Advanced Clinical Research Center

Division of Molecular Therapy分子療法分野

Professor Arinobu Tojo, M.D., D.M.Sc.Associate Professor Satoshi Takahashi, M.D., D.M.Sc.Project Associate Professor Hideaki Kagami, D.D.S., Ph.D.Assistant Professor Muneyoshi Futami, M.D., D.M.Sc.Assistant Professor Seiichiro Kobayashi M.D., D.M.Sc.

教 授 医学博士 東 條 有 伸准教授 医学博士 高 橋 聡特任准教授 医学博士 各 務 秀 明助 教 医学博士 二 見 宗 孔助 教 医学博士 小 林 誠一郎

The main theme of our research is toward the development of novel therapeuticoptions against intractable malignant disorders including leukemia, lymphoma andvarious cancers. For this purpose, we are making every effort to master themechanisms of normal and neoplastic stem cells on the basis of molecular andcellular biology as well as medical informatics. We also try to develop novel thera-pies in the field of regenerative medicine using bone marrow-derived mesenchy-mal stromal cells.(1) Molecular and cellular analysis of hematological malignancies:

Tumor-specific genetic alterations often result in dysregulation of transcriptionfactors and constitutive activation of tyrosine kinases, which appear to be theprimary cause of those tumors. We are studying the molecular and cellular as-pects of hematological malignancies as a model system. Furthermore, westarted clinical sequencing in tight collaboration with Human Genome Centerand Health Intelligence Center to establish a platform for precision medicine.

(2) Development of anti-cancer therapy using recombinant vaccinia virus:Vaccinia virus is a promising vector for oncolytic immunovirotherapy of cancer.For cancer specificity and safety, we introduced some genetic modificationsinto the viral genome by recombinant DNA technique. These include miRNA-regulated expression of B5R, an essential component for virus spreading, anddeletion of thymidine kinase. We are now trying to apply MDVV (miRNA-regu-lated and thymidine kinase-deleted vaccinia virus) to a preclinical model ofmultiple myeloma.

(3) Investigation of cancer stem cells and search for molecular targets for theirelimination:We are focusing on cancer, stem cells, and cancer stem cells. We aim to elu-cidate molecular mechanisms how growth factor signaling regulates tumori-genesis and maintenance of stem cells and cancer stem cells. Moreover, bytaking not only molecular biology but also new bioinformatics approaches, weaim to identify novel cancer biomarkers and molecular targets for cancer ther-apy. Our ultimate goal is to translate them into clinic.

(4) Clinical study of clonal evolution of HTLV-1-infected T cells into leukemia:Adult T-cell leukemia is a T cell malignancy which develops in HTLV-1 infectedindividuals after long latency period. HTLV-1 infected cells are regarded totransform through multi-step oncogenesis process. We are analyzing HTLV-1infected cells in different stages of transformation whose phenotypes such asCD7 and CADM1 expression vary in each stage by sorting them using flow cy-tometer. These analyses will provide useful information regarding molecular

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1. BRAF-V600E mutation in circulating cell-freeDNA is a promising biomarker of high-riskadult Langerhans cell histiocytosis.

Kobayashi M1, Ohno N2, Fukuyama T2, KawamataT2, Uchimaru K2, Tojo A1,2.: 1Division of MolecularTherapy, The Advanced Clinical Research Center,2Department of Hematology/Oncology, IMSUTHospital

Langerhans cell histiocytosis (LCH) and Erdheim-Chester disease (ECD) are rare clonal disorders of ahistiocyte/dendritic cell lineage, and oncogenicBRAF-V600E mutation could be detected in both le-sions from most patients. In patients with variouskinds of cancers, circulating cell-free DNA (cfDNA)contains cancer-derived genomic DNA and hasbeen applied to non-invasive diagnostic procedure.Then, we evaluated BRAF mutation in cfDNA as apotential biomarker of LCH/ECD using allele-spe-cific quantitative polymerase chain reaction (ASQ-PCR). cfDNA was prepared from plasma of 16adult LCH patients including 2 patients compli-cated with ECD, and was subjected to genotypingBRAF alleles by ASQ-PCR, which was specificallydesigned for detection of BRAF-V600E with a 3'-phosphate-modified oligonucleotide blocker. Mu-tant BRAF load was expressed as the percentage ofmutant alleles to total number of alleles. Five out of8 high-risk patients with active lesions were posi-tive for BRAF-V600E, and the mean ratio of mutantalleles to total alleles was 2.43％ (median, 2.01％).Eight pts with inactive lesions and 8 normal partici-pants were negative. Then, in a BRAF-V600E-posi-tive patient, we followed the mutant load duringthe course of initial chemotherapy and confirmedthat the mutant load correlated with the disease ac-tivity. Taken together, ASQ-PCR of BRAF-V600E incfDNA may contribute to planning of risk-basedtreatment as well as monitoring of treatment effi-cacy in LCH/ECD, especially in an active, high-riskgroup.

2. Impact of Ph＋ stem cell burden on clinicalfindings and nilotinib responses in de novoCML-CP: the results from the interim analysisof N-road, a multi-center phase II study.

Tsuda M1, Tojo A1.: 1Division of Molecular Ther-apy, The Advanced Clinical Research Center

A previous study showed that Ph＋ stem cellburden at diagnosis is a prognostic marker of mo-lecular responses (MR) in CML-CP on dasatinib orimatinib. Here, we examined whether this wouldapply to de novo CML-CP in a phase II clinicalstudy (N-road), in which first-line nilotinib is givenfor 24 M. By July 2014, 48 pts were enrolled andBM CD34＋ cell fractions could be evaluated byFACS-FISH analysis at diagnosis in 43 pts, amongthose 39 pts passed 3 M, 39 pts 6 M and 23 pts 12M, respectively. Absolute Ph＋ cell counts inCD34＋CD38－ fraction were estimated in each pa-tient by combining CD34 cell counts, proportion ofCD38－ fraction and percentage of Ph＋ cells. Ph＋CD34＋CD38－ cell counts significantly correlatedwith WBC and inversely with RBC, Hb and per-centage of lymphocytes. Between the 2 groups di-vided by the median cell counts, there were signifi-cant differences in RBC, Hb, Hct, and when dividedby the mean cell counts, there was a significant dif-ference in WBC. MR3.0 rate was 9/39 at 3M, 23/39 at6M and 14/23 at 12M. Although we could not findsignificant differences in MR rate at any checkpoints according to the initial Ph＋ stem cell bur-den, patients with lower number of Ph＋CD34＋CD38－ cells were likely to achieve MR3.0 fasterthan those with higher number of cells. IncreasedPh＋ stem cell burden apparently affects the levelof leukocytosis and anemia at diagnosis, but notMR3.0 rate by 12 M on nilotinib, although it is likelyto extend time to achieve MR3.0.

3. Efficacy and safety of doubly-regulated re-combinant vaccinia virus in a mouse xenograftmodel of multiple myeloma

Futami M1, Nakamura T2, Satoh K1, Tojo A1.: 1Di-vision of Molecular Therapy, The Advanced Clini-

mechanism to develop ATL.(5) Translational research on tissue engineering:

To accomplish this goal, we are focusing on the issues including a) investiga-tion on the environmental effect on tissue regeneration, b) search for mole-cules to affect the growth and differentiation of stem cells, c) clinical studies onbone tissue engineering, and d) development of fundamental technologies forthe widespread use of tissue-engineered products.

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cal Research Center, IMSUT, 2Institute of Regen-erative Medicine and Biofunction, Graduate schoolof Medical Science, Tottori University

Multiple myeloma is a malignancy of plasmacells of the bone marrow. Although the prognosis isvariable, no curative therapy has been defined. Vac-cinia virus is a member of the poxvirus family andis best known as the vaccine that eradicated small-pox in the 1970s. Vaccinia virus infects cancer cellsand kills such cells in a variety of ways. These in-clude direct infection, triggering of immunomedi-ated cell death, and vascular collapse. The potentialof the vaccinia virus as an anti-tumor therapy hasattracted the attention of oncologists. Interestingly,our preliminary experiments revealed that myelomacells were particularly susceptible to vaccinia virus.To exploit this susceptibility and to render vacciniamore myeloma-specific, we generated thymidinekinase-deleted microRNA (miRNA)-regulated vac-cinia viruses in which the essential viral gene B5Rwas regulated by miRNAs of normal human cells.Of the miRNAs examined, let-7a was found to bethe most reliable in terms of regulating viral trans-mission. Exposure to unregulated vaccinia viruskilled myeloma-transplanted SCID mice; the ani-mals succumbed to viral toxicity. In contrast, thethymidine kinase-deleted let7a-regulated virus re-mained localized within myeloma cells, triggeringtumor regression and improving overall survival.In conclusion, a thymidine kinase-deleted let-7a-regulated vaccinia virus was safe and effectivewhen used to treat myeloma. Such recombinantvaccinia viruses are therefore excellent novel candi-dates in the context of anti-myeloma therapy.

4. Advanced HTLV-1 carriers and early-stage in-dolent ATLs are indistinguishable based onthe CADM1 vs. CD7 plot in flow cytometry.

Kobayashi S1, Watanabe E2, Ishigaki T3, Ohno N4,Yuji K4, Nakano K5, Yamochi T5, Watanabe N2,Tojo A1,4, Watanabe T5, Uchimaru K4.: 1Division ofMolecular Therapy, The Advanced Clinical Re-search Center, 2Laboratory of Diagnostic Medi-cine, IMSUT Hospital, 3Division of Stem CellTherapy, Center for Stem Cell Biology and Regen-erative Medicine, 4Department of Hematology/On-cology, IMSUT Hospital, 5Graduate School ofFrontier Sciences, The University of Tokyo

In CD4(＋) cells from peripheral blood of in hu-man T-cell leukemia virus type 1 (HTLV-1)-infectedsubjects, CADM1(－) CD7(＋) (P), CADM1(＋) CD7(dim) (D) and CADM1(＋) CD7(－) (N) subpopula-tions are observed. The D and N subpopulations in-crease as asymptomatic HTLV-1 carriers (AC) pro-gress to indolent adult T-cell leukemia-lymphoma(ATL) and the N subpopulation then expands in

aggressive ATL. In the present study we examinedwhether the analysis can estimate the risk of devel-oping ATL in advanced AC. Peripheral blood sam-ples from AC (N＝41) and indolent ATL patients(N＝19) were analyzed by flow cytometry using theCADM1 versus CD7 plot for CD4(＋) cells and in-verse long PCR (clonality analysis) of FACS-sortedsubpopulations. Almost all AC with a high HTLV-1proviral load (>4 copies/100 cells) had a CADM1(＋) (D＋N) frequency of >10％. AC with 25％ <CADM1(＋) <―50％ contained expanded clones simi-lar to smoldering-type ATL. In many patients in the25％ < CADM1(＋) <―50％ group, the proportion ofabnormal lymphocytes was distributed around the5％ line, which divides AC and smoldering-typeATL in Shimoyama's classification. In conclusion,the CADM1 versus CD7 plot is useful for selectionof putative high-risk AC. The characteristics ofsome AC and smoldering ATL are said to be simi-lar; however, long-term follow up is required andthe clinical outcome (e.g. rate of transformation) ofthese cases should be used to determine whether toinclude them in the same clinical category.

5. Clinical sequencing of malignant as well asrare and undiagnosed hematological disor-ders

Yokoyama K1,7, Yokoyama N2, Kobayashi M3,Yamaguchi K4, Furukawa Y2,4, Yamaguchi R5,Imoto S6, Miyano S5, Tojo A1,3.: 1Department ofHematology/Oncology, IMSUT Hospital, 2Depart-ment of Applied Genomics, IMSUT Hospital, 3Di-vision of Molecular Therapy, Advanced ClinicalResearch Center, 4Division of Clinical Genome Re-search, Advanced Clinical Research Center, 5Labo-ratory of Genome Database, Human Genome Cen-ter, 6Division of Health Medical Data Science,Health Intelligence Center, 7Center for Gene andCell Thrapy

Sequensing of the genome or exome for clinicalapplications such as precise diagnosis and thera-peutic intervention, referred to as clinical sequenc-ing (CS), has now entered medical practice. We or-ganized IMSUT CS team in tight collaboration withHuman Genome Center and Health IntelligenceCenter, and have been establishing a bidirectional(bed to bench and bench to bed) system to integrateclinical and genomic information in hematologicaldisorders, especially in malignant as well as rareand undiagnosed diseases. We mainly performedtarget sequencing on a series of clinical samples byoperating next generation sequencers (NGS) withprimer sets from a disease-oriented and/or more ex-tended panel of genes, and intensively evaluatedthose CS results for identification of driver andcausative genes with the aid of artificial intelligence(AI), IBM-Watson. We have successfully delineated

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the pathogenesis in several tough cases whichcould not be rationally interpreted if CS is notavailable.

