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  • American Society of Hematology  (29)
  • 1
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    Therapies Targeting the MAPK Pathway Improve Bone Marrow (BM) Fibrosis Induced By JAK2V617F
    American Society of Hematology ; 2014
    In:  Blood Vol. 124, No. 21 ( 2014-12-06), p. 162-162
    Details
    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 162-162
    Abstract: In primary myelofibrosis patients, somatic mutations such as JAK2V617F(JAKVF) and MPLW515 that activate JAK-STAT signaling are often seen. Small-molecule JAK2 inhibitors are effective for organomegaly and constitutional symptoms, but the drugs have little effect on BM fibrosis. To clarify the mechanism by which MPN cells with JAK2 mutations cause BM fibrosis, we compared the gene expression patterns of Lin−Sca1+ BM cells in JAK2VF transgenic mice (JAK2VF-TG), which develop myelofibrosis (MF), with that in WT mice. We found that TGFb1 and HOXB4, the target genes of transcription factor USF1 were highly expressed. TGFβ1, which is secreted by hematopoietic cells, is essential for fibrotic development in a murine model of MF (Chagraoui et al. Blood 2002), and increased expression of HOXB4 enhances human megakaryocytic development (Zhong et al. BBRC 2010). To investigate the mechanism of the high expression of these genes downstream of JAK2 signaling, USF1 and a cytokine receptor gene (MPL, EPOR or CSF3R) were co-transfected into 293T cells along with either a TGF-β1/HOXB4 promoter-driven or a STAT5 response element-driven luciferase reporter. Stimulation of MPL with TPO enhanced USF1 transcriptional activity about 3 fold, but stimulation of EPOR with EPO or of CSF3R with G-CSF did not change this activity. However, stimulation with any of the 3 types of cytokines enhanced STAT5 transcriptional activity. JAK2VF upregulated USF1 and STAT5 much more highly than JAK2WT without TPO stimulation. This USF1 upregulation specifically to TPO/MPL signaling was suppressed by a dominant negative mutant of USF1, JAK2 inhibitors (AG490, NS-018) or MEK inhibitors (U0126, PD325901). Inhibition of PI3K or p38MAPK did not affect the USF1 activation. Co-treatment with JAK2 and MEK inhibitors showed a synergistic effect in blocking both USF1 upregulation and STAT5 activation induced by JAK2VF. Next, we tested the MEK inhibitor, PD325901, in combination with the JAK2 inhibitor, NS-018, in the JAK2VF-TG mice. After disease was established 12 weeks after birth, JAK2VF-TG mice were divided into the following 4 groups: vehicle control; PD325901 monotherapy; NS-018 monotherapy; and combined therapy. PD325901 (5 mg/kg) and NS-018 (50 mg/kg) were orally administered once and twice daily, respectively. After 12 weeks of treatment, we evaluated the effect on BM fibrosis. The grading of MF in each group (n = 5-6) was as follows: vehicle control (MF-0: 0/6, MF-1 or 2: 6/6); PD325901 monotherapy (MF-0: 4/5, MF-1 or 2: 1/5); NS-018 monotherapy (MF-0: 0/6, MF-1 or 2: 6/6); and combined therapy (MF-0: 3/6, MF-1 or 2: 3/6). In the 2 groups treated with PD325901, 50~80% of mice showed MF-0. In contrast, in vehicle-treated or NS-018 monotherapy groups, all mice showed MF-1 or 2. Consistent with the MF grading, BM cellularity was significantly increased in the PD325901 monotherapy or combined therapy groups compared with the vehicle-treated group. A significant reduction was seen in the plasma TGFβ1 concentration in the PD325901 monotherapy and combined therapy groups compared with the vehicle-treated group (9.7 ng/ml, 8.1 ng/ml vs. 18.2 ng/ml, respectively). The TGFβ1 concentration in the extracellular fluid of BM (Wagner et al blood 2007) was also significantly reduced (5.6 ng/ml, 6.8 ng/ml vs. 9.1 ng/ml, respectively). BM cellularity and the TGFβ1 concentration in the NS-018 monotherapy group were comparable to those in the vehicle-treated group. Interestingly, megakaryocytes in the PD325901 monotherapy and combined therapy groups were decreased in number and were smaller than those in the vehicle-treated or NS-018 monotherapy groups. Regarding the effect on splenomegaly, spleen weight was significantly reduced in the NS-018 monotherapy and combined therapy groups compared with the vehicle-treated group (0.83 g, 0.69 g vs. 1.18 g, respectively). PD325901 monotherapy had little effect on splenomegaly. It is known that MEK-ERK1/2 pathway is critical in normal megakaryocyte development. In vitro data suggest that JAK2VF activates this pathway downstream of MPL and may contribute to TGFβ1 overproduction and dysmegakaryopoiesis, causing BM fibrosis via transcriptional enhancement of USF1. In vivo data suggest that MEK inhibition has the potential to improve dysmegakaryopoiesis and BM fibrosis. The combined therapy of JAK2 inhibitors with MEK inhibitors might be a promising therapy for improving both splenomegaly and BM fibrosis. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V124.21.162.162
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
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  • 2
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    Potentiated Activation of VLA-4 and VLA-5 Accelerates Proplatelet-Like Formation In Megakaryocytes.
