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Proc Natl Acad Sci U S A. 2015 Dec 8;112(49):15160-5. doi: 10.1073/pnas.1505283112. Epub 2015 Nov 23.

A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity.

Author information

1
Department of Medical Oncology, University Medical Center Groningen, University of Groningen, 9723 GZ Groningen, The Netherlands;
2
Division of Biochemistry, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands;
3
Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Proteos#3-09, Singapore 138673, Republic of Singapore;
4
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;
5
European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen, 9713 AV Groningen, The Netherlands.
6
Department of Medical Oncology, University Medical Center Groningen, University of Groningen, 9723 GZ Groningen, The Netherlands; m.vugt@umcg.nl.

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.

KEYWORDS:

AZD-1775; MK-1775; cell cycle; checkpoint; polyploidy

PMID:
26598692
PMCID:
PMC4679052
DOI:
10.1073/pnas.1505283112
[Indexed for MEDLINE]
Free PMC Article

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