In-cell profiling enables the evaluation of receptor tyrosine activity in a complex environment of regulatory networks that affect signal initiation, propagation and feedback. We used FGF-receptor signaling to identify EGR1 as a locus that strongly responds to the activation of a majority of the recognized protein kinase oncogenes, including 30 receptor tyrosine kinases and 154 of their disease-associated mutants. The EGR1 promoter was engineered to enhance trans-activation capacity and optimized for simple screening assays with luciferase or fluorescent reporters. The efficacy of the developed, fully synthetic reporters was demonstrated by the identification of novel targets for two clinically used tyrosine kinase inhibitors, nilotinib and osimertinib. A universal reporter system for in-cell protein kinase profiling will facilitate repurposing of existing anti-cancer drugs and identification of novel inhibitors in high-throughput screening studies.

Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Biology Faculty of Medicine Masaryk University Brno Czech Republic

Department of Experimental Biology Faculty of Sciences Masaryk University Brno Czech Republic

Department of Internal Medicine Hematology and Oncology Masaryk University Hospital Brno Czech Republic

International Clinical Research Center St Anne's University Hospital Brno Czech Republic

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Bae JH, Schlessinger J. Asymmetric tyrosine kinase arrangements in activation or autophosphorylation of receptor tyrosine kinases. Molecules and Cells. 2010;29:443–448. doi: 10.1007/s10059-010-0080-5. PubMed DOI

Bamborough P. System-based drug discovery within the human kinome. Expert Opinion on Drug Discovery. 2012;7:1053–1070. doi: 10.1517/17460441.2012.724056. PubMed DOI

Buchtova M, Oralova V, Aklian A, Masek J, Vesela I, Ouyang Z, Obadalova T, Konecna Z, Spoustova T, Pospisilova T, Matula P, Varecha M, Balek L, Gudernova I, Jelinkova I, Duran I, Cervenkova I, Murakami S, Kozubik A, Dvorak P, Bryja V, Krejci P. Fibroblast growth factor and canonical WNT/β-catenin signaling cooperate in suppression of chondrocyte differentiation in experimental models of FGFR signaling in cartilage. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 2015;1852:839–850. doi: 10.1016/j.bbadis.2014.12.020. PubMed DOI

Carter EP, Fearon AE, Grose RP. Careless talk costs lives: fibroblast growth factor receptor signalling and the consequences of pathway malfunction. Trends in Cell Biology. 2015;25:221–233. doi: 10.1016/j.tcb.2014.11.003. PubMed DOI

Cortes JE, Kim DW, Pinilla-Ibarz J, le Coutre P, Paquette R, Chuah C, Nicolini FE, Apperley JF, Khoury HJ, Talpaz M, DiPersio J, DeAngelo DJ, Abruzzese E, Rea D, Baccarani M, Müller MC, Gambacorti-Passerini C, Wong S, Lustgarten S, Rivera VM, Clackson T, Turner CD, Haluska FG, Guilhot F, Deininger MW, Hochhaus A, Hughes T, Goldman JM, Shah NP, Kantarjian H, PACE Investigators A phase 2 trial of ponatinib in Philadelphia chromosome-positive leukemias. New England Journal of Medicine. 2013;369:1783–1796. doi: 10.1056/NEJMoa1306494. PubMed DOI PMC

Cox AD, Fesik SW, Kimmelman AC, Luo J, Der CJ. Drugging the undruggable RAS: Mission possible? Nature Reviews Drug Discovery. 2014;13:828–851. doi: 10.1038/nrd4389. PubMed DOI PMC

Cross DA, Ashton SE, Ghiorghiu S, Eberlein C, Nebhan CA, Spitzler PJ, Orme JP, Finlay MR, Ward RA, Mellor MJ, Hughes G, Rahi A, Jacobs VN, Red Brewer M, Ichihara E, Sun J, Jin H, Ballard P, Al-Kadhimi K, Rowlinson R, Klinowska T, Richmond GH, Cantarini M, Kim DW, Ranson MR, Pao W. AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer. Cancer Discovery. 2014;4:1046–1061. doi: 10.1158/2159-8290.CD-14-0337. PubMed DOI PMC

