Found an interesting article on stem cell explaination, just wana to share :
The CML stem cell: Evolution of the progenitor
The success of imatinib mesylate (STI571, Gleevec) in treating chronic myeloid leukemia (CML) is, to date, the crowning achievement of targeted molecular therapy in cancer. Nearly 90% of newly diagnosed patients treated with imatinib in the chronic phase of the disease achieve a complete cytogenetic response. However, more than 95% of these patients retain detectable levels of BCR-ABL mRNA and patients discontinuing imatinib therapy almost invariably relapse, demonstrating that an imatinib insensitive population of leukemia-initiating cells (LICs) persists in nearly all patients. These findings underscore the need for treatments specifically targeting the leukemia-initiating population of CML cells. While mounting evidence suggests that the LIC in the chronic phase of CML is the BCR-ABL positive hematopoietic stem cell, several recent publications suggest that during CML blast crisis, a granulocyte-macrophage progenitor (GMP) population also acquires LIC properties through activation of the ?-catenin pathway. Characterization of these cells and evaluation of their sensitivity to imatinib is critical to our understanding and treatment of CML blast crisis.
Keywords: chronic myeloid leukemia, BCR-ABL, p210, GMP, cancer stem cell, leukemia-initiating cell
The Cancer Stem Cell Hypothesis
It has been more than a decade since John Dick and colleagues first demonstrated that the leukemic potential of most human acute myelogenous leukemias (AML) resides exclusively within the CD34+CD38? population of cells when transplanted into immunocompromised NOD/SCID mice.1 This was the first identification of a putative human cancer stem cell (CSC) and in the years that have followed potential CSCs have been identified in a number of other hematopoietic and solid tumors including tumors of the breast,2 brain,3 colon,4,5 head and neck,6 pancreas,7 lung,8 skin9 and liver10 (reviewed in refs. 11-13). Collectively, these studies have lent support to the cancer stem cell hypothesis, which suggests that in some tumors only a specific subset of tumor cells is capable of driving tumorigenesis.
To demonstrate that a certain tumor follows the cancer stem cell hypothesis, tumors cells are usually sorted into distinct populations based on the expression of cell surface proteins, transplanted into syngeneic or immunocompromised (for human tumors) mice, and assayed for their ability to induce tumorigenesis. For tumors that follow the cancer stem cell hypothesis, it should be possible to sort a distinct population of cells, the cancer stem cell population, that is capable of inducing tumorigenesis in recipient mice and reconstituting the heterogeneity of the original tumor. Furthermore, other purified populations of tumor cells, or a heterogeneous population of tumor cells depleted of the CSCs, should lack the ability to form tumors (Fig. 1).14 Many of the putative cancer stem cells listed above were identified using this experimental strategy.
However, xenotransplantation of human cancer cells into mice presents a number of problems that complicate the identification of cancer stem cells. These include residual immunity in recipient mice and cross-species differences in both cytokine availability and the tissue microenvironment, any of which may lead to the selection of a population of cells that is unique not in its ability to initiate tumors, but rather in its ability to engraft in the recipient mouse. Providing evidence that this is sometimes the case are recent studies of human AML and human melanoma, two cancers previously reported to follow the cancer stem cell hypothesis. While earlier studies had identified rare populations of CSCs for both of these cancers,1,9 more recent studies15,16 demonstrate that transplantation of either type of cancer cells into NOD/SCID mice, which retain a low level of innate immunity,17 dramatically underestimates the frequency of tumor-initiating cells. In the case of human AML, the frequency of tumor-initiating cells in improved mouse models still remains quite low,15 suggesting subsets of AML are still likely to follow the cancer stem cell model. In contrast, transplantation of human melanoma cells under optimized conditions into severely immunocompromised mice suggests that nearly all melanoma cells are capable of initiating tumors in mice,16 indicating that melanoma probably does not follow the cancer stem cell model.
The lesson from studies such as these is that the cells we consider to be cancer stem cells, and even the cancers we consider to be stem cell diseases, will likely change as xenotransplantation models or in vitro assays improve. One alternative to xenotransplantation is to use mouse models of human cancers in which case syngeneic transplants can be used to identify cancer stem cell populations. For cancers where putative CSCs have previously been identified, such experiments will permit a phenotypic comparison of the cancer stem cells identified in mouse models with those identified by xenotransplantation. Recently, this has been done for chronic myelogenous leukemia (CML), where both xenotransplantation studies and studies in a mouse model suggest that during blast crisis, cells of the granulocyte-macrophage progenitor (GMP) population can function as leukemic stem cells. These studies provide additional evidence that CML follows the cancer stem cell model, and in addition provide unique insights into the dynamics of the cancer stem cell population, suggesting that this population is capable of evolving during cancer progression.
