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Blood First Edition Paper, prepublished online May 30, 2008; DOI 10.1182/blood-2008-01-134114
JAK2 stimulates homologous recombination and genetic instability :
potential implication in the heterogeneity of myeloproliferative disorders
Isabelle Plo1,2,3, Mayuka Nakatake1,2,3, Laurent Malivert4, Jean-Pierre de Villartay4, Stéphane
Giraudier1,2,3, Jean-Luc Villeval1,2,3, Lisa Wiesmuller5,William Vainchenker1,2,3*
1- INSERM, UMR790, Villejuif, France
2- Université Paris XI, UMR790, Institut Gustave Roussy, Villejuif, France
3- Institut Gustave Roussy, Villejuif, France
4- INSERM, U768, Hopital Necker-Enfants Malades, Paris, F-75015, France,
5- Department of Obstetrics and Gynaecology of the University of Ulm, Prittwitzstrasse 43,
D-89075 Ulm, Germany.
Running title: Jak2 stimulates homologous recombination
Corresponding author:
William Vainchenker, INSERM U790, Institut Gustave Roussy, 39 Camille Desmoulins,
94805 Villejuif, France. Phone: +33 1 42 11 42 33. Email: [email protected]
Scientific heading: Hematopoiesis
1
Copyright © 2008 American Society of Hematology
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Abstract
The JAK2V617F mutation is frequently observed in classical myeloproliferative disorders and
disease progression is associated with a bi-allelic acquisition of the mutation occurring by
mitotic recombination. In this study, we examined whether JAK2 activation could lead to
increased homologous recombination (HR) and genetic instability. In a Ba/F3 cell line
expressing the erythropoietin (EPO) receptor, mutant JAK2V617F and, to a lesser extent, wild
type (wt) JAK2 induced an increase in HR activity in the presence of EPO without modifying
NHEJ efficiency. Moreover, a marked augmentation in HR activity was found in CD34+
derived cells isolated from patients with Polycythemia Vera or Primitive myelofibrosis in
comparison to control samples. This increase was associated with a spontaneous RAD51 foci
formation. As a result, sister chromatid exchange was 50% augmented in JAK2V617F Ba/F3
cells compared to JAK2wt cells. Moreover, JAK2 activation increased centrosome and ploidy
abnormalities. Finally, in JAK2V617F Ba/F3 cells, we found a 100-fold and 10-fold increase in
mutagenesis at the HPRT and Na/K ATPase loci, respectively. Together, this work highlights
a new molecular mechanism for HR regulation mediated by JAK2 and more efficiently by
JAK2V617F. Our study might provide some keys to understand how a single mutation can give
rise to different pathologies.
2
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Introduction
Myeloproliferative disorders (MPD) are clonal hematopoietic malignancies in which one or
several myeloid lineages (i.e. granulocytic, erythroid and megakaryocytic) are abnormally
amplified. This amplification is thought to result from the deregulation of hematopoietic stem
cells with a downstream selective proliferation advantage in late myeloid differentiation.
MPD are classified by the World Health Organization (WHO) in three categories : (i) chronic
myeloid leukemia (CML), (ii) classical MPD including Polycythemia Vera (PV), Essential
Thrombocythemia (ET) and Primitive Myelofibrosis (PMF) and (iii) unclassified and rare
MPD comprising chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome/chronic
eosinophilic leukemia (HEL/CEL) and other unclassifiable myeloproliferation.
MPD frequently involve the deregulation of a tyrosine kinase due to the acquisition of a
monogenetic abnormality in a hematopoietic stem cell, the archetype of which is BCR/ABL
in CML. Recently, several groups have identified a recurrent acquired mutation in the
tyrosine Janus kinase 2 (JAK2) gene in most cases of PV patients and in about half of ET and
PMF patients
1-5
. JAK2 is a tyrosine kinase that becomes activated downstream of several
cytokine receptors after ligand binding, such as erythropoietin (EPO). JAK2 activation leads
to activation of many signaling pathways including the MAPK, PI3K and STATs pathways.
The V617F point mutation lies in the autoinhibitory JH2 domain of JAK2 and in vitro studies
have demonstrated that the mutation leads to JAK2 autophosphorylation and to the
constitutive activation of downstream pathways 2-4.
Although ET, PV and PMF share some common phenotypic features 6-8, it is surprising that a
single point mutation gives rise to several disorders 9. One possible explanation is the
JAK2V617F « dosage » hypothesis which postulates that the JAK2 mutation could be the
starting point of the three pathologies, and the occurrence of other genetic events, that may
modify JAK2 kinase activity, could explain the heterogeneity among the classical MPD. This
hypothesis is based on mouse models in which low levels of JAK2V617F induces an ET
3
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phenotype, whereas higher expression leads to a PV progressing to myelofibrosis
10,11
. In
addition, JAK2V617F burden in granulocytes and bone marrow samples is markedly low in ET
patients compared to PV and PMF patients12 . This is related to the fact that the JAK2
mutation is mono-allelic in ET patients while the mutation is bi-allelic in most PV patients (at
least in some progenitors) due to a loss of heterozygosity after a mitotic recombination
3,13,14
.
Progression of PV to PMF is accompanied by the expansion of the bi-allelic JAK2 mutated
clone
15
.
Several mechanisms have been proposed that may contribute to the loss of heterozygosity
and/or the acquisition of new genetic abnormalities. Among these, they are DNA-double
strand break repair mechanisms including the homologous recombination (HR) and nonhomologous end-joining (NHEJ) mechanisms. Since both excessive and defective HR and
NHEJ can lead to genome instability, a very precise and acute regulation of these mechanisms
are essential to maintain the necessary equilibrium between stability and diversification of the
genome. Indeed, HR can become deleterious and an uncontrolled HR excess promotes genetic
instability 16 and diseases 17. For instance, HR with crossing over between dispersed repetitive
sequences can lead to a variety of genome rearrangements 16. Gene conversion (a product of
HR) between homolog allele can lead to loss of heterozygosity 18, whereas gene conversion
with a pseudogene, which generally bears stop mutations, can inactivate a functional allele 19.
Moreover, the accumulation of aberrant abortive recombination events can also be toxic
Similar to excessive HR, defects in HR can also result in genetic instability
21-23
expressing a dominant negative form of RAD51, a key protein involved in HR
24
20
.
. Cells
, produce
more tumors when injected into nude mice 21. The level of Rad 51 appears to be an important
parameter for inducing gene instability. Decreased RAD51 protein levels have been reported
in breast carcinomas 25, inc contrast increased levels of RAD51 were detected in other cancer
cells
26-28
and, especially, in cells expressing BCR/ABL or other fusion tyrosine kinase
implicated in hematological diseases 29. Alternatively, deregulation of NHEJ may also lead to
4
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genome rearrangement including translocation, deletion and insertion30.
In this work, we hypothesized that JAK2V617F may deregulate HR and/or NHEJ mechanisms,
which could in turn be responsible for both the loss of heterozygosity of JAK2 and the
acquisition of additional genetic events. Thus, deregulation of such mechanisms could explain
not only the heterogeneity of MPD, but also the evolution of MPD to acute leukemia.
