New Cancer Therapies Based on Thyrosine Kinase Inhibitors Axel Ullrich Max-Planck-Institute of Biochemistry Martinsried, Germany November 7, 2008

Targeted Cancer Therapy „The Dream of the Magic Bullet“ Side Effect-Free Cure of Cancer

Paul Ehrlich 1854 - 1915 Father of Chemotherapy Salvarsan for Treatment of Syphilis Nobel Prize 1908 “Magic Bullet Concept” Ehrlich in his office

Signalling Pathways

Human Epidermal Growth Factor Receptor (EGFR) Size: 170.000 Da Length: 1186 aa 131.000 MW mRNA: 5.8 / 10.5 kb Downward J, Yarden Y, Mayes E, Scrace G, Totty N, Stockwell P, Ullrich A, Schlessinger J, and Waterfield MD (1984). Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature 307. 521-527 Ullrich A, Coussens L, Hayflick JS, Dull TJ, Gray A, Tam AW, Lee J, Yarden Y, Libermann TA, Schlessinger J, Downward J, Bye J, Whittle N, Waterfield MD, and Seeburg PH (1984). Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature 309. 418-425

EGFR / v-erbB EGF-R v-erbB-H v-erbB-ES4 F/S699 F/S699 T/K718 I/V705 H/R811 Q/L840 S/I932 Δ1034 Δ 1042-1062 Y/N1091

RTK-derived Oncogene Products neu v-fms M/V 11 R/Q 91 S/L 301 S/A 374 Y/H 461 P/Q 505 T/A 587 P/L 680 G/D 711 v-erbB-H v-erbB-ES4 v-kit F/S699 T/K718 H/R811 Q/L840 S/I932 Δ1034 Δ 1042-1062 Y/N1091 F/S699 I/V705 Δ AY G/D Δ 34 aa Δ 71 aa Δ 40 aa Δ 40aa

Human EGF Receptor-Related Receptor HER2 / neu / c-erbB2 Size: 185.000 Da Length: 1234 aa 136.000 MW mRNA: 4.8 kb King CR, Kraus MH, and Aaronson SA. (1985) Amplification of a Novel v-erbB-related Gene in a Human Mammary Carcinoma. Science 229, 974-976 Coussens L, Yang-Feng TL, Liao YC, Chen E, Gray A, McGrath J, Seeburg PH, Libermann TA, Schlessinger J, Francke U, Levinson A, and Ullrich A. (1985) Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal localization with neu oncogene. Science 230, 1132-1139 Schechter AL, Hung MC, Vaidyanathan L, Weinberg RA, Yang-Feng TL, Francke U, Ullrich A, and Coussens L (1985). The neu gene: An erbB-homologous gene distinct from and unlinked to the gene encoding the EGF receptor. Science 229. 976-978

“The Herceptin Story” Oncogene Homology-Based Target Discovery Genentech: R. Hudziak M. Shepard B. Fendley L.Coussens P. Carter Herceptin Development Team Clinical Collaborator: D. Slamon, UCLA

HERCEPTIN History Cloning of EGFR cDNA Relation to V-erbB 1984 Slamon et al. Science HER2 gene amplification in breast cancer and correlation with disease progression 1987 1985 HER2 Sequence published Coussens et al. 1989 Hudziak et al. MCB Anti-tumor effect of MAb 4D5 and 2C4 Phase I Rhu MAb 1992 1993 Phase II Phase III 1995 Approval In Europe 2000 1998 FDA 2002 MAb 2C4 In Development

HERCEPTIN Efficacy of Monotherapy in HER2/neu +++ Patients Low (15%) Reminder: Cancer is NOT a Monogenic Disease Moving Disease Target due to Genetic Plasticity of Tumor Cells Combination Therapy HERCEPTIN + - Anthracyclines - Taxotere - Platinum Salts - other MABs - etc

The EGFR Family Signalling Network Transcription factors TGFa (1) EGF (1) Epiregulin (1,4) Beta- cellulin (1) HB-EGF (1,4) Amphi- regulin (1) NRG1 (3,4) NRG2 (4) NRG3 (4) NRG4 (4) LPA Thrombin ET, etc. Ligands Cytokines a b a b Input layer 2 2 4 2 1 Receptor dimers 1 1 4 3 2 4 4 1 2 3 4 1 3 3 3 SRC JAK CBL SHC PLC p21-GDP CRK P13K VAV GRB7 SHP2 GAP GRB2 NCK Adaptors & enzymes SOS p21-GTP RAC Hidden layers AKT RAF PAK Cascades MEK JNKK ABL PKC BAD S6K MAPK JNK Transcription factors JUN SP1 MYC FOS ELK EGR1 STAT Output layer Growth Migration Apoptosis Adhesion Differentiation Yarden and Sliwkowski (2001) Nature Rev. Mol. Cell Biol, 2,127-137.

