This study was initiated based on the assumption that quiescent CML cells and early progenitor cells may differ in their pattern of gene expression from comparable normal cells and that if critical differences could be identified, they would help to reveal the underlying molecular mechanisms responsible for the excessive overproduction of the CML population. It was further hoped that some vulnerable differences would be discovered that would be susceptible to targeting by highly selective drugs, since it was assumed that the quiescent cells would be largely unaffected by existing BCR-ABL inhibitors such as Imatinib and Dasatinib as well as other drugs active against proliferating cells. So far, there has been only one other study attempting this [58
], which was performed using the same methods but using fewer samples (five CML and two normal) and an older microarray platform with considerably less genes represented (14,500 versus 38,500).
Because at least 105 cells were required for each sample for microarray analysis and the enriched CD34+ G0 cells comprised less than 0.02% of the starting cell populations, it was difficult to obtain sufficient cells, especially patient samples, to carry out the study. We initially collected 10 CML samples, but 2 had insufficient RNA so only 8 are included. We also performed microarray analyses on CD34+ G0 and G1/S/G2/M cells enriched from cord blood and normal G-CSF mobilized peripheral blood samples, but excluded them from the final analysis because some of the results differed from cells enriched from normal bone marrow samples and we considered the latter to be more appropriate normal controls. Both the normal and CML quiescent and proliferating fractions consisted of 96–100% CD34+ primitive blast cells and less than 1% of CD34+ G0 cells incorporated BrdU, so almost all of the latter were either in G0 or early G1. Because there are no definitive markers to distinguish stem cells from early progenitor cells, it is unknown what percentage of each was present in the CD34+ G0 fractions, but we presume the majority of cells in both the normal and CML fractions were progenitors in various stages of development. Ideally, we would have preferred to also compare normal and CML stem cells, but were unable to do this because of a lack of clear stem cell markers and insufficient cell numbers.
Some of the genes that were differentially expressed in normal and CML/G0 cells revealed a number of interesting findings which correlate nicely with some of the biological and functional abnormalities previously observed in CML cells. These findings include the following. (i) In keeping with the gene expression data, normal and CML quiescent G0 cells are more highly enriched in primitive cells than the proliferating G1
/M cells. (ii) The CML G0 cells are in a slightly more advanced stage of development than the normal G0 cells, and as previously reported by Graham et al. [58
], the CML CD34+ G0 cells are more similar to the CD34+ proliferating cells than are the normal G0 and G1
/M fractions. (iii) In keeping with their more advanced stage of development and their upregulation of genes involved in DNA replication or part of the mitotic spindle machinery, CML/G0 cells are more poised to begin proliferating than normal G0 cells and are more sensitive to growth stimulation by single cytokines or combinations known to act on early progenitors and stem cells. While normal and CML/G0 cells are almost equally responsive to stimulation by multiple cytokines, the CML cells are triggered into cycle more rapidly. (iv) Once CML quiescent progenitors are stimulated by cytokines to begin proliferating, they undergo further differentiation and maturation more rapidly than normal quiescent progenitors, but both granulopoiesis and erythropoiesis are usually less efficient than in normal hematopoiesis as shown in cloning experiments in which the CML cells form many more small CFU-GM, CFU-E, and BFU-E compared to normal progenitors [14
]. (v) Whereas normal CD34+ cells form almost entirely granulocyte/monocyte clusters and colonies in clonogenic experiments when stimulated by cytokines in the absence of erythropoietin, CML CD34+ G0 cells consistently spontaneously form a combination of GM and erythroid colonies in the absence of EPO. The gene expression data clearly shows that CML/G0 cells have marked overexpression of genes associated with development of the erythrocyte and megakaryocyte lineages, and Graham et al. noted similar findings [58
]. (vi) Prominin-1 (CD133) is the gene most downregulated in CML G0 cells, and there is lower expression of CD133 on the surface of these cells. Cord blood G0 CD 133+ cells form only GM colonies without EPO while CD133− cells form a mixture of GM and erythroid colonies. The downregulation of CD133 appears to be associated with the spontaneous formation of erythroid colonies by CML progenitors in the absence of EPO, but its precise role remains to be better clarified. It has been known for many years that both normal and neoplastic cell populations contain significant numbers of “resting” or quiescent cells that are considerably less sensitive to the damaging effects of irradiation, cytotoxic drugs, and other injurious agents than are proliferating cells [59
]. The dormant state is a protective mechanism that is of crucial importance in enhancing a population's probability of survival under adverse conditions, and early on the concept that dormant cancer cells are important obstacles to curability was widely recognized by both basic and clinical scientists [15
]. If one accepts the premises that almost all lethal cancers originate in adult stem cells or early progenitors functioning as stem cells, that these cells are essential for initiation, maintenance, and expansion of the cancers, and that a large fraction of these cells, like normal stem cells, reside in a quiescent state in which they are resistant to most therapies, then it is obviously important to better understand their derivation and properties, to determine how normal and cancer quiescent stem cells may differ, and to look for ways to develop specific targeted therapies based on these differences.
