Extended Abstracts forthe 47th International Symposium of
the Princess Takamatsu Cancer Research Fund

CURRENT STATUS AND PERSPECTIVE OFCANCER STEM CELL RESEARCH
November 8-10, 2016
Tokyo, Japan
Edited by

Masaki Mori
Michael F. Clarke
Hans Clevers
Koichi Akashi
Hideyuki Saya

Princess Takamatsu Cancer Research Fund

CopyrightⒸ2017 by the Princess Takamatsu Cancer Research Fund
No part of this Extended Abstracts may be reproduced, stored in retrieval
systems or transmitted, in any form or by any means, without permission
from the Princess Takamatsu Cancer Research Fund.

Princess Takamatsu Cancer Research Fund

1-14-15-102 Takanawa, Minato-ku, Tokyo 108-0074, Japan
Telephone: +81-3-3441-0111
Facsimile: +81-3-3441-0112
E-mail:info@ptcrf.or.jp
Printed in Japan
ISSN 2186-8603

B

CONTENTS
KEYNOTE LECTURE

Lgr5 Stem Cell-derived Organoids and Their Applications
Hans Clevers .....................................................................................................................................1

ORGANOIDS AND CANCER STEM CELL MODELS

Organoid-Based Cancer Functional Genomics
Calvin J. Kuo......................................................................................................................................5
Visualization of Human Colon Cancer Stem Cells Using Organoid
Technology
Toshiro Sato ......................................................................................................................................11
Deciphering the Role of Malignant Deaminase Activation in Cancer Stem
Cell Generation
Catriona H. M. Jamieson .........................................................................................................14

LEUKEMIC STEM CELLS

An Autocrine Loop Involving b-Catenin Pathway is Critical for
Development of Human Myeloid Leukemia Stem Cells
Koichi Akashi ..................................................................................................................................18
Innate Lymphoid Cells in the Control of Organ Homeostasis
Andreas Diefenbach ...................................................................................................................21i

Clinical Relevance of Leukemia Stem Cells in AMLJohn E. Dick ......................................................................................................................................23
Essential Roles of Epigenetic Regulator in Acute Myeloid Leukemia Stem
Cells
Issay Kitabayashi ..........................................................................................................................28
The Notch:Myc Signaling Axis in T Cell Development and Transformation
Warren S. Pear ................................................................................................................................32
Molecular Mechanism Regulating Stem Cell Properties Mediated by
Nutrient Signals
Atsushi Hirao ..................................................................................................................................38

SOLID CANCER STEM CELLSNew Strategy for Overcoming Multifaceted Therapy-resistant Cancer
Stem Cells
Hideshi Ishii .....................................................................................................................................42
The Cancer Stem Cell Niche of Squamous Cell Carcinomas: Biology andImpact for Therapeutics
Elaine Fuchs .....................................................................................................................................51

Evidence and Mechanism for the Transdifferentiation from Lung
Adenocarcinoma to Squamous Cell Carcinoma
Hongbin Ji .........................................................................................................................................57
Dynamic Complexity of Glioma Stem Cells
Jeremy N. Rich ...............................................................................................................................62ii

Identification of Critical Drivers of Tumor Maintenance through in Vivo
Functional Genomics
Giulio F. Draetta ............................................................................................................................69

CYP3A5 Mediates Basal and Acquired Therapy Resistance in Different
Subtypes of Pancreatic Ductal Adenocarcinoma
Andreas Trumpp...........................................................................................................................74

TUMOR HETEROGENEITY

Transdifferentiation Approach for Targeting Cancer Stem Cells
Hideyuki Saya ................................................................................................................................80
Mechanisms Controlling Tumor Heterogeneity
Cédric Blanpain .............................................................................................................................83

New Insights into Cellular Plasticity: Targeting Metaplastic Cancers
Thea D. Tlsty....................................................................................................................................88

CANCER STEM CELL SIGNALS

Metabolic Exchange between Stem Cells and Niche Cells
Toshio Suda ......................................................................................................................................95
Hedgehog Signaling in Tissue Renewal and Malignancy
Philip A. Beachy ..........................................................................................................................100

Induction of Cancer Stemness and Drug Resistance by EGFR Inhibitors
and Cellular StressDavid A. Cheresh .......................................................................................................................102iii

Growth Factor Signaling in Cancer Stem-like Cells and Their Niche
Noriko Gotoh ................................................................................................................................105
Imaging Stem Cell Signals in Cancer Heterogeneity and Therapy
ResistanceTannishtha Reya ..........................................................................................................................109
Regulation of Myelopoiesis and Leukemia by Noncoding RNA
Daniel G. Tenen ...........................................................................................................................116

NAKAHARA MEMORIAL LECTURE

Normal and Neoplastic Stem Cells
Irving L. Weissman ...................................................................................................................118

DYNAMICS OF CANCER STEM CELLS

Cell of Origin and Tumor Stem Cells in Mouse Digestive Organ Tumors
Hiroshi Seno...................................................................................................................................130
LGR5+ Stem Cells in Epithelial Homeostasis, Regeneration & Disease of
the Stomach
Nick Barker .....................................................................................................................................133
Cell Cycle Regulation in Cancer Stem Cell
Keiichi I. Nakayama .................................................................................................................136

iv

CONCLUDING KEYNOTE LECTURE

Cancer Stem Cell Biology Enters the Clinic
Michael F. Clarke ........................................................................................................................141

CLOSING REMARKS

Masaki Mori ...................................................................................................................................147

List of Participants ................................................................................................................................151
List of Previous International Symposium Series .......................................................176v

1

LGR5 STEM CELL-DERIVED ORGANOIDS AND THEIRAPPLICATIONS

Hans Clevers

Hubrecht InstituteRoyal Netherlands Academy of Arts and Sciences
University Medical Centre Utrecht
Uppsalalaan 8, 3584 CT Utrecht, the Netherlands(h.clevers@hubrecht.eu)
The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals.

Lgr5/Gpr49 was selected from a panel of intestinal Wnt target genes for its restricted crypt

expression. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling,columnar cells at the crypt base. In addition, Lgr5 was expressed in rare cells in several
other tissues including the hair follicle, mammary gland and stomach. Using an inducible

Cre knock-in allele and the Rosa26-LacZ reporter strain, lineage tracing experiments were

performed in adult mice. The Lgr5 +ve crypt base columnar cell (CBC) generated allepithelial lineages over a 14 month period, implying that it represents the stem cell of thesmall intestine and colon. Similar obserations were made in hair follicles and stomach
epithelium. The expression pattern of Lgr5 suggests that it marks stem cells in multiple

adult tissues and cancers. These predictions have held up and we now know that most if

not all adult epithelia utilize Lgr5-marked, tissue-specfic stem cells for their maintenanceand repair.

Fate mapping of individual crypt stem cells using a multicolor Cre-reporter revealed

that, as a population, Lgr5 stem cells persist life-long, yet crypts drift toward clonality

within a period of 1-6 months. Lgr5 cell divisions occur symmetrically. The cellular

dynamics are consistent with a model in which the resident stem cells double their
numbers each day and stochastically adopt stem or TA fates after cell division.

Intestinal crypts display robust regeneration upon injury. The relatively rare secretory

precursors can replace lost stem cells, but it is unknown if the abundant enterocyte

progenitors that express the Alkaline phosphate intestinal (Alpi) gene also have thiscapacity. We created an Alpi-IRES-CreERT2 (Alpi(CreER)) knockin allele for lineage

2

H. Clevers

tracing. Marked clones consist entirely of enterocytes and are all lost from villus tips withindays. Genetic fate-mapping of Alpi(+) cells before or during targeted ablation of Lgr5-

expressing stem cells generated numerous long-lived crypt-villus "ribbons," indicative ofdedifferentiation of enterocyte precursors into Lgr5(+) stems. By single-cell analysis ofdedifferentiating enterocytes, we observed the generation of Paneth-like cells andproliferative stem cells. We conclude that the highly proliferative, short-lived enterocyte

precursors serve as a large reservoir of potential stem cells during crypt regeneration.

Intestinal cancer is initiated by Wnt pathway-activating mutations in genes such as

APC. As in most cancers, the cell of origin has remained elusive. Deletion of APC in in

Lgr5+ve stem cells leads to their transformation within days. Transformed stem cells remain
located at crypt bottoms, while fueling a growing microadenoma in stomach, small

intestine and colon. These microadenomas display unimpeded growth and develop into

macroscopic adenomas within 4-6 weeks. When APC is deleted in short-lived Transit

Amplifying (TA) cells using a different Cre mouse, the growth of the induced

microadenomas rapidly stalls. Even after 30 weeks, large adenomas are very rare in thesemice. We conclude that stem cell-specific loss of APC results in progressively growing

neoplasia. Moreover, a stem cell/progenitor cell hierarchy is maintained in early stem cell-

derived adenomas, lending support to the “cancer stem cell”-concept.

Lgr5 stem cells are interspersed between terminally differentiated Paneth cells that are

known to produce bactericidal products. We find that Paneth cells are CD24+ and express

EGF, TGF-a, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell maintenancein culture. Co-culturing of sorted stem cells with Paneth cells dramatically improvesorganoid formation (see below for organoid culture conditions). This Paneth cellrequirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth

cells in vivo results in the concomitantloss of Lgr5 stem cells. In colon crypts, CD24+ cells

residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude

that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter
cell, the Paneth cell. In addition, crypt-associated mesenchymal fibroblast-like cells also
provide crucial niche factors, such as Gremlins, R-spondins, EGF and Wnts.

In 2009, we reported the establishment of long-term culture conditions under which

single crypts undergo multiple crypt fission events, whilst simultaneously generating
villus-like epithelial domains in which all differentiated cell types are present. This

involvement culturing of stem calls in 3D matrigel and the addition of EGF, the Wnt

agonist/Lgr5 ligand R-spondin and the BMP inhibitor Noggin. Single sorted Lgr5+ve stem

cells can also initiate these crypt-villus organoids. Tracing experiments indicate that the

Lgr5+ve stem cell hierarchy is maintained in organoids. We concluded that intestinal crypt-

villus units are self-organizing structures, which can be built from a single stem cell in theKeynote Lecture3

absence of a non-epithelial cellular niche. The same technology has now been developed

for human and mouse Lgr5 +ve stem cells from stomach, liver, lung, breast, pancreas,
prostate, ovary, taste bud, esophagus and others. We have since provided evidence thatsuch human organoids can be used to study hereditary disease and cancer of the pertinent

tissues taken from patients. Examples have included Cystic Fibrosis, alpha-1 Anti-trypsin

disease, and cancer of the colon, prostate, pancreas and breast.

Organoids also hold promise for regenerative medicine- and gene therapy strategies. In

a collaboration with the Watanabe lab, we tested the transplantability of cultured mousecolon organoids into superficially damaged mouse colon. The transplanted donor cells

readily integrated into the mouse colon, covering the area that lacked epithelium as a resultof the introduced damage in recipient mice. At 4 weeks after transplantation, the donor-

derived cells constituted a single-layered epithelium, which formed self-renewing crypts
that were functionally and histologically normal. Moreover, we observed long-term (>6months) engraftment with transplantation of organoids derived from a single Lgr5(+) colonstem cell after extensive in vitro expansion. These data have shown the feasibility of colonstem-cell therapy based on the in vitro expansion of a single adult colonic stem cell.

Increased cAMP levels induce rapid swelling of mouse and human intestinal/rectal

organoids by opening the cystic fibrosis transmembrane conductor receptor (CFTR). This

response is lost in organoids derived from cystic fibrosis (CF) patients. We have used the

CRISPR/Cas9 genome editing system to correct the CFTR locus by homologous
recombination in cultured intestinal stem cells of CF patients. The corrected allele isexpressed and fully functional as measured in clonally expanded organoids. This study

provides proof of concept for gene correction by homologous recombination in organoid-

cultured primary adult stem cells derived from patients with a single-gene hereditarydefect.

4

H. Clevers

Hans Clevers, MD, PhD

1986-1989

1989-1991

1991-2002
2002-2012
2002-present
2012-2015

2015-present

Postdoctoral Fellow with Dr. Cox Terhorst, Dana Farber
Cancer Institute, the Harvard Univ.
Assistant professor, Department of Clinical
Immunology, the Univ. Utrecht
Professor in Immunology, the Univ. Utrecht
Director, Hubrecht Institute
Professor in Molecular Genetics, the Univ. Utrecht
President of the Royal Netherlands Academy of Arts
and Sciences (KNAW)
Director, Research of the Princess Maxima Center for
Pediatric Oncology

5

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ORGANOID-BASED CANCER FUNCTIONAL GENOMICS

CalvinCalvin K
Kuouo

Department of Medicine, Hematology Division
Stanford University School of Medicine
Lokey Stem Cell Research Building, 265 Campus Drive, Stanford, CA((cjkuo@stanford.edu)
cjkuo@stanford.edu)
Calvin J. Kuo

Department
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Stanford University School of Medicine
Lokey Stem Cell Research Building, 265 Campus Drive, Stanford, CA 94305, USA
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tract tissues combining the 3D architecture of in vivo tissues withhave generated “organoid” models of wild-type gastrointestinal tract tissues combining the

3D architecture of in vivo tissues with the experimental facility of 2D cell lines, using an air- 1
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within wild-type tissues in a “bottom-up” approach.

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new driver oncogenic events in cancer.

The advent of genome-scale analysis of tumor

DNA, RNA and epigenomes through efforts such as TCGA has created a tremendous

amount of candidate oncogenic events that now require functional validation.

Conventionally, oncogene candidates are evaluated in vitro by manipulation in establishedcancer cell lines that may have been extensively passaged and/or possess numerous

Organoids and Cancer Stem Cell Models

7

epi/genetic alterations, which may in turn confound the ability to functionally assess theincremental transforming ability of a given test locus in question. In contrast, the ability to

propagate wild-type gastrointestinal tissues as organoids generates a cleaner genetic andprogressive and stepwise oncogenic transformation with permutations of Apc knockout,
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8

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- .
Top-down versus bottom-up cancer gene validation strategies

bottom-up cancer gene validation strategies.
-down versus bottom-up
Fig. 5. Top
Top-down
Accordingly, we
usedorganoids
to validatea driveroncogenefrom theoncogene
as a driver
-483
used
have
wehaveAccordingly,
organoids
to miR-483
validateas miR
miR-483
11p15.5 amplicon in human colorectal cancer [1]. Separately, we demonstrated that1
TGFBR2 is a metastasis suppressor gene in gastric cancer.
In both cases, we usedfrom the 11p15.5 amplicon in human colorectal cancer . Separately, we demonstrated
contextual modeling to introduce these candidate genes in a minimally-engineered genetic

background representing the most relevant clinical context [4].
is a metastasis suppressor gene in gastric cancer. In both cases, we usedthat TGFBR2Overall, organoids represent a highly promising experimental approach to cancer

modeling with direct application to functional genomics and novel oncogene discovery.

Further, it is increasingly feasible to apply these methods to primary culture of tumor

minimally-engineered
contextual modeling to introduce these candidate genes in a minimally-engineered
biopsy specimens, with and without stromal components, towards a more holistic in vitro

representing the most relevant clinical context4.
genetic backgroundmodeling of cancer biology and personalized treatment response [5].

Accordingly, we have used organoids to validate miR-483 as a driver oncogene
miR-483
from the 11p15.5 amplicon in human colorectal cancer1. Separately, we demonstrated
that TGFBR2 is a metastasis suppressor gene in gastric cancer. In both cases, we used
contextual modeling to introduce these candidate genes in a minimally-engineered
minimally-engineered
Organoids and Cancer Stem Cell Models

genetic background representing the most relevant clinical context4.
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Figure 6

Novel oncogene validation in organoids

Fig. 6. Novel oncogene validation in organoids.
References
1. Li, X., et al. Oncogenic transformation of diverse gastrointestinal tissues in primaryorganoid culture. Nat Med 20, 769-777 (2014).
2. Ootani, A., et al. Sustained in vitro intestinal epithelial culture within a Wnt-dependent
stem cell niche. Nat Med 15, 701-706 (2009).
3. Neal, J.T. & Kuo, C.J. Organoids as Models for Neoplastic Transformation. Annual
review of pathology 11, 199-220 (2016).
4. Nadauld, L.D., et al. Metastatic tumor evolution and organoid modeling implicate
TGFBR2 as a cancer driver in diffuse gastric cancer. Genome Biol 15, 428 (2014).
5. Cantrell, M.A. & Kuo, C.J. Organoid modeling for cancer precision medicine. Genome Med
7, 32 (2015).

10

C. J. Kuo

Calvin J. Kuo, MD, PhD

1987-1994
1994-1997
1997-2000
2001-2007
2007-20112011-present
2012-present
2015-present
2015-present

MD and PhD, Stanford Univ.
Internship and Residency, Brigham and Women’sClinical Oncology Fellowship, Dana Farber
Assistant Professor of Medicine, Stanford
Associate Professor with tenure, Stanford
Professor of Medicine, Stanford Univ.
Co-Leader for Cancer Biology, Stanford Cancer Center
Vice Chair for Basic and Translational Research,
Department of Medicine, Stanford Univ.
Maureen Lyles D’Ambrogio Professor of Medicine,
Stanford Univ. School of Medicine

11

VISUALIZATION OF HUMAN COLON CANCER STEM CELLSUSING ORGANOID TECHNOLOGY

Toshiro Sato

Department of Gastroenterology, Keio University School of Medicine
35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan(t.sato@keio.jp)

Recent advance in next generation sequencing has been providing better understandingof human colorectal cancers, yet the biological interpretation of genetic mutations in thecontext of malignant progression remains elusive. The difficulty mainly stems from a lackof functional assay system for intestinal epithelium. We identified specific niche

environments supporting mouse small intestinal stem cells, which enabled to grow single

stem cells into ever-growing organoids [1]. The niche factor based organoid culture system
has been broadly applied to various epithelial tissue stem cells from both mouse andhuman with some modifications optimized to the tissue [2,3]. We found that niche
signalling coincides with recurrently mutated oncogenic signalling in human colorectal

cancer, suggesting functional connection between niche signalling and colorectal
carcinogenesis [4]. By the use of CRISPR-Cas9 genome-editing system, we introduced
multiple driver gene mutations into human normal colon organoids [5]. Driver gene (APC,

KRAS, SMAD4, TP53 and PIK3CA) mutant organoids were efficiently enriched by selective

niche factor culture condition, demonstrating niche factor independent growth in theengineered organoids. Upon xenografting, the engineered organoids exhibited robust

tumorigenic capacity in renal subcapsule, but failed to form metastatic colonies in the liver

after injection into spleen [6]. In contrast, when introducing equivalent driver genemutations into chromosome-instable human adenoma organoids, they exhibited aberrant

histologic structure and were capable of forming metastatic colonies. These results indicatethat aberration of niche factor signalling confers niche independent growth capacity, andmetastatic transformation further require genetic lesions other than driver gene mutations.

12

T. SatoThe organoid culture system was applied to human colorectal cancers with 50-60%

establishment efficiency. We optimized isolation and culture protocol for human colorectal

tumours and established human colorectal tumour organoid library (CTOL) encompassing

a range of histological subtypes and clinical stages [7]. Each line was analysed by gene

expression signatures, copy number analysis and exome sequencing. In vitro and in

xenografts, the organoids reproduced the histopathological grade and differentiation
capacity of their parental tumours. Matched pairs of primary and metastatic organoids had

similar genetic profiles and niche factor requirements, but xenograft experiments

demonstrated higher metastatic capacity in the metastasis-derived organoids. Theseobservations underscore the importance of genotype-phenotype analyses at a single-patientlevel.

The cancer stem cell (CSC) theory highlights a rare self-renewing subpopulation of cells

that can rebuild tumours after chemotherapy or metastasis. The existence of human CSCs

is mainly supported by the results of a xeno-transplantation assay using prospectively

isolated cells. However, a direct demonstration of human CSCs in intact cancer tissues hasremained elusive owing to a lack of tractable culture system for human primary cancers.

We generated knock-in human colorectal cancer organoids in which LGR5 or KRT20 gene

expression was visualized by CRISPR-Cas9 mediated genetic knock-in reporter. After

xenografting, patient-derived colon cancer organoids reconstituted stem cell hierarchies

resembling those in the original patient cancer tissues. We also generated LGR5-CreER
knock-in colorectal cancer organoids and their lineage-tracing by fluorescent reporter

revealed the self-renewal and differentiation capacity of LGR5+ tumour cells. In contrast,

KRT20+ cancer cells were mostly post-mitotic and rarely formed tumour colonies. The
selective ablation of LGR5+ cells in LGR5-iCaspase9 knock-in organoids led to tumour
regression. Although residual LGR5 - cancer cells exhibited some compensatory

proliferation, the removal of LGR5+ CSCs constrained efficient tumour regrowth. These
data provide insights into the functional dynamics of CSCs and their potential astherapeutic targets in human colon cancer.

References
1. Sato, T., et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262-265 (2009).
2. Sato, T., et al. Long-term expansion of epithelial organoids from human colon, adenoma,
adenocarcinoma, and Barrett's epithelium. Gastroenterology 141, 1762-1772 (2011).
3. Sato, T., et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts.
Nature 469, 415-418 (2011).

Organoids and Cancer Stem Cell Models

13

4. Sato, T. & Clevers, H. Growing self-organizing mini-guts from a single intestinal stem cell:mechanism and applications. Science 340, 1190-1194 (2013).
5. Fujii, M., Matano, M., Nanki, K. & Sato, T. Efficient genetic engineering of human intestinal organoids using electroporation. Nat Protoc 10, 1474-1485 (2015).
6. Matano, M., et al. Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of
human intestinal organoids. Nat Med 21, 256-262 (2015).
7. Fujii, M., et al. A Colorectal Tumor Organoid Library Demonstrates Progressive Loss ofNiche Factor Requirements during Tumorigenesis. Cell Stem Cell (2016).

Toshiro Sato, MD, PhD

1991-1997
1997-1999
1999-2003
2003-2004
2005-2006
2006-2007
2007-20112011-20132013-present

Keio Univ. School of Medicine
Resident, Keio Univ. Hospital
Graduate School, Keio Univ. School of Medicine
Senior Resident, Keio Univ. Hospital
Chief Physician, Tepco Hospital
Postdoctoral Researcher, Stowers InstitutePostdoctoral Researcher, Hubrecht Institute
Assistant Professor, Keio Univ. School of Medicine
Associate Professor, Keio Univ. School of Medicine

14

DECIPHERING THE ROLE OF MALIGNANT DEAMINASE
ACTIVATION IN CANCER STEM CELL GENERATION

Catriona H. M. Jamieson

Division of Regenerative Medicine and Moores Cancer Center
University of California, San Diego
3855 Health Sciences Drive, La Jolla, California, 92093-0820, USA(cjamieson@ucsd.edu)

Cancer is the leading cause of death for individuals in the US under 85 years of age.

Mortality is most frequently related to cancer progression in the setting of therapeutic
resistance fueled by self-renewing cancer stem cells (CSCs). Thus, there is a pressing unmet
medical need for developing novel strategies for early CSC detection coupled with

innovative treatment modalities that prevent therapeutic resistance. Comprehensive DNA
and whole transcriptome RNA sequencing (RNA-seq) analyses suggest that pre-malignant

stem cell populations acquire age-related deficiencies in DNA damage repair followed by

RNA processing alterations that promote progenitor self-renewal and malignant

transformation.

Recently, RNA-seq analysis revealed that splice isoform signatures could distinguishbenign from malignant hematopoietic stem and progenitor cell (HSPC) aging (Crews et al,

Cell Stem Cell 2016). Splicing deregulation in secondary acute myeloid leukemia (sAML)
leukemia stem cells (LSC) was typified by intron retention and distinctive patterns pro-survival and adhesion splice isoform expression patterns. Spliceosome modulation with

17S-FD-895 reverted splicing to a normal aged HSPC pattern in humanized mouse models

of sAML thereby providing the impetus for translational research efforts to develop thisnovel modulator for clinical trials in relapsed or refractory AML.

Further analysis uncovered inflammatory cytokine networks that drive RNA processing

deregulation by activating adenosine deaminase associated with RNA (ADAR1) duringcancer relapse or progression (Jiang et al, PNAS 2013). Notably, the majority of the ADAR1
mediated RNA editing events occur in the context of double stranded RNA loops formedby primate specific Alu repeat sequences, 5’ and 3’ untranslated regions and microRNAs.

Organoids and Cancer Stem Cell Models

15

Through RNA sequencing, lentiviral overexpression and site-directed mutagenesis, we

discovered that ADAR1 contributes to oncogenic transformation of pre-malignant

progenitors in chronic myeloid leukemia (CML) by impairing let-7 pri-miRNA biogenesis

in response to enhanced JAK2 signaling in malignant niches (Zipeto et al, Cell Stem Cell

2016). Treatment with a selective JAK2 inhibitor, fedratinib, and a BCR-ABL1 inhibitor

reversed malignant RNA editing in a humanized mouse model of blast crisis CML thereby

providing the impetus for development of a combination tyrosine kinase inhibitor (TKI)
strategy. Moreover, we have embarked on intensive research efforts to identify selective

ADAR1 inhibitors that can be used as monotherapy to target CSCs.

In addition to promoting CSC self-renewal, ADAR1 prevented G1 cell cycle transit(Pineda et al, Scientific Reports 2016) typical of CML by editing primary miRNA 26a at the

Drosha cleavage site and thereby impairing biogenesis of precursor and mature miRNA
26a. Lentiviral overexpression of wild-type mir-26a impaired BC CML engraftment in

immunocompromised mice and reversed the G1 transit block thereby providing a RNA
editing driven mechanism of therapeutic resistance in CML (Jiang et al, manuscript in

preparation).

To determine if ADAR1 contributed to CSC driven therapeutic resistance in otherrefractory hematologic malignancies, we analyzed expression and activity of ADAR1 in

multiple myeloma (MM). Notably, copy number amplification of chromosome 1q21 thatencompasses the ADAR1 and IL-6 receptor locus defined a higher risk subset of patientswith MM. These samples harbored a coding region edit in GLI1 that prevented

degradation by SUFU and enhanced in vivo serial transplantation potential of high risk

MM in immunocompromised mouse models suggesting that ADAR1 activation represents

a novel prognostic biomarker and potential therapeutic target in MM (Lazzari et al,
submitted).

Finally, RNA-seq and RNA editing specific PCR revealed that cytidine deaminase

apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC3) transcripts
were key ADAR1 editing targets. As essential components of antiviral immune responses

elicited by inflammatory cytokines, APOBEC deaminases induce cytidine to uridine basechanges that are subsequently read as thymidine (C-to-T). Seminal, reports show recurrent

APOBEC mutagenesis patterns in cancer-associated genes. Moreover, the process ofkataegis, which involves clustering of DNA mutations, is associated with increased
expression of APOBECs capable of inducing DNA base substitutions in tumors. Notably,

BC CML CSCs were typified by increased expression of specific APOBEC family members

and increased editing of APOBEC3D and 3G. Thus, early detection and inhibition ofmalignant RNA and DNA deaminase activity may be vital for eradicating therapy resistant

CSCs in inflammatory microenvironments.

16

C. H. M. Jamieson

References
1. Crews LA, Balaian L, Delos Santos NP, Leu HS, Court AC, Lazzari E, Sadarangani A,
Zipeto MA, La Clair JJ, Villa R, Kulidjian A, Storb R, Morris SR, Ball ED, Burkart MD,
Jamieson CH. RNA Splicing Modulation Selectively Impairs Leukemia Stem Cell
Maintenance in Secondary Human AML. Cell Stem Cell. 2016 Aug 25. pii: S19345909(16)30250-8. doi: 10.1016/j.stem.2016.08.003
2. Zipeto MA, Court AC, Sadarangani A, Delos Santos NP, Balaian L, Chun HJ, Pineda G,
Morris SR, Mason CN, Geron I, Barrett C, Goff DJ, Wall R, Pellecchia M, Minden M,
Frazer KA, Marra MA, Crews LA, Jiang Q, Jamieson CH. ADAR1 Activation Drives
Leukemia Stem Cell Self-Renewal by Impairing Let-7 Biogenesis. Cell Stem Cell. 2016 Jun
7. pii: S1934-5909(16)30088-1. doi: 10.1016/j.stem.2016.05.004. PMID: 27292188
3. Pineda G, Lennon KM, Delos Santos NP, Lambert-Fliszar F, Riso GL, Lazzari E, Marra
MA, Morris S, Sakaue-Sawano A, Miyawaki A, Jamieson CH. Tracking of Normal and
Malignant Progenitor Cell Cycle Transit in a Defined Niche. Sci Rep. 2016 Apr
4;6:23885. doi: 10.1038/srep23885. PMID: 27041210
4. Holm F, Hellqvist E, Mason CN, Ali SA, Delos-Santos N, Barrett CL, Chun HJ, Minden
MD, Moore RA, Marra MA, Runza V, Frazer KA, Sadarangani A, Jamieson CH.
Reversion to an embryonic alternative splicing program enhances leukemia stem cell
self-renewal. Proc Natl Acad Sci USA. 2015 Dec 15;112(50):15444-9. doi:
10.1073/pnas.1506943112. Epub 2015 Nov 30. PMID: 26621726
5. Goff DJ, Recart AC, Sadarangani A, Chun HJ, Barrett CL, Krajewska M, Leu H, LowMarchelli J, Ma W, Shih AY, Wei J, Zhai D, Geron I, Pu M, Bao L, Chuang R, Balaian L,
Gotlib J, Minden M, Martinelli G, Rusert J, Dao KH, Shazand K, Wentworth P, Smith KM,
Jamieson CA, Morris SR, Messer K, Goldstein LS, Hudson TJ, Marra M, Frazer KA,
Pellecchia M, Reed JC, Jamieson CH. A Pan-BCL2 inhibitor renders bone-marrow-resident
human leukemia stem cells sensitive to tyrosine kinase inhibition. Cell Stem Cell. 2013
Mar 7;12(3):316-28. doi: 10.1016/j.stem.2012.12.011. Epub 2013 Jan 17. PMID: 23333150
6. Jiang Q, Crews LA, Barrett CL, Chun HJ, Court AC, Isquith JM, Zipeto MA, Goff DJ,
Minden M, Sadarangani A, Rusert JM, Dao KH, Morris SR, Goldstein LS, Marra MA,
Frazer KA, Jamieson CH. ADAR1 promotes malignant progenitor reprogramming inchronic myeloid leukemia. Proc Natl Acad Sci USA. 2013 Jan 15;110(3):1041-6. doi:
10.1073/pnas.1213021110. Epub 2012 Dec 28. PMID: 23275297
7. Jamieson CH, Ailles LE, Dylla S, Muijtjens M, Jones C, Zehnder JL, Gotlib J, Manz M, Li K,
Keating A, Sawyers C, Weissman IL. Granulocyte-Macrophage Progenitors as
Candidate Leukemic Stem Cells in Blast-Crisis CML. New Engl J Med. 2004 Aug
12;351(7):657-67. PMID: 15306667

Organoids and Cancer Stem Cell Models

Catriona H. M. Jamieson, MD, PhD

1995-1993
1999

2000
2001-2002
2001-2003

2003-2005
2005-20102006-present
2011-20152012-present

2014-present
2014-present
2015-present
2016-present

17Internal Medicine Resident, Univ. of British Columbia
Blood and Marrow Transplant Fellow, Univ. of British
Columbia
Blood and Marrow Transplant Fellow, Stanford Univ.
Hematology Clinical Fellow, Stanford Univ.
Pathology Postdoctoral Fellow, laboratory of Professor
Irving Weissman, Stanford Univ.
Instructor, Hematology, Stanford Univ.
Assistant Professor of Medicine, Univ. of California,
San Diego
Director, Stem Cell Research Program, Moores Cancer
Center
Associate Professor of Medicine,
Hematology/Oncology, UC San Diego
Co-Leader, Hematologic Malignancies Program,
Moores Cancer Center
Chief, Division of Regenerative Medicine
Deputy Director, Sanford Stem Cell Clinical Center
Director, CIRM Alpha Stem Cell Clinic at UC San
Diego
Professor of Medicine, Univ. of California, San Diego

18

AN AUTOCRINE LOOP INVOLVING b-CATENIN PATHWAY IS
CRITICAL FOR DEVELOPMENT OF HUMAN MYELOIDLEUKEMIA STEM CELLS

Koichi Akashi

Department of Medicine and Biosystemic Science
Faculty of Medicine, Kyushu University
3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan(akashi@med.kyushu-u.ac.jp)

Acute myeloid leukemia (AML) originates from self-renewing malignant leukemic stem

cells (LSCs). It has been shown that purified human LSCs can repopulate human AML in
immunodeficient mice after xenogeneic transplantation. Human AML LSCs mainly residein the CD34+CD38- fraction whose phenotype is analogous to normal hematopoietic stem

cells (HSCs). We have reported that the T-cell immunoglobulin mucin-3 (TIM-3) isexpressed on the surface of LSCs in any FAB type of AML with only exception of M3 (acutepromyelocytic leukemia: APL), but it is not expressed in normal human HSCs [1]. Only

TIM-3 + but not TIM-3 - fraction of human AML cells can reconstitute human AML in
immunodeficient mice. We also showed that administration of anti-human TIM-3 killing
antibodies cure immunodeficient mice reconstituted with human AML LSCs. Accordingly,

we have proposed that TIM-3 should be a strong candidate for therapeutic target toeradicate AML LSCs [1].

TIM-3 is a type 1 cell-surface glycoprotein, and was identified originally as a surface

molecule expressed in CD4+ Th1 cells in mice. In human, it is expressed also in a fraction of

T cells, NK cells, monocytes and dendritic cells (DCs). Galectin-9, a ligand of TIM-3, is a b-

galactoside binding lectin that contains 2 carbohydrate recognition domains (CRDs), andbinds to N-terminal immunoglobulin variable (IgV) domain of TIM-3 through its two

CRDs. Ligation of TIM-3 by Galectin-9 has been shown to phosphorylate tyrosine residuesof the cytoplasmic tail of TIM-3, and activate Src family kinases through its Src homology 2(SH2) binding motif in T cells and monocytes. We thus hypothesized that TIM-3 signaling

plays an important role in maintenance or self-renewal of human AML LSCs.