Mechanism-Based Target Identification HER3 Wallasch et al. and Ullrich EMBO J. 1995 HRG-dependent regulation of HER2/neu oncogenic signaling by heterodimerization with HER3 Holbro et al. and Hynes PNAS 2003 The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation Htun van der Horst et al. and Ullrich IJC 2005 Anti-HER3 MAbs inhibit HER3-mediated signaling in breast cancer cell lines resistant to anti-HER2 antibodies

HER3 is a Determinant for Poor Prognosis in Melanoma and a Target for Therapeutic Intervention Markus Reschke Daniela Mihic-Probst Edward Htun van der Horst Pjotr Knyazev Peter J. Wild Markus Hutterer Stefanie Meyer Reinhard Dummer Holger Moch Martin Treder (U3 Pharma) AMGEN

HER3 protein expression in primary melanoma and metastases

HER3 Expression Confers Poor Prognosis for Melanoma Patients Kaplan-Meier Curve Multivariate Analysis HER3 p=0.044 (Hazard ratio: 2,636) Metastases p=0.000 (Hazard ratio: 22,251) Other variables sex age tumor thickness (p=0.065) HER3 low HER3 high Cumulative Survival p = 0.014 Overall Survival

Treatment with Human Anti-HER3 mAb Significantly Reduces pHER3 Levels in BxPC3 Tumor Xenografts IgG1 (25 mpk per mouse) Anti-HER3 mAb pHER3 -Actin U3Pharma/AMGEN

Treatment with Human Anti-HER3 mAbs Results in Inhibition of BxPC-3 Pancreatic Carcinoma Xenografts 100 200 300 400 500 600 700 800 900 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 hIgG1 , 25 mg/kg 2X/wk Anti-HER3 mAb C , 25 mg/kg 2X/wk Anti-HER3 mAb A, 25 mg/kg 2X/wk Anti-HER3 mAb B, 25 mg/kg 2X/wk Gemcitabine, 80mg/kg weekly p<0.0001 vs. hIgG1 Control Time (days) RMANOVA Analysis Blinded Study n=10 per group U3Pharma/AMGEN

Conclusion: HER3- Specific MAbs Have an HER2-independent Inhibitory Effect in Melanoma and Pancreatic Cancer Cell Models May Be Valuable Therapeutic Agents Alone as Well as in Combination with Herceptin, Lapatinib and other Drugs

From PCR-Based Discovery of a Novel RTK to a Multi-Targeted Cancer Drug “Flk-1/VEGFR2 as Target in Anti-Angiogenesis Therapy” MPI for Biochemistry Munich MPI Biochemistry, Martinsried, Germany Birgit Millauer Werner Risau SUGEN Laura Shawver Jerry McMahon Annie Fong Development Team Collaborators Alex Levitzki, Hebrew University, Jerusalem György Kéri, Vichem, Budapest Pharmacia Inc. Pfizer Inc. SUGEN San Francisco

RTK Subclasses EGFR HER2/ neu HER2 HER4 I-R IGF-1R IRR PDGFRα PDGFRβ CSF-1R KIT FLK2/FLT3 VEGFR1 VEGFR2 VEGFR3 FGFR-1 FGFR-2 FGFR-3 FGFR-4 CCK4 TRKA TRKB TRKC MET RON EPHA1- EPHA8 EPHB1- EPHB7 AXL MER TYRO3 TIE TEK RYK DDR-1 DDR-2 RET ROS LTK ALK ROR1 ROR2 MUSK MDK4 AATYK AATYK2 AATYK3

Terman et al. Biochem Biophys Res Commun 1992 Quinn et al. Proc Natl Acad Sci USA 1993 Millauer et al. Cell 1993

Flk-1/VEGFR2: Validation as Anti-Angiogenesis Drug Development Target Receptor for VEGF Specifically Expressed in Endothelial Cells Essential for Tumor Angiogenesis DEVELOPMENT OF SELECTIVE FLK-1 KINASE SMALL MOLECULE INHIBITORS

