OncoRay OncoRay – National Center for National Center for Radiation Research in Oncology, Dresden Beyond the CENTRIC Trial – eyo d t e C C a Integrin Targeting for Tumor Therapy Sensitization Nils Cordes
Cell adhesion and Survival Cress Paula Cordes, Easter, 2008 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
Functions of the ECM Fertilized egg n Lifespan Adulthood Death Rozario & DeSimone. Developmental Biology, 341, 1, 2010, 126–140 Fléchon et al. Reproductive Biology and Endocrinology 2003 1:124 Nishida et al., Invest. Ophthalmol. Vis. Sci. 29:1887–1890, 1988 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
Integrin heterodimers Hynes, Cell, 2000 Legate et al., Genes Dev, 2009 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
Focal adhesion - SIGNALING ECM Extracellular space Extracellular space EGFR Integrin Cell membrane Cell membrane Nck2 PINCH1 ILK Talin Parvin P i Paxillin Grb2 MEK4/7 PP1 FAK Cytosol C t l Akt p130Cas JNK Actin MEK1/2 Schaller and Parsons. Curr Opin Cell Biol. 1994 Burridge and Chrzanowska- Wodnicka Annu Rev Cell Wodnicka. Annu Rev Cell ERK1/2 Dev Biol. 1996 Legate et al., Nat Rev Mol Cell Biol. 2006 Eke and Cordes, Radiother Oncol, 2011 Survival, Proliferation, Differentiation H hl Hehlgans et al., BBA t l BBA reviews, 2007 Vehlow and Cordes, BBA reviews, 2013 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
Overexpression of (various) Integrins HNSCC Lung Lung Pancreas Prostate Breast Breast Brain …. Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
Targeting beta1 integrin ECM Extracellular space Extracellular space EGFR beta1 Integrin Cell membrane Cell membrane X Nck2 PINCH1 ILK Talin Parvin P i Paxillin X Grb2 MEK4/7 PP1 FAK C t Cytosol l X X X Akt p130Cas JNK Actin MEK1/2 X X ERK1/2 Radio- and Chemosensitization Radio and Chemosensitization Anti-invasion Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
beta1 integrin targeting - clonogenicity Eke, Cordes et al., IJROBP, 2012 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
3D procedure 3D lrECM Co = IgG1a AIIB2 = anti- 1 integrin IgG mAb Eke, Cordes et al., JCI, 2012 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
AIIB2 cytotoxicity Co = IgG1a AIIB2 = anti- 1 integrin IgG mAb Eke, Cordes et al., JCI, 2012 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
1 integrin inhibition and radiosensitivity * P < 0.05 ** P < 0.01 Co = IgG1a AIIB2 = anti- 1 integrin IgG mAb Eke, Cordes et al., JCI, 2012 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
Radiosensitization by 1 integrin inhibition * P < 0.05 ** P < 0.01 Cal33 FaDu HSC4 UTSCC5 1 1 1 1 * 0.1 0 1 0.1 0 1 0 1 0.1 0.1 0 1 * * * * ** ** * ** 0.01 0.01 0.01 0.01 ction 0.001 0 001 0 001 0.001 0 001 0.001 0.001 0 001 urviving frac 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 UTSCC8 UTSCC14 UTSCC45 Su 1 1 1 1 1 1 IgG#1 (1 µg/ml) 0.1 * 0.1 0.1 ** AIIB2 (1 µg/ml) * * IgG#1 (10 µg/ml) * ** ** AIIB2 (10 µg/ml) ( µg ) 0 01 0.01 0.01 0.01 0 01 ** * 0.001 0.001 0.001 0 2 4 6 0 2 4 6 0 2 4 6 Radiation dose [Gy] Radiation dose [Gy] Eke, Cordes et al., J Clin Invest, 2012 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
1 Integrin inhibition in vivo UTSCC15 xenograft model g (day 11; post 2.5 mg/kg mAb plus 20 Gy) anti- 1 Co-IgG n = 129 Time (d) to reach: ( ) Co-Ab anti- β 1 Ab + 20 Gy + 20 Gy + 20 Gy 20 Gy ER ER 26 [VB:19; 26] 56 [VB:28; 108] 5-fold starting volume 2.33 P < 0.025 Eke, Cordes et al., JCI, 2012 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
DSB repair upon 1 integrin blocking * P < 0,05 Dickreuter et al., in prep. ** P < 0,01 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
1 Integrin in radioresistance: The mechanisms Phosphoproteome array analysis in vitro in vivo UTSCC15 XF354fl2 SAS UTSCC15 JNK T183/Y185 PKA Calpha/beta T197 p IgG#2 IgG#2 AIIB2 AIIB2 PKCbeta1/2 T500 PKA Cbeta S338 pFAK Y397 FAK Y397 FAK S910 FAK FAK S722 FAK S843 FAK S843 pJNK1 T183/Y185 PKCzeta/lambda T410/T403 PAK1/2/3 S144/S141/S154 JNK1 Src Y529 GSK3alpha S21 pJNK2 T183/Y185 GSK3alpha Y279 JNK2 JNK2 GSK3beta S216 GSK3beta S216 GSK3beta S9 GRK2 S670 -Actin ERK2 T185/Y187 Raf1 S259 0 0.5 1 1.5 2 2.5 3 3.5 Fold change upon 1 integrin inhibition (normalized to total protein expression) Eke, Cordes et al., JCI, 2012 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
