CHIR-124,aNovelPotentInhibitorofChk1,Potentiatesthe ...clincancerres.aacrjournals.org/content/clincanres/13/2/591.full.pdf · 2-M phase checkpoints (for review, ......

CHIR-124, a Novel Potent Inhibitor of Chk1, Potentiates theCytotoxicity of Topoisomerase I Poisons In vitro and In vivoArchie N. Tse,1Katherine G. Rendahl,2 Tahir Sheikh,1Haider Cheema,1Kim Aardalen,2 Millicent Embry,2

Sylvia Ma,3 EdwardJ. Moler,4 ZhiJie Ni,5 Daniel E. Lopes deMenezes,2 Barbara Hibner,2

Thomas G. Gesner,3 and Gary K. Schwartz1

Abstract Purpose: Chk1kinase is a critical regulator of both S and G2-M phase cell cycle checkpoints inresponse to DNA damage. This study aimed to evaluate the biochemical, cellular, and antitumoreffects of a novel Chk1inhibitor, CHIR124.Experimental Design:CHIR-124was evaluated for its ability to abrogate cell cycle checkpoints,to potentiate cytotoxicity, and to inhibit Chk1-mediated signaling inducedby topoisomerase Ipoi-sons in human tumor cell line and xenograft models.Results: CHIR-124 is a quinolone-based small molecule that is structurally unrelated toother known inhibitors of Chk1. It potently and selectively inhibits Chk1 in vitro (IC50 = 0.0003Amol/L). CHIR-124 interacts synergistically with topoisomerase poisons (e.g., camptothecin orSN-38) in causing growth inhibition in several p53-mutant solid tumor cell lines as determinedby isobologram or response surface analysis. CHIR-124 abrogates the SN-38^ induced S andG2-M checkpoints and potentiates apoptosis in MDA-MD-435 breast cancer cells. The abroga-tion of the G2-M checkpoint and induction of apoptosis by CHIR-124 are enhanced by the loss ofp53.We have also shown that CHIR-124 treatment can restore the level of cdc25A protein, whichis normally targeted by Chk1for degradation following DNA damage, indicating that Chk1 signal-ing is suppressed in the presence of CHIR-124. Finally, in an orthotopic breast cancer xenograftmodel, CHIR-124 potentiates the growth inhibitory effects of irinotecan by abrogating the G2-Mcheckpoint and increasing tumor apoptosis.Conclusions: CHIR-124 is a novel and potent Chk1inhibitor with promising antitumor activitieswhen used in combinationwith topoisomerase I poisons.

To ensure high-fidelity transmission of genetic material,dividing cells are equipped with surveillance mechanismsknown as cell cycle checkpoints, which function to delay cellcycle progression when DNA damage is present. Dysregulationof checkpoint function results in genomic instability andrepresents a pathologic hallmark of neoplasia.Chk1 is a serine/threonine kinase that is required for both S

and G2-M phase checkpoints (for review, see refs. 1, 2). In

response to a variety of genotoxic stressors, including replica-tive block from UV light and hydroxyurea and DNA strandbreakage from ionizing radiation and topoisomerase poisons,Chk1 is activated by phosphorylation at Ser317 and Ser345 byupstream kinases (3). Current evidence indicates that ATR isthe primary kinase responsible for Chk1 activation, althoughATM has also recently been implicated as the activating kinasein the case of ionizing radiation–induced DNA damage (4).Chk1 regulates checkpoints by targeting the cdc25 family ofdual-specificity phosphatases, cdc25A at the S phase check-point, and both cdc25A and cdc25C at the G2-M checkpoint,respectively. Phosphorylation of cdc25A by Chk1 at multiplesites results in increased proteosomal degradation of thephosphatase and inability of cdc25A to interact with itscyclin/cyclin-dependent kinase substrates (5–7). Chk1 phos-phorylates cdc25C at Ser216, leading to complex formationwith 14-3-3 proteins and cytoplasmic sequestration of thephosphatase (8). Earlier cell cycle studies have identified a rolefor cdc25A in promoting G1-S transition, whereas cdc25Cand cdc25B (another member of cdc25 phosphatase family)are critical for mitotic entry (8). However, recent data haveindicated that cdc25A also possesses promitotic function (9).Surprisingly, mice lacking both cdc25C and cdc25B are viableand had no demonstrable cell cycle abnormalities, stronglysuggesting that cdc25A alone may be sufficient to promotemitotic entry (10).

Cancer Therapy: Preclinical

Authors’Affiliations: 1Laboratory of New Drug Development, Division of SolidTumor Oncology, Department of Medicine, Memorial Sloan-Kettering CancerCenter, New York, New York and Departments of 2Pharmacology, 3AppliedBiochemistry, 4BioInformatics, and 5Chemistry, Novartis Institutes of BiomedicalResearch (formerly Chiron Corp.), Emeryville, CaliforniaReceived 6/13/06; revised 9/19/06; accepted 9/28/06.The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Note: Supplementary data for this article are available at Clinical Cancer ResearchOnline (http://clincancerres.aacrjournals.org/).A.N.Tse and K.G. Rendahl contributed equally to this work.Current address for B. Hibner: Department of Pharmacology, Millennium Pharma-ceuticals, Cambridge, MA.Requests for reprints: Gary K. Schwartz, Memorial Sloan-Kettering CancerCenter, 1275 York Avenue, NewYork, NY10021. Phone: 212-639-8324; Fax: 212-717-3320; E-mail: [email protected].

F2007 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-06-1424

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Mounting evidence has indicated that abrogation of theChk1-mediated cell cycle checkpoints results in increasedchemosensitivity and radiosensitivity of tumor cells. Becauseof the intrinsic checkpoint defect(s) associated with malignanttransformation, pharmacologic disruption of additional check-points may render tumor cells more susceptible to DNAdamage by cytotoxic chemotherapy or ionizing radiation(4, 11, 12). Theoretically, this can be achieved by targeting adifferent checkpoint of the cell cycle (e.g., inhibiting the G2-Mcheckpoint in tumors that lack a normal G1 checkpoint) or byblocking complementary pathways of the same checkpoint(ref. 13; this report). p53, the tumor suppressor found to bemutated in >50% of human cancers (14), is a critical regulator ofboth the G1 and G2 checkpoints (15). We and others haveshown that inhibition of the Chk1-dependent G2-M checkpointusing the staurosporine analogue UCN-01 (7-hydroxystauro-sporine) selectively sensitizes tumors lacking p53 to thecytotoxic effect of chemotherapy (13, 16). Time-lapse micros-copy studies using GFP-H2B–expressing cells revealed thattumor cells that had escaped the G2-M checkpoint entered anaberrant mitosis (mitotic catastrophe) resulting in eitherapoptosis or fragmentation of chromatin into micronuclei (13).The clinical development of UCN-01 has been hampered by

its unfavorable pharmacokinetic properties and toxicities, suchas hyperglycemia, hypotension, and pulmonary dysfunction,most of which are likely due to effects of the drug thatare unrelated to Chk1 inhibition (17). Thus, identification ofalternative small-molecule Chk1 inhibitors represents a highpriority in this area of drug development. We now presentpreclinical data on a novel, potent, and specific inhibitor ofChk1 [CHIR-124, [(S)-3-(1H-benzo[d]imidazol-2-yl)-6-chloro-4-(quinuclidin-3-ylamino)quinolin-2(1H)-one]. We show thatCHIR-124 abrogates the S and G2-M checkpoints induced bytopoisomerase I poisons and selectively sensitizes tumorslacking p53 function to undergo mitotic death. Furthermore,CHIR-124 enhances the antitumor effect of irinotecan in tumorxenografts by inhibiting the G2-M checkpoint and inducingapoptosis.

