A Novel Tankyrase Inhibitor Decreases Canonical Wnt Signaling in Colon Carcinoma Cells and Reduces Tumor Growth in Conditional APC Mutant Mice

Published OnlineFirst March 22, 2012; DOI: 10.1158/0008-5472.CAN-11-3336

Cancer Therapeutics, Targets, and Chemical Biology Research

Jo Waaler1, Ondrej Machon1,6, Lucie Tumova6, Huyen Dinh1, Vladimir Korinek6, Steven Ray Wilson2, Jan Erik Paulsen3, Nina Marie Pedersen4, Tor J. Eide5, Olga Machonova1,6, Dietmar Gradl7, Andrey Voronkov1, Jens Peter von Kries8, and Stefan Krauss1

Abstract Increased nuclear accumulation of b-catenin, a mediator of canonical Wnt signaling, is found in numerous tumors and is frequently associated with tumor progression and metastasis. Inhibition of Wnt/b-catenin signaling therefore is an attractive strategy for anticancer drugs. In this study, we have identiﬁed a novel small molecule inhibitor of the b-catenin signaling pathway, JW55, that functions via inhibition of the PARP domain of tankyrase 1 and tankyrase 2 (TNKS1/2), regulators of the b-catenin destruction complex. Inhibition of TNKS1/2 poly(ADP-ribosyl)ation activity by JW55 led to stabilization of AXIN2, a member of the b-catenin destruction complex, followed by increased degradation of b-catenin. In a dose-dependent manner, JW55 inhibited canonical Wnt signaling in colon carcinoma cells that contained mutations in either the APC (adenomatous polyposis coli) locus or in an allele of b-catenin. In addition, JW55 reduced XWnt8-induced axis duplication in Xenopus embryos and tamoxifen-induced polyposis formation in conditional APC mutant mice. Together, our ﬁndings provide a novel chemotype for targeting canonical Wnt/b-catenin signaling through inhibiting the PARP domain of TNKS1/2. Cancer Res; 1–11. 2012 AACR.

Introduction tion complex consisting of adenomatous polyposis coli (APC), b a The Wnt/b-catenin signaling pathway is a key regulator in AXIN2, GSK3 , and CK1 (4, 5). Tankyrase 1 and 2 (TNKS1/2) b numerous cellular processes including stem cell maintenance, regulate the stability of the -catenin destruction complex fate decision, and cell-cycle control (1, 2). The main denom- through AXIN2 by poly(ADP-ribosyl)ation (6). Although inator of canonical Wnt signaling, b-catenin, has several TNKS1/2 modulates the destruction complex, this complex cellular functions. At the cell membrane, it is associated with interferes with TNKS. Evidence suggests that TNKS is phos- – b E-cadherin and participates in the formation of the adherens phorylated in a cell-cycle dependent manner by GSK3 (7). junctions (3). In the cytoplasm, b-catenin can form complexes TNKS is furthermore controlled by the ubiquitin E3 ligase with a multitude of proteins, including the b-catenin destruc- RNF146 that destabilizes TNKS1/2, AXIN2, and itself by ubi- quitinylation (8, 9). In the standard model, the destruction complex regulates the sequential phosphorylation of b-catenin Authors' Afﬁliations: 1Oslo University Hospital, SFI-CAST Biomedical by the kinases CK1a at S45 and by GSK3b at S33, S37, and T41. Innovation Center, Unit for Cell Signaling, Forskningsparken, Gaustadal- b leen and Center for Molecular Biology and Neuroscience; 2Department of This leads to polyubiquitination by -TrCP and subsequent Chemistry, University of Oslo; 3Norwegian School of Veterinary Science, degradation of b-catenin by the proteasome (10, 11). Active Department of Food Safety and Infection Biology; 4Institute for Cancer Wnt signaling inhibits the function of the destruction complex, Research, Department of Biochemistry, The Norwegian Radium Hospital, b Stem Cell Innovation Centre, 5Department of Pathology, Rikshospitalet, and -catenin is not phosphorylated at the N-terminus and Oslo University Hospital, Oslo, Norway; 6Institute of Molecular Genetics, degraded, but enters the nucleus. Nuclear uptake of b-catenin 7 Czech Academy of Sciences, Prague, Czech Republic; KIT (Karlsruhe may be further enhanced by the context-dependent C-terminal Institute of Technology) Zoological Institute, Cell and Developmental Biol- ogy, Karlsruhe; and 8Leibniz-Institut Fur€ Molekulare Pharmakologie, FMP, phosphorylation of b-catenin at S675, leading to increased Berlin, Germany nuclear translocation (12). In addition to canonical Wnt sig- Note: Supplementary data for this article are available at Cancer Research naling, a variety of alternative cellular mechanisms may trigger Online (http://cancerres.aacrjournals.org/). altered nuclear accumulation of b-catenin. These mechanisms Corresponding Authors: Jo Waaler, Oslo University Hospital, SFI-CAST include the Hif-1a signaling pathway, downregulation of E- Biomedical Innovation Center, Unit for Cell Signaling, Forskningsparken, cadherin or C-terminal phosphorylation of b-catenin through Gaustadalleen, 21, 0349 Oslo, Norway and Center for Molecular Biology – and Neuroscience, P.O. Box 1105, Oslo N-0317, Norway. Phone: 47-9514- HGF-activated cMet (13 15). Furthermore, PKA-involving 8669; Fax: 47-2295-8150; E-mail: [email protected]; and Stefan mechanisms can lead to C-terminal phosphorylation of b-cate- Krauss, E-mail: [email protected] nin and enhance its nuclear presence (12). Recently it was doi: 10.1158/0008-5472.CAN-11-3336 discovered that the primary cilium diverts Jouberin from the 2012 American Association for Cancer Research. nucleus and limits b-catenin nuclear entry (16).