Activation of quiescent cells following a mitogenic stimulus by serum, cytokines, or other factors is highly complex and involves the coordinated and selective induction of expression and repression of hundreds of genes including specific cyclins, cyclin-dependent kinases (CDKs), and protein kinase inhibitors (PKIs). Many cell cycle genes are transcriptionally silent in quiescent cells, and they express only a limited number of cytokine receptors, and recent studies have shown that siRNAs and microRNAs are also involved in repressing gene transcription and translation in quiescent cells. When one considers the staggering complexity of all of the cytokines and other regulatory factors and cellular interactions that determine whether a quiescent stem cell residing in its protected niche is going to divide while simultaneously deciding what kinds of cells to produce, it is hardly surprising that our present understanding of the mechanisms regulating stem cell behavior is very incomplete. In our analysis, it appears that in many cases, clusters of presumably related genes that are differentially expressed are all associated with a particular stage of development or function, suggesting that their common dysfunction in CML G0 cells may involve aberrant co-regulation.
Most authorities agree that the HSC population is heterogeneous, but it is still uncertain at what level of development the system permits changes to occur in phenotype and functionality, or when the differentiation hierarchy becomes fixed. Circumstantial evidence for both normal and leukemic stem cells favors a heterogenic model in which there is a continuum of stem and early progenitor cells with gradually declining potentials for self-replication, pluripotency, and other stem cell properties, but that some cells may also exhibit flexibility in responding to different stochastic influences in their developmental milieus. Some degree of reversibility may also exist whereby early progenitors can retain or reassume more primitive stem cell properties if needed, such as the ability for more extensive self-replication in order to replace or supplement damaged stem cells. However, it is likely that significant reversibility is restricted to early progenitors and that once they have become committed to differentiate along a particular lineage, it is doubtful that they can revert to functioning as stem cells. With regard to cancer stem cells, some years ago we postulated that most leukemias originate in “limited stem cells” which have more limited pluripotency than normal primitive HSCs, but retain sufficient self-replicating potential to initiate a lethal leukemia [11
]. Many current researchers now agree, although some prefer to call them leukemia or cancer “initiating cells” to distinguish them from true HSCs.
While cells undergoing differentiation and maturation can become temporarily arrested or slowed in their progression through other phases of the cell cycle under certain conditions (e.g., hypoxia, increased cell density, exposure to toxins, cytotoxic drugs, irradiation, or other damaging agents), it is usually only stem cells and primitive progenitors that remain in a quiescent state for extended periods under normal steady-state conditions. Once progenitors become firmly committed to differentiation and maturation, serial cloning studies conducted in vitro [32
] and cytokinetic labeling studies with 3
H-thymidine conducted in vivo [24
] have shown that both normal and leukemic cells usually proceed to undergo a variable but limited number of maturation divisions to produce terminally differentiated cells (which may be highly abnormal in leukemia and other malignancies), and which are incapable of reverting to regain significant self-renewal or other essential stem cell properties. While our in vivo 3
H-thymidine labeling studies were less extensive in patients with lymphomas or solid tumors growing in ascetic form [63
], in cases in which it was possible to distinguish neoplastic cells in differing states of maturity, it appears that once the neoplasticcells become committed to maturation, if the environment is suitable, they usually continue to divide but are only capable of a limited number of divisions before dying spontaneously and are incapable of reproducing the disease. Overall, our in vivo labeling studies strongly suggest that the number of dormant cancer stem and progenitor cells continue to increase as the population of cancer cells expands and that they, thus, constitute a progressively greater obstacle to curative therapy in many types of cancer [11
The constitutive tyrosine kinase activity of the p210bcr-abl
protein causes abnormal phosphorylation of regulatory proteins in numerous interacting signaling pathways [3
]. The overall signaling networks altered by Bcr-Abl are highly complex [68
