Buss Et Al-2011-International Journal of Cancer

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Leukemia stem cells

Eike C. Buss and Anthony D. Ho

Department of Internal Medicine V, Heidelberg University Medical Center, Im Neuenheimer Feld 410, Heidelberg, Germany

Leukemia stem cells (LSCs) might originate from malignant transformation of normal hematopoietic stem cells (HSCs), or

alternatively, of progenitors in which the acquired mutations have re-installed a dysregulated self-renewal program. LSCs are

on top of a hierarchy and generate leukemia cells with more differentiated characteristics. While most leukemia cells are

initially sensitive to chemo- and radiotherapy, LSCs are resistant and are considered to be the basis for disease relapse after

initial response. Albeit important knowledge on LSC biology has been gained from xenogeneic transplantation models

introducing human leukemia cells into immune deficient mouse models, the prospective identification and isolation of human

LSC candidates has remained elusive and their prognostic and therapeutic significance controversial. This review focuses on

the identification, enrichment and characterization of human LSC derived from patients with acute myeloid leukemia (AML).

Experimental data demonstrating the clinical significance of estimating LSC burden and strategies to eliminate LSC will be

summarized. For long-term cure of AML, it is of importance to define LSC candidates and to understand their tumor biology

compared to normal HSCs. Such comparative studies might provide novel markers for the identification of LSC and for the

development of treatment strategies that might be able to eradicate them.

Stem cells in health and in cancerIn analogy to physiological differentiation, a stem cell para-

digm has been proposed for malignant development.1 There

is increasing evidence that human cancers may originate

from transformation of stem cells, or alternatively, from pro-

genitors in which the acquired mutations have re-installed a

dysregulated self-renewal program. The first hints were found

in hematological malignancies where only a small subset of

slowly dividing cells was able to induce transplantable acute

myeloid leukemia.2Since the first description by Lapidot et al.,2 many groups

have confirmed the existence of leukemia stem cells (LSCs)

for acute myeloid leukemia (AML). The engraftment of

human LSC preparations into xenogeneic transplantation

models has been regarded as evidence for the presence of leu-

kemia initiating cells for human AML. In such studies, a

defined number of leukemia cells were administered to

immunodeficient mice and the presence of LSC in the pri-

mary material was retrospectively assessed after engraftment

of human leukemia.3 Albeit relevant knowledge on LSC biol-

ogy has been gained from these sophisticated experiments,

there has been thus far no reliable prospective method for

the estimation of LSC burden.

The abundance of LSC has been associated with clinical

relapse or with refractory disease. This hypothesis has also

remained speculative because published data thus far were

not able to substantiate these claims in a definitive manner.

The prospective identification and isolation of human LSC

candidates have remained elusive and their clinical and prog-

nostic significance controversial.4,5

Similar to normal HSCs, LSCs are characterized by their

ability to self-renew, by their unlimited repopulating poten-

tial, and by the production of a multitude of progeny cellswith more differentiated characteristics, LSCs have also been

reported to express stem cell markers and to survive indefi-

nitely upon serial transplantations in animal models.

Whereas common leukemia blasts multiply prolifically, the

LSC divide slowly.6 The latter characteristic is especially asso-

ciated with their resistance to conventional chemotherapy

strategies that target rapidly dividing cells. Like their normal

counterparts, the LSCs are also extremely rare.

Xenogeneic transplantation modelsThus far, engraftment of human leukemia in an in vivo

xeno-transplantation model represents the ultimate proof forthe concept of LSC. The developmental steps for this animal

model have led in the nineties to the severe combined immu-

nodeficiency (SCID) model7 and thereafter to the successful

transplantation of selected subpopulations of AML cells into

SCID mice.2 This was followed later by the more efficient

NOD/SCID model.3 The transplanted leukemia cells were

highly similar to the original disease from the respective

patient and hence the stem cell nature of the transplanted

cells was considered proven.

