Description of a novel JAK3-P132A mutation in Acute Megakaryoblastic Leukemia and demonstration of previously reported JAK3 mutations in normal subjects

Gain-of-function (GOF) mutations of Janus kinase 2 (JAK2) are frequently seen in myeloproliferative disorders (MPD), meanwhile JAK3 activating substitutions were found in few megakaryocytic cell lines and in primary myeloid leukemia (AMKL). Here, we sought to discover novel leukemogenetic mutations in de novo acute myeloid leukemia of Non-Down Syndrome (NDS) patients by DNA sequencing. A total of 191 normal Caucasian individuals were studied to define single nucleotide polymorphisms (SNP) within the JH2 and JH6 domains. Although known activating substitutions were observed in rare AML [V722I (2/134) or P132T (1/119)], all samples were wild-type (WT) for the oncogenic A572V (119/119). Interestingly, a novel homozygous mutation (P132A) was discovered in a AMKL patient and in vivo studies demonstrated that its ectopic expression was oncogenic in a mouse xenotransplantation model. This study defines a novel JAK3 mutation among N-DS AML patients and demonstrates that normal individual can also display germline JAK3 substitutions, previously proven to have oncogenic properties, in vitro and in vivo. The discovery of these substitutions in normal donors encourages future studies to define new risk factors among MPD patients. INTRODUCTION Acute myeloid leukemia (AML) is relatively rare heterogeneous group of processes characterized by uncontrolled proliferation of clonal neoplastic hematopoietic myeloid elements, which accumulate in the bone marrow, peripheral blood, and often in other lymphoid and non-lymphoid organs, impairing the production of normal blood cells. Among AML, several groups can be distinguished according to cytological, cytochemical and immunophenotyping/differentiation stages [1], or by virtue of unique chromosomal aberrations [2]. Nowadays, it is well established that the molecular stratification of AML patients provides critical information on the clinical progression and evolution, allowing a more objective/quantitative molecular monitoring of the tumor burden, with relevant prediction of clinical relapse rates and/or the delivery of targeted therapies [3]. Like other neoplastic processes, the development of AML is associated with the overtime accumulation of acquired genetic alterations and epigenetic changes. Numerous recurrent structural and numeric cytogenetic aberrations have been identified in unique subset of AML patients [4,5]. The pathogenetic signaling pathways involved in the maintenance of the neoplastic phenotype of AML have in part been elucidated. Critical player appears to be the signal transducer and activator of transcription 5 (STAT5), that is constitutively phosphorylated in at least 70% of AML patients [6], [7], suggesting the presence of deregulated tyrosine kinase (TK) signaling and/or mutated tyrosine kinases. Activating TK mutations have indeed been discovered in a subset of AML samples, as point mutations within the coding region of the c-KIT (5%) gene, mutations or internal tandem duplications of FLT3 (30%), and rare somatic mutations of JAK2, JAK3 and PDGFR. Nevertheless, in many AML it is unclear which molecular mechanisms are responsible for STAT5 phosphorylation, suggesting that other defects may exist. Here, we have searched for the presence of novel putative JAK3 mutations in a well-characterized panel of AML from adult Caucasian individuals. A group of normal control individuals was investigated to underline the presence of JAK3 SNP within this ethnic population. This approach has identified a novel homozygous JAK3 point mutation in position 132 in a single case of AMKL, which can sustain the growth of BaF3 cells in absence of exogenous IL3, in vivo. These findings further confirm that JAK3 mutations may have a tumorigenic role in a small subset of AML patients. Moreover, we have discovered that many of the oncogenic JAK3 mutations seen in Down Syndrome (DS) AMKL can be detected in N-DS AML and in normal individuals as well. These later finding suggests that these substitutions may represent SNP, which may be linked to a higher risk of myeloid disorders. The molecular characterization of JAK3 further stratifies AML patients and might allow the design of novel tailored therapies.. MATERIAL AND METHODS Clinical Samples A panel of 134 well-characterized cases of untreated Acute Myeloid Leukemia (AML 1 to 5), 28 AMKL and 6 AML-M6 were selected among the Hematopathological files of the Pathology Laboratories of the University of Turin, Brescia and the San Raffaele Scientific Institute. Age of AML patients ranged from 23 to 94 years, with a mean of 60 years. Diagnoses were assigned according to the WHO classification and/or the FAB classification. After clinical diagnosis, unutilized fresh peripheral blood and/or bone marrow aspirate samples were used for research. Archived paraffin-embedded materials were utilized for DNA extraction in