Activating alleles of JAK3 in acute megakaryoblastic leukemia
Denise K. Walters,1,2,9Thomas Mercher,3,9Ting-Lei Gu,4,9Thomas O’Hare,1,2Jeffrey W. Tyner,2Marc Loriaux,5
Valerie L. Goss,4Kimberly A. Lee,4Christopher A. Eide,2Matthew J. Wong,2Eric P. Stoffregen,2
Laura McGreevey,6Julie Nardone,4Sandra A. Moore,3John Crispino,7Titus J. Boggon,8
Michael C. Heinrich,2,6Michael W. Deininger,2Roberto D. Polakiewicz,4D. Gary Gilliland,3
and Brian J. Druker1,2,*
1Howard Hughes Medical Institute, Portland, Oregon 97239
2Department of Hematology and Medical Oncology, Oregon Health & Science University, Portland, Oregon 97239
3Brigham and Women’s Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115
4Cell Signaling Technology Inc., 3 Trask Lane, Danvers, Massachusetts 01923
5Department of Pathology, Oregon Health & Science University, Portland, Oregon 97239
6Portland VA Medical Center, Portland, Oregon 97239
7Ben May Institute for Cancer Research, University of Chicago, 924 East 57th Street, Chicago, Illinois 60637
8Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, SHM B-302, New Haven, Connecticut 06520
9These authors contributed equally to this work.
Tyrosine kinases are aberrantly activated in numerous malignancies, including acute myeloid leukemia (AML). To identify
tyrosine kinases activated in AML, we developed a screening strategy that rapidly identifies tyrosine-phosphorylated pro-
teins using mass spectrometry. This allowed the identification of an activating mutation (A572V) in the JAK3 pseudokinase
domain in the acute megakaryoblastic leukemia (AMKL) cell line CMK. Subsequent analysis identified two additional JAK3
alleles, V722I and P132T, in AMKL patients. JAK3A572V, JAK3V722I, and JAK3P132Teach transform Ba/F3 cells to factor-inde-
pendent growth, and JAK3A572Vconfers features of megakaryoblastic leukemia in a murine model. These findings illustrate
the biological importance of gain-of-function JAK3 mutations in leukemogenesis and demonstrate the utility of proteomic
approaches to identifying clinically relevant mutations.
Tyrosine kinases comprise a family of 90 enzymes involved in
the regulation of various cellular processes, including prolifera-
tion, survival, differentiation, and motility (Krause and Van Etten,
2005). The activity of these kinases is normally tightly controlled.
However, tyrosine kinases can become aberrantly activated by
different mechanisms, including point mutation; fusion with un-
related genes that lead to constitutive dimerization and activa-
tion; and in the case of receptor tyrosine kinases, mutation in
the juxtamembrane domain that results in constitutive kinase
activation (Paul and Mukhopadhyay, 2004). Dysregulated tyro-
sine kinases have been shown to have a significant role in
a variety of cancers, including leukemia. In the case of acute
myeloid leukemia (AML), mutations in FLT3 or c-KIT have
been implicated as aberrations that confer a proliferative
advantage to hematopoietic progenitors (Stirewalt and Radich,
2003; Tse et al., 2000; Yamamoto et al., 2001; Yokota et al.,
Despite the importance of dysregulated tyrosine kinases in
cancer, the identification of specific oncogenes within complex
activation pathways is difficult in malignant cells. Recently, pro-
teomic approaches focusing on the phosphotyrosine content of
phorylated proteins can be consistently identified in cancer cell
lines. Indeed, Rush et al. (2005)and Walters et al.(2006) demon-
strated that this methodology is capable of identifying activated
tyrosine kinases even when the signaling pathways are un-
known (Rush et al., 2005; Walters et al., 2006).
A particularly interesting disease in which to search for acti-
vated tyrosine kinases is AML. STAT5 has been found constitu-
tively tyrosine-phosphorylated in the leukemic cells of approxi-
mately 70% of AML patients (Birkenkamp et al., 2001;
Hayakawa et al., 1998). The presence of FLT3 or KIT activating
S I G N I F I C A N C E
We used a mass spectrometry-based screen to identify activating alleles of JAK3 in acute megakaryoblastic leukemia (AMKL). Acti-
pseudokinase domain, providing mechanistic insights into the role of this domain in regulation of the JAK3 catalytic domain. Recent
development of JAK3-selective small molecule inhibitors raises the possibility of therapeutic intervention in this subset of patients.
This study thus provides insights into the molecular pathogenesis of AMKL, suggests that a more broadly based screen for JAK3 muta-
tions in cancer is warranted, and validates high-throughput mass spectrometry of phosphopeptides as a strategy for identification of
therapeutic targets in cancer.
A R T I C L E
CANCER CELL 10, 65–75, JULY 2006 ª2006 ELSEVIER INC. DOI 10.1016/j.ccr.2006.06.00265
mutations can account for STAT5 phosphorylation in up to 35%
of patients. However, the mechanism of constitutive STAT5
phosphorylation remains unclear in a significant percentage of
patients lacking these mutations. As STAT5 tyrosine phos-
phorylation for analysis by phosphopeptide mass spectrometry.
Using this approach, we identified several activated tyrosine
kinases in an acute megakaryoblastic leukemia (AMKL) cell line
that lacked FLT3 or KIT mutations yet possessed constitutive
fied by LC-MS/MS in the activation of STAT5. These studies
guided DNA sequence analysis of candidate genes and enabled
main of JAK3. Correlation of the structural location of A572 with
biochemical data implicates the structural integrity of the pseu-
JAK signaling. These findings provided a rationale for DNA anal-
resulted in identification of two additional JAK3 mutations. Each
JAK3, phosphorylation of STAT5, and transformation of the
hematopoietic cell line Ba/F3 to factor-independent growth.
