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[Cancer Research 65, 9038-9046, October 1, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

The In vitro and In vivo Effects of JNJ-7706621: A Dual Inhibitor of Cyclin-Dependent Kinases and Aurora Kinases

Stuart Emanuel1, Catherine A. Rugg1, Robert H. Gruninger1, Ronghui Lin1, Angel Fuentes-Pesquera1, Peter J. Connolly1, Steven K. Wetter1, Beth Hollister2, Walter W. Kruger2, Cheryl Napier2, Linda Jolliffe1 and Steven A. Middleton1

1 Cancer Therapeutics Research, Johnson & Johnson Pharmaceutical Research & Development, LLC, Raritan, New Jersey and 2 Piedmont Research Center, Morrisville, North Carolina

Requests for reprints: Stuart Emanuel, Johnson & Johnson Pharmaceutical Research & Development, LLC, 1000 Route 202, Raritan, NJ 08869. Phone: 908-704-5815; Fax: 908-704-4996; E-mail: semanuel{at}prdus.jnj.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Modulation of aberrant cell cycle regulation is a potential therapeutic strategy applicable to a wide range of tumor types. JNJ-7706621 is a novel cell cycle inhibitor that showed potent inhibition of several cyclin-dependent kinases (CDK) and Aurora kinases and selectively blocked proliferation of tumor cells of various origins but was about 10-fold less effective at inhibiting normal human cell growth in vitro. In human cancer cells, treatment with JNJ-7706621 inhibited cell growth independent of p53, retinoblastoma, or P-glycoprotein status; activated apoptosis; and reduced colony formation. At low concentrations, JNJ-7706621 slowed the growth of cells and at higher concentrations induced cytotoxicity. Inhibition of CDK1 kinase activity, altered CDK1 phosphorylation status, and interference with downstream substrates such as retinoblastoma were also shown in human tumor cells following drug treatment. Flow cytometric analysis of DNA content showed that JNJ-7706621 delayed progression through G1 and arrested the cell cycle at the G2-M phase. Additional cellular effects due to inhibition of Aurora kinases included endoreduplication and inhibition of histone H3 phosphorylation. In a human tumor xenograft model, several intermittent dosing schedules were identified that produced significant antitumor activity. There was a direct correlation between total cumulative dose given and antitumor effect regardless of the dosing schedule. These results show the therapeutic potential of this novel cell cycle inhibitor and support clinical evaluation of JNJ-7706621.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Under normal growth conditions, cell proliferation is tightly regulated in response to diverse intracellular and extracellular signals. In the eukaryotic cell cycle, a key role is played by the cyclin-dependent kinases (CDK). The combination of catalytic kinase subunits (such as CDK1, CDK2, CDK4, or CDK6) with regulatory cyclin subunits (such as cyclin A, B, D1, D2, D3, or E) results in the assembly of functionally distinct kinase complexes. Coordinated activation of these complexes at specific times drives cells through the cell cycle and ensures the fidelity of cell division (1). Complexes of CDK4 and D-type cyclins govern the early G1 phase of the cell cycle; the activity of the CDK2/cyclin E complex is rate limiting for the G1-to-S phase transition (2), and CDK2/Cyclin A kinase regulates progression through the S phase (3). Biochemical and genetic evidence have shown that the activity of the CDK1/cyclin B complex is required for passage from the G2 phase of the cell cycle into the M phase and for successful completion of mitosis (4, 5). CDK1 phosphorylates a number of proteins including histone H1, DNA polymerase {alpha}, RNA polymerase II, retinoblastoma, p53, nucleolin, cAbl, and lamin A (6, 7). CDKs, their substrates, and regulatory proteins are the target of genetic alteration or overexpression in many human cancers (8, 9) and cell lines (10). Alterations in components of cell cycle signaling pathways are present in ~90% of human cancers (11, 12).

The Aurora kinases have a critical role in controlling chromosome movement and organization. Aurora-A contributes to formation of the mitotic spindle apparatus that guarantees accurate segregation of chromosomes into daughter cells; Aurora-B is required for cytokinesis and proper chromosome architecture during mitosis (13). These kinases are frequently overexpressed and amplified in many human tumors (14).

Accordingly, small-molecule inhibitors of CDK or Aurora activity should prevent the continuous, proliferative growth of cancer cells and provide a way of controlling abnormal mitosis, which would be applicable to a wide range of tumor types (15). A number of small-molecule inhibitors of CDKs have been described (reviewed in ref. 16) and several are currently under evaluation in clinical trials including Flavopiridol, UCN-01, CYC202, and BMS-387032 (17). Recently, relatively selective inhibitors of Aurora kinases have also been reported including ZM447439 (18) and VX-680 (19). In this report, we describe the identification of JNJ-7706621, a [1,2,4]triazole-3,5-diamine dual CDK and Aurora inhibitor with a unique inhibition profile of cell cycle regulatory kinases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents. JNJ-7706621 (Fig. 1A) was synthesized by the Cancer Therapeutics Research team (J&J Pharmaceutical Research & Development). Chemicals were obtained from Sigma (St. Louis, MO) and radioisotopes were from Perkin-Elmer (Boston, MA).



