Researcher Profile and Settings

Master

Affiliation (Master)

• Faculty of Medicine　Internal Medicine　Internal Medicine

Affiliation (Master)

• Faculty of Medicine　Internal Medicine　Internal Medicine

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Profile and Settings

Degree

• MD. Ph.D.（Hokkaido University）

Profile and Settings

• Name (Japanese)

  Nakagawa

• Name (Kana)

  Masao

• Name

  201601014068545301

Alternate Names

http://www.hokudai-hematology.jp/medical/staff/nakagawa/

Achievement

Research Interests

• Immunotherapy   Oncology   Immunology   Lymphoma   

Research Areas

• Life sciences / Hematology and oncology

Awards

• 2108 The Princess Takamatsu Cancer Research Fund, Research Grant
   全ゲノムCRISPRスクリーニング法を用いたT細胞性リンパ腫に対する新規免疫療法の探索 

  受賞者: Masao Nakagawa
• 2019 Nagoya International Cancer Treatment Symposium BMS Award
   

  受賞者: Masao Nakagawa
• 2018 Research Award from Leukemia Research Fund
   

  受賞者: Masao Nakagawa
• 2016 The Fellows Award for Research Excellence (FARE) Travel Award
   

  受賞者: Masao Nakagawa
• 2015 The Center for Cancer Research (CCR) Federal Technology Transfer Award
   

  受賞者: Masao Nakagawa
• 2010 Research Fellowship of Uehara Memorial Foundation
   

  受賞者: Masao Nakagawa

Published Papers

• Whole genome CRISPR screening identifies molecular mechanisms of PD-L1 expression in Adult T-cell leukemia/lymphoma.

  Masahiro Chiba, Joji Shimono, Keito Suto, Takashi Ishio, Tomoyuki Endo, Hideki Goto, Hiroo Hasegawa, Michiyuki Maeda, Takanori Teshima, Yibin Yang, Masao Nakagawa

  Blood 2023/12/24 

  Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis and limited treatment options. Programmed cell death ligand 1(PD-L1) is recognized to be involved in the pathobiology of ATLL. However, what molecules control PD-L1 expression and whether genetic or pharmacological intervention might modify PD-L1 expression in ATLL cells is still unknown. In order to comprehend the regulatory mechanisms of PD-L1 expression in ATLL cells, we performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening in this work. In ATLL cells, we discovered that the neddylation-associated genes NEDD8, NAE1, UBA3, and CUL3 negatively regulated PD-L1 expression while STAT3 positively did so in ATLL cells. We verified, in line with the genetic results, that treatment with the JAK1/2 inhibitor ruxolitinib or the neddylation pathway inhibitor pevonedistat resulted in a decrease in PD-L1 expression in ATLL cells or an increase in it. It is significant that these results held true regardless of whether ATLL cells had the PD-L1 3' structural variant, a known genetic anomaly that promotes PD-L1 overexpression in certain primary ATLL patients. Pevonedistat alone showed cytotoxicity for ATLL cells, but compared to each single modality, pevonedistat improved the cytotoxic effects of the anti-PD-L1 monoclonal antibody Avelumab and chimeric antigen receptor T-cells targeting PD-L1 in vitro. As a result, our work provided insight into a portion of the complex regulatory mechanisms governing PD-L1 expression in ATLL cells and demonstrated the in vitro preliminary preclinical efficacy of PD-L1-directed immunotherapies by using pevonedistat to upregulate PD-L1 in ATLL cells.
• Incidence and course of Epstein-Barr virus viremia after allogeneic hematopoietic stem cell transplant for adult-onset systemic chronic active Epstein-Barr virus disease.

  Preeti Prerna M Vaswani, Masahiro Onozawa, Yuta Hasegawa, Hiroyuki Ohigashi, Takahide Ara, Toshihiro Matsukawa, Atsushi Yasumoto, Souichi Shiratori, Hideki Goto, Masao Nakagawa, Kaoru Kahata, Tomoyuki Endo, Daigo Hashimoto, Takanori Teshima

  Bone marrow transplantation 2023/09/05
• 大腿骨人工骨頭インプラント周囲に発症したALK陰性未分化大細胞型リンパ腫の1例

  森 祐斗, 荒 隆英, 中川 雅夫, 吉田 匠汰, 斎藤 祐美花, 横山 翔大, 松川 敏大, 白鳥 聡一, 遠藤 知之, 豊嶋 崇徳

  臨床血液 (一社)日本血液学会-東京事務局 63 (11) 1592 - 1593 0485-1439 2022/11
• Antiretroviral therapy achieved metabolic complete remission of hepatic AIDS related Epstein-Barr virus-associated smooth muscle tumor.

  Takahide Ara, Tomoyuki Endo, Hideki Goto, Kohei Kasahara, Yuta Hasegawa, Shota Yokoyama, Souichi Shiratori, Masao Nakagawa, Ken Kuwahara, Emi Takakuwa, Satoshi Hashino, Takanori Teshima

  Antiviral therapy 27 (5) 13596535221126828 - 13596535221126828 2022/10 

  Epstein-Barr virus-associated smooth muscle tumor (EBV-SMT) is a rare mesenchymal tumor which occurs in immunocompromised patients. The immune status is an important factor in the treatment of EBV-SMTs, but the efficacy of antiretroviral therapy (ART) is not elucidated in acquired immune deficiency syndrome (AIDS) related EBV-SMTs. Here, we report the first successful case of a 29-year-old man with hepatic AIDS related EBV-SMT treated with ART solely. Positron emission tomography scan was useful for the evaluation of disease status. Recent advances in ART that enables to restore patient's immune status rapidly may change the treatment strategy in AIDS related EBV-SMT.
• Genome-wide CRISPR screens identify CD48 defining susceptibility to NK cytotoxicity in peripheral T-cell lymphomas.

  Masahiro Chiba, Joji Shimono, Takashi Ishio, Norio Takei, Kohei Kasahara, Reiki Ogasawara, Takahide Ara, Hideki Goto, Koh Izumiyama, Satoko Otsuguro, Liyanage P Perera, Hiroo Hasegawa, Michiyuki Maeda, Satoshi Hashino, Katsumi Maenaka, Takanori Teshima, Thomas A Waldmann, Yibin Yang, Masao Nakagawa

  Blood 140 (18) 1951 - 1963 2022/08/03 

  Adult T-cell leukemia/lymphoma (ATLL) is one of the aggressive peripheral T-cell neoplasms with a poor prognosis. Accumulating evidence demonstrates that escape from adaptive immunity is a hallmark for ATLL pathogenesis. However, the mechanisms by which ATLL cells evade NK-cell-mediated immunity have been poorly understood. Here we show that CD48 expression in ATLL cells determines the sensitivity for NK-cell-mediated cytotoxicity against ATLL cells. We performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening using two ATLL derived cell lines and discovered CD48 as one of the best enriched genes whose knockout conferred resistance to YT-1 NK cell line mediated cytotoxicity. The ability of CD48-knockout ATLL cells to evade NK cell effector function was confirmed using human primary NK cells with reduced IFNg induction and degranulation. We found that primary ATLL cells had reduced CD48 expression along with disease progression. Furthermore, other subgroups among aggressive peripheral T-cell lymphomas (PTCL) also expressed lower levels of CD48 than normal T-cells, suggesting that CD48 is a key molecule in malignant T-cell evasion of NK cell surveillance. Thus, this study demonstrates that CD48 expression is likely critical for malignant T-cell lymphoma cell regulation of NK cell mediated immunity and provides a rationale for future evaluation of CD48 as a molecular biomarker in NK cell-associated immunotherapies.
• Gilteritinib enhances graft-versus-leukemia effects against FLT3-ITD mutant leukemia after allogeneic hematopoietic stem cell transplantation.

  Zixuan Zhang, Yuta Hasegawa, Daigo Hashimoto, Hajime Senjo, Ryo Kikuchi, Xuanzhong Chen, Kazuki Yoneda, Tomoko Sekiguchi, Tatsuya Kawase, Hirofumi Tsuzuki, Takashi Ishio, Takahide Ara, Hiroyuki Ohigashi, Masao Nakagawa, Takanori Teshima

  Bone marrow transplantation 57 (5) 775 - 780 2022/05 

  Allogeneic hematopoietic stem cell transplantation (allo-SCT) is a potentially curative therapy for FLT3 internal tandem duplication mutant (FLT3-ITD+) acute myeloid leukemia, but relapse rate is high. A recent study showed that sorafenib, a first generation FLT3 and multikinase inhibitor, enhanced graft-versus-leukemia (GVL) effects against FLT3-ITD+ leukemia via interleukin-15 (IL-15) production. However, it remains to be clarified whether this effect could be mediated by selective FLT3 inhibition. We investigated whether gilteritinib, a selective FLT3 inhibitor, could enhance GVL effects against FLT3-ITD transfected Ba/F3 leukemia (Ba/F3-FLT3-ITD) in mice. Oral administration of gilteritinib from day +5 to +14 after allo-SCT reduced expression of the co-inhibitory receptors PD-1 and TIGIT on donor CD8+ T cells and enhanced IL-15 expression in Ba/F3-FLT3-ITD. Bioluminescent imaging using luciferase-transfected Ba/F3-FLT3-ITD demonstrated that gilteritinib significantly suppressed leukemia expansion after allo-SCT, whereas it did not impact the morbidity or mortality of graft-versus-host disease (GVHD), resulting in significant improvement of overall survival. In conclusion, short-term administration of gilteritinib after allo-SCT enhanced GVL effects against FLT3-ITD+ leukemia without exacerbating GVHD.
• Genome-wide CRISPR screen identifies CDK6 as a therapeutic target in Adult T-cell leukemia/lymphoma.

  Takashi Ishio, Sarvesh Kumar, Joji Shimono, Anusara Daenthanasanmak, Sigrid Dubois, Yuquan Lin, Bonita R Bryant, Michael N Petrus, Emmanuel Bachy, Da Wei Huang, Yandan Yang, Patrick L Green, Hiroo Hasegawa, Michiyuki Maeda, Hideki Goto, Tomoyuki Endo, Takashi Yokota, Kanako C Hatanaka, Yutaka Hatanaka, Shinya Tanaka, Yoshihiro Matsuno, Yibin Yang, Satoshi Hashino, Takanori Teshima, Thomas A Waldmann, Louis M Staudt, Masao Nakagawa

  Blood 139 (10) 1541 - 1556 2021/11/24 

  Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis with current therapy. Here we report genome-wide CRISPR-Cas9 screening of ATLL models, which identified CDK6, CCND2, BATF3, JUNB, STAT3, and IL10RB as genes that are essential for the proliferation and/or survival of ATLL cells. As a single agent, the CDK6 inhibitor palbociclib induced cell cycle arrest and apoptosis in ATLL models with wild type TP53. ATLL models that had inactivated TP53 genetically were relatively resistant to palbociclib owing to compensatory CDK2 activity, and this resistance could be reversed by APR-246, a small molecule activator of mutant TP53. The CRISPR-Cas9 screen further highlighted the dependence of ATLL cells on mTORC1 signaling. Treatment of ATLL cells with palbociclib in combination with mTORC1 inhibitors was synergistically toxic irrespective of the TP53 status. This work defines CDK6 as a novel therapeutic target for ATLL and supports the clinical evaluation of palbociclib in combination with mTORC1 inhibitors in this recalcitrant malignancy.
• High lymphocyte counts before antithymocyte globulin administration predict acute graft-versus-host disease.

