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Current Drug Targets, 2010, 11, 95-110 95
1389-4501/10 $55.00+.00 © 2010 Bentham Science Publishers Ltd.
Targeted Elimination of Leukemia Stem Cells; a New Therapeutic
Approach in Hemato-Oncology
B. ten Cate1, M. de Bruyn1, Y. Wei1,2, E. Bremer1 and W. Helfrich*,1,2
1Department of Surgery, Surgical Research Laboratories, University Medical Center Groningen, University of
Groningen, Groningen, The Netherlands; 2Department of General Surgery, The First Clinical Hospital of Harbin
Medical University, Harbin, China
Abstract: Despite recent advances, treatment of leukemia is often not curative. New insights indicate that this may be
attributable to a small population of therapy-resistant malignant cells with self-renewal capacity and the ability to generate
large numbers of more differentiated leukemia cells. These leukemia-initiating cells are commonly referred to as
Leukemia Stem Cells (LSCs). LSCs are regarded as the root of leukemia origin and leukemia recurrence after seemingly
successful therapy. Not surprisingly therefore, contemporary leukemia research has focused on ways to specifically
eliminate LSCs, leading to the identification of several promising anti-LSC strategies. Firstly, LSCs may be eliminated by
antibody- or ligand-based cell surface delivery of therapeutics such as naked antibodies, immunotoxins, and
immunocytokines. This approach exploits LSC-associated surface antigens, such as CD33, CD44, CD96, CD123 and
CLL-1 for LSC-selective therapy and aims to spare normal hematopoietic stem cells. A second strategy aims to disrupt
the interactions between LSCs and their highly specialized niche. These interactions appear to be pivotal for maintenance
of the stem cell-like characteristics of LSCs. A third strategy centers on the selective modulation of aberrantly activated
signaling pathways central to LSC biology. A fourth strategy, dubbed ‘epigenetic reprogramming’, aims to selectively
reverse ep igenetic alterations that are implicat ed in ontogeny and maintenance of LSCs. In this review , we will discuss the
rationale for these LSCs-targeted strategies and highlight recent advances that may ultimately help pave the way towards
selective LSCs-elimination.
Keywords: Leukemia stem cells, AML, CML, targeting, apoptosis, antibody, epigenetics.
INTRODUCTION
To a varying degree most leukemia types initially
respond well to therapy with partial or even complete
remissions. Unfortunately, after a period of minimal residual
disease (MRD) many patients succumb to refractory relapses
of the disease. Recent insight indicates that the development
of these relapses may be due to the selective continued
survival of a small, but distinct, population of therapy-
resistant tumor-initiating cells, commonly referred to as
Leukemia Stem Cells (LSCs). LSCs are thought to originate
either from normal hematopoietic stem cells (HSCs) or from
more differentiated progenitor cells that have acquired
malignant features. In the latter case, the progenitor cells
have de-differentiated and re-acquired stem cell-like
characteristics via as yet undefined pathways [1, 2]. Both
LSCs and HSCs possess self-renewal capacity but are
relatively quiescent compared to more mature progenitors
cells (see F ig. 1). However, LSCs typically have a much
stronger capacity for cellular expansion than normal HSCs,
probably due to an increase in symmetric self-renewal
activity of LSCs [3].
LSCs were first identified in Acute Myeloid Leukemia
(AML) by John Dick and colleagues [3, 4] and have by now
been found in all major forms of leukemia [3-6]. LSCs are
*Address correspondence to this author at the Department of Surgery,
Surgical Research Laboratories, University Medical Center Groningen,
University of Groningen, Groningen, The Netherlands; Hanzeplein 1, BA44,
9713 GZ Groningen, The Netherlands; Tel:+31 50 361 3733; Fax:+ 31 50
36 32 796; E-mail: w.helfrich@med.umcg.nl
experimentally defined as cells capable of serial engraftment
in immunocompromised mice (see Fig. 1). Importantly, mice
engrafted with these LSCs develop leukemia which resem-
bles the patient’s original disease [7]. During this last decade
substantial progress in the field of LSC research has led to
the identification of many of the hallmark characteristics of
LSCs. Therapy-resistance is typical for LSCs which is
thought to arise from (1) their relative quiescent cellular
activity [8, 9], (2) their array of self-protecting mechanisms
[10], and (3) the highly protective microenvironment in
which the LSCs resides [11].
Since curative treatment of leukemia will most likely
require th e elimination of LSCs, new strategies need to be
designed that overcome the above-described features.
Importantly, in this design care should be taken to spare
normal HSCs. In this respect, strategies that target a distinct
Achilles' heel of the LSC biology appear to be particularly
promising. Here, we review the rationale of different LSCs-
specific strategies and highlight recent advances that may
ultimately lead to selective LSCs-elimination.
LSC CELL SURFACE TARGETING APPROACHES
Cell surface antigens expressed on LSCs may be exp-
loited for antibody- or ligand-based therapeutic approaches.
The ideal target antigen is exclusively and abundantly
expressed on LSCs, thereby maximizing the therapeutic
effect and minimizing off-target effects. Unfortunately, such
an antigen has not been uncovered. However, a series of
surrogate cell surface mark ers have been identified which
have favorable LSCs-restricted expression profiles. These
96 Current Drug Targets, 2010, Vol. 11, No. 1 ten Cate et al.
surrogate markers can be targeted with naked antibodies,
which typically eliminate targeted cells via Antibody
Dependent Cellular Cytotoxicity (ADCC) or Complement
Dependent Cytotoxicity (CDC). However, some antibodies
can directly interfere with the signaling activity by binding to
receptor molecules and sensitize targeted cells to apoptosis
induction [12] or directly induce apoptosis as reported for the
anti-CD20 monoclonal antibody (mAb) Rituximab [13]. The
tumoricidal potential of antibodies can be further enhanced
by conjugating them to cytotoxic drugs or radionuclides.
Alternatively, cell surface molecules may also be targeted
using recombinant forms of their corresponding natural
ligand. Ligand-based approaches have been used to deliver
cytotoxic drugs to the target cell and to trigger apoptosis-
inducing receptors. However, an obvious potential limitation
to antibody-based and ligand-based therapy is the selective
loss of the target antigen during therapy or the presence of
target antigen-negative leukemia cells at the start of therapy.
In this respect, fluctuations in the expression of cell surface
markers on HSCs have been reported [14]. Therefore, the
rational design of combinatorial antibody-based or ligand-
based strategies that target two or more different LSC anti-
gens simultaneously may prove the best way to prevent
escape from therapy. Below, a selection of the most promi-
nent cell surface molecules on LSCs and approaches for
targeting these molecules are briefly reviewed (summarized
in Fig. 2).
LSC Target Antigens
CD33 belongs to immunoglobulin (Ig) superfamily and is
a member of the sialoadhesin family of cellular interaction
Fig. (1). Hypotheses for the ontogeny of leukemia stem cells. LSCs can arise by somatic mutations in normal hematopoietic stem cells
(HSC) or more mature progenitor cells can become malignant, de-differentiate and re-acquire stem cell-lik e characteristics. Currently, LSCs
(typically CD34+CD38-) are defined by the ability to engraft in immunocompromised mice and initiate leukemia resembling the original
patient’s diseas e.
