The RET receptor kinase: mechanism, signaling and therapeutics

by

Taranjit Singh Gujral

A thesis submitted to the Department of Pathology & Molecular Medicine

in conformity with the requirements for

the degree of Doctor of Philosophy

Queen’s University

Kingston, Ontario, Canada

September 2008

Copyright © T.S Gujral. 2008

Abstract

The RET receptor has essential roles in cell survival, differentiation, and proliferation. Oncogenic activation of RET causes the cancer syndrome multiple endocrine neoplasia type 2 (MEN 2), and is a frequent event in sporadic thyroid carcinomas. Multiple endocrine neoplasia 2B (MEN 2B), a subtype of

MEN 2, is caused primarily by a methionine to threonine substitution of residue 918 in the kinase domain of the RET receptor (2B-RET), however the molecular mechanisms that lead to the disease phenotype are unclear. In this study, we show that the M918T mutation causes a 10 fold increase in ATP binding affinity, and leads to a more stable receptor-ATP complex, relative to the wildtype receptor. We also show that 2B-RET can dimerize and become autophosphorylated in the absence of ligand stimulation. Our data suggest that multiple distinct but complementary molecular mechanisms underlie the

MEN 2B phenotype and provide potential targets for effective therapeutics for this disease.

In the second part of the study, we identified a novel β-catenin-RET kinase signaling pathway which is a critical contributor to the development and metastasis of human thyroid carcinoma. We show that RET binds to, and tyrosine phosphorylates, β- catenin and demonstrate that the interaction between RET and β-catenin can be direct and independent of cytoplasmic kinases, such as SRC. As a result of RET-mediated tyrosine phosphorylation, β-catenin escapes cytosolic downregulation by the APC/Axin/GSK3 complex and accumulates in the nucleus, where it can stimulate β-catenin-specific transcriptional programs in a RET-dependent fashion. We show that downregulation of

β-catenin activity decreases RET-mediated cell proliferation, colony formation, and tumour growth in nude mice.

i Finally, we used a structure guided approach to identify and characterize a novel, non-ATP competitive, RET kinase inhibitor; SW-01. We show that SW-01 provides significant RET inhibition in an in vitro kinase assay using purified RET proteins.

Moreover, RET phosphorylation is blocked, or dramatically reduced, in vivo in cells overexpressing active RET. We observe a significant decrease in cell proliferation and colony formation in RET-expressing cells in the presence of SW-01. Together, our data suggest that SW-01 has potential as a novel RET kinase inhibitor with clinical utility.

ii Co-Authorship

This section serves to recognize and document all of the contributions that were made by others to each chapter of this thesis.

Chapter 3: Molecular mechanisms of RET receptor mediated oncogenesis in multiple endocrine neoplasia 2B was published in the journal Cancer Research in 2006. The authors as listed on the paper: Taranjit Gujral, Dr. Vinay Singh, Dr. Zongchao Jia and Dr.

Lois Mulligan. In this paper, I performed all the experiments except for analytical gel filtration which was performed by Dr. Vinay Singh. Drs. Singh and Jia provided help with analysis of biophysical results. I wrote the manuscript which was subsequently edited by Dr. Mulligan.

Chapter 4: A novel RET kinase-β-catenin signaling pathway contributes to tumorigenesis in thyroid carcinoma was published in the journal Cancer Research in

2008. The authors on this paper: Taranjit Gujral, Wendy van Veelen, Douglas

Richardson, Shirley Myers, Jalna Meens, Dr. Dennis Acton, Dr. Mireia Duñach, Dr.

Bruce Elliott, Dr. Jo Höppener, and Dr. Lois Mulligan. I performed all the experiments except for the experiments mentioned below. Wendy Veelen, Dr. Dennis Acton and Dr.

Jo Höppener provided human and mouse MTC data (Figures 4.1and 4.2). Douglas

Richardson performed confocal microscopy (Figure 4.3). Shirley Myers provided technical assistance in qRT-PCR data collection. Jalna Meens and Dr. Bruce Elliott performed the xenograft study (Figure 4.14). Dr. Mireia Duñach provided GST-β-catenin constructs. I wrote the manuscript which was subsequently edited by Dr. Mulligan.

iii

Chapter 5: Identification and characterization of a novel RET tyrosine kinase inhibitor is prepared for submission to the journal of Cancer Research. The authors listed on the paper: Taranjit Gujral, Dr. Vinay Singh, and Dr. Lois Mulligan. I performed all the experiments for this manuscript. Dr. Singh performed in silico docking. I wrote the manuscript which was subsequently edited by Dr. Mulligan.

iv Acknowledgements

First and foremost, I would like to thank Dr. Lois Mulligan for her guidance and patience throughout this project. Without her constant mentorship, encouragement, enlightening feedback this project would not have become the phenomenal learning experience that it has proven to be.

I would also like to thank all the past and present members of the “Mulligan lab”, for their help and constant support. I also wish to acknowledge Drs. Zongchao Jia and

Bruce Elliot for being on my supervisory committee and providing me with constructive feedbacks.

Finally, I would like to thank my family for their support throughout the course of this thesis.

v List of Tables and Figures

Page Abstract i Co-authorship iii Acknowledgements v Table of Contents vi List of Tables and Figures ix Abbreviations List xi

Chapter 1: Introduction 1 1.1 The RET gene 1 1.2 Structure of RET 1 1.3 Activation of RET 3 1.4 RET and Signaling 3 1.5 The role of RET in development 7 1.6 RET and Disease 8 1.6.1 RET and endocrine tumours 8 1.6.2 RET and other associated disease types 16 1.7 RET as a therapeutic target 17 1.7.1 Dimerization inhibitors 18 1.7.2 Tyrosine kinase inhibitors 20 1.7.3 Intracellular signaling inhibitors 24

Chapter 2: Proposed Research 27 2.1 RET-mediated oncogenesis in MEN 2B 27 2.1.1 Rationale 27 2.1.2 Hypothesis 28 2.1.3 Objectives 28 2.2 RET-mediated intracellular signaling 28 2.2.1 Rationale 28 2.2.2 Hypothesis 29 2.2.3 Objectives 29 2.3 Targeting RET for therapeutics 29 2.3.1 Rationale 29 2.3.2 Hypothesis 30 2.3.3 Objectives 30

Chapter 3: Molecular mechanisms of RET receptor mediated oncogenesis 31 in multiple endocrine neoplasia 2B (Gujral et al.,Cancer Res. 2006; 66(22):10741-9) 3.1 Abstract 32 3.2 Introduction 33 3.3 Materials and Methods 35 3.3.1 Expression constructs 35 3.3.2 Expression and purification of WT-RET and 2B-RET 36 proteins

vi 3.3.3 Peptide fingerprinting by mass spectrometry 36 3.3.4 In vitro kinase kinetics 37 3.3.5 Circular dichroism spectroscopy 37 3.3.6 Isothermal titration calorimetry 38 3.3.7 Analytical gel filtration 38 3.3.8 Differential scanning calorimetry 39 3.4 Results 39 3.4.1 Expression and purification of WT and 2B-RET 39 3.4.2 Comparison of WT-RET and 2B-RET structure and activity 42 3.5 Discussion 52 3.6 Acknowledgments 57

Chapter 4: A novel RET kinase-β-catenin signaling pathway contributes to 58 tumorigenesis in thyroid carcinoma (Gujral et al.,Cancer Res. 2008; 68(5): 1338-1346) 4.1 Abstract 59 4.2 Introduction 60 4.3 Materials and Methods 62 4.3.1 Expression constructs 62 4.3.2 Cell culture and transfection 62 4.3.3 Immunoprecipitation, western, and far-western blotting 63 4.3.4 Preparation of cytosolic extracts 64 4.3.5 Glutathione S-transferase (GST)-pull-down assays 64 4.3.6 Reporter assay 64 4.3.7 shRNA production 65 4.3.8 MTT cell proliferation assay 65 4.3.9 Apoptosis assays 65 4.3.10 Soft Agar colony formation assays 66 4.3.11 Confocal microscopy 66 4.3.12 Tumourigenicity in nude mice 66 4.3.13 β-catenin immunohistochemistry 67 4.3.14 Relative quantification by real-Time RT-PCR 68 4.4 Results and Discussion 68 4.4.1 β-catenin nuclear localization in RET-mediated human 68 thyroid tumours 4.4.2 RET associates with and tyrosine phosphorylates β-catenin 70 4.4.3 RET activation induces cytosolic translocation of β-catenin 80 and its escape from the axin regulatory complex 4.4.4 RET-induced β-catenin tyrosine phosphorylation is 80 associated with increased TCF transcriptional activity 4.4.5 β-catenin is required for RET-induced cell proliferation and 83 transformation 4.4.6 Knockdown of β-catenin expression reduces RET-mediated 86 tumour growth and invasiveness in nude mice 4.5 Acknowledgements 95

vii Chapter 5: Identification and characterization of a novel RET tyrosine 96 kinase inhibitor (Manuscript in preparation for Cancer Research) 5.1 Abstract 97 5.2 Introduction 98 5.3 Results and Discussion 100 5.3.1 High throughput in silico screening: identification of SW- 100 01 5.3.2 SW-01 inhibits RET autophosphorylation in vitro and in 102 vivo 5.3.3 Effects of SW-01 on RET-mediated downstream signaling, 104 cellular growth, proliferation and transformation 5.3.4 SW-01 impairs RET-mediated migration of pancreatic 109 cancer cells 5.4 Materials and Methods 111 5.4.1 Small-molecule libraries 111 5.4.2 In-vitro kinase kinetics 111 5.4.3 Cell culture 111 5.4.4 Immunoprecipitations and western blotting 112 5.4.5 MTT cell proliferation assay 112 5.4.6 Soft agar colony formation assay 113 5.4.7 Apoptosis assay 113 5.4.8 Wound healing assay 114

Chapter 6: Discussion 115 6.1 Molecular mechanisms of RET-mediated oncogenesis 115 6.2 RET-mediated downstream signaling 119 6.3 RET as a therapeutic target 123

References 131 Appendix I: Molecular mechanisms of RET-mediated oncogenesis in MEN 2B 144 Appendix II: Summary of qRT-PCR primers and products 154 Appendix III: Composition of Solutions 155

viii List of Tables and Figures

Tables Page Table 1.1 Small-molecule inhibitors of the RET kinase 26 Table 3.1 Kinetic properties of WT-RET and 2B-RET 45 Table 3.2 Calorimetric analysis of the temperature-induced denaturation 51 Table 4.1 Patient information and β-catenin staining patterns for primary and 71 metastazised human MTCs Table 4.2 Gross pathology and histology of tumour lesions 92 Table 5.1 Selectivity of SW-01 105 Table 6.1 Structural effects of MEN 2B RET mutations 117

Figures Page Figure 1.1 Schematic diagram of RET 2 Figure 1.2 Schematic of RET activation 4 Figure 1.3 Schematic of the RET tyrosine kinase domain with different 6 autophosphorylated Figure 1.4 Schematic of RET showing missense mutations associated with 9 HSCR Figure 1.5 Schematic of RET showing missense mutations associated with 11 MEN 2A and MEN 2B Figure 1.6 Schematic of strategies to inhibit RET signaling 19 Figure 1.7 Homology models of RET 22 Figure 3.1 Expression and Purification of GST-fused WT-RET and 2B-RET 40 Figure 3.2 Identification of RET peptides by MALDI-TOF mass spectrometry 41 Figure 3.3 WT-RET and 2B-RET do not differ in overall secondary or tertiary 43 structure Figure 3.4 Thermodynamic analyses of WT-RET and 2B-RET 44 Figure 3.5 Monomer-dimer equilibrium of 2B-RET protein is shifted towards 47 dimers Figure 3.6 2B-RET has increased autokinase activity 48 Figure 3.7 Differential scanning calorimetry (DSC) analyses of WT-RET, and 50 2B-RET Figure 3.8 Model of stable, and transient, dimer formation for WT-RET and 56 2B-RET Figure 4.1 Altered localization of β-catenin in response to activated RET in a 69 murine model of RET-induced MTC Figure 4.2 Localization of β-catenin in human MTCs 72 Figure 4.3 Endogenous RET and β-catenin co-localize at the cell membrane 73 Figure 4.4 RET associates with, and tyrosine phosphorylates, β-catenin 75 Figure 4.5 Recombinant purified RET can directly phosphorylate purified β- 77 catenin in vitro Figure 4.6 RET can phosphorylate β-catenin in the absence of SRC family 78 kinases Figure 4.7 β-catenin tyrosine 654 (Y654) is a major RET-phosphorylation site 79

ix Figure 4.8 RET alters β-catenin localization 81 Figure 4.9 Tyrosine phosphorylated β-catenin escapes the axin regulatory 82 complex in the cytoplasm Figure 4.10 Knockdown of β-catenin and cyclinD1 protein levels by shRNA 84 against β-catenin Figure 4.11 RET activation induces β-catenin-mediated transcription 85 Figure 4.12 β-catenin is required for RET induced proliferation and 87 transformation Figure 4.13 β-catenin has a modest effect on RET-induced cell survival 89 Figure 4.14 RET-mediated tumour outgrowth in nude mice is reduced by 90 shRNA to β-catenin Figure 4.15 Coexpression of RET and endogenous β -catenin promotes regional 91 invasiveness and tumour adhesion to the abdominal wall Figure 4.16 Proposed model of a RET-β-catenin signalling pathway 94 Figure 5.1 High throughput in silico screening: Identification of SW-01 101 Figure 5.2 SW-01 inhibits RET autophosphorylation in vitro and in RET 103 expressing cells Figure 5.3 Effects of SW-01 on RET-mediated cellular proliferation and 107 transformation Figure 5.4 SW-01 causes apoptosis in medullary thyroid carcinoma cells 108 Figure 5.5 SW-01 impairs the ability of MiaPaca2 pancreatic cancer cells to 110 migrate in wound healing assays Figure 6.1 Characterization of RET- β-catenin-E cadherin interaction 120 Figure 6.2 Structure-based inhibition of kinases 125 Figure 6.3 Chemical and pharmacological properties of cyclobenzaprine 126

x Abbreviations List

3D three-dimensional

A-loop activation loop APC adenomatous polyposis coli ATP adenosine triphosphate bp base pair BRAF v-raf murine sarcoma viral oncogene homolog B1 BSA bovine serum albumin

CD circular dichroism spectroscopy C-loop catalytic loop Ct crossing threshold C terminus carboxyl terminus

DMEM Dulbecco’s modified eagle medium DNA deoxyribonucleic acid DOK downstream of tyrosine kinase dNTP deoxy-nucleotide tri-phosphate DSC differential scanning calorimetry DTT dithiothreitol

EDTA ethylenediaminetetraacetate

FACS fluorescence activated cell sorting FBS fetal bovine serum FGFR fibroblast receptor FLT-3 FMS-like tyrosine kinase FMTC familial medullary thyroid carcinoma

GAB1 GRB2-associated binding protein 1 GDF15 growth differentiation factor 15 GDNF glial cell line-derived neurotrophic factor GFL glial cell line-derived neurotrophic factor family ligand GFRα GDNF family receptor alpha GSK-3 glycogen synthase kinase-3 GST glutathione-S-transferase GRB bound protein

HEK human embryonic kidney HPT hyperparathyroidism

xi icRET intracellular portion (amino acid 658 to C- terminus) of RET IPTG isopropylthio-β-D-galactoside IRK receptor kinase ITC isothermal titration calorimetry

Kd dissociation constant

LB Luria- Bertani medium

MALDI matrix-assisted laser desorption/ionization MAPK mitogen-ativated-protein-kinase MEN multiple endocrine neoplasia MEN 2A multiple endocrine neoplasia type 2A MEN 2B multiple endocrine neoplasia type 2B MEN 2B RET RET carrying MEN 2B mutation MTC medullary thyroid carcinoma MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide

NCK non catalytic region of tyrosine kinase

PBS phosphate buffered saline PC pheochromocytoma PCR polymerase chain reaction PDAC pancreatic ductal adenocarcinoma PI3K phosphatidylinositol 3 kinase PLC-γ phospholipase C gamma PMSF phenylmethylsulphonylfluoride PPARγ peroxisome proliferation activated receptor-gamma PTB phosphotyrosine binding PTC papillary thyroid carcinoma qRT-PCR quantitative real time PCR

RET rearranged in transfection RMS root mean square RPI-1 ribose-5-phosphate isomerase RPMI roswell park memorial institute media RTK

xii SDS sodium dodecyl sulfate SDS-PAGE SDS polyacrylamide gel electrophoresis SHC src homology 2 domain-containing transforming protein C SH2 src homology 2 SH3 src homology 3 STAT signal transducer and activator of transcription SYF Src Yes Fyn

TBS tris-buffered saline TBST tris-buffered saline tween 20 TEMED N, N, N’, N’-tetramethylethylenediamine TET tetracycline TK tyrosine kinase TOF time-of-flight

VEGFR vascular endothelial growth factor receptor VGF VGF induced

WCL whole cell lysates WT wild type

xiii Chapter 1:

INTRODUCTION

1.1 The RET gene

The RET (REarranged during Transfection) proto-oncogene encodes a receptor tyrosine kinase which plays important roles in cell growth, differentiation and survival [1-

5]. RET is expressed in cell lineages derived from the branchial arches, the neural crest, and in the tissues of the urogenital system. These include the brain, parasympathetic and sympathetic ganglia, C-cells of the thyroid, adrenal medulla, kidney, and enteric ganglia

[4]. RET occurs in three main isoforms, ranging in size from 1,072 to 1,114 amino acids.

The largest isoform, RET 51, contains 51 unique amino acids at the carboxyl end, RET 43 contains 43 different amino acids, and RET 9 contains 9 different amino acids [6, 7]

(Figure 1.1). The size of RET protein shows a 170 kDa and 150 kDa doublet [8]. Cell fractionation experiments revealed that the 170 kDa protein but not the 150 kDa protein was predominantly in the plasma membrane fraction, indicating that the 170 kDa protein represents the mature glycosylated form of the RET protein present on the cell surface

[8]. The 150 kDa immature form is present in the endoplasmic reticulum and in the cytoplasm.

1.2 Structure of RET

The RET receptor is composed of three domains: an extracellular ligand binding domain, a single transmembrane domain, and an intracellular kinase domain (Figure 1.1)

(Reviewed in [9]). The extracellular domain includes regions with homology to the cadherin family of cell adhesion molecules and a cysteine rich domain, in which there are

27 highly conserved cysteines (Reviewed in [9]). It is thought that these cysteines play a role in forming intramolecular disulfide bonds in RET molecules. The extracellular 1

Figure 1.1 Schematic diagram of RET. RET is composed of three domains: an extracellular ligand binding domain, a single transmembrane domain, and an intracellular kinase domain. The extracellular domain includes four cadherin-like domains and a cysteine rich region. The intracellular domain includes a kinase domain and a carboxyl- terminal domain. The three RET isoform tails of RET 9, RET 43 and RET 51 are indicated.

2 Extracellular Cadherin like Domain Domains

Cysteine rich Domain

Transmembrane Domain

Intracellular Domain Tyrosine kinase Domain RET 9 RET 43 RET 51

2a domain also contains a number of glycosylation sites (13). The intracellular domain of

RET contains a tyrosine kinase domain which plays a role in downstream signaling.

1.3 Activation of RET

RET differs from other receptor tyrosine kinases as it requires a multicomponent complex, rather than a single ligand, to initiate signaling. Activation of RET is a multistep process, involving interaction with a soluble ligand and a non-signaling cell surface bound molecule. RET’s soluble ligands belong to the glial cell line-derived neurotrophic factor (GDNF) family of ligands (GFL), which includes GDNF, , persefin and [10, 11]. The cell surface bound molecules belong to the GDNF family receptor alpha (GFRalpha 1-4) proteins and are attached to the cell membrane by a glycosyl- phosphatidylinositol linkage [12] (Figure 1.2A).

GFL and GFRalpha family members form complexes which, in turn, bind to RET and trigger its activation by causing it to dimerize [12] (Figure 1.2B). This facilitates trans-autophosphorylation of specific tyrosine residues in the RET intracellular domain.

Activation of RET, in turn, stimulates a cascade of intracellular protein interactions.

Phosphotyrosine residues play a critical role in by either providing docking sites for downstream signaling proteins or increasing the receptor catalytic activity [13].

1.4 RET and signaling

RET activates several intracellular signaling cascades, which regulate cell survival, differentiation, proliferation, migration, chemotaxis, morphogenesis, neurite outgrowth and synaptic plasticity (Reviewed in [9]). The intracellular domain of activated

RET contains multiple phosphorylated tyrosines which serve as docking sites for various

SRC-homology 2 (SH2) and phosphotyrosine binding (PTB) domain containing adaptor

3 Figure 1.2 Schematic of RET activation. A. The soluble GDNF family ligands (GDNF, neurturin (NRTN), artemin (ARTN), (PSPN)) bind to non-signaling cell surface bound molecules (GFRalpha 1-4) as shown by 3D images. GDNF and GFRalpha family members form quartenary complex in the presence of calcium. GFRalpha proteins consist of three, or two in the case of GFR4, globular cysteine-rich domains as shown by surface view images. Hypothetical models of GDNF, NRTN, ARTN and PSPN are based on the GDNF crystal structure [14], and hypothetical models of GFRalpha family members are based on the crystal structure of GFRalpha 1 [15] and the structure of the

ARTN–GFRalpha 3 complex [16, 17]. B. The GDNF-GFRalpha quartenary complex in turn, binds to RET forming a hexameric complex as shown in the hypothetical model.

Upon dimerization, the tyrosine kinase domain of RET undergoes a conformational change and many tyrosines in the intracellular domain of RET become autophosphorylated. Images of the RET kinase domain are based on the crystal structure of the RET kinase [18]. A model of the RET extracellular domain was generated using E- cadherin and the laminin gamma 1 chain crystal structures [19]. CLD (1-4): cadherin like domains, CRD: cysteine rich domain.

4 A

B

4a proteins (Figure 1.3). The intracellular domain of RET contains eighteen tyrosine residues: 2 in the juxtamembrane domain, 11 in the kinase domain and 5 in the carboxyl- terminal tail (Figure 1.3). Tyrosine 687 (Y687) in the juxtamembrane domain, Y806,

Y809, Y826, Y864, Y900, Y905, and Y981 in the kinase domain and Y1015, Y1029,

Y1062, Y1090, and Y1096 in the carboxyl-terminal tail have been shown to be autophosphorylated [20, 21]. In addition, phosphorylation of Y687, Y752, Y928, Y905,

Y952, Y981, Y1015, Y1062, and Y1096 has been linked to downstream signaling (Figure

1.3). Each phosphorylated tyrosine on RET binds a specific set of adaptor proteins, resulting in the activation of downstream pathways (Reviewed in [9]).

A summary of pathways that are activated by RET is shown in

Figure 1.3. Phosphorylation of Y687 has been shown to regulate activity of the Rac1- guanine nucleotide exchange factor and play a role in lamellipodia formation [22]. Y752 and Y928 are predicted binding sites for STAT3 [23, 24]. Tyrosine 905 is a docking site for SH2 domain containing adaptor proteins Grb7 and Grb10, which have been linked to the activation of the Mitogen-Activated-Protein-Kinase (MAPK) pathway [25, 26]. Y806,

Y809 and Y900 have been shown to contribute to the catalytic activity of RET kinase

[21]. However, no known substrates which bind at these specific phosphotyrosines have yet been identified. Y952 has been shown to be a binding site for NCK [27]. Y981 is a site of SRC binding [28]. Mutation of Y981 to phenylalanine reduces GDNF-mediated neuronal survival [28]. Tyrosine 1015 is a docking site for phospholipase Cγ which results in enhanced activation of C signaling [29]. Tyrosine 1062, is the most extensively studied phophorylated RET tyrosine and constitutes a docking site for several SH2 or PTB domain containing adapter proteins. Various signal transduction

5 Figure 1.3 Schematic of the RET tyrosine kinase domain with different autophosphorylated tyrosines. Upon dimerization, many tyrosines in the intracellular domain of RET become autophosphorylated. The schematic shows eighteen phosphorylated tyrosines in the RET kinase domain which act as docking sites for various

SH2 or PTB domain-containing substrates such as STAT3, GRB7/10, and SRC.

Phosphorylation of these substrates can then activate various downstream signaling pathways such as JAK-STAT, RAS-ERK, PI3K-AKT pathways, as indicated.

6 6a pathways are activated through pY1062. pY1062 is a docking site for SH2 domains of

SHC and PTB domains of SHC, Shc C (Rai), DOK1, 2, 4, and 5, and FRS2 [30-34]. In addition, Y1062 is also a phospho-independent docking site for ENIGMA, likely through a LIM domain [31, 32, 35-37]. Tyrosine 1096, which is only found in the long isoform of

RET (RET 51), is a binding site for the Grb2 adaptor protein which activates the PI3K and Ras/MAPK pathways [38]. Recently it has been shown that the distinct turnover of

RET 51 and RET 9 is mediated by differential recruitment of the Cbl ubiquitin ligase at

Y1096 and Y1062 [39]. The functional importance of the other known phosphorylated tyrosines, Y826, Y864 in the kinase domain and Y1029 and Y1090 in the C-terminal region have yet to be determined. In addition to SH2 or PTB domain containing proteins,

RET has also been shown to interact with SHANK3, which contains a WW domain, in a phospho-independent manner [40].

1.5 The role of RET in development

RET is expressed mostly in the developing nervous and urogenital systems and plays a crucial role in the development of the enteric nervous system, the kidney, and spermatogenesis [3, 4]. During embryogenesis, RET is expressed in the kidney, parasympathetic nervous system, and central nervous system including motor and catecholaminergic neurons (Reviewed in [9]). In adult tissues, high levels of RET can be observed in the brain, thymus, and testis as well as peripheral, enteric, sympathetic and sensory neurons [41]. The crucial role of RET during development is evident from the observation that mice expressing null mutations in RET lack superior cervical ganglia and the entire enteric nervous system, have agenesis or dysgenesis of the kidney, impaired spermatogenesis, and fewer thyroid C cells [42]. These mice also die shortly after birth

[42].

7 1.6 RET and disease

Both activating and loss of function mutations in the RET proto-oncogene have been associated with a wide array of diseases including Hirschsprung disease (HSCR) and multiple endocrine neoplasia type 2 (MEN 2) [43-46]. HSCR is a congenital abnormality, characterized by absence of intrinsic ganglion cells in the myenteric and submucosal plexuses of the gastrointestinal tract. It has an incidence of 1 in 5000 births [47].

Recognizable mutations in the RET coding sequence account for approximately 50% of familial and 3-7% of sporadic cases of HSCR [48, 49]. RET mutations in HSCR include deletions/truncations and missense point mutations that cause loss of function of the receptor [50].

Point mutations causing HSCR are located throughout the coding sequence of

RET (Figure 1.4). Based on the functional studies [51, 55], mutations causing HSCR in

RET are classified as follows: Class I mutations lie in the extracellular region of RET and cause an impairment of RET transport to the cell membrane [51]. Class II mutations lie in the cysteine rich domain and cause an impairment of RET transport to the cell membrane and covalent dimerization [51]. Class III mutations lie in the tyrosine kinase domain of

RET and confer complete or partial loss of kinase activity. Class IV mutations lie in the

C-terminal regions of RET and disrupt binding of adapter proteins such as SHC on Y1062

[51]. Although the clinical consequences of these point mutations is potentially the same, the structural basis for the inactivation of RET differs depending on the nature and location of the mutation [53].

1.6.1 RET and endocrine tumours

Mutations in RET have been detected in over 95% of families with MEN 2 [54].

