BioIDentification of Alk-associated signaling complexes Ezgi Uçkun Department of Medical Biochemistry and Cell Biology Institute of Biomedicine Sahlgrenska Academy, University of Gothenburg Gothenburg 2022 Cover illustration: Proximity labeling of Alk proximitomes in neuroblastoma cells (bottom left) and the Drosophila larval brain (up right). SK-N-AS cells co- stained with anti-ALK (red) and biotin (green). Drosophila third instar larval brain co-stained with anti-Alk (blue) and biotin (red). Cover design created with BioRender.com by Ezgi Uçkun BioIDentification of Alk-associated signaling complexes © Ezgi Uckun 2022 ezgi.uckun@gu.se ISBN 978-91-8009-969-1 (PRINT) ISBN 978-91-8009-970-7 (PDF) http://hdl.handle.net/2077/72582 Printed in Borås, Sweden 2022 Printed by Stema Specialtryck AB I would like to dedicate this thesis to my family who supported me in every way and believed in me throughout my life. BioIDentification of Alk-associated signaling complexes Ezgi Uçkun Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg Gothenburg, Sweden ABSTRACT Anaplastic lymphoma kinase (Alk) is a receptor tyrosine kinase (RTK) of the insulin receptor family. Alterations in human ALK signaling have been implicated in multiple malignancies including pediatric neuroblastoma. In addition to its role in oncogenesis, previous studies on both invertebrate and vertebrate model organisms have revealed a role for Alk signaling in the central nervous system including axon targeting, synapse development, growth and body size regulation, brain sparing, memory formation and learning, circadian rhythm, and longevity. Although the Alk receptor is associated with a wide range of processes, downstream components of Alk signaling are highly diverse and it is unclear how Alk signaling intersects with different downstream effectors, especially in different tissues. Analysis of Alk- associated signaling complexes in the context of wild-type, active and inactive Alk status in different tissues provides essential information, not only for the development of therapeutic approaches for targeting ALK-driven cancers, but also for understanding its role in neurodevelopmental processes. In this thesis, I have employed BioID-based proximity labeling (PL) to identify components of Alk signaling in both neuroblastoma (NB) cells (Study I) and the Drosophila larval brain (Studies II and III). In Study I, PL was performed in the presence or absence of ALKAL ligand stimulation as well as upon ALK inhibitor treatment. We identified PTPN11/SHP2 and PEAK1 as activity- dependent ALK interactors and functionally investigated the role of the protein tyrosine phosphatase SHP2 in ALK-addicted NB cells. In Study II, we defined the wild-type Alk proximitome by using three different BioID enzyme variants and identified the SHP2 ortholog Corkscrew (Csw) as a downstream component of Alk signaling. In the last study, we performed PL both in the presence or absence of Jeb ligand overexpression as well as in a gain-of- function Alk mutant and identified the LDL receptor related protein 4 (Lrp4) as a negative regulator of Alk activity in the Drosophila brain. Keywords: ALK, neuroblastoma, proximity labeling, BioID, miniTurbo, TurboID ISBN 978-91-8009-969-1 (PRINT) ISBN 978-91-8009-970-7 (PDF) SAMMANFATTNING PÅ SVENSKA Anaplastiskt lymfomkinas (Alk) är ett receptortyrosinkinas (RTK) som ingår i insulinreceptorfamiljen. Felaktig ALK-signalering hos människa är delaktig i flera olika maligniteter, inklusive neuroblastom hos barn. Förutom dess roll i cancer har tidigare studier på både ryggradslösa och ryggradslösa modellorganismer visat att Alk-signalering spelar en central roll i nervsystemet, bland annat i fråga om axon-medierade signaler, synapsutveckling, reglering av tillväxt och kroppsstorlek, i hjärnan, minnesbildning och inlärning, cirkadisk rytm och och inte minst livslängd. Även om ALK-receptorn är förknippad med ett brett spektrum av processer är nedströmskomponenterna i Alk-signalering mycket varierande och det är oklart hur Alk-signalering interagerar med olika nedströmseffektorer, speciellt i olika vävnader. Analys av Alk-medierade signalkomplex i samband med vildtypsuttryck av ALK, både aktiv och inaktiv Alk i olika vävnader ger viktig bas-information, inte bara för utveckling av terapeutiska metoder, utan också för att förstå dess roll i neuroutvecklingsprocesser. I den här avhandlingen använde jag BioID-baserad proximity labeling (PL) för att identifiera komponenter, såkallade intraktionspartners till Alk och Alk- signalering både i neuroblastom celler (NB) (studie I) och i hjärnan hos bananfluge (Drosophila melanogaster) larver (studier II och III). I första studien utfördes PL i närvaro eller frånvaro av ALKAL-ligandstimulering tillsammans med eller utan behandling med ALK-hämmare. Vi identifierade SHP2 och PEAK1 som aktivitetsberoende intraktionspartners till ALK-. Funktionella studier utfördes på proteintyrosinfosfatasets PTPN11/SHP2 roll i ALK-beroende NB-celler. I den andra studiendefinierade vi proximitomet av vildtypen Alk genom att använda tre olika BioID-enzymvarianter och identifierade SHP2-ortologen Corkscrew (Csw) som en nedströmskomponent i Alk-signalering. I den sista studien utförde vi PL både i närvaro eller frånvaro av överuttryck av Jeb-liganden samt i en enzymatisk aktiv Alk-mutant och identifierade LDL-receptorrelaterat protein 4 (Lrp4) som en negativ regulator av Alk-aktiviteten i Drosophilahjärnan. Nyckelord: ALK, neuroblastom, närhet märkning, BioID, miniTurbo, TurboID LIST OF PAPERS This thesis is based on the following studies. I. Uçkun E, Siaw JT, Guan J, Anthonydhason V, Fuchs J, Wolfstetter G, Hallberg B, Palmer RH. BioID-screening identifies PEAK1 and SHP2 as components of the ALK proximitome in neuroblastoma cells. J. Mol. Biol. (2021) II. Uçkun E, Wolfstetter G, Anthonydhason V, Sukumar SK, Umapathy G, Molander L, Fuchs J, Palmer RH. In vivo profiling of the Alk proximitome in the developing Drosophila brain. J. Mol. Biol. (2021) III. Uçkun E, Pfeifer K, Guan J, Wolfstetter G, Anthonydhason V, Palmer RH. Proximity labeling identifies regulators of Alk signaling in the Drosophila CNS. (Manuscript) i LIST OF PAPERS NOT INCLUDED IN THIS THESIS • Uçkun E, Wolfstetter G, Fuchs J, Palmer RH. In vivo characterization of endogenous protein interactomes in Drosophila larval brain, using a CRISPR/Cas9-based strategy and BioID-based proximity labeling. Bio-protocol. (2022) • Mendoza-Garcia P, Basu S, Sukumar SK, Arefin B, Wolfstetter G, Anthonydhason V, Molander L, Uçkun E, Lindehell H, Lebrero-Fernandez C, Larsson J, Larsson E, Bemark M, Palmer RH. DamID transcriptional profiling identifies the Snail/Scratch transcription factor Kahuli as an Alk target in the Drosophila visceral mesoderm. Development. (2021) • Siaw JT, Gabre JL, Uçkun E, Vigny M, Zhang W, Van den Eynden J, Hallberg B, Palmer RH, Guan J. Loss of RET Promotes Mesenchymal Identity in Neuroblastoma Cells. Cancers. (2021) • Wolfstetter G, Pfeifer K, Backman M, Masudi TA, Mendoza- García P, Chen S, Sonnenberg H, Sukumar SK, Uçkun E, Varshney GK, Uv A, Palmer RH. Identification of the Wallenda JNKKK as an Alk suppressor reveals increased competitiveness of Alk-expressing cells. Sci Rep. (2020) ii LIST OF CONTENTS ABBREVIATIONS………………………………………….…………………..…………………….......V 1.INTRODUCTION ............................................................................................ 1 1.1 Mechanisms of signal transduction ...................................................... 1 1.2 Receptor tyrosine kinases (RTKs) .......................................................... 3 1.2.1 RTK structure and physiological activation ................................... 3 1.2.2 Mechanisms of RTK activation in cancer ....................................... 5 1.3 Anaplastic lymphoma kinase (ALK) ....................................................... 5 1.3.1 Biological functions of Alk in Drosophila ....................................... 8 1.3.2 ALK signaling in human cancers .................................................. 10 1.4 The protein tyrosine phosphatase PTPN11/SHP2 ............................... 12 1.4.1 Regulation of SHP2 ...................................................................... 13 1.4.2 Roles of SHP2/Csw in RTK signaling ............................................. 15 1.4.3 SHP2 as therapeutic target .......................................................... 16 1.5 Neuroblastoma.................................................................................... 17 1.5.1 Genomic aberrations and mutations in neuroblastoma ............. 18 1.5.2 ALK in neuroblastoma ................................................................. 19 1.5.3 ALK signaling in neuroblastoma .................................................. 19 1.5.4 Targeting oncogenic ALK ............................................................. 21 1.6 Investigating protein-protein interactions .......................................... 22 1.6.1 Antibody-based affinity purification ........................................... 23 1.6.2 Yeast two-hybrid (Y2H) ................................................................ 24 1.7 Proximity labeling techniques ............................................................. 25 1.7.1 Peroxidase-based proximity labeling .......................................... 26 1.7.2 Biotin ligase-based proximity labeling......................................... 28 2.AIMS……………………………………………… …......……….…….……………………………….31 3.METHODS ................................................................................................... 32 3.1 Cloning and generation of Tet-On BioID NB cell lines………….…………..32 3.2 Western blotting and quantification…………………......... ..….…………….. 32 3.3 Immunostaining of NB cells …………………… .................. ….……………….. 32 3.4 Immunoprecipitation………………………… ......... ……..……......………………..33 3.5 Proliferation assays………………………… ........... …..…….…......………………..33 3.6 Foci formation assays…………………………… ....... ……….…......………………..33 3.7 Generation of endogenous Alk-BioID fusions by CRISPR/Cas9 genome editing in Drosophila……………………….. .......... ….…..……………..34 3.8 Immunostaining of Drosophila embryos………………… ..... …...…………….34 3.9 Immunostaining of third instar larval brains………… ..... …...….…………..35 3.10 Pull-down of biotinylated proteins……………………………………….............35 iii 3.10.1 Preparation of inducible NB cell lysates ..................................... 35 3.10.2 Preparation of Drosophila larval brain lysates ........................... 35 3.10.3 Streptavidin pull-down ............................................................... 36 3.11 Protein digestion, TMT-labeling, liquid chromatography-coupled mass spectrometry (LC/MS)…………………… ............................. …………...………………..36 4.RESULTS AND DISCUSSION ......................................................................... 37 4.1 Paper I ..................................................................................................... 37 4.2 Paper II .................................................................................................... 38 4.3 Paper III ................................................................................................... 41 5.CONCLUSIONS ............................................................................................ 43 5.1 Paper I ..................................................................................................... 43 5.2 Paper II .................................................................................................... 43 5.3 Paper III ................................................................................................... 43 ACKNOWLEDGEMENTS ................................................................................. 44 REFERENCES .................................................................................................. 