Furthermore, JAK3A572Vrecapitulated certain key features of
megakaryoblastic leukemia in a murine model of disease.
Constitutive STAT5 activation in CMK cells
depends on JAK3
The baseline phosphorylation status of a spectrum of human
AML cell lines was assessed, and STAT5 was constitutively
are a megakaryoblastic cell line derived from a patient with
Down’s syndrome and AMKL (Sato et al., 1989). Activating
revealed that the CMK cell line does not possess activating mu-
tations in FLT3 or KIT. To identify the upstream activator of
STAT5, we used a phosphoproteomic approach in which CMK
ases to maximize the number of phosphopeptides identified af-
mass spectrometry (Rush et al., 2005). Using this approach, we
identified a total of 348 nonredundant phosphopeptides corre-
teins (Table S1 in the Supplemental Data available with this
article online). Among these proteins, we detected numerous
phosphorylated peptides corresponding to tyrosine kinases
in two sites, including Y980, within the activation loop—an indi-
cation of JAK3 activation in these cells. Other kinases, such as
TYK2, JAK2, c-Kit, and c-Abl were also found to be tyrosine
phosphorylated in the CMK cell line by mass spectrometry.
Activated JAK proteins are known to directly phosphorylate
STAT proteins (Flores-Morales et al., 1998; Fujitani et al.,
1997). Therefore, we next determined whether the pan-JAK in-
hibitor JAK Inhibitor I would affect the growth and viability of
the CMK cell line. Treatment of CMK cells with the pan-JAK in-
hibitor resulted in a significant decrease in cell proliferation and
viability (Figure 1B). However, treatment of CMK cells with ima-
tinib, which inhibits both c-ABL and c-KIT, had little effect on
CMK cell proliferation (Figure 1C). This indicated that JAK pro-
teins, but not c-KIT or c-ABL, were essential for CMK growth
and viability. Treatment of CMK cells with JAK Inhibitor I also
impacted tyrosine phosphorylation of STAT5, p42/44 mitogen-
activated protein (MAP) kinase, and STAT3 and was associated
with an increase in the apoptotic death rate (Figures 1D and 1E).
This effect could not be attributed to nonspecific toxicity of the
inhibitor, as growth, viability, or STAT5 phosphorylation was not
inhibited by the JAK Inhibitor I in K562 cells that express the
BCR-ABL fusion protein.
We next assessed whether JAK2, JAK3, or TYK2 contributed
an essential role in growth and viability of CMK cells using
a siRNA approach. Expression of JAK2, JAK3, or TYK2 was in-
dividually downregulated with specific siRNAs. Immunoblot
analysis revealed that the expression of these proteins was spe-
following transfection of the respective JAK family member
siRNA into CMK cells (Figure 2A). We observed that downregu-
lation of JAK3, but not JAK2 or TYK2, resulted in inhibition of
CMK cell growth (Figure 2B). Moreover, treatment with JAK3
siRNA resulted in inhibition of STAT5 tyrosine phosphorylation
and increased apoptosis of CMK cells, while downregulation
of JAK2 or TYK2 had no effect (Figures 2A and 2C). In addition,
we observed constitutively phosphorylated JAK3 in CMK cells
following immunoprecipitation and phosphotyrosine immuno-
blot (Figure 2D). These findings indicated that activated JAK3
was essential for growth and survival of CMK cells.
JAK3 contains an activating mutation in CMK cells
We next analyzed JAK3 for activating mutations. Constitutively
phosphorylated JAK3 migrated at the expected molecular
weight in CMK cells (Figure 2), indicating that point mutation
rather than gene rearrangement was a likely mechanism for ac-
zygous C to T mutation at nucleotide position 1774 that is pre-
dicted to result in substitution of valine for alanine at amino
acid position 572 in the JH2 domain of JAK3 (Figure 3A).
To assess the transforming ability of the JAK3A572Vmutation,
JAK3WTor MIG-JAK3A572V. Twenty-four hours after transduc-
tion, GFP-expressing cells were selected by flow cytometry,
plated in liquid culture in the absence of IL-3, and counted daily
to assess IL-3-independent growth. As shown in Figure 3C, ex-
pression of JAK3A572Vconferred IL-3-independent growth to
Ba/F3 cells, whereas JAK3WT-transduced cells retained depen-
dence on IL-3 for proliferation. In addition, JAK3A572Valso con-
not shown). Biochemical analysis in Ba/F3 cells revealed that
JAK3 as well as downstream targets STAT5, phosphoinositol-
3-kinase-Akt, and MAP kinase (p42/44) were each constitutively
phosphorylated in cells transduced with JAK3A572V, but not
in cells transduced with JAK3WT(Figure 3B). As a functional
correlate to the observed increase in JAK3 phosphorylation,
JAK3A572Vexhibited increased kinase activity compared with
JAK3WTin an in vitro immunoprecipitation kinase assay
(Figure S1). Together, these results indicate that the JAK3A572V
mutation results in constitutive activation of the JAK3 tyrosine
kinase and can transform cytokine-dependent hematopoietic
cell lines in vitro.
