The 5th JCA-AACR Special Joint Conference
The Latest Advances in Hematological Cancer Research:
From Basic Science to Therapeutics
July 13th (Wed) – 15th (Fri), 2016
Tokyo Bay Maihama Hotel Club Resort, Chiba, Japan
Organizing Committee Members
Organizing Committee Members
JCA:
Takuro Nakamura (Japanese Foundation for Cancer Research)
Issay Kitabayashi (National Cancer Center)
Shigeru Chiba (University of Tsukuba)
AACR:
Dr. Jonathan D. Licht (University of Florida Health Cancer Center)
Dr. Ross L. Levine (Memorial Sloan Kettering Cancer Center)
Dr. Catriona H. M. Jamieson (University of California, San Diego)
Program and Proceedings
Table of Contents
Contents
Organizing Committee Members....................................... 1
Conference Supporters..................................................... 3
Travel Grants..................................................................... 5
Program at a Glance......................................................... 6
Floor Plan.......................................................................... 8
General Information / Social Program............................... 9
Poster Information........................................................... 10
Meet-the-Expert Evening Information……………………. 11
Conference Program....................................................... 12
Participant List................................................................. 22
Abstracts of Invited Presentations................................... 27
Abstracts of Poster Presentations................................... 55
Author Index of Poster Presentations............................ 109
1
Conference Supporters
Conference Supporters
The JCA and AACR thank the following organizations for
their generous support of this joint conference.
3
Travel Grants
Travel Grants
3 poster presenters of meritorious abstracts have been selected by Conference
Committee members to receive travel grants to attend this conference. The names
of the travel grant awardees, their affiliations, and their poster presentation
numbers are provided below.
Awardees:
Faruk, Mohammed
Ahmadu Bello University, Zaria, Nigeria
(No. 5-4)
Jiang, Qingfei
University of California, San Diego, USA
(No. 1-9)
Luanpitpong, Sudjit
Mahidol University, Bangkok, Thailand
(No. 5-2)
5
Program
Program at
at aa Glance
Glance
Date
Time
Session
Chairperson
July
18:00-18:10
Welcome Address
「Program
at a Glance」ファイル参照
13th
18:10-19:30 Keynote Lectures
Dr. Licht
Dr. Nakamura
Speaker
Title
Disordered Histone
Methylation in Lymphoid
Malignancy
Signaling and Transcriptional
Dr. Takuro Nakamura
Networks in Myeloid
(The Cancer Institute of JFCR)
Leukemogenesis
Dr. Jonathan D. Licht
(The University of Florida)
20:00-22:00 Welcome Party
July
14th
Dr. Atsushi Iwama
(Chiba University)
8:00-9:30
1. Aging and epigenetics
in hematopoietic
malignancies
Dr. Figueora
Dr. Chiba
Dr. Maria E. (Ken) Figueroa
(University of Michigan)
Dr. Shigeru Chiba
(University of Tsukuba)
9:30-9:50
Break
Dr. Ross L. Levine
(Memorial Sloan Kettering
Cancer Center)
9:50-11:20
Deregulated Polycombgroup Gene Functions in
Hematological Malignancies
Epigenetic Deregulation in the
Aging Hematopoietic Stem
Cell
Aging and TET Dioxygenases
in Leukemo/lymphomagenesis
2. Myeloproliferative
Dr. Levine
neoplasms and
Dr. Kitamura
myelodysplastic syndrome
Dr. Toshio Kitamura
(The University of Tokyo)
Dr. Hitoshi Takizawa
(Kumamoto University)
Role of JAK-STAT
Pathway Activation in MPN
Pathogenesis and Therapeutic
Response
The ASXL1 Mutation and
the EZH2 Mutation Induced
Myelodysplastic Syndromes in
Mice via Distinct Mechanisms
Inflammation-induced
Malignant Transformation of
Hematopoietic Stem Cell
11:20-12:20 Lunch and Poster View
12:20-13:50 Poster Presentation
13:50-15:20
3. Recent advances in
lymphoma therapeutics
Dr. Melnick
Dr. Tobinai
Dr. Ari Melnick
(Weill Cornell Medicine)
Epigenetic Circuits and Their
Perturbation in Lymphoid
Neoplasms
Dr. Kensei Tobinai
(National Cancer Center
Hospital)
New Agent Development for
Lymphoid Malignancies in
Japan
Dr. Yasuhito Terui
(Cancer Institute Hospital of
JFCR)
Recent Advances in
Therapeutics for B-cell
Lymphoma
15:20-15:40 Break
Dr. Atsushi Hirao
(Kanazawa University)
15:40-17:40
4. Leukemogenesis:
Leukemia stem cell,
metabolism and novel
targets
Dr. Catriona H. M. Jamieson
(University of California, San
Diego)
Dr. Jamieson
Dr. Kitabayashi
Dr. Koicji Akashi
(Kyushu University)
Dr. Issay Kitabayashi
(National Cancer Center
Research Institute)
18:00-19:00 Meet-the-Expert Evening (all participants)
19:30-21:30 Banquet
6
Molecular Mechanism
Linking Stem Cell Aging
and Tumorigenesis in
Hematopoiesis
Malignant RNA Editing
Activation Drives Cancer Stem
Cell Self-renewal by Impairing
Let-7 Biogenesis
TIM-3 and Its Ligand,
Galectin-9, Constitute an
Autocrine Loop Universally
Critical for Development of
Human Myeloid Leukemia
Stem Cells
Novel Leukemia Stem Celltargeted Therapy for Acute
Myeloid Leukemia Based on
Dual Inhibition of EZH1/EZH2
Date
July
15th
Time
8:30-10:00
Session
5. Genetic and epigenetic
alterations in childhood
leukemia
Chairperson
Dr. Ferrando
Dr. Ito
Speaker
Title
Dr. Junko Takita
(The Univeristy of Tokyo)
Integrated Genetic Analysis
of Pediatric T-cell Acute
Lymphoblastic Leukemia
Dr. Adolfo A. Ferrando
(Columbia University)
Oncogenic Signaling
Pathways and Mechanisms
of Resistance in Acute
Lymphoblastic Leukemia
Dr. Etsuro Ito
(Hirosaki University)
Genetic and Epigenetic
Alterations in Acute
Megakaryoblastic Leukemia in
Down Syndrome
10:00-10:20 Break
Dr. Seishi Ogawa
(Kyoto University)
10:20-11:50
6. Genomic landscapes of
lymphoid malignancies
Dr. Landau
Dr. Mano
Dr. Dan A. Landau
(Weill Cornell Medicine/New
York Genome Center)
Dr. Hiroyuki Mano
(The University of Tokyo)
11:50-12:50
Discovery of DUX4-IGH
Fusion-Type Oncogene in
B-Cell Acute Lymphoblastic
Leukemia of AYA Generation
Lunch and Poster View
Dr. Yutaka Kawakami
(Keio University)
12:50-14:20
A Novel Genetic Mechanism of
Evading Anti-tumor Immunity
in Multiple Human Cancers
Genetic and Epigenetic
Determinant of Chronic
Lymphocytic Leukemia
Evolution
7. Immunotherapy of
hematopoietic neoplasms
Dr. Riddell
Dr. Kawakami
Dr. Hiroyoshi Nishikawa
(National Cancer Center)
Dr. Stanley R. Riddell
(Fred Hutchinson Cancer
Research Center)
Current Progress and Future
Perspectives in Cancer
Immunotherapy: Development
of Personalized Combination
Immunotherapy
Critical Role of Regulatory T
cells in Cancer Immunotherapy
for Hematologic Malignancies
Engineering Safe and Effective
T Cell Therapy for Cancer
14:20-14:40 Break
8. Molecular biology
14:40-15:40 and targets of multiple
myeloma
Dr. Rodger E. Tiedemann
(Princess Margaret Cancer
Dr. Tiedemann Center/University of Toronto)
Dr. Furukawa
Dr. Yusuke Furukawa
(Jichi Medical University)
Intra-Tumor Heterogeneity in
Multiple Myeloma and It's Role
in Therapeutic Resistance and
Relapse
Epigenetic Mechanisms of
Drug Resistance in Multiple
Myeloma
15:40-15:50 Closing Remarks
7
Floor Plan
Poster Presentations
Meet-the-Expert Evening
Lunch
Coffee Break
Amethyst
Opening Remarks
Invited Presentations
Closing Remarks
Emerald
Opal
Welcome Party (July 13th)
Banquet (July 14th)
Ruby
Sapphire
Registration
Shinju
Sango
Cloak
Secretariat
1F Entrance
8
Garnet
Floor Plan
General
Information / Social
Social Program
Program
Conference Venue
Tokyo Bay Maihama Hotel Club Resort
1-7, Maihama, Urayasu-shi, Chiba 279-0031, Japan
Tel: +81-47-355-2411
Fax: +81-47-355-2460
http://www.tbm-clubresort.jp/eng/
Registration and Travel Information Desk
The Registration and Travel Information Desk will be open during the following hours in front of the
Conference rooms:
July 13th (Wed) 16:00 - 21:00
July 14th (Thu)
7:30 - 18:00
July 15th (Fri)
8:00 - 15:00
Name Badge
Conference name badges must be worn at all times while you are in the Conference venue. The badge
will serve as your admission to all sessions and official functions included in your registration fee.
Message Board
Any program changes or urgent announcements from the Secretariat and private messages will be
posted on the message board located in the registration area on the 1st floor. You should check this
board regularly for any messages.
Coffee Break
Coffee service will be available in Room "Amethyst” and “Emerald” (Poster Presentation area) during the
following hours:
July 14th (Thu)
9:30 - 9:50, 15:20 - 15:40
July 15th (Fri)
10:00 - 10:20, 14:20 - 14:40
Lunch
A boxed lunch will be served in Rooms "Amethyst” and “Emerald” (Poster Presentation rooms) during the
following hours:
July 14th (Thu) 11:20 - 12:20
July 15th (Fri)
11:50 - 12:50
Welcome Party
Date & Time: July 13th (Wed)
20:00 - 22:00
Place: Room “Ruby”
Banquet
Date & Time: July 14th (Thu)
19:30 - 21:30
Place: Room “Ruby”
9
Poster
PosterInformation
Information
Session Categories
1. Leukemogenesis: Leukemia stem cell, metabolism and novel targets
2. Aging and
epigenetics in hematopoietic malignancies
Session
Categories
3. Leukemogenesis:
Myeloproliferative Leukemia
neoplasmsstem
and cell,
myelodysplastic
1.
metabolism syndrome
and novel targets
4. Aging
Genomic
malignancies
2.
andlandscapes
epigeneticsofinlymphoid
hematopoietic
malignancies
5. Myeloproliferative
Recent advances in
lymphomaand
therapeutics
3.
neoplasms
myelodysplastic syndrome
6. Genomic
Molecularlandscapes
biology andoftargets
of multiple
myeloma
4.
lymphoid
malignancies
7. Recent
Immunotherapy
neoplasms
5.
advancesofinhematopoietic
lymphoma therapeutics
8. Molecular
Others
6.
biology and targets of multiple myeloma
7. Immunotherapy of hematopoietic neoplasms
Presentation
time
8.
Others
Poster Layout
Poster Layout
Group 1: Odd-numbered posters in each category
12:20 - 13:05,time
July 14th
Presentation
Group 1:
2: Odd-numbered
Even-numberedposters
postersinineach
eachcategory
category
Group
13:05 -- 13:05,
13:50, July
July 14th
14th
12:20
Group 2: Even-numbered posters in each category
Poster
time:
7:30 - 12:00, July 14th
13:05set-up
- 13:50,
July 14th
Poster removal time: 13:00 - 16:00, July 15th
*Any posters
remaining
on- 12:00,
the panels
after 16:00 on July
Poster
set-up time:
7:30
July 14th
15th will
be discarded
by the
secretariat.
Poster
removal
time: 13:00
- 16:00,
July 15th
*Any posters remaining on the panels after 16:00 on July
15th will be discarded by the secretariat.
Room “Amethyst”
Room “Amethyst”
10
Meet-the-Expert Evening Information
Meet-the-Expert Evening Information
Date & Time: 18:00 - 19:00, July 14th
Place:
Session 1
Room “Emerald”
Aging and epigenetics in hematopoietic malignancies
Expert: Dr. Eugenia Figueroa (Univ. of Michigan)
Dr. Shigeru Chiba (Tsukuba Univ.)
Session 2
Myeloproliferative neoplasms and myelodysplastic syndrome
Expert: Dr. Ross L. Levine (MSKCC)
Dr. Toshio Kitamura (Univ. of Tokyo)
Session 3
Recent advances in lymphoma therapeutics
Expert: Dr. Ari Melnick (Weill Cornell Univ.)
Dr. Kensei Tobinai (National Cancer Center)
Session 4
Leukemogenesis (1)
Expert: Dr. Jonathan D. Licht (Univ. Florida)
Dr. Takuro Nakamura (Cancer Inst. of JFCR)
Session 5
Leukemogenesis (2)
Expert: Dr. Catriona HM Jamieson (Univ. California San Diego)
Dr. Issay Kitabayashi (National Cancer Center)
Session 6
Genetic and epigenetic alterations in childhood leukemia
Expert: Dr. Adolfo A. Ferrando (Columbia Univ.)
Dr. Junko Takita (Univ. of Tokyo)
Session 7
Genomic landscapes of lymphoid malignancies
Expert: Dr. Dan A. Landau (Weill Cornel Univ.)
Dr. Seishi Ogawa (Kyoto Univ.)
Session 8
Immunotherapy of hematopoietic neoplasms
Expert: Dr. Stanley R. Riddell (Fred Hutchinson Cancer Research Center)
Dr. Yutaka Kawakami (Keio Univ.)
Session 9
Molecular biology and targets of multiple myeloma
Expert: Dr. Rodger Tiedemann (Ontario Cancer Inst.)
Dr. Yusuke Furukawa (Jichi Univ.)
11
Conference Program
Conference Program
July 13th
18:00-18:10
Welcome Address
18:10-19:30
Keynote Lectures
Co-Chairpersons:
Dr. Licht
Dr. Nakamura
Disordered Histone Methylation in Lymphoid Malignancy
Jonathan D. Licht (The University of Florida Health Cancer Center, USA)
Signaling and Transcriptional Networks in Myeloid Leukemogenesis
Takuro Nakamura (The Cancer Institute, Japanese Foundation for Cancer Research, Japan)
20:00-22:00
Welcome Party
July 14th
8:00-9:30
Session 1. Aging and epigenetics in hematopoietic malignancies
Co-Chairpersons:
Dr. Figueora
Dr. Chiba
Deregulated Polycomb-group Gene Functions in Hematological Malignancies
Dr. Atsushi Iwama (Chiba University, Japan)
Epigenetic Deregulation in the Aging Hematopoietic Stem Cell
Dr. Maria E. (Ken) Figueroa (University of Michigan, USA)
Aging and TET Dioxygenases in Leukemo/lymphomagenesis
Dr. Shigeru Chiba (University of Tsukuba, Japan)
9:30-9:50
Break
9:50-11:20
Session 2.
syndrome
Myeloproliferative
Co-Chairpersons:
Dr. Levine
Dr. Kitamura
12
neoplasms
and
myelodysplastic
Role of JAK-STAT Pathway Activation in MPN Pathogenesis and Therapeutic Response
Dr. Ross L. Levine (Memorial Sloan Kettering Cancer Center, USA)
The ASXL1 Mutation and the EZH2 Mutation Induced Myelodysplastic Syndromes in Mice via
Distinct Mechanisms
Dr. Toshio Kitamura (The University of Tokyo, Japan)
Inflammation-induced Malignant Transformation of Hematopoietic Stem Cell
Dr. Hitoshi Takizawa (Kumamoto University, Japan)
11:20-12:20
Lunch and Poster View
12:20-13:50
Poster Presentation
13:50-15:20
Session 3. Recent advances in lymphoma therapeutics
Co-Chairpersons:
Dr. Melnick
Dr. Tobinai
Epigenetic Circuits and Their Perturbation in Lymphoid Neoplasms
Dr. Ari Melnick (Weill Cornell Medicine, USA)
New Agent Development for Lymphoid Malignancies in Japan
Dr. Kensei Tobinai (National Cancer Center Hospital, Japan)
Recent Advances in Therapeutics for B-cell Lymphoma
Dr. Yasuhito Terui (Cancer Institute Hospital of Japanese Foundation for Cancer Research, Japan)
15:20-15:40
Break
15:40-17:40
Session 4. Leukemogenesis: Leukemia stem cell, metabolism and
novel targets
Co-Chairpersons:
Dr. Jamieson
Dr. Kitabayashi
Molecular Mechanism Linking Stem Cell Aging and Tumorigenesis in Hematopoiesis
Dr. Atsushi Hirao (Kanazawa University, Japan)
Malignant RNA Editing Activation Drives Cancer Stem Cell Self-renewal by Impairing Let-7
Biogenesis
Dr. Catriona H. M. Jamieson (University of California, San Diego, USA)
13
TIM-3 and Its Ligand, Galectin-9, Constitute an Autocrine Loop Universally Critical for
Development of Human Myeloid Leukemia Stem Cells
Dr. Koicji Akashi (Kyushu University, Japan)
Novel Leukemia Stem Cell-targeted Therapy for Acute Myeloid Leukemia Based on Dual Inhibition
of EZH1/EZH2
Dr. Issay Kitabayashi (National Cancer Center Research Institute, Japan)
18:00-19:00
Meet-the-Expert Evening (All participants)
19:30-21:30
Banquet
July 15th
8:30-10:00
Session 5. Genetic and epigenetic alterations in childhood leukemia
Co-Chairpersons:
Dr. Ferrando
Dr. Ito
Integrated Genetic Analysis of Pediatric T-cell Acute Lymphoblastic Leukemia
Dr. Junko Takita (The University of Tokyo, Japan)
Oncogenic Signaling Pathways and Mechanisms of Resistance in Acute Lymphoblastic Leukemia
Dr. Adolfo A. Ferrando (Columbia University, USA)
Genetic and Epigenetic Alterations in Acute Megakaryoblastic Leukemia in Down Syndrome
Dr. Etsuro Ito (Hirosaki University, Japan)
10:00-10:20
Break
10:20-11:50
Session 6. Genomic landscapes of lymphoid malignancies
Co-Chairpersons:
Dr. Landau
Dr. Mano
A Novel Genetic Mechanism of Evading Anti-tumor Immunity in Multiple Human Cancers
Dr. Seishi Ogawa (Kyoto University, Japan)
Genetic and Epigenetic Determinant of Chronic Lymphocytic Leukemia Evolution
Dr. Dan A. Landau (Weill Cornell Medicine/ New York Genome Center, USA)
14
Discovery of DUX4-IGH Fusion-Type Oncogene in B-Cell Acute Lymphoblastic Leukemia of AYA
Generation
Dr. Hiroyuki Mano (The University of Tokyo, Japan)
11:50-12:50
Lunch and Poster View
12:50-14:20
Session 7. Immunotherapy of hematopoietic neoplasms
Co-Chairpersons:
Dr. Riddell
Dr. Kawakami
Current Progress and Future Perspectives in Cancer Immunotherapy: Development of
Personalized Combination Immunotherapy
Dr. Yutaka Kawakami (Keio University, Japan)
Critical Role of Regulatory T Cells in Cancer Immunotherapy for Hematologic Malignancies
Dr. Hiroyoshi Nishikawa (National Cancer Center, Japan)
Engineering Safe and Effective T Cell Therapy for Cancer
Dr. Stanley R. Riddell (Fred Hutchinson Cancer Research Center, USA)
14:20-14:40
Break
14:40-15:40
Session 8. Molecular biology and targets of multiple myeloma
Co-Chairpersons:
Dr. Tiedemann
Dr. Furukawa
Intra-Tumor Heterogeneity in Multiple Myeloma and It’s Role in Therapeutic Resistance and
Relapse
Dr. Rodger E. Tiedemann (Princess Margaret Cancer Center/University of Toronto, Canada)
Epigenetic Mechanisms of Drug Resistance in Multiple Myeloma
Dr. Yusuke Furukawa (Jichi Medical University, Japan)
15:40-15:50
Closing Remarks
15
Poster Presentation
Poster
July 14th 12:20-13:50
Session 1. Leukemogenesis: Leukemia stem cell, metabolism and novel targets
1-1
Targeting the MAP Kinase Pathway in Human Acute Myeloid Leukemia Cells Using a
Recombinant Anthrax Lethal Toxin Inhibits Proliferation through the Rho GTPase
Pathway
Mirvat El-Sibai (Lebanese American University, Lebanon)
1-2
Tetra-O-Methyl Nordihydroguaiaretic Acid Broadly Suppresses Cancer Metabolism and
Synergistically Induces Strong Anticancer Activity in Combination with Etoposide,
Rapamycin and UCN-01
Kotohiko Kimura (Johns Hopkins University, USA)
1-3
CCL3-expressing Basophil-like Leukemia Cells Conduct Leukemic Hematopoiesis in
Chronic Myeloid Leukemia
Tomohisa Baba (Kanazawa University, Japan)
1-4
SCF-type E3 Ligase Fbw7 Plays Pivotal Roles in the Maintenance of the Development and
Commitment in Hematopoietic Cells
Kyoko Kitagawa (Hamamatsu University School of Medicine, Japan)
1-5
Cancelled
1-6
Perturbation of Energy Metabolism by Fatty-acid Derivative Is Effective for Overcoming
Imatinib Resistance in BCR-ABL-harboring Leukemic Cells
Haruka Shinohara (Gifu University, Japan)
1-7
A Novel Molecular-target Therapy for Therapy-resistant AML by Human Anti-ITGA6/B4
Antibody
Kazuhiro Morishita (University of Miyazaki, Japan)
1-8
Cancelled
1-9
RNA Editing Enzyme ADAR1 Regulates Cell Cycle of Hematopoietic Cell
Qingfei Jiang (University of California, San Diego, USA)
1-10
Probing the Hippo Signaling Pathway in Leukemia as a Potential Therapeutic Target
Chanchao Lorthongpanich (Mahidol University, Thailand)
16
1-11
The Roles of Glis2 in Leukemic and Hematopoietic Stem Cells
Emi Takamatsu-Ichihara (National Cancer Center Research Institute, Japan)
1-12
Trib1 Modulates Transcriptional Functions of Hoxa9
Takashi Yokoyama (Japanese Foundation for Cancer Research, Japan)
1-13
The C/EBPβ Transcription Factor Mediates the Effect of IFNα on CML Stem Cells through
Promoting Their Differentiation and Exhaustion
Asumi Yokota (Kyoto University Hospital, Japan)
1-14
A New Role for Meis1 in the Immune Evasion of Myeloid Leukemic Cells
Arnaud Couzinet (Japanese Foundation for Cancer Research, Japan)
1-15
MLL Is Essential for NUP98-HOXA9-Induced Leukemia
Yutaka Shima (National Cancer Center Research Institute, Japan)
1-16
Modeling Tyrosine Kinase Inhibitor-resistant Chronic Myelogenous Leukemia by
Patient-derived Induced Pluripotent Stem Cells
Masashi Miyauchi (The University of Tokyo, Japan)
1-17
Deletion of Bcor Impairs Repopulating Capacity of Hematopoietic Stem Cells but
Promotes Transformation of Progenitor Cells
Shiro Tara (Chiba University, Japan)
1-18
PVPylated Magnetic Metal Oxide Nanoparticles for Drug Delivery to Human Acute Myeloid
Leukemia
Ralph J. Abi-Habib (Lebanese American University, Lebanon)
1-19
Topoisomerase II-targeting Novel Intervention: “Jekyll” Physiology and “Hyde” Pathology
Tsai-Kun Li (National Taiwan University College of Medicine, Taiwan)
1-20
Endogenous MOZ Is Essential for MOZ/MLL-fusion Induced Leukemias and Upregulation
of HoxA9/Meis1
Takuo Katsumoto (National Cancer Center Research Institute, Japan)
1-21
Primary Human AML Cells Utilize Aerobic Respiration Maintained by Enhanced
Expression of ASCT1
Yu Kochi (Kyushu University, Japan)
17
Session 2. Aging and epigenetics in hematopoietic malignancies
2-1
Combined Loss of Tet2/Tet3 Dioxygenases Induces Acute Myeloid Leukemia Sensitive to
Hypomethylating Agents
Koichiro Maie (University of Tsukuba, Japan)
2-2
Tumor Suppressive Role of JMJD5, a JmjC-domain Protein, in Breast Cancer
Akihiko Ishimura (Kanazawa University, Japan)
2-3
A Novel Tumor Suppressor, NDRG2 is Down-regulated by EZH2 Overexpression through
HTLV-1 Infection in ATL Cells
Tomonaga Ichikawa (University of Miyazaki, Japan)
2-4
Ezh2 Loss in Hematopoietic Stem Cells Predisposes Mice to Develop Heterogeneous
Malignancies in an Ezh1-dependent Manner
Makiko Mochizuki-Kashio (Chiba University, Japan)
2-5
DNMT3A Mutation Inhibits Gene-Body Methylation to Maintain Acute Myeloid Leukemia
Yuki Kagiyama (National Cancer Center Research Institute, Japan)
2-6
DNA Methylation of the Human Telomerase Reverse Transcriptase (hTERT) Promoter in
Human T-cell Leukemia Virus Type-1 (HTLV-1) Infected T-cells
Mariko Mizuguchi (Tokyo Medical and Dental University, Japan)
2-7
Clinical Characteristics of TAL1 Super-enhancer Aberrations and STIL-TAL1 Fusion in
Pediatric T Cell Acute Lymphoblastic Leukemia (T-ALL)
Shunsuke Kimura (The University of Tokyo, Japan)
2-8
TIP60 Is Critical for Acute Myeloid Leukemia Induced by MLL Fusions
Kazutsune Yamagata (National Cancer Center Research Institute, Japan)
2-9
Jmjd3, a Histone Demethylase, Is Required for the Functional Integrity of Hematopoietic
Stem Cells in Mice
Yuichiro Nakata (Hiroshima University, Japan)
Session 3. Myeloproliferative neoplasms and myelodysplastic syndrome
3-1
Localization and Characteristic of Nestin-expressing Cells in Human Bone Marrow and
Their Abnormalities in Myelodysplastic Syndromes
Luan Cao Sy (University of Tsukuba, Japan)
18
3-2
The Role of HMGA2 in the Pathogenesis of Myeloproliferative Neoplasms (MPNs)
Koki Ueda (Fukushima Medical University, Japan)
3-3
Combination of ASXL1 and RUNX1 Mutants Induced MDS/AML in Mice with Shorter
Latencies
Reina Nagase (The University of Tokyo, Japan)
3-4
ATP-binding Cassette Transporter G2 (ABCG2) Expression Characterizes Advanced
Myelodysplastic Syndrome Either in Mouse Model or Human Disease
Kimihito C. Kawabata (The University of Tokyo, Japan)
3-5
Functional Analysis of UTX, a Histone H3K27 Demethylase, in Normal Hematopoiesis and
Hematologic Malignancies
Yasuyuki Sera (Hiroshima University, Japan)
Session 4. Genomic landscapes of lymphoid malignancies
4-1
Understanding the Molecular Pathogenesis of Peripheral T-cell Lymphoma by Laser
Microdissection-based Sequencing
Tran B. Nguyen (University of Tsukuba, Japan)
4-2
Genomic Characterization of Primary Central Nervous System Lymphoma
Kazutaka Fukumura (The University of Tokyo, Japan)
Session 5. Recent advances in lymphoma therapeutics
5-1
Hypoxic Resistance by Loss of NDRG2 Expression via Activation of the PI3K/AKT
Signaling Is One of the Main Pathogenic Features in ATLL and Other Cancers
Shingo Nakahata (University of Miyazaki, Japan)
5-2
Redox Status Dictates the Susceptibility of Mantle Cell Lymphoma to Bortezomib
Sudjit Luanpitpong (Mahidol University, Thailand)
5-3
Inhibitory Effect on Lymphoma Cells Proliferation by Regulating Cholesterol Metabolism
Pathway
Yukio Fujiwara (Kumamoto University, Japan)
5-4
Expression and Quantification of Apoptosis Associated Protein (Livin) in a Spectrum of
Lymphomas from Rural Africans in Zaria, Nigeria
Faruk Mohammed (Ahmadu Bello University Teaching Hospital, Nigeria)
19
5-5
Histone Deacetylase Inhibitors Inhibit Metastasis via Restoration of microRNAs and Its
Target CCR6 in Advanced Cutaneous T-cell Lymphoma
Akihro Kitadate (Akita University Graduate School of Medicine, Japan)
5-6
The Essential Role of c-kit-SCF Signaling in the Leukemic Stem Cells Mediated ATL Cell
Propagation and Drug Resistance
Wakako Kuribayashi (Chiba University, Japan)
5-7
Chemotherapy with Hybrid Liposomes for Acute Lymphatic Leukemia Leading to
Apoptosis in vivo
Hideaki Ichihara (Sojo University, Japan)
5-8
Overexpression of CADM1 Enhances Adhesion and Infiltration of ATL Cells
Yuki Kumagai (The University of Tokyo, Japan)
5-9
CCL22, CCL25, CXCL 10 and CXCL11 Chemokine Serum Levels in Patients with Follicular
Lymphoma Who Complicated with Bendamustine-associated Skin Rash
Yoshiharu Kusano (Japanese Foundation for Cancer Research, Japan)
Session 6. Molecular biology and targets of multiple myeloma
6-1
A Novel Role for Interleukin-34 in the Pathogenecity of Multiple Myeloma
Muhammad Baghdadi (Hokkaido University, Japan)
6-2
Antimyeloma Activity of Bromodomain Inhibitors on the Human Myeloma Cell Line U266
via Downregulation of MYCL
Kazuhito Suzuki (Tokyo Medical and Dental University, Japan)
6-3
RC2 Inhibition Sensitizes Myeloma Cells to Proteasome Inhibitors
Ola Rizq (Chiba University Graduate School of Medicine, Japan)
Session 7. Immunotherapy of hematopoietic neoplasms
7-1
Infiltrating Macrophages Express PD-L1 and PD-L2 in Lymphoma Microenvironment
Yoshihiro Komohara (Kumamoto University, Japan)
7-2
High Throughput Dynamic Evaluation of Immune Cytotoxicity in Lymphoid Tumors
through Impedance Analysis
Eiji Okubo (ACEA Biosciences Inc., USA)
7-3
Immunogenicity of Novel Antigen Expressed in Leukemia Stem Cells
Koji Ozawa (Keio University, Japan)
20
7-4
Cytolytic Anti-pan MHC-Class I and Class II mAbs Directly Induce Lymphoma Cell Death
via Large Pore Formation without Complement
Shuji Matsuoka (Juntendo University School of Medicine, Japan)
7-5
Dendritic Cell-based Immunotherapy Induces HTLV-1 Tax-specific CD8+ T Cells and
Reduces Proviral Loads
Atsuhiko Hasegawa (Tokyo Medical and Dental University, Japan)
Session 8. Others
8-1
Whole-Body Imaging with Single Cell Resolution for Unbiased Analysis of Cell Status
Shimpei I. Kubota (The University of Tokyo, Japan)
21
Participant List
Abi-Habib, Ralph Joseph
Lebanese American University, Lebanon
[email protected]
Hirabayashi, Yoko
National Institute of Health Sciences, Japan
[email protected]
Akao, Yukihiro
Gifu University, Japan
[email protected]
Hirao, Atsushi
Kanazawa University, Japan
[email protected]
Akashi, Koichi
Kyushu University, Japan
[email protected]
Ichihara, Hideaki
Sojo University, Japan
[email protected]
Alyahyawi, Alaa Rashed
Barts Cancer Institute, UK
[email protected]
Ichikawa, Tomonaga
University of Miyazaki, Japan
[email protected]
Baba, Tomohisa
Kanazawa University, Japan
[email protected]
Ishimura, Akihiko
Kanazawa University, Japan
[email protected]
Baghdadi, Muhammad
Hokkaido University, Japan
[email protected]
Isshiki, Yusuke
Chiba University, Japan
[email protected]
Cao Sy, Luan
University of Tsukuba, Japan
[email protected]
Ito, Etsuro
Hirosaki University, Japan
[email protected]
Chiba, Shigeru
University of Tsukuba, Japan
[email protected]
Iwama, Atsushi
Chiba University, Japan
[email protected]
Cho, Joong Myung
CrystalGenomics,Inc, Korea
[email protected]
Izumo, Seigo
Takeda Pharmceutical Company Limited, Japan
[email protected]
Couzinet, Arnaud Nicolas
Japanese Foundation for Cancer Research, Japan
[email protected]
Jamieson, Catriona H. M.
