?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 3 March 2008
and long-term immunological and clinical
benefits of this approach need to be thor-
We thank Rogier van Gent, Mette Hazen-
berg, and Jose Borghans for their input.
Address correspondence to: Frank Mie-
dema, University Medical Center Utrecht,
Department of Immunology, Lundlaan
6, PO Box 85090, Utrecht UNK 3508 AB,
The Netherlands. Phone: 31-88-755-7674;
Fax: 31-30-250-4305; E-mail: f.miedema@
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Discovering early molecular determinants
Grover C. Bagby
Departments of Medicine and Molecular and Medical Genetics, Oregon Health and Sciences University,
and Northwest Veterans Affairs Cancer Research Center, Portland, Oregon, USA.
Advances in biotechnology and genomics
have catalyzed enormously important dis-
coveries in the field of cancer biology. Some
of the findings from studies of molecular
pathogenesis have led to the development
of new therapeutic agents that have nota-
bly controlled the growth of malignant
cells in vivo (1). Truly targeted therapy is
that which effectively interdicts a survival
or replication pathway on which the malig-
nant cell depends but normal cells do not.
To develop this kind of therapeutic agent
requires that one first identify such a defect
in a malignant cell population, then devel-
op an agent that attacks it in a specific way.
The target must also be validated in clini-
cal trials. That is, clinical responses must
be attributable to the capacity of the thera-
peutic agent to interdict the function of the
target molecule in the neoplastic cells.
Not only have advances in molecularly
targeted therapy saved thousands of lives
(1), the successes to date have validated the
simple principle that if you understand
it you can fix it. For cancer therapeutics
this idea is widely accepted today and is
supported by massive investments in this
approach by pharmaceutical and biotech-
nology firms. However, while few would
argue with the idea that preventing can-
cer is better than treating it, investigators
in the field of cancer prevention have not
particularly warmed to the notion that the
molecular strategies used for developmen-
tal therapeutics can support their goals as
well. This situation has to change because
there is clear-cut experimental evidence
that the earliest control points for carcino-
genic change in stem cells can be identified
by focusing on stem cell pools under stress.
The natural implication is that by inter-
dicting this stress one might prevent the
evolution of neoplastic clones.
Stressed stem cell pools are
vulnerable to neoplastic change
Most leukemias evolve as clonal out-
growths of single, somatically mutated
HSCs. Studies of patients with inherited
myeloid leukemia;?ELA2, elastase 2;?FA, Fanconi ane-
mia;?MDS, myelodysplastic syndrome; SCN, severe
Conflict?of?interest: The author has declared that no
conflict of interest exists.
Citation?for?this?article: J. Clin. Invest. 118:847–850
848?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 3 March 2008
bone marrow failure syndromes (Table 1),
in which the HSCs are either constitutively
proapoptotic or hypersensitive to apop-
totic cues, suggest that the apoptotic state
itself represents a selective pressure that
favors the evolution of neoplastic clones.
The apoptotic cues that put these “unfit”
(in the Darwinian sense) HSCs at risk have
not been fully defined in these inherited
syndromes, but in the case of the inherited
bone marrow failure syndrome Fanconi
anemia (FA), sufficient evidence has devel-
oped to identify TNF-α as one of the key
pathophysiological factors influencing
clonal evolution in FA HSCs (2).
Up to 40% of children and young adults
with FA (3, 4) will exhibit signs of clonal
evolution in the bone marrow, a process
in which somatically mutated HSCs self-
replicate and give rise to progeny that
overtake the bone marrow, leading to
myelodysplastic syndrome (MDS) and
acute myeloid leukemia (AML). Prior to
the onset of clonal evolution, HSCs and
committed progenitor cells from the bone
marrow of patients bearing inactivating
mutations of two FA complementation
group C (FANCC) alleles undergo apop-
tosis in response to a variety of cytokines
(including TNF-α) at very low doses
— doses that have no capacity to suppress
proliferation and survival of normal pro-
genitors (5, 6). In both murine models and
in humans with FA, when clonal progeny
of somatically mutated stem cells appear
in the marrow, the clones are resistant to
TNF-α (2, 7, 8). These results suggested
that clonal progeny have ascended either
by selection of preexisting covert mutant
stem cells (9) or through adaptive muta-
tions that occur during selective sweeps of
TNF-α through the HSC pool.
