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Blood, Vol. 95 No. 12 (June 15), 2000:
pp. 3891-3899
NEOPLASIA
TEL-JAK2 transgenic mice develop T-cell leukemia
Clémence Carron,
Françoise Cormier,
Anne Janin,
Virginie Lacronique,
Marco Giovannini,
Marie-Thérèse Daniel,
Olivier Bernard, and
Jacques Ghysdael
From the Centre National de la Recherche Scientifique (CNRS) UMR
146-Institut Curie, Centre Universitaire, Orsay, France; Service
Central d'Anatomie et de Cytologie Pathologiques and INSERM U462
Laboratoire Central d'Hématologie, Hopital St. Louis, Paris,
France; and INSERM U434-CEPH, Paris, France.
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Abstract |
We previously reported a fusion between TEL and JAK2
in a t(9;12)(p24;p13) chromosomal translocation in childhood acute
T-cell leukemia. This fusion gene encodes a TEL-JAK2 chimeric protein in which the 336 amino-terminal residues of TEL, including its specific
self-association domain, are fused to the kinase domain of JAK2.
TEL-JAK2 exhibits constitutive activation of its tyrosine kinase
activity which, in turn, confers growth factor-independent proliferation to the interleukin-3-dependent Ba/F3 hematopoietic cell
line. To elucidate the properties of TEL-JAK2 in primary cells and to
create an animal model for TEL-JAK2-induced leukemia, we generated
transgenic mice in which the TEL-JAK2 complementary DNA was placed
under the transcriptional control of the EµSR enhancer/promoter.
TEL-JAK2 founder mice and their transgenic progeny developed fatal
leukemia at 4 to 22 weeks of age. Selective amplification of
CD8-positive T cells was observed in blood, lymph nodes, thymus,
spleen, and bone marrow. Expression of a tyrosine-phosphorylated TEL-JAK2 protein and activation of STAT1 and STAT5 (signal transducer and activator of transcription) were detected in leukemic tissues. TEL-JAK2 diseased mice also displayed invasion of nonhematopoietic organs, including liver, brain, lung, and kidney, by leukemic T cells.
Leukemic organs of founder and transgenic progeny contained a
monoclonal/oligoclonal T-cell population as analyzed by the rearrangement of the TCR locus. Transplantation of TEL-JAK2 leukemic cells in nude mice confirmed their invasive nature. We conclude that
the TEL-JAK2 fusion is an oncogene in vivo and that its
expression in lymphoid cells results in the preferential expansion of
CD8-positive T cells.
(Blood. 2000;95:3891-3899)
© 2000 by The American Society of Hematology.
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Introduction |
Normal hematopoiesis is regulated through the
interaction of cytokine and growth factors with their cognate
receptors. Cytokine receptors are associated with and mediate
ligand-dependent activation of 1 or more of the Janus family of
tyrosine kinases.1 The JAK family is composed of 4 members
in mammals (JAK1, JAK2, JAK3, and TYK2) that are specifically activated
in response to different cytokines. JAKs share regions of homology
designated as JH (Jak homology) segments. Seven JH regions (JH1-JH7)
are described, but with the exception of the JH1 catalytic domain, the
precise function of these regions remains poorly
understood.1 Cytokine-induced activation of JAK kinases
results in the phosphorylation of a number of tyrosine residues
both in the JAK kinase itself and in the cytoplasmic domain of the
associated cytokine receptor. These phosphorylated tyrosine and
adjacent residues serve as docking sites for a variety of intracellular
signaling adaptors and effectors, including specific members of the
STAT (signal transducer and activator of transcription) family of
transcriptional regulators. Recruitment of STATs to the receptor-JAK
signaling complex results in their tyrosine phosphorylation, which
leads to their dimerization, their migration to the nucleus, and their
binding to specific response elements in the promoter region of target
genes.1
JAK2 is the predominant JAK kinase activated in response to interleukin
(IL)-3, granulocyte-macrophage colony-stimulating factor
(GM-CSF), erythropoietin (EPO), thrombopoietin (TPO), IL-5, growth
hormone, and prolactin and is also activated along with another JAK
family member in response to G-CSF, IL-6 and related cytokines, and interferon- .1 JAK2 is essential to
cytokine receptor signaling since its functional inactivation by
homologous recombination suppresses the response of fetal liver cells
to EPO, TPO, IL-3, and GM-CSF and abolishes definitive mouse
erythropoiesis.2,3
Several lines of evidence suggest a potential role of the deregulation
of JAK-STAT pathways in hematologic disorders.4 In
Drosophila, gain of function mutations in the hopscotch locus, which encodes a JAK homologue, results in D-STAT activation and in a
leukemia-like defect.5,6 In mammals, several reports have
demonstrated the constitutive activation of JAKs and STATs in cell
lines transformed by v-Abl and BCR-ABL7 and in cell lines
derived from a variety of human leukemias, including acute lymphoblastic leukemia (ALL), cutaneous T-cell lymphoma, and adult-type leukemia,8-11 as well as in peripheral blood samples of ALL
and acute myeloid leukemia patients.12
Evidence that perturbation of JAK kinase signaling is directly involved
