Hematopoietic and leukemic stem cell

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The hematopoietic system can be symbolized as a pyramid, made up by a numerically expanding hierarchy of cell populations that are progressively restricted in their proliferation and differentiation abilities. The pyramid is headed by the pluripotent hematopoietic stem cell.

All the mature functional cells of the hematopoietic and immune systems originate from a very small number of undifferentiated cells called hematopoietic stem cells (HSCs), which defined by 3 basic characteristics. First, they are able to undergo self-renewal; second, they are able to undergo extensive proliferation; and, third, they are able to differentiate into multiple distinct cell types. These properties provide sufficient primitive cells to sustain haematopoiesis, while generating more mature cells with specialized capacities.

Hematopoietic stem cells (HSCs) were first identified by two Canadian scientists Till and McCulloch in murine bone marrow cells in 1961 (Till and McCulloch, 1961).

The probabilities of asymmetric versus symmetric division of HSC can be stochastically determined or influenced by external signals.

Through the process of asymmetric cell division, a single division can result in the formation of both an identical stem cell and a more mature cell (Ho, 2005).

Recent studies suggest that leukemias have a similar hierarchical organization with only a subset of cells possessing self-renewal capabilities. Such cells are frequently referred to as leukemia stem cells (LSC).

Leukemia stem cells (LSC) exhibit characteristics similar to those of normal HSCs and are thought to arise from normal stem cells through the accumulation of oncogenic insults (4, 5). However, LSCs may also arise from differentiated progenitor cells that have reacquired the capacity for self-renewal (6-8).

Hematopoietic stem cells

The Cell of Origin in CML

CML is a clonal myeloproliferative disorder. CML stem cells are present in CD34+ CD38- cells and contain the Philadelphia chromosome. (Holyoake et al., 2002).

The Philadelphia chromosome contains the chromosomal translocation between the break point cluster region gene and the gene encoding c-Abl (BCR-ABL). The protein products of the BCR-ABL chromosomal translocation are p210BCR-ABL and p190BCR-ABL.

It is difficult to develop a xenograft model of chronic phase human CML because of the persistence of normal HSC in most chronic phase CML patients, and currently the lack of method to selectively isolate the rare CML stem cells. To solve this problem, Eaves et al. recently identified chronic phase patients whose long-term culture-initiating cells were predominantly leukemic and assessed the number and types of the cells they produced in NOD/SCID and NOD/SCID-b2m_/_ mice (Eisterer et al., 2005)

Previous studies using SCID46 and NOD/SCID47 mouse transplant models have been used to determine the engraftment potential of CML stem cells, which share phenotypic markers with their normal counterparts. The majority of CML progenitors were found to have a higher proliferative capacity when compared to normal progenitors,48 which suggested that most CML progenitors were actively cycling. However, more detailed cell cycle analyses of primary CML samples identified a quiescent subpopulation within the CD34+ progenitors.16 In CML, by contrast, the LSCs appear to be very similar if not identical biologically and functionally to the normal HSCs. In chronic phase CML patients, fluorescence in situ hybridization (FISH) demonstrates the presence of the Philadelphia (Ph) chromosome in nearly all myeloid and some B- and T-lymphoid cells [31], indicating that the cell of origin in CML has multilineage differentiation capacity. BCR-ABL transcripts are detected in CD34+CD38_CD90+ cells purified from CML bone marrow [32]; in normal marrow, the CD34+CD38_CD90+ population contains virtually all the HSC activity [23]. Chronic-phase CML patient cells engraft NOD-SCID mice poorly relative to normal HSCs [33], which has hampered the functional characterization of LSCs from this stage of the disease, however, the LSC activity is found within the CD34+CD38- compartment [34].

On the other hand, Huntly and Gilliland [2, 11] demonstrated that BCR-ABL1 lacks the ability to confer SL-IC potential to committed progenitor cells, such as granulocyte-macrophage progenitors. Therefore, it is not clear whether leukemia stem cells in CML phenotypically resemble an SRC as in AML or a committed myeloid progenitor cell. A series of studies have defined the CML stem cell immunophenotype as CD34+ and CD38- [10].

