Targeting the Targets: the EGFR and HER2 stories Dr. Mark Basik

Potential Conflict of Interest Research Grant / – Chemokine Therapeutics

TARGETING THE TARGETS: THE EGFR AND HER2 STORIES Mark Basik MDCM Segal Cancer Center McGill University

OUTLINE EGFR and colorectal cancer HER2 and breast cancer

The HER family of receptors

Mendelsohn, J. et al. J Clin Oncol; 21: Fig 1. Mechanisms of receptor activation

Mendelsohn, J. et al. J Clin Oncol; 21: Fig 2. Epidermal growth factor receptor signaling

EGFR as target Dakocytomation 65-70% colorectal cancers express EGFR (IHC) Correlation with poor prognosis and more aggressive disease

EGFR as target

Anti-EGFR antibodies Cetuximab (Imclone/BMS): chimeric molecule: mouse MoAb vs ligand-binding site of EGFR+human IgG constant region fragment –Approved FDA 2004 for irinotecan-resistant metastatic colorectal cancer Panitumumab (Amgen) :fully human IgG2 MoAb –Approved FDA 2006 for resistant EGFR-expressing metastatic colorectal cancer

EGFR expression not required for response Chung et al, JCO 2005

Phase II Pharmacogenomic exploratory trial 111 patients with metastatic colorectal cancer were enrolled in a trial of cetuximab monotherapy. Transcriptional profiling was conducted on RNA from pre-treatment metastatic site biopsies from all patients to identify genes whose expression correlates with best clinical responses. EGFR, B-RAF and K-RAS mutation analyses and EGFR gene copy number analyses were performed on DNA from pre-treatment biopsies.

Figure 2: mRNA LEVELS OF EGFR LIGANDS EPIREGULIN AND AMPHIREGULIN Amphiregulin CR/PR SD non-responders Epiregulin

Codons 12, 13 in exon 1 FFPE tissue samples PLoS Med. 2005

KRAS mutations and colorectal cancer Activating missense K-Ras mutations –40% colon cancers, 95% pancreatic cancers Approximately 90% of the activating mutations are found in codons 12 (wild-type GGT) and 13 (wild-type GGC) of exon 1 and ~5% in codon 61 (wild-type CAA) located in exon 2 (8– 10). Previous studies from various countries have revealed specific point mutations in codons 12 and 13. The most frequently observed types of mutations are G>A transitions and G>T transversions

Figure 4: K-Ras MUTATION ANALYSIS Disease Control Group (11%) Non-responders (51%) Mutant at K-Ras codon 12 or 13 Wild Type

Figure 3: PROGRESSION-FREE SURVIVAL CURVES Patients Free of Tumor Progression (%) (days) Patients Free of Tumor Progression (%) AREG EREG Patients Free of Tumor Progression (%) Time (days) high low high low K-RAS mutant wild type a c b Khambata-Ford et al, JCO 2007

ab c Figure 5: CETUXIMAB AND K-RAS MODULATE SIGNALING THROUGH EGFR PATHWAY

CLINICAL VALIDATION Predictive vs prognostic biomarkers –Is the marker indicating merely bad disease –How much of the drug effect is real? A randomized clinical trial can definitely establish predictive value of biomarker

Original Article K-ras Mutations and Benefit from Cetuximab in Advanced Colorectal Cancer Christos S. Karapetis, M.D., Shirin Khambata-Ford, Ph.D., Derek J. Jonker, M.D., Chris J. O'Callaghan, Ph.D., Dongsheng Tu, Ph.D., Niall C. Tebbutt, Ph.D., R. John Simes, M.D., Haji Chalchal, M.D., Jeremy D. Shapiro, M.D., Sonia Robitaille, M.Sc., Timothy J. Price, M.D., Lois Shepherd, M.D.C.M., Heather-Jane Au, M.D., Christiane Langer, M.D., Malcolm J. Moore, M.D., and John R. Zalcberg, M.D., Ph.D. N Engl J Med Volume 359(17): October 23, 2008

Kaplan-Meier Curves for Overall Survival According to Treatment Karapetis CS et al. N Engl J Med 2008;359:

Kaplan-Meier Curves for Overall Survival According to K-ras-Mutation Status among Patients Receiving Supportive Care Alone Karapetis CS et al. N Engl J Med 2008;359:

Conclusion Patients with a colorectal tumor bearing mutated K-ras did not benefit from cetuximab, whereas patients with a tumor bearing wild-type K-ras did benefit from cetuximab The mutation status of the K-ras gene had no influence on survival among patients treated with best supportive care alone

BRAF mutations Exclusive of KRAS mutations in CRC 13% of CRCs

Di Nicolantonio, F. et al. J Clin Oncol; 26: Fig 1. KRAS and BRAF mutations correlate with lack of response to treatment with monoclonal antibodies targeting epidermal growth factor receptor

Copyright ©2009 American Association for Cancer Research Sartore-Bianchi, A. et al. Cancer Res 2009;69: Figure 1. Kaplan-Meier cumulative PFS on the basis of PIK3CA and KRAS mutational status and PTEN protein expression in mCRC patients treated with panitumumab and cetuximab

WHY KRAS? KRAS mutation detection FFPE: use enough template DNA Direct sequencing = gold standard (min 20-50% mutant/WT ratio) HRMA to be followed by sequencing

