Anaplastic Lymphoma Kinase mutations and

downstream signalling

Christina Schönherr

Department of Molecular Biology Umeå University Umeå 2012

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Copyright©Christina Schönherr ISBN: 978-91-7459-387-7 Cover front: Christina Schönherr; Cover back: PC12 cells transfected with the ALK gain-of-function mutant hALKF1174S (co-transfected with GFP) give rise to neurite outgrowth. Picture acquired by YasuoYamazaki. Elektronisk version tillgänglig på http://umu.diva-portal.org/ Printed by: Department of Chemistry Printing Service, Umeå University Umeå, Sweden 2012

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To my parents

The capacity to blunder slightly is the real marvel of DNA. Without this special attribute, we would still be anaerobic bacteria and there would be no music. Lewis Thomas

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Table of Contents

TABLE OF CONTENTS 2 ABSTRACT 5 PAPERS INCLUDED IN THIS THESIS 7 ABBREVIATIONS 8 INTRODUCTION 11 1 The Tyrosine Kinase superfamily 11 1.1 Regulation of the activity of Receptor tyrosine kinases 11 2 The RTK Anaplastic Lymphoma Kinase 14 2.1 ALK structure 14 2.2 ALK ligands, signalling and function 16

2.2.1 Drosophila melanogaster ALK 16 2.2.2 Caenorhabditis elegans ALK 18 2.2.3 Danio rerio ALK 18 2.2.4 Mammalian ALK 18

2.3 Oncogenic ALK signalling 20 3 ALK in diseases 23 3.1 ALK translocations 23

3.1.1 Anaplastic Large Cell Lymphoma (ALCL) 23 3.1.2 Inflammatory Myofibroblastic Tumour (IMT) 24 3.1.3 Non-small cell lung cancer (NSCLC) 24 3.1.4 Diffuse large B-cell lymphoma (DLBCL) 25 3.1.5 Renal cell carcinoma 25

3.2 ALK overexpression 26 3.3 Point mutations of ALK 26

3.3.1 Neuroblastoma 26 3.3.2 Genetic hallmarks of neuroblastoma 27 3.3.3 ALK point mutations in neuroblastoma 28 3.3.4 ALK point mutations in other cancers 31

4 Treatments for ALK-positive carcinomas 32 4.1 Kinase inhibitors 33

4.1.1 ALK-specific tyrosine kinase inhibitors 33 4.2 Other approaches to inhibit ALK activity 36 5 The Ras superfamily of small GTPases 37 5.1 Rap1 38 5.2 Rap1 specific regulators 39

5.2.1 Rap1 specific GEFs 39 5.2.2 Rap1 specific GAPs 39

AIMS 41 1 Overall aim 41 2 Specific aims 41

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RESULTS AND DISCUSSION 42 1 Article I: “Anaplastic lymphoma kinase activates the small GTPase

Rap1 via the Rap1-specific GEF C3G in both neuroblastoma and PC12 cells.” 42

1.1 Stimulated ALK activates Rap1 which leads to neurite outgrowth in PC12 cells 42

1.2 Activation of Rap1 downstream of ALK occurs via the Rap1-specific GEF C3G 43

1.3 Rap1 activity is involved in cell proliferation of neuroblastoma cell lines 44 2 Article II: “Appearance of the novel activating F1174S ALK

mutation in neuroblastoma correlates with aggressive tumor progression and unresponsiveness to therapy.” 46

2.1 The ALKF1174S mutant is a ligand-independent gain-of-function mutation and has transforming potential 46

2.2 Ectopic expression of ALKF1174S in the Drosophila eye causes the rough eye phenotype 47

3 Article III: “Activating ALK mutations found in neuroblastoma are inhibited by Crizotinib and NVP-TAE684.” 48

3.1 ALK mutations identified in neuroblastoma are ligand-independent gain-of-function mutations and can be blocked by NVP-TAE684 and crizotinib with different IC50 48

3.2 Ectopic expression of ALK mutants in the Drosophila eye causes the rough eye phenotype 50

4 Article IV: “The Neuroblastoma ALK (I1250T) Mutation is a Kinase-Dead RTK In Vitro and In Vivo.” 52

4.1 The ALKI1250T mutant is not constitutively active in cell culture systems 52 4.2 The ALKI1250T mutant is suggested to act as a dominant-negative receptor 53 4.3 Ectopic expression of ALKI1250T in the Drosophila eye does not cause the

rough eye phenotype 53 4.4 Why is the ALKI1250T mutant inactive? 53 5 Article V: “Anaplastic Lymphoma Kinase (ALK) regulates initiation

of transcription of MYCN in neuroblastoma cells.” 55 5.1 ALK regulates the MYCN promoter in PC12 cells and human

neuroblastoma cell lines 55 5.2 Abrogation of ALK activity results in decreased MYCN mRNA and

proliferation of neuroblastoma cell lines 56 5.3 ALK activity regulates MYCN protein expression 56 5.4 ALK and MYCN co-operate in transforming NIH3T3 cells 58 SUMMARY OF THE MAIN FINDINGS OF THIS THESIS 59 FUTURE PERSPECTIVES 60 ACKNOWLEDGEMENTS 62 REFERENCES 63

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Abstract

The oncogene Anaplastic Lymphoma Kinase (ALK) is a Receptor Tyrosine Kinase (RTK) and was initially discovered as the fusion protein NPM (nucleophosmin)-ALK in a subset of Anaplastic Large Cell Lymphomas (ALCL). Since then more fusion proteins have been identified in a variety of cancers. Further, overexpression of ALK due to gene amplification has been observed in many malignancies, amongst others neuroblastoma, a pediatric cancer. Lately, activating point mutations in the kinase domain of ALK have been described in neuroblastoma patients and neuroblastoma cell lines. In contrast, the physiological function of ALK is still unclear, but ALK is suggested to play a role in the normal development and function of the nervous system.

By employing cell culture based approaches, including a tetracycline-inducible PC12 cell system and the in vivo D. melanogaster model system, we aimed to analyze the downstream signalling of ALK and its role in neuroblastoma. First, we wished to analyze whether ALK is able to activate the small GTPase Rap1 contributing to differentiation/proliferation processes. Activated ALK recruits a complex of the GEF C3G and CrkL and activates C3G by tyrosine phosphorylation. This activated complex is able to activate Rap1 resulting either in neurite outgrowth in PC12 cells or proliferation of neuroblastoma cells suggesting a potential role in the oncogenesis of neuroblastoma driven by gain-of-function mutant ALK. Next, we could show that seven investigated ALK mutations with a high probability of being oncogenic (G1128A, I1171N, F1174L, F1174S, R1192P, F1245C and R1275Q), are true gain-of-function mutations, respond differently to ALK inhibitors and have different transforming ability. Especially the F1174S mutation correlates with aggressive disease development. However, the assumed active germ line mutation I1250T is in fact a kinase dead mutation and suggested to act as a dominant-negative receptor. Finally, ALK mutations are most frequently observed in MYCN amplified tumours correlating with a poor clinical outcome. Active ALK regulates mainly the initiation of MYCN transcription in human neuroblastoma cell lines. Further, ALK gain-of-function mutants and MYCN synergize in transforming NIH3T3 cells.

Overall, somatic mutations appear to be more aggressive than germ line mutations, implying a different impact on neuroblastoma. Further, successful application of ALK inhibitors suggests a promising future for the development of patient-specific treatments for neuroblastoma patients.

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Papers included in this thesis

This thesis is based on the following publications which will be referred to by their roman numerals (I – V). All publications are reproduced with the permission from the journal publishers.

Article I Schonherr C, Yang HL, Vigny M, Palmer RH, Hallberg B. Anaplastic lymphoma kinase activates the small GTPase Rap1 via the Rap1-specific GEF C3G in both neuroblastoma and PC12 cells. Oncogene. 2010 May 13;29(19):2817-30.

Article II Martinsson T, Eriksson T, Abrahamsson J, Caren H, Hansson M, Kogner P, Kamaraj S, Schonherr C, Weinmar J, Ruuth K, Palmer RH, Hallberg B. Appearance of the novel activating F1174S ALK mutation in neuroblastoma correlates with aggressive tumor progression and unresponsiveness to therapy. Cancer Res. 2010 Jan 1;71(1):98-105.

Article III Schonherr C, Ruuth K, Yamazaki Y, Eriksson T, Christensen J, Palmer RH, Hallberg B. Activating ALK mutations found in neuroblastoma are inhibited by Crizotinib and NVP-TAE684. Biochem J. 2011 Dec 15;440(3):405-13.

Article IV Schonherr C, Ruuth K, Eriksson T, Yamazaki Y, Ottmann C, Combaret V, Vigny M, Kamaraj S, Palmer RH, Hallberg B. The Neuroblastoma ALK (I1250T) Mutation Is a Kinase-Dead RTK In Vitro and In Vivo. Transl Oncol. 2011 Aug;4(4):258-65.

Article V Schonherr C, Ruuth K, Kamaraj S, Wang CL, Yang HL, Combaret V, Djos A, Martinsson T, Christensen J, Palmer RH, Hallberg B. Anaplastic Lymphoma Kinase (ALK) regulates initiation of transcription of MYCN in neuroblastoma cells. Oncogene. 2012 Jan 30.

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Abbreviations

ABL V-abl Abelson murine leukemia viral oncogene homolog 1 Akt AKR Thymoma ALCL Anaplastic Large Cell Lymphoma ALK Anaplastic Lymphoma Kinase ALO17 ALK lymphoma oligomerization partner on chromosome 17 Arf ADP-ribosylation factor ATC Anaplastic Thyroid Carcinoma ATIC 5-aminoimidazole-4-carboxamide ribonucleotide

formyltransferase/IMP cyclohydrolase ATP Adenosinetriphosphate BCR Breakpoint Cluster Region C3G Crk SH3 domain-binding Guanine nucleotide exchange factor CAMTA1 Calmodulin-binding transcription activator 1 CARS Cysteinyl-tRNA synthetase CLTC1 Clathrin heavy chain-like 1 CML Chronic Myeloid Leukemia CNS Central Nervous System dALK Drosophila melanogaster Anaplastic Lymphoma Kinase DLBCL Diffuse large B-cell lymphoma Dpp Decapentaplegic Duf Dumbfounded EGFR Epidermal Growth Factor Receptor EML4 Echinoderm microtubule-associated protein like 4 ERK Extracellular signal Regulated Kinase FDA Food and Drug Administration FGF Fibroblast Growth Factor FGFR Fibroblast Growth Factor Receptor Flt3 Fms-like Tyrosine Kinase Receptor-3 FOXO3a Forkhead box O3a FRS2 Fibroblast Growth Factor Receptor Substrate 2 GAP GTPase activating protein GEF Guanine nucleotide exchange factor Grb2 Growth Factor Receptor-bound protein 2 GSK3β Glycogen Synthase Kinase 3β GTPase Guanosine triphosphatase hALK human Anaplastic Lymphoma Kinase Hen-1 Hesitation-1 HER2 Human Epidermal Growth Factor Receptor 2 Hsp90 Heatshockprotein 90 hTERT human telomerase reverse transcriptase

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IgG Immunoglobulin G IMT Inflammatory Myofibroblastic Tumour IR Insulin Receptor IRS-1 Insulin Receptor Substrate 1 JAK3 Janus Kinase-3 Jeb Jelly Belly JNK c-Jun N-terminal Kinase KIF5B Kinesin family member 5B Kirre Kin of irregular chiasm LDL Low-Density Lipoprotein LRP LDL receptor related protein LTK Leukocyte Tyrosine Kinase mALK mouse Anaplastic Lymphoma Kinase MAM Meprin A-5 protein and receptor protein tyrosine phosphatase Mu MAPK Mitogen-Activated Protein Kinase MEK MAPK/ERK Kinase Miple 1 and 2 Midkine and Pleiotrophin MK Midkine MSN Moesin mTOR Mammalian Target of Rapamycin MYCN myc myelocytomatosis viral related oncogene, neuroblastoma

derived MYH9 Non-muscle myosin heavy chain NGF Nerve Growth Factor NIPA Nuclear interacting partner of ALK NPM Nucleophosmin NSCLC Non-small cell lung cancer p130Cas p130 Crk-associated substrate PC12 Pheochromocytoma cells 12 PDGFR Platelet Derived Growth Factor Receptor PI3K Phosphoinositide-3 Kinase PLCγ Phospholipase C γ pp60Src pp60 Sarcoma PPFIBP1 F polypeptide-interacting protein-binding protein 1 PTB Phosphotyrosine-binding PTK Protein Tyrosine Kinase PTN Pleiotrophin PTP Protein Tyrosine Phosphatase Rab Ras-like protein in brain Ran Ras-like nuclear RANBP2 Ran binding protein 2 Rap Ras-proximate RAS Rat sarcoma

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Rho Ras homologous RPTPβ/ζ Receptor Protein Tyrosine Phosphatase β/ζ RTK Receptor Tyrosine Kinase Sar Secretion-associated and Ras-related SCC Squamous cell carcinoma of the esophagus SCD-2 Suppressor of constitutive dauer formation SCF Stem Cell Factor SCLC Small cell lung cancer SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis SEC31L1 SEC31 homologue A (S. cerevisiae) SH2/3 Src homology 2/3 Shc Src homology 2 containing SHH Sonic hedgehog Shp1/2 SH2 domain-containing phosphatase 1/2 SQSTM1 Sequestosome 1 STAT3 and 5 Signal Transducer and Activator of Transcription 3 and 5 TFG TRK-fused gene TGFβ Transforming Growth Factor β TKD Tyrosine Kinase Domain TPM3 and 4 Tropomyosin 3 and 4 TrkA Tropomyosin receptor kinase A VASP Vasodilator-stimulated phosphoprotein VCL Vinculin 17-AAG 17-allyl-amino-demethoxygeldanamycin

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Introduction

1 The Tyrosine Kinase superfamily

In 1906 Levene identified for the first time phosphate in the protein vitellin [3]. Over the years the phosphorylation of serine, threonine or tyrosine has evolved into one of the most common post-translational protein modifications. This enzymatic reaction was first described for the serine phosphorylation of casein in 1954 [4] and is mediated by protein kinases. A couple of years later tyrosine phosphorylation was described [5], followed by the first report of a protein kinase that is able to phosphorylate tyrosine, namely pp60Src, the transforming protein of Rous sarcoma virus [6].

The human genome encodes for 518 different protein kinases which are divided into different groups [7]. One of these groups represents the class of tyrosine kinases, which have been established as key regulators in various cellular functions like proliferation, migration and differentiation. The family of tyrosine kinases is further subdivided into 58 Receptor tyrosine kinases (RTKs) and 32 non-receptor tyrosine kinases [7]. All RTKs share a common domain structure: they contain an extracellular domain comprising a ligand binding region, a transmembrane domain and finally an intracellular domain containing the usually highly conserved kinase domain [2]. Most RTKs are monomers at the cell membrane in the absence of a ligand. However, one exception is the Insulin receptor (IR), which exists as an inactive heterodimer. This heterodimer is activated by structural changes induced by ligand binding resulting in stabilization of an active dimer state [8, 9].

1.1 Regulation of the activity of Receptor tyrosine kinases Generally, activation of RTKs is mediated by ligand-induced dimerization. In

general, a ligand binds simultaneously to two receptor monomers, thereby crosslinking them which stabilizes the formation of an active RTK dimer being able to auto-phosphorylate tyrosines in the kinase domain. To date, there are four different ways of ligand-induced RTK activation (Figure 1). The first includes dimerization mediated by the ligand (Figure 1A). For example, TrkA monomers are dimerized by binding of the dimeric NGF ligand to each receptor molecule in such a way that the two TrkA molecules do not contact each other. Secondly, a ligand mediates dimer formation where the receptor molecules contact each other directly (Figure 1B). This is shown for the stem cell factor (SCF), the KIT ligand, which exists as a homodimer. Each SCF molecule binds to one receptor monomer, thereby crosslinking the two receptors. The third example is about the fibroblast growth factor receptor (FGF) which is crosslinked by a combination of bivalent ligand binding, receptor-receptor contacts and binding of accessory molecules (Figure 1C).

