Selective elimination of leukemia stem cells: Hitting a moving target

Cancer Letters xxx (2012) xxx–xxx

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Mini-review

Selective elimination of leukemia stem cells: Hitting a moving target Leslie A. Crews, Catriona H.M. Jamieson ⇑ Department of Medicine, Stem Cell Program and Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA

a r t i c l e

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Article history: Available online xxxx Keywords: Leukemia Stem cells CML Hematologic malignancies Microenvironment Therapeutics

a b s t r a c t Despite the widespread use of chemotherapeutic cytotoxic agents that eradicate proliferating cell populations, patients suffering from a wide variety of malignancies continue to relapse as a consequence of resistance to standard therapies. In hematologic malignancies, leukemia stem cells (LSCs) represent a malignant reservoir of disease that is believed to drive relapse and resistance to chemotherapy and tyrosine kinase inhibitor (TKIs). Major research efforts in recent years have been aimed at identifying and characterizing the LSC population in leukemias, such as chronic myeloid leukemia (CML), which represents an important paradigm for understanding the molecular evolution of cancer. However, the precise molecular mechanisms that promote LSC-mediated therapeutic recalcitrance have remained elusive. It has become clear that the LSC population evolves during disease progression, thus presenting a serious challenge for development of effective therapeutic strategies. Multiple reports have demonstrated that LSC initiation and propagation occurs as a result of aberrant activation of pro-survival and self-renewal pathways regulated by stem-cell related signaling molecules including b-catenin and Sonic Hedgehog (Shh). Enhanced survival in LSC protective microenvironments, such as the bone marrow niche, as well as acquired dormancy of cells in these niches, also contributes to LSC persistence. Key components of these cell-intrinsic and cell-extrinsic pathways provide novel potential targets for therapies aimed at eradicating this dynamic and therapeutically recalcitrant LSC population. Furthermore, combination strategies that exploit LSC have the potential to dramatically improve the quality and quantity of life for patients that are resistant to current therapies. Ó 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction A prevailing stochastic theory of cancer evolution posits that tumors are composed of rapidly dividing cancer cells that fail to differentiate normally. However, despite the widespread use of cytotoxic agents that eliminate proliferating cell populations, in both normal and malignant tissues, patients suffering from a wide variety of malignancies continue to relapse and develop resistance to standard therapies – the leading causes of cancer related death (http://seer.cancer.gov/statfacts/html/cmyl.html). This serious dilemma has been a critical driving force fueling major research efforts in the last two decades that led to the cancer stem cell (CSC) hypothesis based on early studies in which a small number of self-renewing tumor cells were required to regenerate all aspects of the tumor compared with the stochastic model, which suggested that all cells within the cancer could propagate the malignancy [1,2]. The CSC model posits that CSC possess features characteristic of normal stem cells albeit in a deregulated manner. These deregulated stem cell properties such as enhanced self-re⇑ Corresponding author. Address: Department of Medicine, Moores Cancer Center, University of California, San Diego 3855, Health Sciences Drive, La Jolla, CA 92093-0820, USA. Tel.: +1 858 534 7128; fax: +1 858 822 6288. E-mail address: [email protected] (C.H.M. Jamieson).

newal, survival and dormancy, endow CSC with the capacity to regenerate a tumor and give rise to all cell types found in the original tumor [2]. CSC are distinct from the rapidly dividing bulk tumor cells in that they acquire an enhanced capacity to survive and self-renew, and can also lie dormant in protected tumor niches for long periods of time. Thus, CSC represent a malignant reservoir of disease that is believed to drive chemotherapeutic resistance and relapse.

