The Planar Cell Polarity Pathway Drives Pathogenesis of Chronic Lymphocytic Leukemia by the Regulation of B-Lymphocyte Migration

Published OnlineFirst January 21, 2013; DOI: 10.1158/0008-5472.CAN-12-1752

Cancer Microenvironment and Immunology Research

Marketa Kaucka1, Karla Plevova2,4, Šarka Pavlova2,4, Pavlína Janovska1, Archana Mishra1, Jan Verner2,4, Jirina Prochazkov a1, Pavel Krejcí 1,5,6, Jana Kotaškova2,4, Petra Ovesna3, Boris Tichy2,4, Yvona Brychtova4, Michael Doubek2,4, Alois Kozubík1,5,Jirí Mayer2,4, Šarka Pospíšilova2,4, and Vítezslav Bryja1,5

Abstract The planar cell polarity (PCP) pathway is a conserved pathway that regulates cell migration and polarity in various contexts. Here we show that key PCP pathway components such as Vangl2, Celsr1, Prickle1, FZD3, FZD7, Dvl2, Dvl3, and casein kinase 1 (CK1)-e are upregulated in B lymphocytes of patients with chronic lymphocytic leukemia (CLL). Elevated levels of PCP proteins accumulate in advanced stages of the disease. Here, we show that PCP pathway is required for the migration and transendothelial invasion of CLL cells and that patients with high expression of PCP genes, FZD3, FZD7, and PRICKLE1, have a less favorable clinical prognosis. Our ﬁndings establish that the PCP pathway acts as an important regulator of CLL cell migration and invasion. PCP proteins represent an important class of molecules regulating pathogenic interaction of CLL cells with their microenvironment. Cancer Res; 73(5); 1–11. 2012 AACR.

Introduction proteins) are conserved in evolution and include Vang-like Wnt signaling pathways are crucial for cell-to-cell commu- protein 2 (Vangl2), cadherin EGF LAG 7-pass transmembrane nication, differentiation, and morphogenesis in embryonic G-type receptor (Celsr1), Frizzleds (FZD, in mammals mainly development. Dysfunction or deregulation of Wnt signaling FZD3, FZD6, and FZD7), which act as receptors, and cyto- accounts for a number of developmental defects, inherited plasmic components such as Disheveled (Dvl1-3 in mammals), diseases, and many types of cancer (1). The best-known Wnt Prickle-like proteins (mainly Prickle1), casein kinase 1e (CK1e), pathway—canonical or Wnt/b-catenin pathway—induces and the small GTPases Rho/Rac and downstream kinases b-catenin–dependent activation of T-cell factor/lymphoid ROCK/JNK. PCP proteins regulate cell polarity (3), convergent enhancer factor (TCF/LEF)-mediated transcription. However, extension movements during gastrulation and neurulation, Wnt ligands can activate other so-called noncanonical Wnt and polarity of the hair cells in the inner ear (3, 4). pathways, which are b-catenin–independent and biochemi- In the present study, we investigated the role of PCP proteins cally distinct from the canonical Wnt pathway (2). Among in chronic lymphocytic leukemia (CLL). CLL is characterized þ those, the pathway that regulates planar cell polarity (Wnt/ by clonal expansion and apoptosis dysregulation of CD5 B PCP pathway) is the best known (3). The core components of lymphocytes. Neoplastic CLL cells accumulate in blood, bone the Wnt/PCP pathway (referred here as PCP or polarity marrow, and lymphoid tissue. It is recognized that cell migration and recirculation between peripheral blood and lymphoid niches in bone marrow and lymphoid tissue are important factors contributing to CLL biology (5–7) and that alterations 1 Authors' Affiliations: Institute of Experimental Biology, Faculty of Sci- in survival and proliferation of CLL are affected by their ence, 2CEITEC - Central European Institute of Technology, 3Institute of Biostatistics and Analyses, Masaryk University; 4Center of Molecular interaction with the microenvironment. Here, we show for the Biology and Gene Therapy, Department of Internal Medicine–Hematology first time that the core cassette of the PCP pathway composed and Oncology, University Hospital Brno and Medical Faculty MU; 5Depart- ment of Cytokinetics, Institute of Biophysics, Academy of Sciences of the of Celsr1, Prickle1, Vangl2, Dvl2, Dvl3, FZD3, and FZD7 is Czech Republic, Brno, Czech Republic; and 6Medical Genetics Institute, upregulated in B cells of patients with CLLs. We show that Cedars-Sinai Medical Center, Los Angeles, California PCP proteins, which are conserved regulators of the cell–cell Note: Supplementary data for this article are available at Cancer Research interaction and cell migration, are important mediators of CLL Online (http://cancerres.aacrjournals.org/). chemotactic responses and homing. K. Plevova and Š. Pavlova contributed equally to this work. Materials and Methods Corresponding Author: Vitezslav Bryja, Institute of Biophysics v.v.i., fi Academy of Sciences of the Czech Republic, Kralovopolska 135, Brno Cell isolation, puri cation, and cell treatments CZ-612 65, Czech Republic. Phone: 420-549493291; Fax: 420- Primary B cells from previously untreated patients with 541211214; E-mail: [email protected] CLLs or healthy volunteers were separated using gradient doi: 10.1158/0008-5472.CAN-12-1752 centrifugation followed by non–B-cell depletion (RosetteSep 2012 American Association for Cancer Research. B Cell Enrichment Kit and Human CD3þ Depletion Kit;