Targeted Elimination of Leukemia Stem Cells Current Drug Targets, 2010, Vo l. 11, No. 1 97
molecules [15, 16]. The intracellular domain of CD33
contains an immuno-receptor tyrosine-based inhibitory motif
(ITIM) domain which may function as a negative regulator
of cellular differentiation [17]. CD33 expression is largely
restricted to the myeloid lineage, however some expression
is detected on certain peripheral blood lymphocytes. In
leukemia, CD33 is expressed on blasts in the majority of
AML patients. Importantly, it has been noted that CD33 is
also expressed on AML-LSCs, but not on normal HSCs [18,
19].
CD123 (IL-3R) is the alpha subunit that hetero-
dimerizes with the IL3 chain to form the interleukin-3
receptor. CD123 is constitutively expressed on hematopoie-
tic progenitors cells (HPCs), on which it is involved in
proliferation and differentiation. Furthermore, CD123 is
expressed on LSCs in AML [20], Chronic Myeloid Leuke-
mia (CML), Myelodysplastic Syndrome (MDS) and
Systemic Mastocytosis [18], but not on HSCs [21]. This
expression profile indicates that CD123 might be a suitable
target for the selective elimination of LSCs in various
hematological malignancies.
C-type Lectin-Like Molecule-1 (CLL-1) is a heavily N-
glycosylated transmembrane protein with an intracellular
ITIM domain [22]. CLL-1 is exclusively expressed on cells
of the hematopoietic lineage, including dendritic cells,
granulocytes and monocytes [23]. In leukemia, CLL-1
expression is detected on 92% of all AML cases [23] on
which a high expression at diagnosis correlates with an early
relapse [24]. Importantly, CLL-1 is also expressed on AML-
LSCs, but not on HSCs [24, 25]. In line with these findings,
Fig. (2). Antibody- and ligand-based approaches for the elimination of LSCs. The cell surface molecules CD96, CD123 (IL3R), CLL-1,
CD33 and TRAIL-R1/R2 may be exploited for the selective elimination of LSCs. CD123 targeted strategies include mAb7G3, a fusion
protein comprising an CD123-specific antibody fragment linked to pseudomonas endotoxin A (26292(Fv)-PE38-KDEL) and an IL3:
diphtheria toxin (IL-3:DT) fusion protein. CLL-1 has been targeted with naked antibodies resulting in Complement Dependent Cytotoxicity.
CD33 has been targeted with several experimental therapeutics, including radionuclides, a scFvCD33: sTRAIL fusion protein and the
clinically applied immunotoxin Gemtuzumab Ozogamicin (GO). After CD33-selective binding, GO internalizes and ends up in the
lysosomes. In the acidic milieu of the lysosomes, the calicheamicin moiety is hydrolytically released. Next, calicheamicin translocates to the
nucleus and intercalates with the DNA, resulting in apoptotic cell death. Therapeutic strategies targeting the death-receptors, TRAIL-R1/R2,
include activating anti-TRAIL-R1 and anti-TRAIL-R2 antibodies, recombinant preparations of soluble TRAIL (sTRAIL) and scFv:sTRAIL
fusion proteins. Activation of TRAIL-R1/-R2 receptors results in the recruitment of the adaptor molecule FADD and caspase-8 to the death
domains of these receptors. Assembly of this so-called death-inducing signaling complex (DISC) leads to sequential activation of caspase-8
and caspase-3, ultimately resulting in DNA damage and apoptosis. In some cell types, caspase-8 can cleave Bid (tBid) and thereby activate a
mitochondrial amplification loop. This loop is characterized by cytochrome C release from the mitochondria and subsequent the sequential
activation of caspase-9 and caspase-3.
98 Current Drug Targets, 2010, Vol. 11, N o. 1 ten Cate et al.
it was demonstrated that sorted CD34+/CLL-1+ AML cells
are able to engraft and generate CLL-1+ blasts after trans-
plantation in Non-Obese Diabetic/Severe Combined
Immuno-Deficient (NOD/SCID) mice [24].
CD96 is a transmembrane protein belonging to the Ig
super-family. The extracellular domain of CD96 has three
Ig-like regions and a stalk-like region which can be heavily
O-glycosylated. The only ligand for CD96 identified thus far
is CD155, also known as polio virus receptor [26]. On
normal cells CD96 protein expression appears to be
restricted to T and NK cells, while its expression was also
detected on T cell -Acute Lymphoid Leukemia (ALL) cells
and AML cells [27, 28]. Recently, Hosen and colleagues
identified that comp ared with normal HSCs in AML-LSCs
CD96 mRNA levels are at least 200-fold increased [28].
These mRNA expression data suggests that CD96 protein
may be selectively over-expressed on AML-LSCs. In line
with this finding, they demonstrated that CD96+ AML cells
have a significantly higher engraftment potential then CD96-
AML cells [28]. Therefore, the development of antibody-
based or CD96 ligand-based therapeutics may have potential
for the targeted elimination of CD96+ AML-LSCs.
Targeting LSCs with Naked Antibodies
Indications for the efficacy of targeting CD123+ AML-
LSCs with naked antibodies is demonstrated by a preclinical
study using the CD123 neutralizing antibody, mAb7G3 [29].
Treatment with mAb7G3 inhibited engraftment and homing
of human CD123+ AML-LSCs when injected in NOD/SCID
mice. This effect of mAb7G3 was restricted to AML cells,
since there was only a minor effect towards normal bone
marrow (BM) cells. Furthermore, mAb7G3 treatment of
NOD/SCID mice with pre-established AML reduced both
the disease burden and the ability to transplant AML in
secondary recipient mice (ASH Annual Meeting Abstracts,
20071).
Recently, in vitro treatment with naked anti-CLL-1 mAbs
in the presen ce of serum resulted in efficient Complement
Dependent Cytotoxicity (CDC) towards various CLL-1+
AML cell lines. In lin e with these findings, ex vivo treatment
of primary AML blasts with naked anti-CLL1 mAbs resulted
in CDC in all samples tested. Furthermore, in a xenograft
model one particular anti-CLL-1 mAb reduced the growth of
established tumors by 38% (ASH annual meeting abstracts,
20082). Together, these data indicate that targeting both
CD123 and CLL-1 by naked antibodies might be a promising
approach for the selective elimination of LSCs.
Targeting LSCs with Immunoconjugates
The expression of CD33 in AML has resulted in the
development of several CD33-targeted experimental
therapeutics [30, 31] and the clinically applied anti-AML
1 Lock R, Jin L, Lee E, Ramshaw HS, Busfield S, Peoppl AG, et al. CD123 (IL-3
Receptor alpha Chain) Neutralization by a monoclonal antibody selectively eliminates
human acute myeloid leukemic stem cells. ASH Annual Meeting Abstracts 2007; 110:
161.
