The Aurora family of serine/threonine kinases is important for the regulation of centrosome maturation, chromosome segregation, and cytokinesis during mitosis. Overexpression of Aurora kinases in mammalian cells leads to genetic instability and transformation. Increased levels of Aurora kinases have also been linked to a broad range of human tumors. Here, we describe the properties of CCT129202, a representative of a structurally novel series of imidazopyridine small-molecule inhibitors of Aurora kinase activity. This compound showed high selectivity for the Aurora kinases over a panel of other kinases tested and inhibits proliferation in multiple cultured human tumor cell lines. CCT129202 causes the accumulation of human tumor cells with ≥4N DNA content, leading to apoptosis. CCT120202-treated human tumor cells showed a delay in mitosis, abrogation of nocodazole-induced mitotic arrest, and spindle defects. Growth of HCT116 xenografts in nude mice was inhibited after i.p. administration of CCT129202. We show that p21, the cyclin-dependent kinase inhibitor, is induced by CCT129202. Up-regulation of p21 by CCT129202 in HCT116 cells led to Rb hypophosphorylation and E2F inhibition, contributing to a decrease in thymidine kinase 1 transcription. This has facilitated the use of 3′-deoxy-3′[18F]fluorothymidine-positron emission tomography to measure noninvasively the biological activity of the Aurora kinase inhibitor CCT129202 in vivo. [Mol Cancer Ther 2007;6(12):3147–57]

The Aurora kinase family is a group of cell cycle–regulated serine/threonine kinases that are important for mitosis (14). The activity of all three Aurora kinases peaks at G2 and during mitosis, whereas their expression is low in resting cells (5, 6). Aurora A is a centrosome-associated kinase (7), whereas Aurora B is a “chromosome passenger” kinase that is essential for chromosome segregation and cytokinesis (2, 8). Aurora C is a centrosome-associated kinase, predominantly restricted to germ cells (6), the function of which remains unclear. A variety of Aurora substrates have been identified, of which the most well characterized for Aurora A are p53, TPX2, Ajuba, XIEg5, and D-TACC (9, 10). Aurora B has been reported to be present in a complex with the inner centromere protein and survivin (11, 12). The most well-characterized substrate for Aurora B is histone H3, a structural component of chromatin (13). Overexpression of Aurora A induces abnormal spindle formation, leading to prolonged mitosis and polyploidy (14). Aurora A was first described in human cancer cell lines, but has subsequently been found to be overexpressed in a broad range of human tumors, including primary colorectal carcinoma, gliomas, breast, ovarian, and pancreatic cancers (5, 1518). Aurora B is also overexpressed in human tumors such as gliomas, thyroid carcinoma, seminoma, and colon cancer (1921). A polymorphism in the Aurora A gene has been identified that shows preferential amplification associated with increased aneuploidy in colon cancer (22). We have shown recently that Aurora A mediates phosphorylation of IκBα that induces nuclear factor-κB activation, a novel function of Aurora A, that could potentially lead to carcinogenesis and drug resistance (23, 24).

The overexpression of the Aurora kinases and the association with genetic instability in tumors suggest that a wide range of cancers could respond therapeutically to inhibitors of the Aurora kinases. During the past 3 years, the first generation of Aurora kinase inhibitors, including ZM447439 (25), Hesperadin (26), VX-680 (27; also known as MK-0457), and more recently, PHA-680632 (28) and the specific Aurora A inhibitor MLN8054 (29) have been described. Inhibitors of Aurora kinases kill proliferating cells through apoptosis after accumulation of tetraploid cells generated by the inhibition of cytokinesis and blocking of mitotic histone H3 phosphorylation (2527). VX-680 and PHA-680632 inhibit all three Aurora kinases, exhibit antiproliferative activity, and induce tumor regression in animal models (28, 29), providing a strong support for developing inhibitors against this target. With respect to biomarkers of drug activity in preclinical studies, Aurora kinase inhibitors have been assessed mainly by measuring histone H3 phosphorylation (27, 28). Noninvasive imaging methods can be very advantageous for cancer drug development (30). It is, therefore, desirable to develop and validate noninvasive imaging methods that measure the biochemical effect of Aurora kinase inhibition. Imaging biomarker assays such as [18F]fluorothymidine-positron emission tomography ([18F]FLT-PET) have previously been used successfully to monitor the biological activity of targeted therapies (31).

In this report, we describe the characterization of a representative of a novel class of highly selective Aurora kinase inhibitors CCT129202, which is active on a wide range of human cancer cell lines in vitro and in human xenografts in athymic mice. The molecular mechanism of action of CCT129202 is consistent with the inhibition of Aurora A and Aurora B, as has been shown by monitoring phosphorylation of histone H3 and p53 protein stabilization. We found that CCT129202 induces p21, resulting in the down-regulation of thymidine kinase 1 (TK1) via the Rb pathway. This decrease in TK1 activity provides the opportunity to use [18F]FLT-PET for measuring the biological activity of Aurora inhibitors, and we describe the utility of this approach with CCT129202 in vivo.

Chemistry

CCT129202, a derivative of the piperazinyl imidazo[4,5-b]pyridine scaffold, was synthesized as discussed elsewhere (32).

In vitro Aurora A Kinase Assays

NH2-terminal glutathione S-transferase (GST)-fusion recombinant human Aurora A (aa 118–403), Aurora B (full length), and Aurora C (full length) were expressed in baculovirus, purified, and used in kinase inhibition assays as previously described (32, 33). The selectivity of the compound was tested in a series of in vitro kinase assays using a selected panel of protein kinases (Invitrogen).

