Activation of the in chronic lymphocytic leukemia

Desheng Lu*, Yandong Zhao*, Rommel Tawatao*, Howard B. Cottam*†, Malini Sen*, Lorenzo M. Leoni†‡§, Thomas J. Kipps*‡, Maripat Corr*†, and Dennis A. Carson*†¶

*Rebecca and John Moores UCSD Center and ‡Department of Medicine, University of California at San Diego, La Jolla, CA 92093-0663

Contributed by Dennis A. Carson, December 24, 2003 chronic lymphocytic leukemia (CLL) is characterized by an The goal of this study was to determine whether the canonical accumulation of mature, functionally incompetent B cells. Wnts are Wnt signaling pathway is active in CLL cells, especially in the a large family of secreted glycoproteins involved in cell prolifera- aggressive CLL subgroup. We hypothesized that some of the tion, differentiation, and oncogenesis. The classical Wnt signaling molecular heterogeneity leading to different clinical outcomes in cascade inhibits the activity of the enzyme glycogen synthase CLL cells might be mediated by enhanced expression of Wnt and kinase-3␤, augmenting ␤-catenin translocation to the nucleus, and Fzd family members and augmented signaling through the the transcription of target . Little is known about the poten- ␤-catenin signaling pathway. Hence, we surveyed CLL, normal tial roles of Wnt signaling in CLL. In this study, we quantified the peripheral blood leukocytes, and B cells for their expression expression profiles of the Wnt family, and their cognate patterns of all of the known Wnt and Fzd family members by (Fzd) receptors in primary CLL cells, and determined the real-time PCR. The CLL samples were further subdivided into role of Wnt signaling in promoting CLL cell survival. Wnt3, Wnt5b, groups that expressed mutated and germ-line IgV genes. The Wnt6, Wnt10a, Wnt14, and Wnt16, as well as the Wnt gene expression analyses revealed that several Wnt genes from Fzd3, were highly expressed in CLL, compared with normal B cells. the classical and alternate groups were overexpressed in the Three lines of evidence suggested that the Wnt signaling pathway leukemia cells, compared with normal B cells. Activation of was active in CLL. First, the Wnt͞␤-catenin-regulated transcription Wnt͞␤-catenin signaling with a small-molecule GSK-3␤ inhibi- factor lymphoid-enhancing factor-1, and its downstream target tor enhanced the survival of CLL cells, whereas antagonism of cyclin D1, were overexpressed in CLL. Second, a pharmacological this pathway with an analog of a nonsteroidal antiinflammatory inhibitor of glycogen synthase kinase-3␤, SB-216763, activated drug had the exact opposite effect. These data suggest that the ␤-catenin-mediated transcription, and enhanced the survival of CLL Wnt pathway is not only active in CLL but also can be manip- lymphocytes. Third, Wnt͞␤-catenin signaling was diminished by an ulated pharmacologically. Thus, Wnt signaling may be an at- analog of a nonsteroidal antiinflammatory drug (R-etodolac), at tractive therapeutic target in CLL. concentrations that increased apoptosis of CLL cells. Taken to- gether, these results indicate that Wnt signaling genes are over- Materials and Methods expressed and are active in CLL. Uncontrolled Wnt signaling Cell Isolation and Culture. Protocols for the use of human samples may contribute to the defect in apoptosis that characterizes this were reviewed and approved by the University of California at San malignancy. Diego Institutional Review Board. Blood samples were collected by the Chronic Lymphocytic Leukemia Research