The Journal of Experimental Medicine
JEM © The Rockefeller University Press $30.00
Vol. 205, No. 4, April 14, 2008 751-758 www.jem.org/cgi/doi/
BRIEF DEFINITIVE REPORT
Acute lymphoblastic leukemia (ALL) comprises
a biologically heterogeneous group of clonal
disorders that originate from the uncontrolled
proliferation and expansion of immature lym-
phoblastic cells and are characterized by an ex-
tremely variable clinical outcome ( 1, 2 ). In recent
years, substantial progress has been made toward
understanding the molecular events contributing
to malignant transformation. This has permitted
the recognition of relevant prognostic factors
and risk stratifi cation, and has favored the im-
plementation of therapeutic approaches based on
cytogenetic and molecular lesions ( 2 – 5 ). Despite
Abbreviations used: ALL, acute
lymphoblastic leukemia; B-ALL,
B cell precursor ALL; CM,
conditional medium; DFS,
disease-free survival; DHPLC,
denaturing HPLC; ERK, extra-
cellular signal-regulated kinase;
OS, overall survival; T-ALL,
T cell ALL.
E. Flex and V. Petrangeli contributed equally to this paper.
The online version of this article contains supplemental material.
Somatically acquired JAK1 mutations
in adult acute lymphoblastic leukemia
Elisabetta Flex , 1 Valentina Petrangeli , 1 Lorenzo Stella , 3 Sabina Chiaretti , 4
Tekla Hornakova , 5 Laurent Knoops , 5 Cristina Ariola , 4 Valentina Fodale , 1
Emmanuelle Clappier , 6 Francesca Paoloni , 7 Simone Martinelli , 1
Alessandra Fragale , 2 Massimo Sanchez , 1 Simona Tavolaro , 4
Monica Messina , 4 Giovanni Cazzaniga , 8 Andrea Camera , 9
Giovanni Pizzolo , 10 Assunta Tornesello , 11 Marco Vignetti , 4
Angela Battistini , 2 H é l è ne Cav é , 6 Bruce D. Gelb , 12
Jean-Christophe Renauld , 5 Andrea Biondi , 8 Stefan N. Constantinescu , 5
Robin Fo à , 4 and Marco Tartaglia 1
1 Dipartimento di Biologia Cellulare e Neuroscienze, 2 Dipartimento di Malattie Infettive, Parassitarie e Immunomediate,
Istituto Superiore di Sanit à , Rome, 00161, Italy
3 Dipartimento di Scienze e Tecnologie Chimiche, Universit à “ Tor Vergata, ” Rome, 00133, Italy
4 Dipartimento di Biotecnologie Cellulari ed Ematologia, Universit à “ La Sapienza, ” Rome, 00161, Italy
5 Ludwig Institute for Cancer Research and de Duve Institute, Universit é Catholique de Louvain, Bruxelles, B1200, Belgium
6 D é partement de G é n é tique, H ô pital Robert Debr é , Paris, 75019, France
7 Gruppo Italiano Malattie Ematologiche dell ’ Adulto (GIMEMA) Data Center, GIMEMA Foundation, Rome, 00161, Italy
8 Centro Ricerca M. Tettamanti, Clinica Pediatrica Universit à di Milano Bicocca, Monza, 20052, Italy
9 Dipartimento di Medicina Clinica e Sperimentale, Universit à “ Federico II, ” Napoli, 80131, Italy
10 Divisione di Ematologia, Universit à degli Studi di Verona, Verona, 37100, Italy
11 Divisione di Oncologia Pediatrica, Universit à Cattolica del Sacro Cuore, Rome, 00100, Italy
12 Departments of Pediatrics and Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY 10029
Aberrant signal transduction contributes substantially to leukemogenesis. The Janus kinase 1
( JAK1 ) gene encodes a cytoplasmic tyrosine kinase that noncovalently associates with a
variety of cytokine receptors and plays a nonredundant role in lymphoid cell precursor
proliferation, survival, and differentiation. We report that somatic mutations in JAK1 occur
in individuals with acute lymphoblastic leukemia (ALL). JAK1 mutations were more preva-
lent among adult subjects with the T cell precursor ALL, where they accounted for 18% of
cases, and were associated with advanced age at diagnosis, poor response to therapy, and
overall prognosis. All mutations were missense, and some were predicted to destabilize
interdomain interactions controlling the activity of the kinase. Three mutations that were
studied promoted JAK1 gain of function and conferred interleukin (IL)-3 – independent
growth in Ba/F3 cells and/or IL-9 – independent resistance to dexamethasone-induced
apoptosis in T cell lymphoma BW5147 cells. Such effects were associated with variably
enhanced activation of multiple downstream signaling pathways. Leukemic cells with
mutated JAK1 alleles shared a gene expression signature characterized by transcriptional
up-regulation of genes positively controlled by JAK signaling. Our fi ndings implicate dys-
regulated JAK1 function in ALL, particularly of T cell origin, and point to this kinase as a
target for the development of novel antileukemic drugs.
