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

Molecular pathogenesis and therapy of polycythemia induced in mice by JAK2 V617F.

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.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.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

Essential role for Stat5a/b in myeloproliferative neoplasms induced by BCR-ABL1 and JAK2(V617F) in mice.

STAT5 proteins are constitutively activated in malignant cells from many patients with leukemia, including the myeloproliferative neoplasms (MPNs) chronic myeloid leukemia (CML) and polycythemia vera (PV), but whether STAT5 is essential for the pathogenesis of these diseases is not known. In the present study, we used mice with a conditional null mutation in the Stat5a/b gene locus to determine the requirement for STAT5 in MPNs induced by BCR-ABL1 and JAK2(V617F) in retroviral transplantation models of CML and PV. Loss of one Stat5a/b allele resulted in a decrease in BCR-ABL1-induced CML-like MPN and the appearance of B-cell acute lymphoblastic leukemia, whereas complete deletion of Stat5a/b prevented the development of leukemia in primary recipients. However, BCR-ABL1 was expressed and active in Stat5-null leukemic stem cells, and Stat5 deletion did not prevent progression to lymphoid blast crisis or abolish established B-cell acute lymphoblastic leukemia. JAK2(V617F) failed to induce polycythemia in recipients after deletion of Stat5a/b, although the loss of STAT5 did not prevent the development of myelofibrosis. These results demonstrate that STAT5a/b is essential for the induction of CML-like leukemia by BCR-ABL1 and of polycythemia by JAK2(V617F), and validate STAT5a/b and the genes they regulate as targets for therapy in these MPNs

Essential role for Stat5a/b in myeloproliferative neoplasms induced by BCR-ABL1 and JAK2(V617F) in mice.

STAT5 proteins are constitutively activated in malignant cells from many patients with leukemia, including the myeloproliferative neoplasms (MPNs) chronic myeloid leukemia (CML) and polycythemia vera (PV), but whether STAT5 is essential for the pathogenesis of these diseases is not known. In the present study, we used mice with a conditional null mutation in the Stat5a/b gene locus to determine the requirement for STAT5 in MPNs induced by BCR-ABL1 and JAK2(V617F) in retroviral transplantation models of CML and PV. Loss of one Stat5a/b allele resulted in a decrease in BCR-ABL1-induced CML-like MPN and the appearance of B-cell acute lymphoblastic leukemia, whereas complete deletion of Stat5a/b prevented the development of leukemia in primary recipients. However, BCR-ABL1 was expressed and active in Stat5-null leukemic stem cells, and Stat5 deletion did not prevent progression to lymphoid blast crisis or abolish established B-cell acute lymphoblastic leukemia. JAK2(V617F) failed to induce polycythemia in recipients after deletion of Stat5a/b, although the loss of STAT5 did not prevent the development of myelofibrosis. These results demonstrate that STAT5a/b is essential for the induction of CML-like leukemia by BCR-ABL1 and of polycythemia by JAK2(V617F), and validate STAT5a/b and the genes they regulate as targets for therapy in these MPNs

Two-decade-long trends (1975-1997) in the incidence, hospitalization, and long-term death rates associated with complete heart block complicating acute myocardial infarction: a community-wide perspective

BACKGROUND: The purpose of this community-wide study was to describe a \u3e2-decade-long experience (1975-97) in the incidence and death rates associated with complete heart block (CHB) in patients with acute myocardial infarction (AMI). Limited population-based data exist describing recent, and changes with time therein, incidence and case-fatality rates associated with CHB complicating AMI. METHODS: We conducted an observational study of 9082 metropolitan Worcester, Mass, residents (1990 census = 437,000) hospitalized with validated AMI in all greater Worcester hospitals during 11 1-year periods between 1975 and 1997. RESULTS: Overall, CHB developed in 5.0% of patients with AMI. The incidence rates of CHB declined in the periods studied (6.0% in 1975/78 vs 3.1% in 1997). Declines in the occurrence of CHB were noted in patients with anterior or inferior/posterior MI. These trends remained after adjustment for other factors that might affect the risk of CHB. Patients in whom CHB developed experienced significantly higher hospital death rates than patients in whom CHB did not develop (46.8% vs 14.6%). However, improving trends in the hospital survival rate of patients with CHB were observed between 1975/78 (47.4% surviving) and 1997 (61.3% surviving). Patients in whom CHB developed during hospitalization were not at increased risk for dying after hospital discharge. CONCLUSIONS: Our findings indicate that the incidence of CHB complicating AMI has declined with time. The hospital prognosis of patients in whom CHB developed has improved, but these patients remain at an increased risk of hospital mortality. The long-term prognosis of patients with inferior MI and CHB is similar to that of patients in whom CHB did not develop. Patients with anterior MI and CHB may be at an increased risk of long-term mortality

