Stem cell maintenance and disease progression in chronic myeloid leukemia
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Chronic myeloid leukemia (CML) is a cancer of blood cells driven by the BCR–ABL1 oncogenic protein tyrosine kinase, which is the product of a reciprocal chromosomal translocation known as the Philadelphia chromosome. Discovery of tyrosine kinase inhibitors targeting the BCR–ABL1 kinase revolutionized CML therapy, but these drugs are unable to eradicate the disease due to the presence of a drug-insensitive stem cell population that sustains continued growth of the malignant cells. Resistance to therapies also increases the risk of relapse and disease progression to a more advanced phase. This review discusses emerging issues in CML research, and describes recent progress in elucidating the mechanisms of CML stem cell maintenance and disease progression.
KeywordsChronic myeloid leukemia Stem cell Cancer progression Blast crisis
As we make progress toward effective therapies against cancer, several important issues remain, including therapy resistance, disease relapse following therapy cessation, and disease progression to advanced stages. Decades of studies on acute myeloid leukemia (AML) have provided sufficient evidence to conclude that leukemia is organized as a hierarchy of several distinct leukemic cell types with different degrees of self-renewal and differentiation potential . There is a functionally, and often phenotypically, distinct population of tumor-initiating cells that exhibit the capacity to self-renew, the potential to generate all of the cell types present in the tumor, and the ability to proliferate actively and thereby drive sustained expansion of malignant cells [2, 3]. The tumor-initiating cells, or cancer stem cells, represent a therapy-resistant cell population and are primary drivers of cancer propagation and disease relapse after treatment and/or chemotherapy cessation [4, 5]. Recent studies have also supported the idea that several types of solid tumors, if not all, are a heterogeneous mixture of multiple cell types and are sustained by a sub-population with higher tumorigenic potential [6, 7]. It is thus reasonable and appealing to speculate that targeting a population that can maintain cancer growth may provide clues to effective treatment strategies and, ultimately, a cure to many hematologic malignancies and cancers. Indeed, in the mouse model of MOZ–TIF2-induced AML, forced cell death of the leukemia stem cell population leads to eradication of the disease in vivo . In this Perspective, I will discuss emerging issues and the roles of novel players in stem cell maintenance and disease progression in cancer with a specific focus on chronic myeloid leukemia.
Chronic myeloid leukemia
Patients who show initial resistance to TKI and become resistant to primary TKI therapy are likely to relapse and progress to a more aggressive disease phase called blast crisis (Fig. 1) . Compared with indolent CP-CML in which mature myeloid cells are produced, blast crisis CML (BC-CML) is characterized by the rapid proliferation of differentiation-arrested immature blast cells of either myeloid or lymphoid lineage [13, 14]. Interestingly, differentiation arrest and/or abnormal capacity of differentiation are shared features of hematologic malignancies and solid tumors with poor prognosis. Although the molecular mechanisms driving the transformation of CP to BC-CML are not fully understood, additional genetic mutations, defects in DNA damage repair and telomere maintenance are implicated in this process [13, 14, 15, 16, 17]. Unfortunately, due to its resistance to current therapies, BC-CML remains a major challenge in the treatment of CML.
Recently, it has become clear that while differentiated CML cells are dependent on BCR–ABL1 activity for their survival and therefore remain sensitive to kinase inhibition, CML stem cells do not rely on its activity and persist despite the effective inhibition of the BCR–ABL1 by TKIs [18, 19, 20]. These studies clearly highlight the need for targeting the BCR–ABL1-independent, alternative mechanisms that protect and sustain CML stem cells to develop curative therapies for this hematologic malignancy.