  Souichi Shiratori, Hiroyuki Ohigashi, Takahide Ara, Atsushi Yasumoto, Hideki Goto, Masao Nakagawa, Junichi Sugita, Masahiro Onozawa, Kaoru Kahata, Tomoyuki Endo, Daigo Hashimoto, Takanori Teshima

  Annals of hematology 100 (5) 1321 - 1328 2021/05 

  Antithymocyte globulin (ATG) reduces severe acute and chronic graft-versus-host disease (GVHD) in allogeneic peripheral blood stem cell transplantation (PBSCT). However, risk factors for severe acute GVHD in PBSCT using ATG remain to be determined. We conducted a single-center, retrospective study to analyze the association of acute GVHD requiring systemic corticosteroid (SC-aGVHD) with absolute lymphocyte counts (ALC) before the administration of ATG or conditioning in 53 patients with HLA-matched PBSCT using low-dose thymoglobulin (2 mg/kg) after myeloablative conditioning. The cumulative incidence of SC-aGVHD was 17.0% and ALC before ATG were significantly higher in patients with SC-aGVHD compared to that in patients without it (median, 0.15 × 109/L vs 0.06 × 109/L, P = 0.047). The cumulative incidence of SC-aGVHD was significantly higher in patients with high ALC before ATG (≥ 0.15 × 109/L) than in those with low ALC (38.5% vs 10.0%, P = 0.016). Non-relapse mortality (NRM) was also significantly higher in the high ALC before ATG group than the low ALC before ATG group (2-year NRM: 23.9% vs 6.0%, P = 0.048), leading to worse survival (2-year overall survival: 69.2% vs 83.5%, P = 0.039). Our study suggested that high ALC before ATG is a risk factor for SC-aGVHD.
• A novel model of alternative NF-κB pathway activation in anaplastic large cell lymphoma.

  Hongbo Wang, Wei Wei, Jing-Ping Zhang, Zhihui Song, Yangyang Li, Wenming Xiao, Yijun Liu, Mu-Sheng Zeng, Michael N Petrus, Craig J Thomas, Marshall E Kadin, Masao Nakagawa, Thomas A Waldmann, Yibin Yang

  Leukemia 2020/11/12 

  Aberrant activation of NF-κB is the most striking oncogenic mechanism in B-cell lymphoma; however, its role in anaplastic large cell lymphomas (ALCL) has not been fully established and its activation mechanism(s) remain unclear. Using ALCL cell line models, we revealed the supporting roles for NFKB2 and the NIK pathway in some ALCL lines. To investigate the detailed activation mechanisms for this oncogenic pathway, we performed specifically designed alternative NF-κB reporter CRISPR screens followed by the RNA-seq analysis, which led us to identify STAT3 as the major mediator for NIK-dependent NF-κB activation in ALCL. Consistently, p-STAT3 level was correlated with NFKB2 nuclear accumulation in primary clinical samples. Mechanistically, we found that in NIK-positive ALK- ALCL cells, common JAK/STAT3 mutations promote transcriptional activity of STAT3 which directly regulates NFKB2 and CD30 expression. Endogenous expression of CD30 induces constitutive NF-κB activation through binding and degrading of TRAF3. In ALK+ ALCL, the CD30 pathway is blocked by the NPM-ALK oncoprotein, but STAT3 activity and resultant NFKB2 expression can still be induced by NPM-ALK, leading to minimal alternative NF-κB activation. Our data suggest combined NIK and JAK inhibitor therapy could benefit patients with NIK-positive ALK- ALCL carrying JAK/STAT3 somatic mutations.
• Short-term treatment with imetelstat sensitizes hematopoietic malignant cells to a genotoxic agent via suppression of the telomerase-mediated DNA repair process.

  Daisuke Hidaka, Masahiro Onozawa, Naohiro Miyashita, Shota Yokoyama, Masao Nakagawa, Daigo Hashimoto, Takanori Teshima

  Leukemia & lymphoma 61 (11) 2722 - 2732 2020/11 

  Imetelstat is a specific and competitive inhibitor of telomerase enzymatic activity. We demonstrated that imetelstat could interfere with the DNA repair process and enhance the effect of DNA damaging agents using hematological tumor cell lines. Short-term administration of imetelstat enhanced growth suppression by anticancer agents and radiation. It also upregulated γH2AX expression induced by irradiation. Immunofluorescence staining showed that both human telomerase reverse transcriptase (hTERT) and γH2AX were upregulated and co-localized in the nucleus of peripheral blood mononuclear cells after irradiation, suggesting that hTERT was involved in the DNA-DSB repair process. Imetelstat enhanced growth inhibitory effect of cytotoxic agents in short-term culture without shortening of telomeres, indicating that this effect was attributed by telomere length independent mechanism. Our results suggest that the combination of short-term treatment with imetelstat and cytotoxic agent is a promising strategy to treat a wide variety of hematopoietic malignancies.
• Reduced dose of MTX for GVHD prophylaxis promotes engraftment and decreases non-relapse mortality in umbilical cord blood transplantation

  Souichi Shiratori, Hiroyuki Ohigashi, Shuichiro Takahashi, Takahide Ara, Hideki Goto, Masao Nakagawa, Junichi Sugita, Masahiro Onozawa, Kaoru Kahata, Tomoyuki Endo, Daigo Hashimoto, Takanori Teshima

  Annals of Hematology 99 (3) 591 - 598 0939-5555 2020/03
• A novel model of controlling PD-L1 expression in ALK+ anaplastic large cell lymphoma revealed by CRISPR screening

  Jing-Ping Zhang, Zhihui Song, Hong-Bo Wang, Lang Lang, Yuan-Zhong Yang, Wenming Xiao, Daniel E. Webster, Wei Wei, Stefan K. Barta, Marshall E. Kadin, Louis M. Staudt, Masao Nakagawa, Yibin Yang

  Blood 134 (2) 171 - 185 0006-4971 2019/07/11 [Refereed][Not invited]
   

  The success of programmed cell death protein 1 (PD-1)/PD-L1-based immunotherapy highlights the critical role played by PD-L1 in cancer progression and reveals an urgent need to develop new approaches to attenuate PD-L1 function by gaining insight into how its expression is controlled. Anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma (ALK+ ALCL) expresses a high level of PD-L1 as a result of the constitutive activation of multiple oncogenic signaling pathways downstream of ALK activity, making it an excellent model in which to define the signaling processes responsible for PD-L1 upregulation in tumor cells. Here, using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 library screening, we sought a comprehensive understanding of the molecular effectors required for PD-L1 regulation in ALK+ ALCL. Indeed, we determined that PD-L1 induction is dependent on the nucleophosmin-ALK oncoprotein activation of STAT3, as well as a signalosome containing GRB2/SOS1, which activates the MEK-ERK and PI3K-AKT signaling pathways. These signaling networks, through STAT3 and the GRB2/SOS1, ultimately induce PD-L1 expression through the action of transcription factors IRF4 and BATF3 on the enhancer region of the PD-L1 gene. IRF4 and BATF3 are essential for PD-L1 upregulation, and IRF4 expression is correlated with PD-L1 levels in primary ALK+ ALCL tissues. Targeting this oncogenic signaling pathway in ALK+ ALCL largely inhibited the ability of PD-L1-mediated tumor immune escape when cocultured with PD-1-positive T cells and natural killer cells. Thus, our identification of this previously unrecognized regulatory hub not only accelerates our understanding of the molecular circuitry that drives tumor immune escape but also provides novel opportunities to improve immunotherapeutic intervention strategies.
• Ocular instillation of vitamin A–coupled liposomes containing HSP47 siRNA ameliorates dry eye syndrome in chronic GVHD

  Hiroyuki Ohigashi, Daigo Hashimoto, Eiko Hayase, Shuichiro Takahashi, Takahide Ara, Tomohiro Yamakawa, Junichi Sugita, Masahiro Onozawa, Masao Nakagawa, Takanori Teshima

  Blood Advances 3 (7) 1003 - 1010 2473-9529 2019/04/09 [Refereed][Not invited]
   

  Chronic graft-versus-host disease (GVHD) profoundly affects the quality of life of long-term survivors of allogeneic hematopoietic stem cell transplantation (SCT). The eyes are frequently involved, and dry eye syndrome is the most common manifestation of ocular chronic GVHD. We explored the role of heat shock protein 47 (HSP47) in ocular GVHD and developed a novel antifibrotic topical therapy using vitamin A–coupled liposomes containing HSP47 small interfering RNA (siRNA) against HSP47 (VA-lip HSP47). In a mouse model of chronic GVHD, infiltration of HSP47+ fibroblasts and massive fibrosis surrounding the lacrimal ducts were observed after allogeneic SCT, leading to impaired tear secretion. After ocular instillation, VA-lip HSP47 was distributed to the lacrimal glands, knocked down HSP47 expression in fibroblasts, reduced collagen deposition, and restored tear secretion after allogeneic SCT. Ocular instillation of VA-lip HSP47 also ameliorated established lacrimal gland fibrosis and dry eye syndrome. VA-lip HSP47 eye drops are a promising prophylactic and therapeutic option against dry eye syndrome in chronic GVHD.
• Wilms Tumor 1 Expression at Diagnosis Correlates With Genetic Abnormalities and Polymorphism But Is Not Independently Prognostic in Acute Myelogenous Leukemia: A Hokkaido Leukemia Net Study.

  Daisuke Hidaka, Masahiro Onozawa, Junichi Hashiguchi, Naohiro Miyashita, Kohei Kasahara, Shinichi Fujisawa, Eiko Hayase, Kohei Okada, Souichi Shiratori, Hideki Goto, Junichi Sugita, Masao Nakagawa, Daigo Hashimoto, Kaoru Kahata, Tomoyuki Endo, Satoshi Yamamoto, Yutaka Tsutsumi, Yoshihito Haseyama, Takahiro Nagashima, Akio Mori, Shuichi Ota, Hajime Sakai, Toshimichi Ishihara, Kiyotoshi Imai, Takuto Miyagishima, Yasutaka Kakinoki, Mitsutoshi Kurosawa, Hajime Kobayashi, Hiroshi Iwasaki, Chikara Shimizu, Takeshi Kondo, Takanori Teshima

  Clinical lymphoma, myeloma & leukemia 18 (11) e469-e479  2018/11 

  BACKGROUND: The prognostic effect of Wilms tumor 1 (WT1) expression at the diagnosis of acute myelogenous leukemia (AML) has been controversial. The aim of the present study was to determine the correlations of WT1 expression at the diagnosis of AML with established prognostic alterations. PATIENTS AND METHODS: We analyzed diagnostic bone marrow samples from 252 patients. WT1 expression, single nucleotide polymorphism (SNP) in the WT1 gene (rs16754), and Fms-like tyrosine kinase receptor-3 internal tandem duplication (FLT3-ITD) mutation were analyzed for all patients. The nucleophosmin 1 (NPM1) mutation and CCAAT/enhancer-binding protein-α (CEBPA) double mutation were analyzed for cytogenetically normal (CN)-AML. The KIT mutation was analyzed for core-binding factor AML. RESULTS: Within the cytogenetically favorable prognosis group, WT1 expression in AML with inv(16) or t(15;17) was significantly greater than that in AML with t(8;21). In cases with CN-AML, FLT3-ITD and NPM1 mutations both correlated with greater expression of WT1, and the CEBPA double mutation was related to lower WT1 expression. The existence of both FLT3-ITD and NPM1 mutations showed synergistically greater expression of WT1 in CN-AML. SNP in the WT1 gene (rs16754) was significantly associated with lower expression of WT1. The WT1 levels were not prognostic factors in the total cohort or any cytogenetic group or stratified by SNP status. CONCLUSION: Because WT1 expression has correlated with known prognostic factors, the prognostic effect of WT1 levels could be misunderstood depending on the distribution of the collaborative mutations in each cohort. We have concluded that the prognostic significance of WT1 at the diagnosis of AML is weak compared with the other established prognostic factors.
• Hematogones Predict Better Outcome in Allogeneic Hematopoietic Stem Cell Transplantation Irrespective of Graft Sources

  Takashi Ishio, Junichi Sugita, Takahiro Tateno, Daisuke Hidaka, Eiko Hayase, Souichi Shiratori, Kohei Okada, Hideki Goto, Masahiro Onozawa, Masao Nakagawa, Daigo Hashimoto, Kaoru Kahata, Katsuya Fujimoto, Tomoyuki Endo, Takeshi Kondo, Takanori Teshima

  Biology of Blood and Marrow Transplantation 24 (10) 1990 - 1996 1083-8791 2018/10 [Refereed][Not invited]
   

  Benign precursors of B lymphocytes, termed hematogones, are observed in the regenerative state of hematopoiesis following chemotherapy or allogeneic hematopoietic stem cell transplantation (allo-HSCT). Previous studies have demonstrated that expansion of hematogones correlates with better clinical outcomes after allo-HSCT. We retrospectively analyzed the association between hematogones and clinical outcomes in 309 consecutive patients who underwent allo-HSCT, which is the largest population-based cohort reported so far. The incidence of hematogones was significantly higher in complete remission (CR) patients at the time of transplantation than in non-CR patients, after myeloablative conditioning than after reduced-intensity conditioning, with tacrolimus-based graft-versus-host disease (GVHD) prophylaxis than with cyclosporine-based prophylaxis, and with disease other than malignant lymphoma (all P < .05). Patients with hematogones developed less acute GVHD and infections than did those without them (P < .05). Emergence of hematogones was associated with superior GVHD-free relapse-free survival and lower nonrelapse mortality, and was an independent prognostic factor for overall survival, irrespective of donor sources.
• 成人急性リンパ性白血病におけるIKZF1欠失およびCRLF2発現の解析(Analysis of IKZF1 deletion and CRLF2 expression in adult patients with acute lymphoblastic leukemia)

  橋口 淳一, 小野澤 真弘, 藤澤 真一, 高橋 秀一郎, 宮下 直洋, 早瀬 英子, 白鳥 聡一, 後藤 秀樹, 杉田 純一, 中川 雅夫, 橋本 大吾, 加畑 馨, 遠藤 知之, 山本 聡, 堤 豊, 長谷山 美仁, 永嶋 貴博, 盛 暁生, 太田 秀一, 宮城島 拓人, 柿木 康孝, 黒澤 光俊, 岩崎 博, 近藤 健, 豊嶋 崇徳

  臨床血液 59 (9) 1648 - 1648 0485-1439 2018/09
• The association between the incidence of intestinal graft-vs-host disease and antibiotic use after allogeneic hematopoietic stem cell transplantation.

