Haruna Naito

Antitumor Activity of Small Interfering RNA/Cationic Liposome Complex in Mouse Models of Cancer

Purpose: The RNA interference effect is an alternative to antisense DNA as an experimental method of down-regulating a specific target protein. Although the RNA interference effect, which is mediated by small interfering RNA (siRNA) or micro-RNA, has potential application to human therapy, the hydrodynamic method usually used for rapid administration of oligonucleotides is unsuitable for use in humans. In this study, we have investigated the antitumor activity of a synthetic siRNA, B717, which is sequence specific for the human bcl-2 oncogene, complexed with a novel cationic liposome, LIC-101. Experimental Design: In a mouse model of liver metastasis, we administered B717/LIC-101 by bolus intravenous injection, adjusting the rate and volume of administration to what is feasible in human therapy. In a mouse model bearing prostate cancer in which the cells were inoculated under the skin, B717/LIC-101 was administered subcutaneously around the tumor. Results: The B717/LIC-101 complex inhibited the expression of bcl-2 protein and the growth of tumor cell lines in vitro in a sequence-specific manner in the concentration range of 3 to 100 nmol/L. Furthermore, the complex had a strong antitumor activity when administered intravenously in the mouse model of liver metastasis. B717 (siRNA) was shown to be delivered to tumor cells in the mouse liver, but only when complexed with LIC-101. The complex also inhibited tumor cell growth in the mouse model bearing prostate cancer. Conclusions: By combining siRNA with our cationic liposome, we overcame the difficulty of administering siRNA to animals in ways that can be applied in human therapy. Although our siRNA/liposome complex is not yet in clinical trials, it is expected to provide a novel siRNA therapy for cancer patients.

Efficacy of NS-018, a potent and selective JAK2/Src inhibitor, in primary cells and mouse models of myeloproliferative neoplasms

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.

Effect of NS-018, a selective JAK2V617F inhibitor, in a murine model of myelofibrosis.

A single somatic mutation, V617F, in Janus kinase 2 (JAK2) is one of the causes of myeloproliferative neoplasms (MPNs), including primary myelofibrosis, and the JAK2V617F mutant kinase is a therapeutic target in MPN. However, inhibition of wild-type (WT) JAK2 can decrease the erythrocyte or platelet (PLT) count. Our selective JAK2 inhibitor, NS-018, suppressed the growth of Ba/F3 cells harboring JAK2V617F more strongly than that of cells harboring WT JAK2. The 4.3-fold JAK2V617F selectivity of NS-018 is higher than the 1.0- to 2.9-fold selectivity of seven existing JAK2 inhibitors. NS-018 also inhibited erythroid colony formation in JAK2V617F transgenic mice at significantly lower concentrations than in WT mice. In keeping with the above results, in a JAK2V617F bone marrow transplantation mouse model with a myelofibrosis-like disease, NS-018 reduced leukocytosis and splenomegaly, improved bone marrow fibrosis and prolonged survival without decreasing the erythrocyte or PLT count in the peripheral blood. By exploring the X-ray co-crystal structure of NS-018 bound to JAK2, we identified unique hydrogen-bonding interactions between NS-018 and Gly993 as a plausible explanation for its JAK2V617F selectivity. These results suggest that NS-018 will have therapeutic benefit for MPN patients through both its efficacy and its reduced hematologic adverse effects.

Isolation, Cloning, and Characterization of a Novel Phosphomannan-binding Lectin from Porcine Serum

Mannan-binding protein (MBP) is a C-type serum lectin that is an important constituent of the innate immune defense because it activates the complement system via the lectin pathway. While the pig has been proposed to be an attractive source of xenotransplantable tissues and organs, little is known about porcine MBP. In our previous studies, phosphomannan, but not mannan, was found to be an effective inhibitor of the C1q-independent bactericidal activity of newborn piglet serum against some rough strains of Gram-negative bacteria. In contrast, the inhibitory activities of phosphomannan and mannan were very similar in the case of MBP-dependent bactericidal activity against rough strains of Escherichia coli K-12 and S-16. Based on these findings, we inferred that an MBP-like lectin with slightly or completely different carbohydrate binding specificity might exist in newborn piglet serum and be responsible for the C1q-independent bactericidal activity. Herein we report that a novel phosphomannan-binding lectin (PMBL) of 33 kDa under reducing conditions was isolated from both newborn and adult porcine serum and characterized. Porcine PMBL functionally activated the complement system via the lectin pathway triggered by binding with both phosphomannan (P-mannan) and mannan, which, unlike MBP, was effectively inhibited by mannose 6-phosphate- or galatose-containing oligosaccharides. Our observations suggest that porcine PMBL plays a critical role in the innate immune defense from the newborn stage to adult-hood, and the establishment of a newborn piglet experimental model for the innate immune system studies is a valuable step toward elucidation of the physiological function and molecular mechanism of lectin pathway.