A R T I C L E
CANCER CELL JULY 2006
AMKL patients have activating JAK3 mutations
These findings indicated that activating mutations in JAK3 con-
tribute to the pathogenesis of AMKL. To further address this
possibility, we screened 19 AMKL patient samples, of which 3
were derived from Down’s syndrome and 16 from non-Down’s
syndrome patients. While none of these patients had the
JAK3A572Vmutation, two had other JAK3 mutations, one having
a heterozygous V722I substitution in the JH2 pseudokinase do-
JH6 domain of the receptor binding region (Figure 4A and Table
S2). None of these 19 AMKL patient samples had the common
myeloproliferative disorder mutation JAK2V617F. Of note, as for
the AMKL patient from whom the CMK cell line was derived,
the patient with the JAK3V722Iallele had Down’s syndrome and
harbored a loss-of-function mutation in the GATA-1 gene
(Wechsler et al., 2002). Transduction of Ba/F3 cells with
MIG-JAK3WT, MIG-JAK3V722I, or MIG-JAK3P132Tconfirmed that,
similar to JAK3A572V, the JAK3V722Iand JAK3P132Talleles also
stitutive JAK3 and STAT5 phosphorylation (Figures 4B and 4C).
Modeling of JAK3 JH2 domain mutations
We next developed a structural model to assess the role of the
JAK3 mutations in constitutive kinase activation. The structure
JH2 (pseudokinase) domain lacks several residues critical to
phosphotransferase activity, this domain is expected to adopt
the overall protein architecture characteristic of tyrosine and
serine/threonine kinases. We used the Swiss-model automated
comparative protein modeling server to construct a homology
model of the JAK3 JH2 domain using the crystal structures of
the epidermal growth factor receptor (Stamos et al., 2002), insu-
lin receptor (Parang et al., 2001), Zap-70 (Jin et al., 2004), and
Syk (Atwell et al., 2004) tyrosine kinases (Protein Data Bank ac-
cession codes 1M17, 1GAG, 1U59, and 1XBC, respectively) as
a model. These tyrosine kinases have sequence identity to the
JAK3 JH2 domain search sequence (Q501 to Q812) of 28%,
28%, 33%, and 33%, respectively. All four template crystal
structures for the prediction of the JAK3 JH2 domain are active
conformation tyrosine kinase domains. The model therefore as-
sumes a conformation that is consistent with an active confor-
expected locations of two residues discussed in this study.
A572 is predicted to lie in the pseudokinase domain C helix (Fig-
ure 5). The model predicts that A572 is on the cleft side of the C
helix located at the same position as the catalytic glutamic acid
in active kinase domains. While the preferred conformation of
the JAK3 pseudokinase domain activation loop is not known
formation. Correlation of the structural location of A572 with the
data presented in this paper would suggest a role for the ‘‘cata-
lytic cleft’’ region of the pseudokinase domain in autoregulation.
conformation, potentially packing against A572 in a manner
Figure 1. Constitutively activated STAT5 is caused by the JAK family in CMK cells
A: Cell lines were serum starved for 16 hr. Lysates were subjected to immunoblot analysis for STAT5 and phospho-STAT5.
to an MTS assay for determination of total viable cells. Values represent mean 6 SEM (n = 3).
C: CMK and K562 control cells were treated with increasing concentrations of imatinib. Seventy-two hours later, cells were subjected to an MTS assay for
determination of total viable cells. Values represent mean 6 SEM (n = 3).
D: CMK and K562 control cells were treated as in B. Seventy-two hours later, cells were stained with annexin V-FITC and analyzed by flow cytometry for
determination of apoptotic cells. Values represent mean 6 SEM (n = 3).
E: CMK and K562 control cells were treated as in B. Cell lysates were subjected to immunoblot analysis for total and phospho-STAT5, ERK, and STAT3. *p < 0.05.
A R T I C L E
CANCER CELL JULY 200667
that could be disrupted by an A572V mutation. The second res-
idue that is located in the pseudokinase domain, V722, is pre-
dicted to be located C-terminal to the short kinase fold F helix
(Figure 5). In the model, this residue is expected to be abutting
tive mechanism of activation for the V722I substitution is not
readily apparent from the structural model. Hence, molecular
modeling of the A572V mutation is consistent with functional
data indicating an activating phenotype; however, numerous
JAK3 pseudokinase domain mutations have been identified
that induce loss-of-function phenotypes despite being constitu-
tively phosphorylated (Chen et al., 2000; Notarangelo et al.,
2001). As such, it will be important to assess the position of
these residues in the context of a solved structure of the com-
posite JAK3 JH1/JH2 complex.
JAK3A572Vinduces a lethal hematologic
malignancy in vivo
potential of JAK3A572V. C57BL/6 mice were transplanted with
bone marrow (BM) cells that were retrovirally transduced with
Table 1. Tyrosine residues phosphorylated on kinases in CMK cells
Protein kinase, dual specificity
Protein kinase, ser/thr (nonreceptor)
Protein kinase, tyrosine (nonreceptor)
Protein kinase, tyrosine (receptor)
Tyrosine-phosphorylated kinases identified by mass spectrometry in CMK
cells. x, published sites.
Figure 2. JAK3 is necessary for constitutive STAT5 activation, proliferation,
and survival of CMK cells
A: CMK cells were transfected with nonspecific or JAK2-, JAK3-, or TYK2-spe-
cific siRNA. Forty-eight hours later, lysates were subjected to immunoblot
analysis for JAK2, JAK3, TYK2, phospho-STAT5, and STAT5.
