Aberrant Wnt Signaling in Multiple Myeloma: Molecular Mechanisms and Targeting Options

Leukemia (2019) 33:1063–1075 https://doi.org/10.1038/s41375-019-0404-1

REVIEW ARTICLE

Multiple myeloma gammopathies Aberrant Wnt signaling in multiple myeloma: molecular mechanisms and targeting options

1,2 1,2 1,2 1,2 Harmen van Andel ● Kinga A. Kocemba ● Marcel Spaargaren ● Steven T. Pals

Received: 17 October 2018 / Revised: 18 January 2019 / Accepted: 21 January 2019 / Published online: 15 February 2019 © The Author(s) 2019. This article is published with open access

Abstract Aberrant activation of Wnt/β-catenin signaling plays a central role in the pathogenesis of a wide variety of malignancies and is typically caused by mutations in core Wnt pathway components driving constitutive, ligand-independent signaling. In multiple myelomas (MMs), however, these pathway intrinsic mutations are rare despite the fact that most tumors display aberrant Wnt pathway activity. Recent studies indicate that this activation is caused by genetic and epigenetic lesions of Wnt regulatory components, sensitizing MM cells to autocrine Wnt ligands and paracrine Wnts emanating from the bone marrow niche. These include deletion of the tumor suppressor CYLD, promotor methylation of the Wnt antagonists WIF1, DKK1, DKK3, and sFRP1, sFRP2, sFRP4, sFRP5, as well as overexpression of the co-transcriptional activator BCL9 and the R-

1234567890();,: 1234567890();,: spondin receptor LGR4. Furthermore, Wnt activity in MM is strongly promoted by interaction of both Wnts and R-spondins with syndecan-1 (CD138) on the MM cell-surface. Functionally, aberrant canonical Wnt signaling plays a dual role in the pathogenesis of MM: (I) it mediates proliferation, migration, and drug resistance of MM cells; (II) MM cells secrete Wnt antagonists that contribute to the development of osteolytic lesions by impairing osteoblast differentiation. As discussed in this review, these insights into the causes and consequences of aberrant Wnt signaling in MM will help to guide the development of targeting strategies. Importantly, since Wnt signaling in MM cells is largely ligand dependent, it can be targeted by drugs/antibodies that act upstream in the pathway, interfering with Wnt secretion, sequestering Wnts, or blocking Wnt (co)receptors.

Introduction genes, including Cyclin D1 (CCND1,11q13), FGF3/ MMSET (4p16.3), Cyclin D3 (CCND3, 6p21), MAFC Multiple myeloma (MM) is a clonal expansion of malignant (16q23), and MAFB (20q11). Most of the remaining cases plasma cells in the bone marrow (BM) that is typically are hyperdiploid, with recurrent trisomies of chromosomes accompanied by paraproteinemia, lytic bone lesions, 3, 5, 7, 9, 11, 15, 19, and 21 [2, 3]. Despite this hetero- hypercalcemia, cytopenias, and renal failure. It is preceded geneity, all these primary genetic events are believed to by monoclonal gammopathy of undetermined significance drive cell cycle entry by deregulating D-type cyclins, a (MGUS), an asymptomatic premalignant accumulation of unifying pathogenic event crucial for malignant transfor- clonal plasma cells that shares genetic features with MM mation of plasma cells [4]. In addition, multiple secondary [1]. In ~50% of MGUS and MM patients, recurrent trans- genetic and epigenetic abnormalities have been identified locations involving the immunoglobulin heavy chain (IgH) that drive MM progression. These include general hypo- locus are found, which drive the expression of juxtaposed methylation, gene-specific hypermethylation, mutations in KRAS, NRAS, BRAF, and P53, genetic and epigenetic abnormalities in NF kappa B and Wnt pathway components, and genetic aberrations in the MYC family of onco- * Steven T. Pals genes [2, 3, 5]. Despite the broad landscape of genetic and [email protected] epigenetic abnormalities, virtually all MM tumors are 1 Department of Pathology, Academic Medical Center, University strictly dependent on the BM microenvironment, or niche, of Amsterdam, Amsterdam, The Netherlands for growth and survival [6, 7]. 2 Lymphoma and Myeloma Center Amsterdam (LYMMCARE), The MM microenvironment consists of various extra- Amsterdam, The Netherlands cellular matrix components and cell types, including BM 1064 H. van Andel et al. stromal cells, osteoblasts, osteoclasts, and endothelial Canonical Wnt signaling revolves around the stabili- cells. These cells secrete factors such as interleukin(IL)-6, zation and subcellular localization of the transcriptional insulin-like growth factor (IGF), hepatocyte growth factor coactivator β-catenin (Fig. 1). In the absence of Wnt (HGF) and a proliferation-induced ligand (APRIL), which ligands, β-catenin is continuously phosphorylated by a collectively provide signals essential for growth and sur- destruction complex that includes CK1α, AXIN, APC, vival [6, 8]. Both normal and malignant plasma cells are and GSK3β,whichmarksβ-catenin for ubiquitination and highly decorated with the heparan sulfate proteoglycan proteasomal degradation (Fig. 1, left panel). Engagement (HSPG) syndecan-1, which facilitates communication of a Wnt ligand to one of its cognate Fzd receptors with the BM niche by binding and presenting numerous initiates phosphorylation of the Wnt coreceptors LRP5/6, secreted factors and promoting signal transduction and thereby creating a docking site for AXIN (Fig. 1,right adhesion [9–11]. During disease progression, MM cells panel). Subsequent translocation of AXIN to the plasma continuously interact with and shape the microenviron- membrane disrupts the destruction complex and allows ment to favor tumor growth. This disrupts BM home- stabilization and nuclear translocation of non- ostasis, resulting in cytopenias and lytic bone phosphorylated β-catenin [12, 13]. In cooperation with lesions. Interestingly, the canonical Wnt signaling path- TCF/LEF family transcription factors [16, 17] and other way plays a dual role in the reciprocal interaction between transcriptional coactivators, such as BCL9 [18], this MM cells and the BM niche: (I) the BM microenviron- orchestrates a transcriptional program comprising multi- ment facilitates aberrant activation of canonical Wnt sig- ple targets including MYC and CCND1 (encoding Cyclin naling in MM cells, and thereby plays an important role in D1) [19, 20]. tumorigenesis; (II) MM cells secrete Wnt antagonists In contrast to canonical Wnt signaling, non-canonical which contribute to the development of lytic bone Wnt signaling is independent of LRP5/6 and β-catenin lesions by impairing osteoblast differentiation. In this and plays an important role in regulating cell polarity, review, we examine the causes and biological con- adhesion, and migration. In Wnt/PCP signaling, engage- sequences of aberrant Wnt signaling activity in MM ment of a Wnt ligand to a Fzd receptor results in activa- cells and discuss possible strategies to target the Wnt tion of the small GTPase RhoA and downstream protein pathwayinMM. kinases, including Rho-associated protein kinase (ROCK), which regulates cytoskeletal dynamics by dic- tating the localization of structural proteins such as actin The Wnt signaling pathway [21–23]. In the Wnt/Ca2+ pathway, binding of a Wnt ligand to its receptor results in the release of calcium ions The Wnt cascade represents a highly conserved develop- from the endoplasmic reticulum (ER) via the activation of mental signal-transduction pathway involved in a variety G-proteins, phospholipase C (PLC), and phosphodiester- of cellular processes, including regulation of proliferation, ase (PDE). In turn, elevation of intracellular calcium cell-fate, migration, and cell polarity. There are 19 Wnt levels activates enzymes such as protein kinase C (PKC), genes in the human genome which encode lipid-modified resulting in altered cell motility [24]. secreted glycoproteins, acting as ligands for their cognate Deregulation of canonical Wnt signaling plays a central Frizzled (FZD) receptors. Wnts are relatively unstable and role in malignant transformation and tumorigenesis in a insoluble due to their hydrophobic nature, which con- variety of tissues. This is exemplified by the intestinal strains long-range signaling. As a consequence, they act epithelium. In this archetypal Wnt-dependent tissue Wnt as typical niche or stem cell factors [12, 13]. The lipid signaling is required for stem cell maintenance and, when modification of Wnt proteins involves covalent attach- hyperactivated, is the main driver of malignant transfor- ment of a palmitoyl group, appended by the palmitoyl- mation. Oncogenic Wnt signaling typically results from transferase Porcupine (encoded by PORC), and is required mutations in core pathway components such as APC, β- for signaling and secretion [14, 15]. Wnt signals can be catenin, and AXIN, causing constitutive, ligand- transduced in distinct ways; by a well-defined “canonical” independent signaling [25]. In addition, more recent evi- Wnt/β-catenin pathway, or by either of two β-catenin dence indicates that genetic and epigenetic alterations that independent “non-canonical” Wnt signaling cascades, increase the sensitivity of tumor cells to Wnt ligands can designated the Wnt/PCP and Wnt/Ca2+ signaling path- equally drive tumorigenesis. This is exemplified by genetic way. Whereas some members of the Wnt ligand and Fzd alterations in the ZNRF3/RNF43/R-spondin/LGR axis. receptor families are strictly dedicated to either canonical ZNRF3 and RNF43 are homologous membrane-bound E3 or non-canonical signaling, most Wnts and Fzds are ubiquitin ligases that attenuate signaling by inducing Wnt promiscuously involved in both pathways. In this review, (co)receptor internalization. Their action is antagonized by we will mainly focus on canonical Wnt signaling. leucine-rich repeat-containing G protein-coupled receptor Aberrant Wnt signaling in multiple myeloma: molecular mechanisms and targeting options 1065