Consortium, after cell chronic lymphocytic leukemia (CLL) is the most obtaining informed consent from patients fulfilling diagnostic Bcommon human leukemia. Characterized by a progressive criteria for CLL, at all disease stages. Mononuclear cells were expansion of apparently quiescent B cells, it generally follows an isolated by density-gradient centrifugation over Ficoll͞Hypaque indolent course (1). However, the subset of CLL that has and contained at least 85% B cells as determined by fluorescent germ-line IgV genes, and expresses the ZAP70 tyrosine kinase, CD19 staining. The cells were suspended in RPMI medium 1640, has a more rapid course, usually leading to death within 5 years with 10% FBS, and antibiotics at 37°C, 5% CO2. The human (2–6). The fundamental defect in CLL is thought to be abnormal embryonic kidney cell line HEK293 (American Type Culture regulation of apoptosis, because the circulating leukemia cells Collection) was maintained in DMEM with high glucose supple- have a low growth faction. However, very little is known about mented with 10% FBS and antibiotics. the molecular mechanisms responsible for the prolonged sur- vival of these malignant B cells. RNA Isolation and Real-Time PCR. Total RNA was isolated from 1 ϫ Recent microarray analyses have demonstrated that the 106 cells by TRIzol reagent (Invitrogen). The RNA samples were Wnt3 gene is overexpressed in CLL, compared with normal B further purified by using a Qiagen RNeasy Protect kit (Qiagen, and T cells (7). The Wnt family of secreted glycoproteins Valencia, CA). The mRNA levels were quantified in duplicate by regulate early B cell growth and survival (8, 9). Aberrant real-time RT-PCR on the ABI Prism 7700 sequence detection activation of the Wnt signaling pathway has major oncogenic system (Applied Biosystems) by using the primer sets and reaction effects (10, 11). Binding of Wnt to its receptor complex, conditions in Supporting Methods and Table 2, which are published consisting of a member of the Frizzled (Fzd) family (12–15), as supporting information on the PNAS web site. The data were and in some instances of the low-density lipoprotein-receptor- related (LRP)5 or LRP6 (16–18), leads to stabiliza- tion of ␤-catenin by inhibiting the phosphorylating activity of Abbreviations: CLL, chronic lymphocytic leukemia; Fzd, Frizzled; GSK, glycogen synthase ␤ kinase; TCF, T cell factor; LEF, lymphoid-enhancing factor; LRP, low-density lipoprotein- the glycogen synthase kinase (GSK)-3 . Unphosphorylated receptor-related ; DiOC6, 3,3Ј-dihexyloxacarbocyanine iodide; MTT, 3-[4,5- ␤-catenin accumulates in the cytoplasm and translocates into dimethylthiazol-2-yl]-2,5-dipheyl tetrazolium bromide; PBL, peripheral blood lymphocyte. the nucleus, where it activates target gene expression through †H.B.C., L.M.L., M.C., and D.A.C. are consultants for Salmedix, Inc., a biotechnology com- interacting with T cell (TCF) and lymphoid-enhancing (LEF) pany that is developing R-etodolac for the treatment of cancer. transcription factors (19, 20). This Wnt signaling cascade has §Present address: Salmedix, Inc., 9380 Judicial Drive, San Diego, CA 92121. been termed classical or canonical, in contrast to the recently ¶To whom correspondence should be addressed at: University of California at San Diego, ϩ described Wnt-induced alternative effects on Ca2 uptake and 9500 Gilman Drive, La Jolla, CA 92093-0663. E-mail: [email protected]. protein kinase C activation (21). © 2004 by The National Academy of Sciences of the USA