49 iv ABBREVIATIONS AD - Activation domain ALCL - Anaplastic large cell non-Hodgkin’s lymphoma ALK - Anaplastic lymphoma kinase ALKAL1/2 - ALK and LTK ligand 1/2 AP - Affinity purification APEX - Ascorbate peroxidase ATC - Anaplastic thyroid carcinoma ATR - Ataxia telangiectasia and Rad3-related ATRX - Alpha thalassemia/mental retardation syndrome X-linked Ave - Aveugle BAP - Biotin acceptor peptide BCR - Breakpoint cluster region BioID – Proximity-dependent biotin identification BirA - Biotin protein ligase BRD2 - Bromodomain-containing protein 2 Cas9 - CRISPR-associated protein 9 Cnk - Connector enhancer of kinase suppressor of Ras CNS - Central nervous system CRISPR - Clustered regularly interspaced short palindromic repeats Csw - Corkscrew DBD - DNA binding domain DLBCL - Diffuse large B-cell lymphoma v Dlg1 – Disc large 1 Dos - Daughter of sevenless DUSP4 - Dual specificity protein phosphatase 4 ECD - Extracellular domain ER - Endoplasmic reticulum ERF - ETS domain-containing transcription factor ESCC - Esophageal squamous cell carcinoma ETV - ETS translocation variant FC - Founder cells FGFR - Fibroblast growth factor receptor FOXO - Forkhead box FRS2/3 - Fibroblast growth factor receptor substrate 2/3 GAB1/2 - GRB2 associated binding protein 1/2 GlyR - Glycine-rich region GOF - Gain-of-function GRB2 - Growth factor receptor-bound protein 2 HDR – Homology-directed repair HRP - Horseradish peroxidase IMT - Inflammatory myofibroblastic tumor INRG - International Neuroblastoma Risk Group INSS - International Neuroblastoma Staging System IP - Immunoprecipitation IR - Insulin receptor vi IRS1/2 - Insulin receptor substrate 1/2 JAK - Janus kinase Jeb - Jelly belly Kah - Kahuli LDLa - Low-density lipoprotein receptor class A LOF - Loss-of-function Lrp4 – LDL receptor-related protein 4 LS - LEOPARD syndrome LTK - Leukocyte tyrosine kinase MAM - Meprin, A-5 protein, and receptor protein-tyrosine phosphatase mu MAMTH - Mammalian Membrane Two-Hybrid MB - Mushroom body MELK - Maternal embryonic leucine zipper kinase MS - Mass spectrometry NB - Neuroblastoma Nf1 - Neurofibromatosis 1 NF-B - Nuclear factor-kB NMJ - Neuromuscular junction NS - Noonan syndrome NSCLC - Non-small cell lung cancer ORF - Open reading frame Org-1 - Optomotor-blind-related gene-1 PD-1 - Programmed cell death-1 vii PIK3R1/2 - Phosphoinositide-3-kinase regulatory subunit 1/2 PL - Proximity labeling PLCγ - PhospholipaseCγ POI - Protein of interest PSD - Postsynaptic density PPI - Protein-protein interactions PTB - Phosphotyrosine-binding PTK - Protein tyrosine kinase PTM - Post-translational modification PTPN11 - Protein Tyrosine Phosphatase Non-Receptor Type 11 RCC - Renal cell carcinoma Rg - Rugose RhoGAP15B - Rho GTPase activating protein 15B RMC - Renal medulla carcinoma Rl - Rolled RTK - Receptor tyrosine kinase Sdt - Stardust Sev - Sevenless SFK - Src family kinases SH2 - SRC-Homology 2 SHP2 - SH2 containing protein tyrosine phosphatase 2 SOS1/2 - Son of sevenless homolog 1/2 Spry1/2 - Sprouty 1/2 viii STAT - Signal transducer and activator of transcription SUN2 - Sad1 and UNC84 domain containing 2 Syx1A - Syntaxin 1A TERT - Telomerase reverse transcriptase TKD - Tyrosine kinase domain TKI - Tyrosine kinase inhibitor TM - Transmembrane domain UAS - Upstream activation sequence VM - Visceral mesoderm Y2H - Yeast two-hybrid ix 1. INTRODUCTION 1.1 Mechanisms of signal transduction Cell-cell communication is an important process that mediates signaling and biological function between cells to maintain homeostasis of cells and organs. Cells communicate by mainly four mechanisms: (1) endocrine signaling in which hormones are released by endocrine cells and transported to distant cells via the circulatory system, (2) paracrine signaling, which enables communication between nearby cells through diffusing growth factors, neurotransmitters, and cytokines, (3) juxtracrine signaling which is a contact- dependent signaling via membrane-bound signaling molecules, and (4) autocrine signaling in which cells respond to their own signals by secreting ligands that bind to receptors on the same cell (Trosko and Ruch, 1998, Cooper and Hausman, 2009). Intracellular signal transduction pathways transmit these signals from the extracellular space to the intracellular milieu and thereby activate cellular responses, such as gene expression (Bluthgen and Legewie, 2013). The regulation of these complex, multilayered signaling pathways through reversible covalent modifications (e.g., phosphorylation), allosteric regulation as well as crosstalk with other pathways affects signaling outcome (Hornberg et al., 2006). Signal transduction is facilitated by proteins with intrinsic enzymatic activity in their catalytic domains such as kinases, phosphatases, lipases, and phospholipases as well as non-enzymatic adaptor proteins with conserved interaction domains and unique binding motifs. The activity of these signaling proteins must be tightly regulated to ensure fidelity of a wide range of complex biological functions (Lee and Yaffe, 2016, Borowicz et al., 2020). The enzymatic phosphorylation of proteins was described by Burnett and Kennedy in 1954 for the first time (Burnett and Kennedy, 1954). Soon after, Fischer and Krebs described the conversion of phosphorylase b to phosphorylase a, and the mechanism of phosphorylation/dephosphorylation (Fischer and Krebs, 1955, Krebs and Fischer, 1956). Phosphorylation is a 1 reversible post-translational modification (PTM) that is regulated by kinases and phosphatases, which are responsible for addition or removal of phosphate (PO 3-4 ) groups respectively (Li et al., 2013, Lee and Yaffe, 2016, Fischer and Krebs, 1955, Krebs and Fischer, 1956) (Figure 1). Previous phosphoproteomic studies have mostly focused on phosphorylation of serine (86%), threonine (12%), and tyrosine (2%) since they are stable in low pH conditions suitable for mass spectrometry (MS) applications (Eckhart et al., 1979, Hunter, 1998, Nishi et al., 2014, Olsen et al., 2010, Olsen et al., 2006). As a result of improved technical and analytical approaches in MS, phosphorylation of aspartic acid, glutamic acid, histidine, lysine, arginine, and cysteine residues has also been identified (Fuhs and Hunter, 2017, Attwood et al., 2007, Lapek et al., 2011, Cieśla et al., 2011, Lapek et al., 2015). It is now appreciated that the activity of many proteins is mediated by phosphorylation and dephosphorylation events during signal transduction, making it a crucial mechanism of regulation in a wide range of cellular processes (Ardito et al., 2017). Figure 1: Regulation of protein phosphorylation by kinases and phosphatases. A protein (blue) on the left is phosphorylated by a kinase that catalyzes phosphate (PO43-, depicted in red) transfer to certain residues using ATP as a donor. Phosphorylation can be reverted by phosphatases that remove PO43- by hydrolysis. Created with BioRender.com. 2 1.2 Receptor tyrosine kinases (RTKs) Protein tyrosine kinases (PTKs) are enzymes that transfer the terminal phosphate group of ATP onto tyrosine residues of a substrate. Among the protein tyrosine kinases in the human genome, 58 of them are receptor tyrosine kinases (RTKs) within 20 subfamilies, while 32 are non-receptor tyrosine kinases (Hubbard and Miller, 2007). The less complex Drosophila genome encodes 20 RTKs, most of whom have mammalian orthologs (Mele and Johnson, 2019). RTKs are transmembrane proteins belonging to a large family of cell-surface receptors that regulate cell proliferation, cell survival, differentiation, cell migration, cell division as well as metabolism (Lemmon and Schlessinger, 2010, Blume-Jensen and Hunter, 2001). 1.2.1 RTK structure and physiological activation Although the different RTK subfamilies are involved in distinct cellular processes, they share a similar domain architecture involving a ligand-binding extracellular domain (ECD), a transmembrane (TM) domain and an intracellular region with a juxtamembrane regulatory region and a tyrosine kinase domain (TKD) (Huang, 2021, Hubbard, 1999). While TKDs are conserved, ECDs are diverse in terms of size and functional subdomains, allowing them to bind structurally different ligands (Trenker and Jura, 2020). Aberrant RTK signaling has been associated with development and progression of diseases including diabetes, inflammation, angiogenesis, several developmental conditions, and cancer (Odawara et al., 1989, Almendro et al., 2010, McDonell et al., 2015, Mustonen and Alitalo, 1995, Choura and Rebaï, 2011). In addition to their well-known role in cancer development, RTKs also play important roles in various developmental processes such as cell differentiation, tissue patterning, cell migration through the control of cell shape as well as tissue regeneration and cell survival (Perrimon et al., 2012, Brückner et al., 2004, Hu and Olsen, 2016, Park and Lee, 2015). Studies in model organisms such as the fruit fly Drosophila melanogaster have 3 expanded our knowledge regarding RTK function during development. This is mainly due to the short life cycle of the fly, reduced complexity, availability of genetic tools, as well as high conservation between the Drosophila and human RTK families. (Cheng et al., 2018, Rubin et al., 2000). RTKs are activated by a specific ligand or set of ligands, resulting in conformational changes and recruitment of a second receptor monomer. In many cases, dimerization of two receptor monomers leads to trans- autophosphorylation of tyrosine residues in the TKD activation loop and stabilizes the active conformation (Du and Lovly, 2018, Trenker and Jura, 2020, Mele and Johnson, 2019). Autophosphorylation triggers activation of downstream signaling through recruitment of effector proteins containing SRC-Homology 2 (SH2) and phosphotyrosine-binding (PTB) domains that bind to phosphotyrosine-containing motifs. These effector proteins transmit RTK phosphorylation to downstream signaling pathways (Figure 2) (Mele and Johnson, 2019, Pawson et al., 2001). Figure 2: Schematic representation of RTK activation mechanism. (1) In the absence of its specific ligand, the receptor is inactive. (2) Upon ligand binding, two receptor monomers dimerize, (3) resulting in trans-autophosphorylation of tyrosine residues in the kinase domain. (4) Downstream signaling pathways are activated through recruitment of effector proteins. Created with BioRender.com. 4 1.2.2 Mechanisms of RTK activation in cancer Constitutive activation of RTK signaling allows cells to acquire oncogenic properties and promotes oncogenesis. Four main mechanisms are known to contribute to constitutive RTK activation: (1) genomic amplification of the receptor encoding gene leading to overexpression of the receptor, (2) chromosomal rearrangements involving the receptor encoding gene that result in the generation of fusion oncoproteins with ectopic activity and/or ectopic expression profiles, (3) gain-of-function point mutations within the RTK molecule and (4) aberrant autocrine activation (Du and Lovly, 2018). Overexpression of RTKs often occurs due to increased copy number (such as genomic amplification or gain) and leads to high local concentrations of receptor. Amplification of RTKs occurs in many cancers and is often associated with poor patient outcome. Chromosomal rearrangements have also been described in various cancers and result in formation of RTK fusion oncoproteins which consist of part of RTK and part of the fusion partner (Du and Lovly, 2018). Acquired somatic mutations in RTKs are often clustered into hot spot regions around the DFG motif and nucleotide-binding pocket which are important for ATP binding and catalytic regulation (Lahiry et al., 2010). In some cases, cancer cells can secrete ligands themselves to activate specific RTKs, and this mechanism is known as autocrine activation. Increased local concentrations of ligand causes constitutive RTK activation (Kentsis et al., 2012, Singh and Harris, 2005, Ciardiello and Tortora, 2001). 1.3 Anaplastic lymphoma kinase (ALK) Anaplastic lymphoma kinase (ALK) and the related leukocyte tyrosine kinase (LTK) are RTKs belonging to the insulin receptor (IR) superfamily (Hallberg and Palmer, 2013). ALK was initially described in 1994 as part of a chimeric oncogene generated by a (2;5) chromosomal rearrangement in anaplastic large cell non-Hodgkin’s lymphoma (ALCL) (Morris et al., 1994). Some years later, the full-length ALK receptor was characterized, revealing a gene located on chromosome 2p, encoding a 177 kDa protein which undergoes N-linked 5 glycosylation to form the mature ALK protein of approximately 220 kDa (Iwahara et al., 1997, Morris et al., 1997). The ALK TKD is composed of N-terminal and C-terminal lobes, connected by a hinge region that forms a cavity for ATP binding. The small N-terminal lobe contains the αC helix and glycine loop, while the larger C-terminal lobe contains the activation loop harboring the Y'RAS'YY autophosphorylation motif and the catalytic loop (Figure 3) (Lee et al., 2010, Bossi et al., 2010, Hallberg and Palmer, 2016). Although the TKD domain is highly similar to that of the IR, the extracellular region (ECR) of ALK is unique among the RTKs. The ECR contains two meprin, A-5 protein and receptor protein-tyrosine phosphatase mu (MAM) domains, a low-density lipoprotein receptor class A (LDLa) domain, a ligand-binding TNF-like, glycine-rich region (GlyR) and EGF- like domains (Figure 3) (Iwahara et al., 1997, Morris et al., 1997, Reshetnyak et al., 2021, De Munck et al., 2021). The small secreted FAM150 family proteins, ALKAL1 and ALKAL2, have been identified as ligands of human ALK (Guan et al., 2015, Reshetnyak et al., 2015). Recently, the structure of ALKALs bound to the TNF-like, GlyR and EGF-like portions of the ALK ECR was solved, confirming earlier observations that suggested this portion of the ALK ECR was involved in dimerization and activation of ALK by ligand (Figure 3) (Guan et al., 2015, Reshetnyak et al., 2015, Reshetnyak et al., 2021, De Munck et al., 2021, Li et al., 2021). The ALK RTK has various roles in both normal development and oncogenesis, which will be described in detail in next two chapters, 1.3.1 and 1.3.2. 