Off State On State

RSPO WNT sFRP sFRP

RSPO WNT

ZNRF3/ ZNRF3/ LGR4 FZD LRP5/6 LGR4 FZD LRP5/6 RNF43 RNF43 ub DVL p p AXIN internalization DVL internalization CK1 GSK APC ub AXIN CK1 GSK APC CYLD CYLD β-catenin p β-catenin p proteasomal β-catenin β-catenin p p degradation β-catenin

X BCL9 PYGO β-catenin CCND1 BCL9 MYC PYGO TCF TCF AURKA

Fig. 1 Schematic representation of canonical Wnt signaling. Off state which decreases protein stability. On state (right panel): Binding of a (left panel): In the absence of Wnt ligands, β-catenin is continuously Wnt ligand to its receptor Frizzled induces phosphorylation of the co- phosphorylated by a destruction complex that includes AXIN, APC, receptors LRP5/6, which forms a docking site for AXIN. Subsequent GSK3β, and CK1α, which marks it for proteasomal degradation. In sequestration of AXIN disrupts the destruction complex and allows addition, Wnt signaling is antagonized at multiple levels. First, the stabilization and nuclear translocation of non-phosphorylated β-cate- secreted Wnt inhibitors sFRP and DKK1 prevent activation of Wnt nin. In cooperation with the TCF/LEF family of transcription factors signaling by sequestering Wnt ligands or preventing LRP5/6 phos- and the co-transcriptional activators Pygopus (PYGO) and BCL9, this phorylation, respectively. Second, in the absence of LGR4/R-spondin orchestrates transcription of Wnt target genes. In addition, LGR4/R- signaling, the E3 ubiquitin ligases ZNRF3 and RNF43 antagonize Wnt spondin signaling facilitates signaling by Wnt ligands. Engagement of activity by ubiquitinating Wnt (co)receptors, which induces inter- R-spondin to its receptor LGR4 induces internalization of ZNRF3/ nalization and subsequent degradation. Lastly, the deubiquitinase RNF43, thereby alleviating the negative regulatory role of these E3 CYLD impairs intracellular signal transduction by removing Lys-63- ligases on Wnt receptor stability linked polyubiquitin chains from the adapter protein Disheveled (Dvl),

(LGR)-family receptors in response to cognate R-spondin et al. [5] demonstrating that most primary MMs (pMMs) ligands [26–29] (Fig. 1). Inactivating mutations in ZNRF3/ and human MM cell lines (HMCLs), unlike normal RNF43, as well as gene fusions of R-spondin causing plasma cells, express non-phosphorylated, i.e. active, β- overexpression, have recently been identified in a number of catenin in the nucleus. Moreover, inhibition of Wnt sig- Wnt-driven tumors, including colorectal cancer (CRC) and naling by ectopic expression of a dominant negative TCF4 pancreatic ductal adenocarcinoma [30–32]. These genetic mutant (dnTCF4) in HMCLs suppressed proliferation, alterations render the tumor cells highly sensitive to Wnt implying functional involvement of β-catenin/TCF-medi- ligands by stabilizing Wnt (co-)receptors on the plasma ated transcription. Corroborating these results, other stu- membrane. Importantly, since Wnt activation in tumor cells dies showed that siRNA [33]andshRNA[34] mediated carrying these mutations is still ligand-dependent, it can be silencing of β-catenin or treatment of HMCLs with the targeted by drugs that interfere with Wnt secretion, small molecule Wnt/β-catenin inhibitors PKF115-584 sequester Wnts, or block Wnt receptors. [35], AV-65 [36], BC2059 [37], CGK012 [38], and the FDA-approved Pyrvinium pamoate [39, 40] attenuated cell cycle progression, impaired tumor growth, and Aberrant canonical Wnt signaling in MM markedly increased apoptosis of HMCLs both in vitro and cells in vivo. In addition to attenuating proliferation, silencing of β-catenin increased the sensitivity of the tumor cells to Initial evidence for a role of canonical Wnt signaling in multiple drugs used in the treatment of MM, in particular the pathogenesis of MM came from a study by Derksen to lenalidomide [34, 41]. Gene-expression analysis after 1066 H. van Andel et al. silencing of β-catenin [34] and treatment with PKF115- (Dvl), and members of the PKC family, in particular 584 [35]andCGK012[38] showed downregulation of the PKCμ [50]. In addition, non-canonical Wnt signaling in Wnt target genes CCND1 (encoding cyclin D1) and MYC, MM cells enhanced integrin-mediated heterotypic cell which are known to play a key role in the pathogenesis of adhesion, resulting in cell-adhesion-mediated drug resis- MM [4, 42, 43]. In addition to these established Wnt tance (CAM-DR) of MM cells to doxorubicin [51]. Col- targets, several other genes involved in cell cycle reg- lectively, these studies identified a role of non-canonical ulation were also downregulated, including Aurora Kinase Wnt signaling in regulating migration, dissemination and A(AURKA), which was identified as an important effector CAM-DR of MM cells. of Wnt-driven proliferation in MM [34]. While these data collectively provided solid evidence for a functional role of canonical Wnt/β-catenin signaling in MM pathogen- Paracrine and autocrine Wnt signaling esis, the mechanisms underlying aberrant Wnt activation activation in MM remained incompletely understood. Oncogenic Wnt signaling is typically driven by tumor- Wnts are produced in the BM microenvironment and have cell intrinsic mutations in Wnt pathway components. For been implicated in hematopoietic stem cell (HSC) main- this review, we analyzed the mutational status of a number tenance [52] and early B cell development [53]. Several of Wnt pathway components in published MM datasets and studies have modeled paracrine signaling by activating identified pathogenic mutations in APC, CTNNB1 (encod- Wnt signaling in MM cells with either exogenous Wnt ing β-catenin) and RNF43 that are recurrently found in other ligands or the GSK3 inhibitors lithium chloride (LiCl) and Wnt-driven tumors (Table 1)[44–48]. Furthermore, a 6-bromoindirubin-3-oxomine (BIO) in vitro. These treat- number of mutations were identified of which the patho- ments invariably induced β-catenin stabilization and Wnt genic significance is currently unknown (Table 1). How- reporter activity, indicating that MM cells are well ever, these Wnt pathway mutations occur at low frequency equipped to respond to Wnt ligands. While several studies (3% overall), indicating that alternative mechanisms drive found that Wnt3a-conditioned medium, recombinant illegitimate Wnt activation in the majority of MMs. As Wnt3a or GSK3 inhibition enhanced proliferation of discussed below, several lines of evidence indicate that HMCLs [5, 37], others did not observe this positive effect paracrine Wnts emanating from the BM microenvironment on tumor growth [54–56]. This might be explained by the and/or autocrine Wnts, in concert with loss of negative Wnt different cell lines and compounds used and the pro- pathway regulators, fuel Wnt pathway activation in malig- miscuous role of GSK3 in the regulation of multiple nant plasma cells. signal transduction pathways [57]. Moreover, it should be noted that accumulating evidence in CRC and HSC supports a ‘just-right’ model of Wnt activation, which pro- Non-canonical Wnt signaling in MM cells poses that each individual (tumor) cell has an optimal threshold of Wnt activation [58–60]. This implies that The first evidence for a role of non-canonical Wnt sig- different levels of Wnt activation can have distinct, even naling in MM came from a study by Qiang et al. [49], opposite, cellular effects. Notably, most MMs display demonstrating that Wnt3a induces striking morphological hallmarks of Wnt pathway activation in the absence of changes in MM cells by regulating cytoskeleton dynam- exogenously added Wnts, suggesting that autocrine Wnts ics. Whereas Wnt3a treatment resulted in activation of might be important drivers. Indeed, gene expression both canonical and non-canonical Wnt signaling, these analysis revealed expression of various Wnts (including morphological changes could be blocked by sequestration Wnt3,4,5A,5B,6,7,8A,10A,10B,11,14and16)in of Wnt ligands with sFRP-1 and inhibition of non- pMMs and HMCLs [5, 61, 62]. Furthermore, blocking canonical Wnt signaling by a small molecule inhibitor of Wnt secretion with the small molecule porcupine inhibi- Rho-associated kinases, but not by DKK-mediated inhi- tors IWP-2 and LGK974 [63] or opposing binding of Wnt bition of canonical Wnt signaling. Moreover, activation of ligands to Fzd with secreted Frizzled protein 1 (sFRP1) canonical Wnt signaling by the GSK inhibitor LiCl did [62] decreased β-catenin stabilization, attenuated β-cate- not induce these morphological alterations. This indicated nin/TCF-mediated transcription and impaired expansion that the observed cellular changes were strictly mediated of several HMCLs, confirming the existence of an auto- by non-canonical Wnt signaling. Subsequent studies from crine Wnt signaling loop. Taken together, these data the same lab showed that activation of non-canonical Wnt indicate that aberrant Wnt activation in malignant plasma signaling also enhanced migration and invasion of MM cells can be fueled by autocrine and paracrine Wnt ligands cells, which involved activation of RhoA, Disheveled and regulates proliferation and drug resistance of MM Aberrant Wnt signaling in multiple myeloma: molecular mechanisms and targeting options 1067