in human leukemia was more recently obtained by the demonstration of
the fusion of JAK2 on chromosome 9p24 to TEL, a gene of
the ETS family localized at 12p13, in several cases of ALL and
in a myeloid malignancy to lead to the expression of a TEL-JAK2 fusion
protein.13-15 TEL was initially identified as the
fusion partner of the gene encoding the platelet-derived growth factor- receptor in the t(5;12)(q31;p13) chromosomal translocation in patients with chronic myelomonocytic leukemia.16
Subsequently, several hematopoietic malignancies and, more recently,
congenital fibrosarcoma and mesoblastic nephroma were found to be
associated with specific translocations resulting in the fusion
of TEL to either protein tyrosine kinases or
unrelated transcriptional regulators.14,17-22
TEL shares with other ETS proteins an evolutionarily conserved domain
(ETS domain), which is responsible for its nuclear localization and
specific binding to DNA.16,23 It also shares with a subset of other ETS proteins another domain of 65 amino acid residues known as
the B domain or pointed domain, the function of which is poorly
characterized.24 The B/pointed domain of TEL
presents the unique property to induce the self-association of TEL as
well as that of TEL-derived oncoproteins, including
TEL-JAK2.13,18,25,26 TEL-mediated self-association of
TEL-JAK2 leads to constitutive activation of the tyrosine kinase
activity of its JAK2 moiety.13,15 This, in turn, activates
the transforming properties of TEL-JAK2 as evidenced by its ability to
induce cytokine-independent proliferation of murine BaF/3 cells, a cell
line normally dependent on IL-3 for its survival and
proliferation.13,15
To better understand the pathological process caused by the TEL-JAK2
chimeric protein and thus the biological consequences of abnormal JAK2
activation in vivo, we generated mice expressing an EµSR -TEL-JAK2
transgene corresponding to the fusion gene characterized in the T-cell
ALL case. Our results show that TEL-JAK2 transgenic mice develop a
fatal CD8+ T-cell leukemia at 4 to 22 weeks of age.
TEL-JAK2-induced leukemia is a clonal disease characterized by highly
invasive leukemic cells, which display constitutive activation of STAT1
and STAT5.
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Materials and methods |
Transgene and mice
A pBluescript plasmid containing a hemagglutinin (HA)-tagged version
of TEL-JAK2 complementary DNA13 (cDNA) was linearized by
XbaI and the 3' ends filled in with Klenow polymerase.
After digestion with SalI, the purified insert was subcloned
into EcoRV and SalI-restricted
pEµSR,27 a generous gift of Dr Suzanne Cory. This
vector drives the expression of exogenous cDNA from the EµSR
enhancer/promotor cassette and contains the rabbit globin polyA
addition site, 3' of the inserted exogenous cDNA (Figure 1). The EµSR-TEL-JAK2 construct was
digested by NotI to release the transgene, which was purified
on ELUTIP (Schleicher and Schull, Ecquevilly, France)
before injection into fertilized eggs.
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| Fig 1.
Schematic representation of HA-TEL-JAK2 transgene.
The HA-TEL-JAK2 cDNA13 was inserted 3' of the
Eµ/SR enhancer/promoter (thick hatched and dark boxes), which
allows high-level specific expression in lymphoid cells but only low to
undetectable expression in nonlymphoid tissues.27 The
3' end of the transgene contains the rabbit globin poly(A)
addition site. The self-association domain of TEL is represented by a
black box. The JH1 domain (catalytic domain) of JAK2 is represented by
a thin hatched box.
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Transgenic C57B6/DBA2 animals were generated by pronuclear
microinjection as described previously.28
Transgenic mice were identified by polymerase chain reaction (PCR)
analysis of tail DNA and confirmed by a second round of PCR
and Southern blotting analysis. PCR was performed using the following
primers: forward amplimer: 5'-GGGAAGGGAAGCCCATCAACC-3';
reverse amplimer: 5'-CCGCACTGTAGCACACTCCC-3'.
Cytologic and pathological studies
White blood cell counts were determined using the Unopet lysis
procedure (Becton Dickinson Vacutainer Systems, Le Pont de Claix,
France) from blood harvested from the cavernous sinus. Blood smears and bone marrow imprints were stained with
May-Grünwald-Giemsa (MGG). Mice were fully dissected with
macroscopic analysis of all organs. Spleens and thymuses were weighed.
Spleens, livers, and thymuses were systematically cut into 3 parts, 1 of which was immediately snap-frozen in liquid nitrogen while another
part was fixed in FAA (10% formaldehyde, 75% ethanol, 5% glacial
acetic acid). All organs were fixed for 2 hours in FAA and further
processed for paraffin embedding. Sections 3 µm thick were stained
with hematoxylin-eosin (HE) and trichrome. Histologic analysis focused on the type of cellular infiltration, the pattern of invasion in each
organ, and the extent of the metastatic process in each animal.
Immunofluorescence staining
Immunofluorescence staining was performed on frozen tissue sections
(7 µm of thickness) of organs embedded in Tissue Tek (Sakura Finetek
Bayer, Puteaux, France). The sections were fixed twice in
acetone for 10 minutes at  20°C and kept at 20°C.
