Identification and functional characterization of a cytoplasmic ...

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1099 negative clone in polycythemia patients is present at an earlier stage, perhaps as a primary event. Therefore, we should pay more attention to pathophysiological events related to the JAK2V617F-negative clone in CMPD, including PV. In the ET patients, higher leukocyte and higher hemoglobin level were evident in JAK2V617F-positive patients, indicating that CMPD with JAK2V617F constitutes a common proliferative characteristic. Kralovics et al.3 also noted that CMPD with JAK2V617F showed a higher incidence of secondary myelofibrosis, hemorrhage, thromboembolic events and requirement of treatment. In our PV patients, the presence of JAK2V617F affected hematologic features, whereas homozygous JAK2V617F was linked to clinical features, that is, splenomegaly and tendency toward myelofibrosis. However, it might be difficult to clearly separate patients with heterozygous JAK2V617F from those with homozygous JAK2V617F with a low percentage of cells, owing to technological limitations. Scott et al.8 demonstrated that V617Fhomozygous erythroid progenitors are present in most patients with PV, thus providing the possibility that the difference between heterozygous and homozygous JAK2V617F may represent an occupancy of JAK2 mutation-positive cells. In conclusion, detection of JAK2V617F, especially estimation of cells with mutated alleles, in combination with hematologic features, is important in managing CMPD patients. The presence of JAK2 mutation, either heterozygous or homozygous JAK2V617F, links to pan-myelolysis (leukocytosis, erythrocytosis and thrombocytosis), namely JAK2V617F-positive CMPD. Nevertheless, some possibilities for discrepancy of JAK2V617F detectability are discussed; studies on JAK2V617F-negative clone in CMPD patients might also be important to disclose the pathophysiology of each CMPD category. Although the number of patients examined in the current study is small, our results may suggest that the mutational status of JAK2V617F is associated with clinical features that represent disease progression of various CMPD subtypes.

Acknowledgements We thank Professor J Patrick Barron for review of the manuscript and Mr Kunio Hori and Tohru Makino, NovusGene, Tokyo, for

technical assistance. This work was supported in part by the ‘High-Tech Research Center’ Project from the Ministry of Education, Culture, Sports and Technology (MEXT) (to KO and JHO) and by the ‘University-Industry Joint Research Project’ from MEXT (to KO and JHO).

K Ohyashiki1, Y Aota1, D Akahane1, A Gotoh1 and JH Ohyashiki2 1 First Department of Internal Medicine, Tokyo Medical University, Tokyo, Japan and 2 Intractable Immune System Diseases Research Center, Tokyo Medical University, Tokyo, Japan E-mail: [email protected]

References 1 James C, Ugo V, Le Coue´dic J-P, Staerk J, Delhommeau F, Lacout C et al. A unique clonal JAK2 mutation leading to constitutive signaling causes polycythemia vera. Nature 2005; 434: 1144–1145. 2 Baxter EJ, Scott LM, Campbell PJ, East C, Fouorouclas N, Swanton S et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005; 365: 1054–1061. 3 Kralovics R, Passamonti F, Buser AS, Teo S-S, Tiedt R, Passweg JR et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005; 352: 779–790. 4 Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJP et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005; 7: 387–397. 5 Verstovsek S, Silver RT, Cross NCP, Tefferi A. JAK2V617F mutational frequency in polycythemia vera: 100%, 490%, or less? Leukemia 2006; 20: 2067. 6 Ohyashiki K, Aota Y, Akahane D, Gotoh A, Miyazawa K, Kimura Y et al. The JAK2 V617F tyrosine kinase mutation in myelodysplastic syndrome (MDS) developing myelofibrosis indicates the myeloproliferative nature in a subset of MDS patients. Leukemia 2005; 19: 2359–2360. 7 James C, Delhommeau F, Marzac C, Teyassandier I, Le Coue´dic J-P, Giraudir S et al. Detection of JAK2 V617F as a first intention diagnostic test for erythrocytosis. Leukemia 2006; 20: 350–353. 8 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.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Identification and functional characterization of a cytoplasmic nucleophosmin leukaemic mutant generated by a novel exon-11 NPM1 mutation Leukemia (2007) 21, 1099–1103. doi:10.1038/sj.leu.2404597; published online 15 February 2007

