Inhibition of DDR 1‐ BCR signalling by nilotinib as a new therapeutic strategy for metastatic colorectal cancer

• DOI: 10.15252/emmm.201707918
• Publication Date: 2018-02-12T16:12:54Z
• Authors: Maya Jeitany; Cédric Leroy; Priscillia Tosti; Marie Lafitte; Jordy Le Guet; Valérie Simon; Debora Bonenfant; Bruno Robert; Fanny Grillet; Caroline Mollevi; Safia El Messaoudi; Amaëlle Otandault; Lucile Canterel‐T...; Muriel Busson; Alain R Thierry; Pierre Martineau; Julie Pannequin; Serge Roche; Audrey Sirvent
• Keywords: Signalling

Research Article9 February 2018Open Access Source DataTransparent process Inhibition of DDR1-BCR signalling by nilotinib as a new therapeutic strategy for metastatic colorectal cancer Maya Jeitany CRBM, CNRS, University Montpellier, France Search more papers this author Cédric Leroy Novartis Institutes Biomedical Research, Postfach, Basel, Switzerland Actelion Pharmaceuticals Ltd, Allschwil, Priscillia Tosti Marie Lafitte Jordy Le Guet Valérie Simon Debora Bonenfant Bruno Robert IRCM, INSERM, Fanny Grillet IGF, Caroline Mollevi Safia El Messaoudi Amaëlle Otandault Lucile Canterel-Thouennon Muriel Busson Alain R Thierry Pierre Martineau Julie Pannequin Serge Roche Corresponding Author [email protected] orcid.org/0000-0003-3413-3859 Audrey Sirvent orcid.org/0000-0003-4555-6312 Information Jeitany1,‡, Leroy1,2,3,‡, Tosti1,‡, Lafitte1, Guet1, Simon1, Bonenfant2, Robert4, Grillet5, Mollevi4, Messaoudi4, Otandault4, Canterel-Thouennon4, Busson4, Thierry4, Martineau4, Pannequin5, *,1,‡ and 1CRBM, 2Novartis 3Actelion 4IRCM, 5IGF, ‡These authors contributed equally to work *Corresponding author. Tel: [phone]; E-mail: 434359503; EMBO Mol Med (2018)10:e7918https://doi.org/10.15252/emmm.201707918 PDFDownload PDF article text main figures. Peer ReviewDownload summary the editorial decision including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The clinical management (mCRC) faces major challenges. Here, we show that nilotinib, clinically approved drug chronic myeloid leukaemia, strongly inhibits human CRC cell invasion in vitro reduces their potential intrasplenic tumour mouse models. Nilotinib acts inhibiting kinase activity DDR1, receptor tyrosine collagens, which identified RAS-independent inducer metastasis. Using quantitative phosphoproteomics, BCR DDR1 substrate demonstrated prevents DDR1-mediated phosphorylation on Tyr177, is important maintaining β-catenin transcriptional necessary invasion. inhibition also reduced patient-derived circulating lines. Collectively, our results indicate targeting with may be beneficial patients mCRC. Synopsis Targeting collagens mCRC cells properties paves way promotes nude mice. central DDR1. activation maintains high level progression. pharmacological interest treatment cancer. Introduction Colorectal (CRC) one leading causes malignancy-related death worldwide. Most these cancers are sporadic under control genetic, epigenetic environmental factors. current involves surgical removal primary tumour, often associated chemotherapy. However, recurrence occurs about 50% at distant site, resulting bad prognosis 10% survival 5 years. Therapeutic failure spread, escape disseminate circulation establish secondary lesions organs, mainly liver (Dienstmann et al, 2017). Metastatic behaviour characterized invasive tumour-initiating capacity disseminated (Vanharanta Massague, 2013). Currently, much research focused pathways promote kinases (TK) have emerged determinants (Sirvent 2012; Vanharanta For instance, anti-epidermal growth factor (EGFR) antibodies significantly improve (Lievre 2006, 2008); however, only wild-type RAS (40% cases) hope benefit from treatment. Indeed, many harbour oncogenic mutations components, thus, cascade constitutively activated, independently EGFR. In addition, prolonged EGFR-blockade induces resistance pathway reactivation (Misale 2014). Therefore, there need strategies reduce Clinically drugs designed target well-defined oncogene broader through off-target-dependent mechanisms. belongs agents used clinic inhibit fusion BCR-ABL highly effective against Chronic Myeloid Leukaemia (CML). small-molecule imatinib (an ABL inhibitor) common phase CML (Druker 2001); resistances emerge caused point domain lower inhibitor affinity. has been developed imatinib, notable exception T315I mutant. Interestingly, profiling chemical proteomic methods led discovery highest affinity (Rix 2007). poorly RTK binds ones components extracellular matrix, functions matrix sensor regulate adhesion (Vogel 1997). cross-talks several transmembrane receptors, Notch TGF-β could influence upon collagen stimulation (Leitinger, proliferation, motility invasion, depending type nature microenvironment 2014; Rammal 2016). Curiously, role documented. This seems dispensable most reported cancers, such collective migration squamous carcinoma (Hidalgo-Carcedo 2011), breast (Juin 2014) (Gao Moreover, very little known mechanisms underlying signalling, unusually slow sustained over time One lung where KRAS induce expression sustain tumorigenesis. progression