The RET receptor tyrosine 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 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. 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

  PageAbstract Co-authorship Acknowledgements Table of Contents List of Tables and Figures Abbreviations List Chapter 1: Introduction 1.1 The RET gene 1.2 Structure of RET 1.3 Activation of RET 1.4 RET and Signaling 1.5 The role of RET in development 1.6 RET and Disease 1.6.1 RET and endocrine tumours 1.6.2 RET and other associated disease types 1.7 RET as a therapeutic target 1.7.1 Dimerization inhibitors 1.7.2 Tyrosine kinase inhibitors 1.7.3 Intracellular signaling inhibitors Chapter 2: Proposed Research

2.1 RET-mediated oncogenesis in MEN 2B 2.1.1 Rationale 2.1.2 Hypothesis 2.1.3 Objectives

2.2 RET-mediated intracellular signaling 2.2.1 Rationale 2.2.2 Hypothesis 2.2.3 Objectives

2.3 Targeting RET for therapeutics 2.3.1 Rationale 2.3.2 Hypothesis 2.3.3 Objectives

Chapter 3: Molecular mechanisms of RET receptor mediated oncogenesis

in multiple endocrine neoplasia 2B (Gujral et al.,Cancer Res. 2006; 66(22):10741-9)

3.1 Abstract 3.2 Introduction 3.3 Materials and Methods

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

i iii v vi ix xi 1 1 1 3 3 7 8 8 16 17 18 20 24

27 27 27 28 28 28 28 29 29 29 29 30 30

31

32 33 35 35 36

vi

3.3.3 Peptide fingerprinting by mass spectrometry 3.3.4 In vitro kinase kinetics 3.3.5 Circular dichroism spectroscopy 3.3.6 Isothermal titration calorimetry 3.3.7 Analytical gel filtration 3.3.8 Differential scanning calorimetry

3.4 Results 3.4.1 Expression and purification of WT and 2B-RET 3.4.2 Comparison of WT-RET and 2B-RET structure and activity 3.5 Discussion 3.6 Acknowledgments 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)

4.1 Abstract 4.2 Introduction 4.3 Materials and Methods

4.3.1 Expression constructs 4.3.2 Cell culture and transfection 4.3.3 Immunoprecipitation, western, and far-western blotting 4.3.4 Preparation of cytosolic extracts 4.3.5 Glutathione S-transferase (GST)-pull-down assays 4.3.6 Reporter assay 4.3.7 shRNA production 4.3.8 MTT cell proliferation assay 4.3.9 Apoptosis assays 4.3.10 Soft Agar colony formation assays 4.3.11 Confocal microscopy 4.3.12 Tumourigenicity in nude mice 4.3.13 β-catenin immunohistochemistry 4.3.14 Relative quantification by real-Time RT-PCR

4.4 Results and Discussion 4.4.1 β-catenin nuclear localization in RET-mediated human

thyroid tumours 4.4.2 RET associates with and tyrosine phosphorylates β-catenin 4.4.3 RET activation induces cytosolic translocation of β-catenin

and its escape from the axin regulatory complex 4.4.4 RET-induced β-catenin tyrosine phosphorylation is

associated with increased TCF transcriptional activity 4.4.5 β-catenin is required for RET-induced cell proliferation and

transformation 4.4.6 Knockdown of β-catenin expression reduces RET-mediated

tumour growth and invasiveness in nude mice 4.5 Acknowledgements

36 37 37 38 38 39 39 39 42 52 57

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59 60 62 62 62 63 64 64 64 65 65 65 66 66 66 67 68 68 68

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Chapter 5: Identification and characterization of a novel RET tyrosine kinase inhibitor (Manuscript in preparation for Cancer Research)

5.1 Abstract 5.2 Introduction 5.3 Results and Discussion

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

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

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

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

5.4 Materials and Methods 5.4.1 Small-molecule libraries 5.4.2 In-vitro kinase kinetics 5.4.3 Cell culture 5.4.4 Immunoprecipitations and western blotting 5.4.5 MTT cell proliferation assay 5.4.6 Soft agar colony formation assay 5.4.7 Apoptosis assay 5.4.8 Wound healing assay

Chapter 6: Discussion

6.1 Molecular mechanisms of RET-mediated oncogenesis 6.2 RET-mediated downstream signaling 6.3 RET as a therapeutic target

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

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List of Tables and Figures

Tables   PageTable 1.1 Table 3.1 Table 3.2 Table 4.1 Table 4.2 Table 5.1 Table 6.1

Small-molecule inhibitors of the RET kinase Kinetic properties of WT-RET and 2B-RET Calorimetric analysis of the temperature-induced denaturation Patient information and β-catenin staining patterns for primary and metastazised human MTCs Gross pathology and histology of tumour lesions Selectivity of SW-01 Structural effects of MEN 2B RET mutations

26 45 51 71

92 105 117

Figures   Page

Figure 1.1 Figure 1.2 Figure 1.3 Figure 1.4 Figure 1.5 Figure 1.6 Figure 1.7 Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 Figure 3.5 Figure 3.6 Figure 3.7 Figure 3.8 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7

Schematic diagram of RET Schematic of RET activation Schematic of the RET tyrosine kinase domain with different autophosphorylated tyrosines Schematic of RET showing missense mutations associated with HSCR Schematic of RET showing missense mutations associated with MEN 2A and MEN 2B Schematic of strategies to inhibit RET signaling Homology models of RET Expression and Purification of GST-fused WT-RET and 2B-RET Identification of RET peptides by MALDI-TOF mass spectrometry WT-RET and 2B-RET do not differ in overall secondary or tertiary structure Thermodynamic analyses of WT-RET and 2B-RET Monomer-dimer equilibrium of 2B-RET protein is shifted towards dimers 2B-RET has increased autokinase activity Differential scanning calorimetry (DSC) analyses of WT-RET, and 2B-RET Model of stable, and transient, dimer formation for WT-RET and 2B-RET Altered localization of β-catenin in response to activated RET in a murine model of RET-induced MTC Localization of β-catenin in human MTCs Endogenous RET and β-catenin co-localize at the cell membrane RET associates with, and tyrosine phosphorylates, β-catenin Recombinant purified RET can directly phosphorylate purified β-catenin in vitro RET can phosphorylate β-catenin in the absence of SRC family kinases β-catenin tyrosine 654 (Y654) is a major RET-phosphorylation site