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  		Figure 1. JNJ-7706621 inhibits CDK1 kinase activity in cells. A, chemical structure of JNJ-7706621 (4-[5-amino-1-(2,6-difluoro-benzoyl)-1H-[1,2,4]triazol-3-ylamino]-benzenesulfonamide). B, IC50 value for inhibition of recombinant human CDK1/Cyclin B by JNJ-7706621 was calculated at increasing concentrations of ATP and produced a linear relationship relative to total ATP concentration. All groups were set up with n = 3; bars, SD. C, lysates were prepared from asynchronous HeLa cells and HeLa cells synchronized with nocodazole and treated with either vehicle (0.04% DMSO) or 1 to 4 µmol/L JNJ-7706621. Active CDK1 kinase was immunoprecipitated from lysates and evaluated for CDK1 kinase activity with histone H1 protein as substrate. The phosphorylated histone H1 was separated by SDS-PAGE and the extent of incorporation of 33P-{gamma}-ATP into histone H1 was quantified on a Storm 860 phosphorimager (Molecular Devices, Sunnyvale, CA). D, the same cell lysates used to measure kinase activity were subjected to immunoblot analysis to evaluate the effects of JNJ-7706621 on CDK1 phosphorylation and cell cycle regulatory proteins. Membranes were probed with antibodies to phospho-CDK1 (Tyr15), phospho-CDK1 (Thr161), total CDK1, total cyclin B1, total Wee1, total Myt1, and phospho-retinoblastoma (Ser780). Each membrane was also probed with an antibody to ß-actin as an internal control [only shown for the phospho-CDK1 (Tyr15) blot].

 
Cell culture. Human umbilical vascular endothelial cell, HASMC, and HMVEC were obtained from Cascade Biologicals (Portland, OR). MRC-5 human lung fibroblasts and all other cells were from the American Type Culture Collection (Manassas, VA). Cell culture reagents were obtained from Life Technologies, Inc. (Grand Island, NY). Cells were incubated at 37°C plus 5% CO2 and all cell lines were maintained as exponentially growing cultures in the recommended medium.

In vitro kinase and cell proliferation assays. To identify compounds that inhibited CDK1 kinase activity, a screening method was developed using the CDK1/cyclin B complex purified from baculovirus (NEB, Boston, MA) to phosphorylate a biotinylated peptide substrate containing the consensus phosphorylation site for histone H1, which is phosphorylated in vivo by CDK1 (20). Inhibition of CDK1 activity was measured by observing a reduced amount of 33P-{gamma}-ATP incorporation into the immobilized substrate in streptavidin-coated 96-well scintillating microplates (NEN, Boston, MA). CDK1 enzyme was diluted in 50 mmol/L Tris-HCl (pH 8), 10 mmol/L MgCl2, 0.1 mmol/L Na3VO4, 1 mmol/L DTT, 1% DMSO, 0.25 µmol/L peptide, 0.1 µCi per well 33P-{gamma}-ATP (2,000-3,000 Ci/mmol), and 5 µmol/L ATP in the presence or absence of various concentrations of test compound and incubated at 30°C for 1 hour. The reaction was terminated by washing with PBS containing 100 mmol/L EDTA and plates were counted in a scintillation counter. Linear regression analysis of the percent inhibition by test compound was used to determine IC50 values (GraphPad Prism 3, GraphPad Software, San Diego, CA). The Aurora kinase assays were done with 10 µmol/L ATP and a peptide containing a dual repeat of the kemptide phosphorylation motif. Assays for inhibition of additional kinases were done as described (21). Antiproliferative activity was assessed in a cell proliferation assay measuring 14C-thymidine incorporation into cellular DNA as described (21).

Cell cycle analysis. Cells were stained with propidium iodide for analysis of nuclear DNA content using the CycleTEST PLUS DNA Reagent Kit (Becton Dickinson, San Jose, CA). Stained cells were run on a FACSCalibur (Becton Dickinson) with an excitation wavelength of 488 nm and an emission wavelength of 585 nm. Histograms were analyzed using ModFit (Verity Software House, Topsham, ME) to determine cell cycle distribution.

Cell synchronization. A modified mitotic shake method was used to synchronize cells in G1 (22). Asynchronous cells were tapped to dislodge mitotic cells and the dislodged cells were collected and incubated in fresh medium. Adherent cells were harvested after 4 hours. Cell cycle analysis showed that >93% of the resulting population of cells were in G1 (data not shown). To synchronize cells in G2-M, the microtubule depolymerizing drug nocodazole was used (23). Treatment with 200 nmol/L nocodazole for 16 hours synchronized 98% of the population in G2-M as determined by cell cycle analysis (data not shown).