We found that serum galectin-9 concentration was significantly elevated in AML

Leukemic Stem Cells

19

patients (325+54.7pg/ml, n=20) but not in the healthy individuals (25.2+6.8pg/ml, n=7) or

patients with B cell malignancies (38.6+32.9pg/ml, n=5). Primitive CD34+ AML cells had

abundant galectin-9 protein in their cytoplasms. We then transplanted human CD34+

primitive AML cells into irradiated immunodeficient mice. Strikingly, only in micereconstituted with human AML, but not in those with normal cord blood or human ALL

cells had elevated serum levels of human galectin-9: Serum galectin-9 levels were234.7+69.0pg/ml (n=8) in mice reconstituted with primary human AML cells, whereas
4.64+4.64pg/ml (n=12) in mice with normal human hematopoiesis. These results

collectively suggested that AML cells secreted galectin-9 in vivo in an autocrine manner.
We then performed transcriptome analyses of primary CD34 + TIM-3 + AML cells after

galectin-9 ligation. A pathway enrichment analysis showed that both NF-kB and b-catenin

pathways were activated. In fact, galectin-9 ligation of TIM-3+ human AML cells induced
activation of the NF-kB pathway via ERK and AKT phosphorylation. Activation of ERK

and AKT pathways are known to inhibit the GSK3b activity, and to promote the nucleus

translocation of b-catenin in several cancers. To demonstrate significant nucleus
translocation of b-catenin of primary AML cells, we established a quantitation system byutilizing the Array Scan VTI system (ThermoFisher Scientific, USA). By this system, we

could formally prove that TIM-3 ligation by galectin-9 significantly promoted the nucleus

translocation of b-catenin in primary AML cells [2].

We also have extensively analyzed TIM-3 and galectin-9 interaction in cells frompatients with myeloid malignancies including myelodysplastic syndromes (n=30),

myeloproliferative neoplasms (n=12) and chronic myelogeneous leukemia (n=18).

Strikingly, in all cases, frequencies of CD34+CD38-TIM-3+ cells dramatically increased alongwith disease progression from early/chronic phase to overt leukemias. Furthermore, serum

levels of galectin-9 were also dramatically elevated after leukemic transformation.

Significant nucleus translocation of b-catenin by galectin-9 ligation was also found in these

diseases after leukemic transformation. A recent study has shown that NF-kB and b-

catenin pathways could play a cooperative role in conferring the cancer-stem-cell

properties to non stem cells in intestinal cancer model.

These data collectively suggest that TIM-3 and galectin-9 constitutes a pan-myeloid

autocrine loop to develop malignant stem cells in the vast majority of human myeloid

malignancies [2]. Signaling molecules downstream of TIM-3 and galectin-9 ligation, as wellas surface TIM-3 itself might be good candidates for cancer stem cell-target therapy
common to most myeloid malignancies.

20

K. Akashi

Pre-leukemia

NormalCD34+CD38- IL-3R+CD45RA+LSCs

CD34+CD38- IL-3RlowCD45RA- HSCs
Autocrine

TIM-3

Outgrowth

TIM-3 (-)
Gal-9 secretion(-)

Leukemia

Self-renewal
NF-!B"#-catenin"

Transformation
Autocrine

TIM-3
Gal-9

Outgrowth

TIM-3 (+)
Gal-9 secretion(+)

Self-renewal
NF-!B"#-catenin"

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 toselectively 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 andleukemic progression, Cell Stem Cell 17, 341-52

Koichi Akashi, MD, PhD

1985
1985-1991

1991-1993
1994-1999
2000-20092008-present

Graduated from Faculty of Medicine, Kyushu Univ.
Resident, and Research Fellow, First Department ofInternal Medicine, Kyushu Univ.
Division of Hematology, Sanshinkai General Hospital
Postdoctoral Fellow, Department of Pathology, Stanford
Univ. School of Medicine, (Irving L. Weissman’s lab)
Associate Professor, Dept. of Pathology, Dana-Farber
Cancer Institute, Harvard Medical School
Professor and Chair, Department of Medicine andBiosystemic Science, Kyushu Univ.

21

INNATE LYMPHOID CELLS IN THE CONTROL OF ORGANHOMEOSTASISAndreas Diefenbach

Research Centre of Immunotherapies and Institute of Medical Microbiology and Hygiene
Johannes Gutenberg University Mainz Medical Centre
Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany(diefenbach@uni-mainz.de)

Innate lymphoid cells (ILC) are a recently discovered branch of the lymphoid lineage

that belongs to the innate immune system. ILC are tissue-resident cells that are particulary

numerous in the intestinal lamina propria. While ILC perform traditional functions

associated with immune cells (such as defense against microbial infections), it is an
emerging new paradigm, that ILC are involved in processes not normally linked to theimmune system. For example, ILC play important roles in maintaining tissue homeostasis

and are important regulators of metabolic processes. We have been interrogating two

interconnected signaling networks that are involved in these unconventional functions of

ILC. ILC express the aryl hydrocarbon receptor (AhR), a transcription factor, activity ofwhich is controlled by environmental (e.g., nutrient-derived) metabolites and toxins that

directly initiate transcription of a battery of genes some of which metabolize toxins to non-toxic intermediates. In recent years, the importance of AhR signaling in immune cells has

been receiving attention. For example, mice genetically lacking the AhR in group 3 innate

lymphoid cells (ILC3) showed a profound defect in the maintenance of ILC3 [1,2]. In
addition, AhR signaling in ILC3 is required for the expression of interleukin (IL)-22, an

unusual cytokine that exclusively acts on non-hematopoietic cells (e.g., epithelial cells,

stroma cells) and has been linked to host resistance to certain types of bacterial [3] and viral

infections [4]. Curiously, ILC3 are positioned in proximity to the crypts of Lieberkuhn in thesmall intestine and they produce high levels of IL-22 during steady-state (i.e., in theabsence of infections or inflammation). The effects of IL-22 on epithelial cells have not beenthoroughly explored. ILC3-derived IL-22 has been shown to induce the production of anti-

microbial proteins by the intestinal epithelium, protecting the mucosal layer against

22

A. Diefenbach

pathogenic infections [3]. In addition, IL-22 has been shown to protect crypt-resident stem

cells against damage in a mouse model of graft-versus-host disease, though the IL-22-

regulated targets in stem cells remain unidentified [5,6]. Considering the high conservationof the AhR and its importance for IL-22 production, we investigated the function of the

AhR-IL-22 axis in response to genotoxic stress in intestinal stem cells caused by carcinogens

such as azoxymethane (AOM). Indeed, mice deficient in IL-22 or lacking AhR in ILC3

showed an increased rate of AOM-induced colorectal cancer. Interestingly, IL-22 controlled

molecular circuitry that protects the intestinal stem cell niche from genotoxic stress. The

implications of these finding for the development of human cancer will be discussed.
References

1. Kiss, E.A. et al. Natural aryl hydrocarbon receptor ligands control organogenesis ofintestinal lymphoid follicles. Science 334, 1561-1565 (2011).
2. Kiss, E.A. & Diefenbach, A. Role of the Aryl Hydrocarbon Receptor in Controlling
Maintenance and Functional Programs of RORgammat(+) Innate Lymphoid Cells andIntraepithelial Lymphocytes. Front Immunol 3, 124 (2012).
3. Zheng, Y. et al. Interleukin-22 mediates early host defense against attaching and effacing
bacterial pathogens. Nat Med 14, 282-289 (2008).
4. Hernandez, P.P. et al. Interferon-lambda and interleukin 22 act synergistically for the
induction of interferon-stimulated genes and control of rotavirus infection. Nat Immunol
16, 698-707 (2015).

5. Lindemans, C.A. et al. Interleukin-22 promotes intestinal-stem-cell-mediated epithelial
regeneration. Nature (2015).
6. Gronke, K. & Diefenbach, A. Regenerative biology: Innate immunity repairs gut lining.
Nature 528, 488-489 (2015).

Andreas Diefenbach, MD, PhD

1989-1996
1996-1998
1999-2002
2003-2006

2006-20102006-20132013-present

Medical School, Univ. of Erlangen
Resident Microbiology, Univ. of Erlangen
Postdoc, University of California Berkeley
Assistant Professor, Skirball Institute of Biomolecular
Medicine, New York Univ.
Adjunct Professor, Skirball Institute of Biomolecular
Medicine, New York Univ.
Professor, Univ. of Freiburg
Professor and Chair (Microbiology), JohannesGutenberg-Univ. Mainz

23

CLINICAL RELEVANCE OF LEUKEMIA STEM CELLS IN AMLJohn E. Dick, Liran I. Shlush, Stanley Ng, Amanda Mitchell andJean Wang
Princess Margaret Cancer Centre, University Health Network
Princess Margaret Cancer Research Tower, 101 College Street
Toronto, Ontario, M5G 1L7(jdick@uhnresearch.ca)

AML is organized as an aberrant developmental hierarchy maintained by functionally

Acute myeloid leukemia

distinct leukemia stem cells (LSC). The capacity for self-renewal is the defining hallmark of

all tissue stem cells, responsible for the persistence throughout life of both stem cells andtheir progeny. LSC are the only leukemic cells with sustained self-renewal capacity.

Programs including epigenetic states and the transcription factors and epigenetic modifiers(e.g. Polycomb complexes and miRNA) that govern them are responsible for stem cellproperties. The term “stemness” is increasingly being used in the literature to refercollectively to the integrated functioning of molecular programs that govern and maintainthe stem cell state. LSC exhibit a stemness gene expression program responsible for self-

renewal, quiescence, and resistance to apoptosis. These same properties also drive thecentral role that LSCs play in AML progression, recurrence, and chemotherapy resistance.

A common theme from our studies is the strong similarity between the stemness properties

of HSC and LSC, a finding that is supported by the recent findings that human AML

originates from normal HSC through an intermediate pre-leukemic HSC that possesses aclonal advantage due to mutations in key epigenetic regulation genes such as DNMT3A,

TET1/TET2 and others. However careful comparison of the transcriptional programs

enriched in purified LSC and HSC fractions indicate that there are also differences; LSC

fractions more closely resemble primed or activated HSC/MPP. Indeed, comparison of
LSC and HSC self-renewal, as measured in clonal serial xenograft transplantation assays,suggest that the capacity for LSC self-renewal is greatly enhanced indicating that

regulatory programs must be perturbed during leukemogenesis.

24

J. E. Dick et al.

Despite implementation of combination chemotherapy and hematopoietic cell

Clinical relevance of stemness properties in AMLtransplantation (HCT), AML still remains lethal. Most patients respond to initial induction

achieving complete remission (CR), but most (>50%) will go on to relapse. The initial

success of chemotherapy to seemingly eradicate leukemia, only to have it return in a form

resistant to the original chemotherapy points to the survival of leukemia cells that areresistant and regenerative; properties that rest with LSC. Despite decades of research, thecells that cause relapse have not been precisely identified and characterized making it

difficult to gain mechanistic understanding of why some cells relapse and to develop better

drugs to target them.

Therapeutic strategies in AML rely on the presence or absence of cytogenetic

abnormalities and a small set of gene mutations that broadly define favorable,intermediate, and adverse risk categories. The more adverse subtypes require moreaggressive therapy such as HCT. However, in the favorable and intermediate risk groups,

a subset of patients relapse despite the lack of adverse risk factors. Moreover, patients withcytogenetically- or mutationally-defined adverse risk features and those requiring morethan one cycle of therapy to achieve remission have a worse outcome, with or without

allogeneic transplantation. Thus there is great need for better biomarkers to identify high-risk patients prior to starting induction chemotherapy. We have now achieved

development of the first such biomarker based on LSC stemness that enables rapid andmore accurate risk stratification, not only in the post-remission setting but also prior to

beginning treatment.

We used the AML xenotransplantation model to identify 138 LSC+ and 89 LSC– cell

fractions sorted from 78 AML patients and then through gene expression analysis wederived a 104-gene LSC-specific gene signature. We then applied a statistical-learningalgorithm guided by a clinical dataset from 500 patients to extract the core transcriptional

components of stemness that related to patient outcomes. This approach resulted in ascoring system that robustly predicted outcome and therapy resistance across multiple
AML datasets. Our study identified a 17-gene signature score (LSC17) that is highly

prognostic in 5 independent datasets (n=908) across the broad spectrum of AML patients,

and improves prediction of initial therapy resistance. Patients with high LSC17 scores hadpoor outcomes with current treatment strategies including allogeneic transplantation. The
LSC17 scoring approach was adapted to the clinically-applicable NanoString platform
providing a first-in-class tool that we intend to bring into clinical use to identify AML

patients who do not benefit from standard therapy and should be enrolled in trials

evaluating novel upfront or post-remission therapies.

Leukemic Stem Cells

25

In AML, there is good evidence that resistant cells arise prior to chemotherapy and

Subclonal genetic diversity and the source of relapse in AML

chemotherapy selects these pre-existing cells. Genetic analysis of bulk AML cell

populations from paired diagnosis and relapse samples from the same patient haveestablished that often the major clone at relapse shares some but not all mutations with the

major clone at diagnosis. With deeper sequencing and specific informatic tools, these insilico studies show that the bulk AML population at diagnosis is not uniform; rather it iscomposed of genetically distinct subclones that are related to each other through branching

evolution. In some cases, genetic variants specific to the relapse sample are already present

at low frequencies in the bulk diagnosis sample, suggesting that a subclone already present

at diagnosis generates relapse. However, such in silico depictions of clonal diversity andrelapse origins do not provide insight into the actual cells that bear the relapse mutations

nor do they provide any biological insight into why they have both therapy resistance andregenerative properties.

To more directly identify relapse originating cells, we undertook whole genome

sequencing of 11 paired diagnosis/relapse samples and identified the mutations that wereshared, present only in the bulk diagnosis AML population, or only in the bulk relapse
population. Then these variants were monitored with high sensitivity by digital PCR on 10

populations purified on the basis of HSPC-like or myeloid blast phenotypes and in

multiple xenografts established from the paired samples; we use the xenografts to monitor

the genetic identity of the LSC that initiate AML engraftment. Phylogenetic analysis

showed that, at diagnosis, the LSC compartment is genetically heterogeneous andcomposed of LSC that drive the dominant AML population as well as other multiple small

LSC subclones. We identified relapse-specific variants in these sorted populations or

xenografts derived from the diagnosis sample; thus, in most cases, chemotherapy selects
for preexisting subclones present at diagnosis. Our second major finding was that thecellular origin of relapse differed between samples: one group was termed as having

primitive-relapse origins and the other group as having committed-relapse origins. Thus,

this data is clear evidence that functionally defined LSC are linked to disease recurrence inhuman patients.

Our findings have considerable implications for the design of better ways to monitor

and treat AML patients. The ability to track the cellular origin of relapse will enablebiomarker development to better stratify patients to receive the most appropriate therapies.

Moreover the isolation of diagnosis subclones fated to cause relapse through xenografting
will enable detailed characterization of their vulnerabilities leading to improved therapies.

26

J. E. Dick et al.

References
1. Eppert K, Takenaka K, Lechman ER, Waldron L, Nilsson B, van Galen P, Metzeler KH,
Poeppl A, Ling V, Beyene J,Canty AJ, Danska JS, Bohlander SK, Buske C, Minden MD,
Golub TR, Jurisica I, Ebert BL, Dick JE. Stem cell gene expression programs influence clinical outcome in human leukemia. Nat Med. 2011 Aug 28;17(9):1086-93
2. Notta F, Mullighan CG, Wang JC, Poeppl A, Doulatov S, Phillips LA, Ma J, Minden
MD, Downing JR, Dick JE. Evolution of human BCR-ABL1 lymphoblastic leukaemiainitiating cells, Nature. 2011 Jan 20;469(7330):362-7.
3. Kreso A, Dick JE. Evolution of the cancer stem cell model. Cell Stem Cell. 2014 Mar
6;14(3):275-91.
4. Hope KJ, Jin L, Dick JE. Acute myeloid leukemia originates from a hierarchy ofleukemic stem cell classes that differ in self-renewal capacity. Nat Immunol.
2004;5(7):738-743.
5. Shlush LI, Zandi S, Mitchell A, Chen WC, Brandwein JM, Gupta V, Kennedy JA,
Schimmer AD, Schuh AC, Yee KW, McLeod JL, Doedens M, Medeiros JJ, Marke R, Kim
HJ, Lee K, McPherson JD, Hudson TJ; HALT Pan-Leukemia Gene Panel Consortium,
Brown AM, Trinh QM, Stein LD, Minden MD, Wang JC, Dick JE. Identification of preleukaemic haematopoietic stem cells in acute leukaemia. Nature 2014 Feb
20;506(7488):328-33.
6. Ng SWK, Mitchell A, Kennedy JA, Chen WC, McLeod J, Ibrahimova N, Arruda A,
Popescu A, Gupta V, Schimmer AD, Schuh AC, Yee KW, Bullinger L, Herold T, GörlichD, Büchner T, Hiddemann W, Berdel WE, Wörmann B, Cheok M, Preudhomme C,
Dombret H, Metzeler K, Buske C, Löwenberg B, Valk PJM, Zandstra PW, Minden MD,
Dick JE, and Wang JCY A 17-Gene Stemness Score for Rapid Determination of Risk in
Acute Leukemia. Nature. 2016 Dec 15;540(7633):433-437.
7. Lechman ER, Gentner B, Ng SW, Schoof EM, van Galen P, Kennedy JA, Nucera S,
Ciceri F, Kaufmann KB, Takayama N, Dobson SM, Trotman-Grant A, Krivdova G,
Elzinga J, Mitchell A, Nilsson B, Hermans KG, Eppert K, Marke R, Isserlin R, Voisin V,
Bader GD, Zandstra PW, Golub TR, Ebert BL, Lu J, Minden MD, Wang JCY, Naldini L
and Dick JE miR-126 Regulates Distinct Self-Renewal Outcomes in Normal and
Malignant Hematopoietic Stem Cells. Cancer Cell. 2016;29(2):214-228.

Leukemic Stem Cells

John E. Dick, PhD, FRS

1984

1986-1991

1987-1991

1991-1995

1991-2002

1995-present

2002-present

2007-present

2007-present

27

Post-doctoral Fellow, Ontario Cancer Institute Univ. of
TorontoScientist, Department of Genetics, Research Institute,
Hospital for Sick Children
Assistant Professor, Department of Molecular andMedical Genetics, Univ. of Toronto
Associate Professor, Department of Molecular andMedical Genetics, Univ. of Toronto
Senior Scientist, Department of Genetics, ResearchInstitute, Hospital for Sick Children
Professor, Department of Molecular Genetics, Univ. of
TorontoCanada Research Chair in Stem Cell Biology, Senior
Scientist, Princess Margaret Cancer Centre, Univ.
Health Network
Investigator, McEwen Centre for Regenerative
Medicine, Univ. Health Network
Director, Program in Cancer Stem Cells, Ontario
Institute for Cancer Research, (OICR)28

ESSENTIAL ROLES OF EPIGENETIC REGULATOR IN ACUTE
MYELOID LEUKEMIA STEM CELLS

Issay Kitabayashi

Division of Hematological Malignancy, National Cancer Center Research Institute5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan(ikitabay@ncc.go.jp)

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 dormant cancer stem cells (CSCs), which is

resistant to chemotherapy. Thus, selective targeting of LSCs is a promising strategy forpreventing relapse. Polycomb repressive complexes 1 (PRC1) and 2 (PRC2) are important

epigenetic regulators that maintain the stemness of embryonic and hematopoietic stem

cells. Enhancer of zeste homolog 1 and 2 (EZH1/2) are catalytic components of PRC2,

which trimethylates histone H3 at lysine 27 (H3K27) to repress transcription of target

genes. Mutation and overexpression of EZH1/2 are associated with cancers, including
hematopoietic malignancies.

We found that EZH1/2 are highly expressed in dormant CSCs and are essential for

1. Acute Myeloid Leukemia

maintenance of dormant CSCs. Double deletion of Ezh1/2 induced differentiation of AML
cells and complete remission of AML In AML mice models, which was not achieved bysingle deletion of Ezh1 or Ezh2. Ezh1/2 suppress expression of Cyclin D1/D2 to maintaindormant CSCs. Dormant CSCs dramatically reduced after deletion of both Ezh1 and Ezh2.

Deletion of Ring1A and Ring1B (Ring1A/B) from AML cells caused the loss of self-renewal

ability and induced the expression of numerous genes including Glis2. Overexpression ofGlis2 caused MOZ-TIF2 AML cells to differentiate into mature cells, whereas deletion ofGlis2 expression in Ring1A/B-deficient MOZ-TIF2 cells inhibited differentiation. Therefore,
Ring1A/B regulate and maintain AML stem cells in part by repressing Glis2 expression,

Leukemic Stem Cells

29

which promotes their differentiation. We have developed potent and specific inhibitors

against both EZH1 and EZH2.

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. Taken together, theseresults strongly suggest that dual inhibition of EZH1 and EZH2 is a promising therapeuticstrategy to eradicate CSCs in a wide range of AMLs. The EZH1/2 dual inhibitor was also

effective on xenograft models of mantle cell lymphoma and multiple myeloma. Based onthese results we have initiated Phase I clinical trial of the dual inhibitor in malignant

lymphoma and will also start in AML presently.

Multiple myeloma (MM) is largely incurable as the disease eventually relapses despite

2. Multiple Myeloma

the recent development of novel therapies. Previous reports show that side population (SP)cells comprise myeloma stem cells. Therefore, targeting SP cells may be a promisingstrategy for preventing and treating MM relapse. We found that SP cells expressedsignificantly higher levels of EZH1/2 than non-SP (main population) cells. These results

suggest that overexpression of EZH1/2 is important for maintaining the stemness of MM

cells and that EZH1/2 could be a potential therapeutic target. the EZH1/2 dual inhibitor

suppressed the proliferation of almost all MM cell lines tested, with an IC50 significantly

lower than that of the specific EZH2 inhibitor GSK126. Furthermore, flow cytometry

analysis revealed that the EZH1/2 dual inhibitor significantly depleted the SP cell

population. RNA-seq analysis revealed that the transcriptional profiles of MM cell lines
treated with the EZH1/2 dual inhibitor were characterized by up-regulation of genes

related to the Wnt pathway. qRT-PCR confirmed that expression of Wnt, Frizzled, andProtein kinase C family members increased markedly after exposure to the EZH1/2 dual

inhibitor. Previous studies show that increased activation of the canonical Wnt signaling

pathway down-regulates HSC self-renewal and differentiation. Therefore, we generated b-

catenin-overexpressing MM cells to examine the effects of increased Wnt signaling on MM

cells. Surprisingly, proliferation of these cells was significantly lower than that of control
cells. These results suggested that PRC2 directly targets Wnt signaling, and that over-activation induced by EZH1/2 dual inhibition was responsible for the reduced
proliferation of MM cells.

Oral administration of the EZH1/2 dual inhibitor to mice bearing MM xenografts led tosignificant impairment of subcutaneous tumors. Interestingly, long-term administration of

the drug at lower doses to mice bearing orthotopic xenografts resulted in complete

eradication of minimal residual disease from the bone marrow and complete cure of MM

30

I. Kitabayashi

without any serious side effects. Furthermore, THE EZH1/2 DUAL INHIBITOR treatmentof an orthotopic PDX model derived from a relapsed and heavily pretreated MM patient
led to a reduction in the levels of human immunoglobulins in the serum.

Taken together, these results strongly suggest that dual inhibition of EZH1 and EZH2 isa promising therapeutic approach to eradicating myeloma stem cells and could lead toimportant advances in the treatment of MM.

Mantle cell lymphoma (MCL) is a well-defined and aggressive type of B cell non-

3. Mantle cell lymphoma

Hodgkin’s lymphoma that is genetically characterized by the t(11;14)(q13;q32)
chromosomal translocation, which results in constitutive overexpression of CYCLIN D1.

Although newly developed drugs such as ibrutinib show promising clinical outcomes,

relapsed MCL often acquires drug resistance, which is a critical obstacle to treatment.

Alternative approaches to overcoming the drug resistance of relapsed MCL are urgently

needed. We used a novel dual inhibitor of EZH1/2 to show that inhibiting EZH1/2 is apromising therapeutic strategy for MCL.

First, we developed a xenograft (PDX) mouse model using cells from a heavily

pretreated and relapsed MCL patient, and then orally administered an inhibitor of

EZH1/2, called THE EZH1/2 DUAL INHIBITOR. THE EZH1/2 DUAL INHIBITOR

strongly impaired proliferation of the patient-derived tumors and did not cause any serious
side effects. Additionally, an in vitro assay using MCL cell lines (Mino, JeKo-1, Z-138, andREC-1) showed that THE EZH1/2 DUAL INHIBITOR inhibited the growth of MCL cells,

and that the effect was much more significant than that using the single EZH2 inhibitor(GSK126). The IC50 of THE EZH1/2 DUAL INHIBITOR was about one tenth that of
GSK126. These results strongly suggest that dual inhibition of EZH1/2 could be a
promising therapeutic strategy for relapsed MCL.

Next, to investigate the effect induced by dual inhibition of EZH1/2, we conductedfurther analyses of the MCL cell lines. Cells exposed to THE EZH1/2 DUAL INHIBITOR

showed cell cycle arrest (G1 arrest) along with a dose-dependent reduction in phospho-Rb

and cell differentiation, coupled with increased cell surface expression of hCD138. We thenused RNA-seq analysis of MCL cell lines to compare THE EZH1/2 DUAL INHIBITOR-

treated cells with vehicle-treated cells and found that cell cycle-related signaling was
significantly affected and that a cyclin-dependent kinase inhibitor, CDKN1C (TP57), was

one of the genes most markedly upregulated by THE EZH1/2 DUAL INHIBITOR. ChIP
qPCR of MCL cell lines showed that the CDKN1C locus was strongly marked by H3K27

trimethylation, and that THE EZH1/2 DUAL INHIBITOR induced a significant reductionin the level of this histone marker. Furthermore, administration of THE EZH1/2 DUAL

Leukemic Stem Cells

31

INHIBITOR alone to PDX mice induced increased expression of CDKN1C (as in the in vitro

assay). Thus, dual inhibition of EZH1/2 in MCL induces expression of CDKN1C, which inturn causes cell cycle arrest and reduced growth of MCL.

Taken together, these results strongly suggested that dual inhibition of EZH1 and EZH2

is a promising therapeutic strategy for MCL, illustrating the potential of novel epigenetic

approaches to overcoming drug resistance of relapsed MCL.
References

1. Ogawara Y, Takuo Katsumoto T, Aikawa Y, Shima Y, Kagiyama Y, Soga T, Matsunaga H,
Seki T, Araki K, Kitabayashi I. IDH2 and NPM1 mutations cooperate to activate
Hoxa9/Meis1 and hypoxia pathways in acute myeloid leukemia. Cancer Res. 75:20052016, 2015.
2. Aikawa Y, Yamagata K, Katsumoto T, Shima Y, Shino M, Stanley ER, Cleary ML,
Akashi K, Tenen DG, Kitabayashi I. Essential role of PU.1 in maintenance of MLL-associated leukemia stem cells. Cancer Sci. 106:227-36 2015.
3. Takamatsu-Ichihara E, Kitabayashi I. The roles of Polycomb group proteins inhematopoietic stem cells and hematological malignancies. Int J Hematol., 103:634-42,
2016.

Issay Kitabayashi, PhD

1992-1995
1995-1998

1998-2002

2002-present
2011-20132007-present

Special Researcher for Basic Science, RIKEN
Staff Scientist, National Cancer Center ResearchInstitute
Section Head, National Cancer Center ResearchInstitute
Chief, National Cancer Center Research Institute
Deputy Director, National Cancer Center ResearchInstitute
Guest Professor, Tokyo Univ. of Science32

THE NOTCH:MYC SIGNALING AXIS IN T CELL
DEVELOPMENT AND TRANSFORMATION

Yumi Yashiro-Ohtani and Warren S. Pear

Department of Pathology & Laboratory Medicine
Abramson Family Cancer Research Institute
Perelman School of Medicine at the University of Pennyslvania
421 Curie Blvd, Philadelphia, PA 19104-6160, USA(wpear@mail.med.upenn.edu)
Notch signaling regulates many cell fate decisions in multicellular organisms. Within

the lymphoid compartment, Notch induces both T cell and marginal zone B cell fates.

Activating Notch mutations increase Notch signaling and within the hematolyhmphoidlineages, occur in T cell acute lymphoblastic leukemia (T-ALL) and muiltiple B cell

neoplasms that include mantle cell lymphoma (MCL), chronic lymphocytic leukemia(CLL), splenic marginal zone lymphoma and follicular lymphoma [1-4]. Notch is atransmembrane receptor that normally interacts with a Notch ligand (Jagged, Delta-like) onan adjacent cell [5]. This interaction initiates a series of proteolytic cleavages in theextracellular domain of the Notch receptor, leading to release of the Notch intracellulardomain by a gamma-secretase dependent mechanism. The cleaved intracellular domain of

Notch (ICN) then translocates to the nucleus where it associates with the DNA-binding

factor RBPj (also known as CSL). The Notch:RBPj complex then recruits a member of the

Mastermind family (Maml), forming the Notch transcriptional complex (NTC), which
recruits transcriptional co-activators leading to gene transcription.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of immature T

cells that affects both children and adults. Though current treatments cure ~70% of

pediatric patients, relapsed disease is refractory to treatment and the harsh current

treatments have long-term consequences. T cell transformation results from multiple

genetic events that lead to dysregulated control of cellular differentiation, growth, survival
and metabolism. The most frequently mutated gene in T-ALL is NOTCH1, which

undergoes gain-of-function mutation in ~60% of tumors [6]. In T-ALL, activating Notch

mutations result in either ligand-independent Notch activation and/or decreased Notch

Leukemic Stem Cells

33

degradation, both of which contribute to dysregulated Notch-induced transcription(Figure. 1).

LNR HD
AnkyrinN

C
TM

JͲsecretase inhibitor(GSI)

Figure 1

Oncogenic Notch Muta ons
1. Increase nuclear Notch
2. Decrease Notch turnover
3. Effect is to increase ac vity

Oncogenic Notch mutations in T-ALL frequently target the LNR HD domain, leading to ligand-independent signaling and/or the C-terminal PEST domain, leading to decreased
nuclear Notch1 turnover. The consequence of either mutation is to increase Notrch signaling.

MYC, a transcription factor (TF) intimately tied with cell growth, is one of the mostfrequently amplified genes in multiple human cancers [7]. MYC is induced by manydifferent growth signals, and is thought to be the master transcriptional regulator of cell

growth [8]. The transcriptional program of the MYC oncogene comprises more than 15% of

all cellular genes including regulators of cell proliferation, DNA replication, protein

biosynthesis and metabolism and energy [9-11]. A key function of MYC is to recruit p-Tefb

and elements of the RNA Polymerase machinery, which releases transcriptional pausing[12-14]. MYC rapidly drives transcriptional up-regulation of gene programs critical for cell

growth through these processes. Despite the central role of MYC, drugs directly targeting

this protein have failed for a variety of reasons, thus indirect inhibition via targeting of
MYC activators might be a more viable strategy. [9-11].

Among the well-characterized direct Notch targets in normal and malignant pre-T cells,

Myc is particularly important (Figure. 2). Myc upregulation by Notch is required for pre-T

cells to traverse early developmental checkpoints [15, 16] and for the growth and survivalof T-ALL cells [17-19]. Indeed, ectopic Myc expression is sufficient to rescue multiple

Notch-dependent T-ALL cell lines from Notch inhibition [20]. Although initial studies
showed that NTCs could bind sites within the murine Myc proximal promoter [17, 19, 21],

both my group and the Ferrando lab recently identified a T cell-specific “Notch-dependent

Myc enhancer” (“NDME”) located ~1.3 Mb 3’ of the Myc promoter in mice and humans

34

W. S. Pear et al.

Myc
Relative Myc mRNA

3
2
1
0

Figure 2

DMSO

WASH

Myc is a direct Notch1 transcriptional target. Results of GSI “washout” assay (see Ref Z). In
the presence of GSI, Notch transcription is reduced ~2X (which is the magnitude of theoncogenic effect. Replacing the GSI medium with vehicle alone (“ washout” ) causes
increased Myc transcription, even in the presence of cycloheximide. See Ref. 17 for details.

qFigure 3

GSI

1.4 MbNDME

The Notch Dependent Myc Enhancer (NDME) is located ~1.4 Mb 3’ from the Myc TSS.
Notch1 and RBPj ChIP-Seq tracks are shown from a primary Notch1-induced T-ALL.
Adapted from Ref. 23.

that is duplicated in ~5% of T-ALLs [22, 23] (Figure. 3). The NDME is located within aregulatory region spanning over 500 kb that includes multiple discrete elements marked bya high content of acetylated H3K27 (H3K27Ac), Brd4 (a bromodomain-extraterminal, or

BET protein) and mediator components such as Med1, features that characterize so-called“super-enhancers”, One of the far 3’ elements in this region is an enhancer that regulates

Myc in acute myeloid leukemia cells that is sensitive to small molecule BET inhibitors suchas JQ1 [24]; as a consequence, this element is termed the “BET-dependent Myc enhancer”,

or “BDME”. Significantly, binding of Notch1/RBPJ complexes to the NDME upregulates

H3K27Ac over a ~2 Mb region that includes the Myc promoter and the entire Myc 3’

regulatory region comprising the NDME and BDME [23]. Furthermore, T-ALL cell lines
selected for growth in the presence of GSI maintain Myc expression [25] and looping

between the BDME and the Myc promoter, suggesting that under the selective pressure ofchemotherapy, the BDME can substitute for the NDME to mediate GSI resistance [23](Figure. 4).

In summary, these data show that an evolutionarily conserved T-cell specific Mycenhancer regulates Notch-dependent Myc expression in both developing T cells and T-

ALL. Although, this Myc enhancer is T-cell specific, the T-cell specificity is unlikely toLeukemic Stem Cells

Figure 4

35

GSI resistant T-ALL cells (DND41) are devoid of H3K27Ac at the NDME but not the BDME
(top) and show decreased looping at the NDME but not the BDME (bottom). Adaptedfrom Ref. 23.

occur via Notch signaling as Myc is a direct Notch target in T-ALL, mantle cell lymphoma

and breast cancer [26]. This raises the question of what particular regulatory elements

provide the T-cell specificity. We recently identified the Notch dependent enhancer inmantle cell lymphomas and it is distinct from the T cell NDME (data not shown). Anotherremaining question is how Notch regulates H3K27Ac over such a large region and whether

it also regulates additional epigenetic functions. Finally, although we established thatenhancer use can “switch” under the selective pressure of chemotherapy in cultured cells,

whether this mechanism occurs in vivo remains to be established. Nevertheless, the studies

from my group and others of the Notch:Myc axis reveal how a Notch-dependent superenhancer contributes to both normal T cell development and malignant transformation.

Acknowledgement: These studies were supported by grants from the NIH(P01CA119070, R01AI047833)

References
1. Kridel, R., et al., Whole transcriptome sequencing reveals recurrent NOTCH1 mutations inmantle cell lymphoma. Blood, 2012. 119(9): p. 1963-71.
2. Fabbri, G., et al., Analysis of the chronic lymphocytic leukemia coding genome: role of
NOTCH1 mutational activation. J Exp Med, 2011. 208(7): p. 1389-401.