3118–3123 ͉ PNAS ͉ March 2, 2004 ͉ vol. 101 ͉ no. 9 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308648100 Downloaded by guest on October 1, 2021 Table 1. Expression profile of Wnt and Fzd family in CLL vs. B cells and PBLs B cells PBLs CLL (mutated) CLL (unmutated)

Genes Mean SD Mean SD Mean SD Mean SD

Wnt Wnt-1 14.72 1.17 11.52 0.50 13.34 2.01 14.12 2.50 Wnt-2a 17.81 2.52 23.40 4.02 17.16 1.77 18.80 2.82 Wnt-2b 15.03 1.03 15.11 0.51 14.52 1.75 13.29 2.35 Wnt-3 23.79 3.38 25.24 2.90 14.54 3.44 13.76 3.48 Wnt-3a 26.50 0.80 27.11 0.11 26.74 0.42 27.17 0.71 Wnt-4 15.95 1.08 17.04 1.25 15.67 1.76 15.16 1.98 Wnt-5a 18.32 1.28 13.44 1.22 19.28 4.33 18.68 5.23 Wnt-5b 15.38 0.37 17.33 0.84 15.51 1.91 11.87 2.90 Wnt-6 15.71 1.90 18.71 0.45 12.95 2.14 13.20 1.34 Wnt-7a 18.78 3.61 11.81 0.99 14.09 2.21 15.43 2.94 Wnt-7b 17.81 1.30 25.98 1.99 15.13 3.23 16.40 2.80 Wnt-8a 16.12 1.65 18.27 1.33 14.92 1.12 15.89 1.51 Wnt-8b 16.37 0.85 13.53 0.69 12.79 1.26 13.61 1.31 Wnt-10a 12.22 0.65 15.61 0.59 9.78 1.37 10.52 2.03 Wnt-10b 18.05 0.79 14.91 0.39 15.72 2.04 16.25 2.36 Wnt-11 20.86 2.58 17.36 1.02 21.69 3.30 21.34 3.70 Wnt-14 23.88 3.38 22.59 4.06 14.92 1.49 12.52 2.61 Wnt-16 14.88 2.70 17.11 3.12 9.11 1.95 9.17 2.10 Fzd Fzd-1 16.95 0.78 15.33 0.68 14.92 2.33 15.15 2.74 Fzd-2 14.82 1.59 12.70 0.26 13.14 1.16 12.73 1.47 Fzd-3 11.14 0.22 11.39 0.38 8.31 1.33 8.62 1.34 Fzd-4 16.80 0.81 15.21 0.26 15.67 1.60 16.80 2.18 Fzd-5 12.85 0.33 13.81 0.57 11.36 1.16 12.30 1.35 Fzd-6 10.23 1.25 11.62 1.36 10.43 2.32 9.80 1.67 Fzd-7 16.57 0.87 18.84 1.23 16.24 2.23 15.92 2.65 Fzd-8 13.61 1.14 15.18 0.68 16.50 2.02 16.79 2.21 Fzd-9 15.08 0.65 17.76 0.68 17.27 1.79 18.58 2.63 Fzd-10 24.31 2.73 26.90 0.44 25.43 2.14 25.23 2.75

Total RNA was isolated from healthy peripheral blood B cells (n ϭ 3), healthy PBLs (n ϭ 3), and CLL cells, including 10 mutated and MEDICAL SCIENCES 13 germ-line CLL specimens. Gene expression of Wnt and Fzd family members was detected by real-time PCR. The relative gene expression level was obtained by subtracting the Ct value of 18S (control gene) from the Ct value of the target gene, generating the ⌬Ct value. The Wnt and Fzd family members, which showed significantly greater gene expression in CLL than in normal controls, are indicated in bold (P Յ 0.001 by ANOVA).

analyzed by using the comparative Ct method, where Ct is the cycle vector (Invitrogen). The expression of the Wnt16 and Fzd5 number at which fluorescence first exceeds the threshold. The ⌬Ct vectors was confirmed by transfection of HEK293 cells, followed values from each tissue were obtained by subtracting the values for by immunoblotting or RT-PCR, respectively. 18S Ct from the sample Ct. One difference of Ct value represents a 2-fold difference in the level of mRNA. Cell Transfection. HEK293 cells were transfected in 12-well plates by using FuGENE (Roche, Mannheim, Germany), and ␮ ␮ RT-PCR Analyses. Different pairs of gene-specific primers, based on 0.5 g of reporter plasmid, 0.1–0.2 g of the control plasmid pCMX␤gal, 100–200 ng of the various expression plasmids, GenBank sequences of cloned human LEF and TCF genes, were ␮ used for amplification of cDNA from CLL cells and normal and carrier DNA pBluescriptKSII, for a total of 1 g per well. lymphocytes (Table 3, which is published as supporting information After overnight incubation, the cells were washed and given on the PNAS web site). Reverse transcription was performed with fresh medium containing the different drugs or vehicle. For a SuperScript preamplification kit (Invitrogen). One microgram of luciferase assays, cells were lysed, and light emission was detected in the presence of luciferin by using a microtiter plate RNA and 30 cycles of amplification were used. After electrophore- luminometer (MicroBeta TriLux, Gaithersburg, MD). The sis, the amplicons were visualized under UV light. luciferase values were normalized for variations in transfection ␤ ␤ ͞ efficiency by using the -galactosidase internal control, and Plasmids. The -catenin expression plasmid and the TCF LEF are expressed as fold stimulation of luciferase activity, com- TOPflash reporter plasmid were gifts from H. Clevers (Hub- pared with the designated control cultures. All of the trans- recht Laboratory, Utrecht, The Netherlands). An expression fection results are representative of a minimum of three plasmid for LRP6 (phLRP6-V5) was the kind gift of B. O. independent transfections. Williams (Van Andel Research Institute, Grand Rapids, MI) ␤ (22). pCMX gal was used as a control plasmid (23). Expression Drugs. R-etodolac was prepared by fractional crystallization, plasmids for human Fzd3 (Origene Technologies, Rockville, using a modification of a literature procedure (ref. 24 and MD), Wnt1, Wnt3, Wnt5a, and Wnt5b (Upstate Biotechnology, Supporting Methods). SB-216763 was obtained from Sigma (25). Lake Placid, NY) were purchased. The cDNA for Wnt16 and Fzd5 were amplified from human blood and Jurkat cells, re- Flow Cytometry. CLL cells were plated at a density of 1 ϫ 107 cells spectively, and were then cloned into the pcDNA3 expression per ml and treated with IL-4 (10 ng͞ml,fromR&DSystems),