2 Korver W, Zhao X, Singh S, Pardoux C, Zhao C, Sen S, et al. CLL-1, a receptor
expressed on myeloid leukemic stem cells, is a potential target for Immunotherapy of
AML. ASH Annual Meeting Abstracts 2008; 112: 4003.
immunotoxin Gemtuzumab Ozogamicin (GO). GO consists
of an anti-CD33 mAb that is chemically coupled to a
derivative of the calicheamicintoxin. After CD33-selective
binding, GO internalizes and ends up in the acidic milieu of
lysosomes where the calicheamicin moiety is hydrolytically
released. Subsequently, calicheamicin translocates to the
nucleus and intercalates with the DNA, causing site-specific
double-strand breaks resulting in apoptotic cell death [32]. In
the United States, GO was approved by the FDA for use in
patients over the age of 60 with relapsed AML who are not
considered candidates for standard chemotherapy [33]. In
this subset of patients, GO treatment resulted in a 26%
remission rate, albeit with sometimes considerable tox icity
[34]. Recently, we have demonstrated that the activity of GO
is strongly enhanced when treatment is combined with the
epigenetic modifying drug valproic acid (VPA), a histone
deacetylase inhibitor (HDACi). The co-treatment of AML
cells with GO and VPA f acilitated the interca-lation o f
calicheamicin with the DNA, followed by synergistic
apoptosis induction [35].
It has been reported that in vitro treatment of AML-LSCs
with GO results in a median decrease in cellular viability
with 46%, while normal HSCs were largely unaffected (ASH
Annual Meeting Abstract, 20073). Therefore, it would be
interesting to assess whether the anti-LSCs activ ity of GO
can be further enhanced when treatment is combined with
VPA.
One potential resistance mechanism of AML-LSCs
towards treatment with GO involves the increased expression
and/or activity of drug efflux pumps. In this respect, it
appears that P-glycoprotein (Pgp) expression confers resis-
tance to GO and as such is asso ciated with a worse clinical
response [36]. Therefore, combination treatment of GO and
Pgp inhibitors may enhance the therapeutic effect of GO
towards Pgp-expressing LSCs.
Alternatively, efflux pump-related resistance in AML-
LSCs may be overcome using an anti-CD33 antibody conju-
gated to cytolytic radionuclides like 111In. In vitro expe-
riments w ith an 111In-conjugated anti-CD33 mAb resul-ted in
potent apoptosis in both chemo-resistant CD33+, HL-60
AML cells and in primary AML cells. Indeed, efflux pump-
related resistance was overcome by 111In since these AML
cells expressed several drug efflux pumps including Pgp,
Breast Cancer Resistance Protein 1 and Multidrug Resistance
Protein 1 [37].
Currently, the efficacy of various CD123-targeted immu-
noconjugates is under investigation. In one particular
approach CD123 is targeted by a fusion protein comprising a
CD123-specific single chain fragment of the variable regions
(scFv) antibody fragment that is genetically fused to a
fragment of pseudomonas exotoxin A (PE38). This fusion
protein, designated 26292(Fv)-PE38-KDEL, potently ind-
uced apoptosis in a variety of CD123+ leukemia cell lines
[38]. It remains to be investigated whether 26292(Fv)-PE38-
KDEL can also eliminate CD123+ LSCs.
3 Jawad M, Mony U, Russell N, Pallis M. In vitro chemosensitivity of leukaemic stem
and progenitor cells to gemtuzumab ozogamicin (mylotarg) in AML. ASH Annual
Meeting Abstracts 2007; 110: 650
Targeted Elimination of Leukemia Stem Cells Current Drug Targets, 2010, Vo l. 11, No. 1 99
Targeting LSCs with Ligand-Based Approaches
The cytokine IL3 has been used in fusion protein format
to selectively deliver diphtheria toxin (DT) to CD123+ AML
cells [39]. This IL3-DT fusion protein also selectively
eliminated primitive A ML progenitors, while normal
progenitors where not affected [40]. Importantly, the IL3-DT
fusion protein inhibited engraftment of human CD123+
AML-LSC cells when injected in NOD/SCID mice [40, 41].
Recently, it was demonstrated that the pro-apoptotic activity
of IL3-DT is enhanced when treatment is combined with
Tumor necrosis factor Related Apoptosis Inducing Ligand
(TRAIL) [42]. Currently, a phase I clinical trial with this
IL3-DT fusion protein for a subset of AML and MDS
patients is initiated [41].
Activation of Apoptosis Inducing Receptors
The agonistic TRAIL death receptors, TRAIL-R1 and
TRAIL-R2 (also known as DR4 and DR5, respectively)
belong to the Tumor Necrosis Factor receptor family of
death receptors. Ligation of these death receptors by TRAIL
activates the extrinsic apoptotic pathway (reviewed in [43]).
Hereupon, the adaptor protein FAS associated death domain
(FADD) and the initiator caspase-8 are recruited to the
intracellular death domains of these receptors. Assembly of
this so-called death-inducing signaling complex (DISC)
leads to sequential activation of initiator and effector
caspases and, ultimately, results in apoptotic cell death. In
some cell types, the death-receptor pathway relies on an
additional mitochondrial amplification loop that is activated
by caspase-8-mediated cleavage of the BH3-only interacting
domain death agonist (Bid) to a truncated form. Next,
truncated BID (tBid) activates the mitochondrial pathway
which involves cytochrome C and caspase-9. Intriguingly,
apoptosis resulting from ligation of agonistic TRAIL-Rs
appears to be restricted to malignant cells only, since normal
cells appear to be resistant to TRAIL-R ligation. The mode
of action of this tumor-selectivity of TRAIL-Rs ligation is
still enigmatic.
Currently, activating anti-TRAIL-R1 and anti-TRAIL-R2
antibodies, as well as recombinant preparations of solub le
TRAIL (sTRAIL) are evaluated in clinical trials [44-47]. In
our laboratory, we have developed a series of fusion
proteins, consisting of a tumor-selective scFv antibody
fragment genetically fused to sTRAIL. These scFv:sTRAIL
fusion proteins are designed to enhance tumor-selective cell
surface delivery of sTRAIL (reviewed in [48]). Pre-
clinically, these scFv:sTRAIL fusion proteins demons-trate
potent and target antigen-restricted apoptosis induction. To
this end, we have exploited both antigens expressed on solid
tumors including Epidermal Growth Factor Receptor [49-51]
and Epithelial Cell Adhesion Molecule [52, 53], as well as
antigens selectiv ely expressed on leukemia cells including
CD7 [54], CD19 [55] and CD33 [30].
Very recently, the expression of both TRAIL-R1 and
TRAIL-R2 has been reported on LSCs. Comparing the gene
expression profiles of AML-LSCs and HSCs, Majeti and
colleagues demonstrated an increased expression of TRAIL-
R1 and TRAIL-R2 in LSCs [56]. Furthermore, Yong et al.
demonstrated that in CML non-cycling CD34+ cells have a
significant higher expression of TRAIL-R1 and TRAIL-R2
than cycling CD34+ CML cells [57]. In line with this finding,
sTRAIL significantly inhibited the clonogenic capacity of
CD34+ AML cells without affecting the clonogenic capacity
of normal CD34+ cells [58]. Together, these studies indicate
that TRAIL-R1 and TRAIL-R2 receptors might be suitable
candidates for the selective elimination of LSCs by targeted
apoptosis induction. At present, we are assessing the efficacy
of CD33- and CLL-1-selective scFv:sTRAIL fusion proteins
for targeted apoptosis induction in AML-LSCs.
DISRUPTION OF THE LSCS-NICHE INTERACTION
In 1978, Schofield proposed that HSCs receive
regulatory factors and signals within a specialized niche [59].