36

W. S. Pear et al.

3. Karube, K., et al., Recurrent mutations of NOTCH genes in follicular lymphoma identify a distinctive subset of tumours. J Pathol, 2014. 234(3): p. 423-30.
4. Kiel, M.J., et al., Whole-genome sequencing identifies recurrent somatic NOTCH2 mutations in
splenic marginal zone lymphoma. J Exp Med, 2012. 209(9): p. 1553-65.
5. Hori, K., A. Sen, and S. Artavanis-Tsakonas, Notch signaling at a glance. J Cell Sci, 2013.
126(Pt 10): p. 2135-40.
6. Aster, J.C., W.S. Pear, and S.C. Blacklow, The Varied Roles of Notch in Cancer. Annu Rev
Pathol, 2017. 12: p. 245-275.
7. Wolf, E., et al., Taming of the beast: shaping Myc-dependent amplification. Trends Cell Biol,
2015. 25(4): p. 241-8.
8. Dang, C.V., MYC, metabolism, cell growth, and tumorigenesis. Cold Spring Harb Perspect
Med, 2013. 3(8).
9. Klapproth, K. and T. Wirth, Advances in the understanding of MYC-induced lymphomagenesis.
Br J Haematol, 2010. 149(4): p. 484-97.
10. Lin, C.Y., et al., Transcriptional amplification in tumor cells with elevated c-Myc. Cell, 2012.
151(1): p. 56-67.
11. Nie, Z., et al., c-Myc is a universal amplifier of expressed genes in lymphocytes and embryonicstem cells. Cell, 2012. 151(1): p. 68-79.
12. Eberhardy, S.R. and P.J. Farnham, c-Myc mediates activation of the cad promoter via a postRNA polymerase II recruitment mechanism. J Biol Chem, 2001. 276(51): p. 48562-71.
13. Eberhardy, S.R. and P.J. Farnham, Myc recruits P-TEFb to mediate the final step in the transcriptional activation of the cad promoter. J Biol Chem, 2002. 277(42): p. 40156-62.
14. Rahl, P.B. and R.A. Young, MYC and transcription elongation. Cold Spring Harb Perspect
Med, 2014. 4(1): p. a020990.
15. Liu, H., et al., Notch dimerization is required for leukemogenesis and T-cell development.
Genes Dev, 2010. 24(21): p. 2395-407.
16. Li, X., et al., Oncogenesis of T-ALL and nonmalignant consequences of overexpressing intracellular NOTCH1. J Exp Med, 2008. 205(12): p. 2851-61.
17. Weng, A.P., et al., c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic
leukemia/lymphoma. Genes Dev, 2006. 20(15): p. 2096-109.
18. Sharma, V.M., et al., Notch1 contributes to mouse T-cell leukemia by directly inducing theexpression of c-myc. Mol Cell Biol, 2006. 26(21): p. 8022-31.
19. Palomero, T., et al., NOTCH1 directly regulates c-MYC and activates a feed-forward-looptranscriptional network promoting leukemic cell growth. Proc Natl Acad Sci U S A, 2006.
103(48): p. 18261-6.
20. Chan, S.M., et al., Notch signals positively regulate activity of the mTOR pathway in T-cell acutelymphoblastic leukemia. Blood, 2007. 110(1): p. 278-86.

Leukemic Stem Cells

37

21. Klinakis, A., et al., Myc is a Notch1 transcriptional target and a requisite for Notch1-induced
mammary tumorigenesis in mice. Proc Natl Acad Sci U S A, 2006. 103(24): p. 9262-7.
22. Herranz, D., et al., A NOTCH1-driven MYC enhancer promotes T cell development, transformation and acute lymphoblastic leukemia. Nat Med, 2014. 20(10): p. 1130-7.
23. Yashiro-Ohtani, Y., et al., Long-range enhancer activity determines Myc sensitivity to Notch
inhibitors in T cell leukemia. Proc Natl Acad Sci U S A, 2014. 111(46): p. E4946-53.
24. Shi, J., et al., Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation. Genes Dev, 2013. 27(24): p. 2648-62.
25. Knoechel, B., et al., An epigenetic mechanism of resistance to targeted therapy in T cell acute lymphoblastic leukemia. Nat Genet, 2014. 46(4): p. 364-70.
26. Stoeck, A., et al., Discovery of biomarkers predictive of GSI response in triple-negative breast cancer and adenoid cystic carcinoma. Cancer Discov, 2014. 4(10): p. 1154-67.

Warren S. Pear, MD, PhD

1980
1987

1989
1989-1991

1991-1996

1997-2003

2003-20092009-present
2013-present

2014-present

B.A., Williams College, USA
Ph.D., Tumor Biology (George Klein), Karolinska
Institute
M.D., Univ. of Rochester, USA
Intern and Resident in Pathology, Brigham and
Women's Hospital; Harvard Medical School
Postdoctoral Fellow in laboratory of Professor David
Baltimore, Rockefeller Univ. and Massachusetts
Institute of Technology
Assistant Professor of Pathology and Laboratory
Medicine, Univ. of Pennsylvania
Associate Professor of Pathology and Laboratory
Medicine (tenure), Univ. of Pennsylvania
Gaylord P. and Mary Louise Harnwell Professor of
Pathology and Laboratory Medicine, Univ. of
Pennsylvania
Co-Program Leader, Cancer Immunobiology, Abramson
Cancer Center, Univ. of Pennsylvania
Director, Immunopathology Division, Department of
Pathology & Lab Medicine, Univ. of Pennsylvania

38

MOLECULAR MECHANISM REGULATING STEM CELL
PROPERTIES MEDIATED BY NUTRIENT SIGNALS

Atsushi Hirao

Division of Molecular Genetics, Cancer Research Institute, Kanazawa University
Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan(ahirao@staff.kanazawa-u.ac.jp)

It has been demonstrated that shared mechanisms regulate stem cell properties in both

hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs). Despite the differing

origins of LSCs among different types of leukemia, there appears to be a common

regulatory mechanism governing “stemness”. On the other hand, understanding criticaldifference in regulatory manners between HSCs and LSCs is also important fordevelopment of LSC-specific therapeutic strategy. For example, there are critical differencesin metabolic regulation between these cell types, because normal HSCs utilize glycolysis,

whereas LSCs appear to have low glycolytic flow. Therefore, knowledge of the molecularmechanisms of metabolic regulation may provide novel therapeutic approaches foreradiation of LSCs.

Mammalian/mechanical target of rapamycin (mTOR) is a highly conserved

serine/threonine kinase in response to environmental determinants such as nutrient

availability, energy sufficiency, stress, and growth factor concentration (Figure 1). mTOR

forms two different complexes, called mTOR complex 1 (mTORC1) and mTORC2, andthese complexes have distinct substrate molecules that function in the many biologicalprocesses. mTORC1 consists of several proteins, including an essential component, Raptor,

contributes to promotion of anabolic pathways, i.e., enhancement of protein synthesis

associated with increased energy production [1]. Several clinical studies reported that highmTORC1 activity is co-related with poor prognosis of solid tumor patients, indicating thatthe anabolic processes promote malignant phenotypes of cancer cells. To investigate
physiological roles of mTORC1 in leukemia cells in vivo, we developed several mouse

leukemia models based on drug-inducible Raptor deficient mice. As a result, we revealed

Leukemic Stem Cells

39

an indispensable role of mTORC1 in the development of T-cell leukemia (T-ALL) model

driven by oncogenic Kras and Notch [2]. In contrast, in the acute myeloid leukemia (AML)

model, although Raptor deletion mainly induced apoptosis in majority of leukemia cells,

LSCs survived and maintained in bone marrow long-term [3]. The re-introduction of the

Raptor gene into these Raptor-deficient AML cells induced them to propagate anddifferentiate. Thus, LSCs can survive under the mTORC1 inactivated condition, suggestingthat they do not require the anabolic process for their self-renewal.

O2

Growthfactor,Insulin

Ras
Raf

PI3ͲK
MEK
ERK

CellCycle
Gluconeogenesis
ROS
DNArepair
Autophagy

PIP2
PTEN
HIF1D
D

PIP3

AMP/ATP

AKT

LKB1

AMPK

TSC1ͲTSC2

FOXO

GlucoseAmino
Acid

Rag

Rheb

mTORC2
mTOR

Proteinsynthesis
Fattyaciddegradation
Autophagy

RaptormLST8
PRAS40DEPTOR
Tti1/Tel2

mTORC1
Proteinsynthesis
Glycolysis
Lipidmetabolism
Energyproduction
Autophagy

Figure1

Nutrient-sensing signals for maintenance of LSCs.
mTORC1 is activated by growth factors and amino acids, contributing to catabolic process. In
contrast, depletion of nutrients, including growth factors and glucose, induces activation of
FOXO and AMPK, respectively. These nutrient sensing signals control behavior of LSCs
mediated by metabolic modification.

40

A. Hirao

Depletion of nutrients activates several molecules or pathways that contribute to

catabolic process, including AMPK, FOXO and autophagy. Recent studies have revealed

that such catabolic pathways are critical for maintenance of LSCs. FOXO family, forkheadfamily of transcriptional regulators, contribute to maintenance of tissue stem cells including

HSCs [4]. In C. elegans, exposure to a harsh environmental condition such as nutrient

depletion induces entry into the dauer stage, a reversible condition in which the metabolic

rate is reduced and life span is markedly enhanced, mediated by activation of Daf-16, aortholog of the FOXO family, suggesting that FOXO family members have conserved roles

in stress resistance in a variety of organisms. We and other groups found that FOXO

proteins play critical roles in the resistance of chronic myeloid leukemia (CML) stem cells

to tyrosine kinase inhibitor (TKI) therapy [5]. Based on these findings, we have attempted
to find chemical compounds that inhibit FOXO activity and investigated how

pharmacological inhibition of FOXO pathways affect LSCs. We found that inhibition of

FOXO pathway promotes differentiation of LSCs in vitro and in vivo, through modificationof metabolic status (Figure 2). Taking together with recent knowledge about an essential

role of AMPK in LSC maintenance, catabolic pathways are critical for maintenance of LSCs

in vivo. Therefore, we believe that investigation of molecular functions in these pathways

will lead to the development of successful therapeutics for eradiation of LSCs.

Leukemiastemcell

NonͲLeukemiastemcell

Catabolicpathway

Anabolic pathway

AMPK
FOXO

Differentiation

mTORC1

“Metabolic Dormancy”

ROS
Figure2

Critical roles of catabolic pathways in maintenance of LSCs.
LSCs are maintained with metabolic dormancy, which is characterized by cell cycle arrest,
low oxidative stress and low glycolytic activity. Catabolic molecules, such as AMPK and
FOXO, may contribute to the metabolic dormancy.

Leukemic Stem Cells

41

Reference
1. Hegazy AM, Yamada D, Kobayashi M, Kohno S, Ueno M, Ali MA, Ohta K, Tadokoro Y,
Ino Y, Todo T, Soga T, Takahashi C, Hirao A. Therapeutic strategy for targeting aggressive malignant gliomas by disrupting their energy balance. J Biol Chem. 2016, in press
2. Hoshii T, Kasada A, Hatakeyama T, Ohtani M, Tadokoro Y, Naka K, Ikenoue T, Ikawa T,
Kawamoto H, Fehling HJ, Araki K, Yamamura KI, Matsuda S, Hirao A. Loss of mTOR
complex 1 induces developmental blockage in early T-lymphopoiesis and eradicates T-cell
acute lymphoblastic leukemia cells. Proc Natl Acad Sci USA. 111:3805-10.2014
3. Hoshii T, TadokoroY, Naka K, Ooshio T, Muraguchi T, Sugiyama N, Soga T, Araki K,
Yamamura K, Hirao A. mTORC1 is essential for leukemia-propagation but not stem
cell self-renewal. J Clin Invest. 122:2114-29, 2012.
4. Miyamoto K, Araki YK, Naka K, Arai F, Takubo K, Yamazaki S, Matsuoka S, Miyamoto T,
Ito K, Ohmura M, Chen C, Hosokawa K, Nakauchi H, Nakayama K, Nakayama KI,
Harada M, Motoyama N, Suda T, and Hirao A. Foxo3a is essential for maintenance of thehematopoietic stem cell pool. Cell Stem Cell. 1:101-112: 2007
5. Naka K, Hoshii T, Muraguchi T, Tadokoro Y, Ooshio T, Kondo Y, Nakao S, Motoyama N,
Hirao A. TGFb-FOXO signalling maintains leukaemia-initiating cells in chronic myeoid
leukaemia. Nature, 463:676-80, 2010

Atsushi Hirao, MD, PhD

1988
1994
1995

1997

2001

2002
2005-present

M.D. Jichi Medical Univ.
Ph.D. Univ. of Tokushima
Postdoctoral fellow, Japan Society for the Promotion of
Science (Kumamoto Univ.)
Postdoctoral fellow, Ontario Cancer Institute, Univ. of
TorontoAssistant professor, Institute of Molecular Embryology
and Genetics, Kumamoto Univ.
Assistant professor, Keio Univ. School of Medicine
Professor, Cancer Research Institute, Kanazawa Univ.

42

NEW STRATEGY FOR OVERCOMING MULTIFACETEDTHERAPY-RESISTANT CANCER STEM CELLS

Hideshi Ishii

Cancer Profiling Discovery, Osaka University Graduate School of Medicine
2-2 Yamadaoka, Suita, Osaka 565-0871, Japan(hishii@gesurg.med.osaka-u.ac.jp)

Although numerous advancements in medical science, such as the emergence ofmolecular biology techniques in the 1980s and the completion of the Human Genome
Project in the early 21st century, have led to substantial progress in the treatment of cancer;

however, cancer-related deaths are still increasing. Despite much effort and attention
directed toward cancer treatment, cancer survivors still suffer from poor quality of life,

even in highly developed countries such as Japan, where this is exacerbated by an increasein population aging. International pharmaceutical companies have developed numerous

anticancer agents using molecular targeting strategies and immunotherapies, but resistancedevelops even for these novel medicines. One of the most important factors that make thepresent therapeutic strategies ineffective is tumor heterogeneity. It is now widely acceptedthat a small population of cells within tumors not only contribute to therapy resistance but

also to invasion and metastasis, and represents a main determinant of disease recurrence.

Several recent studies have revealed that these small populations of cells possess

multifaceted characteristics, including self-renewal capacity and multi-functional potential,

aspects that are not present in well-differentiated cancer cells; for at least two decades, such
critical cells have been referred to as cancer stem cells (CSCs). In order to visualize andcollect CSC fractions, we transfected cancer cells with a green fluorescent protein-fused
ornithine decarboxylase (ODC) monitoring cassette. This monitoring system allowed
visualization of cancer cell populations containing therapy-resistant CSCs. The study

revealed that polyamine flux plays a critical role in CSC functions, and polyamine

metabolism is linked to epigenetic regulation of downstream gene expression. Global

profiling studies have revealed that transcription factors are involved in the maintenanceSolid Cancer Stem Cells

43

and function of CSCs. Epigenetic studies, on the other hand, revealed uncharacterizedtranscriptional mechanisms and underscored the significance of molecular profiling in the

discovery of novel therapeutic targets for therapy-resistant cancer cells [1-3]. The novel

approaches may open new avenues, especially for treating gastrointestinal cancers thatpose challenges for treatment.

Figure 1

Therapy-resistant Cancer Cells
Primary tumor heterogeneity is characterized by the presence of actively growing cancer cells
and a small population of dormant CSCs in addition to supportive stromal cells. Afteradministration of anticancer agents and/or surgery, complete tumor remission may beachieved, but therapy-resistant CSCs still remain. In this process, tumor-driving mutations
accumulate, and after some period of time, the tumor relapses and metastasizes to otherorgans.

44

H. Ishii

Figure 2

Therapeutic Strategy against Dormant Slow Cycling Cancer Cells
In tumors, a small fraction of cancer cells (marked red in the figure) proliferate at a slow rate.
The elimination of such cells would result in the suppression and eradication of tumors.
Thus, the development of novel anticancer agents against slow cycling CSCs is highly beneficial to achieve better prognosis through effective cancer treatment.

Figure 3

Single Cell Tracking of Therapy-resistant Cancer Cells
The single cell tracking study indicated that exposure to chemotherapeutic agents results inthe accumulation of dormant or slow cycling cancer cells (shown in red). This approachallowed the identification of the Jumonji domain-containing histone demethylase family
KDM5/Jarid1, which removes tri- and dimethyl groups from lysine 4 on histone H3.

Solid Cancer Stem Cells

Figure 4

45

Jarid1B, a Therapeutic Target for Slow cycling Cancer Cells
Knockdown of Jarid1B using shRNA resulted in the induction of H3K4 tri-methylation andstimulation of p16/INK4A gene expression (upper left). Inhibition of Jarid1B translation
also led to a decrease in the CD44+ cancer stem cell fraction, sensitization of cells to the anticancer agent 5-fluorouracil (FU) (lower left), and suppression of the epithelial mesenchymaltransition (EMT) markers SNAIL (lower right), and VIMENTIN (upper right). The inoculation of Jarid1B-deficient cancer cells in immunocompetent mice resulted in the suppression oftumors in vivo. These data indicate that suppression of Jarid1B expression might be efficientfor the eradication of therapy-resistant cancer stem cells.

46

H. Ishii

Figure 5

Figure 6

Solid Cancer Stem Cells

47

Figure 7
Figure 5
Figure 6

Figure 7

INK Locus in H3K4 Demethylases
The chromatin immunoprecipitation study indicated that Jarid1B interacts with the promoter region of the p16/INK4A gene, whereas LSD1 and Ets proteins do not. The association
with the promoter was particularly high in Zs+-CSCs (Green).

Preferential Activation of the ODC Pathway in CSCs
The study of metabolic labeling with non-radioactive isotopes indicated an increased
polyamine flux in ODC-expressing CSCs compared with non-stem cancer cells (control),suggesting the importance of polyamine flux in the regulation of one-carbon metabolism inCSCs.
H3K4 methylation is Increased in Zs+-CSCs
Free energy calculations by computer simulation of Jarid1B and LSD1 binding interactions
indicated that both these proteins have the ability to bind to polyamines, suggesting thatpolyamines could interfere with the demethylation reactions of these enzymes.

48

H. Ishii

Figure 8

Figure 9

Solid Cancer Stem Cells
Figure 8

Figure 949

Id Stimulates Cancer Metabolism in Zs+-CSCs
The present study allowed the identification of Id genes that play a critical role in themaintenance and progression of cancer stem cells by modulating mitochondrial respirationand glycolysis. Extracellular stimuli such as epidermal growth factor (EGF) stimulate the fluxof polyamines into the one-carbon metabolic pathway, contributing to the specific metabolism of cancer stem cells. The association between Id and c-Myc may be important for thisprocess.

Double-Faced, Metabolite-dependent Epigenetic Response in Therapy-resistant Cancer StemCells
The epigenetic marking of the fourth lysine of histone 3 (H3K4) is important for the maintenance and progression of cancer stem cells. Jarid1B contributes to the control of demethylation at the promoter of the p16/INK4A gene, a cell cycle regulator. LSD1 in turn binds tomultiple enhancer sites and controls the expression of many genes, including the Id family,
which alters tumor metabolism by modifying oxidative phosphorylation (OxPhos) in
CSCs. The present study identifies novel druggable targets for cancer stem cell therapy
and suggests that the discovery of small new compounds acting on the KDM family provides
a breakthrough in the treatment of CSC-related refractory cancers thereby significantly
improves the patients’ clinical outcome.

References
1. Koseki J, Matsui H, Konno M, Nishida N, Kawamoto K, Kano Y, Mori M, Doki Y, Ishii H.
A trans-omics mathematical analysis reveals novel functions of the ornithine metabolic
pathway in cancer stem cells. Sci. Rep. 2016;6:20726.
2. Sueda T, Sakai D, Kawamoto K, Konno M, Nishida N, Koseki J, Colvin S H, Takahashi H,
Haraguchi N, Nishimura J, Hata T, Takemasa I, Mizushima T, Yamamoto H, Satoh T,
Doki Y, Mori M, Ishii H. BRAF V600E inhibition stimulates AMPK-mediatedautophagy in colorectal cancer. Sci. Rep. 2016;6:18949.
3. Colvin H, Nishida N, Konno M, Haraguchi N, Takahashi H, Nishimura J, Hata T,
Kawamoto K, Asai A, Tsunekuni K, Koseki J, Mizushima T, Satoh T, Doki Y, Mori M, Ishii
H. Oncometabolite D-2-hydroxyglurate directly induces epithelial-mesenchymal transition and is associated with distant metastasis in colorectal cancer. Sci. Rep. 2016 (inpress).
(See http://www001.upp.so-net.ne.jp/CancerProfiling/index.htm#, for the previous
publications)

50

H. Ishii

Hideshi Ishii, MD, PhD

1988

1992

1996

2002

2008
2011

2014-present

Graduated from Chiba Univ. School of Medicine(Yoshida’s lab)
Research Resident, National Cancer Center ResearchInstitute (Terada’s lab)
Postdoctoral Fellow, Thomas Jefferson Univ. (Croce’slab)
Lecturer, Jichi Medical Univ. (Furukawa & Ozawa’s
labs)
Associate Professor, Kyusyu Univ. (Mori’s lab)
Endowed Chair Professor, Cancer Frontier Science,
Osaka Univ.
Specially Appointed Professor, Cancer Profiling
Discovery, Osaka Univ.

51

THE CANCER STEM CELL NICHE OF SQUAMOUS CELLCARCINOMAS: BIOLOGY AND IMPACT FOR
THERAPEUTICS

Elaine Fuchs

Howard Hughes Medical Institute, The Rockefeller University
1230 York Avenue, Box 300, New York, NY 10065, USA(fuchs@mail.rockefeller.edu)

Adult tissue stem cells have the ability to self-renew long term and differentiate into one

or more tissues. Many stem cells are used sparingly to replenish cells during normal

homeostasis. However, even stem cells that are quiescent must be able to respond quickly

to injury in order to fuel rapid tissue regeneration. How stem cells balance self-renewal

and differentiation is of fundamental importance to our understanding of normal tissue
maintenance and wound repair. Increasing evidence suggests that the regulatory circuitry

governing this balancing act is at the root of some types of cancers.

The hair follicle is an excellent model system to understand how stem cells remainquiescent during times of minimal wear and tear, how these cells become mobilized during

the cyclical bouts of hair growth and wound-repair, and how the normal process of stem

cell activation goes awry in cancer. Hair follicles have the remarkable capacity to undergo

cycles of growth (anagen), degeneration (catagen) and rest (telogen). In mice, the first

several cycles are synchronized, making hair follicles an ideal system for understanding

how stem cells interact with progeny and heterologous cell types in their niche to transition
between quiescence and active tissue (hair) regeneration.

The quiescent hair follicle stem cells reside in a single layer of the bulge niche, located at

the bottom of the non-cycling portion of the hair follicle. Within the bulge niche is a layerof BMP-expressing differentiated cells that anchor the hair and transmit inhibitory signals

to maintain stem cell quiescence. The stem cells adhere to a basement membrane flanked
by a dermal sheath. Beneath the bulge is a mesenchymal stimulus called the dermal

papilla. Stem cells at the base of the bulge, closest to this stimulus are the first to be

activated at the start of the hair cycle. These cells communicate with the dermal papilla.

52

E. Fuchs

As Valentina Greco, a former postdoc in my lab, now at Yale, showed, the ‘primed’ stem
cells make increasing levels of WNTs; the dermal papilla makes increasing levels of BMP

inhibitory signals, and together these two signals are needed to overcome the inhibitory

BMP signal that otherwise keeps the stem cells in quiescence.

When the activating threshold is reached, the stem cells at the base of the bulge begin toproliferate and generate short-term progeny called matrix cells. The first group of thesecells produces Sonic Hedgehog (SHH). As shown by Ya-Chieh Hsu, a former postdoc in

my lab, now at Harvard, showed, SHH then acts in two ways: first it acts on the dermal

papilla, which elevates BMP inhibitors and pro-activating FGFs; second, it acts on thequiescent upper bulge stem cells, which self-renew and produce a cellular shaft (the outerroot sheath) which pushes the SHH-DP signaling center downward, resulting in a return ofthe bulge to quiescence until the next hair cycle. Meanwhile the signaling center fuels theproliferation of the short lived progeny to produce the hair and its channel, the inner root

sheath. After a period of hair growth, the lower two thirds of the hair follicle differentiates

and degenerates, bringing the DP upward to make contact again with the bulge. In thisway, the hair follicle becomes self-propelling to undergo these cyclical bouts of activity.

Over the years, we developed methods to purify the bulge stem cells and their progeny

and we carried out RNA-seq on these populations straight from fluorescence activating cellsorting (FACS). The stem cells expressed high levels of SOX9, LHX2, NFATc1, FOXC1, IDs,

TCF3 and TCF4 transcription factors, which distinguished them from their committed

counterparts and from the epidermal stem cells residing in the basal layer of the epidermis.

We used conditional knockout technology to ablate each of these factors in the skin. Ourfindings showed that some factors, e.g. NFATc1, IDs and FOXC1, acted downstream from

BMP signaling, and promoted stem cell quiescence. We found that TCF3/4 antagonize

WNT-beta catenin to repress HF fate differentiation, while LHX2 suppresses sebaceous
gland differentiation and SOX represses epidermal differentiation. In carrying out a

combination of in vivo chromatin immunoprecipitation and high throughput DNA

sequencing (ChIP-seq) with antibodies against not only the transcription factors but also

epigenetic marks including H3K27ac, Med1, H3K27me3, H3K4me1, H3K4me3 andH3K36me2, we identified ~350 HFSC genes which are controlled by large open chromatin
domains, or ‘super-enhancers,’ a word coined by Rick Young at MIT. Within these super-enhancers are small regulatory elements, which we call ‘epicenters,’ that contain clusteredbinding sites for the cohort of HFSC transcription factors. In work carried out by one of mygraduate students, Rene Adam, we’ve cloned these regulatory elements and tested them inmice and found that they act to target reporter gene expression to the bulge stem cells.

In looking at the endogenous group of HFSC genes regulated by super-enhancers, wefound that they encode the majority of the stemness genes, including the genes regulating

Solid Cancer Stem Cells

53

SOX9, TCF3/4 and LHX2 as well as Integrins alpha 6 and beta4, LGR5, BMP6 and thereceptors for BMPs, WNTs and FGFs. There are many other human disease genes that arefound on this short-list comprising ~5% of the total number of genes expressed by HFSCs.

Intriguingly, when HFSCs were cultured, their chromatin landscape changed

dramatically as a new cohort of super-enhancers were gained while others were lost. In

seeking mechanisms, we discovered that many HFSC transcription factor genes weresilenced in vitro while new wound-induced transcription factors appeared. Moreover, the

new super-enhancers were regulated by these wound-induced factors, corresponding to

the new proliferative migratory state of the cultured hair follicle stem cells.

This work, led by Rene Adam and Hanseul Yang in my lab, gave us new insights into

the sensitivity of stem cells to their microenvironment and the importance of niche-stem

cell interactions in dictating stem cell behavior. Hanseul along with Daniel Schramek, aformer postdoc in my lab now at the Lunefeld Cancer Center in Toronto, then asked thequestion of how the chromatin landscape changes as hair follicle stem cells acquires themutations that lead to malignant progression. When they carried out ChIP seq analyses on

the tumor-initiating (stem) cells of squamous cell carcinoma chromatin, they unearthed
new super-enhancers regulating oncogenes such as Src and Myc. These super-enhancers

were regulated by a new cohort of transcription factors whose genes were also regulated bysuper-enhancers, thereby generating a feed forward loop. Notably were ETS2 and ELK3,

because these super-enhancer regulating transcription factors are known to bephosphorylated and activated by RAS-MAPK, the pathway upregulated in the SCCs.

Indeed, mice engineered to express a phosphomimic ETS2, the epidermis became

hyperproliferative, invasive and generated a chromatin landscape that was similar to the

SCC state.

Again, these findings underscored the importance of a now new

microenvironment characteristic of the SCC state.

Finally, we looked at how the microenvironment changes during malignant

progression. We discovered that wherever blood vessels invade the stroma and approach

the tumor, the tumor-initiating cancer stem cells at the tumor-stroma interface respond to

TGF-beta brought in by the perivasculature, rich in immune cells such as monocytes. The
outcome is striking—these stem cells become slow-cycling and break down the basement

membrane and invade the stroma. By contrast, stem cells more distant from blood vessels
proliferate rapidly, generating the bulk of the tumor. Does this matter? To test thispossibility, we treated mice with cisplatin, the drug of choice for many human squamouscell carcinomas. Most of the tumor cells died, except for those cancer stem cells responding

to TGF-beta. Moreover, these stem cells evaded the chemotherapy and regrew the cancer

when cisplatin was washed out. This was the work of Naoki Oshimori, who has now
moved on to start his own lab at Oregon Health Science Cancer Center. The studies

54

E. Fuchs

beautifully underscore the importance of heterogeneity in the tumor microenvironment.

I don’t want to leave you with the notion that genetic variation in tumor progression

doesn’t matter. Clearly, it does, as there are hundreds of mutations in these solid tumors.

To this end, we’ve used our powerful in utero lentiviral technology to carry out in somecases, genome-wide screens for oncogenic drivers, tumor suppressors and oncogenic

microRNAs involved in SCC progression. Together, these multipronged approaches

should be useful in our ever growing endeavors to understand the underlying basis forsquamous cell carcinomas, which are not only one of the most common but also one of themost life-threatening cancers world-wide.

References
1. Lay K, Kume T and Fuchs E. FOXC1 maintains the hair follicle stem cell niche and governs stem cell quiescence to preserve long-term tissue-regenerating potential. Proc Natl
Acad Sci USA. 2016 Feb 24. pii: 201601569. [Epub ahead of print] PMID:26912458.
2. Ge Y, Zhang L, Nikolova M, Reva B, Fuchs E. Strand-specific in vivo screen of cancerassociated miRNAs unveils a role for miR-21* in SCC progression. Nat Cell Biol.
18(1):111-21 (2016)
3. Yang H, Schramek D, Adam RC, Keyes BE, Wang P, Zheng D, Fuchs E. ETS familytranscriptional regulators drive chromatin dynamics and malignancy in squamous cell
carcinomas. Elife. Nov 21; 4, e10870, 1-22 (2015).
4. Oshimori N, Oristian D, Fuchs E. TGF-b promotes heterogeneity and drug resistance inSquamous Cell Carcinoma. Cell 160:963-76 (2015).
5. Adam RC, Yang H, Rockowitz S, Larsen SB, Nikolova M, Oristian DS, Polak L, Kadaja M,
Asare A, Zheng D., Fuchs E. Pioneer factors govern super-enhancer dynamics in stem cell
plasticity and lineage choice. Nature 521 366–370 (2015).
6. Genander M, Cook PJ, Ramsköld D, Keyes BE , Mertz AF, Sandberg R, Fuchs E. BMP signaling and its pSMAD1/5 target genes differentially regulate hair follicle stem cell lineages. Cell Stem Cell 15:619-33 (2014).
7. Blanpain C, Fuchs E. Plasticity of epithelial stem cells in tissue regeneration. Science
344:1243-1255 (2014). [cover photo]
8. Hsu YC, Li L, Fuchs E. Transit-amplifying cells orchestrate stem cell activity and tissue
regeneration. Cell 157:935-49 (2014).
9. Lien WH, Polak L, Lin M, Lay K, Zheng D, Fuchs E. In vivo transcriptional governance of
hair follicle stem cells by canonical Wnt regulators. Nature Cell Biology 16: 179-90 (2014).
10. Kadaja M, Keyes B, Lin M, Pasolli HA, Genander M, Polak L, Stokes N, Zheng D, Fuchs E.
SOX9: a Stem Cell transcriptional regulator of secreted niche signaling factors. Genesand Development 28(4):328-41 (2014).

Solid Cancer Stem Cells

55

11. Schramek D, Sendoel A, Segal JP, Beronja S, Heller E, Oristian D, Reva B and Fuchs E. In
vivo RNAi screen unveils myosin-IIa as a tumor suppressor of Squamous Cell
Carcinomas. Science 343:309-13(2014).
12. Beronja S, Janki P, Heller E, Lien W-H, Keyes B, Oshimori N, Fuchs E. Genome-wide
RNAi screens identify physiological regulators of oncogene-dependent epidermal
growth. Nature 501:185-90 (2013).
13. Folgueras, AR, Guo, X, Pasolli, HA, Stokes, N, Polak L, Zheng, D, Fuchs E.
Architectural niche organization by LHX2 Is linked to hair follicle stem cell function.
Cell Stem Cell 13:314-27 (2013).
14. Keyes BE, Segal JP, Heller E, Lien W-H, Chang C-Y, Guo X, Oristian D, Zheng D, Fuchs E.
Nfatc1 orchestrates aging in hair follicle stem cells, Proc. Natl Acad Sci. USA, 110:E4950-9(2013).
15. Lien W-H, Guo X, Polak L, Lawton LN, Young RA, Zheng D, Fuchs E. Genome-wide
maps of histone modifications unwind in vivo chromatin states of the hair follicle lineages. Cell Stem Cell 9, 219-32 (2011).
16. Schober M, Fuchs E. Tumor-Initiating stem cells of squamous cell carcinomas and theircontrol by TGFb and Integrin/FAK signaling. Proc Natl Acad Sci USA, 108,10544-9(2011).
17. Ezhkova E, Lien WH, Stokes N, Pasolli HA, Silva JM, Fuchs E. EZH1 and EZH2 co-govern histone H33-K27 trimethylation and are essential for hair follicle homeostasis andwound repair. Genes Dev, 25,485-98 (2011). [Highlighted in Nature Reviews, Mol Cell Biol. 12,204, 2011]18. Hsu YC, Pasolli HA, Fuchs E. Dynamics between stem cells, niche and progeny. Cell
144,92-105 (2011). [highlighted in Cell Stem Cell 8,8-9, 2011]19. Beronja, B., Livshits G, Williams S, Fuchs E. Rapid functional dissection of genetic networks via non-invasive, tissue-specific transduction of mouse embryos. Nat.Medicine
16,821-7(2010). [cover article]
20. Ezhkova E, Fuchs E. Regenerative medicine: An eye to treating blindness. Nature 466, 5678 (2010).
21. Nguyen H, Merrill BJ, Polak L, Nikolova M, Rendl M, Shaver TM, Pasolli HA, Fuchs E.
Tcf3 and Tcf4 are essential for long-term homeostasis of skin epithelia. Nat Genet
41,1068-75 (2009).
22. Ezhkova E, Pasolli HA, Stokes N, Su I, Tarakhovsky A, Fuchs E. Polycomb protein Ezh2
balances proliferation and differentiation in developing epidermal stem cells. Cell
136,1122-1135 (2009).
23. Greco V, Chen T, Rendl M, Schober M, Pasolli HA, Stoke N, de la Cruz-Racelis J,
Fuchs E. A two step mechanism for stem cell activation during hair regeneration. Cell
Stem Cell, 4,155-169 (2009)

56

E. Fuchs

24. Horsley V., Aliprantis AO, Polak L., Glimcher LH, Fuchs, E. NFTAc1 balances quiescenceand proliferation of skin stem cells. Cell, 132, 299-310 (2008).
25. Guasch G, Schober M, Pasolli HA, Conn EB, Polak L, Fuchs E. Loss of TGF b signaling
destabilizes homeostasis and promotes squamous cell carcinomas in stratified epithelia.
Cancer Cell, 12,313-327 (2007).
26. Rhee H, Polak L, Fuchs E.Lhx2 maintains stem cell character in hair follicles. Science
312,1946-49 (2006).
27. Blanpain, C, Lowry W.E, Geoghegan A, Polak, L, Fuchs E. Self renewal, multipotency andthe existence of two cell populations within an epithelial stem cell niche. Cell 118, 635-648(2004).
28. Tumbar T, Guasch G, Greco V, Blanpain C, Lowry WE, Rendl M, Polak L, Fuchs E.
Defining the epithelial stem cell niche of the skin. [Science Express Dec 11, 2003] Science
303, 359-363 (2004).