University of California, San Diego, USA
[email protected]
El-Sibai, Mirvat
Lebanese American University, Lebanon
[email protected]
Jiang, Qingfei
University of California, San Diego, USA
[email protected]
Ferrando, Adolfo
Columbia University, USA
[email protected]
Kagiyama, Yuki
National Cancer Center Research Institute, Japan
[email protected]
Figueroa, Maria Eugenia
University of Michigan, USA
[email protected]
Kato, Jun-ya
Nara Institute of Science and Technology, Japan
[email protected]
Fujiwara, Yukio
Kumamoto University, Japan
[email protected]
Katoh, Masaru
National Cancer Center, Japan
[email protected]
Fukumura, Kazutaka
The University of Tokyo, Japan
[email protected]
Katsumoto, Takuo
National Cancer Center Research Institute, Japan
[email protected]
Furukawa, Yusuke
Jichi Medical University, Japan
[email protected]
Kawabata, Kimihito Cojin
The University of Tokyo, Japan
[email protected]
Hasegawa, Atsuhiko
Tokyo Medical and Dental University, Japan
[email protected]
Kawakami, Yutaka
Keio University, Japan
[email protected]
22
Kimura, Kotohiko
Johns Hopkins University, USA
[email protected]
Licht, Jonathan D.
The University of Florida Health Cancer Center, USA
[email protected]
Kimura, Shunsuke
The University of Tokyo, Japan
[email protected]
Lorthongpanich, Chanchao
Mahidol University, Thailand
[email protected]
Kitabayashi, Issay
National Cancer Center, Japan
[email protected]
Luanpitpong, Sudjit
Mahidol University, Thailand
[email protected]
Kitadate, Akihiro
Akita University, Japan
[email protected]
Maie, Koichiro
University of Tsukuba, Japan
[email protected]
Kitagawa, Kyok
Hamamatsu University, Japan
[email protected]
Mano, Hiroyuki
The University of Tokyo, Japan
[email protected]
Kitamura, Toshio
The University of Tokyo, Japan
[email protected]
Matsubara, Masahiro
Kyowa Hakko Kirin Co., Ltd., Japan
[email protected]
Kochi, Yu
Kyushu University, Japan
[email protected]
Matsuki, Eri
Tachikawa Hospital, Japan
[email protected]
Komohara, Yoshihiro
Kumamoto University, Japan
[email protected]
Matsuoka, Shuji
Juntendo University School of Medicine, Japan
[email protected]
Kubota, Shimpei I.
The University of Tokyo, Japan
[email protected]
Matsushita, Maiko
Keio University, Japan
[email protected]
Kumagai, Yuki
The University of Tokyo, Japan
[email protected]
Melnick, Ari
Weill Cornell Medicine, USA
[email protected]
Kuribayashi, Wakako
Chiba University, Japan
[email protected]
Minami, Yosuke
Kobe University Hospital, Japan
[email protected]
Kusano, Yoshiharu
Japanese Foundation for Cancer Research, Japan
[email protected]
Miyagi, Satoru
Chiba University, Japan
[email protected]
Landau, Dan A.
Weill Cornell Medicine, USA
[email protected]
Miyata, Tomomi
Kitasato University, Japan
[email protected]
Lee, Doyoung
Crystal Genomics Inc., Korea
[email protected]
Miyauchi, Masashi
The University of Tokyo, Japan
[email protected]
Lee, Sang Yoon
Crystal Genomics Inc., Korea
[email protected]
Mizuguchi, Mariko
Tokyo Medical and Dental University, Japan
[email protected]
Levine, Ross Lawrence
Memorial Sloan Kettering Cancer Center, USA
[email protected]
Mochizuki-Kashio, Makiko
Chiba University, Japan
[email protected]
Li, Tsai-Kun
National Taiwan University College of Medicine, Taiwan
[email protected]
Mohammed, Faruk
Ahmadu Bello University, Nigeria
[email protected]
23
Morishita, Kazuhiro
University of Miyazaki, Japan
[email protected]
Saito, Shior
Jichi Medical University, Japan
[email protected]
Murai, Saomi
Takeda Pharmaceutical Company Limited, Japan
[email protected]
Sakai, Ryuichi
Kitasato University School of Medicine, Japan
[email protected]
Nagase, Reina
The University of Tokyo, Japan
[email protected]
Sera, Yasuyuki
Hiroshima University, Japan
[email protected]
Nakagama, Hitoshi
National Cancer Center, Japan
[email protected]
Shima, Yutaka
National Cancer Center Research Institute, Japan
[email protected]
Nakahata, Shingo
University of Miyazaki, Japan
[email protected]
Shinoda, Daisuke
Chiba University, Japan
[email protected]
Nakamura, Akito
Takeda Pharmaceutical Company Limited, Japan
[email protected]
Shinohara, Haruka
Gifu University, Japan
[email protected]
Nakamura, Takuro
Japanese Foundation for Cancer Research, Japan
[email protected]
Suzuki, Kazuhito
Tokyo Medical and Dental University, Japan
[email protected]
Nakata, Yuichiro
Hiroshima University, Japan
[email protected]
Takamatsu-Ichihara, Emi
National Cancer Center Research Institute, Japan
[email protected]
Nguyen, Tran Bich
University of Tsukuba, Japan
[email protected]
Takano, Junichiro
RIKEN IMS, Japan
[email protected]
Nishikawa, Hiroyoshi
National Cancer Center, Japan
[email protected]
Takita, Junko
The University of Tokyo, Japan
[email protected]
Ogawa, Seishi
Kyoto University, Japan
[email protected]
Takizawa, Hitoshi
Kumamoto University, Japan
[email protected]
Ohtsu, Tomoko
Celgene K.K., Japan
[email protected]
Tanaka, Miwa
Japanese Foundation for Cancer Research, Japan
[email protected]
Okubo, Eiji
ACEA Biosciences Inc., USA
[email protected]
Tara, Shiro
Chiba University, Japan
[email protected]
Ozawa, Koji
Keio University, Japan
[email protected]
Terui, Yasuhito
Japanese Foundation for Cancer Research, Japan
[email protected]
Riddell, Stanley R.
Fred Hutchinson Cancer Research Center, USA
[email protected]
Tiedemann, Rodger
Princess Margaret Cancer Centre, Canada
[email protected]
Rizq, Ola
Chiba University, Japan
[email protected]
Tobinai, Kensei
National Cancer Center Hospital, Japan
[email protected]
Sachithanandan, Sasikala Pallikuppam
National Centre for Biological Sciences, India
[email protected]
Ueda, Koki
Fukushima Medical University, Japan
[email protected]
24
Usui, Noriko
The Jikei University School of Medicine, Japan
[email protected]
Yamagata, Kazutsune
National Cancer Center Research Institute, Japan
[email protected]
Yi, Qing
Cleveland Clinic, USA
[email protected]
Yokota, Asumi
Kyoto University Hospital, Japan
[email protected]
Yokoyama, Takashi
Japanese Foundation for Cancer Research, Japan
[email protected]
Yoshino, Seiko
Japanese Foundation for Cancer Research, Japan
[email protected]
Zhu, Yinghui
Sun Yat-sen University, China
[email protected]
25
Abstracts of Invited Presentations
27
Keynote Lecture
Disordered Histone Methylation in Lymphoid Malignancy
Jonathan D. Licht
The University of Florida Health Cancer Center, Gainesville Florida, USA
Genetic alterations of epigenetic regulators that alter the levels or distribution of methylation of lysine 27 on
histone H3 (H3K27me) is a recurrent feature of a variety of cancers, including multiple myeloma (MM). The
histone demethylase UTX/KDM6A activates gene expression by removing the H3K27me3 repressive histone
mark, counteracting EZH2. UTX inactivating mutations are found in up to 10% of MM; nevertheless, the
mechanisms by which it contributes to disease remain to be elucidated.
Mass spectrometry analysis showed that global levels of H3K27me are not altered after UTX loss or upon its
add-back. Therefore, UTX depletion may alter H3K27me at specific loci. We performed RNA-sequencing
(RNA-seq) on the paired MM cell lines and the add-back system. This analysis revealed ~5,000 genes
differentially expressed between ARP-1 and ARD cells. Re-expression of UTX reversed the expression of
approximately 1,400 genes, most of them being upregulated by UTX. Gene ontology revealed that UTX
regulated JAK-STAT, cadherin, integrin and Wnt pathways. Chromatin immunoprecipitation experiments at
UTX target genes revealed a decrease in H3K27me3 and a concomitant increase in H3K4me3 upon UTX addback, correlating with changes in gene expression.
As loss of UTX leads to a failure in the removal of H3K27me3, we hypothesized that UTX-null cells may be
more dependent on EZH2 to maintain high H3K27me3 levels at specific loci. Treatment of the paired cell lines
with the EZH2 inhibitor GSK343 decreased the viability of UTX-null ARD cells, but had no effect on the UTX
wild-type ARP-1 cells. RNA-seq of ARD cells treated with GSK343 identified ~2,000 genes affected by the
drug, most of them upregulated. These genes partially overlapped with those responsive to UTX addback. Furthermore treatment of UTX deficient cells with GSK343 led to a pattern of de-differentiation of the
plasma cells to resemble germinal center B cells with increased expression of CD20, decreased CD138,
increased expression of transcription factors typical of germinal center B cells which leads to the silencing of
IRF4 and MYC, leading to cell death.
We have also studied a point mutation in NSD2/MMSET, which represent one of the few known gain of
function mutations in histone methyl transferases in cancer. This mutation E1099K is found in up to 15% of
cases of relapsed childhood cute lymphocytic leukemia, increases the activity of this enzyme and leads to a
global increase in H3K36me2 levels in cells with a concomitant decrease in H3k27me3 levels. Using CRISPRmediated gene editing we eliminated this mutant allele, yielding reversal of histone methylation patterns,
deceased growth, altered cellular adhesion and increased susceptibility to chemotherapy. RNA-seq analysis
showed that similar to the UTX mutation NSD2 mutation activated a program of cell adhesion, motility,
signaling and glucocorticoid resistance likely associated with tumor progression. Mice harboring ALL cells
with the E1099K mutation succumbed to disseminated leukemia faster than mice injected with cells in which
the mutation was corrected. Furthermore there was enhanced accumulation of cells with E1099K mutation in
the brain, a common site of relapse in childhood ALL.
In summary deregulation of histone methylation patterns in lymphoid malignancies play an important role in
pathogenesis and progression and rebalancing histone modifications using appropriate inhibitors represent a
potential target for therapy.
28
Keynote Lecture
Signaling and Transcriptioanl Networks in Myeloid Leukemogenesis
Takuro Nakamura
Division of Carcinogenesis, The Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan
Genetic cooperation is essential for leukemia initiating cells to acquire biological abilities to survive and expand
in bone marrow. Such cooperation involves signal transduction and transcriptional modulation in leukemic cells.
Identification and appropriate evaluation of oncogenic activities will therefore provide potential therapeutic
targets for cancer. We have been developing mouse models for human cancer based on the gene transfer to
stem/progenitor cells ex vivo. These approaches facilitate us to reveal unknown functions of the candidate
disease gene in disease progression and to determine the cell-of-origin of cancer.
The TALE-class homeoprotein MEIS1 specifically collaborates with HOXA9 to drive myeloid leukemogenesis.
Although MEIS1 alone has only a moderate effect on cell proliferation in vitro, it is essential for the
development of HOXA9-induced leukemia in vivo. Using murine models of leukemogenesis, we have found
that MEIS1 promotes leukemic cell homing and engraftment in bone marrow and enhances cell-cell interactions
and cytokine-mediated cell migration. We analyzed global DNA binding of MEIS1 in leukemic cells as well as
gene expression alterations in MEIS1-deficent cells and identified synaptotagmin-like 1 (Sytl1, also known as
Slp1) as the MEIS1 target gene that cooperates with Hoxa9 in leukemogenesis. SYTL1 promotes cell migration
via activation of the CXCL12/CXCR4 axis, as SYTL1 determines intracellular trafficking of CXCR4. These
results indicate that MEIS1, through induction of SYTL1, promotes leukemogenesis and supports leukemic cell
homing and engraftment, facilitating interactions between leukemic cells and bone marrow stroma.
Activation of Tribbles proteins is also an important event in HOX-related myeloid leukemogenesis. Tribbles
homolog 1 (Trib1) was identified as a common integration site of the HOXA9/MEIS1 retrovirus in AML. Trib1
is by itself a transforming gene for myeloid cells but also significantly accelerates progression of
Hoxa9/Meis1-induced AML. The strong transforming activity of Trib1 depends on its bi-directional function in
CCAAT/enhancerbinding protein (C/EBPα) degradation and MAPK/ERK activation. Although Trib1 knockout
did not suppress hematopoiesis in mouse bone marrow significantly, increase in mature granulocytes was
observed and promotion of myeloid differentiation was associated with the increased C/EBPα protein. These
data suggest that Trib1 plays an important role in myeloid cell transformation by affecting signal transduction
and modulation of the HOX-mediated transcriptional activities.
29
1. Aging and epigenetics in hematopoietic malignancies
Deregulated Polycomb-group Gene Functions in Hematological Malignancies
Atsushi Iwama
Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Japan
Polycomb-group (PcG) proteins function as transcriptional repressors through histone modifications. They have
been implicated in the maintenance of normal as well as cancer stem cells and thus have been characterized as
oncogenes. Indeed, in mouse AML models, PcG genes promote leukemogenesis. Of great interest, however,
inactivating mutations of polycomb repressive complex (PRC) 2 genes, such as EZH2 and ASXL1, have been
identified in patients with myelodysplastic disorders including myelodysplastic syndrome (MDS) and
MDS/myeloproliferative neoplasms (MPN) overlap disorders. In line with these findings, deletion of Ezh2 alone
was enough to induce MDS and MDS/MPN in mice. Furthermore, Ezh2 loss appeared to cooperate with other
mutations such as TET2, RUNX1 and JAK2V617F in the pathogenesis of MDS, MPN and MDS/MPN.
In contrast to the canonical PRC1, PRC1.1, consisting of Ring1b, Pcgf1, Kdm2b/Fbxl10, and Bcl6 corepressor
(Bcor), represents one of the variant PRC1 complexes. Recently, inactivating mutations of BCOR have also
been implicated in hematological malignancies. We found that deletion of exon 4 of Bcor induces acute T
lymphoblastic leukemia (T-ALL) in mice, suggesting a tumor suppressive function of PRC1.1 in hematopoietic
system. I would like to show several mouse models of hematological malignancies with PcG insufficiency and
discuss the role of PcG genes in hematological malignancies.
30
1. Aging and epigenetics in hematopoietic malignancies
Epigenetic Deregulation in the Aging Hematopoietic Stem Cell
Maria E. Figueroa
University of Michigan Medical School, USA
Maintenance of the hematopoietic stem cell (HSC) pool is crucial for the production of mature blood and bone
marrow cells. With age, there is loss of HSC function, exemplified by a decreased homing ability and an
increased predisposition to differentiate into myeloid rather than lymphoid cells. This age-associated loss of
HSC function contributes to an impaired hematopoietic system; elderly individuals have increased rates of
anemia, loss of adaptive immunity, and an increased risk to develop myeloid malignancies. One such disorder is
Myelodysplastic Syndrome (MDS), a disease that most frequently occurs in elderly individuals. Mutations in
proteins involved in alternative splicing and epigenetic modifiers such as DNMT3A and TET2 are frequently
observed in MDS, and can also occur in healthy elderly individuals. However, little is known about epigenetic
deregulation in the human hematopoietic system with aging, and whether such deregulation predisposes for
MDS. In order to investigate this, we examined epigenetic profiles in FACS purified HSC/HSPCs isolated from
bone marrow from young (18-30 yo) and elderly (65-75 yo) healthy donors. We have found marked epigenetic
differences during normal aging that target key pathways in development and disease. At the meeting we’ll
discuss our findings and their implications for human disease.
31
1. Aging and epigenetics in hematopoietic malignancies
Aging and TET Dioxygenases in Leukemo/lymphomagenesis
Shigeru Chiba
Departmentn of Hematology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
TET1/2/3 are dioxygenases that convert 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC).
Among the genes encoding these enzymes, TET2 is frequently mutated in both myeloid and lymphoid cancers.
As a result, TET2 enzymatic activity is thought to be impaired, which in turn drives leukemo/lymphomagenesis.
Importantly, TET2 mutations are often identified in apparently normal but clonal blood cells in the bood
cancer-carrying patients, as well as in those who do not have blood cancers. In contrast to TET2, mutations are
rare in TET1 and TET3 in any blood cancers. It has, however, been reported that the expression levels of TET1
and TET3 are decreased during aging, while TET2 expression levels are unaffected by aging in both human and
mouse blood cells. These observations, together with the fact that the three TET genes are expressed in a variety
of blood cells in an overlapping manner, imply that impairment of total TET function by mutations and reduced
expression might combinatorially causes initiation of clonal growth. To model such a compound condition,
double deletion of multiple Tet genes in mice is among the possible approaches. To this end, we have generated
Tet2/Tet3 doubly deleted mice, in which floxed Tet2 and Tet3 alleles are deleted by an interferon inducer pIpC.
Tet2 single deletion is known to cause myeloid skewing and development of a myeloproliferative phenotype,
though only after a long latency. Tet3 single deletion is reported to cause any blood cancers. In our observation,
no blood phenotypes were observed in compound heterozygous Tet2+/-Tet3+/- mice. However, Tet2/Tet3 doubly
deleted mice developed fully penetrant aggressive acute myeloid leukemia in a short latency; after pIpC
injection, 50% mice died at 8 weeks and 100% at 12 weeks. The AML cells derived from mice were easily
propagated in culture, and again could develop AML in vivo if injected into mice. A very short latency suggests
that profound impairment of the 5-mC to 5-hmC conversion is sufficient to cause AML without additional gene
mutations; the hypothesis to be confirmed by exome sequencing. It is also of interest to see the phenotypes of
mice with Tet2-/-/Tet3-/+ and Tet2-/+/Tet3-/-. These homozygous/heterozygous mice died much earlier than
heterozygous/heterozygous mice, with 50% of mice with both genotypes being dead developing AML-like
disease around 8 months after pIpC injection. Comparison of Tet2/Tet3 doubly deleted AML cells with
Tet2-/-/Tet3-/+ and Tet2-/+/Tet3-/- AML cells might provide insights into how aging-related impairment of TET
dioxygenase activity affects leukemogenesis.
32
2. Myeloproliferative neoplasms and myelodysplastic syndrome
Role of JAK-STAT Pathway Activation in MPN Pathogenesis and Therapeutic
Response
Maria Kleppe1, Sara Meyer2, Minsuk Kwak3, Priya Koppikar4, Thomas Radimerski5, Rong Fan6, Ross L.
Levine1
1
2
Memorial Sloan Kettering Cancer Center, USA
Huggenberger-Bischoff Cancer Foundation, Switzerland
3
Yale, USA
4
Amgen, USA
5
Novartis, Switzerland
6
Yale, USA
The discovery of JAK2/MPL mutations in the majority of patients with Myeloproliferative Neoplasms (MPN)
led to the development of JAK kinase inhibitors. JAK kinase inhibitors, including ruxolitinib, improve patient
constitutional symptoms and reduce splenomegaly, but do not significantly reduce mutant allele burden. Chronic
exposure to JAK kinase inhibitors results in JAK inhibitor persistence in cell lines, murine models, and patient
samples, which we have shown is due to JAK2-transactivation and persistent JAK-STAT signaling. We have
used murine genetic studies show that MPN cells remain dependent on JAK2 expression for proliferation and
survival. These data suggest that pharmacologic approaches that better inhibit JAK2 activity in MPN cells may
demonstrate increased efficacy in vivo.
All JAK inhibitors in clinical development are type I inhibitors that interact with and inhibit the active
confirmation of the JAK2 kinase. We hypothesized that novel, type II JAK inhibitors that interact with and
inhibit JAK2 in the inactive conformation might retain activity in JAK inhibitor persistent cells and show
increased efficacy in murine MPN models. Signaling studies demonstrated that CHZ868 more potently
attenuated JAK-STAT signaling in JAK2/MPL-mutant cells, with suppression of JAK2 phosphorylation
consistent with a type II mechanism of kinase inhibition. CHZ868 completely suppressed JAK-STAT signaling
in type I JAK inhibitor-persistent cells, and prevented heterodimeric activation of JAK2 by JAK1 and TYK2.
Most importantly, JAK2/MPL-mutant cells which were insensitive to type I JAK inhibitors remained highly
sensitive to CHZ868, demonstrating that type I JAK inhibitor persistence does not confer resistance to type II
inhibitors. CHZ868 showed significant activity in murine models of Jak2V617F-induced polycythemia vera,
and MPLW515L-mutant MF, with normalization of blood counts, stem/progenitor expansion, spleen weights,
and extramedullary hematopoiesis in vivo. Most importantly, CHZ868 resulted in significant reductions of
mutant allele burden in these models.
In addition, it has not been delineated whether the clinical benefits of JAK inhibitors are solely from target
inhibition in MPN cells, or if JAK-STAT inhibition in malignant and non-malignant cells contributes to the
therapeutic response. We show that the levels of circulating cytokines are elevated in MF and that cytokine
production is reduced with JAK inhibitor treatment. Importantly, aberrant cytokine production in MF emanates
from both malignant and non-malignant cells, and that JAK inhibition reduces cytokine production from both
tumor and non-tumor populations. Our data suggest inhibition of JAK-STAT signaling in malignant and
non-malignant cells is required to improve symptoms, reduce disease severity, and to reduce disease progression.
Moreover, potent inhibition of JAK-STAT signaling in malignant and non-malignant cells is warranted to
improve clinical efficacy and outcomes in MPN patients.
33
2. Myeloproliferative neoplasms and myelodysplastic syndrome
The ASXL1 Mutation and the EZH2 Mutation Induced Myelodysplastic
Syndromes in Mice via Distinct Mechanisms
Toshio Kitamura1, Kimihito C Kawabata1, Reina Nagase1, Makoto Saika1, Takeshi Fujino1, Susumu Goyama1,
Omar Abdel-Wahab2, Ross L Levine2, Daichi Inoue1
1
Div of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Japan
2
Memorial Sloan Kettering Cancer Institute, U.S.A.
Myelodysplastc syndromes (MDS) are heterogeneous diseases of hematopoietic stem cells characterized by
abnormal molphologies of myeloid cells, ineffective hematopoiesis and frequent leukemic transformation. Only
a few gene mutations were identified in the past, including Runx1, C/EBPa and Ras mutations. However,
technological advances in the next generation sequensing have identified a variety of novel somatic gene
mutations in MDS patients. These include mutations of epigenetic factors, splicing factors, signaling molecules,
and the molecules of the cohesin complex. Among them, mutations of epigenetic factors and splicing factors are
frequently identified in the early stages of MDS and are thought to be driving mutations. We have focused on
ASXL1 and EZH2 mutations leading to suppression of the functions of polycomb complex 2 (PRC2). Thus,
these mutations are supposed to derepress the expression of genes via decreased H3K27me3, a representative
repressive mark of histone induced by PRC2. To investigate the pathological roles of mutant genes in vivo, bone
marrow cells transduced with the mutated genes derived from patients are transplanted into irradiated mice.
Either an ASXL1 mutation with the C-terminal deletion (ASXL1-MT) or an EZH2 mutation lacking the SET
domain (EZH2-dSET) induced MDS-like diseases in the transplanted mice in a year or so. Interestingly,
although both ASXL1-MT and EZH2-dSET reduce H3K27me3, they induced MDS-like diseases through
distinct mechanisms; while ASXL1-MT derepressed the expression of HoxA9 and A10 as well as miR125a
leading to development of MDS, EZH2-dSET did not induce the expression of these genes. EZH2-dSET
derepressed the expression of ABC-G2 via decreased H3K27me3. Intriguingly, overexpression of ABC-G2
alone did induce an MDS-like disease. Moreover, expression of ABC-G2 is higher in patients with MDS than in
those with other hematological malignancies. We are now investigating how ABC-G2 expression induces MDS.
.
34
2. Myeloproliferative neoplasms and myelodysplastic syndrome
Inflammation-induced Malignant Transformation of Hematopoietic Stem Cell
Hitoshi Takizawa1, Larisa V. Kovtonyuk2, Kristin Frisch2, Markus G. Manz2
1
International Research Center for Medical Sciences, Kumamoto University, Japan
2
Division of Hematology, University Hospital Zurich, Switzerland
Steady state daily hematopoiesis is maintained mianly by highly proliferating hematopoietic progenitors (HPCs)
with lineage committed and limited selfrenewal potential, but rarely by slowly selfrenewing hematopoietic stem
cells (HSCs). Upon naturally-occurring hematopoietic stress such as infection, inflammation, short-lived
immune effector cells are rapidly consumed at the site of hematopoietic challenges, and eventualy replenished
by bone marrow (BM) resident HSCs. A fundamental question is how the local hematopoietic demand is sensed
and translated into hematopoiesis by distally located HSC. HSCs are shown to express subsets of extracellular
inflammatory receptors, and therefore, are able to respond to milieu of inflammatory signals that are locally
induced upon hematopoietic challenges and migrate to BM. Indeed, we have recently demonstrated that in vivo
systemic injection of thrombopoietin, known as HSC maintenance cytokine, activates quiescent HSCs into
self-renewing division and expansion, implying cytokine-mediated HSC activation. Recent studies including
our group have revealed that not only peripheral innate immun cells but also HSCs at the apex of hematopoietic
hierarhy express Toll-like receptor (TLR)-4, a cognate receptor for gram negative bacteria-derived
lipopolysaccharide (LPS). Repetitive systemic application of LPS, a model recapitulating gram negative
bacterial infection, directly activates TLR4 -TRIF-ROS-p38 signalling cascades in HSCs and reduced their
competitive fitness through increased proliferation and genotoxic stress. Intervention of TLR4-mediated
pathways can rescue HSC dysfunction indued upon LPS challenge. These findings highlight hematopoietic
demand-sensing mechanisms of HSC mediated through inflammatory- or pathogen recognition-receptors, and
might explain how chronic inflammation increases risk for malignant transformation of HSCs and development
of malignant HSC clonal diseases, such as myeloproliferative neoplasms, actue myleoid leukemia, as observed
in the population-based studies and animal models.
35
3. Recent advances in lymphoma therapeutics
Epigenetic Circuits and Their Perturbation in Lymphoid Neoplasms
Ari Melnick.
Weill Cornell Medicine, United States
Diffuse Large B-cell Lymphomas and follicular lymphomas (DLBCLs and FLs) are a heterogeneous group of
diseases initiating from germinal center (GC) B cells. GC B cells are uniquely specialized to tolerate rapid
proliferation, and physiological genomic instability in order to generate a diverse set of clones of cells encoding
high affinity antibodies. This accelerated micro-evolutionary process requires the concerted action of
transcription and epigenetic factors that collectively reprogram B cells to overcome many of their normal
checkpoint restraints and tolerate the various stresses associated with affinity maturation. The GC phenotype
poses a significant risk of malignant transformation to B cells, which must accordingly balance suppression of
checkpoints with suppression of pro-oncogenic genes.
Epigenetic regulatory complexes play a critical
role in lymphomagenesis.
The histone methyltransferase (HMT) EZH2, which epigenetically silences genes
through histone 3 lysine 27 methylation is upregulated in normal and malignant GC B cells. EZH2 is often
affected by gain of function mutations in lymphomas that alter its enzymatic specificity. EZH2 mediates GC
formation by transiently suppressing checkpoint genes and terminal differentiation genes through formation of
bivalent chromatin domains.
EZH2 somatic mutations induce germinal center hyperplasia and malignant
transformation, and cooperate with other oncogenes such as BCL2 and BCL6. EZH2 specific inhibitors can
suppress the growth of GC derived lymphoma cells in vitro and in vivo. Another HMT commonly mutated in
lymphomas is KMT2D, which normally mediates H3K4 mono and dimethylation.
KMT2D mutations result
in its loss of function, with consequent disruption of B-cell enhancer networks especially related to genes that
respond to key signaling pathways linked to terminal differentiation.
KMT2D loss results in development of
lymphomas in mice and cooperation with other oncogenes to accelerate transformation.
Collectively,
mutations in EZH2 cause promoters to be stuck in a repressed configuration whereas KMT2 mutation causes
enhancers to be aberrant repressed. These two mechanisms reflect disruption of dynamic epigenetic circuits
that normally transiently prevent key aspects of the B-cell program from manifesting until GC B-cells have
undergone immunoglobulin affinity maturation. Aberrant maintenance of gene silencing locks in the GC
phenotype leading to development of FL and DLBCL.
Patients who have these mutations exhibit
transcriptional repression signatures featuring silencing of genes affected by EZH2 effects at promoters and
KMT2D effects at enhancers respectively. Given the plasticity of the epigenome both of these aberrant
epigenetic programs can be reversed with specific inhibitors, some of which are already in clinical trials.
36
3. Recent advances in lymphoma therapeutics
New Agent Development for Lymphoid Malignancies in Japan
Kensei Tobinai
Department of Hematology, National Cancer Center Hospital, Tokyo, Japan
Current status of new agent development for lymphoid malignancies in Japan will be summarized.
1) Hodgkin lymphoma (HL)
For HL, anti-CD30 antibody-drug conjugate, brentuximab vedotin (BV), has shown its remarkable efficacy
as monotherapy for relapsed or refractory (R/R) patients (Cancer Sci 2014;105:840) and also in combination
with cytotoxic chemotherapeutic regimen, AVD, for untreated patients. The efficacy of the latter novel
combination is being compared with ABVD regimen in a global phase III study, aiming to establish a new
standard regimen for HL (NCT01712490). In addition, recent clinical investigations on anti-PD-1
antibodies, nivolumab and pembrolizumab, have shown high efficacy for R/R HL patients previously
treated with BV. The interim analysis of Japanese phase II study of nivolumab suggested its high efficacy
with acceptable toxicity profiles in patients with BV-resistant R/R HL (ASCO 2016). In addition, a global
phase III study of pembrolizumab vs. BV for R/R HL patients will be soon initiated (NCT02684292).
2) B-cell lymphoid malignancies
Among various emerging new agents under clinical investigation for B-cell lymphoid malignancies, one of
the most promising ones is a Bruton’s tyrosine kinase (BTK) inhibitor influencing B-cell receptor signaling
pathway, such as ibrutinib and ONO/GS-4059. Ibrutinib has shown its remarkable, long-lasting efficacy
with acceptable toxicity profiles for patients with R/R indolent B-cell malignancies including B-cell chronic
lymphocytic leukemia (B-CLL) (Int J Hematol 2016;103:86). Ibrutinib has shown its high efficacy for
R/R MCL in a Japanese phase II study (NCT02169180; JSH 2016), and also is being tested for untreated
MCL in combination with rituximab plus bendamustine as a global phase III study (NCT01776840). In
addition, based on its efficacy as monotherapy for activated B-cell (ABC)-like diffuse large B-cell
lymphoma (DLBCL), the efficacy of ibrutinib combined with R-CHOP regimen is being tested in a global
phase III study for untreated patients (JCO 32:5s, 2014 [suppl; abstr TPS8615]; NCT01855750). Another
group of promising agents for B-cell lymphoid malignancies is a phosphoinositide 3-kinase (PI3 kinase)
inhibitor, including idelalisib and copanlisib. These two agents are being tested as monotherapy and in
combination with rituximab +/- bendamustine for R/R B-cell lymphoid malignancies.
3) Peripheral T-cell lymphoma (PTCL) and adult T-cell leukemia-lymphoma (ATL)
Among various new agents under evaluation for PTCLs, mogamulizumab, a humanized anti-CCR4
antibody engineered to exert potent ADCC by defucosylation, has shown its promising efficacy for R/R
ATL (JCO 2010;28:1591 / JCO 2012;30:837). A subsequent phase II study of mogamulizumab for
relapsed PTCL and cutaneous T-cell lymphoma (CTCL) has also shown its promising efficacy (JCO
2014 ;32:1157). To establish the new standard treatment for untreated aggressive ATL, we conducted a
randomized phase II study of a dose-intensified multiagent regimen (VCAP-AMP-VECP) with or without
mogamulizumab. Higher complete remission rate of the former arm than the latter arm was obtained (52%
vs 33%), meeting the primary endpoint, suggested that VCAP-AMP-VECP plus mogamulizumab is a
reasonable treatment option for untreated aggressive ATL (Br J Haematol 2015;169:672). The results of
these clinical trials have led to the approval of mogamulizumab for relapsed and untreated ATL, and
relapsed PTCL/CTCL in Japan for the first time in the world. Among emerging other new agents targeting
ATL and PTCL, lenalidomide monotherapy has shown its promising efficacy for ATL in phase I and II
studies (Lancet Haematol 2016;3:e107 / ASH 2015;#181), warranting further investigation. Very recently,
based on the promising results in a preclinical study for ATL (Blood. 2016 Jan 15. pii:
blood-2015-08-662593. [Epub ahead of print]), the first-in-human study of EZH dual inhibitor, DS-3201b,
was initiated in Japan for R/R patients with non-Hodgkin lymphoma and ATL.
