Yuji Nakata

Short interfering RNA (siRNA) targeting the Lyn kinase induces apoptosis in primary, and drug-resistant, BCR-ABL1(+) leukemia cells

We studied the effects of Lyn ablation on the survival of drug-resistant chronic myelogenous leukemia (CML) blast crisis cells using siRNA. Lyn siRNA reduced Lyn protein in both normal hematopoietic cells and BCR-ABL1-expressing (BCR-ABL1(+)) blasts by 80–95%. Within 48 h, siRNA-treated BCR-ABL1(+) blasts underwent apoptosis, whereas normal cells remained viable. This increased dependence on Lyn signaling for BCR-ABL1(+) blast survival provides the basis for rational treatment of drug-resistant CML blast crisis, particularly when lymphoid in nature.

c-Myb binds MLL through menin in human leukemia cells and is an important driver of MLL-associated leukemogenesis

Mixed-lineage leukemia (MLL) is a proto-oncogene frequently involved in chromosomal translocations associated with acute leukemia. These chromosomal translocations commonly result in MLL fusion proteins that dysregulate transcription. Recent data suggest that the MYB proto-oncogene, which is an important regulator of hematopoietic cell development, has a role in leukemogenesis driven by the MLL-ENL fusion protein, but exactly how is unclear. Here we have demonstrated that c-Myb is recruited to the MLL histone methyl transferase complex by menin, a protein important for MLL-associated leukemic transformation, and that it contributes substantially to MLL-mediated methylation of histone H3 at lysine 4 (H3K4). Silencing MYB in human leukemic cell lines and primary patient material evoked a global decrease in H3K4 methylation, an unexpected decrease in HOXA9 and MEIS1 gene expression, and decreased MLL and menin occupancy in the HOXA9 gene locus. This decreased occupancy was associated with a diminished ability of an MLL-ENL fusion protein to transform normal mouse hematopoietic cells. Previous studies have shown that MYB expression is regulated by Hoxa9 and Meis1, indicating the existence of an autoregulatory feedback loop. The finding that c-Myb has the ability to direct epigenetic marks, along with its participation in an autoregulatory feedback loop with genes known to transform hematopoietic cells, lends mechanistic and translationally relevant insight into its role in MLL-associated leukemogenesis.

c-Myb contributes to G2/M cell cycle transition in human hematopoietic cells by direct regulation of cyclin B1 expression.

ABSTRACT Myb family proteins are ubiquitously expressed transcription factors. In mammalian cells, they play a critical role in regulating the G1/S cell cycle transition but their role in regulating other cell cycle checkpoints is incompletely defined. Herein, we report experiments which demonstrate that c-Myb upregulates cyclin B1 expression in normal and malignant human hematopoietic cells. As a result, it contributes directly to G2/M cell cycle progression. In cell lines and primary cells, cyclin B1 levels varied directly with c-Myb expression. Chromatin immunoprecipitation assays, mutation analysis, and luciferase reporter assays revealed that c-Myb bound the cyclin B1 promoter preferentially at a site just downstream of the transcriptional start site. The biological significance of c-Myb, versus B-Myb, binding the cyclin B1 promoter was demonstrated by the fact that expression of inducible dominant negative c-Myb in K562 cells accelerated their exit from M phase. In addition, expression of c-Myb in HCT116 cells rescued cyclin B1 expression after B-myb expression was silenced with small interfering RNA. These results suggest that c-Myb protein plays a previously unappreciated role in the G2/M cell cycle transition of normal and malignant human hematopoietic cells and expands the known repertoire of c-myb functions in regulating human hematopoiesis.

c-Myb, Menin, GATA-3, and MLL form a dynamic transcription complex that plays a pivotal role in human T helper type 2 cell development.

GATA-3 and c-Myb are core elements of a transcriptionally active complex essential for human Th2 cell development and maintenance. We report herein mechanistic details concerning the role of these transcription factors in human peripheral blood Th2 cell development. Silencing c-Myb in normal human naive CD4(+) cells under Th2 cell-promoting conditions blocked up-regulation of GATA-3 and interleukin-4, and in effector/memory CD4(+) T cells, decreased expression of GATA-3 and Th2 cytokines. In primary T cells, c-Myb allows GATA-3 to autoactivate its own expression, an event that requires the direct interaction of c-Myb and GATA-3 on their respective binding sites in promoter of GATA-3. Immunoprecipitation revealed that the c-Myb/GATA-3 complex contained Menin and mixed lineage leukemia (MLL). MLL recruitment into the c-Myb-GATA-3-Menin complex was associated with the formation Th2 memory cells. That MLL-driven epigenetic changes were mechanistically important for this transition was suggested by the fact that silencing c-Myb significantly decreased the methylation of histone H3K4 and the acetylation of histone H3K9 at the GATA-3 locus in developing Th2 and CD4(+) effector/memory cells. Therefore, c-Myb, GATA-3, and Menin form a core transcription complex that regulates GATA-3 expression and, with the recruitment of MLL, Th2 cell maturation in primary human peripheral blood T cells.

