Aberrantly expressed LGR4 empowers Wnt signaling in multiple myeloma by hijacking osteoblast-derived R-spondins

Harmen van Andela,b, Zemin Rena,b, Iris Koopmansa,b, Sander P. J. Joostena,b, Kinga A. Kocembaa,b,c, Wim de Laud, Marie José Kerstenb,e, Alexander M. de Bruina,b, Jeroen E. J. Guikemaa,b, Hans Cleversd,1, Marcel Spaargarena,b,2, and Steven T. Palsa,b,1,2

aDepartment of Pathology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; bLymphoma and Myeloma Center Amsterdam (LYMMCARE), 1105AZ Amsterdam, The Netherlands; cDepartment of Medical Biochemistry, Jagiellonian University Medical College, 31008 Krakow, Poland; dHubrecht Institute, 3584CT Utrecht, The Netherlands; and eDepartment of Hematology, Academic Medical Center, University of Amsterdam, 1105AZ Amsterdam, The Netherlands

Contributed by Hans Clevers, November 29, 2016 (sent for review August 10, 2016; reviewed by Roeland Nusse and Karin Vanderkerken)

The unrestrained growth of tumor cells is generally attributed to pathway activation (10). Interestingly, in regulators of Wnt mutations in essential growth control , but tumor cells are also turnover, causing hypersensitivity to Wnt ligands, have re- affected by, or even addicted to, signals from the microenviron- cently been described in a variety of tumors, identifying aberrant Wnt ment. As therapeutic targets, these extrinsic signals may be equally (co)receptor turnover as an alternative mechanism driving oncogenic significant as mutated . In multiple myeloma (MM), a Wnt signaling (17–19). Turnover of Wnt (co)receptors is critically plasma cell , most tumors display hallmarks of active regulated by leucine-rich repeat-containing G -coupled recep- Wnt signaling but lack activating Wnt-pathway mutations, suggest- tor (LGR)-family receptors, which stabilize Wnt (co)receptors in re- ing activation by autocrine Wnt ligands and/or paracrine Wnts sponse to their cognate R-spondin. R-spondin/LGR signaling emanating from the bone marrow (BM) niche. Here, we report a alleviates the inhibitory effect of two homologous membrane-bound pivotal role for the R-spondin/leucine-rich repeat-containing G E3 ubiquitin ligases, ZNRF3 and RNF43, which normally induce Wnt protein-coupled receptor 4 (LGR4) axis in driving aberrant Wnt/ (co)receptor internalization (20–23). This strong positive regulatory β-catenin signaling in MM. We show that LGR4 is expressed by MM effect on Wnt-signaling pathway activation designates the LGR/ plasma cells, but not by normal plasma cells or B cells. This aberrant R-spondin axis as potentially oncogenic, a notion that prompted LGR4 expression is driven by IL-6/STAT3 signaling and allows MM us to explore its possible role in hematologic . Here, cells to hijack R-spondins produced by (pre)osteoblasts in the BM we identify aberrant LGR4/R-spondin signaling as a driver of on- niche, resulting in Wnt (co)receptor stabilization and a dramatically cogenic Wnt/β-catenin signaling in MM. increased sensitivity to auto- and paracrine Wnts. Our study iden- tifies aberrant R-spondin/LGR4 signaling with consequent deregula- Results tion of Wnt (co)receptor turnover as a driver of oncogenic Wnt/ LGR4 Is Aberrantly Expressed in MM. To gain insight into the expres- β-catenin signaling in MM cells. These results advocate targeting sion of LGR4-6 in normal hematopoiesis and in hematologic malig- of the LGR4/R-spondin interaction as a therapeutic strategy in MM. nancies, we initially analyzed publically available -expression

