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A Haploid Genetic Screen Identifies the G1/S Regulatory Machinery As a Determinant of Wee1 Inhibitor Sensitivity

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A Haploid Genetic Screen Identifies the G1/S Regulatory Machinery As a Determinant of Wee1 Inhibitor Sensitivity A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity Anne Margriet Heijinka, Vincent A. Blomenb, Xavier Bisteauc, Fabian Degenera, Felipe Yu Matsushitaa, Philipp Kaldisc,d, Floris Foijere, and Marcel A. T. M. van Vugta,1 aDepartment of Medical Oncology, University Medical Center Groningen, University of Groningen, 9723 GZ Groningen, The Netherlands; bDivision of Biochemistry, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands; cInstitute of Molecular and Cell Biology, Agency for Science, Technology and Research, Proteos#3-09, Singapore 138673, Republic of Singapore; dDepartment of Biochemistry, National University of Singapore, Singapore 117597, Republic of Singapore; and eEuropean Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen, 9713 AV Groningen, The Netherlands Edited by Stephen J. Elledge, Harvard Medical School, Boston, MA, and approved October 21, 2015 (received for review March 17, 2015) The Wee1 cell cycle checkpoint kinase prevents premature mitotic Wee1 kinase at tyrosine (Tyr)-15 to prevent unscheduled Cdk1 entry by inhibiting cyclin-dependent kinases. Chemical inhibitors activity (5, 6). Conversely, timely activation of Cdk1 depends on of Wee1 are currently being tested clinically as targeted anticancer Tyr-15 dephosphorylation by one of the Cdc25 phosphatases drugs. Wee1 inhibition is thought to be preferentially cytotoxic in (7–10). When DNA is damaged, the downstream DNA damage p53-defective cancer cells. However, TP53 mutant cancers do not response (DDR) kinases Chk1 and Chk2 inhibit Cdc25 phos- respond consistently to Wee1 inhibitor treatment, indicating the phatases through direct phosphorylation, which blocks Cdk1 existence of genetic determinants of Wee1 inhibitor sensitivity other activation (11–13). Cdk2 appears to be under similar checkpoint than TP53 status. To optimally facilitate patient selection for Wee1 control and is also phosphorylated by Wee1 on Tyr-15, which inhibition and uncover potential resistance mechanisms, identifica- prevents unscheduled S-phase entry. Conversely, Cdk2 must be tion of these currently unknown genes is necessary. The aim of this dephosphorylated by Cdc25 phosphatases to become active, a study was therefore to identify gene mutations that determine process which is also controlled by the DDR (14, 15). In addition Wee1 inhibitor sensitivity. We performed a genome-wide unbiased to this fast-acting kinase-driven DDR network, a transcriptional functional genetic screen in TP53 mutant near-haploid KBM-7 cells program is activated through p53 stabilization (16). Among the using gene-trap insertional mutagenesis. Insertion site mapping of many p53 target genes, expression of the CDK inhibitor p21 is cells that survived long-term Wee1 inhibition revealed enrichment of induced to mediate a sustained G1/S cell cycle arrest, which SKP2 CUL1 CDK2. G1/S regulatory genes, including , ,and Stable de- makes the G1/S checkpoint largely dependent on p53 (17). pletion of SKP2, CUL1,orCDK2 or chemical Cdk2 inhibition rescued Many human tumors lack functional p53, and consequently γ the -H2AX induction and abrogation of G2 phase as induced by cannot properly arrest at the G1/S transition. TP53-mutant Wee1 inhibition in breast and ovarian cancer cell lines. Remarkably, cancers therefore enter S phase even in the presence of DNA SKP2 CUL1 CDK2 live cell imaging showed that depletion of , ,or did damage and depend strongly on their G2/M checkpoint control not rescue the Wee1 inhibition-induced karyokinesis and cytokinesis for genomic stability. In line with this notion, therapeutic tar- defects. These data indicate that the activity of the DNA replication geting of G2/M checkpoint kinases was proposed to improperly machinery, beyond TP53 mutation status, determines Wee1 inhibitor sensitivity, and could serve as a selection criterion for Wee1-inhibitor Significance eligible patients. Conversely, loss of the identified S-phase genes could serve as a mechanism of acquired resistance, which goes along with Inhibition of Wee1 is considered an attractive anticancer therapy development of severe genomic instability. for TP53 mutant tumors. However, additional factors besides p53 inactivation may determine Wee1 inhibitor sensitivity, which we cell cycle | checkpoint | AZD-1775 | MK-1775 | polyploidy searched for using unbiased functional genetic screening. We discovered that the mutational status of several S-phase genes, recise cell cycle control is critical for proliferating cells, es- including CDK2, determines the cytotoxicity induced by Wee1 Ppecially under