title: CHK1 Inhibition Synergizes with Gemcitabine Initially by Destabilizing the DNA Replication Apparatus.
abstract: Combining cell-cycle checkpoint kinase inhibitors with the DNA-damaging chemotherapeutic agent gemcitabine offers clinical appeal, with a mechanistic rationale based chiefly on abrogation of gemcitabine-induced G2-M checkpoint activation. However, evidence supporting this mechanistic rationale from chemosensitization studies has not been consistent. Here we report a systematic definition of how pancreatic cancer cells harboring mutant p53 respond to this combination therapy, by combining mathematical models with large-scale quantitative biologic analyses of single cells and cell populations. Notably, we uncovered a dynamic range of mechanistic effects at different ratios of gemcitabine and CHK1 inhibitors. Remarkably, effective synergy was attained even where cells exhibited an apparently functional G2-M surveillance mechanism, as exemplified by a lack of both overt premature CDK1 activation and S-phase mitotic entry. Consistent with these findings, S-G2 duration was extended in treated cells, leading to a definable set of lineage-dependent catastrophic fates. At synergistic drug concentrations, global replication stress was a distinct indicator of chemosensitization as characterized molecularly by an accumulation of S-phase cells with high levels of hyperphosphorylated RPA-loaded single-stranded DNA. In a fraction of these cells, persistent genomic damage was observed, including chromosomal fragmentation with a loss of centromeric regions that prevented proper kinetochore-microtubule attachment. Together, our results suggested a "foot-in-the-door" mechanism for drug synergy where cells were destroyed not by frank G2-M phase abrogation but rather by initiating a cumulative genotoxicity that deregulated DNA synthesis.
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Siang-Boon
authors.initials: SB
authors.lastname: Koh
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Courtin
authors.affiliation: Sentinel Oncology Limited, Cambridge, United Kingdom.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Boyce
authors.affiliation: Sentinel Oncology Limited, Cambridge, United Kingdom.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Boyle
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom. Fran.Richards@cruk.cam.ac.uk.
authors.forename: Frances M
authors.initials: FM
authors.lastname: Richards
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Duncan I
authors.initials: DI
authors.lastname: Jodrell
mesh_terms: D000970:Antineoplastic Agents / Q000008:administration & dosage; D002455:Cell Division; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D004249:DNA Damage / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects; D003841:Deoxycytidine / Q000008:administration & dosage / Q000031:analogs & derivatives*; D004357:Drug Synergism; D016195:G2 Phase / Q000187:drug effects; D006801:Humans; D008938:Mitosis / Q000187:drug effects; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000008:administration & dosage; D011494:Protein Kinases / Q000187:drug effects*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D000093542:Gemcitabine
chemical_list: D000970:Antineoplastic Agents; D047428:Protein Kinase Inhibitors; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D003841:Deoxycytidine; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D000093542:Gemcitabine