TRIB2 in normal and malignant hematopoiesis - a transcription factor network

Hematopoiesis is the tightly controlled and complex process in which the entire blood system is formed and maintained by a rare pool of hematopoietic stem cells (HSCs), and its dysregulation results in the formation of leukaemia. TRIB2, a member of the Tribbles family of serine/threonine pseudokinases, has been implicated in a variety of cancers and is a potent murine oncogene that induces acute myeloid leukaemia (AML) in vivo via modulation of the essential myeloid transcription factor CCAAT-enhancer binding protein α (C/EBPα). C/EBPα, which is crucial for myeloid cell differentiation, is commonly dysregulated in a variety of cancers, including AML. Two isoforms of C/EBPα exist - the full-length p42 isoform, and the truncated oncogenic p30 isoform. TRIB2 has been shown to selectively degrade the p42 isoform of C/EBPα and induce p30 expression in AML. In this study, overexpression of the p30 isoform in a bone marrow transplant (BMT) leads to perturbation of myelopoiesis, and in the presence of physiological levels of p42, this oncogene exhibited weak transformative ability. It was also shown by BMT that despite their degradative relationship, expression of C/EBPα was essential for TRIB2 mediated leukaemia. A conditional mouse model was used to demonstrate that oncogenic p30 cooperates with TRIB2 to reduce disease latency, only in the presence of p42. At the molecular level, a ubiquitination assay was used to show that TRIB2 degrades p42 by K48-mediated proteasomal ubiquitination and was unable to ubiquitinate p30. Mutation of a critical lysine residue in the C-terminus of C/EBPα abrogated TRIB2 mediated C/EBPα ubiquitination suggesting that this site, which is frequently mutated in AML, is the site at which TRIB2 mediates its degradative effects. The TRIB2-C/EBPα axis was effectively targeted by proteasome inhibition. AML is a very difficult disease to target therapeutically due to the extensive array of chromosomal translocations and genetic aberrations that contribute to the disease. The cell from which a specific leukaemia arises, or leukaemia initiating cell (LIC), can affect the phenotype and chemotherapeutic response of the resultant disease. The LIC has been elucidated for some common oncogenes but it is unknown for TRIB2. The data presented in this thesis investigate the ability of the oncogene TRIB2 to transform hematopoietic stem and progenitor cells in vitro and in vivo. TRIB2 overexpression conferred in vitro serially replating ability to all stem and progenitor cells studied. Upon transplantation, only TRIB2 overexpressing HSCs and granulocyte/macrophage progenitors (GMPs) resulted in the generation of leukaemia in vivo. TRIB2 induced a mature myeloid leukaemia from the GMP, and a mixed lineage leukaemia from the HSC. As such the role of TRIB2 in steady state hematopoiesis was also explored using a Trib2-/- mouse and it was determined that loss of Trib2 had no effect on lineage distribution in the hematopoietic compartment under steady-state conditions. The process of hematopoiesis is controlled by a host of lineage restricted transcription factors. Recently members of the Nuclear Factor 1 family of transcription factors (NFIA, NFIB, NFIC and NFIX) have been implicated in hematopoiesis. Little is known about the role of NFIX in lineage determination. Here we describe a novel role for NFIX in lineage fate determination. In human and murine datasets the expression of Nfix was shown to decrease as cells differentiated along the lymphoid pathway. NFIX overexpression resulted in enhanced myelopoiesis in vivo and in vitro and a block in B cell development at the pre-pro-B cell stage. Loss of NFIX resulted in disruption of myeloid and lymphoid differentiation in vivo. These effects on stem and progenitor cell fate correlated with changes in the expression levels of key transcription factors involved in hematopoietic differentiation including a 15-fold increase in Cebpa expression in Nfix overexpressing cells. The data presented support a role for NFIX as an important transcription factor influencing hematopoietic lineage specification. The identification of NFIX as a novel transcription factor influencing lineage determination will lead to further study of its role in hematopoiesis, and contribute to a better understanding of the process of differentiation. Elucidating the relationship between TRIB2 and C/EBPα not only impacts on our understanding of the pathophysiology of AML but is also relevant in other cancer types including lung and liver cancer. Thus in summary, the data presented in this thesis provide important insights into key areas which will facilitate the development of future therapeutic approaches in cancer treatment.

The regulation of AMP-activated protein kinase by vascular endothelial growth factor