The research of Jamieson et al [12] revealed higher levels of CD34+Lin- cells in bone marrow from patients with CML in the accelerated or blast phase than in normal bone marrow CML stem cells are CD90+, Thy1+ and Lin-. BC-CML patients have higher levels of the progenitor pool (CD34+ Lin- cells) than other CML patients Notably, seminal studies demonstrated that a quiescent population of CML stem cells (CD34+CD38-CD45RA-CD71-HLA-DRlo) with BCR-ABL kinase domain mutations, detectable prior to initiation of imatinib therapy, gives rise to leukemic cells that persist because they are inherently resistant to imatinib.20,23-33 The Cell of Origin in ALL Although LSCs have most often been associated with myeloid leukemias, recent studies suggest that at least some lymphoid diseases may also arise from malignant stem/progenitor cells. Acute lymphoblastic leukemia (ALL) is characterized by the malignant expansion of immature cells from lymphoid lineages. Approximately 85% of diagnosed ALL cases are a result of the expansion of B-cell precursors, and 15% correspond to T-cell precursor aberrancies.

Further, for Ph+ ALL, functional studies have defined an LSC population. The BCR/ABL translocation is found in 5% to 25% of ALL, the majority of which are B-cell lineage. NOD/SCID xenotransplantation studies were used to assess the functional characteristics of CD34+, CD38- cells from ALL patients carrying the BCR/ABL translocation.51 The phenotypically primitive cells were able to successfully engraft the marrow of experimental animals, but more mature cells could not. This finding demonstrates that malignant cells were generated exclusively by the stem cell population (CD34+/CD38-). Another study in B-cell ALL using patients with known T-cell receptor rearrangements, showed that primitive cell populations (CD34+, CD38-) isolated from remission patients maintained the leukemic T-cell receptor configuration While earlier studies on leukemia initiating or stem cells propose a CD19-negative phenotype in ALL (CD34+CD19- and CD34+CD10- [22, 23]), more recent work demonstrated that also leukemia cells with a committed progenitor phenotype CD34+ CD38low CD19+ have SL-IC potential [24].

Recently, [34] reported serial transplants of three different primary ALL patient samples into an improved strain of immunocompromised (NOD/SCID?c-/-) mice, and the characterization of the cell surface phenotype of the leukemia initiating cells using CD19, CD34, and CD38 markers. These data strongly support the existence also in ALL of a leukemic initiating cell/ALL stem cell, but a functional and phenotypic definition of such cell is currently incomplete.

Signaling pathways and other factors regulate HSC:-

Wnt pathway

Wnt ligands are a family of secreted glycoproteins (19 different Wnt genes in the mouse and human genomes) that are critical for normal development [47].

It is noteworthy that ß-catenin is a key downstream mediator of the canonical Wnt signaling pathway, which is essential for self-renewal of stem cells. Retention of ß- catenin at the stem cell membrane may prevent precocious activation of the Wnt signaling pathway [20].

Wnt stimulation results in accumulation of b-catenin and its translocation to the cell nucleus where it interacts with T-cell factor/lymphoid enhancer factor (TCF/LEF1) to regulate genes which are important in embryonic development and cell proliferation.

Initial experiments that studied the role of Wnt signaling in hematopoiesis showed hematopoietic progenitors from mouse fetal liver displayed a three- to four-fold increase in the ability to form colonies of hematopoietic cells in vitro when cultured with conditioned media containing Wnt5a [50]. A similar experiment performed with lin-, CD34+ human hematopoietic progenitors co-cultured with stromal cells transfected with WNT2B, WNT5A, or WNT10B cDNA showed a 1.4- to 1.8-fold increase in the frequency of primitive CD34+ cells in vitro [48]. Murdoch et al. [71] went on to show that injecting mice with Wnt5a-conditioned media prior to transplant of human umbilical cord blood cells increased engraftment more than three-fold.

Wnt-5A injected in vivo into immunodefi cient mice enhanced engraftment by human CD34??cells (Murdoch et al., 2003).