Elaine Fuchs, PhD

1977-1980

1980-1985
1985-1988
1988-1993

1989-2002
1993-present
2002-present

Postdoctoral Fellow, Massachusetts Institute ofTechnology
Assistant Professor, Univ. of Chicago
Associate Professor, Univ. of Chicago
Associate Investigator, Howard Hughes Medical
Institute
Professor, Univ. of Chicago
Investigator, Howard Hughes Medical Institute
Professor, The Rockefeller Univ.

57

EVIDENCE AND MECHANISM FOR THE
TRANSDIFFERENTIATION FROM LUNG
ADENOCARCINOMA TO SQUAMOUS CELL CARCINOMA

Xiankun Han, Fuming Li, Yijun Gao, Wenjing Zhang, Fei Li,
Haiquan Chen and Hongbin Ji
CAS Key Laboratory of Systems Biology
CAS center for Excellence in Molecular Cell Science
Innovation Center for Cell Signaling Network
Institute of Biochemistry and Cell Biology
Shanghai Institutes for Biological Sciences
Chinese Academy of Sciences
320 Yue Yang Road, Room 711, Shanghai 200031, China(hbji@sibcb.ac.cn)

Lineage transition between lung adenocarcinoma (ADC) and lung squamous cell

carcinoma (SCC) as implicated by clinical observation of mixed adenomatous andsquamous pathologies in lung adenosquamous cell carcinoma (Ad-SCC), remains as afundamental and yet unsolved question. We provide the first in vivo evidence showing the

ADC to SCC transdifferentiation (AST) in three mouse models based on Lkb1 homozygous

deletion: the Kras/Lkb1 (KL) adeno-Cre nasal inhalation mouse model (Figure 1), theKras/Lkb1/Spc-CreERT2 mouse model as well as Kras/Lkb1 lung ADC serialtransplantation in nude mice [1]. In KL mouse model, we find that Lkb1-deficient lung

ADC can progressively transdifferentiate into SCC, via a pathologically mixed Ad-SCC

intermediate. Our data show that lysyl oxidase (Lox) plays an important role in regulating

the AST process. LOX is originally up-regulated by Lkb1 inactivation in mouse lung ADC,
which is known to promote collagen deposition, tumor malignant progression, and evenlung cancer metastasis [2]. However, LOX expression is significantly down-regulated

during the AST process, which results in the reduction of collagen deposition andextracellular matrix (ECM) remodeling. Pharmacological LOX inhibition promotes the AST
process, whereas ectopic Lox expression significantly inhibits this process [1].

We further find that decreased ECM potentially causes the nuclear exportation of YAP,

the major effector downstream of Hippo pathway, which in turn results in down-

regulation of ZEB2 expression [3]. Interestingly, ZEB2 is intrinsically activated to repress

58

H. Ji et al.

DNp63 transcription in lung ADC in a default manner. During the AST process, YAP isinactivated, which in turn relieves ZEB2-mediated default repression of DNp63 andtriggers squamous transdifferentiation. Our data show that overexpression of DNp63

Figure 1

Lkb1-deficient Lung ADC progressively Transdifferentiates into SCC.
(a) Representative histology for AAH, adenoma, ADC and SCC lesions in Kras/Lkb1 mice at a
serial of time points post Ad-Cre treatment. Scale bar, 150 μ m. (b and c) Quantification of
average tumor number (b) and tumor size (c) per mouse for ADC and SCC in Kras/Lkb1 miceat 6 weeks (n=6), 8 weeks (n=12) and 12 weeks (n=8) post Ad-Cre treatment. Data wereshown as mean ± s.e.m. *P <0.05, **P < 0.01, ***P < 0.001. (d) Quantification of individual
tumor size for ADC and SCC in Kras/Lkb1 mice at 10-12 weeks post Ad-Cre treatment. Data
were shown as mean ± s.e.m. *P <0.05, **P < 0.01, ***P < 0.001. (e) Representative mAd-SCC
shown with H&E staining, p63 and SP-C immunohistochemical staining on serial sections.
Scale bar: 150 μ m. (f) Quantification of tumor percentage for mAd-SCC and SCC in Kras/
Lkb1 mice at indicated time points post Ad-Cre treatment. n=8 for each time point. Scale bar:
150 μ m. Data were shown as mean ± s.e.m. *P <0.05, **P < 0.01, ***P < 0.001.

Solid Cancer Stem Cells

59

alone is able to partially drive the squamous transdifferentiation. YAP knockdown

significantly accelerates squamous transdifferentiation, whereas constitutive YAP

activation conversely inhibits this transition (Figure 2). More importantly, ectopic DNp63

expression rescues the inhibitory effect of YAP on squamous transdifferentiation. These

Figure 2

Lineage-specific YAP expression inhibits ADC to SCC transdifferentiation.
(a) Scheme of Ad-Cre treatment in established YKL mouse model. (b) Yap immunohistochemical staining in lung tumors from KL or YKL mice at 10wks post Ad-Cre treatment. Scalebar, 150 μ m. (c) Representative images for H&E and p63 immunohistochemical staining in
KL or YKL mice at 10wks post Ad-Cre treatment. Scale bar, 150 μ m. (d) Quantification of SCC
incidence of KL (n=9) or YKL mice from three SP-C-YAP transgenic lines including YKL-WT32# (n=7) , YKL-S127A-11# (n=6) and YKL-S127A-18# (n=7). (e-f) Tumor number (e) andtumor ratio (f) quantification for ADC and SCC from KL (n=9) or YKL mice from three SP-CYAP transgenic lines including YKL-WT-32# (n=7), YKL-S127A-11# (n=6) and YKL-S127A-18#(n=7). Data are shown as mean ± SEM.*, P <0.05; **, P <0.01.

60

H. Ji et al.

findings establish YAP as an essential barrier for lung cancer cell fate conversion.

It intrigues us that the downstream signaling triggered by LKB1 inactivation such as

LOX up-regulation and collagen deposition is conversely shut down during the AST

process. We find that the oxidative stress triggered by LKB1 inactivation significantly

contributes to the AST process and regulates cancer plasticity [4]. A differential reactive

oxygen species (ROS) level is observed in lung ADC and SCC from KL model. We find that
down-regulation of ROS level through N-acetyl cysteine (NAC) treatment or NRF2

expression inhibits the AST process, highlighting the functional importance of ROS in
regulating cancer plasticity. Our data further show that pentose phosphate pathwayderegulation and impaired fatty acid oxidation collectively contribute to the redox
imbalance and functionally affect the AST process. Similar tumor and redox heterogeneity

are also found in human LKB1-inactivated lung cancer. These data thus uncover a critical

redox control of tumor plasticity which contributes to the AST process through thesignaling reversal.

Interestingly, we find that phenformin treatment, known to effectively inhibit lung ADC

progression in KL model, promotes the AST process and results in the development ofdrug resistance [4]. This indicates that the AST process might represent a novel cellularmechanism for drug resistance. Our work in animal models is further supported by recent

clinical observation about the potential link between AST and drug resistance [5]. Up tonow, a total of 11 cases of EGFR-mutant lung ADC patients have been reported: all of them

are shown to have ADC from first biopsy and initially respond to tyrosine kinase inhibitor(TKI) or chemotherapy very well; however, after the relapse, the second biopsy shows thesquamous pathology frequently maintaining the same TKI-sensitive EGFR mutations [5].

With the efforts to overcome drug resistance conferred by AST, we have establishedover 20 patient derived xenograft (PDX) models in past years and found one with LKB1
inactivating mutation. This LKB1-mutant PDX model develops drug resistance towards
phenformin treatment, potentially through up-regulation of Wnt pathway. This leads us todevelop the combinational treatment of JW-55, the Wnt pathway inhibitor, together withphenformin. We find that this combinational treatment efficiently inhibits the LKB1mutant tumor growth in PDX model.

In summary, we have provided convincing evidence in supporting the AST in animalmodels. Our data have further provided important mechanistic insights into the process of
AST and indicated the AST might represent a novel mechanism for drug resistance.

Moreover, we have provided a potential therapeutic strategy for overcoming drug
resistance in linking to the AST process.

Solid Cancer Stem Cells

61

References
1. Han X, Li F, Fang Z, Gao Y, Li F, Fang R, Yao S, Sun Y, Li L, Zhang W, Ma H, Xiao Q, Ge
G, Fang J, Wang H, Zhang L, Wong KK, Chen H, Hou Y, Ji H*. Transdifferentiation ofLung Adenocarcinoma in mice with Lkb1 Deficiency to Squamous Cell Carcinoma. Nat
Commun, 2014;5:3261.
2. Gao Y, Xiao Q, Ma H, Li L, Liu J, Feng Y, Fang Z, Wu J, Han X, Zhang J, Sun Y, Wu G,
Padera R, Chen H, Wong KK, Ge G*, Ji H*. LKB1 inhibits lung cancer progression
through lysyl oxidase and extracellular matrix remodeling. Proc Natl Acad Sci USA.
2010, 107(44):18892-7.
3. Gao Y, Zhang W, Han X, Li F, Wang X, Wang R, Fang Z, Tong X, Yao S, Li F, Feng Y, Sun
Y, Hou Y, Yang Z, Guan K, Chen H, Zhang L*, Ji H*. YAP inhibits squamous transdifferentiation of Lkb1-deficient lung adenocarcinoma through ZEB2-dependent DNp63
repression. Nat Commun. 2014 Aug 13;5:4629.
4. Li F, Han X, Li F, Wang R, Wang H, Gao Y, Wang X, Fang Z, Zhang W, Yao S, Tong X,
Wang Y, Feng Y, Sun Y, Li Y, Wong KK, Zhai Q, Chen H*, Ji H*. LKB1 Inactivation
Elicits a Redox Imbalance to Modulate Non-small Cell Lung Cancer Plasticity andTherapeutic Response . Cancer Cell. 2015 27(5):698-711.
5. Hou S, Han X, Ji H*. Squamous Transition of Lung Adenocarcinoma and Drug
Resistance. Trends in Cancer. 2016.2 (9). doi:10.1016/j.trecan.2016.08.002

Hongbin Ji, PhD

1995-2000
2000-2004

2004-2007

2007-2007

2007- present

Graduated with Ph.D degree from Shanghai Institute of
Biochemistry and Cell Biology, Chinese Academy of
Sciences
Research associate, Beth Israel Deaconess Medical
Center, Harvard Medical School
Research fellow, Dana-Farber Cancer Institute, Harvard
Medical School
Instructor, Dana-Farber Cancer Institute, Harvard
Medical School
Professor, Shanghai Institute of Biochemistry and Cell
Biology, Chinese Academy of Sciences

62

DYNAMIC COMPLEXITY OF GLIOMA STEM CELLS

Jeremy N. Rich

Department of Stem Cell Biology and Regenerative Medicine
Lerner Research Institute, Cleveland Clinic
NE3-301, Lerner Research Institute, 9500 Euclid Ave, Cleveland, OH 44105, USA(richj@ccf.org)

Recent studies have redefined the designation of gliomas, based on molecularclassification using mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2), chromosome
1p and 19 co-deletion, and mutation of ATRX (Alpha Thalassemia/Mental Retardation

Syndrome X-Linked). Collectively, these mutations appear to resolve adult gliomas into

three different disease states: glioblastoma (wild-type for IDH1/2, with the worst

prognosis), astrocytomas (harboring mutations in IDH1 or IDH2 and ATRX, with anintermediate prognosis), and oligodendrogliomas (harboring mutations in IDH1 or IDH2

and co-deletion of chromosomes 1p and 19, with a relatively better prognosis). In pediatricgliomas, there have been additional driver mutations in histone variants 3.1 and 3.3, as well

as fusion events in BRAF. Collectively, there are now a series of distinct diseases within theoverall rubric of gliomas, which display differences in underlying biology, response totherapy, and prognosis. Within the most common tumor in adults, the glioblastoma, there

have been numerous attempts to better separate patients based on transcriptional profilingand other molecular events (e.g. MGMT promoter methylation), based these efforts have
had limited impact on clinical practice.

Against this background, it is useful to consider the role of the tumor cell hierarchy intumor biology and patient outcome. Gliomas and other brain cancers were among theearly cancer types for which cancer stem cells were defined. There have been bothsubstantial advances and challenges in the field. One area of (unnecessary) conflict has

been in the definition of the cell-of-origin in gliomas. Numerous genetically engineeredmouse models have shown that different cells may yield gliomas, based on moleculardrivers. While the eventual biology of tumors may reflect the cell-of-origin, it remains

Solid Cancer Stem Cells

63

unclear how these findings will be reflected in the tumor hierarchy. Glioblastomas

commonly contain a subset of tumor cells that are self-renewing, express stem cell markers,

and are tumorigenic when transplanted into recipient hosts. Many investigators have used

these criteria as sufficient to define cancer stem cells, but this fails to address the presenceof a hierarchy of tumor initiation. Prospective enrichment and depletion using markers has

been used, but is controversial due to the lack of uniform markers across all tumors, but

CD133 (Prominin1) has been the most widely used. Although not defining characteristics,

we and others have shown the brain tumor stem cells also exhibit a number of otherphenotypes that are clinically relevant, including invasion into normal tissues, promotionof tumor angiogenesis, and modulation of the immune system [1, 2]. Previously, we also

showed that glioma stem cells display a relative resistance to radiotherapy, the mosteffective non-surgical treatment for glioblastoma [3]. Our original report described amechanism linked to activation of the DNA damage checkpoint, but subsequent studies

from our lab and others has enriched these observations to include Notch activation, PARP

activation, and recruitment of meiotic repair mechanisms [3-7]. Therefore, glioma stem

cells appear to a potential important population for designing therapeutic paradigms to
improve the outcome of patients.

Glioblastomas, like most cancers, contain regions that are stressful to the inhabiting

cellular populations, due to the presence of hypoxic, acid, and limited nutrient availability.

We have previously shown that each of these aspects can both maintain glioma stem cells

and induce the acquisition of stem-like features in more differentiated progeny [8-11]. The

hypoxic response in glioma stem cells is accompanied by the specific induction of HIF2 atboth the transcriptional and post-translational regulatory levels, but tumor propagation

depends on the expression of the expression of both HIF1 and HIF2 [9]. Acidic stress is aneven stronger effector of cancer stem cell maintenance than hypoxia and also induces HIF2[10]. Glucose levels in standard cell culture conditions is designed to promote cellular
proliferation, but when we used physiologic levels of glucose (approximately 10% of

standard levels), we found that there was both a relative survival of the glioma stem cells

and an acquisition of stem-like features in differentiated tumor cells [11]. We hypothesizedthat glioma stem cells may have a competitive advantage in extracting glucose from themicroenvironment. We therefore differentially labeled glioma stem cells and differentiatedgliomas cells with fluorescent reporters, implanted them into brain slices and administeredfluorescent glucose. Glioma stem cells display a strong preferential uptake, which we

subsequently linked to the expression of GLUT3, a high affinity glucose transporter

normally expressed by neurons. Targeting GLUT3 expression depletes glioma stem cells

and GLUT3 expression is a negative prognostic factor for glioblastoma patients [11]. In
unpublished studies, we have performed glucose tracing experiments that have revealed a

64

J. N. Richstrong enrichment of de novo purine biosynthetic intermediates (data not shown). We

found that the entire pathway is up-regulated and expression of the components in thispathway portend a poor prognosis (data not shown). Selective uptake in glioma stem cells

extends beyond glucose, as we have found that transferrin, transferrin receptor and theferritins are preferentially expressed in glioma stem cells with strongly increased ironuptake and dependence on the uptake and storage of iron [12]. In additional studies ofglioma stem cell metabolism, we find that glioblastomas display strong transcriptional

regulation of enzymes that act to deplete the available methyl donors, with preferential

expression in the glioma stem cell population (data not shown). Targeting these pathways

attenuates tumor growth, whereas depleting the upstream source of methyl donors(methionine) accelerates tumor growth (data not shown). Finally, the overall structure of

the mitochondria differs between the stem and differentiated tumor cell populations.

Mitochondria in glioma stem cells are fragmented, whereas differentiated progeny containtubular mitochrondria [13]. This process is driven by differential post-translational
regulation of the essential mediator of mitochondrial fission, DRP1 (dynamin-related
protein 1). In glioma stem cells, DRP1 is activated by CDK5 (cyclin-dependent kinase 5),

whereas DRP1 is inhibited in differentiated tumor cells by CAMKII (Ca2+/calmodulin-

dependent protein kinase II) family members [13]. Genetic or pharmacologic targeting of

DRP1 disrupts glioma stem cell maintenance, whereas normal brain cells may be better able
to survival stress with DRP1 inhibition, suggesting that DRP1 may be a therapeutic target

with a high therapeutic index [13]. Collectively, these results and additional ongoingstudies suggest that glioma stem cells display differential metabolic behaviors that offer
potential nodes of fragility to be exploited by novel therapeutic paradigms.

Normal tissue-specific stem cells may reside in multiple niches, either because there are

multiple pools of stem cells or progeny with plasticity of differentiation state. In parallel, in

several studies we and others have found that brain tumor stem cells reside in at least two

distinct niches: a perivascular niche and a hypoxic niche in necrotic regions. In fact, wefind that necrotic regions offer the most effective areas for glioma stem cell derivation(unpublished observations). Vascular proliferation represents a histologic criterion forglioblastoma, and the initial discovery of VEGF (vascular endothelial growth factor) was inpart in glioblastoma. We previously demonstrated that glioma stem cells selectively
promote tumor angiogenesis due to increased VEGF secretion, which was validated byothers and extended to include CXCRL12 (C-X-C motif chemokine 12 (CXCL12), also

known as stromal cell-derived factor 1 (SDF1)) secretion [1]. Subsequently, other groups
have shown that glioma stem cells may contribute structurally to the tumor vasculature

through transdifferentiation towards endothelial cells. However, in collaboration with

Shideng Bao, we found in lineage tracing and targeting studies that glioma stem cells rarelySolid Cancer Stem Cells

65

transdifferentiate towards endothelial cells, but rather become pericytes through attraction

by CXCL12 and then fate transition due to TGF-beta (transforming growth factor-beta) [14].

Based on the recognition that the environment is critical to define glioma stem cellbiology, we have generated glioma organoids [15]. These organoids display structuralcomplexity with an outer region with proliferating cells and frequent SOX2-positive cells

and an inner region with little proliferation and fewer SOX2-positive cells. Radiation

treatment of organoids caused preferential cell death in the outer region with only SOX2-

negative cells undergoing cell death. Implantation of organoid-derived tumor cells into
immunocompromised mice recapitulates the histology of the original tumor from which
they were derived much better than matched tumorspheres grown in serum-free

conditions. Glioblastomas display regional variance in histology, radiographic appearance,

and therapeutic response. Therefore, we have derived tissues from different regions inpatient tumors, revealing distinct organoids with histology maintained upon implantation.

Based on the importance of the in vivo environment, we performed an inducible shRNA

screen for chromatin modifiers (manuscript under re-review) in matched orthotopic brain

tumors and identical cells grown under stem cell (sphere) conditions. Surprisingly, there

were fivefold more hits in vivo than in vitro, and the identity of the hits was almost
completely non-overlapping. The in vivo hits converged onto the enhancer-mediatedpause-release/elongation initiation complex. Matched RNA sequencing (RNA-seq)

revealed in vitro transcriptional profiles aligned with proliferation and metabolism in vitro,whereas in vivo profiles show evidence of cell-cell and microenvironmental interactions.

Super enhancer analysis reveals distinct regulation of critical loci based on growth

conditions and reveal selective dependencies of tumor cells in vivo. These studies raise thespecter that discovery efforts in vitro may not only miss critical tumor maintenance factors,

but may also identify targets that are not essential to in vivo tumor growth.

As noted above, glioblastomas are defined by angiogenesis and pseudopalisading

necrosis. In studies under revision, we demonstrate that these features are associated with

distinct transcriptional programs, with vascular regions showing a Proneural profile andhypoxic regions a Mesenchymal pattern. As these regions harbor glioma stem cells, weinvestigated the epigenetic regulation of these two niches. Proneural, perivascular glioma

stem cells activated EZH2 (Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit),

whereas Mesenchymal glioma stem cells in hypoxic regions expressed BMI1 (BMI1 Proto-

Oncogene, Polycomb Ring Finger) protein, which promoted cellular survival under stress.

Using both genetic and pharmacologic inhibition, we found that Proneural glioma stem

cells are selectively sensitive to EZH2 disruption, whereas Mesenchymal glioma stem cells

are sensitive to BMI1 inhibition. Given that glioblastomas contain both Proneural andMesenchymal glioma stem cells, combined EZH2 and BMI1 targeting proved more

66

J. N. Richeffective than either agent alone both in culture and in vivo, suggesting that strategies thatsimultaneously target multiple epigenetic regulators within glioblastomas may benecessary to overcome resistance to therapies caused by intratumoral heterogeneity.

In conclusion, brain tumor stem cells are important to critical tumor behaviors,

including therapeutic resistance, invasion, and immune modulation. They are regulated bycomplex interactions between the tumor hierarchy and the environment with integration of

signal transduction, metabolism, genetics, and the epigenetic cell state.
References

1. Bao S, Wu Q, Sathornsumetee S, Hao Y, Li Z, Hjelmeland AB, Shi Q, McLendon RE,
Bigner DD, Rich JN. Stem cell-like glioma cells promote tumor angiogenesis throughvascular endothelial growth factor. Cancer Res. 2006 Aug 15;66(16):7843-8.
2. Cheng L, Wu Q, Guryanova OA, Huang Z, Huang Q, Rich JN, Bao S. Elevated invasive
potential of glioblastoma stem cells. Biochem Biophys Res Commun. 2011 Mar
25;406(4):643-8.
3. Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, Dewhirst MW, Bigner DD,
Rich JN. Glioma stem cells promote radioresistance by preferential activation of the
DNA damage response. Nature. 2006 Dec 7;444(7120):756-60.
4. Wang J, Wakeman TP, Lathia JD, Hjelmeland AB, Wang XF, White RR, Rich JN,
Sullenger BA. Notch promotes radioresistance of glioma stem cells. Stem Cells. 2010
Jan;28(1):17-28.
5. Venere M, Hamerlik P, Wu Q, Rasmussen RD, Song LA, Vasanji A, Tenley N, Flavahan
WA, Hjelmeland AB, Bartek J, Rich JN. Therapeutic targeting of constitutive PARP activation compromises stem cell phenotype and survival of glioblastoma-initiating cells. Cell
Death Differ. 2014 Feb;21(2):258-69.
6. Rivera M, Wu Q, Hamerlik P, Hjelmeland AB, Bao S, Rich JN. Acquisition of meiotic DNA
repair regulators maintain genome stability in glioblastoma. Cell Death Dis. 2015 Apr
23;6:e1732.
7. Cheng L, Wu Q, Huang Z, Guryanova OA, Huang Q, Shou W, Rich JN, Bao S. L1CAM
regulates DNA damage checkpoint response of glioblastoma stem cells through NBS1.
EMBO J. 2011 Mar 2;30(5):800-13.
8. Heddleston JM, Li Z, McLendon RE, Hjelmeland AB, Rich JN. The hypoxic microenvironment maintains glioblastoma stem cells and promotes reprogramming towards acancer stem cell phenotype. Cell Cycle. 2009 Oct 15;8(20):3274-84.
9. Li Z, Bao S, Wu Q, Wang H, Eyler C, Sathornsumetee S, Shi Q, Cao Y, Lathia J,
McLendon RE, Hjelmeland AB, Rich JN. Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells. Cancer Cell. 2009 Jun 2;15(6):501-13.

Solid Cancer Stem Cells

67

10. Hjelmeland AB, Wu Q, Heddleston JM, Choudhary GS, MacSwords J, Lathia JD,
McLendon R, Lindner D, Sloan A, Rich JN. Acidic stress promotes a glioma stem cell phenotype. Cell Death Differ. 2011 May;18(5):829-40.
11. Flavahan WA, Wu Q, Hitomi M, Rahim N, Kim Y, Sloan AE, Weil RJ, Nakano I,
Sarkaria JN, Stringer BW, Day BW, Li M, Lathia JD, Rich JN, Hjelmeland AB. Braintumor initiating cells adapt to restricted nutrition through preferential glucose uptake. Nat
Neurosci. 2013 Oct;16(10):1373-82.
12. Schonberg DL, Miller TE, Wu Q, Flavahan WA, Das NK, Hale JS, Hubert CG, Mack SC,
Jarrar AM, Karl RT, Rosager AM, Nixon AM, Tesar PJ, Hamerlik P, Kristensen BW,
Horbinski C, Connor JR, Fox PL, Lathia JD, Rich JN. Preferential Iron Trafficking
Characterizes Glioblastoma Stem-like Cells. Cancer Cell. 2015 Oct 12;28(4):441-55.
13. Xie Q, Wu Q, Horbinski CM, Flavahan WA, Yang K, Zhou W, Dombrowski SM, Huang Z,
Fang X, Shi Y, Ferguson AN, Kashatus DF, Bao S, Rich JN. Mitochondrial control by
DRP1 in brain tumor initiating cells. Nat Neurosci. 2015 Apr;18(4):501-10.
14. Cheng L, Huang Z, Zhou W, Wu Q, Donnola S, Liu JK, Fang X, Sloan AE, Mao Y,
Lathia JD, Min W, McLendon RE, Rich JN, Bao S. Glioblastoma stem cells generate vascular pericytes to support vessel function and tumor growth. Cell. 2013 Mar
28;153(1):139-52.
15. Hubert CG, Rivera M, Spangler LC, Wu Q, Mack SC, Prager BC, Couce M, McLendon RE,
Sloan AE, Rich JN. A Three-Dimensional Organoid Culture System Derived from
Human Glioblastomas Recapitulates the Hypoxic Gradients and Cancer Stem Cell
Heterogeneity of Tumors Found In Vivo. Cancer Res. 2016 Apr 15;76(8):2465-77.

68

J. N. Rich

Jeremy N. Rich, MD, MHS, MBA

1989-1993
1993-1994

1994-1997
1996-1997
1997-1998
1998-1999
2000-2005
2003-2006

2005-2007

2005-20092006-2008

2007-2008

2008-present

2008-present

2008-present

2008-present
2008-present

2013-present
2014-2016

Medicine Doctor (MD), Duke Univ. School of Medicine
Resident, Internal Medicine, The Johns Hopkins
Hospital
Resident, Neurology, The Johns Hopkins Hospital
Chief Resident, Neurology, The Johns Hopkins Hospital
Fellow, Neuro-Oncology, Duke Univ. Medical Center
Associate, Medicine, Duke Univ. Medical Center
Assistant Professor, Medicine, Duke Univ. Medical
CenterAssistant Professor, Neurobiology, Duke Univ. Medical
Center
Associate Professor, Medicine, Duke Univ. Medical
CenterMasters in Health Science (MHS), Clinical ResearchTraining Program, Duke Univ. School of Medicine
Associate Professor, Pharmacology & Cancer Biology,
Duke Univ.
Associate Professor with Tenure, Medicine, Duke Univ.
Medical Center
Adjunct Associate Professor, Neurology, Duke Univ.
Medical Center
Chair and Staff, Stem Cell Biology and Regenerative
Medicine, Cleveland Clinic
Staff; Neurology, Taussig Cancer Center, & Brain
Tumor Center; Cleveland Clinic
Co-Director, National Center for Regenerative Medicine
Professor, Department of Molecular Medicine,
Cleveland Clinic Lerner College of Medicine at Case
Western Reserve Univ.
Director, Cleveland Clinic Brain Tumor Tissue Bank
Health Care Masters in Business Administration(MBA), Baldwin-Wallace Univ.

69

IDENTIFICATION OF CRITICAL DRIVERS OF TUMOR
MAINTENANCE THROUGH IN VIVO FUNCTIONALGENOMICS

Giulio F. Draetta

Institute for Applied Cancer Science
The University of Texas MD Anderson Cancer Center
Unit 1954, 1515 Holcombe Blvd. Houston, TX 77030, USA(gdraetta@mdanderson.org)

Despite truly significant advancements in recent years towards our understanding ofcancer pathogenesis and its underlying genomic complexity, our field is still plagued by aninability to improve around the development of truly effective therapeutic agents. Notable

exceptions are represented by the discovery of certain immune checkpoints-targetingtherapies and molecular targeted therapies that have provided long lasting responses insome disease types, but this certainly is not sufficient.

Several critically important factors have emerged, including the realization thatgenomic, and therefore, functional heterogeneity is a common feature of essentially any
cancer and that conquering the disease requires a better understanding of which cells

within a tumor may be sensitive or resistant to treatment. Rapid adaptation to therapy

because of cell populations within the tumor being resistant to treatment seems torepresent the rule more than an exception in many cases.

Figure 1 Towards functional elucidation of tumor heterogeneity

70

G. F. Draetta

Our laboratory at MD Anderson focuses on the identification of gene products essential

for tumor growth in vivo and on the elucidation of critical dependencies in selected tumor

cell subpopulations. One critical finding has been the discovery that in pancreatic cancer, a

subpopulation of tumor epithelial cells can survive KRAS inactivation, remain dormant in

the tumor and ultimately give rise to recurrence. These cells seem to carry characteristics ofcancer stem cells, being able to aggressively form tumors upon serial transplantation andcarrying surface markers that are characterizing cancer stem cells. Though several different

approaches we could determine that these cells are critically dependent on mitochondrial

function for their survival and that combination inhibition of KRAS signaling, througheither genetic or pharmacological intervention, and of mitochondrial function will resultsin dramatic inhibition of tumor growth [1]. This work has inspired the initiation of a drug

discovery project aimed at identifying potent and selective inhibitors of mitochondrial

oxidative phosphorylation. This compound, IACS-10759, has recently entered the clinic.

Figure 2 Tumor relapse depends on failure to eliminate cells surviving inhibition of KRAS dependent
signalingSolid Cancer Stem Cells

71

Figure 3 The mitochondrial OXPHOS inhibitor IACS-10759 is entering the clinic in multiple indications

Recently we have also discovered another critical vulnerability of pancreatic tumors.

Through in vivo mouse genetics studies and the clinical pathological assessment of humantissues, we have found that certain most aggressive pancreatic adenocarcinomas lose

expression of the chromatin-modifying protein SMARCB1, activate C-Myc and carryalterations of proteostasis and consequently, while being resistant to treatment with KRAS

signaling inhibitors, are sensitive to HSP90 and proteasome inhibitors, as well as to JNK

inhibitor treatment [2]. We plan to conduct clinical trials in this population.

We have also developed an unbiased strategy to identify potential drug discovery

targets in pancreatic adenocarcinoma, as well as in other tumors. Through functional invivo screens performed using both human tumors-derived tissue in xenografts as well as

mouse tumors in syngeneic hosts we have identified several candidate disease drivers andare pursuing the elucidation of their mechanism of action and their suitability as drug

discovery targets [3, 4]. In summary, using complementary genetics approaches, we hope
to continue to contribute to the identification and validation of mechanisms that keep
tumor cells alive and of novel agents that might conquer these devastating diseases.

72

G. F. Draetta

Figure 4 Development of an in vivo genetic screening platform to identify critical vulnerabilities in primary human tumor derived tissue

References
1. Viale A, Pettazzoni P, Lyssiotis CA, Ying H, Sánchez N, Marchesini M, Carugo, A,
Green T, Seth S, Giuliani V, Kost-Alimova M, Muller F, Colla S, Nezi L, Genovese G,
Deem AK, Kapoor A, Yao W, Brunetto E, Kang Y, Yuan M, Asara JM, Wang YA,
Heffernan TP, Kimmelman AC, Wang H, Fleming JB, Cantley LC, DePinho RA, Draetta
GF. Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function.
Nature. 2014 Oct 30;514(7524):628-32. doi:10.1038/nature13611. PubMed PMID:
25119024; PubMed Central PMCID: PMC4376130.
2. Genovese G, Carugo A, Tepper J, Robinson FS, Li L, Svelto M, Nezi L, Corti D, Minelli R,
Pettazzoni P, Gutschner T, Wu CC, Seth S, Akdemir KC, Leo E, Amin S,Molin MD,
Ying H, Kwong LN, Colla S, Takahashi K, Ghosh P, Giuliani V, Muller F, Dey P, Jiang S,
Garvey J, Liu CG, Zhang J, Heffernan TP, Toniatti C, Fleming JB, Goggins MG, Wood LD,
Sgambato A, Agaimy A, Maitra A, Roberts CW, Wang H, Viale A,DePinho RA, Draetta
GF, Chin L. Synthetic vulnerabilities of mesenchymalsubpopulations in pancreatic cancer.
Nature. 2017 Feb 8. doi:10.1038/nature21064. PubMed PMID: 28178232.

Solid Cancer Stem Cells

73

3. Carugo A, Genovese G, Seth S, Nezi L, Rose JL, Bossi D, Cicalese A, Shah PK, Viale A,
Pettazzoni PF, Akdemir KC, Bristow CA, Robinson FS, Tepper J, Sanchez N, Gupta S,
Estecio MR, Giuliani V, Dellino GI, Riva L, Yao W, Di Francesco ME, Green T, D'Alesio C,
Corti D, Kang Y, Jones P, Wang H, Fleming JB, Maitra A, Pelicci PG, Chin L, DePinho RA,
Lanfrancone L, Heffernan TP, Draetta GF. In Vivo Functional Platform Targeting
Patient-Derived Xenografts Identifies WDR5-Myc Association as a Critical Determinant of
Pancreatic Cancer. Cell Rep. 2016 Jun 28;16(1):133-47. doi: 10.1016/j.celrep.2016.05.063.
PubMed PMID: 27320920.
4. Bossi D, Cicalese A, Dellino GI, Luzi L, Riva L, D'Alesio C, Diaferia GR, Carugo A,
Cavallaro E, Piccioni R, Barberis M, Mazzarol G, Testori A, Punzi S, Pallavicini I, Tosti G,
Giacó L, Melloni G, Heffernan TP, Natoli G, Draetta GF, Minucci S, Pelicci P,
Lanfrancone L. In Vivo Genetic Screens of Patient-Derived Tumors Revealed
Unexpected Frailty of the Transformed Phenotype. Cancer Discov. 2016 Jun;6(6):650-63.
doi: 10.1158/2159-8290.CD-15-1200. PubMed PMID: 27179036.

