E2F1 proteolysis via SCF ‐cyclin F underlies synthetic lethality between cyclin F loss and Chk1 inhibition

• DOI: 10.15252/embj.2018101443
• Publication Date: 2019-08-19T10:59:53Z
• Authors: Kamila Burdova; Hongbin Yang; Roberta Faedda; Samuel Hume; Jagat Chauhan; Daniel Ebner; Benedikt M Kessler; Iolanda Vendrell; David H Drewry; Carrow I Wells; Stephanie B Hatch; Grigory L Dianov; Francesca M Buffa; Vincenzo D'Angiol...
• Keywords: Synthetic Lethality

Article19 August 2019Open Access Transparent process E2F1 proteolysis via SCF-cyclin F underlies synthetic lethality between cyclin loss and Chk1 inhibition Kamila Burdova Department of Oncology, Medical Research Council Institute for Radiation University Oxford, UK Search more papers by this author Hongbin Yang Roberta Faedda Samuel Hume Jagat Chauhan Nuffield Clinical Medicine, Ludwig Cancer Research, Headington, Daniel Ebner Target Discovery Institute, Benedikt M Kessler Iolanda Vendrell David H Drewry Structural Genomics Consortium, UNC Eshelman School Pharmacy, North Carolina at Chapel Hill, NC, USA Carrow I Wells Stephanie B Hatch Grigory L Dianov Cytology Genetics, Russian Academy Sciences, Novosibirsk, Federation Novosibirsk State University, Francesca Buffa Vincenzo D'Angiolella Corresponding Author [email protected] orcid.org/0000-0001-8365-9094 Information Burdova1,‡, Yang1,‡, Faedda1, Hume1, Chauhan2, Ebner3, Kessler3, Vendrell1,3, Drewry4, Wells4, Hatch3, Dianov1,5,6, Buffa1 *,1 1Department 2Nuffield 3Nuffield 4Structural 5Institute 6Novosibirsk ‡These authors contributed equally to work *Corresponding author. Tel: [phone]; E-mail: The EMBO Journal (2019)38:e101443https://doi.org/10.15252/embj.2018101443 See also: HI Rösner & CS Sørensen (October 2019) PDFDownload PDF article text main figures. Peer ReviewDownload a summary the editorial decision including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures Info Abstract Cyclins are central engines cell cycle progression in conjunction with cyclin-dependent kinases (CDKs). Among different cyclins controlling progression, does not partner CDK, but instead forms its F-box domain an SCF (Skp1-Cul1-F-box)-type E3 ubiquitin ligase module. Although various substrates have been identified, vulnerabilities cells lacking known. Thus, we assessed viability upon challenging them than 180 kinase inhibitors. screen revealed striking loss. leads DNA replication catastrophe. Replication catastrophe depends on accumulation transcription factor F-depleted cells. We find that controls ubiquitylation degradation during G2/M phase Cyclin restricts activity checkpoint prevent stress. Our findings pave way patient selection clinical use Synopsis A inhibitor adaptor identification as new SCF-Cyclin substrate, whose unrestricted promotes catastrophic stress suppression identify identifies inhibitors primary hits. predisposes toxic effect Chk1i inducing Aberrant damage inhibition. Lack prevents non-degradable CY motif induces death Introduction Cell transitions operated periodic oscillations cyclins, which bind (CDKs) promote phosphorylation target drive progression. Entry into S is initiated activation genes required replication. restrained principally retinoblastoma (Rb) protein, masks transactivation keep it inactive. control Rb crucial unscheduled entry, hallmark cancer. Indeed, common cancer event (Dyson, 2016). co-ordinating E/A B, partnership CDK2 CDK1, respectively, through G2 (Lim Kaldis, 2013). most similar act activator CDKs able (Bai et al, 1994). Instead, F, also known only protein 1 (Fbxo1), founding member family proteins 1996). domain, functional Skp1-Cul1-F-Box (SCF) complex acting ligase. SCFcyclin mediates important genome stability (D'Angiolella 2013; Klein 2015). In addition coordinated action monitored presence checkpoints, restrict CDK execution phases if previous one has completed. Blocks fork single-stranded (ssDNA) behind fork, coating ssDNA-binding RPA represents stimulus ATR kinase. Active elicits response phosphorylating initiating signalling cascade culminates inactivation. exerts function subsequent Cdc25A phosphatase (Busino 2003; Jin Kotsantis 2018). removes inhibitory Tyr15 CDKs, mediated tyrosine Wee1 prompt activation. Upon Chk1, suppressed restrain (Bartek Lukas, 2003). allows repair before committing mitosis restricting until S/G2 transition (Lemmens If blocks cannot be bypassed or repaired, ssDNA gaps can converted double-stranded breaks (DSBs), deleterious could lead gross chromosomal rearrangements resulting death. Given their high proliferative capacity compromised repair, intrinsically higher damage. These observations spurred development number targeting Chk1. being evaluated multiple trials, genetic predispositions would sensitise desensitise fully elucidated. By screening chemogenomic set (KCGS) discover novel lethal interaction observe after promoted E2F1. substrate controlled expressing version E2F1, show exacerbates sensitivity, promoting DSBs Results KCGS Using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9, previously generated knock-out (CCNF K/O) lines (Mavrommati Interestingly, these present profile comparable parental line (Fig EV1A B). To weaknesses