MÉMOIRE DE THÈSE

(1)

IDENTIFICATION AND VALIDATION OF NEW MARKERS AND POTENTIAL THERAPEUTIC TARGETS FOR GASTROINTESTINAL

STROMAL TUMORS IN MURINE MODELS AND IN HUMAN PATHOLOGICAL MATERIAL

Petra GROMOVA

LABORATOIRE DE NEUROPHYSIOLOGIE FACULTÉ DE MÉDECINE

ULB 2011

(2)

(3)

JURY DE THÈSE

Président: Prof. Jacques DEVIERE

(Service de Gastro-entérologie, Hôpital Erasme, ULB, Bruxelles)

Membres Facultaires: Dr. Cédric BLANPAIN

(IRIBHM, Faculté de Médecine, ULB, Bruxelles)

Prof. Pieter DEMETTER

(Service d’Anatomie Pathologique, Hôpital Erasme, ULB, Bruxelles)

Prof. Jean-Luc VAN LAETHEM

(Service de Gastro-entérologie, Hôpital Erasme, ULB, Bruxelles)

Prof. Jean-Christophe NOEL

(Service d’Anatomie Pathologique, Hôpital Erasme, ULB, Bruxelles)

Experts extérieurs: Prof. Patrice DUBREUIL

(Centre de Recherche en Cancérologie de Marseille, U891 INSERM, Institut Paoli-Calmettes, IFR 137, Institut de Cancérologie et d'Immunologie de Marseille, Université de la Méditerranée, Marseille)

Prof. Agnès NOEL

(Biologie des Tumeurs et du Développement, Département des sciences cliniques, Université de Liège, Liège)

Secrétaire/promoteur: Prof. Jean-Marie VANDERWINDEN

(Labo. Neurophysiologie, Faculté de Médecine, ULB, Bruxelles)

Co-promoteur: Prof. Christophe ERNEUX

(IRIBHM, Faculté de Médecine, ULB, Bruxelles)

(4)

(5)

As for the search for truth, I know from my own painful searching, with its many blind alleys, how hard it is to take a reliable step, be it ever so small, towards the understanding of that which is truly significant.

— Albert Einstein —

(6)

(7)

Table of contents

List of abbreviations ... 14

List of figures ... 17

List of tables ... 21

Chapter I Introduction ... 22

1.1. Receptor tyrosine kinase (RTK) KIT ... 23

1.1.1. SCF/KIT signal transduction... 25

1.1.1.1. The RAS/ERK pathway ... 26

1.1.1.2. The phosphatidylinositol-3-kinase pathway ... 26

1.1.1.3. The JAK/STAT pathway ... 27

1.1.1.4. The phospholipase C pathway ... 28

1.1.2. Regulation of KIT signaling ... 28

1.1.3. KIT mutations ... 29

1.2. Interstitial cells of Cajal (ICC) ... 30

1.2.1. ICC and KIT signaling ... 30

1.2.2. ICC distribution along the GI tract ... 31

1.2.3. Roles of ICC in the physiology of GI tract ... 32

1.3. Gastrointestinal stromal tumors (GIST) ... 33

1.3.1. Oncogenic mutations in GIST ... 33

1.3.2. GIST epidemiology ... 35

1.3.3. GIST immunohistochemical markers... 36

1.3.4. Therapeutic targets of GIST ... 38

1.3.5. Novel putative markers/targets of GIST ... 39

1.3.6. GIST murine models ... 40

1.3.6.1. Kit^K641GIST mouse model ... 40

1.3.7. Human GIST cell lines ... 42

1.3.8. Ba/F3 cells KIT signaling model ... 42

1.4. List of references ... 45

(8)

Chapter II

Aims of the study ... 54

2.1. Framework of the study ... 55

Chapter III Kit K641E oncogene upregulates Sprouty homolog 4 and Trophoblast glycoprotein in interstitial cells of Cajal in a murine model of gastrointestinal stromal tumors ... 58

3.1. Introduction ... 59

3.2. Materials and methods... 60

3.2.1. Animals ... 60

3.2.2. Gene expression analysis ... 61

3.2.3. Microarray data analysis ... 61

3.2.4. Comparison of the gene expression profile in KitK641E/K641E antrum with expression profile in human GIST and in mouse ICC ... 62

3.2.5. Real time quantitative PCR ... 62

3.2.6. Immunofluorescence ... 64

3.2.7. Confocal microscopy ... 66

3.3. Results ... 67

3.3.1. Gene expression profiling of KitK641E/K641E antrum ... 67

3.3.2. Confirmation of differential gene expression by qPCR ... 68

3.3.3. Genes upregulated in KitK641E/K641E antrum belong to the gene profiles of murine ICC and human GIST ... 69

3.3.4. Protein expression in Kit-ir ICC ... 72

3.3.4.1. Prkcq/Pkc theta-ir, Pde3a-ir and Gja1/Cx43-ir are present in Kit-ir ICC ... 72

3.3.4.2. Spry4-ir and Tpbg/5T4-ir are detected in Kit-ir cellss only in Kit^K641E ... 76

3.4. Discussion ... 80

Annex 1 Trophoblast glycoprotein (TPBG/5T4) GIST tissue microarray ... 85

(9)

Chapter IV

Oncogenic Kit K641E leads to Spry1 down-regulation and sustained activation of Ras/Erk

pathway in vitro and in vivo ... 88

4.2.1. The Kit^K641E murine model of GIST ... 90

4.2.2. Tissue harvesting ... 91

4.2.3. Immunofluorescence staining ... 91

4.2.5. Ba/F3 Cell model ... 92

4.2.7. Western blotting ... 95

4.2.8. Flow cytometry analysis ... 95

4.3. Results ... 98

4.3.1. In vivo - mouse Kit^K641E GIST model ... 98

4.3.1.1. Spry1 is down-regulated in Kit^K641EGIST mouse model in mRNA and protein level ... 98

4.3.1.2. Erk1/2 is activated in the Kit-ir cells in the antrum of KitK641E/K641E animals ... 99

4.3.2. In vitro - murine Ba/F3 cell model ... 100

4.3.2.1. Spry mRNA expression varies in Ba/F3 cells transfected with WT Kit and Kit oncogenes... 100

4.3.2.2. Kit activation by SCF leads to increase of Spry1; Spry4 mRNA expression in Ba/F3 cells expressing WT Kit and does not alter Spry levels in Kit oncogenic mutants ... 100

4.3.2.3. Ras/Erk pathway downstream of Kit controls Spry1 and Spry4 mRNA expression. ... 102

4.3.2.4. Kit signaling cascade activation and Spry1 and Spry4 protein expression ... 102

4.3.2.5. Effect of acute and sustained SCF stimulation on the level of p-Tyr in Ba/F3 cells transfected with Kit WT and Kit oncogenes ... 104

4.3.2.6. Spry1 mRNA expression in Kit^K641E Ba/F3 cells can be restored by the DNA methyltransferase inhibiting agent 5-Aza/dC ... 106

(10)

Chapter V

Neurotensin receptor 1 is expressed in gastrointestinal stromal tumors but not in interstitial

cells of Cajal ... 112

5.2.1. The Kit^K641Emurine model of GIST ... 114

5.2.2. RNA Extraction and PCR ... 114

5.2.5. Epifluorescence microscopy ... 117

5.2.6. Human GIST tissue micro array ... 117

5.2.7. Immunohistochemistry on FFPE human material ... 119

5.2.8. Human GIST882 cell line ... 120

5.3. Results ... 121

5.3.1. Ntsr1 expression in murine Kit^K641E GIST model ... 121

5.3.1.1. Ntsr1 mRNA expression is up-regulated in Kit^K641E antrum ... 121

5.3.1.2. Ntsr1-ir is not present in WT ICC ... 121

5.3.1.3. Ntsr1-ir is expressed in the hyperplastic layer of Kit-ir cells in Kit^K641E antrum ... 123

5.3.2. NTSR1 expression in human GIST ... 124

5.3.2.1. NTSR1-ir is not present in human KIT-ir ICC ... 124

5.3.2.2. NTSR1-ir is present in human GIST irrespective to the mutation status ... 125

5.3.2.3. NTSR1 - but not Neurotensin (NT) - is expressed in the human GIST882 cells ... 129

5.3.2.4 NTSR1-ir translocates to the nucleus upon agonist stimulation in GIST882 cells ... 129

(11)