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In the nucleus, b-catenin exerts a number of functions, such 10 mmol/L JW55 for RKO cells and 0.1% DMSO or 10, 5, or as binding to the transcription factor TCF/LEF by replacing 1 mmol/L JW55 for SW480 cells. All samples consisted of a Groucho and activation of downstream transcription of genes, minimum of 6 replicates. The plate was incubated in an Incu- including c-Myc, Cyclin D1, and AXIN2 (2). Nuclear b-catenin Cyte (Essen BioScience) inside a cell culture incubator. Images levels are not only determined by the rate of b-catenin import were captured every second hour to monitor proliferation. to the nucleus but also by "nuclear trapping" of b-catenin through factors such as TCF/LEF (17). Colony assay In general, mutations in genes encoding central components A total of 400 SW480, RKO, or HeLa cells were seeded in 6-cm of the Wnt/b-catenin pathway and the destruction complex plates and 1% FBS. The day after, the medium was exchanged lead to the accumulation of nuclear b-catenin and contribute to 0.05% DMSO or 5 mmol/L JW55 for RKO and HeLa cells, and to tumor initiation and progression (18, 19). Wnt activating 0.1% DMSO or 5, 1, or 0.1 mmol/L JW55 for SW480 cells mutations are found in a broad range of solid tumors including (minimum 3 replicates). The cells were grown until colonies colon cancer, gastric cancer, hepatocellular carcinoma, breast appeared in the control plates and medium was changed twice cancer, medulloblastoma, melanoma, non–small cell lung can- a week. The plates were stained and fixed with 0.2% methylene cer, pancreas adenocarcinoma, and prostate cancer (20). It has blue in methanol and washed with PBS. The colonies were been well established that the majority of intestinal neoplasia counted manually with a colony counter (Scienceware). Rep- with deregulated Wnt signaling harbor truncating mutations in resentative pictures of colonies were captured with a Leica MZ both alleles of the APC tumor suppressor gene (21). Apo Stereomicroscope connected to a Leica DC200 camera Considerable efforts have been made to identify drugs and Adobe Photoshop. that inhibit Wnt/b-catenin signaling (18, 22–25). Published inhibitory drugs that interact with biotargets directly asso- Tumor initiation and treatment in ApcCKO/CKO/Lgr5- ciated with canonical Wnt signaling include WAY-316606 CreERT2 mice for SFRP (26), niclosamide for frizzled (27), NCS668036 Seven 12-week old female ApcCKO/CKO/Lgr5-CreERT2 mice for Dsh (28), pyrvinium for CK1 (24), XAV939 and IWR-1 were injected intraperitonally with 25 mg/kg of tamoxifen for TNKS1/2 (4, 22), 2,4-diamino-quinazoline, quercetin, (Sigma) diluted in an ethanol and corn oil (ratio 1:4). The mice b PKF115-584 and ICG-001 for -catenin and its binding to were randomized into 2 groups and treated with either JW55 – TCF or CREB (29 32). (100 mg/kg) or vehicle (DMSO). Daily per oral applications TNKS1/2, in particular, is a promising biotarget for phar- started the day after and continued for 3 weeks. The mouse maceutical reagents (4, 6). TNKS1/2 is not only involved in body weight was measured twice a week. The mice were controlling canonical Wnt signaling but has been associated sacrificed and the intestines were dissected, washed in PBS, with further cellular processes through either protein poly and fixed in formaldehyde [10% solution (v/v) in PBS]. The (ADP-ribosyl)ation (PARsylation) or by protein complex for- small intestines were stained using 1% methylene blue pre- mation. These are (i) telomere maintenance by interacting pared in 10% paraformalaldehyde (PFA)/PBS solution. Small with TRF1 (33), (ii) spindle formation and stabilization by ileum Swiss-rolls were embedded in paraffin sectioned and binding to NuMA (33), and (iii) glucose metabolism by regu- stained with hematoxylin and eosin (H&E). Fixed colons were lating GLUT4 transport through IRAP (33). embedded in paraffin, sectioned and stained with an anti– fi In this work, we have identi ed a novel TNKS inhibitor, b-catenin antibody (610153; BD Transduction Laboratories; JW55, which inhibits the PARP domain of TNKS1/2, leading to see Supplementary Materials and Methods for further details). the stabilization of AXIN2 followed by increased degradation of The number and size of the intestinal lesions were quantified b fi -catenin. JW55 ef ciently decreases canonical Wnt signaling by the Ellipse program (ViDiTo). in colon carcinoma cell lines, in a Xenopus axis duplication For other materials and methods, please see previous assay, and in tamoxifen-induced tumors in ApcCKO/CKOLgr5- þ descriptions (34) and Supplementary Materials and Methods. CreERT2 mice. The identified chemotype JW55 may serve as an attractive start point for the development of novel cancer therapeutics. Results JW55 is a potent and selective inhibitor of the canonical Materials and Methods Wnt pathway To identify chemical compounds that inhibit the canonical Cell lines and luciferase assays Wnt signaling pathway, a library consisting of 37,000 small All cell lines were purchased from American Type Culture molecules (ChemBioNet) was screened using HEK293 cells Collection and maintained and treated according to the sup- that were stably transfected with a SuperTOP-d1EGFP reporter plier's recommendations. Reporter assays were carried out as (34). The vector contains a synthetic TCF-responsive promoter previously described (34). (SuperTOP: 7 x TCF binding sites) that initiates the expression of destabilized d1EGFP upon activation of canonical Wnt IncuCyte cell growth measurement assay signaling (34). Canonical Wnt signaling was activated using A total of 1,000 SW480 or RKO cells were seeded in 96-well conditioned media (CM) from L Wnt3a expressing cells. The plates. The day after, the cell culture medium was exchanged screen resulted in 77 primary hits including the previously to solutions that contained 0.1% dimethyl sulfoxide (DMSO) or described compounds JW67 and JW74 (34) and JW55 (Fig. 1A,