2 4 6 9

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19 22 40 41 43

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Figure 4.8 Figure 4.9 Figure 4.10 Figure 4.11 Figure 4.12 Figure 4.13 Figure 4.14 Figure 4.15 Figure 4.16 Figure 5.1 Figure 5.2 Figure 5.3 Figure 5.4 Figure 5.5 Figure 6.1 Figure 6.2 Figure 6.3

RET alters β-catenin localization Tyrosine phosphorylated β-catenin escapes the axin regulatory complex in the cytoplasm Knockdown of β-catenin and cyclinD1 protein levels by shRNA against β-catenin RET activation induces β-catenin-mediated transcription β-catenin is required for RET induced proliferation and transformation β-catenin has a modest effect on RET-induced cell survival RET-mediated tumour outgrowth in nude mice is reduced by shRNA to β-catenin Coexpression of RET and endogenous β -catenin promotes regional invasiveness and tumour adhesion to the abdominal wall Proposed model of a RET-β-catenin signalling pathway High throughput in silico screening: Identification of SW-01 SW-01 inhibits RET autophosphorylation in vitro and in RET expressing cells Effects of SW-01 on RET-mediated cellular proliferation and transformation SW-01 causes apoptosis in medullary thyroid carcinoma cells SW-01 impairs the ability of MiaPaca2 pancreatic cancer cells to migrate in wound healing assays Characterization of RET- β-catenin-E cadherin interaction Structure-based inhibition of kinases Chemical and pharmacological properties of cyclobenzaprine

81 82

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x

Abbreviations List

3D

three-dimensional

A-loop APC ATP

activation loop adenomatous polyposis coli adenosine triphosphate

bp BRAF BSA

base pair v-raf murine sarcoma viral oncogene homolog B1 bovine serum albumin

CD C-loop Ct C terminus

circular dichroism spectroscopy catalytic loop crossing threshold carboxyl terminus

DMEM DNA DOK dNTP DSC DTT

Dulbecco’s modified eagle medium deoxyribonucleic acid downstream of tyrosine kinase deoxy-nucleotide tri-phosphate differential scanning calorimetry dithiothreitol

EDTA ethylenediaminetetraacetate FACS FBS FGFR FLT-3 FMTC

fluorescence activated cell sorting fetal bovine serum fibroblast growth factor receptor FMS-like tyrosine kinase familial medullary thyroid carcinoma

GAB1 GDF15 GDNF GFL GFRα GSK-3 GST GRB

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

HEK HPT

human embryonic kidney hyperparathyroidism

xi

icRET IPTG IRK ITC

intracellular portion (amino acid 658 to C-terminus) of RET isopropylthio-β-D-galactoside insulin receptor kinase isothermal titration calorimetry

Kd

dissociation constant

LB Luria- Bertani medium MALDI MAPK MEN MEN 2A MEN 2B MEN 2B RET MTC MTT

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

NCK non catalytic region of tyrosine kinase PBS PC PCR PDAC PI3K PLC-γ PMSF PPARγ PTB PTC

phosphate buffered saline pheochromocytoma polymerase chain reaction pancreatic ductal adenocarcinoma phosphatidylinositol 3 kinase phospholipase C gamma phenylmethylsulphonylfluoride peroxisome proliferation activated receptor-gamma phosphotyrosine binding papillary thyroid carcinoma

qRT-PCR

quantitative real time PCR

RET RMS RPI-1 RPMI RTK

rearranged in transfection root mean square ribose-5-phosphate isomerase roswell park memorial institute media receptor tyrosine kinase

xii

SDS SDS-PAGE SHC SH2 SH3 STAT SYF

sodium dodecyl sulfate SDS polyacrylamide gel electrophoresis src homology 2 domain-containing transforming protein C src homology 2 src homology 3 signal transducer and activator of transcription Src Yes Fyn

TBS TBST TEMED TET TK TOF

tris-buffered saline tris-buffered saline tween 20 N, N, N’, N’-tetramethylethylenediamine tetracycline tyrosine kinase time-of-flight

VEGFR VGF

vascular endothelial growth factor receptor VGF nerve growth factor induced

WCL WT

whole cell lysates 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 Domain

Intracellular Domain

Cadherin like Domains

Cysteine rich Domain

Tyrosine kinase Domain

Transmembrane Domain

RET 51

RET 43

RET 9

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, neurturin, persefin and

artemin [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 cell signaling 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), persephin (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 signal transduction 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 protein kinase 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. Imatinib (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 (sunitinib),

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

(sorafenib). 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-kit, 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

(PP2) RET, SRC, YES, FYN, ABL

ZD6474 Vandetanib 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 Motesanib 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 mulligal@post.queensu.ca 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

C

A

ATP + + - -IB: pRETIB: RET

Coomassie

kDa

175

83

62

48

33

GST RET (74 kDa)

GST (26 kDa)

B

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

B

A

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 at 30 o 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 gefitinib 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 mulligal@queensu.ca 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

Membranous Membranous and nuclear

β-catenin

CALC-MEN2BRET MTC

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

(years) MEN 2 disease

phenotype RET Mutation

β-Catenin 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

Nor

mal

ized

luci

fera

se

activ

ity (f

old) *

WT-

RE

T2B

-RE

T

WCL

β-cateninTubulin

Cytosolic

β-catenin

GDNF + + +

Vect

or

IB:

RET

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

β-catenin

CyclinD1Tubulin

shRNA β-catenin - 1 2 3 4 IB:

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: mulligal@post.queensu.ca 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

B

C

D

SW-01 (μM)

SW-01 (μM)

SW-01

A

SW-01 (Cyclobenzaprine

hydrochloride)

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).

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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.

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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

DMSO 1 μm 2 μm

5 μm 10 μm 20 μm

C

107a

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

0

5

10

15

20

25

30

35

40

0 1 2 5 10[SW-01] uM

% A

popt

osis

HEK 293 SKNBE(2)TT

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

ATP in 20 µl kinase buffer (10 mM Tris, 5 mM MgCl2, pH 7.4), at 30oC 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

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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

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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).

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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.

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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].

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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

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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.

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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].

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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

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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-01Name Cyclobenzaprine

Classification •Antidepressive agents, tricyclic •Muscle relaxants, central •Tranquilizing agents

Formula C20-H21-N

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)

Toxicity

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.