Chapter VI

ENDOGLIN/CD105 is expressed in KIT positive cells in the gut and in gastrointestinal

stromal tumors ... 136

6.2.1. The Kit^K641E murine GIST model ... 138

6.2.2. Ba/F3 cell model ... 139

6.2.3. Human GIST882 cell line ... 139

6.2.4. Flow cytometry analysis ... 140

6.2.9. Human GIST tissue micro arrays ... 144

6.2.10. Immunohistochemistry on human material ... 146

6.2.11. Statistical analysis ... 146

6.3. Results ... 147

6.3.1. Endoglin expression in KitK641E/K641E mouse antrum ... 147

6.3.2. Endiglin is expressed in Kit+ cells and in endothelium in the mouse gut wall ... 149

6.3.2. Endoglin expression in human tissue ... 152

6.3.3. Endoglin mRNA expression in Ba/F3 cell is increased by Kit oncogenic mutants but is not controlled by Kit phosphorylation ... 155

6.3.4. Expression of Endoglin mRNA can be restored in Ba/F3 uWT cells by the DNA methyltransferase inhibiting agent 5-Aza/dC ... 157

Chapter VII General discussion ... 164

7.1. Identification of new putative markers/targets for GIST ... 165

7.2. Neurotensin receptor 1 (NTSR1) ... 165

(12)

7.3. Trophoblast glycoprotein (TPBG/5T4) ... 167

7.4. Sprouty homolog family (SPRY) ... 168

7.5. Endoglin (ENG) ... 169

7.6. Concluding words ... 170

Summary ... 176

Résumé ... 180

Acknowledgements ... 184

(13)

14

List of abbreviations

(-) Negative

(+) Positive

(++) Strong Positive

18F-FDG 18F-Fluoro-Deoxyglucose 5-Aza/dC 5’-Aza-2’deoxycytidine

AB Acidic Box

AKT/PKB Oncogenic kinase initially isolated from a transforming mouse retrovirus/

Protein Kinase B

ACP1 Acid Phosphatase 1, soluble AHC Ascending Hierarchic Classification ANGPTl4 Angiopoietin-like 4

APOA2 Apolipoprotein A-II

BLAST Basic Local Alignment Search Tool

CadhD Cadherin-like Domain

CAII Carbonic Anhydrase II

CM Circular Muscle layer

CML Chronic Myeloid Leukemia

CRD Cystein-Rich Domain

CSF1R Colony-Stimulating Factor 1 Receptor

Cyp2b20 Cytochrome P450, family 2, subfamily B, polypeptide 20 Cyp4a14 Cytochrome P450, family 4, subfamily A, polypeptide 14

DAB-Ni Nickel-enhanced DAB

DAG Diacylglycerol

DMP Deep Muscular Plexus

DOG1 Discovered On GIST-1

EAR1 Eosinophil-Associated, Ribonuclease A family, member 1 EAR10 Eosinophil-Associated, Ribonuclease A family 10

EAR3 Eosinophil-Associated, Ribonuclease A family, member 3

EC Extracellular Domain

EGFR Epidermal Growth Factor Receptor EGIST Extra-Gastrointestinal Stromal Tumors

ENG Endoglin

Enpep Glutamyl Aminopeptidase

EphR Ephrin Receptor

ERK Extracellular signal-Regulated Kinase FABP1 Fatty Acid Binding Protein 1, liver FABP4 Fatty Acid Binding Protein 4, adipocyte FACS Flow cytometry analysis

FAMC Factorial Analysis of Multiple Correspondences

FBS Fetal Bovine Serum

FFPE Formalin Fixed Paraffin Embedded

FGF Fibroblast Growth Factor

(14)

15

FGFR Fibroblast Growth Factor Receptor FLT-3 FMS-related tyrosine kinase 3 FNIII Fibronectin Type III-like domain

Gal Galanin

GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase

GEO Gene Expression Omnibus

GI Gastrointestinal tract

GIST Gastrointestinal Stromal Tumors

Gja1/Cx43 Gap Junction membrane channel protein Alpha 1 (Connexin 43, Cx43) GPBAR1 G Protein-Coupled Bile Acid Receptor 1

GPC6 Glypican 6

GPCR G-Protein-Coupled Receptor GPR133 G Protein-Coupled Receptor 133

GRB2 Growth factor Receptor-Bound protein 2

HDAC Histone Deacetylases

HGFR Hepatocyte Growth Factor Receptor

HHT1 Hereditary Hemorrhagic Telangiectasia, type 1 HIF1α Hypoxia Inducible Factor 1 α

HOPX Homeobox only domain

HPF High Power Field

HPRT Hypoxanthine-Guanine Phosphoribosyl Transferase

HZ Hardy-Zuckerman

ICC Interstitial Cells of Cajal

ICC-DMP ICC associated with the Deep Muscular Plexus ICC-IM Intramuscular ICC

ICC-MY ICC in the Myenteric layer

ICC-SM Submucosal ICC

ICH Immunohistochemistry

IF Immunofluorescence

IgD Immunoglubulin-like Domain

IGFBP3 Insulin-like Growth Factor Binding Protein 3

IR Insulin Receptor

ir Immunoreactive

JAK Janus Kinase

JM Juxtamembrane domain

KIT+ KIT-expressing

LD Leucine Domain

L-ENG Long form of Endoglin

LM Longitudinal Muscle layer

LRD Leucine-Rich Domain

MAPK Mitogen-Activated Protein Kinase

MEK1/2 Protein Kinase

MEEBO Mouse Exonic Evidence Based Oligonucleotide

MP Myenteric Plexus

(15)

16

Myl7 Myosin, light polypeptide 7, regulatory

NeoR1 Neomycine R1

NF1 Neurofibromatosis 1

NHS Normal Horse Serum

NL557 Northernlight™ 557

NRQ Normalized Relative Quantities

NT Neurotensin

NT/N Neurotensin/Neuromedin N precursor NTSR1 Neurotensin Receptor 1

OASL2 2'-5' Oligoadenylate Synthetase-Like 2

P Phosphorylated tyrosine residue

P14 two week old mice

PACRG Park2 Co-Regulated

PAP Pancreatitis-Associated Protein

PDE3A Phosphodiesterase 3A

PDGFR Platelet-Derived Growth Factor Receptor PDGFRA Platelet-Derived Growth Factor Receptor Alpha

PET Positron Emission Tomography

PI3-K Phosphatidylinositol-3-Kinase PIP2 Phosphatidylinositol 4, 5-biphosphate PIP3 Phosphatidylinositol 3, 4, 5-triphosphate

PKC Protein Kinase C

Pkrcc/Pkc gama Protein Kinase C, gama Pkrcq/Pkc theta Protein Kinase C theta

PLC Phospholipase C

Prkar2b Protein Kinase, camp Dependent Regulatory, type II beta Prkcn/Prkd3 Protein Kinase C Nu/Protein Kinase D3

PTB Phosphotyrosine Binding domain PTEN Phosphatase and Tensin Homolog

p-Tyr Phospho-Tyrosine

Rab6 Member of RAS oncogene family 6 RAF Serine–theonine protein kinase

RAS Rat Sarcomas oncogene

RASD2 RASD family, member 2

Retnla Resistin Like Alpha

R-PE R-Phycoerythrin

RT Room Temperature

RT-PCR Reverse Transcription PCR

RTK Receptor Tyrosine Kinases

qPCR Real Time Quantitative PCR

S100a8 S100 calcium binding protein A8 (Calgranulin A) S100a9 S100 calcium binding protein A9 (Calgranulin B) SCD1 Stearoyl-Coenzyme A Desaturase 1

