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.

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4106 An activating mutation in the Janus kinase 2 gene (JAK2) (G1849T, which produces JAK2 V617F) occurs at a high frequency in Bcr-Abl-negative myeloproliferative neoplasms (MPNs). JAK2 V617F induces cytokine-independent growth in cell lines and, in murine models, recapitulates much of the pathobiology observed in MPN patients, suggesting that small-molecule inhibitors targeting JAK2 may be therapeutically useful. Some orally bioavailable inhibitors of JAK2 are already in clinical trials. NS-018 is a novel JAK2 inhibitor that inhibits JAK2 enzyme activity with an IC50 value of less than 1 nM. NS-018 shows 30–50-fold selectivity for JAK2 over other JAK-family kinases such as JAK1, JAK3 and TYK2. We tested NS-018 in a murine model of MPN induced by JAK2 V617F. Mice expressing JAK2 V617F controlled by the H2Kb promoter (V617F-TG mice) show an MPN phenotype: leukocytosis, thrombocytosis, progressive anemia, hepatosplenomegaly with extramedullary hematopoiesis, megakaryocyte hyperplasia and bone marrow fibrosis. They also exhibit body weight loss and high mortality compared to wild-type controls. Bone-marrow cells show constitutive activation of STAT5 and cytokine-independent growth of erythroid colony-forming units (CFU-E). NS-018 inhibited cytokine-independent CFU-E growth and constitutive activation of STAT5 in V617F-TG cells in vitro. For in vivo experiments, V617F-TG mice were divided into treatment and vehicle control groups after disease was established at 12 weeks after birth. NS-018 was administered for 24 weeks by oral gavage at doses of 25 mg/kg or 50 mg/kg bid, and the control groups received vehicle only. Mice were monitored by blood counts, and a subset of mice was euthanized for detailed histopathology and fluorescence activated cell sorting analysis. During the study, 12 of 34 mice died in the vehicle group, whereas 1 of 36 mice died in the 50 mg/kg group. There was a statistically significant prolongation of survival in the 50 mg/kg group (p<0.01). Mice treated with NS-018 gained more weight than vehicle-treated mice, and were comparable to wild-type mice. V617F-TG at 12 weeks old showed severe leukocytosis with average white blood cell counts of 24 × 1010/L. After two weeks of NS-018 treatment, the leukocyte count was reduced to 59% in 25 mg/kg group and 39% in the 50 mg/kg group compared to vehicle group, and the effect was maintained until the end of the study. The inhibitory effect of NS-018 on T or B lymphocytes was much less than on myeloid cells. The 50 mg/kg group showed no progression of anemia. NS-018 treatment also improved hepatosplenomegaly in a dose-dependent manner. In the spleen, Mac-1/Gr-1+ myeloid cells associated with extramedullary hematopoiesis were significantly decreased, and B220+ B cells were increased by NS-018 treatment. In correlation with reduction of organ weights and infiltrating myeloid cells, there was also clear evidence of a dose-dependent reduction in the histopathology of extramedullary hematopoiesis in the spleen, liver, and lungs of NS-018-treated mice. In contrast to the improvement in the pathology of these organs, NS-018 had little impact on the progression of fibrosis and megakaryocyte hyperplasia in bone marrow. No significant toxicity was observed in treated mice. In conclusion, NS-018 demonstrated therapeutic efficacy in a murine model of MPN induced by JAK2 V617F. In V617F-TG, which closely mimics human MPN, NS-018 significantly improved survival, body weight loss, hepatosplenomegaly, leukocytosis and anemia progression, thus confirming the viability of a targeted-therapy approach in managing JAK2 V617F positive MPNs. On the basis of these preclinical experiments, NS-018 appears to be an excellent candidate for phase I/II studies in patients with Bcr-Abl-negative MPNs. Disclosures: Nakaya: Nippon Shinyaku Co., Ltd: Employment. Homan:Nippon Shinyaku Co., Ltd: Employment. Kotera:Nippon Shinyaku Co., Ltd: Employment. Shibayama:Nippon Shinyaku Co., Ltd: Employment. Naito:Nippon Shinyaku Co., Ltd: Employment. Shimoda:Nippon Shinyaku Co., Ltd: Research Funding.