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StemCell Technologies; or MACS B cell Isolation Kit II; Miltenyi subsets of patients with CLLs. To confirm that the levels of Biotec) according to the manufacturer's instructions. Flow PCP components are indeed altered in CLLs, we collected cytometry was conducted for evaluation of CD5 and CD19 RNA samples from purified peripheral blood B cells (CD19þ expression on purified cells (tricolor panel: CD45-TRI-COLOR, cell population purity 95%) of control individuals (n ¼ 6) MHCD45065, Invitrogen, CD5-FITC, A08932, CD19-PE, A07769, and patients with CLL (n ¼ 93). qRT-PCR analysis showed Beckman Coulter). For further analyses, samples with the statistically increased mRNA levels of 3 PCP genes, CELSR1, purity higher than 95% B cells were used. The cell line MEC1 PRICKLE1, and FZD7, in CLL samples and identified that 2 was obtained from DSMZ, cell line was tested by DSMZ using other genes VANGL2 and FZD3 have high levels in a subset molecular genetic methods; MEC1 cells were cultured in RPMI- of patients with CLLs [Fig. 1A—normalized to the expression 1640 supplemented with 10% FBS and antibiotics at 37oC and of b2-microglobulin; Supplementary Fig. S1A—normalized to 5% CO2. b-actin]. Subsequent analysis of the protein level by Western blotting Transwell assay confirmed increased protein expression of the PCP proteins The chemotaxis assay was conducted in HTS Transwell-96 Vangl2, Dvl2, Dvl3, Prickle1, and CK1e (Fig. 1B, quantified well plates (Corning Incorporated) with 5.0-mm pore size in Fig. 1C) in patients with CLLs. The level of b-catenin did polycarbonate membranes following the manufacturer's not differ between CLLs and healthy cells (Fig. 1B), and we instructions. A total of 0.5 106 cells (primary CLL or MEC1 failed to detect activated (dephosphorylated) b-catenin (not cells) were seeded in the Transwell upper insert, which was shown), which suggest that the Wnt/b-catenin pathway is either nontreated or coated with a human umbilical vein not active in CLL cells. All CLL samples tested had high ex- endothelial cells monolayer (HUVEC). Cells in the insert were pression of LEF1 and Ror1 (Fig. 1B), which were previously treated with antibodies or inhibitors as described in Supple- identified as abundantly and consistently expressed in CLL mentary Material. Cells were incubated overnight in full medi- cells (9, 10). Flow cytometry detection (Fig. 1D) further con- o um (including 10% fetal calf serum) at 37 C and 5% CO2, and firmed increased cell surface levels of Ror1, FZD3, Celsr1, and after 18 hours the migration toward the chemokine was Vangl2 in comparison to healthy B cells. analyzed by a Coulter Counter (model FN, Coulter Next, we asked whether the levels of PCP proteins are Electronics; Figs. 2A–C, 3A–D, and 4A–C; Supplementary Figs. stable or they change during the disease course. When we S2B and S4E–S4H) or by C6 flow cytometer (Accuri; Figs. 2E– compared samples of 6 patients with CLLs collected at 2 G, 3E–G, and 4D; Supplementary Fig. S2E). The migration index different time points (t1 and t2;Fig.1E),wewereableto was calculated as the number of cells (treated or untreated) detect changes in the levels of PCP proteins in CLL cells from migrating in response to the chemokine divided by the number individual time points. Specifically, we observed a clear of cells migrating toward the control medium only. In case of increase in Dvl2, Dvl3, and Prickle1 levels in patients fol- very variable migration indexes (in primary CLL cells), cell lowed in time, which is quantified in Supplementary Fig. numbers were normalized to the chemokine-only condition S1B. The most prominent changes were observed mainly in and expressed as "relative migration." patients, who progressed from Rai stages (11) 0/I/II to the stage III or IV (see patients C, H, P). The levels of LEF1 and In vivo experiments b-catenin do not change (Fig. 1E). Nonirradiated mice (8- to 16-week-old) nonobese diabetic/ In summary, using several methods, we provide evidence severe combined immunodeficient (NOD/SCID) IL2Rg-null that a host of PCP proteins are overexpressed in B cells of (NSG) mice were used for transplantation experiments. Freshly patients with CLLs, with some accumulating to a greater extent isolated human CLL lymphocytes (25 106 per condition) in advanced stages of the disease. were pretreated as indicated for 20 hours and stained with Calcein AM. Mice underwent transplantation by intraperito- Wnt5a, a PCP ligand, increases migration of primary CLL neal injection of 20 106 of pretreated and calcein AM–stained cells in a chemokine gradient via a Wnt/PCP pathway human CLL lymphocytes in 120 mL of sterile PBS. Recipient It is becoming increasingly evident that the role of micro- mice were analyzed 24 hours after the injection by fluores- environment in the pathogenesis of CLLs is of a crucial cence-activated cell-sorting (FACS) analysis (for details, see importance (7, 12). Chemokines and their receptors, mainly Supplementary Material). CXCL12-CXCR4, CXCL9/10/11-CXCR3, and CCL19/21-CCR7, Further methods including information on patient samples, have been shown to mediate invasiveness and transendothelial quantitative real-time (qRT)-PCR, Western blotting, flow cyto- migration of CLL cells (13–16). In that line, it has been recently metry, nucleofection, and statistics are provided in the Sup- shown that Wnt-5a, which is the major ligand for Ror1/2 plementary Material. receptors and a crucial component of the Wnt/PCP pathway (17–19), promotes cell polarization and directional movement Results of melanoma cells and T cells in a chemokine (CXCL12) Core PCP pathway components are upregulated in CLL gradient (20, 21). patients We thus hypothesized that Wnt5a, which is also expressed in Our earlier microarray expression analysis (8) suggested that CLL cells (Supplementary Fig. S2A) together with receptor PCP components of Wnt/PCP signaling are frequently upregulated protein complexes, may contribute to the pathogenesis of CLLs in CLLs and expressed differently in prognostically distinct by the effects on the polarized migration of CLL cells toward