Reya et al. [72] isolated Lin-, c-kitHI, Sca-1HI and Thy1.1LO (KTSL) cells from mice overexpressing the anti-apoptotic BCL-2 gene and transduced them with a constitutively active form of ß-catenin. This resulted in 20- to 48-fold in vitro expansion of KTSL cells for up to 2 months (compared to control KTSL cells which did not survive past 48 h).

Wnt-5A injected in vivo into immunodefi cient mice enhanced engraftment by human CD34??cells (Murdoch et al., 2003) Reya et al. (2003) showed that overexpression of activated ?-catenin expanded the pool of HSC in long-term culture. Activation of Wnt signaling in HSC increases expression of HOXB4 and Notch1, both implicated in HSC self-renewal.

Reya et al. (2003) showed that overexpression of activated ?-catenin expanded the pool of HSC in long-term culture. He also showed that activation of WNT signaling increases the expression of other transcription factors and cell cycle regulators important in HSC renewal, such as HOXB4 and NOTCH-1.

The essential role of ?-catenin in hematopoiesis has been questioned in studies of mice with Cre-loxP-mediated inactivationed of ?-catenin, since there was no impaired HSC ability to self-renew and differentiate (Cobas et al., 2004).

However, Nemeth and Bodine suggested that an alternative protein, such as ?-catenin, or an alternative signaling pathway, such as the TGF pathway which can also signal through TCF/LEF factors, might be able to partially compensate.


Notch signaling pathway has been demonstrated to play crucial regulatory roles for self-renewal regulation of HSCs [23]. Studies have shown that activation of Notch signaling results in increased numbers of HSCs and hematopoietic progenitors in vitro and in vivo.

Activation of Notch through ligand binding results in proteolytic cleavage of the intracellular domain of Notch, which subsequently transfers to the nucleus where it acts as a transcriptional regulator. It has been shown that Notch signaling mediated by both Delta and Jagged ligands expands the HSC compartment while blocking or delaying terminal myeloid differentiation with a decreased interval in the G1 phase of the cell cycle. (130)

The latter observation has been followed up by more extensive analysis of an interaction between Notch1 and CKI regulation. It has been reported that the basis for Notch influencing G1 may be through alteration in G1-S checkpoint regulator stability, specifically affecting the proteasome degradation of CKI, p27.131 Members of the NOTCH family have critical roles in keeping HSC in an undifferentiated state and may act as gatekeepers for factors governing self-renewal and lineage commitment.

Retrovirus-mediated expression of activated Notch1 enhanced HSC self-renewal, and a similar effect of differentiation inhibition and progenitor/HSC expansion was reported with activated Notch4 (Int3) (Ye et al., 2003).

Bernstein group (Varnum-Finney et al., 2003; Delaney et al., 2005) showed that incubation of murine BM precursors with the Notch ligand Delta1 extracellular domain fused to the Fc portion of human IgG1, together with cytokines (KL, IL-6, IL-11, and Flt3L), inhibited myeloid differentiation and promoted several log increases in precursors capable of short-term lymphoid and myeloid repopulation.

Inhibition of Notch signaling leads to accelerated differentiation of HSC in vitro and depletion of HSC in vivo (Duncan et al., 2005). Duncan et al (2005) found that Notch signalling is active in HSCs in vivo and downregulates as HSCs differentiate.


Hedgehog (Hh) family of proteins comprise three proteins: Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh) and two primary receptors Smoothened (Smo) and Patched (Ptc).

Hedgehog signaling plays essential role in HSC regulation. For instance, recent studies revealed that Hedgehog is a cell cycle regulator in HSCs which control hematopoietic regeneration (Trowbridge et al., 2006).

Sonic hedgehog (SHH) treatment in CD34??cultures induced expansion of human HSC, and Noggin, a specific inhibitor of bone morphogenic protein-4 (BMP-4), inhibited SHH-induced proliferation, indicating that SHH regulates HSC via mechanisms that are dependent on downstream BMP signals (Bhardwaj et al., 2001).

Antibody to Hh blocked cytokine induced proliferation of HSCs, while addition of soluble Shh resulted in an increase in stem cell numbers, as measured by the NOD/SRC assays. The mitogenic effect of Smo was abrogated by Noggin, demonstrating that Shh acts via BMP-4 in primitive HSC proliferation (Bhardwaj et al, 2001).