Materials andMethods

Reagents. CHIR-124 was synthesized at Chiron Corp. (18).Irinotecan (CPT-11; Camptosar) was obtained from PharmaciaUpjohn, Inc. (Kalamazoo, MI), and doxorubicin (Adriamycin) andcisplatin (cisplatin injection) were purchased from American Pharma-ceutical Partners, Inc. (Schaumburg, IL). Camptothecin was purchasedfrom Sigma (St. Louis, MO). SN-38 was a generous gift from Dr. J.Patrick McGovern (formerly at Pharmacia and Upjohn, Inc.) and fromJiangsu Hengrui Medicine Co. (Lianyungang, China). UCN-01 waskindly provided by Dr. Robert Schultz (National Cancer Institute,Bethesda, MD). All drugs were dissolved in dimethyl sulfoxide andstored in aliquots at �20jC.

Cell culture. MDA-MB-435 cells were obtained from Dr. JoshuaFidler of the M.D. Anderson Cancer Center and from the American TypeCulture Collection (Manassas, VA). Cultures were maintained in 50%DMEM high glucose, 50% F-12 medium or 100% DMEM, containing10% heat-inactivated fetal bovine serum, 1% sodium pyruvate, 1% or2% vitamin solution, 1% L-glutamine, and 1% nonessential aminoacids. The parental HCT116 colon carcinoma cell line and its p53-nullderivative (kindly provided by Dr. Bert Vogelstein, Johns HopkinsUniversity, Baltimore, MD) were grown in RPMI 1640 supplementedwith 10% heat-inactivated fetal bovine serum. The MDA-MB-231,SW620, and COLO 205 cell lines used for isobologram studies were

obtained from the American Type Culture Collection and cultured asrecommended.

Kinase selectivity assays. For the CHK1 assay, the kinase domain wasexpressed in Sf9 insect cells, and a biotinylated cdc25c peptidecontaining the consensus Chk1/Chk2 phosphorylation site (*)(biotin-[AHX]SGSGS*GLYRSPSMP-ENLNRPR[CONH2]) was used as thesubstrate. A dilution series of CHIR-124 was mixed with a kinasereaction buffer containing a final concentration of 30 mmol/L Tris-HCl(pH 7.5), 10 mmol/L MgCl2, 2 mmol/L DTT, 4 mmol/L EDTA,25 mmol/L h-glycerophosphate, 5 mmol/L MnCl2, 0.01% bovineserum albumin, 1.35 nmol/L CHK1 kinase domain, 0.5 Amol/L peptidesubstrate, and 1 Amol/L unlabeled ATP, plus 5 nmol/L 33P g-labeledATP (specific activity = 2,000 Ci/mmol). Reactions and detection of thephosphate transfer were carried out by a radioactive method, aspreviously described (19).

For all other kinase assays, the enzymes were expressed in insect cellsor purchased from Upstate Biotechnology (Charlottesville, VA) or NewEngland Biolabs (Ipswich, MA). Reactions were carried out generally aspreviously described (19). See Supplementary Material 1 for details.

Drug interaction by isobologram analysis. MDA-MB-231, MDA-MB-

435, SW-620, and COLO 205 cells in log-phase were plated into 96-well

microplates. CHIR-124 was serially diluted in the presence of six

different concentrations of camptothecin or 0 nmol/L camptothecin.

Camptothecin was also serially diluted in the absence of CHIR-124. The

compounds were added to cells in 96-well dishes and incubated at

37jC for 48 h. Each treatment condition was done in triplicate. Cell

proliferation was monitored by the 3-(4,5-dimethylthiazol-2-yl)-5-

(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H -tetrazolium (MTS),

inner salt (Promega Corp., Madison, WI) assay. MTS inner salt was

added to the microplates, which were incubated for another 3 h, and

absorbance at 490 nm was read on a plate reader. The concentrations of

each drug in the combinations required to produce 50% inhibition

were plotted to generate the isoboles. Isobologram analysis of drug

interaction is based the equation of Loewe additivity (1 = DA/IC50, A +

DB/IC50, B), where IC50, A and IC50, B are the concentrations of drugs

to result in 50% inhibition for each drug alone, and DA and DB

are concentrations of each drug in the combination that yield 50%

overall inhibition (20). A diagonal line indicating Loewe additivity is

included in each graph. Data points that fall below the line indicate

synergy, whereas those that fall above the line (data not shown) would

indicate antagonism.Drug interaction by response surface analysis. Response surface

analysis determines additivity or degree of interaction between twodrugs via exposure of cells to a combinatorial matrix of concentration(reviewed in ref. 21). MDA-MB-435 cells were plated into black 384-well plates (Greiner Bio-One, Inc., Longwood, FL). Cells were exposedto a matrix of drug combinations comprised of 10 CHIR-124concentrations and 8 SN-38 concentrations, for a total of 80combinations. A dilution series of each drug with vehicle was alsorun. The CHIR-124 and SN-38 concentrations consisted of a series of1.4-fold stepwise dilutions from 3.3 � 10�7 to 1.6 � 10�8 mol/L and4-fold stepwise dilutions from 2.7 � 10�6 to 1.6 � 10�10 mol/L,respectively. Each drug was also run in combination with itself as acontrol.

Compounds were added either 24 or 48 h after plating, for a 72- or48-h drug exposure, respectively. CHIR-124 was given as a single48-h treatment, whereas SN-38 was applied every 24 h. Daily reappli-cation of SN-38 was required to generate a meaningful dose-responsecurve (data not shown) due to the short half-life of the drug in serum-containing media (22). This did not significantly change theconcentration of CHIR-124. Drug combinations were tested followingeither simultaneous or sequential addition. In the case of simultaneousaddition, cells were treated with both SN-38 and CHIR-124 for48 h (including a second application of SN-38 24 h later). In the caseof sequential addition, a SN-38 application was given 24 h beforesimultaneous addition of both drugs [i.e., a 72-h exposure of SN-38(three total applications) plus a 48-h exposure of CHIR-124]. Viable cell


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number was determined 96 h after plating using the Cell Titer-Glo assaykit (Promega) and luminescence intensity read with a Victor lumin-ometer (Perkin-Elmer, Wellesley, MA). Cell plating, drug addition, PBSwashes, and the addition of Cell Titer-Glo were all done with an FXrobot (Beckman-Coulter, Durate, CA). The data were analyzed byplotting the deviation from additivity: the difference between theobserved inhibition effect and the effect predicted by the Loeweadditivity model (21),

D ¼ Eobs � Eadd

where D is the deviation from additivity, Eobs is the observed cellnumber normalized to the untreated controls, and Eadd is the additiveeffect predicted by the Loewe additivity model. The variables for theadditive model, the IC50 values and Hill slopes for the individual drugs,were determined using custom software developed by E. Moler(Chiron). A key feature of the Loewe additivity model is that bydefinition, a drug is additive in combination with itself, providing awell-defined control for additivity. Drug interaction is consideredsynergistic or antagonistic when the response surface deviation fromadditivity is greater than +10% or �10%, respectively. The results areillustrated graphically in three dimensions as plots of data points withina color-coded surface. Additive data points are in green (ranging fromblue-green at +10% deviation to green at the zero point and chartreuseat �10%); synergy is represented from blue to violet; and antagonism isrepresented as orange or red. CHIR-124 and SN-38 drug concentrationsare on the x- and y-axes, respectively, and the deviation from additivityis along the z-axis.