6 Figure 3: Domain architecture of human ALK. The ALK extracellular region (ECR) is composed of two meprin, A-5 protein and receptor protein-tyrosine phosphatase mu (MAM) domains, a low-density lipoprotein receptor class A (LDLa) domain and the ligand-binding glycine-rich domain (GRD) containing TNF-like, glycine-rich region (GlyR) and EGF-like region. The transmembrane domain (TM) connects the ECR region with the tyrosine kinase domain (TKD) containing intracellular region. On the top right, the structure of the ALK GRD bound to ALKAL2 (PDB:7LS0) determined by X-ray crystallography is shown. The TNF-like region is depicted in dark blue. The poly-glycine extension and poly-glycine extension loop are indicated in pink and orange respectively. ALKAL2 AD domain was shown in green (Li et al., 2021). On the bottom right, the crystal structure of the ALK TKD (PDB: 3LCS) is shown. The glycine loop (blue), αC helix (red), catalytic loop (teal) and activation loop of the TKD are highlighted. ADP (orange) is shown in the ATP binding site (light blue) (Bossi et al., 2010). Created with BioRender.com. 7 1.3.1 Biological functions of Alk in Drosophila Physiological roles of Alk have been investigated in a range of model systems, from invertebrates to vertebrates. In the interests of space, I will focus on the role of Alk in Drosophila. In Drosophila, Alk is crucial for specification of a subset of myoblasts in the visceral mesoderm (VM) known as muscle founder cells (FCs) which are essential for the formation of midgut muscles during embryonic development (Lorén et al., 2003, Englund et al., 2003, Stute et al., 2004, Lee et al., 2003). Drosophila Alk is activated by a secreted, small LDL-containing, protein named Jelly belly (Jeb) initiating a cascade that results in ERK activation, followed by expression of FC-specific genes including Hand, duf/kirre, optomotor-blind- related gene-1 (org-1), Rho GTPase activating protein 15B (RhoGAP15B) and Kahuli (Kah) (Figure 4) (Zhou et al., 2019, Lorén et al., 2001, Mendoza-García et al., 2017, Lee et al., 2003, Popichenko et al., 2013, Englund et al., 2003, Varshney and Palmer, 2006, Mendoza-Garcia et al., 2021). In the absence of Jeb or Alk, cells of the VM are unable to assume FC fate, leading to failure of gut development and lethality (Englund et al., 2003, Stute et al., 2004, Lee et al., 2003, Lorén et al., 2003). Y2H screening with the Alk intracellular tyrosine kinase containing domain identified the scaffolding molecule connector enhancer of kinase suppressor of Ras (Cnk) scaffolding molecule as direct Alk interactor. Furthermore, Cnk together with its activator Aveugle (Ave), have been defined as essential components of Alk signaling in the VM (Figure 4) (Wolfstetter et al., 2017). 8 Figure 4: Alk signaling in Drosophila visceral founder cells. Alk is activated by Jelly belly (Jeb) and recruits the Cnk/Ave adaptor complex that transmits signals through the Raf/MAPK/ERK cascade. Activation of ERK leads to transcription of founder cell (FC)-specific genes such as duf/kirre, org-1, Hand, RhoGAP15B and eventually reprogramming of visceral mesoderm progenitors as a result of fusion with FCs. Adapted with permission from (Wolfstetter et al., 2017). Created with BioRender.com. 9 Alk is also expressed in the Drosophila central nervous system (CNS) and at the postsynaptic density (PSD) of larval neuromuscular junctions (NMJs). It is involved in various processes during neuronal development, for example, in the developing CNS, Alk activity is required for survival of the lamina L3- neurons and proper targeting of R8-cell axons in the optic lobe (Pecot et al., 2013, Bazigou et al., 2007). Furthermore, increased neuronal Alk activity results in small pupal size phenotype, suggesting a role for Alk in body size regulation and growth. Previous studies identified Drosophila Neurofibromatosis 1 (dNf1) as one of Alk downstream targets involved in size determination during larval development and olfactory learning in adults (Gouzi et al., 2011, Walker et al., 2013). Further, Alk mutants harboring orthologs of human oncogenic mutations display perturbation of neuronal fate in the mushroom body (MB) lineages, identifying a role for Alk in neuronal differentiation (Pfeifer et al., 2022). In addition, Alk and Jeb are required in developing synapses at the NMJ. Here, post-synaptic Alk is activated by pre-synaptic Jeb, and this triggers the Ras/MAPK/ERK cascade, modulating synaptic transmission (Rohrbough and Broadie, 2010). Alk has also been associated with thinness through regulation of lipid and glucose homeostasis (Orthofer et al., 2020). Additional studies have identified a role for Drosophila Alk in brain sparing, sleep regulation, longevity, insulin metabolism and ethanol consumption (Cheng et al., 2011, Lasek et al., 2011, Okamoto and Nishimura, 2015, Bai and Sehgal, 2015). Despite this wide range of functions for neuronal Alk, our understanding of the downstream components and underlying molecular mechanisms is very limited. 1.3.2 ALK signaling in human cancers Gain-of-function mutations, deletions, amplifications, and genomic rearrangements of ALK have been described in wide range of malignancies. ALK activating point mutations are mainly found in neuroblastoma while ALK fusion proteins have been described in anaplastic large cell lymphoma (ALCL), non-small cell lung cancer (NSCLC), inflammatory myofibroblastic tumor (IMT), diffuse large B-cell lymphoma (DLBCL), esophageal squamous cell carcinoma (ESCC), renal medulla carcinoma (RMC), renal cell carcinoma (RCC), 10 breast cancer, colorectal cancer, ovarian cancer and anaplastic thyroid cancer (Hallberg and Palmer, 2016). Overexpression and activation of wild-type ALK has also been described in various cancers including NSCLC, melanoma, neuroblastoma, ovarian cancer, glioblastoma, rhabdomyosarcoma, breast cancer, astrocytoma, Ewing’s sarcoma and retinoblastoma (Dirks et al., 2002, Kruczynski et al., 2012, Perez-Pinera et al., 2007, Palmer et al., 2009, Hallberg and Palmer, 2013). ALK activation by either ALKALs, gain-of-function mutations or overexpression activates various signaling cascades including RAS-MAPK, PI3K-AKT-MEKK2/3-MEK5-ERK5,PI3K-AKT-mTOR/Foxo/GSK3β,phospholipase Cγ (PLCγ)-IP3, CRKL-Rap1 and Janus kinase (JAK)–signal transducer and activator of transcription (STAT) and JUN pathways (Hallberg and Palmer, 2013, Umapathy et al., 2019). ALK also recruits and phosphorylates a number of receptor-associated molecules such as insulin receptor substrate 1 (IRS1), insulin receptor substrate 2 (IRS2), CBL, CRKL, SHC, growth factor receptor- bound protein 2 (GRB2), PLCγ and fibroblast growth factor receptor substrate 2 (FRS2), activating downstream signaling. Activation of ALK downstream signaling pathways triggers transcription of a number of genes involved in various cellular processes including proliferation, survival, differentiation, migration, cell cycle and metastasis (Figure 5) (Hallberg and Palmer, 2016, Umapathy et al., 2019, Van den Eynden et al., 2018, Borenäs et al., 2021). 11 Figure 5: General overview of physiological and oncogenic ALK activation. Wild-type ALK is activated in a ligand-dependent manner. On the other hand, oncogenic ALK activation can be either ligand-dependent (ALK overexpression) or ligand-independent (ALK gain-of-function and ALK fusions) manner. Insulin receptor substrate 1 (IRS1), Insulin receptor substrate 2 (IRS2), SHC, growth factor receptor-bound protein 2 (GRB2), SHP2, C3G, CBL, CRKL and fibroblast growth factor receptor substrate 2 (FRS2) interact with ALK. ALK signals through various pathways including PI3K-AKT-MEKK2/3-MEK5- ERK5, PI3K-AKT-mTOR/Foxo/GSK3β, RAS–MAPK, phospholipase Cγ (PLCγ)-IP3, C3G- Rap1 and JAK-STAT to mediate transcription of number of genes involved in cell proliferation, survival, differentiation, migration, cell cycle and metastasis. ALKALs and ALK point mutations are depicted in yellow and green respectively. Adapted with permission from (Umapathy et al., 2019, Hallberg and Palmer, 2013). Created with BioRender.com. 1.4 The protein tyrosine phosphatase PTPN11 /SHP2 The protein tyrosine phosphatase non-receptor type 11 (PTPN11, also known as SHP2) was initially reported in the 1990s by several groups (Freeman et al., 1992, Adachi et al., 1992, Ahmad et al., 1993). At about the same time, the Drosophila homolog of SHP2, Corkscrew (Csw), was characterized and 12 identified as downstream component of the Torso RTK (Perkins et al., 1992). Since then, numerous studies have demonstrated important roles for SHP2 in cell growth, survival, migration, differentiation, and oncogenic transformation. Deregulation of SHP2 and its activity has been implicated in various developmental diseases as well as in cancer (Lauriol et al., 2015, Ran et al., 2016, Anselmi and Hub, 2020). 1.4.1 Regulation of SHP2 Human SHP2 is a 68 kDa protein ubiquitously distributed in the cytoplasm as well as in the nucleus and mitochondria (Zheng et al., 2009, Lee et al., 2013, Tsutsumi et al., 2013, Salvi et al., 2004, Yuan et al., 2020). It is expressed in wide range of tissues including brain, fat, heart, kidney, and the adrenal gland (Asmamaw et al., 2022). SHP2 has two SH2 domains (referred as N-SH2 and C-SH2) located in the N-terminal, a single central PTP domain and a disordered C-terminal tail. The Drosophila homolog Csw PTP domain exhibits highly similar domain organization and has a 150 aa cysteine and serine-rich insert with unknown function (Figure 6) (Perkins et al., 1992, Neel et al., 2003, Stein-Gerlach et al., 1998). Under basal conditions, the N-SH2 domain occupies the active site of the PTP domain and SHP2 remains inactive (Hof et al., 1998). In the presence of phosphotyrosine-containing substrates, the N- terminal SH2 (N-SH2) domain facilitates the binding of the PTP domain of SHP2 to these specific substrates (Yu et al., 2013). The C-terminal tail of SHP2 contains two phosphorylation sites, Tyr 542 and Tyr 580, which are phosphorylated upon PTK activation, creating binding sites for adaptor proteins. Phosphorylation of these Tyr residues are important for SHP2 activation (Tajan et al., 2015, Asmamaw et al., 2022). 13 Figure 6: Overview of SHP2 and Csw domain structures. Both human SHP2 and Drosophila Csw are composed of two N-terminal SH2 domains followed by a PTP domain and C-terminal tail. The PTP domain of Csw is interrupted by a cysteine and serine-rich insert (shown in light pink) that is approximately 150 amino acids in length. SH2 domains of SHP2 and Csw are depicted in orange and blue respectively. The PTP domain of SHP2 is shown in green while the PTP of Csw is in red. Numbers indicate amino acids. Created with BioRender.com. Wild-type SHP2 shifts between inactive and active conformations and this equilibrium maintains normal phosphatase activity. Loss-of function (LOF) and gain-of-function mutations (GOF) disrupt this equilibrium, thereby affecting phosphatase activity (Pádua et al., 2018, Tartaglia et al., 2006, Tartaglia et al., 2001, Tartaglia et al., 2003, Kontaridis et al., 2006, Keilhack et al., 2005). GOF mutations occur mainly at the interface of the N-SH2 and PTP domains triggering the release of the N-SH2 from the PTP domain and leading to constitutive activation. In contrast, LOF mutations are mainly clustered in the PTP domain and abrogate the catalytic activity of SHP2 (Asmamaw et al., 2022). Germline and somatic mutations have been implicated in multiple developmental disorders such as Noonan syndrome (NS) and LEOPARD syndrome (LS) as well as in several leukemias and solid tumors (Tartaglia et al., 2006, Tartaglia et al., 2003, Keilhack et al., 2005, Dong et al., 2021). 