Targeted Therapies: Radiosensitizing Effects of Kinase Inhibitors

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of Experimental Medicine, McGill University Experimental Therapeutics

1 Targeted Therapies: Radiosensitizing Effects of Kinase Inhibitors Molecular and Clinical Radiobiology Workshop McGill University Health Centre, June 17-19, 2015 Bertrand J. Jean-Claude, PhD Associate Professor, Department of Medicine Division of Experimental Medicine, McGill University Experimental Therapeutics and Metabolism Program Research Institute of the McGill University Health Centre

2 Major obstacles to effective cytotoxic therapy Lack of selectivity for target tissues The genetic complexity of human cancers Resistance to chemotherapeutic agents

3 Classical mechanism of resistance -DA Repair -Drug efflux mechanism (e.g. p-glycoproteins)

4 The emergence of targeted therapy Loss of function of key proteins on a pathway Resistance to apoptosis through activation of anti-apoptotic pathway Signaling redundancy

5 Receptor Tyrosine kinases

6 Epidermal Growth Factor Receptor (EGFR), a unique target HER family (EGFR, Her2, Her3, Her4) Associated with advanced stage, poor prognosis EGFR TKIs and mabs FDA approved Gefitinib on-small cell lung cancer (SCLC) Erlotinib SCLC and locally advanced pancreatic cancer (combination with gemcitabine) Lapatinib metastatic breast cancer (Her2 positive) Cetuximab advanced colorectal cancer, squamous cell head and neck Panitumumab metastatic colorectal cancer 6 Bazley L A and Gullick W J 2005

7 Hepatocyte Growth Factor Receptor: c-met Growth factor receptor overexpressed in many cancers Advanced/metastatic HGF is also overexpressed FDA approved TKI, crizotinib (Alk-mutations in SCLC), also c-met inhibitor Ongoing clinical trials for c-met inhibitors (mabs, ATP-competitive and non-competitive) - Combination therapy - SCLC: gefitinib or erlotinib (EGFR TKI) - Colorectal cancer: panitumumab (EGFR mab) 7 Organ SL and Tsao MS, 2011

8 on-receptor tyrosine kinase, c-src Overexpressed in many solid tumors Activated downstream of several RTKs, Integrins, GPCRs Growth, proliferation and survival through the MAPK, PI3K/Akt and STAT3 pathways Zhang S and Yu D, Trends Pharmacol Sci,

9 The target: EGFR TK Brain tumour Head and neck cancer Breast cancer Lung cancer Prostate cancer Ovarian cancer 2011/05/09 9

10 Quinazolines EGFR tyrosine kinase inhibitors Directed to the ATP-binding sites of these enzymes ex. Iressa, Tarceva O O O O H Iressa TM Tarceva

11 Crystal structure of Tarceva-bound EGFR

12 Molecular Modeling of combi-molecules with basic side chains ASP residue can form a hydrogen bond with the hydrogen on the protonated (basic ligands only) ASP_776 Rachid et al. J. Med Chem 2007

13 Chemoproteomics directed at target profiling Bremer et al. Cancer Res. 65,

14 Table 1. Identification and in vitro characterization of protein kinases targeted by gefitinib Protein kinase Gi no. MW (kda) Gel identification * LC-MS/MS identification Binding assay Kinase assay IC 50 (μmol/l) Aurora A Aurora B BLK Bub1 BRK , ± ,280 + D 11.1 ± ,757 + D 3.1 ± , D , ± 0.06 CaMKII 13.5 ± 1.4 CaMKIIα ,088 + D D CaMKIIβ ,914 + D D CaMKIIγ , D D CaMKIIδ , D D CK1δ ,374 + D 61 ± 7 CK1ε , D CSK , ± 17 EGFR EphB4 Fyn GAK HCK ILK IKKε , ± , D 1.22 ± , D , , ± , D ± , % at 100 μmol/l JK2 Lyn Met p38α RICK Tnk , % at 100 μmol/l , ± , ± , ± , ± , D , ± 0.13

15 Resistance to anti-egfr therapy Structural Alterations Primary and Secondary mutations including: Deletion, point mutations Constitutive activation (EGFR-vIII): lung cancer, breast cancer, glioblastoma Gatekeeper mutations (T790M) Signaling crosstalk Alternate Signaling Pathways Activation of alternate and compensatory signaling pathways: Main cause of clinical failure to tyrosine kinase inhibitors despite their selective targeting properties Amplification of c-met and activation of redundant signaling pathways Overexpression of non-receptor tyrosine kinase c-src Activation of other RTKs including VEGFR, PDGFR, IGF-1R 15

16 Overview of Signaling Crosstalk Redundancy EGFR CROSSTALK c-met Signaling Downstream c-src Signaling Downstream Growth, Proliferation, Survival, Angiogenesis, Invasion, Metastasis 16

17 Overview of our targeting strategy Redundancy EGFR CROSSTALK c-met Signaling Downstream c-src Signaling Downstream Growth, Proliferation, Survival, Angiogenesis, Invasion, Metastasis 17

18 MUTATIOS ASSOCIATED WITH RESISTACE Wheeler et al. ature Clinical Onclogy 2010, 7, 493

19 Phosphorylation sites and functions Wheeler et al. ature Clinical Onclogy 2010, 7, 493

20 The Combi-Targeting Concept bioactive bioactive bioactive Matheson et al. JPET, 2001 Matheson et al. JPET, 2004 H Me SMA41 7 H Me H EGFR TK inhibitory moiety Me H Me DA alkylating moiety H 2 O H SMA52 Me H methyl diazonium Me on-conjugated tautomer EGFR TK inhibition DA alkyation