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Light II cells (Gli1-Luc reporter) that were activated with 50% Shh-CM were not inhibited by 10 or 1 mmol/L JW55. Also, TNFaactivated HEK293 cells containing a transiently transfected NF-kB–Luciferase (NF-kB-Luc) plasmid showed no pathway inhibition in the presence of 10 or 1 mmol/L of JW55 (Fig. 1B and C). The selectivity and specificity of JW55 was further tested in an in vivo Xenopus axis duplication assay, a highly stringent test for examining inhibitors of canonical Wnt signaling. When injected into the ventral blastomeres of 4-cell stage Xenopus laevis embryos, XWnt8 mRNA induced Wnt signaling resulting in the formation of a second body axis. By using selective Wnt inhibitors, the axis duplication can be inhibited and a normal phenotype restored (34). Coinjection of 10 pg XWnt8 and 2 pmol of JW55 resulted in a significant 51% reduction of the axis duplication incidence in developing frog embryos when compared with the DMSO vehicle control (z-test, P ¼ 0.001; Fig. 1D, left). This result provided evidence that JW55 acts as a specific and effective inhibitor of canonical Wnt signaling.

JW55 regulates b-catenin stability at the level of the destruction complex To identify the interference point of JW55 with canonical Wnt signaling, the effect of JW55 was monitored after LiCl- induced activation of the pathway. The activity of GSK3b, which is part of the b-catenin destruction complex, is reduced in the presence of LiCl (35), rendering the N-terminal phosphorylation of b-catenin by GSK3b ineffective. This causes nuclear accumulation of b-catenin and active Wnt signaling. HEK293 cells, transiently transfected with ST-Luc and Renilla, were incubated with 25 mmol/L LiCl and different doses of JW55. The LiCl-activating effect was counteracted by JW55 in a dose-dependent manner with an IC50 value of 360 nmol/L, indicating that JW55 acts at the level or downstream of the destruction complex (Fig. 2A, left and Supplementary Fig. S1B). Next, the effect of JW55 on regulating the activity of b-cate- b fi nin was tested. We used (i) wild-type -catenin and (ii) Figure 1. JW55 speci cally reduces canonical Wnt signaling in reporter b cells and in Xenopus embryos. Reporter controls in 0.1% DMSO. A, left, -catenin with point mutations in the N-terminal phosphor- luciferase reporter activity in HEK293 cells, transiently transfected with ylation sites (S33, S37, T41, and S45) that resists degradation ST-Luc and Renilla plasmids and treated with JW55 at 0.1 to 10 mmol/L. and functions as dominant active (da-Cat). Plasmids contain- þ , 30% Wnt3a-CM; , without Wnt3a-CM. The spotted line shows the ing full-length b-catenin or da-Cat, along with ST-Luc and IC50 value level. Right, chemical structure of JW55. B, effect of 1 and 10 mmol/L JW55 in NIH/3T3 Shh Light II cells. þ, 50% Shh-CM; ,no Renilla, were transiently transfected in HEK293 cells. The stimulation. Cyclopamine: positive control. C, effect of 1 and 10 mmol/L expression of wild-type b-catenin led to an increased ST-Luc JW55 in HEK293 cells transiently transfected with a NF-kB reporter (NF- reporter activity that could be reduced by 55% when the k þ a a B-Luc) and Renilla. , 10 ng/mL TNF ; , without TNF . The results in transfected cells were exposed to 10 mmol/L of JW55 (normality all reporter assays (A–C) show the mean values of at least 3 independent ¼ experiments and error bars present SDs. D, left, quantification of axis test failed, rank sum test: P 0.001; Fig. 2A, right). In contrast, duplication in Xenopus embryos coinjected with 10 pg XWnt8 and 0.5% the activation of the pathway by da-Cat could not be inhibited DMSO (gray) or 10 pg XWnt8 and 2 pmol JW55 (black). The axis by 10 mmol/L JW55, further indicating that JW55 acts at the duplication rate was significantly ( ) reduced after JW55 coinjections level or downstream of the destruction complex (normality test ¼ (P 0.001). Collected data from 3 independent assays are shown. failed, rank sum test: P ¼ 0.457; Fig. 2A, right). Right, representative images of embryos obtained 36 hours after the injection. JW55 inhibits canonical Wnt signaling in colorectal right panel). To evaluate the efficacy and specificity of JW55, cancer cell lines in vitro the compound was tested in 3 different luciferase reporter Mutations in the APC gene, which occur in nearly all assays. Wnt3a-induced HEK293 cells containing a transiently colorectal cancers (CRC; ref. 18), lead to ineffective degradation transfected ST-Luc (SuperTop-luciferase) reporter showed of b-catenin and aberrant upregulation of Wnt signaling. The inhibition by JW55 with an IC50 value of 470 nmol/L (Fig. cell lines SW480 and HCT-15 (mutated in codon 1338 and 1417 1A, left panel and Supplementary Fig. S1A). In contrast, Shh of the APC gene, respectively) were stably transfected with ST-

