Radiobiology: : A changing science. ca Classical Radiobiology. Phenomenology. Modelling. Understanding of Mechanisms. today

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Lee Tyler
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1 Modern Radiobiology State of the Art and Future Perspectives H. Peter Rodemann Division of Radiobiology and Molecular Environmental Research Dept. of Radiation Oncology Eberhard-Karls-University Tübingen

2 Radiobiology: : A changing science Skin alterations Death of cells ca Classical Radiobiology Linear-Quadratic Model of cell inactivation Role of p53 and inter- as well as intracellular signal transduction today Definition of molecular targets Phenomenology Modelling Understanding of Mechanisms Molecular Radiobiology major input to radiation oncology

3 What are the future perspectives? Identification of molecular targets for radiosensitization / radioprotection in the context of their normal and pathological mechanisms Identification of tissue specific target structures on the basis of biological / molecular imaging Identification of genetic markers of individual radiation sensitivity (Genomics / Proteomics) Development of molecular prediction for RT (Theranostics) Application of stem cells to rescue damaged normal tissue

molecular mediated survival targets can be defined to signaling determin

4 Target identification Cytokine signaling determins NT responses If we understand all the details, the most Growth factor and cell relevant matrix molecular mediated survival targets can be defined to signaling determin develop the TT best responses possible therapeutic strategy in RO! Molecular Targeting Strategies

5 Functional molecular imaging high resolution PET-CT / PET-MRI

Genomics DNA microarray 16 17 18 7 Actin DNA

6 Genomics DNA microarray Actin DNA CyclinD1 DNA DHFR DNA 8 RB DNA E2F1 DNA tubulin DNA

7 Prognostic profiling Metastasis Nature, Vol 415, 31 January 2002

8 Proteomics 2D-PAGE (ph range 4-7) of non-irradiated lymphocytes from normal, radiation sen- sitive and radiation resistant identical twins! normal 87 proteins sensitive 12 proteins resistant +15 proteins

9 Theranostics Theranostics: link between diagnostics and therapy DNA-Chips Protein chips Predisposition Profiling Diagnostics Medical Imaging Diagnostics in vitro Diagnostics Molecular Imaging Treatment, Selection / Refinement Pharmacogenomics Image guidance Therapeutic Intervention Home Monitoring Diagnostic Devices Molecular Imaging Treatment Monitoring

10 The Holthusen-Principle Principle is still valid Implementation of available data sets will selectively improve tumor response % Tumor Response % Normal Tissue reaction rel. total dose

11 The Holthusen-Principle Principle is still valid Implementation of available data sets will help to selectively protect normal tissue % Tumor Response % Normal Tissue reaction rel. total dose

12 Molecular Targeting Strategies to improve tumor and prevent normal tissue reponses EXAMPLE 1 : EGF-Receptor antagonists as target to improve tumor response EXAMPLE 2 : TGFß1-production production/signaling as target to prevent radiation- induced fibrosis EXAMPLE 3 : Bowman Birk Protease Inhibitor (BBI) and Phospho- Tyrosine as tools to selectively protect normal tissue

13 EGFR

14 EGFR overexpression in human tumors Solid tumors presenting overexpressed or mutated EGFR H&N % Renal 50-90% Breast 14-91% Esophageal 43-89% Prostate 40-80% NSCLC 40-80% Colorectal 25-77% Gastric 33-74% Ovarian 35-70% Glioma 40-63% Pancreatic 30-50% Bladder 31-48% overexpression / mutation is associated with enhanced receptor signaling massive tumor growth enhanced invasive and metastatic potential and is generally correlated with resistance to chemo-/ radiotherapy

15 EGFR overexpression in human tumors Bonner et al. New Engl J Med H&N tumor patients : 213 RT+Cetuximab (C225) / 211 RT alone Locoregional Control (%) RT plus C225 RT Overall Survival (%) RT plus C225 RT Months Months