Currently, there are indications that these specialized niches
are located in the bone marrow (BM), near the endosteum
[60]. The endosteum is the inner surface located at the bone-
BM interface and consisting mainly of osteoblasts and
osteoclasts. More recent evidence indicates that also vascular
and perivascular cells appear to contribute to these niche
interactions as well (see review [61]). Signals within the
HSCs nich e regulate the normal survival, proliferation and
differentiation of the HSCs. Recently, it was demonstrated
that in leukemia the micromilieu in the HSCs niche appears
to disturbed by the presence of Stem Cell Factor that is
locally secreted by leukemia cells [62]. Furthermore, it has
been demonstrated that intravenously injected AML-LSCs
home to, engraft and subsequently reside in the endosteal
region [63]. Such cellular tropism of AML-LSCs for a
dedicate niche typically involves the coordinated signaling
by various chemokines and adhesion molecules. Neutraliza-
tion or inhibition of these signals may be exploited for the
selective elimination of LSCs. Below, a selection of recent
studies is reviewed that collectively indicate that disruption
of the LSCs-niche interactions may indeed form a
therapeutic option for the elimination of LSCs (summarized
in Fig. 3).
Targeting the Stroma-Derived Factor-1(SDF-1)/CXCR4
Axis
Previously, it has been demonstrated that the chemokine
SDF-1 (CXCL12) as expressed by reticular stromal cells in
the BM is pivotal for homing of HSCs to their niche [64].
SDF-1 binds to the transmembrane receptor CXCR4 which
is expressed on HSCs [64, 65], leukemic blasts and AML-
LSCs [66]. Of note, a high expression of CXCR4 in AML
correlates with a negative prognosis [67, 68]. The pivotal
role of CXCR4 in retention of HSCs in the niche is clearly
demonstrated by the mobilization of HSCs by the CXCR4
antagonist AMD3100 [69].
The interaction of leukemia cells with cell adhesion
molecules in the niche typically reduces their sensitivity to
cytotoxic therapies, a process known as cell adhesion-
mediated drug resistance (CAM-DR). Inhibition of CXCR4
by the specific inhibitors AMD3100 and AMD3465 sensi-
tized AML cells for cytotoxic therapies [70-72]. Most likely,
treatment with the CXCR4 inhibitors mobilizes AML cells,
thereby reducing CAM-DR and sensitizing the AML cells
for cytotoxic therapy.
100 Current Drug Targets, 2010, Vol. 11, No. 1 ten Cate et al.
Tavor and colleagues demonstrated that the anti-CXCR4
mAb12G5 significantly inhibits homing of AML cells to the
BM of recipient mice. Notably, treatment of mice with
established AML with mAb12G5 reduced the leukemic
burden in the BM, blood and spleen. Importantly, similar
treatment did not affect the levels of engrafted HPCs [66].
Additionally, lowering available SDF-1 levels in leuke-
mia might offer a therapeutic strategy. Recently, the medi-
cinal herb-derived compound berberine was shown to inhibit
SDF-1 production by stromal cells, thereby impairing the
migration of AML blasts and AML-LSCs in vitro [73].
Together these results indicate the importance of a properly
functioning SDF-1/CXCR4 axis for homing and retention of
AML blasts and AML-LSCs. Therefore, the SDF-1/CXCR4
axis appears to be a suitable targ et candidate for the selective
elimination of A ML-LSCs.
Targeting Very Late Antigen 4 (VLA-4)
The 1-integr in, VLA-4, has a vital role in the retention
of HPCs in the BM niche by immobilizing these cells [74].
The interaction of VLA-4 on AML cells with fibronectin on
stromal cells initiates the phosphatidylinositol-3-OH kinase
(PI3K) pro-survival pathway. Activation of the PI3K
pathway in AML cells contributes to CAM-DR, which might
explain the observation that VLA-4 expression on AML cells
is correlated with a negative prognosis for AML patients. In
line with this, Matsunaga and colleagues demonstrated that
inhibition of VLA-4 interaction with fibronectin sensitized
AML cells to cytosine arabinoside (Ara-C). This was
demonstrated in a mice model for MRD in which they
applied VLA-4 neutralizing antibodies to prevent the
interaction with fibronectin [75]. Using the same MRD
model, they recently demonstrated that a fibronectin-based
synthetic peptide, designated FNIII14 [76], also sensitized
AML cells to Ara-C [77]. Both approaches may be of
considerable interest for disruption of the LSCs-niche
interaction.
Targeting CD44
CD44 is a transmembrane receptor capable of binding
several ligands, including hyaluronic acid which is abun-
dantly expressed in the endosteal region [78]. On HSCs,
CD44 serves as a homing receptor by interacting with E-
selectin and L-selectin expressed in the BM [79]. Since
CD44 is expressed on both CML-LSCs and AML-LSCs [80,
Fig. (3). D isruption of the LSC-niche interaction. The LSC-niche interaction can be disrupted by targeting VLA-4, CXCR4, CD44 and
Rac1. Binding of LSCs-expressed VLA-4 to fibronectin in the BM initiates the PI3K pro-survival pathway and cell adhesion-mediated drug
resistance (CAM-DR). The VLA-4-fibronectin interaction has been disrupted by mAbs and the synthetic peptide FNIII14. CXCR4 has been
targeted with mAb12G5 and the specific inhibitors AMD3100 and AMD3465. Furthermore, the concentration of the CXCR4 ligand, SDF-1,
can be lowered using berberine. The interaction of CD44 with the niche has been disrupted by mAbH90 and mAbIM7. The compound,
NSC23766, has been utilized to specifically inhibit Rac1.
Targeted Elimination of Leukemia Stem Cells Current Drug Targets, 2010, Vo l. 11, No. 1 101
81], neutralization of CD44-binding might impair the
homing and retention of LSCs in the BM.
In CML, about 95% of the cases is characterized by the
presence of the oncogenic fusion protein Bcr-Abl, a con-
stitutively active mutant tyrosine kinase that causes
uncontrolled cellular proliferation. The Bcr-Abl inhibitor
Imatinib Mesylate induces remissions in the majority of
CML patients. However, Imatinib appears to be unable to
eradicate quiescent CML-LSCs [82], which might explain
why the majority of CML patients relapse upon treatment
discontinuation. Therefore, novel therapeutics for the
selective elimination of Bcr-Abl+ CML-LSCs are needed.
In a series of elegant exper iments, Krause and colleagues
demonstrated that CD44 expressed on Bcr-Abl+ CML-LSCs
is essential for homing of these cells in recipient mice.
Thereto, they performed a competitive homing assay in
which the relative contribution of Bcr-Abl-transduced
CD44+ BM cells and Bcr-Abl-transduced CD44- BM cells in
the development of CML was analyzed. Both cells types
were mixed and injected intravenously in irradiated
recipients mice. After CML development, the animals were
sacrificed and the relative contribution of the two distinct
cell populations was analyzed. This revealed that Bcr-Abl-
transduced CD44+ BM cells contributed 5 times more to the
development of CML than Bcr-Abl-transduced CD44- BM
cells. CML development by the two different cell types was
similar when injected intrafemorally. These results indicate
that CD44 is necessary for homing of intravenously injected
Bcr-Abl transduced cells to th e BM. Importantly, anti-CD4 4
antibody treatment significantly prolonged survival of
NOD/SCID recipient mice upon transplantation with CML-
LSCs [80].
