EGF Receptor Family: Targets for Cancer Therapeutics Prof. Tony Burgess

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1 1 EGF Receptor Family: Targets for Cancer Therapeutics Slide # s Topic 1-6 Introduction to signaling and cancer 7-12 EGF receptor family and ligands EGFR affinity states in solution & cell surface D-structures EGF receptor and erbb d-structure erbb Oligomerization and untethering of the EGFR A unique EGFR monoclonal The EGFR as a target for killing cancer cells 62 Acknowledgements 63 Further reading Tony Burgess burgess@ludwig.edu.au August, 2005 Colon Cancer Cells signaling mutations 2 apc p53 EGFR ras* raf* MLH1 P110α* 3 The screen versions of these slides have full details of copyright and acknowledgements 1

2 apc p53 Integrins ECM ras* MLH1 cripto PTEN P110α* EGFR TGFβ R TGFα raf* N-β-cat c-src 4 Molecular perturbations lead to changes in: adhesion migration location proliferation metabolism differentiation survival 5 If we are to treat cancer successfully, it is essential that we understand the relationship between specific mutations and their biological effects EGFR System Activation, Inhibition and Cancer The screen versions of these slides have full details of copyright and acknowledgements 2

3 Mr EGF 7 Stanley Cohen Incidence of Cancers and EGF Receptor Over-expression in USA Tumour Type New Cases/year Over-expressing EGFR (%) Lung 178, Colo-rectal 131, Breast 181, Head & Neck 30, Brain** 100, Prostate 209, The screen versions of these slides have full details of copyright and acknowledgements 3

4 1) Implications from 3D structural biology for signalling from the EGFR. 2) Inhibiting cancer cell growth by combining agents which inhibit EGF receptor family signalling. 10 Over the last 3 years detailed 3D-structures for the extra-cellular domains of the EGFR, erbb2 and erbb3 have been reported. These 3D-structures were both surprising and fascinating. It is now clear that signalling from this family of receptors involves both conformational change and oligomerization (homo- & hetero-). Understanding the structural changes associated with the activation of EGFR family signaling should offer new opportunities for improving cancer treatment. 11 EGF/EGFR Family >8 Ligands and 4 Receptors TGF-a HB-EGF AMPHIREGULIN EPIREGULIN 12 The screen versions of these slides have full details of copyright and acknowledgements 4

5 EGF receptor and cancer The EGF Receptor Tyrosine kinase can be stimulated in many ways: Normally by its ligands Cross activation, either by induced ligand activation Autocrine cancers TGF-α secretion Mutated receptor cancers EGFR Δ2-7 mutation Amplified /Over-ex pressed receptor cancers EGFR,ErbB2, ErbB3 and ErbB4 over-expression All of the cancer situations are associated with the uncontrolled growth of cancer tissue via a hyperactive tyrosine kinase. EGFR Conundrums 14 One receptor Two Affinities One receptor Multiple ligands Receptor: Monomer Dimer Aggregation Kinase* Truncation Activation EGF Receptor Family Structure/Function erbb2 and erbb3 One EGFR Family member can influence the function of another family member EGF receptor extra-cellular domain (ECD) structure ECD L1 CR1 L2 CR ECD of EGFR 2-7 mutant ECD: Receptor is constitutively active 15 The screen versions of these slides have full details of copyright and acknowledgements 5

6 ECD Analysis of cell surface EGFR ligand affinity Kd1= 30pM Kd2=1nM 16 Extra-cellular domain constructs (ECD) for EGFR L1 L1 CR1 CR1 L2 L2 L1 CR1 L2 EGFR CR2 CR2 segfr Tm 17 Comparison of full length EGFR ECD and st-egfr binding to immobilized hegf Biosensor Analyses ECD EGFR EGFR Bios ensor Resons e (RU) Biosens or Re sponse (RU) Time ( sec) Time (sec) Concentrations :100, 200, 300, 400, 500, 600, 1000nM K D: 90% = 350nM, 10% = 20nM Concentrations : 16, 32, 48, 64, 159, 318, 476nM K D = 23nM 18 Elleman et al, Biochemistry, 2001 The screen versions of these slides have full details of copyright and acknowledgements 6

7 ECD 3D-STRUCTURE Truncated EGFR extra-cellular ligand binding domain (ECD) Residues 1-501: Domains L1, CR1, L2 and initial part of CR2 CRC-Cellular Growth Factors : CSIRO/LICR/WEHI(BRI) Tom Garrett, Colin Ward, Tony Burgess,. Cell, September, ECD EGF Receptor Crystal Structure 20 EGFR_TGF_Crystal_scr.txt ECD 21 Tom Garrett The screen versions of these slides have full details of copyright and acknowledgements 7

