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JAK2 mutation and disease phenotype: a double L611V/V617F in cis mutation of JAK2 is associated with isolated erythrocytosis and increased activation of AKT and ERK1/2 rather than STAT5

Polycythemia vera (PV), a myeloproliferative neoplasm (MPN), is characterized by the presence of a mutated, activated form of the tyrosine kinase JAK2.1 In 95% of cases, PV patients present the V617F mutation in exon 14 (JAK2–V617F) and half of V617F-negative PV patients carry mutations or deletions in exon 12.2 Both types of mutations result in activation of JAK2 and STAT5. Recently the 46/1 (or ‘GGCC’) haplotype of chromosome 9p was found associated with a pre-disposition to JAK2 mutations in the same allele. As this haplotype is relatively frequent, unrecognized mutations of JAK2 may not be rare. Indeed healthy donors were reported positive for JAK2-V617F using nested PCR assays, and the repeated occurrence of the V617F mutation of JAK2 has been demonstrated in essential thrombocythemia (ET) and in PV.3, 4, 5 In addition, in 2 PV patients, JAK2-V617F and mutations in exon 12 of JAK2 co-existed in separate sub-clones originating from a single progenitor in one case, or from unrelated hematopoietic stem cells in the other case.6, 7 V617F-positive PV patients with additional mutation(s) in exon 14 of JAK2 (V615L, C616Y, C618R and D620E) are also evidence of multiple JAK2 mutations.8 As different JAK2 mutants may have different JAK2 activity, the JAK2 mutational status may influence subclone outcome and affect disease phenotype.

To address this question, we studied 3 PV patients found to be carriers of a new mutation in exon 14 of JAK2, leucine 611 changed for a valine (L611V) present in cis with V617F, that resulted in the double JAK2-L611V/V617F mutant. DNA and RNA extracted from purified granulocytes, platelets, CD3+ T-lymphocytes and colonies were analyzed using allele-specific quantitative PCR assays (AS-qPCRs), pyrosequencing and conventional sequencing.9, 10, 11 The primers and probes used are listed in Supplementary Table 1. With informed consent, DNA samples from 10 healthy donors, 199 control patients with idiopathic erythrocytosis (n=22), secondary erythrocytosis (n=148) or splanchnic vein thrombosis (n=29), and 465 patients diagnosed following the 2002 WHO criteria with PV (n=168), ET (n=271) or primary myelofibrosis (n=26) were analyzed, as were DNA from 31 archival samples from MPN transformed into acute myeloid leukemia.

We first investigated the case of patient Na249, who presented with criteria of PV (Table 1) but only 2% JAK2-V617F in granulocyte DNA using a sense V617F AS-qPCR assay.9 Verification of the mutational burden using pyrosequencing detected 20% JAK2-V617F.10 Cloning and sequencing analysis of exon 14 of JAK2 revealed an additional mutation of base 1831 (1831T>G), changing leucine 611 for a valine (L611V) (Figure 1a). Reasoning that the additional mutation would inhibit primer hybridization and subsequent DNA amplification, we re-analyzed granulocyte gDNA from patient Na249 using a second V617F AS-qPCR assay that used anti-sense primers (Supplementary Figure 1) and now found 19% JAK2-V617F. Cloning of PCR products in a sequencing plasmid and pyrosequencing of plasmid DNA from hundreds of bacterial clones established that L611V occurred in cis with V617F. The existence of double, L611V/V617F mutant alleles was confirmed by conventional sequencing of selected plasmid clones.

Table 1 Clinical and biological characteristics of the three PV patients with the double JAK2- L611V/V617F mutation
Figure 1