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Luc and Renilla and incubated at various doses of JW55 for 48 hours. A dose-dependent reduction of luciferase activity was detected in both cell lines. JW55 was effective in the range of 1 to 5 mmol/L in SW480 cells and 0.01 to 5 mmol/L in HCT-15 cells (Fig. 2B, left). Next, HCT116 CRC cells with integrated ST-Luc and Renilla reporters were used to test JW55. HCT116 carries a point mutation in the CK1a-dependent phosphorylation site S45 of one b-catenin allele; however, S45-mutated b-catenin may still be phosphorylated in the remaining GSK3b phosphorylation sites (e.g. S33, S37, and T41), with resulting semi- regulated b-catenin turnover (36). In HCT116 cells, JW55 was effective in the range of 0.01 to 5 mmol/L (Fig. 2B, right). A basal luciferase expression level at approximately 50% was reached after exposure to 5 mmol/L in the CRC cells. Furthermore, the effect of JW55 on the expression of endogenous Wnt target genes was examined by real-time PCR in the cell lines SW480 and DLD-1. Compared with SW480 cells, the APC mutation in DLD-1 cells results in a more extensive C- terminal deletion (37). In SW480 cells, a dose-dependent decline in the expression of the 3 target genes was observed: AXIN2 (25 mmol/L: 55% and 10 mmol/L: 45%), SP5 (25 mmol/L: 38% and 10 mmol/L: 15%) and NKD1 (25 mmol/L: 31% and 10 mmol/L: 7%; Fig. 2C, left). A similar reduction was observed in DLD-1 cells after exposure to 10 mmol/L of JW55 (AXIN2: 72%, SP5: 38% and NKD1: 66%; Fig. 2C, right). Subsequently, a more extensive Illumina gene expression analysis was carried out in JW55-treated SW480 cells. Several Wnt target genes (Wnt homepage) were differently expressed (Supplementary Table S1). Upregulated genes included (Log2 0.5): WISP3, TCF7, PLAUR, EFNB2, and NOTUM. Downregulated genes included (Log2 0.5): AXIN2, NKD1, DKK1, MMP7, ID2, GAST, FZD2, EDN1, CYR61, SOX18 and, similar to our previous observations (34), members of the SPANX gene family.

JW55 destabilizes b-catenin by increasing cytoplasmic AXIN2 levels Previous studies have shown that an increase in AXIN2 steady-state levels induced by TNKS1/2 or CK1a inhibitors is b Figure 2. JW55 inhibits canonical Wnt signaling in CRC cells. A, accompanied by a decrease in -catenin concentrations the control bars are shown in gray and JW55-treated samples are (4, 22, 24, 34). Increased levels of AXIN2 protein promote shown in black. Left, inhibition of GSK3b (25mmol/LLiCl)inHEK293 degradation of b-catenin even in cells with truncated APC cells, transiently transfected with ST-Luc and Renilla, is (4, 22, 38). Western blot analysis of SW480 cells lysates revealed counteracted by 0.1 to 10 mmol/L JW55. þ, 25 mmol/L LiCl; , no LiCl. Controls in 0.1% DMSO. The mean values of 3 independent a dose-dependent increase of cytoplasmic AXIN2 after JW55 assays and SDs are shown. Right, HEK293 cells transiently treatment (range: 10 mmol/L–100 nmol/L; Fig. 3A and Supple- transfected with ST-Luc and Renilla plus full-length b-catenin or da- mentary Fig. S2). Furthermore, an antibody against the active Cat. þ,withb-catenin plasmids; ,withoutb-catenin. Controls in and nonphosphorylated form of b-catenin (active b-catenin, m 0.1% DMSO. Ten mol/L JW55 inhibited luciferase activity induced ABC) identiﬁed reduced levels of b-catenin in the cytoplasm of by ectopic wild-type b-cat (, P < 0.05), whereas da-Cat induction was unaffected. n ¼ total number of measurements from multiple JW55-treated cells (Fig. 3A and Supplementary Fig. S2). In independent assays. The SDs are shown as error bars. B, JW55- addition, a modest reduction of total b-catenin and a substan- mediated reduction of luciferase activity in different CRC cell lines tial decrease of nuclear ABC were observed, and an increase in stably transfected with ST-Luc and Renilla. Left, a dose-dependent phosphorylated b-catenin (pb-catenin) levels was seen indi- reduction of ST-Luc activation was detected in HCT-15 and SW480 b cells harboring mutant APC. Right, JW55-mediated reduction in cating ongoing -catenin degradation (Fig. 3A and Supple- HCT116 cells containing a single allele mutation in S45 of b-catenin. mentary Fig. S2). The mean values and the SE of several independent assays are To gain further insight into the changes in cellular distri- shown. C, 25 or 10 mmol/L JW55 reduced the relative expression of bution of AXIN2 and b-catenin, JW55-treated SW480 cells were AXIN2, SP5,andNKD1 mRNA in the CRC cells SW480 and DLD-1 analyzed by immunoﬂuorescence. A general reduction of total as shown by real-time RT-PCR analysis. The means of b 3 independent experiments are shown along with error bars -catenin, both in the cytoplasmic and nuclear compartments, depicting SDs. was detected at the doses of 5 and 1 mmol/L (equal shutter

