Concepts of human leukemic development - Nature

Oncogene (2004) 23, 7164–7177

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Concepts of human leukemic development Jennifer K Warner1, Jean CY Wang1, Kristin J Hope1, Liqing Jin1 and John E Dick*,1 1

Division of Cell and Molecular Biology, University Health Network and Department of Molecular Genetics and Microbiology, University of Toronto, 620 University Ave, Toronto, ON M5G 2C1, Canada

Two fundamental problems in cancer research are identification of the normal cell within which cancer initiates and identification of the cell type capable of sustaining the growth of the neoplastic clone. There is overwhelming evidence that virtually all cancers are clonal and represent the progeny of a single cell. What is less clear for most cancers is which cells within the tumor clone possess tumorigenic or ‘cancer stem cell’ (CSC) properties and are capable of maintaining tumor growth. The concept that only a subpopulation of rare CSC is responsible for maintenance of the neoplasm emerged nearly 50 years ago. Testing of this hypothesis is most advanced for the hematopoietic system due to the establishment of functional in vitro and in vivo assays for stem and progenitor cells at all stages of development. This body of work led to conclusive proof for CSC with the identification and purification of leukemic stem cells capable of repopulating NOD/SCID mice. This review will focus on the historical development of the CSC hypothesis, the mechanisms necessary to subvert normal developmental programs, and the identification of the cell in which these leukemogenic events first occur. Oncogene (2004) 23, 7164–7177. doi:10.1038/sj.onc.1207933 Keywords: stem cell; NOD/SCID; leukemic stem cell; cancer stem cell; SCID-repopulating cell; SRC; SL-IC

Introduction In hematopoiesis, the ancestral stem cell drives the production of a hierarchy of downstream cells comprised of multilineage and unilineage progenitors, followed by increasingly mature cells more restricted in differentiation capacity, and eventually terminating with the production of fully differentiated functional blood cells. Although hematopoietic stem cells (HSC) are pluripotent, the key property that distinguishes them from the immediate downstream progenitors (which are also pluripotent) is their ability to self-renew, that is, to generate daughter cells with the exact stem cell properties of the parent cell. Self-renewal can be symmetrical, producing two daughter HSC, or asymmetrical,

resulting in one HSC and another downstream progenitor that possesses a reduced capacity for self-renewal, thereby establishing a hierarchy. This progenitor cycles much more rapidly, resulting in marked clonal expansion of cells. Under steady-state conditions, HSC are quiescent or divide slowly, but when they divide they do so asymmetrically, in order to produce the entire complement of cells necessary to maintain blood production over time, without depletion (Dick, 2003b). Under some circumstances such as HSC transplantation, HSC divisions can be mostly symmetrical for a period of time to regenerate the stem cell pool before reverting to asymmetrical. Therefore, normal hematopoietic development is noted for the delicate balance between self-renewal and differentiation. When the processes of self-renewal and differentiation become deregulated or uncoupled, leukemias can result, characterized by an accumulation of immature blast cells that fail to differentiate into functional cells. Like other neoplasms, leukemia arises from the clonal expansion of a single cell (McCulloch et al., 1979; Fialkow et al., 1987b), and is sustained by a leukemic stem cell (LSC). In this review, we will focus on the identification and characterization of the LSC from acute myeloid leukemia (AML), and on emerging information that points to the HSC compartment as the first normal cell that becomes subverted in the leukemogenic process. In particular, we will begin with a brief review of the historical underpinnings that led to the identification of LSC. This body of work, carried out over the course of four decades concomitantly with studies of normal blood development, establishes the paradigm for identification of cancer stem cells (CSC) for any neoplasm. If the CSC hypothesis is applicable for most nonhematologic neoplasms, as recent studies of brain and breast suggest, significant effort should be directed towards identifying CSC for each tumor, determining the cancer-specific pathways that operate within CSC as opposed to the bulk tumor cell population, and developing appropriate CSC-specific therapies.

Identification of heterogeneity within tumors *Correspondence: JE Dick, Toronto General Research Institute, University Health Network and Department of Molecular and Medical Genetics, University of Toronto, Suite 7-700, Princess Margaret Hospital, 620 University Av., Toronto, ON M5G 2C1, Canada; E-mail: [email protected]

The cells comprising a cancerous population have long been known to be heterogeneous. Some of the characteristics by which cells within tumors appear to differ

Human leukemogenesis JK Warner et al

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include cellular morphology, immunophenotype, karyotype, and enzyme expression among others (Heppner, 1984). In AML, there is wide patient-to-patient heterogeneity in the appearance of the leukemic blasts. Conventionally, AML is classified into seven French– American–British (FAB) subtypes corresponding to the maturation stage of the leukemia as assessed by the expression of lineage differentiation markers and morphology of blast populations within individual patients (Bennett et al., 1976, 1985). In contrast to the simplistic view of cancer as uncontrolled cell proliferation, there is marked heterogeneity in the proliferative fraction of cells within a single tumor. For example, in mammary adenocarcinoma from mice it was found that a significant proportion of cells was nondividing and did not incorporate 3 H-thymidine (Mendelsohn, 1962). Similar work in AML showed that the blast population is predominantly noncycling, since this fraction was only marginally able to incorporate 3H-thymidine-50 -triphosphate (Wantzin and Killmann, 1977). Studies by Clarkson et al. (1970) used a continuous infusion of 3H-thymidine into AML patients over a longer period of 8–21 days and showed that 1–12% of cells were noncycling and remained unlabeled over this time period. While this early work clearly identified great variation in proliferative capacity within a neoplasm, these studies relied completely on the growth kinetics of bulk tumor cell populations, making it impossible to assess the capacity of any individual cell to sustain the tumor. Two theories were put forth to explain the marked heterogeneity of tumors and the ability of only a subset of these cells to exhibit significant proliferative potential, and as described below, tumor-initiating potential measured by in vivo or in vitro assays. The stochastic model postulates that each cell within the tumor has an equal but low probability of entering the cell cycle and being able to regrow the tumor (Till et al., 1964; Korn et al., 1973). A cell capable of the extensive proliferation necessary to initiate and sustain tumor growth ultimately undergoes many more divisions than a cell lacking this ability. Therefore, the majority of cells are unable to regrow the tumor because the cumulative probability of undergoing the required number of cell divisions is very low (Reya et al., 2001; Dick, 2003a). The alternate hypothesis proposed by many investigators is a model in which every tumor contains a rare population of functionally distinct CSC (i.e. tumorigenic cells) (Mackillop et al., 1983; Buick and Pollak, 1984). Like normal stem cells, these rare CSC possess the extensive proliferative and self-renewal potentials necessary to create a new tumor and generate a hierarchy of phenotypically diverse downstream cells, which are successively more limited in these properties. This model also stipulates that the descendants of CSC exhibit some remnants of normal differentiation. While the stochastic model postulates that relapses are due to growth of residual cells within the tumor that maintain a fixed low probability of extensive proliferation (therefore any cell is at risk), the hierarchy theory stipulates that the bulk of the tumor population is incapable of initiating tumor