Nucleophosmin (NPM1) mutations occur in 50–60% of adult acute myeloid leukemia (AML) with normal karyotype.1,2 About 40 NPM1 mutations2 have been so far identified, all clustering in exon-12. In spite of molecular heterogeneity, all mutations cause common changes at the C terminus of NPM mutants, i.e. loss of tryptophans 288 and 290 (or 290 alone) and creation of a new nuclear export signal (NES) motif.2 As a consequence, NPM mutants aberrantly accumulates in the cytoplasm of leukaemic

cells;3,4 hence, the term NPMc þ (cytoplasmic-positive) AML.1,2 Here, we report on the identification and functional characterization of a cytoplasmic nucleophosmin mutant generated by a novel exon-11 NPM1 mutation in a patient with AML. Samples from 98 AML patients at diagnosis (Supplementary Table) were analyzed for NPM1 mutations at exons 11 and 12. Denaturing high-pressure liquid chromatography (DHPLC) screening (Supplementary Material 1) identified in one 73year-old male patient a new sequence variant (named Vi2), with eight nucleotides inserted at position 902 in the middle of exon11 (Figure 1a and b). Variant Vi2 was confirmed by allelespecific oligonucleotide polymerase chain reaction (ASO-PCR) Leukemia


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Figure 1 Identification of a novel NPM1 exon-11 Vi2 mutation. (a) Comparison of NPMwt and NPMVi2 mutant nucleotide sequences reveals an 8-bp insertion at position 902. (b) Electropherograms resulting from reverse strand sequencing of mutated and wild-type fragments obtained by ASO-PCR. (c) Bands resulting from ASO-PCR Lane 1: DNA ladder 100 bp; Lane 2: Patient’s cDNA amplified with normal primer; Lanes 3, 4: Control cDNA amplified with normal primer (NP); Lane 5: Mix 1 reaction control; Lane 6: patient’s cDNA amplified with mutation specific primer (ASO); Lanes 7, 8: control cDNA amplified with mutation specific primer (ASO); Lane 9: mix 2 reaction control (Supplementary Material 1).

(Supplementary Material 2). Using the normal primer for amplification, definite bands of 232 bp were detected on agarose gel for the patient and healthy controls; no amplicons were observed in cDNA from normal controls when amplified with allele-specific oligonucleotide (Figure 1c). Nucleotide insertion led to a stop codon at amino acid 275 (Met274Stop). The predicted truncated protein consisted of 274 amino acid residues compared with 294 in wild-type NPM (NPMwt) (Figure 1a). Aberrant cytoplasmic expression is a distinguishing feature of NPM leukaemic mutants generated by exon-12 NPM1 mutations.1–4 To assess whether the exon-11 NPMVi2 mutant is also cytoplasmic, we used pEGFP-C1-NPMwt1 as template to generate the pEGFP-C1-NPMmVi2 construct in which the exon11 NPM-Vi2 mutation is expressed in frame with enhanced green fluorescent protein (eGFP). Plasmids were generated using the QuikChange MultiSite-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA), with primers designed on the following sequence: pEGFP-C1-NPMmVi2, 5’-GTGGAAGC CAAATTCAGGCGCCTATCAATTATGTGAAG. Confocal microscope analysis of NIH-3T3 cells transfected with the pEGFP-C1NPMmVi2 construct (Supplementary Materials 3 and 4) showed Leukemia