MEN 2 is an autosomal dominant disorder which is divided into three subtypes; MEN

8 Figure 1.4 Schematic of RET showing missense mutations associated with HSCR.

Missense mutations causing HSCR are located throughout coding region of RET as shown. Nonsense or premature stop mutations in the extracellular and intracellular domain leading to HSCR are not shown. The different classes of HSCR-causing RET mutations are also indicated [51, 55].

9 9a 2A, MEN 2B, and familial MTC (FMTC), depending on the different tissues involved.

MEN 2A is characterized by medullary thyroid carcinoma (MTC), a tumor of the parafollicular C- cells of the thyroid; pheochromocytoma (PC), a tumor of the chromaffin cells of the adrenal medulla; and hyperparathyroidism (HPT) [56]. MEN 2B, the most aggressive form of the disease, with a 10 year median age of earlier onset than MEN 2A, is also characterized by MTC, and PC, but these patients also have characteristic developmental abnormalities such as a marfanoid habitus, thickened corneal nerves, and ganglioneuromatosis of the buccal membranes and the intestinal tract [57, 58]. The third subtype of MEN 2, FMTC, is characterized by the presence of MTC as the lone disease phenotype [59]. Activating RET mutations causing MEN 2 phenotypes can be generally grouped as extracellular domain mutations (MEN 2A and FMTC), and intracellular kinase domain mutations (MEN 2B and FMTC).

Extracellular domains mutations in RET

The MEN 2A and FMTC sub-phenotypes of MEN 2 are associated with point mutations of any of six cysteine residues (C609, 611, 618, 620, 630, 634) in the extracellular domain of RET. In MEN 2A, codon 634 is most frequently mutated (85%), mostly by a C634R substitution, however in FMTC the mutations are more evenly distributed among the various codons (Figure 1.5).

The cysteine residues in the RET extracellular domain have been suggested to be involved in intramolecular disulfide bonds [44]. Mutation at one of these cysteine residues leaves an unpaired cysteine, which can form intermolecular disulfide bonds with other RET monomers, leading to the permanent dimerization, resulting in constitutive kinase activity and activation of RET [60].

10 Figure 1.5 Schematic of RET showing missense mutations associated with MEN 2A and MEN 2B. MEN 2A is associated with mutations in the extracellular region of RET whereas MEN 2B is caused by mutations in the kinase domain of RET. MEN 2A cause mutations the substitution of extracellular cysteines at codons 609, 611, 618, 620, 630 or

634 with other residues leaving an unpaired cysteine in MEN 2A-RET monomers to form an intermolecular disulfide bond with another MEN 2A-RET monomer. This causes constitutive dimerization and consequent activation of MEN 2A-RET. The MEN 2B mutations cause a conformational change in the kinase domain leading to ligand- independent activation.

11 11a Intracellular domains mutations in RET

MEN 2B mutations

More than 95% of MEN 2B cases are caused by a specific amino acid substitution at residue 918 (M918T) in RET (Figure 1.5). In MEN 2B, it has been proposed that the

M918T mutation may either induce a conformational change of the catalytic core of the kinase domain and activate RET without ligand induced dimerization, or cause alteration in the substrate specificity of RET, or both [60, 61]. It is believed that the mutation

M918T, lies in a loop of the kinase domain that encodes the substrate recognition pocket

[61]. A mutation at this location has been predicted to alter RET conformation so that it associates with substrates preferred by SH2 domain-containing cytoplasmic kinases, such as SRC and ABL, rather than normal RTK substrates [61]. Bocciardi et al. [62] have shown that, compared to wildtype RET, the expression of MEN 2B RET results in the phosphorylation of a set of proteins that interact with Crk and Nck. Specifically, paxillin, a cytoskeletal protein, was found to be dramatically phosphorylated in the presence of

MEN 2B RET (2B-RET) as compared to Wild type (WT-RET). However, it has also been shown that WT-RET can phosphorylate these substrates upon GDNF treatment [63].

Thus, the evidence for the existence of a novel substrate which specifically binds to 2B-

RET is not yet clear.

In addition to M918T, a mutation at codon 883 (A883F) has also been reported in about 3-5% of cases of MEN 2B [64, 65] (Figure 1.5). Concurrent mutations at codons

804/806, or 804/904 have also been reported in single individuals [66] (Figure 1.5).

Alanine 883 also lies in the RET kinase domain, in the C-terminal lobe, between the catalytic site and the activation loop [18]. The substitution of a large, aromatic phenylalanine for the smaller alanine has significant conformational effects on the kinase

12 [67]. It is speculated that this change affects phosphotransfer, thereby contributing to an active conformation in the absence of ligand (Reviewed in [68]). Interestingly, change to a smaller, polar amino acid, threonine, at this site has also been recognized, associated with a much milder, FMTC phenotype, and only in a homozygous state [69], suggesting that the resultant less severe conformational change in RET may be only modestly activating.

Besides alteration in the substrate binding site, tyrosine residues essential for transforming activity are also different between MEN 2A RET and MEN 2B RET. While mutation at Y905 abolishes transforming activity of MEN 2A RET, it does not affect the transforming ability of MEN 2B RET [70]. On the other hand, Y864F and Y952F mutations significantly decreased the transforming ability of MEN 2B RET but had lesser affects on MEN 2A RET transformation [70]. It has also been shown that the Y864F and

Y952F mutations drastically diminished the activity of other MEN 2B RET proteins with the V804M/Y806C or A883F mutation, suggesting that these three mutant proteins (MEN

2B) have similar biological properties [71].

Many studies have also shown that there are differences in MEN 2A RET and

MEN 2B RET with respect to their ability to bind downstream substrates. There is increased GAB1 phosphorylation, and activation of phosphatidylinositol 3 kinase (PI3K) in the presence of MEN 2B RET than MEN 2A RET [72]. Other proteins which have been shown to bind more strongly to MEN 2B RET are SHC [73], DOK1 [31], SRC [74], and NCK [27]. Liu et al. [20] have shown that there is an increase in phosphorylation of

Y1062 in MEN 2B RET, compared to MEN 2A RET and wildtype RET while there is reduced phosphorylation of Y1096 in MEN 2B RET compared to wildtype RET, resulting in decreased GRB2 binding. Thus, these studies suggest that there are

13 differences between MEN 2A and 2B RET proteins in terms of their tyrosine phosphorylation patterns, binding to adaptor proteins, and/ or their ability to activate downstream signaling [20, 61, 75]. Hence, the M918T mutation may lead to the increased activation of known signaling pathways or distinct but as yet unidentified pathway(s) which may account for the phenotypic differences between MEN 2A and

MEN 2B.

FMTC RET mutations

FMTC mutations occur in the intracellular tyrosine kinase domain as well as the extracellular domain. FMTC shares many of the same extracellular mutations as MEN

2A. Substitutions of cysteines at codons 609, 611, 618, 620, 630 and 634 are found in more than 80% of FMTC families [54, 76].

The FMTC mutations occurring in the intracellular domain have usually lower penetrance. Intracellular domain mutations associated with FMTC have been found in codons 768, 804, 790, 791, 891 and 844 [77-82]. Structurally, Leucine 790 and tyrosine

791 lie in the N-terminal lobe of the RET kinase and mutations of these residues may affect ATP binding or inter-lobe flexibility [18] whereas serine 891 lies in the C-terminal lobe of the kinase, adjacent to the activation loop of the kinase facilitating substrate binding and phosphorylation (Reviewed in [68]). 3D molecular modeling has also suggested that substitution of the Y791 and S891 residues, by phenylalanine and alanine, respectively, may alter the position of the activation loop and/or relieve autoinhibition of the kinase [18, 83]. Y791F and S891A mutations constitutively activate multiple signaling pathways including RAS/ERK/STAT, and SRC [83].

Substitution mutations of glutamic acid 768 (E768D) and valine 804 (V804L or

14 V804M), are linked primarily to FMTC and sporadic MTC phenotypes (Reviewed in

[68]). Structurally, E768 lies in the N-terminal lobe of the kinase and the effects of substitution of E768 with a smaller aspartic acid (E768D) are unclear but may alter interactions of the helix in which it lies and the activation loop (Reviewed in [68]). Valine

804 lies in a critical position at the hinge linking the N- and C-terminal lobes of the RET kinase in a conserved hydrophobic pocket where it has roles in hinge flexibility and positioning RET helices for catalysis [18]. Interestingly, V804 appears to be a critical target residue in the efficient binding of small molecule ATP-competitive inhibitors for

RET [18]. The observation that RET proteins containing mutations of V804 are resistant to a range of inhibitors that block kinase activation has suggested that this residue may be an important “gatekeeper” for ATP-binding and normal RET activation (Reviewed in

[68]).

In addition to mutations in RET, translocations involving the RET kinase domain are also frequently observed in papillary thyroid carcinomas (PTC) [84]. PTC is the most common subtype of thyroid cancer, accounting for 80% of all thyroid cases [85]. In these

RET/PTC rearrangements, the transmembrane and extracellular domains of RET are lost, and are replaced by parts of other genes at the 5' end. These genes contain coiled-coil domains with dimerization potential and lead to constitutive, ligand-independent activation of the RET tyrosine kinase domain at the 3' end of the chimeric product

(Reviewed in [86])[87]. To date, 12 different fusion partner genes have been reported to form at least 17 different RET chimeric oncogenes. The most frequent RET gene fusions involve the ELE1 gene (PTC3), and H4 gene (PTC1) (Reviewed in [86]).

15 1.6.2 RET and other associated tumor types

In addition to the oncogenic mutations in RET associated with endocrine tumours, aberrant expression or activation of RET has been associated with other tumour types including pancreatic, and breast cancers.

Pancreatic Cancer: Pancreatic cancer is a very aggressive type of tumor, which is also characterized by an extremely poor prognosis, pronounced invasiveness, and rapid progression (Reviewed in [88]). In 2006, there were 33,730 new cases of pancreatic cancer in the U.S. and 32,300 deaths (American Cancer Society). One of the more aggressive and lethal pancreatic cancers is the RET-associated pancreatic ductal adenocarcinoma (PDAC) [89]. PDAC accounts for approximately 90% of all pancreatic tumors and, depending on the stage of PDAC, 5-year survival rates are between 1 and

3%, with most patients dying within the first year [65-69].

While MTC is associated with activating RET mutations leading to aberrant RET overactivation [3, 29], the overexpression of RET, GFRalpha 1 or GDNF, may also lead to pathological RET overactivation. RET ligands are the GDNF family (GFL) of growth factors. GDNF has been shown to induce pancreatic cell invasiveness by activating matrix metalloproteinase 9 [1, 70-72]. Studies have shown high levels, or increased expression of RET, its GFLs and coreceptors in human PDACs versus normal tissues [73-

75]. Cellular localization of all three molecules is dramatically altered in human PDACs versus normal tissue and is increased in liver metastases [73]. In pancreatic cell lines,

GFL activation of RET may increase cell proliferation and/or cell invasiveness [73-75].

Similarly, in PDAC patients, strong GDNF and RET expression is correlated with invasion and reduced patient survival after surgical resection [74], suggesting that RET

16 may be an important prognostic marker for pancreatic cancer and a potential target for anti-invasion therapy.

The role of RET in PDAC is relatively new and certainly not as well characterized as that in MTC/PTC. However, these recent experimental and clinical data strongly support an important regulatory role for RET and its ligands in PDAC.

Breast cancer: Recently, it has been shown that RET and GFRalpha 1 are overexpressed in a subset of estrogen receptor-positive tumors by screening a tissue microarray of invasive breast tumors [90, 91]. In vitro GDNF stimulation of RET(+)/GFRalpha1(+)

MCF7 breast cancer cells increased cell proliferation and survival, and promoted cell scattering [91]. Moreover, in xenograft models, GDNF expression was found to be up- regulated in the infiltrating endogenous fibroblasts and, to a lesser extent, by the tumor cells themselves [91]. Thus, GDNF could play a role in promoting breast cancer growth by signaling via the RET and GFRalpha complex expressed on tumor cells.

Other cancers – RET/GFRalpha 1 expression has been shown in colorectal cancer cell lines [92]. The expression of the β1 integrin subunit in these cells was significantly enhanced by GDNF [92], suggesting RET-GDNF signaling strongly influences adhesion to, and invasion of ECM proteins in colorectal cancer cells. Additionally, RET has been shown to be overexpressed in lung cancer and neuroblastoma tissue and cell lines [54, 76-

78]. However, the specific roles of RET in these diseases is unclear.

1.7 RET as a therapeutic target

As discussed above, either the mutations in RET or aberrant upregulation of RET signaling plays a critical role in the initiation and progression of multiple tumor types.

Therapeutic options for the management of these diseases are frequently limited and a novel compound that can block these roles would have broad utility. For MTC, radiation

17 therapy and chemotherapy are ineffective [93] and surgery is the primary management for both sporadic and hereditary MTC. Therefore, identification of an inhibitor which works specifically to block RET will provide novel therapeutics to treat MTC. RET signaling can be targeted for inhibition at multiple fronts including inhibitors of dimerization, kinase activity or intracellular signaling pathways (Figure 1.6).

1.7.1 Dimerization inhibitors

Normally, GFL and GFRalpha family members form complexes which, in turn, bind to RET and trigger its activation by causing it to dimerize [12]. In MEN 2A, substitution mutations of one of five cysteine residues in the RET extracellular domain that are involved in intramolecular disulfide bonds, leads to permanent RET dimerization, resulting in constitutive kinase activity [44]. It has also been shown that in MEN 2B, RET can dimerize and become autophosphorylated in the absence of ligand stimulation [94].

Receptor dimerization is a critical step which induces conformational changes in the intracellular domain, leading to tyrosine kinase activity [95, 96]. Thus, targeting RET dimerization is a first line of defense against constitutive RET signaling.

Regions within both the extracellular and intracellular domains of RET have been shown to be important for dimerization [97-99]. It has been suggested that the transmembrane domain (TM) of RTKs plays a passive role in ligand-induced dimerization and consequent activation of the receptor [100]. It has been shown that EGF- induced dimerization is far more efficient for a fragment of EGFR that contains both the extracellular and TM domains than it is for the extracellular domain alone [100]. A point mutation in the transmembrane domain of one receptor, neu/ErbB-2, has been found to enhance its transforming properties by provoking its dimerization and constitutive activation [101, 102]. Thus, a number of studies have studied the role or transmembrane

18 Figure 1.6 Schematic of strategies to inhibit RET signaling. RET signaling can be inhibited by blocking dimerization of RET monomers, or blocking kinase activity of

RET. Finally, intracellular signaling pathways of RET such as PI3K or RAS-ERK pathways can also be blocked to inhibit RET-mediated signaling.

19 19a domain in receptor dimerization and it has been shown that small peptides corresponding to transmembrane region of the receptors, are able to inhibit receptor dimerization leading to inhibition of receptor activation and its associated downstream signalling [103-105].

Whether similar inhibitory approaches also may be applicable to other receptor tyrosine kinases remains to be studied.

In addition to small molecules, oligonucleic acid or peptide molecules, referred to as "aptamers", are beginning to emerge as a class of molecules with which can block receptor dimerization and offer therapeutic potential [106, 107]. These aptamers can be used to identify markers on the surface of a cell type, define the specificity of a cellular state, and allow in vivo targeting for diagnostic and therapeutic applications. These aptamers are specific, have a high affinity for their target, are poorly immunogenic, and can recognize a wide variety of targets [108]. One such neutralizing, nuclease-resistant

D4 aptamer was capable of binding and inhibiting wild-type RET and RET/MEN 2A on the cell surface [109]. Several aptamers are currently under clinical trials including an aptamer for VEGFR [106, 110]. However, the efficacy of D4 as a therapy for RET- associated tumors needs to be established.

1.7.2 Tyrosine kinase inhibitors

Dimerization of RET monomers facilitates ATP binding followed by trans- autophosphorylation of specific tyrosine residues in the RET intracellular domain increasing its catalytic activity [13]. The human genome encodes about 518 protein kinases that share a catalytic domain conserved in sequence and structure but which are notably different in how their catalysis is regulated (Reviewed in [111]). The ATP- binding pocket, which is between the two lobes of the kinase fold, has been the focus of inhibitor design [111] (Figure 1.7). Over the last decade, several tyrosine kinase inhibitors

20 that block ATP binding site have demonstrated therapeutic potential in multiple cancers.

Currently, there are at least eleven ATP-competitive inhibitors in various phases of clinical evaluation (Reviewed in [111]).

Over the last several years, a couple of tyrosine kinase inhibitors have been demonstrated to inhibit RET activity. (Gleevac) has been shown to display inhibitory activity in RET expressing cell lines [112]. Furthermore, it induces RET oncoprotein degradation. However, the IC50 (the concentration that causes 50% reduction in activity) of imatinib necessary to inhibit RET in vitro is high (25–37 µm) [112]. Hence, it is impossible to conclude that imatinib will be a good candidate for systemic therapy of

MTC [112].

The 2-indolinone derivative ribose-5-phosphate isomerase (RPI-1) inhibited RET tyrosine phosphorylation in human TT cells (a MTC-derived cell line harboring a

RETC634W mutation) [113] and RET/MEN 2A and RET/PTC1-expressing NIH3T3 cells with an IC50 in the low micromolar range (3.6 µM) [113]. RPI-1 also inhibited

RET-mediated proliferation, RET protein expression, and the activation of PLCgamma,

ERKs and AKT. RPI-1 also showed antitumor effects in nude mice and could be administered orally [113, 114]. The closely related compound SU11248 (), which is a small molecule tyrosine kinase inhibitior of VEGFR, PDGFR, KIT, FLT-3,

CSF-1, is also a highly active inhibitor of RET (IC50 224 nM) and is currently being evaluated in clinical trials for gastrointestinal stromal tumors and renal cell carcinoma

[115-117]. SU11248 also caused a complete morphological reversion of transformed

RET/PTC3 cells and inhibited the growth of TPC-1 cells that have an endogenous

RET/PTC1 [116].

21 Figure 1.7 Homology models of RET. A, multiple sequence alignment of tyrosine kinase domains of RET and similar RTKs. B, ribbon diagrams of the intracellular domain of WT-RET (residues 709-988) in the autoinhibited and active conformations. Three- dimensional models of RET were created using autoinhibited and activated IRK structures as templates. Positions of the autophosphorylated tyrosines in the activation loop (Y900 and Y905) and of M918 are shown. Residues in the nucleotide binding loop

(orange), catalytic loop (red), activation loop (green), and P+1 loop (yellow) are indicated.

22 22a The pyrazolopyrimidine inhibitors, PP1 and PP2, inhibit RET autophosphorylation [118] as well as induce its degradation [119]. PP1 treatment, causes both MEN 2A RET and MEN 2B RET oncoproteins to translocate from the cell membrane to inner cellular compartments and became rapidly addressed to the degradative pathway [118, 119]. The degradation of RET proteins causes an impairment of RET signalling leading to a reversal of the RET-mediated oncogenic transformation. In addition, PP1 prevented the growth of human papillary thyroid carcinoma cell lines that carry spontaneous RET/PTC1 rearrangements and blocked anchorage-independent growth and tumorigenicity in nude mice of NIH3T3 fibroblasts expressing the RET/PTC3 oncogene [118]. Recently, a co-crystal structure of the RET kinase domain and PP1 revealed that PP1 binds in the ATP binding pocket [18]. The use of PP1, which acts as a degradation-inducing factor, may represent an additional strategy for targeting the RET kinase and holds promise for future MTC therapy [118, 119]. However, PP1 and PP2 also inhibit the SRC family of kinases [120-122]. Therefore, the actions of PP1 and PP2 on oncogenic activity may not depend solely on RET inhibition.

The indolocarbazole derivatives CEP-701 and CEP-751 inhibited RET autophosphorylation in a dose-dependent manner and proliferation of TT cells at concentrations <100 nm [123]. CEP-751 also inhibited tumour growth in nude mice that have been injected with TT cells [123]. Again, CEP-701 is a multi-kinase inhibitor which also inhibits FLT3, Kit, TrkA, EGFR, PDGFR, and VEGFR [124, 125], thus clinical utility of this inhibitor is uncertain.

The selective inhibitor of the VEGFR-2 tyrosine kinase, ZD6474, is a member of the anilinoquinazoline family [126, 127]. ZD6474 targets the enzymatic activity of both

MEN 2 and PTC-related oncogenic RET and has an IC50 of 100 nm [126, 127]. In

23 addition, the compound inhibits tumour growth in RET/PTC transformed NIH3T3 cell xenografts [126]. Because ZD6474 inhibits VEGFR-2, it has antiangiogenic roles as well

[128]. ZD6474 shows evidence of promising clinical activity in patients with hereditary

MTC [129].

Due to their powerful inhibitory effects, RPI-1, SU11248, PP1, PP2, CEP-701, and CEP-751, ZD6474, seem a promising treatment strategy in RET-associated cancer.

Although several of these small molecule RET kinase inhibitors have undergone preclinical testing for efficacy in RET-mediated thyroid neoplasia, none of these agents is

RET-specific (Reviewed [17, 36]). In addition, some constitutively active mutants of RET

(e.g. V804M) are resistant to these inhibitors, because of activating mutations within the

RET sequence critical for inhibitor binding [38]. Combining different (kinase) inhibitors may overcome this resistance. As yet, a small molecule RET inhibitor with high specificity for RET and efficacy for all common RET activating mutants has not been identified.

1.7.3 Intracellular signaling inhibitors

Activated RET transduces signals downstream through several intracellular cascades, which regulate cell survival, differentiation, proliferation, and migration

(Reviewed in [9]) (Figure 1.3). These intracellular pathways offer another avenue for small molecule inhibitors to quench receptor-mediated signaling and its downstream effects. Several of these small molecule inhibitors that target pathways such as RAS/

(RAF) MEK/ERK or PI3K/AKT are under clinical evaluation in tyrosine kinase- associated human cancer (Reviewed in [130]) although none of these agents has yet been investigated for RET-mediated tumours.

24 Of particular interest in RET-associated tumours is the biaryl urea BAY 43–9006

(). BAY 43–9006 is an oral multikinase inhibitor initially developed as a specific inhibitor of BRAF [131, 132]. However, subsequent studies revealed that BAY

43–9006 also inhibits other kinases including VEGFR-2, VEGFR-3, PDGFR, c-, and

FMS-like tyrosine kinase 3 (FLT-3) [133, 134]. In addition, the compound also inhibits

RET [135]. Recently, it was demonstrated that BRAF is a key mediator of the intracellular signalling of oncogenic RET in follicular thyroid cells [136]. The simultaneous inhibition of two levels of signalling, RET and its downstream pathway via

BRAF may offer better treatment and a potential mechanism to circumvent resistance in endocrine tumours. Inhibitors of other downstream signal transduction proteins thus far have not been tested in RET signalling. Nevertheless, they provide interesting possibilities for future research and therapeutic options in RET-associated endocrine malignancies. A summary of small molecule inhibitors of the RET kinase is presented in

Table 1.1.

25

Table 1.1 Small-molecule inhibitors of the RET kinase

Compound Generic name IC50 (RET) Targets RPI-1 20-40 μM RET, RAS PP1, PP2 80 nM (PP1), 100 nM RET, SRC, YES, FYN, ABL (PP2) ZD6474 130 nM RET, VEGFR2, VEGFR3, EGFR CEP-701, CEP-751 <100 nM RET, FLT3, TRK, KIT, EGFR, PDGFR, and VEGFR BAY43-9006 Sorafenib 47 nM VEGFR1,VEGFR2, VEGFR3, PDGFR, FLT3, KIT, FGFR1, RAF, BRAF, p38 MAPK SU-5416, SU011248 Sunitinib 100 nM RET, VEGFR2, PDGFR, FLT3, KIT, FGFR1, CSF1R AMG 706 100 nM RET, VEGFR1, VEGFR2, VEGFR3, PDGFR, FLT3, KIT IC50 (the concentration inhibiting 50% of enzyme activity) values are shown. Kinases with IC50 values below 1,000 nM are listed except for RPI-1.

26 Chapter 2

Proposed Research

Aberrant activation of the RET receptor tyrosine kinase has been implicated in many tumour types [54, 89-92]. To design novel therapeutics for RET-mediated tumours, it is indispensable to understand downstream signaling upon RET activation, and the molecular mechanisms of RET-mediated oncogenesis.

2.1 RET-mediated oncogenesis in MEN 2B

2.1.1 Rationale

More than 95% of MEN 2B cases are caused by a specific amino acid substitution at residue 918 (M918T) in RET. Many studies have predicted that the presence of the

M918T mutation leads to a pattern of tyrosine phosphorylation, adaptor protein binding, and downstream signaling, which is distinct from that associated with wildtype RET [20,

61, 75]. Despite previous predictions, little is understood of the molecular mechanisms underlying the functional changes in RET proteins containing the M918T mutation (2B-

RET), and how such changes may alter RET activation and interactions. Understanding how the 2B-RET mutation lead to ligand-independent activation, requires understanding of how the RET receptor normally functions, and how mutations alter these functions. To date, detailed characterization of the biochemical and biophysical properties of the RET receptor have not been performed. Understanding the biological and biochemical mechanisms by which 2B-RET functions differ from those of normal receptors is essential to our ability to predict and develop anti-cancer therapies.

27 2.1.2 Hypothesis

The biochemical and biophysical changes in the protein caused by 2B-RET mutations may provide insight into RET- mediated oncogenesis. We hypothesize that the

M918T mutation causes conformational changes in the RET kinase, leading to altered stability. These conformational changes may also alter the intrinsic kinase activity of

RET. We address these with the following objectives.

2.1.3 Objectives

• To characterize the properties of the RET tyrosine kinase and its oncogenic variant

(2B-RET) using purified proteins.

• To characterize and compare purified WT-RET and 2B-RET using various

biophysical techniques including circular dichroism and micro-calorimetry.

2. 2 RET-mediated intracellular signaling

2.2.1 Rationale

RET activates several intracellular signaling cascades such as the RAS/ERK,

ERK5, p38MAPK, PI3-kinase, and SRC pathways, which together regulate cell survival, differentiation, and proliferation (Reviewed [9]). Although the RET-mediated cell survival and proliferation pathways have been well characterized, little is known about cascades leading to metastasis in RET-mediated tumours. One of the well characterized pathways for tumour metastasis is the β-catenin signaling pathway. Several studies have examined the expression of E-cadherin/ β-catenin in thyroid tumours [90-92, 137]. It has recently been suggested that several signaling pathways, including the RAS/ERK and

PI3K/AKT pathways converge at β-catenin in thyroid cancer [137], however, the mechanism of this activation is still not clear.

28 2.2.2 Hypothesis

It is hypothesized that that upon RET activation, the β-catenin signaling pathway is activated, directly or indirectly. The RET-β-catenin interaction may play an important role in normal cell-cell interactions, and regulated cell movement.

2.2.3 Objectives

• To investigate the RET- β-catenin interaction in vitro and in vivo.

• To investigate the functional effects of RET-mediated activation of the β-catenin

pathway in vivo.

2. 3 Targeting RET for therapeutics

2.3.1 Rationale

Mutations in the RET kinase, leading to ligand-independent activation, play a critical role in the initiation and progression of multiple tumor types [54, 89-92]. To date, surgery has been the primary management for both sporadic and hereditary MTC, as radiation and chemotherapy have proven ineffective [93]. The causative role of activating

RET mutations in MTC makes RET a rational target for possible treatments of these tumours.

Conventionally, the ATP- binding site of kinases has been the main focus for designing small molecule inhibitors. As this region of kinases is highly conserved, these inhibitors frequently interact with multiple kinase family members. Currently, there are at least eleven ATP-competitive inhibitors in various phases of clinical evaluation, many of which have broad-spectrum inhibitory activity, and are therefore considered less advantageous in terms of achieving clinical specificity. To date, all RET kinase inhibitors

29 that have advanced to clinical trials/preclinical development are “multi-kinase” ATP- competitive inhibitors.

