Activation of Rac1 and the exchange factor Vav3 are involved in
NPM-ALK signaling in anaplastic large cell lymphomas
A Colomba1,2, D Courilleau1,2, D Ramel1,2, DD Billadeau3,4, E Espinos1,2, G Delsol1,2,
B Payrastre1,2and F Gaits-Iacovoni1,2
1INSERM, U563, Centre de Physiopathologie de Toulouse Purpan, Toulouse, France;2Universite ´ Toulouse III Paul-Sabatier, IFR30,
Toulouse, France;3Division of Developmental Oncology Research, Mayo Clinic College of Medicine, Rochester, MN, USA and
4Department of Immunology, Mayo Clinic College of Medicine, Rochester, MN, USA
The majority of anaplastic large cell lymphomas (ALCLs)
express the nucleophosmin-anaplastic lymphoma kinase
(NPM-ALK) fusion protein, which is oncogenic due to its
constitutive tyrosine kinase activity. Transformation by
NPM-ALK not only increases proliferation, but also
modifies cell shape and motility in both lymphoid and
fibroblastic cells. We report that the Rac1 GTPase, a
known cytoskeletal regulator, is activated by NPM-ALK
in ALCL cell lines (Karpas 299 and Cost) and transfected
cells (lymphoid Ba/F3 cells, NIH-3T3 fibroblasts). We
have identified Vav3 as one of the exchange factors
involved in Rac1 activation. Stimulation of Vav3 and
Rac1 by NPM-ALK is under the control of Src kinases. It
involves formation of a signaling complex between NPM-
ALK, pp60c-src, Lyn and Vav3, in which Vav3 associates
with tyrosine 343 of NPM-ALK via its SH2 domain.
Moreover, Vav3 is phosphorylated in NPM-ALK positive
biopsies from patients suffering from ALCL, demonstrat-
ing the pathological relevance of this observation. The use
of Vav3-specific shRNA and a dominant negative Rac1
mutant demonstrates the central role of GTPases in
NPM-ALK elicited motility and invasion.
Oncogene (2008) 27, 2728–2736; doi:10.1038/sj.onc.1210921;
published online 12 November 2007
Keywords: anaplastic large cell lymphomas; NPM-
ALK; Rho GTPases; Vav3
Anaplastic large cell lymphomas (ALCLs), a subtype of
high-grade non-Hodgkin’s lymphomas of T or null
phenotype, are characterized by the aberrant expression
of the oncogenic fusion protein nucleophosmin-anaplas-
tic lymphoma kinase (NPM-ALK) in 75% of cases
(Lamant et al., 1996; Duyster et al., 2001; Falini, 2001).
The constitutive tyrosine kinase activity of NPM-ALK
is responsible for malignant transformation of fibro-
blasts and lymphoid cells and was shown to induce B-
and T-cell lymphomas in transgenic mice (Chiarle et al.,
NPM-ALK-induced transformation depends on the
activation of signaling pathways shared by many
oncogenic tyrosine kinases. Pro-mitogenic functions
include binding of adaptors, such as Shc, Grb2 and
IRS1, to regulators of the Erk pathway, phospholipase
Cg (PLCg), tyrosine phosphatases and the proto-
oncogene pp60c-src(Fujimoto et al., 1996; Bai et al.,
1998; Cussac et al., 2004; Honorat et al., 2006; Marzec
et al., 2007). Antiapoptotic functions are related to the
activation of the survival phosphatidylinositol 3-kinase
(PI3K)/AKT pathway and of the Jak/STAT3–5 module
(Bai et al., 2000; Amin et al., 2003; Chiarle et al., 2005).
However, a hierarchy of downstream signaling, in
terms of importance for transformation, diagnosis and
prognosis of the disease, remains to be established. With
this goal in mind, several large or medium scale
proteomic and transcriptomic studies were undertaken
by several groups (Lamant et al., 2006; Lim and
Elenitoba-Johnson, 2006). New partners have been
found that account for different functions of NPM-
ALK, such as the regulation of mRNA turnover (Fawal
et al., 2006). Among others, proteins regulating cell
features altered in transformed cells, were identified,
leading to investigations of molecules classified as
‘cytoskeleton and motility regulators’ in the context of
ALCLs (Crockett et al., 2004; Cussac et al., 2006).
Ambrogio et al. (2005) reported the association of
(p130Cas) and described its role in actin depolymeriza-
tion and transformation. Along these lines, NPM-ALK
expression alters fibroblasts shape dramatically. They
display an elongated phenotype with extensions similar
to what is observed in PC12 cells transformed by the
native ALK receptor, indicating that the kinase activity
is responsible for the change in morphology.
Our group and others have described modifications of
the expression of regulators of the Rho GTPases
(Crockett et al., 2004; Cussac et al., 2006). We observed
Received 11 May 2007; revised 27 September 2007; accepted 16 October
2007; published online 12 November 2007
Correspondence: Dr F Gaits-Iacovoni, INSERM, U563, Dpt d’Onco-
gene ` se, Signalisation et Innovation the ´ rapeutique, CHU Purpan, BP
3028, Toulouse cedex 3, Midi pyrenees 31024, France.