Lu et al. PNAS ͉ March 2, 2004 ͉ vol. 101 ͉ no. 9 ͉ 3119 Downloaded by guest on October 1, 2021 R-etodolac (300–500 ␮M), SB-216763 (5 ␮M), or vehicle for 48 h. The cells were removed from the plate and incubated for 15 min in medium with 5 ␮g͞ml propidium iodide and 40 nM Ј 3,3 -dihexyloxacarbocyanine iodide (DiOC6), and were then analyzed with a FACSCalibur fluorescence-activated cell sorter (Becton Dickinson). Viable cells had high DiOC6 (FL-1) and low propidium iodide (FL-3) fluorescence, whereas apoptotic cells had low DiOC6 (FL-1) and low propidium iodide (FL-3) fluorescence.

3-[4,5-Dimethylthiazol-2-yl]-2,5-dipheyl Tetrazolium Bromide (MTT)- Based Cell Survival Assay. Briefly, 0.5 ϫ 106 cells CLL cells in 96-well plates were treated with graded concentrations of R- etodolac, SB-216763, or vehicle. After 72 h, 20 ␮lofMTT solution was added to each well. Six hours later, the cells were lysed, and absorbances at 590 and 650 nM were measured. The assays were performed in triplicate, and the results represent the mean values Ϯ SD.

Immunoblotting. Cells were disrupted in lysis buffer and blotted as described in Supporting Methods. Primary antibodies against GSK-3␤ (BD Biosciences), pY216 GSK-3␤ (BD Biosciences), ␤-catenin (Cell Signaling Technology, Beverly, MA), and ␤-actin (Chemicon International) were used. Horseradish peroxidase- conjugated anti-IgG (Santa Cruz Biotechnology) was used as the secondary antibody. Antibody binding was visualized by en- hanced chemiluminescence (ECL; Amersham Pharmacia Life Science, Aylesbury, U.K.).