These efforts in cooperation with pharmaceutical industries will contribute to the further improvement of
therapeutic outcomes of patients with lymphoid malignancies.
37
3. Recent advances in lymphoma therapeutics
Recent Advances in Therapeutics for B-cell Lymphoma
Yasuhito Terui
Department of Hematology Oncology, Cancer Institute Hospital of Japanese Foundation for Cancer Research,
Japan
In the past decade, the treatment for B-cell lymphoma has been developed largely. Rituximab, a chimeric
monoclonal antibody that targets against CD20 molecule, is the first monoclonal antibody approved for the
treatment of B-cell lymphoma. It has been shown that rituximab exerts cytotoxic activities through CDC and
ADCC, and that it directly induces apoptosis. As the other anti-CD20 antibodies, there are ofatumumab and
obinutuzumab, and clinical development is on going. Antibodies against CD22 and CD79b molecules are also
developed for B-cell lymphoma. In addition, the inhibitors targeting apoptosis pathways, proteasome, and
other pathways of B-cell lymphoma are developing as novel drugs for B-cell lymphoma. Especially, BCR
pathway is one of the most crucial pathways, and ibrutinib is a small molecule, once-daily, orally available,
Bruton's tyrosine kinase (BTK) inhibitor that is under development for the treatment of B cell malignancies,
including chronic lymphocytic leukemia, mantle cell lymphoma, and diffuse large B cell lymphoma, as well as
follicular lymphoma. Here, I will discuss about “development of novel drugs for B-cell lymphoma in the present
and the future.
38
4. Leukemogenesis: Leukemia stem cell, metabolism and novel targets
Molecular Mechanism Linking Stem Cell Aging and Tumorigenesis in
Hematopoiesis
Atsushi Hirao
Division of Molecular Genetics, Cancer Research Institute, Kanazawa University, Japan
Stem cell self-renewal is critical for tissue homeostasis, and its dysregulation can lead to organ failure
or tumorigenesis. While self-renewal activity is required for maintenance of normal stem cells, it has
been assumed that oncogenic signals promote tumorigenesis due to abnormal acquisition of
self-renewal capacity. Therefore, we need to better understand the molecular mechanisms regulating
stem cell functions, particularly those distinguishing normal self-renewal activity from
tumor-associated proliferation.
Hematopoietic stem cells (HSCs) are maintained in an undifferentiated quiescent state within a bone
marrow niche. In order to maintain HSC homeostasis over the life of an animal, HSCs must either be
long-lived or self-renew. In either case, the quality of HSCs must be sustained during aging so that
these cells and their progeny are comparable to those derived from HSCs in a young animal. Although
aging process is complicated, mechanisms of HSC aging have been dissected by examining molecules
in stress responses, DNA damage/repair processes, epigenetics, inflammation, cellular senescence and
metabolism. Furtheremore, detailed analysis of the HSC aging may contribute to understanting how
leukemia stem cells are generated, because there are close relationship between aging and
tumorigenesis.
We previously focused on mTOR complex 1 (mTORC1) and forkhead transcription factor FoxO,
which function in control of cellular metabolism, for understanding of HSC homeostasis. In the
quiescent HSCs, the phosphorylation of AKT is down-regulated, associated with activation of FoxO3a.
FoxO3a-deficient HSCs showed increased phosphorylation of p38MAPK, an elevation of ROS,
defective HSC function during aging. Dysregulation of mTORC1 also causes abnormality in quiescent
status, resulting in HSC failure. Thus, appropriate controls of these signaling pathways play a pivotal
role in cell "cycle dormancy" of HSCs. On the other hand, it is also required to consider "cell
proliferation" of HSCs associated with undifferentiated status, so called self-renewal. We focused
regulation of c-Kit signal, because the engagement of the c-Kit receptor by SCF delivers one of the
most important signals governing self-renewal of HSCs. We found that Spred1, a negative regulator of
c-Kit, plays a critical role in regulation of self-renewal activity, HSC aging and leukemogenesis.
In this symposium, I would like to present recent data regarding how fine-turning of self-renewal
activity controls stem cell aging and tumorigenesis.
39
4. Leukemogenesis: Leukemia stem cell, metabolism and novel targets
Malignant RNA Editing Activation Drives Cancer Stem Cell Self-renewal by
Impairing Let-7 Biogenesis
Maria Anna Zipeto1,*, Angela C. Court1,*, Anil Sadarangani1, Nathaniel P. Delos-Santos1, Larisa Balaian1,
Hye-Jung Chun2, Gabriel Pineda1, Sheldon R. Morris1, Cayla N. Mason1, Ifat Geron1, Christian Barrett3, Daniel
J. Goff1, Russell Wall1, Maurizio Pellecchia4, Mark Minden5, Kelly A. Frazer3, Marco A. Marra2, Leslie A.
Crews1, Qingfei Jiang1, Catriona H. M. Jamieson1
1
Division of Regenerative Medicine, Department of Medicine, Moores Cancer Center and Sanford Consortium
for Regenerative Medicine, University of California, San Diego, La Jolla, CA, USA
2
Canada’s Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, BC, Canada
3
Department of Pediatrics, University of California, San Diego, CA, USA
4
Sanford Burnham Medical Research Institute, La Jolla, CA, USA
5
Princess Margaret Hospital, Toronto, ON, Canada
Recently RNA editing, particularly in the context of primate specific Alu sequences that represent 10% of the
human genome, has emerged as an important arbiter of human transcriptomic diversity by introducing novel
splice acceptor sites, editing 5' and 3' UTR sequences and modulating mRNA, microRNA and long non-coding
RNA stability. As the most abundant human RNA editing enzyme, adenosine deaminase associate with RNA
(ADAR) is usually activated in stem cell populations during embryogenesis or as a component of an innate
anti-viral immune response in hematopoietic stem cells. While ADAR-mediated adenosine to inosine (A-to-I)
RNA editing likely evolved to prevent propagation of RNA viruses in primitive stem cell populations,
cumulative RNA sequencing analyses show that ADAR activation contributes to therapeutic resistance and
relapse of a broad array of human malignancies. Recently, we discovered that ADAR1 activation enhances
malignant reprogramming of chronic myeloid leukemia (CML) progenitors into self-renewing cancer stem cells
by inducing missplicing of GSK3thereby preventing -catenin degradation.
Recently, we show that increased JAK2 signaling and BCR-ABL1 amplification in blast crisis (BC) CML
convergence on ADAR1 activation that disrupts the let-7/LIN28B self-renewal asix by imparing let-7
biogenesis. Combined RNA sequencing, qRT-PCR, CLIP-ADAR1, and pri-let-7 mutagenesis data suggest that
ADAR1 promotes self-renewal potential of CML progenitors via let-7 pri-microRNA editing and LIN28B
upregulation. Dual inhibition of JAK2 and BCR-ABL1 restored let-7 biogenesis and reduces cancer stem cell
self-renewal both in vitro and in humanized mouse models of CML. While previous studies have shown that
JAK2 signaling is important in the induction of numerous transcriptional mediators, our discovery of a pivotal
JAK2-ADAR1-let-7 self-renewal axis provides the first mechanistic link between inflammatory cytokine-driven
oncogenic signaling pathways and RNA editing-driven malignant reprogramming of progenitors into LSCs.
Perhaps most importantly, targeted reversal of ADAR1 activity may impede the generation of CSCs in a broad
array of therapeutically recalcitrant malignancies that evolve in inflammatory microenvironments.
40
4. Leukemogenesis: Leukemia stem cell, metabolism and novel targets
TIM-3 and Its Ligand, Galectin-9, Constitute an Autocrine Loop Universally
Critical for Development of Human Myeloid Leukemia Stem Cells
Yoshikane Kikushige, Koichi Akashi
Department of Medicine and Biosystemic Science, Faculty of Medicine, Kyushu University, Japan
Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate
therapeutic target. We have previously reported that the T-cell immunoglobulin mucin-3 (TIM-3) is expressed
on the surface of LSCs in most patients with AML of various FAB types, and that targeting TIM-3 by
anti-TIM-3 monoclonal antibodies could eradicate human AML LSCs in vivo by utilizing xenograft models (1).
We then tested the role of TIM-3 signaling evoked by its ligand, galectin-9 (Gal-9) in the development and
maintenance of human AML LSCs. We found that TIM-3+ AML cells secreted Gal-9 into sera, and the ligation
of TIM-3 by serum galectin-9 positively regulate the self-renewal capacity of TIM-3+ LSCs via co-activation of
NF-kB and β-catenin pathways. We found that this TIM-3/Gal-9 “autocrine stimulatory loop” is involved also
in other types of myeloid leukemias developed from preleukemic malignancies, including myelodysplastic
syndromes (MDS), myeloproliferative neoplasms (MPN) and chronic myelogeneous leukemia (CML).
Strikingly, in all cases, frequencies of CD34+CD38-TIM-3+ cells progressively increased along with disease
progression from early/chronic phase to overt leukemia. These data collectively suggest that TIM-3 and Gal-9
constitutes a pan-myeloid autocrine loop to develop malignant stem cells in the vast majority of human myeloid
malignancies (2). Thus, signaling molecules downstream of TIM-3 and galectin-9 ligation, as well as surface
TIM-3 itself might be good candidates for cancer stem cell-target therapy common to most types of myeloid
malignancies.
(References)
1. Kikushige, Y., Shima, T., Takayanagi, S., Urata, S., Miyamoto, T., Iwasaki, H., Takenaka, K., Teshima, T.,
Tanaka, T., Inagaki, Y. & Akashi, K. (2010) TIM-3 is a promising target to selectively kill acute myeloid
leukemia stem cells. Cell Stem Cell, 7, 708-717.
2. Kikushige Y., Miyamoto T., Yuda J., Tabrizi S.-J., Shima T., Takayanagi S., Niiro H., Yurino A., Miyawaki K.,
Takenaka K., Iwasaki H. & Akashi K. (2015) A TIM-3/Gal-9 autocrine stimulatory loop drives self-renewal of
human myeloid leukemia stem cells and leukemic progression, Cell Stem Cell 17, 341-52
41
4. Leukemogenesis: Leukemia stem cell, metabolism and novel targets
Novel Leukemia Stem Cell-targeted Therapy for Acute Myeloid Leukemia Based
on Dual Inhibition of EZH1/EZH2
Issay Kitabayashi
Division of Hematological Malignancy, National Cancer Center Research Institute, Tokyo, Japan
Despite great progress in curing acute leukemia and malignant lymphoma, survival after relapse remains poor.
The essential cause of the relapse following conventional chemotherapy is a remaining population of
drug-resistant cancer stem cells (CSCs). Thus, selective targeting of LSCs is a promising strategy for preventing
relapse. The polycomb repressive complex 1 (PRC1) and 2 (PRC2) regulate ubiquitination of histone H2A at
lysine 112 (H2AK112) and trimethylation of histone H3 at lysine 27 (H3K27), respectively.
We report here that RING1A/B and EZH1/2, which are catalytic subunits of PRC1 and PRC2, are essential for
maintenance of CSCs. Using Ezh1-null, Ezh2-conditional and their double knock-out mice, we examined the
effects of genetic deletion of EZH1/2 on AML, and found that double deletion of Ezh1/2 induced cell
differentiation and apoptosis more severely than single deletions in vitro in all subtypes of AML. In AML mice
models, deletion of Ezh1/2 induced differentiation of AML cells and complete remission of AML, which was
not achieved by single deletion of Ezh1 or Ezh2. Interestingly, the number of CSCs, especially quiescent CSCs,
dramatically reduced after deletion of both Ezh1 and Ezh2. However, such strong effects were not observed
after deletion of either Ezh1 or Ezh2. Taken together, these results strongly suggest that dual inhibition of EZH1
and EZH2 is a promising therapeutic strategy to eradicate CSCs in a wide range of AMLs.
We have developed potent and specific inhibitors against both EZH1 and EZH2, and found that the EZH1/2
dual inhibitor induced cell differentiation and apoptosis in most subtypes of AML tested in vitro, but a selective
EZH2 inhibitor did not affect the growth and survival of AML cells. Oral administration of the EZH1/2 dual
inhibitor selectively reduced the number of CSCs and prolonged the survival of the AML mice. Moreover,
combination of the EZH1/2 dual inhibitor and Ara-C prolonged survival more dramatically. The dual inhibitor
was also effective on treatment of malignant lymphomas in xenograft models. Based on these results we have
initiated Phase I clinical trial of the dual inhibitor in malignant lymphoma and will also start in AML presently.
42
5. Genetic and epigenetic alterations in childhood leukemia
Integrated Genetic Analysis of Pediatric T-cell Acute Lymphoblastic Leukemia
Junko Takita
Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Japan
T-cell acute lymphoblasic leukemia (T-ALL) is an aggressive malgnancy which accounts for 10-15% of
pediatric leukemia. In addition to distinct clinical characteristics such as an increased propensity for
extramedullary and central nervous system involvement, it is recognized that T-ALL is a heterogeneous disease
with unique biological and molecular features which may be of prognostic relevance. However, genetic basis of
pediatric T-ALL which contributes aggressive phenotype or pregression of disease is still to be elucidated.
Therefore, to discover oncogenic events in the pathogenesis of pediatric T-ALL and to identify novel prognostic
markers, we performed whole- exome sequencing (WES) and transcriptome sequencing (WTS) in 29 and 46
cases, respectively. We also performed targeted deep sequencing and SNP array karyotyping in our cohort. To
detect somatic mutations or fusion transcripts, we used our pipeline “Genomon-exome” and “Genomon-fusion”
algorithm. Subsequently, somatic mutations were validated using deep amplicon sequencing. Candidate fusion
transcripts were validated by reverse - transcription polymerase-chain-reaction and Sanger sequencing.
Recurrent mutations observed in more than 3 cases were detected in NOTCH1 (n = 18, 58%), FBXW7 (n = 7,
23%), PHF6 (n = 5, 16%), GATA3 (n = 4, 13%), MYB (n = 3, 10%), and NRAS (n = 3, 10%), respectively. We
identified previously known fusion genes, such as MLL-ENL (n = 2), CALM-AF10 (n = 2), NUP214-ABL1 (n =
1) and FGFROP1-FGFR1 (n = 1). MLL-ENL is one of the frequent translocation in infant multilineage
leukemia, but also reported in non-infant B cell precursor ALL and T-ALL. FGFR1OP is ubiquitously
expressed, and the predicted chimeric FGFR1OP-FGFR1 protein contains the catalytic domain of FGFR1. It is
thought to promote hematopoietic stem cell proliferation and leukemogenesis through a constitutive
phosphorylation and activation of the downstream pathway of FGFR1. CALM-AF10 leukemia is reported to
increase HOXA cluster gene transcription, we could also confirm elevated HOXA genes expression by FPKM
value. Four SIL-TAL1 fusions were detected in our cohort. Recently, a novel mutation in TAL1 enhancer region
which introduce de novo MYB biding site has been reported. Since this abnormality lead high expression of
TAL1, we also analyzed expression data obtained from WTS. Among 46 specimens, 19 samples showed high
expression of TAL1 (FPKM value ≥5). In those cases, 4 cases had SIL-TAL1 fusions (8%), and 3 cases (6%)
had insertions in enhancer region of TAL1. Subsequent analysis using Gene Set Enrich Analysis (GSEA)
between TAL1 high and low expression samples revealed that “LEE_EARLY_T_LYMPHOCYTE_UP” was
enriched in TAL1 high expression samples (Enrichment score = 0.73, FDR = 0.073). This gene set includes
genes up-regulated at early stages of progenitor T lymphocyte maturation compared to the late stages, and MYB
was included in this gene set. Intriguingly, MYB mutation samples were not represented TAL1 high expression.
TAL1 related rearrangement or enhancer insertion was not detected in the rest of 13 cases with TAL1 high
expression, suggesting that other mechanisms of TAL1 high expression might be exist.
In conclusion, although NOTCH1 and FBXW7 mutations were relatively frequently detected in our series, we
also found recurrent MYB mutations. SIL-TAL1 was known as most frequent rearrangement, TAL1 enhancer
insertions were also frequent in TAL1 overexpressed samples. TAL1 enhancer insertion and MYB mutation was
exclusive, suggesting that TAL1 and MYB have a key role in pediatric T-ALL. Consistent with other reports,
frequent translocations were not observed in T-ALL, and thus, our findings further illustrated the genetic
differences between pediatric T-ALL and other hematological malignancies. Further studies will be necessary to
unravel oncogenic mechanisms that implicated in new therapeutic strategy for pediatric T-ALL.
43
5. Genetic and epigenetic alterations in childhood leukemia
Oncogenic Signaling Pathways and Mechanisms of Resistance in Acute
Lymphoblastic Leukemia
Adolfo A. Ferrando
Institute for Cancer Genetics,Columbia University, USA
Dissecting the genetic and molecular mechanisms driving resistance to therapy in ALL is critical to for the
development of new more effective therapeutic strategies in high risk patients. These studies demonstrate a
complex and dynamic scenario in which specific mutations, deregulation of oncogenic signaling pathways, and
epigenetic and metabolic adaptation contribute to drug resistance. In this context, the development of new
advanced animal models that recapitulate the genetics and natural history of ALL are instrumental for the
discovery and testing of new drugs and drug combinations aimed at overcoming resistance to chemotherapy and
molecularly targeted drugs.
44
5. Genetic and epigenetic alterations in childhood leukemia
Genetic and Epigenetic Alterations in Acute Megakaryoblastic Leukemia in Down
Syndrome
Etsuro Ito1, Kenichi Yoshida2, Tsutomu Toki1, Satoshi Saida3, Kenichiro Watanabe4, Masahiro Nakamura5,
Kiminori, Terui1, Tatsutoshi Nakahata6, Satoru Miyano7, Akira Watanabe4, Seishi Ogawa2
1
2
Department of Pediatrics, Hirosaki University Graduate School of Medicine, Hirosaki, Japan
Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Japan
3
4
Department of Pediatrics, Graduate School of Medicine, Kyoto University, Japan
Department of Hematology and Oncology, Shizuoka Children’s Hospital, Shizuoka, Japan
5
Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, Japan
6
Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Japan
7
Laboratory of DNA Information Analysis, Human Genome Center, Institute of Medical Science, The University
of Tokyo, Japan
Transient abnormal myelopoiesis (TAM) is known as a clonal myeloid proliferation affecting ~10% of
neonatal infants with Down syndrome (DS). Although TAM is self-limiting in the majority of cases, about
20-30% of the survived infants develop acute megakaryoblastic leukemia (AMKL) within 4 years after the
remission. Blast cells in most patients with TAM and DS-AMKL have mutations in exon 2 of the gene coding
for the transcription factor GATA1, which is essential for the normal development of erythroid and
megakaryocytic cells. Investigation into the mechanism of progression from TAM to DS-AMKL can provide the
valuable insight of the evolution of leukemia.
To understand multi-step leukemogenesis in DS, we identified a comprehensive spectrum of gene
mutations in TAM/AMKL cases using whole genome sequencing of four trio samples sequentially obtained at
initial presentation of TAM, during remission and at the subsequent AMKL phase. Whole exome sequencing
was also performed for TAM (n = 15) and AMKL (n = 14) samples. TAM samples had significantly fewer
numbers of somatic mutations compared to AMKL samples with the mean numbers of non-silent somatic
mutations of 1.7 and 5.8 per sample including common GATA1 mutations in whole exome sequencing in TAM
and AMKL cases, respectively. Whereas GATA1 was the only recurrent mutational target in TAM samples, an
additional 8 genes were recurrently mutated in the DS-AMKL samples, including RAD21, STAG2, NRAS, CTCF,
DCAF7, EZH2, KANSL1 and TP53. We then performed targeted deep sequencing of 46 genes including these 8
genes in an extended set of 109 samples including TAM (n = 41), DS-AMKL (n = 49), and non-DS-AMKL (n =
19) using target deep sequencing. We found additional mutations frequently involving cohesin (53%)/CTCF
(20%), epigenetic regulators (45%) and RAS/JAK and other signaling molecules (47%) in DS-AMKL. Cohesin
mutations/deletions involved RAD21, STAG2, NIPBL, SMC1A, and SMC3 in a mutually exclusive manner. DSand non-DS-related AMKL also had substantially different mutational profiles.
Cohesion, CTCF, and other epigenetic regulators, such as EZH2, regulate various epigenetic phenomena
including DNA methylation. We next analyzed global DNA methylation profile of clinical samples including
TAM (n = 61), DS-AMKL (n = 10), non-DS-AMKL (n = 5), using Infiniun 450K DNA methylation array
(Illumina). Although averaged DNA methylation levels of both TAM and DS-AMKL were approximately same,
we observed two distinct clusters delineating TAM and DS-AMKL samples by both hierarchical clustering and
principle component analysis (PCA). We identified differentially methylated cytosines on the promoters
(TAM/AMKL-DMCpGs) among the three groups, and found that most of TAM/AMKL-DMCpGs were
hyper-methylated in DS-AMKL samples compared to TAM, indicating that hyper-methylation of the subset of
CpGs is a hallmark of DS-AMKL. To follow sequential alterations of DNA methylation profile, we established
a xenograft model of the leukemia, which mimicked the progression of TAM to DS-AMKL. We transplanted
TAM blast cells derived from the patient, who developed DS-AMKL later, into NOG mice. One series of the
xenografts exhibited engraftment in the mouse and could passage by serial engraftment in vivo, whereas the
other exhibited engraftment, but could not maintain in the mice by serial transplantation. The global DNA
methylation profile of engrafted cells from the first generation mice was closed to TAM patients, whereas those
from the forth, fifth and sixth generations approached to DS-AMKL patients. Moreover, hyper-methylation of
TAM/AMKL-DMCpGs was also observed in engrafted TAM cells with higher numbers of passages,
demonstrated that the xenograft model mimicked dysregulation of DNA methylation in disease progression of
TAM.
In conclusion, TAM is most likely caused by a single GATA1 mutation in combination with constitutive
trisomy 21. Subsequent AMKL evolves from a TAM clone acquiring additional mutations including
cohesin/CTCF, epigenetic regulators and RAS/JAK and other signaling molecules. Finally, epigenetic
dysregulation is a crucial step for malignant conversion of TAM into DS-AMKL.
45
6. Genomic landscapes of lymphoid malignancies
A Novel Genetic Mechanism of Evading Anti-tumor Immunity in Multiple Human
Cancers
Seishi Ogawa
Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Japan
Successful treatment of many advanced cancer patients using antibodies against programmed cell death 1
(PD-1) and its ligand (PD-L1) has highlighted a critical importance of immune escape in cancer development. In
accordance with this, recent studies relying on revolutionized DNA sequencing revealed that anti-tumor immune
reactions are recruited in many cancer tissues, where cancer cells are clonally selected by acquiring genetic
alterations that enable evading anti-tumor immunity. These findings underscore the critical importance of
utilizing or re-activating anti-tumor immunity for cancer treatment. Here we show a novel genetic mechanism of
immune escape caused by structural variations (SVs) commonly disrupting the 3’ part of the PD-L1 gene.
Widely affecting multiple common cancer types, these SVs invariably lead to a marked elevation of aberrant
PD-L1 transcripts that are stabilized by truncation of the 3’-untranslated region (UTR). Disruption of Pd-l1
3’-UTR enables immune evasion of tumor cells with elevated Pd-l1 expression in vivo, which is effectively
cancelled by Pd-1/Pd-l1 blockade, supporting the role of relevant SVs in clonal selection through immune
evasion. Our findings not only unmask a novel regulatory mechanism of PD-L1 expression, but also suggest a
possibility that PD-L1 3’-UTR disruption could serve as a genetic marker to identify patients who might
potentially benefit from PD-1/PD-L1 blockade therapy.
46
6. Genomic landscapes of lymphoid malignancies
Genetic and Epigenetic Determinants of Chronic Lymphocytic Leukemia
Evolution
Dan A. Landau1,2,3, Eugen Tausch4, Sebastian Böttcher5, Franco Izzo1,2, Chip Stewart3, Ivana Bozic6, Ignaty
Leshchiner3, Daniel Rosebrock3, Amaro N Taylor-Weiner3, Daniel Mertens7, Carrie Sougnez3, Sabrina Kless8,
Michael Kneba9, Matthias Ritgen5, Sandra Kluth10, Jasmin Bahlo10, Anna Fink10, Kirsten Fischer11, Stacey
Gabriel3, Eric Lander3, Hartmut Döhner12, Michael Hallek10, Donna Neuberg13, Martin A. Nowak6, Gad Getz3,
Stephan Stilgenbauer4, Catherine J. Wu3,14
1
Meyer Cancer Center, Weill Cornell Medicine, NY, NY, USA
2
New York Genome Center, NY, NY, USA
3
Broad Institute, Cambridge, MA, USA
4
Department of Internal Medicine III, Ulm University Medical Center, Ulm, Germany
5
Second Department of Medicine, University Hospital of Schleswig-Holstein, Kiel, Germany
6
Harvard University, Program for Evolutionary Dynamics, Cambridge, MA, USA
7
Mechanisms of Leukemogenesis, German Cancer Research Center (DKFZ), Heidelberg, Germany
8
Department of Internal Medicine III, Ulm University, Ulm, Germany
9
Department of Hematology, University Hospital Schleswig-Holstein, Kiel, Germany
10
Department I of Internal Medicine and Center of Integrated Oncology Cologne-Bonn, German CLL Study
Group, University of Cologne, Cologne, Germany
11
University Hospital Cologne, Cologne, Germany
12
University Hospital of Ulm, Ulm, Germany
13
Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA, USA
14
Medical Oncolobgy, Dana-Farber Cancer Institute, Boston, MA, USA
Cancer progression, relapse and resistance are the result of an evolutionary optimization process. Vast
intra-tumoral diversity provides the critical substrate for cancer to evolve and adapt to the selective pressures
provided by effective therapy. Thus, understanding intra-tumoral diversity and evolutionary dynamics will be a
critical step in the development of effective, curative therapies for cancer.
In order to study these questions, we characterized the intra-tumoral genetic heterogeneity of chronic
lymphocytic leukemia (CLL) using massively parallel sequencing of large patient cohorts. These studies have
shown that CLLs contain genetically distinct subpopulations that compete and mold the genetic makeup of the
malignancy. Furthermore, we have demonstrated that this heterogeneity can help predict the future evolutionary
trajectories of CLL, and that higher intra-tumoral heterogeneity in the pre-treatment sample predicts adverse
outcome.
Additionally, in order to comprehensively study cancer evolution, we developed tools to study intra-tumoral
epigenetic heterogeneity, as epigenetic somatic changes are heritable and impact the cellular fitness that is
selected in the evolutionary process. With these tools, we uncovered a central feature of the cancer epigenome:
massive stochastic disorder in methylation patterns.
We have further shown that this stochastic disorder
impacts transcription, evolution and clinical outcome. Thus, methylation changes in cancer may be similar to
the process of genetic diversification, in which stochastic trial and error leads to rare fitness enhancing events.
Ongoing efforts are dedicated to studying the quantitative evolutionary dynamics that enable the relapse clone to
replace the pre-treatment clone. Using deep sequencing with high temporal resolution we determine the therapy
specific clonal fitness with first line chemoimmunotherapy and targeted therapy. These investigations offer a
novel framework for the study of the evolutionary dynamics that underlie disease relapse, directly in patients.
Collectively, these studies demonstrate the tremendous degree of intra-tumoral diversity that fuels cancer
evolution, and highlight the need to integrate intra-tumoral heterogeneity in the development of the next
generation of cancer therapeutics.
47
6. Genomic landscapes of lymphoid malignancies
Discovery of DUX4-IGH Fusion-Type Oncogene
in B-Cell Acute Lymphoblastic Leukemia of AYA Generation
Hiroyuki Mano
Department of Cellular Signaling, Graduate School of Medicine, The University of Tokyo, Japan
Whereas acute lymphoblastic leukemia (ALL) is curable in 80 to 90% of affected children, its prognosis
worsens substantially in adolescents and young adults (AYA, 15–39 years old), despite recent improvements in
pediatric-type chemotherapeutic regimens. Oncogenic fusion genes such as BCR-ABL1 and those involving
KMT2A (also known as MLL) are associated with AYA-ALL at a lower frequency compared with ALL at
younger or older ages. Here we have searched for novel oncogenes in AYA-ALL by performing RNA
sequencing analysis of Philadelphia chromosome–negative AYA-ALL specimens with the use of a
next-generation sequencer. Surprisingly, translocation of the DUX4 gene predominantly to the IGH locus were
frequently identified in B-cell AYA-ALL and were found to result in a high level of expression of DUX4-IGH
fusion proteins. A transplantation assay in mice revealed that expression of DUX4-IGH in pro-B cells was
capable of generating B-cell leukemia in vivo. Additional screening of a second patient cohort revealed that
DUX4 fusions were present in 16.4% of the combined cohorts of AYA generation. Further, gene fusions
involving ZNF384 or MEF2D were also found in 16.4% and 8.2% of such cases, respectively, with both types
of fusion gene also possessing transforming ability. Interestingly DUX4 fusions were preferentially detected in
the AYA generation. Our data thus reveal that DUX4 becomes an oncogenic driver as a result of somatic
chromosomal rearrangements and that AYA-ALL may be a clinical entity distinct from ALL at other ages.
48
7. Immunotherapy of hematopoietic neoplasms
Current Progress and Future Perspectives in Cancer Immunotherapy:
Development of Personalized Combination Immunotherapy
Yutaka Kawakami
Division of Cellular Signaling, Institute for Advanced Medical Research,
Keio University School of Medicine, Japan
Immune-checkpoint blockade and T-cell based adoptive cell therapy have recently shown durable clinical
effects on the patients with various advanced cancers including hematological malignancies (e.g. Hodgkin’s
lymphoma, B cell-ALL/CLL/lymphoma, multiple myeloma). However, there are still many non- or partialresponders. Thus, the identification of biomarkers to select appropriate patients and appropriate
immunotherapies as well as the improvement of immunotherapy efficacy (including converting non-responders
to responders) possibly through combination strategy are needed.
In the development of combined immunotherapies, the following issues targeting different regulation points in
the anti-tumor T-cell responses and their appropriate combination need be considered; 1) identification of
appropriate tumor antigens such as immunogenic mutated antigens expressed also in cancer initiating cells, 2) in
situ tumor destruction to induce immunogenic cancer cell death, 3) enhancement of antigen presenting / T-cell
stimulatory functions of dendritic cells (DC), 4) in vivo activation and expansion of anti-tumor T-cells, 5)
Reversal of cancer-induced immunosuppression. Clinical trials of various combined immunotherapies have
been already in progress particularly along with PD-1/PD-L1 blockade in patients with various cancers. We
have been evaluating various low molecular weight compounds and antibodies which are able to augment
anti-tumor effects of PD-1/PD-L1 blockade through various mechanisms in in vitro human and in vivo murine
tumor models.
Pretreatment immune status varies among cancer patients (e.g. infiltrations of T-cells/B-cells and expresson of
immune-checkpoint molecules in tumors, levels of soluble factors such as cytokines and immune-cells in blood)
and it correlates with responses to various cancer therapies including surgery, chemotherapy, radiation, and
immunotherapies. The immune status may be defined by cancer cell’s genetic characteristics (e.g. immunogenic
passenger mutations, immunosuppressive driver mutations), patients’ immune-reactivity (e.g. SNPs), and
environmental factors (e.g. intestinal microbiota, smoking). Because the immune conditions are different among
cancer types/subpopulations and individual patients, appropriate immune-intervensions may be applied for each
patient.
Altogether, personalized combination therapy based on the evaluation of patients’ immune status may be
exploited for further improvement of current cancer immunotherapies.