130

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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 mulligal@post.queensu.ca 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

Molecular Mechanisms of RET Receptor–Mediated Oncogenesis

in Multiple Endocrine Neoplasia 2B

Taranjit S. Gujral,1Vinay K. Singh,

2Zongchao Jia,

2and Lois M. Mulligan

1

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

Abstract

Multiple endocrine neoplasia 2B (MEN 2B) is an inheritedsyndrome of early onset endocrine tumors and developmentalanomalies. The disease is caused primarily by a methionine tothreonine substitution of residue 918 in the kinase domain ofthe RET receptor (2B-RET); however, the molecular mecha-nisms that lead to the disease phenotype are unclear. In thisstudy, we show that the M918T mutation causes a 10-foldincrease in ATP binding affinity and leads to a more stablereceptor-ATP complex, relative to the wild-type receptor.Further, the M918T mutation alters local protein conforma-tion, correlating with a partial loss of RET kinase auto-inhibition. Finally, we show that 2B-RET can dimerize andbecome autophosphorylated in the absence of ligand stimu-lation. Our data suggest that multiple distinct but comple-mentary molecular mechanisms underlie the MEN 2Bphenotype and provide potential targets for effective thera-peutics for this disease. (Cancer Res 2006; 66(22): 10741-9)

Introduction

The RET (rearranged in transfection) proto-oncogene encodes areceptor tyrosine kinase (RTK) with important roles in cell growth,differentiation, and survival. RET is expressed in cell lineagesderived from the neural crest and in the tissues of the urogenitalsystem (reviewed in ref. 1). Activation of the RET receptor is amultistep process, involving interaction with a soluble ligand of theglial cell line–derived neurotrophic factor (GDNF) family (GFL) anda nonsignaling cell surface–bound coreceptor of the GDNF familyreceptors a (GFRa). GFLs and GFRas form complexes, which, inturn, bind to RET, triggering its dimerization and resulting tyrosinekinase activation (1).

Activating point mutations of RET cause multiple endocrineneoplasia type 2 (MEN 2), an inherited cancer syndrome cha-racterized by medullary thyroid carcinoma (reviewed in ref. 2).The disease has three clinically distinct subtypes, ranging from thelater onset, less severe, familial medullary thyroid carcinoma,characterized only by medullary thyroid carcinoma, to the moresevere MEN 2A, characterized by medullary thyroid carcinoma, theadrenal tumor pheochromocytoma, and parathyroid hyperplasia.MEN 2B, the earliest onset and most aggressive form of MEN 2, ischaracterized by medullary thyroid carcinoma and pheochromo-cytoma, as well as by an array of developmental abnormalities,

including marfanoid habitus, mucosal neuromas, ganglioneuroma-tosis of the intestinal tract, and myelinated corneal nerves (3).Morbidity and early mortality due to MEN 2B are very high.Management of both MEN 2B and sporadic medullary thyroidcarcinoma is by surgical intervention. Treatment is complicated, asmedullary thyroid carcinoma is prone to metastasis and is oftenrefractory to both radiation and chemotherapy (4). Novel strategies,such as kinase inhibitors and small molecules, have as yet largelylacked specificity or efficacy at biologically tolerated dosages (1).Despite early genetic identification, ‘‘cure’’ is reported in <20% ofMEN 2B cases (3).

Although all MEN 2 subtypes arise from mutations of RET, thereare strong genotype/phenotype associations with specific muta-tions identified in each disease subtype. MEN 2A and familialmedullary thyroid carcinoma mutations are primarily substitutionsof one of several cysteine residues in the RET extracellular domain(2). More than 95% of MEN 2B cases are caused by a single germ-line mutation that results in substitution of a threonine for thenormal methionine at residue 918 (M918T) in the RET kinasedomain (5, 6). The same mutation occurs somatically in 50% to70% of sporadic medullary thyroid carcinoma, where it can beassociated with more aggressive disease and poor prognosis (7).The M918T mutation has been predicted either to induce aconformational change in the kinase catalytic core, leading to theactivation of RET without ligand induced dimerization, or to alterthe substrate specificity of RET, so that it preferentially bindssubstrates of cytoplasmic tyrosine kinases, such as SRC, or both(6, 8). Previous studies have predicted that the M918T mutationleads to a pattern of RET tyrosine phosphorylation, adaptor proteinbinding, and downstream signaling that differs in many respectsfrom those associated with wild-type RET (WT-RET; refs. 6, 9). Forexample, in the absence of RET ligand, the M918T MEN 2B mutant(2B-RET) induces phosphorylation of proteins that interact withCRK and NCK, including the cytoskeletal protein paxillin, whichseems to be more phosphorylated in the presence of 2B-RET thanunactivated WT-RET (10). Together, these data have led tospeculation that the M918T mutation may contribute to theincreased activation of known RET signaling pathways or toactivation of distinct, but as yet unidentified, pathways that mayaccount for the earlier onset, broader phenotype, and increasedseverity of MEN 2B (10, 11).

Despite these predictions, the molecular consequences of theM918T mutation, and how this single amino acid substitution leadsto the changes in RET activation or interactions that cause MEN2B, have been difficult to confirm. Thus, elucidation of themechanisms of 2B-RET function and dysfunction in MEN 2Brequire a better understanding of the molecular properties of bothWT-RETand 2B-RETand of the activation and interactions of thesekinases. Here, we have compared the biochemical, thermodynamic,cellular biological, and structural properties of WT-RET and 2B-RET to identify the underlying differences and similarities in thesereceptors. Our data show that the M918T mutation has multiple

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

Requests for reprints: Lois M Mulligan, Department of Pathology, CancerResearch Institute, Botterell Hall Room 329, Queen’s University, Kingston, Ontario,Canada K7L 3N6. Phone: 1-613-533-6000, ext. 77475; Fax: 1-613-533-6830; E-mail:mulligal@post.queensu.ca.

I2006 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-06-3329

www.aacrjournals.org 10741 Cancer Res 2006; 66: (22). November 15, 2006

Research Article

145

distinct and complementary effects on 2B-RET function includingincreasing intrinsic kinase activity, partially releasing kinaseautoinhibition, and facilitating ligand-independent phosphoryla-tion of 2B-RET receptors.

Materials and Methods

Homology modeling. Three-dimensional models of the RET tyrosinekinase domain (residues 709-988) were constructed using the crystalstructure of the autoinhibited and active insulin receptor tyrosine kinase(IRK) as structural templates. RET and IRK sequences were aligned usingCLUSTAL W (12) and manually adjusted for optimal structure prediction.The homology modeling program MODELLER (13) was used to read thealignment files of RET to autoinhibited IRK (Protein Data Bank code: IRK)and active IRK (Protein Data Bank code: 1IR3). Up to 100 hundred modelswere generated for each template, and the ‘‘best fit’’ model was selected asthe model with the lowest value of the MODELLER objective function. The‘‘best’’ active and autoinhibited models of RET were further refined by aseries of energy minimization steps with the Discover module of theInsightII software (Accelrys Software, San Diego, CA). Models were energyminimized using 100 cycles of steepest-descent algorithm and evaluated forstereochemical quality by PROCHECK.