regrowth, and that for a true cure, the tumor stem cells must be eradicated or irreversibly inactivated. The stochastic model suggests that the mechanisms underlying tumorigenicity are apparent in all cells of the tumor and that key properties of the neoplasm can therefore be elucidated by examining the bulk population. In contrast, the hierarchy model postulates that the molecular programs at work in tumor stem cells are unique and impart an inherently tumorigenic potential. Thus, in order to validate the tumor stem cell model, it is necessary to prospectively identify a population of cells within a tumor that can predictably and reliably demonstrate the capacity to regenerate the entire tumor including daughter stem cells and functionally inert tumor cells. If the hierarchy model is true, it should be possible to obtain both a fraction without CSC activity and a fraction enriched for CSC activity. By contrast, the stochastic model predicts that purification is not possible since each cell has equal potential for CSC activity. Thus, this critically important question can only be solved by examining individual neoplastic cells for their functional CSC capacity. The first neoplasm for which these models were rigorously tested was AML, in part because of the detailed knowledge of normal hematopoietic development that was gained over the past four decades. The availability of quantitative assays for individual stem and progenitor cells at all stages of hematopoietic differentiation provides the basis for this detailed understanding and distinguishes this organ system from any other. As technical developments in cell culture methods occurred during the 1950s, it became clear that only a fraction of solid tumor cells had the ability to grow in tissue culture and to form colonies. The pioneering studies of Till and McCulloch (1961) provided the key to modern stem cell biology: the ability to quantitatively assay individual murine stem/ progenitor cells (Becker et al., 1963). Bruce and Van Der Gaag (1963) subsequently showed that only a rare fraction of murine lymphoma cells have the capacity to establish disease in transplanted recipients. For studies of leukemia and specifically human leukemia, the key advance that led to remarkable progress was the development of in vitro agar or methylcellulose clonogenic assays previously optimized for murine hematopoietic progenitors and modified for use with human cells (Pluznik and Sachs, 1965; Bradley and Metcalf, 1966; Ichikawa et al., 1969). The inaugural assay measuring clonogenicity of human neoplastic cells involved the growth of myelomonocytic leukemia cells in vitro (Metcalf et al., 1969). This cell culture technique relies on the principle of immobilization of single cells in a semisolid medium. Once the movement of a cell is restricted, it is possible to evaluate the number of progeny derived from the parent cell following shortterm culture (Metcalf, 1973). Thus, for the first time these assays made possible the quantification of growth potential of individual leukemia cells in a fixed environment. There was clear functional heterogeneity, since only 1 in 102–105 primary AML cells were able to produce small clusters or colonies of cells with blast-like Oncogene


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morphology, despite the addition of stimulating factors. These cells were termed AML colony forming units (AML-CFU) (Moore et al., 1974; Minden et al., 1979). Similarly, the frequency of colony-forming cells in clonogenic assays optimized for solid cancer cells ranges from 1 in 600–5000 for lung carcinoma, ovarian adenocarcinoma, melanoma and neuroblastoma cells (Hamburger and Salmon, 1977). It is important to note that although the cells that read out in the colony forming assay must have the growth potential necessary to generate colonies of 30–40 progeny, these cells may or may not exhibit self-renewal (Mackillop et al., 1983). To measure this critical property, colonies derived from single cells in clonogenic assays can be recovered, pooled, and then replated. The number of colonies generated on the secondary plate gives an indication as to the extent of self-renewal of the initial clonogenic population. These studies showed that the majority of AML-CFU reading out in clonogenic assays had no detectable self-renewal upon replating (Buick et al., 1979). For example, in a test of primary AML patient samples, secondary plating efficiency was below 1% and rarely rose above 10% (McCulloch, 1983). Similarly, low levels of colony forming cell self-renewal were described for solid tumors such as ovarian carcinoma (Buick and MacKillop, 1981). Moreover, the bulk blast population expressed a myriad of differentiation antigens that are lacking on the more immature AML-CFU population and suggests a hierarchical relationship between these two populations (Griffin and Lo¨wenberg, 1986). A limitation of these colony-forming cell assays is that growth of cells is spatially restricted and can only be carried out for short periods (Mackillop et al., 1983). To circumvent these problems, assays were developed involving culturing of AML cells in suspension. The earliest iteration of this technique permitted proliferation for up to 70 days with the requirement that the culture be maintained at very high cell densities (Nara and McCulloch, 1985). An improved suspension culture assay was described by Sutherland et al. (1996) who used an optimized growth factor combination to achieve growth of leukemic populations for extended periods independent of cell concentration. This technique measured the capacity of immature suspension cultureinitiating cells (SC-IC) to endure and generate AMLCFU after 2–8 weeks in suspension. In parallel, this group also investigated the ability of bulk AML cells to maintain growth in long-term cultures containing bone marrow stroma. These cells are termed long-term culture initiating cells (LTC-IC). The frequencies of 8 week SC-IC and 5 week LTC-IC were 0.2–25 in 105 and 1.6–37 in 105 cells, respectively. In every case, cells with the capacity for extended proliferation in these longterm cultures were rarer than AML-CFU. Collectively, these data demonstrate that SC-IC and LTC-IC are distinct from, and have greater clonal longevity than, AML-CFU. Taken together, clonogenic, SC-IC, and LTC-IC assays showed that AML cells as well as cells of other cancer types are heterogeneous in their capacity to sustain growth in vitro. Oncogene

A critical limitation of clonogenic assays is that they cannot test whether a cell can initiate and sustain a leukemia in vivo, that is, whether it is a true LSC. This problem became even more acute with the realization that clonogenic assays for normal cells do not detect the rare HSC, which can only be assayed by repopulation. Similarly, transplantation assays provide a more rigorous test of the growth potential of human tumor populations since the disease can be re-established. In the late 1950s evidence of heterogeneity in in vivo tumorigenic ability was provided by studies in which autologous tumor cells were injected into the thigh of terminal cancer patients. Even at doses of 105–108 cells, only a fraction of patients developed tumor growth at the site of injection. This indicated that within the entire tumor cell population only a small fraction of cells had the capacity to initiate tumors (Southam and Brunschwig, 1960). Since in vivo repopulation assays are the only true measure of CSC and LSC, there was a need to develop in vivo experimental systems that could detect individual human neoplastic cells with the capacity to initiate disease in non-human recipients. Evidence for leukemia stem cells Although xenograft models for human cancer cells have a long history, initial systems did not typically reproduce all elements of the human disease including orthotopic growth, especially for hematological malignancies. The development of robust human-mouse xenograft systems using profoundly immune-deficient mice provided the critical advance that enabled the direct assessment of the malignant stem cell. These were the SCID-leukemia (Lapidot et al., 1994) and NOD/SCID-leukemia (Bonnet and Dick, 1997) xenotransplantation models in which mice were transplanted with leukemic cells from the BM and peripheral blood of AML patients. All FAB subtypes with the exception of M3 engrafted in both strains, with human leukemia cell levels consistently higher in NOD/SCID mice. Subsequent studies with a larger sample size indicated that FAB-M3 could also engraft mice, albeit at somewhat lower levels (Ailles et al., 1999). The resulting leukemic grafts were highly representative of the original patient’s disease, having both identical blast morphology and dissemination profiles. Limiting dilution analysis indicated that the cell capable of initiating AML in NOD/SCID mice (termed SCID-leukemia initiating cell, SL-IC) is present at a frequency of 0.2–100/106 mononuclear cells (Bonnet and Dick, 1997), in contrast to a frequency of 1/100 for AML-CFU. Moreover, the SL-IC population was shown by limiting dilution analysis and serial transplantation to have significant self-renewal capacity compared to normal adult BM. These observations demonstrated that only a very small fraction of AML cells are capable of the extensive self-renewal and proliferation necessary to regenerate the leukemia. This functional assay for LSC provided the basis to directly test the two models of neoplastic development described earlier. It was necessary to determine whether