c-Myb and GATA-3 Cooperatively Regulate IL-13 Expression via Conserved GATA-3 Response Element and Recruit Mixed Lineage Leukemia (MLL) for Histone Modification of the IL-13 Locus

The c-Myb and GATA-3 transcription factors play important roles in T cell development. We recently reported that c-Myb, GATA-3, and Menin form a core transcription complex that regulates GATA-3 expression and ultimately Th2 cell development in human peripheral blood T cells. However, c-Myb roles for Th2 cytokine expression were not demonstrated. In this article, we report that c-Myb and GATA-3 cooperatively play an essential role in IL-13 expression though direct binding to a conserved GATA-3 response element (CGRE), an enhancer for IL-13 expression. c-Myb and GATA-3 were shown to activate the CGRE–IL-13 promoter by ∼160-fold, and mutation of the canonical Myb binding site completely abrogated CGRE enhancer activity. In contrast, mutation of the GATA binding site partially decreased CGRE enhancer activity. GATA-3 did not bind to CGRE when c-myb expression was silenced. c-Myb, GATA-3, Menin, and mixed lineage leukemia (MLL) bound to CGRE in human primary CD4+ effector/memory cells. Moreover, c-myb silencing significantly decreased both methylation of histone H3K4 and acetylation of histone H3K9 at the IL-13 locus in CD4+ effector/memory cells. Therefore, in addition to the strong enhancer effect for the transcription of IL-13, the c-Myb/GATA-3 complex recruits MLL to the CGRE for histone modification of the IL-13 locus during the differentiation of memory Th2 cells.

Effectiveness of Trivalent Inactivated Influenza Vaccine in Children Estimated by a Test-Negative Case-Control Design Study Based on Influenza Rapid Diagnostic Test Results

We assessed vaccine effectiveness (VE) against medically attended, laboratory-confirmed influenza in children 6 months to 15 years of age in 22 hospitals in Japan during the 2013–14 season. Our study was conducted according to a test-negative case-control design based on influenza rapid diagnostic test (IRDT) results. Outpatients who came to our clinics with a fever of 38°C or over and had undergone an IRDT were enrolled in this study. Patients with positive IRDT results were recorded as cases, and patients with negative results were recorded as controls. Between November 2013 and March 2014, a total of 4727 pediatric patients (6 months to 15 years of age) were enrolled: 876 were positive for influenza A, 66 for A(H1N1)pdm09 and in the other 810 the subtype was unknown; 1405 were positive for influenza B; and 2445 were negative for influenza. Overall VE was 46% (95% confidence interval [CI], 39–52). Adjusted VE against influenza A, influenza A(H1N1)pdm09, and influenza B was 63% (95% CI, 56–69), 77% (95% CI, 59–87), and 26% (95% CI, 14–36), respectively. Influenza vaccine was not effective against either influenza A or influenza B in infants 6 to 11 months of age. Two doses of influenza vaccine provided better protection against influenza A infection than a single dose did. VE against hospitalization influenza A infection was 76%. Influenza vaccine was effective against influenza A, especially against influenza A(H1N1)pdm09, but was much less effective against influenza B.

Trivalent inactivated influenza vaccine effective against influenza A(H3N2) variant viruses in children during the 2014/15 season, Japan.

The 2014/15 influenza season in Japan was characterised by predominant influenza A(H3N2) activity; 99% of influenza A viruses detected were A(H3N2). Subclade 3C.2a viruses were the major epidemic A(H3N2) viruses, and were genetically distinct from A/New York/39/2012(H3N2) of 2014/15 vaccine strain in Japan, which was classified as clade 3C.1. We assessed vaccine effectiveness (VE) of inactivated influenza vaccine (IIV) in children aged 6 months to 15 years by test-negative case–control design based on influenza rapid diagnostic test. Between November 2014 and March 2015, a total of 3,752 children were enrolled: 1,633 tested positive for influenza A and 42 for influenza B, and 2,077 tested negative. Adjusted VE was 38% (95% confidence intervals (CI): 28 to 46) against influenza virus infection overall, 37% (95% CI: 27 to 45) against influenza A, and 47% (95% CI: -2 to 73) against influenza B. However, IIV was not statistically significantly effective against influenza A in infants aged 6 to 11 months or adolescents aged 13 to 15 years. VE in preventing hospitalisation for influenza A infection was 55% (95% CI: 42 to 64). Trivalent IIV that included A/New York/39/2012(H3N2) was effective against drifted influenza A(H3N2) virus, although vaccine mismatch resulted in low VE.

