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- 255 - Proc. Hiroshima Univ.RIRBM, 50, 2009 Division of Clinical and Experimental Oncology Department of Molecular Oncology & Leukemia Program Project Professor Toshiya INABA, M.D. Associate professor Hiroya ASOU, M.D. Assistant professor Hirotaka MATSUI, M.D. Assistant professor Daisuke Aki, Ph.D. Postgraduate student Akiko NAGAMACHI Postgraduate student Yuko OZAKI The main purpose of our department is to elucidate the molecular mechanisms of leukemogenesis. are trying to identify key factors that contribute to leukemogenesis from clinical samples. We Elucidation of the mechanisms in radiation-associated leukemia is one of the most important study in our laboratory. In this viewpoint, deletion of the long arm of chromosome 7 is very important because this is not only one of the most frequently detected chromosomal abnormalities found in sporadic myeloid leukemia and myelodysplastic syndrome (MDS), but also strongly implicated in radiation-induced myeloid malignancies. Thus we promoted analysis of candidate responsible genes for 7q deletions, which were isolated by us several years ago. The final goal of our study is to obtain basic data for development of new therapeutical approaches for leukemia by using molecular and cellular biology, as well as by establishing and analyzing gene-targeting mice, which has been performed based on a collaboration with Department of Developmental Oncology. The research projects in this laboratory, which were carried out in 2008 and are being planned for the following years are summarized as follows. 1. Isolation of myeloid tumor suppressor genes from 7q and establishment of an experimental system for large chromosome deletion using iPS cells. Asou H., Inaba T. Purpose: To isolate myeloid-tumor suppressor genes on 7q, we extracted DNA from leukemia samples stocked in RIRBM, and made probes for array-CGH by long-distance PCR technique. We identified candidate genes TITAN (Samd9L) , Kasumi (Samd9), and MIKI (LOC253012), and started to characterize them by using gene-targeting technology and by conventional molecular and cellular biological technique (see 2. to 5.). On the other hand, since there are considerable differences in genomic structure between human and mouse, large chromosome deletions such as 7q- are not easy to analyze using gene-targeting mice. Thus we started to establish an experimental system to analyze the effect of large chromosomal deletion using iPS cells. - 256 - Methods & Results: According to an established procedure by Dr. Yamanaka (Kyoto University), we successfully induced iPS cells from normal human fibroblasts using retroviruses containing each of cDNAs for Myc, Oct3/4, KLF4 or Sox2. We next tried to induce iPS cells from three AML/MDS-derived cells carrying monosomy 7 (FKH-1, OHN-GM, Mono-7). Although these cells showed tendency to aggregate on the feeder cells, they did not form typical iPS cell colony. tried to establish iPS cells from bone marrow mononuclear cells of a MDS patient. We also Although we obtained iPS-like colony formation, colonies grew slowly and we could not replate them. We are now modifying several procedures for induction of iPS cells from hematopoietic cells. 2. Characterization of myeloid tumor suppressor genes on 7q: Miki and CG-NAP, as mitosis regulators Ozaki Y., Asou H., Matsui H., Aki D., Nagamachi A., Inaba T. Purpose: Miki (LOC253012) encodes a protein that localizes to Golgi apparatus in the interphase, centrosomes and mitotic spindles in mitosis, suggesting that downregulation of Miki by 7q deletions contributes to leukemogenesis through disturbance of mitosis. We tried to elucidate detailed molecular mechanisms. Methods & Results: Downregulation of Miki causes impaired maturation and separation of centrosomes, resulting in severe mitotic defect including loss of spindle tension, chromosome scattering and severe chromosome lagging. Consequently, cells undergo pre-anaphase arrest, or form bi-, tri- or even multi-nuclear cells with micronuclei, which are characteristic to MDS. We found that poly(ADP-ribosyl)ation of Miki by Tankyrase-1 is required for binding to spindles. We also found that Miki, Tankyrase-1 and CG-NAP, a giant scaffold protein in centrosome, form a protein complex. Surprisingly human CG-NAP gene is located in 7q21, near Miki gene. We suspected that CG-NAP is a promising candidate for another myeloid tumor suppressor gene in chr. 7. Because tankyrase-1, CG-NAP and Miki localizes to the Golgi apparatus in the interphase, we considered that the function of the complex is transportation of critical pericentriolar materials (PCMs) from the Golgi apparatus to centrosomes and spindles. 