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or not these cells possessed a unique surface phenotype that would consistently distinguish them from the remainder of AML cells. Fractions of AML cells were purified based on the variable expression of CD34 and CD38, and were then transplanted into NOD/SCID mice. In the first seven patients, the SL-IC were consistently found within the primitive CD34 þ CD38– fraction regardless of AML subtype (Lapidot et al., 1994; Bonnet and Dick, 1997). The same cell surface phenotype in normal human hematopoietic cell sources highly enriches for stem cell activity. As with unpurified AML cells, the grafts resulting from transplantation of CD34 þ CD38– cells were shown by flow cytometry to be comprised of aberrantly differentiated blast populations identical in phenotype to those of the original patient. There was no engraftment of CD34 þ CD38 þ cells, which are enriched for AML-CFU, even at cell doses of up to 5 105. Mice transplanted with small numbers of CD34 þ CD38– cells generated large numbers of AMLCFU in vivo. Together, these findings provide direct evidence that AML-CFU are downstream progeny of LSC. These results argue strongly that AML, like normal hematopoiesis, is indeed organized as a hierarchy initiated and maintained by an LSC, which in turn specifies an abnormal differentiation program leading ultimately to the production of terminal blast cells.

CD123. This marker was expressed on 98% of the CD34 þ CD38– cells of 16 AML patients, but was undetectable in normal bone marrow CD34 þ CD38– cells (Jordan et al., 2000). Functional assays confirmed the presence of SL-IC within the CD34 þ CD123 þ cells. The existence of markers such as CD123 homogeneously expressed on LSC from a wide range of AML samples point to a potentially conserved mechanism of leukemogenesis in the stem cell compartment. It will therefore be interesting to elucidate the pathways through which CD123 upregulation or c-kit downregulation occurs. In rare cases of AML, the CD34– fraction as well as the CD34 þ fraction engrafted NOD/SCID mice (Terpstra et al., 1996b; Blair and Sutherland, 2000). However, as with Thy-1, it is not known whether CD34– cells were initially transformed, or whether CD34 expression was lost as a result of the leukemogenic event. Interestingly, CD34 cells isolated from lineagedepleted umbilical cord blood have repopulating capacity in xenografted preimmune sheep (Zanjani et al., 1998) and NOD/SCID mice (Bhatia et al., 1998; Bonnet, 2001), providing proof that this fraction of normal hematopoietic cells contains a rare class of stem cells. This has raised the possibility that the CD34– cell may also be a target of leukemic transformation. On the other hand, if this potentially more primitive population were exempt from transformation, this would provide a means to separate normal HSC and LSC.

Properties of the AML stem cell Functional heterogeneity Phenotype Since LSC in some respects share a similar cell surface phenotype with normal HSC, much emphasis has been placed on further defining the phenotype of the LSC and elucidating the markers that uniquely recognize this cell. Such information is critical both to further our understanding of the leukemogenic process and to facilitate new treatments selective for the LSC and not its normal stem cell counterpart. While the LSC appears to similarly express many of the cell surface markers previously identified for HSC, such as CD34, CD38, HLA-DR, and CD71, several groups have reported that some surface markers are differentially expressed between the two. CD90 (Thy-1) is one marker that potentially distinguishes the HSC and LSC compartments (Blair et al., 1997). Thy-1 is downregulated in normal hematopoiesis as the most primitive stem cells progress toward the progenitor stage. Lack of expression of Thy-1 on LSC might suggest either that the early stem cell does not incur the primary leukemogenic event, or that Thy-1 is downregulated as a result of the acquired genetic change. However, other investigators have since reported several AML cases in which cytogenetically aberrant SL-IC could be found in the CD34 þ CD90 þ population (Brendel et al., 1999; Reya et al., 2003). As opposed to Thy-1, the lack of c-kit expression is a more consistent feature of LSC not shared by normal HSC (Blair and Sutherland, 2000). Perhaps the most promising unique marker of LSC, however, is IL-3 receptor a (IL-3Ra), also known as

Despite efforts to phenotypically characterize LSC on a population level, the functional composition of this compartment and the in vivo fate of individual LSC have not been examined. Our recent work using clonal tracking of retrovirus-transduced human cord blood in NOD/SCID mice demonstrated that the normal human HSC compartment was comprised of individual HSC classes that differ in their repopulating and self-renewal capacities (Guenechea et al., 2000). The phenotypic and biological similarities between the HSC and LSC compartments raise the important question of whether the LSC compartment is also comprised of distinct classes that differ in their proliferation and self-renewal capacities. Using a modified third generation lentivector, which can infect quiescent stem cells under minimal stimulatory conditions, we showed that SL-IC can be transduced with high efficiency, enabling high-resolution in vivo tracking of SL-IC at the single-cell level (Hope et al., 2004). Integration site analysis of serial bone marrow aspirates in transplanted mice revealed that the LSC compartment is heterogeneous with the existence of distinct classes of LSC, including those that could repopulate for only a short period (termed ST SLIC), and others that arose later or persisted for the length of the graft (termed LT SL-IC). Serial transplantation experiments showed conclusively that LT SL-IC can generate ST SL-IC, and that substantial heterogeneity in the self-renewal capacity of the SL-IC population exists. Interestingly, a small fraction of SLIC clones were undetectable in primary mice but became Oncogene


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dominant in secondary or even tertiary recipients. These were termed quiescent long-term SL-IC (qLT SL-IC) since they must divide rarely and/or undergo selfrenewal rather than commitment upon cell division. Together, these studies point to the existence of distinct classes of SL-IC with short- and long-term repopulating ability, and to the extensive heterogeneity in self-renewal potentials within the LSC compartment, suggesting that the SL-IC pool is a hierarchy resembling that of the HSC compartment. LSC, like normal HSC, possess selfrenewal capacity, although the clonal marking data, as well as our prior quantitative analysis by limiting dilution (Bonnet and Dick, 1997), indicate that selfrenewal is disregulated, as LSC possess substantially higher self-renewal capacity than normal SRC. Importantly, LSC still retain the ability to regulate selfrenewal, resulting in a hierarchically organized stem cell pool with marked similarities to the normal HSC compartment. Therefore, the commitment and/or differentiation and self-renewal decisions of LSC are not completely uncoupled, suggesting that the leukemogenic processes do not abolish all the pathways that normally regulate stem cell developmental programs. The finding that the stem cell-specific gene, Bmi-1, continues to play a key role in the self-renewal of both normal and leukemic murine stem cells supports this idea (Dick, 2003b; Lessard and Sauvageau, 2003). As discussed below, this data therefore adds additional support to the hypothesis that in AML the initial target for transformation lies within the HSC compartment and that LSC retain the ability to regulate self-renewal, resulting in a hierarchically organized stem cell pool with marked similarities to the normal HSC compartment. From a clinical standpoint, only a restricted number of longterm, highly self-renewing SL-IC within a functionally heterogeneous SL-IC pool may be responsible for aggressively driving the growth of AML and disease relapse. Identification of novel classes of LSC thus provides new targets for AML therapy. Leukemogenesis The data presented thus far provide support for the concept that rather than abolishing the pathways that normally regulate stem cell developmental programs, leukemogenic processes are layered onto this existing framework. Multiple lines of evidence indicate that tumorigenesis in humans is a multistep process (Hanahan and Weinberg, 2000). In leukemia, multiple acquired genetic changes must occur in order to convert a normal HSC to an LSC. AML is characterized by the accumulation of large numbers of blasts arrested at varying stages of differentiation; thus, a perturbation of the normal differentiation program with maturation arrest is a key event in leukemogenesis. In addition, in order for the leukemic clone to eventually become dominant, changes that confer a proliferative or selfrenewal advantage must occur. Increased cell output could result from increased cell cycling, or from an increase in self-renewal potential of the LSC with Oncogene