6. Maintenance of stemness of breast cancercells by FRS2beta, a feedback inhibitor forHER2-ERK pathway, during mammary tumori-genesis

Machida Y1, Kimura N1, Tojo A1, Gotoh N1,2.: 1Di-vision of Molecular Therapy, The Advanced Clini-cal Research Center, IMSUT, 2Division of CancerCell Biology, Cancer Research Institute of Ka-nazawa University

Inflammatory microenvironment contributes totumorigenesis. Although it is thought that breastcancer stem cells (CSCs) appear and grow in the in-flammatory microenvironment that is called CSCniche, it remains largely unclear how it occurs.Here, we uncovered FRS2beta, a feed-back inhibitorof ERK activity in progenitor cells, plays criticalroles for various cytokine production to increaseprogenitor cell- and CSC- state, and create the CSCniche. Expression levels of FRS2beta were increasedin mammary tumors of MMTV-ErbB2 mice than innormal mammary tissues. Deficiency of FRS2betagreatly delayed mammary tumorigenesis, decreasedmammosphere-forming ability and tumor stroma, acomponent of the CSC niche. Expression levels ofvarious cytokines, including IGF1 and CXCL12,stemness- and stroma-inducing cytokines, respec-tively, were reduced in FRS2beta-mutant mam-mospheres, pre-cancerous mammary and tumor tis-sues. Furthermore, expression levels of FRS2betawere higher in CD44highCD24low CSCs-enrichedpopulation in patient-derived breast cancer tissues.Thus the mechanisms to maintain progenitor cell-state by prevention of excess ERK activity may per-mit production of various cytokines to create CSCniche, and allow appearance and growth of CSCs.These results suggest that treatment with MEK in-hibitors may allow survival of CSCs, raising therisk of recurrence. We provide a rationale of combi-nation therapy targeting both FRS2 and MEK in-hibitors to eradicate tumors.

7. Semaphorin/MICAL3/CRMP2 regulates tumorinitiating activity in human breast cancerstem cells

Kana Tominaga1, Arinobu Tojo1, and Noriko Go-toh1,2: 1Division of Molecular Therapy, The Ad-vanced Clinical Research Center, IMSUT, 2Divi-sion of Cancer Cell Biology, Cancer Research in-stitute of Kanazawa University

The breast cancer stem cells (BCSCs) are thoughtbe a source of tumor cells of breast cancer tissues.

We previously examined heregulin /ErbB3/PI3K sig-naling by using DNA microarray and identifiedmany candidate molecules that potentially functionin cancer stem cells. Among them, we focused onMICAL3, a multidomain signal transduction pro-tein, since it is unknown whether MICAL3 func-tions in cancer stem cells. Knockdown of MICAL3by using shRNA reduced tumor sphere forming ef-ficiency in breast cancer cell lines and patient-de-rived primary breast cancer cells. We also foundthat tumor initiating activity was greatly reduced incells expressing shRNA for MICAL3 by using pa-tient-derived xenograft models (PDXs). Overexpres-sion of the MICAL3 mutant in the FAD-containingmonooxygenase domain reduced tumor sphereforming efficiency. Moreover, upon semaphorine3A stimulation, MICAL3-mediated generation ofH2O2 induced homodimer formation of CRMP2.Thus semaphorin/MICAL3/CRMP2 may play im-portant roles for BCSCs maintenance through gen-eration of H2O2 produced by MICAL3.

8. Clinical study on bone tissue engineering

Kagami H1,2, Inoue M1,2, Nagamura-Inoue T3,Chinzei M4, Yuji K5, Nobuhiro Ohno5, UchimaruK5, Tojo A1,5.: 1Division of Molecular Therapy, TheAdvanced Clinical Research Center, IMSUT, 2De-partment of Oral and Maxillofacial Surgery, Mat-sumoto Dental University, 3Department of CellProcessing and Blood Transfusion, IMSUT Hospi-tal, 4Department of Anesthesia and Surgical Cen-ter, IMSUT Hospital, 5Department of Hematology/Oncology, IMSUT Hospital

Atrophic maxilla or mandible are major obstaclesfor dental implant therapy. For example, severe pe-riodontitis, which is a leading cause of tooth loss inthe elderly, accompanied by significant bone ab-sorption, makes dental implant therapy very diffi-cult if not impossible to perform. Furthermore, al-veolar bone regeneration is also required to im-prove the functional and esthetic aspects of treat-ment outcome. Although use of dental implants isalready an established clinical procedure, there area large number of patients without adequate bonevolume for placement of dental implants. For pa-tients with severe atrophy of alveolar bone, autolo-gous bone grafts from iliac bone, tibial bone, ormandible have been performed. However, these de-structive procedures may not be feasible for all pa-tients. Even when the amount of harvested bone issmall, the procedure is inevitably accompanied byswelling and pain at the donor site. Althoughbioartificial bone substitutes have been frequentlyused, even with biological materials such as demin-eralized freeze-dried allografts or xenogeneic bonesubstitutes, the ability to induce bone regenerationis considered less efficient than native bone. Thus,

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the application is limited. We are carrying out aclinical study of alveolar bone tissue engineeringfor dental implant therapy using bone marrow stro-mal cells (BMSCs), with a goal of eventual commer-

cialization. The study has been approved by the in-stitutional committee and by the Minister of Health,Labour and Welfare of Japan. The clinical studywas sucdesfully carried out for 15 cases.

Publications

Murayama T, Nakaoku T, Nishimura T, TominagaK, Nakata A, Tojo A, Sugano S, Kohno T, GotohN. CD74-NRG1, a fusion gene product, leads toErbB-NFB-IGF2 autocrine/paracrine circuit andconfers cancer stem cell properties. Cancer Res. ac-cepted for publication 2015

Yamamoto H, Lu J, Oba S, Kawamata T, YoshimiA, Kurosaki N, Yokoyama K, Matsushita H,Kurokawa M, Tojo A, Ando K, Morishita K,Katagiri K, Kotani A. miR-133 regulates Evi1 ex-pression in AML cells as a potential therapeutictarget. Sci Rep. 6: 19204, 2016. doi: 10.1038/srep19204.

Kawamata T, Ogawa M, Takei T, Takeda R, Ochi K,Yokoyama K, Fukuyama T, Ohno N, UchimaruK, Tojo A. An adult case of biphenotypic acuteleukemia with t(6;14)(q25;q32). Atlas Gen CytogenOncol Hematol. accepted for publication 2015

Mukai T, Nagamura-Inoue T, Mori Y, Shimazu T,Takahashi A, Tsunoda H, Yamaguchi S, Tojo A.Neurosphere formation enhances the neurogenicdifferentiation potential and migratory ability ofumbilical cord-mesenchymal stromal cells. Cy-totherapy. 18(2): 229-41, 2016 doi: 10.1016/j.jcyt.2015. 10.012.

Hori A, Agata H, Takaoka M, Tojo A, Kagami H.Effect of cell seeding conditions on the efficiencyof in vivo bone formation. Int J Oral Maxillofac Im-plants. 31(1): 232-9, 2016 doi: 10.11607/jomi.4729.

Kamoi, K, Nagata Y, Mochizuki M, Uchimaru K,Tojo A, Ohno-Matsui K. Formation of segmentalrounded nodules during the infiltration of adultT-cell leukemia cells into ocular mucous mem-brane. Cornea. 35(1): 137-9, 2016. doi: 10.1097/ICO.0000000000000689.

Nakata A, Yoshida R, Yamaguchi R, Yamauchi M,Tamada Y, Fujita A, Shimamura T, Imoto S,Higuchi T, Nomura M, Kimura T, Nokihara H,Higashiyama K, Kondoh K, Nishihara H, Tojo A,Yano S, Miyano S, Gotoh N. Elevated -cateninpathway as a novel target for patients with resis-tance to EGF receptor targeting drugs. Sci Rep. 5:13076, 2015 doi: 10.1038/srep13076

He HP, Nagamura-Inoue T, Takahashi A, Mori Y,Yamamoto Y, Shimazu T, Tsunoda H, Tojo A.Immunosuppressive properties of Wharton'sJelly-derived mesenchymal stromal cells in vitro.Int J Hematol. 102(3) 368-78, 2015 doi: 10.1007/s12185-015-1844-7

Akiyama H, Kobayashi A, Ichimura M, Tone H,Nakatani M, Inoue M, Tojo A, Kagami H. Com-

parison of manual and automated cultures ofbone marrow stromal cells for bone tissue engi-neering. J Biosci Bioeng. 120(5): 570-6, 2015 doi:10.1016/j.jbiosc.2015.03.011

Kobayashi S, Watanabe E, Ishigaki T, Ohno N, YujiK, Nakano K, Yamochi T,Watanabe N, Tojo A,Watanabe T, Uchimaru K. Advanced HTLV-1carriers and early-stage indolent ATLs are indis-tinguishable based on the CADM1 vs. CD7 plotin flow cytometry. Cancer Sci. 106(5): 598-603,2015 doi: 10.1111/cas.12639

Konuma T, Kato S, Yuji K, Ohno N, Uchimaru K,Takahashi S, Tojo A. Clearance of blasts from pe-ripheral blood during induction chemotherapyusing exponential decay model predicts completeremission and long-term survival in adult acutemyeloid leukemia. Int J Lab Hematol. 37(3): e59-62, 2015 doi: 10.1111/ijlh.12302

Sato A, Nishida C, Sato-Kusubata K, Ishihara M,Tashiro Y, Gritli I, Shimazu H, Munakata S,Yagita H, Okumura K, Tsuda Y, Okada Y, TojoA, Nakauchi H, Takahashi S, Heissig B, HattoriK. Inhibition of plasmin attenuates murine acutegraft-versus-host disease mortality by suppress-ing the matrix metalloproteinase-9-dependent in-flammatory cytokine storm and effector cell traf-ficking. Leukemia. 29: 145-56, 2015. doi: 10.1038/leu.2014.151

Ishigaki T, Zaike Y, Nojima M, Kobayashi S, OhnoN, Uchimaru K, Tojo A, Nakauchi H, WatanabeN. Quantification of adult T-cell leukemia/lym-phoma cells using simple four-color flow cytome-try. Clin Chem Lab Med. 53(1): 85-93, 2015. doi:10.1515/cclm-2014-0183

Kawasaki T, Sumita Y, Egashira K, Ohba S, KagamiH, Tran SD, Asahina I. Transient exposure to hy-poxic and anoxic oxygen concentrations promoteseither osteogenic or ligamentogenic characteristicsof PDL cells. Biores Open Access . 4(1): 175-87, 2015doi: 10.1089/biores.2014.0049. eCollection 2015.

Zhang Y, Li X, Chihara T, Mizoguchi T, Hori A,Udagawa N, Nakamura H, Hasegawa H, TaguchiA, Shinohara A, Kagami H. Comparing immuno-competent and immunodeficient mice as animalmodels for bone tissue engineering. Oral Dis. 21(5): 583-92, 2015 doi: 10.1111/odi.12319. Epub2015 Mar 30.

Kagami H. The potential use of cell-based therapiesin the treatment of oral diseases. Oral Dis. 2015doi: 10.1111/odi.12320. [Epub ahead of print]

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1. Co-ordinate control of cell division and celldifferentiation of by the Rho family smallGTPases.

Kohtaro Nishimura, Takeshi Fukushima, Toshi-hiko Oki, Toshiyuki Kawashima, Yukinori Mi-noshima, Yosuke Tanaka, Susumu Goyama, andToshio Kitamura.