  Hidaka D, Hayase E, Shiratori S, Hasegawa Y, Ishio T, Tateno T, Okada K, Goto H, Sugita J, Onozawa M, Nakagawa M, Kahata K, Endo T, Hashimoto D, Teshima T

  Clinical transplantation 32 (9) e13361  0902-0063 2018/09 [Refereed][Not invited]
   

  Intestinal microbiota plays an important role in the regulation of allogeneic immune reaction after allogeneic hematopoietic stem cell transplantation (allo-SCT). Intestinal graft-vs-host disease (GVHD) is one of the major causes of mortality after allo-SCT and often complicated with intestinal dysbiosis. Recent studies suggest that antibiotic-induced dysbiosis is a risk factor for intestinal GVHD. We retrospectively evaluated the impacts of antibiotic use on the incidence of intestinal GVHD occurring before day 100 after allo-SCT. Among 213 patients who underwent allo-SCT, 200 patients achieving engraftment were analyzed. Antibiotics were classified into carbapenem, quinolone, penicillin, cephem, and glycopeptide. Among 128 patients who developed acute GVHD, intestinal GVHD developed in 36 patients. Patients with intestinal GVHD received significantly longer administration of carbapenem and glycopeptide compared to those without it in periengraftment period. In multivariate analysis, use of carbapenem for greater than 7 days was associated with an increased risk of intestinal GVHD. However, use of antibiotics for greater than 7 days was not associated with poor overall survival and high nonrelapse mortality. Long use of carbapenem in periengraftment period may be a risk for intestinal GVHD. Prospective studies are required to validate our findings.
• Targeting the HTLV-I-Regulated BATF3/IRF4 Transcriptional Network in Adult T Cell Leukemia/Lymphoma

  Masao Nakagawa, Arthur L. Shaffer, Michele Ceribelli, Meili Zhang, George Wright, Da Wei Huang, Wenming Xiao, John Powell, Michael N. Petrus, Yibin Yang, James D. Phelan, Holger Kohlhammer, Sigrid P. Dubois, Hee Min Yoo, Emmanuel Bachy, Daniel E. Webster, Yandan Yang, Weihong Xu, Xin Yu, Hong Zhao, Bonita R. Bryant, Joji Shimono, Takashi Ishio, Michiyuki Maeda, Patrick L. Green, Thomas A. Waldmann, Louis M. Staudt

  Cancer Cell 34 (2) 286 - 297.e10 1535-6108 2018/08 [Refereed][Not invited]
  
• A multiprotein supercomplex controlling oncogenic signalling in lymphoma

  James D. Phelan, Ryan M. Young, Daniel E. Webster, Sandrine Roulland, George W. Wright, Monica Kasbekar, Arthur L. Shaffer, Michele Ceribelli, James Q. Wang, Roland Schmitz, Masao Nakagawa, Emmanuel Bachy, Da Wei Huang, Yanlong Ji, Lu Chen, Yandan Yang, Hong Zhao, Xin Yu, Weihong Xu, Maryknoll M. Palisoc, Racquel R. Valadez, Theresa Davies-Hill, Wyndham H. Wilson, Wing C. Chan, Elaine S. Jaffe, Randy D. Gascoyne, Elias Campo, Andreas Rosenwald, German Ott, Jan Delabie, Lisa M. Rimsza, Fausto J. Rodriguez, Fayez Estephan, Matthias Holdhoff, Michael J. Kruhlak, Stephen M. Hewitt, Craig J. Thomas, Stefania Pittaluga, Thomas Oellerich, Louis M. Staudt

  Nature 560 (7718) 387 - 391 0028-0836 2018/08 [Refereed][Not invited]
  
• Development of a Fluorescence in Situ Hybridization Probe for Detecting IKZF1 Deletion Mutations in Patients with Acute Lymphoblastic Leukemia

  Junichi Hashiguchi, Masahiro Onozawa, Satoshi Oguri, Shinichi Fujisawa, Masahisa Tsuji, Kohei Okada, Masao Nakagawa, Daigo Hashimoto, Kaoru Kahata, Takeshi Kondo, Chikara Shimizu, Takanori Teshima

  Journal of Molecular Diagnostics 20 (4) 446 - 454 1943-7811 2018/07/01 [Refereed][Not invited]
   

  Intragenic deletion of IKZF1 is a recurrent genomic alteration in acute lymphoblastic leukemia. The deletions are mediated by illegitimate variable(diversity)joining recombination via cryptic recombination signal sequences (RSSs). We developed a fluorescence in situ hybridization (FISH) probe set that can detect any type of IKZF1 deletion, including the commonly deleted exon 4 to 7 region. The probe set consists of a designed probe for the commonly deleted region (Cy3 red) and a bacterial artificial chromosomes clone probe for detecting the 3′ flanking region (Spectrum Green). Intact IKZF1 showed a fusion signal, and the deleted allele showed loss of the red signal (0R1G1F). The FISH probes worked correctly for human leukemic cell lines and clinical samples. One case showed an atypical break-apart signal (1R1G1F). Inverse PCR of the case revealed rearrangement of the excised IKZF1 fragment into a legitimate RSS site at Ig κ on chromosome 2, suggesting a pathogenic role of this recombination-activating gene 1/2-mediated event. In this study, we established FISH probe detecting IKZF1 deletion in a quick, quantitative, and cost-effective manner, and the results provided a novel insight into B-cell receptor editing by rearrangement of a cryptic RSS-mediated genomic fragment in acute lymphoblastic leukemia pathology.
• AMLにおけるWT1発現量と染色体・遺伝子異常の関連

  日高 大輔, 小野澤 真弘, 橋口 淳一, 宮下 直洋, 笠原 耕平, 藤澤 真一, 早瀬 英子, 岡田 耕平, 白鳥 聡一, 後藤 秀樹, 杉田 純一, 中川 雅夫, 加畑 馨, 橋本 大吾, 遠藤 知之, 山本 聡, 堤 豊, 長谷山 美仁, 永嶋 貴博, 盛 暁生, 太田 秀一, 酒井 基, 石原 敏道, 今井 陽俊, 宮城島 拓人, 柿木 康孝, 黒澤 光俊, 小林 一, 岩崎 博, 清水 力, 近藤 健, 豊嶋 崇徳

  臨床血液 (一社)日本血液学会-東京事務局 59 (7) 964 - 964 0485-1439 2018/07
• A novel heterozygous ITGB3 p.T720del inducing spontaneous activation of integrin αIIbβ3 in autosomal dominant macrothrombocytopenia with aggregation dysfunction.

  Naohiro Miyashita, Masahiro Onozawa, Koji Hayasaka, Takahiro Yamada, Ohsuke Migita, Kenichiro Hata, Kohei Okada, Hideki Goto, Masao Nakagawa, Daigo Hashimoto, Kaoru Kahata, Takeshi Kondo, Shinji Kunishima, Takanori Teshima

  Annals of hematology 97 (4) 629 - 640 2018/04 

  We identified a novel heterozygous ITGB3 p.T720del mutation in a pedigree with macrothrombocytopenia exhibiting aggregation dysfunction. Platelet aggregation induced by ADP and collagen was significantly reduced, while ristocetin aggregation was normal. Integrin αIIbβ3 was partially activated in a resting status, but platelet expression of αIIbβ3 was downregulated. Functional analysis using a cell line showed spontaneous phosphorylation of FAK in αIIb/β3 (p.T720del)-transfected 293T cells in suspension conditions. Abnormal cytoplasmic protrusions, membrane ruffling, and cytoplasmic localization of αIIbβ3 were observed in αIIb/β3 (p.T720del)-transfected CHO cells. Such morphological changes were reversed by treatment with an FAK inhibitor. These findings imply spontaneous, but partial, activation of αIIbβ3 followed by phosphorylation of FAK as the initial mechanism of abnormal thrombopoiesis. Internalization and decreased surface expression of αIIbβ3 would contribute to aggregation dysfunction. We reviewed the literature of congenital macrothrombocytopenia associated with heterozygous ITGA2B or ITGB3 mutations. Reported mutations were highly clustered at the membrane proximal region of αIIbβ3, which affected the critical interaction between αIIb R995 and β3 D723, resulting in a constitutionally active form of the αIIbβ3 complex. Macrothrombocytopenia caused by a heterozygous activating mutation of ITGA2B or ITGB3 at the membrane proximal region forms a distinct entity of rare congenital thrombocytopenia.
• T-cell depletion effects of low-dose antithymocyte globulin for GVHD prophylaxis in HLA-matched allogeneic peripheral blood stem cell transplantation.

  Souichi Shiratori, Mizuha Kosugi-Kanaya, Eiko Hayase, Kohei Okada, Hideki Goto, Junichi Sugita, Masahiro Onozawa, Masao Nakagawa, Kaoru Kahata, Daigo Hashimoto, Tomoyuki Endo, Takeshi Kondo, Takanori Teshima

  Transplant immunology 46 21 - 22 2018/02
• Chimeric antigen receptor modified T cells that target chemokine receptor CCR4 as a therapeutic modality for T-cell malignancies

  Liyanage P. Perera, Meili Zhang, Masao Nakagawa, Michael N. Petrus, Michiyuki Maeda, Marshall E. Kadin, Thomas A. Waldmann, Pin-Yu Perera

  AMERICAN JOURNAL OF HEMATOLOGY 92 (9) 892 - 901 0361-8609 2017/09 [Refereed][Not invited]
   

  With the emerging success of treating CD19 expressing B cell malignancies with ex vivo modified, autologous T cells that express CD19-directed chimeric antigen receptors (CAR), there is intense interest in expanding this evolving technology to develop effective modalities to treat other malignancies including solid tumors. Exploiting this approach to develop a therapeutic modality for T cell malignancies for which the available regimens are neither curative, nor confer long term survival we generated a lentivirus-based CAR gene transfer system to target the chemokine receptor CCR4 that is over-expressed in a spectrum of T cell malignancies as well as in CD41CD251Foxp31T regulatory cells that accumulate in the tumor microenvironment constituting a barrier against anti-tumor immunity. Ex vivo modified, donor-derived T cells that expressed CCR4 directed CAR displayed antigen-dependent potent cytotoxicity against patient-derived cell lines representing ATL, CTCL, ALCL and a subset of HDL. Furthermore, these CAR T cells also eradicated leukemia in a mouse xenograft model of ATL illustrating the potential utility of this modality in the treatment of a wide spectrum of T cell malignancies.
• Disseminated toxoplasmosis after hematopoietic stem cell transplantation showing unusual magnetic resonance images