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Figure 1: Ways of Receptor Tyrosine Kinase dimerization. Generally a ligand (red) binds to the extracellular regions of RTKs. The dimerized RTKs undergo structural changes in conformation, leading to RTK activation. (A) A Nerve growth factor interacts with two TrkA receptors without direct contact between the receptor molecules. (B) A stem cell factor dimer crosslinks two KIT receptors which contact each other directly. (C) Two fibroblast growth factor receptors contact each other. In addiction, crosslinking occurs via binding of accessory molecules like heparin or heparin sulfate proteoglycans (white sticks) and ligand binding. (D) Two ErbB receptors dimerize directly. Ligand binding drives conformational changes resulting in stabilization and activation of the receptor dimer. (Figure adapted with permission from [2].

Two monomeric FGF ligands bind the two receptor monomers together with two heparin molecules forming a major complex. These receptor-ligand, receptor-heparin, ligand-heparin and receptor-receptor interactions result in stabilization of the FGFR dimer. The last type of receptor dimerization has been shown for epidermal growth factor receptor (EGFR) family where receptor dimerization is completely receptor mediated (Figure 1D). In the absence of the ligand the receptors are in an intramolecular autoinhibitory state. Bivalent ligand binding induces conformational changes in the receptor, resulting in stabilization and activation of the receptor dimer.

Following ligand-induced dimerization the intracellular tyrosine kinase domain (TKD) of the RTKs is activated by various mechanisms which include the release of cis-autoinhibition of the TKD (Figure 2). Roughly, the TKD is divided into an N- and a C-lobe which form a deep cleft with the active site. Some prominent motifs in the highly dynamic N-lobe include the αC-helix and the glycine-rich loop (G-loop) which is involved in ATP binding. The more rigid C-lobe provides the docking site for the substrate proteins. The catalytic loop contains a conserved HRD motif with the catalytic aspartate. The P+1 loop which is situated C-terminal of the activation loop, binds the substrate at the residue after the phosphorylation site. The activation loop harbours the DFG motif which is involved in magnesium binding and therefore important for catalysis. This activation loop contains critical tyrosines which become auto-phosphorylated upon stimulation.

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Upon ligand activation, one critical tyrosine in the activation loop of one

receptor becomes phosphorylated by its partner, followed by two additional tyrosines. Hence, this trans-phosphorylation disrupts the cis-autoinhibitory interactions and the phosphorylated activation loop changes conformation to an active state (Figure 3A) [2, 10].

Figure 2: Crystal structure of the ALK kinase domain shown in two orthogonal orientations. NT and CT: N- and C-termini of ALK. The N-terminal lobe consists of β-sheets (orange) and the αC-helix (purple). The C-terminal lobe consists of helices (blue). The glycine-rich P-loop is depicted in bright green, the hinge region between the lobes in yellow, the catalytic loop in salmon and the activation loop in red. (Figure adapted with permission from [1].

Figure 3: Ways of inhibition of the intracellular tyrosine kinase domain. The intracellular tyrosine kinase domains contain a C-lobe (light purple), N-Lobe (dark purple or yellow in the active state) and an activation loop (purple or yellow respectively). Inhibition occurs via intramolecular interactions: (A) Activation loop inhibition. (B) Juxtamembrane inhibition. (C) C-terminal tail inhibition. (Figure adapted with permission from [2].

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A further way of autoinhibition is mediated by the juxtamembrane domain as shown e.g. for Flt3 (Figure 3B). The juxtamembrane region binds to the active site of the kinase, stabilizing an inactive conformation. Phosphorylation of critical tyrosines in the juxtamembrane region disrupts the autoinhibitory state and the kinase adopts an active conformation. A third mode of autoinhibition involves the C-terminal tail of the kinase domain (Figure 3C). For instance, in Tie2 the C-terminal tail blocks substrate access to the active site. Auto-phosphorylation of tyrosines in the C-terminal tail may disrupt the autoinhibitory state and thereby result in activation of the kinase [2].

The phosphorylated tyrosines of the activated receptor serve as docking sites for signaling molecules containing Src homology-2 (SH2) and phosphotyrosine-binding (PTB) domains. Hence, receptor activation initiates the activation of various signaling pathways forming a complex signalling network. However, the RTK mediated cell signaling requires strict regulation. This process can occur either via a positive feedback mechanism (e.g. the activity of protein tyrosine phosphatases (PTPs) is temporarily blocked) or via negative feedback mechanisms abrogating RTK mediated cell signaling. This includes for instance direct activation of PTPs, transcription of negative signaling regulators or endocytosis of receptors.

However, despite stringent regulations aberrant activity of tyrosine kinases has been reported in many diseases including cancer [11, 12]. To date, more than 50% of the known RTKs have been implicated in oncogenic malignancies, either by autocrine activation, chromosomal translocations (where the RTK kinase domain is fused to a protein functioning as a dimerizer), overexpression or gain-of-function mutations [2, 11].

2 The RTK Anaplastic Lymphoma Kinase

Anaplastic Lymphoma Kinase (ALK) was described for the first time in 1994 when a novel tyrosine phophorylated protein was found in Anaplastic Large Cell Lymphoma [13, 14]. This protein was identified as the chimeric protein NPM-ALK generated by a translocation between the chromosomes (2;5)(p23;q35). In NPM-ALK, the N-terminal part of nucleophosmin (NPM) is fused to the kinase domain of the novel tyrosine kinase, which received the name ALK after the disease where it was reported for the first time [13].

2.1 ALK structure From the initial discovery in 1994 it took three years until the full length

ALK was identified independently by two groups [15, 16]. Like all RTKs ALK contains a ligand binding extracellular domain, a transmembrane domain and a cytosolic region containing the kinase domain (Figure 4A).

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Together with Leukocyte Tyrosine Kinase (LTK) ALK forms a subgroup

within the Insulin Receptor (IR) superfamily [15, 16]. Human ALK is built up of 1620 amino acids, resulting in a protein of approximately 180 kDa. In SDS-PAGE however, ALK can be detected at 220 kDa due to posttranslational modifications like glycosylations [15, 16]. Mouse ALK consists of 1621 amino acids [15, 16], D. melanogaster ALK of 1701 [17] and C. elegans ALK of 1421 amino acids [18, 19]. The existence of an N-terminal signal peptide is responsible for the transport of ALK to the cell membrane. The extracellular part of ALK contains several domains: two MAM (Meprin A-5 protein and receptor protein tyrosine phosphatase Mu) domains, one LDLa (low density lipoprotein receptor class A) domain and a glycine rich domain (Figure 4A) [15-17, 20]. However, one can only speculate about the functions of those domains. As the LDLa domain mediates binding between LDL receptor and LDL, it might play a role in ALK ligand binding [21, 22]. MAM domains are most likely involved in cell-cell interactions [23] although their role for

Figure 4: Domain structure of ALK and potential tyrosine phosphorylation sites. (A) The extracellular region of ALK contains two Meprin A-5 protein and receptor protein tyrosine phosphatase Mu (MAM) domains, one low density lipoprotein receptor class A (LDL) domain and a glycine rich (G-rich) domain. A transmembrane domain (TMD) connects the extracellular region with the intracellular region containing the protein tyrosine kinase (PTK) domain. The closest family member, Leukocyte Tyrosine Kinase (LTK) is shown with the equivalent regions. In NPM-ALK the PTK is fused to the N-terminal part of nucleophosmin (NPM). (B) The intracellular region of human and mouse ALK comprises the PTK and contains potential tyrosine phosphorylation sites. Tyrosines within the activation loop are indicated in bold. Worth mentioning is the tyrosine 1604 in human ALK that is not present in mouse ALK.

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ALK function is unclear, as is the glycine rich domain. In D. melanogaster however, both the MAM and the glycine rich domains seem to be important for dALK activity, as point mutations in the MAM domain as well as in the glycine rich domain (replacing glycine by acidic amino acids) result in inactive dALK [24].

The ALK domains are conserved across species, with highest similarity in the kinase domain. Although mouse and human ALK are highly similar (87% at protein level) one significant difference has to be mentioned: that is the extra tyrosine at position 1604 which exists only in human ALK and plays most likely a role in tumor progression (Figure 4B) [25]. The activation loop within the kinase domain of ALK contains the auto-phosphorylation motif YxxxYY which exists also in the IR. However in contrast to the IR, where the second tyrosine is first phosphorylated followed by the first and then the third, the order of phosphorylation in ALK is different: here the phosphorylation of the first tyrosine of the YxxxYY motif is predominant in the process of autoactivation. This might be due to the RAS triplet between the tyrosines (Y’RAS’YY) in contrast to IR (Y’ETD’YY) [26, 27].

Recently, the crystal structure of the ALK catalytic domain in complex with ATP competitive inhibitors has been published, providing a great opportunity to investigate structural functional relationships [1, 28].

2.2 ALK ligands, signalling and function

2.2.1 Drosophila melanogaster ALK In D. melanogaster the physiological function has been intensely studied. For

the first time D. melanogaster ALK has been described to be expressed in the developing embryonic mesoderm and central nervous system (CNS), activating the ERK pathway in vivo [17]. During early embryogenesis ALK plays an important role in the development of the visceral musculature of the gut [24]. To date, the only well established ligand for ALK is Jelly Belly (Jeb) which activates dALK in the visceral muscle founder cells, leading to stimulation of the ERK pathway (Figure 5). As a result the founder cells fuse with fusion competent myoblasts, forming the gut musculature [29-33]. As Jeb/dALK signalling is required for the specification of the founder cells, dALK mutant flies lack a functioning gut. The Jeb/dALK mediated ERK activation results in transcription of downstream targets like duf/kirre [29, 31], org [31], hand [34] and dpp [35].

The dALK ligand Jelly Belly is a protein of approximately 61 kDa containing an LDLa domain. Jeb is secreted from the somatic mesoderm and then taken up by the visceral mesoderm [36]. The binding of Jeb to dALK is most likely mediated via the LDLa domain, as Jeb proteins lacking the LDLa domain are unable to bind dALK [31]. Further, two proteins called Miple1 and Miple2 are potential ligands for dALK. Those proteins belong to the midkine/pleiotrophin family of proteins thus the names Miple1 and 2. In D. melanogaster Miple1 has been found to be expressed at high levels in the CNS, while Miple2 is strongly expressed in the developing midgut

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endoderm [37]. Still further studies are required to support the theory about being true dALK ligands.

Figure 5: Drosophila melanogaster and mammalian wild type ALK signalling. Upon ligand binding (for Drosophila ALK it is Jeb) ALK dimerizes, resulting in auto-phosphorylation and the activation of downstream signalling pathways (light grey boxes). For mammalian ALK the natural ligand is still unknown, but midkine and pleiotrophin are suggested ALK ligands. Alternative ways of ALK activation are suggested. One proposes that ALK belongs to the group of dependence receptors. In the absence of a ligand ALK possesses pro-apoptotic properties. Upon extracellular stimulation and intrinsic activation by caspase-mediated cleavage ALK has antiapoptotic activity. The other potential activating mechanism includes the receptor phosphatase RPTPβ/ζ which is inhibited by binding of pleiotrophin, resulting in the overall activation of ALK downstream signalling.

Besides the above named ERK activation mediated by dALK activity, dALK seems to play an important role in the transcription of Dpp (homolog to mammalian TGFβ) and subsequent signaling in the endoderm, resulting in the development of the embryonic endoderm [35]. Further, Jeb/dALK signalling is not only important for the gut development, but is also involved in the anterograde signalling pathway mediating neuronal circuit assembly in the visual system of the fruit fly. Lack of either Jeb or dALK results in misstargeting of R-cell axons during optic lobe maturation [38]. A recent study showed Jeb and ALK localization in developing

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synapses, where Jeb localizes to presynaptic terminals and ALK is concentrated in postsynaptic domains. This Jeb/dALK expression results in anterograde trans-synaptic signalling, important for the synaptic connectivity in the developing motor circuit [39]. Further, during nutrient restriction ALK protects especially the CNS via involvement of the PI3K/Akt pathway [40]. Depletion of ALK in the fruit fly results in increased resistance to the sedating effects of ethanol [41]. Recently, Gouzi et al., showed that dALK is upstream of neurofibromin 1, regulating body size determination and associative learning [42].

2.2.2 Caenorhabditis elegans ALK In C. elegans ALK (T10H9.2) or SCD-2 (suppressor of constitutive dauer

formation) plays a role in synapse stabilization and regulates entry into dauer stage [18, 19]. In 2004 ALK has been identified as a downstream effector of FSN-1, a novel F-box protein, mediating synapse stability [18]. Further, it has been shown that SCD-2 regulates the integration of sensory signals in interneurons [43, 44]. Interestingly, a wild C. elegans strain from a desert oasis revealed a defect in dauer response which is due to a mutation in the scd-2 gene [19]. Further this study suggests that SCD-2 modulates the TGFβ pathway.

Hen-1 has been identified as the SCD-2 ligand [45]. Like the D. melanogaster ALK ligand Jelly Belly, Hen-1 is a secreted protein with an LDL receptor repeat, regulating sensory processing and learning of the neuronal circuit. To date little information is available about the Hen-1/SCD-2 signalling, but the genetic pathway includes the adaptor protein SOC-1 and the MAPK SMA-5 [19].

2.2.3 Danio rerio ALK A study in the zebrafish Danio rerio showed that shady mutants are lacking

iridophores, mirror-like pigment cells derived from the neural crest [46]. Further this study demonstrates that the shady gene encodes the RTK Leukocyte Tyrosine Kinase (LTK). Like D. melanogaster or mammalian ALK the extracellular domain of the zebrafish LTK contains a MAM domain, in contrast to mammalian LTK, which is lacking MAM domains. Therefore D. rerio LTK seems to be closer related to ALK. As the function of mammalian LTK is to date unknown, Lopes et al., show for the first time a role for LTK in vertebrates. A natural ligand for zebrafish LTK has not been identified yet but the fact that LTK is important in the development of iridophores, which are derived from the neural crest, is of great interest as human ALK activating point mutations seem to play an important role in neuroblastoma, a disease derived from neural crest cells (see below).

2.2.4 Mammalian ALK In contrast to D. melanogaster ALK the natural ligand for mammalian ALK

remains a mystery, even about 20 years after the initial identification. Pleiotrophin (PTN) and Midkine (MK) have been reported as activating ligands for mammalian ALK [20, 47, 48]. These small heparin-binding growth factors play a role in neural

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development, survival and tumorigenesis [49, 50]. However, MK and PTN are also able to activate other receptors like RPTPβ/ζ, N-syndecan, LRP and integrins [51-56]. An alternative ALK activating mechanism by PTN involves the receptor phosphatase RPTPβ/ζ: PTN binding inhibits this phosphatase, resulting in activation of the ALK signalling cascade (Figure 5) [57]. However, to date the stimulation of ALK by PTN and MK remains controversial: while some groups proved PTN and MK mediated ALK activation, others have shown contradictory results [20, 48, 58-67]. To sum up, the quest for the natural ligand for ALK is still ongoing.

As the natural ligand for mammalian ALK remains unidentified, wild type ALK needs to be activated artificially, either by substituting the ALK extracellular domain by mouse IgG Fc domain or by activating monoclonal antibodies, resulting in the activation of the MAPK pathway (Figure 5) [64, 68-70]. Activation of the MAPK pathway occurs via the association of Shc and FRS2 with ALK and NPM-ALK respectively [64, 68, 71]. Further, ALK is phosphorylated if the extracellular domain of ALK is replaced by the extracellular part of the epidermal growth factor receptor (EGFR), which results in the activation of PLCγ and PI3K [72]. Stimulation of ALK by activating monoclonal antibodies or PTN leads to the activation of the PI3K/Akt pathway as well, resulting in increased proliferation [58, 66]. Further, Kuo et al., published data showing that MK stimulates ALK which leads to association with IRS-1 and Shc, thereby activating ALK downstream signalling [60]. Further, ALK is able to activate the small GTPase Rap1 by activating its GEF C3G in PC12 and neuroblastoma cells, which will be discussed further in Article I [73].

