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The Preclinical Pharmacology and Therapeutic Activity of the Novel CHK1 Inhibitor SAR-020106

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The Preclinical Pharmacology and Therapeutic Activity of the Novel CHK1 Inhibitor SAR-020106 Published OnlineFirst January 12, 2010; DOI: 10.1158/1535-7163.MCT-09-0938 Research Article Molecular Cancer Therapeutics The Preclinical Pharmacology and Therapeutic Activity of the Novel CHK1 Inhibitor SAR-020106 Michael I. Walton1, Paul D. Eve1, Angela Hayes1, Melanie Valenti1, Alexis De Haven Brandon1, Gary Box1, Kathy J. Boxall1, G. Wynne Aherne1, Suzanne A. Eccles1, Florence I. Raynaud1, David H. Williams2, John C. Reader2, Ian Collins1, and Michelle D. Garrett1 Abstract 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 main- tenance of genomic integrity. Most human tumors lack functional p53 and consequently have compro- mised 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-in- duced 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 autophosphor- ylation 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. ©2010 AACR. Introduction induces a subsequent delay in the cell cycle to facilitate DNA repair and/or apoptosis (2, 3). The observation that Normal cell division is a highly regulated process in- human cancers frequently have defects in p53 function, volving multiple cyclin-dependent kinases (CDK) and through either direct mutations or the disruption of its their appropriate cyclins that control a series of cell cycle regulatory pathway, with a consequent loss of G1-S checkpoints that ensure the fidelity of DNA replication checkpoint control (3, 4) has led to the concept that G2 (1). Genotoxic antitumor drugs cause DNA damage checkpoint inhibitors combined with DNA-damaging and activate cell cycle checkpoints at G1-S,S,andG2- agents may cause aberrant and lethal mitosis in such tu- M, which are required for DNA repair and the mainte- mors, whereas normal tissue would be rescued at the – nance of genomic integrity (2). The transcription factor p53-dependent G1-S checkpoint (2, 5 7). This strategy p53 is a critical checkpoint protein. In the presence of should therefore increase the therapeutic selectivity of damaged DNA, p53 is stabilized and activated, causing several DNA-damaging anticancer drugs, which are still the upregulation of the CDK inhibitor p21, which the mainstay of current cancer treatment. In normal cells, DNA damage is sensed by a com- plex series of signal transduction pathways. The main Authors' Affiliations: 1Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, proximal kinases are the phosphoinositide 3-kinase Sutton, Surrey, United Kingdom and 2Sareum Ltd, Cambridge, United homologues ATM and ATR (2, 8). These transduce sig- Kingdom nals to the effector kinases CHK1 and CHK2, which Note: Supplementary material for this article is available at Molecular then activate a series of cell cycle checkpoints. CHK1 Cancer Therapeutics Online (http://mct.aacrjournals.org/). is predominantly activated through phosphorylation Corresponding Author: Mike Walton, Cancer Research UK Centre for on S317 and S345 catalyzed by ATR (9, 10), and under- Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, 15 Cotswold Road, Sutton, Surrey, SM2 5NG, United goes autophosphorylation on S296 (11, 12). CHK1 is a Kingdom. Phone: 44-0-20-8722-4322; Fax: 44-0-20-8722-4324. E-mail: serine/threonine kinase that is involved in the S-phase [email protected] checkpoint through stabilizing replication forks, and in doi: 10.1158/1535-7163.MCT-09-0938 the G2 checkpoint control through regulating the stabil- ©2010 American Association for Cancer Research. ity and availability of the CDC25 phosphatases (13–15). www.aacrjournals.org 89 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst January 12, 2010; DOI: 10.1158/1535-7163.MCT-09-0938 Walton et al. Numerous studies have shown that UCN-01, a relatively nonselective inhibitor of CHK1, will preferentially sensi- tize p53-defective tumor cells to a variety of DNA- damaging anticancer drugs both in vitro and in vivo (16–18). 