that the NPMVi2 mutant localizes exclusively in the cytoplasm (Figure 2, top left). Cytoplasmic accumulation of exon-12 NPM mutants is dictated by the concerted action of C-terminal mutated tryptophans and the creation of a NES motif.3 Notably, both alterations were also present in the NPMVi2 mutant. In fact, the NPMVi2 mutation resulted in loss of the whole exon-12 of the NPM1 gene (encoding for the C-terminal aromatic region and the tryptophan residues 288 and 290 responsible for nucleolar binding). Moreover, the NESbase version 1.0 program5 identified a putative NES motif with the VxxxFxxLxI sequence (Figure 1a) at the C terminus of the predicted NPMVi2 mutant protein. Therefore, the NPMVi2 mutant has not only lost the ability to target nucleoli, but also gained enforced nuclear export capabilities. Accordingly, the images of pEGFP-NPMVi2transfected cells are the same as those of NPM mutant A (Figure 2, middle left) with which mutant NPMVi2 shares common features. In contrast, they differ from the less frequent mutants retaining tryptophan 288 (e.g. NPM mutant E), which are capable of partial nucleolar localization (Figure 2, bottom left). To prove that the enforced nuclear export capabilities of NPMVi2 mutant are NES-dependent, we transfected NIH-3T3 cells with pEGFP-NPMVi2, incubated them with the Crm1 inhibitor leptomycin B and found that 100% of transfected cells showed exclusive nucleoplasmic localization (Figure 2, top right). This pattern is similar to that observed with NPM mutant A (Figure 2, middle right), but differ from the leptomycin B-induced nucleoplasmic and nucleolar relocation of NPM mutant E (Figure 2, bottom right). These findings prove that export of NPMVi2 mutant is NES-dependent and that the mutant cannot bind to nucleoli, despite artificially raised levels in nucleoplasm. Since the NPMVi2 mutant harbours two physiological N-terminal NES sequences (residues 42–49 and 94–102, respectively2), the specific role of the new NES motif at the C terminus of the mutant cannot be established conclusively in Leptomycin B-based experiments. Therefore, using pEGFP-C1-NPMwt1 as template, we generated a mutant carrying an altered VxxxFxxLxI C-terminal NES sequence of NPMVi2 (Figure 3). In this construct (pEGFP-C1-NPMmVi2-no-NES), the C-terminal putative NES motif of NPMVi2 mutant (VxxxFxxLxI) was disrupted by substitution of phenylalanine (F) and leucine (L) with two guanine (G) residues (F-G and L-G). Plasmids were generated with primers designed on the following sequence: pEGFP-C1-NPMmVi2-no-NES, 5’-GTGGAAGCCAAAGGCAGG CGCGGATCAATTATGTGAAG. Cells transfected with this eGFP-tagged plasmid showed exclusive nucleoplasmic positivity for the recombinant protein (Figure 3, right), demonstrating that VxxxFxxLxI is a functional C-terminal NES and that NPMVi2 mutant export abilities depend upon it. Cell transfection experiments show that NPMVi2 localizes in cytoplasm, but neither provide information as to whether this also occurs in the patient’s AML cells, nor provide information on which haemopoietic cell lineages are involved by the mutation. To address these issues, we immunostained paraffin sections from the patient’s bone marrow biopsy using monoclonal antibodies that recognize fixative-resistant epitopes of NPM1 (Supplementary Material 5). The bone marrow was hypercellular and infiltrated by myeloid blasts (positive for myeloperoxidase and CD68 macrophage-restricted), atypical megakaryocytes and erythroid precursors (Figure 4, left). Leukaemic cells were CD34-negative (not shown) and exhibited cytoplasmic (in addition to nuclear) NPM expression (Figure 4,


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Figure 2 NPMVi2 mutant is cytoplasmic and is relocated into the nucleus by leptomycin B (LMB). Confocal laser microscope analysis of NIH3T3 transfected cells (Supplementary Materials 3 and 4) shows that all eGFP-fused NPM mutants are predominantly cytoplasmic under basal conditions (left panels) but are retained in the nucleus after LMB treatment (LMB, right panels). Only the mutant (NPM-mut E) containing a tryptophan 288 residue at C terminus displays a significant nucleolar localization. Scale bars, 10 mm.

right); C23/nucleolin was not detected because of antigenic denaturation. Notably, the NPMVi2 mutant was detected in the cytoplasm of myeloid blasts, megakaryocytes and immature erythroid precursors (Figure 4, right), indicating that multilineage involvement, a common feature of NPMc þ AML,6 may occur with mutations on NPM1 exons other than exon-12. Our sequence analysis and functional studies reveal that, like NPM leukaemic mutants generated by exon-12 NPM1 mutations,3 cytoplasmic accumulation of NPMVi2 mutant is dictated by the concerted action of tryptophan(s) changes and a new NES motif at the C terminus of the protein. The tryptophan residues

288 and 290 at the NPMVi2 C terminus are missing due to truncation of the last 20 amino acids. This alteration is expected to have the same functional effect on nucleo-cytoplasmic traffic as tryptophan(s) replacement by exon-12 NPM1 mutations. In fact, tryptophan residues are essential for nucleolar binding7 since their absence (or replacement) may favour nuclear export, by reducing mutant binding to nucleoli and increasing affinity for Crm1.3 However, since B23.2, the physiologically truncated NPM isoform present in low amounts in tissues, lacks both tryptophan residues but localizes in nucleoplasm,8 tryptophan loss alone cannot accomplish NPMVi2 mutant delocalization Leukemia


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Figure 3 Disruption of the C-terminal NES (VxxxFxxLxI) blocks nuclear export of NPMVi2 mutant. Confocal analysis of NIH-3T3 cells expressing either eGFP-NPMVi2 (left) or the same mutant after disruption of the NES motif (right). Disruption of the NES impairs the nuclear export of NPMVi2 that localizes in the nucleoplasm. The nucleoli do not contain the mutated protein. Images were collected and reconstructed as described in Supplementary Material 4. Scale bars, 10 mm.