SCF Stem Cell Factor

(16)

17

S-ENG Short form of Endoglin

SH2 Src Homology 2 Domain

SMA Smooth Muscle Actin

SMC Smooth Muscle Cells

SOS Son Of Sevenless

SP Submucosal Plexus

SPRY Sprouty protein family

SPRY1 Sprouty homolog 1

Src Oncogene of the chicken Rous Sarcoma Virus STAT Signal Transducer and Activator of Transcription

STFA1 Stefin A1

SDH Succinate dehydrogenase

DSDHB/C Succinate dehydrogenase, subunits B, C TBS-TX TBS, Containing Triton-X 100

T-ENG Total form of Endoglin

TGF-β Transforming Growth Factor Β TK 1; 2 Tyrosine Kinase domains 1 and 2 TKI Tyrosin Kinase Inhibitors

TM Transmembrane domain

TMA Tissue Micro Arrays

TPBG/5T4 Trophoblast Glycoprotein

TRK Tropomyosin Receptor Kinase

ULB Université Libre de Bruxelles

VEGFR Vascular Endothelial Growth Factor Receptor

WB Western Blotting

WT Wild Type

Footnote: Abbreviations are introduced at their first appearance in each chapter. Names of genes which are referring to mRNA are written in italic, names of proteins are written in regular text.

The names of human genes are written with capital letters while the names of mouse genes are written with the first letter in upper case.

(17)

18

List of figures Chapter I

Figure 1.1. Schematic structure of the main human RTK families ... 23

Figure 1.2. Schematic structure of KIT receptor tyrosine kinase ... 24

Figure 1.3. SCF/KIT signal transduction ... 25

Figure 1.4. Activation of KIT RTK downstream signaling pathways ... 27

Figure 1.5. Regulation of KIT signaling ... 28

Figure 1.6. Schematic diagram illustrating the organization of ICC and smooth muscle cells in the gastric antrum ... 31

Figure 1.7.Kit+ ICC distribution in the mouse gastric antrum ... 32

Figure 1.8. Schematic structure of KIT and PDGFRA receptor tyrosine kinases and distribution of oncogenic mutations in GIST ... 34

Figure 1.9. Kit-ir in normal human colon and in GIST ... 37

Figure 1.10. Kit/Actin immunoreactivity in Kit^K641E mouse model ... 41

Figure 1.11. Level of Kit-ir and p-Tyr-ir in Ba/F3 cells expressing Kit WT and Kit oncogenes 43 Chapter III Figure 3.1. Expression pattern of genes differentially expressed in the antrum of P14 KitK641E/K641E homozygous mice vs Kit^WT/WT littermates ... 67

Figure 3.2. Differences in gene expression confirmed by qPCR ... 69

Figure 3.3. Prkcq/Pkc theta-ir in Kit-ir ICC in the mouse antrum ... 73

Figure 3.4. Pde3a-ir in Kit-ir ICC in the mouse antrum ... 74

Figure 3.5. Gja1/Cx43-ir in Kit-ir ICC in the mouse antrum ... 75

Figure 3.6. Spry4-ir is detected in Kit-ir cells only in Kit^K641E ... 77

Figure 3.7. Spry2-ir is detected in Kit^K641E and Kit^WT/WT in smooth muscle cells ... 78

Figure 3.8. Tpbg/5T4-ir is detected in Kit-ir cells only in Kit^K641E ... 79

(18)

19

Annex 1

Figure A1.1. TPBG/5T4 immunoreactivity in normal human colon and in GIST ... 85 Figure A1.2. TPBG/5T4 and KIT immunohistochemistry on human GIST TMA ... 86

Chapter IV

Figure 4.1. Spry1 expression is dowregulated in KitK641E/K641E

mouse antrum ... 98 Figure 4.2. Erk1/2 is activated in the Kit-ir cells in the antrum of KitK641E/K641E

animals... 99 Figure 4.3. Spry mRNA expression in Ba/F3 cell transfected with WT Kit and Kit oncogenes at

basal level and after SCF stimulation ... 101 Figure 4.4. Spry mRNA expression in Ba/F3 cell transfected with WT Kit and Kit oncogenes after

U0126 treatment ... 102 Figure 4.5. Activation of main pathways downstream of Kit signaling cascade in Ba/F3 cell

transfected with WT Kit and Kit oncogenes at basal level and after SCF stimulation ... 103 Figure 4.6. Acute and chronic SCF stimulation affect the level of p-Tyr in Ba/F3 cells transfected

with Kit WT and Kit oncogenes ... 105 Figure 4.7. Repressed Spry1 expression in Ba/F3-Kit^K641E is restored after treatment with the

methyltransferase inhibitor 5-Aza/dC. ... 106

Chapter V

Figure 5.1. Ntsr1-ir is expressed in the hyperplastic layer of Kit-ir cells in Kit^K641E antrum but not in WT ICC ... 122 Figure 5.2. Ntsr1-ir is expressed in Kit-ir cell clusters in the antrum of adult heterozygous

Kit^WT/K641Emice but not in WT ICC ... 123 Figure 5.3. NTSR1-ir is present in a human GIST with KIT K642E mutation but not in normal

human KIT-ir ICC ... 124 Figure 5.4. NTSR1-ir in human GIST is irrespective to the mutation status ... 125 Figure 5.5. NTSR1-ir patterns in human GIST ... 127 Figure 5.6. NTSR1 is expressed in the human GIST882 cell line and responds to agonist

stimulation ... 130

(19)

20

Chapter VI

Figure 6.1. Eng mRNA expression is upregulated in KitK641E/K641E

antrum ... 148 Figure 6.2. Eng protein expression in Kit^K641E antrum ... 148 Figure 6.3. Eng expression in endothelium and in Kit+ ICC in WT and Kit^K641E antrum ... 149 Figure 6.4. Eng expression in endothelium and in Kit+ ICC in WT mouse small intestine .... 150 Figure 6.5. Eng expression in endothelium and in Kit+ ICC in WT mouse colon ... 151 Figure 6.6. ENG expression in GIST882 cell line... 152 Figure 6.7. ENG-ir in human GIST tissue arrays ... 154 Figure 6.8. Ba/F3 cell lines, Eng mRNA expression is independent of Kit phosphorylation in

vitro ... 156 Figure 6.9. Treatment with the methyltransferase inhibitor 5-Aza/dC increased Eng mRNA

expression in Ba/F3^WT ... 157 Figure 6.10. Expression of Hif1α in Ba/F3 cells and in mouse antrum ... 158

(20)

21

List of tables Chapter III

Table 3.1. Primers used for qPCR ... 63

Table 3.2. Primary and secondary antibodies used for IF ... 65

Table 3.3. Microarray analysis of genes differentially expressed in KitK641E/K641E antrum vs Kit^WT/WT littermates ... 70

Annex 1 Table A1.1. Risk classification and TPBG/5T4-ir association in GIST TMA………….……...87

Chapter IV Table 4.1. Primers used for qPCR ... 94

Table 4.2. Primary and secondary antibodies used for WB, FACS, IF. ... 97

Chapter V Table 5.1. List of primers ... 115

Table 5.2. Clinicopathologic features of the Cleveland Clinic GIST TMA ... 118

Table 5.3. Clinicopathologic features of the SuperBiochips GIST TMA ... 118

Table 5.4. Clinicopathologic characteristics of NTSR1+/KIT- GIST specimens ... 126

Table 5.5. NTSR1-ir on the Cleveland Clinic GIST TMA ... 128

Table 5.6. NTSR1-ir on the SuperBiochips GIST TMA ... 129

Chapter VI Table 6.1. Table of primers ... 141

Table 6.2. Table of antibodies ... 143

Table 6.3. Clinicopathologic features of cases on the Cleveland Clinic GIST TMA ... 145

Table 6.4. Association of Eng-ir with clinicopathologic characteristics in 49 GIST cases ... 153

(21)

Ch C ha a p p te t er r I I

Introduction

Gastrointestinal Stromal Iumors (GIST) are the most common mesenchymal tumors of the gastrointestinal (GI) tract. A breakthrough for diagnosis and treatment of GIST was the recognition of the Receptor Tyrosine Kinase (RTK) KIT expression in these tumors. The majority (but not all) of GIST harbor oncogenic KIT mutations.