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 (19–21). As a part of the development of molecularly targeted kinase inhibitors, particularly those that might not have antitu- mor activity in their own right, it is important to identify appropriate biomarker readouts (22, 23). Consequently, the development of CHK1 inhibitors and the characteriza- Figure 1. Structure of SAR-020106. tion of suitable biomarkers have become an important drug development objective (24–28). In this study, we present the preclinical pharmacology stocks indicated. A2780 cells were stably transfected with and therapeutic utility of SAR-020106, a novel, selective, HPV16E6, and appropriate clones were screened and se- and potent inhibitor of CHK1. We show that SAR- lected essentially as described previously (30). Cells were 020106 can potentiate SN38 and gemcitabine cytotoxi- regularly screened for Mycoplasma using a PCR-based as- in vitro city in several colon cancer cell lines and that say (VenorGem, Minerva Biolabs). this potentiation occurs preferentially in cells that lack p53 function. We further show that SAR-020106 can ab- Kinase Assays rogate an SN38-induced S-phase block and an etopo- CHK1 and CHK2 assay conditions were as described in vitro side-induced S and G2-M arrest .Biomarker previously (31). A kinase profile against 50 different hu- studies showed that SAR-020106 selectively inhibited man kinases was carried out using 10 μmol/L SAR- SN38 or gemcitabine induced autophosphorylation of K 020106 with an ATP concentration equivalent to the m CHK1 at S296, and that this was associated with a loss for each enzyme (Millipore). of CDK1 phosphorylation at Y15, a downstream target γ of CHK1. Increases in H2AX and poly ADP ribose po- Cytotoxicity Assays lymerase (PARP) cleavage were also observed, consis- Cells were plated in 96-well plates (3–6×103 per well) tent with enhanced DNA damage and cell death. or tissue culture flasks (at 2–5×105 per flask) and al- Finally, we present evidence of enhancement of irinote- lowed to attach for 36 h to ensure exponential growth can and gemcitabine therapeutic activity by SAR-020106 at the time of treatment. Cytotoxicity was determined us- in association with CHK1 inhibition in human tumor ing a 96-h (four cell doublings) SRB assay, and GI50 va- xenografts. lues were derived as described previously (32). Materials and Methods G2 Checkpoint Abrogation Assay Measurements of intracellular CHK1 inhibition were Compounds carried out using HT29 and SW620 in a cell-based ELISA SAR-020106 was synthesized as described previously assay. Cells in 96-well plates (1–2×104 per well) were (29). Clinical formulations of gemcitabine and irinotecan treated with etoposide (50 μmol/L × 1 h) to induce a late were obtained from Eli Lilly (Gemzar, Eli Lilly and S-G2 arrest and then were exposed to different concentra- Pfizer (Campto, Pfizer), respectively. SN38, the active tions of the test compound for a further 21 h in medium metabolite of irinotecan, was purchased from LKT containing nocodazole (100 ng/mL) to trap cells in mito- Laboratories. Other reagents were obtained from sis. Cells were then fixed in 4% formaldehyde in PBS for Sigma-Aldrich Chemical Co. 30 min at 4°C and permeabilized with ice-cold methanol for a further 10 min. Cells were washed in PBS, blocked Cells with 5% milk in PBS × 30 min at 37°C, and then treated HT29, SW620, and Colo205 human colon carcinoma overnight with an antibody to mitotic proteins (MPM-2, cell lines were obtained from American Type Culture Upstate) in 5% milk in TBS. Cells were then rinsed in wa- Collection (American Type Culture Collection lot no. ter containing 0.01% Tween 20 and were treated for 1 h 4487729, 3924081, and 57723824, respectively) and with an Eu-labeled secondary antibody (Eu-N1, Perkin- A2780 ovarian carcinoma cells were obtained from Euro- Elmer) at 37°C. The MPM-2 epitope–expressing (i.e., mi- pean Collection of Animal Cell Cultures (Sigma-Aldrich). totic) cells were quantified using a Wallac Victor 1420 Cells were grown in DMEM containing 10% fetal bovine multilabel counter (Perkin-Elmer). IC50 values for mitosis serum (PAA “Gold”) and 5 mmol/L glutamine in a hu- induction were determined using nocodazole as a posi- midified atmosphere of 5% CO2 at 37°C and passaged tive control. The activity index was used as a measure ≤6 mo before the renewal from frozen, early passage of a compound's ability to induce mitosis relative to its 90 Mol Cancer Ther; 9(1) January 2010 Molecular Cancer Therapeutics Downloaded from mct.aacrjournals.org on September 28, 2021.
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