Oncogene (2008) 27, 2728–2736
& 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00
the extinction of the GTPase inhibitor RhoGDI2 in the
proteome of an NPM-ALK(þ) cell line, an effect
shown to correlate with higher metastatic activity and
poor prognosis in bladder cancers (Theodorescu et al.,
2004; Cussac et al., 2006). These findings suggested that
upregulation of Rho GTPases might be of importance
during the progression of the disease. Recently, about 45
proteins were reported to interact with NPM-ALK,
including various Rho GTPase activating proteins
whose pattern of expression was also altered in a study
of the transcriptome of NPM-ALK(þ) cells (Crockett
et al., 2004; Lamant et al., 2006). Rho GTPases mediate
many aspects of cell biology including proliferation,
regulation of the cell survival, polarity, adhesion,
membrane trafficking and motility (Hall, 2005). The
high incidence of overexpression of some GTPases
(RhoA, RhoC, Rac1, Rac3 and Cdc42) or their
regulators in human tumors suggests that GTPases play
a role in carcinogenesis (Sahai and Marshall, 2002). The
most studied members of the family are RhoA, Rac1
and Cdc42, which exert their transformant effects by
regulating cell cycle progression via the cyclin-depen-
dent kinases, and promoting migration and metastasis
through regulation of cytoskeleton dynamics (Sahai and
Marshall, 2002). Rac1 and Cdc42 regulate actin poly-
merization through the Arp2/3 complex with Rac1
involved in the generation of motile structures and
Cdc42 in the establishment of polarity. RhoA organizes
stress fibers predominantly through its effector Rho
kinase (Hall, 1998). In addition, Rho GTPase signaling
was demonstrated to be necessary for the oncogenicity
of other proteins, especially for oncogenes derived from
receptor tyrosine kinases, such as EGFR, IGFR, MET
or RET (Aznar et al., 2004; Titus et al., 2005).
In this study, we demonstrate that Rac1 is activated in
NPM-ALK expressing cells and is regulated by PI3K
and Src family kinases. The Vav3 proto-oncogene is
involved in bridging NPM-ALK and Rac1. The NPM-
ALK chimera forms a multiprotein complex containing
pp60c-src, Lyn and phosphorylated Vav3. Importantly,
we observed activation of Vav3 in tumors from patients
developing ALCL. Altering either Vav3 or the Rac
pathway, by RNA interference or with dominant
negative mutants and toxins, blocked invasion by
NPM-ALK(þ) cells. Altogether, our data demonstrate
a critical role for Rho GTPases in ALCL.
NPM-ALK activates the GTPase Rac1 via PI3K and Src
Activation of the Rho GTPases was first studied in two
NPM-ALK positive ALCL cell lines, Karpas 299
(common type) and Cost (small cells, aggressive variant)
(Falini et al., 1998; Lamant et al., 2004). Pull-down
assays demonstrated that Rac1 was strongly activated in
both cell lines (Figure 1a). NPM-ALK activation can be
monitored by its autophosphorylation on tyrosine 664.
We used two complementary approaches to determine
whether NPM-ALK was responsible for Rac1 activation.
First, we took advantage of the small molecule inhibitor
WHI-154 that inhibits the ALK kinase (Marzec et al.,
2005). Inactivation of NPM-ALK resulted in a marked
decrease in Rac1 activation (Figure 1a). Second, we
examined Rac1 in cell lines of independent origin (NIH-
3T3 fibroblasts and Ba/F3 lymphoid cells) that were
demonstrated to become transformed by stable expres-
sion of ALK oncogenic fusions (Armstrong et al., 2004).
NPM-ALK-dependent Rac1 activation was again ob-
served (Figures 1b and c).
Small GTPase activation requires guanosine exchange
factors (GEFs), which can be regulated by PI3K
products and kinases of the Src family (Hall, 2005).
The p85 regulatory subunit of PI3K and pp60c-srcwere
identified as downstream targets of NPM-ALK. Treat-
ment of serum and IL3-deprived Ba/F3 cells with 25mM
of LY294002 (PI3K inhibitor) or 2mM of SU6656
(indolinone inhibitor of Src kinases) abolished Rac1
activation (Figure 1c), showing that PI3K and Src
kinases are important for NPM-ALK to signal to the
GTPase. We previously demonstrated that NPM-ALK
is a substrate for pp60c-src(Cussac et al., 2004). Hence,
SU6656 treatment reduced NPM-ALK phosphoryla-
tion, making it difficult to conclude on a direct role of
Src on Rac1 activation pathway (Figure 1c). To
overcome this, we used RNA silencing to target pp60c-src
and Lyn, two Src kinases involved in hematological
malignancies (Cussac et al., 2004; Contri et al., 2005;
Thompson et al., 2005), and found that both kinases
could regulate NPM-ALK Y664 autophosphorylation
(not shown). Activation of PAK1 (p21-activated kinase),
a downstream target of Rac1, demonstrated the same
pattern of regulation as the GTPase, as shown with
antibodies to the active phosphorylated form of the
kinase, indicating the functional relevance of Rac1
activation in terms of downstream signaling (Figure 1c).
Finally, evaluation of the activation status of RhoA
and Cdc42 failed to demonstrate significant modifica-
tions in ALCLs and transfected cells (Figures 1d and e).
The proto-oncogene Vav3 is activated downstream of
The Vav proto-oncogenes are the only GEFs with a
structural hallmark of signal transducer proteins repre-
sented by the SH3-SH2-SH3 (Src homology 2 or 3)
module at their C terminus. This unique feature suggests
that they could act as nucleation points for multiple
signaling complexes after being recruited by tyrosine
kinase receptors (Bustelo, 2000; Hornstein et al., 2004).
They are activated by phosphorylation by members of
the Src family, and Vav1 and Vav3 were found to be
associated with NPM-ALK partners, such as Grb2, Shc,
the p85 regulatory unit of PI3K, pp60c-srcand PLCg
(Bustelo, 2001). We therefore checked whether they
could be targets of NPM-ALK by studying the
activating phosphorylation on Y174 of Vav1 and Y173
of Vav3 with specific antibodies. Although some
phosphorylation could be detected, inhibition of ALK
did not affect the level of Vav1 Y174 phosphorylation,
suggesting that NPM-ALK does not regulate Vav1 in
Role of GTPases in NPM-ALK(þ) ALCLs
A Colomba et al
ALCLs (Figure 2). Conversely, Figure 3a shows that
NPM-ALK expression resulted in a robust phosphor-
ylation of Vav3 in both Ba/F3 and NIH-3T3 cells. The
same observation was made in Karpas 299 and Cost
cells where ALK inhibition decreased Vav3 phosphor-
ylation significantly (Figure 3b). We evaluated the status
of Vav3 in protein extracts from four frozen lymph
nodes from patients suffering from NPM-ALK(þ)
ALCLs after immunohistochemistry analysis of the
biopsies (not shown). Again, Vav3 phosphorylation
increased in tumor samples compared with control
lymph nodes or peripheral blood lymphocytes, demon-
strating the pathophysiological relevance of this ob-
servation (Figure 3c).