GIST unique genetic and morphologic similarities indicate that they arise from Interstitial Cells of Cajal (ICC) or an ICC precursor. Development and maintenance of ICC is dependent on KIT signaling. KIT oncogenic mutations in these cells result in the uncontrolled cell growth and GIST formation.

Rapid advances in the understanding of GIST biology and the development of tyrosine kinase inhibitors have epitomized GIST as a paradigm for molecular targeted therapy. However, complete responses to current treatments remain rare and most patients develop resistance over time.

This thesis aims to identify and to validate new putative markers and molecular targets for therapeutic interventions and strategies for GIST management.xxxxxxxxxxxxxxxxxxxxxxx

(22)

23

1.1. Receptor tyrosine kinase (RTK) KIT

In multicellular organisms, regulation and coordination of complex cellular processes like growth, differentiation, migration and apoptosis is essential. RTK play important roles in the regulation of these physiological events. Their aberrant activation has been linked to development and progression of human cancers [1]. On the basis of structural characteristics, RTK can be divided into 20 subfamilies, which share several conserved homologous domains and elements [2] (Fig. 1.1).

Figure 1.1. Schematic structure of the main human RTK families (adapted from: [3])

Abbreviations: Hepatocyte Growth Factor Receptor (HGFR); Epidermal Growth Factor Receptor (EGFR); Insulin Receptor (IR); Platelet Derived Growth Factor Receptor (PDGFR); Fibroblast Growth Factor Receptor (FGFR); Vascular Endothelial Growth Factor Receptor (VEGFR); Tropomyosin Receptor Kinase (TRK); Ephrin Receptor (EphR)

Tyrosine Kinase domain (TK); Cystein-Rich Domain (CRD); Leucine Domain (LD); Fibronectin type III- like domain (FNIII); Acidic Box (AB); Cadherin-like Domain (CadhD); Leucine-Rich Domain (LRD);

Immunoglubulin-like Domain (IgD).

RTK consist of a single transmembrane domain that separates the intracellular tyrosine kinase region from the extracellular portion [4]. Following ligand binding to the extracellular domain, RTK undergo conformational changes that induce and stabilize receptor dimerization. This leads to increased kinase activity and autophosphorylation [1]. Phosphorylation of distinct tyrosine residues of the activated receptor creates binding sites for Src homology 2 (SH2) and

(23)

24

Phosphotyrosine-binding (PTB) domain-containing proteins. Molecules recruited via these binding motifs- are either enzymes that are tyrosine phosphorylated and activated, or adaptor molecules that link RTK activation to downstream signaling pathways [5].

Figure 1.2 Schematic structure of KIT receptor tyrosine kinase.

Structurally, KIT (a.k.a. c-kit) is a member of the type III RTK family, which also includes Platelet Derived Growth Factor Receptor α and β (PDGFRα/β), Colony-Stimulating Factor 1 Receptor (CSF1R) and FMS-related tyrosine kinase 3 (FLT-3) [6;7]. KIT is composed of a glycosylated extracellular ligand-binding domain that is connected to a cytoplasmic region by means of a single transmembrane (TM) domain [8]. The extracellular domain of KIT contains five immunoglubulin-like domains (IgD), in which the second and third membrane distal domains were shown to play a role in ligand recognition [6;7]. The intracytoplasmic catalytic domain includes an ATP-binding site that catalyses receptor autophosphorylation and a phosphotransferase domain which phosphorylates KIT substrates [9]. The hydrophilic kinase insert domain located between these two tyrosine domains is a distinctive feature for several classes of RTK families, suggesting its important role in substrate recognition and signal generation [10] (Fig. 1.2).

(24)

25

1.1.1. SCF/KIT signal transduction

Under physiological conditions, KIT signaling occurs following binding of KIT ligand, Stem Cell Factor (SCF) to the KIT receptor. SCF is a glycoprotein in forming non-covalent homodimer. It is a potent chemoattractant for various KIT+ cell types [11].

SCF binding leads to receptor dimerization and autophosphorylation (Fig. 1.3). This reveals docking sites for SH2 domains which are found in a number of signaling proteins. After SCF binding, KIT recruits and activates a number of intracellular signaling pathways implicated in many biological processes e.g. control of cell proliferation, adhesion, apoptosis, survival and differentiation [8].

Figure 1.3. SCF/KIT signal transduction.

KIT signaling occurs following binding of KIT ligand, Stem Cell Factor (SCF) to the KIT receptor. SCF binding leads to receptor dimerization and autophosphorylation resulting in activation of downstream signal transduction (Fig 1.4) and transcription initiation.

The SCF/KIT system is essential for the development of hematopoietic stem cells, melanocytes, mast cells, germ cells and ICC of the digestive tract. Conversely, aberrant activation of the KIT

(25)

26

signaling pathway has been linked to various human cancers and with developmental disorders [12].

The main pathways triggered by KIT, namely RAS/ERK, PI3-K/AKT, JAK/STAT and PLC pathway [11;13] are presented below (Fig. 1.4).

1.1.1.1. The RAS/ERK pathway

Extracellular signal-Regulated Kinase (ERK) was the first identified Mitogen-Activated Protein Kinase (MAPK). Since then, the critical importance of ERK in cell division, survival and transformation has been demonstrated [14]. Several growth factors, cytokines and proto- oncogenes transduce their growth promoting signals through this pathway. After activation of the small G protein RAS, the serine–theonine kinase RAF is stimulated, leading to the activation of protein kinase MEK1/2. MEK1/2 then phosphorylates the serine/threonine of ERK1/2. Activated ERK translocates into the nucleus where it regulates gene expression by modulating transcription factors [15] (Fig. 1.4).

1.1.1.2. The phosphatidylinositol-3-kinase pathway

Phosphatidylinositol-3-kinase (PI3-K) is implicated in regulation of DNA synthesis, cell survival and chemotaxis, as well as receptor and vesicular trafficking [16]. It is a heterodimeric complex with a regulatory and a catalytic subunit. Activation of PI3K involves association of the SH2 domain of the regulatory subunit with phosphorylated receptors, leading to translocation of PI3K to the plasma membrane and generation of 3'-phospholipids [16].

Downstream targets of 3'-phospholipids include the serine/threonine kinase AKT/PKB. The activity of AKT/PKB is regulated by PI3-K via the synthesis of phosphatidylinositol 3, 4, 5- triphosphate (PIP3) (Fig. 1.4). PTEN antagonizes PI3-K by degrading PIP3 to phosphatidylinositol 4, 5-biphosphate (PIP2). Deregulation of AKT/PKB signaling has been implicated in the progression of different tumors [17].

(26)

27

Figure 1.4. Activation of KIT RTK downstream signaling pathways.

The main pathways triggered by KIT/SCF are RAS/ERK, PI3-/AKT, JAK/STAT pathways and PLC signaling. Their activation results in regulation of important cellular processes like proliferation, survival and migration.