A few months ago, in this journal, Li et
al. (2) reported a model of induced clonal
selection in which FA HSCs exposed to
TNF-α gave rise to cytogenetically abnor-
mal neoplastic clones ex vivo in a mat-
ter of 4–5 weeks. That is, the progeny
of a more fit somatically mutated stem
cell replaced those of the less fit FA cells
because this single stem cell had adapted
to the troublesome apoptotic cue. Our
expectation that this general model will
hold true for some of the other inherited
bone marrow failure syndromes is sup-
ported by the report in mice from Liu,
Link, and colleagues in this issue of the
JCI (10), which suggests that the model
is applicable to HSCs in the marrow of
patients with the inherited bone marrow
failure state known as severe congenital
Clonal evolution in SCN
SCN is a genetically heterogeneous inher-
ited disease frequently associated with
mutations of the neutrophil elastase 2
(ELA2) gene (11). Patients are reliably
responsive to pharmacological doses of
G-CSF, an agent that clearly reduces mor-
bidity and mortality in children with this
disease. The molecular mechanisms link-
ing ELA2 mutations to failure of granulo-
poiesis are unclear, although it has been
reported that myeloid precursor cells
are highly apoptotic owing to constitu-
tive activation of the unfolded protein
response in cells bearing ELA2 mutations
(12, 13). As is the case with most other
inherited bone marrow failure syndromes,
the relative risk of AML or MDS is high
in SCN (see Table 1). Interestingly, trun-
cating mutations of the G-CSF receptor
are common in SCN patients (14) and are
positively associated with clonal evolution
to AML and MDS (15). It had been known
for a number of years that this type of
mutation resulted in conditional hyperac-
tivation of the G-CSF receptor and resis-
tance to apoptosis in myeloid cells (16).
It was not known, however, whether these
activating mutations of the G-CSF recep-
tor played any role in changing the coef-
ficient of selection, specifically in the HSC
pool, an important requirement for early
leukemogenic mutations. In their current
study, Liu, Link, and colleagues (10)?used
a murine model in which HSCs expressed
a truncating mutation of the G-CSF recep-
tor (also known as Csf3r) to?demonstrate
that exogenous G-CSF permitted the
mutant clone to dominate in competitive
repopulation experiments. The G-CSF
receptor possessing the mutation was not
capable of functioning autonomously.
That is, exogenous G-CSF was absolutely
required. These authors also found that
the transcription factor Stat5, known to
be activated in normal cells by G-CSF, was
absolutely required for the mutant HSCs
to be efficient competitive repopulators,
a finding that itself clarifies a number of
prior uncertainties on the role of STAT5
in HSCs. Clones bearing the mutant recep-
tor did not evolve to AML or MDS in the
current study; a finding that recapitulates
the human disease in which even highly
clonal hematopoiesis does not inevita-
bly progress to leukemia (15). Therefore,
studies on the evolution of this mutation
in humans and the obvious consequences
of the mutation in mice strongly suggest
that the G-CSF receptor mutation is an
early event in leukemogenesis.
The role of HSC damage in clonal
The results of this nicely designed study
(10) begin to place clonal evolution in SCN
in the same category as clonal evolution
in FA. In both conditions HSCs are highly
apoptotic, a perfect microenvironmental
setting for the selection of stem cell clones
that have, as a result of somatic mutations
or epigenetic changes, acquired resistance
to factors in the microenvironment that
injure them. In both settings, the somati-
cally mutated HSCs have a high coefficient
of selection because the somatic mutation
enhanced the relationship of the HSC
with its microenvironment. In the case of
FA HSCs, new clones escape cell death by
interdicting apoptotic responses to TNF-α,
precisely the environmental cue to which
the unevolved FA HSCs are hypersensitive
(2). In patients with SCN, neoplastic HSCs
emerge as a result of a G-CSF receptor
mutation. This implies, of course, that the
HSC pool in SCN bone marrow is respond-
ing suboptimally to endogenous G-CSF
Risk of neoplastic complications in patients with inherited bone marrow failure syndromes
Diamond Blackfan anemia
Thrombocytopenia absent radius syndrome
ABy age 40–50 yr; ref. 20.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 3 March 2008
levels. In SCN and FA the pressure for the
emergence of more fit stem cells results
in the selection of clones that are better
adapted for survival in their respective
microenvironments (Figure 1).