2. Defining the leukemia stem cell hierarchy Early reports demonstrated that bulk tumor cells from human acute myeloid leukemia (AML) harbored very low clonogenic potential and formed colonies in vitro at low frequencies [3]. Comprehensive studies in the early 1990s demonstrated that a subpopulation of leukemia cells from AML possessed the capacity to engraft immunocompromised mice and could recapitulate the phenotypic heterogeneity of the original leukemia, thus providing the first evidence of a leukemia-stem cell (LSC). This subpopulation was defined as CD34+CD38 and shared similar cell surface marker expression patterns with normal hematopoietic progenitors, albeit lacking CD90 – a critical hematopoietic stem cell (HSC) marker [4– 6]. These observations supported the contention that in hemato-

0304-3835/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2012.08.006

Please cite this article in press as: L.A. Crews, C.H.M. Jamieson, Selective elimination of leukemia stem cells: Hitting a moving target, Cancer Lett. (2012), http://dx.doi.org/10.1016/j.canlet.2012.08.006

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logic malignancies there exists a LSC hierarchy analogous to normal hematopoiesis that is initiated and maintained by a subset of cancer cells harboring stem cell-like properties [4]. However, the precise molecular mechanisms driving the generation and evolution of these malignant populations have remained elusive and are currently a focus of intense international research efforts. While evidence for the concept of CSC was first demonstrated in AML, some controversy remains as to the existence of and the precise cellular markers that identify CSC in solid tumors. Major research efforts in recent years have focused on identifying and characterizing the CSC population in hematologic malignancies and for solid tumors, and understanding the cell type and context specific events that result in CSC generation. Putative CSC populations have been identified in a number of malignancies, including breast, prostate and pancreatic cancer. A key challenge in elucidating the markers and molecular mechanisms that lead to the generation of CSC has been the relative inability to sequentially sample solid tumors to accurately identify the epigenetic and genetic events that drive malignant acquisition of stem cell properties by tumor cells. Thus hematologic malignancies have provided a unique opportunity to study CSC biology and the molecular evolution of LSC.

3. The molecular evolution of LSC To better understand the molecular evolution of LSC, it is instructive to examine Charles Darwin’s work described in ‘‘On the Origin of Species’’ [7]. He established evolutionary guiding principles over the course of 25 years including variation under nature, variation of species subject to domestication versus in the wild, the struggle for existence and natural selection of species able to adapt to their environment. Rapid cell division alternating with bouts of dormancy during the development of cancer fuels variation and adaptation at a cellular level and CSC arise over time as a result of environmental pressures and become oncogenically addicted to self-renewal and survival signaling pathways on a cellular and molecular scale (Fig. 1). Although cell surface phenotype may vary depending on the type of hematologic malignancy, the cell population responsible for leukemic progression is defined by the functional capacity to self-renew and survive in supportive microenvironments.

While the AML LSC population was initially identified phenotypically as CD34+CD38 , more recent studies have reevaluated this criteria based on observations suggesting that CD34+CD38 or CD34+CD38+ fraction may contain leukemia initiating potential in myeloid malignancies depending on disease stage and the individual patient [8–10]. Similarly, in chronic myeloid leukemia (CML), the cell population involved in chronic phase initiation following BCR–ABL1 expression shares phenotypic marker expression with CD34+CD38 CD90+Lin HSC however with myeloid skewed differentiation potential leading to myeloid progenitor expansion [11–13] (Fig. 1). During blast crisis transformation, myeloid LSC arise from the expanded granulocyte–macrophage progenitor (GMP) population which harbors amplified BCR–ABL1 expression [13]. These malignant GMP are reprogrammed to self-renew via aberrant activation of self-renewal and survival pathways leading to therapeutic resistance [8,13–15] (Fig. 1). Thus, even within one disease in a single patient, the cell population responsible for disease initiation versus progression and relapse differ and are subjected to distinct molecular pressures such as oncogene addiction and cytotoxic therapies, and thus is likely constantly evolving and adapting to survive in the dynamic tumor microenvironment. Then, a critical question that reflects one of the current fundamental dilemmas in CSC biology and cancer therapeutics is: what strategies would be most effective to develop selective anti-cancer treatments that will overcome a moving target such as the LSC population? Based on recent advances in leukemia clinical trials showing promise of a variety of molecular therapies, the short answer to this essential question is that the key to eradicating an evolving population of cells lies in combination therapies that selectively inhibit multiple targets. Ideally, these targets will be specifically activated in the LSC compartment, and pharmacological inhibition will spare normal adult stem cells during the course of treatment. The overall aim of these novel therapeutic approaches is to provide curative strategies for patients that will free them from the burden of a lifetime of pharmacological treatments aggravated by a high risk of disease relapse. Here we will focus on reviewing several major molecular pathways that have shown promise as useful therapeutic targets in selectively eliminating LSC (Fig. 2). Pharmacological and biologic inhibitors of these pathways would complement current standards of leukemia patient care.