Dvorak P, Dvorakova D, Koskova S, Vodinska M, Najvirtova M, Krekac D, Hampl A. Expression and potential role of fibroblast growth factor 2 and its receptors in human embryonic stem cells. Stem Cells. 2005;23:1200–1211. doi: 10.1634/stemcells.2004-0303. PubMed DOI

Finlay MR, Anderton M, Ashton S, Ballard P, Bethel PA, Box MR, Bradbury RH, Brown SJ, Butterworth S, Campbell A, Chorley C, Colclough N, Cross DA, Currie GS, Grist M, Hassall L, Hill GB, James D, James M, Kemmitt P, Klinowska T, Lamont G, Lamont SG, Martin N, McFarland HL, Mellor MJ, Orme JP, Perkins D, Perkins P, Richmond G, Smith P, Ward RA, Waring MJ, Whittaker D, Wells S, Wrigley GL. Discovery of a potent and selective EGFR inhibitor (AZD9291) of both sensitizing and T790M resistance mutations that spares the wild type form of the receptor. Journal of Medicinal Chemistry. 2014;57:8249–8267. doi: 10.1021/jm500973a. PubMed DOI

Forbes SA, Beare D, Gunasekaran P, Leung K, Bindal N, Boutselakis H, Ding M, Bamford S, Cole C, Ward S, Kok CY, Jia M, De T, Teague JW, Stratton MR, McDermott U, Campbell PJ. COSMIC: exploring the world's knowledge of somatic mutations in human cancer. Nucleic Acids Research. 2015;43:D805–D811. doi: 10.1093/nar/gku1075. PubMed DOI PMC

Fountas A, Diamantopoulos LN, Tsatsoulis A. Tyrosine kinase inhibitors and diabetes: A novel treatment paradigm? Trends in Endocrinology & Metabolism. 2015;26:643–656. doi: 10.1016/j.tem.2015.09.003. PubMed DOI

Greig SL. Osimertinib: first global approval. Drugs. 2016;76:263–273. doi: 10.1007/s40265-015-0533-4. PubMed DOI

Harskamp LR, Gansevoort RT, van Goor H, Meijer E. The epidermal growth factor receptor pathway in chronic kidney diseases. Nature Reviews Nephrology. 2016;12:496–506. doi: 10.1038/nrneph.2016.91. PubMed DOI

Hochhaus A, Baccarani M, Deininger M, Apperley JF, Lipton JH, Goldberg SL, Corm S, Shah NP, Cervantes F, Silver RT, Niederwieser D, Stone RM, Dombret H, Larson RA, Roy L, Hughes T, Müller MC, Ezzeddine R, Countouriotis AM, Kantarjian HM. Dasatinib induces durable cytogenetic responses in patients with chronic myelogenous leukemia in chronic phase with resistance or intolerance to imatinib. Leukemia. 2008;22:1200–1206. doi: 10.1038/leu.2008.84. PubMed DOI

Hucthagowder V, Meyer R, Mullins C, Nagarajan R, DiPersio JF, Vij R, Tomasson MH, Kulkarni S. Resequencing analysis of the candidate tyrosine kinase and RAS pathway gene families in multiple myeloma. Cancer Genetics. 2012;205:474–478. doi: 10.1016/j.cancergen.2012.06.007. PubMed DOI PMC

Inglés-Prieto Á, Reichhart E, Muellner MK, Nowak M, Nijman SM, Grusch M, Janovjak H. Light-assisted small-molecule screening against protein kinases. Nature Chemical Biology. 2015;11:952–954. doi: 10.1038/nchembio.1933. PubMed DOI PMC

Jiang T, Zhou C. Clinical activity of the mutant-selective EGFR inhibitor AZD9291 in patients with EGFR inhibitor-resistant non-small cell lung cancer. Translational Lung Cancer Research. 2014;3:370–372. doi: 10.3978/j.issn.2218-6751.2014.08.02. PubMed DOI PMC