2.3.2 Hypothesis

Targeting the substrate binding pocket and/or the activation loop of RET, which are less conserved domains, but critical to kinase activation, may provide more specific inhibition of RET. Identification of an inhibitor which works specifically to block RET function will provide novel therapeutics to treat MTC.

2.3.3 Objectives

• To identify small molecules which may inhibit RET activation by binding at the

substrate binding pocket, using 3D-models of RET

• To characterize RET kinase inhibitor(s) in vitro and in cell-based systems in multiple

tumour model systems.

30 Chapter 3

Molecular Mechanisms of RET Receptor Mediated Oncogenesis in Multiple

Endocrine Neoplasia 2B

Gujral et al.,Cancer Res. 2006; 66(22):10741-9

Taranjit S. Gujral1, Vinay K. Singh2, Zongchao Jia2, Lois M. Mulligan1,*

Departments of Pathology1, & Biochemistry2, Queen's University, Kingston, ON, Canada

K7L 3N6

*To whom correspondence should be addressed Dr Lois M Mulligan Cancer Research Institute Botterell Hall Rm 329 Queen's University Kingston ON Canada K7L 3N6 E-mail [email protected] Phone 1 613 533 6000 X77475 Fax 1 613 533 6830

Running Title: Molecular Mechanisms of MEN 2B

Keywords: MEN 2B, oncogene, receptor tyrosine kinase, RET

Some of the experiments for this publication were done as part of my MSc. thesis. These data are not included in this thesis. The entire study is attached in the form of a reprint as

Appendix I.

31 3.1 Abstract

Multiple endocrine neoplasia 2B (MEN 2B) is an inherited syndrome of early onset endocrine tumours and developmental anomalies. The disease is caused primarily by a methionine to threonine substitution of residue 918 in the kinase domain of the RET receptor (2B-RET), however the molecular mechanisms that lead to the disease phenotype are unclear. In this study, we show that the M918T mutation causes a 10 fold increase in ATP binding affinity, and leads to a more stable receptor-ATP complex, relative to the wildtype receptor. Further, the M918T mutation alters local protein conformation, correlating with a partial loss of RET kinase autoinhibition. Finally, we show that 2B-RET can dimerize and become autophosphorylated in the absence of ligand stimulation. Our data suggest that multiple distinct but complementary molecular mechanisms underlie the MEN 2B phenotype and provide potential targets for effective therapeutics for this disease.

32 3.2 Introduction

The RET (REarranged in Transfection) proto-oncogene encodes a receptor tyrosine kinase (RTK) with important roles in cell growth, differentiation, and survival.

RET is expressed in cell lineages derived from the neural crest, and in the tissues of the urogenital system (Reviewed in [9]). Activation of the RET receptor is a multistep process, involving interaction with a soluble ligand of the glial cell line-derived neurotrophic factor (GDNF) family (GFL) and a non-signaling cell surface-bound coreceptor of the GDNF family receptors alpha (GFRα). GFLs and GFRαs form complexes which, in turn, bind to RET, triggering its dimerization and resulting tyrosine kinase activation [9].

Activating point mutations of RET cause multiple endocrine neoplasia type 2

(MEN 2), an inherited cancer syndrome characterized by medullary thyroid carcinoma

(MTC) (Reviewed in [138]). The disease has three clinically distinct subtypes ranging from the later onset, less severe, familial MTC (FMTC), characterized only by MTC, to the more severe MEN 2A, characterized by MTC, the adrenal tumour pheochromocytoma

(PC), and parathyroid hyperplasia. MEN 2B, the earliest onset and most aggressive form of MEN 2, is characterized by MTC and PC, as well as by an array of developmental abnormalities, including marfanoid habitus, mucosal neuromas, ganglioneuromatosis of the intestinal tract, and myelinated corneal nerves [139]. Morbidity and early mortality due to MEN 2B are very high. Management of both MEN 2B and sporadic MTC is by surgical intervention. Treatment is complicated, as MTC is prone to metastasis and is often refractory to both radiation and chemotherapy [93]. Novel strategies, such as kinase inhibitors and small molecules, have as yet largely lacked specificity or efficacy at

33 biologically tolerated dosages [9]. Despite early genetic identification, “cure” is reported in less than 20% of MEN 2B cases [139].

Although all MEN 2 subtypes arise from mutations of RET, there are strong genotype/phenotype associations with specific mutations identified in each disease subtype. MEN 2A and FMTC mutations are primarily substitutions of one of several cysteine residues in the RET extracellular domain [138]. More than 95% of MEN 2B cases are caused by a single germline mutation that results in substitution of a threonine for the normal methionine at residue 918 (M918T) in the RET kinase domain [61, 140].

The same mutation occurs somatically in 50-70% of sporadic MTC, where it can be associated with more aggressive disease and poor prognosis [141]. The M918T mutation has been predicted either to induce a conformational change in the kinase catalytic core, leading to the activation of RET without ligand induced dimerization, or to alter the substrate specificity of RET, so that it preferentially binds substrates of cytoplasmic tyrosine kinases, such as SRC, or both [60, 61]. Previous studies have predicted that the

M918T mutation leads to a pattern of RET tyrosine phosphorylation, adaptor protein binding, and downstream signaling that differs in a number of respects from those associated with wildtype RET (WT-RET) [61, 75]. For example, in the absence of RET ligand, the M918T MEN 2B mutant (2B-RET) induces phosphorylation of proteins that interact with CRK and NCK, including the cytoskeletal protein paxillin which appears more phosphorylated in the presence of 2B-RET than unactivated WT-RET [62].

Together, these data have led to speculation that the M918T mutation may contribute to the increased activation of known RET signaling pathways or to activation of distinct, but as yet unidentified, pathways that may account for the earlier onset, broader phenotype, and increased severity of MEN 2B [62, 72].

34 Despite these predictions, the molecular consequences of the M918T mutation, and how this single amino acid substitution leads to the changes in RET activation or interactions that cause MEN 2B, have been difficult to confirm. Thus, elucidation of the mechanisms of 2B-RET function and dysfunction in MEN 2B require a better understanding of the molecular properties of both WT-RET and 2B-RET and of the activation and interactions of these kinases. Here, we have compared the biochemical, thermodynamic, cell biological and structural properties of WT-RET and 2B-RET to identify the underlying differences and similarities in these receptors. Our data show that the M918T mutation has multiple distinct and complementary effects on 2B-RET function including increasing intrinsic kinase activity, partially releasing kinase autoinhibition and facilitating ligand-independent phosphorylation of 2B-RET receptors.

3.3 Materials and Methods

Homology modeling, cell culture and transfection, immunoprecipitations and western blotting, Glutathione S-transferase (GST) pull-down assay, SRC kinase assay and

Soft agar colony formation assay were done as part of my Master’s thesis. These methods are described in Appendix I.

3.3.1 Expression constructs

Tetracycline-inducible RET expression constructs, generated by fusing cDNAs encoding a myristylation signal, two dimerization domains and the intracellular portion

(amino acid 658 to C-terminus) of RET (icRET), have been described [142]. Full-length human RET9 cDNA was cloned into pcDNA3.1 (Invitrogen, Burlington, ON). Constructs were validated for binding of known RET substrates (data not shown). Site-specific RET mutants were generated in WT-RET constructs by overlapping PCR, as described [143].

GST-fusion constructs encoding residues 664-1072 of WT-RET and 2B-RET (with the

35 M918T mutation) were generated in a modified pGEX-4T-3 vector. Constructs were verified by direct sequencing (Cortec, Kingston, ON). Expression and GST-fusion constructs for GFRα1 [143], SHC [144], NCK [145], GRB10 [146], SRC (Y527F) [147] and STAT3 [148] have been described.

3.3.2 Expression and purification of WT-RET and 2B-RET proteins

Recombinant GST-WT-RET and GST-2B-RET were expressed in E. coli strain

BL21(RP). Cells were grown in Terrific Broth (Bioshop, Burlington, ON) with 50 µg/ml ampicillin at 37°C and induced with 1mM isopropyl-β-D-thiogalacto-pyranoside. Cells were lysed in PBS with 1 mM PMSF, 10 mM DTT and 0.1 mg/ml DNAseI. Cleared lysates were applied to GSTrap affinity purification columns (Amersham Biosciences,

Baie d'Urfé, QC), washed with PBS, and eluted in PBS containing 50mM reduced glutathione. Protein was extensively dialysed against 5 mM Na2HPO4 (pH 7.0), 50 mM

NaCl and stored in 50% glycerol at -20°C. GST-tags were cleaved using 80 U of thrombin in 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, and

0.01% Triton X-100 for 16 h at 4°C. Cleaved proteins were tested for kinase activity and identity and purity confirmed by matrix-assisted laser desorption ionization mass spectrometry.

3.3.3 Peptide Fingerprinting by Mass Spectrometry

For Mass spectrometry (MS) analyses, Coomassie blue-stained bands of WT-RET and 2B-RET were excised from SDS PAGE gels and digested with trypsin using standard protocols. Samples destined for Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) analysis (Applied Biosystem Voyager DE-PRO instrument) were mixed 1:1 (v/v) with 5 mg/ml α-cyano-4-hydroxy-cinnamic acid (Sigma) matrix in 50%

36 (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic acid. Calibration was done externally using bovine serum albumin as calibrant. Raw data were analysed using the computer software provided by the manufacturer (Applied Biosystem) and reported as monoisotopic masses. The top 50 most intense peaks were submitted for the database search. Search parameters were set at trypsin with up to one missed cleavage, and variable oxidation of methionine.

3.3.4 In vitro kinase kinetics

Approximately 5-20 µg of purified GST-WT-RET and GST-2B-RET proteins were incubated with 1 mM ATP in 10 mM Tris, 5 mM MgCl2 (pH 7.4), at 30°C for 30 minutes. Reactions were terminated by boiling in SDS-PAGE sample buffer. Samples were resolved on 10% acrylamide gels, and western blotted with appropriate antibodies.

For kinetic analyses, densitometry was performed on western blots to assess phosphorylation levels under varying conditions of ATP. KM values were calculated from the Michaelis-Menton equation fitted using a nonlinear least-squares regression kinetics program [149]. Kinetic values are the means of three separate measurements and are reproducible within ±10% SE of the mean value.

3.3.5 Circular dichroism spectroscopy (CD)

CD was performed on an OLIS RSM CD spectrophotometer (Bogart, Jefferson,

GA) at 25°C using purified recombinant GST-WT-RET and GST-2B-RET proteins.

Protein concentrations and path lengths used for far and near-UV CD analyses were 30

µM and 0.1 cm, and 150 µM and 1 cm, respectively. Each CD plot represented an average of 30 accumulated scans. Plots were baseline corrected for buffer. Observed ellipticity [θ] was normalized for protein concentration using the equation [θ] = θ x

37 100/ncl, where n is the number of amino acids in the protein, c is the molar protein concentration and l is the path length of the cell (cm). Percentage secondary structure was calculated using the web-based program CDNN [150].

3.3.6 Isothermal titration calorimetry (ITC)

ITC was performed with a VP-ITC calorimeter (Microcal, Northampton, MA).

Protein solutions for ITC were dialyzed extensively against kinase buffer (pH 7.0).

Injections (6µl x 50) of 2 mM ATP were made into a reaction cell containing 1.43 ml of the 50 µM protein solution. The heat of dilution was determined to be negligible in separate titrations of the ATP into the buffer solution. Calorimetric data were analysed with ORIGIN 5 software (MicroCal). Binding parameters, such as number of binding sites, binding constant, and binding enthalpy of bound ligand, were determined by fitting the experimental binding isotherms.

3.3.7 Analytical Gel Filtration

Hydrodynamic dimensions of WT-RET and 2B-RET were measured by analytical gel filtration, performed on a Superose-12 10/300 GL column attached on an AKTA

FPLC (Amersham Biosciences, Piscataway, NJ). Columns were calibrated using a high and low molecular weight gel filtration calibration kit (Amersham Biosciences,

Piscataway, NJ). Purified recombinant RET proteins were cleaved with thrombin to remove GST tags, as described. Protein solutions (~0.5 mg/ml) were loaded onto columns equilibrated with PBS. Elution using PBS was carried out isocratically at a flow rate of

0.5 ml/min and monitored by absorbance at 280 nm. Aliquots from each 5 ml fraction were analyzed by western blotting, as described.

38 3.3.8 Differential scanning calorimetry (DSC)

Thermal denaturation studies used a VP-DSC calorimeter (Microcal) and covered a temperature range of 20°C to 120°C at a rate of 45°C/h. Prior to analysis, samples were dialyzed overnight against kinase buffer. The dialysis buffer was used to obtain a baseline. Protein-buffer scans were performed at a final protein concentration of 50µM.

Data analysis was performed using Microcal Origin 5 software.

3.4 Results

3.4.1 Expression and purification of WT and 2B-RET

To study the mechanism of oncogenesis mediated by an MEN 2B mutation

(M918T) in RET, understanding the difference between WT and 2B-RET proteins is critical. For this purpose, we generated constructs for WT-RET (GST-WT-RET) and 2B-

RET (GST-2B-RET) intracellular domains (AA665-C-terminus) linked to GST-tags

(Figure 3.1A). GST fusion proteins were purified using an E. coli expression system and yielded approximately 4 mg of RET protein per one litre culture. Purity of the expressed proteins was determined by SDS-PAGE and coomassie blue staining (Figure 3.1B). The evaluated molecular mass on SDS–PAGE was 74 kDa. We confirmed the functionality of

RET fusion proteins by in vitro kinase assay followed by immunoblotting for pRET

(Figure 3.1C). Finally, identity of the RET proteins was confirmed using mass spectrometry (Figure 3.2). RET fragments were confirmed by comparing the mass values obtained to the theoretical values calculated from known amino acid sequences using the

MASCOT database and search engine (Matrix Science, London, UK).

39 Figure 3.1 Expression and Purification of GST fused WT-RET and 2B-RET. A.

Schematic representation of WT-RET and 2B-RET constructs generated for overexpresion of intracellular domain (AA665-C-terminus) linked to GST-tags. B.

Purification profile of GST-fused RET. Representative SDS-PAGE showing near homogeneity purification of GST WT-RET as revealed by staining with coomassie blue.

Proteins were expressed in E. coli BL21RP cells, and purified as detailed in

“Experimental procedure”. C. Autoradiogram showing kinase activity of WT and 2B-

RET confirmed by non-radioactive in vitro kinase assay followed by immunoblotting for pRET.

40 A

B kDa

175

83 GST RET (74 kDa) 62

48

33 GST (26 kDa) Coomassie

C

ATP + + - - RET IB: pRET IB: RET

40a Figure 3.2 Identification of RET peptides by MALDI-TOF mass spectrometry. Mass spectrometric analysis of tryptic peptides is shown. GST WT-RET treated with ATP was digested with trypsin and the resulting peptide samples were analyzed by MALDI-TOF mass spectrometry. MS data were submitted to the MASCOT search engine. The inset table shows the identified peptide sequences from the WT-RET protein.

41 41a 3.4.2 Comparison of WT-RET and 2B-RET structure and activity

In order to identify the molecular mechanisms underlying MEN 2B, we first investigated whether the WT-RET and the oncogenic 2B-RET mutant differed in overall structure. We used circular dichroism (CD), a form of spectroscopy used to determine the secondary structure of molecules, to compare WT-RET and 2B-RET. Common secondary structure motifs exhibit distinctive CD spectra in the far-UV range while near-UV provides a fingerprint of protein tertiary structure. Using purified recombinant WT-RET and 2B-RET proteins, we compared the relative fraction of each molecule that was in alpha-helix, beta-sheet (antiparallel/parallel), beta-turn, or other (random) conformations.

Far and near-UV CD spectra of WT-RET and 2B-RET had profiles corresponding to globular proteins with defined secondary structure, comparable with those of typical kinases (Figure 3.3). Both far- and near-UV CD spectra of 2B-RET were superimposable on those of WT-RET, suggesting that there were no significant global changes in the secondary or tertiary structure of RET due to the M918T mutation.

We next asked whether WT-RET and 2B-RET differed in intrinsic properties such as ATP binding, thermostability, and enzyme kinetics. We used isothermal titration calorimetry (ITC), a thermodynamic technique for the evaluation of interactions between different molecules, to compare the ATP binding affinity of WT-RET and 2B-RET using purified recombinant proteins (Figure 3.4). In ITC, heat released upon interaction of two molecules can be used to determine dissociation constant (Kd), reaction stoichiometry

(N), and thermodynamic parameters including binding enthalpy (∆H) (Table 3.1). We found that the ATP equilibrium dissociation constant (Kd) for 2B-RET (15.3µM) was significantly lower than that of WT-RET (192µM), indicating that 2B-RET has more than

42 Figure 3.3 WT-RET and 2B-RET do not differ in overall secondary or tertiary structure. Far-UV circular dichroism (CD) spectra (A) and near-UV spectra (B) are shown for purified recombinant WT-RET (solid line) and 2B-RET (dashed line) proteins.

Average CD plots for at least 30 accumulated scans are shown. Percentage secondary structural composition was calculated using the web-based program CDNN [150].

43 A

B

43a Figure 3.4 Thermodynamic analyses of WT-RET and 2B-RET. Isothermal Titration

Calorimetry (ITC) analysis of in vitro ATP binding to WT-RET (left) and 2B-RET

(right). Upper panels show the raw heat change elicited by successive injections of ATP into a solution containing WT-RET or 2B-RET. Bottom panels depict the normalized integration data (kcal/mol ATP) as a function of the molar ratio of ATP to WT-RET, or

2B-RET, with a least-squares fit of the data (red line).

44 44a

Table 3.1 Kinetic properties of WT-RET and 2B-RET

ATP binding to WT-RET and 2B-RET was measured by ITC. Titrations were performed o at 30 C in kinase buffer ((10 mM Tris, 5 mM MgCl2, pH 7.4). Binding parameters such as the number of binding sites (N), the binding constant (Kd), and the binding enthalpy of bound ligand (∆H) were determined by fitting the experimental binding isotherms.

Protein Kinetics (KM) Stoichiometry (N) Kd ∆H WT-RET 188.2 ± 17 µM 1.11 192.0 µM -1.04 E5 2B-RET 105.0 ± 11 µM 2.00 15.3 µM -4.95 E4

KM: Michaelis-Menten constant- substrate (ATP) concentration required for 1/2 maximal activity Kd: dissociation constant ∆H: binding enthalpy

45 10 fold greater affinity for ATP (Table 3.1). Consistent with this, the heat of enthalpy was also more favorable for ATP binding to 2B-RET than WT-RET. As anticipated for RTKs, stoichiometry calculated by ITC suggested a single ATP binding site for WT-RET receptor monomers (N=1.11). Interestingly however, 2B-RET appeared to be associated with two ATP molecules (N=2), suggesting that these molecules may associate in solution to form dimers that associate with two ATP molecules (Table 3.1). We further confirmed this using analytical gel filtration, which allows size separation of dimers and monomers in solution. Our data confirmed the presence of significantly more dimers for

2B-RET than WT-RET (Figure 3.5).

We compared the kinetic properties of purified WT-RET and 2B-RET, in in vitro kinase assays (Figure 3.6). When kinase activity was compared over a range of ATP concentrations, we found that WT-RET required much higher ATP concentrations (KM

188.2 ± 17µM) to achieve the same level of autophosphorylation as 2B-RET (KM 105 ±

11µM) (Table 3.1). The M918T mutation appeared to increase the stability of the enzyme-ATP complex, and relatively strengthened ATP-binding. The result is a significant overall increase in the kinase activity of 2B-RET, as compared to WT-RET.

Consistent with this increased ATP binding, we found that 2B-RET appeared more highly autophosphorylated, both in the presence and absence of ligand stimulation, as compared to activated WT-RET (Appendix I). Further, when we investigated known RET substrates

(e.g. Paxillin, SHC, STAT3, NCK1, GRB10), we found that although each of these bound both WT-RET and 2B-RET, all bound more 2B-RET, as we would expect due to the higher relative levels of 2B-RET autophosphorylation. However, when relative differences in autophosphorylation levels were taken into account, we found no

46 Figure 3.5 Monomer-dimer equilibrium of 2B-RET protein is shifted towards dimers. Analytical gel filtration analysis of RET monomers and dimers was performed using purified recombinant RET proteins from which the GST-tag had been cleaved.

Higher molecular weight proteins appear in earlier fractions on the size exclusion column.

Peaks corresponding to dimer and monomer elution are indicated. Aliquots from each of eluted fractions 3, 4 and 5 were separated by SDS-PAGE and immunoblotted for RET, as shown in the inset. This figure was provided by Dr. Vinay Singh.

47 47a Figure 3.6 2B-RET has increased autokinase activity. Kinetic properties of WT-RET and 2B-RET were compared in non-radioactive in vitro kinase assays using our purified recombinant proteins over a range of ATP concentrations. The Michaelis-Menten equation was used to fit the data for in vitro autophosphorylation of WT-RET (top) and

2B-RET (bottom). A representative example from two independent experiments in the presence of varying ATP concentrations is shown. Corresponding Hill plots of these data are presented in the inset.

48 48a differences in the binding of any tested substrate to phospho WT-RET or phospho 2B-

RET (Appendix I).

To assess more localized changes in interactions between residues and domains due to the M918T mutation, we next used differential scanning calorimetry (DSC) to compare the overall thermal stability and structural flexibility of WT-RET and 2B-RET conformations over a range of temperatures (20-120°C) and in the presence or absence of

ATP (Figure 3.7). In the absence of ATP, we found that WT-RET was more conformationally stable or rigid than 2B-RET, as indicated by a higher melting temperature (midpoint of denaturation transition [Tm]= 65.10°C) (Table 3.2). The relatively higher energy requirement for denaturation is consistent with the tightly folded, more stable conformation of an autoinhibited (inactive) kinase structure [151, 152]. 2B-

RET had a lower melting temperature (Tm 62.65°C), suggesting a relatively more flexible protein conformation, with a less rigid overall structure. Interestingly, in the presence of

ATP, we found that WT-RET and 2B-RET had similar melting temperatures (Table 3.2), indicating that the conformations of ATP-bound (active) WT-RET and 2B-RET enzymes were more similar than those of their autoinhibited forms. The lower melting temperature of the active forms was consistent with the thermodynamic effects of the release of autoinhibition in other kinases [151, 152]. Together, these data suggested that, in addition to altered kinase activity, changes in intramolecular interactions and a potential reduction in conformational rigidity, might play significant roles in the functional effects of the

M918T mutation in 2B-RET.

49 Figure 3.7 Differential scanning calorimetry (DSC) analyses of WT-RET, and 2B-

RET. Thermal denaturation was determined over a temperature range of 20-120°C in the absence (top) or presence (bottom) of ATP. Midpoint of denaturation transition (Tm) was calculated using a least-squares fit of the data (dashed lines) and a two-peak model, corresponding to RET and the attached GST-tag. There was no difference in the denaturation of the GST-tag under any experimental condition (Tm 60.52°C).

50 50a

Table 3.2 Calorimetric analysis of the temperature-induced denaturation

Differential scanning calorimetry was performed in the presence and absence of ATP to measure the relative stability of WT RET and 2B RET. Data analysis was performed using Microcal Origin 5.0 software.

Protein Tm Tm + ATP WT-RET 65.10 ± 0 °C 50.11 ± 2.49 °C

2B-RET 62.65 ± 0.45°C 50.34 ± 1.76 °C

Tm: midpoint of denaturation transition, melting temperature

51 3.5 Discussion

MEN 2B is the most severe form of multiple endocrine neoplasia type 2, characterized by early onset endocrine tumours and a broad range of developmental abnormalities [139]. More than 95% of MEN 2B is caused by a methionine to threonine substitution of residue 918 in the kinase domain of RET [140] and the identical mutation occurs in more than 50% of sporadic MTC, where it has been associated with a poorer prognosis [141]. Despite predictions as to the mechanisms of this mutation, experimental confirmation of its functional effects on RET has been lacking. In this study, we have characterized the thermodynamic and kinetic properties of the RET kinase and investigated the proposed molecular mechanisms underlying the M918T mutation. Our data indicate that this mutation has multiple related but distinct effects on the RET receptor.

Our data suggest that the primary effects of the M918T mutation may be to enhance the intrinsic kinase activity of 2B-RET. We found that the affinity of 2B-RET for

ATP was more than 10 fold greater than that of WT-RET (Kd 15.3µM vs 192µM, respectively). Further, WT-RET required a much higher concentration of ATP to achieve the same levels of autophosphorylation seen with 2B-RET (KM 188µM vs 105µM), suggesting that the 2B-RET-ATP complex was significantly more stable. Together, these effects would significantly increase the relative kinase activity of 2B-RET. As we would predict from these biophysical studies, we showed that 2B-RET is more highly autophosphorylated than is activated WT-RET, and binds and phosphorylates correspondingly more of its substrates in multiple cell types (Appendix I). Our data also confirmed that this is an intrinsic property of 2B-RET and was not dependent on the

52 presence of other kinases, such as SFKs, to activate RET since in vitro analyses using purified recombinant proteins and in vivo confirmation in SYF cells clearly showed that

2B-RET achieves the same high level of autophosphorylation, irrespective of the presence of SFKs (Appendix I).

The increase in 2B-RET ATP binding is intriguing, as the M918T mutation is located in the substrate binding region of the receptor, distant from the sequences of the

ATP binding cleft (Appendix I). Mutations that specifically increase ATP binding (as opposed to other activation mechanisms) and alter downstream signaling have been identified in oncogenic forms of other RTKs, such as EGFR, but these mutations are primarily localized within or close to the ATP binding cleft, where they impact directly on interactions between the receptor and ATP [153, 154]. The location of M918, distant from this region, suggested a novel effect on RTK conformation that might contribute to altered ATP binding. Our initial analyses using CD did not identify overall global differences in the secondary or tertiary structures of WT-RET and 2B-RET (Table 3.1;

Figure 3.3), suggesting to us that the M918T mutation might cause more local changes in interactions, possibly affecting RET autoinhibition, that indirectly affected ATP binding.

In the absence of activation, receptor tyrosine kinase monomers adopt a closed

“autoinhibited” conformation in which the activation loop blocks access to the substrate- binding pocket. This conformation is very energetically stable (high Tm) due to tight intramolecular interactions that result in a rigid conformation. Upon activation, the kinase becomes much more flexible as autoinhibiting interactions are released, and it takes on a more open conformation (lower Tm). Consistent with these predictions, when we compared thermal denaturation profiles generated by DSC, we found that WT-RET had a higher Tm, suggesting a more rigid conformation, while 2B-RET had a lower Tm,

53 suggesting a more flexible, partially open conformation. In the presence of ATP, Tm was much lower for both receptors, consistent with the more flexible, open conformation of an activated kinase. Together, these data suggested that 2B-RET has a more flexible conformation that may reflect partial loss of autoinhibition.

