THERAPIES CIBLEES en cancérologie Nicolas Mounier Onco-Hématologie CHU Nice – Hôpital l’Archet

Introduction 6 altérations clefs Mouvement de la cancérologie classique Identification de l’organe/tumeur initiale Diagnostic microscopique Vers cancérologie moléculaire Entité tumorale clinico-biologique Thérapeutiques ciblées 6 altérations clefs Immortalité Auto-suffisance pr croissance Insensibilité inhibiteur Résistance apoptose Angiogénèse Pouvoir métastasiant

Traitements ciblés en cours de développement en cancérologie Les cibles => quelques exemples Perspectives Zoom sur les toxicités

Les grandes familles de thérapies ciblées

Les cibles 1 La signalisation cellulaire 2 L’angiogenèse Les voies de l’apoptose Le cycle cellulaire Les cibles épigénétiques La méthylation du DNA L’acétylation des histones L’immunité anti-tumorale

1- La signalisation : quels niveaux de ciblage ?

Extracellular Cytoplasme TK receptors TK receptors Jak Src Src Ras PDK Membrane Jak Src PI3K Grb2 P P Cytoplasme Src P P P SOS P STAT STAT STAT Abl P STAT Ras Raf ERK MEK PDK P Akt STAT STAT mTor P p70S6K p53 p53 Gene Transcription Cell Growth Proteins translation Cell Cycle Apoptosis Apoptosis

1- La signalisation : quels niveaux de ciblage ? Ligand extracellulaire : VEGF Récepteur membranaire : HER 1, HER 2, CD20, IGF1, CTLA4, CXCR4 Protéines sous membranaires : Farnésyl-transférases Transduction intracellulaire : Voie PI3K (m-TOR) , Voie PKC (enzastaurin) Transcription : bortezomid

Ciblage des récepteurs Par des anticorps : Rituximab, Trastuzumab, Cetuximab Pertuzumab (HER 1, HER 2), IMCA 12 et CP 751,871 (IGF1-R) Par des petites molécules : Imatinib, Gefitinib, Erlotinib Lapatinib (EGF-R, HER-2) AEE 788 (EGF-R, HER-2, VEGF R2) AMN 107 (KIT, PDGFR) PP1 (Src, Abl) SU 6656 (Src)

Ciblage de la transduction intracellulaire La voie PI3K et mTOR : rapamycine, RAD 001, CCI 779 La voie PKC : PKC 412, enzastaurin La voie Ras : ARRAY 142 886 (ERK ½), inhibiteurs de B-Raf

2- L’angiogenèse : quand les cibles se mélangent… Angiogénèse : Indispensable à la survie et à la croissance tumorale Médiée par des cytokines, des protéases et des facteurs de croissance Ciblée par des anticorps ou des inhibiteurs de Kinases

8. Endothelial Cell Proliferation, Migration, and Differentiation Following degradation of the ECM, endothelial cells begin to proliferate in response to tumor-derived growth factors such as VEGF and migrate toward the growth factor stimulus. Cell adhesion molecules involved in cell-cell or cell-matrix interactions, such as the integrins, help mediate endothelial cell migration. Many of the integrins bind to the Arg-Gly-Asp (RGD) tripeptide sequence found on a number of matrix proteins, allowing endothelial cells to interact with a variety of ECM components.64 The adhesion receptor integrin avb3 is critical for the differentiation, maturation, and survival of blood vessels.65 Present on the surface of activated endothelial cells, avb3 enables the cells to spread, assists them in forming protrusions, and participates in lumen formation.65 Growth factors such as FGF increase the expression of avb3 on endothelial cells.43

Targeted therapy strategy Inhibiting a single pathway (growth factor and/or receptor) Bevacizumab – VEGF2 Inhibiting multiple pathways (growth factors and/or receptors) Imatinib – PDGF, KIT (receptor for the stem cell factor) SU11248 – VEGF, PDGF, KIT, colony stimulating factor 1 receptor, and FLT-3

VEGF Family of Ligands and Receptors VEGF- A121 VEGF- A145 VEGF- A165 VEGF- A189 VEGF- A206 VEGF- B167 VEGF- B186 PlGF- 1,2 VEGF- C VEGF- D VEGF- E Y Y s-s s-s X X VEGFR1 (Flt-1) NRP-1 VEGFR2 (Flk-1/KDR) VEGFR3 (Flt-4) NRP-2 The VEGF receptor system is composed of three different receptors – VEGFR-1, -2, and -3. These receptors are stimulated by various ligands. The most dominant and important is the VEGF-A and its isoforms, which stimulate either R-2 or R-1. R-1 has its unique ligands that stimulate VEGF-B and placental growth factor. R-2 has its specific ligand called VEGF-E, which is a viral-derived ligand. VEGF-C and -D stimulate specifically R-3 as well as R-2. R-1 and R-2 are important for vasculogenesis and angiogenesis, and R-3 is important for lymphangiogenesis. The approach that ImClone has taken to target the R-2 receptor, which is thought to be the most important receptor in tumor angiogenesis, is developing an antibody against the receptor. This antibody blocks the ability of ligands to bind to the receptor. The alternative approaches are antibodies such as bevacizumab, which bind to ligand VEGF-A, or soluble receptors, which can also bind ligands, or tyrosine kinase inhibitors that inhibit the intrinsic kinase activity upon stimulation of the receptor. The important functions of VEGF in physiological and pathological angiogenesis are permeability, proliferation, and migration. VEGF-A modulates the release of blood-borne elements from the vessel. It causes the stimulation of endothelial cells from preexisting vessels. VEGF-A will induce the migration of these endothelial cells from preexisting vessels and exert a protective effect on immature vascular beds protecting endothelial cells from undergoing apoptosis. Vasculogenesis Angiogenesis Lymphangiogenesis

