A Novel Synthetic Inhibitor of CDC25 Phosphatases: BN82002

[CANCER RESEARCH 64, 3320–3325, May 1, 2004] A Novel Synthetic Inhibitor of CDC25 Phosphatases: BN82002

Marie-Christine Brezak,1 Muriel Quaranta,2 Odile Monde´sert,2 Marie-Odile Galcera,1 Olivier Lavergne,1 Fre´de´ric Alby,2 Martine Cazales,2 Ve´ronique Baldin,2 Christophe Thurieau,1 Jeremiath Harnett,1 Christophe Lanco,1 Philip G. Kasprzyk,1,3 Gregoire P. Prevost,1 and Bernard Ducommun2 1IPSEN, Institut Henri Beaufour, Les Ulis Cedex, France; 2Laboratoire de Biologie Cellulaire et Moleculaire du Controle de la Proliferation-Centre National de la Recherche Scientifique UMR5088-IFR109 “Institut d’Exploration Fonctionnelle des Ge´nomes,” Universite´ Paul Sabatier, Toulouse, France; and 3IPSEN, Biomeasure, Milford, Massachusetts

ABSTRACT B at mitosis (2) where it dephosphorylates tyrosine 15 and threonine 14. It also plays a role in the control of the initiation of S phase (5). CDC25 dual-specificity phosphatases are essential regulators that de- The elucidation of the specific role of each isoform at specific stages phosphorylate and activate cyclin-dependent kinase/cyclin complexes at of the cell cycle is a major issue that is still currently under investi- key transitions of the cell cycle. CDC25 activity is currently considered to be an interesting target for the development of new antiproliferative gation. agents. Here we report the identification of a new CDC25 inhibitor and Overexpression of CDC25A and B, but not C, has been observed in the characterization of its effects at the molecular and cellular levels, and a variety of cancers (i.e., breast, ovary, head and neck, and colon) with in animal models. a striking association with tumor aggressiveness and poor prognosis BN82002 inhibits the phosphatase activity of recombinant human (6–9). This overexpression of CDC25A has been observed concom- CDC25A, B, and C in vitro. It impairs the proliferation of tumoral cell itantly with the loss of other regulators such as p27 in high-grade lines and increases cyclin-dependent kinase 1 inhibitory tyrosine phospho- B-cell non-Hodgkin’s lymphomas and may, therefore, possibly con- rylation. In synchronized HeLa cells, BN82002 delays cell cycle progres- tribute to their aggressivity (10). Targeted overexpression of CDC25B sion at G -S, in S phase and at the G -M transition. In contrast, BN82002 1 2 in mice is responsible for an increased sensitivity to the carcinogen arrests U2OS cell cycle mostly in the G phase. Selectivity of this inhibitor 1 agent DMBA (11, 12). CDC25B is also overexpressed in neurons of is demonstrated: (a) by the reversion of the mitotic-inducing effect observed in HeLa cells upon CDC25B overexpression; and (b) by the partial Alzheimer’s disease patients, and it has been suggested that this might reversion of cell cycle arrest in U2OS expressing CDC25. We also show be responsible for an inappropriate activation of mitotic CDK/cyclin that BN82002 reduces growth rate of human tumor xenografts in athymic complexes resulting in the neurodegenerative process (13). CDC25 nude mice. phosphatases are therefore considered to be relevant physiopatholog- BN82002 is a original CDC25 inhibitor that is active both in cell and ical targets supporting the identification and the characterization of animal models. This greatly reinforces the interest in CDC25 as an chemical inhibitors (14, 15). anticancer target. However, over the last few years, only a very limited number of CDC25 inhibitors have been described in the literature. Menadione INTRODUCTION has been reported to be a noncompetitive inhibitor of recombinant CDC25 phosphatase and to induce a cell cycle delay in treated cells The cell cycle is controlled by evolutionarily conserved mecha- (15–17). More potent quinone derivatives such as NSC663284 and nisms that ensure the strict regulation of the division process and the NSC95397 have also been identified (18). These molecules display accurate segregation of the genome in two daughter cells. These competitive kinetics against CDC25 and inhibit tumor cell prolifera- mechanisms are able to integrate intra- and extracellular signals and, tion (19, 20). SC-␣␣delta 9 was identified in the screening of a small therefore, play a central role in the control of cell proliferation. Cdc25 molecule library as a potent and selective inhibitor of the CDC25 dual-specificity phosphatase was initially identified in yeast model family of dual-specificity phosphatases (21). However, it has been organisms as an essential cell cycle regulator (1). Its activity is reported recently that structural resemblance of SC-␣␣delta 9 to required at mitosis to dephosphorylate and activate Cdc2 (cyclin- phospholipids allows it to inhibit cellular phospholipase C and is dependent kinase; CDK1), a well-known key cell cycle regulator. responsible for its antiproliferative effects (22). Indolyldihydroxiqui- Three human homologues, CDC25A, B, and C, have subsequently nones have been shown to effectively compete with CDC25 substrate been identified (reviewed in Ref. 2). They share common structural in vitro and to inhibit cell proliferation (23). A few other products properties, and the ability to dephosphorylate critical tyrosine and such as the steroidal derivative dithiocarboxy cholestanol (24) and threonine residues. Their known substrates are CDKs associated with natural products such as dysidiolide have also been reported to inhibit their cyclin regulatory subunits. Dephosphorylation by CDC25 phos- CDC25 activity in vitro and to cause inhibition of HL60 cell prolif- phatases allows CDK activation and the subsequent induction of cell eration (25, 26). cycle progression. CDC25A is involved in the control of the G -S 1 Here, we report the identification of a new inhibitor of CDC25 transition where it dephosphorylates the CDK2/cyclin A complex (3) phosphatase and demonstrate that it impairs cell cycle progression and and probably also acts in the control of entry into mitosis (4). reduces tumor growth in animal models. This compound represents a CDC25B has been proposed to play the role of the starter of mitosis promising chemical structure active both in cell and animal models, by dephosphorylating and activating CDK2/cyclin A and CDK1/ and, therefore, greatly reinforces the interest in the CDC25 cell cycle cyclin B (2). CDC25C is recognized as an activator of CDK1/cyclin regulator as an anticancer target.

