title: Cytokinetic effects of Wee1 disruption in pancreatic cancer.
abstract: The Wee1 kinase, which is activated in response to DNA damage, regulates exit from G2 through inhibitory phosphorylation of Cdk1/Cdc2, and is an attractive drug target. However, recent work has highlighted effects of Cdk2 phosphorylation by Wee1 on movement through S-phase, suggesting the potential to sensitize to S-phase specific agents by Wee1 inhibitors. In this paper we applied multiparametric flow cytometry to patient-derived pancreatic cancer xenograft tumor cells to study the cell cycle perturbations of Wee1 disruption via the small molecule inhibitor MK-1775, and genetic knockdown. We find that in vitro treatment with MK-1775, and to a lesser degree, Wee1 RNA transcript knockdown, results in the striking appearance of S-phase cells prematurely entering into mitosis. This effect was not seen in vivo in any of the models tested. Here, although we noted an increase of S-phase cells expressing the damage response marker γH2AX, treatment with MK-1775 did not significantly sensitize cells to the cytidine analog gemcitabine. Treatment with MK-1775 did result in a transient but large increase in cells expressing the mitotic marker phosphorylated H3S10 that reached a peak 4 hours after treatment. This suggests a role for Wee1 regulating the progression of genomically unstable cancer cells through G2 in the absence of extrinsically-applied DNA damage. A single dose of 8Gy ionizing radiation resulted in the time-dependent accumulation of Cyclin A2 positive/phosphorylated H3S10 negative cells at the 4N position, which was abrogated by treatment with MK-1775. Consistent with these findings, a genome-scale pooled RNA interference screen revealed that toxic doses of MK-1775 are suppressed by CDK2 or Cyclin A2 knockdown. These findings support G2 exit as the more significant effect of Wee1 inhibition in pancreatic cancers.
authors.affiliation: a Ontario Cancer Institute/Princess Margaret Cancer Center , Toronto , Ontario , Canada.
authors.forename: Qing
authors.initials: Q
authors.lastname: Chang
authors.affiliation: b Donnelly Centre for Cellular and Biomolecular Research, Department of Molecular Genetics , University of Toronto , Toronto , Ontario , Canada.
authors.forename: Megha
authors.initials: M
authors.lastname: Chandrashekhar
authors.affiliation: a Ontario Cancer Institute/Princess Margaret Cancer Center , Toronto , Ontario , Canada.
authors.forename: Troy
authors.initials: T
authors.lastname: Ketela
authors.affiliation: b Donnelly Centre for Cellular and Biomolecular Research, Department of Molecular Genetics , University of Toronto , Toronto , Ontario , Canada.
authors.forename: Yaroslav
authors.initials: Y
authors.lastname: Fedyshyn
authors.affiliation: b Donnelly Centre for Cellular and Biomolecular Research, Department of Molecular Genetics , University of Toronto , Toronto , Ontario , Canada.
authors.forename: Jason
authors.initials: J
authors.lastname: Moffat
authors.affiliation: a Ontario Cancer Institute/Princess Margaret Cancer Center , Toronto , Ontario , Canada.
authors.forename: David
authors.initials: D
authors.lastname: Hedley
mesh_terms: D000818:Animals; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D056751:Cyclin A2 / Q000235:genetics*; D051357:Cyclin-Dependent Kinase 2 / Q000235:genetics*; D004249:DNA Damage / Q000187:drug effects; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D051379:Mice; D008938:Mitosis / Q000187:drug effects / Q000235:genetics; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D010190:Pancreatic Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D011720:Pyrazoles / Q000008:administration & dosage; D011743:Pyrimidines / Q000008:administration & dosage; D011744:Pyrimidinones; D023041:Xenograft Model Antitumor Assays
chemical_list: C503721:CCNA2 protein, human; D018797:Cell Cycle Proteins; D056751:Cyclin A2; D009687:Nuclear Proteins; D011720:Pyrazoles; D011743:Pyrimidines; D011744:Pyrimidinones; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; C549567:adavosertib
keywords: G2 checkpoint; flow cytometry; ionizing radiation; pancreatic cancer; patient-derived xenograft; γH2AX