Cell Culture, Immunoblotting, and Cell Cycle Analysis

Human colon cancer cell lines (HCT116, Colo205, SW620, HT29, KW12), ovarian tumor cell lines (A2780, OVCAR8), a breast tumor cell line MDA-MB-157, and HeLa cervical tumor cells were maintained in DMEM with 10% FCS, penicillin, streptomycin, and glutamine, whereas the B-myelomonocytic leukemia cell line (MV4-11; American Type Culture Collection) was maintained in Iscove's modified Dulbecco's medium with 4 mmol/L l-glutamine supplemented with 10% FCS, penicillin, and streptomycin at 37°C and 5% CO2. Immunoblotting was assessed as previously described (23). For cell cycle analysis, cells were treated with inhibitors for 24 h in the absence or presence of amphidicolin (1 μg/mL) or nocodazole (50 ng/mL) and harvested with 5 mmol/L EDTA-PBS, then fixed in 85% ice-cold ethanol overnight and stained with propidium iodide (Sigma), and analyzed on Beckman Coulter Cytomics FC500. Antibodies used were IAK1 (Aurora A; BD Bioscience), α-tubulin (Sigma), poly(ADP-ribose) polymerase (PARP; Cell Signaling Technology), p53 (Oncogene), ezrin (gift from Prof. C. Isacke, The Breakthrough Breast Cancer Research Centre, London, United Kingdom), MPM2 and Ser10 phosphorylated histone H3 (Upstate Biotechnology), total histone H3 and TK1 (Abcam), p21, total and hypo-phosphorylated Rb (BD Bioscience), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Chemicon International).

Histone Extraction

Cells were harvested and resuspended in lysis buffer [10 mmol/L Tris (pH, 7.4), 50 mmol/L sodium bisulfite, 1% Triton ×100, 10 mmol/L MgCl2, and 8.6% sucrose] and then dounce homogenized for 1 min. Cell nuclei were pelleted at 1,500 × g for 10 min at 4°C. Nuclei were washed 3× in lysis buffer and once in wash buffer [10 mmol/L Tris (pH 7.5), 13 mmol/L EDTA]. The nuclei were then resuspended in ice-cold distilled water and 0.4 N sulfuric acid and incubated on ice for 1 h. Samples were pelleted at 20,000 × g for 10 min at 4°C. The resulting supernatant was added to acetone, incubated overnight at −20°C, pelleted at 20,000 × g for 10 min at 4°C, and air dried, and the extracted histone were resuspended in 50 μL distilled water. The proteins were quantified using Bio-Rad Bradford dye and ran on SDS-PAGE gels.

Cell Viability Assay

The effects of CCT129202 on cell proliferation were analyzed with the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma) according to the manufacturer's instructions. Cells were plated in 96-well plates at 2,500 per well and were treated with a range of 0 to 50 μmol/L of CCT129202 for 72 h. The absorbance was measured at 570 nm using the Wallac VICTOR2TM 1420 Multilabel Counter (PerkinElmer).

Cell-based Enzyme-Linked Immunosorbent Assay (CELISA)

Cells were seeded at 8,000 cells per well in sterile 96-well plates 24 h before the addition of inhibitors. Cells were harvested at the indicated times and fixed in 3% paraformaldehyde/0.25% glutaradehyde/0.25% Triton X-100 for 30 min at 37°C, followed by blocking with 5% nonfat milk for 30 min at 37°C. Cells were incubated with anti–phospho-histone H3 Ser10 antibody (Upstate Biotechnology) or anti-p53 antibody (Merck Chemicals Ltd.) for 1 h at 37°C, then with Eu-labeled secondary antibody (PerkinElmer) for 1 h at 37°C. DELFIA enhancement solution was added, and plate was read at 615 nmol/L on Wallac VICTOR2TM 1420 Multilabel Counter (PerkinElmer).

Immunofluorescence Microscopy

Cells were grown overnight on coverslips and treated with inhibitors for 24 h. Cells were washed with PBS and fixed with 4% paraformaldehyde for 1 h at 37°C and permeabilized with 0.5% Triton ×100, followed by 1 h incubation with anti–α-tubulin antibody at room temperature and 1 h incubation with FITC-labeled anti-mouse antibody at room temperature. DNA was stained with TO-PRO-3 (Invitrogen). Coverslips were mounted on microscope slides and fluorescence visualized with Leica SP1 confocal microscope.

Gene Expression cDNA Microarrays

RNA was harvested using the ABI PRISM 6100 Nucleic Acid PrepStation (Applied Biosystems). The quantity was determined with the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies). The CyScribe Post-Labeling Kit (GE Healthcare) was used to label 5 μg of total RNA, according to the manufacturer's recommendations, except that the reverse transcription reaction was carried out overnight. The control samples (untreated cell lines) were labeled with Cy3, and the test samples (treated with CCT129202) were labeled with Cy5. cDNA microarrays were done according to the procedure outlined in ref. (34) (Supplementary Materials and Methods).4

4

Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

In vivo Pharmacokinetics

Mice (female Balb/C, ages 6–8 weeks) received an i.v. dose of CCT129202·3HCl (5 mg kg−1) in 10% DMSO, 5% Tween 20 in saline. After administration, mice were killed at 5, 15, and 30 min and 1, 2, 4, 6, and 24 h. Blood was removed by cardiac puncture and centrifuged to obtain plasma samples. Plasma samples (100 μL) were added to the analytic internal standard (CCT129127, a 6-H analogue of CCT129202; IS), followed by protein precipitation with 300 μL acetonitrile. Following centrifugation (1,200 × g, 20 min, 4°C), the resulting supernatants were analyzed for CCT129202 levels by LCMS using a reverse-phase Synergi Max-RP (Phenomenex, 50 × 2.1 mm) analytic column and positive ion mode ESI MRM on a Waters 2795 liquid chromatography system coupled to a Quattro Ultima triple quadrupole mass spectrometer (Micromass Ltd.). In vitro metabolic stability was assessed by the incubation of CCT129202 (1 μmol/L) with male CD1 mouse liver microsomes (1 mg/mL) protein in the presence of NADPH (2 mmol/L), NADH (2 mmol/L), and MgCl2 (10 mmol/L) in PBS (10 mmol/L) at 37°C. Samples were taken from the incubation mixture at 0, 15, and 30 min and added directly to 3× v/v acetonitrile containing IS. Samples were centrifuged as described for plasma samples before analysis on a Thermo Finnigan LC system consisting of an online degassing system (Alltech Associates), P4000 pump, AS3000 autosampler, and SN4000 system controller interface (SpectraSystems, Thermo Separation Products) coupled to an iontrap MS (LCQ Classic) with Xcalibur data handling system (version 1.1). Control incubations were generated by the omission of NADPH and NADH from the incubation reaction. The percentage compound remaining was determined after analysis by liquid chromatography–mass spectrometry (LCMS).