Abbreviations:phosphorylated tyrosine residue (P); Phosphatidylinositol 3- kinase (PI3K); oncogenic kinase initially isolated from a transforming mouse retrovirus/ Protein Kinase B (Akt/PKB); phospholipase C (PLC);

Protein Kinase C (PKC); oncogene of the chicken Rous sarcoma virus (Src);

Son Of Sevenless (Sos); Growth Factor Receptor-Bound protein 2 (Grb2); rat sarcomas oncogene (Ras);

serine/threonine-protein kinase (Raf);

Extracellular-Regulated Kinase / Mitogen-Activated Protein Kinase (ERK/MAPK); Janus kinase (JAK);

Signal Transducer and Activator of Transcription (STAT).

1.1.1.3. The JAK/STAT pathway

The Janus kinases (JAK) are cytoplasmic tyrosine kinases that are rapidly activated by ligand binding to cytokine receptors or RTK. Important targets of activated JAK are the Signal Transducers and Activators of Transcription (STAT) proteins. Seven STAT family members have been identified; STAT 1–6 with STAT 5 having isoforms a and b. STAT are a group of transcription factors that have a central DNA binding domain followed by a SH2 domain and a C-terminal transactivation domain. Activated JAK phosphorylate the cytoplasmic STAT, which then undergo a SH2 domain–dependent dimerization and subsequent translocation to the nucleus (Fig. 1.4). Nuclear STAT regulates expression of responsive genes by binding to their promoters [18].

(27)

28

1.1.1.4. The phospholipase C pathway

Phospholipase C (PLC) mediated signaling is implicated in differentiation, division, motility, survival, egg fertilization and immune response. PLC interacts with the phosphorylated Y730 residue of KIT. PLC hydrolyzes phosphatidylinositol-4, 5-bisphosphate (PIP2), generating diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). DAG then binds and activates conventional and novel Protein Kinase C (PKC) isoforms, whereas IP3 induces release of Ca²⁺ from intracellular stores [19] (Fig. 1.4).

1.1.2. Regulation of KIT signaling

Activation of KIT signaling pathways is tightly regulated at multiple steps. Commonly, over-activation or impaired down-regulation of RTK signaling is associated with cell growth and tumor formation. Activation of RTK by their growth factors not only leads to propagation of the signals, but also to initialization of multiple attenuation mechanisms, to ensure that once triggered, signals are shut off after an appropriate period of time [20]. Upon ligand binding,

Figure 1.5. Regulation of KIT signaling. (adapted from: [20])

A: Ligand-induced KIT receptor ubiquitination and degradation mediated by the ubiquitin ligase c-Cbl.

B: Inhibitory proteins that counteract downstream signaling. Activation of Ras/ERK pathway leads to the induction of the Sprouty (SPRY) negative-feedback loop. The role of the phosphatidylinositol phosphatase PTEN as a specific attenuator of the KIT-PI3K-AKT pathway is also ilustrated.

B

A

(28)

29

receptor-ligand complexes are endocytosed via clathrin-coated pits. The receptor could be recycled to the cell surface or targeted to lysosomes for degradation [21-23]. Moreover, the receptor is subjected to nonlysosomal degradation involving polyubiquitination of the protein.

C-Cbl has been shown to execute degradation of KIT receptor through its E3 ubiquitin ligase activity [24]. Subsequently, transcription of late attenuators, such as Sprouty protein family (Spry) and Phosphatase and Tensin Homolog (PTEN), is induced and act to further dampen the triggered signals [20] (Fig.1.5)

1.1.3. KIT mutations

KIT signaling regulates normal cellular processes in several cell types (ICC, mast cells, melanocytes, gonads, stem cells). Its deregulation by lost or gain of function mutations is involved in the development and progression of human diseases, including cancers. Activating mutations of c-kit were first described in a feline model, induced by the Hardy-Zuckerman 4- feline sarcoma virus encoding the transforming oncogene v-kit, a mutated viral homolog of c-kit [6].

In human KIT gain-of-function mutations can result in GIST [25], chronic myeloproliferative disorders [26] and myelogenous leukemia [27]. KIT mutation leading to constitutive activation of the receptor, results in proliferative disorders of mast cells (mastocytosis) in the peripheral blood, skin and spleen [28].

KIT loss of function results in piebaldism. This is a rare autosomal dominant disorder of melanocyte development characterized by a congenital white forelock and multiple symmetrical hypopigmented or depigmented macules. It is consequence of lack of melanocytes in the affected skin and hair follicles as a result of KIT mutations [29].

This thesis centers on KIT expression in the ICC and in the GIST which are though to arise from this cell type as a consequence of KIT oncogenic mutation.

(29)

30

1.2. Interstitial cells of Cajal (ICC)

ICC were originally described by the Spanish neuroanatomist Santiago Ramon y Cajal in 1893. They were categorized by large, oval, nuclei with little perinuclear cytoplasm with spindle or stellate shape. Two cellular networks in the musculature of the small intestine were described. One between the muscle layers, associated with (Auerbach's) Myenteric Plexus (MP), the other associated with the Deep Muscular Plexus (DMP) [30;31]. Cajal regarded these cells as primitive neurons and part of the gut autonomic nervous system. For a long time, the identification of ICC was cumbersome, with imprecise criteria and their function remained speculative. Prior to 1960, more than 200 light microscopy studies of ICC were published, reflecting conflicting opinions on the identification, distribution and significance of ICC as a separate cell type [32]. Later, transmission electron microscopy studies demonstrated that ICC are neither neurons nor glial cells and the similarities between ICC and fibroblasts were emphasized [33]. In the 1980´s - 90´s, ICC emerged as ultrastructurally distinct cell types intercalated between nerves and Smooth Muscle Cells (SMC) [34-36]. Use of animal models with developmental lesions in ICC networks, caused by loss of function of KIT has led to the conclusions that ICC are mesenchymal cells without clear muscle or neural differentiation [32;37].

1.2.1. ICC and KIT signaling

Study of animal models with a loss of function of Kit RTK and the use of Kit inhibitors has revealed probably the most important advance in interstitial cell biology. It was recognized that KIT receptor is expressed in ICC in the GI tract and that the functional development of ICC networks depends on signaling via the KIT receptor pathways. KIT was rapidly adopted as the diagnostic light microscopy marker for ICC [38]. Immunolabeling of KIT receptor has provided an efficient way to identify ICC in several species, including human, guinea pig, mouse, rat and birds [32;39]. Noteworthy, KIT has been shown to be present in ICC during development and in mature tissue, but it is not present in any other mesenchymal cells of the intestines [40;41].

(30)

31

1.2.2. ICC distribution along the GI tract

The musculature of the gastrointestinal tract is organized into two principal layers; the inner Circular Muscle (CM) layer beneath the mucosa and the outer Longitudinal Muscle (LM) layer. The myenteric plexus is located in the space between the circular and longitudinal muscle.

ICC are scattered in the muscle layers and also clustered around MP throughout the GI tract [39]

(Fig 1.6).

Figure 1.6. Schematic diagram illustrating the organization of in the gastric antrum. (Adapted from [42])

Smooth Muscle Cells (SMC) are arranged in the longitudinal direction (LM) and the circular direction (CM) of the stomach. ICC are distributed between the intermittent spaces of SMC arround myenteric plexis (MP) (ICC-MY) and within the SM locate intramuscular ICC (ICC-IM).

Based on their localization, ICC could be divided into four subtypes: ICC in the myenteric layer (ICC-MY), which form a network between the circular muscle and longitudinal muscle layers surrounding the neuronal MP; intramuscular ICC (ICC-IM) within the LM and CM; submucosal ICC (ICC-SM) which form networks surrounding the Submucosal Plexus (SP); and in the small intestine, ICC associated with the deep muscular plexus (ICC-DMP) which form a network around DMP in the circular muscle close to the mucosa [32;37].

