Improved Tumor Control Through Circadian Clock Induction By

Research Article

Improved Tumor Control through Circadian Clock Induction by Seliciclib, a Cyclin-Dependent Kinase Inhibitor Ida Iurisci,1,2 Elisabeth Filipski,1 Jens Reinhardt,3 Ste´phane Bach,3 Athos Gianella-Borradori,4 Stefano Iacobelli,2 Laurent Meijer,3 and Francis Le´vi1

1Institut National de la Sante´et de la Recherche Me´dicale, U776 ‘‘Rythmes Biologiques et Cancers,’’ Hoˆpital Paul Brousse, Villejuif Cedex, France; Universite´Paris Sud, Orsay, France; 2Department of Oncology and Neurosciences, Universita` ‘‘G. D’Annunzio,’’ Chieti, Italy; 3Centre National de la Recherche Scientifique, Cell Cycle Group, UPS-2682 & UMR-7151, Roscoff Cedex, France; and 4Cyclacel Ltd., Dundee, United Kingdom

Abstract cancer patients (4, 5). Taken together, these findings support a The circadian timing system and the cell division cycle are close link between the circadian timing system and the cell division frequently deregulated in cancer. The therapeutic relevance of cycle, which is frequently deregulated in cancer cells. the reciprocal interactions between both biological rhythms The circadian timing system efficiently orchestrates the was investigated using Seliciclib, a cyclin-dependent kinase physiology of living organisms to match environmental or imposed (CDK) inhibitor (CDKI). Mice bearing Glasgow osteosarcoma 24-hour cycles (6). Most cells in the brain and peripheral tissues received Seliciclib (300 mg/kg/d orally) or vehicle for 5 days contain a molecular clock consisting of at least 12 specific clock at Zeitgeber time (ZT) 3, 11, or 19. On day 6, tumor mRNA genes in mammals. This molecular clock rhythmically controls the f f 24-hour expression patterns were determined for clock genes transcriptional activity of 10% of the genome, 10% of which (Per2, Rev-erba, and Bmal1) and clock-controlled cell cycle are proliferation-related genes (7). Among the 12 genes that a genes (c-Myc, Wee1, cyclin B1, and CDK1) with quantitative constitute the molecular clock, Per2, Bmal1, and Rev-erb play a reverse transcription-PCR. Affinity chromatography on immo- central role. Thus, mice with a null mutation in these genes bilized Seliciclib identified CDK1/CDK2 and extracellular displayed profound alterations of the circadian phenotype in signal-regulated kinase (ERK) 1/ERK2, CDK7/CDK9, and constant darkness. Indeed, the rest-activity rhythm was ablated in casein kinase CK1E as Seliciclib targets, which respectively Per2 or Bmal1 mutants. Rev-erba mutation shortened the period regulate cell cycle, transcription, and circadian clock in length of this rhythm with wide interindividual variations and Glasgow osteosarcoma. Seliciclib reduced tumor growth by dramatically altered the phase-shifting response to light (6, 8, 9). 55% following dosing at ZT3 or ZT11 and by 35% at ZT19 The intracellular clock mechanisms involve interacting positive compared with controls (P < 0.001). Tolerability was also best and negative transcriptional feedback loops that drive recurrent at ZT3. Mean transcriptional activity of Rev-erba, Per2, and rhythms in the RNA levels of these key components. High levels of Bmal1 was arrhythmic in the tumors of untreated mice. Bmal1 mRNA and protein promote the formation of BMAL1:- Seliciclib induced rhythmic clock gene expression patterns CLOCK heterodimers that bind to the E-box sequences in the a with physiologic phase relations only after ZT3 dosing. c-Myc promoter of clock genes Per2 and Rev-erb and activate their a and Wee1 mRNAs displayed synchronous circadian rhythms in transcription. In turn, Rev-erb negatively regulates Bmal1 the tumors of control mice receiving vehicle only but not in transcription, whereas PER2:CRY1 complexes inhibit the transcrip- those of mice given the drug. Seliciclib further enhanced Wee1 tion of their own genes by interfering with CLOCK:BMAL1 (6, 8, 9). expression irrespective of dosing time, an effect that rein- In previous studies, we found that the circadian expression pattern of core clock genes Per2, Bmal1, and Rev-erba was severely forced G2-M gating. Seliciclib also inhibited CK1E, which determines circadian period length. The coordination of clock altered in Glasgow osteosarcoma, a rapidly proliferating tumor gene expression patterns in tumor cells was associated with transplanted in mice (10, 11). Further disruption of the circadian best antitumor activity of Seliciclib. The circadian clock and timing system with ablation of the hypothalamic pacemaker or its upstream regulators represent relevant targets for CDKIs. chronic jet lag accelerated malignant progression of this tumor (Cancer Res 2006; 66(22): 10720-8) (10–12). At least three molecular mechanisms link molecular clock with Introduction the cycling of cell division. The molecular clock controls Wee1 transcription through an E-box-mediated mechanism (13). Wee1 Large epidemiologic studies have shown an increased incidence negatively controls the activity of cyclin-dependent kinase (CDK) of breast or colorectal cancers in populations exposed to prolonged 1/cyclin B1, which regulates the G2-M transition. In addition, the shift work, frequent transmeridian flights, or light exposure at night BMAL1:CLOCK heterodimers repress c-Myc transcription through (1–3). These latter conditions profoundly alter circadian rhythms. E-box-mediated reactions in the c-Myc gene P1 promoter and In addition, a tight relationship has been established between mPer2 can suppress c-Myc expression indirectly by stimulating circadian rhythm disruption and quality of life and even survival of Bmal1 transcription (8). In general, knocking down the molecular clock modified transcription patterns of genes involved in the cell cycle regulation, which translated into genomic instability, thus Requests for reprints: Francis Le´vi, Institut National de la Sante´etdela Recherche Me´dicale, U776 ‘‘Rythmes Biologiques et Cancers’’ (Universite´Paris XI), favoring malignant progression or growth (8, 11). Finally, Per1 Paul Brousse Hospital, 94800 Villejuif, France. Phone: 33-1-45-59-38-55; Fax: 33-1-45-59- as well as Per2 and possibly other clock genes also control DNA 36-02; E-mail: [email protected]. I2006 American Association for Cancer Research. repair through interactions with ataxia-telangiectasia mutated and doi:10.1158/0008-5472.CAN-06-2086 mdm2 (14, 15).

