Anaplastic Lymphoma Kinase
mutations and
downstream signalling
Christina Schönherr
Department of Molecular Biology
Umeå University
Umeå 2012
1
Copyright©Christina Schönherr
ISBN: 978-91-7459-387-7
Cover front: Christina Schönherr; Cover back: PC12 cells transfected with the ALK gain-of-function
mutant hALKF1174S (co-transfected with GFP) give rise to neurite outgrowth. Picture acquired by Yasuo
Yamazaki.
Elektronisk version tillgänglig på http://umu.diva-portal.org/
Printed by: Department of Chemistry Printing Service, Umeå University
Umeå, Sweden 2012
2
To my parents
The capacity to blunder slightly is the real marvel of DNA. Without this special
attribute, we would still be anaerobic bacteria and there would be no music.
Lewis Thomas
1
Table of Contents
TABLE OF CONTENTS
ABSTRACT
PAPERS INCLUDED IN THIS THESIS
ABBREVIATIONS
INTRODUCTION
1
The Tyrosine Kinase superfamily
1.1 Regulation of the activity of Receptor tyrosine kinases
2
The RTK Anaplastic Lymphoma Kinase
2.1 ALK structure
2.2 ALK ligands, signalling and function
2.2.1
Drosophila melanogaster ALK
2.2.2
Caenorhabditis elegans ALK
2.2.3
Danio rerio ALK
2.2.4
Mammalian ALK
2.3 Oncogenic ALK signalling
3
ALK in diseases
3.1 ALK translocations
3.1.1
Anaplastic Large Cell Lymphoma (ALCL)
3.1.2
Inflammatory Myofibroblastic Tumour (IMT)
3.1.3
Non-small cell lung cancer (NSCLC)
3.1.4
Diffuse large B-cell lymphoma (DLBCL)
3.1.5
Renal cell carcinoma
3.2 ALK overexpression
3.3 Point mutations of ALK
3.3.1
Neuroblastoma
3.3.2
Genetic hallmarks of neuroblastoma
3.3.3
ALK point mutations in neuroblastoma
3.3.4
ALK point mutations in other cancers
4
Treatments for ALK-positive carcinomas
4.1 Kinase inhibitors
4.1.1
ALK-specific tyrosine kinase inhibitors
4.2 Other approaches to inhibit ALK activity
5
The Ras superfamily of small GTPases
5.1 Rap1
5.2 Rap1 specific regulators
5.2.1
Rap1 specific GEFs
5.2.2
Rap1 specific GAPs
AIMS
1
Overall aim
2
Specific aims
2
2
5
7
8
11
11
11
14
14
16
16
18
18
18
20
23
23
23
24
24
25
25
26
26
26
27
28
31
32
33
33
36
37
38
39
39
39
41
41
41
RESULTS AND DISCUSSION
1
Article I: “Anaplastic lymphoma kinase activates the small GTPase
Rap1 via the Rap1-specific GEF C3G in both neuroblastoma and
PC12 cells.”
1.1 Stimulated ALK activates Rap1 which leads to neurite outgrowth in PC12
cells
1.2 Activation of Rap1 downstream of ALK occurs via the Rap1-specific
GEF C3G
1.3 Rap1 activity is involved in cell proliferation of neuroblastoma cell lines
2
Article II: “Appearance of the novel activating F1174S ALK
mutation in neuroblastoma correlates with aggressive tumor
progression and unresponsiveness to therapy.”
2.1 The ALKF1174S mutant is a ligand-independent gain-of-function mutation
and has transforming potential
2.2 Ectopic expression of ALKF1174S in the Drosophila eye causes the rough
eye phenotype
3
Article III: “Activating ALK mutations found in neuroblastoma are
inhibited by Crizotinib and NVP-TAE684.”
3.1 ALK mutations identified in neuroblastoma are ligand-independent gainof-function mutations and can be blocked by NVP-TAE684 and crizotinib
with different IC50
3.2 Ectopic expression of ALK mutants in the Drosophila eye causes the
rough eye phenotype
4
Article IV: “The Neuroblastoma ALK (I1250T) Mutation is a KinaseDead RTK In Vitro and In Vivo.”
4.1 The ALKI1250T mutant is not constitutively active in cell culture systems
4.2 The ALKI1250T mutant is suggested to act as a dominant-negative receptor
4.3 Ectopic expression of ALKI1250T in the Drosophila eye does not cause the
rough eye phenotype
4.4 Why is the ALKI1250T mutant inactive?
5
Article V: “Anaplastic Lymphoma Kinase (ALK) regulates initiation
of transcription of MYCN in neuroblastoma cells.”
5.1 ALK regulates the MYCN promoter in PC12 cells and human
neuroblastoma cell lines
5.2 Abrogation of ALK activity results in decreased MYCN mRNA and
proliferation of neuroblastoma cell lines
5.3 ALK activity regulates MYCN protein expression
5.4 ALK and MYCN co-operate in transforming NIH3T3 cells
SUMMARY OF THE MAIN FINDINGS OF THIS THESIS
FUTURE PERSPECTIVES
ACKNOWLEDGEMENTS
REFERENCES
42
42
42
43
44
46
46
47
48
48
50
52
52
53
53
53
55
55
56
56
58
59
60
62
63
3
4
Abstract
The oncogene Anaplastic Lymphoma Kinase (ALK) is a Receptor Tyrosine
Kinase (RTK) and was initially discovered as the fusion protein NPM
(nucleophosmin)-ALK in a subset of Anaplastic Large Cell Lymphomas (ALCL).
Since then more fusion proteins have been identified in a variety of cancers. Further,
overexpression of ALK due to gene amplification has been observed in many
malignancies, amongst others neuroblastoma, a pediatric cancer. Lately, activating
point mutations in the kinase domain of ALK have been described in neuroblastoma
patients and neuroblastoma cell lines. In contrast, the physiological function of ALK
is still unclear, but ALK is suggested to play a role in the normal development and
function of the nervous system.
By employing cell culture based approaches, including a tetracyclineinducible PC12 cell system and the in vivo D. melanogaster model system, we
aimed to analyze the downstream signalling of ALK and its role in neuroblastoma.
First, we wished to analyze whether ALK is able to activate the small GTPase Rap1
contributing to differentiation/proliferation processes. Activated ALK recruits a
complex of the GEF C3G and CrkL and activates C3G by tyrosine phosphorylation.
This activated complex is able to activate Rap1 resulting either in neurite outgrowth
in PC12 cells or proliferation of neuroblastoma cells suggesting a potential role in
the oncogenesis of neuroblastoma driven by gain-of-function mutant ALK. Next, we
could show that seven investigated ALK mutations with a high probability of being
oncogenic (G1128A, I1171N, F1174L, F1174S, R1192P, F1245C and R1275Q), are
true gain-of-function mutations, respond differently to ALK inhibitors and have
different transforming ability. Especially the F1174S mutation correlates with
aggressive disease development. However, the assumed active germ line mutation
I1250T is in fact a kinase dead mutation and suggested to act as a dominant-negative
receptor. Finally, ALK mutations are most frequently observed in MYCN amplified
tumours correlating with a poor clinical outcome. Active ALK regulates mainly the
initiation of MYCN transcription in human neuroblastoma cell lines. Further, ALK
gain-of-function mutants and MYCN synergize in transforming NIH3T3 cells.
Overall, somatic mutations appear to be more aggressive than germ line
mutations, implying a different impact on neuroblastoma. Further, successful
application of ALK inhibitors suggests a promising future for the development of
patient-specific treatments for neuroblastoma patients.
5
6
Papers included in this thesis
This thesis is based on the following publications which will be referred to by
their roman numerals (I – V). All publications are reproduced with the permission
from the journal publishers.
Article I
Schonherr C, Yang HL, Vigny M, Palmer RH, Hallberg B. Anaplastic lymphoma
kinase activates the small GTPase Rap1 via the Rap1-specific GEF C3G in both
neuroblastoma and PC12 cells. Oncogene. 2010 May 13;29(19):2817-30.
Article II
Martinsson T, Eriksson T, Abrahamsson J, Caren H, Hansson M, Kogner P,
Kamaraj S, Schonherr C, Weinmar J, Ruuth K, Palmer RH, Hallberg B.
Appearance of the novel activating F1174S ALK mutation in neuroblastoma
correlates with aggressive tumor progression and unresponsiveness to therapy.
Cancer Res. 2010 Jan 1;71(1):98-105.
Article III
Schonherr C, Ruuth K, Yamazaki Y, Eriksson T, Christensen J, Palmer RH,
Hallberg B. Activating ALK mutations found in neuroblastoma are inhibited by
Crizotinib and NVP-TAE684. Biochem J. 2011 Dec 15;440(3):405-13.
Article IV
Schonherr C, Ruuth K, Eriksson T, Yamazaki Y, Ottmann C, Combaret V, Vigny
M, Kamaraj S, Palmer RH, Hallberg B. The Neuroblastoma ALK (I1250T)
Mutation Is a Kinase-Dead RTK In Vitro and In Vivo. Transl Oncol. 2011
Aug;4(4):258-65.
Article V
Schonherr C, Ruuth K, Kamaraj S, Wang CL, Yang HL, Combaret V, Djos A,
Martinsson T, Christensen J, Palmer RH, Hallberg B. Anaplastic Lymphoma Kinase
(ALK) regulates initiation of transcription of MYCN in neuroblastoma cells.
Oncogene. 2012 Jan 30.
7
Abbreviations
ABL
Akt
ALCL
ALK
ALO17
Arf
ATC
ATIC
ATP
BCR
C3G
CAMTA1
CARS
CLTC1
CML
CNS
dALK
DLBCL
Dpp
Duf
EGFR
EML4
ERK
FDA
FGF
FGFR
Flt3
FOXO3a
FRS2
GAP
GEF
Grb2
GSK3β
GTPase
hALK
Hen-1
HER2
Hsp90
hTERT
8
V-abl Abelson murine leukemia viral oncogene homolog 1
AKR Thymoma
Anaplastic Large Cell Lymphoma
Anaplastic Lymphoma Kinase
ALK lymphoma oligomerization partner on chromosome 17
ADP-ribosylation factor
Anaplastic Thyroid Carcinoma
5-aminoimidazole-4-carboxamide ribonucleotide
formyltransferase/IMP cyclohydrolase
Adenosinetriphosphate
Breakpoint Cluster Region
Crk SH3 domain-binding Guanine nucleotide exchange factor
Calmodulin-binding transcription activator 1
Cysteinyl-tRNA synthetase
Clathrin heavy chain-like 1
Chronic Myeloid Leukemia
Central Nervous System
Drosophila melanogaster Anaplastic Lymphoma Kinase
Diffuse large B-cell lymphoma
Decapentaplegic
Dumbfounded
Epidermal Growth Factor Receptor
Echinoderm microtubule-associated protein like 4
Extracellular signal Regulated Kinase
Food and Drug Administration
Fibroblast Growth Factor
Fibroblast Growth Factor Receptor
Fms-like Tyrosine Kinase Receptor-3
Forkhead box O3a
Fibroblast Growth Factor Receptor Substrate 2
GTPase activating protein
Guanine nucleotide exchange factor
Growth Factor Receptor-bound protein 2
Glycogen Synthase Kinase 3β
Guanosine triphosphatase
human Anaplastic Lymphoma Kinase
Hesitation-1
Human Epidermal Growth Factor Receptor 2
Heatshockprotein 90
human telomerase reverse transcriptase
IgG
IMT
IR
IRS-1
JAK3
Jeb
JNK
KIF5B
Kirre
LDL
LRP
LTK
mALK
MAM
MAPK
MEK
Miple 1 and 2
MK
MSN
mTOR
MYCN
MYH9
NGF
NIPA
NPM
NSCLC
p130Cas
PC12
PDGFR
PI3K
PLCγ
pp60Src
PPFIBP1
PTB
PTK
PTN
PTP
Rab
Ran
RANBP2
Rap
RAS
Immunoglobulin G
Inflammatory Myofibroblastic Tumour
Insulin Receptor
Insulin Receptor Substrate 1
Janus Kinase-3
Jelly Belly
c-Jun N-terminal Kinase
Kinesin family member 5B
Kin of irregular chiasm
Low-Density Lipoprotein
LDL receptor related protein
Leukocyte Tyrosine Kinase
mouse Anaplastic Lymphoma Kinase
Meprin A-5 protein and receptor protein tyrosine phosphatase Mu
Mitogen-Activated Protein Kinase
MAPK/ERK Kinase
Midkine and Pleiotrophin
Midkine
Moesin
Mammalian Target of Rapamycin
myc myelocytomatosis viral related oncogene, neuroblastoma
derived
Non-muscle myosin heavy chain
Nerve Growth Factor
Nuclear interacting partner of ALK
Nucleophosmin
Non-small cell lung cancer
p130 Crk-associated substrate
Pheochromocytoma cells 12
Platelet Derived Growth Factor Receptor
Phosphoinositide-3 Kinase
Phospholipase C γ
pp60 Sarcoma
F polypeptide-interacting protein-binding protein 1
Phosphotyrosine-binding
Protein Tyrosine Kinase
Pleiotrophin
Protein Tyrosine Phosphatase
Ras-like protein in brain
Ras-like nuclear
Ran binding protein 2
Ras-proximate
Rat sarcoma
9
Rho
RPTPβ/ζ
RTK
Sar
SCC
SCD-2
SCF
SCLC
SDS-PAGE
SEC31L1
SH2/3
Shc
SHH
Shp1/2
SQSTM1
STAT3 and 5
TFG
TGFβ
TKD
TPM3 and 4
TrkA
VASP
VCL
17-AAG
10
Ras homologous
Receptor Protein Tyrosine Phosphatase β/ζ
Receptor Tyrosine Kinase
Secretion-associated and Ras-related
Squamous cell carcinoma of the esophagus
Suppressor of constitutive dauer formation
Stem Cell Factor
Small cell lung cancer
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis
SEC31 homologue A (S. cerevisiae)
Src homology 2/3
Src homology 2 containing
Sonic hedgehog
SH2 domain-containing phosphatase 1/2
Sequestosome 1
Signal Transducer and Activator of Transcription 3 and 5
TRK-fused gene
Transforming Growth Factor β
Tyrosine Kinase Domain
Tropomyosin 3 and 4
Tropomyosin receptor kinase A
Vasodilator-stimulated phosphoprotein
Vinculin
17-allyl-amino-demethoxygeldanamycin
Introduction
1
The Tyrosine Kinase superfamily
In 1906 Levene identified for the first time phosphate in the protein vitellin
[3]. Over the years the phosphorylation of serine, threonine or tyrosine has evolved
into one of the most common post-translational protein modifications. This
enzymatic reaction was first described for the serine phosphorylation of casein in
1954 [4] and is mediated by protein kinases. A couple of years later tyrosine
phosphorylation was described [5], followed by the first report of a protein kinase
that is able to phosphorylate tyrosine, namely pp60Src, the transforming protein of
Rous sarcoma virus [6].
The human genome encodes for 518 different protein kinases which are
divided into different groups [7]. One of these groups represents the class of tyrosine
kinases, which have been established as key regulators in various cellular functions
like proliferation, migration and differentiation. The family of tyrosine kinases is
further subdivided into 58 Receptor tyrosine kinases (RTKs) and 32 non-receptor
tyrosine kinases [7]. All RTKs share a common domain structure: they contain an
extracellular domain comprising a ligand binding region, a transmembrane domain
and finally an intracellular domain containing the usually highly conserved kinase
domain [2]. Most RTKs are monomers at the cell membrane in the absence of a
ligand. However, one exception is the Insulin receptor (IR), which exists as an
inactive heterodimer. This heterodimer is activated by structural changes induced by
ligand binding resulting in stabilization of an active dimer state [8, 9].
1.1
Regulation of the activity of Receptor tyrosine kinases
Generally, activation of RTKs is mediated by ligand-induced dimerization. In
general, a ligand binds simultaneously to two receptor monomers, thereby
crosslinking them which stabilizes the formation of an active RTK dimer being able
to auto-phosphorylate tyrosines in the kinase domain. To date, there are four
different ways of ligand-induced RTK activation (Figure 1). The first includes
dimerization mediated by the ligand (Figure 1A). For example, TrkA monomers are
dimerized by binding of the dimeric NGF ligand to each receptor molecule in such a
way that the two TrkA molecules do not contact each other. Secondly, a ligand
mediates dimer formation where the receptor molecules contact each other directly
(Figure 1B). This is shown for the stem cell factor (SCF), the KIT ligand, which
exists as a homodimer. Each SCF molecule binds to one receptor monomer, thereby
crosslinking the two receptors. The third example is about the fibroblast growth
factor receptor (FGF) which is crosslinked by a combination of bivalent ligand
binding, receptor-receptor contacts and binding of accessory molecules (Figure 1C).
11
Figure 1: Ways of Receptor Tyrosine Kinase
dimerization.
Generally a ligand (red) binds to the
extracellular regions of RTKs. The dimerized
RTKs
undergo
structural
changes
in
conformation, leading to RTK activation.
(A) A Nerve growth factor interacts with two
TrkA receptors without direct contact between
the receptor molecules.
(B) A stem cell factor dimer crosslinks two KIT
receptors which contact each other directly.
(C) Two fibroblast growth factor receptors
contact each other. In addiction, crosslinking
occurs via binding of accessory molecules like
heparin or heparin sulfate proteoglycans (white
sticks) and ligand binding.
(D) Two ErbB receptors dimerize directly.
Ligand binding drives conformational changes
resulting in stabilization and activation of the
receptor dimer.
(Figure adapted with permission from [2].
Two monomeric FGF ligands bind the two receptor monomers together with
two heparin molecules forming a major complex. These receptor-ligand, receptorheparin, ligand-heparin and receptor-receptor interactions result in stabilization of
the FGFR dimer. The last type of receptor dimerization has been shown for
epidermal growth factor receptor (EGFR) family where receptor dimerization is
completely receptor mediated (Figure 1D). In the absence of the ligand the receptors
are in an intramolecular autoinhibitory state. Bivalent ligand binding induces
conformational changes in the receptor, resulting in stabilization and activation of
the receptor dimer.
Following ligand-induced dimerization the intracellular tyrosine kinase
domain (TKD) of the RTKs is activated by various mechanisms which include the
release of cis-autoinhibition of the TKD (Figure 2). Roughly, the TKD is divided
into an N- and a C-lobe which form a deep cleft with the active site. Some
prominent motifs in the highly dynamic N-lobe include the αC-helix and the
glycine-rich loop (G-loop) which is involved in ATP binding. The more rigid C-lobe
provides the docking site for the substrate proteins. The catalytic loop contains a
conserved HRD motif with the catalytic aspartate. The P+1 loop which is situated Cterminal of the activation loop, binds the substrate at the residue after the
phosphorylation site. The activation loop harbours the DFG motif which is involved
in magnesium binding and therefore important for catalysis. This activation loop
contains critical tyrosines which become auto-phosphorylated upon stimulation.
12
Figure 2: Crystal structure of the ALK kinase domain shown in two orthogonal orientations.
NT and CT: N- and C-termini of ALK. The N-terminal lobe consists of β-sheets (orange) and the αChelix (purple). The C-terminal lobe consists of helices (blue). The glycine-rich P-loop is depicted in
bright green, the hinge region between the lobes in yellow, the catalytic loop in salmon and the
activation loop in red.
(Figure adapted with permission from [1].
Upon ligand activation, one critical tyrosine in the activation loop of one
receptor becomes phosphorylated by its partner, followed by two additional
tyrosines. Hence, this trans-phosphorylation disrupts the cis-autoinhibitory
interactions and the phosphorylated activation loop changes conformation to an
active state (Figure 3A) [2, 10].
Figure 3: Ways of inhibition of the
intracellular tyrosine kinase domain.
The intracellular tyrosine kinase domains
contain a C-lobe (light purple), N-Lobe (dark
purple or yellow in the active state) and an
activation loop (purple or yellow respectively).
Inhibition occurs via intramolecular interactions:
(A) Activation loop inhibition.
(B) Juxtamembrane inhibition.
(C) C-terminal tail inhibition.
(Figure adapted with permission from [2].
13
A further way of autoinhibition is mediated by the juxtamembrane domain as
shown e.g. for Flt3 (Figure 3B). The juxtamembrane region binds to the active site
of the kinase, stabilizing an inactive conformation. Phosphorylation of critical
tyrosines in the juxtamembrane region disrupts the autoinhibitory state and the
kinase adopts an active conformation. A third mode of autoinhibition involves the
C-terminal tail of the kinase domain (Figure 3C). For instance, in Tie2 the Cterminal tail blocks substrate access to the active site. Auto-phosphorylation of
tyrosines in the C-terminal tail may disrupt the autoinhibitory state and thereby
result in activation of the kinase [2].
The phosphorylated tyrosines of the activated receptor serve as docking sites
for signaling molecules containing Src homology-2 (SH2) and phosphotyrosinebinding (PTB) domains. Hence, receptor activation initiates the activation of various
signaling pathways forming a complex signalling network. However, the RTK
mediated cell signaling requires strict regulation. This process can occur either via a
positive feedback mechanism (e.g. the activity of protein tyrosine phosphatases
(PTPs) is temporarily blocked) or via negative feedback mechanisms abrogating
RTK mediated cell signaling. This includes for instance direct activation of PTPs,
transcription of negative signaling regulators or endocytosis of receptors.
However, despite stringent regulations aberrant activity of tyrosine kinases
has been reported in many diseases including cancer [11, 12]. To date, more than
50% of the known RTKs have been implicated in oncogenic malignancies, either by
autocrine activation, chromosomal translocations (where the RTK kinase domain is
fused to a protein functioning as a dimerizer), overexpression or gain-of-function
mutations [2, 11].
2
The RTK Anaplastic Lymphoma Kinase
Anaplastic Lymphoma Kinase (ALK) was described for the first time in 1994
when a novel tyrosine phophorylated protein was found in Anaplastic Large Cell
Lymphoma [13, 14]. This protein was identified as the chimeric protein NPM-ALK
generated by a translocation between the chromosomes (2;5)(p23;q35). In NPMALK, the N-terminal part of nucleophosmin (NPM) is fused to the kinase domain of
the novel tyrosine kinase, which received the name ALK after the disease where it
was reported for the first time [13].
2.1
ALK structure
From the initial discovery in 1994 it took three years until the full length
ALK was identified independently by two groups [15, 16]. Like all RTKs ALK
contains a ligand binding extracellular domain, a transmembrane domain and a
cytosolic region containing the kinase domain (Figure 4A).
14
Figure 4: Domain structure of ALK and potential tyrosine phosphorylation sites.
(A) The extracellular region of ALK contains two Meprin A-5 protein and receptor protein tyrosine
phosphatase Mu (MAM) domains, one low density lipoprotein receptor class A (LDL) domain and a
glycine rich (G-rich) domain. A transmembrane domain (TMD) connects the extracellular region with
the intracellular region containing the protein tyrosine kinase (PTK) domain. The closest family
member, Leukocyte Tyrosine Kinase (LTK) is shown with the equivalent regions. In NPM-ALK the
PTK is fused to the N-terminal part of nucleophosmin (NPM).
(B) The intracellular region of human and mouse ALK comprises the PTK and contains potential
tyrosine phosphorylation sites. Tyrosines within the activation loop are indicated in bold. Worth
mentioning is the tyrosine 1604 in human ALK that is not present in mouse ALK.
Together with Leukocyte Tyrosine Kinase (LTK) ALK forms a subgroup
within the Insulin Receptor (IR) superfamily [15, 16]. Human ALK is built up of
1620 amino acids, resulting in a protein of approximately 180 kDa. In SDS-PAGE
however, ALK can be detected at 220 kDa due to posttranslational modifications
like glycosylations [15, 16]. Mouse ALK consists of 1621 amino acids [15, 16], D.
melanogaster ALK of 1701 [17] and C. elegans ALK of 1421 amino acids [18, 19].
The existence of an N-terminal signal peptide is responsible for the transport of
ALK to the cell membrane. The extracellular part of ALK contains several domains:
two MAM (Meprin A-5 protein and receptor protein tyrosine phosphatase Mu)
domains, one LDLa (low density lipoprotein receptor class A) domain and a glycine
rich domain (Figure 4A) [15-17, 20]. However, one can only speculate about the
functions of those domains. As the LDLa domain mediates binding between LDL
receptor and LDL, it might play a role in ALK ligand binding [21, 22]. MAM
domains are most likely involved in cell-cell interactions [23] although their role for
15
ALK function is unclear, as is the glycine rich domain. In D. melanogaster however,
both the MAM and the glycine rich domains seem to be important for dALK
activity, as point mutations in the MAM domain as well as in the glycine rich
domain (replacing glycine by acidic amino acids) result in inactive dALK [24].
The ALK domains are conserved across species, with highest similarity in the
kinase domain. Although mouse and human ALK are highly similar (87% at protein
level) one significant difference has to be mentioned: that is the extra tyrosine at
position 1604 which exists only in human ALK and plays most likely a role in tumor
progression (Figure 4B) [25]. The activation loop within the kinase domain of ALK
contains the auto-phosphorylation motif YxxxYY which exists also in the IR.
However in contrast to the IR, where the second tyrosine is first phosphorylated
followed by the first and then the third, the order of phosphorylation in ALK is
different: here the phosphorylation of the first tyrosine of the YxxxYY motif is
predominant in the process of autoactivation. This might be due to the RAS triplet
between the tyrosines (Y’RAS’YY) in contrast to IR (Y’ETD’YY) [26, 27].
Recently, the crystal structure of the ALK catalytic domain in complex with
ATP competitive inhibitors has been published, providing a great opportunity to
investigate structural functional relationships [1, 28].
2.2
ALK ligands, signalling and function
2.2.1 Drosophila melanogaster ALK
In D. melanogaster the physiological function has been intensely studied. For
the first time D. melanogaster ALK has been described to be expressed in the
developing embryonic mesoderm and central nervous system (CNS), activating the
ERK pathway in vivo [17]. During early embryogenesis ALK plays an important
role in the development of the visceral musculature of the gut [24]. To date, the only
well established ligand for ALK is Jelly Belly (Jeb) which activates dALK in the
visceral muscle founder cells, leading to stimulation of the ERK pathway (Figure 5).
As a result the founder cells fuse with fusion competent myoblasts, forming the gut
musculature [29-33]. As Jeb/dALK signalling is required for the specification of the
founder cells, dALK mutant flies lack a functioning gut. The Jeb/dALK mediated
ERK activation results in transcription of downstream targets like duf/kirre [29, 31],
org [31], hand [34] and dpp [35].
The dALK ligand Jelly Belly is a protein of approximately 61 kDa containing
an LDLa domain. Jeb is secreted from the somatic mesoderm and then taken up by
the visceral mesoderm [36]. The binding of Jeb to dALK is most likely mediated via
the LDLa domain, as Jeb proteins lacking the LDLa domain are unable to bind
dALK [31]. Further, two proteins called Miple1 and Miple2 are potential ligands for
dALK. Those proteins belong to the midkine/pleiotrophin family of proteins thus the
names Miple1 and 2. In D. melanogaster Miple1 has been found to be expressed at
high levels in the CNS, while Miple2 is strongly expressed in the developing midgut
16
endoderm [37]. Still further studies are required to support the theory about being
true dALK ligands.
Figure 5: Drosophila melanogaster and mammalian wild type ALK signalling.
Upon ligand binding (for Drosophila ALK it is Jeb) ALK dimerizes, resulting in auto-phosphorylation
and the activation of downstream signalling pathways (light grey boxes). For mammalian ALK the
natural ligand is still unknown, but midkine and pleiotrophin are suggested ALK ligands. Alternative
ways of ALK activation are suggested. One proposes that ALK belongs to the group of dependence
receptors. In the absence of a ligand ALK possesses pro-apoptotic properties. Upon extracellular
stimulation and intrinsic activation by caspase-mediated cleavage ALK has antiapoptotic activity. The
other potential activating mechanism includes the receptor phosphatase RPTPβ/ζ which is inhibited by
binding of pleiotrophin, resulting in the overall activation of ALK downstream signalling.
Besides the above named ERK activation mediated by dALK activity, dALK
seems to play an important role in the transcription of Dpp (homolog to mammalian
TGFβ) and subsequent signaling in the endoderm, resulting in the development of
the embryonic endoderm [35]. Further, Jeb/dALK signalling is not only important
for the gut development, but is also involved in the anterograde signalling pathway
mediating neuronal circuit assembly in the visual system of the fruit fly. Lack of
either Jeb or dALK results in misstargeting of R-cell axons during optic lobe
maturation [38]. A recent study showed Jeb and ALK localization in developing
17
synapses, where Jeb localizes to presynaptic terminals and ALK is concentrated in
postsynaptic domains. This Jeb/dALK expression results in anterograde transsynaptic signalling, important for the synaptic connectivity in the developing motor
circuit [39]. Further, during nutrient restriction ALK protects especially the CNS via
involvement of the PI3K/Akt pathway [40]. Depletion of ALK in the fruit fly results
in increased resistance to the sedating effects of ethanol [41]. Recently, Gouzi et al.,
showed that dALK is upstream of neurofibromin 1, regulating body size
determination and associative learning [42].
2.2.2 Caenorhabditis elegans ALK
In C. elegans ALK (T10H9.2) or SCD-2 (suppressor of constitutive dauer
formation) plays a role in synapse stabilization and regulates entry into dauer stage
[18, 19]. In 2004 ALK has been identified as a downstream effector of FSN-1, a
novel F-box protein, mediating synapse stability [18]. Further, it has been shown
that SCD-2 regulates the integration of sensory signals in interneurons [43, 44].
Interestingly, a wild C. elegans strain from a desert oasis revealed a defect in dauer
response which is due to a mutation in the scd-2 gene [19]. Further this study
suggests that SCD-2 modulates the TGFβ pathway.
Hen-1 has been identified as the SCD-2 ligand [45]. Like the D. melanogaster
ALK ligand Jelly Belly, Hen-1 is a secreted protein with an LDL receptor repeat,
regulating sensory processing and learning of the neuronal circuit. To date little
information is available about the Hen-1/SCD-2 signalling, but the genetic pathway
includes the adaptor protein SOC-1 and the MAPK SMA-5 [19].
2.2.3 Danio rerio ALK
A study in the zebrafish Danio rerio showed that shady mutants are lacking
iridophores, mirror-like pigment cells derived from the neural crest [46]. Further this
study demonstrates that the shady gene encodes the RTK Leukocyte Tyrosine
Kinase (LTK). Like D. melanogaster or mammalian ALK the extracellular domain
of the zebrafish LTK contains a MAM domain, in contrast to mammalian LTK,
which is lacking MAM domains. Therefore D. rerio LTK seems to be closer related
to ALK. As the function of mammalian LTK is to date unknown, Lopes et al., show
for the first time a role for LTK in vertebrates. A natural ligand for zebrafish LTK
has not been identified yet but the fact that LTK is important in the development of
iridophores, which are derived from the neural crest, is of great interest as human
ALK activating point mutations seem to play an important role in neuroblastoma, a
disease derived from neural crest cells (see below).
2.2.4 Mammalian ALK
In contrast to D. melanogaster ALK the natural ligand for mammalian ALK
remains a mystery, even about 20 years after the initial identification. Pleiotrophin
(PTN) and Midkine (MK) have been reported as activating ligands for mammalian
ALK [20, 47, 48]. These small heparin-binding growth factors play a role in neural
18
development, survival and tumorigenesis [49, 50]. However, MK and PTN are also
able to activate other receptors like RPTPβ/ζ, N-syndecan, LRP and integrins [5156]. An alternative ALK activating mechanism by PTN involves the receptor
phosphatase RPTPβ/ζ: PTN binding inhibits this phosphatase, resulting in activation
of the ALK signalling cascade (Figure 5) [57]. However, to date the stimulation of
ALK by PTN and MK remains controversial: while some groups proved PTN and
MK mediated ALK activation, others have shown contradictory results [20, 48, 5867]. To sum up, the quest for the natural ligand for ALK is still ongoing.