SOMATIC JAK1 MUTATIONS IN ALL | Flex et al.
( Fig. 1 A and Table I ). To verify that the nonsynonymous
substitutions identifi ed in patients for whom nonleukemic
DNA was not available were not gene variants occurring in
the population, 335 population-matching control individuals
were analyzed, and none harbored the 611A > T (Lys204Met),
2635C > A (Arg879Ser), 2635C > T (Arg879Cys), and 2636G > A
(Arg879His) changes or other defects altering those codons.
Although the T-ALL – restricted occurrence of three distinct
substitutions aff ecting Arg 879 (3/38 versus 0/335; Fisher ’ s
exact probability < 0.001) further supported the relevance
of the substitution of this residue, we could not exclude that
the 611A > T change might represent a private neutral variant.
The two remaining missense changes, 184A > G (Ile62Val) and
1078C > T (Arg360Trp), were deemed nonpathogenic vari-
ants, as they were observed in nonleukemic cells of aff ected
patients or in unaff ected control subjects.
All mutations occurred as heterozygous changes and af-
fected conserved residues within the FERM, SH2, pseudoki-
nase, and kinase domains ( Fig. 1 B ). In two cases, DHPLC
profi les and electropherograms indicated that the mutant al-
lele might be present in only a fraction of leukemic cells,
suggesting that these lesions did not represent early events
during leukemogenesis but were acquired during disease pro-
gression ( Fig. 1 C ). Remarkably, mutations were relatively
common among individuals with T-ALL (18.4% of cases,
95% CI = 7.7 – 34.3%), whereas they occurred in only three
patients with B-ALL (3.4% of cases, 95% CI = 0.7 – 9.6%;
Table I ). Such a diff erence in mutation prevalence between
cohorts was statistically signifi cant (Fisher ’ s exact probability =
0.003). DHPLC screening performed on aff ected exons by
using pooled DNAs excluded loss of the normal allele and a
homozygous condition for a gene variant caused by mitotic
recombination in all cases.
To investigate the prevalence of JAK1 mutations among
pediatric ALL cases, genomic DNA from BM obtained at di-
agnosis was scanned for mutations in aff ected exons. No le-
sion was observed within the B-ALL cohort ( n = 85), whereas
a nonsynonymous 1957C > T transition (Leu653Phe) was
identifi ed in 1 of 49 subjects with T-ALL (2.0% of cases, 95%
CI = 0.05 – 10.9%). This mutation was not observed in the
BM obtained from the patient at the time of remission, indi-
cating that it was somatically acquired in the leukemic cells.
Overall, these results indicated that JAK1 gene mutations
occur in ALL and are more frequently observed among adult
individuals with involvement of the T cell lineage.
Functional consequences of somatic JAK1 mutations
To examine the eff ects of the identifi ed mutations on protein
function, WT JAK1 or a mutant form (A634D, R724H, and
R879C) was expressed transiently in JAK1-defective human
fi brosarcoma U4C cells, and endogenous STAT1 phosphory-
lation was compared basally and after stimulation with IFN- ?
( Fig. 2 A ). Consistent with previous studies ( 10, 11 ), untrans-
fected cells lacking functional JAK1 did not exhibit STAT1
phosphorylation in response to IFN- ? . All JAK1 mutants
promoted an enhanced response to the ligand compared with
these accomplishments, long-term survival in adults with ALL
remains largely unsatisfactory, making the design of novel an-
tileukemic drugs tailored to specifi c biological targets a current
priority ( 4, 6 ).
The four mammalian members of the JAK family (JAK1,
JAK2, JAK3, and TYK2) are nonreceptor tyrosine kinases
functioning as signal transducers to control cellular prolifera-
tion, survival, and diff erentiation ( 7, 8 ). JAK proteins associ-
ate constitutively with a variety of cytokine receptors lacking
intrinsic kinase activity, and promote signal fl ow by phos-
phorylating tyrosyl residues of activated receptors to allow the
recruitment and activation of STAT proteins. They share a
complex multidomain structure characterized by a tyrosine
kinase domain at the C terminus, which is fl anked by a catalyti-
cally inactive pseudokinase domain with regulatory function.
