2849 Analysis of the cancer gene targeting of clinical kinase inhibitor drugs by combining cellular and biochemical profiling Joost C.M. Uitdehaag , Jeroen A.D.M. de Roos , Antoon M. van Doornmalen , Martine B.W. Prinsen , Jos de Man , Yoshinori Tanizawa , Yusuke Kawase , Kohichiro Yoshino , Rogier C. Buijsman , Guido J.R. Zaman 2 rd 1 Netherlands Translational Research Center B.V., Molenstraat 110, 5342 CC Oss, The Netherlands; Carna Biosciences Inc., 3 Floor, BMA, 1-5-5 Minatojima-Minamimachi, Kobe, Japan. T: +31412700500 W: www.ntrc.nl and www.carnabio.com E: [email protected] 1 1 1 1 2 Introduction Bosutinib Bosutinib Ponatinib Ponatinib Cabozantinib Vandetanib Dabrafenib Cabozantinib Ruxolitinib Dabrafenib Erlotinib Ruxolitinib Gefitinib Erlotinib Lapatinib Gefitinib 0 100 imatinib gefitinib dabrafenib 10 ABL inhibitors 1 0,1 0,1 A 1 10 100 Pazopanib 20 Regorafenib 60 40 100 Erlotinib 60 Gefitinib 40 20 0 20 Lapatinib 0 0.1 ● SMAD4 ● VHL NOTCH MAP2K4 MSH2 CDKN2a.P14. ABL TP53 ● MAP2K4 MYC 0.1 BRAF STK11 APC SMARCA4 MEK inh. 3,00 2,50 2,00 1,50 1,00 0,50 SMAD4 1 0,00 0.01 increased biochemical selectivity (selectivity entropy) 0.1 FBXW7 KRAS CDKN2A RB1 PIK3CA MYC 1 MLH1 drug IC50 effect TP53 FBXW7 PIK3CA MLH1 NOTCH PIK3KR1 RB1 PTEN 10 1 100 0.1 1 KRAS SMARCA4 APC CDKN2a.P14. BRAFABL PIK3KR1STK11 NRASCTNNB1 VHL PTEN drug IC50 effect 10 Figure 5 Anova analysis for drug response markers reveals novel associations for MEK and EGFR inhibitors. ABL 1,E-08 p-value vemurafenib 0,001 Everolimus (mTOR) Temsirolimus (mTOR) Fasudil (ROCK) 0,1 1 0,001 1,E-06 1,E-04 trametinib 1,E-02 dasatinib everolimus 1,E+00 1 1000 drug IC50 effect 0,001 1,E-03 cabozantinib ponatinib axitinib nilotinib sorafenib dasatinib 1,E-02 afatinib lapatinib vandetanib 1,E-01 pazopanib bosutinib 1 erlotinib 1000 1,E+00 0,1 trametinib dasatinib drug IC50 effect 1 drug IC50 effect Figure 6 Comparison of targeting efficacy of marketed BRAF, ABL and EGFR inhibitors. Figure 1 Inhibitors used in this study and their biochemical selectivity profiles. • Cell line panel profiling reveals that MEK inhibitors can be beneficial in β-catenin mutant cancers and EGFR inhibitors in SMAD4 mutant cancers gefitinib 1,E-10 0,01 Crizotinib (ALK) 1,E-04 imatinib dabrafenib Conclusions EGFR 1,E-12 0,000001 p-value other kinase inhibitors 80 3,50 CDKN2A CTNNB1 NRAS mTOR inh. 200 4,00 Afatinib 0.01 ● mTOR inh. ABL inhibitors B 0,0001 80 2000 BRAF 0,00001 Afatinib gefitinib Figure 4 Correlation between the biochemical and cellular activities of inhibitors: (A) potency (B) selectivity. Sorafenib Vandetanib 100 Trametinib 0.01 dabrafenib 1000 biochemical potency (target IC50, nM) Sunitinib EGFR inhibitors imatinib RAF inhibitors EGFR inhibitors 0.001 p−value EGFR inhibitors 20000 0.001 p−value 1000 decreasing general cytooxicity (average IC50 in the cell panel (nM)) most potent IC50 in the cell panel (nM) RAF inhibitors 10000 Figure 3 Clustering of cell proliferation responses ( logIC50) In red non kinase drugs. 