Genotoxic antitumor agents continue to be the mainstay of current cancer chemotherapy. These drugs cause DNA damage and activate numerous cell cycle checkpoints facilitating DNA repair and the maintenance of genomic integrity. Most human tumors lack functional p53 and consequently have compromised G1-S checkpoint control. This has led to the hypothesis that S and G2-M checkpoint abrogation may selectively enhance genotoxic cell killing in a p53-deficient background, as normal cells would be rescued at the G1-S checkpoint. CHK1 is a serine/threonine kinase associated with DNA damage–linked S and G2-M checkpoint control. SAR-020106 is an ATP-competitive, potent, and selective CHK1 inhibitor with an IC50 of 13.3 nmol/L on the isolated human enzyme. This compound abrogates an etoposide-induced G2 arrest with an IC50 of 55 nmol/L in HT29 cells, and significantly enhances the cell killing of gemcitabine and SN38 by 3.0- to 29-fold in several colon tumor lines in vitro and in a p53-dependent fashion. Biomarker studies have shown that SAR-020106 inhibits cytotoxic drug–induced autophosphorylation of CHK1 at S296 and blocks the phosphorylation of CDK1 at Y15 in a dose-dependent fashion both in vitro and in vivo. Cytotoxic drug combinations were associated with increased γH2AX and poly ADP ribose polymerase cleavage consistent with the SAR-020106–enhanced DNA damage and tumor cell death. Irinotecan and gemcitabine antitumor activity was enhanced by SAR-020106 in vivo with minimal toxicity. SAR-020106 represents a novel class of CHK1 inhibitors that can enhance antitumor activity with selected anticancer drugs in vivo and may therefore have clinical utility. Mol Cancer Ther; 9(1); 89–100

Normal cell division is a highly regulated process involving multiple cyclin-dependent kinases (CDK) and their appropriate cyclins that control a series of cell cycle checkpoints that ensure the fidelity of DNA replication (1). Genotoxic antitumor drugs cause DNA damage and activate cell cycle checkpoints at G1-S, S, and G2-M, which are required for DNA repair and the maintenance of genomic integrity (2). The transcription factor p53 is a critical checkpoint protein. In the presence of damaged DNA, p53 is stabilized and activated, causing the upregulation of the CDK inhibitor p21, which induces a subsequent delay in the cell cycle to facilitate DNA repair and/or apoptosis (2, 3). The observation that human cancers frequently have defects in p53 function, through either direct mutations or the disruption of its regulatory pathway, with a consequent loss of G1-S checkpoint control (3, 4) has led to the concept that G2 checkpoint inhibitors combined with DNA-damaging agents may cause aberrant and lethal mitosis in such tumors, whereas normal tissue would be rescued at the p53-dependent G1-S checkpoint (2, 57). This strategy should therefore increase the therapeutic selectivity of several DNA-damaging anticancer drugs, which are still the mainstay of current cancer treatment.

In normal cells, DNA damage is sensed by a complex series of signal transduction pathways. The main proximal kinases are the phosphoinositide 3-kinase homologues ATM and ATR (2, 8). These transduce signals to the effector kinases CHK1 and CHK2, which then activate a series of cell cycle checkpoints. CHK1 is predominantly activated through phosphorylation on S317 and S345 catalyzed by ATR (9, 10), and undergoes autophosphorylation on S296 (11, 12). CHK1 is a serine/threonine kinase that is involved in the S-phase checkpoint through stabilizing replication forks, and in the G2 checkpoint control through regulating the stability and availability of the CDC25 phosphatases (1315). Numerous studies have shown that UCN-01, a relatively nonselective inhibitor of CHK1, will preferentially sensitize p53-defective tumor cells to a variety of DNA-damaging anticancer drugs both in vitro and in vivo (1618). More recently, inhibition of CHK1 function by knockdown of protein expression by small interfering RNA and several more selective CHK1 inhibitors have confirmed these early observations (1921). As a part of the development of molecularly targeted kinase inhibitors, particularly those that might not have antitumor activity in their own right, it is important to identify appropriate biomarker readouts (22, 23). Consequently, the development of CHK1 inhibitors and the characterization of suitable biomarkers have become an important drug development objective (2428).

In this study, we present the preclinical pharmacology and therapeutic utility of SAR-020106, a novel, selective, and potent inhibitor of CHK1. We show that SAR-020106 can potentiate SN38 and gemcitabine cytotoxicity in several colon cancer cell lines in vitro and that this potentiation occurs preferentially in cells that lack p53 function. We further show that SAR-020106 can abrogate an SN38-induced S-phase block and an etoposide-induced S and G2-M arrest in vitro. Biomarker studies showed that SAR-020106 selectively inhibited SN38 or gemcitabine induced autophosphorylation of CHK1 at S296, and that this was associated with a loss of CDK1 phosphorylation at Y15, a downstream target of CHK1. Increases in γH2AX and poly ADP ribose polymerase (PARP) cleavage were also observed, consistent with enhanced DNA damage and cell death. Finally, we present evidence of enhancement of irinotecan and gemcitabine therapeutic activity by SAR-020106 in association with CHK1 inhibition in human tumor xenografts.

Compounds

SAR-020106 was synthesized as described previously (29). Clinical formulations of gemcitabine and irinotecan were obtained from Eli Lilly (Gemzar, Eli Lilly and Pfizer (Campto, Pfizer), respectively. SN38, the active metabolite of irinotecan, was purchased from LKT Laboratories. Other reagents were obtained from Sigma-Aldrich Chemical Co.

Cells

HT29, SW620, and Colo205 human colon carcinoma cell lines were obtained from American Type Culture Collection (American Type Culture Collection lot no. 4487729, 3924081, and 57723824, respectively) and A2780 ovarian carcinoma cells were obtained from European Collection of Animal Cell Cultures (Sigma-Aldrich). Cells were grown in DMEM containing 10% fetal bovine serum (PAA “Gold”) and 5 mmol/L glutamine in a humidified atmosphere of 5% CO2 at 37°C and passaged ≤6 mo before the renewal from frozen, early passage stocks indicated. A2780 cells were stably transfected with HPV16E6, and appropriate clones were screened and selected essentially as described previously (30). Cells were regularly screened for Mycoplasma using a PCR-based assay (VenorGem, Minerva Biolabs).

Kinase Assays

CHK1 and CHK2 assay conditions were as described previously (31). A kinase profile against 50 different human kinases was carried out using 10 μmol/L SAR-020106 with an ATP concentration equivalent to the Km for each enzyme (Millipore).

