Ruxolitinib inhibits PCM1-JAK2 in vitro and in vivo. (A) Bone marrow smear at diagnosis, showing hypereosinophilia and marked hypergranularity of neutrophils and their precursors. (B) FISH on a cytogenetic marrow specimen at diagnosis. Dual color JAK2 break-apart FISH was performed using the following probes: RP11 307I14–RP11 125K10–RP11 509D8 (Spectrum Green) and RP11 140C18–RP11 635N21 (Spectrum Orange), as described.5 The t(8;9) leads to translocation of the green signal to der(8), while the red probes and a tiny little green signal are retained on the der(9) (solid arrows). The same break-apart pattern is observed in interphase cells (dotted arrows). (C) Dose-response curves of Ba/F3 cells expressing PCM1-JAK2, ETV6-JAK2, SEC31A-JAK2, or JAK2-V617F (left panel). Cells were treated for 24 hours with various concentrations of ruxolitinib, diluted in DMSO immediately before use. The proliferation relative to untreated controls is shown. The growth of wild type Ba/F3 in the presence of IL-3, and various concentrations of ruxolitinib is also shown (curves fitted with GraphPad Prism 5). Western blot analysis of 2 × 106 PCM1-JAK2–transformed Ba/F3 cells after treatment with ruxolitinib for 90 minutes (right panel). Gel electrophoresis was performed using NuPage Bis-Tris 4%-12% gels (Invitrogen). Immunoblotting was done with anti-phospho-JAK2, anti-JAK2, anti-phospho-STAT5, anti-STAT5a (Cell Signaling Technology), and anti–mouse/anti–rabbit peroxidase-labeled antibodies (Amersham Biosciences). (D) Evolution of peripheral blood counts before and after initiation of ruxolitinib. The top panel depicts the evolution of peripheral leukocyte count (blue) and absolute eosinophil count (pink) over time. The middle panel shows hemoglobin levels (red) and platelet count (blue) over time. Erythrocyte or platelet transfusions are shown as red and blue dots above the x-axis. The average daily dose (mg) of hydroxyurea and ruxolitinib are shown in the bottom panel. Start of ruxolitinib treatment is time point 0. (E) Evolution of bone marrow karyotype and FISH. Bars show the relative proportion of t(8;9)-positive metaphases (red) versus normal metaphases (green). The number of t(8;9)-positive metaphases versus normal metaphases is indicated above each bar. Pink lines indicate the percentage of interphase nuclei with a break-apart pattern in these marrow specimens.
