Preclinical characterization of Aurora kinase inhibitor R763/AS703569 identified through an image-based phenotypic screen

J Cancer Res Clin Oncol (2010) 136:99–113
DOI 10.1007/s00432-009-0641-1
ORIGINAL PAPER

Preclinical characterization of Aurora kinase inhibitor R763/AS703569 identiWed through an image-based phenotypic screen

John McLaughlin · Vadim Markovtsov · Hui Li · Steve Wong · Marina Gelman · Yanhong Zhu · Christian Franci · D. Wayne Lang · Erlina Pali · Joe Lasaga · Caroline Low · Feifei Zhao · Betty Chang · Tarikere L. Gururaja · Weiduan Xu · Muhammad Baluom · David Sweeny · David Carroll · Arvinder Sran · Sambaiah Thota · Manjeet Parmer · Angela Romane · George Clemens · Elliott Grossbard · Kunbin Qu · Yonchu Jenkins · Taisei Kinoshita · Vanessa Taylor · Sacha J. Holland · Ankush Argade · Rajinder Singh · Polly Pine · Donald G. Payan · Yasumichi Hitoshi

Received: 12 September 2008 / Accepted: 23 June 2009 / Published online: 17 July 2009© Springer-Verlag 2009

AbstractPurpose Aurora kinases play a key role in mitotic pro-gression. Over-expression of Aurora kinases is found inseveral human cancers and correlated with histologicalmalignancy and clinical outcomes. Therefore, Aurorakinase inhibitors should be useful in the treatment of can-cers.Methods Cell-based screening methods have an advan-tage over biochemical approaches because hits can be opti-mized to inhibit targets in the proper intracellular context.We developed a novel Aurora kinase inhibitor R763/AS703569 using an image-based phenotypic screen. Theanti-proliferative eVect was examined in a panel of tumorcell lines and primary cells. The eYcacy was determined ina broad panel of xenograft models.

Results R763/AS703569 inhibits Aurora kinases, alongwith a limited number of other kinases including FMS-related tyrosine kinase 3 (FLT3), and has potent anti-prolif-erative activity against many cell types accompanyingunique phenotypic changes such as enlarged cell size, endo-reduplication and apoptosis. The endoreduplication cycleinduced by R763/AS703569 was irreversible even after thecompound was withdrawn from the culture. Oral adminis-tration of R763/AS703569 demonstrated marked inhibitionof tumor growth in xenograft models of pancreatic, breast,colon, ovarian, and lung tumors and leukemia. An acutemyeloid leukemia cell line MV4-11, which carries a FLT3internal tandem duplication mutation, is particularly sensi-tive to R763/AS703569 in vivo.Conclusions R763/AS703569 is a potent inhibitor ofAurora kinases and exhibited signiWcant anti-proliferativeactivity against a wide range of tumor cells both in vitroand in vivo. Inhibition of Aurora kinases has the potentialto be a new addition to the treatment of cancers.

Keywords Cytokinesis · Mitosis · Neoplasm · Aurora kinases · Anti-neoplastic agents · Protein kinases · Protein kinase inhibitors

Introduction

The most potent anti-cancer drugs used today target divid-ing cells, since uncontrolled cell division is a hallmark ofcancer cells. The success of drugs derived from Vinca alka-loids and Taxanes has proved that mitosis is a cell cyclephase that is vulnerable during tumor cell growth. Thesedrugs interrupt microtubule dynamics through cross-linkingtubulins or disrupting tubulin polymerization, and activatethe mitotic checkpoint that leads to massive cell death

J. McLaughlin, V. Markovtsov, H. Li, and S. Wong contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s00432-009-0641-1) contains supplementary material, which is available to authorized users.

J. McLaughlin · V. Markovtsov · H. Li · S. Wong · M. Gelman · Y. Zhu · C. Franci · D. W. Lang · E. Pali · J. Lasaga · C. Low · F. Zhao · B. Chang · T. L. Gururaja · W. Xu · M. Baluom · D. Sweeny · D. Carroll · A. Sran · S. Thota · M. Parmer · G. Clemens · E. Grossbard · K. Qu · Y. Jenkins · T. Kinoshita · V. Taylor · S. J. Holland · A. Argade · R. Singh · P. Pine · D. G. Payan · Y. Hitoshi (&)Rigel Pharmaceuticals Inc., 1180 Veterans Boulevard, South San Francisco, CA 94080, USAe-mail: [email protected]

A. RomaneEMD Serono Inc., One Technology Place, Rockland, MA 02370, USA

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http://dx.doi.org/10.1007/s00432-009-0641-1

100 J Cancer Res Clin Oncol (2010) 136:99–113

(Zhou and Giannakakou 2005). However, targeting micro-tubules causes undesired side eVects such as neurologic,cardiovascular, or mucocutaneous toxicities, becausemicrotubules are critical components of a variety of cellularstructures that govern important physiological functions.In addition, some of these drugs have unfavorable physi-cochemical properties for therapeutic use (Zhou andGiannakakou 2005; Haskell 2001). Therefore, researchershave been looking for alternative mitotic targets to reducethe risk of the side eVects and to change the unfavorableproperties. Recently, several mitotic regulators have beenidentiWed and validated as drug targets, which includeaurora kinases.

Aurora kinases A, B, and C have highly conserved cata-lytic sites and are involved in the progression of mitosis ormeiosis, although the function of Aurora kinase C is lesswell understood (Carmena and Earnshaw 2003; Marumotoet al. 2005). Aurora kinase A plays a key role in centro-some maturation and separation while Aurora kinase B isimportant for chromosome condensation, spindle attach-ment, and cytokinesis. Expression and activity of theseAurora kinases peak during the G2/M phase and becomelow during the G0/G1 and S phases. Ectopic expression ofAurora kinase A in mammalian cells leads to ampliWcationof centrosomes, induction of aneuploidy, and transforma-tion (BischoV et al. 1998; Zhou et al. 1998). Over-expres-sion of Aurora kinases A and B are found in manymalignant tumors (BischoV et al. 1998; Zhou et al. 1998;Sen et al. 1997, 2002; Tanner et al. 2000; Sakakura et al.2001; Watanabe et al. 2002; Gritsko et al. 2003; Moreno-Bueno et al. 2003; Jeng et al. 2004; Rojanala et al. 2004;Yakushijin et al. 2004; Buschhorn et al. 2005; Kurai et al.2005; Tanaka et al. 2005; Lee et al. 2006; Tatsuka et al.1998; Takahashi et al. 2000; Araki et al. 2004; ChieY et al.2004, 2006; Smith et al. 2005; Sorrentino et al. 2005). Inmany cases, over-expression of Aurora kinase A was due toampliWcation of chromosome 20q13 region (Zhou et al.1998; Sen et al. 1997, 2002; Tanner et al. 2000; Sakakuraet al. 2001; Watanabe et al. 2002; Gritsko et al. 2003;Moreno-Bueno et al. 2003; Rojanala et al. 2004). Over-expression of Aurora kinase A was correlated with histo-logical malignancy and clinical outcomes (Tanner et al.2000; Sakakura et al. 2001; Sen et al. 2002; Tanaka et al.1999, 2005; Miyoshi et al. 2001; Neben et al. 2004). Simi-larly, over-expression of Aurora kinase B was correlatedwith high-grade tumors and poor prognosis (Kurai et al.2005; ChieY et al. 2006). However, there are some con-Xicting points of view: (1) over-expression of Aurorakinase A in early stage/low-grade ovarian tumors (Gritskoet al. 2003); (2) association of loss of Aurora kinase A andprogression of in situ to ductal invasive breast carcinoma(Hoque et al. 2003); and (3) no correlation between Aurorakinase A expression and the tumor size, lymph node status,

hormone receptor status, or survival rates (Royce et al.2004).