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Based on the evidence for signaling pathways shared by the France) for 3 weeks before tumor inoculation (10). An adequate synchro- circadian clock and the cell cycle (8, 11–16), we have used the CDK nization of mice by these lighting regimens was assessed by the occurrence inhibitor (CDKI) Seliciclib (also known as R-roscovitine, CYC202; of a normal circadian variation in rectal temperature before treatment ref. 17) as a pharmacologic tool to investigate the therapeutic initiation. The experiment involved 132 B6D2F1 male mice bearing Glasgow osteosarcoma. Nine days after tumor inoculation, when overall tumor relevance of cross-talks between the circadian clock and the cell weight reached 100 to 400 mg, Seliciclib (300 mg/kg/d) was given via oral division cycle in tumors grown in mice. Seliciclib is a 2,6,9- gavage once daily during 5 days to a total of 96 mice at one of three different trisubstituted purine in clinical phase II development. This drug times referred to light onset [hours after light onset (HALO)], so-called competes with ATP for the binding to the catalytic site of CDKs Zeitgeber time (ZT): ZT3, ZT11, or ZT19 (32 mice per dosing ZT). ZT3 and and induces transient growth arrest in both G1-S and G2-M by ZT11 were located during the light span when mice usually rest; ZT19 inhibiting CDK2/cyclin E and CDK1/cyclin B1, respectively (18). It corresponded to the dark span when mice are usually active. Thirty-six mice also interferes with transcription by inhibiting CDK7/cyclin H and [12 per circadian time (CT)] received vehicle (50 mmol/L HCl). After the last CDK9/cyclin T, two kinases involved in the phosphorylation of the drug injection, all the animals were exposed to constant darkness during COOH-terminal repeat domain of RPB1, the largest subunit of RNA 24 hours, then they were sacrificed at CT5, CT11, CT17, or CT23, and two polymerase II. It further interacts with the casein kinase 1 (CK1) perpendicular tumor diameters were measured with a caliper and tumors were dissected (Fig. 1). This procedure avoids any masking effect of light on family (19, 20), which plays a central role in circadian clock q y circadian rhythmic patterns and provides a reliable estimate of CT in groups function. Indeed, CK1 or CK1 mutations or alterations produce of individual mice within the initial 3 days in constant darkness because the circadian disorders of rest-activity or sleep-wakefulness rhythms circadian phase of B6D2F1 mice in constant darkness only drifted by 10 both in rodents and in humans (6, 21). In particular, phosphory- to 30 minutes daily (23). The samples (30 mg) were frozen immediately in lation S662 of hPER is required for the serial phosphorylation of liquid nitrogen and stored at À80jC until RNA extraction. Circadian COOH-terminal serines by CK1q, and an S662G missense mutation expression patterns of mPer2, mBmal1, mRev-erba, mWee1, mCyclin B1, causes familial advanced sleep phase syndrome in a dominant mCDK1, and mC-Myc were measured with reverse transcription-PCR using fashion (22). LightCycler instrument (Roche Diagnostics, Meylan, France). There were In the current study, the antitumor efficacy of Seliciclib and its eight treated and three control animals per sampling time in each group. effects on molecular clock and cell division cycle genes in tumor Drugs were investigated in mice with Glasgow osteosarcoma, a rapidly Seliciclib was obtained from Cyclacel Ltd. (Dundee, United Kingdom). It growing tumor known to be regulated by the circadian timing was diluted in 50 mmol/L HCl and administered via oral gavage once daily system (10–12). during 5 days. Controls received 50 mmol/L HCl. Tumor Model Materials and Methods Glasgow osteosarcoma tumor was kindly provided by the Research Centre The study was conducted in accordance with the guidelines approved for of Aventis Pharma (Vitry sur Seine, France). It was transplanted as bilateral animal experimental procedures by the French Ethical Committee (decree s.c. fragments (3 Â 3 mm) in each flank of the recipient mouse. Tumor length 87-848). and width (mm) were measured with a caliper daily and tumor weight (mg) was computed as follows: tumor weight = (length Â width2) / 2 (10). Animals and Synchronization B6D2F1 male mice, 5 to 6 weeks old, were purchased from Janvier Real-time Quantitative PCR (Le Genest St Isle, France). All mice were synchronized with an alternation Total RNA was extracted from frozen tissue specimens of tumor using of 12 hours of light and 12 hours of darkness in an autonomous chronobio- RNeasy Mini kit (Qiagen S.A., Courtaboeuf, France) according to the logical animal facility (Jouan-Thermo Electron LED S.A.S., Saint-Herblain, manufacturer’s instructions. Contaminating genome DNA was eliminated