Transduction of mouse HSC with constitutively activated STAT3 enhanced HSC self-renewal under stimulated but not homeostatic conditions, while a dominant negative form of STAT3 suppressed self-renewal (Chung et al., 2006).

The STAT5 pathway is activated strongly following ligand binding to the erythropoietin and IL-3 receptors and weakly following FL binding to Flt3. However, constitutively activating mutants of Flt3, found in ~25% of human acute myeloid leukemia, are associated with strong activation of STAT5 (reviewed in Moore, 2005).

A constitutively activated double mutant of STAT5a [STAT5a(1*6)] transduced into CD34??cells promoted enhanced HSC self-renewal, as measured by CAFC assay, and promoted enhanced erythroid differentiation relative to myeloid (Schuringa et al., 2004).

To evaluate their role in normal and leukemic stem cells, Kato et al. (2005) recently transfected constitutively active STATs mutants to activate STAT signaling selectively in HSCs. They found that activation of STAT5 in HSCs led to a dramatic expansion of multipotential progenitors and promoted HSC self-renewal ex vivo. In a mouse model of myeloproliferative disease (MPD), sustained STAT5 activation in HSCs but not in multipotential progenitors induced fatal MPD, indicating that the capacity of STAT5 to promote self-renewal of HSCs is crucial to MPD development. These findings indicate a specific role for STAT5 in self-renewal of normal as well as leukemic stem cells.

More tractable stem cell systems such as mouse ES cells and Drosophila germ line stem cells identified a role for STAT transcription factors in promoting self-renewal.107,108 STAT transcription factors are the effectors of the JAK-STAT signaling pathway, suggesting that instructive mechanisms can be important for the regulation of self-renewal. Roles for STAT transcription factors in the regulation of HSC activity weresuggested in two recent studies. First, HSCs in STAT5a?/? STAT5b?/??double-knockout mice displayed a cell-intrinsic defect in long-term repopulating activity.109,110 Second, mice deficient in STAT1 and STAT3 signaling because of a carboxy terminal deletion of the gp130 receptor subunit have increased numbers of immature multipotent progenitor cells.111


The Polycomb-group transcriptional repressor gene Bmi-1 has been implicated in HSC maintenance, and loss of function studies showed profound defects in HSC (Park et al., 2003). Bmi-1 overexpression down-regulated expression of p16 and p19Arf, which are encoded by ink4a, and enhanced HSC symmetrical division, resulting in expansion of multipotent progenitors in vitro and enhanced HSC repopulation in vivo (Jacobs et al., 1999).

Bmi-1 represents one of the best-characterized Polycomb family members with respect to function within multiple stem cell types. Bmi-1-deficient mice exhibit a reduction in phenotypically-defined HSCs; these cells engraft poorly and exhaust prematurely in serial transplantation experiments. Both gain- and loss-of-function experiments have demonstrated that Bmi-1 plays a role in HSC self-renewal, with an initial focus on the role of Bmi-1 as a negative regulator of the CDK inhibitor, p16INK4A 47-49

Recent evidence suggests that the Polycomb-family transcription factor Bmi-1 is a positive regulator of self-renewal.105,106

The BMI-1 oncogene is a member of the Polycomb group ring finger (PCGF) gene family. Its transcriptional activity was shown to be high in HSC and progressively down-regulated during hematopoietic differentiation (Dimri et al., 2002).

It is highly expressed in purified HSC and its expression declines with differentiation (Park et al., 2003). Bmi-1 seems to regulate stem cell renewal by modulating other genes that are important in cellular functions such as proliferation, survival, and lineage commitment (Park et al., 2003).

The fact that Bmi-1-deficient mice develop hypocellular BM and die at <2 months of age led to the suspicion that Bmi-1 is involved in the maintenance of the stem cell pool. The impairment in the stem cell self-renewal machinery in Bmi-1-deficient mice may be mediated via p16 (ink 4A) and/or p19 (ARF), which are known to be repressed by Bmi-1 and mediate cell cycle arrest or apoptosis respectively (Park et al, 2003).