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speed; Supplementary Fig. S3). Confocal microscopy (equal shutter speeds) revealed, in accordance with the Western blot analysis, that the levels of cytoplasmic AXIN2 were signiﬁcantly increased (Fig. 3B) and large protein foci, probably represent- ing accumulated destruction complexes, were observed (Fig. 3B, arrows). Clusters of colocalized cytoplasmic b-catenin and AXIN2 have previously been detected in SW480 cells after treatment with the Wnt antagonist JW74 (34). Similar to JW74, enhanced phosphorylation and resulting degradation of b-catenin after JW55 exposure seemed to be orchestrated by stabilization of AXIN2 in the destruction complex (Fig. 3 and 5).

JW55 speciﬁcally inhibits the PARsylation activity of TNKS1 and TNKS2 By inhibiting the PARP domain of TNKS1/2, XAV939, IWR-1, and JW74 prevent auto-PARsylation of TNKS1/2 and PARsyla- tion of AXIN2 (4, 22, 39, 40). This leads to a stabilization of AXIN2, an accumulation of proteins in the destruction complex followed ultimately by an increased degradation of b-catenin (4, 22). To test whether JW55 decreased canonical Wnt signaling by directly inhibiting the PARP domain of TNKS1/2, we carried out biochemical assays for moni- toring the activity of TNKS1/2 and PARP. JW55 decreased auto-PARsylation of TNKS1/2 in vitro with IC50 values of 1.9 mmol/L and 830 nmol/L, respectively (Fig. 4A and

Figure 3. JW55 mediates increased AXIN2 stability and induces b-catenin degradation. A, Western blot analysis of lysates from SW480 cells after incubations with JW55 (24 hours). Controls in 0.1% DMSO). Antibodies against AXIN2, ABC (active b-catenin), and pb-catenin (N-terminal phosphorylated b-catenin) were used. ACTIN (cytoplasmic) or LAMIN B1 (nuclear extracts) documented equal protein loading. Full-length and uncropped blots are shown in Supplementary Fig. S2. The blots show Figure 4. JW55 speciﬁcally inhibits TNKS1, TNKS2 but not PARP1 in representative data derived from multiple experiments. B, cellular biochemical assays. A, JW55 B, XAV939 (logarithmic scale). C, the redistribution of b-catenin and stabilization of AXIN2 in SW480 cells calculated IC50 values are displayed in the table. The mean values incubated with 5 mmol/L JW55 (48 hours). The arrows show clustered represent 2 independent experiments and the error bars show SDs. D, AXIN2 and b-catenin. The confocal microscopy images are immunoblotting of TNKS1/2 after exposures of SW480 cells to JW55, representative examples from one of several independent experiments. XAV939, or IWR-1.

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Figure 5. Scheme showing the proposed effect of JW55 on protein stability in the b-catenin destruction complex. The red arrow indicates nuclear translocation of b-catenin and expression Wnt target genes in a Wnt on state. The blue arrows indicate TNKS, AXIN2, and RNF146 degradation in untreated cells. The black arrows depict the inhibition of the PARP domain of TNKS by JW55, leading to stabilization of the destruction complex, increased N-terminal phosphorylation of b-catenin, and degradation in the proteasome.