The adaptive responses can occur in two
ways. Rarely, mutant FA HSCs correct the
inherited mutation on one of two mutant
alleles. This results in clonal reversion
(mosaicism) (17) and, in some cases, com-
plete resolution of bone marrow failure
(18). The second pathway of adaptation
reflects a kind of molecular “work around”
and aberrant activation of antiapoptotic
programs. This second adaptive pathway
is more maladaptive with respect to the
whole patient because the clonal progeny
are too resistant to apoptotic cues for their
own good. They survive when a normal cell
would have reasons not to (for example,
when high-level genetic damage should
induce apoptotic responses). These mutant
cells are more apt to suffer a concatenation
of additional mutations and are therefore
more likely to evolve to frank leukemic
stem cell clones.
The results reported in this issue by
Liu et al. (10) help to define some unique
The selection coefficient in HSC pools influences clonal evolution. Potentially leukemogenic
translocations can be found in normal individuals who do not go on to develop leukemia (9).
Shown here are somatically mutated HSCs (black) in the context of normal bone marrow (normal
HSCs are shown in gray) and in the bone marrow of a patient with an inherited bone marrow
failure syndrome (genetically unfit HSCs are shown in red). The coefficient of selection for the
somatically mutated HSC in normal hematopoiesis is low so the mutant HSC is not significantly
more fit. However, when the large population of HSCs is genetically unfit, the same mutant clone
has a better opportunity to gain a foothold. Here, the somatically mutated HSCs in the normal
and abnormal hematopoietic states have precisely the same mutation. The somatic mutant HSC
in the company of the genetically unfit HSCs has a higher selection coefficient, not because of an
autonomous attribute, but because the surrounding stem cells are highly unfit. The unfit HSCs
are purged from the bone marrow (t1) and are replaced by the progeny of the somatic mutant
(this population expands at t2). The data reported in this issue of the JCI by Liu et al. (10) dem-
onstrate that the truncation G-CSF receptor mutation is capable of influencing the expansion of
HSCs in the context of G-CSF treatment in mice. Although key studies remain to be performed
in human cells, the results do provide a likely pathophysiological mechanism (G-CSF–induced
STAT5 activation via a conditionally mutated receptor) for clonal HSC expansion in patients with
SCN, expansions that seem to be required very early in the leukemogenic process. t, time.
investigative opportunities. For example,
whether the ELA2 mutation interferes
with G-CSF signaling specifically in HSCs
should be tested directly. Positive results
would explain how the coefficient of
selection for G-CSF receptor mutations
would be sufficiently high to account for
clonal evolution in vivo. Secondly, because
activated STAT5 multimerizes and then
translocates to the nucleus to function
as a transcription factor, experimentally
defining promoter binding sites unique
to stem cells bearing the G-CSF receptor
mutation may help identify gene products
that directly enhance stem cell fitness.
Third, experiments on clonal progeny
that have evolved without a G-CSF recep-
tor mutation might be targeted to factors
that modulate G-CSF activity. For exam-
ple, might loss-of-function mutations
involving suppressors of cytokine signal-
ing (19) be involved in these less frequent
cases? Most importantly, by defining the
full scope and order of adaptive somatic
mutations we can begin to contemplate
experiments designed to enhance HSC
fitness, thereby reducing the likelihood
of clonal selection of potentially neo-
plastic stem cells. It is time to exploit the
investigative strategies that have so nicely
served developmental therapeutics teams
and turn the methods to a bold new aim
— molecularly targeted leukemia preven-
tion for patients at high risk.
I thank Laura Hays, Johanna Svahn, Gabri-
elle Meyers, Qishen Pang, Susan Olson,
Robb Moses, Markus Grompe, and D.
