contribute to the resistance of the residual sub-population of AML cells that causing disease relapse. The current models helped to overcome the obstacles facing the in-depth studying of these rare AML cells due to the difficulty in obtaining them from clinical samples. Finally, the molecular findings of this study paved the way to potentially important future directions of research that could help to achieve the ultimate goal of eradicating AML cells and preventing relapse.

Aurora kinases play an essential role in orchestrating chromosome alignment, segregation and cytokinesis during mitotic progression, with both aurora-A and B frequently over-expressed in a variety of human malignancies including those of leukaemic origin. Acute myeloid leukaemia (AML) is a heterogeneous clonal disorder of haematopoietic progenitor cells whose prognosis is particularly poor and where standard induction therapy has changed little over the past thirty years. This thesis evaluated the effects of AZD1152-hQPA (barasertib-hQPA), a highly selective inhibitor of aurora-B kinase, in AML cell lines and primary samples. Inhibition of phospho-Histone H3 (pHH3) on serine 10 can be used as a biomarker for AZD1152-hQPA activity and an assay was optimized to measure pHH3 in our cell lines and primary samples. AZD1152-hQPA inhibited pHH3 in our cell lines resulting in polyploid cells, apoptosis, and cell death, irrespective of cellular p53 status. Over-expression of the ATP-binding cassette (ABC) drug transporter proteins P-glycoprotein (Pgp) and Breast cancer resistance protein (BCRP) is a major obstacle for chemotherapy in many tumour types with Pgp conferring particularly poor prognosis in AML. A cell line which over-expresses Pgp was developed by selecting for daunorubicin (DNR) resistance in OCI-AML3 cells. Pgp and also BCRP expressing AML cell lines were found to be resistant to AZD1152-hQPA and it was found that AZD1152-hQPA is effluxed by these transporters. pHH3 inhibition by low dose AZD1152-hQPA was seen in all of the primary samples tested with Pgp and BCRP positive samples being less sensitive. However, 50% inhibition of pHH3 by AZD1152-hQPA was achieved in 94.6% of these samples. The FLT3-ITD-expressing MOLM-13 and MV4-11 cell lines were particularly sensitive to AZD1152-hQPA. Internal tandem duplications (ITDs) within the FLT3 tyrosine kinase receptor are found in approximately 25% of AML patients and are associated with a poor prognosis. It was demonstrated that AZD1152-hQPA directly targets phosphorylated FLT3 in the FLT3-ITD cell lines along with inhibiting its downstream target pSTAT5. FLT3-ITD primary samples were particularly sensitive to clonogenic inhibition and pSTAT5 down-regulation after treatment with AZD1152-hQPA compared with FLT3 wild-type (WT) samples.

The homeobox transcription factor PROX1 is the mammalian ortholog of the Drosophila gene Prospero. Expression of PROX1 in a subset of venous endothelial cells changes their fate to lymphatic endothelial cells (LEC). PROX1 is required for lymphatic development as Prox1 null mice lack all lymphatic vasculature. PROX1 has been shown to have cell-type dependent roles in regulating the cell cycle. We hypothesize that PROX1 functions as a key cell cycle regulator in LECs and promotes their cell cycle progression. In this study, immunocytochemistry, western blotting and luciferase assays were used to characterize PROX1 mediated activation of the mouse Ccne1 promoter. Following deletion of the Prospero 1 domain (PD1∆), the resulting PROX1 protein is localized to both the nucleus and the cytoplasm. We have determined that PROX1 requires both E2F binding sites located in the Ccne1 promoter to activate transcription of the gene. We observed that siRNA knockdown of Prox1 reduced CYCLIN E1 protein levels as well as decreased cellular proliferation in LECs. In contrast, overexpression of a version of PROX1 in which the homeodomain and Prospero domain 2 (HDPD2Δ) were deleted increased CYCLIN E1 protein levels in human umbilical vein endothelial cells (HUVEC), but resulted in the arrest of cells in the G1 phase. We have also established that PROX1 is phosphorylated in primary human LECs. We have shown a role for the PD1 domain in mediating PROX1 subcellular localization and we have observed that the expression of the HDPD2Δ version of PROX1 blocks proliferation in HUVECs. We are the first to demonstrate a role for PROX1 as a transcriptional co-activator and to establish that PROX1 is phosphorylated in LECs.