Received 12/19/03; revised 2/11/04; accepted 2/13/04. Grant support: Grant from La Ligue Nationale Contre le Cancer (Equipe Labelise´e 2001; B. Ducommun). MATERIALS AND METHODS The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with Preparation of BN82002. 5-(Dimethylamino)-2-hydroxy-3-me´thoxybenz- 18 U.S.C. Section 1734 solely to indicate this fact. aldehyde (Ref. 27; 1 g, 5.12 mmol) was added dropwise to a mixture of Requests for reprints: Bernard Ducommun, LBCMCP-Centre National de la Recher- N-methyl-2-(4-nitrophenyl)ethylamine hydrochloride (1.22 g, 5.63 mmol) and che Scientifique UMR5088-IFR109 “Institut d’Exploration Fonctionnelle des Ge´nomes,” Universite´Paul Sabatier, Toulouse, 31062 France. Phone: 33-5-61-55-81-10; Fax: 33-5- triethylamine (1.1 ml, 7.7 mmol) in anhydrous methanol (30 ml) at room 61-55-81-09; E-mail: [email protected]. temperature under argon, and the resulting mixture was stirred for 18 h. 3320

Sodium borohydride (0.213 g, 5.63 mmol) was then added, and stirring was azan formation (Roche Diagnostic). Human tumor cell lines were seeded in maintained for 4 additional hours. The reaction mixture was then partitioned 96-well plates at day 0 in DMEM supplemented with 10% FCS, 50,000 between cold water (10 ml) and dichloromethane (50 ml), and the aqueous units/liter penicillin, and 50 mg/liter streptomycin. At day 1, cells were treated phase was additionally extracted with dichloromethane. The combined organic for 96 h with increasing concentrations of drug (up to 50 ␮M). The number of layers were dried over magnesium sulfate, filtered, and concentrated to give seeded cells for each cell line was determined previously, allowing control 1.10 g of a brown viscous oil. Purification by chromatography over silica gel cells to be in growth phase at the end of the experiment. These experiments using 3% methanol in dichloromethane gave 0.626 g (34%) of a brown solid: were performed twice with eight determinations per tested concentration. For mp 92–93°C. 1H NMR (DMSO) d 8.90 (s, 1H), 8.15–8.12 (d, J ϭ 8.7, 2H), each compound, values falling in the linear part of the sigmoid curve were ϭ 7.52–7.50 (d, J 8.7, 2H), 6.28 (s, 1H), 6.04 (s, 1H), 3.70 (s, 3H), 3.57 (s, 2H), included in a linear regression analysis and were used to estimate the IC50. 2.94 (t, J ϭ 7.1, 2H), 2.74–2.70 (m, 8H), 2.21 (s, 3H). Flow Cytometry Analysis of DNA Content. Cells were subjected to flow Cell Culture Conditions. Human parental cell lines were purchased from cytometry analysis of their DNA content after propidium iodide staining as American Type Culture Collection (Rockville, MD). They were cultivated in described previously (30). Data were collected on a BD FACSCalibur cytom- DMEM with 10% FCS, glutamine, and penicillin/streptomycin at 37°C in 5% eter and analyzed using the Modfit software (Verity Software).