Expression constructs. Tetracycline-inducible RET expression con-structs, generated by fusing cDNAs encoding a myristylation signal, twodimerization domains, and the intracellular portion (amino acid 658 to

COOH terminus) of RET (icRET), have been described (14). Full-lengthhuman RET9 cDNA was cloned into pcDNA3.1 (Invitrogen, Burlington,Ontario, Canada). Constructs were validated for binding of known RETsubstrates (data not shown). Site-specific RET mutants were generated in

WT-RET constructs by overlapping PCR, as described (15). GlutathioneS-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

pGEX-4T-3 vector. Constructs were verified by direct sequencing (Cortec,Kingston, Ontario, Canada). Expression and GST fusion constructs forGFRa1 (15), SHC (16), NCK (17), GRB10 (18), SRC (Y527F; ref. 19), and signaltransducers and activators of transcription 3 (STAT3; ref. 20) have been

described.Protein purification and biophysical analyses. Purification of GST-

RET proteins and biophysical analyses are described in Supplementary

Methods.

Cell culture and transfection. HEK293 cells expressing the reverse-tetracycline transcriptional activator (Tet-on)(BD Biosciences, Mississauga,

Ontario, Canada) were grown in DMEM (Invitrogen) supplemented with

10% fetal bovine serum (Sigma, Oakville, Ontario, Canada) and 1 Ag/mL

doxycycline. SYF cells (19) were grown in the same medium withoutdoxycycline. Constructs were transiently transfected into cells using

Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruc-

tions. Full-length RETconstructs were cotransfected with a GFR1 expressionconstruct (15) and treated with 100 ng/mL GDNF (Promega, Madison, WI)

for 15 minutes before harvesting. AP20187 dimerizer (1 Amol/L; ARIAD,

Cambridge, MA) was added to induce icRET dimerization 30 minutes before

harvesting.Immunoprecipitations and Western blotting. Total protein lysates

were harvested 48 hours after transfection and suspended in 20 mmol/L

Tris-HCl (pH 7.8), 150 mmol/L NaCl, 1 mmol/L sodium orthovanadate, 1%

Igepal, 2 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 10 Ag/mLaprotonin, and 10 Ag/mL leupeptin (15). Protein assays were carried out

using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL).

RET expression was detected using the C-19 antibody (Santa CruzBiotechnology, Santa Cruz CA) and RET tyrosine phosphorylation was

detected using an anti-phospho-RET antibody (Cell Signaling, Beverly, MA)

that specifically recognizes phosphorylation of primary tyrosine residue

Y905 (21) and an anti-phoshotyrosine antibody, pY99 (Sigma), which wasalso used to detect phospho-paxillin. For immunoprecipitations, lysates

were incubated with a 1:50 dilution of the appropriate primary antibody

with agitation for 2 hours at 4jC, mixed with Protein AG (Santa Cruz

Biotechnology), and incubated on ice for 2 hours with shaking.

Immunoprecipitates were pelleted, washed, and resuspended in SDS-PAGEsample buffer. Samples were denatured, separated on 10% SDS-PAGE gels,

and transferred to nitocellulose membranes (Bio-Rad, Mississauga, Ontario,

Canada; ref. 15). For our low-stringency nonreducing conditions, lysates

were prepared in sample buffer in the absence of h-mercaptoethanol andseparated on 6% SDS-PAGE gels, without any denaturation step.

GST pull-down assays. GST fusion proteins expressed in E. coli were

eluted with a polyprep column (Bio-Rad) in 100 mmol/L glutathione elution

buffer. For GST pull-down assays, 5 Ag of GST fusion protein and GSTsepharose beads (Amersham) were incubated with whole-cell lysates at 4jCfor 3 hours with agitation. Bound proteins were resolved by SDS-PAGE as

described above.

SRC kinase assay. Transfected SYF cells were harvested after 24 hoursand immunoprecipitated with anti-RET or anti-SRC (Cell Signaling)

antibodies. SRC kinase assay was done using a SRC kinase kit according

to the manufacturer’s instructions (Cell Signaling; ref. 22). The SRC kinasespecific activity was calculated from the specific counts (total counts minus

nonspecific counts). Nonspecific counts were determined by doing parallel

assays in the absence of immunoprecipitates.

Soft agar colony formation assay. Soft agar colony formation assayswere done as described (23). Briefly, HEK293 cells were transiently

transfected with each of the icRET constructs or a control plasmid (pTRE2).

Approximately 5 � 104 cells were resuspended in 0.2% top agar in medium

and plated on 0.4% bottom agar in medium. AP20187 (100 nmol/L) wasadded in both the lower and upper layers and culture medium

supplemented with 100 nmol/L AP20187 was added to the top layer every

2 to 3 days. Colonies were counted after 2 weeks. Statistical significance wasconfirmed by one-way ANOVA.

Results

Comparison of WT-RET and 2B-RET structure and activity.To identify the molecular mechanisms underlying MEN 2B, we firstinvestigated whether the WT-RET and the oncogenic 2B-RETmutant differed in overall structure. We used circular dichroism(CD), a form of spectroscopy used to determine the secondarystructure of molecules, to compare WT-RET and 2B-RET. Commonsecondary structure motifs exhibit distinctive CD spectra in the far-UV range whereas near-UV provides a fingerprint of proteintertiary structure. Using purified recombinant WT-RETand 2B-RETproteins, we compared the relative fraction of each molecule thatwas in a-helix, h-sheet (antiparallel/parallel), h-turn, or other(random) conformations. Far-UV and near-UV CD spectra of WT-RET and 2B-RET had profiles corresponding to globular proteinswith defined secondary structure, comparable with those of typicalkinases (Supplementary Fig. S1). Both far-UV and near-UV CDspectra of 2B-RET were superimposable on those of WT-RET,suggesting that there were no significant global changes in thesecondary or tertiary structure of RET due to the M918T mutation.

