Skip to main content
NCBI home page
Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2009 Jul 17;136(1):99–113. doi: 10.1007/s00432-009-0641-1

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

John McLaughlin 1, Vadim Markovtsov 1, Hui Li 1, Steve Wong 1, Marina Gelman 1, Yanhong Zhu 1, Christian Franci 1, D Wayne Lang 1, Erlina Pali 1, Joe Lasaga 1, Caroline Low 1, Feifei Zhao 1, Betty Chang 1, Tarikere L Gururaja 1, Weiduan Xu 1, Muhammad Baluom 1, David Sweeny 1, David Carroll 1, Arvinder Sran 1, Sambaiah Thota 1, Manjeet Parmer 1, Angela Romane 2, George Clemens 1, Elliott Grossbard 1, Kunbin Qu 1, Yonchu Jenkins 1, Taisei Kinoshita 1, Vanessa Taylor 1, Sacha J Holland 1, Ankush Argade 1, Rajinder Singh 1, Polly Pine 1, Donald G Payan 1, Yasumichi Hitoshi 1,✉
PMCID: PMC11827822  PMID: 19609559

Abstract

Purpose

Aurora kinases play a key role in mitotic progression. Over-expression of Aurora kinases is found in several human cancers and correlated with histological malignancy and clinical outcomes. Therefore, Aurora kinase inhibitors should be useful in the treatment of cancers.

Methods

Cell-based screening methods have an advantage over biochemical approaches because hits can be optimized to inhibit targets in the proper intracellular context. We developed a novel Aurora kinase inhibitor R763/AS703569 using an image-based phenotypic screen. The anti-proliferative effect was examined in a panel of tumor cell lines and primary cells. The efficacy was determined in a broad panel of xenograft models.

Results

R763/AS703569 inhibits Aurora kinases, along with a limited number of other kinases including FMS-related tyrosine kinase 3 (FLT3), and has potent anti-proliferative activity against many cell types accompanying unique phenotypic changes such as enlarged cell size, endoreduplication and apoptosis. The endoreduplication cycle induced by R763/AS703569 was irreversible even after the compound was withdrawn from the culture. Oral administration of R763/AS703569 demonstrated marked inhibition of tumor growth in xenograft models of pancreatic, breast, colon, ovarian, and lung tumors and leukemia. An acute myeloid leukemia cell line MV4-11, which carries a FLT3 internal tandem duplication mutation, is particularly sensitive to R763/AS703569 in vivo.

Conclusions

R763/AS703569 is a potent inhibitor of Aurora kinases and exhibited significant anti-proliferative activity against a wide range of tumor cells both in vitro and in vivo. Inhibition of Aurora kinases has the potential to be a new addition to the treatment of cancers.

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.

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

Introduction

The most potent anti-cancer drugs used today target dividing cells, since uncontrolled cell division is a hallmark of cancer cells. The success of drugs derived from Vinca alkaloids and Taxanes has proved that mitosis is a cell cycle phase that is vulnerable during tumor cell growth. These drugs interrupt microtubule dynamics through cross-linking tubulins or disrupting tubulin polymerization, and activate the mitotic checkpoint that leads to massive cell death (Zhou and Giannakakou 2005). However, targeting microtubules causes undesired side effects such as neurologic, cardiovascular, or mucocutaneous toxicities, because microtubules are critical components of a variety of cellular structures that govern important physiological functions. In addition, some of these drugs have unfavorable physicochemical properties for therapeutic use (Zhou and Giannakakou 2005; Haskell 2001). Therefore, researchers have been looking for alternative mitotic targets to reduce the risk of the side effects and to change the unfavorable properties. Recently, several mitotic regulators have been identified and validated as drug targets, which include aurora kinases.

Aurora kinases A, B, and C have highly conserved catalytic sites and are involved in the progression of mitosis or meiosis, although the function of Aurora kinase C is less well understood (Carmena and Earnshaw 2003; Marumoto et al. 2005). Aurora kinase A plays a key role in centrosome maturation and separation while Aurora kinase B is important for chromosome condensation, spindle attachment, and cytokinesis. Expression and activity of these Aurora kinases peak during the G2/M phase and become low during the G0/G1 and S phases. Ectopic expression of Aurora kinase A in mammalian cells leads to amplification of centrosomes, induction of aneuploidy, and transformation (Bischoff et al. 1998; Zhou et al. 1998). Over-expression of Aurora kinases A and B are found in many malignant tumors (Bischoff 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; Chieffi et al. 2004, 2006; Smith et al. 2005; Sorrentino et al. 2005). In many cases, over-expression of Aurora kinase A was due to amplification of chromosome 20q13 region (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; Rojanala et al. 2004). Over-expression of Aurora kinase A was correlated with histological 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). Similarly, over-expression of Aurora kinase B was correlated with high-grade tumors and poor prognosis (Kurai et al. 2005; Chieffi et al. 2006). However, there are some conflicting points of view: (1) over-expression of Aurora kinase A in early stage/low-grade ovarian tumors (Gritsko et al. 2003); (2) association of loss of Aurora kinase A and progression of in situ to ductal invasive breast carcinoma (Hoque et al. 2003); and (3) no correlation between Aurora kinase 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 specific small interfering RNAs (siRNA) or an anti-Aurora kinase A antibody caused 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, followed by polyploidy and subsequent cell death (Terada et al. 1998). A number of small molecule inhibitors of Aurora kinases are currently under evaluation in preclinical studies (Barabasz et al. 2006; Chan et al. 2007; Ditchfield et al. 2003; Emanuel et al. 2005; Fancelli et al. 2005; Hauf et 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; Wilkinson et al. 2007). Aurora kinase inhibitors hold promise for a new paradigm in cancer therapy.

Biochemical kinase assays are broadly used as a primary screen for drug development. These assays are often carried out in vitro with isolated kinase domains and synthetic peptides as substrates. Hits identified in this manner often require further optimization for activity in the cell due to low potency against a physiological conformation of the kinases, poor ability to penetrate membranes, or low metabolic stability. Cell-based approaches enable rapid selection and optimization of the compounds that can inhibit kinases in their physiological environment. Using a high content screening assay, we have established the methods to measure the effects of compounds on the progression of cells through the cell cycle. Here, we report on the identification and development of a novel Aurora kinase inhibitor, R763/AS703569, through a unique image-based high content screen. R763/AS703569 shows pronounced anti-tumor activities in vitro and in vivo in a wide range of tumor cell types. We demonstrate that R763/AS703569 induces caspase-3-dependent mitotic cell death and endoreduplication, followed by irreversible cell cycle arrest at G2/M/tetraploid (4N) and Octaploid (8N) DNA content. Significant anti-tumor activity is observed in several xenograft models including an acute myeloid leukemia (AML) model. R763/AS703569 should be a very useful agent in the treatment of cancers.

Methods

Cell lines, primary cells, and cell culture

Tumor cells were obtained from the American Type Culture Collection (ATCC) (Rockville, MD) and the German Collection of Microorganisms and Cell Cultures (DSMZ) (Braunschweig, Germany) and cultured according to the suppliers’ instructions. For the colony assays, patient-derived tumor cell lines established at Oncotest GmbH (Freiburg, Germany) were used. Human primary cells except for human primary lymphocytes and cultured human primary mast cells were purchased from Cambrex (San Diego, CA) and maintained according to the supplier’s instructions. Human primary lymphocytes were obtained from healthy volunteers. Cultured human primary mast cells were established from human umbilical cord blood CD34+ 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 (Perkin Elmer Life Science, Norwalk, CT) in duplicate replicates. The compound dilutions for the six-point screens (from 10 to 0.41 μM, threefold dilution) were performed manually. Following incubation with the compound for 48 h, cells were fixed with 2.0% paraformaldehyde (Sigma-Aldrich Chemicals, St. Louis, MO) in phosphate-buffered saline (PBS) (Ca2+/Mg2+-free) for 30 min, washed with PBS, stained for 60 min with a 6 ng/mL solution of 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Molecular Probes Inc., Eugene, OR) in PBS. Cells were stored at 4°C in PBS for at least 16 h before imaging to allow stain to equilibrate. A Zeiss Axiovert S100 inverted fluorescent microscope, equipped with a Plan-NEOFLUAR 10× objective (Carl Zeiss Inc., Thornwood, NY) and a Hamamatsu Lightningcure 200 Mercury-Xenon light source with the Omega Optical XF57 quad filter (Hamamatsu Photonics, Japan), was used for capturing images. Nine images per well were taken, in an adjacent grid pattern, in each well of the 96-well plates of treated tumor cells. Images were analyzed in a 12-bit format using segmentation and morphological routines contained in the Image Pro software package (Media Cybernetics Inc., Bethesda, MD). The half maximal effective concentration (EC50) curve fitting 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 was plotted and smoothed using the Lowess method (Hutcheson and Matthew 1995). Apoptosis was assessed by manual inspection of the cell cycle profile and fragmented nuclei, as assessed by the imaging analysis. Endoreduplication was assessed by manual inspection based upon enlarged nuclei and presence of the 8N population. 3,000 compounds were screened with this assay, all of which were synthesized at Rigel Pharmaceuticals Inc. The primary data such as nuclear images and DNA intensity, and the processed information such as cell cycle analysis, assessment of apoptosis, and endoreduplication, and EC50s are stored in the database.