Similar impaired repopulation has been reported in Rae28 (another polycomb gene)-deficient mice following transplantation of fetal liver (Ohta et al, 2002).

The HOX Family of Hematopoietic Regulators

HOX genes (including A, B, C, D clusters) encode transcription factors that are important regulators of hematopoiesis, and HOXB4 over-expression in particular has been identified to be able to drive high-level ex vivo HSC expansion.

It has been shown that the number of HSC in mice transplanted with virally HOXB4 transduced HSC are significantly higher (14-fold on average) than in mice transplanted with normal untransduced mice [30].

In 2002, Antonchuk et al. demonstrated the potency of HOXB4 to enable high-level ex vivo HSPCs expansion [31]. It has been further shown that over-expression of HOXB4 can result in a rapid and extensive in vitro expansion of highly polyclonal HSCs that retain full lympho-myeloid repopulating potential and have enhanced in vivo regenerative potential. Recently, Zhang et al. demonstrated that HOXB4 over-expression led to significantly increased CD341 cell expansion and infusion of the HOXB4 expanded cells resulted in superior engraftment in vivo [32]. More recently, a decoy peptide containing the YPWM motif of HOX protein also appeared to increase the number of CD341 cells and enhance HOX function [33].

Although the exact mechanism of HOX is not fully understood, evidence suggests that HOXB4 may elicit an in vitro differentiation delay in bone morrow cells [34]. In contrast to HOXB4, over-expression of the other three HOX gene family members, HOXB6, HOXB9, and HOXB10, not only expands HSPCs but also perturbs cell differentiation [35-37].

Cocultures of human CD34+CD38) cells on stromal cells secreting HoxB4 underwent a 20-fold increase in long-term initiating cells (LTC-IC) and 2.5-fold increase in SRC (Amsellem et al, 2003). Similar results were obtained when mouse BM cells were grown on stromal cells engineered to express HoxB4 preceded by the human immunodeficiency virus (HIV)-transactivating protein, TAT (Krosl et al, 2003).

HOXB4 promotes the expansion of HSC without losing their ability to differentiate into normal lymphoid and myeloid cells (Sauvageau et al., 1995). It is abundantly expressed in HSC but declines as terminal differentiation proceeds (Sauvageau et al., 1995).

The most promising results so far were obtained after overexpressing the homeobox transcription factor HoxB4 in HSCs. Endogenous HoxB4 is expressed in a variety of developing embryonic and adult tissues including a highly enriched hematopoietic progenitor-stem cell population.112 Retroviral overexpression of HoxB4 in mouse HSCs resulted in a 50-fold amplification of HSCs, initially without identifiable anomalies in the peripheral blood of HoxB4-transduced mice.113 Moreover, HoxB4-mediated expansion of HSCs could be accomplished ex vivo,114 and expression of HoxB4 was able to confer adulttype HSC engraftment potential to hematopoietic progenitors generated from mouse ES cells.115 Although promising, translation of these results into novel therapeutic approaches will require circumventing the genetic manipulation required to overexpress HoxB4 and an understanding of why, in some systems, HoxB4 expression compromises lymphoid or multilineage differentiation.116,117 HOXB4 plays a critical role in promoting HSC self-renewal and engraftment (reviewed in Sauvageau et al., 2004; Moore, 2005). Combinations of early-acting cytokines increased HOXB4 promoter activity in primitive hematopoietic cells, and Tpo acting via Mpl and p38MAPK increased HoxB4 expression two- to threefold in primitive hematopoietic cells (Kirito et al., 2003).

Retroviral-mediated ectopic expression of HOXB4 resulted in a rapid increase in proliferation of murine HSC both in vivo (1000-fold increase in transduced HSC in a murine transplant model) and in vitro (40-fold expansion of murine HSC), with retention of lymphomyeloid repopulating potential and enhanced regenerative capability in mice (reviewed in Sauvageau et al., 2004). However, high levels of HOXB4 expression in human umbilical cord blood (CB) CD34??cells were recently reported either to increase proliferation of HSC and inhibit differentiation (Schiedlmeier et al., 2003) or to direct the cells toward a myeloid differentiation program rather than increasing proliferation (Brun et al., 2003). These studies suggest that in human hematopoietic progenitors, HOXB4 affects cell fate decisions (self-renewal, differentiation, or a differentiation block) in a concentration-dependent manner.