Characterization of the double L611V/V617F mutation of JAK2.(a) Localization and configuration in cis of the L611V and V617F mutations in exon 14 of JAK2. Exon 14 of JAK2 was amplified by PCR from patient genomic DNA using iProof HF master mix (Biorad, Hercules, CA, USA). PCR products were cloned in the pCR4 sequencing vector (Invitrogen, Carlsbad, CA, USA) and transformed in TOP10 bacteria (Invitrogen). Individual bacterial colonies were picked and grown overnight at 37 °C in 96 well plates with ampicillin (100 μg ml−1) medium, without shaking. A total of 2 μl of bacterial cultures were used as template for PCR with primers JAK200F and JAK200Rbio (see Supplementary Table 1 for sequences). After PCR the biotinylated strand was captured on streptavidin sepharose beads (Amersham Biosciences, Uppsala, Sweden) and annealed with a pyrosequencing primer JAK1831S. Pyrosequencing was carried out using PSQ HS 96 Gold SNP Reagents and the PSQ HS 96 pyrosequencing machine (Qiagen, Valencia, CA, USA).10 Mutational status at nucleotides 1831 T>G and 1849 G>T was analyzed by the pyrosequencing PSQ HS 96A1.2 software. In selected clones, plasmid DNA was purified and the pyrosequencing results were confirmed by sequencing at the MD Anderson Cancer Center DNA Core Facility using ABI Big Dye terminator cycle chemistry. (b) Proportion of JAK2-mutated erythroid colonies grown from blood progenitors of PV patients carrying the double JAK2-L611V/V617F mutation. Colony assays were carried out in methylcellulose-based media (H4531, Stem Cell Technologies, Vancouver, BC, Canada) using frozen peripheral blood mononuclear cell (PBMC), plated at 50 000 per ml. Cultures were incubated at 37 °C without erythropoietin (Epo) for 7 days to obtain Epo-independent erythroid colonies (EEC). Between days 7 and 10, Epo was added to the EEC cultures to obtain colonies of sufficient size for DNA analysis. At day 14, after the identification of colonies under microscope, erythroid colonies were picked one by one and analyzed by allele-specific quantitative PCR assays (AS-qPCR) following DNA extraction. All JAK2-mutated colonies, representing 12% (Na249), 30% (Na382) and 0% (Di362) of the colonies, were heterozygous for the double L611V/V617F mutation. GRA%mut: % of JAK2-L611V/V617F-mutated alleles in granulocyte genomic DNA. Nbr: number of single erythroid colonies analyzed for each patient. (c) Transient expression and functional analysis of the L611V, V617F and L611V/V617F mutant forms of JAK2 in BaF-3/Epo-R cells. Plasmids pCR2.1 containing the cDNA of JAK2-WT and (gift from Dr Jan Cools) and JAK2-V617F served as matrices for L611V- and L611V/V617F-directed mutagenesis. PCRs were carried out with the relevant L611 or V611 primers (see Supplementary Table 1) using Pfu Ultra DNA polymerase (Stratagene, La Jolla, CA, USA). Mutagenesis was checked by sequencing then another Pfu PCR was performed to extract JAK2 cDNAs (WT, L611V, V617F and L611V/V617F). Purified PCR products were cloned into expression vector pcDNA3.1. For functional analyses, 107 murine BaF-3/Epo-R cells grown in RPMI with 2% fetal calf serum (FCS) and 1 IU ml−1 Epo were transfected with 25 μg of pcDNA3.1 containing JAK2-WT and mutant cDNA using the Amaxa Nucleofector device. After transfection, cells were grown in RPMI medium supplemented with 2% FCS and 1 IU ml−1 Epo for 24 h. Transfected cells were then washed, deprived of Epo for 5 h, then stimulated with Epo (25 IU ml−1) for 10 min at 37 °C. Cells were then harvested, washed twice in PBS and lysed in RIPA buffer. Successful mRNA expression of single and double JAK2 mutant cDNAs was verified using RT-qPCR in aliquots of transfected cells. For intra-cellular signalling studies, 25 μg of proteins were loaded on a 10% polyacrylamide SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were incubated with antibodies specific for p-JAK2, p-STAT5, p-ERK1/2 and p-AKT (Cell Signalling, Danvers, MA, USA) or β-actin (Millipore Corporation, Billerica, MA, USA), then stripped and re-probed with antibodies specific for total JAK2 (Millipore Corporation, Billerica, MA, USA), STAT5 (BD Bioscience, San Jose, CA, USA), ERK1/2 and AKT (Cell Signalling, Danvers, MA, USA). Blots were revealed using the BM Chemiluminescence Blotting Substrate (Roche, Mannheim, Germany). Results shown are representative of four series of transient transfections.

Pyrosequencing and AS-qPCR assays specific for L611V were designed and DNA samples from the 465 MPN and 209 control patients were screened (Supplementary Table 2). Blasts from 31 transformed MPN were also tested. L611V was detected in 2 (1.8%) PV patients, Na382 and Di362, considered V617F-negative. Using the anti-sense V617F AS-qPCR assay, they were both found positive (27 and 28% V617F, respectively). Sequencing of cloned alleles confirmed that L611V also occurred in cis with V617F for these patients. No other mutation was detected using sequencing and high-resolution melting curve analysis of exons 12 and 14 of JAK2.

The predominance of L611V/V617F alleles in granulocytes was confirmed by pyrosequencing of 100 clones per patient: Na249: 15%; Na382: 23%; and Di362: 26%.10 It is interesting to note that single mutant alleles were also found, representing 0.9% of alleles for V617F (4/429 of cloned PCR products from genomic DNA). These results were consistent with the 2% V617F alleles detected in granulocyte DNA of patient Na249 by the sense V617F AS-qPCR assay, which covers the sequence coding for L611 and is thus unable to amplify double mutated alleles. Altogether, this indicated the co-existence of two separate clones, one carrying JAK2-V617F singly, the other carrying the double JAK2-L611V/V617F mutation. Of note, a low (1.5–3%) representation of V617F alleles had been reported in 1 PV patient with additional JAK2 mutations in exon 12.6 Hence the presence of JAK2-V617F does not ensure clone expansion, implying that other factors intervene in the expansion of JAK2-mutated progenitors: perhaps a defective bone marrow micro-environment, abnormal cytokine stimulation or the congenital or acquired genetic background. Regarding the latter, patients Na382 and Di362 were found positive, each in a heterozygous manner, for the rs12343867 polymorphism of JAK2 intron 14 characteristic of the 46/1 haplotype associated with a pre-disposition to JAK2 mutations. L611V/V617F and rs12343867 were on the same allele in 13/13 (Na382) and 10/10 (Di362) clones sequenced (Supplementary Figure 1). Yet patient Na249 (with three JAK2 mutations) was negative for the 46/1 haplotype.