Figure 1. Study design. Seliciclib or vehicle was given at three different ZTs (3, 11, and 19) for 5 consecutive days in mice whose circadian timing system was synchronized with an alternation of 12 hours of light and 12 hours of darkness (the light/dark Zeitgeber). Starting 24 hours after the last administration, liver and tumor were sampled at four different endogenous CTs (5, 11, 17, and 23). For this purpose, the mice were switched to constant darkness after the last dose was delivered. This procedure avoided a possible masking effect of light on the endogenous tumor rhythms that were assessed.

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Figure 2. Dosing time-dependent effects of Seliciclib on tumor growth. Tumor weight change as a function of Seliciclib administration time. ., controls; o, treated at ZT3; E, treated at ZT11; D, treated at ZT19. Treatment started on day 9 after tumor inoculation. Arrows, days of drug injection. Thirty-six controls and 32 mice were allocated to each treatment group. Points, mean; bars, SE. Differences between controls and animals treated at ZT3, ZT11, or ZT19 were validated with ANOVA (P < 0.001).

by treatment with RNase-Free DNase Set kit (Qiagen). First-step reverse Bead buffer: Tris (50 mmol/L; pH 7.4), 5 mmol/L NaF, 250 mmol/L NaCl, transcription was done with the High-Capacity cDNA Archive kit (Applied 5 mmol/L EDTA, 5 mmol/L EGTA, 0.1% NP40, 10 Ag/mL of leupeptin, Biosystems, Courtaboeuf, France) according to the manufacturer’s instruc- aprotinin, and soybean trypsin inhibitor, and 100 Amol/L benzamidine. tions. RNA (5 Ag) was reverse transcribed using multiscribe (Applied Purification and identification of Seliciclib-interacting proteins Biosystems). from tumors. Glasgow osteosarcoma samples were sonicated in homoge- Second-step real-time PCR was done in LightCycler using QuantiTect nization buffer. Homogenates were centrifuged for 10 minutes at 14,000 Â g SYBR Green PCR kit (Qiagen). All the PCR primers were obtained from at 4jC. The supernatant was recovered, assayed for protein content [Bio-Rad Invitrogen Life Technologies (Cergy Pontoise, France). Primer sequences (Marnela Coquette, France) protein assay], and immediately loaded batch were designed to avoid the formation of secondary structures with Primer3 wise on the affinity matrix. algorithm and were as follows: Per2, 5¶-AGAGCACAGGGTCTGGAGGA-3¶ Seliciclib and N6-methyl-Seliciclib (control, kinase-inactive analogue) and 5¶-TGGAACACAGGTAGGGGGTAAG-3¶; Bmal1, 5¶-GGCTGGACGAAGA- beads were obtained as described (20, 24). Just before use, 10 AL of packed CAATGAGC-3¶ and 5¶-GTTGTGGAACCATGTGCGAGT-3¶;Rev-erba,5¶- Seliciclib beads were washed with 1 mL of bead buffer and resuspended in GGCTGATTCTTCACACACACAC-3¶ and 5¶-GGTCTTGGGGTGGCTA- 600 AL of this buffer. The tumor extract supernatant (3 mg total proteins) TACTG-3¶;c-Myc,5¶-TGATGTGGTGTCTGTGGAGAAG-3¶ and 5¶- was then added; the tubes were rotated at 4jC for 30 minutes. After a brief CGTAGTTGTGCTGGTGAGTGG-3¶; Wee1, 5¶-GAAACAAGACCTGCCAAAA- spin at 10,000 Â g and removal of the supernatant, the beads were washed GAA-3¶ and 5¶-GCATCCATCTAACCTCTTCACAC-3¶; CDK1, 5¶-GGCAGTT- four times with bead buffer before addition of 60 AL2Â Laemmli sample CATGGATTCTTCACTC-3¶ and 5¶-GCCAGTTTGATTGTTCCTTTGTC-3¶; and buffer. Following heat denaturation for 3 minutes, the bound proteins were cyclin B1, 5¶-TCTTGACAACGGTGAATGGACAC-3¶ and 5¶-CATTCTTAGC- analyzed by SDS-PAGE and Western blotting or silver staining as described CAGGTGCTGCAT-3¶. The primer sequence for time-invariant 36B4 gene below. used for normalization was 5¶-GACCTCACTGAGATTCGGGATA-3¶ and 5¶-GGTCCTAGACCAGTGTTCTGAGC-3¶. Electrophoresis and Western Blotting A four-step experimental run protocol was used: denaturation of cDNA Antibodies. The following antibodies were obtained from commercial (15 minutes at 95jC) followed by amplification and quantification program sources: anti-CKIa (1:500, 1 hour; BD Transduction Laboratories, Erembo- repeated 45 times (15 seconds at 95jC, 25 seconds of primers annealing at deyem, Belgium), anti-CK1q (1:1,000, 1 hour; Cell Signaling Technology, 60jC, for all the primers used, 15 seconds at 72jC of extension/synthesis Beverly, MA), anti-extracellular signal-regulated kinase (ERK) 1/ERK2 with a single fluorescence measurement) at each cycle, melting curve (1:4,000, 1 hour; Sigma, Saint Quentin Fallavier, France), anti-PSTAIRE program (15 seconds at 65jC with a heating rate of 0.1jC per second and a (1:3,000, 1 hour; Sigma), anti-CDK7 (1:500, 1 hour; Santa Cruz Biotechnology, continuous fluorescence measurement), and cooling program down to Santa Cruz, CA), anti-CDK9 (1:500, 1 hour; Santa Cruz Biotechnology), and 40jC. The intra-assay and inter-assay variation coefficients were <1% and anti-cyclin H (1:500, 1 hour; Santa Cruz Biotechnology). Anti-PDXK was <4%, respectively. The real-time PCR efficiency of each transcript was generated as previously reported (18), and anti-ahCaMKII (RU16) was validated by construction of serial dilution curves and calculated from the prepared and characterized at The Rockefeller University (New York, NY). À slope according to the established equation: E =10[ 1/slope]. It was close to Electrophoresis and Western blotting. Following heat denaturation for 1.9 in all PCRs. The specificity of the PCR products was assessed by melting 3 minutes, the proteins purified with the Seliciclib matrix were resolved by curve analysis and by agarose gel electrophoresis to confirm that the size of 10% SDS-PAGE (0.7-mm-thick gels) followed by immunoblotting analysis or the product corresponded to that of the expected amplicon and to check silver staining using an Amersham Biosciences/GE Healthcare (Orsay, the presence of nonspecific products. The relative expression of the target France) SDS-PAGE silver staining kit. For immunoblotting, proteins were transcripts was normalized to the corresponding 36B4 cDNA. transferred to 0.45-Am nitrocellulose filters (20, 24). These were blocked with 5% low fat milk in TBS-Tween 20, incubated for 1 hour with antibodies, Affinity Chromatography on Immobilized Seliciclib and analyzed by enhanced chemiluminescence (Amersham Biosciences). Buffers Homogenization buffer: h-Glycerophosphate (60 mmol/L), 15 mmol/L CK1 Assays p-nitrophenylphosphate, 25 mmol/L MOPS (pH 7.2), 15 mmol/L EGTA, Construction and expression of glutathione S-transferase-axin 15 mmol/L MgCl2, 1 mmol/L DTT, 1 mmol/L sodium vanadate, 1 mmol/L fragment fusion protein. An axin fragment was generated by PCR using NaF, 1 mmol/L phenylphosphate, 10 Ag leupeptin/mL, 10 Ag aprotinin/mL, the plasmid pET-mouse Axin (290-827; a generous gift from Frank Costantini, 10 Ag soybean trypsin inhibitor/mL, and 100 Amol/L benzamidine. Department of Genetics and Development, Columbia University, New York,