Assessment for apoptosis and micronucleation in vitro. Log-phasecells were plated onto 60-mm dishes in duplicate and allowed to adherefor 36 to 48 h. Following drug treatment, both adherent andnonadherent cells were harvested, fixed in 3% paraformaldehyde, andstained with 4¶,6¶-diamidino-2-phenylindole (Sigma). In experimentsinvolving sequential therapy, floating cells were collected afterincubation with the first drug and were added back to the plate forsubsequent treatment. The incidence of apoptosis and micronucleationwas determined by examining the nuclear morphology of cells underfluorescence and scored for those with condensed fragmentedchromatin and multiple (z3) interphase nuclei, respectively. At least400 cells were counted for each sample.

Cell cycle analysis. Biparameter flow cytometry was done asdescribed previously (13). Samples were analyzed on a FACScan(Becton Dickinson, Franklin Lakes, NJ) for DNA content and mitoticindex following labeling with the mitosis-specific antibody MPM-2.

Immunoblot analysis. Twenty-five to 50 Ag of lysate protein werefractionated by SDS-PAGE and transferred onto polyvinylidenedifluoride Immobilon membranes (Millipore, Billerica, MA). Blotswere blocked with 5% nonfat milk and probed with antibodies againstChk1, cdc25A (both from Santa Cruz Biotechnology, CA), phospho-

Chk1 (Ser317), phospho-cdc25C (Ser216; both from Cell Signaling,Beverly, MA), cdc25A, and a-tubulin (both from Sigma). Boundprimary antibodies were detected with horseradish peroxidase–conjugated secondary antibodies (ICN/Jackson ImmunoResearch,West Grove, PA) and visualized with enhanced chemiluminescencereagent (Amersham Pharmacia, Piscataway, NJ). The levels of expres-sion were quantified using Kodak 1D Image Analysis Software(Rochester, NY).

In vivo antitumor activity studies. Severe combined immunodefi-cient mice harboring MDA-MD-435 tumor xenografts were randomizedinto the following treatment groups of 10: vehicle (captisol) alone,5 mg/kg CPT-11, 10 mg/kg CHIR-124, 20 mg/kg CHIR-124, 5 mg/kgCPT-11 plus 10 mg/kg CHIR-124, or 5 mg/kg CPT-11 plus 20 mg/kgCHIR-124. Treatment was initiated on the day after randomization (day1). CPT-11 was given i.p. daily (four times daily) �5 on days 1 to 5,whereas CHIR-124 was given orally four times daily � 6 on days 2 to 7in captisol. Percent tumor growth inhibition was defined as % T/C ,where T = the treatment group mean, and C = the control group mean.In a similar study, tumors harvested from mice sacrificed on day 4 oftreatment were examined for apoptosis by terminal deoxynucleotidyltransferase–mediated nick-end labeling staining and for mitotic indexby immunofluorescence labeling with phospho-histone H3 antibody.See Supplementary Material 2 for details.

Results

CHIR-124 is a potent and selective inhibitor of Chk1kinase. CHIR-124 is a lead member of a novel series ofquinolone derivatives identified upon screening a diversechemical library for Chk1 inhibitors (Fig. 1A; ref. 18). CHIR-124 is structurally distinct from the prototypical Chk1 inhibitor

Fig. 1. Chemical structures of (A) CHIR-124 and (B) UCN-01.

Table 1. Kinase selectivity profile of CHIR-124

Kinase IC50 (Mmol/L)

Cell cycle kinasesCHK1 KD 0.0003CHK2 0.6974Cdk4/cyclin D 2.05CDC2/cyclin B 0.5057Cdk2/cyclin A 0.1911

Receptor tyrosine kinasescMET >3bFGFR 2.01FGFR3 1.29VEGFR2 FLK1 0.5779VEGFR1 FLT1 0.4636PDGFR 0.0066FLT3 0.0058

Serine/threonine kinasesPKCa 0.58PKAh I 2.25PKCh II 0.58PKCg 0.11ERK2 4.31PKA 0.1031GSK3 0.0233

Non–receptor tyrosine kinasesCABL 14.88LCK 0.1583FYN 0.0988

Abbreviations: KD, kinase domain; Cdk2, cyclin-dependent kinase2; bFGFR, basic fibroblast growth factor receptor; PKC, proteinkinase C; VEGFR, vascular endothelial growth factor receptor;ERK, extracellular signal-regulated kinase.

CHIR-124 Inhibits Chk-1and SensitizesTumor Cells




UCN-01 (Fig. 1B). In an in vitro kinase assay, CHIR-124showed potent inhibitory activity against the kinase domain ofrecombinant Chk1 with an IC50 of 0.0003 Amol/L, a value thatwas 2,000-fold lower than that against Chk2 (0.70 Amol/L;Table 1). Co-crystal structure of the kinase domain of Chk1in complex with CHIR-124 shows occupation of the ATP-binding site of the enzyme by the inhibitor (18). Assuming acompetitive mode of inhibition and using a Km of 105 Amol/Lof the enzyme for ATP, the K i of CHIR-124 was 0.0003 Amol/L.In addition, CHIR-124 is 500- to 5,000-fold less active againstother cell cycle kinases, such as cyclin-dependent kinase 2/cyclin A (0.19 Amol/L), cdc2/cyclin B (0.51 Amol/L), andcyclin-dependent kinase 4/cyclin D (2.1 Amol/L; Table 1).When tested against a panel of representative kinases, CHIR-124 showed good selectivity against members of the receptortyrosine kinases, non–receptor tyrosine kinases, and serine/threonine kinases, with the exception of FLT3 and platelet-derived growth factor receptor, which were inhibited at anf20-fold higher concentration of CHIR-124 when comparedwith Chk1 (Table 1). Of note, CHIR-124 has only weakinhibitory effects on the classic protein kinase C isoforms a, h,and g when compared with UCN-01, which has been reportedto be a potent inhibitor against these kinases with publishedIC50s in the 29 to 34 nmol/L range (23).Growth inhibition by topoisomerase I poisons in combination