Wade Clapp for instructive discussions,
Susan Bagby for her encouragement, and
my laboratory team for their hard work
and dedication. I have appreciated the
long-standing support of my laboratory
by the Department of Veterans Affairs,
the NIH, and the OHSU Cancer Institute.
I also thank the dedicated staff, founders,
executive committee, and scientific adviso-
ry board of the Fanconi Anemia Research
Fund Inc. for their extraordinary efforts
Address correspondence to: Grover C.
Bagby, Portland Veterans Affairs Medi-
cal Center, Molecular Oncology Research
Laboratory, Mailstop: R&D2, Building
103, Room E221B, 3710 SW Veterans Hos-
pital Road, Portland, Oregon 97239, USA.
Phone: (503) 494-0524; Fax: (503) 494-7086;
850?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 118 Number 3 March 2008
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for leukemia prevention. Hematology. 2007:40–46.
4. Rosenberg, P.S., Greene, M.H., and Alter, B.P. 2003.
Cancer incidence in persons with Fanconi anemia.
5. Bagby, G.C., and Alter, B.P. 2006. Fanconi anemia.
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6. Haneline, L.S., et al. 1998. Multiple inhibitory
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and covert leukemic clones are generated during
normal fetal development. Proc. Natl. Acad. Sci. U. S. A.
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a strong clonal HSC advantage via activation of
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Currying favor for the heart
Jonathan A. Epstein
Department of Cell and Developmental Biology, Cardiovascular Institute, and Institute for Regenerative Medicine,
University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Epigenetics is a term used to describe fea-
tures of DNA packaging and assembly
that modify cellular process and are sta-
bly maintained when cells divide, but do
not involve changes in DNA sequence.
DNA is maintained within the nucleus
in an ordered and dynamic structure in
association with other proteins, includ-
ing histones. The complex of DNA and
associated proteins is known as chroma-
tin, and an exciting area of active research
involves the epigenetic regulation of chro-
matin structure by enzymes that modify
histones. Among these enzymes are those
that add or remove acetyl groups on lysine
tails of histones. Enzymes that remove ace-
tyl groups are called histone deacetylases
(HDACs), and those reactions are reversed
by histone acetyl transferases (HATs),
which include p300 and CREB-binding
protein (CBP). In general, HDACs act as
transcriptional repressors, since removal
of acetyl groups allows the chromatin to
pack more tightly, and access of transcrip-
tion factors to promoters is restricted.
Conversely, HATs tend to function as acti-
vators of gene expression.
HDACs have recently been implicated as
important regulators of cardiac homeosta-
sis (1). There are at least 11 mammalian
HDACs that compose the so-called class
1 and class 2 families, in addition to more
distantly related families (2). Inactivation of
some of the class 2 HDACs in mice results
in cardiac hypertrophy and subsequent
heart failure (1, 3, 4). On the other hand,
inactivation of a class 1 HDAC, HDAC2,
results in resistance to cardiac hypertro-
phy (5), which suggests that class 1 and
class 2 HDACs may play opposing roles (6).
Interestingly, chemical HDAC inhibitors,
which block both classes, tend to block
hypertrophic responses (6–8). In this issue
of the JCI, 2 papers examine the effects of
curcumin on the heart and conclude that
this commonly available spice blocks HAT
activity and prevents cardiac hypertrophy
and failure in rodent models (9, 10).
Curcumin and cardiac hypertrophy
Curcumin is a polyphenol responsible for
the yellow color of the curry spice turmeric.
It has relatively poor bioavailability when
taken orally, but also low toxicity (11). It
has been touted to possess a myriad of ben-
eficial activities, and clinical trials have been
conducted in patients with cancer, rheu-
matoid arthritis, cystic fibrosis, inflamma-
tory bowel disease, psoriasis, pancreatitis,
and other disorders (11–13). Limited data
suggest that it possesses antitumor, anti-
oxidant, and antiinflammatory activities.
Nonstandard?abbreviations?used: HAT, histone acetyl
transferase; HDAC, histone deacetylase; MEF, myocyte
Conflict?of?interest: The author has declared that no
conflict of interest exists.
Citation?for?this?article: J. Clin. Invest. 118:850–852