CDK1 kinase activity in HeLa cell lysates. To eliminate variations in CDK1 kinase activity due to cell cycle position, the cell population was first synchronized in G2-M. Synchronized cells were then treated for 8 hours with either 200 nmol/L nocodazole plus vehicle or 200 nmol/L nocodazole plus 1 to 4 µmol/L JNJ-7706621 and cell cycle analysis was done to confirm that cells remained in G2-M arrest. Immunocomplexes were recovered from 1 mg of total cell lysate by incubation overnight with agarose conjugated total CDK1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and evaluated for CDK1 kinase activity with 1.5 µg of histone H1 substrate (Upstate Biotechnology, Lake Placid, NY) as described (24).

Immunoblot analysis. Lysates of asynchronous HeLa cells or cells synchronized in G2-M and treated with either nocodazole plus JNJ-7706621 or nocodazole plus vehicle, were separated by SDS-PAGE, transferred to nitrocellulose, and probed with the following antibodies: Wee1 (Santa Cruz Biotechnology); total CDK1, phospho-CDK1 (Thr161), phospho-CDK1 (Tyr15), Myt1, and phospho-retinoblastoma (Ser780) (Cell Signaling, Beverly, MA); cyclin B1 (BD PharMingen, San Diego, CA); phospho-Histone H3 (Ser10) (Upstate Biotechnology); and actin (Sigma). Secondary antibodies and enhanced chemiluminescence detection reagents were from Amersham Biosciences (Piscataway, NJ).

Colony formation assay. HeLa cells were plated at various densities in 90-mm diameter Petri dishes and exposed to JNJ-7706621 for 48 hours. Cells were then washed in PBS and incubated an additional 7 days in drug-free medium. Cells were fixed in 95% ethanol, stained with 0.5% crystal violet, and colonies containing >50 cells manually counted.

Apoptosis analysis. Detection of externalized phosphatidylserine on cell membranes and assessment of cell viability was done by dual staining with Annexin V and propidium iodide. U937 cells were treated with vehicle alone or JNJ-7706621 for 24 hours. Cells (5 x 105) were stained with Annexin V-FITC conjugate (Oncogene, San Diego, CA) for 15 minutes followed by propidium iodide and immediately analyzed flow cytometrically.

Human tumor xenograft studies. The effect of JNJ-7706621 on the growth of tumors in female nu/nu mice was done as previously reported (21). Briefly, animals were implanted s.c. with 1 mm3 A375 tumor fragments in the hindflank. After tumors reached 62 to 126 mg, groups were pair matched. Animals were given JNJ-7706621 or vehicle control starting on day 1. The tumor growth delay method was followed where each animal was euthanized when its neoplasm reached a predetermined size of 2.0 g. All statistical analyses were conducted using unpaired t tests at a P level of 0.05 (two tailed).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibition of cell cycle kinase activity in vitro. JNJ-7706621 (Fig. 1A) is a pan-CDK kinase inhibitor with highest potency on CDK1 and CDK2 but also exhibits activity towards CDK3, CDK4, and CDK6 (Table 1). For the in vitro CDK1 kinase assay, a Km for ATP of 17.12 ± 1.03 µmol/L was determined (data not shown) and the assay was carried out with 5 µmol/L total ATP. The IC50 value for inhibition of CDK1 by JNJ-7706621 at increasing concentrations of ATP was calculated and a linear relationship relative to the concentration of ATP present in the reaction was observed (Fig. 1B). This result is consistent with an ATP-competitive mode of inhibition of CDK1. In addition, JNJ-7706621 showed inhibition of Aurora-A and Aurora-B but had no activity at the highest concentration tested on the Plk1 or Wee1 serine/threonine kinases (Table 1). Therefore, its antiproliferative effects are likely attributable to the inhibition of a unique subset of kinases with important regulatory roles in cell growth and cell division.


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  		Table 1. In vitro kinase and cell proliferation assays IC50 (µmol/L)

 
Kinase selectivity profile. When assayed against a panel of representative tyrosine and serine/threonine kinases, JNJ-7706621 showed some inhibition of the VEGF-R2 and FGF-R2 tyrosine kinase receptors and also inhibition of GSK3ß (Table 1). It was less potent but still exhibited submicromolar inhibition of the VEGF-R3, Tie2, and FGF-R1 receptor tyrosine kinases and was relatively inactive on the remainder of the kinases profiled.

Effects of JNJ-7706621 on cell proliferation. JNJ-7706621 showed potent growth inhibition in vitro on all human cancer cell types examined with IC50 values ranging from 112 to 514 nmol/L (Table 1). The compound was several fold less potent at inhibiting growth of normal cell types, including fibroblast, smooth muscle, and endothelial cells, where IC50 values ranged from 3.67 to 5.42 µmol/L (Table 1). The IC50 for JNJ-7706621 was nearly identical in both the sensitive MES-SA and the P-glycoprotein overexpressing MES-SA/Dx5 cell line (25), indicating that the antiproliferative effect of this compound was not modulated by P-glycoprotein overexpression (Table 1).