Chronic Myelogenous Leukemia
Chronic myelogenous leukemia is a myeloproliferative disorder of clonal origin with an annual incidence of about 1 in 50,000 per year.18 CML patients usually present in the chronic phase of the disease during which there is a gradual expansion of mature myeloid cells in the bone marrow and peripheral blood (Fig. 2). Without treatment, patients inevitably progress (on average 4-6 years after diagnosis) through an accelerated phase of disease to a terminal acute phase known as blast crisis, characterized by a massive increase in undifferentiated blasts that can be either myeloid or lymphoid in nature (Fig. 2). Major insight into the origin of CML came from the work of Nowell and Hungerford, who in 1960 described a common chromosomal abnormality in CML patient samples and suggested a "causal relationship between the chromosome abnormality observed and chronic granulocytic leukemia".19 This unique chromosome, known as the Philadelphia chromosome (Ph), was later shown to be a reciprocal translocation between the long arms of chromosomes 9 and 22 [t(9:22)(q34;q11)]20 and leads to the fusion of the breakpoint cluster (BCR) and human ABL1 genes. The resulting BCR-ABL mRNA produces a constitutively active form of the Abl kinase and this kinase activity is required for BCR-ABL induced transformation, proving Nowell and Hungerford right, and providing an invaluable molecular marker for the leukemic cells of CML patients.18,21,22
Evolution of Leukemic Stem Cells in CML Disease Progression. In normal hematopoiesis, the hematopoietic stem cell (HSCs) gives rise to all other lineages of hematopoietic cells. In CML chronic phase (CP) patients, expression of BCR-ABL in the HSC compartment leads to an expansion of the myeloid lineage resulting in an abnormal number of mature granulocytes. The HSC likely functions as the leukemia-initiating cell during CML-CP. Transition to blast crisis (BC) involves additional genetic and epigenetic alterations leading to the accumulation of immature blasts. Progression to blast crisis also results in the acquisition of self-renewal potential by a GMP population with elevated ?-catenin activity. In a mouse model, as few as fifty cells from the BCR-ABL-transformed GMP compartment can initiate a CML-like disease. GMP from CML-BC patients also initiate leukemia when transplanted into immunocompromised mice. Arrows mark critical events in disease progression and self-renewal potential. Dotted circles indicate BCR-ABL-positive cells. (HSC, hematopoietic stem cell; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; Gr, granulocyte; Mac, macrophage).
Lessons from Imatinib Treatment of CML
The identification of BCR-ABL as the molecular entity responsible for CML led to a search for small molecule inhibitors of the Abl kinase and ultimately resulted in the discovery of imatinib mesylate as a drug that was extremely effective at inhibiting the proliferation of BCR-ABL expressing cells both in vitro and in vivo.23 Clinical trials of imatinib soon followed,24-26 and a follow-up study of newly diagnosed CML patients reported that 93% of imatinib-treated patients remained free of disease progression after 5 years.27 However, despite the incredible success of the drug, imatinib fails to completely eradicate BCR-ABL positive cells from patients,28,29 and patients who discontinue treatment (even those with a complete molecular response), almost invariably relapse.30-34 The inevitable conclusion is that while imatinib is incredibly successful at eliminating the bulk tumor, it is unable to eliminate at least some fraction of leukemic stem cells.
Furthermore, while imatinib is able to sustain a prolonged remission in patients diagnosed and treated in chronic phase, it is much less effective in controlling disease progression in patients treated during accelerated phase or blast crisis.35-37 Even patients who do respond in the advanced stages of the disease tend to quickly relapse,36,37 often due to point mutations within the BCR-ABL protein that render the kinase insensitive to imatinib.38-40 The cellular events that lead to both the increased aggressiveness of blast crisis tumors and the presence and/or rapid emergence of imatinib resistant clones are not well understood at present. However, it seems probable that during the prolonged chronic phase, genetic mutations accumulate that ultimately drive the selection of a more malignant and genetically diverse tumor population.41
Collectively, the clinical data from imatinib-treated patients highlight two important issues. Most importantly, CML is a stem cell disease and any curative therapy must eradicate the CML stem cell. Secondly, there is need to identify additional molecular and cellular hallmarks of CML blast crisis in order to improve treatment of advanced stage patients. Interestingly several recent studies suggest that one important difference between chronic phase and blast crisis CML is the acquisition of a new population of leukemic stem cells in blast crisis.42
Leukemic Stem Cells in Chronic Phase CML