Mouse models of CML and insights for cancer stem cell research
Another model of CML takes advantage of genetically engineered mice expressing the BCR–ABL1 transgene. Several distinct transgenic strains are available [29, 30, 31, 32], and among those, a tetracycline(Tet)-regulated binary transgenic model is the most promising (Fig. 2b). This model carries the BCR–ABL1 transgene under the control of a Tet-responsive element as well as a second transgene encoding a Tet-regulated transactivator protein (tTA) driven by the SCL gene enhancer. The combination of these two transgenes results in Tet-regulated BCR–ABL1 expression in HSCs. BCR–ABL1 gene expression can be induced by the removal of doxycycline, a tetracycline analog, leading to a CML-like disease characterized by neutrophilia and splenomegaly. Using the retroviral/transplantation model, CML stem cells have been identified in the population with a Lin− Sca-1+ c-Kit+ (LSK) phenotype . In a recent study using the binary BCR–ABL1-inducible model, Passegue and colleagues found that CML stem cells share the same cell surface phenotype as long-term HSCs (LSK Flk2− CD48− CD150+) . Although the transplantation of BCR–ABL1-expressing cells with a short-term HSC phenotype (LSK Flk2− CD48− CD150−) can cause transient myeloid hyperplasia, these cells are unable to induce CML-like disease in vivo. In contrast, as few as 50 BCR–ABL1+ long-term HSCs lead to robust and long-lasting engraftment, and most of the engrafted mice eventually develop CML with an average latency of 4 months, demonstrating that CML stem cells reside exclusively in the long-term HSC compartment.
The most physiologically relevant mouse model currently available is a xenograft model of human primary CML cells (Fig. 2c). Flow-sorted primary CD34+ leukemia cells from patient bone marrow specimens are transplanted into immune-compromised recipient mice, such as NOD–SCID mice [35, 36]. Although the model remains technically challenging due to low efficiencies of long-term engraftment and/or biologic variations observed in primary leukemia specimens, development of the novel immunodeficient mouse strain NSG (NOD–SCID IL-2Rγ-chain-deficient) has generated an improved model with robust engraftment, providing a better way to analyze human CML stem cells in vivo and test the effectiveness of novel therapies against human CML .
Emerging players mediating stem cell maintenance and disease progression in CML
In the past decade, multiple signaling pathways have been identified as critical regulators of CML pathogenesis by the use of the aforementioned mouse models of CML. These include classic developmental pathways such as Wnt/beta-catenin and Hedgehog [38, 39, 40], as well as the multifunctional signaling scaffold protein β-arrestin , the promyelocytic leukemia protein (PML), transcriptional regulators of FoxO and HIF1α [42, 43, 44] and the arachidonate 5-lipoxygenase Alox5  (well reviewed in [46, 47, 48]). Discussed below are the emerging players that sustain stem cells and drive cancer progression in CML.
Oncogenic RNA binding proteins
Pioneering work on hnRNP E2 clearly showed that its gene expression is highly upregulated in human BC-CML compared with CP-CML, and that the hnRNP E2 protein binds to C/EBPα mRNA to repress its translation . The transcription factor C/EBPα is essential for granulocyte differentiation, and functional inactivation of C/EBPα results in a block to myeloid differentiation. Thus, suppression of C/EBPα mRNA translation by upregulated hnRNP E2 protein is one of the mechanisms of differentiation arrest leading to the expansion of immature myeloid cells in BC-CML. A recent study has shown that miR-328, one of the microRNAs (miRNAs) downregulated during CML progression, competes with C/EBPα mRNA for binding to hnRNP E2 . Forced expression of miR-328 increases C/EBPα protein levels in BC-CML cells and functionally impairs the clonogenic potential of human BC-CML cells, indicating that miR-328 functions as a decoy RNA to modulate hnRNP E2 activity.
RBPs also mediate regulation of miRNA biogenesis. The RNA binding protein Lin28 binds to let-7 miRNA precursors and inhibits their processing and maturation. While the let-7 family of miRNAs is abundant in differentiated cells, its expression is repressed in undifferentiated pluripotent ES cells and human cancers . Consistent with its role in let-7 miRNA maturation, Lin28 is overexpressed in a broad range of human cancers . Lin28 expression is more frequent in BC-CML specimens than in CP-CML, and the inhibition of Lin28 expression by RNA interference in K562 cells restores the levels of mature let-7 miRNA and impairs the proliferative and clonogenic capacities with concomitant induction of cellular differentiation, suggesting that targeting the Lin28-let-7 axis might delay or block the blast crisis transformation.
