ALBERT

Description: A single somatic mutation, V617F, in Janus kinase 2 (JAK2) is one of the causes of myeloproliferative neoplasms (MPN), including primary myelofibrosis, and the mutant kinase JAK2V617F is a therapeutic target in MPN. However, inhibition of wild-type JAK2 (JAK2WT) can decrease the red blood cell (RBC) or platelet count. Therefore, a JAK2 inhibitor that produces a smaller reduction in the RBC and platelet counts in the therapeutic window would have clinical benefit. NS-018 is a potent and selective inhibitor of JAK2 and Src-family kinases which is currently in an early-phase clinical trial for MPN. To compare the inhibitory effect of NS-018 on JAK2WT and JAK2V617F in the cell, we assessed the antiproliferative activity of NS-018 against Ba/F3 cells expressing murine JAK2WT or JAK2V617F. NS-018 suppressed the growth of Ba/F3-JAK2V617F cells with an IC50 value of 470 nM, whereas it suppressed the growth of Ba/F3-JAK2WT cells stimulated with IL-3 with an IC50value of 2000 nM. Thus, NS-018 showed 4.3-fold selectivity for Ba/F3-JAK2V617F over Ba/F3-JAK2WT cells (V617F/WT ratio). Other JAK2 inhibitors also showed selectivity for Ba/F3-JAK2V617F over Ba/F3-JAK2WT cells, though their selectivity was lower. For example, INCB018424 (ruxolitinib) and TG101348 showed V617F/WT ratios of 2.0 and 1.5, respectively. Among the eight JAK2 inhibitors tested, NS-018 showed the highest selectivity for JAK2V617F cells. NS-018 also inhibited erythroid colony formation in JAK2V617F transgenic mice at significantly lower concentrations than in wild-type mice. To assess the ability of NS-018 to selectively inhibit JAK2V617F-harboring cells in vivo, we established a JAK2V617F bone marrow transplantation (BMT) mouse model. NS-018 was administered by oral gavage twice a day for 40 days at a dose of 50 mg/kg. When assessment was carried out 50 days after the start of the study, NS-018 was found to have significantly prolonged the survival of JAK2V617F BMT mice, decreased their splenomegaly and restored their disrupted splenic architecture. NS-018 also partially suppressed bone marrow fibrosis in JAK2V617F BMT mice. All vehicle-treated mice that had survived to the study endpoint had mild-to-moderate reticulin fibrosis, whereas all mice treated with NS-018 had slight-to-little reticulin fibrosis, except for one mouse with mild fibrosis. Although vehicle-treated JAK2V617F BMT mice showed marked leukocytosis, NS-018 treatment achieved a 95% suppression of this increase. In spite of the marked effects of NS-018 in JAK2V617F BMT mice described above, NS-018 treatment had not decreased the RBC or reticulocyte count after 50 days of administration. JAK2V617F BMT mice showed a 78% decrease in the platelet count compared with control mice, and NS-018 treatment did not further decrease the count. To better understand the ability of NS-018 to preferentially inhibit the mutated form of JAK2, we explored the X-ray co-crystal structure of NS-018 bound to activated JAK2 and focused on the flipped carbonyl group of Gly933, which is located immediately N-terminal to the DFG (Asp-Phe-Gly) motif in the activation loop of JAK2. We identified two kinds of hydrogen-bonding interactions between NS-018 and the carbonyl group of Gly993: water-mediated hydrogen bonding involving a nitrogen atom of NS-018 and a CH•••O hydrogen bond involving an aromatic CH of NS-018. The unique mode of binding of NS-018 to activated JAK2 provides a plausible explanation for its JAK2V617F selectivity. In summary, NS-018 preferentially inhibited the growth of JAK2V617F-harboring cells over JAK2WT-harboring cells. NS-018 was also effective against leukocytosis, splenomegaly, and bone marrow fibrosis, and prolonged survival in JAK2V617F BMT mice with no reduction in the RBC or platelet counts. These characteristics of NS-018 may be explained at least in part by its unique mode of binding to the activated form of JAK2. NS-018 may have therapeutic benefit for MPN patients in virtue of its simultaneous satisfaction of the two requirements of efficacy and reduced hematologic adverse effects. Disclosures: Nakaya: Nippon Shinyaku: Employment. Naito:Nippon Shinyaku: Employment. Niwa:Nippon Shinyaku: Employment. Horio:Nippon Shinyaku: Employment.