As the natural ligand for mammalian ALK remains unidentified, wild type
ALK needs to be activated artificially, either by substituting the ALK extracellular
domain by mouse IgG Fc domain or by activating monoclonal antibodies, resulting
in the activation of the MAPK pathway (Figure 5) [64, 68-70]. Activation of the
MAPK pathway occurs via the association of Shc and FRS2 with ALK and NPMALK respectively [64, 68, 71]. Further, ALK is phosphorylated if the extracellular
domain of ALK is replaced by the extracellular part of the epidermal growth factor
receptor (EGFR), which results in the activation of PLCγ and PI3K [72]. Stimulation
of ALK by activating monoclonal antibodies or PTN leads to the activation of the
PI3K/Akt pathway as well, resulting in increased proliferation [58, 66]. Further, Kuo
et al., published data showing that MK stimulates ALK which leads to association
with IRS-1 and Shc, thereby activating ALK downstream signalling [60]. Further,
ALK is able to activate the small GTPase Rap1 by activating its GEF C3G in PC12
and neuroblastoma cells, which will be discussed further in Article I [73].
Currently, the physiological function of mammalian ALK is unknown.
However, as ALK mRNA is expressed widely in the nervous system during mouse
embryogenesis, in the developing chick central nervous system, as well as in
developing dorsal root ganglia in rat, ALK is suggested to function in the
development of the nervous system [15, 16, 74-78]. This suggested role is further
strengthened by ALK protein expression in tissue samples of the human central
nervous system [79] as well as in cell culture systems: several groups showed in in
vitro cell culture studies that activated ALK induces neuronal differentiation in
PC12 cells, which is mediated via several signalling pathways downstream of ALK
[63, 64, 68-70, 80]. ALK’s role in the nervous system is further fortified by mouse
studies. The study by Bilsland et al., shows that ALK mutant mice show enhanced
basal hippocampal progenitor proliferation and dopaminergic signalling in the
frontal cortex. Further, those mice performed better in object-recognition tests [81].
Weiss et al., reported that ALK mutant mice possess reduced neurogenesis, have
lower anxiety levels and have improved spatial memory which is LTK dependent,
indicating genetic interactions between ALK and LTK [82]. The fact, that ALK
seems to play an important role in neurodevelopment suggests that ALK might be
involved in neurological or mental disorders. Indeed, polymorphisms within the
intracellular domain of ALK have been correlated with increased susceptibility to
schizophrenia [83].
19
Additionally, like dALK, mouse ALK mutants show increased ethanol
tolerance indicating that those behavioral responses are evolutionary conserved [41].
An elegant mouse study by Lasek et al., showed that ALK expression is
transcriptionally regulated by LMO (LIM-domain only) and ERα (estrogen receptor
α), which has impact on behavioral responses to cocaine [84].
A different function for ALK is proposed by Mourali et al., who suggest that
ALK belongs to the group of dependence receptors. In ALK expressing Jurkat and
13.S.1.24 rat neuroblast cells they show that in the absence of a ligand ALK
possesses proapoptotic activity. However, upon extracellular stimulation as well as
intrinsic activation by caspase-mediated cleavage at D1160 ALK has anti-apoptotic
properties (Figure 5) [65].
2.3
Oncogenic ALK signalling
As ALK was initially discovered as the fusion protein NPM-ALK most
knowledge about ALK signalling has been gained from studies on NPM-ALK. The
major signalling pathways downstream of ALK include the PLCγ, the PI3K/Akt, the
MAPK and the JAK/STAT pathways (Figure 6):
Via its SH2-domain, PLCγ binds to the tyrosine at position 664 on NPMALK, thereby phosphorylating and activating PLCγ. This activation of PLCγ by
NPM-ALK results in transformation in transfected cell culture systems, contributing
to the mitogenic activity of NPM-ALK [25].
NPM-ALK activates the Akt pathway via interaction with the p85 subunit of
PI3K, leading to anti-apoptotic signalling, transformation and tumour growth [8587]. Further, activation of the PI3K/Akt pathway by NPM-ALK does not only result
in phosphorylation of mTOR but regulates also the transcription of FOXO3a target
genes, e.g. cyclin D2, Bin-1 and p27kip1 [88-90]. Recently it has been shown that
NPM-ALK mediated activation of the PI3K/Akt pathway regulates the
phosphorylation of Serine 9 at GSK3β, thereby inhibiting GSK3β activity. This
leads to accumulation of CDC25A and Mcl-1, resulting in enhanced oncogenesis
[91].
The third major pathway downstream of NPM-ALK is the MAPK pathway.
The activated receptor interacts with IRS-1, Shc and Grb2, which results in the
assembly and stimulation of downstream signalling [89, 92-94].
Another pathway activated by NPM-ALK is the JAK/STAT pathway. It has
been demonstrated by several groups that STAT3 is phosphorylated and thereby
activated by NPM-ALK, a process which can be blocked by ALK inhibitors [95100]. However, the exact mechanism of how NPM-ALK is able to activate STAT3
remains unclear. Some studies report an association of JAK3 with NPM-ALK and
JAK3 inhibition leads to reduced STAT3 activation [99, 101, 102]. On the other
hand, some data suggest that ALK binds and activates STAT3 directly involving the
first tyrosine in the YxxxYY motif [27]. Regulators of the JAK/STAT pathway like
Shp1, which has been shown to interact with NPM-ALK, and protein phosphatase
20
2A are abnormally expressed in ALK-positive ALCL [100, 103, 104]. The role of
STAT3 in the pathogenesis of NPM-ALK positive ALCL has been confirmed,
however the involvement of STAT5 in NPM-ALK mediated oncogenicity is less
established. While some groups suggest an NPM-ALK mediated activation of
STAT5B, which leads to apoptosis and cell-cycle arrest, others could not show
STAT5 activation [99, 100, 105]. However, STAT5 might not only promote
oncogenicity, but there is evidence that STAT5A might act as a tumour suppressor
in ALK-positive ALCL cell lines [106].
Figure 6: Oncogenic ALK signalling.
The requirement of ligand binding for downstream signalling of constitutively active ALK either by
point mutations or amplification is most likely not required. While the downstream signalling of ALK
translocations is fairly well investigated, in case of amplified and mutated ALK several potential
downstream signalling pathways need to be confirmed (grey font).
21
Besides the above named major signalling pathways further downstream
targets of NPM-ALK have been identified. NPM-ALK activates the small GTPase
Cdc42 thereby controlling cell shape and proliferation of ALCL [107]. Another
GTPase downstream of NPM-ALK is Rac1 which is activated by the exchange
factor Vav3 [108]. Vav3 binds to Y343 of NPM-ALK via its SH2-domain and is
phosphorylated. The activation of Vav3/Rac1 is involved in motility and invasion of
NPM-ALK positive ALCL. Further, NPM-ALK’s transforming potential is
mediated by p130 Crk-associated substrate (p130Cas) via Grb2 [109]. Also Src
kinases like pp60Src are involved in NPM-ALK positive ALCL cell proliferation,
while the tyrosine phosphatase Shp2 binds to NPM-ALK thereby mediating cell
growth and migration [110, 111]. NIPA (nuclear interacting partner of ALK), a
ubiquitin E3 ligase, binds to NPM-ALK resulting in anti-apoptotic signalling [112,
113]. NPM-ALK is also able to activate JNK, leading to lymphoma development in
mice as well as cell-cycle progression and oncogenesis in ALCL cell lines [114,
115]. Further, JNK participates in the blocking of the p53 tumor suppressor pathway
downstream of NPM-ALK [116]. An elegant study by Singh et al., suggested a
cooperation between the Sonic Hedgehog (SHH) and the PI3K pathway downstream
of NPM-ALK: here, PI3K activation by NPM-ALK regulates SHH/GLI1 signalling
resulting in synergistic effects that contribute to NPM-ALK’s oncogenicity in ALCL
[117]. Additional novel ALK targets and interacting proteins like Dok2, IRS1, SHC,
Crk, CrkL, STAT3, VASP and ATIC were identified using proteomics-based
approaches [92, 118-120]. Various ALK regulated genes, e.g. the anti-apoptotic
protein BCL2A and the transcription factor C/EBPβ, were identified in screens of
the transcriptomes of ALCL cell lines [121, 122]. Further, a recent report identified
for the first time that serine phosphorylation of NPM-ALK is also important for
ERK and JNK signalling and therefore contributes to the oncogenic potential of
NPM-ALK [123].
Due to its initial discovery, NPM-ALK has been investigated most
intensively. The other known fusion proteins are thought to mediate downstream
signals in a similar manner, although this has not been proven for all and some
differences have been reported: e.g. ATIC-ALK interacts with Grb2 and Shc, while
TGF-ALK binds to Grb2, Shc and PLCγ [124, 125]. Further, KIF5B-ALK, an ALK
fusion protein detected in Non-small cell lung cancer, activates STAT3 and Akt
[126]. Investigation of NPM-ALK, TPM3-ALK, TFG-ALK, CLTC-ALK and
ATIC-ALK revealed different activation of signalling pathways like PI3K/Akt,
resulting in different transforming and tumourigenic potential [127]. One particular
ALK fusion protein – EML4-ALK – has received great attention in the last couple
of years, thereby increasing the knowledge of this ALK fusion protein [128, 129].
EML4-ALK possesses transforming potential [128, 129], and treatment with the
ALK specific inhibitor NVP-TAE684 results in apoptosis in lung cancer cells via
the ERK/BIM and STAT3/survivin signalling pathways [130]. Blocking EML4ALK signalling by ALK specific inhibitors resulted in the development of new
therapeutic treatments for lung cancer patients [131].
22
However, oncogenic ALK signalling derives not only from fusion proteins,
but also from ALK overexpression and activating point mutations which will be
discussed below [76, 128, 132-136]. The activity of some of these mutant ALK
receptors can be blocked by ALK-specific inhibitors [76, 78, 133, 134].
3
ALK in diseases
Although the physiological function of ALK in mammals is still unclear, the
role of ALK in the development and onset of several diseases has been well
established. In 1994 a screen from patients with Anaplastic Large Cell Lymphoma
(ALCL) revealed a new fusion protein – NPM-ALK – where the N-terminal part of
nucleophosmin is fused to the kinase domain of ALK as a result of a t(2;5)(p23;q35)
translocation [13]. Since this initial discovery a plethora of ALK fusion proteins
have been described in inflammatory myofibroblastic tumours (IMT) [137], Nonsmall cell lung cancer (NSCLC) [138, 139], diffuse large B-cell lymphomas
(DLBCLs) [140], renal cell carcinoma [141] and squamous cell carcinoma of the
esophagus (SCC) [142, 143]. Besides translocation ALK overexpression [59, 144149] as well as point mutations [128, 132-135] have been reported in several cancers
(Figure 7).
3.1
ALK translocations
3.1.1 Anaplastic Large Cell Lymphoma (ALCL)
Most studies regarding ALK have been performed in ALCL. This disease,
which was described in 1985 for the first time [150], is a type of Non-Hodgkin’s
lymphoma arising from T-cells. ALCL is characterized by large horseshoe shaped
nuclei and the expression of CD30. Further, ALK is expressed in 60 – 80% of
ALCL, which is mainly observed in children and young adults [151-153]. The fact
that ALK positive ALCL patients have a higher 5-year survival rate compared to
ALK negative ALCL patients, makes ALK expression an important prognostic
factor [153-157]. However, some reports described the detection of NPM-ALK in
blood and lymphoid tissue of healthy persons, raising the question of whether NPMALK on its own is sufficient to promote tumorigenesis [158, 159]. Since the initial
discovery of NPM-ALK, numerous other fusion partners for ALK have been
reported in ALCL (Figure 7): Tropomyosin 3 and 4 (TPM3/4) [160-162], TRKfused gene (TFG) [124, 163], 5-aminoimidazole-4-carboxamide ribonucleotide
formyltransferase/IMP cyclohydrolase (ATIC) [125, 164, 165], Clathrin heavy
chain-like 1 (CLTC1) [166], Moesin (MSN) [167, 168], ALK lymphoma
oligomerization partner on chromosome 17 (ALO17) [169] and Non-muscle myosin
heavy chain (MYH9) [170]. This great variety of fusion partners mirrors the high
23
ability of the ALK locus to undergo recombination, although the reason for this
process is still poorly understood.
3.1.2 Inflammatory Myofibroblastic Tumour (IMT)
IMT belongs to the class of “Inflammatory Pseudotumors”. These tumors
occur mostly in young individuals, although they may also appear in older patients
[171]. Tumours are situated mostly in soft tissues, most commonly in lung, abdomen
and retroperitoneum, although they can be located anywhere in the body [172].
Further, inflammatory infiltrates containing plasma cells and lymphocytes are
detected in IMTs [171]. Griffin et al., reported the first appearance of ALK in IMT
in 1999, describing a 2p23 chromosomal rearrangement and ALK expression in
IMT, suggesting ALK’s involvement in solid tumours for the first time [137]. Since
then, several additional ALK fusion partners in IMT have been described (Figure 7):
Tropomyosin 3 and 4 (TPM3/4) [173], 5-aminoimidazole-4-carboxamide
ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC) [174], Clathrin heavy
chain-like 1 (CLTC1) [175, 176], RAN binding protein 2 (RANBP2) [177],
Cysteinyl-tRNA synthetase (CARS) [169, 178], SEC31 homologue A (S. cerevisiae)
(SEC31L1) [179] and F polypeptide-interacting protein-binding protein 1
(PPFIBP1) [180]. Altogether, about 50% of all IMTs appear to have ALK
rearrangements and in accordance with ALCL, ALK positive IMTs seem to occur
mostly in younger individuals [137, 173, 181, 182].
3.1.3 Non-small cell lung cancer (NSCLC)
Lung cancer is the most widespread cause of cancer death with 1.4 million
deaths/year worldwide (2008) [183]. Lung cancer is split into two major subgroups:
Small cell lung cancer (SCLC) and Non-small cell lung cancer (NSCLC). The latter
one accounts for about 80% of all lung cancers and responds poorly to conventional
cancer treatments. In 2007 a novel fusion protein, EML4-ALK, where the Nterminal domain of echinoderm microtubule associated protein like 4 is fused to the
ALK kinase domain, was described in NSCLC [138, 139]. Since then, further
variants of EML4-ALK have been reported in NSCLC [138, 139, 184, 185]. EML4ALK occurs in approximately 3 – 13% of all lung tumours and in about 5% of all
NSCLC [139, 186-192]. Further ALK fusion partners in NSCLC have been
identified: TRK-fused gene (TFG) [138] and kinesin family member 5B (KIF5B)
[126, 185], although those fusion proteins are not as common as EML4-ALK [131,
193]. Interestingly, ALK translocations and mutations in other known oncogenes
like EGFR or KRAS seem to be mutually exclusive in NSCLC patients [186, 188,
190, 194, 195]. However, Sasaki et al., identified a subset of NSCLC patients (3/50;
6%) harbouring ALK rearrangements together with EGFR activating mutations
[196].
24
Figure 7: Schematic overview of ALK aberrations in cancer.
ALK fusion proteins, in which the kinase domain of ALK is fused to the N-terminal portion of various
proteins, have been described in numerous cancers. Moreover, secondary mutations in the context of ALK
fusions have been described. ALK overexpression has been reported in a number of cancer types. ALK
point mutations have been found mainly in neuroblastoma, where most of the mutations are situated within
the kinase domain of ALK, but also in ALK gene of NSCLC and ATC origin. The structural model shows
the ALK kinase domain with the C-helix (orange), the P-loop (green), activation loop (blue) and catalytic
loop (yellow) highlighted. Red balls indicate verified point mutations observed in neuroblastoma patients.
The three most common sites of point mutations in neuroblastoma are indicated. Abbreviations for the
ALK fusion partners are explained in the chapter “ALK translocations”.
3.1.4 Diffuse large B-cell lymphoma (DLBCL)
The most frequent ALK fusion partner in DLBCL is clathrin (CLTC) [155,
166, 197-200]. ALK-positive DLBCLs express epithelial membrane antigen (EMA),
immunoglobulin light chains, CD38 and CD138, but do not express many B- and Tcell markers like CD30 antigen [155, 197, 198]. DLBCLs are associated with a poor
clinical outcome and respond poorly to chemotherapy [201, 202]. Other fusion
proteins described in some DLBCL cases are NPM-ALK and SQSTM1
(Sequestosome 1)-ALK [203-205]. Further, an insertion of a 3’-ALK sequence at
chromosome 4q22-24 has been reported in DLBCL, although the ALK fusion
partner is unknown so far [206].
3.1.5 Renal cell carcinoma
Recently, the fusion product of ALK and vinculin (VCL) has been detected in
young patients diagnosed with renal cell carcinoma [141]. This (2;10) translocation
results in the VCL-ALK fusion product which seem to be important for
development and onset of renal medullary carcinoma [207]. Recently, TPM3- and
25
EML4-ALK have been detected in cases of adult renal cell carcinoma [208].
However, as only few reports have been published so far, further investigation of
this patient population for the existence of ALK fusions will be very interesting.
Despite the increasing number of different ALK fusion partners and different
cancer types harbouring ALK rearrangements, the various fusion proteins have
several common features: (1) the promoter of the ALK partner protein drives
transcription of the fusion protein; (2) the ALK partner protein seems to determine
the cellular localization, i.e. rather than being inserted into the cell membrane ALK
fusion proteins are localized in the cytosol; and (3) oligomerization of the ALK
partner protein leads to auto-phosphorylation and thereby activation of the ALK
kinase domain, resulting in downstream signalling events that lead to biological
outcomes involved in tumourigenesis.
3.2
ALK overexpression
ALK overexpression has been described in a variety of different cancers like
thyroid carcinoma, NSCLC, breast cancer, melanoma, neuroblastoma, glioblastoma,
astrocytoma, retinoblastoma, Ewing’s sarcoma and rhabdomyosarcoma (Figure 7)
[59, 148, 149]. Further, ALK expression has been reported in leiomyosarcoma,
peripheral nerve sheath tumours and malignant fibrous histocytoma [209].
Additional discussion of ALK overexpression especially in neuroblastoma will be
covered in the chapter “Genetic hallmarks of neuroblastoma”.
3.3
Point mutations of ALK
3.3.1 Neuroblastoma
Neuroblastoma is the most common solid extracranial childhood cancer,
being responsible for approximately 15% of all pediatric cancer deaths [210]. This
disease derives from neural crest cells of the sympaticoadrenal lineage and can
hence appear throughout the sympathetic nervous system. Most primary tumours are
located within the abdomen, followed by neck, chest and pelvis. However, despite
improved clinical treatments, the long-term survival rate for children with high-risk
neuroblastoma still ranges below 40% [211]. According to the International
Neuroblastoma Risk Group (INRG) staging system, neuroblastoma can be divided
into different stages (1, 2, 3, 4 and 4S according to the old staging system and L1,
L2, M and MS according to the new one) depending on several criteria, e.g. age
(patient <18 month have a more favourable prognosis), ploidy and MYCN
amplification status (correlated with poor prognosis).
26
3.3.2 Genetic hallmarks of neuroblastoma
The most common genetic hallmarks of neuroblastoma are deletions of parts
of chromosome arms 1p and 11q, gain of parts of 17q, triploidy and MYCN
amplification [134, 212-215].
Deletions of 1p correlate with MYCN amplification and advanced disease
stage [212]. In particular, allelic loss at 1p36 predicts disease progression but not
overall survival [216]. Attempts are carried out to identify putative tumor suppressor
genes that are deleted within this region. One tumor suppressor candidate is the
calmodulin binding transcription activator 1 (CAMTA1). Ectopic CAMTA1
expression resulted in decreased cell proliferation, colony formation and suppressed
growth of tumor xenografts. Further, CAMTA1 induced differentiation in
neuroblastoma cell lines, suggesting that CAMTA1 is a potential tumor suppressor
in neuroblastoma [217].
Allelic loss of 11q is inversely correlated with 1p deletion and is rarely seen
in tumors with amplified MYCN. However, loss of 11q is associated with decreased
event-free survival [212]. One candidate gene within the region that is frequently
deleted is the putative tumor suppressor gene TSLC1/IGSF4/CADM1 (Tumor
suppressor in lung cancer 1/Immunoglobulin superfamily 4/Cell adhesion molecule
1). TSLC1 expression levels are reduced in unfavorable neuroblastomas and are
assocated with poor prognosis. In addition, TSLC1 reduced proliferation of a
neuroblastoma cell line, suggesting that TSLC1 might be a tumor suppressor gene
involved in neuroblastoma [218].
Another hallmark represents the gain of parts of 17q. The breakpoints vary,
but gain of a region from 17q22-qter suggests a selective advantage due to dosage
effects of one or more genes [219]. One example is the overexpression of survivin,
an inhibitor of apoptosis [220].
Measuring the DNA content of neuroblastomas serves as an important
prognostic marker. Roughly, the DNA content can be divided into two groups: neardiploid or hyperdiploid (often near triploid). Triploidy seems to be favourable and is
associated with less aggressive tumors, while near-diploidy is more often seen in
malignant neuroblastomas [210, 212].
One characteristic hallmark of neuroblastoma is the amplification of the
MYCN gene locus on chromosome 2p23-24 (~24% of all cases), correlating with
poor survival [212, 221, 222]. MYCN belongs to the family of MYC protooncogenes, which comprises also c-MYC and MYCL. All MYC proteins are basic
helix-loop-helix transcription factors forming a heterodimeric complex with Max,
which can bind to E-box sequences (CACGTG) (reviewed in [223]). Generally,
MYC plays a crucial role during embryonic development. While c-MYC is
expressed abundantly, MYCN expression is very restricted to certain tissues during
development (reviewed in [224]). Interestingly, c-MYC and MYCN appear to be
complementarily expressed, suggesting also a difference in function. However,
according to a mouse study MYCN is able to replace c-MYC during development
[225].
27
Like all MYC proteins, MYCN participates in the regulation of many cellular
processes like cell proliferation, growth, protein synthesis, metabolism but also
apoptosis and differentiation [226]. However, amplification or overexpression of
MYCN contributes to the development of several cancers, often originating from
tissues with normal MYCN expression. These tumors include beside the above
mentioned neuroblastoma also retinoblastoma, glioblastoma and small cell lung
cancer [227-229]. Further, neuroblastoma cell lines harbouring MYCN amplification
show increased proliferation, downregulation of angiogenesis inhibitors, blocked
terminal differentiation and enhanced invasive potential [212, 222, 230, 231].
Therefore, MYC is a double-edged sword: on the one hand MYC is crucial for
development and many cellular processes, on the other hand deregulated MYC
exhibits detrimental effects. However, MYC alone has no transforming capacity,
rather it needs some companions. In murine cells, the oncoproteins MYC and Ras
cooperate in cellular transformation, while more events are necessary in human cells
[232, 233]. Human cell transformation requires both the inactivation of tumor
suppressor genes and unlimited replication ability (hTERT activation) together with
oncogene activation. Generally, amplified MYCN appears to be a key factor in the
development of neuroblastoma. However, despite these findings, it has been
suggested that MYCN amplification alone is not sufficient to initiate tumour
formation [234].
Expression of full-length ALK in neuroblastoma was described for the first
time in 2000 by Lamant et al., [235]. Subsequent studies described ALK
overexpression as a result of genetic amplification both in neuroblastoma cell lines
and patient samples. This alk amplification leads to ALK activation, which probably
is involved in the development or the onset of this disease, correlating with a poor
prognosis of neuroblastoma patients [144-147, 236]. Interestingly, alk amplification
in neuroblastoma tumours is often observed together with MYCN [132, 237, 238].
Further, mutation-independent ALK overexpression in neuroblastoma patients
correlates with a poor prognosis, further strengthening the role for ALK
overexpression in neuroblastoma [147]. Interestingly, ALK protein levels do not
necessarily correlate with ALK mRNA levels and/or genetic alterations, suggesting
that alternative mechanisms other than mutations or amplification regulate ALK
expression levels in neuroblastoma [147]. Moreover, Schulte et al., reported that
neuroblastomas with mutated ALK exhibited elevated ALK expression levels
compared with wild type ALK [236]. Further, neuroblastomas with enhanced wild
type ALK resemble neuroblastomas with mutated ALK in clinical and molecular
phenotypes, suggesting that high levels of wild type ALK may contribute to the
malignancy of neuroblastoma [236].
3.3.3 ALK point mutations in neuroblastoma
It was not until the year 2008 that activating ALK point mutations were
described in both familial and sporadic neuroblastoma as well as neuroblastoma cell
lines (Table 1) [128, 132-135]. Most of these mutations are situated within or
28
adjacent to the ALK kinase domain and are suggested to be gain-of-function
mutations [239, 240]. siRNA mediated knockdown of ALK expression decreased
proliferation of neuroblastoma cell lines [135], while expression of F1174L and
K1062M ALK mutants in NIH3T3 cells and nude mice resulted in rapid formation
of subcutaneous tumours, demonstrating the oncogenic and transforming potential
of these ALK mutants [128]. To date several ALK point mutants have been
experimentally investigated to determine whether they are true gain-of-function
mutations [135]. Indeed, several groups including our own could demonstrate that
the majority of ALK point mutations are constitutively active and can be inhibited
by small ALK-specific inhibitors like NVP-TAE684 or crizotinib, which will be
discussed explicitly in Article II and III [128, 241, 242]. Surprisingly, one somatic
mutation – I1250T – which has been identified in two neuroblastoma patients, is in
fact inactive and might possibly act as a dominant negative RTK, as discussed in
detail in Article IV [243]. Further, several silent mutations have been described in
neuroblastoma patients [236]. However, the three major mutational hotspots are
F1174, R1275 and F1245; notably, mutations at position F1174 appear only in
somatic tumours [213, 239]. A recent meta-analysis of neuroblastoma by Brouwer et
al., reported also that ALK gain-of-function mutation occured at a frequency of
6.9% in investigated neuroblastoma tumours. Further, when comparing ALK
mutation frequency in relation to genomic subtype, ALK mutations were most
frequently observed in MYCN amplified tumours (8.9%), correlating with poor
clinical outcome [213]. Based on this report one could strongly assume that a
connection between ALK and MYCN expression exists. Indeed, as will be discussed
explicitly in Article V, ALK is able to regulate the initiation of the transcription of
MYCN in neuroblastoma cell lines [244]. Recently two cases of congenital
neuroblastoma carrying somatic, heterozygous ALK mutations (F1174L and
F1245V, respectively) showed severe encephalopathy and brainstem abnormalities,
suggesting that abnormal ALK activation is detrimental to the development of the
central nervous system [245]. Further, the F1174L and R1275Q ALK variants were
reported to exhibit impaired maturation with defective N-linked glycosylation [246].
The importance of N-linked glycosylation of ALK was further strengthed by a report
by Del Grosso et al., where inhibition of N-linked glycosylation results in reduced
ALK phosphorylation, subsequent decreased downstream signalling and impaired
cell viability in ALK mutated/amplified neuroblastoma cells [247]. An additional
method of aberrant ALK activation is the expression of a truncated form of ALK in
neuroblastoma [248]. An in-frame deletion spanning exons 2 and 3 results in the
expression of a 208 kDa form of ALK, which is auto-phosphorylated. This ALK
variant retains mainly in the endoplasmatic reticulum and has transforming ability.
29
a.a.
mutation
Table 1: Point mutations found in human ALK
disease
G/S targeted region
phenotype
ref.
?
First MAM domain
?
[249]
S413N
V597A
H694R
Burkitt’s
lymphoma (CL)
lung cancer
lung cancer
lung cancer
S
S
S
?
?
G-O-F
[76]
[76]
[76]
G881D
C1021Y
lung cancer
Osteosarcoma (CL)
S
?
?
?
[76]
[249]
K1062M
NB
ND
?
[128]
T1087I
NB
G
?
[128]
D1091N
NB
S
?
[135]
A1099T
NB
?
?
NP
G1123S/D
?
G-O-F
[250]
G1128A
Resistant in SHSY5Y
NB
G-O-F
[135, 242]
T1151M
1151Tins
L1152R
C1156Y
NB
NSCLC
NSCLC
NSCLC
G
?
?
ND
?
Sec. mut.
Sec. mut.
Sec. mut.
[133]
[251]
[196]
[184]
M1166R
I1170T/S
I1171N
F1174L
NB
NB
NB
NB
S
ND
S
S
First MAM domain
Second MAM domain
Between
second
MAM and G-rich
domain
G-rich domain
Extracellular domain,
close
to
transmembrane
domain
Juxtamembrane
domain
Juxtamembrane
domain
Juxtamembrane
domain
Juxtamembrane
domain
Loop in between β1
and β2
Loop in between β1
and β2
End of β3 sheet
End of β3 sheet
End of β3 sheet
Loop in between β3
and αC
αC-helix
αC-helix
αC-helix
End of αC-helix
?
?
G-O-F
G-O-F
F1174S
F1174I
F1174C
F1174V
R1192Q
S
G, S
S
S
?
End of αC-helix
End of αC-helix
End of αC-helix
End of αC-helix
Loop in between β4
and β5
G-O-F
?
?
?
?
R1192P
NB
NB
NB
NB
Uterine
leiomyosarcoma
(CL)
NB
[135]
[236]
[135, 242]
[128, 132,
133, 135]
[241]
[132, 135]
[128]
[128, 134]
[249]
G
G-O-F
[135, 242]
L1196M
L1198F
NSCLC
ATC
ND
ND
Sec. mut.
G-O-F
[184]
[136]
L1198P
ND
G-O-F
[250]
G1201E
Resistant EML4ALK in Ba/F3, SHSY5Y
ATC
Loop in between β4
and β5
gatekeeper
Loop in between β4
and β5
Loop in between β4
and β5
G-O-F
[136]
G1202R
NSCLC
?
Sec. mut.
[251]
D1203N
Resistant EML4ALK in Ba/F3
ND
Loop in between β4
and β5
Loop in between β4
and β5
Loop in between β4
and β5
G-O-F
[250]
R412C
30
G
ND
S1206Y
NSCLC
?
Loop in between β4
and β5
αE helix
Catalytic loop
Catalytic loop
-2 to HRD, catalytic
loop
-2 to HRD, catalytic
loop
-2 to HRD, catalytic
loop
-2 to HRD, catalytic
loop
+1 to HRD, catalytic
loop
Kinase domain
Sec. mut.
[251]
A1234T
Y1239H
L1240V
F1245C
NB
lung cancer
NB
NB
S
S
S
S
?
?
?
G-O-F
?
[133]
[76]
[236]
[133, 135,
242]
[132]
F1245I
NB
S
F1245L
NB
S
?
[128, 132]
F1245V
NB
S
?
[133, 135]
I1250T
NB
G
KD
[133, 135,
243]
[249]
A1252V
?
G1269A
R1275Q
Carcinoma of the
endometrium (CL)
NSCLC
NB
Kinase domain
+2 to DFG, activation
loop
+2 to DFG, activation
loop
A-loop,
NPM-ALK
(Y338) essential for
kinase activity [76]
?
G-O-F
R1275L
NB
ND
Y1278H
?
Y1278S
Resistant in SHSY5Y,
only
together
with
G1123S/D
NB, lung cancer
S
D1311A
E1384K
Lung cancer (CL)
lung cancer
?
S
1464STOP
NB
ND
K1518N
Lung cancer (CL)
?
K1525E
Upper respiratory
tract
adenocarcinoma
(CL)
?
ND
G, S
?
?