B: CMK and K562 control cells were transfected with siRNA as in A. Seventy-
two hours later, cells were subjected to an MTS assay for determination of
total viable cells. Values represent mean 6 SEM (n = 7).
C: CMK and K562 control cells were transfected with siRNA as in A. Seventy-
two hours later, cells were stained with annexin V-FITC for determination of
apoptotic cells. Values represent mean 6 SEM (n = 6).
D: Cell lines were serum starved for 16 hr. Lysates were immunoprecipitated
with a JAK3-specific antibody, and immunoprecipitates were subjected to
immunoblot analysis for phosphotyrosine and JAK3. *p < 0.05.
A R T I C L E
CANCER CELL JULY 2006
either MIG-JAK3WTor MIG-JAK3A572V. JAK3A572Vmice ex-
hibited significantly decreased survival compared with JAK3WT
mice (Figure 6A) and showed several phenotypic characteristics
of megakaryoblastic leukemia. There was splenomegaly in
JAK3A572Vversus JAK3WTanimals (476 6 94 mg versus 98 6
6 mg, respectively). Multiparameter flow cytometry showed an
approximate 8-fold expansion in the megakaryocyte-erythroid
progenitors (MEP), defined as lineage2, c-KIT+, Sca12,
JAK3A572Vcompared with JAK3WTanimals. As a functional cor-
relate of this finding, we observed an expansion in megakaryo-
cyte colony formation in JAK3A572Vcompared with JAK3WTan-
imals (Figure 6B). In addition, we detected abnormally high
numbers of megakaryocytes infiltrated in the spleen and liver
of JAK3A572Vtransplanted mice as determined by staining for
the megakaryocyte-specific marker von Willebrand factor
(vWF) (Figure 6D and Figure S2B). To further characterize the ef-
fect of JAK3A572Von megakaryocyte maturation, we assessed
terminal differentiation by ex vivo culture of bone marrow cells
from wild-type or mutant mice for 4 days in the presence
of thrombopoietin (TPO) and stem cell factor (SCF). In both
JAK3A572Vand JAK3WT, mature proplatelet-forming megakar-
yocytes were observed in the cultures. Nevertheless, ploidy
analysis on CD41+ cells showed a significant decrease in the
median ploidy of megakaryocytes from JAK3A572Vcompared
to JAK3WTmice, and an increase in megakaryocytes in S phase
(Figure 6C). Despite the increased incidence ofmegakaryocytes
in spleen and liver, JAK3A572Vmice exhibited normal platelet
counts in peripheral blood (Figure S2C). Also, secondary trans-
plantation of either bone marrow or spleen cells into sublethally
irradiated mice did not result in visible disease in the recipient
animals (data not shown). Taken together, these findings indi-
cate that JAK3A572Vrecapitulates several, but not all, of the
phenotypic characteristics of AMKL, including splenomegaly,
expansion of the megakaryocyte progenitors, and impaired
megakaryocyte differentiation as assessed by analysis of poly-
In addition to the megakaryocyte phenotype in JAK3A572V-
transduced animals, we observed marked leukocytosis that
was comprised primarily of lymphocytes (Figure 6D). There
was also lymphocytic infiltration of bone marrow, liver, spleen,
nophenotypic analysis did reveal a marked expansion of CD8+
cells in the thymus, peripheral blood, bone marrow, and spleen
in JAK3A572Vcompared with JAK3WTanimals (Figure 6F and
data not shown). These findings are consistent with a coexistent
in the megakaryocytic lineage induced by JAK3A572V.
Using a screening strategy involving phosphopeptide immuno-
precipitation followed by LC-MS/MS mass spectrometry, we
have identified a JAK3 mutation, JAK3A572V, associated with
human cancer in the megakaryoblastic cell line CMK. These
findings prompted DNA sequence analysis of primary cells
Figure 3. JAK3 mutation causes constitutive
JAK3 activity in CMK cells
A:The JAK3 JH2 domain was sequenced, reveal-
ing an alanine to valine substitution at amino
acid position 572.
B: Ba/F3 cells were transduced with MIG-JAK3WT
or MIG-JAK3A572V. GFP-expressing cells were se-
lected by flow cytometry, cultured for 5–8 days
in IL-3-containing media, and serum starved for
4 hr prior to biochemical analysis. Lysates were
immunoprecipitated with anti-JAK3 followed by
immunoblot analysis for phosphotyrosine or
JAK3 or immunoprecipitated with anti-STAT5
followed by immunoblot analysis for phospho-
STAT5 or STAT5. Immunoblot analyses for p42/44
C: Ba/F3 cells were infected and sorted as in B.
Cells were then incubated in media without IL-3
for 5 days and counted using trypan blue every
day fordetermination oftotalviablecells.Values
represent mean 6 SEM (n = 3). *p < 0.05.
A R T I C L E
CANCER CELL JULY 200669
derived from patients with AMKL and identified two additional
activating alleles of JAK3: JAK3V722Iand JAK3P132T. Expression
growth that is associated with constitutive activation of the re-
spective JAK3 allele in the absence of cytokine, and activation
of known downstream effectors of JAK signaling, including
STAT5, ERK, and AKT.
associated with leukemia, several studies have previously sug-
gested an association of JAK3 with cancer phenotypes in the
absence of known mutations. Initially, JAK3 expression was
found preferentially in hematopoietic cells, including samples
of B cell lymphomas, supporting a possible involvement of
JAK3 in transformation of hematopoietic cells (Tortolani et al.,
1995). Also, JAK3 splice variants were discovered in cancer
cells from hematopoietic and epithelial origin, and JAK3 was
found to be important for activation of the proto-oncogenes c-
fos, c-myc, and bcl-2 (Kawahara et al., 1995; Lai et al., 1995).