The determination of the number of leukemic stem cells

is usually performed by limiting-dilution experiments. A

Key words: Leukemia, Stem cells, HSC, LSC, ALDH

DOI: 10.1002/ijc.26318

History:Received 13 Apr 2011; Accepted 12 Jul 2011; Online 27 Jul

2011

Correspondence to:Anthony D. Ho, Department of Internal

Medicine V, Heidelberg University Medical Center, Im Neuenheimer

Feld 410, 69120 Heidelberg, Germany,

E-mail: [email protected]

SpecialSection

Paper

Int. J. Cancer: 129, 23282336 (2011) VC 2011 UICC

International Journal of Cancer

IJC


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defined number of cells in log-scale reductions were seeded

in colony-formation or transplantation studies and the

growth or engraftment is subsequently assessed and extrapo-

lated to estimate the original stem/progenitor cell number.

Early in vivo experiments estimated a frequency of AML-ini-

tiating cells of about 0.4 per 105 mononuclear cells.2 This

was further refined by the demonstration of a self-renewal

capacity by serial transplantations. The most recent improve-

ments of this model system include murine models with

increased immunodeficiency8,9 and increased permissiveness

to normal as well as leukemia human cells by expression of

transgenes for human growth-factors in these animals.10

Methods derived from these experiments have become blue-prints for cancer stem cell research derived from other

tissues.

Albeit the relevance of LSC has been suggested by exten-

sive experiments in animal models, translation of this knowl-

edge into the clinic has faced two major challengescontro-

versy in the identification of LSC candidates and

subsequently their prospective separation, as well as the defi-

nition of their biologic properties as compared to normal

HSC (Table 1).

Surface antigen markers for enrichment of myeloid

LSCIn analogy to identification of human HSC, most investiga-tors have used surface antigen markers to separate and enrich

the LSC subset from the primary leukemia population. The

usual marker profile to distinguish and select the leukemic

stem cell population is based on CD34 as starting point. This

antigen is expressed on most HSC, although there is probably

a small fraction of dormant and primitive HSC without this

antigen. CD34 is also expressed on committed progenitor

cells and then lost in the further hematopoietic differentiation

process. Thus, it is not exclusive for stem cells. Similarly,

CD34 is expressed on a majority of AML stem cells. A com-

monly used surface antigen marker for myeloid differentia-

tion is CD38, which can also be found on several nonmyeloid

cell types and which is absent from HSC. The significance of

CD38-expression for enrichment of LSC is controversial. In

most of the initial studies, LSCs were defined by a

CD34CD38 phenotype, but recently Taussiget al. demon-

strated that seven of seven tested individual AML samples

could engraft from CD34CD38 cells. However, the cell

dose required for engraftment of CD34CD38 cells in ani-

mal models was in the range of 106 cells per animal, whereas

that of CD34CD38 was in a much lower range.16

This marker combination CD34/CD38 was able to

enrich leukemia-initiating cells or LSC, as first demonstratedby Lapidot et al. in 1994. In this seminal publication, the

injection of 2 105 CD34/CD38 AML cells into immuno-

deficient SCID mice was sufficient to initiate an AML in the

transplanted recipients.2 Given the recent development of

more permissive immunodeficient mouse models and trans-

plantation techniques, considerably lower numbers of LSC

candidates with as few as 2001000 cells per animal have

been reported to result in stable engraftment of AML.17,18

Other authors have also indicated that LSC could be

enriched in the cell fraction that is CD33 or CD9019,20 or

CD34CD90.21 A different approach is the staining with

broad intracellular dyes. The most prominent is Hoechst

33342, a mitochondrial dye that is also a substrate for ATP-

binding cassette (ABC)-type transporters. The Hoechst

33342low population is termed side-population and has stem

cell features both in healthy and malignant cells.22

Pitfalls of using surface antigen markersMost correlative studies have demonstrated LSC activity in

xenograft studies within the CD34/CD38 fraction. Taussig

et al.,23 however, recently demonstrated that the phenotype

of LSC from NPM-mutated AML is characterized by low

CD34 expression and is different to that reported for

Table 1. Development of blood stem cell and leukemia stem cell assays

Year Assay Authors

1961 Spleen colony forming cells (CFU-S) as progenitors after transplantation intoirradiated recipient mice

Till and McCulloch11

1965/66 Clonogenic assays in semisolid media for blood progenitor cells (CFU-GM, CFU-B) Pluznik and Sachs12;