In search for key molecules that prevent murineM1 leukemic cells from undergoing IL-6-induceddifferentiation into macrophages, we isolated an an-tisense cDNA that encodes full-length mouseMgcRacGAP through functional cloning. In humanHL-60 leukemic cells, overexpression of the humanMgcRacGAP induced differentiation to macro-phage. Interestingly, MgcRacGAP localized to thenucleus in interphase, accumulated to the mitoticspindle in metaphase, and was condensed in themidbody during cytokinesis. Moreover, the GAPactivity of MgcRacGAP was required for comple-tion of cytokinesis. We also found that MgcRacGAPis phosphorylated by Aurora B at the midbody. In-triguingly, this phosphorylation induced the Rho-GAP activity of MgcRacGAP, which was critical forcompletion of cytokinesis. We identified S387 as a

phosphorylation site responsible for the acquire-ment of Rho-GAP activity during cytokinesis at themidbody. On the other hand, MgcRacGAP mainlylocalizes in the nucleus in the interphase. We dem-onstrated that MgcRacGAP directly bound tran-scription factors STAT3 and STAT5, and enhancedtranscriptional activation of STAT proteins as a RacGAP. MgcRacGAP was found to harbor functionalNLS and works as a nuclear chaperon togetherwith Rac1.We found using an MgcRacGAP-GFP fusion

protein that MgcRacGAP expression increased inthe early G1 phase in parallel with or even earlierthan Geminin, suggesting that MgcRacGAP mayplay roles in G1 check point. In addition, wehave recently identified that APCCDH1 targetsMgcRacGAP for destruction by ubiquitination. Insummary, our results indicate that MgcRacGAPplays distinct roles depending on the cell cyclethereby co-ordinating control of cell division anddetermination of cell fate, implicating multiple lev-els of regulation of MgcRacGAP including phos-phorylation and ubiquitination in distinct biologicalroles in different cell cycles. Now we plan to gener-ate a transgenic mouse expressing MgcRacGAP-GFP (or -mVenus) fusion protein in hematopoietic

Advanced Clinical Research Center

Division of Cellular Therapy細胞療法分野

Professor Toshio Kitamura, M.D., D.M.Sc.Associate Professor Susumu Goyama, M.D., D.M.Sc.Assistant Professor Tomofusa Fukuyama, M.D., D.M.Sc.Assistant Professor Yosuke Tanaka, M.D., D.M.Sc.

教 授 医学博士 北 村 俊 雄准教授 医学博士 合 山 進助 教 医学博士 福 山 朋 房助 教 医学博士 田 中 洋 介

Our major projects are (1) Co-ordinate control of cell division and differentiation bya crosstalk between JAK/STAT and small GTPases, (2) Molecular targeted thera-pies, and (3) Elucidation of molecular basis of leukemia, hematological malignan-cioes.

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stem cells and/or progenitors to examine the rela-tionship between cell division and cell fate.

2. Molecular targeting therapies using smallmolecule compounds

Akiho Tsuchiya, Reina Nagase, ToshiyukiKawashima, Yukinori Minoshima, SusumuGoyama, and Toshio Kitamura:

STAT3 is frequently activated in many cancersand leukemias, and is required for transformationof NIH3T3 cells. Therefore, we have started search-ing for STAT3 inhibitors. We established an effi-cient screening protocol for identification of STAT3inhibitors. Through the screening of a library ofsmall molecule compounds, we found the com-pounds RJSI-1 and RJSI-2 that inhibited STAT3 acti-vation. RJSI-2 also inhibited activation of STAT1,STAT5, JAK1 and JAK2. On the other hand, RJSI-1inhibited nuclear transport of phosphorylated STATproteins, implicating a novel mechanism in inhibit-ing STAT proteins. We have also shown that thesecompounds are effective in a tumor-burden mousemodel. In addition, we collaborated with a US-based biotech company in modification of RSJI-1for optimization to develop anti-cancer drugs, andhave developed JP1156 that kills the tumor cellsmore efficiently both in vitro and in vivo withmuch lower IC50.In addition to STAT3 inhibitors, we have recently

started a new project to develop STAT5 inhibitorsin collaboration with a pharmaceutical company. Tothis end, we have developed a screening method tosearch for STAT5 inhibitors. In addition to STAT3/5inhibitors, we have started several collaborationswith several domestic and global pharmaceuticalcompanies to evaluate the efficacies of a variety ofmolecular targeted therapies in our establishedmouse MDS/AML/MPN models.

3. Molecular basis of acute leukemia, myelodys-plastic syndromes (MDS), MDS overt leuke-mia, and myeloproliferative neoplasms (MPN).

Daichi Inoue, Reina Nagase, Makoto Saika, Take-shi Fujino, Yasutaka Hayashi, Shuji Asada, KojinC Kawabata, Naoko Watanabe, Yukiko Komeno,Naoko Kato, Yutaka Enomoto, Toshihiko Oki,Yuka Harada1, Hironori Harada1, Tetsuya No-saka2, Jiro Kitaura3, Yosuke Tanaka, TomofusaFukuyama, Susumu, Goyama, and Toshio Kita-mura: 1Department of Hematology/Oncology, Jun-tendo University, 2Mie University School of Medi-cine, and 3Allergy Center, Juntendo University.

Recent progress using high-speed sequencing hasidentified mutations in genes that are not catego-rized to class I and class II mutations, including

epigenetic factors, and splicing factors. We have re-cently established two MDS models induced byASXL1 mutations and EZH2 mutations; mice trans-planted with bone marrow cells expressing C-termi-nal truncating mutants of ASXL1 or EZH2 derivedfrom MDS patients developed MDS-like diseases ina year or two. Concerning the molecular mecha-nisms, the ASXL1 mutant (ASXL1-MT) suppressedPRC2 functions, leading to the derepression of pos-terior HoxA genes and miR125a via inhibition ofH3K27 trimethylation. While expression of poste-rior HoxAs is known to contribute transformationof hematopoietic cells, miR125a is a well-known on-cogenic micro RNA, in particular for hematologicalmalignancies. In addition to known target genes ofmiR125a, we have identified Clec5a/MDL1. Wehave also found that Clec5a is required for granulo-cytic differentiation of 32D cells, implicating itsdownregulation in the pathogenesis of MDS.ASXL1 mutations are frequently associated withSETBP1 mutations (SETBP1-MT) that stabilizeSETBP1 and SET oncoprotein, leading to activationof the PI3K/Akt pathway. In the BMT model, com-bination of ASXL1-MT and SETBP1-MT inducedAML with much shorter latencies. GSEA indicatedthat the TGF beta pathway was profoundly inhib-ited, implying the inhibition of the TGF beta path-way in leukemic transformation of MDS. Furtherexperiment is now under way to clarify the molecu-lar mechanisms by which the TGF beta pathwaywas inhibited.We have recently established Rosa26-knock-in

mice for ASXL1-MT and the EZH2 mutant, and arenow characterizing them. We have also started anew project on AML1-ETO where we have identi-fied a new isoform with stronger leukemogenic ac-tivities.

4. Molecular pathogenesis of AML1-ETO andMLL-fusion leukemias.

Susumu Goyama, Toshio Kitamura, Nicolas N.Nassar4, Joseph S. Palumbo4, James C. Mulloy4:4Cincinnati Children's Hospital Medical Center

Using human and mouse models for AML1-ETOand MLL-fusion leukemias, we have elucidatednew molecular aspects in pathogenesis and pro-gression of acute myeloid leukemia (AML). The t(8;21)-related AML1-ETO fusion protein is one of themost common genetic aberrations in AML. Clinicaldata suggest that CBL mutations are a frequentevent in t(8;21) AML, but the role of CBL in AML1-ETO-induced leukemia has not been elucidated. Wedemonstrate that CBL mutations collaborate withAML1-ETO to expand human CD34＋ cells both invitro and in a xenograft model. CBL depletion byshRNA also promotes the growth of AML1-ETOcells, demonstrating the inhibitory function of en-

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dogenous CBL in t(8;21) AML. Mechanistically, lossof CBL function confers hyper-responsiveness tothrombopoietin and enhances STAT5/AKT/ERK/Srcsignaling in AML1-ETO cells. Interestingly, wefound the protein tyrosine phosphatase UBASH3B/Sts-1, which is known to inhibit CBL function, isupregulated by AML1-ETO through transcriptionaland miR-9-mediated regulation. UBASH3B/Sts-1depletion induces an aberrant pattern of CBL phos-phorylation and impairs proliferation in AML1-ETO cells. The growth inhibition caused byUBASH3B/Sts-1 depletion can be rescued by ectopicexpression of CBL mutants, suggesting thatUBASH3B/Sts-1 supports the growth of AML1-ETOcells partly through modulation of CBL function.Thus, our study reveals a role of CBL in restrictingmyeloid proliferation of human AML1-ETO-in-duced leukemia, and identifies UBASH3B/Sts-1 as apotential target for pharmaceutical intervention.MLL-fusion leukemia is an aggressive form of

leukemia carrying chimeric fusion of the MLL gene.We previously showed that the combined loss ofRunx1/Cbfb inhibited the development of MLL-AF9-induced leukemia. However, c-kit＋/Gr-1－ cells re-mained viable in Runx1/Cbfb-deleted MLL-AF9

cells, indicating that suppressing RUNX activitymay not eradicate the most immature leukemiastem cells (LSCs). We found upregulation of severalhemostasis-related genes, including the thrombin-activatable receptor PAR-1, in Runx1/Cbfb-deletedMLL-AF9 cells. Similar to the effect of Runx1/Cbfb-deletion, PAR-1 overexpression induced CDKN1A/p21 expression and attenuated proliferation inMLL-AF9 cells. To our surprise, PAR-1-deficiencyalso prevented leukemia development induced by asmall number of MLL-AF9 LSCs in vivo. Re-expres-sion of PAR-1 in PAR-1-deficient cells combinedwith a limiting-dilution transplantation assay dem-onstrated the cell-dose dependent role of PAR-1 inMLL-AF9 leukemia: PAR-1 inhibits rapid leukemicproliferation when there are a large number ofLSCs, while a small number of LSCs need PAR-1for their efficient growth. Mechanistically, PAR-1increased adherance properties of MLL-AF9 cellsand promoted their engraftment to bone marrow.Together, these data revealed a multifaceted rolefor PAR-1 in leukemogenesis, and highlight this re-ceptor as a potential target to eradicate primitiveLSCs in MLL-fusion leukemia.

Publications

Inoue, D., Nishimura, K., Matsumoto, M., Nagase,R., Saika, M., Fujino, T., Nakayama, K-I. and Ki-tamura, T. (2016) Truncation mutants of ASXL1observed in myeloid malignancies are expressedat detectable protein levels. Exp. Hematol. 44:172-176.

Matsukawa, T., Izawa, K., Isobe, M., Takahashi, M.,Maehara, A., Yamanishi, Y., Kaitani, A., Oku-mura, K., Teshima, T., ＊Kitamura, T. and ＊Ki-taura, J. (2016) Ceramido-CD300f binding sup-presses experimental colitis by inhibiting ATP-mediated mast cell activation. Gut 65: 777-787.

Goyama, S., Schibler, J., Gasilina, A., Shrestha, M.,Lin, S., Link, KA., Chen, J., Whitman, SP., Bloom-field, C.D., Nicolet, D., Assi, S.A., Ptasinska, A.,Heidenreich, O., Bonifer, C., Kitamura, T., Nas-sar, N.N., Mulloy, J.C. (2016) UBASH3B/Sts-1-CBL axis regulates myeloid proliferation in hu-man preleukemia induced by AML1-ETO. Leuke-mia 30: 728-739

Kitamura, T., Watanabe-Okochi, N., Enomoto, Y.,Nakahara, F., Oki, T., Komeno, Y., Kato, N.,Doki, N., Uchida, T., Kagiyama, Y., Togami, K.,Kawabata, K.C., Nishimura, K., Hayashi, Y., Na-gase, R., Saika, M., Fukushima, T., Asada, S., Fu-jino, T., Izawa, Y., Horikawa, S., Fukuyama, T.,Tanaka, Y., Ono, R., Goyama, S., Nosaka, T., Ki-taura, J., and Inoue D. (2016) Novel working hy-pothesis for pathogenesis of hematological malig-nancies: combination of mutations-induced cellu-

lar phenotypes determines the disease (cMIP-DD). J. Biochem. 159: 17-25.

Inoue, D., Nishimura, K., Kozuka-Hata, H., Oyama,M. and Kitamura, T. (2015) The stability of epige-netic factor ASXL1 is regulated through ubiquiti-nation and USP7-mediated deubiquitination. Leu-kemia 29: 2257-2260.

Togami, K., Kitaura, J., Uchida, T., Inoue, D.,Nishimura, K., Kawabata, K.C., Nagase, R.,Horikawa, S., Izawa, K., Fukuyama, T., Naka-hara, F., Oki, T., Harada, Y., Harada, H.,Aburatani, H. and Kitamura, T. (2015) C-terminalmutant of C/EBP (C/EBP-Cm) down-regulatesM-CSF receptor which is a potent accelerator inthe progression of AML with C/EBP-CmB. Exp.Hematol. 43: 300-308.