Animal Efficacy Studies

For efficacy studies, human HCT116 colon carcinoma xenografts were established from the inoculation of 2 × 106 cells in the bilateral flanks of female NCr athymic mice, 6 to 8 weeks old. CCT129202 was dissolved in DMSO and injected i.p in vehicle, which comprised 10% DMSO, 5% Tween 20, and 85% sterile saline at 0.1 mL/10 g body weight. Dosing with CCT129202 commenced when tumors were well established (∼5 mm mean diameter); control animals received an equivalent volume of vehicle. Mouse body weights and condition were monitored throughout the study. Tumors were measured thrice weekly across two perpendicular diameters and tumor volume (V) was calculated using the formula V = 4/3π [(d1 +d2)/4]3 (35). At the end of the study, tumors were excised, weighed, and snap frozen in liquid nitrogen for pharmacokinetic and pharmacodynamic analyses. Serum and tumor samples were collected at 2, 4, and 6 h after the final dose.

Molecular Biomarker Analysis

The levels of total histone H3 and phosphorylated histone H3 were determined using an electrochemiluminescent immunoassay (Meso Scale Discovery, MSD system). Tumor xenografts were homogenized with the PreCellys 24 (Bertin Technologies) in xenograft lysis buffer [1% NP40, 1% sodium deoxycholate, 0.1% SDS, 50 mmol/L Tris (pH, 7.5), 1 mmol/L EDTA, 150 mmol/L NaCl, 1 mmol/L β-glycerophosphate, 2 mmol/L phenylmethylsulfonyl fluoride, 10 mmol/L NaF, mini complete protease inhibitor (Roche)]. Homogenates were pelletted, and the resulting supernatant was used for the analysis. Standard 96-well single-spot MA6000 MSD plates were pre-coated overnight with pan-histone antibody (Chemicon) at 4°C, followed by 1 h incubation with 3% MSD Blocker A at room temperature on shaker and 2 h incubation with 10 μg per well of tumor sample at room temperature also on shaker. MSD plates were then incubated with anti-total histone H3 antibody (Abcam) or anti–phospho-histone H3 Ser10 antibody (Upstate Biotechnology) for 2 h at room temperature on a shaker, followed by MSD anti-rabbit sulfo-tagged detection antibody for 1 h at room temperature also on a shaker. MSD read buffer was added, and the fluorescence was read immediately on a MSD Sector 6000. The amount of phosphorylated histone H3 was normalized to the total amount of histone H3 to obtain the relative histone H3 phosphorylation.

PET Imaging

[18F]FLT was synthesized and characterized as previously reported (31). Before imaging studies, size-matched tumor-bearing mice were randomized into four cohorts (day 2 vehicle and CCT129202-treated groups, and day 7 vehicle and CCT129202-treated groups) and given daily doses of CCT129202 at 100 mg/kg body weight or vehicle i.p. for 2 (three doses) or 7 days (eight doses). The last drug dose was injected 2 h before imaging. The scanning procedure and the image analysis have been described previously (31). Tumor radioactivity uptake normalized that of heart at 60 min post-injection (NUV60) was used for comparison. The area under the nTAC (AUC) was calculated as the integral from 0 to 60 min. The fractional retention of tracer (FRT) at 60 min relative to that at 1.5 min was also calculated.

All procedures involving animals were done in accordance with National Home Office regulations under the Animals (Scientific Procedures) Act 1986 and within guidelines set out by the Institute's Animal Ethics Committee and the United Kingdom Coordinating Committee for Cancer Research's Ad hoc Committee on the Welfare of Animals in Experimental Neoplasia (36).

Identification and Characterization of CCT129202: A Representative of a Novel Class of Potent and Selective of Aurora Kinase Inhibitors

It is valuable to develop a range of Aurora inhibitors based on different chemical backbones. We have previously described the development of an assay for Aurora A activity suitable for high-throughput screening (33). The assay was successfully used for screening of ∼70,000 compounds for Aurora A inhibition. One identified inhibitor was further modified to produce the imidazopyridine inhibitor CCT129202 (ref. 32; Fig. 1A). In in vitro kinase assays using purified recombinant proteins, CCT129202 inhibited Aurora A, Aurora B, and Aurora C, with IC50 values of 0.042 ± 0.022, 0.198 ± 0.05, and 0.227 ± 0.064 μmol/L, respectively. To define the biochemical mechanism of action of CCT129202, we examined the effect of increasing concentration of ATP on the inhibitory activity of the compound using steady-state analysis. The results showed that the CCT129202 is an ATP-competitive inhibitor of recombinant Aurora A kinase with a Ki of 49.8 nmol/L (data not shown). The compound was tested in a series of in vitro kinase assays using different recombinant kinases. CCT129202 showed high selectivity for the Aurora kinases over a broad range of 13 other kinases tested at 1 μmol/L concentration (Fig. 1B). We evaluated the activity of CCT129202 against different human tumor cell lines, and this compound was found to induce apoptosis with half-maximal growth inhibition (GI50) values that ranged between 0.08 and 1.7 μmol/L (Fig. 1C). All cell lines tested showed increased expression levels of Aurora A and Aurora B in comparison with the expression levels of the two proteins in normal tissue controls (refs. 5, 28, and data not shown). Aurora A and Aurora B levels were affected only at very high concentrations of the inhibitor (>5× GI50) possibly due to cell toxicity (data not shown). We did not find an obvious relationship between the sensitivity of cells in the growth inhibition assay and the protein levels of the Aurora kinases within the cell lines analyzed. Differences in sensitivity might rather be associated with different genetic backgrounds of the cell lines, such as lack of proteins associated with Aurora kinases (28).