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þ Figure 1. PCP genes and proteins are upregulated in patients with CLL. A, expression of the indicated genes from CD19 cells of healthy individualsÀÁ (control; n ¼ 6) and patients with CLLs (n ¼ 93) was analyzed by qRT-PCR. Data represent the fold difference relative to the reference gene 2DCt b2- microglobulin. B, the amounts of the indicated PCP proteins were analyzed by Western blotting in B cells of healthy controls and patients with CLLs. One lane represents one individual. C, densitometric quantification of data in B. D, the cell surface expression of the PCP proteins Ror1, FZD3, Celsr1, and Vangl2 in CD19þ B cells of healthy individual and patients with CLLs were analyzed by flow cytometry. E, Western blotting of Dvl2, Dvl3, Prickle1, LEF1, Ror1, and b-catenin in B cells from 6 patients, where each patient has been taken and analyzed at 2 different time points (t1 and t2). b-Actin served as the loading control. The Rai clinical stages of each patient at t1 and t2, and the time between t1 and t2 are indicated. Mean timespan between t1 and t2 is 1,308 days. , P < 0.001; , P < 0.05. n.s., not significant (Student t test). www.aacrjournals.org Cancer Res; 73(5) March 1, 2013 OF3

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Figure 2. PCP proteins regulate migration of primary CLL cells in a chemokine gradient. A, migration of primary CLL cells (expressed as migration index) in the presence of CXCL12, Wnt5a, and CXCL12/ Wnt5a was analyzed using Transwell migration chambers (n ¼ 6). The observed increase is dependent on the activity of CK1 (n ¼ 3; B) and on presence of the Ror1 cell surface receptor (aRor1, Ror1-blocking antibody; n ¼ 4; C). Both CK1 inhibition and Ror1 blocking antibody clearly interfere with Wnt/PCP pathway as shown by their ability to reduce the Wnt- induced mobility shift of Dvl3 (full arrowhead, open arrowhead— non-shifted Dvl3; D). E–G, the effects of the inhibitors of the Rho-associated kinase (Y27632; E), Rac1 (F), and porcupine (IWP-2; G) on the chemotaxis driven by Wnt5a/CXCL12 were tested by Transwell assays. Values represent mean SEM. Statistical differences were tested by one- way ANOVA and Tukey post hoc test (, P < 0.05; , P < 0.01; , P < 0.001). n.s., not signiﬁcant.

chemokines. To test this prediction, we studied primary cells ulates its activity in the PCP pathway (19, 23–25). Treatment from patients with CLLs for their ability to respond to CXCL12 with the CK1 inhibitor D4476 (CK1 inh. I; Fig. 2B; ref. 26) in the presence and absence of recombinant Wnt5a (200 significantly reduced the positive effects of Wnt5a on the ng/mL) using Transwell migration chambers (16, 22). As we chemotaxis toward CXCL12. To exclude that these effects are show in Fig. 2A, CXCL12 alone increased migration of CLL cells, due to the activation of the Wnt/b-catenin pathway, we tested whereas Wnt5a alone had no effect. In combination with in primary CLL cells the ability of Wnts to activate sensitive CXCL12, Wnt5a significantly increased migration of CLLs cells TCF/LEF (TopFlash) luciferase reporter (27) and analyzed the compared with all other experimental conditions. A similar expression of bona fide Wnt/b-catenin target genes AXIN2 and effect was seen for CXCL10, CXCL11, and CCL21 but not for DKK1 by quantitative PCR. As we show in Supplementary Fig. CXCL9 and CCL19 (Supplementary Fig. S2B). Importantly, the S2C and S2D, no experimental treatments, including treatment Wnt5a-induced synergism was dependent on the activity of with Wnt3a, a typical ligand of the Wnt/b-catenin pathway, CK1, which phosphorylates a key PCP protein Dvl and reg- trigger the expression from TCF/LEF-responsive promoters.

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Figure 3. Regulation of MEC1 cell migration by PCP proteins. A, migration of MEC1 cells was analyzed in presence of CXCL12, CCL19, and CCL21 (200 ng/mL) with or without Wnt5a using Transwell migration assays. Wnt5a does not further promote migration driven by CXCL12, CCL19, and CCL21. B–G, MEC1 migration triggered by CCL21 and/or CCL19 chemokines is blocked by CK1 inhibitor D4476 (CK1 inh. I; 100 mmol/L; B), PF670462 (CK1 inh. II; 50 mmol/L; C), knockdown of Dvl2, the efficiency of knockdown was determined by Western blot analysis and quantified by densitometry (Dvl2/actin; D), inhibition of the Rho-associated kinase (Y27632; E), and the inhibitor of endogenous Wnt production (inhibitor of porcupine IWP-2; G). The inhibitorof Rac1 (F) does not have an effect. Values represent mean SEM. Statistical differences were tested by one-way ANOVA and Tukey post hoc test (, P < 0.05; , P < 0.01; , P < 0.001; n.s., not significant). Each experiment has been carried out in at least 3 independent replicates.

This strongly suggests that Wnt/b-catenin pathway is not cytoskeletal rearrangements and cell migration. As we show involved in the observed phenotypes. in Fig. 2E, the inhibition of RhoA-driven signaling by Y27632, The effects similar to the inhibition of CK1 have been an inhibitor of Rho-associated ROCK protein kinase (29) but observed, when we blocked the putative Wnt5a receptor in not the inhibition of Rac1 by a Rac1 inhibitor NSC23766 the PCP complex, Ror1, with the goat Ror1 blocking antibody (Fig. 2F; ref. 30), blocked the migration of primary CLL cells. (aRor1) but not with the control goat IgG (Fig. 2C). The No inhibitors used in this section affect the viability of prim- efficiency of this antibody, which binds Ror1 and promotes ary CLL cells, as assessed by WST1 test, and the effects on its internalization, has been shown earlier (28). Both D4476 chemotaxis thus do not reflect differences in the condition of (CK1 inh. I) and aRor1 antibody clearly inhibited activation of CLL cells (Supplementary Fig. S3). PCP pathway in primary CLL cells as shown by their ability to The experiments above suggest that PCP pathway is block phosphorylation-dependent shift of endogenous Dvl3 capable to promote and is required for the efficient chemo- (PS-Dvl3), which is a hallmark of Dvl3 activation in the non- tactic migration of primary CLL cells. To further elaborate canonical Wnt pathway (Fig. 2D; ref. 19). on the mechanism of the interaction between Wnt/PCP and In the Wnt/PCP pathway, Dvl mediates the activation of the chemokine signaling, we tested functionally the role of small GTPases RhoA and Rac1, which trigger subsequent the CXCR4 (the receptor for CXCL12) and the role of

