Overview of Wnt signalingThe Wnt family of glycoproteins signals through a "canonical" or "noncanonical" pathway ( Figure 1). In the absence of Wnt's, glycogen synthase kinase-␤ (GSK3␤), Axin, adenomatous polyposis coli (APC), and casein kinase I␣ (CKI␣) form a ␤-catenin destruction complex. 1 CKI␣ phosphorylates ␤-catenin at Ser45, and then GSK3␤ phosphorylates ␤-catenin at Ser33/Ser37/Thr41. Phosphorylated ␤-catenin is recognized by ␤-transducin repeat protein, ubiquitinated, and degraded by proteasomes. In contrast, activation of canonical Wnt signaling occurs when a Wnt protein binds to one of the 10 members of the frizzled (FZD) receptor family; Wnt-FZD then binds low-density lipoprotein-related protein-5 (LRP5) or LRP6. The resultant complex activates Dishevelled (Dvl), a protein that attracts Axin away from the destruction complex and antagonizes the ability of GSK3␤ to phosphorylate ␤-catenin, thereby preventing destruction complex formation. If ␤-catenin is not degraded, it translocates to the nucleus where it binds to the transcription factor T-cell factor-4 (TCF4) and enhances target gene expression. 1 Canonical Wnt's promote caveolin-dependent LRP internalization and facilitate its interaction with Axin. 2 In contrast, DKK1 binds to LRP5/6 causing the receptor to attract a Kremen; this interaction promotes clathrin-mediated internalization, thereby inactivating LRP, though some data suggest that Kremen may not be essential for DKK1-mediated Wnt inhibition. 3,4 Moreover, R-Spondins amplify the activity of canonical Wnt's while antagonizing DKK1-mediated interaction with LRP and Kremen. 5 Wise and SOST are also secreted Wnt inhibitors that bind to and inactivate LRP. 6,7 Wnt inhibitory factor (WIF) proteins, which are structurally similar to the extracellular portion of the Derailed/Ryk class of transmembrane Wnt receptors, and secreted forms of frizzled proteins (sFRP, sizzled, and FrzB) act by directly binding Wnt molecules and can function as Wnt inhibitors, but may also stabilize Wnt's and facilitate Wnt signaling. 8,9 DKK1 regulates bone development and accrual and maintenance of bone mass Bone marrow-derived mesenchymal stem cells (MSC) can differentiate into adipocytes, chondrocytes, or osteoblasts. Although the precise orchestration of Wnt signaling during bone development is dependent on complex microenvironmental cues, data from several groups suggest that Wnt3a, Wnt5a, Wnt7b, and Wnt10b are central to osteoblast differentiation (Figure 2). 10-14 Increased ␤-catenin is found in cells committed to the osteoblast lineage, and loss of ␤-catenin in osteoblast precursor cells results in reduced bone deposition. 15 In addition to promoting osteoblast commitment, canonical Wnt signaling inhibits adipocyte differentiation, primarily through accrual of stabilized ␤-catenin and subsequent transactivation of TCF-responsive genes. 16,17 However, noncanonical Wnt signaling through Wnt5a inactivates peroxisome proliferatoractivated receptor-␥ (PPAR␥), a key adipogenic transcription factor and activates Ru...

We have sequenced 463 presenting cases of myeloma entered into the UK Myeloma XI study using whole exome sequencing. Here we identify mutations induced as a consequence of misdirected AID in the partner oncogenes of IGH translocations, which are activating and associated with impaired clinical outcome. An APOBEC mutational signature is seen in 3.8% of cases and is linked to the translocation mediated deregulation of MAF and MAFB, a known poor prognostic factor. Patients with this signature have an increased mutational load and a poor prognosis. Loss of MAF or MAFB expression results in decreased APOBEC3B and APOBEC4 expression, indicating a transcriptional control mechanism. Kataegis, a further mutational pattern associated with APOBEC deregulation, is seen at the sites of the MYC translocation. The APOBEC mutational signature seen in myeloma is, therefore, associated with poor prognosis primary and secondary translocations and the molecular mechanisms involved in generating them.

