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Eradication of Leukemia Stem Cells as a New Goal of
Therapy in Leukemia
Farhad Ravandi and Zeev Estrov
Abstract
Leukemias have traditionally been classified and treated on the basis of phenotypic characteristics,
such as morphology and cell-surface markers, and, more recently, cytogenetic aberrations. These
classification systems are flawed because they do not take into account cellular function. The
leukemia cell population is functionally heterogeneous: it consists of leukemia stem cells (LSC)
and mature leukemia cells that differentiate abnormally to varying extents. Like normal hematopoietic stem cells, LSCs are quiescent and have self-renewal and clonogenic capacity. Because
they are quiescent, LSCs do not respond to cell cycle ^ specific cytotoxic agents used to treat
leukemia and so contribute to treatment failure.These cells may undergo mutations and epigenetic
changes, further leading to drug resistance and relapse. Recent data suggest that mature leukemia cells may acquire LSC characteristics, thereby evading chemotherapeutic treatment and
sustaining the disease. Ongoing research is likely to reveal the molecular mechanisms responsible
for LSC characteristics and lead to novel strategies for eradicating leukemia.
The hematopoietic system is thought to originate from
pluripotent hematopoietic stem cells (HSC) capable of
producing a hierarchy of downstream multilineage and unilineage progenitor cells that differentiate into mature cells
(1, 2). HSCs have self-renewal and clonogenic abilities and
can differentiate into multiple lineages. HSCs with the capacity for both long-term and short-term repopulation of the
mouse hematopoietic system have been characterized. These
rare cells give rise to progenitor cells, which are destined to
generate fully differentiated cells. HSC self-renewal is thought
to be either symmetrical, producing two daughter HSCs, or
asymmetrical, producing an identical HSC and a progenitor
with diminished self-renewal capacity but with the ability to
enact clonal expansion and maintain the circulating blood cell
population (1 – 3).
It has recently been hypothesized that leukemogenesis, and,
for that matter, carcinogenesis, arises from neoplastic stem
cells. Leukemia stem cells (LSC) exhibit characteristics similar
to those of normal HSCs and are thought to arise from normal
stem cells through the accumulation of oncogenic insults (4, 5).
However, LSCs may also arise from differentiated progenitor
cells that have reacquired the capacity for self-renewal (6 – 8).
Like normal HSCs, LSCs give rise to differentiated daughter cells
that lose their self-renewal capacity. However, defects in their
cellular machinery usually eliminate their ability to differentiate
fully into morphologically and phenotypically mature cells.
As a result, the leukemic population consists of undifferentiated
and variably differentiated leukemia cells. The degree of
differentiation of leukemia cells has traditionally formed the
basis of the morphologic classification of leukemias (9, 10).
The generally accepted paradigm of leukemogenesis rests on
the theory that leukemia arises from a single cell and is
maintained by a small population of LSCs (5, 11, 12). Studies
of acute myeloid leukemia (AML) have shown that only 0.1%
to 1% of AML cells have the capacity to initiate leukemia when
injected into severe combined immunodeficient mice (4, 13). It
has long been recognized that, like solid tumors, AML consists
of a heterogeneous population of cells with a small percentage
of noncycling, quiescent cells (14, 15). Two models of leukemogenesis have long been proposed. According to the stochastic model, leukemia consists of a homogeneous population
of immature cells and a few cells that can either self-renew or
proliferate in a stochastic manner (3, 16, 17). According to
the hierarchy model, leukemia consists of a heterogeneous
population, within which only a small percentage of LSCs
sustain the disease. Recently, a third model was proposed.
According to this model, mature leukemia cells can dedifferentiate and regain LSC capacity (6, 8, 18). Whereas the first two
models hold that only LSCs sustain leukemia, the third model
allows that mature cells can regain self-renewal capacity,
thereby sustaining the disease.
Despite the development of multiple new agents that are
effective at reducing the tumor burden in patients with
leukemia, relapse continues to be the most common cause
of death, particularly in patients with AML. Current efforts
directed at detecting and quantifying minimal residual disease
are based on the assumption that eradication of all cells capable
of sustaining the disease will improve treatment outcomes.
Modern techniques for detecting minimal residual disease, such
Authors’ Affiliation: Department of Leukemia, The University of Texas M.D.
Anderson Cancer Center, Houston,Texas
Received 8/29/05; revised 10/12/05; accepted 11/3/05.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Farhad Ravandi, Department of Leukemia, Unit 428, The
University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard,
Houston, TX 77030. Phone : 713-745-0394; Fax : 713-745-4612; E-mail:
fravandi@ mdanderson.org.