Cytotoxicity Assays

Cells were plated in 96-well plates (3–6 × 103 per well) or tissue culture flasks (at 2–5 × 105 per flask) and allowed to attach for 36 h to ensure exponential growth at the time of treatment. Cytotoxicity was determined using a 96-h (four cell doublings) SRB assay, and GI50 values were derived as described previously (32).

G2 Checkpoint Abrogation Assay

Measurements of intracellular CHK1 inhibition were carried out using HT29 and SW620 in a cell-based ELISA assay. Cells in 96-well plates (1–2 × 104 per well) were treated with etoposide (50 μmol/L × 1 h) to induce a late S-G2 arrest and then were exposed to different concentrations of the test compound for a further 21 h in medium containing nocodazole (100 ng/mL) to trap cells in mitosis. Cells were then fixed in 4% formaldehyde in PBS for 30 min at 4°C and permeabilized with ice-cold methanol for a further 10 min. Cells were washed in PBS, blocked with 5% milk in PBS × 30 min at 37°C, and then treated overnight with an antibody to mitotic proteins (MPM-2, Upstate) in 5% milk in TBS. Cells were then rinsed in water containing 0.01% Tween 20 and were treated for 1 h with an Eu-labeled secondary antibody (Eu-N1, Perkin-Elmer) at 37°C. The MPM-2 epitope–expressing (i.e., mitotic) cells were quantified using a Wallac Victor 1420 multilabel counter (Perkin-Elmer). IC50 values for mitosis induction were determined using nocodazole as a positive control. The activity index was used as a measure of a compound's ability to induce mitosis relative to its cytotoxicity (i.e., ratio of the G2 checkpoint abrogation IC50 and SRB cytotoxicity GI50).

Potentiation Assay

Cells were exposed to a fixed concentration of the cytotoxic agent that inhibited growth by 50% relative to untreated controls (GI50 dose) in combination with increasing concentrations of the CHK1 inhibitor in a 96-h SRB assay. The ability of SAR-020106 to enhance genotoxic cell killing was expressed as a potentiation index (PI), which was the ratio of GI50 for the CHK1 inhibitor alone and GI50 for the CHK1 inhibitor in combination with a cytotoxic agent.

Immunoblotting

Drug-treated cells were lysed in a buffer containing 50 mmol/L HEPES (pH 7.4) 250 mmol/L NaCl, 0.1% NP40, 1 mmol/L DTT, 1 mmol/L EDTA, 1 mmol/L NaF, 10 mmol/L β-glycerophosphate, 100 mmol/L NaVO4, and 1× Complete proteinase inhibitor tablet per 10-mL buffer (Roche). Tumor samples were homogenized using a buffer containing 50 mmol/L Tris (pH 7.4), 1 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, 1 mmol/L NaF, 1 mmol/L NaVO4, 5 μmol/L Fenvalerate, 5 μmol/L Vbphen, 10 mg/mL TLCK, 1× Complete inhibitor tablet per 10-mL buffer (Roche), protease inhibitor cocktail, and phosphatase inhibitor 1 and 2 (Sigma-Aldrich; ref. 33). Protein concentrations were measured by Bradford assay. Proteins samples (50 μg) were denatured in Laemmli buffer and were separated on precast 10% or 16% Tris-glycine gels (Novex, Invitrogen). Samples were transferred to polyvinylidene difluoride membranes, which were incubated in a blocking buffer [5% dried milk in 50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl and 0.1% Tween 20] for 1 h, probed with primary antibody in blocking buffer overnight at 4°C, and detected with HRPO conjugated secondary antibody (Bio-Rad). Proteins were visualized with enhanced chemiluminescence reagents (Pierce, Thermo Fisher) on Hyperfilm (GE Healthcare) using a Compact X4 developer (Xograft). Antibodies used were as follows: pS296 CHK1, pS317 CHK1, pY15 CDK1, total CDK1, cleaved PARP (CST), Total CHK1 (SC), pS139 H2AX, total H2AX (Upstate), and glyceraldehyde-3-phosphate dehydrogenase (Chemicon, Millipore).

Cell Cycle Analysis

Cells were stained with bromodeoxyuridine (BrdUrd) and PI as described previously (34). Cells were pulsed with 10 μmol/L BrdUrd for 30 min before harvesting and fixation in 70% ethanol. Subsequently, cells were acid denatured in 2 mol/L HCl containing trypsin to obtain nuclei. These were stained with a primary antibody to BrdUrd (Clone Bu20a, DAKO) and then conjugated to a FITC-labeled secondary antibody (Sigma-Aldrich) and stained with PI overnight. Cells were analyzed on a BD LSR II flow cytometer (Becton Dickinson). Cell cycle analysis was carried out on gated single-cell events using WinMDI 2.8.

Mice and Antitumor Studies

BALB/c mice were obtained from Charles River. Nude mice (CrTac:Ncr-Fox1nu) were bred in-house and mice ages 6 to 10 wk were used. Mice were allowed access to food (rodent diet no. 5002, Lab Diet) and water ad libitum. Tumor cells were inoculated s.c. in to both flanks of the recipient mice and palpable tumors were treated at an average diameter of 0.38 to 0.47 cm. Animals were randomized into treatment groups of six to eight mice and drugs were administered either i.p. or i.v. at 0.01 mL/g as appropriate. SAR-020106 was dissolved in 10% DMSO and 5% Tween 20 in saline and was administered i.p. either alone or 1 h before or 24 h after the cytotoxic drug administration. Irinotecan was administered on days 0, 7, and 14 at 12.5 mg/kg i.p. and SAR-020106 was administered on days 0, 1, 7, 8, 14, and 15 at 40 mg/kg i.p. either alone or in combination. In the latter case, the CHK1 inhibitor was administered 1 h before and 24 h after irinotecan. Similarly, gemcitabine was administered alone on days 0, 3, 6, and 9 at 60 mg/kg i.v. and SAR-020106 was administered alone on days 0, 3, 6, and 9 at 40 mg/kg i.p. For combination studies, gemcitabine was administered as above except that SAR-020106 was administered either 1 h before or 24 h after the cytotoxic agent. Mice were monitored daily and tumor size and animal weight were measured every 2 or 3 d. Tumor volumes were calculated from two orthodiagonal measurements and growth delay estimates were derived from growth curves at 300% tumor volume, a value that all tumors grew up to or through. Animals were treated in accordance with local and national animal welfare guidelines (35).