Studies by Fialkow et al. first demonstrated in 1967 that CML was a clonal disease originating from a pluripotent hematopoietic cell.43 Further studies by he and his colleagues demonstrated that this target cell was, at least in some cases, the totipotent hematopoietic stem cell (HSC).44,45 The fact that HSCs could be the target of the BCR-ABL translocation, along with the fact that these cells are capable of self renewal, led to speculation that the Ph+ HSC was the CML stem cell. Since it was known that at least some fraction of CML stem cells are resistant to imatinib, as indicated by patient relapse following discontinuation of imatinib,30-34 the speculation that the CML stem cell was the HSC led to an investigation of the imatinib sensitivity of Ph+ HSCs. These studies demonstrated that HSCs from CML patients are in fact less sensitive to imatinib, strongly suggesting these cells are the leukemic stem cell in chronic phase.29,46 More conclusive evidence that the HSC functions as the leukemic stem cell in most patients was provided by a recent study using QPCR analysis of sorted cell populations from imatinib-treated patients. This study showed that, in chronic phase patients with major molecular responses, residual BCR-ABL transcripts were detectable almost exclusively in the HSC compartment.28 In addition to these studies, recent mathematical models based on clinical data also support the fact that the Ph+ HSC is the CML stem cell.47,48
Leukemic Stem Cells in Blast Crisis CML
While it's possible that a single CSC could drive tumorigenesis throughout the life of a tumor, it's also possible that increasing genetic instability within the tumor population could lead to the selection of additional CSCs populations. This latter possibility seems to be the case during the advanced stages of CML (Fig. 2), where genetic instability has been well documented.41
It has been shown that granulocyte macrophage progenitors (GMPs) from patients with advanced CML (accelerated phase or blast crisis) have the ability to form colonies in repeated passages in vitro demonstrating that they can acquire the ability to self-renew.49 Furthermore, the levels of active ?-catenin (which is known to be important for stem cell self-renewal12) in these GMP showed a direct correlation with disease progression and enforced expression of an inhibitor of the ?-catenin pathway significantly decreased GMP colony formation. Thus, GMPs likely acquire the ability to self-renew through improper activation of the ?-catenin pathway. While this initial work was all conducted in vitro, the same group has now shown that leukemic GMPs from blast crisis patients are capable of serially transplanting leukemia into immunocompromised mice with an efficiency even greater than that of leukemic HSCs,42 showing that cells within the GMP population can function as leukemic stem cells in vivo.
While results from xenotransplantation studies must be interpreted cautiously (as discussed above), a similar finding has recently been reported in a syngeneic mouse model of CML blast crisis.50 In these studies, infection of a pluripotent hematopoietic progenitor line with BCR-ABL (p210) resulted in the expansion of the GMP population in vitro (Fig. 3A). Transplantation of 5 × 103 of these GMP into congenic mice generated a transplantable blast crisis-like leukemia in 4/4 mice while only 1/7 mice transplanted with 5 × 103 non-GMP developed disease (the incidence of leukemia in the mouse transplanted with non-GMP may reflect the presence of leukemic HSCs in the non-GMP fraction) (Fig. 3B). Limiting dilution experiments showed that as few as 50 leukemic GMPs were able to generate disease, suggesting that the GMP population is highly enriched for leukemic stem cells. In addition, both the in vitro cultured GMP and the GMP isolated from leukemic mice displayed elevated levels of ?-catenin activity (Fig. 3), suggesting that these GMP may have acquired self-renewal capabilities in a manner similar to that of GMP from CML blast crisis patients.
BCR-ABL-transformed GMP as myeloid leukemic stem cells. (A) E2A?/? murine hematopoietic progenitor cells (mHPC) were infected with BCR-ABLp210-IRES-GFP or GFP retrovirus. Expression of p210 leads to the expansion of the GMP population and this population of cells contain elevated ?-catenin activity. ( Transplantation of p210/mHPC, but not GFP/mHPC, into congenic mice results in a CML-like disease. Sorting p210/mHPC into GMP and non-GMP prior to transplantation reveals that the leukemia-initiating cells reside primarily in the GMP population. The leukemia resulting from the non-GMP population may be a result of leukemic HSCs in the non-GMP fraction. Bold arrows indicate cell sorting strategies.
Previous studies have suggested that ?-catenin might play an important role in the progression of CML to myeloid blast crisis. It was recently reported that while wild-type mice transduced with p210-infected mouse bone marrow readily develop an acute myeloid disease resembling CML blast crisis,51 mice lacking ?-catenin in the hematopoietic compartment rarely develop myeloid disease, instead generally succumbing to acute lymphoid leukemias.52 Thus, ?-catenin appears to facilitate the progression of advanced myeloproliferative disease. Furthermore, a recent study that used microarray analysis to characterize the changes associated with CML progression found that the ?-catenin pathway was one of the pathways most deregulated in blast crisis patients.53 It is tempting to speculate that the involvement of ?-catenin in the progression of CML is due to its role in conferring self-renewal properties to the GMP population during the advanced stages of the disease.