multiple myeloma | Wnt signaling | LGR4 | R-spondins | osteoblast Significance

ultiple myeloma (MM) in most patients is an incurable Multiple myeloma (MM) cells are highly dependent on signals Mhematologic malignancy characterized by the accumula- provided by the bone marrow (BM) niche for growth and survival. tion of clonal plasma cells in the bone marrow (BM). Despite the Most MMs display hallmarks of active Wnt signaling, but lack ac- wide variety of underlying structural and numerical genomic tivating Wnt-pathway mutations, suggesting activation by auto- abnormalities (1–3), virtually all MMs are highly dependent on a crine Wnt ligands and/or paracrine Wnts emanating from the BM protective BM microenvironment, or “niche,” for growth and niche. In this study we uncover a pivotal role for the leucine-rich survival (4). Understanding the complex reciprocal interaction repeat-containing -coupled receptor 4 (LGR4)/R-spondin between MM cells and the BM microenvironment is critical for axis in facilitating activation of Wnt signaling in MM. LGR4 is the development of new targeted therapies. expressed by most MMs, but not by healthy plasma cells, and is In MM, aberrant activation of the canonical Wnt pathway drives regulated by STAT3 signaling. LGR4 expression allows MMs to proliferation and is associated with disease progression, dissemi- respond to (pre)osteoblast–derived R-spondins, resulting in stabi- nation, and (5–9). Because MMs with hallmarks lization of Wnt receptors and greatly enhanced sensitivity to auto- of active Wnt signaling do not harbor mutations that typically and paracrine Wnt ligands. These results advocate targeting of underlie constitutive Wnt pathway activation, this oncogenic Wnt proximal Wnt signaling in MM. pathway activity was proposed to involve autocrine and/or para- crine Wnt ligands (6, 8, 10). Wnts are lipid-modified glycoproteins Author contributions: H.v.A., M.S., and S.T.P. designed research; H.v.A., Z.R., I.K., S.P.J.J., K.A.K., that function as typical niche factors because they are relatively A.M.d.B., and J.E.J.G. performed research; W.d.L. and H.C. contributed new reagents/analytic unstable and insoluble due to their hydrophobic nature, which tools; H.v.A., K.A.K., M.J.K., M.S., and S.T.P. analyzed data; and H.v.A. and S.T.P. wrote the paper. constrains long-range signaling (11). Binding of a Wnt ligand to its Reviewers: R.N., Stanford University School of Medicine; and K.V., Vrije Universiteit receptor (Fzd) initiates a signaling cascade that ultimately Brussel. results in stabilization and nuclear translocation of the Wnt ef- The authors declare no conflict of interest. fector β-catenin. In cooperation with TCF/LEF family transcrip- Freely available online through the PNAS open access option. tion factors this orchestrates a transcriptional program (12, 13), 1To whom correspondence may be addressed. Email: [email protected] or s.t.pals@ comprising targets such as MYC and CCND1 (Cyclin D1) that play amc.uva.nl. crucial roles in the pathogenesis of MM (14–16). Aberrant Wnt 2M.S. and S.T.P. contributed equally to this work. signaling in typically results from mutations in APC, β-catenin This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (CTNNB1), or AXIN that drive constitutive, ligand-independent 1073/pnas.1618650114/-/DCSupplemental.

376–381 | PNAS | January 10, 2017 | vol. 114 | no. 2 www.pnas.org/cgi/doi/10.1073/pnas.1618650114 Downloaded by guest on September 25, 2021 LGR4 (218326_s_at) silencing of STAT3 or ectopic expression of a dominant *** negative STAT3 mutant (DN-STAT3), significantly decreased 600 both baseline and IL-6–induced LGR4 expression (Fig. 3B and Fig. 400 S3 D and E). Taken together, these findings indicate that STAT3 signaling instigates aberrant LGR4 expression in MM. 250 R-spondins Potentiate Wnt Signaling in MM in a LGR4-Dependent 200 Manner. Functionally, R-spondin binding to LGR4 facilitates Wnt 150 pathway activation by stabilization of Wnt (co)receptors, thereby increasing the sensitivity to Wnt ligands without activating Wnt 100 signaling itself (20, 21). To establish whether the LGR4/R-spondin

microarray signal axis is instrumental in regulating Wnt signaling in MM cells, we 50 initially assessed phosphorylation of the serine 1490 residue of low- 0 density lipoprotein receptor-related protein 6 (LRP6) (Fig. S4A), which sequesters Axin to the plasma membrane (30), and the s s ls s ls ls L L L L Cs el FL MM consequent stabilization and nuclear localization of β-catenin (Fig. ast last c AM CML ALL AL CL bl cyte cel T- B- B cells ro ab BMP T T DLBC 4A). As expected, stimulation with Wnt3a alone induced LRP6 e ro B Cel ïv nt ry 4+ 8+ phosphorylation and nuclear translocation of β-catenin in all MM Cent Na Ce mo Plasm CD CD cell lines tested (Fig. 4A and Fig. S4A). However, stimulation with Me R-spondin alone did not result in Wnt pathway activation in the majority of cell lines (Fig. 4A and Fig. S4A), but modestly increased Fig. 1. LGR4 expression in normal hematopoiesis and hematologic malignan- cies. Analysis of LGR4 mRNA expression in publically available microarray data- Wnt signaling in LME-1 (a 3.1-fold increase in pLRP6 and a 1.4-fold sets. AML: acute myeloid leukemia; B-ALL: B-cell acute lymphoblastic leukemia; BMPC: bone marrow plasma cells; CLL: chronic lymphoid leukemia; CML: chronic myeloid leukemia; DLBCL: diffuse large B-cell lymphoma; FL: follicular lymphoma; 1.25 T-ALL: T-cell acute lymphoblastic leukemia. Data were normalized by global A scaling to 100. ***P ≤ 0.001 using one-way ANOVA with Bonferroni correction. 1.00