CD146 is a cell adhesion molecule that has been shown to regulate cell adhesion, migration and proliferation of different cell types. It is highly expressed in blood endothelial cells (BECs), but is only lowly expressed in lymphatic endothelial cells (LECs). The PROX1 homeobox gene is a master regulator of lymphangiogenesis and its expression is necessary and sufficient to drive venous endothelial cells into a LEC phenotype. The highly permeable nature of the lymphatic vessels may partially derive from PROX1 mediated repression of CD146 transcription. We hypothesize that PROX1 promotes lymphatic differentiation by repressing CD146 transcription. In gain of function studies, Human Umbilical Vein Endothelial Cells (HUVECs) were infected with adenoviruses encoding EGFP, wild type PROX1 (AdProx1) or a Homeo-Prospero domain deleted version of PROX1 (AdHDPD), which cannot bind DNA. In order to knockdown PROX1, LECs were transfected with PROX1 specific siRNA. When compared to EGFP infected HUVECs, AdProx1 infected HUVECs had decreased CD146 expression both at protein and mRNA levels. In contrast, AdHDPD infected HUVECs had increased levels of CD146 expression. In support of a role for PROX1 in repressing CD146, PROX1 siRNA transfected LECs express higher levels of CD146 as compared to mock transfected LECs or LECs transfected with control siRNA. Based on these results, we predict that CD146 expression is kept at basal levels by an unknown repressor bound to the CD146 promoter. By interacting with this unknown repressor, PROX1 further represses CD146 expression. On the other hand, the DNA binding-deficient ΔHDPD version of PROX1 binds the unknown repressor and sequesters it from the CD146 promoter, thereby relieving the repression of CD146 expression in ECs. Different levels of CD146 expression between BECs and LECs might reflect the structural and functional differences between blood and lymphatic vessels. Since CD146 plays a critical role in EC adhesion, regulation of CD146 expression in ECs might be one of the key factors regulating vessel permeability.

The homeobox transcription factor PROX1 is the mammalian ortholog of the Drosophila gene Prospero. Expression of PROX1 in a subset of venous endothelial cells changes their fate to lymphatic endothelial cells (LEC). PROX1 is required for lymphatic development as Prox1 null mice lack all lymphatic vasculature. PROX1 has been shown to have cell-type dependent roles in regulating the cell cycle. We hypothesize that PROX1 functions as a key cell cycle regulator in LECs and promotes their cell cycle progression. In this study, immunocytochemistry, western blotting and luciferase assays were used to characterize PROX1 mediated activation of the mouse Ccne1 promoter. Following deletion of the Prospero 1 domain (PD1∆), the resulting PROX1 protein is localized to both the nucleus and the cytoplasm. We have determined that PROX1 requires both E2F binding sites located in the Ccne1 promoter to activate transcription of the gene. We observed that siRNA knockdown of Prox1 reduced CYCLIN E1 protein levels as well as decreased cellular proliferation in LECs. In contrast, overexpression of a version of PROX1 in which the homeodomain and Prospero domain 2 (HDPD2Δ) were deleted increased CYCLIN E1 protein levels in human umbilical vein endothelial cells (HUVEC), but resulted in the arrest of cells in the G1 phase. We have also established that PROX1 is phosphorylated in primary human LECs. We have shown a role for the PD1 domain in mediating PROX1 subcellular localization and we have observed that the expression of the HDPD2Δ version of PROX1 blocks proliferation in HUVECs. We are the first to demonstrate a role for PROX1 as a transcriptional co-activator and to establish that PROX1 is phosphorylated in LECs.

CD146 is a cell adhesion molecule that has been shown to regulate cell adhesion, migration and proliferation of different cell types. It is highly expressed in blood endothelial cells (BECs), but is only lowly expressed in lymphatic endothelial cells (LECs). The PROX1 homeobox gene is a master regulator of lymphangiogenesis and its expression is necessary and sufficient to drive venous endothelial cells into a LEC phenotype. The highly permeable nature of the lymphatic vessels may partially derive from PROX1 mediated repression of CD146 transcription. We hypothesize that PROX1 promotes lymphatic differentiation by repressing CD146 transcription. In gain of function studies, Human Umbilical Vein Endothelial Cells (HUVECs) were infected with adenoviruses encoding EGFP, wild type PROX1 (AdProx1) or a Homeo-Prospero domain deleted version of PROX1 (AdHDPD), which cannot bind DNA. In order to knockdown PROX1, LECs were transfected with PROX1 specific siRNA. When compared to EGFP infected HUVECs, AdProx1 infected HUVECs had decreased CD146 expression both at protein and mRNA levels. In contrast, AdHDPD infected HUVECs had increased levels of CD146 expression. In support of a role for PROX1 in repressing CD146, PROX1 siRNA transfected LECs express higher levels of CD146 as compared to mock transfected LECs or LECs transfected with control siRNA. Based on these results, we predict that CD146 expression is kept at basal levels by an unknown repressor bound to the CD146 promoter. By interacting with this unknown repressor, PROX1 further represses CD146 expression. On the other hand, the DNA binding-deficient ΔHDPD version of PROX1 binds the unknown repressor and sequesters it from the CD146 promoter, thereby relieving the repression of CD146 expression in ECs. Different levels of CD146 expression between BECs and LECs might reflect the structural and functional differences between blood and lymphatic vessels. Since CD146 plays a critical role in EC adhesion, regulation of CD146 expression in ECs might be one of the key factors regulating vessel permeability.

