Aiming at the Wrong Target? Discussion of Abstracts CRA3507 (Alberts) and 3508 (Goldberg)

1. Aiming at the Wrong Target?Discussion of Abstracts CRA3507 (Alberts) and 3508 (Goldberg) Louis M. Weiner, MD Lombardi Comprehensive Cancer Center Georgetown University Medical Center

2. Potential ConflictsRelevant to this Presentation • Consultant, Stock Options • Merrimack Pharmaceuticals • Research Support • Amgen

3. The Facts • Cetuximab does not improve adjuvant therapy cure rates of resected Stage III colorectal cancer • Trends towards inferior outcomes • True for KRAS-wild type (Abstract CRA 3507) and KRAS-mutant (Abstract 3508) tumors • No significant concerns raised by study design or execution of the clinical trial • These results are definitive • Cetuximab toxicity may interfere with delivery of chemotherapy • Not likely that results will improve if other clinical trial designs are tested

4. EGFR This Should Have Worked! • Cetuximab (and related antibodies (i.e., panitumumab) have modest activity in refractory colorectal cancer • Single-agent • Combined with chemotherapy • KRAS mutations identify patients who will not benefit • Minimally active chemotherapy (i.e., 5-FU + LV) for metastatic colon cancer has benefit in the adjuvant setting How did we miss the target?

5. Cetuximab – Two Major Mechanisms of Action • Antibody-dependent Cellular Cytotoxicity • Signaling Perturbation

6. Possible Explanations and Implications Tumor Cell Antibody Killer Cell NK, Macrophage, Neutrophil • Antibody-Dependent Cellular Cytotoxicity (ADCC) not relevant • True if EGFR effectively targeted • Not relevant if EGFR+ cells are not the correct targets for colon cancer metastasis Antigen Fc Domain

7. Possible Explanations and Implications • ADCC is not relevant • EGFR Signaling is complicated • Robust EGFR resistance networks

8. Metastasis Proliferation EGFR: a central and heavily targeted pathway Cetuximab Panitumumab EGFR Cell membrane Erlotinib Gefitinib Lapatinib Shc “Downstream” Signaling proteins Grb2 PI3-K Sos-1 Ras AKT Raf MEKK-1 MEK mTOR MKK-7 ERK JNK Cancer- Relevant outputs Survival signaling Angiogenesis

9. Some Molecular Determinants of Clinical Benefit of Anti-EGFR Therapies

10. The EGFR Signaling Network is Vast and Complicated

11. Making Sense of the ComplexityDefining the EGFR Network 638 Genes Astsaturov et al, Science Signaling, In Press

12. Gene Nucleus Cell DNA mRNA siRNA Exploring the EGFR functional network with siRNA-based genomics X Protein 638-element siRNA library created to target each gene in the EGFR signaling network Astsaturov et al, Science Signaling, In Press

13. Gene Nucleus Cell DNA mRNA siRNA Exploring the EGFR functional network with siRNA-based genomics X Protein siRNA libraries target the expression of selected genes and permit study of the effects of targeted gene knockdown on cell function Astsaturov et al, Science Signaling, In Press

14. Synthetic Lethal Screening of an EGFR Network-directed siRNA Library Cell Line Seeded into Multi-well Plate – Each Well is Precoated with siRNA Against One Gene siRNA for 638 Genes Introduced into Cells Distinct Gene Knocked Down in Cells Growing in Each Well Lethal Phenotype? ± EGFR inhibitor (IC30) Confirm, Validate and Map Hits “Hit” Astsaturov et al, Science Signaling, In Press

15. EGFR Network Determinants of Response to EGFR Inhibition • 61 validated “hits” identify the EGFR “resistance space” in multiple cell lines • Expected and unexpected mediators of resistance • Knockdown of KRAS modestly sensitizes cells to EGFR inhibition Astsaturov et al, Science Signaling, In Press

16. Possible Explanations and Implications • ADCC is not relevant • EGFR Signaling is complicated • Robust EGFR resistance networks • EGFR is not a relevant target in colon cancer metastasis

17. FTS binding protein FAP Snail FSP-1 Slug N-cadherin SIP1 Vimentin a-SMA Fibronectin Twist b-catenin Goosecoid Ob-cadherin LEF-1 Syndecan-1 FOXC2 miR10b miR21 EGFR Syndecan E-cadherin MUC1 Cytokeratin Desmoplakin ZO-1 a1 (IV) collagen Laminin-1 miR200 family Entactin Right Target, Wrong Setting?Epithelial-Mesenchymal Transition (EMT) Adapted from Kalluri & Weinberg, J Clin Invest 119: 1420-8, 2009

18. EGFR Established Metastasis Primary Tumor Metastasis EGFR  pAKT  E-Cadherin PI3K  Vimentin IGF1R  EGFR  E-Cadherin  Vimentin Is EGFR an Appropriate Target when Cells Undergo EMT? Druggable Targets EMT Epithelial

19. EGFR Established Metastasis Primary Tumor Metastasis High High Low Relative Sensitivity to Cetuximab “EMT status may be a broadly applicable indicator of sensitivity to EGFR inhibitors.” (Barr et al, Clin Exp Metastasis (2008) 25:685–693)

20. Summary and Implications • Cetuximab therapy does not prevent metastasis following resection of Stage III colon cancer • Likely to be true for panitumumab and other anti-EGFR antibodies • EGFR-directed monoclonal antibodies should not be used in Stage III colon cancer-directed adjuvant therapy regimens • Numerous genes contribute to resistance to EGFR antagonism • May underlie resistance to cetuximab • Combination signaling inhibitor strategies are needed

21. Summary and Implications • Cancer cell populations exhibit epithelial-mesenchymal transition (EMT) • Colon cancer metastasis may not be dependent upon signaling through EGFR • Targeting EMT-related processes may be a better approach to inhibit colon cancer metastasis

22. Cancer cell populations exhibit epithelial-mesenchymal transition (EMT) Colon cancer metastasis may not be dependent upon signaling through EGFR Targeting EMT-related processes may be a better approach to inhibit colon cancer metastasis • A better understanding of colon cancer biology will inevitably lead to better treatments that • Target the right cells at the right time • Effectively disrupt one or more targets that are required for the function of crucial signaling networks Summary and Implications