Inoue, D., Kitaura, J., Matsui, H., Hou, H-A, Chou,W-C, Nagamachi, A., Kawabata, K.C., Togami,K., Nagase, R., Horikawa, S., Saika, M., Micol, J-P., Hayashi, Y., Harada, Y., Harada, H., Inaba, T.,Tien, H-F., Abdel-Wahab, O., and Kitamura, T.(2015) SETBP1 mutations drive leukemic transfor-mation in ASXL1-murtated MDS. Leukemia 29:847-857.

Miyadera, H., Ohashi, J., Lernmark, A., Kitamura,T., and Tokunaga, K. (2015) Autoimmune suscep-tible HLA alleles encode unstable proteins. J.Clin. Invest. 125: 275-291.

Goyama, S., Wunderlich, M., and Mulloy, J.C.(2015) Xenograft models for normal and malig-

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nant stem cells. Blood. 125: 2630-2640.Matsuo, H., Goyama, S., Kamikubo, Y., and Adachi,S. (2015) The subtype-specific features of EVI1and PRDM16 in acute myeloid leukemia. Haema-tologica. 100: e116-117.

Goyama, S., Huang, G., Kurokawa, M., and Mulloy,J.C. (2015) Posttranslational modifications ofRUNX1 as potential anticancer targets. Oncogene.34: 3483-3492.

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I. Clinical development of anti-programmeddeath 1 (PD-1) Ab in melanoma patients

Hideaki Tahara

Check-point blockades, which block the regula-tory pathways of CTL activation with antagonisticantibodies to promote immunological responses,have been shown to be effective for various typesof cancer in the clinical trials. Among them, anti-PD-1 antibodies have been particularly drawing at-tention of the oncologists.Nivolumab (ONO-4538/BMS-936558/MDX-1106)

is a fully human monoclonal IgG4 antibody(HuMAb) against PD-1 which has high affinity forPD-1 (Kd 2.6 nM) and block cross-linkage to bothPD-L1 (B7-H1) and L2. Based on good safety pro-files and promising anti-tumor effects in phase Itrial for recurrent solid tumor patients, we initiatedphase II study of nivolumab as a pharmaceutical-supported trial to treat melanoma patients in Japan.The results of such trial have shown the significantanti-tumor effects and manageable side-effects, andnivolumab has become the first government-ap-proved drug in the world as a PD1 related drug.We are now analyzing the immunological parame-ters to further develop this powerful agent.

II. Augmentation of immune checkpoint block-ade therapy with IL-18

Zhifeng Ma1,2, Hideaki Tahara, and Haruki Oka-mura1: 1Laboratory of Tumor Immunology andImmunotherapy, 2Department of Orthopaedic Sur-gery, Hyogo College of Medicine, Nishinomiya,Hyogo, 663-8501, Japan

Immune responses that lead to anti-tumor effectsrequire both activation of effector cells and reduc-tion of inhibitory elements of the immune system.Although animal models and recent clinical trialsdemonstrated that immune checkpoint blockade en-hanced effector cell responses and tumor rejection,further development and improvement of cancerimmunotherapy is necessary to achieve morefavorable objective responses. In this study, we ex-amined the effect of IL-18 on the treatment of peri-toneal dissemination of CT-26 mouse colon carci-noma cell line harnessing anti-programmed deathligand-1 (PD-L1) monoclonal antibody (mAb) (PD-L1) and/or anti-cytotoxic T lymphocyte-associatedantigen-4 (CTLA-4) mAb (CTLA-4). The treatmentof mice with PD-L1 and/or CTLA-4 significantlyenhanced their survival. However, when combinedwith IL-18, PD-L1 and/or CTLA-4 provided

Advanced Clinical Research Center

Division of Bioengineering臓器細胞工学分野

Professor Hideaki Tahara, M.D., Ph.D.Senior Assistant Professor Hiroaki Uchida, M.D., Ph.D.Assistant Professor Marimo Sato-Matsushita, Ph.D.

教 授 医学博士 田 原 秀 晃講 師 医学博士 内 田 宏 昭助 教 学術博士 松下（佐藤）まりも

Our division has been conducting basic research projects for development of inno-vative cancer therapy using immunologic and gene therapy approaches. The re-agents, modalities, and concepts developed in this division have been clinicallyapplied as translational research projects. We believe that bidirectional informationexchange between the bench and the bed side would be one of the most impor-tant requirements for the successful development of novel and effective therapies.

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much greater therapeutic benefits. IL-18 allowed ac-cumulation of precursor of mature natural killer(pre-mNK) cells in the peritoneal cavity, when in-jected with these check-point inhibitors. The resultsstrongly suggested that IL-18 promoted therapeuticeffects of immune checkpoint blockade against peri-toneal dissemination of carcinoma through accumu-lation of pre-mNK cells, memory-type CD8＋ Tcells, and deletion of CD4＋CD25＋ T cells. Ourstudy provides a basis for cancer immunotherapythat would involve combination of cytokines acti-vating innate immunity and immune checkpointblockade.

III. Development of cancer immunotherapy usingthe blockade of MFG-E8

Marimo Sato-Matsushita, Yu Mizote, Mika Ue-matsu-Hamada, Hideaki Tahara

The secreted protein, milk fat globule epidermalgrowth factor-8 (MFG-E8), stimulates disease pro-gression through coordinated v3 integrin signal-ing in tumor and host cells. MFG-E8 enhances tu-mor cell survival, invasion, and angiogenesis, andcontributes to local immune suppression.We have shown that systemic MFG-E8 blockade

cooperates with cytotoxic chemotherapy, molecu-larly targeted therapy, and radiation therapy to in-duce destruction of various types of establishedmouse tumors. The combination treatments evokeextensive tumor cell apoptosis that is coupled to ef-ficient dendritic cell cross-presentation of dying tu-mor cells. Our previous findings suggest that sys-temic MFG-E8 blockade might intensify the antitu-mor activities of existing therapeutic regimensthrough coordinated cell-autonomous and immune-mediated mechanisms also in human. In order toapply these findings to treat cancer patients, wehave developed antibodies specific to the humanMFG-E8. These antibodies include the one withblocking activity on MFG-E8 functions and the onesuitable for immune-staining of human tissue. Weare currently investigating the human situations re-lated to MFG-E8 and have found that strong ex-pression of MFG-E8 in the tumor cells has signifi-cant impact on the survival of certain types of can-cer patients (manuscript in preparation). Further-more, we are now in the process of developing thisagent for clinical application.

IV. Development of novel gene and cell therapyagainst cancer via T-cell immune checkpointblockade.

Marimo Sato-Matsushita, Yoshihiro Hayakawa,Hideo Yagita, Hideaki Tahara

We have reported for anti-tumor effects and

mechanisms of IL-23, which is a cytokine secretedby dendritic cells, and have been trying to developnovel and effective cancer immunotherapy. Re-cently, we have been focused on T-cell suppressingpathway of immune responses against cancer in-cluding CTLA-4, PD-1, and TIM-3. These immunecheckpoints have been blocked using antagonisticantibodies against them to enhance the anti-tumorimmune response of gene therapy using cytokineswith or without dendritic cell administration. Atthe same time, the mechanisms of such combinationtherapies have been investigated.

V. IL-17-producing NK1.1－ CD27－ T cells pro-mote tumor malignant progression by induc-ing inflammatory microenvironment.

Yoshihiro Hayakawa3, Yoshitaka Kimura4, Ma-rimo Sato-Matsushita, Hideaki Tahara: 3Institutenatural Medicine, University of Toyama, 4The Uni-versity of Tokyo

Inflammatory microenvironment is an essentialcomponent of tumors and important for carcino-genesis and metastasis of tumor cells, however, theprecise details of inflammatory immune responsesto promote tumor malignant progression are stillunclear. To characterize such tumor-promoting in-flammatory immune responses, we employ aunique in vivo model in which low tumorigeniccell line QR-32 acquires high malignant phenotypeafter exposure to host inflammatory responses in-duced by an inflammation initiator. By using thismodel, we investigated the role of inflammatory cy-tokines IL-17 and IFN in tumor malignant progres-sion process. We demonstrated that IL-17 and IFNplayed positive and negative roles, respectively, inthe malignant progression of tumor cells and IL-17played a predominant role in this process. Adop-tive transfer of inflammatory cells from wild-typemice into IL-17-deficient mice recovered in vivoprogression of QR-32 cells and the exact source ofIL-17 within such inflammatory cells was deter-mined as NK1.1－ CD27－ T cells. Furthermore,CD11b＋ Ly-6G＋ neutrophils infiltrated into the in-flammatory site primed by IL-17-producing NK1.1－CD27－ T cells in the presence of QR-32 and IL-17played an important role for maintaining such tu-mor-associated inflammatory microenvironment.Collectively, our data clearly implicate that the in-flammatory tumor microenvironment triggered byIL-17-producing NK1.1－ CD27－ T cells is impor-tant for tumor malignant progression. We are nowfurther characterizing T cells in the inflammatorymicroenvironment promoting tumor malignant pro-gression and exploring the components for down-stream inflammatory immune responses triggeredby IL-17.

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VI. Treatment of malignant pleural mesotheliomausing replication-defective recombinant ade-noviral vector expressing the suppressor ofcytokine signaling 3 (SOCS3). (Manufactureof the viral vector for preclinical studies innon-human primates)

Tetsuji Naka5, Hiroyuki Mizuguchi6, TakafumiNakamura7, Hisako Katano8, Hiroaki Uchida,Takuma Suzuki, Hideaki Tahara: 5Laboratory forImmune Signal, National Institute of BiomedicalInnovation, Osaka, Japan, 6Laboratory of Bio-chemistry and Molecular Biology, GraduateSchool of Pharmaceutical Sciences, Osaka Univer-sity, 7Tottori University, 8University of Tokyo

In collaboration with the research team, we haveprepared the replication-defective recombinant ade-noviral vector expressing the suppressor of cy-tokine signaling 3 (SOCS3), AdSOCS3 for treatmentof malignant pleural mesothelioma. We have sup-ported the vector production using Vector Facilityin IMSUT utilizing the master and working cellbanks of 293 cells, which we established previously.The purified final products have been used for pre-clinical study in monkey. We have also started tosupport the safety and biodistribution studies forAdSOCS3 in the context of intrapleural or intrave-nous administration in a mouse model. Based onthese results, we are now in the phase of preparingthe phase I study for the patients with malignantpleural mesothelioma using this strategy.

VII. Development of fully retargeted herpes sim-plex virus (HSV) vectors for oncolytic vi-rotherapy

Hiroaki Uchida, Yu Okubo, Tomoko Shibata,Takuma Suzuki, Hitomi Ikeda, Tomomi Tanaka,Tomoki Shiroyama, Hideaki Tahara

Herpes simplex virus (HSV) vectors are promis-ing agents for oncolytic virotherapy. Uchida estab-lished a fully retargeted HSV platform that medi-ates virus entry exclusively via tumor-associatedantigens in the lab of Prof. Joseph Glorioso at theUniversity of Pittsburgh. Entry of HSV is initiatedby the binding of glycoprotein D (gD) to one of itsreceptors, herpesvirus entry mediator (HVEM) ornectin-1. This interaction results in a conformationalchange in gD, triggering sequential activation of gHand gB to execute fusion between the viral enve-

lope and cell membranes. We inserted a number ofdifferent single-chain antibodies (scFv) into the re-targeted HSV platform that encodes a gD ablatedfor binding to natural receptors and a gB contain-ing entry-enhancing mutations we previously iden-tified. As a result, we observed specific virus entryinto cells expressing the cognate target antigen foreach of the retargeted constructs. Our results indi-cate the adaptability of our system to different tar-geting ligands, leading to a new generation ofbroadly applicable and effective oncolytic HSV vec-tors.