Inhibition of CDK1 kinase activity in cells. The ability of JNJ-7706621 to inhibit CDK1 activity in cells is shown in Fig. 1C. Asynchronous cells had a low level of CDK1 kinase activity. Cells synchronized in G2-M and treated with vehicle alone exhibited a high level of CDK1 kinase activity, likely resulting from being growth arrested in G2-M and not due to microtubule depolymerization or direct modulation of CDK1 kinase activity by nocodazole (26). G2-M synchronized cells treated with JNJ-7706621 showed a dose-responsive decrease in CDK1 kinase activity with >50% inhibition from 1 µmol/L treatment.

Effects of JNJ-7706621 on CDK1 regulation in cells. The effects of JNJ-7706621 treatment on CDK1 phosphorylation status and cell cycle regulatory proteins were characterized by immunoblot analysis. CDK1 activity is positively regulated by an activating phosphorylation at Thr161 (27) and is maintained in an inactive state by inhibitory phosphorylations on Tyr15 and Thr14 mediated by Wee1 and Myt1. In the asynchronous HeLa cell population, most of the CDK1 was inactive with Tyr15 in the phosphorylated state (Fig. 1D). Cells synchronized in G2-M and treated with vehicle alone had a very low level of Tyr15 phosphorylation with most of the CDK1 exhibiting the activating Thr161 phosphorylation (Fig. 1D). G2-M synchronized cells treated with JNJ-7706621 showed low levels of the activating Thr161 and high levels of the inhibitory Tyr15 phosphorylation (Fig. 1D). These results are consistent with the reduction in CDK1 kinase activity observed after JNJ-7706621 treatment (Fig. 1C). The amount of total CDK1 protein did not change following nocodazole synchronization or drug treatment (Fig. 1D). Cyclin B1 levels were low in the asynchronous cell population and elevated in cells treated with nocodazole (Fig. 1D) as expected for cells arrested in G2-M (3). Wee1 protein levels were lower in nocodazole synchronized cells than in asynchronous cells (Fig. 1D). Expression of Wee1 is regulated by CDK1 and Plk1 catalyzed phosphorylation during mitosis, which targets it for ubiquitin-mediated degradation (28). A slower migrating species in nocodazole-arrested cells (Fig. 1D) is presumably hyperphosphorylated Wee1. Cells treated with JNJ-7706621 had higher levels of Wee1 and no hyperphosphorylated Wee1 compared with cells treated with vehicle alone (Fig. 1D), possibly as a result of inhibition of CDK1 kinase activity as has been described for the CDK inhibitor butyrolactone (28). Plk1 function is not affected by JNJ-7706621 treatment (Table 1); thus, the mechanism of targeting Wee1 for degradation is only partially inhibited. Myt1 kinase is also involved in the negative regulation of CDK1 by catalyzing the Tyr15 and Thr14 inhibitory phosphorylations, and during mitosis, Myt1 is inactivated by hyperphosphorylation (29). In asynchronous cells, Myt1 was present in an unphosphorylated faster migrating form (Fig. 1D). Cells synchronized in G2-M and treated with vehicle alone have Myt1 in its slower migrating hyperphosphorylated form, whereas cells synchronized in G2-M and treated with JNJ-7706621 express Myt1 in its active state where CDK1 kinase activity is inhibited (Fig. 1D). Retinoblastoma was found to be essentially unphosphorylated in asynchronous cultures of HeLa cells and highly hyperphosphorylated in the nocodazole-arrested cells (Fig. 1D). The hyperphosphorylation of retinoblastoma was prevented in JNJ-7706621-treated cells, which may result not only from inhibition of CDK1/cyclin B, the principal retinoblastoma kinase in mitotic cells (7), but also due to inhibition of other CDK isoforms by this compound given that inhibition of retinoblastoma phosphorylation at multiple residues was also indicated by the reduction of a wide band to a uniform single band following drug treatment.

JNJ-7706621 can induce a cytostatic or cytotoxic effect in HeLa cells. HeLa cells treated with 0.5 µmol/L JNJ-7706621 for 72 hours continued to proliferate at a reduced rate (Fig. 2A, {triangledown}). Cells treated with 1 µmol/L JNJ-7706621 maintained a cytostatic growth profile (no net growth; Fig. 2A, {triangleup}). Cells treated with 2 or 3 µmol/L JNJ-7706621 also maintained a cytostatic growth profile to 24 hours and thereafter viable cell numbers decreased below the number originally plated indicating a cytotoxic effect (Fig. 2A, {circ} and {square}).