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NY). The PCR product was cloned into pGEX-4T1 (Amersham Biosciences). supernatant, the beads were washed thrice with bead buffer and once in The fusion protein was expressed in Escherichia coli BL21 (Stratagene, kinase assay buffer. For the kinase assays, 50 Amol/L of the CK1-specific Amsterdam, the Netherlands) after induction with 0.5 mmol/L isopropyl-L- peptide substrate RRKHAAIGpSAYSITA (pS stands for phosphorylated thio-h-D-galactopyranoside at 32jC for 3 hours. Bacteria resuspended in PBS serine; gift of F. Meggio and L. Pinna, Universita`di Padova, Dipartimento [containing protease inhibitors aprotinin, soybean protease inhibitor, and di Chimica Biologica, Padova, Italy; ref. 24), 15 Amol/L ATP containing leupeptin (10 Ag/mL each) and 100 Amol/L benzamidine] were lysed by 3 ACi/mL [g-33P]ATP, and kinase assay buffer were added to the beads as well sonication or using a French press at 160 bar. After centrifugation at 10,000 Â as the indicated concentrations of Seliciclib (with the solvent DMSO added to g for 10 minutes, the supernatant was recovered and the glutathione 10%). The reaction was carried out for 15 minutes at 30jC with frequent S-transferase (GST)-axin fragment fusion protein was purified on glutathione mixing, and the reaction was stopped by cooling on ice. The reaction mixture agarose matrix (Sigma) for 1 hour in bead buffer. The beads were washed in was spotted on phosphocellulose paper (Whatman p81, Oxon, bead buffer and stored until further usage on ice. United Kingdom) filters, which were washed four times for 5 minutes in 1% Kinase assay buffer. h-Glycerophosphate (60 mmol/L), 30 mmol/L phosphoric acid. Scintillation was measured in a Packard Instruments p-nitrophenylphosphate, 25 mmol/L MOPS (pH 7.0), 5 mmol/L EGTA, (Rungis, France) scintillation counter after addition of 1 mL scintillation fluid. 15 mmol/L MgCl2, 1 mmol/L DTT, and 0.1 mmol/L sodium vanadate. CK1 purification and activity determination. The tumor extract Statistical Analyses supernatant (0.5 mg/assay) was loaded on glutathione agarose beads For each studied variable, means and standard errors were calcu- saturated with GST-axin and incubated with bead buffer for 1 hour under lated. Intergroup differences were compared statistically using multiple- constant rotation at 4jC. After a brief spin at 10,000 Â g and removal of the way ANOVA. Statistical significance of circadian rhythmicity was validated