Statistical Analysis. The gene expression levels in CLL, CLL subsets, and normal B cells were compared by ANOVA. Only a P value Յ0.001 was considered significant because of the number of genes compared (Ϸ40). Results Wnt and Fzd Genes Expressed by Normal and CLL B Cells. Several Wnt family member transcripts were detectable in normal and ma- Fig. 1. Relative expression of Wnts and Fzd3 in CLL cells. The relative mRNA lignant B cells (Table 1). In the CLL cells, Wnt16 and Wnt10a levels of Wnt3 (A), Wnt5b (B), Wnt6 (C), Wnt10a (D), Wnt14 (E), Wnt16 (F), and were most abundant, with ⌬Ct values of 10 or below. Compared Fzd3 (G) were detected by using real-time PCR in normal peripheral blood B with normal B cells, the CLL samples had significantly higher cells (n ϭ 3), unfractionated PBLs (n ϭ 3), and CLL cells, including mutated levels of Wnt3, Wnt5b, Wnt6, Wnt10a, Wnt14, and Wnt16 (Fig. (mut) (n ϭ 10), and unmutated (unmut) (n ϭ 13) CLL specimens. The relative 1). Notably, the aggressive CLL subset with germ-line IgV genes expression levels were determined by normalizing the ⌬Ct values against the ⌬ expressed higher levels of Wnt3, Wnt5b, and Wnt14 than did the average Ct values for normal B cells for the specified genes. mutated samples. Both Fzd3 and Fzd6 were prominent in normal and malignant B cells. Fzd3 levels were significantly higher in stream targets of Wnt and ␤-catenin signaling (30, 31). The CLL CLL than in normal B cells (Fig. 1). In addition, LRP5 and LRP6 cells had an Ϸ3-fold higher level of cyclin D1 mRNA than did mRNA transcripts were readily detectable in the CLL cells normal B cells (Fig. 3B). (mean ⌬Ct values of 13.2 Ϯ 2.5 and 16 Ϯ 1.6, respectively). ␤ ͞ GSK-3 Inhibition Enhances CLL Survival. The phosphorylation of Wnt3 Activates the TCF LEF Promoter. The HEK293 cell line was ␤ ␤ transfected with the TCF and ␤-catenin dependent TOPflash -catenin by GSK-3 marks it for ubiquitination and degradation. Inhibition of this activity allows unbound ␤-catenin to accumulate reporter plasmid, and with various combinations of Wnt, Fzd, ͞ and LRP expression plasmids (Fig. 2). Transfection with a Wnt3 in the cytoplasm, translocate into the nucleus, and activate TCF LEF-dependent transcription. In HEK293 cells that were cotrans- plasmid resulted in a small increase in reporter gene activity, ␤ ␤ which was augmented by the transfection with the LRP6 core- fected with the TOPflash and -catenin plasmids, the GSK-3 ceptor (Fig. 2A). Further synergy was seen after cotransfection inhibitor SB-216763 substantially enhanced reporter gene activity with Fzd3 or Fzd5 expression plasmids (Fig. 2B). The activation (Fig. 4A). CLL cells treated with the same compound had less active ␤ of the TOPflash reporter was specific to only certain Wnt genes, GSK-3 , as demonstrated by diminished tyrosine 216-phosphory- as reported (26–28). Thus, the Wnt1 and Wnt3 plasmids stim- lation detectable by immunoblotting (Fig. 4B). Moreover, incuba- ␤ ulated the reporter, whereas Wnt16, Wnt5a, and Wnt5b did not, tion of CLL cells with the GSK-3 inhibitor at concentrations up under the conditions tested (Fig. 2C). to 7 ␮M improved their survival in culture, and reduced apoptotic cell death (Fig. 4 C and D). LEF1 and Cyclin D1 Overexpression in CLL. The canonical Wnt signaling pathway is mediated by transcription factors of the Inhibition of ␤-Catenin Signaling Diminishes Cell Survival. Various TCF͞LEF family (29). Whereas LEF1 transcripts were barely nonsteroidal antiinflammatory drugs have been reported to detectable in normal B cells, the CLL samples expressed this inhibit ␤-catenin signaling, independent of cyclooxygenase in- gene in abundance (Fig. 3A). TCF7 was not detectable in any of hibition (32, 33). The R-enantiomer of the nonsteroidal antiin- the samples tested. The cyclin D genes are established down- flammatory drug etodolac (R-etodolac), which lacks cyclooxy-

3120 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308648100 Lu et al. Downloaded by guest on October 1, 2021 Fig. 3. Elevated expression of the LEF1 and cyclin D1 in CLL cells compared with normal B cells. (A) The indicated TCF transcription factor family members were amplified by RT-PCR in normal B cells and CLL specimens. (B) Cyclin D1 expression in normal B cells, PBLs, and CLL cells. The expression of cyclin D1 was assessed by real-time PCR. The relative expression level of cyclin D1 was determined by normalizing the ⌬Ct value against the B cell value. The gene expression in B cells was arbitrarily set to 1. Cyclin D1 levels were significantly higher in the CLL cells, compared with normal lymphocytes (P Ͻ 0.0005 by MEDICAL SCIENCES ANOVA).