Simultaneously, the group of John Dick obtained similar
results by targeting CD44 in AML. Treatment with the
CD44-activating mAbH90 inhibited AML-LSCs homing to
the BM niche and altered the fate of the AML-LSCs
resulting in an efficient and selective elimination of th e
AML-LSCs [81]. Together, these studies clearly indicate a
rationale for targeting CD44 to selectively eliminate CML-
LSCs and AML-LSCs. Therefore, an evaluation of the
efficacy of targeting CD44 in both CML and AML patients
seems warranted.
Targeting Rac
The Rac subfamily of the Rho-family of GTP-ases is
implicated in cell adhesion and motility. The family consists
of three members Rac1, Rac2 and Rac3. Rac1 is ubiquitously
expressed [83], while Rac2 expression is limited to the
hematopoietic system [84] and Rac3 is selectively expressed
in the brain [85]. Rac1-/- HSCs and progenitors are unable to
reconstitute hematopoiese in irradiated recipient mice upon
transplantation [86]. Moreover, it has been shown that Rac is
overexpressed on CD34+ cells derived from AML patients.
Rac appears to contribute to an enhanced migration and
adhesion capacity of CD34+ cells to BM stromal cells.
Gao and colleagues [87] generated a Rac1 inhibitor,
designated NSC23766, that significantly delayed leukemia
development in a murine tumor model [88] and strongly
reduced the clonal expansion of CD34+ AML cells [89].
Together, these promising data warrants further pre-clinical
assessment of Rac1 inhibition for the elimination of LSCs.
TARGETING ABERRANT MOLECULAR PATH-
WAYS
Both, HSCs and LSCs rely on similar molecular
pathways for their maintenance and self-renewal. However,
in LSCs these pathways are often aberrantly regulated. LSCs
appear to be particular dependent on these deregulated
pathways to maintain their malignant phenotype. This strong
dependence indicates that therapeutic intervention in these
pathways might be exploitab le for the selectiv e elimination
of LSCs. Thus far, several of these deregulated pathways
have been identified (e.g. the Hedgehog and Wnt pathways).
The selective targeting of these pathways may yield
promising anti-LSCs activity.
Compounds such as arsenic trioxide and parthenolide
have been assessed for their capacity to induce apoptosis in
LSCs. However, the pathways in which these compounds
intervene are currently not completely understood. There-
fore, further delineation of their specific molecular targets
may contribute to development of more and rationally
designed LSCs targeted therapeutics. Below, we provide a
selection of aberrantly regulated pathways present in LSCs
and a selection of chemical compounds that may have
potential for the elimination of LSCs (summarized in Fig. 4).
The Hedgehog (Hh) Pathway
The Hh pathway is involved in HSCs self-renewal [90]
and is initiated by binding of one of the three Hh ligands,
Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) or desert
Hedgehog (Dhh) to the transmembrane receptor Patched.
Upon binding of Hh to Patched the inhibiting effect of
Patched on the transmembrane receptor Smoothened (Smo)
is relieved (reviewed in [91]). Next, Smo can activate Hh
targets g enes via the Gli family of transcription factors.
Amongst these Hh targets genes are CyclinD/E [92] and B-
cell-specific Moloney murine leukemia virus insertion site 1
(Bmi-1) [93] which are involved in cell proliferation and
self-renewal, respectively. The Hh pathway can be inhibited
by the chemical compound cyclopamine, which stabilizes
Smo in its inactive form [94].
In a very recent study conducted by Zhao and colleagues
the role of Smo in Bcr-Abl+ CML-LSCs was analyzed [95].
Thereto, Bcr-Abl-transduced normal HPCs and Bcr-Abl-
transduced Smo-/- HPCs were transplanted in irradiated
recipient mice after which the development of CML was
compared. More than 90% of the mice transplanted with
Bcr-Abl-transduced normal HPCs developed CML. In
contrast, only 47% of the mice developed CML when
transplanted with Bcr-Abl-transduced Smo-/- HPCs. When
the Bcr-Abl-transduced HSCs were transplanted in mice, all
recipient mice died from CML within four weeks. Howev er,
when these transplanted mice were treated with cyclo-
pamine, 60% of the animals were still alive after 7 weeks.
Furthermore, CML cells freshly isolated from cyclopamine-
treated mice were unable to effectively repopulate secondary
recipients. This corroborates with the strong reduction (up to
14-fold) of CML-LSCs in cyclopamine-treated mice.
Addition ally, cyclopamine treatment of human CML cell
102 Current Drug Targets, 2010, Vol. 11, No. 1 ten Cate et al.
lines and primary CML samples reduced their clonogenic
capacity. Importantly, the authors indicate that Smo inhibi-
tion by cyclopamine may also impaired the propagation of
Imatinib-resistant human CML cells in xenografts [95].
The Wnt pathway
Wnt is implicated in HSCs self-renewal and prolif eration
[96] and is overexpressed in many different types of human
cancers [97]. Thus far, three distinct Wnt signaling pathways
have been described of which the canonical pathway is most
prominently involved in leukemia. The key mediator of the
canonical pathway is -catenin. Normally, -catenin is deg-
raded rapidly after synthesis by the multiprotein destruction
complex (MDC), which tags -catenin for proteasomal deg-
radation. The MDC consists of axin, adenomatous polyposis
coli (APC) and glycogen-synthase kinase 3 (GSK3). Upon
binding of Wnt to its receptors Frizzled and LRP5/6, the
MDC becomes destabilized and therefore unable to tag -
catenin for degradation. Consequently, -catenin accumu-
lates and is able to translocate to the nucleus. In the nucleus
-catenin complexes with T-cell factor (TCF), B cell
Fig. (4). Targeting aberrantly regulated pathways and epigenetic reprogramming Aberrantly regulated Wnt and Hedgehog pathways
have been identified in LSCs and therefore might be suitable candidates for therapeutic intervention. At the cell surface, binding of Wnt to
LRP5/6 and Frizzled destabilizes the Multiprotein Destruction Complex (MDC), which in the absence of Wnt signaling facilitates the
degradation of -catenin. As a result of Wnt binding to its receptors, -catenin accumulates and translocates to the nucleus where it
complexes with TCF, Bcl-9 and pygo to induce the expression of several genes including c-myc, cyclin-D and CD44. Extracellularly, the
Wnt signaling pathway can be inhibited by the scavengers sFRP, Wif1 and Cerberus and the DKK-family members which prevent Wnt
binding to its receptor. Intracellularly, Wnt signaling can be inhibited by the compounds CGP049090 and PKF115-584, which disrupt the
TCF/-catenin complex. The Hedgehog pathway is initiated by binding of a ligand (Shh, Ihh or Dhh) to the receptor Patched. Hereupon,
Smoothened becomes activated and via the Gli family of transcription factors induces the expression of several target genes including
CyclinD/E and Bmi-1. The Hedgehog pathway can be inhibited by cyclopamine which stabilizes Smoothened in its inactive form. Binding of
Growth factors (GF) to Growth Factor Receptors (GFR) or binding of VLA-4 to fibronectin induces activation of PI3K. This pro-survival
signaling pathway is characterized by the conversion of PIP2 to PIP3 and subsequent downstream signaling via several proteins, including
mTOR. Normally, PTEN functions as an inhibitor of the PI3K pathway. Pharmacologically, both rapamycin and arsenic trioxide (via PML)
can alter mTOR activ ation. The enzymes ALDH1A1 and ALDH3A1 have been implicated in Multi D rug Resistance (MDR) and can be
inhibited by DEAB and ATRA. The anti-leukemia activity of parthenolide (PTL) is characterized by the inhibition of NF-B and the
generation of ROS. The compound TDZD-8 can also inhibit NF-B activation and might influence LSCs membrane integrity. Frequently,
gene expression in leukemic cells is altered by epigenetic reprogramming. Therefore, reprogramming the epigenetic status using decitabine
or HDACi appears promising for the elimination of LSCs.