CO2 atmosphere. HeLa cells were transfected using Exgen-500 (Euromedex) Human Tumor Xenografts in Nude Mice. Cells of the human pancreatic following the manufacturer’s instructions. Cell cycle synchronization was carcinoma cell line MIA PaCa-2 were injected s.c. into the flank of female achieved as described previously (28). The U2OS cell line conditionally athymic NCr-nu/nu mice, 4–6 weeks old. Tumors were allowed to reach a expressing hemagglutinin (HA)-tagged CDC25B1 (under the control of the volume of 100 mm3. Once tumors were established, treatment was started by tet-promoter) is described previously (29). Expression of CDC25B was i.p. route as indicated in the figure legend. Six animals/group were treated by achieved by tetracycline removal form the culture media. BN82002 dissolved in DMSO. Tumor measurements and animal weights were In Vitro Enzymatic Assay. The activity of a maltose binding protei- monitored and recorded twice per week. Animal care was in accordance with CDC25 recombinant enzyme produced in bacteria (GTP Technology, Labe`ge, institutional guidelines. France) was monitored using 3-O-methylfluorescein phosphate (OMFP) or fluoresceine diphosphate. The assay was performed in 384-well plates in a RESULTS final volume of 50 ␮l. The maltose binding protein-CDC25 proteins were kept in elution buffer [20 mM Tris-HCl (pH 7.4), 250 mM NaCl, 1 mM EDTA, 1 mM BN82002 Inhibits the Activity of Recombinant CDC25 Phos- DTT, and 10 mM Maltose]. They were diluted in assay buffer [50 mM Tris-HCl phatases. BN82002, a novel CDC25 inhibitor, was identified in the (pH 8.2), 50 mM NaCl, 1 mM DTT, and 20% glycerol]. The final concentra- course of a molecular library screening based on the inhibition of the tions were 158 ng/well for CDC25A, 84 ng/well for CDC25B2, 168 ng/well enzymatic activity of a recombinant CDC25C phosphatase. BN82002 for CDC25B3, 350 ng/well for CDC25C, and 70 ng/well for the catalytic inhibits in vitro the activity of CDC25C phosphatase in a concentra- domain of CDC25C (CDC25C-cat). Products were studied in concentration tion dose manner with an IC of 5.4 ␮M. Menadione used as positive response up to 40 ␮M. The reaction was initiated by addition of 500 ␮M of 50 ␮ O-methylfluorescein phosphate. After4hat30°C, 3-O-methylfluorescein control leads to an inhibition of 18.8 M. The inhibitory effect of fluorescent emission was measured with Victor 2 (Perkin-Elmer; excitation BN82002 and menadione was also observed when only the activity of filter: 475 nm and emission filter: 510 nm). CDC25C catalytic domain is challenged leading to IC50 values also in CD45 enzyme activity was monitored using either the synthetic substrate the micromolar range. In addition, other CDC25 family members OMFP or its natural substrate. For testing on OMFP substrate at 500 ␮M, the including CDC25A and the CDC25B2–3 variants (31) were inhibited CD45 enzyme (SE-135; BIOMOL, Plymouth Meeting, PA) was diluted at 40 by both compounds within the same range (Table 1). Selectivity was ng/wells in assay buffer [50 mM HEPES (pH 7.2), 1 mM EDTA, 1 mM DTT, also investigated on the CD45 tyrosine phosphatase. BN82002 was and 0.05% NP40. Concentration response of products were studied up to 40 found to be 20-fold less active on CD45 using the peptidic substrate ␮ M for Menadione (M-5750; Sigma, St. Louis, MO) and BN82002. After 2 h pp60c-src (see “Materials and Methods”) than on CDC25C using the at 30°C, 3-O-methylfluorescein fluorescent emission