16 EGFR signaling Cytoplasmic EGFR pathways Nuclear EGFR pathways PLC PI3K Ras STATs Nuclear Targets E2F1 STAT3 Cyclin D1 B-Myb inos Cell Survival, Proliferation Tumorigenesis, Metastasis Chemo- & Radioresistance G1-S-Progression Proliferation Proliferation Metastasis

17 Activated EGF receptor and its cellular consequences ligand - + -IR +2Gy P-EGFR P-EGFR Ras PI3K STAT Raf AKT MEK mtor MAPK Proliferation Cell Cycle Control Survival Proliferation Radioresistance

18 Targeting EGFR signaling small molecule tyrosine kinase inhibitor (SMI) mtor EGFR- TK PI3K AKT monoclonal antibody (mab) STAT Ras Raf MEK MAPK Proliferation Cell Cycle Control Survival Proliferation Radioresistance Radiosensitization

19 C225 leads to radiosensitization in vitro and in vivo Harari et al IJROBP 49: Krause et al Radiother. Oncol. 74: Clonogenic assay in vitro H&N SCC cell lines Tumor Control Study in vivo FaDu tumor xenograft model

20 Effect of C225 on localisation of DNA-PK Huang et al Clin. Cancer Res. 6:

21 Radiation-induced EGFR signaling Güven et al. J Biol Chem 2001 Genes Chromos Cancer 2003 PLC PI3K Ras STATs Dittmann et al. J Biol Chem 2005 Radiother Oncol 2005 Radiother Oncol 2007 Int J Rad Oncol Biol Phys 2008 Radiother Oncol 2008 DNA-PK DNA-Repair via NHEJ DNA-PK DNA-Repair via NHEJ Toulany et al. Radiother Oncol 2005a Radiother Oncol 2005b Clin Cancer Res 2006 Mol Cancer Res 2007 Rodemann et al. Sem Rad Oncol 2007 Int J Rad Biol 2007 Radioresistance Radioresistance

22 C225 prevents radiation-induced induced nuclear translocation of EGFR and activation of DNA-PK Dittmann et al. J. Biol. Chem C225 EGFR EGFR DNA-PK DNA-DSB? REPAIR

23 C225 mediates impaired DNA-DSB repair and enhances radiation sensitivity Dittmann et al. J. Biol. Chem. 2005

24 Specific inhibition of EGFR-tyrosine kinase activity Ligand Ligand BIBX1382BS TKI R R K K TKI highly selective inhibitor of EGFR tyrosine kinase chemical class: pyrimido-[5,4-d]- pyrimidine-2,8-diamine,n8-(3- chloro-4-fluorophenyl) binds directly to intracellular tyrosine-kinase domain inhibits ligand-induced cell growth - erbb1-cells IC 50 = 0.01 µm - erbb2-cells IC 50 = 1.0 µm

25 Differential radiation response of Ras wt or Ras mt tumor cells to BIBX1382BS Toulany et al. Clin. Cancer Res Single dose irradiation K-RAS mt A549 cells K-RAS wt FaDu cells 1 Clonogenic fraction 0.1 A549 Controls BIBX FaDu Controls BIBX X-ray dose, Gy X-ray dose, Gy

26 Differential radiation response of Ras wt or Ras mt tumor cells to BIBX1382BS Toulany et al. Radiother. Oncol Fractionated dose irradiation (4 x 2 Gy) K-Ras mutated cell lines K-Ras wildtype cell lines SF(± SD) IR BIBX,IR Total Dose (Gy) 0.01 (2 Gy fractions) IR BIBX,IR Total Dose (Gy) DMF for Ras mt cell lines A549, MDA-MB231, PC3 No effect in normal Ras cell lines FaDu, HTB35/SiHa, HH4DD

27 Autocrine activation of EGFR-PI3K-AKT signaling in K-RAS mt cells Toulany et al Radiother. Oncol. 76: EGFR- TK Ras Ras Amphiregulin TGFa PI3K Raf AKT MEK Radioressistance Relative SF ( ± SD ) HTB-35 CM, 2 Gy A549 CM, 2 Gy vehicle, 2 Gy MAPK Proliferation CREB