a prolonged lifespan. A central aim of CLL research is to discover the factors that both induce the growth and sustain the lifespan of the malignant B cells, and then to develop therapeutic agents that can interfere specifically with their actions. As potential candidates, we investigated the expression and func- Fig. 2. Role of different Wnts and Fzds in activating ␤-catenin͞TCF-mediated tional role of the Wnt and Fzd receptor families in CLL. transcription. HEK293 cells were cotransfected with the TOPflash reporter The 19 known human Wnt genes are secreted growth factors construct and a ␤-galactosidase vector, along with the indicated plasmids. All that bind to 10 or more Fzd receptors, to induce a complex cells were harvested 48 h after transfection, and cell extracts were assayed for signaling cascade that regulates cell growth and differentiation luciferase and ␤-galactosidase activities. The ␤-galactosidase activity was used during embryogenesis. Considering that many arise from to confirm consistent transfection efficiencies (data not shown). (A) Wnt3- immature cells, it seemed logical that one or more Wnt genes activated TCF-response elements in the presence of LRP6. HEK293 cells were could be overexpressed and functionally important in some cotransfected with Wnt3 and LRP6 plasmids. (B) Fzd3 and Fzd5 enhanced Wnt3-mediated transcription. HEK293 cells were cotransfected with expres- malignancies. sion plasmids for Wnt3, LRP6, Fzd3, and Fzd5, as indicated. (C) Unlike Wnt1 Our experiments showed that six of 19 Wnt genes (Wnt 3, and Wnt3, expression of the Wnt5a, Wnt5b, and Wnt16 genes did not activate Wnt5b, Wnt6, Wnt10a, Wnt14, and Wnt16) were significantly the TCF-response element. Vectors expressing Wnt1, Wnt3, Wnt5a, Wnt5b, overexpressed in CLL, compared with normal B cells. Fzd3 and and Wnt16 were cotransfected into HEK293 cells in the presence of both LRP6 its coreceptors LRP5 and 6 were also abundantly expressed in and Fzd5. the malignant lymphocytes, as was the Wnt-regulated transcrip- tion factor LEF1. Three CLL-specific Wnt genes (Wnt3, Wnt5b, and Wnt14) were present at higher levels in the CLL subgroup genase inhibitory activity (34, 35), reduced TOPflash activity in with germ-line IgV genes than in the subgroup with mutated V HEK293 cells (Fig. 5A). The same concentrations of R-etodolac genes, with Wnt5b showing the greatest disparity. ͞␤ that inhibited Wnt -catenin signaling also shortened the in vitro A previous microarray study identified Wnt3 as a ‘‘signature’’ survival of the CLL cells (Fig. 5 B–D). gene in CLL (7). The homologous gene, Wnt3a, promoted the proliferation of mouse pro-B cells in the bone marrow by Discussion initiating signaling events leading to ␤-catenin-dependent acti- The expansion of a malignantly transformed cell clone is the vation of the transcription factor LEF1 (8). Further tissue outcome of an abnormal balance between proliferation and analyses suggested that most of the Wnt3a was expressed by bone death. In CLL, the abnormal lymphocytes grow slowly, but have marrow stromal cells. The fact that mice genetically deficient in