was measured. The nat- OMFP synthetic substrate. Similarly, Menadione was Ͼ20-fold less ural substrate pp60c-src COOH-terminal phosphoregulatory peptide (P301; BIOMOL) was diluted at 5.7 ␮g/well and the CD45 enzyme between 0.94 and active on CD45 than on CDC25C. The chemical structure of 1.25 ng/well in assay buffer 2. The assays were made in 384-well plates in a BN82002 presented in Fig. 1 is a novel tertiary (2-hydroxy-3-me- final volume of 20 ␮l. Concentration response of products were studied up to thoxy-5-dimethylamino-1-benzyl)amine that can be considered orig- 80 ␮M for BN82002 or up to 160 ␮M for Menadione. After1hat30°C, the free inal compared with known CDC25 inhibitors (15, 23). phosphate released from substrate due to enzymatic cleavage was quantified BN82002 Inhibits in Vitro Human Tumor Cell Proliferation. using BIOMOL Green reagent, reading absorbance at 620 nm. The effect of BN82002 on cell proliferation was evaluated in vitro on Western Blot Analysis. Cells were treated for 1 h with BN82002, washed several human tumor cell lines (Table 2). Menadione, which has been with PBS, and refed with fresh medium. After 24 h the cells were washed, reported to inhibit cell proliferation, was used as a control. All of the scraped, and lysed in lysis buffer [50 mM HEPES (pH 7.5), 10 mM NaCl, 1% examined cell lines were sensitive to BN82002 and Menadione in a Triton X 100, 10% Glycerol, 5 mM MgCl ,1mM EDTA, 1 mM sodium 2 concentration-dependent manner in the low micromolar range. The orthovanadate, and antiprotease mixture; Roche Diagnostic, Mannheim, Ger- many]. Lysates were obtained after centrifugation at 14,000 ϫ g for 10 min. most sensitive was the pancreatic cancer cell line MIA PaCa-2 with an ␮ Protein concentration was determined using Bio-Rad DC protein assay. Protein IC50 of 7.2 M, and the less sensitive cell line was the colon cancer concentration was adjusted to 20 ␮g/well. After electrophoresis on 12% SDS-polyacrylamide Tris/glycine gels (Bio-Rad, Marnes la coquette, France) Table 1 BN82002 inhibits purified recombinant members of CDC25 family and transfer to nitrocellulose membrane (Hybond-C; Amersham, Les Ulis, Enzyme inhibition was assayed on recombinant MBP-CDC25A, MBP-CDC25C, France) Western blotting was performed with rabbit polyclonal anti Phospho- MBP-CDC25C catalytic domain, MBP-CDC25B2, and MBP-CDC25B3 proteins using CDK1 [phospho-cdc2 (Tyr15) antibody, 9111L; Cell Signaling Technology, 3-O-methylfluorescein phosphate as substrate. The IC50 and the SD were calculated from Beverly, MA] diluted at 1:1300 and with secondary antibody (antirabbit at least two independent experiments with 8 determinations per tested concentration (see “Materials and Methods” for details). IgG-horseradish peroxidase, sc2030; Santa Cruz Biotechnology, Santa Cruz, Ϯ ␮ CA) diluted at 1:40,000. Western blotting detection system ECLϩ (Amer- IC50 ( )SD M sham) was performed and the membranes were exposed to Kodak BioMax Menadione BN82002 Light film (Sigma-Aldrich, Saint Quentin Fallavier, France). Images were CDC25A 7.1 (1.1) 2.4 (1.2) digitized using BioProfil scanner (Vilbert Lourmat). CDC25B2 14.2 (2.6) 3.9 (0.01) Cell Proliferation Assay. The inhibition of cell proliferation was deter- CDC25B3 19.6 (5.7) 6.3 (4.1) mined using a colorimetric assay WST1 based on cleavage of the tetrazolium CDC25C 18.8 (2.9) 5.4 (3.4) salt WST1 by mitochondrial dehydrogenases in viable cells, leading to form- CDC25C-cat 9.1 (1.7) 4.6 (0.5) 3321