Giulio F. Draetta, MD, PhD

1974-1981
1981-1985
1983-1989
1985-1989
1989-1992

1992-1995
1995-2004
1999-2004
2004-2008

2008-20112011-present

MD, Univ. of Naples
Graduate Diploma, Univ. of Naples
Fogarty Fellow, NCI, NIH
Robertson Fellow, Senior Staff Fellow, Cold Spring
Harbor Laboratory
Investigator, European Molecular Biology Laboratory(EMBL)
Vice-President, research, Miotix Inc.
Division Director, European Institute of Oncology
Executive Director and Site Head, Pharmacia Discovery
Research Europe
Vice-President and Worldwide basic Franchise Head,
Oncology, Merck Research Laboratories
Deputy Director, Belfer Institute for Applied Cancer
Science, and Chief Research Business Officer, DanaFarber Cancer Institute
Professor of Genomic Medicine and Director, Institutefor Applied Cancer Science, The Univ. of Texas MD
Anderson Cancer Center

74

CYP3A5 MEDIATES BASAL AND ACQUIRED THERAPYRESISTANCE IN DIFFERENT SUBTYPES OF PANCREATICDUCTAL ADENOCARCINOMA

Elisa M. Noll1,2, Martin R. Sprick1,3* and Andreas Trumpp1,2,3*

Heidelberg Institute for Stem Cell Technology and Experimental Medicine(HI-STEM gGmbH) , Heidelberg, Germany
2
Division of Stem Cells and Cancer
German Cancer Research Center (DKFZ) and DKFZ-ZMBH Alliance, Heidelberg, Germany
3German Cancer Consortium (DKTK), Heidelberg, Germany* joined last-authorship
Correspondence: a.trumpp@dkfz.de (A.T.), Martin.Sprick@hi-stem.de (M.R.S.)
1

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with dismal

prognosis[1]. In both Europe and the USA pancreatic cancer is the fourth leading cause ofcancer death[2,3]. Treatment with gemcitabine[4], FOLFIRINOX scheme[5] or the albumin-

paclitaxel conjugate nab-paclitaxel[6] only offer a modest increase in overall survival.

Despite extensive testing of targeted therapies in clinical trials, thus far all of the examinedcompounds confer little or no survival benefit in unselected cohorts of PDAC

patients[1,7,8]. Although patient stratification according to molecular characteristics has

not yet been performed in clinical trials for PDAC, molecular subtypes for PDAC wererecently described[9-12]. We established patient-derived models representing the full

spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-

like PDAC subtypes, and identified two markers—HNF1A and KRT81—that enablestratification of tumors into different subtypes by immunohistochemistry (Figure 1a, b)[13].

Patients bearing tumors of these subtypes show substantial differences in overall survival(Figure 1c)[13].

While resistance of PDAC to therapy is well described 1 , little is known about themolecular mechanisms mediating it. Members of the cytochrome P450 (CYP) enzyme

family have been previously only investigated with regard to a role in systemic drug
metabolism[14,15] or their up- or down-regulation in solid tumors compared to normal

tissues[16]. Thus, the functional role and impact of CYPs on tumor-cell autonomous drug

resistance remains largely unknown[16,17]. We could show that the three PDAC subtypes

significantly differ in drug sensitivity, with the exocrine-like subtype being resistant totyrosine kinase inhibitors and paclitaxel[13]. The xenobiotic biotransformation enzyme,

Solid Cancer Stem CellsaPatient
PDAC

d

KRT81

HNF1A

KRT81

HNF1A

Serum-free
culture (PACO)

c

PACO derived
tumor (DT)

KRT81

HNF1A

DN
(Classical)

HNF1A+(Exocrine)

KRT81+(QM-PDA)Classical (PACO2) Exocrine (PACO10) QM-PDA (PACO7)b

1° Xenograft
(PT)

75

1.0

HNF1A+ (Exocrine)
DN (Classical)

Fraction survival

0.8

KRT81+ (QM-PDA)

0.6

0.4

0.2

0.0
0

20

40

60

80

100

120

Time after surgey (Months)

Figure 1 Subtype stratification of PDAC models and patients by two markers[13].

(a) Schematic overview of the experimental workflow used to generate orthotopic
xenografts and PACO cells. H&E staining of a human PDAC tumor, the corresponding
first passage xenograft (PT), phase contrast image of the derived cell line (PACO10) and therespective derived xenograft (DT). Scale bar, 100 μM. (b) KRT81 and HNF1A immunofluorescence staining on PACO lines from the three different subtypes (n = 3). Scale bar, 50 μM.
(c) KRT81 and HNF1A immunostaining on sections from a TMA of individuals withPDAC (n = 241). Scale bar, 100 μM. (d) Kaplan-Meier analysis of overall survival of subjects
with PDAC (n = 217). Tumor sections on the TMA were retrospectively subtyped into
three groups based on KRT81 and HNF1A expression as determined by immunostaining(HNF1A+: n = 46; DN: n = 92; KRT81+: n = 79). P value was determined by log-rank test.

76

A. Trumpp et al.

cytochrome P450 3A5 (CYP3A5), metabolizes these compounds in tumor cells of theexocrine-like subtype, and pharmacological or small hairpin RNA (shRNA)-mediated
CYP3A5 inhibition sensitizes tumor cells to these drugs (Figure 2)[13]. Additionally,

retrospective analysis of a large patient cohort confirmed that CYP3A5 is predominantly

found in those patient tumors classified as exocrine-like[13]. Whereas the hepatocyte
nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced

CYP3A5 upregulation is mediated by the nuclear receptor NR1I2[13]. Interfering withthese regulatory mechanisms may provide an alternative approach to suppress thisCYP3A5 mediated resistance pathway. CYP3A5 also contributes to acquired drug

400

shCYP3A5 + erlotinib

n.s.

200

100

**

0

2

2,500

4

6

8

Time (d)

10

12

14

1,500

shScr + vehicle
shScr + paclitaxel

2,000

shCYP3A5 + vehicle
shCYP3A5 + paclitaxel

1,500
n.s.
1,000

500

0

Figure 2

*
0

2

4

6

8

10

Time (d)

12

14

16

18

Exocrine (PACO14) / round II

tumor volume normalized to day 0 (%)

Exocrine (PACO14) / round I

shCYP3A5 + vehicle

300

0

b

shScr + vehicle
shScr + erlotinib

tumor volume normalized to day 0 (%)

tumor volume normalized to day 0 (%)

Exocrine (PACO14)ashScr + vehicle
shScr + paclitaxel
shCYP3A5 + vehicle
shCYP3A5 + paclitaxel

1,000

n.s.

500

0

**
0

2

4

6

8

Time (d)

10

12

14

CYP3A5 mediates drug resistance in exocrine-like PDAC cells in vivo[13].
(a) Growth curves of PDAC xenografts from exocrine-like shScr and shCYP3A5 cells, treated
for two cycles of 5 days with erlotinib (100 mg/kg) and 2 days recovery. (b) Growth curves of
PDAC xenografts from exocrine-like shScr and shCYP3A5 cells treated with two cycles of 5days paclitaxel (2 mg/kg) and 2 days recovery followed by 4 days of paclitaxel (left panel,round I). Cells from one xenograft per treatment group were re-injected and treated fortwo cycles of 5 days paclitaxel and 2 days recovery (right panel, round II). Tumor volume
was measured with a digital caliper. Shown are tumor volumes normalized to baseline(day 0) and depict mean ± SEM. P values were determined at the end point using onesided Mann-Whitney U test. (n = 6 mice per treatment group; *P < 0.05; **P < 0.01; n.s. = notsignificant).

Solid Cancer Stem Cells

77

resistance in some QM-PDA and classical PDAC cases. In other cases CYP3A5independent pathways mediate acquired drug resistance in QM-PDA and classical cells.

However, the mechanistic details of the pathways mediating the observed resistance are

yet to be determined. To note, CYP3A5 is also highly expressed in several additional
malignancies[13]. Taken together, these findings designate CYP3A5 as predictor of therapy

response and as a tumor cell-autonomous detoxification mechanism that must be overcome

to prevent drug resistance (Figure 3).

Figure 3

CYP3A5 mediates basal and acquired drug resistance in different subtypes of PDAC.
Patient-derived models of pancreatic ductal adenocarcinoma (PDAC) represent the full
spectrum of previously identified quasi-mesenchymal (QM-PDA), exocrine-like and classicalsubtypes. According to the expression of the surrogate markers HNF1A and KRT81, PDAC
models and tumors can be stratified into three distinct biological groups, which areenriched in the respective PDAssigner genes{Collisson, 2011 #5}. These subtypes significantly differ in drug sensitivity; with the exocrine-like subtype being resistant to the small
molecule drugs erlotinib, dasatinib and paclitaxel. The observed resistance can be attributed
to the cytochrome P450 3A5 (CYP3A5) enzyme, which actively metabolizes and detoxifies
these small molecule drugs. Pharmacological or short hairpin RNA (shRNA)-mediated
CYP3A5 inhibition sensitizes tumor cells to erlotinib, dasatinib and paclitaxel in vitro and invivo. Hence, CYP3A5, previously mainly implicated in systemic drug metabolism in hepatocytes, plays a critical role in mediating resistance to various small molecule drugs inexocrine-like tumor cells. Additionally, CYP3A5 contributes to acquired drug resistance insome cases of the QM-PDA and classical PDAC subtype after long-term treatment withsmall molecule drugs. Also CYP3A5-independent pathways mediate acquired drug resistance in QM-PDA and classical cells. However, the mechanistic details of the pathways
mediating the observed resistance are yet to be determined. Taken together, CYP3A5 not only
mediates drug resistance in the exocrine-like subtype, but also contributes to acquired
drug resistance in the some cases of the QM-PDA and classical PDAC subtypes.

78

A. Trumpp et al.

This work was supported by the German Bundesministerium für Bildung und

Acknowledgements

Forschung (BMBF) funded e:Med consortium “PANC-STRAT” (grants 01ZX1305B,

01ZX1305C), the Helmholtz pre-clinical comprehensive cancer center (PCCC) and theDietmar Hopp Foundation.

References
1. Hidalgo, M. Pancreatic cancer. The New England journal of medicine 362, 1605-1617 (2010).
2. Malvezzi, M., Bertuccio, P., Levi, F., La Vecchia, C. & Negri, E. European cancer mortality predictions for the year 2014. Annals of oncology : official journal of the European Society for
Medical Oncology / ESMO (2014).
3. Siegel, R.L., Miller, K.D. & Jemal, A. Cancer statistics, 2016. CA: a cancer journal for clinicians 66, 7-30 (2016).
4. Burris, H.A., 3rd, et al. Improvements in survival and clinical benefit with gemcitabine as
first-line therapy for patients with advanced pancreas cancer: a randomized trial.
Journal of clinical oncology : official journal of the American Society of Clinical Oncology 15, 24032413 (1997).
5. Conroy, T., et al. FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. The
New England journal of medicine 364, 1817-1825 (2011).
6. Von Hoff, D.D., et al. Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine. The New England journal of medicine 369, 1691-1703 (2013).
7. Vincent, A., Herman, J., Schulick, R., Hruban, R.H. & Goggins, M. Pancreatic cancer.
Lancet 378, 607-620 (2011).
8. Moore, M.J., et al. Erlotinib plus gemcitabine compared with gemcitabine alone inpatients with advanced pancreatic cancer: a phase III trial of the National Cancer
Institute of Canada Clinical Trials Group. Journal of clinical oncology : official journal of theAmerican Society of Clinical Oncology 25, 1960-1966 (2007).
9. Biankin, A.V. & Maitra, A. Subtyping Pancreatic Cancer. Cancer Cell 28, 411-413 (2015).
10. Collisson, E.A., et al. Subtypes of pancreatic ductal adenocarcinoma and their differing
responses to therapy. Nat. Med. 17, 500-U140 (2011).
11. Moffitt, R.A., et al. Virtual microdissection identifies distinct tumor- and stroma-specificsubtypes of pancreatic ductal adenocarcinoma. Nature genetics 47, 1168-1178 (2015).
12. Bailey, P., et al. Genomic analyses identify molecular subtypes of pancreatic cancer.
Nature 531, 47-52 (2016).
13. Noll, E.M., et al. CYP3A5 mediates basal and acquired therapy resistance in different
subtypes of pancreatic ductal adenocarcinoma. Nat. Med. 22, 278-287 (2016).

Solid Cancer Stem Cells

79

14. Guengrich, F.P. Cytochrome P450 Enzymes. in Comprehensive Toxicology (Second
Edition), Vol. 9 (ed. McQueen, C.A.) 43-76 (Elesvier Ltd., 2010).
15. Rochat, B. Role of cytochrome P450 activity in the fate of anticancer agents and in drug
resistance: focus on tamoxifen, paclitaxel and imatinib metabolism. Clinical pharmacokinetics 44, 349-366 (2005).
16. Bruno, R.D. & Njar, V.C.O. Targeting cytochrome P450 enzymes: A new approach in anticancer drug development. Bioorganic & Medicinal Chemistry 15, 5047-5060 (2007).
17. Michael, M. & Doherty, M.M. Drug metabolism by tumours: its nature, relevance andtherapeutic implications. Expert Opinion on Drug Metabolism & Toxicology 3, 783-803(2007).

Andreas Trumpp, PhD

1993-1993

1994-2000
2000-2008

2005-2008
2006-present
2008-present

2008-present

Postdoctoral research, European Molecular Biology
Laboratory (EMBL), with Dr. Rolf Zeller
Postdoctoral research, Univ. of California San Francisco
in the Laboratories of Prof. J. Michael Bishop and Prof.
Gail R. Martin
Head, “Genetics and Stem Cell Laboratory” at the Swiss
Institute for Experimental Cancer Research (ISREC) in
Epalinges/Lausanne, Switzerland
Professor (PATT) for Molecular Oncology and Stem
Cell Biology, Ecole Polytechnique Fédérale deLausanne (EPFL)
Coordinator of the Research Program “Cell andTumorbiology, Deutsches Krebsforschungszentrum(DKFZ)
Professor and Head of the Division of Stem Cell andCancer, DKFZManaging Director, Heidelberg Institute for Stem Cell
Technology and Experimental Medicine (HI-STEM
gGmbH)

80

TRANSDIFFERENTIATION APPROACH FOR TARGETINGCANCER STEM CELLS

Hideyuki Saya

Division of Gene Regulation, Institute for Advanced Medical Research (IAMR)
School of Medicine, Keio University
35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan(hsaya@a5.keio.jp)

Cancer stem cells (CSCs) are a subset of tumor cells that are responsible for initiating

and maintaining the disease. In the clinical point of view, the most importantcharacteristics of CSCs include their resistance to various therapeutic interventions.

However, the underlying mechanisms of the resistance remain unclear. To address thisquestion, we established a mouse osteosarcoma (OS) model by overexpressing c-MYC inbone marrow stromal cells (BMSCs) derived from Ink4a/Arf (-/-) mice (Figure 1) [1]. Single-

cell cloning revealed that c-MYC-expressing BMSCs are composed of two distinctly

c-MYC

BM
Bone marrowstromal cells(BMSC)C57BL/6
Ink4a/Arf KO

IP
inoculation

C57BL/6/XQJ

C
Osteo.

V

Vessel/LYHU

Figure 1

Chondro.

Development of mouse osteosarcoma cells. c-MYC gene was introduced into the mousebone marrow stromal cells (BMSCs) derived from Ink4a/Arf knock-out C57BL/6 mice byretroviral transfer, and we enriched infected cells positive for green fluorescent protein(GFP). GFP positive cells were inoculated in syngeneic C57BL/6 mice. The pathologicalanalysis of the tumor developed after the inoculation shows heterogenous tumor cell composition. All mice inoculated the tumor cells had metastasis of osteosarcoma cells in lung andliver.

Tumor Heterogeneity81

different clones: highly tumorigenic cells (termed AX cells), similar to bipotent-committed

osteochondral progenitor cells, and low-tumorigenic tripotent cells (termed AO cells),

similar to mesenchymal stem cells. We found that the loss of adipogenic potential is an
essential event for AO cells to become AX-like tumorigenic cells. Although AO cells are nottumorigenic as far as they maintain the adipogenic differentiation potential, AO cells were

found to be highly resistant to chemotherapeutic agents such as Adriamycin. Then, AO

cells survived after the chemotherapy eventually change to AX-like cells by losing theiradipogenic differentiation potential to become tumorigenic. Therefore, our understandingof regulatory mechanisms of adipocyte differentiation would greatly contribute to control

OS tumorigenesis.

We have recently found a novel regulatory mechanism of adipocyte differentiation.

Regulation of the transcriptional coactivator MKL1 (megakaryoblastic leukemia 1) by actin

cytoskeleton dynamics drives adipocyte differentiation mediated by peroxisome

proliferator–activated receptor γ (PPAR γ ), a master transcriptional regulator of
adipogenesis [2]. We found that disruption of actin stress fibers through the inactivation of

RhoA-ROCK signaling induces the rapid increase in monomeric G-actin, leading to theinteraction of G-actin with MKL1, which prevents nuclear translocation of MKL1 andallows expression of PPAR γ followed by adipogenic differentiation (Figure 2). Ourfindings thus provide new mechanistic insight into the relation between the actin dynamics

and transcriptional regulation during cellular differentiation.

Based on this discovery, we attempted to induce adipocyte differentiation in OS stem

cells by treatment with Rho kinase inhibitors. Rho kinase inhibitors induced adipocyte

differentiation of OS stem cells and significantly suppressed their in vitro growth and invivo tumorigenesis. Our findings suggest that induction of trans-differentiation of cancer

F-actin

G-actinActin depolymerizer
Rho/ROCK inhibitor
MKL1

nucleusMKL1X

PPARJJ

Figure 2

ON

OFF

X

PPARJJ

Mechanism of adipocyte differentiation driven by actin cytoskeleton dynamics.

82

H. Sayastem cells by regulating actin cytoskeleton dynamics is a potential approach for sometumor types.

References
1. Shimizu T, Ishikawa T, Sugihara E, Kuninaka S, Miyamoto T, Mabuchi Y, Matsuzaki Y,
Tsunoda T, Miya F, Morioka H, Nakayama R, Kobayashi E, Toyama Y, Kawai A,
Ichikawa H, Hasegawa T, Okada S, Ito T, Ikeda Y, Suda T, and Saya H: c-MYC overexpression with loss of Ink4a/Arf transforms bone marrow stromal cells into osteosarcoma
accompanied by loss of adipogenesis. Oncogene 29: 5687-5699, 2010
2. Nobusue H, Onishi N, Shimizu T, Sugihara E, Oki Y, Sumikawa Y, Chiyoda T, Akashi K,
Saya H and Kano K: Regulation of MKL1 via actin cytoskeleton dynamics drives
adipocyte differentiation. Nat Commun 5: 3368, 2014

Hideyuki Saya, MD, PhD

1987-1988

1988-1994

1994-2006

2007-present

2015-present

Postdoctoral Fellow, Brain Tumor Research Center,
University of California, San Francisco
Assistant Professor, Department of Neuro-Oncology
M.D. Anderson Cancer Center
Professor and Chairman, Department of Tumor
Genetics and Biology, Kumamoto Univ. School of
Medicine
Professor, Institute for Advanced Medical Research
Keio Univ. School of Medicine
Director, Clinical and Translational Research Center
Keio Univ. Hospital

83

MECHANISMS CONTROLLING TUMOR HETEROGENEITY

Cédric Blanpain

WELBIO, Université Libre de Bruxelles, IRIBHM
808 Route de Lennik – Campus Erasme, 1070 Bruxelles, Belgium(Cedric.blanpain@ulb.ac.be)

For the vast majority of cancers, the cell at the origin of tumour initiation is still

unknown [1]. Here, we used mouse genetics to identify cells at the origin of basal cell

carcinoma (BCC), which is one of the most frequent types of cancer in human and resultsfrom the activation of hedgehog signalling pathway [2]. Using mice conditionallyexpressing constitutively active Smoothened mutant (SmoM2), we activated hedgehog

signalling in different cellular compartments of the skin epidermis, and determined inwhich epidermal compartments hedgehog activation induces BCC formation. Activationof SmoM2 in hair follicle bulge stem cells (SCs) and their transient amplifying progenies

did not induce cancer formation, demonstrating that BCC do not originate from bulge SC

as previously thought. Using clonal analysis, we found that BCC arise from long-term
resident cells of the interfollicular epidermis and the upper infundibulum. Our studies

uncover the cells at the origin of BCC in mice and demonstrate that expression ofdifferentiation markers in tumour cells is not necessary predictive of the cancer initiating
cells [3, 4].

The skin interfollicular epidermis (IFE) is the first barrier against the external

environment and its maintenance is critical for survival [5]. Quantitative analysis of clonalfate data and proliferation dynamics demonstrate the existence of two distinct proliferativecell compartments composed of slow-cycling SC and committed progenitors (CPs), both ofwhich undergo population asymmetric self-renewal. However, following wounding, only

SCs contribute substantially to the repair and long-term regeneration of the tissue, while
CP cells make a minimal and transient contribution [6].

84

C. Blanpain

The changes that occur in cell dynamics following oncogenic mutation leading to thedevelopment of invasive tumors are currently unknown. Here, using skin epidermis as amodel, we assessed the impact of oncogenic Hedgehog (HH) signalling in distinct cell

populations and their capacity to induce basal cell carcinoma (BCC), the most frequentcancer in humans. We found that only K14-CreER targeted Stem Cells (SCs), and not Inv-

CreER targeted committed progenitors (CPs), were competent to initiate tumor formation

upon oncogenic HH signalling in the different skin regions. Interestingly, this difference

was due to the hierarchical organization of the tumor growth in oncogene targeted SCs,characterized by increased symmetric self-renewing division, but also to a higher resistanceof oncogene-targeted SCs to apoptosis and growth arrest compared to oncogene-targeted

CP. Combined, these properties induced a much more rapid cell expansion in oncogene-targeted SCs, allowing them to progress to BCCs. Our work reveals that the capacity of

oncogene-targeted cells to induce tumor formation is not only dependent on the long term

survival and increased fitness of these cells, but also depends on the specific clonal
dynamics of the cancer cell of origin [7] (Figure 1).

Figure 1

Stem cells but not progenitors are competent to initiate BCC formation
Activation of oncogene in SCs leads to the generation of BCC due to an increase in symmetric renewal and resistance to apoptosis. However, activation of p53 in SmoM2-expressing CPs
restricts the progression of dysplastic clones to BCC by promoting apoptosis and cell cycle
arrest. Deletion of p53 in CPs allows them to progress into BCC.

Tumor Heterogeneity

85

Squamous cell carcinoma (SCC) represents the second most frequent skin cancer. The

cellular origin of SCC remains controversial [1]. Here, we used mouse genetics todetermine the epidermal cell lineages at the origin of SCC. Using mice conditionallyexpressing a constitutively active KRas mutant (G12D) and an inducible CRE in different

epidermal lineages, we activated Ras signaling in different cellular compartments of theskin epidermis, and determined from which epidermal compartments Ras activationinduces squamous tumor formation. Expression of mutant KRas in hair follicle bulge stem

cells (SCs) and their immediate progeny (hair germ and outer root sheath), but not in theirtransient amplifying matrix cells, led to benign squamous skin tumor (papilloma).

Expression of KRas G12D in interfollicular epidermis also led to papilloma formation,
demonstrating that squamous tumor initiation is not restricted to the hair follicle lineages.

Whereas no malignant tumor was observed following KRasG12D expression alone, expressionof KRasG12D combined with the loss of p53 induced invasive SCC. Our studies demonstratethat different epidermal lineages including bulge SC are competent to initiate papilloma

formation and that multiple genetic hits in the context of oncogenic KRas are required forthe development of invasive SCC [8].

Epithelial to mesenchymal transition (EMT) in cancer cells has been associated withmetastasis, tumor stemness and resistance to therapy. Here, we developed geneticallyengineered mouse models allowing lineage tracing and activation of the same oncogenic

hits in different epidermal lineages, and assessed whether the cancer cell of origin controls

EMT in mouse skin squamous cell carcinoma (SCC). Surprisingly, while SCCs arising fromthe interfollicular epidermis consisted essentially of well-differentiated tumors, SCCs that

arose from hair follicle lineages exhibited very frequently EMT. Transplantation assays

showed the intrinsic priming of HF derived tumor epithelial cells to undergo EMT.

Transcriptional and chromatin profiling of the different tumor cell populations and theircell of origin unravelled the changes in the chromatin landscape and gene regulatory

network associated with tumorigenesis and EMT and demonstrated the importance of

epigenetic and transcriptional priming of the cancer cell of origin to undergo EMT upon
oncogenic transformation [9] (Figure 2).

86

C. Blanpain

Figure 2

The cancer cell of origin primes tumour-initiating cells to undergo EMT
Squamous cell carcinomas (SCCs) derived from interfollicular epidermis (IFE) are generally
well-differentiated, while hair follicle (HF) stem cell-derived SCCs frequently exhibit EMT.
Transcriptional and chromatin profiling revealed IFE and HF tumor-initiating cells possess
distinct epigenetic landscapes and gene regulatory networks that prime the cancer cell of origin to undergo EMT during tumorigenesis.

References
1. Blanpain C (2013) Tracing the cellular origin of cancer. Nat Cell Biol.
2. Epstein EH (2008) Basal cell carcinomas: attack of the hedgehog. Nat Rev Cancer
8(10):743-754.
3. Kass Youseff K, Van Keymeulen A, Lapouge G, Beck B, Achouri Y, Michaux C,,
Sotiropoulou P and Blanpain C. (2010) Identification of the cell lineage at the origin ofbasal cell carcinoma. Nat Cell Biol 12(3):299-305.

Tumor Heterogeneity87

4. Youssef KK, Lapouge G, Bouvrée K, Rorive S, Brohée S, Appelstein O, Larsimont JC,
Sukumaran V, Van de Sande B, Pucci D, Dekoninck S, Berthe JV, Aerts S, Salmon I, Del
Marmol V, Blanpain C. (2012) Adult interfollicular tumour-initiating cells are reprogrammed into an embryonic hair follicle progenitor-like fate during basal cell carcinoma
initiation. Nat Cell Biol 14(12):1282-1294.
5. Blanpain C & Fuchs E (2009) Epidermal homeostasis: a balancing act of stem cells in theskin. Nat Rev Mol Cell Biol 10(3):207-217.
6. Mascré G, Dekoninck S, Drogat B, Youssef Kk, Brohée S, Sotiropoulou P, Simons BD, andBlanpain C. (2012) Distinct contribution of stem and progenitor cells to epidermal
maintenance. Nature 489(7415):257-262.
7. Sánchez-Danés A, Hannezo E, Larsimont JC, Liagre M, Youssef KK, Simons B, Blanpain
C. (2016) Defining the clonal dynamics leading to mouse skin tumour initiation. Nature
536(7616):298-303.
8. Lapouge G, Kass Youssef K, Vokaer B, Achouri Y, Michaux C, Sotiropoulou PA,
Blanpain C. (2011) Identifying the cellular origin of squamous skin tumors. Proceedings of
the National Academy of Sciences of the United States of America 108(18):7431-7436.
9. Latil M, Nassar D, Beck B, Boumahdi S, Wang L, Brisebarre A, Dubois C, Nkusi E,
Lenglez S, Checinska A, Vercauteren Drubbel A, Devos M, Declercq W, Yi R, Blanpain C.
(2016) Cell-Type-Specific Chromatin States Differentially Prime Squamous Cell
Carcinoma Tumor-Initiating Cells for Epithelial to Mesenchymal Transition. Cell Stem Cellin press.

Cédric Blanpain, MD, PhD

1995-97

1997-2001

2001
2001-2002
2002
2002-2006

2006-20122012-20132013-present

Residency in Internal Medicine, Hôpital Erasme,
Université Libre de Bruxelles (ULB)
Research Fellow of the Belgian National Research
Scientific Fund (F.N.R.S.) at the Interdisciplinary
Research Institute (IRIBHM), Medical School, ULB
PhD in Medical Science, ULB
Residency in Medical Genetics, Hôpital Erasme, ULB
Board Certified Internal Medicine, ULB
Post-doctoral fellow in the Laboratory of Elaine Fuchs,
The Rockefeller University
Tenure Assistant Professor and independent groupleader of the Belgian Research National Scientific Fund
(F.N.R.S.) at the Interdisciplinary Research Institute(IRIBHM), ULB
Full professor, ULB
Professor, ULB

88

NEW INSIGHTS INTO CELLULAR PLASTICITY:TARGETING METAPLASTIC CANCERS

Thea D. Tlsty, Somdutta Roy, Philippe Gascard, Tony Caruso and Deng Pan
Department of Pathology
University of California, San Francisco, School of Medicine
513 Parnassus Avenue, Room HSW 513, Box 0511, San Francisco, CA 94143, USA(thea.tlsty@ucsf.edu)

Cellular plasticity is a universal and fundamental biological process that generates non-Cellular Plasticity

genetic variation and allows cells to explore multiple phenotypic states. Embryonic stem

cells (ESCs) exhibit cellular plasticity during differentiation. Ensuing epigenetic changes

then dictate cell fate and generate differentiated progeny. Cellular plasticity can also be
activated by cellular stress responses that are initiated upon wound healing. Current

research is now indicating that one population of regenerative cells responds tohomeostatic cues and keeps tissue in balance while an independent population of cells

responds to wounding cues and regenerates functional tissue after damage [1-3]. In areasof chronic wounding, caused by repeated irritation, injury or inflammation, cells respondby generating metaplastic tissues. Metaplastic tissues are healthy “normal” tissues that areformed in inappropriate sites. For example, exposure of esophageal tissue to repeated acidreflux injures the squamous esophageal cells which are then replaced by intestinal tissue[4]. The intestinal tissue is properly differentiated to secrete protective mucins from goblet

cells thereby protecting the underlying cells from acid damage. The origin of the cells thatgive rise to metaplastic tissues is unknown. The extent of cellular plasticity isunderappreciated as a mechanism to generate tumor heterogeneity independently ofgenetic mutations. Cellular plasticity allows the adoption of altered cellular states that canfuel metastasis (epithelial to mesenchymal transition; EMT) as well as drug resistance(persister or drug-tolerant cells) [5].

Interestingly, cellular plasticity is activated by conditions known to promotemalignancy and in places where malignancy often arises, such as areas of chronicTumor Heterogeneity

89

wounding. The cells that exhibit plasticity must be able to bypass proliferative arrest that is

induced by negative growth signals as well as exhibit multi-lineage potential. Here we
describe our studies to isolate such cells and characterize a rare population of human cells

that exhibit cellular plasticity under stress conditions.

The majority of cells within an injured tissue activate cell cycle checkpoint controls andRare Cells Can Bypass Negative Growth or Injury Signals and Exhibit Cellular Plasticity

proliferative arrest until resolution of cellular damage releases this inhibition or apoptosis

removes these cells from the injured field [6]. Only cells with compromised cell cycle

checkpoints can continue to proliferate in the presence of damage signals. We reasoned

that cells exhibiting plasticity would reveal or unveil themselves under stress conditions
and make their evaluation possible. This proved to be correct.

Evaluation of primary isolates of disease-free human breast tissue unmasked a cell

population which, when placed in culture conditions that activate stress responses, exhibits

cellular plasticity. The major population, human mammary epithelial cells (HMEC), is

composed of luminal and myoepithelial cells. These cells undergo cell cycle arrest when

they encounter stress conditions and remain in a static state for an extended period of time.

Strikingly, a rare subpopulation of cells could be identified by a reversible repression of p16

transcription [7]. Realizing that repression of p16 activity was a defining characteristic ofpluripotent and adult stem cell populations [8], we evaluated this population of cells fortheir ability to exhibit stem cell traits or plasticity. Pluripotent cells (human embryonic stem

cells (hESCs), induced Pluripotent Stem Cells (iPSCs)) and adult tissue-specific stem cells

have the potential to self-renew and generate multiple lineage derivatives. Surprisingly,

we found that the rare population of p16-repressed cells we had identified had the ability
to self-renew and generate mature cells of all three embryonic lineages (Figure 1), far

exceeding the plasticity of tissue-specific cell types that one might have expected [7].

Further study of these endogenous Plastic Somatic (ePS) cells revealed that, when

activated, they expressed canonical pluripotent factors including co-incident expression of
Sox2, Oct3/4 and Nanog (SON) and existed in multiple tissues of the adult human body.

We identified cell surface markers and growth conditions that allowed prospectiveenrichment and isolation of these rare ePS cells. Rare cells (0.15% of breast parenchyma),
expressing NT5E/CD73 and EpCAM but devoid of expression of THY1/CD90, wereextensively characterized. We showed that ePS cells coincidently express SON within the

nucleus when in their activated state at levels similar to those measured for hESCs (Figure

2 and [7]) and, when placed in differentiation conditions, can generate glial and neuronalcells, bone, cartilage, adipocytes, cardiomyocytes, endothelial, pancreatic and intestinal

cells (Figure 1). When SON is expressed, ePS cells demonstrate (a) pluripotency without

90

T. D. Tlsty et al.

ePS

Figure 1

breastcartilage

Characterization of endogenous Plastic Somatic Cells

intestine

culture in vitro, (b) clonal evidence of pluripotency in vitro and in vivo, (c) cell type-specificgene expression and (d) functionality of all three lineage derivatives (ectodermal: secretionof human milk in transplanted mice, mesodermal: lipid-accumulating adipocytes, tubule-forming endothelial cells and beating cardiomyocytes, and endodermal: intestinal goblet

cells). Finally, exclusion of cell-cell fusion or contamination events (through Short Tandem

Repeat (STR) analysis and karyotyping of multiple cell populations before and after

differentiation) confirms the origin of the cells. Together, these studies provide

morphological, molecular and functional evidence of lineage plasticity of these cells. TheePS cells are distinct from hESCs, iPS cells and adult stem cells as assayed by expression

profiling, functional tests, growth conditions and expression of cell surface markers [7]. For
example, when we isolated tissue-specific breast stem cells using previously publishedmarkers [9], the isolated cells easily generated breast derivatives but failed to makemesodermal or endodermal derivatives.

Notably, we also demonstrated the mortal and non-malignant state of the ePS cells

isolated from breast tissue obtained from multiple disease-free women of various ages andethnicities. The ePS cells exhibit normal diploid 46,XX karyotypes, low telomerase

Tumor Heterogeneity

91

expression and activity and ultimately enter replicative senescence, distinguishing themfrom immortal, genomically unstable tumor cells and from hESCs (Figure 2). While ePS

cells exhibit enough proliferation potential to generate fully expanded and differentiatedtissues in the context of (non-malignant) teratomas, their mortality suggests a short-term

contribution to tissue function under yet-to-be-determined conditions such as tissue repair

during wound healing. We have found evidence of these cells in multiple tissues of thehuman body (Pan and Caruso, unpublished results). Finally, colleagues have provided
independent validation that these cells can be isolated and exhibit the characteristics

pop. doublings

reported in Roy et al. [7].

