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Abstract
Combining DNA-damaging drugs with DNA checkpoint inhibitors is an emerging strategy to manage cancer. Checkpoint kinase 1 inhibitors (CHK1is) sensitize most cancer cell lines to DNA-damaging drugs and also elicit single-agent cytotoxicity in 15% of cell lines. Consequently, combination therapy may be effective in a broader patient population. Here, we characterized the molecular mechanism of sensitization to gemcitabine by the CHK1i MK8776. Brief gemcitabine incubation irreversibly inhibited ribonucleotide reductase, depleting dNTPs, resulting in durable S phase arrest. Addition of CHK1i 18 h after gemcitabine elicited cell division cycle 7 (CDC7)- and cyclin-dependent kinase 2 (CDK2)-dependent reactivation of the replicative helicase, but did not reinitiate DNA synthesis due to continued lack of dNTPs. Helicase reactivation generated extensive single-strand (ss)DNA that exceeded the protective capacity of the ssDNA-binding protein, replication protein A. The subsequent cleavage of unprotected ssDNA has been termed replication catastrophe. This mechanism did not occur with concurrent CHK1i plus gemcitabine treatment, providing support for delayed administration of CHK1i in patients. Alternative mechanisms of CHK1i-mediated sensitization to gemcitabine have been proposed, but their role was ruled out; these mechanisms include premature mitosis, inhibition of homologous recombination, and activation of double-strand break repair nuclease (MRE11). In contrast, single-agent activity of CHK1i was MRE11-dependent and was prevented by lower concentrations of a CDK2 inhibitor. Hence, both pathways require CDK2 but appear to depend on different CDK2 substrates. We conclude that a small-molecule inhibitor of CHK1 can elicit at least two distinct, context-dependent mechanisms of cytotoxicity in cancer cells.
Highlights
Combining DNA-damaging drugs with DNA checkpoint inhibitors is an emerging strategy to manage cancer
We previously reported that concurrent Checkpoint kinase 1 inhibitors (CHK1is) only slightly sensitized cells to a 6-h gemcitabine treatment, while delaying addition of CHK1i until 18 h resulted in much greater cytotoxicity [5, 6]
Gemcitabine alone potently induced CHK1–pSer-296 compared with control, which was reversed by CHK1i when administered either concurrently or 18 h later (Fig. 1B)
Summary
Combining DNA-damaging drugs with DNA checkpoint inhibitors is an emerging strategy to manage cancer. Checkpoint kinase 1 inhibitors (CHK1is) sensitize most cancer cell lines to DNA-damaging drugs and elicit single-agent cytotoxicity in 15% of cell lines. We have previously shown that checkpoint kinase 1 inhibitors (CHK1i) can sensitize cancer cells to several DNA-damaging agents [3,4,5,6]. It remains unclear how CHK1i increases the cytotoxic effect of each source of DNA damage. CHK1i can induce premature activation of cyclin-dependent kinase 1 (CDK1) in S phase-arrested cells leading to premature mitosis [15,16,17] Another area of investigation has been the restart of stalled replication following recovery from the genotoxic insult [18]. We have investigated these different mechanisms in a variety of cell lines with various phenotypes to obtain a more comprehensive analysis of the mechanism of sensitivity
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