We next asked whether WT-RET and 2B-RET differed in intrinsicproperties such as ATP binding, thermostability, and enzymekinetics. We used isothermal titration calorimetry, a thermody-namic technique for the evaluation of interactions betweendifferent molecules, to compare the ATP binding affinity of WT-RET and 2B-RET using purified recombinant proteins (Supple-mentary Fig. S2A). In isothermal titration calorimetry, heatreleased on interaction of two molecules can be used to determinedissociation constant (Kd), reaction stoichiometry (N), and thermo-dynamic variables including binding enthalpy (DH ; Table 1). Wefound that the ATP equilibrium dissociation constant (Kd) for2B-RET (15.3 Amol/L) was significantly lower than that of WT-RET(192 Amol/L), indicating that 2B-RET has >10-fold greater affinityfor ATP (Table 1). Consistent with this, the heat of enthalpy wasalso more favorable for ATP binding to 2B-RET than WT-RET.

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As anticipated for RTKs, stoichiometry calculated by isothermaltitration calorimetry suggested a single ATP binding site for WT-RET receptor monomers (N = 1.11). Interestingly, however, 2B-RETseemed to be associated with two ATP molecules (N = 2),suggesting that these molecules may associate in solution to formdimers that associate with two ATP molecules (Table 1). Wefurther confirmed this using analytic gel filtration, which allowssize separation of dimers and monomers in solution. Our dataconfirmed the presence of significantly more dimers for 2B-RETthan WT-RET (Supplementary Fig. S3).

We compared the kinetic properties of purified WT-RET and 2B-RET in in vitro kinase assays (Supplementary Fig. S4). When kinaseactivity was compared over a range of ATP concentrations, wefound that WT-RET required much higher ATP concentrations (KM

188.2 F 17 Amol/L) to achieve the same level of autophosphor-ylation as 2B-RET (KM 105 F 11 Amol/L; Table 1). The M918Tmutation seemed to increase the stability of the enzyme-ATPcomplex and relatively strengthened ATP-binding. The result is asignificant overall increase in the kinase activity of 2B-RET ascompared with WT-RET.

Consistent with this increased ATP binding, we found that 2B-RET seemed to be more highly autophosphorylated both in thepresence and absence of ligand stimulation as compared withactivated WT-RET (Fig. 1). Further, when we investigated knownRET substrates (e.g., paxillin, SHC, STAT3, NCK1, and GRB10), wefound that although each of these bound both WT-RET and 2B-RET, all bound more 2B-RET, as we would expect due to the higherrelative levels of 2B-RET autophosphorylation. However, whenrelative differences in autophosphorylation levels were taken intoaccount, we found no differences in the binding of any testedsubstrate to phospho-WT-RET or phospho-2B-RET (Fig. 1).

To assess more localized changes in interactions betweenresidues and domains due to the M918T mutation, we next useddifferential scanning calorimetry to compare the overall thermalstability and structural flexibility of WT-RET and 2B-RETconformations over a range of temperatures (20-120jC) and inthe presence or absence of ATP (Supplementary Fig. S2B). In theabsence of ATP, we found that WT-RET was more conformationallystable or rigid than 2B-RET, as indicated by a higher meltingtemperature [midpoint of denaturation transition (Tm), 65.10jC;Table 1]. The relatively higher energy requirement for denaturationis consistent with the tightly folded, more stable conformation ofan autoinhibited (inactive) kinase structure (24, 25). 2B-RET had alower melting temperature (Tm, 62.65jC), suggesting a relativelymore flexible protein conformation with a less rigid overall

structure. Interestingly, in the presence of ATP, we found thatWT-RET and 2B-RET had similar melting temperatures (Table 1),indicating that the conformations of ATP-bound (active) WT-RETand 2B-RET enzymes were more similar than those of theirautoinhibited forms. The lower melting temperature of the activeforms was consistent with the thermodynamic effects of the releaseof autoinhibition in other kinases (24, 25). Together, these datasuggested that, in addition to altered kinase activity, changes inintramolecular interactions and a potential reduction in confor-mational rigidity might play significant roles in the functionaleffects of the M918T mutation in 2B-RET.Homology modeling of WT-RET and 2B-RET. To provide a

framework for our structural and functional comparisons of WT-RET and 2B-RET, we generated three-dimensional homologymodels for RET. A BLAST search, using the amino acid sequenceof the RET tyrosine kinase domain (residues 709-988), identifiedmultiple similar RTKs with significant homology (Fig. 2A). The RETtyrosine kinase domain shares 53%, 52%, and 39% homology,respectively, with that of fibroblast growth factor (FGF) receptor 1,FGF receptor 2, and IRK. Of these, IRK was the only kinase forwhich the crystal structure is available for both active andautoinhibited forms, providing templates for homology modeling.

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) DH Tm (jC) Tm + ATP (jC)

WT-RET 188.2 F 17 1.11 192.0 �1.04 � 10�5 65.10 F 0 50.11 F 2.492B-RET 105.0 F 11 2.00 15.3 �4.95 � 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.

Figure 1. 2B-RET is a more active kinase than WT-RET. HEK293 cellsexpressing GFRa1 and either WT-RET or 2B-RET were treated with GDNF andeither immunoprecipitated with an anti-RET or anti-paxillin antibody or subjectedto GST-pull down using purified GST-tagged NCK-SH2 domain or GRB10-SH2domain as indicated. Lysates or precipitated samples were resolved on 6%SDS-PAGE gels and immunoblotted with appropriate antibodies as indicated.

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Thus, we generated three-dimensional homology models of thekinase domain for each of WT-RET and 2B-RET in both their activeand autoinhibited forms based on the corresponding IRKstructures. Ribbon diagrams of the tyrosine kinase domain ofRET show a bilobed structure that is common to all protein kinases(Fig. 2B).

Methionine 918 lies within the substrate-binding pocket of RET,in the P+l loop, adjacent to the activation loop (Fig. 2), andsubstitution of this residue with thronine seems to alter the shapeof the substrate-binding pocket in our models (SupplementaryFig. S5). This would most likely reflect changes in amino acidinteractions caused by replacement of a large methionine residuewith a smaller, polar, threonine residue. Our models show that, inthe autoinhibited form of WT-RET, M918 interacts with residues inthe activation (I913) and P+1 (P914) loops and with neighboringresidues further downstream (Y928, S922, and L923), which in turninteract with P+1 and catalytic loop residues, forming a tightlyclosed, autoinhibited structure (Fig. 3A). On activation of WT-RET,interactions between M918 and some of these residues (S922 andY928) are reduced in strength or lost (predicted distance between

residues exceeds 3.5 A) while new interactions (V915) form,resulting in less tightly constrained, more open conformation.Interestingly, in the autoinhibited 2B-RET, T918 does not interactwith I913, P914, or Y928, although it does interact with V915, andretains the interactions with S922 and L923 that are seen inautoinhibited WT-RET (Fig. 3A). Thus, T918 in autoinhibited 2B-RET retains some of the normal interactions of M918 inautoinhibited WT-RET but also acquires some of the propertiesand interactions associated with the active WT-RET.Functional analyses of mutant RET proteins. Our models

suggested that the M918T mutation may loosen the tightinteractions among residues in the activation, catalytic, and P+1loops thought to maintain WT-RET in an autoinhibited conforma-tion. If relaxation of autoinhibition is a significant contributor toaberrant 2B-RET function, we would predict that mutations ofother residues involved in these interactions in WT-RET wouldmimic the effects of M918T and lead to active receptors withsimilar properties to 2B-RET. Based on predictions from ourmodels, we selected four critical residues (I913, P914, S922, andY928; Fig. 3A) that interact with M918, and generated alanine