CML, GMP and the CSC Hypothesis
The available data strongly suggest that there are some cancers that follow the cancer stem cell hypothesis and some that do not. In addition, there may be cancers in which the types of cells capable of initiating tumorigenesis change as the disease progresses. Especially for cancers that initially follow the cancer stem cell hypothesis, where only a fraction of the cells are tumor-initiating, it seems reasonable to expect that the selection pressure for cells with a growth or survival advantage may give rise to additional populations of tumor-initiating cells. Even limiting the discussion to leukemia, one can find tumors that fall into each of the above groups.
As discussed earlier, most acute myeloid leukemias (AML) appear to adhere to the cancer stem cell hypothesis.1,15 In contrast, it has been shown that for certain types of high-risk childhood acute lymphoblastic leukemia (ALL) all sorted populations of cells are able to induce tumorigenesis in immunocompromised mice.54 Importantly, the tumors that arise from these sorted populations all recapitulate the heterogeneity of the original tumor. This demonstrates the remarkable plasticity of these tumor cells and convincingly shows that these tumors do not follow the cancer stem cell hypothesis. The studies described in this review, on the other hand, demonstrate that CML is a malignancy in which the tumor-initiating population of cells evolves as the disease progresses. In chronic phase, the tumor-initiating cell is the Ph+ HSC, which functions as a cancer stem cell. As the disease progresses and additional genetic mutations occur, the GMP population acquires self-renewal potential and the ability to function as a second CML stem cell (Fig. 3). The specific genetic alterations that endow GMP with the ability to self-renew remain largely unknown, although a recent study suggests that, at least in some patients, self-renewal may result from missplicing of GSK3-? and a resultant increase in ?-catenin activity.42
While progression of CML leads to the evolution of a second cancer stem cell, it also seems possible that a cancer initially following the cancer stem cell hypothesis would progress to the point where cells of the bulk tumor acquire self-renewal. Thus, the more advanced disease may no longer follow the cancer stem cell hypothesis. In this regard, studies of Ph+ ALL are quite interesting. Studies using xenotransplantation of human Ph+ ALL cells have identified a rare population of committed progenitors that function as CSCs.55,56 However, a recent mouse model of Ph+ ALL generated in an Arf?/? background demonstrated that nearly all cells in this model are able to initiate tumorigenesis in syngeneic transplants.57 This may indicate that events such as ARF loss can confer tumor-initiating properties to the bulk tumor. As loss of the ARF locus is frequently observed in Ph+ ALL patients,58 it would be interesting to determine if tumors from such patients contain a higher percentage of LICs than those from patients with an intact ARF locus.
Studies such as these on Ph+ ALL, as well as the aforementioned studies on CML, underscore the need to consider the stage of the disease when characterizing cancer stem cells as the tumor-initiating population of cells may evolve as the disease progresses.
CML Stem Cells and CML Therapy
Most conventional cancer therapies target the bulk tumor. The most important implication of the cancer stem cell model is that treatment of the bulk tumor may be insufficient to cure disease if a cancer stem cell population is spared. Nowhere has this been manifest more clearly than in the case of CML, where treatment with imatinib is incredibly effective in eliminating the bulk tumor, but fails to eradicate all leukemic stem cells. The failure of imatinib to eradicate these cells is likely due to an inability to eliminate the quiescent fraction of Ph+ HSCs.28,29,46,59 Thus, therapeutics that target this population are of significant interest.
The observation that GMPs can function as cancer stem cells in blast crisis, where imatinib is largely ineffective, raises the intriguing question of whether these cells might also be intrinsically insensitive to imatinib or whether they might acquire resistance during their evolution to cancer stem cells. In this regard, QPCR analysis of chronic phase patients with major molecular responses to imatinib rarely detect BCR-ABL transcripts in the GMP population, suggesting Ph+ GMP are not intrinsically resistant to imatinib.28 However, we have observed that GMPs in a murine model of CML are capable of proliferating in the presence of imatinib, suggesting that blast crisis GMP may be resistant to the drug (Minami et al. manuscript in preparation). Thus, additional studies will be needed to determine if leukemic GMP contribute to the imatinib-insensitivity of blast crisis CML.
While the differences between CSCs and cells of the bulk tumor may prevent CSCs from being eliminated by therapies that target the bulk tumor, these differences may also provide unique therapeutic targets. Therefore, the identification of cancer stem cells may open the door to new targeted therapies as the differences between the cancer stem cell, the bulk tumor, and normal cells are realized. The observation that the leukemic GMPs in CML blast crisis largely depend on the ?-catenin pathway for self-renewal point to this pathway as one attractive therapeutic target. Future studies with purified populations of HSCs and GMPs from patients with CML will be essential to identifying additional differences amenable to therapeutic intervention.