Description: Although the Abelson (Abl) tyrosine kinase inhibitor imatinib mesylate has improved the treatment of breakpoint cluster region–Abl (Bcr-Abl)–positive leukemia, resistance is often reported in patients with advanced-stage disease. Although several Src inhibitors are more effective than imatinib and simultaneously inhibit Lyn, whose overexpression is associated with imatinib resistance, these inhibitors are less specific than imatinib. We have identified a specific dual Abl-Lyn inhibitor, NS-187 (elsewhere described as CNS-9), which is 25 to 55 times more potent than imatinib in vitro. NS-187 is also at least 10 times as effective as imatinib in suppressing the growth of Bcr-Abl–bearing tumors and markedly extends the survival of mice bearing such tumors. The inhibitory effect of NS-187 extends to 12 of 13 Bcr-Abl proteins with mutations in their kinase domain but not to T315I. NS-187 also inhibits Lyn without affecting the phosphorylation of Src, Blk, or Yes. These results suggest that NS-187 may be a potentially valuable novel agent to combat imatinib-resistant Philadelphia-positive (Ph+) leukemia.

Description: Chemical modifications of imatinib mesylate made with the guidance of molecular modeling yielded several promising compounds. Among them, we selected a compound denoted NS-187 (elsewhere described as CNS-9) on the basis of its affinity to Abl, and also to Lyn, which may be involved in imatinib-resistance (Figure). The most striking structural characteristic of NS-187 is its trifluoromethyl (CF3) group at position 3 of the benzamide ring. The presence of the CF3 group strengthened the hydrophobic interactionss of the molecule with the hydrophobic pocket of Abl. Another possible merit of the CF3 group is that it may fix the conformation of the drug by hindering its rotation at the 4-position of the benzamide ring; as a result, a CF3-bearing molecule may be more potent than more flexible compounds such as imatinib. In fact, NS-187 was 25–55 times more potent than imatinib in vitro and and at least 10 times more potent than in vivo. NS-187 also inhibited the phosphorylation and growth of all Bcr-Abl mutants tested except T315I at physiological concentrations. Another special feature of NS-187, in addition to its increased affinity to Abl is its unique spectrum of inhibitory activity against protein kinases. At a concentration of 0.1 μM, NS-187 inhibited only four of 79 tyrosine kinases, that is, Abl, Arg, Fyn, and Lyn. Notably, at 0.1 μM NS-187 did not inhibit PDGFR, Blk, Src or Yes. The IC50 values of NS-187 for Abl, Src and Lyn were 5.8 nM, 1700 nM and 19 nM, respectively, and those of imatinib were 106 nM, 〉10,000 nM and 352 nM, respectively. These findings indicate that NS-187 acts as a Bcr-Abl/Lyn inhibitor. In this respect, NS-187 may stand out among other novel Abl tyrosine kinase inhibitors, because BMS-354825 inhibits all members of the Src family, while AMN-107 inhibits none of the Src-family kinases. Our proposed docking models of the NS-187/Abl complex support the notion that NS-187 is more specific for Lyn than for Src. The amino acid at position 252 is either Gln or Cys in Src-family proteins. NS-187 inhibited the Gln252-bearing proteins Abl, Fyn and Lyn but had lower activity against the Cys252-bearing Src and Yes. This is probably because Gln, unlike Cys, readily forms hydrogen bonds. The distinguishing characteristic of NS-187, its high affinity for and specific inhibition of Abl and Lyn, may be useful in the treatment of Bcr-Abl-positive leukemia patients. Figure Figure