F 2006 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-05-1879
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Leukemia Stem Cells as Therapeutic Targets
as PCR, are not sensitive enough to detect all residual leukemic
cells. Therefore, a state of absolute disease eradication cannot
be determined with the available tests. For example, disease
recurrence has been reported after discontinuation of therapy
with imatinib mesylate in patients with chronic myelogenous
leukemia who have achieved a complete molecular remission
(19). On the other hand, minimal residual disease will not
lead to disease recurrence if the residual leukemic cells are not
disease-sustaining or if the residual LSCs remain dormant
(20, 21). Here, we examine the role of LSCs as applied to
treatment strategies aimed at curing leukemia.
Defining LSCs
The phenotypic and functional properties of normal HSCs
have been extensively studied (22, 23). Several combinations of
cell-surface markers, such as Lin Thy+CD34+CD38 /low, have
been used to identify populations that are enriched with
HSCs (24). Other techniques include the extrusion of Hoechst
dye 33342 to identify side population cells, the use of
immature cell-surface markers such as CD133 and CD150,
and the use of Bodipy aminoacetaldehyde to assess aldehyde
dehydrogenase activity (25 – 30). It was shown that, for most
AML subtypes (with the exception of acute promyelocytic
leukemia), the cells that initiate the disease in non-obese
diabetic/severe combined immunodeficient mice have a
CD34+CD38 or CD34+CD38+/low phenotype (4, 31). However, leukemic cells bearing these phenotypes are dissimilar
functionally and include both short-term and long-term
leukemia-initiating cells (Fig. 1). Several studies have suggested
that HSCs and LSCs share some cell-surface markers but not
others. For example, HSCs and LSCs both express CD34
but not CD71 and HLA-DR. However, Thy-1 (CD90) and c-Kit
(CD117) are expressed on HSCs but not on LSCs, and CD123
and interleukin-3 receptor-a are expressed on LSCs but not
on HSCs (31 – 33). Remarkably, cytogenetically abnormal
leukemia-initiating cells have been found in the CD34+CD90+
population from several patients with AML and, in rare
cases, CD34 cells as well as CD34+ cells have successfully
engrafted and initiated human leukemia in mice (34 – 36). In
acute promyelocytic leukemia, unlike in other myeloid leukemias, the characteristic translocation has been observed in
CD34 CD38+ but not in CD34+CD38 cells (4, 37). Therefore,
a universal phenotype for LSCs may not exist and patientto-patient variations in cell-surface protein expression may be
the rule.
Of note, a similar heterogeneity exists in HSC gene
expression: genes thought to form a stem cell gene signature
have been identified; however, dissimilar data suggest that the
existence of such a signature may be premature (38 – 40).
Because stem cell – specific properties, such as self-renewal,
quiescence, and proliferation, are not governed by genes that
are specific to stem cells, it was proposed that there is a ‘‘stem
cell state’’ rather than a ‘‘stem cell portrait’’ in the hematopoietic
system (41). The stem cell state may represent a transient and
potentially reversible state that cells can assume in response to
the correct trigger. Using a similar concept, one can argue that
there is an ‘‘LSC state’’ rather than an ‘‘LSC phenotype,’’ a
concept that is supported by the well-described lineage
‘‘infidelity’’ in human leukemias (42). Although defining the
sets of conditions that pertain to the LSC state may allow the
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Fig. 1. Proposed concepts and properties of leukemic stem cells (2, 5). SL-IC,
SCID-leukemia initiating cell; AML-CFU, AML colony-forming units.
development of strategies to eliminate LSCs or render them
inconsequential, the identification of specific genes and surface
markers expressed solely by LSCs may be difficult or
impossible.
Molecular Pathways Regulating HSCs and LSCs
It is generally accepted that self-renewal is the hallmark
property of stem cells in both normal and neoplastic tissues.
Recent research has delineated molecular pathways that
regulate the self-renewal capacity of HSCs. Overexpression
and knockout experiments have identified several genes,
transcription factors, and cell cycle regulators that modulate
the self renewal and differentiation of HSCs (43, 44).
Genes such as SCL, GATA-2, LMO-2, and AML-1 (also known
as CBFA2 or RUNX1) govern the transcriptional regulation
of early hematopoiesis, and the deregulation of these genes
through chromosomal aberrations leads to the genesis of
several hematopoietic malignancies. For example, the gene
encoding the transcription factor SCL is the most frequent target
of chromosomal rearrangements in children with T-cell acute
lymphoblastic leukemia (45). SCL is normally expressed in
HSCs and immature progenitors and is down-regulated as
differentiation proceeds. As a result of chromosomal translocations, SCL is inappropriately expressed and, through
collaboration with other oncoproteins, initiates malignant
transformation (46). Similarly, transcriptional activation of
the AML-1 gene is required for definitive hematopoiesis. As a
result of translocation t(8;21), the fusion protein AML-ETO,
one of the most frequent chromosomal abnormalities in AML,
is generated (47). Constitutive expression of AML-ETO has been
shown to increase the frequency of self-renewal in stem cells
(48). Of interest, such increased self-renewal is of no apparent
pathogenic consequence, presumably because secondary mutations are necessary for the expression of the leukemic
phenotype (49).