Table 1 Overview of genetic and epigenetic abnormalities in MM that induce or facilitate canonical Wnt activation Gene CDS change AA change Mutation type Clinical signiﬁcance Recurrence Frequency

Classic Wnt pathway mutations [44–48] APC nr S1465fs Frameshift Pathogenic Recurrent in CRC 1/133 APC 6976C>G R2326G Missense Likely pathogenic Germline mutation in 1/84 FAP APC 718A>T T240S Missense Unknown Not previously 1/84 reported APC 5369G>A R1790K Missense Unknown Not previously 1/14 reported APC 497insAa T166fs Frameshift Pathogenic Not previously nr reported AXIN1 2089C>T P697S Missense Likely pathogenic Sporadic in HCC 1/203 AXIN2 944C>T T315M Missense Likely pathogenic Sporadic in GC 1/203 CTNNB1 nr T41I Missense Pathogenic Recurrent in CRC and 1/133 PC RSPO/LGR4/RNF43/ZNRF3 mutations [46] RSPO2 219G>T E73D Missense Unknown Not previously 1/203 reported RSPO2 257G>A R86Q Missense Likely pathogenic Recurrent in CRC and 1/203 PC RNF43 1813G>T A605S Missense Unknown Not previously 1/203 reported RNF43 1976delG G659fs Frameshift Pathogenic Recurrent in CRC and 1/203 PC ZNRF3 2419G>C A807P Missense Unknown Not previously 1/203 reported ZNRF3 68T>G V23G Missense Unknown Not previously 1/203 reported Promotor methylation of secreted Wnt antagonists [61, 62, 65] Abnormalities in LGR4 [63] Gene pMM HMCLs mRNA overexpression 86% (30/35) APC 18% (9/50) 25% (1/4) WIF 22% (11/50) 50% (2/4) Abnormalities in CYLD [44– 48, 68–70] DKK1 33% (3/12) 67% (4/6) Mutation 3% (22/744) DKK3 16% (8/50) 50% (2/4) Deletion 17% (nr/nr) sFRP1 27% (33/123) 78% (7/9) 16q deletion 35% (40/114) sFRP2 52% (38/73) 56% (5/9) sFRP3/FRZB nd nd Abnormalities in BCL9 [69, 72, 78–80] sFRP4 7% (8/123) 56% (5/9) mRNA overexpression 60% (38/64) sFRP5 6% (7/123) 67% (6/9) Low miR30 expression 60% (47/78) 1q gain 36% (192/530) Clinical signiﬁcance and recurrence is based on previous publications on these speciﬁc mutations and/or occurrence of these mutations in the Catalog of Somatic Mutations in Cancer (COSMIC) database (www.cancer.sanger.uc.uk) CDS coding DNA sequence, AA amino acid, nr not reported aPatient with germline mutation in APC (attenuated form of familial adenomatous polyposis) that developed MM

cells by inducing expression of Wnt target genes, such as MM that reinforce this Wnt pathway activation, either by CCND1, MYC,andAURKA. As discussed below, several directly enhancing signaling by Wnt ligands or by facil- genetic and epigenetic alterations have been identiﬁed in itating intracellular signal transduction. 1068 H. van Andel et al.

and was associated with advanced disease stage (Table 1 mutated / deleted DKK1 overexpressed SOST and Fig. 2). Over 60% of these patients displayed epigenetic epigenetically silenced silencing of multiple Wnt antagonist [61, 62, 65]. Reverting WIF sFRP Wnt inhibitor this promotor methylation by the demethylating agent decitabine resulted in re-expression of these antagonists rosmantuzumab ipafricept vantictumab and mitigated Wnt signaling, demonstrating that their WNT silencing indeed facilitates Wnt activation in MM cells. In RSPO WNT addition to epigenetic silencing of Wnt antagonists, aberrant ZNRF3/ expression of the R-spondin receptor LGR4 was identiﬁed LGR4 FZD LRP5/6 RNF43 as a cause of increased responsiveness of MM cells to Wnt ub DVL p p ligands [63]. Analysis of LGR4 expression revealed it is

