title: Wee1 Kinase Inhibitor AZD1775 Radiosensitizes Hepatocellular Carcinoma Regardless of TP53 Mutational Status Through Induction of Replication Stress.
abstract: Wee1 kinase inhibitors are effective radiosensitizers in cells lacking a G1 checkpoint. In this study we examined the potential effect of Wee1 kinase inhibition on inducing replication stress in hepatocellular carcinoma (HCC).
  
  
  
  Five independent datasets from the Oncomine database comparing gene expression in HCC compared to normal tissue were combined and specific markers associated with Wee1 sensitivity were analyzed. We then performed a series of in vitro experiments to study the effect of Wee1 inhibition on irradiated HCC cell lines with varying p53 mutational status. Clonogenic survival assays and flow cytometry using anti-γH2AX and phospho-histone H3 antibodies with propidium iodide were performed to study the effect of AZD1775 on survival, cell cycle, and DNA repair. Additionally, nucleoside enriched medium was used to examine the effect of altering nucleotide pools on Wee1 targeted radiation sensitization.
  
  
  
  Our analysis of the Oncomine database found high levels of CDK1 and other cell cycle regulators indicative of Wee1 sensitivity in HCC. In our in vitro experiments, treatment with AZD1775 radiosensitized and chemosensitized Hep3B, Huh7, and HepG2 cell lines and was associated with delayed resolution of γH2AX foci and the induction of pan-nuclear γH2AX staining. Wee1 inhibition attenuated radiation-induced G2 arrest in the Hep3B (TP53 null) and Huh7 (TP53 mutant) cell lines but not in the TP53 wild-type cell line HepG2. Supplementation with nucleosides reversed the radiation-sensitizing effect of AZD1775 and reduced the amount of cells with pan-nuclear γH2AX staining after radiation.
  
  
  
  Radiation sensitization with Wee1 inhibition occurs in cells regardless of their p53 mutational status. In this study we show for the first time that replication stress via the overconsumption of nucleotides plays an important role in AZD1775-induced radiation sensitization.
authors.affiliation: Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan. Electronic address: kcuneo@umich.edu.
authors.forename: Kyle C
authors.initials: KC
authors.lastname: Cuneo
authors.affiliation: Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan.
authors.forename: Meredith A
authors.initials: MA
authors.lastname: Morgan
authors.affiliation: Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan.
authors.forename: Mary A
authors.initials: MA
authors.lastname: Davis
authors.affiliation: Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan.
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Parcels
authors.affiliation: Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan.
authors.forename: Joshua
authors.initials: J
authors.lastname: Parcels
authors.affiliation: Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan.
authors.forename: David
authors.initials: D
authors.lastname: Karnak
authors.affiliation: Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan.
authors.forename: Caila
authors.initials: C
authors.lastname: Ryan
authors.affiliation: Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan.
authors.forename: Na
authors.initials: N
authors.lastname: Liu
authors.affiliation: Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Maybaum
authors.affiliation: Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan.
authors.forename: Theodore S
authors.initials: TS
authors.lastname: Lawrence
mesh_terms: D016203:CDC2 Protein Kinase / Q000032:analysis; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology / Q000532:radiotherapy*; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors*; D004249:DNA Damage; D004261:DNA Replication; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects; D016158:Genes, p53; D056945:Hep G2 Cells; D006657:Histones / Q000032:analysis; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology / Q000532:radiotherapy*; D009154:Mutation; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011720:Pyrazoles / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D011744:Pyrimidinones; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D013312:Stress, Physiological
chemical_list: D018797:Cell Cycle Proteins; C468783:H2AX protein, human; D006657:Histones; D009687:Nuclear Proteins; D011720:Pyrazoles; D011743:Pyrimidines; D011744:Pyrimidinones; D011838:Radiation-Sensitizing Agents; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human; D016203:CDC2 Protein Kinase; C549567:adavosertib