– – Supplementary Fig. S1C). However, in contrast to XAV939 37.3% 38.8%) and increased the number of cells in G2 M phase (ref. 4; Fig. 4A and Supplementary Fig. S1D), but similar to (from 34.3%–39%; Fig. 6A). IWR-1 (4), JW55 exhibited no inhibition of PARP1 at doses Next, we monitored the cell proliferation kinetics using up to 20 mmol/L (Fig. 4A). Although further details may IncuCyte. SW480 cells were exposed to 10, 5, or 1 mmol/L of emerge, present evidence suggests that JW55 decreases JW55 for 9 days, and the confluency was measured every 2 canonical Wnt signaling by specifically inhibiting the PARP hours. All doses of JW55 reduced SW480 cell growth and the domain of TNKS1/2 (Fig. 5), although leaving the activity of most robust effect was detected at a dose of 10 mmol/L JW55. the PARP domain of at least PARP1 unaffected. Molecular The confluency was reduced to 55% relative to the DMSO docking of JW55 into the adenosine site of the human PARP control at the end of experiment (Fig. 6B). In parallel, the domain of TNKS2 using the structure of the PARP domain in CRC cell line RKO, which contains wild-type APC and complex with IWR-1 (X-ray, PDB code 3UA9) reveals that the b-catenin and exerts Wnt-independent cell growth, was used binding site and position of JW55 in the model is similar to as a control. RKO cells reached 100% confluency within 7 IWR-1 (40), but different from XAV939 (Supplementary Fig. days and were not affected by treatment with 10 mmol/L S4). Binding of JW55 to recombinant TNKS2 protein was also JW55 (Fig. 6B). observed by using a fluorescence polarization competition Furthermore, proliferation of SW480 cells was examined by assay, with a structurally similar but inactive analog as a consecutive passages in the presence of 5, 2.5, or 1 mmol/L negative control (ref. 41; Supplementary Fig. S5). Interest- JW55. A dose-dependent decrease of cell numbers over 3 ingly, opposite to the effect of XAV939 (4), a decrease in passages was noted in SW480 cells in the presence of JW55, endogenous TNKS1/2 levels was detected after exposure to whereas HeLa cells (Wnt-independent cervical cancer cells) JW55 and IWR-1 in SW480 cells. remained unaffected (Fig. 6C). Finally, SW480 cells were grown under low serum conditions (1% FBS) along with various JW55 reduces growth of SW480 colon cancer cells concentrations of JW55 (5, 1, and 0.1 mmol/L) and the forma- CRC cells can enter cell-cycle arrest as a result of antago- tion of colonies was quantified. We observed a concentration- nized canonical Wnt signaling (42–44). Various proliferation dependent reduction of colony numbers in SW480 cells and no assays were carried out to see whether JW55-mediated inhi- reduction in colony numbers in the control cell lines HeLa and bition of canonical Wnt signaling would affect CRC cell growth. RKO when cultured in 5 mmol/L JW55 (Fig. 6D, left). All SW480 First, SW480 cells were incubated with 10 mmol/L JW55 and colonies, which formed in the presence of JW55, were sub- labeled with bromodeoxyuridine (BrdUrd) and propidium stantially smaller (Fig. 6D, right). iodide (PI). The subsequent flow cytometry cell-cycle analysis Taken together, these data showed that JW55-mediated revealed that JW55 treatment lowered the proportion of cells in inhibition of canonical Wnt signaling resulted in reduced the S phase (from 28.4% in DMSO-treated controls to 22.2%), cell-cycle progression, proliferation, and colony formation in raised moderately the cell fraction in the G1 phase (from the CRC cell line SW480 in vitro.

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Figure 6. Inhibition of canonical Wnt signaling by JW55 promotes cell-cycle arrest and specifically reduces proliferation in SW480 CRC cells. A, left, fluorescence-activated cell sorting scatter plots showing SW480 cells labeled with BrdUrd and PI after incubation in 10 mmol/L or 0.1% DMSO. The different cell-cycle phases, G1, S, and G2–M, are gated in 3 different compartments. Right, table shows a representative percent cell-cycle phase distribution (mean values) of cells after incubation with JW55 or DMSO. B, cell growth curve as measured by IncuCyte shows a concentration-dependent decrease of proliferation in SW480 cells compared with the Wnt-independent CRC control cell line RKO. SW480 and RKO cells were incubated with 0.1% DMSO or exposed to JW55. The plot shows the mean value of 2 independent experiments and all relative SDs are below 20%. C, relative cell count (%) of SW480 and Wnt-independent HeLa cells under serial passages in JW55. The cell count of control cells cultured in 0.05% DMSO was defined as 100%. The graphs depict the mean values of several experiments and the error bars show the SEs. D, a colony assay that shows a specific decrease in colony formation of SW480 cells cultured in various doses of JW55 and 1% DMSO. Left, the bar chart shows a dose-dependent reduction of SW480 colonies and unaffected growth of the control cell lines HeLa and RKO. Controls in 0.05% DMSO. The graphs show the mean values of several measurements from multiple experiments along with error bars depicting SDs. Right, the images from a representative assay show that the general SW480 colony sizes are smaller in increased doses of JW55.