3. Characterization of myeloid tumor suppressor genes on 7q: Miki as an endosomal transportation regulator Aki D., Asou H., Ozaki Y., Matsui H., Nagamachi A., Inaba T. Purpose: Miki (LOC253012) encodes a protein that localizes to the Golgi apparatus in the interphase. Moreover, using yeast two hybrid technology, Miki was demonstrated to form a complex with proteins involved in endosomal transportation system, such as GGA1 (Golgi-localized γ-ear-containing ARF binding protein 1) or VAC14. These data prompted us to elucidate possible roles of Miki in the interphase. Methods & Results: Miki was detected in lysosomes and late endosomes, in addition to trans-Golgi network. Intriguingly, downregulation of Miki in HeLa cells reduced the proteolysis of EGF receptor-ligand complex by lysosome and subsequently sustained signals from the receptor-ligand complexes, such as activation of Ras pathways. These data suggested that downregulation of Miki may contribute to leukemogenesis through an additional mechanism, i.e., abnormal upregulation of cytokine signal pathways. - 257 - 4. Characterization of myeloid tumor suppressor genes on 7q: Kasumi and Titan using gene-targeting mouse Nagamachi A., Matsui H., Ozaki Y., Asou H., Aki D., Inaba T., Miyazaki K. (Dept. of Developmental Oncology), Honda H (Dept. of Developmental Oncology). Purpose: Human Kasumi and Titan locate adjacent each other and share a common gene structure with a 70% amino acid identity, while mouse has Titan gene alone. To study the contribution of Titan to leukemogenesis, we generated Titan-deficient mice. Methods & Results: titan–/– mice were normally born and no hematological abnormalities were observed, suggesting that additional gene alterations are required for leukemia development. Retrovirus infection induced acute leukemia in homozygous (titan–/–) and heterozygous (titan+/–) mice with higher morbidity and mortality than in wild-type (titan+/+) littermates. Leukemias developed in titan+/+ mice were mainly of T-cell lineage. By contrast, those developed in titan–/– and titan+/– mice were negative for lymphoid markers but expressed various combination of cell surface markers for myeloid (Gr1), monocytic (Mac1), erythyroid (Ter119) and megakaryocytic (CD61) progenitors. These data suggested that leukemias developed in titan-deficient mice represent stem cell malignancy rather than AML. Inverse PCR detected two common integration sites specific for titan–/– and titan+/– mice, which induced deregulated expression of a transcription factor, Evi1, and a histone demethylase, Fbxl10. These results demonstrated that haploinsufficiency and deficiency of Titan predispose leukemia development through inhibiting TGFβ-mediated signaling or epigenetic change. 5. Biochemical characterization of myeloid tumor suppressor genes on 7q: Kasumi and Titan Matsui H., Nagamachi A., Ozaki Y., Asou H., Aki D., Inaba T. Kikuchi A. (Dept. of Biochemistry, Hiroshima University) Purpose: Because Titan and Kasumi share no homology to any other genes and carry no functional motifs, biochemical function of Titan/Kasumi is largely unknown. Recently, biallelic point mutations of Kasumi gene are known to cause Normophosphatemic Familial Tumoral Calcinosis, a rare inherited disease characterized by massive deposition of calcified tumors, which are formed by the impairment of wound healing due to suppression of cell migration. In addition, one paper suggests implication of Titan/Kasumi in the Wnt signal transduction pathways. These data prompted us to elucidate biochemical function of Titan/Kasumi that contributes to leukemogenesis. Methods & Results: By mass spectometry, we found that mouse Titan bounds to Flightless 1 (Fli1), an actin severing protein, and co-localizes with Fli1 in cytoplasm. Titan-/- fibroblasts showed disorganized actin structures with markedly reduced cell migration. These data suggest that Titan regulates actin remodeling through the control of Fli1 activity. Recently Fli1 was reported to be involved in the Wnt signal pathways. We are currently investigating possible roles of Titan/Kasumi in the Wnt signal pathways, which is known to regulate growth and differentiation of hematopoietic stem cells. List of Contributions A. Original Papers 1. Aki, D., Minoda, Y.