Targeted therapy strategy Inhibiting a single pathway (growth factor and/or receptor) Becizumab – VEGF2 Inhibiting multiple pathways (growth factors and/or receptors) Imatinib – PDGF, KIT (receptor for the stem cell factor) Sunitinib– VEGF, PDGF, KIT, colony stimulating factor 1 receptor, and FLT-3

Human kinome ABL, KIT, PDGFR ABL Imatinib Sorafenib (BAY43-9006) Valatanib (PTK787) Sunitinib (SU11248) Manning G et al. Science 2002; 298:1912

Perspectives Les agents multicibles Les combinaisons d’agents ciblés (Bévacizumab + Cétuximab ; trastuzumab + cetuximab) Les combinaisons avec les cytotoxiques Mais surtout, l’individualisation des choix thérapeutiques par le profil génétique des tumeurs (pharmacogénomique) et celui de l’individu (pharmacogénétique)

Zoom sur les monoclonaux Acm intégralement humains Acm murins 1975 Acm chimériques 1984 Acm humanisés 1988-1991 Acm intégralement humains 1994-1999 Domaines constants VH et VL humains Rituximab, Infliximab, Cetuximab… Systèmes d’expression « display » Souris transgéniques Adalimumab CDR grafting Bevacizumab Trastuzumab… Hybridomes Muromomab

Cancers du colon Cancers du rein Cancers digestifs Cancers du sein AcM thérapeutiques en 2006 Cancers du colon Cancers du rein Cancers digestifs Cancers du sein Cancer ORL Lymphomes

Rituximab : mécanismes d’action présumés CD20 FcRIIIa CD20 apoptose Effecteur cytotoxique (macrophage, NK) LYMPHOME B CD20 ADCC Souris KO FcR-g Complément C1q CDC Souris KO C1q

CHOP21 vs R-CHOP21 GELA Trial , n=399 Coiffier et al., NEJM, 2002 OS 399 patients with Diffuse large B cell lymphoma 60 to 80 years 5 yr update, Feugier et al, JCO 2005

Does Rituximab impact on survival by overcoming chemo-resistance due to Bcl-2 gene familly ? Part de la tumeur part de l’hote Polymorphisme P Cinetique, P Dynamique Fig. 2. Rituximab-mediated inhibition of the Raf-1/MEK/ERK1/2 signaling pathway in non-AIDS-related NIH cell lines. The scheme illustrates that B-NHL cell lines have a constitutively activated Raf-1/MEK/ERK1/2 pathway resulting in activation of the transcription factor AP-1. Activation of AP-1 results in transcriptional up-regulation of Bclx and thereby in chemoresistance. Upon treatment with rituximab, there is inhibition of Ras and downstream the Raf-1/ MEK/ERK1/2 pathway resulting in inhibition of AP-1 and Bcl-xL transcription and thereby in reversal of drug resistance. The regulation of rituximab-mediated effects is via the induction of RKIP expression which in turn inhibits the Raf-1/MEK/ERK1/2 pathway. The rituximab-mediated effects have been corroborated by the use of specific, small-molecule inhibitors. Fig. 3. Rituximab-mediated inhibition of the NF-B signaling pathway in non-AIDS-related NHL cell lines. This scheme illustrates that B-NHL cell lines have a constitutively activated NIK/IKK/IB-/NF-B pathway resulting in the activation of the transcription factor NF-B. The activation of NF-B results in the transcriptional upregulation of Bclx and resulting in chemoresistance. Upon treatment with rituximab, there is inhibition of Ras and downstream the NIK/IKK/IB-/NF-B pathway resulting in inhibition of NF-B and Bcl-xL transcription and in reversal of drug resistance. The regulation of rituximab-mediated effects is via the induction of RKIP expression which in turn inhibits the NIK/IKK/IB-/NF-B pathway. The rituximab-mediated effects have been corroborated by the use of specific, small-molecule inhibitors. Bonavida B, Oncogene 2005

Bcl-2 DLBCL subset analysis n=292 Bcl2 +, n=193 P=0.01 5 yr update, Mounier, Haematologica 2006 A Bcl2 - n=99 P=0.6 5 yr update, Mounier, Haematologica 2006

