Download Division of Clinical and Experimental Oncology Department of

- 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)