Currently, the physiological function of mammalian ALK is unknown. However, as ALK mRNA is expressed widely in the nervous system during mouse embryogenesis, in the developing chick central nervous system, as well as in developing dorsal root ganglia in rat, ALK is suggested to function in the development of the nervous system [15, 16, 74-78]. This suggested role is further strengthened by ALK protein expression in tissue samples of the human central nervous system [79] as well as in cell culture systems: several groups showed in in vitro cell culture studies that activated ALK induces neuronal differentiation in PC12 cells, which is mediated via several signalling pathways downstream of ALK [63, 64, 68-70, 80]. ALK’s role in the nervous system is further fortified by mouse studies. The study by Bilsland et al., shows that ALK mutant mice show enhanced basal hippocampal progenitor proliferation and dopaminergic signalling in the frontal cortex. Further, those mice performed better in object-recognition tests [81]. Weiss et al., reported that ALK mutant mice possess reduced neurogenesis, have lower anxiety levels and have improved spatial memory which is LTK dependent, indicating genetic interactions between ALK and LTK [82]. The fact, that ALK seems to play an important role in neurodevelopment suggests that ALK might be involved in neurological or mental disorders. Indeed, polymorphisms within the intracellular domain of ALK have been correlated with increased susceptibility to schizophrenia [83].

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Additionally, like dALK, mouse ALK mutants show increased ethanol tolerance indicating that those behavioral responses are evolutionary conserved [41]. An elegant mouse study by Lasek et al., showed that ALK expression is transcriptionally regulated by LMO (LIM-domain only) and ERα (estrogen receptor α), which has impact on behavioral responses to cocaine [84].

A different function for ALK is proposed by Mourali et al., who suggest that ALK belongs to the group of dependence receptors. In ALK expressing Jurkat and 13.S.1.24 rat neuroblast cells they show that in the absence of a ligand ALK possesses proapoptotic activity. However, upon extracellular stimulation as well as intrinsic activation by caspase-mediated cleavage at D1160 ALK has anti-apoptotic properties (Figure 5) [65].

2.3 Oncogenic ALK signalling As ALK was initially discovered as the fusion protein NPM-ALK most

knowledge about ALK signalling has been gained from studies on NPM-ALK. The major signalling pathways downstream of ALK include the PLCγ, the PI3K/Akt, the MAPK and the JAK/STAT pathways (Figure 6):

Via its SH2-domain, PLCγ binds to the tyrosine at position 664 on NPM-ALK, thereby phosphorylating and activating PLCγ. This activation of PLCγ by NPM-ALK results in transformation in transfected cell culture systems, contributing to the mitogenic activity of NPM-ALK [25].

NPM-ALK activates the Akt pathway via interaction with the p85 subunit of PI3K, leading to anti-apoptotic signalling, transformation and tumour growth [85-87]. Further, activation of the PI3K/Akt pathway by NPM-ALK does not only result in phosphorylation of mTOR but regulates also the transcription of FOXO3a target genes, e.g. cyclin D2, Bin-1 and p27kip1 [88-90]. Recently it has been shown that NPM-ALK mediated activation of the PI3K/Akt pathway regulates the phosphorylation of Serine 9 at GSK3β, thereby inhibiting GSK3β activity. This leads to accumulation of CDC25A and Mcl-1, resulting in enhanced oncogenesis [91].

The third major pathway downstream of NPM-ALK is the MAPK pathway. The activated receptor interacts with IRS-1, Shc and Grb2, which results in the assembly and stimulation of downstream signalling [89, 92-94].

Another pathway activated by NPM-ALK is the JAK/STAT pathway. It has been demonstrated by several groups that STAT3 is phosphorylated and thereby activated by NPM-ALK, a process which can be blocked by ALK inhibitors [95-100]. However, the exact mechanism of how NPM-ALK is able to activate STAT3 remains unclear. Some studies report an association of JAK3 with NPM-ALK and JAK3 inhibition leads to reduced STAT3 activation [99, 101, 102]. On the other hand, some data suggest that ALK binds and activates STAT3 directly involving the first tyrosine in the YxxxYY motif [27]. Regulators of the JAK/STAT pathway like Shp1, which has been shown to interact with NPM-ALK, and protein phosphatase

20

2A are abnormally expressed in ALK-positive ALCL [100, 103, 104]. The role of STAT3 in the pathogenesis of NPM-ALK positive ALCL has been confirmed, however the involvement of STAT5 in NPM-ALK mediated oncogenicity is less established. While some groups suggest an NPM-ALK mediated activation of STAT5B, which leads to apoptosis and cell-cycle arrest, others could not show STAT5 activation [99, 100, 105]. However, STAT5 might not only promote oncogenicity, but there is evidence that STAT5A might act as a tumour suppressor in ALK-positive ALCL cell lines [106].

Figure 6: Oncogenic ALK signalling. The requirement of ligand binding for downstream signalling of constitutively active ALK either by point mutations or amplification is most likely not required. While the downstream signalling of ALK translocations is fairly well investigated, in case of amplified and mutated ALK several potential downstream signalling pathways need to be confirmed (grey font).

21

Besides the above named major signalling pathways further downstream targets of NPM-ALK have been identified. NPM-ALK activates the small GTPase Cdc42 thereby controlling cell shape and proliferation of ALCL [107]. Another GTPase downstream of NPM-ALK is Rac1 which is activated by the exchange factor Vav3 [108]. Vav3 binds to Y343 of NPM-ALK via its SH2-domain and is phosphorylated. The activation of Vav3/Rac1 is involved in motility and invasion of NPM-ALK positive ALCL. Further, NPM-ALK’s transforming potential is mediated by p130 Crk-associated substrate (p130Cas) via Grb2 [109]. Also Src kinases like pp60Src are involved in NPM-ALK positive ALCL cell proliferation, while the tyrosine phosphatase Shp2 binds to NPM-ALK thereby mediating cell growth and migration [110, 111]. NIPA (nuclear interacting partner of ALK), a ubiquitin E3 ligase, binds to NPM-ALK resulting in anti-apoptotic signalling [112, 113]. NPM-ALK is also able to activate JNK, leading to lymphoma development in mice as well as cell-cycle progression and oncogenesis in ALCL cell lines [114, 115]. Further, JNK participates in the blocking of the p53 tumor suppressor pathway downstream of NPM-ALK [116]. An elegant study by Singh et al., suggested a cooperation between the Sonic Hedgehog (SHH) and the PI3K pathway downstream of NPM-ALK: here, PI3K activation by NPM-ALK regulates SHH/GLI1 signalling resulting in synergistic effects that contribute to NPM-ALK’s oncogenicity in ALCL [117]. Additional novel ALK targets and interacting proteins like Dok2, IRS1, SHC, Crk, CrkL, STAT3, VASP and ATIC were identified using proteomics-based approaches [92, 118-120]. Various ALK regulated genes, e.g. the anti-apoptotic protein BCL2A and the transcription factor C/EBPβ, were identified in screens of the transcriptomes of ALCL cell lines [121, 122]. Further, a recent report identified for the first time that serine phosphorylation of NPM-ALK is also important for ERK and JNK signalling and therefore contributes to the oncogenic potential of NPM-ALK [123].

Due to its initial discovery, NPM-ALK has been investigated most intensively. The other known fusion proteins are thought to mediate downstream signals in a similar manner, although this has not been proven for all and some differences have been reported: e.g. ATIC-ALK interacts with Grb2 and Shc, while TGF-ALK binds to Grb2, Shc and PLCγ [124, 125]. Further, KIF5B-ALK, an ALK fusion protein detected in Non-small cell lung cancer, activates STAT3 and Akt [126]. Investigation of NPM-ALK, TPM3-ALK, TFG-ALK, CLTC-ALK and ATIC-ALK revealed different activation of signalling pathways like PI3K/Akt, resulting in different transforming and tumourigenic potential [127]. One particular ALK fusion protein – EML4-ALK – has received great attention in the last couple of years, thereby increasing the knowledge of this ALK fusion protein [128, 129]. EML4-ALK possesses transforming potential [128, 129], and treatment with the ALK specific inhibitor NVP-TAE684 results in apoptosis in lung cancer cells via the ERK/BIM and STAT3/survivin signalling pathways [130]. Blocking EML4-ALK signalling by ALK specific inhibitors resulted in the development of new therapeutic treatments for lung cancer patients [131].

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However, oncogenic ALK signalling derives not only from fusion proteins, but also from ALK overexpression and activating point mutations which will be discussed below [76, 128, 132-136]. The activity of some of these mutant ALK receptors can be blocked by ALK-specific inhibitors [76, 78, 133, 134].

3 ALK in diseases

Although the physiological function of ALK in mammals is still unclear, the role of ALK in the development and onset of several diseases has been well established. In 1994 a screen from patients with Anaplastic Large Cell Lymphoma (ALCL) revealed a new fusion protein – NPM-ALK – where the N-terminal part of nucleophosmin is fused to the kinase domain of ALK as a result of a t(2;5)(p23;q35) translocation [13]. Since this initial discovery a plethora of ALK fusion proteins have been described in inflammatory myofibroblastic tumours (IMT) [137], Non-small cell lung cancer (NSCLC) [138, 139], diffuse large B-cell lymphomas (DLBCLs) [140], renal cell carcinoma [141] and squamous cell carcinoma of the esophagus (SCC) [142, 143]. Besides translocation ALK overexpression [59, 144-149] as well as point mutations [128, 132-135] have been reported in several cancers (Figure 7).

3.1 ALK translocations

3.1.1 Anaplastic Large Cell Lymphoma (ALCL) Most studies regarding ALK have been performed in ALCL. This disease,

which was described in 1985 for the first time [150], is a type of Non-Hodgkin’s lymphoma arising from T-cells. ALCL is characterized by large horseshoe shaped nuclei and the expression of CD30. Further, ALK is expressed in 60 – 80% of ALCL, which is mainly observed in children and young adults [151-153]. The fact that ALK positive ALCL patients have a higher 5-year survival rate compared to ALK negative ALCL patients, makes ALK expression an important prognostic factor [153-157]. However, some reports described the detection of NPM-ALK in blood and lymphoid tissue of healthy persons, raising the question of whether NPM-ALK on its own is sufficient to promote tumorigenesis [158, 159]. Since the initial discovery of NPM-ALK, numerous other fusion partners for ALK have been reported in ALCL (Figure 7): Tropomyosin 3 and 4 (TPM3/4) [160-162], TRK-fused gene (TFG) [124, 163], 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC) [125, 164, 165], Clathrin heavy chain-like 1 (CLTC1) [166], Moesin (MSN) [167, 168], ALK lymphoma oligomerization partner on chromosome 17 (ALO17) [169] and Non-muscle myosin heavy chain (MYH9) [170]. This great variety of fusion partners mirrors the high

23

ability of the ALK locus to undergo recombination, although the reason for this process is still poorly understood.

3.1.2 Inflammatory Myofibroblastic Tumour (IMT) IMT belongs to the class of “Inflammatory Pseudotumors”. These tumors

occur mostly in young individuals, although they may also appear in older patients [171]. Tumours are situated mostly in soft tissues, most commonly in lung, abdomen and retroperitoneum, although they can be located anywhere in the body [172]. Further, inflammatory infiltrates containing plasma cells and lymphocytes are detected in IMTs [171]. Griffin et al., reported the first appearance of ALK in IMT in 1999, describing a 2p23 chromosomal rearrangement and ALK expression in IMT, suggesting ALK’s involvement in solid tumours for the first time [137]. Since then, several additional ALK fusion partners in IMT have been described (Figure 7): Tropomyosin 3 and 4 (TPM3/4) [173], 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC) [174], Clathrin heavy chain-like 1 (CLTC1) [175, 176], RAN binding protein 2 (RANBP2) [177], Cysteinyl-tRNA synthetase (CARS) [169, 178], SEC31 homologue A (S. cerevisiae) (SEC31L1) [179] and F polypeptide-interacting protein-binding protein 1 (PPFIBP1) [180]. Altogether, about 50% of all IMTs appear to have ALK rearrangements and in accordance with ALCL, ALK positive IMTs seem to occur mostly in younger individuals [137, 173, 181, 182].

3.1.3 Non-small cell lung cancer (NSCLC) Lung cancer is the most widespread cause of cancer death with 1.4 million

deaths/year worldwide (2008) [183]. Lung cancer is split into two major subgroups: Small cell lung cancer (SCLC) and Non-small cell lung cancer (NSCLC). The latter one accounts for about 80% of all lung cancers and responds poorly to conventional cancer treatments. In 2007 a novel fusion protein, EML4-ALK, where the N-terminal domain of echinoderm microtubule associated protein like 4 is fused to the ALK kinase domain, was described in NSCLC [138, 139]. Since then, further variants of EML4-ALK have been reported in NSCLC [138, 139, 184, 185]. EML4-ALK occurs in approximately 3 – 13% of all lung tumours and in about 5% of all NSCLC [139, 186-192]. Further ALK fusion partners in NSCLC have been identified: TRK-fused gene (TFG) [138] and kinesin family member 5B (KIF5B) [126, 185], although those fusion proteins are not as common as EML4-ALK [131, 193]. Interestingly, ALK translocations and mutations in other known oncogenes like EGFR or KRAS seem to be mutually exclusive in NSCLC patients [186, 188, 190, 194, 195]. However, Sasaki et al., identified a subset of NSCLC patients (3/50; 6%) harbouring ALK rearrangements together with EGFR activating mutations [196].

24

Apr

p

lo

A

Figure 7: Schematic overview of ALK aberrations in cancer. LK fusion proteins, in which the kinase domain of ALK is fused to the N-terminal portion of various oteins, have been described in numerous cancers. Moreover, secondary mutations in the context of ALK

fusions have been described. ALK overexpression has been reported in a number of cancer types. ALK oint mutations have been found mainly in neuroblastoma, where most of the mutations are situated within

the kinase domain of ALK, but also in ALK gene of NSCLC and ATC origin. The structural model shows the ALK kinase domain with the C-helix (orange), the P-loop (green), activation loop (blue) and catalytic

op (yellow) highlighted. Red balls indicate verified point mutations observed in neuroblastoma patients. The three most common sites of point mutations in neuroblastoma are indicated. Abbreviations for the

LK fusion partners are explained in the chapter “ALK translocations”.

3.1.4 Diffuse large B-cell lymphoma (DLBCL) The most frequent ALK fusion partner in DLBCL is clathrin (CLTC) [155,

166, 197-200]. ALK-positive DLBCLs express epithelial membrane antigen (EMA), immunoglobulin light chains, CD38 and CD138, but do not express many B- and T-cell markers like CD30 antigen [155, 197, 198]. DLBCLs are associated with a poor clinical outcome and respond poorly to chemotherapy [201, 202]. Other fusion proteins described in some DLBCL cases are NPM-ALK and SQSTM1 (Sequestosome 1)-ALK [203-205]. Further, an insertion of a 3’-ALK sequence at chromosome 4q22-24 has been reported in DLBCL, although the ALK fusion partner is unknown so far [206].

3.1.5 Renal cell carcinoma Recently, the fusion product of ALK and vinculin (VCL) has been detected in

young patients diagnosed with renal cell carcinoma [141]. This (2;10) translocation results in the VCL-ALK fusion product which seem to be important for development and onset of renal medullary carcinoma [207]. Recently, TPM3- and

25

EML4-ALK have been detected in cases of adult renal cell carcinoma [208]. However, as only few reports have been published so far, further investigation of this patient population for the existence of ALK fusions will be very interesting.

Despite the increasing number of different ALK fusion partners and different cancer types harbouring ALK rearrangements, the various fusion proteins have several common features: (1) the promoter of the ALK partner protein drives transcription of the fusion protein; (2) the ALK partner protein seems to determine the cellular localization, i.e. rather than being inserted into the cell membrane ALK fusion proteins are localized in the cytosol; and (3) oligomerization of the ALK partner protein leads to auto-phosphorylation and thereby activation of the ALK kinase domain, resulting in downstream signalling events that lead to biological outcomes involved in tumourigenesis.

3.2 ALK overexpression ALK overexpression has been described in a variety of different cancers like

thyroid carcinoma, NSCLC, breast cancer, melanoma, neuroblastoma, glioblastoma, astrocytoma, retinoblastoma, Ewing’s sarcoma and rhabdomyosarcoma (Figure 7) [59, 148, 149]. Further, ALK expression has been reported in leiomyosarcoma, peripheral nerve sheath tumours and malignant fibrous histocytoma [209]. Additional discussion of ALK overexpression especially in neuroblastoma will be covered in the chapter “Genetic hallmarks of neuroblastoma”.