Kant SG, Cervenkova I, Balek L, Trantirek L, Santen GW, de Vries MC, van Duyvenvoorde HA, van der Wielen MJ, Verkerk AJ, Uitterlinden AG, Hannema SE, Wit JM, Oostdijk W, Krejci P, Losekoot M. A novel variant of FGFR3 causes proportionate short stature. European Journal of Endocrinology. 2015;172:763–770. doi: 10.1530/EJE-14-0945. PubMed DOI

Krejci P, Murakami S, Prochazkova J, Trantirek L, Chlebova K, Ouyang Z, Aklian A, Smutny J, Bryja V, Kozubik A, Wilcox WR. NF449 is a novel inhibitor of fibroblast growth factor receptor 3 (FGFR3) signaling active in chondrocytes and multiple myeloma cells. Journal of Biological Chemistry. 2010;285:20644–20653. doi: 10.1074/jbc.M109.083626. PubMed DOI PMC

Krejci P, Pejchalova K, Wilcox WR. Simple, mammalian cell-based assay for identification of inhibitors of the Erk MAP kinase pathway. Investigational New Drugs. 2007;25:391–395. doi: 10.1007/s10637-007-9054-7. PubMed DOI

Krutá M, Šeneklová M, Raška J, Salykin A, Zerzánková L, Pešl M, Bártová E, Franek M, Baumeisterová A, Košková S, Neelsen KJ, Hampl A, Dvořák P, Rotrekl V. Mutation frequency dynamics in HPRT locus in culture-adapted human embryonic stem cells and induced pluripotent stem cells correspond to their differentiated counterparts. Stem Cells and Development. 2014;23:2443–2454. doi: 10.1089/scd.2013.0611. PubMed DOI PMC

Kubo T, Kuroda Y, Shimizu H, Kokubu A, Okada N, Hosoda F, Arai Y, Nakamura Y, Taniguchi H, Yanagihara K, Imoto I, Inazawa J, Hirohashi S, Shibata T. Resequencing and copy number analysis of the human tyrosine kinase gene family in poorly differentiated gastric cancer. Carcinogenesis. 2009;30:1857–1864. doi: 10.1093/carcin/bgp206. PubMed DOI

Kweon SM, Cho YJ, Minoo P, Groffen J, Heisterkamp N. Activity of the Bcr GTPase-activating domain is regulated through direct protein/protein interaction with the Rho guanine nucleotide dissociation inhibitor. Journal of Biological Chemistry. 2008;283:3023–3030. doi: 10.1074/jbc.M705513200. PubMed DOI

Laederich MB, Horton WA. FGFR3 targeting strategies for achondroplasia. Expert Reviews in Molecular Medicine. 2012;14:e11. doi: 10.1017/erm.2012.4. PubMed DOI

Lemmon MA, Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell. 2010;141:1117–1134. doi: 10.1016/j.cell.2010.06.011. PubMed DOI PMC

Loots GG, Ovcharenko I. rVISTA 2.0: evolutionary analysis of transcription factor binding sites. Nucleic Acids Research. 2004;32:W217–W221. doi: 10.1093/nar/gkh383. PubMed DOI PMC

Meloche S, Pouysségur J. The ERK1/2 mitogen-activated protein kinase pathway as a master regulator of the G1- to S-phase transition. Oncogene. 2007;26:3227–3239. doi: 10.1038/sj.onc.1210414. PubMed DOI

Modugno M. New resistance mechanisms for small molecule kinase inhibitors of Abl kinase. Drug Discovery Today: Technologies. 2014;11:5–10. doi: 10.1016/j.ddtec.2013.12.001. PubMed DOI

Naski MC, Wang Q, Xu J, Ornitz DM. Graded activation of fibroblast growth factor receptor 3 by mutations causing achondroplasia and thanatophoric dysplasia. Nature Genetics. 1996;13:233–237. doi: 10.1038/ng0696-233. PubMed DOI

Ni Q, Titov DV, Zhang J. Analyzing protein kinase dynamics in living cells with FRET reporters. Methods. 2006;40:279–286. doi: 10.1016/j.ymeth.2006.06.013. PubMed DOI