0.75 datasets. In normal bone marrow and subsets, as well as in most hematologic malignancies, LGR4-6 mRNA was vir- 0.50

tually undetectable (Fig. 1 and Fig. S1A). Interestingly, however, mRNA expression LGR4 expression was present in the majority of primary MMs 0.25

(pMMs) (Fig. 1) at levels comparable to those in intestinal tissues LGR4 0.00 (Fig. S1B), in which the role of LGR/R-spondin signaling is well 1 6 1 L 63 GC ast M3 established(24).Nosignificant differences in LGR4 expression were bl U266U XG XG3 L3 Naïve pMM1pMM2pMM3pMM4 ANB LME- HEK293 Pre-GC Memory observed between MMs from previously defined molecular sub- RPMI8226 NCI-H929 groups (Fig. S1C) (25). Notably, no significant expression of LGR5 or B Plasma LGR6 was detected in MM samples (Fig. S1D). Quantitative PCR hBMPC#1 hBMPC#2 hBMPC#3 hBMPC#4 (qPCR) analysis confirmed LGR4 mRNA expression in most human multiple myeloma cell lines (HMCLs) and pMMs (Fig. 2A). In contrast, LGR4 mRNA was virtually absent in normal B-cell subsets, + + − i.e., naive B cells (CD19 /IgD /CD38 ), pregerminal center B cells + + + + (pGC, CD19 /IgD /CD38 ), germinal center B cells (GC, CD19 / − + + − − isotype IgD /CD38 ), memory B cells (CD19 /IgD /CD38 ), and plas- pMM#1 pMM#2 pMM#3 pMM#4 + − LGR4 mablasts (CD19 /IgD /CD38hi) isolated from tonsils (Fig. 2A). No LGR5 and LGR6 mRNA was detected in pMM, HMCLs, and developing B cells by qPCR (Fig. S2A). In agreement with the mRNA data, flow cytometric analysis using an LGR4-specific monoclonal antibody (mAb) (Fig. S2B) revealed that LGR4 protein is expressed on the majority of pMMs and HMCLs (Fig. 2 LGR4 B–D) but, importantly, not on human bone marrow-derived cells (hBMPCs) (Fig. 2B) or normal B-cell subsets (Fig. S2C). LGR5 ** XG-1 ANBL-6 protein expression was not detected in pMMs and HMCLs (Fig. C 1500 D S2D). These findings indicate that LGR4 expression in MM is a hallmark of malignant transformation and not a reflection of 1000 differentiation status or lineage commitment.

LGR4 Expression Is Transcriptionally Regulated by STAT3 Signaling. 500 STAT3 signaling is almost invariably activated in MM as a result LGR4 isotype

– MeanGeom LGR4 of of auto- or paracrine stimulation by IL-6 (26 28) and was recently CAS9-control shown to regulate LGR4 expression in osteosarcoma (29). We 0 CAS9-sgLGR4 therefore investigated whether STAT3 signaling also mediates hBMPCs MM MEDICAL SCIENCES aberrant LGR4 expression in MM. As expected, IL-6 treatment Fig. 2. LGR4 is expressed in MM plasma cells, but not in normal plasma cells or of the HMCL XG-1 and pMM cells induced phosphorylation of B-cell subsets. (A) LGR4 mRNA expression in HEK293T cells, normal B-cell subsets the tyrosine 705 residue of STAT3, indicating pathway activation (n = 5), pMMs (n = 4), and HMCLs (n = 9), analyzed by qPCR and normalized to (Fig. S3 A and B). Interestingly, IL-6 treatment of HMCLs and TBP.(B) LGR4 protein expression in hBMPCs and pMMs. (C) Quantification of pMM cells and ectopic expression of a constitutively active (CA) geometric mean of LGR4 FACS stainings in hBMPC (n = 4) and MM (n = 12) STAT3 mutant in XG-1 induced robust LGR4 mRNA and pro- samples. Unpaired Student’s t-test with Mann–Whitney correction was used for tein expression (Fig. 3 and Fig. S3 B and C). Conversely, inhi- statistical analysis; **P ≤ 0.01. (D) LGR4 protein expression in control transduced bition of STAT3 signaling, either by inducible shRNA-mediated (black) or CRISPR/CAS9-mediated LGR4 KO HMCLs (red).