resultant expansion of the LSC pool. Although most immature progenitor and mature myeloid blood lineages are short-lived, alterations in the cell death pathways resulting in a survival benefit would also cause increased cell numbers of aberrant cells. Finally, given the greatly increased proliferative capacity of LSC, they must eventually acquire a mechanism by which to maintain telomeric DNA if they are to continue replicating indefinitely. Each of these changes – blocked differentiation, increased proliferation, altered self-renewal capacity, increased survival, and telomere maintenance – will be discussed in turn. Differentiation A common feature to all AML cases is arrested aberrant differentiation leading to an accumulation of more than 20% blast cells in the bone marrow (Gilliland and Tallman, 2002). As discussed earlier, traditional FAB classification of AML is based on the morphology and immunophenotype of these blast cells, with each subtype corresponding to a maturation stage (Bennett et al., 1976, 1985). More recently, the World Health Organization has adopted a classification system that considers not only morphology, but also the specific molecular lesions that are present (Harris et al., 1999). More than 80% of myeloid leukemias are associated with at least one chromosomal rearrangement (Pandolfi, 2001), and over 100 different chromosomal translocations have been cloned (Gilliland and Tallman, 2002). Frequently, these translocations involve genes encoding transcription factors that have been shown to play an important role in hematopoietic lineage development. Thus, alteration of the transcriptional machinery appears to be a common mechanism leading to arrested differentiation (Pandolfi, 2001; Tenen, 2003), representative examples of which will be discussed here. One transcription complex frequently targeted is the core-binding factor (CBF), consisting of the proteins AML1 and CBFb. AML1 is involved in early hematopoiesis with embryonic lethality in AML1/ mice, although the specific functions are not well understood (Speck and Gilliland, 2002). The chromosomal translocation t(8;21) is present in 12–15% of cases of AML, and is associated with the FAB M2 subtype (Speck and Gilliland, 2002). The resulting fusion protein, AML1-ETO, is a dominant-negative inhibitor of CBF transcriptional regulation, resulting in repression of AML1 target genes (Meyers et al., 1995). Another common chromosomal rearrangement, inv(16), is associated with 8–10% of cases of AML, specifically M4Eo (Speck and Gilliland, 2002). Cloning of the breakpoint for this translocation identified the fusion protein CBFb-SMMHC (Liu et al., 1993). Similar to AML1-ETO, CBFb-SMMHC functions as a dominant-negative inhibitor of AML1 transcriptional activity (Lutterbach et al., 1999). To date, there have been more than 12 different translocations identified that target the CBF complex, accounting for 25% of all AML (Speck and Gilliland, 2002). In addition, loss of function mutations in AML1 have been found in 20% of cases of AML M0, the most immature


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subtype (Osato et al., 1999; Preudhomme et al., 2000). It is interesting to note that loss of CBF function can result in different subtypes of AML. One explanation for this might be the way in which the complex is inhibited. With AML1-ETO, the ETO moiety recruits the nuclear co-repressor complex to the promoters of AML1 target genes (Gelmetti et al., 1998; Lutterbach et al., 1998), whereas with CBFb-SMMHC, the AML1 protein is sequestered to actin filaments in the cytoskeleton (Adya et al., 1998). This difference in localization may affect the ability of other transcription factors to partially compensate for the loss of AML1 transcriptional activation, leading to maturation arrest at different stages. The emergence of CBF as a common target in leukemogenesis raises the possibility of development of therapies directed at restoring the function of the CBF complex. Such an approach has proved successful for acute promyelocytic leukemia (APL; FAB M3), which accounts for 10% of cases of AML and is characterized by rearrangements of the gene encoding RARa. The therapeutic use of all-trans retinoic acid has proved successful in APL, inducing complete, although not durable, remission in most patients through the degradation of RARa fusion proteins and subsequent differentiation of leukemic blasts (Miller and Waxman, 2002). The subject of transcriptional and differentiation therapies has been reviewed elsewhere (Pandolfi, 2001; Miller and Waxman, 2002) and will not be discussed in detail here. It is becoming clear, however, that with increased understanding of the molecular changes involved in AML, the large number of different translocations that can occur may be distilled down to disruption of a few key pathways, facilitating the development of directed therapies in the future. Although a block in differentiation represents a necessary step in the development of leukemia, it is likely not sufficient. Transgenic mice expressing PMLRARa display abnormalities in myeloid differentiation, but only develop leukemia after long latency and with limited penetrance (Brown et al., 1997; Grisolano et al., 1997; He et al., 1997). Similarly, transgenic mice expressing a conditional AML1-ETO oncogene, or chimeric for CBFb-SMMHC expression, do not develop leukemia without the addition of chemical mutagens (Castilla et al., 1999; Higuchi et al., 2002). In primary human hematopoietic cells, expression of fusion oncoproteins involving transcription factors, such as TLSErg and AML1-ETO, leads to aberrant differentiation, but does not fully establish a leukemogenic program (Pereira et al., 1998; Mulloy et al., 2003). Taken together, this evidence suggests that alterations in transcriptional regulation leading to aberrant myeloid differentiation may be a necessary step in leukemogenesis, but that additional events are required to confer the growth and survival advantages that are necessary to progress to AML. Proliferation In order for a malignant clone to become dominant, it must acquire a significant growth advantage over