*1, Yoshida, H.*1, Watanabe, S. *1, Yoshida, R. *1, Takaesu, G. *1, Chinen, T. *1, Inaba, T., Hikida, M. *2, Kurosaki,T.*2, Saeki, K. *1, Yoshimura, A. *1(*1Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, *2 RIKEN Research Center for Allergy and Immunology) Peptidoglycan and lipopolysaccharide activate PLCγ2, leading to enhanced cytokine production in macrophages and dendritic cells. Genes Cells 13: 199-208, 2008.(I) - 258 - 2. Watanabe-Okochi, N.*1, Kitaura, J.*1, Ono, R.*1, Harada, H.*2, Harada, Y.*3, Komeno, Y.*1, Nakajima, H.*1, Nosaka, T.*1, Inaba T, Kitamura, T. *1 (*1Division of Cellular Therapy, Advanced Clinical Research Center , The Institute of Medical Science, The University of Tokyo, Hematology and Oncology, *3International *2Department of Radiation Information Center) AML1 mutations induced MDS and MDS/AML in a mouse BMT model. Blood 111: 4297-4308, 2008.(I) 3. Ectopic cyclin D1 overexpression increases chemosensitivity but not cell proliferation in multiple myeloma. Kuroda, Y.*1, Sakai, A.*1, Tsuyama, N. *2, Katayama, Y. *3, Munemasa, S. *1, Asaoku, H. *3, Okikawa, Y. *1, Nakaju, N. *1, Mizuno ,M. *4, Ogawa, K. *5, Nishisaka, T. *6, Matsui, H., Tanaka, H. *1, Kimura, A*1. (*1Department of Hematology and Oncology, Medicine, Hiroshima University, *3Department *2 Department of Analusis Molecular of Internal Medicine, Hiroshima Red-Cross Hospital; *4Department of Blood Transfusion Service, *5 Clinical Hospital; *6 Department of Pathology and Laboratory Medicine, Hiroshima Prefectural Hospital) Int Pathology, Hiroshima University J Oncol 33: 1201-1213, 2008.(I) 4. Ras-mediated up-regulation of survivin expression in cytokine-dependent murine pro-B lymphocytic cells. Shinjyo, T.*1, Kurosawa, H. *2, Miyagi, J. *1, Ohama, K. *1, Masuda, M. *1, Nagasaki, A, *1, Matsui, H., Inaba, T., Furukawa, Y. *3, Takasu, N. *1 (*1Department of Endocrinology and Metabolism, Department of Internal Medicine, University of the Ryukyu, School of Medicine, *2Department Molecular of Pediatrics, Dokkyo Medical University,*3Division of Stem Cell Research Center of Medicine, Jichi Medical School) Tohoku J Exp Med 216: 25-34, 2008.(I) 5. Inaba, T., Asou, H. Inaba T., Asou H. chromosome 7. edited by Tomonaga M. Myeloid tumor suppressor genes on the long arm of Basic and clinical studies on myelodysplastic syndrome. pp.55-62, Iyaku Journal Co.Ltd. Osaka, 2008 6. Asou H. Overview of Myelodysplastic Syndrome. Experimental Medicine: 27(1) pp103-108(2009.01) B. Meetings 1. Ozaki Y., Matsui H., Aki, D., Nagamachi A., Asou H., Inaba T. Functional analysis of CG-NAP, a candidate for causative gene of radiation-induced leukemia. The 49th Annual Meeting of Late Effects of Atomic Bomb, Nagasaki, June 8, 2008.(R)(G) 2. Nagamachi, A., Asou H., Matsui H., Miyazaki K.*1, Yamasaki N. *1, Inaba T., Honda, H. *1 (*1Department of Developmental Biology) Frequent leukemia development in Titan-deficient mice infected by retrovirus at neonate. The 49th Annual Meeting of Late Effects of Atomic Bomb, Nagasaki, June 8, 2008. (R)(A)(G) 3. Asou, H., Matsui, H., Nagamachi, A., Aki, D., Ohsugi, M*1, Inaba, T. (*1The Institute of Medical Science, The University of Tokyo) Downregulation of the chromosome 7 gene Miki induces mitotic disturbance characteristic of myelodysplastic syndrome. The annual meeting of the Japanease Society for Cell Biology, Yokohama, June 29, 2008. (R)(G) 4. Nagamachi A., Asou H., Aki D., Ozaki Y., Miyazaki K.*1, Yamasaki N. *1, Oda H. *2, Inaba T., Honda, H. *1 (*1Department of Developmental Biology, *2Department of Pathology, Tokyo Women’s Medical University) Deletion of TITAN contributes to leukemogenesis by TGFb signaling suppression and epigenetic changes. The 70th Annual Meeting of The Japanese Journal of Clinical Hematology, Kyoto International Conference Center, Kyoto, Oct.10-12, 2008.(R)(A)(G) 5. Matsui H., Nagamachi A., Ozaki Y., Aki D., Asou H., Honda H.