van Andel et al. PNAS | January 10, 2017 | vol. 114 | no. 2 | 377 Downloaded by guest on September 25, 2021 A pMM1 pMM2 pMM3 production. Wnt secretion is dependent on palmitoylation by the porcupine enzyme, encoded by the PORC gene (31, 32), and 3 5 *** recently developed small-molecule porcupine inhibitors (IWP-2, ** 4 LGK974) efficiently block this Wnt secretion (18, 33, 34). Both porcupine inhibitors strongly inhibited the robust stabilization of 2 3 β-catenin and Wnt reporter activation observed upon stimula- expression expression tion of LME-1 with R-spondin alone (Fig. 4C and Fig. S5B), thus 2 1 confirming the existence of an autocrine Wnt loop.

mRNA Taken together, these results demonstrate that R-spondins facili- 1 R4 tate auto- and paracrine Wnt signaling in MM in an LGR4-de- LGR4 mRNA 0 LG 0 pendent manner and suggest that Wnt (co)receptor levels are limiting - IL-6 - IL-6 - IL-6 in establishing high levels of Wnt-pathway activation in MM cells.

iso iso iso β - IL-6 - IL-6 - IL-6 Inhibition of Proximal Wnt/ -Catenin Signaling Impairs Proliferation + IL-6 + IL-6 + IL-6 in a Subset of HMCLs. Downstream inhibition of Wnt/β-catenin signaling by a dominant negative form of TCF4 (dnTCF4) and shRNA-mediated silencing of β-catenin, BCL9-like , or small-molecule Wnt inhibitors has been previously shown to impair MM proliferation and expansion (6, 7, 9, 35). To confirm and extend these data, we expressed dnTCF4 in the HMCLs LME-1, RIES, and XG-1 from a bicistronic vector containing a LGR4 LGR4 LGR4 fluorescent marker. Cells expressing dnTCF4 were rapidly out- B competed by cells that did not express dnTCF4 and incorporated less BrdU, confirming a role for Wnt signaling in MM cell ex- iso iso iso pansion (Fig. 5A and Fig. S6 A and B). Interestingly, in LME-1 - IL-6 ctrl ctrl + IL-6 CA- DN- and RIES, inhibition of Wnt secretion by small-molecule por- ST3 ST3 cupine inhibitors also impaired growth (Fig. 5B), whereas XG-1 cells were unaffected (Fig. S6C), suggesting that the LME-1 and RIES cell lines require autocrine Wnt secretion. Indeed, LME-1 cells displayed detectable levels of autocrine Wnt signaling (Fig. 4 and Fig. S5B) whereas RIES cells were found to express ex- traordinarily high levels of LGR4 (Fig. S6D). As shown in Fig. LGR4 LGR4 LGR4 5C, doxycycline-inducible, shRNA-mediated silencing of LGR4 (Fig. S6D) significantly impaired expansion of LME-1 and RIES Fig. 3. LGR4 expression in MM is under transcriptional control of STAT3 (Fig. 5C), whereas silencing LGR4 in XG-1 had no apparent signaling. (A) qPCR for LGR4 mRNA (Top) and flow cytometry analysis of effect on cell growth (Fig. S6E). Taken together, these data indicate LGR4 (Bottom) in three pMMs treated with or without IL-6 for 24 h. Error ± ≤ ≤ functional involvement of (autocrine) Wnts and the R-spondin/ bars indicate SEM of three independent experiments; **P 0.01 and ***P LGR4 axis in the growth of a subset of MMs. 0.001 using unpaired Student’s t-test. (B) Flow cytometry analysis of LGR4 in XG-1 cells treated with or without IL-6 (Left), transduced with CA-STAT3 R-Spondins Are Produced in the BM Niche by (Pre)osteoblasts. (Middle), or transduced with DN-STAT3 (Right). Wnts have been shown to regulate hematopoietic stem cell homeostasis and early B-cell development in a paracrine manner, and Wnt increase in nuclear β-catenin,Fig.4A and Fig. S4A), reflecting expression in the BM niche is well established (36, 37). However, autocrine production of Wnts by the latter cells (see below). Si- the source of R-spondins remains to be defined. Prime candidates multaneous stimulation