CYLD AXIN expressed by the majority of MMs, but not by normal CK1 GSK APC plasma cells (Table 1). LGR4 is transcriptionally regulated XAV939 by IL-6/STAT3 signaling and allows MM cells to respond IWR endoplasmic G007/G244-LM Pyrvinium to R-spondins, which are produced in the BM micro- reticulum WNT environment by cells of the osteoblast lineage [63]. β-catenin Engagement of R-spondin by LGR4 alleviates Wnt (co) LGK974 β-catenin ETC-159 receptor internalization by ZNRF/RNF43, resulting in sta- β-catenin PORC C59 β-catenin IWP-2 bilization of Wnt (co)receptors and greatly increased sen- – BC2059 sitivity of tumor cells to Wnt ligands [26 29] (Fig. 1, right panel). Accordingly, while Wnt ligands alone only moder- WNT palmitoyl ately activate Wnt signaling in HMCLs, addition of recombinant R-spondin or (pre)osteoblast-conditioned PRI-724 media dramatically enhances signaling by both autocrine ICG-001 PYGO and paracrine Wnt ligands. Moreover, silencing of LGR4 in β-catenin CCND1 SAH-BCL9 BCL9 TBP MYC HMCLs secreting autocrine Wnt ligands impaired pro- PKF115-584 TCF AURKA liferation in vitro, indicating functional involvement of LGR4 in mediating Wnt-driven tumorigenesis [63]. Interestingly, we recently reported that syndecan-1 Fig. 2 Schematic overview of genetic and epigenetic abnormalities in Wnt pathway components in MM and drugs that target canonical Wnt (CD138) acts as a key mediator of both R-spondin/ signaling. Aberrant canonical Wnt signaling pathway activation in LGR4 signaling and signaling by Wnt ligands in MM [66]. MM cells is facilitated by epigenetic silencing (blue), overexpression Syndecan-1 is a HSPG that is strongly expressed by normal (green), or mutation (yellow) of multiple Wnt pathway components. and malignant plasma cells and consists of a core protein Notably, a variety of antibodies, small molecule, and peptides have been recently developed that target Wnt signaling at multiple distinct decorated with covalently bound heparan sulfate chains that levels (red, see also Table 2) can promiscuously bind and present a variety of growth factors and cytokines [9–11]. Silencing of the enzyme exostosin 1 (EXT1), which is required for heparan sulfate Epigenetic silencing of Wnt antagonists, chain synthesis, mitigated binding of both Wnts and R- aberrant expression of LGR4 and HSPG spondins to MM cells and attenuated Wnt pathway activa- facilitate ligand-induced Wnt signaling tion. Moreover, silencing of EXT1 attenuated the growth of in MM MM tumors both in vitro and in vivo, which could be rescued by ectopic expression of a constitutive active β- Gene-specific promotor methylation is frequently observed catenin mutant or by c-MYC expression, indicating invol- in MM and can contribute to tumorigenesis by selectively vement of canonical Wnt signaling [66, 67]. and reversibly suppressing transcription of tumor suppressor genes, such as p16/INK4a (encoded by CDKN2A) [64]. Interestingly, epigenetic silencing of core Wnt path- Deletion of the tumor suppressor CYLD, way components and/or of secreted Wnt antagonists is sumoylation of β-catenin, and frequently present in MM and likely plays a key role in overexpression the oncogene BCL9 facilitating Wnt activation. Hypermethylation of the pro- hyperactivates Wnt signaling MM motor of APC or at least one of the secreted Wnt antagonists WIF1, DKK1, DKK3, and sFRP1, sFRP2, sFRP4, and The above described epigenetic alterations enhance the sFRP5 was detected in the majority of pMMs and HMCLs sensitivity of MM cells to Wnt ligands by affecting Aberrant Wnt signaling in multiple myeloma: molecular mechanisms and targeting options 1069 membrane proximal regulators of the Wnt signaling cas- malignant plasma cells [79]. In functional studies, shRNA- cade. However, aberrant expression of intracellular Wnt mediated knockdown of BCL9 in HMCLs suppressed Wnt pathway regulators CYLD and BCL9 can also strongly signaling and attenuated tumor growth in vitro and in vivo. promote Wnt signaling in MM. Loss of the tumor sup- In addition, BCL9 silencing decreased the migration of pressor CYLD, by gene deletion and/or inactivating muta- tumor cells and suppressed expression of vascular endo- tions, is among the most common genetic abnormalities in thelial growth factor (VEGF) [79]. Similar results were MM and is associated with a poor prognosis [46, 47, 68–72] obtained by suppressing BCL9 function with ectopic (Table 1). CYLD is a deubiquitinase that decreases stability expression of miR-30–5p [79] or treatment with a BCL9- of target proteins by specifically removing Lys63-linked derived peptide that specifically disrupts the BCL9/β–cate- polyubiquitin chains, and acts as a negative regulator of NF nin interaction [80], supporting the notion that BCL9 kappa B and Wnt signaling [73–75]. In Wnt signaling, overexpression mediates Wnt-driven tumorigenesis in MM. CYLD targets and destabilizes the adapter protein Dvl, In addition to BCL9, mucin1-C (MUC1-C) was recently which impairs signal transduction (Fig. 1). Gene-expression also reported to act as transcriptional co-activator in Wnt analysis in pMM revealed a negative correlation between signaling, increasing β-catenin occupancy on the MYC CYLD mRNA expression and the strength of a Wnt target promoter and forming a complex with β-catenin and TCF4, gene signature. In addition, gene-set-enrichment analysis thereby driving MYC transcription [81]. showed enrichment of Wnt pathway genes in CYLD low Collectively, the above-described studies support a compared to CYLD high MM patients. These data suggest model in which loss of Wnt antagonists or aberrant that loss of CYLD facilitates Wnt signaling in MM cells. expression of positive Wnt regulators sensitizes MM cells to Indeed, functional studies demonstrated that silencing of autocrine and paracrine Wnt ligands. This results in CYLD in MM cells enhances β-catenin stabilization and β- hyperactivation of canonical Wnt signaling and enhances catenin/TCF-mediated transcription, while reintroduction of proliferation, migration, and drug resistance of MM cells. CYLD in MM cells that display a bi-allelic loss of the CYLD locus suppresses Wnt activity and impairs proliferation. Thus, loss of CYLD can promote Wnt pathway Wnt signaling in MM bone disease activation and drive proliferation in MM [71]. In addition to ubiquitination, sumoylation has also been Osteolytic lesions are present in the vast majority of MM implicated in the deregulation of canonical Wnt signaling in patients and are frequently accompanied by highly debili- MM [76]. Increased protein sumoylation has been pre- tating symptoms such as bone pain and pathological frac- viously observed in MM and is associated with adverse tures. Current evidence indicates that MM cells induce lytic patient outcome [77]. siRNA-mediated silencing of SUMO1 bone disease by secreting paracrine factors that both insti- in MM cells enhanced β-catenin phosphorylation and gate osteoclastic bone resorption and impair bone formation decreased β-catenin-mediated transcription. Moreover, this by attenuating osteoblast differentiation [82]. was accompanied by decreased proliferation and increased Canonical Wnt signaling is required for osteoblast apoptosis of MM cells, which could be rescued by ectopic development and plays a key role in bone homeostasis. expression of a constitutive active β-catenin mutant. These This is illustrated by rare hereditary diseases characterized results indicate that sumoylation of β-catenin can enhance by increased or decreased bone mass. Loss of the Wnt/β-catenin signaling in MM cells [76]. Similar to CYLD osteocyte-specific secreted Wnt antagonist sclerostin (Van deletion, aberrant expression of the transcriptional coacti- Buchem disease or sclerosteosis) or mutations in the Wnt vator BCL9 has also been linked to hyperactivation of the coreceptor LRP5, which prevent binding of the Wnt Wnt pathway in MM. BCL9 functions by recruiting the antagonists sclerostin and DKK1, have been shown to transcriptional coactivator Pygopus to the β-catenin/TCF cause increased bone mass. Conversely, loss-of-function complex, which is required for transcription of Wnt target mutations of LRP5 have been identified in osteoporosis- genes [18]. Gene-expression analysis revealed that BCL9 is pseudoglioma syndrome (OPPG), which is characterized overexpressed in approximately 60% of MMs, while BCL9 by decreased bone mass [83]. A landmark study by Tian expression is negligible in normal plasma cells (Table 1). et al. [84] identified secretion of the Wnt antagonist Amplification of chromosome 1q, which contains the BCL9 DKK1 by MM cells as a key mechanism underlying MM locus, is recurrently observed in MM. However, 1q gain did bone disease. Gene-expression analysis revealed that most not significantly correlate to BCL9 mRNA expression, MMs express DKK1, in contrast to normal plasma cells or suggesting that other mechanisms drive BCL9 expression MGUS. DKK1 expression strongly correlated with the [72, 78]. Indeed, loss of microRNA miR30–5p, which tar- presence of lytic bone lesions. Functional studies indi- gets the 3′UTR of BCL9, has recently been identified as cated that DKK1-mediated inhibition of canonical Wnt important mechanism underlying BCL9 overexpression in signaling in osteoblast contributes to the development of 1070 H. van Andel et al. osteolytic lesions by attenuating osteoblast differentiation bone marrow Wnt [84, 85]. MM In addition to DKK1, the secreted Wnt inhibitors sFRP2 [86], sFRP3/FRZB [87, 88] and sclerostin [89, 90] have also been reported to be overexpressed in MM. Of note, only DKK1 and sFRP3/FRZB expression levels were sig- IL-6 nificantly correlated to the presence of lytic bone disease BAFF DKK1 MSC/ [91]. Intriguingly, expression of DKK1 as well as of other R-spondin sFRPs osteoblast Wnt Sclerostin osteoblast secreted Wnt antagonists is largely restricted to early dis- progenitor ... Wnt signaling ease stages. During disease progression, expression of these Wnt antagonists is frequently lost due to promoter methylation. This suggests that Wnt antagonists exert disease bone stage-specific functions [61, 62, 65]. Following the identi- fi Fig. 3 Schematic representation of inhibition of osteoblast differ- cation of secreted Wnt antagonists as drivers of lytic bone entiation by MM-derived Wnt antagonists. The MM-derived secreted disease, the therapeutic potential of activating Wnt signaling Wnt antagonists DKK1, sFRP, and sclerostin attenuate Wnt signaling in osteoblasts was studied in various murine in vivo MM in osteoblast precursors, which impairs osteoblasts differentiation and models. Stimulation of Wnt signaling in the tumor micro- contributes to the development of osteolytic lesions. Inhibition of osteoblast differentiation creates a tumor permissive environment, environment either by ectopic expression of Wnt3a in since osteoblast precursors secrete high levels of IL-6, BAFF, and HMCLs [54] or by systemic administration of Wnt3a, other cytokines and growth factors. In addition, immature osteoblasts GSK3-inhibitors [56] or an anti-DKK1 monoclonal anti- secrete R-spondin, which protects LGR4-expressing MM cells against body (mAb) [92, 93] was found to increase osteoblast secreted Wnt antagonists by enhancing sensitivity of tumor cells to autocrine and paracrine Wnt ligands numbers, enhance bone formation, and prevent the development of osteolytic lesions. Collectively, these results provided strong evidence for a key role of deregulated Wnt Conclusions and therapeutic perspectives signaling in the development of osteolytic lesions. Inter- estingly, some studies reported that the increase in bone As discussed in this review, current evidence indicates that formation in these models was accompanied by a decrease canonical Wnt signaling plays a dual and disease stage- in tumor burden, which seems to contradict the notion that specific role in the pathogenesis of MM. At early disease Wnt activation drives proliferation of MM cells. However, stages when MM cells are strictly confined to the BM, they additional experiments revealed that the GSK3 inhibitor secrete various Wnt antagonists, which attenuate osteoblast LiCl only decreased tumor growth in the BM, while the differentiation and thereby contribute to the development of growth of subcutaneously inoculated MM cells was osteolytic lesions. Since osteoblast precursors secrete high enhanced [56]. Ectopic expression of dnTCF4 in the MM levels of cytokines and growth factors that promote MM cells blocked the LiCl-induced growth of subcutaneous cell growth and survival this creates a tumor permissive tumors, confirming that it was indeed driven by Wnt sig- environment (Fig. 3). During MM disease progression, the naling. Furthermore, ectopic expression of Wnt3a in MM secreted Wnt antagonists are subject to epigenetic silencing, cells or treatment of MM-bearing mice with Wnt3a [54]or which facilitates Wnt pathway activation and Wnt-driven with anti-DKK1 mAbs [92, 93] was reported to decrease proliferation of MM cells. growth of intramedullary MMs but did not suppress the Oncogenic Wnt activity is typically driven by mutations growth of subcutaneously inoculated MMs. However, in in key Wnt pathway components, but these pathway these cases no growth acceleration of subcutaneous tumors intrinsic mutations are rare in MM (Table 1). Instead, Wnt was observed. These data suggest that MM-derived Wnt pathway activation in MM is largely fueled by autocrine antagonists indirectly enhance tumorigenesis by suppres- and paracrine Wnt ligands and promoted by epigenetic sing osteoblast differentiation. Consistent with this scenario, silencing of secreted Wnt antagonist, overexpression of the it was reported that osteoblasts precursors secrete significant co-receptor LGR4 and the transcriptional co-activator higher levels of IL-6, IL-10, BAFF, HGF, and VEGF, BCL9, expression of HSPGs, sumoylation of β-catenin suggesting that suppression of osteoblast differentiation and mutation/deletion of the negative regulator CYLD enhances tumor growth by creating a tumor permissive (Table 1 and Fig. 2). This indicates that illegitimate Wnt microenvironment [55, 92, 94] (Fig. 3). Of note, we activation in MM results from ‘releasing the breaks’ rather recently reported that immature osteoblasts also secrete high than ‘hitting the gas’. Aberrant Wnt pathway activation in levels of R-spondin, which may protect LGR4-expressing MM cells mediates proliferation by inducing expression of MM cells against Wnt inhibition by secreted Wnt cell cycle genes, such as CCND1, MYC, and AURKA and antagonist [63]. causes drug resistance. Of note, deregulation of the cell Aberrant Wnt signaling in multiple myeloma: molecular mechanisms and targeting options 1071