Targeted Elimination of Leukemia Stem Cells Current Drug Targets, 2010, Vo l. 11, No. 1 103
lymphoma 9 (Bcl-9) and pygo to induce TCF target genes
expression [98], including the oncogenes C-MYC [99],
Cyclin-D1 [100] and the cell surface antigen CD44 [101].
In AML, several oncogenic fusion proteins, such as
AML-1⁄eight twenty one (AML1-ETO), promyelocytic
leukemia ⁄ retinoic acid receptor alpha (PML⁄RAR) and
promyelocytic leukemia zinc finger (PLZF⁄RAR) [102] can
activate the Wnt signaling pathway. Furthermore, evidence is
emerging that indicate an aberrant Wnt signaling pathway in
LSCs [56] (extensively reviewed in ref. [103]). In this
respect, it has been shown that -catenin plays a pivotal role
in self-renewal of both HSCs as well as CML-LSCs [104].
Furthermore, very recently -catenin has been implicated in
the survival of Imatinib resistant CML-LSCs in Bcr-Abl
induced CML [105]. Therefore, modulation of Wnt signaling
in the LSCs may contribute to their selective elimination.
There are several possibilities to inhibit the Wnt
signaling pathway. Wnt signaling can be inhibited by
extracellular proteins (reviewed in [106]) which scavenge
Wnt and thereby prevent Wnt from binding to its receptors.
These Wnt binding proteins include secreted Frizzled-related
proteins (sFRP), Wnt inhibitory factor 1 (wif-1) and
Cerberus. Moreover, members of the Dickkopf (DKK)
family can directly bind the Wnt receptor and thereby
prevent Wnt-induced signaling. In this respect, very recently
it appeared that the anti-proliferative effects of human
mesenchymal stem cells towards CML cells is at least partly
attributable to secretion of DKK-1[107]. Additionally, also
very recently two natural compounds, CGP049090 and PKF
115-584 [108], have been identified that disrupt the TCF/-
catenin complex. In AML these compounds possess potent
tumoricidal activity [109]. It would be worthwhile to assess
the possibilities of inhibition of the Wnt signaling pathway
for the elimination of LSCs.
Aldehyde Dehydrogenases (ALDH) Enzymes
ALDH are a group of cytosolic enzymes that catalyzes
the conversion of intracellular aldehydes. The ALDH
superfamily consists of seventeen members [110] of which
two members, cytosolic aldehyde dehydrogenase class-1A1
(ALDH1A1) and class-3A1 (ALDH3A1), have been impli-
cated in tumor drug resistance against oxazaphosphorines
(e.g. cyclophosphamide) [111]. ALDH enzyme activity,
including the activity of ALDH1A1 and ALDH3A1, can be
assessed using activatable fluorescent substrates. ALDH
enzyme activity measurements enables the identification and
isolation of HSCs [112], breast cancer stem cells [113], liver
cancer stem cells [114] and AML-LSCs [115]. Manipulation
of ALDH1A1 and ALDH3A1 expression or activity may be
used to sensitize AML-LSCs to chemotherapy, especially to
oxazaphosphorines. In this respect, the ALDH1 inhibitor,
diethylaminobenzaldehyde (DEAB) might be utilized for
sensitizing AML-LSCs to treatment with chemotherapeutic
agents. Unfortunately, DEAB appeared to b e very instable in
vivo [116]. Therefore, the development of novel DEAB-
analogues with enhanced stability is warranted. All-trans
Retinoic Acid (ATRA), an established clinically applied
anti-leukemia agent, appears to downregulate both
ALDH1A1 and ALDH3A1 which may be beneficial for the
selective elimination of LSCs [111]. In this respect,
treatment of Acute Promyelocytic Leukemia (APL) and
AML cells with ATRA resulted in differentiation of tumor
cells and stimulation of cell cycling [117]. Therefore, we
suggest that the combination treatment of oxazaphosphorines
and ATRA may have potential for the elimination of LSCs.
Phosphatase and Tensin Homologue (PTEN)
PTEN is the second most commonly mutated gene in
human malignancies, including hematopoietic malignancies
[118, 119]. Normally, PTEN inhibits cell proliferation and
cell survival by negatively regulating the PI3K pathway
[120]. In this respect, Yilmaz and colleagues recently
compared th e effect of PTEN inactivation in HSCs and LSCs
[121]. Conditional deletion of PTEN resulted in the
development of myeloproliferative disorder in 17 out of 19
mice which in the majority culminated in leukemia. In th e
HSCs, deletion of PTEN was characterized by an elevated
cell cycle progression and an impaired replenishment of the
HSCs pool, thus finally resulting in a depletion of HSCs.
Apparently, PTEN deletion facilitates leukemogenesis while
it depletes HSCs. These effects of PTEN deletion appear to
be mediated by mammalian target of rapamycin (mTOR),
since treatment with the mTOR inhibitor rapamycin reversed
the effects. Therefore, rapamycin treatment seems to be a
promising approach for the selective elimination of LSCs
while sparing HSCs.
However, Ito and colleagues recently reported on a
contradictory effect of rapamycin [122]. They investigated
the role of the tumor-suppressor promyelocytic leukemia
protein (PML) in CML. Their results demonstrated that
PML-deficient CML-LSCs are less quiescent than wildtype
CML-LSCs, resulting in LSCs exhaustion. Furthermore,
deletion of PML in HSCs also results in exhaustion and con-
sequently impairment in long-term hematopoietic recons-
titution. Since PML deficiency in both HSCs and LSCs is
characterized by an increased mTOR activity they assessed
the effect of rapamycin treatment. Here, rapamycin rescued
the typical PML-deficient phenotype, thus exhaustion of the
CML-LSCs and HSCs was prevented. Together, these
seemingly contradictory results of mTOR inhibition in the
elimination of LSCs indicate that further delineation of the
role of mTOR in LSCs is needed.
Of note, since arsenic trioxide is known to decrease PML
levels, Ito and colleagues continued with assessing arsenic
trioxide for the elimination of CML-LSCs [122]. Treatment
with arsenic trioxide resulted in the exit of the CML-LSCs
from quiescence and therefore the CML-LSCs might become
exhausted and more vulnerable to chemotherapeutics. Impor-
tantly, this cell cycle entry appeared to be more profound in
LSCs than in HSCs, thereby generating a possible
therapeutic window for the treatment of CML-LSCs by
arsenic trioxide.