In addition, we investigated whether Src kinases were
involved in Vav3 phosphorylation. Treatment of Ba/F3
cells with SU6656 resulted in a decreased signal
(Figure 4a, left panel). We then used RNA interference
to target pp60c-srcand Lyn. RNA silencing of pp60c-src
demonstrated that it was responsible for Vav3 phos-
phorylation in this model (Figure 4a, right panel).
Finally, we transfected cells with the L211Q inactive
PBD as described in Materials and methods in (a) Karpas 299 and Cost cells treated for 1h with 15mM of the NPM-ALK inhibitor
WHI-154, and (b) Control or NPM-ALK expressing NIH-3T3 cells, (c) Rac1 activation is dependent on PI3K and Src. Control or
NPM-ALK expressing Ba/F3 cells were treated for 30min with 25mM of the PI3K inhibitor LY294002 or 2mM of the Src inhibitor
SU6656. Active Rac1-GTP and total Rac1 in the extracts were assessed by western blotting with antibodies to Rac1. The activation of
NPM-ALK was followed with antibodies to the phosphorylated Y664 (pNPM-ALK(Y664)) and NPM-ALK level with ALK
antibody. Phosphorylation of Rac effectors PAK1/2 was followed with anti-phospho-PAK1/2 (S199–204/S192–197) antibodies. a-
Tubulin was used as loading control. (d) and (e) Karpas 299, Cost, Ba/F3 and NIH-3T3 cells were treated as above then levels of active
Cdc42 and active RhoA were evaluated by pull-down experiments with GST-PBD and GST-RBD, respectively. Active Cdc42-GTP,
RhoA-GTP, total Cdc42 and RhoA in the extracts were assessed by western blotting with antibodies to Cdc42 and RhoA. Data are
representative of three to five experiments.
Rac1 is activated downstream of NPM-ALK. Levels of active Rac1 were evaluated by pull-down experiments with GST-
Role of GTPases in NPM-ALK(þ) ALCLs
A Colomba et al
mutant of Vav3 containing a point mutation in the
exchange DH (Dbl homology) domain (Figure 4b). In
addition, we depleted Vav3 with specific short hairpin
RNAs (shRNAs) (Figure 4c). In both cases, Rac1GTP
levels were reduced, showing that functional Vav3 is
required for Rac1 activation downstream of NPM-
The SH2 domain of Vav3 drives its association with
Immunoprecipitations with an antibody directed against
ALK demonstrated that a complex containing phos-
phorylated Vav3, pp60c-srcand Lyn co-precipitated with
the active oncogene (Figure 5a). In all cases, a decrease
(but not abrogation) in the association is observed upon
WHI-154 treatment suggesting that activation of NPM-
ALK is important in the stabilization of the complex
(Figure 5a). Figure 5b shows that the reciprocal
immunoprecipitation of Vav3 also led to the same
results. Overexpression of Vav3 mutants in NPM-ALK
transfected cells confirmed the association of NPM-
ALK, Vav3 and active Src kinases in this model
(Figure 5c). Indeed, the Y173F mutant that was shown
to adopt an open conformation facilitating access to
partners and to GTPases (Llorca et al., 2005), binds to
NPM-ALK strongly (Figure 5c). Interestingly, when we
expressed the R697A mutant with a disabled phospho-
tyrosine binding SH2 domain, no binding to NPM-ALK
was observed, demonstrating that the SH2 domain is
crucial for the interaction (Figure 5c).
ALK expressing cells and in tumors from patients. The activating
phosphorylation on Y173 of Vav3 was measured in total lysates
from (a) control or NPM-ALK expressing Ba/F3 cells or NIH-3T3
cells, and (b) Karpas 299 and Cost cells treated for 1h with 15mM
WHI-154. Lysates were immunoblotted with anti-pVav3 (Y173),
anti-pNPM-ALK (Y664), anti-ALK or anti-Vav3 antibodies as
loading control. Data are representative of three experiments. (c)
Total lysates from two frozen control (LN) or four NPM-ALK
positive lymph nodes and peripheral blood lymphocytes (PBL)
from two healthy donors were immunoblotted with anti-pVav3
(Y173) or anti-human Vav3 antibodies.
The exchange factor Vav3 is phosphorylated in NPM-
NPM-ALK. (a) NPM-ALK expressing Ba/F3 cells were treated for
30min with 2mM SU6656 or 1h with 15mM WHI-154 (left panel) or
nucleofected with nothing (control) or 4mM of siRNA targeting
pp60c-srcor Lyn (right panel). Total cell lysates were immunoblotted
with the indicated antibodies. Tubulin was used as a loading
control. (b) Control or NPM-ALK expressing NIH-3T3 cells were
nontransfected (NT) or transfected with a plasmid expressing the
GEF inactive L211Q human Vav3 mutant. Active Rac1-GTP was
measured by pull-down experiments. Expression of the Vav3
mutant was assessed in total lysates with antibodies to human
Vav3. (c) NPM-ALK expressing NIH-3T3 cells were not trans-
fected (NT) or transfected with control vector (control) or a vector
expressing short hairpin RNA (shRNA) to Vav3. After 40h, levels
of Rac1-GTP were assessed by pull-downs. The level of Vav3 was
measured with anti-mouse Vav3.