14 1.4.2 Roles of SHP2/Csw in RTK signaling As a regulator of multiple RTKs, SHP2 promotes signaling pathways that are involved in various processes including cell proliferation, survival, migration, differentiation and metabolism (Song et al., 2022). Once stimulated by growth factors or cytokines, RTKs dimerize and phosphorylate SHP2 on tyrosine residues. Phosphorylated SHP2 either dephosphorylates downstream effectors to activate downstream signaling or acts as a phosphatase-independent adaptor (Guo and Xu, 2020). The Ras/ERK pathway is one of the major signaling pathways that is mediated by SHP2 through multiple mechanisms. Downstream of EGFR, SHP2 prevents Ras inactivation by dephosphorylating RasGAP docking sites on EGFR and Gab1 (Agazie and Hayman, 2003, Montagner et al., 2005). Another way by which SHP2 promotes ERK activation is via dephosphorylation of negative regulators of Ras, such as Sprouty 1 (Spry1) and Spry2 (Hanafusa et al., 2004, Jarvis et al., 2006, Pan et al., 2010). Dissociation of the Spry/GRB2 complex promotes binding of the FRS2 adaptor to GRB2, thereby leading to activation of ERK (Hanafusa et al., 2002). It also regulates Src family kinases (SFKs) through dephosphorylation of negative regulators, such as paxillin and Cbp/PAG, and potentiates the Ras/ERK cascade (Ren et al., 2004, Zhang et al., 2004). SHP2 can also act as an adaptor and binds to GRB2 to recruit the GRB2/SOS complex to phosphotyrosine docking sites on RTKs, thereby activating Ras/ERK signaling (Li et al., 1994, Schlessinger, 2000, Ran et al., 2016). SHP2 also modulates PI3K/AKT, JAK/STAT, programmed cell death-1 (PD-1), nuclear factor-kB (NF-B), Hippo and Wnt/β-catenin pathways (You et al., 1999, Wu et al., 2001, Zhang et al., 2002, Qu, 2002, Yu et al., 2000, Tsutsumi et al., 2013, Tang et al., 2018, Zhang et al., 2018). In Drosophila, the SHP2 homolog Csw was initially identified as a downstream component of the Torso RTK which is the key determinant of terminal patterning during embryogenesis (Perkins et al., 1992, Duffy and Perrimon, 1994). Csw directly binds to activated Torso and recruits daughter of sevenless (Dos) and the GRB2 homolog Drk in a complex with Sos (a 15 RasGEF) to the membrane, promoting the exchange of Ras-bound GDP and activating downstream effectors Raf, MEK/Dsor1 and ERK/Rolled (Rl). Inhibition of transcriptional repressors by ERK/Rl leads to activation of gap genes, tailless and huckebein (Lu et al., 1993, Cleghon et al., 1998, Sopko and Perrimon, 2013). Csw also acts downstream of the Sevenless (Sev) and EGFR RTKs during the specification of R7 photoreceptor neurons. A similar cassette of signaling proteins including Csw transmits signals upon activation of both Sev and EGFR. Csw directly binds to Dos which also recruits Drk/Sos and activates the Ras/Raf/ERK cascade, followed by transcriptional activation of lozenge and prospero enhancers for specification of R7 cell fate (Herbst et al., 1996, Hamlet and Perkins, 2001, MacDougall and Waterfield, 1996, Sopko and Perrimon, 2013, Feller et al., 2002). Similar to mammalian SHP2, Csw also dephosphorylates Spry proteins that act as a negative regulator of RTK signaling and thereby potentiates the Ras/Raf/ERK cascade (Jarvis et al., 2006). Furthermore, several studies have shown a role for SHP2/Csw in metabolism and life span through regulation of insulin signaling (Tajan et al., 2014, Ruzzi et al., 2020). LOF mutations in csw resulted in decreased insulin signaling and extended life span (Ruzzi et al., 2020). 1.4.3 SHP2 as a therapeutic target Given the critical role of SHP2 downstream of multiple RTKs, SHP2 inhibition has been investigated as a potential therapeutic approach for treatment of RTK-driven cancers as well as in the context of preventing acquired resistance (Prahallad et al., 2015, Torres-Ayuso and Brognard, 2018, Xia et al., 2021, Asmamaw et al., 2022). Combination of SHP2 inhibitors with anticancer drugs or small molecule inhibitors of RTKs resulted in synergistic inhibition of tumor growth (Prahallad et al., 2015, Kano et al., 2021, Sun et al., 2019, Chen et al., 2020, Pudelko et al., 2020, Song et al., 2022, Uçkun et al., 2021, Karaca Atabay et al., 2022, Cai et al., 2022). To date, various small molecule SHP2 inhibitors have been developed, including catalytic site inhibitors, allosteric site inhibitors, PROTAC-based degraders, and inhibitors of SHP2 protein-protein interactions (Asmamaw et al., 2022). Here, I will briefly introduce two allosteric SHP2 inhibitors employed in our studies. 16 SHP099 is a recently developed, highly specific allosteric SHP2 inhibitor that stabilizes SHP2 in an inactive conformation (Chen et al., 2016, Ran et al., 2016). SHP099 binds to the interface of N-SH2 domain and the PTP domain, thereby blocking the binding of phosphotyrosine-containing substrates and catalytic activity (Chen et al., 2016). In preclinical studies, SHP099 has demonstrated good efficacy in inhibiting RTK-driven cancers, either alone or in combination with TKIs (Chen et al., 2016, Dardaei et al., 2018, Wong et al., 2018, Ahmed et al., 2019, Cai et al., 2022, Uçkun et al., 2021). RMC-4550 is another allosteric inhibitor of SHP2 that has a similar mechanism of inhibition, stabilizing the auto-inhibited conformation of SHP2. It is also highly selective for SHP2 over other phosphatases (Nichols et al., 2018). In cancers harboring Ras-GTP dependent BRAF mutant, nucleotide-cycling KRAS mutant and NF1 loss, RMC-4550 suppressed Ras/MAPK signaling and inhibited tumor growth, suggesting that SHP2 inhibition is a promising approach for treatment of Ras- driven and NF1-associated tumors (Nichols et al., 2018, Harigai et al., 2022). 1.5 Neuroblastoma Neuroblastoma (NB) is the most common pediatric extracranial solid tumor, causing approximately 15% of childhood cancer-related deaths (Maris et al., 2007, Park et al., 2010, Zafar et al., 2021). NB develops exclusively in young children with the median age of 17-18 months (Johnsen et al., 2018). It can occur anywhere along the developing sympathetic nervous system, including the adrenal glands (47%), abdomen (24%), thoracic region (15%), pelvis (3%) and neck (2.7%) (Vo et al., 2014). NB is highly heterogenous, and clinical outcome can range from spontaneous regression to metastatic high-risk disease with poor prognosis (Johnsen et al., 2018). The International Neuroblastoma Staging System (INSS) is a post-surgical staging system based on age, tumor histology, DNA ploidy, chromosomal aberrations, and genetic characteristics such as MYCN amplification and TRKA overexpression (Brodeur et al., 1993). Patients classified as stage 1 and 2 have non-metastatic, resectable tumors located in a certain area while stage 3 tumors are unresectable with regional lymph node metastasis. Stage 4 is 17 classified as advanced stage disease with distant metastatic tumors. Stage 4S NB represents localized small primary tumors in patients under 12 months with dissemination restricted to liver, skin and/or bone marrow. Patients with stage 4S NB represent a more favorable group with good prognosis and tumors often undergo spontaneous regression (Matthay, 1998, Nickerson et al., 2000, Brodeur, 2018). In addition, a pre-treatment risk classification system, the International Neuroblastoma Risk Group (INRG), was developed in 2009. Based on INSS stage, age, histology, differentiation status, MYCN amplification status and segmental chromosome aberrations, NB is classified in 4 groups: (i) very low, (ii) low, (iii) intermediate, and (iv) high-risk (Cohn et al., 2009, Monclair et al., 2009, Irwin et al., 2021). The overall survival for low- risk and medium-risk neuroblastoma is 80-90% (Johnsen et al., 2018). Treatment for high-risk patients includes surgical resection of primary tumor, high dose chemotherapy, radiotherapy, stem cell transplantation, immunotherapy as well as combination treatments. In spite of recent advances in treatment modalities, the 5-year overall survival rate for patients with high-risk NB is below 50%. The majority of patients with high-risk NB relapse, and overall survival is below 10% with relapsed or refractory metastatic NB (Valteau-Couanet et al., 2014, Ladenstein et al., 2008, Fransson et al., 2020, Guan et al., 2021). 1.5.1 Genomic aberrations and mutations in neuroblastoma Investigation of genomic aberrations and mutations associated with NB has allowed identification of prognostic markers as well as pathways that contribute to NB initiation and progression. Most NB cases arise sporadically, while hereditary NB accounts for 1-2% (Matthay et al., 2016). Mutation burden is low in NB and the most common somatic mutations are observed in ALK, PTPN11/SHP2, TERT and ATRX. Furthermore, mutations in NF1, RAS, RAF, PTPN11/SHP2 and FGFR are more frequently found in patients with relapsed NB (Eleveld et al., 2015, Guan et al., 2021). In addition, chromosomal aberrations are frequently observed in NB and include MYCN amplification, chromosome 1p deletion, 17q gain, 2p gain and 11q deletion (Bown et al., 1999, Maris and Matthay, 1999, Mora et al., 2000, Carén et al., 2010, 18 Szewczyk et al., 2019, Javanmardi et al., 2019, Brady et al., 2020, Guan et al., 2021). 1.5.2 ALK in neuroblastoma ALK mutations are observed in both primary and relapsed tumors and are associated with poor prognosis, especially in intermediate and high-risk NB cases (Martinsson et al., 2011, Schleiermacher et al., 2014, Bresler et al., 2014). Activating ALK mutations in NB are mainly located in the intracellular region harboring the TKD and represent 6-12% of sporadic NB cases. The majority of these ALK point mutations (85%) are clustered in three, so-called hot spot residues in the TKD: R1275, F1174 and F1245 (Hallberg and Palmer, 2013, Bresler et al., 2014). Amplification of MYCN is one of the main prognostic factors in NB and is associated with 20-25% of NB cases (Brodeur et al., 1984, Maris et al., 2007, Campbell et al., 2017). MYCN amplification strongly promotes NB cell proliferation and an aggressive NB phenotype, indicating poor prognosis (Seeger et al., 1985, Campbell et al., 2017). Meta-analysis of NB tumors described a correlation between ALK GOF mutations and MYCN amplification in high-risk NB (De Brouwer et al., 2010). In addition, studies have shown that ALK regulates MYCN transcription and protein stability through PI3K/AKT/mTOR and MAPK pathways (Berry et al., 2012, Schönherr et al., 2012). Furthermore, ALK GOF mutations accelerate MYCN-driven NB in both cell lines and animal models, indicating a strong cooperation between ALK and MYCN (Schönherr et al., 2012, Heukamp et al., 2012, Berry et al., 2012, Zhu et al., 2012, Cazes et al., 2014, Borenäs et al., 2021). Since both ALK and MYCN are located at chromosome 2p.23 and 2p.24 respectively, chromosome 2p gain (30% of NB cases) often involves both MYCN and ALK and is predictive of poor survival (Jeison et al., 2010, Javanmardi et al., 2019). 1.5.3 ALK signaling in neuroblastoma Several studies have addressed mechanisms of ALK signaling in NB and identified several ALK downstream components. Phophosphoproteomic 19 analysis in NB cells upon ALK inhibition revealed decreased phosphorylation of several adaptor proteins such as fibroblast growth factor receptor substrate 2/3 (FRS2/3), insulin receptor substrate 2 (IRS2), SHC1 to 3, and GRB2 associated binding protein 1/2 (GAB1/2) as well as other proteins including SHP2 , ataxia telangiectasia and Rad3-related (ATR) protein, breakpoint cluster region (BCR) protein, maternal embryonic leucine zipper kinase (MELK), bromodomain-containing protein 2 (BRD2) and dual specificity protein phosphatase 4 (DUSP4) and also members of the ETS translocation variant (ETV), forkhead box (FOXO), and ETS domain-containing transcription factor families, indicating their importance in the ALK signaling. Further functional analysis validated a role of ETV3 and ETV4 in ALK signaling since loss of either transcription factor inhibited proliferation of ALK-addicted NB cells. In addition, DUSP4 and FOXO3 were also validated as downstream components regulated by ALK (Van den Eynden et al., 2018). Another important hit from this phosphoproteomic analysis was the Sad1 and UNC84 domain containing 2 (SUN2), that highlighted ALK as a potential regulator of ATR. Indeed, ALK-driven NB cells were highly sensitive to ATR inhibition, and a combination of ALK and ATR inhibitors resulted in complete elimination of tumor in NB mouse models, indicating its potential as a therapeutic target (Szydzik et al., 2021). In another study, integrated proteomics approaches upon ALK TKI treatments identified SHC1/3, GAB1/2, IRS2, GRB2, son of sevenless homolog 1 /2 (SOS1/2) as well as phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) and PIK3R2, SHP2 and the adaptor protein SH2B2 as ALK downstream components. Among these proteins, IRS2 was confirmed as a mediator of cell survival through the PI3K-AKT-FOXO3 axis in NB cells (Emdal et al., 2018). SHP2 has been further analyzed in the context of NB. In zebrafish models of NB, it has been shown that transgenic overexpression of a SHP2 mutant cooperates with MYCN and increases tumor penetrance (Zhang et al., 2017). In addition, BioID-based proximity labeling of ALK in NB cells identified SHP2 as an ALK interactor. Further analysis showed that ALK-driven NB cells are 20 sensitive to SHP2 inhibition, and that combined inhibition of ALK and SHP2 synergistically inhibits NB growth (Uçkun et al., 2021). A recent study demonstrated that the sensitivity of NB cells to SHP2 inhibition correlates with NF1 mutational status. Moreover, SHP2 inhibition in high-risk NB models suppressed tumor growth, indicating that targeting SHP2 could be effective against NB (Cai et al., 2022). 