Table 2 Overview of monoclonal antibodies, peptides, and small molecule inhibitors that target canonical Wnt signaling Name Target Study phase Trial number

Monoclonal antibodies/peptides Vantictumab (OMP-18R5) [99] Frizzled receptors Phase I NCT01973309, NCT01345201, NCT02005315, NCT0195700784 Ipafricept (OMP-54F28) [100] Wnt ligands (Fzd8-Fc) Phase I NCT02092363, NCT02050178, NCT02069145, NCT01608867 Rosmantuzumab (OMP-131R10) R-spondin 3 Phase I NCT02482441 [101] SAH-BCL9 [80] BCL9/β-catenin interaction Preclinical Small molecules LGK974 [30, 102] Porcupine (Wnt secretion) Phase I NCT01351103, NCT02278133 ETC-159 [103] Porcupine (Wnt secretion) Phase I NCT02521844 C59 [104] Porcupine (Wnt secretion) Preclinical IWP-2 [105] Porcupine (Wnt secretion) Preclinical XAV939 [106] Tankyrase (Axin Preclinical stabilization) IWR [105] Tankyrase (Axin Preclinical stabilization) G007/G244-LM [97] Tankyrase (Axin Preclinical stabilization) Pyrvinium [40] CK1α activation Preclinicala BC2059 [37] β-catenin Phase I NCT03459469 PKF115–584 [35] TCF/β–catenin interaction Preclinical ICG-001 [108] CBP/β–catenin interaction Preclinical PRI-724 [109] CBP/β–catenin interaction Phase I/II NCT01764477, NCT01606579 AV-65 [36] Unknown Preclinical CGK012 [38] Unknown Preclinical aPyrvinium is FDA-approved as an anti-helminthic drug cycle is a unifying pathogenic event required for malignant MM pathogenesis. Activation of this signaling cascade transformation of plasma cells [4]. involves signaling true RhoA/ROCK and PKC family Given the role of DKK1 in the development of lytic bone members, and mediates migration, invasion, and disease, a mAb targeting DKK1 has been developed and has adhesion of MM cells, resulting in tumor dissemination and been tested in clinical trials. Initial reported results indicate CAM-DR. that anti-DKK1 treatment is well tolerated; thus far effects The central role of the Wnt pathway in a wide variety of on MM bone disease and disease outcome have not been malignancies has led to the development and (pre)clinical reported [95]. Importantly, anti-DKK1 treatment will likely testing of many monoclonal antibodies, and small molecule not only increase Wnt signaling in osteoblasts but also in inhibitors, targeting the Wnt signaling cascade at multiple the tumor cells. Furthermore, apart from DKK-1 MM cells distinct levels (Fig. 2 and Table 2)[30, 80, 99–109]. express a number of additional Wnt antagonists that will not Despite the fact that Wnt signaling is essential for many be targeted using this strategy. Since DKK1 and other Wnt homeostatic functions, these drugs have proven to be sur- antagonists present bonafide transcriptional feedback targets prisingly well tolerated. Importantly, the Wnt signaling of Wnt signaling in many tissues, it will be important to cascade in MM is essentially intact and Wnt activation is determine if they are also transcriptionally regulated by Wnt largely ligand dependent. This implies that Wnt signaling signaling in MM (see also wnt.stanford.edu) [96–98]. If so, can be targeted by drugs that act on membrane proximal in targeting Wnt signaling in MM cells may not only decrease the pathway, such as Porcupine inhibitors interfering with proliferation and drug resistance, but also suppress secretion secretion of Wnts, or mAbs and small molecules/peptides of Wnt antagonist, thus preventing osteoblast inhibition and that sequester Wnts or block Wnt receptors. In addition, MM bone disease. given its central role in regulating the sensitivity of tumor In addition to canonical Wnt signaling, several studies cells to Wnt ligands, blocking LGR4/R–spondin interaction have indicated a role of non-canonical Wnt signaling in with mAbs or other compounds is a potentially attractive 1072 H. van Andel et al. strategy to target Wnt signaling in MM (Fig. 2 and Table 2). 7. Kawano Y, Moschetta M, Manier S, Glavey S, Görgün GT, Furthermore, targeting the BCL9/β-catenin complex pre- Roccaro AM, et al. Targeting the bone marrow microenviron- – sents a potentially promising therapeutic strategy to inhibit ment in multiple myeloma. Immunol Rev. 2015;263:160 72. 8. Chen Z, Orlowski RZ, Wang M, Kwak L, McCarty N. Osteo- canonical Wnt signaling in MM cells, since BCL9 is blastic niche supports the growth of quiescent multiple myeloma exclusively expressed by malignant plasma cells but not by cells. Blood. 2014;123:2204–8. normal plasma cells and many other cell types [10]. Since 9. Reijmers RM, Spaargaren M, Pals ST. Heparan sulfate pro- most MM display hallmarks of Wnt activation, irrespective teoglycans in the control of B cell development and the pathogenesis of multiple myeloma. FEBS J. 2013;280:2180–93. of the genetic background, targeting the Wnt pathway is 10. Derksen PW, Keehnen RM, Evers LM, van Oers MH, Spaar- potentially of benefit for a large group of MM patients. garen M, Pals ST. Cell surface proteoglycan syndecan-1 med- Further studies are required to define the best strategy for iates hepatocyte growth factor binding and promotes Met – targeting canonical Wnt signaling in MM, possibly by signaling in multiple myeloma. Blood. 2002;99:1405 10. 11. Sanderson RD, Yang Y. Syndecan-1: a dynamic regulator uncoupling Wnt signaling in MM cells from Wnt activation of the myeloma microenvironment. Clin Exp Metastas-. in the BM niche. 2008;25:149–59. 12. Clevers H, Loh KM, Nusse R. Stem cell signaling. An integral Acknowledgements Supported by grants from the Dutch Cancer program for tissue renewal and regeneration: Wnt signaling and Society and Lymph&Co. stem cell control. Science. 2014;346:1248012. 13. Nusse R, Clevers H. Wnt/β-catenin signaling, disease, and – Compliance with ethical standards emerging therapeutic modalities. Cell. 2017;169:985 99. 14. Willert K, Brown JD, Danenberg E, Duncan AW, Weissman IL, Reya T, et al. Wnt proteins are lipid-modified and can act as stem Conflict of interest The authors declare that they have no conflict of cell growth factors. Nature. 2003;423:448–52. interest. 15. Kadowaki T, Wilder E, Klingensmith J, Zachary K, Perrimon N. The segment polarity gene porcupine encodes a putative multi- Publisher’s note: Springer Nature remains neutral with regard to transmembrane protein involved in Wingless processing. Genes jurisdictional claims in published maps and institutional affiliations. Dev. 1996;10:3116–28. 16. Behrens J, von Kries JP, Kühl M, Bruhn L, Wedlich D, Gros- schedl R, et al. Functional interaction of beta-catenin with the Open Access This article is licensed under a Creative Commons transcription factor LEF-1. Nature. 1996;382:638–42. Attribution 4.0 International License, which permits use, sharing, 17. Molenaar M, van de Wetering M, Oosterwegel M, Peterson- adaptation, distribution and reproduction in any medium or format, as Maduro J, Godsave S, Korinek V, et al. XTcf-3 transcription long as you give appropriate credit to the original author(s) and the factor mediates beta-catenin-induced axis formation in Xenopus source, provide a link to the Creative Commons license, and indicate if embryos. Cell . 1996;86:391–9. changes were made. The images or other third party material in this 18. Kramps T, Peter O, Brunner E, Nellen D, Froesch B, Chatterjee article are included in the article’s Creative Commons license, unless S, et al. Wnt/wingless signaling requires BCL9/legless-mediated indicated otherwise in a credit line to the material. If material is not recruitment of pygopus to the nuclear beta-catenin-TCF com- included in the article’s Creative Commons license and your intended plex. Cell . 2002;109:47–60. use is not permitted by statutory regulation or exceeds the permitted 19. He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa use, you will need to obtain permission directly from the copyright LT, et al. Identification of c-MYC as a target of the APC path- holder. To view a copy of this license, visit http://creativecommons. way. Science. 1998;281:1509–12. org/licenses/by/4.0/. 20. Tetsu O, McCormick F. Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature. 1999;398:422–6. 21. Fujisawa K, Fujita A, Ishizaki T, Saito Y, Narumiya S. Identi- References fication of the Rho-binding domain of p160ROCK, a Rho- associated coiled-coil containing protein kinase. J Biol Chem. 1996;271:23022–8. 1. Palumbo A, Anderson K. Multiple myeloma. N Engl J Med. 22. Klingensmith J, Nusse R, Perrimon N. The Drosophila segment 2011;364:1046–60. polarity gene dishevelled encodes a novel protein required for 2. Manier S, Salem KZ, Park J, Landau DA, Getz G, Ghobrial IM. response to the wingless signal. Genes Dev. 1994;8:118–30. Genomic complexity of multiple myeloma and its clinical 23. Strutt DI, Weber U, Mlodzik M. The role of RhoA in tissue implications. Nat Rev Clin Oncol. 2017;14:100–13. polarity and frizzled signalling. Nature. 1997;387:292–5. 3. Morgan GJ, Walker BA, Davies FE. The genetic architecture of 24. Kühl M, Sheldahl LC, Park M, Miller JR, Moon RT. The Wnt/ multiple myeloma. Nat Rev Cancer. 2012;12:335–48. Ca2+pathway: a new vertebrate Wnt signaling pathway takes 4. Bergsagel PL, Kuehl WM, Zhan F, Sawyer J, Barlogie B, shape. Trends Genet. 2000;16:279–83. Shaughnessy J, et al. Cyclin D dysregulation: an early and unifying 25. Clevers H, Nusse R. Wnt/beta-catenin signaling and disease. Cell pathogenic event in multiple myeloma. Blood . 2005;106:296–303. . 2012;149:1192–205. 5. Derksen PW, Tjin E, Meijer HP, Klok MD, MacGillavry HD, 26. de Lau W, Barker N, Low TY, Koo BK, Li VS, Teunissen H, van Oers MH, et al. Illegitimate WNT signaling promotes pro- et al. Lgr5 homologues associate with Wnt receptors and mediate liferation of multiple myeloma cells. Proc Natl Acad Sci USA. R-spondin signalling. Nature. 2011;476:293–7. 2004;101:6122–7. 27. Carmon KS, Gong X, Lin Q, Thomas A, Liu Q. R-spondins 6. Hideshima T, Mitsiades C, Tonon G, Richardson PG, Anderson function as ligands of the orphan receptors LGR4 and LGR5 to KC. Understanding multiple myeloma pathogenesis in the bone regulate Wnt/beta-catenin signaling. Proc Natl Acad Sci USA. marrow to identify new therapeutic targets. Nat Rev Cancer. 2011;108:11452–7. 2007;7:585–98. Aberrant Wnt signaling in multiple myeloma: molecular mechanisms and targeting options 1073