with CHIR-124 is synergistic in a variety of cancer celllines. Treatment of cells with type I topoisomerase poisons,such as camptothecin and its derivatives, generate double-stranded DNA breaks in a replication-dependent manner(24, 25), resulting in activation of both the S and G2-Mcheckpoints where Chk1 plays a critical role. We thereforestudied the in vitro effect of a matrix of camptothecin andCHIR-124 combinations in a number of human cancer celllines, including breast carcinoma (MDA-MB-231 and MDA-MB-435) and colon carcinoma (SW-620 and Colo205), all ofwhich are mutant for p53. The antiproliferative effect oftreatment was evaluated using an MTS inner salt assay, anddrug interaction was assessed by the isobologram method. Asynergistic interaction between camptothecin and CHIR-124was shown in all four cell lines (Fig. 2A). The IC50s of thecombined drugs represent lower concentrations of each drugthan would be predicted by Loewe additivity equation. Inaddition, we observed synergy between CHIR-124 and cisplatinin MDA-MB-435 cells and with doxorubicin in MDA-MD-231cells (data not shown).To further evaluate the combined effect of a topoisomerase I

poison and CHIR-124 on MDA-MB-435 cells, we did responsesurface (21) analysis with CHIR-124 and SN-38, an activemetabolite of the clinically important drug irinotecan (Fig. 2B).This methodology allows a more rigorous evaluation of druginteractions than the isobologram technique. When cells weresimultaneously exposed to a matrix of 80 different concentra-tion combinations of CHIR-124 and SN-38 for 48 h, significantsynergy or >10% deviation from additivity was observed(Fig. 2B, e) in the concentration ranges of z4.2 � 10�8 mol/Lfor SN-38 and z6.0 � 10�8 mol/L for CHIR-124. These valuesoverlap and fall below the IC50s for SN-38 (1.2 � 10�7 mol/L)and CHIR-124 (2.2 � 10�7 mol/L), respectively. Sequentialaddition of SN-38 followed 24 h later by CHIR-124 (72- and48-h exposure, respectively), resulted in >35% deviation fromadditivity (Fig. 2B, f). This occurred in the concentration range

of z4.2 � 10�8 for SN-38, close to the IC50 for this drug (8.5 �10�8 mol/L), and over the entire dilution series of CHIR-124.Thus, synergy was most pronounced at higher concentrations ofSN-38 and over a wide range of concentrations of CHIR-124(Fig. 2B, f). Importantly, control plates of SN-38 plus SN-38 at48 or 72 h (Fig. 2B, g and h) or CHIR-124 plus CHIR-124 at48 h (Fig. 2B, g-i) show a flat surface of data points within 10%deviation from additivity, as expected.CHIR-124 abrogates the SN-38–induced S and G2-M phase

cell cycle checkpoints. Having established that there is synergybetween CHIR-124 and topoisomerase I poisons in inhibitingthe proliferation of human cancer cells, we set out to determinethe mechanistic basis for this interaction. A 24-h treatment ofMDA-MB-435 cells with 100 nmol/L CHIR-124 or 100 nmol/LUCN-01 resulted in no appreciable changes in the cell cycledistribution of these cells (Fig. 3A). Treatment with 20 nmol/LSN-38 for 24 h caused an S phase arrest as evidenced by theaccumulation of cells with >2N and V4N DNA content as wellas the loss of MPM-2 positive mitotic cells (Fig. 3A). However,when cells were exposed concurrently to SN-38 and CHIR-124,this S-phase delay was abolished as the majority of cells hadcompleted replication and appeared as cells with 4N DNAcontent at 24 h (Fig. 3A). As expected, UCN-01, the knownChk1 inhibitor, also abrogated the S-phase checkpoint inducedby SN-38 (Fig. 3A). Following removal of SN-38 andincubation in drug-free medium (ND) over time (SN !ND24 h), these cells progressed through the S phase buteventually arrested in G2 without mitotic entry (Fig. 3B).However, when MDA-MB-435 cells were treated sequentially

Fig. 2. Synergism between topoisomerase I poisons and CHIR-124 in humancancer cell lines expressing mutant p53. A, isobolograms were done with breastand colon carcinoma cell lines treated with camptothecin (CPT) and CHIR-124.Theindicated cell lines were exposed to the single agents camptothecin or CHIR-124or a matrix of dilutions of the two drugs simultaneously for 48 h. Isoboles wereobtained by plotting the combined concentrations of each drug required to producea 50% growth inhibition. Dashed lines, hypothetical additive drug effect; concavecurves, synergy. B, response surface analysis of a matrix of SN-38 and CHIR-124concentrations applied to MDA-MB-435 cells.





with SN-38 for 24 h followed by graded concentrations ofCHIR-124 (20, 50, and 100 nmol/L), a dose-dependentincrease in mitotic index was observed, which was mostapparent at the 8 h time point, indicating an abolishment ofthe SN-38–induced G2-M checkpoint by CHIR-124 (Fig. 3B).The extent of G2-M checkpoint abrogation induced by 100nmol/L CHIR-124 was comparable with that seen with anequimolar concentration of UCN-01 (Fig. 3B). All mitotic(MPM-2 positive) cells had a 4N DNA content, indicating thatcells that had escaped the G2-M checkpoint and entered mitosisdid so only after completion of DNA replication (data notshown).Enhancement of apoptosis by sequential treatment with SN-38

followed by CHIR-124. We next examined whether G2-Mcheckpoint release observed in MDA-MB-435 cells treated withSN-38 and CHIR-124 resulted in apoptotic cell death. Single-agent treatment with 20 nmol/L SN-38 or 100 nmol/L CHIR-124 for 24 h was ineffective in causing apoptosis in these cells(Fig. 3C). Sequential treatment with SN-38 followed by CHIR-124 caused a marked increase in apoptotic cells (up to 32 F 2%for SN ! CHIR24 h; Fig. 3C) in a time-dependent manner. Theextent of apoptosis observed was comparable with that inducedby SN-38 followed by 100 nmol/L UCN-01 (Fig. 3C).Sequential treatment with either CHIR-124 or UCN-01 also

resulted in a mild increase in interphase cells with micronuclei,consistent with mitotic catastrophe previously described inHCT116 colon cancer cells treated sequentially with SN-38 andUCN-01 (Fig. 3C; ref. 13).Selective abrogation of the DNA damage–induced G2-M

checkpoint and induction of cell death by CHIR-124 in p53-nullHCT116 cells. We have previously shown that HCT116 coloncancer cells that lack p53 are more prone to undergoing G2-Mcheckpoint abrogation and apoptosis by UCN-01 than parentalcells containing wild-type p53 (13). We therefore compared theintegrity of the G2-M checkpoint between parental HCT116 andits isogenic derivative that lacked p53 following combinedtreatment with SN-38 and CHIR-124. Our data indicated thatp53-null cells were selectively more susceptible to undergoingabrogation of the G2-M checkpoint after sequential treatmentwith SN-38 followed by CHIR-124 when compared withparental cells (51% versus 15 % mitosis at 8 h following theaddition of CHIR-124; Supplementary Material 3A). Further-more, under our experimental conditions, single-agent SN-38,CHIR-124, or SN-38 followed by drug-free medium resulted inminimal apoptosis (<5%) in both cell lines (SupplementaryMaterial 3B). However, sequential treatment with SN-38followed by CHIR-124 resulted in enhanced induction ofapoptosis and micronucleation in p53-null cells (23% and

Fig. 2 Continued. E, simultaneous addition of SN-38 and CHIR-124 for 48 h resulted in significant synergy, as determined by a >10 % deviation from additivity.The degree ofdrug interaction is color coded (see Materials and Methods). F, sequential addition of SN-38 for 24 h followed by a further 48-h exposure to SN-38 plus CHIR-124 resultedin synergy as indicated by the >30% deviation from additivity at higher concentrations of SN-38 and over a wide range of CHIR-124 concentrations.G, H, and I, additivitycontrols for SN-38 plus SN-38 for 48 and 72 h, respectively, and for CHIR-124 plus CHIR-124 for 48 h.