conducted drug assess differences CCNF K/O (HeLa; Fig 1A). end, used KCGS, origins traced well-utilised collections PKIS PKIS2 (Elkins 2016; 2017). simple premise publicly available, well annotated potent selective disease-relevant phenotypic assays efficient elucidate biology uncover dependencies (Jones Bunnage, current consists 188 small molecule cover 221 kinases. None members broadly kinome active, facilitating deconvolution. was run duplicate average replicate correlation (Pearsons') 0.9467 Z-Factor 0.6040, demonstrating good dynamic range DMSO (vehicle-negative control) (chemo-toxic-positive robust reproducibility replicates. inhibitors, specifically cells, normalised results using Z-score each then expressed difference ΔZ. graph 1A additional data Table highlight compounds sensitive resistant (Dataset EV1). obtained CCT244747, (Walton 2012). killed identified VE-822, (Charrier 2011). Similarly, parallel screen, sensitivity two other unrelated structure, PF477736 AZD7762, observed 1B; Blasina 2008; Zabludoff 2008). Click here expand figure. Figure EV1. (related 1) HeLa detected FACS DAPI. distribution untreated combined phospho-histone H3 Serine 10 EdU staining. survival RPE transfected non-targeting siRNA siNC (negative siCyc treatment (LY2603618) indicated concentrations compared DMSO-treated (NT). measured resazurin treated (expressed relative proliferation %). Cells were concentrations. U-2-OS Data information: presented mean ± SD, least three independent experiments. P-values (*P < 0.05, **P 0.005) calculated paired two-tailed t-test. Download figure PowerPoint 1. (KSGS) 384-well format. After 72 h, resazurin. Flowchart representation (left). plotted acquired μM concentration (right). 0.1 concentration. (0) UCN-01 Differences specific log10 scale measure IC50. Number left 24, 48 h indicated. (n.s. = non-significant). measurements dead %) propidium iodide staining (LY2603618). seeded F. Twenty-four hours transfection, 3 days measured. LY2603618 standard deviation (SD), 0.005, ***P 0.0005, ****P 0.00005) NT DMSO. Top hits comparing Compound name Δ z-score 100 nM More C10 VE-822 −0.61484 B17 CCT251545 −0.56548 CDK8, CDK19 I03 GSK2336394 −0.4252 PIK3CB Less A20 GW784752X 1.016439 GSK3b A21 GW580509X 1.052113 VEGFR2 E22 GW296115X 1.443446 PDGFR B08 CCT244747 −2.88143 CHK1 E16 BI00614644 −0.56505 MAPKAPK2, MAPKAPK5, PIM3, PHKG2 I05 SB-725317 −0.55738 CDK2/cyclinA, LTK C11 SB-590885 1.036447 B-raf B03 JNK-IN-7 1.164645 JNK 1.180556 PDGFRB D15 GW814408X 1.364238 H16 GSK1070916 1.572064 Aurora D12 CA93.0 2.072805 GAK1 H19 GW807982X −1.06323 I20 TPKI-39 −0.86181 CIT, KIT, PDGFRB, CSF1R, PDGFRA, DDR1, FLT1, TIE2 C13 GSK1838705 −0.73301 IGF1R, IR G14 PFE-PKIS 0.838916 TNK2, TYRO3, YES, FGFR1,2,&3 H11 CCT241533 0.859239 CHK2 G09 GW440135 0.866643 NEK7, MEK5, VEGFR2, NEK9, FLT4 confirm PF47736 AZD7762 target, tested UCN-01, structurally decreased 1C). LY2603618, very (King 2014), viability. IC50 normal > μM, whereas had 400 nM, accounting significant fold across 1D). establish due defects K/O. Overall, 72-h time-frame 1E). However, stopped proliferating 1E), corresponding increase 1F). exclude possibility phenotype induced limited lines, knockdown siRNA. 500 caused where levels diminished significantly reduced increased 1G H). addition, non-transformed RPE-1 showed EV1C). Since upstream surprising above VE-821 killing K/O, although doses less pronounced EV1D). Similar depletion EV1E). depletion. investigated further. Loss causes ssDNA, exacerbated over time active saturates exposed nucleases. Nucleases convert DSBs, DNA-PK ATM DSB repair. markers indication (Toledo gain insights mechanism inhibition, inhibitor. contrast (S) 33, surrogate marker activation, 6 already S33 2 2A). While attributed RPA-coated further associated formation leading γ-H2AX, S4 S8 KAP1 S824 16-h point reported line, LY2603618. Activation continued thereafter, accumulating profoundly 16 24 It worth mentioning overall unchanged indicating events rather change total reproduced EV2A). 2. (h) harvested lysed SDS. Indicated resolved SDS–PAGE Western blot (WB). TFIIH loading control. Percentage (%) γ-H2AX-positive G1 Fluorescence Activated Sorting (FACS). considered having DAPI 2n negative. replicating (EdU+) points. treatment. 4n negativity. Intensity γ-H2AX fluorescence units (RFU) early Chk1i. Mean intensity from EdU+ 2N content. EdU+. Overall EdU− gated cells; phase; pH3 S10-negative S10 positive experiments SD. Representative plots versus RPA2 signal (h). Blue labelled dots represent Relates Materials section details representation. biological replicates SD (B–D) RFU (E, F). EV2. Figs 3) 20 Early content % mid-late Mid-late Half hour harvesting, incorporated (10 final concentration). click reaction, γ-H2Ax immunostaining, analysis. indeed encountered catastrophe, analysed chromatin bound accordance induction profound 2B–D) points, progressed (Figs 2E EV2B C). At treatment, about 15% found highly negative, 2-4n 2G). arrested phase, likely extensive excessive starting EV2D). Consistent observation reached peak generation conversion (high loading) (γ-H2AX) confined highest degree chromatin-loaded 2H). shape plot nuclear ratio following consistent 2H)