JW55 reduces tumor development in conditional Apc in the small intestine. To evaluate the JW55-mediated decrease knockout mice of intestinal tumor development in vivo, ApcCKO/CKOLgr5- þ Recently, the leucine-rich repeat containing G-protein–cou- CreERT2 mice were injected intraperitoneally with a 25 pled receptor 5 (Lgr5) was established as a specific marker for mg/kg single dose of tamoxifen. A day after, daily per oral intestinal epithelium stem cells (ISC). The knockin mouse Lgr5- applications of JW55 (100 mg/kg; 3 females) or vehicle (DMSO; EGFP-IRES-CreERT2, further referred to as Lgr5-CreERT2, 4 females) were initiated. The dose of 100 mg/kg was chosen to expresses a tamoxifen-regulated variant of Cre recombinase counteract the rapid liver metabolism of JW55 as indicated by that is under the control of the Lgr5 locus (45). The the human liver microsome stability analysis (t1/2 ¼ 10.1 Cre-mediated excision of the floxed exon 14 in mice with minutes; Supplementary Fig. S7A). No measurable effects on conditional Apc alleles (CKO) changes the reading frame mouse body weight were noticed throughout the experiment downstream of the deletion. This results in the production of period (Supplementary Fig. S7B). After 21 days, the mice were a truncated and nonfunctional 580 amino acid (aa) Apc sacrificed and the dissected intestines were embedded in polypeptide (46) that relates to the homozygous mutations paraffin and sectioned. Immunohistochemical staining seen in the intestinal cancer model mouse ApcMin (multiple revealed that the neoplastic lesions expressed b-catenin and intestinal neoplasia, Min; ref. 47). Lgr5-CreERT2 mice were the mouse ISC marker Ephrin type-B receptor 2 (EphB2), intercrossed with ApcCKO/CKO animals and multiple tumors indicating aberrant activation of canonical Wnt signaling in were observed after tamoxifen injections in the small intestine the tumor tissue (Fig. 7B; refs. 44, 48–50). The b-catenin– þ (ileum) and the colon of ApcCKO/CKOLgr5-CreERT2 mice (Fig. stained colon adenomas contrasted with the surrounding 7A and Supplementary Fig S6). In the colon, fewer tumors healthy mucosa, and image analysis software (Ellipse) was developed when compared with the general adenoma progress used to quantify the number and areas of b-catenin–positive

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lesions in the colon (Supplementary Fig. S7C). As it was PARP domain of TNKS1/2 with low molecular inhibitors, such impossible to distinguish the borders of individual tumors in as XAV939, IWR-1, and JW74 (4, 34), leads to the stabilization of the small intestine (ileum), only the total tumor area per mouse the destruction complex that triggers increased degradation of was recorded. In the ileum, a significant reduction of the total b-catenin (19). As predicted for a TNKS inhibitor, we observed tumor area was observed after JW55 injections (mean: 2.93 a massive cytoplasmic accumulation of AXIN2 after treatment mm2 and median: 2.95 mm2) when compared with the control with 500 nmol/L JW55, followed by a significant reduction of group (mean: 8.84 mm2 and median: 9.51 mm2; normality test b-catenin levels in vitro (Fig. 5). A similar stabilization of AXIN2 failed, rank sum test: P ¼ 0.003; Fig. 7A and C, top panel). In the was also seen with JW74, XAV939, and IWR-1 (4, 22, 34). colon, the tumor count was substantially reduced in JW55- However, despite the substantial increased specificity of treated mice (mean: 9.7 and median: 6.0) when evaluated JW55 to the PARP domain of TNKS1/2 when compared with against the control group (mean: 33.8 and median: 26.0; nor- XAV939, we cannot rule out that JW55 may affect additional mality test failed, rank sum test: P ¼ 0.057; Fig. 7C, bottom mechanisms that contribute to the observed effects in colon þ panel). Furthermore, a significant decrease of the total tumor cancer cell lines and in ApcCKO/CKOLgr5-CreERT2 mice. For area was noticed after injections with JW55 (mean: 0.022 mm2) instance pyrvinium decreases Wnt signaling both by stabilizing when compared with the control group (mean: 0.154 mm2; AXIN2 and through inhibition of CK1a (24). Furthermore, in Students t test: P ¼ 0.009; Fig. 7C, bottom panel). The area of complementation to altering canonical Wnt/b-catenin signal- single tumors was significantly reduced after injections with ing, a JW55-mediated inhibition of TNKS1/2 may induce JW55 (mean: 0.0025 mm2) when compared with the untreated additional intracellular effects including altered telomere group (mean: 0.0049 mm2; Students t test: P ¼ 0.043; Fig. 7C, maintenance or spindle formation. bottom panel). Interestingly, the proportion of cells that The in vivo treatment of tamoxifen-induced polyposis in þ expressed Ki67, a marker of proliferating and ISC-like cells ApcCKO/CKOLgr5-CreERT2 mice JW55 showed a profound (50), was substantially decreased in adenomas exposed to JW55 effect on tumor progression. In comparison with the well- when compared with tumors that developed in the control established ApcMin mice (47), this mouse model provides a mice (Fig. 7B, panel h and i). clearer assay for scoring adenoma formation because the Importantly, when examining the healthy mucosa with tumor induction and treatment are time controlled. However, antibodies against various epithelial cell populations, such as as adenoma induction is rather strong, it is difficult to assess EphB2 for ISCs and Krt20 for terminally differentiated epithe- later stages of CRC development as mouse mortality does not lial cells (50), we did not observe changes in its morphology, allow long-term tracking of the tumors. Thus, the presented proliferation, or differentiation after exposure to JW55 (Fig. data further solidify the functional implication of PARsylation 7D). In summary, the results obtained from JW55-treated by TNKS1/2 in a malignant transformation of the colon and the conditional Apc knockout mice indicated that in vivo small- small intestine. molecular inhibition of TNKS1/2 leads to decreased canonical Interestingly, in our setting, the exposure of progenitor cells Wnt signaling followed by reduced adenoma induction and in a normal crypt to JW55 did not exert visible alterations in the possibly progression of adenoma carcinoma. intact mucosa as measured with the markers EphB2, Ki67, and Krt20. However, we cannot exclude that a longer treatment may cause reduction in cell turnover in the intestine epithe- Discussion lium (45). The efficient in vivo reduction of adenomas in mice Altered properties of Wnt/b-catenin signaling are enabling and CRC growth in vitro by a tankyrase-specific inhibitor factors for a multitude of diseases, including cancer. Among further strengthens the importance of TNKS and b-catenin as known interference points in the canonical Wnt signaling potential therapeutic biotargets. pathway, the PARP domains of TNKS1/2 seem to be a partic- TNKS1/2isamemberofafamilyof5PARPproteinsin ular suitable target for inhibiting the pathway. Blocking the humans, including PARP1, which is an extensively explored