Figure 1.

CCT129202 is a selective Aurora kinase inhibitor that inhibits proliferation of several human tumor cell lines. A, chemical structure of CCT129202 [2-(4-(6-chloro-2-(4-(dimethylamino)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)-N-(thiazol-2-yl)acetamide], a derivative of the piperazinyl imidazo[4,5-b]pyridine scaffold. B, activity of the CCT129202, against a panel of protein kinases at a concentration of 1 μmol/L. C, cell growth inhibition assays of CCT129202 in a panel of human tumor cell lines.

Figure 1.

CCT129202 is a selective Aurora kinase inhibitor that inhibits proliferation of several human tumor cell lines. A, chemical structure of CCT129202 [2-(4-(6-chloro-2-(4-(dimethylamino)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)-N-(thiazol-2-yl)acetamide], a derivative of the piperazinyl imidazo[4,5-b]pyridine scaffold. B, activity of the CCT129202, against a panel of protein kinases at a concentration of 1 μmol/L. C, cell growth inhibition assays of CCT129202 in a panel of human tumor cell lines.

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CCT129202 Induces Aberrant Mitosis that Leads to Apoptosis

It was of interest to test the effect of CCT129202 on the cell cycle progression, growth, and viability of human tumor cells. We examined the effects of CCT129202 on the cell cycle profile of the HCT116 human colon tumor cell line by flow cytometry. CCT129202 caused the accumulation of HCT116 cells with ≥4N DNA content (Fig. 2A). Furthermore, a sub-G1 cell population can be seen at 24 h after CCT129202 treatment, which is indicative of apoptosis (Fig. 2A). These results were confirmed by an increase in cleaved PARP in treated cells (Fig. 2B).

Figure 2.

CCT129202 induces accumulation of cells with ≥4N DNA and apoptosis. A, HCT116 cells were treated with 700 nmol/L (2 × GI50) of CCT129202 for 24 and 48 h or 0.1% DMSO vehicle control. DNA content was assessed by flow-cytometric analysis of cells labeled with propidium iodine (FL3Lin; top). Percentage of HCT116 cells in the sub-G1, G1, S, G2-M, and >4N DNA following treatment with 700 nmol/L of CCT129202 for 24 and 48 h or 0.1% DMSO vehicle (bottom). M1, sub-G1; M2, G1; M3, S; M4, G2-M; M5, >4N DNA. B, detection of apoptosis in HCT116 cells with cleaved PARP was assessed by immunoblotting. Tubulin was used as a loading control. GI, growth inhibition.

Figure 2.

CCT129202 induces accumulation of cells with ≥4N DNA and apoptosis. A, HCT116 cells were treated with 700 nmol/L (2 × GI50) of CCT129202 for 24 and 48 h or 0.1% DMSO vehicle control. DNA content was assessed by flow-cytometric analysis of cells labeled with propidium iodine (FL3Lin; top). Percentage of HCT116 cells in the sub-G1, G1, S, G2-M, and >4N DNA following treatment with 700 nmol/L of CCT129202 for 24 and 48 h or 0.1% DMSO vehicle (bottom). M1, sub-G1; M2, G1; M3, S; M4, G2-M; M5, >4N DNA. B, detection of apoptosis in HCT116 cells with cleaved PARP was assessed by immunoblotting. Tubulin was used as a loading control. GI, growth inhibition.

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It has been reported that the inhibition of Aurora A results in G2-M accumulation and spindle defects (29), whereas inhibition of Aurora B abrogates the mitotic checkpoint leading to aneuploidy due to cytokinesis failure (25). We examined the effects of CCT129202 on the cell cycle status of aphidicolin-synchronized cells by determining DNA content by flow cytometry. Figure 3A shows the DNA content of cells in each phase of the cell cycle. Approximately 28% of CCT129202-treated HCT116 cells remain temporarily in G2-M 12 h after the release from aphidicolin compared with 16% in G2-M in the untreated cells (P = 0.0009; Fig. 3B). Moreover, HCT116 cells treated with CCT129202 showed abrogation of nocodazole arrest 2 to 4 h earlier than untreated cells (Fig. 3C), consistent with Aurora B inhibition (25). Similar results were obtained with paclitaxel-arrested HCT116 cells (data not shown). CCT129202 abrogated paclitaxel-induced arrest of HCT116 cells 6 h earlier than nontreated cells.

Figure 3.

Effect of CCT129202 on cell cycle and cell morphology. A, HCT116 cells were released from aphidicolin-induced G1-S block in the presence of 350 nmol/L (GI50) of CCT129202 or DMSO vehicle. DNA content of cells collected at the indicated time points was assessed by flow cytometry analysis of cells labeled with propidium iodine. B, percent of HCT116 cells in G2-M phase of the cell cycle (P = 0.0009). C, HCT116 cells were treated with nocodazole alone or in combination with 350 nmol/L CCT129202. At the indicated time points, cells were lysed, and the MPM2 phosphorylation was assessed by immunoblotting. Tubulin was used as a loading control. D, HCT116 cells were treated with CCT129202 or vehicle control (0.1% DMSO) for 24 h. α-Tubulin staining (green) was assessed by immunocytochemistry. 4′,6-Diamidino-2-phenylindole staining (blue) indicates the DNA content. Bar, 1 cm = 25 μm.

