JAK2 activation is the driver mechanism in negative myeloproliferative neoplasms (MPN), i. 13] Recent data have documented the existence of MF HSC residing in the spleen with increased transplantation capacity in ZM 336372 comparison to blood or marrow HSC [14]. Cues promoting this MF HSC circulation and homing to extramedullary territories have not been completely characterized. Interestingly, a recent study has shown that the MF splenic environment is characterized by an increased level of intact and functional CXCL12 that can contribute to the localization of MF CD34+ cells to the spleen [15]. CXCR4, the receptor for CXCL12, is a master regulator of cell trafficking in normal and pathological settings [16C18]. CXCR4 promotes HSC retention within the BM microenvironment [19, 20], and CXCR4 inhibitors ZM 336372 induce neutrophil and HSC mobilization [21, 22]. These cells are mobilized from BM, but also from EMH sites [23]. Similarly, CXCR4 antagonists specifically target malignant ZM 336372 leukemic precursors, present both in BM and EM tissues [24, 25]. These data are consistent with CXCL12 expression in BM and extramedullary organs [26, 27], and indicate that CXCL12/CXCR4 ZM 336372 axis is involved in the dynamic interplay between HSC and multiple different tissue compartments. Several alterations of the CXCL12/CXCR4 axis have been identified in MF, including the abnormal processing of CXCL12 in a pathological environment [28] and a decreased expression of CXCR4 through hypermethylation of the gene promoter [29C31]. Nevertheless, MF CD34+ cells demonstrate higher migration compared to normal control peripheral blood (PB) CD34+ cells [32]. Thus, even in a context of low CXCR4 expression, a gain of function of CXCR4 characterizes MF CD34+ cells, which may favor their maintenance within the bone marrow microenvironment and extramedullar sites. It is presently unknown whether there is a crosstalk between JAK2 oncogenic activation and CXCL12/CXCR4 signaling, which may be involved in cell trafficking and EMH. Here, we show that JAK2 activation by both oncogenic events and exogenous cytokines synergize with CXCL12/CXCR4 pathway to induce chemotaxis and signaling via PI3K activation. Altogether, these results suggest that JAK2 inhibitors can reduce cell trafficking by decreasing the CXCL12/CXCR4 activity. This may be part of their therapeutic activity, which mainly targets EMH. Moreover, these data provide a rationale to explore the therapeutic activity of combined therapy between JAK2 inhibitors and CXCR4 antagonists or PI3K inhibitors. RESULTS MPLW515L expression increases the chemotactic response to CXCL12 To investigate a possible crosstalk between JAK2 and CXCR4 signaling, we expressed the gain-of-function migratory responses to CXCL12 of PB CD34+ cells were analyzed TRUNDD in 33 MF patients (20 PMF, 6 post-ET/MF and 7 post-PV/MF). An increased chemotactic response to CXCL12 was noticed in MF CD34+ cell samples (Figure ?(Figure6A)6A) which was in average greater than that observed with CD34+ cells from different controls (mobilized PB, non-mobilized PB and BM) (Figure ?(Figure6B).6B). However, a heterogeneous migratory response to CXCL12 was ZM 336372 noticed, as some patient samples migrated in a similar manner as the controls. Pretreatment of MF CD34+ cells with the CXCR4 inhibitor TN140 inhibited CXCL12-induced migration (Figure ?(Figure6C)6C) demonstrating that this migratory response is CXCR4 specific. CXCR4 expression was heterogeneous among MF CD34+ cells (Figure 6D, 6E) and was slightly, but significantly correlated with the chemotactic response (r=0.4994, P= 0.0068) (Figure ?(Figure6F).6F). Thus, MF CD34+ cells exhibit a strong chemotactic response to CXCL12 even in case of low or intermediate CXCR4 membrane expression, suggesting that the CXCL12/CXCR4 signaling pathway is over activated in MF patients. Figure 6 Chemotaxis of MF CD34+ cells in response to CXCL12 To determine the role of oncogenic JAK2, we specifically knocked down JAK2V617F in MF CD34+ cells using a specific shRNA against JAK2V617F. The specificity of this shRNA was.