Efficacy of R723, a Potent and Selective JAK2 Inhibitor, in JAK2V617F-Induced Murine MPD Model.

Abstract 3897

Poster Board III-833

The V617F mutation of JAK2 (JAK2-V617F) occurs at a high frequency in Philadelphia chromosome-negative myeloproliferative diseases (MPDs). JAK2-V617F leads to constitutive activation of the JAK2 kinase, causing cytokine independent growth in cell lines and development of MPD in mice. These data suggest that aberrant activation of JAK2 plays a pivotal role in the pathophysiology of MPDs and targeting JAK2-V617F may be therapeutically useful. Some orally bioavailable inhibitors of JAK2 are already in clinical trials. R723 is a potent and highly selective (in vitro kinase assay: JAK2 IC₅₀=2nM, JAK1 IC₅₀ >1μM, JAK3 IC₅₀ =24nM) small molecule inhibitor of JAK2. We tested R723 in a murine model of MPD induced by JAK2-V617F. H2K_b promoter-controlled JAK2-V617F expressing mice (V617F-TG) show extreme leukocytosis, thrombocytosis and progressive anemia. They also show hepato-splenomegaly with extramedullary hematopoiesis. Megakaryocytes are predominant in bone marrow and new formation of bony trabeculae and accumulation of fibers are seen. In vitro analysis of bone marrow cells shows constitutive activation of STAT5 and formation of cytokine-independent growth of erythroid colony-forming units (CFU-E). They exhibit high mortality compared to wild-type controls. At first, we assessed the effect of R723 on wild-type (WT) and V617F-TG bone marrow cells in vitro. R723 inhibited cytokine-independent CFU-E growth and constitutive activation of STAT5 of V617F-TG cells. R723 also inhibited cytokine-dependent colony growth and cytokine-induced STAT5 activation in both WT and V617F-TG cells at the same level. Next, we assessed the effect of R723 in vivo. On development of MPD, V617F-TG mice were divided into treatment or vehicle control groups. R723 was administered by oral gavage at 35mg/kg or 70mg/kg bid for 16 weeks, whereas the control groups received vehicle only. Mice were followed by blood counts and a subset of mice was euthanized for detailed histopathology and FACS analysis. In treated mice, there was a significant reduction in leukocyte count in both groups, and a reduction in platelet count in the high dose group. There was no improvement in anemia. They showed a dose-dependent-reduction of hepato-splenomegaly. Histopathology and FACS analysis revealed a reduction of myeloid cells, with partially restored architecture in spleen. In bone marrow, R723 had little effect on progression of fibrosis and megakaryocyte hyperplasia. During the time course of study, 6 out of 23 mice died in the vehicle group, whereas 1 out of 26 mice died in the 70mg/kg group. There was a statistically significant prolongation of survival in the 70mg/kg group (p<0.05). R723 shows therapeutic efficacy in a murine model of MPD induced by JAK2-V617F, and could become the basis for a next generation of potent and selective compounds targeting JAK2-dependent MPDs.