4-benzyl, 2-methyl, 1,2,4-thiadiazolidine, 3,5 dione
(TDZD-8)
TDZD-8 was designed as a non-ATP competitive
inhibitor of GSK3 for the treatment of Alzheimer’s disease
[123]. However, Guzman and colleagues uncovered potent
tumoricidal activity of TDZD-8 towards leukemia cells. In a
series of experiments they demonstrated that TDZD-8
exhibit strong anti-leukemia effects towards AML, CLL,
104 Current Drug Targets, 2010, Vol. 11, N o. 1 ten Cate et al.
ALL and blast crisis CML cells, without apparent toxic
effects towards normal BM cells. Since a panel of other
GSK3 inhibitors did not possess any comparable activity,
the anti-leukemia effect is probably not governed by the
inhibition of GSK3. This notion is supported by the
observation that -catenin, of which the degradation is in
part mediated by GSK3, is elevated in CML-LSCs.
Moreover, very recently GSK3 missplicing was observed in
CML-LSCs and not in HSCs [124], together indicating that
GSK3 is dispensable for leukemogenesis.
Importantly, TDZD-8 induced apoptosis in AML-LSCs
and CML-LSCs without affecting HSCs [125]. In line with
this, TDZD-8 specifically reduced the engraftment potential
of AML-LSCs and not of HSCs. TDZD-8 is a highly
hydrophobic agent which may result in a rapid loss of
membrane integrity in leukemic cells by TDZD-8 insertions
in the cellular membrane. The absence of this eff ect in HSCs
might indicate that there is crucial difference in the
membrane composition of LSCs and HSCs [125], which
might open novel avenues for the selective elimination of
LSCs.
Parthenolide (PTL)
PTL is a compound, derived from the plant feverfew,
which eliminates cancer cells via reactive oxygen species
and the inhibition of NF-kB [126-128]. Guzman and
colleagues analyzed the effects of PTL and Ara-C on AML
blasts and AML-LSCs. Apoptosis induction in the AML
blasts by Ara-C was more pronounced than by PTL.
However, PTL potently induced apoptosis in AML-LSCs,
whereas Ara-C exhibited only minimal apoptotic activity.
Importantly, the PTL concentration necessary for significant
AML-LSCs elimination had only a modest effect on HSCs
[129]. Moreover, PTL treatment significantly reduced the
engraftment potential of A ML-LSCs, while PTL treatment
did not affected the engraftment of HSCs [129].
Despite the favorable characteristics, the clinical appli-
cation of PTL is probably hampered by its poor water
solubility. Therefore, Guzman and colleagues generated a
series of parthenolide analogues aiming to identify an active
compound with improved pharmacological properties [130].
Recently, they identified a novel compound, designated
dimethylamino analog of parthenolide (DMAPT), which
exhibits similar activity as PTL. However, when formulated
as a fumarate salt, DMAPT had 1000-fold enhanced water
solubility, resulting in a 70% bioavailability of orally applied
DMAPT [130]. In contrast to PTL, oral administration of
DMAPT did result in plasma concentrations sufficient to
exert an anti-leukemia effect in both rodent and canine
models.
EPIGEN ETIC REPROGRAMMING OF LSCS
The step-wise conversion of a normal cell to a leukemic
cell typically involves the activation of oncogenes and anti-
apoptotic genes and the inactivation of tumor suppressor and
proapoptotic genes [131]. Frequently, gene expression in
leukemic cells is altered by epigenetic reprogramming
(reviewed in [132]).
Epigenetic reprogramming is characterized by various
distinct chromatin modifications. Chromatin consists of
histone proteins and DNA. The central unit of the chromatin
structure is an octamer of core histone proteins, referred to as
the nucleosome. The distance between adjacent nucleosomes
and thus the compaction of the chromatin in between,
defines two types of chromatin. Euchromatin is the open
type of chromatin with a relative low compaction which
allows access of the transcription machinery to the D NA,
while heterochromatin is the condensed form in which the
transcription machinery can not access the DNA and reflects
an inactive state. There are several types of modifications
that can alter the accessibility of the transcription machinery
to the DNA. The best studied are DNA and histone
methylation which are associated with heterochromatin and
histone acetylation which is associated with euchromatin.
Normal stems cells rely on Polycomb group (PcG)
proteins to repress genes encoding transcription factors
required for differentiation including the homeotic (HOX)
genes [133]. It is postulated that methylation of promoter
regions of those repressed genes could lock in stem cell
phenotypes and initiate abnormal clonal expansion and
thereby predispose to cancer [134, 135]. Repression of genes
by the PcG proteins is initiated by the Polycomb Repressor
Complex 2 (PRC2) containing the histone methyltransferase
EZH2, after which the Polycomb Repressor Complex 1
(PRC1) further maintains the repression. The PRC1
comprises amongst others Bmi-1, which is essential for self-
renewal of HSCs [136]. Of note, in both AML and CML
patients Bmi-1 is overexpressed compared to normal CD34+
BM cells. Patients with an elevated Bmi-1 expression have
less chance of achieving complete remission, higher chance
of relapse and reduced duration of survival [137, 138].
Moreover, very recently Wang and colleagues have shown
that the dysregulation of a chromatin-binding plant homeo-
domain (PHD) finger can cause hematological malignancies
[139]. Normally, PHD fingers respond to certain histone
methylation states [140, 141]. In AML however, chromo-
somal translocations are observed which results in the fusion
of a PHD finger to a common leukemia fusion partner called
NUP98 [142-144]. Wang and colleagues compared the
transcriptome profile of BM progenitor cells transduced with
such a fusion protein with control cells. This analysis
indicated that in the cells transduced with this fusion protein
several genes were upregulated including HOXA9 and
MEIS1 [145]. Lessard and Sauvageau have shown that
enforced expression of HOXA9/MEIS1 can induce
leukemia, which is dependent on the expression of Bmi-1
[146], suggesting that Bmi-1 inhibitors may contribute to the
elimination of LSCs.
However, also drugs with a more general epigenetic
modification activity may have clinical relevance in
attacking LSCs. This is evident from the clinical application
of the demethylating agent decitabine, which is approved for
the treatment of MDS. Decitabine inhibits DNA methyltrans-
ferases (DNMT) and therefore has a broad range of action by
tipping the balance towards euchromatin. Intriguingly,
decitabine exerts differential effects in LSCs and HSCs,
which is probably due to differences in their epigenomic
status. In LSCs treatment with decitabine results in
differentiation, whereas decitabine treatment of HSCs results
Targeted Elimination of Leukemia Stem Cells Current Drug Targets, 2010, Vo l. 11, No. 1 105
in enhanced self-renewal (ASH Annual Meeting Abstracts,
20084). Furthermore, it was demonstrated that decitabine
treatment reactivated more genes in bladder cancer cells than
in normal fibroblasts [147]. These studies indicate that
general epigenetic modifications can exert differential effects
in LSCs and HSCs, which might be utilized for the selective
elimination of LSCs.