VIII. Establishment of highly functional mono-clonal antibodies through novel screeningmethods for targeted cancer therapy

Hiroaki Uchida, Miki Yamaguchi9, Hitomi Ikeda,Yu Okubo, Tomomi Tanaka, Hideaki Tahara:9Department of Molecular Medicine, Research In-stitute for Frontier Medicine, Sapporo MedicalUniversity School of Medicine

Monoclonal antibodies (mAbs) have become anestablished therapeutic modality in clinical oncol-ogy. In order to identify cell-surface molecules thatmay be useful for targeting various types of cancer,our group established a unique screening approachthat employs an adenoviral vector harboring fiberproteins engineered to bind antibodies, Adv-FZ33.This approach led to the successful identification ofan array of potential target molecules for cancertreatment. Immunotoxins (antibody-drug conju-gates; ADC) are a promising class of cancer thera-peutics composed of a cytotoxic agent linked cova-lently to a cancer-targeted antibody. To systemati-cally hunt for cell-surface molecules that may be ef-ficiently targeted by immunotoxins, our group cre-ated another method for screening highly func-tional cancer-targeted mAbs and cognate antigens.The receptor-binding domain of the Diphtheriatoxin (DT) was replaced with the antibody-bindingdomain (3C) derived from the Streptococcal proteinG. The resultant mutated toxin protein (DT-3C) wasused for selection of mAbs for specific cell killingactivity as components of immunotoxins. Our novelscreening system is advantageous in that the se-lected antibodies bind to intact cancer cells withhigh affinity and get internalized efficiently, whichhas been critically required for therapeutic applica-tions but elusive thus far.

Publications

1. Miyagawa, Y., Marino, P., Verlengia, G., Uchida,H., Goins, W.F., Yokota, S., Geller, D.A.,Yoshida, O., Mester, J., Cohen, J.B. and Glorioso,

J.C. Herpes simplex viral-vector design for effi-cient transduction of nonneuronal cells withoutcytotoxicity. Proc. Natl. Acad. Sci. U. S. A. 112:

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E1632-1641, 2015.2. Nishii, Y., Yamaguchi, M., Kimura, Y., Hase-

gawa, T., Aburatani, H., Uchida, H., Hirata, K.and Sakuma, Y. A newly developed anti-Mucin13 monoclonal antibody targets pancreatic ductaladenocarcinoma cells. Int. J. Oncol. 46: 1781-1787,2015.

3. Mazzacurati, L., Marzulli, M., Reinhart, B., Miya-gawa, Y., Uchida, H., Goins, W.F., Li, A., Kaur,B., Caligiuri, M., Cripe, T., Chiocca, N., Amanku-lor, N., Cohen, J.B., Glorioso, J.C. and Grandi, P.

Use of miRNA response sequences to block off-target replication and increase the safety of anunattenuated, glioblastoma-targeted oncolyticHSV. Mol. Ther. 23: 99-107, 2015.

4. Ma Z, Li W, Yoshiya S, Xu Y, Hata M, Eldarav-ish Y, Markova T, Yamanishi K, Yamanishi H,Tahara H, Tanaka Y, and Okamura H. Augmen-tation of immune checkpoint blockade therapywith IL-18. Clin. Can. Res. (in Press)

5. 内田宏昭，田原秀晃．大腸癌の遺伝子治療・ウイルス療法．日本臨床．１０７７：５６９―５７３，２０１５．

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1. Identification of novel molecular targets forthe treatment of human cancers

Kiyoshi Yamaguchi, Yoichi Furukawa, Paul Sheri-dan1, Rui Yamaguchi1, Seiya Imoto2, and SatoruMiyano1: 1Laboratory of DNA Information Analy-sis, Human Genome Center, 2Division of HealthMedical Data Science, Health Intelligence Center,IMSUT

Accumulated evidence has demonstrated that ab-errant epigenetic modifications are implicated incarcinogenesis. Bromodomain has been known as aprotein interaction module that recognizes ace-tylated lysine residues within histone and non-his-tone proteins. Through this interaction, protein con-taining a bromodomain(s) conducts the assembly ofnuclear factor complexes to their target sites onchromatin, resulting in the transcriptional activa-tion. We recently found that bromodomain contain-ing 8 (BRD8) was frequently accumulated in col-orectal cancer. Although BRD8 is a component inTRRAP/TIP60-histone acetyltransferase complex, itsbiological role and function are largely unknown.We are investigating the biological role of BRD8 incancer cells through integrative analysis of large-scale gene expression and ChIP-seq data. Since bro-

modomain-proteins have emerged as druggable tar-gets, small molecule inhibitor targeting the bromo-domain of BRD8 might be a novel therapeutic ap-proach for the treatment of colorectal cancer.

2. Cancer drug discovery through a large chemi-cal library screening

Kiyoshi Yamaguchi, Yoichi Furukawa

We recently developed a cell-based reporter as-say system to screen effectively and specificallycompounds and molecules that affect the transcrip-tional activity of the target molecule or signal trans-duction pathway. Applying this assay system, wecarried out a high-throughput screening of Wnt in-hibitors using a chemical library containing 20,000compounds, and have identified several candidatesmall molecules. We have started the studies oftheir mode of action, and structure-activity relation-ship in collaboration with a group in the Universityof Tokyo. Furthermore, we are going to expand ourscreening of chemical libraries with the help ofDrug Discovery Initiative in the University of To-kyo.

Advanced Clinical Research Center

Division of Clinical Genome Research臨床ゲノム腫瘍学分野

Professor Yoichi Furukawa M.D., Ph.D.Associate Professor Tsuneo Ikenoue M.D., Ph.D.Project Senior Assistant Professor Kiyoshi Yamaguchi Ph.D.

教 授 医学博士 古 川 洋 一准教授 医学博士 池 上 恒 雄特任講師 薬学博士 山 口 貴世志

We have been working on the following five projects, 1) development of noveltherapeutic strategies of human cancer, 2) development of anticancer drugsthrough a screening of chemical libraies, 3) establishment and investigation ofmouse models of human cancer, 4) elucidation of genetic characteristics of humantumors and mechanisms of their development, and 5) clinical sequence for the im-plementation of genomic medicine. These projects are aimed to develop strategiesfor better diagnosis, effective treatment, and prevention of human cancer.

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3. Establishment and investigation of novelmouse models of human cancer

Tsuneo Ikenoue and Yoichi Furukawa

Genetically engineered mice are useful tools forstudying human diseases, including cancer. In thisproject, we have successfully established a mousemodel of intrahepatic cholangiocarcinoma (ICC) byliver-specific Kras activation and Pten deletion. Toinvestigate the origin of the tumor cells in thismodel, we have taken advantage of lineage tracingsystem using tamoxifen-inducible Cre mice andRosa-GFP reporter mice. Furthermore, we have in-vestigated the molecular mechanisms how Kras ac-tivation and Pten deletion induce ICC in this mousemodel.We have also generated a mouse strain carrying

a conditional knockin allele of the Fbxw7 gene,which is frequently mutated in human colon andbile duct cancer. Using this strain, we are now try-ing to establish novel mouse models of these tu-mors. Intensive investigation of these modelsshould provide better understanding of their car-cinogenesis and facilitate the development of newtherapies to these cancers.

4. Elucidation of genetic characteristics of hu-man tumors and mechanisms of their devel-opment

Kiyoshi Yamaguchi, Tsuneo Ikenoue, Yoichi Fu-rukawa

We analyzed genetic alterations in Japanese ex-trahepatic biliary tract cancer (BTC) and pseudo-myxoma peritonei of the colon (PMP) using multi-plex PCR-based targeted enrichment and next-gene-ration sequencing (NGS).In the genetic analysis of BTC, we analyzed a to-

tal of 27 tumors and their matched non-canceroustissues, and identified frequent mutations in TP53(14/27), KRAS (6/27), PIK3CA (6/27), and SMAD4(6/27). Interestingly, the frequency of the PIK3CAmutation was higher compared with CaucasianBTC cases. This result may suggest that activationof the PI3K-AKT pathway in addition to the abro-gation of p53, SMAD4, and RAS-MAPK pathwaysmay play a crucial role in the carcinogenesis ofJapanese BTC.In the PMP study, we analyzed 18 PMPs contain-

ing 10 low-grade tumors (DPAMs) and 8 high-grade tumors (PMCAs). As a result, a total of 35 so-matic mutations were identified. Frequent muta-tions were identified in KRAS (14/18) and GNAS(8/18), but their frequencies were not significantlydifferent between DPAMs and PMCAs. On theother hand, TP53 mutations were found in PMCAs(3/8), but not in the DPAMs. PIK3CA and AKT1

mutations were also identified in two PMCAs, butnot in the DPAMs. These results suggest that KRASand/or GNAS mutations are common genetic fea-tures of PMP, and that mutations in TP53 and/orgenes related to the PI3K-AKT pathway may rendermalignant properties to PMP. These data may beuseful for the understanding of tumor characteris-tics, and facilitate the development of personalizedmedicine for PMP.

5. Clinical sequence for the implementation ofgenomic medicine

Kiyoshi Yamaguchi, Tsuneo Ikenoue, Yoichi Fu-rukawa, Mitsuhiro Komura1, Eigo Shimizu1, RuiYamaguchi1, Tetsuo Shibuya2, Satoru Miyano1,2,Takanori Hasegawa3, Seiya Imoto3, Masayuki Ko-bayashi4, Kazuaki Yokoyama4, Arinobu Tojyo4,Koichiro Yuji5: 1Laboratory of DNA InformationAnalysis, 2Laboratory of Sequence Analysis, Hu-man Genome Center, 3Division of Health MedicalData Science, Health Intelligence Center, 4Divisionof Molecular Therapy, 5Division of InternationalAdvanced Medical Research, Advanced ClinicalResearch Center

Next-generation sequencing (NGS) has enabledus to analyze the comprehensive human genome,and facilitated the identification of germlinechanges responsible for hereditary diseases and so-matic alterations in human neoplasms. In collabora-tion with Human Genome Center, Health Intelli-gence Center, and Advanced Clinical Research Cen-ter, we have been working on the following pro-jects; 1) the determination of germline mutations inpatients suspected of hereditary colon tumor, 2) ap-plication of a cognitive computing system, namelyIBM Watson Genomic Analytics (WGA), for thepersonalized medicine. These projects are aimed touse the information of personal genome and/or can-cer genome in clinic, and apply the data for theirdiagnosis and treatment.In the first project, we carried out three different

NGS, namely targeted sequencing, whole exome se-quencing, or whole genome sequencing for ten pa-tients with colonic polyposis. In the patients, wepreviously failed to identify pathological mutationswithin the 5′two-thirds region of the APC gene bySanger sequencing. However, NGS successfullyidentified pathological mutations in three of the tenpatients; two were mosaic mutations in APC , andthe other was a very rare mutation in the 3′termi-nal region of APC . These data have corroboratedthe usefulness of NGS in genetic diagnosis.In the second project, we generated a pipeline to

apply genomic data to IBM WGA. After written in-formed consent was obtained from the patientswith pseudomyxoma peritonei (PMP), they wereenrolled in this study. Genetic alterations in their

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tumor were determined by NGS and the data weresubsequently analyzed by WGA. The results ofWGA including predicted driver mutations andsuggestion of actionable drugs were discussed in

Tumor Board, which is composed of clinical se-quencing members. Evaluation of the results is nowongoing.

Publications

1. Yamaguchi, K., Komura, M., Yamaguchi, R.,Imoto, S., Shimizu, E., Kasuya, S., Shibuya, T.,Hatakeyama, S., Takahashi, N., Ikenoue, T.,Hata, K., Tsurita, G., Shinozaki, M., Suzuki, Y.,Sugano, S., Miyano, S. and Furukawa, Y. Detec-tion of APC mosaicism by next-generation se-quencing in an FAP patient. J. Hum. Genet. 60(5): 227-231, 2015.

2. Ikenoue, T., Yamaguchi, K., Komura, M., Imoto,S., Yamaguchi, R., Shimizu, E., Kasuya, S.,Shibuya, T., Hatakeyama, S., Miyano, S. andFurukawa, Y. Attenuated familial adenomatouspolyposis with desmoids by an APC mutation.Hum. Genome Variation. 2: 15011, 2015.

3. Iwaizumi, M., Tao, H., Yamaguchi, K., Yamada,H., Shinmura, K., Kahyo, T., Yamanaka, Y.,Kurachi, K., Sugimoto, K., Furukawa, Y. andSugimura, H. A novel APC mosaicism in a pa-tient with familial adenomatous polyposis.Hum Genome Variation. 2: 15057, 2015.

4. Kulak, O., Chen, H., Holohan, B., Wu, X., He,H., Borek, D., Otwinowski, Z., Yamaguchi, K.,Garofalo, L.A., Ma, Z., Wright, W., Chen, C.,Shay, J.W., Zhang, X. and Lum, L. Disruptionof Wnt/-catenin signaling and telomeric short-ening are inextricable consequences oftankyrase inhibition in human cells. Mol CellBiol. 35(14): 2425-2435, 2015.