50 ␮M BN82002, a concentration that fully inhibits cell proliferation, the cell cycle distribution was only modestly affected with a slight

decrease in S phase and an increase in cells containing both a G1 and aG2 DNA content, suggesting that the cells treated with BN82002 were arrested at various stages of the cell cycle. To analyze the effect of BN82002 on cell cycle progression in more detail, we made use of HeLa cells that were synchronized in late G -phase by double thymidine block and release. As shown in Fig. Fig. 1. Chemical structure of BN82002. 1 4A, left panel, after double thymidine block these cells were fully Ͼ synchronized with 80% of the population containing a G1 phase Table 2 BN82002 inhibits in vitro growth of human tumor cell lines Growth inhibition was assayed on human pancreatic carcinoma cell line (Mia PaCa-2), human prostate carcinoma (DU-145), human glioblastoma (U-87 MG), human prostate carcinoma (LNCaP), human colon carcinoma cell line (HT-29), human osteosarcoma (U2OS), and HeLa cells using the WST1 cell proliferation assay. The IC50 and the SE reported are calculated from at least two independent experiments with 8 determinations per tested concentration (see “Materials and Methods” for details). ␮ IC50 ( M) (SE) Cell lines Menadione BN82002 Mia PaCa-2 6.2 (0.15) 7.2 (0.9) DU-145 10.6 (0) 13.5 (0.8) U-87 MG 5.3 (1.3) 13.9 (3.5) LNCaP 15 (2.2) 22.4 (3.1) HT-29 9.8 (2.6) 32.6 (8.9) U2OS 6.3 (1.9) 11.2 (0.6) HeLa 15.5 (0.5) 23 (4.5)

␮ HT-29 with an IC50 of 32.6 M. The range of activity is very similar Fig. 2. BN82002 inhibits CDC25 activity in vitro and in vivo. A, Mia PaCa-2 cell were to the one observed with menadione (5–15 ␮M). treated with BN82002 at the indicated concentration for 1 h. Cells were lysed 24 h later BN82002 Inhibits Endogenous CDC25 Phosphatase Activity in and processed for Western blot analysis using antibodies against tyrosine 15 phosphorylated cyclin-dependent kinase 1 and actin. B, U2OS cells expressing hemagglutinin epitope Whole Cells. We investigated the molecular bases of the inhibitory tagged CDC25B under the control of the tetracycline-repressible promoter were grown for activity of BN82002 on cultured cells by monitoring its effect on the 16 h in the absence of tetracycline to allow CDC25 expression. Cells were treated with BN82002 at the indicated concentration for 2 h then harvested. Cell extracts were prepared tyrosine phosphorylation status of a natural CDC25C substrate, the and subjected to immunoprecipitation with antihemagglutinin antibodies. The phospha- CDK1 kinase. Indeed, the tyrosine 15 dephosphorylation of CDK1 is tase activity of CDC25 was assayed on the immunoprecipitate. fully dependent on the activity of the CDC25C phosphatase. MIA PaCa-2 cells established from a human pancreatic cancer were treated with increasing concentrations of BN82002 for 1 h. Twenty-four h later, the cells were lysed and processed for Western blot analysis using an antibody specific for the tyrosine 15-phosphorylated form of the CDK1 kinase. Actin was used as loading control. As shown in Fig. 2A, BN82002 at 50 ␮M and 100 ␮M induced an accumulation of the tyrosine 15-phosphorylated form of CDK1. The inhibition of CDK-1 dephosphorylation suggests that BN82002 efficiently down-regulates the activity of the CDC25C phosphatase toward CDK1 in cultured MIA PaCa-2 cells. To additionally characterize the effect of BN82002 on the activity of CDC25, we made use of a human osteosarcoma cell line U2OS that has been engineered to express a HA epitope-tagged CDC25B phosphatase under the control of the tetracycline repressible promoter. As already reported (29), CDC25B phosphatase was expressed at a de- tectable level starting from 12 h after tetracycline removal from the culture medium. After 16 h of CDC25B expression these cells were treated for 2 h with BN82002 (5 or 50 ␮M) then harvested. Cell extracts were subjected to immunoprecipitation with anti-HA antibodies, and the phosphatase activity of CDC25B was assayed. As shown in Fig. 2B, in cells treated with 50 ␮M BN82002, the activity of CDC25B was inhibited by ϳ60%. Taken together, these results indicate that treatment with BN82002 inhibits CDC25 phosphatase activity in cultured cells. The effects of this compound on cell cycle progression were therefore examined. BN82002 Inhibits Cell Cycle Progression. HeLa cells, a conven- Fig. 3. BN82002 impairs cell cycle progression. HeLa exponentially growing cells ient model for cell cycle studies, were used to investigate by flow were treated with 50 ␮M BN82002 for 24 h. Cells were harvested and subjected to flow cytometry analysis of their DNA content after propidium iodide staining. Data were cytometry after propidium iodide staining the effect of BN82002 on collected on a BD FACSCalibur and analyzed using the Modfit software (Verity soft- cell cycle progression. As shown in Fig. 3, after 24 h of treatment by ware). 3322