It is interesting to note that at the time of diagnosis the presentation of JAK2-L611V/V617F patients (Table 1) was similar to those of most patients with JAK2 exon 12 mutations.2 The 3 female patients showed a high hematocrit (>61.5%), normal leukocyte counts and normal or low platelet counts and no splenomegaly; 2/3 patients had endogenous erythroid colonies. JAK2-L611V/V617F was present at the time of diagnosis for patients Na249 and Di362; for patient Na382, it was discovered after interruption of pipobroman. The two JAK2 mutations were acquired, as the blood lymphocytes were wild-type.

Pyrosequencing and AS-qPCR analysis found similar proportions (15–28%) of L611V/V617F alleles in granulocyte DNA from the 3 patients. However, in platelet cDNA, studied for 2 patients, L611V/V617F alleles were either absent or reduced by half compared with granulocyte cDNA. Among erythroid colonies, only 0–30% carried L611V/V617F, always in a heterozygous fashion, the rest being wild type (Figure 1b). Thus for L611V/V617F patients, hematopoiesis remained predominantly ‘wild-type JAK2’. Clonal cells with the double L611V/V617F mutation were sensitive to cytoreductive treatment, even after three decades of evolution (Na382). Patient Na249 responded to hydroxyurea with almost complete disappearance of JAK2-mutated alleles (<1% in granulocyte DNA after 21 months of treatment). Conversely, 16 months after interruption of pipobroman treatment of patient Na382, the L611V/V617F allelic ratio was raised from 5 to 27%, remaining stable after 33 months.

The effects of L611V, V617F and double L611V/V617F mutations on the function of JAK2 were analyzed using transient transfection of murine BaF-3/EpoR cells, which depend on erythropoietin (Epo) for their growth. Immunoblotting analysis (Figure 1c) confirmed the tyrosine phosphorylation of JAK2, STAT5, AKT and ERK1/2 in response to Epo, and the activation of JAK2 by V617F, as assessed by the constitutive tyrosine phosphorylation of JAK2 and increased phosphorylation of JAK2, STAT5 and AKT in response to Epo, compared with JAK2-WT. The single JAK2-L611V mutant showed tyrosine phosphorylation of STAT5 and AKT similar to JAK2-WT; phosphorylation of JAK2 and ERK1/2 in response to Epo was low. The double L611V/V617F mutant revealed greater constitutive and Epo-stimulated phosphorylation of JAK2, AKT and ERK1/2, compared with both wild-type JAK2 and JAK2-V617F, but low activation of STAT5.

In summary, subsets of PV patients with or without the 46/1 haplotype may present several sub-clones carrying different mutations of JAK2, unrecognized unless sensitive assays with sense and anti-sense primers are used. The presence of JAK2 mutations, functionally silent or activating, does not ensure clone expansion. Certain activating mutations may alter JAK2 function differently than V617F and may be associated with a distinct disease phenotype. The double L611V/V617F mutation increased the activation of JAK2, AKT and ERK1/2 but not STAT5 and was found associated with isolated erythrocytosis.


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We are indebted to colleagues of the Clinical Hematology Departments of the University Hospitals of Nantes and Dijon for providing patient samples; to Dr Radek Skoda (Basel, Switzerland) for murine BaF-3/Epo-R cells and JAK2-V617F cDNA; to Dr Serge Carillo for JAK2 exon 12 mutation analysis; to Dr Isabelle Corre for expert advice; and to Mrs Danielle Pineau for excellent technical help. The study was supported by grants from the Ligue Nationale contre le Cancer (Comité de Loire-Atlantique, Comité du Morbihan, Comité d’Ille-et-Vilaine) and from the Association pour la Recherche contre le Cancer (ARC). CC and MB benefited from scholarships from the French Ministry of Research.

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Correspondence to S Hermouet.

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Supplementary Information accompanies the paper on the Leukemia website

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Cleyrat, C., Jelinek, J., Girodon, F. et al. JAK2 mutation and disease phenotype: a double L611V/V617F in cis mutation of JAK2 is associated with isolated erythrocytosis and increased activation of AKT and ERK1/2 rather than STAT5. Leukemia 24, 1069–1073 (2010).

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