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CHK1 Inhibition Synergizes with Gemcitabine Initially by Destabilizing the DNA Replication Apparatus.


Type

Article

Change log

Authors

Koh, Siang-Boon 
Courtin, Aurélie 
Boyce, Richard J 
Boyle, Robert G 
Richards, Frances M 

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.

Description

Keywords

Antineoplastic Agents, Cell Division, Cell Line, Tumor, Checkpoint Kinase 1, DNA Damage, DNA Replication, Deoxycytidine, Drug Synergism, G2 Phase, Humans, Mitosis, Pancreatic Neoplasms, Phosphorylation, Protein Kinase Inhibitors, Protein Kinases, Tumor Suppressor Protein p53, Gemcitabine

Journal Title

Cancer Res

Conference Name

Journal ISSN

0008-5472
1538-7445

Volume Title

75

Publisher

American Association for Cancer Research (AACR)
Sponsorship
Cancer Research UK (CB4270)
Cancer Research UK (15678)
This study was funded by Cancer Research UK via Institute Senior Group Leader funding (C14303/A17197) to DI Jodrell, and by Sentinel Oncology through an award from Innovate UK.