Cell-based kinase assays

(1) Aurora kinase B inhibition: A549 cells were synchronized with nocodazole (Sigma-Aldrich Chemicals), followed by 1 h of treatment with R763/AS703569. The cells were fixed with 2% paraformaldehyde (Sigma-Aldrich Chemicals) and stained with the anti-phospho-histone H3 serine 10 antibody (Cell Signaling Technology, Beverly, MA) and DAPI. The phospho-histone H3 serine 10 positive cells were imaged using the Zeiss Axiovert S100 inverted fluorescent microscope equipped with the Hamamatsu CCD camera (Carl Zeiss Inc., Hamamatsu Photonics.) The positivity was calculated and the EC50 curve fitting was executed with MathLab software version 6.5 (MathWorks Inc.). (2) FLT3 inhibition: FLT3 in MV4-11 cells is constitutively activated due to an FLT3 internal tandem duplication (ITD) mutation. MV4-11 cells were incubated with R763/AS703569 for 1 h. Cells were lysed in lysis buffer [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, protease inhibitor cocktail]. FLT3 was immunoprecipitated using anti-human FLT3 antibody (S-18) (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and protein A/G agarose (VWR International, San Francisco, CA). Precipitated proteins were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were probed with anti-phosphotyrosine (4G10) antibody (Upstate Biotechnology Inc., Waltham, MA) or with anti-FLT3 antibody to control for loading. EC50 was estimated according to the intensity of phosphotyrosine on FLT3. (3) Vascular endothelial growth factor receptor 2 (VEGFR2) inhibition: Human umbilical vein endothelial cells were pre-incubated with R763/AS703569 and the cells were stimulated with the 165 amino acid residue variant of VEGF, VEGF165 (R&D Systems Inc., Minneapolis, MN). The cells were fixed, permeabilized, and incubated with anti-human phospho-VEGFR2 tyrosine 1175 antibody (pY1175) (Cell Signaling Technology), followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA), and the chemiluminescent reagent and chemiluminescence was measured. The cells were next stained with Crystal Violet (Sigma-Aldrich Chemicals), for the relative cell number, and the staining intensity was measured for normalization of each chemiluminescence reading. (4) Insulin receptor kinase, epidermal growth factor (EGF) receptor kinase, and AKT inhibition: Hela cells were pre-incubated with R763/AS703569 for 1 h and stimulated with anti-Axl antibody (R&D Systems Inc.), human insulin (Upstate Biotechnology), or EGF (VWR International, San Francisco, CA). For inhibition of the constitutively active AKT pathway by R763/AS703569, a phosphatase and tensin homolog (PTEN) null cell line, MDA-MB-231 was used. The cells were fixed, permeabilized, and stained with anti-human phospho-AKT threonine 473 (pT473) antibody (Cell Signaling Technology), followed by HRP-conjugated goat anti-rabbit IgG antibody and the chemiluminescent reagent. Chemiluminescence was measured and normalized as described above. (5) Adenosine monophosphate (AMP)-activated protein kinase (AMPK) inhibition: A549 cells were pre-incubated with R763/AS703569 and stimulated with NaN3. The cells were fixed, permeabilized, and stained with anti-human phospho-acetyl-CoA carboxylase (ACC) serine 79 (pS79) antibody (Cell Signaling Technology), followed by HRP-conjugated goat anti-rabbit IgG antibody and the chemiluminescent reagent. Chemiluminescence was measured and normalized as described above. (6) Transforming growth factor (TGF)-beta-activated kinase 1 (TAK1) and c-JUN kinase (JNK) pathway inhibition: A549 cells were plated onto a 96-well tissue culture plate 1 day before the assay. Cells were pre-incubated with R763/AS703569 for 2 h and stimulated with interleukin-1 (IL-1) β for TAK1 activation or Anisomycin for JNK/p38 mitogen-activated protein kinase (MAPK) activation at their final concentrations of 10 ng/mL and 25 μg/mL, respectively, for 20 min at 37°C. Following stimulation, cells were lysed in the radio-immunoprecipitation assay (RIPA) buffer for 30 min at room temperature. Phosphorylation of stress-activated protein kinase (SAPK)/JNK was detected using the PathScan phospho-SAPK/JNK (pT183/pY185) Sandwich ELISA kit (Cell Signaling Technology, Beverly, MA). Specificity of all antibodies used in the cell-based assays described above was tested in Western blotting assays and correlation between these assays and the Western blotting assays was confirmed.

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

The effects of R763/AS703569 on proliferation of suspension cells were assessed using the Chemiluminescent BrdU ELISA cell proliferation assay kit (Roche Applied Sciences, Chicago, IL).

Colony formation assay

Solid human tumor xenografts were maintained at Oncotest Inc., GmbH, Freiburg, Germany, and mechanically and enzymatically disaggregated for the colony assays.

The clonogenic assay was performed according to a modified two-layer soft agar assay (Hamburger and Salmon 1977). Colonies were counted with an automatic image analysis system (OMNICON 3600, Biosys GmbH) after staining with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride. Human umbilical cord blood cells were used for the primary cell control. Cells were treated with R763/AS703569 in triplicate at six concentrations and EC50 value was determined by plotting compound concentration versus relative colony count.

Biochemical assays

In vitro biochemical assays with Aurora kinases A, B, and C for the half maximal inhibitory concentration (IC50) estimation were performed at Invitrogen Corporation (Carlsbad, CA). Briefly, R763/AS703569, the peptide/kinase mixture, and adenosine triphosphates (ATP) were incubated for 60 min. The development reagent that cleaved non-phosphorylated peptides was added and the reaction mixture was incubated for 60 min. The phosphorylated peptides maintained fluorescence resonance energy transfer (FRET) after the reaction. The ratio of donor emission to acceptor emission was used to quantitate reaction progress. For the ATP competitive assay, Aurora kinase A and its peptide substrate Kemptide were purchased from Upstate Biotechnology and the activity in vitro was assessed in a standard radioactive kinase assay. The kinase reaction mixture was blotted on P81 paper and scanned using 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 for 48 h with R763/AS703569. After drug treatment, cells were incubated at 37°C for 4 h with BrdU and fixed with ice-cold 95% ethanol. Nuclei were purified by incubating the fixed cells with 0.08% pepsin (in 0.1 N HCl). Nuclei were stained with anti-BrdU antibody labeled with fluorescein isothiocyanate (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 a FACScalibur. (2) Apoptosis assay: cells were treated with R763/AS703569 for 24 or 48 h. The cells were suspended in annexin-binding buffer and stained with FITC annexin V. Then PI was added to the cells. The stained cells were analyzed using a FACScalibur.

In vivo efficacy studies

Animal studies were carried out in the USA in accordance with the Institutional Animal Care and Use Committee of Rigel Pharmaceuticals Inc. and in Germany in accordance with the guidelines of the German Animal Health and Welfare Act (Tierschutzgesetz). R763/AS703569 was prepared in saline and orally (p.o.) administered once daily (q.d.). Mice were treated daily for 3 days with various doses of R763/AS703569, followed by a 4-day rest period. This cycle was repeated four times. Tumor dimensions and body weights were measured twice weekly. Vernier calipers were used to measure tumors in two planes (the longest dimension, L and the dimension perpendicular to L, W), and tumor volume (V) was calculated as V = (L × W 2)/2. For MiaPaCa-2 study, female CB17 Severe Combined Immunodeficiency (SCID) mice (Charles River Laboratories, Hollister, CA) were injected subcutaneously in the right flank with 2-5 × 106 cells. Treatment was started after tumors reached a mean size of 200 mm3. For the study using human ovarian carcinoma cell line, NCI-ADR, female NMRI nu/nu mice were inoculated with 1 × 107 NCI-ADR cells into the right flank. The treatment was started after tumors reached 50 mm3. For the MOLT4 study, female CB17 non-obese diabetic (NOD)/SCID mice (Jackson Laboratories, Hollister, CA) were pretreated with cyclophosphamide for 48 h and inoculated by intravenous tail vein injection with 5 × 106 leukemia cells. The treatment was started 2 weeks after inoculation. After four cycles of 3 days on and 4 days off treatment, bone marrow cells were harvested from the treated mice, stained with anti-human CD45 and CD71 antibodies, and analyzed for percentages of leukemic cells using a FACScalibur. For the MV4-11 study, CB17 NOD/SCID mice were injected subcutaneously in the right flank with 5 × 106 cells in Matrigel. The treatment was started after tumors reached 300 mm3. For survival studies, an animal was killed if body weight loss was >20% of its body weight at the start of drug treatment, 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 with either vehicle or various doses of R763/AS703569. Tumors were 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 (Cell Signaling, Beverly, MA), biotinylated goat anti-mouse IgG antibody (Biocare, Concord, CA), and HRP-conjugated Streptavidin. Digital pictures were taken using a 20× objective and cells positive for phosphorylation of histone H3 were counted.

Statistical analysis

Statistical analyses of R763/AS703569-treated groups against vehicle groups were performed with ANOVA and Bonferroni post-test using the Prism software (Graphpad Software Inc., CA). In some experiments, Student’s t test was performed manually. P < 0.05 was considered statistical significant.

Results

Cell-based approach for designing Aurora kinase inhibitors

Inhibition of Aurora kinases by specific siRNAs and small molecule inhibitors results in a potent anti-proliferative effect with unique phenotypic changes such as endoreduplication, enlarged multi-lobed nuclei, and apoptosis. Therefore, we devised a high content assay based upon the number of nuclei, DNA content, and nuclear morphology to detect the unique phenotypic changes in the lung cancer cell lines, A549 and H1299. Small molecules exhibiting anti-proliferative effects with these phenotypic changes were selected and confirmed with traditional biochemical and cell signaling approaches. Through this cell-based approach, we obtained R763/AS703569, the chemical structure of which is described in detail in WO 2005118544 A2.

