1. HER1/EGFR and HER2/ErbB2 pathways O. Segatto, Regina Elena Cancer Institute

2. RTK activity regulates cellular programmes crucial to cell transformation and tumour progression The hallmarks of cancer

3. RTKs have intrinsic tyrosine kinase activity which is activated upon ligand binding RTKs are bistable systems, i.e. they transit from an “off” to an “on” state with no intermediate states Kinase domain Extracellular domain COOH tail Ligand binding induces kinase activation and receptor self-phosphorylation on specific Tyr residues

4. Specific p-Tyr sites generated by RTK autophosphorylation initiate downstream signalling by acting as docking sites for cellular proteins containing SH2 or PTB domains. All SH2 and some PTB domains bind to p-Tyr. Specificity of molecular recognition is dictated by residues C-terminal to p-Tyr for SH2 domains and N-terminal to p-Tyr for PTB domains. The human genome encodes 139 SH2-containing and 49 PTB-containing proteins.

6. EGFR PLC -  IP 3 + DAG Activation of downstream signalling: enzymes containing an SH2 domain bind to pY-EGFR and are relocated to the cell surface Relocation onto the EGFR allows PLC-  to be activated via Tyr phosphorilation and “induced proximity” PIP 2

7. RAS EGFR PI-3K-AKTRAF-ERK SOS GRB2 Activation of downstream signalling: SH2 adaptors bind to pY-EGFR and relocate enzymes to the cell surface RAS GTP (active) GDP (inactive)

8. Direct plasmamembrane-to-nucleus signalling through engagement of STAT proteins

9. SH2 and PTB domains are present in enzymes, adaptors and membrane-bound scaffolding proteins

12. Formazione del “signalosoma” su RTK: perché? Concentrazione di effettori enzimatici in membrana Attivazione rapida e reversibile di funzioni enzimatiche Attivazione integrata di vie di segnalazione multiple Amplificazione del segnale Polarizzazione del segnale

13. RTK activation needs to be tightly controlled Aberrant RTK activity is linked to cell transformation

14. Focus Understanding the “design principles” of ErbB activation, as deviation from these design principles may underline oncogenic conversion of ErbB RTKs

15. Intramolecular interactions lock receptors in an inactive conformation, which is released upon ligand binding Restricted ligand availability limits receptor activation Feedback inhibition restricts receptor activity Key design principles a) on-demand-only activation b) prevention of unwanted activation c) tight monitoring of receptor activity Operational principles

16. Intramolecular interactions lock EGF receptors in a monomeric inactive conformation, which is released upon ligand binding Part 1: structural transitions in the extracellular domain

17. Xuewu Zhang et al., Cell, Volume 125, Issue 6, 1137-1149, 13 June 2006Volume 125, Issue 6 Intramolecular interactions lock receptors in an inactive conformation, which is released upon ligand binding Part 2: structural transitions in the catalytic domain

18. Ligand-induced EGFR dimerization releases the intra-molecular inhibition of EGFR kinase via inter-molecular allosteric activation

19. Intramolecular interactions lock receptors in an inactive conformation, which is released upon ligand binding Restricted ligand availability limits receptor activation Feedback inhibition restricts receptor activity Operational principles

20. Restricted ligand availability limits receptor activation ErbB ligands are synthesised by stromal cells as membrane-bound precursors. Cleavage by proteases releases the soluble form

21. Intramolecular interactions lock receptors in an inactive conformation, which is released upon ligand binding Restricted ligand availability limits receptor activation Feedback inhibition restricts receptor activity Operational principles

22. P P P P P P P P PTP X EGFR inhibitors Feedback inhibition restricts EGF receptor activity

23. Downregulation depletes receptors and ligands, leading to cellular refractoriness to further homologous stimulation Inducible feedback regulators? Feedback inhibition restricts receptor activity

24. MIG6 is an inducible feedback inhibitor that suppresses EGFR catalytic activation by binding to a dimer interface located in the COOH lobe of the EGFR kinase domain Xuewu Zhang, Kerry A. Pickin, Ron Bose, Natalia Jura, Philip A. Cole & John Kuriyan Nature 450, 741-744(29 November 2007)

25. wt KO KO + Gefitinib

26. EGFR signalling activity is the result of a dynamic equilibrium between mechanisms of signal generation and signal extinction

27. RTKs are bistable systems, i.e. they transit from an “off” to an “on” state with no intermediate states Intramolecular interactions lock receptors in an inactive conformation, which is released upon ligand binding Restricted ligand availability limits receptor activation Feedback inhibition restricts receptor activity Oncogenic conversion of EGFR and ERBB2 releases the receptors from these constrains, thus allowing unabated signalling activity

28. ErbB receptors at work: the network context

29. Thomas Jefferson, United States Declaration of Independence, 1776 … whereas ErbB receptors, ligands and ligand-receptor combinations are not!

