Leukemia stem cells - Wiley Online Library

IJC International Journal of Cancer

Leukemia stem cells Eike C. Buss and Anthony D. Ho

Special Section Paper

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 cancer In analogy to physiological differentiation, a stem cell paradigm has been proposed for malignant development.1 There is increasing evidence that human cancers may originate from transformation of stem cells, or alternatively, from progenitors 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.2 Since 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 leukemia 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 primary material was retrospectively assessed after engraftment of human leukemia.3 Albeit relevant knowledge on LSC biology has been gained from these sophisticated experiments, there has been thus far no reliable prospective method for the estimation of LSC burden.

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]

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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 prognostic significance controversial.4,5 Similar to normal HSCs, LSCs are characterized by their ability to self-renew, by their unlimited repopulating potential, and by the production of a multitude of progeny cells with more differentiated characteristics, LSCs have also been reported to express stem cell markers and to survive indefinitely upon serial transplantations in animal models. Whereas common leukemia blasts multiply prolifically, the LSC divide slowly.6 The latter characteristic is especially associated with their resistance to conventional chemotherapy strategies that target rapidly dividing cells. Like their normal counterparts, the LSCs are also extremely rare.

Xenogeneic transplantation models Thus far, engraftment of human leukemia in an in vivo xeno-transplantation model represents the ultimate proof for the concept of LSC. The developmental steps for this animal model have led in the nineties to the severe combined immunodeficiency (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

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Year

Assay

Authors

1961

Spleen colony forming cells (CFU-S) as progenitors after transplantation into irradiated 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 vivo demonstration 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

null

mice with extended immunodeficiency and

Shultz et al.9

2000

Development of NOD/LtSz-Rag1 higher engraftment rates

2005

Development of NOD/SCID/IL2R?null mice with extended immunodeficiency and higher engraftment rates

Ishikawa et al.8

2010

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

Wunderlich et al.10

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 extrapolated to estimate the original stem/progenitor cell number. Early in vivo experiments estimated a frequency of AML-initiating 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 improvements 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 blueprints for cancer stem cell research derived from other tissues. Albeit the relevance of LSC has been suggested by extensive experiments in animal models, translation of this knowledge into the clinic has faced two major challenges—controversy in the identification of LSC candidates and subsequently their prospective separation, as well as the definition of their biologic properties as compared to normal HSC (Table 1).

Surface antigen markers for enrichment of myeloid LSC In analogy to identification of human HSC, most investigators 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 comC 2011 UICC Int. J. Cancer: 129, 2328–2336 (2011) V

monly used surface antigen marker for myeloid differentiation 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 CD34þCD38 phenotype, but recently Taussig et al. demonstrated that seven of seven tested individual AML samples could engraft from CD34þCD38þ cells. However, the cell dose required for engraftment of CD34þCD38þ cells in animal models was in the range of 106 cells per animal, whereas that of CD34þCD38 was in a much lower range.16 This marker combination CD34þ/CD38 was able to enrich leukemia-initiating cells or LSC, as first demonstrated by Lapidot et al. in 1994. In this seminal publication, the injection of 2 105 CD34þ/CD38 AML cells into immunodeficient SCID mice was sufficient to initiate an AML in the transplanted recipients.2 Given the recent development of more permissive immunodeficient mouse models and transplantation techniques, considerably lower numbers of LSC candidates with as few as 200–1000 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 CD33þ or CD90þ19,20 or CD34þCD90.21 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 ATPbinding 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 markers Most correlative studies have demonstrated LSC activity in xenograft studies within the CD34þ/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

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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 also reports 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 CD90þ

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.

unselected AML. In some samples, the LSC were exclusively CD34, whereas other samples had both CD34 and CD34þ LSC. Their study has suggested that there is greater heterogeneity in the phenotype of LSC than previously thought. There are 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 display 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 detection 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 candidates Based 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 dehydrogenase (ALDH)bright/CD34high subset had the highest leukemic long-term culture initiating cell (LTC-IC) frequencies as compared to other subsets. In a leukemia LTC-IC assay of individually 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 characteristic of the original AML. We found that a varying proportion of colonies showed the original cytogenetic aberrations. Our observation 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 conditions, survival and growth of normal HSCs are preferentially favored over that of LSCs, in analogy to engraftment experiments in xeno-transplantation models.27,28

20,21

, there are also reports on CD34þCD90

21

LSC

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 preliminary experiments exploiting the expression of typical aberrant markers on LSC candidates are promising (unpublished 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 and leukemia stem cells In a series of experiments, our group has provided evidence that other parameters such as divisional kinetics and asymmetric 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 parameter. With this technology, we were able to isolate a very limited number of leukemia cells that fulfilled some of the criteria for LSC candidates. However, we were not able to recover an adequate number for functional studies (Ho AD, unpublished 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 NADþ-dependent irreversible reaction. ALDH can be detected by activation of the dye aldefluor and its high fluorescence then signifies hematopoietic and leukemic stem cells (Fig. 1). ALDHbright cells have also been identified in hematopoietic stem cells (HSCs) C 2011 UICC Int. J. Cancer: 129, 2328–2336 (2011) V