with BN82002 or with DMSO as a control. As shown in Fig. 4A, right

panels, cells that were treated in G1 phase with BN82002 remained longer in G1 phase and were delayed in their progression to S phase. At 10 h, most of the treated cells were still in S phase, whereas the

control population was already in G2 phase. When cells were treated with BN82002 in S phase (Fig. 4B, right panel), they also displayed

a significant delay in their progression to G2 phase. At 10 h, most of the cells were still in S phase, whereas the control population had

already passed mitosis (G2 to G1 DNA content) and entered the next cell cycle. Finally, HeLa cells that were treated in G2 phase with BN82002 also displayed a major delay in their progression to mitosis

(Fig. 4C, right panel). After 10 h, control cells were almost all in G1 phase, whereas half of the BN82002-treated cells were still in the G2 phase. From this set of experiments, we concluded that BN82002 is able to target and to delay cell cycle progression at different stages. CDC25 Overexpression Partially Reverts the Effects of BN82002 on Cell Cycle Progression. The U2OS cell line conditionally expressing CDC25B described above was used to investigate whether the expression of a high level of CDC25B (29) was able to counteract the ability of BN82002 to impair cell cycle progression. In contrast to what we observed with HeLa cells (Fig. 3), U2OS cells treated for 24 h with 50 ␮M BN82002 (in the presence of tetracycline to repress CDC25B expression), mostly (88%) arrested

their cell cycle with a G1 DNA content (Fig. 5). When treatment with 50 ␮M BN82002 was applied to cells that were grown previously for 20 h in the absence of tetracycline to allow CDC25B expression, the

effect of BN82002 was partially reversed with only 65% of cells in G1 and an ϳ30% population that was still in S phase. Expression of CDC25B had no significant effect on the control DMSO-treated cells. BN82002 Reverts CDC25B Mitotic-Inducing Effect. It has been demonstrated previously that the overexpression of CDC25B in HeLa cells results in illegitimate and premature entry into an abnormal mitotic process with cells displaying a phenotype described as premature chromosome condensation (32). We made use of this observation to set up a cellular assay allowing us to monitor the activity of the BN82002 inhibitor on a biological feature that is directly dependent on CDC25 activity. HeLa cells were transiently transfected with a HA-epitope tagged CDC25B-expressing plasmid and were treated 12 h later for an additional 12-h duration with 50 ␮M BN82002. Cells were fixed and examined by immunofluorescence after HA and 4Ј,6- diamidino-2-phenylindole staining. The percentage of abnormal mitotic figures was determined among the transfected cells expressing CDC25B. Twenty three percent of the CDC25B transfected cells