The more flexible structure predicted for 2B-RET may be due in part a reduction in the sum of tight molecular interactions between residues in the activation loop, catalytic loop, and substrate binding P+1 loop that are important in maintaining autoinhibition (Appendix I). Together, our functional and thermodynamic analyses, and our previously shown molecular models [53, 94] suggest that the M918T mutation causes a local conformational change in the 2B-RET kinase that partially releases autoinhibition, increasing ATP-binding. However, structural release of substrate blockade alone cannot account for potentiated 2B-RET function. We have previously shown that, upon ligand induced activation, both WT-RET and 2B-RET form high molecular weight complexes, but that 2B-RET and 2A-RET also form these dimers in the absence of ligand under low stringency non-reducing conditions (Appendix I). Further, while the ratio of RET dimers and monomers are similar for 2A-RET and 2B-RET, the proportion of phosphoRET in dimers is greater for 2A-RET than 2B-RET. All RTKs are thought to exist in equilibrium between monomeric and dimeric pools, even in the absence of ligand [155, 156]. In general, the dimeric forms are non-productive interactions as these RTKs are locked in the autoinhibited state, and their formation is transient and energetically unfavorable

(Appendix I). For wildtype receptors, extracellular ligand binding stabilizes the formation of active dimers and leads to kinase stimulation. In 2A-RET, intermolecular disulphide bonds link extracellular domain residues and mimic this effect, leading to constitutively active RET dimers [60, 157]. Interestingly, 2A-RET dimers appear to be stronger than

54 those between 2B-RET monomers, which were detected in the absence of denaturation but were abolished by even a brief denaturation step (not shown) (Appendix I). As previously described, in the presence of the M918T mutation, 2B-RET adopts an intermediate, partially open conformation, although it remains conformationally unlikely that these monomers can self-phosphorylate (Appendix I). However, the stoichiometry of

2B-RET-ATP-binding, and our analytical gel filtration data indicating that purified 2B-

RET forms dimers, as well as the detection of phospho-2B-RET in high molecular weight protein complexes in the absence of ligand stimulation, suggest that the monomer:dimer equilibrium is shifted for 2B-RET, increasing the pool of transient dimers (Figure 3.8).

These complexes enable transphosphorylation of RET receptors, followed by phosphorylation of downstream targets, but in the absence of conventional ligand and co- receptor, complexes are not as stable as dimers formed by 2A-RET or activated WT-RET.

As a result, complex formation is transient and leads to a pool of phosphorylated monomeric 2B-RET, dissociated from the higher molecular weight complexes that may also be able to initiate downstream signals (Figure 3.8). We would thus predict that the sum of both active dimers and monomers may also contribute, in part, to the increased activity of 2B-RET that we observe.

In summary, we have shown that a combination of mechanisms including increased kinase activity, partial release of autoinhibition, and a relative increase in ligand independent formation of activated monomers and dimers, all contribute to 2B-RET activity. Our data suggest that the effect of these distinct mechanisms on kinase activity may be at least partly additive, which may contribute to the relative severity and broader phenotype of MEN 2B as compared to that of other MEN 2 forms. Understanding the molecular mechanisms of 2B-RET has important implications for development of

55 Figure 3.8 Model of stable, and transient, dimer formation for WT-RET and 2B-

RET In the presence of ligand, stable dimers form between RET monomers, leading to autophosphorylation and downstream signaling (top). Transient dimer formation, while predicted to occur at equilibrium for many receptors, would not promote autophosphorylation and signaling for autoinhibited wildtype receptors (middle). In the presence of the M918T mutation, altered conformation stabilizes these dimers, altering the equilibrium of monomers and dimers, while the open conformation of the activation loop permits autophosphorylation and activation of 2B-RET dimers (bottom).

56 56a therapeutics with some specificity for mutant RET forms. Pharmacological small molecule inhibitors which target ATP binding sites have well-proven clinical utility but also have potential for broad side effects related to normal functions of the targeted kinase. As RET is known to be an important neuronal survival receptor [28], therapeutic targeting of mutant receptors, while sparing the wildtype molecule, would be advantageous. Mutations of EGFR that increase ATP binding have been shown to increase sensitivity to the inhibitor in non-small cell lung cancer [154], while

MET molecules with a M1268T mutation that corresponds to M918T, have proven more sensitive to ATP blocking inhibitors [158]. Thus, specific targeting of ATP binding in

RET may be a valuable tool to aid in the development of anticancer therapies for both

MEN 2B and for sporadic tumours that harbor this mutation.

3.6 Acknowledgments

We would like to thank Rob Campbell and Kim Munro for assistance and J

McGlade, L LaRose, J Duyster, B Elliott and J Darnell for providing us with expression constructs. This research was funded by the Canadian Institutes of Health Research, and the Canadian Cancer Society (LMM). TSG is the recipient of CIHR traineeships in

Transdisciplinary Cancer Research and Protein Function Discovery.

57

Chapter 4

A novel RET kinase-β-catenin signaling pathway contributes to tumorigenesis in thyroid carcinoma

Gujral et al.,Cancer Res. 2008; 68(5): 1338-1346

Taranjit S. Gujral1, Wendy van Veelen2, Douglas S. Richardson1, Shirley M. Myers1, Jalna A. Meens1, Dennis S. Acton2, Mireia Duñach3, Bruce E. Elliott1, Jo W.M. Höppener2, and Lois M. Mulligan1*

1Division of Cancer Biology and Genetics, Cancer Research Institute, Queen’s University, Kingston, ON, Canada; 2Department of Metabolic and Endocrine Diseases, University Medical Center Utrecht, Utrecht, The Netherlands; 3Unitat de Biofísica, Departament de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, Spain.

Running title: RET activates the β-catenin pathway

Keywords: RET/ β-catenin/ tumour metastasis/ thyroid tumours/ MEN 2

*To whom correspondence should be addressed: Dr Lois M Mulligan Cancer Research Institute Botterell Hall Rm 329 Queen's University Kingston ON Canada K7L 3N6 E-mail [email protected] Phone 1 613 533 6000 X77475 Fax 1 613 533 6830

58 4.1 Abstract

The RET receptor tyrosine kinase has essential roles in cell survival, differentiation, and proliferation. Oncogenic activation of RET causes the cancer syndrome multiple endocrine neoplasia type 2 (MEN 2), and is a frequent event in sporadic thyroid carcinomas. However, the molecular mechanisms underlying RET’s potent transforming and mitogenic signals are still not clear. Here, we demonstrate that nuclear localization of

β-catenin is frequent in both thyroid tumours and their metastases from MEN 2 patients, suggesting a novel mechanism of RET-mediated function, through the β-catenin signaling pathway. We show that RET binds to, and tyrosine phosphorylates, β-catenin and demonstrate that the interaction between RET and β-catenin can be direct and independent of cytoplasmic kinases, such as SRC. As a result of RET-mediated tyrosine phosphorylation, β-catenin escapes cytosolic downregulation by the APC/Axin/GSK3 complex and accumulates in the nucleus, where it can stimulate β-catenin-specific transcriptional programs in a RET-dependent fashion. We show that downregulation of

β-catenin activity decreases RET-mediated cell proliferation, colony formation, and tumour growth in nude mice. Together, our data show that a β-catenin-RET kinase pathway is a critical contributor to the development and metastasis of human thyroid carcinoma.

59 4.2 Introduction

The RET receptor is required for development of urogenital and neural crest derived cell types [9]. Under normal cellular conditions, RET is activated by binding of both glial cell line-derived neurotrophic factor (GDNF) ligands and cell surface bound co- receptors of the GDNF family receptor α (GFRα) proteins [10]. However, oncogenic activation of RET, by germline point mutations, leads to ligand-independent constitutive kinase activity, giving rise to the inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN 2). MEN 2 is characterized by medullary thyroid carcinoma (MTC), a tumour of thyroid C-cells; and the adrenal tumour pheochromocytoma (PC), as well as other less common tumor and developmental phenotypes (Reviewed [138]). MTC is the predominant disease feature, with early onset tumours and metastases to lymph nodes and distant organs [159]. RET activation contributes to stimulation of RAS-ERK, JNK, PI3K, p38MAPK, SRC, STAT and ERK5 signaling cascades (Reviewed [9]). However, the identity of the critical secondary oncogenic signals involved in the broad and early metastatic pattern of RET-mediated MTC is still largely unknown.

β-catenin is a ubiquitously expressed multifunctional protein that plays important roles in cell adhesion and signal transduction [160]. At the plasma membrane, β-catenin associates with E-cadherin and α-catenin in linking the cytoskeleton and adherens junctions, while in the nucleus it acts as a mediator of transcription through other DNA binding proteins such as TCF/LEF family members (Reviewed [161]). Cytosolic free β- catenin interacts with the adenomatous polyposis coli (APC) and axin proteins to form a complex, which in turn recruits glycogen synthase kinase-3 (GSK3) and casein kinase, to

60 form a destruction complex that serine/threonine phosphorylates β-catenin and targets it to the proteosome (Reviewed [137]).

Abnormal expression, or localization of β-catenin has been reported in many tumour types [160, 162], and β-catenin-mediated loss of cell/cell adhesion has been implicated in anchorage-independent cell growth and cancer metastasis [162]. The best characterized mechanism leading to β-catenin-mediated signal transduction is through activation of the WNT pathway by binding of WNT proteins to frizzled and LRP family cell surface receptors [163]. However, β-catenin signaling can also be induced in response to overexpression or activation of tyrosine kinases in a WNT-independent fashion, and both these pathways converge to target β-catenin to the nucleus and induce expression of a similar set of β-catenin-specific target genes [164-166]. β-catenin tyrosine phosphorylation causes its dissociation from membrane-associated E-cadherin, leading to accumulation of a pool of free cytoplasmic β-catenin [167]. This can, in turn, increase the amount of β-catenin reaching the nucleus where it acts as a transcription factor, upregulating expression of genes involved in cell migration, growth, differentiation, and survival [162]. Tyrosine phosphorylation of β-catenin, followed by functional downregulation of E-cadherin-mediated cell-cell contact, is potentially critical in initiating cell migration in both normal physiological processes and in tumor metastasis

[167].

Loss of membrane-associated β-catenin, often with an accompanying relative increase in cytosolic or nuclear expression, has been noted in anaplastic and poorly differentiated thyroid carcinomas, and in thyroid papillary microcarcinoma [168-170].

However, β-catenin had not been previously investigated in RET-mediated tumour

61 development and metastasis. Here, we report that RET interacts with, and activates β- catenin, and that a RET-β-catenin signaling pathway plays roles in RET-mediated tumour growth, invasion and metastasis.

4.3 Materials and Methods

4.3.1 Expression Constructs

Expression constructs for full-length human RET, GFRα1, and mutant RET constructs have been previously described [94, 142, 143]. Intracellular (ic)RET expression constructs, generated by fusing cDNA encoding a myristylation signal, two dimerization domains, and the intracellular portion (amino acid 658 to the C-terminus) of

RET, have been previously described [142]. Axin and wildtype β-catenin expression constructs were a gift from Dr. J. Woodgett (Ontario Cancer Research Institute, Toronto,

Canada). GST-tagged expression constructs for WT and mutant β-catenin have been reported [171, 172].

4.3.2 Cell Culture and Transfection

The human neuroblastoma cell line SH-SY-5Y was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, Oakville, ON). All other cell lines were grown in Dulbecco’s Modified Eagle’s Medium (DMEM)

(Invitrogen, Burlington, ON), supplemented with 10% FBS. Medium for HEK293-TET-

ON cells, used to induce icRET, was further supplemented with 1 µg/ml doxycycline.

HEK293 cells were transiently transfected with the indicated expression constructs using

Lipofectamine 2000 (Invitrogen, Burlington, ON), according to the manufacturer’s instructions. RET activation was induced by addition of 100 ng/ml of GDNF (Promega,

62 Madison, WI) for full length RET, or with 1 µM AP20187 dimerizer (ARIAD

Pharmaceuticals, Cambridge, MA) for icRET, for the time periods indicated.

4.3.3 Immunoprecipitation, Western, and Far-Western Blotting

Whole cell lysates (WCL) were harvested 48 hours after transfection, and suspended in lysing buffer (20 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 mM sodium orthovanadate, 1% Igepal, 2 mM EDTA, 1 mM PMSF, 10 µg/ml aprotonin and 10 µg/ml leupeptin) [143]. Protein concentration was determined by BCA protein assay kit, according to the manufacturer’s instructions (Pierce Biotechnology, Rockford, IL). For immunoprecipitations, lysates were incubated with a 1:50 dilution of the appropriate primary antibody for 2 hours at 4°C with agitation. Complexes were collected on Protein

AG beads (Santa Cruz Biotechnology, Santa Cruz, CA) by centrifugation at 13,000 rpm, washed 3 times with lysing buffer, and resuspended in laemmli buffer. Protein samples were denatured at 99°C for 5 mins, separated on 10% SDS-PAGE gels, and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA), as previously described [94, 143].

Antibodies used included: anti-RET (c19), anti-ubiquitin (N19), and anti-myc-tag

(NE10), antibodies (Santa Cruz Biotechnology, Santa Cruz CA), anti-phospho-RET

(pY905) antibody (Cell Signaling, Beverly, MA), anti-β-catenin antibody (BD

Biosciences, Mississauga, ON), and anti-V5 tag (axin) (Invitrogen, Burlington ON). An anti-phosphotyrosine antibody (pY99; Santa Cruz) was used to detect tyrosine phosphorylation of β-catenin. For far-western analyses, protein lysates were immunoprecipitated for RET and resolved and western blotted, as above. Membranes was incubated for 2 hrs at 4°C in a 0.1% Tween-20/TBS solution containing a probe of

1µg/ml GST-β-catenin, GST alone, or no probe. After three washes, bound proteins were

63 detected with anti-GST antibody (Santa Cruz Biotechnology, Santa Cruz CA) or immunoblotted for RET.

4.3.4 Preparation of Cytosolic Extracts

Cells were harvested by gentle centrifugation, washed twice with PBS, and suspended in ice-cold hypotonic buffer (20 mM Hepes-KOH, pH 7.0, 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, 250 mM sucrose, and protease inhibitors) [173]. After incubation on ice for 15 min, cells were disrupted by dounce homogenization. Nuclei were pelleted by centrifugation and cytosolic fractions were isolated by collection of the supernatant.

4.3.5 Glutathione S-transferase (GST)-Pull-down Assays

GST-fusion proteins expressed in E. coli, were eluted with 100 mM glutathione elution buffer using a polyprep column (Bio-Rad, Hercules, CA), as described previously

[94]. For GST pull-down assays, 5 µg of GST-fusion protein and GST-sepharose-beads

(GE Healthcare/Amersham Biosciences, Baie d'Urfé, QC) were incubated with whole cell lysates at 4°C for 3 hours, with agitation. Bound proteins were eluted by boiling in laemmli sample buffer containing 2-mercaptoethanol, and resolved by SDS-PAGE and western blotting, as described above.

4.3.6 Reporter Assay

For dual-luciferase reporter assays, TOPFLASH or FOPFLASH vectors (Upstate

Biotechnology, Lake Placid, NY) and pRL-TK control were co-transfected into HEK293 cells stably expressing icRET or empty vector. Luciferase activity was measured with a

Dual-Luciferase reporter kit (Promega, Madison, WI).

64 4.3.7 ShRNA Production

Four different β-catenin shRNAs, in pLKO.1 lentiviral vectors, were obtained from Open Biosystems (Huntsville, AL). Lentiviral particles containing the different β- catenin shRNA constructs were grown in 293T packaging cells by transfecting a three- plasmid packaging system [174] according to the manufacturer’s instructions.

Supernatants were collected 48 and 72 hours after transfection, filtered, and pooled. NIH

3T3 stably expressing the oncogenic 2A-RET (C634R) were infected with lentiviral constructs or an heterologous lentiviral control, and polyclonal stable cell lines were generated.

4.3.8 MTT Cell Proliferation Assay

MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assays were performed as described [175]. Briefly, HEK293 cells transiently expressing RET or empty vector, seeded in 6-well plates were either infected with lentiviral β-catenin shRNA or untreated. Cells were grown in medium supplemented with

100 ng/ml GDNF. After 3 days, MTT was added to a final concentration of 250 µg/ml for

2 h at 37°C. Culture medium was removed and formazan crystals were dissolved in

DMSO. Reduced MTT was measured spectrophotometrically at 570 nm. Statistical significance was calculated by one-way ANOVA.

4.3.9 Apoptosis Assays

NIH 3T3 cells stably expressing 2A-RET were infected with lentiviral constructs for β-catenin shRNA or control vector and cultured for 48 hours. Cells were harvested, fixed in absolute ethanol, and treated with RNaseA at 37oC overnight. Fixed cells were incubated in 5 mg/ml propidium iodide for 15 minutes at room temperature, and cell

65 cycle analysis performed using an EPICS ALTRA HSS flow cytometer (Beckman

Coulter, Mississauga ON). Statistical significance was calculated by one-way ANOVA.

4.3.10 Soft Agar Colony Formation Assays

Soft agar colony formation assays were performed as described previously [94].

Briefly, NIH 3T3 expressing 2A-RET or K758M constructs were infected with lentiviral vectors for β-catenin shRNA or control. Approximately 5 x 104 cells were resuspended in

0.2% top agar in culture medium, and plated on 0.4% bottom agar in medium. Culture medium was supplemented every 2 to 3 days. Colonies were counted after 14 days, and statistical significance was confirmed by one-way ANOVA.

4.3.11 Confocal Microscopy

SH-SY-5Y neuroblastoma cells were cultured on glass coverslips coated with

0.2% gelatin. 24 hours prior to fixation, 10 nM retinoic acid was added to the culture medium. Cells were serum starved for 3 hours, then fixed in 3% paraformaldehyde for 40 minutes at room temperature. Cells were then washed in phosphate-buffered saline, permeablised with 0.15% Triton-X-100, and blocked for 30 minutes in 3% BSA. Cells were double-immunostained with primary antibodies specific for β-catenin (BD

Biosciences) and RET (c19, Santa Cruz Biotechnology), and species-matched secondary antibodies labeled with Alexa 594 or 488, respectively. Coverslips were mounted on glass slides in Mowiol mounting medium, and observed using a Leica TCS-SP2 confocal microscope. Individual channels were overlayed using Image J software and colocalization was determined using the Image J RG2B colocalization plug-in.

4.3.12 Tumourigenicity in Nude Mice

All in vivo experiments were performed using 6-8 week old athymic nude mice

(NIH, Bethesda, U.S.A). Experiments were performed in duplicate using a minimum of 5

66 animals/treatment group. Mice were maintained in laminar flow rooms with constant temperature and humidity. Experimental protocols were approved by the Ethics

Committee for Animal Care (Queen’s University Kingston, Canada). NIH 3T3 parental cells, cells stably expressing 2A-RET constructs, or polyclonal cell lines expressing 2A-

RET and β-catenin shRNA (described above) were inoculated subcutaneously into the right flank of the mice. Cells (2 X106 in suspension) were injected on day 0, and tumor growth was followed every 2-3 days by tumour diameter measurements using vernier calipers. Tumour volumes (V) were calculated using the formula: V = AB2/2 (A = axial diameter; B =rotational diameter). Mice were sacrificed at day 14, and tumour tissues were excised for protein extraction and histology. Tumour tissue was homogenized in 10 volumes homogenization buffer (20 mM Tris-HCl, pH 7.8, 150 mM NaCl, 1 mM sodium orthovanadate, 2 mM EDTA, 1 mM PMSF, 10 µg/ml aprotonin and 10 µg/ml leupeptin).

Tissue debris was removed by centrifugation, the supernatant collected, and cells lysed at a final concentration of 1% Triton X-100. Samples were centrifuged to remove insoluble material. Tissue for histology was fixed in neutral buffered formalin and processed by routine methods. Paraffin embedded sections of 5µm were stained with haematoxylin and eosin for histologic examination.

4.3.13 β-catenin Immunohistochemistry

Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissues. Tumour tissue from the CALC-MEN2BRET mice was excised, fixed, and paraffin embedded, by routine methods. Human MTC samples were obtained from the

Department of Pathology of the University Medical Center Utrecht, Utrecht, The

Netherlands. Paraffin sections (6µm) were blocked with 0.5% CASEIN blocking reagent

(PerkinElmer Life Sciences, Waltham, MA, USA) in 0.1% Triton-X100 in PBS, and

67 treated with 1.5% hydrogen peroxide to inhibit endogenous peroxidase activity. Sections were incubated with a mouse monoclonal anti-β-catenin antibody (BD Biosciences, San

Jose, CA, USA), 1:50 dilution, for one hour at room temperature. Slides were incubated with peroxidase-labeled rabbit anti-mouse secondary antibody (Dako, Glostrup,

Denmark), 1:100 dilution, for 30 minutes at room temperature and subsequently with peroxidase-labeled swine anti-rabbit antibody (Dako), 1:100 dilution, for 30 minutes at room temperature. Finally, sections were incubated with DAB and counterstained with

Mayer’s haematoxylin. Nuclear staining was considered positive when one or more positive nuclei were observed in each microscopic field (40x) [176]. Negative control experiments were performed by omitting the primary antibody.

4.3.14 Relative Quantification by Real-Time RT-PCR (qRT-PCR)

Relative differences between gene transcript levels were confirmed using the

QuantiTect SYBR Green RT-PCR kit (Qiagen, Mississauga, ON) and a SmartCycler II

(Cepheid, Sunnyvale, CA). Primer and PCR product information are found in Appendix

II. The qRT-PCR assays were repeated at least three times. Crossing threshold (Ct) values were taken at the same threshold for each experiment; folds expression were averaged and mean fold change, relative to the empty vector control, calculated.

4.4 Results and Discussion

4.4.1 β-catenin nuclear localization in RET-mediated human thyroid tumours

In preliminary immunohistochemical experiments, using MTCs from CALC-

MEN2BRET transgenic mice, which express a constitutively active, oncogenic RET mutant (2B-RET) [177], we found that 6 out of 7 tumours had nuclear localization of β- catenin (Figure 4.1), suggesting a role for β-catenin signaling in these tumours and a

68 Figure 4.1 Altered localization of β-catenin in response to activated RET in a murine model of RET-induced MTC. Representative micrographs of tumour tissue from

CALC-MEN2BRET transgenic mice that express the activated RET mutant 2B-RET and endogenous β-catenin. Nuclear β-catenin localization was detected in 6 out of 7 MTCs, using a mouse monoclonal anti-β-catenin antibody (BD Biosciences, San Jose, CA,

USA). Membranous and nuclear β-catenin expression are indicated by white and black arrow heads, respectively. A black arrow (right panel) indicates a dividing cell. Scale bars represent 20 µm. This figure was provided by Wendy Veelen, Dr. Dennis Acton and Dr.

Jo Höppener.

69 CALC-MEN2BRET MTC

β-catenin

Membranous Membranous and nuclear

69a relationship between RET activation and β-catenin nuclear localization. Further, in human MTC samples from twenty MEN 2- patients with known oncogenic RET mutations (Table 4.1), 8 had nuclear β-catenin expression in a subset of cells, heterogeneously spread throughout the tumours (Figure 4.2A). In addition, although some MTCs showed β-catenin expression at the cellular membrane, it was less prominent, particularly in tumours with strong nuclear β-catenin expression (Figure

4.2A). Association of nuclear staining with loss of membranous staining, has also been reported in other cancers, such as colorectal carcinomas [176]. Nuclear localization of β- catenin was not detected in normal or hyperplastic C-cells, in mouse or human tissues.

Interestingly, nuclear localization was more prevalent in metastases (5/7 cases) than in primary MTCs (3/13 cases) (Figure 4.2B, Table 4.1), suggesting an association of more aggressive or advanced MTC disease stage and activation of β-catenin. Conversely, in the absence of oncogenic activation of RET, we showed that the endogenous RET and β- catenin proteins colocalized at the plasma membrane, in the neuroblastoma cell line SH-

SY-5Y (Figure 4.3). Together, these observations indicated that β-catenin signalling may play an important role in progression of RET-mediated tumours and suggested that a novel RET- β-catenin signalling mechanism could be taking place.

4.4.2 RET associates with and tyrosine phosphorylates β-catenin

As tyrosine phosphorylation of β-catenin by several kinases has been correlated with tumorigenesis and metastasis [165-167], we investigated the association of RET with β-catenin. In TT cells, a line derived from a human MTC expressing endogenous

RET with an activating mutation (2A-RET), we found that RET induced phosphorylation of endogenous β-catenin. Similarly, in cells co-transfected with RET and β-catenin

70 Table 4.1: Patient information and β-catenin staining patterns for primary and metastasized human MTCs.

MTC Age MEN 2 disease RET Mutation β-Catenin (years) phenotype staining Primary 4 MEN 2A C634W 5 MEN 2A C634R 5 MEN 2A C634G 7 MEN 2A C634R 8 MEN 2A C634R 10 MEN 2A C634G 13 MEN 2A C634R 14 MEN 2A C634Y nuclear 26 MEN 2A C634Y nuclear 30 MEN 2A C634R 34 MEN 2A C634G nuclear 36 MEN 2A C634W 37 MEN 2A C634R Metastasis 26 MEN 2B M918T nuclear 30 MEN 2A C634Y 35 MEN 2B M918T nuclear 38 MEN 2A C618S nuclear 45 MEN 2A C618S nuclear 54 MEN 2A C634R nuclear 76 MEN 2A C634W

71 Figure 4.2 Localization of β-catenin in human MTCs. A) Paraffin sections of human primary MTCs (left and right panels) and an MTC metastasis (middle panel) from MEN 2 patients were stained for β-catenin. Membranous and nuclear β-catenin expression is indicated with white and black arrow heads, respectively. The scale bars represent 20 µm.

B) Dendrogram showing distribution of nuclear β-catenin expression in our panel of human primary MTCs and metastases from 20 MEN 2 patients with well characterized oncogenic RET mutations. This figure was provided by Wendy Veelen, Dr. Dennis Acton and Dr. Jo Höppener.

72 A

B

72a

Figure 4.3 Endogenous RET and β-catenin co-localize at the cell membrane. SH-SY-

5Y neuroblastoma cells expressing endogenous RET and β-Catenin were serum starved, and fixed in 3% (w/v) paraformaldehyde/PBS. Cells were double immunostained for

RET (A, D) and β-Catenin (B, E). Panel C is a merged image of A, and B. Images D, and

E and F are enlargements of the dashed boxes shown in A, B and C, respectively. Panel G represents image F after application of a co-localization filter (RG2B colocalization –

Image J) which replaces pixels containing signal in both the red and green channel with a grey-scale pixel of similar intensity. This figure was provided by Douglas Richardson.

73 RET

β-catenin

Merge

73a expression constructs, we showed that treatment with the RET ligand GDNF also induced phosphorylation of β-catenin (Figure 4.4A). Immunoprecipitation of β-catenin, and immunoblotting with appropriate antibodies, showed that both endogenous and transiently expressed β-catenin and RET associate in complexes (Figure 4A). These complexes could also be detected by immunoprecipitation of RET and immunoblotting for β-catenin (not shown). Our data show that both the ligand-activated wildtype RET

(WT-RET) and the oncogenic mutants, 2A-RET and 2B-RET, induce tyrosine- phosphorylation of β-catenin (Figure 4.4A). In the absence of GDNF, the constitutively active 2B-RET protein, but not WT-RET, was able to induce significant β-catenin tyrosine phosphorylation (Figure 4.4A). Further, a catalytically-compromised RET mutant, K758M [94], was unable to induce β-catenin tyrosine phosphorylation, even in the presence of GDNF stimulation, suggesting a RET kinase-dependent mechanism of β- catenin phosphorylation (Figure 4.4A). Interestingly, however, K758M RET was still able to associate with β-catenin, in a phosphorylation-independent fashion, suggesting a constitutive association between RET and β-catenin.

Although β-catenin may associate with receptor kinases and can be tyrosine phosphorylated in cells stimulated by their ligands, EGFR is the only receptor known to directly phosphorylate β-catenin [178]. Other receptors may induce activation of SRC, which in turn can phosphorylate β-catenin, as well as other proteins of the adherens junctions [167]. To determine whether β-catenin tyrosine phosphorylation could result from a direct interaction with RET, we performed in vitro kinase assays using purified

RET kinase [94] and purified recombinant GST-tagged β-catenin [171]. We found that

74 Figure 4.4 RET associates with, and tyrosine phosphorylates, β-catenin. A) RET induces tyrosine phosphorylation of β-catenin. TT cells expressing endogenous 2A-RET and β-catenin, and HEK293 cells transiently co-transfected with GFRα1, RET (WT-RET,

2B-RET, K758M), and myc- tagged β-catenin constructs, in the presence (GDNF for 15 minutes) or absence of ligand stimulation, were immunoprecipitated for β-catenin and immunoblotted with appropriate antibodies. B). RET and β-catenin interact directly.