In addition, peripheral T lymphocytes infected and transformed
with HTLV-1, a major cause of adult T cell leukemia, were sub-
sequently shown to exhibit constitutively phosphorylated
JAK3, and this event was shown to correlate with IL-2-indepen-
dent growth (Migone et al., 1995; Xu et al., 1995). Finally, oligo-
merization of JAK3 induced by fusion with TEL, although an ar-
tificial construct not found in patients so far, has been shown to
constitutively activate JAK3, resulting in factor-independent
growth of BaF3 cells as well as activation of downstream signal-
ing partners, including STAT1, -3, and -5 (Lacronique et al.,
2000). In this manuscript, we identified activating, mutated al-
leles of JAK3 in patients with leukemia and demonstrated that
constitutively activated JAK3 can induce a lethal hematopoietic
malignancy in vivo.
Several inferences on the mechanism of activation of JAK3
canbe drawnfor thetwomutations residingintheJAK3JH2do-
main, A572V and V722I. Deletion of the JH2 domain in experi-
mental systems results in constitutive activation of JH1 (Sahar-
inen and Silvennoinen, 2002). These observations have led to
the hypothesis that JH2, which is nearly identical in amino acid
sequence to JH1, but lacks catalytic activity, serves an autoinhi-
bitory function. In support of this hypothesis, there are other ex-
amples of activating alleles in the JH2 domain of JAK family
members that include the E695K Hopscotch allele in the single
Drosophila JAK, and the JAK2V617Fallele observed in the major-
ity of cases of polycythemia vera, myeloid metaplasia with mye-
lofibrosis, and essential thrombocythemia (Baxter et al., 2005;
ine etal.,2005;Zhaoetal.,2005).TheE695K Hopscotchalleleis
predicted to disrupt a salt bridge and result in loss of structure,
and thereby loss of autoinhibitory activity of the JH2 domain. A
predicted structure of the JAK2 JH1 and JH2 domains suggests
transform BaF3 cells
A: JAK3 was sequenced in 19 megakaryoblastic
AML patients (3 with Down’s syndrome, 16 non-
Down’s syndrome), and two mutations, V722I
and P132T, were identified.
B: Ba/F3 cells were transduced and sorted as in
described in Figure 3, and biochemical analysis
of Ba/F3 cells infected with JAK3WT, JAK3V722I,
and JAK3P132Twas performed. Cells were serum
starved for 4 hr, and lysates were immunoprecip-
itated with anti-JAK3 and subjected to immuno-
noprecipitated with anti-STAT5 and subjected to
immunoblot analysis for phospho-STAT5 or STAT5.
C: Sorted cells were incubated in media without
IL-3 for 12 days and subjected to an MTS assay
Values represent mean 6 SEM (n = 3). *p < 0.05.
A R T I C L E
CANCER CELL JULY 2006
that the JH1 and JH2 regions may interact to prevent kinase
activation (Lindauer et al., 2001) and predicts that residues
V617–E621 of JAK2 are important for autoinhibition. However,
V617 is conserved between JAK1, JAK2, and TYK2, but not
JAK3. Indeed, substitution of the homologous M592 residue
with phenylalanine in the context of JAK3 does not result in ki-
nase activation (Staerk et al., 2005). Our structural modeling
suggests that the A572V substitution is on the cleft side of the
C helix at the same position as the catalytic glutamic acid resi-
due in active kinase domains and that there may be a role for
the catalytic cleft region of the pseudokinase domain in autore-
gulation of kinase activity. However, the crystallographic struc-
ture of the respective JH1–JH2 domains of JAK family members
will be required to formally test these hypotheses.
Expression of JAK3A572Vin a murine bone marrow transplant
model induces several features of AMKL that include spleno-
megaly, increase in megakaryocyte-erythroid progenitors, and
of AMKL,including bone marrow fibrosis andserially transplant-
able disease, were not observed, suggesting that full AMKL
transformation may require other cooperating mutations. Con-
sistent with this hypothesis, two of three JAK3 mutations were
found in samples from patients with Down’s syndrome and
AMKL that also harbored an additional chromosome 21 and
a GATA-1 mutation resulting in expression of a truncated pro-
tein. Recent reports indicate that GATA-1 mutations alone do
not cause AMKL in a murine knockin model but result in abnor-
mal transient proliferation of megakaryocyte progenitors during
embryonic development (Li et al., 2005). Taken together, our
mutations in the pathogenesis of AMKL, including GATA-1
mutant alleles or oncogenic events phenocopying trisomy 21.
lymphoproliferative disorder. As demonstrated by studies on
JAK3-deficient mice and genotypic analyses of severe com-
bined immunodeficiency disorders, JAK3 signaling plays an im-
portant role in T cell development, proliferation, and function by
virtue of association with the common gamma chain of a spec-
trum of T cell-specific cytokine receptors that include IL-2, -4,
-7, -9, -15, and -21 (Brown et al., 1999; Johnston et al., 1994;
Miyazaki et al., 1994; Noguchi et al., 1993; Nosaka et al.,
1995; Pesu et al., 2005; Russell et al., 1994; Sohn et al., 1998;
row transplantation assay enables retroviral transduction of the
full spectrum of myeloid and lymphoid hematopoietic progeni-
phoproliferative disorderin this murineBMTmodel, whereas the
JAK2V617Fallele associated with myeloproliferative disorders
does not (Wernig et al., 2006). This observation suggests that
JAK3 mutations may also be relevant in the pathogenesis of
T cell lymphoproliferative disorders.