Vav3 participates in Rac1 activation downstream of
phosphorylation of Vav1. The activating phosphorylation on Y174
of Vav1 was measured on Vav1 immunoprecipitates from Karpas
299 and Cost cells treated for 1h with 15mM WHI-154. Proteins
present in the immunoprecipitates were analysed by western
blotting with polyclonal anti-Vav1 and anti-pVav1 (Y174) anti-
bodies. The activation of NPM-ALK was followed with the anti-
pNPM-ALK(Y664) antibody, loading was assessed with the ALK
antibody. Data are representative of two experiments.
Modification of NPM-ALK activity does not affect
Role of GTPases in NPM-ALK(þ) ALCLs
A Colomba et al
To determine which NPM-ALK tyrosine residue was
required to bind Vav3, we co-transfected HEK293 cells
with wild-type Vav3 and different mutants of NPM-
ALK, in which targeted tyrosines were replaced by
phenylalanines (Y338F, Y342F, Y343F, Y418F (dock-
ing for pp60c-src) and Y664F (docking for PLCg)
mutants). Our data indicate that among the tested
mutants, only Y343F had an impact on Vav3 binding
(Figure 5d). pp60c-srcis not a physical intermediate of
Vav3 and NPM-ALK association since mutation of its
binding site (Y418F) had no effect on the association.
Interestingly, assessment of the intrinsic tyrosine kinase
activity of NPM-ALK with the anti-pNPM-ALK(Y664)
antibody confirmed that three of the mutants, namely,
Y338F, Y342F and Y343F, had reduced kinase activity
(Duyster et al., 2001).The fact that two of them are still
capable of binding Vav3 indicates that Y343 is probably
phosphorylated by another kinase still to identify.
Small GTPases drive NPM-ALK-induced migration
NPM-ALK was reported to affect cell morphology,
adhesion and migration in various cell types. Those
functions have clearly been attributed to GTPases of the
Rho family in many normal cell types and in tumoral
cells (Sahai and Marshall, 2002; Hall, 2005). In invasion
assays through 3D Matrigel chambers, transfection of
adherent fibroblasts with the dominant negative mutant
from Karpas 299 and Cost cells treated for 1h with 15mM WHI-154. Proteins present in the immunoprecipitates were analysed by
western blotting with the indicated antibodies. (b) Vav3 was immunoprecipitated from Karpas 299 and Cost cells with anti-Vav3
antibodies. Proteins in the complexes were analysed as described above. (c) Control or NPM-ALK expressing NIH-3T3 cells were
nontransfected (NT), or transfected with wild-type human Vav3 (WT), active open Y173F Vav3, GEF inactive L211Q Vav3 or SH2
defective R697A mutants. Twenty-four hours after transfection, proteins in NPM-ALK immunoprecipitates were analysed by
immunoblotting. Expression of the mutants in total lysates was assessed with anti-human Vav3 antibody. (d) HEK293 cells were
transfected with vectors expressing wild-type Vav3 and NPM-ALK mutated on the indicated tyrosines. Twenty-four hours after
transfection, proteins in NPM-ALK immunoprecipitates were analysed with the indicated antibodies. Data are representative of two to
Vav3 forms a complex with NPM-ALK and Src kinases. (a) NPM-ALK was immunoprecipitated with ALK1 antibodies
Role of GTPases in NPM-ALK(þ) ALCLs
A Colomba et al
Rac1T17N completely blocked cellular migration of
NIH-3T3 NPM-ALK cells (Figure 6a). Similarly,
depletion of Vav3 by RNA interference also reduced
migration, confirming that Vav3/Rac1 signaling plays a
major role in mediating NPM-ALK effects on cellular
migration and invasion in vitro (Figure 6b).
We report the PI3K- and Src-dependent activation of
Rac1 downstream of NPM-ALK in two ALCL cell lines
(Karpas 299 and Cost), Ba/F3 and NIH-3T3 transfected
cells. To find the link between NPM-ALK and Rac, we
focused on the Vav family of GEFs (Vav1, Vav2 and
Vav3). In addition to their GEF activity, Vavs have the
unique feature of acting as docking proteins, which
makes them excellent candidates for relaying NPM-
ALK functions (Bustelo, 2001). Indeed, these GEFs are
major regulators of lymphocyte function, and Vav1 and
Rac were involved in BCR-ABL signaling in leukemia
(Bassermann et al., 2002; Cho et al., 2005). Over-
expression of active Vav1 and Vav3 mutants have
phenotypes reminiscent of NPM-ALK transformed cells
(Movilla and Bustelo, 1999; Zeng et al., 2000; Hornstein
et al., 2004). Although Vav2 was shown to act down-
stream of tyrosine kinases such as the EGF and PDGF
receptors, there are major differences between Vav2 and
NPM-ALK signaling. Specifically, overexpression of
Vav2 generates strong stress fibers while NPM-ALK
cells are characterized by a disappearance of actin cables
(Liu and Burridge, 2000; Ambrogio et al., 2005).
