CHK1 is a multifunctional protein kinase integral to both the cellular response to DNA damage and control of the number of active replication forks. CHK1 inhibitors are currently under investigation as chemopotentiating agents due to CHK1's role in establishing DNA damage checkpoints in the cell cycle. Here, we describe the characterization of a novel CHK1 inhibitor, LY2606368, which as a single agent causes double-stranded DNA breakage while simultaneously removing the protection of the DNA damage checkpoints. The action of LY2606368 is dependent upon inhibition of CHK1 and the corresponding increase in CDC25A activation of CDK2, which increases the number of replication forks while reducing their stability. Treatment of cells with LY2606368 results in the rapid appearance of TUNEL and pH2AX-positive double-stranded DNA breaks in the S-phase cell population. Loss of the CHK1-dependent DNA damage checkpoints permits cells with damaged DNA to proceed into early mitosis and die. The majority of treated mitotic nuclei consist of extensively fragmented chromosomes. Inhibition of apoptosis by the caspase inhibitor Z-VAD-FMK had no effect on chromosome fragmentation, indicating that LY2606368 causes replication catastrophe. Changes in the ratio of RPA2 to phosphorylated H2AX following LY2606368 treatment further support replication catastrophe as the mechanism of DNA damage. LY2606368 shows similar activity in xenograft tumor models, which results in significant tumor growth inhibition. LY2606368 is a potent representative of a novel class of drugs for the treatment of cancer that acts through replication catastrophe.

Interference with DNA damage checkpoints has been demonstrated preclinically to be a highly effective means of increasing the cytotoxicity of a number of DNA-damaging cancer therapies. Cell cycle arrest at these checkpoints protects injured cells from apoptotic cell death until DNA damage can be repaired. In the absence of functioning DNA damage checkpoints, cells with damaged DNA may proceed into premature mitosis followed by cell death. A key protein kinase involved in activating and maintaining the S and G2/M checkpoints is Chk1. Pharmacological inhibition of Chk1 in the absence of p53 functionality leads to abrogation of DNA damage checkpoints and has been shown preclinically to enhance the activity of many standard of care chemotherapeutic agents. LY2603618 is a potent and selective small molecule inhibitor of Chk1 protein kinase activity in vitro (IC(50) = 7 nM) and the first selective Chk1 inhibitor to enter clinical cancer trials. Treatment of cells with LY2603618 produced a cellular phenotype similar to that reported for depletion of Chk1 by RNAi. Inhibition of intracellular Chk1 by LY2603618 results in impaired DNA synthesis, elevated H2A.X phosphorylation indicative of DNA damage and premature entry into mitosis. When HeLa cells were exposed to doxorubicin to induce a G2/M checkpoint arrest, subsequent treatment with LY2603618 released the checkpoint, resulting in cells entering into metaphase with poorly condensed chromosomes. Consistent with abrogation of the Chk1 and p53-dependent G2/M checkpoint, mutant TP53 HT-29 colon cancer cells were more sensitive to gemcitabine when also treated with LY2603618, while wild-type TP53 HCT116 cells were not sensitized by LY2603618 to gemcitabine. Treatment of Calu-6 human mutant TP53 lung cancer cell xenografts with gemcitabine resulted in a stimulation of Chk1 kinase activity that was inhibited by co-administration of LY2603618. By all criteria, LY2603618 is a highly effective inhibitor of multiple aspects of Chk1 biology.

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Pharmacological inhibition of CHK1 in the absence of p53 functionality leads to abrogation of the S and G2/M DNA damage checkpoints. We report the preclinical therapeutic activity of LY2603618 (CHK1 inhibitor) at inhibiting CHK1 activation by gemcitabine and enhancing in vivo efficacy. The in vivo biochemical effects of CHK1 inhibition in the absence or presence of DNA damage were measured in human tumor xenograft models. Colon, lung and pancreatic xenografts models were treated with gemcitabine, LY2603618, or gemcitabine plus LY2603618. Gemcitabine treatment alone induced a significant increase in CHK1 autophosphorylation over untreated tumors. Co-administration of LY2603618 with gemcitabine showed a clear inhibition of CHK1 autophosphorylation for at least 24 h. Combining LY2603618 with gemcitabine resulted in an increase in H2AX serine 139 phosphorylation, indicating a corresponding increase in damaged DNA in the tumors. LY2603618 abrogated the S-phase DNA damage checkpoint in Calu-6 xenograft tumors treated with gemcitabine but did not significantly alter the G2/M checkpoint. Combining gemcitabine with LY2603618 resulted in a significant increase in tumor growth inhibition in Calu-6, HT-29 and PAXF 1869 xenografts over gemcitabine treatment alone. The best combination efficacy occurred when LY2603618 was given 24 h following dosing with gemcitabine. LY2603618 worked effectively to remove the S-phase DNA damage checkpoint and increase the DNA damage and the antitumor activity of gemcitabine treatment.

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