], indeed reaching a level of complexity that some observers have likened to Heisenberg's uncertainty principle in quantum mechanics. Nevertheless, although the specific signaling changes responsible for each of the biological abnormalities that have been described are still incompletely defined, it is highly likely that the faulty signaling disrupts multiple interactive networks that normally tightly regulate the orderly well-coordinated processes of proliferation, differentiation, and maturation in normal hematopoiesis. This misregulation can probably explain all the abnormalities observed in early-stage CML including the initial overproduction of GM progenitors, the imbalanced lineage apportionment, the inefficiency of production of both granulocytes and erythrocytes, and all the other more subtle dysplastic morphological, biochemical, and functional changes that have been described [25
Gleevec and some of the newer Bcr-Abl inhibitors are highly effective in globally inhibiting the increased tyrosine phosphorylation of multiple proteins involved in these signaling pathways [25
] and since Bcr-Abl is usually the sole mutation in early stage CML, the progenitors are restored to near normal behavior when the kinase is adequately inhibited. At higher concentrations, the Abl TKI inhibitors are lethal to both fresh primary CML progenitors and Bcr-Abl-driven cell lines while at still higher concentrations, they also kill normal progenitors and cell lines not driven by Bcr-Abl, the exact normal: CML lethal concentration ratios depending on the particular cells and inhibitors.
However, as suggested by the usual relatively slow induction of remissions in CML patients over the course of weeks or months, it is doubtful if the BCR-ABL inhibitors when administered in clinically tolerable doses actually kill many of the proliferating CML progenitors and precursors. Rather, by inhibiting Bcr-Abl's constitutive tyrosine kinase activity, they at least transiently restore the cells to functioning more normally, and in so doing the CML progenitors cease excessive cell production, presumably by reacquiring the ability to respond properly to quorum sensing signals that ensure maintenance of normal homeostatic cell density limits. Meanwhile, the later CML progenitors which are already committed to differentiation continue to proceed through a limited number of maturation divisions and then die as terminally differentiated cells, just as do normal cells. After the body burden of leukemic cells has been sufficiently reduced, the residual normal stem cells are released from the CML cells' (poorly understood) inhibitory effects and resume production, usually resulting in a complete hematologic or cytogenetic remission. The BCR-ABL inhibitors are clearly a very important advance since they are able to induce durable remissions in the majority of CML patients in chronic phase, but they are not usually curative since most patients relapse if therapy is discontinued, probably because quiescent CML stem/progenitor cells are not killed by the drugs and are able to reproduce the disease [9
]. Several mechanisms of resistance have also been well described as noted earlier [7
Thus, while Gleevec and other Bcr-Abl TKIs are very effective in the early stage of CML in largely eliminating the majority of proliferating Ph+ progenitor and precursor cells, more attention should be given to seeking ways to selectively kill the quiescent leukemia stem and progenitor cells. While our own studies so far have not revealed any specific vulnerable targets, it is important that the search be continued. The alterations in gene expression described here must be confirmed in a larger number of patients, and if possible with further technological advances, in still more highly enriched populations of early progenitors and stem cells. As the search proceeds, the significance of some of the differences in gene expression reported here may become clearer and eventually lead to discovery of new ways to selectively kill the quiescent CML stem and progenitor cells. For a number of reasons, it has become increasingly difficult to obtain large enough samples of CML cells to carry out the rigorous procedures required to isolate sufficient numbers of highly enriched stem and progenitor cells for further studies, so this is another important issue that must be addressed.
In a broader sense, it is perhaps even more important to design similar therapeutic strategies for other types of cancer. Much of the recent development of anticancer drugs has been directed towards producing different classes of drugs that block segments of one or more signaling pathways that are known to be dysregulated by the particular initiating mutation(s) commonly found in different types of cancer. However in advanced malignancies it is often difficult to distinguish the importance of the primary causative mutation(s) compared to that of secondary or still later (passenger) mutations and the situation may become further complicated by multiple epigenetic changes. A huge number of new drugs of different classes are now available, but few cause complete or durable remissions and they are almost never curative. Greater emphasis should therefore be placed on more clearly identifying and whenever possible selectively targeting the primary driving mutations in the cancer stem or early progenitor cells in early stage disease, and also in developing new strategies to selectively kill the quiescent stem or progenitor cells that escape most current therapies.