with R-spondin and Wnt ligands dramat- are osteoblasts, which have recently been suggested to use auto- ically increased LRP6 phosphorylation and β-catenin stabilization/ crine R-spondin for their differentiation (38) and, moreover, are translocation in all LGR4-positive HMCLs tested, but not in the key constituents of the MM niche (39, 40). We therefore analyzed LGR4-negative cell line L363 (Fig. 4A and Fig. S4A). To further R-spondin mRNA expression in primary human osteoblasts study the role of LGR4 in amplification of Wnt signaling by (hOB) by qPCR. As expected, these cells expressed high levels of R-spondin, we stably transduced HMCLs with a β-catenin– the osteoblastic markers ALPL (alkaline phosphatase), BGLAP sensitive fluorescent Wnt-reporter (TOP-GFP), which coexpresses (osteocalcin), and COL1A1 (collagen 1A1) (Fig. S7A). Interest- a histone2B(H2B)/mCherry fusion protein as a transduction marker ingly, mRNA of all four R-spondins was readily detectable in (Fig. S4B), combined with either inducible shRNA-mediated si- primary hOB, with RSPO2 and RSPO3 showing the highest ex- lencing (Fig. S4C) or CRISPR/Cas9-mediated gene disruption pression levels (Fig. 6A). To extend these data and allow func- + (Fig. 2D) of LGR4. FACS-sorted TOP-GFP MM cells displayed tional studies, we analyzed R-spondin expression in preosteoblast − higher levels of nuclear β-catenin compared with TOP-GFP cells, cell lines and in in vitro-differentiated osteoblasts, marked by confirming specificity of the reporter (Fig. S4B). In line with the high expression of the osteoblastic markers alkaline phosphatase observed increase in LRP6 phosphorylation and β-catenin stabi- (akp2) and osteocalcin (bglap) and high alkaline phosphatase (AP) lization, R-spondin robustly amplified Wnt reporter activity in the activity (Fig. S7 B and C). Rspo2 and rspo3 mRNA was readily presence of Wnt ligands (Fig. 4B and Fig. S4C). Similar results detectable in both preosteoblasts and osteoblasts (Fig. S7D). were obtained upon transient transfection of the TOPflash lucif- Surprisingly, R-spondin expression was markedly reduced in erase reporter, which was activated up to 200-fold by simultaneous transition from preosteoblast to osteoblast. (Fig. S7D). Function- stimulation with Wnt3a and R-spondin (Fig. S5A). Importantly, ally, (pre)osteoblast-conditioned media facilitated autocrine Wnt shRNA-mediated silencing of LGR4 and CRISPR/Cas9-mediated signaling in LME-1 and robustly synergized with exogenous Wnts LGR4 gene disruption abolished the potentiating effect of in activating Wnt signaling in all cell lines tested (Fig. 6B and Fig. R-spondins on Wnt-pathway activation (Fig. 4B and Fig. S4C), S7E). However, by itself, and even if combined with exogenous indicating a crucial role for LGR4. R-spondins, (pre)osteoblast-conditioned media lack the capacity In LME1 cells, Wnt receptor stabilization by R-spondin to activate Wnt signaling in OPM2 and XG-3 cells, indicating that increased Wnt pathway activity in the absence of exogenous functional Wnts are absent in (pre)osteoblast-conditioned media Wnts (Fig. 4 A and B and Fig. S4A), suggesting autocrine Wnt (Fig. 6B and Fig. S7E). These results identify (pre)osteoblasts as a

378 | www.pnas.org/cgi/doi/10.1073/pnas.1618650114 van Andel et al. Downloaded by guest on September 25, 2021 A ANBL-6 LME-1 nucleus cytoplasm nucleus cytoplasm R-spondin - +-+ - +-+ - +-+ - +-+ Wnt3a --++ --++ --++ --++

β-catenin 92 kDa

Lamin A/C 69/62 kDa

β-tubulin 55 kDa 1.0 1.0 4.7 19.4 1.0 1.1 9.4 23.3 1.0 1.4 1.0 4.9 1.0 1.9 1.3 3.8 B C LME-1 TOP-GFP 10 *** 8 ***