49
7. Immunotherapy of hematopoietic neoplasms
Critical Role of Regulatory T Cells in Cancer Immunotherapy
for Hematologic Malignancies
Hiroyoshi Nishikawa1,2
1
Division of Cancer Immunology, Research Institute/EPOC, National Cancer Center, Japan
2
Department of Immunology, Nagoya University Graduate School of Medicine, Japan
There are two types of tumor antigens: tumor-specific antigens (TSAs), which are either oncogenic viral
proteins or abnormal proteins that stem from somatic mutations (neoantigens), and tumor-associated antigens
(TAAs), which are highly or aberrantly expressed normal proteins. It is not yet determined how CD8+ T cells
specific for each antigen contribute to clinical tumor regression and whether activation of these self vs non-self
antigen specific CD8+ T cells are controlled differently. Natural FoxP3+CD4+ regulatory cells (Tregs) inhibit
anti-tumor immune responses. To address the differences of Treg suppression, Melan-A (a representative TAA)and CMV (a surrogate antigen of TSA)-specific CD8+ T cells were stimulated in the presence of Tregs.
Melan-A-specific CD8+ T cells activated with Tregs underwent one cell division and then stopped further
proliferation. Tregs render Melan-A-specific T cells anergic (i.e., hypo-proliferative and cytokine
hypo-producing upon antigen re-stimulation) by controlling the co-stimulatory function of antigen-presenting
cells. These Melan-A-specific CD8+ T cells were identified by CCR7/CTLA-4 co-expression and contributed to
the tolerance of tumor (self)-antigens. In sharp contrast, while CMV-specific CD8+ T cells proliferated in the
presence of Tregs, they highly expressed co-inhibitory molecules such as PD-1, suggesting the importance of
anti-PD-1 mAb for resurgence of anti-tumor activity by neoantigen-specific CD8+ T cells.
In the treatment of hematologic malignancies, some anti-cancer reagents specifically targeting malignant cells
such as tyrosine kinase inhibitors (TKIs) cross-react with immune cells including T cells. Molecular-targeted
therapies against these molecules may therefore modulate anti-tumor immune responses as well, indicating the
importance of evaluating immune responses. FoxP3+CD4+ T cells can be dissected into three subpopulations by
the expression levels of FoxP3 and CD45RA: FoxP3loCD45RA+ cells (naïve Tregs), FoxP3hiCD45RA- cells
(effector Tregs, eTregs), and FoxP3loCD45RA- non-Tregs. One of TKIs, imatinib, showed selective depletion of
highly suppressive effector Tregs in CML patients in complete molecular remission whereas this was not seen in
paitients who failed to enter molecular remission. The former group concurrently increased the number of
effector- or memory-type CD8+ T cells producing multiple cytokines. In vitro, imatinib induced apoptosis
predominantly in effector Tregs, augmenting CD8+ T-cell responses against various tumor antigens in healthy
individuals and cancer patients. Therefore, it is becoming clear that there is a need to consider immune
responses in a wide range of cancer treatments.
50
7. Immunotherapy of hematopoietic neoplasms
Engineering Safe and Effective T Cell Therapy for Cancer
Stanley Riddell1,2, Turtle, C1,2, Maloney D1,2, Liu L1, Srivastava S1, Balakrishnan A1, Salter A1, Veatch J1,
Cabonov A1, Hill T1
1
Fred Hutchinson Cancer Research Center, USA
2
University of Washington, USA
Advances in understanding the mechanisms by which cancers evade immune recognition have led to new
immunotherapies that have the potential to transform the management of many human cancers. T lymphocytes
are critical to adaptive immunity to pathogens and tumors because of their longevity and ability to clonally
expand, and are increasingly being manipulated for the treatment of cancer. Advances in gene transfer methods
using viral and non-viral delivery have made it possible to introduce genes into T cells that encode natural T cell
or synthetic receptors that bind to molecules on the surface of cancer cells, and redirect the specificity of the T
cell to mediate tumor cell destruction. Several groups, including our own have shown that the adoptive transfer
of T cells that are genetically engineered to express a synthetic chimeric antigen receptor (CAR) that targets the
B cell lineage molecule CD19 exert potent antitumor activity in patients with chemotherapy refractory B-cell
malignances including acute lymphoblastic leukemia, chronic lymphocytic leukemia and non-Hodgkin’s
lymphoma. T cells exist in different subsets with cell intrinsic properties that affect their ability to persist and
proliferate after adoptive transfer. By selecting T cells with optimal attributes for genetic modification and
adoptive therapy, eradication of massive tumor burdens can be accomplished in patients with remarkably low
doses of engineered T cells, and with predictable T cell expansion kinetics and toxicity. A remaining challenge is
to identify target molecules that are expressed on common human cancers and to design receptors that safely
target them so that adoptive immunotherapy with genetically redirected T cells can be broadly applied. This
obstacle may be overcome by novel receptor designs that provide multifunctionality to the engineered T cells
including the ability to be eliminated in the event of serious toxicities. Thus, advances in understanding the
biology of T cells and their intrinsic properties combined with those in cell engineering provide new
opportunities to treat cancer with therapeutic T cells.
51
8. Molecular biology and targets of multiple myeloma
Intra-Tumor Heterogeneity in Multiple Myeloma
and It’s Role in Therapeutic Resistance and Relapse
Rodger E. Tiedemann1,2
1
Princess Margaret Cancer Centre, Toronto, ON, Canada
2
University of Toronto, Toronto, ON, Canada
Multiple Myeloma (MM) is a mature B cell neoplasm characterized by the accumulation of
immunoglobulin-secreting plasma cells (PCs) within the bone marrow. Despite treatment advances, MM
remains incurable in the majority of patients and is over represented in current cancer death rates. The lack of
reliable cure in MM, despite the achievement of deep clinical responses with proteasome inhibitors (PIs),
immunomodulatory drugs (IMiDs) and myeloablative melphalan, implicates the presence of intra-tumoral
heterogeneity and the existence of drug-resistant cells with tumorigenic capacity. However the molecular and
cellular basis of this intra-tumoral heterogeneity remains only partially understood. Using a sequential FACS
and single cell immunoflourescence-FISH strategy we have identified clonal tumor progenitor subpopulations
within the bone marrow of MM patients that recapitulate the physiologic maturation stages between post
germinal centre B cells and plasma cells. These clonal MM subpopulations include tumor cells that resemble
CD20+ CD27+ CD38- CD138 -Irf4- Xbp1s- memory B cells, CD38+ CD138- Irf4- Xbp1s- pre-plasmablasts, CD38+
CD138- Irf4+ XBP1s+ plasmablasts and tumor-bulk CD38+ CD138+ IRF4+ Xbp1s+ plasma cells. Importantly,
compared to tumor bulk plasma cells, early Xbp1s- tumor progenitor cells appear innately resistant to mainstay
treatment with proteasome inhibitors (PI). Mechanistically, the absence of Ire1-Xbp1 signaling in these
primitive MM cells appears to be associated with secretory immaturity. As Xbp1s- MM cells produce less
secretory immunoglobulin, a principal determinant of endoplasmic reticulum (ER) load in PCs, they experience
less ER stress than Xbp1s+ cells and are thus predictably less susceptible to induction of cytotoxic ER stress
when ER-associated degradation (ERAD) is inhibited by PIs. Significantly, small numbers of Xbp1s- tumor
cells can be detected in most MMs and as these progenitor cells are innately insensitive to PI-induced cytotoxic
ER stress, these observations can account for a universal low level resistance that is inherent in the pathogenesis
and residual progenitor structure of MM and that prevents PI-induced cure. Whilst these findings may account
for some clinical features of MM, at present it remains unknown whether or not Xbp1s- MM tumor progenitors
are fully malignant or if they possess the capacity required to re-initiate MM disease. Conceivably, the non PC
cells in MM may instead represent a counterpart to the clonal hematopoiesis and pre-malignant pre-leukemic
stem cells identified in AML, CLL and HCL; or they may alternatively represent de-differentiated PCs.
Furthermore, although this heterogeneity of tumor cell maturation may contribute to therapeutic evasion, MM
tumors also commonly contain multiple genetic subclones that show tidal dominance under therapeutic pressure
and that can also facilitate treatment escape. Notably, this genetic heterogeneity is often assumed to be mutually
exclusive with functional diversity in tumor cell phenotype such as that described above, but in MM we show
these processes to be orthogonal and non-exclusive. When MM genetic subclones with common recurrent
secondary chromosomal structural abnormalities (such as del 13, del 17p, +1q or del 1p) are identified amongst
tumor bulk PCs, similar subclones may also be identified amongst non-PC Irf4- Xbp1s- MM subpopulations,
suggesting that tumor progenitor cells may play a role in the genetic evolution of tumors and may represent a
reservoir for subclonal relapse. In support of a model in which MM PCs can be replenished from MM
progenitor cells, we have found that primary Xbp1s- MM progenitors possess the ability to differentiate in vitro
into mature Xbp1s+ MM plasma cells. Notably, however, in contrast to a model in which MM PCs are
continuously replenished by MM progenitors, we have also identified some genetic events that appear to show
restricted representation amongst tumor PCs, consistent with MM PCs comprising a self-sustaining tumorigenic
population in its own right. Overall, these data support a model in which Xbp1s- progenitor subpopulations in
MM represent a reservoir of innately PI-resistant cells that, following eradication of tumorigenic PCs, may give
rise to clonally-related clinical relapse via MM progenitor cell differentiation. Overall these findings imply that
future treatment strategies in MM may need to better address tumor progenitor cells, whilst also erradicating
MM PCs and all subclonal lineages, in order to achieve deeper treatment responses and cure for patients.
52
8. Molecular biology and targets of multiple myeloma
Epigenetic Mechanisms of Drug Resistance in Multiple Myeloma
Yusuke Furukawa, M.D.
Division of Stem Cell Regulation, Center for Molecular Medicine, Jichi Medical University, Japan
Multiple myeloma is caused by neoplastic transformation of terminally differentiated B-lymphocytes, named
plasma cells, with capability of producing immunoglobulin. As their normal counterpart, multiple myeloma
cells mainly proliferate and survive within a bone marrow milieu via physical and functional interactions with
bone marrow stromal cells (BMSCs). The interaction with BMSCs also confers anti-cancer drug resistance to
myeloma cells via two overlapping mechanisms. First, BMSCs produce soluble factors, such as interleukin-6
and insulin-like growth factor-1 (IGF-1), to activate the signal transduction pathways leading to drug resistance
in myeloma cells. Second, BMSCs up-regulate the expression of cell cycle inhibitors (CDK inhibitors p21 and
p27), anti-apoptotic members of the Bcl-2 family (Bcl-2 and Mcl-1) and ABC family drug transporters in
myeloma cells upon direct adhesion. These forms of drug resistance are termed soluble factor-mediated drug
resistance (SFM-DR) and cell adhesion-mediated drug resistance (CAM-DR), respectively, and are causally
related each other to make multiple myeloma one of the most reluctant malignancies. Elucidation of the
mechanisms underlying drug resistance may greatly contribute to the advancement of cancer therapies and thus,
is the subject of current extensive investigations. Given the feasibility of therapeutic application, we have
focused on the epigenetic aspect of CAM-DR in multiple myeloma. We found that proteasome inhibitors were
able to overcome CAM-DR by disrupting VLA-4-mediated interaction of myeloma cells with BMSCs (1).
Down-regulation of VLA-4 is not NF-kB-dependent but via epigenetic mechanisms because the inhibition of
histone deacetylases (HDACs) reproduced this process (2). Proteasome inhibitors repress transcription of class I
HDAC genes through caspase-8-mediated cleavage of Sp1 transcriptional activator (3, 4). Furthermore, we
identified trimethylation of histone H3 at lysine-27 (H3K27me3) as a critical histone modification for CAM-DR
in multiple myeloma. Cell adhesion counteracted drug-induced hypermethylation of H3K27 via inactivating
phosphorylation of the H3K27-specific methyltransferase EZH2, leading to the sustained expression of
anti-apoptotic genes including IGF1, BCL2 and HIF1A. Inhibition of the IGF-1R/PI3K/Akt pathway was able to
reverse CAM-DR by promoting EZH2 dephosphorylation and H3K27 hypermethylation both in vitro and in
refractory murine myeloma models (5). These findings provide a rationale for the inclusion of epigenetic drugs,
such as HDAC inhibitors and histone demethylase inhibitors, in combination chemotherapy to increase the
therapeutic index in multiple myeloma.
Related publications:
1) Noborio-Hatano K, Kikuchi J, Takatoku M, et al.: Bortezomib Overcomes Cell Adhesion-mediated Drug
Resistance through Downregulation of VLA-4 Expression in Multiple Myeloma. Oncogene 28: 231-242, 2009.
2) Sripayap P, Nagai T, Hatano, K, et al. Romidepsin Overcomes Cell Adhesion-mediated Drug Resistance in
Multiple Myeloma Cells. Acta Haematol. 132: 1-4, 2014.
3) Kikuchi J, Wada T, Shimizu R, et al. Histone Deacetylases Are Critical Targets of Bortezomib-induced
Cytotoxicity in Multiple Myeloma. Blood 116: 406-417, 2010.
4) Kikuchi J, Yamada S, Koyama D, et al. The Novel Orally Active Proteasome Inhibitor K-7174 Exerts
Anti-myeloma Activity in Vitro and in Vivo by Down-regulating the Expression of Class I Histone Deacetylases.
J. Biol. Chem. 288: 25593-25602, 2013.
5) Kikuchi J, Koyama D, Wada T, Izumi T, Hofgaard PO, Bogen B, Furukawa Y. Phosphorylation-mediated
EZH2 Inactivation Promotes Drug Resistance in Multiple Myeloma. J. Clin. Invest. 125: 4375-4390, 2015.
53
Abstracts of Poster Presentations
55
1-1
Targeting the MAP Kinase Pathway in Human Acute Myeloid Leukemia Cells
Using a Recombinant Anthrax Lethal Toxin Inhibits Proliferation through the Rho
GTPase Pathway
Saleh Al-Dimassi, Ralph Abi-Habib, Mirvat El-Sibai
Lebanese American University, Natural Science, Beirut, Lebanon
Around 15,000 new case of Acute Myeloid Leukemia are diagnosed each year with a fatality rate of 65%. The
poor prognosis rate is due to the high proliferative and quick progressive characteristics of AML. Previous
studies in our laboratory showed that proliferation in AML cells can be inhibited through the inhibition of the
mitogen-activated protein kinase (MAPK) pathway using a recombinant anthrax lethal toxin (LeTx). In this
study, we examined the effect of Rho GTPases, downstream from the MAPK pathway, on cell proliferation and
apoptosis. We tested potency of LeTx on a panel of 11 human AML cell lines. Seven cell lines showed cytotoxic
responses to LeTx and by the specific mitogen-activated protein/extracellular signal–regulated kinase kinase 1/2
(MEK1/2) inhibitor U0126, indicating that LeTx-induced cell death is mediated through the
MEK1/2–extracellular signal–regulated kinase (ERK1/2) branch of the MAPK pathway. This cytotoxicity was
mimicked in cells transfected with siRNA against the Rho GTPases RhoA and Cdc42 but not Rac. This effect
was reversed in cells that were treated with LeTx and transfected with constitutively active Cdc42 or RhoA
constructs showing that the effect of LeTx is mediated through the Rho GTPases. We also looked at the
activation of Cdc42 and Rho in a pull-down assay and the activation in these cells was significantly reduced
after treatment with LeTx proving that the MAPK pathway is activating Cdc42 and RhoA in these cells.
In this study, we have shown that a majority of AML cell lines are sensitive to the LF-mediated inhibition of the
MAPK pathway and that this was, at least in part, mediated through RhoA and Cdc42 and their downstream
effectors.
Keywords: Anthrax toxin, AML, MAPK, RhoA, Cdc42.
56
1-2
Tetra-O-Methyl Nordihydroguaiaretic Acid Broadly Suppresses Cancer
Metabolism and Synergistically Induces Strong Anticancer Activity in
Combination with Etoposide, Rapamycin and UCN-01
Kotohiko Kimura, Ru Chih C Huang
Department of Biology, Johns Hopkins University, Baltimore, M.D., U.S.A.
We have been developing an anticancer drug, Tetra-O-methyl nordihydroguaiaretic acid (M4N), for many years.
M4N is currently in Phase I/II clinical trials in patients with various advanced cancer. The results of these
clinical trials so far indicate that M4N has a certain degree of anticancer activity, but was unable to induce
remission in any of these patients with advanced cancers. One of the most frequently attempted strategy to
increase anticancer efficacy of chemotherapy drugs is combination treatment with one or more appropriately
selected drugs. An important finding from the clinical trials with M4N is that the toxicity of the drug was very
low. Patients were able to tolerate high doses of M4N with minimal side-effects, which make this drug very
suitable for being use in multidrug treatments (for instance the LD50 of M4N for mice is greater than 1000mg/kg
while that of NDGA is only 75mg/kg). In this study the ability of M4N to induce rapid cell death in combination
with Etoposide, Rapamycin, or UCN-01 was examined in LNCaP cells, both in cell culture and animal
experiments. Mice treated with M4N drug combinations with either Etoposide or Rapamycin showed no
evidence of tumor and had a 100% survival rate 100 days after tumor implantation. By comparison all other
vehicles or single drug treated mice failed to survive longer than 30 days after implantation. This synergistic
improvement of anticancer effect was also confirmed in more than 20 cancer cell lines including several
leukemic cell lines (L428 Hodgkin lymphoma cells, K528 myelogenous leukemia cells). In LNCaP cells, M4N
was found to reduce cellular ATP content, and suppress NDUFS1 expression while inducing hyperpolarization
of mitochondrial membrane potential. M4N-treated cells lacked autophagy with reduced expression of BNIP3
and ATG5. To understand the mechanisms of this anticancer activity of M4N, the effect of this drug on three
cancer cell lines (LNCaP prostatic, AsPC-1 pancreatic, and L428 leukemic cells) was further examined via
transcriptome and metabolomics analyses. Metabolomic results showed that there were reductions of 26
metabolites essential for energy generation and/or production of cellular components in common with these
three cell lines following 8 hours of M4N treatment. Deep RNA sequencing analysis demonstrated that there
were sixteen genes whose expressions were found to be modulated following 6 hours of M4N treatment
similarly in these three cell lines. Six out of these 16 genes were functionally related to the 26 metabolites
described above. One of these up-regulated genes encodes for CHAC1, a key enzyme affecting the stress
pathways through its degradation of glutathione. In fact M4N was found to suppress glutathione content and
induce reactive oxygen species production. The data overall indicate that M4N has profound specific negative
impacts on a wide range of cancer metabolisms supporting the use of M4N combination treatments for cancers
of various origins including leukemia.
57
1-3
CCL3-expressing Basophil-like Leukemia Cells Conduct Leukemic Hematopoiesis
in Chronic Myeloid Leukemia
Tomohisa Baba, Yamato Tanabe, Naofumi Mukaida
Cancer Research Institute, Kanazawa University, Japan
Accumulating evidence suggests that an inflammatory chemokine, CCL3, has multiple functions in
hematopoietic system besides its pro-inflammatory activities. CCL3 can in vitro inhibit the proliferation of
hematopoietic stem/progenitor cells (HSPCs). Based on this unique function, CCL3 is also called as “stem cell
inhibitor”. Thus, CCL3 can potentially influence the homeostasis of HSPCs. In sharp contrast, CCL3 hardly
affects leukemia initiating cells (LICs) in chronic myeloid leukemia (CML) although they share several
characteristic capabilities including self-renewal and cellular quiescence, with normal HSPCs. Thus, CCL3 can
be a potent mediator to induce the dominant proliferation of LICs in the CML BM. However, there is a dearth of
studies precisely demonstrating the major cellular source of CCL3 in BM and the contribution of
endogenously-produced CCL3 to the CML pathophysiology.
Basophilia is a frequently observed hematological abnormality in CML but its pathophysiological roles are
undefined. Herein, our meticulous analysis revealed that basophil-like leukemia cells are a major cellular source
of CCL3 in BM of murine CML model. Moreover, CCL3-expressing basophil-like leukemia cells accumulated
in CML BM and basophil-derived CCL3 preferentially acted on the normal HSPCs, resulting in their suppressed
proliferation. As a consequence, LICs expanded dominantly in BM during the initiation process of CML. Indeed,
the ablation of CCL3 or the depletion of basophils markedly retarded the CML development in mouse CML
model. These observations would imply that intra-BM basophil expansion can favor leukemia-tropic
hematopoiesis in CML by providing CCL3, a potent inhibitor of normal hematopoiesis and that
basophil-derived CCL3 can be a novel target molecule for the treatment of CML.
58
1-4
SCF-type E3 Ligase Fbw7 Plays Pivotal Roles in the Maintenance of the
Development and Commitment in Hematopoietic Cells
Kyoko Kitagawa, Masatoshi Kitagawa
Department of Moblecular Biology, Hamamatsu University School of Medicine, Japan
The levels of many cellular proteins are regulated by transcriptional control and protein degradation. The
ubiquitin–proteasome system participates in the selective and rapid degradation of cellular proteins. Besides, it
has pleiotropic functions and contributes to various biological processes. The specificity of the ubiquitylation
process is achieved by E3 ligase. F-box proteins form SCF (SKP1-Cullin1-F-box protein)-type E3 ligases,
besides, many studies have reported the deregulation of F-box proteins in human cancers. In particular, Fbw7 is
sometimes mutated in human tumors including haematopoietic marignaicies such as T-ALL and functions as a
tumor suppressive E3 ligase targeting ongogenic proteins such as c-Myc, Notch, MCL-1 and NFB2.
Moreover, we identified c-Myb, GATA2 and GATA3 as the novel substrates of Fbw7. All of them play critical
roles in the development and commitment of hematopoietic cells. In many cases of the reported Fbw7 substrates,
phosphorylation of a consensus phospho-binding motif, termed Cdc4 phosphodegron (CPD) is critical for the
regulation of their degradation, moreover, it is often phosphorylated by GSK3. Nevertheless three novel targets
were degraded in a CPD phosphorylation-dependent manner, the functional kinases were divergent. The CPD
motif in c-Myb was modified by GSK3 like the previously reported substrates, while the responsive kinases of
GATA2 and GATA3 were CDK1 and CDK2, respectively. These observations suggest that regulatory signals in
addition to GSK3 participate in the diverse regulation of Fbw7 substrates.
Depletion of Fbw7 by RNA interference delayed turnover and increased the abundance of GATA2 and c-Myb
in myeloid leukemia cells, moreover suppressed the transcriptional level of γ-globin, which receives
transcriptional repression from c-Myb. Conditional Fbw7-depletion using Cre recombinase under control of the
Mx1 gene promoter increased GATA2 levels in mice hematopoietic stem cells and myeloid progenitors at the
early stage. In particular, the increase in a GATA2 level was correlated with a decrease in a c-Kit high
expressing population of myeloid progenitor cells. On the other hand, Conditional inactivation of Fbw7 in
mouse T-cell by Cre recombinase activity under the control of the Lck promoter development resulted in
reduced thymic CD4 single-positive (SP) and splenic CD4+ and CD8+ cell proportions. Fbw7 deficiency skewed
CD8 SP lineage differentiation, which exhibited a higher incidence of apoptosis. We also observed augmented
GATA3 in CD4/CD8 double negative stage 4, CD4 SP, and CD8 SP lineages in Fbw7-deficient thymocytes.
These observations suggest that Fbw7-mediated GATA3 regulation contributes to the precise differentiation of
T-cell lineages.
These results support an importance of the precise expression of Fbw7 protein for the maintenance of the
development and commitment in the hematopoietic cells, besides, they imply a significance of lineage specific
phosphorylation of Fbw7-targets via the distinct signaling pathways for their degradation. It is thought that
deregulation of the Fbw7-mediated degradation of diverse hematopoietic transcription factors participate in
various hematopoietic marignancies.
59
1-6
Perturbation of Energy Metabolism by Fatty-acid Derivative Is Effective for
Overcoming Imatinib Resistance in BCR-ABL-harboring Leukemic Cells
Haruka Shinohara1, Yosuke Minami2, Tomoki Naoe3, Yukihiro Akao1
1
United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Japan
2
Department of Transfusion Medicine and Cell Therapy, Kobe University Hospital, Japan
3
National Hospital Organization Nagoya Medical Center, Japan
Imatinib, a BCR-ABL-kinase inhibitor, has brought marked clinical improvement for the treatment of patients
with chronic myeloid leukemia (CML) or Ph-positive acute lymphoblastic leukemia (ALL). However, further
improvement is needed to prevent relapses due to residual or resistant leukemic cells.
Cancer cells efficiently use limited energy sources by modulating cellular signaling and reprograming metabolic
pathways, thus, drugs targeting cellular metabolism improve the efficacy of therapy. We recently reported
medium-chain fatty-acid derivative AIC-47 is expected as a novel anti-cancer agent for the treatment of CML.
In human CML K562 and KCL-22 cells, the anti-cancer activity of AIC-47 was approximately the same as that
of etoposide. AIC-47 and imatinib in combination exhibited a significant synergic cytotoxicity. In contrast,
AIC-47 did not affect the growth of mitogen-stimulated human blood lymphocytes. Moreover, we characterized
the effects of AIC-47 and imatinib from the perspective of cancer-specific energy metabolism. Imatinib
inhibited only the phosphorylation of BCR-ABL; whereas AIC-47 suppressed the expression of the protein itself.
We found new knowledge that BCR-ABL modulated the Warburg effect, which is cancer-specific glucose
metabolism. The Warburg effect is achieved through regulated expression of pyruvate kinase isoforms, PKM1
and PKM2, by alternative splicers including polypyrimidine tract-binding protein 1 (PTBP1). Both AIC-47 and
imtinib induced the switching of PKM isoforms from PKM2 to PKM1 through the down-regulation of PTBP1,
resulting in perturbation of glycolysis. Glucose metabolism is a central source of energy for cancer cells,
however, impared glycolysis can be compensated by fatty-acid oxidation (FAO; beta-oxidation) through
up-regulation of carnitine palmitoyltransferase 1C (CPT1C), which is the rate-limiting enzyme of FAO. The
expression level of CPT1C was up-regulated in imatinib-treated cells; whereas it was decreased in
AIC-47-treated cells. These results indicated that imatinib activated FAO in stead of impared the Warburg
effect; however, AIC-47 inhibited FAO independent of the perturbation of the Warburg effect.
In this study, we examined the effects of AIC-47 on imatinib-insensitive cells, CD34-positive stem cell-like
fraction and BCR-ABL mutant cells. As a result, AIC-47 exhibited significant cytotoxicity in cultures of
CD34-positive fraction of Ph-positive ALL cells. Most leukemic stem cells are not in the cell-cycle; and they are
much less sensitive to imatinib. Our data indicated that leukemic stem cells would make a better use of FAO
than non-stem leukemia cells, resulting in acquired insensitivity to imatinib. We considered that the inhibition of
FAO by AIC-47 caused cytotoxicity even in CD34-positive stem cell-like fraction. AIC-47 also led to
significant reduction in the viability of cells harbouring BCR-ABL-kinase mutation, including T315I. Either
wild type- or T315I-BCR-ABL cells, AIC-47 induced the switching of PKM isoforms from PKM2 to PKM1,
resulting in the growth inhibition. Our findings suggested that AIC-47 would be useful agent for combination
treatment with imatinib, overcoming imatinib resistance, and stopping imatinib.
60
1-7
A Novel Molecular-target Therapy for Therapy-resistant AML by Human
Anti-ITGA6/B4 Antibody
Kazuko Kaneda1, Yusuke Saito1, Akira Suekane1, Shingo Nahakata1, Gene Kurosawa2, Koichi Sato3, Yukio
Sudo4, Kazuhiro Morishita1
1
University of Miyazaki, Japan
Fujita Health University, Japan
3
Perseus Proteomics Inc., Japan
2
We have previously shown that integrin α6/β4 (ITGA6/B4) complex works as one of the main adherent
molecules in the bone marrow niche for acute myeloid leukemia (AML) cells with EVI1 high expression (EVI1
high
AML). The highly adherent ability of EVI1high AML cells was dependent on the binding of the ITGA6/B4
complex to laminin 332, which resulted in chemoresistance (Plosone 2011). The high expression of ITGA6 and
ITGB4 were transcriptionally regulated by EVI1 and GATA2. Moreover, ITGA6/B4 is also found as one of
the important molecules expressed in leukemia stem cells from therapy-resistant AML and 25% of AML with
normal karyotype present with poor prognosis. Therefore, to develop novel molecular targeted therapy to
chemoresistant AML with high ITGA6/B4 expression, we isolated several clones of anti-human ITGA6/B4
antibodies by phage display method derived from human IgG mRNA. Among them, treatment of EVI1high AML
cells with anti-human ITGA6/B4 antibodies inhibited their adhesion ability to laminin 332 and recovered
sensitivity to anticancer drugs. Moreover, treatment of anti-human ITGA6/B4 antibodies with standard
chemotherapy to the immunodeficient NOG mice subcutaneously transplanted with EVI1high AML cells could
reduce the tumor growth significantly. After transplantation of leukemia cells to NOG mice through the vein, the
treatment of ITGA6/B4 antibody significantly reduced the numbers of leukemia cells in the bone marrow,
resulting in extended life span of mice with anticancer drug. Therefore, the treatment of ITGA6/B4 antibody
may improve the outcome of the patients with therapy-resistant AML, combining with conventional
chemotherapy.
61
1-9
RNA Editing Enzyme ADAR1 Regulates Cell Cycle of Hematopoietic Cell
Maria Anna Zipeto1,2, Qingfei Jiang1,2, Heather Leu1,2, Nathan Delos Santos1,2, Etienne Atien2, Sheldon Morris1,
Catriona Jamieson1,2
University of California, San Diego, USA
Compelling murine studies demonstrate that adenosine-to-inosine (A-to-I) RNA editing mediated by adenosine
deaminase associated with RNA1 (ADAR1) is vital for both fetal and adult hematopoiesis. While genetic
ablation of ADAR1 editase leads to murine embryonic lethality due to severe defects in erythropoiesis,
conditional deletion in the hematopoietic system impairs maintenance indicative of cell type and context
specific roles for ADAR1 in cell fate specification and self-renewal. We have previously shown that
inflammation-responsive ADAR1 plays important roles in both stem cell differentiation and self-renewal in
CML (chronic myeloid leukemia) disease progression. Though we have established ADAR1-mediated RNA
editing as a novel therapeutic target for treating CML, we do not yet understand the underlying mechanism of
RNA editase’s involvement in normal hematopoiesis. In our new study, we describe ADAR1’s role in cell cycle
regulation of normal hematopoietic stem cell (HSC) and its molecular editing targets.
Our results demonstrated that ADAR1 induces G0 to G1 phase transition in normal hematopoietic stem cells, as
demonstrated by elevated expression of Ki67, reduced DiR signal, and in vivo cord blood engraftment. Cell
cycle qRT-qPCR microarray and whole transcriptome RNA-sequencing analysis indicates that CDKN1a
expression level is reduced by >80% in normal cord blood CD34+ cells over-expressing ADAR1. However,
there are no direct A-to-I editing observed in CDKN1a transcript by whole transcriptome sequencing. We found
that miR-26a-5p, a miRNA frequently downregulated in leukemia, is inhibited by ADAR1-mediated RNA
editing in normal cord blood CD34+ cells. Lentiviral expression of mature miR26-5p enhances CDKN1a
expression, inhibits cord blood proliferation in vivo, as well as reduces HSC self-renewal in colony-formation
assay. Importantly, ADAR1 directly binds and edits the DROSHA cleavage site of pri-miR26a, thereby prevent
proper miR26a-5p biogenesis.
Our finding suggests carefully regulated A-to-I editing by ADAR1 is essential for the maintenance of proper cell
proliferation in HSC. For future study, it will be interesting to investigate if the elevated expression of ADAR1
in CML BC LSC contributes to false regulation of cell cycle that leads to the expansion of malignant leukemia
stem cells.