5. Kulak, O., Yamaguchi, K. and Lum, L. Identifi-cation of therapeutic small-molecule leads incultured cells using multiplexed pathway re-porter readouts. Chemical Biology-Methods andProtocols. Methods Mol Biol. 1263: 3-14, 2015.

6. Suzuki, H., Hirata, Y., Suzuki, N., Ihara, S.,

Sakitani, K., Kobayashi, Y., Kinoshita, H., Hay-akawa, Y., Yamada, A., Watabe, H., Tateishi, K.,Ikenoue, T., Yamaji, Y. and Koike, K. Charac-terization of a new small bowel adenocarci-noma cell line and screening of anti-cancer drugagainst small bowel adenocarcinoma. Am J Pa-thol. 185(2): 550-562, 2015.

7. Sakitani, K., Hirata, Y., Hikiba, Y., Hayakawa,Y., Ihara, S., Suzuki, H., Suzuki, N., Serizawa,T., Kinoshita, H., Sakamoto, K., Nakagawa, H.,Tateishi, K., Maeda, S., Ikenoue, T., Kawazu, S.and Koike, K. Inhibition of autophagy exertsanti-colon cancer effects via apoptosis inducedby p53 activation and ER stress. BMC Cancer.15: 795, 2015.

8. Noguchi, R., Yano, H., Gohda, Y., Suda, R.,Igari, T., Ohta, Y., Yamashita, N., Yamaguchi,K., Terakado, Y., Ikenoue, T. and Furukawa, Y.Molecular profiles of high-grade and low-gradepseudomyxoma peritonei. Cancer Med. 2015 inpress.

9. Noguchi R, Yamaguchi K, Ikenoue T, TerakadoY, Ohta Y, Yamashita N, Kainuma O, Yokoi S,Maru Y, Nagase H, Furukawa Y. Genetic altera-tions in Japanese extrahepatic biliary tract can-cer. Oncology Letters, 2016 in press.

10. Mohri, D., Ijichi, H., Miyabayashi, K., Taka-hashi, R., Kudo, Y., Sasaki, T., Asaoka, Y.,Tanaka, Y., Ikenoue, T., Tateishi, K., Tada, M.,Isayama, H. and Koike K. A potent therapeuticsfor gallbladder cancer by combinatorial inhibi-tion of the MAPK and mTOR signaling net-works. J Gastroenterol, 2016 in press.

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Creation of novel recombinant oncolytic HSV-1

The use of genetically-engineered oncolytic vi-ruses is a novel therapeutic strategy for cancer.Various kinds of virus have been studied world-wide as oncolytic viruses, but genetically engi-neered HSV-1 is particularly useful because of fol-lowing favorable characteristics: (1) It shows littletoxicity to normal tissues, and there exist theoreticalbackgrounds for tumor cell selectivity. (2) The viralgenome is stable. (3) It can efficiently infect widerange of tumor types and exhibits a potent onco-lytic activity. (4) Cell-to-cell spread is minimally af-fected by circulating antiviral antibodies. (5) Inflam-matory reactions to the virus are generally mildand repeated administrations are possible. (6) Thereare antiviral drugs available to terminate viral rep-lication when undesired events occur. (7) Antitu-mor immune responses are elicited in the course ofoncolytic activities by the virus. (8) The large sizeof HSV-1 genome (～152kb) allows insertion oflarge or multiple foreign genes.Conventional homologous recombination tech-

niques had required time-consuming processes tocreate new recombinant oncolytic HSV-1. We haveestablished an innovative recombinant HSV-1 con-

struction system using bacterial artificial chromo-some and two sets of recombinases (Cre/loxP andFLP/FRT). This system allows rapid generation ofmultiple new recombinant HSV-1 with desired se-quences inserted into a specific locus.Application of oclolytic HSV-1 for malignant

glioma is a major study interest in our laboratory.In addition, in vitro and in vivo tumor models ofother cancers, including renal cancer, prostate can-cer, bladder cancer, malignant mesothelioma,tongue cancer, esophageal cancer, colon cancer,lung cancer, breast cancer, nasopharyngeal cancer,cholangiocarcinoma, malignant melanoma, and ma-lignant lymphoma have also been used for testingefficacy and safety.

Studies using glioma-derived cancer stem cells

There exists a small population of tumor-initiat-ing, stem-like cells within the tumor. Because can-cer stem-like cells (CSC) are reported to be resistantto current therapies and responsible for recurrence,a novel approach that can eliminate CSCs is neededto cure the disease. We currently use glioma-de-rived CSCs to study new therapeutic approachesincluding oncolytic virus therapy using genetically

Advanced Clinical Research Center

Division of Innovative Cancer Therapy先端がん治療分野

Professor Tomoki Todo, M.D., Ph.D.Associate Professor Yasushi Ino, M.D., Ph.D.Project Associate Professor Minoru Tanaka, M.D., Ph.D.Senior Assistant Professor Hiroyuki Momota, M.D., Ph.D.

教 授 医学博士 藤 堂 具 紀准教授 医学博士 稲 生 靖特任准教授 医学博士 田 中 実講 師 医学博士 百 田 洋 之

The major research topic of our laboratory is to develop oncolytic virus therapiesfor various malignant tumors. Oncolytic viruses are designed so that they can in-fect, replicate selectively within, and destroy tumor cells. G47, a recombinant, tri-ple-mutated oncolytic herpes simplex virus type 1 (HSV-1), exhibits potent anti-tu-mor efficacy while maintaining safety. Two clinical trials using G47 are currentlybeing conducted at IMSUT Hospital.

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engineered HSV-1. G47 has been shown to killCSCs efficiently. Novel oncolytic HSV-1 that exhibit

high efficacy for tumors rich in CSCs have beencreated and are being evaluated.

Publications

1. Takai H, Masuda K, Hiraoka K, Echizen K,Koyama-Nasu R, Nasu-Nishimura Y, Ogawa H,Kozuka-Hata H, Oyama M, Todo T, Ino Y, Mu-kasa A, Saito N, Toyoshima C, Shirahige K, Aki-yama T: 5-Hydroxymethylcytosine plays a criti-cal role in glioblastomagenesis by recruiting themethylosome. Cell Reports 9(1): 48-60, 2014(Epub 2014 Oct 2. doi: 10.1016/j.celrep.2014.08.071).

2. Tanaka S, Nakada M, Yamada D, Nakano I,Todo T, Ino Y, Hoshii T, Tadokoro Y, Ohta K,Ali MAE, Hayashi Y, Hamada JI, Hirao A.Strong therapeutic potential of -secretase inhibi-tor MRK003 for CD44-high and CD133-lowglioblastoma initiating cells. J Neurooncol 121(2):239-50, 2015 (doi: 10.1007/s11060-014-1630-z.Epub 2014 Oct 8).

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1. Assistance of Clinical Trials/TRs at ResearchHospital

Minako Kouno, Riyo Owada, Makiko Karasawa,Masanori Nojima, Fumitaka Nagamura

In Research Hospital, we work together withstaffs of Center for Translational Research. The as-sistance of Translational (Clinical) Research Coordi-nators is indispensable for the conduct of clinicaltrials, especially for TR. The activities of Coordina-tors are results of the collaboration between Divi-sion of Advanced Medicine Promotion and Centerfor Translational Research. In 2014, we supported 4investigator-initiated investigational new drug ap-plication (IND) clinical trials and 2 non-IND clinicalstudies.

2. Scholastic Program for the Graduate Stu-dents of Nurses in the Area of TranslationalResearch.

Minako Kouno, Riyo Owada,, Fumitaka Naga-mura

TR is the early phase of clinical trials, which ap-plied the developments of basic researches for pa-

tients with incurable and/or life-threatening dis-eases. Highly educated nurses are indispensable forthe conducts of TRs in terms of the protection ofparticipants in TRs and the conducts of scientifi-cally appropriate TRs. We developed the scholasticprogram for the graduate students of nurses in thearea of TR. We planned and implemented the one-week program to foster the expert research nurseaimed at the graduate students. It consists of thelectures on the feature points of TR (e.g. ethicalconsiderations of TR, and the role of researchnurse), role-plays of Institutional Review Board andobtaining Informed Consent, case conference, andthe experience of the actual operations. We evalu-ated the reports and the questionnaires from thestudents to explore the degree of their understand-ings and satisfactions for this program. These re-ports and questionnaires were analyzed. Generally,our program meets the demands of the students,however, the improvement of the content on theexperience of the actual operations is the next issue.

3. Management of "Translational Research Net-work Program" of Japan Agency for MedicalResearch and Development.

Makiko Karasawa, Hiroshi Yasui, Fumitaka Naga-

Advanced Clinical Research Center

Division of Advanced Medicine Promotion先端医療開発推進分野

Professor Fumitaka Nagamura, M.D., D.M.Sc 教 授 医学博士 長 村 文 孝

Division of Advanced Medicine Promotion was established in 2011. Our mission isto assist the clinical development and the conduct of clinical trials, especially fortranslational researches. For this purpose, it is critical to discover the new "seeds"and to eradicate many blockades until the clinical utilization. In this sense, our roleis the translation from the results of basic science of our Institute to the conduct ofclinical trials in the IMSUT Hospital. In IMSUT Hospital, we work together withstaffs of Center for Translational Research. Concurrently, for the reduction ofblockades during translational researches, we engage in research on RegulatoryScience.

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mura

Ministry of Education, Culture, Sports, Scienceand Technology launched "Translational ResearchNetwork Program" to promote translational re-searches based on the results of basic science at aca-demia. This grogram was transferred to JapanAgency for Medical Research and Development in

2015 and has been expected to support TRs frombasic science to seek obtaining intellectual propertyto early stage of clinical trial. In 2015, we supported17 basic researches, 15 preclinical studies, and 6clinical studies. The number of studies we assisthas been increasing year by year. Organization re-inforcement is the urgent problem.

Publications

1. 長村文孝 ウイルス療法のガイドライン・ガイダンスについて 実験医学 印刷中

2. Nagamura F. Collaboration between academia

for the development of translational research.Nihon Yakugaku Zasshi. 145: 211-15, 2015

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1. Fusion HBx form HBV integrant might affectER stress response and be associated withHCC

Ryosuke Muroyama, Kaku Goto, Yasuo Matsu-bara, Ryo Nakagawa, Sayuri Morimoto, SayakaIto, Naoya Kato

Backgrounds/Objectives: Hepatitis B virus (HBV)is a major risks factor associated with hepatocellu-lar carcinoma (HCC), and HBV integration hasbeen suggested to be associated with hepatocarcino-genesis. However, its molecular mechanisms re-mains unclear. In this study, we identified fusionHBx from HBV integrant in human hepatoma cellline, and investigated its role in hepatocarcinogene-sis.Methods: 1) We identified fusion HBx translated

from HBV integrant in Hep3B cells, which con-sisted of 3′-truncated HBx following 61 amino ac-ids from human sequences, and established stablyknocked-down (KD) cells against fusion HBx bysiRNA. 2) Using KD cells, we examined the effectof fusion HBx on cell growth, invasion ability andtumorigenicity in vivo. 3) We examined the expres-sion change of mRNAs in KD cells using microar-ray, and gene set enrichment analysis (GSEA) wasperformed to investigate the signature of fusion

HBx.Results: 1) We established KD cells in which fu-

sion HBx was disappeared by immunofluorescence.2) In KD cells, cell proliferation and invasion abilitywas reduced. In addition, KD cells could not de-velop any visible tumor in nude mice when we in-jected KD cells subcutaneously into nude mice. 3)We identified 305 up-regulated and 115 down-regu-lated genes in KD cells by more than two folds. InGSEA, up-regulated genes in KD cells were en-riched in endoplasmic reticulum (ER) stress re-sponse.Conclusions: Fusion HBx translated from HBV

integrant might affect ER stress response and playan important role in hepatocarcinogenesis

2. Small molecules for MICA regulation

Kaku Goto1 , Masahisa Jinushi2 , RyosukeMuroyama1, Wenwen Li1, Ryo Nakagawa1, SayakaIto1, Yasuo Matsubara1, Yasushi Tanoue1,3, ToshioFujisawa1,4, Naoya Kato: 1Division of AdvancedGenome Medicine, IMSUT; 2Institute for Ad-vanced Medical Research, Keio University Gradu-ate School of Medicine; 3Department of Gastroen-terology and Hepatology, JCHO Tokyo TakanawaHospital; 4Department of Gastroenterology, NTTMedical Center Tokyo

Advanced Clinical Research Center

Division of Advanced Genome Medicine先端ゲノム医学分野

Associate Professor Naoya Kato, M.D., Ph.D.Project Assistant Professor Ryosuke Muroyama, M.D., Ph.D.