Figure 4 Aberrant cytoplasmic NPM expression in leukaemic cells of different lineages carrying the NPMVi2 mutation. Left: Bone marrow paraffin section showing myeloid blasts (double short arrows), a displastic megakaryocyte (short arrow) and proerythroblasts (long arrow) (haematoxylin–eosin; 800). Right: Section from the same case exhibits multilineage involvement. Cytoplasmic NPM is present in myeloid blasts (double short arrows), a dysplastic megakaryocyte (short arrow) and proerythroblast (long arrow); a normal residual haemopoietic cell exhibits the expected NPM nuclear positivity (arrowhead) (APAAP; hematoxylin counterstaining; 800).

into cytoplasm. The additional force ensuring nuclear export and cytoplasmic accumulation is the new NES (VxxxFxxLxI) at the NPMVi2 mutant C terminus. The VxxxFxxLxI NES sequence is that of a typical Rev-type NES, which is defined by a short stretch of closely spaced leucine or other hydrophobic residues, that is, isoleucine, methionine, valine or phenylalanine.5 Although functionally active, the VxxxFxxLxI NES is slightly different from those identified at C terminus of exon-12 NPM mutants.1–4 This points to heterogeneity of C terminal NES motifs deputed to ensure nuclear export of NPM mutants. Thus, we have shown that alterations at C-terminal NPMVi2 mutant mimic those of exon-12 NPM mutants. Since mutations at different exons of NPM1 are all associated with aberrant cytoplasmic NPM expression, they seem to be designed to export mutants into cytoplasm, further supporting the view that cytoplasmic NPM dislocation is a critical step in leukaemogenesis. NPM mutants bind and delocalize endogenous NPM and also the ARF protein into cytoplasm,8 interfering with a proper p53 response. Furthermore, an imbalance in the nuclear– cytoplasmic NPM ratio could alter other functions of NPMwt.8 However, the exact role of NPM mutants in leukaemogenesis still remains unknown. Leukemia

Our patient also shared several biological and clinical features with NPMc þ AML carrying NPM1 exon-12 mutations, i.e. normal karyotype, M4-FAB morphology, CD34 negativity and involvement of myeloid, erythroid and megakaryocytic series.1,2 He harboured a NPM1 mutation without FLT3-ITD, a molecular combination that has been associated with a relatively good prognosis in AML with normal karyotype.2 However, the prognostic value of this association cannot be established in our patient. In fact, because of age and cardiac problems, he received only supportive therapy and died of infectious complications five months after diagnosis. Our findings raise the question about the incidence of NPM1 exon-11 mutations among AML patients. In a previous study correlating immunohistochemistry with molecular analysis of NPM1 mutations, cytoplasmic NPM could predict with 100% accuracy the presence of NPM1 mutations, and all 200 NPM cytoplasmic-positive AMLs harboured NPM1 mutations at exon-12.4 Thus, the mutation described in this study (and other mutations that may putatively involve NPM1 exons other than exon-12) must be extremely rare and clinically of scarce importance. Nevertheless, it is necessary to establish guidelines for detecting these rare NPM1 mutation variants. If using cDNA


as template, DHPLC allows the analysis of amplicons including more than one exon at time. For this reason, DHPLC assay is a good approach suitable to identify genetic aberrations occurring outside the specific mutational hot spot. Only the samples showing an abnormal chromatogram are subsequently sequenced. As an alternative, immunohistochemistry that detects, through cytoplasmic dislocation on NPM, ‘all types’ of NPM1 mutations,3,4 could serve as first step for directing further molecular studies, i.e. restricting analysis for NPM1 exons other than exon12 to AML cases that may turn out to be NPM cytoplasmicpositive in the absence of exon-12 NPM1 mutations.

Acknowledgements Supported by the Associazione Vicentina Leucemie e Linfomi (AVILL), Associazione Italiana per la Ricerca sul Cancro (AIRC) and the Federazione Monte dei Paschi di Siena. EA was recipient of a grant from ‘Fondazione Progetto Ematologia’ – Vicenza. NB is a recipient of a fellowship from FIRC (Federazione Italiana per la Ricerca sul Cancro). We are grateful to Dr A Montaldi for cytogenetic analysis, Dr S Roberti for statistical analysis, Dr GA Boyd for editorial assistance and Mrs I Frasson for excellent secretarial assistance. B Falini applied for a patent of the clinical use of NPM mutants.