10 Cabozantinib 40 0 Imatinib Figure 2 Clustering of the response in kinase activity assays. %-inhibition at 10 µM inhibitor concentration. 100000 10,000 nM MOLT−4 Jurkat E6.1 CCRF−CEM SR PA−1 AU−565 FaDu CAL 27 C−33 A U−87 MG U−2 OS SW480 MeWo Hs 578T A−172 769−P LoVo BxPC−3 ACHN A375 SK−N−AS A−427 MG−63 A−549 NCI−H460 HCT−116 LS 174T SW620 SW48 RPMI−7951 K−562 A−204 SJCRH30 NCI−H82 AN3 CA HCT−15 DLD−1 A−498 786−0 OVCAR−3 J82 BT−20 BT−549 SHP−77 20 Trametinib (MEK) 60 Axitinib 0% inh. Vemurafenib Everolimus Imatinib Temsirolimus Vemurafenib ABL 40 Vemurafenib 80 0% inh. 1 nM ABL 60 Dabrafenib 100 VEGFR2 100 VEGFR2 PDGFR / VEGFR inhibitors RAF / MEK inhibitors 80 0 PDGFRα Bosutinib 20 PDGFRα 0 40 BRAF KIT EGFR HER2 20 Ponatinib Tofacitinib BRAF KIT EGFR HER2 Nilotinib 60 ROCK1 40 Ruxolitinib ROCK1 60 Dasatinib 80 Dabrafenib Cisplatin Ruxolitinib Vemurafenib Tofacitinib Fasudil Nilotinib Imatinib Pazopanib Sorafenib Cabozantinib Etoposide Fluorouracil Axitinib Ponatinib Bosutinib Sunitinib Crizotinib Afatinib Vandetanib Regorafenib Erlotinib Lapatinib Gefitinib Dasatinib Everolimus Bortezomib Vincristine Docetaxel Trametinib Temsirolimus Doxorubicin_v3 Doxorubicin_v1 Doxorubicin_v2 Lapatinib Tofacitinib Afatinib Fasudil Tofacitinib Trametinib Fasudil Everolimus Temsirolimus Trametinib JAK2 MET ALK 80 Imatinib 100 100% 100%inh. inh. Afatinib JAK2 MET ALK 100 1 Regorafenib Sorafenib Sorafenib Vandetanib • In addition, all kinase inhibitor drugs were profiled in a panel of highly reproducible proliferation assays on 44 well-characterized cell 2 lines (Oncolines™) at NTRC JAK inhibitors 1 Nilotinib Nilotinib Pazopanib Pazopanib Regorafenib • All kinase inhibitor drugs were profiled in a panel of more than 300 kinase activity assays at Carna 1 Biosciences ABL inhibitors 2 Sunitinib Sunitinib Crizotinib Crizotinib Axitinib Axitinib Dasatinib Dasatinib • We have systematically compared the biochemical and cellular potency and selectivity of all kinase inhibitor drugs on the market • The results allow comparison of existing therapies, and define hypotheses for specific target populations for new therapies 2 p-value 1 References 1. Kitagawa et al. Activity-based kinase profiling of approved tyrosine kinase inhibitors. Genes to cells 18: 110-122 (2013). 2. Uitdehaag et al. Comparison of the cancer gene targeting and biochemical selectivities of all targeted kinase inhibitors approved for clinical use. PLOS ONE 9: e92146 (2014). N E T H E R L A N D S T R A N S L AT I O N A L R E S E A R C H C E N T E R B . V. 10 • Biochemical selectivity and potency are prime determinants of the target efficacy of kinase inhibitor drugs • Integrated biochemical and cellular profiling can help to prioritize compounds and provide directions for optimization
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