1 integrin-FAK interaction In situ Proximity ligation assay Eke, Cordes et al., JCI, 2012 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
1 Integrin inhibition affects FAK Gabarra-Niecko et al., Biochem J. 2002 Eke, Cordes et al., JCI, 2012 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
1 Integrin inhibition affects FAK * P < 0.05 ** P < 0.01 * Increase of radioresistance Reduction of AIIB2 susceptibility Gabarra-Niecko et al., Biochem J. 2002 Co = IgG1a AIIB2 = anti- 1 integrin IgG mAb Eke, Cordes et al., JCI, 2012 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
1 Integrin inhibition affects FAK * P < 0.05 ** P < 0.01 * Increase of radioresistance Reduction of AIIB2 susceptibility Gabarra-Niecko et al., Biochem J. 2002 Co = IgG1a AIIB2 = anti- 1 integrin IgG mAb Eke, Cordes et al., JCI, 2012 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
1 Integrin inhibition affects FAK * P < 0.05 ** P < 0.01 * Increase of radioresistance Reduction of AIIB2 susceptibility Gabarra-Niecko et al., Biochem J. 2002 Co = IgG1a AIIB2 = anti- 1 integrin IgG mAb Eke, Cordes et al., JCI, 2012 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
Cell rounding upon 1 integrin blocking Eke, Cordes et al., JCI, 2012 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
FAK interacts with Cortactin UTSCC15 UTSCC15 IP: IgG IP: FAK pCTTN Y421 CTTN FAK -Actin CTTN CTTN Eke et al., J Clin Invest. 2012 Apr 2;122(4):1529-40 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
Modified Cortactin by 1 integrin inhibition In situ Proximity ligation assay IP IP – Mass Spec approach M S h Cortactin : Cortical actin binding protein: - cytoplasmic expression - polymerization and rearrangement of polymerization and rearrangement of actin cytoskeleton Eke, Cordes et al., JCI, 2012 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
Proper FAK-Cortactin interaction critical for radiation cell survival P371A/P374A P873A/P876A/P879A Kinase FAK-Cort 1 FAK-Cort 3 Kinase FERM FAT GFP FERM FAT GFP domain domain P642A/P645A/P648A P973A/P976A FAK-Cort 2 Kinase FAK-Cort 4 Kinase FERM FAT GFP FERM FAT GFP domain domain UTSCC15 UTSCC15 1 g fraction GFP FAK wt * FAK-Cort 1 * CTTN FAK-Cort 2 FAK-Cort 2 Surviving 1.2 ** n.s. precipitation FAK-Cort 3 1.0 * el. CTTN FAK-Cort 4 0.8 0.6 ** ** ** S ** re Co-p 0.4 ** ** 0.1 0.2 0 2 4 6 0.0 Radiation dose [Gy] Eke et al., J Clin Invest. 2012 Apr 2;122(4):1529-40 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
Proper FAK-Cortactin interaction critical for radiation cell survival P371A/P374A P873A/P876A/P879A Kinase FAK-Cort 1 FAK-Cort 3 Kinase FERM FAT GFP FERM FAT GFP domain domain P642A/P645A/P648A P973A/P976A FAK-Cort 2 Kinase FAK-Cort 4 Kinase FERM FAT GFP FERM FAT GFP domain domain UTSCC15 UTSCC15 1 g fraction GFP FAK wt * FAK-Cort 1 * CTTN FAK-Cort 2 FAK-Cort 2 Surviving 1.2 ** n.s. precipitation FAK-Cort 3 1.0 * el. CTTN FAK-Cort 4 0.8 0.6 ** ** ** S ** re Co-p 0.4 ** ** 0.1 0.2 0 2 4 6 0.0 Radiation dose [Gy] *p < 0.05; **p < 0.01 Eke et al., J Clin Invest. 2012 Apr 2;122(4):1529-40 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
Proper FAK-Cortactin interaction critical for radiation cell survival P371A/P374A P873A/P876A/P879A Kinase FAK-Cort 1 FAK-Cort 3 Kinase FERM FAT GFP FERM FAT GFP domain domain P642A/P645A/P648A P973A/P976A FAK-Cort 2 Kinase FAK-Cort 4 Kinase FERM FAT GFP FERM FAT GFP domain domain UTSCC15 UTSCC15 1 g fraction GFP FAK wt * FAK-Cort 1 * CTTN FAK-Cort 2 FAK-Cort 2 Surviving 1.2 ** n.s. precipitation FAK-Cort 3 1.0 * el. CTTN FAK-Cort 4 0.8 0.6 ** ** ** S ** re Co-p 0.4 ** ** 0.1 0.2 0 2 4 6 0.0 Radiation dose [Gy] *p < 0.05; **p < 0.01 Eke et al., J Clin Invest. 2012 Apr 2;122(4):1529-40 Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013
Molecular and Cellular Radiobiology | Cordes | NCT Symposium 2013 Our model
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