28 Role of AKT in regulating DNA-PK Toulany et al., Clin Cancer Res 2006 Toulany et al., Mol Cancer Ther 2008 in press EGFR/erbB-1 EGFR-spec.TK-.TK-inh. (BIBX) PI3K LY Post 2 Gy (min) P-Akt IP: P-Akt PDK1/2 Akt P-T2609 IR (2 Gy) Akt P-T2609 DNA-PK con sirna Akt sirna Akt- sirna P Akt P DNA-PK API Surviving fraction DNA-PK control BIBX Gy Gy DNA-DSB repair is through markedly non inhibited! homologous RADIOSENSITIZATION endjoining NHEJ

29 EGFR / PI3K inhibitors mediate enhanced residual DNA-damage Toulany et al. Clin. Cancer Res γ-h2ax-foci indicating residual DNA-DSB 24h after IR K-Ras mt A549 K-Ras wt FaDu A Gy A549 + BIBX + 6 Gy Number of γ H2AX foci per cell Gy BIBX / 2 Gy 6 Gy BIBX / 6 Gy LY/ 6 Gy 2 Gy BIBX / 2 Gy 6 Gy BIBX / 6 Gy LY/ Gy

30 Importance of EGFR-signaling for DNA-DSB repair in K-Ras mt tumor cells EGFR- TK PI3K EGFR- TK Ras Raf nuclear translocation AKT Apoptosis MEK MAPK EGFR-TK DNA-PK DNA-PK DNA-DSB repair (NHEJ) Proliferation

31 Importance of EGFR-signaling for DNA-DSB repair in K-Ras mt tumor cells EGFR- TK PI3K EGFR- TK C225 BIBX LY Ras Raf nuclear translocation API-59 AKT Apoptosis MEK MAPK EGFR-TK DNA-PK DNA-PK DNA-DSB repair (NHEJ) repair (NHEJ) Proliferation enhanced radiation sensitivity

32 TGFß

33 Tissue responses resulting in radiation-induced induced fibrosis of the lung (Rübe et al. 2004) Bronchial Epithelium Inflammatory cytokines Alveolar macrophages Alveoli Pneumo- cytes Capillary Endothelial cells Fibrogenic Cytokines TGF-ß PDGF, TGF-ß Inflammatory process FIBROSIS Collagen Fibroblasts Fibrocytes

34 TGFß1-signaling and potential targets for intervention LTGFβ1 LTGFβ1 Mn-SOD ROS / Amifostine Epperly et al / TGFβ1-Gene Vujaskovic expression 2002 TF Smad 2/3 Smad4 Smad 2/3 GTCAGAC Amifostine Vujaskovic ROS et al proteolytic protease activation inhibitors e.g. Furin Haase et al P P P P anti-tgfβ1-ab TGF TGFβ1 Hakenjos active et al 2000 TGFβ1 R2 R1 Smad4 Kinase-Inh. R2 R1 Wiegman et al Smad7 P Smad 2/3 Smad 2/3 Smad4 Smad 2/3 TGFβ1 P P P P P P Smad 2 Smad 3 gene expression, e.g. p21, p27, collagen

35 Radiation-induced induced lung fibrosis (Vujaskovic et al. 2002) Rat lung Total collagen production TGFß1 production before RT 6 month after IR (SD 28 Gy)

36 Amifostine prevents manifestation of radiation-induced induced fibrosis of the lung by inhibiting TGF-ß production 150 mg/kg amifostine 30 min before IR (Vujaskovic et al. 2002) activation of proteolytic enzymes Amifostine ROS LTGFß TGFß1 gene expression TGFß

37 BBI / ptyr

Cys Lys Cys Pro Cys Cys 10 Asp Asn Cys Pro Cys Phe Ser Gln Ala Ala Gln Glu 50 Val Thr 60 Glu