  Takahiro Tateno, Masahiro Onozawa, Junichi Hashiguchi, Takashi Ishio, Sayaka Yuzawa, Satomi Matsuoka, Mizuha Kosugi-Kanaya, Kohei Okada, Souichi Shiratori, Hideki Goto, Taichi Kimura, Junichi Sugita, Masao Nakagawa, Daigo Hashimoto, Kaoru Kahata, Katsuya Fujimoto, Tomoyuki Endo, Takeshi Kondo, Shinya Tanaka, Satoshi Hashino, Takanori Teshima

  TRANSPLANT INFECTIOUS DISEASE 19 (4) 1398-2273 2017/08 [Refereed][Not invited]
   

  We herein report a patient who had disseminated toxoplasmosis after hematopoietic stem cell transplantation showing atypical clinical presentation and neuroimaging. Parkinsonism symptoms such as muscle rigidity, bradykinesia, tremor, and postural instability were initial manifestations. Magnetic resonance imaging showed diffuse symmetrical lesions of bilateral basal ganglia lacking ringed enhancement. Post-mortem analysis revealed multiple tachyzoites of Toxoplasma gondii in the basal ganglia, mid brain, cerebellum, and cardiac muscle.
• Cytokine receptor signaling is required for the survival of ALK- anaplastic large cell lymphoma, even in the presence of JAK1/STAT3 mutations

  Jing Chen, Yong Zhang, Michael N. Petrus, Wenming Xiao, Alina Nicolae, Mark Raffeld, Stefania Pittaluga, Richard N. Bamford, Masao Nakagawa, Sunny Tianyi Ouyang, Alan L. Epstein, Marshall E. Kadin, Annarose Del Mistro, Richard Woessner, Elaine S. Jaffe, Thomas A. Waldmann

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114 (15) 3975 - 3980 0027-8424 2017/04 [Refereed][Not invited]
   

  Activating Janus kinase (JAK) and signal transducer and activator of transcription (STAT) mutations have been discovered in many T-cell malignancies, including anaplastic lymphoma kinase (ALK)-anaplastic large cell lymphomas (ALCLs). However, such mutations occur in a minority of patients. To investigate the clinical application of targeting JAK for ALK-ALCL, we treated ALK-cell lines of various histological origins with JAK inhibitors. Interestingly, most exogenous cytokine-independent cell lines responded to JAK inhibition regardless of JAK mutation status. JAK inhibitor sensitivity correlated with the STAT3 phosphorylation status of tumor cells. Using retroviral shRNA knockdown, we have demonstrated that these JAK inhibitor-sensitive cells are dependent on both JAK1 and STAT3 for survival. JAK1 and STAT3 gain-of-function mutations were found in some, but not all, JAK inhibitor-sensitive cells. Moreover, the mutations alone cannot explain the JAK1/STAT3 dependency, given that wildtype JAK1 or STAT3 was sufficient to promote cell survival in the cells that had either JAK1or STAT3 mutations. To investigate whether other mechanisms were involved, we knocked down upstream receptors GP130 or IL-2R gamma. Knockdown of GP130 or IL-2R. induced cell death in selected JAK inhibitor-sensitive cells. High expression levels of cytokines, including IL-6, were demonstrated in cell lines as well as in primary ALK-ALCL tumors. Finally, ruxolitinib, a JAK1/2 inhibitor, was effective in vivo in a xenograft ALK-ALCL model. Our data suggest that cytokine receptor signaling is required for tumor cell survival in diverse forms of ALK-ALCL, even in the presence of JAK1/STAT3 mutations. Therefore, JAK inhibitor therapy might benefit patients with ALK-ALCL who are phosphorylated STAT3(+).
• Regulation of normal B-cell differentiation and malignant B-cell survival by OCT2

  Daniel J. Hodson, Arthur L. Shaffer, Wenming Xiao, George W. Wright, Roland Schmitz, James D. Phelan, Yandan Yang, Daniel E. Webster, Lixin Rui, Holger Kohlhammer, Masao Nakagawa, Thomas A. Waldmann, Louis M. Staudt

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 113 (14) E2039 - E2046 0027-8424 2016/04 [Refereed][Not invited]
   

  The requirement for the B-cell transcription factor OCT2 (octamer-binding protein 2, encoded by Pou2f2) in germinal center B cells has proved controversial. Here, we report that germinal center B cells are formed normally after depletion of OCT2 in a conditional knockout mouse, but their proliferation is reduced and in vivo differentiation to antibody-secreting plasma cells is blocked. This finding led us to examine the role of OCT2 in germinal center-derived lymphomas. shRNA knockdown showed that almost all diffuse large B-cell lymphoma (DLBCL) cell lines are addicted to the expression of OCT2 and its coactivator OCA-B. Genome-wide chromatin immunoprecipitation (ChIP) analysis and gene-expression profiling revealed the broad transcriptional program regulated by OCT2 that includes the expression of STAT3, IL-10, ELL2, XBP1, MYC, TERT, and ADA. Importantly, genetic alteration of OCT2 is not a requirement for cellular addiction in DLBCL. However, we detected amplifications of the POU2F2 locus in DLBCL tumor biopsies and a recurrent mutation of threonine 223 in the DNA-binding domain of OCT2. This neomorphic mutation subtly alters the DNA-binding preference of OCT2, leading to the transactivation of noncanonical target genes including HIF1a and FCRL3. Finally, by introducing mutations designed to disrupt the OCT2-OCA-B interface, we reveal a requirement for this protein-protein interface that ultimately might be exploited therapeutically. Our findings, combined with the predominantly B-cell-restricted expression of OCT2 and the absence of a systemic phenotype in our knockout mice, suggest that an OCT2-targeted therapeutic strategy would be efficacious in both major subtypes of DLBCL while avoiding systemic toxicity.
• Targeting Non-proteolytic Protein Ubiquitination for the Treatment of Diffuse Large B Cell Lymphoma

  Yibin Yang, Priscilla Kelly, Arthur L. Shaffer, Roland Schmitz, Hee Min Yoo, Xinyue Liu, Da Wei Huang, Daniel Webster, Ryan M. Young, Masao Nakagawa, Michele Ceribelli, George W. Wright, Yandan Yang, Hong Zhao, Xin Yu, Weihong Xu, Wing C. Chan, Elaine S. Jaffe, Randy D. Gascoyne, Elias Campo, Andreas Rosenwald, German Ott, Jan Delabie, Lisa Rimsza, Louis M. Staudt

  CANCER CELL 29 (4) 494 - 507 1535-6108 2016/04 [Refereed][Not invited]
   

  Chronic active B cell receptor (BCR) signaling, a hallmark of the activated B cell-like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), engages the CARD11-MALT1-BCL10 (CBM) adapter complex to activate I kappa B kinase (IKK) and the classical NF-kappa B pathway. Here we show that the CBM complex includes the E3 ubiquitin ligases cIAP1 and cIAP2, which are essential mediators of BCR-dependent NF-kappa B activity in ABC DLBCL. cIAP1/2 attach K63-linked polyubiquitin chains on themselves and on BCL10, resulting in the recruitment of IKK and the linear ubiquitin chain ligase LUBAC, which is essential for IKK activation. SMAC mimetics target cIAP1/2 for destruction, and consequently suppress NF-kB and selectively kill BCR-dependent ABC DLBCL lines, supporting their clinical evaluation in patients with ABC DLBCL.
• Gain-of-function CCR4 mutations in adult T cell leukemia/lymphoma

  Masao Nakagawa, Roland Schmitz, Wenming Xiao, Carolyn K. Goldman, Weihong Xu, Yandan Yang, Xin Yu, Thomas A. Waldmann, Louis M. Staudt

  JOURNAL OF EXPERIMENTAL MEDICINE 211 (13) 2497 - 2505 0022-1007 2014/12 [Refereed][Not invited]
   

  Adult T cell leukemia/lymphoma (ATLL) is an aggressive malignancy caused by human T cell lymphotropic virus type-I (HTLV-I) without curative treatment at present. To illuminate the pathogenesis of ATLL we performed whole transcriptome sequencing of purified ATLL patient samples and discovered recurrent somatic mutations in CCR4, encoding CC chemokine receptor 4. CCR4 mutations were detected in 14/53 ATLL samples (26%) and consisted exclusively of nonsense or frameshift mutations that truncated the coding region at C329, Q330, or Y331 in the carboxy terminus. Functionally, the CCR4-Q330 nonsense isoform was gain-of-function because it increased cell migration toward the CCR4 ligands CCL17 and CCL22, in part by impairing receptor internalization. This mutant enhanced PI(3) kinase/AKT activation after receptor engagement by CCL22 in ATLL cells and conferred a growth advantage in long-term in vitro cultures. These findings implicate somatic gain-of-function CCR4 mutations in the pathogenesis of ATLL and suggest that inhibition of CCR4 signaling might have therapeutic potential in this refractory malignancy.
• Clonal heterogeneity of lymphoid malignancies correlates with poor prognosis

  Miyuki Suguro, Noriaki Yoshida, Akira Umino, Harumi Kato, Hiroyuki Tagawa, Masao Nakagawa, Noriko Fukuhara, Sivasundaram Karnan, Ichiro Takeuchi, Toby D. Hocking, Kotaro Arita, Kennosuke Karube, Shinobu Tsuzuki, Shigeo Nakamura, Tomohiro Kinoshita, Masao Seto

  CANCER SCIENCE 105 (7) 897 - 904 1347-9032 2014/07 [Refereed][Not invited]
   

  Clonal heterogeneity in lymphoid malignancies has been recently reported in adult T-cell lymphoma/leukemia, peripheral T-cell lymphoma, not otherwise specified, and mantle cell lymphoma. Our analysis was extended to other types of lymphoma including marginal zone lymphoma, follicular lymphoma and diffuse large B-cell lymphoma. To determine the presence of clonal heterogeneity, 332 cases were examined using array comparative genomic hybridization analysis. Results showed that incidence of clonal heterogeneity varied from 25% to 69% among different types of lymphoma. Survival analysis revealed that mantle cell lymphoma and diffuse large B-cell lymphoma with clonal heterogeneity showed significantly poorer prognosis, and that clonal heterogeneity was confirmed as an independent predictor of poor prognosis for both types of lymphoma. Interestingly, 8q24.1 (MYC) gain, 9p21.3 (CDKN2A/2B) loss and 17p13 (TP53, ATP1B2, SAT2, SHBG) loss were recurrent genomic lesions among various types of lymphoma with clonal heterogeneity, suggesting at least in part that alterations of these genes may play a role in clonal heterogeneity.
• Clinical impact of cycling the administration of antibiotics for febrile neutropenia in Japanese patients with hematological malignancy

  S. Hashino, L. Morita, H. Kanamori, M. Takahata, M. Onozawa, M. Nakagawa, T. Kawamura, F. Fujisawa, K. Kahata, K. Izumiyama, M. Yonezumi, K. Chiba, T. Kondo, M. Asaka

  EUROPEAN JOURNAL OF CLINICAL MICROBIOLOGY & INFECTIOUS DISEASES 31 (2) 173 - 178 0934-9723 2012/02 [Refereed][Not invited]
   

  Despite the availability of newer classes of antibiotics, infection with multi-drug-resistant bacteria is a serious problem. To suppress the appearance of multi-drug-resistant bacteria and to avoid severe infection derived from febrile neutropenia (FN), we conducted cycling the administration of antibiotics for FN in patients with hematological malignancy. The treatment protocol consisted of the administration of four antibiotics each for 3 months in 1 year. The above regimen was repeated for 4 years. A total of 193 patients were registered in the protocol. The mean duration of the administration of cycling antibiotics was 5.9 days (range: 1-16 days). The frequency of FN before the study and during the study was unchanged until the third year, but decreased significantly in the fourth year. The frequency of detection of multi-drug-resistant bacteria in the first year was the same as that before the study was started, but dramatically decreased after the second year. Bacteriological treatment success rates were similar in each trimester and each year. The effective rate was not statistically different in each trimester and each year. We conclude that cycling the administration of antibiotics in patients with FN is useful for suppressing the appearance of multi-drug-resistant bacteria and for obtaining excellent clinical efficacy.
• Synergistic effect of Bcl2, Myc and Ccnd1 transforms mouse primary B cells into malignant cells