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  		Figure 2. JNJ-7706621 inhibits cell growth and induces apoptosis. A, exponentially growing HeLa cells were treated with DMSO only ({blacksquare}) or with 0.5 µmol/L ({triangledown}), 1 µmol/L ({triangleup}), 2 µmol/L ({circ}), or 3 µmol/L ({square}) JNJ-7706621 in DMSO. Cells were harvested by trypsinization at the indicated times and live cell number determined with trypan blue staining. Total cell population (monolayer cells and cells in the medium) was counted. All groups were set up with n = 3; bars, SD. B, exponentially growing HeLa cells were treated with 1 µmol/L ({triangleup} and {blacktriangleup}) or 3 µmol/L ({square} and {blacksquare}) JNJ-7706621 for 48 hours. Cells (monolayer cells and cells in medium) were then washed twice in PBS (arrow), refed, and maintained in the presence of either 1 µmol/L ({triangleup}) JNJ-7706621, 3 µmol/L ({square}) JNJ-7706621, or 0.03% DMSO ({blacktriangleup} and {blacksquare}). Live cell numbers of the total population were determined as described above. All groups were set up with n = 3; n SD. C, colony formation in HeLa cells exposed to JNJ-7706621 for 48 hours and then incubated an additional 7 days in drug-free medium. All groups were set up with n = 3; bars, SD. D, U937 cells were exposed to JNJ-7706621 for 24 hours, stained with Annexin V-FITC and propidium iodide (PI), and analyzed flow cytometrically.

 
Reversibility of growth effects of JNJ-7706621 is concentration dependent. To investigate the reversibility of growth effects following exposure to JNJ-7706621, HeLa cells were exposed to either a cytostatic (1 µmol/L) or cytotoxic (3 µmol/L) dose of JNJ-7706621 for 48 hours (Fig. 2A). Cells were then washed twice with PBS (Fig. 2B, arrow) and maintained in the presence of either 1 or 3 µmol/L JNJ-7706621 or vehicle alone (Fig. 2B). Washed cells maintained in the presence of 1 µmol/L drug showed no net growth up to 144 hours (Fig. 2B, {triangleup}), whereas those maintained in the presence of 3 µmol/L drug continued to die, and by the 144-hour time point, almost all the cells were dead (Fig. 2B, {square}). When cells treated with 1 µmol/L drug for 48 hours were washed and placed in fresh medium containing vehicle alone, they resumed exponential growth (Fig. 2B, {blacktriangleup}). When cells treated with 3 µmol/L drug for 48 hours were washed and placed in fresh medium containing vehicle alone, they experienced a lag period of 48 hours before resuming exponential growth (Fig. 2B, {blacksquare}). These results indicate the reversibility of the effects of JNJ-7706621 on cell growth is dependent on the concentration of the initial exposure and JNJ-7706621 can have a lasting effect on cells in vitro.

Inhibition of colony formation in HeLa cells. To determine the effects of JNJ-7706621 on long-term survival, the ability of HeLa cells to form colonies after drug exposure was evaluated. A 48-hour exposure to 1 µmol/L JNJ-7706621 inhibited colony formation by 50% and a 48-hour exposure to 3 µmol/L drug inhibited colony formation by 95.5% (Fig. 2C).

Induction of apoptosis in human cancer cells. To investigate if JNJ-7706621 treatment induces apoptosis, U937 cells were examined for alterations in the membrane phospholipid, phosphatidylserine. In cells treated with vehicle alone, only a small percentage of the population was in early or late apoptosis (Fig. 2D). When cells were treated with 0.5 or 1 µmol/L drug for 24 hours, an increase in the numbers of early and late apoptotic cells was observed along with an increase in dead cells (Fig. 2D). Cells exposed to higher concentrations of drug (2-4 µmol/L) for 24 hours showed greater increases in both late apoptotic and dead cells (Fig. 2D). These data show that the extent of apoptosis induced in cells exposed to JNJ-770662 depends on drug concentration and length of exposure.

JNJ-7706621 delays exit from G1, arrests cells in G2-M, and induces endoreduplication. To more precisely define the cell cycle effects of JNJ-7706621, HeLa cells were synchronized in G1 by mitotic shake and treated with various concentrations of compound. Cells were harvested at specific times after drug treatment and cell cycle analysis done. Control cells (treated with DMSO only) entered the S phase 4 hours after G1 synchronization (Fig. 3A). However, cells treated with 3 µmol/L JNJ-7706621 did not enter the S phase until 16 hours after G1 synchronization (Fig. 3A). Figure 3B depicts time of S-phase entry of cells treated with various concentrations of drug and shows that JNJ-7706621 delayed entry of cells into the S phase in a dose-dependent manner. The time to complete the S phase was ~8 hours in both control cells and 3 µmol/L drug-treated cells (Fig. 3A). A total S-phase transition time of 8 hours was also seen in cells treated with 1 and 2 µmol/L drug (data not shown). Therefore, JNJ-7706621 has no effect on S-phase progression in HeLa cells. As shown in Fig. 3C, JNJ-7706621 arrests cells in G2-M. Cells synchronized in G1 are arrested in G2-M after 24 hours of drug treatment. At 3 µmol/L drug, cells were still arrested in G2-M after 72 hours of drug exposure, whereas at 2 µmol/L drug, cells began to move out of G2-M between 36 and 48 hours and a population of cells with a >4N DNA content emerged at 72 hours. The observed endoreduplication suggests that JNJ-7706621 also has an effect on Aurora kinase activity (18, 19).