3.3 Point mutations of ALK

3.3.1 Neuroblastoma Neuroblastoma is the most common solid extracranial childhood cancer,

being responsible for approximately 15% of all pediatric cancer deaths [210]. This disease derives from neural crest cells of the sympaticoadrenal lineage and can hence appear throughout the sympathetic nervous system. Most primary tumours are located within the abdomen, followed by neck, chest and pelvis. However, despite improved clinical treatments, the long-term survival rate for children with high-risk neuroblastoma still ranges below 40% [211]. According to the International Neuroblastoma Risk Group (INRG) staging system, neuroblastoma can be divided into different stages (1, 2, 3, 4 and 4S according to the old staging system and L1, L2, M and MS according to the new one) depending on several criteria, e.g. age (patient <18 month have a more favourable prognosis), ploidy and MYCN amplification status (correlated with poor prognosis).

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3.3.2 Genetic hallmarks of neuroblastoma The most common genetic hallmarks of neuroblastoma are deletions of parts

of chromosome arms 1p and 11q, gain of parts of 17q, triploidy and MYCN amplification [134, 212-215].

Deletions of 1p correlate with MYCN amplification and advanced disease stage [212]. In particular, allelic loss at 1p36 predicts disease progression but not overall survival [216]. Attempts are carried out to identify putative tumor suppressor genes that are deleted within this region. One tumor suppressor candidate is the calmodulin binding transcription activator 1 (CAMTA1). Ectopic CAMTA1 expression resulted in decreased cell proliferation, colony formation and suppressed growth of tumor xenografts. Further, CAMTA1 induced differentiation in neuroblastoma cell lines, suggesting that CAMTA1 is a potential tumor suppressor in neuroblastoma [217].

Allelic loss of 11q is inversely correlated with 1p deletion and is rarely seen in tumors with amplified MYCN. However, loss of 11q is associated with decreased event-free survival [212]. One candidate gene within the region that is frequently deleted is the putative tumor suppressor gene TSLC1/IGSF4/CADM1 (Tumor suppressor in lung cancer 1/Immunoglobulin superfamily 4/Cell adhesion molecule 1). TSLC1 expression levels are reduced in unfavorable neuroblastomas and are assocated with poor prognosis. In addition, TSLC1 reduced proliferation of a neuroblastoma cell line, suggesting that TSLC1 might be a tumor suppressor gene involved in neuroblastoma [218].

Another hallmark represents the gain of parts of 17q. The breakpoints vary, but gain of a region from 17q22-qter suggests a selective advantage due to dosage effects of one or more genes [219]. One example is the overexpression of survivin, an inhibitor of apoptosis [220].

Measuring the DNA content of neuroblastomas serves as an important prognostic marker. Roughly, the DNA content can be divided into two groups: near-diploid or hyperdiploid (often near triploid). Triploidy seems to be favourable and is associated with less aggressive tumors, while near-diploidy is more often seen in malignant neuroblastomas [210, 212].

One characteristic hallmark of neuroblastoma is the amplification of the MYCN gene locus on chromosome 2p23-24 (~24% of all cases), correlating with poor survival [212, 221, 222]. MYCN belongs to the family of MYC proto-oncogenes, which comprises also c-MYC and MYCL. All MYC proteins are basic helix-loop-helix transcription factors forming a heterodimeric complex with Max, which can bind to E-box sequences (CACGTG) (reviewed in [223]). Generally, MYC plays a crucial role during embryonic development. While c-MYC is expressed abundantly, MYCN expression is very restricted to certain tissues during development (reviewed in [224]). Interestingly, c-MYC and MYCN appear to be complementarily expressed, suggesting also a difference in function. However, according to a mouse study MYCN is able to replace c-MYC during development [225].

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Like all MYC proteins, MYCN participates in the regulation of many cellular processes like cell proliferation, growth, protein synthesis, metabolism but also apoptosis and differentiation [226]. However, amplification or overexpression of MYCN contributes to the development of several cancers, often originating from tissues with normal MYCN expression. These tumors include beside the above mentioned neuroblastoma also retinoblastoma, glioblastoma and small cell lung cancer [227-229]. Further, neuroblastoma cell lines harbouring MYCN amplification show increased proliferation, downregulation of angiogenesis inhibitors, blocked terminal differentiation and enhanced invasive potential [212, 222, 230, 231]. Therefore, MYC is a double-edged sword: on the one hand MYC is crucial for development and many cellular processes, on the other hand deregulated MYC exhibits detrimental effects. However, MYC alone has no transforming capacity, rather it needs some companions. In murine cells, the oncoproteins MYC and Ras cooperate in cellular transformation, while more events are necessary in human cells [232, 233]. Human cell transformation requires both the inactivation of tumor suppressor genes and unlimited replication ability (hTERT activation) together with oncogene activation. Generally, amplified MYCN appears to be a key factor in the development of neuroblastoma. However, despite these findings, it has been suggested that MYCN amplification alone is not sufficient to initiate tumour formation [234].

Expression of full-length ALK in neuroblastoma was described for the first time in 2000 by Lamant et al., [235]. Subsequent studies described ALK overexpression as a result of genetic amplification both in neuroblastoma cell lines and patient samples. This alk amplification leads to ALK activation, which probably is involved in the development or the onset of this disease, correlating with a poor prognosis of neuroblastoma patients [144-147, 236]. Interestingly, alk amplification in neuroblastoma tumours is often observed together with MYCN [132, 237, 238]. Further, mutation-independent ALK overexpression in neuroblastoma patients correlates with a poor prognosis, further strengthening the role for ALK overexpression in neuroblastoma [147]. Interestingly, ALK protein levels do not necessarily correlate with ALK mRNA levels and/or genetic alterations, suggesting that alternative mechanisms other than mutations or amplification regulate ALK expression levels in neuroblastoma [147]. Moreover, Schulte et al., reported that neuroblastomas with mutated ALK exhibited elevated ALK expression levels compared with wild type ALK [236]. Further, neuroblastomas with enhanced wild type ALK resemble neuroblastomas with mutated ALK in clinical and molecular phenotypes, suggesting that high levels of wild type ALK may contribute to the malignancy of neuroblastoma [236].

3.3.3 ALK point mutations in neuroblastoma It was not until the year 2008 that activating ALK point mutations were

described in both familial and sporadic neuroblastoma as well as neuroblastoma cell lines (Table 1) [128, 132-135]. Most of these mutations are situated within or

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adjacent to the ALK kinase domain and are suggested to be gain-of-function mutations [239, 240]. siRNA mediated knockdown of ALK expression decreased proliferation of neuroblastoma cell lines [135], while expression of F1174L and K1062M ALK mutants in NIH3T3 cells and nude mice resulted in rapid formation of subcutaneous tumours, demonstrating the oncogenic and transforming potential of these ALK mutants [128]. To date several ALK point mutants have been experimentally investigated to determine whether they are true gain-of-function mutations [135]. Indeed, several groups including our own could demonstrate that the majority of ALK point mutations are constitutively active and can be inhibited by small ALK-specific inhibitors like NVP-TAE684 or crizotinib, which will be discussed explicitly in Article II and III [128, 241, 242]. Surprisingly, one somatic mutation – I1250T – which has been identified in two neuroblastoma patients, is in fact inactive and might possibly act as a dominant negative RTK, as discussed in detail in Article IV [243]. Further, several silent mutations have been described in neuroblastoma patients [236]. However, the three major mutational hotspots are F1174, R1275 and F1245; notably, mutations at position F1174 appear only in somatic tumours [213, 239]. A recent meta-analysis of neuroblastoma by Brouwer et al., reported also that ALK gain-of-function mutation occured at a frequency of 6.9% in investigated neuroblastoma tumours. Further, when comparing ALK mutation frequency in relation to genomic subtype, ALK mutations were most frequently observed in MYCN amplified tumours (8.9%), correlating with poor clinical outcome [213]. Based on this report one could strongly assume that a connection between ALK and MYCN expression exists. Indeed, as will be discussed explicitly in Article V, ALK is able to regulate the initiation of the transcription of MYCN in neuroblastoma cell lines [244]. Recently two cases of congenital neuroblastoma carrying somatic, heterozygous ALK mutations (F1174L and F1245V, respectively) showed severe encephalopathy and brainstem abnormalities, suggesting that abnormal ALK activation is detrimental to the development of the central nervous system [245]. Further, the F1174L and R1275Q ALK variants were reported to exhibit impaired maturation with defective N-linked glycosylation [246]. The importance of N-linked glycosylation of ALK was further strengthed by a report by Del Grosso et al., where inhibition of N-linked glycosylation results in reduced ALK phosphorylation, subsequent decreased downstream signalling and impaired cell viability in ALK mutated/amplified neuroblastoma cells [247]. An additional method of aberrant ALK activation is the expression of a truncated form of ALK in neuroblastoma [248]. An in-frame deletion spanning exons 2 and 3 results in the expression of a 208 kDa form of ALK, which is auto-phosphorylated. This ALK variant retains mainly in the endoplasmatic reticulum and has transforming ability.

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Table 1: Point mutations found in human ALK a.a. mutation

disease G/S targeted region phenotype ref.

R412C Burkitt’s

lymphoma (CL) ? First MAM domain ? [249]

S413N lung cancer S First MAM domain ? [76] V597A lung cancer S Second MAM domain ? [76] H694R lung cancer S Between second

MAM and G-rich domain

G-O-F [76]

G881D lung cancer S G-rich domain ? [76] C1021Y Osteosarcoma (CL) ? Extracellular domain,

close to transmembrane domain

? [249]

K1062M NB ND Juxtamembrane domain

? [128]

T1087I NB G Juxtamembrane domain

? [128]

D1091N NB S Juxtamembrane domain

? [135]

A1099T NB ? Juxtamembrane domain

? NP

G1123S/D Resistant in SH-SY5Y

? Loop in between β1 and β2

G-O-F [250]

G1128A NB G Loop in between β1 and β2

G-O-F [135, 242]

T1151M NB G End of β3 sheet ? [133] 1151Tins NSCLC ? End of β3 sheet Sec. mut. [251] L1152R NSCLC ? End of β3 sheet Sec. mut. [196] C1156Y NSCLC ND Loop in between β3

and αC Sec. mut. [184]

M1166R NB S αC-helix ? [135] I1170T/S NB ND αC-helix ? [236] I1171N NB S αC-helix G-O-F [135, 242] F1174L NB S End of αC-helix G-O-F [128, 132,

133, 135] F1174S NB S End of αC-helix G-O-F [241] F1174I NB G, S End of αC-helix ? [132, 135] F1174C NB S End of αC-helix ? [128] F1174V NB S End of αC-helix ? [128, 134] R1192Q Uterine

leiomyosarcoma (CL)

? Loop in between β4 and β5

? [249]

R1192P NB G Loop in between β4 and β5

G-O-F [135, 242]

L1196M NSCLC ND gatekeeper Sec. mut. [184] L1198F ATC ND Loop in between β4

and β5 G-O-F [136]

L1198P Resistant EML4-ALK in Ba/F3, SH-SY5Y

ND Loop in between β4 and β5

G-O-F [250]

G1201E ATC ND Loop in between β4 and β5

G-O-F [136]

G1202R NSCLC ? Loop in between β4 and β5

Sec. mut. [251]

D1203N Resistant EML4-ALK in Ba/F3

ND Loop in between β4 and β5

G-O-F [250]

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S1206Y NSCLC ? Loop in between β4 and β5

Sec. mut. [251]

A1234T NB S αE helix ? [133] Y1239H lung cancer S Catalytic loop ? [76] L1240V NB S Catalytic loop ? [236] F1245C NB S -2 to HRD, catalytic

loop G-O-F [133, 135,

242] F1245I NB S -2 to HRD, catalytic

loop ? [132]

F1245L NB S -2 to HRD, catalytic loop

? [128, 132]

F1245V NB S -2 to HRD, catalytic loop

? [133, 135]

I1250T NB G +1 to HRD, catalytic loop

KD [133, 135, 243]

A1252V Carcinoma of the endometrium (CL)

? Kinase domain ? [249]

G1269A NSCLC ND Kinase domain ? [252] R1275Q NB G, S +2 to DFG, activation

loop G-O-F [128, 132-

135] R1275L NB ND +2 to DFG, activation

loop ? [134]

Y1278H Resistant in SH-SY5Y, only together with G1123S/D

? A-loop, NPM-ALK (Y338) essential for kinase activity [76]

G-O-F [250]

Y1278S NB, lung cancer S A-loop, NPM-ALK (Y338) essential for kinase activity [76]

? [76, 134]

D1311A Lung cancer (CL) ? Kinase domain ? [249] E1384K lung cancer S C-terminal end of

kinase domain G-O-F [76]

1464STOP NB ND C-terminal to kinase domain

? NP

K1518N Lung cancer (CL) ? C-terminal end of kinase domain

? [249]

K1525E Upper respiratory tract adenocarcinoma (CL)

? C-terminal end of kinase domain

? [249]

Silent mutations

[236]

ATC: Anaplastic Thyroid Carinoma; CL: Cell line; G-O-F: Gain-of-function; G/S: Germline/Somatic; KD: Kinase Dead; Sec. mut.: Secondary Mutation; NB: Neuroblastoma; ND: Not determined; NP: Not published; NSCLC: Non-small cell lung cancer; Targeted regions in kinase domain according to Bossi et al., [28]. ALK mutations investigated and discussed in this thesis are underlined.

3.3.4 ALK point mutations in other cancers Since the initial reports, ALK point mutations have not only been found in

neuroblastoma, but have also been described in other cancers like lung cancer and Anaplastic thyroid cancer (ATC), as well as in various cancer cell lines, including Burkitt’s lymphoma and osteosarcoma cell lines. In ATC, the novel ALK point

31

mutations L1198F and G1201E are constitutively active [136]. In lung cancer ALK mutations have also been observed in the extracellular domain of ALK. Most extracellular ALK mutations possess weak oncogenic potential, except the mutation H694R that is highly transforming, although the exact mechanism behind the increased activating nature of this mutation remains unclear [76]. Recently, two newly identified ALK mutations are of clinical importance. In a NSCLC sample isolated from a crizotinib-treated patient two secondary mutations in the kinase domain of EML4-ALK – C1156Y and L1196M – were identified. L1196M is in the gatekeeper site and as expected showed resistance to crizotinib therapy [184, 193]. However, as C1156Y is not located at the gatekeeper position but in the loop between β3 and the αC-helix, the mechanism of this secondary mutation is still unclear [184]. Additional ALK point mutations in NSCLC patient samples were identified by Katayama et al., [251]. Those mutations include 1151Tins, G1202R and S1206Y and, like the previous secondary mutations, are localized near the ATP binding pocket of ALK. Recently, Sasaki et al., described a further secondary ALK mutation, L1152R, found in a crizotinib-resistant cell line. This mutation also showed resistance to the structurally unrelated ALK-specific inhibitor NVP-TAE684 [196]. On the other hand, other ALK mutants identified in various cancer cell lines were not tyrosine phosphorylated and were unable to drive foci formation compared to the F1174L ALK mutant [249]. These findings suggest that those described ALK mutations might represent “passenger” mutations.

Since the occurrence of secondary ALK mutations exhibiting resistance towards crizotinib treatment, efforts are being made to identify further ALK mutations that might possibly show resistance to ALK inhibitors. Indeed, Heuckmann et al., screened cell lines for mutations showing resistance to crizotinib and/or NVP-TAE684 [250]. While some crizotinib resistant mutations were highly sensitive to NVP-TAE684, two novel EML4-ALK mutations (L1198P and D1203N) showed resistance to both ALK-specific inhibitors. These results suggest differences in therapeutic efficacy depending on ALK inhibitors and ALK mutations. However, these mutations have not been found in cancer patients yet.

4 Treatments for ALK-positive carcinomas

Depending on type and onset of disease, the commonly used therapeutical approaches include different combinations of surgery, radiation therapy and chemotherapy. However, in addition to the above named “traditional” methods, alternative therapies are starting to be employed. As ALK is an established oncogene in many different cancers by now, this RTK serves as an excellent target for treatment by e.g. kinase inhibitors.

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4.1 Kinase inhibitors The development of tyrosine kinase inhibitors has influenced the treatment of

cancer patients significantly. One famous example is Gleevec (Imatinib), targeting BCR-ABL in chronic myeloid leukemia (CML) [253, 254]. Besides inhibiting ABL, Gleevec also blocks the RTKs c-Kit and PDGFR [255, 256]. Further established RTK inhibitors are Gefitinib and Erlotinib, which block the activity of EGFR (ErbB1) and are currently used in the treatment of NSCLC patients [257].

