A role for Wnt signaling and Adrenomedullin and signaling Wnt for A role Myeloma Multiple of niche in the pathogenesis marrow The bone The bone marrow niche in the pathogenesis of Multiple Myeloma A role for Wnt signaling and Adrenomedullin pracownia DTP i grafiki www.przygotowalniaDTP.pl ISBN 978-83-63861-16-2 Kinga Anna Kocemba okladka_nowa.indd 1-3 2014-01-20 11:57:46 The bone marrow niche in the pathogenesis of Multiple Myeloma A role for Wnt signaling and Adrenomedullin The bone marrow niche in the pathogenesis of Multiple Myeloma A role for Wnt signaling and Adrenomedullin Kinga Anna Kocemba The research described in this thesis was funded by the Dutch Cancer Society Editing Przygotowalnia Pracownia DTP i Grafiki Graphic design and typesetting Przygotowalnia Pracownia DTP i Grafiki Cover: Antelope Canyon, Arizona, USA by Kinga Anna Kocemba © Copyright by Kinga Anna Kocemba ISBN 978-83-63861-16-2 Amsterdam–Kraków 2014 Pracownia DTP i Grafiki ul. Łużycka 71c/9 30–693 Kraków [email protected] www.przygotowalniaDTP.pl Moim rodzicom To my parents The bone marrow niche in the pathogenesis of Multiple Myeloma A role for Wnt signaling and Adrenomedullin academisch proefschrift ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus prof. dr. D.C. van den Boom ten overstaan van een door het college voor promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel op donderdag 6 maart 2014, te 14:00 uur door Kinga Anna Kocemba geboren te Oświęcim, Polen Promotiecommissie promotor Prof. dr. S.T. Pals co-promotor Dr. M. Spaargaren overige leden Dr. M.M. Maurice Prof. dr. C.J.M. van Noesel Prof. dr. M.H.J. van Oers Prof. dr. P. Sonneveld Prof. dr. K. Vanderkerken Faculteit der Geneeskunde Contents 9 chapter 1 General introduction 37 chapter 2 Wnt signaling reaching gale force during multiple myeloma progression 59 chapter 3 Transcriptional silencing of the Wnt-antagonist DKK1 by promoter methylation is associated with enhanced Wnt signaling in advanced multiple myeloma 87 chapter 4 Loss of CYLD expression unleashes Wnt signaling in multiple myeloma and is associated with aggressive disease 111 chapter 5 N-cadherin-mediated interaction with multiple myeloma cells inhibits osteoblast differentiation 143 chapter 6 The hypoxia target adrenomedullin is aberrantly expressed in multiple myeloma and promotes angiogenesis 201 chapter 7 Summary and discussion 213 chapter 8 Nederlandse samenvatting Acknowledgments Curriculum vitae Chapter 11 General introduction B CELL DEVELOPMENT AND B-lineaGE MALIGNANCIES The development of B cells is characterized by sequential molecular events regu- lated by B-lineage specific transcription factors. During early development in the 1 bone marrow, B cells acquire the ability to express cell surface-bound immuno- globulins, which constitutie the antigen-binding part of the B cell receptor (BCR) multiprotein complex.1 BCR diversity, required for antigen recognition, is gen- erated by somatic recombination, combining various gene segments within the 3 immunoglobuline (Ig) heavy and light chain loci. The heavy chain is assembled from Variable (V), Diversity (D) and Joining (J) gene segments that somatically 4 recombine with the Constant (C) region exons, generating unique immunoglobu- lins of diverse antigenic specificity. The light chain variable region (which can be of 5 kappa or lambda type) is composed of only a V and J gene segment. The diversity of the IgV regions is further enhanced by addition of nucleotides at the junctions 6 of the segments. Upon successful rearrangement of Ig light and heavy chain genes, a complete IgM molecule is expressed at the cell surface, which is required for sur- 7 vival. Non self-reactive immature B cells exit the bone marrow, whereas self-reac- tive immature B cells undergo apoptosis (clonal deletion), or generate a new B cell receptor by receptor editing.2,3,4,5 The immature B cell then migrate to the second- ary lymphoid organs, i.e. lymph nodes, spleen and mucosa-associated lymphoid tissue (MALT), to complete the process of maturation. Upon antigen encounter, these naive B cells may undergo antigen-specific B cell differentiation. Initially, antigen is internalized by the BCR and processed peptides are presented by MHC class II molecules to Th cells. This interaction with Th cells provides the B cells with co-stimulatory signals, crucial for further differentiation. B cells may now differen- tiate into short-lived plasmablast outside of the germinal center (GC), or migrate into a primary follicle to initiate the formation of a GC.6,7 Germinal centers can be 11 Chapter 1 subdivided in two distinct zones, called the dark and light zones. Rapidly prolifer- ating B cells (centroblasts) form the dark zone of the GC. In this zone, somatic hy- permutation (SHM) of the immunoglobulin genes of the B cells takes place.8–10 This involves introduction of point mutations by the enzyme called