    American Society of Hematology ; 2010
    In:  Blood Vol. 116, No. 21 ( 2010-11-19), p. 2585-2585
    Details
    In: Blood, American Society of Hematology, Vol. 116, No. 21 ( 2010-11-19), p. 2585-2585
    Abstract: Abstract 2585 Several lines of reports have suggested that mature magakaryocytes (MKs) form long cytoplasmic processes containing platelets (PLT) organelles from which PLT break off due to blood flow pressures in bone marrow (BM). These cytoplasmic processes were termed ‘proplatelet'. MKs differentiated from hematopoietic stem cells by in vitro culture also develop similar processes, referred to as ‘proplatelet-like formation (PPF)'. It has been already reported that fibronectin (FN) and phorbol 12-myristate 13-acetate (PMA) are essential for inducing PPF in MKs using CHRF-288 human megakaryoblastic cell line (Jiang F et al. Blood 99, 2002). FN plays important roles in megakaryocytopoiesis through the FN-receptors. The role of adhesive interactions with FN in BM stroma and FN-receptor beta1-integrins has been reported in proliferation, differentiation and maintenance of megakaryocytic lineage cells. However, the substantial role of these FN-receptors and their functional assignment in PPF are not yet fully understood. We first investigated the effects of beta1-integrins on PPF using CHRF-288 cells, which express alpha4beta1-integrin (VLA-4) and alpha5beta1-integrin (VLA-5) as FN-receptors. When the cells were cultured on FN for 3 days, PMA prompted PPF in a dose-dependent manner. While nearly 15% of the cells displayed PPF with PMA (100 ng/mL), no cells cultured with FN alone or PMA alone exhibited PPF. PPF induced by FN plus PMA combination (FN/PMA) was abrogated by addition of anti-alpha4-integrin monoclonal antibodies (mAb) plus anti-alpha5-integrin mAb combination, but not by the addition of anti-alpha4-integrin mAb alone or anti-alpha5-integrin mAb alone. Thus, the adhesive interaction with FN via VLA-4 and VLA-5 were responsible for PPF. We next investigated the effect of TNIIIA2, which enhances the adhesive interaction between FN and beta1-integrins, in PPF induced by FN/PMA. TNIIIA2 (RSTDLPGLKAATHYTITIRGVC) is a 22-mer peptide derived from the 14th FN type III-like (FNIII) repeat in tenascin (TN)-C molecule which we found recently, and it induces the conformational change necessary for functional activation of beta1-integrins (Fukai F et al. J Biol Chem 282, 2007; J Biol Chem 284, 2009). The PPF induced by FN/PMA was highly accelerated when CHRF-288 cells were enforced adhering to FN by treatment with TNIIIA2 (25 microg/mL). More than 45% of the cells displayed PPF with FN/PMA plus TNIIIA2 combination (FN/PMA/TNIIIA2). Blocking experiments using anti-beta1-integrin mAbs indicated that adhesive interaction with FN via VLA-4 and VLA-5 was also responsible for acceleration of PPF induced by FN/PMA/TNIIIA2. On the other hand, control peptide, TNIIIA2mutant (RSTDLPGLKAATHYTATARGVC) did not accelerate PPF induced by PMA/FN. The calculated yield of the cells with PPF induced by FN/PMA/TNIIIA2 was 2.5-fold more than that induced by FN/PMA. We have previously established ‘a three-phase serum-free culture system' to generate large amount of PLT from human cord blood CD34+ cells (Matsunaga T et al. Stem cells 24, 2006). A study on the effect of TNIIIA2 on our ‘three-phase serum-free culture system' is now underway. Finally, we investigated signal transduction pathways responsible for PPF induced by FN/PMA. While FN/PMA induced activation of extracellular signal-regulated protein kinase 1 (ERK1/2), FN alone or PMA alone did not induce ERK1/2 activation. The results was in accordance with the data previously reported by Jiang et at (Blood 99, 2002). TNIIIA2 strongly enhanced activation of ERK1/2 by FN/PMA. However, c-Jun amino-terminal kinase 1 (JNK1), p38 and phosphoinositide-3 kinase (PI3K)/Akt were not stimulated by FN/PMA even in the presence of TNIIIA2. Thus, enhanced activation of ERK1/2 by FN/PMA/TNIIIA2 was responsible for acceleration of PPF by FN/PMA. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V116.21.2585.2585
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2010
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 3
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    R723, a selective JAK2 inhibitor, effectively treats JAK2V617F-induced murine myeloproliferative neoplasm
    American Society of Hematology ; 2011
    In:  Blood Vol. 117, No. 25 ( 2011-06-23), p. 6866-6875
    Details
    In: Blood, American Society of Hematology, Vol. 117, No. 25 ( 2011-06-23), p. 6866-6875
    Abstract: The activating mutations in JAK2 (including JAK2V617F) that have been described in patients with myeloproliferative neoplasms (MPNs) are linked directly to MPN pathogenesis. We developed R723, an orally bioavailable small molecule that inhibits JAK2 activity in vitro by 50% at a concentration of 2nM, while having minimal effects on JAK3, TYK2, and JAK1 activity. R723 inhibited cytokine-independent CFU-E growth and constitutive activation of STAT5 in primary hematopoietic cells expressing JAK2V617F. In an anemia mouse model induced by phenylhydrazine, R723 inhibited erythropoiesis. In a leukemia mouse model using Ba/F3 cells expressing JAK2V617F, R723 treatment prolonged survival and decreased tumor burden. In V617F-transgenic mice that closely mimic human primary myelofibrosis, R723 treatment improved survival, hepatosplenomegaly, leukocytosis, and thrombocytosis. R723 preferentially targeted the JAK2-dependent pathway rather than the JAK1- and JAK3-dependent pathways in vivo, and its effects on T and B lymphocytes were mild compared with its effects on myeloid cells. Our preclinical data indicate that R723 has a favorable safety profile and the potential to become an efficacious treatment for patients with JAK2V617F-positive MPNs.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2010-01-262535
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2011
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 4
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    Absence of Somatically Acquired JAK1 Mutations in Adult T-Cell Leukemia/Lymphoma.