[252]
[128, 132135]
[134]
G-O-F
[250]
A-loop,
NPM-ALK
(Y338) essential for
kinase activity [76]
?
[76, 134]
Kinase domain
C-terminal end of
kinase domain
C-terminal to kinase
domain
C-terminal end of
kinase domain
C-terminal end of
kinase domain
?
G-O-F
[249]
[76]
?
NP
?
[249]
?
[249]
Silent
mutations
[236]
ATC: Anaplastic Thyroid Carinoma; CL: Cell line; G-O-F: Gain-of-function; G/S: Germline/Somatic;
KD: Kinase Dead; Sec. mut.: Secondary Mutation; NB: Neuroblastoma; ND: Not determined; NP: Not
published; NSCLC: Non-small cell lung cancer; Targeted regions in kinase domain according to Bossi
et al., [28]. ALK mutations investigated and discussed in this thesis are underlined.
3.3.4 ALK point mutations in other cancers
Since the initial reports, ALK point mutations have not only been found in
neuroblastoma, but have also been described in other cancers like lung cancer and
Anaplastic thyroid cancer (ATC), as well as in various cancer cell lines, including
Burkitt’s lymphoma and osteosarcoma cell lines. In ATC, the novel ALK point
31
mutations L1198F and G1201E are constitutively active [136]. In lung cancer ALK
mutations have also been observed in the extracellular domain of ALK. Most
extracellular ALK mutations possess weak oncogenic potential, except the mutation
H694R that is highly transforming, although the exact mechanism behind the
increased activating nature of this mutation remains unclear [76]. Recently, two
newly identified ALK mutations are of clinical importance. In a NSCLC sample
isolated from a crizotinib-treated patient two secondary mutations in the kinase
domain of EML4-ALK – C1156Y and L1196M – were identified. L1196M is in the
gatekeeper site and as expected showed resistance to crizotinib therapy [184, 193].
However, as C1156Y is not located at the gatekeeper position but in the loop
between β3 and the αC-helix, the mechanism of this secondary mutation is still
unclear [184]. Additional ALK point mutations in NSCLC patient samples were
identified by Katayama et al., [251]. Those mutations include 1151Tins, G1202R
and S1206Y and, like the previous secondary mutations, are localized near the ATP
binding pocket of ALK. Recently, Sasaki et al., described a further secondary ALK
mutation, L1152R, found in a crizotinib-resistant cell line. This mutation also
showed resistance to the structurally unrelated ALK-specific inhibitor NVPTAE684 [196]. On the other hand, other ALK mutants identified in various cancer
cell lines were not tyrosine phosphorylated and were unable to drive foci formation
compared to the F1174L ALK mutant [249]. These findings suggest that those
described ALK mutations might represent “passenger” mutations.
Since the occurrence of secondary ALK mutations exhibiting resistance
towards crizotinib treatment, efforts are being made to identify further ALK
mutations that might possibly show resistance to ALK inhibitors. Indeed,
Heuckmann et al., screened cell lines for mutations showing resistance to crizotinib
and/or NVP-TAE684 [250]. While some crizotinib resistant mutations were highly
sensitive to NVP-TAE684, two novel EML4-ALK mutations (L1198P and D1203N)
showed resistance to both ALK-specific inhibitors. These results suggest differences
in therapeutic efficacy depending on ALK inhibitors and ALK mutations. However,
these mutations have not been found in cancer patients yet.
4
Treatments for ALK-positive carcinomas
Depending on type and onset of disease, the commonly used therapeutical
approaches include different combinations of surgery, radiation therapy and
chemotherapy. However, in addition to the above named “traditional” methods,
alternative therapies are starting to be employed. As ALK is an established
oncogene in many different cancers by now, this RTK serves as an excellent target
for treatment by e.g. kinase inhibitors.
32
4.1
Kinase inhibitors
The development of tyrosine kinase inhibitors has influenced the treatment of
cancer patients significantly. One famous example is Gleevec (Imatinib), targeting
BCR-ABL in chronic myeloid leukemia (CML) [253, 254]. Besides inhibiting ABL,
Gleevec also blocks the RTKs c-Kit and PDGFR [255, 256]. Further established
RTK inhibitors are Gefitinib and Erlotinib, which block the activity of EGFR
(ErbB1) and are currently used in the treatment of NSCLC patients [257].
4.1.1 ALK-specific tyrosine kinase inhibitors
As ALK seems play a more and more important role in many cancers, the use
of ALK-specific inhibitors should influence the treatment of a variety of cancers in a
positive way. One of the first ALK-specific inhibitors is NVP-TAE684 targeting the
ATP binding pocket of ALK. According to initial tests and an extensive screen of
various human cancer cell lines, NVP-TAE684 not only blocked proliferation of
ALK-positive ALCL cells, but of neuroblastoma and NSCLC cell lines as well [96,
258]. Since then several studies reported that tumours induced by constitutively
active ALK in vivo as well as cells expressing ALK gain-of-function mutations
demonstrate sensitivity towards NVP-TAE684, including both ALK translocations
and neuroblastoma cells harboring ALK point mutations [76, 133, 259-261].
Interestingly, the study by Duijkers et al., reports that neuroblastoma cell lines with
mutated ALK express ALK on higher mRNA and protein levels and respond better
to ALK inhibitors [260]. However, recently NVP-TAE684 was shown to effectively
inhibit the leucine-rich repeat kinase 2 (LRRK2) which is involved in Parkinson’s
disease [262].
To date, numerous other ALK inhibitors have been developed (Table 2)
(reviewed in [131]). Amongst all ALK-specific inhibitors crizotinib (PF-2341066)
has undergone a very impressive and rapid development to an anticancer drug since
the first report in 2007. Like NVP-TAE684, crizotinib is an ATP-competitive small
molecule inhibitor as well, targeting not only ALK but also c-Met [263, 264].
Already three years later, in 2010, first results of clinical trials in NSCLC patients
harbouring EML4-ALK were very promising, resulting in recent FDA approval
under the name Xalkori [193, 265-267]. Currently, phase III trials are ongoing and
despite some reports of certain ALK mutations, especially secondary mutations, in
EML4-ALK positive NSCLC exhibiting crizotinib resistance [184, 196, 268, 269],
the results indicate a 64% overall survival of crizotinib-treated ALK-positive
patients after two years [270]. Further, Bresler et al., reported that cell lines
harbouring the F1174L ALK mutation were relatively resistant to crizotinib due to
an increased affinity to ATP binding, an effect that might be overcome with higher
doses of crizotinib and/or higher-affinity inhibitors [271]. Also an IMT patient
harbouring the RANBP-ALK translocation responded partially to crizotinib while
two ALK-positive ALCL patients had complete response to crizotinib [272, 273].
Altogether, those promising reports support the therapeutical use of crizotinib for
ALK-positive cancer patients.
33
The AP26113 inhibitor was reported to block ALK activity in crizotinibresistant NSCLC cell lines harbouring EML4-ALK with the gatekeeper mutation
L1196M [261]. Another orally available inhibitor blocking ALK with the gatekeeper
mutation, and hence inhibiting EML4-ALK L1196M promoted cell proliferation, is
CH5424802 (AF802) [274, 275].
Two further potent small molecule tyrosine kinase inhibitors are X-378 and
X-396, showing high specificity towards ALK [276]. Particularly X-396 has been
shown to block EML4-ALK harboring the crizotinib-resistant mutations L1196M
and C1156Y. Further, combining X-396 with the mTOR inhibitor rapamycin
inhibited growth in a synergistic manner, suggesting an improved approach for the
treatment of ALK-positive cancer patients. Another highly potent and orally active
ALK inhibitor is CEP-28122 which shows anti-tumour activity in ALK-positive
ALCL, NSCLC and neuroblastoma [277]. Further interesting ALK inhibitors exist,
although little is known about them so far. GSK1838705A has been shown to block
ALK as well as IGF-IR and IR, thereby blocking the proliferation of cancer cell
lines and growth of tumour xenografts in nude mice [278]. While PHA-E429 has
been reported as a crystal structure with the ALK kinase domain [28], F91873 and
F91874 were identified as multikinase inhibitors showing activity against ALK in a
biochemical screen [279]. ASP3026 is in Phase I clinical trials for ALK related
malignancies, as is LDK378 from Novartis. However, little information exists about
the pre-clinical compound 3-39 from Novartis.
With regard to the development of secondary ALK mutations in samples
showing crizotinib-resistance, the need for novel inhibitors showing inhibitory
activity against ALK persists. Indeed, besides the above named second generation
ALK inhibitors CH5424802 and ASP-3026, new molecules are being described,
such as NMS-E628 [280], SJ-08-0025 [281], tetrahydropyridopyrazines [282],
piperidine carboxamides [283] and compounds from structural-based virtual
screening approaches [284]. Further, several groups are attempting to design and
modify ALK inhibitors based on structural insights of the kinase domain ([285] and
reviewed in [286]). Altogether, it will be interesting to see the development of new,
more potent and specific ALK inhibitors as well as the results of the clinical trials.
34
Company
Pfizer
Novartis
Inhibitor
Crizotinib
(PF-2341066,
Xalkori)
NVP-TAE684
LDK378
3-39
AF802
(CH5424802)
Table 2: ALK inhibitors
Clinical
G/W
trial,
phase
NCT00932893,
III
NCT01154140,
III
No
NCT00939770,
I/II
No
NCT01121588,
I/II
No
NCT00932451,
II
NCT00585195,
I
N/A
NCT01283516,
I
Pre-clinical
JapicCTI101264,
I/II
No
No
No
Yes
Yes
Yes
Aims of investigation
Crizotinib vs standard of care in
patients with advanced NSCLC
Randomized, open-label study of the
efficacy and safety of Crizotinib vs
Pemetrexed/Cisplatin or Pemetrexed
Carboplatin in previously untreated
patients
Young patients with relapsed or
refractory solid tumors, ALCL, CNS
or NBs
Safety and efficacy in patients with
tumors except NSCLC that are
ALK-positive
Safety and efficacy in NSCLC
patients
Safety in patients with advanced
malignancy
Not developed
Safety in ALK-positive/genetic
abnormal tumors, no available data
Safety, tolerability and
pharmacokinetic in NSCLC
patients with ALK-fusion
gene
II.
Efficacy and safety of AF802
IPI-504*
NCT01228435,
N/A
Inhibitor of Hsp90, which protects
Infinity
II
other proteins from being destroyed,
possibly also EML4-ALK fusion
proteins in NSCLC patients
ASP3026
NCT01284192,
ND
Safety and tolerability of ASP3026.
Astella
I
No pre-clinical data available but
aim for advanced malignancies, Bcell lymphoma, solid tumors and
ALK
AP-26113
NCT01449461,
Yes
AP-26113 abrogates Crizotinib
Ariad
I/II
resistant mutations in EML4-ALK.
Safety,
tolerability,
pharmacokinetics and preliminary
anti-tumor activity
X-396
Pre-clinical
Yes
X-396 inhibits two ALK point
Xcovery
mutations, C1156Y and L1196M,
works in synergy with rapamycin.
May initiate clinical trials by the end
of 2011
GSK-1838705A
Pre-clinical
Yes
Abrogates ALK and growth of
GlaxoALCL, some NBs and a subset of
Smith-Kline
NSCLC
ALCL: Anaplastic Large Cell Lymphoma; G/W: refers to ability to inhibit gateway mutation;
NB: Neuroblastoma; N/A: Not applicable; ND: Not determined; IPI-504 (marked with *) is not an
ALK inhibitor, but an Hsp90 inhibitor.
Chugai
I.
35
4.2
Other approaches to inhibit ALK activity
Besides small molecular inhibitors there are various other strategies to block
ALK activity. One approach is the reduction of ALK mRNA, resulting in decreased
ALK protein levels. This is a very attractive idea, especially when considering the
appearance of secondary ALK mutations mediating inhibitor resistance. Indeed,
neuroblastoma cell lines which have been transduced by lentiviral shRNA targeting
ALK exhibit decreased proliferation and undergo apoptosis. Further, mouse studies
show that liposomal siALK delivery to neuroblastoma cells results in cell growth
arrest, apoptosis, inhibited angiogenesis and prolonged survival [287, 288]. Similar
results were reported in NPM-ALK positive ALCL, where siRNA mediated
downregulation of ALK resulted in cell cycle arrest, apoptosis in vitro as well as
tumour growth inhibition and regression in vivo [289, 290]. Further, in ALCL, a
combination of ALK gene silencing together with ERK-inhibitor U0126 treatment
resulted in a synergistic growth inhibition [291]. Another approach to block the
growth of ALCL is the DNA vaccination of mice against ALK. This effect is further
enhanced by combination with chemotherapy resulting in prolonged survival of
mice harbouring ALK-positive lymphomas [292]. In glioblastoma, where both PTN
and ALK are upregulated, a knockdown of both PTN and ALK mediated by
ribozymes, blocked cell proliferation in vitro and diminished tumour growth in a
xenograft model in vivo [293].
Another way to inhibit ALK activity is the use of inhibitory antibodies. One
well-known example is Trastuzumab (Herceptin), a monoclonal antibody binding to
HER2, which prolongs life in patients with HER2-positive breast cancers [294]. In
cell culture experiments inhibitory antibodies targeting ALK successfully reduce
ALK downstream signalling and induce cytotoxicity in neuroblastoma cell lines [63,
295]. However, this inhibitory antibody is not suitable for treatment of cancers
harbouring ALK fusion proteins, but might serve as an alternative for cancer
patients with ALK amplification of gain-of-function mutations like in
neuroblastoma. To date, no such inhibitory antibodies are in clinical trials.
Direct targeting of ALK might be the preferential approach for therapy of
ALK-positive cancers, but this might not be sufficient due to the likely development
of secondary mutations. However, to overcome resistance to inhibitors, additional
targeting of proteins that bind to ALK or are activated by ALK represents an
additional therapeutic approach. One example of an additional target is Hsp90 which
is shown to interact with ALK in ALCL. Inactivation of Hsp90 with 17-allyl-aminodemethoxygeldanamycin (17-AAG) results in degradation of NPM-ALK and
apoptosis in ALCL cell lines [296]. IPI-504 is another Hsp90 inhibitor which
showed promising effects in NSCLC patients harbouring EML4-ALK. Further, IPI504, being already in Phase II Clinical Trials, might be a convenient drug for
treating EML4-ALK positive NSCLC patients that show crizotinib-resistance due to
the secondary ALK mutations L1196M [128, 261, 297-299]. Several studies have
used JAK3 inhibitors like AG490, WHI-P131 and WHI-P154 in NPM-ALK positive
ALCL. These inhibitors have been reported to directly block the activity of ALK,
36
though in a modest way [97, 101]. Another inhibitor of JAK3/STAT3 signaling,
cucurbitacin I, has been shown to induce apoptosis and proteasomal degradation of
NPM-ALK in ALCL cells [102].
5
The Ras superfamily of small GTPases
As described earlier ALK activates the MAPK pathway, strongly suggesting
that small GTPases like Ras might be involved in downstream signalling. As this
potential activation has not been shown to date, we aimed to investigate the
activation of small GTPases downstream of ALK. Indeed, stimulated wild type ALK
activates Ras with a peak at 15 minutes post stimulation, confirming the
assumptions (Figure 8A).
Figure 8: Stimulated ALK activates the small GTPases Ras and Rap1.
Wild type mouse ALK expressing tet-on PC12 cells were stimulated with 1 µg/ml mAb46 or 50 ng/ml
EGF respectively for the indicated times. Precleared cell lysates were incubated with (A) GST-Raf-RBD
or (B) GST-RalGDS beads. Bound Ras or Rap1 proteins were analyzed by immunoblotting. Ras or
Rap1 in whole cell lysates (WCL) was used as a loading control and detection of p-ERK was used as a
control for mALK stimulation. The experiment shown in (A) was performed by Lovisa Olofsson.
The Ras superfamily of small guanosine triphosphatases (GTPases) contains
over 150 human members with evolutionarily conserved orthologs in other species
and regulates many different cellular functions [300, 301]. The Ras superfamily is
divided into five subgroups: Ras, Rho, Rab, Sar/Arf and Ran. Rat sarcoma (Ras)
oncogene proteins are the founding members of the Ras family and were initially
discovered as transforming genes of the Harvey and Kirsten murine sarcoma viruses
[302, 303]. Later, their role as oncogenes with transforming capacity in human
cancer was established [304-306]. Additionally, a third ras gene with transforming
potential was identified in neuroblastoma-derived DNA, named NRAS [307, 308].
Since then, further members like Rap and Ral were added to this family, all playing
critical roles in human oncogenesis by being involved in proliferation, growth and
differentiation. Members of the Ras homologous (Rho) family like Rho, Rac and
Cdc42 are mainly regulating the cytoskeleton, e.g. by promoting actin stress fiber
formation, lamellipodium and filopodium formation as well as membrane ruffling.
37
The largest branch of the superfamily is comprised of Ras-like proteins in brain
(Rab) which harbour a central function in vesicular transport. A further subfamily
being involved in the regulation of vesicular transport is the Sar/Arf (Secretionassociated and Ras-related/ADP-ribosylation factor) family. Members of this
subfamily regulate the transport of cop-coated vesicles between the endoplasmatic
reticulum and the Golgi. The last subfamily is formed of Ras-like nuclear (Ran)
proteins which are the most abundant small GTPases in the cell and function in the
nucleocytoplasmic transport of RNA and proteins.
However, the mechanism of action and regulation is the same for all Ras
superfamily small GTPases (Figure 9).
Figure 9: Mechanism of action and regulation
of small GTPases.
In the GDP-bound state small GTPases are in an
inactive conformation. Guanine-nucleotide
exchange factors (GEFs) induce the release of
GDP which is replaced by the more abundant
GTP resulting in an active conformation. In this
state, small GTPases can interact with effector
proteins, resulting in various biological effects.
GTPase-activating proteins (GAPs) contain a
catalytic group for GTP hydrolysis, thereby
accelerating the intrinsic GTPase activity which
is very low in small GTPases.
Small GTPases possess high-affinity binding for GDP and GTP. In the GDPbound state, the small GTPases are inactive. With the help of guanine-nucleotideexchange factors (GEFs) GTP binds to the small GTPases which are thereby
activated and can activate biological downstream effectors. In order to return to the
inactive GDP-bound state GTPase-activating proteins (GAPs) accelerate the
intrinsic GTPase activity which is very low in small GTPases (reviewed in [301,
309]).
5.1
Rap1
Rap1 (Ras-proximate 1) belongs to the Ras family forming a subgroup of the
Ras superfamily of GTPases. Originally, Rap1 was identified as Krev-1 which was
able to suppress the phenotype of K-ras transformed fibroblasts, and therefore
thought to antagonize Ras signalling [310]. However, over the years several reports
revealed multiple functions of Rap1: e.g. it is involved in the regulation of cell
polarity, integrin-mediated adhesion, secretion and cell-cell junction formation. Like
all GTPases, the activity of Rap1 is controlled by activating GEFs and inhibiting
GAPs [311]. Further, Rap1 seems to function in neurite outgrowth and neuronal
polarization by mediating sustained activation of the MAPK pathway as well as
regulating the cytoskeleton [312-315]. Rap1 also plays a role in the regulation of
cell proliferation, indicating that Rap1 might be involved in oncogenesis. Indeed,
38
upon overexpression, Rap1 induces oncogenic transformation in Swiss-3T3 cells
[316] and has been reported to be activating in metastatic mammary carcinomas,
metastasis and during melanoma tumorigenesis [317, 318]. Further, Rap1 seems to
play a role in thyroid mitogenesis and has been suggested to participate in thyroidstimulating hormone-stimulated thyroid tumorigenesis [319, 320]. According to a
recent report, Rap1 also plays a role in the proliferation and migration of endothelial
cells as well as vasculatization, further suggesting a participation in oncogenesis
[321]. However on the contrary, Lin et al., reported that Rap1 seems to suppress
oncogenesis [322]. To date, a clear function for Rap1 in the development and/or
onset of neuroblastoma has not been determined yet.
5.2
Rap1 specific regulators
5.2.1 Rap1 specific GEFs
Rap1 is activated by certain GEFs, like C3G, the Epac family proteins, CDGEFs and PDZ-GEFs ([323-329] and reviewed in [309]). One particular Rap1
specific GEF, namely C3G, possesses an essential function in vascular maturation
and the development of the nervous system during mouse embryogenesis [330-333].
In PC12 cells knockdown of C3G mediated by siRNA results in decreased neurite
outgrowth and in human neuroblastoma cells C3G has been suggested to play a role
in differentiation and survival [334-336]. Besides C3G, a further Rap1 specific GEF
possessing oncogenic capacity is the atypical RapGEF DOCK4, displaying dual
activity toward both Rap1 and Rac [337]. Further, the Drosophila C3G orthologue is
a Rap1-specific GEF as well and is involved in maintaining muscle integrity during
larval stages [338].
5.2.2 Rap1 specific GAPs
As Rap1 possesses a very low intrinsic GTPase activity, its inactivation is
dependent on GTP hydrolysis by Rap1 specific GAPs, e.g. Rap1GAP and members
of the Spa1 family ([339, 340] and reviewed by [309]). Although Rap1 belongs to
the Ras family there are some differences in the process of hydrolysis. Instead of a
glutamine (Gln61 in Ras) Rap1 contains a threonine (Thr61) which is important
rather for binding than catalysis. Moreover, Rap1GAP does not use an arginine
finger to stabilize the transition state in hydrolysis. Rather, Rap1GAP inserts an
asparagine thumb containing the catalytic asparagine into the active site of Rap1
[341-343]. A product of a familial tuberous sclerosis gene named tuberin displays in
vitro Rap1GAP activity as well and induces benign tumors upon deletion [344].
Decreased Rap1GAP expression levels result in enhanced Rap1 activity leading to
increased cell migration and invasive behavior as shown in prostate cancer cell lines
[345]. A recent study by Kim et al., further proved that low expression levels of
Rap1GAP result in increased invasion of renal cell carcinoma [346]. Moreover,
39
several reports suggested that Rap1GAP might act as a tumour suppressor gene in
some tumours like thyroid tumours, epithelial tumours and melanoma [347-353].
Overall, deregulated Rap1 signalling is involved in a variety of malignancies
like leukemia, neuroblastoma and prostate cancer (reviewed by [354]).
40
Aims
1
Overall aim
The general aim of this thesis was to elucidate the function ALK in the
development and onset of neuroblastoma. We intended to find answers to the
following questions, whether ALK point mutations found both in neuroblastoma cell
lines and patient samples are truly constitutively active, if they are potentially
involved in the progression of this disease, whether these ALK mutants can be
blocked by small ALK-specific inhibitors and which ALK downstream signaling
pathways are involved.
2
Specific aims
More specifically, in article I the aim is to investigate whether ALK activates
small GTPases, in particular Rap1 and the potential involvement in oncogenesis.
In article II we want to investigate whether the newly reported ALKF1174S mutation
is constitutively active and whether it responds to treatment.
Article III approaches the question whether the ALK point mutations found in
neuroblastoma cell lines and patients are constitutively active and if so, whether the
downstream signalling can be blocked by ALK-specific inhibitors. Further, we aim
to investigate whether these mutations are involved in disease progression.
Article IV focuses on one particular ALK point mutation found in neuroblastoma,
namely ALKI1250T, which was initially suggested to be constitutively active. In this
paper we investigate whether the ALKI1250T mutation is truly constitutively active.
Article V aims to determine whether ALK can regulate the initiation of MYCN
transcription in neuroblastoma.
41
Results and Discussion
1
Article I: “Anaplastic lymphoma kinase activates the small
GTPase Rap1 via the Rap1-specific GEF C3G in both
neuroblastoma and PC12 cells.”
As most reports have been studying ALK signalling mediated by NPM-ALK,
we wished to gain more information about the cell signalling of the wild type RTK.
As small GTPases regulate various cellular processes like growth, differentiation
and migration, we addressed the question whether small GTPases could serve as
downstream targets of ALK.
1.1
Stimulated ALK activates Rap1 which leads to neurite outgrowth
in PC12 cells
In particular, we focused on whether mouse ALK can activate Rap1. In order
to address this question, we used a tetracycline-inducible PC12 cell system, which
was generated in our lab. Upon induction with doxycycline, PC12 cells express wild
type mouse ALK (mALK) which can be stimulated with an agonist monoclonal
antibody, mAb46, resulting in the activation of ALK and its downstream signalling,
as well as neurite outgrowth in PC12 cells [63, 70]. Indeed, stimulation of mALK
with mAb46 resulted in activated Rap1, whose activation pattern is highly similar to
the one of ERK phosphorylation.
In order to clarify that mALK is able to activate Rap1 we employed the ALKspecific inhibitor NVP-TAE684 (recently also identified as an effective inhibitor of
leucine-rich repeat kinase 2 [262]) which has been shown to block NPM-ALKdriven cell proliferation and downstream signalling targets like ERK [96]. While
NVP-TAE684 could not block NGF-mediated TrkA receptor signalling, it was able
to inhibit mAb-mediated mALK activation as indicated by reduced ERK
phosphorylation. Further, pretreatment with NVP-TAE684 before mAb46
stimulation abrogated Rap1-GTP levels efficiently.
As Rap1 is required for ERK activation [312, 314, 315] we examined whether
this requirement is also valid downstream of ALK. Abrogation of Rap1 activity
either by siRNA or by overexpression of Rap1GAP had no impact on reduction of
ERK phosphorylation downstream of TrkA or mALK. However, pre-treatment of
cells with siRap1 resulted in prolonged ERK-phosphorylation upon NGF
stimulation, which is in agreement with a previous report [355].
Further, Rap1 is involved in neurite outgrowth in PC12 cells upon NGF
stimulation (control; no ALK expression) and upon stimulation in ALK-expressing
PC12 cells. Neurite outgrowth was decreased by reduced Rap1 expression levels
and by abrogated Rap1 activity due to Rap1GAP overexpression. This finding is in
42
agreement with a previous report indicating that overexpression of constitutively
active Rap1 leads to neurite outgrowth in PC12 cells [356].
1.2
Activation of Rap1 downstream of ALK occurs via the Rap1specific GEF C3G
Having established a link between ALK and Rap1, we wanted to investigate
what happens “in between”. As the Rap1-specific GEF C3G has been shown to play
a role in neurite outgrowth in PC12 cells [312, 315], we looked into whether mALK
was able to activate C3G. By immunoprecipitation we could detect a constitutive
interaction between C3G and CrkL which was independent of mALK stimulation
and in accordance with a previous study by Feller et al., [357]. Further, activated
mALK coimmunoprecipitated with either C3G or CrkL, indicating the formation of
a protein complex consisting of ALK, C3G and CrkL.
In order to investigate whether ALK can activate C3G, we investigated the
tyrosine phosphorylation status of C3G after mALK stimulation. Indeed, after ALK
stimulation mediated by mAb46, we could detect tyrosine phosphorylation of C3G
which is consistent with previous studies reporting C3G activation in response to
several exogenous stimuli [358, 359]. Altogether, activated mALK recruits a
complex consisting of C3G and CrkL, followed by tyrosine phosphorylation and
thereby activation of C3G.
Next, we wanted to address the function of C3G in ALK-induced neurite
outgrowth. Endogenous C3G levels were reduced by two independent siRNAs
which resulted in decreased neurite outgrowth mediated by NGF induction in ALKnonexpressing cells and by mAb46 stimulation in ALK-expressing PC12 cells.
However, the decrease in neurite outgrowth by siC3G was not as pronounced as
with siRap1, suggesting that other potential mechanisms may activate Rap1 and/or
that the reduction of C3G expression levels was not sufficient. Our results are in line
with previous studies also reporting decreased neurite outgrowth upon knockdown
of Rap1 or C3G levels in PC12 or neuroblastoma cells [334, 336]. Interestingly,
mALK-expressing PC12 cells that were not transfected with siC3G appeared to
express more C3G upon stimulation with mAb46. This observation is in agreement
with a study in the neuroblastoma cell line IMR32 by Radha et al., although the
precise molecular mechanisms are unclear [336].
Further, to confirm that C3G activates Rap1 in mALK-expressing PC12 cells,
we performed a Rap1 pulldown after transfection with siC3G. Indeed, knockdown
of C3G results in decreased Rap1 activation in mALK-expressing PC12 cells,
indicating that mALK activates C3G which in turn activates Rap1 and has a function
in neurite outgrowth.
Overall, we conclude that Rap1 activity is not absolutely required for ERK
phosphorylation, but that it is necessary for neurite outgrowth in our model system.
Therefore, other molecular mechanisms not including Rap1 might lead to the
prolonged ERK phosphorylation downstream of stimulated mALK. According to a
43
previous report the FRS2 adaptor protein is recruited to activated ALK, suggesting
that a complex consisting of ALK/FRS2/CrkL/C3G might be formed. Further, one
has to keep in mind that comparing similar results, e.g. PC12 cells and Rap1
activation, is quite difficult due to genetic composition and variation of different
PC12 cell clones.
1.3
Rap1 activity is involved in cell proliferation of neuroblastoma cell
lines
So far we proved that ALK regulates Rap1 in a very controlled inducible cell
culture model. However, this provides no clue about how significant the function of
Rap1 is in ALK signalling under physiological conditions or in tumor development.
Rap1 can also be activated by cAMP, which inhibits or increases cell proliferation
depending on the cell type. These cAMP mediated effects on cell proliferation are
suggested to be mediated by Rap1, which can cooperate either antagonistically or
synergistically with Ras, depending on the cell type [309, 360, 361]. Although no
activating mutations in Rap1 have been identified in tumours so far [362-364], Rap1
could still contribute to oncogenesis via other mechanisms, like overexpression or
downregulation of Rap1 or any regulators up- or downstream of Rap1 [316, 337,
345, 350, 365]. For instance, several reports suggest that C3G inhibits cell
proliferation [366-368]. In the neuroblastoma cell line IMR32, Radha et al., showed
that NGF-mediated activation or overexpression of C3G results in cell-cycle arrest,
implying that the C3G/Rap1 pathway contributes to the survival of the
neuroblastoma cell line IMR32 [336]. Together with the first reports of activating
ALK point mutations in neuroblastoma [128, 132-135], these studies suggest that
there might be a relevant connection between ALK and Rap1 in neuroblastoma and
that the contribution of the C3G/Rap1 pathway in the growth of neuroblastoma cell
lines may depend on ALK. In order to investigate this theory, we employed the
neuroblastoma cell lines SK-N-SH and SH-SY5Y, which both harbour the
ALKF1174L point mutation. Abrogation of Rap1 activity by either siRap1 or
overexpression of Rap1GAP decreased proliferation of both neuroblastoma cell
lines [258]. Additionally, in SH-SY5Y cells mAb46 mediated stimulation of cell
proliferation was blocked by reduced Rap1 expression levels, which could not be
observed in SK-N-SH cells. This difference between those two neuroblastoma cell
lines which both harbour the ALKF1174L gain-of-function mutation, might further
depend on the genetic disposition. Altogether, these results suggest that the
C3G/Rap1 pathway is not only important for ALK-mediated PC12 cell
differentiation, but that this pathway is also involved in the regulation of
neuroblastoma cell proliferation.
Conclusively, we propose the following model (Figure 10): Activated ALK
forms a complex together with C3G and CrkL (and maybe further potential adaptor
proteins like FRS2), in which C3G becomes tyrosine phosphorylated and therefore
44
activated. This activated complex is able to activate Rap1 which results in neurite
outgrowth in PC12 cells or proliferation of neuroblastoma cells.
Figure 10: Model of ALK mediated activation
of the small GTPase Rap1.