30. Some ligands do not lead to EGFR degradation: TGF  and Epiregulin drive complete and fast EGFR recycling, Amphiregulin drives both fast and slow EGFR recycling. These ligands may be continuously re-used by the cell and also allow the EGFR to escape down- regulation. High gain of signalling potency

31. ErbB2 and ErbB3 are non-authonomous receptors: ErbB2 does not bind to any known ligand, whereas ErbB3 has no kinase activity. Under physiological conditions ErbB2 and ErbB3 signal only in the context of ligand-induced heterodimers

32. ErbB2/HER2: an atypical RTK Sequence divergence in the extracellular region generates a) inability to bind ligand b)extended conformation of the dimerization arm ErbB2 is the hierarchically dominant ErbB receptor in dimer assembly ErbB2 is refractory to endocytosis/downregulation EGFR:ErbB2 heterodimers gain signalling potency due to decelerated ligand off-rates and refractoriness to endocytosis/downregulation ErbB2 is a powerful signal amplifier

33. Strength and duration of signals generated by ErbB RTKs depend on the nature of the ligand:dimer combination

34. Citri et al. Nature Reviews Molecular Cell Biology 7, 505–516 (July 2006) Towards the system level…

35. Oncogenic conversion of EGFR and ERBB2 in human tumours: mechanisms and therapeutic opportunities Oncogenic conversion is caused by genetic lesions, which drive the constitutive signalling activity of EGFR and ERBB2. Oncogenic signalling by EGFR and ERBB2 differs in quantitative and qualitative terms from physiological signalling. This may create a state of “oncogene addiction” and cause tumour cells to become exquisitively sensitive to drugs that target EGFR and/or ERBB2.

36. Inactive wtEGFRL858R EGFR mutant EGFR mutations in lung cancer generate constitutively active kinases Yun, C-H et al., 2007, Cancer Cell, 11:217-227

37. Mutations cause constitutive activation of the EGFR kinase Mutations are associated to EGFR copy gain Mutations render EGFR refractory to down-regulation Mutations sensitize tumour cells to EGFR kinase inhibitors Compound effects of mutational activation of EGFR in NSCLC

38. ERBB2 is activated by gene amplification and attendant over-expression – mutational activation is very rare Case study: breast cancer, ERBB2 subtype ERBB2 overexpression drives constitutive homo-dimerization ERBB2 over-expression is associated to increased ERBB3 expression, with ERBB3 being a necessary signalling subunit of ERBB2

39. RTKs are bistable systems, i.e. they transit from an “off” to an “on” state with no intermediate states Intramolecular interactions lock receptors in an inactive conformation, which is released upon ligand binding MUTATIONS, OVEREXPRESSION Restricted ligand availability limits receptor activation AUTOCRINE PRODUCTION of LIGANDS Feedback inhibition restricts receptor activity REFRACTORINESS to DOWN-REGULATION (several mechanisms) Oncogenic conversion of EGFR and ERBB2 releases the receptors from these constrains, thus allowing unabated signalling activity

40. Quantity (strength and duration)… … but also quality

43. Signal promiscuity imposed by ERBB2 average: 7.2 average: 17 Jones, RB et al., 2006 Nature, 439:168-174

44. Over-expression modifies the quality of signals generated by EGFR and ERBB2 Jones, RB et al., 2006 Nature, 439:168-174

45. Genetic lesions of EGFR and ERBB2 in human tumours lead to constitutive signalling activity which differs from physiological signalling in quantitative and qualitative terms

46. Cetuximab inhibits ligand-dependent activation of EGFR

47. Targeting ERBB2 oncogenic signalling with therapeutic antibodies

48. Kinase inhibitors target the ATP-binding pocket of EGFR and ErbB2

49. Yun, C-H et al., 2007, Cancer Cell, 11:217-227 Mutations in the EGFR kinase may increase its affinity for competitive ATP inhibitors such as Gefitinib Different mutations may display different sensitivity to competitive ATP inhibitors: shall drugs get personal?

50. Targeting the ErbB network, rather than any individual ErbB RTK may result in a therapeutic advantage Citri et al. Nature Reviews Molecular Cell Biology 7, 505–516 (July 2006)

51. Conclusions Oncogenic signalling by EGFR and ErbB2 originates from the subversion of key regulatory principles of receptor activation Oncogenic conversion grants EGFR and ErbB2 full operational autonomy as well as evasion from negative regulation Targeting EGFR and ErbB2 in tumours must take into account the “design principles” of oncogenic signalling by EGFR and ErbB2, including aberrant network activation

52. In memoria di Matthias Kraus, 1956-2009

54. X X

56. Citri et al. Nature Reviews Molecular Cell Biology 7, 505–516 (July 2006)

59. Inactive wtEGFR