Previously identified JAK3 mutations have been loss-of-func-
tion mutations associated with a severe combined immunodefi-
ciency. These observations indicate that inhibition of JAK3
would be predicted to inhibit T cell function and have spawned
an effort to develop JAK3 inhibitors as immunomodulatory
agents in autoimmune disorders that include asthma and aller-
gies (Malaviya et al., 1999), type I diabetes (Cetkovic-Cvrlje
et al., 2003; Tian et al., 1998), allograft rejection (Hall, 1991;
Stepkowski et al., 2002), and amyotrophic lateral sclerosis
(Cetkovic-Cvrlje and Tibbles, 2004). This study suggests that
JAK3 inhibitors that are currently in development for treatment
of autoimmune disease (Borie et al., 2004; Changelian et al.,
2003; O’Shea et al., 2004) may also have clinical activity in
AMKL patients with mutant activating alleles of JAK3, and po-
tentially in T cell lymphoma/leukemia. Our murine model should
provide a useful tool for assessing these potential therapeutic
Another important aspect of this study was the use of a mass
spectrometry-based strategy to identify novel therapeutic tar-
gets in cancer. Recent data indicate that using high-throughput
DNA sequence analysis of the tyrosine and serine-threonine ki-
nome may yield few cancer alleles (Davies et al., 2005). Mass
spectrometric assessment of phosphopeptides that denote
constitutive activation of signal transduction pathways may pro-
vide a useful surrogate screen for functional activation of cancer
disease alleles that are, in turn, potential therapeutic targets.
in human cancer and indicate that JAK3 contributes to the
both types of leukemias for activating JAK3 mutations.
Cell lines and reagents
AML-193, GDM-1, CMK, K562, HEL, CHRF-288-11, and Ba/F3 cells were
obtained from the German National Resource Centre for Biological Material
(DSMZ). GF-D8 cells were generously provided by Dr. Orietta Spinelli, Labo-
Figure 5. Homology model of the JH2 pseudokinase domain of human JAK3
The model was constructed using the Swiss-Model automated protein
modeling server using the JAK3 pseudokinase domain and the crystal
structures of epidermal growth factor receptor, insulin receptor, Zap-70,
and Syk tyrosine kinases (Protein Data Bank accession codes 1M17,
1GAG, 1U59, and 1XBC, respectively) as a model. Although several residues
critical forkinase activity arenotconserved, thepseudokinasedomain isex-
pected to retain the essential features of the protein kinase fold, including
the smaller b sheet-rich N lobe and the larger, mostly helical C lobe. The lo-
cations of A572 and V722 are indicated with red spheres. A572 is expected
to lie within the kinase fold C helix, and V722 is expected to be C-terminal to
the kinase fold F helix. The figure was made using the program PYMOL
A R T I C L E
CANCER CELL JULY 200671
supplemented with 10% FCS, 1 unit/ml penicillin G, and 1 mg/ml streptomy-
KIT and FLT3 mutational analysis
Mutational analysis was performed as previously described (Goemans et al.,
2005; O’Farrell et al., 2003).
Phosphopeptides were prepared using PhosphoScan Kit (Cell Signaling
Analysis by LC-MS/MS mass spectrometry
Peptides in the immunoprecipitation eluate (40 ml) were concentrated and
separated from eluted antibody using Stop and Go extraction tips (Stage-
Tips) (Rappsilber et al., 2003). Peptides were eluted from the microcolumns
with 1 ml of 60% MeCN, 0.1% TFA into 7.6 ml of 0.4% acetic acid/0.005%
heptafluorobutyric acid (HFBA). The sample was loaded onto a 10 cm 3
75 mm PicoFrit capillary column (New Objective) packed with Magic C18
AQ reversed-phase resin (Michrom Bioresources) using a Famos autosam-
pler with an inert sample injection valve (Dionex). The column was developed
delivered at 280 nl/min (Ultimate, Dionex). Tandem mass spectra were
collected in a data-dependent manner with an LTQ ion trap mass spectrom-
eter (ThermoFinnigan), using a top-ten method, a dynamic exclusion repeat
count of 1, and a repeat duration of 10 s. TurboSequest (ThermoFinnigan)
searches were done against the NCBI human database released on August
24, 2004 (containing 27,175 proteins), allowing for tyrosine phosphorylation
(Y + 80) and oxidized methionine (M + 16) as dynamic modifications. Se-
quence assignments were accepted based on the score-filtering criteria
described previously (Rush et al., 2005), with the exception that assignments
having score characteristics of false-positive assignments, determined by
searching against a database of reversed protein sequences (Peng et al.,
2003), were not accepted.
Patient sample collection, sequencing, and mutational analysis
samples were as follows: exon 4 forward: 50-GTAAAACGACGGCCAGT
GGGGTCAGCCCAGGATTG-30, reverse: 50-CAGGAAACAGCTATGACCGC
CCTGGGTCATAGGAACAC-30; exon 16 forward: 50-GTAAAACGACGGCC
AGTACCCCCACTTTGACAGAAGG-30, Reverse: 50-CAGGAAACAGCTATG
2.5 (SoftGenetics, State College, PA). All clinical samples were obtained with
Figure 6. In vivo expression of JAK3A572Vcauses a lethal hematopoietic malignancy with megakaryoblastic features
A: MSCV retroviruses expressing either JAK3WT-GFPor JAK3A572V-GFPwere used to transduce bone marrow cells from wild-type C57BL/6 mice. Transduced bone
marrow cells were then transplanted into lethally irradiated C57BL/6 recipient mice, and survival was monitored (n = 10).