5
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Materials and Methods
Materials
Fetal bovine serum (FBS), murine recombinant thrombopoietin (TPO) and IL-6 were from
Stem Cell Technologies (Meylan, France). Liquid cell culture media, including Iscove’s
Modified Dulbecco Medium (IMDM) and Dulbecco Modified Eagle Medium (DMEM), were
from Invitrogen (Cergy Pontoise, France). Human recombinant EPO, SCF, FLT3L and IL3
were generous gifts from Amgen (Neuilly, France) and recombinant TPO from Kirin (Tokyo,
Japan). Recombinant human IL6 was provided by S. Burstein (Oklahoma City, OK, USA).
Restriction enzymes were purchased from Fermentas (St Leon-Rot, Germany).
Plasmids, DNA manipulations, production of retroviruses
The HR-EGFP/3’EGFP plasmid for HR measurements was constructed by insertion of the
puromycin resistance gene, one acceptor gene cassette (HR-EGFP), one spacer cassette
(Hygromycin B), and one donor gene (3’EGFP) in series into multiple cloning site of the
retroviral vector p5NM
31
. The acceptor gene substrate was designed such that the I-SceI
recognition sequence replaced 4 bp (HR-EGFP) and the donor gene 3'EGFP was designed
such that the ATG was mutated.
Murine and human JAK2V617F and wild-type JAK2 (JAK2wt) pMEGIX retroviral vectors
were previously described 32. The KS-TEL-JAK2 plasmid was a gift from Dr. V. Lacronique
(Paris, France). These three plasmids were subcloned into the retroviral pREX-CD4 plasmid
kindly provided by Dr. S. Constantinescu (Brussels, Belgium)
33
. Retroviral particles were
produced by transfection of 293EBNA cells with 3 different plasmids: pgag-pol (packaging
plasmid), pVSV-G (coding for the VSV-G protein envelope) and pREX containing JAK2
constructs, in the presence of lipofectamine 2000 (Invitrogen), according to the
manufacturer's instructions.
The I-SceI gene was inserted into a HIV-derived lentiviral vector (pRRL sin PGK WPRE;
Généthon, Evry, France). Lentivirus particles were produced by transfection of 293T cells
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with 3 different plasmids: pCMV (packaging plasmid), pMD2G (coding for the VSV-G
protein envelope) and pRRL/PGK-I-Sce1 in the presence of lipofectamine 2000 (Invitrogen),
according to the manufacturer's instructions. Viral stocks were stored at -80oC.
Cell lines
The murine, pro B Ba/F3-EPOR cell line and derivative cell lines were cultured in RPMI
medium (Invitrogen) complemented with 10% FBS (Stem Cell Technologies) and in the
presence of 1U/mL EPO. Parental, JAK2wt- or JAK2V617F- expressing Ba/F3-EPOR cells
were obtained by infecting cells with human or murine JAK2V617F and wild-type JAK2
(JAK2wt) pMEGIX retroviral vectors
32
. The Ba/F3-HR2 cell line was obtained by
electroporation of Ba/F3-EPOR cells with the HR-EGFP/3’EGFP plasmid and through a
selection of clones grown in methylcellulose in the presence of EPO and puromycin
(1µg/mL). Intrachromosomal integration was verified by PCR relying on the following
oligonucleotides PCR-2-1 (5'-TACACAAATCGCCCGCAGAAGC-3') and PCR-2-2 (5'CTGTCTTTAACAAATTGGACTAATCG-3') as previously described
31
. PCR conditions
were 5 min at 94°C, then 35 cycles at 92°C for 60 sec, 60°C for 60 sec and 72°C for 120 sec,
followed with a final extension step of 72°C for 7 min.
Recombination assays in Ba/F3-HR2 cell lines and in CD34-positive cells from patients.
For the Ba/F3-HR2 cell line, cells were plated at 4 x 104 cells per well in 96-well plates and
infected with pREX-CD4 constructs containing the murine mutated or wt JAK2. For CD34+
cells isolated from patients and controls, cells were immunopurified and amplified in medium
containing SCF/IL3/EPO for 5 days. Then, cells were plated at 4 x 104 cells per well in 96well plates and infected with the HR substrate. Five days after infection, cells were washed
and fixed in PBS/2% PAF for 15 min at room temperature and GFP+ cells were detected by
flow cytometry analysis using a FACSort.
Analysis of error-prone NHEJ in Ba/F3-HR2 cell lines
Ba/F3-HR2 cells were infected with pREX-CD4 constructs containing JAK2 and infected
7
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with I-SceI virus. Non infected cells served as controls. GFP- cells were then sorted by a
FACSDIVA to exclude HR events and PCR was performed on genomic DNA with the
oligonucleotides PCR-1-1 and PCR-1-2 as previously described
31
. PCR products were then
digested with I-SceI or with XhoI. Band intensities of uncleaved 2.3 kb PCR fragments were
quantified and corrected for background and loading. Error-prone NHEJ was estimated by
calculating the difference between the corrected band intensities for I-SceI and for the
digestion control with XhoI.
In Vitro NHEJ Assay
Whole-cell extracts (WCE) preparation and in vitro NHEJ assay were performed using a
procedure adapted from Baumann34,35. Briefly, after washing in 1× PBS, cells were lysed
through three freeze/thaw cycles in LB buffer (25 mM Tris [pH 7.5], 333 mM KCl, 1.3 mM
EDTA, 4 mM DTT, protease and phosphatase inhibitors). Lysates were incubated for 20 min
at 4°C and cleared by centrifugation. Supernatants were dialyzed against 1× E buffer (20 mM
Tris [pH 8.0], 20% glycerol, 0.1 M K(OAc), 0.5 mM EDTA, 1 mM DTT). WCE were kept
frozen (-80°C) until use. For NHEJ assay, 15 µg of WCE was incubated (10 µl reaction) with
25 ng of linear DNA (EcoRI digested pEGFPN2) or 50 ng of linear DNA (EcoRI digested
pBluescript) in 1× LigB (250 mM Tris [pH 8.0], 300 mM K(OAc), 2.5 mM Mg(OAc)2, 5
mM ATP, 5 mM DTT, 0.5 mg/ml BSA, 1 µg/ml IP6) for 2 hr at 37°C. Reactions were then
treated with 1 µl RNase (1 mg/ml) for 5 min at RT and with 2 µl of 5× deproteination solution
(10 mg/ml Proteinase K, 2.5% SDS, 50 mM EDTA, 100 mM Tris [pH 7.5]) for 30 min at
55°C. After migration of the samples in 0.7% agarose, the gels were stained with SYBRGreen (30 min), and fluorescence was detected via a FluorImager.
Western blot analysis
Cells were washed in PBS and lysed in denaturing loading dye buffer for 20 min at 4°C.
Samples were boiled for 5 min and subjected to Western blot analysis using anti-JAK2, anti
[pY705]STAT5 antibodies (Cell Signaling Technology, Ozyme, Saint Quentin, France), anti-
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RAD51 (Santa Cruz, TEBU, Le Perray en Yvelines, France) and anti-β actin antibodies.
Antibodies were visualized using the ECL detection kit (GE Healthcare).