    American Society of Hematology ; 2009
    In:  Blood Vol. 114, No. 22 ( 2009-11-20), p. 1921-1921
    Details
    In: Blood, American Society of Hematology, Vol. 114, No. 22 ( 2009-11-20), p. 1921-1921
    Abstract: Abstract 1921 Poster Board I-944 Background: Janus kinase 1 (JAK1) plays a critical role in lymphocyte proliferation and differentiation. Somatic JAK1 mutations are found in 18% of adult precursor T acute lymphoblastic leukemias (T-ALL). Some of the mutations were shown to induce the phosphorylation of JAK1 and STAT5 and lead to cytokine-independent proliferation. These data suggest that dysregulation of JAK1 can be involved in the development or progression of T-ALL (Flex et al. J Exp Med. 2008;205:751-758). Adult T-cell leukemia/lymphoma (ATLL) is a type of T-cell neoplasm, and the activation of JAK/STAT is sometimes observed in the tumor cells. Therefore, we investigated JAK1 mutations in ATLL patients. Patients and methods: Twenty Japanese ATLL patients whose percentage of peripheral abnormal lymphocytes was greater than 30% total cell count were sequentially enrolled into the study from 2000 to 2007. Diagnosis of ATLL was made on the basis of clinical features and laboratory characteristics. All cases tested positive for the serum anti-HTLV-1 antibody. The diagnosis was confirmed by observing monoclonal insertion of the HTLV-1 viral genome into leukemia cells by Southern blot hybridization. Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved at -80°C. These PBMCs were thawed and genomic DNA was isolated using standard protocol. The entire coding sequence of the JAK1 gene (exons 2 through 25) was amplified by the polymerase chain reaction (PCR) method. The sequence of PCR primers were kindly provided by Dr. Marco Tartaglia (Istituto Superiore di Sanità, Roma, PhD). The nucleotide sequences were determined by fluorescent dye chemistry sequencing and analyzed by sequencing analysis software. By referencing the assembled sequence in the Ensembl genome database, the presence of homozygous mutations was first checked and then candidates for heterozygous mutations or single nucleotide polypeptides (SNPs) on each allele were screened by comparing the ratio of different bases calculated with the height of the peaks seen from sequencing to the reference genome when the ratio was between 0.15 and 1.0. Result: The percentage of abnormal lymphocytes ranged from 30-90%, and the mean value was 55.4%. The mean value of WBC and lymphocyte number was 40.5×109/L and 33.4×109/L, respectively. The mean value of LDH, Ca2+ or sIL-2R was 609 IU/L, 11.4 mg/dL, or 54748 U/mL, respectively. According to Shimoyama criteria (Shimoyama et al. Br J Haematol. 1991;79:428-437), 19 cases were diagnosed as acute-type ATLL, and one case was diagnosed as chronic-type ATLL. The surface markers of all but one abnormal PBMC were CD3+CD4+CD8-CD25+. In that one exception, loss of CD4 expression was observed. We examined the entire coding sequence of the JAK1 gene in 20 ATLL patients and identified no nonsynonymous or nonsense mutations and five types of silent substitutions in 12 cases. All silent substitutions were synonymous SNPs, as determined from referencing the base sequence in the Ensembl genome database. In the ATLL patients examined, the genotype frequency (%) is c546-AA/AG/GG, 97.5/2.5/0; c1590-CC/CT/TT, 97.5/2.5/0; c2049-CC/CT/TT, 50/50/0; c2097-CC/CG/GG, 95/5/0; c2199-AA/AG/GG, 60/40/0. There is no statistical difference in genotype frequency pattern of these SNPs, between the Japanese ATLL patients examined and the general Asian population on the Ensembl database. Conclusion: Mutations in the coding region of JAK1 do not associate with either activation of the JAK/STAT pathway or leukemogenesis in ATLL. We only examined the coding region of JAK1, and the regulatory region of JAK1 remains to be investigated. Further investigation including downstream signaling molecules and inhibitory molecules in the JAK/STAT signaling pathway is necessary to clarify the mechanism contributing to the leukemogenesis of ATLL. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V114.22.1921.1921
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2009
    detail.hit.zdb_id: 1468538-3
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  • 5
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    Landscape of Genetic Alterations in Adult T-Cell Leukemia/Lymphoma
    American Society of Hematology ; 2014
    In:  Blood Vol. 124, No. 21 ( 2014-12-06), p. 75-75
    Details
    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 75-75
