Specific MicroRNA Deregulated in Myleoproliferative Neoplasm Are Regulated by JAK2V617F and May Contribute to Aberrant Hematopoiesis.

Abstract 965

The myeloproliferative neoplasms (MPN), PV, ET and IMF, harbor the same gain-of-function mutation JAK2V617F at a high frequency (∼100%, 70% and 50% respectively). Accumulating evidence suggest that JAK2V617F may not be the initiating event in MPN, and other genetic anomalies play an important role in MPN pathogenesis. We hypothesized that miRNA deregulation contributes to the development of MPN. To test this idea, miRNA expression in CD34+ cells isolated from 8 patient samples (4 PV with JAK2V617F, 3 ET with wild-type JAK2 and 1 IMF with unknown JAK2 status) and 4 healthy controls was determined using a Taqman Low Density Array (TLDA) representing 667 known miRNAs. PV (JAK2V617F) and ET cases (JAK2WT) showed 14 and 78 differentially expressed miRNAs, respectively, when compared to controls. 6 miRNAs were commonly deregulated in PV and ET, while the majority were unique to each disease type. When all MPN patients were grouped and compared to controls, 28 miRNAs were significantly deregulated (p<0.05). These miRNAs differ from those previously reported to be differentially expressed in the peripheral blood of PV patients. Among these 28, mir-214 was down-regulated and mir-410, mir-22* and mir-505* were up-regulated most consistently. Several miRNAs, including mir-135b, mir-542-5p, mir-149, mir-133b and mir-134 were undetectable in normal CD34+ cells and activated in MPN patients. We further hypothesized that some miRNAs are regulated through the action of the mutant JAK2V617F kinase. To test this, miRNA levels were assessed by TaqMan array in HEL and UKE-1 cells (harboring JAK2V617F) treated with 2 μM JAK inhibitor I (Calbiochem) for 20h before RNA extraction. In parallel, miRNA expression as determined by TLDA in TF-1 cells rendered cytokine independent by stable expression of JAK2V617F was compared to that of control TF-1 cells, both cultured overnight in the absence of cytokines. A total of 24 miRNAs were significantly deregulated (>2 fold) in at least two cell line systems. To test which deregulated miRNAs in MPN patients were JAK2 responsive, JAK2 activity was manipulated in HEL and TF-1 cells as described above, and the expression of miRNAs was determined by individual Taqman miRNA assays. mir-1, mir-200a, mir-9, mir-133b, mir-22* and mir-155 were responsive to manipulation of JAK2 activity. miR-155 expression was repressed 50% with the inhibition of JAK2 in HEL cells and stimulated almost 2 fold with the overexpression of JAK2V617F in TF-1 cells. By contrast, mir-214 (downregulated in MPN) and mir-134 (upregulated in MPN) were not responsive to manipulation of JAK2V617F activity in either the gain or loss-of-function systems. To further confirm the ability of JAK2V617F to regulate specific miRNAs, lineage negative (lin-) murine marrow progenitor cells were transduced with JAK2V617F or empty vector, allowed to form colonies for 7 days and miRNA levels in the colonies were determined. Again miR-200a, miR-9 and miR-22* and miR-155 were responsive to JAKV617F overexpression, while mir-134 was not. Transduction of lineage negative murine marrow progenitor cells with a lentiviral vector harboring mir-155 yielded a 30% increase in a myeloid colony formation in vitro. The effect is consistent with the reported ability of mir-155 to induce myeloproliferation in mice. Transduction of marrow progenitors with miR-133b, which is activated in MPN patients, responsive to JAK2V617F manipulation and not previously reported to have a role in hematopoiesis, led to an increase in both erythroid and myeloid colony formation. Taken together we conclude that at least 4 miRNAs are deregulated in CD34+ cells of MPN patients as a result of aberrant JAK2 activity. Two of these tested so far have a role in hematopoiesis. Part of the action of JAK2V617F in myeloproliferation may be mediated by specific miRNA, thus representing alternative therapeutic targets in MPN.