  Masao Nakagawa, Shinobu Tsuzuki, Keiichiro Honma, Osamu Taguchi, Masao Seto

  HAEMATOLOGICA-THE HEMATOLOGY JOURNAL 96 (9) 1318 - 1326 0390-6078 2011/09 [Refereed][Not invited]
   

  Background A synergistic effect resulting from a combination of BCL2 and MYC or MYC and CCND1 has been implicated in human B-cell lymphomas. Although the identification of other cooperative genes involved is important, our present understanding of such genes remains scant. The objective of this study was to identify the additional cooperative gene(s) associated with BCL2 and MYC or MYC and CCND1. First, we assessed whether Bcl2, Myc and Ccnd1 could cooperate. Next, we developed a synergism-based functional screening method for the identification of other oncogene(s) that act with Bcl2 and Myc. Design and Methods Growth in culture, colony formation and oncogenicity in vivo were assessed in mouse primary B cells exogenously expressing various combinations of Bcl2, Myc and Ccnd1. For the functional screening, Bcl2- and Myc-expressing primary B cells were infected with a retroviral cDNA library. Inserted cDNA of transformed cells in culture were then identified. Results Primary B cells exogenously expressing Bcl2, Myc and Ccnd1 showed factor-independent growth ability, enhanced colony-forming capability and aggressive oncogenicity, unlike the cases observed with the expression of any combination of only two of the genes. We identified CCND3 and NRAS as cooperative genes with Bcl2 and Myc through the functional screening. Conclusions Bcl2, Myc and Ccnd1 or Bcl2, Myc and CCND3 synergistically transformed mouse primary B cells into aggressive malignant cells. Our new synergism-based method is useful for the identification of synergistic gene combinations in tumor development, and may expand our systemic understanding of a wide range of cancer-causing elements.
• Identification of FOXO3 and PRDM1 as tumor-suppressor gene candidates in NK-cell neoplasms by genomic and functional analyses

  Kennosuke Karube, Masao Nakagawa, Shinobu Tsuzuki, Ichiro Takeuchi, Keiichiro Honma, Yasuhiro Nakashima, Norio Shimizu, Young-Hyeh Ko, Yasuo Morishima, Koichi Ohshima, Shigeo Nakamura, Masao Seto

  BLOOD 118 (12) 3195 - 3204 0006-4971 2011/09 [Refereed][Not invited]
   

  Oligo-array comparative genomic hybridization (CGH) and gene-expression profiling of natural killer (NK)-cell neoplasms were used in an effort to delineate the molecular pathogenesis involved. Oligoarray CGH identified two 6q21 regions that were most frequently deleted (14 of 39 or 36%). One of these regions included POPDC3, PREP, PRDM1, ATG5, and AIM1, whereas the other included LACE1 and FOXO3. All genes located in these regions, except for POPDC3 and AIM1, were down-regulated in neoplastic samples, as determined by gene-expression analysis, and were therefore considered to be candidate tumor-suppressor genes. A20 and HACE1, the well-known tumor-suppressor genes located on 6q21-23, were included as candidate genes because they also demonstrated frequent genomic deletions and down-regulated expression. The Tet-Off NK cell line NKL was subsequently established for functional analyses. Seven candidate genes were transduced into Tet-Off NKL and forced re-expression was induced. Re-expression of FOXO3 and PRDM1 suppressed NKL proliferation, but this was not the case after re-expression of the other genes. This effect was confirmed using another NK cell line, SNK10. Furthermore, genomic analyses detected nonsense mutations of PRDM1 that led to functional inactivation in one cell line and one clinical sample. PRDM1 and FOXO3 are considered to play an important role in the pathogenesis of NK-cell neoplasms. (Blood. 2011;118(12):3195-3204)
• Clonal evolution of adult T-cell leukemia/lymphoma takes place in the lymph nodes

  Akira Umino, Masao Nakagawa, Atae Utsunomiya, Kunihiro Tsukasaki, Naoya Taira, Naoyuki Katayama, Masao Seto

  BLOOD 117 (20) 5473 - 5478 0006-4971 2011/05 [Refereed][Not invited]
   

  Adult T-cell leukemia/lymphoma (ATLL) is the neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). We performed oligo-array comparative genomic hybridization (CGH) against paired samples comprising peripheral blood (PB) and lymph node (LN) samples from 13 patients with acute ATLL. We found that the genome profiles of the PB frequently differed from those of the LN samples. The results showed that 9 of 13 cases investigated had a log2 ratio imbalance among chromosomes, and that chromosome imbalances were more frequent in LN samples. Detailed analysis revealed that the imbalances were likely caused by the presence of multiple subclones in the LN samples. Five of 13 cases showed homozygous loss regions in PB samples, which were not found in the LN samples, indicating that tumors in the PB were derived from LN subclones in most cases. Southern blot analysis of TCR gamma showed that these multiple subclones originated from a common clone. We concluded that in many ATLL cases, multiple subclones in the LNs originate from a common clone, and that a selected subclone among the LN subclones appears in the PB. (Blood. 2011; 117(20):5473-5478)
• Diversity of genome profiles in malignant lymphoma

  Masao Seto, Keiichiro Honma, Masao Nakagawa

  CANCER SCIENCE 101 (3) 573 - 578 1347-9032 2010/03 [Refereed][Not invited]
   

  Characteristic chromosome translocations are associated with specific disease entities, and are known to play a pivotal role in lymphoma development. Chromosome translocation alone, however, is not sufficient to produce tumors. Factors including the microenvironment and epigenetic and genetic alterations other than chromosome translocations have been shown to play a role in lymphoma development. Follicular lymphoma cells proliferate in close contact with follicular dendritic cells. Mucosa-associated lymphoid tissue (MALT) lymphoma cells proliferate at the marginal zone area of reactive follicles which are formed by preceding chronic inflammation. The importance of genetic alterations other than chromosome translocation has been recognized since the introduction of array comparative genomic hybridization (array CGH). Variations in the genomic profile among patients with the same disease entity have been found by array CGH analyses. These variations indicate that multiple genetic pathways leading to the development of lymphomas may exist and hence result in the variable clinicopathological features observed. (Cancer Sci 2010; 101: 573-578)
• TNFAIP3/A20 functions as a novel tumor suppressor gene in several subtypes of non-Hodgkin lymphomas

  Keiichiro Honma, Shinobu Tsuzuki, Masao Nakagawa, Hiroyuki Tagawa, Shigeo Nakamura, Yasuo Morishima, Masao Seto

  BLOOD 114 (12) 2467 - 2475 0006-4971 2009/09 [Refereed][Not invited]
   

  The constitutive activation of nuclear factor-kappa B (NF-kappa B) has been implicated in tumorigenesis of lymphoid malignancies. We have previously shown that chromosome 6q was frequently deleted in ocular marginal zone B-cell lymphoma and identified TNFAIP3/A20, a negative regulator of NF-kappa B pathways, as the primary target for 6q deletion. In the study reported here, we extended the analysis to other subsets of non-Hodgkin lymphomas and found that A20 is frequently deleted in mantle cell lymphoma and diffuse large B-cell lymphoma. Importantly, A20 promoter methylation or gene mutation is also frequently detected in these lymphomas, raising the possibility that inactivation of A20 may be involved in lymphomagenesis. To address this question, we conducted overexpression experiments in lymphoma cell lines with A20 deletion and down-regulated expression of A20 with an siRNA technique in Epstein-Barr virus-infected lymphoblastoid cell lines. These experiments found that overexpression of A20 induced apoptosis and silencing of A20 was associated with resistance to apoptosis and enhanced clonogenicity. The cells with down-regulated A20 exhibited enhanced NF-kappa B activities, which may account for the observed effects. These results indicate that our study provides a novel insight into molecular mechanisms leading to lymphoma and that specific targeting of NF-kappa B pathways may be advantageous for treatment. (Blood. 2009; 114: 2467-2475)
• Metric learning for DNA microarray data analysis

  Ichiro Takeuchi, Masao Nakagawa, Masao Seto

  INTERNATIONAL WORKSHOP ON STATISTICAL-MECHANICAL INFORMATICS 2009 (IW-SMI 2009) 197 1742-6588 2009 [Refereed][Not invited]
   

  In many microarray studies, gene set selection is an important preliminary step for subsequent main task such as tumor classification, cancer subtype identification, etc. In this paper, we investigate the possibility of using metric learning as an alternative to gene set selection. We develop a simple metric learning algorithm aiming to use it for microarray data analysis Exploiting a property of the algorithm, we introduce a novel approach for extending the metric learning to be adaptive. We apply the algorithm to previously studied microarray data on malignant lymphoma subtype identification.
• Array CGH analysis of PTCL-U reveals a distinct subgroup with genetic alterations similar to lymphoma-type ATLL.

  Nakagawa M, Nakagawa-Oshiro A, Karnan S, Tagawa H, Utsunomiya A, Nakamura S, Takeuchi I, Ohshima K, Seto M

  Clin Cancer Res 15 (1) 30 - 38 1557-3265 2009/01 [Refereed][Not invited]
   

  PURPOSE:Peripheral T-cell lymphoma, unspecified (PTCL-U) comprises histopathologically and clinically heterogeneous groups. The purpose of this study was to identify subgroups with distinct genetic, histopathologic, and prognostic features.
  EXPERIMENTAL DESIGN:
  We used array comparative genomic hybridization (CGH) for high-resolution analysis of 51 PTCL-U patients and the array data for examining possible correlations of histopathologic and clinical features. Moreover, we compared the genetic, histopathologic, and prognostic features of the PTCL-U cases with those of 59 cases of lymphoma-type adult T-cell leukemia/lymphoma (ATLL).
  RESULTS:We identified 32 regions with frequent genomic imbalance, 1 region with high copy number gain at 14q32.2, and 1 region with homozygous loss at 9p21.3. Gains of 7p and 7q and loss of 9p21.3 showed a significant association with poor prognosis. PTCL-U cases with genomic imbalance showed distinct histopathologic and prognostic features compared with such cases without alteration and a marked genetic, histopathologic, and prognostic resemblance to lymphoma-type ATLL.
  CONCLUSIONS:The array CGH enabled us to identify the frequently altered genomic regions with strong prognostic power among PTCL-U cases. A correlative analysis using the array CGH data disclosed a subgroup in PTCL-U with genomic alterations and with histopathologic and clinical relevance. In addition to histopathologic similarity, the strong genetic and prognostic resemblance between PTCL-U cases with genomic imbalance detected by array CGH and lymphoma-type ATLL seems to support the notion that the former may constitute a distinct PTCL-U subgroup.
• The potential of copy number gains and losses, detected by array-based comparative genomic hybridization, for computational differential diagnosis of B-cell lymphomas and genetic regions involved in lymphomagenesis

  Ichiro Takeuchi, Hiroyuki Tagawa, Akira Tsujikawa, Masao Nakagawa, Miyuki Katayama-Suguro, Ying Guo, Masao Seto

  Haematologica-The Hematology Journal 94 (1) 61 - 69 0390-6078 2009/01 [Refereed][Not invited]
   

  Background The differentiation of biologically and clinically different malignant lymphoma diseases or subtypes is crucial because it leads to better prognostication and therapeutic decision-making. Attempts have been made at subtype classification for diagnosing lymphomas on the basis of gene-expression profiling. Although array-based comparative genomic hybridization (array CGH) has identified a characteristic genomic alteration pattern for each disease entity, it has not been clear whether each patient with certain genomic alterations can be classified by array CGH data. Design and Methods Data on copy number gains and losses for 46 diffuse large B-cell lymphomas and 29 mantle cell lymphomas were used. The gene expressions of the diffuse large B-cell lymphomas cases were profiled and hierarchical clustering revealed that 28 of them were of the activated B-cell type and 18 were of the germinal center-B-cell type. Using these data, we developed a computer algorithm to classify lymphoma diseases or subtypes on the basis of copy number gains and losses. Results The method correctly classified 88% of the diffuse large B-cell lymphomas and mantle cell lymphomas, and 83% of the activated B-cell and germinal center-B-cell subtypes. These results demonstrate that copy number gains and losses detected by array CGH can be used for classifying lymphomas into biologically and clinically distinct diseases or subtypes. Conclusions Our computer algorithm based on array CGH data successfully classified diffuse large B-cell lymphomas and mantle cell lymphomas and activated B-cell and germinal center-B-cell subtypes with high accuracy. An important finding is that the regions automatically identified by the computer algorithm were located in the critical regions that are likely to be involved in the development of lymphoma.
• Array Comparative Genomic Hybridization Analysis of PTCL-U Reveals a Distinct Subgroup with Genetic Alterations Similar to Lymphoma-Type Adult T-Cell Leukemia/Lymphoma