Suppression of Aurora kinase A by speciWc small inter-fering RNAs (siRNA) or an anti-Aurora kinase A antibodycaused mitotic abnormalities (Marumoto et al. 2003),which led to inhibition of tumor growth in vitro and in vivo(Hata et al. 2005). Similarly, over-expression of kinase-inactive Aurora kinase B resulted in cytokinesis failure, fol-lowed by polyploidy and subsequent cell death (Teradaet al. 1998). A number of small molecule inhibitors ofAurora kinases are currently under evaluation in preclinicalstudies (Barabasz et al. 2006; Chan et al. 2007; DitchWeldet al. 2003; Emanuel et al. 2005; Fancelli et al. 2005; Haufet al. 2003; Heron et al. 2006; Jung et al. 2006; Soncini et al.2006; Warner et al. 2006) or clinical trials (Carpinelli et al.2007; Harrington et al. 2004; Manfredi et al. 2007; Wilkinsonet al. 2007). Aurora kinase inhibitors hold promise for anew paradigm in cancer therapy.

Biochemical kinase assays are broadly used as a primaryscreen for drug development. These assays are often carriedout in vitro with isolated kinase domains and synthetic pep-tides as substrates. Hits identiWed in this manner oftenrequire further optimization for activity in the cell due tolow potency against a physiological conformation of thekinases, poor ability to penetrate membranes, or low meta-bolic stability. Cell-based approaches enable rapid selectionand optimization of the compounds that can inhibit kinasesin their physiological environment. Using a high contentscreening assay, we have established the methods to mea-sure the eVects of compounds on the progression of cellsthrough the cell cycle. Here, we report on the identiWcationand development of a novel Aurora kinase inhibitor, R763/AS703569, through a unique image-based high contentscreen. R763/AS703569 shows pronounced anti-tumoractivities in vitro and in vivo in a wide range of tumor celltypes. We demonstrate that R763/AS703569 inducescaspase-3-dependent mitotic cell death and endoreduplica-tion, followed by irreversible cell cycle arrest at G2/M/tet-raploid (4N) and Octaploid (8N) DNA content. SigniWcantanti-tumor activity is observed in several xenograft modelsincluding an acute myeloid leukemia (AML) model. R763/AS703569 should be a very useful agent in the treatment ofcancers.

Methods

Cell lines, primary cells, and cell culture

Tumor cells were obtained from the American Type Cul-ture Collection (ATCC) (Rockville, MD) and the GermanCollection of Microorganisms and Cell Cultures (DSMZ)(Braunschweig, Germany) and cultured according to the

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J Cancer Res Clin Oncol (2010) 136:99–113 101

suppliers’ instructions. For the colony assays, patient-derived tumor cell lines established at Oncotest GmbH(Freiburg, Germany) were used. Human primary cellsexcept for human primary lymphocytes and cultured humanprimary mast cells were purchased from Cambrex (SanDiego, CA) and maintained according to the supplier’sinstructions. Human primary lymphocytes were obtainedfrom healthy volunteers. Cultured human primary mastcells were established from human umbilical cord bloodCD34+ progenitor cells (AllCells, LLC, Berkeley, CA)using the method described in US patent no. 7070996.

Image-based proliferation assays

Cells were plated in ViewPlate96 96-well plates (PerkinElmer Life Science, Norwalk, CT) in duplicate replicates.The compound dilutions for the six-point screens (from 10to 0.41 �M, threefold dilution) were performed manually.Following incubation with the compound for 48 h, cellswere Wxed with 2.0% paraformaldehyde (Sigma-AldrichChemicals, St. Louis, MO) in phosphate-buVered saline(PBS) (Ca2+/Mg2+-free) for 30 min, washed with PBS,stained for 60 min with a 6 ng/mL solution of 4�,6-diami-dino-2-phenylindole dihydrochloride (DAPI) (MolecularProbes Inc., Eugene, OR) in PBS. Cells were stored at 4°Cin PBS for at least 16 h before imaging to allow stain toequilibrate. A Zeiss Axiovert S100 inverted Xuorescentmicroscope, equipped with a Plan-NEOFLUAR 10£objective (Carl Zeiss Inc., Thornwood, NY) and a Ham-amatsu Lightningcure 200 Mercury-Xenon light sourcewith the Omega Optical XF57 quad Wlter (Hamamatsu Pho-tonics, Japan), was used for capturing images. Nine imagesper well were taken, in an adjacent grid pattern, in eachwell of the 96-well plates of treated tumor cells. Imageswere analyzed in a 12-bit format using segmentation andmorphological routines contained in the Image Pro soft-ware package (Media Cybernetics Inc., Bethesda, MD).The half maximal eVective concentration (EC50) curveWtting was executed with MathLab software version 6.5(MathWorks Inc., Natick, MA). For cell cycle analysis,DNA content of each nucleus in the sample images wasplotted and smoothed using the Lowess method (Hutchesonand Matthew 1995). Apoptosis was assessed by manualinspection of the cell cycle proWle and fragmented nuclei,as assessed by the imaging analysis. Endoreduplication wasassessed by manual inspection based upon enlarged nucleiand presence of the 8N population. 3,000 compounds werescreened with this assay, all of which were synthesized atRigel Pharmaceuticals Inc. The primary data such asnuclear images and DNA intensity, and the processed infor-mation such as cell cycle analysis, assessment of apoptosis,and endoreduplication, and EC50s are stored in the data-base.

Cell-based kinase assays

(1) Aurora kinase B inhibition: A549 cells were synchro-nized with nocodazole (Sigma-Aldrich Chemicals), fol-lowed by 1 h of treatment with R763/AS703569. The cellswere Wxed with 2% paraformaldehyde (Sigma-AldrichChemicals) and stained with the anti-phospho-histone H3serine 10 antibody (Cell Signaling Technology, Beverly,MA) and DAPI. The phospho-histone H3 serine 10 positivecells were imaged using the Zeiss Axiovert S100 invertedXuorescent microscope equipped with the HamamatsuCCD camera (Carl Zeiss Inc., Hamamatsu Photonics.) Thepositivity was calculated and the EC50 curve Wtting wasexecuted with MathLab software version 6.5 (MathWorksInc.). (2) FLT3 inhibition: FLT3 in MV4-11 cells is consti-tutively activated due to an FLT3 internal tandem duplica-tion (ITD) mutation. MV4-11 cells were incubated withR763/AS703569 for 1 h. Cells were lysed in lysis buVer[1% TX-100, 10 mM Tris–HCl pH 7.4, 150 mM NaCl,2 mM MgCl2, 0.1 mM ethylene diamine tetraacetic acid(EDTA), 25 mM NaF, 1 mM sodium vanadate, proteaseinhibitor cocktail]. FLT3 was immunoprecipitated usinganti-human FLT3 antibody (S-18) (Santa Cruz Biotechnol-ogy Inc., Santa Cruz, CA) and protein A/G agarose (VWRInternational, San Francisco, CA). Precipitated proteinswere separated on sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) gels and transferred topolyvinylidene diXuoride (PVDF) membranes. The mem-branes were probed with anti-phosphotyrosine (4G10) anti-body (Upstate Biotechnology Inc., Waltham, MA) or withanti-FLT3 antibody to control for loading. EC50 was esti-mated according to the intensity of phosphotyrosine onFLT3. (3) Vascular endothelial growth factor receptor 2(VEGFR2) inhibition: Human umbilical vein endothelialcells were pre-incubated with R763/AS703569 and thecells were stimulated with the 165 amino acid residue vari-ant of VEGF, VEGF165 (R&D Systems Inc., Minneapolis,MN). The cells were Wxed, permeabilized, and incubatedwith anti-human phospho-VEGFR2 tyrosine 1175 antibody(pY1175) (Cell Signaling Technology), followed by horse-radish peroxidase (HRP)-conjugated goat anti-rabbit IgGantibody (Jackson ImmunoResearch Laboratories Inc.,West Grove, PA), and the chemiluminescent reagent andchemiluminescence was measured. The cells were nextstained with Crystal Violet (Sigma-Aldrich Chemicals), forthe relative cell number, and the staining intensity wasmeasured for normalization of each chemiluminescencereading. (4) Insulin receptor kinase, epidermal growth fac-tor (EGF) receptor kinase, and AKT inhibition: Hela cellswere pre-incubated with R763/AS703569 for 1 h and stim-ulated with anti-Axl antibody (R&D Systems Inc.), humaninsulin (Upstate Biotechnology), or EGF (VWRInternational, San Francisco, CA). For inhibition of the