Immunofluorescence analysis
RAD51 foci were analyzed as described
36
. Briefly, after cytocentrifugation, smears were
fixed in absolute methanol for 15 min at -20°C and then rinsed in ice-cold acetone for a few
seconds. After three washes with PBS, the slides were incubated with blocking solution (PBS
with 5% BSA and 0.1% Tween 20) for 30 min and then incubated with a rabbit anti-RAD51
antibody (1/100) (Oncogene, VWR, Fontenay sous Bois, France) in a humidified incubator
for 2 hrs. The slides were then washed in PBS another three times for 10 min each and
incubated for 1 hr with Alexa 488-conjugated anti-rabbit IgG appropriately diluted with PBS.
After three additional washes with PBS, the preparations were counterstained with DAPI for 1
min. The slides were mounted with Fluoromount-G mounting medium. The number of
RAD51 foci per cell was measured and cells with more than 5 foci were considered as
positive. For centrosome analysis, cells were fixed and permeabilized during 15 min in cold
methanol followed by 10 sec in acetone. Centrosomes were counted in metaphases using an
anti-gamma-tubulin antibody (Sigma).
Patients
Peripheral blood samples were collected from PV and PMF patients. For PV, diagnoses were
made according to the modified Polycythemia Vera Study Group (PVSG) criteria 37, and the
Italian criteria for PMF
38
. Only JAK2V617-positive patients were selected after analyzing
mutational status using fluorescent competitive probes for quantitative real-time PCR on an
ABI 7500 (Applied Biosystems, Foster City, CA), as reported previously
4,32
. Control
peripheral blood samples were collected from patients undergoing cytapheresis. The study
was approved by the Local Research Ethics Committee from the Hôtel-Dieu and the Henri
Mondor hospitals and informed consent was obtained from each patient in accordance with
the Declaration of Helsinki (Paris, France).
9
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Purification and in vitro amplification of CD34+ cells
Mononuclear cells were separated over a Ficoll density gradient and CD34+ cells were
purified by a double-positive magnetic cell sorting system (AutoMACS, Miltenyi Biotec,
Paris, France), according to the manufacturer's recommendations. CD34+ cells were amplified
for 5 days in IMDM with penicillin/streptomycin/glutamine, alpha-thioglycerol, bovine serum
albumin (BSA), a mixture of sonicated lipids and insulin-transferrin, in the presence of 5
recombinant human cytokines (25 ng/ SCF, 100U/mL IL3, 10 ng/mL IL6, 10 ng/mL FLT3-L
and 10 ng/mL TPO).
Chromosomes
Cells were incubated with colchicine (50 µg/mL) for 2 hrs, collected and incubated in KCl
(12.5 mM) for 20 min at 37°C. Cells were fixed overnight in methanol/acetic acid (3/1 v/v),
washed three times in methanol/acetic acid, spread on cold microscope slides, and stained
with 3% Giemsa in 1.5% phosphate buffer pH 6.7. Metaphases were analyzed by microscopy.
Sister chromatid exchange assay
A total of 3.5 x 105 cells were plated in a 24-well plate containing 4 µg/mL of BrdU. After
two cell divisions in the presence of BrdU (30 hrs), colcemid (Sigma) was added at a final
concentration of 0.5 µg/mL for 2 hrs. Cells were then washed with PBS, trypsinized, collected
and centrifuged. Cells in pellet were dispersed in 10 mL of 0.075 M KCl and incubated for
15 min at 37°C. Before centrifugation, 100 µL of the fixative (methanol–acetic acid, 3:1) was
added. The pellet was suspended in the fixative and incubated overnight at 4°C. Cells were
then centrifuged, washed in a fresh fixative and dropped onto wet slides. The slides were
covered for 20 min with 10 µg/mL of Hoechst 33258 (Sigma) dissolved in water and
subsequently incubated in a solution containing 0.3 M NaCl and 30 mM sodium citrate at pH
6.3 (SSC 2x, pH 6.3) for 2 hrs under UV light. The slides were plunged in SSC 2x, pH 6.3 at
60°C for 15 min and stained with 1.5% Giemsa for 3–5 min.
Mutagenesis measurements
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A fluctuation analysis was used to estimate spontaneous mutagenesis. For each cell line
analyzed, several independent cultures were plated and cultured to confluence. Cells were
then counted and one part was used for plating efficiency estimation. The remaining cells
were plated in methylcellulose with EPO and the mutant colonies were selected by either 2
mM ouabain or 20 µM 6-TG. The number of ouabain- or 6-TG-resistant clones allowed us to
calculate the mutagenesis frequency. The rate of recombination or mutagenesis per cell per
generation was calculated by using the fluctuation analysis of Luria and Delbrück
39,40
.
11
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Results
JAK2 activation induces an increase in HR
The Ba/F3 cell line expressing the EPO receptor was electroporated with the HR vector (HREGFP/3’EGFP) (Figure 1A)
31
and puromycin resistant clones were isolated in
methylcellulose. Several clones were amplified, genomic DNA was extracted and PCR was
performed to verify vector integration (Figure 1B). This intrachromosomal HR vector
monitors all kinds of HR events, including HR events associated with and without crossingover and single-strand annealing events, by measuring the frequency of GFP+ cells. To study
the impact of JAK2 activation on HR, Ba/F3-HR2 cells were infected with either JAK2wt,
JAK2V617F, TEL/JAK2 or the empty vector. Overexpression of different forms of JAK2 and
ectopic expression of TEL/JAK2 were verified by Western blotting using an anti-JAK2
antibody (Figure 1C). Then, we checked JAK2 activity by measuring STAT5 phosphorylation
by western-blotting using an anti-[pY705]STAT5 antibody. In Ba/F3-HR2 cells, EPO
stimulated a transient phosphorylation of STAT5, whereas phosphorylation was more
pronounced and prolonged in JAK2wt overexpressing cells. In contrast, a spontaneous
STAT5 phosphorylation and sustained activation were observed in JAK2V617F overexpressing
cells (Figure 1D). Moreover, in the presence of EPO, JAK2V617F and to a lesser extent
JAK2wt induced a 20- and 5-fold increase in spontaneous HR, respectively, compared to
Ba/F3-HR2 parental cells (Figure 1E). As previously described, TEL/JAK2 induced an
increase in HR
41
. Importantly, when we infected cells with a lentiviral vector encoding the
rare-cutting meganuclease I-SceI, which targets a unique double-strand break (DSB) in the
HR vector
42
, induced-HR increased significantly in JAK2V617F and TEL/JAK2
overexpressing cells compared to Ba/F3-HR2 parental cells (Figure 1F).
RAD51 is a key protein involved in HR, whose expression is deregulated by an ectopic
expression of BCR/ABL and TEL/JAK2
29,41
. Surprisingly, we did not find any significant
changes in RAD51 protein levels in Ba/F3 cells expressing human or murine JAK2 constructs
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(Figure 2A and data not shown).
After a genotoxic stress, RAD51 assembles into nuclear DNA repair foci
36,43
. Thus, we
counted the number of cells with RAD51 foci (Figure 2C) and the number of foci per cell
(Figure 2D) after immunostaining with an anti-RAD51 antibody. As a positive control, we
treated Ba/F3-EPOR cells with Mitomycin C (MMC) for 3 hrs and found a 5-fold increase in
the number of cells with RAD51 foci. As a confirmation of our observations that HR was
increased by JAK2 activation, we observed in unchallenged cells a significant increase both in
the number of cells harboring nuclear RAD51 foci and in the number of foci/cell in Ba/F3JAK2V617F and to a lesser extent in Ba/F3-JAK2wt compared to parental cells (Figure 2B, C
and D). Since the amount of RAD51 is known to vary during cell cycle, we verified whether
the Ba/F3-JAK2V617F cell cycle was not altered and found no difference compare to the
control cell line (Figure 2E). Altogether, the present results show that activation of JAK2
greatly stimulates HR in the Ba/F3-EPOR cell line.