20%, respectively) compared with parental cells (14% and 3%,respectively; Supplementary Material 3B). Thus, consistent withthe results of selective abrogation of the G2-M checkpoint byCHIR-124 in p53-null cells (Supplementary Material 3A), theloss of p53 also rendered these cells more susceptible to thecytotoxic effect of combined SN-38 and CHIR-124.Suppression of the Chk1 signaling pathway by CHIR-124. In

response to DNA damage induced by topoisomerase I poison,Chk1 is activated by phosphorylation at Ser317 and Ser345 byATR (3). An important downstream target of activated Chk1 isthe dual-specificity phosphatase cdc25A, which promotes cellcycle progression through both the G1-S and G2-M transitions(26). Phosphorylation of cdc25A by Chk1 on several NH2-terminal sites resulted in accelerated proteosomal degradationof the phosphatase (5, 6). We therefore examined the levels ofphospho-Chk1 and cdc25A in HCT116 p53�/� cells treatedwith the SN-38 and CHIR-124 combination. As expected, SN-38 treatment resulted in a 2.5-fold increase in activatingphosphorylation of Chk1 at Ser317 (ref. 3; Fig. 4). Total Chk1protein levels remained fairly constant under these conditions.Treatment with SN-38 alone for 24 h or followed by a drugwashout for serial time points resulted in a marked decrease of

cdc25A to 20% of untreated control level (Fig. 4). Incubationwith 200 nmol/L CHIR-124 led to a 2.5-fold elevated level ofcdc25A above that of the untreated control (Fig. 4), a findingthat is in accord with the concept that some basal level ofChk1 activity is present during an unperturbed cell cycle topromote cdc25A turnover (4, 6). The down-regulation ofcdc25A induced by SN-38 was completely restored by con-current or sequential treatment with CHIR-124, providing bio-chemical evidence that CHIR-124 inhibited the Chk1-mediateddestruction of cdc25A in whole cells (Fig. 4). Although it isfrequently stated that Chk1 phosphorylates Ser216 of cdc25Cfollowing DNA damage, this site is constitutively phosphory-lated even in untreated cells (Fig. 4). Treatment with SN-38only increased Ser216 phosphorylation further by 30% when itsexpression was normalized to total cdc25C (Fig. 4). Thespecificity of the phospho-antibody has been confirmed usingHCT116 cells that overexpress GFP-fused wild-type cdc25C orcdc25C mutated at Ser216 (Ser ! Ala; data not shown). Whencompared with SN-38 followed by drug-free medium at3 h (SN24 h ! ND3 h), we did not observe any appreciabledecrease in Ser216 phosphorylation following the addition ofCHIR-124 (SN24 h ! CHIR3 h). Similarly, there was only amodest 30% reduction of phosphorylation at a later time point(SN24 h ! CHIR6 h) when cells had already entered mitosis asevidenced by an increase in phospho-histone H3, raising thepossibility that the loss of Ser216 phosphorylation may repre-sent the consequence rather than the cause of mitotic entry.Conversely, the effect of CHIR-124 on cdc25A levels was appa-rent as early as 3 h (SN24 h ! CHIR3 h), providing a temporallinkage between restoration of cdc25A level and abrogation ofthe G2-M checkpoint (Fig. 4). Whereas total cdc25C levels didnot change with treatment, the protein became hyperphos-phorylated during mitosis, resulting in an upward mobilityshift on the gel, consistent with previous report on cdc25Chyperphosphorylation during mitosis (Fig. 4, arrow; refs. 8, 27).CHIR-124 in combination with CPT-11 shows potentiation of

tumor growth inhibition and increased apoptosis in vivo. Todetermine whether CHIR-124 enhances the effect of top-oisomerase 1 poisons in vivo , we evaluated the Chk1 inhibitorin combination with CPT-11 in an orthotopic breast carcinomamodel. Severe combined immunodeficient mice implanted inthe mammary fat pad with MDA-MB-435 cells were random-ized into the following treatment groups: vehicle alone, dailydosing with the single-agent CPT-11 at 5 mg/kg on days 1 to 5,daily dosing of 10 or 20 mg/kg CHIR-124 alone on days 2 to 7,or 5 mg/kg CPT-11 in combination with either dose of CHIR-124. Thus, in the two combination groups, CPT-11 was givenalone on day 1, and CHIR-124 was given 4 h following CPT-11on subsequent dosing days. CHIR-124 at either 10 or 20 mg/kgdid not have a significant effect on tumor growth whencompared with the vehicle-treated group at the end of the study(day 36; Fig. 5A). However, when animals were treatedsequentially with CPT-11 followed by CHIR-124, there waspotentiation of tumor growth inhibition relative to CPT-11treatment alone. At the end of the study, there was a significantdifference between mean tumor volumes of the CHIR-124alone groups and the CPT-11 plus CHIR-124 dose-matchedcombination groups (P < 0.05; Fig. 5A). The % tumor growthinhibition on day 36 in the groups dosed with 5 mg/kg CPT-11and the combination groups dosed with 5 mg/kg CPT-11plus 10 mg/kg CHIR-124 or 5 mg/kg CPT-11 plus 20 mg/kg

Fig. 3. Abrogation of cell cycle checkpoints and induction of apoptosis byCHIR-124 in MDA-MD-435 cells. A, asynchronous MDA-MD-435 cells weretreated for 24 h with single agent 20 nmol/L SN-38, 100 nmol/LCHIR-124, or100 nmol/LUCN-01, concurrently with SN-38 and CHIR-124, or concurrently withSN-38 and UCN-01. Cell cycle distribution was analyzed by biparameter flowcytometry for DNA content and mitotic index (M) as described in Materials andMethods.