4.1.1 ALK-specific tyrosine kinase inhibitors As ALK seems play a more and more important role in many cancers, the use

of ALK-specific inhibitors should influence the treatment of a variety of cancers in a positive way. One of the first ALK-specific inhibitors is NVP-TAE684 targeting the ATP binding pocket of ALK. According to initial tests and an extensive screen of various human cancer cell lines, NVP-TAE684 not only blocked proliferation of ALK-positive ALCL cells, but of neuroblastoma and NSCLC cell lines as well [96, 258]. Since then several studies reported that tumours induced by constitutively active ALK in vivo as well as cells expressing ALK gain-of-function mutations demonstrate sensitivity towards NVP-TAE684, including both ALK translocations and neuroblastoma cells harboring ALK point mutations [76, 133, 259-261]. Interestingly, the study by Duijkers et al., reports that neuroblastoma cell lines with mutated ALK express ALK on higher mRNA and protein levels and respond better to ALK inhibitors [260]. However, recently NVP-TAE684 was shown to effectively inhibit the leucine-rich repeat kinase 2 (LRRK2) which is involved in Parkinson’s disease [262].

To date, numerous other ALK inhibitors have been developed (Table 2) (reviewed in [131]). Amongst all ALK-specific inhibitors crizotinib (PF-2341066) has undergone a very impressive and rapid development to an anticancer drug since the first report in 2007. Like NVP-TAE684, crizotinib is an ATP-competitive small molecule inhibitor as well, targeting not only ALK but also c-Met [263, 264]. Already three years later, in 2010, first results of clinical trials in NSCLC patients harbouring EML4-ALK were very promising, resulting in recent FDA approval under the name Xalkori [193, 265-267]. Currently, phase III trials are ongoing and despite some reports of certain ALK mutations, especially secondary mutations, in EML4-ALK positive NSCLC exhibiting crizotinib resistance [184, 196, 268, 269], the results indicate a 64% overall survival of crizotinib-treated ALK-positive patients after two years [270]. Further, Bresler et al., reported that cell lines harbouring the F1174L ALK mutation were relatively resistant to crizotinib due to an increased affinity to ATP binding, an effect that might be overcome with higher doses of crizotinib and/or higher-affinity inhibitors [271]. Also an IMT patient harbouring the RANBP-ALK translocation responded partially to crizotinib while two ALK-positive ALCL patients had complete response to crizotinib [272, 273]. Altogether, those promising reports support the therapeutical use of crizotinib for ALK-positive cancer patients.

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The AP26113 inhibitor was reported to block ALK activity in crizotinib-resistant NSCLC cell lines harbouring EML4-ALK with the gatekeeper mutation L1196M [261]. Another orally available inhibitor blocking ALK with the gatekeeper mutation, and hence inhibiting EML4-ALK L1196M promoted cell proliferation, is CH5424802 (AF802) [274, 275].

Two further potent small molecule tyrosine kinase inhibitors are X-378 and X-396, showing high specificity towards ALK [276]. Particularly X-396 has been shown to block EML4-ALK harboring the crizotinib-resistant mutations L1196M and C1156Y. Further, combining X-396 with the mTOR inhibitor rapamycin inhibited growth in a synergistic manner, suggesting an improved approach for the treatment of ALK-positive cancer patients. Another highly potent and orally active ALK inhibitor is CEP-28122 which shows anti-tumour activity in ALK-positive ALCL, NSCLC and neuroblastoma [277]. Further interesting ALK inhibitors exist, although little is known about them so far. GSK1838705A has been shown to block ALK as well as IGF-IR and IR, thereby blocking the proliferation of cancer cell lines and growth of tumour xenografts in nude mice [278]. While PHA-E429 has been reported as a crystal structure with the ALK kinase domain [28], F91873 and F91874 were identified as multikinase inhibitors showing activity against ALK in a biochemical screen [279]. ASP3026 is in Phase I clinical trials for ALK related malignancies, as is LDK378 from Novartis. However, little information exists about the pre-clinical compound 3-39 from Novartis.

With regard to the development of secondary ALK mutations in samples showing crizotinib-resistance, the need for novel inhibitors showing inhibitory activity against ALK persists. Indeed, besides the above named second generation ALK inhibitors CH5424802 and ASP-3026, new molecules are being described, such as NMS-E628 [280], SJ-08-0025 [281], tetrahydropyridopyrazines [282], piperidine carboxamides [283] and compounds from structural-based virtual screening approaches [284]. Further, several groups are attempting to design and modify ALK inhibitors based on structural insights of the kinase domain ([285] and reviewed in [286]). Altogether, it will be interesting to see the development of new, more potent and specific ALK inhibitors as well as the results of the clinical trials.

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Table 2: ALK inhibitors Company Inhibitor Clinical

trial, phase

G/W Aims of investigation

NCT00932893, III

No Crizotinib vs standard of care in patients with advanced NSCLC

NCT01154140, III

No Randomized, open-label study of the efficacy and safety of Crizotinib vs Pemetrexed/Cisplatin or Pemetrexed Carboplatin in previously untreated patients

NCT00939770, I/II

No Young patients with relapsed or refractory solid tumors, ALCL, CNS or NBs

NCT01121588, I/II

No Safety and efficacy in patients with tumors except NSCLC that are ALK-positive

NCT00932451, II

No Safety and efficacy in NSCLC patients

Pfizer Crizotinib (PF-2341066, Xalkori)

NCT00585195, I

No Safety in patients with advanced malignancy

NVP-TAE684 N/A Not developed LDK378 NCT01283516,

I Yes Safety in ALK-positive/genetic

abnormal tumors, no available data

Novartis

3-39 Pre-clinical Yes Chugai AF802

(CH5424802) JapicCTI-101264, I/II

Yes I. Safety, tolerability and pharmacokinetic in NSCLC patients with ALK-fusion gene

II. Efficacy and safety of AF802 Infinity IPI-504* NCT01228435,

II N/A Inhibitor of Hsp90, which protects

other proteins from being destroyed, possibly also EML4-ALK fusion proteins in NSCLC patients

Astella ASP3026 NCT01284192, I

ND Safety and tolerability of ASP3026. No pre-clinical data available but aim for advanced malignancies, B-cell lymphoma, solid tumors and ALK

Ariad AP-26113 NCT01449461, I/II

Yes AP-26113 abrogates Crizotinib resistant mutations in EML4-ALK. Safety, tolerability, pharmacokinetics and preliminary anti-tumor activity

Xcovery X-396 Pre-clinical Yes X-396 inhibits two ALK point mutations, C1156Y and L1196M, works in synergy with rapamycin. May initiate clinical trials by the end of 2011

Glaxo-Smith-Kline

GSK-1838705A Pre-clinical Yes Abrogates ALK and growth of ALCL, some NBs and a subset of NSCLC

ALCL: Anaplastic Large Cell Lymphoma; G/W: refers to ability to inhibit gateway mutation; NB: Neuroblastoma; N/A: Not applicable; ND: Not determined; IPI-504 (marked with *) is not an ALK inhibitor, but an Hsp90 inhibitor.

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4.2 Other approaches to inhibit ALK activity Besides small molecular inhibitors there are various other strategies to block

ALK activity. One approach is the reduction of ALK mRNA, resulting in decreased ALK protein levels. This is a very attractive idea, especially when considering the appearance of secondary ALK mutations mediating inhibitor resistance. Indeed, neuroblastoma cell lines which have been transduced by lentiviral shRNA targeting ALK exhibit decreased proliferation and undergo apoptosis. Further, mouse studies show that liposomal siALK delivery to neuroblastoma cells results in cell growth arrest, apoptosis, inhibited angiogenesis and prolonged survival [287, 288]. Similar results were reported in NPM-ALK positive ALCL, where siRNA mediated downregulation of ALK resulted in cell cycle arrest, apoptosis in vitro as well as tumour growth inhibition and regression in vivo [289, 290]. Further, in ALCL, a combination of ALK gene silencing together with ERK-inhibitor U0126 treatment resulted in a synergistic growth inhibition [291]. Another approach to block the growth of ALCL is the DNA vaccination of mice against ALK. This effect is further enhanced by combination with chemotherapy resulting in prolonged survival of mice harbouring ALK-positive lymphomas [292]. In glioblastoma, where both PTN and ALK are upregulated, a knockdown of both PTN and ALK mediated by ribozymes, blocked cell proliferation in vitro and diminished tumour growth in a xenograft model in vivo [293].

Another way to inhibit ALK activity is the use of inhibitory antibodies. One well-known example is Trastuzumab (Herceptin), a monoclonal antibody binding to HER2, which prolongs life in patients with HER2-positive breast cancers [294]. In cell culture experiments inhibitory antibodies targeting ALK successfully reduce ALK downstream signalling and induce cytotoxicity in neuroblastoma cell lines [63, 295]. However, this inhibitory antibody is not suitable for treatment of cancers harbouring ALK fusion proteins, but might serve as an alternative for cancer patients with ALK amplification of gain-of-function mutations like in neuroblastoma. To date, no such inhibitory antibodies are in clinical trials.

Direct targeting of ALK might be the preferential approach for therapy of ALK-positive cancers, but this might not be sufficient due to the likely development of secondary mutations. However, to overcome resistance to inhibitors, additional targeting of proteins that bind to ALK or are activated by ALK represents an additional therapeutic approach. One example of an additional target is Hsp90 which is shown to interact with ALK in ALCL. Inactivation of Hsp90 with 17-allyl-amino-demethoxygeldanamycin (17-AAG) results in degradation of NPM-ALK and apoptosis in ALCL cell lines [296]. IPI-504 is another Hsp90 inhibitor which showed promising effects in NSCLC patients harbouring EML4-ALK. Further, IPI-504, being already in Phase II Clinical Trials, might be a convenient drug for treating EML4-ALK positive NSCLC patients that show crizotinib-resistance due to the secondary ALK mutations L1196M [128, 261, 297-299]. Several studies have used JAK3 inhibitors like AG490, WHI-P131 and WHI-P154 in NPM-ALK positive ALCL. These inhibitors have been reported to directly block the activity of ALK,

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though in a modest way [97, 101]. Another inhibitor of JAK3/STAT3 signaling, cucurbitacin I, has been shown to induce apoptosis and proteasomal degradation of NPM-ALK in ALCL cells [102].

5 The Ras superfamily of small GTPases

As described earlier ALK activates the MAPK pathway, strongly suggesting that small GTPases like Ras might be involved in downstream signalling. As this potential activation has not been shown to date, we aimed to investigate the activation of small GTPases downstream of ALK. Indeed, stimulated wild type ALK activates Ras with a peak at 15 minutes post stimulation, confirming the assumptions (Figure 8A).

Figure 8: Stimulated ALK activates the small GTPases Ras and Rap1. Wild type mouse ALK expressing tet-on PC12 cells were stimulated with 1 µg/ml mAb46 or 50 ng/ml EGF respectively for the indicated times. Precleared cell lysates were incubated with (A) GST-Raf-RBD or (B) GST-RalGDS beads. Bound Ras or Rap1 proteins were analyzed by immunoblotting. Ras or Rap1 in whole cell lysates (WCL) was used as a loading control and detection of p-ERK was used as a control for mALK stimulation. The experiment shown in (A) was performed by Lovisa Olofsson.

The Ras superfamily of small guanosine triphosphatases (GTPases) contains over 150 human members with evolutionarily conserved orthologs in other species and regulates many different cellular functions [300, 301]. The Ras superfamily is divided into five subgroups: Ras, Rho, Rab, Sar/Arf and Ran. Rat sarcoma (Ras) oncogene proteins are the founding members of the Ras family and were initially discovered as transforming genes of the Harvey and Kirsten murine sarcoma viruses [302, 303]. Later, their role as oncogenes with transforming capacity in human cancer was established [304-306]. Additionally, a third ras gene with transforming potential was identified in neuroblastoma-derived DNA, named NRAS [307, 308]. Since then, further members like Rap and Ral were added to this family, all playing critical roles in human oncogenesis by being involved in proliferation, growth and differentiation. Members of the Ras homologous (Rho) family like Rho, Rac and Cdc42 are mainly regulating the cytoskeleton, e.g. by promoting actin stress fiber formation, lamellipodium and filopodium formation as well as membrane ruffling.

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The largest branch of the superfamily is comprised of Ras-like proteins in brain (Rab) which harbour a central function in vesicular transport. A further subfamily being involved in the regulation of vesicular transport is the Sar/Arf (Secretion-associated and Ras-related/ADP-ribosylation factor) family. Members of this subfamily regulate the transport of cop-coated vesicles between the endoplasmatic reticulum and the Golgi. The last subfamily is formed of Ras-like nuclear (Ran) proteins which are the most abundant small GTPases in the cell and function in the nucleocytoplasmic transport of RNA and proteins.

However, the mechanism of action and regulation is the same for all Ras superfamily small GTPases (Figure 9).

Figure 9: Mechanism of action and regulation of small GTPases. In the GDP-bound state small GTPases are in an inactive conformation. Guanine-nucleotide exchange factors (GEFs) induce the release of GDP which is replaced by the more abundant GTP resulting in an active conformation. In this state, small GTPases can interact with effector proteins, resulting in various biological effects. GTPase-activating proteins (GAPs) contain a catalytic group for GTP hydrolysis, thereby accelerating the intrinsic GTPase activity which is very low in small GTPases.

Small GTPases possess high-affinity binding for GDP and GTP. In the GDP-

bound state, the small GTPases are inactive. With the help of guanine-nucleotide-exchange factors (GEFs) GTP binds to the small GTPases which are thereby activated and can activate biological downstream effectors. In order to return to the inactive GDP-bound state GTPase-activating proteins (GAPs) accelerate the intrinsic GTPase activity which is very low in small GTPases (reviewed in [301, 309]).

5.1 Rap1 Rap1 (Ras-proximate 1) belongs to the Ras family forming a subgroup of the

Ras superfamily of GTPases. Originally, Rap1 was identified as Krev-1 which was able to suppress the phenotype of K-ras transformed fibroblasts, and therefore thought to antagonize Ras signalling [310]. However, over the years several reports revealed multiple functions of Rap1: e.g. it is involved in the regulation of cell polarity, integrin-mediated adhesion, secretion and cell-cell junction formation. Like all GTPases, the activity of Rap1 is controlled by activating GEFs and inhibiting GAPs [311]. Further, Rap1 seems to function in neurite outgrowth and neuronal polarization by mediating sustained activation of the MAPK pathway as well as regulating the cytoskeleton [312-315]. Rap1 also plays a role in the regulation of cell proliferation, indicating that Rap1 might be involved in oncogenesis. Indeed,

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upon overexpression, Rap1 induces oncogenic transformation in Swiss-3T3 cells [316] and has been reported to be activating in metastatic mammary carcinomas, metastasis and during melanoma tumorigenesis [317, 318]. Further, Rap1 seems to play a role in thyroid mitogenesis and has been suggested to participate in thyroid-stimulating hormone-stimulated thyroid tumorigenesis [319, 320]. According to a recent report, Rap1 also plays a role in the proliferation and migration of endothelial cells as well as vasculatization, further suggesting a participation in oncogenesis [321]. However on the contrary, Lin et al., reported that Rap1 seems to suppress oncogenesis [322]. To date, a clear function for Rap1 in the development and/or onset of neuroblastoma has not been determined yet.

5.2 Rap1 specific regulators

5.2.1 Rap1 specific GEFs Rap1 is activated by certain GEFs, like C3G, the Epac family proteins, CD-

GEFs and PDZ-GEFs ([323-329] and reviewed in [309]). One particular Rap1 specific GEF, namely C3G, possesses an essential function in vascular maturation and the development of the nervous system during mouse embryogenesis [330-333]. In PC12 cells knockdown of C3G mediated by siRNA results in decreased neurite outgrowth and in human neuroblastoma cells C3G has been suggested to play a role in differentiation and survival [334-336]. Besides C3G, a further Rap1 specific GEF possessing oncogenic capacity is the atypical RapGEF DOCK4, displaying dual activity toward both Rap1 and Rac [337]. Further, the Drosophila C3G orthologue is a Rap1-specific GEF as well and is involved in maintaining muscle integrity during larval stages [338].