JAK2 activation has no significant impact on NHEJ efficiency and fidelity.
We then wanted to determine the impact of JAK2 activation on NHEJ since both NHEJ and
HR can lead to genome rearrangement. First, we analyzed in vitro end-joining of two
linearized plasmid DNA by using cell-free extracts
34,35
(Figure 3A and B). This DNA-end
ligation assay, which results in the formation of DNA concatemers requires a functional
NHEJ apparatus, as demonstrated by the absence of DNA oligomers when using extracts from
a DNA Ligase IV deficient cell line (N114) versus parental lymphoid cell line (Nalm6). When
using extracts from Ba/F3-EPOR, Ba/F3-JAK2wt or Ba/F3-JAK2V617F cell lines, we found no
significant changes in DNA end-joining between the three Ba/F3 cell lines (Figure 3A and B).
We then investigated the NHEJ fidelity by means of the intrachromosomal substrate (HREGFP/3’EGFP). Ba/F3-HR2 cells were infected or not with I-SceI virus and EGFP- cells were
sorted to exclude HR events. In the remaining cells, error-free religation may have
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reconstituted the I-SceI site after cleavage, whereas error-prone NHEJ may have caused loss
of the I-SceI site. Therefore, we performed PCR reactions as indicated in Figure 3C and
cleaved the resulting fragments with I-SceI and as a control digest with XhoI. Error-prone
NHEJ was estimated by quantification of the intensities of the uncleaved 2.3 kb bands for ISceI and XhoI digests (corrected for background), subsequent calculation of the fraction of
the 2.3 kb fragment in total DNA (loading), and comparison of specific I-SceI versus control
XhoI digest values (Figure 3D). The data from three experiments did not indicate significant
differences between the three cell lines regarding specific I-SceI site alteration, which is
indicative of error-prone NHEJ. Altogether, these results indicate that JAK2 activation has no
impact on both NHEJ efficiency and fidelity.
JAK2 activation induces genetic instability
We further investigated whether the JAK2-induced hyper-recombination phenotype could be
associated with genetic instability. Indeed, previous reports have shown that HR mutants for
RAD51, XRRC2 or XRCC3 lead to centrosome and ploidy defects 21,44,45. For this reason, we
measured the numbers of centrosomes in mitosis of the different cell lines by
immunofluorescence using an anti-γ-tubulin antibody (Figure 4A). The Ba/F3-EPOR cells
exhibited a low number of mitosis (3.2%) where more than two centrosomes could be seen.
However, a higher occurrence of centrosome abnormalities was recorded in the Ba/F3JAK2V617F cell lines (12.4% of mitosis) (p<0.001)(Figure 4B). To a lesser degree, centrosome
abnormalities were found in 6.9% of mitosis in the Ba/F3-JAK2wt cell lines, a percentage
slightly elevated compared to the parental cell line (p=0.0816). These results show that JAK2
activation induces an increase in the number of centrosomes during mitosis. Since centrosome
defects should lead to aneuploidy, we measured cell ploidy. As shown in Figure 4C, a
significantly increased number of aneuploid cells was seen for Ba/F3-JAK2wt and Ba/F3JAK2V617F cells as compared to the parental cell line as indicated by different distribution
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(P<0.01). Indeed, we observed both an increase in the number of metaphases with less than
39 chromosomes (corresponding to a loss of chromosome) as well as of metaphases with
more than 39 chromosomes (corresponding to a gain of chromosome) (Figure 4D). Thus,
aneuploidy corresponded to both gain and loss of chromosomes and was consistent with the
increased centrosome abnormalities detected after JAK2 activation. In addition, we also
observed a significant increase in metaphase corresponding to a tetraploid content in Ba/F3JAK2V617F cells (16.4%) and Ba/F3-JAK2wt (6.1%) versus control Ba/F3 (2.8%).
We also evaluated the genomic instability by measuring the sister chromatid exchange (SCE),
which is the result of HR associated with crossing-over mechanism. An increase (56%) in
spontaneous SCE in Ba/F3-JAK2V617F metaphases was observed compared to Ba/F3 cells and
Ba/F3-JAK2wt (Figure 4E). As SCE involves HR, this result also fits with a JAK2-induced
hyper-recombination phenotype.
Altogether, these results suggest that JAK2 activation may produce genetic instability.
JAK2V617F induces a mutator phenotype
The above data show that JAK2 activation deregulates the HR pathway. Consequently, one
prediction would be that mutagenesis should be increased in cells expressing ectopic JAK2.
For that reason, we measured spontaneous mutagenesis at two different loci: the Na+/K+ATPase membrane gene and the HPRT gene that leads to ouabain resistance and 6thioguanine (6-TG) resistance, respectively, in mutant cells. Whereas Na+/K+-ATPase
membrane mutants reflect only point mutations, HPRT mutants monitor both point mutations,
deletions and insertions
46
. We first measured spontaneous mutagenesis in the HPRT locus.
Spontaneous mutagenesis was calculated by fluctuation analysis using the Luria and Delbrück
or the Capizzi and Jameson assays
39,40
. In Ba/F3-EPOR cells, JAK2V617F led to a 100-fold
increase in the spontaneous rate of mutagenesis per cell per generation compared to control or
Ba/F3-JAK2wt cells (Table I). We then measured spontaneous mutagenesis in the Na+/K+-
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ATPase membrane locus. At this locus, JAK2V617F induced a 10-fold increase in the
spontaneous rate of mutagenesis per cell per generation compared to control or Ba/F3JAK2wt cells (Table I). Altogether, these findings show that JAK2V617F generates a mutator
phenotype that can be potentially associated with punctual mutations, deletion and/or
insertion.
JAK2 activation induces a drug resistance towards HR-inducing agents.
Ba/F3 parental or Ba/F3 cells expressing either JAK2wt or JAK2V617F were treated for one hr
with genotoxic drugs and, viable cells were counted by trypan blue exclusion test 48 hrs later.
First, the cell lines were treated with the DNA cross-linking drug MMC since HR is
stimulated in BCR/ABL-expressing cells exhibiting resistance to MMC, on one hand, and
sensitivity to this drug is a hallmark of a defective HR pathway, on the other hand
29
.
Expression of JAK2V617F in Ba/F3 cells resulted in a 4-fold to 5-fold increased resistance to
MMC (IC50= 3.0 µM) compared to parental Ba/F3 (IC50= 0.7 µM) or Ba/F3-JAK2wt (IC50=
0.6 µM) cells, respectively (Figure 5A). Second, we treated the cell lines with bleomycin
since this agent generates DNA double strand breaks that can be repaired by HR mechanism
47,48
. The expression of JAK2V617F in Ba/F3 cells resulted in a 13-fold increased resistance to
bleomycin (IC50= 9.0 µM) compared to parental Ba/F3 cells (IC50= 0.7 µM) (Figure 5B).
