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

Cancer Res. 2015 Sep 1;75(17):3583-95. doi: 10.1158/0008-5472.CAN-14-3347. Epub 2015 Jul 3.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents / administration & dosage
  • Cell Division
  • Cell Line, Tumor
  • Checkpoint Kinase 1
  • DNA Damage / drug effects
  • DNA Replication / drug effects
  • Deoxycytidine / administration & dosage
  • Deoxycytidine / analogs & derivatives*
  • Drug Synergism*
  • G2 Phase / drug effects
  • Humans
  • Mitosis / drug effects
  • Pancreatic Neoplasms / drug therapy*
  • Pancreatic Neoplasms / genetics
  • Pancreatic Neoplasms / pathology
  • Phosphorylation / drug effects
  • Protein Kinase Inhibitors / administration & dosage
  • Protein Kinases / drug effects*
  • Tumor Suppressor Protein p53 / genetics

Substances

  • Antineoplastic Agents
  • Protein Kinase Inhibitors
  • TP53 protein, human
  • Tumor Suppressor Protein p53
  • Deoxycytidine
  • gemcitabine
  • Protein Kinases
  • CHEK1 protein, human
  • Checkpoint Kinase 1