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constitutively active AKT pathway by R763/AS703569, aphosphatase and tensin homolog (PTEN) null cell line,MDA-MB-231 was used. The cells were Wxed, permeabili-zed, and stained with anti-human phospho-AKT threonine473 (pT473) antibody (Cell Signaling Technology), fol-lowed by HRP-conjugated goat anti-rabbit IgG antibodyand the chemiluminescent reagent. Chemiluminescencewas measured and normalized as described above. (5)Adenosine monophosphate (AMP)-activated protein kinase(AMPK) inhibition: A549 cells were pre-incubated withR763/AS703569 and stimulated with NaN3. The cells wereWxed, permeabilized, and stained with anti-human phos-pho-acetyl-CoA carboxylase (ACC) serine 79 (pS79)antibody (Cell Signaling Technology), followed by HRP-conjugated goat anti-rabbit IgG antibody and the chemilu-minescent reagent. Chemiluminescence was measured andnormalized as described above. (6) Transforming growthfactor (TGF)-beta-activated kinase 1 (TAK1) and c-JUNkinase (JNK) pathway inhibition: A549 cells were platedonto a 96-well tissue culture plate 1 day before the assay.Cells were pre-incubated with R763/AS703569 for 2 h andstimulated with interleukin-1 (IL-1) � for TAK1 activationor Anisomycin for JNK/p38 mitogen-activated proteinkinase (MAPK) activation at their Wnal concentrations of10 ng/mL and 25 �g/mL, respectively, for 20 min at 37°C.Following stimulation, cells were lysed in the radio-immu-noprecipitation assay (RIPA) buVer for 30 min at roomtemperature. Phosphorylation of stress-activated proteinkinase (SAPK)/JNK was detected using the PathScan phos-pho-SAPK/JNK (pT183/pY185) Sandwich ELISA kit (CellSignaling Technology, Beverly, MA). SpeciWcity of allantibodies used in the cell-based assays described abovewas tested in Western blotting assays and correlationbetween these assays and the Western blotting assays wasconWrmed.

5-Bromo-2-deoxyuridine (BrdU) proliferation assay

The eVects of R763/AS703569 on proliferation of suspen-sion cells were assessed using the Chemiluminescent BrdUELISA cell proliferation assay kit (Roche Applied Sci-ences, Chicago, IL).

Colony formation assay

Solid human tumor xenografts were maintained at OncotestInc., GmbH, Freiburg, Germany, and mechanically andenzymatically disaggregated for the colony assays.

The clonogenic assay was performed according to amodiWed two-layer soft agar assay (Hamburger and Salmon1977). Colonies were counted with an automatic imageanalysis system (OMNICON 3600, Biosys GmbH) afterstaining with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-

tetrazolium chloride. Human umbilical cord blood cellswere used for the primary cell control. Cells were treatedwith R763/AS703569 in triplicate at six concentrations andEC50 value was determined by plotting compound concen-tration versus relative colony count.

Biochemical assays

In vitro biochemical assays with Aurora kinases A, B, andC for the half maximal inhibitory concentration (IC50)estimation were performed at Invitrogen Corporation(Carlsbad, CA). BrieXy, R763/AS703569, the peptide/kinase mixture, and adenosine triphosphates (ATP) wereincubated for 60 min. The development reagent thatcleaved non-phosphorylated peptides was added and thereaction mixture was incubated for 60 min. The phosphory-lated peptides maintained Xuorescence resonance energytransfer (FRET) after the reaction. The ratio of donor emis-sion to acceptor emission was used to quantitate reactionprogress. For the ATP competitive assay, Aurora kinase Aand its peptide substrate Kemptide were purchased fromUpstate Biotechnology and the activity in vitro wasassessed in a standard radioactive kinase assay. The kinasereaction mixture was blotted on P81 paper and scannedusing PhosphorImager Typhoon 9410 (GE Healthcare Bio-Sciences, Piscataway, NJ).

Flow cytometry analysis for the cell cycle and apoptosis

(1) Cell cycle analysis: tumor cell lines were treated for48 h with R763/AS703569. After drug treatment, cells wereincubated at 37°C for 4 h with BrdU and Wxed with ice-cold95% ethanol. Nuclei were puriWed by incubating the Wxedcells with 0.08% pepsin (in 0.1 N HCl). Nuclei werestained with anti-BrdU antibody labeled with Xuoresceinisothiocyanate (FITC) and incubated on ice overnight.RNase A was added to the nuclei, then propidium iodide(PI) was added. Cell cycle analysis was performed using aFACScalibur. (2) Apoptosis assay: cells were treated withR763/AS703569 for 24 or 48 h. The cells were suspendedin annexin-binding buVer and stained with FITC annexin V.Then PI was added to the cells. The stained cells were ana-lyzed using a FACScalibur.

In vivo eYcacy studies

Animal studies were carried out in the USA in accordancewith the Institutional Animal Care and Use Committee ofRigel Pharmaceuticals Inc. and in Germany in accordancewith the guidelines of the German Animal Health and Wel-fare Act (Tierschutzgesetz). R763/AS703569 was preparedin saline and orally (p.o.) administered once daily (q.d.).Mice were treated daily for 3 days with various doses of

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R763/AS703569, followed by a 4-day rest period. Thiscycle was repeated four times. Tumor dimensions and bodyweights were measured twice weekly. Vernier caliperswere used to measure tumors in two planes (the longestdimension, L and the dimension perpendicular to L, W), andtumor volume (V) was calculated as V = (L £ W2)/2. ForMiaPaCa-2 study, female CB17 Severe Combined Immu-nodeWciency (SCID) mice (Charles River Laboratories,Hollister, CA) were injected subcutaneously in the rightXank with 2-5 £ 106 cells. Treatment was started aftertumors reached a mean size of 200 mm3. For the studyusing human ovarian carcinoma cell line, NCI-ADR,female NMRI nu/nu mice were inoculated with 1 £ 107

NCI-ADR cells into the right Xank. The treatment wasstarted after tumors reached 50 mm3. For the MOLT4study, female CB17 non-obese diabetic (NOD)/SCID mice(Jackson Laboratories, Hollister, CA) were pretreated withcyclophosphamide for 48 h and inoculated by intravenoustail vein injection with 5 £ 106 leukemia cells. The treat-ment was started 2 weeks after inoculation. After fourcycles of 3 days on and 4 days oV treatment, bone marrowcells were harvested from the treated mice, stained withanti-human CD45 and CD71 antibodies, and analyzed forpercentages of leukemic cells using a FACScalibur. For theMV4-11 study, CB17 NOD/SCID mice were injected sub-cutaneously in the right Xank with 5 £ 106 cells in Matri-gel. The treatment was started after tumors reached300 mm3. For survival studies, an animal was killed if bodyweight loss was >20% of its body weight at the start of drugtreatment, if the tumor size of an animal was ¸2000 mm3,if the tumor ulcerated, or if the animal became moribund.