62
1-10
Probing the Hippo Signaling Pathway in Leukemia as a Potential Therapeutic
Target
Chanchao Lorthongpanich1, Nittaya Jiamvoraphong1, Chuti Latowammathron1, Sudjit Luanpitpong1, Yaowalak
U-pratya1,2, Surapol Issaragrisil1,2
1
Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University,
Bangkok, Thailand
2
Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University,
Bangkok, Thailand
The Hippo pathway is an evolutionally conserved protein kinase cascade involved in cell growth control. It has
been shown that cell-cell contact activates Hippo signaling activity resulting in cell proliferation inhibition and
apoptotic gene activation. These mechanisms can, therefore, control cell growth. Inactivation of the Hippo
pathway leads to unlimited cell growth and cancer development. It has been reported that inactivation of Hippo
signaling pathway was observed in several types of human cancers with a high frequency, which suggests that
the Hippo pathway could be a potent target for developing anticancer drugs. To date, several approaches are
being tested for targeting the Hippo pathway in several types of cancer except for myeloid leukemia. To
determine whether Hippo pathway plays role in leukemia, we investigated the expression of Hippo component
genes and the activation state of Hippo pathway in acute and chronic myeloid leukemia. Result showed
down-regulation of several up-stream mediators, known as tumor suppressor gene, but up-regulation of
oncogenes such as YAP and TAZ. These results clearly suggested the role of Hippo pathway in controlling
leukemic cell growth. We further observed that, even without cell contact, the Hippo pathway is active in both
acute and chronic myeloid leukemia and this activation is independent of the canonical LATS kinase. Our
results significantly contribute a step towards developing the Hippo pathway as a therapeutic target for leukemia
therapy.
63
1-11
The Roles of Glis2 in Leukemic and Hematopoietic Stem Cells
Emi Takamatsu-Ichihara, Haruko Shima, Kazutsune Yamagata, Shuhei Fujita, Yukiko Aikawa, Issay
Kitabayashi
National Cancer Center Research Institute, Division of Hematological Malignancy, Tokyo, Japan
Gli similar 2 (Glis2) is a Kruppel-like zinc finger transcription factor with high homology to the Gli family.
Recently, CBFA2T3 (MTG16)-Glis2 fusion gene resulting from an inversion of chromosome 16 was identified
in acute megakaryoblastic leukemia (AMKL). We previously showed that expression of Glis2 is suppressed by
polycomb-group repressive complex1 (PRC1), which is essential for development and maintenance of
MOZ-TIF2 and MLL-AF10 induced acute myeloid leukemia (AML). Forced expression of Glis2 induced
differentiation and apoptotic cell death of AML cells, suggesting that suppression of Glis2 by PRC1 is important
to maintain AML stem cells. To clarify the mechanism of leukemic cell differentiation by Glis2, we compared
gene expression profiles of AML cells forcibly expressing Glis2 and empty control vector. Among several
target genes that were elevated by overexpression of Glis2, we focused on Ets transcription factor Spi-B known
to be essential for the differentiation of intestinal microfold cells. Forced expression of Spi-B in AML cells
induced alteration of colony and cellular morphology and up-regulated expression of macrophage markers such
as CD14, CD68, or F4/80, indicating that Spi-B stimulated differentiation of AML progenitors into macrophage.
Therefore, Glis2 may regulate leukemic stem cell differentiation, at least in part, through up-regulation of Spi-B
gene expression. By contrast, forced expression of Glis2 in c-Kit-positive hematopoietic progenitor/stem cells
inhibited cell differentiation and retained their colony-forming capability in methylcellulose culture. Therefore,
Glis2 may play an essential role in maintenance of hematopoietic progenitor/stem cells. Our results shows that
the function of Glis2 in AML cells differs from that in normal hematopoietic progenitor/stem cells, suggesting
the possibility that activation of Glis2 may be a therapeutic target for AML.
64
1-12
Trib1 Modulates Transcriptional Functions of Hoxa9
1
1
1
1
1
1
Takashi Yokoyama , Seiko Yoshino , Miwa Tanaka , Yukari Yamazaki , Tomoko Takahara , Aya Nakamura ,
2
2
1
Shuichi Tsutsumi , Hiroyuki Aburatani , Takuro Nakamura
1
2
Div. Carcinogenesis, Cancer Inst., JFCR, Japan
Genome Science Div., RCAST, Univ. of Tokyo, Tokyo, Japan
Tribbles family genes encode pseudokinase proteins that are well conserved during evolution and regulate cell
proliferation, migration and morphogenesis. We have identified murine Trib1 as a collaborator of Hoxa9/Meis1
in myeloid leukemogenesis and have found that it by itself acts as a leukemia disease gene through enhancement
of MAP kinase signaling and degradation of C/EBPa. The Trib1 knockout mouse exhibits significant increase of
mature neutrophils associated with increase of C/EBPa protein and up-regulation of its target genes.
In order to clarify Trib1’s functional role in cooperation with Hoxa9, hematopoietic cells of Trib1 KO or wild
type mice were transformed by Hoxa9. Biological phenotypes of Hoxa9-immortalized cells with different Trib1
expression status, Trib1hi, Trib1lo and Trib1null, were compared. These cell lines exhibited immature myeloid
phenotypes. Overexpression of Trib1 induced increase of CD34 and decrease of Mac-1/Gr-1 expression.
Enhanced proliferation and DNA synthesis was also observed in the Trib1hi cell. The Trib1hi phenotype was
associated with decrease of C/EBPa protein and enhancement of ERK phosphorylation. Gene expression
profiling showed that three cell types were subdivided into mutually independent clusters. Gene set enrichment
analysis (GSEA) showed enrichment of gene sets associated with cell cycle as well as C/EBPa target genes.
Genome-wide DNA binding region of Hoxa9 and C/EBPa were then assessed in Trib1hi, Trib1lo and Trib1null.
ChIP-Seq analyses showed 5,153, 1,764 and 3,081 Hoxa9 binding peaks in Trib1hi, Trib1lo and Trib1null,
respectively, and 1,106 peaks were common in three cell types. There were also cell type-specific Hoxa9
binding peaks with 3,119 in Trib1hi as the most frequent peaks. Frequent overlapping of Hoxa9 and C/EBPa
binding peaks (1,862 peaks) was observed in Trib1null cells. Interestingly, 606 of 1,862 C/EBPa/Hoxa9
co-binding peaks disappeared and 766 C/EBPa-specific binding peaks were occupied by Hoxa9 in Trib1hi cells.
These data suggest that Trib1 significantly modulates transcriptional regulation of C/EBPa and Hoxa9 target
genes through C/EBPa degradation. Alterations of Hoxa9 binding according to the Trib1 expression level is
associated with modulation of Histone H3K9me3, K27me3 and H3K27Ac binding. Potentially important Hoxa9
and C/EBPa target genes were searched in nearest neighbor genes of 766 C/EBPa binding peaks replaced by
Hoxa9 in Trib1hi, of which expression was 1.5-fold up- or down-regulated by Trib1 overexpression. Erbb3, Kit,
Cd34, Cd44, Cdk6 and E2f3 are such Hoxa9 target candidates up-regulated by Trib1 overexpression. Also, Idh1,
Tet2 and Vcam1 are down-regulated genes by Trib1 overexpression. These results suggested that Trib1 might
alter cell cycle progression and cellular differentiation by modulating Hoxa9-mediated transcriptional program
and by reppressing C/EBPa target tumor suppressor genes.
65
1-13
The C/EBPβ Transcription Factor Mediates the Effect of IFNα
on CML Stem Cells through Promoting Their Differentiation and Exhaustion
Asumi Yokota1, Hideyo Hirai1, Yoshihiro Hayashi2, Ryuichi Sato3, Hiroko Adachi3, Fumiko Sato3,
Atsushi Sato1, Akihiro Tamura1, Masaki Iwasa1, 4, Aya Fujishiro1, 4, Tsukimi Shoji1, Takahiro Kashiwagi1,
Yasuo Miura1, Masakazu Nakano3, Kei Tashiro3, Taira Maekawa1
1
2
Dept. Transfusion Med. & Cell Therapy, Kyoto Univ. Hospital, Kyoto, Japan
Div. Experimental Hematology & Cancer Biology, Cincinnati Children’s Hospital Medical Center, OH, USA
3
Dept. Genomic Medical Sciences, Kyoto Prefectural Univ. Med., Kyoto, Japan
4
Div. Gastroenterology & Hematology, Shiga Univ. Medical Science, Shiga, Japan
[Backgrounds and purpose of this study]
CCAAT/enhancer binding protein β (C/EBPβ) is a transcription factor playing an essential role for emergency
granulopoiesis. Under stressed conditions including infections and cytokine stimulations, prompt and sufficient
supply of granulocytes is evoked by C/EBPβ, which promotes both proliferation and myeloid differentiation of
hematopoietic stem/progenitor cells (Hirai H et al, Nat Immunol 2006; Satake S, Hirai H et al, J Immunnol
2012). We have previously shown that C/EBPβ is upregulated by BCR-ABL-STAT5 signaling in chronic-phase
chronic myeloid leukemia (CML), and C/EBPβ promotes myeloid differentiation of CML stem cells (Hayashi Y,
Hirai H et al, Leukemia 2013).
In this study, we investigated the mechanism for STAT5-mediated upregulation of C/EBPβ, and propose a novel
therapeutic approach targeting CML stem cells by upregulating their C/EBPβ expression.
[Results]
First, we searched for proximal promoter or distal enhancer region of Cebpb, which is responsible for induction
of C/EBPβ expression by STAT5. By ChIP-seq analysis, we identified novel BCR-ABL-responsive STAT5
binding sites in 3’ distal region of Cebpb. Interestingly, two STAT5 consensus motifs (TTCNNNGAA) are
tandemly located in this region, which is highly conserved among various species including human. These
results suggest that this region could be an important enhancer for BCR-ABL-dependent expression of C/EBPβ.
Next, we explored which cytokine signaling could efficiently induce C/EBPβ expression using a hematopoietic
stem cell line, EML cells. We found that IFNα rapidly phosphorylated STAT5 as well as STAT1 and STAT3, and
increased C/EBPβ expression irrespective of the presence of BCR-ABL. Notably, IFNα also recruited STAT5 to
3’ distal enhancer region of Cebpb, suggesting that this enhancer regulates cytokine-responsive C/EBPβ
expression.
In order to clarify C/EBPβ-mediated effects of IFNα, LSK cells from WT or C/EBPβ KO mouse bone marrow
(BM) were transduced with BCR-ABL, and subjected to the serial replating assay in the presence or absence of
IFNα. IFNα promoted differentiation of CML stem cells toward myeloid cells, and reduced their
colony-forming ability. In contrast, C/EBPβ-deficient CML stem cells retained their immature status and
colony-forming ability even in the presence of IFNα.
Finally, we evaluated the in vivo effect of IFNα on CML stem cells by performing serial BM transplantation.
BCR-ABL-transduced mouse BM cells were transplanted to the 1st recipients, and these mice were
administrated with PolyI:C, which induces IFNα production in vivo. Frequencies and absolute numbers of CML
stem cells in BM of the 1st recipients were significantly decreased by administration of PolyI:C, and this effect
was completely abolished when C/EBPβ was absent in CML stem cells.
[Conclusions]
Collectively, STAT5 regulates C/EBPβ expression in response to BCR-ABL signaling and IFNα stimulation,
most likely through 3’ distal enhancer identified in this study. Furthermore, IFNα has therapeutic effects through
promoting differentiation and exhaustion of CML stem cells, and C/EBPβ plays critical roles in these processes.
66
1-14
A New Role for Meis1 in the Immune Evasion of Myeloid Leukemic Cells
Arnaud Couzinet, Takashi Yokoyama, Takuro Nakamura
The Cancer Institute, Japanese Foundation for Cancer Research (JFCR), Japan
Acute Myeloid Leukemia (AML) is the most frequent type of leukemia and accounts for ~90% of all acute
leukemia in adults. The five-year survival rate of patients with AML is the poorest of all leukemia (only 40%),
which highlights the urgent necessity to improve therapy, and thereby implies a better comprehension of the
cellular and molecular mechanisms governing the onset and progression of the disease.
The hallmark mutations associated with human leukemia are nonrandom chromosomal translocations. One such
translocation involves MLL (Mixed Lineage Leukemia), which has been shown to be fused with over 60
different partner genes and is associated with unfavorable survival. One consequence of MLL protein
chimerization is the constitutive upregulation of two proteins : Hoxa9 and Meis1. The overexpression of these
two transcription factors has been found to be sufficient for leukemia onset.
Interestingly, while overexpression of Hoxa9 is sufficient for inducing immortalization of hematopoietic stem
cells in vitro, these Hoxa9-overexpressing cells are however unable to induce AML in vivo, demonstrating that
the mere immortalization of cells does not confer the ability to induce leukemia. Indeed, for proper and robust in
vivo propagation of leukemic cells, Meis1 co-expression (with Hoxa9) is critically required, suggesting that
Meis1 regulates fundamental genes for in vivo adaptation of Hoxa9-transformed cells.
Since the role of Meis1 in AML is still elusive, the purpose of the project is therefore to uncover the role of
Meis1, by identifying its critical target genes. We recently found that Sytl1 (synaptotagmin-like 1) is a direct
target gene of Meis1. Furthermore, we found that overexpression of Sytl1 in Hoxa9-overexpressing leukemic
cells was sufficient to bypass Meis1 absence and restored the ability of leukemic cells to engraft in bone marrow.
Therefore, Meis1 is critical for cells engraftment through Sytl1 expression.
Interestingly, however, Hoxa9/Sytl1-overexpressing cells survived only temporarily and disappeared gradually
in vivo, suggesting that engraftment ability is not sufficient for long-term survival. We then found that the
gradual disappearance of Hoxa9/Sytl1-overexpressing cells is due to an immune system attack. Indeed,
Hoxa9/Sytl1-overexpressing cells inoculated into immune deficient mice (NOD/SCID or RAG2-/- mice)
engrafted and developed in a similar pattern than Hoxa9/Meis1-overexpressing cells. Therefore, Meis1 confers
the ability for AML cells to evade immune surveillance. In other words, Meis1 protects AML cells from the
immune attack, revealing Meis1 as an immune shield.
In summary, we identified at least two roles for Meis1 :
(i) Meis1 expression confers bone marrow engraftment ability through Sytl1 upregulation,
(ii) Meis1 attributes leukemic cells an evasion capacity from immune surveillance through a still unidentified
factor.
Both of these Meis1 functions must be achieved jointly for the proper onset of Hoxa9/Meis-mediated AML.
Therefore, annihilating the immune evasion function of AML cells by chemical regulation of Meis1 target genes
may serve as a future strategy for immunotherapy of AML patients with MLL translocation.
67
1-15
MLL Is Essential for NUP98-HOXA9-Induced Leukemia
Yutaka Shima, Minori Yumoto, Issay Kitabayashi
Division of Hematological Malignancy, National Cancer Center Research Institute, Japan
NUP98, a component of the nuclear pore complex (NPC), plays an important role in molecular trafficking
between the cytoplasm and the nucleus. NUP98 has two Phe-Gly (FG) repeat domains, which are characteristic
of NPC proteins. The NUP98 gene is rearranged and fused to several partner genes, such as HOXA9 and DDX10,
in acute myeloid leukemia (AML) and myelodysplastic syndromes. Leukemia with NUP98 rearrangement is
associated with poor prognosis. However, the molecular mechanism of leukemogenesis induced by
NUP98-fusions remains unclear.
To investigate the role of NUP98 in leukemogenesis, we generated a set of deletion mutants of NUP98-HOXA9
lacking each FG repeat domain. These mutants were transduced into normal murine progenitor cells and their
activity in the immortalization of murine cells was examined by colony-formation assays. Deletion of the
second FG domain of the NUP98 moiety of NUP98-HOXA9 fusion protein resulted in lack of cell
immortalization activity. Deletion of the second FG domain of the NUP98 moiety also resulted in a dramatic
delay in AML induction when cells were transplanted into mice, suggesting that the second FG repeat domain of
the NUP98 moiety is crucial for cell immortalization in vitro and leukemogenesis in vivo. qPCR and ChIP
analyses showed that the FG repeat domain is necessary for Hoxa gene activation and recruitment to the Hoxa
gene locus. To identify interacting proteins of the FG repeat domain of NUP98-HOXA9, protein complexes
containing full-length and mutant NUP98-HOXA9 were purified. Mass spectrometry analysis showed that
full-length NUP98-HOXA9, but not the mutant lacking the second FG repeat, interacted with MLL, which is a
methyltransferase and targets lysine 4 of histone H3. ChIP analysis also showed co-localization of
NUP98-HOXA9 and MLL at the Hoxa gene locus in NUP98-HOXA9-expressing cells. Furthermore, analysis
of Mll null mice showed that MLL is essential for the recruitment of NUP98-HOXA9 to the Hoxa gene locus
and for NUP98-HOXA9-induced cell immortalization and leukemogenesis. These results suggest that NUP98
fusion proteins interact with MLL to activate HOXA genes during leukemogenesis.
68
1-16
Modeling Tyrosine Kinase Inhibitor-resistant Chronic Myelogenous Leukemia by
Patient-derived Induced Pluripotent Stem Cells
Masashi Miyauchi, Shunya Arai, Akira Honda, Sho Yamazaki, Keisuke Kataoka, Akihide Yoshimi, Kazuki
Taoka, Keiki Kumano, Mineo Kurokaw
Department of Hematology and Oncology, Graduate School of Medicine, The University of Tokyo, Japan
Since the emergence of tyrosine kinase inhibitor (TKI), imatinib, long-term survival of patients with chronic
myelogenous leukemia (CML) has been dramatically improved. However, not only imantib but also 2nd
generation TKIs including nilotinib and dasatinib have not cured CML, mainly due to TKIs-resistant CML stem
cells. CML stem cells are quite difficult to analyze because they represent an extremely minor population of
CML cells. To overcome this obstacle, we established integration-free induced pluripotent stem cells (iPSCs)
from bone marrow of two patients with CML in chronic phase (CML-CP) and analyzed
hematopoietic-differentiated cells from CML-iPSCs.
To efficiently establish iPSCs from CML patient samples, purified CD34+ cells from bone marrow of patients
with CML-CP were expanded for 2 days in alpha-MEM medium supplemented with fetal bovine serum, stem
cell factor (SCF), FLT-3 ligand, interleukin (IL)-3, IL-6 and thrombopoietin (TPO). The plasmid mixture, which
encoded OCT3/4, SOX2, KLF4, L-MYC, LIN28, EBNA1, and short hairpin RNA for TP53 was electroporated
into 2 x 105 CD34+ cells. The cells were then cultured with mouse embryonic fibroblast for 20-30 days and we
obtained embryonic stem (ES) cell-like colonies from two patients with CML. Of the ES cell-like colonies, we
confirmed teratoma formation and not exogenous but endogenous expression of pluripotent cell marker genes
including OCT3/4, SOX2, KLF4, L-MYC, and LIN28 in RT-PCR. CML-specific oncogene expression of
BCR-ABL and chromosomal translocation of t(9;22)(q34;q11.2) were also shown in the colonies, demonstrating
that integration-free CML-iPSCs from two patients were established.
To analyze hematopoietic-differentiated cells from CML-iPSCs, CML-iPSCs were cultured with C3H10T1/2
stromal cells in IMDM medium containing vascular endothelial growth factor for 15-17 days and then we
obtained CML pre-hematopoietic progenitor cells (pre-HPCs), immature hematopoietic-differentiated cells
phenotypically defined by CD34+/CD45- among CD43+ hematopoietic-differentiated cells. In semisolid culture
with methocult H4434 classic, pre-HPCs from CML-iPSCs gave rise to comparable numbers of myeloid and
erythroid colonies to those from normal-iPSCs. In the liquid culture with SCF, FLT-3 ligand, IL-3, IL-6 and
TPO, pre-HPCs from CML-iPSCs exhibited increased cell proliferation compared with those from
normal-iPSCs, which was canceled by imatinib. However, even in the presence of imatinib, pre-HPCs from
CML-iPSCs did not undergo apoptosis and kept on growing, in marked contrast to differentiated hematopoietic
cells (CD34-/CD45+). These findings indicate that pre-HPCs from CML-iPSCs not only recapitulate CML
disease phenotype but also show multipotent capacity and resistance against imatinib, principal features of CML
stem cells.
Compared with differentiated hematopoietic cells, gene expression enrichment analysis for pre-HPCs derived
from CML-iPSCs demonstrated that several gene sets related with HOXA9 targets and TGF- signaling
pathway were enriched, which were well known to be involved in the maintenance of hematopoietic stem cells
and TKI-resistance, respectively. Quantitative RT-PCR revealed that pre-HPCs from CML-iPSCs had elevated
level of expression of MEIS1, which regulated metabolism of hematopoietic stem cells, and IL1RL1, which was
already reported as a gene associated with imatinib-resistance. In addition, gene expression analysis for
pre-HPCs from CML-iPSCs identified candidate genes, which mark TKI-resistant CML cells and potentially
become novel therapeutic targets. In conclusion, pre-HPCs derived from CML-iPSCs, as a powerful model of
CML stem cells, have the potential to identify a novel marker of TKI-resistant CML cells.
69
1-17
Deletion of Bcor Impairs Repopulating Capacity of Hematopoietic Stem Cells
but Promotes Transformation of Progenitor Cells
Shiro Tara1, Tomoyuki Tanaka1, Yaeko Nakajima-Takagi1, Motohiko Oshima1, Atsunori Saraya1, Haruhiko
Koseki2, Atsushi Iwama 1
1
Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Japan
Labolatory for Developmental Genetics, RIKEN Research Center for Integrative Medical Science, Japan
2
BCL6 co-repressor (BCOR) was identified as co-repressor of BCL-6 and also a component of a variant polycomb
repressive complex (PRC) 1, which mono-ubiquitylates H2A at K119 and inhibits its target gene expression.
Recently, recurrent inactivating mutations of BCOR have been identified in various hematological disorders such
as acute myloid leukemia (AML), myelodysplastic syndrome (MDS), and aplastic anemia (AA). Here, we
analyzed the male mice deficient for Bcor exon4 (BcorΔE4/y), which generates a short form of Bcor that cannot
bind to Bcl-6..
First we evaluated BcorΔE4/y hematopoietic stem cell (HSC) repopulation capacity using bone marrow
transplantation (BMT) assays. Toal bone marrow cells from Cre-ERT control and Cre-ERT;Bcorfl/fl mice
(CD45.2) were transplanted to lethally irradiated CD45.1 recipient mice. After engraftment, conditional gene
deletion was induced by tamoxifen injection. In BMT with CD45.1 total bone marrow competitor cells, the
deletion of Bcor (BcorΔE4/y), but not Bcl6 (Bcl6Δ/Δ) compromised repopulating capacity of HSCs. In
non-competitive BMT model, BcorΔE4/y mice showed a propensity to develop lethal T-cell acute lymphoblastic
leukemia (T-ALL) up to 30~40% after the conditional deletion of Bcor during the observation priod of 10 months.
In contrast to HSCs, however, BcorΔE4/y thymocytes showed a higher proliferative capacity in culture.
Unexpectedly, BcorΔE4/y hematopoietic progenitor cells maintained H2AK119ub1 levels, suggesting that Bcor
exon 4 is not essential for the catalytic activity of variant PRC1. These results define Bcor as a novel regulator of
HSCs and uncover its differentiation stage-specific function. Our findings also provide the evidence of its tumor
suppressor role in hematopoiesis, implicating inactivating mutations of BCOR in the pathogenesis of
hematological disorders.
70
1-18
PVPylated Magnetic Metal Oxide Nanoparticles for Drug Delivery to
Human Acute Myeloid Leukemia
Kheireddine El-Boubbou1,2, Daniel Azar3, Ralph J. Abi-Habib3
1
Department of Basic Sciences, King Saud bin Abdulaziz University for Health Sciences, King Abdulaziz
Medical City, National Guard Health Affairs, Kingdom of Saudi Arabia
2
King Abdullah International Medical Research Center (KAIMRC), National Guard Hospital, Kingdom of
Saudi Arabia
3
Department of Natural Sciences, School of Arts and Sciences, Lebanese American University, Lebanon
Drug delivery using functional magnetic nanoparticles for the treatment of acute myeloid leukemia (AML) has
not been significantly investigated yet. In this study, we report the development of a chemotherapeutic
formulation made of polymeric-stabilized multifunctional magnetic metal oxide nanoparticles (M3NPs) of
ferrites loaded with the anticancer drug Doxorubicin as a promising drug-carrier delivery system for the
treatment of AML. M3NP-drug conjugates (Doxironide) simultaneously exhibited high drug content, maximized
fluorescence, and excellent release properties. Fast release profiles of Doxorubicin were designed to achieve
sufficient toxic concentrations in AML cells in relatively short periods of time. Nanoparticulate uptake and cell
death following addition of Doxironide were evaluated in four different types of human AML cell lines: ML-2,
HL-60, TF1-vRaf, and Mono-Mac-1. While the unloaded NPs were not toxic to any of the cells tested,
Doxironide was found to be highly potent against all four AML cell lines tested, albeit at different inhibition
concentrations, with IC50 values ranging from 0.5 to 4.5 µM Doxorubicin content. Cellular uptake experiments
showed significant uptake of Doxironide by the different cell lines at all time-points compared to that of
unlabeled control NPs, with a detectable increase in the first 4 hours post-treatment and increasing with time.
TEM images showed that Doxironide nanoparticles were taken up by the cells via membrane-bound vesicles,
and shuttled into the cytoplasm releasing doxorubicin intracellularly. Live confocal microscopy imaging
confirmed that Doxorubicin was indeed delivered to the nucleus in relatively short periods of time, however, no
nanoparticles were found to be present in the nucleus as evident from TEM microscopy. These results
demonstrate that Doxironide is highly potent against AML cells and indicate that the observed cytotoxic effects
are dependent on the uptake of NPs by cells followed by Doxorubicin release and translocation to the nucleus.
Importantly, this targeted payload may enhance the effectiveness of the drug in AML patients and may further
allow physicians to image leukemic cells exposed to Doxironide. This can potentially open new opportunities
for in vivo AML therapeutic strategies.
71
1-19
Topoisomerase II-targeting Novel Intervention: “Jekyll” Physiology
and “Hyde” Pathology
Po-Ku Chen1, Yi-Song Chen1, Huang-Ling Hsu1, Nei-Li Chen2, Tsai-Kun Li1
1
Graduate Institute of Microbiology, Graduate Institute of Molecular Biology and Biochemistry, National Taiwan
University College of Medicine, Taiwan
DNA topoisomerase II isozymes ((Top2 and Top2) are double-edged swords, those essential enzymes
resolve the topological problems arising from DNA metabolism and cellular processes by the generation of
transient double-stranded break (DSB, an evolutionary vulnerable point). Despite functions of Top2 are
important for ensuring genomic integrity, the ability to induce enzyme-mediated DSB (i.e. Top2-targeting) is an
effective strategy for cancer chemotherapeutics. Notably, our collaborative effects have revealed potential
sources for tumor specificity of human Top2 (hTop2)-directed anticancer drugs that is primarily attributed to
their hTop2-targeting ability, while targeting to the hTop2 isozyme is mainly responsible for clinical side
effects; leading to novel therapeutic intervention based on Top2 isozyme-specific targeting! In addition, various
naturally encoded toxins and metabolites have also been shown to exhibit the Top2-targeting ability. In this
regard, our lab is the 1st one to demonstrate that oxidative and nitrosylation stress can activate Top2-mediated
DSBs, cell death and inflammation-associated tumorigenesis. These findings lead to the discoveries of oxidative
and thiol modifications of Top2 proteins as a novel way to induce formation of Top2 cleavable complex
(Top2cc) and a novel pathological involvement of Top2s, especially the Top2 isozyme in inflammation-caused
DNA breakage, mutagenesis and carcinogenesis as well as for other clinic side effects (such as cardiotoxicity
nd
and 2 leukemia) and tumor heterogeneity. These molecular and biochemical studies have not only allowed a
fundamental understanding of physiological functions and pathological roles of the Top2 isozymes, but also
provided therapeutic interventions via refined targeting of hTop2 isozymes. Based on above findings, our team
has designed, synthesized and screened compounds for new hTop2-targeting leads for anticancer therapeutics
against leukemia, prostate and/or cancer small cell lung cancer as well hTop2-preferred catalytic inhibitors for
therapy-related cardiotoxicity and other side effects, including hepatotoxicity and myelo-suppression. Both the
pharmacological and toxicological assessments of novel lead-to-candidate development of various
Top2-targeting compounds will also be discussed.
72
1-20
Endogenous MOZ Is Essential for MOZ/MLL-fusion Induced Leukemias and
Upregulation of HoxA9/Meis1
Takuo Katsumoto1, Kazutsune Yamagata1, Yoko Ogawara1, Takuro Nakamura2, Ryo Goitsuka3 Issay
Kitabayashi1
1
Div. of Hematological Malignancy, National Cancer Center, Res. Inst., Tokyo, Japan
Div. of Carcinogenesis, Cancer Inst., Japanese Foundation for Cancer Res., Tokyo, Japan
3
Div. of Devlopment & Aging, Res. Inst., for Biol. Science, Tokyo University of Science, Chiba, Japan
2
Monocytic leukemia Zinc finger protein (MOZ) is Myst-type histone acetyltransferase involved in chromosome
translocations associated with FAB M4 or M5 subtypes of acute myelogenous leukemia (AML). We have
revealed that MOZ is essential for self-renewal of hematopoietic stem cells (HSCs) and for expression of
HoxA9/Meis1 genes that were constitutively activated in MOZ/MLL fusion leukemia. To clarify the roles of
MOZ in malignant hematopoiesis, we introduced leukemia-associated fusion genes, such as MOZ-TIF2 and
MLL-AF10, into fetal liver hematopoietic stem/progenitor cells (HSPCs) prepared from MOZ+/- or -/- fetus by
retrovirus system. At 5 days after infection, the cells expressing fusion genes were cultured in methylcellulose
medium or were transplanted into sub-lethal irradiated recipient mice. The MOZ+/- HSPCs expressing
MOZ-TIF2 or MLL-AF10 expressed increased levels of HoxA9/Meis1 and repeatedly formed numerous
colonies in vitro and induced AML in vivo 2-3 month after transplantation. Although the MOZ-/- HSPCs
expressing MOZ-TIF2 also formed numerous colonies and expressed increased levels of HoxA9, AML
development was impared and Meis1 expression was severely diminished. Neither colony formation and AML
development nor HoxA9/Meis1 expression was induced in MOZ-/- HSPCs expressing MLL fusion gene.
To reveal the molecular mechanism, we performed chromatin immune-precipitation analysis at HoxA9/Meis1
locus in MOZ fusion leukemia cells. The experiments demonstrated that MOZ-fusion was accumulated at
HoxA9 locus but not at Meis1 locus in the MOZ-/- leukemia cells. Active histone modification marks were
decreased and repressive marks were elevated at Meis1 locus in MOZ-/- leukemia cells. These data suggested
that endogenous MOZ plays a essential role to recruite MOZ-fusion and sustain active chromatin status at
Meis1 locus.
We also analyzed roles of Meis1 in MOZ-fusion induced AML development. When Meis1 was ectopically
introduced in MOZ-/- HSPCs, AML development induced by MOZ-fusion was recovered. In contrast, AML
development was impared by deletion of conditional deletion of Meis1. Flow cytometry analysis demonstrated
that Lineage-, Sca-1-, c-Kit+ FcgR+ fraction that were containing in leukemia initiating cells, was decreaced by
deletion of conditional deletion of Meis1. Succesive colony formation was not affected by Meis1 deletion.
These results suggested that Meis1 was critical for AML development induced by MOZ-fusion gene.
Finally, we also investigated in roles of MOZ in HoxA9 expression. We introduced MOZ fusion gene in HSCs
or common myeloid progenitrs (CMPs) collected from E14.5 MOZ-/- fetal liver. Although MOZ-/- HSCs
expressing MOZ fusion could persistently generate colonies and showed high level of HoxA9 expression as
shown above, when MOZ fusion genes were introduced into MOZ-/- CMPs, colony formation and expression
of not only Meis1 but also of HoxA9 were abolished. This data suggested that MOZ was also important for
induction of HoxA9 expression and immortatlzation of myeloid progenitor cells by MOZ-fusion gene.
Taken together, these results suggested that endogenous MOZ plays an essential role in MOZ/MLL
fusion-induced AML development through maintenance of active chromatin status at target gene locus to be
able to bind fusion gene. Our findings indicate that MOZ is a potential target for AML therapies.