Moreover, JAK2wt induced a resistance to bleomycin but to a lesser extent than JAK2V617F
(IC50= 4.6 µM). Importantly, under these treatment conditions (1 hr-exposure), neither typical
morphological features of apoptosis nor caspase-3 cleavage were found in contrast to the
situation after continuous exposure with these agents (Figures 5C and D). Altogether, these
results show that a strong activation of JAK2 induces a resistance against genotoxic agents
rather due to increased repair than decreased apoptosis.
16
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Elevated HR activity and RAD51 nuclear foci in PV and MF patients.
In order to confirm the hyper-recombination phenotype observed in the JAK2V617F-expressing
cell line, we measured nuclear RAD51 foci in cells from JAK2V617F-positive patients (at least
heterozygous for JAK2V617F in granulocytes at the diagnosis). For this purpose, CD34+ cells
were immunopurified from the blood of 5 PV patients and 6 MF (5 PMF and 1 post-PV MF)
patients. CD34+ cells were then amplified for 5 days with 5 cytokines (SCF/IL3/FLT3L/IL6/TPO) and the number of cells presenting RAD51 foci was counted for each patient
(Figure 6A). CD34+ cells from G-CSF-mobilized healthy donors were used as a control. We
observed that PV and MF cells displayed a high number of cells with RAD51 foci compared
to mobilized blood (Figure 6B). We next investigated the HR activity in CD34+ cells from the
blood of 3 PV patients, 2 PMF patients and 3 mobilized donors. CD34+ cells were amplified
for 5 days with 3 cytokines (SCF/IL3/EPO) and infected with the retrovirus containing the
HR substrate. Importantly, no difference in the proliferation was found in CD34+ cells from
healthy donors versus patients under these culture conditions (Figure 6C). In these
experiments and as measured by the % of GFP+ cells, we observed that cells derived from PV
and MF patients displayed a higher HR activity compared to mobilized blood (Figure 6D).
Taken together, our data indicate that JAK2V617F also induced a hyper-recombination
phenotype in MPD patients.
17
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Discussion
In the present study, we have investigated the effect of the JAK2V617F mutation on HR to
understand whether this could lead to genetic instability and explain disease progression. The
point mutation lies in exon 14 of the JAK2 gene and results in a valine to phenylalanine
substitution at position 617 (V617F) in the pseudo-kinase domain of JAK2. This mutation
induces a spontaneous activation of the tyrosine kinase, which seems to require a
homodimeric type I cytokine receptor. However the presence of such a receptor does not
appear totally indispensable, 4,32,49 but a receptor such as the erythropoietin receptor (EPO-R)
facilitates the transforming effects of JAK2V617F 49,50. Our results show, for the first time, that
activation of an overexpressed JAK2wt in the presence of EPO resulted in an increase in both
HR activity and RAD51 nuclear foci formation in the Ba/F3-EPOR cell line. Overexpression
of JAK2V617F in Ba/F3-EPOR cells had an even more potent effect probably due to its stronger
activity compared to JAK2wt (Figure 1D). Importantly, the concomitant increase in
spontaneous HR activity and RAD51 nuclear foci formation was also observed in CD34+
derived cells from JAK2V617F positive PV and PMF patients indicating a hyper-recombination
phenotype. These results are consistent with previous data showing that strong JAK2
activation by TEL/JAK2 fusion tyrosine kinase stimulated HR
41
, a result confirmed in the
present work. Furthermore, our findings also fit with the BCR/ABL-induced hyperrecombination phenotype observed in CML 29 and, therefore, strengthen the assumption that
numerous kinases are capable of interfering with HR.
Although the increase in HR measured intrachromosomally correlated with RAD51 focus
formation, we were unable to detect change in RAD51 protein levels by JAK2 activation in
contrast to CML, 29. However, these differences in RAD51 levels associated with BCR/ABL
versus JAK2 activation may be related to differences in the cell cycle distribution, since low
levels of RAD51 are found in G0/G1 followed by an increase during S and G2/M
51
.
Consistent with the stable RAD51 expression, we did not observe marked cell cycle changes
18
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in cells expressing either JAK2wt or JAK2V617Fcompared to control cells.
Since deregulation in NHEJ can also lead to genome rearrangements
30
and since HR and
NHEJ might be competitive mechanisms52, we also investigated the impact of JAK2
activation on NHEJ mechanism, but did not find any significant change either in the
efficiency or in the fidelity of NHEJ. These results represent the first marked difference in the
regulation of DNA repair between CML and JAK2V617F-positive-MPD since BCR/ABL is
able to greatly activate NHEJ 53.
Since excess in HR can promote genetic instability 16,17, the hyper-recombination phenotype
induced by JAK2V617F may explain both the heterogeneity of MPD and disease progression.
Moreover, since gene conversion between homologs is a product of HR and leads to loss of
heterozygosity 18,54, the JAK2V617F-induced hyper-recombination phenotype might account for
the loss of heterozygosity in 90% of PV patients 3. The work by Kralovics et al. suggests that
mitotic recombination is the major mechanism for loss of heterozygosity 3. Importantly,
mitotic recombination requires HR and is facilitated by a hyper-recombination state.
However, the exact mechanism by which JAK2V617F controls the hyper-recombination
remains to be investigated. One can speculate that – in analogy to BCR/ABL- this mechanism
could be indirect through the activation of MAPK, PI3K or STAT5 or via direct tyrosine
phosphorylation of RAD51 29.
In vertebrate cells, dysfunction of HR can result in genome instability 21,22,44,45 and increased
mutagenesis 55. Defective centrosome duplication leads to multipolar mitotic cells resulting in
aberrant chromosome segregation and aneuploidy, a hallmark of tumor cells. Interestingly,
this study also revealed that activation of JAK2 generates centrosome abnormalities
associated with chromosomal instability. This situation is highly consistent with JAK2 impact
on HR which leads to genetic instability as monitored by mutagenesis assays. Mutagenesis
results further suggested that JAK2V617F not only generates point mutations but also increases
deletions and/or insertions based on the differences between the inducible mutation rates
19
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obtained in Na+/K+ ATPase and HPRT loci. It is worthwhile noting that only JAK2V617F was
able to stimulate mutagenesis suggesting that a strong activation of the kinase is necessary to
generate this mutator phenotype. Alternatively JAK2V617F may phosphorylate other substrates
than JAK2wt. Thus, JAK2V617F -induced chromosomal instability and mutagenesis may
explain the numerous different cytogenetic abnormalities found in rare cases of ET, PV
(around 10%) and more frequently in MF (20% to 40%) 56-58.
As previously reported in BCR/ABL-expressing cells
29
, our study also provides
evidences that JAK2V617F induces resistance to genotoxic agents such as MMC and bleomycin
which are considered to be good inducers of HR
44
. Importantly, we excluded the fact that
JAK2 activation induced-resistance was due to decreased apoptosis since no apoptosis was
found under our conditions of treatments. The discrepancy found in the resistance against
these two agents may result from the fact that they differentially damage DNA. MMC is a
DNA crosslinker that may stimulate HR and nucleotide excision repair (NER). Bleomycin
works through free-radicals and causes DNA strand breaks 47, in which at least HR, NHEJ or
base excision repair (BER) could be implicated. It remains to be determined whether
JAK2V67F may also modify other DNA repair mechanisms such as NER or BER. Moreover,
the differences in the resistance observed between JAK2wt and JAK2V67F may come from
their respective activities as shown in figure 1D. One can hypothesize that this resistance
could be due either to accelerated DNA repair of the genotoxic lesions by HR and/or to the
mutator phenotype induced by JAK2V617F. Our findings may have major implications for
future strategies in predicting the individual response of post-MPD leukemia patients to
chemotherapeutic treatments, depending on the type of drug as well as the dosage applied.