Figure 3.

Effect of CCT129202 on cell cycle and cell morphology. A, HCT116 cells were released from aphidicolin-induced G1-S block in the presence of 350 nmol/L (GI50) of CCT129202 or DMSO vehicle. DNA content of cells collected at the indicated time points was assessed by flow cytometry analysis of cells labeled with propidium iodine. B, percent of HCT116 cells in G2-M phase of the cell cycle (P = 0.0009). C, HCT116 cells were treated with nocodazole alone or in combination with 350 nmol/L CCT129202. At the indicated time points, cells were lysed, and the MPM2 phosphorylation was assessed by immunoblotting. Tubulin was used as a loading control. D, HCT116 cells were treated with CCT129202 or vehicle control (0.1% DMSO) for 24 h. α-Tubulin staining (green) was assessed by immunocytochemistry. 4′,6-Diamidino-2-phenylindole staining (blue) indicates the DNA content. Bar, 1 cm = 25 μm.

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Aurora A and Aurora B play an important role in centrosome maturation, spindle formation, and cytokinesis (7). Therefore, inhibition of Aurora kinases disturbs the progression of cells through normal mitosis, resulting in aberrant mitotic spindles. To determine the effects of CCT129202 on spindle formation, HCT116 or HeLa cells were incubated with DMSO or CCT129202 for 24 h, and the morphology of mitotic spindles was examined by immunofluorescence microscopy. As expected, in DMSO-treated cells, normal metaphase and anaphase spindles were readily apparent, as shown using an α-tubulin antibody (Fig. 3D). CCT129202-treated HCT116 (Fig. 3D) and HeLa cells (data not shown) induced the formation of abnormal mitotic spindles, with various degrees of chromosome alignment defects. This phenotype is consistent with the inhibition of Aurora kinase activity as shown by the use of small interfering RNA (siRNA) and other Aurora kinase inhibitors (25, 29).

CCT129202 Decreases Histone H3 Phosphorylation and Causes p53 Stabilization

Aurora B has been shown to phosphorylate histone H3 at Ser10 (13). Inhibition of histone H3 phosphorylation by CCT129202 was confirmed in several human tumor cell lines (data not shown). Using a Ser10 phospho-specific α-histone H3 antibody, we have developed an assay (CELISA), in which we measure the inhibition of H3 phosphorylation after inhibitor treatment. Time course experiments in HCT116 cells treated with CCT129202 showed that the inhibitory effect on the phosphorylation of the histone H3 occurred as early as 15 min after treatment (Fig. 4A). These results are consistent with the inhibition of Aurora B by CCT129202.

Figure 4.

CCT129202 reduces phosphorylation of histone H3, stabilizes p53, and inhibits growth of HCT116 human colon cancer xenografts in athymic mice. A, HCT116 cells treated with CCT129202, and the relative levels of phosphorylated histone H3 was assessed at the indicated time points by CELISA (left). HCT116 cells were treated with different concentrations of CCT129202 or 0.1% DMSO for 8 and 24 h, and the levels of total histone H3 and phosphorylated histone H3 were assessed by immunoblotting after histone extraction (right). B, HCT116 cells were treated with 350 nmol/L CCT129202, 160 nmol/L VX-680, or 0.1% DMSO control for 24 h, and the levels of p53 were assessed by CELISA (left). HCT116 cells treated with different concentrations of CCT129202 or 0.1% DMSO control at 24 h and the levels of p53 were assessed by immunoblotting. GAPDH was used as a loading control (right). C, athymic mice bearing HCT116 human colon cancer xenografts were treated i.p. with a single dose of 100 mg/kg of CCT129202 or 0.1% DMSO vehicle control, and the phosphorylation of histone H3 was assessed in tumors at different time points by electrochemiluminescence immunoassay. D, athymic mice bearing established HCT116 tumors were treated i.p. with either vehicle control (▪) or CCT129202 (○) at a dose of 100 mg/kg/day for 9 d. n = 5 per group. Points, mean tumor volumes; bars, SE. GI, growth inhibition.

Figure 4.

CCT129202 reduces phosphorylation of histone H3, stabilizes p53, and inhibits growth of HCT116 human colon cancer xenografts in athymic mice. A, HCT116 cells treated with CCT129202, and the relative levels of phosphorylated histone H3 was assessed at the indicated time points by CELISA (left). HCT116 cells were treated with different concentrations of CCT129202 or 0.1% DMSO for 8 and 24 h, and the levels of total histone H3 and phosphorylated histone H3 were assessed by immunoblotting after histone extraction (right). B, HCT116 cells were treated with 350 nmol/L CCT129202, 160 nmol/L VX-680, or 0.1% DMSO control for 24 h, and the levels of p53 were assessed by CELISA (left). HCT116 cells treated with different concentrations of CCT129202 or 0.1% DMSO control at 24 h and the levels of p53 were assessed by immunoblotting. GAPDH was used as a loading control (right). C, athymic mice bearing HCT116 human colon cancer xenografts were treated i.p. with a single dose of 100 mg/kg of CCT129202 or 0.1% DMSO vehicle control, and the phosphorylation of histone H3 was assessed in tumors at different time points by electrochemiluminescence immunoassay. D, athymic mice bearing established HCT116 tumors were treated i.p. with either vehicle control (▪) or CCT129202 (○) at a dose of 100 mg/kg/day for 9 d. n = 5 per group. Points, mean tumor volumes; bars, SE. GI, growth inhibition.

