Splenectomy Normalizes Hematocrit in Murine Polycythemia Vera

Splenic enlargement (splenomegaly) develops in numerous disease states, although a specific pathogenic role for the spleen has rarely been described. In polycythemia vera (PV), an activating mutation in Janus kinase 2 (JAK2V617) induces splenomegaly and an increase in hematocrit. Splenectomy is sparingly performed in patients with PV, however, due to surgical complications. Thus, the role of the spleen in the pathogenesis of human PV remains unknown. We specifically tested the role of the spleen in the pathogenesis of PV by performing either sham (SH) or splenectomy (SPL) surgeries in a murine model of JAK2V617F-driven PV. Compared to SH-operated mice, which rapidly develop high hematocrits after JAK2V617F transplantation, SPL mice completely fail to develop this phenotype. Disease burden (JAK2V617) is equivalent in the bone marrow of SH and SPL mice, however, and both groups develop fibrosis and osteosclerosis. If SPL is performed after PV is established, hematocrit rapidly declines to normal even though myelofibrosis and osteosclerosis again develop independently in the bone marrow. In contrast, SPL only blunts hematocrit elevation in secondary, erythropoietin-induced polycythemia. We conclude that the spleen is required for an elevated hematocrit in murine, JAK2V617F-driven PV, and propose that this phenotype of PV may require a specific interaction between mutant cells and the spleen

Efficacy of NS-018, a potent and selective JAK2/Src inhibitor, in primary cells and mouse models of myeloproliferative neoplasms

Aberrant activation of Janus kinase 2 (JAK2) caused by somatic mutation of JAK2 (JAK2V617F) or the thrombopoietin receptor (MPLW515L) plays an essential role in the pathogenesis of myeloproliferative neoplasms (MPNs), suggesting that inhibition of aberrant JAK2 activation would have a therapeutic benefit. Our novel JAK2 inhibitor, NS-018, was highly active against JAK2 with a 50% inhibition (IC50) of <1 n, and had 30–50-fold greater selectivity for JAK2 over other JAK-family kinases, such as JAK1, JAK3 and tyrosine kinase 2. In addition to JAK2, NS-018 inhibited Src-family kinases. NS-018 showed potent antiproliferative activity against cell lines expressing a constitutively activated JAK2 (the JAK2V617F or MPLW515L mutations or the TEL–JAK2 fusion gene; IC50=11–120 n), but showed only minimal cytotoxicity against most other hematopoietic cell lines without a constitutively activated JAK2. Furthermore, NS-018 preferentially suppressed in vitro erythropoietin-independent endogenous colony formation from polycythemia vera patients. NS-018 also markedly reduced splenomegaly and prolonged the survival of mice inoculated with Ba/F3 cells harboring JAK2V617F. In addition, NS-018 significantly reduced leukocytosis, hepatosplenomegaly and extramedullary hematopoiesis, improved nutritional status, and prolonged survival in JAK2V617F transgenic mice. These results suggest that NS-018 will be a promising candidate for the treatment of MPNs

Analysis of jak2 catalytic function by peptide microarrays: The role of the JH2 domain and V617F mutation

Janus kinase 2 (JAK2) initiates signaling from several cytokine receptors and is required for biological responses such as erythropoiesis. JAK2 activity is controlled by regulatory proteins such as Suppressor of Cytokine Signaling (SOCS) proteins and protein tyrosine phosphatases. JAK2 activity is also intrinsically controlled by regulatory domains, where the pseudokinase (JAK homology 2, JH2) domain has been shown to play an essential role. The physiological role of the JH2 domain in the regulation of JAK2 activity was highlighted by the discovery of the acquired missense point mutation V617F in myeloproliferative neoplasms (MPN). Hence, determining the precise role of this domain is critical for understanding disease pathogenesis and design of new treatment modalities. Here, we have evaluated the effect of inter-domain interactions in kinase activity and substrate specificity. By using for the first time purified recombinant JAK2 proteins and a novel peptide micro-array platform, we have determined initial phosphorylation rates and peptide substrate preference for the recombinant kinase domain (JH1) of JAK2, and two constructs comprising both the kinase and pseudokinase domains (JH1-JH2) of JAK2. The data demonstrate that (i) JH2 drastically decreases the activity of the JAK2 JH1 domain, (ii) JH2 increased the Kmfor ATP (iii) JH2 modulates the peptide preference of JAK2 (iv) the V617F mutation partially releases this inhibitory mechanism but does not significantly affect substrate preference or Kmfor ATP. These results provide the biochemical basis for understanding the interaction between the kinase and the pseudokinase domain of JAK2 and identify a novel regulatory role for the JAK2 pseudokinase domain. Additionally, this method can be used to identify new regulatory mechanisms for protein kinases that provide a better platform for designing specific strategies for therapeutic approaches