Toxicité Ac Monoclonaux (mab) Relargage cytokinique Allergie Effets de classe : Hémato, immunosuppression HTA, retard cicatrisation Inhibiteur des kinases (inib) Nécrose tumorale Rétention hydro sodée, T cutané Métabolique: Hypothyroïdie, diabète digestif Et à long terme ?

mab

VEGF Biology & Avastin effects Arterial Thromboembolism mab VEGF Biology & Avastin effects Mechanism of anti-VEGF efficacy Mechanism of anti-VEGF adverse effects VEGF biology Avastin effects VEGF induces endothelial cell survival “anti-vascular” VEGF induces EC permeability “normalization” VEGF induces EC proliferation, migration “anti-angiogenic” VEGF biology Avastin effects Maintenance of vascular tone Hypertension Kidney mesangial cell survival factor Proteinuria Vessel wall biology - plaque stabilization Arterial Thromboembolism Wound contraction, granulation, closure  Wound healing

-Trouble de la cicatrisation ( 10 à 20% ) : Avastin Tolérance -Trouble de la cicatrisation ( 10 à 20% ) : - Saignement post opératoire - Retard de cicatrisation Hemorragies (4%) : Saignement muqueux , epistaxis ( 20-40 %) Tumoral ( 9%) « Terrain dépendant «  ( NSCLC epidermoide) Perforations intestinales ( 1.5 à 2%) « terrain dépendant » Cancer colorectal. Cancer de l’ovaire Maladie inflammatoire intestinale ( RCH Maladie de Crohn) Diverticulose sigmoidienne Ulcere gastrique evolutif mab

- Thromboses veineuse (?) - Asthenie mab Avastin Tolérance (2) -Hypertension artérielle ( 10 à 15% dont 0.7% imposant l’arrêt de Beva ) -Protéinurie (25 à 30%) -Thromboses artérielles ( 2% ) Risque augmenté chez les patients agés ( > 65 ans ) - Thromboses veineuse (?) - Asthenie - Migraine ( Toxicite aigue dose limitante en phase I 20 mg/kg ) Effets en rapport avec l’action sur la cible VEGF

Physiopathologie de l’hypertension -Thrombose mab Physiopathologie de l’hypertension -Thrombose Hypertension ( J.Yang Clinical Cancer Research): -Action du VEGF sur la « NO synthase « endotheliale -NO synthase est impliquée dans la vasodilatation et vasoconstriction et régulée par le VEGF / IL² Thrombose ( Kabbinavar JCO 2003) - Augmentation de marqueur d’activation de la coagulation ( facteur VIII Von Willebrand ) - Participation de la chimiotherapie ?

Physiopathologie de la protéinurie mab Physiopathologie de la protéinurie Etudes cliniques : Protéinurie 25% Etudes précliniques impact rénal ( Singe, Lapin) : Non informatives -Pas d’accumulation de Beva au niveau du rein -Pas de modification de la fonction rénale -Pas d’aggravation de l’insuffisance rénale ( modele Cisplatine ) Hypothése pour la protéinurie - Cellules mesangiales capillaires riches en VEGF ( pôle arteriel du glomérule ) - Modification de la perméabilité glomérulaire ( syndrome néphrotique ) Effet réversible sans insuffisance rénale

Refractory to INF and IL2 5 previous lines of chemotherapy inib Evidence of Tumor Necrosis Patient aged 50 Renal cell carcinoma Refractory to INF and IL2 5 previous lines of chemotherapy CT-Scan after 4 cycles Surgical Resection of adrenal metastasis after 5 months of treatment with SU11248 Total central necrosis with no viable cancer cells Peripheral area surrounding necrosis with viable cancer cells

inib Tumor Necrosis SU11248 Tumor type Type of Doses (preexisting conditions) complications 100 mg q2d Renal (local recurrence) None 50 mg daily Cervix (irradiated area) Rectal fistula 75 mg daily Rectum (irradiated area) Bladder and rectal fistula UCNT (necrotic lung metastasis) Infection in necrotic areas GIST (peritoneal metastasis) Peritonitis and toxic death 100 mg daily Renal (local recurrence) None (preventive surgery)

inib

Reversible Hair Depigmentation inib Reversible Hair Depigmentation

Sunitinib blocks KIT in animal and human Microphtalmia that controls the Tyrosinase gene expression Pro-melanin MAPKs melanin Pigmentation + - + / - KIT c-kit antibody and SU11248 lead to depigmented gray or white hair with a decreased melanocyte proliferation and differentiation in mice

Perspectives de développement des thérapie ciblées Au diagnostic : il existe des facteurs pronostiques => Base pour essais thérapeutiques et intégration des facteurs pronostiques biologiques Pour le suivi : il existe méthodes statistiques => Etudes multivariées et séquentielles La question : Bouleversement des traitements Mise en place de nouvelles stratégies - Ac monoclonaux, petites molécules - Induction, adjuvant - Consolidation, entretien