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We next looked for molecular evidence that CCT129202 targets Aurora A in cells. It has been reported that Aurora A phosphorylates p53 at Ser315 (37). This phosphorylation leads to p53 degradation in an MDM2-dependent manner. We used this information to test if CCT129202 also inhibited Aurora A in cells. Using the DO1 antibody against p53 protein, we have developed a CELISA assay in which p53 stabilization can be measured after inhibitor treatment. HCT116 cells treated for 24 h with CCT129202 or the previously described Aurora kinase inhibitor VX-680 (27) showed p53 stabilization compared with the DMSO-treated control HCT116 cells (Fig. 4B). These results are consistent with the inhibition of Aurora A by CCT129202.

CCT129202 Inhibits Phosphorylation of Histone H3 in HCT116 Tumor Xenografts in Athymic Mice and Causes Tumor Growth Inhibition

Before investigating the therapeutic activity of CCT129202 in HCT116 human tumor xenografts in athymic mice, the pharmacokinetic properties of the compound were first assessed in non–tumor-bearing mice. Following i.v. administration of 5 mg/kg of CCT129202 to mice, the compound had a reasonable half-life of 0.94 h and a low clearance (0.07 L/h). The volume of distribution was 0.053 L, and the plasma concentrations were above IC50 for up to 3 h. In vitro metabolism studies showed that CCT129202 was metabolically stable, with only a ∼25% decrease in parent compound following incubation with mouse liver microsomes for 30 min. This was in good agreement with the clearance measured in vivo.

Tumor levels of CCT129202 were determined following a single dose of 100 mg/kg to athymic mice bearing s.c. HCT116 colon cancer xenografts. We examined the inhibition of histone H3 phosphorylation as a biomarker of Aurora B inhibition in vivo. Reduction of histone H3 phosphorylation by ∼50% was observed in tumors 30 min following the administration of CCT129202 as measured by electroluminescent immunoassay (Fig. 4C), demonstrating the inhibition of Aurora B kinase. The effect started to disappear 2 h after treatment, compatible with the half-life of the compound.

Based on these promising pharmacokinetic-pharmacodynamic results, we next examined the effects of CCT129202 on the growth of HCT116 xenografts. CCT129202 was given i.p at 100 mg/kg once a day for a period of 9 days to HCT116 tumor-bearing athymic mice. Significant tumor growth inhibition was observed compared with the vehicle-treated mice (% treated versus control, 57.7; P = 0.0355, Mann Whitney U test; Fig. 4D). CCT129202 was well tolerated, with <5% body weight changes compared with vehicle controls.

CCT129202 Causes p21 Up-regulation, Rb Hypophosphorylation, and H2F-Dependent TK1 Down-regulation

To identify genes that are deregulated by the Aurora kinase inhibitor CCT129202 and that may be potential additional biomarkers, we did a gene expression microarray analysis in HCT116 human colon tumor cells. HCT116 cells were treated for different time periods with 1× GI50 CCT129202 or with vehicle (0.1% DMSO). cDNA microarrays of 28K cDNA clones were used to analyze gene expression changes. The complete list is supplied as supplementary data (Supplementary Table S1).4 These comprise E2F-regulated genes, together with a group of genes that play a significant role in mitosis. A decrease in expression of mitotic checkpoint genes such as BUB1, BUB3, MAD2L1, and TTK was observed in response to CCT129202, most likely as a consequence of mitotic abrogation caused by the inhibition of Aurora B (Supplementary Table S1; Supplementary Fig. S1).4 Among the genes we found to show expression changes in HCT116 cells treated with CCT129202 was the cyclin-dependent kinase (cdk) inhibitor p21, a regulator of the Rb/E2F pathway. The differential regulation of p21 was confirmed at the protein level using immunoblot assays. A time-dependent up-regulation of p21 was observed following CCT129202 treatment (Fig. 5A). The specificity of the effect of CCT129202 on the p21 regulation was tested using the inactive analogue CCT129127 (2-(4-(2-(4-(dimethylamino)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)-N-(thiazol-2-yl)acetamide). HCT116 cells were treated with equimolar concentrations of the active or inactive analogues. p21 up-regulation was only observed in the HCT116 cells treated with CCT129202 (Fig. 5B).

Figure 5.

CCT129202 up-regulates p21, inhibits Rb phosphorylation, and decreases TK1 expression. A, HCT116 cells were treated with 350 nmol/L CCT129202 at different time points or 50 ng/mL nocodazole for 24 h, and p21 expression was assessed at the indicated time points by immunoblotting. B, HCT116 cells were treated with different concentrations of CCT129202, an equimolar concentration of the inactive analogue CCT129127, 50 ng/mL nocodazole, or 0.1% DMSO vehicle control for 24 h, and p21 expression was assessed by immunoblotting. C, HT29 and HeLa cells were treated with different concentrations of CCT129202, nocodazole, or 0.1% DMSO control for 24 h, and p21 expression was assessed by immunoblotting. D, HCT116 cells were treated with different concentrations of CCT129202, nocodazole, or 0.1% DMSO control for 24 h, and the p21, hypophosphorylated Rb (p-Rb), total Rb (pp-Rb), and TK1 expression levels were assessed by immunoblotting. GAPDH and tubulin were used as loading control. GI, growth inhibition.

Figure 5.

CCT129202 up-regulates p21, inhibits Rb phosphorylation, and decreases TK1 expression. A, HCT116 cells were treated with 350 nmol/L CCT129202 at different time points or 50 ng/mL nocodazole for 24 h, and p21 expression was assessed at the indicated time points by immunoblotting. B, HCT116 cells were treated with different concentrations of CCT129202, an equimolar concentration of the inactive analogue CCT129127, 50 ng/mL nocodazole, or 0.1% DMSO vehicle control for 24 h, and p21 expression was assessed by immunoblotting. C, HT29 and HeLa cells were treated with different concentrations of CCT129202, nocodazole, or 0.1% DMSO control for 24 h, and p21 expression was assessed by immunoblotting. D, HCT116 cells were treated with different concentrations of CCT129202, nocodazole, or 0.1% DMSO control for 24 h, and the p21, hypophosphorylated Rb (p-Rb), total Rb (pp-Rb), and TK1 expression levels were assessed by immunoblotting. GAPDH and tubulin were used as loading control. GI, growth inhibition.