Factors such as TGF&szlig;-1 and macrophage inhibitory protein-1a?(MIP-1a) have been noted to play a role in dampening hematopoietic cell growth kinetics.43,44 Particularly, TGF&szlig;-1 has been shown to be able to selectively inhibit the growth of HSCs and progenitor cells.45-48 TGF?-1 has been documented to have varied effects on hematopoietic cells including enhancement of granulocytes proliferation in response to granulocyte-macrophage colony stimulating factor117 or inhibition of progenitor cell responsiveness to other growth promoting cytokines.118

TGF?-1 has been extensively characterized as a dominant negative regulator of hematopoietic cell proliferation including inhibiting primitive progenitor cells.44,49,121,122 Antisense TGF?-1 or neutralizing antibodies of TGF?-1 have been used to induce quiescent stem cells into the cell cycle and to augment retroviral gene transduction in conjunction with downregulation of p27 in human CD34+ subsets.44,50,101 Based on the roles of CKIs in hematopoietic cells as described previously, the link between TGF?-1 and CKIs in stem cell regulation has been recently addressed. TGF?-1-induced cell cycle arrest has been shown to be mediated through p15, p21, or p27 in multiple cell lines or cell types, including human epithelial cell lines,123,124 fibroblast cells,125 and colon126 and ovary cancer cell lines.127

Recently, it was proposed that p21 and p27 are key downstream mediators for TGF?-1 in hematopoietic cells,121,128

TGF??maintains HSC in a quiescent or slowly cycling state, and this cell-cycle arrest was linked to up-regulation of the cyclin-dependent kinase inhibitor p57KIP2 in primary human hematopoietic cells (Fig. 49.2) (Scandura et al., 2004). Fortunel et al. (2003) attributed the TGF??affect in part to down-modulation of cell surface expression of tyrosine kinase receptors cKit, Flt3, and IL-6R and the Tpo receptor Mpl. This negative regulatory role of TGF??has been challenged by Larsson et al. (2003), who showed that TGF?- R1-null mice had normal in vivo hematopoiesis and a normal HSC cell-cycle distribution and did not differ in long-term HSC repopulating potential as compared to wildtype animals.


Telomeres are regions of double stranded DNA, consisting of repetitive T2AG3 sequences, found at chromosome ends, and were first described as early as the 1930s as essential components that stabilize chromosome ends.21

More recently, three further critical observations have been made. First, telomere length decreases with every cell division,24 second, telomeres from older tissues are short compared to younger tissues25 and third, telomere length is reduced in tumours as compared to adjacent normal tissue.26

Telomere length is maintained by a balance of processes that lead to shortening or lengthening of the telomere sequence. The main factor, which elongates telomeres, is the enzyme telomerase. Low levels of telomerase expression are seen in primitive HSC and in lymphocytes.29-32

However, in tissue culture, when human cell lines are immortalized, telomerase expression is greatly upregulated and high levels of telomerase are also seen in 90 percent of human cancers.33

AML Stem Cell Properties

1- Phenotype.

It is important to differentiate between HSC and LSC phenotype markers to guarantee therapeutics strategies targeted against the LSC not HSC.

LSC and HSC share many of the cell surface markers such as CD34, CD38, HLA-DR, and CD71. Blair et al. demonstrated that the majority of AML blasts lack the expression of Thy1 (CD90), which differentiate primitive AML progenitor cells from normal hematopoietic progenitor cells (28). Same with c-kit (CD117), the lack of c-kit expression is a more consistent feature of LSC that is not shared by normal HSC.

Jordan et al. showed that the interleukin-3 receptor a chain (IL-3a) also called (CD123) is a unique marker for human AML stem cells (101). This marker was found to be expressed on 98% of the CD34+CD38- AML cells. Hosen et al identified CD96 as a surface marker on the majority of CD34+CD38- AML cells, with minimal expression on normal CD34+38- cells (102).