normal cells. Gilliland has proposed a two-hit model in which both a block in differentiation and an increase in proliferation (and/or apoptosis) contribute to leukemogenesis (Dash and Gilliland, 2001), and in which the proliferative advantage is often provided by activation of a receptor tyrosine kinase. Support for this model comes from the fact that two mitogenic growth factor receptor tyrosine kinases, Flt-3 and c-kit, are frequently mutated in AML (Reilly, 2003). Activating mutations in Flt-3 include internal tandem duplication of the juxtamembrane domain and the D835 point mutation in the activation loop. These have been identified in 17–34% and 7–10% of AML, respectively (Nakao et al., 1996; Yokota et al., 1997; Abu-Duhier et al., 2001; Yamamoto et al., 2001; Zheng et al., 2004). An additional 50% of AML samples express high levels of wild-type Flt-3 (Birg et al., 1992; Carow et al., 1996), and Flt-3 ligand (FL) induces proliferation of AML cells in vitro (Dehmel et al., 1996; Drexler et al., 1999). Coupled with the finding that all AML samples express detectable levels of FL, and that co-expression of Flt-3 and FL can generate an autocrine loop (Zheng et al., 2004), these data suggest that activation of this signaling pathway leading to increased proliferation plays an important role in the pathogenesis of AML. This increased proliferation, however, does not occur in all cells of the leukemic clone since the leukemic blasts that make up the majority of the clone have only limited proliferative capacity (Wantzin and Killmann, 1977). Similarly, studies in mice showed that 5-fluorouacil, a cell cycle active drug, ablated AML-CFU, but was unable to ablate AML cells with long term repopulating capacity (Terpstra et al., 1996a), suggesting that LSC also do not exhibit increased proliferation. Additional support for the quiescence of LSC was provided by cell cycle analysis of immunophenotypically defined primitive leukemic cells (Guzman et al., 2001). Thus, neither the stem cell population nor the leukemic blasts in AML are highly proliferative; instead, it appears that progenitors downstream from LSC are the cells in which this change is manifest. Investigation of CFC and LTCIC from primary AML samples showed that a higher proportion of these cells is cycling as compared to samples from normal individuals (Guan and Hogge, 2000). This may be due in part to autocrine signaling since normal progenitors from AML patients also show increased cycling (Guan and Hogge, 2000), suggesting paracrine effects from neighboring leukemic progenitors. Increased proliferation of AML progenitor cells but not LSC is consistent with the data reported for c-kit. This growth factor receptor tyrosine kinase is expressed in as many as 80% of cases of AML (Ikeda et al., 1991; Lyman and Jacobsen, 1998), and is mutated in a small proportion of these (Gari et al., 1999). The majority of AML samples expressing c-kit are capable of proliferating in response to its ligand, stem cell factor (SCF) (Ikeda et al., 1991; Lyman and Jacobsen, 1998). However, LSC capable of initiating leukemia in NOD/ SCID mice lack c-kit expression. Furthermore, proliferation of CD34 þ c-kit– AML cells is dependent on SCF, Oncogene


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and results in the production of c-kit þ cells (Blair and Sutherland, 2000). These data suggest that LSC that are c-kit differentiate to c-kit þ progenitors, which in turn can rapidly proliferate in response to SCF. An intriguing concept that arises from the two-hit model discussed above is that of cooperation between the two hits of altered differentiation and increased proliferation. Leukemias that have mutations affecting the CBF complex, specifically t(8;21) or inv(16), are highly enriched for mutations in c-kit (Gari et al., 1999; Beghini et al., 2000). In contrast, Flt-3 mutations in these same leukemias are rare (Schnittger et al., 2002; Thiede et al., 2002). These data suggest that in some cases the differentiation and proliferation events may cooperate to induce leukemia. Self-renewal One key feature of LSC that is shared with normal HSC is the ability to self-renew. Self-renewal is distinct from proliferation, as it involves a cell fate decision that is made upon cell division. From a theoretical standpoint, the continued expansion of the leukemic clone suggests that self-renewal is deregulated, resulting in LSC expansion due to a higher proportion of symmetrical stem cell fate outcomes. In vivo transplantation studies suggested that the self-renewal potential of LSC is significantly higher than normal HSC (Bonnet and Dick, 1997; Cashman et al., 1997), and the clonal tracking studies of AML stem cells provided conclusive evidence for LSC self-renewal (Hope et al., 2004). Determining the genetic mechanisms that regulate the self-renewal capacity of stem cell populations is critical to the understanding of how normal HSC and LSC function to maintain normal hematopoiesis and the leukemic clone, respectively, and may aid in the development of novel therapies. The molecular regulators are coming into view with the linking of Bmi-1 to the self-renewal machinery (Dick, 2003b). As discussed in more detail in this issue (see Lessard, Faubert and Sauvageau), Bmi-1 expression is confined to the very immature populations of BM cells in both human and mouse (Lessard et al., 1998; Park et al., 2002), as well as in the CD34 þ fraction of AML (Lessard and Sauvageau, 2003). Additionally, Bmi-1 controls the expression of Hox genes that when overexpressed in HSC lead to their expansion and eventual transformation (Sauvageau et al., 1995; Thorsteinsdottir et al., 1997; Kroon et al., 1998). In the experimental model of murine AML generated by co-expression of Hoxa9 and Meis1 in fetal liver (FL) cells, the leukemia generated in wild-type and Bmi-1/ mice was markedly different. Although AML was generated in both recipients, it was only possible to serially passage the AML in wild-type mice, indicating that self-renewal of murine LSC in this model requires Bmi-1 (Lessard and Sauvageau, 2003). Similar experiments involving serial transplantation of BM and FL cells from Bmi-1 knockout mice confirmed that this molecule is also a determinant of self-renewal in normal murine HSC (Park et al., 2003). As we argue below, the fact that selfrenewal is regulated by the same gene in both LSC and Oncogene

HSC provides strong molecular support for the origin of LSC from within the HSC compartment. It will now be imperative to examine whether Bmi-1 plays a similar role in normal and leukemic human HSC. Survival Evasion of apoptosis allows preleukemic cells to persist and acquire further mutations. Genetic events that alter differentiation, such as those discussed earlier, can frequently also confer a survival benefit. For example, PML-RARa protects human progenitor cells from apoptosis upon growth factor withdrawal (Grignani et al., 2000). Similarly, CBFb-SMMHC can reduce apoptosis in response to DNA damaging agents by suppressing p53 induction (Britos-Bray et al., 1998). However, a block in differentiation is not always associated with increased survival. In fact, a high level of apoptosis is the key biological feature that distinguishes myelodysplastic syndromes (MDS) from AML (Albitar et al., 2002). Progression from MDS to AML is associated with a relative increase in expression of antiapoptotic versus proapoptotic Bcl-2 family members (Davis and Greenberg, 1998), highlighting the role that survival genes play in leukemogenic progression. A role for Bcl-2 in leukemogenesis was confirmed by the generation of PML-RARa/BCL-2 doubly transgenic mice (Kogan et al., 2001). These mice develop leukemia at an accelerated rate and with increased frequency compared to mice transgenic for PML-RARa alone, demonstrating that Bcl-2 can cooperate with PML-RARa to initiate leukemia. However, a latent period of up to 7 months was observed in the mice, suggesting that additional genetic alterations are still required to fully transform these cells and generate leukemia. Another protein implicated in survival pathways in leukemia is the IL-3 receptor (IL-3R) (Testa et al., 2004). More than 80% of AML samples express IL-3R, and 45% have elevated expression of the IL-3Ra chain compared to normal CD34 þ cells (Budel et al., 1989; Testa et al., 2002). As mentioned above, SL-IC have been found to express IL-3Ra, a characteristic not shared by normal HSC (Jordan et al., 2000). Thus, upregulation of IL-3Ra may be a common survival mechanism in AML. Stimulation of IL-3R initiates numerous signaling pathways with effects including the activation of the Bcl-2 family member Mcl-1, as well as the antiapoptotic transcription factor NFkB (Wang et al., 2003; Guthridge et al., 2004). NFkB is constitutively activated in the majority of primary AML samples, and notably in quiescent LSC populations, but not in normal HSC. Downregulation of NFkB in vitro leads to apoptosis of leukemic but not normal CD34 þ CD38– cells, suggesting that NFkB is a key survival factor for LSC (Guzman et al., 2001). Activation of survival pathways may also account for resistance to treatment in some cases of AML. For example, Bcl-2 expression in AML is associated with a poor response to chemotherapeutic agents (Campos et al., 1993). Therefore, treatment modalities that target antiapoptotic pathways may be useful to include in