R763/AS703569 showed potent anti-proliferative effects in A549 lung cancer cells, with an average EC50 of 0.007 μM (n = 8) and phenotypic changes such as enlarged cell size and endoreduplication (enlarged multi-lobed nuclei and >4N DNA content) (Fig. 1a). The phenotypic changes were clear even at the EC50 of R763/AS703569. These changes were consistent with the phenotypes induced by specific siRNAs against Aurora kinase B (data not shown). In vitro biochemical assays demonstrated that R763/AS703569 inhibited Aurora kinases A, B, and C with IC50 values of 4.0, 4.8, and 6.8 nM, respectively. An excess amount of ATP reduced the level of inhibition of Aurora kinase A by R763/AS703569 in vitro, indicating that R763/AS703569 is an ATP competitive inhibitor (data not shown). The compound was tested in a panel of in vitro kinase assays using different recombinant kinases (supplemental Table 1). Aurora kinase A, Abelson murine leukemia viral oncogene homolog 1 (ABL1), FLT1 (VEGFR1), and FLT3 were more than 90% inhibited by 100 nM of R763/AS703569. Inhibition of Aurora kinase B was confirmed by an image-based Aurora kinase-dependent assay detecting intracellular phosphorylation of histone H3 serine 10 (Fig. 1b; Table 1). R763/AS703569 reduced the phosphorylation during mitosis with an average EC50 of 14 nM (n = 5). The selectivity of R763/AS703569 was assessed using a series of cell-based kinase assays, which measured the activation of kinases using antibodies against phosphorylated sites of specific substrates (Table 1). R763/AS703569 exhibited potent inhibition of FLT3 activation mediated by the ITD mutation and VEGF165-induced VEGFR2 activation. Inhibition of Aurora kinase activity in tumor cells had at least 10-fold selectivity over inhibition of AMPK, AXL, AKT, and SAPK activity. The selectivity of R763/AS703569 was also assessed in a series of cell-based counter assays (supplemental Table 2). R763/AS703569 was selective toward inhibition of tumor cell growth. R763/AS703569 did not show potent inhibition of immune cell activation mediated by many stimuli tested, while it potently inhibited tumor growth.

Fig. 1.

Fig. 1

Open in a new tab

a Induction of enlarged nuclei and 4N/8N arrest by R763/AS703569. A549 cells were incubated with 0.007 μM R763/AS703569 for 48 h. Nuclear morphology and DNA intensity were captured with an inverted fluorescent microscope after cells were fixed and stained with DAPI. DNA content of each nucleus was plotted and smoothed using the Lowess method for cell cycle analysis. b Inhibition of 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 μM, 10 points, threefold dilution). The cells were fixed and stained with FITC-labeled anti-phospho-histone H3 serine 10 antibody and DAPI. The number of cells, cell cycle profile, and phospho-histone H3 serine 10 positive cells were measured in the image-based assay. The images obtained at 0.0003, 0.003, 0.031, 0.28, and 2.5 μM are shown in the figure

Table 1.

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

Kinase assays EC50 (μM) 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
Open in a new tab

The effect of R763/AS703569 on intracellular phosphorylation of specific substrates was measured in ELISA format assays using anti-phosphoprotein antibodies. The effect of R763/AS703569 on a variety of cellular activities was analyzed in order to check the selectivity of the compound. EC50s were generated by curve fitting of activities using Matlab version 6.5 (MathWorks Inc., MA). The fold differences between off target activities of the inhibitor and inhibition of Aurora B kinase activity in cells or A549 proliferation are shown as “window”. EGFR EGF receptor kinase

Anti-proliferative effect of Aurora kinase inhibitor

Table 2 summarizes the EC50 values for a panel of tumor cell lines and three different types of primary cells that were determined using both the image-based proliferation assay and BrdU incorporation assay. R763/AS703569 potently inhibited tumor and primary cell growth in the low nanomolar range. All cell lines and primary cells except for human mammary epithelial cells showed an EC50 of less than 100 nM. Colo205, MiaPaCa-2, Hela, and MV4-11 were the most sensitive to R763/AS703569. The dividing primary cells were also sensitive to R763/AS703569, although the EC50s were higher than those of the tumor cells. The difference in sensitivity of tumor cells versus dividing primary cells in vitro could be due to slower growth and/or intact cell cycle checkpoints in primary cells. In addition, we addressed the effect of R763/AS703569 on survival of non-dividing primary cells (Table 2, bottom). The cell numbers of all four types of primary cells remained constant up to 10 μM of R763/AS703569 and there was no apparent apoptosis in these cells as judged from cell appearance (i.e., no cytoplasmic blebbing or nuclear fragmentation) and the cell cycle profile (no sub-G1 population) (data not shown).

Table 2.

Anti-proliferative effect of R763/AS703569 in a panel of tumor cell lines and primary cells

Human cell line Origin EC50 (μM)
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
Open in a new tab

EC50 values were determined in the image-based assay. In addition, the effect of R763/AS703569 on survival of non-dividing cells such as astrocytes, hepatocytes, cardiomyocytes and differentiated skeletal myoblasts was assessed in the image-based assay. EC50s were generated by curve fitting of cell numbers or chemiluminescence using Matlab version 6.5 (MathWorks Inc., MA)

aEC50 values were determined in the BrdU assay

The ability of R763/AS703569 to inhibit tumor growth was examined in colony forming assays using 64 different human tumor xenografts in vitro (Fig. 2). R763/AS703569 was active against various tumor types. The mean EC50 value was 0.09 μM. A majority of lung, breast, and renal tumors had EC50 values below the mean value. R763/AS703569 showed pronounced activity at less than one-tenth of the mean EC50 value in 9 out of 14 lung cancers, in 4 out of 12 mammary cancers, in 3 out of 3 renal cancers, in 1 out of 5 melanomas, and 1 out of 2 pancreatic cancers. Two small cell lung carcinomas turned out to be very sensitive to R763/AS703569, with EC50s of 1 and 2 nM. Colony formation of hematopoietic stem cells from umbilical cord blood was also inhibited. These results indicate that R763/AS703569 potently inhibits a colony forming activity of tumor cells from a variety of tumor types.

Fig. 2.

Fig. 2

Open in a new tab

Inhibition of tumor colony formation in vitro by R763/AS703569. Cells from a wide variety of solid tumor xenografts that were directly derived from human primary tumors and maintained in nude 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 EC50 value, 10× mean value, and 0.1× mean value are indicated as horizontal bars. ATCC-derived cell lines used in this assay are SF268 glioblastoma (EC50 = 1.185 μM), HT29 colon adenocarcinoma (EC50 = 0.027 μM), K562 chronic myelogenous leukemia (EC50 = 0.010 μM), A549 lung carcinoma (EC50 = 0.004 μM), L363 plasma cell leukemia/lymphoma (EC50 = 0.007 μM), RAJI Burkitt’s lymphoma (EC50 = 0.017 μM), MCF7 breast carcinoma (EC50 = 3.7 μM), MDA-MB-231 breast carcinoma (EC50 = 0.024 μM), MX1 breast carcinoma (EC50 = 0.005 μM), DU-145 prostate carcinoma (EC50 = 0.024 μM), and MRIH1579 prostate carcinoma (EC50 = 0.005 μM)

Induction of apoptosis and endoreduplication by R763/AS703569

We tested cell cycle profiles and apoptosis induction in a panel of tumor cell lines to see which phenotype (endoreduplication vs. apoptosis) becomes dominant after R763/AS703569 treatment (Table 3). In all cell lines tested except for Colo205, treatment with R763/AS703569 resulted in endoreduplication within 48 h, and these cells were arrested at 4N and 8N DNA content after the treatment. In the case of Colo205, detection of endoreduplication was very difficult because there were few live cells remaining after treatment. In H1299, DU-145, and PC-3, >8N DNA content was observed. Interestingly, Colo205, Hela, and MiaPaCa-2 cells underwent massive apoptosis following R763/AS703569 treatment and reached over 50% cell death by 48 h, while the majority of H1299, A549, PC-3, and DU-145 cells remained viable after 48 h of R763/AS703569 treatment. We further tested caspase-3 dependency of cell death after R763/AS703569 treatment with 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 positive after the treatment. DEVD-FMK inhibited R763/AS703569-induced cell death and a majority of R763/AS703569 treated cells remained annexin V and PI negative. The live cells cultured with R763/AS703569 and DEVD-FMK became larger in size. Cell cycle analysis showed that most of these live cells had 8N DNA content, suggesting that the tumor cells entered the endoreduplication cycle after apoptosis was blocked. Overall, all the tested tumor cell lines treated with R763/AS703569 entered the endoreduplication cycle, which can result from Aurora kinase B inhibition. Taken together, these tumors were clearly divided into two groups: apoptosis-dominant and apoptosis-resistant cell lines.

Table 3.

Induction of endoreduplication and apoptosis by R763/AS703569

Cell line Tissue origin R763 (μM) 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
Open in a new tab

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

Fig. 3.