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Figure 4. Regulation of transendothelial migration of CLL cells by PCP pathway. A–D, transendothelial invasion of primary human CLL lymphocytes and MEC1 cells was tested in the Transwell assays coated with the HUVEC endothelial cell monolayer and is indicated as the migration index. The level of invasion of human primary CLL lymphocytes (A) was analyzed in the presence of chemokine CCL19, Wnt5a, or a combination of CCL19 and Wnt5a. Wnt5a strongly promotes CCL19-induced invasion of CLL cells through the endothelial monolayer. B–D, the positive effect of CCL19 on transendothelial migration of MEC1 cells is fully blocked by the addition of the D4476 (CK1 inh. I; B). siRNA-mediated knockdown of Dvl2 (C) and the inhibition of Rho-associated kinase (Y27632) and endogenous Wnt production (IWP-2; D). Statistical differences were tested by one-way ANOVA and Tukey post hoc test (, P < 0.05; , P < 0.01; , P < 0.001). Each experiment has been carried out in at least 3 independent replicates.

endogenous Wnts in CLL chemotaxis. Interestingly, CXCR4- treatment with Wnt5a did not further promote the promi- blocking antibody was unable to inhibit CLL migration gratory effect of chemokines (Fig. 3A) and Wnt5a-blocking (Supplementary Fig. S2E), whereas the disruption of the antibody did not block the migration (Supplementary Fig. endogenous Wnt production by the porcupine inhibitor S4E) despite the high endogenous expression of Wnt5a (see IWP-2 (31) reduced CLL migration (Fig. 2G). Given the fact Supplementary Fig. S4A and S4D) Inhibition of CK1e,a that CXCL12-CXCR4 signaling does not regulate Wnt-5a kinase, which is required for PCP signaling due to its role expression and Wnts do not regulate expression of CXCR4 in the phosphorylation of Dvl, with 2 unrelated inhibitors in CLL cells (see Supplementary Fig. S2F), we conclude that D4476 (CK1 inh. I) and PF670462 (CK1 inh. II; 50 mmol/L; (i) the effects do not take place at the level of transcription, ref. 34) blocked the chemotacticresponseofMEC1cells (ii) that the autocrine stimulation by Wnts contributes to the toward CCL21 and CCL19 (Fig. 3B and C). It can be expected CLL chemotaxis. and (iii) that the chemotactic response is that CK1 blocks CLL migration via phosphorylation of the not critically mediated by CXCR4. key PCP protein Dvl. Indeed, even partial Dvl2 knockdown efficiently reduced the motility of MEC1 cells (Fig. 3D). PCP protein Dvl and its kinase CK1« are required for the Similarly to primary CLL cells, even in MEC1, the chemotaxis migration in the chemokine gradient was blocked by the inhibitors of Rho-associated kinase To study the role of Wnt/PCP pathway in CLL migration in ROCK (Fig. 3E) and by the inhibitor of Wnt secretion more detail and to overcome the natural variability among IWP-2 (Fig. 3F) but not by the inhibitor of Rac1 (Fig. 3G). patients and limitations in the experimental manipulation These data suggest that endogenous Wnts (but not uniquely with primary CLL cells, we carried out further experiments Wnt5a), Dvl2, and likely the whole PCP machinery activating in MEC1 cells (32). MEC1 is a well-defined cell line derived from Rho and ROCK kinase is required for the polarized migration a patients with CLLs, which recapitulates many aspects of the of CLL cells. CLL biology and is used as the transplantation model of CLL (33). Quantitative analysis of mRNA (WNT5A, CELSR1, PRICK- PCP proteins regulate chemokine-driven LE1, FZD3, FZD7; Supplementary Fig. S4A) and protein levels transendothelial invasion of CLL cells (Ror1, Vangl2, Dvl2, Dvl3, CK1e; Supplementary Fig. S4B) In the human body, cells behave in a complex environment showed that MEC1 cells express most PCP genes in high levels, and have to pass via extracellular matrix or endothelial bar- although the levels of Ror1 were relatively low (Supplementary riers, which is a process known as cell invasion. We have tested Fig. S4B). the role of PCP proteins in the invasion of CLL cells by the MEC1 cells were unable to respond to CXCL12 but analysis of their invasion through a layer of HUVEC. In this responded clearly to CCL19 and CCL21 in a dose-dependent experimental system, Wnt5a significantly promoted transen- manner (Fig. 3A; Supplementary Fig. S4C). Interestingly, dothelial migration of primary patient CLL cells in a gradient of