Figure 3. Effects of Seliciclib treatment on mRNA expression patterns of clock genes. A to C, relative mRNA levels for Rev-erba (A), Per2 (B), and Bmal1 (C) in the tumors of mice after 5 days of treatment with Seliciclib at ZT3 (solid line) or vehicle (dashed line). The relative mRNA expression of each gene of interest was normalized to that of 36B4. Points, mean of eight to nine (controls) or six to eight (treated) mice; bars, SE. X axis, hatched rectangles, subjective light span; black rectangles, dark span. To better visualize the rhythm during the 24-hour period, data obtained at CT5 were repeated 24 hours later. No significant 24-hour rhythm was found in controls. Treatment with Seliciclib at ZT3 induced circadian rhythm in Rev-erba (P from cosinor = 0.007), Per2 (P = 0.0004), and Bmal1 (P = 0.08). D, twenty-four-hour means of mRNA levels in controls and in mice treated at ZT3, ZT11, or ZT19. Columns, mean of 24 to 30 tumors; bars, SE. Data are expressed as % of mean mRNA levels of controls. In mice treated at ZT3, expression was significantly decreased for Rev-erba (P = 0.004, ANOVA; *, P = 0.009, post hoc test) and increased for Bmal1 (P = 0.006, ANOVA; *, P = 0.0038, post hoc test). www.aacrjournals.org 10723 Cancer Res 2006; 66: (22). November 15, 2006

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Cancer Research with Cosinor analysis (11). This method characterizes a rhythm by variables of the fitted cosine function best approximating all data. Periods s = 24 hours and s = 12 hours were considered a priori. The rhythm characteristics estimated by this linear least squares method include the mesor (rhythm-adjusted mean), the double amplitude (difference between minimum and maximum of fitted cosine function), and the acrophase (time of maximum in fitted cosine function, with light onset as phase reference). A rhythm was detected if the null hypothesis was rejected with P < 0.05. Differences in lethal toxicity were compared with m2 Fisher’s exact test. All standard statistical tests were done using Statistical Package for the Social Sciences version 11.5 for Windows software (SPSS, Chicago, IL).