Additionally, these data may implicate that JAK2V617F can induce resistance to JAK2
inhibitors via the mutator phenotype.
In conclusion, our data show that strong activation of JAK2V617F stimulates HR, centrosome
and ploidy abnormalities, and induces a mutator phenotype and a resistance against genotoxic
20
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agents. Taken together, this study suggests for the first time that the heterogeneity of MPD
features and its evolution into secondary leukemia could be in part ascribed to JAK2V617Finduced genetic instability.
Authorship
Contribution: I. Plo performed all the experiments, wrote the manuscript and designed the
study. M.Nakatake assisted IP in all experiments. L. Malivert and J.P. de Villartay did the
NHEJ efficiency experiments and contributed to the discussion. Lisa Wiesmuller provide the
HR vector and contributed to the discussion, S. Giraudier and J.L Villeval collected the
samples of PV and PMF patients, W. Vainchenker designed the study, supervised the work,
wrote the manuscript and was responsible for the final draft of the manuscript.
Conflict-of-interest disclosure: The authors declare no potential conflict of interest.
Correspondence: William Vainchenker, INSERM U790, Institut Gustave Roussy, 39 Camille
Desmoulins, 94805 Villejuif, France; e-mail: [email protected].
ACKNOWLEDGEMENTS
This work was supported by grants from Ligue Nationale Contre le Cancer “Equipe labellisée
2007, INCa (projets libres 2006) and INSERM. IP was funded by the Fondation pour la
Recherche Médicale. MN was funded by a post-doctoral fellowship from Ligue Nationale
Contre le Cancer.
REFERENCES
1.Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of the tyrosine kinase JAK2
in human myeloproliferative disorders. Lancet. 2005;365:1054-1061.
2.
James C, Delhommeau F, Marzac C, et al. Detection of JAK2 V617F as a first
intention diagnostic test for erythrocytosis. Leukemia. 2006;20:350-353.
3.
Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in
myeloproliferative disorders. N Engl J Med. 2005;352:1779-1790.
4.
Levine RL, Wadleigh M, Cools J, et al. Activating mutation in the tyrosine kinase
JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with
21
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
myelofibrosis. Cancer Cell. 2005;7:387-397.
5.
Zhao R, Xing S, Li Z, et al. Identification of an acquired JAK2 mutation in
polycythemia vera. J Biol Chem. 2005;280:22788-22792.
6.
Spivak JL. Diagnosis of the myeloproliferative disorders: resolving phenotypic
mimicry. Semin Hematol. 2003;40:1-5.
7.
Spivak JL. The chronic myeloproliferative disorders: clonality and clinical
heterogeneity. Semin Hematol. 2004;41:1-5.
8.
Spivak JL, Barosi G, Tognoni G, et al. Chronic myeloproliferative disorders.
Hematology Am Soc Hematol Educ Program. 2003:200-224.
9.
Kaushansky K. On the molecular origins of the chronic myeloproliferative disorders: it
all makes sense. Blood. 2005;105:4187-4190.
10.
Lacout C, Pisani DF, Tulliez M, Gachelin FM, Vainchenker W, Villeval JL.
JAK2V617F expression in murine hematopoietic cells leads to MPD mimicking human PV
with secondary myelofibrosis. Blood. 2006;108:1652-1660.
11.
Tiedt R, Hao-Shen H, Sobas MA, et al. Ratio of mutant JAK2-V617F to wild type
JAK2 determines the MPD phenotypes in transgenic mice. Blood. 2007.
12.
Lippert E, Boissinot M, Kralovics R, et al. The JAK2-V617F mutation is frequently
present at diagnosis in patients with essential thrombocythemia and polycythemia vera.
Blood. 2006;108:1865-1867.
13.
Dupont S, Masse A, James C, et al. The JAK2 617V>F mutation triggers
erythropoietin hypersensitivity and terminal erythroid amplification in primary cells from
patients with polycythemia vera. Blood. 2007;110:1013-1021.
14.
Scott LM, Scott MA, Campbell PJ, Green AR. Progenitors homozygous for the V617F
mutation occur in most patients with polycythemia vera, but not essential thrombocythemia.
Blood. 2006;108:2435-2437.
15.
Passamonti F, Rumi E, Pietra D, et al. Relation between JAK2 (V617F) mutation
status, granulocyte activation, and constitutive mobilization of CD34+ cells into peripheral
blood in myeloproliferative disorders. Blood. 2006;107:3676-3682.
16.
Bertrand P, Saintigny Y, Lopez BS. p53's double life: transactivation-independent
repression of homologous recombination. Trends Genet. 2004;20:235-243.
17.
Purandare SM, Patel PI. Recombination hot spots and human disease. Genome Res.
1997;7:773-786.
18.
Haigis KM, Dove WF. A Robertsonian translocation suppresses a somatic
recombination pathway to loss of heterozygosity. Nat Genet. 2003;33:33-39.
19.
Amor M, Parker KL, Globerman H, New MI, White PC. Mutation in the CYP21B
gene (Ile-172----Asn) causes steroid 21-hydroxylase deficiency. Proc Natl Acad Sci U S A.
1988;85:1600-1604.
20.
Gangloff S, Soustelle C, Fabre F. Homologous recombination is responsible for cell
death in the absence of the Sgs1 and Srs2 helicases. Nat Genet. 2000;25:192-194.
21.
Bertrand P, Lambert S, Joubert C, Lopez BS. Overexpression of mammalian Rad51
does not stimulate tumorigenesis while a dominant-negative Rad51 affects centrosome
fragmentation, ploidy and stimulates tumorigenesis, in p53-defective CHO cells. Oncogene.
2003;22:7587-7592.
22.
Deans B, Griffin CS, O'Regan P, Jasin M, Thacker J. Homologous recombination
deficiency leads to profound genetic instability in cells derived from Xrcc2-knockout mice.
Cancer Res. 2003;63:8181-8187.
23.
Sonoda E, Sasaki MS, Buerstedde JM, et al. Rad51-deficient vertebrate cells
accumulate chromosomal breaks prior to cell death. Embo J. 1998;17:598-608.
24.
Shinohara A, Ogawa H, Matsuda Y, Ushio N, Ikeo K, Ogawa T. Cloning of human,
mouse and fission yeast recombination genes homologous to RAD51 and recA. Nat Genet.
1993;4:239-243.
22
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
25.
Yoshikawa K, Ogawa T, Baer R, et al. Abnormal expression of BRCA1 and BRCA1interactive DNA-repair proteins in breast carcinomas. Int J Cancer. 2000;88:28-36.
26.
Maacke H, Jost K, Opitz S, et al. DNA repair and recombination factor Rad51 is overexpressed in human pancreatic adenocarcinoma. Oncogene. 2000;19:2791-2795.