Notredame C, Higgins DG, Heringa J. T-Coffee: a novel method for fast and accurate multiple sequence alignment. Journal of Molecular Biology. 2000;302:205–217. doi: 10.1006/jmbi.2000.4042. PubMed DOI

Passos-Bueno MR, Wilcox WR, Jabs EW, Sertié AL, Alonso LG, Kitoh H. Clinical spectrum of fibroblast growth factor receptor mutations. Human Mutation. 1999;14:115–125. doi: 10.1002/(SICI)1098-1004(1999)14:2<115::AID-HUMU3>3.0.CO;2-2. PubMed DOI

Regot S, Hughey JJ, Bajar BT, Carrasco S, Covert MW. High-sensitivity measurements of multiple kinase activities in live single cells. Cell. 2014;157:1724–1734. doi: 10.1016/j.cell.2014.04.039. PubMed DOI PMC

Scuto A, Krejci P, Popplewell L, Wu J, Wang Y, Kujawski M, Kowolik C, Xin H, Chen L, Wang Y, Kretzner L, Yu H, Wilcox WR, Yen Y, Forman S, Jove R. The novel JAK inhibitor AZD1480 blocks STAT3 and FGFR3 signaling, resulting in suppression of human myeloma cell growth and survival. Leukemia. 2011;25:538–550. doi: 10.1038/leu.2010.289. PubMed DOI PMC

ten Freyhaus H, Dumitrescu D, Berghausen E, Vantler M, Caglayan E, Rosenkranz S. Imatinib mesylate for the treatment of pulmonary arterial hypertension. Expert Opinion on Investigational Drugs. 2012;21:119–134. doi: 10.1517/13543784.2012.632408. PubMed DOI

Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA, Kinzler KW. Cancer genome landscapes. Science. 2013;339:1546–1558. doi: 10.1126/science.1235122. PubMed DOI PMC

Wang B, Chen J, Santiago FS, Janes M, Kavurma MM, Chong BH, Pimanda JE, Khachigian LM. Phosphorylation and acetylation of histone H3 and autoregulation by early growth response 1 mediate interleukin 1beta induction of early growth response 1 transcription. Arteriosclerosis, Thrombosis, and Vascular Biology. 2010;30:536–545. doi: 10.1161/ATVBAHA.109.193821. PubMed DOI

Wendt DJ, Dvorak-Ewell M, Bullens S, Lorget F, Bell SM, Peng J, Castillo S, Aoyagi-Scharber M, O'Neill CA, Krejci P, Wilcox WR, Rimoin DL, Bunting S. Neutral endopeptidase-resistant C-type natriuretic peptide variant represents a new therapeutic approach for treatment of fibroblast growth factor receptor 3-related dwarfism. Journal of Pharmacology and Experimental Therapeutics. 2015;353:132–149. doi: 10.1124/jpet.114.218560. PubMed DOI

Yang SH, Sharrocks AD, Whitmarsh AJ. Transcriptional regulation by the MAP kinase signaling cascades. Gene. 2003;320:3–21. doi: 10.1016/S0378-1119(03)00816-3. PubMed DOI

Yi SJ, Lee HT, Groffen J, Heisterkamp N. Bcr/Abl P190 interaction with Spa-1, a GTPase activating protein for the small GTPase Rap1. International Journal of Molecular Medicine. 2008;22:453–458. doi: 10.3892/ijmm_00000042. PubMed DOI

Yosaatmadja Y, Silva S, Dickson JM, Patterson AV, Smaill JB, Flanagan JU, McKeage MJ, Squire CJ. Binding mode of the breakthrough inhibitor AZD9291 to epidermal growth factor receptor revealed. Journal of Structural Biology. 2015;192:539–544. doi: 10.1016/j.jsb.2015.10.018. PubMed DOI

Zhao X, Ghaffari S, Lodish H, Malashkevich VN, Kim PS. Structure of the Bcr-Abl oncoprotein oligomerization domain. Nature structural biology. 2002;9:117–120. doi: 10.1038/nsb747. PubMed DOI

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