Preferential Nuclear Accumulation of JAK2V617F In CD34+ but Not Granulocytic, Megakaryocytic or Erythroid Cells of Patients with Philadelphia-Negative Myeloproliferative Neoplasia

Abstract 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.