Fig. 3

Open in a new tab

a Inhibition of R763/AS703569-mediated cell death by a caspase-3 inhibitor, DEVD-FMK. Colo205 cells were cultured in 50 nM R763/AS703569 containing media for 48 h with or without 50 μM DEVD-FMK. An apoptosis assay (annexin V/PI staining) and BrdU cell cycle analysis (anti BrdU antibody/PI staining) were performed to assess the effect 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 sulfoxide (DMSO) (vehicle) was used for a negative control. b Irreversible 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 incubation with R763/AS703569 free media for 24–96 h. Percentage of cells within each gate is indicated next to the gate. 0.05% DMSO (vehicle) was used for a negative control

We are particularly interested in the fate of cells arrested at 4N or 8N DNA content after R763/AS703569 treatment. A549 cells were treated with R763/AS703569 for 144 h or for 48 h followed by R763/AS703569 withdrawal for 96 h. As shown in Fig. 3b, R763/AS703569 treatment for 48 and 144 h caused 4N and 8N arrest in A549 cells and no cells in the S phase (BrdU positive) were observed. We did not observe cell cycle arrest at more than 8N DNA content in A549 cells during the 144-h treatment. After washing out R763/AS703569, a majority of A549 cells remained in an arrested state at 4N and 8N DNA content and few cells were observed in the S phase (BrdU positive) with 4N/8N DNA content. Similar results were obtained when human primary umbilical vein endothelial cells were used (data not shown). H1299 cells (p53 null) were also examined in order to check involvement of p53 in irreversible cell cycle arrest at 4N and 8N DNA content. After the 144-h treatment, a majority of the cells were in the sub-G0/G1 peak, which is indicative of apoptosis, and there were few cells in G1, S, G2/M or >8N phase of the cell cycle. After washing out R763/AS703569, a majority of the treated cells stayed BrdU negative (data not shown). These results suggested that R763/AS703569-induced 4N and 8N arrest is irreversible, and p53 may not be a major contributor to the irreversible cell cycle arrest.

In vivo efficacy of R763/AS703569

The anti-tumor activity of R763/AS703569 was examined in human pancreatic tumor (MiaPaCa-2) xenografts implanted subcutaneously in SCID mice. Clearance (Cl), volume of distribution (Vss), half-life (T 1/2), and oral bioavailability (%F) of R763/AS703569 in SCID mice are 14.5 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 treatment group/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 the initial volume. However, the tumors were surrounded by massive amounts of fibrous tissue and eosinophilic substances, suggesting that histological regression was achieved (Fig. 4c). Inhibition of tumor growth was paralleled by a reduction of histone H3 phosphorylation. Inhibition 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 final dosing (supplemental Figure 1).

Fig. 4.

Fig. 4

Open in a new tab

Potent anti-tumor activity of R763/AS703569 against human solid tumor cell lines in vivo. Mice were orally treated consecutively for 3 days with various doses of R763/AS703569, followed by a 4-day rest interval. This cycle was repeated four times (shown as arrows). In all animal studies, a 4-week treatment with R763/AS703569 was well tolerated with a minimum body weight loss at the highest dose (less than 10% in all experiments). At the end of the four cycles (including the last 4 day resting), gross and microscopic examination of different tissues from the R763/AS703569-treated mice showed lack of any significant alterations. Toxicity profiling of all tested tissues, including bone marrow and gastrointestinal tract, confirmed that there was no difference between orally treated mice and mice treated through the intra-peritoneal route. No sign of mechanism-independent toxicity of the compound was observed. a SCID mice bearing MiaPaCa-2 pancreatic cancer 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 as mean tumor volume ± standard error of measurement (SEM) (small bar) of 10 mice/group. b SCID mice bearing NCI-MDR tumors were treated 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 tumor volume ± SEM (small bar) of 10 mice/group. Two out of ten mice (the 7 mg/kg day dosing group), and seven out of ten mice (the 10 mg/kg day dosing group) showed clear reduction of tumor volume less than the original volume. The two-sample t test was applied to evaluate the data against the control. Asterisks (*, **, and ***) next to the symbols represent P values of <0.05, <0.01, and <0.001, respectively. c Histological analysis of R763/AS703569-treated tumors. MiaPaCa-2 tumors from mice treated with vehicle or R763/AS703569 20 mg/kg day for 4 weeks were harvested and stained with hematoxylin and eosin

R763/AS703569 was also tested in a slow growing and adriamycin-resistant tumor (NCI-ADR) xenograft model in nude mice (Fig. 4b). Tumor growth was significantly suppressed by the treatment with R763/AS703569 (7 and 10 mg/kg day) in comparison with the control group. Two out of ten mice in the 7 mg/kg day dosing group and seven out of ten mice in 10 mg/kg day dosing group showed clear reduction of tumor volume. Similarly, R763/AS703569 also exhibited potent anti-tumor activity in colon cancer Colo205, ovarian cancer A2780, and lung cancer LXFE 211 (Oncotest GmbH) xenograft models (supplemental Table 3).

The anti-tumor activity of R763/AS703569 was examined in MOLT-4 leukemia cells implanted i.v. in NOD/SCID mice. The total number of bone marrow cells was reduced to 5–10-fold after R763/AS703569 treatment compared to vehicle controls. As shown in Fig. 5a, R763/AS703569 treatment significantly reduced the percentages of leukemia cells (mean% ± standard deviation (SD) = 9.6 ± 9.5, n = 9, P < 0.001), while the control group had a large number of leukemia cells in the bone marrow (mean% ± SD = 61.7 ± 17.7, n = 9). In three out of nine treated mice, leukemic cells were not detected (<0.1%). Hematopoietic cells in bone marrow had a normal cell cycle profile even though leukemia cells were largely diminished. A dose-dependent increase in lifespan was observed in animals treated intermittently at doses of 7.5, 10 and 15 mg/kg R763/AS703569 (data not shown).

Fig. 5.

Fig. 5

Open in a new tab

Potent anti-tumor activity of R763/AS703569 against human leukemia cell lines in vivo. We used an intermittent schedule of administration as described in materials and methods. a MOLT-4 leukemia cells 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 marrow cells from NOD/SCID mice bearing Molt4 leukemia cells were harvested after four cycles of the R763/AS703569 treatment. Leukemic cells were identified as human CD45 positive and human CD71 positive cells with a FACScalibur. Data are presented as % leukemic cells in individual mice and mean ± SD (n = 9). The two-sample t test was applied for statistical significance. b MV4-11 leukemia cells, which carry a FLT3 ITD mutation, were injected subcutaneously into NOD/SCID mice. The mice bearing MV4-11 leukemia cells were treated with vehicle control (rhombus, solid line), R763/AS703569 7.5 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 repeated four times (shown as arrows). Data are presented as mean tumor volume ± SEM (small bar) (n = 10/group). c Survival of NOD/SCID mice 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 an animal was ≥2,000 mm3, if the tumor ulcerated, or if the mice became moribund. d Reduction of phosphorylated histone H3 expression in MV4-11 xenografts by R763/AS703569. Phospho-histone H3 positive cells were counted and data are presented as the number of phospho-histone H3 positive cells per field (20× magnification) (n = 12 fields per tumor, 3 tumors/group). Hatched bars, dotted bars, and open bars represents mean ± SD of vehicle, R763/AS703569 7.5 mg/kg treated and R763/AS703569 15 mg/kg day treated mice, respectively. The number of phospho-histone H3 positive cells per field in the treated groups of each time point was compared against the vehicle control of the same time point. Asterisks (*, **, and ***) on top of the bars represent P values of <0.05, <0.01, and <0.001, respectively

A myeloid leukemia line (MV4-11), which carries a FLT3 ITD mutation, was examined in a xenograft study. R763/AS703569 produced pronounced dose-dependent anti-tumor activity (Fig. 5b). P values of the vehicle group versus 7.5, 10, 12.5 and 20 mg/kg day of the R763/AS703569-treated groups were <0.001. Partial regressions were noted in 67, 92, and 83% of the animals treated at dose levels of 10, 12.5, and 20 mg/kg, respectively, and 17% of the animals demonstrated undetectable tumor level at 20 mg/kg day dosing. R763/AS703569 treatment was stopped at day 28 and survival of the treated mice was examined (Fig. 5c). Increase in lifespan was observed in all R763/AS703569-treated groups, while all mice in the control groups died within 35 days. P values of the vehicle group versus 7.5, 10, 12.5 and 20 mg/kg day of the R763/AS703569 treated groups were <0.0001. Supporting these results, inhibition of tumor growth was paralleled by a reduction in histone H3 phosphorylation in the xenografts (Fig. 5d). At 15 mg/kg day dosing, histone H3 phosphorylation was still suppressed 48 h after the final dosing event. In addition, complete inhibition of FLT3 tyrosine-phosphorylation was seen up to 48 h after the final dosing in tumor lysates from mice treated with 15 mg/kg R763/AS703569, which is consistent with reduction of histone H3 phosphorylation (data not shown).

Discussion

In this paper, we report the development of a novel Aurora kinase inhibitor R763/AS703569 through an image-based phenotypic screen. R763/AS703569 exhibits a potent anti-proliferative effect in a wide range of tumor cell types with unique phenotypic changes such as enlarged cell size, irreversible endoreduplication, and apoptosis. Inhibition of tumor growth was demonstrated in a broad panel of in vivo xenograft models. Several solid tumors, including pancreatic cancers and small cell lung carcinomas, were particularly sensitive to R763/AS703569. In addition, R763/AS703569 was extremely effective in a FLT3 ITD mutant leukemia cell line, MV4-11. R763/AS703569 could be a potent anti-tumor agent for the treatment of a wide variety of cancers.