After incubation for 20 minutes in phosphate-buffered saline (PBS)
containing 10% fetal calf serum (FCS), sections were incubated for 1 hour at room temperature with different antibodies at the appropriate dilution in PBS containing 3% FCS. After rinsing twice in PBS-3% FCS,
samples were examined using a Zeiss microscope. Monoclonal antibodies
(Mab) specific for CD45R/B220 (RA3-6B2 clone, Phar-Mingen Becton Dickinson, Le Pont de Claix, France), Thy 1.2 (53-2.1 clone; PharMingen), CD4 (H129.19 clone, PharMingen), CD8a
(53-6.7 clone; PharMingen), CD3 (145-2C11 clone, PharMingen),
Mac1 chain (M1/70 clone; PharMingen), and GR-1 (RB6-8C5 clone;
PharMingen) conjugated with either fluorescein isothiocyanate (FITC) or
phycoerythrin (PE) were used. Control staining was performed with FITC-
or PE-conjugated rat immunoglobulin (Ig)G2a monoclonal immunoglobulin
isotype standard (R35-95 clone; PharMingen). When polyclonal antibody
specific for the HA epitope (Santa Cruz Biotechnology, Santa Cruz, CA) was used, a second incubation with FITC- or PE-conjugated antirabbit immunoglobulins (Sigma, Saint Quentin Fallavier, France)
was performed for 1 hour.
Flow cytometric analysis and isolation of specific lymphocyte
subclasses
Single-cell suspensions from lymph nodes, thymus, spleen, and bone
marrow were prepared in RPMI medium containing 10% FCS. After
centrifugation for 5 minutes at 1200g, cells were resuspended in FCS containing 10% DMSO and frozen at 80°C. After
thawing and centrifugation, aliquots of 106 cells were
resuspended in PBS containing 3% FCS and 0.1% sodium azide and
processed for immunostaining. Monoclonal and polyclonal antibodies were
added at the appropriate dilution in suspension buffer, and samples
were incubated for 1 hour at 4°C in the dark. Each sample was
rinsed twice in PBS/FCS, and cells were resuspended in PBS-0.1% sodium
azide. In costaining experiments to detect cytoplasmic HA-TEL-JAK2,
cells were fixed in 4% paraformaldehyde for 20 minutes at 4°C and
rinsed twice in PBS containing 0.5% BSA and 0.1% saponin before
incubation with a rabbit antibody specific for the influenza HA epitope
(Santa Cruz Biotechnology) for 1 hour. Cells were rinsed twice in PBS
containing 0.5% BSA and 0.1% saponin, incubated for 1 hour with
antirabbit-FITC-conjugated antibody, and then rinsed twice in
PBS-0.5% BSA. Cells were resuspended in PBS-0.1% azide before
cytometric analysis. All cytometric analysis was performed on
FACsCalibur (Becton Dickinson). A total of 10 000 events were acquired
and analyzed using the CellQuest software (Becton Dickinson).
CD8+ cells and CD4+CD8+ cells were
magnetically separated using the MACS system (Miltenyi Biotec, Paris,
France). Leukemic cells were incubated with
Microbeads-conjugated antimouse CD4 monoclonal antibody (Miltenyi
Biotec) in PBS containing 0.5% BSA for 15 minutes at 8°C.
CD4+ cells were obtained by positive selection on
LS+ columns and eluted. These cells were more than 90%
CD4+CD8+. Unretained cells were subsequently
chromatographed through CS columns to remove trace amounts of
CD4+ T cells. The resulting population was more than 90%
CD8+CD4 .
Northern blot and Southern blot analyses
Total RNA from different tissues was extracted in
guanidium isothiocyanate and purified as
described.29 Fifteen micrograms of total RNA were loaded on
1% agarose gels containing paraformaldehyde, transferred to
nitrocellulose (Nytran Plus, Schleicher & Schull), fixed by UV
irradiation, and hybridized with 32P-labeled DNA probes.
After washing, blots were analyzed by exposure on X-AR films (Kodak,
Européenne d'Imagenie Scientifique, Massy, France).
Probes were made using the Rediprime II Kit (Amersham, Les Ulis,
France). The TEL-specific probe was obtained using the 1-kilobase (kb) EcoRI-EcoRV fragment of TEL-JAK2 cDNA;
the JAK2-specific probe was derived from the 1.1-kb
EcoRV-HindIII fragment of TEL-JAK2 cDNA. For Southern
blot analyses, 10 µg of high molecular weight DNA obtained from mouse
tissues were digested by HindIII and processed according to
standard procedures.30 Hybridization was carried out in
0.5-mol/L Na2HPO4, pH 7.2; 7% sodium dodecyl
sulfate (SDS); 1 mM of ethylenediaminetetraacetic acid (EDTA); and
radiolabeled probes at 68°C. Washes were carried out in
40 mmol/L Na2HPO4, pH 7.2, and 1% SDS
at 68°C.31 The probe was a murine J2.6 286-base pair ClaI- PstI fragment.32
Immunoprecipitation and Western blot analyses
A total of 107 cells from spleen, thymus,
and lymph nodes were rinsed twice in ice-cold PBS, lysed in 1 mL of
radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-HCl, pH 7.4;
0.1 M NaCl; 0.001 M EDTA; 1% Triton X-100; 0.5% sodium deoxycholate;
0.1% SDS; 1% aprotinin [Sigma]; 100 µg/mL phenylmethylsulfonyl
fluoride [Sigma]; and 0.1% leupeptin [Sigma]) and centrifuged at
15 000g for 30 minutes. Immunoprecipitation analyses were
performed using either a rabbit antiserum directed against amino acids
1110 to 1129 of JAK2 (anti-JAK2; Santa Cruz Biotechnology) or a rabbit
antiserum to the first 52 amino acids of TEL.33
Immunoprecipitates were rinsed 3 times in RIPA buffer and once in
TNE buffer (10 mM Tris-HCl, pH 7.4; 0.1 M NaCl;
0.001 M EDTA). Immunoblotting analyses were performed after
electrophoretic transfer of immunoprecipitated proteins separated by
SDS-PAGE followed by incubation with a monoclonal antibody specific for
the influenza HA epitope (12CA5; Boehringer Mannheim, Meylan,
France) or the 4G10 monoclonal antibody directed to
phosphotyrosine (Upstate Biotechnology Euromedex Souffelweyersheim, France). Bands were visualized using the ECL
chemiluminescence kit (Amersham).