The Musashi gene (Msi) encodes an RBP originally identified as a developmental regulator of neural cell fate in the fruit fly. Later studies showed that there are two paralogous Msi genes in mammals (Msi1 and Msi2), and that the Msi1 gene expression marks tissue stem cells in brain, eye, and intestine . Msi1 binds to and inhibits the translation of the mRNA for Numb, which specifies a differentiated cell fate in neural progenitors and HSCs [54, 55]. The functional importance of Msi1 in neural stem cells, however, remains elusive. Msi1 inactivation alone results in barely appreciable defects during embryonic neural development, which may be due to functional redundancies among the two paralogs . The second gene, Msi2, is predominantly expressed in the most immature HSC compartment within the hematopoietic organ, and during CML disease progression, Msi2 expression levels are significantly elevated in the blast crisis phase . Importantly, NUP98–HOXA9 can trigger Msi2 expression, which in turn represses Numb expression. Genetic loss of Msi2 results in increased Numb levels and, as a consequence, functionally impairs the development and propagation of BC-CML in vivo. Interestingly, Msi2 is not only highly upregulated during human CML progression, but is also a marker for poorer prognosis in human BC-CML and de novo AMLs [57, 58]. Collectively, these studies define the essential role of the RNA binding factor Msi2 as a molecular driver of cancer progression by regulating the balance between stem cell self-renewal and differentiation.
SIRT1 histone deacetylase
Histone deacetylases (HDACs) are among the promising candidates for targeted therapies in CML . A recent study identified a non-canonical class III HDAC protein, SIRT1 as an exciting new target for eradicating CML stem cells . SIRT1, the founding member of the Sirtuin family of proteins, is a mammalian homolog of the yeast protein Silent Information Regulator 2 (Sir2), and exerts a powerful influence on a variety of cellular processes, including DNA repair, cell cycle, metabolism and aging in diverse organisms . Unlike canonical HDACs, SIRT1 requires the coenzyme nicotinamide adenine dinucleotide to deacetylate its targets, and it is not inhibited by pan-HDAC inhibitors . In the CD34+ population from human CP- and BC-CML cells, SIRT1 is expressed at higher levels than in the CD34+ population in normal bone marrow . SIRT1 downregulation by RNA interference or pharmacological inhibition of its activity by tenovin-6, a selective small molecule inhibitor of SIRT1, induces cell death and impairs the proliferation of the quiescent, TKI-insensitive CML stem cell population in culture. SIRT1 inhibition ex vivo reduces the growth of CML cells and compromises the long-term engraftment of human CD34+ CML cells upon transplantation into immunodeficient NSG mice. Spurred by the successful inhibition of CML stem cell growth in vitro, Li et al. tested whether tenovin-6 could impair the CML development in vivo by using the Tet-regulated BCR–ABL1 transgenic model. Although imatinib treatment alone reduced tumor burden, it failed to impact the CML stem cells. In contrast, combined treatment of imatinib and tenovin-6 effectively inhibited the maintenance of the BCR–ABL1+ long-term HSC population and significantly prolonged overall survival after discontinuation of the drug treatments. These results suggest that targeting SIRT1 could provide a novel therapeutic approach for the eradication of the CML stem cells.
Recent basic and translational research has contributed to our understanding of the biology of CML stem cells and has uncovered several potential candidates for targeted therapies in the myeloid leukemia. An accumulating body of evidence supports the idea that the properties of CML stem cells are shared by a wide array of cancer stem cells in other hematologic malignancies and solid tumors. Thus, the knowledge gained thus far from studies of CML is likely to have broader implications and extend to other cancers.
This work was supported in part by fellowships to T.I. from the Interdisciplinary Stem Cell Training Program by the California Institute of Regenerative Medicine, and from the Astellas Foundation for Research on Metabolic Disorders. I would like to thank Dr. Tannishtha Reya and the members of the Reya Laboratory for fruitful discussions, thoughtful insights and critical reading of this manuscript.
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