Accessibility of small molecule inhibitors to their active sites in vitro and in vivo may not be identical because of potential activators or associated molecules in cells. In the case of Aurora kinases, there are crucial associated molecules [TPX2 (Kufer et al. 2002) and Ajuba (Hirota et al. 2003) for Aurora kinase A, and the inner centromere protein, INCENP (Adams et al. 2000; Kaitna et al. 2000) and Survivin (Wheatley et al. 2001) for Aurora kinase B], which determine proper localization and activation of Aurora kinases. According to the structures of the complexes of Aurora kinase A with TPX2 (Bayliss et al. 2003) and Aurora kinase B with INCENP (Sessa et al. 2005), activation of Aurora kinases A and B requires conformational changes induced by TPX2 and INCENP. For this reason, tracking the activity of small molecule inhibitors using only in vitro kinase assays may be misleading. As an illustration, during our drug development efforts, we identified several compounds that potently inhibit Aurora kinases in biochemical assays but not in the cell, even though the compounds potently inhibited other kinases in the cell (data not shown). Consequently, we used our high content screening assay to both quantify compound inhibition of cell cycle progression and verify phenotypically that the mechanism of action was mediated through Aurora kinases. This phenotype-based approach was complemented with traditional biochemical and cell signaling approaches. The activity of the inhibitors identified using cell-based approaches may be already optimized to inhibit the targets in the proper intracellular environment. Thus, cell-based screening approaches could have a great advantage over conventional biochemical screening, although all hits from cell-based screens require detailed deconvolution studies to identify the precise target(s) or mode through which they are acting.

R763/AS703569 induced endoreduplication and apoptosis to various degrees in p53 wild-type and deficient tumors, indicating that induction of endoreduplication and apoptosis by R763/AS703569 is independent of the p53 status. Interestingly, p53 deficient cell lines, as well as Hela cells in which p53 is inactivated by E6 expression, had more pronounced phenotypes than p53 wild-type tumor cells. To further investigate the contribution of p53 to observed phenotypes, we treated p53 wild-type cell lines A549, 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 A549 tumor cells resulted in an increased number of cells showing endoreduplication following R763/AS703569 treatment. In contrast, inactivation of p53 in HCT-116 and U2OS did not further increase the number of endoreduplicated cells after R763/AS703569 treatment, although both parental and p53 inactivated tumor lines showed marked endoreduplication. These results indicate that p53 may not be critical for induction and enhancement of endoreduplication in some tumors. Gizatullin et al. (2006) reported that endoreduplication and apoptosis were regulated by a p53 target gene, p21waf1/cip1 after inhibition of Aurora kinases by VX-680 and that the cell lines lacking efficient activation of p21, such as U2OS, showed endoreduplication and apoptosis but VX-680 did not enhance the phenotypes, even when p53 was inactivated. This indicates that the status of cell cycle inhibitors such as p21waf1/cip1 may play a role in induction of endoreduplication and apoptosis by R763/AS703569, although it is still not clear if other cell cycle inhibitors are also responsible for the phenotype.

Colo205, which demonstrated an apoptosis-dominant phenotype after R763/AS703569 treatment, showed enhanced endoreduplication with R763/AS703569 when caspase-3 was inhibited. Expression of apoptosis regulators such as Bcl-2 family or inhibitors of apoptosis (IAPs) might be involved in the apoptosis sensitivity. In addition, we investigated the fate of the cells arrested at 4N or 8N DNA content after R763/AS703569 treatment. We found that 4N and 8N cell cycle arrest induced by R763/AS703569 is irreversible. Interestingly, the irreversibility is also observed in p53 null H1299 cells, suggesting that a p53-independent mechanism might be involved in this process. Irreversible arrest after endoreduplication may be a desirable feature for anti-cancer agents.

Expression levels of drug targets or multidrug resistance genes can establish sensitivity of tumors to chemotherapeutics. However, we did not observe any clear correlation between the mRNA expression level of Aurora kinases and sensitivity of tumors to R763/AS703569 among the tumor cell lines tested (data not shown). This needs to be further examined 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 is supported by the efficacy in a drug-resistant NCI-ADR tumor xenograft model. Expression levels of Aurora kinases or multidrug resistance genes may not be a critical factor for sensitivity. Since Aurora kinase inhibitors can suppress tumor cell growth when the cells enter the mitotic phase, rapidly proliferating cells should be more sensitive to R763/AS703569. To our surprise, slow growing and drug-resistant NCI-ADR tumor models also demonstrated significant tumor regression after R763/AS703569 treatment. R763/AS703569 seems to target both slow and rapidly dividing tumor cells in vivo. Although the mechanism of tumor sensitivity to the compound is not clear at this moment, how would patients be selected for treatment with this compound? The status of cell cycle regulators, proapoptotic genes, or anti-apoptotic molecules in the tumors might be used as markers for sensitivity. However, this question needs to be answered through further analyses regarding tumor sensitivity to R763/AS703569.

Inhibition of Aurora kinases A and B resulted in phenotypes identical to inactivation of Aurora kinase B: the formation of multinucleated cells due to the failure of cytokinesis and down-regulation of the histone H3 serine 10 phosphorylation followed by mitotic cell death (Ditchfield et al. 2003; Fancelli et al. 2005; Hauf et al. 2003; Jung et al. 2006; Harrington et al. 2004). Inactivation of Aurora kinase B bypasses requirement of Aurora kinase A, because activation of the mitotic checkpoint induced by inhibition of Aurora kinase A is dependent on Aurora kinase B activity (Yang et al. 2005). Most compounds reported today are inhibitors of all three Aurora kinases, which show phenotypes due to inhibition of Aurora kinase B. Since the phenotypes are consistent with inhibition of Aurora kinase B, down-regulation of the histone H3 serine 10 phosphorylation 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 been developed (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. In sharp contrast to pan-Aurora kinase and Aurora kinase B inhibitors, ML8054 induced spindle defects, G2/M accumulation without inducing endoreduplication, and massive mitotic cell death (Manfredi et al. 2007; Hoar et al. 2007), supporting different roles of both kinases in mitosis. Because of the difference, there are some arguments about which Aurora kinase would be a preferred target. Aurora kinase B could be the preferred target because its inhibition leads to mitotic catastrophe and significant suppression of tumor growth in vivo (Wilkinson et al. 2007). On the other hand, over-expression of Aurora kinase A is seen in many human cancers (Marumoto et al. 2005) and selective inhibition of Aurora kinase A potently induces apoptosis (Hata et al. 2005; Manfredi et al. 2007). Aurora kinase A on its own could also be an attractive target. However, an Aurora kinase A and B inhibitor, VX-680, failed to kill cells expressing the drug-resistant Aurora kinase B mutant, suggesting inhibition of Aurora kinase A may not be sufficient (Cochran 2008; Girdler et al. 2008). The answer regarding which Aurora kinase could be a better target may emerge shortly since many of Aurora kinase inhibitors are currently being evaluated in clinical studies.

It might be beneficial for R763/AS703569 to inhibit other kinases in addition to Aurora kinases. Selective inhibitors might be less toxic but may also lead to drug resistance more easily, while broad kinase inhibitors might be more toxic but may be more resistant to drug-resistant mutants or have more potency from inhibiting other targets. While Aurora kinase inhibitors suppress tumor cell growth through the mitotic phase, inhibitors of receptor tyrosine kinases are effective by targeting different phases of the cell cycle such as G0/G1 to S transition. One could speculate that combination therapy with receptor tyrosine kinase and Aurora kinase inhibitors would be useful for the treatment of several cancers. R763/AS703569 inhibited FLT3 in addition to Aurora kinases in vitro and in vivo and demonstrated an excellent efficacy in xenograft studies against the FLT3 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-2 tumors, suggesting that inhibiting both FLT3 and Aurora kinases may have a much stronger impact on tumor cell growth and survival by targeting multiple phases of the cell cycle. Further experiments should be performed using highly selective inhibitors of FLT3 or Aurora kinases in order to clarify if inhibition of both FLT3 and Aurora kinases is beneficial.

R763/AS703569 also potently inhibits VEGFR2 in cells. VEGFR2 is a growth factor receptor tyrosine kinase, which transmits an important signal for angiogenesis. Release of its ligand, VEGF, causes an endothelial cell to survive, migrate, or differentiate. Bevacizumab, a monoclonal antibody against VEGF, and Sunitinib, a small molecule inhibitor of VEGFR2, demonstrated the usefulness of targeting VEGFR2 in colorectal (Hurwitz et al. 2004), renal (Escudier et al. 2007; Motzer et al. 2006; Yang et al. 2003), and lung cancers (Sandler et al. 2006). Therefore, R763/AS703569 is expected to have a potent anti-tumor effect partly due to inhibition of angiogenesis, which might be a bonus for the treatment of cancer. Importantly, bevacizumab has been associated with hypertension, proteinuria, and some other complications (Hurwitz et al. 2004; Yang et al. 2003; Kabbinavar et al. 2005a, b; Scappaticci et al. 2005; Chu et al. 2007). Having anti-VEGFR2 activity could be potentially useful, but toxicity needs to be carefully monitored during the treatment.

In conclusion, we have identified a potent Aurora kinase inhibitor, R763/AS703569, through an image-based high content screen. R763/AS703569 has a significant anti-proliferative effect in a wide range of tumor cell types with unique phenotypic changes such as enlarged cell size, endoreduplication, and apoptosis. The phenotypes induced by R763/AS703569 are consistent with inhibition of Aurora kinase B. Tumor regression has been demonstrated in some xenograft models with a well-tolerated dose of R763/AS703569. Our studies indicate that inhibition of Aurora kinases is a feasible approach toward many types of cancers and R763/AS703569 should be useful in the treatment of the disease. Currently, phase I clinical studies with R763/AS703569 are underway.

Electronic supplementary material

Below is the link to the electronic supplementary material.

