title: Combined inhibition of Wee1 and Chk1 gives synergistic DNA damage in S-phase due to distinct regulation of CDK activity and CDC45 loading.
abstract: Recent studies have shown synergistic cytotoxic effects of simultaneous Chk1- and Wee1-inhibition. However, the mechanisms behind this synergy are not known. Here, we present a flow cytometry-based screen for compounds that cause increased DNA damage in S-phase when combined with the Wee1-inhibitor MK1775. Strikingly, the Chk1-inhibitors AZD7762 and LY2603618 were among the top candidate hits of 1664 tested compounds, suggesting that the synergistic cytotoxic effects are due to increased S-phase DNA damage. Combined Wee1- and Chk1-inhibition caused a strong synergy in induction of S-phase DNA damage and reduction of clonogenic survival. To address the underlying mechanisms, we developed a novel assay measuring CDK-dependent phosphorylations in single S-phase cells. Surprisingly, while Wee1-inhibition alone induced less DNA damage compared to Chk1-inhibition, Wee1-inhibition caused a bigger increase in S-phase CDK-activity. However, the loading of replication initiation factor CDC45 was more increased after Chk1- than Wee1-inhibition and further increased by the combined treatment, and thus correlated well with DNA damage. Therefore, when Wee1 alone is inhibited, Chk1 suppresses CDC45 loading and thereby limits the extent of unscheduled replication initiation and subsequent S-phase DNA damage, despite very high CDK-activity. These results can explain why combined treatment with Wee1- and Chk1-inhibitors gives synergistic anti-cancer effects.
authors.affiliation: Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, N-0310, Norway.
authors.forename: Sissel
authors.initials: S
authors.lastname: Hauge
authors.affiliation: Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, N-0310, Norway.
authors.forename: Christian
authors.initials: C
authors.lastname: Naucke
authors.affiliation: Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, N-0310, Norway.
authors.forename: Grete
authors.initials: G
authors.lastname: Hasvold
authors.affiliation: Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, N-0310, Norway.
authors.forename: Mrinal
authors.initials: M
authors.lastname: Joel
authors.affiliation: Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, N-0310, Norway.
authors.forename: Gro Elise
authors.initials: GE
authors.lastname: Rødland
authors.affiliation: Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, N-0310, Norway.
authors.forename: Petras
authors.initials: P
authors.lastname: Juzenas
authors.affiliation: Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, N-0310, Norway.
authors.forename: Trond
authors.initials: T
authors.lastname: Stokke
authors.affiliation: Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, N-0310, Norway.
authors.forename: Randi G
authors.initials: RG
authors.lastname: Syljuåsen
mesh_terms: D000072283:A549 Cells; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004249:DNA Damage; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics; D004357:Drug Synergism; D005434:Flow Cytometry; D006801:Humans; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010671:Phenylurea Compounds / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011719:Pyrazines / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011744:Pyrimidinones; D016196:S Phase / Q000187:drug effects* / Q000235:genetics; D013876:Thiophenes / Q000494:pharmacology; D014508:Urea / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: C532363:3-(carbamoylamino)-5-(3-fluorophenyl)-N-(3-piperidyl)thiophene-2-carboxamide; C113571:CDC45 protein, human; D018797:Cell Cycle Proteins; C582547:LY2603618; D009687:Nuclear Proteins; D010671:Phenylurea Compounds; D047428:Protein Kinase Inhibitors; D011719:Pyrazines; D011720:Pyrazoles; D011743:Pyrimidines; D011744:Pyrimidinones; D013876:Thiophenes; D014508:Urea; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D018844:Cyclin-Dependent Kinases; C549567:adavosertib
keywords: CDK activity; DNA damage; cancer treatment; checkpoint kinase inhibitors; replication stress