Evolution of JAK2 V617F-induced polycythemia to “spent” phase with myelofibrosis.

<div><p>(A) Box-style plots of hematocrit (red squares, left axis) and reticulocyte counts (blue triangles, right axis) in a cohort (<i>n</i> = 12) of Balb/c recipients of syngeneic JAK2 V617F-transduced BM, followed for over eight months after transplantation.</p> <p>Similar data were observed for a B6 cohort (data not shown).</p> <p>(B) Increasing fibrosis (demonstrated by reticulin staining) in spleen (left panels) and BM (right panels) of representative JAK2 V617F recipients at about 3 months (middle panels) and 7 months (bottom panels) after transplantation.</p> <p>Note the marked increase in reticulin staining at 7 months in the JAK2 V617F recipients, but not in recipients of JAK2 WT-transduced BM (top panels).</p> <p>(C): Efficient transfer of the PV-like MPD by transplantation of BM from primary mice sacrificed either in the early, polycythemic phase (left, <i>n</i> = 3, sacrificed 72–167 days post-transplant) or the late, myelofibrotic phase (right, <i>n</i> = 2, sacrificed 208 days post-transplant), to lethally irradiated syngeneic secondary recipients (<i>n</i> = 6 for early phase and <i>n</i> = 4 for late phase).</p> <p>The graphs depict mean hematocrit (black, left axis), reticulocyte count (white, right axis) and peripheral blood leukocyte count (grey, right axis) of the donors at the time of sacrifice, and of the recipients at day 30–70 post-transplant.</p> <p>For transplants performed in the late phase of the disease, the hematocrit and reticulocyte counts of recipients were significantly higher than of the donors (<i>P</i> = 0.0407 and <i>P</i> = 0.0337, respectively, unpaired <i>t</i>-test), while there was no significant difference between donors and recipients transplanted in the early phase.</p></div

Effect of JAK2 V617F on leukocyte and platelet counts.

<div><p>(A) Peripheral blood leukocyte counts for the three cohorts in Balb/c and B6 backgrounds as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000018#pone-0000018-g001" target="_blank">Figure 1</a>.</p> <p>The difference in leukocyte counts between JAK2 V617F and JAK2 WT recipients was significant for the Balb/c cohort (<i>P</i> = 0.0039) but not for B6 (<i>P</i> = 0.0542, unpaired <i>t</i>-test).</p> <p>The hatched portion of the histogram represents the percentage of the total leukocyte count comprised by neutrophils.</p> <p>(B) Wright-Giemsa-stained cytospins of peripheral blood leukocytes from representative mice with BCR-ABL-induced CML-like MPD (left) or JAK2 V617F-induced PV-like MPD (right).</p> <p>Note the predominance of mature neutrophils and lack of immature myeloid elements (myelocytes and promyelocytes) in the JAK2 V617F recipient.</p> <p>(C) Platelet counts for the groups in (A).</p> <p>There was no significant difference in platelet counts between the three cohorts in either stain.</p> <p>(D) GFP fluorescence (left panels) and phase contrast images (right panels) of purified megakaryocytes from recipients of JAK2 V617F-transduced BM (top panels) or normal control mice (bottom panels).</p> <p>Note the GFP fluorescence in the majority of megakaryocytes from JAK2 V617F recipients.</p> <p>(E) Tail bleeding time (Balb/c cohort) for the three groups.</p> <p>The mean bleeding time (bar) of JAK2 V617F recipients was significantly longer than that of vector or JAK2 WT recipients (<i>P</i> = 0.0328 and <i>P</i> = 0.0002, respectively, unpaired <i>t</i>-test).</p></div

JAK2 V617F induces polycythemia through autonomous overproduction of erythrocytes.