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  		Figure 3. JNJ-7706621 delays exit from G1, arrests cells in G2-M, and induces endoreduplication. A, HeLa cells were synchronized in G1 by mitotic shake and treated with JNJ-7706621. Cell cycle analysis showed that control cells (exposed to vehicle alone) began to enter the S phase 4 hours after G1 synchronization. Cells treated with 3 µmol/L JNJ-7706621 did not enter the S phase until 16 hours after G1 synchronization. S-phase transit time was identical in both control cells and drug treated cells. B, in G1 synchronized cells, JNJ-7706621 delayed S-phase entry in a dose-dependent manner. C, cell cycle analysis showed that G1 synchronized cells exposed to 2 or 3 µmol/L drug were predominantly in G2-M phase after 24 hours of treatment. Cells exposed to 2 µmol/L JNJ-7706621 began to move out of G2-M between 36 and 48 hours and an 8N population emerged by 72 hours. Cells exposed to 3 µmol/L JNJ-7706621 remained arrested in G2-M out to 72 hours. D, level of histone H3 phosphorylation on Ser10 was analyzed by Western blotting in lysates of asynchronous HeLa cells and HeLa cells synchronized with nocodazole and treated with either vehicle or JNJ-7706621. Band intensity was quantified on a Lumi-Imager F1 (Roche Diagnostics, Indianapolis, IN) and normalized to the level of ß-actin. E, HeLa cells were synchronized in G2-M with nocodazole and exposed to nocodazole plus 0.5 µmol/L JNJ-7706621 for an additional 24 hours. Cell cycle analysis showed a large population of cells with DNA contents >4N. Cells exposed to nocodazole plus vehicle alone were maintained in G2-M with no emergence of DNA polyploidy (data not shown).

 
Inhibition of Aurora kinases by JNJ-7706621. To provide evidence that JNJ-7706621 inhibited Aurora activity in cells, the effect on histone H3, a direct downstream target of the Aurora kinases, was evaluated. Histone H3 is involved in chromosome condensation and phosphorylation on Ser10 by Aurora-B kinase is thought to be crucial for proper chromosome alignment and cytokinesis (18, 30). Lysates of asynchronous HeLa cells and cells synchronized in G2-M and treated for 8 hours with either JNJ-7706621 or vehicle were evaluated for levels of phospho-histone H3 (Ser10) by immunoblotting. Nocodazole-synchronized cells exhibited a high level of histone H3 phosphorylation relative to untreated asynchronous cells. Cells synchronized with nocodazole and treated with 0.5 µmol/L JNJ-7706621 exhibited ~60% inhibition of histone H3 phosphorylation and cells exposed to higher amounts of drug had levels equivalent to asynchronous cells (Fig. 3D). These results show that JNJ-7706621 suppressed histone H3 phosphorylation on Ser10 in vitro indicating that Aurora-B kinase was inhibited. Aurora kinase inhibitors have been reported to compromise nocodazole-induced spindle checkpoint activation; cells arrested with nocodazole for prolonged periods have been shown to exit G2-M when treated with the Aurora inhibitor, ZM447439 (18). To test whether JNJ-7706621 had a similar effect on arrested cells, HeLa cells were treated with nocodazole for 16 hours and then treated for an additional 24 hours with either nocodazole plus JNJ-7706621 or nocodazole plus vehicle. Cells were exposed to 0.5 µmol/L JNJ-7706621 as this concentration does not induce a cell cycle arrest (data not shown) but has a significant effect on histone H3 phosphorylation (Fig. 3D). In cells treated with nocodazole plus vehicle, cell cycle analysis showed that G2-M arrest was maintained throughout the 40-hour treatment period (data not shown). In cells treated with nocodazole plus 0.5 µmol/L JNJ-7706621, significant numbers of cells were released from G2-M arrest and endoreduplicated resulting in ~33% of the population with DNA contents >4N (Fig. 3E). These results show that JNJ-7706621 can compromise checkpoint arrest induced by nocodazole and suggest that the compound has an inhibitory effect on Aurora-B kinase.

Human tumor xenograft studies with intermittent dosing. The antitumor efficacy of JNJ-7706621 was examined in an A375 melanoma human tumor xenograft model. Two dose levels, 100 and 125 mg/kg, were evaluated and mean tumor size was calculated from six animals per group. Figure 4A shows tumor sizes for the 125 mg/kg dose under various schedules. Daily dosing was the most efficacious and caused tumor regression; however, this schedule could only be tolerated for 22 days before toxicity emerged (Table 2). There were five treatment-related deaths in this dose group; all occurred between days 22 and 39 and were not preceded by detectable weight loss. The 7 on/7 off schedule was nearly as effective as the daily dosing regimen with 93% tumor growth inhibition (TGI) and all animals survived to the end of the study (Fig. 4A; Table 2). The next most effective schedule at 125 mg/kg was 7 on/14 off (88% TGI) followed by Q3D and Q4D, with 69% and 43% TGI, respectively, and all these schedules were well tolerated (Fig. 4A; Table 2). Identical dosing schedules were applied to evaluate the 100 mg/kg dose and the same pattern of efficacy was observed (Table 2). Several schedules and dose combinations resulted in equivalent efficacy. For example, the 125 mg/kg 7 on/7 off schedule and the 100 mg/kg QD schedule produced identical TGI values of 93% (Fig. 4B; Table 2). Figure 4C compares two different dosing schedules at the same dose level. Tumor growth was nearly flat under the QD regimen ({circ}), whereas under the 7 on/7 off schedule, a pattern of tumor inhibition and regrowth was observed ({bullet}). A reduction in tumor size was apparent during periods of dosing (days 1-7, 14-21, and 29-35), and tumor regrowth was observed during periods of nondosing (days 8-14, 22-28, and 36-41). However, at a slightly higher dose level of 125 mg/kg but under the same schedule of 7 on/7 off, there was a persistent effect evident during dosing holidays with very little tumor regrowth (Fig. 4B). Analysis of the relationship between tumor size and dose indicates that the amount of inhibition of tumor growth was proportional to the total cumulative dose, regardless of the schedule. Figure 4D shows the average tumor size versus the total cumulative dose calculated on day 11 of the study. This relationship held true for analysis done at any time during the study (data not shown). These results identify suitable dosing regimens that could be applied in clinical trials.