Chemical Structure of SU5416 - An Inhibitor of VEGF Receptor Kinase SU5416 3-[2, 4-dimethylpyrrol-5-yl methylidenyl]-2-indolinone

SU5 416 Response in AIDS Kaposi’s Sarcoma Protocol 5416.003, Patient #003, R-P Before treatment with SU541 After treatment with SU5416

Patient 018/JZH: SU5416 Response Facial Lesions

SU5416 Highly Selective Efficacious in Mouse Models Anti - Metastatic Sub – Optimal Pharmacological Properties Efficacious in Phase I Kaposi-Sarcoma Trial Colon Cancer Trial Terminated

Biochemical Effects of SU6668 a VEGFR-2, PDGFR, & FGFR RTK Inhibitor Flk-1 PDGFRb FGFR-1 EGFR Ki Km (ATP) Ki Km (ATP) Ki Km (ATP) IC50 SU5416 SU6668 0.16 2.1 0.43 0.32 0.008 6.2 19.5 1.2 2.6 >100 As is shown in this table, SU6668 is very potent against PDGF receptor tyrosine kinase. SU6668 is more potent at inhibiting FGFR than SU5416 with only a slight loss in activity against Flk-1/KDR. Mean Ki and Km values are shown (mM) Both compounds exhibit competitive (with respect to ATP) inhibition Both compounds also inhibit ligand-dependent phosphorylation of c-Kit

SU11248 A Multitargeted Kinase Inhibitor Receptor Ligand Tumor Expression PDGFR PDGF Glioma, HCC, NSCLC, OvCa, CaP, melanoma, CMML*, MDS* VEGFR VEGF CRC, OvCa, RCC, Melanoma, NSCLC, Sarcomas, Breast, NET KIT SCF GIST*, AML*, Melanoma, SCLC, Breast, OvCa, Cervix, Mastocytoma* FLT3 FLT3 Ligand AML*, MDS*, T-ALL, Neurological * Proliferation driven by mutant receptor

Anti-Angiogenic Activity SU11248 Oral Multi-Targeted Receptor Tyrosine Kinase Inhibitor with Anti-Tumor and Anti-Angiogenic Activity

From Mono- to Multi-Targeted Kinase Inhibitors

Eric Raymond et al., 2002 NCI/EORTC/AACR SU11248 Exhibits Cytoreductive Activity in Diverse Tumors of Patients in Phase I Clinical Trials Baseline Week 4 Patient with metastatic renal cell carcinoma Phase I trials ongoing at multiple international sites Patients heavily pre-treated, with progressive disease at entry Confirmed partial responses observed SU11248 is well tolerated most common toxicities seen in patients are fatigue, GI, and hematologic toxicity Eric Raymond et al., 2002 NCI/EORTC/AACR

Sunitinib in Metastatic RCC Before Treatment After 4 weeks of Sunitinib Lung Lesion Response in RCC; Courtesy of Dr. Ronald Bukowski, Cleveland Clinic Foundation Sunitinib is New First-Line Therapy in Metastatic Clear Cell Renal Cell Carcinoma (RCC) Adverse Side Effects are Tolerable

FDA approves SUTENT for Treatment of Gleevec-Resistant GIST and RCC SU 11248/SUTENT January 26, 2006 FDA approves SUTENT for Treatment of Gleevec-Resistant GIST and RCC July 19,2006 EMEA Approval for Europe

SUTENT History Flk-1 shown to be VEGF-R (Millauer et al., Quinn et al.) Dominant negative VEGFR-2 inhibits tumor angiogenesis and-growth in vivo (Millauer et al.) SU5416 inhibits tumor growth in vivo (Fong et al.) SU11248 orally active multi-targeted drug (O‘Farrell et al.) SUTENT approval by FDA and EMEA (Pfizer) 1993 1994 1999 2003 2006

and Metastasis Formation Receptor-Tyrosine-Kinases Drivers of Cancer Progression Activated Receptor Ligand Binding Receptor- Tyrosine-Kinase Cell Membrane Y P Tyrosine Kinase Domain Signal Transduction Migration Proliferation Survival Tumor Growth and Metastasis Formation

Multi-Targeting = Blocking Multiple Tyrosine Kinases Simultaneously Ligand Binding Activated Receptors Y P Y P Receptor Tyrosine Kinase Sunitinib    Proliferation Survival Migration Tumor Growth and Metastasis Formation