Other transcription factors such as the Homeobox (Hox)
genes, including HoxB4, and the Wnt signaling pathway have
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recent years suggest that a small population of LSCs, through
their HSC-like properties, survive chemotherapy and sustain
the disease (3, 66). High levels of ATP-binding cassette
transporters have been reported in both normal and cancer
stem cells (68, 69). Indeed, HSCs, but not lineage-committed
progenitors, express high levels of genes responsible for
multidrug resistance-related transporters (70). As mentioned
earlier, the efflux of fluorescent dyes such as Hoechst 33342 has
been used to isolate HSCs (25, 71). Similarly, LSCs either
inherently possess drug resistance mechanisms or acquire them
through mutations (66).
Other properties of LSCs are also likely to contribute to
drug resistance and relapse. Stem cell progenies such as longterm culture-initiating cells and AML colony-forming units
are in an active cell cycle (72, 73) whereas several studies
have clearly shown that LSCs remain quiescent (65, 74 – 76).
For example, a study by Guzman et al. (75) showed that as
much as 96% of the LSC population, as defined by the phenotype CD34+CD38 CD123+, were in the G0 phase of the cell
cycle. This resting status of the putative LSCs protects them
from the commonly used cell cycle – specific chemotherapeutic
agents.
It is likely that secondary events, such as the development of
mutations, further contribute to the intrinsic resistant properties of LSCs. In a multistep pathogenic process, quiescent
LSCs may carry the initial mutagenic event leading to genomic
instability and the induction of secondary mutations that are
responsible for a more resistant phenotype. Alternatively,
random secondary mutations or mutations occurring as a
result of selective pressure caused by therapy may contribute to
disease progression or resistance. This has been seen in patients
with chronic myelogenous leukemia (CML) treated with
imatinib mesylate, in whom mutations of the ATP-binding site
of BCR-ABL are well documented (77).
well-described roles in regulating the self-renewal and differentiation of HSCs (35, 50). HoxB4 promotes the expansion
of HSCs whereas they retain their ability to differentiate into
normal lymphoid and myeloid cells (51). It is abundantly
expressed in HSCs but declines as terminal differentiation
proceeds (51). Of note, deregulated expression of Hox family
members such as HoxA9 is commonly observed in AML
(52, 53). The Wnt signaling pathway has been shown to be
critical to the development of several organs and recent studies
have illustrated its important role in the regulation of hematopoietic stem and progenitor cell function (35, 54). Overexpression of h-catenin, a downstream activator of the Wnt
signaling pathway, expands the transplantable HSC pool in
long-term cultures (3). Furthermore, activation of Wnt signaling also increases the expression of other transcription factors
and cell cycle regulators important in HSC renewal, such as
HoxB4 and Notch-1 (35, 55).
The Notch/Jagged pathway is important in regulating the
integration of extracellular regulatory signals controlling
HSC fate (56). Ligand binding leads to proteolytic cleavage
and transport of the intracellular domain of Notch into the
nucleus, where it functions as a transcription activator.
Members of the Notch family have critical roles in keeping
HSCs in an undifferentiated state and may act as a gatekeeper
for factors governing self-renewal and lineage commitment
(57). Of interest, the gene encoding the Notch receptor was
originally identified as the gene rearranged by recurrent
chromosomal translocations in some patients with T-cell acute
lymphoblastic leukemia (58). Other transcription factors and
cell cycle regulators associated with oncogenesis, such as Bmi-1
and Sonic hedgehog (Shh), may play roles in the regulation
of proliferation of both HSCs and LSCs (59, 60). Bmi-1 is
a member of the Polycomb family of genes thought to be
responsible for the preservation of gene silencing (61). It is
highly expressed in purified HSCs and its expression declines
with differentiation (62). It seems to regulate stem cell renewal
by modulating other genes that are important in cellular
functions such as proliferation, survival, and lineage commitment (62). Bmi-1 has an essential role in regulating the
proliferative potential of leukemic stem cells (63). Although
direct evidence for the role of Shh in the regulation of stem cell
renewal is lacking, in vitro studies have shown increased selfrenewal of HSCs in response to Shh albeit in combination with
other growth factors (64).
Therefore, differential expression of several transcription
factors controls the fate of HSCs and plays a critical role in
the determination of self-renewal, differentiation, and lineage
commitment. These pathways are under the control of various
intracellular stimuli as well as cytokines and stromal factors
from adjacent cells in the bone marrow microenvironment.
Further studies of these transcription factors in HSCs and the
mechanisms causing their deregulation are likely to provide us
with better targets for development of disease-specific therapies.
