title: A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity.
abstract: The Wee1 cell cycle checkpoint kinase prevents premature mitotic entry by inhibiting cyclin-dependent kinases. Chemical inhibitors of Wee1 are currently being tested clinically as targeted anticancer drugs. Wee1 inhibition is thought to be preferentially cytotoxic in p53-defective cancer cells. However, TP53 mutant cancers do not respond consistently to Wee1 inhibitor treatment, indicating the existence of genetic determinants of Wee1 inhibitor sensitivity other than TP53 status. To optimally facilitate patient selection for Wee1 inhibition and uncover potential resistance mechanisms, identification of these currently unknown genes is necessary. The aim of this study was therefore to identify gene mutations that determine Wee1 inhibitor sensitivity. We performed a genome-wide unbiased functional genetic screen in TP53 mutant near-haploid KBM-7 cells using gene-trap insertional mutagenesis. Insertion site mapping of cells that survived long-term Wee1 inhibition revealed enrichment of G1/S regulatory genes, including SKP2, CUL1, and CDK2. Stable depletion of SKP2, CUL1, or CDK2 or chemical Cdk2 inhibition rescued the γ-H2AX induction and abrogation of G2 phase as induced by Wee1 inhibition in breast and ovarian cancer cell lines. Remarkably, live cell imaging showed that depletion of SKP2, CUL1, or CDK2 did not rescue the Wee1 inhibition-induced karyokinesis and cytokinesis defects. These data indicate that the activity of the DNA replication machinery, beyond TP53 mutation status, determines Wee1 inhibitor sensitivity, and could serve as a selection criterion for Wee1-inhibitor eligible patients. Conversely, loss of the identified S-phase genes could serve as a mechanism of acquired resistance, which goes along with development of severe genomic instability.
authors.affiliation: Department of Medical Oncology, University Medical Center Groningen, University of Groningen, 9723 GZ Groningen, The Netherlands;
authors.forename: Anne Margriet
authors.initials: AM
authors.lastname: Heijink
authors.affiliation: Division of Biochemistry, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands;
authors.forename: Vincent A
authors.initials: VA
authors.lastname: Blomen
authors.affiliation: Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Proteos#3-09, Singapore 138673, Republic of Singapore;
authors.forename: Xavier
authors.initials: X
authors.lastname: Bisteau
authors.affiliation: Department of Medical Oncology, University Medical Center Groningen, University of Groningen, 9723 GZ Groningen, The Netherlands;
authors.forename: Fabian
authors.initials: F
authors.lastname: Degener
authors.affiliation: Department of Medical Oncology, University Medical Center Groningen, University of Groningen, 9723 GZ Groningen, The Netherlands;
authors.forename: Felipe Yu
authors.initials: FY
authors.lastname: Matsushita
authors.affiliation: Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Proteos#3-09, Singapore 138673, Republic of Singapore; Department of Biochemistry, National University of Singapore, Singapore 117597, Republic of Singapore;
authors.forename: Philipp
authors.identifier: http://orcid.org/0000-0002-7247-7591
authors.initials: P
authors.lastname: Kaldis
authors.affiliation: European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen, 9713 AV Groningen, The Netherlands.
authors.forename: Floris
authors.initials: F
authors.lastname: Foijer
authors.affiliation: Department of Medical Oncology, University Medical Center Groningen, University of Groningen, 9723 GZ Groningen, The Netherlands; m.vugt@umcg.nl.
authors.forename: Marcel A T M
authors.initials: MA
authors.lastname: van Vugt
mesh_terms: D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors*; D016193:G1 Phase / Q000235:genetics; D006238:Haploidy; D006801:Humans; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D016196:S Phase / Q000235:genetics
chemical_list: D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human
keywords: AZD-1775; MK-1775; cell cycle; checkpoint; polyploidy