The JAK2V617 mutation induces constitutive activation and agonist hypersensitivity in basophils of polycythemia vera.

BACKGROUND: The JAK2V617F mutation has been associated with constitutive and enhanced activation of neutrophils, while no information is available concerning other leukocyte subtypes. DESIGN AND METHODS: We evaluated correlations between JAK2V617F mutation and the count of circulating basophils, the number of activated CD63(+) basophils, their response in vitro to agonists as well as the effects of a JAK2 inhibitor. RESULTS: We found that basophil count was increased in patients with JAK2V617F -positive myeloproliferative neoplasms, particularly in those with polycythemia vera, and was correlated with the V617F burden. The burden of V617F allele was similar in neutrophils and basophils from patients with polycythemia vera, while total JAK2 mRNA content was remarkably greater in the basophils; however, the content of JAK2 protein in basophils was not increased. The number of CD63(+) basophils was higher in patients with polycythemia vera than in healthy subjects or patients with essential thrombocythemia or primary myelofibrosis and was correlated with the V617F burden. Ultrastructurally, basophils from patients with polycythemia vera contained an increased number of granules, most of which were empty suggesting cell degranulation in vivo. Ex vivo experiments revealed that basophils from patients with polycythemia vera were hypersensitive to the priming effect of interleukin-3 and to f-MLP-induced activation; pre-treatment with a JAK2 inhibitor reduced polycythemia vera basophil activation. Finally, we found that the number of circulating CD63(+) basophils was significantly greater in patients suffering from aquagenic pruritus, who also showed a higher V617F allele burden. CONCLUSIONS: These data indicate that the number of constitutively activated and hypersensitive circulating basophils is increased in polycythemia vera, underscoring a role of JAK2V617F in these cells’ abnormal function and, putatively, in the pathogenesis of pruritus

Tetraspanin CD9 participates in dysmegakaryopoiesis and stromal interactions in primary myelofibrosis

Primary myelofibrosis is characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in the bone marrow and spleen. The expression of CD9, a tetraspanin known to participate in megakaryopoiesis, platelet formation, cell migration and interaction with stroma, is deregulated in patients with primary myelofibrosis and is correlated with stage of myelofibrosis. We investigated whether CD9 participates in the dysmegakaryopoiesis observed in patients and whether it is involved in the altered interplay between megakaryocytes and stromal cells. We found that CD9 expression was modulated during megakaryocyte differentiation in primary myelofibrosis and that cell surface CD9 engagement by antibody ligation improved the dysmegakaryopoiesis by restoring the balance of MAPK and PI3K signaling. When co-cultured on bone marrow mesenchymal stromal cells from patients, megakaryocytes from patients with primary myelofibrosis displayed modified behaviors in terms of adhesion, cell survival and proliferation as compared to megakaryocytes from healthy donors. These modifications were reversed after antibody ligation of cell surface CD9, suggesting the participation of CD9 in the abnormal interplay between primary myelofibrosis megakaryocytes and stroma. Furthermore, silencing of CD9 reduced CXCL12 and CXCR4 expression in primary myelofibrosis megakaryocytes as well as their CXCL12-dependent migration. Collectively, our results indicate that CD9 plays a role in the dysmegakaryopoiesis that occurs in primary myelofibrosis and affects interactions between megakaryocytes and bone marrow stromal cells. These results strengthen the “bad seed in bad soil” hypothesis that we have previously proposed, in which alterations of reciprocal interactions between hematopoietic and stromal cells participate in the pathogenesis of primary myelofibrosis

miRNA-mRNA integrative analysis in primary myelofibrosis CD34+ cells: role of miR-155/JARID2 axis in abnormal megakaryopoiesis

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by megakaryocyte (MK) hyperplasia, bone marrow fibrosis, and abnormal stem cell trafficking. PMF may be associated with somatic mutations in JAK2, MPL, or CALR. Previous studies have shown that abnormal MKs play a central role in the pathophysiology of PMF. In this work, we studied both gene and microRNA (miRNA) expression profiles in CD34(+) cells from PMF patients. We identified several biomarkers and putative molecular targets such as FGR, LCN2, and OLFM4. By means of miRNA-gene expression integrative analysis, we found different regulatory networks involved in the dysregulation of transcriptional control and chromatin remodeling. In particular, we identified a network gathering several miRNAs with oncogenic potential (eg, miR-155-5p) and targeted genes whose abnormal function has been previously associated with myeloid neoplasms, including JARID2, NR4A3, CDC42, and HMGB3. Because the validation of miRNA-target interactions unveiled JARID2/miR-155-5p as the strongest relationship in the network, we studied the function of this axis in normal and PMF CD34(+) cells. We showed that JARID2 downregulation mediated by miR-155-5p overexpression leads to increased in vitro formation of CD41(+) MK precursors. These findings suggest that overexpression of miR-155-5p and the resulting downregulation of JARID2 may contribute to MK hyperplasia in PMF

Ruxolitinib is an effective treatment for CALR-positive patients with myelofibrosis

Approximately 60–65% of patients with myelofibrosis carry mutations in the Janus kinase 2 gene (JAK2) and 5–10% carry mutations in the thrombopoietin receptor gene, MPL (Vainchenkeret al, 2011). Recently, somatic mutations in the calreticulin gene (CALR) were identified in most patients lacking JAK2 or MPL mutations (Klampflet al, 2013; Nangalia et al, 2013). In preclinical models, overexpression o fCALR mutations resulted in cytokine-independent prolifera-tion of Ba/F3 cells and activation of signal transducer and activator of transcription 5 (STAT5) (Klampflet al, 2013). Fedratinib, a JAK2 inhibitor, suppressed these effects in vitro (Klampfl et al, 2013) and reduced palpable spleen length and symptoms in two patients with myelofibrosis (Passamonti et al , 2014). These results suggest that inhibition of abnormal JAK/STAT signalling represents a therapeutic option for CALR-mutated patients