Their N-terminal half contains a FERM homology domain,
which is implicated in receptor binding and possibly regulates
the catalytic activity of the kinase, and an adjacent SH2-like
domain. In contrast to their conserved structure, increasing
experimental data indicate that JAK family members prefer-
entially associate with a diverse subset of cytokine receptors,
each diff erentially expressed by individual cell lineages and
tissues, facilitating specifi city in function ( 9 ). Among them,
JAK1 plays an essential and nonredundant role in mediat-
ing biological responses induced by a specifi c subgroup of
cytokines controlling lymphoid cell precursor development
( 10 ). Jak1 ? / ? mouse pups exhibit a thymus that is markedly
reduced in size, which is associated with a severe decrease in
cellularity. Jak1 loss of function is also associated with pro-
found abnormalities in the B cell compartment caused by a
block in diff erentiation at the pro – B/pre – B cell transition
step, resulting in a defi cit in the production of mature B lym-
phocytes ( 10 ). Based on its crucial role in lymphocyte pro-
liferation, survival, and diff erentiation, we hypothesized that
up-regulation of JAK1 signaling might contribute to malig-
nancies of the lymphoid lineage. In this study, we report that
somatic activating JAK1 mutations occur among adults with
T cell precursors ALL, and are associated with poor response
to therapy and overall prognosis.
RESULTS AND DISCUSSION
JAK1 mutation analysis in ALL
To explore possible contributions of somatic JAK1 gene mu-
tations in ALL, genomic DNA samples from BM aspirates of
adult subjects with B cell precursor ALL (B-ALL; n = 88) and
T cell ALL (T-ALL; n = 38) obtained at diagnosis and before
therapy were screened for mutations in the entire JAK1 coding
region using denaturing HPLC (DHPLC). Direct sequencing
of variant elution profi les allowed the identifi cation of 37 in-
tronic and exonic changes, including 9 nonsynonymous vari-
ants observed in 14 individuals ( Table I and Table S1, available
Among the missense defects, genotyping of genomic DNAs
from BM obtained during remission demonstrated the somatic
origin of the 1535C > T (Ser512Leu), 1901C > A (Ala634Asp),
and 2171G > A (Arg724His) changes in the leukemic clones
JEM VOL. 205, April 14, 2008
BRIEF DEFINITIVE REPORT
Jak1 protein was observed in cultures maintained in the
presence of IL-3 ( Fig. 2 D ). Notably, we did not observe
Jak1 phosphorylation in parental and transduced Ba/F3 cells
basally and cultured in 2% WEHI-3B cell CM; however,
phosphorylation was appreciable in A634D and R724H Jak1-
transduced cells after selection (7-d culture in absence of IL-3;
IL-9 protects the T cell lymphoma BW5147 cell line
against dexamethasone-induced apoptosis ( 12 ), an eff ect that
is dependent on Jak1-mediated Stat3 and Stat5 activation ( 13 ).
To demonstrate the activating role of the ALL-associated
JAK1 mutations in a diff erent cellular system, BW5147 cells
were transduced with WT Jak1 or each of the selected
mutants to evaluate their eff ect on this stress response. GFP-
expressing cells were isolated by fl ow cytometry, cultured in
the presence of cyclosporine A and dexamethasone, with or
without IL-9, and [ 3 H]thymidine incorporation was deter-
mined to assess proliferation ( Fig. 2 E ). Three independent
experiments documented that expression of the A634D and
R879C Jak1 mutants, but not R724H Jak1, conferred in-
creased growth to cells basally. Of note, whereas transduced
cells exhibited comparable responses to high levels of IL-9,
those expressing the A634D and R879C Jak1 mutants were
more responsive to low levels of the cytokine. Expression of
the A634D Jak1, but not that of the R879C mutant, was as-
sociated with enhanced phosphorylation of Jak1, Stat3, and
Stat5 basally (unpublished data).
WT JAK1. Of note, basal STAT1 phosphorylation was ob-
served in cells expressing the A634D mutant, suggesting
ligand-independent up-regulation of the kinase. Consistent
with this, expression of the A634D mutant resulted in an
essentially constitutive STAT1 transcriptional activation,
whereas a statistically signifi cant increase in STAT1 activity
was observed in U4C cells expressing both the R724H and
R879C mutants, basally and after stimulation ( Fig. 2 B ).
To further assess the ability of mutations to up-regulate
signal fl ow, we transduced Ba/F3 cells with WT Jak1 or each
of the selected mutants to evaluate whether their expression
induced autonomous growth of cytokine-dependent cells.