HEK293 cells transiently expressing WT-RET (+) or vector (-), and myc-tagged β- catenin, were treated with GDNF, as above. Cell lysates were immunoprecipitated with anti-RET antibody (c-19), western blotted and incubated with purified recombinant GST-

β-catenin (top), or GST alone (middle), or no probe (bottom) and immunoblotted with either an anti-GST or anti-RET antibody, as indicated.

75 75a the purified RET-kinase can be activated in the presence of ATP and can directly phosphorylate β-catenin in vitro (Figure 4.5). As endogenous wildtype RET and β-catenin proteins colocalize at the plasma membrane (Figure 4.3), we confirmed that they could directly interact by far-western assays. We showed that purified recombinant GST-β- catenin could interact with immunoprecipitated WT-RET, while a purified GST control could not, in direct far-western assays (Figure 4.4B). In vivo, we further showed that the

RET- β-catenin interaction can occur independent of SRC, using co- immunoprecipitations in SYF-/- cells, which lack the SRC family kinases SRC, YES, and

FYN (Figure 4.6). Our data show that RET can associate with, and directly phosphorylate, β-catenin and that the tyrosine phosphorylation of β-catenin is not dependent on SRC activation.

The pattern of tyrosine residue phosphorylation of β-catenin has been shown to be kinase specific. Activation of tyrosine kinases FYN, FER, and MET, leads to phosphorylation at Y142, while Y654 can be phosphorylated directly by EGFR or SRC

[165, 166, 171, 178, 179]. In pull-down assays using GST-tagged β-catenin tyrosine mutants for either Y142 (Y142F) or Y654 (Y654F) [172], we showed that wild type β- catenin, and to a lesser extent the Y142F mutant, were phosphorylated by WT-RET, but that Y654F β-catenin was not phosphorylated (Figure 4.7) suggesting that Y654 was the major site for RET-mediated β-catenin phosphorylation. Tyrosine 654 lies within the putative E-cadherin binding region of β-catenin, and the bulkier phosphorylated residue has been shown to reduce β-catenin binding affinity for E-cadherin leading to dissociation of β-catenin from the membrane [167, 179].

76 Figure 4.5 Recombinant purified RET can directly phosphorylate purified β-catenin in vitro. Purified GST-tagged RET protein (5 µg) , was incubated with GST-WT β- catenin (5 µg) in the presence of ATP for 30 min at 30° C. Samples were subjected to

SDS-PAGE and immunoblotted with an anti-phosphotyrosine antibody (pY99). Purified

RET is phosphorylated, as shown previously [94]. In the presence of an active RET kinase, β-catenin is also phosphorylated, suggesting that β-catenin acts a RET kinase substrate.

77 GST-β-catenin + - +

GST-RET - + +

β-catenin

RET

IB: pY

77a Figure 4.6 RET can phosphorylate β-catenin in the absence of SRC family kinases.

SYF-/- cells (lacking SRC family kinases) were transiently co-transfected with myc- tagged β-catenin and the indicated RET construct or vector alone. Cell lysates were immunoprecipitated for β-catenin and immunoblotted with appropriate antibodies.

78 78a Figure 4.7 β-catenin tyrosine 654 (Y654) is a major RET-phosphorylation site. Cell lysates from HEK293 cells stably expressing WT-RET or empty vector were pulled-down with purified WT or mutant GST-β-catenin. Complexes were immunoblotted with anti- phosphotyrosine or anti- β-catenin antibodies. WCL- whole cell lysates.

79 79a 4.4.3 RET activation induces cytosolic translocation of β-catenin and its escape from the axin regulatory complex

Accumulation of cytosolic free β-catenin is tightly regulated by the

APC/axin/GSK3 complex, which binds β-catenin and leads to its serine/threonine phosphorylation, targeting it for ubiquitination, and proteosomal degradation [180]. We have shown that RET and β-catenin co-localize at the plasma membrane (Figure 4.3), but that activation of RET leads to a relative increase in β-catenin in the cytosol (Figure

4.8A). Notably, the cytosolic level was not further enhanced by the more active 2B-RET kinase, perhaps suggesting that increased RET activity may enhance nuclear β-catenin levels preferentially. Interestingly, in the presence of active RET, we found a relative decrease in β-catenin associated with the APC/axin/GSK3 complex in immunoprecipitations of axin, which interacts directly with β-catenin in this complex

(Figure 4.9A). Further, there was an accompanying relative decrease in ubiquitinated β- catenin (Figure 4.9B), suggesting that RET-mediated tyrosine phosphorylation of β- catenin protects it from degradation. This would be consistent with data on RON and

MET which also suggest that tyrosine phosphorylation of β-catenin inhibits its interactions with the APC/axin/GSK3 complex [165].

4.4.4 RET-induced β-catenin tyrosine phosphorylation is associated with increased

TCF transcriptional activity

β-catenin also has important signalling roles in the nucleus, mediated through its interactions with the TCF family of transcription factors, and other transcriptional

80 Figure 4.8 RET alters β-catenin localization. A) Cytosolic β-catenin-levels are relatively increased upon expression and activation of RET. Whole cell lysates (WCL) and cytosolic cell fractions from HEK293 cells expressing GFRα1 and RET (WT-RET or

2B-RET), or empty vector, were immunoblotted for RET, β-catenin, or a tubulin control.

B) β-catenin mediates TCF-4 transcriptional activity in RET expressing cells. HEK293 cells transiently transfected with TOPFLASH or FOPFLASH and pRL-TK reporters and inducible icRET constructs were treated with dimerizer for 2h to induce RET activation, and a dual-luciferase assay was performed. Results are the means of three independent experiments ±sd of fold differences.

81 A B

* Vector WT-RET 2B-RET GDNF + + + IB: Cytosolic β-catenin Tubulin β-catenin activity (fold) WCL

RET Normalized luciferase

81a Figure 4.9 Tyrosine phosphorylated β-catenin escapes the axin regulatory complex in the cytoplasm. A) RET activation reduces the association of β-catenin and axin.

HEK293 cells transiently expressing GFRα1 and either WT-RET, or empty vector, were immunoprecipitated with an antibody to axin and immunoblotted for axin or β-catenin.

The relative interaction of β-catenin and axin in the presence and absence of RET activation is shown (left) and the relative quantitation is indicated (right). B) β-catenin ubiquitination is reduced in the presence of activated RET. HEK293 whole cell lysates, as described in A) above, were immunoprecipitated with an anti-ubiquitin antibody and immunoblotted for β-catenin. Relative levels of β-catenin ubiquitination, in the presence or absence of ligand stimulated RET, are indicated (left) and the relative quantitation is indicated (right). Each comparison was repeated a minimum of twice and a representative example is shown.

82 A

B

82a regulators, and the subsequent activation of target genes, such as cyclinD1 [162, 165, 166,

178]. Initially, to determine whether RET activation could induce a β-catenin transcriptional program, we used the well-characterized TOPFLASH (TCF binding site) and FOPFLASH (mutated TCF binding site) luciferase reporters [165, 181] for detection of β-catenin/TCF-mediated transcription. In cells stimulated with GDNF, TCF-reporter activity was significantly increased (relative to FOPFLASH) in the presence of WT-RET but not in the presence of the kinase-dead mutant (Figure 4.8B), suggesting that a RET- dependent induction of a β-catenin/TCF transcriptional program may occur.

4.4.5 β-catenin is required for RET-induced cell proliferation and transformation

The role of RET activation in stimulation of β-catenin-mediated transcription, prompted us to investigate whether β-catenin was required for known RET-mediated processes using shRNA-knock-down of β-catenin. We evaluated four lentivirus-produced

β-catenin shRNA constructs, pooled those producing the most significant knock-down of

β-catenin and its target, cyclinD1 (Figure 4.10), and used these to generate polyclonal β- catenin-deficient cell lines. We used quantitative real time PCR (qRT-PCR) to evaluate the effect of loss of β-catenin on expression of a panel of known RET-target genes, previously identified in gene expression analyses [143, 182, 183]. RET activation has been shown to increase expression of cyclinD1 and our data demonstrate that both RET and β-catenin are required to induce this in HEK 293 cells (Figure 4.11; Figure 4.10).

Knock-down of β-catenin expression had no significant effect on RET expression or on expression of control transcripts (e.g. axin) not known to be modulated by either RET or

β-catenin (Figure 4.11). However, it did block, or significantly reduce, RET-induced

83 Figure 4.10 Knockdown of β-catenin and cyclinD1 protein levels by shRNA against

β-catenin. HEK293 cells expressing 4 different lentiviral- produced shRNAs (1-4) or lentiviral control (-) were monitored for β-catenin and cyclinD1 expression by western blotting, using appropriate antibodies. shRNAs 3 and 4 were pooled for use in the described analyses.

84 shRNA β-catenin - 1 2 3 4 IB: β-catenin CyclinD1 Tubulin

84a Figure 4.11 RET activation induces β-catenin-mediated transcription. RET and β- catenin have overlapping transcriptional target gene patterns. HEK293 cells stably expressing WT-RET and transiently expressing β-catenin shRNA or a control vector, were treated with GDNF, as above. RNA was isolated after 3 hours and subjected to qRT-

PCR, using appropriate primers. Relative changes in expression are shown in comparison to 0h treatment conditions. Fold changes were averaged and expressed as a mean fold change ±sd.

85 85a upregulation of a subset of RET targets, including CyclinD1, JunB, and EGR1, in cells expressing both activated RET and shRNA for β-catenin (Figure 4.11A). Other RET- targets were unaffected (e.g. GDF15, VGF), consistent with a β-catenin –independent mechanism of RET-mediated stimulation. Interestingly, a RET- β-catenin signaling pathway appeared to be important to induction of cell proliferative targets, including immediate early genes cyclinD1, EGR1 and JunB, but had less impact on targets associated with more differentiated cell functions, such as GDF15 (growth differentiation factor 15) and VGF (VGF nerve growth factor induced). Together, our data show that

RET activation may stimulate a β-catenin transcriptional program but that not all RET- targets require β-catenin for expression.

4.4.6 Knockdown of β-catenin expression reduces RET-mediated tumour growth and invasiveness in nude mice

As tyrosine phosphorylation of β-catenin by other kinases has been shown to divert its function from cell adhesion to increased signaling roles [167], and as we found a correlation of β-catenin nuclear expression and metastasis of MTC, our gene expression data led us to postulate that RET-induced activation of the β-catenin pathway may contribute to cell proliferation and tumourigenesis. Using the 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay, we showed that

GDNF-treatment of HEK293 cells significantly increased cell proliferation in the presence of WT-RET but did not affect cells expressing the kinase dead K758M RET mutant, or an empty vector (Figure 4.12A). Similarly, GDNF did not stimulate RET- mediated proliferation in the presence of β-catenin shRNA (Figure 4.12A). A significant

86 Figure 4.12 β-catenin is required for RET induced proliferation and transformation.

A) HEK293 cells transiently expressing the indicated RET construct or vector alone were treated with GDNF and with shRNA against β-catenin, or a control, and cell proliferation was measured by MTT assay. Means of at least 3 replicates ±sd are shown. B) Down- regulation of β-catenin reduces RET induced transformation. NIH 3T3 cells expressing constructs for an oncogenic (2A-RET) or a kinase dead (K758M) form of RET were infected with β-catenin shRNA or control lentivirus (CTL) and grown on soft agar.

Colony formation was assessed in a minimum of three replicate experiments and is expressed as mean colony number ±sd.

87 A

B

87a decrease in colony formation induced by the oncogenic mutant 2A-RET was also detected in soft agar anchorage-independent growth assays in the presence of β-catenin shRNA

(Figure 4.12B). While β-catenin shRNA expression did increase apoptosis, the level of apoptotic cell death remained quite low in RET-expressing cells in the presence of β- catenin shRNA (Figure 4.13), suggesting that a RET-β-catenin signaling pathway has roles in cell proliferation but is not as critical for cell survival.

We next used our β-catenin-shRNA knock-down model to investigate the role of

β-catenin in RET-mediated oncogenesis. NIH 3T3 cells stably expressing the 2A-RET oncogenic receptor, with or without β-catenin shRNA, were injected subcutaneously into athymic mice and the ability of cells to form tumour outgrowths was monitored (Figure

4.14A). NIH 3T3 cells expressing 2A-RET grew rapidly in this model, producing measurable tumours by day 9 (Figures 4.14A, 4.14B), while parental NIH 3T3 cells produced no detectable lesions (Figure 4.14B). In the presence of shRNA directed against β-catenin, tumour growth was slower with a notable lag in exponential growth phase. Tumours derived from cells expressing 2A-RET and control vector were nodular and adherent to the abdominal wall with frequent regional invasion and, in some cases, spreading to visceral organs such as kidney (Figure 4.15; Table 4.2). In contrast, tumours derived from cells expressing 2A-RET and β-catenin shRNA were encapsulated, with no adhesion or invasion to surrounding tissues. The average volume of tumours was more than 2-fold less (P<0.001) in the animals receiving cells containing both 2A-RET and β- catenin shRNA as compared to 2A-RET and control vector, and this effect was correlated with a reduction in β-catenin protein in the corresponding primary tumour tissue (Figure

88 Figure 4.13 β-catenin has a modest effect on RET-induced cell survival. NIH 3T3 cells stably expressing 2A-RET and infected with β-catenin shRNA or control lentivirus, as above, were fixed and stained with propidium iodide and percentage apoptotic cells estimated by flow cytometry. Assays were performed in triplicate and means ±sd are shown.

89 89a Figure 4.14 RET-mediated tumour outgrowth in nude mice is reduced by shRNA to

β-catenin. A) NIH 3T3 cells stably expressing 2A-RET together with β-catenin shRNA or a control vector, were injected into the right flank of nude mice, and tumour volume

(mm3) was measured every 2-3 days. Asterisk (*) denotes statistical significance, p <

0.05, denotes mouse which had to be euthanized due to excessive tumour volume. B)

Photographs of representative animals injected with parental NIH 3T3 cells (left), or cells expressing 2A-RET and endogenous β-catenin (middle), or 2A-RET with β-catenin shRNA (right) are shown. Arrows indicate tumours. 2A-RET and endogenous β-catenin coexpression leads to larger, more nodular tumours, compared to 2A-RET in presence of

β-catenin shRNA. C) RET and β-catenin expression in primary tumours. Proteins extracted from snap-frozen tumour samples were subjected to western blotting with the indicated antibodies.

90 A

B C

90a Figure 4.15 Coexpression of RET and endogenous β-catenin promotes regional invasiveness and tumour adhesion to the abdominal wall. Representative hematoxylin and eosin stained micrographs of tumour tissue from mice injected with cells co- expressing 2A-RET and endogenous β-catenin (panels A-C) or cells expressing 2A-RET and β-catenin shRNA (panels D, E) are shown. Panels B and E are higher magnifications of boxed regions shown in A, and D, respectively. Histology of the 2A-RET tumour, infiltrating into the walls of the kidney, is shown in panel C. The scale bars represent 100

µm in panels A, C, and D, and 50 m in panels B and E. Inv: invasive tumour cells, ad: tumour adhesion to surrounding tissue. This figure was provided by Dr. Dr. Bruce Elliott.

91 91a

Table 4.2. Gross pathology and histology of tumour lesions.

Group Adhesion to abdominal wall Regional invasion* NIH 3T3 (parental) - - 2A-RET 5/5 (100%) 3/5 (60%) 2A-RET + β-catenin shRNA 1/5 (20%) 0/5 (0%) * Regional invasion through abdominal wall with occasional spreading to visceral organs.

92 4.14C). Together, these data suggest that the highly tumorigenic potential of oncogenic

RET mutants is, in part, mediated through a β-catenin signaling cascade.

These observations establish a novel signaling pathway linking the RET receptor tyrosine kinase to β-catenin. We show that RET and β-catenin can interact directly, leading to RET-mediated β-catenin tyrosine phosphorylation, nuclear translocation, and induction of a RET-β-catenin transcriptional program (Figure 4.16). In cell-based models, our data confirm that β-catenin is an important contributor to RET-induced cell proliferation and colony formation. In vivo, our data showing nuclear β-catenin localization in primary human MTC samples are consistent with activation of a RET-β- catenin pathway in these tumours. Nuclear localization was significantly more common in

MTC metastases (p<.001) than in primary tumours from MEN 2 patients. Further, β- catenin nuclear localization was observed in 2/2 cases of MTC metastasis bearing the 2B-

RET mutation (M918T), the most transforming RET mutant, associated with the most aggressive clinical form of MEN 2, MEN 2B [94], as well as in 6/7 MTCs from the

CALC-MEN2BRET mice which bear the corresponding RET mutation (Figure 4.1). Our mouse transplantation model was consistent with a role for β-catenin in tumour outgrowth and invasiveness, suggesting that activation of the β-catenin signaling pathway by an active oncogenic RET mutant can increase the relative rate of tumour growth and permit infiltration across tissue layers. Together, these data suggest that β-catenin nuclear localization may be an important marker of tumour progression or advanced disease in human MTC. Interestingly, in familial adenomatous polyposis patients, inactivating mutations of APC that lead to increased nuclear β-catenin are also associated with

93 Figure 4.16 Proposed model of a RET-β-catenin signalling pathway. β-catenin is constitutively associated with RET at the cell membrane. Upon ligand stimulation of

RET, β-catenin is tyrosine phosphorylated and may dissociate from the membrane.

Tyrosine phosphorylated β-catenin appears to escape cytosolic regulation by the

Axin/GSK3/APC regulatory complex, which degrades free β-catenin, and moves to the nucleus. In the nucleus, β-catenin acts as a transcriptional regulator of genes for growth, and survival that have also been shown to be modulated by RET.

94 94a increased risk for papillary thyroid carcinoma, tumours which are also associated with activating RET mutations [138, 184], suggesting that there could be a role for a RET- mediated upregulation of β-catenin in these tumours as well. Targeting β-catenin pathways in thyroid cancer may in future be another avenue for therapeutic intervention in these diseases. Thus, understanding the cellular mechanisms by which RET activates the β-catenin pathway may provide potentially broad insight into the pathogenesis of multiple thyroid tumour types.

In summary, we have identified a novel RET-β-catenin signalling pathway, which is a critical contributor to enhanced cell proliferation and tumour progression in thyroid cancer. Our studies show that RET induces β-catenin-mediated transcription, cell proliferation, and transformation in vitro and that β-catenin nuclear localization and the resultant RET mediated β-catenin signalling is a key secondary event in tumour growth and spreading in vivo. This novel interaction suggests a mechanism that may underlie the broad and early metastatic potential of MTC. Our data suggest a previously unrecognized role for β-catenin signalling that may have implications for tyrosine kinase-mediated tumourigenesis in multiple tumour types.

4.5 Acknowledgements

We would like to thank Dr. M.R. Canninga (Dept. Pathology, UMC Utrecht) for providing the human MTC material. We are grateful to Drs. J. Woodgett, P. Greer, and J.

MacLeod for providing us with constructs. We would like to thank Drs. S. Peng, P. Greer, and D. LeBrun and N. Kaur for helpful discussion.

95 Chapter 5

Identification and characterization of a novel RET tyrosine kinase inhibitor.

Taranjit S. Gujral1, 2, Vinay K. Singh3, and Lois M. Mulligan1,2* 1Division of Cancer Biology and Genetics, Queen’s Cancer Research Institute, 2Departments of Pathology & Molecular Medicine and 3Biochemistry, Queen’s University, Kingston, ON, Canada. K7L 3N6.

Manuscript in Preparation (Priority Report in Cancer Research)

*To whom correspondence should be addressed Dr. Lois M Mulligan, Department of Pathology and Molecular Medicine, Cancer Research Institute, Botterell Hall Rm 329, Queen’s University, Kingston, ON, Canada K7L 3N6. E-mail: [email protected] Phone: 1 613 533 6000 Ext 77475 Fax: 1 613 533 6830

Keywords: RET, kinase inhibitor, homology modeling, receptor tyrosine kinase

96 5.1 Abstract

Mutations in RET are the single most common, genetic events in the genesis of familial and sporadic thyroid neoplasia. Gain-of-function mutations in RET have been implicated in more than 50% of sporadic medullary thyroid carcinoma (MTC), and all families with multiple endocrine neoplasia type 2 (MEN 2). MEN 2 is caused by either mutations in the extracellular region, or by mutations in the kinase domain of RET, leading to constitutive ligand-independent activation of RET. RET mutations are also found in a significant proportion of adrenal tumours, primarily pheochromocytomas, while increased expression of RET is noted in neuroblastoma, lung tumours, seminomas, and other cancers, making RET an exploitable target for therapeutic intervention. To date, surgery is the only intervention for the treatment of MTC. In this study, we used a structure guided approach to identify and characterize a novel, non-ATP competitive,

RET kinase inhibitor; SW-01. We show that SW-01 provides significant RET inhibition in an in vitro kinase assay using purified RET proteins. Moreover, RET phosphorylation is blocked, or dramatically reduced, in vivo in cells overexpressing active RET. Similarly, stimulation of RET downstream signals is reduced in the presence of SW-01. We observe a significant decrease in cell proliferation and colony formation in RET- expressing cells in the presence of SW-01. Together, our data suggest that SW-01 has potential as a novel RET kinase inhibitor with clinical utility.

97 5.2 Introduction

The RET (REarranged during Transfection) proto-oncogene, which is expressed in neural crest-derived cell lineages, encodes a receptor tyrosine kinase that plays important roles in cell growth, differentiation and survival [1-5]. Under normal cellular conditions,

RET is activated by binding of both glial cell line-derived neurotrophic factor (GDNF) ligands and cell surface bound co-receptors of the GDNF family receptor α (GFRα) proteins [10]. Activation of RET, in turn, leads to autophosphorylation of intracellular tyrosines, which stimulates a cascade of intracellular protein interactions [13].

Gain-of-function mutations in RET have been implicated in more than 50% of sporadic Medullary Thyroid Carcinoma (MTC) and all families with Multiple Endocrine

Neoplasia type 2 (MEN 2) [54], which is characterized by MTC, as well as pheochromocytoma (PC), either with or without hyperparathyroidism. MEN 2 is caused by either mutations in the extracellular region or mutations in the kinase domain of RET leading to constitutive ligand-independent activation of RET. RET mutations are also found in a significant proportion of the adrenal tumour, PC, while increased expression of

RET or it’s ligand (GDNF) is noted in neuroblastoma, lung tumours, pancreatic ductal carcinoma (PDAC) and others [89, 185-188]. In PDAC patients, strong GDNF and RET expression has been correlated with invasion and poor patient survival after surgical resection [89, 185-187], suggesting that RET may be an important prognostic marker for pancreatic cancer and a potential target for anti-invasion therapy. Recently, it has been shown that RET and GFRalpha are overexpressed in a subset of estrogen receptor- positive tumors by screening a tissue microarray of invasive breast tumors [90, 91].

RET/GFRalpha-1 expression has been shown in colorectal cancer cell lines [92].

98 Therapeutic options for the management of RET-associated diseases are frequently limited. For MTC, radiation therapy and chemotherapy are ineffective [93], thus, surgery is the primary management for both sporadic and hereditary MTC. The cure rate of PDAC is significantly less than 25% [189, 190]. The causative role of activated

RET mutants or over-expression makes RET a rational target for possible treatments of these tumor types.

Over the last several years, a few tyrosine kinase inhibitors have been demonstrated to inhibit RET activity [118, 119, 126, 135]. Some of these small molecule antagonists block access to the highly conserved ATP binding site, and thus inhibit a broad spectrum of kinases, including RET [115, 118, 126, 135]. However, some constitutively active mutants of RET are resistant to these inhibitors because of activating mutations of RET within regions that are critical for inhibitor binding [191]. Several small molecules are currently in clinical trials for thyroid cancer [192]. A summary of small molecule inhibitors of the RET kinase is presented in Table 1.1. ZD6474 is a

VEGFR inhibitor, which also has activity against RET (IC50 130 nM)[126-128].

SU11248 (sunitinib), which is an oral small molecule tyrosine kinase inhibitior of

VEGFR, PDGFR, KIT, FLT-3 is also a highly active inhibitor of RET (IC50 224 nM)

[115-117]. In addition, BAY 43–9006 is an oral multikinase inhibitor initially developed as a specific inhibitor of BRAF [131, 132] that also inhibits other kinases including

VEGFR-2, VEGFR-3, PDGFR, c-kit, FLT-3 [133, 134] and RET [135]. Moreover, 17-

AAG is an HSP90 inhibitor that acts indirectly by blocking a key chaperone responsible for RET maturation [193].

As of yet, a small molecule RET inhibitor with high specificity for RET, and with efficacy for all common RET activating mutants, has not been identified. In this study, we

99 have identified and characterized a novel RET kinase inhibitor, which we have called

SW-01 (cyclobenzaprine hydrochloride). We show that SW-01 provides significant RET inhibition in in vitro kinase assays (307nM) using purified RET proteins [94]. Moreover,

RET phosphorylation is blocked or dramatically reduced in vivo in cells overexpressing

RET. We observe a significant decrease in cell proliferation and colony formation in

RET-expressing cells in the presence of SW-01. Together, our data suggest that SW-01 is a novel RET kinase inhibitor with potential clinical utility.

5.3 Results and Discussion

5.3.1 High throughput in silico screening: Identification of SW-01

We have previously developed 3D-models of the RET kinase domain [94], that are in close agreement with predictions based on crystal structures of RET [18] and other related kinases [94]. We used these models to identify the functional effects of activating

RET mutations [94]. We have further used these validated models to perform a virtual screen for small molecules that bind active sites of RET, using a structure-guided docking strategy (FlexX module of SYBYL program) and a diversity set library of small molecule structural information freely available from the Developmental Therapeutics Program at

NCI/NIH, USA (http://dtp.nci.nih.gov/). This diversity set contained 1900 small molecules representing the maximal pharmacophore coverage of the NCI/NIH repository set of more than 140,000 non-discreet synthetic and natural products from many sources.

We identified 238 small molecules that docked to RET at various sites. Following knowledge based filtering, we identified three of these compounds (NSC 10777, 78206,

27476) with predicted binding to the RET activation loop, a region with low homology to other kinases but critical to RET activation, which we considered might provide both effective and specific RET inhibition (Figure 5.1A). The existing small molecule

100 Figure 5.1. High throughput in silico screening: Identification of SW-01. A. A ribbon diagram of the intracellular domain of WT-RET (residues 709-988) in the active conformation is shown. Using a RET structure-guided docking strategy, we identified three compounds (NSC 10777, 78206, 27476) that bind active sites of RET. The three compounds are shown to dock at the activation loop and substrate binding region. In addition, they do not occupy or interfere with the residues in the ATP-binding pocket. B.

Surface view of docked NSC78206 (SW-01) in the substrate binding pocket of RET kinase (left). SW-01 makes contacts with two autophosphorylated tyrosines in the activation loop (Y900 and Y905) of RET (right).

101 A

B

101a inhibitors for RET bind to the ATP-binding segment, thus, activating RET mutants that interfere with the ATP binding (e.g. V804M, V804L) are resistant to inhibition by these molecules [126, 135, 191]. The methodology we have used to identify

RET inhibitors may avoid this problem. We have selected for inhibitors that bind to critical residues within the RET activation loop that are unique to RET and should therefore be highly selective for this kinase (Figure 5.1). These sequences do not include, or directly interact with any residues known to be mutated in activated RET and thus no known RET mutants are predicted to have a priori resistance to our inhibitor.

