4089 Constitutive activation of JAK2 by chromosomal translocations or a point mutation is a frequent event in hematological malignancies particularly in Philadelphia-negative MPN. Recently, Dawson et al. identified a novel nuclear role of JAK2 in the phosphorylation of Tyr 41 of histone H3 leading to chromatin displacement of HP1a in hematopoietic cell lines and in the CD34+ cells collected from the peripheral blood of one PMF patient with JAK2V617F mutation. To determine whether the V617F mutation observed in MPN patients affects the sub-cellular localization of JAK2, we first analyzed by confocal immunofluorescence (CIF) microscopy and Western Blot (WB), K562 cells stably transfected with pMSCV-puroJAK2V617F or pMSCV-puroJAK2. The results confirm the nuclear and cytoplasmic localization of JAK2 in K562 as reported by Dawson et al. However, we consistently observed a much stronger nuclear signal in the cells expressing JAK2V617F than in those carrying wt JAK2 suggesting that the mutation leads to a preferential accumulation of JAK2V617F in the nucleus. To determine whether there is a preferential nuclear translocation of JAK2V617F in vivo, we analyzed by CIF microscopy and WB the total BM cells of 10 JAK2V617F-positive MPN patients (ET n=4, PV n=3, PMF n=3, allele burden median: 56%, 70%, 72% respectively) and of 5 MPN patients with wt JAK2 (PMF n= 2, ET n=3). We found a strong nuclear signal in mononucleated cells of 10 of 10 JAK2V617F-positive patients but not in those with wt JAK2. The JAK2 signal was observed almost exclusively in the nucleus suggesting a predominantly nuclear homing of JAK2V617F. To identify the phenotype of these cells, we used fluorescence activated cell sorting (FACS) to isolate CD34+, CD15+, CD41+ and CD71+ fractions from the BM of three JAK2V617F-positive MPN patients (1 ET, 1 PV, 1 early PMF). We found nuclear JAK2 in CD34+ positive cells collected from BM only in V617F mutated patients. No obvious nuclear signal was detected in differentiated granulocytic, megakaryocytic and erythroid cells obtained from the patients (n=15). To determine whether the block of JAK2 activity could interfere with nuclear localization of JAK2, we incubated JAK2V617F and JAK2 expressing K562 with the selective JAK2 inhibitor AG490. At the IC50 dose (25 uM) and after 3 h of incubation, CIF images showed the JAK2 redistribution in the vast majority of V617F expressing K562 and the replacement in the cytoplasm but not in wt cells. By QRT-PCR we demonstrated that the V617F mutation strongly up-regulates LMO2 expression in K562 and in CD34+ cells. In our assay, the addiction of AG490 progressively and completely restore LMO2 levels in V617F expressing K562. Our data corroborate recently published results of a nuclear localization of JAK2 in hematopoietic cells and they also extend these findings by showing that in all subtypes of MPN patients JAK2V617F accumulates in the nucleus of progenitor CD34+ cells while remains mostly in the cytoplasm of their differentiated progeny. The chromatin alterations due to the preferential accumulation of JAK2V617F in the nucleus correlates with a significant increase in LMO2 expression in cell lines and in sorted CD34+ cells. The selective JAK2 inhibitor AG490 is able to revert nuclear JAK2 and normalize LMO2 levels in vitro, suggesting how the block in JAK2 nuclear translocation could be a new treatment strategy for JAK2 mutated patients. Disclosures: No relevant conflicts of interest to declare.
Our results suggest that there is a protective effect of U83836E in ischemia-reperfusion injury, in that tissue damage due to oxidative stress is reduced, thus ameliorating renal function impairment.
IntroductionThe discovery of an acquired somatic mutation in the JAK2 gene resulting in a Val-to-Phe substitution at position 617 (JAK2V617F) has substantially modified our molecular knowledge of myeloproliferative neoplasia (MPN). 1-7 Recently, Dawson et al 8 identified in hematopoietic cell lines a novel nuclear role of JAK2 in the phosphorylation of Tyr 41 of histone H3, leading to chromatin displacement of heterochromatin protein 1␣ (HP1␣) with a consequent reduction of the potential tumor suppressive functions of (HP1␣). It is proposed that these changes result in erratic mitotic recombination and transcription deregulation of several JAK2-regulated genes including LMO2. In this work we find that in contrast to cells with normal JAK2 in which the protein is detected predominantly in the cytoplasm, JAK2 is mostly nuclear in V617F-positive CD34 ϩ cells. However, this nuclear localization is no longer observed in V617F-positive differentiated cells. After expressing JAK2V617F in K562 cells, we observe a similar preferential accumulation of JAK2 in the nucleus in contrast to untransfected and wild-type (wt) JAK2-expressing cells in which the protein is found in the cytoplasm. The mutated-JAK2 nuclear translocation is mainly reverted by the addiction of the JAK2 inhibitor AG490. Methods Cell culturesK562 were grown as previously described. 8 Transfection was performed by AMAXA electroporation, and stable cell lines were obtained by puromycin selection (3 g/mL). Leptomycin B was added to a final concentration of 1M, and the cells were harvested after 24 hours of incubation. K562 were differentiated with 10nM phorbol 12-myristate 13-acetate (PMA). PlasmidspMSCV-puro-JAK2 and pMSCV-puro-JAK2V617F were kindly provided by Dr J. Cools (Center for Human Genetics, KU Leuven, Belgium). Confocal immunofluorescence microscopyThe BM fractions of CD34 ϩ , CD15 ϩ , CD41 ϩ , CD71 ϩ cells and the K562 cells were prepared as previously described. 8 Confocal laser images were captured with a laser scanning LSM 510 META microscope (Zeiss) equipped with a 403 oil-immersion lens. PatientsBM aspirates and peripheral blood (PB) samples were obtained from 15 patients newly diagnosed with MPN according to the WHO criteria. Informed consent was obtained in accordance with the Declaration of Helsinki, and samples were obtained with approval from the Italian ethics committee. Cell sortingBM or PB mononucleated cells were incubated with the antibodies CD15-allophycocyanin, CD41-fluorescein isothiocyanate, CD71-peridinin chlorophyll protein complex, and CD34-phycoerythrin (BD Biosciences), analyzed, and sorted with a fluorescence-activated cell sorter FACSAria (BD Biosciences). ASO-PCR and RT-PCRThe JAK2V617F mutation was identified by allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) as previously described. For personal use only. on May 10, 2018. by guest www.bloodjournal.org From Green quantitative reverse-transcription (QRT)-PCR was performed as previously described. 8 Cell fractionation and immunoblottingCytoplasmic an...
A letter to the editor is presented related to the case where GATA1 is overexpressed in patients with essential thrombocythemia and polycythemia vera, but not in patients with primary myelofibrosis or chronic myelogenous leukemia
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.