GFP-expressing Ba/F3 cells were selected by fl ow cytometry,
cultured in 5 or 0.5% WEHI-3B cell conditional medium
(CM) as a source of IL-3, as well as in absence of the cytokine,
and counted to assess proliferation ( Fig. 2 C ). Three indepen-
dent experiments indicated that expression of the A634D and,
with less effi ciency, R724H Jak1 mutants conferred IL-3 –
independent growth to cells, whereas cells expressing WT Jak1
or the R879C Jak1 mutant retained dependence on the cyto-
kine for survival. Of note, cells expressing each of the three
mutants exhibited enhanced growth in response to IL-3.
Consistent with these fi ndings, Ba/F3 cells expressing the
A634D Jak1 mutant exhibited enhanced Stat5, Akt, and ex-
tracellular signal-regulated kinase (ERK) phosphorylation ba-
sally and after stimulation, whereas a higher phosphorylation
level of these signal transducers in cells expressing the R724H
Table I. List of nonsynonymous JAK1 changes identifi ed in subjects with ALL
Cohort and lineageNumber of
Exon Amino acid
184A > G
611A > T
1901C > A
2171G > A
184A > G
1078C > T
1535C > T
1901C > A
2171G > A
2635C > A
2635C > T
2636G > A
11957C > T 13Leu653Phe Pseudokinase Mutation d
a This change was observed in unaffected individuals.
b This change was not present among 335 population-matching control individuals.
c This change was present at remission.
d This change was not present at remission.
e This change was not present at remission in the one individual analyzed.
f This subject carried a concomitant 2171G → A change.
SOMATIC JAK1 MUTATIONS IN ALL | Flex et al.
kinase domain at the interface with the kinase domain in JAK2
(Val 617 ) and JAK3 (Ala 572 ) promote increased catalytic activity
basally (Fig. S1, available at http://www.jem.org/cgi/content/
full/jem.20072182/DC1) ( 18 – 20 ). Our model also predicts
that residues Lys 204 and Ser 512 would perturb the SH2 – FERM
interdomain interaction because they are located at the inter-
face between these domains, approximately facing each other.
This fi nding is noteworthy because it has been proposed that
JAK1 ’ s SH2 domain does not function as a phosphotyrosyl-
binding domain, but instead plays a structural role in stabilizing
the conformation of the FERM domain ( 21 ), which mediates
its association to cytokine receptors and exerts an as yet unchar-
acterized restraint on catalytic function ( 22, 23 ). The molecular
mechanism through which these mutations aff ect JAK1 func-
tion remains to be explained. Structural and functional conse-
quences were not obvious for the activating changes aff ecting
residues Arg 724 and Arg 879 .
Gene expression profi le analysis in blasts with a mutated
Total RNA was available from blasts of 5 JAK1 mutation-
positive (S512L, A634D, and R724H) and 11 mutation-
negative subjects of the T-ALL cohort. Unsupervised cluster-
ing based on 1,345 probe sets selected by nonspecifi c fi ltering
clustered expression profi les of mutation-positive blasts into
two clusters, suggesting contribution of JAK1 mutations to
Overall, these data indicated that the three selected leuke-
mia-associated JAK1 mutants are hypermorphs, with A634D
Jak1 having a seemingly stronger eff ect, and that diff erent
mechanisms are likely to be involved in their cell context –
related gain of function.
Molecular modeling of JAK1 and location
of affected residues
To look at the structural causes resulting in JAK1 functional
up-regulation, we generated a model of JAK1 structure be-
cause no crystallographic information was available for this
protein. Energy-minimized models of each of the four do-
mains were generated separately by homology to available
crystallographic structures of proteins with similar sequences
and overall fold. The quaternary arrangement of the four do-
mains was then determined by superimposing the models on a
predicted three-dimensional structure of JAK2 ( 14 ). Accord-
ing to the superimposed structure, Ala 634 and Leu 653 are placed
on the surface of the pseudokinase domain involved in the in-
teraction with the kinase domain ( Fig. 1 B ). Based on the evi-
dence supporting a negative regulatory role of the pseudokinase
domain on catalytic function of JAK proteins ( 15 – 17 ), the
pathogenetic mechanism of the A634D and L653F changes is
predicted to involve a looser interdomain interaction, relaxing
inhibitory control on the kinase activity. Consistent with this
hypothesis, substitution of two residues located in the pseudo-
Figure 1. Somatic JAK1 mutations in ALL. (A) Representative electropherograms showing the occurrence of somatically acquired JAK1 mutations in
subjects with T-ALL. In all cases, mutations were observed at diagnosis (top), but were undetectable during remission (bottom). (B) JAK1 domain structure
and location of affected residues. The predicted amino acid substitutions resulting from the JAK1 mutations are positioned below the diagram of the
protein with its functional domains indicated (left) and shown in JAK1 three-dimensional modeled structure (right). (C) Electropherograms showing the
occurrence of mutations in a fraction of leukemic cells of two individuals with T-ALL. In both patients, the mutant allele constituted only a portion of the
amplifi ed fragment from BM obtained at diagnosis (blasts > 70% of total cells; top). The heterozygous status of each subject for an intragenic polymor-
phic site (bottom) is shown for comparison.