    Abstract: Adult T-cell leukemia/lymphoma (ATL) is a distinct form of peripheral T-cell lymphoma, which is etiologically associated with human T-cell leukemia virus type 1 (HTLV-1) infection during early infancy. Although HTLV-1 can effectively immortalize human T cells, there is a long latency period of ~50 years before the onset of ATL, suggesting that HTLV-1 infection alone may be insufficient for the development of ATL, but additional acquired genetic events that accumulate during the later life are essential for the development of ATL. However, such somatic alterations underlying the pathogenesis of ATL have not been fully elucidated. To obtain a complete registry of genetic alterations in ATL, we performed an integrated genetic study, in which whole-genome/exome and RNA sequencing (RNA-seq) was performed together with array-based methylation and genomic copy number analysis among a cohort of 50 paired ATL samples, followed by extensive validation using targeted deep sequencing of detected mutations in 〉 400 follow-up samples. Compared with other lymphoid malignancies, ATL cells carried higher numbers of mutations, copy number alterations, and rearrangements than in other lymphoid malignancies, suggesting the presence of global genomic instability in ATL. In addition to previously reported mutational targets in ATL (TP53,TCF8, and FAS) and known targets frequently mutated in other lymphoid malignancies (CARD11, GATA3, IRF4, POT1, and RHOA), we identified a variety of highly recurrent mutations affecting previously unknown mutational targets, many of which are involved in T-cell development, activation and migration, immunosurveillance, and transcriptional regulation. Molecular and functional analysis using human T-cell leukemia cell lines showed that some of these novel mutations actually augment T-cell receptor signaling, validating their biological significance in ATL. A comparison of mutations among disease subtypes revealed that several subtype-specific mutations, including TP53, CD58, IRF4 and TBL1XR1 mutations in acute and lymphoma types, and STAT3mutation in chronic and smoldering types, suggesting that different oncogenic mechanisms underlie different ATL subtypes. Furthermore, ATL cells had a distinct pattern of copy number changes and genomic rearrangements. Interestingly, their gene targets showed a significant overlap to mutational targets. Surprisingly, somatic focal deletions involving the 14q31.1 locus were observed in all the cases examined by whole-genome sequencing and therefore are thought to uniquely characterize ATL genomes, although their gene targets remained to be identified. Like other regions also frequently deleted in ATL, such as 7q31.1 and 1p21.3 loci, these deletions were thought to reflect high levels of genetic instability. Finally and conspicuously, pathway analysis revealed that multiple genes involved in the Tax interactome were systematically altered in ATL, although Tax itself underwent gene silencing in most cases. These data suggested that ATL cells can escape from cytotoxic T-lymphocytes by silencing immunogenic Tax expression, while developing alternative oncogenic mechanisms through acquiring somatic mutations or copy number alterations in the Tax-related pathway. Our findings suggest that deregulated T-cell functionalities caused by genetic alterations, especially those associated with HTLV-1 Tax oncoprotein, are central to ATL pathogenesis, and provide a novel clue to contrive new diagnostics and therapeutics for this intractable disease. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V124.21.75.75
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
    detail.hit.zdb_id: 1468538-3
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  • 6
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    Impact Of TET2 Deficiency On MPN Harboring JAK2V617F Mutation
    American Society of Hematology ; 2013
    In:  Blood Vol. 122, No. 21 ( 2013-11-15), p. 478-478
    Details
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 478-478
    Abstract: JAK2V617F (JAK2VF) is the most frequent mutation in myeloproliferative neoplasms (MPN), and its role has been demonstrated in mouse models. Actually, JAK2VF transgenic (JAK2VF Tg) mice generated by us induce lethal MPN (Shide et al. Leukemia 2008). Recently, mutations of epigenetic regulator such as TET2 are also frequently identified in MPN, and several TET2 knock out or knock down (TET2KD) mouse models are generated. We previously analyzed TET2KD mice (Ayu17-449) (Shide et al. Leukemia 2012). TET2KD fetal liver (FL) or bone marrow (BM) cells showed a growth advantage over Wt BM cells, with increased self-renewal capacity of hematopoietic stem cells; however TET2KD mice didn’t develop MPN, and its role in MPN remained unclear. To explore the role of TET2 deficiency in MPN harboring JAK2VF, we examined the cooperative effect, using these mutant mice. Materials and methods (1) Mice and collection of test cells. JAK2VF Tg mice (C57BL/6, Ly5.2) and TET2KD mice (Ayu17-449, C57BL/6, Ly5.2) were used. We crossed them, and collected JAK2Wt-TET2Wt (Wt-Wt), JAK2Wt-TET2KD (Wt-KD), JAK2VF-TET2Wt (VF-Wt), and JAK2VF-TET2KD (VF-KD) FL cells. (2) Non-competitive repopulation assay (NCRA). FL cells (Ly5.2, 1x106 cells) were transplanted into lethally irradiated recipients (Ly5.1) without competitor cells. Recipients were analyzed by complete blood counts, flow cytometry, colony-forming assay, colony-replating assay, pathology at 20-28 weeks post-transplantation, and overall survival. (3) Competitive repopulation assay (CRA) and serial BM transplantation (sBMT). FL cells (Ly5.2, 1x106 cells) were transplanted into lethally irradiated recipients (Ly5.1) with competitor Wt BM cells (Ly5.1, 5x106 cells), and sBMT was performed by 1x106 BM cells of