Bradley and Metcalf13

1977 Stroma-based cultivation methods for in vitro identification of LTC-ICs Dexter et al.14

1989 In vitro stroma-based limiting dilution cobblestone area forming cell (CAFC)assay for blood progenitor and stem cells

Ploemacher et al.15

1994 First in vivodemonstration of a human AML-initiating cell in immunodeficient SCID mice Lapidot et al.2

1997 Transplantation of AML stem cells in more immunodeficient NOD/SCID mice Bonnet and Dick3

2000 Development of NOD/LtSz-Rag1null mice with extended immunodeficiency andhigher engraftment rates

Shultz et al.9

2005 Development of NOD/SCID/IL2R?null mice with extended immunodeficiency andhigher engraftment rates

Ishikawa et al.8

2010 Development of NOD/SCID/IL2R?null mice plus transgenetic expression of human growthfactors SCF, GM-CSF and IL-3 with higher permissiveness for human myeloid cells

Wunderlich et al.10

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unselected AML. In some samples, the LSC were exclusively

CD34, whereas other samples had both CD34 and CD34

LSC. Their study has suggested that there is greater heteroge-

neity in the phenotype of LSC than previously thought. Thereare indications that LSC might change their phenotype and

LSC might even be found in multiple fractions with different

intensities of CD34 and CD38 expressions.

Hence present evidence indicates that leukemia blasts dis-

play extremely diverse molecular and phenotypic features,

which are reflected correspondingly in their LSCs. In some

cases, aberrant markers are identified that can be very useful

for sorting strategies and are also clinically relevant for detec-

tion of minimal residual diseases. These aberrant surface

antigens are markers of lymphoid cells that are usually not

found on healthy myeloid cells, often T-cell antigens like

CD4 or CD7. The antibody panel used for characterization of

LSC therefore often comprises other markers such as CD90(Thy-1), CD117 (c-kit) and CD123 (IL3RA). Especially

CD123 is in many cases a strong marker for leukemic stem

cells24 (Table 2).

Heterogeneity of the LSC candidatesBased on the evidence listed in the previous paragraph, LSCs

are probably fairly heterogeneous and using surface markers

alone is not adequate for enrichment of LSC candidates.

Our group has recently demonstrated that the aldehyde de-

hydrogenase (ALDH)bright/CD34high subset had the highest leu-

kemic long-term culture initiating cell (LTC-IC) frequencies as

compared to other subsets. In a leukemia LTC-IC assay of indi-vidually sorted cells, i.e., single cell LTC-IC assay of each of

these aforementioned subsets of ALDHbright cells, the progeny

cell-colonies were assessed for cytogenetic markers characteris-

tic of the original AML. We found that a varying proportion of

colonies showed the original cytogenetic aberrations. Our ob-

servation indicated that the LSCs are heterogeneous and that

some of the LSC might not bear the characteristic cytogenetic

marker at all. Another possibility is that under in vitro condi-

tions, survival and growth of normal HSCs are preferentially

favored over that of LSCs, in analogy to engraftment experi-

ments in xeno-transplantation models.27,28

Nevertheless, our results have provided evidence that

varying proportions of residual HSC might be found in LSC

preparations. Future challenge will include the separation of

normal HSC versus LSC from the same individual and pre-liminary experiments exploiting the expression of typical

aberrant markers on LSC candidates are promising (unpub-

lished results). In conjunction with index sorting and single

cell deposition, the functional and molecular characteristics

of these LSC as well as HSC candidates from the same

patient can be compared in future studies.