Next, we evaluated the ability of these compounds to interfere with the activation of RET in vitro. We found that one of these compounds NSC 78206 (SW-01) inhibited

RET autophosphorylation significantly. The surface view of SW-01 docked in the substrate binding pocket of RET is shown in Figure 5.1B. Based on the SW-01-RET model complex, SW-01 makes contacts with two tyrosines in the RET activation loop,

Y900 and Y905 (Figure 5.1B). We predict SW-01 interferes with the mobility of the activation loop, a critical step for kinase activation (Figure 5.1B).

5.3.2 SW-01 inhibits RET autophosphorylation in vitro and in vivo

We further investigated the effect of SW-01 on RET autophosphorylation both in vitro and in cells lines expressing active RET. We found that SW-01, provided significant

RET inhibition in in vitro kinase assays (307 nM) using purified recombinant RET proteins that have previously been functionally validated [94] (Figure 5.2B). We measured the autokinase activity of RET using a phospho-RET antibody, pY905, which recognizes phosphorylation of RET at this residue. Y905 is a primary phosphotyrosine which lies in the activation loop of the kinase and is critical for RET autophosphorylation

102 Figure 5.2. SW-01 inhibits RET autophosphorylation in vitro and in RET expressing cells. A. The chemical structure of SW-01. SW-01 is a tricyclic antidepressant named cyclobenzaprine hydrochloride. B. SW-01 inhibits RET autokinase activity in an in vitro kinase assay. Western blot showing the decrease in the autokinase activity of RET, measured using a phospho-RET antibody (pY905). The same blots were re-probed with pan RET antibody to detect the amount of RET protein C. SW-01 blocks RET activation in HEK293 cells stably expressing RET. Western blots of samples from HEK293 cells treated with DMSO control or increasing concentrations of SW-01, show inhibition of

RET autophosphorylation. The same blot was re-probed with pan-RET antibody to measure the amount of total RET. D. SW-01 blocks RET-mediated activation of ERK without affecting its expression levels. Western blots of whole cell lysates from HEK293 cells treated with increasing concentrations of SW-01 (1-20 µM), immunoblotted with

RET, ERK and pERK antibodies as indicated. The amount of pERK is drastically reduced with increased SW-01, while the amount of RET and ERK remain the same.

103 A

SW-01 (Cyclobenzaprine hydrochloride) B SW-01 (μM)

C

SW-01 (μM)

D SW-01

103a and subsequent activation [18]. The phosphorylation of RET was also measured using generic phosphotyrosine antibody (pY99) (data not shown). The same blots were re- probed with a pan-RET antibody to detect the amount of RET protein in order to show that SW-01 does not affect RET protein expression (Figure 5.2B). Thus, SW-01 does not affect the expression levels of RET but inhibits its autokinase activity.

To evaluate the efficacy of SW-01 in vivo, HEK293 cells over-expressing activated RET were treated with varying concentrations of SW-01 for 2 hours. We found that RET phosphorylation was blocked, or dramatically reduced upon SW-01 treatment in a concentration-dependent manner (Figure 5.2C). Also, SW-01 did not affect expression levels of RET, as shown. Thus, we identified SW-01 as a novel RET inhibitor which blocks its auto-kinase activity both in vitro and in cells expressing activated RET.

5.3.3 Effects of SW-01 on RET-mediated downstream signalling, cellular growth, proliferation and transformation

Upon activation, RET transduces downstream signals through phosphorylation of multiple intracellular tyrosines, and has been shown to activate RAS-ERK, JNK, PI3K, p38MAPK, SRC and ERK5 signalling pathways [9]. These signalling pathways result in cell proliferation, cell differentiation, or transformation [194, 195]. We therefore investigated the effect of SW-01 on these downstream signalling pathways. Our data show that SW-01 blocks RET-mediated activation of ERK without affecting its expression levels (Figure 5.2 D). However, parental HEK293 cells, which do not express

RET, treated with similar concentration of SW-01 did not show any effect of ERK or

AKT pathways (data not shown). This indicates that the inhibition of ERK pathway is

RET-dependent (Table 5.1).

104 Table 5.1. Selectivity of SW-01. We evaluated the effect of SW-01 on other kinases. We found that even at 10µM, SW-01 does not inhibit the MET, and c-KIT receptor tyrosine kinases, FPS/FER, and SRC cytoplasmic tyrosine kinases and AKT, ERK, MEK1/2 serine/threonine kinases. “++” indicates inhibition of activity by more than 50% measure by phospho-specifc antibodies, “--“ indicates no significant inhibition was observed upon

SW-01 treatment.

105 Receptor Tyrosine Kinase

Cytoplasmic Tyrosine Kinase

Cytoplasmic Ser/Thr Kinase

105a We next compared the cell proliferation and transformation that can be induced by

RET in the absence or presence of varying concentrations of SW-01 (Figure 5.3). A decrease in RET- induced cell proliferation and colony formation were shown using the

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation and soft agar anchorage-independent growth assays, respectively (Figures 5.3A and B).

Inhibition of both cell proliferation and transformation was proportional to the decrease in kinase activity of RET upon treatment with SW-01. Further, treatment with SW-01 for

24 hours induced morphological changes including cell rounding in TT cells which are known to be dependent on oncogenic RET [196] (Figure 5.3C). Higher concentrations of

SW-01 resulted in alterations of cell-to-cell and cell-matrix adhesions indicating possible cell death or growth arrest. Thus, SW-01 not only blocks RET auto-kinase activity, but also RET-mediated downstream signalling and biological effects of RET activation.

We next investigated whether RET inhibition by SW-01 can cause apoptosis. We used multiple cell model systems including embryonic kidney cells (HEK293), thyroid cancer cells (TT) and a neuroblastoma SKNBE (2) cell line. Using propidium iodide staining and fluorescence activated cell sorting (FACS) analysis, our data show that there was an increase in subG1 population of cells, which indicates the percentage of apoptotic cells in the presence of SW-01. We found that inhibition of RET autophosphorylation by

SW-01 leads to apoptosis in TT cells, but not in HEK293, and SKNBE (2) cells expressing RET (Figure 5.4). This is consistent with the fact that TT cell survival is

RET-dependent [196]. Together, our data suggest that SW-01 may have potential as a novel RET kinase inhibitor.

106 Figure 5.3. Effects of SW-01 on RET-mediated cellular proliferation and transformation. A. SW-01 blocks RET-mediated cell proliferation in HEK293 cells.

HEK293 cells stably expressing RET were treated with SW-01 or DMSO control, and cell proliferation was measured by MTT assays. The data are presented relative to DMSO control. Means of at least three replicates ±sd are shown. Statistical significance was calculated by one-way ANOVA. Asterick (*) indicates statistical significance (p<0.05).

B. SW-01 inhibits RET-mediated colony formation in soft agar assays. A relative decrease in RET-induced colony formation was also shown in soft agar anchorage- independent assays. Colony formation was assessed relative to DMSO control in a minimum of three replicate experiments. Statistical significance was calculated by one- way ANOVA. Asterick (*) indicates statistical significance (p<0.05). C. Morphological changes in TT cells caused by treatment with SW-01. TT cells treated with DMSO or varying concentration of SW-01 for 24 hours (10X) magnification show cell rounding and decreased cell-cell contact.

107 A

B

C

DMSO 1 μm 2 μm

107a 5 μm 10 μm 20 μm Figure 5.4. SW-01 causes apoptosis in medullary thyroid carcinoma cells. The representative graph of percentage apoptosis measured using propidium iodide staining and FACS analysis in multiple cell model systems show that inhibition of RET autophosphorylation by SW-01 leads to apoptosis in TT cells, but not in HEK293, and

SKNBE (2) cells expressing RET.

108 40 HEK 293 35 SKNBE(2) 30 TT 25 20 15 10 % Apoptosis 5 0 012510 [SW-01] uM

108a 5.3.4 SW-01 impairs RET-mediated migration of pancreatic cancer cells

RET ligand (GDNF) has been shown to induce pancreatic cell invasiveness in cell based system and in vivo [89, 185-187]. We next compared the cell migration that can be induced by RET activation in the absence or presence of SW-01 in a wound healing assay

(Figure 5.5). In this assay, cell monolayers, when wounded or scratched, respond to the disruption of cell-cell contacts by healing the wound through a combination of cell proliferation and cell migration [197, 198]. Our data show that GDNF induction caused an increase in the rate of cell migration of MiaPaca2 pancreatic cancer cells. However, this was compromised when the same cells were treated with SW-01 (Figure 5.5). When treated with SW-01, even in the presence of GDNF, MiaPaca2 cells migrated at a similar speed to untreated cells. This suggests that SW-01 impairs the ability of pancreatic cells to migrate in response to GDNF.

In conclusion, we have identified a novel RET kinase inhibitor, SW-01. Our structural data suggest that SW-01 is non-ATP competitive and binds in the activation region of the RET kinase. We show that SW-01 inhibits RET autophosphorylation and blocks RET-mediated growth and transformation and migration in multiple cell model systems. The preclinical findings reported here suggest that SW-01 offers a potential treatment strategy for multiple tumour types.

109 Figure 5.5. SW-01 impairs the ability of MiaPaca2 pancreatic cancer cells to migrate in wound healing assays. A. Representative images of the wound diameter acquired at 0,

2, 12 and 24 hours in the wound healing assay are shown. MiaPaca2 cells were either un- induced (top), or induced with GDNF (middle). GDNF induced cells were also treated with SW-01 (bottom). B. A graph of wound closure over time. Images collected from six different wound diameter were quantified. Cells growing in the presence of GDNF showed faster migration than in the absence of GDNF. The diameter of wound space remaining over time indicates that cell migration was inhibited in the presence of GDNF and SW-01, as compared to cell migration in the presence of GDNF alone. Percentages calculated from at least five replicates ±sd are shown. Statistical significance was calculated by one-way ANOVA. Asterick (*) denotes statistical significance (p<0.05).

110 A

B

* * * *

110a 5.4 Materials and Methods

5.4.1 Small-molecule libraries

Small-molecule libraries used in this study were from the NCI/NIH [199]. The structural diversity set (1,990 molecules) represents the structural diversity of 140,000 molecules. Detailed information about the composition of these molecules is available elsewhere [199]. Inhibitor analogues tested in this study were provided by the Drug

Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of

Cancer Treatment and Diagnosis (NCI) database (Bethesda, MD).

5.4.2 In-vitro kinase kinetics

Approximately 5-20 µg of purified GST-RET proteins were incubated with 1 mM

o ATP in 20 µl kinase buffer (10 mM Tris, 5 mM MgCl2, pH 7.4), at 30 C for 30 minutes.

The kinase reactions were terminated by boiling the samples in SDS-PAGE sample buffer

(20 mM Tris-HCl [pH 7.8], 150 mM NaCl, 1 mM sodium orthovanadate, 1% Igepal, 2 mM EDTA, 1 mM PMSF, 10 µg/ml aprotonin, and 10 µg/ml leupeptin). Samples were resolved on 10% acrylamide gels by SDS-PAGE followed by western blotting.

Phosphorylation was detected using a pan-phosphotyrosine (pY99) antibody or RET phospho-specific antibody (pY905) (Cell Signaling, Beverly, MA).

5.4.3 Cell culture

TT and Miapaca2 cells were grown in Dulbecco’s Modified Eagle’s Medium

(DMEM) (Invitrogen), supplemented with 10% fetal bovine serum (FBS) (Sigma,

Oakville, ON). The human neuroblastoma cell line SKNBE(2) was maintained in RPMI

1640 medium supplemented with 10% (FBS). HEK293 cells expressing the reverse- tetracycline transcriptional activator (Tet-on)(BD Biosciences, Mississauga, ON) and intracellular (ic)RET expression constructs (previously described [142]) were grown in

111 DMEM supplemented with 10% FBS. To induce expression of icRET in HEK293 Tet-on cells, media was further supplemented with 1 µg/ml doxycycline. 1 µM AP20187 dimerizer (ARIAD, Cambridge, MA) was added to induce intracellular RET (icRET) dimerization 30 minutes before harvesting.

5.4.4 Immunoprecipitations and western blotting

Total protein lysates were harvested 48 hours after transfection and suspended in

20 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 mM sodium orthovanadate, 1% Igepal, 2 mM EDTA, 1 mM PMSF, 10 µg/ml aprotonin, and 10 µg/ml leupeptin [143]. Protein assays were carried out using the BCA protein assay kit (Pierce, Rockford, IL) according to manufacturer’s instructions.

RET expression was detected using the C-19 antibody (Santa Cruz Biotechnology,

Santa Cruz CA) and RET tyrosine phosphorylation was detected using an anti- phosphoRET antibody (pY905) (Cell Signaling, Beverly, MA) that specifically recognizes phosphorylation of primary tyrosine residue Y905 [21]. For immunoprecipitations, lysates were incubated with a 1:50 dilution of the appropriate primary antibody with agitation for 2 hours at 4°C, mixed with Protein AG (Santa Cruz) and incubated on ice for 2 hours, with shaking. Immunoprecipitates were pelleted, washed, and resuspended in SDS-PAGE sample buffer. Samples were denatured, separated on 10% SDS-PAGE gels and transferred to nitocellulose membranes (Bio-Rad,

Mississauga, ON) [143].

5.4.5 MTT cell proliferation assay

MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assays were performed according to the method described in [175]. Briefly, HEK293 cells

112 stably expressing WT-RET, were seeded in 6-well plates. One half of the cells expressing

WT-RET were treated with inhibitors for the time periods indicated. All cells were subsequently treated with 100 ng/ml GDNF (Promega, Madison, WI) and seeded into a

96 well plates. After 3 days of incubation under normal culture conditions, MTT was added at a final concentration of 250 µg/ml (0.6 mM) and incubated for another 2 h at

37°C. The mitochondria of viable cells can reduce MTT to formazan, which was dissolved with 150 µl DMSO. Reduced MTT was then measured spectrophotometrically in a microtiter plate reader at 570 nm. The statistical significance was calculated by one- way ANOVA.

5.4.6 Soft Agar Colony Formation Assay

Soft agar colony formation assays were performed as described previously [94,

200]. Briefly, RET expressing HEK293 cells were treated with DMSO or inhibitors for the time indicated. Approximately 5 x 104 cells were resuspended in 0.2% top agar in medium, and plated on 0.4% bottom agar in medium. Culture medium was supplemented to the top layer every 2 to 3 days. Colonies were counted after 2 weeks, and statistical significance was confirmed by one-way ANOVA.

5.4.7 Apoptosis assay

Cells were cultured, and treated with DMSO or inhibitors for the time period indicated. After several washes of PBS, cells were harvested, and 1-2 x 106 cells were resuspended in 1mL of PBS. The cells were then fixed in absolute ethanol, treated with

20 µL RNaseA and incubated at 37oC overnight. 100µg propidium iodide was added, and samples were incubated for 15 minutes at room temperature. Cell cycle analysis was performed using an EPICS ALTRA HSS flow cytometer (Beckman Coulter, Mississauga

ON).

113 5.4.8 Wound healing assay

Cell migration was measured by the scratch wound healing assay [201]. Cells were grown to confluent monolayers in 35mm plastic dishes and scratched with a sterile syringe needle. The cell migration was evaluated in the presence of or absence of GDNF

(100 ng/ml) and SW-01 by imaging the wound diameter every hour until wound closure.

114 Chapter 6

DISCUSSION

Activating mutations, aberrant up-regulation or rearrangements in the RET proto- oncogene have been implicated in many tumour types [54, 89, 185-188]. Understanding the biological and biochemical mechanisms by which such changes result in altered function is essential to our ability to predict and develop anti-cancer therapies.

Unequivocally, since its discovery, the RET kinase has been the focal point for developing targeted therapies for the treatment of these diseases. To design novel RET based therapeutic options, it is essential to understand the molecular mechanisms of RET- mediated oncogenesis and RET-mediated downstream signalling.

6.1 Molecular mechanisms of RET-mediated oncogenesis

To understand the oncogenesis mediated by an activating mutation in the RET proto-oncogene, it is critical to identify the biochemical and biophysical changes in the

RET protein caused by such mutations. We have shown experimentally that the M918T mutation partially activates the RET receptor, thereby causing constitutive activation [94]

. Our experimental data suggest that 2B-RET causes a conformation change in RET leading to a more stabilized active form that is intermediate between inactive WT and active WT-RET forms (Table 3.2, Figure 3.7). According to our micro-calorimetry data,

2B-RET has increased affinity for ATP, which could be associated with the “positive” conformational changes caused by the M918T mutation (Table 3.1, Figure 3.4). Our data suggest a previously unrecognized mechanism that may explain the consistently higher levels of phosphorylation of 2B-RET than WT, which has been recognized in our lab and others [75, 94, 202].

115 Although, the M918T mutation accounts for most cases of MEN 2B, there are other mutations reported in the RET kinase that cause MEN 2B [64, 65] . In 2%–3% of patients with MEN 2B, the “genotype” A883F can be found [64, 65]. Recently, a double mutation at codons 804 and 806 (V804M/Y806C) in cis was reported [66]. In addition, a report of patients presenting with “atypical” MEN 2B harboring a germline double point mutation in the RET proto-oncogene in cis (V804M and S904C) has also been published

[203]. Although the clinical manifestations associated with all of these mutations are similar, little attempt has been made to investigate this similarity at the molecular level.

Now that we understand the molecular mechanism of M918T-harbouring RET, it will be interesting to study the structural- functional effects of other MEN 2B-causing RET mutations.

Structurally, it has been suggested that A883F (between the N- and C-lobes), and

V804M/Y806C (within the linker region), are likely to affect interlobe flexibility, which may favor maintenance of an active RET kinase conformation [18]. Based on our 3D homology modeling data [53], and available crystal structures [18] as well as our functional data, it is plausible that all MEN 2B-causing RET mutations affect the local structure to alter its autoinhibition and stabilize RET in its active state. Table 6.1 summarizes our predicted structural effects of RET mutations causing MEN 2B. Thus, the more aggressive phenotype of MEN 2B may be directly correlated with the increased intrinsic RET-kinase activity caused by these mutations.

In a preliminary study, we have investigated the functional effects of all the above

MEN 2B-causing RET mutations. Our data show that all MEN 2B RET proteins are more phosphorylated than WT-RET [202] (data not shown). The increased autophosphorylation of these proteins translates to increased substrate binding as seen with M918T RET. This

116

117 suggests that all MEN 2B-causing RET mutations alter its function towards more activated forms with possibly enhanced intrinsic kinase activity. It would also be interesting to conduct biophysical and kinetic analyses of all MEN 2B RET proteins to investigate if all the other MEN 2B-causing RET mutations also lead to increases in their affinity for ATP, as seen with M918T RET. If all MEN 2B-causing RET mutants share similar properties functionally, distinct forms of treatment for MEN 2B patients can be suggested, especially MTC patients displaying metastasis in distant organs.

As we gradually understand the structure-function alterations in the RET protein caused by activating mutations, we can begin to design novel therapeutics, aiming to target only the mutant RET and leaving WT-RET alone. Since 2B-RET proteins are intrinsically more active proteins, it would require more potent or higher concentrations small molecule inhibitors to quench 2B-RET autophosphorylation compared to WT-RET.

Alternatively, if all MEN 2B-causing RET mutations cause local conformational changes, then a small molecule inhibitor which can interfere with these changes might selectively block 2B-RET. Our data also showed that the 2B-RET mutant was able to form higher molecular weight complexes in the absence of ligand stimulation (Figure 3.5, Appendix

1). Thus, a small agent which can block the formation of these complexes might selectively inhibit activation of 2B-RET.

Although RET kinase is a potential target for small molecule inhibitors, targeting

RET-mediated downstream signalling is also an option to specifically inhibit RET- mediated oncogenesis.

118 6.2 RET-mediated downstream signalling

RET, upon activation, plays a critical role in several intracellular signalling cascades such as RAS/ERK, ERK5, p38MAPK, PLCγ, PI3-kinase, JNK, STAT, and SRC cascades (Reviewed [9]) (Figure 1.3). These well characterized pathways regulate RET- mediated cell survival, differentiation, proliferation, migration, and chemotaxis

(Reviewed in [137]). In addition, several studies have examined the expression of E- cadherin/ β-catenin in thyroid tumours [168, 204-206]. It has recently been suggested that multiple signalling pathways including RAS/ERK, PI3K/AKT, and the peroxisome proliferation activated receptor-gamma (PPARγ) pathways converge at β-catenin in thyroid cancer [137], however the mechanism of activation of β-catenin has now been partially elucidated (Chapter 4) [200].

We have identified a novel RET-β-catenin signalling pathway, which is a critical contributor to enhanced cell proliferation and tumour progression in medullary thyroid cancer [200]. Our data suggest a previously unrecognized role for β-catenin signalling that may have implications for tyrosine kinase-mediated tumourigenesis in multiple tumour types.

We have shown that RET and β-catenin can interact directly in vitro and in vivo

(Figure 4.3B, 4.4). Although RET is known to interact with SH2 or PTB domains containing proteins, how it interacts with an ARM repeat containing protein like β-catenin directly still needs to be elucidated. In order to characterize these interactions further, it is crucial to identify the specific residues involved in RET-β-catenin interactions. In our preliminary studies, using N- or C-terminal deletion constructs of β-catenin [172] we showed that RET interacts with N-terminal ARM domains, while our control E-cadherin requires the presence of more C-terminal repeats (Figure 6.1). In RET, we have shown

119 Figure 6.1 Characterization of RET-β-catenin-E-cadherin interaction. A. Schematic showing the domain structure of β-catenin. Known phosphotyrosines and regions required for binding of interacting proteins on β-catenin are indicated. For functional studies, we used 1-12 ARM and 1-6 ARM domain β-catenin deletion proteins. B. RET interacts with the N-terminal region of β-catenin whereas E-cadherin binds to the C-terminal region.

Using deletion mutants of β-catenin, we show that ARM domains 1-6 can bind RET but not to E-cadherin in a GST-pull down assay. C. RET associates with E-cadherin. HEK

293 cells expressing RET or empty vector were harvested and immunoprecipitated (IP) with E-cadherin. Our data shows that RET can be co-immunoprecipitated with E- cadherin.

120 A

B C

120a that the β-catenin binding region(s) lie within the intracellular domain (amino acids 665-

1061) (Figure 4.3B, 4.4). Once a minimal critical region of RET-β-catenin interaction is established, we can generate site-specific mutants in functional analyses to dissect the role of β-catenin in these RET-mediated cellular proliferation, transformation and tumour formation.

Further, in addition to RET, β-catenin has been shown to associate with the

EGFR, c-Erb-2, or MET receptor kinases and β-catenin is tyrosine phosphorylated in cells stimulated by ligands for these receptors [207, 208]. However, EGFR is the only receptor known to directly phosphorylate β-catenin [178]. If a region of interaction is mapped on the RET kinase, the existence or conservation of this region in other kinases is worth exploring. Thus, the activation of β-catenin signalling pathway may be a shared downstream event among various kinases. The broader RTK-β-catenin pathway may play distinct roles that may depend upon the expression of kinases involved specific to the tissue types. This may suggest a cross-talk between RTKs and proteins in adhesion complexes regulating various cellular processes.

The formation and regulation of cadherin-catenin adhesion complexes is critical to normal cell-cell interactions and the maintenance of tissue integrity. Dissociation from these complexes is an important step in the epithelial mesenchymal transition that underlies the metastatic process [209]. However, it has been recently been shown that the disruption of cell-cell contacts alone does not enable metastasis [210]. Further, it has been reported that β-catenin is necessary, but not sufficient, for induction of metastatic phenotype [210]. In addition to the disruption of E-cadherin-mediated cell-cell contacts, induction of multiple transcription factors, such as, Twist, is necessary for E-cadherin loss–induced metastasis [210].

121 Our data shows that RET can directly phosphorylate β-catenin, leading to its loss from adhesion complexes and that this correlates with an increase in RET-mediated proliferation, and transformation (Figure 4.8). However, in view of the above mentioned recent findings, the loss of β-catenin from adhesion complexes might not be the only event upon RET activation contributing to tumourigenesis. Whether β-catenin contributes to RET-mediated tumour formation due to loss of β-catenin from cell-cell interaction complexes or due to an increase in an oncogenic transcription profile of β-catenin still needs to be elucidated.

The role of RET-β-catenin interaction in the catenin-cadherin adhesion complex is also unclear. It is also unclear whether interaction of RET and β-catenin is necessary or sufficient for RET to associate with the catenin-cadherin complex. Our preliminary results show that RET may also associate, either directly or indirectly with E-cadherin

(Figure 6.1). Our data, and the literature, suggest that RET, like MET and EGFR, may inhibit the catenin-cadherin adhesion complex or be closely associated with it, acting as a mechanism of regulating phosphorylation levels of the complex [167]. To address this, it is essential to characterize interactions between RET and the major component proteins of the adhesion complex that have been previously shown to be targets of tyrosine phosphorylation (β-catenin E- cadherin, p120catenin, α-catenin). It is plausible that RET can phosphorylate some or all of the proteins in this complex directly or indirectly.

Perhaps RET-mediated phosphorylation of these proteins can contribute to alteration of cell-cell adhesion which may play a role in RET-mediated tumorigenesis.

As described above, oncogenic activation of receptor tyrosine kinases provides important targets for directed therapies. Since the activation of a RET-mediated β-catenin signalling pathway contributes to cellular proliferation and invasion in tumours, targeting

122 this pathway provides an option for the development of novel therapeutics. Targeting the

RET-β-catenin signalling pathway selectively may provide a better treatment strategy than more promiscuous signalling pathways such as RAS/ERK or PI3K/AKT pathways.

A RET-β-catenin signalling pathway provides more precise targets through either agents which disrupt tyrosine phosphorylation of β-catenin or agents which disrupt nuclear translocation of β-catenin, thereby blocking the β-catenin-mediated transcription profile.

There are several small molecule inhibitors of β-catenin signalling which show promising antiproliferative effects in colon, liver, and lung cancer cells [211-213]. These agents may in future have potential for the treatment of RET-associated phenotypes, such as MTC.

We found that β-catenin expression in the nucleus is associated with metastatic cases of MEN 2, suggesting β-catenin nuclear localization is a late-stage event. It is plausible that the nuclear expression of β-catenin could be a prognostic marker in thyroid cancer. Similar correlation has been seen previously in breast and pancreatic cancers

[214, 215]. Thus, RET- β-catenin signalling confers not only a novel therapeutic target but also a prognostic marker for the management of MEN 2.

6.3 RET as a therapeutic target

As we gradually understand the mechanisms of RET activation and its downstream signalling, there is a potential for developing novel therapeutic strategies targeting RET-mediated downstream signalling. One of the approaches, is targeting the

RET kinase itself for therapeutic purposes. Currently, there are at least seven small molecule inhibitors of RET which are in various phases of clinical evaluation for RET- mediated tumour types [192], but none of these molecules are RET-specific (Table 1.1).

Majority of these are ATP-competitive small molecule inhibitors. Not surprisingly, RET can be inhibited by small molecule antagonists that block access to the ATP-binding site,

123 a highly conserved domain in many kinases (Figure 6.2). As a result, constitutively activating mutants of the RET “gatekeeper residue” (eg V804M) in the ATP-binding site, are resistant to these inhibitors [191].

Compounds such as Sunitinib, Sorafenib, and ZD6474 can block a broad spectrum of kinases, including RET (Table 1.1). These molecules have objective response rates of

20-30% in clinical trials for thyroid cancer [216]. These broad spectrum kinases have also been linked to increased cardiac toxicity [217]. As yet, a small molecule RET inhibitor with high specificity for RET, efficacy for all common RET activating mutants, and low levels of associated toxicities has not been identified.