B: Splenocytes from transplanted JAK3WTand JAK3A572Vwere plated onto collagen-based culture containing Tpo, IL-11, IL-3, and IL-6. Colonies were stained
for acetylcholinesterase activity and counted after 7 days. Values represent mean 6 SEM (n = 3).
C: Bone marrow cells from JAK3WTor JAK3A572Vtransplanted mice were cultured in the presence of Tpo and SCF for 4 days. They were subsequently stained
with anti-CD41 antibody and propidium iodide (PI) and analyzed by flow cytometry. Shown are PI histograms of CD41-positive cells.
D: Spleens were harvested from JAK3WTor JAK3A572Vtransplanted mice at 120 days posttransplantation. Splenic sections were subjected to immunohisto-
chemistry for von Willebrand factor (vWF) and analyzed by light microscopy.
(n = 5).
A R T I C L E
CANCER CELL JULY 2006
informed consent with approval by the University of Chicago or Brigham and
Women’s Hospital Institutional Review Board.
siRNA and siRNA transfection
JAK2, JAK3, and TYK2 SMARTpool siRNA duplexes were purchased from
Dharmacon Research, Inc. A nonspecific SMARTpool siRNA was used as
a control. Cells were transfected with the various siRNAs via electroporation.
a square-wave electroporator (BTX Genetronics), incubated at room temper-
ature for 30 min, and transferred to 96-well, 24-well, and/or 6-well plates
Flow cytometry and immunoblotting
Detection of apoptosis was performed as previously described (Walters
et al., 2005). Detection of total and phosphorylated protein targets was
performed using standard immunoprecipitation and immunoblotting proce-
dures. Antibodies used were anti-phospho-STAT5, anti-phospho-Akt,
anti-Akt, anti-phospho-p42/44, and anti-p42/44 (Cell Signaling Technology,
Beverly, MA); anti-STAT5 and anti-TYK2 (BD Transduction Laboratories,
BD Pharmingen, San Diego, CA); anti-JAK3 and anti-phospho-JAK3 (Santa
Cruz Biotechnology, Inc., Santa Cruz, CA); and anti-phosphotyrosine 4G10
(Upstate Biotechnologies). Megakaryocyte/erythrocyte progenitor (MEP)
analysis was performed using a previously described protocol (Na Nakorn
et al., 2002) modified to accommodate the presence of GFP in sorted cells.
blood cells were lysed using RBC Lysis buffer (Gentra).
Cells were stained in five steps: (1) rat anti-mouse CD34 antibody; (2) PE
Cy5.5-conjugated goat anti-rat antibody (Caltag); (3) biotin-conjugated
FCgRII/III antibody, APC-conjugated cKit antibody (eBioscience), and PE
Cy7-conjugated lineage markers antibodies (CD3, CD4, CD8, B220, CD19,
Ter119, and Gr1); (5) propidium iodide. Cells were blocked with rat immuno-
globulin between steps 2 and 3 and were washed with PBS (4ºC) between
each step. Lineage-negative cells (2 3 104) were analyzed. MEPs were de-
fined as lineage2, c-KIT+, Sca12, CD34low, and CD16/CD32low. Acquisi-
with Flowjo software.
Generation of JAK3A572V, JAK3V722I, and JAK3P132Tmutants
and MTS assay
A cDNA encoding the S isoform of human JAK3 was prepared from a pur-
chased clone encoding the M isoform of human JAK3 (Origene, Rockville,
MD) (Lai et al., 1995). The required C-terminal region for the S isoform of
JAK3 was amplified by RT-PCR using cDNA from the CMK cell line, then di-
rectionally inserted as a 1.0 kb SrfI-XbaI fragment. Following transfer of the
resulting JAK3 (S isoform) cDNA to pRC/CMV as a 4.7 kb NotI fragment,
the A572V, V722I, and P132T mutations were introduced using the GeneTai-
lor site-directed mutagenesis system (Invitrogen, Carlsbad, CA) and then
transferred into the MSCV-IRES-EGFP (MIG) vector. Full-length sequencing
confirmed the identities of the respective cDNAs. Primer sequences are as
RT-PCR, forward: 50-CGCTAGGCGACAATACAGGT-30, reverse: 50-ATAA
TTCTAGACACATATGCCCATCTG-30, mutagenesis (JAK3A572V) forward: 50-
GCATGGAGTCATTCCTGGAAGTAGCGAGCTTG-30, (JAK3A572V) reverse:
50-CTTCCAGGAATGACTCCATGCAGTTCTTGTG-30, (JAK3P132T) forward: 50-
GTGCTATCCTTGACCTGACAGTCCTGGAGCACCTC-30, (JAK3P132T) reverse:
50-GAGGTGCTCCAGGACTGTCAGGTCAAGGATAGCAC-30, (JAK3V722I) for-
ward: 50-GGGAAGTGTTTAGTGGCATCACCATGCCCATCAGT-30, (JAK3V722I)
reverse: 50-ACTGATGGGCATGGTGATGCCACTAAACACTTCCC-30. Ba/F3
cells were transduced with viral supernatants from 293T cells transfected
with either MIG-JAK3WT, MIG-JAK3A572V, MIG-JAK3V722I, or MIG-JAK3P132T. Cells
were selected for expression of GFP 24 hr after infection by cell sorting,
and GFP-positive cells were incubated in the absence of IL-3 for 5–12
days. Viable cell number was determined every 1–2 days by counting with
trypanblue.The MTSassaysusing JAKInhibitorIandsiRNAwereperformed
72 hr after treatment.