432_2009_641_MOESM1_ESM.tif (1.5MB, tif)

Supplemental Figure 1. Reduction of phosphorylation of histone H3 (pHH3) in MIA-PaCa2 tumor tissues by R763/AS703569. Mice were treated with 20 mg/kg day and tumors were harvested for immunohistochemical analysis as shown in “Methods”. Total number of pHH3 positive nuclei was counted manually. The data are shown as mean ± SD of three tumors per time point after final dosing of R763/AS703569 (TIFF 1,520 kb)

432_2009_641_MOESM2_ESM.tif (1.5MB, tif)

Supplemental Table 1. Activity of R763/AS703569 against a panel of protein concentration of 100 nM. The selectivity of the compound was tested in a series of in vitro biochemical kinase assays using a selected panel of protein kinases (Upstate Biotechnology Inc., Waltham, MA). Based on the IC50 value of R763/AS703569 against Aurora kinase A in vitro (4 nM), a 25-fold higher concentration of the compound was chosen for the assays. In vitro biochemical kinase assay for VEGFR2 was done separately (TIFF 1,520 kb)

432_2009_641_MOESM3_ESM.tif (1.5MB, tif)

Supplemental Table 2. The effect of R763/AS703569 on variety of cell activities. The effect of R763/AS703569 on a variety of cellular activities was analyzed. EC50s were generated by curve fitting of activities using Matlab version 6.5 (MathWorks Inc., MA). (1) For the mast cell activation assays, cultured human primary mast cells were sensitized with human IgE (Cortex Biochem Inc., San Leandro, CA) followed by incubation with Rabbit anti-human IgE antibody (Bethyl Laboratories Inc., Montgomery, TX). Tryptase release was quantified by cleavage of the synthetic peptide substrate, Z-Ala-Lys-Arg-amidomethylcoumarin (2 trifluoroacetic acid) (Enzyme Systems Product Inc., Livermore, CA). (2) Jurkat cells were pre-incubated at 37°C for 1 h and stimulated with anti-T cell receptor beta chain monoclonal antibody (C305) (BD Biosciences, Mansfield, MA) or Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich Chemicals) for 20 h. The cells were stained by Allophycocyanin (APC)-conjugated anti-CD69 antibody (Caltag Laboratories Inc., Burlingame, CA). The geometric mean of APC fluorescence was measured on a FACScalibur (BD Biosciences, San Jose, CA). (3) A549 cells were pre-incubated with R763/AS703569 for 1 h and cultured with tumor necrosis factor alpha (TNFα) (Peprotech Inc., Rocky Hill, NJ), IL-1β (Peprotech. Inc.) or Interferon gamma (IFNγ) (Peprotech. Inc.) for 24 h. The cells were resuspended in PBS and stained with an APC-conjugated monoclonal mouse anti-human intercellular adhesion molecule 1 (ICAM-1, CD54) antibody (BD Biosciences). Surface ICAM-1 expression was analyzed by flow cytometry. The geometric means were used for EC50 estimation. (4) Hela cells expressing Luciferase-fused inhibitor of kappa light chain gene enhancer in B cells (IkB) protein were plated in a 96-well tissue culture plate 1 day before the assay. The cells were pre-incubated with R763/AS703569 for 2 h and stimulated with 2 ng/mL IL-1β for 30 min at 37°C. Following stimulation, the amount of the luciferase reporter was measured using the luciferase assay system (Promega Corporation, Madison, WI) (TIFF 1,520 kb)

432_2009_641_MOESM4_ESM.tif (1.5MB, tif)

Supplemental Table 3. Potent anti-tumor activity of R763/AS703569 against human solid tumor cell lines in vivo. The studies were performed as described in “Methods”. Briefly, MIA-PaCa2, Colo205, A2780 and LXFE 211 tumor cells were implanted in immunocompromised mice. The studies were initiated when tumors reached to a mean size of 100–300 mm3. Various doses of R763/AS703569 was administered once daily orally (PO) or intraperitoneally (IP). 2–4 cycles of the intermittent schedules were used as shown in the table. %T/C values were calculated using the following formula (Tx − Ti) × 100/(Tcx − Tci). Tx means tumor volume of the treated group on day x, Ti means initial tumor volume of the treated group, Tcx means tumor volume of the control group on day x, Tci means initial tumor volume of the control group. X is 28 for MIA PaCa2 and Colo205, and 15 for A2780 and LXFE 211 (TIFF 1,520 kb)