conditions of genomic stress. Modulation of inhibition. Notably, we found that Wee1 inhibition induces two the cell cycle checkpoint machinery is therefore often proposed distinct phenotypes: accumulation of DNA damage in S phase and as a therapeutic strategy to potentiate anticancer therapy (1). karyokinesis/cytokinesis failure during mitosis. Stable depletion Therapeutic inhibition of checkpoint kinases can deregulate cell of S-phase genes only reversed the formation of DNA damage, cycle control and improperly force cell cycle progression, even in but did not rescue karyokinesis/cytokinesis failure upon Wee1 the presence of DNA damage. Chemical inhibitors for several cell inhibition. Thus, inactivation of nonessential S-phase genes can cycle checkpoint kinases have been developed. Preclinical research, overcome Wee1 inhibitor resistance, while allowing the survival however, has shown that the efficacy of therapeutic checkpoint of genomically instable cancer cells. inhibition is context-sensitive and depends on the genetic make-up of an individual cancer (2, 3). Therefore, to optimally implement Author contributions: A.M.H. and M.A.T.M.v.V. designed research; A.M.H., V.A.B., X.B., F.D., F.Y.M., F.F., and M.A.T.M.v.V. performed research; X.B. and P.K. contributed new such novel inhibitors in the clinic, the molecular characteristics that reagents/analytic tools; A.M.H., V.A.B., X.B., P.K., and M.A.T.M.v.V. analyzed data; and determine inhibitor activity need to be identified to select eligible A.M.H. and M.A.T.M.v.V. wrote the paper. patients and to anticipate on mechanisms of acquired resistance. The authors declare no conflict of interest. In response to cellular insults like DNA damage, cells activate This article is a PNAS Direct Submission. cell cycle checkpoints to arrest proliferation at the G1/S or G2/M Data deposition: Sequence data have been deposited at the NCBI Sequence Read Archive, transition. These checkpoints operate by controlling the in- www.ncbi.nlm.nih.gov/sra (accession no. SRP065601). hibitory phosphorylation on cyclin-dependent kinases (CDKs), 1To whom correspondence should be addressed. Email: [email protected]. key drivers of the cell cycle (4). Most of the current knowledge This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. concerns the regulation of Cdk1, which is phosphorylated by the 1073/pnas.1505283112/-/DCSupplemental. 15160–15165 | PNAS | December 8, 2015 | vol. 112 | no. 49 www.pnas.org/cgi/doi/10.1073/pnas.1505283112 Downloaded by guest on September 28, 2021 force p53-defective cells to progress through the cell cycle in and SKOV3 (Fig. S1A). Based on these results, we selected the the presence of DNA damage (1). IC95 dose of MK-1775 (300 nM) to study the kinetics of cell death Wee1 is one of the G2/M cell cycle checkpoint kinases for which induced by Wee1 inhibition in KBM-7 cells. Time-course analysis selective chemical inhibitors have been developed as anticancer of Cdk1 phosphorylation at Tyr-15 revealed that Wee1 inhibition drugs (3, 18). Inhibition of Wee1 potentiates DNA-damaging was effective within 15 min after administration (Fig. S1B). Also, chemotherapeutics and radiotherapy and has cytotoxic effects as Wee1 inhibition resulted in reduced levels of Cdk2 phosphoryla- a single agent (19–22). The consensus view is that Wee1 inhibi- tion at Tyr-15, and elevated γ-H2AX levels as described (25). MK- tion facilitates tumor cell killing through G2/M checkpoint in- 1775-induced cell death was visible from 6 h of treatment onwards, activation, which would catalyze mitotic catastrophe (23–25). As as evidenced by PARP cleavage (Fig. S1B) and the appearance of expected, Wee1 inhibition is synergistic with DNA-damaging cells with sub-G1 DNA content (Fig. S1C). agents, specifically in TP53-mutant cancer cell lines (3). However, Due to the effective induction of apoptosis within 48 h of in- a recent study indicated that only a subset of TP53-mutant pa- cubation (Fig. S1C), and near-complete cell death within 72 h tient-derived pancreatic cancer xenografts showed benefit from (Fig. S1A), we were able to use this setup for screening gene Wee1 inhibition (21). This indicates that molecular determinants mutations that confer resistance to Wee1 inhibitor treatment. other than TP53 mutation status control the cytotoxic effects of Approximately 60 million KBM-7 cells were randomly muta- Wee1 inhibition, but these determinants are currently unknown. genized using retroviral delivery of gene-trap virus. After muta- To improve cancer patient selection for Wee1 inhibitor treat- genesis, KBM-7 cells were treated with Wee1 inhibitor, and the ment, to uncover possible mechanisms of resistance, and to help surviving cells were allowed to grow colonies for 14 d. In- understand how Wee1 inhibitors mediate cytotoxicity, we aimed tegration sites of ∼80,000
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