JAK2 V617F Mutation Induces a Myeloproliferative Disorder in Mice. (2005)

Abstract Background: An activating mutation (V617F) in the JH2 domain of the Jak2 kinase is found in 74%–97% of patients with polycythemia vera (PV), 23%–57% of patients with essential thrombocythemia and 35%–57% of patients with idiopathic myelofibrosis. We studied the effects of this mutant in murine hematopoietic cells in vitro and in vivo. Materials and Methods: Murine wild type (Jak2-WT) and V617F mutant JAK2 (Jak2-V617F) were cloned into the MIGR1-IRES-GFP retroviral vector. BaF3 cells stably expressing mutant and wild type Jak2 were generated by electroporation followed by prolonged culture in IL-3 containing media and FACS selection for GFP-positive cells. Proliferation and viability assays were performed in graded concentrations of IL-3. Bone marrow from 5-FU treated Balb/c mice were infected with JAK2-V617F, JAK2-WT and empty vector retrovirus and injected into lethally irradiated recipients. The mice were monitored by observation and blood counts. Plasma levels of erythropoietin (EPO) were determined by ELISA on day 44 after transplantation. 1-way analysis of variance (ANOVA) was used to compare differences between cell line and hematologic parameters. The relationship between EPO levels and Hct/Hgb was analysed by Pearson correlation. Results: BaF3 cells expressing JAK2-V617F showed enhanced proliferation in response to murine IL-3 compared to cells expressing Jak2-WT (p&amp;lt;0.001) and parental cells (p= 0.009). Similarly, there was a trend for increased viability (p=0.071 vs. Jak-WT and p=0.059 vs. parental cells). Sudden complete IL-3 withdrawal induced &amp;gt;95% cell death, followed by outgrowth of IL-3-independent lines after 20 days. Mice transplanted with Jak2-V617F showed increased median hematocrit (Hct), hemoglobin (Hgb) and median corpuscular volume (MCV) compared to Jak2-WT and empty vector mice (table). There was a trend for increased white cell counts (WBC). Median EPO levels were 65 pg/ml in the Jak2-V617F mice compared to 88 pg/ml in the Jak2-WT and 131 pg/ml in the empty vector controls (p= 0.032). There was a significant correlation between EPO and Hct (p = 0.011, r = −0.677) and Hgb (p= 0.002, r= −0.770) across the three groups of mice. On day 44 post transplant one Jak2-V617F mouse developed trilineage MPD with a Hct of 56.8%, leukocytosis (22.6 x 103/ul) and thrombocytosis (1004 x 103/ul). The peripheral blood smear showed neutrophilia, elevated platelets and multiple nucleated red cells. Necropsy revealed hepatosplenomegaly. Conclusions: (i) JAK2-V617F induces hypersensitivity to IL-3 in BaF3 cells. Complete IL-3 independence was seen only after “crisis” and prolonged culture, suggesting additional mutations may be required. (ii) In a murine transduction-transplantation model JAK2-V617F induces MPD closely mimicking PV. Studies are in progress to determine whether additional phenotypes may be observed in a larger cohort of animals. JAK2-V617F (n= 4) JAK2- WT (n= 4) MIGR-1 vector-control (n= 5) P value (V617F vs. WT) P value (V617F vs. empty vector) Median blood parameters (range) in groups of mice 44 days after transplant Hct (%) 51.1 (44.6–56.8) 43.7 (42.8–45) 43.6 (40.4–45.2) P = 0.015 P= 0.011 Hgb (g/dl) 16.2 (14.6–17.6) 14.6 (14.2–15.4) 14.2 (13–15) P= 0.033 P= 0.010 MCV (fl) 49.5 (47–54) 46.2 (46–47) 46.2 (45–48) P= 0.041 P= 0.031 WBC (103/ul) 9.6 (3.8–22.6) 5.5 (3.6–9.4) 3.4 (2.2–4.6) ns P= 0.096 EPO (pg/ml) 65 (44–98) 88 (80–112) 131 (95–231) ns P= 0.032