selected cases (28 AMKL samples). A total of 191 peripheral blood mononuclear cell collections from normal blood donors were included as controls (Blood Bank Collection, Turin). A general informed consent was obtained according to the local ethical committees guidelines of each participating Institution. Samples were numerically identified maintaining patients’ anonymity. DNA And RNA Extraction Formalin-fixed, paraffin-embedded tissue blocks were also used for the study. Formalin-fixed, paraffin-embedded samples were de-paraffinized with a xylene-ethanol protocol [8]. Briefly, tissue specimens were incubated overnight at 55°C in lysis buffer with Proteinase K (20 mg/mL), and DNA was obtained by phenolisopropanol extraction. Total RNA was extracted from cells or tissues and used as template for the cDNA transcription accordingly to the BIOMED guidelines [8,9]. Polymerase Chain Reaction Amplification And DNA Sequencing Analysis The entire JAK3 JH2 domain (including A572V and V722I) and the exon 3 of the JH6 domain, spanning the amino acid site P132T, were sequenced. Amplification of JAK3 exons was performed by polymerase chain reaction (PCR) with specific oligonucleatide primers (Table 1 of supplementary material). DNA sequencing was executed using the cDNA [AML (100) and control sample (191)] or the genomic DNA from fixed tissue bone marrow biopsies [AML-7 (28) or AML6 (6)]. Sequencing reactions were carried out using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA), and an ABI 310 automated capillary system (Applied Biosystems) (see supplementary methods). cDNA Constructs And Mutagenesis The JAK3 cDNA (Clone: HsCD00021445, Harvard Medical School) was subcloned into the pcDNA3 (Invitrogen) expression vector at EcoRV site. JAK3, JAK3 and JAK3 mutated (mut) cDNA were generated by PCR-based mutagenesis (Stratagene, La Jolla, CA) and confirmed by DNA sequencing. JAK3 and mut-JAK3 cDNA were then subcloned (HindIII/XhoI) into a EGFP retroviral vector (Pallino) [10]. The oligonucleotide primers list used for the JAK3 mutagenesis is available in the Table II of supplementary methods. Cell Culture And DNA Transfection Human embryonic kidney (HEK) 293T, NIH3T3 and murine IL-3-dependent BaF3 cells were cultured, respectively, in Dulbecco modified Eagle medium (DMEM) or in RPMI 1640 supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 μg/mL streptomycin,. WEHI-conditioned medium (10%) was used as a source of IL-3. Cells were transfected using the Effectene kit, following the manufacture’s instructions (Qiagen, Valencia, CA). For the focus assay, NIH3T3 cells (2.5x10/100 mm plate) were first transfected (2 μg of total DNA), then selected with G418 (400 μg/ml, pcDNA3 vectors) or puromycin (2 μg/ml, retroviral vectors). Three weeks post transfection, NIH3T3 cells were fixed and stained with crystal violet. Murine IL-3-dependent BaF3 cells were electroporated using Cell Line Nucleofector Kit V (Amaxa Biosystems, USA) and selected with puromycin (2 μg/mL, 5 days). For cytokine independent growth assays, JAK3 BaF3 cells were electroporated, then extensively washed with PBS, and subsequently resuspended in complete media in absence of IL-3 (seeded at 10/ml in duplicate in 12-well plates). Viable cells were determined by trypan blue or by TMRM staining exclusion [11]. Western Immunoblotting Analysis BaF3 and electroporated JAK3 BaF3 cells were cultured in RPMI 1640 medium with or without IL3 (12h). NIH3T3 cells were transiently transfected with the different JAK3 constructs and harvested 48h after transfection. Cells were lysed (4°C for 30 min.) using a buffer containing 20 mM TrisHCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM NaF, 1 mM Na3VO4, and protease inhibitors (Roche, Mannheim, Germany). Cell lysates were collected by centrifugation at 15,000 x g, and supernatants were analyzed for protein concentration with a Bio-Rad Dc protein assay kit (Bio-Rad Laboratories) and stored at -80° C. Forty micrograms of proteins were run on SDS-PAGE under reducing conditions. For immunoblotting, proteins were separated by SDS/PAGE, transferred to nitrocellulose and incubated with the specific antibody. Immune complexes were detected with secondary antimouse or antirabbit peroxidase-conjugated antibodies (Amersham) and visualized by enhanced chemiluminescence reagent (Amersham) according to the manufacturer's. The following antibodies were used: anti-ALK (1:4000; Zymed, Seattle, WA), monoclonal antiphospho-Tyr (PY20; 1:1000; Transduction Laboratories, Lexington, KY, USA), anti-phospho-Stat3 (Y705) (1:1000; Cell Signaling Technology), anti-Stat3 (1:1000; Cell Signaling Technology), antiphospho-Erk1/2 (1:500; Cell Signaling Technology), anti-Erk1/2 (1:1000; Santa Cruz Biotechnology), anti-phospho-AKT (1:1000, Cell Signaling Technology), anti-AKT (1:1000; Cell Signaling Technology), anti-phospho-Shc (Y317) (1:1000; Cell Signaling Technology), anti-Shc (1:1000; Santa Cruz Biotechnology), anti-Stat5 (1:1000 Cell Signaling Technology), anti-phospho