Wild type ALK is activated by monoclonal
antibodies like mAb46, resulting in autophosphorylation and activation. Activated ALK
recruits the Rap1-specific GEF C3G and CrkL
(and maybe further potential adaptor protein like
FRS2). In this complex C3G becomes tyrosine
phosphorylated and thereby activated which
results in the activation of Rap1. This results in
neurite outgrowth in PC12 cells or proliferation
of neuroblastoma cell lines. Overexpression of
Rap1GAP abrogates Rap1 activity and hence the
above named downstream effects.
45
2
Article II: “Appearance of the novel activating F1174S ALK
mutation in neuroblastoma correlates with aggressive tumor
progression and unresponsiveness to therapy.”
A young Swedish patient was diagnosed with neuroblastoma and was treated
accordingly. However, about eight months after diagnosis, a biopsy of the tibial
bone showed viable tumor cells showing positive staining for phospho-ALK.
Despite intensive care this patient succumbed to disease ten months after initial
diagnosis. However, investigation of tumor genetics revealed a loss of
heterozygosity on chromosome 2p which includes the ALK gene locus. DNA
sequencing of the ALK gene detected a homozygous mutation (T3521C) which
results in the missense mutation F1174S.
2.1
The ALKF1174S mutant is a ligand-independent gain-of-function
mutation and has transforming potential
By employing several cell culture based systems we wanted to biochemically
evaluate the ALKF1174S mutant. Doxycycline-inducible PC12 cell lines stably
transfected with human wild type ALK and ALKF1174S were developed. While wild
type ALK needs to be activated by the monoclonal antibody mAb31, no further
stimulation with an agonist antibody is needed for activation of the ALKF1174S
mutant as indicated by tyrosine phosphorylation of ALK and ERK phosphorylation
as a downstream target. Moreover, both ALK and ERK phosphorylation are
abrogated by the ALK-specific small molecule inhibitor NVP-TAE684. These
results were further confirmed by transient transfection of ALKF1174S and ALKF1174L
which show similar ALK and ERK phosphorylation. Therefore, we can conclude
that the ALKF1174S mutant is constitutively active, which might contribute to the
severe disease progression of this particular neuroblastoma patient.
A further method to investigate whether the ALKF1174S mutant is truly
constitutively active, is to measure the neurite outgrowth in PC12 cells. Previously,
we and others have shown that activated ALK leads to the extension of neurites in
PC12 cells [64, 69, 70, 369]. Indeed, transient transfection of PC12 cells with
human ALKF1174S or ALKF1174L results in neurite outgrowth, while human wild type
ALK needs to be stimulated with monoclonal antibody mAb31 in order to mediate
neurite outgrowth. However, expression of unstimulated wild type ALK results in
minor neurite formation, which might be due to overexpression. This result further
strengthens the hypothesis, that ALKF1174S is a ligand-independent gain-of-function
mutation.
Finally, the transforming potential of the ALKF1174S mutant was assessed.
NIH3T3 cells were transfected, and in contrast to the wild type receptor expression
of both human ALKF1174S and ALKF1174L, mediates foci formation. This result
demonstrates the transforming ability of the ALKF1174S mutant.
46
2.2
Ectopic expression of ALKF1174S in the Drosophila eye causes the
rough eye phenotype
Based on this finding, the aim was to examine the nature of this ALK
mutation, i.e. whether it was the major cause for the rapid tumor progression. First
indications were obtained in cell culture based systems. Another approach is the use
of a different model system, namely D. melanogaster. Upon ectopic expression of
human wild type ALK in the Drosophila eye this RTK is expressed in the
developing photoreceptors of the eye without any noticeable phenotype, similar to
the controls. As the ligand-dependent wild type ALK apparently is not activated by
endogenous Drosophila ligands, therefore providing a clean background,
overexpression of ALK in the eye serves as an optimal tool for the analysis of
potential activating ALK mutants. Indeed, ectopic expression of human ALKF1174S
or ALKF1174L (the latter one serving as a positive control [128]) results in the rough
eye phenotype, indicating ligand-independent RTK activation. As no human ALK
ligand is present in the Drosophila eye these results confirm that the ALKF1174S
mutant is constitutively active in vivo.
To sum up, the results demonstrate that the novel human ALKF1174S mutant is
a ligand-independent gain-of-function mutation, correlating with aggressive
neuroblastoma development. Further, treatment with ALK-specific inhibitors like
NVP-TAE684 indicates that the use of such inhibitors might be beneficial for the
treatment of neuroblastoma patients. In addition, this case report shows that initial
screening of the first tumor biopsy is not sufficient and that further genetic analyses,
in particular of the ALK locus, are required in order to get more insight into the
development of neuroblastoma and subsequently gain more information about the
treatment of patients.
47
3
Article III: “Activating ALK mutations found in
neuroblastoma are inhibited by Crizotinib and NVPTAE684.”
In this article we aimed to investigate whether certain ALK point mutations
found in neuroblastoma patients are truly constitutively active and if they are
involved in the progression of this disease.
According to one of the early publications of ALK point mutations in
neuroblastoma by Mossé et al., we initially selected seven mutations with the
highest probability of being activating mutations, i.e. with the highest probability
that the amino acid change results in an oncogenic activation (Figure 11) [135]. This
article discusses six of them (human ALKG1128A, ALKI1171N, ALKF1174L, ALKR1192P,
ALKF1245C and ALKR1275Q), while one mutation (human ALKI1250T) is dealt with in
Article IV.
Figure 11: Model of ALK kinase domain
showing the investigated point mutations.
The structural model shows the ALK kinase
domain with the C-helix (orange), the P-loop
(green), activation loop (blue) and catalytic loop
(yellow) highlighted. Red balls indicate verified
point mutations observed in neuroblastoma
patients. The investigated neuroblastoma point
mutations in this thesis are indicated.
3.1
ALK mutations identified in neuroblastoma are ligandindependent gain-of-function mutations and can be blocked by
NVP-TAE684 and crizotinib with different IC50
Initially, we investigated whether the above named mutations in human ALK
were also conserved in mouse ALK. Therefore we created these mutations at the
equivalent sites in mouse ALK, which are: mALKG1132A, mALKI1175N, mALKF1178L,
mALKR1196P, mALKF1249C and mALKR1279Q. In order to compare the activity of these
ALK mutants we created PC12 cell clones with doxycycline-inducible ALK
expression. As a control we used wild type mouse ALK, which upon stimulation
with the activating monoclonal antibody mAb46, becomes tyrosine phosphorylated
and activates downstream signalling targets like ERK and PKB/Akt. However,
doxycycline-induced expression of mALK mutants results in tyrosine
phosphorylation of the RTK and activation of downstream signalling even in the
absence of an activating antibody. Additionally, all six investigated ALK mutants
48
result in STAT3 activation which is in contrast to wild type mALK. Therefore, all
six investigated ALK mutants are activated ligand-independently and stimulate
downstream targets like STAT3, PKB/Akt and ERK. In order to compare the
relative ALK activity between the mutants we assessed the ratio of phospho-ALK to
total ALK which seems to differ only slightly between the mutants, although it
seems that mALKF1178L, mALKR1196P and mALKG1132A are slightly more active than
the other mutants. Some difference in downstream signalling can also be observed.
For example, mALKG1132A seems to phosphorylate STAT3 to a higher extent than
mALKI1175N and mALKF1249C with comparable levels of ALK expression. However,
as individual cell clones were selected for the expression of mALK mutants, with
this employed cell culture model it is difficult to draw any conclusion about the
activity of those ALK mutants in reality.
To further verify that the ALK mutations are constitutively active we
employed the neurite outgrowth assay in PC12 cells. Neurite outgrowth mediated by
stimulated wild type mALK can be blocked by the ALK-specific inhibitor NVPTAE684 [64, 69, 70, 73]. All six expressed mALK mutants are able to induce
neurite outgrowth ligand-independently, which can be blocked upon addition of the
inhibitor, although to various degrees at low doses of inhibitor. The mALKR1279Q
mediated neurite outgrowth was completely inhibited with 30 nM NVP-TAE684,
while the neurite outgrowth induced by mALKI1175N, mALKR1196P and mALKF1249C
was only blocked to about 50% by NVP-TAE684. In contrast, treatment of
mALKG1132A and mALKF1178L resulted only in a small decrease of neurite outgrowth,
which is in agreement with the suggested higher activity of those mutants. However,
all uninduced cell clones exhibited a certain level of background neurite outgrowth
which might be due to leakage or the presence of a so far unknown endogenous
ligand in the case of unstimulated wild type mALK cells.
Next, we wanted to confirm that the human ALK mutations also respond to
treatment with ALK-specific inhibitors. In addition to NVP-TAE684 we included
crizotinib (PF-2341066), a dual inhibitor of c-MET and ALK, as this inhibitor is
clinically used in contrast to NVP-TAE684 [263, 264]. In order to exclude any
effects due to selection of individual PC12 cell clones and different ALK
expression, we transiently transfected PC12 cells with human wild type or mutated
ALK. In agreement with the inducible PC12 cell lines expressing mALK, stimulated
human wild type ALK induces neurite outgrowth, while the ALK mutants mediate
robust neurite outgrowth ligand-independently. In addition to NVP-TAE684,
treatment with crizotinib also blocks neurite formation.
As NVP-TAE684 seems to have toxic effects, we continued to use only
crizotinib, which has no obvious toxicity. In order to compare the different response
of these six ALK mutants toward crizotinib, we employed the widely used Ba/F3
cell system. Interestingly, some differences between the ALK mutants in their
ability to mediate proliferation in the absence of IL-3 could be observed. For each
ALK mutant three independent transfections were performed, and from each
transfection we obtained four IL-3-independent cell lines for ALKF1174L, ALKR1192P
49
and ALKR1245C. However, ALKG1128A only resulted in three cell lines. To our
surprise, ALKI1171N and ALKR1275Q were unable to compensate for IL-3 depletion
and no Ba/F3 cell lines could be obtained, which contradicts results found in a
previous study [133]. One possible explanation for these divergent results might be
the different transfection methods used: our study employed transient transfection,
while George et al., used retroviral infection of Ba/F3 cells. However, the fact that
we were unable to get Ba/F3 cell lines with ALKI1171N and ALKR1275Q might
represent a further indication that these two mutations have less transforming
potential than the others, which is not sufficient to drive IL-3-independent Ba/F3
cell proliferation. Further, crizotinib blocked the proliferation of all four Ba/F3 cell
lines. Subsequent calculations of the IC50 revealed that ALKF1174L and ALKR1192P
required higher doses of crizotinib than ALKG1128A and ALKR1245C. These results
could be verified by Western Blot analysis detecting ALK phosphorylation at
position Y1278, the first tyrosine of the YxxxYY motif that is necessary for ALK’s
auto-activation and the transforming ability of NPM-ALK [27], as well as ERK
phosphorylation.
Next, we investigated the transforming potential of these activating human
ALK mutations in NIH3T3 cells. While neither vector control nor ALK wild type
gave rise to foci formation, all investigated ALK mutations drive foci formation
although to a different extent. hALKG1128A, hALKI1171N, hALKR1192P and hALKR1275Q
showed comparatively weak foci formation, while hALKF1174L and hALKF1245C
resulted in strong foci formation. However, hALKI1171N and hALKR1275Q were able to
transform NIH3T3 cells but could not drive IL-3-independent Ba/F3 cell
proliferation, which may be due to the fact that these ALK mutations only have
transforming potential in cells that are immortalized by other crucial genetic events
in neuroblastoma.
To sum up, both mouse and human ALK mutants respond to NVP-TAE684
and crizotinib treatment. The activity of the ALK mutants is inhibited by crizotinib,
although different doses are required. Further, all investigated human ALK
mutations have transforming potential though to different degrees.
3.2
Ectopic expression of ALK mutants in the Drosophila eye causes
the rough eye phenotype
To further examine if ALK mutations are truly constitutively active, they
were overexpressed in the developing photoreceptors of the Drosophila eye. We
chose to analyse hALKF1174L and hALKR1275Q which represent the most common
mutations found in neuroblastoma patients [213]. Similar to the previous study
(Article II), expression of hALK wild type did not cause any obvious phenotype in
the eye and resembled controls [241]. Expression of hALKF1174L and hALKR1275Q
caused the rough eye phenotype, similar to hALKF1174S, indicating their ligandindependent activation. In agreement with previous results, hALKF1174L caused a
more severe phenotype than hALKR1275Q. Both ALK mutants, especially
50
hALKR1275Q, responded to NVP-TAE684 treatment as indicated by the improvement
of the rough eye phenotype. However, the effect of crizotinib treatment on the rough
eye phenotype was less pronounced, which might reflect the higher toxicity of NVPTAE684.
In summary, all six investigated human ALK mutations with the highest
predictions of being oncogenic are indeed true gain-of-function mutations. In
particular, one mutant, namely hALKR1275Q, which has been found both in germ line
and somatic tumor DNA samples [133-135], seems to respond better to treatment
with ALK inhibitors like NVP-TAE684 or crizotinib. Further, the obtained results
suggest that the various mutations might have a different impact on development,
onset and severity of neuroblastoma. Generally, we see a trend that somatic ALK
mutations like ALKF1174L or ALKF1245C seem to be more aggressive and respond less
to ALK inhibitor treatment than germ line mutations like ALKG1128A or ALKR1192P
[135]. This is an important piece of information regarding patient-specific
treatments for ALK-positive neuroblastoma with ALK-specific inhibitors like
crizotinib.
51
4
Article IV: “The Neuroblastoma ALK (I1250T) Mutation is
a Kinase-Dead RTK In Vitro and In Vivo.”
Initially, we analysed seven ALK point mutations with the highest probability
of being oncogenic [135]: the six previously discussed mutations (Article III) and an
additional mutation, namely hALKI1250T. This mutation was identified as a germ line
mutation with a high probability of being oncogenic [135].
4.1
The ALKI1250T mutant is not constitutively active in cell culture
systems
The equivalent mutation was created in mouse ALK (mALKI1254T) and stably
transfected into PC12 cells. However, when selected PC12 cell clones were screened
for inducible ALK expression, no neurite outgrowth could be observed, in contrast
to the other investigated mutations (Article III), despite ALK expression. These
initial observations suggested that this mutation might not be ligand-independent,
but act like the wild type ALK, i.e. the mALKI1254T mutation can be activated by a
monoclonal antibody. While wild type ALK activated by monoclonal antibody
mAb46 is tyrosine phosphorylated, activates the downstream target ERK and
induces neurite outgrowth, surprisingly mALKI1254T cannot be stimulated by the
activating monoclonal antibody mAb46. These unexpected results were confirmed
both by transient transfection of PC12 cells with human ALKI1250T as well as by
tetracycline-inducible PC12 cell clones expressing human ALK.
As hALKI1250T cannot be activated by monoclonal antibodies, the question
was raised whether this mutation might be localized differently compared to the wild
type receptor. Mazot et al., reported that ALK is not only localized on the cell
membrane, but also at intracellular compartments like the endoplasmic reticulum
and Golgi [246]. Accordingly to this report, we could detect both wild type ALK
and ALKI1250T in the endoplasmic reticulum and the Golgi apparatus, but also on the
cell membrane in transiently transfected HEK293 cells, therefore being accessible to
antibody stimulation.
Next we wanted to assess whether this mutant displays any transforming
potential. While the gain-of-function mutation hALKF1174S mediates foci formation,
both wild type human ALK and hALKI1250T were unable to transform NIH3T3 cells.
So far we can conclude that despite cell membrane localization the
I1250T
hALK
mutation has no detectable tyrosine phosphorylation activity, is unable
to activate downstream signalling like ERK, cannot induce neurite outgrowth and
has no transforming potential.
52
4.2
The ALKI1250T mutant is suggested to act as a dominant-negative
receptor
Genetically it might be possible that the hALKI1250T mutation is only on one
copy of the ALK locus while the other one is wild type or contains a gain-of-function
mutation like hALKF1174S. In order to answer this question we co-transfected PC12
cells with wild type hALK together with hALKI1250T. In this experiment, hALKI1250T
seems to have a dominant-negative effect as indicated by reduced ERK
phosphorylation on stimulated wild type ALK. This finding was confirmed by
further results using different experimental approaches: (1) ERK phosphorylation is
reduced upon co-transfection of hALKF1174S with hALKI1250T in PC12 cells. (2)
Transfection of the neuroblastoma cell line CLB-GE (harbouring ALKF1174V) [134]
with FLAG-tagged hALKI1250T results in reduced ERK phosphorylation. (3)
Transient transfection of PC12 cells with FLAG-tagged hALKI1250T and untagged
wild type ALK revealed that FLAG-hALKI1250T is not detectably tyrosine
phosphorylated. (4) Co-transfection of PC12 cells with wild type ALK and
ALKI1250T, followed by stimulation of ALK results in decreased ALK-mediated
neurite outgrowth in the presence of ALKI1250T. Altogether, these results suggest that
the expression of hALKI1250T might potentially act as a dominant-negative receptor
when it is expressed together with active ALK receptors.
4.3
Ectopic expression of ALKI1250T in the Drosophila eye does not
cause the rough eye phenotype
As for the previously discussed mutations (Article II and III) we wanted to
investigate the activating potential of hALKI1250T by ectopical expression in the
Drosophila eye. As described earlier (Article II and III), overexpression of wild type
human ALK does not cause the rough eye phenotype, therefore providing a clean
phenotypic background. Serving as a positive control for a gain-of-function
mutation, ectopic expression of hALKF1174S results in the rough eye phenotype (see
also Article II). However, ectopic expression of hALKI1250T neither leads to
destruction of normal tissue morphology in the fly eye nor disrupts the organization
of ommatidial units. This mutation rather shows a similar phenotype to wild type
human ALK and controls, thereby suggesting that this mutant is not ligandindependent as initially predicted.
4.4
Why is the ALKI1250T mutant inactive?
However, the question why the ALKI1250T mutation is a kinase dead mutation
still remains to be answered. Recently, structural studies by Bossi et al., and Lee et
al., elucidating the crystal structure of the ALK kinase domain have shed some light
on the understanding of the mechanistics of ALK mutations [1, 28]. Using the
published crystal structure of the ALK kinase domain as a basis we suggest a
mechanistic explanation for the inactivity of ALKI1250T. The mutation at amino acid
53
position 1250 is located in the catalytic loop of the kinase domain and is highly
conserved in the active site of protein kinases [1, 28]. These large, hydrophobic
amino acids are included in the conserved catalytic loop sequence HRDI/LAARN,
influencing the hydrophobic spine as well [10]. According to the ALK crystal
structure forms contacts with hydrophobic residues from helix 1 and 2 (I1233,
I1268, F1315) of the C-lobe. These interactions help to anchor the catalytic loop in a
correct position in respect to the DFG loop and ATP. By replacing the hydrophobic
isoleucine at position 1250 with threonine, which harbours a polar side chain, the
hydrophobic contact/anchorage is probably weakened which most likely destabilizes
the whole active site of ALK. Another hypothesis is that the I1250T mutation
influences the interaction of the side chain of H1247 with the carbonyl oxygen of
D1270 from the DFG motif, which could result in a destabilization of the kinase
domain.
Overall, our structural hypothesis together with our obtained results are in
stark contrast with earlier studies reporting that the ALKI1250T mutation is
constitutively active and promotes oncogenicity [28, 135]. Therefore, the ALKI1250T
mutation does not seem to be a driving force in the development and progression of
neuroblastoma, implying that patients should be treated accordingly. However, it
might be possible that ALKI1250T has a biological role in the presence of wild type or
constitutively active ALK, i.e. that it acts as a dominant-negative receptor according
to our results. Indeed, such kind of “cross-activation” has been reported for kinase
dead BRAF and oncogenic RAS [370] or for ligand-induced trans-phosphorylation
of receptors [371, 372]. However, this putative biological function of ALKI1250T
needs to be validated by in vivo studies. On the other hand, one could argue that a
dominant-negative mutation on one copy of the ALK locus in humans might have a
minor effect as ALK knockout mice do not show any major phenotype ([373], BH
and RHP unpublished results).
54
5
Article V: “Anaplastic Lymphoma Kinase (ALK) regulates
initiation of transcription of MYCN in neuroblastoma cells.”
In neuroblastoma amplification of the MYCN locus is one of the genetic
hallmarks [212]. ALK gain-of-function mutations occur in about 6.9% of
investigated neuroblastoma tumours according to a recent meta-analysis [213].
Further, this analysis revealed that ALK mutations occur in approximately 8.9% of
MYCN amplified tumours, correlating with a poor clinical outcome [213]. Therefore
we proposed the hypothesis that activated ALK might influence MYCN in
neuroblastoma.
5.1
ALK regulates the MYCN promoter in PC12 cells and human
neuroblastoma cell lines
Initially we investigated whether ALK is able to regulate the MYCN
promoter. PC12 cells were co-transfected with human ALK and MYCNP-luciferase
which contains approximately 600 bp before the transcription start site [374]. We
observed that unstimulated wild type ALK induced a 5-fold increase of luciferase
activity which might be caused by ALK overexpression. Stimulation with the
agonist monoclonal antibody mAb46 further increased the luciferase activity 2 – 3fold. Further, co-transfection with hALKF1174L and hALKR1275Q, the two most
common ALK mutations in neuroblastoma, resulted in luciferase activity. To verify,
that the luciferase activity was caused by ALK activity, we employed two ALKspecific inhibitors, NVP-TAE684 and crizotinib. Both inhibitors abrogate luciferase
activity mediated by the ALK gain-of-function mutations and stimulated wild type
ALK. Interestingly, inhibition of MYCNP-luciferase activity was less pronounced in
hALKF1174L expressing PC12 cells, indicating that this mutation responds less to
ALK-specific inhibitors [271]. Effective inhibition of ALK activity was analyzed by
immunoblotting for phospho-ERK. Similar results were obtained using our PC12
cell clones which upon induction with doxycycline express wild type or mutant
ALK (Article II – IV).
Next, we decided to investigate whether ALK is able to regulate the MYCN
promoter in the background of neuroblastoma. We used several neuroblastoma cell
lines with different genetic backgrounds: CLB-GA (MYCN non-amplified, 1p
deletion, 11q deletion, 17q gain, non-amplified ALK containing ALKR1275Q
mutation), CLB-GE (MYCN amplified, 1p deletion, 17q gain, amplified ALK
containing ALKF1174V) and CLB-BAR (amplified MYCN and ALK, 1p deletion, 17q
gain) [134, 375, 376]. Transfection of these cell lines with MYCNP-luciferase
resulted in increased luciferase activity which could be reduced by treatment with
NVP-TAE684 or crizotinib. Although the use of these neuroblastoma cell lines is
more relevant to neuroblastoma than the controlled PC12 cell system, the
interpretation and comparison of results using these cell lines is slightly problematic
due to their different genetic backgrounds. The CLB-BAR cell line, for instance,
55
expresses constitutively activated ALK, although no point mutations have been
identified in the kinase domain, suggesting that other mechanisms somehow lead to
ALK activation in this cell line [134]. An additional neuroblastoma cell line, IMR32
(MYCN non-amplified, exon 2 – 4 ALK amplified, 1p deletion, 17q gain), was
investigated. Stimulation with mAb46 led to a modest increase in MYCNP-luciferase
activity which could be blocked with crizotinib. To conclude, ALK regulates the
initiation of transcription of the MYCN upstream promoter in PC12 cells and
neuroblastoma cell lines.
5.2
Abrogation of ALK activity results in decreased MYCN mRNA
and proliferation of neuroblastoma cell lines
In order to further examine which role ALK plays in the initiation of MYCN
transcription we quantified the relative MYCN mRNA content by qRT-PCR. The
neuroblastoma cell lines CLB-GA, -GE and –BAR showed reduced MYCN mRNA
levels compared with untreated cells upon treatment with NVP-TAE684 and
crizotinib as well as by siRNA mediated knockdown of ALK expression levels.
Stimulation with the activating antibody mAb46 did not increase relative MYCN
mRNA levels in these cell lines with constitutive active ALK. IMR32 cells showed
a modest increase of MYCN mRNA upon stimulation with mAb46, which was
reduced upon treatment with the ALK inhibitors. In order to show that the inhibitor
concentrations used truly inhibit ALK activity, we assessed cell proliferation.
Proliferation of CLB-GA, -GE and –BAR was inhibited by 250 nM crizotinib or 50
nM NVP-TAE684 respectively, suggesting that ALK activity is an important factor
contributing to neuroblastoma cell proliferation. These results were confirmed by
siRNA mediated reduction of ALK expression levels. However, proliferation of
IMR32 cells was only slightly inhibited by 250 nM crizotinib, implying that this cell
line is not as dependent on ALK activity as the others. But higher doses of crizotinib
(500 nM) inhibited cell proliferation. Although crizotinib is a FDA-approved drug
for the treatment of ALK-positive NSCLC patients, crizotinib is also an effective
inhibitor of c-Met which is also involved in tumour progression. However,
according to control experiments we can conclude that c-Met has no important
function in this study.
Overall, inhibition of ALK activity in neuroblastoma cell lines with
constitutively active ALK leads to reduced transcription of MYCN mRNA.
5.3
ALK activity regulates MYCN protein expression
Next, we wanted to investigate what effect inhibition of ALK activity has on
MYCN protein levels in neuroblastoma cell lines. In order to detect MYCN in CLBGA cells, which have non-amplified MYCN, twice as much cell lysate was loaded.
Treatment with crizotinib or NVP-TAE684 (data not shown) decreased MYCN
expression significantly, which was confirmed with siRNAs targeting ALK. Further,
56
crizotinib treatment blocked ALK activity as indicated by decreased
phosphorylation of ALK Y1278, which is important for the autoactivation of ALK
[27]. Like in the previous experiments, IMR32 cells needed to be treated with
mAb46 in order to observe a modest increase in MYCN and ALK activity, which
could be inhibited by crizotinib treatment.
According to earlier studies high levels of MYCN are suggested to increase
expression of Aurora kinase A mRNA directly or indirectly, and MYCN
overexpression increases Aurora kinase A expression in SH-EP cells [377, 378].
Therefore, if MYCN is a transcriptional downstream target of ALK, we expect to see
decreased Aurora kinase A expression when ALK is inhibited. Indeed, crizotinib
treatment resulted in decreased expression levels of Aurora kinase A in all
investigated neuroblastoma cell lines. Further, inhibition of ALK reduced the
phosphorylation of PKB/Akt and ERK, confirming these signalling proteins as
downstream targets of ALK. As a control the neuroblastoma cell lines were treated
with PKB inhibitor LY294002 and ERK inhibitor U0126, which resulted in
decreased MYCN expression levels. However, ERK inhibition in the IMR32 cells
did not result in a pronounced decrease of MYCN levels. Generally the stability and
degradation of MYCN appears to be regulated by several signalling pathways
involving post-translational modifications like phosphorylation and ubiquitination,
all forming a complex network [379]. MYCN is stabilized upon phosphorylation at
Ser62, now being able to enter the nucleus and act as a transcription factor [380]. If
in addition to Ser62, MYCN is phosphorylated at Thr58 mediated by GSK3-β in
complex with Pin1, PP2A and Axin, MCYN is stabilized [379, 381, 382]. However,
phosphorylation at Thr58 alone represents a signal for the recruitment of the
ubiquitin ligase Fbxw7, resulting in ubiquitination and proteasomal degradation of
MYCN. This process is suggested to occur at low levels of PI3K activity [378]. On
the other hand, proteasomal degradation of MYCN can be blocked by recruitment of
Aurora kinase A [378]. Further, PKB/Akt activates mTORC1, which downregulates
the phosphatase PP2A, thereby blocking dephosphorylation of MYCN at Thr58
which leads to increased stability of MYCN. According to the report by Chesler et
al., inhibition of PI3K activity destabilizes MYCN, blocking neuroblastoma
progression as shown in the TH-MYCN neuroblastoma mouse model [383].
In order elucidate the effect of ALK on MYCN expression and stability in
neuroblastoma cells, we blocked de novo protein biosynthesis by treatment with
cycloheximide. Following addition of cycloheximide we could detect a decrease of
MYCN protein but not MYCN mRNA in CLB-GE and CLB-BAR cells. In CLB-GA
the decrease was not as pronounced which might be due to the fact that MYCN in
non-amplified in this cell line. Further, treatment with MG-132, a proteasome
inhibitor [384], increased MYCN expression levels, while treatment with crizotinib
prior to MG-132 resulted in lower expression levels. Overall, we can conclude that
active ALK primarily influences the transcription of MYCN in neuroblastoma cell
lines, although post-transcriptional regulation cannot be excluded.
57
5.4
ALK and MYCN co-operate in transforming NIH3T3 cells
In previous studies we reported that the human ALK gain-of-function
mutations ALKF1174S, ALKF1174L and ALKR1275Q have transforming potential in
NIH3T3 cells (Article II and III). As activated ALK drives initiation of MYCN
transcription, we planned to investigate whether ALK and MYCN have a synergistic
effect on NIH3T3 cell transformation. Expression of ALKF1174L or ALKR1275Q
mediates foci formation, while expression of MYCN or wild type ALK alone does
not result in transformation. However, ALKF1174L or ALKR1275Q together with
MYCN result in a robust increase in transformation compared with mutated ALK
alone. Hence, constitutively active ALK together with MYCN results in more potent
transforming potential.
Altogether, we could demonstrate that activated ALK, either by stimulated
wild type or by gain-of-function mutations, activates the initiation of MYCN
transcription. Blocking ALK activity either by crizotinib, NVP-TAE684 or siRNA
resulted in reduced MYCN mRNA, MYCN protein levels and decreased
proliferation of neuroblastoma cell lines. Further, co-expression of ALK gain-offunction mutants together with MYCN results in a synergistic effect on NIH3T3 cell
transformation. The latter finding might be important for patients with chromosome
2 amplification, i.e. having MYCN amplification together with wild type or
constitutively active ALK mutants, resulting in extremely poor clinical outcome.
This is supported by a previous report by Berthier et al., describing a relationship
between ALK and MYCN expression and MYCN amplification repectively [385].
Overall, this study provides further evidence for ALK as an important player in the
development and progression of neuroblastoma. Additionally, this report suggests
that ALK is a promising target in treating neuroblastoma patients harbouring MYCN
and ALK amplification.
Recently, an elegant study by Zhu et al., reported results in accordance to ours. They
generated transgenic zebrafish models which developed neuroblastoma upon MYCN
overexpression in the fish analog of the adrenal medulla. Zebrafish overexpression
ALK or ALKF1174L on its own did not result in tumour formation. However, fish
overexpressing both MYCN and ALKF1174L showed increased tumour onset and
penetrance as a result of the synergistic effects of the two oncogenes. Further, Liu et
al., propose that ALK gain-of-function mutations might be the “initiating” events in
neuroblastoma development and need a “second hit” like MYCN
amplification/overexpression in order to achieve full transformation of cells [386,
387].
58
Summary of the main findings of this thesis
Article I
• ALK activates the small GTPase Rap1 via C3G/CrkL which can be blocked by
the small ALK-specific inhibitor NVP-TAE684.
• Rap1 activation does not seem to be crucial for ALK mediated ERK
phosphorylation in PC12 cells.
• ALK-mediated activation of the C3G/CrkL/Rap1 pathway leads to neurite
outgrowth in PC12 cells and proliferation in neuroblastoma cell lines.
Article II
• The human ALKF1174S mutant found in a neuroblastoma patient is a gain-offunction mutant with transforming potential, correlating with aggressive disease
development.
Article III
• Six investigated ALK mutations (hALKG1128A, hALKI1171N, hALKF1174L,
hALKR1192P, hALKF1245C and hALKR1275Q) found in neuroblastoma patients with
a high probability of being oncogenic, are truly constitutively active.
• These ALK mutations respond with different IC50 to the ALK inhibitors NVPTAE684 and crizotinib and possess different transforming potential.
• These ALK gain-of-function mutations are suggested to have a different impact
on development, onset and severity of neuroblastoma.