JEM VOL. 205, April 14, 2008
BRIEF DEFINITIVE REPORT
known to be positively modulated by JAK/STAT signaling,
including IRF1 , SOCS3 , ISG15 , ISGF3G , IFI44L , and
IRF7 , were overrepresented in all the JAK1 mutation-positive
subjects, further supporting the gain-of-function role of the
ALL-associated JAK1 lesions.
Clinical relevance of somatic JAK1 mutations
The clinical relevance of JAK1 mutations within the adult
T-ALL cohort was investigated. Although no statistically
distinct major mechanisms of deregulation (Fig. S2, available at
Notably, supervised gene expression analysis revealed a dis-
tinctive expression signature shared by leukemic cells with a
mutated JAK1 gene ( Fig. 3 A ) based on the expression of
133 diff erentially expressed probe sets, consisting of 112 dif-
ferentially expressed genes, the majority of them being over-
expressed in JAK1 mutation-positive samples (Table S2).
Among the up-regulated genes, those whose transcription was
Figure 2. Functional effects of leukemia-associated JAK1 mutations. (A) STAT1 phosphorylation assays. Basal and IFN- ? – stimulated endogenous
STAT1 phosphorylation in JAK1-defective U4C cells transiently transfected with WT JAK1 or selected mutants. Blots are representative of three experi-
ments. (B) STAT1 activation assays. Basal and IFN- ? – stimulated endogenous STAT1 transcriptional activity in JAK1-defective U4C cells transiently cotrans-
fected with p-GAS-Luc and phRL-TK constructs, and WT JAK1 or a mutant allele. STAT1-induced luciferase gene expression levels were determined by
measuring the luciferase activity (CPS, counts per second) normalized to the activity of the Renilla luciferase, using a dual-luciferase reporter assay sys-
tem. Activity ratios are expressed as the mean of three replicates ± the SD. (C) Ba/F3 survival assays. WT or mutant Jak1 -transduced Ba/F3 cells were
grown in the absence of IL-3 (top) or with 0.5 or 5% WEHI-3B cell CM as source of IL-3 (bottom). Cell numbers (mean of three replicates ± the SD) were
counted at the indicated time points (top) or at day 3 of culture (bottom). (D) Stat5, Akt, and ERK phosphorylation assays. Endogenous Stat5 Tyr694, Akt
Ser473, and ERK1/2 Thr202/Tyr204 phosphorylation levels from lysates of Ba/F3 cells transduced with WT Jak1 or a mutant form and cultured without
IL-3 (left) or with 2% WEHI-3B cell CM as the source of IL-3 (right). Activation of Stat5 (pStat5/Stat5), AKT (pAkt/Akt), and ERK1/2 (pERK/ERK) is expressed
as a multiple of activation in untransduced cells. Blots are representative of at least three experiments performed. (E) BW5147 proliferation assays. WT or
mutant Jak1 transduced BW5147 cells were grown in presence of 200 μ g/ml dexamethasone and 50 μ g/ml cyclosporine A, in absence (left) or with differ-
ent concentrations (right) of IL-9. After 72 h, proliferation was measured after [ 3 H]thymidine was added to the cultures (6 h). Data are shown as the
means of three replicates ± the SD. For convenience, the amino acid changes affecting residues Ala 633 , Arg 723 , and Arg 878 of the murine Jak1 protein
(encoded by the pMX-Jak1-IRES-GFP constructs used to transduce the Ba/F3 and BW5147 cell lines) are indicated according to the homologous residues
in human JAK1.
SOMATIC JAK1 MUTATIONS IN ALL | Flex et al.
signifi cance of these associations (DFS: HR= 6.20, 95% CI =
1.32 – 29.09, P = 0.02; OS: HR = 2.82, 95% CI = 1.07 – 7.48,
P = 0.04), and excluded a signifi cant contribution of patients ’
age (DFS: HR = 0.99, 95% CI = 0.93 – 1.05, P = 0.64; OS:
HR= 1.01, 95% CI = 0.97 – 1.05, P = 0.60).
In the adult T-ALL cohort, the mutation status for NRAS ,
KRAS , NOTCH1 , and PTEN was also assessed (unpub-
lished data). Among the JAK1 mutation-positive cases, no
defect was observed in the NRAS , KRAS , and PTEN genes,
although such mutations had a relatively low prevalence in
the entire adult T-ALL cohort ( RAS genes: 11% of cases,
95% CI = 2.9 – 24.8%; PTEN : 14% of cases, 95% CI = 4.5 –
28.8%). In contrast, heterozygous mutations in NOTCH1
were observed in all JAK1 mutation-positive individuals.