1.5.4 Targeting oncogenic ALK Detection of ALK aberrations and oncogenic ALK signaling in various human cancers has led to the development of ALK tyrosine kinase inhibitors (TKIs) such as crizotinib, ceritinib, alectinib, brigatinib, entrectinib and lorlatinib (Umapathy et al., 2019). Some of these inhibitors are already in clinical use for treatment of ALK-positive cancers. (Lin et al., 2017, Rothenstein and Chooback, 2018, Millett et al., 2018). Here, I will briefly introduce the inhibitors employed in my studies. Crizotinib was first ALK-directed TKI to enter clinical trials and was approved in 2011 by the FDA for the treatment of ALK fusion-positive NSCLC (Kwak et al., 2010, Umapathy et al., 2019). Further clinical studies showed that crizotinib was more effective than chemotherapy for patients with previously treated, advanced ALK fusion-positive NSCLC (Shaw et al., 2013, Solomon et al., 2014). It has also been tested in other ALK-positive cancers such as pediatric and adult ALCL, neuroblastoma and IMT. Although clinical response to crizotinib in ALCL was good, it was disappointing in neuroblastoma and IMT patients (Mossé et al., 2013, Gambacorti Passerini et al., 2014, Mossé et al., 2017). In spite of good initial responses against crizotinib, patients with ALK fusion-positive NSCLC generally develop resistance either due to secondary mutations that arise within the TKD domain, ALK amplification or activation of bypass signaling pathways (Lin et al., 2017). In addition, crizotinib was found to be ineffective on brain metastases of ALK fusion-positive NSCLC as a result of low penetration of crizotinib into the CNS (Maillet et al., 2013). To improve inhibition of ALK 21 activity in the brain and efficacy towards secondary resistance mutations, next-generation ALK TKIs were developed. Lorlatinib is a third generation ALK/ROS1 TKI that penetrates the blood- brain barrier (Solomon et al., 2018). Studies in both in vitro and in vivo models demonstrated that lorlatinib is more effective compared to other ALK TKIs and potently inhibits ALK mutants resistant to crizotinib and second- generation TKIs (Lin et al., 2017, Shaw et al., 2017, Umapathy et al., 2019). Since preclinical investigation showed high overall potency, superior intracranial/CNS efficacy, and good safety profile, it was approved by FDA for treatment of ALK fusion-positive metastatic NSCLC (Zou et al., 2015, Umapathy et al., 2019). Furthermore, lorlatinib has been shown to effectively inhibit tumor growth in ALK-addicted NB xenograft models, suggesting investigation of lorlatinib as therapeutic option in ALK-positive NB (Guan et al., 2016, Infarinato et al., 2016, Szydzik et al., 2021). Lorlatinib is currently in Phase I clinical trials (NCT03107988) for the treatment of relapsed or refractory ALK-positive high-risk NB patients (Goldsmith et al., 2022). 1.6 Investigating protein-protein interactions Genome sequencing of more than 200 organisms revealed that phenotypic complexity cannot simply be explained by genome composition (Keskin et al., 2016). Alternative splicing is one of the major drivers of phenotypic complexity since more than 90% of all human genes are estimated to generate different mRNA isoforms (Kim et al., 2014, Wang et al., 2008). Post- translational modifications also contribute to complexity, as do protein- protein interactions (Keskin et al., 2016). It is estimated that more than 80 % of proteins interact with other proteins molecules to form complexes and transmit signals within cells to mediate various cellular pathways (Yeger- Lotem and Sharan, 2015, Berggård et al., 2007). Protein-protein interactions (PPIs) are often context-specific and dependent on cell type, cellular environment, or conditions. Perturbations in cross-talk between proteins within complexes are associated with many diseases including cancer, making it essential to understand PPI networks in different contexts (Qin et al., 2021). 22 Conventional strategies to analyze PPIs include biochemical methods such as antibody-based affinity purifications and genetic methods such as the yeast two hybrid system (Y2H) (Petschnigg et al., 2011). 1.6.1 Antibody-based affinity purification Antibody-based affinity purification is one of the most widely used in vitro methods to study antigen-antibody, protein–protein or protein–ligand interactions (LaCava et al., 2016). Affinity purification (AP) is performed by employing specific antibodies and is often referred to as immunoprecipitation. Antibodies can be targeted either to endogenous proteins or an epitope tag fused to the protein of interest (Dunham et al., 2012). Immunoprecipitation involves capturing protein antigens by means of an antigen-specific antibody, followed by purification of antigen-antibody complexes along with their stable interaction partners using insoluble resins such as protein A or G agarose (DeCaprio and Kohl, 2020, Bonifacino et al., 2016, DeCaprio and Kohl, 2017). General immunoprecipitation protocols consist of: (1) sample preparation, including harvesting cells/tissues expressing the protein of interest and lysis to solubilize proteins; (2) addition of specific antibodies targeting the protein of interest (POI) itself or epitope tag and POI-antibody complex formation; (3) addition of protein A/G beads; (4) elution of the POI together with stably interacting proteins from beads after a series of washes, and (5) detection of protein complexes by western blotting or analysis by mass spectrometry (MS) (Figure 7) (Dunham et al., 2012). 23 Figure 7: Schematic workflow of antibody-based affinity purification. (1) Cells expressing the protein of interest (POI) are lysed. (2) Antibodies specific to the POI or an epitope tag are added to allow protein-antibody complex formation. (3) Protein A/G beads are added, and samples are washed to remove unbound proteins. (4) POI- interacting protein complexes are eluted from beads and (5) protein-protein interactions (PPIs) are detected by western blotting or mass spectrometry (MS). Created with BioRender.com. 1.6.2 Yeast two-hybrid (Y2H) The yeast two-hybrid (Y2H) screening technique was developed as a result of the elucidation of the structure of Gal4, a transcriptional activator in Saccharomyces cerevisiae, in 1986. It was shown that in the presence of galactose, Gal4 could specifically bind to a UAS (upstream activation sequence) and activate transcription. When Gal4 was separated into N- terminal and C-terminal domains, the N-terminal facilitated binding but was unable to activate transcription, indicating that the C-terminal domain was critical for transcriptional activation. Furthermore, independently produced N-terminal DNA binding domains (DBD) and C-terminal transcriptional activation domains (AD) could interact and form fully functional Gal4 (Fields and Song, 1989, Keegan et al., 1986). In the classical Y2H system, two proteins that potentially interact with each other, X (bait) and Y (prey), are fused to DBD and AD domains of Gal4. 24 The resulting X-DBD fusion binds to UAS in the promoter. Interaction of X and Y reconstitute Gal4, thereby recruiting RNA polymerase II and driving reporter gene transcription (Figure 8) (Fields and Song, 1989, Brückner et al., 2009, Parrish et al., 2006). Over the years, different variations of Y2H systems have been developed to allow entire cellular proteome and high-throughput Y2H screening using open reading frames (ORFs) and cDNA libraries have become a valuable tool to establish PPI networks in vivo (Rajagopala, 2015, Brückner et al., 2009). Figure 8: Schematic overview of the classical Y2H system. The bait protein (X) is fused to the DNA binding domain of Gal4, and the prey protein (Y) is fused to the transcriptional activation domain (AD). On the left, two proteins of interest do not interact, therefore Gal4 is not functional, and the reporter gene is not transcribed. On the right, interaction of two proteins reconstitutes functional Gal4, leading to reporter gene transcription. Created with BioRender.com. 1 .7 Proximity labeling techniques Conventional methods such as AP and Y2H have allowed the discovery of dynamically organized protein complexes and definition of interactome networks in different model systems (Qin et al., 2021). In spite of recent advances in AP, loss of weak and transient interactions remains an important limitation. It is also restricted by availability of antibodies specific to the POI and is challenging with insoluble proteins. Furthermore, it is only possible to test whether there is interaction between two suspected interaction partners, and it is therefore unsuitable for high-throughput screening 25 (Dunham et al., 2012). On the other hand, Y2H allows high-throughput screening of millions of interaction combinations (Brückner et al., 2009, Trigg et al., 2017, Parrish et al., 2006). Traditional Y2H approaches require direct interaction and translocation of interacting proteins to the nucleus, limiting its usefulness in assaying membrane-associated proteins. To overcome this limitation, truncated proteins are often used (Brückner et al., 2009, Qin et al., 2021). Another method developed for identification of PPIs of membrane proteins is the Mammalian Membrane Two-Hybrid (MAMTH), which is based on a split ubiquitin approach. MAMTH allows identification of weak/transient PPIs of membrane proteins in their native mammalian cell environment (Petschnigg et al., 2014, Saraon et al., 2017). While Y2H is a powerful technique, false positives due to protein overexpression as well as false negatives as a result of steric restrictions are still common challenges (Qin et al., 2021). Proximity labeling (PL) offers an alternative method for mapping protein interaction networks in vivo and has been employed extensively in living cells as well as in organisms (Samavarchi-Tehrani et al., 2020, Mair and Bergmann, 2022, Zhang et al., 2021, Christopher et al., 2022). PL methods mainly involve two types of engineered biotinylating enzymes, peroxidases and biotin ligases (Xu et al., 2021). Here the biotinylating enzyme of choice is fused to the POI and the resulting POI-enzyme fusion is expressed either in inducible/stable cell lines or in organisms which are manipulated by CRISPR-Cas9 based knock- in or as transgenic constructs allowing overexpression in specific tissues. Expression results in biotinylation of proteins in the vicinity of the POI, which can subsequently be isolated by streptavidin beads and identified by MS (Han et al., 2018, Zafra and Piniella, 2022). 1.7.1 Peroxidase-based proximity labeling Peroxidase-based methods employ horseradish peroxidase (HRP) (Kotani et al., 2008) or the engineered ascorbate peroxidase (APEX or its variant APEX2) (Lam et al., 2015, Martell et al., 2012, Rhee et al., 2013). In this technique, cells are supplemented with biotin-phenol substrate and H2O2 (1 min), 26 resulting in oxidation of biotin-phenol to a phenoxy-biotin. Short lived phenoxy-biotin radicals label electron-rich amino acid residues (Tyr, Trp, Cys and His) in neighboring proteins (Figure 9) (Zafra and Piniella, 2022). The reaction is stopped by simply removing H2O2 and addition of quenching buffer (Chen and Perrimon, 2017). HRP is 44 kDa enzymatic tag stabilized by four disulfide bonds and two Ca2+ ion-binding sites which are disrupted in reducing environments such as the cytosol, thus restricting its use in intracellular PPI studies. HRP exhibits high activity in oxidizing environments such as the extracellular space, ER or Golgi lumen, thus making its use limited to PL applications on the surface of the cells or in the secretory pathway (Xu et al., 2021, Chen and Perrimon, 2017, Qin et al., 2021). HRP can utilize range of substrates including fluorescein arylazide, biotin arylazide and biotin-tyramide which are often referred to as biotin-phenol (Honke and Kotani, 2012, Iwamaru et al., 2016, Jiang et al., 2012, Rees et al., 2017). Furthermore, HRP conjugated to a specific antibody can also be used for PL purposes (Li et al., 2014). APEX is a 28 kDa engineered ascorbate peroxidase which is originally from plants and uses the same labeling reaction as HRP (Hung et al., 2014, Rhee et al., 2013, Xu et al., 2021). APEX was first applied in the mitochondrial matrix and permits PL with only 1-min H2O2 incubation (Rhee et al., 2013). Later, an improved variant APEX2 harboring one additional mutation (A134P) was developed using yeast display-based directed evolution, exhibiting higher catalytic activity and sensitivity for PL (Lam et al., 2015). Unlike HRP, APEX and APEX2 lack disulfide bonds and calcium ions. Since both remain catalytically active in reducing cytosolic environments, they have become useful for labeling in intracellular compartments (Chen and Perrimon, 2017, Rhee et al., 2013). The time required for labeling by HRP, APEX and APEX2 is very short (minute scale), making these enzymes the ideal choice to capture dynamic processes in the interactome with high temporal resolution (Zhou and Zou, 2021). Over the past 9 years, APEX-based approaches have enabled mapping of proximal proteomes in different cell compartments such as mitochondrial matrix, outer mitochondrial and endoplasmic reticulum (ER) membranes, 27 primary cilia, ER-plasma membrane as well as in organisms such as Drosophila and yeast (Chen et al., 2015, Hung et al., 2017, Hung et al., 2014, Jing et al., 2015, Hwang and Espenshade, 2016, Hwang et al., 2016). However, certain limitations such as the requirement of H2O2, which may result in toxicity and low permeability of biotin-phenol radicals, has restricted its use in vivo (Qin et al., 2021). Figure 9: Peroxidase-based proximity labeling. Peroxidases, either horseradish peroxidase (HRP) or engineered ascorbate peroxidases (APEX, APEX2) (depicted in orange) are fused to the protein of interest (POI, grey). In the presence of H2O2, these enzymes catalyze the rapid oxidation of biotin-phenol to a biotin-phenoxyl radical, resulting in biotin labeling (shown in red) of electron-rich amino acid residues (Tyr, Trp, Cys, His) of proteins in the close vicinity. Created with BioRender.com. 1.7.2 Biotin ligase-based proximity labeling Biotin ligase-based PL employs derivatives of the E.coli biotin ligase BirA (BioID, BioID2, miniTurbo, TurboID, AirID, microID and microID2) that are fused to the POI (Zhou and Zou, 2021, Johnson et al., 2022, Kubitz et al., 2022, Kubitz et al., 2022). Wild-type BirA is a 35 kDa protein that generates reactive biotinyl-AMP from biotin in the presence of ATP and labels lysine residue of specific substrates such as carboxylases. BirA keeps the biotinyl-AMP in the active site until a specific substrate or short acceptor peptide is available (Chapman-Smith and Cronan Jr, 1999, Beckett et al., 1999, Chen and 28 Perrimon, 2017). Since it is highly specific for its substrate, BirA-mediated labeling has been used to validate PPIs. To study interaction between two proteins, bait protein is tagged with BirA, and prey protein is fused to biotin acceptor peptide (BAP). Upon interaction of two proteins, the prey protein becomes biotinylated (Fernández-Suárez et al., 2008). To enhance promiscuous labeling, a mutant form of BirA enzyme harboring a R118G mutation (often referred as BirA* or BioID) was initially generated. In the presence of biotin and ATP, the enzyme converts biotin to biotinyl-AMP and the R118G mutation in the active site significantly reduces affinity for reactive biotinyl-AMP, resulting in premature release and labeling on lysine residues of proteins in close vicinity estimated to be 10 nm (Figure 10). (Choi-Rhee et al., 2004, Roux et al., 2012, Kim et al., 2014). Later in 2016, a new variant of BioID originated from A. aeolicus, referred to as BioID2, was generated. Compared to the original BioID, BioID2 is smaller (27 kDa) as it lacks N- terminal DNA binding domain and harbors a R40G mutation. BioID2 requires less biotin for labeling and enables higher labeling efficiency (Kim et al., 2016). BioID and BioID2 employ biotin, which is non-toxic, and thereby highly suitable for in vivo applications. However, both approaches require at least several hours of incubation with biotin to efficiently label proximal proteomes because of low catalytic activity. To overcome this limitation, Branon et al. (2018) developed two new variants, called TurboID and miniTurbo, by yeast display-based directed evolution. TurboID (35 kDa) and miniTurbo (28 kDa) variants displayed 22-fold and 10-fold higher catalytic activity respectively compared to BioID. Both new variants enabled labeling in only 10 minutes and the resulting proximal proteome was comparable to 18 h labeling with BioID in terms of size and specificity (Branon et al., 2018). Furthermore, compared to the first generation BioID which was isolated from E. coli and requires 37oC for optimal activity, miniTurbo and TurboID were developed in yeast which grows at 30oC, therefore enabling efficient labeling in organisms (such as flies, worms, and zebrafish) with lower optimal culture temperatures (Xiong et al., 2021, Branon et al., 2018, Sanchez and Feldman, 2021, Zhang et al., 2021, Uçkun et al., 2021, Rosenthal et al., 2021). Both miniTurbo and TurboID variants utilize endogenous biotin and exhibited some level of 29 activity even in the absence of exogenous biotin, indicating that control of the labeling window is rather difficult. An additional concern is that high constitutive expression of these enzymes may deplete endogenous biotin and cause toxicity (Zafra and Piniella, 2022, Branon et al., 2018). Furthermore, two studies have reported some stability issues with miniTurbo (Branon et al., 2018, May et al., 2020). Recently, several new enzymes, AirID, microID and microID2, engineered to improve stability and decrease labeling background, have been reported. However, their potential use in labeling of proximal proteins in vivo has not been explored in intact organisms (Kubitz et al., 2022, Johnson et al., 2022, Kido et al., 2020). Figure 10: Biotin ligase-based proximity labeling. A protein of interest (POI, grey) is fused to promiscuous variants of the biotin ligase BirA (depicted in purple). In the presence of biotin and ATP, biotin is converted to a reactive biotin-5’AMP anhydride, resulting in biotinylation (shown in red) of Lys residues in neighboring proteins. Created with BioRender.com. 30 2. AIMS In this thesis, we aimed to identify and characterize downstream components of ALK signaling in model systems, including NB cell lines and D. melanogaster, employing BioID-based proximity labeling. Specific aims Study I: In this study, we aimed to identify novel components of ALK signaling in NB cells. To achieve this, we generated two inducible ALK BioID expressing NB cell lines and performed proximity labeling both upon ALKAL ligand stimulation and ALK inhibitor treatment. Study II: Here, our purpose was to identify a neuronal Alk proximitome in Drosophila using both first and second generation BioID enzyme variants. We generated flies harboring an endogenously tagged Alk locus with either BirA*, miniTurbo or TurboID (AlkBirA*, AlkTurboID and AlkminiTurbo) by CRISPR/Cas9 genome editing and performed proximity labeling from Drosophila larval brain tissues. Study III: In the last study, our aim was to identify and characterize activity-dependent components of neuronal Alk signaling in the Drosophila larval brain. To achieve this, we performed proximity labeling in Alk::TurboID either in the presence or absence of ectopically expressed Jeb ligand as well as in a TurboID-tagged gain-of-function mutant of Alk (AlkY1355S::TurboID). 31 3. MATERIALS AND METHODS 3.1 Cloning and generation of Tet-On BioID NB cell lines Double strand DNA fragments (gBlocks, Integrated DNA Technologies) including a short linker (Gly-Ser-Ala-Thr x4) and the BirA* coding sequence were initially cloned into pcDNA3.1-ALK for BioID experiments in HEK293 cells. To generate inducible BirA* expressing NB cells, the GSATx4 linker-BirA* fusion was cloned into the pLVX-TRE3G lentiviral vector. To generate inducible Alk-BirA* cell lines, the ALK-GSATx4-BirA* fusion was subcloned from pcDNA3.1-ALK-GSATx4-BirA* into the pLVX-TRE3G lentiviral vector. Lentivirus production was done in HEK293T cells using transfection reagents and lentiviral packaging plasmids. Two NB cell lines, SK-N-AS and SK-N-BE(2), were transduced with lentivirus supernatant. Transduced cells were selected with 1µg/ml puromycin. Two inducible SK-N-AS.BirA* and SK-N-BE(2).BirA* control cell lines and two ALK-BirA* expressing, SK-N-AS.ALK-BirA* and SK-N- BE(2).ALK-BirA*, cell lines were established. 3.2 Western blotting and quantification NB cells were lysed with RIPA buffer and protein concentrations for each sample determined by BCA assay. Samples were run on SDS polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membrane followed by blocking either with 5% w/v nonfat dry milk or 5% BSA in TBST buffer. After blocking, membranes were blotted with primary antibody overnight at 4 °C. The following day, after washing in TBST, membranes were incubated with secondary antibodies for 1 hour at room temperature and subsequently developed with ECL western blotting substrate. Quantification was done using Image Studio™ Lite software. 3.3 Immunostaining of NB cells NB cells were seeded on collagen coated cover glasses. After incubation with biotin (50 µM) for 24 hours, cells were washed with PBS and fixed with 4% formaldehyde. After washing with PBS, permeabilization was done with either 32 0.1% Triton X-100 for 3 min or 0.1% Saponin for 5 min followed by blocking with 1% FBS for 40 minutes at room temperature. Primary antibody incubation was done for 1.5 hours at room temperature in blocking buffer. Cells were washed with PBS and incubated with secondary antibodies for 30 min at room temperature in the dark. Samples were mounted with Fluoromount-G. VECTASTAIN Elite ABC reagent was employed to detect biotinylated molecules. Images were acquired with a Zeiss Celldiscoverer7 imaging system. 3.4 Immunoprecipitation 0.5 - 1 mg whole cell lysate was employed for immunoprecipitation experiments in inducible NB cells. Either 4 µg ALK or SHP2 antibody and 25 µl of Dynabeads Protein A beads was added to each NB lysate, followed by incubation overnight at 4 °C. The following day, samples were washed with lysis buffer and bound proteins were eluted in 2X Laemmli buffer by heating for 5 minutes at 95 °C prior to SDS-PAGE and western blotting. For anti-HA immunoprecipitation, 500 µg of lysate from third instar larval brains was incubated with HA antibody overnight at 4 °C. On the next day, 30 µl of Protein G Sepharose beads were added on samples and incubated for 3 hours at 4 °C, followed by several washes with lysis buffer. 2X Laemmli buffer were added on samples and heated for 5 minutes at 95 °C for elution of bound proteins. Samples were then analyzed by SDS-PAGE and western blot. 3.5 Proliferation assays Cells were seeded on 96-well plates in triplicate and cultured overnight prior to treatment with either SHP2 or ALK inhibitor or both in combination. Cells were scanned every 12 hours for five days at 37°C with a Sartorius Incucyte. Cell confluency was calculated using Sartorius Incucyte S3 software. 3.6 Foci formation assay Cells were seeded in 6-well plates and treated with either SHP2 or ALK inhibitor or both in combination for 14 days. Cells were washed in PBS, air- 33 dried, fixed with methanol, and stained with 0.2% crystal violet. Plates were washed, air-dried and scanned using a Toshiba Studio 2505AC. 3.7 Generation of endogenous Alk BioID fusions by CRISPR/Cas9 genome editing in Drosophila Suitable CRISPR target sites in the C-terminal region of the Alk coding sequence were selected using the flyCRISPR Target Finder tool. Target guide RNAs were then generated by PNK-phosphorylating and annealing target site- specific sense and antisense oligos followed by cloning into the pU6-BbsI- chiRNA plasmid. gBlock DNA fragments containing either BirAR118G or miniTurbo or TurboID coding sequences as well as sequences for the GSATx4 linker and HA tag, gene-specific homology arms for CRISPR/Cas9-mediated homology-directed repair (HDR), and by short flanking repeats for Gibson assembly cloning were obtained from Integrated DNA Technologies. These DNA fragments were then cloned into pBlueScript II SK (-) using the NEB HiFi DNA assembly kit. gRNA plasmid and the donor construct were injected into embryos expressing Cas9 in their germ line (BestGene Inc.). Screening of positive HDR events was done by PCR of F2-males, after mating them to 2nd chromosome balancer females. The resulting AlkBirA*, AlkTurboID and AlkminiTurbo lines were also confirmed by sequencing. 3.8 Immunostaining of Drosophila embryos Flies were transferred to embryo collection cages and embryos collected for 8 h during the day/overnight at 25 °C. Embryos were transferred onto mesh and dechorionated in 3% sodium-hypochlorite solution. After dechorionation, embryos were washed and fixed in 4% buffered formaldehyde solution for 40 minutes. 500 µl of methanol and heptane was added after removing FA buffer and embryos were quickly vortexed to remove the vitelline membrane. After washing with 0.3% NP-40/PBS buffer, samples were blocked in 5 % goat serum for 30 minutes, followed by primary antibody incubation at 4 °C overnight. The next day, samples were washed with 0.3 % NP-40/PBS buffer and incubated with the appropriate secondary antibodies for 2 hours in the dark at room temperature. Samples were cleared in an ethanol dilution series (50, 70, 34 100%) prior to mounting with methyl salicylate. Images were acquired with Zeiss LSM800 confocal microscope. 3.9 Immunostaining of Drosophila third instar larval brains Third instar larval brains were dissected in ice-cold PBS and fixed in 4 % formaldehyde for 30 min at room temperature. Following fixation, samples were washed with PBS and permeabilized in 1 % Triton X-100 /PBS buffer for 10 minutes. After washing with 0.5 % Triton X-100 buffer, samples were blocked in either 5 % goat serum or BSA for at least 30 minutes. Primary antibodies were added in blocking buffer and samples were incubated at 4 °C overnight. The following day, samples were washed three times with 0.5 % Triton X-100/PBS buffer and incubated with the appropriate secondary antibodies for 2 hours in the dark at room temperature. Samples were mounted with Fluoromount-G and images acquired either with Zeiss LSM800 confocal microscope or Zeiss Celldiscoverer7 imaging system. 3.10 Pull-down of biotinylated proteins 3.10.1 Preparation of inducible NB cell lysates For each sample/condition, 3 x 106 cells were seeded on 15 cm dishes. Two confluent 15 cm dishes of cells were lysed in RIPA buffer, and samples centrifuged for 20 min at 14000 rpm at 4°C. Protein concentration was determined by BCA assay and 5 mg of total protein was used for streptavidin pull-downs. 