Description: Abstract 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

Description: Background Cell signaling mediated by the JAK2-STAT (Janus kinase 2-signal transducer and activator of transcription) pathway plays a critical role in hematopoiesis, and its aberrant activation is associated with the progression of hematological malignancies. For instance, somatic mutations in the JAK2 gene that lead to constitutive activation of STATs are involved in the pathogenesis of myeloproliferative neoplasms (MPN) and refractory anemia with ringed sideroblasts with thrombocytosis, one of the myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN, U). In addition, insufficient inactivation of the JAK-STAT pathway due to repression of SOCS-1 (suppressor of cytokine signaling 1) through hypermethylation of the SOCS-1 gene is involved in the disease progression of high-risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) (Brakensiek et al.; Br. J. Haematol. 2005;130:209). These observations prompted us to investigate the effect of the JAK2-selective inhibitor NS-018, which is in an early-phase clinical trial for MPN, on the colony formation by bone marrow hematopoietic progenitor cells from high-risk de novo MDS patients and the phosphorylation status of STAT3 in these cells. Methods Bone marrow mononuclear cells (BMMNCs) from six MDS patients and three healthy volunteers were collected with informed consent in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Board. The MDS subtypes of the six patients included two RCMD, three RAEB-1 and one RAEB-2 according to the WHO classification. All MDS patients had complex cytogenetic abnormalities and their prognostic risks were defined to be high (N=1) or very high (N=5) in the IPSS-R. Commercially available normal human CD34+ BM cells were also examined for comparison. Cells were incubated in methylcellulose medium containing cytokines with or without NS-018. Burst forming unit-erythroid (BFU-E) and colony forming unit-granulocyte/macrophage (CFU-GM) were scored on day 14 of culture. Colony-forming cells were then collected and subjected to western blotting analysis. Results We first examined the colony-formation capacity of MDS-derived BMMNCs. CFU-GM colony formation was observed in four of six MDS samples, although the absolute CFU-GM numbers from MDS BMMNCs were less than those from normal BMMNCs and normal CD34+BM cells. MDS-derived BMMNCs produced larger numbers of CFU-GM colonies (78–99% of total colonies) than BFU-E, similar to the findings usually observed in AML, whereas normal BMMNCs and normal CD34+BM cells formed virtually the same numbers of CFU-GM colonies and BFU-E colonies. These results suggest that BMMNCs from four of the MDS patients enrolled in this study had characteristics similar to AML BMMNCs, although they showed less proliferation than AML BMMNCs. We next examined the effect of NS-018 on CFU-GM colony formation. NS-018 treatment decreased the numbers of CFU-GM colonies from MDS-derived BMMNCs in a dose-dependent manner, and this effect was significantly more potent against MDS-derived than against normal cells (57.2% inhibition in MDS cells vs. 12.8% inhibition in normal cells at 0.5 mM NS-018; see Figure). These results indicate that NS-018 is preferentially efficacious in inhibiting CFU-GM formation from BMMNCs from high risk MDS. In addition, the level of phospho-STAT3 in MDS-derived colony-forming cells was twice as high as in colony-forming cells from normal BMMNCs, and 1.0 mM NS-018 completely suppressed the phosphorylation of STAT3 in CFU-GM colony-forming cells from MDS. Conclusion Our results show for the first time that a JAK2 inhibitor, NS-018, potently suppresses the formation of MDS colonies. NS-018 could be a new therapeutic option for high-risk MDS patients. Disclosures: Kodama: Nippon Shinyaku: Employment. Naito:Nippon Shinyaku: Employment. Nakaya:Nippon Shinyaku: Employment. Taniwaki:Novartis: Honoraria.

Description: Abstract 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 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.

Description: Abstract 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.

Description: We have identified a specific dual Bcr-Abl/Lyn inhibitor, NS-187 (elsewhere described as CNS-9), which is 25–55 times more potent than imatinib against wild type Bcr-Abl in vitro. To evaluate the potential of NS-187 as a therapeutic agent, we assessed its in vivo activity. When Balb/c mice were given NS-187 orally at a dose of 30 mg/kg, the pharmacokinetic parameters were as follows: Tmax, 2 h; Cmax, 586 ng/ml; AUC0-∝, 2999 ng•h/ml; T1/2, 1.0 h; and bioavailability value (BA), 33%. The maximal tolerated dose (MTD) of NS-187 in Balb/c or Balb/c-nu/nu mice was 200 mg/kg/day (100 mg/kg, twice daily). To test the effect of NS-187 on in vivo tumor growth, Balb/c-nu/nu mice were injected subcutaneously with Bcr-Abl-positive KU812 cells on Day 0 and given NS-187 or imatinib orally twice a day from Day 7 to Day 17. At 20 mg/kg/day, imatinib inhibited tumor growth slightly, while at 200 mg/kg/day, it inhibited tumor growth almost completely. In contrast, at only 0.2 mg/kg/day NS-187 significantly inhibited tumor growth, while at 20 mg/kg/day it completely inhibited tumor growth without any adverse effects. The body weights of the treated tumor-bearing mice were not significantly different from those of untreated mice, even at a dosage of 200 mg/kg/day NS-187. Thus, NS-187 was at least 10-fold more potent than imatinib in vivo with complete inhibition of tumor growth as the end-point. We also tested the ability of NS-187 to suppress tumor growth in another murine tumor model, namely, Balb/c-nu/nu mice intravenously transplanted with BaF3 cells harboring wild type Bcr-Abl. The mice were treated orally with NS-187 or imatinib for 11 days starting on Day 1. All eight untreated mice and all eight mice treated with 400 mg/kg/day imatinib had died by Day 25 due to leukemic cell expansion, and NS-187 significantly prolonged the survival of the mice in a dose-dependent manner. We next examined the ability of NS-187 to block the in vivo growth of BaF3 cells harboring one of the Abl point-mutants M244V, G250E, Q252H, Y253F, T315I, M351T and H396P in Balb/c-nu/nu mice. These mice were treated with NS-187 or imatinib for 11 days starting on Day 1. NS-187 at 200 mg/kg/day significantly prolonged the survival of mice inoculated with BaF3 cells harboring any of these mutants except T315I compared with untreated or imatinib-treated mice (see Figure for an example). Thus, NS-187 was more potent than imatinib and could override the point-mutation-based imatinib-resistance mechanism in vivo. The efficacy and safety of NS-187 for Ph+ leukemias is expected to be verified by early-phase clinical trials. Figure Figure