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future therapeutic regimens. Antisense Bcl-2 has already been shown to be effective in sensitizing AML blasts to chemotherapy (Konopleva et al., 2000). It may also be possible to exploit the differential expression of IL-3Ra by normal HSC and LSC to selectively target and kill LSC. Telomere maintenance Cells in culture have a finite replicative lifespan due to the erosion of telomeric DNA with successive rounds of cell division (Hayflick, 1997). Unlike most normal human somatic cells, hematopoietic stem and progenitor cells exhibit some telomerase activity (Broccoli et al., 1995; Engelhardt et al., 1997). Nevertheless, telomeres in normal hematopoietic cells shorten with age (Rufer et al., 1999) and following transplantation (Rufer et al., 2001), suggesting that the telomerase activity in progenitor cells is not sufficient to completely prevent telomere shortening with proliferation. Given the greatly increased proliferative capacity of LSC, one of the essential elements of leukemogenesis is the acquisition of a mechanism to maintain telomere function and thereby provide limitless replicative potential (Hanahan and Weinberg, 2000). Increased levels of telomerase activity have been observed in more than 70% of AML cases (Ohyashiki et al., 2002). Despite this, telomeres in AML blasts are generally shorter than those in normal cells (Ohyashiki et al., 2002), reflecting continued telomere attrition during expansion of the leukemic clone. In normal hematopoiesis, telomerase expression is associated with entry into the cell cycle and active proliferation (Engelhardt et al., 1997; Yui et al., 1998). Thus, the increased telomerase activity observed in AML may simply reflect the higher proportion of cells that are actively cycling, and not upregulation of enzyme expression. Critically short telomeres, and not average telomere length, trigger genomic instability and lead to decreased cell viability (Hemann et al., 2001). Therefore, telomerase in LSC may be acting to preferentially maintain the shortest telomeres (Liu et al., 2002). Alternatively, telomerase upregulation may occur as a late event in leukemogenesis after telomeres have shortened due to leukemic proliferation. In addition, genetic changes involving alterations in the expression of other telomere-associated proteins may also be involved. In summary, while the genetic events that occur during leukemogenesis can induce changes that fall into the broad areas discussed above, frequently one event may have multiple effects on the leukemogenic program. Moreover, the temporal order in which these alterations need to occur is unclear. As mentioned, translocations such as PML-RARa can affect apoptosis as well as differentiation (Grignani et al., 1993, 2000; Testa et al., 1998). In addition, IL-3Ra upregulation may lead to increased proliferation as well as survival of AML blast cells (Testa et al., 2002), and there is evidence that the apoptosis related proteins Bcl-2, Bcl-XL, and p53 play a role in hematopoietic differentiation (Haughn et al., 2003; Matas et al., 2004). Studies are complicated by the fact that genes can have different effects in different cell

systems. For instance, whereas PML-RARa leads to survival in myeloid precursor cells, it can induce apoptosis in some hematopoietic cell lines (Grignani et al., 1993, 2000; Ferrucci et al., 1997). Also, a mutant form of the transcription factor C/EBPa identified in AML has been shown to block granulopoiesis in human CD34 þ cells, but promote granulopoiesis in lineagedepleted mouse cells (Schwieger et al., 2004). Future studies involving combinations of genes in the correct cellular environments, ideally primary human cells, are required in order to fully elucidate the essential components involved in leukemogenesis. The cell of origin in AML As discussed above, AML is maintained by a small population of LSC with extensive proliferative capacity and self-renewal potential. This raises a question that has been an ongoing focus of investigation: which target cell in the normal hematopoietic hierarchy acquires the necessary transforming mutations to become an LSC from which the leukemic clone originates? Two disparate models regarding the nature of the target cell have been proposed to account for the observed phenotypic heterogeneity between AML patients. One model postulates that many cell types in the hematopoietic hierarchy, including committed progenitors, are susceptible to transformation (Griffin and Lo¨wenberg, 1986; Fialkow et al., 1987a). Acquired leukemogenic mutations alter the normal developmental program, resulting in the expansion of abnormal cells that are blocked at a particular stage of differentiation reflective of the target cell. This model predicts that the degree of differentiation of the target cell will influence the phenotype of the resultant leukemic blasts; thus, the heterogeneity observed among AML samples is due to the fact that different target cells are transformed in different patients. The second model proposes that the genetic events that lead to leukemic transformation occur in primitive cells only (McCulloch, 1983; Lapidot et al., 1994; Bonnet and Dick, 1997). In this case, the phenotype of the leukemic blasts is influenced mainly by the nature of the specific transforming events and the subsequent effects on the developmental program of the target cell. Thus, the observed heterogeneity is due to the genetic changes that are acquired rather than to the degree of lineage commitment of the target cell. Importantly, this model predicts that there will be relatively little variability in the phenotype of LSC among different patients. These models represent the extreme case. An intermediate model would be that the initial event occurs in the stem cell to create a ‘preleukemic’ stem cell that is still able to differentiate down multiple lineages, and that additional oncogenic alterations occur in downstream progenitors to create the LSC (Reya et al., 2001). Descriptive studies Initial studies to identify the target cell for leukemogenesis in AML used two general approaches to assess stem Oncogene


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cell involvement. The first approach taken in early studies employed various methods to determine whether cells from different lineages were part of the leukemic clone; involvement of multiple lineages would suggest that the leukemia originated from a primitive cell capable of multilineage differentiation. This idea had the underlying assumption that normal development as well as leukemic growth could arise from the same cell, either because the leukemogenic event(s) was only active in the myeloid lineage or that the initial leukemogenic event still permitted normal development, but that subsequent events occurred only in the affected myeloid lineage. The first demonstration of erythroid lineage involvement was provided by investigators using radioactive-iron labeling to show that labeled erythroid precursors carried the leukemic karyotype in three cases of AML (Blackstock and Garson, 1974). Subsequently, Fialkow and co-workers studied X chromosome inactivation patterns (XCIP) to determine lineage involvement in female AML patients who were heterozygous at the G6PD locus (Fialkow et al., 1981, 1987a; Ferraris et al., 1985). The results of their studies were conflicting, with some patients showing the same skewed (nonrandom) XCIP in their leukemic blasts as well as in erythroid cells, platelets, and in one case B lymphoid cells, while other patients seemed to have involvement of only the granulocytic/monocytic lineages. Similar findings were reported in an additional study assessing karyotypic abnormalities in bone marrow cells from AML patients stained with lineage antibodies (Keina¨nen et al., 1988). Together, these data suggest that AML is a heterogeneous disease, originating in some cases from a primitive multipotential cell, and in other cases from a committed progenitor restricted to granulocytic/monocytic differentiation. There are several limitations to the use of XCIP to study lineage involvement in AML (see Gale and Linch, 1998 for a review). First, due to the occurrence of constitutive skewing and tissue specificity of XCIP, it is difficult to obtain appropriate controls. Second, acquired skewing of XCIP occurs with increasing age, particularly in myeloid cells (Busque et al., 1996; Gale et al., 1997), rendering interpretation of skewed XCIP in the elderly difficult. Third, clonality studies are not suited to detect rare populations due to the low sensitivity of the techniques used; minor clonal populations on a polyclonal background are not detected. Finally, analysis of the clonality of mature populations will not be accurate if differentiation of LSC to certain lineages is suppressed because of the nature of specific leukemogenic mutations (McCulloch, 1983). This latter possibility is supported by the finding that in two AML patients, BFU-E grown in vitro had skewed XCIP and appeared to be part of the leukemic clone, but in both patients circulating red cells were nonclonal, suggesting that a multipotential cell was transformed but that differentiation to the erythroid lineage was suppressed in vivo (Fialkow et al., 1987a). Thus, lineage restriction could be more apparent than real. The second approach used to identify the cell of origin in AML involved a combination of immunophenotyping Oncogene