February 2nd, 2009

Anti-EGFR TKIs (lung cancer)

INTRINSIC RESISTANCE TO CETUXIMAB MUTATIONS & MUTATIONS LOW AUTOCRINE SIGNALING

Acquired resistance to cetuximab Cell line model (NSCLC) 6 months of cetuximab Increased steady-state EGFR expression due to altered traffic and degradation Activation of HER2, HER3 and cMET No clinical trial material for acquired resistance Wheeler DL et al. Oncogene 2008

ErbB-2/Her-2/neu amplification in breast cancer 20-30% Amplification 17q12 Associated with: 1) Adverse prognosis 2) Aggressive tumors Effect: cell proliferation, survival, angiogenesis, migration 17 ErbB-2

TRASTUZUMAB (HERCEPTIN) Humanized anti-ErbB-2 monoclonal antibody Recognizes extracellular portion of ErbB-2 Mechanisms of Action –???? –Receptor endocytosis and degradation –Decreased AKT signaling (PTEN activation) –ADCC –Induction of apoptosis –Cell cycle arrest –Inhibition angiogenesis

Trastuzumab response rate Monotherapy (metastatic breast cancer): 12-34% response Median duration 9 months Most patients with initial response acquire resistance after 1 year

Mechanisms of intrinsic resistance 1.Mechanisms involving ErbB-2: Direct implication of ErbB-2 function or localization Inhibition of trastuzumab binding to ErbB-2 (e.g. MUC4) Increased circulating ECD-HER2 (not clinically validated) Increased expression of p95 form of HER2 (partially clinically validated)

Mechanisms of intrinsic resistance 2. Mechanisms involving altered downstream or compensatory signaling pathways Downregulation of p27 Loss of PTEN (expression/mutation) (partially clinically validated) Activation of IGF-1R Upregulation of TGF-alpha Increase in heat shock protein function

siRNA screen: PIK3CA/PTEN Berns, Cancer Cell 2007

INTRINSIC vs ACQUIRED Is it the same mechanism? The issue of cell number? –Few resistant cells: acquired –Many resistant cells: intrinsic More difficult to study acquired Resistance both in vitro and clinically: No clinical material

Chan CT et al, Breast Cancer Res Treatment, 2005

pAkt Akt  -tubulin 0.2  M Hcptn 1  M Hcptn BT0.2J1E __ + __ __ + __ __ __ + 24 hrs AKT activated in Herceptin- resistant cells

Ritter et al, CCR 2007 Acquired resistance to herceptin: in vivo

DARPP-32 expression in trastuzumab-resistant cells DARPP-32  -tubulin BT0.2J* __ + __ __ __ + 1E* 0.2J __ __ __ 1E __ + + __ __ __ 0.2  M Hcptn 1  M Hcptn

DARPP-32 Dopamine and cAMP regulated phosphoprotein of MW 32 Protein phosphatase 1 inhibitor (PP1) Protein kinase A inhibitor (PKA)

T-Darpp is required and sufficient for resistance to trastuzumab in BT-474 cells Hamel S et al. Breast Cancer Res Treat 2009

DARPP-32 activates AKT

DARPP-32 expression in primary breast cancers Hamel S et al. Breast Cancer Res Treat 2009 DARPP-32: anti-apoptotic effect (AKT-BCL2) Belkhiri et al, Cancer Res. 2008

Other causes of acquired resistance to herceptin Co-operating factors –CXCR4/DARPP32 –EGFR/DARPP32 Context dependence (ER+ vs ER-) GENE ONTOLOGY Term SKBR3 UP P value gene changed BT474 1F P value gene changed Heat shock protein binding Alcohol metabolism Wnt receptor signaling Lysosome/lytic vacuole Monosaccharide metabolism Unfolded protein binding Metal ion transport Cysteine protease inhibitor activity cAMP mediated signaling Amine receptor activity

HERCEPTIN RESISTANCE Intrinsic: Mutation + HER pathway alterations Extrinsic: different pathways activated Functional overlap AKT the common factor

LAPATINIB: resistance small molecule ErbB2 Tk inhibitor Intrinsic: PIK3CA mutations and PI3K hyperactivation (Eichhorn et al, Cancer Res 2009) Acquired: activation of ER signaling (Xia et al, PNAS 2006)

How to overcome acquired resistance Target the resistance factor –PI3K/mTor (e.g. NVP-BEZ235), geldanamycin Combine with vertical or horizontal inhibitors (e.g. IGF-1R, mTOR…) CONTINUE anti-Her therapy –Lapatinib (affects MAPK > PI3K pathways) –Pertuzumab –Continue herceptin? –HER3 as a target

HER3: required for AKT-P Hsieh and Moasser, Br J Cancer 2007

Continuing Herceptin: double clinical response Von Minckwitz et al, Journal Clinical Oncology March 2009

Continuing anti-HER2 therapy?

Di Nicolantonio, F. et al. J Clin Oncol; 26: Fig 4. (A and B) The colorectal cancer cell line DiFi was transduced with either an empty vector or a BRAF V600E-encoding lentiviral vector

Tumor heterogeneity: an essential component of acquired resistance? Moroni, Lancet Oncology 2005

The analysis of tumor subclones MOLECULAR PROFILING: The cancer stem cell?

THANK YOU A. Bouchard S. Hamel C. Ferrario A. Aguilar-Mahecha D.Mauro S. Khambata-Ford