Moreover, Vav2 was described as being more prone to
activate RhoA than Vav1 and Vav3, which fits with its
action on the actin cytoskeleton but does not match the
pattern of NPM-ALK-induced GTPase activation since
RhoA activity was fairly weak and not affected by the
oncogene. These observations do not favor a role of
Vav2 in NPM-ALK transformation. Moreover, we
demonstrated that no difference in Vav1 activating
phosphorylationwas observedwhen NPM-ALK
Percent of migrating cells
Percent of migrating cells
cells were transfected with pcDNA3-EGFP-Rac1T17N or the control vector. Thirty hours after transfection, cells were plated on
Matrigel-coated inserts and the number of migrating GFP positive cells was evaluated as percentage of control cells. Lysates were
immunoblotted with anti-GFP to control EGFP-Rac1T17N expression. (b) Cells were transfected with the short hairpin RNAs
(shRNA) Vav3 targeting vector or with the control vector and analysed as above. Lysates were immunoblotted with anti-mouse Vav3
to control Vav3 silencing. Control, cells transfected with the control vector; shRNA Vav3, cells transfected with the Vav3 targeting
vector. The levels and the activation of NPM-ALK were assessed with anti-ALK and anti-pNPM-ALK(Y664) antibodies. Data are
expressed as mean±s.e.m.
Small GTPases are required for NPM-ALK-dependent migration in vitro. (a). Control or NPM-ALK expressing NIH-3T3
Role of GTPases in NPM-ALK(þ) ALCLs
A Colomba et al
activity was challenged. Conversely, Vav3 is phosphory-
lated on the activating Y173 downstream of NPM-ALK
in ALCL and transfected cells as well as in lymph node
biopsies originating from patients suffering from NPM-
ALK(þ) lymphomas, suggesting that this pathway
could be a valid target in the human pathology. Because
we demonstrated that it is ALK kinase activity that
drives Rac1 activation, it is likely that other transloca-
tions involving the ALK kinase domain would also
activate Rac1. A broader study will determine if
activation of Vav3/Rac1 is a common feature of
neoplasia resulting from deregulated ALK activity.
It is now well accepted that pathways regulated by
Rho GTPases are very important in human cancers.
Their role in tumorigenesis was first demonstrated in
fibroblasts overexpressing dominant positive forms of
RhoA, Rac1 or Cdc42. Not only was their proliferation
deregulated, but they also induced lung metastasis when
subcutaneously grafted in mice. Since then, they were
shown to be necessary for transformation evoked by
oncogenes such as Ras or receptor tyrosine kinases
(Sahai and Marshall, 2002; Titus et al., 2005). Beside
their effects on cell proliferation, GTPases are central
modulators of the actin and microtubule cytoskeleton,
cell adhesion and motility (Hall, 1998). Using an EGFP
tagged version of the dominant negative mutant
Rac1T17N, we demonstrated that small GTPases drove
NPM-ALK elicited migration. In fact, the balance
between Rac, Cdc42 and RhoA activities determines
cell morphology and migration. In ALCLs and NPM-
ALK transfected cells, Rac1 activity was strong while
RhoA-GTP was difficult to detect. Indeed, it was
demonstrated that Rac activation can downregulate
RhoA, leading to the dissolution of focal adhesions and
increasing cell motility (Rottner et al., 1999). We
observed a 10-fold decrease in the number of focal
adhesions associated with the expression of NPM-ALK
in NIH-3T3 fibroblasts (not shown), the biological
impact of a cross-regulation between Rac1 and RhoA
in ALCL is currently under investigation. Interestingly,
when we depleted cells for Vav3 using RNA inter-
ference, we also observed a blockade of migration
through 3D Matrigel that was less intense than
84.25% for Rac1T17N). This Rac1 mutant acts by
sequestering the upstream Rho GEFs and thereby
inhibiting endogenous Rho GTPases. It then affects
more than one GEF/GTPase couple and its stronger
effect on migration suggests several GEFs act down-
stream of NPM-ALK. Accordingly, the use of the Rac
GEF inhibitor NSC23766 (Gao et al., 2004), reported
not to act on Vav, resulted in a blockade of migration
similar to Vav3 depletion (61.28% of inhibition). We are
currently investigating the synergy between different
GEFs and their regulatory pathways in ALCLs.
Recently, small GTPases have become potential
candidates for anticancer therapy. Different types of
natural molecules and synthetic drugs that inactivate
GTPases or their effectors are available, some of them
show striking antineoplastic or antimetastatic activity
(Aznar et al., 2004; Fritz and Kaina, 2006). We have
for shVav3 versus
tested Clostridium toxins (Petit et al., 2003; Genth et al.,
2006). Both Toxin B from Clostridium difficile (affects
Rac, RhoA and Cdc42) and LT-IP82 from Clostridium
sordellii (specific for Rac, Ras, Ral and Rap) affected
NPM-ALK-induced migration but also blocked prolif-
eration, demonstrating that the role of GTPases down-
stream of NPM-ALK is broader than migration and
invasion (Colomba, A and Gaits-Iacovoni, F., unpub-
lished). Altogether, our observations demonstrate a
central role for Rac1 in NPM-ALK positive lympho-
mas. These data illustrate how an oncogene, here NPM-
ALK, can potentiate its transforming activity by
recruiting proto-oncogenes (such as pp60c-srcand Vav3)
in its vicinity. Interestingly, both the common type and a
small cell variant of ALCL display the same pattern of
activation of Rho GTPases, indicating that targeting
GTPases could be a therapeutic avenue valid to treat not
only the primary disease, but also prevent relapses of the
most aggressive subtypes.