activation-induced cytidine deaminase (AID), which deaminates cytidines to uracil. This results in C-T and G-A transitions in the V region of immunoglobulin (Ig) genes, creat- ing Ig variants with altered affinity for a particular antigen.11 The mutated B cells (centrocytes) migrate to the light zone of the GC. Here, they re-encounter antigen presented by the follicular dendritic cells (FDC), and a selection process, based on the affinity of the BCR for antigen, is initiated. Depending on the strength of BCR signal, B cells will receive a survival and proliferation signal from the FDCs and Th cells, or will die by apoptosis.12–14 High affinity B cells will process antigen and present this to antigen-specific T cells,15 which provide stimulatory signals (e.g. T cell receptor (TCR)/CD3-MHC class II, CD40-CD40L, CD80/86-CD28 and several cytokines),6,13,16–18 resulting in class switch recombination (CSR) and further differentiation into either memory B cells or plasma cells.9,17,18 VDJ recombination (VDJR), as well as (CSR) and SHM, involve the genera- tion of DNA breaks, potentially dangerous events that predispose to chromosomal translocations Indeed, the DNA breaks that are induced by VDJR, CSR and SHM coincide with the sites of chromosomal translocations that involve the IgH or IgL loci in many lymphoid malignancies.19–21 For instance, chromosomal translocation that involve the IgH switch region and the partner oncogene (e.g. BCL-2, BCL6, c-MYC, CCND1, CCND3, FGFR3-MMSET and MAFC), have been identified in follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), Burkitt’s lymphoma and multiple myeloma (MM).22–25 Moreover, during the SHM process in the GC, AID can introduce mutations in non-immunoglobulin genes, e.g. the oncogenes BCL6, MYC, PIM1, PAX5.19,26 In general, each lymphoma subtype resembles a B cell trapped at the particular stage of B cell development (Figure 1), as determined by the presence or absence of somatic hypermutation and the gene expression profile.27,28 For instance, for Burkitt lymphoma, follicular lymphoma, and the germinal center B cell-like (GCB) sub- type of diffuse large B cell lymphoma (DLBCL), the normal cellular counterpart is the germinal center B cell,29,30 whereas the activated B cell-like (ABC) subtype of DLBCL resembles post-germinal center B cells/plasmablasts.29 Mucosa-associated lymphoid tissue (MALT) lymphomas are extra nodal in origin and phenotypically related to post-germinal center marginal zone B cells.31 Hairy cell leukemia has mutated Ig genes and class switched Ig heavy chains, along with a gene expression profile pointing to post-germinal center memory B cell as a cell of origin.32 12 General introduction 1 Figure 1. Normal B cell development and the related stages of B cell malignancy Schematic representation of B cell differentiation. The malignant counterparts are indicated by italic font. BM-bone marrow, ALL-acute lymphoblastic leukemia, MALT-mucosa associated lymphoid tis- sue, MCL-mucosa associated lymphoid tissue, DLBCL-diffuse large B cell lymphoma, CLL-chronic 3 lymphocytic leukemia, FL-follicular lymphoma, MM-multiple myeloma. 4 MULTIPLE MYELOMA 5 Multiple myeloma (MM) is characterized by a clonal proliferation of plasma cells 6 (PCs) in the bone marrow (BM), often associated with pancytopenia and osteolyt- ic bone disease. It is one of the most frequent hematological cancers and remains 7 largely incurable, despite high-dose chemotherapy with additional stem cells sup- port. The presence of somatic hypermutation of the immunoglobulin variable region genes in MM cells indicates that the cell of origin in myeloma is a post-ger- minal center B cell.33,34 In the majority of cases MM arises from a pre-malignant expansion of plasma cells called monoclonal gammopathy of undetermined sig- nificance (MGUS), with a prevalence of approximately 3% and 5% in people older than 50 and 70 years, respectively.35,36 Based on chromosomal studies, two main cytogenetic subgroups can be discriminated: a group characterized by a high in- cidence of five recurrent IgH translocations and loss of chromosome 13/13q14; and a hyperdiploid group, characterized by multiple trisomies.24 The recurrent translocations in the non-hyperdiploid group involve the IgH switch region 14q32 13 Chapter 1 and the translocation partners: cyclin D1 (CCND1) (11q13), cyclin D3 (CCND3) (6p21), MAFC (16q23), FGFR3 and MMSET (4p16), and MAFB (20q11).37 Hyperdiploid myeloma is characterized by trisomies of multiple odd chromosomes (3, 5, 7, 9, 11, 15, 19, and 21). Together with t(11;14), hyperdiploidy confers a rela- tively favorable prognosis, whereas MAFC, MAFB, or FGFR3/MMSET activa- tion and deletion of chromosome 13 and/or 17 are associated with a poor prog- nosis.38,39 Interestingly, there is no single genetic event that distinguishes MGUS from MM. However, several genetic aberrations have been associated with disease progression.