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

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substitution mutants at each position on a WT-RET background.As Y928 has been shown to act as a phosphospecific binding site inRET-mediated STAT signaling (26), we also generated a Y928Fmutant, which would not bind these substrates but which wouldnot alter the overall shape of the substrate-binding pocket. We usedpreviously described RET expression constructs (14) that containthe intracellular domain of RET linked to dimerization domainsand a membrane localization signal (icRET) that can be activatedby treatment with a bivalent dimerizing agent to induce receptordimerization (27). We found that mutant RET proteins wereexpressed at slightly lower levels than either WT-RET or 2B-RET(Fig. 3B), as has previously been noted for RET site-specificmutants (e.g., refs. 21, 28, 29). However, although WT-RET, 2B-RET,and the I913A and S922A mutant proteins were phosphorylated atrelatively similar levels on induction of RET dimerization whenexpression was taken into account, there was some reduction inthe relative phosphorylation levels of Y928F and Y928A RETmutants and almost complete loss of phosphorylation of the P914Amutant (Fig. 3B). Binding efficiency of WT-RET and RET mutantsfor a GST-tagged SHC-SH2 domain, a known RET substrate,correlated with autophosphorylation levels, with reduced or absentsubstrate binding for I914A and Y928A mutants (Fig. 3B).

We next tested the effects of mutating the predicted RET criticalresidues on anchorage-independent growth in soft agar colonyformation assays. As predicted from our kinase activity data, wefound that cells expressing 2B-RET formed significantly more

colonies than those expressing a ligand/dimerizer activated WT-RET, or any of the critical residue mutants (P < 0.01; Fig. 3C).Interestingly, the I913A mutant, as well as the P914A and Y928ARET mutants, had significantly reduced colony-forming ability ascompared with either 2B-RET (P < 0.01) or WT-RET (P < 0.05), andnot appreciably different from an empty vector control (pTRE).However, despite expressing relatively less RET protein, the Y928Fmutant had a comparable colony forming efficiency to activatedWT-RET, whereas the S922A mutant had an increased colonyforming efficiency relative to WT-RET (P < 0.05) and intermediateto that of activated WT-RET and 2B-RET (Fig. 3C). Together, thesedata show that substitution of I913 or P914 and the Y928Amutation do not mimic the M918T mutation, but in factsignificantly impair one or all RET functions, suggesting that theseresidues have important structural or functional roles in additionto participating in the tight interactions that maintain theautoinhibited conformation of RET. Conversely, the Y928F andS922A mutations, which are predicted to reduce the sum ofinteractions between residues in the substrate binding pocket,catalytic loop, and activation loop (Fig. 3A), seem to retainsignificant autophosphorylation ability, substrate binding, andtransforming potential, consistent with the type of functionaleffects associated with the M918T mutation in 2B-RET. These datasuggest that mutations that reduce the tight interactions seen inthe autoinhibited form of WT-RET can contribute to the activationor 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 expressionand 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. HEK293cells were transfected with the indicated constructs and treated with dimerizer as described. Protein lysates and GST-pull downs were resolved on 10% SDS-PAGEgels and immunoblotted with anti-RET or anti-phospho-RET antibodies. C, soft agar colony formation assays were done in HEK293 cells expressing the indicatedRET constructs or empty vector (pTRE) as described. Colony forming efficiencies for each construct were compared with numbers of colonies formed in the presence of2B-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 threeindependent 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. Previousstudies have suggested that the M918T mutation might also alter2B-RET substrate recognition such that it preferentially bindssubstrates of cytoplasmic kinases, such as SRC (6). To determinewhether 2B-RET was more able to act on SRC substrates than wasWT-RET, we examined their relative ability to phosphorylate anormal SRC substrate peptide (KVEKIGEGTYGVVYK, derived fromp34cdc2 ; ref. 22). As SRC itself is also a known RET substrate (30), weassessed RET-mediated phosphorylation of SRC substrates in SYFcells, which do not express the three SRC family kinases SRC, YES,or FYN (19), to avoid contamination of our assays by SRC kinaseactivity. Using a series of full-length RET expression constructs, wefirst confirmed that, on RET activation with GDNF and GFRa1,both WT-RET and 2B-RET were autophosphorylated (Supplemen-tary Fig. S6) and, further, that 2B-RET was more highlyautophosphorylated than WT-RET in SYF cells. Our data indicatethat SRC family kinases are not required for RET phosphorylationand, further, that phosphorylation by SRC is not a major causeof the higher 2B-RET phosphorylation. Interestingly, followingreceptor activation, both WT-RET and 2B-RET phosphorylatedthe p34cdc2 SRC substrate peptide (Supplementary Fig. S6B).Moreover, as seen for other RET substrates, the level of substratephosphorylation correlated well with the relative levels of auto-phosphorylation of WT-RET and 2B-RET and was comparable tothat of an activated SRC control. Similar results were observedusing both a specific anti-phospho-RET antibody (pY905) and apan-phosphotyrosine (pY99) antibody (not shown). When wecontrolled for the different autophosphorylation levels of WT-RET and 2B-RET, we saw no significant difference in relative SRCsubstrate phosphorylation (Supplementary Fig. S6B), suggestingthat intrinsic differences in recognition of this specific SRCsubstrate by WT-RET and 2B-RET are minimal.Monomeric and dimeric RET proteins. Previous studies have