Materials and methods
Reagents and antibodies
Peripheral lymph nodes biopsies were obtained from patients
diagnosed for NPM-ALK(þ) ALCLs at the Department of
Pathology of Toulouse University Medical Center (France),
after informed consent. The study was approved by the
institutional review board of the Purpan’s Hospital, Toulouse,
France. Cell culture reagents were purchased from Invitrogen
(Carlsbad, CA, USA). SU6656 and WHI-154 were from
Calbiochem (San Diego, CA, USA). All other chemicals were
from Sigma-Aldrich (St Louis, MO, USA). Antibodies used
were ALK1 (DakoCytomation, Glostrup, Denmark); Rac1,
human Vav3 and mouse Vav3 (Upstate Biotechnology, Lake
Placid, NY); a-tubulin (Sigma-Aldrich); ALK, pPAK1/2
(S199–204/S192–197), pSrc (Y416), pALK (Y1604) that
(Y664) (Cell Signaling Technologies, Beverly, MA, USA);
pVav3(Y173) (Biosource International); Vav1, pVav1(Y174),
Cdc42, RhoA, c-src (clone H-12), Lyn and GFP (Santa Cruz
Biotechnologies Inc., Santa Cruz, CA, USA). Horseradish
peroxidase-conjugated goat anti-mouse and goat anti-rabbit
immunoglobulin antisera were from Promega (Madison, WI,
Cell culture, plasmids and transfection
HEK293 and NIH-3T3 fibroblasts were maintained in
Dulbecco’s modified Eagle’s medium and Ba/F3 lymphoid
cells in RPMI 1640 containing 2ngml?1murine recombinant
IL-3 (mrIL3) (R&D Systems Europe, Abingdon, UK). Media
were supplemented with 10% fetal calf serum (FCS),
100Uml?1penicillin, 100mgml?1streptomycin and 0.5mgml?1
geneticin (G418) for cells stably expressing NPM-ALK.
Human ALCLs cell lines Karpas 299 and Cost were cultured
in Iscove’s modified Dulbecco’s medium supplemented with
15% FCS. Before immunoprecipitation, GTPases activation
and western blot studies, Karpas 299, Cost and Ba/F3 cells
were serum and IL3 starved overnight and NIH-3T3 cells were
maintained in 2% serum overnight. NIH-3T3 were transfected
using Lipofectamine (Invitrogen) and HEK293 with Effectene
(Qiagen, Valencia, CA, USA) according to the manufacturer’s
nucleofected using the Amaxa technology (Amaxa, Koeln,
Role of GTPases in NPM-ALK(þ) ALCLs
A Colomba et al
Germany). Briefly, 2?106cells were nucleofected using the
Amaxa solution kit V, 4mM of siRNA smartpool pp60c-srcor
Lyn (Dharmacon Inc., Lafayette, CO, USA) and the program
X-01 following the Amaxa guidelines. Cells were lysed 48h
post nucleofection. pcDNA3 containing wild-type NPM-ALK
or the Y338F, Y342F, Y343F, Y418F and Y664F mutants
were already described (Duyster et al., 2001; Cussac et al.,
2004). pcDNA3-EGFP-Rac1T17N was a generous gift from
Dr K Hahn (University of North Carolina, Chapel Hill,
NC, USA). pCI2.F.hVav3.WT, pCI2.F.hVav3.Y173F, pCI2.F.
hVav3.L211Q, pCDNA3.F.hVav3R697A, and pCMS3.H1P
and pCMS3.H1P.shVav3 that contain a separate transcrip-
tional cassette driving Green Fluorescent Protein (GFP)
expression allowing easy identification of transfected cells
were already described (Zakaria et al., 2004; Charvet et al.,
Cell lysis, immunoprecipitation and immunoblotting
Total proteins were extracted with lysis buffer (50mM Tris-
base pH 8, 150mM NaCl, 5mM EGTA, 1% Nonidet P-40,
1mM PMSF, 25mM NaF, 2mM Na3VO4, 10mgml?1leupeptin
and 2mgml?1aprotinin). For lysates from NPM-ALK positive
or negative lymph nodes from ALCLs patients, frozen tissues
were sonicated in detergent-free lysis buffer, cleared by
centrifugation and proteins in the supernatant quantitated
with the Bio-Rad protein assay (Bio-Rad, Munich, Germany).
For immunoprecipitations, clarified homogenates were incu-
bated overnight at 41C with suited antibodies and a mix of
protein A/G sepharose beads. After washes, proteins were
eluted with Laemmli buffer and analysed by SDS–PAGE
followed by western blotting on Immobilon-P membranes
(Millipore, Billerica, MA, USA). Immunoreactive bands were
detected by chemiluminescence with the SuperSignal detection
system (Pierce Chemical Co, Rockford, IL, USA).
GTPases pull-down assays
The amounts of GTP-bound active Rac1, Cdc42 or RhoA
were determinated by pull-down as previously described
(Benard and Bokoch, 2002). Briefly, cells were lysed in ice-
cold lysis buffer (50mM Tris-base pH 7.4, 500mM NaCl,
10mM MgCl2, 2.5mM EGTA, 1% Triton X-100, 0.5% sodium
deoxycholate, 0.1% SDS, 1mM PMSF, 50mM NaF, 1mM
Na3VO4, 10mgml?1leupeptin and 2mgml?1aprotinin) and
clarified lysates were incubated with 30mg GST-PBD (PAK1
Binding Domain for Rac1 and Cdc42) or GST-RBD
(Rhotekin Binding Domain for RhoA) bound to glutathione
sepharose at 41C for 30min. Beads were washed with 50mM
Tris-base pH 7.4, 150mM NaCl, 1mM MgCl2, 5mM EGTA,
1% triton X-100 and GTP-bound GTPases eluted with
Laemmli buffer and subjected to SDS–PAGE followed by
Cell invasion assay
To assess the role of Rac1 and Vav3 in migration, NIH-3T3
cells were transfected with pCMS3.H1P (expressing GFP),
pCMS3.H1P.shVav3 (expressing GFP and shRNA targeting
Vav3) or pcDNA3.EGFP.Rac1T17N. After 30h, cells were
seeded in 24-well plates on biocoated Matrigel Invasion
Chambers that consisted in a 8mm-size pore filter coated with
a reconstituted basal membrane matrix (Becton Dickinson,
Mountain View, CA, USA). Migration proceeded for 17h at
371C. Then, cells on the upper side of the filters were scrapped
with a cotton swab and cells positive for GFP were scored. A
correction index was applied to the raw values, it corresponds
to the growth of the various transfectants over 17h. Results
are expressed as percentages of control cells expressing GFP
We thank Dr Popoff for Clostridium lethal toxins. We are
grateful to Dr H Tronche ` re, Dr S Manenti, Dr MP Gratacap,
Dr C Racaud-Sultan and Dr M Plantavid for helpful
discussions. AC and DR were financed by the ‘Ministe ` re de
la Recherche et de la Technologie’ and the ‘Association pour la
Recherche sur le Cancer’. This work was supported by grants
from the INSERM, ARC, ARECA, La Ligue contre le
Cancer, the ‘Cance ´ ropo ˆ le Grand Sud-Ouest’ and the ‘Institut
National du Cancer’ (INCa), the Re ´ gion Midi-Pyre ´ ne ´ es and
the ‘Po ˆ le de Compe ´ titivite ´ Cancer-Bio Sante ´ ’.