Pharmacokinetics

Drugs were extracted from plasma and tissue homogenates (25% w/v in water) using three volumes of acetonitrile containing internal standard. Drug concentrations were determined using reverse-phase high performance liquid chromatography and mass spectrometry analysis. Briefly, drugs were separated on a UPLC, BEH, C18 column (Waters), using gradient elution (9 mmol/L ammonium acetate in acetonitrile to 100% acetonitrile over 3 min and held for 1.6 min) with a flow rate of 0.4 mL/min at 55°C. SAR-020106 was identified and quantified by mass spectrometry analysis (MRM transition of 383.1–86.1 at 15 kV collision energy using an Agilent 1200 LC system coupled to an Agilent 6410 triple quadrupole mass spectrometer with electrospray ionization in positive ionization mode). Noncompartmental pharmacokinetic parameters were determined using WinNonLin software version 5.2.

Statistics

Statistical significance (*, P < 0.05; **, P < 0.01; and ***, P < 0.001) was determined using unpaired, one-tailed, t tests as appropriate, with GraphPad Prism 4 software.

Potent and Selective Inhibition of CHK1 by SAR-020106

SAR-020106 (Fig. 1) was shown to be a potent inhibitor of recombinant human CHK1 in vitro with an IC50 of 13.3 ± 1.3 nmol/L (mean ± SD, n = 3). Ki determination confirmed that the compound was acting as an ATP mimetic (Ki = 10.9 nmol/L). The corresponding IC50 values for CHK2 and CDK1 were >10 μmol/L. Kinase profiling with 10 μmol/L SAR-020106 showed minimal cross-reactivity with Aurora A, CDK1, CDK2, and CHK2 (>75% remaining activity compared with 2% for CHK1) and with 38 other kinases. Inhibition of Flt3, IRAK4, Met, MST2, p70S6K, Ret, RSK1, and Trk A (<20% activity remaining; see Supplementary Table 1) was observed.

Figure 1.

Structure of SAR-020106.

Figure 1.

Structure of SAR-020106.

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SAR-020106 Abrogates Genotoxic-Induced Cell Cycle Arrest

A series of experiments were carried out to explore the ability of SAR-020106 to abrogate DNA damage–induced cell cycle checkpoints. The use of BrdUrd allowed the resolution of G1 from early S checkpoint and late S from G2-M checkpoints, which cannot always be resolved using simple PI-based analyses. Initial experiments were carried out in SW620 colon carcinoma cells following SN38 treatment. Figure 2A and B show that there were minimal effects of SAR-020106 alone on cell cycle distribution up to a concentration of at least 1 μmol/L. A concentration of 20 nmol/L SN38 alone caused a profound S-phase arrest at 24 hours (83.6%) with only 1.8% in G1 and 10.7% in G2-M with the remaining 3.9% cells in a noncycling S-phase (S'). The combination of SN38 with SAR-020106 (0.1 μmol/L) caused a marked abrogation of this SN38-induced S-phase block (35.8% S phase) with a subsequent increase in G2-M to 59.8%. Increasing concentrations of SAR-020106 up to 1 μmol/L seemed to cause only minor changes in this S-phase population but a slight decrease in G2-M with a correspondingly small increase in G1. Consequently, there was clear evidence that SAR-020106 caused abrogation of SN38-induced S-phase arrest. A summary of the cell cycle effects obtained in HT29 colon carcinoma cells treated with etoposide (50 μmol/L × 1 hour) and subsequently exposed to SAR-020106 for 23 hours is shown in Fig. 2C. Etoposide treatment alone induced a late S-phase and G2-M arrest relative to controls (52.8% and 49.3%, respectively, in drug-treated cells) and 0.1 μmol/L SAR-020106 caused a profound abrogation of this S-phase block and a slight increase in the G2-M population (19.1% and 55.2%, respectively). An increase in concentration to 0.5 μmol/L SAR-020106 partially abrogated this G2-M block with a corresponding increase in G1 and S', with minimal changes in this distribution occurring at higher doses (1 μmol/L). These data clearly show that SAR-020106 abrogated an etoposide-induced S and G2 arrest.

Figure 2.

Effects of SAR-020106 on drug-induced cell cycle arrest in SW620 and HT29 colon cancer cells. A, effects of different concentrations of SAR-020106 alone (0.1, 0.5, and 1 μmol/L) or in combination with a fixed concentration of SN38 (20 nmol/L) for 24 h on cell cycle distribution in SW620 cells. Histograms, cell cycle distribution as measured by propidium iodide (PI) DNA staining; dot plots, DNA synthesis as measured by BrdUrd incorporation. Cell cycle distribution was assessed by BrdUrd distribution (G1, S, S', and G2-M) as shown and described in Materials and Methods. B, quantification of the effects of different concentrations of SAR-020106 on SN38-induced (20 nmol/L) cell cycle arrest in SW620 cells at 24 h. ▪, G1-G0 phase;

graphic
, S phase; □, S' phase;
graphic
, G2-M phase. C, quantification of the effects of different doses of SAR-020106 on etoposide induced (50 μmol/L ×1 h) cell cycle arrest in HT29 cells 23 h following cytotoxic exposure. Symbols as in B. Columns, mean derived from three independent experiments; bars, SD.

Figure 2.

Effects of SAR-020106 on drug-induced cell cycle arrest in SW620 and HT29 colon cancer cells. A, effects of different concentrations of SAR-020106 alone (0.1, 0.5, and 1 μmol/L) or in combination with a fixed concentration of SN38 (20 nmol/L) for 24 h on cell cycle distribution in SW620 cells. Histograms, cell cycle distribution as measured by propidium iodide (PI) DNA staining; dot plots, DNA synthesis as measured by BrdUrd incorporation. Cell cycle distribution was assessed by BrdUrd distribution (G1, S, S', and G2-M) as shown and described in Materials and Methods. B, quantification of the effects of different concentrations of SAR-020106 on SN38-induced (20 nmol/L) cell cycle arrest in SW620 cells at 24 h. ▪, G1-G0 phase;

graphic
, S phase; □, S' phase;
graphic
, G2-M phase. C, quantification of the effects of different doses of SAR-020106 on etoposide induced (50 μmol/L ×1 h) cell cycle arrest in HT29 cells 23 h following cytotoxic exposure. Symbols as in B. Columns, mean derived from three independent experiments; bars, SD.