28. Hao HX, Xie Y, Zhang Y, Charlat O, Oster E, Avello M, et al. reduces myeloma sensitivity to single-agent bortezomib therapy. ZNRF3 promotes Wnt receptor turnover in an R-spondin- Blood. 2014;123:632–9. sensitive manner. Nature. 2012;485:195–200. 46. Lohr JG, Stojanov P, Carter SL, Cruz-Gordillo P, Lawrence MS, 29. Koo BK, Spit M, Jordens I, Low TY, Stange DE, van de Wetering Auclair D, et al. Widespread genetic heterogeneity in multiple M, et al. Tumour suppressor RNF43 is a stem-cell E3 ligase that myeloma: implications for targeted therapy. Cancer Cell. induces endocytosis of Wnt receptors. Nature. 2012;488:665–9. 2014;25:91–101. 30. Jiang X, Hao HX, Growney JD, Woolfenden S, Bottiglio C, Ng 47. Bolli N, Avet-Loiseau H, Wedge DC, Van Loo P, Alexandrov N, et al. Inactivating mutations of RNF43 confer Wnt depen- LB, Martincorena I, et al. Heterogeneity of genomic evolution dency in pancreatic ductal adenocarcinoma. Proc Natl Acad Sci and mutational profiles in multiple myeloma. Nat Commun. USA. 2013;110:12649–54. 2014;5:2997. 31. Wu J, Jiao Y, Dal Molin M, Maitra A, de Wilde RF, Wood LD, 48. Bolli N, Li Y, Sathiaseelan V, Raine K, Jones D, Ganly P, et al. et al. Whole-exome sequencing of neoplastic cysts of the pancreas A DNA target-enrichment approach to detect mutations, copy reveals recurrent mutations in components of ubiquitin-dependent number changes and immunoglobulin translocations in multiple pathways. Proc Natl Acad Sci USA. 2011;108:21188–93. myeloma. Blood Cancer J. 2016;6:e467. 32. Seshagiri S, Stawiski EW, Durinck S, Modrusan Z, Storm EE, 49. Qiang YW, Endo Y, Rubin JS, Rudikoff S. Wnt signaling in B- Conboy CB, et al. Recurrent R-spondin fusions in colon cancer. cell neoplasia. Oncogene. 2003;22:1536–45. Nature. 2012;488:660–4. 50. Qiang YW, Walsh K, Yao L, Kedei N, Blumberg PM, Rubin JS, 33. Ashihara E, Kawata E, Nakagawa Y, Shimazaski C, Kuroda J, et al. Wnts induce migration and invasion of myeloma plasma Taniguchi K, et al. Beta-catenin small interfering RNA suc- cells. Blood. 2005;106:1786–93. cessfully suppressed progression of multiple myeloma in a 51. Kobune M, Chiba H, Kato J, Kato K, Nakamura K, Kawano Y, mouse model. Clin Cancer Res. 2009;15:2731–8. et al. Wnt3/RhoA/ROCK signaling pathway is involved in 34. Dutta-Simmons J, Zhang Y, Gorgun G, Gatt M, Mani M, adhesion-mediated drug resistance of multiple myeloma in an Hideshima T, et al. Aurora kinase A is a target of Wnt/beta- autocrine mechanism. Mol Cancer Ther. 2007;6:1774–84. catenin involved in multiple myeloma disease progression. 52. Reya T, O’Riordan M, Okamura R, Devaney E, Willert K, Nusse Blood. 2009;114:2699–708. R, et al. Wnt signaling regulates B lymphocyte proliferation 35. Sukhdeo K, Mani M, Zhang Y, Dutta J, Yasui H, Rooney MD, through a LEF-1 dependent mechanism. Immunity. 2000;13:15–24. et al. Targeting the beta-catenin/TCF transcriptional complex in 53. Reya T, Duncan AW, Ailles L, Domen J, Scherer DC, Willert K, the treatment of multiple myeloma. Proc Natl Acad Sci USA. et al. A role for Wnt signalling in self-renewal of haematopoietic 2007;104:7516–21. stem cells. Nature. 2003;423:409–14. 36. Yao H, Ashihara E, Strovel JW, Nakagawa Y, Kuroda J, Nagao 54. Qiang YW, Shaughnessy JD Jr, Yaccoby S. Wnt3a signaling R, et al. AV-65, a novel Wnt/β-catenin signal inhibitor, suc- within bone inhibits multiple myeloma bone disease and tumor cessfully suppresses progression of multiple myeloma in a mouse growth. Blood. 2008;112:374–82. model. Blood Cancer J. 2011;1:e43. 55. Gunn WG, Conley A, Deininger L, Olson SD, Prockop DJ, 37. Savvidou I, Khong T, Cuddihy A, McLean C, Horrigan S, Gregory CA. A crosstalk between myeloma cells and marrow Spencer A. β-catenin inhibitor BC2059 is efficacious as mono- stromal cells stimulates production of DKK1 and interleukin-6: therapy or in combination with proteasome inhibitor bortezomib a potential role in the development of lytic bone disease and in multiple myeloma. Mol Cancer Ther. 2017;16:1765–78. tumor progression in multiple myeloma. Stem Cells. 38. Choi PJ, OY, Her JH, Yun E, Song GY, Oh, et al. Anti- 2006;24:986–91. proliferative activity of CGK012 against multiple myeloma 56. Gunn WG, Krause U, Lee N, Gregory CA. Pharmaceutical cells via Wnt/β-catenin signaling attenuation. Leuk Res. inhibition of glycogen synthetase kinase-3β reduces multiple 2017;60:103–8. myeloma-induced bone disease in a novel murine plasmacytoma 39. Thorne CA, Hanson AJ, Schneider J, Tahinci E, Orton D, Cse- xenograft model. Blood. 2011;117:1641–51. lenyi CS, et al. Small-molecule inhibition of Wnt signaling 57. Cohen P, Frame S. The renaissance of GSK3. Nat Rev Mol Cell through activation of casein kinase 1α. Nat Chem Biol. Biol. 2001;2:769–76. 2010;6:829–36. 58. Albuquerque C, Breukel C, van der Luijt R, Fidalgo P, Lage P, 40. Xu F, Zhu Y, Lu Y, Yu Z, Zhong J, Li Y, et al. Anthelmintic Slors FJ, et al. The ‘just-right’ signaling model: APC somatic pyrvinium pamoate blocks Wnt/β-catenin and induces apoptosis mutations are selected based on a specific level of activation of in multiple myeloma cells. Oncol Lett. 2018;15:5871–8. the beta-catenin signaling cascade. Hum Mol Genet. 41. Bjorklund CC, Ma W, Wang ZQ, Davis RE, Kuhn DJ, Kornblau 2002;11:1549–60. SM, et al. Evidence of a role for activation of Wnt/beta-catenin 59. Christie M, Jorissen RN, Mouradov D, Sakthianandeswaren A, signaling in the resistance of plasma cells to lenalidomide. J Biol Li S, Day F, et al. Different APC genotypes in proximal and Chem. 2011;286:11009–20. distal sporadic colorectal cancers suggest distinct WNT/β-catenin 42. Dechow T, Steidle S, Götze KS, Rudelius M, Behnke K, signalling thresholds for tumourigenesis. Oncogene. Pechloff K, et al. GP130 activation induces myeloma and col- 2013;32:4675–82. laborates with MYC. J Clin Invest. 2014;124:5263–74. 60. Luis TC, Ichii M, Brugman MH, Kincade P, Staal FJ. Wnt 43. Selvanayagam P, Blick M, Narni F, van Tuinen P, Ledbetter signaling strength regulates normal hematopoiesis and its DH, Alexanian R, et al. Alteration and abnormal expression deregulation is involved in leukemia development. Leukemia. of the c-myc oncogene in human multiple myeloma. Blood. 2012;26:414–21. 1988;71:30–5. 61. Kocemba KA, Groen RW, van Andel H, Kersten MJ, Mahtouk 44. Tao H, Shinmura K, Yamada H, Maekawa M, Osawa S, K, Spaargaren M, et al. Transcriptional silencing of the Wnt- Takayanagi Y, et al. Identification of 5 novel germline APC antagonist DKK1 by promoter methylation is associated with mutations and characterization of clinical phenotypes in Japanese enhanced Wnt signaling in advanced multiple myeloma. PLoS patients with classical and attenuated familial adenomatous One. 2012;7:e30359. polyposis. BMC Res Notes. 2010;3:305. 62. Chim CS, Pang R, Fung TK, Choi CL, Liang R. Epigenetic 45. Mulligan G, Lichter DI, Di Bacco A, Blakemore SJ, Berger A, dysregulation of Wnt signaling pathway in multiple myeloma. Koenig E, et al. Mutation of NRAS but not KRAS significantly Leukemia. 2007;21:2527–36. 1074 H. van Andel et al.