27.
Maacke H, Opitz S, Jost K, et al. Over-expression of wild-type Rad51 correlates with
histological grading of invasive ductal breast cancer. Int J Cancer. 2000;88:907-913.
28.
Raderschall E, Stout K, Freier S, Suckow V, Schweiger S, Haaf T. Elevated levels of
Rad51 recombination protein in tumor cells. Cancer Res. 2002;62:219-225.
29.
Slupianek A, Schmutte C, Tombline G, et al. BCR/ABL regulates mammalian RecA
homologs, resulting in drug resistance. Mol Cell. 2001;8:795-806.
30.
Guirouilh-Barbat J, Huck S, Bertrand P, et al. Impact of the KU80 pathway on NHEJinduced genome rearrangements in mammalian cells. Mol Cell. 2004;14:611-623.
31.
Akyuz N, Boehden GS, Susse S, et al. DNA substrate dependence of p53-mediated
regulation of double-strand break repair. Mol Cell Biol. 2002;22:6306-6317.
32.
James C, Ugo V, Le Couedic JP, et al. A unique clonal JAK2 mutation leading to
constitutive signalling causes polycythaemia vera. Nature. 2005;434:1144-1148.
33.
Royer Y, Menu C, Liu X, Constantinescu SN. High-throughput gateway bicistronic
retroviral vectors for stable expression in mammalian cells: exploring the biologic effects of
STAT5 overexpression. DNA Cell Biol. 2004;23:355-365.
34.
Baumann P, West SC. DNA end-joining catalyzed by human cell-free extracts. Proc
Natl Acad Sci U S A. 1998;95:14066-14070.
35.
Buck D, Malivert L, de Chasseval R, et al. Cernunnos, a novel nonhomologous endjoining factor, is mutated in human immunodeficiency with microcephaly. Cell.
2006;124:287-299.
36.
Haaf T, Golub EI, Reddy G, Radding CM, Ward DC. Nuclear foci of mammalian
Rad51 recombination protein in somatic cells after DNA damage and its localization in
synaptonemal complexes. Proc Natl Acad Sci U S A. 1995;92:2298-2302.
37.
Michiels JJ, Barbui T, Finazzi G, et al. Diagnosis and treatment of polycythemia vera
and possible future study designs of the PVSG. Leuk Lymphoma. 2000;36:239-253.
38.
Barosi G, Ambrosetti A, Finelli C, et al. The Italian Consensus Conference on
Diagnostic Criteria for Myelofibrosis with Myeloid Metaplasia. Br J Haematol.
1999;104:730-737.
39.
Capizzi RL, Jameson JW. A table for the estimation of the spontaneous mutation rate
of cells in culture. Mutat Res. 1973;17:147-148.
40.
Luria SE, Delbruck M. Mutations of Bacteria from Virus Sensitivity to Virus
Resistance. Genetics. 1943;28:491-511.
41.
Slupianek A, Hoser G, Majsterek I, et al. Fusion tyrosine kinases induce drug
resistance by stimulation of homology-dependent recombination repair, prolongation of
G(2)/M phase, and protection from apoptosis. Mol Cell Biol. 2002;22:4189-4201.
42.
Liang F, Han M, Romanienko PJ, Jasin M. Homology-directed repair is a major
double-strand break repair pathway in mammalian cells. Proc Natl Acad Sci U S A.
1998;95:5172-5177.
43.
Aten JA, Stap J, Krawczyk PM, et al. Dynamics of DNA double-strand breaks
revealed by clustering of damaged chromosome domains. Science. 2004;303:92-95.
44.
Daboussi F, Thacker J, Lopez BS. Genetic interactions between RAD51 and its
paralogues for centrosome fragmentation and ploidy control, independently of the sensitivity
to genotoxic stresses. Oncogene. 2005;24:3691-3696.
45.
Griffin CS. Aneuploidy, centrosome activity and chromosome instability in cells
deficient in homologous recombination repair. Mutat Res. 2002;504:149-155.
46.
Dass SB, Heflich RH, Casciano DA. The mutagenic response at the ouabain resistance
locus in T cells of mice exposed to N-ethyl-N-nitrosourea parallels the response at the Hprt
23
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
locus and correlates with mutation target size. Carcinogenesis. 1997;18:2233-2237.
47.
Letavayova L, Markova E, Hermanska K, et al. Relative contribution of homologous
recombination and non-homologous end-joining to DNA double-strand break repair after
oxidative stress in Saccharomyces cerevisiae. DNA Repair (Amst). 2006;5:602-610.
48.
Moore CW, McKoy J, Dardalhon M, Davermann D, Martinez M, Averbeck D. DNA
damage-inducible and RAD52-independent repair of DNA double-strand breaks in
Saccharomyces cerevisiae. Genetics. 2000;154:1085-1099.
49.
Funakoshi-Tago M, Pelletier S, Moritake H, Parganas E, Ihle JN. Jak2 FERM domain
interaction with the Erythropoietin Receptor regulates Jak2 kinase activity. Mol Cell Biol.
2007.
50.
Lu X, Levine R, Tong W, et al. Expression of a homodimeric type I cytokine receptor
is required for JAK2V617F-mediated transformation. Proc Natl Acad Sci U S A.
2005;102:18962-18967.
51.
Chen F, Nastasi A, Shen Z, Brenneman M, Crissman H, Chen DJ. Cell cycledependent protein expression of mammalian homologs of yeast DNA double-strand break
repair genes Rad51 and Rad52. Mutat Res. 1997;384:205-211.
52.
Allen C, Kurimasa A, Brenneman MA, Chen DJ, Nickoloff JA. DNA-dependent
protein kinase suppresses double-strand break-induced and spontaneous homologous
recombination. Proc Natl Acad Sci U S A. 2002;99:3758-3763.
53.
Gaymes TJ, Mufti GJ, Rassool FV. Myeloid leukemias have increased activity of the
nonhomologous end-joining pathway and concomitant DNA misrepair that is dependent on
the Ku70/86 heterodimer. Cancer Res. 2002;62:2791-2797.
54.
Haigis KM, Caya JG, Reichelderfer M, Dove WF. Intestinal adenomas can develop
with a stable karyotype and stable microsatellites. Proc Natl Acad Sci U S A. 2002;99:89278931.
55.
Lambert S, Lopez BS. Inactivation of the RAD51 recombination pathway stimulates
UV-induced mutagenesis in mammalian cells. Oncogene. 2002;21:4065-4069.
56.
Bench AJ, Cross NC, Huntly BJ, Nacheva EP, Green AR. Myeloproliferative
disorders. Best Pract Res Clin Haematol. 2001;14:531-551.
57.
Gangat N, Strand J, Lasho TL, et al. Cytogenetic studies at diagnosis in polycythemia
Vera: Clinical and JAK2V617F allele burden correlates. Eur J Haematol. 2007.
58.
Tefferi A, Mesa RA, Schroeder G, Hanson CA, Li CY, Dewald GW. Cytogenetic
findings and their clinical relevance in myelofibrosis with myeloid metaplasia. Br J Haematol.
2001;113:763-771.