• Somatic mutations like ALKF1174L or ALKF1245C appear to be more aggressive
than germ line mutations like ALKG1128A or ALKR1192P.
Article IV
• The hALKI1250T mutation, which was initially discovered as a germ line mutation
with a high probability of being oncogenic, is in fact a kinase dead mutation and
cannot be activated by monoclonal antibodies.
• Results suggest that this mutation acts as a dominant-negative receptor.
Article V
• ALK regulates the initiation of MYCN transcription both in PC12 and human
neuroblastoma cell lines.
• Abrogation of ALK activity decreases proliferation of neuroblastoma cell lines
and MYCN mRNA as well as protein expression.
• Co-expression of ALK gain-of-function mutants together with MYCN results in a
synergistic effect on NIH3T3 transformation, which suggests an extremely poor
clinical outcome for neuroblastoma patients with the appropriate genetic
background.
• ALK is shown to be an important player in the development and progression of
neuroblastoma and might therefore serve as an excellent therapeutical target.
59
Future perspectives
In the last couple of years ALK appeared to be more and more clinically
important in the development and progression of cancer, in particular neuroblastoma
and NSCLC. Hence, targeting ALK exhibited potential as an effective and more
specific treatment of ALK-positive cancer patients. Since the discovery of ALK
point mutations, it has become increasingly obvious that the type of ALK mutation,
i.e. germ line vs somatic mutations and driver vs passenger mutations, might have
an impact on the choice and success of patient specific treatment.
The development of ALK inhibitors has undergone great progress with very
promising results from the first clinical trials for patients with ALK-positive cancers
which has resulted in FDA approval of crizotinib under the name Xalkori only four
years after the first discovery. However, this is no reason to lean back and relax: the
appearance of crizotinib-resistant secondary ALK mutations demands the
development of second generation ALK inhibitors to overcome this resistance.
However, according to Katayama et al., secondary ALK mutations do not represent
the only mechanism for crizotinib resistance. Amplification of the ALK locus serves
as another resistance mechanism as well as “diverted” signalling via EGFR autophosphorylation and KIT amplification [251]. Therefore, a combinatorial therapy
involving ALK inhibitors and inhibitors targeting other RTKs or downstream
signalling proteins will most likely be required for the future therapy of ALKpositive cancer patients. Indeed, Tanizaki et al., reported recently, that the
combination of the ALK-specific inhibitor NVP-TAE684 and the MEK inhibitor
AZD6244 induced apoptosis and inhibition of STAT3 and ERK pathways in EML4ALK positive NSCLC cells, that were unresponsive to NVP-TAE684 treatment
alone [388]. An additional strategy for ALK-positive neuroblastoma therapy is the
ALK-targeted immunotherapy including crizotinib and ALK-targeting antibodies
[295]. This combinatorial therapy using tyrosine kinase inhibitors and antibodies has
been successfully tested for NSCLC expressing erlotinib-resistant EGFRT790M,
inducing tumor regression [389]. The ALKF1174L mutant which is resistant to
crizotinib, behaves fairly similar to EGFRT790M [271], suggesting such kind of dual
therapy might be beneficial for neuroblastoma patients harbouring the ALKF1174L
mutant.
A futuristic therapeutical approach for neuroblastoma patients harbouring
amplified ALK, or ALK gain-of-function mutations together with amplified MYCN,
might involve the use of an ALK inhibitor combined with liposomes containing
siRNA targeting MYCN. However, recent studies proposed therapeutical
approaches including indirect targeting of MYCN. The PI3K/mTOR inhibitor NVPBEZ235 has been shown to drive MYCN degradation in tumour cells, which
resulted in decreased tumour proliferation and angiogenesis [390]. Further, the
authors suggest that NVP-BEZ235 should be clinically tested in neuroblastoma
patients with MYCN amplification. Another therapeutical target could be Aurora
60
kinase A which is able to prevent proteasomal degradation of MYCN [378].
Recently, the Aurora kinase inhibitor CCT137690 has been shown to reduce MYCN
expression and block proliferation of MYCN-amplified neuroblastoma cell lines
[391]. Hence, a combination of an ALK and PI3K/mTOR or Aurora kinase A
inhibitor could serve as an alternative therapy especially for MYCN-amplified
neuroblastoma patients with ALK gain-of-function mutations.
In spite of the growing prominent role of ALK in oncogenesis, many
questions regarding wild type ALK still persist. Since the initial discovery of ALK
in the 1990s the natural ligand of ALK still remains a mystery. The identification of
the Drosophila ALK ligand Jeb provided one clue: Jeb is not conserved in
mammals. So what is the ligand for mammalian ALK? Linked to this question is
another one: what is the physiological function? To date one can only speculate that
ALK might be involved in the nervous system. The mouse model appears to be the
method of choice to study the physiological role of mammalian ALK. However,
ALK knockout mice have no significant phenotype, except that they are slightly
smaller and show certain behavioral and neurochemical alterations ([81, 82, 392],
BH and RHP unpublished results).
In general, many questions and challenges remain and new ones will emerge.
Great efforts will be made to tackle those tasks, on the one hand trying to understand
the physiological function of wild type ALK and finding the natural ligand, on the
other hand trying to elucidate the genetic variability of ALK-positive cancer
patients. This allows us to look into an optimistic future of individual cancer therapy
based on the patients’ genetic background and might contribute to winning the “War
on Cancer”.
61
Acknowledgements
During my PhD journey many people have crossed my way, contributing to
this thesis and making it possible, as well as sharing highs and lows.
Most importantly, finishing this PhD thesis would not have been possible
without the support of my supervisors Bengt and Ruth. Thank you for giving me
this great opportunity of doing my PhD in your labs, for your ever lasting
enthusiasm and optimism and all valuable lessons I have learnt!
Rutan: thanks for your immense expertise, invaluable advice and discussions
within and outside science! All past and present group members, especially HaiLing for setting up the PC12 cell system. Lovisa Olofsson for initiating the Rap1
project. The RHP group for sharing many journal clubs and group meetings.
A great thanks to all collaborators and co-authors who contributed to the
work.
Particular thanks to the technical and administrative personnel, especially
Maria, Ethel, Anitha, Johnny, Marek and Media & Dishes: thank you for taking
care of administrative issues, orders, computer issues, solutions and heaps of glass
ware!
During my time at the department many people have crossed my way, sharing
teaching, taking courses, lunch breaks, Fika, parties, IKSU classes, … Thank you all
(past and present members) at the Molecular Biology Department for a nice
time!
People outside the Department and outside the “science world”, i.e. the “real
world”: Emma: thank you for helping me to get started in Sweden and for being a
very good friend. No one can beat our Skype dates ;-) Matilda: thanks for a nice
time with hiking and skiing trips, rafting, IKSU and for being my friend. Thanks to
the German gang at the Department: without you my German would have
deteriorated! The book club: yes, we really tried to discuss the books besides other
matters! ☺ The craft night gang: one time in the future my craft project will be
finished! ;-) Thanks to the orchestras, Umeå Musiksällskap and Hemvärnets
Musikkår Umeå, for providing great distraction from science. Jana, thanks for
introducing me to HvMk Umeå!
Thanks to my friends back in Germany for keeping in contact over the
distance and over all the years: Ulrike, Antje, Mirjam, and Stefan.
Finally, thanks to all people not mentioned, but not forgotten.
Last but not least, I would like to thank my family. Especially Mum and
Dad, for always believing in me, giving me all the support I could ever ask for and
for always being there for me. You are the best!
62
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
Lee CC, Jia Y, Li N, Sun X, Ng K, Ambing E, Gao MY, Hua S, Chen C, Kim S, Michellys PY,
Lesley SA, Harris JL, Spraggon G. Crystal structure of the ALK (anaplastic lymphoma kinase)
catalytic domain. Biochem J 2010 Aug 27;430(3):425-37.
Lemmon MA, Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 2010 Jun
25;141(7):1117-34.
Levene PA, Alsberg CL. The cleavage products of vitellin. J Biol Chem 1906;2(1):127-33.
Burnett G, Kennedy EP. The enzymatic phosphorylation of proteins. J Biol Chem 1954
Dec;211(2):969-80.
Eckhart W, Hutchinson MA, Hunter T. An activity phosphorylating tyrosine in polyoma T
antigen immunoprecipitates. Cell 1979 Dec;18(4):925-33.
Hunter T, Sefton BM. Transforming gene product of Rous sarcoma virus phosphorylates
tyrosine. Proc Natl Acad Sci U S A 1980 Mar;77(3):1311-5.
Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. The protein kinase complement
of the human genome. Science 2002 Dec 6;298(5600):1912-34.
Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 2000 Oct 13;103(2):211-25.
Ward CW, Lawrence MC, Streltsov VA, Adams TE, McKern NM. The insulin and EGF
receptor structures: new insights into ligand-induced receptor activation. Trends Biochem Sci
2007 Mar;32(3):129-37.
Kornev AP, Taylor SS. Defining the conserved internal architecture of a protein kinase. Biochim
Biophys Acta 2010 Mar;1804(3):440-4.
Blume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature 2001 May 17;411(6835):35565.
Lengyel E, Sawada K, Salgia R. Tyrosine kinase mutations in human cancer. Curr Mol Med
2007 Feb;7(1):77-84.
Morris SW, Kirstein MN, Valentine MB, Dittmer KG, Shapiro DN, Saltman DL, Look AT.
Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma.
Science 1994 Mar 4;263(5151):1281-4.
Shiota M, Fujimoto J, Semba T, Satoh H, Yamamoto T, Mori S. Hyperphosphorylation of a
novel 80 kDa protein-tyrosine kinase similar to Ltk in a human Ki-1 lymphoma cell line, AMS3.
Oncogene 1994 Jun;9(6):1567-74.
Iwahara T, Fujimoto J, Wen D, Cupples R, Bucay N, Arakawa T, Mori S, Ratzkin B,
Yamamoto T. Molecular characterization of ALK, a receptor tyrosine kinase expressed
specifically in the nervous system. Oncogene 1997 Jan 30;14(4):439-49.
Morris SW, Naeve C, Mathew P, James PL, Kirstein MN, Cui X, Witte DP. ALK, the
chromosome 2 gene locus altered by the t(2;5) in non-Hodgkin's lymphoma, encodes a novel
neural receptor tyrosine kinase that is highly related to leukocyte tyrosine kinase (LTK).
Oncogene 1997 May 8;14(18):2175-88.
Loren CE, Scully A, Grabbe C, Edeen PT, Thomas J, McKeown M, Hunter T, Palmer RH.
Identification and characterization of DAlk: a novel Drosophila melanogaster RTK which drives
ERK activation in vivo. Genes Cells 2001 Jun;6(6):531-44.
Liao EH, Hung W, Abrams B, Zhen M. An SCF-like ubiquitin ligase complex that controls
presynaptic differentiation. Nature 2004 Jul 15;430(6997):345-50.
Reiner DJ, Ailion M, Thomas JH, Meyer BJ. C. elegans anaplastic lymphoma kinase ortholog
SCD-2 controls dauer formation by modulating TGF-beta signaling. Curr Biol 2008 Aug
5;18(15):1101-9.
Stoica GE, Kuo A, Aigner A, Sunitha I, Souttou B, Malerczyk C, Caughey DJ, Wen D,
Karavanov A, Riegel AT, Wellstein A. Identification of anaplastic lymphoma kinase as a
receptor for the growth factor pleiotrophin. J Biol Chem 2001 May 18;276(20):16772-9.
Daly NL, Scanlon MJ, Djordjevic JT, Kroon PA, Smith R. Three-dimensional structure of a
cysteine-rich repeat from the low-density lipoprotein receptor. Proc Natl Acad Sci U S A 1995
Jul 3;92(14):6334-8.
Fass D, Blacklow S, Kim PS, Berger JM. Molecular basis of familial hypercholesterolaemia
from structure of LDL receptor module. Nature 1997 Aug 14;388(6643):691-3.
Beckmann G, Bork P. An adhesive domain detected in functionally diverse receptors. Trends
Biochem Sci 1993 Feb;18(2):40-1.
63
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
64
Loren CE, Englund C, Grabbe C, Hallberg B, Hunter T, Palmer RH. A crucial role for the
Anaplastic lymphoma kinase receptor tyrosine kinase in gut development in Drosophila
melanogaster. EMBO Rep 2003 Aug;4(8):781-6.
Bai RY, Dieter P, Peschel C, Morris SW, Duyster J. Nucleophosmin-anaplastic lymphoma
kinase of large-cell anaplastic lymphoma is a constitutively active tyrosine kinase that utilizes
phospholipase C-gamma to mediate its mitogenicity. Mol Cell Biol 1998 Dec;18(12):6951-61.
Donella-Deana A, Marin O, Cesaro L, Gunby RH, Ferrarese A, Coluccia AM, Tartari CJ,
Mologni L, Scapozza L, Gambacorti-Passerini C, Pinna LA. Unique substrate specificity of
anaplastic lymphoma kinase (ALK): development of phosphoacceptor peptides for the assay of
ALK activity. Biochemistry 2005 Jun 14;44(23):8533-42.
Tartari CJ, Gunby RH, Coluccia AM, Sottocornola R, Cimbro B, Scapozza L, Donella-Deana
A, Pinna LA, Gambacorti-Passerini C. Characterization of some molecular mechanisms
governing autoactivation of the catalytic domain of the anaplastic lymphoma kinase. J Biol Chem
2008 Feb 15;283(7):3743-50.
Bossi RT, Saccardo MB, Ardini E, Menichincheri M, Rusconi L, Magnaghi P, Orsini P, Avanzi
N, Borgia AL, Nesi M, Bandiera T, Fogliatto G, Bertrand JA. Crystal structures of anaplastic
lymphoma kinase in complex with ATP competitive inhibitors. Biochemistry 2010 Aug
17;49(32):6813-25.
Englund C, Loren CE, Grabbe C, Varshney GK, Deleuil F, Hallberg B, Palmer RH. Jeb signals
through the Alk receptor tyrosine kinase to drive visceral muscle fusion. Nature 2003 Oct
2;425(6957):512-6.
Klapper R, Stute C, Schomaker O, Strasser T, Janning W, Renkawitz-Pohl R, Holz A. The
formation of syncytia within the visceral musculature of the Drosophila midgut is dependent on
duf, sns and mbc. Mech Dev 2002 Jan;110(1-2):85-96.
Lee HH, Norris A, Weiss JB, Frasch M. Jelly belly protein activates the receptor tyrosine kinase
Alk to specify visceral muscle pioneers. Nature 2003 Oct 2;425(6957):507-12.
Martin BS, Ruiz-Gomez M, Landgraf M, Bate M. A distinct set of founders and fusioncompetent myoblasts make visceral muscles in the Drosophila embryo. Development 2001
Sep;128(17):3331-8.
Stute C, Schimmelpfeng K, Renkawitz-Pohl R, Palmer RH, Holz A. Myoblast determination in
the somatic and visceral mesoderm depends on Notch signalling as well as on
milliways(mili(Alk)) as receptor for Jeb signalling. Development 2004 Feb;131(4):743-54.
Varshney GK, Palmer RH. The bHLH transcription factor Hand is regulated by Alk in the
Drosophila embryonic gut. Biochem Biophys Res Commun 2006 Dec 29;351(4):839-46.
Shirinian M, Varshney G, Loren CE, Grabbe C, Palmer RH. Drosophila Anaplastic Lymphoma
Kinase regulates Dpp signalling in the developing embryonic gut. Differentiation 2007
Jun;75(5):418-26.
Weiss JB, Suyama KL, Lee HH, Scott MP. Jelly belly: a Drosophila LDL receptor repeatcontaining signal required for mesoderm migration and differentiation. Cell 2001 Nov
2;107(3):387-98.
Englund C, Birve A, Falileeva L, Grabbe C, Palmer RH. Miple1 and miple2 encode a family of
MK/PTN homologues in Drosophila melanogaster. Dev Genes Evol 2006 Jan;216(1):10-8.
Bazigou E, Apitz H, Johansson J, Loren CE, Hirst EM, Chen PL, Palmer RH, Salecker I.
Anterograde Jelly belly and Alk receptor tyrosine kinase signaling mediates retinal axon
targeting in Drosophila. Cell 2007 Mar 9;128(5):961-75.
Rohrbough J, Broadie K. Anterograde Jelly belly ligand to Alk receptor signaling at developing
synapses is regulated by Mind the gap. Development 2010 Oct;137(20):3523-33.
Cheng LY, Bailey AP, Leevers SJ, Ragan TJ, Driscoll PC, Gould AP. Anaplastic lymphoma
kinase spares organ growth during nutrient restriction in Drosophila. Cell 2011 Aug
5;146(3):435-47.
Lasek AW, Giorgetti F, Berger KH, Tayor S, Heberlein U. Lmo Genes Regulate Behavioral
Responses to Ethanol in Drosophila melanogaster and the Mouse. Alcohol Clin Exp Res 2011
Sep;35(9):1600-6.
Gouzi JY, Moressis A, Walker JA, Apostolopoulou AA, Palmer RH, Bernards A, Skoulakis EM.
The receptor tyrosine kinase alk controls neurofibromin functions in Drosophila growth and
learning. PLoS Genet 2011 Sep;7(9):e1002281.
Li YX, Wang Y, Hu YO, Zhong JX, Wang DY. Modulation of the assay system for the sensory
integration of 2 sensory stimuli that inhibit each other in nematode Caenorhabditis elegans.
Neurosci Bull 2011 Apr;27(2):69-82.
Shinkai Y, Yamamoto Y, Fujiwara M, Tabata T, Murayama T, Hirotsu T, Ikeda DD, Tsunozaki
M, Iino Y, Bargmann CI, Katsura I, Ishihara T. Behavioral choice between conflicting
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
alternatives is regulated by a receptor guanylyl cyclase, GCY-28, and a receptor tyrosine kinase,
SCD-2, in AIA interneurons of Caenorhabditis elegans. J Neurosci 2011 Feb 23;31(8):3007-15.
Ishihara T, Iino Y, Mohri A, Mori I, Gengyo-Ando K, Mitani S, Katsura I. HEN-1, a secretory
protein with an LDL receptor motif, regulates sensory integration and learning in Caenorhabditis
elegans. Cell 2002 May 31;109(5):639-49.
Lopes SS, Yang X, Muller J, Carney TJ, McAdow AR, Rauch GJ, Jacoby AS, Hurst LD,
Delfino-Machin M, Haffter P, Geisler R, Johnson SL, Ward A, Kelsh RN. Leukocyte tyrosine
kinase functions in pigment cell development. PLoS Genet 2008 Mar;4(3):e1000026.
Reiff T, Huber L, Kramer M, Delattre O, Janoueix-Lerosey I, Rohrer H. Midkine and Alk
signaling in sympathetic neuron proliferation and neuroblastoma predisposition. Development
2011 Nov;138(21):4699-708.
Stoica GE, Kuo A, Powers C, Bowden ET, Sale EB, Riegel AT, Wellstein A. Midkine binds to
anaplastic lymphoma kinase (ALK) and acts as a growth factor for different cell types. J Biol
Chem 2002 Sep 27;277(39):35990-8.
Kadomatsu K, Muramatsu T. Midkine and pleiotrophin in neural development and cancer.
Cancer Lett 2004 Feb 20;204(2):127-43.
Muramatsu T. Midkine and pleiotrophin: two related proteins involved in development,
survival, inflammation and tumorigenesis. J Biochem 2002 Sep;132(3):359-71.
Maeda N, Nishiwaki T, Shintani T, Hamanaka H, Noda M. 6B4 proteoglycan/phosphacan, an
extracellular variant of receptor-like protein-tyrosine phosphatase zeta/RPTPbeta, binds
pleiotrophin/heparin-binding growth-associated molecule (HB-GAM). J Biol Chem 1996 Aug
30;271(35):21446-52.
Meng K, Rodriguez-Pena A, Dimitrov T, Chen W, Yamin M, Noda M, Deuel TF. Pleiotrophin
signals increased tyrosine phosphorylation of beta beta-catenin through inactivation of the
intrinsic catalytic activity of the receptor-type protein tyrosine phosphatase beta/zeta. Proc Natl
Acad Sci U S A 2000 Mar 14;97(6):2603-8.
Muramatsu H, Zou K, Sakaguchi N, Ikematsu S, Sakuma S, Muramatsu T. LDL receptorrelated protein as a component of the midkine receptor. Biochem Biophys Res Commun 2000
Apr 21;270(3):936-41.
Muramatsu H, Zou P, Suzuki H, Oda Y, Chen GY, Sakaguchi N, Sakuma S, Maeda N, Noda M,
Takada Y, Muramatsu T. alpha4beta1- and alpha6beta1-integrins are functional receptors for
midkine, a heparin-binding growth factor. J Cell Sci 2004 Oct 15;117(Pt 22):5405-15.
Nakanishi T, Kadomatsu K, Okamoto T, Ichihara-Tanaka K, Kojima T, Saito H, Tomoda Y,
Muramatsu T. Expression of syndecan-1 and -3 during embryogenesis of the central nervous
system in relation to binding with midkine. J Biochem 1997 Feb;121(2):197-205.
Raulo E, Chernousov MA, Carey DJ, Nolo R, Rauvala H. Isolation of a neuronal cell surface
receptor of heparin binding growth-associated molecule (HB-GAM). Identification as Nsyndecan (syndecan-3). J Biol Chem 1994 Apr 29;269(17):12999-3004.
Perez-Pinera P, Zhang W, Chang Y, Vega JA, Deuel TF. Anaplastic lymphoma kinase is
activated through the pleiotrophin/receptor protein-tyrosine phosphatase beta/zeta signaling
pathway: an alternative mechanism of receptor tyrosine kinase activation. J Biol Chem 2007 Sep
28;282(39):28683-90.
Bowden ET, Stoica GE, Wellstein A. Anti-apoptotic signaling of pleiotrophin through its
receptor, anaplastic lymphoma kinase. J Biol Chem 2002 Sep 27;277(39):35862-8.
Dirks WG, Fahnrich S, Lis Y, Becker E, MacLeod RA, Drexler HG. Expression and functional
analysis of the anaplastic lymphoma kinase (ALK) gene in tumor cell lines. Int J Cancer 2002 Jul
1;100(1):49-56.
Kuo AH, Stoica GE, Riegel AT, Wellstein A. Recruitment of insulin receptor substrate-1 and
activation of NF-kappaB essential for midkine growth signaling through anaplastic lymphoma
kinase. Oncogene 2007 Feb 8;26(6):859-69.
Lu KV, Jong KA, Kim GY, Singh J, Dia EQ, Yoshimoto K, Wang MY, Cloughesy TF, Nelson
SF, Mischel PS. Differential induction of glioblastoma migration and growth by two forms of
pleiotrophin. J Biol Chem 2005 Jul 22;280(29):26953-64.
Mathivet T, Mazot P, Vigny M. In contrast to agonist monoclonal antibodies, both C-terminal
truncated form and full length form of Pleiotrophin failed to activate vertebrate ALK (anaplastic
lymphoma kinase)? Cell Signal 2007 Dec;19(12):2434-43.
Moog-Lutz C, Degoutin J, Gouzi JY, Frobert Y, Brunet-de Carvalho N, Bureau J, Creminon C,
Vigny M. Activation and inhibition of anaplastic lymphoma kinase receptor tyrosine kinase by
monoclonal antibodies and absence of agonist activity of pleiotrophin. J Biol Chem 2005 Jul
15;280(28):26039-48.
65
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
66
Motegi A, Fujimoto J, Kotani M, Sakuraba H, Yamamoto T. ALK receptor tyrosine kinase
promotes cell growth and neurite outgrowth. J Cell Sci 2004 Jul 1;117(Pt 15):3319-29.
Mourali J, Benard A, Lourenco FC, Monnet C, Greenland C, Moog-Lutz C, Racaud-Sultan C,
Gonzalez-Dunia D, Vigny M, Mehlen P, Delsol G, Allouche M. Anaplastic lymphoma kinase is
a dependence receptor whose proapoptotic functions are activated by caspase cleavage. Mol Cell
Biol 2006 Aug;26(16):6209-22.
Powers C, Aigner A, Stoica GE, McDonnell K, Wellstein A. Pleiotrophin signaling through
anaplastic lymphoma kinase is rate-limiting for glioblastoma growth. J Biol Chem 2002 Apr
19;277(16):14153-8.
Yanagisawa H, Komuta Y, Kawano H, Toyoda M, Sango K. Pleiotrophin induces neurite
outgrowth and up-regulates growth-associated protein (GAP)-43 mRNA through the
ALK/GSK3beta/beta-catenin signaling in developing mouse neurons. Neurosci Res 2010
Jan;66(1):111-6.
Degoutin J, Vigny M, Gouzi JY. ALK activation induces Shc and FRS2 recruitment: Signaling
and phenotypic outcomes in PC12 cells differentiation. FEBS Lett 2007 Feb 20;581(4):727-34.
Souttou B, Carvalho NB, Raulais D, Vigny M. Activation of anaplastic lymphoma kinase
receptor tyrosine kinase induces neuronal differentiation through the mitogen-activated protein
kinase pathway. J Biol Chem 2001 Mar 23;276(12):9526-31.
Yang HL, Eriksson T, Vernersson E, Vigny M, Hallberg B, Palmer RH. The ligand Jelly Belly
(Jeb) activates the Drosophila Alk RTK to drive PC12 cell differentiation, but is unable to
activate the mouse ALK RTK. J Exp Zoolog B Mol Dev Evol 2007 May 15;308(3):269-82.
Chikamori M, Fujimoto J, Tokai-Nishizumi N, Yamamoto T. Identification of multiple SNTbinding sites on NPM-ALK oncoprotein and their involvement in cell transformation. Oncogene
2007 May 3;26(20):2950-4.
Piccinini G, Bacchiocchi R, Serresi M, Vivani C, Rossetti S, Gennaretti C, Carbonari D, Fazioli
F. A ligand-inducible epidermal growth factor receptor/anaplastic lymphoma kinase chimera
promotes mitogenesis and transforming properties in 3T3 cells. J Biol Chem 2002 Jun
21;277(25):22231-9.
Schonherr C, Yang HL, Vigny M, Palmer RH, Hallberg B. Anaplastic lymphoma kinase
activates the small GTPase Rap1 via the Rap1-specific GEF C3G in both neuroblastoma and
PC12 cells. Oncogene 2010 May 13;29(19):2817-30.
Degoutin J, Brunet-de Carvalho N, Cifuentes-Diaz C, Vigny M. ALK (Anaplastic Lymphoma
Kinase) expression in DRG neurons and its involvement in neuron-Schwann cells interaction.
Eur J Neurosci 2009 Jan;29(2):275-86.
Hurley SP, Clary DO, Copie V, Lefcort F. Anaplastic lymphoma kinase is dynamically
expressed on subsets of motor neurons and in the peripheral nervous system. J Comp Neurol
2006 Mar 10;495(2):202-12.
Wang YW, Tu PH, Lin KT, Lin SC, Ko JY, Jou YS. Identification of oncogenic point mutations
and hyperphosphorylation of anaplastic lymphoma kinase in lung cancer. Neoplasia 2011
Aug;13(8):704-15.
Vernersson E, Khoo NK, Henriksson ML, Roos G, Palmer RH, Hallberg B. Characterization of
the expression of the ALK receptor tyrosine kinase in mice. Gene Expr Patterns 2006
Jun;6(5):448-61.
Yan X, Kennedy CR, Tilkens SB, Wiedemeier O, Guan H, Park JI, Chan AM. Cooperative
Cross-Talk between Neuroblastoma Subtypes Confers Resistance to Anaplastic Lymphoma
Kinase Inhibition. Genes Cancer 2011 May;2(5):538-49.
Pulford K, Lamant L, Morris SW, Butler LH, Wood KM, Stroud D, Delsol G, Mason DY.
Detection of anaplastic lymphoma kinase (ALK) and nucleolar protein nucleophosmin (NPM)ALK proteins in normal and neoplastic cells with the monoclonal antibody ALK1. Blood 1997
Feb 15;89(4):1394-404.
Gouzi JY, Moog-Lutz C, Vigny M, Brunet-de Carvalho N. Role of the subcellular localization of
ALK tyrosine kinase domain in neuronal differentiation of PC12 cells. J Cell Sci 2005 Dec
15;118(Pt 24):5811-23.
Bilsland JG, Wheeldon A, Mead A, Znamenskiy P, Almond S, Waters KA, Thakur M,
Beaumont V, Bonnert TP, Heavens R, Whiting P, McAllister G, Munoz-Sanjuan I. Behavioral
and neurochemical alterations in mice deficient in anaplastic lymphoma kinase suggest
therapeutic potential for psychiatric indications. Neuropsychopharmacology 2008 Feb;33(3):685700.
Weiss JB, Xue C, Benice T, Xue L, Morris SW, Raber J. Anaplastic Lymphoma Kinase and
Leukocyte Tyrosine Kinase: Functions and genetic interactions in learning, memory and adult
neurogenesis. Pharmacol Biochem Behav 2012 Jan;100(3):566-74.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
Kunugi H, Hashimoto R, Okada T, Hori H, Nakabayashi T, Baba A, Kudo K, Omori M,
Takahashi S, Tsukue R, Anami K, Hirabayashi N, Kosuga A, Tatsumi M, Kamijima K, Asada T,
Harada S, Arima K, Saitoh O. Possible association between nonsynonymous polymorphisms of
the anaplastic lymphoma kinase (ALK) gene and schizophrenia in a Japanese population. J
Neural Transm 2006 Oct;113(10):1569-73.
Lasek AW, Gesch J, Giorgetti F, Kharazia V, Heberlein U. Alk is a transcriptional target of
LMO4 and ERalpha that promotes cocaine sensitization and reward. J Neurosci 2011 Oct
5;31(40):14134-41.
Bai RY, Ouyang T, Miething C, Morris SW, Peschel C, Duyster J. Nucleophosmin-anaplastic
lymphoma kinase associated with anaplastic large-cell lymphoma activates the
phosphatidylinositol 3-kinase/Akt antiapoptotic signaling pathway. Blood 2000 Dec
15;96(13):4319-27.
Polgar D, Leisser C, Maier S, Strasser S, Ruger B, Dettke M, Khorchide M, Simonitsch I, Cerni
C, Krupitza G. Truncated ALK derived from chromosomal translocation t(2;5)(p23;q35) binds to
the SH3 domain of p85-PI3K. Mutat Res 2005 Feb 15;570(1):9-15.
Slupianek A, Nieborowska-Skorska M, Hoser G, Morrione A, Majewski M, Xue L, Morris SW,
Wasik MA, Skorski T. Role of phosphatidylinositol 3-kinase-Akt pathway in
nucleophosmin/anaplastic lymphoma kinase-mediated lymphomagenesis. Cancer Res 2001 Mar
1;61(5):2194-9.
Gu TL, Tothova Z, Scheijen B, Griffin JD, Gilliland DG, Sternberg DW. NPM-ALK fusion
kinase of anaplastic large-cell lymphoma regulates survival and proliferative signaling through
modulation of FOXO3a. Blood 2004 Jun 15;103(12):4622-9.
Marzec M, Kasprzycka M, Liu X, El-Salem M, Halasa K, Raghunath PN, Bucki R, Wlodarski P,
Wasik MA. Oncogenic tyrosine kinase NPM/ALK induces activation of the rapamycin-sensitive
mTOR signaling pathway. Oncogene 2007 Aug 16;26(38):5606-14.
Vega F, Medeiros LJ, Leventaki V, Atwell C, Cho-Vega JH, Tian L, Claret FX, Rassidakis GZ.