Given the high prevalence of NOTCH1 defects observed
in the cohort (70% of cases, 95% CI = 53.0 – 84.1%), this
association was not statistically signifi cant (P = 0.06). This
observation, however, suggests that activation of JAK1 and
NOTCH1 transduction pathways might cooperate in T-ALL
pathogenesis and/or progression. No signifi cant diff erence
in response to therapy or outcome was observed between
NOTCH1 mutation-positive and -negative patients. Inter-
estingly, NOTCH1 and PTEN mutations exhibited a mutu-
ally exclusive distribution because none of the fi ve subjects
carrying PTEN lesions had a NOTCH1 defect (Fisher ’ s exact
probability = 0.001).
Aberrant JAK1 function and leukemogenesis
Functional up-regulation of two members of the JAK fam-
ily, JAK2 and JAK3, has recently been discovered in myelo-
proliferative disorders and other malignancies of the myeloid
lineage ( 18 – 20, 24 ). The JAK2 V617F amino acid change
occurs in the majority of polycythemia vera cases and in
? 50% of individuals with essential thrombocythemia or
idiopathic myelofi brosis. The available data support the
view that this recurrent change, which aff ects the pseudoki-
nase domain of the protein, induces constitutive activation
of the kinase and hypersensitivity to cytokines. Similarly,
three JAK3 hypermorphic alleles promoting cytokine inde-
pendence in Ba/F3 cells have been identifi ed in acute mega-
karyoblastic myeloid leukemia. In this study, we showed that
somatically acquired activating JAK1 mutations occur in
ALL, particularly in adults, further emphasizing the impor-
tance of JAK-mediated signaling dysregulation in leukemo-
genesis and extending the spectrum of hematologic malignancies
associated with aberrant activation of this signal transduc-
tion pathway. Even though the molecular mechanisms by
which individual JAK1 mutations promote gain of function
are likely to be diverse and remain to be fully characterized,
modeling and biochemical data are consistent with the view
that, similar to what has been observed for somatic leuke-
mia-associated JAK2 and JAK3 defects, most mutations
would interfere with the autoinhibitory control on the cat-
alytic activity. For most mutations, this eff ect would be
achieved by triggering local structural rearrangements in re-
gions involved in interdomain interactions between the
signifi cant diff erence was observed in white blood cell counts,
gender distribution, or association with specifi c chromosomal
rearrangements, patients with a mutated JAK1 allele tended
to have a more advanced age at diagnosis (median = 40.6 vs.
24.2; P < 0.01), which was consistent with the lower preva-
lence of mutations identifi ed among children and adolescents
with T-ALL included in the study. Comparison of the
response to therapy between JAK1 mutation-positive and
-negative patients indicated a higher percentage of cases
exhibiting resistance to induction therapy in the former
(43 vs. 20%), although this diff erence did not reach statistical
signifi cance caused by the relatively small size of the study
cohort. Consistent with that fi nding, a statistically signifi cant
reduced disease-free survival (DFS; median =8.7 vs. 20.5 mo;
P = 0.01) and overall survival (OS; median = 10.6 vs. 32.5;
P < 0.01) was observed among JAK1 mutation-positive pa-
tients ( Fig. 3 B ). Multivariate analysis confi rmed the statistical
Figure 3. Gene expression profi les and clinical relevance of
somatic JAK1 mutations in adult T-ALL. (A) Supervised hierarchical
clustering of gene expression profi les performed on blasts from 16 adult
T-ALL patients, with (orange) or without (green) a JAK1 mutation.
(B) Kaplan-Meier estimates of DFS (top) and OS (bottom) in subjects with
(red) or without (black) a JAK mutation. Multivariate analysis confi rmed
the statistical signifi cance of the reduced DFS and OV among JAK1 mutation-
positive patients, and excluded a signifi cant contribution of the more
advanced age of these subjects.
JEM VOL. 205, April 14, 2008
BRIEF DEFINITIVE REPORT
tagged JAK1 cDNA cloned in pRc/CMV vector provided by S. Pellegrini
(Pasteur Institute, Paris, France). JAK1-defective human fi brosarcoma U4C
cells (provided by S. Pellegrini) were maintained in DME supplemented
with 10% heat-inactivated FCS and antibiotics. To evaluate endogenous
STAT1 phosphorylation, after starvation (24 h), transiently transfected cells
were stimulated with IFN- ? (1,000 U/ml, 15 min) or left unstimulated.