CHIR-124 was 14%, 34%, and 53%, respectively. Finally, ani-mals in all groups were healthy and did not manifest any signsof toxicity or significant body weight loss (defined as >5% ofbody weight).Apoptosis induction in MDA-MB-435 xenograft tumors from

mice treated with vehicle alone, 5 mg/kg CPT-11, 20 mg/kg

CHIR-124 alone, or the combination of 5 mg/kg CPT-11 plus20 mg/kg CHIR-124 was assessed by terminal deoxynucleotidyltransferase–mediated nick-end labeling staining on day 4.Occasional terminal deoxynucleotidyl transferase–mediatednick-end labeling–positive cells were observed in the tumorsfrom the vehicle and CHIR-124–treated animals (1.8 F 0.4%

Fig. 3 Continued. B, cells were treated with 20 nmol/L SN-38 for 24 h followed by either drug-free medium (ND), increasing concentrations of CHIR-124 or100 nmol/LUCN-01. Cells were harvested at the indicated time points and processed for flow cytometry analysis. Representative of two independent experiments.





and 2.0 F 0.6%, respectively, group; Fig. 5B and D). Thepercentage of labeled cells was similar in tumors obtained fromCPT-11–treated animals (2.3 F 1.0%; Fig. 5B and D) but wasmarkedly increased in tumors derived from animals treatedwith the drug combination (12.4 F 1.9%; Fig. 5B and D). Thepercentage of positive staining in these tumors was significantlydifferent from that in tumors from animals treated with eithersingle agent (P < 0.01; Fig. 5D). These results indicate that thepotentiation of the tumor growth inhibitory effect of CPT-11 byCHIR-124 is associated with an increase in apoptosis inductionin the tumors.To assess the pharmacodynamic effect of CHIR-124 in vivo,

we also examined the mitotic index in tumor sections obtainedfrom animals treated with either agent or the combination ofCPT-11 and CHIR-124, using the antibody against the mitoticmarker phospho-histone H3. Treatment with CPT-11 resultedin a decrease in the number of mitoses per low power fieldcompared with control (17 versus 28, P = 0.06), consistent withactivation of the G2-M checkpoint (Fig. 5C and D). Impor-tantly, cotreatment with CHIR-124 reversed the suppressionof phospho-H3 staining induced by CPT-11 (34 versus 17,P = 0.005; Fig. 5B and D), indicating abrogation of the G2-Mcheckpoint by CHIR-124 in the xenografts.

Discussion

Dysregulation of cell cycle checkpoints is now recognized asa salient feature of the malignant transformation process.Checkpoint dysfunction in tumors provides an opportunity fordeveloping a therapeutic strategy that combines conventionalcancer treatment with inhibitors of cell cycle checkpoints. Thisis based on the proposition that pharmacologic disruption ofcheckpoint function may selectively sensitize tumors withintrinsic checkpoint defects to genotoxic stress imparted bychemotherapy or radiation. Chk1 is considered a potentialtarget for such a combination approach. In this report, wepresent data on the molecular pharmacology of a potent andselective Chk1 inhibitor (CHIR-124) in combination withtopoisomerase I poisons.

Our data indicate that CHIR-124 is a biologically active Chk1inhibitor. First, in an in vitro biochemical assay, CHIR-124shows potent inhibitory activity against the kinase domain ofrecombinant human Chk1 and is highly selective for Chk1

Fig. 3 Continued. C, enhancement of apoptosis by sequential treatmentwith SN-38 followed by CHIR-124. MDA-MD-435 cells were treated withsingle agent 20 nmol/L SN-38,100 nmol/LCHIR-124, or100 nmol/LUCN-01for 24 h, or sequentially with SN-38 for 24 h followedby drug-freemedium (ND), CHIR-124, or UCN-01for the indicated times. Apoptosisand micronucleation were determined by examining the nuclearmorphology of fixed cells under fluorescence after 4¶,6¶-diamidino-2-phenylindole staining. Points, averages of three independent experiments(mean); bars, SD.

Fig. 4. Suppression of the Chk1signaling pathway by CHIR-124. HCT116 p53-nullcells were treated with 20 nmol/L SN-38 or 200 nmol/LCHIR-124 alone, or incombination for 24 h, or sequentially with SN-38 for 24 h followed by drug-freemedium (ND) or CHIR-124 for various times. Phospho-Chk1at Ser317 and cdc25Alevels were used as surrogate markers for Chk1activation and Chk1-dependentsignaling response to DNA damage, respectively. Phospho-histone H3was used asa mitotic marker. Hyperphosphorylation of cdc25C during mitosis resulted in anupward motility shift (arrow). Representative of two independent experiments.





relative to several other kinases, including Chk2. Second, CHIR-124 inhibits Chk1 function in cells. It abrogates the S and G2-Mcheckpoints induced by SN-38 in the p53-defective MDA-MB-435 cell line at concentrations comparable with that of theprototypical Chk1 inhibitor UCN-01. Third, CHIR-124 treat-ment reverses the suppression of cdc25A by SN-38 both incombination and sequentially, indicating that cellular signalingof Chk1 is altered by CHIR-124. Interestingly, although CHIR-124 effectively abolishes the G2-M checkpoint induced by DNAdamage, we did not observe consistent inhibition of the level ofSer216 phosphorylation in cdc25C. This is in contrast to whatinvestigators have previously reported with UCN-01 (28) but isconsistent with a recent study using the selective Chk1 inhibitorCEP-3891, which also abrogates the G2-M checkpoint withoutaltering Ser216 phosphorylation (29). Overall, it seems thatreversal of DNA damage–induced suppression of cdc25Aprotein level by CHIR-124 represents a more reliable pharma-codynamic indicator of Chk1 inhibition.We have shown that CHIR-124 interacts synergistically

with camptothecin in four different p53-deficient carcinomacell lines The combined effect of CHIR-124 and a top-oisomerase I poison was further examined in vivo in MDA-MB-435 xenografts. CHIR-124 alone was well tolerated bythe animals, as expected, but did not inhibit tumor growth.However, CHIR-124 potentiated the antitumor activity ofCPT-11 and enhanced induction of apoptosis. To ourknowledge, these results represent the first demonstrationof potentiation of the antitumor activity of a cytotoxic agentby a specific Chk1 inhibitor in vivo . A significant, although

Fig. 5. Potentiation of the antitumor effect of CPT-11by CHIR-124 in vivo.A, CHIR-124 potentiates the growth inhibitory effect of CPT-11in a human breastcarcinoma xenograft model. MDA-MB-435 cells were implanted in the mammaryfat pad of 8- to10-week-old female severe combined immunodeficient mice.Animals were randomized into the indicated groups after reaching a mean tumorvolume of 200 mm3 F 10%. CPT-11and CHIR-124 were dosed either alone or incombination. CPT-11was given daily at 5 mg/kg on days1to 5 (four times daily� 5;filled arrows), whereas10 mg/kg CHIR-124, 20 mg/kg CHIR-124, or vehicle weredosed daily on days 2 to 7 (four times daily � 6; open arrows).