6 TOP-GFP

4 FOP-GFP Wnt activity 2

0 R-spondin R-spondin -+--+-+--+-+--+R-spondin Wnt3a Wnt3a -----+++ +-----IWP-2 ------++++LGK974

Fig. 4. R-spondins potentiate Wnt/β-catenin signaling in MM in an LGR4-dependent manner. (A) Analysis of subcellular distribution of β-catenin in HMCLs after treatment with recombinant R-spondin 2, Wnt3a, or both by Western blot is representative of five independent experiments. β-Tubulin (cytoplasm) and lamin A/C (nucleus) served as fractionation and loading controls. Densitometric quantification (β-catenin/lamin A/C for nuclear fractions and β-catenin/ β-tubulin for cytoplasmic fractions) relative to unstimulated conditions is shown below. (B) Flow cytometry analysis of Wnt activity in TOP-GFP–transduced HMCLs treated with R-spondin 2, Wnt3a, or both after CRISPR/CAS9-mediated LGR4 KO. Wnt activity is plotted as the percentage of mCherry/TOP-GFP double- positive live cells. Error bars indicate ± SEM of three independent experiments in triplicate; ***P ≤ 0.001 using one-way ANOVA with Bonferroni correction. (C) Flow cytometry analysis of Wnt activity in TOP-GFP– or FOP-GFP (control)–transduced LME-1 cells treated with the porcupine inhibitors LGK974 or IWP-2 and stimulated with recombinant R-spondin 2. Error bars indicate ± SEM of three independent experiments in triplicate; ***P ≤ 0.001 using one-way ANOVA with Bonferroni correction.

potential source of R-spondin in the BM niche that may facilitate been shown to drive oncogenic Wnt signaling (45, 46). Thus, these Wnt/β-catenin signaling in MM via aberrantly expressed LGR4. mutations might represent alternative mechanisms driving Wnt signaling in a subset of MMs. Discussion The seminal finding of our study is that LGR4 is expressed by Wnt/β-catenin pathway activation in MM is associated with dis- the majority of pMMs and HMCLs, whereas normal bone mar- ease progression and drug resistance and mediates MM cell row plasma cells and B cells are completely devoid of LGR4. proliferation (5–9). Because classical Wnt-pathway–activating This designates aberrant LGR4 expression as a hallmark of mutations have not been described in MM, aberrant Wnt activity malignancy and suggests that it can contribute to MM patho- was proposed to be driven by the BM microenvironment. This genesis. Importantly, LGR4 expression is independent of the Wnt pathway activation may be further boosted by epigenetic loss specific molecular MM subgroups, indicating that neither of the of Wnt pathway inhibitors, including sFRPs and DKK1 (8, 41, 42), common MM subgroup-specific genomic alterations drives as well as by mutational inactivation of CYLD, a gene encoding LGR4 expression. Rather, we found that STAT3 signaling, which a deubiquiting enzyme that negatively regulates Wnt signaling in is activated in the vast majority of MMs and is well known for its MM (43), all of which are common events in advanced MM. role in inflammation and cancer, mediates aberrant LGR4 ex- However, the exact mechanism(s) driving Wnt pathway activation pression (26–28). This finding is of great interest because it links in MM remains undefined. Our current study demonstrates that oncogenic Wnt activity in MM to inflammation and deregulated Wnt pathway activation in MM is facilitated by a proximal event in signaling. Although a wide variety of and the Wnt pathway, i.e., by aberrant expression of LGR4. This en- growth factors are capable of activating STAT3, activation of ables MM cells to hijack R-spondins produced by (pre)osteoblasts STAT3 in MM typically results from auto- or paracrine IL-6 in the MM niche, thereby deregulating Wnt (co)receptor turnover signaling (26, 28). Indeed, we found that IL-6 stimulation leads and causing hypersensitivity of MM cells to autocrine and para- to STAT3-mediated LGR4 up-regulation. Recently, Dechow crine Wnts (Fig. S8). Interestingly, analysis of primary MM exome et al. (27) reported that chronic STAT3 activation cooperates sequencing data from Lohr et al. (2) revealed that RNF43 and with MYC, which is a transcriptional target of β-catenin (16), in ZNRF3, the E3 ligases that are subject to inhibition by LGR/ the development of MM. This underscores the importance of MEDICAL SCIENCES R-spondin signaling, harbor mutations in primary MM at low fre- simultaneous activation of STAT3 signaling and Wnt/β-catenin quency (together 4/203, 2%). Mutations in these genes have been signaling in MM pathogenesis. shown to drive Wnt signaling in other tumors (17–19); however, Wnts are produced by BM stromal cells and play a role in the the effect of these specific mutations on Wnt signaling in MM control of hematopoietic stem cell homeostasis and early B-cell remains to be established. In addition, in analyzing the data of development (36, 37). Likewise, Wnt signaling in MM can be Mulligan et al. (44) involving targeted sequencing of established activated by paracrine Wnt ligands emanating from the BM niche, oncogenes, we identified a T41I in β-catenin (CTNNB1) which is mimicked in vitro by stimulation with exogenous Wnts. In and a frameshift mutation in APC (S1465fs*3), both of which have addition, gene-expression studies showed overexpression of Wnts