Lysed samples were analyzed by 10% SDS-PAGE, transferred to a polyvi-
nylidene difl uoride membrane (Pierce Chemical Co.) and probed with anti –
phospho-Stat1 (9171; Cell Signaling Technology), anti-Stat1 (9172; Cell
Signaling Technology), and anti-Jak1 (3332; Cell Signaling Technology)
antibodies. Endogenous STAT1 transcriptional activity was assessed by lucif-
erase transactivation assays in cells cotransfected with a p-GAS-Luc con-
struct, switched to serum-starvation medium (8 h), and then stimulated with
IFN- ? (1,000 U/ml, 16 h) or left unstimulated. STAT1-induced luciferase
expression was assessed and normalized using a dual luciferase reporter assay
system (Promega) and a phRL-TK plasmid constitutively expressing the Re-
Each of the three leukemia-associated mutations was also introduced
in the murine Jak1 cDNA cloned in the bicistronic retroviral vector pMX-
IRES-GFP. Constructs were transfected into Phoenix or BOSC packag-
ing cells to produce retroviruses, and murine Ba/F3 (maintained in RPMI
1640 medium containing 10% FCS, 1% l-glutamine, and 10% WEHI-3B
cell CM) and BW5147 (maintained in Iscove-Dulbecco’s medium supple-
mented with 10% FBS, 0.55 mM l-arginine, 0.24 mM l-asparagine, and
1.25 mM l-glutamine) cells were infected with retroviral supernatants. GFP-
positive populations were purifi ed by fl ow-cytometric sorting, and then ex-
panded. Equal GFP expression levels of transduced cells were confi rmed by
FACS analysis (Fig. S3, available at http://www.jem.org/cgi/content/full/
jem.20072182/DC1). Transduced Ba/F3 cells were cultured in the absence
or presence of IL-3 (0.5 or 5% WEHI-3B cell CM) for assaying cytokine
independence and response, and viable cells were counted by Trypan blue
exclusion. Proliferation of transduced BW5147 cells cultured in the presence
of 50 μg/ml cyclosporine A and 200 μg/ml dexamethasone, in the presence
or absence of mIL-9, was determined using [3H]thymidine incorporation
assay. In brief, 3,000 cells were seeded in 96-well plates (200 μl volume
medium), and after 72 h, [3H]thymidine was added to the cultures for 6 h.
Cells were then collected on microfi lter plates, and thymidine incorporation
was measured after addition of 25 μl of liquid scintillant. For Western blot
studies, Ba/F3 cells were starved in RPMI 1640 medium containing 1%
BSA (5 h), and then stimulated with 2% WEHI-3B cell CM (30 min) or
left unstimulated, whereas BW5417 cells were starved in Iscove-Dulbecco’s
medium (12 h) and left unstimulated or stimulated with 100 U/ml mIL-9 for
15 min. Evaluation of Jak1, Akt, Stat3, Stat5, and ERK1/2 expression and
phosphorylation levels was performed using anti–phospho-Jak1, anti-Jak1,
anti–phospho-Akt, anti-Akt, anti–phospho-Stat3, anti-Stat3, anti–phospho-
Stat5, anti-Stat5, anti–phospho-p44/42 ERK, and anti-p44/42 ERK anti-
bodies (all from Cell Signaling Technology).
Structural models of the kinase, pseudokinase, and SH2 and FERM do-
mains of JAK1 were generated as described in the online supplemental infor-
Statistical analysis. Confidence intervals of proportions (at 95% level)
were calculated based on the binomial distribution. The probabilities of
OS and DFS were estimated using the Kaplan-Meier method. The log-
rank test was used to compare treatment effect and risk factor cate-
gories. All tests were two-sided, accepting P ≤ 0.05 as indicating a
statistically significant difference. Cox proportional hazard models, in-
cluding age and JAK1 as variables, were used to perform multivariate
analyses for OS and DFS. The SAS software (SAS Institute) was used
for the analysis.
Online supplemental material. Methodologies and approaches used to
generate the JAK1 molecular modeling and to perform and analyze adult
T-ALL leukemic cell gene expression profi les (including cited references) are
provided in the Supplemental Materials and methods. Table S1 is a list of the
pseudokinase and kinase domains or the FERM and SH2
domains (Fig. S1).
JAK1 is expressed widely and participates in intracellular
signaling elicited by class II cytokine receptors and receptors
that use the gp130 or ? c receptor subunit. Although the he-
matopoietic defects in Jak1 ? / ? mice were restricted to the
lymphoid cell compartment as a result of an impaired re-
sponse to IL-7 ( 10 ) and the present fi ndings indicate a cell-
context dependence of somatically acquired JAK1 mutations ’
contribution to leukemogenesis, we cannot exclude the in-
volvement of this kinase in other malignancies. We speculate
that a concomitant genetic event, including a mutation af-
fecting other members of the JAK family, might synergize
with the JAK1 defect to promote aberrant cell proliferation
and/or survival in a cell-specifi c context. This hypothesis is
currently under investigation.