Fig. 4. BN82002 impairs cell cycle progression of synchronized HeLa cells. HeLa cells were synchronized in G1 phase by thymidine double-block then treated immediately (A)or released for either4h(B)or8h(C) to progress in S phase or in G2 phase, respectively, before the addition of BN82002. A–C, cells synchronized in G1,S,orG2 were treated during the indicated time with 50 ␮M BN82002, DMSO, or not treated (mock cells). Cells were harvested and subjected to flow cytometry analysis of their DNA content after propidium iodide staining. Data were collected on a BD FACSCalibur and analyzed using the Modfit software. Fig. 5. Expression of CDC25 partially reverts the BN82002-induced cell cycle arrest. U2OS cells expressing hemagglutinin epitope-tagged CDC25B under the control of the DNA content. Four h after release (Fig. 4B, left panel) most of the tetracycline repressible promoter were grown for 20 h in the presence (ϩTet)orinthe absence (ϪTet) of tetracycline to allow CDC25B expression. Cells were subsequently cells were in S phase (70%), and at 8 h the majority (65%) were in G2 treated with the BN82002 or DMSO (control) for 24 h, then harvested and subjected to phase (Fig. 4C, left panel). These G1-, S- and G2-synchronized pop- flow cytometry analysis of their DNA content after propidium iodide staining. Data were ulations (Fig. 4, A, B, and C, respectively) were treated for 3 and 10 h collected on a BD FACS Calibur and analyzed using the Modfit software. 3323