Activation of mammalian target of rapamycin signaling pathway contributes to tumor cell
survival in anaplastic lymphoma kinase-positive anaplastic large cell lymphoma. Cancer Res
2006 Jul 1;66(13):6589-97.
McDonnell SR, Hwang SR, Basrur V, Conlon KP, Fermin D, Wey E, Murga-Zamalloa C, Zeng
Z, Zu Y, Elenitoba-Johnson KS, Lim MS. NPM-ALK signals through glycogen synthase kinase
3beta to promote oncogenesis. Oncogene 2011 Dec 19.
Crockett DK, Lin Z, Elenitoba-Johnson KS, Lim MS. Identification of NPM-ALK interacting
proteins by tandem mass spectrometry. Oncogene 2004 Apr 8;23(15):2617-29.
Fujimoto J, Shiota M, Iwahara T, Seki N, Satoh H, Mori S, Yamamoto T. Characterization of
the transforming activity of p80, a hyperphosphorylated protein in a Ki-1 lymphoma cell line
with chromosomal translocation t(2;5). Proc Natl Acad Sci U S A 1996 Apr 30;93(9):4181-6.
Riera L, Lasorsa E, Ambrogio C, Surrenti N, Voena C, Chiarle R. Involvement of Grb2 adaptor
protein in nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-mediated signaling and
anaplastic large cell lymphoma growth. J Biol Chem 2010 Aug 20;285(34):26441-50.
Amin HM, McDonnell TJ, Ma Y, Lin Q, Fujio Y, Kunisada K, Leventaki V, Das P, Rassidakis
GZ, Cutler C, Medeiros LJ, Lai R. Selective inhibition of STAT3 induces apoptosis and G(1) cell
cycle arrest in ALK-positive anaplastic large cell lymphoma. Oncogene 2004 Jul
15;23(32):5426-34.
Galkin AV, Melnick JS, Kim S, Hood TL, Li N, Li L, Xia G, Steensma R, Chopiuk G, Jiang J,
Wan Y, Ding P, Liu Y, Sun F, Schultz PG, Gray NS, Warmuth M. Identification of NVPTAE684, a potent, selective, and efficacious inhibitor of NPM-ALK. Proc Natl Acad Sci U S A
2007 Jan 2;104(1):270-5.
Marzec M, Kasprzycka M, Ptasznik A, Wlodarski P, Zhang Q, Odum N, Wasik MA. Inhibition
of ALK enzymatic activity in T-cell lymphoma cells induces apoptosis and suppresses
proliferation and STAT3 phosphorylation independently of Jak3. Lab Invest 2005
Dec;85(12):1544-54.
Wan W, Albom MS, Lu L, Quail MR, Becknell NC, Weinberg LR, Reddy DR, Holskin BP,
Angeles TS, Underiner TL, Meyer SL, Hudkins RL, Dorsey BD, Ator MA, Ruggeri BA, Cheng
M. Anaplastic lymphoma kinase activity is essential for the proliferation and survival of
anaplastic large-cell lymphoma cells. Blood 2006 Feb 15;107(4):1617-23.
Zamo A, Chiarle R, Piva R, Howes J, Fan Y, Chilosi M, Levy DE, Inghirami G. Anaplastic
lymphoma kinase (ALK) activates Stat3 and protects hematopoietic cells from cell death.
Oncogene 2002 Feb 7;21(7):1038-47.
67
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
110.
111.
112.
113.
114.
115.
116.
117.
68
Zhang Q, Raghunath PN, Xue L, Majewski M, Carpentieri DF, Odum N, Morris S, Skorski T,
Wasik MA. Multilevel dysregulation of STAT3 activation in anaplastic lymphoma kinasepositive T/null-cell lymphoma. J Immunol 2002 Jan 1;168(1):466-74.
Amin HM, Medeiros LJ, Ma Y, Feretzaki M, Das P, Leventaki V, Rassidakis GZ, O'Connor SL,
McDonnell TJ, Lai R. Inhibition of JAK3 induces apoptosis and decreases anaplastic lymphoma
kinase activity in anaplastic large cell lymphoma. Oncogene 2003 Aug 21;22(35):5399-407.
Shi X, Franko B, Frantz C, Amin HM, Lai R. JSI-124 (cucurbitacin I) inhibits Janus kinase3/signal transducer and activator of transcription-3 signalling, downregulates nucleophosminanaplastic lymphoma kinase (ALK), and induces apoptosis in ALK-positive anaplastic large cell
lymphoma cells. Br J Haematol 2006 Oct;135(1):26-32.
Hegazy SA, Wang P, Anand M, Ingham RJ, Gelebart P, Lai R. The tyrosine 343 residue of
nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) is important for its interaction with
SHP1, a cytoplasmic tyrosine phosphatase with tumor suppressor functions. J Biol Chem 2010
Jun 25;285(26):19813-20.
Khoury JD, Rassidakis GZ, Medeiros LJ, Amin HM, Lai R. Methylation of SHP1 gene and loss
of SHP1 protein expression are frequent in systemic anaplastic large cell lymphoma. Blood 2004
Sep 1;104(5):1580-1.
Nieborowska-Skorska M, Slupianek A, Xue L, Zhang Q, Raghunath PN, Hoser G, Wasik MA,
Morris SW, Skorski T. Role of signal transducer and activator of transcription 5 in
nucleophosmin/ anaplastic lymphoma kinase-mediated malignant transformation of lymphoid
cells. Cancer Res 2001 Sep 1;61(17):6517-23.
Zhang Q, Wang HY, Liu X, Wasik MA. STAT5A is epigenetically silenced by the tyrosine
kinase NPM1-ALK and acts as a tumor suppressor by reciprocally inhibiting NPM1-ALK
expression. Nat Med 2007 Nov;13(11):1341-8.
Ambrogio C, Voena C, Manazza AD, Martinengo C, Costa C, Kirchhausen T, Hirsch E,
Inghirami G, Chiarle R. The anaplastic lymphoma kinase controls cell shape and growth of
anaplastic large cell lymphoma through Cdc42 activation. Cancer Res 2008 Nov 1;68(21):8899907.
Colomba A, Courilleau D, Ramel D, Billadeau DD, Espinos E, Delsol G, Payrastre B, GaitsIacovoni F. Activation of Rac1 and the exchange factor Vav3 are involved in NPM-ALK
signaling in anaplastic large cell lymphomas. Oncogene 2008 Apr 24;27(19):2728-36.
Ambrogio C, Voena C, Manazza AD, Piva R, Riera L, Barberis L, Costa C, Tarone G, Defilippi
P, Hirsch E, Boeri Erba E, Mohammed S, Jensen ON, Palestro G, Inghirami G, Chiarle R.
p130Cas mediates the transforming properties of the anaplastic lymphoma kinase. Blood 2005
Dec 1;106(12):3907-16.
Cussac D, Greenland C, Roche S, Bai RY, Duyster J, Morris SW, Delsol G, Allouche M,
Payrastre B. Nucleophosmin-anaplastic lymphoma kinase of anaplastic large-cell lymphoma
recruits, activates, and uses pp60c-src to mediate its mitogenicity. Blood 2004 Feb
15;103(4):1464-71.
Voena C, Conte C, Ambrogio C, Boeri Erba E, Boccalatte F, Mohammed S, Jensen ON, Palestro
G, Inghirami G, Chiarle R. The tyrosine phosphatase Shp2 interacts with NPM-ALK and
regulates anaplastic lymphoma cell growth and migration. Cancer Res 2007 May 1;67(9):427886.
Bassermann F, von Klitzing C, Munch S, Bai RY, Kawaguchi H, Morris SW, Peschel C,
Duyster J. NIPA defines an SCF-type mammalian E3 ligase that regulates mitotic entry. Cell
2005 Jul 15;122(1):45-57.
Ouyang T, Bai RY, Bassermann F, von Klitzing C, Klumpen S, Miething C, Morris SW, Peschel
C, Duyster J. Identification and characterization of a nuclear interacting partner of anaplastic
lymphoma kinase (NIPA). J Biol Chem 2003 Aug 8;278(32):30028-36.
Leventaki V, Drakos E, Medeiros LJ, Lim MS, Elenitoba-Johnson KS, Claret FX, Rassidakis
GZ. NPM-ALK oncogenic kinase promotes cell-cycle progression through activation of
JNK/cJun signaling in anaplastic large-cell lymphoma. Blood 2007 Sep 1;110(5):1621-30.
Turner SD, Tooze R, Maclennan K, Alexander DR. Vav-promoter regulated oncogenic fusion
protein NPM-ALK in transgenic mice causes B-cell lymphomas with hyperactive Jun kinase.
Oncogene 2003 Oct 30;22(49):7750-61.
Cui YX, Kerby A, McDuff FK, Ye H, Turner SD. NPM-ALK inhibits the p53 tumor suppressor
pathway in an MDM2 and JNK-dependent manner. Blood 2009 May 21;113(21):5217-27.
Singh RR, Cho-Vega JH, Davuluri Y, Ma S, Kasbidi F, Milito C, Lennon PA, Drakos E,
Medeiros LJ, Luthra R, Vega F. Sonic hedgehog signaling pathway is activated in ALK-positive
anaplastic large cell lymphoma. Cancer Res 2009 Mar 15;69(6):2550-8.
118.
119.
120.
121.
122.
123.
124.
125.
126.
127.
128.
129.
130.
131.
132.
133.
134.
Boccalatte FE, Voena C, Riganti C, Bosia A, D'Amico L, Riera L, Cheng M, Ruggeri B, Jensen
ON, Goss VL, Lee K, Nardone J, Rush J, Polakiewicz RD, Comb MJ, Chiarle R, Inghirami G.
The
enzymatic
activity
of
5-aminoimidazole-4-carboxamide
ribonucleotide
formyltransferase/IMP cyclohydrolase is enhanced by NPM-ALK: new insights in ALKmediated pathogenesis and the treatment of ALCL. Blood 2009 Mar 19;113(12):2776-90.
Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ, Zhang H, Zha XM, Polakiewicz RD,
Comb MJ. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotechnol
2005 Jan;23(1):94-101.
Sjostrom C, Seiler C, Crockett DK, Tripp SR, Elenitoba Johnson KS, Lim MS. Global proteome
profiling of NPM/ALK-positive anaplastic large cell lymphoma. Exp Hematol 2007
Aug;35(8):1240-8.
Lamant L, de Reynies A, Duplantier MM, Rickman DS, Sabourdy F, Giuriato S, Brugieres L,
Gaulard P, Espinos E, Delsol G. Gene-expression profiling of systemic anaplastic large-cell
lymphoma reveals differences based on ALK status and two distinct morphologic ALK+
subtypes. Blood 2007 Mar 1;109(5):2156-64.
Piva R, Pellegrino E, Mattioli M, Agnelli L, Lombardi L, Boccalatte F, Costa G, Ruggeri BA,
Cheng M, Chiarle R, Palestro G, Neri A, Inghirami G. Functional validation of the anaplastic
lymphoma kinase signature identifies CEBPB and BCL2A1 as critical target genes. J Clin Invest
2006 Dec;116(12):3171-82.
Wang P, Wu F, Zhang J, McMullen T, Young LC, Ingham RJ, Li L, Lai R. Serine
phosphorylation of NPM-ALK, which is dependent on the auto-activation of the kinase
activation loop, contributes to its oncogenic potential. Carcinogenesis 2011 Feb;32(2):146-53.
Hernandez L, Bea S, Bellosillo B, Pinyol M, Falini B, Carbone A, Ott G, Rosenwald A,
Fernandez A, Pulford K, Mason D, Morris SW, Santos E, Campo E. Diversity of genomic
breakpoints in TFG-ALK translocations in anaplastic large cell lymphomas: identification of a
new TFG-ALK(XL) chimeric gene with transforming activity. Am J Pathol 2002
Apr;160(4):1487-94.
Trinei M, Lanfrancone L, Campo E, Pulford K, Mason DY, Pelicci PG, Falini B. A new variant
anaplastic lymphoma kinase (ALK)-fusion protein (ATIC-ALK) in a case of ALK-positive
anaplastic large cell lymphoma. Cancer Res 2000 Feb 15;60(4):793-8.
Wong DW, Leung EL, Wong SK, Tin VP, Sihoe AD, Cheng LC, Au JS, Chung LP, Wong MP.
A novel KIF5B-ALK variant in nonsmall cell lung cancer. Cancer 2011 Jun 15;117(12):2709-18.
Armstrong F, Duplantier MM, Trempat P, Hieblot C, Lamant L, Espinos E, Racaud-Sultan C,
Allouche M, Campo E, Delsol G, Touriol C. Differential effects of X-ALK fusion proteins on
proliferation, transformation, and invasion properties of NIH3T3 cells. Oncogene 2004 Aug
12;23(36):6071-82.
Chen Y, Takita J, Choi YL, Kato M, Ohira M, Sanada M, Wang L, Soda M, Kikuchi A, Igarashi
T, Nakagawara A, Hayashi Y, Mano H, Ogawa S. Oncogenic mutations of ALK kinase in
neuroblastoma. Nature 2008 Oct 16;455(7215):971-4.
Chen Z, Sasaki T, Tan X, Carretero J, Shimamura T, Li D, Xu C, Wang Y, Adelmant GO,
Capelletti M, Lee HJ, Rodig SJ, Borgman C, Park SI, Kim HR, Padera R, Marto JA, Gray NS,
Kung AL, Shapiro GI, Janne PA, Wong KK. Inhibition of ALK, PI3K/MEK, and HSP90 in
murine lung adenocarcinoma induced by EML4-ALK fusion oncogene. Cancer Res 2010 Dec
1;70(23):9827-36.
Takezawa K, Okamoto I, Nishio K, Janne PA, Nakagawa K. Role of ERK-BIM and STAT3survivin signaling pathways in ALK inhibitor-induced apoptosis in EML4-ALK-positive lung
cancer. Clin Cancer Res 2011 Apr 15;17(8):2140-8.
Hallberg B, Palmer RH. ALK and NSCLC: Targeted therapy with ALK inhibitors. F1000 Med
Rep 2011;3:21.
Caren H, Abel F, Kogner P, Martinsson T. High incidence of DNA mutations and gene
amplifications of the ALK gene in advanced sporadic neuroblastoma tumours. Biochem J 2008
Dec 1;416(2):153-9.
George RE, Sanda T, Hanna M, Frohling S, Luther W, 2nd, Zhang J, Ahn Y, Zhou W, London
WB, McGrady P, Xue L, Zozulya S, Gregor VE, Webb TR, Gray NS, Gilliland DG, Diller L,
Greulich H, Morris SW, Meyerson M, Look AT. Activating mutations in ALK provide a
therapeutic target in neuroblastoma. Nature 2008 Oct 16;455(7215):975-8.
Janoueix-Lerosey I, Lequin D, Brugieres L, Ribeiro A, de Pontual L, Combaret V, Raynal V,
Puisieux A, Schleiermacher G, Pierron G, Valteau-Couanet D, Frebourg T, Michon J, Lyonnet S,
Amiel J, Delattre O. Somatic and germline activating mutations of the ALK kinase receptor in
neuroblastoma. Nature 2008 Oct 16;455(7215):967-70.
69
135.
136.
137.
138.
139.
140.
141.
142.
143.
144.
145.
146.
147.
148.
149.
150.
151.
70
Mosse YP, Laudenslager M, Longo L, Cole KA, Wood A, Attiyeh EF, Laquaglia MJ, Sennett R,
Lynch JE, Perri P, Laureys G, Speleman F, Kim C, Hou C, Hakonarson H, Torkamani A, Schork
NJ, Brodeur GM, Tonini GP, Rappaport E, Devoto M, Maris JM. Identification of ALK as a
major familial neuroblastoma predisposition gene. Nature 2008 Oct 16;455(7215):930-5.
Murugan AK, Xing M. Anaplastic thyroid cancers harbor novel oncogenic mutations of the
ALK gene. Cancer Res 2011 Jul 1;71(13):4403-11.
Griffin CA, Hawkins AL, Dvorak C, Henkle C, Ellingham T, Perlman EJ. Recurrent
involvement of 2p23 in inflammatory myofibroblastic tumors. Cancer Res 1999 Jun
15;59(12):2776-80.
Rikova K, Guo A, Zeng Q, Possemato A, Yu J, Haack H, Nardone J, Lee K, Reeves C, Li Y, Hu
Y, Tan Z, Stokes M, Sullivan L, Mitchell J, Wetzel R, Macneill J, Ren JM, Yuan J, Bakalarski
CE, Villen J, Kornhauser JM, Smith B, Li D, Zhou X, Gygi SP, Gu TL, Polakiewicz RD, Rush J,
Comb MJ. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung
cancer. Cell 2007 Dec 14;131(6):1190-203.
Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S, Fujiwara S, Watanabe H,
Kurashina K, Hatanaka H, Bando M, Ohno S, Ishikawa Y, Aburatani H, Niki T, Sohara Y,
Sugiyama Y, Mano H. Identification of the transforming EML4-ALK fusion gene in non-smallcell lung cancer. Nature 2007 Aug 2;448(7153):561-6.
Arber DA, Sun LH, Weiss LM. Detection of the t(2;5)(p23;q35) chromosomal translocation in
large B-cell lymphomas other than anaplastic large cell lymphoma. Hum Pathol 1996
Jun;27(6):590-4.
Debelenko LV, Raimondi SC, Daw N, Shivakumar BR, Huang D, Nelson M, Bridge JA. Renal
cell carcinoma with novel VCL-ALK fusion: new representative of ALK-associated tumor
spectrum. Mod Pathol 2011 Mar;24(3):430-42.
Du XL, Hu H, Lin DC, Xia SH, Shen XM, Zhang Y, Luo ML, Feng YB, Cai Y, Xu X, Han YL,
Zhan QM, Wang MR. Proteomic profiling of proteins dysregulted in Chinese esophageal
squamous cell carcinoma. J Mol Med 2007 Aug;85(8):863-75.
Jazii FR, Najafi Z, Malekzadeh R, Conrads TP, Ziaee AA, Abnet C, Yazdznbod M, Karkhane
AA, Salekdeh GH. Identification of squamous cell carcinoma associated proteins by proteomics
and loss of beta tropomyosin expression in esophageal cancer. World J Gastroenterol 2006 Nov
28;12(44):7104-12.
Miyake I, Hakomori Y, Misu Y, Nakadate H, Matsuura N, Sakamoto M, Sakai R. Domainspecific function of ShcC docking protein in neuroblastoma cells. Oncogene 2005 Apr
28;24(19):3206-15.
Miyake I, Hakomori Y, Shinohara A, Gamou T, Saito M, Iwamatsu A, Sakai R. Activation of
anaplastic lymphoma kinase is responsible for hyperphosphorylation of ShcC in neuroblastoma
cell lines. Oncogene 2002 Aug 29;21(38):5823-34.
Osajima-Hakomori Y, Miyake I, Ohira M, Nakagawara A, Nakagawa A, Sakai R. Biological
role of anaplastic lymphoma kinase in neuroblastoma. Am J Pathol 2005 Jul;167(1):213-22.
Passoni L, Longo L, Collini P, Coluccia AM, Bozzi F, Podda M, Gregorio A, Gambini C,
Garaventa A, Pistoia V, Del Grosso F, Tonini GP, Cheng M, Gambacorti-Passerini C, Anichini
A, Fossati-Bellani F, Di Nicola M, Luksch R. Mutation-independent anaplastic lymphoma kinase
overexpression in poor prognosis neuroblastoma patients. Cancer Res 2009 Sep 15;69(18):733846.
Salido M, Pijuan L, Martinez-Aviles L, Galvan AB, Canadas I, Rovira A, Zanui M, Martinez A,
Longaron R, Sole F, Serrano S, Bellosillo B, Wynes MW, Albanell J, Hirsch FR, Arriola E.
Increased ALK gene copy number and amplification are frequent in non-small cell lung cancer. J
Thorac Oncol 2011 Jan;6(1):21-7.
van Gaal JC, Flucke UE, Roeffen MH, de Bont ES, Sleijfer S, Mavinkurve-Groothuis AM,
Suurmeijer AJ, van der Graaf WT, Versleijen-Jonkers YM. Anaplastic lymphoma kinase
aberrations in rhabdomyosarcoma: clinical and prognostic implications. J Clin Oncol 2012 Jan
20;30(3):308-15.
Stein H, Mason DY, Gerdes J, O'Connor N, Wainscoat J, Pallesen G, Gatter K, Falini B, Delsol
G, Lemke H, Schwarting R, Lennert K. The expression of the Hodgkin's disease associated
antigen Ki-1 in reactive and neoplastic lymphoid tissue: evidence that Reed-Sternberg cells and
histiocytic malignancies are derived from activated lymphoid cells. Blood 1985 Oct;66(4):84858.
Amin HM, Lai R. Pathobiology of ALK+ anaplastic large-cell lymphoma. Blood 2007 Oct
1;110(7):2259-67.
152.
153.
154.
155.
156.
157.
158.
159.
160.
161.
162.
163.
164.
165.
166.
167.
168.
Stein H, Foss HD, Durkop H, Marafioti T, Delsol G, Pulford K, Pileri S, Falini B. CD30(+)
anaplastic large cell lymphoma: a review of its histopathologic, genetic, and clinical features.
Blood 2000 Dec 1;96(12):3681-95.
Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW. WHO
Classification of Tumours of Haematopoietic and Lymphiod Tissues. 4th Edition ed. Bosman FT,
editor. Lyon: International Agency for Research on Cancer 2008.
Falini B, Pileri S, Zinzani PL, Carbone A, Zagonel V, Wolf-Peeters C, Verhoef G, Menestrina F,
Todeschini G, Paulli M, Lazzarino M, Giardini R, Aiello A, Foss HD, Araujo I, Fizzotti M,
Pelicci PG, Flenghi L, Martelli MF, Santucci A. ALK+ lymphoma: clinico-pathological findings
and outcome. Blood 1999 Apr 15;93(8):2697-706.
Gascoyne RD, Aoun P, Wu D, Chhanabhai M, Skinnider BF, Greiner TC, Morris SW, Connors
JM, Vose JM, Viswanatha DS, Coldman A, Weisenburger DD. Prognostic significance of
anaplastic lymphoma kinase (ALK) protein expression in adults with anaplastic large cell
lymphoma. Blood 1999 Jun 1;93(11):3913-21.
Savage KJ, Harris NL, Vose JM, Ullrich F, Jaffe ES, Connors JM, Rimsza L, Pileri SA,
Chhanabhai M, Gascoyne RD, Armitage JO, Weisenburger DD. ALK- anaplastic large-cell
lymphoma is clinically and immunophenotypically different from both ALK+ ALCL and
peripheral T-cell lymphoma, not otherwise specified: report from the International Peripheral TCell Lymphoma Project. Blood 2008 Jun 15;111(12):5496-504.
Shiota M, Nakamura S, Ichinohasama R, Abe M, Akagi T, Takeshita M, Mori N, Fujimoto J,
Miyauchi J, Mikata A, Nanba K, Takami T, Yamabe H, Takano Y, Izumo T, Nagatani T, Mohri
N, Nasu K, Satoh H, Katano H, Fujimoto J, Yamamoto T, Mori S. Anaplastic large cell
lymphomas expressing the novel chimeric protein p80NPM/ALK: a distinct clinicopathologic
entity. Blood 1995 Sep 1;86(5):1954-60.
Maes B, Vanhentenrijk V, Wlodarska I, Cools J, Peeters B, Marynen P, de Wolf-Peeters C. The
NPM-ALK and the ATIC-ALK fusion genes can be detected in non-neoplastic cells. Am J Pathol
2001 Jun;158(6):2185-93.
Trumper L, Pfreundschuh M, Bonin FV, Daus H. Detection of the t(2;5)-associated NPM/ALK
fusion cDNA in peripheral blood cells of healthy individuals. Br J Haematol 1998
Dec;103(4):1138-44.
Lamant L, Dastugue N, Pulford K, Delsol G, Mariame B. A new fusion gene TPM3-ALK in
anaplastic large cell lymphoma created by a (1;2)(q25;p23) translocation. Blood 1999 May
1;93(9):3088-95.
Meech SJ, McGavran L, Odom LF, Liang X, Meltesen L, Gump J, Wei Q, Carlsen S, Hunger
SP. Unusual childhood extramedullary hematologic malignancy with natural killer cell properties
that contains tropomyosin 4--anaplastic lymphoma kinase gene fusion. Blood 2001 Aug
15;98(4):1209-16.
Siebert R, Gesk S, Harder L, Steinemann D, Grote W, Schlegelberger B, Tiemann M,
Wlodarska I, Schemmel V. Complex variant translocation t(1;2) with TPM3-ALK fusion due to
cryptic ALK gene rearrangement in anaplastic large-cell lymphoma. Blood 1999 Nov
15;94(10):3614-7.
Hernandez L, Pinyol M, Hernandez S, Bea S, Pulford K, Rosenwald A, Lamant L, Falini B, Ott
G, Mason DY, Delsol G, Campo E. TRK-fused gene (TFG) is a new partner of ALK in
anaplastic large cell lymphoma producing two structurally different TFG-ALK translocations.
Blood 1999 Nov 1;94(9):3265-8.
Colleoni GW, Bridge JA, Garicochea B, Liu J, Filippa DA, Ladanyi M. ATIC-ALK: A novel
variant ALK gene fusion in anaplastic large cell lymphoma resulting from the recurrent cryptic
chromosomal inversion, inv(2)(p23q35). Am J Pathol 2000 Mar;156(3):781-9.
Ma Z, Cools J, Marynen P, Cui X, Siebert R, Gesk S, Schlegelberger B, Peeters B, De WolfPeeters C, Wlodarska I, Morris SW. Inv(2)(p23q35) in anaplastic large-cell lymphoma induces
constitutive anaplastic lymphoma kinase (ALK) tyrosine kinase activation by fusion to ATIC, an
enzyme involved in purine nucleotide biosynthesis. Blood 2000 Mar 15;95(6):2144-9.
Touriol C, Greenland C, Lamant L, Pulford K, Bernard F, Rousset T, Mason DY, Delsol G.
Further demonstration of the diversity of chromosomal changes involving 2p23 in ALK-positive
lymphoma: 2 cases expressing ALK kinase fused to CLTCL (clathrin chain polypeptide-like).
Blood 2000 May 15;95(10):3204-7.
Tort F, Campo E, Pohlman B, Hsi E. Heterogeneity of genomic breakpoints in MSN-ALK
translocations in anaplastic large cell lymphoma. Hum Pathol 2004 Aug;35(8):1038-41.
Tort F, Pinyol M, Pulford K, Roncador G, Hernandez L, Nayach I, Kluin-Nelemans HC, Kluin
P, Touriol C, Delsol G, Mason D, Campo E. Molecular characterization of a new ALK
71
169.
170.
171.
172.
173.
174.
175.
176.
177.
178.
179.
180.
181.
182.
183.
184.
185.
186.
72
translocation involving moesin (MSN-ALK) in anaplastic large cell lymphoma. Lab Invest 2001
Mar;81(3):419-26.
Cools J, Wlodarska I, Somers R, Mentens N, Pedeutour F, Maes B, De Wolf-Peeters C, Pauwels
P, Hagemeijer A, Marynen P. Identification of novel fusion partners of ALK, the anaplastic
lymphoma kinase, in anaplastic large-cell lymphoma and inflammatory myofibroblastic tumor.
Genes Chromosomes Cancer 2002 Aug;34(4):354-62.
Lamant L, Gascoyne RD, Duplantier MM, Armstrong F, Raghab A, Chhanabhai M, RajcanSeparovic E, Raghab J, Delsol G, Espinos E. Non-muscle myosin heavy chain (MYH9): a new
partner fused to ALK in anaplastic large cell lymphoma. Genes Chromosomes Cancer 2003
Aug;37(4):427-32.
Gleason BC, Hornick JL. Inflammatory myofibroblastic tumours: where are we now? J Clin
Pathol 2008 Apr;61(4):428-37.
Coffin CM, Watterson J, Priest JR, Dehner LP. Extrapulmonary inflammatory myofibroblastic
tumor (inflammatory pseudotumor). A clinicopathologic and immunohistochemical study of 84
cases. Am J Surg Pathol 1995 Aug;19(8):859-72.
Lawrence B, Perez-Atayde A, Hibbard MK, Rubin BP, Dal Cin P, Pinkus JL, Pinkus GS, Xiao
S, Yi ES, Fletcher CD, Fletcher JA. TPM3-ALK and TPM4-ALK oncogenes in inflammatory
myofibroblastic tumors. Am J Pathol 2000 Aug;157(2):377-84.
Debiec-Rychter M, Marynen P, Hagemeijer A, Pauwels P. ALK-ATIC fusion in urinary bladder
inflammatory myofibroblastic tumor. Genes Chromosomes Cancer 2003 Oct;38(2):187-90.
Bridge JA, Kanamori M, Ma Z, Pickering D, Hill DA, Lydiatt W, Lui MY, Colleoni GW,
Antonescu CR, Ladanyi M, Morris SW. Fusion of the ALK gene to the clathrin heavy chain
gene, CLTC, in inflammatory myofibroblastic tumor. Am J Pathol 2001 Aug;159(2):411-5.
Patel AS, Murphy KM, Hawkins AL, Cohen JS, Long PP, Perlman EJ, Griffin CA. RANBP2
and CLTC are involved in ALK rearrangements in inflammatory myofibroblastic tumors. Cancer
Genet Cytogenet 2007 Jul 15;176(2):107-14.
Ma Z, Hill DA, Collins MH, Morris SW, Sumegi J, Zhou M, Zuppan C, Bridge JA. Fusion of
ALK to the Ran-binding protein 2 (RANBP2) gene in inflammatory myofibroblastic tumor.
Genes Chromosomes Cancer 2003 May;37(1):98-105.
Debelenko LV, Arthur DC, Pack SD, Helman LJ, Schrump DS, Tsokos M. Identification of
CARS-ALK fusion in primary and metastatic lesions of an inflammatory myofibroblastic tumor.
Lab Invest 2003 Sep;83(9):1255-65.
Panagopoulos I, Nilsson T, Domanski HA, Isaksson M, Lindblom P, Mertens F, Mandahl N.
Fusion of the SEC31L1 and ALK genes in an inflammatory myofibroblastic tumor. Int J Cancer
2006 Mar 1;118(5):1181-6.
Takeuchi K, Soda M, Togashi Y, Sugawara E, Hatano S, Asaka R, Okumura S, Nakagawa K,
Mano H, Ishikawa Y. Pulmonary inflammatory myofibroblastic tumor expressing a novel fusion,
PPFIBP1-ALK: reappraisal of anti-ALK immunohistochemistry as a tool for novel ALK-fusion
identification. Clin Cancer Res 2011 May 15;17(10):3341-8.
Coffin CM, Patel A, Perkins S, Elenitoba-Johnson KS, Perlman E, Griffin CA. ALK1 and p80
expression and chromosomal rearrangements involving 2p23 in inflammatory myofibroblastic
tumor. Mod Pathol 2001 Jun;14(6):569-76.
Cook JR, Dehner LP, Collins MH, Ma Z, Morris SW, Coffin CM, Hill DA. Anaplastic
lymphoma kinase (ALK) expression in the inflammatory myofibroblastic tumor: a comparative
immunohistochemical study. Am J Surg Pathol 2001 Nov;25(11):1364-71.
Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer
J Clin 2011 Mar-Apr;61(2):69-90.
Choi YL, Soda M, Yamashita Y, Ueno T, Takashima J, Nakajima T, Yatabe Y, Takeuchi K,
Hamada T, Haruta H, Ishikawa Y, Kimura H, Mitsudomi T, Tanio Y, Mano H. EML4-ALK
mutations in lung cancer that confer resistance to ALK inhibitors. N Engl J Med 2010 Oct
28;363(18):1734-9.