We have identified a novel RET kinase inhibitor, which we have called SW-01

(Cyclobenzaprine). To increase the specificity of interactions, and avoid the highly conserved ATP-binding region, we selected compounds with predicted binding to the

RET activation loop, that we considered might provide effective RET inhibition (Figure

5.1). As this region has only 30-54% homology with other similar tyrosine kinases we predicted that compounds binding in this region would have much better selectivity than those binding to the ATP binding site (63-75% homology) (Figure 6.2). Treatment of thyroid or pancreatic carcinoma cell lines with SW-01 induced morphological changes including cell rounding and decreased cell-cell contact, and led to a significant decrease in cell growth and proliferation (Figures 5.3, 5.5). Together, our data suggested that SW-

01 has clinical potential as a RET kinase inhibitor and that further preclinical characterization is required to assess its value.

SW-01 is a well-characterized compound that is currently prescribed for muscle spasms and acute muscle pain (Figure 6.3) [218]. It has been used in humans for over 30 years and thus its safety and efficacy have been extensively documented in clinical trials

124 Figure 6.2 Structure based inhibition of kinases. Ribbon diagram of RET tyrosine kinase domain (left). The Majority of the small molecule inhibitors of kinases bind in the

ATP binding pocket which is highly conserved among kinases (upper right). We targeted the activation loop or /and substrate binding pocket which are less conserved among kinases and has function specific roles. Targeting these regions in theory will lead to identification of compounds with better selectivity. Orange: ATP binding region, Red:

Catalytic loop, Green: Activation loop, Yellow: Substrate binding pocket.

125 125a

Figure 6.3 Chemical and pharmacological properties of cyclobenzaprine.

Cyclobenzaprine is a tricyclic antidepressant and muscle relaxant. The toxicity profile in mice has been well characterized [219].

126 SW-01 Name Cyclobenzaprine

Classification •Antidepressive agents, tricyclic •Muscle relaxants, central •Tranquilizing agents Formula C20-H21-N Toxicity

Organism Test Type Route Reported Dose (Normalized Dose)

mouse LD50 intraperitoneal 90mg/kg (90mg/kg)

mouse LD50 intravenous 36mg/kg (36mg/kg)

mouse LD50 oral 250mg/kg (250mg/kg)

126a and in the larger general population [220]. As a result of its long-term use for other pathologies, the risk of adverse effects associated with SW-01 for RET-associated phenotypes is relatively low. This offers a tremendous competitive advantage over other broad-spectrum RET inhibitors that are in early clinical trials since these compounds may yet demonstrate unexpected toxic side effects. Further, a PubChem

(http://pubchem.ncbi.nlm.nih.gov/) database search identified 109 family member compounds with similar structures to SW-01. Although the IC50 of SW-01 both in vitro and in cell based systems is in lower micro-molar range, it can be further improved using structure-based systematic drug design.

Although we have shown that SW-01 blocks RET activity both in in vitro assays and in cell based readouts for cell proliferation, apoptotic cell death and cell migration

(Figure 5.3, 5.5), the possibility of impact of SW-01 on tumour growth or metastasis in in vivo models has not been addressed. Based on data in the NCI in vivo screening database,

SW-01 was not efficacious in a melanoma and two leukemia cancer mouse models

(http://dtp.nci.nih.gov/docs/invivo/invivoscreen.html). It is not surprising that these tumours did not respond to SW-01 since they do not generally express RET [221, 222].

Thus, to address the role of SW-01 in vivo, the effect of SW-01 on the growth of the MTC derived TT cells in a nude mouse tumor formation assay should be investigated.

This line provides the best-characterized model for evaluating RET inhibitors in vivo and has been the primary model used to evaluate other potential inhibitors tested in MTC

[123, 135]. Further, the effect of RET-inhibition on metastasis of pancreatic carcinoma cell lines can also be evaluated to further validate the role of RET in progression of pancreatic cancer.

127 In addition to testing SW-01 as a primary therapeutic agent in thyroid and pancreatic cancer models, the potential role of SW-01 as an adjuvant to other therapies in these diseases can also be evaluated. There are currently no curative systemic treatments for MTC, although several small molecule inhibitors of RTKs are currently undergoing phase II trials (ZD6474, Sunitinib) [192]. Standard of care in pancreatic cancer is treatment with gemcitibine, although adjuvant therapies with this have been largely ineffective in improving overall survival [223]. The effects of combinatorial treatment with SW-01 with irinotecan, a topoisomerase inhibitor which has been effectively used in combination with other chemotherapeutics and which has been shown to increase RET inhibition by another kinase inhibitor CEP-751 [224] and with gemcitibine in cell based assays can be investigated. Now that we better understand RET-mediated downstream signalling, a combination of SW-01 and a β-catenin inhibitor can potentially also offer enhanced effect than single agent alone.

128 Summary

To dissect the molecular mechanisms of MEN 2B, using various biophysical methodologies, we showed that the M918T mutation causes a 10-fold increase in ATP binding affinity, and leads to a more stable receptor-ATP complex, relative to the wildtype receptor. Further, 2B-RET can dimerize and become autophosphorylated in the absence of ligand stimulation. Together, these suggest that multiple distinct but complementary molecular mechanisms underlie the MEN 2B phenotype and provide potential targets for effective therapeutics for this disease.

Further, to understand the dissemination of RET-mediated oncogenic signals in the cells, we identified a novel β-catenin-RET kinase signaling pathway which is believed to drive the development and metastasis of human thyroid carcinoma. We showed that

RET binds to, and tyrosine phosphorylates, β-catenin causing its dissociation from cell membrane. As a result of RET-mediated tyrosine phosphorylation, β-catenin escapes cytosolic downregulation by the APC/Axin/GSK3 complex and accumulates in the nucleus, where it can stimulate β-catenin-specific transcriptional programs in a RET- dependent fashion. We show that downregulation of β-catenin activity decreases RET- mediated cell proliferation, colony formation, and tumour growth in nude mice. This pathway provides an additional target for therapeutics for the treatment of RET-mediated tumorigenesis.

Understanding the biological and biochemical mechanisms by which mutations alter structure and function of RET has enhanced our ability to predict and develop anti- cancer therapies. We used a structure-guided approach to identify and characterize a novel, non-ATP competitive, RET kinase inhibitor; SW-01. We show that SW-01 provides significant RET inhibition in an in vitro kinase assay using purified RET

129 proteins. Moreover, RET phosphorylation is blocked, in vivo in cells overexpressing active RET. We observe a significant decrease in cell proliferation and colony formation in RET-expressing cells in the presence of SW-01. Together, our data suggest that SW-01 has potential as a novel RET kinase inhibitor with clinical utility.

Thus, the research presented here provided significant insight into the molecular mechanisms of RET-mediated oncogenesis in MEN 2B, and RET-mediated downstream signalling via RET-β-catenin pathway. Further, an identification and characterization of a novel small molecule inhibitor for RET kinase provides potential for therapeutics.

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142 216. Ball, D.W., Medullary thyroid cancer: therapeutic targets and molecular markers. Curr Opin Oncol, 2007. 19(1): p. 18-23. 217. Strevel, E.L., D.J. Ing, and L.L. Siu, Molecularly targeted oncology therapeutics and prolongation of the QT interval. J Clin Oncol, 2007. 25(22): p. 3362-71. 218. Katz, W.A. and J. Dube, Cyclobenzaprine in the treatment of acute muscle spasm: review of a decade of clinical experience. Clin Ther, 1988. 10(2): p. 216-28. 219. Stewart, J., M.P. Charest, and F. Herr, A Pharmacological Investigation Of Potential Antidepressants Of The -Type. J Med Chem, 1963. 6: p. 338-9. 220. Spiller, H.A., et al., Five-year multicenter retrospective review of cyclobenzaprine toxicity. J Emerg Med, 1995. 13(6): p. 781-5. 221. Takaya, K., et al., Expression of the RET proto-oncogene in normal human tissues, pheochromocytomas, and other tumors of neural crest origin. J Mol Med, 1996. 74(10): p. 617-21. 222. Nakamura, T., et al., Expression of the ret proto-oncogene product in human normal and neoplastic tissues of neural crest origin. J Pathol, 1994. 172: p. 255- 260. 223. Li, D., et al., Pancreatic cancer. Lancet, 2004. 363(9414): p. 1049-57. 224. Strock, C.J., et al., Activity of irinotecan and the tyrosine kinase inhibitor CEP- 751 in medullary thyroid cancer. J Clin Endocrinol Metab, 2006. 91(1): p. 79-84.

143 Appendix I

Molecular Mechanisms of RET Receptor Mediated Oncogenesis in Multiple Endocrine

Neoplasia 2B

Gujral et al.,Cancer Res. 2006; 66(22):10741-9

Taranjit S. Gujral1, Vinay K. Singh2, Zongchao Jia2, Lois M. Mulligan1,*

Departments of Pathology1, & Biochemistry2, Queen's University, Kingston, ON, Canada

K7L 3N6

*To whom correspondence should be addressed Dr Lois M Mulligan Cancer Research Institute Botterell Hall Rm 329 Queen's University Kingston ON Canada K7L 3N6 E-mail [email protected] Phone 1 613 533 6000 X77475 Fax 1 613 533 6830

Running Title: Molecular Mechanisms of MEN 2B

Keywords: MEN 2B, oncogene, receptor tyrosine kinase, RET

144 Research Article

Molecular Mechanisms of RET Receptor–Mediated Oncogenesis in Multiple Endocrine Neoplasia 2B

Taranjit S. Gujral,1 Vinay K. Singh,2 Zongchao Jia,2 and Lois M. Mulligan1

Departments of 1Pathology and 2Biochemistry, Queen’s University, Kingston, Ontario, Canada

Abstract including marfanoid habitus, mucosal neuromas, ganglioneuroma- tosis of the intestinal tract, and myelinated corneal nerves (3). Multiple endocrine neoplasia 2B (MEN 2B) is an inherited Morbidity and early mortality due to MEN 2B are very high. syndrome of early onset endocrine tumors and developmental Management of both MEN 2B and sporadic medullary thyroid anomalies. The disease is caused primarily by a methionine to carcinoma is by surgical intervention. Treatment is complicated, as threonine substitution of residue 918 in the kinase domain of medullary thyroid carcinoma is prone to metastasis and is often the RET receptor (2B-RET); however, the molecular mecha- refractory to both radiation and chemotherapy (4). Novel strategies, nisms that lead to the disease phenotype are unclear. In this such as kinase inhibitors and small molecules, have as yet largely study, we show that the M918T mutation causes a 10-fold lacked specificity or efficacy at biologically tolerated dosages (1). increase in ATP binding affinity and leads to a more stable Despite early genetic identification, ‘‘cure’’ is reported in <20% of receptor-ATP complex, relative to the wild-type receptor. MEN 2B cases (3). Further, the M918T mutation alters local protein conforma- Although all MEN 2 subtypes arise from mutations of RET, there tion, correlating with a partial loss of RET kinase auto- are strong genotype/phenotype associations with specific muta- inhibition. Finally, we show that 2B-RET can dimerize and tions identified in each disease subtype. MEN 2A and familial become autophosphorylated in the absence of ligand stimu- medullary thyroid carcinoma mutations are primarily substitutions lation. Our data suggest that multiple distinct but comple- of one of several cysteine residues in the RET extracellular domain mentary molecular mechanisms underlie the MEN 2B (2). More than 95% of MEN 2B cases are caused by a single germ- phenotype and provide potential targets for effective thera- line mutation that results in substitution of a threonine for the peutics for this disease. (Cancer Res 2006; 66(22): 10741-9) normal methionine at residue 918 (M918T) in the RET kinase domain (5, 6). The same mutation occurs somatically in 50% to Introduction 70% of sporadic medullary thyroid carcinoma, where it can be The RET (rearranged in transfection) proto-oncogene encodes a associated with more aggressive disease and poor prognosis (7). receptor tyrosine kinase (RTK) with important roles in cell growth, The M918T mutation has been predicted either to induce a differentiation, and survival. RET is expressed in cell lineages conformational change in the kinase catalytic core, leading to the derived from the neural crest and in the tissues of the urogenital activation of RET without ligand induced dimerization, or to alter system (reviewed in ref. 1). Activation of the RET receptor is a the substrate specificity of RET, so that it preferentially binds multistep process, involving interaction with a soluble ligand of the substrates of cytoplasmic tyrosine kinases, such as SRC, or both glial cell line–derived neurotrophic factor (GDNF) family (GFL) and (6, 8). Previous studies have predicted that the M918T mutation a nonsignaling cell surface–bound coreceptor of the GDNF family leads to a pattern of RET tyrosine phosphorylation, adaptor protein receptors a (GFRa). GFLs and GFRas form complexes, which, in binding, and downstream signaling that differs in many respects turn, bind to RET, triggering its dimerization and resulting tyrosine from those associated with wild-type RET (WT-RET; refs. 6, 9). For kinase activation (1). example, in the absence of RET ligand, the M918T MEN 2B mutant Activating point mutations of RET cause multiple endocrine (2B-RET) induces phosphorylation of proteins that interact with neoplasia type 2 (MEN 2), an inherited cancer syndrome cha- CRK and NCK, including the cytoskeletal protein paxillin, which racterized by medullary thyroid carcinoma (reviewed in ref. 2). seems to be more phosphorylated in the presence of 2B-RET than The disease has three clinically distinct subtypes, ranging from the unactivated WT-RET (10). Together, these data have led to later onset, less severe, familial medullary thyroid carcinoma, speculation that the M918T mutation may contribute to the characterized only by medullary thyroid carcinoma, to the more increased activation of known RET signaling pathways or to severe MEN 2A, characterized by medullary thyroid carcinoma, the activation of distinct, but as yet unidentified, pathways that may adrenal tumor pheochromocytoma, and parathyroid hyperplasia. account for the earlier onset, broader phenotype, and increased MEN 2B, the earliest onset and most aggressive form of MEN 2, is severity of MEN 2B (10, 11). characterized by medullary thyroid carcinoma and pheochromo- Despite these predictions, the molecular consequences of the cytoma, as well as by an array of developmental abnormalities, M918T mutation, and how this single amino acid substitution leads to the changes in RET activation or interactions that cause MEN 2B, have been difficult to confirm. Thus, elucidation of the mechanisms of 2B-RET function and dysfunction in MEN 2B Note: Supplementary data for this article are available at Cancer Research Online require a better understanding of the molecular properties of both (http://cancerres.aacrjournals.org/). Requests for reprints: Lois M Mulligan, Department of Pathology, Cancer WT-RET and 2B-RET and of the activation and interactions of these Research Institute, Botterell Hall Room 329, Queen’s University, Kingston, Ontario, kinases. Here, we have compared the biochemical, thermodynamic, Canada K7L 3N6. Phone: 1-613-533-6000, ext. 77475; Fax: 1-613-533-6830; E-mail: cellular biological, and structural properties of WT-RET and 2B- [email protected]. I2006 American Association for Cancer Research. RET to identify the underlying differences and similarities in these doi:10.1158/0008-5472.CAN-06-3329 receptors. Our data show that the M918T mutation has multiple

www.aacrjournals.org 10741 Cancer Res 2006; 66: (22). November145 15, 2006 Cancer Research distinct and complementary effects on 2B-RET function including Immunoprecipitates were pelleted, washed, and resuspended in SDS-PAGE increasing intrinsic kinase activity, partially releasing kinase sample buffer. Samples were denatured, separated on 10% SDS-PAGE gels, autoinhibition, and facilitating ligand-independent phosphoryla- and transferred to nitocellulose membranes (Bio-Rad, Mississauga, Ontario, tion of 2B-RET receptors. Canada; ref. 15). For our low-stringency nonreducing conditions, lysates were prepared in sample buffer in the absence of h-mercaptoethanol and separated on 6% SDS-PAGE gels, without any denaturation step. Materials and Methods GST pull-down assays. GST fusion proteins expressed in E. coli were Homology modeling. Three-dimensional models of the RET tyrosine eluted with a polyprep column (Bio-Rad) in 100 mmol/L glutathione elution kinase domain (residues 709-988) were constructed using the crystal buffer. For GST pull-down assays, 5 Ag of GST fusion protein and GST structure of the autoinhibited and active insulin receptor tyrosine kinase sepharose beads (Amersham) were incubated with whole-cell lysates at 4jC (IRK) as structural templates. RET and IRK sequences were aligned using for 3 hours with agitation. Bound proteins were resolved by SDS-PAGE as CLUSTAL W (12) and manually adjusted for optimal structure prediction. described above. The homology modeling program MODELLER (13) was used to read the SRC kinase assay. Transfected SYF cells were harvested after 24 hours alignment files of RET to autoinhibited IRK (Protein Data Bank code: IRK) and immunoprecipitated with anti-RET or anti-SRC (Cell Signaling) and active IRK (Protein Data Bank code: 1IR3). Up to 100 hundred models antibodies. SRC kinase assay was done using a SRC kinase kit according were generated for each template, and the ‘‘best fit’’ model was selected as to the manufacturer’s instructions (Cell Signaling; ref. 22). The SRC kinase the model with the lowest value of the MODELLER objective function. The specific activity was calculated from the specific counts (total counts minus ‘‘best’’ active and autoinhibited models of RET were further refined by a nonspecific counts). Nonspecific counts were determined by doing parallel series of energy minimization steps with the Discover module of the assays in the absence of immunoprecipitates. InsightII software (Accelrys Software, San Diego, CA). Models were energy Soft agar colony formation assay. Soft agar colony formation assays minimized using 100 cycles of steepest-descent algorithm and evaluated for were done as described (23). Briefly, HEK293 cells were transiently stereochemical quality by PROCHECK. transfected with each of the icRET constructs or a control plasmid (pTRE2). 4 Expression constructs. Tetracycline-inducible RET expression con- Approximately 5 Â 10 cells were resuspended in 0.2% top agar in medium structs, generated by fusing cDNAs encoding a myristylation signal, two and plated on 0.4% bottom agar in medium. AP20187 (100 nmol/L) was dimerization domains, and the intracellular portion (amino acid 658 to added in both the lower and upper layers and culture medium COOH terminus) of RET (icRET), have been described (14). Full-length supplemented with 100 nmol/L AP20187 was added to the top layer every human RET9 cDNA was cloned into pcDNA3.1 (Invitrogen, Burlington, 2 to 3 days. Colonies were counted after 2 weeks. Statistical significance was Ontario, Canada). Constructs were validated for binding of known RET confirmed by one-way ANOVA. substrates (data not shown). Site-specific RET mutants were generated in WT-RET constructs by overlapping PCR, as described (15). Glutathione Results S-transferase (GST) fusion constructs encoding residues 664 to 1,072 of WT- RET and 2B-RET (with the M918T mutation) were generated in a modified Comparison of WT-RET and 2B-RET structure and activity. pGEX-4T-3 vector. Constructs were verified by direct sequencing (Cortec, To identify the molecular mechanisms underlying MEN 2B, we first Kingston, Ontario, Canada). Expression and GST fusion constructs for investigated whether the WT-RET and the oncogenic 2B-RET GFRa1 (15), SHC (16), NCK (17), GRB10 (18), SRC (Y527F; ref. 19), and signal mutant differed in overall structure. We used circular dichroism transducers and activators of transcription 3 (STAT3; ref. 20) have been (CD), a form of spectroscopy used to determine the secondary described. structure of molecules, to compare WT-RET and 2B-RET. Common Protein purification and biophysical analyses. Purification of GST- secondary structure motifs exhibit distinctive CD spectra in the far- RET proteins and biophysical analyses are described in Supplementary UV range whereas near-UV provides a fingerprint of protein Methods. Cell culture and transfection. HEK293 cells expressing the reverse- tertiary structure. Using purified recombinant WT-RET and 2B-RET tetracycline transcriptional activator (Tet-on)(BD Biosciences, Mississauga, proteins, we compared the relative fraction of each molecule that Ontario, Canada) were grown in DMEM (Invitrogen) supplemented with was in a-helix, h-sheet (antiparallel/parallel), h-turn, or other 10% fetal bovine serum (Sigma, Oakville, Ontario, Canada) and 1 Ag/mL (random) conformations. Far-UV and near-UV CD spectra of WT- doxycycline. SYF cells (19) were grown in the same medium without RET and 2B-RET had profiles corresponding to globular proteins doxycycline. Constructs were transiently transfected into cells using with defined secondary structure, comparable with those of typical Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruc- kinases (Supplementary Fig. S1). Both far-UV and near-UV CD tions. Full-length RET constructs were cotransfected with a GFR1 expression spectra of 2B-RET were superimposable on those of WT-RET, construct (15) and treated with 100 ng/mL GDNF (Promega, Madison, WI) suggesting that there were no significant global changes in the for 15 minutes before harvesting. AP20187 dimerizer (1 Amol/L; ARIAD, secondary or tertiary structure of RET due to the M918T mutation. Cambridge, MA) was added to induce icRET dimerization 30 minutes before harvesting. We next asked whether WT-RET and 2B-RET differed in intrinsic Immunoprecipitations and Western blotting. Total protein lysates properties such as ATP binding, thermostability, and enzyme were harvested 48 hours after transfection and suspended in 20 mmol/L kinetics. We used isothermal titration calorimetry, a thermody- Tris-HCl (pH 7.8), 150 mmol/L NaCl, 1 mmol/L sodium orthovanadate, 1% namic technique for the evaluation of interactions between Igepal, 2 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 10 Ag/mL different molecules, to compare the ATP binding affinity of WT- aprotonin, and 10 Ag/mL leupeptin (15). Protein assays were carried out RET and 2B-RET using purified recombinant proteins (Supple- using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL). mentary Fig. S2A). In isothermal titration calorimetry, heat RET expression was detected using the C-19 antibody (Santa Cruz released on interaction of two molecules can be used to determine Biotechnology, Santa Cruz CA) and RET tyrosine phosphorylation was dissociation constant (Kd), reaction stoichiometry (N), and thermo- detected using an anti-phospho-RET antibody (Cell Signaling, Beverly, MA) dynamic variables including binding enthalpy (DH; Table 1). We that specifically recognizes phosphorylation of primary tyrosine residue K Y905 (21) and an anti-phoshotyrosine antibody, pY99 (Sigma), which was found that the ATP equilibrium dissociation constant ( d)for A also used to detect phospho-paxillin. For immunoprecipitations, lysates 2B-RET (15.3 mol/L) was significantly lower than that of WT-RET were incubated with a 1:50 dilution of the appropriate primary antibody (192 Amol/L), indicating that 2B-RET has >10-fold greater affinity with agitation for 2 hours at 4jC, mixed with Protein AG (Santa Cruz for ATP (Table 1). Consistent with this, the heat of enthalpy was Biotechnology), and incubated on ice for 2 hours with shaking. also more favorable for ATP binding to 2B-RET than WT-RET.

Cancer Res 2006; 66: (22). November 15, 2006 10742 www.aacrjournals.org146 Molecular Mechanisms of MEN 2B

Table 1. Kinetic and thermodynamic properties of WT-RET and 2B-RET proteins

Protein Kinetics Thermodynamics

Isothermal titration calorimetry Differential scanning calorimetry

KM (Amol/L) Stoichiometry (N) Kd (Amol/L) DHTm (jC) Tm + ATP (jC)

À WT-RET 188.2 F 17 1.11 192.0 À1.04 Â 10 5 65.10 F 0 50.11 F 2.49 À 2B-RET 105.0 F 11 2.00 15.3 À4.95 Â 10 4 62.65 F 0.45 50.34 F 1.76

NOTE: KM, Michaelis-Menten constant—substrate (ATP) concentration required for 1/2 maximal activity. Kd, dissociation constant. DH, binding enthalpy. Tm, midpoint of denaturation transition, melting temperature.

As anticipated for RTKs, stoichiometry calculated by isothermal structure. Interestingly, in the presence of ATP, we found that titration calorimetry suggested a single ATP binding site for WT- WT-RET and 2B-RET had similar melting temperatures (Table 1), RET receptor monomers (N = 1.11). Interestingly, however, 2B-RET indicating that the conformations of ATP-bound (active) WT-RET seemed to be associated with two ATP molecules (N = 2), and 2B-RET enzymes were more similar than those of their suggesting that these molecules may associate in solution to form autoinhibited forms. The lower melting temperature of the active dimers that associate with two ATP molecules (Table 1). We forms was consistent with the thermodynamic effects of the release further confirmed this using analytic gel filtration, which allows of autoinhibition in other kinases (24, 25). Together, these data size separation of dimers and monomers in solution. Our data suggested that, in addition to altered kinase activity, changes in confirmed the presence of significantly more dimers for 2B-RET intramolecular interactions and a potential reduction in confor- than WT-RET (Supplementary Fig. S3). mational rigidity might play significant roles in the functional We compared the kinetic properties of purified WT-RET and 2B- effects of the M918T mutation in 2B-RET. RET in in vitro kinase assays (Supplementary Fig. S4). When kinase Homology modeling of WT-RET and 2B-RET. To provide a activity was compared over a range of ATP concentrations, we framework for our structural and functional comparisons of WT- K found that WT-RET required much higher ATP concentrations ( M RET and 2B-RET, we generated three-dimensional homology 188.2 F 17 Amol/L) to achieve the same level of autophosphor- models for RET. A BLAST search, using the amino acid sequence ylation as 2B-RET (KM 105 F 11 Amol/L; Table 1). The M918T of the RET tyrosine kinase domain (residues 709-988), identified mutation seemed to increase the stability of the enzyme-ATP multiple similar RTKs with significant homology (Fig. 2A). The RET complex and relatively strengthened ATP-binding. The result is a tyrosine kinase domain shares 53%, 52%, and 39% homology, significant overall increase in the kinase activity of 2B-RET as respectively, with that of (FGF) receptor 1, compared with WT-RET. FGF receptor 2, and IRK. Of these, IRK was the only kinase for Consistent with this increased ATP binding, we found that 2B- which the crystal structure is available for both active and RET seemed to be more highly autophosphorylated both in the autoinhibited forms, providing templates for homology modeling. presence and absence of ligand stimulation as compared with activated WT-RET (Fig. 1). Further, when we investigated known RET substrates (e.g., paxillin, SHC, STAT3, NCK1, and GRB10), we found that although each of these bound both WT-RET and 2B- RET, all bound more 2B-RET, as we would expect due to the higher relative levels of 2B-RET autophosphorylation. However, when relative differences in autophosphorylation levels were taken into account, we found no differences in the binding of any tested substrate to phospho-WT-RET or phospho-2B-RET (Fig. 1). To assess more localized changes in interactions between residues and domains due to the M918T mutation, we next used differential scanning calorimetry to compare the overall thermal stability and structural flexibility of WT-RET and 2B-RET conformations over a range of temperatures (20-120jC) and in the presence or absence of ATP (Supplementary Fig. S2B). In the absence of ATP, we found that WT-RET was more conformationally stable or rigid than 2B-RET, as indicated by a higher melting T j temperature [midpoint of denaturation transition ( m), 65.10 C; Table 1]. The relatively higher energy requirement for denaturation Figure 1. 2B-RET is a more active kinase than WT-RET. HEK293 cells is consistent with the tightly folded, more stable conformation of expressing GFRa1 and either WT-RET or 2B-RET were treated with GDNF and an autoinhibited (inactive) kinase structure (24, 25). 2B-RET had a either immunoprecipitated with an anti-RET or anti-paxillin antibody or subjected T j to GST-pull down using purified GST-tagged NCK-SH2 domain or GRB10-SH2 lower melting temperature ( m, 62.65 C), suggesting a relatively domain as indicated. Lysates or precipitated samples were resolved on 6% more flexible protein conformation with a less rigid overall SDS-PAGE gels and immunoblotted with appropriate antibodies as indicated.

www.aacrjournals.org 10743 Cancer Res 2006; 66: (22). November147 15, 2006 Cancer Research

Figure 2. Homology modeling of RET. A, multiple sequence alignment of tyrosine kinase domains of RET and similar RTKs. B, ribbon diagrams of the intracellular domain of WT-RET (residues 709-988) in the autoinhibited and active conformations. Three-dimensional models of RET were created using autoinhibited and activated IRK structures as templates. Positions of the autophosphorylated tyrosines in the activation loop (Y900 and Y905) and of M918 are shown. Residues in the nucleotide binding loop (orange), catalytic loop (red), activation loop (green), and P+1 loop (yellow) are indicated.