Figure 2

p59

60

p52

55
50
45
40

p52

p59

ePS cells express SON, yet, are mortal, expand ~59 population doublings, maintain low
telomerase activity, exhibit a normal diploid karyotype and arrest in G1

We have pursued these unexpected observations in several directions. First, we foundStudying The Regulation and Biological Relevance of ePS Cells In Vitro and In Vivothat ePS cells need the activated expression of SON to exhibit pluripotency; knockdown of
any of these three genes ablates the plastic potential of ePS cells and triggers a caspase-dependent apoptotic response [10]. Second, we determined that the well-known signaling

pathways that regulate and maintain pluripotency in hESCs and iPSCs (the activin A andFGF2 pathways) are necessary but not sufficient to activate or maintain SON expression

and plasticity in ePS cells and that additional unique signaling pathways are required to92

T. D. Tlsty et al.

activate the SON program. One of these pathways relies on CD73 enzymatic activity. We

have now defined the signaling necessary and sufficient to activate SON and plasticity inePS cells [10] (Figure 3). Thus, ePS cells can enter into a pluripotent state using pathways

that are novel and not utilized by hESC or iPSC.

ePS cell
Activin A

Adora2bcAMP/PKA
Smads CD73

IL6

Stat3 Oct4AA CD73

IL6

Sox2

Nanog

Adenosin
e
AMP

CD39

ADPorATP

MEK/ErK
PI3K X

FGF2R
FGF2
Pluripotency

Figure 3

Novel Circuitry Controls Pluripotency in ePS Cells

The most lethal of tumors are the rare metaPLASTIC tumors that are highly

Potential in Disease?

undifferentiated and exhibit embryonic properties of cell movement (as seen in metastasis)and heterogeneity (known to drive drug resistance). Strikingly, these tumors exhibit themost aggressive phenotypes. A metaplastic breast cancer can contain, not only malignant

breast tissue, but also additional malignant tissues such as bone, pancreas and cartilage

tissue derivatives – all in one localized area (Figure 4). Since previous studies
demonstrated that these multiple tissue types all contain the same mutations andchromosomal abnormalities (for example identical mutations in p53), investigators havelong postulated that they arose from a single pluripotent cell that became malignant [11](Figure 4). Until now, that cell had been hypothetical.

The characterization of ePS cells described above suggests the hypothesis that ePS cells

may provide a source of cells and/or factors that contribute to the unusual phenotypic andbehavioral features that typify metaplastic tissues in areas of chronic wounding and theirTumor Heterogeneity93

associated carcinosarcomas. If correct, we hypothesize that ePS cells may ultimately be

used for modeling these disease states and that understanding their biology may provide

critical leads in screening for useful drugs to prevent or intervene in the generation of lethal

malignancies that contain metaplastic tissue derivatives. Since metaplastic tumors areresistant to all known therapeutics, an unmet need in this field is to find a therapeutic
approach that can be used for patients suffering from metaplastic cancers.

HAPLN1ALIZARIN
RED

Identical cytogenetic and molecular abnormalites…
p53 mutations, chromosomal translocations, etc.
Figure 4

Teixeira et al., 1998; Thompson et al., 1996; Geyer et al., 2010; Lien et al., 2004

Lineage Plasticity in Metaplastic Breast Carcinoma

References
1. Roth S1, Franken P, Sacchetti A, Kremer A, Anderson K, Sansom O, Fodde R. Paneth cells
in intestinal homeostasis and tissue injury. PLoS One. 2012;7(6):e38965.
2. Ito M, Liu Y, Yang Z, Nguyen J, Liang F, Morris RJ, Cotsarelis G. Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis. Nat
Med. 2005;11:1351-4.
3. Plikus MV, Gay DL, Treffeisen E, Wang A, Supapannachart RJ, Cotsarelis G. Epithelial
stem cells and implications for wound repair. Semin Cell Dev Biol. 2012;23:946-53.
4. Modiano N, Gerson LB. Barrett's esophagus: Incidence, etiology, pathophysiology, prevention and treatment. Ther Clin Risk Manag. 2007;3(6):1035-145.
5. Sharma SV, Lee DY, Li B, Quinlan MP, Takahashi F, Maheswaran S, McDermott U,
Azizian N, Zou L, Fischbach MA, Wong KK, Brandstetter K, Wittner B, Ramaswamy S,
Classon M, Settleman A chromatin-mediated reversible drug-tolerant state in cancer
cell subpopulations. J. Cell. 2010;141:69-80.

94

T. D. Tlsty et al.

6. Livingstone LR, White A, Sprouse J, Livanos E, Jacks T, Tlsty TD. Altered cell cycle
arrest and gene amplification potential accompany loss of wild-type p53. Cell.
1992;70:923-35.
7. Roy S, Gascard P, Dumont N, Zhao J, Pan D, Petrie S, et al. Rare somatic cells fromhuman breast tissue exhibit extensive lineage plasticity. Proc Natl Acad Sci USA.
2013;110:4598-603.
8. Park IK, Morrison SJ, Clarke MF. Bmi1, stem cells, and senescence regulation. J Clin
Invest. 2004;113:175-9.
9. Lim E, Vaillant F, Wu D, Forrest NC, Pal B, Hart AH, et al. Aberrant luminal progenitors
as the candidate target population for basal tumor development in BRCA1 mutation
carriers. Nat Med. 2009;15:907-13.
10. Pan D, Roy S, Gascard P, Zhao J, Chen-Tanyolac C, Tlsty TD. SOX2, OCT3/4 and
NANOG expression and cellular plasticity in rare human somatic cells requires CD73.
Cell Signaling, 2016; 28: 1923-1932.
11. Thompson, L., Chang, B. and Barsky, S.H. Monoclonal Origins of Malignant Mixed
Tumors (Carcinosarcomas) The American Journal of Surgical Pathology, 1996;20, p277285.

Thea D. Tlsty, PhD

1976-1980
1981-1985

1985-1994

1995-present

Graduated with a Ph.D. from Program in Molecular andCellular Biology, Washington Univ., School of
Medicine
Postdoctoral Fellow, Department of Biology, Stanford
Univ.
Assistant and Associate Professor of Pathology, Univ.
of North Carolina, Chapel Hill
Professor of Pathology, Univ. of California, San
Francisco

95

METABOLIC EXCHANGE BETWEEN STEM CELLS ANDNICHE CELLS

Keiyo Takubo1,Daiki Karigane2 and Toshio Suda3,4

Department of Stem Cell Biology, Research InstituteNational center for Global Health and Medicine1
Keio University School of Medicine2
Cancer Science Institute, National University of Singapore3
14 Medical Drive, #12-01, Singapore 117599
International Research Center for Medical Sciences, Kumamoto University4(sudato@keio.jp)

Hematopoietic stem and progenitor cells (HSPCs) maintain the hematopoietic system

for an organism’s entire lifetime under homeostatic or in stress settings. At steady state,long term hematopoietic stem cells (LT-HSCs), the most undifferentiated cells in thehematological system, are kept quiescent or in G0 of the cell cycle. Hematological stresses

deplete the body of blood cells, and if stresses remain below the lethal dose, bone marrow(BM) LT-HSCs enter the cell cycle and robustly repopulate the entire hematopoietic system

via multi-lineage differentiation and self-renewal. Such hematological repopulation

activity is termed “stress hematopoiesis,” an activity essential for homeostatic maintenanceof blood production. Understanding stress hematopoiesis is also critical to establish

efficient methods enabling expansion in vitro of HSPCs, since forced activation of the cell

cycle by cytokine supplementation induces a stress-like cellular state. p38MAPK inhibitionis thought to suppress aberrant HSPC proliferation and protect HSPCs from exhaustion or

replicative senescence in stress settings or in the context of damage associated with aging.

However, genetic evidence supporting this hypothesis is still limited.

To identify the predominant p38MAPK isozyme in BM compartments, we assessed

expression of mRNAs encoding the four p38MAPK isozymes by qPCR of cDNAs from LT-

HSCs, short-term (ST)-HSCs, multipotent progenitors (MPPs), lineage markers- c-Kit+ Sca-1-(LKS-) myeloid progenitors, and lineage markers+ (Lin+) fractions of wild-type BM. Amongthem, p38 α was highly expressed in BM mononuclear cells (BMMNCs), and p38 α

expression levels were significantly higher in HSPC fractions than in fractions ofdifferentiated cells. To test p38 α function, we took an inducible conditional knockout

approach using the CAG-CreERT2:p38 α flox/flox (p38 α fl/fl) mouse, in which p38 α deletion in

96

T. Suda et al.

vivo (p38 α Δ / Δ ) is induced globally by intraperitoneal tamoxifen administration.
Frequencies of various primitive hematopoietic cells (LT-HSCs, ST-HSCs, MPPs, andprogenitors) were also indistinguishable in p38 α +/+ and p38 α Δ /Δ BM. Thus p38 α isdispensable for steady state hematopoiesis in young mice, despite the fact that it isexpressed in wild-type hematopoietic cells. Given the function of p38 α in the context ofstress, we monitored how tolerant p38 α Δ /Δ HSCs were of hematological stress byperforming serial BMT. We observed lower relative PB and BM chimerism in the p38 α Δ/Δcompared to the p38 α + / + cell-transplanted group in the primary and secondarytransplantation. We next evaluated survival rate after injection of mice of both genotypes

with 5-fluorouracil (5-FU), which eliminates differentiated hematopoietic cells and induces

proliferation of quiescent HSCs. After a single administration of 5-FU, p38 α Δ /Δ mice
showed higher mortality than did p38 α +/+ mice. Taken together, p38 α loss in mice resultsin defects in stem cell capacity during stress hematopoiesis in vivo.

Next, we focused on cell cycle progression after BMT using the fluorescent label

carboxyfluorescein succinimidyl ester (CFSE). CFSE-labeled cells lose fluorescence at eachcell division; thus CFSE intensity reflects cell division history of transplanted cells after

BMT. For this analysis we stained freshly isolated p38 α +/+ or p38 αΔ/Δ BMMNCs with CFSE

and transplanted them into lethally-irradiated mice. We then analyzed donor-derived LTHSCs after BMT. CFSE fluorescence levels were significantly higher in mice transplantedwith p38 α Δ /Δ compared to p38 α +/+ cells from days 1 to 3 after BMT, whereas CFSE

fluorescence levels prior to BMT were identical between genotypes. These defects wereseen not only in LT-HSCs but also in ST-HSCs and MPPs. Taken together, loss of p38 α

results in defective control of LT-HSC cell cycle progression in stressed settings. Given thataltered metabolic activities can change cell cycle status [1,2], we asked whether p38 α

regulation of a particular metabolic pathway could initiate HSPC cycling under stressconditions. To do so, we collected p38 α+/+ or p38 αΔ/Δ LSK cells either at steady state or after

BMT and analyzed metabolites using mass spectrometry. Among metabolites surveyed,
we focused on glycine and aspartic acid as they were the only two of four metabolites

assessed whose levels increased in p38 α Δ /Δ cells. Levels of both increased in p38 α Δ /Δ as
compared with p38 α +/+ LSK cells after BMT. Also, mice transplanted with p38 α Δ /Δ

compared with p38 α +/+ LSK cells showed lower levels of allantoin, a product of purine
catabolism. These findings suggest that p38 α loss suppresses purine metabolism during

stress hematopoiesis. Expression of both inosine-5’-monophosphate dehydrogenase(Impdh) 2, the rate-limiting enzyme of guanosine monophosphate (GMP) synthesis, andguanosine monophosphate synthetase (Gmps) was significantly decreased in p38 α Δ /Δ

relative to p38 α +/+ LT-HSCs on day 1 after BMT.

Cancer Stem Cell Signals

97

To identify how p38 α regulates purine enzymes, we searched open-access databases fortranscription factors capable of binding to promotor regions of genes that encode purine-

related enzymes and are highly expressed in HSCs. Among candidates, we found that the

microphthalmia-associated transcription factor (Mitf) commonly binds promoters of

purine-related genes and is highly expressed in HSCs. To evaluate Mitf function in vivo

and in vitro, we used a mouse line homozygous for the vitiligo-associated spontaneous

Mitf mutation (Mitfvit/vit), a point mutant encoding a protein with reduced transcriptional
activity. In vitro EdU incorporation into LT-HSCs decreased in Mitfvit/vit relative to Mitf+/+
LT-HSCs and also transplanted Mitfvit/vit LT-HSCs showed higher CFSE-fluorescence,suggesting delayed proliferation immediately after BMT. We also evaluated expression of

Impdh and Gmps by qPCR. Expression levels of Impdh2 and Gmps significantly decreasedin Mitf v i t / v i t relative to Mitf + / + LT-HSCs, an effect similar to that seen following
transplantation of p38 αΔ/Δ LT-HSCs.

To verify that Impdh2 functions as a p38 α effector in HSC proliferation in vitro and invivo, we transduced LSK cells with retrovirus expressing Impdh2. Impdh2 overexpression
restored short-term EdU incorporation and CFSE fluorescence levels in p38 α Δ/Δ LSK cells.
Next, Impdh2-overexpressing LT-HSCs were sorted and transplanted into lethally-

irradiated recipient mice along with competitor MNCs. Impdh2 overexpression rescued
short-term repopulation defects in p38 α Δ /Δ LT-HSCs to levels seen in non-mutant cells.

However, Impdh2-overexpression did not rescue long-term repopulation defects. Next, we

asked whether Impdh2 functions as a Mitf effector in HSC proliferation. Impdh2

overexpression restored short-term EdU incorporation in Mitfvit/vit Lin- cells. Unlike the casein p38 α Δ /Δ LT-HSCs, Impdh2 overexpression restored both short- and long-term

repopulation defects seen following Mitfvit/vit transplantation. We conclude that after
transplantation, p38 α promotes HSC cycling through Mitf-dependent Impdh2 expression.

In summary, we have identified p38 α as a cell cycle and metabolic regulator of HSPCs

in hematological stress. p38 α is required for HSPC cell cycle initiation, especially during

stress settings including transplantation, bone marrow recovery, and ex vivo proliferation[3]. In addition, hematological stresses activate p38 α -dependent purine metabolism toinitiate cycling of quiescent HSCs. Artificial regulation of the p38 α /Mitf/purine

metabolism axis could be a novel modulator of HSPCs during hematological stress or exvivo expansion (Figure 1) [3]. This study reinforces the importance of cellular metabolism
in stem cell regulation and is the first study of metabolic requirements of HSPCs during

stress.

98

T. Suda et al.

Figure 1

Regulation of stress hematopoiesis by p38α-activated purine metabolism

References
1. Takubo K, Goda N, Yamada W, Iriuchishima H, Ikeda E, Kubota Y, Shima H, Johnson RS,
Hirao A, Suematsu M, Suda T. Regulation of the HIF-1alpha level is essential forhematopoietic stem cells. Cell Stem Cell. 2010;7:391-402.
2. Suda T, Takubo K, Semenza GL: Metabolic regulation of hematopoietic stem cells in the
hypoxic niche. Cell Stem Cell 9: 298-310, 2011
3. Takubo K, Nagamatsu G, Kobayashi CI, Nakamura-Ishizu A, Kobayashi H, Ikeda E,
Goda N, Rahimi Y, Johnson RS, Soga T, Hirao A, Suematsu M, Suda T. Regulation of glycolysis by Pdk functions as a metabolic checkpoint for cell cycle quiescence inhematopoietic stem cells. Cell Stem Cell. 2013;12:49-61.
4. Karigane D, Kobayashi H, Morikawa T, Ootomo Y, Sakai M, Nagamatsu G, Kubota Y,
Goda N, Matsumoto M, Nishimura EK, Soga T, Otsu K, Suematsu M, Okamoto S, Suda T,
Takubo K. p38 α Activates Purine Metabolism to Initiate Hematopoietic Stem/
Progenitor Cell Cycling in Response to Stress. Cell Stem Cell. 2016;19:192-204.

Cancer Stem Cell Signals

Toshio Suda, MD, PhD

1974

1974

1978

1982

1983

1991

1992

2002- 2015

2015-present

2015-present

99

Graduated from Yokohama City Univ. School of
Medicine
Junior and Senior Resident, Kanagawa Children's
Medical Center
Research Associate, Division of Hematopoiesis,
Institute of Hematology, Jichi Medical School
Research Associate, Department of Medicine, Medical
Univ. of South Carolina (Dr. Makio Ogawa's Lab)
Assistant Professor, Division of Hemopoiesis, Instituteof Hematology, Jichi Medical School
Associate Professor, Division of Hematology,
Department of Medicine, Jichi Medical School
Professor, Department of Cell Differentiation, Instituteof Molecular Embryology and Genetics, Kumamoto
Univ. School of Medicine
Professor, Developmental Biology, The Sakaguchi
Laboratory, Keio Univ. School of Medicine
Professor, Cancer Science Institute of Singapore,
National Univ. of Singapore
Director and Distinguished Professor, International
Research Center for Medical Sciences, Kumamoto
Univ.

100

HEDGEHOG SIGNALING AND THE STEM CELL NICHE IN
TISSUE RENEWAL AND MALIGNANCY

Philip A. Beachy

Departments of Biochemistry and of Developmental Biology
Institute for Stem Cell Biology and Regenerative Medicine
Stanford Cancer Institute and Howard Hughes Medical Institute
265 Campus Drive, Room G3120a
Stanford University School of Medicine
Stanford, CA 94305, USA(pbeachy@stanford.edu)

The Hedgehog signaling pathway plays a critical role in the postnatal control of tissue-

specific stem cell activity for the repair of injured tissues and maintenance of tissue

integrity. In organs such as the urinary bladder, epithelial injury triggers augmented
expression of the Hedgehog signal, which then elicits signals from underlying stromal cells

that in turn trigger epithelial cell proliferation and differentiation. This feedback between

tissue layers enables cells of the undamaged stromal layer to serve as a stem cell niche that

templates the maintenance and repair of damaged epithelia. Surprisingly, in the context ofmalignancy, this feedback signaling from stromal to epithelium serves to restrain tumor
growth and progression. This protective effect is due to Hedgehog induction of stromal

signals, often BMP family proteins, that induce epithelial differentiation and thus prevent

further tumor growth and spread. Similar roles for Hedgehog signaling in restraining

cancer growth have been found in other endodermal malignancies, such as those arisingfrom pancreas and colon.

Cancer Stem Cell Signals

Philip A. Beachy, PhD

1980-1986
1986-1988

101

Ph.D., Stanford University
Staff Associate, Department of Embryology, Carnegie
Institution of Washington
Johns Hopkins University School of Medicine
1988-1993
Assistant Professor, Department of Molecular Biology
and Genetics
1993-1998
Associate Professor, Department of Molecular Biology
and Genetics
1998-2006
Professor, Department of Molecular Biology andGenetics
2004-2006
Professor, Department of Oncology
Howard Hughes Medical Institute (HHMI)
1988-present Investigator, Johns Hopkins University School of
Medicine, Stanford University School of Medicine
Stanford University School of Medicine
2006-present Professor, Department of Developmental Biology
2007-present Ernest and Amelia Gallo Professor in the School of
Medicine
2011-present Professor, Department of Biochemistry

102

INDUCTION OF CANCER STEMNESS AND DRUGRESISTANCE BY EGFR INHIBITORS AND CELLULAR STRESSDavid A. Cheresh

Moores Cancer Center, Sanford Consortium for Regenerative Medicine and
UC San Diego School of Medicine
7880 Torrey Pines Scenic Drive, #0695, La Jolla, CA 92037-0695, USA(dcheresh@ucsd.edu)

Previous studies in our lab revealed that integrin avb3 become upregulated on bloodvessels during tumor angiogenesis. We found that targeting this integrin with antagonists

could block angiogenesis. Our current findings reveal that avb3 expression leads toendothelial cell reprogramming to an immature state and following its down regulation

endothelial cells mature into vascular structures. However, avb3 also plays a role in tumor

growth and metastasis. Here we show that avb3 expression on tumor cells is upregulated

in response to drug treatment or microenvironemental stress. Tumor cells encounterstresses including therapeutic intervention and overcome these stresses to achieve diseaseprogression and metastasis. Previous work has established that cancer therapy can selectfor the expansion of drug resistant cancer stem cells or induce an epithelial-tomesenchymal transition leading to tumor progression. We recently reported that epithelial

cancers exposed to receptor tyrosine kinase inhibitors such as Erlotinib showed an

upregulation of integrin αub3 on the cell surface. We found that αub3 was both necessary
and sufficient to account for cancer stemness and drug resistance by initiation a

KRAS/Ralb/TBK1/NFkB pathway (Seguin et al, Nature Cell Biology, 2014). In fact, αub3

formed a membrane complex with the multivalent lectin Galectin 3 allowing it to drive
KRAS addiction and a dependency on micropinocytosis for nutrient uptake and survival.

A clinically active Galectin 3 inhibitor was able to selectively destroy αub3 positive drug
resistant lung tumors by suppressing micropinocytosis in these cells.

While αub3 is necessary and sufficient to account for erlontib resistance and tumor

stemness it is not clear whether αub3 expression was due to a selection of preexisting drug

resistant cells or the induction of αub3 gene expression due to drug treatment or cellularCancer Stem Cell Signals

103stress. Here we show the surprising finding that EGFR inhibitor treatment represents one

form of cellular stress such as hypoxia, nutrient deprivation, oxidative stress that directly

induces the reprogramming of epithelial carcinoma cells, converting them to a stresstolerant, stem-like, drug-resistant state. Each of these forms of stress induces integrin b 3(ITGB3) transcription that is necessary and sufficient to drive expression of pluripotency

maintaining factors OCT4 and NANOG, representing a plastic state that endows cells withstress tolerance and drug resistance. These reprogrammed cells can be detected in regionsof tumor hypoxia and are highly enriched in the circulation of tumor bearing mice after

acquired resistance to systemic therapy. Mechanistically, stress induces a series of histone

modifications that promote an open chromatin state on the b 3 promoter, allowing bindingof the transcription factor HNF4A that is itself induced by stress. Histone acetyltransferase

or demethylase inhibitors not only block stress-mediated histone modifications on the b 3

promoter to prevent the induction of b 3, but block the induction of OCT4/NANOG andsensitize cells to the effects of stress. Our findings reveal b 3 both as a marker and mediatorof stress tolerance and cellular reprogramming that drives tumor progression, likely
accounting for its enrichment on metastatic tumor lesions. The induction of b 3 and
HNF4A by stress highlights the ability of epithelial tumor cells to rapidly adapt totherapeutic intervention, thus challenging the notion that drug resistance primarily resultsfrom a small population of pre-existing stress-tolerant cells. Consequently, this work
reveals how certain anti-cancer therapies may accelerate cancer progression by
upregulating integrin b 3 as an adaptive response to stress.
Reference

1. Seguin L, Kato S, Franovic A, Camargo MF, Lesperance J, Elliott KC, Yebra M, Mielgo A,
Lowy AM, Husain H, Cascone T, Diao L, Wang J, Wistuba II, Heymach JV, Lippman SM,
Desgrosellier JS, Anand S, Weis SM and Cheresh DA. A b 3 integrin-KRAS-RalB complex
drives tumor stemness and resistance to EGFR inhibition. Nature Cell Biol 16: 457-468,
PMCID: 4105198, 2014

104

D. A. Cheresh

David A. Cheresh, PhD

1978-1982
1982-1984

1984-1985

1985-1989

1989-1996

1996-2005

2005-20122005-20132006-present

2010-present

2012-present

2013-present

Graduated from Univ. of Miami
Postdoctoral Fellow, Research Institute of Scripps
ClinicSenior Research Associate, Research Institute of
Scripps Clinic
Assistant Professor, Department of Immunology, The
Scripps Research Institute
Associate Professor, Department of Immunology, The
Scripps Research Institute
Professor, Department of Immunology, The Scripps
Research Institute
Professor, Department of Pathology, Moores Cancer
Center, Univ. of California, San Diego (UCSD)
Director for Translational Research, Moores Cancer
Center, UCSD
Vice Chair for Research, Department of Pathology,
UCSD
Associate Director, Institute of Engineering in Medicine(IEM), UCSD
Distinguished Professor, Department of Pathology,
UCSD School of Medicine
Associate Director for Innovation and Industry
Alliances, Moores UCSD Cancer Center

105

GROWTH FACTOR SIGNALING IN CANCER STEM-LIKE
CELLS AND THEIR NICHE

Noriko Gotoh

Division of Cancer Cell Biology, Cancer Research Institute, Kanazawa University
Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan(ngotoh@staff.kanazawa-u.ac.jp)

Cancer stem cells (CSCs) have characteristics in common with normal stem cells fromtumor-prone tissue. For instance, CSCs can self-renew and simultaneously produce

differentiated daughter cells that proliferate strongly until they reach their final
differentiated state. Apparent differences also exist between CSCs and normal stem cells.

The latter are maintained under tight homeostatic regulation and are passively protected in

the surrounding microenvironment or stem cell niche in adult tissues. However, the former

may actively contribute to tumor formation and may use the CSC niche for their own
survival and proliferation. It is now believed that many solid tumors have cancer stem-likecells.

Growth factor signaling plays important roles for a variety of biological aspects inphysiological and pathological conditions, including tumorigenesis. It is thought that

inflammatory microenvironment creates a pro-tumorigenic state through production of

growth factors or cytokines. However, it is obscure how growth factor- or cytokine-

signaling contributes to cancer stem-like cells and the CSC niche.

We found that growth factor signaling plays critical roles for the communication

between cancer stem-like cells and their niche in order to maintain the cancer stem-likecells in the CSC niche. We have established conditions of culturing patient-derived tumor

spheres and generated patient-derived xenograft (PDX) models derived from the breast

cancer tissues. By using these patient-derived cells and PDXs, we uncovered the various
key mechanisms mediated by signaling through NFkB, IGF1 receptor, beta-catenin,

semaphorin, FRS2beta, growth differentiation factor (GDF) 15, MTHFD2, and so on. There

are various mechanisms: autocrine-paracrine mechanisms, regulation of stemness,

106

N. Gotohregulation of symmetric-asymmetric division and regulation of metabolism. Cancer stem-

like cells appear to survive and divide by using these mechanisms in order to adapt in the

different conditions of the CSC niche. Among them, we have been able to identify propermolecular targets for cancer stem-like cells to eradicate tumors. In this symposium I would

like to talk about several key mechanisms we have recently identified for maintenance ofcancer stem-like cells in the CSC niche.

1. Many breast cancer patients suffer from relapse that is potentially due to cancer stem

cells that are not eliminated by treatment. Not only cancer stem cells do not easily
disappear, but also cancer cells appear to have plasticity by which they undergo

dedifferentiation and even acquire cancer stem cell properties! However, it is largely

unknown how cancer stemness is maintained. Here we have shown the fundamental

mechanisms to stabilize cancer stemness. Since the HER2/3-PI3K-NFkB pathway isimportant for cancer stemness, we examined expression of downstream molecules in thispathway by comprehensive analysis of gene expression profiles over time after addition of

the HER3 ligand heregulin (HRG) that is also called as neuregulin (NRG)1. Insulin-likegrowth factor 2 (IGF2) was identified as a key downstream molecule, since anti-IGF2

antibody treatment blocked tumor sphere formation, a characteristic of stemness, evenunder conditions where other growth factors/cytokines were present. IGF2-PI3K signaling

induced tumor sphere formation and enhanced the expression of genes favoring stemness,

including the transcription regulator ID1 and IGF2 itself. Consistent with these data, ID1

and the IGF2 receptor IGF-1R were expressed at high levels in cancer stem cell populationof the breast cancer PDXs. Moreover, ID1 knockdown suppressed IGF2-induced
expression of IGF2. Finally, treatment with anti-IGF1/2 antibodies blocked tumorigenesis

derived from the IGF1Rhigh CSC-enriched population in a PDX model. Thus, NF k B may

trigger IGF2-ID1-IGF2 positive feedback circuits that allow cancer stem-like cells to appear.

Then, they may become addicted to the circuits. Since the circuits are the Achilles’ heels of

CSCs, it will be critical to break them for eradication of CSCs.


/0,


/0,


!"#$


HER2


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%0


IGF1R


HER3

!"#$

()*(*+
 


NF!
!B

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,:(;)0-1.(8+1<(-1778


!"#$

698'6=;6889:@5:A >?698'6=;6889:@5:A@'


%&'

",-.


%&'

"12

25678'9:7;:659:6<<8925678'9:7;:659:6<<89()* (*+

925678'9:7;:6 9925678'9:7;:6598'6=9:6<<89


"34


!"#$
",-.

()*
 (*+



!B
NF!


%0
EGFR



HER2

;?;@'$=>"#5

Tumor angiogenesis
angiogenesis
Cancer associated fibroblasts (CAFs)(CAFs)


Cancer Stem Cell Signals

107

2. Cancer stem cells (CSCs) are thought to be responsible for the initiation andrecurrence of tumors. Therefore, targeting molecules that have a critical role inmaintenance of CSCs would be a useful strategy. CD74-NRG1 fusion gene was identified

in 5-15% of in invasive mucinous adenocarcinoma of the lung, a malignant type of lungadenocarcinoma. CD74-NRG1 protein contains the CD74 transmembrane domain and the

EGF-like domain of the NRG1/HRG protein, suggested to mediate juxtacrine signals

through HER2:HER3 receptors. In this study, we expressed cDNA of CD74-NRG1 fusion
gene in H322 human lung cancer cell line and BT20 human breast cancer cell line by using alentivirus system, and investigated whether this fusion gene is involved in the promotionof CSC phenotype using a CSC assay. First, we examined the self-renewal ability of CD74-

NRG1 expressing breast cancer cells by performing the sphere forming assay. CD74-NRG1

expressing cancer cells were able to form tumor spheres without adding any growth

factors, while cells infected with the lentivirus carrying control vector were not. Then, we

analyzed the population of BCSCs by flow cytometry using CD44 and CD24 antibody. The

percentages of CD44high/CD24-/low CSC-enriched population increased from 1.94% to 9.47%(variant 1) or 8.21% (variant 2). These results suggest that expression of CD74-NRG1 fusion

gene promotes cancer stem cell properties and is involved in stem cell function of several

types of cancers including lung and breast cancer.

108

N. Gotoh

References
1. Tominaga K, Shimamura T, Kimura N, Murayama T, Matsubara D, Kanauchi H, Niida A,
Shimizu S, Nishioka K, Tsuji E, Yano M, Sugano S, Shimono Y, Ishii H, Saya H, Mori M,
Akashi K, Tada K, Ogawa T, Tojo A, Miyano S, Gotoh N.: Addiction to the IGF2-ID1-IGF2
circuit for maintenance of the breast cancer stem-like cells. Oncogene, online publication,
22 August 2016.
2. Murayama T, Nakaoku T, Enari T, Nishimura T, Tominaga K, Nakata A, Tojo A,
Sugano S, Kohno T, Gotoh N.: Oncogenic fusion gene CD74-NRG1 confers cancer stem
cell-like properties in lung cancer through a IGF2 autocrine/paracrine circuit, Cancer
Res, 76, 974-983, 2016.
3. Nakata A, Yoshida R, Yamaguchi R, Yamauchi M, Tamada Y, Fujita A, Shimamura T,
Imoto S, Higuchi T, Nomura M, Kimura T, Nokihara H, Higashiyama K, Kondoh K,
Nishihara H, Tojo A, Yano S, Miyano S, Gotoh N.: Elevated beta-catenin pathway as anovel target for patients with resistance to EGF receptor targeting drugs. Sci Rep, 5,13076, 2015.
4. Hinorara K, Kobayashi S, Kanauchi H, Shimizu S, Nishioka K, Tsuji E, Tada K,
Umezawa K, Mori M, Ogawa T, Inoue J, Tojo A. & Gotoh N: ErbB/NF-kB signaling
controls mammosphere formation in human breast cancer. Proc. Natl. Acad. Sci., USA, 109,6584-6589, 2012.

Noriko Gotoh, MD, PhD

1983-1989
1989-1991

1993-1998

1998-2001

2001-2003

2003-2005

2005-2006

2007-20132013-present

Graduated from Kanazawa Univ., School of Medicine
Resident for Internal Medicine, The Univ. of Tokyo
Hospital
Assistant Professor, Institute of Medical Science, The
Univ. of Tokyo
Visiting Scientist, New York Univ., School of Medicine(Prof. Joseph Schlessinger)
Assistant Professor, Institute of Medical Science, The
Univ. of Tokyo
Lecturer, Institute of Medical Science, The Univ. ofTokyo
Associate Professor, Institute of Medical Science, The
Univ. of Tokyo
Project Associate Professor, Division of Systems
Biomedical Technology, Institute of Medical Science,
The Univ. of Tokyo
Professor, Division of Cancer Cell Biology, Cancer
Research Institute, Kanazawa Univ.

109

IMAGING STEM CELL SIGNALS IN CANCERHETEROGENEITY AND THERAPY RESISTANCE

Tannishtha Reya

Departments of Pharmacology and Medicine
Sanford Consortium for Regenerative Medicine andMoores Comprehensive Cancer Center
University of California San Diego School of Medicine
La Jolla, CA 92093, USA(treya@ucsd.edu)
Malignant progression of pancreatic adenocarcinoma is critically dependent on the

RNA binding prorein Musahi [1]. Pancreatic cancer is a disease for which treatment israrely curative and in developed countries, it is the fourth leading cause of cancer-related

deaths [2]. Because patients are asymptomatic at early stages, by the time a diagnosis is

made, standard treatments have limited impact [3-5]. Four genes are commonly altered inpancreatic cancer: activating mutations of KRAS2 are found in greater than 90% of tumors,while the tumor suppressors p16/INK4A, p53, and SMAD4 [6-10] are frequently
inactivated by mutation, deletion or epigenetic silencing. These mutations have proved

difficult to target and currently no targeted therapies are available for use in pancreaticcancer. In the absence of targetable driver mutations, defining downstream epigenetic

programs that are critical signaling nodes for disease progression could identify new
therapeutic targets.

Pancreatic cancer progresses from a more differentiated non-invasive precursor stage

called pancreatic intraepithelial neoplasia (PanIN) to a more undifferentiated andmalignant invasive adenocarcinoma [11]. Although the aberrant proliferation and survival
in pancreatic cancer cells have been targeted by several approaches 12 , the molecular

programs that disable differentiation events during pancreatic cancer progression are notas well understood. This undifferentiated state is, however, a hallmark of adenocarcinoma

and as integral to its maintenance as proliferation and survival; thus, identifying thepathways that mediate this differentiation arrest can be an important avenue forunderstanding the basis of pancreatic cancer progression and may reveal new
vulnerabilities. To address this, we have focused on signals that can antagonize

110

T. Reyadifferentiation in stem and progenitor cells during normal development. One particularly

powerful but little studied signal is the RNA binding protein Musashi (Msi), a moleculethat governs stem cell fate. Msi is an evolutionarily conserved RNA binding protein
originally identified in drosophila [13]. A gene duplication event in teleosts led to thecreation of two Msi family members, Msi1 and Msi2. Mouse Msi1 and mouse Msi2 share a

75% similarity in amino acid sequence with the highest sequence similarity (85%) in thecritical RNA Recognition Motifs [14,15]. Each family member displays 95-99% sequencesimilarity between their mouse and human homologs [16]. Msi is highly expressed in
mammalian stem and progenitor cells across many tissues, such as the brain, mammary

glands and the gut, and has been long used as a marker of immaturity in many contexts[17-19]. However, its functional impact is only beginning to emerge: genetic loss offunction models of Msi1 or Msi2 have shown that Msi signaling is important for
maintaining stem cells in the mammalian nervous system [20], and, more recently, innormal and malignant hematopoiesis [21-23]. Thus, whether and how Msi contributes toadenocarcinoma development in primary pancreatic and other solid cancers and whether it

may be a tractable therapeutic target remains unknown.