*1, Inaba T. (*1 Department of Developmental Biology) Titan(Samd9L), a novel myeloid tumor suppressor on 7q, activates actin - 259 - remodeling. The 70th Annual Meeting of The Japanese Journal of Clinical Hematology, Kyoto International Conference Center, Kyoto, Oct.10-12, 2008. (R)(G) 6. Asou H., Ozaki Y., Aki D., Nagamachi A., Matsui H., Inaba T. CG-NAP that encodes centrosomal protein is a candidate myeloid-tumor suppressor gene on 7q. The 70th Annual Meeting of The Japanese Journal of Clinical Hematology, Kyoto International Conference Center, Kyoto, Oct.10-12, 2008. (R)(G) 7. Kurosawa H.*1, Okuya M.*1, Kikuchi J.*2, Furukawa Y. *2, Inukai T. *3, Aki D., Matsui H., Inaba T., Matsushita T.*1, Satou Y.*1, Ogisawa S.*1, Fukushima K.*1, Sugita K.*1, Arisaka O.*1 (*1Dokkyo Medical University, *2 Jichi Medical University, *3 University of Yamanashi, Faculty of Medicine) Inhibition of caspase independent apoptosis in t(17;19)-ALL. The 70th Annual Meeting of The Japanese Journal of Clinical Hematology, Kyoto International Conference Center, Kyoto, Oct.10-12, 2008. 8. Tanigasaki H.*1, Takahashi Y.*1, Asou H., Inaba T., Kojima S.*1 (*1Nagoya University, Faculty of Pediatrics) Analysis of monosomy 7 associated tumor suppressor gene in JMML. The 70th Annual Meeting of The Japanese Journal of Clinical Hematology, Kyoto International Conference Center, Kyoto, Oct.10-12, 2008. 9. Asou H., Ozaki Y., Nagamachi A., Aki D., Matsui H., Inaba T. Restoration of nornal mitosis and chromosome instability: a possible therapeutic approach for MDS. The 67th Annual Meeting of the Japanese Cancer Association, Nagoya Congress Center, Oct.28-30, 2008. (R)(G) 10. Matsui H., Nagamachi A., Ozaki Y., Aki D., Asou H., Honda H., Inaba T. Titan(Samd9L), a novel myeloid tumor suppresor on 7q, activates actin remodeling. The 67th Annual Meeting of the Japanese Cancer Association, Nagoya Congress Center, Oct.28-30, 2008. (R)(G) 11. Nagamachi A., Asou H., Matsui H., Ozaki Y., Aki D., Miyazaki K. *1, Yamasaki N. *1, Miyazaki M. *2, Wolff L. *3, Koller R. *3, Oda H. *4, Inaba T., Honda H. *1 (*1Department of Developmental Biology, *2Department *3NCI/NIH, of Immunology, Guraduate School of Biomedical Science, Hiroshima University, Bethesda, MD., *4Department of Pathology, Tokyo Women’s Medical University) Haploinsufficiency and deficiency of a 7q gene Titan (Samd9L) predispose leukemia development in cooperation with deregulated expression of a transcription factor, Evi1, or a histone demethylase, Fbxl10. The 50th Annual Meeting of American Society of Hematology, December 6-9, 2008, Moscone Center, San Francisco, CA. (R)(A)(G) 12. Asou H., Ozaki Y., Nagamachi, A., Aki D., Matsui H., Inaba T. Identification of two 7q genes encoding centrosomal proteins as myeloid tumor-suppressor candidates. The 50th Annual Meeting of American Society of Hematology, December 6-9, 2008, Moscone Center, San Francisco, CA. (R)(G) 13. Aki D., Takemura Y.*1, Nagamachi A., Honda, H.*2, Ozaki Y., Matsui H., Asou H., Inaba T. (*1The University of Tokyo, *2 Department of Developmental Biology) The function of the chromosome 7 gene Miki in intracellular membrane traffic. The 31st Annual Meeting of the Japanese Biochemistry and Molecular Biology, Kobe, Dec.9-12, 2008. (R)(G) 14. Ozaki Y., Matsui H., Nagamachi A., Asou H., Inaba T. Identification and characterization of Tankyrase1/Miki/CG-NAP protein complex that promotes centrosomal maturation. The 31st Annual Meeting of the Japanese Biochemistry and Molecular Biology, Kobe, Dec.9-12, 2008. (R)(G) C. Others 1. Toshiya Inaba: Characterization of genes isolated from chromosome-arm 7q frequently deleted in AML and MDS. Saitama Hematology Seminar, Omiya (200.6.25) - 260 - 2. Hirotaka Matsui, Toshiya Inaba: Towards establishment of experimental systems for analysis of contribution of large chromosomal deletions to MDS and AML using iPS cells. Annual Meeting of the Development of new age therapy for MDS, the Ministry of Health, Labor and Welfare of Japan, Tokyo (2008.11.22) (R)(G) 3. Hiroya Asou, Toshiya Inaba: Characterization of genes isolated from chromosome-arm 7q frequently deleted in AML and MDS. Annual Meeting of the Development of new age therapy for MDS, the Ministry of Health, Labor and Welfare of Japan, Tokyo (2009.1.20) (R)(G) 4. Toshiya Inaba: Cooperation of 7q deletion and Fbxl10, a histone demethylase, in myeloid leukemogenesis. Kyoto Hematology Seminar, Kyoto (2009.2.27) 5. Hiroya Asou: -7/7q- and myelodysplastic syndrome. Kobe forum on advance in hematology, Kobe (2009.3.7)