准教授 医学博士 加 藤 直 也特任助教 医学博士 室 山 良 介

Our major goal is to establish personalized medicine for patients with infectiousdiseases or cancers, especially those of gastrointestinal and hepatic fields, basedon human or pathogenic microbe genome information.

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A natural killer (NK) group 2D (NKG2D) ligandMHC class I polypeptide-related sequence A(MICA) was identified to be a genetic susceptibilityfactor for hepatitis C virus (HCV)-induced hepato-cellular carcinoma (HCC) in our genome-wide asso-ciation study (Kumar V et al., Nat Genet 2011).Lower levels of MICA expression were associatedwith the elevated risk of HCC development in pa-tients, and preventive effects of MICA expressionon hepatocarcinogenesis were suggested. We there-fore aimed to find drugs for regulation of MICAexpression. Our new MICA promoter reporter sys-tem detected an anti-cancer agent as the top hit in ascreen for an FDA-approved drug library. The drugtreatment indeed upregulated the expression ofMICA in HCC cells, boosting NK cell-mediated cy-totoxicity in coculture and inhibiting tumor growthvia NK cells in vivo. The mode of MICA expressioninduction and further possibilities of pharmacologi-cal modulation of MICA are currently investigated.Findings are expected to lead to novel immuno-therapies for prevention and elimination of HCC(Goto K et al., J Gastroenterol 2015; Goto K et al.,Nippon Rinsho 2015).

3. The AMPK-related kinase SNARK in viralhepatitis and hepatocellular carcinoma

Kaku Goto1, Raymond T. Chung2, Naoya Kato1:1Division of Advanced Genome Medicine, IMSUT;2GI Unit, Massachusetts General Hospital, Har-vard Medical School

Our genome-wide RNAi screen for host cellularcofactors for hepatitis C virus (HCV) replication(Tai AW et al., Cell Host Microbe 2009) identifiedsucrose, non-fermenting 1/AMP-activated proteinkinase-related kinase (SNARK) to be a proviralgene. We then revealed that SNARK supportedHCV replication by its phosphorylation and phos-photransferase activity and conversely SNARK ex-pression level was upregulated by HCV infection inpatients and cell culture, interfering with intracellu-lar signalings. These effects were abrogated bySNARK kinase inhibitor, raising SNARK as an ef-fective target of therapies against the virus andpathogenesis (Goto K et al., J Hepatol 2013). Ourinvestigation into substrates, interactors, and signal-ings targeted by the kinase is underway, clarifyinginvolvement of SNARK in hepatitis virus patho-genesis and hepatocellular carcinoma developmentwith pharmacological regulation of the kinase activ-ity examined further.

4. Decreased miR-425 induced inflammatory cy-tokine production in CD4＋ T cells of primarybiliary cirrhosis via upregulating N-Ras ex-pression

Ryo Nakagawa1,2, Ryosuke Muroyama1, KazuhikoKoike2, Sayaka Ito1, Keiko Takano2, Kaku Goto1,Masahiro Nakano2, Chisato Saeki2, Yasuo Mat-subara1, Naoya Kato1, Mikio Zeniya2: 1Division ofAdvanced Genome Medicine, IMSUT; 2Depart-ment of Gastroenterology and Hepatology, TheJikei University School of Medicine

Background: Primary biliary cirrhosis (PBC) is anautoimmune liver disease of unknown pathogene-sis. Moreover, therapeutic targets for the autoim-munity of PBC are not yet found. Since CD4＋ Tcells are known to play a pivotal role in the immu-nological disorder of PBC, we analyzed microRNA(miRNA) and mRNA of CD4＋ T cells integratedlyto reveal its pathogenesis.Methods: Clinically and pathologically diagnosed

7 PBC patients and 7 healthy controls, who agreedto provide samples with written informed consent,were enrolled in this study. Then, we analyzed theexpression profile of miRNA and mRNA in CD4＋ Tcells of PBC patients and controls by microarrayand qRT-PCR using a gene set enrichment analysis(GSEA). The biological function of differentially ex-pressed and GSEA-enriched miRNAs was evalu-ated by miRNA overexpression assay, reporter as-say, and CD3 stimulation assay using culturedcells.Results: An integral miRNA-mRNA analysis re-

vealed 4 decreased miRNAs (miR-181a, -181b, -374b, -425) coordinately dysregulated T cell receptor(TCR) signaling pathway in CD4＋ T cells of PBC.Especially, N-Ras in the upper stream of TCR sig-naling pathway was targeted by 4 decreased miR-NAs. In vitro assays revealed miR-425 downregu-lated N-Ras expression and therefore suppressedproduction of inflammatory cytokines (IL-2, IL-10,and IFN-).Conclusion: Decreased miR-425 in CD4＋ T cells

upregulates N-Ras and induces the inflammationthrough dysregulation of TCR signaling pathway inPBC. The restoration of decreased miR-425 and/ordownregulation of N-Ras expression in CD4＋ Tcells could be novel therapeutic options for PBC.

5. Novel zinc finger protein in gastrointestinaltract

Yasuo Matsubara1,2, Kazuaki Takahashi2, Masa-hiro Arai2, Jun Miwa2, Shunji Mishiro2, RyosukeMuroyama1, Kaku Goto1, Ryo Nakagawa1, SayakaIto1, Sayuri Morimoto1, Naoya Kato1: 1Division ofAdvanced Genome Medicine, IMSUT; 2Depart-ment of Medical Science, Toshiba General Hospi-tal

The gastrointestinal tract has definite anatomicaland functional boundaries between its contiguoussegments. Because some human cancers arise in a

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background of tissue metaplasia, e.g. Barrett'sesophagus and intestinal metaplasia of the stomach,it is important to clarify the molecular and cellularbasis of region formation and preservation. Somegenetic markers that delimit gastrointestinalboundaries have been reported, but it is still un-known how such boundaries are established andmaintained.We identified ZNF-114-like hypothetical protein

in the gastric biopsy specimen by mass spectrome-try. Its mRNA sequence and other mRNAs withsimilar sequences were determined by RACE. Theexpression vector was constructed and transfectionwas performed. Apparent phenotype change wasnot revealed in transfected cultured cells of uppergastrointestinal tract. Knock-down studies will beexecuted for further functional analysis.

6. H. pylori in HIV-infected patients.

Yasuo Matsubara1,2, Ryosuke Muroyama1,2, KakuGoto1, Ryo Nakagawa1, Sayaka Ito1, Sayuri Mori-moto1, Naoya Kato1,2: 1Division of AdvancedGenome Medicine, IMSUT: 2Department of Ad-vanced Medical Science, IMSUT Hospital

The prevalence of H. pylori infection in the HIV-infected patients is to be elucidated. Some studiesshowed higher rates of infection in HIV-negativepatients than HIV-positive. Ones One of hypothesesis an appropriate amount of CD4＋ cells is neededfor colonization of H. pylori . We investigated H. py-lori in HIV-infected patients using biopsy speci-mens taken by upper gastrointestinal endoscopy.Rate of H. pylori infection diagnosed by light mi-croscopy was lower in HIV-positive subjects. Therewas not significant relation between blood CD4count and H. pylori infection.

7. HBx-binding compounds are potential targetfor treatment of HBV-induced HCC

Sayuri Morimoto, Sayaka Ito, Ryosuke Muro-yama, Ryo Nakagawa, Kaku Goto, Yasuo Ma-tsubara, Naoya Kato

Chronic Hepatitis B virus (HBV) infection is arisk factor for developing hepatocellular carcinoma(HCC). Hepatitis B virus X protein (HBx) plays animportant role in the development of HCC in HBV-infected patients. HBx has a variety of functionssuch as control of HBV replication, transcriptionaltrans-activating ability on cellular genes, and abilityto induce apoptosis of cells. a compound that bindsto HBx and modifies its ability to perform thesefunctions could be a potential drug for treatment ofHBV-induced HCC. Therefore, we constructed glu-tathione S-transferase (GST)-tagged HBx proteinsfor screening chemical compounds. To confirm that

the presence of GST does not affect the function ofHBx, GST-tagged HBx's trans-activating ability wascompared to that of HBx by luciferase assay inHeLa cells transfected with HBx or GST-HBx har-boring plasmids. GST-HBx showed a comparablelevel of trans-activation to that of HBx, so the fu-sion protein was amplified and purified for use inscreening. Compounds that bind to GST-HBx willbe further analyzed.

8. Detection of HCV NS5A L31/Y93 mutationsconferring treatment resistance

Sayaka Ito1, Kaku Goto1, Yasushi Tanoue1,2, Ryo-suke Muroyama1, Yasuo Matsubara1, Wenwen Li1,Ryo Nakagawa1, Toshio Fujisawa1,3, ShinshoYoshiba2, Naoya Kato1: 1Division of AdvancedGenome Medicine, The Institute of Medical Sci-ence, The University of Tokyo; 2Department ofGastroenterology and Hepatology, JCHO TokyoTakanawa Hospital; 3Department of Gastroen-terology, NTT Medical Center Tokyo

Background and aims: Recently interferon-freeregimen for HCV infection was attained by thecombination treatment with NS5A inhibitor andprotease inhibitor. However, amino acid mutationsconferring resistance to NS5A inhibitor were identi-fied in the N-terminal region of NS5A protein. Wehere focused on the resistance mutations at L31 andY93 of NS5A reported in genotype 1b HCV infec-tion, and examined their emergence in patients us-ing our newly established detection system. Meth-ods: Following the isolation of viral RNA from pa-tient serum and its reverse transcription, the regioncovering the mutations was amplified by nestedPCR. Subsequently L31/Y93 mutations were investi-gated by direct sequencing. Zero Blunt TOPO PCRCloning Kit was used for the construction of plas-mids encoding NS5A fragments with L31F or Y93H. Results: Reverse transcribed viral RNA from se-rum of HCV-infected patient was used for thenested PCR with primers designed based on HCVgenotype 1b genome sequences. Then we identifiedthe primer set with the highest efficiency and speci-ficity in amplification and subsequent direct se-quencing. The established method was capable ofsensing each mutation in the mixture of the plas-mids encoding L31F or Y93H, with the individualmutation content of 25％ or more and in a mixingratio-dependent manner. So far L31F/M and Y93Hwere observed in 5 (5％) and 12 (12％) out of 102cases, respectively, and both emerged in the twobreakthrough cases without L31/Y93 mutations be-fore the treatment with daclatasvir and asnaprevir.Conclusions: We newly generated a detection sys-tem of NS5A inhibitor resistance mutations in NS5A of HCV from patient serum. Our system is ex-pected to be conducive for designing treatment

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strategies to evade emergence of resistant virusesand achieve SVR.

9. Induction of MICA expression by anti-cancerdrugs for novel HCC immunotherapy

Yasushi Tanoue1,2, Kaku Goto1, Toshio Fujisawa1,3,Sayaka Ito1, Ryosuke Muroyama1, Ryo Nakagawa1,Yasuo Matsubara1, Naoya Kato1: 1Division of Ad-vanced Genome Medicine, The Institute of Medi-cal Science, The University of Tokyo; 2Departmentof Gastroenterology and Hepatology, JCHO TokyoTakanawa Hospital; 3Department of Gastroen-terology, NTT Medical Center Tokyo

Identification of an anti-tumor ligand MHC classI polypeptide-related sequence A (MICA) as a ge-netic susceptibility factor for HCV-induced hepato-cellular carcinoma (HCC) in our genome-wide asso-ciation study (Kumar V et al., Nat Genet 2011) indi-cated the hepatocarcinogenesis-preventive potentialof MICA expression. In our screen for an FDA-ap-proved drug library using a luciferase reporter sys-tem, an anti-cancer agent was found to stronglyupregulate MICA promoter activity, suggesting fur-ther potencies of related drugs. Currently theireffects on MICA biogenesis and anti-HCC activitiesof NK cell are examined, with novel HCC immuno-therapies in view.