Molecular Pathogenesis and Therapy of Polycythemia Induced in Mice by JAK2 V617F

BACKGROUND: A somatic activating mutation (V617F) in the JAK2 tyrosine kinase was recently discovered in the majority of patients with polycythemia vera (PV), and some with essential thrombocythemia (ET) and chronic idiopathic myelofibrosis. However, the role of mutant JAK2 in disease pathogenesis is unclear. METHODS AND FINDINGS: We expressed murine JAK2 WT or V617F via retroviral bone marrow transduction/transplantation in the hematopoietic system of two different inbred mouse strains, Balb/c and C57Bl/6 (B6). In both strains, JAK2 V617F, but not JAK2 WT, induced non-fatal polycythemia characterized by increased hematocrit and hemoglobin, reticulocytosis, splenomegaly, low plasma erythropoietin (Epo), and Epo-independent erythroid colonies. JAK2 V617F also induced leukocytosis and neutrophilia that was much more prominent in Balb/c mice than in B6. Platelet counts were not affected in either strain despite expression of JAK2 V617F in megakaryocytes and markedly prolonged tail bleeding times. The polycythemia tended to resolve after several months, coincident with increased spleen and marrow fibrosis, but was resurrected by transplantation to secondary recipients. Using donor mice with mutations in Lyn, Hck, and Fgr, we demonstrated that the polycythemia was independent of Src kinases. Polycythemia and reticulocytosis responded to treatment with imatinib or a JAK2 inhibitor, but were unresponsive to the Src inhibitor dasatinib. CONCLUSIONS: These findings demonstrate that JAK2 V617F induces Epo-independent expansion of the erythroid lineage in vivo. The fact that the central erythroid features of PV are recapitulated by expression of JAK2 V617F argues that it is the primary and direct cause of human PV. The lack of thrombocytosis suggests that additional events may be required for JAK2 V617F to cause ET, but qualitative platelet abnormalities induced by JAK2 V617F may contribute to the hemostatic complications of PV. Despite the role of Src kinases in Epo signaling, our studies predict that Src inhibitors will be ineffective for therapy of PV. However, we provide proof-of-principle that a JAK2 inhibitor should have therapeutic effects on the polycythemia, and perhaps myelofibrosis and hemostatic abnormalities, suffered by MPD patients carrying the JAK2 V617F mutation

Efficacious Intermittent Dosing of a Novel JAK2 Inhibitor in Mouse Models of Polycythemia Vera

A high percentage of patients with the myeloproliferative disorder polycythemia vera (PV) harbor a Val617→Phe activating mutation in the Janus kinase 2 (JAK2) gene, and both cell culture and mouse models have established a functional role for this mutation in the development of this disease. We describe the properties of MRLB-11055, a highly potent inhibitor of both the WT and V617F forms of JAK2, that has therapeutic efficacy in erythropoietin (EPO)-driven and JAK2V617F-driven mouse models of PV. In cultured cells, MRLB-11055 blocked proliferation and induced apoptosis in a manner consistent with JAK2 pathway inhibition. MRLB-11055 effectively prevented EPO-induced STAT5 activation in the peripheral blood of acutely dosed mice, and could prevent EPO-induced splenomegaly and erythrocytosis in chronically dosed mice. In a bone marrow reconstituted JAK2V617F-luciferase murine PV model, MRLB-11055 rapidly reduced the burden of JAK2V617F-expressing cells from both the spleen and the bone marrow. Using real-time in vivo imaging, we examined the kinetics of disease regression and resurgence, enabling the development of an intermittent dosing schedule that achieved significant reductions in both erythroid and myeloid populations with minimal impact on lymphoid cells. Our studies provide a rationale for the use of non-continuous treatment to provide optimal therapy for PV patients

Epigenetic abnormalities in myeloproliferative neoplasms: a target for novel therapeutic strategies

The myeloproliferative neoplasms (MPNs) are a group of clonal hematological malignancies characterized by a hypercellular bone marrow and a tendency to develop thrombotic complications and to evolve to myelofibrosis and acute leukemia. Unlike chronic myelogenous leukemia, where a single disease-initiating genetic event has been identified, a more complicated series of genetic mutations appear to be responsible for the BCR-ABL1-negative MPNs which include polycythemia vera, essential thrombocythemia, and primary myelofibrosis. Recent studies have revealed a number of epigenetic alterations that also likely contribute to disease pathogenesis and determine clinical outcome. Increasing evidence indicates that alterations in DNA methylation, histone modification, and microRNA expression patterns can collectively influence gene expression and potentially contribute to MPN pathogenesis. Examples include mutations in genes encoding proteins that modify chromatin structure (EZH2, ASXL1, IDH1/2, JAK2V617F, and IKZF1) as well as epigenetic modification of genes critical for cell proliferation and survival (suppressors of cytokine signaling, polycythemia rubra vera-1, CXC chemokine receptor 4, and histone deacetylase (HDAC)). These epigenetic lesions serve as novel targets for experimental therapeutic interventions. Clinical trials are currently underway evaluating HDAC inhibitors and DNA methyltransferase inhibitors for the treatment of patients with MPNs