3.10.2 Preparation of Drosophila third instar larval brain lysates Freshly hatched first instar larvae were transferred to fly food supplemented with 100 µM biotin and fed until wandering third instar stage. Approximately 150 larval brains were dissected per sample and lysed with RIPA buffer. The protein concentration of each sample was determined by BCA assay and 1- 1.5 mg protein was used for streptavidin pull-downs. 35 3.10.3 Streptavidin pull-down Depending on starting protein concentration, 300-600 µL Streptavidin Dynabeads (Thermo Fisher Scientific) were added to tube placed on a magnetic rack. After 3 minutes, the supernatant was removed, protein lysate was added, and samples rotated overnight at 4°C. The next day, beads were washed with wash buffer 1 (2% (w/v) SDS), wash buffer 2 (0.1% (w/v) deoxycholic acid, 1% Triton X-100, 1 mM EDTA, 500 mM NaCl, 50 mM HEPES pH 7.5) and wash buffer 3 (0.5% (w/v) deoxycholic acid, 0.5% NP-40, 1 mM EDTA, 250 mM LiCl, 10 mM Tris-Cl pH 7.4) for 8 min at room temperature, respectively. Beads were washed four times with 1.5 mL 50 mM Tris-Cl pH 7.4 to remove detergents. After the final wash, 10 % of resuspended beads were saved for western blotting analysis and the remaining 90 % was submitted for MS analysis. 3.11 Protein digestion, TMT-labeling and liquid chromatography coupled mass spectrometry (LC-MS) analysis Streptavidin beads were washed with 50 mM TEAB and digested with 0.5 µg LysC (Promega) in 100 µl 50 mM TEAB for 3h in 37 °C. Reduction and alkylation were done in 5 mM TCEP and 10 mM MMTS respectively for 30 min in room temperature. Following reduction and alkylation step, a second digestion was performed with 0.3 µg Pierce MS grade Trypsin (Thermo Fisher Scientific) overnight at 37°C. The following day, beads were pelleted using a magnetic rack and supernatant transferred to new tube. Peptides were labelled using Tandem Mass Tag reagents (TMTpro 16-plex, Thermo Fisher Scientific) and were fractionated to either 5 or 10 fractions using a Pierce High pH Reversed- Phase Peptide Fractionation Kit (Thermo Scientific). Samples were dried in vacuum centrifuge and dissolved in 15 μl of 3% acetonitrile, 0.1% formic acid for LC-MS analysis. Fractions were analyzed on an Orbitrap FusionTM LumosTM TribridTM mass spectrometer interfaced with an Easy-nLC1200 liquid chromatography system. 36 4. RESULTS AND DISCUSSION 4.1 Paper I BioID-screening identifies PEAK1 and SHP2 as components of the ALK proximitome in neuroblastoma cells. In this study, we employed BioID-based proximity labeling to identify novel ALK-associated signaling components in NB cells. We generated SK-N-A-S and SK-N-BE(2) Tet-On inducible NB cell lines expressing either ALK-BirA* fusion or BirA* and validated expression of BirA*, ALK-BirA* fusion as well as increased biotinylation upon induction with doxycycline. We also confirmed that the ALK-BirA* fusion was functional and able to activate ALK signaling upon ligand stimulation in these inducible NB cells. Upon ALKAL2 stimulation, ALK downstream signaling components, pERK and pAKT were activated while signaling was abrogated after treatment with the ALK TKI lorlatinib as expected. To define the proximal proteome of ALK, SK-N-AS.ALK-BirA* and SK-N- BE(2).ALK-BirA*, cells were stimulated with ALKAL2 in the presence or absence of lorlatinib, followed by pull-down of biotinylated proteins and analysis by LC-MS3. In both SK-N-AS.ALK-BirA* and SK-N-BE(2). ALK-BirA* cells, several known downstream components of ALK signaling as well as novel candidates were enriched when compared with BirA* expressing control cells. In order to define activity-dependent ALK signaling components, SK-N-AS.ALK-BirA* and SK-N-BE(2).ALK-BirA* cells were stimulated with ALKAL2 for 24 hours in the presence or absence of lorlatinib. Among proteins which are enriched upon ALKAL2 induction and decreased after lorlatinib treatment, we validated PEAK1 and SHP2 as ALK interactors by anti-ALK co- immunoprecipitations. We also showed that their interaction was enriched upon ALKAL2 stimulation and abrogated by lorlatinib, confirming that it is activity dependent. Since PTPN11/SHP2 is commonly mutated in NB, we focused on the ALK-SHP2 interaction. To validate SHP2 as ALK downstream target, we investigated SHP2 phosphorylation upon ALK ligand stimulation and inhibitor treatment. In ALK-addicted NB cells, ALK inhibition caused a 37 decrease of SHP2 and ERK1/2 phosphorylation that was increased upon ALKAL1 stimulation. To investigate the effect of ALK and SHP2 inhibition on proliferation of NB cells, we employed two independent SHP2 inhibitors, SHP009 and RMC-4550. Inhibition of SHP2 with either inhibitor led to decreased phosphorylation of ERK1/2 and reduced NB cell proliferation. Furthermore, we accessed the effect of treating cells with the SHP0009 SHP2 inhibitor, alone or in combination with ALK inhibitor treatment, assessing cell viability and foci forming ability. Although SHP009 moderately decreased cell growth, combination of SHP009 with lorlatinib synergistically inhibited proliferation of NB cells. Further supporting the role of SHP2 in NB, a recent study demonstrated that treatment of high-risk in vivo NB models with SHP2 inhibitor results in significant inhibition of tumor growth (Cai et al., 2022). In addition, it will be interesting to explore other candidates which have not previously linked to ALK signaling in the context of NB. In summary, in this work we were able to define an ALK proximal proteome by employing BioID in two different NB cell lines, identifying previously known ALK interactors as well as novel components of ALK signaling. We further validated PEAK1 and SHP2 as ALK interactors and investigated SHP2 as downstream target of ALK. Our study showed that SHP2 activity contributes to the survival of ALK-addicted NB cells and that combined inhibition of ALK and SHP2 synergistically abrogates proliferation of NB cells, suggesting SHP2 as a potential therapeutic for ALK-driven NB. 4.2 Paper II In vivo profiling of the Alk proximitome in the developing Drosophila brain In this paper, we employed proximity labeling by using different BirA* biotin ligase variants to define components of Alk signaling in the Drosophila CNS. To achieve this, we endogenously tagged the C-terminal of Alk in locus with the original BioID enzyme BirA* as well as the next generation miniTurbo and TurboID BirA variants using CRISPR/Cas9 genome editing. We initially investigated whether these three Alk-BirA* variants exhibit normal levels of Alk expression and activity. In all three Alk-BirA variants, AlkBirA*, AlkTurboID and 38 AlkminiTurbo, founder cell specification in the embryonic VM, which is dependent on Alk activity, was normal. Additionally, expression of Alk, HA- tagged BirA enzyme variants as well as biotinylation levels were assessed in AlkBirA*, AlkTurboID and AlkminiTurbo larval brains. Compared with w1118 controls, AlkBirA* brains showed only slightly higher levels of biotinylation while significantly higher levels of biotinylation were detected in both AlkTurboID and AlkminiTurbo brain extracts. We further investigated the effect of biotin feeding in these flies with biotin-supplemented food and showed that feeding resulted in higher biotin labeling as expected in AlkTurboID and AlkminiTurbo compared to non-fed flies. Since more efficient biotinylation was observed in both AlkTurboID and AlkminiTurbo larval brains, we also investigated biotin labeling in visceral mesoderm (VM) of embryos, where Alk is expressed during embryonic development. The VM of AlkTurboID and AlkminiTurbo exhibited high biotin signals, while negligible biotin signal was detected in AlkBirA*. Taken together, our data show that TurboID and miniTurbo allow efficient biotinylation even during embryogenesis when biotin feeding is not possible. Next, we performed proximity labeling in AlkBirA*, AlkTurboID and AlkminiTurbo third instar larval brains. While we observed only a few significantly enriched proteins in AlkBirA*, we identified 142 and 140 proteins enriched in AlkTurboID and AlkminiTurbo respectively with a 90% overlap between them. Among proteins which were enriched in both AlkTurboID and AlkminiTurbo, we selected five candidates for urther investigation: Stardust (Sdt), Disc large 1 (Dlg), Syntaxin 1A (Syx1A), Corkscrew (Csw) and Rugose (Rg), based on availability of reagents. In all cases, we were able to confirm their co-expression with Alk in CNS, particularly in the larval ventral nerve cord. Since we had previously identified Csw homolog SHP2 as an ALK interactor in NB cells, we focused on Csw for further analysis. We were able to detect Csw in HA-pulldowns from AlkTurboID larval brain lysates, confirming Csw as an Alk interactor. We also generated Csw expressing clones in the larval wing disc and showed that Alk and Csw indeed colocalize on the membrane. To investigate role of Csw upon Alk signaling in the VM, we expressed dominant-negative Csw in the developing embryonic mesoderm and assessed dpERK activation. Expression of dominant-negative Csw resulted in increased number of dpERK-negative 39 cells in the founder cell row compared to control embryos. Although dpERK signal was present at the beginning of germ band retraction in controls, it was barely visible in embryos expressing dominant-negative Csw, suggesting that dominant-negative Csw is not able to abrogate ERK activation but rather decreases the duration of the signal. Furthermore, we also investigated whether Csw is required for Alk signaling in the brain. Since increased Alk activity in the larval brain results in small size phenotype, we overexpressed Csw wild-type and dominant-negative transgenes with pan-neuronal driver and assessed size modification. Upon expression of dominant negative Csw, we observed rescue of the small size phenotype caused by Jeb overexpression. This suggested that Csw is a downstream modulator of Alk signaling output in Drosophila. Recently, other studies in flies, worms, zebrafish, and plants employing either endogenous or inducible TurboID fusions, have also shown that TurboID-based labeling can effectively identify protein interactors (Mair et al., 2019, Shinoda et al., 2019, Artan et al., 2021, Rosenthal et al., 2021). However, precise control of the TurboID-labeling window is challenging in intact organisms and background labeling remains a limitation. To improve spatial specificity, split versions of TurboID have been generated. In this approach, activity of two inactive split fragments is restored when they are in proximity (Chen et al., 2022, Takano and Soderling, 2021, Cho et al., 2020). It would be interesting to generate Alk-split TurboID fusions and perform PL to detect interactors upon dimerization and activation of the receptor. In summary, this work provided the first comparative study of a single genetic loci modified with all three variants of BirA*. Our results clearly show that TurboID and miniTurbo generate more efficient proximity labeling in vivo when compared with the first generation BirA* enzyme. Using both miniTurbo and TurboID, we were able to define an Alk proximitome in the Drosophila larval CNS that includes potential candidates important in Alk signaling. Among these candidates, we have characterized the role of Csw in Alk signaling in the larval brain. 