the recipients at every 12 weeks post-transplantation. Recipients which were not selected as the donors were analyzed. (4) Analyses of adult mutant mice. Mice were bred in BDF1 background and analyzed at 20 or more weeks of age, as well as the recipients in NCRA. (5) Statistical analysis. Results were presented as means±S.D. Two-tailed Student’s t-test and log-rank test were used. Result In NCRA, both recipients transplanted with VF-Wt cells and VF-KD cells developed MPN with increase in WBC and Plt, decrease in Hb, fibrosis in BM and spleen, and extramedullary hematopoiesis (EMH) of lung and liver; and the latter developed more severe MPN and died earlier: VF-Wt (n=10) vs. VF-KD (n=10); WBC (x104/µl), 4.2±1.6 vs. 7.3±3.3 (p 〈 0.05); peripheral blood (PB) myeloid cells (%), 59.6±9.7 vs. 71.9±8.2 (p 〈 0.05); liver weight (g), 1.15±0.22 vs. 1.48±0.22 (p 〈 0.01); spleen weight (g), 0.26±0.11 vs. 0.52±0.19 (p 〈 0.01): VF-Wt (n=36) vs. VF-KD (n=30); mean survival time (weeks), 36 vs. 39 (p 〈 0.05). In colony-forming assay, number of CFU-GM was more increased in VF-KD cells than VF-Wt cells: VF-Wt (n=9) vs. VF-KD (n=9); colonies/2x104 BM cells, 107±37 vs. 157±46 (p 〈 0.05). In colony-replating assay, VF-Wt BM cells lost replating capacity by 3rd to 5th passage; VF-KD BM cells retained replating capacity beyond 5th passage: VF-Wt (n=9) vs. VF-KD (n=6); number of colonies in 4th passage, 5.9±6.8 vs. 896±613 (p 〈 0.01). In CRA, all recipients transplanted with VF-Wt cells (n=9) or VF-KD cells (n=9) showed ≥ 70% test cell-derived PB chimerism, and developed MPN with fibrosis and EMH at 12 weeks. In 2nd BMT, 4/9 recipients transplanted with VF-Wt cells showed ≥ 35% PB chimerism at 12 weeks. Six recipients were analyzed at 12-16weeks, and no one (0/6) showed pathological findings of MPN. Whereas, 7/9 recipients transplanted with VF-KD cells showed ≥ 35% PB chimerism. Five recipients were analyzed, and 3/5 developed MPN with fibrosis and EMH: VF-Wt (n=6) vs. VF-KD (n=5); liver weight (g), 1.00±0.12 vs. 1.39±0.15 (p 〈 0.002); spleen weight (g), 0.069±0.019 vs. 0.20±0.097 (p 〈 0.05). In analyses of adult mutant mice, both VF-Wt mice and VF-KD mice developed MPN, and disease severities or colony-replating capacities are similar tendencies as those in transplantation model. Conclusion TET2 deficiency increases severity of MPN harboring JAK2VF. TET2 deficiency enhances disease initiating potential of JAK2VF-MPN stem cells. TET2 deficiency is considered to be critical for both onset and progression of MPN harboring JAK2V617F. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V122.21.478.478
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 7
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    Depletion of Neoplastic CD11b Positive Cells in Jak2V617F Mutant Mice Reduced Fibrocytes in Bone Marrow and Improved Myelofibrosis
    American Society of Hematology ; 2019
    In:  Blood Vol. 134, No. Supplement_1 ( 2019-11-13), p. 310-310
    Details
    In: Blood, American Society of Hematology, Vol. 134, No. Supplement_1 ( 2019-11-13), p. 310-310
    Abstract: Myelofibrosis (MF) associated with myeloproliferative neoplasms (MPN) has been considered to be a reactive phenomenon caused by mesenchymal stromal cells (MSCs) stimulated by cytokines such as TGFb-1 overproduced by neoplastic megakaryocytes (MKs) and platelets. TGFb-1 stimulates non-neoplastic mesenchymal cells to produce collagen and fibronectin and to induces bone marrow (BM) fibrosis. However, the involvement of neoplastic fibrocyte in MF has recently been reported (Verstovsek et al. JEM 2016), and among blood cells, monocytes in particular are considered to be the main source of neoplastic fibrocytes. In this study, we assesed the role of neoplastic fibrocytes using a mouse model of MPN induced by Jak2V617F (Shide et al. Leukemia 2008). First, the distribution of neoplastic fibrocyte in the BM of Jak2V617F transgenic (TG) mice was examined. We transplanted wild-type (WT) or Jak2V617F TG cells (B6-CD45.2), together with WT BM cells (B6-CD45.1) into irradiated WT recipient mice (B6-CD45.1). Only recipient mice transplanted with a mixture of Jak2V617F cells and WT cells developed BM fibrosis. In immunofluorescent staining of fibrotic BM, cells expressing the fibrocyte marker CD45/Collagen-1(Col-1) were observed much more than cells expressing the fibroblast marker CD90(usually positive for MSCs)/Col-1. As for CD45/Col-1 positive cells, cells expressing CD45.2/Col-1 were much more than cells expressing CD45.1/Col-1, clearly indicating that these cells were derived from Jak2V617F mutant blood cells. On the other hand, in the BM of recipient mice transplanted with control WT cells, few cells expressing CD45/Col-1 or CD90/Col-1 were present. To examine the differentiation ability of Jak2V617F blood cells to fibrocytes directly, peripheral blood (PB) mononuclear cells (MNC) of Jak2V617F mice or WT mice were cultured in vitro. After 5 days of culture, PB MNCs from Jak2V617F mice differentiated into mature fibrocytes exhibiting a long spindle shape with Col-1 expression. On the other hand, there were very few fibrocytes differentiated from PB MNC from WT mice. Next, we depleted