E Albiero1, D Madeo1, N Bolli2, I Giaretta1, E Di Bona1, MF. Martelli2, I Nicoletti3, F Rodeghiero1 and B Falini2 1 Department of Cell Therapy and Haematology, San Bortolo Hospital, Vicenza, Italy; 2 Section of Haematology and Immunology, University of Perugia, IBIT Foundation, Perugia, Italy and

3

Institute of Internal Medicine, University of Perugia, Perugia, Italy E-mails: [email protected] or [email protected]

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References 1 Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med 2005; 352: 254–266. 2 Falini B, Nicoletti I, Martelli MF, Mecucci C. Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin (NPMc+ AML): biological and clinical features. Blood 2007; 109: 874–885. 3 Falini B, Bolli N, Shan J, Martelli MP, Liso A, Pucciarini A et al. Both carboxy-terminus NES motif and mutated tryptophan(s) are crucial for aberrant nuclear export of nucleophosmin leukemic mutants in NPMc+ AML. Blood 2006; 107: 4514–4523. 4 Falini B, Martelli MP, Bolli N, Bonasso R, Ghia E, Pallotta MT et al. Immunohistochemistry predicts nucleophosmin (NPM) mutations in acute myeloid leukemia. Blood 2006; 108: 1999–2005. 5 la Cour T, Gupta R, Rapacki K, Skriver K, Poulsen FM, Brunak S. NESbase version 1.0: a database of nuclear export signals. Nucleic Acids Res 2003; 31: 393–396. 6 Pasqualucci L, Liso A, Martelli MP, Bolli N, Pacini R, Tabarrini A et al. Mutated nucleophosmin detects clonal multilineage involvement in acute myeloid leukemia: Impact on WHO classification. Blood 2006; 108: 4146–4155. 7 Nishimura Y, Ohkubo T, Furuichi Y, Umekawa H. Tryptophans 286 and 288 in the C-terminal region of protein B23.1 are important for its nucleolar localization. Biosci Biotechnol Biochem 2002; 66: 2239–2242. 8 Grisendi S, Mecucci C, Falini B, Pandolfi PP. Nucleophosmin and cancer. Nat Rev Cancer 2006; 6: 493–505.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Polycythemia associated with the JAK2V617F mutation emerged during treatment of chronic myelogenous leukemia Leukemia (2007) 21, 1103–1104. doi:10.1038/sj.leu.2404591; published online 15 February 2007

A gain-of-function mutation at codon 617(V617F) of the Janus kinase 2 (JAK2) gene has been reported in chronic myeloproliferative diseases. The JAK2V617F results in a valine to phenylalanine substitution causing constitutive activation of the JAK–STAT pathway. This in turn leads to proliferation of the hematopoietic cells.1 Jelinek et al.2 recently published results of a study of the first large series of Philadelphia chromosome (Ph)positive chronic myelogenous leukemia (CML) patients with the JAK2V617F mutation. The mutation was not found in 99 CML patients, including 55 imatinib-resistant patients. Recently, we encountered a JAK2V617F- and Ph-positive CML patient being treated with imatinib that evolved to erythrocytosis needing phlebotomy during cytogenetic complete remission (CCR). The patient was a 43-year-old man diagnosed as chronic phase CML with a 49% hematocrit (Ht). Cytogenetic analysis revealed a karyotype of 46, XY, t(9;22)(q34;q11) in all the metaphases(20/20). The type of BCR-ABL was b2a2. He was treated with interferon a-2b (IFN-a) followed by hematological

remission. One year later he showed leukocytosis again and was diagnosed as ‘transformation to accelerated phase’ with no additional chromosomal aberration. Because there was no HLA-matched donor, he could not receive hematopoietic stem cell transplantation. Thus, he was treated with IFN-a and hydroxyurea to control CML for another 5 years. After imatinib was approved in Japan, IFN-a and hydroxyurea were stopped and imatinib therapy was started. One month following commencement of imatinib therapy, hematological remission was achieved and CCR was obtained at 33 months. Two months before imatinib administration, intriguingly, erythrocytosis developed. Although imatinib was effective for CML, the red blood cell count gradually increased. As the Ht value exceeded 50%, phlebotomy has been performed since then intermittently. To investigate the mechanism of erythrocytosis, search for the mutation and quantify the level of the JAK2V617F, real-time quantitative polymerase chain reaction (RQ–PCR) was performed using a Gene Amp 5700 sequence detection system (Applied Biosystems, CA, USA). For JAK2 gene mutational and quantitative analysis, genomic DNA of the subject’s bone marrow mononuclear cells was isolated using the QIAamp DNA Mini Kit (QIAGEN, Stanford, CA, USA). Allele-specific Leukemia