Strategies to Overcome LSC Resistance to Therapy
As implied by the multistep theory of carcinogenesis, LSCs
are likely to have the fewest number of molecular aberrations in
the population of the malignant cells and, as such, biologically
most similar to normal HSCs. The primary challenge in
developing treatment strategies targeted toward LSCs is to
identify proapoptotic stimuli that spare the normal HSCs while
exerting the desired effect on LSCs.
A primary concern in the development of tumor stem cell –
specific drugs is to overcome the inherent drug efflux pumps
that are highly expressed in LSCs. Several agents effective
in inhibiting the ATP-binding cassette transporters have been
studied and found to have limited clinical efficacy (78, 79). The
biggest obstacle to this approach is the similarly high
expression of these transporters in normal HSCs, making them
equally susceptible to the inhibitors (66). As such, strategies
directed at pathways that specifically regulate LSC survival
would probably be more fruitful (80). The search for
identification of survival pathways that are preferentially
overexpressed in LSCs is ongoing and several recent studies
have described means of differential activation of apoptosis
mechanisms in LSCs (75, 81, 82).
The transcription factor nuclear factor nB was found to
be constitutively activated in LSCs but not in normal HSCs
(75). Therefore, nuclear factor nB inhibitors were added to
The Role of LSCs in Drug Resistance and Relapse
Normal HSCs possess several characteristics that protect
them from potential insults. LSCs have similar properties,
including quiescence (65), resistance to drugs and toxins
through the expression of ATP-associated transporters (66),
and resistance to apoptotic stimuli (67). Data accumulated in
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Leukemia Stem Cells as Therapeutic Targets
used to monitor minimal residual disease. The clinical benefits
of early identification of minimal residual disease using these
techniques remain uncertain and are under investigation.
Despite significant progress, relapse continues to be the
most prominent factor responsible for the failure of therapy
in leukemia, particularly in AML. Recent insights into the
nature of normal and malignant stem cells have led to the
identification of quiescent and drug-resistant LSCs as the likely
minimal residual disease candidates responsible for relapse.
In the appropriate setting, assays such as PCR may identify
residual cells expressing leukemia-specific molecular abnormality; however, these techniques cannot currently distinguish
LSCs from their more mature progeny. As such, further research
should be focused on better defining the LSC state.
Characterization of the molecular and biological features of
the cells that initiate and maintain leukemia is an essential step
in the development of novel agents effective against the disease.
Skeptics may ignore the above-mentioned evidence and argue
that LSCs are simply leukemia cells previously referred to as
resting in the G0 phase of the cell cycle. Others may concentrate
their efforts on identifying a ‘‘stem cell phenotype,’’ thus
ignoring the ‘‘stem cell state’’ and the potential fluidity of
LSCs (a process in which the leukemia progenitors regain stem
cell properties). Future studies of LSC biology, concentrating
on the function of LSCs rather than strictly on their phenotype,
are likely to identify new molecular targets and effective
treatment modalities directed at LSCs.
antileukemic agents such as idarubicin in experimental models
(75, 81). Recently, single-agent parthenolide, a potent
inhibitor of nuclear factor nB, was found to induce apoptosis
in AML and CML LSCs and progenitors while sparing normal
HSCs (82). Notably, parthenolide was much more selective
in eliminating LSCs and sparing normal hematopoietic cells
than was the standard chemotherapy agent cytarabine (82).
Constitutive activation of the phosphatidylinositide-3 kinase is
also necessary for the survival of LSCs and its pharmacologic
inhibition by LY294002 leads to a dose-dependent decrease in
survival (83).
The fate of LSCs depends on the relative expression of
transcription factors and their regulation, usually by aberrant
signaling pathways (84, 85). Although it has been proposed
that recruitment of LSCs from the G0 to the S phase of the cell
cycle might contribute to their eradication by cell cycle – specific
cytotoxic agents, it is possible that prolonging the ‘‘quiescent
phase’’ could be beneficial. One could envision a scenario in
which LSCs are maintained in a state of hibernation, thereby
prolonging relapse-free survival. The best evidence for this
possibility is the existence of patients with clinically distinct
‘‘indolent’’ and ‘‘proliferative’’ forms of AML as well as the wide
variation in the duration of relapse-free interval in individual
patients.
Future Directions
Over the past several decades, the mainstay of leukemia
therapy (and treatment of other tumors) has been to induce a
complete remission and to consolidate this with further courses
of chemotherapy. Recently, flow cytometry and PCR have been
Acknowledgments
We thank David Galloway for editing this manuscript.
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Eradication of Leukemia Stem Cells as a New Goal of
Therapy in Leukemia
Farhad Ravandi and Zeev Estrov
Clin Cancer Res 2006;12:340-344.
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