Acknowledgments

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

Footnotes

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

References

  1. Adams RR, Wheatley SP, Gouldsworthy AM, Kandels-Lewis SE, Carmena M, Smythe C et al (2000) INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow. Curr Biol 10(17):1075–1078 [DOI] [PubMed] [Google Scholar]
  2. Araki K, Nozaki K, Ueba T, Tatsuka M, Hashimoto N (2004) High expression of Aurora-B/Aurora and Ipll-like midbody-associated protein (AIM-1) in astrocytomas. J Neurooncol 67(1–2):53–64 [DOI] [PubMed] [Google Scholar]
  3. Barabasz A, Foley B, Otto JC, Scott A, Rice J (2006) The use of high-content screening for the discovery and characterization of compounds that modulate mitotic index and cell cycle progression by differing mechanisms of action. Assay Drug Dev Technol 4(2):153–163 [DOI] [PubMed] [Google Scholar]
  4. Bayliss R, Sardon T, Vernos I, Conti E (2003) Structural basis of Aurora-A activation by TPX2 at the mitotic spindle. Mol Cell 12(4):851–862 [DOI] [PubMed] [Google Scholar]
  5. Bischoff JR, Anderson L, Zhu Y, Mossie K, Ng L, Souza B et al (1998) A homologue of Drosophila aurora kinase is oncogenic and amplified in human colorectal cancers. EMBO J 17(11):3052–3065 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buschhorn HM, Klein RR, Chambers SM, Hardy MC, Green S, Bearss D et al (2005) Aurora-A over-expression in high-grade PIN lesions and prostate cancer. Prostate 64(4):341–346 [DOI] [PubMed] [Google Scholar]
  7. Carmena M, Earnshaw WC (2003) The cellular geography of aurora kinases. Nat Rev Mol Cell Biol 4(11):842–854 [DOI] [PubMed] [Google Scholar]
  8. Carpinelli P, Ceruti R, Giorgini ML, Cappella P, Gianellini L, Croci V et al (2007) PHA-739358, a potent inhibitor of Aurora kinases with a selective target inhibition profile relevant to cancer. Mol Cancer Ther 6(12 Pt 1):3158–3168 [DOI] [PubMed] [Google Scholar]
  9. Chan F, Sun C, Perumal M, Nguyen QD, Bavetsias V, McDonald E et al (2007) Mechanism of action of the Aurora kinase inhibitor CCT129202 and in vivo quantification of biological activity. Mol Cancer Ther 6(12 Pt 1):3147–3157 [DOI] [PubMed] [Google Scholar]
  10. Chieffi P, Troncone G, Caleo A, Libertini S, Linardopoulos S, Tramontano D et al (2004) Aurora B expression in normal testis and seminomas. J Endocrinol 181(2):263–270 [DOI] [PubMed] [Google Scholar]
  11. Chieffi P, Cozzolino L, Kisslinger A, Libertini S, Staibano S, Mansueto G et al (2006) Aurora B expression directly correlates with prostate cancer malignancy and influence prostate cell proliferation. Prostate 66(3):326–333 [DOI] [PubMed] [Google Scholar]
  12. Chu TF, Rupnick MA, Kerkela R, Dallabrida SM, Zurakowski D, Nguyen L et al (2007) Cardiotoxicity associated with tyrosine kinase inhibitor sunitinib. Lancet 370(9604):2011–2019 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cochran AG (2008) Aurora A: target invalidated? Chem Biol 15(6):525–526 [DOI] [PubMed] [Google Scholar]
  14. Ditchfield C, Johnson VL, Tighe A, Ellston R, Haworth C, Johnson T et al (2003) Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores. J Cell Biol 161(2):267–280 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Emanuel S, Rugg CA, Gruninger RH, Lin R, Fuentes-Pesquera A, Connolly PJ et al (2005) The in vitro and in vivo effects of JNJ-7706621: a dual inhibitor of cyclin-dependent kinases and aurora kinases. Cancer Res 65(19):9038–9046 [DOI] [PubMed] [Google Scholar]
  16. Escudier B, Koralewski P, Pluzanska A, Ravaud A, Bracarda S, Szczylik C et al (2007) A randomized, controlled, double-blind phase III study (AVOREN) of bevacizumab/interferon-α2a vs placebo/interferon-α2a as first-line therapy in metastatic renal cell carcinoma. In: Annual meeting of the American society of clinical oncology 2007, Chicago, IL, 1–5 June 2007
  17. Fancelli D, Berta D, Bindi S, Cameron A, Cappella P, Carpinelli P et al (2005) Potent and selective Aurora inhibitors identified by the expansion of a novel scaffold for protein kinase inhibition. J Med Chem 48(8):3080–3084 [DOI] [PubMed] [Google Scholar]
  18. Girdler F, Sessa F, Patercoli S, Villa F, Musacchio A, Taylor S (2008) Molecular basis of drug resistance in aurora kinases. Chem Biol 15(6):552–562 [DOI] [PubMed] [Google Scholar]
  19. Gizatullin F, Yao Y, Kung V, Harding MW, Loda M, Shapiro GI (2006) The Aurora kinase inhibitor VX-680 induces endoreduplication and apoptosis preferentially in cells with compromised p53-dependent postmitotic checkpoint function. Cancer Res 66(15):7668–7677 [DOI] [PubMed] [Google Scholar]
  20. Gritsko TM, Coppola D, Paciga JE, Yang L, Sun M, Shelley SA et al (2003) Activation and overexpression of centrosome kinase BTAK/Aurora-A in human ovarian cancer. Clin Cancer Res 9(4):1420–1426 [PubMed] [Google Scholar]
  21. Hamburger AW, Salmon SE (1977) Primary bioassay of human tumor stem cells. Science 197(4302):461–463 [DOI] [PubMed] [Google Scholar]
  22. Harrington EA, Bebbington D, Moore J, Rasmussen RK, Ajose-Adeogun AO, Nakayama T et al (2004) VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nat Med 10(3):262–267 [DOI] [PubMed] [Google Scholar]
  23. Haskell C (2001) Cancer treatment, 5th edn. Elsevier, Philadelphia [Google Scholar]
  24. Hata T, Furukawa T, Sunamura M, Egawa S, Motoi F, Ohmura N et al (2005) RNA interference targeting aurora kinase A suppresses tumor growth and enhances the taxane chemosensitivity in human pancreatic cancer cells. Cancer Res 65(7):2899–2905 [DOI] [PubMed] [Google Scholar]
  25. Hauf S, Cole RW, LaTerra S, Zimmer C, Schnapp G, Walter R et al (2003) The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint. J Cell Biol 161(2):281–294 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Heron NM, Anderson M, Blowers DP, Breed J, Eden JM, Green S et al (2006) SAR and inhibitor complex structure determination of a novel class of potent and specific Aurora kinase inhibitors. Bioorg Med Chem Lett 16(5):1320–1323 [DOI] [PubMed] [Google Scholar]
  27. Hirota T, Kunitoku N, Sasayama T, Marumoto T, Zhang D, Nitta M et al (2003) Aurora-A and an interacting activator, the LIM protein Ajuba, are required for mitotic commitment in human cells. Cell 114(5):585–598 [DOI] [PubMed] [Google Scholar]
  28. Hoar K, Chakravarty A, Rabino C, Wysong D, Bowman D, Roy N et al (2007) MLN8054, a small-molecule inhibitor of Aurora A, causes spindle pole and chromosome congression defects leading to aneuploidy. Mol Cell Biol 27(12):4513–4525 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hoque A, Carter J, Xia W, Hung MC, Sahin AA, Sen S et al (2003) Loss of aurora A/STK15/BTAK overexpression correlates with transition of in situ to invasive ductal carcinoma of the breast. Cancer Epidemiol Biomarkers Prev 12(12):1518–1522 [PubMed] [Google Scholar]
  30. Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W et al (2004) Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 350(23):2335–2342 [DOI] [PubMed] [Google Scholar]
  31. Hutcheson, Matthew C (1995) Trimmed resistant weighted scatterplot smooth. Thesis (MS), Department of Statistics, Cornell University, Ithaca, NY
  32. Jeng YM, Peng SY, Lin CY, Hsu HC (2004) Overexpression and amplification of Aurora-A in hepatocellular carcinoma. Clin Cancer Res 10(6):2065–2071 [DOI] [PubMed] [Google Scholar]
  33. Jung FH, Pasquet G, Lambert-van der Brempt C, Lohmann JJ, Warin N, Renaud F et al (2006) Discovery of novel and potent thiazoloquinazolines as selective Aurora A and B kinase inhibitors. J Med Chem 49(3):955–970 [DOI] [PubMed] [Google Scholar]
  34. Kabbinavar FF, Hambleton J, Mass RD, Hurwitz HI, Bergsland E, Sarkar S (2005a) Combined analysis of efficacy: the addition of bevacizumab to fluorouracil/leucovorin improves survival for patients with metastatic colorectal cancer. J Clin Oncol 23(16):3706–3712 [DOI] [PubMed] [Google Scholar]
  35. Kabbinavar FF, Schulz J, McCleod M, Patel T, Hamm JT, Hecht JR et al (2005b) Addition of bevacizumab to bolus fluorouracil and leucovorin in first-line metastatic colorectal cancer: results of a randomized phase II trial. J Clin Oncol 23(16):3697–3705 [DOI] [PubMed] [Google Scholar]
  36. Kaitna S, Mendoza M, Jantsch-Plunger V, Glotzer M (2000) Incenp and an aurora-like kinase form a complex essential for chromosome segregation and efficient completion of cytokinesis. Curr Biol 10(19):1172–1181 [DOI] [PubMed] [Google Scholar]
  37. Kufer TA, Sillje HH, Korner R, Gruss OJ, Meraldi P, Nigg EA (2002) Human TPX2 is required for targeting Aurora-A kinase to the spindle. J Cell Biol 158(4):617–623 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Kurai M, Shiozawa T, Shih HC, Miyamoto T, Feng YZ, Kashima H et al (2005) Expression of Aurora kinases A and B in normal, hyperplastic, and malignant human endometrium: Aurora B as a predictor for poor prognosis in endometrial carcinoma. Hum Pathol 36(12):1281–1288 [DOI] [PubMed] [Google Scholar]
  39. Lee EC, Frolov A, Li R, Ayala G, Greenberg NM (2006) Targeting aurora kinases for the treatment of prostate cancer. Cancer Res 66(10):4996–5002 [DOI] [PubMed] [Google Scholar]
  40. Manfredi MG, Ecsedy JA, Meetze KA, Balani SK, Burenkova O, Chen W et al (2007) Antitumor activity of MLN8054, an orally active small-molecule inhibitor of Aurora A kinase. Proc Natl Acad Sci USA 104(10):4106–4111 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Marumoto T, Honda S, Hara T, Nitta M, Hirota T, Kohmura E et al (2003) Aurora-A kinase maintains the fidelity of early and late mitotic events in Hela cells. J Biol Chem 278(51):51786–51795 [DOI] [PubMed] [Google Scholar]
  42. Marumoto T, Zhang D, Saya H (2005) Aurora-A—a guardian of poles. Nat Rev Cancer 5(1):42–50 [DOI] [PubMed] [Google Scholar]
  43. Miyoshi Y, Iwao K, Egawa C, Noguchi S (2001) Association of centrosomal kinase STK15/BTAK mRNA expression with chromosomal instability in human breast cancers. Int J Cancer 92(3):370–373 [DOI] [PubMed] [Google Scholar]
  44. Moreno-Bueno G, Sanchez-Estevez C, Cassia R, Rodriguez-Perales S, Diaz-Uriarte R, Dominguez O et al (2003) Differential gene expression profile in endometrioid and nonendometrioid endometrial carcinoma: STK15 is frequently overexpressed and amplified in nonendometrioid carcinomas. Cancer Res 63(18):5697–5702 [PubMed] [Google Scholar]
  45. Motzer RJ, Michaelson MD, Redman BG, Hudes GR, Wilding G, Figlin RA et al (2006) Activity of SU11248, a multitargeted inhibitor of vascular endothelial growth factor receptor and platelet-derived growth factor receptor, in patients with metastatic renal cell carcinoma. J Clin Oncol 24(1):16–24 [DOI] [PubMed] [Google Scholar]
  46. Neben K, Korshunov A, Benner A, Wrobel G, Hahn M, Kokocinski F et al (2004) Microarray-based screening for molecular markers in medulloblastoma revealed STK15 as independent predictor for survival. Cancer Res 64(9):3103–3111 [DOI] [PubMed] [Google Scholar]
  47. Rojanala S, Han H, Munoz RM, Browne W, Nagle R, Von Hoff DD et al (2004) The mitotic serine threonine kinase, Aurora-2, is a potential target for drug development in human pancreatic cancer. Mol Cancer Ther 3(4):451–457 [PubMed] [Google Scholar]
  48. Royce ME, Xia W, Sahin AA, Katayama H, Johnston DA, Hortobagyi G et al (2004) STK15/Aurora-A expression in primary breast tumors is correlated with nuclear grade but not with prognosis. Cancer 100(1):12–19 [DOI] [PubMed] [Google Scholar]
  49. Sakakura C, Hagiwara A, Yasuoka R, Fujita Y, Nakanishi M, Masuda K et al (2001) Tumour-amplified kinase BTAK is amplified and overexpressed in gastric cancers with possible involvement in aneuploid formation. Br J Cancer 84(6):824–831 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sandler A, Gray R, Perry MC, Brahmer J, Schiller JH, Dowlati A et al (2006) Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med 355(24):2542–2550 [DOI] [PubMed] [Google Scholar]
  51. Scappaticci FA, Fehrenbacher L, Cartwright T, Hainsworth JD, Heim W, Berlin J et al (2005) Surgical wound healing complications in metastatic colorectal cancer patients treated with bevacizumab. J Surg Oncol 91(3):173–180 [DOI] [PubMed] [Google Scholar]
  52. Sen S, Zhou H, White RA (1997) A putative serine/threonine kinase encoding gene BTAK on chromosome 20q13 is amplified and overexpressed in human breast cancer cell lines. Oncogene 14(18):2195–2200 [DOI] [PubMed] [Google Scholar]
  53. Sen S, Zhou H, Zhang RD, Yoon DS, Vakar-Lopez F, Ito S et al (2002) Amplification/overexpression of a mitotic kinase gene in human bladder cancer. J Natl Cancer Inst 94(17):1320–1329 [DOI] [PubMed] [Google Scholar]
  54. Sessa F, Mapelli M, Ciferri C, Tarricone C, Areces LB, Schneider TR et al (2005) Mechanism of Aurora B activation by INCENP and inhibition by hesperadin. Mol Cell 18(3):379–391 [DOI] [PubMed] [Google Scholar]
  55. Smith SL, Bowers NL, Betticher DC, Gautschi O, Ratschiller D, Hoban PR et al (2005) Overexpression of aurora B kinase (AURKB) in primary non-small cell lung carcinoma is frequent, generally driven from one allele, and correlates with the level of genetic instability. Br J Cancer 93(6):719–729 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Soncini C, Carpinelli P, Gianellini L, Fancelli D, Vianello P, Rusconi L et al (2006) PHA-680632, a novel Aurora kinase inhibitor with potent antitumoral activity. Clin Cancer Res 12(13):4080–4089 [DOI] [PubMed] [Google Scholar]
  57. Sorrentino R, Libertini S, Pallante PL, Troncone G, Palombini L, Bavetsias V et al (2005) Aurora B overexpression associates with the thyroid carcinoma undifferentiated phenotype and is required for thyroid carcinoma cell proliferation. J Clin Endocrinol Metab 90(2):928–935 [DOI] [PubMed] [Google Scholar]
  58. Takahashi T, Futamura M, Yoshimi N, Sano J, Katada M, Takagi Y et al (2000) Centrosomal kinases, HsAIRK1 and HsAIRK3, are overexpressed in primary colorectal cancers. Jpn J Cancer Res 91(10):1007–1014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Tanaka T, Kimura M, Matsunaga K, Fukada D, Mori H, Okano Y (1999) Centrosomal kinase AIK1 is overexpressed in invasive ductal carcinoma of the breast. Cancer Res 59(9):2041–2044 [PubMed] [Google Scholar]
  60. Tanaka E, Hashimoto Y, Ito T, Okumura T, Kan T, Watanabe G et al (2005) The clinical significance of Aurora-A/STK15/BTAK expression in human esophageal squamous cell carcinoma. Clin Cancer Res 11(5):1827–1834 [DOI] [PubMed] [Google Scholar]
  61. Tanner MM, Grenman S, Koul A, Johannsson O, Meltzer P, Pejovic T et al (2000) Frequent amplification of chromosomal region 20q12–q13 in ovarian cancer. Clin Cancer Res 6(5):1833–1839 [PubMed] [Google Scholar]
  62. Tatsuka M, Katayama H, Ota T, Tanaka T, Odashima S, Suzuki F et al (1998) Multinuclearity and increased ploidy caused by overexpression of the aurora- and Ipl1-like midbody-associated protein mitotic kinase in human cancer cells. Cancer Res 58(21):4811–4816 [PubMed] [Google Scholar]
  63. Terada Y, Tatsuka M, Suzuki F, Yasuda Y, Fujita S, Otsu M (1998) AIM-1: a mammalian midbody-associated protein required for cytokinesis. EMBO J 17(3):667–676 [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Warner SL, Bashyam S, Vankayalapati H, Bearss DJ, Han H, Mahadevan D et al (2006) Identification of a lead small-molecule inhibitor of the Aurora kinases using a structure-assisted, fragment-based approach. Mol Cancer Ther 5(7):1764–1773 [DOI] [PubMed] [Google Scholar]
  65. Watanabe T, Imoto I, Katahira T, Hirasawa A, Ishiwata I, Emi M et al (2002) Differentially regulated genes as putative targets of amplifications at 20q in ovarian cancers. Jpn J Cancer Res 93(10):1114–1122 [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Wheatley SP, Carvalho A, Vagnarelli P, Earnshaw WC (2001) INCENP is required for proper targeting of Survivin to the centromeres and the anaphase spindle during mitosis. Curr Biol 11(11):886–890 [DOI] [PubMed] [Google Scholar]
  67. Wilkinson RW, Odedra R, Heaton SP, Wedge SR, Keen NJ, Crafter C et al (2007) AZD1152, a selective inhibitor of Aurora B kinase, inhibits human tumor xenograft growth by inducing apoptosis. Clin Cancer Res 13(12):3682–3688 [DOI] [PubMed] [Google Scholar]
  68. Yakushijin Y, Hamada M, Yasukawa M (2004) The expression of the aurora-A gene and its significance with tumorigenesis in non-Hodgkin’s lymphoma. Leuk Lymphoma 45(9):1741–1746 [DOI] [PubMed] [Google Scholar]
  69. Yang JC, Haworth L, Sherry RM, Hwu P, Schwartzentruber DJ, Topalian SL et al (2003) A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med 349(5):427–434 [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Yang H, Burke T, Dempsey J, Diaz B, Collins E, Toth J et al (2005) Mitotic requirement for aurora A kinase is bypassed in the absence of aurora B kinase. FEBS Lett 579(16):3385–3391 [DOI] [PubMed] [Google Scholar]
  71. Zhou J, Giannakakou P (2005) Targeting microtubules for cancer chemotherapy. Curr Med Chem Anticancer Agents 5(1):65–71 [DOI] [PubMed] [Google Scholar]
  72. Zhou H, Kuang J, Zhong L, Kuo WL, Gray JW, Sahin A et al (1998) Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation. Nat Genet 20(2):189–193 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