In addition, recent studies suggest that LSCs from most AMLs express the early myeloid antigen CD33 [27] and the novel antigen C-type lectin-like molecule-1 (CLL-1) [28]. Although CD33 might also be expressed on some normal HSCs [27], CLL-1 is present exclusively on malignant CD34+ CD38- cells [15].

Another cell-surface target on LSCs is CD44. CD44 is overexpressed on both AML and CML LSCs relative to normal HSC. expression of CD13 [14] are specifically expressed on AML stem cells but not on normal SRC .

2- Cell Cycle

Cell cycle status can be used to differentiate between LSC and other AML blast, where most of the AML-colony forming units (AML-CFU) are active cells and LSCs are quiescent cells.

Guan et al. showed that AML stem cells reside mostly in a quiescent cell cycle state, analogous to their normal hematopoietic stem cell counterparts. This observation is important because most therapeutic approaches to leukemia are directed towards actively cycling populations. The quiescent nature of LSCs indicates that standard chemotherapy drugs will not generally be effective against AML stem cells. [14] The relative quiescence of LSCs may be a major factor contributing to relapse [8].

To analyze nuclear factor-B (NF-?B) in primitive AML cells, Guzman et al. isolated CD34+ CD38- CD123+ cells; these cells were not stimulated to reflect the status of primitive AML cells. Further assessment with flow cytometry revealed the average proportion of cells in G0 corresponds to 96%±2.3%. (38)

Another evidence for quiescent LSCs was reported by Terpstra et al,19 who showed that treatment with the cycle active drug 5-fluorouracil was not effective in ablating AML cells in SCID mice transplanted with primary leukemic specimens.

3- Regulation of Cell Death and Self-Renewal

LSC and normal HSC share the ability to self-renew, thus it is important to understand how HSC and LSC maintain normal hematopoiesis and leukemogenecity, respectively, and this would explain the relapse of AML cases Guzman et al. has shown that primitive AML cells aberrantly express the tumour suppressor genes interferon regulatory factor 1 (IRF1) and death-associated protein kinase (DAPK) with increase expression of transcription factor NF- ?&szlig; (129&999), but not in CD34+/CD38- cells from normal specimens. Normal hematopoietic stem cells do not show activation of NF-?B. This is only a leukemia pecific phenomenon. He reasoned that transcriptional activators NF-?B might lie upstream of IRF-1. NF- ?&szlig; had been shown to exhibit anti-apoptotic activity in a variety of cancers (39-42). Furthermore, it was shown that NF- ?&szlig; is activated in the majority of primary AML specimens and LSC quiescent cells; however, it is not activated in their normal counterpart, suggesting this transcription factor is a key survival factor and a leukemia specific phenomenon for the LSC (142).

Another important pathway implicated in LSC survival is signaling via the PI3 kinase pathway. PI3 kinase activity has been reported for a large percentage of primary AML specimens. It has been demonstrated loss of LSC as a result of treatment with drugs that inhibit PI3 kinase activity. (Xu et al., 2003)

Other studies have demonstrated the important role that disregulated wnt pathway signaling plays in leukemia and LSC biology (23-25).

Bmi-1 is not required for the formation of HSCs or LSCs, but is required for their maintenance [13;18].

Previous studies have demonstrated that the Bmi-1 is necessary for self renewal of both HSC and LSC. (45) Park et al. demonstrated that there was no detectable self renewal of adult HSCs as a result of defect in Bmi-1-/- mice. (49)

Targeting LSC Self-Renewal

Wnt Signaling in Leukemia


As the role of wnt pathway found to promote normal stem cell self renewal, it has been found also to promote LSC proliferation (Yilmaz et al., 2006).

Several recent papers have demonstrated the important role that disregulated wnt pathway signaling plays in leukemia and LSC biology.23-25

Furthermore, very recently, research has shown that progression to blast crisisCMLis associated with missplicing of GSK-3b in granulocyte-macrophage progenitors, allowing unphosphorylated b-catenin to contribute to self-renewal (Abrahamsson et al. 2007).