The ICC make close contacts with nerves and form gap junctions with neighboring muscle cells [32]. The pattern of ICC distribution is fairly consistent among species but varies significantly among the different regions of the digestive tract (Fig 1.7). The cell shape and arrangement of ICC is mainly determined by their relationships to local nerve plexuses, the orientation of the smooth muscle layer in which they are contained and the frequency of connections between ICC themselves [43].

(31)

32

Figure 1.7. Kit+ ICC distribution in the mouse gastric antrum.

The interstitial cells of Cajal (ICC) network is labeled with anti-Kit antibody in red; neuronal marker PGP9.5 is labeled in green. Figures are oriented with the longitudinal muscle layer (LM) up and the circular muscle layer (CM) down. Scale bar: 20 microns.

1.2.3. Roles of ICC in the physiology of GI tract

ICC are often referred to as the “pacemaker cells” because they are able to generate and propagate the electrical slow waves of depolarization which are transmitted through the gut smooth musculature. [44-46]. This “pacemaker activity” underlies the spatio-temporal organization of the peristaltic activity of the GI tract. The initiation of pacemaker activity in the ICC is caused by release of Ca²⁺ from inositol 1,4,5-trisphosphate (IP(3)) receptor-operated stores, uptake of Ca²⁺ into mitochondria and the development of unitary currents. Summation of unitary currents causes depolarization and activation of a dihydropyridine-resistant Ca²⁺

conductance that entrains pacemaker activity in a network of ICC, resulting in the active propagation of slow waves. Slow wave frequency is regulated by a variety of physiological agonists and conditions and shifts in pacemaker dominance can occur in response to both neural and nonneural inputs [37].

In addition, ICC could transduce inputs from the enteric nervous system in GI smooth muscles [37;47;48], however the place of ICC in neurotransmition is still debated [32]. ICC appear intimately close to nerves containing various neurotransmitters and express immunoreactivity for various receptors to neuropeptides and neurotransmitters. The neurochemical coding for the innervation thus appears multiple [32]. ICC may play additional role as stretch sensors [49;50]

or vagal afferent mechanoreceptors [51;52]. However, further evidence is required to support these findings.

(32)

33

1.3. Gastrointestinal stromal tumors (GIST)

Shortly after identification of KIT as marker for ICC, mutations in the KIT receptor were reported in GIST [25;53;54]. Based on genetic and morphologic similarities it was shown that GIST arise from ICC or an ICC precursor cell. The ability to delineate GIST as a particular type of mesenchymal cells based on KIT-ir and the possibility to inhibit KIT activation by relatively specific compounds have revolutionized GIST diagnosis and therapeutics.

1.3.1. Oncogenic mutations in GIST

Oncogenic gain-of-function mutations of KIT were the first described and are the most prevalent [25]. Afterwards, activating mutations in PDGFRA were also found, as an alternative pathogenetic event in GIST [55;56]. A number of studies identifying of KIT and/or PDGFA mutations in human cancers have been published [57-59]. The KIT and PDGFRA genes are located in chromosome 4q12 [60]. Both encode type III receptor tyrosine kinases sharing closely related structural features(see above §1.1). Oncogenic mutations in KIT and PDGFRA lead to the constitutive (ligand-independent) activation of the receptor and downstream signaling pathways (see above §1.1.1), which causes alterations in cell cycle, protein translation, metabolism and apoptosis [61].

Approximately 85% of GIST harbor oncogenic KIT mutations and 7% contain oncogenic PDGFRA mutations. The majority (70%) of KIT mutations involve the JM domain (exon 11).

These are intragenic deletion and insertion mutations that do not affect the reading frame, or missense mutations. Tandem repeat mutations are infrequently seen in the distal part of KIT exon 11. Alterations in extracellular domain (exon 9) harbor about 9% of cases. GIST with an exon 9 mutation (insertion of six base pairs) frequently arise in the small intestine and are high- risk tumors. Primary KIT mutations (mostly point mutations) can also occur in exon 13 (TK 1) and exon 17 (TK 2) (2%) [62] (Fig. 1.8)[63].

(33)

34

Exon 9 (~ 9%)

Exon 11 (~ 70%) Exon 13 (~ 1%)

Exon 17 (~ 1%)

JM TM

TK1 EC

TK2

Exon 12 (<1%) Exon 14 (< 1%)

Exon 18 (~ 8%)

KIT PDGFRA

Exon 9 (~ 9%)

Exon 11 (~ 70%) Exon 13 (~ 1%)

Exon 17 (~ 1%)

JM TM

TK1 EC

TK2

Exon 12 (<1%) Exon 14 (< 1%)

Exon 18 (~ 8%)

KIT PDGFRA

Figure 1.8. Schematic structure of KIT and PDGFRA receptor tyrosine kinases and distribution of oncogenic mutations in GIST.

The percentage of mutations in KIT/PDGFRA exons in human GIST is given under parenthesis.

Abbreviations: extracellular domain (EC), juxtamembrane (JM) domain, tyrosine kinase domains 1 and 2 (TK1, 2)

PDGFRA mutations involve mainly (8%) exon 18 (TK2) and rarely exon 14 (TK1) (< 1%) or exon 12 (JM) (< 1%) (Fig. 1.8). Most GIST with PDGFRA mutations occur primarily in the stomach and are often associated with a epitheloid phenotype and KIT-negative immunoreactivity [62]. KIT and PDGFRA oncogenic mutations are essentially mutually exclusive. It is likely that these tumours derive from different precursors. PDGFRA and KIT receptors are coexpressed in the same precursor cells in the murine gut, yet they could differentiate into different cell types, namely: the KIT+ ICC and the KIT - / PDGFRA+

fibroblast-like cells [64]. Several studies using immunohistochemistry have shown that PDGFRA often co-localizes with markers of fibroblasts or fibroblast-like cells such as CD34 [65;66], SK3 [67;68] and vimentin [69].

In roughly 10% of GIST, no mutations in the “hot spots” are found even if they are KIT/PDGFRA positive [70-72]. They are generally ranked as Wild Type (WT), which may simply reflect limitations of the mutation analysis. Recently, mutations in proto-oncogene BRAF (BRAF V600E) were identified in 13% of adult WT GIST [73] and mutations in succinate

(34)

35

dehydrogenase (SDH) subunits B, C (SDHB/C) were reported in 12% of patients with WT GIST [74]. It is very likely that other new mutations will be discovered in the near future in WT GIST.

Nowadays, the problesm encountered in GIST management are mainly caused by secondary RTK mutations which are often developed during chemothery and lead to resistance to the treatment. Secondary KIT kinase mutations are nonrandomly distributed single nucleotide substitutions affecting codons in the ATP binding pocket (exons 13 and 14) and the kinase activation loop (exon 17 and 18). The mechanisms of secondary RTK mutations and the quest for more effective therapy thus represent important fields of investigation in GIST [75].

1.3.2. GIST epidemiology

The exact incidence of GIST is hard to determine since GIST as a separate diagnosis has been established quite recently. Recent population-based studies, performed in different countries, vary in the incidence estimation. Even though reported incidences of GIST are low (from 10 to 20 cases per million of inhabitants per year) [62;63] the prevalence of GIST is higher, since many patients live with the disease unnoticed for many years. GIST constitute between 0.1% and 1% of all gastrointestinal malignant tumours and are the most common mesenchymal tumor of the gastrointestinal tract. They usually present in older adults, with the median age around 60 years, with a slight male predominance. Rarely (1-2%) pediatric GIST cases also occur. These are associated with a marked female predominance, preferentially located in the stomach and are considered as a separate clinicopathologic diagnosis. Sporadic GIST are most common, but families with germ-line KIT mutations in GIST have been described. There is an increased tendency for GIST to develop in patients with neurofibromatosis 1 (NF1) [76;77] andCarney-Stratakis syndrome [78;79]. NF1-GIST patients are on average 10 years younger than patients with common somatic disease. These GIST usually exhibit a very low mitotic index and express KIT, usually in the absence of KIT and PDGFRA mutations [62;63]. Carney-Stratakis syndrome is caused by inactivating germline mutations in SDHB, -C, or –D [80;81] predisposing affected individuals to GIST,

(35)

36

paraganglioma and pulmonary chondroma [78;79]. The clinical features include occurrence at a young age, female predilection, tumor multifocality, slow growth, frequent metastasis, lack of response to imatinib treatment and sometimes fatal outcome. In addition, this subset of GIST occurs mainly in the gastric antrum, shows predominantly epithelioid morphology and lacks KIT/PDGFRA mutations [62;63].