Sunitinib

Sunitinib   Tumor Angiogenesis   

SUTENT/Sunitinib going forward…. Comprehensive Kinase Interaction Profile Cell Line-Characteristic Biological Responses Identification of Therapeutically Relevant Kinase Targets (Clinical Response Prediction Signature) Identification of Kinase Targets Responsible for Side Effects (Fatigue, Cardiotoxicity etc.) Resistance Formation

Molecular Targets in Cancer Receptor Tyrosine Kinase RAS-GDP RAF-1 SHC BAD SOS GRB-2 14-3-3 Nucleus BCLXL Mitochondria SRC P STAT1+3 RAS-GTP SAPK AKT RAS-GAP DOK ? MYC PI3K MEK1/2 MAPK mTOR SU11248 (Pfizer) Zarnestra (JNJ) CT2584 (CTI) RAD001 (Novartis) SKI 606(Wyeth) BAY43-9006 (Bayer) Imatinib (Novartis) BMS354825 (Bristol-MS)

Future of Individualized Cancer Therapy Diagnostic Data Traditional Pathological Parameter Tumor Gene Expression Analysis Oncogene Mutation Profile Tumor Markers Germline SNP Profile

Future of Individualized Cancer Therapy Multi-Targeted Kinase Inhibitors (Gleevec, SUTENT, Nexavar….) Traditional Chemotherapy (Taxol, Anthracyclines…..) Target Specific Monoclonal Antibodies (Herceptin, HER3 Ab…..) Combinatorial Treatment Hormonal therapy Radiotherapy

Acknowledgements SUTENT Response Prediction Project Michaela Bairlein Henrik Daub (MPI for Biochemistry) Cooperations: György Kéri (Vichem, Hungary) Singapore OncoGenome Project (Singapore) Matthias Mann (Dept. Proteomics, MPI for Biochemistry)

Acknowledgements Axl Project Yixiang Zhang Pjotr Knyazev Yuri Cheburkin Yuri Knyazev Henrik Daub MPIB-DM Kirti Sharma MPIB-DM Vichem Chemie, Hungary György Kéri László Örfi Istvan Szabadkai

THANK YOU FOR YOUR ATTENTION

Michaela Bairlein et al. 2008 Proteomic Characterization of SUTENT/Sunitinib Maleate Activity on Cancer Cell Lines Michaela Bairlein et al. 2008

Target-Identification via Affinity Chromatography and Mass Spectrometry modified SUTENT SUTENT provided by G. Kéri Coupling to ECH-Sepharose S1 S2 In-gel Trypsin digestion FPLC- affinity chromatography LC/MS (LTQ)-Orbitrap Coomassie Gel cell lysate Eluates 1-3 10 cancer cell lines of different tissue origin (3x breast, 1x pancreas, 1x prostate, 2x glioblastoma, 3x melanoma) - 2 affinity columns in series (0.3 & 3 mM ligand concentration) - elution: 1. specific: 1 mM free SUTENT; 10 mM ADP; 10 mM AMP-PNP 2. SDS (0,5%, RT) - tryptic ”in gel digestion”  LC/MS - qualitative data analysis in MASCOT  target- comparison between columns & cell lines

Targets bound to 0.3mM-column: Kinase Affinity Spectrum in Cancer Cell Lines breast cancer cell lines  identified kinases 38 24 40 0.3mM column 3mM Hs578T (102 kinases in total) 36 23 56 MDA-MB-435S (115 kinases in total) 52 37 MDA-MB-231 (129 kinases in total) Targets bound to 0.3mM-column: e.g.: PDGFRa/b cKit VEGFR2 CSF-1R AuroraA AuroraB Ron Axl Ret Nek2 FAK Cdc2 RSK1/2

Receptor Tyrosine Kinases (RTKs) SUTENT-Responsive Receptor Tyrosine Kinases (RTKs) = identified RTKs (0.3mM SUTENT column)

Summary SUTENT… … is a multi-targeted kinase-inhibitor with an interactome of >100 kinases … has an inhibitory effect on cancer cell proliferation … induces cancer cell apoptosis … interferes with cancer cell migration and invasiveness … inhibits autophosphorylation of several RTKs in cellular kinase assays

Gas/Axl System Hafizi S, et al.. Cytokine Growth Factor Rev. 2006, 17(4): 295-304