4107 A somatic point mutation of Janus Kinase 2 (JAK2) tyrosine kinase (JAK2 V617F) has been shown to occur at a high frequency in myeloproliferative neoplasm (MPN) patients. JAK2 V617F is a constitutively activated kinase that activates the JAK/STAT signaling pathway and dysregulates cell growth and function. These findings suggest that the inhibition of aberrant JAK2 activation has a therapeutic benefit. Our novel JAK2 inhibitor, NS-018, is highly active against JAK2 with an IC50 value of less than 1 nM, and it has 30–50-fold selectivities for JAK2 over other JAK-family kinases such as JAK1, JAK3 and Tyk2. We determined the X-ray structure of JAK2 in complex with NS-018. An Asp-Phe-Gly (DFG) motif is located at the N-terminus of the activation loop and regulates ATP binding. The resolved X-ray structure showed that NS-018 bound to JAK2 in the “DFG-in” active conformation. A molecular modeling study indicated that NS-018 would hardly bind to JAK2 in the “DFG-out” inactive conformation. In accordance with the structural analysis, NS-018 preferentially suppressed the growth of bone-marrow cells expressing activated JAK2. Thus, NS-018 reduced in a dose-dependent manner the number of erythroid colony-forming units (CFU-E) derived from bone-marrow cells taken from JAK2 V617F transgenic mice, but had only a limited effect on the number of colonies from wild-type mice (Figure A). NS-018 had no effect on the number of granulocyte-macrophage colony-forming units (CFU-GM) from either mouse strain. Furthermore, NS-018 showed potent antiproliferative activity against Ba/F3 cells expressing JAK2 V617F with an IC50 value of <100 nM but showed only minimal cytotoxicity against most other hematopoietic and non-hematopoietic cell lines (IC50 >3 μ M). In a mouse Ba/F3-JAK2 V617F leukemia model, NS-018 significantly prolonged survival during repeated oral administrations at 6.25 mg/kg bid and reduced splenomegaly at doses as low as 1.5 mg/kg bid. NS-018 was well tolerated at dosages of more than 100 mg/kg bid. In conclusion, NS-018 is a potent JAK2 inhibitor which preferentially inhibits an activated form of JAK2 and has potent in vitro and in vivo efficiency in preclinical studies. NS-018 is expected to be suitable for the treatment of MPN caused by aberrant JAK2 activation and its effectiveness will be verified by early-phase clinical investigations in the near future. JAK2 V617F preferential inhibition of erythrocyte colony growth Bone-marrow cells were collected from femurs of JAK2 V617F transgenic mice and same-strain BDF1 wild-type mice. (a) To detect CFU-E colonies, cells were treated with NS-018 in semisolid methylcellulose containing erythropoietin (EPO) and cell clusters were counted after incubation for two days. (b) To detect CFU-GM colonies, cells were treated with NS-018 in semisolid methylcellulose containing EPO, interleukin-3 (IL-3), IL-6 and stem cell factor and colonies were counted on day 7. Disclosures: Nakaya: Nippon Shinyaku Co., Ltd: Employment. Naito:Nippon Shinyaku Co., Ltd: Employment. Homan:Nippon Shinyaku Co., Ltd: Employment. Sugahara:Nippon Shinyaku Co., Ltd: Employment. Horio:Nippon Shinyaku Co., Ltd: Employment. Niwa:Nippon Shinyaku Co., Ltd: Employment. Shimoda:Nippon Shinyaku Co., Ltd: Research Funding.

2900 Multiple myeloma (MM) is a clonal B-cell malignancy characterized by the accumulation of malignant plasma cells in the bone marrow, and it finally leads to osteolytic bone destruction and impaired hematopoiesis. The pathophysiology of MM is closely linked with the bone marrow microenvironment, which consists of various cell components including bone marrow stromal cells (BMSCs) and bone marrow endothelial cells as well as osteoclasts and osteoblasts. Interaction between MM cells and BMSCs through cell-adhesion molecules confers drug resistance to myeloma cells and stimulates the release of cytokines such as interleukin-6 (IL-6) and receptor activator of NF-kappaB ligand (RANKL) from BMSCs. IL-6 is a major cytokine which enhances cell proliferation and promotes the survival of MM cells by downstream signaling through Janus kinase (JAK) and signal transducer and activator of transcription (STAT). RANKL triggers osteoclast differentiation and activation leading to bone resorption, lytic bone lesion, and osteopenia. The Src family kinases (SFKs), c-Src and Fyn, mediate signaling by cell-adhesion molecules and the RANKL receptor, and they play important roles in cell adhesion and osteoclast differentiation. NS-018 is a potent and selective dual JAK2/SFKs inhibitor which is under phase1/2 clinical development for the treatment of myelofibrosis. A previous study has shown that NS-018 inhibits JAK2, c-Src and Fyn kinases with IC50 values of 0.72, 6.0 and 7.3 nmol/L, respectively. NS-018 also inhibits JAK2 and SFKs in cells as evidenced by its antiproliferative effect against Ba/F3 cells expressing constitutively activated JAK2 and NIH3T3 cells transformed by v-Src. In the present study, the ability of NS-018 to inhibit JAK2/STAT3 signaling was examined in IL-6-responsive human MM cell lines such as U266, RPMI 8226, and PCM6. NS-018 suppressed IL-6-induced phosphorylation of STAT3 in a dose-dependent manner at concentrations greater than 100 nmol/L. In addition, NS-018 inhibited the IL-6-enhanced the proliferation of PCM6 cells at concentrations similar to those required to inhibit STAT3 phosphorylation. To assess whether SFKs inhibition by NS-018 could contribute to an improvement in MM pathology, we next investigated the effect of NS-018 on the adhesion of myeloma cells to cell-adhesion molecules and on osteoclast formation. NS-018 (100 nmol/L) inhibited the adhesion of RPMI 8226 cells to collagen type 1 and VCAM-1 by about 40%. NS-018 also suppressed RANKL-induced differentiation of human osteoclast precursor cells to mature osteoclasts. The numbers of TRAP-positive multinucleated osteoclasts were reduced to about one-half at 100 nmol/L NS-018 and none were observed at 1000 nmol/L. NS-018 similarly suppressed the differentiation of murine RAW264.7 cells to mature osteoclasts. The suppression of cell adhesion and osteoclast formation by NS-018 could both be mediated by c-Src and/or Fyn inhibition, because both inhibitory effects were observed with a typical SFKs inhibitor but not with a typical JAK2 inhibitor. In conclusion, NS-018 reduced IL-6-enhanced myeloma cell proliferation through inhibition of the JAK2/STAT3 signaling pathway and suppressed cell adhesion and osteoclast formation through inhibition of c-Src and/or Fyn. These results suggest that NS-018 has a dual mechanism of action in MM by simultaneously blocking the JAK2/STAT3 and SFKs pathways. Treatment with NS-018 is a potential new therapeutic option to improve the complex pathological condition of patients with MM. Disclosures: No relevant conflicts of interest to declare.