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

Potential Conflicts Relevant to this Presentation Consultant, Stock Options –Merrimack Pharmaceuticals Research Support –Amgen

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

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 EGFR How did we miss the target?

Cetuximab – Two Major Mechanisms of Action 1.Antibody-dependent Cellular Cytotoxicity 2.Signaling Perturbation

Possible Explanations and Implications Tumor Cell Killer Cell NK, Macrophage, Neutrophil Antibody 1.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

Possible Explanations and Implications 1.ADCC is not relevant 2.EGFR Signaling is complicated –Robust EGFR resistance networks

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

Some Molecular Determinants of Clinical Benefit of Anti-EGFR Therapies Determinant(s)InfluenceReferences KRAS MutationNegative J Clin Onc 25: 3230, 2007 J Clin Onc 26: 374, 2008 BRAF Mutation, PTEN Loss Negative J Clin Onc 27: 5924, 2009 J Clin Onc 27: 2622, 2009 Amphiregulin or Epiregulin Expression Positive J Clin Onc 25: 3230, 2007 J Clin Onc 27: 5068, 2009 EGFR Gene Amplification Positive J Clin Onc 27: 5924, 2009

The EGFR Signaling Network is Vast and Complicated

Making Sense of the Complexity Defining the EGFR Network 638 Genes Astsaturov et al, Science Signaling, In Press

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

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

siRNA for 638 Genes Introduced into Cells Lethal Phenotype? “Hit” Synthetic Lethal Screening of an EGFR Network-directed siRNA Library Confirm, Validate and Map Hits Cell Line Seeded into Multi-well Plate – Each Well is Precoated with siRNA Against One Gene Distinct Gene Knocked Down in Cells Growing in Each Well ± EGFR inhibitor (IC 30 ) Astsaturov et al, Science Signaling, In Press

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

Possible Explanations and Implications 1.ADCC is not relevant 2.EGFR Signaling is complicated –Robust EGFR resistance networks 3.EGFR is not a relevant target in colon cancer metastasis

Right Target, Wrong Setting? Epithelial-Mesenchymal Transition (EMT) Adapted from Kalluri & Weinberg, J Clin Invest 119: , 2009 EGFR Syndecan E-cadherinMUC1 CytokeratinDesmoplakin ZO-1  1 (IV) collagen Laminin-1miR200 family Entactin FTS binding protein FAPSnail FSP-1Slug N-cadherinSIP1 Vimentin  -SMA FibronectinTwist  -cateninGoosecoid O  -cadherinLEF-1 Syndecan-1FOXC2 miR10bmiR21

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

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

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

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 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 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