63. van Andel H, Ren Z, Koopmans I, Joosten SP, Kocemba KA, de 80. Takada K, Zhu D, Bird GH, Sukhdeo K, Zhao JJ, Mani M, Lau W, et al. Aberrantly expressed LGR4 empowers Wnt sig- et al. Targeted disruption of the BCL9/β-catenin complex naling in multiple myeloma by hijacking osteoblast-derived R- inhibits oncogenic Wnt signaling. Sci Transl Med. spondins. Proc Natl Acad Sci USA. 2017;114:376–81. 2012;4:148ra117. 64. Gonzalez-Paz N, Chng WJ, McClure RF, Blood E, Oken MM, 81. Tagde A, Rajabi H, Bouillez A, Alam M, Gali R, Bailey S, et al. Van Ness B, et al. Tumor suppressor p16 methylation in multiple MUC1-C drives MYC in multiple myeloma. Blood. myeloma: biological and clinical implications. Blood. 2016;127:2587–97. 2007;109:1228–32. 82. O’Donnell EK, Raje NS. Myeloma bone disease: pathogenesis 65. Jost E, Gezer D, Wilop S, Suzuki H, Herman JG, Osieka R, et al. and treatment. Clin Adv Hematol Oncol. 2017;15:285–95. Epigenetic dysregulation of secreted frizzled-related proteins in 83. Baron R, Kneissel M. WNT signaling in bone homeostasis and multiple myeloma. Cancer Lett. 2009;281:24–31. disease: from human mutations to treatments. Nat Med. 66. Ren Z, van Andel H, de Lau W, Hartholt RB, Maurice MM, 2013;19:179–92. Feb Clevers H, et al. Syndecan-1 promotes Wnt/β-catenin signaling 84. Tian E, Zhan F, Walker R, Rasmussen E, Ma Y, Barlogie B, in multiple myeloma by presenting Wnts and R-spondins. Blood. et al. The role of the Wnt-signaling antagonist DKK1 in the 2018;131:982–94. development of osteolytic lesions in multiple myeloma. N Engl J 67. Reijmers RM, Groen RW, Rozemuller H, Kuil A, de Haan- Med. 2003;349:2483–94. Kramer A, Csikós T, et al. Targeting EXT1 reveals a crucial role 85. Heath DJ, Chantry AD, Buckle CH, Coulton L, Shaughnessy JD for heparan sulfate in the growth of multiple myeloma. Blood. Jr, Evans HR, et al. Inhibiting Dickkopf-1 (Dkk1) removes 2010;115:601–4. suppression of bone formation and prevents the development of 68. Walker BA, Leone PE, Chiecchio L, Dickens NJ, Jenner MW, osteolytic bone disease in multiple myeloma. J Bone Miner Res. Boyd KD, et al. A compendium of myeloma-associated chro- 2009;24:425–36. mosomal copy number abnormalities and their prognostic value. 86. Oshima T, Abe M, Asano J, Hara T, Kitazoe K, Sekimoto E, Blood. 2010;116:e56–65. et al. Myeloma cells suppress bone formation by secreting a 69. Walker BA, Boyle EM, Wardell CP, Murison A, Begum DB, soluble Wnt inhibitor, sFRP-2. Blood. 2005;106:3160–5. Dahir NM, et al. Mutational spectrum, copy number changes, 87. Davies FE, Dring AM, Li C, Rawstron AC, Shammas MA, and outcome: results of a sequencing study of patients with O’Connor SM, et al. Insights into the multistep transformation of newly diagnosed myeloma. J Clin Oncol. 2015;33:3911–20. MGUS to myeloma using microarray expression analysis. Blood. 70. Keats JJ, Fonseca R, Chesi M, Schop R, Baker A, Chng WJ, 2003;102:4504–11. et al. Promiscuous mutations activate the noncanonical NF- 88. De Vos J, Couderc G, Tarte K, Jourdan M, Requirand G, Delteil kappaB pathway in multiple myeloma. Cancer Cell. MC, et al. Identifying intercellular signaling genes expressed in 2007;12:131–44. malignant plasma cells by using complementary DNA arrays. 71. van Andel H, Kocemba KA, de Haan-Kramer A, Mellink CH, Blood. 2001;98:771–80. Piwowar M, Broijl A, et al. Loss of CYLD expression unleashes 89. Colucci S, Brunetti G, Oranger A, Mori G, Sardone F, Specchia Wnt signaling in multiple myeloma and is associated with G, et al. Myeloma cells suppress osteoblasts through sclerostin aggressive disease. Oncogene. 2017;36:2105–15. secretion. Blood Cancer J. 2011;1:e27. 72. Carrasco DR, Tonon G, Huang Y, Zhang Y, Sinha R, Feng B, 90. McDonald MM, Reagan MR, Youlten SE, Mohanty ST, Seck- et al. High-resolution genomic profiles define distinct clinico- inger A, Terry RL, et al. Inhibiting the osteocyte-specific protein pathogenetic subgroups of multiple myeloma patients. Cancer sclerostin increases bone mass and fracture resistance in multiple Cell. 2006;9:313–25. myeloma. Blood. 2017;129:3452–64. 73. Brummelkamp TR, Nijman SM, Dirac AM, Bernards R. Loss of 91. Kristensen IB, Christensen JH, Lyng MB, Møller MB, Pedersen the cylindromatosis tumour suppressor inhibits apoptosis by L, Rasmussen LM, et al. Expression of osteoblast and osteoclast activating NF-kappaB. Nature. 2003;424:797–801. regulatory genes in the bone marrow microenvironment in 74. Kovalenko A, Chable-Bessia C, Cantarella G, Israël A, Wallach multiple myeloma: only up-regulation of Wnt inhibitors SFRP3 D, Courtois G. The tumour suppressor CYLD negatively reg- and DKK1 is associated with lytic bone disease. Leuk Lym- ulates NF-kappaB signalling by deubiquitination. Nature. phoma. 2014;55:911–9. 2003;424:801–5. 92. Fulciniti M, Tassone P, Hideshima T, Vallet S, Nanjappa P, 75. Tauriello DV, Haegebarth A, Kuper I, Edelmann MJ, Henraat M, Ettenberg SA, et al. Anti-DKK1 mAb (BHQ880) as a potential Canninga-van Dijk MR, et al. Loss of the tumor suppressor therapeutic agent for multiple myeloma. Blood. 2009;114:371–9. CYLD enhances Wnt/beta-catenin signaling through K63-linked 93. Yaccoby S, Ling W, Zhan F, Walker R, Barlogie B, Shaugh- ubiquitination of Dvl. Mol Cell. 2010;37:607–19. nessy JD Jr.. Antibody-based inhibition of DKK1 suppresses 76. Huang HJ, Zhou LL, Fu WJ, Zhang CY, Jiang H, Du J, et al. β- tumor-induced bone resorption and multiple myeloma growth catenin SUMOylation is involved in the dysregulated prolifera- in vivo. Blood. 2007;109:2106–11. tion of myeloma cells. Am J Cancer Res. 2014;5:309–20. 94. Reagan MR, Ghobrial IM. Multiple myeloma mesenchymal stem 77. Driscoll JJ, Pelluru D, Lefkimmiatis K, Fulciniti M, Prabhala cells: characterization, origin, and tumor-promoting effects. Clin RH, Greipp PR, et al. The sumoylation pathway is dysregulated Cancer Res. 2012;18:342–9. in multiple myeloma and is associated with adverse patient 95. Iyer SP, Beck JT, Stewart AK, Shah J, Kelly KR, Isaacs R, et al. outcome. Blood. 2010;115:2827–34. A Phase IB multicentre dose-determination study of BHQ880 78. Mani M, Carrasco DE, Zhang Y, Takada K, Gatt ME, Dutta- in combination with anti-myeloma therapy and zoledronic Simmons J, et al. BCL9 promotes tumor progression by con- acid in patients with relapsed or refractory multiple myeloma ferring enhanced proliferative, metastatic, and angiogenic prop- and prior skeletal-related events. Br J Haematol. 2014; erties to cancer cells. Cancer Res. 2009;69:7577–86. 167:366–75. 79. Zhao JJ, Lin J, Zhu D, Wang X, Brooks D, Chen M, et al. miR- 96. Niida A, Hiroko T, Kasai M, Furukawa Y, Nakamura Y, 30-5p functions as a tumor suppressor and novel therapeutic tool Suzuki Y, et al. DKK1, a negative regulator of Wnt signaling, is by targeting the oncogenic Wnt/β-catenin/BCL9 pathway. Can- a target of the beta-catenin/TCF pathway. Oncogene. cer Res. 2014;74:1801–13. 2004;23:8520–6. Aberrant Wnt signaling in multiple myeloma: molecular mechanisms and targeting options 1075