Divisional kinetics and ALDH activity of normal andleukemia stem cellsIn a series of experiments, our group has provided evidence

that other parameters such as divisional kinetics and asym-

metric divisions might facilitate the identification of the most

primitive HSC. Other authors have shown that, in analogy to

normal HSC, LSC have comparable slow divisional kinetics

and the ability for extensive self-renewal.29,30 We and others

have reported that a slow dividing fraction (PKHbright) of

HSC is superior to a fast dividing fraction (PKHdim) in

reconstituting the NOD/SCID mouse not only with myeloid

cells, but also T cells and B cells.27,31

In preliminary experiments, we attempted to isolate LSC

based on slow divisional kinetics using dilution of PKH

membrane dye or dilution of cytosolic carboxyfluorescein

succinimidyl ester (CFSE) dye during divisions as a parame-

ter. With this technology, we were able to isolate a very lim-

ited number of leukemia cells that fulfilled some of the crite-ria for LSC candidates. However, we were not able to recover

an adequate number for functional studies (Ho AD, unpub-

lished results, 2011).27

Another recent development is the use of the enzyme

ALDH as a marker for primitive HSC.4,25,32 ALDH is a group

of enzymes catalyzing the conversion of a broad range of

aldehydes to the corresponding acid via a NAD-dependent

irreversible reaction. ALDH can be detected by activation of

the dye aldefluor and its high fluorescence then signifies he-

matopoietic and leukemic stem cells (Fig. 1). ALDHbright cells

have also been identified in hematopoietic stem cells (HSCs)

Table 2. Antigens and functional markers for flow-cytometry based stem cell sorting25,26

Staining target Description

CD34 Marker antigen for hematopoietic stem and progenitor cells should be positive and alsoreports on CD34 stem cells and leukemic stem cells23

CD38 Maturation marker, normal HSC should be negative for this marker

CD33 Myeloid lineage marker

CD90 (Thy-1) HSC and myeloid LSC are usually CD90 20,21, there are also reports on CD34CD90 21 LSC

CD117 (c-kit) The receptor (tyrosine kinase) for stem cell factor regularly positive on HSC and myeloid LSC

CD123 (IL3RA) Often a strong marker for LSC24

CLL-1 C-type lectin-like molecule-1, a stem cell marker, also for myeloid LSC23

Hoechst 33342 Intracellular dye, stem cells are low22, analysed as a so-called side-population

ALDH High activity of ALDH in myeloid LSC25,26 and also HSC; see also Figure 1.

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2330 Leukemia stem cells



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from umbilical cord blood.3335 High ALDH activity has

been reported to delineate distinct CD34 or CD133 stem

cell subsets that are more primitive than the ALDH-negative

fractions.32,36,37 These normal HSCs have been reported to

demonstrate LTC-IC and NOD/SCID mouse repopulating

activity.

Using ALDH activity and low side-scatter pattern as a pa-

rameter, Cheunget al. reported that LSC candidates could be

isolated from patients with AML.25 They showed that these

cells were able to engraft in the NOD/SCID mouse modeland induced human AML growth. In another study reported

by Pearce et al., the ALDHbright and CD34 subpopulation in

AML largely overlapped, but a significant amount of ALDH-bright cells with LSC characteristics did not express the

CD34CD38/low phenotype.4

Our group has shown that LSC candidates could be repro-

ducibly enriched by combining the markers ALDH and

CD34.26 Functional studies in vitro have demonstrated that

ALDHbright cells from AML patients divided slowly, were

more adhesive to the stroma, gave rise to LTC-IC and leuke-

mia colonies that showed the same cytogenetic aberrations as

the parent leukemia.27 Moreover, ALDHbright/CD34 cells,

when compared with ALDHbright cells, yielded similar results.

Schubert et al. have demonstrated that in the NOD/SCID

mouse model, repopulating human AML initiating cells were

recovered from ALDHbright as well as from slow dividing

(PKHbright) cells.27 Divisional kinetics was determined by the

membrane dye (PKH) dilution method, as described previ-

ously by our group and other authors.31,38 Comparing all

these methods for enrichment of LSC candidates, we found

that isolation using ALDH activity, either alone or in combi-nation with CD34 expression, yielded comparable results

according to functional parameters. The efficiency of isolation

of viable ALDHbright cells for functional experiments was,

however, consistently higher than using other methods.