<div><p>(A–C): Hematocrit (A), blood hemoglobin (B), and reticulocyte counts (C) from cohorts of Balb/c or B6 mice transplanted with syngeneic BM cells transduced with empty vector (green, <i>n</i> = 2), or retrovirus expressing murine JAK2 WT (blue, <i>n</i> = 10) or JAK2 V617F (red, <i>n</i> = 15).</p> <p>In the case of the B6 cohorts, untransplanted mice (“Normal”, <i>n</i> = 4) were used instead of empty vector controls.</p> <p>The difference between JAK2 V617F and JAK2 WT recipients was significant (unpaired <i>t</i>-test) for hematocrit (<i>P</i><0.0001 for Balb/c and <i>P</i> = 0.0071 for B6), hemoglobin (<i>P</i> = 0.0011 for Balb/c and <i>P</i> = 0.0024 for B6), and reticulocytes (<i>P</i><0.0001 for Balb/c and <i>P</i> = 0.0195 for B6), while there were no significant differences between vector and JAK2 WT recipients.</p> <p>(D) Reticulocyte stains of peripheral blood from recipients of BM transduced with JAK2 WT (left) or JAK2 V617F (right).</p> <p>(E–F): Erythrocyte survival curves (E) and red cell mass determination (F) for the three groups (Balb/c background).</p> <p>The red cell mass of JAK2 V617F recipients was significantly greater than that of recipients of vector- or JAK2 WT-transduced BM (<i>P</i> = 0.0068 and <i>P</i> = 0.0009, respectively, unpaired <i>t</i>-test).</p> <p>(G) Plasma Epo levels for the three groups (B6 background).</p> <p>The mean Epo level (bar) of JAK2 V617F recipients was significantly lower than that of recipients of vector- or JAK2 WT-transduced BM (<i>P</i> = 0.0048 and <i>P</i> = 0.0151, respectively, unpaired <i>t</i>-test).</p> <p>(H) Number of CFU-E colonies from normal BM (green) or spleen of JAK2 V617F recipients (red), expressed as percent of maximal colony number (Balb/c background).</p> <p>Data for JAK2 WT recipients were similar to normal mice (data not shown).</p> <p>All studies were carried out between 6 and 10 weeks post-transplantation.</p></div

Polycythemia induced by JAK2 V617F is independent of Src kinases.

<div><p>(A–C): Hematocrit (A), blood hemoglobin (B), and reticulocyte counts (C) from normal (–) B6 mice (green), B6 recipients of B6 WT BM transduced with retrovirus expressing murine JAK2 WT (blue) or JAK2 V617F (red), and B6 <i>Lyn</i>^−/−<i>Hck</i>^−/−<i>Fgr</i>^−/− BM transduced with retrovirus expressing JAK2 V617F (orange).</p> <p>The difference between recipients of JAK2 V617F-transduced <i>Lyn</i>^−/−<i>Hck</i>^−/−<i>Fgr</i>^−/− BM and recipients of JAK2 WT-transduced WT BM was significant (unpaired <i>t</i>-tests) for hematocrit (<i>P</i> = 0.0009), hemoglobin (<i>P</i> = 0.0007), and reticulocytes (<i>P</i> = 0.0068), while the corresponding differences between recipients of JAK2 V617F-transduced BM from WT and <i>Lyn</i>^−/−<i>Hck</i>^−/−<i>Fgr</i>^−/− donors were not significant.</p> <p>(D) Western blot analysis of extracts of primary myeloerythroid cells from individual normal (lanes 1–3) B6 mice, recipients of WT BM transduced with JAK2 WT retrovirus (lanes 4–5), recipients of WT BM transduced with JAK2 V617F retrovirus (lanes 6–9), and recipients of <i>Lyn</i>^−/−<i>Hck</i>^−/−<i>Fgr</i>^−/− BM transduced with JAK2 V617F retrovirus (lanes 10–14).</p> <p>The membrane was immunoblotted with antibody recognizing the phosphorylated activation loop tyrosine (Y146 homolog) of c-Src, Lyn, Hck, Fyn, Lck, and Yes (top panel), and subsequently blotted with antibody recognizing total c-Src, Fyn, Yes, and Fgr (bottom panel).</p></div