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CCL19 (Fig. 4A), to a lesser extent in the gradient of CXCL12 PCP-high patient cohorts show worse clinical (Supplementary Fig. S4F) but showed no effect in the gradient characteristics of CCL21 (Supplementary Fig. S4G). This suggests that coop- The expression as well as functional data suggested that PCP eration between Wnt/PCP and chemokine signaling is not proteins are strongly associated with CLL cell behavior. In the limited to chemotaxis but takes place also in the process of next step, we analyze the clinical significance of PCP protein transendothelial invasion. expression by analysis of samples from 93 previously untreated To check whether invasion is controlled by PCP signaling, patients (identical to patients analyzed in Fig. 1A). The nor- specifically via CK1e-Dvl2-Rho pathway (as shown in Fig. 3 for malized expression of FZD3, FZD7, CELSR1, VANGL2, and chemotaxis), we have carried out another set of experiments in PRICKLE1 assessed by qRT-PCR (see also Fig. 1A) was corre- MEC1 cells. O the 3 tested chemokines, only CCL19 was able to lated to the clinical parameters. induce transendothelial migration of MEC1 cells (Supplemen- Importantly, the increased expression of FZD3, FZD7, tary Fig. S4H). This response has not been promoted by Wnt5a VANGL2, and PRICKLE1 correlated positively with unmutated but has been almost completely abolished by CK1 inhibition immunoglobulin heavy chain (IGHV; Table 1), which is strong- (Fig. 4B). Similar to the basal chemotaxis, the downregulation ly associated with worse prognosis. In contrast, patients with of the key PCP protein Dvl2 by siRNA (Fig. 4C) as well as unfavorable chromosomal aberrations del11q22-23 (ATM) or inhibition of ROCK kinase (by Y27632) and porcupine (by IWP- del17p13 (TP53) did not show significantly higher expression 2; Fig. 4D) efficiently reduced also transendothelial invasion of of PCP genes. However, it should be noted that the number MEC1 cells. In summary, these results show that PCP proteins of patients with del11q22-23 (n ¼ 17; 18%) or del17p13 (n ¼ 7; mediate both migration/chemotaxis and chemokine-driven 7.5%) was rather low and insufficient for robust statistical invasion of CLL cells. analysis. Next, we analyzed the association of PCP gene expression PCP proteins regulate CLL homing in vivo with the treatment-free survival (TFS) using Kaplan–Meier To test the role of PCP proteins in CLL cells in vivo,we survival curves. Patients were sorted into 2 groups (high vs. transplanted primary CLL cells into NSG mice (Fig. 5A), low) based on the mRNA levels (as determined by qRT-PCR) which lack mature T cells, B cells, and functional natural of individual PCP genes (FZD3, FZD7, CELSR1, VANGL2,and killer cells (35). Primary CLL cells efficiently home in NSG PRICKLE1). The cutoff for each gene was set up as the value mice and can be detected mainly in spleen, liver, and bone corresponding to 75% percentile for the expression levels in marrow. Interestingly, pretreatment with the casein kinase 1 normal B cells from the periphery (see Fig. 1A and below). inhibitor D4476 decreased homing to spleen, liver, and bone TFS analysis was conducted in both groups of patients. marrow without any effect on the apoptosis of the cells (Fig. Importantly, FZD3-, FZD7-, and PRICKLE1-high patient 5B and C; for typical dot plots for apoptosis assay, see cohorts had significantly (Breslow test, P < 0.05) worse Supplementary Fig. S5A). This experiment suggests that prognosis than the group of patients with low expression inhibition of CK1 diminishes the ability of CLL cells to of these genes (Fig. 6). Specifically, the median TFS of FZD3- migrate and colonize murine tissues. Interference with the low (cutoff 1.2) patients was 40 months compared with 17 Ror1- and FZD7-driven signaling by treatment with anti- months in FZD3-highgroup,54versus25monthsforFZD7 bodies directed against Ror1 and FZD7 (for validation of low versus high (cutoff 2.0), and 54 versus 31 months for anti-FZD7 antibody, see Supplementary Fig. S5B) did not PRICKLE1-low versus PRICKLE1-high (cutoff 1.0) patient affect the survival of CLL cells either (Fig. 5D). Importantly, cohorts. Very similar data were obtained when we con- treatment with the blocking anti-Ror1 antibody (28) effi- ducted the analysis with the data normalized to b-actin ciently eliminated homing of CLL cells from most patients to (Supplementary Fig. S6). the spleen (Fig. 5E). The homing to bone marrow could not These findings suggest that mainly more aggressive CLL be determined because treatment with any antibody (even cells with unmutated IGHV use PCP pathway to regulate their control IgG) for unknown reasons completely abolished chemotaxis toward chemokines. Indeed, when we looked at migration to bone marrow in vivo (data not shown). Liver unmutated IGHV patient subset only, we have not observed infiltration was diminished only for some patients (Fig. 5F). any statistically significant difference in survival curves Treatment with the anti-FZD7 antibody did not show any between PCP-high and -low patient cohorts (data not shown). effect on the CLL cells in liver (Fig. 5F); however, decreased In summary, the expression analyses in patients with CLLs spleen infiltration by CLL cells occurred in a subset of showed that (i) the expression of key PCP genes correlates with patients (Fig. 5E). Interestingly, the patient subset respond- clinically important mutational status of IGHV and (ii) on the ing to anti-FZD7 antibody had mutated p53, a strong marker clinical sample show the relevance of processes controlled by of aggressive disease, whereas the patients not responding to PCP pathway for disease pathogenesis. anti-FZD7 antibody treatment had wild-type (wt) p53. In summary, these results show that CK1 activity and the Discussion cell surface receptors Ror1 and FZD7 regulate biologic In the present study we, for the first time, show that (i) core properties of CLL cells in vivo. These observations are PCP components such as Vangl2, Prickle1, CK1e, Dvl, and consistent with the in vitro results and further strengthen Celsr1 are upregulated in CLLs, (ii) PCP pathway components the conclusion that PCP proteins act as regulators of CLL help regulate chemotaxis and transendothelial migration in biology and pathogenesis. the chemokine gradient and in vivo homing of CLL cells, and

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Figure 5. PCP proteins affect engraftment of CLL cells in vivo. A, scheme of the experiment. Typical FACS dot blot is shown (see gate G1 for transplanted cells.). B, the level of apoptosis in primary CLL cells after dimethyl sulfoxide (DMSO)/D4476 (CK1 inh. I) was determined by ﬂow cytometric analysis of the mitochondrial membrane potential using tetramethylrhodamine ethyl ester dye in combination with the green calcein staining. C, proportion of CLL cells (number of positive cells/number of total cells in the organ) treated with DMSO or D4476 (CK1 inh. I) that were recovered from the spleen (Ci), liver (Cii), and bone marrow (Ciii) of transplanted mice. D, treatment with the aRor1 and aFZD7 antibodies does not induce apoptosis as determined by tetramethylrhodamine ethyl ester analysis. E and F, proportion of CLL cells, when treated with a nonspeciﬁc antibody (IgG) or antibodies directed against Ror1 and FZD7, were recovered from the mouse spleen (E) or liver (F). Please note that samples responding to aFZD7 come from patients with mutated p53 (p53 MUT). B and D, graphs show mean SEM. C and E, one symbol on the graph represents one patient. , P < 0.05, Wilcoxon paired t test (C) or one-way ANOVA and Tukey post hoc test (E).