Biomarker studies

Mice were treated p.o.q.d. for three consecutive days witheither vehicle or various doses of R763/AS703569. Tumorswere harvested 2, 4, 8, 24 and 48 h after the last dose.Immunohistochemical analysis was performed using anti-human phospho-histone H3 (Ser10) antibody 6G3 (CellSignaling, Beverly, MA), biotinylated goat anti-mouse IgGantibody (Biocare, Concord, CA), and HRP-conjugatedStreptavidin. Digital pictures were taken using a 20£objective and cells positive for phosphorylation of histoneH3 were counted.

Statistical analysis

Statistical analyses of R763/AS703569-treated groupsagainst vehicle groups were performed with ANOVA andBonferroni post-test using the Prism software (GraphpadSoftware Inc., CA). In some experiments, Student’s t testwas performed manually. P < 0.05 was considered statisti-cal signiWcant.

Results

Cell-based approach for designing Aurora kinase inhibitors

Inhibition of Aurora kinases by speciWc siRNAs and smallmolecule inhibitors results in a potent anti-proliferativeeVect with unique phenotypic changes such as endoredupli-cation, enlarged multi-lobed nuclei, and apoptosis. There-fore, we devised a high content assay based upon thenumber of nuclei, DNA content, and nuclear morphology todetect the unique phenotypic changes in the lung cancercell lines, A549 and H1299. Small molecules exhibitinganti-proliferative eVects with these phenotypic changeswere selected and conWrmed with traditional biochemicaland cell signaling approaches. Through this cell-basedapproach, we obtained R763/AS703569, the chemicalstructure of which is described in detail in WO 2005118544A2.

R763/AS703569 showed potent anti-proliferative eVectsin A549 lung cancer cells, with an average EC50 of0.007 �M (n = 8) and phenotypic changes such as enlargedcell size and endoreduplication (enlarged multi-lobednuclei and >4N DNA content) (Fig. 1a). The phenotypicchanges were clear even at the EC50 of R763/AS703569.These changes were consistent with the phenotypesinduced by speciWc siRNAs against Aurora kinase B (datanot shown). In vitro biochemical assays demonstrated thatR763/AS703569 inhibited Aurora kinases A, B, and C withIC50 values of 4.0, 4.8, and 6.8 nM, respectively. An excessamount of ATP reduced the level of inhibition of Aurorakinase A by R763/AS703569 in vitro, indicating that R763/AS703569 is an ATP competitive inhibitor (data notshown). The compound was tested in a panel of in vitrokinase assays using diVerent recombinant kinases (supple-mental Table 1). Aurora kinase A, Abelson murine leuke-mia viral oncogene homolog 1 (ABL1), FLT1 (VEGFR1),and FLT3 were more than 90% inhibited by 100 nM ofR763/AS703569. Inhibition of Aurora kinase B was con-Wrmed by an image-based Aurora kinase-dependent assaydetecting intracellular phosphorylation of histone H3 serine10 (Fig. 1b; Table 1). R763/AS703569 reduced the phos-phorylation during mitosis with an average EC50 of 14 nM(n = 5). The selectivity of R763/AS703569 was assessedusing a series of cell-based kinase assays, which measuredthe activation of kinases using antibodies against phosphor-ylated sites of speciWc substrates (Table 1). R763/AS703569 exhibited potent inhibition of FLT3 activationmediated by the ITD mutation and VEGF165-inducedVEGFR2 activation. Inhibition of Aurora kinase activity intumor cells had at least 10-fold selectivity over inhibition ofAMPK, AXL, AKT, and SAPK activity. The selectivity ofR763/AS703569 was also assessed in a series of cell-basedcounter assays (supplemental Table 2). R763/AS703569

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was selective toward inhibition of tumor cell growth. R763/AS703569 did not show potent inhibition of immune cellactivation mediated by many stimuli tested, while itpotently inhibited tumor growth.

Anti-proliferative eVect of Aurora kinase inhibitor

Table 2 summarizes the EC50 values for a panel of tumor celllines and three diVerent types of primary cells that were deter-mined using both the image-based proliferation assay andBrdU incorporation assay. R763/AS703569 potently inhibitedtumor and primary cell growth in the low nanomolar range.All cell lines and primary cells except for human mammaryepithelial cells showed an EC50 of less than 100 nM. Colo205,MiaPaCa-2, Hela, and MV4-11 were the most sensitive toR763/AS703569. The dividing primary cells were also sensi-tive to R763/AS703569, although the EC50s were higher thanthose of the tumor cells. The diVerence in sensitivity of tumorcells versus dividing primary cells in vitro could be due toslower growth and/or intact cell cycle checkpoints in primarycells. In addition, we addressed the eVect of R763/AS703569on survival of non-dividing primary cells (Table 2, bottom).The cell numbers of all four types of primary cells remainedconstant up to 10 �M of R763/AS703569 and there was noapparent apoptosis in these cells as judged from cell appear-ance (i.e., no cytoplasmic blebbing or nuclear fragmentation)and the cell cycle proWle (no sub-G1 population) (data notshown).

The ability of R763/AS703569 to inhibit tumor growthwas examined in colony forming assays using 64 diVerenthuman tumor xenografts in vitro (Fig. 2). R763/AS703569was active against various tumor types. The mean EC50

value was 0.09 �M. A majority of lung, breast, and renaltumors had EC50 values below the mean value. R763/

Fig. 1 a Induction of enlarged nuclei and 4N/8N arrest by R763/AS703569. A549 cells were incubated with 0.007 �M R763/AS703569 for 48 h. Nuclear morphology and DNA intensity were cap-tured with an inverted Xuorescent microscope after cells were Wxed andstained with DAPI. DNA content of each nucleus was plotted andsmoothed using the Lowess method for cell cycle analysis. b Inhibitionof histone H3 serine 10 phosphorylation by R763/AS703569. A549

cells were synchronized with nocodazole and treated with R763/AS703569 for 1 h (0.0001–2.5 �M, 10 points, threefold dilution). Thecells were Wxed and stained with FITC-labeled anti-phospho-histoneH3 serine 10 antibody and DAPI. The number of cells, cell cycle pro-Wle, and phospho-histone H3 serine 10 positive cells were measured inthe image-based assay. The images obtained at 0.0003, 0.003, 0.031,0.28, and 2.5 �M are shown in the Wgure

Table 1 Selectivity of R763/AS703569 assessed in a panel of cell-based kinase assays

The eVect of R763/AS703569 on intracellular phosphorylation of spe-ciWc substrates was measured in ELISA format assays using anti-phos-phoprotein antibodies. The eVect of R763/AS703569 on a variety ofcellular activities was analyzed in order to check the selectivity of thecompound. EC50s were generated by curve Wtting of activities usingMatlab version 6.5 (MathWorks Inc., MA). The fold diVerencesbetween oV target activities of the inhibitor and inhibition of Aurora Bkinase activity in cells or A549 proliferation are shown as “window”.EGFR EGF receptor kinase

Kinase assays EC50 (�M) Window (fold)

Aurora B 0.014 1

FLT3 0.011 1

VEGFR2 0.027 2

AMPK 0.201 14

Insulin R 0.255 18

AXL 0.324 23

TAK1 0.579 41

AKT 0.713 51

EGFR 1.491 107

JNK/MAPKs 7.429 531

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AS703569 showed pronounced activity at less than one-tenth of the mean EC50 value in 9 out of 14 lung cancers, in4 out of 12 mammary cancers, in 3 out of 3 renal cancers, in1 out of 5 melanomas, and 1 out of 2 pancreatic cancers.Two small cell lung carcinomas turned out to be very sensi-tive to R763/AS703569, with EC50s of 1 and 2 nM. Colonyformation of hematopoietic stem cells from umbilical cordblood was also inhibited. These results indicate that R763/AS703569 potently inhibits a colony forming activity oftumor cells from a variety of tumor types.