73
1-21
Primary Human AML Cells Utilize Aerobic Respiration
Maintained by Enhanced Expression of ASCT1
Yu Kochi1, Yoshikane Kikushige1, Toshihiro Miyamoto1, Koichi Akashi1,2
1
Department of Medicine and Biosystemic Science, Kyushu Univ., Japan
2
Center for Cellular and Molecular Medicine, Kyushu Univ., Japan
Alterations of metabolic pathways have been reported in mouse acute myeloid leukemia (AML) models, but
there is little information on metabolism of primary human AML cells. Here, we performed metabolome
analysis of purified human CD34+ AML stem/progenitor cells (n=16) and CD34+ normal human hematopoietic
stem/progenitor cells (HSPCs) (n=5) using highly sensitive CE-QqQMS assay. In glycolysis, CD34+ AML cells
had a significantly high pyruvate/lactate ratio as compared to normal HSPCs, indicating the preferential
utilization of aerobic respiration in AML. To investigate aerobic/anaerobic proportions of metabolism, we
directly measured the O2 consumption rate (OCR) and the extracellular acidification rate (ECAR) of CD34+
AML cells and CD34+ HSPCs using Flux Analyzer System from seahorse bioscience. Consistent with
metabolome data, CD34+ AML cells displayed high OCR/ECAR ratio, a good index for the discrimination of
aerobic/anaerobic respiration pattern. These results collectively suggested that CD34+ AML cells predominantly
utilize aerobic respiration. Aerobic respiration produces reactive oxygen species (ROS), and therefore,
antioxidant activity should be critical for AML cells. In fact, we also found that CD34+ AML cells had
glutathione (GSH), a primary intracellular antioxidant, at levels much higher than normal HSPCs. To clarify the
machinery for the increased GSH production in AML, we investigate expression of amino acid transporter
because cysteine uptake is important for GSH synthesis. Interestingly, among known human cystine/cysteine
transporters including ASCT1, ASC1 and xCT, only ASCT1 is highly expressed in CD34+ AML cells, but it is
not expressed in normal HSCs. Collectively, human AML cells predominantly utilize aerobic respiration that is
supported by a high level of GSH, and the high level of GSH is maintained presumably by AML-specific
expression of ASCT1. These data suggest that ASCT1 could be a useful molecule to target AML
stem/progenitor cell.
74
2-1
Combined Loss of Tet2/Tet3 Dioxygenases Induces Acute Myeloid Leukemia
Sensitive to Hypomethylating Agents
Koichiro Maie1, Mamiko Sakata-Yanagimoto1, Motohiko Oshima2, Yaeko Nakajima-Takagi2, Hirotaka Matsui3,
Takayasu Kato1, Hideharu Muto1, Enguerran Mouly4, Olivier A. Bernard4, Haruhiko Koseki5, Atsushi Iwama2,
Shigeru Chiba1
1
2
Department of Hematology, University of Tsukuba, Japan
Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Japan
3
Department of Molecular Laboratory Medicine, Kumamoto University, Japan
4
INSERM U1170, Institut Gustave Roussy, France
5
RIKEN Research Center for Allergy and Immunology, Japan
TET enzymes are methylcytosine dioxygenases that regulate gene expression by oxidizing methylcytosine and
hydroxymethyl/formylcytosine. Although TET2 mutation is recurrently found in various myeloid malignancies, it
has remained unclear whether the loss of catalytic function of TET dioxygenases indeed contributes to the
pathogenesis. We first retrieved RNA-seq data from The Cancer Genome Atlas database and found that the
expression of TET3 was positively correlated with TET2 in human acute myeloid leukemia (AML) patient
samples. Based on this finding, we established Tet2 and Tet3 double conditional knockout mouse model.
Combined loss of Tet2 and Tet3, but not single loss of either Tet2 or Tet3 induced fully penetrant, lethal acute
myeloid leukemia. Tet2/Tet3 double knockout hematopoietic stem and progenitor cells exhibited methylation and
gene expression profiles distinct from single knockout cells. Rescue of methylcytosine dioxigenase activity by
overexpression of the catalytic domain of human wild-type TET2, but not of a corresponding loss-of-function
mutant markedly reduced cell proliferation. Treatment of hypomethylating agents decitabine inhibited cell
proliferation in vitro, and azacitidine delayed the progression of AML in vivo. These results suggest substantial
reduction in TET dioxygenase activity plays an important role in AML pathogenesis.
75
2-2
Tumor Suppressive Role of JMJD5, a JmjC-domain Protein, in Breast Cancer
Akihiko Ishimura1, 2, Shoichiro Tange3, Minoru Terashima1 and Takeshi Suzuki1, 2
1
2
Div. Functional Genomics, Cancer Research Institute, Kanazawa Univ., Japan
Unit Mol. Therapeutic Target, Inst. Frontier Sci. Initiative, Kanazawa Univ., Japan
3
Res. Education Ctr. for Innovative & Preventive Med., Kanazawa Univ., Japan
In order to identify novel genes involved in leukemia/lymphoma, we have performed the retroviral insertional
mutagenesis in BXH2 and AKXD mice. Using mutant mice for Bloom (Blm) gene that shows genomic
instability, we have cloned Jmjd5 as a candidate tumor suppressor gene (Suzuki et al., EMBO J., 2006). Jmjd5 is
a nuclear protein that contains JmjC domain, a motif for histone demethylases. To investigate the physiological
role of Jmjd5, we generated Jmjd5-deficient mice (Ishimura et al., Development, 2012) and found that the
expression of a subset of p53-regulated genes was up-regulated in Jmjd5-deficient embryos at E 8.25 without
the induction of Trp53 expression. Thus, we have recently reported that Jmjd5 is a novel regulator of p53
signaling during mouse embryogenesis (Ishimura et al., Cell Tissue Res., 2016). However, it is difficult to study
whether Jmjd5 works as a tumor suppressor in mice, because Jmjd5-deficient mice show embryonic lethality at
the mid-gestation stage. Next, we generated Jmjd5-hypomorphic mouse embryonic fibroblasts (Jmjd5neo/neo
MEFs) to further investigate an intrinsic mechanism of Jmjd5 at cellular level. Jmjd5neo/neo MEFs proliferated
more slowly than wild-type MEFs, similar to the growth phenotype of Jmjd5-deficient embryos. This growth
retardation was significantly rescued under Trp53-null genetic background (Jmjd5neo/neo; Trp53Δ/Δ). Interestingly,
when Jmjd5neo/neo; Trp53Δ/Δ MEFs were cultured on non-adherent plate in serum-free medium, Jmjd5neo/neo;
Trp53Δ/Δ MEFs but not Trp53Δ/Δ MEFs could acquire sphere-forming activity that was an indicator of cancer
stem-like cell’s characters, although the growth ratio of Jmjd5neo/neo; Trp53Δ/Δ MEFs was less than Trp53Δ/Δ
MEFs. The result suggested that tumor suppressive role of Jmjd5 might be dependent on a specific cellular
context (e.g. ablation of Trp53).
On the other hand, we used TCGA (The Cancer Genome Atlas) database to obtain RNA-seq gene expression
profiles from various cancer patients and compared the expression of JMJD5 between cancer and normal tissues.
The result showed that the expression of JMJD5 was significantly down-regulated in triple-negative (TN) breast
cancer patients, known as an aggressive subtype of breast cancers, compared with normal breast epithelial
tissues (Normal) and other breast cancer types (nonTN). Knockdown of JMJD5 in T47D cells, a luminal-type
breast cancer cell line, lead to the elevated sphere-forming activity, whereas cell growth assay indicated no
significant difference between JMJD5-knockdown and control cells. Taken together, these results strongly
suggested a tumor suppressive role of JMJD5 in TN breast cancers. We will propose a role of JMJD5 repression
during breast cancer malignancies.
76
2-3
A Novel Tumor Suppressor, NDRG2 is Down-regulated by EZH2 Overexpression
through HTLV-1 Infection in ATL Cells
Tomonaga Ichikawa, Shingo Nakahata, Kazuhiro Morishita
Division of Tumor and Cellular Biochemistry, Department of Medical Sciences, University of Miyazaki, Japan
Recently, we identified N-myc downstream-regulated gene 2 (NDRG2) as a novel tumor suppressor gene in
adult T-cell leukemia/lymphoma (ATL) and other cancer. NDRG2 was a PTEN-binding protein that recruits
protein phosphatase 2A (PP2A) to promote the dephosphorylation of PTEN, resulting in the suppression of the
PI3K-AKT and NF-kappaB signal transduction pathways. It was considered that NDRG2 is a stress-inducible
gene and plays an important role in host defenses or immune responses to Human T-cell leukemia virus type 1
(HTLV1) infection. However, The molecular mechanism of NDRG2 expression regulation in cancer cells
remains poorly understood. HTLV-1 oncogenic protein Tax induced NDRG2 expression at 6-12 hours in JPX9
inducible expression of Tax by Zinc. The expression of NDRG2 was increased by Tax-induced NF-kappaB
bound to the NDRG2 promoter in JPX9 at the early expression of Tax. Furthermore, the long time expression of
Tax for 3-5 days induced the decrease of NDRG2 expression, along with the increase of Polycomb group
protein Enhancer of zeste homolog 2 (EZH2) and its histone modification H3K27me3 that occupy the NDRG2
promoter. EZH2 expression was up-regulated by the binding of Tax-drived NF-kappaB to the promoter region
of EZH2 in JPX9. EZH2 and H3K27me3 mark were over-exppresed in ATL patient sample and cell lines. The
knockdown of EZH2 through shEZH2 and EZH2 inhibitor exhibited the restoration of NDRG2 expression and
the suppression of cell proliferation in ATL cell lines. We suggested that while NDRG2 inhibited
HTLV-1induced aberrant signal transduction pathway through the up-regulation of NDRG2 expression at the
initial infection, the down-regulation of NDRG2 by the epigenetic modification of EZH2 and H3K27me3
involved in the sustained activation of signaling pathways to induce the disruption of host defenses or immune
responses, resulting in the initiation and progression of tumor development after latent HTLV-1 infection.
77
2-4
Ezh2 Loss in Hematopoietic Stem Cells Predisposes Mice to Develop
Heterogeneous Malignancies in an Ezh1-dependent Manner
Makiko Mochizuki-Kashio1,2, Kazumasa Aoyama1, Goro Sashida1,3, Motohiko Oshima1, Changshan Wang1,4,
Atsushi Iwama1
1
2
Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Japan
Laboratory of Chromatin Metabolism and Epigenetics, Graduate School of Science, Chiba University, Japan
3
International Research Center for Medical Sciences, Kumamoto University, Japan
4
College of Life Sciences, Inner Mongolia University, China
Recent genome sequencing revealed inactivating mutations in EZH2, which encodes an enzymatic component
of polycomb repressive complex 2 (PRC2), in patients with myelodysplastic syndrome (MDS),
myeloproliferative neoplasms (MPN) and MDS/MPN overlap disorders. We herein demonstrated that the
hematopoietic-specific deletion of Ezh2 in mice induced heterogeneous hematopoietic malignancies.
Myelodysplasia was detected in mice following the deletion of Ezh2, and resulted in the development of MDS
and MDS/MPN. Thrombocytosis was induced by Ezh2 loss and sustained in some mice with myelodysplasia.
Although less frequent, Ezh2 loss also induced T-cell acute lymphoblastic leukemia and the clonal expansion of
B-1a B cells. Gene expression profiling showed that PRC2 target genes were de-repressed upon the deletion of
Ezh2 in hematopoietic stem and progenitor cells, but were largely repressed during the development of MDS
and MDS/MPN, suggestive of the compensatory function of Ezh1, another enzymatic component of PRC2. The
deletion of Ezh1 alone did not cause dysplasia or any hematological malignancies in mice, but abolished the
repopulating capacity of hematopoietic stem cells when combined with Ezh2 loss. These results revealed an
essential role of Ezh1 in the pathogenesis of hematopoietic malignancies induced by Ezh2 insufficiency, and
highlighted the differential functions of Ezh1 and Ezh2 in hematopoiesis.
78
2-5
DNMT3A Mutation Inhibits Gene-Body Methylation to Maintain Acute Myeloid
Leukemia
Yuki Kagiyama, Yoko Ogawara, Issay Kitabayashi
Division of Hematological Malignancy, National Cancer Center Research Institute, Japan
DNA methyltransferase 3A (DNMT3A) catalyzes de novo DNA methylation and regulates self-renewal and
differentiation in hematopoietic stem cell. Acute myeloid leukemia (AML) is a hematopoietic disease
characterized by abnormal proliferation of myeloid cells with differentiation blocked. DNMT3A mutations are
detected in about 20% of AML patients and other hematological malignancies, with the majority of mutations
affecting a single amino acid, Arg882 (R882), in the catalytic domain, and often accompany mutation of other
genes such as NPM, IDH1/2 and FLT3 simultaneously. It has been reported that DNMT3AR882H, one of the most
frequent DNMT3A mutants, inhibits active DNMT3A tetramer. However, the crucial molecular mechanism
how DNMT3A mutants contribute to the onset of AML is yet to be revealed.
We have established an AML model with DNMT3AR882H by bone marrow (BM) transplantation transduced with
four mutant genes (NIDF; NPMc, IDH2R140Q, DNMT3AR882H and FLT3-ITD) in NPM+/- mouse BM cells (Y
Ogawara et al., 2015 Cancer Res.). To analyze roles of DNMT3AR882H in AML, we modified this model to a
DNMT3AR882H floxed-AML model, in which DNMT3AR882H can be conditionally deleted using the
tamoxifen-induced ERT2-Cre/loxP system.
We comfirmed that AML was efficiently induced in NI(D)floxF-mice as did NIDF-mice. To investigate the roles
of DNMT3AR882H in maintenance of AML, NI(D)floxF-AML mice were administrated with tamoxifen to delete
DNMT3AR882H. The deletion of DNMT3AR882H prolonged the survival of NI(D)floxF-AML mice, but it did not
affect the survival of conventional NIDF-AML mice. Morphological analysis indicated that DNMT3AR882H
deletion induced differentiation of the cells into mature granulocytes and erythroblast-like cells. Flow
cytometric analysis showed that CD11b+/Gr-1high cells and Ter119+ cells were increased, and that the leukemic
granulocyte/macrophage progenitor (L-GMP) population containing leukemia stem cells (LSCs) was
significantly decreased by DNMT3AR882H deletion. Transplantation analysis demonstrated that LSC activity was
severely decreased by DNMT3AR882H deletion. These data indicates that DNMT3AR882H blocks cell
differentiation to maintain LSCs.
Gene expression profiling showed that sets of genes associated with hematopoietic cell differentiation
(platelet-specific genes, Gata1-target genes and genes up-regulated in mature blood cells) were up-regulated by
DNMT3AR882H deletion. Interestingly, comprehensive DNA methylation analysis suggested that the deletion of
DNMT3AR882H promoted gene-body methylations, which are associated with activation of gene expression. In
particular, gene bodies of Fog1, Xpo7 and Dapk2, which are expressed in megakaryocyte and/or erythrocyte,
were hypermethylated and their expressions were increased by DNMT3AR882H deletion.
Fog1 is a cofactor for Gata1 that critically regulates megakaryo/erythropoiesis. Gata1-target genes and
platelet-specific genes were up-regulated by DNMT3AR882H deletion in association with the differentiation of the
leukemic cells. Thus, our data suggest that DNMT3AR882H blocks megakaryocyte/erythroid differentiation by
inducing gene-body hypomethylation of the target genes such as Fog1.
79
2-6
DNA Methylation of the Human Telomerase Reverse Transcriptase (hTERT)
Promoter in Human T-cell Leukemia Virus Type-1 (HTLV-1) Infected T-cells
Mariko Mizuguchi1, Toshifumi Hara2, Manami Yoshita-Takahashi1, Yuetsu Tanaka3, Takuya Fukushima4,
Masataka Nakamura1
1
Human Gene Sciences Center, Tokyo Medical and Dental University, Japan
Division of Virology, Niigata University Graduate School of Medical and Dental Sciences, Japan
3
Department of Immunology, Graduate School and Faculty of Medicine, University of the Ryukyus, Japan
4
Laboratory of Hematoimmunology, School of Health Sciences, Faculty of Medicine, University of the Ryukyus,
Japan
2
Abberant expression of the human telomerase reverse transcriptase (hTERT) gene is related to tumorigenesis.
We recently found that the transcription factor Krüppel-like factor 2 (KLF2) repressed hTERT transcription
through its binding to a DNA element encompassing CpG sequences in the hTERT promoter in normal human
resting T-cells. KLF2 binding to the hTERT promoter was inhibited by DNA methylation of the KLF2 binding
site, presumably resulting in expression of the hTERT gene in leukemic T-cell lines.
Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignant disease of peripheral T-cells caused by
human T-cell leukemia virus type-1 (HTLV-1) infection. To confirm DNA methylation of the hTERT promoter
in HTLV-1 infected T-cells, we examined three kinds of HTLV-1 infected cells: in vitro HTLV-1 transformed
cell lines, ATL-derived cell lines and primary PBLs from ATL patients. The hTERT promoter was highly
methylated in in vitro HTLV-1 transformed. Clones with methylated and unmethylated hTERT promoters
coexisted in each ATL-derived cell line. Those two cell lines constitutively expressed hTERT mRNA. DNA
methylation of the hTERT promoter was not seen in ATL patient-derived PBLs as well as normal T-cells. These
results suggest that primary ATL cells differ from in vitro HTLV-1 transformed and ATL-derived cell lines in
that DNA methylation of the hTERT promoter and hTERT expression.
80
2-7
Clinical Characteristics of TAL1 Super-enhancer Aberrations and STIL-TAL1
Fusion in Pediatric T Cell Acute Lymphoblastic Leukemia (T-ALL)
Shunsuke Kimura1, Masafumi Seki1, Kenichi Yoshida2, Yuichi Shiraishi3, Kenichi Chiba3, Hiroko Tanaka3,
Hiromichi Suzuki2, Keisuke Kataoka2, Motohiro Kato4, Katsuyoshi Koh5, Ryoji Hanada5, Toshihiko Imamura6,
Nobutaka Kiyokawa4, Akira Ohara7, Yasuhide Hayashi8, Satoru Miyano3, Seishi Ogawa2, Junko Takita1
1
Department of Pediatrics, The University of Tokyo, Japan
Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Japan
3
Human Genome Center Institute of Medical Science, The University of Tokyo, Japan
4
Department of Pediatric Hematology and Oncology Research, National Research Institute for Child Health
and Development, Japan
5
Department of Hematology and Oncology, Saitama Children’s Medical Center, Japan
6
Department of Pediatrics, Kyoto Prefectural University of Medicine, Japan
7
Department of Pediatrics, Toho University, Japan
8
Gunma Children's Medical Center, Japan
2
Background
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic tumor derived from malignant
transformation of T cell progenitor, accounting for 10 to 15% of newly diagnosed pediatric ALL cases.
TAL1 deletion on chromosome 1p33 is found in 20 to 25% of cases of T-ALL, which results in overexpression
of TAL1 by mediating fusion with STIL. Recently, a TAL1 super-enhancer (TAL1-SE) abnormality was reported,
which shows aberrant expression of TAL1 without gene fusion. Clinical characteristics of TAL1-SE aberrations
and STIL-TAL1 have been poorly reported.
Methods
Mutation analysis was performed by target capture sequencing including TAL1-SE region in 92 pediatric T-ALL
cases, and screening for STIL-TAL1 fusion by reverse transcription PCR.
Results and Discussion
TAL1-SE and STIL-TAL1 fusion were found in 5 (5.4%) and 13 cases (14%), respectively, and were mutually
exclusive. Three year overall survival (3y OS) of TAL1-SE, STIL-TAL1 fusion and the others was 80%, 91%
and 67%, and relapse was observed in 60%, 25% and 34%, respectively. STIL-TAL1 fusion cases showed
obviously high white blood cell (WBC) counts at diagnosis (>100×106/l, 73%). All the patients with TAL1-SE
were under 10 years old. Although TAL1-SE patients had high rate of relapse, 2 out of 3 relapsed patients were
rescued by treatment, which is different from other relapsed T-ALL patients whose prognosis is extremely
unfavorable. Amoung patients whose clinical data was available, all patients with STIL-TAL1 fusion (n = 7)
showed poor prednisone response (over 1,000/µl of blast at day 8), though they showed low relapse rate and
favorable OS (3y OS 86%), which might be resulted from high WBC counts at diagnosis.
Conclusion
A total of 92 cases with pediatric T-ALL were screened for TAL1-SE and STIL-TAL1 fusions. TAL1-SE and
STIL-TAL1 showed favorable outcome, though a poor predonisone response was observed in STIL-TAL1 fusion
patients and high relapse rate in TAL1-SE, which might be helpful for development a new therapeutic strategy
for these patients.
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2-8
TIP60 Is Critical for Acute Myeloid Leukemia Induced by MLL Fusions
Kazutsune Yamagata, Ykiko Aikawa, Mika Shino, Issay Kitabayashi
Division of Hematological Malignancy, National Cancer Center Research Institute, Tokyo, Japan
Chromosome translocation involving the mixed lineage leukemia (MLL) gene which generates an in-frame
fusion gene of the MLL 5′-region and partner genes, is a common rearrangement in acute myeloid and lymphoid
leukemia that is associated with poor prognosis. Knock-in and retroviral transduction studies show that
MLL-fusion results in constitutive activation of the transcription of target genes such as Hoxa9 and Meis1
during the development of leukemia. Recent studies show that several transcription regulators, such as Dot1L,
Cbx8, PAF1, and AEP/EAP complexes, are required for the leukemogenic activity of MLL-fusion; however, the
underlying mechanisms remain elusive.
To clarify the mechanism of epigenetic regulation by MLL-fusions, we established a novel leukemia model by
generating a conditional MLL-AF10 fusion gene, MLL-AF10 flox, in which the 3′-AF10 region is deleted by
4-OHT-activated Cre-ERT recombinase, resulting in inactivation of MLL-AF10 flox. Mouse hematopoietic
stem/progenitor cells (c-kit+) were immortalized by retroviral transduction of MLL-AF10 flox and cultured in
vitro or transplanted into irradiated recipient mice to induce AML in vivo. Treatment of MLL-AF10 flox cells
with 4-OHT in vitro to inactivate MLL-AF10 flox downregulated Hoxa9 expression and markedly decreased
colony-forming ability. In addition, the inactivation of MLL-AF10 flox rapidly decreased the acetylation level of
the histone H2A variant H2A.Z on the Hoxa9 locus. These results suggest that MLL-AF10, possibly together
with a histone acetyltransferase (HAT), regulates the acetylation of H2A.Z on the Hoxa9 locus. To identify the
HAT responsible for H2A.Z acetylation induced by MLL-AF10, protein complexes associated with
H2A.Z-containing nucleosomes were purified, resulting in the identification of Tip60, a MYST-type HAT in a
complex with H2A.Z. MLL-AF10 physically interacted with Tip60 via the AF10 C-terminal portion of
MLL-AF10. ChIP analysis showed that MLL-AF10 and Tip60 co-localize on the Hoxa9 locus in
MLL-AF10-transformed cells (MLL-AF10 cells). Furthermore, conditional deletion of Tip60 in MLL-AF10
(Tip60 Flox/Flox, Cre-ERT2) cells dramatically downregulated Hoxa9 expression and resulted in the
accumulation of unacetylated H2A.Z on the Hoxa9 locus. Consistent with these data, in vitro acetylation
analysis showed that Tip60 directly acetylates H2A.Z. To assess the role of Tip60 in leukemia development in
vivo, MLL-AF10 (Tip60 Flox/Flox, Cre-ERT2) leukemia cells were injected into recipient mice. Animals
receiving intraperitoneal injection of tamoxifen to delete Tip60 failed to develop MLL-AF10 leukemia. These
data indicate that Tip60 is required for the development of MLL-AF10 leukemia and suggest that MLL-AF10
recruits Tip60 to acetylate H2A.Z on the Hoxa9 locus.
The effect of H2A.Z acetylation on Hoxa9 expression was examined by purifying nucleosomes containing
acetylation-deficient 3KR H2A.Z (which mimics unacetylated H2A.Z), in which lysines 4, 7, and 11 were
substituted by arginine. 3KR H2A.Z preferentially formed nucleosomes with histone H3 trimethylation at lysine
27, which is catalyzed by polycomb repressive complex 2 (PRC2). This finding suggests that nucleosomes
including unacetylated H2A.Z are the preferential targets of PRC2. Loss of Tip60 in MLL-AF10 cells resulted
in decreased levels of acetylated H2A.Z on the Hoxa9 locus and the recruitment of Ezh2 (a catalytic subunit of
PRC2) and increased histone H3 K27 trimethylation. Taken together, these data indicate that Tip60 is a critical
factor in the development of MLL-AF10 leukemia. MLL-AF10 may maintain an active chromatin state on its
target genes by recruiting Tip60, which acetylates H2A.Z to prevent PRC2 recruitment and gene silencing. On
the other hand, unacetylated H2A.Z may be a signal for PRC2 recruitment, which would be induced as a result
of Tip60 loss or inactivation of MLL-AF10.
82
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Jmjd3, a Histone Demethylase, Is Required for the Functional Integrity of
Hematopoietic Stem Cells in Mice
Yuichiro Nakata, Takeshi Ueda, Kenichiro Ikeda, Norimasa Yamasaki, Hiroaki Honda
Department of Disease Model, Research Institute for Radiation Biology and Medicine, Hiroshima University,
Japan
The complex process of hematopoiesis involves the interplay between lineage-specific transcription factors and
a series of epigenetic marks, including covalent histone tail modifications. Among these, trimethylation of
histone H3 at Lys27 (H3K27me3) is associated with repressed gene expression. Jumonji domain-containing 3
(Jmjd3) is a histone demethylase specifically for H3K27me2/3 and highly expressed in primary LSK
(lineage-negative (Lin-), Sca-1+, c-Kit+) hematopoietic stem cells (HSCs) and myeloid cells. A recent study
demonstrated increased expression of Jmjd3 in CD34+ bone marrow cells in myelodysplastic syndrome and
suggested an involvement of Jmjd3 in tumor pathogenesis.
To investigate the role of Jmjd3 in the hematopoietic system, we generated and analyzed Jmjd3 conditional
knockout (cKO) mice. Acquired deletion of Jmjd3 significantly decreased LSK and myeloid progenitor cells in
adult bone marrow. In addition, competitive and serial bone marrow transplantation assay revealed impaired
self-renewal and reconstitution ability of HSCs in the cKO mice. These results indicate that Jmjd3 required for
the HSC integrity in a cell autonomous manner. To elucidate the link between Jmjd3 and hematologic
malignancies, c-kit+ bone marrow cells were retrovirally transduced with MLL-AF9 fusion oncogene. The cKO
cells displayed a significantly decreased number of colonies by MLL-AF9 compared with those of the controls,
which suggests jmjd3 contributes to MLL-AF9 leukemia development. We are currently investigating how these
issues would lead to therapeutic target for cancer.
83
3-1
Localization and Characteristic of Nestin-expressing Cells in Human Bone
Marrow and Their Abnormalities in Myelodysplastic Syndromes
Luan Cao Sy1, Naoshi Obara2, Tatsuhiro Sakamoto1, Takayasu Kato2, Hidekazu Nishikii2, Satoshi Ikeda3, Keiko
Suzuki3, Shigeru Chiba2
1
Department of Hematology, Graduate School of Comprehensive Human Sciences, University of Tsukuba,
Japan
2
Department of Hematology, Faculty of Medicine, University of Tsukuba, Japan
3
Department of Pathology, Tsuchiura Kyodo General Hospital, Japan
Background: Nestin-expressing cells (NeC) in the bone marrow (BM) have function as niche cells for
hematopoietic stem cells (HSCs). In mouse BM, two distinct subsets of NeC are reported: those localized
within/proximity of arterioles and the others around sinusoids, with the emphasis that the former consist of the
HSC niche. However, the location of NeC in human BM and their roles as a component of HSCs niche remain
to be clarified. Studies of NeC in human BM are limited, and no information is available about the anatomical
distribution.
Methods: Paraffin-embedded BM biopsy samples from patients with myelodysplastic syndromes (MDS) were
immunostained with antibodies against 6 markers: nestin, CD34, laminin, α-smooth muscle actin (αSMA), glial
fibrillary acidic protein (GFAP), and neurofilament heavy (NFH). BM biopsy specimens from patients with
malignant lymphoma without BM invasion were analyzed as a normal control.
Results: NeC were found at multiple locations at distinct contexts. NeC that were associate with the arteriolar
structure were found at three locations; the highest expression was demonstrated in the intimal layer cells,
followed by those in smooth muscle and adventitial layer. Another type of NeC was identified without
association with vascular structures, albeit at a low frequency and weak nestin staining. In some MDS samples
with hypercellular BM, there was a marked increase in NeC that are unassociated with the vascular structures. A
portion of these increased NeC co-expressed GFAP. These cells potentially represent Schwann cells because
some of them surrounded NFH-stained structure.
Discussion: A variety of stromal cells in human BM express nestin at different levels. A part of GFAP(+)
Schwann cells along with the arteriolar adventitial layer appear to express nestin. These cells might be the origin
of those which are increased in BM of patients with MDS. It is to be elucidated whether increased sympathetic
nervous structure is involved in pathophysiology of MDS.
84
3-2
The Role of HMGA2 in the Pathogenesis of Myeloproliferative Neoplasms (MPNs)
Koki Ueda1, Kazuhiko Ikeda1,2, Kazuei Ogawa1, Yuko Hashimoto3, Soji Morishita4, Norio Komatsu4, Kotaro
Shide5, Kazuya Shimoda5, Atsushi Iwama6, Yasuchika Takeishi1
1
2
Dept. Cardiology and Hematology, Fukushima Medical Univ., Fukushima, Japan
Dept. Blood Transfusion and Transplantation Immunology, Fukushima Medical University, Fukushima, Japan
3
Dept. Diagnostic Pathology, Fukushima Medical University, Fukushima, Japan
4
Dept. Hematology, Juntendo University School of Medicine, Tokyo, Japan
5
Dept. Gastroenterology and Hematology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
6
Dept. Cellular and Molecular Medicine, Chiba University Graduate School of Medicine, Chiba, Japan
MPNs are driven by mutations in JAK2, CALR and MPL. Mutations in epigenetic modifiers and aberrant
expressions of microRNAs may also play crucial roles, but their downstream are largely unknown. We have
recently shown high HMGA2 mRNA level in almost all patients with myelofibrosis (MF) and a quarter patients
with other MPNs. To elucidate mechanisms that increase HMGA2 level in them, we here performed deep
sequencing and qPCR, which revealed correlation of high HMGA2 level with either mutations in EZH2 and
ASXL1 or reduced let7. Thus, to clarify if HMGA2 affect JAK2V617F+ hematopoiesis, we crossed
HMGA2-overexpressing mice (Hmga2-Tg) with JAK2V617F transgenic mice (JAK2VF-Tg) and obtained
Hmga2-JAK2VF mice (double-Tg). At 3 months old, leukocytosis, thrombocytosis, anemia and splenomegaly
were most severe in double-Tg compared with Hmga2-Tg or JAK2VF-Tg. Hmga2-Tg and JAK2VF-Tg survived
for over a year, but all double-Tg died within 5 months. Lineage-Sca1+Kit+ cells were most frequent in
double-Tg followed by ∆Hmga2, indicating HMGA2 contributes to expansion of JAK2V617F+ hematopoietic
stem cells (HSC). In competitive/serial transplants, ∆Hmga2 and double-Tg cells steadily expanded, while
JAK2VF cells were decreased and eventually rejected in 3rd transplant. Thus, HMGA2 may accelerate
proliferative hematopoiesis harboring JAK2V617F with expanding MPN HSC. In fact, endogenous HMGA2 is
upregulated in JAK2VF with conditional EZH2 KO (EZH2-/-JAK2VF). To confirm the role of HMGA2, we are
testing in vivo effect of HMGA2 KO and seeking commonly deregulated genes with double-Tg by RNA-seq in
EZH2-/-JAK2VF.