Figure 7. JW55 treatment decreases development of adenomas in conditional Apc knockout mice. A, representative microscopy images showing hematoxylin and eosin (H&E)-stained sections of Swiss-rolls (the distal part of the ileum is centered) showing an extensive decrease in adenoma development in the small intestine of JW55-treated (100 mg/kg) mice when compared with control mice (DMSO). The right panel shows the morphology þ of a wild-type (wt) ileum. B, tumors in the colon of ApcCKO/CKOLgr5-CreERT2 mice displaying high levels of b-catenin and EphB2, which indicates active Wnt signaling and an ISC-like phenotype. The expression of b-catenin (d, e), EphB2 (f, g), and the proliferation marker Ki67 (h, i) is shown in the colon of vehicle (DMSO) or JW55-treated mice. Microphotographs in d and e are shown in more detail in d' and e', respectively. The sections were counterstained with hematoxylin nuclear stain. Epithelial lesions are indicated by black arrows. C, the graphs illustrate quantification of tumor area, þ count, and size from the ileum (H&E stained) and colon (b-catenin labeled) of JW55-treated or untreated ApcCKO/CKOLgr5-CreERT2 mice. The histograms display the mean values and the error bars show SDs. The top panel shows a statistically significant () decrease of average total tumor area in the ileum subsequent to the JW55 treatment (P ¼ 0.003). Bottom panels, left, the colon tumor count was substantially () reduced after JW55 exposure (P ¼ 0.057). Middle, the mean total tumor area of the colon was significantly () reduced (P ¼ 0.043). Right, the mean area of one tumor was significantly ()reduced(P ¼ 0.003). D, the phenotype of nonadenomatous colons is not affected by JW55 (right panels) when compared with wild-typetissue(wt,leftpanels). Representative pictures are shown. The sections werestainedwithKi67(landm)andEphB2(jandk)thatlabel proliferating and ISC-like cells localized toward the bottom of the crypts. Cytokeratin 20 (Krt20; n and o) identifies terminally differentiated epithelial cells that are found at the apical surface of the mucosa. The black bars in the bottom right corners of the images represent 1 mm in (a, b, and c), 0.2 mm in (d, e), and 0.1 mm in other images.

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target for drug development (33). Particularly in the BRCA1/ Acknowledgments 2 mutant background, PARP1 inhibitors exhibit therapeutic The authors thank R. Kemler for da-Cat plasmid, R. Moon for ST-Luc plasmid, and C. Erdmann for helping with the high-throughput screen as well effects in vitro and in vivo. Because PARP proteins are as A. Philimonenko for help with the image evaluation using the Ellipse involved in a range of biologic functions, including genome software. maintenance, the selectivity of PARP inhibitors may be an issue in the therapeutic setting. Similar to XAV939, JW55 inhibits auto-PARsylation of TNKS1 and TNKS2 in a bio- Grant Support J. Waaler, O. Machon, O. Machonova, H. Dinh, and S. Krauss were chemical assay. However, in contrast to XAV939, we see no supported by the Research Council of Norway, CRI program, and Helse evidence for the inhibition of PARP1 by JW55, making this Sør-st, grant 2010031; O. Machon and O. Machonova were supported by grant Agency of the Czech Republic (GACR P305/12/2042); L. Tumova and V. chemotype attractive for further development toward a Korinek were supported by qChIP/chip06 project from the Ministry of TNKS1/2 selective inhibitor. It remains to be determined Education, Youth and Sports of the Czech Republic (B06077), and the in which settings a preferred PARP1 inhibitor such as for institutional grant from the Academy of Sciences of the Czech Republic (AV0Z50520514). instance olaparib, a broad PARP inhibitor such as XAV939, The costs of publication of this article were defrayed in part by the or a selective TNKS inhibitor such as JW55, JW74, and IWR-1 payment of page charges. This article must therefore be hereby marked may be the favorable choice. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Disclosure of Potential Conﬂicts of Interest The described chemical compound may have commercial value if further Received October 13, 2011; revised February 15, 2012; accepted March 7, 2012; investigated. published OnlineFirst March 22, 2012.

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www.aacrjournals.org Cancer Res; 2012 OF11

A Novel Tankyrase Inhibitor Decreases Canonical Wnt Signaling in Colon Carcinoma Cells and Reduces Tumor Growth in Conditional APC Mutant Mice

Jo Waaler, Ondrej Machon, Lucie Tumova, et al.

Cancer Res Published OnlineFirst March 22, 2012.

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