38 Bowman-Birk Birk Proteinase Inhibitor (BBI) and ptyr 30 1 Asn Asp Asp Glu Ser Leu Ser Ser 40 Arg Cys Met His Asp Lys Cys Ile 20 Ser Ser Arg Cys Pro Ser Cys Ala Pro Gln Cys Lys Ala Pro Cys Cys Lys Cys Pro Cys Cys 10 Asp Asn Cys Pro Cys Phe Ser Gln Ala Ala Gln Glu 50 Val Thr 60 Glu Cys Tyr Asp Ser Lys Ile Cys Thr Asp Phe Asp Asp Lys Glu Asn 70 Leu Ser Tyr Pro phospho-tyrosine (ptyr)

39 BBI affects normal and tumorigenic human fibroblasts differentially (Dittmann et al.1995) Normal human skin fibroblasts Transformed, tumorigenic human skin fibroblasts HSF1 HH4dd wildtype p53 mutated p53

40 BBI affects p53 wt and p53 mt tumor cells differentially (Dittmann et al.1998) lung adeno carcinoma cell line A549 presenting wildtype p53 lung adeno carcinoma cell line A549 transfected w/ mutated p53 wildtype p53: functional mutated p53: non-functional

41 Stimulation of DNA-DSB-repair by P-Tyr in p53 wt cells (Dittmann et al ) γ-h2ax-focus assay was used for determination of residual DNA-DSB 24 h post IR fibroblasts, p53 wt 0 2 6

42 Mode of action of BBI and ptyr pre-irradiation treatment with BBI / ptyr EGFR Dittmann et al. Radiother. Oncol 2008 Wanner et al. Radiother. Oncol 2008 Dittmann et al. Radiother. Oncol 2007 Dittmann et al. Int. J. Rad. Biol Dittmann et al. Curr. Med. Chem Dittmann et al. Int. J. Cancer 2001 P p53wt? Activation of DNA- repair machinery Enhanced cell survival post IR

43 BBI effect in vivo: normal tissue vs. tumor response (Dittmann et al. 2005) Leg contracture assay Tumor growth delay assay C3H-mice were treated 3x with different doses of BBI at day 2, day -1, and day 0 before IR; both hind legs were irradiated w/ SD of 55 Gy. Leg contracture was quantified up to 120 days post IR.

44 Current impact of proposed perspectives Identification of molecular targets for radiosesiti- zation / radioprotection in the context of their nor- mal and pathological mechanisms Identification of tissue specific target structures on the basis of biological / molecular imaging Identification of genetic markers of individual radiation sensitivity (Genomics / Proteomics) Development of molecular prediction for RT (Theranostics) Application of stem cells to rescue damaged normal tissue

Thanks to the co-workers...... current staff Klaus Dittmann, PhD Petra Ohneseit, PhD Mahmoud Toulany, PhD Nadine Hoffmann, Techn. Simone Keinath, Techn. Heidi Löffler, Techn. Claus Mayer, Techn.

45 Thanks to the co-workers current staff Klaus Dittmann, PhD Petra Ohneseit, PhD Mahmoud Toulany, PhD Nadine Hoffmann, Techn. Simone Keinath, Techn. Heidi Löffler, Techn. Claus Mayer, Techn. Urszula Florczak, PhD-Stud. Gerd Krötzinger, MD-Stud. Julia Mihatsch, PhD-Stud. Minjma Minjgee, PhD-Stud. Christine Rothmund, PhD-Stud. Gabriele Wanner, PhD-Stud.... former postdocs, PhD- / MD-students, and guest scientists... and to you! Marcel Bläse / Anke Burger / Anke Binder Lydia Hakenjos / Oliver Haase / Carsten Herskind / Sonja Nolte / Andreas von Pfeil / Erwin Wiegman / Weiguo Xu

EGFR: fundamenteel en klinisch

EGFR: fundamenteel en klinisch Guido Lammering MAASTRO Clinic Maastricht, NL What is EGFR?? The EGFR some facts 1186 amino acids 170 kda Expressed by all cells of epithelial origin Increased activation