GIST may arise anywhere in the gut wall, however most of them occur in the stomach (60 - 70%) or small intestine (22%). They are found less commonly in the colon and rectum (8%) or other sites (10%). Extra-gastrointestinal stromal tumors (EGIST) were reported in mesentery, omentum, retroperitoneum, liver, gall bladder, urinary bladder and vagina. Most of the GIST of the stomach are located in the gastric body (40%), followed by the gastric antrum (25%) and the pylorus (20%). Around 60% of them are submucosal, 30% are subserosal and the remaining 10% are intramural [62;63].

GIST are frequently asymptomatic and when they produce symptoms, they are usually not specific, which hinders their early detection. For this reason many cases (50-60%) are discovered at a locally advanced stage or with distant metastasis. Occasionally, they are also detected incidentally during a workup for an unrelated condition. The most frequent symptoms are: abdominal pain of variable duration, gastrointestinal bleeding, anaemia due to occult blood loss, abdominal mass and loss of weight. Metastases occur mostly in the liver and abdominal cavity. Although they usually do not metastasize to lymph nodes, they can spread to lungs and bones in advanced stage [62;63].

1.3.3. GIST immunohistochemical markers

The immunohistochemical demonstration of KIT immunoreactivity (-ir) is is currently regarded as essential for GIST diagnostic [82] (Fig. 1.9). Nevertheless, 15% of GIST show low or undetectable level of KIT expression. GIST which fail to react with KIT antibodies may remain undiagnosed, however they might still respond to the therapy. These are mainly KIT WT cases or GIST harboring mutations in PDGFRA [83;84].

(36)

37

Other commonly used, but less sensitive and specific, markers used for diagnosis are CD34, h- caldesmon and Smooth Muscle Actin (SMA). CD34 is expressed in approximately 80% of gastric tumors, whereas h-caldesmon is expressed in 60% of GIST and SMA in 30%. Further S- 100, desmin and cytokeratin are rarely expressed in GIST [85-88]. Recently, Discovered on GIST-1 (DOG1), Protein kinase C theta (PKC theta) and Carbonic anhydrase II (CAII) were proposed as diagnostic markers especially in KIT negative GIST cases [89-92]. These genes are also expressed in normal KIT-ir ICC [89;91;93] and belong to the gene expression profile of Kit-ir ICC in the mouse small intestine [94].

Usually, Formalin Fixed Paraffin Embedded (FFPE) tissue is used for immunolabeling.

However, commercially available antibodies often do not show reproducible immunohistochemical results, or show high background staining and therefore have limited diagnostic utility [95]. Thus, KIT-negative GIST still remains a diagnostic challenge [62].

Figure 1.9. Kit-ir in normal human colon and in GIST (Scale bar: 20 microns.)

(37)

38

1.3.4. Therapeutic targets of GIST

Targeting oncogenic KIT and PDGFRA by receptor Tyrosin Kinase Inhibitors (TKI) like imatinib mesilate (a.k.a. STI571) and sunitinib malate (a.k.a. SU11248), are currently the only treatment for inoperable or metastatic GIST. Imatinib mesilate was originally identified in a screen for PDGFR inhibitors [96] and was first clinically used to target the BCR-ABL fusion kinase in patients with Chronic Myeloid Leukemia (CML). It was approved by the FDA for this indication in 2000. In the same year, the first GIST patient was treated with imatinib and the drug gained FDA approval for GIST in 2002 [97;98]. GIST emerged as the paradigm for successful targeted therapy, since up to 85% of patients that receive the drug achieve disease control. Median overall survival for patients with metastatic GIST raised from 19 months - in the pre-imatinib era - to 50 months in treated patients [99-101].However, primary resistance to imatinib has been demonstrated in GIST harboring KIT exon 11 mutation (5%), as well as in GIST with KIT exon 9 mutation (16%) and in the KIT WT GIST (23%). Patients with PDGFRA mutation of exon 18 (D842V) have been shown to be resistant to imatinib as well [62;102].

After imatinib failure, patients are treated with sunitinib, a second-line GIST therapy approved by the FDA in 2006 [103;104]. Sunitinib is a multikinase inhibitor that targets KIT, PDGFRs, VEGFRs 1-3, FLT3 and RET [105]. The response rate to sunitinib was 65% (7% partial response, 58% stable disease) in a phase III placebo-controlled clinical trial [106].

Moreover, it has been shown that population of GIST cells may enter quiescence after imatinib or sunitinib treatment. Quiescent cells remain metabolically active, but are withdrawn from the cell division cycle which renders them intrinsically resistant to numerous chemotherapeutic agents [107]. Hence causing refractory disease and/or relapse, if they harbor changes associated with resistance. GIST patients remain on imatinib or sunitinib for a long period of time.

Noteworthy, 44-67% GIST patient develop secondary KIT mutations which are resistant to commonly used TKI therapies and relapse within 2 years [75]. Sunitinib has been shown to be effective against secondary mutations located in the ATP binding pocket (exon 13 and 14) but not against mutations in the kinase activation loop (exon 17 and 18) [62;75].

(38)

39

1.3.5. Novel putative markers/targets for GIST

Clinical results mentioned above (see §1.4.3/4) make clear that development of new diagnostic and therapeutic strategies for GIST management remains essential. New, improved KIT kinase inhibitors with higher affinity and with a more favorable activity spectrum (e.g.

Masitinib[108], Nilotinib[109;110], Sorafenib[111], etc) are currently being developed or have already entered clinical trials.

In addition to the classical direct interaction with the ATP-binding pocket of KIT or PDGFRA and subsequent inhibition of its kinase activity, it may be possible to target GIST through several unrelated mechanisms. Compounds targeting RTK downstream pathways may be especially beneficial for combination therapy, either with KIT/PDGFRA inhibitors or with each other. A number of PI3K inhibitors, AKT inhibitors, mTOR inhibitors and RAS/ERK inhibitors are currently in clinical trials [112]. Other emerging targets are histone deacetylases (HDAC) and the ubiquitin-proteasome machinery. Inhibiting HDAC promotes chromatin relaxation, leading to increased transcription of cell cycle inhibitory genes and therapeutic effect. HDAC inhibitors have recently shown promising results in preclinical model of GIST and are currently undergoing phase I clinical trials. Proteasome inhibitor bortezomib can effectively enhance stability of various regulatory proteins and induce cell death in imatinib-sensitive as well as imatinib-resistant GIST cell lines and may have as well potentially beneficial effect in GIST patients [113]. The above mentioned targets are however rather unspecific therapeutic approaches, not designed specifically for GIST.

Several microarray studies searching for GIST specific markers/targets have been performed, comparing different soft tissue tumors [114;115], different GIST mutation status and/or anatomic sites [116-118] or imatinib treated and untreated GIST samples [119]. Nevertheless, all recently reported, new specific diagnostic markers and/or therapeutic targets (e.g. DOG1, [92;93], PKC theta [89;90], CAII [91]) for GIST are also expressed in normal human KIT-ir ICC and belong to the gene expression profile of Kit-ir ICC in the mouse small intestine [94].

(39)

40

Their presence in GIST thus likely reflects just ICC origin in the GIST tumors and their targeting is thus not optimalas normal ICC will also be hit.