Takeuchi K, Choi YL, Togashi Y, Soda M, Hatano S, Inamura K, Takada S, Ueno T, Yamashita
Y, Satoh Y, Okumura S, Nakagawa K, Ishikawa Y, Mano H. KIF5B-ALK, a novel fusion
oncokinase identified by an immunohistochemistry-based diagnostic system for ALK-positive
lung cancer. Clin Cancer Res 2009 May 1;15(9):3143-9.
Inamura K, Takeuchi K, Togashi Y, Hatano S, Ninomiya H, Motoi N, Mun MY, Sakao Y,
Okumura S, Nakagawa K, Soda M, Choi YL, Mano H, Ishikawa Y. EML4-ALK lung cancers are
characterized by rare other mutations, a TTF-1 cell lineage, an acinar histology, and young onset.
Mod Pathol 2009 Apr;22(4):508-15.
187.
188.
189.
190.
191.
192.
193.
194.
195.
196.
197.
198.
199.
200.
201.
202.
203.
204.
Inamura K, Takeuchi K, Togashi Y, Nomura K, Ninomiya H, Okui M, Satoh Y, Okumura S,
Nakagawa K, Soda M, Choi YL, Niki T, Mano H, Ishikawa Y. EML4-ALK fusion is linked to
histological characteristics in a subset of lung cancers. J Thorac Oncol 2008 Jan;3(1):13-7.
Koivunen JP, Mermel C, Zejnullahu K, Murphy C, Lifshits E, Holmes AJ, Choi HG, Kim J,
Chiang D, Thomas R, Lee J, Richards WG, Sugarbaker DJ, Ducko C, Lindeman N, Marcoux JP,
Engelman JA, Gray NS, Lee C, Meyerson M, Janne PA. EML4-ALK fusion gene and efficacy of
an ALK kinase inhibitor in lung cancer. Clin Cancer Res 2008 Jul 1;14(13):4275-83.
Martelli MP, Sozzi G, Hernandez L, Pettirossi V, Navarro A, Conte D, Gasparini P, Perrone F,
Modena P, Pastorino U, Carbone A, Fabbri A, Sidoni A, Nakamura S, Gambacorta M, Fernandez
PL, Ramirez J, Chan JK, Grigioni WF, Campo E, Pileri SA, Falini B. EML4-ALK rearrangement
in non-small cell lung cancer and non-tumor lung tissues. Am J Pathol 2009 Feb;174(2):661-70.
Shaw AT, Yeap BY, Mino-Kenudson M, Digumarthy SR, Costa DB, Heist RS, Solomon B,
Stubbs H, Admane S, McDermott U, Settleman J, Kobayashi S, Mark EJ, Rodig SJ, Chirieac LR,
Kwak EL, Lynch TJ, Iafrate AJ. Clinical features and outcome of patients with non-small-cell
lung cancer who harbor EML4-ALK. J Clin Oncol 2009 Sep 10;27(26):4247-53.
Shinmura K, Kageyama S, Tao H, Bunai T, Suzuki M, Kamo T, Takamochi K, Suzuki K,
Tanahashi M, Niwa H, Ogawa H, Sugimura H. EML4-ALK fusion transcripts, but no NPM-,
TPM3-, CLTC-, ATIC-, or TFG-ALK fusion transcripts, in non-small cell lung carcinomas.
Lung Cancer 2008 Aug;61(2):163-9.
Wong DW, Leung EL, So KK, Tam IY, Sihoe AD, Cheng LC, Ho KK, Au JS, Chung LP, Pik
Wong M. The EML4-ALK fusion gene is involved in various histologic types of lung cancers
from nonsmokers with wild-type EGFR and KRAS. Cancer 2009 Apr 15;115(8):1723-33.
Hallberg B, Palmer RH. Crizotinib--latest champion in the cancer wars? N Engl J Med 2010 Oct
28;363(18):1760-2.
Kim HR, Shim HS, Chung JH, Lee YJ, Hong YK, Rha SY, Kim SH, Ha SJ, Kim SK, Chung
KY, Soo R, Kim JH, Cho BC. Distinct clinical features and outcomes in never-smokers with
nonsmall cell lung cancer who harbor EGFR or KRAS mutations or ALK rearrangement. Cancer
2012 Jun 30;118(3):729-39.
Soda M, Takada S, Takeuchi K, Choi YL, Enomoto M, Ueno T, Haruta H, Hamada T,
Yamashita Y, Ishikawa Y, Sugiyama Y, Mano H. A mouse model for EML4-ALK-positive lung
cancer. Proc Natl Acad Sci U S A 2008 Dec 16;105(50):19893-7.
Sasaki T, Koivunen J, Ogino A, Yanagita M, Nikiforow S, Zheng W, Lathan C, Marcoux JP, Du
J, Okuda K, Capelletti M, Shimamura T, Ercan D, Stumpfova M, Xiao Y, Weremowicz S,
Butaney M, Heon S, Wilner K, Christensen JG, Eck MJ, Wong KK, Lindeman N, Gray NS,
Rodig SJ, Janne PA. A Novel ALK Secondary Mutation and EGFR Signaling Cause Resistance
to ALK Kinase Inhibitors. Cancer Res 2011 Sep 15;71(18):6051-60.
De Paepe P, Baens M, van Krieken H, Verhasselt B, Stul M, Simons A, Poppe B, Laureys G,
Brons P, Vandenberghe P, Speleman F, Praet M, De Wolf-Peeters C, Marynen P, Wlodarska I.
ALK activation by the CLTC-ALK fusion is a recurrent event in large B-cell lymphoma. Blood
2003 Oct 1;102(7):2638-41.
Delsol G, Lamant L, Mariame B, Pulford K, Dastugue N, Brousset P, Rigal-Huguet F, al Saati T,
Cerretti DP, Morris SW, Mason DY. A new subtype of large B-cell lymphoma expressing the
ALK kinase and lacking the 2; 5 translocation. Blood 1997 Mar 1;89(5):1483-90.
Gesk S, Gascoyne RD, Schnitzer B, Bakshi N, Janssen D, Klapper W, Martin-Subero JI,
Parwaresch R, Siebert R. ALK-positive diffuse large B-cell lymphoma with ALK-Clathrin fusion
belongs to the spectrum of pediatric lymphomas. Leukemia 2005 Oct;19(10):1839-40.
Reichard KK, McKenna RW, Kroft SH. ALK-positive diffuse large B-cell lymphoma: report of
four cases and review of the literature. Mod Pathol 2007 Mar;20(3):310-9.
Beltran B, Castillo J, Salas R, Quinones P, Morales D, Hurtado F, Riva L, Winer E. ALKpositive diffuse large B-cell lymphoma: report of four cases and review of the literature. J
Hematol Oncol 2009;2:11.
Laurent C, Do C, Gascoyne RD, Lamant L, Ysebaert L, Laurent G, Delsol G, Brousset P.
Anaplastic lymphoma kinase-positive diffuse large B-cell lymphoma: a rare clinicopathologic
entity with poor prognosis. J Clin Oncol 2009 Sep 1;27(25):4211-6.
Adam P, Katzenberger T, Seeberger H, Gattenlohner S, Wolf J, Steinlein C, Schmid M, MullerHermelink HK, Ott G. A case of a diffuse large B-cell lymphoma of plasmablastic type
associated with the t(2;5)(p23;q35) chromosome translocation. Am J Surg Pathol 2003
Nov;27(11):1473-6.
Onciu M, Behm FG, Downing JR, Shurtleff SA, Raimondi SC, Ma Z, Morris SW, Kennedy W,
Jones SC, Sandlund JT. ALK-positive plasmablastic B-cell lymphoma with expression of the
NPM-ALK fusion transcript: report of 2 cases. Blood 2003 Oct 1;102(7):2642-4.
73
205.
206.
207.
208.
209.
210.
211.
212.
213.
214.
215.
216.
217.
218.
219.
220.
74
Takeuchi K, Soda M, Togashi Y, Ota Y, Sekiguchi Y, Hatano S, Asaka R, Noguchi M, Mano H.
Identification of a novel fusion, SQSTM1-ALK, in ALK-positive large B-cell lymphoma.
Haematologica 2011 Mar;96(3):464-7.
Stachurski D, Miron PM, Al-Homsi S, Hutchinson L, Harris NL, Woda B, Wang SA.
Anaplastic lymphoma kinase-positive diffuse large B-cell lymphoma with a complex karyotype
and cryptic 3' ALK gene insertion to chromosome 4 q22-24. Hum Pathol 2007 Jun;38(6):940-5.
Marino-Enriquez A, Ou WB, Weldon CB, Fletcher JA, Perez-Atayde AR. ALK rearrangement
in sickle cell trait-associated renal medullary carcinoma. Genes Chromosomes Cancer 2011
Mar;50(3):146-53.
Sugawara E, Togashi Y, Kuroda N, Sakata S, Hatano S, Asaka R, Yuasa T, Yonese J, Kitagawa
M, Mano H, Ishikawa Y, Takeuchi K. Identification of anaplastic lymphoma kinase fusions in
renal cancer: Large-scale immunohistochemical screening by the intercalated antibody-enhanced
polymer method. Cancer 2012 Jan 17.
Cessna MH, Zhou H, Sanger WG, Perkins SL, Tripp S, Pickering D, Daines C, Coffin CM.
Expression of ALK1 and p80 in inflammatory myofibroblastic tumor and its mesenchymal
mimics: a study of 135 cases. Mod Pathol 2002 Sep;15(9):931-8.
Maris JM, Hogarty MD, Bagatell R, Cohn SL. Neuroblastoma. Lancet 2007 Jun
23;369(9579):2106-20.
Matthay KK, Villablanca JG, Seeger RC, Stram DO, Harris RE, Ramsay NK, Swift P, Shimada
H, Black CT, Brodeur GM, Gerbing RB, Reynolds CP. Treatment of high-risk neuroblastoma
with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cisretinoic acid. Children's Cancer Group. N Engl J Med 1999 Oct 14;341(16):1165-73.
Brodeur GM. Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer 2003
Mar;3(3):203-16.
De Brouwer S, De Preter K, Kumps C, Zabrocki P, Porcu M, Westerhout EM, Lakeman A,
Vandesompele J, Hoebeeck J, Van Maerken T, De Paepe A, Laureys G, Schulte JH, Schramm A,
Van Den Broecke C, Vermeulen J, Van Roy N, Beiske K, Renard M, Noguera R, Delattre O,
Janoueix-Lerosey I, Kogner P, Martinsson T, Nakagawara A, Ohira M, Caron H, Eggert A,
Cools J, Versteeg R, Speleman F. Meta-analysis of neuroblastomas reveals a skewed ALK
mutation spectrum in tumors with MYCN amplification. Clin Cancer Res 2010 Sep
1;16(17):4353-62.
Michels E, Vandesompele J, De Preter K, Hoebeeck J, Vermeulen J, Schramm A, Molenaar JJ,
Menten B, Marques B, Stallings RL, Combaret V, Devalck C, De Paepe A, Versteeg R, Eggert
A, Laureys G, Van Roy N, Speleman F. ArrayCGH-based classification of neuroblastoma into
genomic subgroups. Genes Chromosomes Cancer 2007 Dec;46(12):1098-108.
Vandesompele J, Baudis M, De Preter K, Van Roy N, Ambros P, Bown N, Brinkschmidt C,
Christiansen H, Combaret V, Lastowska M, Nicholson J, O'Meara A, Plantaz D, Stallings R,
Brichard B, Van den Broecke C, De Bie S, De Paepe A, Laureys G, Speleman F. Unequivocal
delineation of clinicogenetic subgroups and development of a new model for improved outcome
prediction in neuroblastoma. J Clin Oncol 2005 Apr 1;23(10):2280-99.
Maris JM, Weiss MJ, Guo C, Gerbing RB, Stram DO, White PS, Hogarty MD, Sulman EP,
Thompson PM, Lukens JN, Matthay KK, Seeger RC, Brodeur GM. Loss of heterozygosity at
1p36 independently predicts for disease progression but not decreased overall survival
probability in neuroblastoma patients: a Children's Cancer Group study. J Clin Oncol 2000
May;18(9):1888-99.
Henrich KO, Bauer T, Schulte J, Ehemann V, Deubzer H, Gogolin S, Muth D, Fischer M,
Benner A, Konig R, Schwab M, Westermann F. CAMTA1, a 1p36 tumor suppressor candidate,
inhibits growth and activates differentiation programs in neuroblastoma cells. Cancer Res 2011
Apr 15;71(8):3142-51.
Ando K, Ohira M, Ozaki T, Nakagawa A, Akazawa K, Suenaga Y, Nakamura Y, Koda T,
Kamijo T, Murakami Y, Nakagawara A. Expression of TSLC1, a candidate tumor suppressor
gene mapped to chromosome 11q23, is downregulated in unfavorable neuroblastoma without
promoter hypermethylation. Int J Cancer 2008 Nov 1;123(9):2087-94.
Lastowska M, Cotterill S, Bown N, Cullinane C, Variend S, Lunec J, Strachan T, Pearson AD,
Jackson MS. Breakpoint position on 17q identifies the most aggressive neuroblastoma tumors.
Genes Chromosomes Cancer 2002 Aug;34(4):428-36.
Islam A, Kageyama H, Takada N, Kawamoto T, Takayasu H, Isogai E, Ohira M, Hashizume K,
Kobayashi H, Kaneko Y, Nakagawara A. High expression of Survivin, mapped to 17q25, is
significantly associated with poor prognostic factors and promotes cell survival in human
neuroblastoma. Oncogene 2000 Feb 3;19(5):617-23.
221.
222.
223.
224.
225.
226.
227.
228.
229.
230.
231.
232.
233.
234.
235.
236.
237.
238.
239.
240.
241.
242.
Schmidt ML, Lukens JN, Seeger RC, Brodeur GM, Shimada H, Gerbing RB, Stram DO, Perez
C, Haase GM, Matthay KK. Biologic factors determine prognosis in infants with stage IV
neuroblastoma: A prospective Children's Cancer Group study. J Clin Oncol 2000
Mar;18(6):1260-8.
Seeger RC, Brodeur GM, Sather H, Dalton A, Siegel SE, Wong KY, Hammond D. Association
of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. N Engl J
Med 1985 Oct 31;313(18):1111-6.
Westermark UK, Wilhelm M, Frenzel A, Henriksson MA. The MYCN oncogene and
differentiation in neuroblastoma. Semin Cancer Biol 2011 Oct;21(4):256-66.
Hurlin PJ. N-Myc functions in transcription and development. Birth Defects Res C Embryo
Today 2005 Dec;75(4):340-52.
Malynn BA, de Alboran IM, O'Hagan RC, Bronson R, Davidson L, DePinho RA, Alt FW. Nmyc can functionally replace c-myc in murine development, cellular growth, and differentiation.
Genes Dev 2000 Jun 1;14(11):1390-9.
Eilers M, Eisenman RN. Myc's broad reach. Genes Dev 2008 Oct 15;22(20):2755-66.
Hui AB, Lo KW, Yin XL, Poon WS, Ng HK. Detection of multiple gene amplifications in
glioblastoma multiforme using array-based comparative genomic hybridization. Lab Invest 2001
May;81(5):717-23.
Lee WH, Murphree AL, Benedict WF. Expression and amplification of the N-myc gene in
primary retinoblastoma. Nature 1984 May 31-Jun 6;309(5967):458-60.
Nau MM, Brooks BJ, Jr., Carney DN, Gazdar AF, Battey JF, Sausville EA, Minna JD. Human
small-cell lung cancers show amplification and expression of the N-myc gene. Proc Natl Acad
Sci U S A 1986 Feb;83(4):1092-6.
Goodman LA, Liu BC, Thiele CJ, Schmidt ML, Cohn SL, Yamashiro JM, Pai DS, Ikegaki N,
Wada RK. Modulation of N-myc expression alters the invasiveness of neuroblastoma. Clin Exp
Metastasis 1997 Mar;15(2):130-9.
Hatzi E, Breit S, Zoephel A, Ashman K, Tontsch U, Ahorn H, Murphy C, Schweigerer L, Fotsis
T. MYCN oncogene and angiogenesis: down-regulation of endothelial growth inhibitors in
human neuroblastoma cells. Purification, structural, and functional characterization. Adv Exp
Med Biol 2000;476:239-48.
Boehm JS, Hession MT, Bulmer SE, Hahn WC. Transformation of human and murine
fibroblasts without viral oncoproteins. Mol Cell Biol 2005 Aug;25(15):6464-74.
Land H, Parada LF, Weinberg RA. Tumorigenic conversion of primary embryo fibroblasts
requires at least two cooperating oncogenes. Nature 1983 Aug 18-24;304(5927):596-602.
Weiss WA, Aldape K, Mohapatra G, Feuerstein BG, Bishop JM. Targeted expression of MYCN
causes neuroblastoma in transgenic mice. EMBO J 1997 Jun 2;16(11):2985-95.
Lamant L, Pulford K, Bischof D, Morris SW, Mason DY, Delsol G, Mariame B. Expression of
the ALK tyrosine kinase gene in neuroblastoma. Am J Pathol 2000 May;156(5):1711-21.
Schulte JH, Bachmann HS, Brockmeyer B, Depreter K, Oberthur A, Ackermann S, Kahlert Y,
Pajtler K, Theissen J, Westermann F, Vandesompele J, Speleman F, Berthold F, Eggert A, Brors
B, Hero B, Schramm A, Fischer M. High ALK receptor tyrosine kinase expression supersedes
ALK mutation as a determining factor of an unfavorable phenotype in primary neuroblastoma.
Clin Cancer Res 2011 Aug 1;17(15):5082-92.
Bagci O, Tumer S, Olgun N, Altungoz O. Copy number status and mutation analyses of
anaplastic lymphoma kinase (ALK) gene in 90 sporadic neuroblastoma tumors. Cancer Lett 2012
Apr 1;317(1):72-7.
Subramaniam MM, Piqueras M, Navarro S, Noguera R. Aberrant copy numbers of ALK gene
is a frequent genetic alteration in neuroblastomas. Hum Pathol 2009 Nov;40(11):1638-42.
Azarova AM, Gautam G, George RE. Emerging importance of ALK in neuroblastoma. Semin
Cancer Biol 2011 Oct;21(4):267-75.
Palmer RH, Vernersson E, Grabbe C, Hallberg B. Anaplastic lymphoma kinase: signalling in
development and disease. Biochem J 2009 Jun 15;420(3):345-61.
Martinsson T, Eriksson T, Abrahamsson J, Caren H, Hansson M, Kogner P, Kamaraj S,
Schonherr C, Weinmar J, Ruuth K, Palmer RH, Hallberg B. Appearance of the novel activating
F1174S ALK mutation in neuroblastoma correlates with aggressive tumor progression and
unresponsiveness to therapy. Cancer Res 2010 Jan 1;71(1):98-105.
Schonherr C, Ruuth K, Yamazaki Y, Eriksson T, Christensen J, Palmer RH, Hallberg B.
Activating ALK mutations found in neuroblastoma are inhibited by Crizotinib and NVPTAE684. Biochem J 2011 Dec 15;440(3):405-13.
75
243.
244.
245.
246.
247.
248.
249.
250.
251.
252.
253.
254.
255.
256.
257.
258.
259.
260.
261.
76
Schonherr C, Ruuth K, Eriksson T, Yamazaki Y, Ottmann C, Combaret V, Vigny M, Kamaraj
S, Palmer RH, Hallberg B. The Neuroblastoma ALK(I1250T) Mutation Is a Kinase-Dead RTK
In Vitro and In Vivo. Transl Oncol 2011 Aug;4(4):258-65.
Schonherr C, Ruuth K, Kamaraj S, Wang CL, Yang HL, Combaret V, Djos A, Martinsson T,
Christensen JG, Palmer RH, Hallberg B. Anaplastic Lymphoma Kinase (ALK) regulates
initiation of transcription of MYCN in neuroblastoma cells. Oncogene 2012 Jan 30.
De Pontual L, Kettaneh D, Gordon CT, Oufadem M, Boddaert N, Lees M, Balu L, Lachassinne
E, Petros A, Mollet J, Wilson LC, Munnich A, Brugière L, Delattre O, Vekemans M, Etchevers
H, Lyonnet S, Janoueix-Lerosey I, Amiel J. Germline Gain-of-Function Mutations of ALK
Disrupt Central Nervous System Development. Human Mutation. [Rapid Communication]
2011;32(3):272-6.
Mazot P, Cazes A, Boutterin MC, Figueiredo A, Raynal V, Combaret V, Hallberg B, Palmer
RH, Delattre O, Janoueix-Lerosey I, Vigny M. The constitutive activity of the ALK mutated at
positions F1174 or R1275 impairs receptor trafficking. Oncogene 2011 Apr 28;30(17):2017-25.
Del Grosso F, De Mariano M, Passoni L, Luksch R, Tonini GP, Longo L. Inhibition of N-linked
glycosylation impairs ALK phosphorylation and disrupts pro-survival signaling in neuroblastoma
cell lines. BMC Cance r2011 Dec 22;11(1):525.
Okubo J, Takita J, Chen Y, Oki K, Nishimura R, Kato M, Sanada M, Hiwatari M, Hayashi Y,
Igarashi T, Ogawa S. Aberrant activation of ALK kinase by a novel truncated form ALK protein
in neuroblastoma. Oncogene 2012 Jan 16.
McDuff FK, Lim SV, Dalbay M, Turner SD. Assessment of the transforming potential of novel
anaplastic lymphoma kinase point mutants. Mol Carcinog 2011 Nov 15.
Heuckmann JM, Holzel M, Sos ML, Heynck S, Balke-Want H, Koker M, Peifer M, Weiss J,
Lovly CM, Grutter C, Rauh D, Pao W, Thomas RK. ALK mutations conferring differential
resistance to structurally diverse ALK inhibitors. Clin Cancer Res 2011 Dec 1;17(23):7394-401.
Katayama R, Shaw AT, Khan TM, Mino-Kenudson M, Solomon BJ, Halmos B, Jessop NA,
Wain JC, Yeo AT, Benes C, Drew L, Saeh JC, Crosby K, Sequist LV, Iafrate AJ, Engelman JA.
Mechanisms of Acquired Crizotinib Resistance in ALK-Rearranged Lung Cancers. Sci Transl
Med 2012 Feb 8;4(120):120ra17.
Doebele RC, Pilling AB, Aisner D, Kutateladze TG, Le AT, Weickhardt AJ, Kondo KL,
Linderman DJ, Heasley LE, Franklin WA, Varella-Garcia M, Camidge DR. Mechanisms of
Resistance to Crizotinib in Patients with ALK Gene Rearranged Non-Small Cell Lung Cancer.
Clin Cancer Res 2012 Mar 1;18(5):1472-82.
Druker BJ. Translation of the Philadelphia chromosome into therapy for CML. Blood 2008 Dec
15;112(13):4808-17.
Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, Zimmermann J, Lydon
NB. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive
cells. Nat Med 1996 May;2(5):561-6.
Buchdunger E, Cioffi CL, Law N, Stover D, Ohno-Jones S, Druker BJ, Lydon NB. Abl proteintyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and
platelet-derived growth factor receptors. J Pharmacol Exp Ther 2000 Oct;295(1):139-45.
Buchdunger E, Zimmermann J, Mett H, Meyer T, Muller M, Regenass U, Lydon NB. Selective
inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine
kinase inhibitor of the 2-phenylaminopyrimidine class. Proc Natl Acad Sci U S A 1995 Mar
28;92(7):2558-62.
Arora A, Scholar EM. Role of tyrosine kinase inhibitors in cancer therapy. J Pharmacol Exp
Ther 2005 Dec;315(3):971-9.
McDermott U, Iafrate AJ, Gray NS, Shioda T, Classon M, Maheswaran S, Zhou W, Choi HG,
Smith SL, Dowell L, Ulkus LE, Kuhlmann G, Greninger P, Christensen JG, Haber DA,
Settleman J. Genomic alterations of anaplastic lymphoma kinase may sensitize tumors to
anaplastic lymphoma kinase inhibitors. Cancer Res 2008 May 1;68(9):3389-95.
Cerchietti L, Damm-Welk C, Vater I, Klapper W, Harder L, Pott C, Yang SN, Reiter A, Siebert
R, Melnick A, Woessmann W. Inhibition of anaplastic lymphoma kinase (ALK) activity provides
a therapeutic approach for CLTC-ALK-positive human diffuse large B cell lymphomas. PLoS
One 2011;6(4):e18436.
Duijkers FA, Gaal J, Meijerink JP, Admiraal P, Pieters R, de Krijger RR, van Noesel MM.
Anaplastic lymphoma kinase (ALK) inhibitor response in neuroblastoma is highly correlated
with ALK mutation status, ALK mRNA and protein levels. Cell Oncol (Dordr) 2011
Oct;34(5):409-17.
Katayama R, Khan TM, Benes C, Lifshits E, Ebi H, Rivera VM, Shakespeare WC, Iafrate AJ,
Engelman JA, Shaw AT. Therapeutic strategies to overcome crizotinib resistance in non-small
262.
263.
264.
265.
266.
267.
268.
269.
270.
271.
272.
273.
274.
275.
276.
277.
278.
cell lung cancers harboring the fusion oncogene EML4-ALK. Proc Natl Acad Sci U S A 2011
May 3;108(18):7535-40.
Zhang J, Deng X, Choi HG, Alessi DR, Gray NS. Characterization of TAE684 as a potent
LRRK2 kinase inhibitor. Bioorg Med Chem Lett 2012 Mar 1;22(5):1864-9.
Christensen JG, Zou HY, Arango ME, Li Q, Lee JH, McDonnell SR, Yamazaki S, Alton GR,
Mroczkowski B, Los G. Cytoreductive antitumor activity of PF-2341066, a novel inhibitor of
anaplastic lymphoma kinase and c-Met, in experimental models of anaplastic large-cell
lymphoma. Mol Cancer Ther 2007 Dec;6(12 Pt 1):3314-22.
Zou HY, Li Q, Lee JH, Arango ME, McDonnell SR, Yamazaki S, Koudriakova TB, Alton G,
Cui JJ, Kung PP, Nambu MD, Los G, Bender SL, Mroczkowski B, Christensen JG. An orally
available small-molecule inhibitor of c-Met, PF-2341066, exhibits cytoreductive antitumor
efficacy through antiproliferative and antiangiogenic mechanisms. Cancer Res 2007 May
1;67(9):4408-17.
access.data.fda.gov. Approval letter for the use of Xalkori in ALK-positive NSCLC patients.
2011 http://wwwaccessdatafdagov/drugsatfda_docs/appletter/2011/202570s000ltrpdf2011.
Camidge DR, Hirsch FR, Varella-Garcia M, Franklin WA. Finding ALK-positive lung cancer:
what are we really looking for? J Thorac Oncol 2011 Mar;6(3):411-3.
Kwak EL, Bang YJ, Camidge DR, Shaw AT, Solomon B, Maki RG, Ou SH, Dezube BJ, Janne
PA, Costa DB, Varella-Garcia M, Kim WH, Lynch TJ, Fidias P, Stubbs H, Engelman JA, Sequist
LV, Tan W, Gandhi L, Mino-Kenudson M, Wei GC, Shreeve SM, Ratain MJ, Settleman J,
Christensen JG, Haber DA, Wilner K, Salgia R, Shapiro GI, Clark JW, Iafrate AJ. Anaplastic
lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010 Oct
28;363(18):1693-703.
Sasaki T, Okuda K, Zheng W, Butrynski J, Capelletti M, Wang L, Gray NS, Wilner K,
Christensen JG, Demetri G, Shapiro GI, Rodig SJ, Eck MJ, Janne PA. The neuroblastomaassociated F1174L ALK mutation causes resistance to an ALK kinase inhibitor in ALKtranslocated cancers. Cancer Res 2010 Dec 15;70(24):10038-43.
Sasaki T, Rodig SJ, Chirieac LR, Janne PA. The biology and treatment of EML4-ALK nonsmall cell lung cancer. Eur J Cancer 2011 Jul;46(10):1773-80.
Shaw AT, Yeap BY, Solomon BJ, Riely GJ, Gainor J, Engelman JA, Shapiro GI, Costa DB, Ou
SH, Butaney M, Salgia R, Maki RG, Varella-Garcia M, Doebele RC, Bang YJ, Kulig K, Selaru
P, Tang Y, Wilner KD, Kwak EL, Clark JW, Iafrate AJ, Camidge DR. Effect of crizotinib on
overall survival in patients with advanced non-small-cell lung cancer harbouring ALK gene
rearrangement: a retrospective analysis. Lancet Oncol 2011 Oct;12(11):1004-12.
Bresler SC, Wood AC, Haglund EA, Courtright J, Belcastro LT, Plegaria JS, Cole K,
Toporovskaya Y, Zhao H, Carpenter EL, Christensen JG, Maris JM, Lemmon MA, Mosse YP.
Differential inhibitor sensitivity of anaplastic lymphoma kinase variants found in neuroblastoma.
Sci Transl Med 2011 Nov 9;3(108):108ra14.
Butrynski JE, D'Adamo DR, Hornick JL, Dal Cin P, Antonescu CR, Jhanwar SC, Ladanyi M,
Capelletti M, Rodig SJ, Ramaiya N, Kwak EL, Clark JW, Wilner KD, Christensen JG, Janne PA,
Maki RG, Demetri GD, Shapiro GI. Crizotinib in ALK-rearranged inflammatory myofibroblastic
tumor. N Engl J Med 2010 Oct 28;363(18):1727-33.
Gambacorti-Passerini C, Messa C, Pogliani EM. Crizotinib in anaplastic large-cell lymphoma.
N Engl J Med 2011 Feb 24;364(8):775-6.
Kinoshita K, Asoh K, Furuichi N, Ito T, Kawada H, Hara S, Ohwada J, Miyagi T, Kobayashi T,
Takanashi K, Tsukaguchi T, Sakamoto H, Tsukuda T, Oikawa N. Design and synthesis of a
highly selective, orally active and potent anaplastic lymphoma kinase inhibitor (CH5424802).
Bioorg Med Chem 2012 Feb 1;20(3):1271-80.
Sakamoto H, Tsukaguchi T, Hiroshima S, Kodama T, Kobayashi T, Fukami TA, Oikawa N,
Tsukuda T, Ishii N, Aoki Y. CH5424802, a selective ALK inhibitor capable of blocking the
resistant gatekeeper mutant. Cancer Cell 2011 May 17;19(5):679-90.
Lovly CM, Heuckmann JM, de Stanchina E, Chen H, Thomas RK, Liang C, Pao W. Insights into
ALK-driven cancers revealed through development of novel ALK tyrosine kinase inhibitors.
Cancer Res 2011 Jul 15;71(14):4920-31.
Cheng M, Quail MR, Gingrich DE, Ott GE, Lu L, Wan W, Albom MS, Angeles TS, Aimone
LD, Cristofani F, Machiorlatti R, Abele C, Ator MA, Dorsey BD, Inghirami GG, Ruggeri B.
CEP-28122, a Highly Potent and Selective Orally Active Inhibitor of Anaplastic Lymphoma
Kinase with Antitumor Activity in Experimental Models of Human Cancers. Mol Cancer Ther
2012 Mar;11(3):670-9.
Sabbatini P, Korenchuk S, Rowand JL, Groy A, Liu Q, Leperi D, Atkins C, Dumble M, Yang J,
Anderson K, Kruger RG, Gontarek RR, Maksimchuk KR, Suravajjala S, Lapierre RR, Shotwell
77
279.
280.
281.
282.
283.
284.
285.
286.
287.
288.
289.
290.
291.
292.
293.
294.
295.
296.
78
JB, Wilson JW, Chamberlain SD, Rabindran SK, Kumar R. GSK1838705A inhibits the insulinlike growth factor-1 receptor and anaplastic lymphoma kinase and shows antitumor activity in
experimental models of human cancers. Mol Cancer Ther 2009 Oct;8(10):2811-20.