Results CT dependency of therapeutic activity. Treatment with Seliciclib or vehicle was initiated 9 days after tumor inoculation, when the mean tumor weight (FSE) was 278 mg (F12; range, 141-458 mg). Daily oral treatment was given to different groups of mice at one of three dosing times separated by 8 hours: ZT3 HALO, ZT11, or ZT19. At the end of treatment, mean tumor weight was reduced by 55% in the animals receiving Seliciclib at ZT3 or ZT11 and by 35% in those treated at ZT19 compared with untreated controls (P < 0.001, ANOVA; Fig. 2). Seliciclib toxicity was the lowest following dosing at ZT3 (lethal toxicity rate, 3.1%) compared with ZT11 (6.2%) or ZT19 (21.9%; P = 0.07, Fisher’s exact test). Seliciclib induction of clock gene expression rhythms in tumors. No significant rhythmic transcription was found for any of the three clock genes (Rev-erba, Per2, and Bmal1) tested in the tumors of untreated mice. Seliciclib dosing at ZT3 induced rhythmic expression of Rev-erba (P = 0.007), Per2 (P = 0.0004), and Bmal1 (P = 0.08) with 5.2-, 3.8-, and 2.4-fold respective peak to trough differences (Fig. 3A-C). The zenith in circadian Bmal1 mRNA accumulation in the tumors of mice treated at ZT3 cor- related well with the circadian nadir in Rev-erba mRNA, a typical feature in healthy tissues (9). In addition, Seliciclib nearly halved the 24-hour mean mRNA expression of Rev-erba and doubled that of Bmal1 in the tumors of mice treated at ZT3 (Fig. 3D), an effect that was not seen at other dosing times. The tumor circadian clock was modeled through an adjustment of 24-hour cosine functions to the expression data of the three core clock genes. The tumor clock in untreated mice was clearly disrupted (Fig. 4B) compared with that in healthy liver (Fig. 4A) a Figure 4. Cosinor-based model of molecular clock in tumor as a function where the acrophases occur at 5.20 (95% CI, 2.40-8.00) for Rev-erb , of Seliciclib dosing time. A, physiologic clock. The adjustment of a cosine curve 14.20 (95% CI, 10.20-18.20) for Per2, and 23.40 (95% CI, 21.40-1.40) with a 24-hour period to mRNA expression of Rev-erba (blue line), Per2 for Bmal1 (11). In mice given Seliciclib at ZT3, both the double (red line), and Bmal1 (green line) allowed to build a model of the circadian clock in healthy liver of B6D2F1 mice (data from 11). This model establishes amplitude and the acrophases of the three clock genes in tumor physiologic dynamic relations with maxima occurring at CT6 for Rev-erba, CT15 became similar to those in healthy liver; for example, acrophases for Per2, and CT23 for Bmal1. B to E, tumor clock. The same method was were respectively located at 5.30 (95% CI, 2.20-8.40), at 11.20 (95% applied to clock gene data obtained in the current study for control tumors (B) and for tumors from mice treated with Seliciclib at ZT3 (C), ZT11 (D), or ZT19 CI, 9.00-13.40), and at 22.40 (95% CI, 19.10-2.10; Fig. 4C). Seliciclib (E). Physiologic phase relations between expression patterns of the three dosing at ZT11 also resulted in double amplitudes in clock gene clock genes, similar to those in healthy liver, were induced by treatment at ZT3 expression that were similar to those in healthy liver, yet the or at ZT11 but not at ZT19. acrophases were ill placed; for example, at 11.20 (95% CI, 9.50- 12.50) for Rev-erba, at 17.50 (95% CI, 16.40-19.00) for Per2, and at Seliciclib alteration of cell cycle gene expression patterns in 5.20 (95% CI, 1.10-9.30) for Bmal1 (Fig. 4D). Finally, Seliciclib tumors. c-Myc and Wee1 mRNAs displayed synchronous circadian administration at ZT19 achieved double amplitudes in clock genes rhythms in control tumors (P from cosinor = 0.03 and 0.01, that were nearly half of those in mice treated at ZT3 or ZT11. The respectively) with acrophases occurring at 9.00 (95% CI, 4.30-13.30) acrophases were nearly opposite as those found in normal liver; for and 11.50 (95% CI, 8.20-15.20). Seliciclib treatment at ZT3 advanced example, at 18.40 (95% CI, 14.40-23.20) for Rev-erba, at 23.50 (95% the acrophase of c-Myc to 3.50 (95% CI, 23.30-8.10) and ablated CI, 20.10-3.30) for Per2, and at 12.10 (95% CI, 7.00-17.20) for Bmal1 Wee1 rhythm (Fig. 5A and B). Both rhythms were suppressed in (Fig. 4E). mice treated at ZT11 or ZT19 (data not shown). No rhythm was

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Targeting Clock Genes Improves Cancer Control observed for CDK1 or cyclin B1 expression in the tumors of control Discussion or treated mice (data not shown). Seliciclib doubled the expression CDK pathways represent promising targets as cancer therapeu- of Wee1 (P = 0.002) and CDK1 (P = 0.06) compared with controls tics (25). Seliciclib, like flavopiridol, another CDKI in clinical and mildly increased cyclin B1, with largest increments in the mice testing, has shown activity against several human tumor cell lines dosed at ZT3 (Fig. 5C). both in vitro and following engraftment in nude mice. Surprisingly, Main cell cycle, transcription, and circadian clock target however, no report mentions any activity of these compounds in molecules of Seliciclib in tumors. Affinity chromatography was the mouse tumor models that are currently used for preclinical done on Glasgow osteosarcoma extracts using immobilized testing (26, 27). Overall, clinical trials have revealed modest activity Seliciclib, with immobilized CDK-inactive N6-methyl-Seliciclib as of both compounds against solid tumors (25). Yet a marked efficacy a control matrix (Fig. 6A; refs. 20, 24). SDS-PAGE followed by silver of flavopiridol was recently uncovered in patients with refractory staining and Western blotting with various antibodies revealed the chronic lymphocytic leukemia receiving combined bolus and expected targets [i.e., CDK1/CDK2 (jointly detected with anti- intermittent infusional delivery (28). These and other results have PSTAIRE), ERK1/ERK2, calmodulin-dependent kinase 2 isoforms, emphasized the marked schedule dependency of CDKI efficacy, and pyridoxal kinase (PDXK); Fig. 6B]. In addition, this method also possibly influenced by the cyclic nature of the CDK target pathways identified Seliciclib interactions with CK1q but not CK1a. Both themselves. Thus, the rhythmic assembly and dissociation of CDKs CDK7, and its activatory cyclin H, and CDK9 were also detected on and cyclins drive the physiologic course of the cell division cycle, Seliciclib beads. None of these proteins, except PDXK, interacted a biological clock in its own. Furthermore, the cell division cycle with control N6-methyl-Seliciclib agarose (Fig. 6B). closely interacts with the circadian clock, which controls several Seliciclib inhibited the catalytic activity of CK1 following affinity cell and organism functions along the 24-hour time scale (8, 13–16, purification of Glasgow osteosarcoma fragments from untreated 29). In particular, the circadian clock controls Wee1 expression, A mice. The IC50 was 18 mol/L (Fig. 6C), in agreement with prior thus gating G2-M transition, a checkpoint that is also largely studies on other tissue extracts (20, 24). affected by Seliciclib (13).

