Curative therapeutic options for a number of immunological disorders remain to be established, and approaches for identifying drug candidates are relatively limited. Furthermore, phenotypic screening ...methods using induced pluripotent stem cell (iPSC)-derived immune cells or hematopoietic cells need improvement. In the present study, using immortalized monocytic cell lines derived from iPSCs, we developed a high-throughput screening (HTS) system to detect compounds that inhibit IL-1β secretion and NLRP3 inflammasome activation from activated macrophages. The iPSCs were generated from a patient with neonatal onset multisystem inflammatory disease (NOMID) as a model of a constitutively activated NLRP3 inflammasome. HTS of 4,825 compounds including FDA-approved drugs and compounds with known bioactivity identified 7 compounds as predominantly IL-1β inhibitors. Since these compounds are known inflammasome inhibitors or derivatives of, these results prove the validity of our HTS system, which can be a versatile platform for identifying drug candidates for immunological disorders associated with monocytic lineage cells.
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.
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.
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.
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.
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.
Abstract 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.
Serum mannan binding protein (MBP), a mannose/N-acetylglusosamine-specific lectin, is important in innate immunity. In order to elucidate the mechanism underlying the wide intra- and interracial ...variety in the MBP serum level, we have studied the transcriptional regulation of human MBP. Rapid amplification of cDNA ends (5' RACE) analysis of Hep G2 RNA indicated the presence of a novel exon, designated as “exon 0, ” upstream of previously identified exon 1 Taylor, M.E. et al. (1989) Biochenu J. 262, 763–771. Two MBP mRNAs with different sized 5'-noncoding regions were detected: the longer transcript starts at exon 0 and the shorter one at exon 1. Promoter analysis involving a luciferase assay vector revealed that the transcript starting from exon 1 predominates over that starting from exon 0. In addition, a hepatocyto-specific nuclear factor, (HNF)-3, which is known to control the expression of hepatocyto-specific genes, up-regulates the transcription of human MBP from exon 1, while a glucocorticoid, which is known to up-regulate acute phase proteins, markedly suppresses MBP transcription. Recently, polymorphisms were found to occur in the promoter region at two positions Madsen, H.O. et al. (1995) J. ImmunoL 155, 3013–3020. Functional promoter analysis indicated that three haplotype variants as to these positions, HY, LY, and LX, exhibit high, medium and low promoter activity, respectively, in accordance with the results of a previous population study.
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.
The myelodysplastic syndromes (MDS) are a group of stem cell disorders characterized by dysplasia of one or more hematopoietic cell lineages and a risk of progression to acute myeloid leukemia. The ...cytidine analog azacitidine (Vidaza), a hypomethylating agent, improves survival in patients with MDS, but its mechanism of action is not well understood.
The effects of azacitidine on the MDS-derived cell line SKM-1 were investigated by DNA methylation assay, cell proliferation assay, and a subcutaneous xenograft mouse model.
Azacitidine and decitabine induced hypomethylation of the tumor suppressor gene cyclin-dependent kinase 4 inhibitor B (CDKN2B) in SKM-1 cells, whereas the deoxycytidine analog cytarabine did not. Azacitidine and decitabine also inhibited SKM-1 cell growth in vitro. In the mouse xenograft model, azacitidine significantly suppressed tumor growth.
Inhibition of DNA methyltransferase by azacitidine contributes to its antiproliferative and antitumor effects against SKM-1 cells and may explain its clinical efficacy in MDS.
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.
Nakaya:Nippon Shinyaku: Employment. Naito:Nippon Shinyaku: Employment. Niwa:Nippon Shinyaku: Employment. Horio:Nippon Shinyaku: Employment.
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