and genotype analysis to detect cells carrying characteristic cytogenetic abnormalities in phenotypically defined primitive and mature cell populations. Two groups defined the CD34 þ CD33 fraction of AML cells as more immature than the CD34 þ CD33 þ fraction based on in vitro assays, and used G6PD isozyme studies (Bernstein et al., 1992) or in situ hybridization (ISH) (Raymakers et al., 1995) to show that in most of the patients studied, CFC derived from the more primitive fraction were nonclonal. This would suggest that the leukemia originated from a more mature progenitor, or alternatively that transformation of a primitive cell resulted in clonal expansion only upon progression to a more committed stage of differentiation. However, two other studies using cytogenetics and fluorescence in situ hybridization (FISH) came to a different conclusion: they found that in all AML samples tested, cytogenetically abnormal cells were present in the primitive CD34 þ CD38 fraction (Haase et al., 1995; Mehrotra et al., 1995). Furthermore, in five patients both lymphoid and erythroid cells carried the cytogenetic markers (Mehrotra et al., 1995). These data suggest that in fact leukemic transformation occurs at the level of immature multipotential CD34 þ CD38 cells. These findings are supported by two reports of patients with t(8;21) AML expressing the AML1-ETO fusion gene. Miyamoto et al. (1996, 2000) used RT–PCR analysis of remission bone marrow from these patients to show that AML1-ETO transcripts were present in primitive CD34 þ Thy-1 þ CD38/lo cells, lymphoid cells, and in multiple lineages of clonogenic progenitors, suggesting that the t(8;21) translocation occurs at the level of stem cells capable of multilineage differentiation. Many CFC derived from these primitive cells differentiated normally, while the CFC from the CD34 þ CD38 þ cells were mainly leukemic, suggesting that AML1-ETO expression resulted in a pre-LSC, and that additional events occurred in the more committed progenitor pool (Miyamoto et al., 2000). However, the additional events could also have occurred in the primitive fraction resulting in the expansion of progenitors. The LSC activity of these fractions has not yet been reported. Functional studies All of these studies, however, were limited by the absence of a direct link between a cell carrying a cytogenetic abnormality and a test of whether that cell has the ability to initiate and maintain the disease in vivo (that is, whether the cell is an LSC). Two recent papers have sought to address this issue by studying the leukemias induced by transduction of MLL fusion genes into defined murine myeloid progenitors and HSC (Cozzio et al., 2003; So et al., 2003). Many MLLassociated leukemias in humans, especially in children, are characterized by coexpression of myeloid and lymphoid antigens, resulting in a mixed lineage phenotype. Two mechanisms have been proposed to explain the aberrant expression of lymphoid markers in some cases of AML. Mixed lineage leukemias may result from ‘lineage infidelity’, whereby leukemic transformation


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leads to aberrant expression of lineage-associated genes (McCulloch, 1983); according to this model, biphenotypic leukemias could potentially arise from committed progenitors. Alternatively, leukemic transformation of primitive cells that have not yet undergone lineage restriction, and that transiently express markers of multiple lineages (‘lineage promiscuity’), may lead to maturation arrest and the propagation of a mixed lineage phenotype in leukemic blasts (Greaves et al., 1986). This latter idea is supported by the recent proposition of Enver and Greaves (1998) that in HSC the chromatin of many lineage affiliated genes is open resulting in low level expression (i.e. globin, myeloperoxidase). Commitment and differentiation result in the progressive silencing of all genes except those affiliated with one lineage. This ‘stem cell preview’ model is consistent with the lineage promiscuity model: primitive stem cells could become transformed resulting in the abnormal retention of the stem cell state where lineage affiliated genes are still expressed. So et al. (2003) found that transduction of MLLGAS7 into murine HSC or multipotent progenitors (MPP) resulted in the production of biphenotypic progenitors and induction of mixed lineage leukemias in transplanted mice, while transduction of lineagerestricted common myeloid progenitors (CMP) or common lymphoid progenitors (CLP) did not, suggesting that multipotential but not committed progenitors are targets for induction of mixed lineage leukemias by MLL. However, using a different fusion partner of MLL, ENL, the same group showed that transduction of murine HSC, CMP, and granulocyte/monocyte progenitors (GMP) led to transformation in vitro and initiation of AML in vivo with arrest at an identical stage of myelomonocytic differentiation regardless of the target cell population used (Cozzio et al., 2003). These results imply that myeloid leukemias induced by an MLL oncogene can be initiated in committed progenitors. However, titration studies revealed that the more primitive the target cell population, the fewer transduced cells were required to induce leukemia in mice, and that even transplantation with large doses resulted in a limited number of clones arising. This suggests that MLL-ENL transformed CMP and GMP are not equivalent to transformed HSC, and that significant functional heterogeneity may exist within the pools of transformed cells. Several further observations can be made regarding these important experiments. First, despite targeting of MLL oncogenes to MPP, specific fusion partners seem to heavily influence the phenotype of the induced leukemias. For example, MLL-ENL appears much less effective than MLL-GAS7 in inducing outgrowth of biphenotypic progenitors and instead favors myeloid transformation in vivo. Thus, the phenotype of the blast cells is determined primarily by the specific transforming events. This is consistent with a model in which primitive cells are the targets for leukemic transformation in AML. Second, MLL-ENL did not induce any detectable expansion in the HSC population, but selectively induced a large expansion of downstream