Clinical significance of identifying LSC candidatesUsing ALDH activity and low side-scatter pattern as a pa-

rameter, Cheunget al. reported that LSC candidates could be

isolated from 43 cases of AML.25 They then showed that the

presence of LSC was associated with adverse cytogenetic

markers, a strong leukemic engraftment in the NOD/SCID

Figure 1. Example for the sorting of an ALDH leukemia stem cell population. FACS gating of a stained sample of blood or bone marrow

cells. (a) Gating on a population of intact leukocytes in an FSC/SSC plot ( size/granularity). (b) Gating on propidium iodide (PI) negative

cell ( living cells). (c) Gating on Aldefluor-positive cells as a marker for ALDH-activity, in fluorescence channel FL1. There are about 2.4%

ALDH stem cells within the mononuclear cells of this sample. (d) As a control for specificity by inhibition of ALDH activity. (Ran, Schubert,

Eckstein; Reproduced with kind permission.39)

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mouse model and probably with a poorer prognosis. Simi-

larly, Pearce et al. showed that engraftment potential after i.v.

transplantation of unmanipulated AML cells (107 to 108) into

irradiated NOD/SCID immunodeficient mice correlated sig-

nificantly with poor prognosis40; 51% of 59 cases in this

study engrafted successfully and possible associations withhoming capacities, white blood cell, FLT-3 and nucleophos-

min mutations, CXCR4-expression were excluded. Neverthe-

less, engraftment of AML samples with cytogenetic aberra-

tions (five of five), as well as a strong relationship between

engraftment in NOD/SCID mice and poor overall survival

(OS) (13 of 25) in these patients indicated the clinical rele-

vance of engraftment data in the NOD/SCID model.

In a series of articles using a combination of surface

markers and side scatter characteristics, the group of Schuur-

huis has reported that detection of high frequencies of LSC

in the marrow of patients with AML at diagnosis or in remis-

sion is associated with poor survival.5 Van Rhenen et al.41

suggested that aberrant marker patterns are expressed on theCD34/CD38 cells in patients with AML which permitted

the separation of malignant from the normal stem cell com-

partments from the same patient. Using a combination of

forward and side scatter behaviors in flow cytometric studies

as well as expression of CD34 and aberrant markers for

detection of LSC, Terwijn et al.14,42 have documented that

detection of residual LSC in remission bone marrow predicts

relapse in patients with AML and represents an independent

prognostic factor in the identification of poor prognostic

patients. Albeit the experimental approaches were different,

the observations by these authors are compatible with the

notion that the frequency of LSC candidates at the time of

diagnosis correlates with poor prognosis.

In a recent study in 101 patients with AML, we have

demonstrated that ALDHbright cells in the marrow as a surro-

gate marker for LSC candidates at the time of diagnosis of

AML is an independent prognostic factor predicting refrac-

tory disease and poor clinical outcome.28 A remarkable find-

ing is the significant relationship between levels of LSC can-

didates at the time of diagnosis with the persistence of

leukemia blasts in the marrow after the first induction chem-

otherapy (Ran et al., manuscript in preparation).

Univariate and multivariate Cox regression analyses on

relapse free survival (RFS) as well as on OS further confirmed

the relevance of LSC candidates for long-term outcome. Inunivariate models, high frequencies of LSC candidates repre-

sent significant prognostic factors for decreased RFS as well

as decreased OS. Similarly, genetic factors were also relevant

prognostic factors for progression free survival and OS. In

the multivariate model (stepwise regression analysis), fre-

quency of ALDHbright cells was the strongest prognostic

marker affecting OS. Cytogenetic prognostic markers showed

no strong effect on OS in the multivariate model. Our obser-

vations support the notion that higher levels of LSC candi-

dates are associated with resistant disease and that LSC can-

not be eliminated by conventional chemotherapy alone.28

Interactions between LSC and bone marrow nicheThe essential role of the interactions between stroma and tu-