Electrophoretic mobility shift assays
Whole-cell extracts were prepared by lysing single-cell suspensions
from mouse tissues in 20 mmol/L HEPES, pH 7.9; 20% glycerol; 50 mM
KCl; 400 mmol/L NaCl; 1 mM EDTA; 1 mM dithiothreitol;
1-mmol/L Na3VO4; 10-mmol/L
4-nitrophenylphosphate, and protease inhibitors as in RIPA buffer.
After 3 cycles of freezing and thawing, extracts were centrifuged at
30 000g for 10 minutes at 4°C. Equal amounts of each
extract (15 µg total protein) were incubated with either the m67SIE
(Stat Inducible Element) or -casein STAT-specific oligonucleotide
probes and protein-DNA complexes separated from the free probe as
previously described.34 For supershift experiments, 1.5 µL anti-STAT1 (Transduction Laboratories, Lexington, KY)
or anti-STAT5 antibodies35 were included in the binding reaction.
Transplantation of leukemic cells into nude mice
About 5×106 to
10×106 cells were obtained from leukemic
tissues, resuspended in RPMI medium without serum, and injected
subcutaneously into 6- to 8-week-old Swiss nu/nu mice.
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Results |
Lymphoid leukemia in TEL-JAK2 transgenic mice
A cDNA encoding an HA-tagged version of the TEL-JAK2 protein
corresponding to the fusion gene identified in a case of acute lymphoblastic T-cell leukemia13 was inserted downstream of
the EµSR promoter/enhancer construct27 (Figure 1). The
EµSR/TEL-JAK2 transgene was released by NotI digestion,
purified, and injected into zygotes that were transplanted into
pseudopregnant females.
Fifteen transgenic founder mice for the EµSR/HA-TEL-JAK2 construct
were obtained. Six died between 4 and 20 weeks of age, and 6 other
animals were found moribund between 6 and 13 weeks and killed for
pathological analysis. Because the early onset of pathological symptoms
was rapidly followed by death, F1 transgenic animals could be obtained
from only 2 founders (Nos. 3 and 71). Founder animals as well as their
progeny derived from serial backcrosses with C57B6 mice developed the
same symptoms and were therefore used for subsequent analyses when
reaching the terminal stage of the disease.
Macroscopic examination of diseased mice revealed typical symptoms of
lymphoid malignancy. As illustrated in Figure
2A and detailed in Table
1, all diseased animals exhibited a marked splenomegaly, with prominent lymph nodes enlargement in most cases. Thymic enlargement was also a hallmark of the disease, occurring in the
progeny of founder No. 71 and in 2 other founders examined (Nos. 60 and
65; Table 1). Diseased mice also displayed a 2-fold to more than
200-fold increase in circulating white blood cells compared with
control littermates (Table 1).
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| Fig 2.
: Histopathological and cytologic analysis of TEL-JAK2
transgenic mice.
(A) Macroscopic appearance of an F1 transgenic mouse showing
hyperplasia of spleen (Sp); thymus (Th); and cervical (C), axillary
(A), and femoral (F) lymph nodes. (B) May-Grünwald-Giemsa (MGG)
staining of blood smears of transgenic mice. Note the morphology
resembling that of prolymphocytes, the size heterogeneity, and the
presence of a mitotic cell. (C-E) Histopathology of hematopoietic
organs. (C) Thymus showing invasion of the perilobular capsule (white
arrows) (HE). (D) Spleen; note the loss of its normal architecture
(HE). (E) Bone marrow; note infiltration by leukemic cells (MGG). (F
and G) Histopathology of nonhematopoietic organs (HE) showing
infiltration of leukemic cells in the (F) pericentrolobular area and
sinusoids of the liver (HE) and (G) brain; subarachnoid area (HE). (H)
Kidney; infiltration of leukemic cells around peritubular capillaries
(HE).
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Blood smears showed elevated levels of cells resembling T-cell
prolymphocytes with a high nucleocytoplasmic ratio, irregular nuclei
with slightly condensed chromatin, and a scant basophilic cytoplasm
(Figure 2B). Occasional mitosis was also observed (Figure 2B).
Histologic examination of diseased animals showed massive infiltration
of thymus, spleen, lymph nodes, and bone marrow with these abnormal
cells. Immunohistochemical analysis showed that the lymphoid cells in
enlarged lymph nodes were essentially of T-cell origin as evidenced by
Thy1.2 (Figure 3A) and CD3 expression (data
not shown). Occasional small clusters of
B220+IgMThy1.2
B cells could, however, be detected (Figure 3B and data not
shown). Thymuses displayed local or, most often, complete architectural disorganization with homogeneous sheets of leukemic cells in both the
cortical and medullary areas. Occasionally, leukemic cells also
disrupted the thymic peripheral capsule (Figure 2C). The normal splenic
architecture was also disrupted in diseased animals with a large
expansion of leukemic cells in the white pulp area (Figure 2D).