Other general epigenetic modulators are HDACi such as
suberoylanilide hydroxamic acid (SAHA), 4-phenylbutyric
acid (PBA) and VPA. HDACi can also tip the balance
toward euchromatin and have shown to induce apoptosis in
leukemia (see Fig. 4). In clinical trials VPA monotherapy
was well tolerated, but showed disappointing anti-leukemia
effects (reviewed in [148]). However, combination treatment
of VPA with other anti-cancer agents showed significan tly
enhanced and sometimes synergistic anti-tumor effects [35,
149-151].
MicroRNA’s (miRs) are small, non-coding, single stran-
ded RNA sequences that regulate gene expression by binding
to mRNA molecules. Interestingly, upon co-treatment with
decitabine and PBA, miRs appear to be differential reac-
tivated in cancer cells and normal cells [152]. Saito and
colleagues have shown that the transcription of one parti-
cular miR, miR-127, was increased upon combination treat-
ment of decitabine and PBA. Subsequently, they identified
that a predicted target of miR-127, BCL6, was downre-
gulated [152]. This indicates that not only epigenetic
silenced protein-coding sequences, but also miRs can be
reactivated upon treatment with demethylating agents and
HDACi. This opens novel avenues for modifying proteins
levels in LS Cs and therefore may be exploited to eliminate
LSCs.
CONCLUSIONS AND PERSPECTIVES
The specific elimination of LSCs in hemato-oncology is
a research area still in its infancy. Currently, researchers are
at the brink of identifying biological processes imperative to
LSCs biology. Elucidation of these LSCs-specific charac-
teristics is of eminent importance for the rational design of
novel LSCs targeted therapies. It is anticipated that the
successful elimination of LSCs will have tremendous impact
on leukemia therapy. Several strategies appear promising for
the elimination of LSCs, includ ing targeting LSC-selective
cell surface antigens, disrupting LSCs-niche interactions,
targeting aberrantly regulated pathways and epigenetic
reprogramming of LSCs. Several studies utilizing these
strategies have generated promising pre-clinical results.
However, the vast majority of the data is obtained by xeno-
grafting LSCs in severely immunocompromised recipient
mice. It is evident that the data obtained using these models
can not be extrapolated directly to the clinical situation.
Furthermore, it is likely that multifaceted therapy combining
several strategies will be the most effectiv e approach.
Therefore, rational designed combinatorial approaches and
multimodality targeting of LSCs are warranted. Subsequent,
these combinatorial strategies have to prove to be safe and
effective in clinical trials.
4 Negrotto S, Hu Z, Link K, Duong H, Schade AE, Maciejewski JP, et al. Differentia-
tion-chronology specific function of DNMT1 and selective anti-leukemia stem-cell
therapy. ASH Annual Meeting Abstracts 2008; 112: 201.
ACKNOWLEDGEMENTS
Supported by grants from the Dutch Cancer Society
(2005-3358 and 2007-3784) to W.H.
ABBREVIATION S
ALDH = Aldehyde dehydrogenases
ALDH1A1 = Aldehyde Dehydrogenase class-1A1
ALDH3A1 = Aldehyde Dehydrogenase class-3A1
ALL = Acute Lymphoid Leukemia
AML = Acute Myeloid Leukemia
AML1-ETO = AML-1⁄ eight twenty one
AML-LSCs = Acute Myeloid Leukemia Leukemia
Stem Cells
APC = Adenomatous Polyposis Coli
APL = Acute Promyelocytic Leukemia
Ara-C = Cytosine Arabinoside
ATRA = All-trans Retinoic Acid
Bcl-9 = B Cell Lymphoma 9
Bcr-Abl = Breakpoint cluster region-abelson
Bid = BH3-only interacting domain death
agonist
BM = bone marrow
Bmi-1 = B-cell-specific Moloney murine
leukemia virus insertion site-1
CAM-DR = Cell Adhesion-Mediated Drug
Resistance
CD = Cluster of Differentiation
CDC = Complement Dependent Cytotoxicity
CLL = Chronic Lymphoid Leukemia
CLL-1 = C-type Lectin-Like Molecule-1
CML = Chronic Myeloid Leukemia
CML-LSCs = Chronic Myeloid Leukemia Leukemia
Stem Cells
DEAB = Diethylamino-benzaldehyde
Dhh = Desert Hedgehog
DISC = Death Induced Signaling Complex
DKK = Dickkopf
DMAPT = Dimethylamino Analog of Parthenolide
DNA = Deoxyribonucleic acid
DNMT = DNA methyltransferase
DR = Death Receptor
DT = Diphtheria Toxin
FDA = Federal Drug Administration
FADD = FAS associated death domain
GO = Gemtuzumab Ozogamicin
106 Current Drug Targets, 2010, Vol. 11, N o. 1 ten Cate et al.
GSK3 = Glycogen-synthase kinase 3
HAT = Histone Acetyltransferase
HDACi = Histone Deactylase inhibitor
Hh = Hedgehog
HPCs = Hematopoietic Progenitor Cells
HSCs = Hematopoietic Stem Cells
Ig = Immunoglobulin
Ihh = Indian Hedgehog
IL-3R = Interleukin 3 Receptor
ITIM = Immunoreceptor tyrosine-based
inhibitory motif
LRP5/6 = Lipoprotein receptor-related protein 5/6
LSCs = Leukemia Stem Cells
mAb = Monoclonal Antibody
MDC = Multiprotein Destruction Complex
MDS = Myelodysplastic Syndrome
miRs = microRNA’s
mTOR = Mammalian target of rapamycin
NF-kB = Nuclear Factor kappa B
NK cell = Natural Killer cell
NOD/SCID = Non-Obese Diabetic/ Severe Combined
Immunodeficient
PBA = 4-phenylbutyric acid
PcG = Polycomb group
PE38 = Pseudomonas Exotoxin Fragment of
38 kilodalton
Pgp = P-glycoprotein
PIP2 = Phosphatidylinositol (3,4)-bisphosphate
PIP3 = Phosphatidylinositol (3,4,5)-
trisphosphate
PI3K = Phosphatidylinositol-3-OH kinase
PLZF ⁄RAR = Promyelocytic leukemia zinc finger⁄
retinoic acid receptor alpha
PML = Promyelocytic leukem ia protein
PML⁄RAR = Promyelocytic leukemia ⁄ retinoic acid
receptor alpha
PRC1 = Polycomb Repressor Complex 1
PRC2 = Polycomb Repressor Complex 2
PTEN = Phosphatase and tensin homologue
PTL = Parthenolide
SAHA = Suberoylanilide hydroxamic acid
scFv = Single chain fragment of the variable
regions
SDF-1 = Stroma-derived factor-1
Shh = Sonic Hedgehog
Smo = Smoothened
SP = Side Population
tBid = Truncated form of Bid
TCF = T-cell factor
TDZD-8 = 4-benzyl, 2-methyl, 1,2,4-
thiadiazolidine, 3,5 dione
TNFR = Tumor Necrosis Factor Receptor
TRAIL = Tumor necrosis factor Related Apoptosis
Inducing Ligand
TRAIL-R1 = TRAIL Receptor 1
TRAIL-R2 = TRAIL Receptor 2
VLA-4 = Very Late Antigen 4
wif-1 = Wnt inhibitory factor 1
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Received: May 19, 2009 Revised: June 19, 2009 Accepted: August 24, 2009