(iii) PCP-high patient cohorts show worse clinical parameters Our data suggest that PCP pathway contributes to the such as TFS. Our ﬁndings suggest that PCP proteins affect CLL CLL pathogenesis mainly via regulation of chemotactic pathogenesis via regulation of chemokine-driven migration responses to chemokines, which are the leading mediators and as such inﬂuence disease progression. of the interaction between CLL cells and their microenvi-

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Table 1. The expression of PCP genes determined by qRT-PCR in the patient cohorts with mutated (IGHV MUT) and unmutated (IGHV UNMUT) immunoglobulin heavy chain

IGHV MUT IGHV UNMUT Gene name (n ¼ 39) (n ¼ 54) P (Mann–Whitney test) FZD7 2.697 (0.9081) 3.674 (0.6834) 0.0075 FZD3 0.7684 (0.07373) 1.217 (0.1130) 0.0054 VANGL2 4.937 (2.137) 11.12 ( 6.119) 0.007 PRICKLE1 1.942 (0.2322) 2.978 (0.2950) 0.0113

NOTE: Numbers indicate the mean, SEM; n, number of patients; P, statistical signiﬁcance of the difference between both cohorts determined by Mann-Whitney test.

ronment (for review, see ref. 36). Components of the PCP leukemia and (ii) the first implication for the role of Celsr1 in machinery thus become an interesting target for the novel human cancer. PCP signaling has been, however, function- therapies, which interfere with the communication between ally implicated earlier in the metastatic process of various CLL cells and their environment, similarly to the inhibitors solid tumors with the best defined role in melanoma, breast, of chemokine receptor or B-cell receptor (BCR) signaling and gastric cancer (40–42). It has been convincingly shown, pathways (37–39). In agreement with these data, our obser- mainly in melanoma, that PCP pathway increases the capac- vations show that PCP-controlled migration is mainly rel- ity of tumor cells to migrate and invade into surrounding evant for the CLL cells with unmutated IGHV, which were tissue (43). Our data support the possibility that PCP path- shown to be more dependent on chemokine and BCR way mediates physical aspects of cell migration via its effects signaling. on cytoskeleton and that chemokine signaling can serve as Our data provide to our best knowledge (i) the first the navigator of invading cells into their final destination. evidence for the increased levels of the conserved proteins Interesting prediction of this hypothesis is that PCP pathway unique for the PCP pathway—Prickle1, Vangl2, Celsr1—in is dynamically and temporarily used by migrating immune

Figure 6. Expression of PCP genes deﬁnes CLL progression. Patients were sorted into 2 groups (high vs. low expression; cutoff indicated by the line in the upper graph) based on the mRNA levels of individual PCP genes (FZD3, FZD7, and PRICKLE1), and Kaplan–Meier survival curves (TFS) were plotted and survival analysis was conducted in both groups of patients. PRICKLE1-(A),FZD3- (B), and FZD7-high (C) patient cohorts had a signiﬁcantly (Breslow test, P < 0.05) worse prognosis than the group of patients with low expression of these genes.

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Kaucka et al.

cells during the immune response. This interesting topic A. Mishra, J. Verner, J. Prochazkov a, J. Kotaškova, B. Tichy, Y. Brychtova, M. Doubek, J. Mayer clearly exceeds the current study and has to be tested in Analysis and interpretation of data (e.g., statistical analysis, biostatistics, future. computational analysis): M. Kaucka, S. Pavlova, A. Mishra, J. Prochazkova, š In summary, our findings provide strong evidence that core P. Krejcí, J. Kota kova, P. Ovesna, B. Tichy, V. Bryja Writing, review, and/or revision of the manuscript: M. Kaucka, K. Plevova, components of the Wnt/PCP pathway play an important role S. Pavlova, A. Mishra, A. Kozubík, J. Mayer, Š. Pospíšlova, V. Bryja in CLL pathogenesis via regulation of CLL migration. We for Administrative, technical, or material support (i.e., reporting or orga- fi nizing data, constructing databases): K. Plevova, J. Kotaškova, A. Kozubík, the rst time show the biologic importance of the crosstalk J. Mayer between chemokine and PCP signaling, which might have Study supervision: P. Krejcí, Š. Pospíšlova, V. Bryja implication for understanding of the biology of invasiveness in other tumors. Given the fact that Wnt/PCP pathway is an Grant Support evolutionary conserved regulator of cell polarity and migra- This work was supported by grants from the Czech Science Foundation (204/ 09/H058, 301/11/0747), Ministry of Health of the Czech Republic (NT11217-5/ tion, our data further emphasize the role of cell migration in the 2010, NT 13493-4/2012), Ministry of Education, Youth and Sports of the Czech development of CLLs. Republic (MSM0021622430), Masaryk University Rector's programme to support MU student's creative work (MUNI/E/0128/2009, MUNI/A/0784/2011), Europe- an Regional Development Fund (CZ.1.07/2.3.00/20.0180, CZ.1.05/1.1.00/02.0068, Disclosure of Potential Conflicts of Interest CZ.1.07/2.3.00/20.0045), and by an EMBO Installation Grant. P. Krejcí is sup- No potential conflicts of interest were disclosed. ported by the grants Kontakt LH12004 (Ministry of Education) and P305/11/0752 (Czech Science Foundation). The costs of publication of this article were defrayed in part by the payment Authors' Contributions of page charges. This article must therefore be hereby marked advertisement Conception and design: M. Kaucka, P. Krejcí, Š. Pospíšlova, V. Bryja in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Development of methodology: M. Kaucka, P. Krejcí Acquisition of data (provided animals, acquired and managed patients, Received May 7, 2012; revised November 29, 2012; accepted December 12, 2012; provided facilities, etc.): M. Kaucka, K. Plevova, S. Pavlova, P. Janovska, published OnlineFirst January 21, 2013.