Infiltrating lymphoid cells also infringed upon follicle boundaries to
invade the red pulp. Immunohistochemical staining of spleen sections
with both anti-B220 and anti-Thy1.2 Mabs revealed a profound
disorganization of the lymphoid follicles as evidenced by the
disappearance of B-cell centers and the invasion of leukemic T cells
(data not shown). Bone marrow from diseased mice displayed increased
cellularity resulting from its invasion by leukemic cells (Figure 2E).
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| Fig 3.
Infiltrating lymphoid cells in TEL-JAK2 transgenic mice
are T cells.
A lymph node section from a diseased mouse was double stained with
anti-Thy1.2-FITC antibody and anti-B220-PE antibody and examined for
expression of Thy1.2 (A) or B220 (B). A nontransgenic liver section (C)
and the liver section from a diseased mouse (D) were stained with
anti-Thy1.2 antibody.
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Nonhematopoietic organs, including liver, brain, lung, kidney, adrenal
gland, salivary gland, thyroid gland, and ovary, were also infiltrated
by leukemic cells. Pericentrolobular, portal, and sinusoidal
infiltration of leukemic cells occurred in the liver with, in the most
severe cases, a nearly complete destruction of the hepatic tissue
(Figure 2F). Anti-Thy1.2 staining demonstrated the T-cell nature of
these infiltrating cells (Figure 3D). Anti-B220 staining showed no
evidence of B-cell infiltration (data not shown). Brains were edematous
with an infiltration of the subarachnoid space with leukemic cells,
further illustrating their invasive behavior (Figure 2G). In the
kidney, foci of infiltrated leukemic cells surrounded the peritubular
capillaries (Figure 2H).
Enlarged lymph nodes and infiltrated livers showed no evidence of
myelomonocytic cells, as demonstrated by the lack of cells staining
with GR1 and Mac1 (data not shown).
Flow cytometric analysis
The cell surface markers of the expanded lymphoid compartment in
diseased transgenic animals were further characterized by flow
cytometric analysis. Figure 4A illustrates
the results obtained from a representative transgenic animal (No. 71-4)
and its sibling control (No. 71-2). In lymph nodes, more than 90% of
the cells were T cells as evidenced by Thy1.2 staining (data not
shown). Analysis of CD4 and CD8 expression demonstrated the presence of a mixed population of CD4+CD8+ double-positive
(DP) cells, an immunophenotype normally only seen in the thymus, and of
CD4CD8+ (CD8+
single-positive [SP]) T cells (Figure 4A). More than 90% of lymph nodes T cells were therefore CD8+.
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| Fig 4.
: CD4 and CD8 expression in TEL-JAK2 leukemic cells.
(A) Single-cell suspensions from lymph nodes (LN), thymus (Th), spleen
(Sp), and bone marrow (BM) were obtained from a transgenic leukemic
animal (71-4) and its sibling control (71-2). (B) Single-cell
suspensions obtained from a tumor generated in nude mice (Nu18; Table
3) were sorted as CD8+ SP and
CD4+CD8+ DP as described in "Materials and
methods." The CD4 and CD8 expression pattern of unsorted cells
(left), CD8+ SP cells (middle), and DP cells (right) was
determined by flow cytometry. For each experiment, 106
cells were incubated with anti-CD4-PE (Y-axis) and anti-CD8-FITC
(X-axis) and analyzed by flow cytometry. The results were computed from
the acquisition of 10 000 events. The relative percentage of each
subpopulation (CD4+CD8;
CD4CD8+; and
CD4+CD8+) is indicated in the corresponding
area of the diagrams.
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A drastic increase of the CD4CD8+
compartment also characterized the thymus of diseased animals, with
30% to 65% of thymocytes being CD8+ SP as compared with
about 10% in controls (Table 2 and Figure 4A). Of note, a fraction of the CD8+ cells displayed a
larger size as compared with the CD8+ cells from control
littermates (data not shown). This size heterogeneity was also observed
in blood leukemic cells (Figure 2B). Double staining demonstrated that
CD8+ T leukemic cells in the thymus expressed the TCR
and CD3 at low levels (data not shown).
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Table 2.
Cell surface marker analysis of leukemic cells obtained
from thymus, spleen, and bone marrow of TEL-JAK2 transgenic animals
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The T-cell population was also expanded in spleen and bone marrow of
diseased mice as evidenced by Thy1.2 staining (Table 2). Double
staining with antibodies to CD4 and CD8 showed an increase both of the
DP compartment and the CD8+ SP compartment (Figure 4A and
Table 2).
Expression of TEL-JAK2 in the leukemic cells of transgenic mice
leads to the activation of STAT1 and STAT5 DNA binding activity
To ascertain that the leukemic cells of TEL-JAK2 transgenic mice
expressed the fusion gene, Northern blot analysis was performed on
tumor material and on control tissue from nontransgenic littermates. Figure 5 shows that the expected 3.2-kb
TEL-JAK2 transcript was specifically detected in the spleen, thymus,
and lymph nodes from diseased transgenic mice. This transcript was
expressed at levels similar to those of the endogenous TEL messenger
RNA (mRNA) and was detected using a TEL-specific or a JAK2-specific
probe (Figure 5A). Expression of the HA-TEL-JAK2 fusion protein in
leukemic cells of transgenic animals was analyzed by double-staining
flow cytometry using antibodies specific to Thy1.2 and the HA epitope, respectively. Figure 5B clearly shows the expression of HA-TEL-JAK2 in
the Thy1.2+ cells from transgenic thymus and lymph nodes.