xenograft models, suggesting an kinase-dependent function (Ambrogio mechanism modulates currently unknown. report additional metastasis formation. We mechanism. By phosphoproteomic analysis, then involved maintenance showed can model lines originating tumours cells. advanced CRC. Results displays anti-metastatic found TKI (100 nM) panel Boyden chamber assays, irrespective KRAS/BRAF status (Fig 1A). Similar were obtained 3D spheroid assays HCT116 embedded I 1B). deduced IC50 was 15–30 nM range 1C) comparable inhibitory potency leukaemic (Weisberg 2005). evaluated vivo using mice, injected spleen recipient animals colonize via hepatic portal vein. A daily regimen 50 mg/kg dose shows anti-leukaemic experimental models 2005), inhibited formation treated compared untreated controls (vehicle) 1D). Thus, Figure 1. anti-invasive Percentage chambers containing 1 mg/ml Matrigel indicated incubated 100 (mean ± SEM; n = 3). assays. or DMSO (control) seeded upper compartment 24 h spheroids 72 imaged phase-contrast microscopy. histogram percentage migrating normalized condition set 100% 3 independent experiments six replicates; *P < 0.05 Student's t-test). quantification shown (C). Dose–response curve effect (relative control) HT29 (▲) (■) concentrations mice (n 11/group). After 4 weeks treatment, livers removed. Representative images (black arrowheads metastasis) index vehicle (oral administration) 11/group; data available online figure. Data [emmm201707918-sup-0005-SDataFig1.pdf] Download figure PowerPoint analysed molecular anti-tumour As not mutated cancer, speculated involvement other approach To test hypothesis, first assessed well expressed active all tested lines, increased EV1A). depletion two different shRNAs capacities KRAS-mutated BRAF-mutated 2A–C). silencing after inoculation 2D). confirmed large decrease DNA (ctDNA) biomarker (Mouliere 2013; Fig Conversely, overexpression SW620 2E F) development DDR1-overexpressing (SW620-DDR1 cells) (mock transfected) 2G). agreement, ctDNA inoculated (mock) These Click here expand EV1. collagen-mediated A. Collagen (pY DDR1). B, C. phosphorylation. Western blotting protein lysates 40 μg/ml 18 incubation times nilotinib. 2. A–D. shRNA (A) infected vectors expressing blotting. Invasion (B) (C) 7 (C); **P 0.01, ***P 0.001 representative image each line shown. (D) 12/group). weeks, group, (ng/ml plasma) animal 0.05; 0.01 E–G. (E) viruses. (F) viruses 3; (G) 12 mock group 20 group). relative 2 [emmm201707918-sup-0006-SDataFig2.pdf] mediated next determined activities increasing doses (0.8–100 slow, but persistent increase induced EV1B). 1–8 range, tightly correlated its Surprisingly, addition 15 min enough abrogate overnight EV1C), regulated phosphatases leukaemia mutation threonine 315 into bulkier isoleucine appeared conserved TK because corresponding (T701I) I-induced mutant 50-fold (WT) 3A). assess DDR1-depleted retroviruses express kinase-dead (KD) T701I levels similar those endogenous 3B) examined response Expression T701I, KD rescued lost 3C, DMSO). indicates essential 3C). Similarly, prevented observed nodules 3D). corroborate action, overexpressed infection) promotion fully abolished 3E). Finally, block already nodules. experiments, SW620-DDR1 luciferase started day 7, when luciferase-positive metastases detectable. setting, decreased 3F). monitoring signal surrogate marker burden (Appendix S1). highlight 3. Biochemical analysis nilotinib-resistant Tyrosine HEK293T transfected constructs stimulated presence concentration B–D. abrogated blot (wild type, WT; T701I; dead, KD). 4; 5/group) starting post-injection administration). E, F. overexpress spleen, 21 14 group) mg/kg/d indicated, [emmm201707918-sup-0007-SDataFig3.pdf] Quantitative analyses identify novel gain insight targeted revealed moderate, content (4–18 h) activation, measured DDR1-Tyr792 autophosphorylation. absence (shDDR1) suggests it might involve receptors EV2A). On hand, had no MAPK AKT EV2A), indicating collagen-stimulated RAS-independent. result inability stimulate EV2B). addressed whether, like 2016), activity. MAPKs MEK collagen-induced EV2C). thus consistent EV2. A, B. Depletion abolishes (pTyr) BCR, (Col I) times. (treatment 10 μM PD98059 during does impact signalling. D. metastases. described 3F. performed global phosphotyrosine peptide immunopurification followed label-free mass spectrometry-based (Rush 2005; 2015). molecules comparing profiles unstimulated cells, h, period corresponds maximal before lysis acute 4A B). With approach, 229 289 unique tyrosine-phosphorylated peptides biological 105 83 proteins (Dataset EV1). PEAK1 reproducibly phosphorylated 4C). Tyr484 juxtamembrane Tyr792 Tyr796 loop. confirms activated stimulation. pseudokinase PEAK1, previously (Wang 2010), Tyr462 Tyr177. essentially context BCR-ABL-transformed (Deininger 2000). low number events moderate detected did detect any effectors systems, SHP2, SHC, CDC42, JAK2 STAT3 Leitinger, Gao downstream substrates CRK CRKL analysis; modified stimulation, they cascade. 4. identifies Workflow casca