432_2009_641_MOESM1_ESM.tif (1.5MB, tif)

Supplemental Figure 1. Reduction of phosphorylation of histone H3 (pHH3) in MIA-PaCa2 tumor tissues by R763/AS703569. Mice were treated with 20 mg/kg day and tumors were harvested for immunohistochemical analysis as shown in “Methods”. Total number of pHH3 positive nuclei was counted manually. The data are shown as mean ± SD of three tumors per time point after final dosing of R763/AS703569 (TIFF 1,520 kb)

432_2009_641_MOESM2_ESM.tif (1.5MB, tif)

Supplemental Table 1. Activity of R763/AS703569 against a panel of protein concentration of 100 nM. The selectivity of the compound was tested in a series of in vitro biochemical kinase assays using a selected panel of protein kinases (Upstate Biotechnology Inc., Waltham, MA). Based on the IC50 value of R763/AS703569 against Aurora kinase A in vitro (4 nM), a 25-fold higher concentration of the compound was chosen for the assays. In vitro biochemical kinase assay for VEGFR2 was done separately (TIFF 1,520 kb)

432_2009_641_MOESM3_ESM.tif (1.5MB, tif)

Supplemental Table 2. The effect of R763/AS703569 on variety of cell activities. The effect of R763/AS703569 on a variety of cellular activities was analyzed. EC50s were generated by curve fitting of activities using Matlab version 6.5 (MathWorks Inc., MA). (1) For the mast cell activation assays, cultured human primary mast cells were sensitized with human IgE (Cortex Biochem Inc., San Leandro, CA) followed by incubation with Rabbit anti-human IgE antibody (Bethyl Laboratories Inc., Montgomery, TX). Tryptase release was quantified by cleavage of the synthetic peptide substrate, Z-Ala-Lys-Arg-amidomethylcoumarin (2 trifluoroacetic acid) (Enzyme Systems Product Inc., Livermore, CA). (2) Jurkat cells were pre-incubated at 37°C for 1 h and stimulated with anti-T cell receptor beta chain monoclonal antibody (C305) (BD Biosciences, Mansfield, MA) or Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich Chemicals) for 20 h. The cells were stained by Allophycocyanin (APC)-conjugated anti-CD69 antibody (Caltag Laboratories Inc., Burlingame, CA). The geometric mean of APC fluorescence was measured on a FACScalibur (BD Biosciences, San Jose, CA). (3) A549 cells were pre-incubated with R763/AS703569 for 1 h and cultured with tumor necrosis factor alpha (TNFα) (Peprotech Inc., Rocky Hill, NJ), IL-1β (Peprotech. Inc.) or Interferon gamma (IFNγ) (Peprotech. Inc.) for 24 h. The cells were resuspended in PBS and stained with an APC-conjugated monoclonal mouse anti-human intercellular adhesion molecule 1 (ICAM-1, CD54) antibody (BD Biosciences). Surface ICAM-1 expression was analyzed by flow cytometry. The geometric means were used for EC50 estimation. (4) Hela cells expressing Luciferase-fused inhibitor of kappa light chain gene enhancer in B cells (IkB) protein were plated in a 96-well tissue culture plate 1 day before the assay. The cells were pre-incubated with R763/AS703569 for 2 h and stimulated with 2 ng/mL IL-1β for 30 min at 37°C. Following stimulation, the amount of the luciferase reporter was measured using the luciferase assay system (Promega Corporation, Madison, WI) (TIFF 1,520 kb)

432_2009_641_MOESM4_ESM.tif (1.5MB, tif)

Supplemental Table 3. Potent anti-tumor activity of R763/AS703569 against human solid tumor cell lines in vivo. The studies were performed as described in “Methods”. Briefly, MIA-PaCa2, Colo205, A2780 and LXFE 211 tumor cells were implanted in immunocompromised mice. The studies were initiated when tumors reached to a mean size of 100–300 mm3. Various doses of R763/AS703569 was administered once daily orally (PO) or intraperitoneally (IP). 2–4 cycles of the intermittent schedules were used as shown in the table. %T/C values were calculated using the following formula (Tx − Ti) × 100/(Tcx − Tci). Tx means tumor volume of the treated group on day x, Ti means initial tumor volume of the treated group, Tcx means tumor volume of the control group on day x, Tci means initial tumor volume of the control group. X is 28 for MIA PaCa2 and Colo205, and 15 for A2780 and LXFE 211 (TIFF 1,520 kb)

Articles from Journal of Cancer Research and Clinical Oncology are provided here courtesy of Springer

RESOURCES