mor cells43 as well as normal HSC and the marrow niche for

maintenance of stem cell properties has been reported exten-

sively. Within this context, adhesion molecules binding HSC

to the cellular determinants play a vital role.44,45

Most of theevidence has been derived from studies in animal models. In

all the engraftment studies using immunodeficient animal

models, the murine bone marrow environment represents a

substitute niche that is suboptimal and might not be extrapo-

lated for engraftment of HSC or LSC in the human marrow

microenvironment. The results from animal models, espe-

cially on the interactions between human stem cells and the

marrow niche, should therefore be validated using human

cells. Within this context, the human mesenchymal stromal

cell (MSC) preparations might represent a more appropriate

surrogate. Problems of the effect of the microenvironment

and of the chosen host model have been discussed in detail

by Rosen and Jordan.46In analogy, LSCs are maintained in a dormant state and

protected by the niche from cytotoxicity of chemotherapeutic

agents.47,48 Using HSC and MSC, derived from human marrow

as a surrogate model for the interaction between stem cells and

their niche, we have shown that direct contact between stem

cells and the microenvironment is essential in regulating asym-

metric divisions and promoting stem cell renewal.44,4952

To characterize the interactions between human HSC and

stromal cells Wuchter et al. have systematically analysed the

homotypic cell-cell contact among HSC and MSC.53 Although

among HSC, defined as CD34/CD38 cells, no prominent

junctions of cell-cell contacts were evident, remarkable junc-

tions and junction complexes were found between MSC. The

mesenchymal cells were interconnected by occasional gap

junctions and two morphotypes of adhering junctions, i.e.,

typical puncta adhaerentia and an abundant and elaborate

form of variously sized, invaginating villi-to-vermiform junc-

tion complex (complexus phalloides).

Using an immunodeficient mouse model, Ishikawa et al.

have demonstrated that CD34CD38 AML LSC home and

engraft to the osteoblast rich endosteal area of the animals.17

Results from our group have demonstrated that co-culture of

leukemia blasts or LSC with human MSC as a surrogate

niche model would increase the resistance of these cells

against chemotherapeutic agents.

27

Other authors havereported the significant role of the adhesive mechanisms

between LSC and the bone marrow niche, leading to dor-

mancy and drug resistance of the LSC.48 The adhesion mole-

cule CD44 might also play an essential role, as targeting of

CD44 might eradicate human myeloid LSC.54

Special features of lymphoid LSCThe first phenotype of acute lymphoid leukemia (ALL) stem

cells was described as immature CD34/CD19 cells.55,56 This

was later challenged by other authors who reported ALL-ini-

tiating cells in the CD34/CD19 subset.57,58 Lymphoid LSC

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candidates derived from patients characterized by t(9;22) or

t(4;11) might be found in the CD34/CD19 subset.57,59 Serial

transplantation studies have suggested that human lymphoid

LSC with more mature phenotypes were able to repopulate and

propagate B-precursor ALL.60,61 Konget al. reported that leu-

kemia initiating activity could be found both in the CD34

/CD38/CD19 as well as in the CD34/CD38/CD19 subset.

Thus, lymphoid LSCs, similar to the myeloid counterparts, are

fairly heterogeneous.60

By enumeration of copy number alterations (CNAs) of

the ETV6-RUNX1 fusion product in individually analyzed

LSC derived from childhood ALL, Anderson et al. have iden-

tified distinctive genetic signatures of subclones and their fre-

quencies, based on which they have inferred the evolutionary

architecture of the leukemia subclones.62 By monitoring FISH

stainings, the ETV6-RUNX1 (TEL-AML1) fusion at different

time points of the disease and by serial transplantations into

immunodeficient NOD/SCID/IL2Rcnull mice, they have dem-

onstrated clonal diversity and evolution in the LSC depart-ment and concluded that clonal architecture is subject to

alterations at diagnosis and in relapse.62

Notta et al. have demonstrated the genetic diversity of leu-

kemia initiating cells and they reported that many diagnostic

samples from patients with ALL contain multiple genetically

distinct subclones of LSC.63 This group focused on the evolu-

tion of adult Philadelphia positive (BCR-ABL positive) ALL-

initiating cells. With copy-number alteration (CNA) profiling,

they could demonstrate several subclones at diagnosis that

could be followed through repopulation studies in immunode-

ficient mice. During the repopulation process, the contributions

of subclones changed and smaller ones could outgrow the ini-

tial major subclone. Their findings implicate that there are

probably genetically distinct subclones at the LSC level that

undergo clonal evolution processes during the disease process.