5.2.2 Rap1 specific GAPs As Rap1 possesses a very low intrinsic GTPase activity, its inactivation is

dependent on GTP hydrolysis by Rap1 specific GAPs, e.g. Rap1GAP and members of the Spa1 family ([339, 340] and reviewed by [309]). Although Rap1 belongs to the Ras family there are some differences in the process of hydrolysis. Instead of a glutamine (Gln61 in Ras) Rap1 contains a threonine (Thr61) which is important rather for binding than catalysis. Moreover, Rap1GAP does not use an arginine finger to stabilize the transition state in hydrolysis. Rather, Rap1GAP inserts an asparagine thumb containing the catalytic asparagine into the active site of Rap1 [341-343]. A product of a familial tuberous sclerosis gene named tuberin displays in vitro Rap1GAP activity as well and induces benign tumors upon deletion [344]. Decreased Rap1GAP expression levels result in enhanced Rap1 activity leading to increased cell migration and invasive behavior as shown in prostate cancer cell lines [345]. A recent study by Kim et al., further proved that low expression levels of Rap1GAP result in increased invasion of renal cell carcinoma [346]. Moreover,

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several reports suggested that Rap1GAP might act as a tumour suppressor gene in some tumours like thyroid tumours, epithelial tumours and melanoma [347-353].

Overall, deregulated Rap1 signalling is involved in a variety of malignancies like leukemia, neuroblastoma and prostate cancer (reviewed by [354]).

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Aims

1 Overall aim

The general aim of this thesis was to elucidate the function ALK in the development and onset of neuroblastoma. We intended to find answers to the following questions, whether ALK point mutations found both in neuroblastoma cell lines and patient samples are truly constitutively active, if they are potentially involved in the progression of this disease, whether these ALK mutants can be blocked by small ALK-specific inhibitors and which ALK downstream signaling pathways are involved.

2 Specific aims

More specifically, in article I the aim is to investigate whether ALK activates small GTPases, in particular Rap1 and the potential involvement in oncogenesis.

In article II we want to investigate whether the newly reported ALKF1174S mutation is constitutively active and whether it responds to treatment.

Article III approaches the question whether the ALK point mutations found in neuroblastoma cell lines and patients are constitutively active and if so, whether the downstream signalling can be blocked by ALK-specific inhibitors. Further, we aim to investigate whether these mutations are involved in disease progression.

Article IV focuses on one particular ALK point mutation found in neuroblastoma, namely ALKI1250T, which was initially suggested to be constitutively active. In this paper we investigate whether the ALKI1250T mutation is truly constitutively active.

Article V aims to determine whether ALK can regulate the initiation of MYCN transcription in neuroblastoma.

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Results and Discussion

1 Article I: “Anaplastic lymphoma kinase activates the small GTPase Rap1 via the Rap1-specific GEF C3G in both neuroblastoma and PC12 cells.”

As most reports have been studying ALK signalling mediated by NPM-ALK, we wished to gain more information about the cell signalling of the wild type RTK. As small GTPases regulate various cellular processes like growth, differentiation and migration, we addressed the question whether small GTPases could serve as downstream targets of ALK.

1.1 Stimulated ALK activates Rap1 which leads to neurite outgrowth in PC12 cells In particular, we focused on whether mouse ALK can activate Rap1. In order

to address this question, we used a tetracycline-inducible PC12 cell system, which was generated in our lab. Upon induction with doxycycline, PC12 cells express wild type mouse ALK (mALK) which can be stimulated with an agonist monoclonal antibody, mAb46, resulting in the activation of ALK and its downstream signalling, as well as neurite outgrowth in PC12 cells [63, 70]. Indeed, stimulation of mALK with mAb46 resulted in activated Rap1, whose activation pattern is highly similar to the one of ERK phosphorylation.

In order to clarify that mALK is able to activate Rap1 we employed the ALK-specific inhibitor NVP-TAE684 (recently also identified as an effective inhibitor of leucine-rich repeat kinase 2 [262]) which has been shown to block NPM-ALK-driven cell proliferation and downstream signalling targets like ERK [96]. While NVP-TAE684 could not block NGF-mediated TrkA receptor signalling, it was able to inhibit mAb-mediated mALK activation as indicated by reduced ERK phosphorylation. Further, pretreatment with NVP-TAE684 before mAb46 stimulation abrogated Rap1-GTP levels efficiently.

As Rap1 is required for ERK activation [312, 314, 315] we examined whether this requirement is also valid downstream of ALK. Abrogation of Rap1 activity either by siRNA or by overexpression of Rap1GAP had no impact on reduction of ERK phosphorylation downstream of TrkA or mALK. However, pre-treatment of cells with siRap1 resulted in prolonged ERK-phosphorylation upon NGF stimulation, which is in agreement with a previous report [355].

Further, Rap1 is involved in neurite outgrowth in PC12 cells upon NGF stimulation (control; no ALK expression) and upon stimulation in ALK-expressing PC12 cells. Neurite outgrowth was decreased by reduced Rap1 expression levels and by abrogated Rap1 activity due to Rap1GAP overexpression. This finding is in

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agreement with a previous report indicating that overexpression of constitutively active Rap1 leads to neurite outgrowth in PC12 cells [356].

1.2 Activation of Rap1 downstream of ALK occurs via the Rap1-specific GEF C3G Having established a link between ALK and Rap1, we wanted to investigate

what happens “in between”. As the Rap1-specific GEF C3G has been shown to play a role in neurite outgrowth in PC12 cells [312, 315], we looked into whether mALK was able to activate C3G. By immunoprecipitation we could detect a constitutive interaction between C3G and CrkL which was independent of mALK stimulation and in accordance with a previous study by Feller et al., [357]. Further, activated mALK coimmunoprecipitated with either C3G or CrkL, indicating the formation of a protein complex consisting of ALK, C3G and CrkL.

In order to investigate whether ALK can activate C3G, we investigated the tyrosine phosphorylation status of C3G after mALK stimulation. Indeed, after ALK stimulation mediated by mAb46, we could detect tyrosine phosphorylation of C3G which is consistent with previous studies reporting C3G activation in response to several exogenous stimuli [358, 359]. Altogether, activated mALK recruits a complex consisting of C3G and CrkL, followed by tyrosine phosphorylation and thereby activation of C3G.

Next, we wanted to address the function of C3G in ALK-induced neurite outgrowth. Endogenous C3G levels were reduced by two independent siRNAs which resulted in decreased neurite outgrowth mediated by NGF induction in ALK-nonexpressing cells and by mAb46 stimulation in ALK-expressing PC12 cells. However, the decrease in neurite outgrowth by siC3G was not as pronounced as with siRap1, suggesting that other potential mechanisms may activate Rap1 and/or that the reduction of C3G expression levels was not sufficient. Our results are in line with previous studies also reporting decreased neurite outgrowth upon knockdown of Rap1 or C3G levels in PC12 or neuroblastoma cells [334, 336]. Interestingly, mALK-expressing PC12 cells that were not transfected with siC3G appeared to express more C3G upon stimulation with mAb46. This observation is in agreement with a study in the neuroblastoma cell line IMR32 by Radha et al., although the precise molecular mechanisms are unclear [336].

Further, to confirm that C3G activates Rap1 in mALK-expressing PC12 cells, we performed a Rap1 pulldown after transfection with siC3G. Indeed, knockdown of C3G results in decreased Rap1 activation in mALK-expressing PC12 cells, indicating that mALK activates C3G which in turn activates Rap1 and has a function in neurite outgrowth.

Overall, we conclude that Rap1 activity is not absolutely required for ERK phosphorylation, but that it is necessary for neurite outgrowth in our model system. Therefore, other molecular mechanisms not including Rap1 might lead to the prolonged ERK phosphorylation downstream of stimulated mALK. According to a

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previous report the FRS2 adaptor protein is recruited to activated ALK, suggesting that a complex consisting of ALK/FRS2/CrkL/C3G might be formed. Further, one has to keep in mind that comparing similar results, e.g. PC12 cells and Rap1 activation, is quite difficult due to genetic composition and variation of different PC12 cell clones.

1.3 Rap1 activity is involved in cell proliferation of neuroblastoma cell lines So far we proved that ALK regulates Rap1 in a very controlled inducible cell

culture model. However, this provides no clue about how significant the function of Rap1 is in ALK signalling under physiological conditions or in tumor development. Rap1 can also be activated by cAMP, which inhibits or increases cell proliferation depending on the cell type. These cAMP mediated effects on cell proliferation are suggested to be mediated by Rap1, which can cooperate either antagonistically or synergistically with Ras, depending on the cell type [309, 360, 361]. Although no activating mutations in Rap1 have been identified in tumours so far [362-364], Rap1 could still contribute to oncogenesis via other mechanisms, like overexpression or downregulation of Rap1 or any regulators up- or downstream of Rap1 [316, 337, 345, 350, 365]. For instance, several reports suggest that C3G inhibits cell proliferation [366-368]. In the neuroblastoma cell line IMR32, Radha et al., showed that NGF-mediated activation or overexpression of C3G results in cell-cycle arrest, implying that the C3G/Rap1 pathway contributes to the survival of the neuroblastoma cell line IMR32 [336]. Together with the first reports of activating ALK point mutations in neuroblastoma [128, 132-135], these studies suggest that there might be a relevant connection between ALK and Rap1 in neuroblastoma and that the contribution of the C3G/Rap1 pathway in the growth of neuroblastoma cell lines may depend on ALK. In order to investigate this theory, we employed the neuroblastoma cell lines SK-N-SH and SH-SY5Y, which both harbour the ALKF1174L point mutation. Abrogation of Rap1 activity by either siRap1 or overexpression of Rap1GAP decreased proliferation of both neuroblastoma cell lines [258]. Additionally, in SH-SY5Y cells mAb46 mediated stimulation of cell proliferation was blocked by reduced Rap1 expression levels, which could not be observed in SK-N-SH cells. This difference between those two neuroblastoma cell lines which both harbour the ALKF1174L gain-of-function mutation, might further depend on the genetic disposition. Altogether, these results suggest that the C3G/Rap1 pathway is not only important for ALK-mediated PC12 cell differentiation, but that this pathway is also involved in the regulation of neuroblastoma cell proliferation.

Conclusively, we propose the following model (Figure 10): Activated ALK forms a complex together with C3G and CrkL (and maybe further potential adaptor proteins like FRS2), in which C3G becomes tyrosine phosphorylated and therefore

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activated. This activated complex is able to activate Rap1 which results in neurite outgrowth in PC12 cells or proliferation of neuroblastoma cells.

Figure 10: Model of ALK mediated activation of the small GTPase Rap1. Wild type ALK is activated by monoclonal antibodies like mAb46, resulting in auto-phosphorylation and activation. Activated ALK recruits the Rap1-specific GEF C3G and CrkL (and maybe further potential adaptor protein like FRS2). In this complex C3G becomes tyrosine phosphorylated and thereby activated which results in the activation of Rap1. This results in neurite outgrowth in PC12 cells or proliferation of neuroblastoma cell lines. Overexpression of Rap1GAP abrogates Rap1 activity and hence the above named downstream effects.

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2 Article II: “Appearance of the novel activating F1174S ALK mutation in neuroblastoma correlates with aggressive tumor progression and unresponsiveness to therapy.” A young Swedish patient was diagnosed with neuroblastoma and was treated

accordingly. However, about eight months after diagnosis, a biopsy of the tibial bone showed viable tumor cells showing positive staining for phospho-ALK. Despite intensive care this patient succumbed to disease ten months after initial diagnosis. However, investigation of tumor genetics revealed a loss of heterozygosity on chromosome 2p which includes the ALK gene locus. DNA sequencing of the ALK gene detected a homozygous mutation (T3521C) which results in the missense mutation F1174S.

2.1 The ALKF1174S mutant is a ligand-independent gain-of-function mutation and has transforming potential By employing several cell culture based systems we wanted to biochemically

evaluate the ALKF1174S mutant. Doxycycline-inducible PC12 cell lines stably transfected with human wild type ALK and ALKF1174S were developed. While wild type ALK needs to be activated by the monoclonal antibody mAb31, no further stimulation with an agonist antibody is needed for activation of the ALKF1174S mutant as indicated by tyrosine phosphorylation of ALK and ERK phosphorylation as a downstream target. Moreover, both ALK and ERK phosphorylation are abrogated by the ALK-specific small molecule inhibitor NVP-TAE684. These results were further confirmed by transient transfection of ALKF1174S and ALKF1174L which show similar ALK and ERK phosphorylation. Therefore, we can conclude that the ALKF1174S mutant is constitutively active, which might contribute to the severe disease progression of this particular neuroblastoma patient.

A further method to investigate whether the ALKF1174S mutant is truly constitutively active, is to measure the neurite outgrowth in PC12 cells. Previously, we and others have shown that activated ALK leads to the extension of neurites in PC12 cells [64, 69, 70, 369]. Indeed, transient transfection of PC12 cells with human ALKF1174S or ALKF1174L results in neurite outgrowth, while human wild type ALK needs to be stimulated with monoclonal antibody mAb31 in order to mediate neurite outgrowth. However, expression of unstimulated wild type ALK results in minor neurite formation, which might be due to overexpression. This result further strengthens the hypothesis, that ALKF1174S is a ligand-independent gain-of-function mutation.

Finally, the transforming potential of the ALKF1174S mutant was assessed. NIH3T3 cells were transfected, and in contrast to the wild type receptor expression of both human ALKF1174S and ALKF1174L, mediates foci formation. This result demonstrates the transforming ability of the ALKF1174S mutant.

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2.2 Ectopic expression of ALKF1174S in the Drosophila eye causes the rough eye phenotype Based on this finding, the aim was to examine the nature of this ALK

mutation, i.e. whether it was the major cause for the rapid tumor progression. First indications were obtained in cell culture based systems. Another approach is the use of a different model system, namely D. melanogaster. Upon ectopic expression of human wild type ALK in the Drosophila eye this RTK is expressed in the developing photoreceptors of the eye without any noticeable phenotype, similar to the controls. As the ligand-dependent wild type ALK apparently is not activated by endogenous Drosophila ligands, therefore providing a clean background, overexpression of ALK in the eye serves as an optimal tool for the analysis of potential activating ALK mutants. Indeed, ectopic expression of human ALKF1174S or ALKF1174L (the latter one serving as a positive control [128]) results in the rough eye phenotype, indicating ligand-independent RTK activation. As no human ALK ligand is present in the Drosophila eye these results confirm that the ALKF1174S mutant is constitutively active in vivo.

To sum up, the results demonstrate that the novel human ALKF1174S mutant is a ligand-independent gain-of-function mutation, correlating with aggressive neuroblastoma development. Further, treatment with ALK-specific inhibitors like NVP-TAE684 indicates that the use of such inhibitors might be beneficial for the treatment of neuroblastoma patients. In addition, this case report shows that initial screening of the first tumor biopsy is not sufficient and that further genetic analyses, in particular of the ALK locus, are required in order to get more insight into the development of neuroblastoma and subsequently gain more information about the treatment of patients.

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3 Article III: “Activating ALK mutations found in neuroblastoma are inhibited by Crizotinib and NVP-TAE684.”

In this article we aimed to investigate whether certain ALK point mutations found in neuroblastoma patients are truly constitutively active and if they are involved in the progression of this disease.

According to one of the early publications of ALK point mutations in neuroblastoma by Mossé et al., we initially selected seven mutations with the highest probability of being activating mutations, i.e. with the highest probability that the amino acid change results in an oncogenic activation (Figure 11) [135]. This article discusses six of them (human ALKG1128A, ALKI1171N, ALKF1174L, ALKR1192P, ALKF1245C and ALKR1275Q), while one mutation (human ALKI1250T) is dealt with in Article IV.

Figure 11: Model of ALK kinase domain showing the investigated point mutations. The structural model shows the ALK kinase domain with the C-helix (orange), the P-loop (green), activation loop (blue) and catalytic loop (yellow) highlighted. Red balls indicate verified point mutations observed in neuroblastoma patients. The investigated neuroblastoma point mutations in this thesis are indicated.