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  		Figure 4. JNJ-7706621 is efficacious in a human tumor xenograft model under intermittent dosing regimens. A, JNJ-7706621 was given i.p. formulated in a nanocrystal suspension at 125 mg/kg under various schedules as indicated and tumor growth was measured over a 42-day period. Control animals were treated with vehicle alone (dotted line). B, several schedules and dose combinations resulted in equivalent efficacy. JNJ-7706621 given daily at 100 mg/kg ({circ}) showed equivalent activity to compound dosed at 125 mg/kg on a 7 on/7 off schedule ({blacksquare}). Solid bars on the x-axis indicate periods of dosing for the 125 mg/kg 7 on/7 off regimen and gaps show dosing holidays. C, compound given at 100 mg/kg showed greater efficacy in groups receiving daily doses ({circ}) than in groups on a 7 on/7 off schedule ({bullet}). A pattern of tumor inhibition during periods of dosing and tumor regrowth during nondosing was observed in the 7 on/7 off schedule. Solid bars on the x-axis indicate periods of dosing for the 100 mg/kg 7 on/7 off regimen and gaps show dosing holidays. D, when total cumulative dose was plotted against mean tumor size, there was a linear correlation between total amount of compound given and the size of the tumors regardless of the schedule of administration. Data from day 11 of the study is shown where r2 = 0.913, but the trend continued throughout the study. For all treatment groups (n = 6) and for control group (n = 10).

 

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  		Table 2. In vivo activity of JNJ-7706621 in A375 melanoma human tumor xenograft model under intermittent dosing regimens

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
JNJ-7706621 is a promising antitumor agent that inhibits cell growth, activates apoptosis, and induces cytotoxicity in human cancer cells. This activity is a result of potent inhibition of several CDKs and Aurora kinases. When IC50 values for JNJ-7706621 are compared with published values for other CDK inhibitors, JNJ-7706621 is a more potent inhibitor of CDK1, CDK2, and CDK4 than BMS-387032 (31) and exhibits superior potency to Flavopiridol on CDK1 and CDK2 but shows less activity towards CDK4 and CDK6 than Flavopiridol (17). JNJ-7706621 showed moderate inhibition of several receptor tyrosine kinases involved in angiogenesis including VEGF-R2, FGF-R2, VEGF-R3, Tie2, and FGF-R1 that may contribute to its antitumor activity, especially in the A375 melanoma model as this cell type expresses VEGF-R2 and secretes the vascular endothelial growth factor (VEGF) ligand (32). The GSK3 kinases are phylogenetically most closely related to the CDKs and consequently, most CDK inhibitors have been found to be good inhibitors of GSK3ß due to the similarity of the ATP binding domain in the GSK family. In fact, both UCN-01 and Flavopiridol inhibit GSK3ß with nearly equal potency to CDK1 (33). In contrast, JNJ-7706621 is markedly more potent towards CDK1 than it is to GSK3ß.

JNJ-7706621 shows potent antiproliferative activity in all cancer cell types evaluated, regardless of p53, retinoblastoma status, or P-glycoprotein expression level, and is several fold less potent at inhibiting normal cell growth. The in vitro potency of JNJ-7706621 against various human cancer cell types is comparable with Flavopiridol and BMS-387032 (17, 31). The differential antiproliferative activity on normal cells relative to human cancer cell lines could be partially due to the longer cycling time of the primary cell types. The population doubling time of human umbilical vascular endothelial cell cells has been reported to be 92 hours (34), HASMC cells 70 to 85 hours (35), HMVEC cells 72 hours (36), and MRC-5 cells 51 hours (37). The cancer cell lines cycle much faster, with doubling times of 18 to 19 hours in HeLa cells (38), 16 to 18 hours for HCT116 (39), and 16 hours for A375 (40). This ability to target rapidly cycling cells may translate to a wider therapeutic index in the clinic. In vivo, there may be even less of an effect on endothelial and other cell types that are not actively cycling. The antiproliferative activity of the compound was not affected by P-glycoprotein overexpression and resulted in nearly identical IC50 values in genetically matched drug-sensitive and drug-resistant cells. In contrast, BMS-387032 has been reported to act as a substrate of P-glycoprotein (41).