Close modal

We next investigated whether p21 up-regulation by CCT129202 was also seen in cell lines with deregulated p53. In HT29 and HeLa cells, p53 is mutated or inactivated, respectively. HT29 and HeLa cells were treated with different concentrations of CCT129202 for 24 h. Although the overall p21 protein levels were less than in HCT116 cells, which has wild-type p53, up-regulation of p21 was seen in both cell lines, indicating that a minor p53-independent inhibition also takes place (Fig. 5C). Through its inhibitory effects on cdks, p21 decreases the phosphorylation of the Rb protein. When hypophosphorylated, Rb is in a complex with the transcription factor E2F and thereby inhibits the activity of E2F in regulating proteins that play a role in the S-phase entry. To examine the effect of CCT129202 on the p21/Rb pathway, we first examined the phosphorylation status of the Rb protein. Figure 5D shows that, in CCT129202-treated HCT116 cells, the Rb protein is hypophosphorylated in a concentration-dependent manner. One of the genes known to be regulated by E2F is that encoding TK1 (38). We therefore examined the effect of CCT129202 on TK1 levels in HCT116 cells. As shown in Fig. 5D, TK1 expression was decreased in a concentration-dependent manner in HCT116 cells. The down-regulation of TK1 levels was also confirmed in p53-mutant HT29 cells (Supplementary Fig. S2).4 To further confirm that the deregulation of p21/Rb/E2F pathway was a consequence of Aurora inhibition, we also tested the known pan-Aurora inhibitor VX-680. HCT116 cells treated with this inhibitor showed similar kinetics of p21 up-regulation, Rb hypophosphorylation, and TK1 down-regulation, consistent with the view that this effect is due to Aurora inhibition (Supplementary Fig. S3).4 The inactive analogue CCT129127 showed no effect on Rb phosphorylation or TK1 in HCT116 cells (data not shown). In summary, these data suggest that Aurora kinase inhibition affects the p21/Rb/E2F pathway, and that the effect on TK1 expression might be used as a biomarker of pathway inhibition downstream of Aurora inhibition by CCT129202.

CCT129202 Decreases [18F]FLT Uptake

The alterations described above in p21, phosphorylation of Rb and TK1 levels that are mediated by CCT129202, together with the data demonstrating that TK1 is required for [18F]FLT uptake in vivo (39), led us to hypothesize that [18F]FLT-PET might be used to monitor the biological activity of CCT129202 in vivo. [18F]FLT-PET images of mice bearing HCT116 colon cancer xenografts are shown in Fig. 6A. The data illustrated are intensity-normalized images from vehicle control and CCT129202-treated mice. Whereas no obvious difference in image intensity was observed between CCT129202-treated and controls at the early time point (day 2), there was a marked difference at day 7. CCT129202 reduced the retention of [18F]FLT-derived radioactivity at day 7, as illustrated by the quantitative normalized time versus radioactivity curves (nTACs; Fig. 6B), in keeping with a reduction in proliferation with treatment. All three parameters of [18F]FLT retention (mean NUV60, FRT, and AUC) were unchanged on day 2 of CCT129202 treatment, but were significantly reduced at day 7 compared with control (Fig. 6C). Of note, NUV60, FRT, and AUC for tumor 15 shown in Fig. 6A (day 7, CT129202) were in the lower 50% of the respective mean day 7 CCT129202-treated values.

Figure 6.

[18F]FLT-PET studies of CCT129202. A, transverse (0.5 mm) PET images of vehicle and CCT129202-treated athymic mice bearing HCT116 human colon cancer xenografts on the days indicated. B, summary normalized time versus radioactivity curves in HCT116 tumors under the different treatment conditions. For each mouse, five image slices of tumor were averaged for each of the 19 time points and normalized to the mean activity from five slices of the heart region (blood volume). Data were mean ± SD for four to six mice. C, summary of kinetic parameters—NUV60, FRT, and AUC—determined as described in Materials and Methods for the different conditions of day 2 vehicle, day 2 CCT129202, day 7 vehicle, and day 7 CCT129202 (2V, 2D, 7V, and 7D, respectively). Bar, SD. *, P < 0.02 (Mann-Whitney test). D, athymic mice bearing HCT116 human colon cancer xenografts were treated i.p. with 100 mg/kg of CCT129202 or vehicle control at days 2 and 7, and the TK1 protein levels were assessed in tumors by immunoblotting on the days indicated. GAPDH was used as loading control.

Figure 6.

[18F]FLT-PET studies of CCT129202. A, transverse (0.5 mm) PET images of vehicle and CCT129202-treated athymic mice bearing HCT116 human colon cancer xenografts on the days indicated. B, summary normalized time versus radioactivity curves in HCT116 tumors under the different treatment conditions. For each mouse, five image slices of tumor were averaged for each of the 19 time points and normalized to the mean activity from five slices of the heart region (blood volume). Data were mean ± SD for four to six mice. C, summary of kinetic parameters—NUV60, FRT, and AUC—determined as described in Materials and Methods for the different conditions of day 2 vehicle, day 2 CCT129202, day 7 vehicle, and day 7 CCT129202 (2V, 2D, 7V, and 7D, respectively). Bar, SD. *, P < 0.02 (Mann-Whitney test). D, athymic mice bearing HCT116 human colon cancer xenografts were treated i.p. with 100 mg/kg of CCT129202 or vehicle control at days 2 and 7, and the TK1 protein levels were assessed in tumors by immunoblotting on the days indicated. GAPDH was used as loading control.