3.1 ALK mutations identified in neuroblastoma are ligand-independent gain-of-function mutations and can be blocked by NVP-TAE684 and crizotinib with different IC50 Initially, we investigated whether the above named mutations in human ALK

were also conserved in mouse ALK. Therefore we created these mutations at the equivalent sites in mouse ALK, which are: mALKG1132A, mALKI1175N, mALKF1178L, mALKR1196P, mALKF1249C and mALKR1279Q. In order to compare the activity of these ALK mutants we created PC12 cell clones with doxycycline-inducible ALK expression. As a control we used wild type mouse ALK, which upon stimulation with the activating monoclonal antibody mAb46, becomes tyrosine phosphorylated and activates downstream signalling targets like ERK and PKB/Akt. However, doxycycline-induced expression of mALK mutants results in tyrosine phosphorylation of the RTK and activation of downstream signalling even in the absence of an activating antibody. Additionally, all six investigated ALK mutants

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result in STAT3 activation which is in contrast to wild type mALK. Therefore, all six investigated ALK mutants are activated ligand-independently and stimulate downstream targets like STAT3, PKB/Akt and ERK. In order to compare the relative ALK activity between the mutants we assessed the ratio of phospho-ALK to total ALK which seems to differ only slightly between the mutants, although it seems that mALKF1178L, mALKR1196P and mALKG1132A are slightly more active than the other mutants. Some difference in downstream signalling can also be observed. For example, mALKG1132A seems to phosphorylate STAT3 to a higher extent than mALKI1175N and mALKF1249C with comparable levels of ALK expression. However, as individual cell clones were selected for the expression of mALK mutants, with this employed cell culture model it is difficult to draw any conclusion about the activity of those ALK mutants in reality.

To further verify that the ALK mutations are constitutively active we employed the neurite outgrowth assay in PC12 cells. Neurite outgrowth mediated by stimulated wild type mALK can be blocked by the ALK-specific inhibitor NVP-TAE684 [64, 69, 70, 73]. All six expressed mALK mutants are able to induce neurite outgrowth ligand-independently, which can be blocked upon addition of the inhibitor, although to various degrees at low doses of inhibitor. The mALKR1279Q mediated neurite outgrowth was completely inhibited with 30 nM NVP-TAE684, while the neurite outgrowth induced by mALKI1175N, mALKR1196P and mALKF1249C was only blocked to about 50% by NVP-TAE684. In contrast, treatment of mALKG1132A and mALKF1178L resulted only in a small decrease of neurite outgrowth, which is in agreement with the suggested higher activity of those mutants. However, all uninduced cell clones exhibited a certain level of background neurite outgrowth which might be due to leakage or the presence of a so far unknown endogenous ligand in the case of unstimulated wild type mALK cells.

Next, we wanted to confirm that the human ALK mutations also respond to treatment with ALK-specific inhibitors. In addition to NVP-TAE684 we included crizotinib (PF-2341066), a dual inhibitor of c-MET and ALK, as this inhibitor is clinically used in contrast to NVP-TAE684 [263, 264]. In order to exclude any effects due to selection of individual PC12 cell clones and different ALK expression, we transiently transfected PC12 cells with human wild type or mutated ALK. In agreement with the inducible PC12 cell lines expressing mALK, stimulated human wild type ALK induces neurite outgrowth, while the ALK mutants mediate robust neurite outgrowth ligand-independently. In addition to NVP-TAE684, treatment with crizotinib also blocks neurite formation.

As NVP-TAE684 seems to have toxic effects, we continued to use only crizotinib, which has no obvious toxicity. In order to compare the different response of these six ALK mutants toward crizotinib, we employed the widely used Ba/F3 cell system. Interestingly, some differences between the ALK mutants in their ability to mediate proliferation in the absence of IL-3 could be observed. For each ALK mutant three independent transfections were performed, and from each transfection we obtained four IL-3-independent cell lines for ALKF1174L, ALKR1192P

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and ALKR1245C. However, ALKG1128A only resulted in three cell lines. To our surprise, ALKI1171N and ALKR1275Q were unable to compensate for IL-3 depletion and no Ba/F3 cell lines could be obtained, which contradicts results found in a previous study [133]. One possible explanation for these divergent results might be the different transfection methods used: our study employed transient transfection, while George et al., used retroviral infection of Ba/F3 cells. However, the fact that we were unable to get Ba/F3 cell lines with ALKI1171N and ALKR1275Q might represent a further indication that these two mutations have less transforming potential than the others, which is not sufficient to drive IL-3-independent Ba/F3 cell proliferation. Further, crizotinib blocked the proliferation of all four Ba/F3 cell lines. Subsequent calculations of the IC50 revealed that ALKF1174L and ALKR1192P required higher doses of crizotinib than ALKG1128A and ALKR1245C. These results could be verified by Western Blot analysis detecting ALK phosphorylation at position Y1278, the first tyrosine of the YxxxYY motif that is necessary for ALK’s auto-activation and the transforming ability of NPM-ALK [27], as well as ERK phosphorylation.

Next, we investigated the transforming potential of these activating human ALK mutations in NIH3T3 cells. While neither vector control nor ALK wild type gave rise to foci formation, all investigated ALK mutations drive foci formation although to a different extent. hALKG1128A, hALKI1171N, hALKR1192P and hALKR1275Q showed comparatively weak foci formation, while hALKF1174L and hALKF1245C resulted in strong foci formation. However, hALKI1171N and hALKR1275Q were able to transform NIH3T3 cells but could not drive IL-3-independent Ba/F3 cell proliferation, which may be due to the fact that these ALK mutations only have transforming potential in cells that are immortalized by other crucial genetic events in neuroblastoma.

To sum up, both mouse and human ALK mutants respond to NVP-TAE684 and crizotinib treatment. The activity of the ALK mutants is inhibited by crizotinib, although different doses are required. Further, all investigated human ALK mutations have transforming potential though to different degrees.

3.2 Ectopic expression of ALK mutants in the Drosophila eye causes the rough eye phenotype To further examine if ALK mutations are truly constitutively active, they

were overexpressed in the developing photoreceptors of the Drosophila eye. We chose to analyse hALKF1174L and hALKR1275Q which represent the most common mutations found in neuroblastoma patients [213]. Similar to the previous study (Article II), expression of hALK wild type did not cause any obvious phenotype in the eye and resembled controls [241]. Expression of hALKF1174L and hALKR1275Q caused the rough eye phenotype, similar to hALKF1174S, indicating their ligand-independent activation. In agreement with previous results, hALKF1174L caused a more severe phenotype than hALKR1275Q. Both ALK mutants, especially

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hALKR1275Q, responded to NVP-TAE684 treatment as indicated by the improvement of the rough eye phenotype. However, the effect of crizotinib treatment on the rough eye phenotype was less pronounced, which might reflect the higher toxicity of NVP-TAE684.

In summary, all six investigated human ALK mutations with the highest predictions of being oncogenic are indeed true gain-of-function mutations. In particular, one mutant, namely hALKR1275Q, which has been found both in germ line and somatic tumor DNA samples [133-135], seems to respond better to treatment with ALK inhibitors like NVP-TAE684 or crizotinib. Further, the obtained results suggest that the various mutations might have a different impact on development, onset and severity of neuroblastoma. Generally, we see a trend that somatic ALK mutations like ALKF1174L or ALKF1245C seem to be more aggressive and respond less to ALK inhibitor treatment than germ line mutations like ALKG1128A or ALKR1192P [135]. This is an important piece of information regarding patient-specific treatments for ALK-positive neuroblastoma with ALK-specific inhibitors like crizotinib.

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4 Article IV: “The Neuroblastoma ALK (I1250T) Mutation is a Kinase-Dead RTK In Vitro and In Vivo.”

Initially, we analysed seven ALK point mutations with the highest probability of being oncogenic [135]: the six previously discussed mutations (Article III) and an additional mutation, namely hALKI1250T. This mutation was identified as a germ line mutation with a high probability of being oncogenic [135].

4.1 The ALKI1250T mutant is not constitutively active in cell culture systems The equivalent mutation was created in mouse ALK (mALKI1254T) and stably

transfected into PC12 cells. However, when selected PC12 cell clones were screened for inducible ALK expression, no neurite outgrowth could be observed, in contrast to the other investigated mutations (Article III), despite ALK expression. These initial observations suggested that this mutation might not be ligand-independent, but act like the wild type ALK, i.e. the mALKI1254T mutation can be activated by a monoclonal antibody. While wild type ALK activated by monoclonal antibody mAb46 is tyrosine phosphorylated, activates the downstream target ERK and induces neurite outgrowth, surprisingly mALKI1254T cannot be stimulated by the activating monoclonal antibody mAb46. These unexpected results were confirmed both by transient transfection of PC12 cells with human ALKI1250T as well as by tetracycline-inducible PC12 cell clones expressing human ALK.

As hALKI1250T cannot be activated by monoclonal antibodies, the question was raised whether this mutation might be localized differently compared to the wild type receptor. Mazot et al., reported that ALK is not only localized on the cell membrane, but also at intracellular compartments like the endoplasmic reticulum and Golgi [246]. Accordingly to this report, we could detect both wild type ALK and ALKI1250T in the endoplasmic reticulum and the Golgi apparatus, but also on the cell membrane in transiently transfected HEK293 cells, therefore being accessible to antibody stimulation.

Next we wanted to assess whether this mutant displays any transforming potential. While the gain-of-function mutation hALKF1174S mediates foci formation, both wild type human ALK and hALKI1250T were unable to transform NIH3T3 cells.

So far we can conclude that despite cell membrane localization the hALKI1250T mutation has no detectable tyrosine phosphorylation activity, is unable to activate downstream signalling like ERK, cannot induce neurite outgrowth and has no transforming potential.

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4.2 The ALKI1250T mutant is suggested to act as a dominant-negative receptor Genetically it might be possible that the hALKI1250T mutation is only on one

copy of the ALK locus while the other one is wild type or contains a gain-of-function mutation like hALKF1174S. In order to answer this question we co-transfected PC12 cells with wild type hALK together with hALKI1250T. In this experiment, hALKI1250T seems to have a dominant-negative effect as indicated by reduced ERK phosphorylation on stimulated wild type ALK. This finding was confirmed by further results using different experimental approaches: (1) ERK phosphorylation is reduced upon co-transfection of hALKF1174S with hALKI1250T in PC12 cells. (2) Transfection of the neuroblastoma cell line CLB-GE (harbouring ALKF1174V) [134] with FLAG-tagged hALKI1250T results in reduced ERK phosphorylation. (3) Transient transfection of PC12 cells with FLAG-tagged hALKI1250T and untagged wild type ALK revealed that FLAG-hALKI1250T is not detectably tyrosine phosphorylated. (4) Co-transfection of PC12 cells with wild type ALK and ALKI1250T, followed by stimulation of ALK results in decreased ALK-mediated neurite outgrowth in the presence of ALKI1250T. Altogether, these results suggest that the expression of hALKI1250T might potentially act as a dominant-negative receptor when it is expressed together with active ALK receptors.

4.3 Ectopic expression of ALKI1250T in the Drosophila eye does not cause the rough eye phenotype As for the previously discussed mutations (Article II and III) we wanted to

investigate the activating potential of hALKI1250T by ectopical expression in the Drosophila eye. As described earlier (Article II and III), overexpression of wild type human ALK does not cause the rough eye phenotype, therefore providing a clean phenotypic background. Serving as a positive control for a gain-of-function mutation, ectopic expression of hALKF1174S results in the rough eye phenotype (see also Article II). However, ectopic expression of hALKI1250T neither leads to destruction of normal tissue morphology in the fly eye nor disrupts the organization of ommatidial units. This mutation rather shows a similar phenotype to wild type human ALK and controls, thereby suggesting that this mutant is not ligand-independent as initially predicted.

4.4 Why is the ALKI1250T mutant inactive? However, the question why the ALKI1250T mutation is a kinase dead mutation

still remains to be answered. Recently, structural studies by Bossi et al., and Lee et al., elucidating the crystal structure of the ALK kinase domain have shed some light on the understanding of the mechanistics of ALK mutations [1, 28]. Using the published crystal structure of the ALK kinase domain as a basis we suggest a mechanistic explanation for the inactivity of ALKI1250T. The mutation at amino acid

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position 1250 is located in the catalytic loop of the kinase domain and is highly conserved in the active site of protein kinases [1, 28]. These large, hydrophobic amino acids are included in the conserved catalytic loop sequence HRDI/LAARN, influencing the hydrophobic spine as well [10]. According to the ALK crystal structure forms contacts with hydrophobic residues from helix 1 and 2 (I1233, I1268, F1315) of the C-lobe. These interactions help to anchor the catalytic loop in a correct position in respect to the DFG loop and ATP. By replacing the hydrophobic isoleucine at position 1250 with threonine, which harbours a polar side chain, the hydrophobic contact/anchorage is probably weakened which most likely destabilizes the whole active site of ALK. Another hypothesis is that the I1250T mutation influences the interaction of the side chain of H1247 with the carbonyl oxygen of D1270 from the DFG motif, which could result in a destabilization of the kinase domain.

Overall, our structural hypothesis together with our obtained results are in stark contrast with earlier studies reporting that the ALKI1250T mutation is constitutively active and promotes oncogenicity [28, 135]. Therefore, the ALKI1250T mutation does not seem to be a driving force in the development and progression of neuroblastoma, implying that patients should be treated accordingly. However, it might be possible that ALKI1250T has a biological role in the presence of wild type or constitutively active ALK, i.e. that it acts as a dominant-negative receptor according to our results. Indeed, such kind of “cross-activation” has been reported for kinase dead BRAF and oncogenic RAS [370] or for ligand-induced trans-phosphorylation of receptors [371, 372]. However, this putative biological function of ALKI1250T needs to be validated by in vivo studies. On the other hand, one could argue that a dominant-negative mutation on one copy of the ALK locus in humans might have a minor effect as ALK knockout mice do not show any major phenotype ([373], BH and RHP unpublished results).

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5 Article V: “Anaplastic Lymphoma Kinase (ALK) regulates initiation of transcription of MYCN in neuroblastoma cells.”

In neuroblastoma amplification of the MYCN locus is one of the genetic hallmarks [212]. ALK gain-of-function mutations occur in about 6.9% of investigated neuroblastoma tumours according to a recent meta-analysis [213]. Further, this analysis revealed that ALK mutations occur in approximately 8.9% of MYCN amplified tumours, correlating with a poor clinical outcome [213]. Therefore we proposed the hypothesis that activated ALK might influence MYCN in neuroblastoma.

5.1 ALK regulates the MYCN promoter in PC12 cells and human neuroblastoma cell lines Initially we investigated whether ALK is able to regulate the MYCN

promoter. PC12 cells were co-transfected with human ALK and MYCNP-luciferase which contains approximately 600 bp before the transcription start site [374]. We observed that unstimulated wild type ALK induced a 5-fold increase of luciferase activity which might be caused by ALK overexpression. Stimulation with the agonist monoclonal antibody mAb46 further increased the luciferase activity 2 – 3-fold. Further, co-transfection with hALKF1174L and hALKR1275Q, the two most common ALK mutations in neuroblastoma, resulted in luciferase activity. To verify, that the luciferase activity was caused by ALK activity, we employed two ALK-specific inhibitors, NVP-TAE684 and crizotinib. Both inhibitors abrogate luciferase activity mediated by the ALK gain-of-function mutations and stimulated wild type ALK. Interestingly, inhibition of MYCNP-luciferase activity was less pronounced in hALKF1174L expressing PC12 cells, indicating that this mutation responds less to ALK-specific inhibitors [271]. Effective inhibition of ALK activity was analyzed by immunoblotting for phospho-ERK. Similar results were obtained using our PC12 cell clones which upon induction with doxycycline express wild type or mutant ALK (Article II – IV).

Next, we decided to investigate whether ALK is able to regulate the MYCN promoter in the background of neuroblastoma. We used several neuroblastoma cell lines with different genetic backgrounds: CLB-GA (MYCN non-amplified, 1p deletion, 11q deletion, 17q gain, non-amplified ALK containing ALKR1275Q mutation), CLB-GE (MYCN amplified, 1p deletion, 17q gain, amplified ALK containing ALKF1174V) and CLB-BAR (amplified MYCN and ALK, 1p deletion, 17q gain) [134, 375, 376]. Transfection of these cell lines with MYCNP-luciferase resulted in increased luciferase activity which could be reduced by treatment with NVP-TAE684 or crizotinib. Although the use of these neuroblastoma cell lines is more relevant to neuroblastoma than the controlled PC12 cell system, the interpretation and comparison of results using these cell lines is slightly problematic due to their different genetic backgrounds. The CLB-BAR cell line, for instance,

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expresses constitutively activated ALK, although no point mutations have been identified in the kinase domain, suggesting that other mechanisms somehow lead to ALK activation in this cell line [134]. An additional neuroblastoma cell line, IMR32 (MYCN non-amplified, exon 2 – 4 ALK amplified, 1p deletion, 17q gain), was investigated. Stimulation with mAb46 led to a modest increase in MYCNP-luciferase activity which could be blocked with crizotinib. To conclude, ALK regulates the initiation of transcription of the MYCN upstream promoter in PC12 cells and neuroblastoma cell lines.