References 1. Clevers H. Wnt/beta-catenin signaling in development and disease. 14. Hartmann TN, Grabovsky V, Wang W, Desch P, Rubenzer G, Wollner S, Cell 2006;127:469–80. et al. Circulating B-cell chronic lymphocytic leukemia cells display 2. Semenov MV, Habas R, Macdonald BT, He X. SnapShot: noncanonical impaired migration to lymph nodes and bone marrow. Cancer Res Wnt signaling pathways. Cell 2007;131:1378. 2009;69:3121–30. 3. Seifert JR, Mlodzik M. Frizzled/PCP signalling: a conserved mecha- 15. Redondo-Munoz J, Jose Terol M, Garcia-Marco JA, Garcia-Pardo A. nism regulating cell polarity and directed motility. Nat Rev Genet Matrix metalloproteinase-9 is up-regulated by CCL21/CCR7 interac- 2007;8:126–38. tion via extracellular signal-regulated kinase-1/2 signaling and is 4. Torban E, Kor C, Gros P. Van Gogh-like2 (Strabismus) and its role in involved in CCL21-driven B-cell chronic lymphocytic leukemia cell planar cell polarity and convergent extension in vertebrates. Trends invasion and migration. Blood 2008;111:383–6. Genet 2004;20:570–7. 16. Richardson SJ, Matthews C, Catherwood MA, Alexander HD, Carey 5. Caligaris-Cappio F, Ghia P. Novel insights in chronic lymphocytic BS, Farrugia J, et al. ZAP-70 expression is associated with enhanced leukemia: are we getting closer to understanding the pathogenesis ability to respond to migratory and survival signals in B-cell chronic of the disease? J Clin Oncol 2008;26:4497–503. lymphocytic leukemia (B-CLL). Blood 2006;107:3584–92. 6. Chiorazzi N, Rai KR, Ferrarini M. Chronic lymphocytic leukemia. N Engl 17. Nishita M, Itsukushima S, Nomachi A, Endo M, Wang Z, Inaba D, J Med 2005;352:804–15. et al. Ror2/Frizzled complex mediates Wnt5a-induced AP-1 acti- 7. Zenz T, Mertens D, Kuppers R, Dohner H, Stilgenbauer S. From vation by regulating Dishevelled polymerization. Mol Cell Biol pathogenesis to treatment of chronic lymphocytic leukaemia. Nat Rev 2010;30:3610–9. Cancer 2010;10:37–50. 18. Minami Y, Oishi I, Endo M, Nishita M. Ror-family receptor tyrosine 8. Kotaskova J, Tichy B, Trbusek M, Francova HS, Kabathova J, Mal- kinases in noncanonical Wnt signaling: their implications in devel- cikova J, et al. High expression of lymphocyte-activation gene 3 opmental morphogenesis and human diseases. Dev Dyn 2010; (LAG3) in chronic lymphocytic leukemia cells is associated with .239:1–15 unmutated immunoglobulin variable heavy chain region (IGHV) gene 19. Bryja V, Schulte G, Rawal N, Grahn A, Arenas E. Wnt-5a in- and reduced treatment-free survival. J Mol Diagn 2010;12:328–34. duces Dishevelled phosphorylation and dopaminergic differ- 9. Baskar S, Kwong KY, Hofer T, Levy JM, Kennedy MG, Lee E, et al. entiation via a CK1-dependent mechanism. J Cell Sci 2007; Unique cell surface expression of receptor tyrosine kinase ROR1 in 120:586–95. human B-cell chronic lymphocytic leukemia. Clin Cancer Res 20. Witze ES, Litman ES, Argast GM, Moon RT, Ahn NG. Wnt5a control of 2008;14:396–404. cell polarity and directional movement by polarized redistribution of 10. Fukuda T, Chen L, Endo T, Tang L, Lu D, Castro JE, et al. Antisera adhesion receptors. Science 2008;320:365–9. induced by infusions of autologous Ad-CD154-leukemia B cells iden- 21. Ghosh MC, Collins GD, Vandanmagsar B, Patel K, Brill M, Carter A, tify ROR1 as an oncofetal antigen and receptor for Wnt5a. Proc Natl et al. Activation of Wnt5A signaling is required for CXC chemokine Acad Sci U S A 2008;105:3047–52. ligand 12-mediated T-cell migration. Blood 2009;114:1366–73. 11. Rai KR, Sawitsky A, Cronkite EP, Chanana AD, Levy RN, Pasternack 22. Redondo-Munoz J, Escobar-Diaz E, Samaniego R, Terol MJ, Gar- BS. Clinical staging of chronic lymphocytic leukemia. Blood 1975; cia-Marco JA, Garcia-Pardo A. MMP-9 in B-cell chronic lympho- 46:219–34. cytic leukemia is up-regulated by alpha4beta1 integrin or CXCR4 12. Ghia P, Chiorazzi N, Stamatopoulos K. Microenvironmental inﬂuences engagement via distinct signaling pathways, localizes to podo- in chronic lymphocytic leukaemia: the role of antigen stimulation. somes, and is involved in cell invasion and migration. Blood J Intern Med 2008;264:549–62. 2006;108:3143–51. 13. Vaisitti T, Aydin S, Rossi D, Cottino F, Bergui L, D'Arena G, et al. CD38 23. Witte F, Bernatik O, Kirchner K, Masek J, Mahl A, Krejci P, et al. increases CXCL12-mediated signals and homing of chronic lympho- Negative regulation of Wnt signaling mediated by CK1-phosphorylat- cytic leukemia cells. Leukemia 2010;24:958–69. ed Dishevelled via Ror2. Faseb J 2010;24:2417–26.