85
3-3
Combination of ASXL1 and RUNX1 Mutants Induced MDS/AML in Mice with
Shorter Latencies
Reina Nagase1, Daichi Inoue2, Makoto Saika1, Hsin-An Hou3, Wen-Chien Chou4, Kimihito Cojin Kawabata1,
Hironori Harada5, Akinori Kanai6, Susumu Goyama1, Hiroaki Honda7, Hwei-Fang Tien3, Toshio Kitamura1
1
Dvision of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Japan
Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, USA
3
Division of Hematology, Department of Internal Medicine, National Taiwan University Hospital, College of
Medicine, National Taiwan University, Taiwan
4
Departments of Laboratory Medicine, National Taiwan University Hospital, College of Medicine, National
Taiwan University, Taiwan
5
Department of Hematology, Juntendo University School of Medicine, Japan
6
Department of Molecular Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima
University, Japan
7
Department of Disease Model, Research Institute for Radiation Biology and Medicine, Hiroshima University,
Japan
2
Recently, advances in sequencing technologies have identified a large number of novel somatic mutations in
MDS patients, including mutations in moluecules involved in epigenetic regulation and splicing. We previously
reported that bone marrow (BM) cells transduced with mutants of an epigenetic regulator ASXL1 (ASXL1-MT)
induced an MDS-like disease in the transplanted mice by inhibiting PRC2 functions in a dominant-negative
fashion. It was reported that ASXL1 mutations are closely associated with mutations in RUNX1, both of which
are independent poor prognostic factors. Thus, we attempted to clarify how these mutations cooperate in
inducing MDS. First, we found that mutations in the transactivation domain of RUNX1 with premature stop,
such as S291fs, were significantly enriched among ASXL1-mutated MDS. Second, 32Dcl3 cells transduced
with both ASXL1-MT and RUNX1-S291fs displayed differentiation block and myeloid dysplasia after G-CSF
treatment. Third, in the mouse bone marrow transplantation (BMT) model, the mice with both mutants
developed profound macrocytic anemia compared to the control mice by 9 months (P < 0.001), although the
overall survival was not affected. Finally, the BM cells transduced with the both mutants significantly decreased
erythroid colony output. Next, to mimic the clinical condition, we generated Asxl1-MT conditional knock-in
(Asxl1-MT-KI) mice where Asxl1-MT is inserted downstream of Rosa26 promoter. Clonal hematopoiesis with
somatic mutations in ASXL1 as well as DNMT3A and TET2 is frequently observed in healthy elderly, and is
associated with increased risk of hematologic malignancies. This suggests that ASXL1 mutations could work as
a driver mutation in myeloid malignancies. Asxl1-MT-KI mice could be a good model to investigate the human
clonal hematopoiesis and pre-MDS pathogenesis. The Asxl1-MT-KI mice were crossed with the hematopoietic
specific Vav-cre transgenic mice and Asxl1-MT proteins are expressed exclusively in hematopoietic cells. In the
BMT model, Asxl1-MT-KI-BM cells transduced with RUNX1-S291fs induced macrocytic anemia by 3 months
(P < 0.001) and died of MDS/AML in 5 months. We are now investigating the underlying molecular
mechanisms by which ASXL1-MT and RUNX1-S291fs collaborate in inducing MDS/AML.
86
3-4
ATP-binding Cassette Transporter G2 (ABCG2) Expression Characterizes
Advanced Myelodysplastic Syndrome Either in Mouse Model or Human Disease
Kimihito C Kawabata1, Yasutaka Hayashi1, Daichi Inoue1,2, Hiroko Sakurai3, Jiro Kitaura4, Susumu Goyama1,
Yuka Harada5, Hironori Harada3, Hiroyuki Aburatani6, Toshio Kitamura1
1
Institute of Medical Science, Division of Cellular Therapy, The University of Tokyo, Japan
2
Memorial Sloan-Kettering Cancer Center, USA
3
Department of Hematology, School of Medicine, Juntendo University, Japan
4
Atopy Research Center, Juntendo University, Japan
5
Department of Clinical Laboratory Medicine, Bunkyo Gakuin University, Japan
6
Affiliation,Country Genome Science Division, Research Center for Advanced Science and Technology, The
University of Tokyo, Japan
A histone H3 Lysine 27 (H3K27)-methyltransferase, enhancer of zeste homolog 2(EZH2) is known as a
tumor-associated gene. Physiological role of EZH2 is an enzymatic component of polycomb repressive complex
2 (PRC2) to inhibit expression of target genes. EZH2 plays tumor-suppressive roles in myeloid malignancies.
We have generated a short-form EZH2 that lacks the catalytic SET domain (EZH2-dSET). Using this
EZH2-dSET we could produce serially transplantable MDS-like disease with leukemic transformation
(secondary AML). Microarray analysis using the MDS-like bone marrow cells enabled us to identify novel
targets of EZH2 in MDS tumorigenesis, including ATP-binding cassette (ABC) transporters. Intriguingly, with
Abcg2 expression alone, primary bone marrow cells could produce an MDS-like cytopenic disease in our BMT
model. With all in vitro assays and microarray analyses using those primary mouse bone marrow cells, how the
Abcg2-expressing MDS cells maintain stem/ progenitor activity was estimated. In clinical specimens, ABCG2
high expressions were specific to MDS and associated with advanced cases, while ABCG2 expression was low
in secondary AML. Furthermore, in vitro and live imaging experiments show that stromal components of those
MDS-like mice are converted and have influences on either normal or MDS hematopoietic cells. In conclusion,
MDS is characterized by higher expression of ABCG2 achieved by mechanisms including inhibition of EZH2.
And those cells are supposed to behave as potent tumor-initiating/ stem cells with conversion of bone marrow
microenvironments.
87
3-5
Functional Analysis of UTX, a Histone H3K27 Demethylase, in Normal
Hematopoiesis and Hematologic Malignancies
Yasuyuki Sera1, Takeshi Ueda2, Yu-ichiro Nakata1, Ken-ichiro Ikeda1, Norimasa Yamasaki1, Hideaki Oda3, Akiko
Nagamachi4, Akinori Kanai4, Toshio Suda5, Keiyo Takubo6, Hiroaki Honda 1
1
Department of Disease Model, Research Institute for Radiation Biology and Medicine, Hiroshima University,
Japan
2
Department of Biochemistry, Faculty of Medicine, Kinki University, Japan
3
Department of Pathology, Tokyo Women’s Medical University, Japan
4
Department of Molecular Oncology and Leukemia Program Project, Research Institute for Radiation Biology
and Medicine, Hiroshima University, Japan
5
Cancer Science Institute, National University of Singapore, Singapore
6
Department of Stem Cell Biology, Research Institute, National Center for Global Health and Medicine, Japan
Trimethylation of lysine 27 on histone H3 (H3K27me3) is regarded as a repressive histone mark that functions as
gene silencing and plays important roles especially in development and tissue differentiation. While methylation
of H3K27 is mediated by mediated by polycomb repressive complex 2 (PRC2), demethylation of H3K27 is
mainly regulated by two distinct histone demethylases, JMJD3 and UTX. Recent studies reported somatic
mutations of UTX in human cancers including hematologic malignancies, such as myelodysplastic syndrome
and myeloproliferative neoplasms (MDS/MPN). To elucidate functional roles of UTX in normal hematopoiesis
and leukemogenesis, we generated UTX conditional knockout mice. UTX-deficient mice exhibited
hematopoietic abnormalities, such as leukocytosis, thrombocytopenia, and tri-lineage dysplasia that are
reminiscent of MDS/MPN. Analysis of hematopoietic tissues revealed abnormal differentiation of
hematopoietic cells and extramedullary hematopoiesis. In addition, competitive repopulation assays
demonstrated that UTX-deficient hematopoietic stem cells (HSCs) possessed a significantly reduced
reconstituion activity compared to control HSCs. Moreover, transcriptome analysis of UTX-deficient
hematopoietic progenitor cells and HSCs (HSPCs) revealed upregulation of oxidative phosphorylation pathway
and exhibited accumulation of reactive oxygen species (ROS) by 5FU treatment. To investigate possible
contribution of UTX deficiency to leukemogenesis, retroviral insertional mutagenesis was employed. Virus
infection induced various types of leukemias in UTX-deficienc mice with much higher incidence than controls,
indicating that deficiency of UTX predisposes to leukemic transformation. Inverse PCR identified six retroviral
common integration sites (CISs), and bone marrow transplantation experiments demonstrated that
overexpression of Sox4, a CIS gene, cooperated with UTX deficiency to develop leukemia. These results
indicated that UTX plays essential roles in normal hematopoiesis and deficiency of UTX induces MDS/MPN
that progresses to leukemia in cooperation with additional gene alterations.
88
4-1
Understanding the Molecular Pathogenesis of Peripheral T-cell Lymphoma by
Laser Microdissection-based Sequencing
Tran B. Nguyen1, Mamiko Sakata-Yanagimoto1,2,3, Yukitsugu Asabe1, Kenichi Yoshida4, Koji Izutsu5, 6, Naoya
Nakamura7, Kengo Takeuchi8, Seishi Ogawa4, Shigeru Chiba1,2,3
1
Department of Hematology, Faculty of Medicine, University of Tsukuba, Japan
Department of Hematology, Comprehensive Human Biosciences, University of Tsukuba, Japan
3
Department of Hematology, University of Tsukuba Hospital, Japan
4
Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Japan
5
Department of Hematology, Toranomon Hospital, Japan
6
Okinaka Memorial Institute for Medical Research, Japan
7
Department of Pathology, Tokai University School of Medicine, Japan
8
Pathology Project for Molecular Targets, The Cancer Institute, Japanese Foundation for Cancer Research,
Japan
2
Angioimmunoblastic T-cell lymphoma (AITL) is a subtype of peripheral T-cell lymphoma (PTCL),
characterized by generalized lymphadenopathy and autoimmune-like manifestations. Regarding genetic lesions
of AITL, frequent mutations in TET2, IDH2, DNMT3A and RHOA have been identified. Among these, TET2
and DNMT3A mutations were identified in the stem/progenitor cells of some PTCL patients. In contrast, we
described that G17V RHOA mutations were confined to the tumor cell-enriched population based on the
analysis of a few cases. To further clarify the molecular pathogenesis of AITL, we performed targeted
sequencing for 71 genes in 87 samples, then, distribution of mutations were analyzed using pooled cells
collected by laser-assisted microdissection in 19 out of the 87 samples, followed by amplicon sequencing.
Recurrent mutations were newly found in NAV2, ODZ1 (4/87 [4.6%] for each); COL19A1, FAT2, MTERF3,
NOTCH1 (3/87 [3.4 %] for each); and B2M, HMCN1, LAMA2, MLL2, TET3 (2/87 [2.3%] for each). We found
that the TET2 and DNMT3A mutations were identified in both programmed death-1 (PD-1)-positive and
CD20-positive cells in 15 out of 16 cases, while the G17V RHOA and R172 IHD2 mutations were confined to
the PD-1-positive cells in all 10 and 4 samples, respectively, that harbored the mutations. Most of the newly
identified mutations were similarly classified into those demonstrated in both PD-1- and CD20-positive cells,
and those in only PD-1-positive cells. However, we found some mutations only in the CD20-positive but not in
the PD1-positive cells. Detection of B cell-specific mutations suggests premalignant status of B cells in AITL,
in which B-cell lymphoma occasionally develops. The distribution of IDH2 mutations was among our interests.
R172 IDH2 mutations were found to co-exist with TET2 mutations in the PD-1-positive cells, but not in the
CD20-positive cells. This suggests that IDH2 mutations are actually co-present with TET2 mutations but only in
the tumor cells, rather than in the hematopoietic stem/progenitor cells.
89
4-2
Genomic Characterization of Primary Central Nervous System Lymphoma
Kazutaka Fukumura1, Akitake Mukasa2, Yoshitaka Narita3, Ryo Nishikawa4, Motoo Nagane5, Hiroyuki Mano6,7
1
Laboratory of Clinical Sequence, Department of Computational Biology and Medical Sciences, Graduate
School of Frontier Sciences, The University of Tokyo, Japan
2
Department of Neurosurgery, Graduate School of Medicine, The University of Tokyo, Japan
3
Department of Neurosurgery and Neuro-Oncology, National Cancer Center Hospital, Japan.
4
Department of Neuro-Oncology/Neurosurgery, Saitama International Medical Center, Saitama Medical
University, Japan
5
Department of Neurosurgery, School of Medicine, Kyorin University Faculty of Medicine, Japan
6
Department of Cellular Signaling, Graduate School of Medicine, The University of Tokyo, Japan
7
Department of Cellular Signaling, Graduate School of Medicine, The University of Tokyo, Japan
Primary central nervous system lymphoma (PCNSL) is a rare malignancy confined to the central nervous
system (CNS), and majority of PCNSL is pathologically classified as diffuse large B-cell lymphoma (DLBCL).
We have now performed whole-exome sequencing for 41 tumor tissues of DLBCL-type PCNSL and paired
normal specimens and also RNA-sequencing for 30 tumors, revealing a very high frequency of nonsynonymous
somatic mutations in PIM1 (100 %), BTG2 (92.7 %), and MYD88 (85.4 %). Many genes in the NF-κB pathway
are concurrently mutated within the same tumors. Further, focal deletion or somatic mutations in the HLA genes
are associated with poor prognosis. Copy number amplification and overexpression of genes at chromosome
7q35 were both found to predict short progression-free survival as well. Oncogenic mutations in GRB2 were
also detected, the effects of which in cultured cells were attenuated by inhibitors of the downstream kinases
MAP2K1 and MAP2K2. Individuals with tumors positive for MYD88 mutations also harbored the same
mutations at a low frequency in peripheral blood mononuclear cells, suggesting that MYD88 mutation-positive
precancerous cells originate outside of the CNS and develop into lymphoma after additional genetic hits that
confer adaptation to the CNS environment.
90
5-1
Hypoxic Resistance by Loss of NDRG2 Expression via Activation of the PI3K/AKT
Signaling Is One of the Main Pathogenic Features in ATLL and Other Cancers
Shingo Nakahata1, Tomonaga Ichikawa1, Yusuke Saito1, Tomohiko Taki2, Masafumi Taniwaki3, Kazuhiro
Morishita1
1
Division of Tumor and Cellular Biochemistry, Department of Medical Sciences, University of Miyazaki, Japan
2
Department of Molecular Diagnostics and Therapeutics, Kyoto Prefectural University of Medicine, Japan
3
Department of Molecular Hematology and Oncology, Kyoto Prefectural University of Medicine, Japan
Adult T-cell leukemia/lymphoma (ATLL) is an aggressive malignancy of the peripheral T-cells caused by
human T-lymphotropic virus type 1 with extremely poor prognosis. We have recently reported that the N-myc
downstream-regulated gene 2 (NDRG2) gene on chromosome 14q11 is frequently inactivated by genomic
deletion and/or epigenetic silencing in ATLL (Nat Commun, 2014). Down-regulation of NDRG2 expression in
ATLL cells induces constitutive activation of the PI3K/AKT pathway via the enhanced phosphorylation of
PTEN at the Ser380/Thr382/Thr383 (STT) cluster. NDRG2 interacts with PTEN and recruits PP2A to PTEN to
promote dephosphorylation of PTEN-STT. Because the expression of NDRG2 is induced by cellular stresses
such as hypoxia, in this study, we investigated whether NDRG2 plays a role in the regulation of hypoxia
responses through the regulation of PI3K/AKT pathway. We found that NDRG2 acts as a novel negative
feedback regulator of the PI3K/AKT pathway. Upon the activation of PI3K, the downstream kinase SGK1
phosphorylates NDRG2 at Se332 and promotes the recruitment of PP2A to PTEN, resulting in the
dephosphorylation of PTEN-STT and suppression of the activation of AKT. Hypoxic treatment of
non-cancerous HacaT cells expressing NDRG2 in normal growth media exhibited decreased phosphorylation of
PTEN-STT and AKT-Ser473, which was accompanied by decreased cell growth. On the other hand, ATL cell
lines with low NDRG2 expression under hypoxia showed high proliferation ability and sustained activation of
PI3K/AKT, which was reversed by ectopic expression of NDRG2. These results suggest that NDRG2 might
regulate the PI3K/AKT pathway during hypoxia to control cell survival, cell growth or apoptosis and
down-regulation of NDRG2 in ATLL cells can partly contribute to the resistance to hypoxia, which may play a
role in the pathogenesis of ATLL.
91
5-2
Redox Status Dictates the Susceptibility of Mantle Cell Lymphoma to Bortezomib
Sudjit Luanpitpong, Nawin Chanthra, Paweorn Angsutararux, Surapol Issaragrisil
Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University,
Thailand
Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin B-cell lymphoma with disappointing 5-year
survival rate. While proteasome inhibitor bortezomib (BTZ) has remarkably improved therapeutic outcome of
relapsed and refractory MCL, substantial numbers of MCL patients are either intrinsic or acquired resistance to
BTZ. An increased reactive oxygen species (ROS) is observed in the microenvironment of various aggressive
tumors, including B-cell lymphoma, but how ROS affects MCL cellular behaviors is still limit. Superoxide
anion (O2−) is the primary free radical generated from aerobic cellular metabolism of tumor cells themselves
and/or neighboring cells. Here, we investigated the roles of O2− in the regulation of MCL aggressive cancer
phenotypes. Using various known small molecule inhibitors and donor of cellular O2−, we revealed for the first
time an inverse correlation between the level of O2− and aggressive phenotypes. O2− inhibits MCL clonal
exapasion, cell proliferation and sensitizes MCL to apoptosis induced by BTZ. We further observed that
expression of anti-apoptotic Mcl-1 is a favorable target of O2−. Using pharmacological inhibition and gene
manipulation, we verified that Mcl-1 is responsible for apoptosis sensitizing effect by O2−. These findings
identify an important role of redox status of the cells in determining their aggressive phenotypes, which are
imperative to a better understanding of therapeutic resistance and pathophysiology of aggressive B-cell
lymphoma.
92
5-3
Inhibitory Effect on Lymphoma Cells Proliferation by Regulating Cholesterol
Metabolism Pathway
Yukio Fujiwara1, Hasita Horlad1, Daisuke Niino2, Yutaka Okuno3, Yoshitaka Kikukawa3, Masao Matsuoka4,
Motohiro Takeya1, Yoshihiro Komohara1
1
Department of Cell Pathology, Graduate School of Medical Scuences, Kumamoto University, Japan
2
Department of Pathology, Graduate School of Biomedical Scuences, Nagasaki University, Japan
3
Department of Hematology, Rheumatology and Infectious Disease, Graduate School of Medical Scuences,
Kumamoto University, Japan
4
Laboratoy of Virus Control, Instisute for Virus Research, Kyoto University, Japan
Cholesterol is one of main component composed of cell wall membrane, and also known as a substrate of bile
acid and steroidogenic hormones. Free and esterified cholesterol is containing in chylomicrons, very low density
lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL) in blood stream, and
LDL is known to be prominent carrier of cholesterol in blood. As cancer cells grow and proliferate, cholesterol
has to be up-taken in cancer cells and cholesterol metabolism is up-regulated in cancer cells. Since free
cholesterol is harmful to cells via unknown mechanisms, free cholesterol has to be quickly esterified by ACAT.
Based on this mechanism, many researchers tried to treat the cancer cells by ACAT inhibitor, however, limited
anti-cancer effect of ACAT inhibitors was observed. Although the reasons of this limited anti-cancer effect has
been uncovered, we suggested that blocking the cholesterol efflux showed more effective for anti-cancer therapy.
SR-BI is expressed on cell surface membrane of many cells including cancer cells, and involved in cholesterol
efflux via HDL. Therefore we suggest the SR-B1 as a novel target for anti-cancer therapy.
It is well known that many vacuoles are detected in cytoplasm in Burkkit lymphoma cells, and this vacuole is
one of important finding for the morphological diagnosis. Interestingly, we newly noticed that this “vacuole” is
often observed in high grade B-cell and T-cell lymphoma, and esterified cholesterol is a main component of this
“vacuole”. In our unpublished data, SR-BI was found to be overexpressed on lymphoma cell lines. High grade
lymphoma cell might be more sensitive to inhibition of cholesterol metabolism. Based on these background, we
hypothesized SR-BI inhibitor and ACAT inhibitor are effective to high grade lymphoma cells in which
cholesterol metabolism is up-regulated.
In the present study, we revealed that SR-BI inhibitor significantly inhibited lymphoma cell proliferation and
ACAT inhibitor also inhibited lymphoma cell proliferation. Furthermore, synergistic effect of SR-BI inhibitor
and ACAT inhibitor was observed in in vitro study and the administration of SR-BI inhibitor suppressed
lymphoma progression in tumor-bearing mice model. Therefore, those data indicates that the application of
SR-BI inhibitors and ACAT inhibitors is a potential new anti-lympoma therapy targeting cholesterol metabolism
pathway in lymphoma.
93
5-4
Expression and Quantification of Apoptosis Associated Protein (Livin) in a
Spectrum of Lymphomas from Rural Africans in Zaria, Nigeria
Sirajo Mohammed Aminu1, Mohammed Faruk2, Yawale Iliyasu2, Abdullahi Mohammed2, Sani Ibrahim4,
Mohammed Sani Shehu1, Ahmed Adamu3, Abdullahi Jubril Randawa5, Adamu Abdullahi6, Adoke Kasimu
Umar
1
Department of Haematology and Blood Transfusion, Ahmadu Bello University, Teaching Hospital, Nigeria
2
Department of Pathology, Ahmadu Bello University Teaching Hospital, Nigeria
3
Department of Surgery, Ahmadu Bello University Teaching Hospital, Nigeria
4
Department of Biochemistry, Ahmadu Bello University, Nigeria
5
Department of Obstetrics and Gynaecology, Ahmadu Bello University Teaching Hospital Zaria, Nigeria
6
Affiliation,Country Department of Radiology/Radiotherapy, Ahmadu Bello University Teaching Hospital,
Nigeria
Hematologic malignancies are a group of cancers characterized by abnormal growth of cells of hematopoietic
origin. Resistance to chemotherapy in hematologic malignancies is one of the obstacles that affects prognosis
negatively and overall survival of patients. One of the factors implicated in chemoresistances are the presence of
Anti-apoptotic protein of which Livin is one. Livin protein is one of the most widely studied members of
antiapoptotic protein family. It is a protein with one baculoviral IAP repeat and a COOH-terminal RING zinc
finger domain which, previous studies though not in Nigeria showed that it is upregulated in lymphomas. Herein,
we report the expression of Livin protein, in various subtypes of malignant lymphomas in Zaria, Nigeria using
immunohistochemistry and immunofluorescence from 84 cases. These include leukaemia-lymphoma, Burkitt’s,
Hodgkin’s lymphoma and other sub types of Non-Hodgkin lymphoma. Immunohistochemistry and
immunofluorescence were performed on 5-μm sections from formalin-fixed paraffin-embedded tissue samples
from the subjects using Anti-BIRC7 monoclonal antibody for BIRC7/Livin protein expression. The results
showed positive expression of the Livin protein on all the tissue blocks. The Livin protein expression on
Hodgkin lymphoma sections was more defined compared to Burkitt’s lymphoma and leukaemia-lymphoma (p
<0.05). The results show strong significant expression of the Livin protein in diffuse large B cell lymphoma
compared to other non-Hodgkin lymphoma subtypes in this study (p <0.05). However, there was no significant
expression of the Livin protein when comparing diffuse non-Hodgkin lymphoma (centrocytic type) and chronic
small cell lymphoma (p >0.05).
Upregulated expression of Livin protein in non-Hodgkin lymphoma subtypes
may imply that the protein can serve as a possible prognostic marker/target in the management of
haematological malignancies. This needs further research.
Key words: Expression, Quantification, Livin, Lymphoma
94
5-5
Histone Deacetylase Inhibitors Inhibit Metastasis via Restoration of microRNAs
and Its Target CCR6 in Advanced Cutaneous T-cell Lymphoma
Akihro Kitadate1, Fumito Abe1, Sho Ikeda1, Junsuke Yamashita2, Hiroki Nakanishi3, Naoto Takahashi1,
Tomomitsu Myagaki4, Makoto Sugaya4, Hiroyuki Tagawa1
1
Department of Hematology, Nephrology, and Rheumatology, Akita University Graduate School of Medicine,
Japan
2
Division of Bioscience Center, Radioisotope, Akita University, Japan
3
Affiliation, Country Research Center for Biosignal, Akita University, Japan
4
Department of Dermatology, The University of Tokyo, Japan
Background: We have shown that a tumor suppressive microRNA, miR-150 inhibits metastasis via combining
"seed sequence" of messenger RNA of a chemokine receptor CCR6 in advanced cutaneous T-cell lymphoma
(CTCL) (Blood 2014; Oncotarget 2016). Because histone deacetylase inhibitor (HDACi), vorinostat yielded
excellent outcome for treating advanced CTCL, HDACi might have a potential to reduce metastasis capability
of CTCL via targeting miR-150 and CCR6. Indeed we previously showed that a tumor suppressive miRNA,
miR-16 was epigenetically downregulated and HDACi restored its expression leading to induce cellular
senescence or apoptosis in various T-cell lymphomas (Oncogene 2015). This led us to consider that HDACis
might upregulate variety of tumor suppressive miRNAs and there might be deep association of restoration for
some critical gene(s) or/and miRNA(s) expression with HDACis in CTCL oncogenesis. In this study, we
focused on to investigate relationship between miRNAs and HDACis, especially focused on to detect CCR6
associated miRNA(s).
Methods: To investigate whether HDACis could inhibit metastasis against advanced CTCL, we used CTCL cell
lines (My-La, HH and HUT78) for functional analysis whose subcutaneous transplantation into NOD/Shi-scid
IL-2γnul mice (CTCL mice) show with shortened survival and multiple metastases. To investigate expression
changes of miRNAs by HDACis, we conducted miRNA microarray. To validate in vivo effects of HDACi or
miR-150, we used the CTCL mouse model. qRT-PCR of miR-150 was also examined against primary early and
advanced CTCL.
Results: We demonstrated that pan-HDACis (vorinostat and panobinostat) inhibited migration of CTCL cells
with downregulating CCR6. When we examined miRNA array against CTCL cells treated with and without
respective HDACi, 168 miRNAs were commonly upregulated by pan-HDACis in My-La, HH and HUT78.
Among these 168 miRNAs, 23 miRNAs that possesses seed sequence of CCR6 showed upregulation by
HDACis treatment. Among them, although miR-24-3p, miR-96, miR-150 and miR-196 have potential to
downregulate CCR6 with migration inhibition, only miR-150 was likely to have biological significance because
of its highly expression in normal T-cells with downregulation of advanced CTCL samples. These data strongly
suggested that most likely target miRNA of pan-HDACis in metastasis-inhibition of advanced CTCL is
miR-150. Finally, we conducted in vivo experiment to examine whether miR-150 indeed inhibit tumor
metastasis. We demonstrated that administration of miR-150 against CTCL model mice led to prolong their
survivals.
Conclusion: miR-150 and CCR6 are essential targets of pan-HDAC inhibitors in advanced CTCL. Our results
provide rational reasons for using pan-HDACi against metastatic CTCL. Furthermore, these results suggest that
miR-150 could be not only powerful biomarker for molecular diagnosis, and predictive of metastasis, but also
novel therapeutic molecule in advanced CTCL.
95
5-6
The Essential Role of c-kit-SCF Signaling in the Leukemic Stem Cells
Mediated ATL Cell Propagation and Drug Resistance
Wakako Kuribayashi1,2, Takuo Mizukami1, Kazuya Takizawa, Kazuya Takizawa1, Kenji Sugata 3, Madoka
Kuramitsu1, Kiyoko Nojima 2, Haruka Momose 2, Atsushi Iwama 2, Masao Matsuoka 3, Isao Hamaguchi 2
1
2
Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Japan
Department of Safety Research on Blood and Biological Products, National Institute of Infectious Disease,
Japan
3
Laboratory of Virus Control, Institute for Virus Research, Kyoto University, Japan
Adult T-cell leukemia (ATL) is a malignant disease caused by infection with human T-lymphotropic virus type 1
(HTLV-1). The response to chemotherapy of ATL is extremely poor because of intrinsic or acquired drug
resistance. Thus, we hypothesized the existence of chemotherapy resistant leukemic stem cells (LSCs) in ATL.
LSCs were reported in various tumor and leukemia, and we have previously identified ATL stem cell candidates
in an ATL model using Tax transgenic (Tax-Tg) mice (Yamazaki et al., Blood 2009). Recently, HTLV-1 bZIP
factor (HBZ) was also identified as an important factor in the development of ATL. HBZ transgenic (HBZ-Tg)
mice also show ATL-like disease, and are thought to be an ATL model mouse (Satou et al., PLoS Pathog, 2011).
We aimed to identify and characterize ATL stem cells in the HBZ-Tg mouse to clarify the clonal evolution of
ATL stem cells and molecular mechanisms underlying ATL development and drug resistance.
First, we transplanted 1×107 Ht48 cells derived ATL model using HBZ-Tg mice intraperitoneally into C57BL/6
mice to assess tumorigenicity. The transplanted mice showed massive splenomegaly and died within 25 days,
and the ATL cells had infiltrated into multiple tissues. Next, to evaluate serial transplantability of Ht48 cells, we
performed 13th consecutive serial transplantations. Ht48 cells regenerated the ATL-like disease and
reconstituted the original phenotypes such as CD4-CD8+, CD4+CD8+, and CD4-CD8- after each transplantation.
Those results indicated Ht48 cells included ATL stem cells that showed the tumorgeniety and the ability of
self-renewal.
Next, we performed side population (SP) analyses to identify the drug resistance cells in Ht48 cells. As a result,
we detected SP cells with high drug resistance existed among Ht48 cells. Interestingly, more than 40% of c-kit+
cells were enriched with SP cells, while less than 5% of c-kit+ cells compared the other population. As expected,
both SP and c-kit+ cells showed a high proliferative activity in vitro and in vivo, and these cells could
differentiate into all lineages of ATL cells (CD4-CD8+, CD4+CD8+, and CD4-CD8-) in vivo.
Because ATL stem cells of Tax-Tg and HBZ-Tg showed c-kit+, we hypothesized c-kit – c-kit ligand (stem cell
factor; SCF) plays an important role to mediate ATL cells propathogen. As we expected, a neutralizing antibody
against SCF inhibited ATL stem cell proliferation in vitro. In adition, Sl/Sld mice lacked membrane-bound SCF
did not develop ATL-like disease after the transplantation of Ht48 cells. Together with these results, we
concluded that c-kit play a major role to maintain ATL stem cell functions such as drug resistance and high
tumorigenicity in Ht48 cells.
96
5-7
Chemotherapy with Hybrid Liposomes for Acute Lymphatic Leukemia Leading to
Apoptosis in vivo
Hideaki Ichihara, Masayo Umebayashi, Yoko Matsumoto
Division of Applied Life Science, Graduate School of Engineering, Sojo University, Japan
We have produced hybrid liposomes (HL) composed of vesicular and micellar molecules.1 The
physical properties of HL such as shape, size, membrane fluidity, and the temperature of phase transition can be
controlled by changing the constituents and compositional ratios. Significnatry prolonged survival have been
obtained in rats glioma model treated with HL including antitumor drugs without side effects.2 HL without
drugs have been also effective for inhibiting the growth of various tumor cells in vitro and in vivo using animal
model of carcinoma.3-4 No toxicity of HL was observed in normal rats in vivo without any side effects.5
Successful clinical chemotherapy with drug-free HL to patients with lymphoma has been reported after passing
the committee of bioethics.5 We have elucidated the mechanistic details for apoptosis of tumor cells induced by
HL6 and the correlation between antitumor effects and membrane fluidity of HL7. However, apoptotic pathway
and therapeutic effects of HL for human acute lymphatic leukemia (ALL, MOLT-4) cells in vivo have not yet
been elucidated.
In this study, we investigated the therapeutic effects of HL-25 composed of
L-α-dimyristoyl-phosphatidylcholine (DMPC) and polyoxyethylene(25)dodecyl ether (C12(EO)25) using model
mice of carcinoma after the inoculation of human ALL (MOLT-4) cells in vivo.
We prepared HL-25 by the method of sonication of a mixture containing 95 mol% DMPC and 5 mol%
C12(EO)25 using bath type sonicater in 5 % glucose solution at 45 ºC with 300 W, and filtered with a 0.20 μm
cellulose acetate filter. We examined the morphology of HL-25 composed of 95 mol % DMPC and 5 mol %
C12(EO)25 on the basis of dynamic light scattering measurements. Hydrodynamic diameter (dhy) of HL-25 was
about 50 nm, which was preserved for a period of remained stable for more than one month. On the other hand,
DMPC liposomes were unstable and precipitated after 14 days. It is worthy to note that HL-25 having a
diameter of 50 nm could avoid the reticular endothelial system (RES) and should be appropriate for in vivo and
clinical applications after the intravenous administration.