Lu et al. PNAS ͉ March 2, 2004 ͉ vol. 101 ͉ no. 9 ͉ 3121 Downloaded by guest on October 1, 2021 Fig. 4. Activation of ␤-catenin͞TCF signaling and enhancement of CLL survival by a GSK-3␤ inhibitor. (A) SB-216763 activates the TCF-response elements. HEK293 cells were transfected with the TOPflash reporter and with or without the expression plasmid for ␤-catenin. Transfected cells were treated with the GSK-3␤ inhibitor SB-216763 (5 ␮M) or with the DMSO vehicle control for 24 h. (B) SB-216763 increased ␤-catenin levels by inhibiting GSK-3␤ in CLL cells. CLL cells from four specimens were incubated with 5 ␮M SB-216763 or with the positive control survival factor IL-4 (10 ng͞ml) for 48 h. Whole-cell lysates were immunoblotted with the indicated antibodies. (C) The prosurvival activity of SB-216763 in CLL cells. CLL cells (0.5 ϫ 106 cells per well) were incubated with SB-216763 for 72 h before determinations of viability by MTT assay. The mean incremental survival measured in triplicate and the SD are shown. (D) SB-216763 protected CLL cells from apoptosis. CLL cells were incubated with 5 ␮M SB-216763 or 10 ng͞ml recombinant human IL-4 for 48 h. The mitochondrial transmembrane potential was measured by flow cytom- Fig. 5. R-etodolac partially blocked canonical Wnt signaling and induced etry by using the dye, DiOC6. Three specimens of CLL cells were analyzed. apoptosis of CLL cells. (A) R-etodolac partially blocked ␤-catenin- and Wnt3͞ LRP6-mediated transcription in a dose-dependent manner. The ␤-catenin- and TCF͞LEF-regulated TOPflash reporter plasmid was cotransfected into HEK293 LEF1 had defects in pro-B cell proliferation and survival impli- cells with expression plasmids for ␤-catenin, Wnt3, and LRP6. The transfected cated the Wnt pathway in the regulation of immature B cell cells were then treated with R-etodolac. (B) Cytotoxic effects of R-etodolac in development. However, there was little or no expression of CLL cells. CLL cells were incubated with R-etodolac, and viability was measured Wnt3a or LEF1 in mature germinal center B cells (8). by the MTT assay after 72 h. Representative results for three different patients are shown. (C) Proapoptotic effects of R-etodolac in CLL cells. CLL cells, PBLs, Wnt16 was originally discovered as a gene that is overex- ␮ pressed in leukemia and normal pro-B cells (36, 37). Our and B cells were incubated with 250 M R-etodolac for 24 and 48 h. Apoptosis was measured by flow cytometry by using DiOC6͞PI staining. (D) Two repre- investigations revealed that Wnt16 was similarly overabundant in sentative examples of the proapoptotic effect of R-etodolac are shown. CLL cells, compared with normal B cells. In transfection studies with HEK293 cells, Wnt3 activated the canonical ␤-catenin Wnt signaling pathway. However, Wnt16 did not induce TCF͞LEF- for immature B cells and their malignant counterparts, its dependent transcription, even after overexpression of Fzd and function in lymphocyte proliferation and survival remains to be LRP6 genes. Thus, whereas Wnt16 appears to be a marker established.

3122 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308648100 Lu et al. Downloaded by guest on October 1, 2021 Fzd3 was the predominant Fzd gene in CLL cells. However, inhibited the transcription of a ␤-catenin-dependent TCF͞LEF both CLL and normal B cells did express other Fzd genes. reporter gene in HEK293 cells, and at the same concentrations, Recently, Fzd gene expression has been examined in other B cell diminished the in vitro survival of CLL cells. malignancies (9). Myeloma cells were found to express multiple The expression of all of the components of the canonical Wnt Fzd genes, whereas B lymphoma cell lines expressed predomi- signaling cascade in CLL cells, together with the observed effects nantly Fzd3. Unlike the B lymphoma cell lines, our experiments of ␤-catenin activators and inhibitors, implicate the Wnt pathway revealed that the LRP5 and LRP6 genes were readily detectable in CLL pathogenesis, and suggest that it could play a role in in primary CLL cells. extending the lifespan of the malignant B cells. If CLL cell The synthesis of a growth factor receptor and ligand by the survival is more dependent on Wnt pathway activation than is same malignant cell is a well established autocrine pathway in the the survival of normal cells, then Wnt evolution of cancer. The CLL clones expressed both Wnt growth antagonists could have therapeutic benefit in the disease. Even factors and the major elements of the canonical Wnt signaling if Wnt signaling is necessary for the survival of some normal pathway. However, the detection of a complete Wnt͞Fzd͞LRP͞ ␤ ͞ cells, the Wnt and Fzd genes that are overexpressed in CLL -catenin LEF1 gene expression signature in CLL cells did not could represent attractive targets for the development of more prove that the canonical Wnt signaling pathway was functional. ␤ specific pharmacologic antagonists, including antibodies and We used pharmacologic modulators of -catenin-dependent cytotoxins (38). function to study the effects of Wnt signaling on CLL survival. A GSK-3␤ inhibitor, which would effectively release active ␤ We thank Dr. Laura Rassenti, Michael Rosenbach, and Kathy Pekny for -catenin, and mimic constitutive canonical Wnt signaling, their assistance. This work was supported in part by National Institutes prolonged the survival of the malignant B cells. of Health Grants CA23100, AR44850, CA81534, and GM23200 and the Several compounds related to the nonsteroidal antiinflamma- University of California Biotechnology Strategic Targets for Alliances in tory drugs have been reported recently to inhibit ␤-catenin Research Project (BioSTAR). T.J.K. and D.A.C. are members of the stability or function (32, 33). The R-enantiomer of etodolac Chronic Lymphocytic Leukemia Research Consortium.

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