monocytes, the main source of fibrocytes, and observed its effects on BM fibrosis in vivo. Jak2V617F TG mice were mated with CD11b-diphtheria toxin receptor (DTR) TG mice (Duffield et al. JCI 2005) to obtain Jak2V617F/CD11b-DTR double TG mice. Mice transplanted with BM cells from Jak2V617F/CD11b-DTR double TG mice (hereinafter called Jak2V617F/CD11b-DTR mice) exhibit leukocytosis, thrombocytosis, anemia, splenomegaly, and BM fibrosis with increased megakaryocytes. Jak2V617F/CD11b-DTR mice was administered diphtheria toxin (DT) intraperitoneally to deplete monocytes. One day after DT administration, the number of PB monocytes (CD11b+/F4/80+) drastically decreased in Jak2V617F/CD11b-DTR mice, and the reduction of monocyte was maintained by every-other-day DT administration. After 8 weeks DT treatment, mice were sacrificed and analyzed. As a control group, Jak2V617F/CD11b-DTR mice treated with PBS were examined. DT treatment drastically decreased the number of neoplastic fibrocytes expressing CD45.2/Col-1 in BM and spleen of Jak2V617F/CD11b-DTR mice compared with control mice treated with PBS. Consistently, reticulin fibers were eliminated almost completely and collagen fibers almost fully disappeared in BM, which led to a reversal of the decrease in BM cellularity, although the number of MKs was not affected. Similar findings were observed in the spleen, although not completely. Plasma TGF-b1 level were about 2-fold higher in Jak2V617F/CD11b-DTR mice than in WT mice. Neoplastic monocyte depletion significantly decreased TGF-b1 level. Since MK numbers did not change, this indicates that fibrocytes are one of the main sources of TGF-b1. In other features of MF in Jak2V617F/CD11b-DTR mice, splenomegaly was ameliorated by DT treatment. Microscopic analysis revealed an improvement in the damaged spleen architecture and the disappearance of splenic fibrosis. In summary, most collagen-producing cells in BM were neoplastic fibrocytes in Jak2V617F-induced MPN, indicating that neoplastic fibrocytes played an essential role and mesenchymal fibroblasts had a minor contribution in fibrosis in MPN. Depletion of neoplastic monocytes also improved splenomegaly as well as BM fibrosis in mice, and this cell fraction could be a promising therapeutic target. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2019-130042
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2019
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  • 8
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    Next-Generation Sequencing Reveal Proviral Genome and Transcriptome in Adult T-Cell Leukemia/Lymphoma
    American Society of Hematology ; 2015
    In:  Blood Vol. 126, No. 23 ( 2015-12-03), p. 3882-3882
    Details
    In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 3882-3882
    Abstract: Background: Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell neoplasm caused by human T-cell leukemia virus type-1 (HTLV-1) retrovirus infection. As for its pathogenesis, viral products, such as Tax and HBZ, play indispensable roles and their oncogenic mechanisms have been extensively studied. Recently, we have performed an integrated genetic study of a large number of ATL cases and revealed the entire landscape of somatic mutations, copy number alterations, and gene fusions in ATL. However, the detailed analysis of HLTV-1 integration using next-generation sequencing has not been performed so far. In this study, combining whole-genome and RNA sequencing data, we delineated the effect of HTLV-I integration on viral and cellular transcription. Patients and Methods: We performed WGS and RNA-seq for 48 and 57 ATL cases, respectively. All the analyses of the sequencing data were performed using our in-house pipelines. We analyzed HTLV-1 proviral genomic structure and the effect of HTLV-1 integration on viral and cellular transcription. Results: A cardinal feature of ATL genome is HTLV-1 integration, which was precisely located in all the cases analyzed by WGS. Multiple proviral integration sites were detected in 12 cases (total, 62 HTLV-1 integrations sites). The provirus integration was clonal in the architecture inferred from somatic mutations, and apparently randomly integrated into the host genome as previously reported. Within the HTLV-1 genome, frequent 5' proviral segment (gag/pol/env loci) deletions and/or sense gene (gag/pol/env/tax/rex/p13/p30) mutations were observed, which seem to cause defective viral replication/production, whereas HBZ gene was maintained in all the cases. RNA-seq revealed that HTLV-1 integration in ATL cells was associated with aberrant transcription. In general, viral transcripts were predominantly derived from the antisense strand, whereas sense transcription was largely suppressed, leading to global silencing of the sense genes. Especially, in contrast to the ubiquitous HBZ expression (antisense strand), tax expression (sense strand) was almost completely lost in all but one case, which exceptionally exhibited high expression of both tax and HBZ. Strikingly, in most cases, the antisense transcripts were not terminated in 5'-long terminal repeat (LTR), but read through into the juxtaposed cellular genome, extending into up to 50 