97. González-Sancho JM, Aguilera O, García JM, Pendás-Franco N, 103. Madan B, Ke Z, Harmston N, Ho SY, Frois AO, Alam J, et al. Peña C, Cal S, et al. The Wnt antagonist DICKKOPF-1 gene is a Wnt addiction of genetically defined cancers reversed by downstream target of beta-catenin/TCF and is downregulated in PORCN inhibition. Oncogene. 2016;35:2197–207. human colon cancer. Oncogene. 2005;24:1098–103. 104. Proffitt KD, Madan B, Ke Z, Pendharkar V, Ding L, Lee MA, 98. Chamorro MN, Schwartz DR, Vonica A, Brivanlou AH, Cho et al. Pharmacological inhibition of the Wnt acyltransferase KR, Varmus HE. FGF-20 and DKK1 are transcriptional targets PORCN prevents growth of WNT-driven mammary cancer. of beta-catenin and FGF-20 is implicated in cancer and devel- Cancer Res. 2013;73:502–7. opment. EMBO J. 2005;24:73–84. 105. Chen B, Dodge ME, Tang W, Lu J, Ma Z, Fan CW, et al. 99. Gurney A, Axelrod F, Bond CJ, Cain J, Chartier C, Donigan L, Small molecule-mediated disruption of Wnt-dependent signaling et al. Wnt pathway inhibition via the targeting of Frizzled in tissue regeneration and cancer. Nat Chem Biol. 2009;5:100–7. receptors results in decreased growth and tumorigenicity of 106. Huang SM, Mishina YM, Liu S, Cheung A, Stegmeier F, human tumors. Proc Natl Acad Sci USA. 2012;109:11717–22. Michaud GA, et al. Tankyrase inhibition stabilizes axin and 100. Jimeno A, Gordon M, Chugh R, Messersmith W, Mendelson D, antagonizes Wnt signalling. Nature. 2009;461:614–20. Dupont J, et al. A first-in-human Phase I study of the anticancer 107. Lau T, Chan E, Callow M, Waaler J, Boggs J, Blake RA, et al. A stem cell agent ipafricept (OMP-54F28), a decoy receptor for novel tankyrase small-molecule inhibitor suppresses APC Wnt ligands, in patients with advanced solid tumors. Clin Cancer mutation-driven colorectal tumor growth. Cancer Res. Res. 2017;23:7490–7. 2013;73:3132–44. 101. Katoh M, Katoh M. Molecular genetics and targeted therapy of 108. Emami KH, Nguyen C, Ma H, Kim DH, Jeong KW, Eguchi M, WNT-related human diseases (review). Int J Mol Med. et al. A small molecule inhibitor of beta-catenin/CREB-binding 2017;40:587–606. protein transcription [corrected]. Proc Natl Acad Sci USA. 102. Liu J, Pan S, Hsieh MH, Ng N, Sun F, Wang T, et al. 2004;101:12682–7. Targeting Wnt-driven cancer through the inhibition of porcupine by 109. Lenz HJ, Kahn M. Safely targeting cancer stem cells via selective LGK974. Proc Natl Acad Sci Usa. 2013;110:20224–9. catenin coactivator antagonism. Cancer Sci. 2014;105:1087–92.