Ambrogio C, Voena C, Manazza AD, Piva R, Riera L, Barberis L
et al. (2005). p130Cas mediates the transforming properties of the
anaplastic lymphoma kinase. Blood 106: 3907–3916.
Amin HM, Medeiros LJ, Ma Y, Feretzaki M, Das P, Leventaki V
et al. (2003). Inhibition of JAK3 induces apoptosis and decreases
anaplastic lymphoma kinase activity in anaplastic large cell
lymphoma. Oncogene 22: 5399–5407.
Armstrong F, Duplantier MM, Trempat P, Hieblot C, Lamant L,
Espinos E et al. (2004). Differential effects of X-ALK fusion
proteins on proliferation, transformation, and invasion properties of
NIH3T3 cells. Oncogene 23: 6071–6082.
Aznar S, Fernandez-Valeron P, Espina C, Lacal JC. (2004). Rho
GTPases: potential candidates for anticancer therapy. Cancer Lett
Bai RY, Dieter P, Peschel C, Morris SW, Duyster J. (1998).
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
Bai RY, Ouyang T, Miething C, Morris SW, Peschel C, Duyster J.
(2000). Nucleophosmin-anaplastic lymphoma kinase associated with
anaplastic large-cell lymphoma activates the phosphatidylinositol 3-
kinase/Akt antiapoptotic signaling pathway. Blood 96: 4319–4327.
Bassermann F, Jahn T, Miething C, Seipel P, Bai RY, Coutinho S
et al. (2002). Association of Bcr-Abl with the proto-oncogene Vav is
implicated in activation of the Rac-1 pathway. J Biol Chem 277:
Benard V, Bokoch GM. (2002). Assay of Cdc42, Rac, and Rho
GTPase activation by affinity methods. Methods Enzymol 345: 349–
Bustelo XR. (2000). Regulatory and signaling properties of the Vav
family. Mol Cell Biol 20: 1461–1477.
Bustelo XR. (2001). Vav proteins, adaptors and cell signaling.
Oncogene 20: 6372–6381.
Charvet C, Canonigo AJ, Billadeau DD, Altman A. (2005). Membrane
localization and function of Vav3 in T cells depend on its association
with the adapter SLP-76. J Biol Chem 280: 15289–15299.
Chiarle R, Gong JZ, Guasparri I, Pesci A, Cai J, Liu J et al. (2003).
NPM-ALK transgenic mice spontaneously develop T-cell lympho-
mas and plasma cell tumors. Blood 101: 1919–1927.
Chiarle R, Simmons WJ, Cai H, Dhall G, Zamo A, Raz R et al. (2005).
Stat3 is required for ALK-mediated lymphomagenesis and provides
a possible therapeutic target. Nat Med 11: 623–629.
Cho YJ, Zhang B, Kaartinen V, Haataja L, de Curtis I, Groffen J et al.
(2005). Generation of rac3 null mutant mice: role of Rac3 in Bcr/
Abl-caused lymphoblastic leukemia. Mol Cell Biol 25: 5777–5785.
Role of GTPases in NPM-ALK(þ) ALCLs
A Colomba et al
Contri A, Brunati AM, Trentin L, Cabrelle A, Miorin M, Cesaro L Download full-text
et al. (2005). Chronic lymphocytic leukemia B cells contain
anomalous Lyn tyrosine kinase, a putative contribution to defective
apoptosis. J Clin Invest 115: 369–378.
Crockett DK, Lin Z, Elenitoba-Johnson KS, Lim MS. (2004).
Identification of NPM-ALK interacting proteins by tandem mass
spectrometry. Oncogene 23: 2617–2629.
Cussac D, Greenland C, Roche S, Bai RY, Duyster J, Morris SW et al.
(2004). Nucleophosmin-anaplastic lymphoma kinase of anaplastic
large-cell lymphoma recruits, activates, and uses pp60c-src to
mediate its mitogenicity. Blood 103: 1464–1471.
Cussac D, Pichereaux C, Colomba A, Capilla F, Pont F, Gaits-
Iacovoni F et al. (2006). Proteomic analysis of anaplastic lymphoma
cell lines: identification of potential tumour markers. Proteomics 6:
Duyster J, Bai RY, Morris SW. (2001). Translocations involving
anaplastic lymphoma kinase (ALK). Oncogene 20: 5623–5637.
Falini B. (2001). Anaplastic large cell lymphoma: pathological,
molecular and clinical features. Br J Haematol 114: 741–760.
Falini B, Bigerna B, Fizzotti M, Pulford K, Pileri SA, Delsol G et al.
(1998). ALK expression defines a distinct group of T/null
lymphomas (‘ALK lymphomas’) with a wide morphological
spectrum. Am J Pathol 153: 875–886.
Fawal M, Armstrong F, Ollier S, Dupont H, Touriol C, Monsarrat B
et al. (2006). A ‘liaison dangereuse’ between AUF1/hnRNPD and
the oncogenic tyrosine kinase NPM-ALK. Blood 108: 2780–2788.