Induction of apoptosis and endoreduplication by R763/AS703569

We tested cell cycle proWles and apoptosis induction ina panel of tumor cell lines to see which phenotype

(endoreduplication vs. apoptosis) becomes dominant afterR763/AS703569 treatment (Table 3). In all cell lines testedexcept for Colo205, treatment with R763/AS703569resulted in endoreduplication within 48 h, and these cellswere arrested at 4N and 8N DNA content after the treat-ment. In the case of Colo205, detection of endoreduplica-tion was very diYcult because there were few live cellsremaining after treatment. In H1299, DU-145, and PC-3,>8N DNA content was observed. Interestingly, Colo205,Hela, and MiaPaCa-2 cells underwent massive apoptosisfollowing R763/AS703569 treatment and reached over50% cell death by 48 h, while the majority of H1299, A549,PC-3, and DU-145 cells remained viable after 48 h ofR763/AS703569 treatment. We further tested caspase-3dependency of cell death after R763/AS703569 treatmentwith a caspase-3 inhibitor, Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-FMK (DEVD-FMK) (Fig. 3a). More than 70%of Colo205 cells were annexin V positive and/or PI positiveafter the treatment. DEVD-FMK inhibited R763/AS703569-induced cell death and a majority of R763/AS703569 treated cells remained annexin V and PI nega-tive. The live cells cultured with R763/AS703569 andDEVD-FMK became larger in size. Cell cycle analysisshowed that most of these live cells had 8N DNA content,suggesting that the tumor cells entered the endoreduplica-tion cycle after apoptosis was blocked. Overall, all thetested tumor cell lines treated with R763/AS703569 enteredthe endoreduplication cycle, which can result from Aurorakinase B inhibition. Taken together, these tumors wereclearly divided into two groups: apoptosis-dominant andapoptosis-resistant cell lines.

We are particularly interested in the fate of cells arrestedat 4N or 8N DNA content after R763/AS703569 treatment.A549 cells were treated with R763/AS703569 for 144 h orfor 48 h followed by R763/AS703569 withdrawal for 96 h.As shown in Fig. 3b, R763/AS703569 treatment for 48 and144 h caused 4N and 8N arrest in A549 cells and no cells inthe S phase (BrdU positive) were observed. We did notobserve cell cycle arrest at more than 8N DNA content inA549 cells during the 144-h treatment. After washing outR763/AS703569, a majority of A549 cells remained in anarrested state at 4N and 8N DNA content and few cellswere observed in the S phase (BrdU positive) with 4N/8NDNA content. Similar results were obtained when humanprimary umbilical vein endothelial cells were used (data notshown). H1299 cells (p53 null) were also examined inorder to check involvement of p53 in irreversible cell cyclearrest at 4N and 8N DNA content. After the 144-h treat-ment, a majority of the cells were in the sub-G0/G1 peak,which is indicative of apoptosis, and there were few cells inG1, S, G2/M or >8N phase of the cell cycle. After washingout R763/AS703569, a majority of the treated cells stayedBrdU negative (data not shown). These results suggested

Table 2 Anti-proliferative eVect of R763/AS703569 in a panel oftumor cell lines and primary cells

EC50 values were determined in the image-based assay. In addition, theeVect of R763/AS703569 on survival of non-dividing cells such asastrocytes, hepatocytes, cardiomyocytes and diVerentiated skeletalmyoblasts was assessed in the image-based assay. EC50s were gener-ated by curve Wtting of cell numbers or chemiluminescence using Mat-lab version 6.5 (MathWorks Inc., MA)a EC50 values were determined in the BrdU assay

Human cell line Origin EC50 (�M)

A549 Non small cell lung carcinoma 0.007

A549a Non small cell lung carcinoma 0.004

AsPC-1 Pancreatic adenocarcinoma 0.008

Colo205 Colorectal adenocarcinoma 0.006

DU-145 Prostate carcinoma 0.015

H1299 Non small cell lung carcinoma 0.018

Hela Cervical carcinoma 0.008

HL60a Acute promyelocytic leukemia 0.007

MiaPaCa-2 Pancreatic carcinoma 0.002

MOLT-4a Acute lymphoblastic leukemia 0.006

MV-4-11a Biphenotypic B myelomonocytic leukemia

0.003

OVCAR-3 Ovarian adenocarcinoma 0.017

PC-3 Prostate adenocarcinoma 0.019

SU.86.86 Pancreatic ductal carcinoma 0.019

U2OS Osteosarcoma 0.011

Human primary cells (dividing cells)

Mammary epithelial cells 0.16

Umbilical vein endothelial cells 0.031

Human primary cells (non-dividing cells)

Astrocytes >10

Cardiomyocytes >10

Hepatocytes >10

Myoblasts >10

Myotubes >10

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that R763/AS703569-induced 4N and 8N arrest is irrevers-ible, and p53 may not be a major contributor to the irrevers-ible cell cycle arrest.

In vivo eYcacy of R763/AS703569

The anti-tumor activity of R763/AS703569 was examinedin human pancreatic tumor (MiaPaCa-2) xenograftsimplanted subcutaneously in SCID mice. Clearance (Cl),volume of distribution (Vss), half-life (T1/2), and oral bio-availability (%F) of R763/AS703569 in SCID mice are14.5 mL/min kg, 1.85 L/kg, 2.49 h and 37%, respectively.When R763/AS703569 was administered at 15 and 20 mg/kg, the % change in mean tumor volume for treatmentgroup/change in control group was 25.5% (n = 10,P < 0.001) and 10.5% (n = 10, P < 0.001), respectively(Fig. 4a). The treated tumor volume was not lower than theinitial volume. However, the tumors were surrounded bymassive amounts of Wbrous tissue and eosinophilic sub-stances, suggesting that histological regression wasachieved (Fig. 4c). Inhibition of tumor growth was paral-leled by a reduction of histone H3 phosphorylation. Inhibi-tion of this phosphorylation in tumor tissues was greatest(>95% at 20 mg/kg day dosing) at the 2 and 4 h time points,and recovered by 24 h after the Wnal dosing (supplementalFigure 1).

R763/AS703569 was also tested in a slow growing andadriamycin-resistant tumor (NCI-ADR) xenograft model innude mice (Fig. 4b). Tumor growth was signiWcantly sup-pressed by the treatment with R763/AS703569 (7 and10 mg/kg day) in comparison with the control group. Two

out of ten mice in the 7 mg/kg day dosing group and sevenout of ten mice in 10 mg/kg day dosing group showed clearreduction of tumor volume. Similarly, R763/AS703569also exhibited potent anti-tumor activity in colon cancerColo205, ovarian cancer A2780, and lung cancer LXFE211 (Oncotest GmbH) xenograft models (supplementalTable 3).