The present study was designed to identify novel GIST markers/targets which would not be expressed in the normal ICC. We have used in vivo Kit^K641E GIST murine model as well as in vitro systems: murine Ba/F3 and human GIST882 cell lines, which are described below.

1.3.6. GIST murine models

Murine GIST models provide a powerful approach to further understand oncogenic KIT signaling and search for potential molecular targets. Furthermore, such models may be useful for the preclinical testing of new therapeutic strategies. Murine GIST models retain histology, biology and genetic characteristics similar to human GIST.

Three knock-in in vivo mouse models of human GIST with KIT mutation have been generated:

Kit-Asp818Tyr [120], Kit-del-Val558 [121] and Kit-Lys641Glu [122]. In Kit^delV558 heterozygous mice patchy hyperplasia of Kit-positive cells is evident within the myenteric plexus of the entire GI tract. Kit-Asp818Tyr and Kit-Lys641Glu models appear quite similar, with a diffuse hyperplasia of Kit+ cells, more marked in homozygous animals and the development of GIST mainly in the antrum and cecum [120-122].

1.3.6.1. Kit ^K641 GIST mouse model

In this study we used Kit^K641E mouse GIST model, as it was available to us on a collaborative base with Dr. Brian Rubin; Anatomic Pathology and Molecular Genetics, Cleveland Clinic, Cleveland, OH. KIT mutation in position 642 in exon 13 was originally identified in sporadic GIST [123]. Later a germ line mutation KIT 642 has been reported in some familial GIST patients [124;125]. This mutation affects the catalytic domain I of KIT, resulting in a Lys-to-Glu substitution at codon 642 of human KIT. KIT K642E mutation is associated with hyperplasia of the ICC layer and GIST formation [125]. Transgenic mice harboring Kit K641E - the murine homolog of the human mutation KIT K642E - have been

(40)

41

generated by a knock-in gene targeting strategy. Kit^K641E mutant mice provide an in vivo GIST model, presenting with massive hyperplasia of Kit+ cells, especially in the antrum and cecum, recapitulating the human GIST with KIT K642E mutation [122].

Figure 1.10. Kit/Actin immunoreactivity in Kit^K641E mouse model (S. Ralea and J.M. Vanderwinden, unpublished results)

Kit-ir is significantly elevated in Kit^K641E mutant animals. In P14 KitK641E/K641E

homozygous mice longitudinal muscle layer is replaced by hyperplasic layer of Kit+ cell. There are no ICC cells in the circular muscle layer. In P14 and adult heterozygous Kit^WT/K641E, ICC are present in both the circular and longitudinal muscle layers and are often forming Kit+ clusters.

Whereas Kit^WT/K641E heterozygous mice are viable and asymptomatic up to advanced age in our colony, most KitK641E/K641E

homozygous mice died before weaning i.e. around 3 weeks after birth. In the antrum of P14 KitK641E/K641E

homozygous mice, the whole longitudinal smooth muscle cell layer is virtually replaced by a hyperplasic layer of Kit+ cells. There are no Kit+

(41)

42

ICC in the circular muscle layer. In heterozygous P14 animals, ICC are present in both SM and CM and are often forming Kit+ clusters. Heterozygous adult animals exhibit diffuse hyperplasia of Kit+ cells in the gut (Fig. 1.10).

1.3.7. Human GIST cell lines

Several human GIST cell lines have been reported. GIST430 and GIST48 were established from GIST that had progressed during imatinib therapy after initial clinical response. These cell lines exhibit moderate sensitivity to imatinib and contain heterozygous primary KIT exon 11 mutations, accompanied by secondary imatinib-resistance mutations in exons 13 and 17, respectively [126]. GIST62 and GIST-T1 were also derived from a KIT+

GIST with a KIT exon 11 in-frame mutation and 57-bp deletion in exon 11 respectively [126;127]. The GIST62 cell line (despite retaining the activating KIT mutation in all cells) expresses virtually undetectable levels of KIT transcript and protein [126]. All these cell lines are however instable and unpractical to use (Rubin BP, personal communication).

The sole homozygous, stable GIST cell line, broadly used for in vitro studies is GIST882.

GIST882 was established from an untreated, human GIST with a homozygous missense K642E mutation in KIT exon 13.Imatinib potently suppressed proliferation and induced apoptosis in this cell lines [128]. The GIST882 cell line was kindly provided to us by Dr. Jonathan A.

Fletcher; Harvard Medical School, Boston, MA.

1.3.8. Ba/F3 cells: model of KIT signaling pathways

Ba/F3 cells - a murine interleukin-3 (IL3) dependent pro-B cell line - were used to model the KIT signaling pathway in this study [125;129]. The cellular context in lymphoid cells is presumably very different from mesenchymal ICC and this tool, albeit valuable for studying signaling pathways downstream of KIT, is by no means a replica of the functions of KIT+ ICC in situ. Murine Kit^WT, Kit^K641E, Kit^del559and Kit^D814E were stably expressed in Ba/F3 cells

(42)

[125;129]. Ba/F3 Kit^WT cells require SCF stimulation for IL3 independent growth. Kit receptor is phosphorylated after SCF binding leading to activati

BaF3 expressing oncogenic Kit to grow autonomously in minimal is constitutively phosphorylated and d

receptor phosphorylation could be inhibited with TKI i.e. imatininb (Fig. 1.11). The ability to modulate activation of Kit receptor in BaF3 cells could thus provide an interesting

signaling model [125;129].

Figure 1.11. Level of Kit-ir and p A: At basal state, 24.5% of Ba/F3 with SCF, the percentage of Kit/p B: In Kit oncogenic mutants, Kit/p Ba/F3-Kit^K641E, Ba/F3-Kit^del559 and Ba/F3 C: Inhibition of Kit phosporylation by

43

cells require SCF stimulation for IL3 independent growth. Kit receptor is phosphorylated after SCF binding leading to activation of downstream signaling.

Kit mutants, affecting different domains in the Kit receptor

autonomously in minimal medium in absence of IL3 or SCF. Kit receptor in these cells is constitutively phosphorylated and downstream Kit pathways are activated. Kit oncogenic receptor phosphorylation could be inhibited with TKI i.e. imatininb (Fig. 1.11). The ability to modulate activation of Kit receptor in BaF3 cells could thus provide an interesting

ir and p-Tyr-ir in Ba/F3 cells expressing Kit WT and Kit oncogenes.

: At basal state, 24.5% of Ba/F3-Kit^WTcells were Kit/p-Tyr double positive. After acute stimulation with SCF, the percentage of Kit/p-Tyr positive cells surged to 56.8%.

Kit/p-Tyr double positive cells represent 69.2%, 61.8% and 49.7% in and Ba/F3-Kit^D814E, respectively.

: Inhibition of Kit phosporylation by imatinib mesylate (STI571) in Kit^K641E oncogeni

cells require SCF stimulation for IL3 independent growth. Kit^WT on of downstream signaling.

mutants, affecting different domains in the Kit receptor, are able . Kit receptor in these cells pathways are activated. Kit oncogenic receptor phosphorylation could be inhibited with TKI i.e. imatininb (Fig. 1.11). The ability to modulate activation of Kit receptor in BaF3 cells could thus provide an interesting in vitro

Ba/F3 cells expressing Kit WT and Kit oncogenes.

Tyr double positive. After acute stimulation 69.2%, 61.8% and 49.7% in

oncogenic mutant.

(43)

44

GIST epitomize a paradigm for molecular targeted therapy and lot of studies were performed seeking for better treatments. We designed here an original strategy to identify new GIST markers/therapeutic targets. A detailed framework of the study is described in the following chapter (chapter 2). Three parts of this study have already been published in peer review journals and are presented in chapters 3, 5 and 6. We are currently preparing another manuscript (chapter 4) for publication. All the results are discussed and the perspectives are outlined in chapter 7.