Gene Expression Analysis towards Target Identification AXL Overexpression of the RTK Axl correlates with cancer cell line invasivity of breast cancer cell lines Basis for the development of an anti-metastasic cancer drug

Identification of AXL as a Gene Characteristic for Invasive Breast Cancer Non-invasive normal

Discovery of Axl Tyrosine Kinase Inhibitors Yixiang Zhang et al. 2008

Screening for Axl Tyrosine Kinase Inhibitors Nested Chemical Library First compound library (216 compounds) (18 chemical families) Identify active chemical family Second compound library (99 compounds) (7 chemical families) Identify active compounds Third compound library (220 compounds) (106 compounds belong to Quinazoline family) Structure-activity relationship of compounds Cooperation with György Kéri (Vichem, Hungary)

Active Compounds Some compounds of quinoline, quinazoline, pyrimidines, quinoxaline and Indolin-2-ones families have inhibitory activities against auto-phosphorylation of AXL (IC50<10µM).

SKI-606 (Wyeth, clinical phase II) Derivative of NA80x1 with Stronger Inhibitory Activity on Axl Phosphorylation, Migration and Invasion NA80x1 SKI-606 (Wyeth, clinical phase II)

Inhibitory Effects of SKI-606 on the Phosphorylation of Receptor Tyrosine Kinase AXL Kinase assay IC50=0.56±0.08µM MDA-MB-435s cells Hs578T cells, IC50 = 0.34± 0.04µM

Cellular Specificity Profile of Compounds VI17525 and VI17614 Potential targets: 32 kinases Src family members Abl family members Axl 8 more tyrosine kinases (FAK, 4 Eph family members) 9 STE group of kinases (6 MAP4K/STE20 kinase family members and 2 MAP2K family members Other kinases

Summary AXL: NA80x1 and SKI-606 are Submicromolar AXL Kinase Inhibitors They Inhibit More than 32 Other Tyr- and Ser/Thr Kinases SKI-606 is in Phase II Clinical Trials for CML and other Cancers (Wyeth) Starting Point for Medicinal Chemistry Towards a Tyrosine Kinase Inhibitor with an Even Better Target Inhibition Profile

Genentech Inc. CMM Singapore A*STAR MPI for Biochemistry Munich Biopolis

NA80x1 A 3-quinolinecarbonitrile Compound with Strong Inhibitory Activity on Axl Phosphorylation, Cell Migration and Invasion

Inhibitory Effects of NA80x1 on the Phosphorylation of Receptor Tyrosine Kinase AXL Kinase assay IC50= 12.67± 0.45µM MDA-MB-435s cells Hs578T cells, IC50=4,11± 1,47µM

Axl is a Promising Target for Invasive Breast Cancer Therapy AXL is expressed in highly invasive breast cancer cells, but not in breast cancer cells of low invasivity Ectopic expression of AXL is sufficient to confer a highly invasive phenotype to weakly invasive MCF7 breast cancer cells Experimental inhibition of AXL signaling by dominant-negative mutant, Axl shRNA or Axl antibody decreases motility and invasivity of highly invasive breast cancer cells Overexpression of dominant-negative Axl suppresses brain tumor cell growth and invasivity, and prolongs survival in a mouse xenograft model Vajkoczy P, et al.. PNAS, 2006

Henrik Daub et al. KinaTor Technology Definition of Kinase Inhibitor Specificity Affinity column enrichment factors in the order of 10‘000 signalling proteins < 2% of cellular proteome

RTK Axl : Relevance for Cancer In 1991, first identified as Axl/Ufo/Ark Janssen et al. 1991 – O’Bryan et al. 1991 – Rescigno et al. 1991 Axl overexpression has been reported in several types of human cancers, including glioblastoma, colon, prostate, melanoma, thyroid, breast and lung carcinomas

Targeted Cancer Drug Development Kinase Inhibitors So far: Identification of Potential Targets OncoGenomis, Oncoproteomics, Rational Pathway Analysis Validation in vitro / Model Systems Clinical Validation Development of Interfering Agent in vitro /whole cell screening Medicinal Chemistry Optimization Preclinical / Clinical Testing

Targeted Cancer Drug Development Kinase Inhibitors New: Structurally Diverse Kinase Inhibitor Library HCS of Multiple Cancer Cell Lines Hit / Lead Kinase Inhibition Profile Medicinal Chemistry Preclinical / Clinical Testing