shown that mutant forms of RET containing an MEN 2A typecysteine substitution mutation in the extracellular domain (2A-RET) form dimers or higher molecular weight protein complexesconstitutively without ligand stimulation, whereas WT-RET doesnot in the absence of ligand (31, 32). The active form of 2B-REThas been predicted to be either a constitutive dimer or cis-phosphorylating monomer (33). We compared the abilities of full-length WT-RET, 2A-RET, and 2B-RET to form higher molecularweight protein complexes under very low-stringency nonreducingconditions (Fig. 4). In the presence of GDNF, we found both WT-RET and 2B-RET primarily in high molecular weight complexes,presumed to include dimerized receptors and interacting proteins(Fig. 4A). However, in the absence of ligand, and without any formof protein denaturation (e.g., boiling), WT-RET is found primarilyas monomers with minimal complex formation (Fig. 4B), whereasa significant fraction of 2B-RET and 2A-RET are present in highermolecular weight protein complexes. When we compared thephosphorylation of WT-RET, 2B-RET, and 2A-RET, we found that,in the absence of ligand, 2B-RET and 2A-RET are morephosphorylated than WT-RET (Fig. 4B), and that the highermolecular weight protein complexes formed by both 2B-RET and2A-RET are phosphorylated and of similar size. Further, whereasphospho-RET is found in the high molecular weight complexes forboth 2A-RET and 2B-RET, the phosphodimer/phosphomonomerratio is higher for 2A-RET than 2B-RET, suggesting that a greaterproportion of phospho-2B-RET occurs as phosphomonomersunder nonreducing conditions. Under reducing conditions, thehigher molecular weight protein complexes were not seen (Fig. 4B)

whereas a modest denaturation step, such as boiling samplesbefore SDS-PAGE, eliminated all but 2A-RET dimers (not shown).Thus, our data suggest that 2B-RET exists both as activatedmonomers and as dimers but that these dimers are less stablethan those formed by 2A-RET through formation of covalentintermolecular bonds.

Discussion

MEN 2B is the most severe form of multiple endocrine neoplasiatype 2, characterized by early onset endocrine tumors and a broadrange of developmental abnormalities (3). More than 95% of MEN2B is caused by a methionine to threonine substitution of residue918 in the kinase domain of RET (5) and the identical mutationoccurs in >50% of sporadic medullary thyroid carcinoma, whereit has been associated with a poorer prognosis (7). Despitepredictions about the mechanisms of this mutation, experimentalconfirmation of its functional effects on RET has been lacking. Inthis study, we have characterized the thermodynamic and kineticproperties of the RET kinase and investigated the proposedmolecular mechanisms underlying the M918T mutation. Our dataindicate that this mutation has multiple related but distinct effectson the RET receptor.

Our data suggest that the primary effects of the M918T mutationmay be to enhance the intrinsic kinase activity of 2B-RET. Wefound that the affinity of 2B-RET for ATP was >10-fold greater thanthat of WT-RET (Kd 15.3 versus 192 Amol/L, respectively). Further,WT-RET required a much higher concentration of ATP to achievethe same levels of autophosphorylation seen with 2B-RET (KM 188versus 105 Amol/L), suggesting that the 2B-RET-ATP complex wassignificantly more stable. Together, these effects would significantlyincrease the relative kinase activity of 2B-RET. As we would predictfrom these biophysical studies, we showed that 2B-RET is morehighly autophosphorylated than activated WT-RET, and binds andphosphorylates correspondingly more of its substrates in multiplecell types (Figs. 1 and 3; Supplementary Fig. S6). Our data alsoconfirmed that this is an intrinsic property of 2B-RET and was notdependent on the presence of other kinases, such as SRC familykinases, to activate RET because in vitro analyses using purifiedrecombinant proteins and in vivo confirmation in SYF cells clearlyshowed that 2B-RET achieves the same high level of autophos-phorylation, irrespective of the presence of SRC family kinases(Supplementary Fig. S6).

The increase in 2B-RET-ATP binding is intriguing, as the M918Tmutation is located in the substrate binding region of the receptor,distant from the sequences of the ATP binding cleft (Fig. 2).Mutations that specifically increase ATP binding (as opposed toother activation mechanisms) and alter downstream signaling havebeen identified in oncogenic forms of other RTKs, such as epidermalgrowth factor receptor (EGFR), but these mutations are primarilylocalized within or close to the ATP binding cleft, where theydirectly affect the interactions between the receptor and ATP(34, 35). The location of M918, distant from this region, suggested anovel effect on RTK conformation that might contribute to alteredATP binding. Our initial analyses using CD did not identify overallglobal differences in the secondary or tertiary structures of WT-RETand 2B-RET (Table 1; Supplementary Fig. S1), suggesting that theM918T mutation might cause more local changes in interactions,possibly affecting RETautoinhibition, which indirectly affected ATPbinding. In the absence of activation, RTK monomers adopt a closed‘‘autoinhibited’’ conformation in which the activation loop blocks

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access to the substrate-binding pocket. This conformation is veryenergetically stable (high Tm) due to tight intramolecular inter-actions that result in a rigid conformation. On activation, the kinasebecomes much more flexible as autoinhibiting interactions arereleased, and it takes on a more open conformation (lower Tm).Consistent with these predictions, when we compared thermaldenaturation profiles generated by differential scanning calorime-try, we found that WT-RET had a higher Tm, suggesting a more rigidconformation, whereas 2B-RET had a lower Tm, suggesting a moreflexible, partially open conformation. In the presence of ATP, Tm wasmuch lower for both receptors, consistent with the more flexible,open conformation of an activated kinase. Together, these datasuggested that 2B-RET has a more flexible conformation that mayreflect partial loss of autoinhibition.

The more flexible structure predicted for 2B-RET may be due, inpart, to a reduction in the sum of tight molecular interactionsbetween residues in the activation loop, catalytic loop, andsubstrate binding P+1 loop, which are important in maintainingautoinhibition (Fig. 3). Our homology model and functional data oncritical residues suggest some interactions that may contribute tothis process. We found that mutation S922A significantly

potentiates colony formation over an activated WT-RET, which ispredicted to act in a similar fashion to 2A-RET receptors (ref. 36;P < 0.05, one way ANOVA), mimicking, at least in part, the effect ofthe M918T mutation in 2B-RET (Fig. 3C). Consistent with this,mutations of serine 922, to either phenylalanine or proline, havebeen detected in sporadic medullary thyroid carcinoma (37, 38),indicating that mutations of this residue may also be transformingin vivo . Conversely, whereas I913A and Y928A mutants retainedsome ability to autophosphorylate and bind substrate, they did notpromote growth in soft agar assays, even on ligand stimulation(Fig. 3), suggesting a partial loss of some RET functions. These dataare consistent with the M918T mutation having multiple distincteffects on RET structure and function, only some of which aremimicked by these critical residue mutations.

