This file contains all supplementary figures: Figure S1. LY2606368 is an ATP competitive inhibitor of CHK1. Figure S2. LY2606368 is a potent catalytic and phenotypic inhibitor of CHK1. Figure S3. LY2606368 inhibits DNA damage-dependent autophosphorylation of CHK2. Figure S4. LY2606368 does not inhibit RSK activity in cells Figure S5. Treatment of Calu-6 lung cancer cells with LY2606368 results in rapid accumulation of DNA strand breaks in early S-phase. Figure S6. Timelapse photomicroscopy of LY2606368 treated HeLa cells confirms that S-phase transit is required for aberrant mitosis and cell death. Figure S7. Staurosporine causes rapid appearance of apoptotic TUNEL staining, but not H2AX serine 139 phosphorylation.
ARTICLE ABSTRACT
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. Mol Cancer Ther; 14(9); 2004–13. ©2015 AACR.