kb downstream therefrom (read-through transcript). Moreover, in 11 sites of intragenic proviral integration, aberrantly spliced fusion transcripts were observed between LTR and the affected gene, and more commonly associated with antisense (n = 9) than sense (n = 2) integration, accompanied by upregulated cellular gene expression. In other cases (n = 3), fusion transcripts were also generated between HBZ and an exon of highly expressed cellular gene adjacent to the integration site. These results indicate the potential significance of antisense transcription and aberrant fusion transcripts with host genome sequences during ATL development. Although the precise role of these novel aberrant antisense transcripts remains unknown, antisense transcripts containing the LTR region has been implicated in NF-κB activation, which is a hallmark of ATL pathogenesis. Conclusion: In summary, combining WGS and RNA-seq data, we demonstrated the global silencing of sense-oriented viral transcripts (including Tax) and the predominance of aberrant antisense-directed transcription, which often involved cellular gene expression, including aberrant fusion transcripts between host and viral genomes (read-through and aberrantly spliced fusion transcripts). These results suggest that antisense transcription and abnormal virus-host fusion transcripts play pivotal roles in the pathogenesis of ATL. Disclosures Tobinai: Gilead Sciences: Research Funding. Miyazaki:Kyowa-Kirin: Honoraria, Research Funding; Celgene Japan: Honoraria; Sumitomo Dainippon: Honoraria; Chugai: Honoraria, Research Funding; Shin-bio: Honoraria.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V126.23.3882.3882
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
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  • 9
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    Prognostic Relevance of Integrated Genetic Profiling in Adult T-Cell Leukemia/Lymphoma
    American Society of Hematology ; 2015
    In:  Blood Vol. 126, No. 23 ( 2015-12-03), p. 2643-2643
    Details
    In: Blood, American Society of Hematology, Vol. 126, No. 23 ( 2015-12-03), p. 2643-2643
    Abstract: Adult T-cell leukemia/lymphoma (ATL) is a distinct subtype of peripheral T-cell neoplasms associated with human T-cell leukemia virus type-1 retrovirus. ATL includes a heterogeneous group of patients in terms of pathological and clinical features as well as prognosis, suggesting the presence of underlying molecular pathogenesis that could explain such heterogeneity among patients. Recently, we performed an integrated molecular analysis of a large number of ATL cases and delineated a comprehensive registry of gene mutations and other genetic/epigenetic lesions in ATL. In this study, we investigated possible correlations between these genetic/epigenetic lesions and clinical/pathological phenotypes in a large set of ATL patients, with a special focus on the impact of mutations and copy number alterations (CNAs) on clinical outcome. We analyzed a total of 361 ATL samples, including acute (n = 192), lymphoma (n = 66), chronic (n = 89), and smoldering (n = 14) subtypes, for recurrent mutations and CNAs. Each subtype had characteristic genetic/epigenetic features, suggesting a distinct molecular pathogenesis therein. Aggressive (acute and lymphoma) subtypes were characterized by a higher number of mutations and CNAs including focal amplifications/deletions, hyperploid status, and CIMP phenotype, compared with indolent (chronic and smoldering) tumors. Two mutations (TP53 and IRF4) and eight focal deletions involving 1p13 (CD58), 6p21 (HLA-B), 9p21 (CDKN2A), 10p11 (CCDC7), 13q32 (GPR183), 16q23 (WWOX), 17p13 (TP53), and 19q13 (CEBPA), were more common in aggressive ATL than in indolent ATL. In contrast, showing a similar mutational distribution to those found in large granular lymphocytic leukemia, STAT3 mutations were characteristic of the indolent diseases. Gene set enrichment analysis of RNA-seq data showed a significant enrichment of MYC pathway and genes regulating cell cycle and DNA repair in upregulated genes in aggressive ATL. Next, we assessed the impact of mutations and CNVs on prognosis among 215 ATL cases, for which survival data were available. In the entire cohort, mutation in CCR4 and IRF4, focal amplification in 9p24 (CD274) and 14q32 (BCL11B), and focal deletion in 9p21 (CDKN2A) were found to be significant predictors of poor overall survival, after adjustment for disease subtype and age. Multivariate analysis revealed that disease subtype (aggressive vs. indolent) was the most significant predictor of clinical outcome in ATL. Subsequent multivariate analysis according to disease subtype showed that within the patients with aggressive ATL, older age (≥ 70 years), CCR4 mutations, and 9p24 amplification were independently associated with an adverse outcome. Based on the number of the risk factors they owned, patients with aggressive ATL were classified into three categories showing marked difference in 3-year overall survival (OS) (P 〈 0.001): those with no risk factors (OS, 32%), with one risk factor (18%), and with two or more (0%). Among the patients with indolent ATL, we found IRF4 and