Figure 5. Effects of Seliciclib treatment on mRNA expression patterns of clock-controlled cell cycle genes. Relative mRNA levels for c-Myc (A) and Wee1 (B) in the tumors of mice after 5 days of treatment with Seliciclib at ZT3 (solid line) or vehicle (dashed line). The relative mRNA expression of each gene of interest was normalized to that of 36B4. Points, mean of eight to nine (controls) or six to eight (treated) mice; bars, SE. X axis, hatched rectangles, subjective light span; black rectangles, dark span. To better visualize the rhythm during the 24-hour period, data obtained at CT5 were repeated 24 hours later. The circadian rhythms of c-Myc and Wee1 in control tumors (P from cosinor = 0.003 and 0.001, respectively) were abolished in mice treated with Seliciclib. C, twenty-four-hour means of mRNA levels in controls and in mice treated at ZT3, at ZT11, or at ZT19. Columns, mean of 24 to 30 tumors; bars, SE. Data are expressed as % of mean mRNA levels of controls. In mice treated at ZT3, Wee1 expression was more than doubled compared with controls (P = 0.004, ANOVA; *, P = 0.008, post hoc test). Cyclin B1 was also increased (P = 0.0018, ANOVA). Seliciclib treatment also significantly increased CDK1 mRNA expression at all dosing times (P < 0.0001, ANOVA; *, P < 0.0001, post hoc test).

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Figure 6. Targets of Seliciclib in Glasgow osteosarcoma tumors grown in mice. A to B, affinity chromatography purification on immobilized Seliciclib. A, tumor extracts were loaded on immobilized N6-methyl-Seliciclib or immobilized Seliciclib. B, beads were extensively washed and the bound proteins were analyzed by SDS-PAGE followed by silver staining and Western blotting with antibodies directed against CaMKII, CKIq, CKIa, ERK1/ERK2, PDXK, PSTAIRE, CDK9, CDK7, and cyclin H. C, Seliciclib inhibition of CKI purified from Glasgow osteosarcoma by affinity chromatography on axin fragment beads. Tumor extracts were loaded on immobilized axin fragment. After extensive washing, CKI activity was assayed with a CKI-specific peptide substrate in the presence of increasing concentrations of Seliciclib. Mean (.) of three independent determinations (o).

In our study, we confirmed the antitumor efficacy of Seliciclib in gene rhythms in Glasgow osteosarcoma (10, 11). Previous studies mice with Glasgow osteosarcoma, a rapidly growing mouse tumor. also revealed that the more the clock gene patterns were altered, The cellular effects of Seliciclib result from combined actions on the faster tumors grew and developed (10–12, 14). In the current several protein targets. Seliciclib arrests cell cycle through the study, Seliciclib induced rhythmic expression of the three main inhibition of CDK1 and CDK2, which respectively gate G2-M and clock genes in the tumor. This induction persisted for at least G1-S transitions (17, 26, 30). The cell cycle effects are further 48 hours after the last dose. Treatment in early light, when the drug reinforced by the inhibition of CDK7/cyclin H, an activator of was most effective, induced near-normal clock gene expression CDKs, and that of CDK9/cyclin T by Seliciclib. Thus, this drug may patterns with phase relations similar to those reported in healthy also act as a general transcription inhibitor because the two latter organs. In the liver and in other peripheral organs of mice, the molecular targets are essential regulators of RNA polymerase II circadian peak in mRNA synthesis usually occurs near the middle (31). The transient Seliciclib-induced inhibition of CDK7/CDK9- of the light span for Rev-erba, shortly after light-dark transition for dependent transcription leads to the down-regulation of short-lived Per2, and in the second half of the dark span for Bmal1 (11). mRNAs and proteins of survival factors Mcl-1, XIAP, and survivin Seliciclib dosing near mid-dark, when the drug was least effective, (32, 33). Such broad effects of Seliciclib on the cell cycle and produced abnormal phase relations between clock gene expression transcription machineries could depend on drug dose and schedule rhythms, a situation that results in a nonfunctional clock. and/or tumor cells characteristics. Our study identified the The ablated rhythms in clock gene expression in the tumor expected molecular targets of this drug in Glasgow osteosarcoma tissue of untreated mice could result from an impairment of the following purification on Seliciclib-immobilized sepharose beads. molecular clock within each individual cancer cell or from an These results supported the known mechanisms of antitumor altered synchronization of the molecular clocks in each malignant effects of Seliciclib in this tumor model. cell. Indeed, individual transformed fibroblasts and hepatoma cells A circadian rhythm in Seliciclib tolerability was found with least remained self-sustained circadian oscillators with slightly different lethal toxicity corresponding to drug dosing at ZT3, in the early rest periods when cultured without any synchronizing signal (38, 39). span. Highest toxicity occurred when the drug was given at ZT19, Even dividing fibroblasts passed information on their own near the middle of the activity span. This chronotolerance was circadian phase to their daughter cells (40). Thus, daily Seliciclib consistent with that reported earlier for cytotoxic chemotherapeu- could reset the clocks of malignant cells, an effect also achieved tic drugs with different mechanisms of action, such as oxaliplatin, with glucocorticoids and meal timing (11, 39, 41). However, irinotecan (34), docetaxel (35), or gemcitabine (36). Seliciclib also significantly modified the 24-hour mean expression Furthermore, the efficacy of Seliciclib against Glasgow osteosar- of Rev-erba and Bmal1 in opposite directions without affecting that coma clearly depended on dosing time. After five daily doses, the of Per2, a finding consistent with the down-regulation of Bmal1 by drug was most active following administration during the rest span Rev-erba (6, 8, 9). Such effect could not result from the mere compared with dosing during the activity span. Such coincidence synchronization of the molecular clocks in distinct malignant cells. of chronoefficacy and chronotolerance was in good agreement Rather, a direct intervention of Seliciclib on the clock mechanisms with prior results obtained in this and other mouse tumor models itself must be considered. Thus, in mouse liver, this agent both (34–37). modified the mean 24-hour mean expression of these genes and The therapeutic relevance of the molecular interactions between altered their rhythm pending on dosing time (42). the circadian clock and the cell division cycle was investigated. No In the current study, the induction of a functional molecular significant rhythmic transcription of core clock genes Rev-erba, clock in tumor was associated with the best antitumor activity of Per2, and Bmal1 was found in the tumors of control mice, thus the drug. Seliciclib did not alter mean expression of c-Myc but confirming previous observations of damped or suppressed clock enhanced that of Wee1. This effect could decrease tumor cell