Fig. 5 Continued. B, increased apoptosisin tumor xenograft from animals treatedwith both CPT-11and CHIR-124 relative toeach single agent. Apoptosis was assessedby terminal deoxynucleotidyl transferase ^mediated nick-end labeling staining intumors harvested frommice on day4 receiving the indicated treatments daily.Black arrows, terminal deoxynucleotidyltransferase ^ mediated nick-end labeling ^positive cells. Representative sections ata magnification of �400.





less profound, abrogation of the G2-M checkpoint, asmeasured by the induction of mitotic entry, was observedin MDA-MD-435 xenografts derived from mice treated withCPT-11 and CHIR-124 compared with that obtained in thesame cells treated in culture with SN-38 and CHIR-124. Thisdiscrepancy may stem from differences in the timing oftumor sampling or in drug exposure between in vivo andin vitro studies.We have previously shown that the cytotoxicity induced by

disruption of the Chk1-mediated S and G2-M checkpointsinvolves disparate mechanisms (13). Thus, abrogation of the S-phase checkpoint leads to an amplified DNA damage responsecharacterized by an enhanced induction of p53, p21, and g-H2AX, whereas inhibition of the G2-M checkpoint results in anaberrant mitosis and increased apoptosis (13). Pharmacologicmanipulation of the latter checkpoint is of particular interestbecause p53-deficient cells are more sensitive than wild-typecells in undergoing premature G2-M entry, aberrant mitosis,and subsequent apoptosis (ref. 13 and this report).Current evidence has indicated that following mitotic failure,

the ensuing reproductive cell death can proceed by severalmechanisms: (a) apoptosis induction either during aberrantmitosis or in micronucleated cells that have exited mitosis (13),(b) sustained cell cycle arrest due to activation of the p53-dependent endoreduplication checkpoint (30, 31), or (c)unknown mechanisms leading to demise of polypoid giantcells, which can possibly involve cellular senescence.6 Notably,

our current results show that cell fate after G2-M checkpointabrogation seems to differ between cell lines. For example,HCT116 p53�/� cells that have escaped the G2-M checkpointdevelop either apoptosis or micronucleation, whereas MDA-MB-435 cells that undergo checkpoint abrogation culminateprimarily in apoptosis.

Fig. 5 Continued. C, abrogation of the CPT-11induced G2-M checkpoint by CHIR-124. Mitotic cellswere labeledusing the rabbit polyclonal antibody againstphospho-H3 (green), and chromatin was stained with4¶,6¶-diamidino-2-phenylindole (blue). Representativefluorescence micrographs at amagnification of �100.

Fig. 5 Continued. D, quantitation of apoptotic and mitotic cells in tumor sections.Percentage apoptosis andmitotic index per low-powered field in different treatmentgroups were determined as described in Materials and Methods. Differencesbetween groups were compared using paired t test. Columns, mean; bars, SD.6 A. N.Tse and G. K. Schwartz, unpublished data.





Several unresolved issues remain regarding the use of Chk1inhibitors in cancer therapy. First, because Chk1 is an essentialcheckpoint kinase required for maintaining genomic stability,one theoretical concern is whether Chk1 inhibition mightproduce any deleterious effects in the renewal of normal stemcells in various tissues. The consequence of Chk1 inhibition inan unperturbed cell cycle has recently been addressed (32).Interference of Chk1 function using small-molecule inhibitorsor by small interfering RNA resulted in increased initiation ofDNA replication, an ATR-dependent DNA damage response,and frank DNA breaks (32). However, our mouse xenograftstudies have indicated that CHIR-124, when given as a singleagent for five consecutive days, is well tolerated and results in noappreciable tumor growth inhibition, yet enhances the anti-tumor activity of CPT-11 in animals. These results suggests thatthe effect of Chk1 inhibition on S-phase cells in the absence ofDNA damage, if any, does not seem to affect the short-termproliferative potential of both normal and tumor cells in vivo .Second, it is generally believed that the function of

checkpoint delay following genotoxic stress is to allow timefor repair before any DNA damage becomes irreversibly fixedafter cell division. However, the relationship between check-point abrogation and chemosensitivity or radiosensitivity is notentirely clear. A recent report has shown a direct linkagebetween Chk1 signaling and the homologous recombinationrepair pathway, suggesting that the enhanced cytotoxicityobserved with Chk1 inhibition following DNA damage mayalso be mediated by regulation of the homologous recombi-nation repair system by Chk1 (33).Third, as with all combination therapies, the sequence of

administration of the individual agents may critically affect

treatment outcome. The importance of schedule was shown inour in vitro studies in which concurrent treatment with atopoisomerase I poison and Chk1 inhibitor targeted primarilythe S-phase checkpoint, whereas sequential exposure favoredinhibition of the G2-M checkpoint. Of note, in our in vivostudies in MDA-MD-435 xenografts where potentiation of theantitumor effect of CPT-11 by CHIR-124 was shown, CPT-11was given alone on the 1st day and 4 h before CHIR-124thereafter. We have not examined other treatment sequences orvaried the time interval between drug administrations; addi-tional studies are required to determine the optimal treatmentschedule in vivo.In summary, we have shown that CHIR-124 is a potent and

selective Chk1 inhibitor. CHIR-124 disrupts the S and G2-Mcheckpoints by interfering with Chk1 intracellular signalingand enhances the antitumor activity of topoisomerase I poisonsboth in vitro and in vivo . Furthermore, tumor cells that lack p53function are more susceptible to undergoing G2-M checkpointabrogation upon treatment with CHIR-124, validating theconcept of combining Chk1 inhibitors with DNA-damagingagents as a therapeutic strategy to sensitize tumors with intrinsiccheckpoint defects. Newer generations of Chk1 inhibitors usingdifferent chemical scaffolds (34) and alternate means to down-regulate Chk1 function (35)7 have been reported and arepoised for clinical testing. Our current data show that CHIR-124 is a promising Chk1 inhibitor that warrants furtherinvestigation to understand its therapeutic potential.

Acknowledgments

We thankTrina Slabiak for her excellent technical help in performing the isobolo-gram assays, Dr. Mithat Gonen for providing advice in statistical analysis, Dr.WilliamHaag for development of the initial response surface analysis software, and Drs.Nancy Pryer andDirkMendel for their support and critical readingof themanuscript.7 Tse et al., in preparation.

References1. Bartek J, Lukas J. Chk1and Chk2 kinases in check-point control and cancer. Cancer Cell 2003;3:421^9.

2. Zhou BB, Bartek J.Targeting the checkpoint kinases:chemosensitization versus chemoprotection. Nat RevCancer 2004;4:216^25.

3. Zhao H, Piwnica-Worms H. ATR-mediated check-point pathways regulate phosphorylation and activa-tion of human Chk1. Mol Cell Biol 2001;21:4129^39.

4. Sorensen CS, Syljuasen RG, Falck J, et al. Chk1 reg-ulates the S phase checkpoint by coupling thephysio-logical turnover and ionizing radiation-inducedaccelerated proteolysis of Cdc25A. Cancer Cell2003;3:247^58.

5.Mailand N, Falck J, Lukas C, et al. Rapid destructionof human Cdc25A in response to DNA damage. Sci-ence 2000;288:1425^9.

6. Zhao H,WatkinsJL, Piwnica-Worms H. Disruption ofthe checkpoint kinase 1/cell division cycle 25A path-way abrogates ionizing radiation-induced S and G2checkpoints. Proc Natl Acad Sci U S A 2002;99:14795^800.