5.2 Abrogation of ALK activity results in decreased MYCN mRNA and proliferation of neuroblastoma cell lines In order to further examine which role ALK plays in the initiation of MYCN

transcription we quantified the relative MYCN mRNA content by qRT-PCR. The neuroblastoma cell lines CLB-GA, -GE and –BAR showed reduced MYCN mRNA levels compared with untreated cells upon treatment with NVP-TAE684 and crizotinib as well as by siRNA mediated knockdown of ALK expression levels. Stimulation with the activating antibody mAb46 did not increase relative MYCN mRNA levels in these cell lines with constitutive active ALK. IMR32 cells showed a modest increase of MYCN mRNA upon stimulation with mAb46, which was reduced upon treatment with the ALK inhibitors. In order to show that the inhibitor concentrations used truly inhibit ALK activity, we assessed cell proliferation. Proliferation of CLB-GA, -GE and –BAR was inhibited by 250 nM crizotinib or 50 nM NVP-TAE684 respectively, suggesting that ALK activity is an important factor contributing to neuroblastoma cell proliferation. These results were confirmed by siRNA mediated reduction of ALK expression levels. However, proliferation of IMR32 cells was only slightly inhibited by 250 nM crizotinib, implying that this cell line is not as dependent on ALK activity as the others. But higher doses of crizotinib (500 nM) inhibited cell proliferation. Although crizotinib is a FDA-approved drug for the treatment of ALK-positive NSCLC patients, crizotinib is also an effective inhibitor of c-Met which is also involved in tumour progression. However, according to control experiments we can conclude that c-Met has no important function in this study.

Overall, inhibition of ALK activity in neuroblastoma cell lines with constitutively active ALK leads to reduced transcription of MYCN mRNA.

5.3 ALK activity regulates MYCN protein expression Next, we wanted to investigate what effect inhibition of ALK activity has on

MYCN protein levels in neuroblastoma cell lines. In order to detect MYCN in CLB-GA cells, which have non-amplified MYCN, twice as much cell lysate was loaded. Treatment with crizotinib or NVP-TAE684 (data not shown) decreased MYCN expression significantly, which was confirmed with siRNAs targeting ALK. Further,

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crizotinib treatment blocked ALK activity as indicated by decreased phosphorylation of ALK Y1278, which is important for the autoactivation of ALK [27]. Like in the previous experiments, IMR32 cells needed to be treated with mAb46 in order to observe a modest increase in MYCN and ALK activity, which could be inhibited by crizotinib treatment.

According to earlier studies high levels of MYCN are suggested to increase expression of Aurora kinase A mRNA directly or indirectly, and MYCN overexpression increases Aurora kinase A expression in SH-EP cells [377, 378]. Therefore, if MYCN is a transcriptional downstream target of ALK, we expect to see decreased Aurora kinase A expression when ALK is inhibited. Indeed, crizotinib treatment resulted in decreased expression levels of Aurora kinase A in all investigated neuroblastoma cell lines. Further, inhibition of ALK reduced the phosphorylation of PKB/Akt and ERK, confirming these signalling proteins as downstream targets of ALK. As a control the neuroblastoma cell lines were treated with PKB inhibitor LY294002 and ERK inhibitor U0126, which resulted in decreased MYCN expression levels. However, ERK inhibition in the IMR32 cells did not result in a pronounced decrease of MYCN levels. Generally the stability and degradation of MYCN appears to be regulated by several signalling pathways involving post-translational modifications like phosphorylation and ubiquitination, all forming a complex network [379]. MYCN is stabilized upon phosphorylation at Ser62, now being able to enter the nucleus and act as a transcription factor [380]. If in addition to Ser62, MYCN is phosphorylated at Thr58 mediated by GSK3-β in complex with Pin1, PP2A and Axin, MCYN is stabilized [379, 381, 382]. However, phosphorylation at Thr58 alone represents a signal for the recruitment of the ubiquitin ligase Fbxw7, resulting in ubiquitination and proteasomal degradation of MYCN. This process is suggested to occur at low levels of PI3K activity [378]. On the other hand, proteasomal degradation of MYCN can be blocked by recruitment of Aurora kinase A [378]. Further, PKB/Akt activates mTORC1, which downregulates the phosphatase PP2A, thereby blocking dephosphorylation of MYCN at Thr58 which leads to increased stability of MYCN. According to the report by Chesler et al., inhibition of PI3K activity destabilizes MYCN, blocking neuroblastoma progression as shown in the TH-MYCN neuroblastoma mouse model [383].

In order elucidate the effect of ALK on MYCN expression and stability in neuroblastoma cells, we blocked de novo protein biosynthesis by treatment with cycloheximide. Following addition of cycloheximide we could detect a decrease of MYCN protein but not MYCN mRNA in CLB-GE and CLB-BAR cells. In CLB-GA the decrease was not as pronounced which might be due to the fact that MYCN in non-amplified in this cell line. Further, treatment with MG-132, a proteasome inhibitor [384], increased MYCN expression levels, while treatment with crizotinib prior to MG-132 resulted in lower expression levels. Overall, we can conclude that active ALK primarily influences the transcription of MYCN in neuroblastoma cell lines, although post-transcriptional regulation cannot be excluded.

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5.4 ALK and MYCN co-operate in transforming NIH3T3 cells In previous studies we reported that the human ALK gain-of-function

mutations ALKF1174S, ALKF1174L and ALKR1275Q have transforming potential in NIH3T3 cells (Article II and III). As activated ALK drives initiation of MYCN transcription, we planned to investigate whether ALK and MYCN have a synergistic effect on NIH3T3 cell transformation. Expression of ALKF1174L or ALKR1275Q mediates foci formation, while expression of MYCN or wild type ALK alone does not result in transformation. However, ALKF1174L or ALKR1275Q together with MYCN result in a robust increase in transformation compared with mutated ALK alone. Hence, constitutively active ALK together with MYCN results in more potent transforming potential.

Altogether, we could demonstrate that activated ALK, either by stimulated wild type or by gain-of-function mutations, activates the initiation of MYCN transcription. Blocking ALK activity either by crizotinib, NVP-TAE684 or siRNA resulted in reduced MYCN mRNA, MYCN protein levels and decreased proliferation of neuroblastoma cell lines. Further, co-expression of ALK gain-of-function mutants together with MYCN results in a synergistic effect on NIH3T3 cell transformation. The latter finding might be important for patients with chromosome 2 amplification, i.e. having MYCN amplification together with wild type or constitutively active ALK mutants, resulting in extremely poor clinical outcome. This is supported by a previous report by Berthier et al., describing a relationship between ALK and MYCN expression and MYCN amplification repectively [385]. Overall, this study provides further evidence for ALK as an important player in the development and progression of neuroblastoma. Additionally, this report suggests that ALK is a promising target in treating neuroblastoma patients harbouring MYCN and ALK amplification. Recently, an elegant study by Zhu et al., reported results in accordance to ours. They generated transgenic zebrafish models which developed neuroblastoma upon MYCN overexpression in the fish analog of the adrenal medulla. Zebrafish overexpression ALK or ALKF1174L on its own did not result in tumour formation. However, fish overexpressing both MYCN and ALKF1174L showed increased tumour onset and penetrance as a result of the synergistic effects of the two oncogenes. Further, Liu et al., propose that ALK gain-of-function mutations might be the “initiating” events in neuroblastoma development and need a “second hit” like MYCN amplification/overexpression in order to achieve full transformation of cells [386, 387].

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Summary of the main findings of this thesis

Article I • ALK activates the small GTPase Rap1 via C3G/CrkL which can be blocked by

the small ALK-specific inhibitor NVP-TAE684. • Rap1 activation does not seem to be crucial for ALK mediated ERK

phosphorylation in PC12 cells. • ALK-mediated activation of the C3G/CrkL/Rap1 pathway leads to neurite

outgrowth in PC12 cells and proliferation in neuroblastoma cell lines.

Article II • The human ALKF1174S mutant found in a neuroblastoma patient is a gain-of-

function mutant with transforming potential, correlating with aggressive disease development.

Article III • Six investigated ALK mutations (hALKG1128A, hALKI1171N, hALKF1174L,

hALKR1192P, hALKF1245C and hALKR1275Q) found in neuroblastoma patients with a high probability of being oncogenic, are truly constitutively active.

• These ALK mutations respond with different IC50 to the ALK inhibitors NVP-TAE684 and crizotinib and possess different transforming potential.

• These ALK gain-of-function mutations are suggested to have a different impact on development, onset and severity of neuroblastoma.

• Somatic mutations like ALKF1174L or ALKF1245C appear to be more aggressive than germ line mutations like ALKG1128A or ALKR1192P.

Article IV • The hALKI1250T mutation, which was initially discovered as a germ line mutation

with a high probability of being oncogenic, is in fact a kinase dead mutation and cannot be activated by monoclonal antibodies.

• Results suggest that this mutation acts as a dominant-negative receptor.

Article V • ALK regulates the initiation of MYCN transcription both in PC12 and human

neuroblastoma cell lines. • Abrogation of ALK activity decreases proliferation of neuroblastoma cell lines

and MYCN mRNA as well as protein expression. • Co-expression of ALK gain-of-function mutants together with MYCN results in a

synergistic effect on NIH3T3 transformation, which suggests an extremely poor clinical outcome for neuroblastoma patients with the appropriate genetic background.

• ALK is shown to be an important player in the development and progression of neuroblastoma and might therefore serve as an excellent therapeutical target.

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Future perspectives

In the last couple of years ALK appeared to be more and more clinically important in the development and progression of cancer, in particular neuroblastoma and NSCLC. Hence, targeting ALK exhibited potential as an effective and more specific treatment of ALK-positive cancer patients. Since the discovery of ALK point mutations, it has become increasingly obvious that the type of ALK mutation, i.e. germ line vs somatic mutations and driver vs passenger mutations, might have an impact on the choice and success of patient specific treatment.

The development of ALK inhibitors has undergone great progress with very promising results from the first clinical trials for patients with ALK-positive cancers which has resulted in FDA approval of crizotinib under the name Xalkori only four years after the first discovery. However, this is no reason to lean back and relax: the appearance of crizotinib-resistant secondary ALK mutations demands the development of second generation ALK inhibitors to overcome this resistance. However, according to Katayama et al., secondary ALK mutations do not represent the only mechanism for crizotinib resistance. Amplification of the ALK locus serves as another resistance mechanism as well as “diverted” signalling via EGFR auto-phosphorylation and KIT amplification [251]. Therefore, a combinatorial therapy involving ALK inhibitors and inhibitors targeting other RTKs or downstream signalling proteins will most likely be required for the future therapy of ALK-positive cancer patients. Indeed, Tanizaki et al., reported recently, that the combination of the ALK-specific inhibitor NVP-TAE684 and the MEK inhibitor AZD6244 induced apoptosis and inhibition of STAT3 and ERK pathways in EML4-ALK positive NSCLC cells, that were unresponsive to NVP-TAE684 treatment alone [388]. An additional strategy for ALK-positive neuroblastoma therapy is the ALK-targeted immunotherapy including crizotinib and ALK-targeting antibodies [295]. This combinatorial therapy using tyrosine kinase inhibitors and antibodies has been successfully tested for NSCLC expressing erlotinib-resistant EGFRT790M, inducing tumor regression [389]. The ALKF1174L mutant which is resistant to crizotinib, behaves fairly similar to EGFRT790M [271], suggesting such kind of dual therapy might be beneficial for neuroblastoma patients harbouring the ALKF1174L mutant.

A futuristic therapeutical approach for neuroblastoma patients harbouring amplified ALK, or ALK gain-of-function mutations together with amplified MYCN, might involve the use of an ALK inhibitor combined with liposomes containing siRNA targeting MYCN. However, recent studies proposed therapeutical approaches including indirect targeting of MYCN. The PI3K/mTOR inhibitor NVP-BEZ235 has been shown to drive MYCN degradation in tumour cells, which resulted in decreased tumour proliferation and angiogenesis [390]. Further, the authors suggest that NVP-BEZ235 should be clinically tested in neuroblastoma patients with MYCN amplification. Another therapeutical target could be Aurora

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kinase A which is able to prevent proteasomal degradation of MYCN [378]. Recently, the Aurora kinase inhibitor CCT137690 has been shown to reduce MYCN expression and block proliferation of MYCN-amplified neuroblastoma cell lines [391]. Hence, a combination of an ALK and PI3K/mTOR or Aurora kinase A inhibitor could serve as an alternative therapy especially for MYCN-amplified neuroblastoma patients with ALK gain-of-function mutations.

In spite of the growing prominent role of ALK in oncogenesis, many questions regarding wild type ALK still persist. Since the initial discovery of ALK in the 1990s the natural ligand of ALK still remains a mystery. The identification of the Drosophila ALK ligand Jeb provided one clue: Jeb is not conserved in mammals. So what is the ligand for mammalian ALK? Linked to this question is another one: what is the physiological function? To date one can only speculate that ALK might be involved in the nervous system. The mouse model appears to be the method of choice to study the physiological role of mammalian ALK. However, ALK knockout mice have no significant phenotype, except that they are slightly smaller and show certain behavioral and neurochemical alterations ([81, 82, 392], BH and RHP unpublished results). In general, many questions and challenges remain and new ones will emerge. Great efforts will be made to tackle those tasks, on the one hand trying to understand the physiological function of wild type ALK and finding the natural ligand, on the other hand trying to elucidate the genetic variability of ALK-positive cancer patients. This allows us to look into an optimistic future of individual cancer therapy based on the patients’ genetic background and might contribute to winning the “War on Cancer”.

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Acknowledgements

During my PhD journey many people have crossed my way, contributing to this thesis and making it possible, as well as sharing highs and lows.

Most importantly, finishing this PhD thesis would not have been possible without the support of my supervisors Bengt and Ruth. Thank you for giving me this great opportunity of doing my PhD in your labs, for your ever lasting enthusiasm and optimism and all valuable lessons I have learnt! Rutan: thanks for your immense expertise, invaluable advice and discussions within and outside science! All past and present group members, especially Hai-Ling for setting up the PC12 cell system. Lovisa Olofsson for initiating the Rap1 project. The RHP group for sharing many journal clubs and group meetings.

A great thanks to all collaborators and co-authors who contributed to the work. Particular thanks to the technical and administrative personnel, especially Maria, Ethel, Anitha, Johnny, Marek and Media & Dishes: thank you for taking care of administrative issues, orders, computer issues, solutions and heaps of glass ware! During my time at the department many people have crossed my way, sharing teaching, taking courses, lunch breaks, Fika, parties, IKSU classes, … Thank you all (past and present members) at the Molecular Biology Department for a nice time! People outside the Department and outside the “science world”, i.e. the “real world”: Emma: thank you for helping me to get started in Sweden and for being a very good friend. No one can beat our Skype dates ;-) Matilda: thanks for a nice time with hiking and skiing trips, rafting, IKSU and for being my friend. Thanks to the German gang at the Department: without you my German would have deteriorated! The book club: yes, we really tried to discuss the books besides other matters! ☺ The craft night gang: one time in the future my craft project will be finished! ;-) Thanks to the orchestras, Umeå Musiksällskap and Hemvärnets Musikkår Umeå, for providing great distraction from science. Jana, thanks for introducing me to HvMk Umeå!

Thanks to my friends back in Germany for keeping in contact over the distance and over all the years: Ulrike, Antje, Mirjam, and Stefan. Finally, thanks to all people not mentioned, but not forgotten.

Last but not least, I would like to thank my family. Especially Mum and Dad, for always believing in me, giving me all the support I could ever ask for and for always being there for me. You are the best!

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