To assess the contribution of Msi in pancreatic carcinogenesis, we created knock-in

fluorescent reporter mouse strains that allow visualization and tracking of Msi+ cells in vivo

and crossed them to the Ras/p53 driven mouse models of pancreatic cancer. This served asa unique new tool for image based and functional tracking and showed with remarkable

precision that Msi reporter activity marks cancer stem cells that propagate adenocarcinoma(Figure 1), circulating tumor cells with preferential capacity to metastasize, and cells thatare gemcitabine resistant. The fact that Msi+ cells are such a “high risk” population

highlighted the need for targeting them: deletion of either Msi1 or Msi2 in geneticallyengineered mouse models delayed tumor initiation (Figures 2 and 3) and dramatically
impaired transition from PanIN to adenocarcinoma, and markedly improved survival,suggesting that disease progression is deeply dependent on Msi signaling (Figures 4 and 5).

Figure 1

Survival curves of mice orthotopically transplanted with Msi-Reporter+ or Msi-ReporterKPffC pancreatic tumor cells reflect the tumor-propagating ability of Msi-expressing cells.

Cancer Stem Cell Signals

Figure 3

111

Figure 2

MRI images of normal, WT-KPffC andMsi1-/-KPffC mice, with 3-dimensional
volume rendering of the tumor mass
shown in red. Tumors are significantly
smaller in the absence of Msi1.

Figure 4

Survival curves of mice orthotopically transplanted with WT or Msi1-/- KPffC pancreatictumor cells demonstrate improved survival in the absence of Msi1.

Figure 5

Survival curves of WT and Msi2-/- KPffC mice demonstrate improved survival in theabsence of Msi2.

MRI images of normal, WT-KPffC
and Msi2-/- KPffC mice, with 3dimensional volume rendering of
the tumor mass shown in red.
Tumors are significantly smallerin the absence of Msi2.

112

T. Reya

Mechanistically, MSI1 and MSI2 controlled the expression of shared and distinct genetic
programs; among these, Msi could bind the 3’UTR and regulate expression of the proto-

oncogene c-MET, which could rescue defects triggered by Msi loss. In human lines, Msi

expression generally rose with aggressiveness, and drove enhanced growth. Msi inhibition

not only blocked growth of these lines, but also adenocarcinoma growth in patient-derived

pancreatic cancer xenografts, demonstrating that Msi is important in the context of multiple

and varied somatic mutations that occur in human disease. Finally, to explore thetranslational potential of this work, and define if Msi can be targeted with a deliverable

small molecule, we used a newly emerging paradigm to block its expression. Thus wedesigned, screened, and developed therapeutic antisense oligonucleotides against Msi that

effectively downregulated Msi expression and blocked growth of established primarypancreatic tumors in vivo (Figure 6). Our data suggests that MSI-directed ASOs should beconsidered for further development, not only for potential use in pancreatic cancer, but also

for broad use in other aggressive cancers with highly upregulated Msi expression, such as

glioblastoma and breast cancer. Finally, we also found that Msi rises in both humanpancreatitis and in caerulein induced mouse models of the disease, raising the intriguing

possibility that blocking Msi via ASO delivery could prevent or reduce risk of progression

from pancreatitis to pancreatic cancer and thus could contribute to prevention efforts aswell. In the long term, defining Msi as a new target in pancreatic cancer together withmethods to block this pathway could provide a new approach to control cancer growth andprogression.

Figure 6

Msi1-ASO delivery reduces growth of established pancreatic tumor in vivo.

Cancer Stem Cell Signals

113

References
1. Fox, R. G., Lytle, N. K., Jaquish, D. V., Park, F. D., Ito, T., Bajaj, J., Koechlein, C. S.,
Zimdahl, B., Yano, M., Kopp, J. L., Kritzik, M., Sicklick, J. K., Sander, M., Grandgenett, P.
M., Hollingsworth, M. A., Shibata, S., Pizzo, D., Valasek, M. A., Sasik, R., Scadeng, M.,
Okano, H., Kim, Y., MacLeod, A. R., Lowy, A. M. & Reya, T. Image-based detection andtargeting of therapy resistance in pancreatic adenocarcinoma. Nature 534, 407-411,(2016).
2. Jemal, A., Bray, F., Center, M. M., Ferlay, J., Ward, E. & Forman, D. Global cancer statistics. CA: a cancer journal for clinicians 61, 69-90, (2011).
3. Paulson, A. S., Tran Cao, H. S., Tempero, M. A. & Lowy, A. M. Therapeutic advances inpancreatic cancer. Gastroenterology 144, 1316-1326, (2013).
4. Rhim, A. D., Mirek, E. T., Aiello, N. M., Maitra, A., Bailey, J. M., McAllister, F., Reichert,M., Beatty, G. L., Rustgi, A. K., Vonderheide, R. H., Leach, S. D. & Stanger, B. Z. EMT anddissemination precede pancreatic tumor formation. Cell 148, 349-361, (2012).
5. Yachida, S. & Iacobuzio-Donahue, C. A. The pathology and genetics of metastatic pancreatic cancer. Archives of pathology & laboratory medicine 133, 413-422, (2009).
6. Almoguera, C., Shibata, D., Forrester, K., Martin, J., Arnheim, N. & Perucho, M. Most
human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell 53, 549-554,(1988).
7. Hahn, S. A., Schutte, M., Hoque, A. T., Moskaluk, C. A., da Costa, L. T., Rozenblum, E.,
Weinstein, C. L., Fischer, A., Yeo, C. J., Hruban, R. H. & Kern, S. E. DPC4, a candidate
tumor suppressor gene at human chromosome 18q21.1. Science 271, 350-353, (1996).
8. Jones, S., Zhang, X., Parsons, D. W., Lin, J. C., Leary, R. J., Angenendt, P., Mankoo, P.,
Carter, H., Kamiyama, H., Jimeno, A., Hong, S. M., Fu, B., Lin, M. T., Calhoun, E. S.,
Kamiyama, M., Walter, K., Nikolskaya, T., Nikolsky, Y., Hartigan, J., Smith, D. R.,
Hidalgo, M., Leach, S. D., Klein, A. P., Jaffee, E. M., Goggins, M., Maitra, A., IacobuzioDonahue, C., Eshleman, J. R., Kern, S. E., Hruban, R. H., Karchin, R., Papadopoulos, N.,
Parmigiani, G., Vogelstein, B., Velculescu, V. E. & Kinzler, K. W. Core signaling pathways
in human pancreatic cancers revealed by global genomic analyses. Science 321, 18011806, (2008).
9. Redston, M. S., Caldas, C., Seymour, A. B., Hruban, R. H., da Costa, L., Yeo, C. J. &Kern, S. E. p53 mutations in pancreatic carcinoma and evidence of common involvement of homocopolymer tracts in DNA microdeletions. Cancer research 54, 3025-3033,(1994).

114

T. Reya

10. Schutte, M., Hruban, R. H., Geradts, J., Maynard, R., Hilgers, W., Rabindran, S. K.,
Moskaluk, C. A., Hahn, S. A., Schwarte-Waldhoff, I., Schmiegel, W., Baylin, S. B., Kern, S.
E. & Herman, J. G. Abrogation of the Rb/p16 tumor-suppressive pathway in virtually all
pancreatic carcinomas. Cancer research 57, 3126-3130, (1997).
11. Hruban, R. H., Adsay, N. V., Albores-Saavedra, J., Compton, C., Garrett, E. S.,
Goodman, S. N., Kern, S. E., Klimstra, D. S., Kloppel, G., Longnecker, D. S., Luttges, J. &Offerhaus, G. J. Pancreatic intraepithelial neoplasia: a new nomenclature and classification
system for pancreatic duct lesions. The American journal of surgical pathology 25, 579-586,(2001).
12. Li, D., Xie, K., Wolff, R. & Abbruzzese, J. L. Pancreatic cancer. Lancet 363, 1049-1057,(2004).
13. Nakamura, M., Okano, H., Blendy, J. A. & Montell, C. Musashi, a neural RNA-binding
protein required for Drosophila adult external sensory organ development. Neuron 13, 6781, (1994).
14. Sakakibara, S., Nakamura, Y., Satoh, H. & Okano, H. Rna-binding protein Musashi2:
developmentally regulated expression in neural precursor cells and subpopulations ofneurons in mammalian CNS. The Journal of neuroscience : the official journal of the Society for
Neuroscience 21, 8091-8107, (2001).
15. Siddall, N. A., Kalcina, M., Johanson, T. M., Monk, A. C., Casagranda, F., Been, R. P.,
McLaughlin, E. A. & Hime, G. R. Drosophila Rbp6 is an orthologue of vertebrate Msi-1
and Msi-2, but does not function redundantly with dMsi to regulate germline stem cell
behaviour. PloS one 7, e49810, (2012).
16. Good, P., Yoda, A., Sakakibara, S., Yamamoto, A., Imai, T., Sawa, H., Ikeuchi, T., Tsuji, S.,
Satoh, H. & Okano, H. The human Musashi homolog 1 (MSI1) gene encoding thehomologue of Musashi/Nrp-1, a neural RNA-binding protein putatively expressed in
CNS stem cells and neural progenitor cells. Genomics 52, 382-384, (1998).
17. Okano, H., Imai, T. & Okabe, M. Musashi: a translational regulator of cell fate. Journal ofcell science 115, 1355-1359, (2002).
18. Okano, H., Kawahara, H., Toriya, M., Nakao, K., Shibata, S. & Imai, T. Function ofRNA-binding protein Musashi-1 in stem cells. Experimental cell research 306, 349-356,(2005).
19. Sutherland, J. M., McLaughlin, E. A., Hime, G. R. & Siddall, N. A. The Musashi family of
RNA binding proteins: master regulators of multiple stem cell populations. Advances inexperimental medicine and biology 786, 233-245, (2013).

Cancer Stem Cell Signals

115

20. Sakakibara, S., Nakamura, Y., Yoshida, T., Shibata, S., Koike, M., Takano, H., Ueda, S.,
Uchiyama, Y., Noda, T. & Okano, H. RNA-binding protein Musashi family: roles for
CNS stem cells and a subpopulation of ependymal cells revealed by targeted disruption
and antisense ablation. Proceedings of the National Academy of Sciences of the United States ofAmerica 99, 15194-15199, (2002).
21. de Andres-Aguayo, L., Varas, F., Kallin, E. M., Infante, J. F., Wurst, W., Floss, T. & Graf, T.
Musashi 2 is a regulator of the HSC compartment identified by a retroviral insertion
screen and knockout mice. Blood 118, 554-564, (2011).
22. Hope, K. J., Cellot, S., Ting, S. B., MacRae, T., Mayotte, N., Iscove, N. N. & Sauvageau, G.
An RNAi screen identifies Msi2 and Prox1 as having opposite roles in the regulation ofhematopoietic stem cell activity. Cell stem cell 7, 101-113, (2010).
23. Ito, T., Kwon, H. Y., Zimdahl, B., Congdon, K. L., Blum, J., Lento, W. E., Zhao, C.,
Lagoo, A., Gerrard, G., Foroni, L., Goldman, J., Goh, H., Kim, S. H., Kim, D. W., Chuah,C., Oehler, V. G., Radich, J. P., Jordan, C. T. & Reya, T. Regulation of myeloid leukaemia
by the cell-fate determinant Musashi. Nature 466, 765-768, (2010).

Tannishtha Reya, PhD

1997-1999
1999-2001
2001-2008

2006-20102008-20102011-present
2011-present

2011-present

2015-present

2015-present

Postdoctoral Fellow, Univ. of California, San Francisco
Postdoctoral Fellow, Stanford Univ.
Assistant Professor, Department of Pharmacology &Cancer Biology, Duke Univ. Medical Center
Co-Director, Stem Cell and Regenerative Medicine
Program, Duke Univ. Medical Center
Associate Professor, Department of Pharmacology &Cancer Biology, Duke Univ. Medical Center
Adjunct Associate Professor, Department ofPharmacology & Cancer Biology, Duke Univ. Medical
CenterAdjunct Professor, Sanford Burnham Prebys Medical
Discovery Institute
Professor, Department of Pharmacology, Univ. of
California, San Diego School of Medicine
Professor, Department of Medicine, Univ. of California,
San Diego School of Medicine
Co-Director, Biomedical Sciences Graduate Program,Cancer Biology Track, Univ. of California, San Diego
School of Medicine

116

REGULATION OF MYELOPOIESIS AND LEUKEMIA BY
NONCODING RNA

Daniel G. Tenen

Cancer science Institute, National University of Singapore
14 Medical Drive, Singapore 117599(daniel.tenen@nus.edu.sg)

Tight regulation of myeloid transcription factors PU.1 and C/EBPa is necessary forproper hematopoietic stem cell function and granulopoiesis, and dysregulation of thesegenes contributes to leukemia. We have focused on regulation of these two genes bynoncoding RNAs (ncRNAs). In the case of PU.1, we previously described a long antisense

RNA that is initiated from a discrete promoter in intron 3 and extends past thetranscription start site. This transcript is expressed at highest levels in T cells, in which

PU.1 is not expressed, and can inhibit PU.1 protein. siRNAs which target this antisensetranscript can increase PU. 1 mRNA and protein, and induce differentiation of leukemic

cells. A second nuclear long noncoding RNA locus is initiated in the upstream regulatory

element (URE), extending greater than 10 kb toward the transcription start site. We are

testing the function of the URE enhancer RNA using human BAC transgenics in whichtranscription terminators have been used to abrogate expression of the noncoding RNA.

In the case of C/EBP alpha, we identified a ncRNA extending beyond thepolyadenylation signal. In contrast to PU.1, this extracoding transcript correlates positively
with C/EBP alpha mRNA, and siRNA knockdown of the ncRNA leads to a decrease in

C/EBP alpha mRNA and increase in methylation of the locus. Overexpression of thisncRNA leads to an increase in expression of C/EBP alpha in a cell line (K562) in which
C/EBP alpha is methylated and not expressed.

In summary, we have initiated studies of long noncoding RNAs in both the PU.1 and

C/EBP alpha genes. The function of these RNAs appears to be completely different. In thecase of PU.1, an antisense noncoding RNA downregulates PU.1 expression. In the case of
C/EBP alpha, an extracoding RNA inhibits methylation of the locus and increases mRNA

Cancer Stem Cell Signals

117

levels. Targeting the PU.1 antisense RNAs with shRNAs or inducing gene selective
demethylation of tumor suppressors using RNAs represent novel approaches to inducing

differentiation of leukemic cells.

Daniel G. Tenen, MD

1967-1977
1971-1975
1975-1982
1977-1982

1982-1983

1978-1986
1986-1993

1994-1999

1999-present
2008-present

2008-present

B.A. Univ. of California, Los Angeles
M.D. Harvard Medical School
Residency in Internal Medicine, Peter Bent Brigham
Hospital
Research Fellow in Medicine, Dana Farber Cancer
Institute
Fellow in Medical Oncology, Dana Farber Cancer
Institute
Instructor in Medicine, Harvard Medical School
Assistant Professor of Medicine, Harvard Medical
SchoolAssociate Professor of Medicine, Harvard Medical
SchoolProfessor of Medicine, Harvard Medical School
Distinguished Professor of Medicine, National Univ. ofSingaporeDirector, Cancer Science Institute, National Univ. ofSingapore

118

NORMAL AND NEOPLASTIC STEM CELLS

Irving L. Weissman

Stanford Institute for Stem Cell Biology and Regenerative Medicine
Ludwig Center for Cancer Stem Cell Research at Stanford
Developmental Biology, Neurosurgery and Biology, Stanford University
265 Campus Drive W, Room G3167, Stanford, CA 94305, USA(irv@stanford.edu)
Blood-forming stem cells are responsible for the development and lifelong maintenanceof normal blood and for blood diseases such as leukemia (Figure 1) [1]. Thus, tounderstand leukemia, one should examine the changes occurring in the blood-forming
stem cell.

The cell surface molecules that define HSC have been well described in the scientific

literature. In mice, these cells are positive for Thy1-lo, CD150, c-kit, Sca-1; and negative for

B220, Mac-1, Gr-1, CD3, 4, 8, Ter119, Flk2, CD34 [2]. In humans, HSC are positive for
CD34, Thy1-lo, CD150, c-kit; and negative for CD3, CD4, CD8, CD10, CD14, CD15, CD16,

CD19, CD20, CD 38, glycophorin A [3]. However, at least in mice these markers were notsufficient to define and completely purify long term HSC [4]. We recently found that a

Figure 1

Blood formation by Hematopoietic Stem Cells [HSCs].

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gene called HoxB5 is expressed on only a subset of the cells we have previously defined as

HSC. To test the utility of this marker, we generated a reporter mouse in which cells
express mCherry along with the HoxB5 gene. Among the cells previously defined as HSC,

only a small fraction were positive (high or low) for HoxB5. We assessed thedevelopmental outcome of the different cell subsets by transplantation into myeloablated

recipient mice and found that the HoxB5-positive (high and low) cells, but not

HoxB5–negative, could reconstitute hematopoiesis for the life of the animal. Therefore,

HoxB5 is not only a marker for blood-forming stem cells, but defines the true long-term[LT] HSC [4].

These reporter mice allowed us to visualize LT-HSC in their bone marrow niche for the

first time and ask: where are stem cells in the blood-forming tissue? We published thatwithin the bone marrow, 94% of the HoxB5 + blood-forming stem cells are attached to theoutside of one kind of blood vessel, the bone marrow sinusoids (Figure 2). Hence, this areaaround the HoxB5+ cell has to be the bone marrow niche. Given this finding, the firstpublished claims that the hematopoietic niche was located next to the bone or endosteium,cannot be explained, because no HoxB5+ cells were found close to the bone. Other claims

that the niche was next to the arterioles also cannot be the explanation, because the HoxB5+

cells were only detected next to venous sinusoids or venules. A quantitative analysis

showed that there were only about 100 LT-HSC in each mouse femur. In sum, we haveestablished the identity and location of the long-term stem cells [4].

Many years ago we became interested in the clinical importance of blood-forming stem

cells. We analyzed blood-forming tissue—bone marrow or mobilized peripheral blood(circulating blood into which HSC have been mobilized from the bone marrow). Bonemarrow and mobilized blood were and still are used in autologous transplantations incancer patients. In this scenario, a sample of mobilized blood is removed from the patient;very high dose combination chemotherapy is administered, which removes most of the

Figure 2

HoxB5+ blood forming stem cells (arrows) found next to bone marrow sinusoidal vessels [4].

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I. L. Weissman

patient’s blood-forming tissue and most of the cancer cells throughout the body; and thenthe mobilized blood is injected back into the same patient to restore blood-forming

function. We thought that since we found cancer cells contaminating the mobilized blood

this approach could not be effective at curing cancer. When we purified blood-forming

stem cells from the mobilized blood and transplanted those into patients, we had decreased

the numbers of contaminating cancer cells by a factor of 250,000 (Figure 3) and had

removed T cells [5]. We then conducted a clinical trial (initiated 20 years ago): we purified

stem cells from mobilized blood in women with breast cancer that had metastasized to thelymph nodes and distant tissues, such as liver, lung, and bone. No surgery or radiotherapy

could eliminate the disease, so a cure required high-dose chemotherapy. Figure 4 shows

the long-term results of that trial, comparing transplants of HSC purified from mobilized

blood vs. transplants of unpurified mobilized blood [6]. Amazingly, in 2011 and 2017, one

third of the pure HSC-transplanted patients are still alive and free of disease. Therefore,

Figure 3

Removal of contaminating cancer cells from blood-forming tissue grafts. [5]Figure 4

Survival of patients with metastatic breast cancer treated with high dose combination
chemotherapy followed by autologous transplantation of purified blood-forming stem
cells from mobilized blood VS. unpurified mobilized blood [6].

Nakahara Memorial Lecture

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when high-dose chemotherapy followed by autologous transplantation of blood-forming

tissue is used to treat diseases (such as leukemia, lymphoma, metastatic breast cancer, andmelanoma), one should use cancer free purified HSC. Stanford University is opening apure stem-cell transplantation laboratory and clinic and we intend to extend those initialclinical trials.

We also examined the role of purified blood-forming stem cells in transplantations fromone person to another (donor to recipient). In Figure 5, on the left, when T cells from thedonor contaminate the transplant, they attack the tissues in the recipient causing multiorgan system graft versus host disease (GVHD). These patients have to be contained in anearly germ-free environment, while immunosuppressive drugs that increase the risk ofinfections are used to decrease the severity of GVHD. Amongst the T cells that are causing

GVHD, some are usefully combating the cancer. This was the forerunner of current T-celltherapy for cancer. If we wish to cure diseases of the blood-forming system with donor-to-

recipient (allogeneic) transplantation, using purified HSC will not cause GVHD, and thus

not necessitate immunosuppression. Moreover, allogeneic HSC transplantation inducestransplantation tolerance. Therefore any organ or cells from the HSC donor could be

subsequently transplanted to the same recipient and tolerated without immunosuppression[7,8]. In sum, purified HSC transplantation eliminates GVHD and enables other tissuetransplantation without immunosuppressive drugs. This would expand the use of HSC

transplantation to treat blood diseases such as severe combined immunodeficiency (SCID),

thalassemia and sickle cell anemia. It could also cure, as we showed in animal models,

genetic autoimmune diseases such as type I diabetes [9] and systemic lupus erythematosus[10] by replacing a blood-forming system prone to autoimmunity with a healthy one(Figure 5, right) [7,8].

Typically blood-forming tissue transplantation requires prior conditioning of the patientwith high-dose radiation and chemotherapy to eliminate the existing blood-forming cells.

Figure 5

HSC transplantation from a healthy donor to a recipient. [7-10]

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I. L. Weissman

Because this radiation is toxic to other systems, we have sought to develop, through the useof antibodies, a less toxic conditioning method to enable transplantation. As Figure 6

shows, anti c-kit antibody (ACK2; which targets the recipient’s blood-forming stem cells),

and anti-CD4 and anti-CD8 antibodies (which target recipient T cells), are not on their own

sufficient to eliminate those cells in mice and allow a transplant to engraft [11]. However,combining these antibodies with anti-CD47 antibody allows the purified donor’s blood-

forming stem cells to engraft in an unrelated recipient, leading to full reconstitution of all

blood cell types without GVHD (Figure 6). In sum, we want to limit the use of cytotoxic

conditioning and radiation conditioning in the future, and use far less toxic antibody-only

conditioning. Because of the fact that HSC from a donor induce tolerance in the recipient toall other tissues from that same donor, in the far future, we want to use cells rather thanwhole tissues or organs from a donor to generate HSC and other needed tissue-forming
stem cells to replace diseased or damaged tissue for life.

To understand the role of HSC in the development of leukemia we first analyzed thegenetic makeup of leukemia cells from patients in Japan after the atom bomb [12]. In
AML1-ETO leukemia, the of leukemia stem cells had the distinctive AML1-ETO

chromosomal translocation plus other mutations. Furthermore, the AML1-ETO

translocation had to occur in a stem cell, because, the initial genetic changes that we foundin leukemias do not change a non–self-renewing cell into a self-renewing cell. We

Figure 6

Successful engraftment of a mouse transplant from a donor to recipient (with minor mismatch) using antibody conditioning rather than radiation conditioning. (Antibodies: anti-c-kit[ACK2], anti-CD4, anti-CD8, anti-CD47) [11].

Nakahara Memorial Lecture123

hypothesized that therefore the only cells that must accumulate the changes and pass themon to daughter cells, are the self-renewing stem cells [13]. If this were true, one could traceback from the leukemia stem cell (Figure 7, bottom row, cells marked “1-7”), and find aclone that has n-1 (in this case, 6) mutations, another with n-2, another with n-3, etc.

Therefore, knowing all the mutations in the leukemia from a particular patient could allow

one to determine the order of the mutations. Another important point: the number of stem

cells and the number of niches are constant. Thus, in order for a stem cell clone with agiven mutation or set of mutations to advance to a point where it is dangerous, it must out-

compete the normal stem cells for limited niche sites.

We tested this hypothesis by sequencing the DNA to find mutations that were in theleukemia but not in normal tissue (Figure 8, top half). We prepared DNA primers thatwould identify the normal or mutant stem cells when we analyzed one stem cell at a time

[14,15]. The bottom row of Figure 8 shows some of the mutations in the first patient whosebone marrow we analyzed. The first mutation was TET2 Y1649stop, which stopped theprotein from being expressed, leaving haploexpression of TET2. The second mutation(T1884A) was in the second allele of that TET2 gene, leading to no expression of this gene.

We could find mutant cells in bone marrow from anywhere in that patient, because not

only did the clone expand, but, like normal blood-forming stem cells, it migrated from one

bone marrow cavity to another. Moreover, the mutation that was associated with the initial

Figure 7

Schematic diagram of the development of leukemia. The numerals superimposed over the
blood-forming stem cells (1, 1-2, 1-3, etc.) indicate the number of mutations present [13].

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Figure 8(Top) Strategy for analyzing leukemia stem cells compared to normal blood forming stem
cells (HSCs) and other normal stem cells to identify sequence of mutations leading toleukemia. (Bottom) Example of the results of this analysis applied to a patient. (Stripedand hatched stem cells acquired mutations that did not lead to proliferation; final mutation ingrey cells [leukemia stem cells] led to proliferation) [14].

diagnosis—FLT3-ITD (internal tandem repeat)—was not found in the stem cell clone but

only in the leukemia stem cell derived from that clone. In looking at 21 other patients, we

could generalize: the initiating mutations are in those genes that are required for normal
differentiation from the stem cell toward a blood cell.

A knockout of TET2 lowers thefrequency of differentiation but does not limit proliferation, so the cells can compete for theniche. The last mutated gene in different patients (e.g., K-ras, N-ras, FLT3) appears as thelast step and allows massive increase in proliferation, with perhaps self-renewal. Thus, theinitial mutations have prepared the cells to finally become leukemic once the last mutation

occurred [14]. This important principle also probably applies to other cancer in any tissues

with stem cells and progenitor cells.

Elimination of all cells in a cancer except the cancer stem cell will allow the disease to

grow. Therefore, successful therapy must eliminate all the cancer stem cells. Thus, we

wanted to know whether there are targets on leukemia stem cells but not normal stem cells.
The very first we discovered was CD47, a signal that protects a cell that expresses it fromphagocytosis by macrophages. Tht is, CD47 is a "don't eat me" signal to macrophages. As

shown in Figure 9, we proposed that in order for an aging normal or cancer cell to survive,its "don't eat me" signal must be overcoming a contrary "eat me" signal that the cell

expresses. To study this, we transplanted human leukemias from patients into the blood-

forming system of immune deficient mice and allowed them to engraft and grow (Figure10). [16] When we then treated for 2 weeks with a blocking antibody to CD47, 90% of theleukemias were cured. In 10% of cases the leukemia cells managed to escape elimination
by macrophages.

CD47 was also in breast cancer. As shown in Figure 11, when breast cancer stem cells

isolated by Michael Clarke were transplanted into an immune deficient mouse and allowedNakahara Memorial Lecture

Figure 9125

Expression of CD47 on myeloid leukemia cells contributes to pathogenesis by providing a"don't eat me" signal to macrophages that counteracts other "eat me" signals on theleukemia cells [16].

Figure 10 Anti-CD47 antibody treatment depletes human acute myeloid leukemia transplanted andallowed to take over the bone marrow of mice. (Right panel: arrows point to macrophages
containing leukemia cells) [16].

Figure 11 Anti-CD47 antibodies inhibit the growth of breast cancer tumors transplanted frompatients into mice. [17]126

I. L. Weissman

to grow into a tumor, treatment with a blocking antibody to CD47 cleared the tumor. In
fact for every type of cancer we transplanted into a mouse and then treated with anti-CD47

antibody, if the tumor was small enough, it was cleared; if it was not small enough, at least

the metastases were eliminated [17].

There are two parts to the strategy to increase phagocytic removal of tumor cells: block

their "don't eat me" signal and provide or strengthen their “eat me" signal. Figure 12 showsthat antibodies like rituximab (anti-CD 20) have a binding site on the back of the antibody

for the phagocytic receptor—the high affinitity Fc receptor—on macrophages. When an

aggressive human lymphoma was transplanted into an immune deficient mouse andallowed to grow, treatment with rituximab plus anti-CD47 antibody (but neither agent

alone) cured the lymphoma [18].

Figure 12 Combination therapy with anti-CD47 antibody + cancer-targeting monoclonal antibodies(e.g., rituximab [anti-CD 20]). (SIRPa=receptor for CD47; FcR = receptor for Fc fragment ofanti-CD20 antibody) [18,19].

In summary, by taking stem cells as a way of looking at the biology of the human blood-

forming system (and later brain-forming, breast-forming, etc.), we discovered that by

purifying the cells you can begin to understand the events that would: allow a normal stem

cell to regenerate the system; allow physicians to switch from chemotherapy and radiation

conditioning to less toxic antibody conditioning prior to performing transplant; and allow

us to compare cancer stem cells to normal tissue stem cells to discover therapeutic targets
like CD47.

Nakahara Memorial Lecture

127

References
1. Weissman IL. Stem cells: units of development, units of regeneration, and units in evolution. Cell. 2000;100(1):157-68. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10647940
2. Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mousehematopoietic stem cells. Science. 1988;241(4861):58-62. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=2898810
3. Baum CM, Weissman IL, Tsukamoto AS, Buckle AM, Peault B. Isolation of a candidate
human hematopoietic stem-cell population. Proc Natl Acad Sci USA. 1992;89(7):2804-8.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=1372992
4. Chen JY, Miyanishi M, Wang SK, Yamazaki S, Sinha R, Kao KS, Seita J, Sahoo D,
Nakauchi H, Weissman IL. Hoxb5 marks long-term haematopoietic stem cells andreveals a homogenous perivascular niche. Nature. 2016;530(7589):223-7. 4854608.
http://www.ncbi.nlm.nih.gov/pubmed/26863982
5. Negrin RS, Atkinson K, Leemhuis T, Hanania E, Juttner C, Tierney K, Hu WW,
Johnston LJ, Shizurn JA, Stockerl-Goldstein KE, Blume KG, Weissman IL, Bower S,
Baynes R, Dansey R, Karanes C, Peters W, Klein J. Transplantation of highly purified
CD34+Thy-1+ hematopoietic stem cells in patients with metastatic breast cancer. Biol
6.

7.

8.

9.

Blood Marrow Transplant. 2000;6(3):262-71. http://www.ncbi.nlm.nih.gov/entrez/
query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10871151
Muller AM, Kohrt HE, Cha S, Laport G, Klein J, Guardino AE, Johnston LJ, StockerlGoldstein KE, Hanania E, Juttner C, Blume KG, Negrin RS, Weissman IL, Shizuru JA.
Long-term outcome of patients with metastatic breast cancer treated with high-dose
chemotherapy and transplantation of purified autologous hematopoietic stem cells. Biol
Blood Marrow Transplant. 2012;18(1):125-33. http://www.ncbi.nlm.nih.gov/entrez/
query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=21767515
Shizuru JA, Jerabek L, Edwards CT, Weissman IL. Transplantation of purified
hematopoietic stem cells: requirements for overcoming the barriers of allogeneic
engraftment. Biol Blood Marrow Transplant. 1996;2(1):3-14. http://www.ncbi.nlm.nih.
gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9078349
Shizuru JA, Weissman IL, Kernoff R, Masek M, Scheffold YC. Purified hematopoietic stem
cell grafts induce tolerance to alloantigens and can mediate positive and negative T cellselection. Proc Natl Acad Sci USA. 2000;97(17):9555-60. http://www.ncbi.nlm.nih.
gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10920206
Beilhack GF, Scheffold YC, Weissman IL, Taylor C, Jerabek L, Burge MJ, Masek MA,
Shizuru JA. Purified allogeneic hematopoietic stem cell transplantation blocks diabetes
pathogenesis in NOD mice. Diabetes. 2003;52(1):59-68. http://www.ncbi.nlm.nih.
gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12502494

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10. Smith-Berdan S, Gille D, Weissman IL, Christensen JL. Reversal of autoimmune disease inlupus-prone New Zealand black/New Zealand white mice by nonmyeloablative transplantation of purified allogeneic hematopoietic stem cells. Blood. 2007;110(4):1370-8.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17435112
11. Chhabra A, Ring AM, Weiskopf K, Schnorr PJ, Gordon S, Le AC, Kwon HS, Ring NG,
Volkmer J, Ho PY, Tseng S, Weissman IL, Shizuru JA. Hematopoietic stem cell transplantation in immunocompetent hosts without radiation or chemotherapy. Sci Transl
Med. 2016;8(351):351ra105. http://www.ncbi.nlm.nih.gov/pubmed/27510901
12. Miyamoto T, Weissman IL, Akashi K. AML1/ETO-expressing nonleukemic stem cells inacute myelogenous leukemia with 8;21 chromosomal translocation. Proc Natl Acad Sci
USA. 2000;97(13):7521-6.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=
Retrieve&db=PubMed&dopt=Citation&list_uids=10861016
13. Rossi DJ, Jamieson CH, Weissman IL. Stems cells and the pathways to aging and cancer.
Cell. 2008;132(4):681-96.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=
Retrieve&db=PubMed&dopt=Citation&list_uids=18295583
14. Jan M, Chao MP, Cha AC, Alizadeh AA, Gentles AJ, Weissman IL, Majeti R.
Prospective separation of normal and leukemic stem cells based on differential expressionof TIM3, a human acute myeloid leukemia stem cell marker. Proc Natl Acad Sci USA.
2011;108(12):5009-14. 3064328. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=21383193
15. Jan M, Snyder TM, Corces-Zimmerman MR, Vyas P, Weissman IL, Quake SR, Majeti R.
Clonal evolution of preleukemic hematopoietic stem cells precedes human acute
myeloid leukemia. Sci Transl Med. 2012;4(149):149ra18. 4045621. http://www.ncbi.nlm.
nih.gov/pubmed/22932223
16. Majeti R, Chao MP, Alizadeh AA, Pang WW, Jaiswal S, Gibbs KD, Jr., van Rooijen N,
Weissman IL. CD47 is an adverse prognostic factor and therapeutic antibody target on
human acute myeloid leukemia stem cells. Cell. 2009;138(2):286-99. PMC2726837.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19632179
17. Willingham SB, Volkmer JP, Gentles AJ, Sahoo D, Dalerba P, Mitra SS, Wang J,
Contreras-Trujillo H, Martin R, Cohen JD, Lovelace P, Scheeren FA, Chao MP,
Weiskopf K, Tang C, Volkmer AK, Naik TJ, Storm TA, Mosley AR, Edris B, Schmid SM,
Sun CK, Chua MS, Murillo O, Rajendran P, Cha AC, Chin RK, Kim D, Adorno M,
Raveh T, Tseng D, Jaiswal S, Enger PO, Steinberg GK, Li G, So SK, Majeti R, Harsh GR,
van de Rijn M, Teng NN, Sunwoo JB, Alizadeh AA, Clarke MF, Weissman IL. The
CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target forhuman solid tumors. Proc Natl Acad Sci USA. 2012;109(17):6662-7. 3340046.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=22451913

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18. Chao MP, Alizadeh AA, Tang C, Myklebust JH, Varghese B, Gill S, Jan M, Cha AC,
Chan CK, Tan BT, Park CY, Zhao F, Kohrt HE, Malumbres R, Briones J, Gascoyne RD,
Lossos IS, Levy R, Weissman IL, Majeti R. Anti-CD47 antibody synergizes with rituximab
to promote phagocytosis and eradicate non-Hodgkin lymphoma. Cell. 2010;142(5):699713. 2943345. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=
PubMed&dopt=Citation&list_uids=20813259
19. Weiskopf K, Ring AM, Ho CC, Volkmer JP, Levin AM, Volkmer AK, Ozkan E, Fernhoff
NB, van de Rijn M, Weissman IL, Garcia KC. Engineered SIRPalpha Variants as
Immunotherapeutic Adjuvants to Anticancer Antibodies. Science. 2013;341(6141):88-91.
3810306. http://www.ncbi.nlm.nih.gov/pubmed/23722425

Irving L. Weissman, MD

1965-1967
1969-1974
1974-1981

1981-present
1986-2001

1989-present
1990-1992

1990-present

2002-present

2004- present
2005-2008
2007-present

Postdoctoral Fellow, Henry S. Kaplan, Stanford Univ.
Assistant Professor, Department of Pathology, Stanford
Univ.
Associate Professor, Department of Pathology, Stanford
Univ.
Professor, Department of Pathology, Stanford Univ.
Chairman, Stanford Univ. Immunology Program(degree-granting)
Professor, Department of Developmental Biology,
Stanford Univ.
Investigator, Howard Hughes Medical Institute,
Stanford Univ.
Professor, Department of Biology, Stanford Univ. (bycourtesy)
Director, Stanford Institute for Stem Cell Biology andRegenerative Medicine
Professor, Department of Neurosurgery, Department ofMedicine, Stanford Univ. Medical Center (by courtesy)
Director, Stanford Cancer Center
Director, Stanford Ludwig Center for Cancer Stem Cell
Research and Medicine

130

CELL OF ORIGIN AND TUMOR STEM CELLS IN MOUSEDIGESTIVE ORGAN TUMORS

Hiroshi Seno

Department of Gastroenterology and Hepatology
Kyoto University Graduate School of Medicine
54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan(seno@kuhp.kyoto-u.ac.jp)

Accumulating evidence suggests the existence of tumor stem cells that resemble normal

stem cells. There is great interest in tumor stem cells as potential therapeutic targets;however, cancer therapies targeting tumor stem cells are limited. One of the drawbacks isthat tumor stem cell markers are often shared by normal stem cells; thus, therapies thattarget these markers may cause severe injury to normal tissues. In this respect, we reported

that doublecortin-like kinase 1 (Dclk1) is a unique marker that distinguishes tumor stem
cells from normal stem cells in the intestine [1]. In Dclk1creERT2/+; Rosa26-LacZ; ApcMin/+ mice,intestinal tumors were occupied by LacZ-labeled progeny tumor cells derived from Dclk1-

expressing tumor stem cells with scattered LacZ expression in the normal intestinal mucosa

after tamoxifen injection. Consistent with this, specific ablation of Dclk1-expressing tumor

stem cells resulted in a marked regression of polyps without apparent damage to thenormal intestine. Therefore, targeting Dclk1-expressing cells appears to be a potential
therapeutic strategy against intestinal tumors.