8 ECD Tom Garrett 22 ECD 23 ECD CR1-loop Loss of high affinity binding Hot saturation Cold saturation 24 The screen versions of these slides have full details of copyright and acknowledgements 8

9 ECD Shige Yokoyama and his colleagues published their structure for the full-length EGFR-ECD (including all of CR2 and bound EGF) in the same issue of Cell. In all respects their structure corresponded to our 3-D structure for EGFR So all was solved we knew how the EGFR bound ligand and initiated intracellular kinase activation 25 But there was another twist: Leahy and Cho also reported the structure and position of the erbb3-ecd, including the CR2 domain 26 HER3 ECD: Leahy and Cho, Science The screen versions of these slides have full details of copyright and acknowledgements 9

10 In March 2003 Kate Ferguson and Mark Lemmon reported the 3D-structure for an EGFR EGF complex formed at low ph 28 EGF Receptor1-621:TGF-a Crystal Structure, ph5 29 EGFR_TGF_Crystal with CR2= Ferguson et al, Molecular Cell, March 2003 erbb2 30 The screen versions of these slides have full details of copyright and acknowledgements 10

11 erbb D- Structure 31 Garrett et al, Molecular Cell, March 2003 see also Cho & Leahy, Nature, 2003 for full length erbb2-ecd ErbB2 L1 & L2 In close contact Precludes ligand binding 33 The screen versions of these slides have full details of copyright and acknowledgements 11

12 R411 N281 Y280 Hinge region stabilized by long-range H-bonding and hydrophobic contacts E ERBB2 35 erbb2 EGFR erbb2 EGFR 36 The screen versions of these slides have full details of copyright and acknowledgements 12

13 erbb2 full-length ECD Cho and Leahy Nature, March ErbB2 ErbB2 is unable to bind EGF-like ligands The ErbB2 ECD is fixed in the dimerizing conformation ErbB2 -ECD is unlikely to homodimerize. ErbB2 would be more likely to heterodimerize & act as the preferred signalling partner for the other EGFR Family members 38 Current View of Activation Mechanism 39 The screen versions of these slides have full details of copyright and acknowledgements 13

14 CR-1 domain (cysteine rich) L1-domain (b solenoid) L2-domain (b solenoid) EGF Transmembrane domain CR-2 domain (cysteine rich) C-terminal phosphorylation sites 40 Kinase domain EGF Receptor Activation Movie 41 Let s dwell on the nature of the EGFR at the cell surface 42 Cohen Carpenter Schlessinger Yarden Jovin Bastiens Gullick Hynes Staros Walker Moriki Clayton The screen versions of these slides have full details of copyright and acknowledgements 14

15 At the cell surface EGFR is complicated EGFR = ECD-Tm-Jm-Kinase-C-terminus In solution: ECD by itself ECD truncated forms Kinase domain ( truncated forms) EGFR in solution ( ie with Tx-100) At the cell surface: EGFR normal levels or overexpressed EGFR in the presence of erbb2, erbb3 and/or erbb4 EGFR in the presence of ligand Inside the cell EGFR In solution: 44 Removal of CR2 domain increases conformational flexibility and ligand affinity EGFR in solution with ligand: = ligand Requires High [ECD] & Ligand Fc High affinity (~ 100pM) 45 Low affinity ( ~ 500nM) The screen versions of these slides have full details of copyright and acknowledgements 15

16 On the cell surface? Low affinity Inside-out signalling High affinity? 46 No detectable EGFR kinase activity On the cell surface in the presence of ligand??? High Affinity dimer High Affinity, tetramer 47 No EGFR kinase activity Kinase activated Epitope for mab 806 The screen versions of these slides have full details of copyright and acknowledgements 16

17 Capturing the ligand induced untethering of EGFR on the cell surface Binding* Antibody mab 528 mab806 Cells Stimulus WT nil WT EGF 93 3 Y246W nil Y246W EGF *EGFR expressed on Baf/3 cells, measured antibody binding after pretreatment with EGF Walker F, et al, J Biol Chem, 2004 EGFR inhibitors synergize with cytotoxic drugs to kill cells stimulated by activated EGFR. 50 Mutant EGF Receptor Induces Resistance to Cytotoxic Drugs in Brain Tumours (U87MG) Nagane,M et al, PNAS vol 95:5724, 1998 LICR, San Diego U87MG-Δ(2-7)-EGFR U87MG 51 The screen versions of these slides have full details of copyright and acknowledgements 17