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G2 Checkpoint Abrogation and Potentiation of Cytotoxicity by SAR-020106

A cellular assay for measuring functional CHK1 inhibition and G2 checkpoint abrogation was developed. An acute etoposide exposure (50 μmol/L × 1 hour) was used to arrest cells in a late S-G2 checkpoint before abrogation with different concentrations of SAR-020106 in the presence of nocodazole. This allowed mitotic cells to be collected and quantified in a cell-based ELISA assay. This G2 checkpoint abrogation assay in HT29 and SW620 showed that SAR-020106 was capable of abrogating etoposide-induced cell cycle arrest with an IC50 of 55 ± 19 nmol/L and 91 ± 51 nmol/L, respectively (mean ± SD, n = 5; Fig. 3A). Table 1 shows that SAR-020106 was relatively nontoxic with a GI50 of 0.48 μmol/L in HT29 and 2 μmol/L in SW620, resulting in an activity index of 8.7 and 22, respectively. Furthermore, it enhanced the cytotoxicity of both SN38 and gemcitabine in HT29 cells to a similar degree (Table 1; Fig. 3B), giving a PI of 3. By contrast, greater potentiation occurred in SW620 and Colo205 with gemcitabine compared with SN38 (Table 1). The ability of SAR-020106 to selectively sensitize p53-deficient cells to genotoxic chemotherapy was assessed using an A2780 isogenic cell line in which p53 was functionally inactivated by the overexpression of HPV16E6. The PI values for parental A2780 (wild-type p53) with SN38 and gemcitabine were 1.4 and 3.6, respectively, whereas the corresponding values in A2780E6 (nonfunctional p53) were 3.2 and 16.2, giving a p53−/− selectivity of 2.3- and 4.5-fold, respectively.

Figure 3.

SAR-020106 abrogates a G2 arrest and potentiates the cytotoxicity of gemcitabine and SN38 in vitro. A, G2 checkpoint abrogation assay. HT29 cells in 96-well plates were treated with etoposide (50 μmol/L ×1 h) followed by either no addition (▿), nocodazole (▾; 100 ng/mL), or SAR-020106 (○) at the concentrations indicated, in the presence of nocodazole (100 ng/mL) for 21 h. Cells were also treated with nocodazole alone (100 ng/mL) as a positive control for mitosis (♦). Cells were fixed and stained for the mitotic marker MPM2 as described in Materials and Methods. B, cytotoxicity and potentiation assay. HT29 cells were treated in 96-well plates with either: SAR-020106 alone (○) at the concentrations indicated or, a fixed concentration of SN38 (▾; GI50) together with different concentrations of SAR-020106, or a fixed concentration of gemcitabine (•; GI50) combined with different concentrations of SAR-020106. Cells were fixed and stained with SRB after 96 h of drug exposure, and GI50 values determined as described in Materials and Methods. Points, mean for four replicates; bars, SD. Similar results were obtained in repeat experiments.

Figure 3.

SAR-020106 abrogates a G2 arrest and potentiates the cytotoxicity of gemcitabine and SN38 in vitro. A, G2 checkpoint abrogation assay. HT29 cells in 96-well plates were treated with etoposide (50 μmol/L ×1 h) followed by either no addition (▿), nocodazole (▾; 100 ng/mL), or SAR-020106 (○) at the concentrations indicated, in the presence of nocodazole (100 ng/mL) for 21 h. Cells were also treated with nocodazole alone (100 ng/mL) as a positive control for mitosis (♦). Cells were fixed and stained for the mitotic marker MPM2 as described in Materials and Methods. B, cytotoxicity and potentiation assay. HT29 cells were treated in 96-well plates with either: SAR-020106 alone (○) at the concentrations indicated or, a fixed concentration of SN38 (▾; GI50) together with different concentrations of SAR-020106, or a fixed concentration of gemcitabine (•; GI50) combined with different concentrations of SAR-020106. Cells were fixed and stained with SRB after 96 h of drug exposure, and GI50 values determined as described in Materials and Methods. Points, mean for four replicates; bars, SD. Similar results were obtained in repeat experiments.

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Table 1.

Summary of the cytotoxicity and potentiation of genotoxic drugs by the CHK1 inhibitor SAR-020106 in vitro

CellsGI50 (μmol/L)GenotoxicCombination GI50 (μmol/L)PI*
HT29 0.47 ± 0.19 (n = 11) SN38 0.19 ± 0.10 (n = 5) 3.1 ± 1.6 (n = 5) 
Gem 0.18 ± 0.089 (n = 7) 3.0 ± 1.2 (n = 7) 
SW620 2.0 ± 0.93 (n = 4) SN38 0.62 ± 0.12 (n = 4) 3.3 ± 0.81§ (n = 4) 
Gem 0.20 ± 0.10 (n = 4) 12 ± 4.9 (n = 4) 
Colo205 3.5 ± 0.57 (n = 3) SN38 1 ± 0.36 (n = 3) 4.1 ± 1.8 (n = 3) 
Gem 0.13 ± 0.036 (n = 3) 29 ± 9.1 (n = 3) 
A2780C 0.45 ± 0.09 (n = 5) SN38 0.37 ± 0.12 (n = 4) 1.4 ± 0.59 (n = 4) 
Gem 0.12 ± 0.0057 (n = 3) 3.6 ± 0.17§ (n = 3) 
A2780E6 0.29 ± 0.034 (n = 5) SN38 0.11 ± 0.054 (n = 4) 3.2 ± 1.4 (n = 4) 
Gem 0.02 ± 0.0081 (n = 3) 16.2 ± 5.6 (n = 3) 
CellsGI50 (μmol/L)GenotoxicCombination GI50 (μmol/L)PI*
HT29 0.47 ± 0.19 (n = 11) SN38 0.19 ± 0.10 (n = 5) 3.1 ± 1.6 (n = 5) 
Gem 0.18 ± 0.089 (n = 7) 3.0 ± 1.2 (n = 7) 
SW620 2.0 ± 0.93 (n = 4) SN38 0.62 ± 0.12 (n = 4) 3.3 ± 0.81§ (n = 4) 
Gem 0.20 ± 0.10 (n = 4) 12 ± 4.9 (n = 4) 
Colo205 3.5 ± 0.57 (n = 3) SN38 1 ± 0.36 (n = 3) 4.1 ± 1.8 (n = 3) 
Gem 0.13 ± 0.036 (n = 3) 29 ± 9.1 (n = 3) 
A2780C 0.45 ± 0.09 (n = 5) SN38 0.37 ± 0.12 (n = 4) 1.4 ± 0.59 (n = 4) 
Gem 0.12 ± 0.0057 (n = 3) 3.6 ± 0.17§ (n = 3) 
A2780E6 0.29 ± 0.034 (n = 5) SN38 0.11 ± 0.054 (n = 4) 3.2 ± 1.4 (n = 4) 
Gem 0.02 ± 0.0081 (n = 3) 16.2 ± 5.6 (n = 3) 

NOTE: Cytotoxicity (GI50) was determined by SRB assay. See Materials and Methods and Materials for further details.

*PI is the ratio of GI50/combination GI50. Values are mean ± SD of n independent determinations.