The anti-tumor activity of R763/AS703569 was exam-ined in MOLT-4 leukemia cells implanted i.v. in NOD/SCID mice. The total number of bone marrow cells wasreduced to 5–10-fold after R763/AS703569 treatment com-pared to vehicle controls. As shown in Fig. 5a, R763/AS703569 treatment signiWcantly reduced the percentagesof leukemia cells (mean% § standard deviation(SD) = 9.6 § 9.5, n = 9, P < 0.001), while the control grouphad a large number of leukemia cells in the bone marrow(mean% § SD = 61.7 § 17.7, n = 9). In three out of ninetreated mice, leukemic cells were not detected (<0.1%).Hematopoietic cells in bone marrow had a normal cellcycle proWle even though leukemia cells were largelydiminished. A dose-dependent increase in lifespan wasobserved in animals treated intermittently at doses of 7.5,10 and 15 mg/kg R763/AS703569 (data not shown).

A myeloid leukemia line (MV4-11), which carries aFLT3 ITD mutation, was examined in a xenograft study.R763/AS703569 produced pronounced dose-dependentanti-tumor activity (Fig. 5b). P values of the vehicle groupversus 7.5, 10, 12.5 and 20 mg/kg day of the R763/AS703569-treated groups were <0.001. Partial regressionswere noted in 67, 92, and 83% of the animals treated atdose levels of 10, 12.5, and 20 mg/kg, respectively, and

Fig. 2 Inhibition of tumor colony formation in vitro by R763/AS703569. Cells from a wide variety of solid tumor xenografts thatwere directly derived from human primary tumors and maintained innude mice at Oncotest Inc. (open circles), and xenografts of ATCC-derived tumor cells (closed circles), were used for the clonogenic assays.Each circle represents an EC50 value of each cell line. The mean EC50value, 10£ mean value, and 0.1£ mean value are indicated as horizon-tal bars. ATCC-derived cell lines used in this assay are SF268 glio-

blastoma (EC50 = 1.185 �M), HT29 colon adenocarcinoma (EC50 =0.027 �M), K562 chronic myelogenous leukemia (EC50 = 0.010 �M),A549 lung carcinoma (EC50 = 0.004 �M), L363 plasma cell leukemia/lymphoma (EC50 = 0.007 �M), RAJI Burkitt’s lymphoma(EC50 = 0.017 �M), MCF7 breast carcinoma (EC50 = 3.7 �M), MDA-MB-231 breast carcinoma (EC50 = 0.024 �M), MX1 breast carcinoma(EC50 = 0.005 �M), DU-145 prostate carcinoma (EC50 = 0.024 �M),and MRIH1579 prostate carcinoma (EC50 = 0.005 �M)

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17% of the animals demonstrated undetectable tumor levelat 20 mg/kg day dosing. R763/AS703569 treatment wasstopped at day 28 and survival of the treated mice wasexamined (Fig. 5c). Increase in lifespan was observed in allR763/AS703569-treated groups, while all mice in the con-trol groups died within 35 days. P values of the vehiclegroup versus 7.5, 10, 12.5 and 20 mg/kg day of the R763/AS703569 treated groups were <0.0001. Supporting theseresults, inhibition of tumor growth was paralleled by areduction in histone H3 phosphorylation in the xenografts(Fig. 5d). At 15 mg/kg day dosing, histone H3 phosphory-lation was still suppressed 48 h after the Wnal dosing event.In addition, complete inhibition of FLT3 tyrosine-phos-phorylation was seen up to 48 h after the Wnal dosing intumor lysates from mice treated with 15 mg/kg R763/AS703569, which is consistent with reduction of histoneH3 phosphorylation (data not shown).

Discussion

In this paper, we report the development of a novel Aurorakinase inhibitor R763/AS703569 through an image-basedphenotypic screen. R763/AS703569 exhibits a potent anti-proliferative eVect in a wide range of tumor cell types withunique phenotypic changes such as enlarged cell size, irre-versible endoreduplication, and apoptosis. Inhibition oftumor growth was demonstrated in a broad panel of in vivoxenograft models. Several solid tumors, including pancre-atic cancers and small cell lung carcinomas, were particu-larly sensitive to R763/AS703569. In addition, R763/

AS703569 was extremely eVective in a FLT3 ITD mutantleukemia cell line, MV4-11. R763/AS703569 could be apotent anti-tumor agent for the treatment of a wide varietyof cancers.

Accessibility of small molecule inhibitors to their activesites in vitro and in vivo may not be identical because ofpotential activators or associated molecules in cells. In thecase of Aurora kinases, there are crucial associated mole-cules [TPX2 (Kufer et al. 2002) and Ajuba (Hirota et al.2003) for Aurora kinase A, and the inner centromere pro-tein, INCENP (Adams et al. 2000; Kaitna et al. 2000) andSurvivin (Wheatley et al. 2001) for Aurora kinase B],which determine proper localization and activation ofAurora kinases. According to the structures of the com-plexes of Aurora kinase A with TPX2 (Bayliss et al. 2003)and Aurora kinase B with INCENP (Sessa et al. 2005), acti-vation of Aurora kinases A and B requires conformationalchanges induced by TPX2 and INCENP. For this reason,tracking the activity of small molecule inhibitors using onlyin vitro kinase assays may be misleading. As an illustration,during our drug development eVorts, we identiWed severalcompounds that potently inhibit Aurora kinases in bio-chemical assays but not in the cell, even though the com-pounds potently inhibited other kinases in the cell (data notshown). Consequently, we used our high content screeningassay to both quantify compound inhibition of cell cycleprogression and verify phenotypically that the mechanismof action was mediated through Aurora kinases. This phe-notype-based approach was complemented with traditionalbiochemical and cell signaling approaches. The activity ofthe inhibitors identiWed using cell-based approaches may be

Table 3 Induction of endoreduplication and apoptosis by R763/AS703569

Percentage of apoptotic cells after R763/AS703569 treatment for 24 and 48 h was identiWed as annexin V positive cells. Induction of endoredupli-cation after R763/AS703569 treatment for 48 h were estimated as accumulation of cells at 4 N and 8 N DNA content. ND, Not determined

Cell line Tissue origin R763 (�M) Apoptosis Endoreduplication

@24 h (%) @48 h (%) 4N (%) 8N (%)

A549 Lung 0 4.4 7.4 5 <0.1

0.05 7.5 14.5 73.3 7.4

H1299 Lung 0 16.7 4.7 2.6 <0.1

0.05 11.1 7.1 5.2 33.2

Hela Cervix 0 19.7 6.8 8.7 <0.1

0.05 23.5 47.5 41.5 18.3

Colo205 Colon 0 22.9 7.2 3.1 <0.1

0.05 55.9 >70 ND ND

MiaPaCa-2 Pancreas 0 13.1 5.6 7.6 <0.1

0.05 19.8 61.8 32.8 13.3

DU-145 Prostate 0 17 19.2 7.8 <0.1

0.05 16.6 15.3 9.1 40.2

PC-3 Prostate 0 28.7 9.9 12.3 <0.1

0.05 22 16.8 17.7 27.9

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already optimized to inhibit the targets in the properintracellular environment. Thus, cell-based screeningapproaches could have a great advantage over conventionalbiochemical screening, although all hits from cell-basedscreens require detailed deconvolution studies to identifythe precise target(s) or mode through which they are acting.