NS-018 reduces myeloma cell proliferation and suppresses osteolysis through inhibition of the JAK2 and Src signaling pathways

Dear Editor, Multiple myeloma (MM) is a clonal B-cell malignancy characterized by the infiltration of malignant plasma cells into the bone marrow, and it eventually leads to impaired hematopoiesis, the production of high levels of monoclonal immunoglobulin, and osteolytic bone destruction. Bone pain and compression fractures caused by bone lesions not only severely impair the quality of life of MM patients, but also are associated with an ∼20% increase in the risk of death. The pathophysiology of MM is closely linked with the bone marrow microenvironment. Bone marrow stromal cells release cytokines, such as interleukin-6 (IL-6) which promote the proliferation and survival of myeloma cells, whereas myeloma cells secrete osteoclast differentiation factors, such as receptor activator of nuclear factor-kappa B ligand (RANKL), causing osteolysis. NS-018 is a potent, ATP-competitive small-molecule inhibitor of Janus kinase 2 (JAK2) and Src, which is under development for the treatment of myeloproliferative neoplasm. In the present study, we investigated the inhibitory effect of NS-018 on the IL-6/JAK2/STAT3 (signal transducer and activator of transcription) and Src signaling pathways in myeloma cell lines and its biological effects in a mouse model of MM. We previously reported that NS-018 selectively inhibits JAK2 and Src-family kinases in in vitro kinase assays. In the present study, we explored the mechanism of this inhibition in terms of molecular interactions at the atomic level. We began by comparing the modes of binding of NS-018 to Src kinase and JAK2 (Fig. 1a). An in silico study of the docking of NS-018 into the X-ray crystallographic structure of Src kinase showed NS-018 in close proximity to the Ala residue immediately N-terminal to the AspPhe-Gly (DFG) motif in the activation loop (Fig. 1a, right panel). In JAK2, the position corresponding to this Ala is occupied by a Gly residue, which is important in the binding of NS-018 to JAK2 (ref. Fig. 1a, left panel). The presence of a small residue at this position (Gly in JAK2 and Ala in Src kinase) allows the binding of NS-018 to both of these kinases. To test the inhibitory effects of NS018 on Src signaling at the cellular level, NIH3T3 cells overexpressing v-Src were incubated with NS-018 and the phosphorylation of Src and its downstream effector focal adhesion kinase (FAK) were analyzed by western blotting. NS-018 suppressed the phosphorylation of Src at concentrations of 10 nmol/L or more and of FAK at 100 nmol/L or more (Fig. 1b). IL-6 is one of the most widely studied cytokines that is elevated in MM patients, and it plays a critical role in the proliferation and survival of myeloma cells. Because the JAK/STAT pathway is the major downstream pathway of IL-6 signaling, JAK inhibitors such as ICNB16562, CYT387, and AZD1480 inhibit IL-6-induced MM cell survival. To investigate the inhibitory effect of NS-018 on the JAK/STAT signaling pathway, we investigated whether NS-018 inhibited the IL-6-induced proliferation and survival of myeloma cells. First, to examine the responsiveness of MM or plasma cell leukemia cell lines to IL-6, we treated PCM6, U266, RPMI 8226, KMM-1, and ARH-77 cells with IL-6, and analyzed the phosphorylation status of STAT3 by western blotting. In the absence of IL-6, STAT3 phosphorylation was

NS-018, a selective JAK2 inhibitor, preferentially inhibits CFU-GM colony formation by bone marrow mononuclear cells from high-risk myelodysplastic syndrome patients.

JAK2/STAT signaling promotes survival and expansion of myelodysplastic syndrome (MDS) clones, but little is known about the potential of JAK2/STAT as a therapeutic target in MDS. We investigated the effect of NS-018, a novel antagonist for JAK2, on the colony-forming ability of bone marrow mononuclear cells (BMMNCs) from high-risk MDS patients. NS-018 decreased colony-forming unit-granulocyte/macrophage (CFU-GM) colony numbers from MDS-derived BMMNCs in a dose-dependent manner, and this effect was significantly more potent than against normal BMMNCs. In addition, NS-018 suppressed the phosphorylation of STAT3 in colony-forming cells from MDS patients. Collectively, NS-018 could be a new therapeutic option for high-risk MDS.