P < 0.05 significantly different from unity.

P < 0.01 significantly different from unity.

§P < 0.001 significantly different from unity.

Inhibition of CHK1 Autophosphorylation and the Cell Cycle Pathway by SAR-020106

The characterization of biomarker changes associated with target inhibition is an important aspect of molecularly targeted drug development (23). Figure 4A shows that phosphorylation at S296 of CHK1, a site of autophosphorylation, is barely detectable in unperturbed cells, but is markedly induced in response to SN38 treatment for 24 hours in SW620 cells. This response is thought to be catalyzed by the DNA damage–induced activation of CHK1 providing a potential biomarker of CHK1 activity (11, 12). The combination of SN38 with increasing concentrations of SAR-020106 was shown to decrease this signal in a concentration-dependent fashion with complete suppression at ≥1 μmol/L SAR-020106. These changes in CHK1 phosphorylation were associated with minimal alterations in total protein expression. As shown by the previous data, a critical response of cells to DNA damage is cell cycle arrest. In particular, phosphorylation of tyrosine 15 on CDK1 (pY15) blocks the activity of this kinase, causing arrest at the G2-M transition. CHK1 controls this arrest through regulating CDC25 phosphatase activity (14, 36, 37), and consequently, pY15 CDK1 can be regarded as a downstream biomarker for CHK1 activity and G2-M arrest. Figure 4A shows that SN38 caused an increase in pY15 CDK1 consistent with a cell cycle arrest, whereas SAR-020106 alone had no effect. By contrast, the combination of SN38 and SAR-020106 blocked the induction of pY15 at ≥1 μmol/L SAR-020106, consistent with the loss of the pS296 CHK1 signal. In addition, the band shift of CDK1, which is indicative of Y15 phosphorylation, was also inhibited at concentrations of SAR-020106 that are ≥1 μmol/L. Interestingly, combinations of SN38 and SAR-020106 at ≥0.1 μmol/L caused an increase in S139 H2AX phosphorylation (γH2AX), a signal associated with DNA double-strand breaks (38) and there was a corresponding increase in PARP cleavage, a marker of cell death (39), at concentrations ≥1 μmol/L SAR-020106. Figure 4B shows that a concentration of 20 nmol/L gemcitabine (which induced pS296 CHK1 and pY15 CDK1) combined with SAR-020106 for 24 hours in SW620 cells caused similar effects to combinations with SN38, although S296 CHK1 phosphorylation seemed to be slightly more sensitive to SAR-020106 inhibition and γH2AX was induced ≥1 μmol/L SAR-020106. Similar results were obtained in HT29 cells following 24 hours of treatment with SN38 or gemcitabine (data not shown). These results show that SAR-020106 is an inhibitor of genotoxic drug–induced CHK1 activation in human tumor cells in vitro.

Figure 4.

Effect of SAR-020106 on SN38 and gemcitabine-induced biomarker changes in SW620 colon cancer cells. A, cells were treated with SN38 (100 nmol/L) alone, with SAR-020106 alone (5 μmol/L), or with a fixed concentration of SN38 (100 nmol/L) in combination with increasing concentrations of SAR-020106 for 24 h. B, cells were treated with gemcitabine (20 nmol/L) alone, with SAR-020106 alone (5 μmol/L), or with a fixed concentration of gemcitabine in combination with different concentrations of SAR-020106 for 24 h. SAR-020106 was administered 1 h before the cytotoxic agent. Protein expression was characterized by Western blotting using 50 μg samples per lane as described in Materials and Methods. CHK1 phosphorylated at S296 (PS296) was used as a biomarker of CHK1 inhibition, pY15 CDK1 as a downstream biomarker of CHK1 inhibition, and pS139 (γ) H2AX and cleaved PARP as biomarkers of DNA damage and apoptosis, respectively. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Similar results were obtained in repeat experiments.

Figure 4.

Effect of SAR-020106 on SN38 and gemcitabine-induced biomarker changes in SW620 colon cancer cells. A, cells were treated with SN38 (100 nmol/L) alone, with SAR-020106 alone (5 μmol/L), or with a fixed concentration of SN38 (100 nmol/L) in combination with increasing concentrations of SAR-020106 for 24 h. B, cells were treated with gemcitabine (20 nmol/L) alone, with SAR-020106 alone (5 μmol/L), or with a fixed concentration of gemcitabine in combination with different concentrations of SAR-020106 for 24 h. SAR-020106 was administered 1 h before the cytotoxic agent. Protein expression was characterized by Western blotting using 50 μg samples per lane as described in Materials and Methods. CHK1 phosphorylated at S296 (PS296) was used as a biomarker of CHK1 inhibition, pY15 CDK1 as a downstream biomarker of CHK1 inhibition, and pS139 (γ) H2AX and cleaved PARP as biomarkers of DNA damage and apoptosis, respectively. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Similar results were obtained in repeat experiments.

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Pharmacokinetics of SAR-020106 in Mice

The preliminary pharmacokinetic properties of SAR-020106 were determined in nontumor-bearing BALB/c mice to ensure that tissue drug concentrations were adequate for CHK1 inhibition in vivo. Figure 5A and Table 2 summarize the pharmacokinetic characteristics of SAR-020106 following administration at 5 mg/kg either i.v. or i.p. Peak plasma concentration was 1.35 μmol/L after i.v. administration with a relatively short half-life and high clearance and volume of distribution giving an AUC0-∞ of 703 nmol/L h. Following i.p. administration, the plasma elimination time course was comparable with that following i.v. administration (data not shown), but the peak drug concentration was 3-fold lower, the clearance was 2-fold slower, and the volume of distribution was 4-fold higher (Table 2). This resulted in an AUC0-∞ of 334 nmol/L h giving, an i.p. bioavailability of 47.5%. Interestingly, following i.p. administration, muscle tissue uptake was higher than in plasma and there was evidence of tissue retention as shown by the 5- to 6-fold longer half-life and correspondingly lower clearance. Consequently, muscle drug exposure was much higher than in plasma with an AUC0-∞ of 2615 nmol/L h and a tissue/plasma ratio of 8:1. Other tissues showed tissue/plasma ratios of 10:1 to 50:1 following i.v. administration, consistent with tissue drug accumulation and retention at this dose. Assuming linear pharmacokinetics, a dose of 40 mg/kg i.p. would be expected to result in tissue exposures of 21 to 130 μmol/L h, which equals or exceeds that required for biomarker modulation in vitro (24 μmol/L h; see Fig. 3). Figure 5B shows that SAR-020106 tumor concentrations greatly exceeded plasma drug concentrations with tumor/plasma ratios of 47:1 and 85:1 at 6 and 24 hours, respectively, following 40 mg/kg i.p. Moreover, this dose gave tumor drug concentrations that greatly exceeded those required for G2 checkpoint abrogation in vitro (IC50, 55 nmol/L) for at least 24 hours, consistent with checkpoint abrogation in tumor cells in vivo (see Fig. 5B). Plasma binding studies established that SAR-020106 was 94.4% bound to mouse plasma proteins.