R763/AS703569 induced endoreduplication and apopto-sis to various degrees in p53 wild-type and deWcienttumors, indicating that induction of endoreduplication andapoptosis by R763/AS703569 is independent of the p53status. Interestingly, p53 deWcient cell lines, as well as Helacells in which p53 is inactivated by E6 expression, hadmore pronounced phenotypes than p53 wild-type tumorcells. To further investigate the contribution of p53 toobserved phenotypes, we treated p53 wild-type cell linesA549, HCT-116, and U2OS, and their variants expressing

either a dominant negative p53 or E6, with R763/AS703569 (data not shown). Inactivation of p53 in A549tumor cells resulted in an increased number of cells show-ing endoreduplication following R763/AS703569 treat-ment. In contrast, inactivation of p53 in HCT-116 andU2OS did not further increase the number of endoredupli-cated cells after R763/AS703569 treatment, although bothparental and p53 inactivated tumor lines showed markedendoreduplication. These results indicate that p53 may notbe critical for induction and enhancement of endoreduplica-tion in some tumors. Gizatullin et al. (2006) reported thatendoreduplication and apoptosis were regulated by a p53target gene, p21waf1/cip1 after inhibition of Aurora kinasesby VX-680 and that the cell lines lacking eYcient activa-tion of p21, such as U2OS, showed endoreduplication andapoptosis but VX-680 did not enhance the phenotypes,

Fig. 3 a Inhibition of R763/AS703569-mediated cell death by a caspase-3 inhibitor, DEVD-FMK. Colo205 cells were cul-tured in 50 nM R763/AS703569 containing media for 48 h with or without 50 �M DEVD-FMK. An apoptosis assay (annexin V/PI staining) and BrdU cell cycle analysis (anti BrdU antibody/PI staining) were per-formed to assess the eVect of R763/AS703569. Percentage of cells in each gate is shown at the corner of the quadrant or next to the gate. 0.05% dimethyl sulfox-ide (DMSO) (vehicle) was used for a negative control. b Irre-versible cell cycle arrest at 4N and 8N DNA content in A549 cells. Cells were treated with R763/AS703569 for 48–144 h or for 48 h followed by incuba-tion with R763/AS703569 free media for 24–96 h. Percentage of cells within each gate is indi-cated next to the gate. 0.05% DMSO (vehicle) was used for a negative control

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even when p53 was inactivated. This indicates that the sta-tus of cell cycle inhibitors such as p21waf1/cip1 may play arole in induction of endoreduplication and apoptosis byR763/AS703569, although it is still not clear if other cellcycle inhibitors are also responsible for the phenotype.

Colo205, which demonstrated an apoptosis-dominantphenotype after R763/AS703569 treatment, showedenhanced endoreduplication with R763/AS703569 whencaspase-3 was inhibited. Expression of apoptosis regulatorssuch as Bcl-2 family or inhibitors of apoptosis (IAPs) mightbe involved in the apoptosis sensitivity. In addition, weinvestigated the fate of the cells arrested at 4N or 8N DNAcontent after R763/AS703569 treatment. We found that 4Nand 8N cell cycle arrest induced by R763/AS703569 is irre-versible. Interestingly, the irreversibility is also observed inp53 null H1299 cells, suggesting that a p53-independent

mechanism might be involved in this process. Irreversiblearrest after endoreduplication may be a desirable feature foranti-cancer agents.

Expression levels of drug targets or multidrug resistancegenes can establish sensitivity of tumors to chemotherapeu-tics. However, we did not observe any clear correlationbetween the mRNA expression level of Aurora kinases andsensitivity of tumors to R763/AS703569 among the tumorcell lines tested (data not shown). This needs to be furtherexamined with a larger set of data. Interestingly, R763/AS703569 turns out to be active against Hela cells over-expressing multidrug resistance gene 1 (MDR1). This issupported by the eYcacy in a drug-resistant NCI-ADRtumor xenograft model. Expression levels of Aurorakinases or multidrug resistance genes may not be a criticalfactor for sensitivity. Since Aurora kinase inhibitors can

Fig. 4 Potent anti-tumor activity of R763/AS703569 against humansolid tumor cell lines in vivo. Mice were orally treated consecutivelyfor 3 days with various doses of R763/AS703569, followed by a 4-dayrest interval. This cycle was repeated four times (shown as arrows). Inall animal studies, a 4-week treatment with R763/AS703569 was welltolerated with a minimum body weight loss at the highest dose (lessthan 10% in all experiments). At the end of the four cycles (includingthe last 4 day resting), gross and microscopic examination of diVerenttissues from the R763/AS703569-treated mice showed lack of any sig-niWcant alterations. Toxicity proWling of all tested tissues, includingbone marrow and gastrointestinal tract, conWrmed that there was nodiVerence between orally treated mice and mice treated through the in-tra-peritoneal route. No sign of mechanism-independent toxicity of thecompound was observed. a SCID mice bearing MiaPaCa-2 pancreaticcancer cells were treated with vehicle control (rhombus, solid line),R763/AS703569 15 mg/kg day (circle, dashed line) or R763/

AS703569 20 mg/kg day (pentagon, dashed line). Data are shown asmean tumor volume § standard error of measurement (SEM) (smallbar) of 10 mice/group. b SCID mice bearing NCI-MDR tumors weretreated with vehicle (rhombus, solid line), R763/AS703569 7 mg/kg day (open squares, dashed line), or R763/AS703569 10 mg/kg day(open triangles, dashed line). Data are shown as mean tumorvolume § SEM (small bar) of 10 mice/group. Two out of ten mice (the7 mg/kg day dosing group), and seven out of ten mice (the 10 mg/kg day dosing group) showed clear reduction of tumor volume lessthan the original volume. The two-sample t test was applied to evaluatethe data against the control. Asterisks (*, **, and ***) next to the sym-bols represent P values of <0.05, <0.01, and <0.001, respectively. cHistological analysis of R763/AS703569-treated tumors. MiaPaCa-2tumors from mice treated with vehicle or R763/AS703569 20 mg/kg day for 4 weeks were harvested and stained with hematoxylin andeosin

123


suppress tumor cell growth when the cells enter the mitoticphase, rapidly proliferating cells should be more sensitiveto R763/AS703569. To our surprise, slow growing anddrug-resistant NCI-ADR tumor models also demonstratedsigniWcant tumor regression after R763/AS703569 treat-ment. R763/AS703569 seems to target both slow and rap-idly dividing tumor cells in vivo. Although the mechanismof tumor sensitivity to the compound is not clear at thismoment, how would patients be selected for treatment withthis compound? The status of cell cycle regulators, proa-poptotic genes, or anti-apoptotic molecules in the tumors

might be used as markers for sensitivity. However, thisquestion needs to be answered through further analysesregarding tumor sensitivity to R763/AS703569.