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We investigated the effect of CCT129202 on tumor TK1 protein levels in HCT116 tumor xenografts in treated mice. Figure 6D shows TK1 protein levels in pretreatment tumors on day 7 vehicle- and CCT129202-treated HCT116 tumors. TK1 levels decreased after treatment with CCT129202. These findings are consistent with the reduction of the [18F]FLT uptake as determined by PET.

Because Aurora kinases are overexpressed in a variety of human tumors leading to deregulation of mitosis, they represent important targets for anticancer drug development. We have discovered the novel ATP-competitive imidazopyridine inhibitor CCT129202, a representative of a new class of small-molecule inhibitors with potent activity against Aurora kinases. The compound showed no substantial cross-reactivity with a panel of other kinases tested, indicating considerable selectivity. CCT129202 inhibited the growth of a range of human tumor cell lines. The sensitivity might be associated with the inhibition of all Aurora kinases, in particular Aurora A and Aurora B, in addition to a different genetic background among the cell lines. First, there is evidence that links Aurora A with p53 in carcinogenesis. It has been shown that Aurora A phosphorylates p53, leading to its inactivation either by degradation or by suppression of its transcriptional activity (37, 40). Our own results are consistent with these previous studies because we showed that inhibition of Aurora A by CCT129202 resulted in the stabilization of p53 levels in tumor cells. Second, Aurora B has been shown to phosphorylate histone H3 at Ser10, and phosphorylation of histone H3 is associated with the induction of neoplastic transformation (41). Consistent with this, we showed that CCT129202 is a potent inhibitor of the phosphorylation of histone H3 in tumor cells. Antitumor activity was seen in human tumor xenografts at a dose of 100 mg/kg, with evidence of Aurora inhibition provided by decreased phosphorylation of histone H3. Moreover, CCT129202-treated HCT116 or HeLa cells were able to abrogate both the temporarily induced mitotic arrest due to Aurora A inhibition or nocodazole-induced mitotic arrest. This abrogation was possibly due to the reduction in the expression of mitotic checkpoint proteins such as BubR1, Bub1 Mad2L1, and TTK that we observed upon treatment with CCT129202. When the mitotic checkpoint is compromised, cells can exit mitosis without undergoing cytokinesis with ≥4N DNA content. The observed abnormal mitotic spindles and multinucleation of tumor cells treated with CCT129202 resembles the phenotype induced in cells by inhibiting Aurora A and Aurora B using siRNA or other chemical inhibitors (25, 29).

Pharmacodynamic markers are essential for the rational development of molecular therapeutics (30, 42), including Aurora inhibitors. They can be used to show proof of concept for the proposed mechanism of drug action in phase I studies, as well as to help select the optimal dose schedule. Given the desirability of avoiding invasive methods, particularly involving tumor biopsies (30, 42), it would be of interest to develop noninvasive imaging methods to measure the biological activity of Aurora kinase inhibitors. Here, we have shown, using a molecular profiling approach, that p21 is induced in tumor cells by Aurora inhibitors. Although the p21 induction was more intense in cells with wild-type p53, human tumor cells with mutated or inactivated p53 also showed up-regulation of p21. This suggests that the effect is mainly p53 dependent, with a minor p53-independent component. Based on these findings, we examined the effect of CCT129202 on the pathway downstream of p21, in particular the Rb/E2F pathway, to identify potential end points. Of particular interest was that the TK1 protein was modulated by CCT129202. TK1 is required for [18F]FLT uptake in vivo (39). Therefore, we hypothesized that we may be able to visualize a decrease in [18F]FLT uptake using [18F]FLT-PET following the inhibition of Aurora kinases by CCT129202. As predicted, a decrease in [18F]FLT was shown. [18F]FLT-PET is a validated preclinical and clinical noninvasive imaging approach commonly used to measure tumor cell proliferation (31). Taken together, these observations support the in vivo use of [18F]FLT imaging for detecting the biological effects of CCT129202. CCT129202 was used in vivo at doses that were associated with the inhibition of phosphorylation of histone H3, up-regulation of p21, and reduction of tumor growth. In addition, the significant reduction of NUV60, FRT, and AUC at day 7, as measured by PET, mirrored the changes in TK1 protein levels.

Although more potent inhibitors of Aurora have been described (27), CCT129202 is representative of an imidazopyridine novel chemical series. Aurora inhibitors based on distinct chemical scaffolds could have potential advantages, for example, in term of selectivity and therapeutic index, and it is important that the different Aurora inhibitory chemotypes are advanced into the clinic. We are continuing to optimize this new series, in particular to improve in vivo potency. The biomarkers reported here will be used for this. In addition, this should be suitable for use with other Aurora inhibitors.

In conclusion, Aurora is now recognized as an important molecular target for anticancer drug discovery. In this study, we have shown that the imidazopyridine CCT129202 is a novel, potent, and selective Aurora kinase inhibitor in vitro and in vivo. Molecular and biochemical changes such as p21 induction, hypophosphorylation of pRB, reduction in TK1 protein, and histone H3 phosphorylation are associated with CCT129202 treatment of human tumor cells and xenografts. In addition, CCT129202 induces a reduction in tumor [18F]FLT retention, detectable by noninvasive PET imaging. These favorable attributes of CCT129202 warrant further investigation of the imidazopyridine compound class as therapeutic agents. The molecular, biochemical, and imaging methods described in this report could form an integral part of the preclinical and early clinical evaluation of this and other classes of Aurora kinase inhibitors.

Grant support: Programme grants from Cancer Research UK (C309/A8274 and C2536/A5708) and Breakthrough Breast Cancer.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Chroma Therapeutics (Oxford, United Kingdom) for providing the kinome profiling results, D. Moffat and A. Drummond from Chroma Therapeutics, A. Ashworth and C. Isacke from The Breakthrough Breast Cancer Research Centre, and all the members of the Cancer Drug Target Discovery Team, The Institute of Cancer Research, for critically reading and discussing the manuscript. We also thank Sharon Gowan for help with tumor sample processing.

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