myelomonocytic leukemic progeny (Cozzio et al., 2003). Thus, while the oncogene may be present in the stem cell it only disrupts the developmental program in the downstream progeny. This may provide a mechanism to explain previous observations in humans that clonal progenitors can be found in the more mature CD34 þ CD33 þ fraction but not in the immature CD34 þ CD33– fraction in some cases of AML (Bernstein et al., 1992; Raymakers et al., 1995). MLL-ENL also appears able to rapidly induce self-renewal in restricted, short-lived progenitors, and thus may be able in some cases to initiate leukemia through transformation of more restricted progenitors (see below). Finally, although a full discussion is beyond the scope of this review, it should be noted that while murine studies provide a very powerful model, transformation of murine progenitors might not accurately model leukemogenesis in humans in vivo due to some fundamental differences in tumor biology between mice and humans. For instance, mouse cells become immortalized at a much higher frequency than do human cells (Kraemer et al., 1986; Shay and Wright, 1989), and repression of telomerase activity likely functions as a tumor suppressor mechanism in humans but not in short-lived species such as mice (Wright and Shay, 2000). The NOD/SCID xenotransplantation assay provides a model system with which to directly test the ability of human AML cells to initiate leukemia in vivo and overcomes the inherent limitations of in vitro assays. Several lines of evidence suggest that AML arises from the primitive HSC pool. First, several groups found that the cells capable of initiating leukemia in mice can be prospectively identified and purified, and have a primitive immunophenotype (CD34 þ CD38 or CD34 þ CD71–HLA-DR–) with similarities to normal HSC regardless of the subtype of AML studied or the immunophenotype of the majority of the leukemic blasts (Lapidot et al., 1994; Bonnet and Dick, 1997; Blair et al., 1998). One limitation of these studies is the potential for bias where only those AML samples that have stem cell involvement can actually grow. Moreover, a much larger number of samples need to be tested to more accurately reflect the diversity of AML. Second, the fact that the LSC compartment is comprised of different classes of LSC organized as a stem cell hierarchy similar to the normal HSC compartment provides additional strong support. A regulated self-renewal process lies at the heart of both stem cell hierarchies suggesting they are related. The finding that murine normal HSC and LSC both require Bmi-1 supports this contention. Acute promyelocytic leukemia: an exception to the rule? One possible exception to the primitive cell model may be APL (for a review, see, Grimwade and Enver, 2004). APL is characterized by chromosomal translocations involving 17q21, leading to rearrangements of the gene encoding RARa. The vast majority of APL cases are associated with the PML-RARa fusion gene resulting from t(15;17) (Kakizuka et al., 1991). In support of the view that APL may originate from committed Oncogene


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progenitors rather than stem cells, a frequently cited study reported that PML-RARa transcripts could be detected in CD34 þ CD38 þ but not CD34 þ CD38– cells by RT– PCR, suggesting that primitive hematopoietic cells are not involved in the leukemic process in APL (Turhan et al., 1995). However, a more recent study using FISH demonstrated that the PML-RARa fusion gene was present in the majority of CD34 þ CD38 cells (Edwards et al., 1999), suggesting that transcription of PML-RARa is suppressed in CD34 þ CD38– cells and providing evidence that at least in some cases APL arises in primitive cells. This is further supported by recent data that the T-lineage-affiliated CD2 locus lies within an open chromatin domain in primary APL blasts regardless of the surface expression of CD2 (Grimwade et al., 2002), suggesting that the cell of origin is a primitive multipotential cell (see lineage promiscuity, above). A second argument used to support the hypothesis that APL arises from committed progenitors is the lack of engraftment of leukemic cells from three APL patients in NOD/SCID mice (Bonnet and Dick, 1997). However, a subsequent report showed engraftment of four out of five APL samples studied, although the level of engraftment was lower compared to other subtypes of AML (Ailles et al., 1999). Overall, the accumulated evidence would seem to support a model in which primitive HSC are the targets for leukemic transformation in human AML, and the phenotype of the resulting leukemia is influenced by the nature of the specific transforming events. Two key properties of stem cells, self-renewal and longevity, likely play an important role in the stepwise and progressive conversion of normal HSC to LSC. Selfrenewal is a central feature of both normal HSC and LSC; thus leukemogenesis would presumably involve accumulation of a greater number of mutations in more committed progenitors lacking intrinsic self-renewal capacity, as compared to stem cells in which this machinery is already active. Second, it is reasonable to assume that it would be easier to accumulate the number of genetic changes required for leukemogenesis in HSC that persist throughout life and are longer-lived, than in more mature cells with limited lifespan. For example, unless acquisition of self-renewal capacity is a relatively early event in the leukemic transformation of a restricted progenitor, that cell will be lost before full transformation can occur. This model does not exclude the possibility that the early, initiating mutations may occur in HSC and the final transforming events in downstream progenitors, or that the acquired genetic changes in HSC may exert their effects in downstream cells leading to expansion of the progenitor pool without expansion of the target cell pool.

Emerging concepts and future directions Identification of the hierarchical structure of AML has important implications for future research as well as for the development of novel therapies. In order to learn Oncogene

more about the nature of the events involved in the initiation and progression of leukemia, research must focus on the rare subset of cells that is able to initiate and maintain the leukemia, and not on the blast population that makes up the majority of the leukemic clone. The phenotypic description of LSC now enables their purification and will facilitate identification of genes that are preferentially expressed in these primitive cells compared to normal HSC, for example through gene expression profiling (Guzman et al., 2001). A better understanding of the biology of LSC will also aid in the design of novel therapies targeted to their unique properties. The failure of current therapeutic regimens to achieve permanent cures and prevent relapses is likely related to the resistance and persistence of LSC. Deeper insight into the genetic changes that occur in LSC will lead to improvements in drug efficacy and specificity, for example through targeting of molecules, such as NFkB, that provide survival signals to leukemic but not normal stem cells (Guzman et al., 2001; see also Jordan and Guzman, in this issue). Recent studies in solid tumors indicate that the concept of cancer as a hierarchy that is initiated and maintained by a rare population of stem cells may have broader implications beyond the field of hematopoiesis. Al-Hajj et al. (2003) were able to prospectively isolate a minor phenotypically distinct subset of breast cancer cells (Lin–CD44 þ CD24–/low) that was able to form mammary tumors in NOD/SCID mice, while cells with alternate phenotypes were nontumorigenic. The tumorigenic cells could be serially passaged and were able to generate tumor heterogeneity, demonstrating the ability to self-renew and to produce differentiated, nontumorigenic progeny. Thus, like AML, breast cancer appears to be organized as a hierarchy that is driven by a rare population of cells that retain remnants of normal developmental programs. Two recent reports have also suggested the existence of brain CSC, although conclusive proof must await demonstration that the cells in question are able to generate new tumors in vivo that exhibit evidence of differentiation and self-renewal (Hemmati et al., 2003; Singh et al., 2003). The increasing evidence that rare stem cells drive formation of a number of different tumor types raises the possibility that CSC may well be at the apex of all neoplastic systems and that the processes that subvert normal cells into CSC may be central to the pathogenesis of cancer in general. For example, as discussed above, Bmi-1 is important in the regulation of selfrenewal in both normal and leukemic HSC (Lessard and Sauvageau, 2003; Park et al., 2003). A recent study has shown that Bmi-1 is also required for the self-renewal of stem cells in the peripheral and central nervous systems (Molofsky et al., 2003). Together, these data highlight the important role that Bmi-1 plays in the regulation of self-renewal in diverse types of stem cells. A greater understanding of the biology of CSC will be gained by further dissecting this and other conserved pathways that may become deregulated in the process of tumorigenesis.


7175 Acknowledgements This work was supported by the National Cancer Institute of Canada (NCIC) with funds from the Canadian Cancer Society, Canadian Institutes for Health

Research, Stem Cell Network of the National Centres of Excellence, Canadian Genetic Diseases Network of the National Centres of Excellence, and a Canada Research Chair.

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