Fritz G, Kaina B. (2006). Rho GTPases: promising cellular targets for
novel anticancer drugs. Curr Cancer Drug Targets 6: 1–14.
Fujimoto J, Shiota M, Iwahara T, Seki N, Satoh H, Mori S et al.
(1996). 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 USA 93:
Gao Y, Dickerson JB, Guo F, Zheng J, Zheng Y. (2004). Rational
design and characterization of a Rac GTPase-specific small molecule
inhibitor. Proc Natl Acad Sci USA 101: 7618–7623.
Genth H, Huelsenbeck J, Hartmann B, Hofmann F, Just I, Gerhard R.
(2006). Cellular stability of Rho-GTPases glucosylated by Clostri-
dium difficile toxin B. FEBS Lett 580: 3565–3569.
Hall A. (1998). Rho GTPases and the actin cytoskeleton. Science 279:
Hall A. (2005). Rho GTPases and the control of cell behaviour.
Biochem Soc Trans 33: 891–895.
Honorat JF, Ragab A, Lamant L, Delsol G, Ragab-Thomas J. (2006).
SHP1 tyrosine phosphatase negatively regulates NPM-ALK tyr-
osine kinase signaling. Blood 107: 4130–4138.
Hornstein I, Alcover A, Katzav S. (2004). Vav proteins, masters of the
world of cytoskeleton organization. Cell Signal 16: 1–11.
Lamant L, De Reynies A, Duplantier MM, Rickman DS, Sabourdy F,
Giuriato S et al. (2006). Gene expression profiling of systemic
anaplastic large cell lymphoma reveals differences depending on
ALK status and two distinct morphological ALK+ subtypes. Blood
Lamant L, Espinos E, Duplantier M, Dastugue N, Robert A, Allouche
M et al. (2004). Establishment of a novel anaplastic large-cell
lymphoma-cell line (COST) from a ‘small-cell variant’ of ALCL.
Leukemia 18: 1693–1698.
Lamant L, Meggetto F, al Saati T, Brugieres L, de Paillerets BB,
Dastugue N et al. (1996). High incidence of the t(2;5)(p23;q35)
translocation in anaplastic large cell lymphoma and its lack of
detection in Hodgkin’s disease. Comparison of cytogenetic analysis,
reverse transcriptase-polymerase chain reaction, and P-80 immu-
nostaining. Blood 87: 284–291.
Lim MS, Elenitoba-Johnson KS. (2006). Mass spectrometry-based
proteomic studies of human anaplastic large cell lymphoma. Mol
Cell Proteomics 5: 1787–1798.
Liu BP, Burridge K. (2000). Vav2 activates Rac1, Cdc42, and RhoA
downstream from growth factor receptors but not beta1 integrins.
Mol Cell Biol 20: 7160–7169.
Llorca O, Arias-Palomo E, Zugaza JL, Bustelo XR. (2005). Global
conformational rearrangements during the activation of the GDP/
GTP exchange factor Vav3. EMBO J 24: 1330–1340.
Marzec M, Kasprzycka M, Liu X, Raghunath PN, Wlodarski P,
Wasik MA. (2007). Oncogenic tyrosine kinase NPM/ALK induces
activation of the MEK/ERK signaling pathway independently of c-
Raf. Oncogene 26: 813–821.
Marzec M, Kasprzycka M, Ptasznik A, Wlodarski P, Zhang Q, Odum
N et al. (2005). Inhibition of ALK enzymatic activity in T-cell
lymphoma cells induces apoptosis and suppresses proliferation and
STAT3 phosphorylation independently of Jak3. Lab Invest 85:
Movilla N, Bustelo XR. (1999). Biological and regulatory properties of
Vav-3, a new member of the Vav family of oncoproteins. Mol Cell
Biol 19: 7870–7885.
Petit P, Breard J, Montalescot V, El Hadj NB, Levade T, Popoff M
et al. (2003). Lethal toxin from Clostridium sordellii induces
apoptotic cell death by disruption of mitochondrial homeostasis in
HL-60 cells. Cell Microbiol 5: 761–771.
Rottner K, Hall A, Small JV. (1999). Interplay between Rac and Rho
in the control of substrate contact dynamics. Curr Biol 9: 640–648.
Sahai E, Marshall CJ. (2002). RHO-GTPases and cancer. Nat Rev
Cancer 2: 133–142.
Theodorescu D, Sapinoso LM, Conaway MR, Oxford G, Hampton
GM, Frierson Jr HF. (2004). Reduced expression of metastasis
suppressor RhoGDI2 is associated with decreased survival for
patients with bladder cancer. Clin Cancer Res 10: 3800–3806.
Thompson MA, Stumph J, Henrickson SE, Rosenwald A, Wang Q,
Olson S et al. (2005). Differential gene expression in anaplastic
lymphoma kinase-positive and anaplastic lymphoma kinase-nega-
tive anaplastic large cell lymphomas. Hum Pathol 36: 494–504.
Titus B, Schwartz MA, Theodorescu D. (2005). Rho proteins in
cell migration and metastasis. Crit Rev Eukaryot Gene Expr 15:
Zakaria S, Gomez TS, Savoy DN, McAdam S, Turner M, Abraham
RT et al. (2004). Differential regulation of TCR-mediated gene
transcription by Vav family members. J Exp Med 199: 429–434.
Zeng L, Sachdev P, Yan L, Chan JL, Trenkle T, McClelland M et al.
(2000). Vav3 mediates receptor protein tyrosine kinase signaling,
regulates GTPase activity, modulates cell morphology, and induces
cell transformation. Mol Cell Biol 20: 9212–9224.
Role of GTPases in NPM-ALK(þ) ALCLs
A Colomba et al