Figure 5.

Pharmacokinetic properties and pharmacodynamic effects of SAR-020106 in combination with irinotecan in vivo. A, pharmacokinetics of SAR-020106 in nontumor-bearing BALB/c mice following 5 mg/kg i.p. bolus administration in plasma (○) and muscle tissue (•). Points, mean for three mice per time point; bars, SD. Drug concentrations and pharmacokinetic parameters were determined as described in Materials and Methods. B, concentrations of SAR-020106 occurring in plasma (white columns) and SW620 tumors (black columns) 6 and 24 h following initial drug administration. Dashed line, the IC50 concentration of SAR-020106 required for cellular G2 checkpoint abrogation determined in vitro. Tumor-bearing nude mice were given SAR-020106 (40 mg/kg i.p.) 1 h before a fixed dose of irinotecan (50 mg/kg i.p.). Columns, mean for three mice per time point; bars, SD. C, biomarker changes occurring in SW620 tumor xenografts from B 24h following treatment with either irinotecan alone or in combination with SAR-020106. Tumor-bearing animals were given either vehicle alone, irinotecan alone (25 or 50 mg/kg i.p.), or combined with a fixed dose of SAR-020106 (40 mg/kg i.p.) 1 h before irinotecan administration. Animals were sacrificed 6 or 24 h following initial drug treatment and tumors were recovered and snap frozen. Tumor protein expression was characterized by Western blotting using 50 μg samples per lane as described in Materials and Methods. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Similar results were obtained in repeat experiments. D, densitometric quantification of the immunoblots shown in C. ▪, pS296 CHK1; □, total CHK1;

graphic
, pY15 CDK1; and
graphic
, total CDK1. Statistics: *, P < 0.05; ***, P < 0.001 significantly different from irinotecan treatment alone.

Figure 5.

Pharmacokinetic properties and pharmacodynamic effects of SAR-020106 in combination with irinotecan in vivo. A, pharmacokinetics of SAR-020106 in nontumor-bearing BALB/c mice following 5 mg/kg i.p. bolus administration in plasma (○) and muscle tissue (•). Points, mean for three mice per time point; bars, SD. Drug concentrations and pharmacokinetic parameters were determined as described in Materials and Methods. B, concentrations of SAR-020106 occurring in plasma (white columns) and SW620 tumors (black columns) 6 and 24 h following initial drug administration. Dashed line, the IC50 concentration of SAR-020106 required for cellular G2 checkpoint abrogation determined in vitro. Tumor-bearing nude mice were given SAR-020106 (40 mg/kg i.p.) 1 h before a fixed dose of irinotecan (50 mg/kg i.p.). Columns, mean for three mice per time point; bars, SD. C, biomarker changes occurring in SW620 tumor xenografts from B 24h following treatment with either irinotecan alone or in combination with SAR-020106. Tumor-bearing animals were given either vehicle alone, irinotecan alone (25 or 50 mg/kg i.p.), or combined with a fixed dose of SAR-020106 (40 mg/kg i.p.) 1 h before irinotecan administration. Animals were sacrificed 6 or 24 h following initial drug treatment and tumors were recovered and snap frozen. Tumor protein expression was characterized by Western blotting using 50 μg samples per lane as described in Materials and Methods. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Similar results were obtained in repeat experiments. D, densitometric quantification of the immunoblots shown in C. ▪, pS296 CHK1; □, total CHK1;

graphic
, pY15 CDK1; and
graphic
, total CDK1. Statistics: *, P < 0.05; ***, P < 0.001 significantly different from irinotecan treatment alone.

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Table 2.

Summary of SAR-020106 pharmacokinetics in BALB/c mice following 5 mg/kg i.v. or i.p.

RouteTissueTmax (h)Cmax (nmol/L)T1/2 (h)Cl (L/h)Vz (L)AUC0-∞ (nmol/L h)
I.V. Plasma N/A 1350 0.658 0.371 0.353 703 
I.P. Plasma <0.083 415 0.995 0.781 1.12 334 
I.P. Muscle 0.5 644 5.59 0.100 0.806 2615 
RouteTissueTmax (h)Cmax (nmol/L)T1/2 (h)Cl (L/h)Vz (L)AUC0-∞ (nmol/L h)
I.V. Plasma N/A 1350 0.658 0.371 0.353 703 
I.P. Plasma <0.083 415 0.995 0.781 1.12 334 
I.P. Muscle 0.5 644 5.59 0.100 0.806 2615 

NOTE: Nonparametric pharmacokinetic parameters were determined using PC Nonlin software with six to seven time points and two to three mice per time point.

Abbreviation: N/A, not available.

Inhibition of DNA Damage–induced CHK1 Biomarkers by SAR-020106 In vivo

Having shown biomarker modulation in vitro and established that SAR-020106 exhibited pharmacokinetics that were consistent with potentially active tumor drug exposures, SAR-020106 was combined with the topoisomerase 1 inhibitor irinotecan to explore its effects on CHK1 biomarker expression in vivo. Figure 5C and D show that irinotecan at 25 and 50 mg/kg i.p. caused a marked increase in CHK1 autophosphorylation at S296 in SW620 tumors, 24 hours following drug administration, compared with controls. In addition, irinotecan also enhanced Y15 CDK1 phosphorylation consistent with a drug-induced cell cycle arrest in these tumor cells at 24 hours. The addition of SAR-020106 at 40 mg/kg caused a significant reduction in the pS296 CHK1 signal at 24 hours with minimal effects on total CHK1 levels consistent with CHK1 inhibition in vivo (Fig. 5D). Moreover, there was also a significant reduction in pY15 CDK1 signal intensity following SAR-020106 treatment, suggesting that irinotecan cell cycle arrest may have been abrogated in vivo (Fig. 5D).

