Multiple myeloma: The role of glycosylation in disease development and progression.

Abstract

Multiple myeloma (MM) is a clonal plasma cell disorder that is characterized by skeletal destruction, renal failure, anaemia and hypercalcaemia. MM cells are intimately associated with the bone marrow microenvironment, with an array of adhesive interactions taking place between the bone marrow extracellular matrix (ECM) and the surface of MM cells. The considerable heterogeneity in the behaviour of MM is partially governed by differences in gene expression. As MM progresses further alterations in gene expression ensue, drug resistance increases and some cases develop bone marrow microenvironmental independence with spread to extra-medullary sites, possibly due to changes in adhesion properties. Despite recent advances in therapy MM remains an incurable disease. In particular, little progress has been made in patients with high-risk disease, in whom median survival remains less than 3 years (1). Knowledge of the events that underlie disease progression in MM is incomplete. Using chromosomal analysis and high throughput (HTP) genomic approaches important prognostic information can be obtained, stratifying patients into low and high-risk groups. These technologies, however do not give an account of the entire milieu within which the malignant plasma cells survive and proliferate. Considerable information may be overlooked using these approaches alone, particularly regarding the impact of PTMs on the ability of plasma cell surface proteins to interact with the surrounding microenvironment. The composition of the bone marrow microenvironment is also an important consideration if we are to acquire a deep understanding of what factors are at play, which may influence MM cell proliferation and survival within this supportive niche. PTMs are covalent, generally enzymatic, modifications that occur during or after the biosynthesis of proteins and serve to increase the functional diversity of proteins. These modifications include, but are not limited to, phosphorylation, ubiquitination, methylation, acetylation, glycosylation and proteolysis. This diverse array of protein modifications influences almost all aspects of normal and disease state cell biology. Several of these PTMs occur aberrantly in MM and have been implicated in the development of drug resistance (2-4). When considering MM cells within the bone marrow microenvironment it is important to consider PTMs that may functionally alter the properties of cell surface proteins, which act as an interface between the malignant cell and other bone marrow niche cells. One such PTM is glycosylation, which results in the alteration of sugar moieties on cell surface proteins, which can in turn alter their adhesive properties. Glycosylation is a stepwise process of covalent attachment of oligosaccharide chains to proteins, and alterations in this process have been associated with malignant transformation. Altered glycosylation is a universal feature of cancer cells and alterations in this process have been associated with a more aggressive phenotype in several solid and haematological malignancies. The glycosylation pattern of a cell can change rapidly depending on the cell-cell interaction, local microenvironment and immunological milieu. This process may significantly influence disease biology and risk of progression in MM plasma cells, where the cells are known to be dependent on bone marrow stromal cells and the bone marrow microenvironment for survival and proliferation signals. Many cytokines and adhesion molecules, critical for MM survival, are glycoproteins and depend on glycosylation for their normal function. The role of glycosylation in the interaction between the ECM and MM cells has not yet been defined. The study of the ECM in MM and other cancers has been slowed in the past by the lack of high throughput technologies that allow for rapid and detailed profiling of large insoluble ECM proteins. In MM adhesive interactions between MM cells and the ECM are known to confer drug resistance and may offer protection to MM cells from therapeutic agents. Therefore knowledge of the composition of the ECM is needed in order to further advance the identification of therapeutic targets to over come these mechanisms. The hypothesis of the work outlined in this thesis is that differential transcriptional regulation of glycosylation-associated genes, and consequent differences in glycosylation of plasma cell surface proteins, plays a role in the pathobiology of MM. The primary aim of this body of work is to explore the role of glycosylation in MM and to advance our understanding of the alterations that occur in this process in malignant plasma cells. A secondary aim of this work is to more clearly define the alterations that occur in the ECM of MM, as an important component of the bone marrow microenvironment, where glycosylated cell surface proteins are widely present The work presented in this thesis demonstrates that altered sialylation influences homing and survival of MM cells in the bone marrow niche invivo. Knockdown studies demonstrate that reduction in alpha 2,3 linked sialic acid on the surface of MM cells results in decreased tumour burden and prolonged survival in xenograft murine models. This study highlights the importance of altered glycosylation, particularly sialylation, in MM progression and metastasis. Further studies were undertaken to interrogate the bone marrow tumour microenvironment in MM, and a novel proteomics platform was applied, which facilitated the identification and annotation of ECM proteins in this disease. This work demonstrates that the tumour ECM is remodeled at an early stage in MM development in humans and this process continues as the disease progresses. The body of work presented serves to advance the current knowledge of the MM bone marrow microenvironment and the role of glycosylation in this disease.