van Andel et al. PNAS | January 10, 2017 | vol. 114 | no. 2 | 379 Downloaded by guest on September 25, 2021 in MM (6, 8), suggesting . Our functional A RSPO1-4 expression B studies show that R-spondin/LGR4 interaction strongly amplifies LME-1 TOP-GFP the response to exogeneous/paracrine Wnts. Moreover, the finding 0.7 20 that R-spondin can also potentiate Wnt signaling in the absence of 0.6 *** exogenously added Wnts, and that small-molecule inhibitors of 15 0.2 y * Wnt secretion mitigate this effect, firmly establishes the existence it of an autocrine Wnt-signaling loop in a subset of MMs. Down- 10 activ control stream inhibition of Wnt signaling was previously shown to impair – 0.1 osteoblast MM proliferation (5 7, 9, 35). Interestingly, our current results Wnt 5 indicate that a subset of HMCLs are also sensitive to proximal Wnt pathway inhibition by porcupine inhibitors and LGR4 si- relative mRNA expression 0.0 0 lencing. MM cell lines are almost invariably derived from MM R2 R3R1R4 R2 R3R1R4 - cells in a leukemic phase of the disease at which the malignant R-spondin -+ - + - primary human Jurkat Wnt3a --+ --+ cells have lost their dependence upon the BM niche. By contrast, osteoblasts however, the vast majority of primary MMs is strictly dependent upon a protective BM niche for growth and survival. Therefore, Fig. 6. (Pre)osteoblast-derived R-spondins facilitate auto- and paracrine primary MM will most likely be more vulnerable to inhibition of Wnt signaling. (A)qPCRanalysisofRSPO1-4 mRNA levels in primary hu- Wnts and/or R-spondins emanating from the BM niche. man osteoblasts from two independent donors. Jurkat cells served as a Whereas the presence of Wnt ligands in the BM niche is well negative control. (B) Analysis of Wnt activity in TOP-GFP–transduced LME-1 established, expression of R-spondins in the BM microenviron- cells treated with R-spondin 2 or low levels of Wnt3a (25 ng/mL) in the ment thus far has remained largely unexplored. Because autocrine absence (white) or presence (black) of osteoblast-conditioned medium (KS483). Error bars indicate ± SEM of three independent experiments in triplicate; *P ≤ 0.05 and ***P ≤ 0.001 using one-way ANOVA with Bonferroni correction.

A LME-1 RIES 125 125 R-spondins have been suggested to regulate differentiation of os- 100 100 teoblasts (38), which are important constituents of the MM niche 75 ** 75 * (39, 40), we hypothesized that osteoblasts could be a major source of R-spondins driving oncogenic Wnt signaling. Corroborating 50 50 *** *** this hypothesis, we found expression of several R-spondins in 25 control *** 25 control (pre)osteoblasts and demonstrated that (pre)osteoblast-conditioned dn.TCF4 dn.TCF4 rel.% transduced cells 0 rel.% transduced cells 0 media robustly synergized with Wnt ligands in activating Wnt 0 1234567 0 1234567 signaling. Notably, we observed that R-spondin expression de- time (days) time (days) creases during differentiation of preosteoblasts to osteoblasts. This suggests a scenario in which active suppression of osteo- LME-1 RIES B 600 400 blast differentiation by MM cells (47) may lead to increased local levels of R-spondins, creating a feed-forward loop that

th 300 drives Wnt signaling in the tumor cells. 400 In summary, we have uncovered a previously unknown role for

growth * β * * 200 the LGR4/R-spondin axis in regulating Wnt/ -catenin in MM ve grow ve * i cells. Importantly, we demonstrate that the R-spondin effects are 200 at 100 relati rel fully dependent on LGR4, implying that, unlike in intestinal epi- thelium where LGR4 and LGR5 have redundant functions, 0 0 therapeutic targeting of LGR4 in MM will be sufficient to abolish 2 4 2 R-spondin–mediated Wnt signaling amplification. These findings K97 DMSO IWP- DMSO IWP- LGK974 LG identify LGR4/R-spondin signaling as a therapeutic target for MM patients. LME-1 RIES C 900 400 shNT shNT Materials and Methods