Antitumor Effects of SAR-020106 in Combination with Irinotecan or Gemcitabine

The ability of SAR-020106 to potentiate the antitumor activity of irinotecan and gemcitabine in vivo was assessed in SW620 xenografts. Figure 6A shows that there were minimal effects of either irinotecan or SAR-020106 alone in SW620 tumors at the doses used. The mean time for control tumors to reach 300% of their initial treatment size was 6.2 days. By comparison SAR-020106 alone–treated tumors (40 mg/kg i.p.) and irinotecan alone–treated tumors (12.5 mg/kg i.p.) reached this size after a mean of 6.3 and 6.9 days, (P = 0.159 and 0.460, respectively). However, there was a clear decrease in tumor growth associated with the combination with tumors reaching 300% by 12.5 days, resulting in a significant growth delay relative to irinotecan alone of 5.6 days (P < 0.01) and an increase in growth delay of 5.5 days. Two mice were culled on days 15 and 18 in the SAR-020106 treatment alone group due to tumor growth. There was minimal body weight loss associated with this combination giving a nadir on day 11, and a mean body weight of 96.4% of the initial weight. The combination of gemcitabine with SAR-020106 in SW620 confirmed that SAR-020106 alone at 40 mg/kg i.p. had negligible antitumor activity (Fig. 6B), with a mean time for tumors to reach 300% of their initial treatment size of 7.0 compared with 6.2 days in controls (P = 0.204). Gemcitabine alone had a clear antitumor effect with tumors taking 10.1 days to reach 300%, giving a significant growth delay of 3.9 days relative to controls (P < 0.001). The combination of gemcitabine and SAR-020106 was more potent when the two agents were administered simultaneously rather than with a 24-hour delay following the cytotoxic. The mean time for tumors to reach 300% of the initial treatment size was 14.7 and 12.5 days for the simultaneous and delayed combination, respectively, resulting in growth delays of 8.5 and 6.3 days relative to control and a significant increase in growth delay of 4.6 days (P < 0.01) and 2.4 days (P = 0.0514) relative to gemcitabine alone. This represents an increase in growth delay of 3.8 days for the simultaneous treatment, which is a doubling of the antitumor effect of gemcitabine alone. One mouse died in the simultaneous combination group on day 18. Once again this synergistic antitumor interaction was associated with minimal weight loss with a nadir on day 16 and a mean body weight of 93.3% of the initial weight.

Figure 6.

Antitumor effects of combining SAR-020106 with different cytotoxic agents in nude mice bearing SW620 xenograft tumors. A, combination of SAR-020106 with irinotecan. Vehicle alone (•) and irinotecan alone (◊; 12.5 mg/kg i.p.) were administered on the days shown by arrows. ▿, SAR-020106 alone (40 mg/kg i.p.); ▾, irinotecan and SAR-020106 combined (SAR-020106 was administered 1 h before irinotecan using the single agent doses). B, combination of SAR-020106 with gemcitabine. •, vehicle alone; ◊, gemcitabine alone (60 mg/kg i.v.); ▿, SAR-020106 alone (40 mg/kg i.p.); ♦, gemcitabine and SAR-020106 combined at single agent doses (SAR-020106 1 h before gemcitabine); or ▾, combination with SAR-020106 administered 24 h following gemcitabine. Points, mean (n = 10–16); bars, SEM. Tumor size and animal body weight was assessed as described in Materials and Methods.

Figure 6.

Antitumor effects of combining SAR-020106 with different cytotoxic agents in nude mice bearing SW620 xenograft tumors. A, combination of SAR-020106 with irinotecan. Vehicle alone (•) and irinotecan alone (◊; 12.5 mg/kg i.p.) were administered on the days shown by arrows. ▿, SAR-020106 alone (40 mg/kg i.p.); ▾, irinotecan and SAR-020106 combined (SAR-020106 was administered 1 h before irinotecan using the single agent doses). B, combination of SAR-020106 with gemcitabine. •, vehicle alone; ◊, gemcitabine alone (60 mg/kg i.v.); ▿, SAR-020106 alone (40 mg/kg i.p.); ♦, gemcitabine and SAR-020106 combined at single agent doses (SAR-020106 1 h before gemcitabine); or ▾, combination with SAR-020106 administered 24 h following gemcitabine. Points, mean (n = 10–16); bars, SEM. Tumor size and animal body weight was assessed as described in Materials and Methods.

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These studies were undertaken to characterize the preclinical pharmacology of SAR-020106 and to identify genotoxic agents, which in combination with SAR-020106 exhibited enhanced antitumor activity in human tumor xenografts. As a part of these studies, it was important to identify and monitor suitable biomarkers of CHK1 inhibition and to establish any pharmacokinetic-pharmacodynamic relationships both in vitro and in vivo. This is a particularly important aspect of CHK1 inhibitor development as they are unlikely to exhibit any antitumor activity as single agents. The identification and use of these biomarkers allow the integrity of the CHK1 inhibitor/target pathway to be monitored and maintained throughout the preclinical and clinical evaluation process. Furthermore, this approach allows the construction of a “pharmacologic audit trail” that will facilitate further drug development and interpretation of clinical trials (23).

SAR-020106 is a novel, selective, and potent CHK1 inhibitor. This compound was initially assayed against recombinant human CHK1 and was shown to be a potent ATP competitive inhibitor. Kinase profiling data confirmed that there was minimal activity against the functionally related kinase CHK2 and the cell cycle kinase CDK1 (>7,000-fold selectivity). This was considered advantageous as the therapeutic utility of CHK2 is still unclear (40), and inhibiting CDK1 would interfere with cell cycle progression and prevent G2 checkpoint abrogation (41). There was some cross-reactivity with several receptor tyrosine kinases such as Flt3, Ret, Met, and Trk A, but it has been argued that sustained inhibition of receptor tyrosine kinases is required for a pharmacologic effect and is therefore unlikely to occur with the relatively short exposures required for checkpoint abrogation (25).

Abrogation of an etoposide-induced G2 checkpoint and induction of mitosis was quantified in a cell-based ELISA assay. These data together with the cell cycle results (Fig. 2) confirmed that SAR-020106 abrogates an etoposide-induced G2 arrest in HT29 and SW620 cells with similar potencies, which were 9- and 22-fold lower than the corresponding GI50 values giving a potential therapeutic margin. The apparent discrepancy between the similar cellular CHK1 kinase inhibition and the different cytotoxicity of SAR-020106 in these two cell lines suggests that cytotoxicity is not directly associated with CHK1 inhibitory activity. Similar checkpoint abrogation activity was reported for PF00477736 (25), with the dual CHK1 and CHK2 kinase inhibitor AZD7762 giving a slightly lower value (24). The ability of SAR-020106 to potentiate the cytotoxicity of SN38 (the active metabolite of irinotecan, a topoisomerase 1 inhibitor) and gemcitabine (an antimetabolite) was evaluated in several colon tumor cell lines in vitro. In general, the cytotoxicity of gemcitabine was potentiated to a greater extent than SN38, although the degree of potentiation may depend on compound concentration and schedule as well as genetic background. Importantly, the use of an E6-transfected cell line with compromized p53 function, confirmed that CHK1 inhibition by SAR-020106 selectively enhanced genotoxic cell killing in a p53−/− dependent fashion, with gemcitabine once again showing consistently greater sensitization than SN38 in this model. Other studies have also shown that several CHK1 inhibitors can potentiate SN38 and gemcitabine cytotoxicity in vitro with gemcitabine exhibiting the greatest potentiation (2427).