Thus, we generated three-dimensional homology models of the residues exceeds 3.5 A˚) while new interactions (V915) form, kinase domain for each of WT-RET and 2B-RET in both their active resulting in less tightly constrained, more open conformation. and autoinhibited forms based on the corresponding IRK Interestingly, in the autoinhibited 2B-RET, T918 does not interact structures. Ribbon diagrams of the tyrosine kinase domain of with I913, P914, or Y928, although it does interact with V915, and RET show a bilobed structure that is common to all protein kinases retains the interactions with S922 and L923 that are seen in (Fig. 2B). autoinhibited WT-RET (Fig. 3A). Thus, T918 in autoinhibited 2B- Methionine 918 lies within the substrate-binding pocket of RET, RET retains some of the normal interactions of M918 in in the P+l loop, adjacent to the activation loop (Fig. 2), and autoinhibited WT-RET but also acquires some of the properties substitution of this residue with thronine seems to alter the shape and interactions associated with the active WT-RET. of the substrate-binding pocket in our models (Supplementary Functional analyses of mutant RET proteins. Our models Fig. S5). This would most likely reflect changes in amino acid suggested that the M918T mutation may loosen the tight interactions caused by replacement of a large methionine residue interactions among residues in the activation, catalytic, and P+1 with a smaller, polar, threonine residue. Our models show that, in loops thought to maintain WT-RET in an autoinhibited conforma- the autoinhibited form of WT-RET, M918 interacts with residues in tion. If relaxation of autoinhibition is a significant contributor to the activation (I913) and P+1 (P914) loops and with neighboring aberrant 2B-RET function, we would predict that mutations of residues further downstream (Y928, S922, and L923), which in turn other residues involved in these interactions in WT-RET would interact with P+1 and catalytic loop residues, forming a tightly mimic the effects of M918T and lead to active receptors with closed, autoinhibited structure (Fig. 3A). On activation of WT-RET, similar properties to 2B-RET. Based on predictions from our interactions between M918 and some of these residues (S922 and models, we selected four critical residues (I913, P914, S922, and Y928) are reduced in strength or lost (predicted distance between Y928; Fig. 3A) that interact with M918, and generated alanine

Cancer Res 2006; 66: (22). November 15, 2006 10744 www.aacrjournals.org148 Molecular Mechanisms of MEN 2B substitution mutants at each position on a WT-RET background. colonies than those expressing a ligand/dimerizer activated WT- As Y928 has been shown to act as a phosphospecific binding site in RET, or any of the critical residue mutants (P < 0.01; Fig. 3C). RET-mediated STAT signaling (26), we also generated a Y928F Interestingly, the I913A mutant, as well as the P914A and Y928A mutant, which would not bind these substrates but which would RET mutants, had significantly reduced colony-forming ability as not alter the overall shape of the substrate-binding pocket. We used compared with either 2B-RET (P < 0.01) or WT-RET (P < 0.05), and previously described RET expression constructs (14) that contain not appreciably different from an empty vector control (pTRE). the intracellular domain of RET linked to dimerization domains However, despite expressing relatively less RET protein, the Y928F and a membrane localization signal (icRET) that can be activated mutant had a comparable colony forming efficiency to activated by treatment with a bivalent dimerizing agent to induce receptor WT-RET, whereas the S922A mutant had an increased colony dimerization (27). We found that mutant RET proteins were forming efficiency relative to WT-RET (P < 0.05) and intermediate expressed at slightly lower levels than either WT-RET or 2B-RET to that of activated WT-RET and 2B-RET (Fig. 3C). Together, these (Fig. 3B), as has previously been noted for RET site-specific data show that substitution of I913 or P914 and the Y928A mutants (e.g., refs. 21, 28, 29). However, although WT-RET, 2B-RET, mutation do not mimic the M918T mutation, but in fact and the I913A and S922A mutant proteins were phosphorylated at significantly impair one or all RET functions, suggesting that these relatively similar levels on induction of RET dimerization when residues have important structural or functional roles in addition expression was taken into account, there was some reduction in to participating in the tight interactions that maintain the the relative phosphorylation levels of Y928F and Y928A RET autoinhibited conformation of RET. Conversely, the Y928F and mutants and almost complete loss of phosphorylation of the P914A S922A mutations, which are predicted to reduce the sum of mutant (Fig. 3B). Binding efficiency of WT-RET and RET mutants interactions between residues in the substrate binding pocket, for a GST-tagged SHC-SH2 domain, a known RET substrate, catalytic loop, and activation loop (Fig. 3A), seem to retain correlated with autophosphorylation levels, with reduced or absent significant autophosphorylation ability, substrate binding, and substrate binding for I914A and Y928A mutants (Fig. 3B). transforming potential, consistent with the type of functional We next tested the effects of mutating the predicted RET critical effects associated with the M918T mutation in 2B-RET. These data residues on anchorage-independent growth in soft agar colony suggest that mutations that reduce the tight interactions seen in formation assays. As predicted from our kinase activity data, we the autoinhibited form of WT-RET can contribute to the activation found that cells expressing 2B-RET formed significantly more or transforming potential of RET.

Figure 3. Mutations of RET critical residues have variable functional effects. A, schematic diagram of inter-residue interactions in the autoinhibited and active WT-RET (top) and autoinhibited 2B-RET (bottom). Solid lines, van der Waals forces of interaction; dotted lines, hydrogen bonding. All distances are measured in angstroms. Residues in the nucleotide-binding loop (orange), catalytic loop (red), activation loop (green), and P+1 loop (yellow) are indicated. B, Western blot analysis of expression and phosphorylation of WT-RET, 2B-RET, and other RET critical residue mutants, and their ability to pull down GST-tagged SHC-SH2 domain fusion proteins. HEK293 cells were transfected with the indicated constructs and treated with dimerizer as described. Protein lysates and GST-pull downs were resolved on 10% SDS-PAGE gels and immunoblotted with anti-RET or anti-phospho-RET antibodies. C, soft agar colony formation assays were done in HEK293 cells expressing the indicated RET constructs or empty vector (pTRE) as described. Colony forming efficiencies for each construct were compared with numbers of colonies formed in the presence of 2B-RET and WT-RET. *, P < 0.01; **, P < 0.05, significant differences in colony number, as compared with 2B-RET or WT-RET, respectively. Columns, mean of three independent experiments; bars, SD. Western blot data presented in (B) are representative of RET expression levels seen in our colony formation assays.

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Characterization of 2B-RET preferred substrates. Previous whereas a modest denaturation step, such as boiling samples studies have suggested that the M918T mutation might also alter before SDS-PAGE, eliminated all but 2A-RET dimers (not shown). 2B-RET substrate recognition such that it preferentially binds Thus, our data suggest that 2B-RET exists both as activated substrates of cytoplasmic kinases, such as SRC (6). To determine monomers and as dimers but that these dimers are less stable whether 2B-RET was more able to act on SRC substrates than was than those formed by 2A-RET through formation of covalent WT-RET, we examined their relative ability to phosphorylate a intermolecular bonds. normal SRC substrate peptide (KVEKIGEGTYGVVYK, derived from cdc2 p34 ; ref. 22). As SRC itself is also a known RET substrate (30), we assessed RET-mediated phosphorylation of SRC substrates in SYF Discussion cells, which do not express the three SRC family kinases SRC, YES, MEN 2B is the most severe form of multiple endocrine neoplasia or FYN (19), to avoid contamination of our assays by SRC kinase type 2, characterized by early onset endocrine tumors and a broad activity. Using a series of full-length RET expression constructs, we range of developmental abnormalities (3). More than 95% of MEN first confirmed that, on RET activation with GDNF and GFRa1, 2B is caused by a methionine to threonine substitution of residue both WT-RET and 2B-RET were autophosphorylated (Supplemen- 918 in the kinase domain of RET (5) and the identical mutation tary Fig. S6) and, further, that 2B-RET was more highly occurs in >50% of sporadic medullary thyroid carcinoma, where autophosphorylated than WT-RET in SYF cells. Our data indicate it has been associated with a poorer prognosis (7). Despite that SRC family kinases are not required for RET phosphorylation predictions about the mechanisms of this mutation, experimental and, further, that phosphorylation by SRC is not a major cause confirmation of its functional effects on RET has been lacking. In of the higher 2B-RET phosphorylation. Interestingly, following this study, we have characterized the thermodynamic and kinetic receptor activation, both WT-RET and 2B-RET phosphorylated properties of the RET kinase and investigated the proposed cdc2 the p34 SRC substrate peptide (Supplementary Fig. S6B). molecular mechanisms underlying the M918T mutation. Our data Moreover, as seen for other RET substrates, the level of substrate indicate that this mutation has multiple related but distinct effects phosphorylation correlated well with the relative levels of auto- on the RET receptor. phosphorylation of WT-RET and 2B-RET and was comparable to Our data suggest that the primary effects of the M918T mutation that of an activated SRC control. Similar results were observed may be to enhance the intrinsic kinase activity of 2B-RET. We using both a specific anti-phospho-RET antibody (pY905) and a found that the affinity of 2B-RET for ATP was >10-fold greater than K A pan-phosphotyrosine (pY99) antibody (not shown). When we that of WT-RET ( d 15.3 versus 192 mol/L, respectively). Further, controlled for the different autophosphorylation levels of WT- WT-RET required a much higher concentration of ATP to achieve RET and 2B-RET, we saw no significant difference in relative SRC the same levels of autophosphorylation seen with 2B-RET (KM 188 substrate phosphorylation (Supplementary Fig. S6B), suggesting versus 105 Amol/L), suggesting that the 2B-RET-ATP complex was that intrinsic differences in recognition of this specific SRC significantly more stable. Together, these effects would significantly substrate by WT-RET and 2B-RET are minimal. increase the relative kinase activity of 2B-RET. As we would predict Monomeric and dimeric RET proteins. Previous studies have from these biophysical studies, we showed that 2B-RET is more shown that mutant forms of RET containing an MEN 2A type highly autophosphorylated than activated WT-RET, and binds and cysteine substitution mutation in the extracellular domain (2A- phosphorylates correspondingly more of its substrates in multiple RET) form dimers or higher molecular weight protein complexes cell types (Figs. 1 and 3; Supplementary Fig. S6). Our data also constitutively without ligand stimulation, whereas WT-RET does confirmed that this is an intrinsic property of 2B-RET and was not not in the absence of ligand (31, 32). The active form of 2B-RET dependent on the presence of other kinases, such as SRC family has been predicted to be either a constitutive dimer or cis- kinases, to activate RET because in vitro analyses using purified phosphorylating monomer (33). We compared the abilities of full- recombinant proteins and in vivo confirmation in SYF cells clearly length WT-RET, 2A-RET, and 2B-RET to form higher molecular showed that 2B-RET achieves the same high level of autophos- weight protein complexes under very low-stringency nonreducing phorylation, irrespective of the presence of SRC family kinases conditions (Fig. 4). In the presence of GDNF, we found both WT- (Supplementary Fig. S6). RET and 2B-RET primarily in high molecular weight complexes, The increase in 2B-RET-ATP binding is intriguing, as the M918T presumed to include dimerized receptors and interacting proteins mutation is located in the substrate binding region of the receptor, (Fig. 4A). However, in the absence of ligand, and without any form distant from the sequences of the ATP binding cleft (Fig. 2). of protein denaturation (e.g., boiling), WT-RET is found primarily Mutations that specifically increase ATP binding (as opposed to as monomers with minimal complex formation (Fig. 4B), whereas other activation mechanisms) and alter downstream signaling have a significant fraction of 2B-RET and 2A-RET are present in higher been identified in oncogenic forms of other RTKs, such as epidermal molecular weight protein complexes. When we compared the growth factor receptor (EGFR), but these mutations are primarily phosphorylation of WT-RET, 2B-RET, and 2A-RET, we found that, localized within or close to the ATP binding cleft, where they in the absence of ligand, 2B-RET and 2A-RET are more directly affect the interactions between the receptor and ATP phosphorylated than WT-RET (Fig. 4B), and that the higher (34, 35). The location of M918, distant from this region, suggested a molecular weight protein complexes formed by both 2B-RET and novel effect on RTK conformation that might contribute to altered 2A-RET are phosphorylated and of similar size. Further, whereas ATP binding. Our initial analyses using CD did not identify overall phospho-RET is found in the high molecular weight complexes for global differences in the secondary or tertiary structures of WT-RET both 2A-RET and 2B-RET, the phosphodimer/phosphomonomer and 2B-RET (Table 1; Supplementary Fig. S1), suggesting that the ratio is higher for 2A-RET than 2B-RET, suggesting that a greater M918T mutation might cause more local changes in interactions, proportion of phospho-2B-RET occurs as phosphomonomers possibly affecting RET autoinhibition, which indirectly affected ATP under nonreducing conditions. Under reducing conditions, the binding. In the absence of activation, RTK monomers adopt a closed higher molecular weight protein complexes were not seen (Fig. 4B) ‘‘autoinhibited’’ conformation in which the activation loop blocks

Cancer Res 2006; 66: (22). November 15, 2006 10746 www.aacrjournals.org150 Molecular Mechanisms of MEN 2B

Figure 4. Formation of high molecular weight protein complexes by WT-RET, 2A-RET, and 2B-RET. A, ligand-induced dimerization leads to RET localization in high molecular weight protein complexes under nonreducing conditions. Whole-cell lysates isolated from GDNF-treated HEK293 cells expressing GFRa1 and full-length WT-RET or 2B-RET were resolved on 6% SDS-PAGE gels under nonreducing (left) or reducing (right) conditions and immunoblotted for RET. B, in the absence of ligand, autophosphorylated 2A-RET and 2B-RET, but not WT-RET, are found in high molecular weight complexes. Cell lysates, prepared as above without GDNF- treatment, were resolved under nonreducing (left) or reducing (below) conditions and immunoblotted for RET or phospho-RET. Lysates prepared for cells expressing 2A-RET, known to associate with high molecular weight complexes (33), were used for complex size comparison. Right, graphical representation of the relative ratio of dimer to monomer shown in the representative immunoblots on the left.

access to the substrate-binding pocket. This conformation is very potentiates colony formation over an activated WT-RET, which is energetically stable (high Tm) due to tight intramolecular inter- predicted to act in a similar fashion to 2A-RET receptors (ref. 36; actions that result in a rigid conformation. On activation, the kinase P < 0.05, one way ANOVA), mimicking, at least in part, the effect of becomes much more flexible as autoinhibiting interactions are the M918T mutation in 2B-RET (Fig. 3C). Consistent with this, released, and it takes on a more open conformation (lower Tm). mutations of serine 922, to either phenylalanine or proline, have Consistent with these predictions, when we compared thermal been detected in sporadic medullary thyroid carcinoma (37, 38), denaturation profiles generated by differential scanning calorime- indicating that mutations of this residue may also be transforming try, we found that WT-RET had a higher Tm, suggesting a more rigid in vivo. Conversely, whereas I913A and Y928A mutants retained conformation, whereas 2B-RET had a lower Tm, suggesting a more some ability to autophosphorylate and bind substrate, they did not T flexible, partially open conformation. In the presence of ATP, m was promote growth in soft agar assays, even on ligand stimulation much lower for both receptors, consistent with the more flexible, (Fig. 3), suggesting a partial loss of some RET functions. These data open conformation of an activated kinase. Together, these data are consistent with the M918T mutation having multiple distinct suggested that 2B-RET has a more flexible conformation that may effects on RET structure and function, only some of which are reflect partial loss of autoinhibition. mimicked by these critical residue mutations. The more flexible structure predicted for 2B-RET may be due, in Together, our models and our functional and thermodynamic part, to a reduction in the sum of tight molecular interactions analyses suggest that the M918T mutation causes a local conforma- between residues in the activation loop, catalytic loop, and tional change in the 2B-RET kinase that partially releases auto- substrate binding P+1 loop, which are important in maintaining inhibition, increasing ATP-binding. However, structural release of autoinhibition (Fig. 3). Our homology model and functional data on substrate blockade alone cannot account for potentiated 2B-RET critical residues suggest some interactions that may contribute to function. Our data further show that, on ligand induced activation, this process. We found that mutation S922A significantly both WT-RET and 2B-RET form high molecular weight complexes,

www.aacrjournals.org 10747 Cancer Res 2006; 66: (22). November151 15, 2006 Cancer Research but that 2B-RET and 2A-RET also form these dimers in the absence seem to be stronger than those between 2B-RET monomers, which of ligand under low-stringency nonreducing conditions (Fig. 4). were detected in the absence of denaturation but were abolished by Further, whereas the ratios of RET dimers and monomers are similar even a brief denaturation step (not shown). As described above, our for 2A-RET and 2B-RET, the proportion of phospho-RET in dimers data suggest that, in the presence of the M918T mutation, 2B-RET is greater for 2A-RET than for 2B-RET. All RTKs are thought to exist adopts an intermediate, partially open conformation, although it in equilibrium between monomeric and dimeric pools, even in remains conformationally unlikely that these monomers can self- the absence of ligand (39, 40). In general, the dimeric forms are phosphorylate. However, the stoichiometry of 2B-RET-ATP binding nonproductive interactions as these RTKs are locked in the and our analytic gel filtration data indicating that purified 2B-RET autoinhibited state, and their formation is transient and energeti- forms dimers, as well as the detection of phospho-2B-RET in high cally unfavorable (Fig. 5). For wild-type receptors, extracellular molecular weight protein complexes in the absence of ligand ligand binding stabilizes the formation of active dimers and leads to stimulation, suggest that the monomer-dimer equilibrium is shifted kinase stimulation. In 2A-RET, intermolecular disulfide bonds link for 2B-RET, increasing the pool of transient dimers (Figs. 4 and 5). extracellular domain residues and mimic this effect, leading to These complexes enable transphosphorylation of RET receptors, constitutively active RET dimers (8, 41). Interestingly, 2A-RET dimers followed by phosphorylation of downstream targets; however, in the absence of conventional ligand and coreceptor, complexes are not as stable as dimers formed by 2A-RETor activated WT-RET. As a result, complex formation is transient and leads to a pool of phosphory- lated monomeric 2B-RET, dissociated from the higher molecular weight complexes that may also be able to initiate downstream signals (Fig. 5). We would thus predict that the sum of both active dimers and monomers may also contribute, in part, to the increased activity of 2B-RET that we observe. Finally, previous studies have suggested that 2B-RET may also recognize novel substrates or have a shift in substrate preference, allowing it to activate additional, or alternative, signaling path- ways distinct from the targets of WT-RET (6, 10). However, these studies generally compared 2B-RET with unactivated WT-RET before the recognition of GDNF as the RET ligand. In the presence of ligand stimulation, we did not detect differences in the nature of WT-RET and 2B-RET substrates, and the relative differences in binding or phosphorylation of substrates correlated completely with the relative levels of WT-RET and 2B-RET autophosphor- ylation (Figs. 1 and 3; Supplementary Fig. S6). Substrates such as paxillin (Fig. 1), previously thought to be unique to 2B-RET (10), also seem to bind ligand-stimulated WT-RET, although at lower levels. We also did not detect a preference for binding of the SRC cdc2 substrate p34 by 2B-RET, although we cannot exclude differ- ences in binding of WT-RET and 2B-RET to other SRC substrates. Together, these data are consistent with studies of RET in Drosophila, which failed to detect novel 2B-RET specific substrates (42), and, with the lack of novel substrates identified to date in numerous studies, suggest that activation of additional signaling pathways through strong interactions with uniquely 2B-RET substrates may not be a major contributing component of 2B-RET onco-activity. In summary, we have shown that a combination of mechanisms, including increased kinase activity, partial release of autoinhibition, and a relative increase in ligand-independent formation of activated monomers and dimers, contributes to 2B-RET activity. Our data suggest that the effect of these distinct mechanisms on kinase activity may be at least partly additive, which may contribute to the relative severity and broader phenotype of MEN 2B as compared with that of other MEN 2 forms. Understanding the molecular mechanisms of 2B-RET has important implications Figure 5. Model of stable, and transient, dimer formation for WT-RET and for development of therapeutics with some specificity for mutant 2B-RET. In the presence of ligand, stable dimers form between RET monomers, RET forms. Pharmacologic small-molecule inhibitors that target leading to autophosphorylation and downstream signaling (top). Transient dimer formation, while predicted to occur at equilibrium for many receptors, ATP binding sites have well-proven clinical usefulness but also would not promote autophosphorylation and signaling for autoinhibited have potential for broad side effects related to normal functions of wild-type receptors (middle). In the presence of the M918T mutation, altered the targeted kinase. As RET is known to be an important neuronal conformation stabilizes these dimers, altering the equilibrium of monomers and dimers, whereas the open conformation of the activation loop permits survival receptor (30), therapeutic targeting of mutant receptors, autophosphorylation and activation of 2B-RET dimers (bottom). while sparing the wild-type molecule, would be advantageous.

Cancer Res 2006; 66: (22). November 15, 2006 10748 www.aacrjournals.org152 Molecular Mechanisms of MEN 2B

Mutations of EGFR that increase ATP binding have been shown to Acknowledgments increase sensitivity to the inhibitor gefitinib in non–small-cell lung Received 9/8/2006; accepted 9/19/2006. cancer (35), whereas MET molecules with a M1268T mutation that Grant support: Canadian Institutes of Health Research and the Canadian Cancer corresponds to M918T have proved more sensitive to ATP blocking Society (L.M. Mulligan), and Canadian Institutes of Health Research traineeships in Transdisciplinary Cancer Research and Protein Function Discovery (T.S. Gujral). inhibitors (43). Thus, specific targeting of ATP binding in RET may The costs of publication of this article were defrayed in part by the payment of page be a valuable tool to aid in the development of anticancer therapies charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. for both MEN 2B and for sporadic tumors that harbor this We thank Rob Campbell and Kim Munro for assistance, and J. McGlade, L. LaRose, mutation. J. Duyster, B. Elliott, and J. Darnell for providing us with expression constructs.

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Appendix II

Summary of qRT-PCR primers and products

Gene Gene Name NCBI Dir Primer sequence Product symbol identifier Size (bp) CTNNB1 Catenin, beta 1 NM_001904 F 5’-TGT TCG TGC ACA TCA GGA TAC C-3’ 134 R 5’-ACA TCC CGA GCT AGG ATG TGA AG-3’ RET REarranged in Transfection AJ297349 F 5’-AAT TTG GAA AAG TGG TCA AGG C-3’ 186 R 5’-CTG CAG GCC CCA TAC AAT-3’ CCND1 Cyclin D1 NM_053056 F 5’-CGA GAA GCT GTG CAT CTA CA-3’ 123 R 5’-AAT GAA ATC GTG CGG GGT CA-3’ JUNB Jun B proto-oncogene NM_002229 F 5’-TCT GCA CAA GAT GAA CCA CG-3’ 129 R 5’-TAG CTG CTG AGG TTG GTG TA-3’ EGR1 Early Growth Response 1 NM_001964 F 5’-AGA TGA TGC TGC TGA GCA ACG-3’ 216 R 5’-ATG TCA GGA AAA GAC TCT GCG GT-3’ GDF15 Growth differentiation factor 15 NM_004864 F 5’-TGG GAA GAT TCG AAC ACC GA-3’ 203 R 5’-TCG TGT CAC GTC CCA CGA CCT TGA-3’ VGF VGF nerve growth factor NM_003378 F 5’-ACC TCT CGC GTC GTG ACA CCA-3’ 104 inducible R 5’-AAC CCG TTG ATC AGC AGA AG-3’ AXIN1 Axin 1 NM_003502 F 5’-AGC ATC GTT GTG GCG TAC T-3’ 158 R 5’-ACA GTC AAA CTC GTC GCT CA-3’

Dir: direction. bp: base pair F: forward. R: reverse

154 Appendix III Composition of Solutions

SDS- PAGE Gels

10% running 6% running 10 % acrylamide 6 % acyrlamide 0.031 M Tris-HCl pH 8.8 0.031 M Tris-HCl pH 8.8 0.1 % SDS 0.1 % SDS 0.1 % ammonium persulfate 0.1 % ammonium persulfate 0.05 mM TEMED 0.1 mM TEMED

Stacking 1.7 % acrylamide 0.01 M Tris-HCl pH 6.8 0.1 % SDS 0.1 % ammonium persulfate 0.09 nM TEMED

10X TBS 100 mM Tris pH 7.5 1M NaCl

TBST 10 mM Tris pH 7.5 100 mM NaCl 0.1 % tween 20

Gel running buffer 25 mM Tris 190 mM glycine 0.001% SDS

Transfer buffer 25 mM Tris 190 mM glycine 0.2 % methanol

155 10X CMF 1.48 M NaCl 0.05 M KCl 8.7 M Na2PO4 0.032 M NaHCO3 2.2 mM KH2PO4 0.06 M glucose

10X Western lyzing buffer 200 mM Tris-HCl, pH 7.8 1.5 M NaCl 10 % Igepal 20 mM Na2EDTA

Laemmli buffer (2X) 250 mM Tris-HCl pH 6.8 4% SDS 10% glycerol 0.006% bromophenol blue 2% Beta-mercaptoethanol

Kinase buffer 10 mM Tris-HCl (pH 7.4) 5 mM MgCl2

LB medium 1% Bacto-tryptone 0.5% Bacto-yeast 1% NaCl pH 7.0 autoclave

LB agar As per medium before autoclaving, add 1.5% Bacto-agar

SOB medium 2% Bacto-tryptone 0.5% Bacto-yeast 0.05% NaCl pH 7.0 autoclave before use, add 0.5% 2M MgCl2

156 Gel loading buffer 0.05% bromophenol blue 40% sucrose 0.1 M EDTA 0.5% SDS

10X TPE 0.9 M Tris 0.01% orthophosphoric acid 20 mM EDTA

2YT medium 1.6% bacto-tryptone 1% yeast extract 0.5% NaCl pH 7.0

Elution buffer 1.5 M Tris-HCl pH 8.0 100 mM glutathione 1M NaCl

10X PBS 1.4 M NaCl 0.03 M KCl 0.04 M Na2PO4 0.02 M KH2PO4 pH to 7.4

Coomassie blue stain 0.2% coomassie blue 50% methanol 7% acetic acid

Coomassie blue de-stain 5% methanol 7% glacial acetic acid

Dialysis buffer (for RET purification) 5mM Na2HPO4 (pH 7.0) 50mM NaCl

Tissue homogenization buffer 20 mM Tris-HCl (pH 7.8) 150 mM NaCl 1 mM sodium orthovanadate

157 2 mM EDTA 1 mM PMSF 10 ug/ml aprotonin 10 ug/ml leupeptin

Thrombin cleavage buffer 50mM Tris–HCl (pH 7.5) 150mM NaCl, 1mM EDTA 1mM DTT 0.01% Triton X-100

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 0.6mM MTT

Hypotonic buffer for cytosolic extracts 20 mM Hepes-KOH (pH 7.0) 10 mM KCl 1.5 mM MgCl2 1 mM sodium EDTA 1 mM sodium EGTA 1 mM dithiothreitol 250 mM sucrose

158