18 U87MG- (2-7)-EGFR cells in vitro 52 Nagane,M et al, PNAS 95:5724, 1998,LICR San Diego U87MG- (2-7)-EGFR xenografts In vivo this combination is active, but not so potent Tumor Volume (mm) Days Post Inoculation vehicle 400 ug AG mg/kg Cisplatin AG Cisplatin Optimization of the EGFR inhibitor cytotoxic combination and the treatment protocols can lead to more effective tumour killing. 54 The screen versions of these slides have full details of copyright and acknowledgements 18

19 U87MG EGFR Xenografts 55 TUMOUR VOLUME (mm) DAYS POST INOCULATION Rod Luwor, Terry Johns & Andrew Scott LICR, Melbourne AG1478 (500 µg): Days 8, 10, 12, 15, 17, 19 Temozolomide (5 mg/kg): Days 8, 15 PBS AG1478 Temozolomide AG Temo The LICR has developed an antibody mab 806 which has specificity for the -EGFR and a sub-population of over-expressed EGFR Rod Luwor, Terry Johns & Andrew Scott. LICR, Melbourne The screen versions of these slides have full details of copyright and acknowledgements 19

20 Treatment of A431 Xenografts with mabs 806 and 528 Tumour Volume (mm 3 ) Vehicle 0.5mg mg mg mg Control Treated 58 Antibody Administration -2-7 EGFR is spontaneously active Xenograft responses Control mab Unfortunately, despite some success, EGFR inhibitors and antagonists have not been as effective in humans. Signalling Therapeutics require a better understanding of mechanisms of synergy an increased knowledge of growth factor action & cytotoxic drug cell killing kinetics 60 The screen versions of these slides have full details of copyright and acknowledgements 20

21 61 Our current research includes : Development of AG1478 as a therapeutic agent. Development of EGFR501-Fc as a ligand trap. Development of site specific EGFR family antibodies: eg CR1-loop specific antibodies for EGFR, erbb2,erbb3 and erbb4. Discovery of small molecule antagonists for the ECDs of EGFR and erbb2. Clinical trials with the humanized form of the anti-egfr antibody mab 806. EGF receptor signalling during the cell cycle. Inhibiting multiple signalling pathways. Collaborators At LICR Francesca Walker, Suzie Orchard Ed Nice, Julie Rothacker Andrew Scott, Rod Luwor, Terry Johns Colleagues at CSIRO, & WEHI : Colin Ward and Tom Garrett 62 Alex Levitzki and Web Cavenee Further Reading This brief list of references is selected to direct you to further information about the EGFR family and cancer. It is not intended to be a complete bibliography, rather a pointer into the literature. 63 T. Holbro, G. Civenni, and N. E. Hynes. The ErbB receptors and their role in cancer progression. Exp.Cell Res. 284 (1):99-110, Terrance G. Johns, Rodney B. Luwor, Carmel Murone, Francesca Walker, Janet Weinstock, Angela A. Vitali, Rushika M. Perera, Achim A. Jungbluth, Elisabeth Stockert, Lloyd J. Old, Edouard C. Nice, Antony W. Burgess, and Andrew M. Scott Antitumor efficacy of cytotoxic drugs and the monoclonal antibody 806 is enhanced by the EGF receptor inhibitor AG1478 Proc Natl Acad Sci U S A. 2003; 100(26): Lilach M. Friedman, Ariel Rinon, Bilha Schechter, Ljuba Lyass, Sara Lavi, Sarah S. Bacus, Michael Sela, and Yosef Yarden Synergistic down-regulation of receptor tyrosine kinases by combinations of mabs: Implications for cancer immunotherapy Proc Natl Acad Sci U S A. 2005; 102(6): R. N. Jorissen, F. Walker, N. Pouliot, T. P. Garrett, C. W. Ward, and A. W. Burgess. Epidermal growth factor receptor: mechanisms of activation and signalling. Exp.Cell Res. 284 (1):31-53, A. W. Burgess, H. S. Cho, C. Eigenbrot, K. M. Ferguson, T. P. Garrett, D. J. Leahy, M. A. Lemmon, M. X. Sliwkowski C. W. Ward, and S. Yokoyama. An open-and-shut case? Recent insights into the activation of EGF/ErbB receptors. Mol.Cell 12 (3): , Francesca Walker, Suzanne G. Orchard, Robert N Jorissen, Nathan E. Hall, Hui-Hua Zhang, Peter A. Hoyne, Timothy E.Adams, Terrance G. Johns, Colin Ward, Thomas P.J Garrett, Hong-Jian Zhu, Maureen Nerrie, Andrew M. Scott, Edouard C. Nice and Antony W. Burgess CR1/CR2 interactions modulate the functions of the cell-surface Epidermal Growth Factor Receptor. J Biol Chem, 2004 May 21;279(21): The screen versions of these slides have full details of copyright and acknowledgements 21

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