The c-Myb target gene neuromedin U functions as a novel cofactor during the early stages of erythropoiesis.

The requirement of c-Myb during erythropoiesis spurred an interest in identifying c-Myb target genes that are important for erythroid development. Here, we determined that the neuropeptide neuromedin U (NmU) is a c-Myb target gene. Silencing NmU, c-myb, or NmUs cognate receptor NMUR1 expression in human CD34(+) cells impaired burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) formation compared with control. Exogenous addition of NmU peptide to NmU or c-myb siRNA-treated CD34(+) cells rescued BFU-E and yielded a greater number of CFU-E than observed with control. No rescue of BFU-E and CFU-E growth was observed when NmU peptide was exogenously added to NMUR1 siRNA-treated cells compared with NMUR1 siRNA-treated cells cultured without NmU peptide. In K562 and CD34(+) cells, NmU activated protein kinase C-βII, a factor associated with hematopoietic differentiation-proliferation. CD34(+) cells cultured under erythroid-inducing conditions, with NmU peptide and erythropoietin added at day 6, revealed an increase in endogenous NmU and c-myb gene expression at day 8 and a 16% expansion of early erythroblasts at day 10 compared to cultures without NmU peptide. Combined, these data strongly support that the c-Myb target gene NmU functions as a novel cofactor for erythropoiesis and expands early erythroblasts.

Three-season effectiveness of inactivated influenza vaccine in preventing influenza illness and hospitalization in children in Japan, 2013-2016

OBJECTIVES We assessed the vaccine effectiveness (VE) of inactivated influenza vaccine (IIV) in children 6 months to 15 years of age in 2015/16 season. In addition, based on the data obtained during the three seasons from 2013 to 2016, we estimated the three-season VE in preventing influenza illness and hospitalization. METHODS Our study was conducted according to a test-negative case-control design (TNCC) and as a case-control study based on influenza rapid diagnostic test results. RESULTS During 2015/16 season, the quadrivalent IIV was first used in Japan. The adjusted VE in preventing influenza illness was 49% (95% confidence interval [CI]: 42-55%) against any type of influenza, 57% (95% CI: 50-63%) against influenza A and 34% (95% CI: 23-44%) against influenza B. The 3-season adjusted VE was 45% (95% CI: 41-49%) against influenza virus infection overall (N = 12,888), 51% (95% CI: 47-55%) against influenza A (N = 10,410), and 32% (95% CI: 24-38%) against influenza B (N = 9232). An analysis by age groups showed low or no significant VE in infants or adolescents. By contrast, VE was highest in the young group (1-5 years old) and declined with age thereafter. The 3-season adjusted VE in preventing hospitalization as determined in a case-control study was 52% (95% CI: 42-60%) for influenza A and 28% (95% CI: 4-46%) for influenza B, and by TNCC design, it was 54% (95% CI: 41-65%) for influenza A and 34% (95% CI: 6-54%) for influenza B. CONCLUSION We demonstrated not only VE in preventing illness, but also VE in preventing hospitalization based on much larger numbers of children than previous studies.

Altered cellular immunity in transgenic mice with T cell-specific expression of human D4-guanine diphosphate-dissociation inhibitor (D4-GDI)

D4-GDI, a Rho guanosine diphosphate (GDP) dissociation inhibitor, is preferentially expressed in hematopoietic tissues and binds to a small GTP-binding protein, Rho, and inhibits GDP dissociation from Rho. We identified point mutations in the D4-GDI gene in human leukemic cells. We therefore investigated the functions of D4-GDI and mutated D4-GDI in T cells. Transgenic mice (Tg) harboring human wild-type and mutant D4-GDI transgenes driven by the lck promoter were generated. Cellular immunity responses against cytozoic pathogens were examined. The cytoskeletal organization in the CD3+T cells and the proliferation of splenocytes by Con A were investigated in both Tg and littermates (LMs). Granuloma formation by bacille Calmette-Guerin was impaired in the wild-type D4-GDI Tg. On the other hand, the number of granulomas of the mutated D4-Tg was significantly higher. Infection with Listeria was more rapidly fatal to wild-type D4-GDI Tg than to LMs, while the survival of mutated D4-GDI Tg was prolonged. The CD3+T cells in wild-type D4-GDI Tg showed an impairment in the formation of stress fibers on anti-CD3 antibody-coated plates, whereas the cytoskeletal organization in CD3+T cells of the mutated D4-GDI Tg was augmented. The proliferation of splenocytes after Con A stimulation was higher in the mutated D4-GDI Tg than in the LMs. D4-GDI may have important functions, such as induction of T cell migration, adhesion and/or proliferation in inflammatory foci, in cellular immunity responses to cytozoic pathogens.