The function of the vascular endothelium is to maintain vascular homeostasis, by providing an anti-thrombotic, anti-inflammatory and vasodilatory interface between circulating blood and the vessel wall, meanwhile facilitating the selective passage of blood components such as signaling molecules and immune cells. Dysfunction of the vascular endothelium is implicated in a number of pathological states including atherosclerosis and hypertension, and is thought to precede atherogenesis by a number of years. Vascular endothelial growth factor A (VEGF) is a crucial mitogenic signaling molecule, not only essential for embryonic development, but also in the adult for regulating both physiological and pathological angiogenesis. Previous studies by our laboratory have demonstrated that VEGF-A activates AMP-activated protein kinase (AMPK), the downstream component of a signaling cascade important in the regulation of whole body and cellular energy status. Furthermore, studies in our laboratory have indicated that AMPK is essential for VEGF-A-stimulated vascular endothelial cell proliferation. AMPK activation typically stimulates anabolic processes and inhibits catabolic processes including cell proliferation, with the ultimate aim of redressing energy imbalance, and as such is an attractive therapeutic target for the treatment of obesity, metabolic syndromes, and type 2 diabetes. Metabolic diseases are associated with adverse cardiovascular outcomes and AMPK activation is reported to have beneficial effects on the vascular endothelium. The mechanism by which VEGF-A stimulates AMPK, and the functional consequences of VEGF-A-stimulated AMPK activation remain uncertain. The present study therefore aimed to identify the specific mechanism(s) by which VEGF-A regulates the activity of AMPK in endothelial cells, and how this might differ from the activation of AMPK by other agents. Furthermore, the role of AMPK in the pro-proliferative actions of VEGF-A was further examined. Human aortic and umbilical vein endothelial cells were therefore used as a model system to characterise the specific effect(s) of VEGF-A stimulation on AMPK activation. The present study reports that AMPK α1 containing AMPK complexes account for the vast majority of both basal and VEGF-A-stimulated AMPK activity. Furthermore, AMPK α1 is localized to the endoplasmic reticulum when sub-confluent, but translocated to the Golgi apparatus when cells are cultured to confluence. AMPK α2 appears to be associated with a structural cellular component, but neither α1 nor α2 complexes appear to translocate in response to VEGF-A stimulation. The present study confirms previous reports that when measured using the MTS cell proliferation assay, AMPK is required for VEGF-A-stimulated endothelial cell proliferation. However, parallel experiments measuring cell proliferation using the Real-Time Cell Analyzer xCELLigence system, do not agree with these previous reports, suggesting that AMPK may in fact be required for an aspect of mitochondrial metabolism which is enhanced by VEGF-A. Studies into the mitochondrial activity of endothelial cells have proved inconclusive at this time, but further studies into this are warranted. During previous studies in our laboratory, it was suggested that VEGF-A-stimulated AMPK activation may be mediated via the diacylglycerol (DAG)-sensitive transient receptor potential cation channel (TRPCs -3, -6 or -7) family of ion channels. The present study can neither confirm, nor exclude the expression of TRPCs in vascular endothelial cells, nor rule out their involvement in VEGF-A-stimulated AMPK activation; more specific investigative tools are required in order to characterise their involvement. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP)-stimulated Ca2+ release from acidic intracellular organelles is not required for AMPK activation by VEGF-A. Despite what is known about the mechanisms by which AMPK is activated, far less is known concerning the downregulation of AMPK activity, as observed in human and animal models of metabolic disease. Phosphorylation of AMPK α1 Ser485 (α2 Ser491) has recently been characterised as a mechanism by which the activity of AMPK is negatively regulated. We report here for the first time that VEGF-A stimulates AMPK α1 Ser485 phosphorylation independently of the previously reported AMPK α1 Ser485 kinases Akt (protein kinase B) and ERK1/2 (extracellular signal-regulated kinase 1/2). Furthermore, inhibition of protein kinase C (PKC), the activity of which is reported to be elevated in metabolic disease, attenuates VEGF-A- and phorbol 12-myristate 13-acetate (PMA)-stimulated AMPK α1 Ser485 phosphorylation, and increases basal AMPK activity. In contrast to this, PKC activation reduces AMPK activity in human vascular endothelial cells. Attempts to identify the PKC isoform responsible for inhibiting AMPK activity suggest that it is one (or more) of the Ca2+-regulated DAG-sensitive isoforms of PKC, however cross regulation of PKC isoform expression has limited the present study. Furthermore, AMPK α1 Ser485 phosphorylation was inversely correlated with human muscle insulin sensitivity. As such, enhanced AMPK α1 Ser485 phosphorylation, potentially mediated by increased PKC activation may help explain some of the reduced AMPK activity observed in metabolic disease.

Improving protein yield from mammalian cells by manipulation of stress response pathways

Monoclonal antibodies are a class of therapeutic that is an expanding area of the lucrative biopharmaceutical industry. These complex proteins are predominantly produced from large cultures of mammalian cells; the industry standard cell line being Chinese Hamster Ovary (CHO) cells. A number of optimisation strategies have led to antibody titres from CHO cells increasing by a hundred-fold, and it has been proposed that a further bottleneck in biosynthesis is in protein folding and assembly within the secretory pathway. To alleviate this bottleneck, a CHO-derived host cell line was generated by researchers at the pharmaceutical company UCB that stably overexpressed two critical genes: XBP1, a transcription factor capable of expanding the endoplasmic reticulum and upregulating protein chaperones; and Ero1α, an oxidase that replenishes the machinery of disulphide bond formation. This host cell line, named CHO-S XE, was confirmed to have a high yield of secreted antibody. The work presented in this thesis further characterises CHO-S XE, with the aim of using the information gained to lead the generation of novel host cell lines with more optimal characteristics than CHO-S XE. In addition to antibodies, it was found that CHO-S XE had improved production of two other secreted proteins: one with a simple tertiary structure and one complex multi-domain protein; and higher levels of a number of endogenous protein chaperones. As a more controlled system of gene expression to unravel the specific roles of XBP1 and Ero1α in the secretory properties of CHO-S XE, CHO cells with inducible overexpression of XBP1, Ero1α, or a third gene involved in the Unfolded Protein Response, GADD34, were generated. From these cell lines, it was shown that more antibody was secreted by cells with induced overexpression of XBP1; however, Ero1α and GADD34 overexpression did not improve antibody yield. Further investigation revealed that endogenous XBP1 splicing was downregulated in the presence of an abundance of the active form of XBP1. This result indicated a novel aspect of the regulation of the activity of IRE1, the stress-induced endoribonuclease responsible for XBP1 splicing. Overall, the work described in this thesis confirms that the overexpression of XBP1 has an enhancing effect on the secretory properties of CHO cells; information which could contribute to the development of host cells with a greater capacity for antibody production.