basis for the development of new classes of CDC25-directed therapeutic agents (see Introduction). The work presented here focuses on the characterization of BN82002, a new inhibitor of CDC25 phosphatases discovered in the course of a compound library screen set up to identify small molecules able to inhibit in vitro the catalytic activity of a recombinant CDC25C phosphatase. BN82002 shows activity similar to Menadione, but is less active than the compounds published recently from the Lazo et al. (19). However, based on the recent reviews describing CDC25 inhibitors, BN82002 represents an original structure that is chemically accessible. The active site of CDC25 contains a catalytic cysteine (39). BN82002, like Menadione and NSC663284 (18), might inactivate CDC25 by inducing disulfide bond linkage through quinone redox reaction. We have found that addition of Glutathion (20 mM) together with BN82002 or Menadione to the CDC25C enzyme assay increased the Menadione IC by 3.9-fold and BN82002 IC by 4.3-fold. Fig. 6. Growth inhibition of human pancreas tumor Mia PaCa-2 in nude mice treated 50 50 with BN82002 by i.p. route. Cells of the human pancreatic carcinoma cell line Mia PaCa-2 Glutathion by itself had no effect. Glutathion might prevent the link of were injected s.c. into the flank of female athymic mice. Tumors were allowed to reach BN82002 or Menadione on the catalytic cysteine in the active site of 3 a volume of 100 mm . Once tumors were established, treatment was started by i.p. route the enzyme. as 15 mg/kg daily during 10 days (once daily ϫ 10) or 40 mg/kg weekly for 3 weeks (once weekly ϫ 3). As reported by Rudolph et al. (40), the specificity of CDC25 phosphatases for their CDK substrates resides within the catalytic domain itself and in region(s) outside the phosphate binding site of the displayed abnormal condensed chromatin (premature chromosome enzyme. On the basis of this observation, we made use of a full-length condensation) 24 h after transfection. DMSO had no influence on this recombinant CDC25C protein to evaluate the inhibitory activity of result. In cells treated with BN82002, the percentage of premature BN82002. However, using OMFP as substrate, no significant differ- chromosome condensation cells was only 12%, which represents a ence could be observed between the inhibition of the full-length 45% inhibition of the mitosis-inducing effect of CDC25B. This result CDC25C enzyme and the corresponding catalytic domain. Our initial indicates that treatment with BN82002 is able to counteract the attempts to set up a robust assay with a natural CDK-cyclin phospho- mitotic inducing activity of the CDC25B phosphatase. This observa- rylated substrate were not successful (data not shown). Consequently, tion indicates that in cultured cells CDC25 is indeed a target for the the assays reported here were only performed with artificial sub- BN82002 compound. strates. In addition, we found that the ratio of selectivity for one In Vivo Growth Inhibition of MIA PaCa-2 Cells in Athymic compound on two different phosphatases could vary considerably Nude Mice. On the basis of the in vitro results, the activity of upon minor changes of the experimental parameters (i.e., pH) or 4 BN82002 was evaluated in athymic mice xenografted with the human artificial ligand choice. On the basis of these technical limitations, no pancreatic cell MIA PaCa-2. Drug treatment was initiated when the additional efforts were made to perform selectivity studies on isolated tumors reached a volume of 100 mm3 and continued once a day for 10 recombinant phosphatases. As an alternative, we have explored the days at 15 mg/kg or once a week for 3 weeks at 40 mg/kg. BN82002 effects of BN82002 on whole cells where the natural endogenous stabilized tumor growth during the once daily ϫ 10 treatment and substrate CDK-cyclin complexes are present in a biological context. induced a delay in the once weekly ϫ 3 schedule. The benefit of this We present several lines of evidence, which strongly suggest that stabilization was still visible after the arrest of the treatment (Fig. 6). BN82002 targets CDC25 phosphatases in cultured cells. First, in MIA No toxicity in terms of weight loss and no animal death were noted PaCa-2 cells, CDK1 tyrosine phosphorylation is increased by treat- during the course of the experiments. In contrast, no activity of ment with BN82002, indicating that CDC25 phosphatase activities menadione was observed up to 100 mg/kg in this model. that are essential to dephosphorylate and activate CDK1 have been inhibited. The high BN82002 concentration required for affecting CDK1 phosphorylation is likely to be related to the treatment duration DISCUSSION that is shorter (1 h) that in the proliferation assay (96 h). Second, A number of laboratories are currently evaluating the possibility of treatment with BN82002 diminishes the CDC25 activity that can be targeting cell cycle regulators as a new strategy in cancer therapy (33, immunoprecipitated from cultured cells, suggesting that the activity of 34). Indeed, cell-cycle deregulation is a hallmark of tumor cells, and the phosphatase has been inhibited by the compound. Third, BN82002 a number of neoplasias show abnormal expression or mutation of key efficiently reverts the aberrant mitotic-inducing effect observed in a regulators (35). Over the past few years, CDK inhibition has been of specific biological assay based on the phenotype observed upon considerable interest because of the essential role of these enzymes in CDC25B overexpression in HeLa cells. Finally, we observed that the the control of cell cycle progression. Several potent CDK inhibitors growth inhibitory effect of BN82002 is partly counteracted by the have been identified and characterized, and few of them are currently conditional expression of a CDC25 phosphatase in the U2OS cell line, undergoing Phase I and II clinical trials (reviewed in Refs. 36, 37). suggesting that a large excess of CDC25 is able to titrate out the Because of their critical role in the activation of CDKs, CDC25 inhibitor. phosphatases are also considered attractive putative targets for the It is not surprising that the cell cycle of HeLa cells treated with development of new chemotherapeutic drugs and are currently the BN82002 is globally affected with, as shown in Fig. 4, a delay in object of drug discovery efforts in pharmaceutical companies (15, 38). progression at various stages. This observation is in accordance with Most of the inhibitors described thus far lack the efficacy that is required to enter clinical trials. However, they provide a structural 4 Unpublished observations. 3324

BN82002 being active against several members of the CDC25 phos- 12. Yao Y, Slosberg ED, Wang L, et al. Increased susceptibility to carcinogen-induced phatase family (variants B and C). Thus, cell cycle inhibition is mammary tumors in MMTV- Cdc25B transgenic mice. Oncogene 1999;18:5159–66. 13. Vincent I, Bu B, Hudson K, Husseman J, Nochlin D, Jin L. Constitutive Cdc25B observed at the various stages of the cell cycle where these enzymes tyrosine phosphatase activity in adult brain neurons with M phase-type alterations in Alzheimer’s disease. Neuroscience 2001;105:639–50. are required. Why U2OS cells are only arrested in G1 phase is unclear 14. Zhang ZY. Protein tyrosine phosphatases: structure and function, substrate specific- and remains to be investigated. ity, and inhibitor development. Annu Rev Pharmacol Toxicol 2002;42:209–34. We have shown here that BN82002 delays tumor growth in xe- 15. Prevost G, Brezac M, Goubin F, et al. Inhibitors of CDC25 phosphatases. 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