Together, our models and our functional and thermodynamicanalyses suggest that the M918T mutation causes a local conforma-tional change in the 2B-RET kinase that partially releases auto-inhibition, increasing ATP-binding. However, structural release ofsubstrate blockade alone cannot account for potentiated 2B-RETfunction. Our data further show that, on ligand induced activation,both WT-RET and 2B-RET form high molecular weight complexes,

Figure 4. Formation of high molecularweight protein complexes by WT-RET,2A-RET, and 2B-RET. A, ligand-induceddimerization leads to RET localization inhigh molecular weight protein complexesunder nonreducing conditions. Whole-celllysates isolated from GDNF-treatedHEK293 cells expressing GFRa1 andfull-length WT-RET or 2B-RET wereresolved on 6% SDS-PAGE gels undernonreducing (left ) or reducing (right )conditions and immunoblotted forRET. B, in the absence of ligand,autophosphorylated 2A-RET and 2B-RET,but not WT-RET, are found in highmolecular weight complexes. Cell lysates,prepared as above without GDNF-treatment, were resolved undernonreducing (left ) or reducing (below )conditions and immunoblotted for RET orphospho-RET. Lysates prepared for cellsexpressing 2A-RET, known to associatewith high molecular weight complexes (33),were used for complex size comparison.Right, graphical representation of therelative ratio of dimer to monomer shown inthe representative immunoblots on the left.

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but that 2B-RET and 2A-RET also form these dimers in the absenceof ligand under low-stringency nonreducing conditions (Fig. 4).Further, whereas the ratios of RET dimers and monomers are similarfor 2A-RET and 2B-RET, the proportion of phospho-RET in dimersis greater for 2A-RET than for 2B-RET. All RTKs are thought to existin equilibrium between monomeric and dimeric pools, even inthe absence of ligand (39, 40). In general, the dimeric forms arenonproductive interactions as these RTKs are locked in theautoinhibited state, and their formation is transient and energeti-cally unfavorable (Fig. 5). For wild-type receptors, extracellularligand binding stabilizes the formation of active dimers and leads tokinase stimulation. In 2A-RET, intermolecular disulfide bonds linkextracellular domain residues and mimic this effect, leading toconstitutively active RET dimers (8, 41). Interestingly, 2A-RET dimers

seem to be stronger than those between 2B-RET monomers, whichwere detected in the absence of denaturation but were abolished byeven a brief denaturation step (not shown). As described above, ourdata suggest that, in the presence of the M918T mutation, 2B-RETadopts an intermediate, partially open conformation, although itremains conformationally unlikely that these monomers can self-phosphorylate. However, the stoichiometry of 2B-RET-ATP bindingand our analytic gel filtration data indicating that purified 2B-RETforms dimers, as well as the detection of phospho-2B-RET in highmolecular weight protein complexes in the absence of ligandstimulation, suggest that the monomer-dimer equilibrium is shiftedfor 2B-RET, increasing the pool of transient dimers (Figs. 4 and 5).These complexes enable transphosphorylation of RET receptors,followed by phosphorylation of downstream targets; however, in theabsence of conventional ligand and coreceptor, complexes are not asstable 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 molecularweight complexes that may also be able to initiate downstreamsignals (Fig. 5). We would thus predict that the sum of both activedimers and monomers may also contribute, in part, to the increasedactivity of 2B-RET that we observe.

Finally, previous studies have suggested that 2B-RET may alsorecognize 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, thesestudies generally compared 2B-RET with unactivated WT-RETbefore the recognition of GDNF as the RET ligand. In the presenceof ligand stimulation, we did not detect differences in the natureof WT-RET and 2B-RET substrates, and the relative differences inbinding or phosphorylation of substrates correlated completelywith the relative levels of WT-RET and 2B-RET autophosphor-ylation (Figs. 1 and 3; Supplementary Fig. S6). Substrates such aspaxillin (Fig. 1), previously thought to be unique to 2B-RET (10),also seem to bind ligand-stimulated WT-RET, although at lowerlevels. We also did not detect a preference for binding of the SRCsubstrate p34cdc2 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 inDrosophila , which failed to detect novel 2B-RET specific substrates(42), and, with the lack of novel substrates identified to date innumerous studies, suggest that activation of additional signalingpathways through strong interactions with uniquely 2B-RETsubstrates may not be a major contributing component of2B-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 ofactivated monomers and dimers, contributes to 2B-RET activity.Our data suggest that the effect of these distinct mechanisms onkinase activity may be at least partly additive, which maycontribute to the relative severity and broader phenotype of MEN2B as compared with that of other MEN 2 forms. Understandingthe molecular mechanisms of 2B-RET has important implicationsfor development of therapeutics with some specificity for mutantRET forms. Pharmacologic small-molecule inhibitors that targetATP binding sites have well-proven clinical usefulness but alsohave potential for broad side effects related to normal functions ofthe targeted kinase. As RET is known to be an important neuronalsurvival receptor (30), therapeutic targeting of mutant receptors,while sparing the wild-type molecule, would be advantageous.

Figure 5. Model of stable, and transient, dimer formation for WT-RET and2B-RET. In the presence of ligand, stable dimers form between RET monomers,leading to autophosphorylation and downstream signaling (top ). Transientdimer formation, while predicted to occur at equilibrium for many receptors,would not promote autophosphorylation and signaling for autoinhibitedwild-type receptors (middle ). In the presence of the M918T mutation, alteredconformation stabilizes these dimers, altering the equilibrium of monomersand dimers, whereas the open conformation of the activation loop permitsautophosphorylation and activation of 2B-RET dimers (bottom ).

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Mutations of EGFR that increase ATP binding have been shown toincrease sensitivity to the inhibitor gefitinib in non–small-cell lungcancer (35), whereas MET molecules with a M1268T mutation thatcorresponds to M918T have proved more sensitive to ATP blockinginhibitors (43). Thus, specific targeting of ATP binding in RET maybe a valuable tool to aid in the development of anticancer therapiesfor both MEN 2B and for sporadic tumors that harbor thismutation.

Acknowledgments

Received 9/8/2006; accepted 9/19/2006.Grant support: Canadian Institutes of Health Research and the Canadian Cancer

Society (L.M. Mulligan), and Canadian Institutes of Health Research traineeships inTransdisciplinary Cancer Research and Protein Function Discovery (T.S. Gujral).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We 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.

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Appendix II

Summary of qRT-PCR primers and products

Gene symbol

Gene Name NCBI identifier

Dir Primer sequence Product 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 inducible

NM_003378 F 5’-ACC TCT CGC GTC GTG ACA CCA-3’ 104 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

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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

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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

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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

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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

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