800 shLGR4 300 shLGR4 For additional information on methods, see SI Materials and Methods. owth 700 growth Plasmids and Cloning. pTRIPZ-shLGR4 and pTRIPZ-shSTAT3 were constructed * 200 ve ** as previously described (48). In short, a 97-mer template was amplified by PCR 600 and inserted in the XhoI/EcoRI site of the pTRIPZ vector (Thermo Scientific). 100 relati relative gr relative Targeting sequences were GCGTAATCAAATCTACCAAAT (hLGR4) and GCA- 100 CAATCTACGAAGAATCAA (hSTAT3). pLentiCrispr-sgLGR4 was constructed by 0 0 inserting sgLGR4 (GCAGCTGCGACGGCGACCGT, chr11:27493733–27493752, -+-+dox -+-+dox hg19) in pLentiCrispr (Addgene) as previously described (49). TOP-GFP. Fig. 5. Inhibition of Wnt signaling by suppression of β-catenin–mediated mCh/FOP-GFP.mCh (Addgene 35491 and 35492) were kind gifts from David , inhibition of Wnt secretion, or silencing of LGR4 impairs MM Horst, Ludwig-Maximilians-Universität München (LMU), Munich (50). pEF. cell expansion. (A) Percentage of MM cells transduced with control (black) or STAT3DN.Ubc.GFP (24984), pEF.STAT3C.Ubc.GFP (24983), and EdTC (dnTCF4, dnTCF4 (red), relative to t = 0, in a 7-d time course. Error bars indicate ± SEM 24310) were obtained from Addgene. of three independent transductions. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 using unpaired Student’s t-test. (B) Flow cytometry analysis of the effect of Microarray Data Analysis. Datasets presented in Fig. 1 and Fig. S1 A and B were the small-molecule Wnt inhibitors IWP-2 and LGK974 on HMCL expansion obtained from the publically available Amazonia! web atlas (51) and nor- after 4 d of culture, relative to day 0. Error bars indicate ± SEM of three malized by global scaling to 100 and include samples representing naive B cells independent transductions. *P ≤ 0.05 using unpaired Student’s t-test. (n = 5), centroblasts (n = 4), centrocytes (n = 4), memory B cells (n = 5), plas- (C) Flow cytometry analysis of the effect of doxycycline-induced shRNA- mablasts (n = 5), bone marrow plasma cells (n = 5) (52), CD4+ Tcells(n = 4), mediated silencing of LGR4 on MM cell expansion at day 4 of culture, rela- CD8+ T cells (n = 6) (53), chronic myeloid leukemia (n = 35) (54), acute myeloid tive to day 0. Error bars indicate ± SEM of three independent experiments in leukemia (n = 89) (55, 56), B-cell acute lymphoblastic leukemia (n = 82) (57) triplicate. *P ≤ 0.05 and **P ≤ 0.01 using unpaired Student’s t-test. (Lugo-Trampe et al. GSE10820), T-cell acute lymphoblastic leukemia (n = 54)

380 | www.pnas.org/cgi/doi/10.1073/pnas.1618650114 van Andel et al. Downloaded by guest on September 25, 2021 (Winter et al. GSE14615), diffuse large B-cell lymphoma (n = 51) (58), follicular hyperdyploid (n = 47), low bone disease (n = 23), MAF/MAFB (n = 13), lymphoma (n = 20) (59), chronic lymphoid leukemia (n = 124) (54, 60–62), MMSET (n = 36), myeloid gene signature (n = 71), and proliferation (n = multiple myeloma (n = 30), and intestinal tissues (n = 8) (Roth et al. GSE7307). 21) subgroups. The microarray dataset presented in Fig. S1 C and D contained gene-expression data of primary MM samples divided into subgroups based on molecular ACKNOWLEDGMENTS. This study was supported by a grant from the Dutch profiling that was normalized as described (25). Molecular subgroups Cancer Society (AMC-2011-5205) and the European Union Framework were previously identified and include the CD1 (n = 18), CD2 (n = 26), Programme 7, OVER-MYR.

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