OF10 Cancer Res; 73(5) March 1, 2013 Cancer Research

PCP Proteins Drive Pathogenesis of CLL

24. Strutt H, Price MA, Strutt D. Planar polarity is positively regulated by 35. Shultz LD, Lyons BL, Burzenski LM, Gott B, Chen X, Chaleff S, et al. casein kinase I epsilon in Drosophila. Curr Biol 2006;16:1329–36. Human lymphoid and myeloid cell development in NOD/LtSz-scid 25. Klein TJ, Jenny A, Djiane A, Mlodzik M. CKIepsilon/discs overgrown IL2R gamma null mice engrafted with mobilized human hemopoietic promotes both Wnt-Fz/beta-catenin and Fz/PCP signaling in Dro- stem cells. J Immunol 2005;174:6477–89. sophila. Curr Biol 2006;16:1337–43. 36. Burger JA. Chemokines and chemokine receptors in chronic lympho- 26. Rena G, Bain J, Elliott M, Cohen P. D4476, a cell-permeant inhibitor of cytic leukemia (CLL): from understanding the basics towards thera- CK1, suppresses the site-specific phosphorylation and nuclear exclu- peutic targeting. Semin Cancer Biol 2010;20:424–30. sion of FOXO1a. EMBO Rep 2004;5:60–5. 37. Hoellenriegel J, Meadows SA, Sivina M, Wierda WG, Kantarjian H, 27. KorinekV,BarkerN,MorinPJ,vanWichenD,deWegerR, Keating MJ, et al. The phosphoinositide 30-kinase delta inhib- Kinzler KW, et al. Constitutive transcriptional activation by a itor, CAL-101, inhibits B-cell receptor signaling and chemo- beta-catenin-Tcf complex in APC-/- colon carcinoma. Science kine networks in chronic lymphocytic leukemia. Blood 2011;118: 1997;275:1784–7. 3603–12. 28. Kaucka M, Krejci P, Plevova K, Pavlova S, Prochazkova J, Janovska P, 38. Friedberg JW, Sharman J, Sweetenham J, Johnston PB, Vose JM, et al. Post-translational modifications regulate signalling by Ror1. Acta Lacasce A, et al. Inhibition of Syk with fostamatinib disodium has Physiol (Oxf) 2011;203:351–62. significant clinical activity in non-Hodgkin lymphoma and chronic 29. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, et al. lymphocytic leukemia. Blood 2010;115:2578–85. Calcium sensitization of smooth muscle mediated by a Rho-associ- 39. Herman SE, Gordon AL, Wagner AJ, Heerema NA, Zhao W, Flynn JM, ated protein kinase in hypertension. Nature 1997;389:990–4. et al. Phosphatidylinositol 3-kinase-delta inhibitor CAL-101 shows 30. Gao Y, Dickerson JB, Guo F, Zheng J, Zheng Y. Rational design and promising preclinical activity in chronic lymphocytic leukemia by characterization of a Rac GTPase-specific small molecule inhibitor. antagonizing intrinsic and extrinsic cellular survival signals. Blood Proc Natl Acad Sci U S A 2004;101:7618–23. 2010;116:2078–88. 31. Chen B, Dodge ME, Tang W, Lu J, Ma Z, Fan CW, et al. Small molecule- 40. Weeraratna AT, Jiang Y, Hostetter G, Rosenblatt K, Duray P, Bittner M, mediated disruption of Wnt-dependent signaling in tissue regeneration et al. Wnt5a signaling directly affects cell motility and invasion of and cancer. Nat Chem Biol 2009;5:100–7. metastatic melanoma. Cancer Cell 2002;1:279–88. 32. Stacchini A, Aragno M, Vallario A, Alfarano A, Circosta P, Gottardi D, 41. Pukrop T, Klemm F, Hagemann T, Gradl D, Schulz M, Siemes S, et al. MEC1 and MEC2: two new cell lines derived from B-chronic et al. Wnt 5a signaling is critical for macrophage-induced invasion lymphocytic leukaemia in prolymphocytoid transformation. Leuk Res of breast cancer cell lines. Proc Natl Acad Sci U S A 2006;103: 1999;23:127–36. 5454–9. 33. Bertilaccio MT, Scielzo C, Simonetti G, Ponzoni M, Apollonio B, Fazi C, 42. Kurayoshi M, Oue N, Yamamoto H, Kishida M, Inoue A, Asahara T, et al. et al. A novel rag2-/-{gamma}c-/–xenograft model of human CLL. Expression of Wnt-5a is correlated with aggressiveness of gastric Blood 2010;115:1605–9. cancer by stimulating cell migration and invasion. Cancer Res 34. Badura L, Swanson T, Adamowicz W, Adams J, Cianfrogna J, Fisher K, 2006;66:10439–48. et al. An inhibitor of casein kinase I epsilon induces phase delays in 43. O'Connell MP, Fiori JL, Xu M, Carter AD, Frank BP, Camilli TC, et al. The circadian rhythms under free-running and entrained conditions. orphan tyrosine kinase receptor, ROR2, mediates Wnt5A signaling in J Pharmacol Exp Ther 2007;322:730–8. metastatic melanoma. Oncogene 2009;29:34–44.

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The Planar Cell Polarity Pathway Drives Pathogenesis of Chronic Lymphocytic Leukemia by the Regulation of B-Lymphocyte Migration

Markéta Kaucká, Karla Plevová, Sárka Pavlová, et al.

Cancer Res Published OnlineFirst January 21, 2013.

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