10. IL-13R2 as a novel prognostic biomarkerfor human pancreatic cancer

Toshio Fujisawa1,2 Yasushi Tanoue1,3, Kaku Goto1,Sayaka Ito1, Ryosuke Muroyama1, Ryo Nakagawa1,Yasuo Matsubara1, Naoya Kato1: 1Division of Ad-vanced Genome Medicine, IMSUT; 2Departmentof Gastroenterology, NTT Medical Center Tokyo;3Department of Gastroenterology and Hepatology,JCHO Tokyo Takanawa Hospital

Interleukin-13 Receptor 2 (IL-13R2) is knownas a cancer testis antigen and a potential target forcancer immunotherapy. IL-13R2 is overexpressedin about 70％ pancreatic cancer samples and in-volved in cancer invasion and metastasis. We inves-tigated a possible correlation between IL-13R2 ex-pression in pancreatic cancer and patient prognosis.Totally, 236 samples of surgically resected pan-

creatic cancer tissue (adenocarcinoma) were ob-tained from two different institutions. Sampleswere immunohistochemically stained for IL-13R2expression and its intensity was evaluated by total4 investigators. The expression of IL-13R2 wascorrelated with patient characteristics includingprognosis and clinicopathological parameters.As a result, 63％-68％ of pancreatic cancer sam-

ples overexpressed IL-13R2. Kaplan-Meier sur-vival analysis revealed that patients with IL-13R2

positive pancreatic cancer had significantly lowersurvival compared to patients with IL-13R2 nega-tive cancer (P＜0.0057 and 0.0002). On multivariateanalysis, only IL-13R2 expression and UICC-stageswere identified as common prognostic factor atboth institutions. Interestingly, tumors that had sig-nificant nerve invasion frequently correlated withhigh IL-13R2 expression. IL-13R2 could be aprognostic biomarker of surgically resected pancre-atic cancer patients. Because IL-13R2 expression isrelated with invasion to nerve, immunotherapy tar-geting IL-13R2 might not only prolong patientsurvival but also suppress symptoms by nerve in-vasion including cancer pain.Samples used in this study were all Japanese.

Therefore, we will perform additional analysis us-ing TCGA data from US national cancer institute,and confirm the interracial universality of the re-sults in the next year.

11. Development of anti-metastasis treatmenttargeting IL-13R2 for pancreatic cancer

Toshio Fujisawa1,2 , Kaku Goto1 , RyosukeMukoyama1, Naoya Kato1: 1Division of AdvancedGenome Medicine, IMSUT; 2Department of Gas-troenterology, NTT Medical Center Tokyo

Pancreatic cancer is an aggressive disease withonly limited therapeutic options available. Inter-leukin-13 receptor 2 chain (IL-13R2), which is ahigh-affinity receptor for IL-13, is overexpressed ina variety of human solid cancers including pancre-atic cancer. We have previously reported that his-tone acetylation was a key mechanism to controlIL-13R2 expression in pancreatic cancer cells, andhistone deacetylation (HDAC) inhibitors upregu-lated IL-13R2 expression and enhanced the effi-cacy of immunotoxin targeting IL-13R2. On theother hand, IL-13R2 helps invasion and metastasisof pancreatic cancer through activation of extracel-lular signal-regulated kinase 1/2 and activator pro-tein-1 (AP-1) nuclear factors. We, thereupon, hy-pothesized that histone acetyltransferase (HAT) in-hibitors, which oppositely works from HDAC in-hibitors, and AP-1 inhibitors decrease IL-13R2 ex-pression and metastasis of pancreatic cancer. Lastyear, we examined IL-13R2 expression in pancre-atic cancer cell lines and picked up two cell lines,HS766T and MIA-PaCa2, because of their aggres-sive metastasis. And we took notice of three HATinhibitors for inhibiting IL-13R2 expression. Thisyear, we examined the efficacy of these drugs forinhibiting IL-13R2 expression in the pancreaticcancer cells. Epigallocatechin gallate showed thestrongest effect for IL-13R2 inhibition and it waspromising to prevent pancreatic cancer metastasis.Continuously, the possibility of the drugs for inhi-bition of cancer invasion and metastasis was exam-

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ined. All three drugs showed a certain measure ofinhibiting effects for cancer invasion in vitro. Aswell as IL-13R2 inhibition, Epigallocatechin gallate

most strongly prevent pancreatic cancer invasionand metastasis. For confirming these effects, we arenow planning animal study of pancreatic cancer.

Publications

Goto K, Kato N. MICA SNPs and the NKG2D sys-tem in virus-induced HCC. J Gastroenterol 2015;50(3): 261-72

Li W, Goto K, Matsubara Y, Ito S, Murayama R, LiQ, Kato N. The characteristic changes in hepatitisB virus X region for hepatocellular carcinoma: Acomprehensive analysis based on global data.

PLoS One 2015; 10(5): e0125555Chen J, Jin Y, Wang H, Wei S, Chen D, Ying L,Zhou Q, Li G, Li J, Gao J, Kato N, Hu W, Li Y,Wang Y. A haplotype of two novel polymor-phisms in -Sarcoglycan gene increases risk of di-lated cardiomyopathy in mongoloid population.PLoS One 2015; 10(12): e0145602

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1. Immuno-gene therapy with CD19-directedCAR-modified T cells (CD19-CAR-T cells) foradult patients with relapsed and refractory B-precursor acute lymphoblastic leukemia (B-ALL)

Sumimasa Nagai, and Keiya Ozawa

It has been reported that CD19-CAR-T gene ther-apy is highly effective for relapsed and refractory B

cell malignancies, especially B-ALL. In order to de-velop this novel promising gene therapy in Japan,we are currently preparing Japanese multicenterclinical trial of CD19-CAR-T cell therapy for adultpatients with relapsed and refractory B-ALL. CD19-CAR-T gene therapy for malignant B-cell lym-phoma was conducted in one patient at Jichi Medi-cal University Hospital as Phase I/II clinical re-search.

Publications

1. Nagai, S., and Ozawa, K.: Clinical trial designsto obtain marketing authorization of drugs forhaematologic malignancy in Japan, the EU andthe US. Br. J. Haematol. [Epub ahead of print]

2. Nagai, S., Urata, M., Sato, H., Mikami, M.,Kuga, W., Yanagihara, R., Miyamoto, D.,Suzuki, Y., and Shikano, M.: Evolving Japaneseregulations on companion diagnostics. Nat.Biotechnol. 34(2): 141-144, 2016.

3. Muroi, K. , Miyamura, K. , Okada, M. ,Yamashita, T., Murata, M., Ishikawa, T., Uike,N., Hidaka, M., Kobayashi, R., Imamura, M.,Tanaka, J., Ohashi, K., Taniguchi, S., Ikeda, T.,

Eto, T., Mori, M., Yamaoka, M., and Ozawa, K.:Bone marrow-derived mesenchymal stem cells(JR-031) for steroid-refractory grade III or IVacute graft-versus-host disease: a phase II/IIIstudy. Int. J. Hematol. [Epub ahead of print]

4. Nakata, M., Yamamoto, S., Okada, T., Daram-bazar, G., Okano, H., Ozawa, K., and Yada, T.:IL-10 gene transfer upregulates arcuate POMCand ameliorates hyperphagia, obesity and dia-betes by substituting for leptin. Int. J. Obes.(Lond). [Epub ahead of print]

5. Kasahara, Y., Tateishi, Y., Hiraoka, Y., Otsuka,A., Mizukami, H., Ozawa, K., Sato, K., Hidema,

Advanced Clinical Research Center

Division of Genetic Therapeutics遺伝子治療開発分野

Professor Keiya Ozawa, M.D., D.M.Sc.Project Professor Shinichi Muramatsu, M.D., Ph.D.Senior Assistant Professor Sumimasa Nagai, M.D., Ph.D.

教 授 医学博士 小 澤 敬 也特任教授（兼務，非常勤） 医学博士 村 松 慎 一講 師 医学博士 永 井 純 正

The main project of our division is to promote clinical development of novel genetherapy for cancer and chronic intractable diseases. We are currently engaged inclinical development of immuno-gene therapy with chimeric antigen receptor(CAR)-modified T cells for relapsed and refractory hematological malignancies

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S., and Nishimor, K.: Role of the oxytocin re-ceptor expressed in the rostral medullary raphein thermoregulation during cold conditions.Front. Endocrinol. (Lausanne). 6: 180. 2016.

6. Maeda, H., Fukuda, S., Kameda, H., Murabe,N., Isoo, N., Mizukami, H., Ozawa, K., andSakurai, M.: Corticospinal axons make directsynaptic connections with spinal motoneuronsinnervating forearm muscles early during post-natal development in the rat. J. Physiol. 594(1):189-205, 2016.

7. Kasahara, T., Takata, A., Kato, T.M., Kubota-Sakashita, M., Sawada, T., Kakita, A., Mizu-kami, H., Kaneda, D., Ozawa, K., and Kato, T.:Depression-like episodes in mice harboringmtDNA deletions in paraventricular thalamus.Mol. Psychiatry. 21(1): 39-48, 2016.

8. Nagai, S., Yanagihara, R., and Kishioka, Y.Japanese regulatory authority's perspective onbiosimilars. Lancet Oncol. 16: e101, 2015.

9. Sadakane, O., Masamizu, Y., Watakabe, A.,Terada, S., Ohtsuka, M., Takaji, M., Mizukami,H., Ozawa, K., Kawasaki, H., Matsuzaki, M.,and Yamamori, T.: Long-term two-photon cal-cium imaging of neuronal populations withsubcellular resolution in adult non-human pri-mates. Cell Rep. 13(9): 1989-1999, 2015.

10. Sadakane, O., Watakabe, A., Ohtsuka, M.,Takaji, M., Sasaki, T., Kasai, M., Isa, T., Kato,G., Nabekura, J., Mizukami, H., Ozawa, K.,Kawasaki, H., and Yamamori, T.: In vivo two-photon imaging of dendritic spines in marmo-set neocortex (1,2,3). eNeuro. 2(4): 0019-15, 2015.

11. Onishi, A., Akimoto, T., Urabe, M., Hirahara, I.,Muto, S., Ozawa, K., Nagata, D., and Kusano,E.: Attenuation of methylglyoxal-induced peri-toneal fibrosis: immunomodulation by inter-leukin-10. Lab. Invest. 95(12): 1353-1362, 2015.

12. Hirokawa, M., Sawada, K., Fujishima, N., Tera-

mura, M., Bessho, M., Dan, K., Tsurumi, H.,Nakao, S., Urabe, A., Fujisawa, S., Yonemura,Y., Kawano, F., Oshimi, K., Sugimoto, K., Ma-tsuda, A., Karasawa, M., Arai, A., Komatsu, N.,Harigae, H., Omine, M., Ozawa, K., Kurokawa,M. and PRCA Collaborative Study Group.:Long-term outcome of patients with acquiredchronic pure red cell aplasia (PRCA) followingimmunosuppressive therapy: a final report ofthe nationwide cohort study in 2004/2006 by theJapan PRCA collaborative study group. Br. J.Haematol. 169(6): 879-886, 2015.

13. Kamiyama, T., Kameda, H., Murabe, N.,Fukuda, S., Yoshioka, N., Mizukami, H.,Ozawa, K., and Sakurai, M.: Corticospinal TractDevelopment and Spinal Cord InnervationDiffer between Cervical and Lumbar Targets. J.Neurosci. 35(3): 1181-1191, 2015.

14. Suzuki, T., Oh, I., Ohmine, K., Meguro, A.,Mori, M., Fujiwara, S., Yamamoto, C., Nagai, T.,and Ozawa, K.: Distribution of serum erythro-poietin levels in Japanese patients with myelo-dysplastic syndromes. Int. J. Hematol. 101(1):32-36, 2015.

15. Tsukahara, T., Iwase, N., Kawakami, K.,Iwasaki, M., Yamamoto, C., Ohmine, K., Uchi-bori, R., Teruya, T., Ido, H., Saga, Y., Urabe, M.,Mizukami, H., Kume, A., Nakamura, M.,Brentjens, R., and Ozawa, K.: The Tol2 trans-poson system mediates the genetic engineeringof T-cells with CD19-specific chimeric antigenreceptors for B-cell malignancies. Gene Ther. 22(2): 209-215, 2015.

16. Watakabe, A., Ohtsuka, M., Kinoshita, M.,Takaji, M., Isa, K., Mizukami, H., Ozawa, K.,Isa, T., and Yamamori, T.: Comparative analy-ses of adeno-associated viral vector serotypes 1,2, 5, 8 and 9 in marmoset, mouse and macaquecerebral cortex. Neurosci. Res. 93: 144-157, 2015.

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