The fusion protein was also expressed in B220+ lymph node
cells (data not shown). The immunoreactive protein detected by
fluorescence-activated cell sorter analysis corresponds to the expected
80-kd full-length TEL-JAK2 protein as evidenced by Western blot
analysis of cellular extracts from thymus, spleen, and lymph nodes from
diseased transgenic animals, using either anti-HA or anti-TEL
antibodies (Figure 5C, upper panel). Furthermore, in line with our
previous observations in model cellular systems,13 the
immunoprecipitated TEL-JAK2 protein was also detected by a phosphotyrosine-specific antibody, demonstrating constitutive activation of its tyrosine kinase activity in TEL-JAK2 leukemic cells
(Figure 5C, lower panel).
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| Fig 5.
Expression of HA-TEL-JAK2 transgene.
(A) Northern blot analysis. Total mRNA from diseased spleen (lane 1),
thymus (lane 2), lymph nodes (lane 3), and from a control spleen (lane
4) were hybridized as indicated with probes specific for TEL or JAK2.
The TEL-JAK2 transgene mRNA is indicated by a white arrowhead. Bands
corresponding to the endogenous TEL and JAK2 transcripts are indicated
by gray and black arrowheads, respectively. (B) Flow cytometric
detection of HA-TEL-JAK2 protein expression in diseased thymus and
lymph nodes. Double labeling with mouse anti-Thy1.2-PE and rabbit
anti-HA plus antirabbit Ig-FITC antibodies revealed that
Thy1.2+ cells express the HA epitope. For the thymus,
FITC-fluorescence intensity in gated Thy1.2+ cells is
represented for a transgenic animal (71-10, dark surface) and for a
control animal (71-6, open surface). For diseased lymph nodes, labeling
is with the anti-HA rabbit antibody plus antirabbit Ig-FITC (dark
surface) or with antirabbit Ig-FITC alone (open surface). (C) Western
blot analysis of cellular extracts from lymph nodes, thymus, and spleen
from diseased and control animals. Extracts corresponding to the same
number of cells from each organ were immunoprecipitated by anti-JAK2
antibody, separated by SDS-PAGE, and blotted on nitrocellulose. Blots
were analyzed with either the anti-HA (12CA5, Boehringer) or
anti-TEL23 antibodies (upper panel) or with the 4G10
phosphotyrosine-specific antibody (lower panel). Lanes 1 and 2: lymph
nodes from transgenic animals 71-4 and 71-18. Lanes 3 and 5: thymus
from transgenic mice 65 and 71-18. Lane 6: spleen from transgenic mouse
71-18. Lanes 4 and 7: control thymus and spleen, respectively. The
HA-TEL-JAK2 protein is indicated by a white arrowhead.
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TEL-JAK2-induced transformation of model cell lines is associated with
the activation of STAT1 and STAT5 DNA binding
activity.13,15 To investigate whether STAT transcriptional
regulators were also activated in leukemic tissues of TEL-JAK2
transgenic mice, STAT DNA binding activity was analyzed by
electrophoretic mobility shift assay using either the
m67SIE probe, which is specific for STAT1, or
the casein probe, which binds STAT5 with high affinity and STAT1
with lower affinity. Whole-cell extracts obtained from spleen or lymph
nodes of a leukemic mouse or from control spleen were incubated with
the SIE or casein probe, and protein/DNA complexes were resolved by
nondenaturing gel electophoresis. Figure 6
shows that nuclear extracts from TEL-JAK2 leukemic samples generated a
specific retarded complex when using the SIE probe. This complex corresponds to specific binding to the probe since it was competed by a
100-fold molar excess of unlabeled SIE oligonucleotide used as
competitor but not by a nonspecific competitor (Figure 6A, upper
panel). This complex results from the binding of STAT1 since it was
supershifted by a STAT1-specific antibody (Figure 6A, upper panel). A
major complex was detected in leukemic cells using the casein
probe, which was specifically competed by a 100-fold molar excess of
unlabeled casein oligonucleotide (Figure 6B, lower panel). This
complex results from STAT5 binding since it was specifically
supershifted by a STAT5-specific antibody. We conclude that both STAT1
and STAT5 are activated in the leukemic cells of TEL-JAK2 transgenic
mice.
View larger version (55K):
[in this window]
[in a new window]
| Fig 6.
TEL-JAK2 leukemic cells express activated STAT1 and
STAT5.
Electrophoretic mobility shift assays were carried out with either the
m67SIE probe (upper panel) or the casein probe (lower panel) and
whole-cell extracts from the spleen and lymph nodes of a diseased
transgenic animal (71-45) or from a control spleen. Positions of the
STAT/DNA complexes are indicated by open arrowheads. Specific and
nonspecific competitors were added as indicated. For supershift
experiments, anti-STAT1 or anti-STAT5 antibodies were added as
indicated. Supershifted complexes are indicated by black arrowheads.
|
|
TEL-JAK2-induced leukemia is clonal or oligoclonal
To analyze whether TEL-JAK2 leukemic cells represented a polyclonal
or monoclonal population, DNA from thymuses, lymph nodes, and
infiltrated livers of leukemic animals were digested with HindIII and compared with similarly digested DNA from a control thymus for rearrangement of the TCR gene locus, using a J2 probe. Figure 7A shows that leukemic cells of
founder mice Nos. 30 and 65 and of the F1 progeny of transgenic mouse
No. 71 show clonal or oligoclonal TCR rearrangements. Of note, the
same clone was found to be present in the leukemic cells of different
organs in the same animal (Figure 7A). We conclude that, despite its early onset, TEL-JAK2-induced leukemia is likely to require the activation of secondary genetic events.