TP53 mutations, 9p24 amplification, and deletions in 9p21 and 10p11 were independently associated with reduced survival. Interestingly, these alterations, except for 9p24 amplification, were also identified as genes more frequent in aggressive ATL. More importantly, based on these risk factors, the patients with indolent ATL can be classified into two categories showing very different prognostic profiles: patients with no risk factors (OS, 89%) and those with one or more risk factors (21%) (P 〈 0.001, HR = 16.8, 95% CI:5.4-52.5), suggesting that patients with indolent ATL having a genetic feature of the aggressive subtypes might genetically and biologically represent a distinct subset, which should be better managed as having an aggressive disease. Among these poor prognostic factors, 9p24 amplification and CCR4 mutation are especially interesting, because these lesions might be plausible targets of available agents, including anti-PD1/PD-L1 and anti-CCR4 antibodies. In conclusion, based on the comprehensive genetic profiling, we demonstrated that the known subtypes of ATL can be further classified into genetically and biologically distinct subsets of tumors characterized by discrete sets of genetic lesions and substantially different prognosis. Our results suggest that molecular profiling can improve the prediction of prognosis in ATL patients and better guide therapy. Disclosures Tobinai: Gilead Sciences: Research Funding. Miyazaki:Shin-bio: Honoraria; Chugai: Honoraria, Research Funding; Sumitomo Dainippon: Honoraria; Celgene Japan: Honoraria; Kyowa-Kirin: Honoraria, Research Funding. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V126.23.2643.2643
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2015
    detail.hit.zdb_id: 1468538-3
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  • 10
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    Mogamulizumab for ATLL in Clinical Practice
    American Society of Hematology ; 2016
    In:  Blood Vol. 128, No. 22 ( 2016-12-02), p. 2998-2998
    Details
    In: Blood, American Society of Hematology, Vol. 128, No. 22 ( 2016-12-02), p. 2998-2998
    Abstract: Introduction Adult T-cell leukemia/lymphoma (ATLL) is an aggressive peripheral T cell neoplasm that is resistant to conventional chemotherapy and carries a poor prognosis. The effect of mogamulizumab, an immunoglobulin (Ig) G1 monoclonal antibody targeting CCR4 for ATLL cells, was reported in a previous phase 2 study in which mogamulizumab monotherapy was evaluated in relapsed ATLL patients. The overall response rate (ORR), median progression-free survival (PFS) and median overall survival (OS) were 50%, 5.2 and 13.7 months, respectively. It was not stated whether these values were derived in the real world or in clinical practice. Here we evaluate the clinical impact of mogamulizumab treatment in CCR-4-positive aggressive ATLL patients in clinical practice. Patients and methods We retrospectively analyzed 101 CCR-4-positive ATLL patients who received at least one cycle of mogamulizumab infusion between March, 2012 and April, 2016 in 7 facilities in Miyazaki prefecture, an HTLV-1 endemic area in Southwestern Japan. The ORR, PFS, OS and adverse effects (AEs) were evaluated. We next compared OS in patients with at least one course of mogamulizumab therapy with that in historical control patients without mogamulizumab therapy. Results Of the 101 patients, 92 were evaluable for treatment response, survival and AEs. The median age was 70 years old (range; 45 to 90), and 52 patients (51%) were more than 70 years old. According to Shimoyama's criteria, 66 patients were classified as acute type, 32 as lymphoma type, and 3 as chronic type. All 3 chronic-type ATLL patients had at least one unfavorable risk factor. Of the 101 patients, 96 had refractory or relapsed ATLL when mogamulizumab treatment was started, and the prior treatments consisted of VCAP-AMP-VECP, CHOP, DeVIC or CHASE therapy, with an average of 2 courses. In the 5 remaining cases, mogamulizumab was administered as the initial therapy for ATLL. Mogamulizumab was administered as monotherapy in 87 cases (86%), and as combination therapy with other drugs in 14 cases (14%). The ORR was 37%, including a complete remission rate of 19%. The median PFS and OS were 1.8 and 4.2 months, respectively. Among the 101 patients treated with mogamulizumab, only 26 (26%) fulfilled the inclusion criteria of the phase 2 clinical study. Among patients who met those inclusion criteria, the median PFS and OS were 6.0 and 8.4 months, respectively. The use of mogamulizumab improved OS in clinical practice. The median OS of patients receiving mogamulizumab therapy was 12 months, whereas that of patients who did not receive mogamulizumab in the historical cohort was 8.4 months. Hematologic toxicity and skin rash were the most common AEs, and both were manageable Conclusion Mogamulizumab therapy showed clinically meaningful activity in ATLL patients, with an acceptable toxicity in clinical practice. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V128.22.2998.2998
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2016
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