7.Uto K, InoueD, Shimuta K, NakajoN, Sagata N. Chk1,but not Chk2, inhibits Cdc25 phosphatases by a novelcommonmechanism. EMBO J 2004;23:3386^96.

8. Peng CY, Graves PR, Thoma RS,Wu Z, ShawAS,Piwnica-Worms H. Mitotic and G2 checkpoint con-trol: regulation of 14^3-3 protein binding by phos-phorylation of Cdc25C on serine-216. Science 1997;277:1501^5.

9. Chen MS, Ryan CE, Piwnica-Worms H. Chk1kinasenegatively regulates mitotic function of Cdc25A

phosphatase through 14^3-3 binding. Mol Cell Biol2003;23:7488^97.

10. Ferguson AM,White LS, Donovan PJ, Piwnica-WormsH. Normal cell cycle and checkpoint responsesinmice and cells lacking Cdc25Band Cdc25C proteinphosphatases. Mol Cell Biol 2005;25:2853^60.

11.Wang Q, Fan S, Eastman A,Worland PJ, SausvilleEA, O’Connor PM. UCN-01: a potent abrogator of G2checkpoint function in cancer cells with disruptedp53. JNatl Cancer Inst 1996;88:956^65.

12. Shao RG, Cao CX, ShimizuT, O’Connor PM, KohnKW, PommierY. Abrogation of an S-phase checkpointand potentiation of camptothecin cytotoxicity by 7-hydroxystaurosporine (UCN-01) in human cancer celllines, possibly influencedby p53 function. Cancer Res1997;57:4029^35.

13. Tse A, Schwartz G. Potentiation of cytotoxicity oftopoisomerase I poison by concurrent and sequentialtreatment with the checkpoint inhibitor 7-hydroxys-taurosporine involves disparate mechanisms resultingin either p53-independent clonogenic suppression orp53-dependent mitotic catastrophe. Cancer Res2004;64:6635^44.

14. Hollstein M, Hergenhahn M, Yang Q, Bartsch H,Wang ZQ, Hainaut P. New approaches to understand-ing p53 gene tumor mutation spectra. Mutat Res1999;431:199^209.

15. Bunz F, Dutriaux A, Lengauer C, et al. Requirementfor p53 and p21to sustain G2 arrest after DNA dam-age. Science1998;282:1497^501.

16. Bunch RT, Eastman A. Enhancement of cisplatin-

induced cytotoxicity by 7-hydroxystaurosporine(UCN-01), a new G2-checkpoint inhibitor. Clin CancerRes 1996;2:791^7.

17. Sausville EA, Arbuck SG, Messmann R, et al. PhaseI trial of 72-hour continuous infusion UCN-01 inpatients with refractory neoplasms. J Clin Oncol2001;19:2319^33.

18.Ni ZJ, Barsanti P, Brammeier N, et al. 4-(Aminoalky-lamino)-3-benzimidazole-quinolinones as potentCHK-1 inhibitors. Bioorg Med Chem Lett 2006;16:3121^4.

19.Trudel S, Li ZH,Wei E, et al. CHIR-258, a novel,multi-targeted tyrosine kinase inhibitor for the potentialtreatment of t(4;14) multiple myeloma. Blood 2005;105:2941^8.

20. Tallarida RJ. Drug synergism: its detection andapplications. J Pharmacol Exp Ther 2001;298:865^72.

21. GrecoWR, Bravo G, ParsonsJC.The search for syn-ergy: a critical review from a response surface per-spective. Pharmacol Rev1995;47:331^85.

22.CummingsJ, BoydG,MacphersonJS, et al. Factorsinfluencing the cellular accumulation of SN-38 andcamptothecin. Cancer Chemother Pharmacol 2002;49:194^200.

23. Seynaeve CM, Kazanietz MG, Blumberg PM,Sausville EA,Worland PJ. Differential inhibition of pro-tein kinase C isozymes by UCN-01, a staurosporineanalogue. Mol Pharmacol1994;45:1207^14.

24.Holm C, CoveyJM, Kerrigan D, PommierY.Differen-tial requirement of DNA replication for the cytotoxicity





of DNA topoisomerase I and II inhibitors in Chinesehamster DC3F cells. Cancer Res1989;49:6365^8.

25. HsiangYH, Liu LF,Wall ME, et al. DNA topoisomer-ase I-mediated DNA cleavage and cytotoxicity ofcamptothecin analogues. Cancer Res 1989;49:4385^9.

26. Lam MH, Rosen JM. Chk1versus Cdc25: chkingone’s levels of cellular proliferation. Cell Cycle 2004;3:1355^7.

27. Kohn EA, Ruth ND, Brown MK, Livingstone M,Eastman A. Abrogation of the S phase DNA damagecheckpoint results in S phase progression or prema-ture mitosis depending on the concentration of 7-hydroxystaurosporine and the kinetics of Cdc25Cactivation. JBiol Chem 2002;277:26553^64.

28.Graves PR,Yu L, SchwarzJK, et al.The Chk1protein

kinase and the Cdc25C regulatory pathways are tar-gets of the anticancer agent UCN-01. J Biol Chem2000;275:5600^5.

29. Syljuasen RG, Sorensen CS, NylandstedJ, Lukas C,Lukas J, Bartek J. Inhibition of Chk1 by CEP-3891accelerates mitotic nuclear fragmentation in responseto ionizing Radiation. Cancer Res 2004;64:9035^40.

30. Lanni JS, Jacks T. Characterization of the p53-dependent postmitotic checkpoint following spindledisruption. Mol Cell Biol 1998;18:1055^64.

31. Motwani M, Li X, Schwartz GK. Flavopiridol, acyclin-dependent kinase inhibitor, prevents spindle in-hibitor-induced endoreduplication in human cancercells. Clin Cancer Res 2000;6:924^32.

32. Syljuasen RG, Sorensen CS, Hansen LT, et al. Inhi-bition of human Chk1 causes increased initiation of

DNA replication, phosphorylation of ATR targets, andDNA breakage. Mol Cell Biol 2005;25:3553^62.

33. Sorensen CS, Hansen LT, DziegielewskiJ, et al.Thecell-cycle checkpoint kinase Chk1is required formam-malianhomologous recombination repair. Nat CellBiol2005;7:195^201.

34.Wang GT, Li G, Mantei RA, et al. 1-(5-Chloro-2-alkoxyphenyl)-3-(5-cyanopyrazin-2-yl)ureas [cor-rection of cyanopyrazi] as potent and selectiveinhibitors of Chk1 kinase: synthesis, preliminarySAR, and biological activities. J Med Chem 2005;48:3118^21.

35. Arlander SJ, Eapen AK,Vroman BT, McDonald RJ,Toft DO, Karnitz LM. Hsp90 inhibition depletes Chk1and sensitizes tumor cells to replication stress. J BiolChem 2003;278:52572^7.





2007;13:591-602. Clin Cancer Res Archie N. Tse, Katherine G. Rendahl, Tahir Sheikh, et al.

In vivo and In vitroCytotoxicity of Topoisomerase I Poisons CHIR-124, a Novel Potent Inhibitor of Chk1, Potentiates the

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