40 4.3 Paper III Proximity labeling identifies regulators of Alk signaling in the Drosophila CNS Our previous work described the core Alk proximitome in the Drosophila CNS. In this study, we employed TurboID-based labeling of the Alk proximitome under different conditions to identify activity-dependent Alk-associated signaling complexes. To do this we used two approaches: (1) pan-neuronal Jeb ligand overexpression and (2) use of an activated gain-of-function AlkY1555S::TurboID mutant. In the first approach, proximity labeling was performed in the presence or absence of ectopically expressed Jeb ligand, followed by analysis of biotin-labeled proteins by LC-MS3. Compared to controls, we detected enrichment of Alk, the previously described Alk interactors Nf1 and Csw, and several Alk proximitome components identified in our previous study. We identified 86 proteins enriched upon Jeb overexpression Alk::TurboID background compared to Alk::TurboID, including proteins associated with Alk signaling, such as Csw and Chico, as well as novel candidates including low- density lipoprotein receptor-related protein 4 (Lrp4). To complement this dataset, we also endogenously tagged an activated AlkY1355S mutant with TurboID (referred here as AlkYS::TurboID). We initially showed that Alk expression and biotinylation levels in the AlkYS::TurboID larval CNS were comparable to those observed in the Alk::TurboID CNS. In addition, we confirmed that AlkYS::TurboID flies exhibited similar phenotypes to AlkY1355S, such as reduced pupal size and ectopic expression of Mamo protein in the mushroom body (MB) lineages of the CNS, indicating that the TurboID fusion did not affect AlkY1355S function. Employing TurboID-labeling, we identified 785 proteins that were commonly enriched in both wild-type Alk::TurboID and AlkYS::TurboID compared to control. Among these, 101 proteins including Cnk, Ksr, Csw, the adaptor proteins Crk and Drk, which are known to be involved in Alk signaling, as well as novel candidates, were enriched in AlkYS::TurboID compared to wild-type Alk::TurboID CNS. 41 One novel candidate, Lrp4, was one of the most abundant proteins enriched in both datasets. Previous studies have described Lrp4 as a synaptic organizer and regulator of synaptic function in Drosophila. We initially investigated the expression of Lrp4 in our previous larval CNS single cell sequencing (sc-RNA seq) dataset and detected high Lrp4 expression specifically in Alk-expressing cell clusters, especially in mature neurons. We also investigated endogenous Lrp4 expression by immunostaining and confirmed that Alk and Lrp4 co-localize in the larval CNS. Futhermore, we analyzed a previously generated Lrp4 loss of function mutant, Lrp4Dalek. Interestingly, Lrp4Dalek displayed a pupal size phenotype reminiscent of the Alk gain-of-function phenotypes, suggesting that Lrp4 might be a negative regulator of Alk signaling. Reduction in pupal size was also observed upon Lrp4 knockdown via two independent RNAi constructs. Additionally, overexpression of Lrp4 was able to partially rescue the size phenotype caused by increased Alk activity, indicating that Lrp4 might function as negative regulator of Alk. We further assessed Alk activity in larval brain by antibodies that detect phosphorylated Y1278 of human Alk, which is conserved in the activation loop of Drosophila Alk, observing that Lrp4Dalek mutants displayed slightly higher phosphorylated Alk levels compared to control. In addition, our previous finding that AlkY1355S mutants display ectopic Mamo expression in MB γ neurons, prompted us to also analyze Mamo in Lrp4Dalek mutants. Strikingly, we detected that a high proportion of γ neurons ectopically expressed Mamo in Lrp4Dalek mutants. Overall, these data suggest Lrp4 is a negative regulator of Alk signaling in the CNS. 42 5. CONCLUSIONS 5.1 Paper I  We described a human ALK proximitome in NB cells by BioID-based proximity labeling.  Proximity labeling upon ALKAL2 stimulation and lorlatinib treatment revealed activity-dependent changes in the ALK proximitome.  SHP2 and PEAK1 were validated as ALK interactors in NB cells.  The SHP2 protein tyrosine phosphatase acts downstream of ALK, and combined inhibition synergizes to abrogate proliferation in NB cells. 5.2 Paper II  We identified components of the Drosophila larval brain Alk proximitome employing proximity labeling with three different BioID enzyme variants.  We reported the first comparison of a single genetic loci modified with all three BioID enzyme variants, BirA*, miniTurbo and TurboID.  We showed that miniTurbo and TurboID variants are more efficient in labeling of proximal proteins than BirA*.  Both miniTurbo and TurboID variants exhibit proximity labeling during embryogenesis even in the absence of exogenous biotin.  We identified the SHP2 homolog, Corkscrew as an Alk interactor and regulator of Alk signaling in Drosophila. 5.3 Paper III  We describe dynamic changes in the Alk proximitome in response to Alk activation in the Drosophila larval CNS.  We identified both known and novel components of Alk signaling in the larval CNS.  We further characterized Lrp4 as a negative regulator of Alk signaling in the CNS. 43 ACKNOWLEDGEMENTS Hi there, I know this is the first page you read in this thesis � From the bottom of my heart, I thank all the people who are part of my PhD journey, and I am grateful that our paths crossed. Firstly, I would like to thank my supervisor Ruth Palmer for her tremendous support and guidance throughout my PhD. Your enthusiasm towards science and constant motivation always inspired me. Looking back, I realize how much I learned, not just about the field of Alk but also about new techniques. Although you have a very busy schedule, you always had time for me to discuss experiments and results, and also help with manuscript preparations. To these, I am extremely grateful. You also encouraged me to go to courses and conferences to learn new things and meet with other people in the field, which helped me to build connections. You supported me in every way to be a better researcher and I cannot thank you enough. A big thanks also to my co-supervisor Georg Wolfstetter for his help and guidance in my projects. Your knowledge about Drosophila genetics and development impressed me over these years. Whenever I need a suggestion, whether for an experimental plan or to solve a problem, you always gave me so many ideas and I appreciate that. I am also very grateful for your critical comments and suggestions during manuscript preparation, as well as preparation for this thesis. Thank you, Bengt Hallberg, for your valuable comments and suggestions during my project presentations. I also appreciate your inputs and critical reading during manuscript preparations. To Anne Uv, many thanks for your help during preparation for half time as my committee member and your suggestions on my projects during our joint lab meetings. Next, I would like to thank current members of the Palmer and Hallberg lab. I am grateful that I had a chance to work in such a hardworking international team. 44 Dear Kathrin, when I started working in this lab, I asked you almost everything about immunostaining and you shared your tips. Thanks to you, I have got nice stainings ever since. Over these years, we also have spent some fun times outside of the lab having lunches, fikas and yoga sessions that I will always remember. Thank you so much for inspiring me to do yoga. I have really benefited from it. My dear friend, Tafheem, thank you for the fun times in the fly lab, discussions, lunches and many fikas together. I also very much appreciate your emotional support during hard times. I wish you all the best. Sanjay, thank you for being such a helpful and caring friend. During these years, we had fun times in the fly lab discussing pretty much anything. I will remember those talks and laughs. Good luck on the “Sparkly” project and I wish you all the best for your PhD. You will do great! Dear Vimala, it was very nice to collaborate with you on my projects and I appreciate your help with bioinformatics analysis. Thank you also for your patience and support during manuscript preparation. To Hisae, thank you for cooking fly food every week and keep our flies happy! Marcus, thanks for being a great office buddy. I also appreciate your effort on organizing lab dinners. Thanks to you it’s finally happening! I wish you all best for your PhD. To Jikui, I appreciate your help and suggestions, especially on NB cell culture experiments. We had a good collaboration over these years, and I learned a lot from you. Thank you! Joachim, my good friend, I always admired your positivity and your passion for science. It was a pleasure to collaborate with you for our ALK-SHP2 paper and I think it turned out great. Thank you also for fun times, sightseeing and fishing, during the course in Tromso. I wish you all the best for your future career. To my friend Ganesh, thank you for all the help and guidance in cell culture and valuable tips in western blotting. I also appreciate your suggestions about my project during lab talks. Our lab manager Dan, thank you for lunches, fun conversations, and Friday beers. I also very much appreciate your help with ordering and help to organize things in the lab. Badrul, I always enjoyed our talks in the fly room about our projects and future plans. Many thanks for being very kind and understanding. I wish you all the best in your future career. Thanks Tzu-Po and Wei- Yun for being so nice and helpful all the time. I appreciate your ideas and suggestions during my project talks. My friend Malik, thanks to you “oh lord” stuck in my mind 45 and every time something unexpected happens, I say that. Joking aside, you are a good friend who always put a smile on my face, and I will always remember you and your delicious fikas. I wish you all the happiness. Yash, it was nice to meet you and thank you for your friendship. I wish you a good start in your new position at AstraZeneca. Jonatan, thank you for good conversations and after works on Friday. I wish you all the best for your PhD. Thanks, Linnea, for being a DJ of the fly lab and having always positive attitude. I wish you success in your studies. Edit, I wish you good luck with your PhD. New member of our lab Joel, welcome and I wish you all the best. Next, I would also like to thank former members of the Palmer and Hallberg lab who I truly missed during past years. To my Joanna, thank you for your enormous support throughout these years. You are an amazing friend who is kind, loving, and always caring. I will always remember the times we spent together, many lunches, little fikas during incubations, ice-cream breaks in the botanic garden, and our weekend trip to Poland. During these years, you were always there for me when i needed the most, I will never forget that. My dear Patricia, Pato, thank you for your invaluable friendship, support, and encouragement during these years. You are great person with a lot of positive energy, and I missed having you around. We had great time together not only in the lab but also outside, at afterworks, cocktail parties and joint birthday parties. Hope we have many more to come! To Diana, thank you for introducing me to cell culture and western blotting for the first time and sharing your expertise. I also appreciate our time together outside the lab, spontaneous afterworks, lunches, cosy Christmas dinner in your place and swimming sessions. Thanks Wasi for being a good and caring friend. I always remember the time when we went to a course in Tromso, we had a great time together. I wish you all the best in your career. Hannah, thank you for being such a good office mate and friend. During the first years of my PhD, we had so much fun in comedy nights and 90s parties. I wish you all the happiness in life. Maite, I am glad we met and thank you for sharing your protocol and giving me tips for immunostaining of cells. 46 I also want to thank other colleagues for making Medicinaregatan 9 a great workplace. A beloved member of my Turkish gang, Direniş, I am very glad i met you during the first years of our PhD. Thank you for your support and fun times during spontaneous lunches and fikas at work, parties, and raki nights. I wish you all the best for your PhD and I already know you will do great! Ainsley, thanks for your positivity and fun conversations in the microscopy room. Thank you Jameel and Andy for good conversations during lunch times. I also would like to thank to my friends who are current and former members of Sahlgrenska Cancer Center. My lovely Polish ladies, Dorota and Agnieszka, I am very grateful that Joanna introduced me to you in the beginning of our studies. During the past two years, we also became neighbours and had a lot of fun together having parties, lunches and fikas. I could not ask better aunties for Jojo and he specifically thanks for cat sittings. I also appreciate your suggestions and support regarding all PhD related processes. Canlarim beybilerim, Ebru ve Pinar, iyi ki sizinle yollarimiz burada kesişmiş ve iyi ki sizi tanimişim. Doktoramin ilk iki senesini şenlendirdiğiniz ve hep yanimda olduğunuz için size çok teşekkür ederim. Lahmacun partilerimizi ve fikalarimizi çok özledim. Umarim ilerleyen zamanlarda tekrarlariz. My lovely Hana, thanks for all fun times including lunches, fikas and lahmacun dates after work. You were the one of the persons that understand what I am going through with all these permit issues and thank you for emotional support. Thanks to members of Proteomics Core Facility, especially Johannes Fuchs and Carina Sihlbom for their technical support and collaboration in my projects during PhD. Special thanks to my partner Christian who supported me during the last two years of my PhD journey and helped me to go through the hard times. I am grateful for your constant love and care. Love you. 47 Finally, I would like to thank my family for their huge support throughout my studies. Canim annem Birsel ve babam İbrahim, sizin desteğiniz ve inanciniz bu güne kadar bana hep güç, kuvvet verdi. Sizin çalişkanliğiniz ve azminiz yaşamimda hep bana örnek oldu. Herşey icin teşekkür ediyorum. Çekirdek Uçkun ailemin diğer üyeleri Gülcişim ve Yeliz ablam, bu süreçte hep yanimda olduğunuz için ve sevginiz için size teşekkür ediyorum. Canim anneannem Yüksel, uzakta da olsak sevgini, desteğini dualarini hep hissettim. Bunun için teşekkür ediyorum. Hepinizi çok seviyorum. 48 REFERENCES 1. ADACHI, M., SEKIYA, M., MIYACHI, T., MATSUNO, K., HINODA, Y., IMAI, K. & YACHI, A. 1992. Molecular cloning of a novel protein-tyrosine phosphatase SH-PTP3 with sequence similarity to the src-homology region 2. FEBS Lett, 314, 335-9. 2. AGAZIE, Y. M. & HAYMAN, M. J. 2003. Molecular mechanism for a role of SHP2 in epidermal growth factor receptor signaling. Mol Cell Biol, 23, 7875- 86. 3. AHMAD, S., BANVILLE, D., ZHAO, Z., FISCHER, E. H. & SHEN, S. H. 1993. 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