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q proliferation as a result of improved control of G2-M gating. The synchrony in functional clocks through transient inhibition of CK1 enhanced Wee1 transcription was consistent with the doubling of within a permissive time window (ZT3 or ZT11). Simultaneous Bmal expression because Wee1 is unidirectionally controlled by inhibition of CDK7 by Seliciclib would further block the gene CLOCK:BMAL1 (13). The observed increase in CDK1 expression expression of CK1y/CK1q, which is otherwise constitutively likely resulted from a feedback loop where inhibition of CDK expressed (49). enzymatic activity by Seliciclib stimulated CDK mRNA synthesis. In conclusion, the antitumor effect of Seliciclib in mice was Overall, the delivery of Seliciclib at ZT3 both induced near found to be circadian rhythm dependent, reaching an optimum physiologic rhythms in clock gene expression and enhanced the efficacy when dosing was done near the beginning of the rest span. expression of G2-M genes in tumor. Seliciclib dosing at other times This maximal antitumor activity coincided with a minimal toxicity. induced abnormal rhythms in clock gene expression and/or weaker The mechanisms underlying this favorable drug effect involve activation of G2-M gating genes. Two possible mechanisms could interactions of Seliciclib with regulators of cell cycle, transcription, account for Seliciclib effects on the circadian clock. Firstly, CK1y/ and circadian rhythm. Results also suggest that cross-talks between CK1q, a key determinant of circadian period (21, 43–45), could be both biological oscillators represent a relevant dynamic target for inhibited by Seliciclib (19, 20, 46). Our study further confirmed such cancer therapeutics and further support the consideration of effect on CK1y/CK1q activity from Glasgow osteosarcoma. appropriate circadian timing for CDKIs. Seliciclib more specifically targeted CK1q, a kinase known to bind to and to phosphorylate Per proteins, leading to their ubiquitina- Acknowledgments tion and degradation. Inhibition of CK1q impairs Per2 degradation and nuclear translocation and results in increased Bmal1 Received 6/7/2006; revised 8/18/2006; accepted 9/20/2006. Grant support: Association pour la Recherche sur le Temps Biologique et la transcription (6, 43), an effect consistent with the average doubling Chronothe´rapie(F. Le´vi),Association pour la Recherche sur le Cancer (grant Nj3342), in Bmal expression that resulted from Seliciclib dosing at ZT3 in and Paul Brousse Hospital Villejuif, France (F. Le´vi);European Union through the our study. Secondly, highly coordinated sequential transcription is Network of Excellence BioSim, contract no. LSHB-CT-2004-005137 (F. Le´vi)and FP6- 2002-Life Sciences & Health, PRO-KINASE Research Project (L. Meijer); Canceropole a major mechanism of circadian rhythms. Seliciclib could affect the Grand-Ouest, France (L. Meijer); Cyclacel Ltd., Dundee, United Kingdom; and generation of circadian rhythmicity through moderate and Universita’‘‘G. D’Annunzio,’’Chieti,Italy (fellowship to I. Iurisci). The costs of publication of this article were defrayed in part by the payment of page transient time-dependent inhibition of CDK7 and CDK9. Indeed, charges. This article must therefore be hereby marked advertisement in accordance the reversible inhibitor of transcription, 5,6-dichloro-h-1-riboben- with 18 U.S.C. Section 1734 solely to indicate this fact. zimidazole, mimicked the circadian rhythm alteration that was We thank Christophe Desterke and Caroline Le Bousse-Kerdiles for their precious help and advice in molecular biology experiments; Delphine Cre´pinand Jennyfer produced with Seliciclib in a molluscan model (47, 48). Thus, daily Carrie`refor excellent technical assistance; and Jan Filipski, Urs Albrecht, Ueli Schibler, Seliciclib could act as a strong resetter of tumor cells that have lost and Nicolas Cermakian for helpful and in-depth scientific discussions on these results.

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Ida Iurisci, Elisabeth Filipski, Jens Reinhardt, et al.

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