Multiple myeloma (MM) is characterized by osteolytic bone lesions (OBL) that arise as a consequence of osteoblast inactivation and osteoclast activation adjacent to tumor foci within bone. Wnt signaling in osteoblasts regulates osteoclastogenesis through the differential activation and inactivation of Receptor Activator of Nuclear factor Kappa B Ligand (RANKL) and osteoprotegerin (OPG), positive and negative regulators of osteoclast differentiation, respectively. We demonstrate here that MM cell-derived DKK1, a soluble inhibitor of canonical Wnt signaling, disrupted Wnt3a-regulated OPG and RANKL expression in osteoblasts. Confirmed in multiple independent assays, we show that pretreatment with rDKK1 completely abolished Wnt3a-induced OPG mRNA and protein production by mouse and human osteoblasts. In addition, we show that Wnt3a-induced OPG expression was diminished in osteoblasts cocultured with a DKK1-expressing MM cell line or primary MM cells. Finally, we show that bone marrow sera from 21 MM patients significantly suppressed Wnt3a-induced OPG expression and enhanced RANKL expression in osteoblasts in a DKK1-dependent manner. These results suggest that DKK1 may play a key role in the development of MM-associated OBL by directly interrupting Wnt-regulated differentiation of osteoblasts and indirectly increasing osteoclastogenesis via a DKK1-mediated increase in RANKL-to-OPG ratios. (Blood. 2008;112:196-207) IntroductionBone destruction, a cardinal feature of multiple myeloma (MM), results from an uncoupling of osteoclast and osteoblast activities adjacent to intramedullary tumor foci. [1][2][3] Osteoclasts are activated by binding of the receptor activator of nuclear factor kappa B ligand (RANKL) 416 to its cognate receptor, RANK, whereas osteoprotegerin (OPG), 7 a soluble member of the tumor necrosis receptor super-family, acts as a naturally occurring decoy receptor that competes with RANK for binding of RANKL. 8 The balance of these 2 molecules plays a critical role in the control of osteoclastogenesis. MM cells likely stimulate expression of RANKL and suppress expression of OPG by osteoblasts or their progenitors. 9,10 Increased serum levels of RANKL and decreased levels of OPG have been associated with a poor prognosis in MM. 11 Restoring the RANKL/OPG imbalance by RANKL antagonist or recombinant OPG not only reduces MM-associated bone lesions but also halts disease progression in animal models. 10,[12][13][14] Mechanistically, regulation of osteoclastogenesis by osteoblast-derived OPG [15][16][17]18,19 involves Wnt signaling, a pathway that is regulated by a large number of antagonists, including the Dickkopf family, 20 secreted frizzled-related proteins (sRFPs), 21,22 and sclerostin. 23 DKK1 blocks maturation of osteoblasts and formation of mineralized matrix by antagonizing the canonical Wnt pathway through it binding to LRP5/6 and Kremen. [24][25][26][27][28] Germ line inactivating mutations in the Wnt coreceptor LRP5 causes the osteoporosis-pseudoglioma syndrome (OPPG), 29 whereas a high bone m...

Wnts comprise a family of secreted proteins that interact with receptors consisting of a Frizzled (Fz) family member alone or complexed with LDL receptor-related proteins (LRP5/6). Wnt signaling plays a crucial role in both development and differentiation, and activation of a 'canonical' Wnt pathway resulting in b-catenin stabilization is associated with several types of human cancers. To date, little is known about potential Wnt signaling in mature lymphocytes or lymphoid neoplasia. Herein, we have analysed Wnt signaling in mature B cells (lymphomas) and plasma cells (multiple myeloma). Both Fz and LRP5/6 mRNAs were expressed in myeloma lines, but LRP5/6 were not observed in lymphomas. In myelomas, a canonical Wnt signaling pathway was activated following treatment with Wnt-3a as assessed by accumulation of bcatenin, but b-catenin levels actually decreased in lymphoma cells. Wnt-3a treatment further led to striking morphological changes in myeloma cells accompanied by rearrangement of the actin cytoskeleton. Morphological changes were associated with a second Wnt pathway dependent on Rho activation. These results suggest that Wnt responsiveness is a stage-specific phenomenon in Bcell development and that the morphological changes associated with Wnt signaling may play a role in the motility and metastatic potential of myeloma cells.

In multiple myeloma cells, insulinlike growth factor-I (IGF-I) activates 2 distinct signaling pathways, mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI-3K), leading to both proliferative and antiapoptotic effects. However, it is unclear through which of these cascades IGF