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| Fig 7.
TCR gene rearrangement in TEL-JAK2 leukemic cells.
(A) DNA was extracted from the thymus (T) from founder mouse 65, from 4 F1 animals of transgenic line 71 (71-11, 71-24, 71-10, and 71-4), and
from a control thymus. Lymph nodes (LN) from transgenic mice 30 and
71-4 and the liver (L) from mouse 65 were also analyzed. DNA were
digested by HindIII and analyzed by Southern blot
using a j2-specific probe. An arrow points to germline
configuration. TCR-rearranged bands (indicated by stars) are
prominant in all leukemic samples. (B) DNA was extracted from a control
thymus (lane 1), from the thymus of a diseased transgenic mouse 71-18 (lane 2), from nude mice tumors obtained following transplantation
either of the leukemic cells of animal 71-18 (Nu18, lane 3) or
following secondary transplantation of Nu18 tumor cells (lanes 4 and
5), from nude mice tumors obtained from the transplantation of sorted
SP (lanes 6-8) or DP cells (lane 11), and from the sorted CD8+
SP and DP populations derived from transplanted tumors Nu18/SP4
and Nu18/DP1 (lanes 9-10 and 12-13) (see also Table 3). DNA were
digested by HindIII and analyzed by Southern blot
using a j2-specific probe. Germline configuration is indicated by an
arrow. TCR-rearranged bands (indicated by stars) are prominant in
all leukemic samples. (C) DNA was extracted from a control thymus (lane
1), from the thymus of diseased transgenic mouse 71-24 (lane 2), from
nude mice tumors obtained either following transplantation of the
leukemic cells of animal 71-24 (Nu24, lane 3) or following secondary
transplantation of Nu24 tumor cells (Nu24/1, lane 4), from nude mice
tumors obtained from the transplantation of sorted SP (Nu24/SP1, lane
7), and from the sorted DP and CD8+ SP populations derived
from either transplanted tumors Nu24/1 (lanes 5 and 6) or Nu24/SP1
(lanes 8 and 9) (see also Table 3). DNA were digested by
HindIII and analyzed by Southern blot using a
j2-specific probe. Germline configuration is indicated by an arrow.
TCR-rearranged bands (indicated by stars) are prominant in all
leukemic samples.
|
|
CD4+CD8+ DP cells and CD8+
SP cells are transplantable to nude mice
The malignant nature of the TEL-JAK2-induced T-cell leukemia was
further borne out by the rapid engraftment of these leukemic cells
following their serial transplantation into nude mice and their
subsequent invasion of the spleen and lymph nodes of transplanted animals (Table 3 and data not shown).
Transplanted tumors show the same TCR gene locus rearrangement as
the original tumor DNA, showing that both resulted from the expansion
of the same clonal population (Figure 7B, compare lanes 2 to 3-5, and
Figure 7C, compare lanes 15 to 16-17). As leukemic cells from
transgenic animals, transplanted tumor cells are composed of a mixture
of CD8+ SP and DP T cells, suggesting that both populations
represented the phenotypic progression of a single clone (Figure 4B and
Table 3). To analyze this in further detail, CD8+ SP and DP
leukemic cells obtained from transgenic animals 71-24 and 71-43 as well
as from transplanted mice Nu18 and Nu24 (Table 3) were purified (Figure
4B) and transferred into nude mice. Table 3 shows that both sorted DP
and CD8+ SP cells efficiently transplanted to generate a
mixed population of DP and CD8+ SP cells in secondary
recipients (Table 3). As shown in Figure 7B and 7C, the same TCR
rearrangement was observed in both the CD8+ SP and DP
populations obtained from the transfer of purified DP and CD8+
SP leukemic cells. These results show that the CD8+
SP and DP leukemic cells originate from the same clone. They
also indicate that TEL-JAK2 leukemia may affect an intermediate-stage cell population ranging from immature CD8+ SP to
CD4+CD8+ DP T cells.
View this table:
[in this window]
[in a new window]
|
Table 3.
Immunophenotype of leukemic cells generated by unsorted,
sorted CD8+ SP, or sorted
CD4+CD8+ DP populations following
transplantation in nude mice
|
|
| |
Discussion |
In this study, we describe a transgenic mouse model for
TEL-JAK2-induced leukemia. The TEL-JAK2 fusion gene used here
was initially identified in a childhood T-cell leukemia in relapse and
was shown to encode a fusion protein between the first 336 amino acid
residues of TEL and the kinase domain (JH1) of JAK2.13 Our
results show that directed expression of TEL-JAK2 to the lymphoid lineage using an EµSR enhancer/promoter reproducibly induces a
rapid, fatal T-cell leukemia in mice with preferential transformation of CD8+ cells.