  Masao Nakagawa, Aya Nakagawa-Oshiro, Sivasundaram Karnan, Hiroyuki Tagawa, Atae Utsunomiya, Shigeo Nakamura, Ichiro Takeuchi, Koichi Ohshima, Masao Seto

  CLINICAL CANCER RESEARCH 15 (1) 30 - 38 1078-0432 2009/01 [Refereed][Not invited]
   

  Purpose: Peripheral T-cell lymphoma, unspecified (PTCL-U) comprises histopathologically and clinically heterogeneous groups. The purpose of this study was to identify subgroups with distinct genetic, histopathologic, and prognostic features. Experimental Design: We used array comparative genomic hybridization (CGH) for high-resolution analysis of 51 PTCL-U patients and the array data for examining possible correlations of histopathologic and clinical features. Moreover, we compared the genetic, histopathologic, and prognostic features of the PTCL-U cases with those of 59 cases of lymphoma-type adult T-cell leukemia/lymphoma (ATLL). Results: We identified 32 regions with frequent genomic imbalance, 1 region with high copy number gain at 14q32.2, and 1 region with homozygous loss at 9p21.3. Gains of 7p and 7q and loss of 9p21.3 showed a significant association with poor prognosis. PTCL-U cases with genomic imbalance showed distinct histopathologic and prognostic features compared with such cases without alteration and a marked genetic, histopathologic, and prognostic resemblance to lymphoma-type ATLL. Conclusions: The array CGH enabled us to identify the frequently altered genomic regions with strong prognostic power among PTCL-U cases. A correlative analysis using the array CGH data disclosed a subgroup in PTCL-U with genomic alterations and with histopathologic and clinical relevance. In addition to histopathologic similarity, the strong genetic and prognostic resemblance between PTCL-U cases with genomic imbalance detected by array CGH and lymphoma-type ATLL seems to support the notion that the former may constitute a distinct PTCL-U subgroup.
• Graft-versus-tumor effect after reduced-intensity allogeneic hematopoietic stem cell transplantation in a patient with advanced colon cancer

  Satoshi Hashino, Sumiko Kobayashi, Mutsumi Takahata, Masahiro Onozawa, Masao Nakagawa, Takahito Kawamura, Fumie Fujisawa, Koh Izumiyama, Kaoru Kahata, Takeshi Kondo, Masahiro Asaka

  INTERNATIONAL JOURNAL OF CLINICAL ONCOLOGY 13 (2) 176 - 180 1341-9625 2008/04 [Refereed][Not invited]
   

  A 27-year-old man with advanced colon cancer that was resistant to conventional chemoradiotherapies was treated with reduced-intensity allogeneic peripheral blood stem cell transplantation (PBSCT). After obtaining complete donor-type chimerism, there was an apparent graft-versus-tumor effect accompanied by severe hepatic graft-versus-host disease (GVHD) showing hyperbilirubinemia, resulting in a stable disease condition that lasted for 18 months, which had not been seen previously in his previous disease history. The antitumor effect observed in this patient was insufficient for the patient to achieve complete remission, because the disease was at an already widespread and treatment-resistant stage. He finally died of hepatic failure due to extensive liver GVHD 65 months after the diagnosis of the advanced colon cancer and 29 months after the allogeneic PBSCT. Prospective studies are necessary to achieve better clinical results in patients with advanced colon cancer.
• [Array comparative genomic hybridilyzation analysis for unclassified peripheral T-cell lymphoma revealed possible subgrouping].

  Nakagawa M

  [Rinsho ketsueki] The Japanese journal of clinical hematology The Japanese Society of Hematology 49 (3) 165 - 170 0485-1439 2008/03 [Refereed][Not invited]
  
• Administration of micafungin as prophylactic antifungal therapy in patients undergoing allogeneic stem cell transplantation.

  Hashino S, Morita L, Takahata M, Onozawa M, Nakagawa M, Kawamura T, Fujisawa F, Kahata K, Izumiyama K, Yonezumi M, Chiba K, Kondo T, Asaka M

  International journal of hematology 1 87 (1) 91 - 97 0925-5710 2008/01 [Refereed][Not invited]
   

  Invasive fungal infection is one of the major causes of death in neutropenic patients undergoing allogeneic stem cell transplantation (SCT). Although prophylactic antifungal therapy with fluconazole (FLCZ) has become the standard care for these patients, there remains a need for more effective and cost-beneficial alternative drugs. We conducted a prospective study to evaluate the usefulness of the administration of micafungin (MCFG) as a prophylactic antifungal therapy for patients undergoing allogeneic SCT. The results were compared with previous data for patients who had received FLCZ. A total of 44 patients who underwent allogeneic SCT were enrolled in the study. Data from 29 patients who received allogeneic SCT using prophylactic FLCZ before this study were used as historical control data. Underlying diseases included acute leukemia (n = 16), non-Hodgkin's lymphoma (n = 11), myelodysplastic syndrome (n = 6), and others (n = 11) in the MCFG group and acute leukemia (n = 18), chronic myelogenous leukemia (n = 6), and others (n = 5) in the FLCZ group. The median durations of administration of MCFG and FLCZ were 36 and 34 days, respectively. Prophylactic success, defined as the absence of proven, probable, and possible invasive fungal infection (IFI) until the end of prophylactic therapy was achieved in 36 (87.8%) of the 41 evaluated patients in the MCFG group and in 65.5% of the patients in the FLCZ group (P = 0.038). No patients in the MCFG group showed proven or probable IFI, whereas proven or probable IFI was observed in three patients in the FLCZ group. Four patients in the MCFG group required dose escalation due to febrile neutropenia. Although one patient in the MCFG group required the discontinuation of MCFG due to allergic skin eruption (grade 2), none of the other patients in either group required dose reduction due to adverse effects. Although the study design was not a prospective randomized trial, our results indicate that the administration of MCFG at a daily dose of 100 mg is promising for prophylactic antifungal therapy in patients undergoing allogeneic SCT.
• TNFAIP3 is the target gene of chromosome band 6q23.3-q24.1 loss in ocular adnexal marginal zone B cell lymphoma

  Keiichiro Honma, Shinobu Tsuzuki, Masao Nakagawa, Sivasundaram Karnan, Yoshifusa Aizawa, Won Seog Kim, Yoon-Duk Kim, Young-Hyeh Ko, Masao Seto

  GENES CHROMOSOMES & CANCER 47 (1) 1 - 7 1045-2257 2008/01 [Refereed][Not invited]
   

  The genomic aberrations in extra nodal marginal zone B cell lymphoma vary according to their anatomical origin. This polarization is a reflection of the participation of different genes in the lymphomagenesis of marginal zone B cell lymphoma. We previously demonstrated by means of genome-wide array comparative genomic hybridization (CGH) that the genomic profile of ocular adnexal marginal zone B cell lymphoma is distinct from that of pulmonary or nodal marginal zone B cell lymphoma. The novel finding was a recurrent deletion of a 2.9-Mb region at chromosome band 6q23.3-q24.1, including homozygous loss, in ocular adnexal marginal zone B cell lymphoma. For a more detailed examination of the deletions of 6q23.3-24.1, we used contig bacterial artificial chromosome (BAC) array CGH, containing 24 BAC clones covering the 2.9-Mb region, to analyze nine cases with 6q23.3-q24.1 loss. We narrowed the minimal common region down to a length of 586 kb with two genes and four expressed sequence tags (ESTs). All of these genes and ESTs were subjected to RT-PCR and real-time quantitative RT-PCR. Correlation between genomic loss and expression level was found only for TNFAIP3, demonstrating that TNFAIP3 is a target gene of 6q deletion in ocular adnexal marginal zone B cell lymphoma. TNFAIP3 is an inhibitor of NF-kB signaling so that loss of this gene may play an important role in lymphomagenesis and suggests that TNFAIP3 may act as a tumor suppressor gene in ocular adnexal marginal zone B cell lymphoma. This article contains Supplementary Material available at http://www.interscience. wiley.com/jpages/1045-2257/suppmat. (c) 2007 Wiley-Liss, Inc.
• Chromosomal imbalances are associated with outcome of Helicobacter pylori eradication in t(11;18)(q21;q21) negative gastric mucosa-associated lymphoid tissue lymphomas

  Noriko Fukuhara, Tsuneya Nakamura, Masao Nakagawa, Hiroyuki Tagawa, Ichiro Takeuchi, Yasushi Yatabe, Yasuo Morishima, Shigeo Nakamura, Masao Seto

  GENES CHROMOSOMES & CANCER 46 (8) 784 - 790 1045-2257 2007/08 [Refereed][Not invited]
   

  Approximately 70% of gastric mucosa-associated lymphoid tissue (MALT) lymphomas can be successfully treated with H. pylori eradication. The translocation t(11;18)(q21;q21) characteristic of MALT lymphoma is recognized as a marker for H. pylori independency, but this marker is found in only a half of the MALT lymphomas resistant to H. pylori eradication. Detailed analyses of the genomic features of eradication resistant as well as responsive groups are important for understanding their molecular basis. We performed array-based comparative genomic hybridization (array-CGH) for 29 gastric MALT lymphomas treated with H. pylori eradication. These comprised ten cases of t(11; 18) positive MALT, nine cases of t(11; 18) negative MALT with H. pylori dependency, and ten cases of t(11;18) negative MALT with H. pylori independency. Array-CGH analysis demonstrated that no significant genetic alterations were found in t(11; 18) positive MALT lymphomas, but numerous genomic alterations were detected in t(11; 18) negative MALT lymphomas. Many of these alterations were similar to those found in diffuse large B-cell lymphoma with trisomy 3 being the most recurrent alteration. Within the t(11;18) negative MALT lymphoma without large cell components group, genomic imbalances occurred more frequently in the H. pylori independent than in the H. pylori dependent group (P = 0.02). Genomic imbalances are associated with H. pylori independency in t(11;18) negative gastric MALT lymphomas. They may thus play an important role in the development of H. pylori independency.
• Successful reduced-intensity stem cell transplantation with cord blood for a poor-prognosis adult with refractory chronic active Epstein-Barr virus infection

  Masao Nakagawa, Satoshi Hashino, Mutsumi Takahata, Takahito Kawamura, Fumie Fujisawa, Kaoru Kahata, Takeshi Kondo, Masahiro Imamura, Sachiko Ando, Masahiro Asaka

  INTERNATIONAL JOURNAL OF HEMATOLOGY 85 (5) 443 - 445 0925-5710 2007/06 [Refereed][Not invited]
   

  A 56-year-old woman with a poor-prognosis chronic active Epstein-Barr virus (CAEBV) infection underwent reduced-intensity stem cell transplantation (RIST) using cryopreserved cord blood (CB). Administration of EBV-seronegative CB cells following a reduced-intensity conditioning regimen was effective and well tolerated. Complete remission with no symptoms, low titers of EBV-related antibodies, and an undetectable level of EBV DNA in peripheral blood mononuclear cells continued for 16 months after RIST This report is the first of successful RIST with CB for an adult with CAEBV infection. The results also show that a graft-versus-CAEBV effect can be achieved in an allogeneic hematopoietic stem cell transplantation setting.
• Relationship between preexisting anti-varicella-zoster virus (VZV) antibody and clinical VZV reactivation in hematopoietic stem cell transplantation recipients