In cells, 1 µmol/L JNJ-7706621 reduced kinase activity of CDK1 immunocomplexes >50% and to baseline levels at ≥2 µmol/L. The difference observed between cellular potency and in vitro potency is not surprising for an ATP-competitive kinase inhibitor. In the in vitro kinase assay, JNJ-7706621 exhibited an IC50 of 5 nmol/L at 1 µmol/L ATP and 85 nmol/L at 100 µmol/L ATP (Fig. 1B) and would be expected to be less potent in cells where ATP concentrations can reach 1 mmol/L. When cells were treated with JNJ-7706621, the CDK1 protein displayed high levels of the inhibitory Tyr15 phosphorylation and low levels of the activating Thr161 phosphorylation. The cdc25C phosphatase which is responsible for removing the Thr14 and Tyr15 phosphates is activated in the M phase by phosphorylation of its NH2-terminal regulatory domain by CDK1 (27); thus, its ability to dephosphorylate these residues may be compromised in drug-treated cells. Similarly, this compound may interfere with CDK7 directly or through a feedback loop to influence Thr161 phosphorylation. Wee1 protein stability is reportedly regulated by feedback from CDK1 and also by other kinases such as Plk1. The degradation of Wee1 is likely not as complete in cells treated with JNJ-7706621 due to CDK1 inhibition. CDK1 is able to catalyze phosphate addition on both serine and threonine residues of Myt1, and although this does not directly reduce its kinase activity (29), Myt1 hyperphosphorylation could be blocked as a result of CDK1 inhibition. The inhibition of retinoblastoma hyperphosphorylation in nocodazole-arrested and JNJ-7706621-treated cells likely occurs through inhibition of multiple CDKs. Although the Ser780 residue has been shown to be phosphorylated by CDK4 (42), the retinoblastoma protein detected by this phospho-specific antibody also seems to be phosphorylated at several additional residues, which is catalyzed by other CDKs (43).

The principal effects of this compound on cells stem from its ability to delay transit through the cell cycle and induce a G2-M arrest. These effects are consistent with JNJ-7706621 acting primarily as a CDK inhibitor, although additional mechanisms are also functioning such as inhibition of Aurora kinases. Potent inhibition of CDK1/Cyclin B results in the expected mitotic arrest phenotype, but the delay of G1-phase progression observed in G1 synchronized cells reflects inhibition of other CDK family members that control G1 and S phases (CDK2/Cyclin E or CDK4, CDK3 and CDK6/Cyclin D). The inhibition of GSK3ß may be another factor in the G1-S delay as this kinase has been reported to be involved in Cyclin D1 degradation (44). JNJ-7706621 shows significant inhibition of Aurora-A, which is required for activation of CDK1/CyclinB in mitosis and is essential for recruitment of CDK1/Cyclin B to centrosomes (45), as well as Aurora-B. It should be noted that the DNA polyploidy expected to occur as a result of Aurora-B inhibition may not be apparent at all drug concentrations due to CDK inhibition which takes place upstream from Aurora-mediated functions and precludes endoreduplication events. Endoreduplication was observed in cells treated with 2 µmol/L but not 3 µmol/L JNJ-7706621 and only after incubations of ≥72 hours presumably because cells were released from G2-M arrest following exposure to 2 µmol/L compound but were held in G2-M in the presence of 3 µmol/L compound thus masking any effects on chromosome segregation due to Aurora-B inhibition. However, when cells were first treated with nocodazole to arrest the population in G2-M and then treated with concentrations of JNJ-7706621 that did not induce G2-M arrest, a large proportion of the cells underwent endoreduplication. This shows that JNJ-7706621 can compromise nocodazole-induced spindle checkpoint activation. Furthermore, the phosphorylation of histone H3 on Ser10 was inhibited by drug treatment. This phosphorylation event and the phenotype of compromised spindle checkpoint function is mediated through Aurora-B (18, 30); thus, this effect could be attributed to Aurora-B inhibition by JNJ-7706621.

Tumor xenograft studies showed that efficacy improved as dose and frequency of administration increased. However, tolerability issues limited the doses and schedules that could be applied in rodents. By manipulating the dose and schedule, equivalent activity could be achieved while minimizing adverse effects of the compound. From these studies, the optimal schedule to follow can be selected and used as a starting point for clinical trials.

We have described a novel kinase inhibitor that shows activity against multiple CDK family members and Aurora kinases. In preclinical models, the effects on cell cycle progression, modulation of CDK and Aurora regulatory pathways, and induction of apoptosis indicate the profile of a promising antineoplastic agent. These findings support further clinical investigation to determine the therapeutic potential of this compound.


   Acknowledgments
 
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.

Received 3/16/05. Revised 7/20/05. Accepted 7/22/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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