Recent studies have also shown that digestive organ tumors are complex and can bederived from both normal tissue stem cells and differentiated cells. Another factor

complicating the tumor hierarchy is that genetic or epigenetic alterations caused byintrinsic and extrinsic events conceal the identity of the “cell of origin” of tumors including

tumor stem cells. This raises the question of whether “cell of origin” affects the biologicalbehaviors of tumors in the presence of the same genetic or epigenetic alterations.

In this symposium, I presented the examples of pancreatic tumors in the association

with Dclk1 and SWI/SNF chromatin remodeling complexes. In the pancreas, pancreaticacinar cells with oncogenic Kras formed pancreatic intra-epithelial neoplasia (PanIN) via

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131

acinar-to-ductal metaplasia (ADM). Additional inactivation or loss of p53 led PanIN toprogress to pancreatic ductal adenocarcinoma (PDA). During this process, Kras activationin Dclk1-expressing duct or islet cells did not affect their proliferation. However, undercaerulein-induced inflammatory conditions, Kras activation in Dclk1-expressing cells

resulted in PanIN formation. Furthermore, Brg1, a subunit of SWI/SNF complexes foundin both eukaryotes and prokaryote, in acinar cells suppressed the development of ADM
and PanIN as in the case in intestinal tumors [2, 3]. In contrast, loss of Brg1 in pancreatic

duct cells cooperated with oncogenic Kras to form intraductal papillary mucinous

neoplasm (IPMN), a distinct precursor that progresses to PDA as in the case in PanIN. As

well, loss of Arid1a, another subunit of SWI/SNF complexes, also showed resembling

phenotypes. Importantly, IPMN-derived PDA was less lethal than PanIN-derived PDA inboth mouse and human.

In addition, Brg1 deletion inhibited Kras-dependent PanIN

development from adult acinar cells, but promoted Kras-driven preneoplastictransformation in adult duct cells. Thus, “cell of origin” may skew the initiation,

developmental process, and prognosis of PDA even in the presence of the same genetic

changes.

Based on these data, we discussed how digestive organ tumors are initiated anddevelop in a “cell of origin” context dependent manner especially focusing on the pancreas.

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H. Seno.

References
1. Nakanishi Y, Seno H, Fukuoka A, Ueo T, Yamaga Y, Maruno T, Nakanishi N, Kanda K,
Komekado H, Kawada M, Isomura A, Kawada K, Sakai Y, Yanagita M, Kageyama R,
Kawaguchi Y, Taketo MM, Yonehara S, Chiba T. Dclk1 distinguishes between tumor
and normal stem cells in the intestine. Nat Genet. 2013;45:98-103.
2. von Figura G, Fukuda A, Roy N, Liku ME, Morris Iv JP, Kim GE, Russ HA, Firpo MA,
Mulvihill SJ, Dawson DW, Ferrer J, Mueller WF, Busch A, Hertel KJ, Hebrok M. The
chromatin regulator Brg1 suppresses formation of intraductal papillary mucinous neoplasm and pancreatic ductal adenocarcinoma. Nat Cell Biol. 2014;16:255-267.
3. Takada Y, Fukuda A, Chiba T, Seno H. Brg1 plays an essential role in development andhomeostasis of the duodenum through regulation of Notch signaling. Development.
2016;143:3532-3539.

Hiroshi Seno, MD, PhD

1991
1991
1992
1995
2001
2005
2008
2015-present

Graduated from Kyoto Univ., Faculty of Medicine
Resident, Kyoto Univ. Hospital
Resident, Kurashiki Central Hospital
Staff, Shizuoka General Hospital
Instructor, Kyoto Univ.
Research Associate, Washington Univ., USA
Senior Lecturer, Kyoto Univ.
Professor, Kyoto Univ.

133

LGR5+ STEM CELLS IN EPITHELIAL HOMEOSTASIS,
REGENERATION & DISEASE OF THE STOMACH

Nick Barker

A*STAR Institute of Medical Biology, 138648 Singapore
Centre for Regenerative Medicine, The University of Edinburgh
Edinburgh, EH16 4TJ, UK(Nicholas.barker@imb.a-star.edu.sg)

The gastric epithelium continuously self-renews throughout life, driven by limited

reservoirs of resident Lgr5+ adult stem cells [1-5]. In vivo ablation of Lgr5+ cells severely

impairs epithelial homeostasis in both the pyloric antrum and the corpus. Transcriptome

analysis of the pyloric Lgr5+ stem cells reveals novel gastric stem cell-specific markers thatcan be used to selectively target cancer-causing mutations to the Lgr5 + stem cellcompartment in the stomach as a means of evaluating their contribution to gastric cancer

initiation. These new pyloric stem cell markers include membrane-expressed genes thatcan be used to isolate human gastric stem cells for therapeutic applications. Genes
including AQP5 are currently being validated as stem cell markers via in vivo lineage

tracing using new AQP5-EGFP-ires-CreERT2 mouse models. Using antibodies against
AQP5, we can isolate endogenous pyloric stem cells from wild-type mice that are highly

enriched for Lgr5 expression and selectively generate gastric organoids in ex vivo culture.

Future efforts will be directed at using anti-AQP5 antibodies to isolate the equivalent

human pyloric stem cells for evaluation via organoid assay. We hope that this will provide

a source of pure human stomach stem cells for regenerative medicine applications in theclinic.

We additionally document Lgr5 expression on a subset of Chief cells in the corpus. In

vivo lineage tracing using a novel Lgr5-2A-CreERT2 model and ex vivo organoid culture
assays reveals these to be a damage-inducible stem cell population contributing toepithelial repair following Parietal cell atrophy. In comparison with other publishedmarkers such as Troy, Sox2 and Mist1, Lgr5 exhibits the most restricted expression patternin both mouse and human corpus. Conditional mutation of these damage-inducible Lgr5+

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N. Barker

corpus stem cells drives the formation of SPEM, a pre-cancerous lesion considered to be theprecursor to invasive gastric cancer in humans, identifying these cells as a source of gastriccancer. We also present a new mouse model of human metastatic gastric cancer,

incorporating the first stomach-specific Cre line, Cldn-18-CreERT2. Using this Cre toselectively introduce conditional mutations into the stomach epithelium efficiently drives

invasive, metastatic gastric cancer throughout the glandular region. These new mousemodels of human gastric cancer will be employed to derive invaluable mechanistic insight

into gastric cancer initiation and progression, for evaluating potential Lgr5+ cancer stem

cells and as an accurate pre-clinical model for evaluating novel gastric cancer drugs.

Finally, we present preliminary data on the identification of new Lgr5+ stem cell
populations and their evaluation as cancer cell origins in various tissues using a new Lgr5-

2A-CreERT2 mouse model.

References
1. Ng A, Tan S, Singh G, Rizk P, Swath Y, Tan TZ, Huang RY-U, Leushacke M, & Barker N.
Lgr5 Marks Stem/Progenitor Cells in Ovary and Tubal Epithelia. Nat Cell Biol 2014;
16:745
2. Barker N, de Lau W, Low TY, Koo B-K, Li V, Teunissen H, Kujala P, Haegebarth A,
Peters PJ, van de Wetering M, Stange DE, van Es J, Guardavaccaro D, Schasfoort R,
Mohri Y, Nishimori K, Mohammed S, Heck SM, Heck AJR and Clevers H. Lgr5
homologs associate with Wnt receptors and mediate Rspondin signaling. Nature, 2011;
476:293
3. Barker N, Huch, M, Kujala P, van de Wetering M, Snippert HJ, van Es JH, Sato T,
Stange DE, Begthel H, van den Born M, Danenberg E, van den Brink S, Korving J, Abo A,
Peters PJ, Wright N, Poulsom, R and Clevers H. Lgr5+ve Stem Cells Drive Self-Renewal in
the Pyloric Stomach and Build Long-Lived Pyloric Gastric Units In-Vitro. Cell Stem Cell
2010; 6:25
4. Barker N, Ridgway RA, van Es JH, van de Wetering M, Begthel H, van den Born M,
Danenberg E, Clarke AR, Sansom OJ, and Clevers H. Crypt stem cells as the Cells-ofOrigin of Intestinal Cancer. Nature, 2009; 457:608
5. Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, Haegebarth A,
Korving J, Begthel H, Peters PJ, Clevers H. Identification of stem cells in small intestineand colon by marker gene Lgr5. Nature 2007; 449:1003

Dynamics of Cancer Stem Cells

Nick Barker, PhD

1992-1995
1995-2001

2001-2005

2006-20102010-20142015-present

PhD, University of Reading, UK
Postdoc, University Medical Center Utrecht, theNetherlands
Staff Scientist, Semaia Pharmaceuticals BV, theNetherlands
Staff Scientist, Hubrecht Institute, Utrecht, theNetherlands
Senior Principal Investigator, IMB, Singapore
Research Director, Institute of Medical Biology,
Singapore

135

136

CELL CYCLE REGULATION IN CANCER STEM CELL

Keiichi I. Nakayama

Medical Institute of Bioregulation, Kyushu University
3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan(nakayak1@bioreg.kyushu-u.ac.jp)

Most cancer stem cells (CSCs) are quiescent, and this characteristic provides these cellswith resistance to conventional anticancer therapies that preferentially target dividing cells.

CSCs that persist in spite of therapies may result in relapses and metastases. Therefore,

elucidation of the mechanism by which CSCs maintain quiescence is critical for thedevelopment of new therapeutic approaches toward the elimination of cancer.

Fbxw7 is the F-box protein component of an SCF-type ubiquitin ligase, in which it

functions as a receptor responsible for the recognition of many targets including c-Myc andNotch. We found that Fbxw7 plays a pivotal role in maintenance of quiescence inleukemia-initiating cells (LICs) of chronic myeloid leukemia (CML) (Figure 1). Ourfindings reveal that ablation of Fbxw7 in a mouse model of CML results in accumulation ofc-Myc and disruption of quiescence in LICs. Furthermore, we demonstrate that Fbxw7deficient LICs are sensitive to currently available anticancer drugs and combination
therapy with Fbxw7 depletion and these drugs is able to eradicate LICs, leading to adecreased relapse rate and a significant survival advantage. Finally, we present data thatsuch combination therapy is also effective for human CML LICs, supporting our conclusionthat Fbxw7 is a promising target for the treatment of human leukemia. Therefore,

combination of Fbxw7 suppression and anti-cancer drugs may provide the basis for new
approaches to eradication of CSCs.

However, we found that Fbxw7 suppression might increase the rate of metastasis. We

generated mice that are deficient in Fbxw7 specifically in bone marrow–derived cells (Fbw7cKO mice). Intravenous injection of melanoma (B16/F10 or B16/F1) cells or of lung cancer(Lewis lung carcinoma) cells into these mutant mice revealed that lung metastasis wasDynamics of Cancer Stem Cells

137

enhanced and that the animals died sooner compared with injected wild-type controls.

Orthotopic transplantation of E0771 murine breast cancer cells also showed that metastasis

to the lungs was markedly enhanced in Fbw7 cKO mice, whereas the size of the primarytumors did not differ significantly between the mutant and wild-type animals (Figure 2).

Deletion of Fbxw7 in mouse bone marrow-derived stromal cells induced theaccumulation of Notch and consequent transcriptional activation of the CCL2 gene.

Increased serum levels of the chemokine CCL2 in Fbxw7-deficient mice resulted in the

recruitment of both monocytic myeloid-derived suppressor cells and macrophages andpromoted metastatic tumor growth (Figure 3).

Administration of propagermanium, an antagonist of the CCL2 receptor CCR2, blocked

the enhancement of metastasis in Fbxw7-deficient mice. Furthermore, FBXW7 expressionin peripheral blood was associated with the serum CCL2 concentration and prognosis in

human breast cancer patients. These results suggest that modulation of the Fbxw7-Notch-

CCL2 axis may provide the basis for new approaches to suppression of cancer metastasis(Figure 4).

Figure 1

The Fbxw7 – c-Myc axis is pivotal for maintenance of quiescence (G0 phase) and stemness inFigure 1. The
Fbxw7–c-Myc
axis is pivotal
maintenanceof quiescence(G0 phase
LICs. Combinationof Fbxw7
ablation and anticancer
drugs isfor
effective
for LIC eradicationbypurging
LICs
fromquiescence.
and stemness in LICs. Combination of Fbxw7 ablation and anticancer drugs is effectivefor LIC eradication by purging LICs from quiescence.

138

K. I. Nakayama

Figure 2

Fbxw7 deletion in bone marrow promotes cancer metastasis in an intravenous tumor cell

Figure 3

Model for the promotion of cancer metastasis by loss of Fbxw7 in the host environment.

Figuretransplantation
2. Fbxw7 deletionin bone marrow promotes cancer metastasis in an intravenous
model (melanoma and lung cancer) and in an orthotopic transplantation
tumormodel
cell transplantation
model (melanoma and lung cancer) and in an orthotopic(breast cancer).
transplantation model (breast cancer).

Figure3. Modelfor thebypromotion
oftocancer
metastasisofbyitslossof Fbxw7causedin theby
host
Excessive
signaling
Notch1 due
the impairment
degradation
Fbxw7
environment.
Excessive
signaling production
by Notch1 ofdue
to theimpairment
its degradation
ablation gives
rise to increased
CCL2
by bonemarrow –ofderived
mesenchymal
stem(MSCs).
The
consequent
recruitmentof myeloid-derived
suppressor
causedby cells
Fbxw7
ablationgives
rise to increased
productionof CCL2 by bonemarrow–cells
2
(MDSCs)and tumorstem
associatedfacilitates
metastatic oftumor
growth.
derivedmesenchymal
cells macrophages(MSCs). The(TAMs)consequent
recruitment
myeloidderived suppressor cells (MDSCs) and tumor associated macrophages (TAMs) facilitates
metastatic tumor growth.

Dynamics of Cancer Stem Cells

Figure 4

139

Propagermanium prevents cancer metastasis induced by CCL2 overproduction.

Figure Propagermanium
4. Propagermanium
prevents
cancermetastasis
inducedby CCL2
is a commerciallyavailable
drug approvedfor hepatitis
B treatment,
andoverproduction.
Propagermanium
is a commercially available drug approved forhas been used
for more than 20 years.
hepatitis B treatment, and has been used for more than 20 years.
References

1. Nakayama KI, Nakayama K. Ubiquitin ligases: cell-cycle control and cancer. Nature
Rev. Cancer 6(5): 369-381, 2006.
2. Matsuoka S, Oike Y, Onoyama I, Iwama A, Arai F, et al. Fbxw7 acts as a critical fail-safe
against premature loss of hematopoietic stem cells and development of T-ALL. Genes
Dev. 22(8): 986-991, 2008.
3. Takeishi S, Matsumoto A, Onoyama I, Naka K, Hirao A, et al. Ablation of Fbxw7 eliminates leukemia-initiating cells by preventing quiescence. Cancer Cell 23(3): 347-361,
2013.
4. Yumimoto K, Akiyoshi S, Ueo H, Sagara Y, Onoyama I, et al. F-box protein FBXW7
inhibits cancer metastasis in a non-cell-autonomous manner. J. Clin. Invest. 125(2): 621635, 2015.



4

140

K. I. Nakayama

Keiichi I. Nakayama, MD, PhD

1980-1986

1986-1990

1990-1995

1995-1996
1996-present

Graduated from Tokyo Medical and Dental Univ.
School of Medicine
Graduated from Juntendo Univ. Graduate School of
Medicine
Post-doctoral fellow, Howard Hughes Medical Institute,
Washington Univ. School of Medicine
Senior Scientist, Nippon Roche Research Center
Professor, Medical Institute of Bioregulation, Kyushu
Univ.

141

CANCER STEM CELL BIOLOGY ENTERS THE CLINIC

Shung Cai, Yohei Shimono, Maddalena Adorno and Michael F. Clarke
Stanford Institute for Stem Cell Biology and Regenerative Medicine
265 Campus Drive, Stanford, CA 94305, USA(mfclarke@stanford.edu)

Most common cancers, such as cancers of the breast and colon, arise in organs that

contain a small population of stem cells that constantly replenish the mature cells of thetissue. Stem cells are defined by the ability to divide and give rise to a new stem cell (self-

renewal), as well as the ability to give rise to the differentiated cells of an organ, and thus
are the only long-lived cell population in many tissues. It is becoming increasingly

apparent that cancers utilize stem cell pathways, particularly self-renewal pathways.
Indeed, these stem cell pathways contribute to critical features of cancers.

Because cancers arise in tissues with stem cells and stem cell pathways contribute to themorbidity and mortality of cancers, it is important to understand self-renewal pathways.

Hematopoiesis is the best understood stem cell system. Although the details of eachorgans stem cell and differentiation hierarchy functions might differ, understanding thehematopoietic stem cell (HSC) can serve as a foundation for understanding the othertissues. In the blood, transplantation of a single HSC can regenerate the blood system of amouse for life, and can generate thousands more stem cells which can also regenerate theblood system [1]. No other hematopoietic cell has this capability. The unique ability to self-

renew underlies the ability of an HSC to maintain the blood system. Bmi1 was the firstgene that was identified that regulates self-renewal. It is a member of the polycomb family,

and it regulates self renewal by repressing senescence, apoptosis, and differentiation

pathways specifically in stem cells, but not in more differentiated progeny (Figure 1) [2].

Subsequent work has shown that Bmi1 is required for the self-renewal of brain, mammary
gland and mesenchymal stem cells [3, 4].

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M. F. Clarke et al.

Figure 1

Bmi1 is necessary for HSC self-renewal. Bmi1 represses the Tp53, Cdkn2a (p16) and Hox differentiation pathways specifically in HSCs. Loss of Bmi1 results in exhaustion of the HSC
pool.

Although expression of Bmi1 is necessary for the self-renewal of stem cells frommultiple tissues, it is not sufficient. Thus, a major question is why Bmi1 expresssion in cells

other than stem cells cannot self-renew. Yohei Shimono et al. looked in the mammary

gland, and found that other than stem cells, most epithelial cells in the mammary glandstem express members of the miR-200 microRNA family. He found that miR-200 targets

Bmi1 and prevents the self renewal of the stem cells [5]. Similarly, Adorno et al. found that

expression of Usp16, which modulates Bmi1 by removing the Bmi1-mediatedubiquitination of proteins, is expressed at higher levels in non stem cells [6]. This showsthat stem cells can be defined by two properties. First, by the expression of genes such as

Bmi1 which are required for self-renewal. Second, by the lack of expression of genes suchas miR-200 and Usp16 that prevent self renewal (Figure 2).

Recent evidence suggests that colon and breast cancers arise from normal stem or earlyprogenitor cells [7]. Our laboratory was the first to isolate breast and colon cancer stem

cells, demonstrating that these cells are critical for the growth and metastasis [8, 9]. Thesefindings have implications for the treatment of these cancers. We reasoned that thefrequency of cancer stem cells in early tumors could be used to identify high-risk Stage-II

Concluding Keynote Lecture

Figure2

143

Bmi1 modulators prevent self-renewal in non stem cell populations. Bmi1 is expressed in the
HSC and its immediate non-stem cell progenitor, the MPP. The MPP expresses Usp16 andmiR-200, which modulate Bmi1 and prevent the self-renewal of the MPP.

colon carcinomas patients who need adjuvant treatment after surgery. Microarray-based

multi-gene expression signatures derived from stem/progenitor cells hold promise, but are

difficult to implement in clinical practice. We therefore used a novel bioinformatics
approach to search for robust, individual biomarkers of colon epithelial differentiation

across gene-expression arrays. Based on the availability of a clinical-grade diagnostic

assay, the top candidate was tested for association with disease-free survival (DFS) andbenefit form adjuvant chemotherapy using subgroup analysis on a collection of

independent and retrospective cohorts of colon cancer patients. The transcription factor

CDX2 ranked first in our screen. A small population of colon carcinomas lacking CDX2
expression (CDX2neg) were associated with reduced 5-year DFS independently of stage,

grade, age or gender, in both discovery (n=32/466, 6.9%; HR=3.44, 95%CI=1.60-7.38,

p=0.002) and validation datasets (n=38/314, 12.0%; HR=2.42, 95%CI=1.36-4.29, p=0.003).
The difference in 5-year DFS between CDX2 neg and CDX2 pos tumors was statisticallysignificant in Stage-II patients (discovery dataset: 49% vs. 87%, p=0.003; validation dataset:

51% vs. 80%, p=0.004). Stage-II CDX2neg tumors treated with adjuvant chemotherapy wereassociated with improved 5-year DFS as compared to surgery alone in a pooled database of

all patient cohorts (chemotherapy vs. no-chemotherapy, 56% vs. 91%, p=0.006). Lack of

CDX2 expression appears to identify a subset of high-risk Stage-II colon cancer patients

who appear to benefit from adjuvant chemotherapy. To our knowledge, this is the firsttime that the investigation of cancer stem cells has led to improvement of patient outcomes

and has informed therapeutic decisions in the clinic (Figure 3) [10].

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M. F. Clarke et al.

Figure3

CDX2 identifies patients who benefit from therapy. A) Most patients with stage II coloncancer do not benefit from adjuvant chemotherapy (top line). B) Patients with CDX2 minus
tumors do benefit from chemotherapy (top line). C) When patients with CDX2 minus
tumors are removed from the dataset, there is virtually no benefit from adjuvant
chemotherapy. Note that there still might be an unidentified small group of patients who dobenefit. The top line in each panel is patients who received adjuvant therapy.

In summary, stem cell self-renewal pathways are used by cancer cells to maintaintumors and enable them to metastasize. Recent evidence demonstrates that cancer stem

cell biology indeed has clinical significance. In can be used to identify patients with poor

prognosis tumors. More importantly, in can be used to inform therapeutic decisions andsave lives. Finally, identifying drugs that more effectively target cancer stem cells should

improve clinical outcomes and save lives.

References
1. Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mousehematopoietic stem cells. Science. 1988;241(4861):58-62. PubMed PMID: 2898810.
2. Park IK, Qian D, Kiel M, Becker MW, Pihalja M, Weissman IL, Morrison SJ, Clarke MF.
Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells.
Nature. 2003;423(6937):302-5. PubMed PMID: 12714971.
3. Molofsky AV, Pardal R, Iwashita T, Park IK, Clarke MF, Morrison SJ. Bmi-1 dependence
distinguishes neural stem cell self-renewal from progenitor proliferation. Nature.
2003;425(6961):962-7. PubMed PMID: 14574365.

Concluding Keynote Lecture145

4. Pietersen AM, Evers B, Prasad AA, Tanger E, Cornelissen-Steijger P, Jonkers J, vanLohuizen M. Bmi1 regulates stem cells and proliferation and differentiation of committed
cells in mammary epithelium. Curr Biol. 2008;18(14):1094-9. Epub 2008/07/19. doi:
S0960-9822(08)00874-9 [pii]5. 10.1016/j.cub.2008.06.070. PubMed PMID: 18635350.
6. Shimono Y, Zabala M, Cho RW, Lobo N, Dalerba P, Qian D, Diehn M, Liu H, Panula SP,
Chiao E, Dirbas FM, Somlo G, Pera RA, Lao K, Clarke MF. Downregulation of miRNA200c links breast cancer stem cells with normal stem cells. Cell. 2009;138(3):592-603.
Epub 2009/08/12. doi: S0092-8674(09)00850-2 [pii]7. 10.1016/j.cell.2009.07.011. PubMed PMID: 19665978; PMCID: 2731699.
8. Adorno M, Sikandar S, Mitra SS, Kuo A, Nicolis Di Robilant B, Haro-Acosta V, Ouadah Y,
Quarta M, Rodriguez J, Qian D, Reddy VM, Cheshier S, Garner CC, Clarke MF. Usp16
contributes to somatic stem-cell defects in Down's syndrome. Nature. 2013;501(7467):
380-4. doi: 10.1038/nature12530. PubMed PMID: 24025767; PMCID: PMC3816928.
9. Barker N, Ridgway RA, van Es JH, van de Wetering M, Begthel H, van den Born M,
Danenberg E, Clarke AR, Sansom OJ, Clevers H. Crypt stem cells as the cells-of-origin of
intestinal cancer. Nature. 2008. Epub 2008/12/19. doi: nature07602 [pii]10. 10.1038/nature07602. PubMed PMID: 19092804.
11. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA. 2003;100(7):3983-8.
PubMed PMID: 12629218.
12. Dalerba P, Dylla SJ, Park IK, Liu R, Wang X, Cho RW, Hoey T, Gurney A, Huang EH,
Simeone DM, Shelton AA, Parmiani G, Castelli C, Clarke MF. Phenotypic characterizationof human colorectal cancer stem cells. Proc Natl Acad Sci USA. 2007;104(24):10158-63.
PubMed PMID: 17548814.
13. Dalerba P, Sahoo D, Paik S, Guo X, Yothers G, Song N, Wilcox-Fogel N, Forgo E,
Rajendran PS, Miranda SP, Hisamori S, Hutchison J, Kalisky T, Qian D, Wolmark N,
Fisher GA, van de Rijn M, Clarke MF. CDX2 as a Prognostic Biomarker in Stage II andStage III Colon Cancer. N Engl J Med. 2016;374(3):211-22. doi: 10.1056/NEJMoa1506597.
PubMed PMID: 26789870; PMCID: PMC4784450.

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M. F. Clarke et al.

Michael F. Clarke, MD

1980-1983

1983-1986

1986-1992

1992-1998

1992-2005

1992-2005

1998-2005

2003-2005

2005-present

Clinical Associate, Medicine Branch, National Cancer
Institute, National Institutes of Health
Staff Fellow, National Institute of Arthritis, Diabetes,and Digestive and Kidney Diseases
Assistant Professor, Internal Medicine, Department of
Hematology and Oncology, Univ. of Michigan
Associate Professor, Internal Medicine, Department of
Hematology and Oncology, Univ. of Michigan
Attending Physician, Univ. of Michigan Bone Marrow
Transplant Unit
Attending Physician, Univ. of Michigan, Lymphoma
ClinicProfessor, Internal Medicine, Department of
Hematology and Oncology, Univ. of Michigan
Professor of Cell and Development Biology, Univ. of
Michigan
Professor of Internal Medicine, The Karel and Avice
Beekhuis Endowed Professorship in Cancer Biology,
Associate Director of the Stanford Institute for Stem
Cell Biology and Regenerative Medicine

147

CLOSING REMARKS

Masaki Mori

Department of Gastroenterological Surgery
Osaka University Graduate School of Medicine
2-2 Yamadaoka, Suita, Osaka 565-0871, Japan(mmori@gesurg.med.osaka-u.ac.jp)

The 47th international symposium of the Princess Takamatsu Cancer Research Fund provided an

excellent opportunity for scientists to appreciate where we stand regarding cancer stem cell research,

as well as highlighting the real prospect of treating cancer by targeting cancer stem cells. It wasmost encouraging to see the emerging evidence supporting the original notion that cancer stem cells

are responsible for treatment resistance and disease recurrence, and that at least in some cancers,
targeting cancer stem cells could cure what would otherwise be a terminal disease. Here are some ofthe highlights from the symposium.

Large scale clinical studies show that haematological and solid cancers with signatures ofstemness are more likely to recur and be associated with worse prognosis, supporting the original
hypothesis that cancer stem cells are responsible for treatment resistance and cancer recurrence.

Thus stemness can also serve as a useful biomarker, and CDX2 expression was found to be a usefulmarker in stage-II colon cancer for identifying individuals who would benefit from adjuvant

chemotherapy.

The organoid system and the development of novel approaches to studying cancer stem cells insitu through creative tracing methods have allowed us to understand about the origin and fate ofcancer stem cells, namely that cancer stem cells appear to arise from transformed stem or pluripotent

cells. Investigation of the origin of metaplastic cancers also showed that they originate from normal
pluripotent cells.

Recent success with immune check point inhibitors for cancer treatment have refocused ourattention to the role of the immune system in carcinogenesis. Cancers are thought to arise throughthe evasion of immune-surveillance that would normally destroy transformed cells. If we are able toanswer how the transformed stem cells (i.e. the cancer stem cells) are able evade the immune system

148

M. Moriin the first place, we may be able to effectively treat and prevent cancer. One such mechanism

appears to be the expression of the “don’t eat me” protein CD47 on the cell surface of cancer cells

and cancer stem cells, preventing macrophages from attacking these cells. Blocking CD47 was able

to eliminate human cancer stem cells, and this approach is currently undergoing investigation in aclinical trial.

It was very much evident from the lectures that we now know much more about the biology ofcancer stem cells, and this has lead to many other promising strategies that could target cancer stem

cells. The targeting of quiescence, which is one of the main features of cancer stem cells through the
suppression of Fbxw7, the F-box protein component of an SCF-type ubiquitin ligase, was one

elegant example of making leukemia-initiating cells in chronic myeloid leukemia become moresensitive to the currently available anticancer drugs. Others have directly targeted the epigenetic

mechanisms involved with the maintenance of stemness and/or chemoresistance, thereby inducing

differentiation or transdifferentiation of cancer stem cells. Cancer stem cells were found to belocated in certain places within tumours, reinforcing the importance of the cancer stem cell niche,

and other studies demonstrated the importance of the metabolic crosstalk between the cancer stem

cells and their niche.

We now have a marker, DCLK1, that can reliably differentiate cancer stem cells from normal

stem cells, which asides from research purposes, could allow for precise treatment of cancer whileleaving the healthy tissues intact. That organoids required mutations only in four genes (APC,

SMAD4, TP53 and KRAS) to generate intestinal cancer was most intriguing and provides support
that cancer stem cells could be targeted through the manipulation of a finite number of signalling
pathways.

Much has therefore been achieved in the field of cancer stem cells in the last few years, and we

sincerely hope that the cancer stem cell concept will be put through its final test one day soon, that is

to target them in cancers to hopefully achieve long-term cure in patients. One concern shared bymany is the plasticity of stemness, and most importantly the ability of differentiated cancer cells torevert to cancer stem cells. Therefore, the eradication of cancer stem cells alone may only betransient and insufficient.

We may also need to be wary of the way in which cancer stem cell therapy is evaluated, given it

may not necessarily lead to the disappearance of tumours. Instead, the long-lasting growth inhibitionof tumours may be a more appropriate and meaningful endpoint. Also, we may need to generatereliable animal models of cancer recurrence in order to evaluate cancer stem cell therapy.

On behalf of the organisers I would like to thank the Princess Takamatsu Cancer Research Fundfor hosting this international symposium, and our distinguished speakers and guests for making this
symposium a great success.

Closing Remarks

Masaki Mori, MD, PhD

1986-1987

1987-1991

1991-1993

1993-1994

1994-1998

1998-2008

2008-present

149

Assistant Professor, Department of Pathology, Kyushu
Univ.
Surgeon and Assistant Professor, Department ofSurgery II, Kyushu Univ.
Research Fellow, Dana-Farber Cancer Institute, Harvard
Medical School
Surgeon and Assistant Professor, Department ofSurgery II, Kyushu Univ.
Chief of Surgical Oncology and Associate Professor,
Department of Surgery, Medical Institute of
Bioregulation, Kyushu Univ.
Chairman Professor of Surgery, Medical Institute of
Bioregulation, Kyushu Univ.
Chairman Professor of Surgery, Graduate School of
Medicine, Osaka Univ.