Although 70 – 80% of pediatric patients with either B- or
T-ALL have excellent long-term response to intensive
combination chemotherapy, adult patients exhibit a less fa-
vorable outcome ( 4, 25 ). In B- ALL, such a poor prognosis
has been associated in part with the presence of BCR / ABL
or ALL1 / AF4 gene rearrangements. In contrast, the unfa-
vorable outcome of adult patients with T-ALL has not con-
clusively been attributed to any cytogenetic lesion, albeit
the prognostic relevance of aberrant ERG and TLX1 gene
expression and NOTCH1 mutations has recently been re-
ported ( 26 – 28 ). The present work provides the fi rst evidence
that JAK1 gene defects are associated with a poor response
to therapy, frequent relapse of the disease, and reduced OS,
identifying such mutations as a novel informative prognos-
tic marker occurring in a sizable proportion of adult T-ALL.
Although studies on larger cohorts are required to deter-
mine more precisely the clinical relevance and prognostic
value of JAK1 defects in adult and pediatric ALL, our fi nd-
ings provide a rationale for the development of novel thera-
peutic approaches tailored at interfering with JAK1 signaling,
encouraging studies aimed at testing the effi cacy and side
eff ects of JAK1 inhibitors in the management of adult
T-ALL patients .
MATERIALS AND METHODS
All cohorts and molecular analyses. Cohorts studied included patients
enrolled in the Gruppo Italiano Malattie Ematologiche dell ’ Adulto 0496 and
2000 (adults), and Associazione italiana ematologia ed oncologia pediatrica
ALL 2000 (children and adolescents) clinical trials. Written informed con-
sent for genetic analyses was obtained from all subjects according to the
Declaration of Helsinki. BM mononuclear cells were isolated by density
gradient centrifugation, and then cryopreserved. Genomic DNA was iso-
lated in a standard fashion. Exons 26, 27, and 34 of the NOTCH1 gene were
analyzed by direct sequencing, whereas the entire JAK1 and PTEN coding
regions and exons 1 and 2 of NRAS and KRAS were screened by DHPLC
(Wave 2100 System; Transgenomic) and sequencing. Primer sequences are
available upon request. Gene expression profi ling methodology is described
in the Supplemental materials and methods (available at http://www.jem
Functional studies. The mutations resulting in the A634D, R724H, and
R879C changes were introduced by site-directed mutagenesis in a VSV-
758 Download full-text
SOMATIC JAK1 MUTATIONS IN ALL | Flex et al.
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intronic or synonymous JAK1 changes identifi ed in the study. Table S2 is a
list of diff erentially expressed genes in JAK1 mutation-positive versus muta-
tion-negative adult subjects with T-ALL. Fig. S1 shows the JAK1 domain
structure and the location of JAK1, JAK2, and JAK3 residues reported to be
mutated in myeloproliferative disorders and leukemias. Fig. S2 shows the
unsupervised hierarchical clustering of gene expression profi les of blasts from
16 adult T-ALL patients, with or without a JAK1 mutation. Fig. S3 shows
the GFP expression levels of purifi ed Ba/F3 and BW5417 cells transduced
with a bicistronic retroviral vector pMX-Jak1-IRES-GFP coding for WT
Jak1 or one of the three generated mutants. The online version of this article
is available at http://www.jem.org/cgi/content/full/jem.20072182/DC1.
We are indebted to the patients who participated in the study and physicians of
the GIMEMA network who referred the subjects and provided BM specimens for
the study. Thanks are due to Sandra Pellegrini (Pasteur Institute, Paris, France)
for providing the JAK1-defective U4C cell line and the construct encoding the
human VSV-tagged JAK1 protein, and Romano Kroemer (Sanofi -Aventis, Centre de
Recherche de Paris, Vitry-sur-Seine, France) for making available the coordinates of
the JAK2 model.
This work was supported by grants from Ricerca Oncologica Project of
Integrated Program “ Multidimensional classifi cation of lymphohematopoietic
malignancies “ (to M. Tartaglia) and Associazione Italiana per la Ricerca sul Cancro
(AIRC; to R. Foa). EF is supported by a fellowship from Associazione ONLUS “ Morgan
Di Gianvittorio per la cura e la ricerca nei tumori e leucemie in et à pediatrica. ”
The authors declare no competing fi nancial interests.
Submitted: 10 October 2007
Accepted: 4 March 2008
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