Inhibition of Aurora kinases A and B resulted in pheno-types identical to inactivation of Aurora kinase B: theformation of multinucleated cells due to the failure of cyto-kinesis and down-regulation of the histone H3 serine 10phosphorylation followed by mitotic cell death (DitchWeldet al. 2003; Fancelli et al. 2005; Hauf et al. 2003; Jung et al.2006; Harrington et al. 2004). Inactivation of Aurora kinaseB bypasses requirement of Aurora kinase A, because

Fig. 5 Potent anti-tumor activity of R763/AS703569 against humanleukemia cell lines in vivo. We used an intermittent schedule of admin-istration as described in materials and methods. a MOLT-4 leukemiacells were implanted i.v. in NOD/SCID mice. Treatment with R763/AS703569 began 14 days after i.v. inoculation of the leukemia cells.The majority of MOLT-4 cells repopulated in bone marrow. Bone mar-row cells from NOD/SCID mice bearing Molt4 leukemia cells wereharvested after four cycles of the R763/AS703569 treatment. Leuke-mic cells were identiWed as human CD45 positive and human CD71positive cells with a FACScalibur. Data are presented as % leukemiccells in individual mice and mean § SD (n = 9). The two-sample t testwas applied for statistical signiWcance. b MV4-11 leukemia cells,which carry a FLT3 ITD mutation, were injected subcutaneously intoNOD/SCID mice. The mice bearing MV4-11 leukemia cells weretreated with vehicle control (rhombus, solid line), R763/AS7035697.5 mg/kg day (open square, dashed line), R763/AS703569 10 mg/kg day (open triangle, dashed line), R763/AS703569 12.5 mg/kg day(circle, dashed line) or R763/AS703569 20 mg/kg day (pentagon,

dashed line). The intermittent treatment of R763/AS703569 was re-peated four times (shown as arrows). Data are presented as mean tumorvolume § SEM (small bar) (n = 10/group). c Survival of NOD/SCIDmice bearing MV4-11 leukemia cells after four rounds of the R763/AS703569 treatment. Mice were killed if body weight loss was >20%of its body weight at the start of drug treatment, if the tumor size of ananimal was ¸2,000 mm3, if the tumor ulcerated, or if the mice becamemoribund. d Reduction of phosphorylated histone H3 expression inMV4-11 xenografts by R763/AS703569. Phospho-histone H3 positivecells were counted and data are presented as the number of phospho-histone H3 positive cells per Weld (20£ magniWcation) (n = 12 Weldsper tumor, 3 tumors/group). Hatched bars, dotted bars, and open barsrepresents mean § SD of vehicle, R763/AS703569 7.5 mg/kg treatedand R763/AS703569 15 mg/kg day treated mice, respectively. Thenumber of phospho-histone H3 positive cells per Weld in the treatedgroups of each time point was compared against the vehicle control ofthe same time point. Asterisks (*, **, and ***) on top of the bars rep-resent P values of <0.05, <0.01, and <0.001, respectively

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activation of the mitotic checkpoint induced by inhibitionof Aurora kinase A is dependent on Aurora kinase B activ-ity (Yang et al. 2005). Most compounds reported today areinhibitors of all three Aurora kinases, which show pheno-types due to inhibition of Aurora kinase B. Since the phe-notypes are consistent with inhibition of Aurora kinase B,down-regulation of the histone H3 serine 10 phosphoryla-tion may be used as a pharmacodynamic marker of R763/AS703569, which works well in preclinical models.

Recently, selective inhibitors of both Aurora kinase B,AZD1152, and Aurora kinase A, MLN8054, have beendeveloped (Manfredi et al. 2007; Wilkinson et al. 2007;Hoar et al. 2007), although their advantage over pan-Aurora kinase inhibitors is not clear at this moment. Insharp contrast to pan-Aurora kinase and Aurora kinase Binhibitors, ML8054 induced spindle defects, G2/M accu-mulation without inducing endoreduplication, and massivemitotic cell death (Manfredi et al. 2007; Hoar et al. 2007),supporting diVerent roles of both kinases in mitosis.Because of the diVerence, there are some arguments aboutwhich Aurora kinase would be a preferred target. Aurorakinase B could be the preferred target because its inhibitionleads to mitotic catastrophe and signiWcant suppression oftumor growth in vivo (Wilkinson et al. 2007). On the otherhand, over-expression of Aurora kinase A is seen in manyhuman cancers (Marumoto et al. 2005) and selective inhibi-tion of Aurora kinase A potently induces apoptosis (Hataet al. 2005; Manfredi et al. 2007). Aurora kinase A on itsown could also be an attractive target. However, an Aurorakinase A and B inhibitor, VX-680, failed to kill cellsexpressing the drug-resistant Aurora kinase B mutant, sug-gesting inhibition of Aurora kinase A may not be suYcient(Cochran 2008; Girdler et al. 2008). The answer regardingwhich Aurora kinase could be a better target may emergeshortly since many of Aurora kinase inhibitors are currentlybeing evaluated in clinical studies.

It might be beneWcial for R763/AS703569 to inhibitother kinases in addition to Aurora kinases. Selective inhib-itors might be less toxic but may also lead to drug resis-tance more easily, while broad kinase inhibitors might bemore toxic but may be more resistant to drug-resistantmutants or have more potency from inhibiting other targets.While Aurora kinase inhibitors suppress tumor cell growththrough the mitotic phase, inhibitors of receptor tyrosinekinases are eVective by targeting diVerent phases of the cellcycle such as G0/G1 to S transition. One could speculatethat combination therapy with receptor tyrosine kinase andAurora kinase inhibitors would be useful for the treatmentof several cancers. R763/AS703569 inhibited FLT3 inaddition to Aurora kinases in vitro and in vivo and demon-strated an excellent eYcacy in xenograft studies against theFLT3 ITD mutant AML cell line, MV4-11. Interestingly,the length of the pHH3 inhibition by R763/AS703569 in

MV4-11 tumors is much longer than that in MiaPaCa-2tumors, suggesting that inhibiting both FLT3 and Aurorakinases may have a much stronger impact on tumor cellgrowth and survival by targeting multiple phases of the cellcycle. Further experiments should be performed usinghighly selective inhibitors of FLT3 or Aurora kinases inorder to clarify if inhibition of both FLT3 and Aurorakinases is beneWcial.

R763/AS703569 also potently inhibits VEGFR2 in cells.VEGFR2 is a growth factor receptor tyrosine kinase, whichtransmits an important signal for angiogenesis. Release ofits ligand, VEGF, causes an endothelial cell to survive,migrate, or diVerentiate. Bevacizumab, a monoclonal anti-body against VEGF, and Sunitinib, a small molecule inhib-itor of VEGFR2, demonstrated the usefulness of targetingVEGFR2 in colorectal (Hurwitz et al. 2004), renal (Escudieret al. 2007; Motzer et al. 2006; Yang et al. 2003), and lungcancers (Sandler et al. 2006). Therefore, R763/AS703569is expected to have a potent anti-tumor eVect partly dueto inhibition of angiogenesis, which might be a bonus forthe treatment of cancer. Importantly, bevacizumab hasbeen associated with hypertension, proteinuria, and someother complications (Hurwitz et al. 2004; Yang et al. 2003;Kabbinavar et al. 2005a, b; Scappaticci et al. 2005; Chuet al. 2007). Having anti-VEGFR2 activity could be poten-tially useful, but toxicity needs to be carefully monitoredduring the treatment.

In conclusion, we have identiWed a potent Aurora kinaseinhibitor, R763/AS703569, through an image-based highcontent screen. R763/AS703569 has a signiWcant anti-pro-liferative eVect in a wide range of tumor cell types withunique phenotypic changes such as enlarged cell size, endo-reduplication, and apoptosis. The phenotypes induced byR763/AS703569 are consistent with inhibition of Aurorakinase B. Tumor regression has been demonstrated in somexenograft models with a well-tolerated dose of R763/AS703569. Our studies indicate that inhibition of Aurorakinases is a feasible approach toward many types of cancersand R763/AS703569 should be useful in the treatment ofthe disease. Currently, phase I clinical studies with R763/AS703569 are underway.

Acknowledgments We thank Caroline Sula and Jorge Victorino fortechnical assistance, Dr. Tomas Sun and Dr. David Lau for consistentsupport for the project and Dr. Jim Diehl for critical reading of themanuscript. We also thank Mr. Niko Bausch and Dr. Heinz H. Fiebig,Oncotest GmbH, for helpful discussion and support.

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