Two other cases of TEL-JAK2 fusion genes have been described so far in
human leukemia, each associated with a specific phenotype. These genes
encode fusion proteins containing additional JAK2 sequences besides the
JH1 domain. Specifically, the TEL-JAK2 protein encoded in the pre-B ALL
case also contains part of the JH2 domain, whereas the fusion protein
found in an atypical CML contains the entire JH2 domain.14
Because the JH2 domain appears to be involved in JAK kinase specificity
presumably via protein-protein interactions,36,37 the
presence of additional JAK2 sequences in these latter fusion proteins
could be instrumental in determining the phenotype of the respective leukemias.
The transgenic mouse model for TEL-JAK2-induced leukemia described
here is distinct from that reported recently following transplantation
in syngeneic animals of mouse bone marrow cells infected with a
MSCV-TEL-JAK2 retrovirus, which developed a mixed myeloid-lymphoid
leukemia with short latency.15 Because the same TEL-JAK2
fusion gene was used in both studies, the basis for this difference is
likely to originate either from the type of promoter used to drive
TEL-JAK2 expression (EµSR vs MSCV LTR) or from the fact that
retroviral-mediated infection in tissue culture is relatively
unrestricted in terms of the cell lineage and differentiation stage of
targeted cells. Both studies converge, however, to demonstrate that
TEL-JAK2 is a powerful oncogene in vivo and that, within the lymphoid
lineage, TEL-JAK2 preferentially transforms T cells.
The best characterized signaling pathway situated downstream of JAK
kinases is the activation of transcription factors of the STAT
family.1 As Ba/F3 cells transformed by
TEL-JAK2,13,15 leukemic cells of TEL-JAK2 transgenic mice
also show constitutive activation of STAT1 and STAT5. Recent studies
have shown that inactivation of both STAT5a and STAT5b by homologous
recombination in mice, although without detectable effect on thymocyte
development, has a profound effect on peripheral T-cell
proliferation.38,39 Specifically, peripheral T cells
lacking STAT5a/b failed to proliferate in response to engagement of the
T-cell receptor (TCR) by antigen both in the presence and absence of
IL-2, a phenotype that correlates with their inability to express genes
involved in cell cycle progression.39 Independent studies
have shown that STAT5 is rapidly phosphorylated on tyrosine residues
following TCR activation and that inhibition of STAT5 function by
expression of a dominant negative form of STAT5 interferes with
antigen-induced T-cell proliferation.40 Constitutive
activation of STAT5 in TEL-JAK2 leukemic cells could therefore bypass
the normal signaling pathways controlled by specific receptors to
induce their uncontrolled proliferation. This notion is consistent with
the fact that the transforming properties in Ba/F3 cells of TEL fusion
proteins modeled on TEL-JAK2 but containing instead the kinase domain
of either JAK1, JAK3, or TYK2 correlates with the activation of STAT5
but not with that of STAT1 or STAT3.34 It is also
consistent with the fact that enforced expression of a gain of function
STAT5 mutant is sufficient to induce cell survival and factor
independent proliferation of hematopoietic cell lines41,42 and with the fact that D-STAT was found to be important to
the leukemia-like disease associated with gain of function mutant of
Hopscotch in Drosophila.6
Although additional studies are required to establish this point, our
transplantation studies suggest that TEL-JAK2 leukemia results mostly
from the expansion of an immature CD8+ T-cell compartment.
We cannot exclude, however, that TEL-JAK2 expression may also interfere
with the maturation of T cells along the CD4 lineage. This property
might also result from the constitutive activation of STAT5 because
mice lacking STAT5a/b show with time a decrease of CD8 SP cells
relative to CD4 SP cells.39 The availability of a mouse
model for TEL-JAK2-induced leukemia and of STAT5a/b-deficient mice
should allow us to directly assess whether the leukemogenic properties
of TEL-JAK2 indeed depend on STAT5 activation. This model will also be
useful to identify additional downstream events or complementary
functions essential to TEL-JAK2-induced leukemia.
| |
Acknowledgments |
The authors thank Radcliffe G. Lopez, Anthony Boureux, Isabelle Cremer,
and Lionel Larue for their help with some of the experiments; Maryvonne
Williame for expert technical assistance; Marc-Henri Stern and L. Larue
for discussions; Suzanne Cory and M.H. Stern for reagents; and the SEAT
of CNRS for their handling of transgenic animals.
| |
Footnotes |
Submitted July 12, 1999; accepted February 1, 2000.
Supported by the CNRS, Institut National pour la Santé et
la Recherche Médicale (INSERM), Institut Curie, Ligue Nationale contre le Cancer (Axe Oncogenèse), Association pour la Recherche sur le Cancer, and an EU Biomed Concerted Action. C.C. supported by
predoctoral fellowships from the MESR and Ligue Nationale Contre le Cancer.
Reprints: Jacques Ghysdael, CNRS UMR146-Institut Curie, Centre
Universitaire, Bat. 110, 91405 Orsay, France; e-mail: Jacques.Ghysdael{at}curie.u-psud.fr.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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M. H.-H. Nguyen, J. M.-Y. Ho, B. K. Beattie, and D. L. Barber
TEL-JAK2 Mediates Constitutive Activation of the Phosphatidylinositol 3'-Kinase/Protein Kinase B Signaling Pathway
J. Biol. Chem.,
August 24, 2001;
276(35):
32704 - 32713.
[Abstract]
[Full Text]
[PDF]
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Abstract
Introduction