24
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
25
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
Figure legends
Figure 1. JAK2 stimulates HR in Ba/F3 cell lines. A/ HR substrate (HR-EGFP/3’EGFP)
contains a tandem repeat of two inactive EGFP genes, HR-EGFP and 3’EGFP. When the ISceI endonuclease is expressed, a DSB is introduced at the I-SceI site in the HR-EGFP gene.
Recombination restores a functional EGFP gene and the recombinant cells can be monitored
by fluorescence detection methods. Importantly, since the 3’EGFP cassette is only deleted at
the 5' end, EGFP-fluorescence not only monitors gene conversion events associated with or
without crossing-over, but also single strand annealing. B/ Ba/F3-EPOR cells were
electroporated with HR substrate and the puromycin resistant colonies were isolated on
methylcellulose. Genomic DNA was prepared and intrachromosomal integration was verified
by PCR as detailed in Material and Methods. C/D Ba/F3-HR2 were infected with the empty
retroviral vector or viruses encoding JAK2wt, JAK2V617F or TEL-JAK2, (C) JAK2 protein
levels were analyzed by Western-blotting using an anti-JAK2 antibody or (D) STAT5
phosphorylation was detected after EPO stimulation (10U/ml) at various times by westernblotting analysis using an anti- [pY 705]STAT5 antibody. E/ GFP+ cells were measured by
flow cytometry analysis to determine HR. Percentages of GFP+ cells are given as the mean
±SD of at least 6 independent experiments. F/ Cells were infected or not with a lentivirus
encoding I-SceI and induced HR was calculated as the difference between the % of GFP
+
cells infected with I-SceI and the % of GFP+ cells non-infected for each conditions. Results
are given as the mean ±SD of 4 independent experiments and * represents statistical
significance (p<0.05) using student t test.
Figure 2. JAK2 promotes RAD51 focus formation in Ba/F3 cell lines. A/ Ba/F3-EPOR
cells were infected with the empty retroviral vector or viruses encoding JAK2wt or JAK2V617F
and RAD51 protein levels were analyzed by Western-blotting using an anti-RAD51 antibody.
B/ The figure illustrates cells with RAD51 foci (green) after immunostaining in spontaneous
26
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condition. Alternatively, Ba/F3-EPOR cells were treated for 3 hrs with MMC as a positive
control. C/D The percentage of cells with RAD51 foci (C) or the number of RAD51 foci per
cell (D) were calculated after immunostaining with RAD51 antibody. Two hundred cells were
counted in three independent experiments, the percentages are the mean reported ±SD (n=3)
and * represents statistical significance (p<0.05) using student t test. E/ Cell cycle
distributions of Ba/F3-EPOR, Ba/F3-JAK2wt, Ba/F3-JAK2V617F cells in culture conditions
containing EPO were analyzed by cytometry analysis after propidium iodide staining.
Figure 3. NHEJ efficiency and fidelity in Ba/F3 cell lines. WCE from a DNA Ligase
IVdeficient cell line (N114) and parental lymphoid cell line (Nalm6) and Ba/F3-EPOR cells
were incubated with the linearized plasmids pEGFPN2 (A) or pBluescript (B) for in vitroanalysis of DNA end ligation activities. The reaction products were run on agarose gel and
stained with SYBR-Green. Multimerized DNA molecules (nX) were formed. Linearized
plasmid can also be found in supercoiled state (sc). C/ Scheme of PCR products and the
expected sizes of these fragments after restriction enzyme cleavage for the analysis of errorprone NHEJ. D/ Ba/F3-HR2 cells were infected or not with I-SceI virus, incubated for 96 hrs
for DSB repair to take place, EGFP+ cells were excluded by FACSsort, genomic DNA
isolated, and PCR analysis performed. A representative gel shows the PCR fragments with
and without I-SceI and XhoI digestion for the three Ba/F3 cell lines.
Figure 4. JAK2V617F induces centrosome and chromosome abnormalities.
A/ This Figure illustrates centrosome abnormalities in mitosis. Examples of Ba/F3-JAK2V617F
cells with two, three or four centrosomes visualized by immunofluorescence with an anti-γ
tubulin antibody (upper panels). The lower panels illustrate the corresponding Hoechst
staining showing the cell nucleus. B/ The percentage of cells in metaphase with extra
centrosomes (>2) was measured in Ba/F3-expressing JAK2wt or JAK2V617F cells or in
parental cell lines. At least 200 cells in metaphase were counted and significant difference
was analyzed using χ2 tests. * represents significant differences (p<0.001) compared with
27
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Ba/F3 control cell lines. C/ The ploidy of the different cell lines was analyzed. At least 100
metaphases per cell line were analyzed and a non parametric ANOVA was used for statistical
significance of the distribution. Significant difference (p<0.01) was found in Ba/F3
expressing JAK2wt and JAK2V617F compared with Ba/F3 control cell lines. D/ Histograms
represent the percentage of cells with 39 chromosomes and less or more than 39
chromosomes in Ba/F3 cell lines. E/ This Figure illustrates an example of metaphase spread
from Ba/F3-EPOR cells displaying SCE. Then, SCE were counted on metaphases in each cell
lines. At least 50 metaphases were counted and a non parametric ANOVA was used for
statistical significance of the distribution. Significant difference (p<0.001) was found in
Ba/F3 expressing JAK2V617F compared with Ba/F3 control cell lines.
Figure 5. JAK2V617F induces resistance towards genotoxic agents.
Cells were treated for 1 hr-exposure with various concentrations of (A) MMC or (B)
bleomycin and cell viability was assessed by trypan blue exclusion 48 hrs later. * represents
statistical significance (p<0.05) using student t test. C/ Morphological features were analyzed
after DAPI staining either in untreated cells or in 1hr-treated cells to identify potentially
apoptotic cells. Continuous MMC treatment for 48 hours was used as a positive control (% of
apoptotic cells >0% is indicated in the images). D/ Cleavage of caspase 3 was analysed by
western-blotting using an anti-caspase 3 antibody 48 after treatment.
Figure 6. JAK2V617F induces a hyper-recombination phenotype in cells from PV and MF
patients.
A/B Cells were cultured for 5 days in medium containing SCF, IL3, IL6, FLT3-L and TPO.
Cells were immunostained with an anti-RAD51 antibody (A) and the percentage of cells with
RAD51 foci was calculated (B). At least 60 cells were counted for each patient (PV, n=5; MF,
n=6); and for mobilized donors (n=6). Horizontal line corresponds to the mean. C/D CD34+
cells were immunopurified either from the blood of either JAK2V617F positive-patients or from
the blood of G-CSF-mobilized donors and were cultured in medium containing SCF, IL3,
28
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EPO. C/ The number of cells was counted during 7 days. D/ At day 5, cells were infected
with the retrovirus containing HR substrate (HR-EGFP/3’EGFP) and GFP+ cells were
measured 5 days later by cytometry analysis. Error bars represent standard error.
29
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Prepublished online May 30, 2008;
doi:10.1182/blood-2008-01-134114
JAK2 stimulates homologous recombination and genetic instability :
potential implication in the heterogeneity of myeloproliferative disorders
Isabelle Plo, Mayuka Nakatake, Laurent Malivert, Jean-Pierre de Villartay, Stephane Giraudier, Jean-Luc
Villeval, Lisa Wiesmuller and William Vainchenker
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