In vitro kinase assay
JAK3WTand JAK3A572Vwere immunoprecipitated using standard proce-
dures from Ba/F3 cells described above using a polyclonal JAK3 antibody
(Santa Cruz). Immunoprecipitates were incubated in kinase assay buffer
(Cell Signaling) with a JAK3-specific substrate (Upstate) according to manu-
Bone marrow transplant and animal analysis
Viral supernatants were obtained as previously described (Schwaller et al.,
1998). Five days prior to bone marrow procurement, 8- to 10-week-old
C57BL/6 (obtained from Taconic) mice were injected intraperitoneally with
150 mg/kg of 5-FU. On day 0, primary bone marrow cells were collected
from femurs and tibiae and cultured overnight in RPMI 1640 supplemented
with 10% FBS and IL-3, IL-6, and SCF. Cells were transduced with identical
titer viral supernatants on day 1 and day 2. Each time, cells were centrifuged
for 90 min at 2500 rpm at 30ºC in the presence of virus, returned to the incu-
bator for 3–4 hr, and resuspended into fresh media containing cytokines. Af-
buffer, and 1 3 106cells were injected in the tail veins of lethally irradiated
C57BL/6 mice. Eye-bleeds were obtained using EDTA-coated capillary
tubes, and blood counts were performed within 30 min on a HemaVet
eral blood, and lungs were harvested from transplantedmice, sectioned, and
stained with hematoxylin and eosin. Histopathology was analyzed by light
microscopy. Spleen and liver sections were stained by standard immunohis-
tochemistry procedures with an antibody specific for von Willebrand factor
(Dako Cytomation). Approval for the use of animals in this study was granted
bythe Children’s Hospital BostonAnimal Care andUse Committeeunder the
protocol number A04-03-029.
Megakaryocyte colony-forming assay and ploidy analysis
For megakaryocyte colony-forming assays, primary spleen cells were col-
lected from transplanted animals and subsequently treated with red blood
cell lysis buffer (Puregene, Gentra systems). Cells (1 3 105) were mixed
with MegaCult-C (StemCell Technologies), containing Tpo, IL-11, IL-3, and
IL-6 and plated onto two double chamber culture slides, and colonies were
stained for acetylcholinesterase and enumerated after 8 days. For ploidy
analysis, bone marrow cells from transplanted animals were cultured
4 days in RPMI 1640 supplemented with 10% FBS, 10 ng/ml mouse SCF,
and 10 ng/ml mouse Tpo prior to analysis. Cells were first stained with
CD41 antibody, followed by staining with APC-conjugated secondary anti-
body. Cells were then incubated 30 min in a 0.1% sodium citrate solution
containing 50 mg/ml of RNase A and 50 mg/ml of propidium iodide. Analysis
of ploidy was performed on CD41+ cells.
Model of JAK3 JH2 domain structure
The Swiss-Model automated comparative protein modeling server (http://
swissmodel.expasy.org/) was used to create an energy-minimized model
oftheJH2domain ofJAK3(Schwede etal., 2003).The sequence forresidues
Q501 to Q812 of JAK3 was submitted to Swiss-Model for a first approach
mode search using the Protein Data Bank (http://www.pdb.org/) deposited
structures for epidermal growth factor receptor (Stamos et al., 2002), insulin
receptor (Parang et al., 2001), Zap-70 (Jin et al., 2004), and Syk (Atwell et al.,
2004) tyrosine kinases (Protein Data Bank accession codes 1M17, 1GAG,
1U59, and 1XBC, respectively) as three-dimensional templates (sequence
identity 28%, 28%, 33%, and 33%, respectively). An energy-minimized
model of the JH2 domain of JAK3 was determined and manually checked
for global reasonableness. The locations of A572 and V722 in this model cor-
the equivalent residues. Further modeling runs using autoinhibited tyrosine
kinase domain crystal structures as templates confirmed the location of
these residues (data not shown).
Survival curves were analyzed for significance by the log-rank test. All other
statistical analyses were performed using paired Student’s t test. Values
were considered statistically significant when p < 0.05.
The Supplemental Data include two supplemental figures and two supple-
mental tables and can be found with this article online at http://www.
A R T I C L E
CANCER CELL JULY 2006 73
This study was supported by Howard Hughes Medical Institute (D.G.G. and
B.J.D.), a grant from the Doris Duke Charitable Foundation (D.G.G. and
B.J.D.), the Leukemia and Lymphoma Society (D.G.G. and B.J.D.), the NIH
(D.G.G.), and a Veterans Affairs Merit Review Grant (M.C.H.). Funding was
also provided by Cell Signaling for the authors at Cell Signaling. T.M. is a re-
J.W.T. is supported byan OHSU NIH Cancer Biology Training Grant. T.J.B. is
supported by an American Society of Hematology Basic Research Scholar
Award. T.-L.G., V.L.G., K.A.L., J.N., and R.D.P. are employees of Cell Signal-
Received: February 28, 2006
Revised: April 21, 2006
Accepted: June 1, 2006
Published: July 17, 2006
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