At present, it is unclear which cytotoxic agents are most efficacious in combination with CHK1 inhibition. Nevertheless, the genotoxic agents that are most prominently reported in publications on CHK1 inhibition and show marked potentiation are irinotecan and gemcitabine. Both of these agents are reported to be involved in activating the intra–S-phase checkpoint through stalled replication forks (42, 43), and CHK1 is required for stabilizing these forks and preventing chromosomal breaks as well as reinitiating replication (13, 44). Our cell cycle studies showed that both SN38 and etoposide caused S-phase arrest that was completely abrogated by SAR-020106. Moreover, in vitro potentiation experiments and biomarker studies clearly show that this effect was associated with increased cell killing and induction of γH2AX and PARP cleavage with SN38. Although a correlation between CHK1 inhibition and premature mitosis and enhanced cell killing has been reported for camptothecins and gemcitabine (45, 46), there is still some controversy about the exact mechanism of increased cytotoxicity. For example, it has recently been reported that there is a better correlation between inhibition of the Rad 51 repair protein response and sensitization than checkpoint abrogation for gemcitabine and a CHK1 inhibitor (PD-321852) in pancreatic cancer cells (47). Consequently, the cellular response to a genotoxic agent and CHK1 inhibitor combination may depend on numerous factors including the type of genotoxic agent used, the checkpoint response involved, the DNA repair capacity, and the genetic background of the tumor cell. One fascinating corollary to this is the recent observation that tumor cyclin B1 expression may predict efficacy for CHK1 inhibitors (46).

The pharmacokinetics of SAR-020106 showed that the drug was concentrated and retained in tissues with a large AUC0-∞ relative to plasma. Consequently, tumor concentrations exceeding those required for CHK1 inhibition were readily achieved following a dose of 40 mg/kg i.p. The protein binding was moderately high but did not seem to prevent active drug concentrations from being achieved in vivo, and tissue drug accumulation seemed unimpaired. Despite these observations, it is possible that active drug concentrations may be required for at least one cell cycle in tumors and either repeat drug administration or drug infusion may be necessary for maximum antitumor effects in vivo.

Biomarker studies with SN38 and gemcitabine in vitro showed enhanced CHK1 autophosphorylation on S296 consistent with DNA damage–induced CHK1 activation (12). SAR-020106 inhibited this effect in a concentration-dependent manner with complete inhibition of >1 μmol/L × 24 hours in both colon tumor cell lines. Tyrosine 15 CDK1 phosphorylation was also modulated in a similar fashion, indicating that CHK1-mediated abolition of genotoxic drug induced cell cycle arrest. Biomarker changes consistent with CHK1 inhibition contributing to increased DNA damage and cell death specifically in the SAR-020106 treated combinations were also detected. Perhaps more significantly, irinotecan readily induced S296 CHK1 and pY15 CDK1 phosphorylation in tumor xenografts after 24 hours of exposure, confirming CHK1 activation and a cell cycle arrest. SAR-020106 was able to inhibit these effects, confirming that CHK1 inhibition and abrogation of the cell cycle arrest could be achieved in vivo under these conditions. Subsequent antitumor studies with relatively modest doses of irinotecan and gemcitabine showed that SAR-020106 could clearly potentiate the antitumor effects of these genotoxic agents in a synergistic fashion. These results support the use of pS296 CHK1 and 15Y CDK1 as biomarkers of CHK1 inhibition and confirm the relationship between CHK1 inhibition and the enhanced antitumor activity of SN38 in vivo. Reassuringly, there was minimal weight loss or antitumor effects of SAR-020106 alone, despite reports of the lethal effects of CHK1 knockouts in mouse embryos (48). The combination of either genotoxic agent with SAR-020106 was also well tolerated with minimal body weight loss, suggesting that intermittent inhibition of CHK1 may have minimal toxic side effects. Interestingly our studies showed that the simultaneous combination of gemcitabine with SAR-020106 was more efficacious than gemcitabine followed 24 hours later by SAR-020106. Although we have shown that CHK1 inhibition by SAR-020106 gives a synergistic antitumor response with irinotecan and gemcitabine, an equally critical question relates to the ability of CHK1 inhibition to facilitate tumor cures. Other studies have shown cytotoxic anticancer drug potentiation with different CHK1 inhibitors (2427) and there is also evidence of increased cure rates (24). The mechanism by which CHK1 inhibitors enhance anticancer drug cell killing is still unclear and the role and efficiency of checkpoint abrogation versus the inhibition of DNA damage repair remains to be resolved.

In conclusion, this study shows that SAR-020106 is a selective, potent CHK1 inhibitor. The cytotoxicity of SN38 and gemcitabine can be enhanced in several different human tumor cell lines in vitro by SAR-020106 and this is associated with biomarker changes and cell cycle effects consistent with CHK1 inhibition. Perhaps most importantly, we show that SAR-020106 can enhance the antitumor effects of both irinotecan and gemcitabine in vivo with appropriate biomarker changes and minimal toxicity. These results support further CHK1 inhibitor development and clinical evaluation.

M.I. Walton, P.D. Eve, A. Hayes, M. Valenti, A. De Haven Brandon, G. Box, K.J. Boxall, G.W. Aherne, S.A. Eccles, F.I. Raynaud, I. Collins, and M.D. Garrett are employees of The Institute of Cancer Research that has a commercial interest in CHK1 inhibitors. J.C. Reader is an employee of Sareum Ltd, which has a commercial interest in CHK1 inhibitors. Sareum PLC is a wholly owned subsidiary of Sareum Holdings PLC, of which J.C. Reader is a shareholder. D.H. Williams is a former employee of Sareum Ltd and is a shareholder of Sareum Holdings PLC. Both Sareum and the Institute of Cancer Research have been involved in a commercial collaboration with Cancer Research Technology Ltd to discover and develop inhibitors of CHK1.

We thank Gowri Vijayaraghavan for the help with the flow cytometry studies and the Cell Cycle Control Team for useful discussions.

Grant support: Cancer Research UK [CUK] grant number C309/A8274. We also acknowledge NHS funding to the NIHR Biomedical Research Centre.

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.

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