  Masahiro Onozawa, Satoshi Hashino, Mutsumi Takahata, Fumie Fujisawa, Takahito Kawamura, Masao Nakagawa, Kaoru Kahata, Takeshi Kondo, Shuichi Ota, Junji Tanaka, Masahiro Imamura, Masahiro Asaka

  JOURNAL OF CLINICAL MICROBIOLOGY 44 (12) 4441 - 4443 0095-1137 2006/12 [Refereed][Not invited]
   

  Reactivation of latent varicella-zoster virus (VZV), presenting as localized zoster or as disseminated infection, is a common and potentially serious complication in hematopoietic stem cell transplantation (HSCT) recipients. We retrospectively studied anti-VZV immunoglobulin G titers by the immune adherence hemagglutination method after HSCT and also studied VZV DNA by real-time PCR during clinical VZV reactivation using cryopreserved serum samples. No significant difference was found between anti-VZV titers in 13 patients with VZV infection (localized zoster in 11 patients and disseminated zoster in 2 patients) and in 13 subjects without VZV infection at each time point after HSCT. Preexisting anti-VZV titers of disseminated zoster cases tended to be lower than those of localized zoster cases (P = 0.10). Serum VZV DNA copy numbers at the onset of disseminated zoster cases tended to be higher than those of localized zoster cases (P = 0.09). A strong inverse correlation was found between preexisting anti-VZV titer and serum VZV DNA at onset (r = -0.90, P = 0.006). In HSCT recipients, preexisting antibody does not prevent the development of VZV reactivation but may contribute to decreased viral load at onset, resulting in a mild clinical course.
• MALT1 contains nuclear export signals and regulates cytoplasmic localization of BCL10

  M Nakagawa, Y Hosokawa, M Yonezumi, K Izumiyama, R Suzuki, S Tsuzuki, M Asaka, M Seto

  BLOOD 106 (13) 4210 - 4216 0006-4971 2005/12 [Refereed][Not invited]
   

  MALT1, BCL10 (B-cell lymphoma 10), and AP12 (apoptosis inhibitor 2)-MALT1 are key molecules in mucosa-associated lymphoid tissue (MALT) lymphomagenesis. We previously reported that MALT1 and AP12-MALT1 were localized only in cytoplasm, where we suggested that both molecules were likely to be active. In the study presented here, we further examined the localization-determining region by generating various mutants and were able to demonstrate that there were nuclear export signal (NES)-containing domains in the MALT1 C-terminal region. The use of leptomycin B, an NES-specific inhibitor, demonstrated that both MALT1 and AP12-MALT1 were predominantly retained in the nuclei, indicating that these molecules were shuttling between nucleus and cytoplasm in an NES-dependent manner. It was also found that MALT1 was involved in the nuclear export of BCL10, which is originally localized in both nucleus and cytoplasm. These results correlate well with the nuclear BCL10 expression pattern in both t(1;14) and t(11;18) MALT lymphomas. The nucleocytoplasmic shuttling of MALT1 and BCL10 complex may indicate that these molecules are involved not only in the nuclear factor kappa B (NF-kappa B) pathway but also in other biologic functions in lymphocytes.
• AP12-MALT1 fusion protein induces transcriptional activation of the API2 gene through NF-kappa B binding elements: Evidence for a positive feed-back loop pathway resulting in unremitting NF-kappa B activation

  Y Hosokawa, H Suzuki, M Nakagawa, TH Lee, M Seto

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 334 (1) 51 - 60 0006-291X 2005/08 [Refereed][Not invited]
   

  t(11;18)(q21;q21) is a characteristic as well as the most frequent chromosomal translocation in mucosa-associated lymphoid tissue (MALT) type lymphoma, and this translocation results in a fusion transcript, API2-MALT1. Although API2-MALT1 has been shown to enforce activation of NF-kappa B signaling, the transcriptional target genes of this fusion protein remains to be identified. Our analyses of the API2-MALT transfectants suggested that one of the target genes may be the apoptotic inhibitor API2 gene. Luciferase reporter assays with deletion and mutational constructs of the API2 promoter and electrophoretic mobility shift assays established that API2-MALT1 induces transcriptional activation of the API2 gene through two NF-kappa B binding elements. Moreover, supershift experiments indicated that these elements are recognized by the NF-kappa B p50/p65 heterodimer. Taken together, our results strongly indicated that API2-MALT1 possesses a novel mechanism of self-activation by up-regulating its own expression in t(11;18)(q21;q21)-carrying MALT lymphomas, highlighting a positive feedback-loop pathway resulting in unremitting NF-kappa B activation. (c) 2005 Elsevier Inc. All rights reserved.
• Stability and subcellular localization of API2-MALT1 chimeric protein involved in t(11;18) (q21;q21) MALT lymphoma

  K Izumiyama, M Nakagawa, M Yonezumi, Y Kasugai, R Suzuki, H Suzuki, S Tsuzuki, Y Hosokawa, M Asaka, M Seto

  ONCOGENE 22 (50) 8085 - 8092 0950-9232 2003/11 [Refereed][Not invited]
   

  t(11;18) (q21;q21) is a chromosomal aberration specific to low-grade mucosa-associated lymphoid tissue (MALT) lymphoma, and generates the chimeric product apoptosis inhibitor 2 (API2)-MALT1, which has been suggested to play an important role in MALT lymphomagenesis. However, little is known about the characteristics of API2, MALT1, and API2-MALT1 proteins. We therefore investigated the subcellular localization and stability of these products. API2 was localized in the nucleus and the cytoplasm, and MALT1 and API2-MALT1 in the cytoplasm only. Western blot analysis showed that the products of API2 and MALT1 were unstable, while the API2-MALT1 product was stable. The API2 deletion mutants at the end of the C-terminal and the MALT1 deletion mutants at the end of the N-terminal were stable compared with the full-length products. These results indicate that the domains responsible for protein instability are located in the end of the C-terminal of API2 and in that of the N-terminal of MALT1, and also that API2-MALT1 became stable because it lacks these domains. It has been suggested that NF-kappaB activation plays an important role in the tumorigenesis of MALT lymphoma. Our findings further suggest that the stabilized expression of API2-MALT1 products may continuously stimulate the NF-kappaB activating pathway, thus leading to MALT lymphomagenesis.

MISC

• Letermovir Is Effective for Prevention of Cytomegalovirus Reactivation in HLA-Haploidentical Peripheral Blood Stem Cell Transplantation with Post-Transplant Cyclophosphamide

  Takahide Ara, Yuta Hasegawa, Hiroyuki Ohigashi, Souichi Shiratori, Atsushi Yasumoto, Hideki Goto, Junichi Sugita, Masahiro Onozawa, Masao Nakagawa, Kaoru Kahata, Daigo Hashimoto, Takanori Teshima  BLOOD  138-  2021/11  

  0
• Fluid retention after allogeneic hematopoietic stem cell transplantation has a poor prognosis

  宮下直樹, 須藤啓斗, 日高大輔, 大東寛幸, 荒隆英, 白鳥聡一, 安本篤史, 後藤秀樹, 杉田純一, 小野澤真弘, 中川雅夫, 遠藤知之, 橋本大吾, 豊嶋崇徳  日本造血細胞移植学会総会プログラム・抄録集  43rd-  2021
• CRISPR screen identifies CD48 as a key molecule for evasion from NK cell surveillance in PTCL

  千葉雅尋, 下埜城嗣, 須藤啓斗, 石尾崇, 後藤秀樹, 橋野聡, 豊嶋崇徳, 中川雅夫  日本血液学会学術集会抄録(Web)  83rd-  2021
• 妊娠後期に診断された発作性夜間ヘモグロビン尿症に対してeculizumabを導入した1例

  小島 圭祐, 荒 隆英, 遠藤 知之, 高橋 承吾, 米田 和樹, 横山 翔大, 笠原 耕平, 白鳥 聡一, 後藤 秀樹, 中川 雅夫, 森川 守, 山本 準也, 豊嶋 崇徳  臨床血液  60-  (11)  1583  -1583  2019/11
• AMLにおけるWT1発現量と染色体・遺伝子異常の関連

  日高 大輔, 小野澤 真弘, 橋口 淳一, 宮下 直洋, 笠原 耕平, 藤澤 真一, 早瀬 英子, 岡田 耕平, 白鳥 聡一, 後藤 秀樹, 杉田 純一, 中川 雅夫, 加畑 馨, 橋本 大吾, 遠藤 知之, 山本 聡, 堤 豊, 長谷山 美仁, 永嶋 貴博, 盛 暁生, 太田 秀一, 酒井 基, 石原 敏道, 今井 陽俊, 宮城島 拓人, 柿木 康孝, 黒澤 光俊, 小林 一, 岩崎 博, 清水 力, 近藤 健, 豊嶋 崇徳  臨床血液  59-  (7)  964  -964  2018/07  [Not refereed][Not invited]
  
• ステロイド抵抗性急性移植片対宿主病に対するヒト間葉系幹細胞療法の後方視的解析

  白鳥 聡一, 早瀬 英子, 岡田 耕平, 後藤 秀樹, 杉田 純一, 小野澤 真弘, 中川 雅夫, 加畑 馨, 橋本 大吾, 遠藤 知之, 近藤 健, 豊嶋 崇徳  日本輸血細胞治療学会誌  64-  (2)  452  -452  2018/04  [Not refereed][Not invited]
  
• 造血幹細胞移植後に発症したトキソプラズマ症の2症例

  立野 貴大, 石尾 崇, 橋口 淳一, 小杉 瑞葉, 白鳥 聡一, 杉田 純一, 小野澤 真弘, 中川 雅夫, 藤本 勝也, 豊嶋 崇徳  臨床血液  58-  (11)  2302  -2302  2017/11  [Not refereed][Not invited]
  
• Frequent gain-of-function CCR4 mutations in adult T-cell leukemia/lymphoma (ATLL)

  Masao Nakagawa, Roland Schmitz, Wenming Xiao, Carolyn K. Goldman, Weihong Xu, Yandan Yang, Xin Yu, Thomas A. Waldmann, Louis M. Staudt  CANCER RESEARCH  75-  2015/08  [Not refereed][Not invited]
  
• A gain-of-function CCR4 mutations in adult T-cell leukemia/lymphoma (ATL) enhance the chemotactic abilities and P13K/AKT activation

  Masao Nakagawa, Roland Schmitz, Wenming Xiao, Yandan Yang, Thomas A. Waldmann, Louis M. Staudt  RETROVIROLOGY  12-  2015/08  [Not refereed][Not invited]
  
• A Gain-of-Function CCR4 Mutations in Adult T-Cell Leukemia/Lymphoma (ATLL) Enhance the Chemotactic Abilities and PI3K/AKT Activation

  Masao Nakagawa, Roland Schmitz, Wenming Xiao, Carolyn K. Goldman, Weihong Xu, Yandan Yang, Thomas A. Waldmann, Louis M. Staudt  BLOOD  124-  (21)  2014/12  [Not refereed][Not invited]
  
• IDENTIFICATION OF FOXO3 AND PRDM1 AS TUMOR SUPPRESSOR GENE CANDIDATES IN NK CELL NEOPLASMS BY THE COMBINATION OF GENOMIC AND FUNCTIONAL ANALYSES

  K. Karube, M. Nakagawa, S. Tsuzuki, Y. Ko, S. Nakamura, M. Seto  ANNALS OF ONCOLOGY  22-  100  -101  2011/06  [Not refereed][Not invited]
  
• Array Comparative Genomic Hybridization Revealed Polyclonality In Acute Type Adult T-Cell Leukemia/Lymphoma and PTCL NOS.