Kruczynski A, Mayer P, Marchand A, Vispe S, Fournier E, Annereau JP, Brel V, Barret JM,
Delsol G, Imbert T, Fahy J, Bailly C. Antitumor activity of pyridoisoquinoline derivatives
F91873 and F91874, novel multikinase inhibitors with activity against the anaplastic lymphoma
kinase. Anticancer Drugs 2009 Jun;20(5):364-72.
Ardini E, Menichincheri M, De Ponti C, Amboldi N, Saccardo MB, Texido G, Russo M, Orsini
P, Bandiera T, Lombardi Borgia A, Isacchi A, Pesenti E, Colotta F, Magnaghi P, Galvani A,
Medical N. Characterization of NMS-E628, a small molecule inhibitor of anaplastic lymphoma
kinase with antitumor efficacy in ALK-dependent lymphoma and non-small cell lung cancer
models. Mol Cancer Ther 2009;8:(Suppl I):A243.
Jake Slavish P, Jiang Q, Cui X, Morris SW, Webb TR. Design and synthesis of a novel tyrosine
kinase inhibitor template. Bioorg Med Chem 2009 May 1;17(9):3308-16.
Milkiewicz KL, Weinberg LR, Albom MS, Angeles TS, Cheng M, Ghose AK, Roemmele RC,
Theroff JP, Underiner TL, Zificsak CA, Dorsey BD. Synthesis and structure-activity
relationships of 1,2,3,4-tetrahydropyrido[2,3-b]pyrazines as potent and selective inhibitors of the
anaplastic lymphoma kinase. Bioorg Med Chem 2010 Jun 15;18(12):4351-62.
Bryan MC, Whittington DA, Doherty EM, Falsey JR, Cheng AC, Emkey R, Brake RL, Lewis
RT. Rapid Development of Piperidine Carboxamides as Potent and Selective Anaplastic
Lymphoma Kinase Inhibitors. J Med Chem 2012 Feb 23;55(4):1698-705.
Okamoto M, Kojima H, Saito N, Okabe T, Masuda Y, Furuya T, Nagano T. Virtual screening
and further development of novel ALK inhibitors. Bioorg Med Chem 2011 May 15;19(10):308695.
Kinoshita K, Ono Y, Emura T, Asoh K, Furuichi N, Ito T, Kawada H, Tanaka S, Morikami K,
Tsukaguchi T, Sakamoto H, Tsukuda T, Oikawa N. Discovery of novel tetracyclic compounds as
anaplastic lymphoma kinase inhibitors. Bioorg Med Chem Lett 2011 Jun 15;21(12):3788-93.
Li R, Morris SW. Development of anaplastic lymphoma kinase (ALK) small-molecule inhibitors
for cancer therapy. Med Res Rev 2008 May;28(3):372-412.
Di Paolo D, Ambrogio C, Pastorino F, Brignole C, Martinengo C, Carosio R, Loi M, Pagnan G,
Emionite L, Cilli M, Ribatti D, Allen TM, Chiarle R, Ponzoni M, Perri P. Selective Therapeutic
Targeting of the Anaplastic Lymphoma Kinase With Liposomal siRNA Induces Apoptosis and
Inhibits Angiogenesis in Neuroblastoma. Mol Ther 2011 Dec;19(12):2201-12.
Di Paolo D, Brignole C, Pastorino F, Carosio R, Zorzoli A, Rossi M, Loi M, Pagnan G, Emionite
L, Cilli M, Bruno S, Chiarle R, Allen TM, Ponzoni M, Perri P. Neuroblastoma-targeted
nanoparticles entrapping siRNA specifically knockdown ALK. Mol Ther 2011 Jun;19(6):113140.
Hsu FY, Zhao Y, Anderson WF, Johnston PB. Downregulation of NPM-ALK by siRNA causes
anaplastic large cell lymphoma cell growth inhibition and augments the anti cancer effects of
chemotherapy in vitro. Cancer Invest 2007 Jun;25(4):240-8.
Piva R, Chiarle R, Manazza AD, Taulli R, Simmons W, Ambrogio C, D'Escamard V, Pellegrino
E, Ponzetto C, Palestro G, Inghirami G. Ablation of oncogenic ALK is a viable therapeutic
approach for anaplastic large-cell lymphomas. Blood 2006 Jan 15;107(2):689-97.
Ito M, Zhao N, Zeng Z, Chang CC, Zu Y. Synergistic growth inhibition of anaplastic large cell
lymphoma cells by combining cellular ALK gene silencing and a low dose of the kinase inhibitor
U0126. Cancer Gene Ther 2010 Sep;17(9):633-44.
Chiarle R, Martinengo C, Mastini C, Ambrogio C, D'Escamard V, Forni G, Inghirami G. The
anaplastic lymphoma kinase is an effective oncoantigen for lymphoma vaccination. Nat Med
2008 Jun;14(6):676-80.
Grzelinski M, Steinberg F, Martens T, Czubayko F, Lamszus K, Aigner A. Enhanced
antitumorigenic effects in glioblastoma on double targeting of pleiotrophin and its receptor ALK.
Neoplasia 2009 Feb;11(2):145-56.
Hudis CA. Trastuzumab--mechanism of action and use in clinical practice. N Engl J Med 2007
Jul 5;357(1):39-51.
Carpenter EL, Haglund EA, Mace EM, Deng D, Martinez D, Wood AC, Chow AK, Weiser
DA, Belcastro LT, Winter C, Bresler SC, Asgharzadeh S, Seeger RC, Zhao H, Guo R,
Christensen JG, Orange JS, Pawel BR, Lemmon MA, Mosse YP. Antibody targeting of
anaplastic lymphoma kinase induces cytotoxicity of human neuroblastoma. Oncogene 2012 Jan
23.
Bonvini P, Gastaldi T, Falini B, Rosolen A. Nucleophosmin-anaplastic lymphoma kinase (NPMALK), a novel Hsp90-client tyrosine kinase: down-regulation of NPM-ALK expression and
297.
298.
299.
300.
301.
302.
303.
304.
305.
306.
307.
308.
309.
310.
311.
312.
313.
314.
315.
316.
317.
318.
tyrosine phosphorylation in ALK(+) CD30(+) lymphoma cells by the Hsp90 antagonist 17allylamino,17-demethoxygeldanamycin. Cancer Res 2002 Mar 1;62(5):1559-66.
Neal JW, Sequist LV. Exciting new targets in lung cancer therapy: ALK, IGF-1R, HDAC, and
Hh. Curr Treat Options Oncol 2010 Jun;11(1-2):36-44.
Normant E, Paez G, West KA, Lim AR, Slocum KL, Tunkey C, McDougall J, Wylie AA,
Robison K, Caliri K, Palombella VJ, Fritz CC. The Hsp90 inhibitor IPI-504 rapidly lowers
EML4-ALK levels and induces tumor regression in ALK-driven NSCLC models. Oncogene
2011 Jun 2;30(22):2581-6.
Sequist LV, Gettinger S, Senzer NN, Martins RG, Janne PA, Lilenbaum R, Gray JE, Iafrate AJ,
Katayama R, Hafeez N, Sweeney J, Walker JR, Fritz C, Ross RW, Grayzel D, Engelman JA,
Borger DR, Paez G, Natale R. Activity of IPI-504, a novel heat-shock protein 90 inhibitor, in
patients with molecularly defined non-small-cell lung cancer. J Clin Oncol 2010 Nov
20;28(33):4953-60.
Colicelli J. Human RAS superfamily proteins and related GTPases. Sci STKE 2004 Sep
14;2004(250):RE13.
Wennerberg K, Rossman KL, Der CJ. The Ras superfamily at a glance. J Cell Sci 2005 Mar
1;118(Pt 5):843-6.
Harvey JJ. An Unidentified Virus Which Causes the Rapid Production of Tumours in Mice.
Nature 1964 Dec 12;204:1104-5.
Kirsten WH, Mayer LA. Morphologic responses to a murine erythroblastosis virus. J Natl
Cancer Inst 1967 Aug;39(2):311-35.
Der CJ, Krontiris TG, Cooper GM. Transforming genes of human bladder and lung carcinoma
cell lines are homologous to the ras genes of Harvey and Kirsten sarcoma viruses. Proc Natl
Acad Sci U S A 1982 Jun;79(11):3637-40.
Parada LF, Tabin CJ, Shih C, Weinberg RA. Human EJ bladder carcinoma oncogene is
homologue of Harvey sarcoma virus ras gene. Nature 1982 Jun 10;297(5866):474-8.
Santos E, Tronick SR, Aaronson SA, Pulciani S, Barbacid M. T24 human bladder carcinoma
oncogene is an activated form of the normal human homologue of BALB- and Harvey-MSV
transforming genes. Nature 1982 Jul 22;298(5872):343-7.
Hall A, Marshall CJ, Spurr NK, Weiss RA. Identification of transforming gene in two human
sarcoma cell lines as a new member of the ras gene family located on chromosome 1. Nature
1983 Jun 2-8;303(5916):396-400.
Shimizu K, Goldfarb M, Suard Y, Perucho M, Li Y, Kamata T, Feramisco J, Stavnezer E, Fogh
J, Wigler MH. Three human transforming genes are related to the viral ras oncogenes. Proc Natl
Acad Sci U S A 1983 Apr;80(8):2112-6.
Bos JL, de Rooij J, Reedquist KA. Rap1 signalling: adhering to new models. Nat Rev Mol Cell
Biol 2001 May;2(5):369-77.
Kitayama H, Sugimoto Y, Matsuzaki T, Ikawa Y, Noda M. A ras-related gene with
transformation suppressor activity. Cell 1989 Jan 13;56(1):77-84.
Raaijmakers JH, Bos JL. Specificity in Ras and Rap signaling. J Biol Chem 2009 Apr
24;284(17):10995-9.
Kao S, Jaiswal RK, Kolch W, Landreth GE. Identification of the mechanisms regulating the
differential activation of the mapk cascade by epidermal growth factor and nerve growth factor in
PC12 cells. J Biol Chem 2001 May 25;276(21):18169-77.
Schwamborn JC, Puschel AW. The sequential activity of the GTPases Rap1B and Cdc42
determines neuronal polarity. Nat Neurosci 2004 Sep;7(9):923-9.
Wu C, Lai CF, Mobley WC. Nerve growth factor activates persistent Rap1 signaling in
endosomes. J Neurosci 2001 Aug 1;21(15):5406-16.
York RD, Yao H, Dillon T, Ellig CL, Eckert SP, McCleskey EW, Stork PJ. Rap1 mediates
sustained MAP kinase activation induced by nerve growth factor. Nature 1998 Apr
9;392(6676):622-6.
Altschuler DL, Ribeiro-Neto F. Mitogenic and oncogenic properties of the small G protein
Rap1b. Proc Natl Acad Sci U S A 1998 Jun 23;95(13):7475-9.
Gao L, Feng Y, Bowers R, Becker-Hapak M, Gardner J, Council L, Linette G, Zhao H,
Cornelius LA. Ras-associated protein-1 regulates extracellular signal-regulated kinase activation
and migration in melanoma cells: two processes important to melanoma tumorigenesis and
metastasis. Cancer Res 2006 Aug 15;66(16):7880-8.
Hemmeryckx B, van Wijk A, Reichert A, Kaartinen V, de Jong R, Pattengale PK, GonzalezGomez I, Groffen J, Heisterkamp N. Crkl enhances leukemogenesis in BCR/ABL P190
transgenic mice. Cancer Res 2001 Feb 15;61(4):1398-405.
79
319.
320.
321.
322.
323.
324.
325.
326.
327.
328.
329.
330.
331.
332.
333.
334.
335.
336.
337.
338.
339.
80
Hong K, Lou L, Gupta S, Ribeiro-Neto F, Altschuler DL. A novel Epac-Rap-PP2A signaling
module controls cAMP-dependent Akt regulation. J Biol Chem 2008 Aug 22;283(34):23129-38.
Lou L, Urbani J, Ribeiro-Neto F, Altschuler DL. cAMP inhibition of Akt is mediated by
activated and phosphorylated Rap1b. J Biol Chem 2002 Sep 6;277(36):32799-806.
Li W, Jin B, Cornelius LA, Zhou B, Fu X, Shang D, Zheng H. Inhibitory effects of Rap1GAP
overexpression on proliferation and migration of endothelial cells via ERK and Akt pathways. J
Huazhong Univ Sci Technolog Med Sci 2012 Dec;31(6):721-7.
Lin Y, Mettling C, Chou C. Rap1-suppressed tumorigenesis is concomitant with the interference
in ras effector signaling. FEBS Lett 2000 Feb 11;467(2-3):184-8.
de Rooij J, Boenink NM, van Triest M, Cool RH, Wittinghofer A, Bos JL. PDZ-GEF1, a
guanine nucleotide exchange factor specific for Rap1 and Rap2. J Biol Chem 1999 Dec
31;274(53):38125-30.
de Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A, Bos JL. Epac
is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 1998
Dec 3;396(6710):474-7.
Ebinu JO, Bottorff DA, Chan EY, Stang SL, Dunn RJ, Stone JC. RasGRP, a Ras guanyl
nucleotide- releasing protein with calcium- and diacylglycerol-binding motifs. Science 1998 May
15;280(5366):1082-6.
Gotoh T, Hattori S, Nakamura S, Kitayama H, Noda M, Takai Y, Kaibuchi K, Matsui H, Hatase
O, Takahashi H, et al. Identification of Rap1 as a target for the Crk SH3 domain-binding guanine
nucleotide-releasing factor C3G. Mol Cell Biol 1995 Dec;15(12):6746-53.
Kawasaki H, Springett GM, Mochizuki N, Toki S, Nakaya M, Matsuda M, Housman DE,
Graybiel AM. A family of cAMP-binding proteins that directly activate Rap1. Science 1998 Dec
18;282(5397):2275-9.
Kawasaki H, Springett GM, Toki S, Canales JJ, Harlan P, Blumenstiel JP, Chen EJ, Bany IA,
Mochizuki N, Ashbacher A, Matsuda M, Housman DE, Graybiel AM. A Rap guanine nucleotide
exchange factor enriched highly in the basal ganglia. Proc Natl Acad Sci U S A 1998 Oct
27;95(22):13278-83.
Yamashita S, Mochizuki N, Ohba Y, Tobiume M, Okada Y, Sawa H, Nagashima K, Matsuda
M. CalDAG-GEFIII activation of Ras, R-ras, and Rap1. J Biol Chem 2000 Aug
18;275(33):25488-93.
van den Berghe N, Cool RH, Horn G, Wittinghofer A. Biochemical characterization of C3G: an
exchange factor that discriminates between Rap1 and Rap2 and is not inhibited by
Rap1A(S17N). Oncogene 1997 Aug 14;15(7):845-50.
Voss AK, Britto JM, Dixon MP, Sheikh BN, Collin C, Tan SS, Thomas T. C3G regulates
cortical neuron migration, preplate splitting and radial glial cell attachment. Development 2008
Jun;135(12):2139-49.
Voss AK, Gruss P, Thomas T. The guanine nucleotide exchange factor C3G is necessary for the
formation of focal adhesions and vascular maturation. Development 2003 Jan;130(2):355-67.
Voss AK, Krebs DL, Thomas T. C3G regulates the size of the cerebral cortex neural precursor
population. Embo J 2006 Aug 9;25(15):3652-63.
Hisata S, Sakisaka T, Baba T, Yamada T, Aoki K, Matsuda M, Takai Y. Rap1-PDZ-GEF1
interacts with a neurotrophin receptor at late endosomes, leading to sustained activation of Rap1
and ERK and neurite outgrowth. J Cell Biol 2007 Aug 27;178(5):843-60.
Mitra A, Kalayarasan S, Gupta V, Radha V. TC-PTP dephosphorylates the guanine nucleotide
exchange factor C3G (RapGEF1) and negatively regulates differentiation of human
neuroblastoma cells. PLoS One 2011;6(8):e23681.
Radha V, Rajanna A, Gupta RK, Dayma K, Raman T. The guanine nucleotide exchange factor,
C3G regulates differentiation and survival of human neuroblastoma cells. J Neurochem 2008
Dec;107(5):1424-35.
Yajnik V, Paulding C, Sordella R, McClatchey AI, Saito M, Wahrer DC, Reynolds P, Bell DW,
Lake R, van den Heuvel S, Settleman J, Haber DA. DOCK4, a GTPase activator, is disrupted
during tumorigenesis. Cell 2003 Mar 7;112(5):673-84.
Shirinian M, Popovic M, Grabbe C, Varshney G, Hugosson F, Bos H, Rehmann H, Palmer RH.
The Rap1 guanine nucleotide exchange factor C3G is required for preservation of larval muscle
integrity in Drosophila melanogaster. PLoS One 2010;5(3):e9403.
Kurachi H, Wada Y, Tsukamoto N, Maeda M, Kubota H, Hattori M, Iwai K, Minato N. Human
SPA-1 gene product selectively expressed in lymphoid tissues is a specific GTPase-activating
protein for Rap1 and Rap2. Segregate expression profiles from a rap1GAP gene product. J Biol
Chem 1997 Oct 31;272(44):28081-8.
340.
341.
342.
343.
344.
345.
346.
347.
348.
349.
350.
351.
352.
353.
354.
355.
356.
357.
358.
359.
360.
361.
Polakis PG, Rubinfeld B, Evans T, McCormick F. Purification of a plasma membrane-associated
GTPase-activating protein specific for rap1/Krev-1 from HL60 cells. Proc Natl Acad Sci U S A
1991 Jan 1;88(1):239-43.
Chakrabarti PP, Daumke O, Suveyzdis Y, Kotting C, Gerwert K, Wittinghofer A. Insight into
catalysis of a unique GTPase reaction by a combined biochemical and FTIR approach. J Mol
Biol 2007 Apr 6;367(4):983-95.
Daumke O, Weyand M, Chakrabarti PP, Vetter IR, Wittinghofer A. The GTPase-activating
protein Rap1GAP uses a catalytic asparagine. Nature 2004 May 13;429(6988):197-201.
Scrima A, Thomas C, Deaconescu D, Wittinghofer A. The Rap-RapGAP complex: GTP
hydrolysis without catalytic glutamine and arginine residues. EMBO J 2008 Apr 9;27(7):114553.
Wienecke R, Konig A, DeClue JE. Identification of tuberin, the tuberous sclerosis-2 product.
Tuberin possesses specific Rap1GAP activity. J Biol Chem 1995 Jul 7;270(27):16409-14.
Bailey CL, Kelly P, Casey PJ. Activation of Rap1 Promotes Prostate Cancer Metastasis. Cancer
Res 2009 May 26;69(12):4962-8.
Kim WJ, Gersey Z, Daaka Y. Rap1GAP regulates renal cell carcinoma invasion. Cancer Lett
2012 Jan 19;320(1):65-71.
Nellore A, Paziana K, Ma C, Tsygankova OM, Wang Y, Puttaswamy K, Iqbal AU, Franks SR,
Lv Y, Troxel AB, Feldman MD, Meinkoth JL, Brose MS. Loss of Rap1GAP in papillary thyroid
cancer. J Clin Endocrinol Metab 2009 Mar;94(3):1026-32.
Tsygankova OM, Feshchenko E, Klein PS, Meinkoth JL. Thyroid-stimulating hormone/cAMP
and glycogen synthase kinase 3beta elicit opposing effects on Rap1GAP stability. J Biol Chem
2004 Feb 13;279(7):5501-7.
Tsygankova OM, Ma C, Tang W, Korch C, Feldman MD, Lv Y, Brose MS, Meinkoth JL.
Downregulation of Rap1GAP in human tumor cells alters cell/matrix and cell/cell adhesion. Mol
Cell Biol 2010 Jul;30(13):3262-74.
Tsygankova OM, Prendergast GV, Puttaswamy K, Wang Y, Feldman MD, Wang H, Brose MS,
Meinkoth JL. Downregulation of Rap1GAP contributes to Ras transformation. Mol Cell Biol
2007 Oct;27(19):6647-58.
Zhang L, Chenwei L, Mahmood R, van Golen K, Greenson J, Li G, D'Silva NJ, Li X, Burant
CF, Logsdon CD, Simeone DM. Identification of a putative tumor suppressor gene Rap1GAP in
pancreatic cancer. Cancer Res 2006 Jan 15;66(2):898-906.
Zheng H, Gao L, Feng Y, Yuan L, Zhao H, Cornelius LA. Down-regulation of Rap1GAP via
promoter hypermethylation promotes melanoma cell proliferation, survival, and migration.
Cancer Res 2009 Jan 15;69(2):449-57.
Zuo H, Gandhi M, Edreira MM, Hochbaum D, Nimgaonkar VL, Zhang P, Dipaola J,
Evdokimova V, Altschuler DL, Nikiforov YE. Downregulation of Rap1GAP through epigenetic
silencing and loss of heterozygosity promotes invasion and progression of thyroid tumors.
Cancer Res 2010 Feb 15;70(4):1389-97.
Minato N, Hattori M. Spa-1 (Sipa1) and Rap signaling in leukemia and cancer metastasis.
Cancer Sci 2009 Jan;100(1):17-23.
Zwartkruis FJ, Wolthuis RM, Nabben NM, Franke B, Bos JL. Extracellular signal-regulated
activation of Rap1 fails to interfere in Ras effector signalling. EMBO J 1998 Oct
15;17(20):5905-12.
Vossler MR, Yao H, York RD, Pan MG, Rim CS, Stork PJ. cAMP activates MAP kinase and
Elk-1 through a B-Raf- and Rap1-dependent pathway. Cell 1997 Apr 4;89(1):73-82.
Feller SM, Knudsen B, Hanafusa H. Cellular proteins binding to the first Src homology 3 (SH3)
domain of the proto-oncogene product c-Crk indicate Crk-specific signaling pathways. Oncogene
1995 Apr 20;10(8):1465-73.
de Jong R, van Wijk A, Heisterkamp N, Groffen J. C3G is tyrosine-phosphorylated after
integrin-mediated cell adhesion in normal but not in Bcr/Abl expressing cells. Oncogene 1998
Nov 26;17(21):2805-10.
Ichiba T, Hashimoto Y, Nakaya M, Kuraishi Y, Tanaka S, Kurata T, Mochizuki N, Matsuda M.
Activation of C3G guanine nucleotide exchange factor for Rap1 by phosphorylation of tyrosine
504. J Biol Chem 1999 May 14;274(20):14376-81.
Ribeiro-Neto F, Urbani J, Lemee N, Lou L, Altschuler DL. On the mitogenic properties of
Rap1b: cAMP-induced G(1)/S entry requires activated and phosphorylated Rap1b. Proc Natl
Acad Sci U S A 2002 Apr 16;99(8):5418-23.
Schmitt JM, Stork PJ. Cyclic AMP-mediated inhibition of cell growth requires the small G
protein Rap1. Mol Cell Biol 2001 Jun;21(11):3671-83.
81
362.
363.
364.
365.
366.
367.
368.
369.
370.
371.
372.
373.
374.
375.
376.
377.
378.
379.
380.
381.
382.
383.
82
Gyan E, Frew M, Bowen D, Beldjord C, Preudhomme C, Lacombe C, Mayeux P, Dreyfus F,
Porteu F, Fontenay M. Mutation in RAP1 is a rare event in myelodysplastic syndromes.
Leukemia 2005 Sep;19(9):1678-80.
Matsuse M, Mitsutake N, Rogounovitch T, Saenko V, Nakazawa Y, Rumyantsev P, Lushnikov
E, Suzuki K, Yamashita S. Mutation analysis of RAP1 gene in papillary thyroid carcinomas.
Endocr J 2009 Mar;56(1):161-4.
Zemojtel T, Penzkofer T, Duchniewicz M, Zwartkruis FJ. HRap1B-retro: a novel human
processed rap1B gene blurs the picture? Leukemia 2006 Jan;20(1):145-6; author reply 6-7.
Ishida D, Kometani K, Yang H, Kakugawa K, Masuda K, Iwai K, Suzuki M, Itohara S,
Nakahata T, Hiai H, Kawamoto H, Hattori M, Minato N. Myeloproliferative stem cell disorders
by deregulated Rap1 activation in SPA-1-deficient mice. Cancer Cell 2003 Jul;4(1):55-65.
Guerrero C, Martin-Encabo S, Fernandez-Medarde A, Santos E. C3G-mediated suppression of
oncogene-induced focus formation in fibroblasts involves inhibition of ERK activation, cyclin A
expression and alterations of anchorage-independent growth. Oncogene 2004 Jun
17;23(28):4885-93.
Schmitt JM, Stork PJ. PKA phosphorylation of Src mediates cAMP's inhibition of cell growth
via Rap1. Mol Cell 2002 Jan;9(1):85-94.
Stork PJ. Does Rap1 deserve a bad Rap? Trends Biochem Sci 2003 May;28(5):267-75.
Greene LA, Kaplan DR. Early events in neurotrophin signalling via Trk and p75 receptors. Curr
Opin Neurobiol 1995 Oct;5(5):579-87.
Heidorn SJ, Milagre C, Whittaker S, Nourry A, Niculescu-Duvas I, Dhomen N, Hussain J, ReisFilho JS, Springer CJ, Pritchard C, Marais R. Kinase-dead BRAF and oncogenic RAS cooperate
to drive tumor progression through CRAF. Cell 2010 Jan 22;140(2):209-21.
Bellot F, Crumley G, Kaplow JM, Schlessinger J, Jaye M, Dionne CA. Ligand-induced
transphosphorylation between different FGF receptors. EMBO J 1991 Oct;10(10):2849-54.
Chen WS, Lazar CS, Poenie M, Tsien RY, Gill GN, Rosenfeld MG. Requirement for intrinsic
protein tyrosine kinase in the immediate and late actions of the EGF receptor. Nature 1987 Aug
27-Sep 2;328(6133):820-3.
Pulford K, Morris SW, Turturro F. Anaplastic lymphoma kinase proteins in growth control and
cancer. J Cell Physiol 2004 Jun;199(3):330-58.
Gualdrini F, Corvetta D, Cantilena S, Chayka O, Tanno B, Raschella G, Sala A. Addiction of
MYCN amplified tumours to B-MYB underscores a reciprocal regulatory loop. Oncotarget 2010
Aug;1(4):278-88.
Combaret V, Turc-Carel C, Thiesse P, Rebillard AC, Frappaz D, Haus O, Philip T, Favrot MC.
Sensitive detection of numerical and structural aberrations of chromosome 1 in neuroblastoma by
interphase fluorescence in situ hybridization. Comparison with restriction fragment length
polymorphism and conventional cytogenetic analyses. Int J Cancer 1995 Apr 10;61(2):185-91.
Schleiermacher G, Janoueix-Lerosey I, Combaret V, Derre J, Couturier J, Aurias A, Delattre O.
Combined 24-color karyotyping and comparative genomic hybridization analysis indicates
predominant rearrangements of early replicating chromosome regions in neuroblastoma. Cancer
Genet Cytogenet 2003 Feb;141(1):32-42.
Berwanger B, Hartmann O, Bergmann E, Bernard S, Nielsen D, Krause M, Kartal A, Flynn D,
Wiedemeyer R, Schwab M, Schafer H, Christiansen H, Eilers M. Loss of a FYN-regulated
differentiation and growth arrest pathway in advanced stage neuroblastoma. Cancer Cell 2002
Nov;2(5):377-86.
Otto T, Horn S, Brockmann M, Eilers U, Schuttrumpf L, Popov N, Kenney AM, Schulte JH,
Beijersbergen R, Christiansen H, Berwanger B, Eilers M. Stabilization of N-Myc is a critical
function of Aurora A in human neuroblastoma. Cancer Cell 2009 Jan 6;15(1):67-78.
Gustafson WC, Weiss WA. Myc proteins as therapeutic targets. Oncogene 2010 Mar
4;29(9):1249-59.
Sjostrom SK, Finn G, Hahn WC, Rowitch DH, Kenney AM. The Cdk1 complex plays a prime
role in regulating N-myc phosphorylation and turnover in neural precursors. Dev Cell 2005
Sep;9(3):327-38.
Arnold HK, Sears RC. Protein phosphatase 2A regulatory subunit B56alpha associates with cmyc and negatively regulates c-myc accumulation. Mol Cell Biol 2006 Apr;26(7):2832-44.
Arnold HK, Zhang X, Daniel CJ, Tibbitts D, Escamilla-Powers J, Farrell A, Tokarz S, Morgan
C, Sears RC. The Axin1 scaffold protein promotes formation of a degradation complex for cMyc. EMBO J 2009 Mar 4;28(5):500-12.
Chesler L, Schlieve C, Goldenberg DD, Kenney A, Kim G, McMillan A, Matthay KK, Rowitch
D, Weiss WA. Inhibition of phosphatidylinositol 3-kinase destabilizes Mycn protein and blocks
malignant progression in neuroblastoma. Cancer Res 2006 Aug 15;66(16):8139-46.
384.
385.
386.
387.
388.
389.
390.
391.
392.
Lee DH, Goldberg AL. Proteasome inhibitors: valuable new tools for cell biologists. Trends Cell
Biol 1998 Oct;8(10):397-403.
Berthier A, Piqueras M, Villamon E, Berbegall A, Tadeo I, Castel V, Navarro S, Noguera R.
Anaplastic lymphoma kinase expression in neuroblastomas and its relationship with genetic,
prognostic, and predictive factors. Hum Pathol 2011 Feb;42(2):301-2.
Liu Z, Thiele CJ. ALK and MYCN: When Two Oncogenes Are Better than One. Cancer Cell
2012;21:325-6.
Zhu S, Lee J, Guo F, Shin J, Perez-Atayde AR, Kutok JL, Rodig SJ, Neuberg DS, Helman D,
Feng H, Stewart RA, Wang W, George RE, Kanki JP, Look AT. Activated ALK Collaborates
with MYCN in Neuroblastoma Pathogenesis. Cancer Cell 2012;21:362-73.
Tanizaki J, Okamoto I, Takezawa K, Sakai K, Azuma K, Kuwata K, Yamaguchi H, Hatashita E,
Nishio K, Janne PA, Nakagawa K. Combined effect of ALK and MEK inhibitors in EML4-ALKpositive non-small-cell lung cancer cells. Br J Cancer 2012 Feb 14;106(4):763-7.
Regales L, Gong Y, Shen R, de Stanchina E, Vivanco I, Goel A, Koutcher JA, Spassova M,
Ouerfelli O, Mellinghoff IK, Zakowski MF, Politi KA, Pao W. Dual targeting of EGFR can
overcome a major drug resistance mutation in mouse models of EGFR mutant lung cancer. J Clin
Invest 2009 Oct;119(10):3000-10.
Chanthery YH, Gustafson WC, Itsara M, Persson A, Hackett CS, Grimmer M, Charron E,
Yakovenko S, Kim G, Matthay KK, Weiss WA. Paracrine signaling through MYCN enhances
tumor-vascular interactions in neuroblastoma. Sci Transl Med 2012 Jan 4;4(115):115ra3.
Faisal A, Vaughan L, Bavetsias V, Sun C, Atrash B, Avery S, Jamin Y, Robinson SP, Workman
P, Blagg J, Raynaud FI, Eccles SA, Chesler L, Linardopoulos S. The aurora kinase inhibitor
CCT137690 downregulates MYCN and sensitizes MYCN-amplified neuroblastoma in vivo. Mol
Cancer Ther 2011 Nov;10(11):2115-23.
Pulford K, Lamant L, Espinos E, Jiang Q, Xue L, Turturro F, Delsol G, Morris SW. The
emerging normal and disease-related roles of anaplastic lymphoma kinase. Cell Mol Life Sci
2004 Dec;61(23):2939-53.
83