21 Semin Radiat Oncol ; 20:

22 Semin Radiat Oncol ; 20:

24 POLYPHARMACOLOGICAL APPROACH T1 A B A B H 2 O A B A + B T1 T2 T1 T2 T2

25 The Combi-Targeting Concept bioactive bioactive bioactive Matheson et al. JPET, 2001 Matheson et al. JPET, 2004 H Me SMA41 7 H Me H EGFR TK inhibitory moiety Me H Me DA alkylating moiety H 2 O H SMA52 Me H methyl diazonium Me on-conjugated tautomer EGFR TK inhibition DA alkyation

26 Cascade Release Model

27 Western blot analysis (DU145, AGT-proficient) RB24 EGF EGF M Phosphotyrosine EGFR

28 Comet tail moment, percent control Single cell electrophoresis (DU145, AGTproficient) 2000 RB24 TEM Concentration( M)

29 Binary EGFR-DA Targeting RB24

30 Stress response and DA repair (XRCC1) EGF XRCC1 XRCC1 EGFR MEK1 SAPK Erk MEK1 DA damage RB10 RB10 TEM TEM RB24 RB24 EGF XRCC1 a-tubulin

31 DA repair (XRCC1) EGF XRCC1 XRCC1 EGFR MEK1 SAPK Erk MEK1 DA damage LCAP-EGFR RB24 RB24 TEM TEM EGF XRCC1 Tubulin

32 Stress Response Yacoub et al. Mutation Res. 2005

33 Repair kinetics, percent control DA repair kinetics (DU145, AGT-proficient) (2 h drug exposure) 1000 RB24 TEM Time (h)

34 Effects of RB24 on BAD activation in DU145 cells +EGF -EGF +EGF ( M) Phospho-BAD (Ser136) BAD

35 Percentage apoptosis after 24h exposure Apoptosis (Annexin V) Control TEM RB24 TEM RB24 TEM RB24 TEM RB24 SB PD98059 U0126

36 Table 1. Antiproliferative effects of RB24 and TEM alone or in combination with cell signaling inhibitors in the prostate carcinoma cell lines DU145 and LCaP. Compounds DU145 (IC 50, μm) a LCAP (IC 50, μm) a RB24 12 ± ± 0.9 RB10 38 ± ± 1.8 Gefitinib 49 ± ± 2.3 RB24 + O6-BG 7 ± 1.4 RB24 + SB ± 1.7 RB24 + PD ± ± 0.07 RB24 + U ± ± 0.06 TEM 486 ± ± 15.0 TEM + O6-BG 55 ± 1.1 TEM + SB ± 9.6 TEM + PD ± ± 5.2 TEM + U ± ± 6.8 TEM + gefitinib 59 ± ± 2.6 TEM + gefitinib + U ± ± RB24 + TEM + U ± 0.21 RB24 + gefitinib + U ± 0.53

Fig. 6 Schematic representation of the effect of combi-molecule RB24 on multiple signaling pathways. Ranjita Banerjee, Ying Huang, Qiyu Qiu, James P. Mcnamee, Gina Belinsky, Bertrand J.

38 Fig. 6 Schematic representation of the effect of combi-molecule RB24 on multiple signaling pathways. Ranjita Banerjee, Ying Huang, Qiyu Qiu, James P. Mcnamee, Gina Belinsky, Bertrand J. Jean-Claude The combi-targeting concept: Mechanism of action of the pleiotropic combi-molecule RB24 and discovery of a novel cell signaling-based combination principle Cellular Signalling, Volume 23, Issue 4, 2011,

39 DRUG DESIG RECOMMEDATIOS - To target EGFR-DA + IR ( Muanza s and Jean-Claude s groups, Int J Radiat Oncol Biol Phys, 2015, 92, To target MEK-EGFR + IR - To target c-met-egfr + IR -To target Met-MEK-EGFR +IR -To target MEK-DA + IR -To target MEK-Met-DA + IR - To target EGFR nuclear transport??

40 Design of a novel molecule O OH OMe AL530 O H 2 l O Cl Clinical mustard Chlorambucil (CBL) Cl H O Me H Me O degradation + O OMe H 2 O PD98059 MEK inhibitor Cl Mustard DA damaging species PD98059 MEK inhibitor 40

41 41 Future prospects Hypoxia area Target 1 Target 2 Target 1 Target 2

42 Proposed ideal multitargeted drugs for combination with radiation 42

43 SUGGESTED READIGS uclear EGFR shuttling induced by ionizing radiation is regulated by phosphorylation at residue Thr654 Edited by Veli-Pekka Lehto FEBS Letter, 2010, 3878 Requirement of Tyr992 and Tyr1173 in phosphorylation of the epidermal growth factor receptor by ionizing radiation and modulation by SHP2 Lisa-Marie Sturla, George Amorino, Michael S. Alexander, Ross B. Mikkelsen, Kristoffer Valerie and Rupert K. Schmidt-Ullrich JBC 2005 Radiation-induced Dimer Formation of EGFR: Implications for the Radiosensitizing Effect of Cetuximab MAKOTO KIYOZUKA1, TETSUO AKIMOTO1,2, MIKA FUKUTOME1, ATSUSHI MOTEGI1,2 and ORIO MITSUHASH Anti cancer research 2014

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