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Figure 1. Example for the sorting of an ALDHþ 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 (¼ size/granularity). (b) Gating on propidium iodide (PI) negative cell (¼ living cells). (c) Gating on Aldefluor-positive cells as a marker for ALDH-activity, in fluorescence channel FL1. There are about 2.4% ALDHþ 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)

from umbilical cord blood.33–35 High ALDH activity has been reported to delineate distinct CD34þ or CD133þ 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 parameter, Cheung et 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 model and induced human AML growth. In another study reported by Pearce et al., the ALDHbright and CD34þ subpopulation in AML largely overlapped, but a significant amount of ALDHbright cells with LSC characteristics did not express the CD34þCD38/low phenotype.4 Our group has shown that LSC candidates could be reproducibly 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 leukemia colonies that showed the same cytogenetic aberrations as C 2011 UICC Int. J. Cancer: 129, 2328–2336 (2011) V

the parent leukemia.27 Moreover, ALDHbright/CD34þ 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 previously 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 combination 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 candidates Using ALDH activity and low side-scatter pattern as a parameter, Cheung et 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


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mouse model and probably with a poorer prognosis. Similarly, 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 significantly with poor prognosis40; 51% of 59 cases in this study engrafted successfully and possible associations with homing capacities, white blood cell, FLT-3 and nucleophosmin mutations, CXCR4-expression were excluded. Nevertheless, engraftment of AML samples with cytogenetic aberrations (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 relevance 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 Schuurhuis has reported that detection of high frequencies of LSC in the marrow of patients with AML at diagnosis or in remission is associated with poor survival.5 Van Rhenen et al.41 suggested that aberrant marker patterns are expressed on the CD34þ/CD38 cells in patients with AML which permitted the separation of malignant from the normal stem cell compartments 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 surrogate marker for LSC candidates at the time of diagnosis of AML is an independent prognostic factor predicting refractory disease and poor clinical outcome.28 A remarkable finding is the significant relationship between levels of LSC candidates at the time of diagnosis with the persistence of leukemia blasts in the marrow after the first induction chemotherapy (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. In univariate models, high frequencies of LSC candidates represent 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), frequency of ALDHbright cells was the strongest prognostic marker affecting OS. Cytogenetic prognostic markers showed no strong effect on OS in the multivariate model. Our observations support the notion that higher levels of LSC candidates are associated with resistant disease and that LSC cannot be eliminated by conventional chemotherapy alone.28

Leukemia stem cells

Interactions between LSC and bone marrow niche The essential role of the interactions between stroma and tumor cells43 as well as normal HSC and the marrow niche for maintenance of stem cell properties has been reported extensively. Within this context, adhesion molecules binding HSC to the cellular determinants play a vital role.44,45 Most of the evidence 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 extrapolated for engraftment of HSC or LSC in the human marrow microenvironment. The results from animal models, especially 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.46 In 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 asymmetric divisions and promoting stem cell renewal.44,49–52 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 CD34þ/CD38 cells, no prominent junctions of cell-cell contacts were evident, remarkable junctions 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 junction complex (complexus phalloides). Using an immunodeficient mouse model, Ishikawa et al. have demonstrated that CD34þCD38 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 have reported the significant role of the adhesive mechanisms between LSC and the bone marrow niche, leading to dormancy and drug resistance of the LSC.48 The adhesion molecule CD44 might also play an essential role, as targeting of CD44 might eradicate human myeloid LSC.54

Special features of lymphoid LSC The first phenotype of acute lymphoid leukemia (ALL) stem cells was described as immature CD34þ/CD19 cells.55,56 This was later challenged by other authors who reported ALL-initiating cells in the CD34þ/CD19þ subset.57,58 Lymphoid LSC C 2011 UICC Int. J. Cancer: 129, 2328–2336 (2011) V

candidates derived from patients characterized by t(9;22) or t(4;11) might be found in the CD34þ/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 Kong et al. reported that leukemia initiating activity could be found both in the CD34þ/ CD38þ/CD19þ as well as in the CD34þ/CD38/CD19þ 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 identified distinctive genetic signatures of subclones and their frequencies, 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 demonstrated clonal diversity and evolution in the LSC department and concluded that clonal architecture is subject to alterations at diagnosis and in relapse.62 Notta et al. have demonstrated the genetic diversity of leukemia 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 evolution of adult Philadelphia positive (BCR-ABL positive) ALLinitiating cells. With copy-number alteration (CNA) profiling, they could demonstrate several subclones at diagnosis that could be followed through repopulation studies in immunodeficient mice. During the repopulation process, the contributions of subclones changed and smaller ones could outgrow the initial 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 LSC The 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 HSC unharmed. 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 (NFjB), 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 LSC in vitro and in vivo. A compound that has attracted attention is parthenolide, which is extracted from the herb feverfew. Preclinical experiments have demonstrated remarkable activity against LSC. The C 2011 UICC Int. J. Cancer: 129, 2328–2336 (2011) V

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 adhesion between LSC and their bone marrow niche.70,71 Mobilization or priming of dormant LSC should thus release LSC from their protective microenvironment. First indications that this strategy might work have been suggested by Lo¨wenberg 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 new experimental data by Ishikawa and colleagues.18 The recent development 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 interferon-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 Pandolfi 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 inhibition of PML by the already clinically used arsenic trioxide (As2O3) together with conventional chemotherapy lead to apoptosis 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

Conclusions It is of utmost importance to understand the mechanisms of interaction between cellular niche and HSCs versus LSCs to provide a basis for the development of more efficient treatment strategies. The application of such principles might induce long-term cure as they could eradicate the ultimate source of leukemia.

Acknowledgement The 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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