We examined inhibitory effects of intravenous treatment with HL-25 on the growth of tumor in
subcutaneous xenograft model mice of ALL. The more remarkable reduction rate of 40 % (p < 0.01) in tumor
volume was obtained in mice treated in HL-25, in contrast with that of 25 % (p < 0.05) in mice treated with
DMPC liposomes. Furthermore, statistical significance between DMPC liposomes and HL-25 was obtained (p <
0.05). It is noteworthy that a remarkable reduction of tumor volume was obtained in model mice of ALL
intravenously treated with HL-25 without drugs after subcutaneously inoculating MOLT-4 cells.8
We examined the induction of apoptosis by HL-25 for solid tumor in model mice of ALL using the TUNEL
method. Brown color was observed in the tumor cells of mice after the treatment with HL-25, although the
apoptotic cells were not observed in the group treated with DMPC liposomes. These results indicate that HL-25
have remarkable inhibitory effects along with apoptosis on the growth of MOLT-4 cells. Signal transduction of
apoptosis by HL-25 for MOLT-4 cells in vivo was examined. No activity of caspase-8, caspase-9 or caspase-3
by HL-25 was obtained on the basis of caspase fluorometric protease assay. To elucidate the apoptotic pathway
which pass directly through mitochondria with independent caspase cascade, we examined participation of
apoptosis inducing factor (AIF). Many AIF positive cells for MOLT-4 cells treated with HL-25 were observed
apart from the cases of DMPC liposomes and control group. These results suggest that the mitochondorial
pathway could be implicated in the apoptosis induced by the HL-25 for MOLT-4 cells. 8
We examined the therapeutic effects of HL-25 using mice of ALL with peritoneal dissemination after
the intraperitoneal treatment with HL-25. The median survival time for mice treated with HL-25 was higher
than 420 days without death, although that for mice in the control group was 84.7  2.2. On the other hand, the
median survival time for mice treated with DMPC was 385  62. It is noteworthy that a significantly prolonged
survival (> 400 %, p<0.01) was obtained in the mice treated with HL-25. These results indicate that HL-25
could strongly inhibit the growth of MOLT-4 cells in vivo. 8
The results in this study should be advantageous in the chemotherapy for patients with acute lymphatic
leukemia in the near future clinical applications.
[1] J. Am. Chem. Soc., 110, 1588 (1988). [2] Cancer Res., 56, 3986 (1996). [3] Bioorg. Med. Chem. Lett. 16,
782 (2006). [4] Int. J. Pharm., 372, 162 (2009). [5] Anticancer Res., 34, 4701 (2014). [6] Int. J. Cancer, 115, 377
(2005). [7] ACS Med. Chem. Lett., 2, 275 (2011). [8] Int. J. Pharm., 406, 173 (2011).
97
5-8
Overexpression of CADM1 Enhances Adhesion and Infiltration of ATL Cells
Yuki Kumagai, Gan Siew Pey, Shigefumi Murakami, Misaki Noguchi, Mika Sakurai, Takeshi Ito,
Yoshinori Murakami
Divesion of Molecular Pathology, Institute of Medical Science the University of Tokyo, Japan
Adult T-cell Leukemia (ATL) is a neoplastic disease of CD4+ T cells which is triggered by infection of human
T-cell Leukemia Virus Type 1 (HTLV-1). The estimated incidences of ATL in HTLV-1 carriers are 6-7% for
males and 2-3% for females with long latency of more than 35 years. ATL is well known to infiltrate into
various organs, such as skin, lung, liver, and lymph nodes. Although several characteristic genetic and
epigenetic alterations have been identified in the development and malignant progression of ATL, the molecular
mechanism involved in its highly infiltrative features remains unknown. Comprehensive gene expression
analysis of acute-type ATL cells in comparison with normal CD4+ and CD4+CD45RO+ T cells revealed that
expression of the CADM1 (Cell Adhesion Molecule 1) gene was most significantly upregulated in ATL cells.
CADM1 is now clinically used as a specific surface marker of ATL cells and HTLV-1 infected cells by FACS
analysis.
CADM1 is a member of the immunoglobulin superfamily proteins and serves as a cell adhesion molecule in
cell-cell interaction. CADM1 consists of extracellular domain with three immunoglobulin-like loops, a
trans-membrane domain and a short cytoplasmic domain containing a 4.1 binding motif and a PDZ binding
motif. Interestingly, CADM1 was initially identified as a tumor suppressor gene in non-small lung cancer and
the expressison of CADM1 is downregulated in advanced stages of various cancers of epithelial origin. In
contrast, above findings suggest that CADM1 works as an oncogene in ATL cells. In fact, the introduction of
CADM1 into ATL cells lacking CADM1 enhances subcutaneous tumor growth and organ infiltration into the
liver and lung in immunodeficient NOG mice. To understand the mechanistic feature of CADM1-dependent
organ infiltration, we have previously demonstrated that CADM1 interacts with Tiam1 (T-lymphoma invasion
and metastasis 1), which acts as a guanine nucleotide exchange factor (GEF) of Rac. We have also reported that
CADM1 interacts with Tiam1 through its cytoplasmic PDZ binding motif, activates Rac pathway, and induces
lamellipodia formation in HTLV-1 infected cell lines.
In this study, to investigate the precise role of CADM1 in organ infiltration of ATL cells, we depleted CADM1
expresssion in ATL-derived ATN1 cells and HTLV-1-infected MT2 cells using CADM1 shRNA. In both ATN1
and MT2 cells, knockdown of CADM1 inhibited cell adhesion to human umbilical vein endothelial cells
(HUVEC) and transendothelial migration across HUVEC without affecting cell growth. Furthermore,
knockdown of CADM1 suppressed extravasation and tumor formation of MT2 cells in the liver after
intravenous injection into NOG mice. These data suggest that CADM1 promotes liver infiltration of ATL cells
through enhancing adhesion to endothelial cells and transendothelial migration.
To evaluate CADM1-mediated cell extension and to identify the CADM1-mediated signaling pathway, we have
previously established a cell-based method, in which CADM1-expressing cells are overlayed on the glass coated
with CADM1-Fc flagments. CADM1-expressinig cells adhere to the glass plate and show extendedmorphology
through the trans-homophilic interaction of CADM1. Using this method, we screened the antagonistic or
agonistic chemical compounds of the pathway in CADM1-mediated cell adhesion and extension and found that
PI3K inhibitors, LY294002 and BYL718, suppressed CADM1-mediated cell adhesion and cell extension
without affecting cell growth activity. These data suggest that PI3K signaling pathway involved in adhesion and
extension of CADM1-expressing cells.
In conclusion, we propose that CADM1 promotes organ infiltration of ATL cells through adhesion to and
extension on the endothelial cells activating Tiam1 and PI3K signaling pathway independently or
synergistically.
98
5-9
CCL22, CCL25, CXCL 10 and CXCL11 Chemokine Serum Levels in Patients with
Follicular Lymphoma Who Complicated with Bendamustine-associated Skin Rash
Yoshiharu Kusano1,2, Yasuhito Terui1,2, Ryoko Kuniyoshi2, Kiyohiko Hatake1,2
1
Department of Hematology and Oncology, Cancer Institute Hospital, Japanese Foundation for Cancer
Research, Tokyo, Japan
2
Devison of Clinical Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research,
Tokyo, Japan
Background
regimen containing bendamustine and rituximab is an effective standard regimen for indolent and aggressive
B-cell lmyphomas as R-CHOP. Skin rash is a bendamustine-specific adverse event but a mechanism of
bendamustine-associated skin rash (BSR) has been unknown. The most case of BSR is mild; however, several
resports of Stevens Johnson synderome (SJS) has reported previously. Terui Y et al (in submission)
demonstrated that several serum chemokines are correlated with BSR.
Methods
We evaluated serum CCL22, CCL25, CXCL10, and CXCL11 level in patients, who were treated with
bendamustine suffered a BSR including in our hospital. Blood samples before the treatment and at diagnosis of
BSR were used. The levels of each chemokine were analyzed by using Human TECK/MDC Quantikine ELISA
kit. The procedure was following. First, add 100 µL of Assay Diluent to each well. Next, Add 100 µL of Standard,
control, or sample to each well. Standard ranged 125 - 4000 pg/mL. Cover with a plate sealer, and incubate at
2-8 °C for 2 hours. Aspirate each well and wash, repeating the process 3 times for a total of 4 washes. Add 200 µL
of Conjugate to each well. Cover with a new plate sealer, and incubate at 2-8 °C for 2 hours. Aspirate and wash 4
times. Add 200 µL Substrate Solution to each well. Incubate at room temperature for 30 minutes in the
circumstance without light. Add 50 µL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set
wavelength correction to 540 nm. Results obtained using naturally expressed human MDC showed linear curves
that were parallel to the standard curves obtained using the Quantikine kit standards.
Results
Overall, plasma samples of 13 patients with BSR were analyzed. Twelve patients had grade 1 or 2 rash
according to CTCAE version IV and one patient experience SJS. The transitions of the four chemokines before
and after the onset of BSR of 13 patients are shown in Figure, respectively. The chemokine levels after
treatment of BSR were measured several times in only the SJS patient. In almost all patients, the transitions of
chemokines moved from pretreatment level to the diagnosis of BSR level. CCL22 inclined to decrease from
pretreatment level, which was also observed in SJS case. In contrast, CCL25, CXCL10, and CXCL11 tened to
have highest level at onset of BSR. In the SJS patient, these three levels decreased as BSR was improved by
treatment. The levels of CXCL10 and CXCL11 moved more dynamically than others.
Conclusion
Our study demonstrated that CCL22, CCL25, CXCL10, and CXCL11 were the marker of BSR. Especially, both
CXCL10 and CXCL11 are the best markers of BSR in the point of view of dynamic movement in proportion to
the manifestation of BSR.
99
6-1
A Novel Role for Interleukin-34 in the Pathogenecity of Multiple Myeloma
Muhammad Baghdadi, Haruka Wada, Ken-ichiro Seino
Division of Immunobiology, Institute for Genetic Medicine, Hokkaido University, Japan
Multiple myeloma (MM) is a lethal human cancer characterized by a clonal expansion of malignant plasma cells
in the bone marrow and induction of osteoclastogenesis which results in bone destruction. Accordingly, MM
patients frequently develop bone disease characterized with severe bone pain, osteolytic lesions and pathologic
fractures. Previous studies have suggested that a dysregulated production of numerous cytokines in the bone
marrow microenvironment of MM patients results in the uncoupling of bone cell activity and triggering of
uncontrolled osteoclast formation. In this report, we identify interleukin-34 (IL-34) as a novel factor produced
by MM cells which importantly contributes to MM-induced osteoclast formation. IL-34 is a newly discovered
cytokine which binds to Colony Stimulating Factor 1 Receptor (CSF1R) and regulates macrophage and
osteoclast differentiation. IL-34 was found to be expressed in the murine multiple myeloma cell line
(MOPC315.BM cells) at mRNA and protein levels. IL-34 expression was enhanced when MOPC315.BM cells
were co-cultured with bone marrow stromal cells (BMSCs). Supernatants of MM-BMSC coculture induced
osteoclast differentiation from bone marrow precursors in vitro, which was significantly impaired by the
targeting of IL-34 by siRNA or α-IL-34 neutralizing antibodies. Similarly, IL-34 was also found to be expressed
in human myeloma cell lines in addition to myeloma cells from myeloma patients. Together, we identify a novel
role for IL-34 in MM-induced osteoclastogenesis, suggesting it as a promising target in future therapeutic
strategies of Multiple Myeloma.
Collaborators:
Professor Bjarne Bogen, Oslo University
Professor Masahiro Abe, Tokushima University
Professor Takanori Teshima, Hokkaido University
100
6-2
Antimyeloma Activity of Bromodomain Inhibitors on the Human Myeloma Cell
Line U266 via Downregulation of MYCL
Kazuhito Suzuki1,2, Yasuhiro Arakawa1, Kouhei Yamamoto2, Hisashi Yamada3, Noriko Usui4, Keisuke Aiba1,
Masanobu Kitagawa2
1
Department of Comprehensive Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical
and Dental University, Japan
2
Core Research Facilities for Basic Science Division of Molecular Genetics, The Jikei University School of
Medicine, Japan
3
Division of Clinical Oncology/Hematology, Department of Internal Medicine, The Jikei University School of
Medicine, Japan
4
Division of Transfusion Medicine, The Jikei University School of Medicine, Japan
Introduction
Multiple myeloma is a clonal human plasma cell neoplasm and an incurable disease although several novel
agents have improved patient outcomes in the last decade. Small-molecule inhibitors of the bromodomain and
extra-terminal (BET) family of bromodomain proteins, so-called BET inhibitors, suppress expression of c-MYC,
a master regulatory factor of cell proliferation in myeloma cells. BET inhibitors induce G1 arrest in human
myeloma cell lines via downregulation of c-MYC. U266, a human myeloma cell line, expresses the MYCL gene
but not the c-MYC gene. Our purpose was to investigate the antimyeloma activity of BET inhibitors on U266
cells via downregulation of MYCL.
Methods
Four human myeloma cell lines, U266, RPMI8226, MM1S, and KMS11, were used in this study. Two BET
inhibitors, I-BET151 and JQ1, were tested. Cell proliferation assay was performed with MTS terazolium. Cell
cycle and apoptosis were evaluated by flow cytometry. Regulation of genes in U266 and KMS11 cells with and
without exposure to I-BET151 were analysed by microarray. MYCL1, c-MYC, and MYCN gene levels were
analysed by qRT-PCR.
Results
U266 cell proliferation decreased to 61.5% and 54.0% of the control after incubation with 500 nM I-BET151 for
72 and 96 h and to 53.5%, and 56.4% of control after incubation with 500 nM JQ1 for 72 and 96 h by MTS
terazolium, respectively. BET inhibitors induced cell cycle arrest at G1 phase in U266 cells, but did not induce
apoptosis by flow cytometry. According to GSEA, MYC-related genes were significantly downregulated in
U266 cells treated with I-BET151. The MYCL1 was expressed in U266 cells whereas c-MYC and MYCN were
not by the qRT-PCR. In contrast, c-MYC was expressed although the MYCL1 was not expressed in the other
myeloma cell lines. Incubation of U266 with I-BET151 induced downregulation of MYCL1. Pathway analysis
showed that several cell cycle-related transcription factors were downregulated in both U266 and KMS11 cells
exposed to I-BET151. In contrast, c-MYC expression was downregulated only in KMS11 cells treated with
I-BET151.
Conclusion
BET inhibitors induce G1 arrest and interfere with the proliferation of U266 myeloma cells, which express
MYCL but not c-MYC. BET inhibitors might be active in cancers that express MYCL but not c-MYC.
101
6-3
PRC2 Inhibition Sensitizes Myeloma Cells to Proteasome Inhibitors
Ola Rizq1, Naoya Mimura2, Motohiko Oshima1, Atsunori Saraya1, Shuhei Koide1, Yuko Kato1, Kazumasa
Aoyama1, Changshan Wang1,3, Tetsuhiro Chiba4, Tohru Iseki2, Chiaki Nakaseko5, Atsushi Iwama1
1
Department of Cellular and Molecular Medicine, Chiba University Graduate School of Medicine, Japan
2
Department of Transfusion Medicine and Cell therapy, Chiba University Hospital, Japan
3
College of Life Sciences, Inner Mongolia University, Hohhot, China
4
Department of Gastroenterology and Nephrology, Chiba University Graduate School of Medicine, Japan
5
Department of Hematology, Chiba University Hospital, Japan
EZH2, the catalytic component of polycomb repressive complex 2 (PRC2), catalyzes the trimethylation of
lysine 27 of histone H3 generating H3K27me3 which leads to transcriptional repression of target genes. EZH2
overexpression and inactivating mutations of the histone demethylase UTX have been reported in multiple
myeloma (MM). In this study we investigated the impact of the use of UNC1999, a dual inhibitor of EZH2 and
its homolog EZH1, alone and in combination with proteasome inhibitors on MM cells in vivo and in vitro. First
we used lentiviral vectors to knock down EZH2 and confirmed that EZH2 is indispensable for MM cells. Next,
we studied the effect of dual inhibition of EZH2 and EZH1 on MM cells. UNC1999 induced cytotoxicity in
MM cell lines as well as in primary cells from MM patients. Next, we examined the activity of the combination
of UNC1999 and proteasome inhibitors. We found strong synergism between UNC1999 and bortezomib.
UNC1999 enhanced the cytotoxicity induced by bortezomib in MM cell lines and primary cells from MM
patients. We used an MM.1S xenograft mouse model to test the effect of the combination in vivo. The
combination therapy significanlty reduced the size of the tumors as compared to the control and either single
agent with prolongation of the survival of mice. Of interest, EZH2 overexpression in MM cell lines induced
resistance to bortezomib, which was overcome by dual inhibition of EZH2 and EZH1, suggesting that the mode
of PRC2 activity holds the key to the efficacy of bortezomib. Of note, the dual inbibition had a much better
effect in combination with bortezomib than an EZH2-specific inhibitor, suggesting a role for EZH1 inhibition in
the synergistic effect of the combination therapy. RNA-sequence analysis revealed that EZH2 expression in MM
cells was significantly downregulated by bortezomib. Nevertheless, the net PRC2 activity was unchanged as
evident from the maintenance of H3K27me3 levels and sustained silencing of PRC2 target genes. These
findings suggest that EZH1 together with the residual EZH2 complements EZH2 inhibition by bortezomib.Thus
dual inhibition of EZH2 and EZH1 sensitizes myeloma cells to bortezomib. Taken together, our study
demostrates that the combination of a dual inhibitor of EZH2 and EZH1 with proteasome inhibitors could be a
novel potential therapeutic option to improve the outcome of MM patients.
102
7-1
Infiltrating Macrophages Express PD-L1 and PD-L2 in Lymphoma
Microenvironment
Yoshihiro Komohara1, Hasita Horlad1, Chaoya Ma1, Shinya Endo2, Yoshitaka Kikukawa2, Yutaka Okuno2,
Masao Matsuoka3, Motohiro Takeya1
1
Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
2
Department of Hematology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
3
Laboratory of Virus Control, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, Japan
The immune escape and tolerance in tumor microenvironment are closely involved in tumor
progression, and are caused by T-cell exhaustion mediated the inhibitory signal of immune checkpoint
molecules including programmed death-1 (PD-1) and cytotoxic T-lymphocyte associated protein 4
(CTLA-4). In the present study, we investigated the PD-1 ligand 1 (PD-L1) expression in lymphoma
microenvironment using paraffin embossed tissue samples, and then the detail mechanism of
up-regulation of PD-L1/2 on macrophages were studied using human macrophages and lymphoma cell
lines. We found that macrophages in lymphoma tissues expressed PD-L1 in almost all cases of adult
T-cell leukemia/lymphoma (ATLL), follicular lymphoma, and diffuse large B-cell lymphoma. Cell
culture studies revealed that conditional medium of ATL-T and SLVL cell lines induced increased
expression of PD-L1/2 on macrophages. Cytokine array analysis showed IL-27 was secreted from
these cell lines, and IL-27 was found to induce PD-L1/2 overexpression. PD-L1/2 overexpression on
macrophages stimulated with conditional medium and IL-27 was significantly abrogated by inhibition
of signal transducer and activator of transcription 3 (Stat3). In this study, we could not reveal the
clinical significance of PD-L1/2 expression on lymphoma, however, PD-L1/2 expression on
macrophages are potentially involved in immune suppression in lymphoma microenvironment.
103
7-2
High Throughput Dynamic Evaluation of Immune Cytotoxicity in Lymphoid
Tumors through Impedance Analysis
Eiji Okubo, Fabio Cerignoli, Biao Xi, Garret Guenther, Wei Tang, Chris Radecke, Leyna Zhao, Lincoln Muir,
Yama Abassi
ACEA Biosciences Inc., San Diego, CA, USA
Immunotherapy is one of the most promising approaches in cancer treatment, allowing specific elimination of
cancer cells through activation of the immune response. However, despite the necessity of in vitro
characterization of efficacy and potency of reagents and protocols before moving to more expensive animal
models and Phase I and II clinical studies, current in vitro assays are difficult to implement in high throughput
environment and are mainly based on end point methodologies that are unable to capture the full dynamic of the
immune response. Here we present an adaptation of impedance technology to measure immune-mediated cell
killing in B lymphoid tumor cells. Impeance-based technology detects alterations in cell adhesion, morphology
and cell number as variations of an impedance-related parameter, denominated Cell Index (CI), by measuring
changes in conductance of microelectrodes embedded in 96 and 384-wells cell culture plates. The technology
permits continuous monitoring of cell viability and the unaltered cells are also available for secondary end point
assays that can better clarify the biological mechanism involved in the response. To allow measurement of
cancer B lymphocytes killing we developed a strategy where the bottom of the wells are coated with an
anti-CD40 antibody. Such coating allows specific adhesion and retention of B cells and measurement of changes
in impedance that are proportional to cell number. Using combinations of Bispecific T Cell Engagers (BiTE)
antibodies and purified T lymphocytes, or Natural Killer (NK) cells expressing Chimeric Antigen Receptors
(CAR) against CD19, we were able to demonstrate specific killing of tumor B cells. The results were also
confirmed by flow cytometry. Overall, our results demonstrate the value of the impedance-based approach in
measuring the full dynamic of cytotoxic response over the temporal scale, a feature otherwise impossible to
access with more canonical end point assays like 51Cr release or flow cytometry. Moreover, the availability of
384-wells format and minimal sample handling makes the technology ideal for testing therapeutic protocols in a
personalized medicine environment.
104
7-3
Immunogenicity of Novel Antigen Expressed in Leukemia Stem Cells
Koji Ozawa1, Maiko Matsushita1, Shinya Yokoe1, Saori Kanchi1, Akane Uchiumi1, Daiju Ichikawa1, Eri Matsuki3,
Masatoshi Sakurai3, Daiki Karigane3, Hidenori Kasahara3, Yutaka Kawakami2, Shinichiro Okamoto3, Yutaka
Hattori1
1
2
Division of Clinical Physiology and Therapeutics, Facuty of Pharmacy, Keio University, Japan
Institute for Advanced Medical Research, 3Division of Hematology, School of Medicine, Keio University,
Japan
3
Division of Hematology, School of Medicine, Keio University, Japan
Background and objective: The dramatic success of tyrosine kinase inhibitors (TKIs) has led us to the next
therapeutic goal of chronic myelogenous leukemia (CML), which is treatment-free survival. However, about
half of patients eventually relapse after cessation of TKIs, and leukemic stem cells (LSCs), which are resistant
to TKIs, are considered to be the cause of this disease recurrence. Thus, it is crucial to develop novel therapy
targeting LSCs for the cure of CML. We previously identified KU-MEL9 as a highly expressed antigen in
several hematological malignancies. In this study, we investigated whether KU-MEL9 could serve as a
therapeutic target for CML.
Method & Result: The expression of KU-MEL9 gene and the protein in LSCs (CD34+CD38- fraction) of CML
cell lines and patients' bone marrow (BM) samples was confirmed by quantitative PCR and
immunocytochemical staining. We observed high expression of KU-MEL9 in both LSCs and non-LSCs of
KU812 (CML cell line) or BM mononuclear cells from CML patients. On the other hand, CD34+ BM cells
obtained from healthy donor did not express KU-MEL9. In addition, expression of this gene was up-regulated
by addition of 5-aza-2’-deoxycytidine only in CML cells. We then induced KU-MEL9-specific cytotoxic T
lymphocytes (CTLs) by stimulating lymphocytes from healthy donors with KU-MEL9 49-57 peptide, an
HLA-A24:02-restricted epitope. CD107a mobilization assay showed that these CTLs preferentially recognized
CD34+ BM cells from CML patients. Moreover, we detected KU-MEL9-specific CTL in peripheral blood of the
CML patients who remained in complete molecular remission after cessation of TKIs by KU-MEL9-specific
dextramer staining.
Discussion: Our data suggests that KU-MEL9 is a novel LSC antigen. T cell immunity against this antigen may
play an important role in eliminating LSCs of CML after discontinuation of TKIs. Immunotherapy targeting this
antigen in combination with demethylating agent following TKI cessation might also be an attractive
therapeutic strategy for the cure of CML.
105
7-4
Cytolytic Anti-pan MHC-Class I and Class II mAbs Directly Induce Lymphoma
Cell Death via Large Pore Formation without Complement
Shuji Matsuoka, Hino Okio
Department of pathology and Oncology, Juntendo University School of Medicine, Japan
A newly established mouse anti-pan HLA class II mAb (4713) triggered cytoskeleton-dependent, but
complement- and caspase-independent, cell death in Hodgkin Lymphoma cell lines, Burkitt lymphoma cell lines,
and advanced adult T-cell leukemia cell lines. Intravenous injection of mAb 4713 in tumor-bearing SCID mice
improved survival significantly.
We previously reported that Rat anti-mouse pan MHC class I mAb, RE2 induced cytoskeleton-dependent, but
complement- and caspase-independent cell death of lymphoma cells and activated lymphocytes. RE2 mAb
rescued mice from fulminant hepatitis by killing activated CD8 cytotoxic T cells and NKT cells.
Treatments with these mAbs (anti-pan MHC class I mAb and anti-pan HLA class II mAb) induced the
formation of large pores on the surface of target lymphoma cells within 30 min. This finding suggests that the
cell death process induced by these anti-pan MHC mAbs may involve the same death signals stimulated.
Antibodies against MHC molecules may be promising therapeutic device for hematological cancer.
106
7-5
Dendritic Cell-based Immunotherapy Induces HTLV-1 Tax-specific CD8+ T Cells
and Reduces Proviral Loads
Atsuhiko Hasegawa1, Satomi Ando1, Yuji Murakami1, Yasuhiro Maeda2, Youko Suehiro3, Mari Kannagi1
1
Department of Immunotherapeutics, Tokyo Medical and Dental University, Tokyo, Japan
2
Department of Hematology, Osaka Minami Medical Center, Osaka, Japan
3
Department of Cell Therapy, National Kyushu Cancer Center, Fukuoka, Japan
Human T-cell leukemia virus type-1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATL),
an aggressive CD4+ T-cell malignancy with a poor prognosis. About 5% of infected individuals develop ATL
although most remain asymptomatic throughout their lives. Based on a large cohort study in Japan, higher
proviral load (PVL; more than 4% of PBMC), advanced age (more than 40 years old), and family history of
ATL are regarded as a risk factor for the development of ATL. In HTLV-1 infection as well as other viral
infections, HTLV-1-specific CD8+ cytotoxic T lymphocytes (CTL) play an important role in controlling the
expansion of infected cells. However, we previously reported that not only ATL patients but also some
asymptomatic carriers had severely impaired functions of CD8+ T cells specific for HTLV-1 Tax, the major
target antigen for HTLV-1-specific CTL. The functions of Tax-specific CD8+ T cells tended to be inversely
correlated with PVL. Such weak HTLV-1-specific CTL responses may cause the elevation of PVL, leading to
the increased possibility to transform an infected cell into a leukemic cell. Therefore, vaccines to induce
HTLV-1-specific CD8+ T cell responses would be required to contain HTLV-1-infected cells.
Denritic cells (DC) are the professional antigen-presenting cells, which induce primary immune responses, and
potentiate the effector functions of previously primed memory T cells. However, it was reported that
plasmacytoid DC (pDC), one of the important cells for acquired immunity, was functionally reduced in
HTLV-1-infected individuals and PVL was inversely correlated with the functionality of pDC, suggesting that
DC in some asymptomatic carriers with higher PVL may be functionally impaired. Therefore, monocyte-derived
DC (MoDC)-based immunotherapy would be one of the good candidates to induce functional HTLV-1-specific
CD8+ T cells if functional DC can be generated from monocytes in HTLV-1-infected individuals.
In this study, to develop prophylactic vaccines to prevent the onset of ATL, we first examined the capacity of
MoDC to stimulate antigen-specific autologous CD8+ T cells from HTLV-1-infected individuals. MoDC
generated from most infected individuals, including ATL patients was found to have the capability of
stimulating the expansion of CMV-specific autologous CD8+ T cells, indicating that MoDC from infected
individuals could be used as a tool for DC-based immunotherapy. We next evaluated the effects of CTL epitope
peptide (Tax180-188)-pulsed dendritic cell (Tax/DC) vaccine on HTLV-1-specific CD8+ T cell responses and
PVL, using a persistently HTLV-1-infected rat model showing elevated PVL and weak HTLV-1-specific T cell
responses. IFN production and in vitro expansion of Tax-specific splenic CD8+ T cells were observed in
HTLV-1-infected rats after Tax/DC vaccination while no or little Tax-specific CD8+ T cell responses were seen
in infected rats without the vaccination. Futhermore, the vaccinated rats were found to have significantly lower
PVL compared to infected rats without the vaccination. It is of note that, among vaccinated rats, half of rats
showing higher Tax-specific CD8+ T cell expansion carried undetectable PVL, suggesting that the Tax/DC
vaccine might reduce PVL through induction of functional Tax-specific CD8+ T cells. Our results suggest that
DC-based immunotherapy might be a useful strategy to reduce the risk for ATL development in asymptomatic
carriers having higher PVL and weak HTLV-1-specific CD8+ T cell responses.
107
8-1
Whole-Body Imaging with Single Cell Resolution
for Unbiased Analysis of Cell Status
Shimpei I Kubota1, Kazuki Tainaka1,2,3, Takeru Q Suyama1, Etsuo A Susaki1,2,3, Hiroki R Ueda1,2,3
1
Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, Japan
2
Laboratory for Synthetic Biology, RIKEN Quantitive Biology Center, Japan
3
CREST, Japan Science and Technology Agency, Japan
To reveal sophisticated life systems of multicellular organism, we need to focus the most essential unit of life
“cell”. Whole-body imaging with single-cell resolution will enable to reveal these systems such as cellular
dynamics which has been a fundamental challenge in biology. Previous clearing methods focused on
homogenizing mismatched refractive indices of individual tissues and lipid removal, enabling reductions in
opacity. However these methods is not unable to clear the whole body because of the light absorbance by
endogenous chromophores. Here, we show that aminoalcohols in CUBIC reagents decolorize the blood by
efficiently eluting the heme chromophore. Direct transcardial perfusion of CUBIC reagents with a 10 day to 2
week clearing protocol decolorized nearly transparent almost all organs of adult mice as well as the entire body
of infant and adult mice. This CUBIC-perfusion protocol enables rapid whole-body and whole-organ imaging
with single-cell resolution by using light-sheet fluorescent microscopy. The CUBIC protocol is also applicable
to 3D pathology, anatomy, and immunohistochemistry of various organs. These results suggest whole-body
imaging with single-cell resolution will lead to organism-level systems biology. Because Optical clearing
techniques can be applicable to deep imaging of bone marrow, our imaging methods will enable us to analyze
comprehensive dynamics of individual cells of metastatic cancer, including hematological cancer.
108
Author Index of Poster Presentations
Abi-Habib, Ralph Joseph
1-18
Lorthongpanich, Chanchao
1-10
Baba, Tomohisa
1-3
Luanpitpong, Sudjit
5-2
Baghdadi, Muhammad
6-1
Maie, Koichiro
2-1
Cao Sy, Luan
3-1
Matsuoka, Shuji
7-4
1-14
Miyauchi, Masashi
1-16
El-Sibai, Mirvat
1-1
Mizuguchi, Mariko
2-6
Fujiwara, Yukio
5-3
Mochizuki-Kashio, Makiko
2-4
Fukumura, Kazutaka
4-2
Mohammed, Faruk
5-4
Hasegawa, Atsuhiko
7-5
Morishita, Kazuhiro
1-7
Ichihara, Hideaki
5-7
Nagase, Reina
3-3
Ichikawa, Tomonaga
2-3
Nakahata, Shingo
5-1
Ishimura, Akihiko
2-2
Nakata, Yuichiro
2-9
Jiang, Qingfei
1-9
Nguyen, Tran Bich
4-1
Kagiyama, Yuki
2-5
Okubo, Eiji
7-2
1-20
Ozawa, Koji
7-3
Couzinet, Arnaud Nicolas
Katsumoto, Takuo
Kawabata, Kimihito Cojin
3-4
Rizq, Ola
6-3
Kimura, Kotohiko
1-2
Sera, Yasuyuki
3-5
Kimura, Shunsuke
2-7
Shima, Yutaka
1-15
Kitadate, Akihiro
5-5
Shinohara, Haruka
1-6
Kitagawa, Kyoko
1-4
Suzuki, Kazuhito
6-2
Kochi, Yu
1-21
Takamatsu-Ichihara, Emi
1-11
Komohara, Yoshihiro
7-1
Tara, Shiro
1-17
Kubota, Shimpei I.
8-1
Ueda, Koki
3-2
Kumagai, Yuki
5-8
Yamagata, Kazutsune
2-8
Kuribayashi, Wakako
5-6
Yokota, Asumi
1-13
Kusano, Yoshiharu
5-9
Yokoyama, Takashi
1-12
Li, Tsai-Kun
1-19
109
MEMO
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