  Akira Umino, Masao Nakagawa, Atae Utsunomiya, Kunihiro Tsukasaki, Naoyuki Katayama, Masao Seto  BLOOD  116-  (21)  1277  -1277  2010/11  [Not refereed][Not invited]
  
• Comparison of ATLL and PTCL, NOS in terms of genomic imbalance by using array CGH analysis

  中川 雅夫, 中川-大城 綾, 瀬戸 加大  血液・腫瘍科  61-  (1)  45  -51  2010/07
• Cross-talk between JAK-STAT and NF-κB pathway in diffuse large B cell lymphoma

  本間 圭一郎, 中川 雅夫, 瀬戸 加大  血液・腫瘍科  58-  (2)  182  -188  2009/02
• Molecular pathogenesis of MALT lymphoma with t(11;18)

  中川 雅夫, 本間 圭一郎, 瀬戸 加大  血液・腫瘍科  58-  (1)  49  -56  2009/01
• Ocular adnexal marginal zone B cell lymphoma has characteristic deletion at chromosome band 6q23.3-24.1 whose target is TNFAIP3

  Keiichiro Honma, Shinobu Tsuzuki, Masao Nakagawa, Sivasundaram Kaman, Won-Seog Kim, Yoon-Duk Kim, Young-Hyeh Ko, Masao Seto  BLOOD  110-  (11)  777A  -778A  2007/11  [Not refereed][Not invited]
  
• 胃MALTリンパ腫のH.pylori除菌反応性とゲノム異常

  福原 規子, 中村 常哉, 田川 博之, 中川 雅夫, 谷田部 恭, 森島 泰雄, 中村 栄男, 瀬戸 加大  日本リンパ網内系学会会誌  47-  99  -99  2007/05  [Not refereed][Not invited]
  
• Array CGH analysis for PTCL-U revealed two genetically distinct subgroups.

  Masao Nakagawa, Aya Oshiro, Hiroyuki Tagawa, Sivasundaram Karnan, Shinobu Tsuzuki, Koichi Ohshima, Shigeo Nakamura, Masao Seto  BLOOD  108-  (11)  582A  -582A  2006/11  [Not refereed][Not invited]
  
• Molecular pathogenesis of MALT lymphoma: two signaling pathways underlying the antiapoptotic effect of API2-MALT1 fusion protein

  M Nakagawa, M Seto, Y Hosokawa  LEUKEMIA  20-  (6)  929  -936  2006/06  [Not refereed][Not invited]
   

  At least three recurrent chromosomal translocations, t(11;18)(q21;q21), t(1;14)(p22;q32), t(14;18)(q32;q21), involving the API2-MALT1 fusion protein, BCL10 and MALT1, have been implicated in the pathogenesis of mucosa-associated lymphoid tissue (MALT) lymphoma. Several lines of evidence indicated that both BCL10 and MALT1 are required for nuclear factor kappa B (NF-kappa B) activation by antigen receptor stimulation in lymphocytes, and API2-MALT1 can bypass this BCL10/MALT1 signaling pathway. Nuclear factor kappa B activation may contribute to antiapoptotic effect through NF-kappa B-mediated upregulation of apoptotic inhibitor genes. We recently demonstrated that API2-MALT1 can induce transactivation of the API2 gene through NF-kappa B activation, thus highlighting a positive feedback-loop mechanism of self-activation by upregulating its own expression in t(11;18) MALT lymphomas. We also demonstrated that API2-MALT1 possesses an antiapoptotic effect, in part, through its direct interaction with apoptotic regulators. These findings therefore led us to hypothesize that the antiapoptotic effect by API2-MALT1 may be mediated by its interaction with apoptotic regulators, on the one hand, and by NF-kappa B-mediated upregulation of apoptotic inhibitor genes on the other. We also found that BCL10 and MALT1 are shuttling between nucleus and cytoplasm, and that MALT1 can regulate the subcellular location of BCL10.
• MALT1 contains nuclear export signals and regulates cytoplasmic localization of BCL10

  M Nakagawa, Y Hosokawa, R Suzuki, S Tsuzuki, M Seto  ANNALS OF ONCOLOGY  16-  92  -93  2005/06  [Not refereed][Not invited]
  
• Requirement of Src kinases Lyn,Hck and Fgr for BCR-ABL 1-induced B-lymphoblastic leukemia but not chronic myeloid leukemia.Hu Y,et al.Nat Genet 2004;36:453-61.PMID:15098032--複数のフィラデルフィア染色体陽性疾患(Ph〔+〕)のなかでPh〔+〕B-ALLに特異的な造腫瘍機構の解明とその治療に対する臨床応用に足がかりをつける論文

  中川 雅夫, 鈴木 律朗  Mebio oncology  1-  (3)  86  -88  2004/11
• Subcellular localization of AP12, MALT1, and AP12-MALT1 chimeric proteins involved in t(11;18)(q21;q21) MALT lymphoma.

  M Nakagawa, K Izumiyama, M Yonezumi, Y Kasugai, R Suzuki, H Suzuki, S Tsuzuki, Y Hosokawa, M Seto  BLOOD  102-  (11)  182B  -182B  2003/11  [Not refereed][Not invited]
  

Research Projects

• Investigation of molecular mechanisms for constitutive IRF4 expression in T-cell malignancies

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2021/04 -2024/03 

  Author : 中川 雅夫

   

  目的１）成人T細胞性白血病リンパ腫におけるIRF4の恒常的高発現が維持される分子メカニズムの解明とその制御方法の開発 ・ CRISPRゲノムワイド遺伝子ノックアウトによるIRF4蛋白発現スクリーニング： 成人T細胞性白血病リンパ腫患者由来細胞株に対して19,114遺伝子を標的としたsgRNAライブラリーをレンチウイルスで感染させ、フローサイトメーターでIRF4低発現分画をソートする。ソート前と比較してソート後に増加した細胞でノックアウトされていた遺伝子を同定する計画で、この予備実験を終えた。 ・ 化合物ライブラリーによるIRF4蛋白発現スクリーニング: IRF4発現変化の検出系として、フローサイトメーターを用いたHigh throughput screening (HTS)の系を確立した。北大化合物ライブラリー（約3000個の既存薬を搭載）の中から、成人T細胞性白血病リンパ腫患者由来細胞株のIRF4発現低下をもたらす候補化合物を同定した。 ・ 機能的エンハンサー解析:成人T細胞性白血病リンパ腫細胞株と臨床検体のATACseqからIRF4遺伝子座周辺のopen chromatin領域の解析を進めた。 目的２）T細胞性腫瘍全体におけるIRF4恒常的発現異常の分布と頻度 ・ 組織マイクロアレイ作成：北日本血液研究会(http://www.njhsg.com)と連携し、T細胞性腫瘍の組織マイクロアレイを作成した。
• Identification of novel inhibitors for essential genes in cell proliferation and survival in T-cell lymphomas

  AMED：Division of Strategic Planning and Evaluation (The iD3 Booster)

  Date (from‐to) : 2021/10 -2023/09 

  Author : Masao Nakagawa
• CRISPR screening for identification of molecular targets of T/NK cell lymphomas

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2018/04 -2021/03 

  Author : Nakagawa Masao

   

  In this study, we performed genome-wide CRISPR-Cas9 screens in multiple T/NK lymphoma cell lines for identifying essential genes for cellular proliferation/survival in a series of T/NK cell lymphoma cell lines. Positive and negative control sgRNAs were correctly identified through the entire screening, confirming the validity of the screening results. We identified genes which were selectively essential for each diease group in T/NK cell lymphomas. Some among the essential genes were confirmed in the follow-up functional experiments. All together, we established an entire list of candidate genes for molecular targeted therapies in T/NK cell lymphomas.
• Grant-in-Aid for Scientific Research (C), Research Project Number:18K08313

  JSPS：

  Date (from‐to) : 2018 -2020 

  Author : Masao Nakagawa
• Identification of molecular biomarkers for chemotherapy in T-cell lymphomas

  Research Grant from Astellas Foundation for Research on Metabolic Disorders：研究助成金

  Date (from‐to) : 2019 

  Author : Masao Nakagawa
• Research Grant from Suhara Memorial Foundation

  Date (from‐to) : 2018 

  Author : Masao Nakagawa
• Project of Translational and Clinical Research Core Centers, Translational Research Network Program.

  AMED：

  Date (from‐to) : 2018 

  Author : Masao Nakagawa
• Project of Translational and Clinical Research Core Centers, Translational Research Network Program.

  AMED：

  Date (from‐to) : 2017 

  Author : Masao Nakagawa
• Research Grant from Takada Research Foundation.

  Date (from‐to) : 2017 

  Author : Masao Nakagawa
• Research Grant from SENSHIN Medical Research Foundation.

  Date (from‐to) : 2017 

  Author : Masao Nakagawa
• 新規スクリーニング法を用いた機能的T細胞リンパ腫がん遺伝子の網羅的探索

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2011 -2011 

  Author : 中川 雅夫

   

  ATLL(成人T細胞性リンパ腫白血病)リンパ腫型は、日本で最も多いT細胞性リンパ腫である。HTLV-1ウイルスの感染がその成立の前提条件ではあるが、それだけでは腫瘍化せず、何らかの付加的遺伝子異常が必須と考えられる。しかしながら、リンパ腫の病態形成に必須の遺伝子は未知である。 本研究はT細胞性リンパ腫形成に働く遺伝子を、機能的スクリーニングにより網羅的に検索することを目的とする。具体的には、ATLLリンパ腫型細胞株からレトロウイルスcDNAライブラリーを作製し、正常のT細胞にライブラリーを導入し、形質転換能、マウスへの移植による造腫瘍能を指標に、リンパ腫形成に必須の遺伝子を探索するものである。 本年度は、その手法を確立するために以下の研究を行い、一定の成果を収めた。 (1)マウスの胎児肝細胞から造血細胞を純化し、OP9-DL1ストローマ細胞と共培養することにより、T細胞をin vitroで分化・増殖させることに成功した。 (2)次に、この共培養のシステムにおいて、レトロウイルスを感染させることにより、T細胞に遺伝子を導入することに成功した。T細胞はCD3陰性からCD4陽性CD8陽性、さらにはCD4単独陽性と各分化段階の細胞が見られ、T細胞性リンパ腫の発生段階に対応する任意の細胞に遺伝子を発現させることが可能であった。 (3)そこで、この共培養を応用したレトロウイルスによるT細胞への遺伝子導入システムが、T細胞性リンパ腫の発がん研究に応用できるかどうかを、モデル系で検討した。この目的のために、T細胞に活性型RasやAkt,Myc,ERGなどの既知のがん原性遺伝子を導入し、T細胞の増殖動態を観察した。その結果、いずれの場合もT細胞の増殖が確認でき、特にRas,Mycで強い作用を観察した。 以上から、共培養を応用した本システムがT細胞性リンパ腫研究に応用可能であることが明らかとなった。
• Grant-in-Aid for Young Scientists (B), Research Project Number:23791095

  JSPS：

  Date (from‐to) : 2011 

  Author : Masao Nakagawa
• Identification of molecular basis of acute-and lymphoma-type adult T-cell leukemia/lymphoma

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2009 -2010 

  Author : NAKAGAWA Masao

   

  Acute-type adult T-cell leukemia/lymphoma (ATLL) cases frequently have lymph node lesions. I performed detailed genomic analysis and clarified; 1) a part of such cases had genomic alterations similar to those frequently found in lymphoma-type ATLL, 2) multiple subclones with various additional genomic alterations were frequently found in a lymph node of individual cases and a selected subclone among the lymph node subclones appeared in peripheral blood. These new findings indicate that a part of acute-type ATLL cases should be categorized as lymphoma-type ATLL with leukemic change.
• Grant-in-Aid for Young Scientists (B), Research Project Number:21790930

  JSPS：

  Date (from‐to) : 2009 -2010 

  Author : Masao Nakagawa
• Array CGH analytics and gene expression analytics revealed distinct subgroups in Peripheral T cell lymphomas

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2007 -2008 

  Author : NAKAGAWA Masao

   

  末梢性T細胞リンパ腫(PTCL)の中で、明確な疾患名に診断できない症例は末梢性T細胞リンパ腫・分類不能型(PTCL-U)として扱われる。PTCL-Uは未知の疾患が混在していると考えられるが、明確な報告はなかった。本研究ではPTCL-U51症例にアレイCGH法によるゲノム異常解析を行い、ゲノム異常様式・予後・病理像すべてに相関する二つの疾患群に分類できることをはじめて明らかにした。また二つのうち片方の疾患群は、別のPTCLである成人T細胞性白血病リンパ腫・リンパ腫型とよく似た均質な一群であることも明らかにした。
• Grant-in-Aid for Young Scientists (B), Research Project Number:19790678

  JSPS：

  Date (from‐to) : 2007 -2008 

  Author : Masao Nakagawa