Therapies directed against LSCThe ultimate goal of research in LSC is to induce long-term

cure for patients with AML by treatment strategies that will

enable us to eradicate the LSC. Different treatment principles

have been proposed in this respect.64,65

Molecules targeting proteins or pathways that are essential

and specific for survival and maintenance of LSC could be an

ideal approach.66 Thus far, there is little evidence for agents

that show specific effect against LSC, leaving normal HSCunharmed. The combined inhibition of two specific pathways

together has been proven to be effective in eradicating LSC in

animal models. The first pathway is the induction of oxidative

stress combined with the inhibition of nuclear factor jB (NF-

jB), physiologically transmitting survival signals. Guzman et

al.67 have reported that the proteasome inhibitor MG-132

might successfully inhibit both these processes and have led

to the preferential apoptosis of LSCin vitro and in vivo.

A compound that has attracted attention is parthenolide,

which is extracted from the herb feverfew. Preclinical experi-

ments have demonstrated remarkable activity against LSC. The

mechanism of cytotoxic activity was traced back to the inhibition

of NF-jB and exertion of cellular oxidative stress.68 To define

the underlying mechanisms, a genetic expression profile was

generated from parthenolide-treated LSC. These results were

then exploited for generation of a search pattern for expression

databases to identify substances with similar molecular effects.By means of this innovative method, two further agents have

been identified: celastrol and 4-hydroxy-2-nonenal (HNE),

which have been shown to effectively eradicate AML cells69 as

well as their corresponding progenitors and stem cells.

Another approach is influencing the pertubation of the ad-

hesion between LSC and their bone marrow niche.70,71 Mobili-

zation or priming of dormant LSC should thus release LSC

from their protective microenvironment. First indications that

this strategy might work have been suggested by Lowenberg

et al. In this clinical trial, G-CSF has been administered before

chemotherapy to patients with AML as a priming strategy.72

The interpretation of the results of this study has remained

controversial. Recently the concept was strengthened by newexperimental data by Ishikawa and colleagues.18 The recent de-

velopment of another molecule, plerixafor, a potent CXCR4

antagonist and modulator, might be promising. Pre-clinical

studies in animal models have shown encouraging results48,73

and a clinical trial in AML is already activated.74

A novel approach to make leukemic cells vulnerable to

chemotherapy is by forcing them into cell cycle by inducing

stress.75 One possible stress mechanism is the use of inter-

feron-a (IFN-a) for chronic myeloid leukemia (CML). Based

on investigations in animal models, Trumpp et al. suggested

that the application of IFN-a before treatment with 5-FU

might lead to an exhaustion of the dormant stem cell pool in

murine model.59,76

A further approach was presented by the group of Pan-

dolfi et al. who demonstrated the essential role of the PML

tumor suppressor protein already well known from acute

promyelocytic leukemia (APL) for the maintenance of

healthy HSC as well as CML LSC.77 Consequently, an inhibi-

tion of PML by the already clinically used arsenic trioxide

(As2O3) together with conventional chemotherapy lead to ap-

optosis of LSC and increased survival of mice in a transgenic

mouse model mimicking CML. Further targeted approaches

against CML stem cells are discussed.74,78

ConclusionsIt is of utmost importance to understand the mechanisms of

interaction between cellular niche and HSCs versus LSCs to pro-

vide a basis for the development of more efficient treatment strat-

egies. The application of such principles might induce long-term

cure as they could eradicate the ultimate source of leukemia.

AcknowledgementThe authors wish to thank Dan Ran, Mario Schubert, Volker Eckstein and

the team of the Bone Marrow Laboratory of the Department of Internal

Medicine V, Medical Center of the University of Heidelberg, for providing

the figures for this manuscript.

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