Background. Haematopoietic stem cell transplantation (HSCT) is an intensive procedure associated with psychological distress particularly during the first weeks (acute phase). Based on the self-regulatory model of adjustment to illness, a preparatory group intervention was developed aiming at alleviating distress by reducing negative perceptions of HSCT and fostering helpful coping. Aims. The present study aimed to evaluate the feasibility of delivering the intervention and of conducting a trial to assess its efficacy. It also aimed to explore the applicability of the self-regulatory model in HSCT. Methods. Participants were adults from consecutive referrals at two transplant centres. Half were randomised to the intervention and half to treatment as usual at each site. Psychological distress, HSCT perceptions, and coping were assessed at baseline (following consent), on transplant day, two weeks, and four weeks after transplantation. Results. Of 99 eligible patients, 45 consented. Main barriers included inability to consent prior to transplantation, competing priorities, being unwell, and long travel distance. Of 21 participants randomised to intervention, five attended. Main barriers included being unable to attend prior to transplantation and having competing priorities. Groups could not be held sufficiently frequently to enable attendance prior to transplantation, as randomising participants to the control group prevented sufficient accrual at each site. Anxiety peaked two weeks following transplantation but depression increased throughout the acute phase. Intervention effects were small but sample sizes for a full trial appeared feasible. Negative perceptions of HSCT and use of a range of coping styles (including styles considered helpful) predicted higher distress throughout the period. Conclusions. The findings revealed considerable barriers to delivering a group-based intervention and conducting a trial to assess its effectiveness. This highlighted a need for better integration with routine care and alternative trial procedures. However, the findings illustrated complex psychological needs during the acute phase of HSCT and the role of negative HSCT perceptions and unhelpful coping in underpinning distress.

Relapse in AML is thought to arise from dormant leukaemic cells that are characterised by low RNA synthesis activity, protected by the bone marrow (BM) niche, and may evade the effects of chemotherapeutic drugs. Our aim was to investigate agents which might be able to overcome chemoprotection by targeting the intrinsic apoptosis pathway. We developed in vitro assays to identify and characterise the dormant AML cells using combinations of markers, including the cell-division marker PKH26, leukaemia-associated phenotypes (LAPs), and dormancy markers. In a dormancy model based on 12-day AML/stroma co-culture, we have shown that the expression of some aberrant phenotypes can persist for several days. Also, after 12 days, some of the CD34+, PKH26high (dormant), and LAP+ (leukaemic) cells maintained their primitiveness and were still clonogenic. Furthermore, our chemosensitivity data showed that novel agents TG02, and BH3 mimetics ABT-737 and ABT-199, which inhibit the B-cell lymphoma 2 (BCL-2) family of anti-apoptotic molecules, could efficiently target BM niche-mediated chemoresistance, which is thought to be one of the main obstacles to traditional chemotherapy. We explored various candidate dormancy markers based on the low RNA, non-proliferative profile of dormant cells. Among those tested, the RNA synthesis marker Pyronin Y (PY), and an antibody to the transferrin receptor CD71 were the most reproducible in terms of marker expression and stability. We endeavoured to characterise cell dormancy on the molecular level by investigating gene expression in the PYlow (dormant) and PYhigh (proliferating) subsets and have obtained limited results. In summary, this study has identified and partly characterised dormant AML cells by development of in vitro assays, and has shown chemosensitivity to novel agents TG02, ABT-737 and ABT-199 in dormant leukaemia cells.

The lymphatic system complements the blood circulatory system in absorption and transport of nutrients, and in the maintenance of homeostasis. Historically, the angiogenesis field has advanced faster and farther than the field of lymphangiogenesis. The discovery of lymphatic markers and the emerging evidence implicating the lymphatic system as a central player in a variety of pathological conditions has attracted research interest and driven the field forward. Research efforts have produced the observation that regulators of the blood endothelium are frequently members of the same protein families of regulators of the lymphatic endothelium. More importantly, these regulators do not act discretely, restricting their regulatory activities to one endothelial cell (EC) type. Two examples of regulators that behave in this manner are the VEGF and the Angiopoietin families of proteins, which have cell-type-dependent effects on EC processes such as migration, proliferation and survival. The study of these regulators therefore requires an in vitro EC system capable of accommodating the simultaneous characterization of the signaling pathways downstream of these shared molecular regulators in venous, arterial and lymphatic endotheliums. To build such an in vitro system, I isolated and validated lymphatic, venous, and arterial ECs derived from vessels of bovine mesentery. The proteomes of the three cell types were comparatively studied using two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometric identification. The three cell types were used in a subtractive immunization scheme for the production of a monoclonal antibody selectively reactive to a potentially novel surface protein marker of lymphatic ECs. The studies recorded herein all share the common goal of identifying and characterizing unique molecular signatures that distinguish lymphatic ECs from blood ECs, and that may underline the cellular biology of the lymphatic endothelium as distinct from the blood endothelium.