In AML, studies have shown that there is constitutive activation of the Wnt pathway (Simon et al. 2005) and that translocation products, e.g., AML1-ETO, PML-RARa, and PLZF- RARa activate the Wnt signaling pathway in hematopoietic cells (Muller-Tidow et al. 2004).

The Hedgehog Pathway in Leukemia Recent microarray studies by our group (Graham et al. 2007) and others (Radich et al. 2006) provide evidence that the SHH pathway is active in CML stem cells and that the SHH pathway becomes progressively more activated from chronic phase, through accelerated phase to blast crisis (Radich et al. 2006). Furthermore, in this study, activation of the SHH pathway correlated with CD34 expression, suggesting upregulation within CML stem and primitive progenitor cells.

Abnormal Hedgehog signaling may also be a feature of AML and MDS (Merchant et al. 2007).

The Notch Pathway in Leukemia


As the role of Notch pathway found to promote normal stem cell self renewal, it has been found also to promote LSC proliferation (Yilmaz et al., 2006).

Notch 1 is found to be constitutively activated in patients with the t(7:9) chromosomal translocation. This involves high expression of a constitutively activated form of Notch 1 by the promoter and enhancer elements regulating the &szlig;-chain of the T-cell receptor (Fig. 1B). Although this distinctive chromosomal translocation is found in <1% of T-cell acute lymphoblastic leukemia cases, >50% of T-cell acute lymphoblastic leukemia patients carry somatic activating point mutations of Notch 1 (24,25).

A gain-of function mutation is very common in the NOTCH1 receptor in T-ALL. This increased NOTCH signaling results in increased T-cell differentiation and selfrenewal in hematopoietic progenitors, leading to transformation to T-ALL (Aster et al. 2008).

NF-kB in Leukemia

NF-kB is constitutively activated in LSCs in AML and recently studies have focussed on attempting to eradicate AML stem cells using proteasome inhibitors which induce apoptosis in AML stem cells in association with inhibition of NF-kB and activation of p-53-related genes (Guzman et al. 2002). In addition, the naturally occurring small-molecule inhibitor parthenolide also causes apoptosis in LSC in AML and blast crisis CML, again through inhibition of NF-kB and activation of p-53 and also increased production of reactive oxygen species (Guzman et al. 2005, 2007b).

The HOX Gene Family in Leukemia


Notably, deregulated expression of HOX family members such as HOXA9 is commonly observed in AML (Lawrence et al., 1999).


HOX genes have been linked with the development of acute leukemia, and chromosomal translocations between NUP98 and HOXA9 or HOXD13 are reported in AML (Borrow et al. 1996; Raza-Egilmez et al. 1998), with overexpression ofHOXA9 carrying a particularly poor prognosis (Golub et al. 1999).

Overexpression of HOX11 has been reported in T-ALL (Hatano et al. 1991).

It has been proposed that self-renewal in LSCs may be regulated by HOX-dependent pathways (Argiropoulos and Humphries 2007).

The Polycomb Gene BMI-1 in Leukemia


BMI-1 has an essential role in regulating the proliferative potential of leukemic stem cells (Lessard & Sauvageau, 2003) Recent experiments with Bmi-1-/- mice showed that leukemic (AML) stem/progenitor cells lacking Bmi-1 were unable to engraft and proliferate and displayed signs of differentiation and apoptosis. Conversely, the reconstitution of the Bmi-1 gene was found to completely abrogate these proliferative defects (8).

The Role of PTEN in Leukemia

A recent study has shown that dependence on the tumor suppressor gene PTEN separates HSCs from LSCs (Yilmaz et al. 2006). In an in vivo mouse model, conditional deletion of PTEN resulted in a myeloproliferative disorder which progressed to acute leukemia over a number of weeks and also induced acute leukemia in recipient mice in amouse transplantation model.

Telomerase in Leukemia Studies have shown that telomeres are shorter and telomerase activity higher in CML LSCs compared to normal HSCs (Brummendorf et al. 2000). This raises the possibility of exploiting differences in telomerase activity to target LSCs.