Fig. 1. An ‘‘Origin of Species’’ based model of LSC evolution. The molecular evolution of hematopoietic stem cells (HSC) into LSC initiates with the BCR–ABL translocation occurring at the stem cell level, followed by the expansion of the multipotent progenitor (MPP) population and myeloid progenitors. The acquisition of additional signal transduction abnormalities such as BCL-2 activation or b-catenin or Wnt signaling dependence allows enhanced survival and/or self-renewal capacity of the granulocyte– macrophage progenitor (GMP) population. These cell-intrinsic alterations and extracellular niche-derived cues apply selective pressures that favor the expansion and survival of LSC in supportive microenvironments.

Please cite this article in press as: L.A. Crews, C.H.M. Jamieson, Selective elimination of leukemia stem cells: Hitting a moving target, Cancer Lett. (2012), http://dx.doi.org/10.1016/j.canlet.2012.08.006

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need for development of innovative, targeted therapeutic strategies to eradicate this malignant population. Useful combinatorial therapeutic approaches targeting LSC could involve drugs that selectively inhibit pathways or hubs of signaling activity regulating such stem cell survival and self-renewal activities. Cumulative research efforts suggest that activation of apoptosis pathways (including mitochondrial pro-survival and pro-apoptotic factors), autophagy [28], Wnt/b-catenin and glycogen synthase kinase-3b (GSK3b) signaling, the Hedgehog (Hh) pathway, and/or epigenetic, metabolic and molecular chaperone pathways may be key cell-intrinsic factors that fuel LSC persistence and TKI resistance [29,30], while cell-extrinsic factors that support LSC maintenance may involve intercellular signaling mechanisms that promote cell migration and interactions with protective hematopoietic microenvironments such as the bone marrow niche [26].

5. Targeting LSC through cell-intrinsic pathways Fig. 2. Targeting stem cell pathways in LSC. In addition to TKI therapies that represent current standards of care in CML treatment, both cell-extrinsic (nichederived cues such as inflammatory stimuli) and cell-intrinsic pathways (cell survival/apoptosis, proliferation, differentiation, and self-renewal potential) should be targeted in future therapeutic strategies aimed at eradicating LSC.

4. CML as a paradigm for understanding CSC biology Although relatively rare (1.0–1.3 cases/100,000 people in the United States, www.lls.org), CML represents an important paradigm for understanding the molecular evolution of cancer because it was the first cancer to be associated with a diagnostic molecular mutation, BCR–ABL, and the first target of molecular therapy [16]. Diagnosis of this disorder is based on the detection of the characteristic genetic abnormality wherein a translocation occurs between chromosomes 9 and 22, generating the Philadelphia chromosome which results in production of the BCR–ABL fusion protein tyrosine kinase [17–20]. Targeted therapies for the treatment of CML using tyrosine kinase inhibitors (TKI) that block BCR–ABL activity have advanced in recent years to include not only imatinib but also second-generation TKIs such as dasatinib and nilotinib. Although a striking reduction in mortality has occurred in patients treated early in chronic phase CML with TKI therapy, ultimately a significant fraction of treated patients develop therapeutic resistance attributed to the persistence of malignant progenitors [21,22]. Compelling studies, such as the STIM trial, in which a majority of CML patients relapsed within 12 months of TKI discontinuation, suggest that new therapeutic strategies are required to provide molecular cures for this disease [23]. Recent studies have uncovered critical information regarding the mechanisms driving TKI-resistance at a cellular and microenvironmental level. Evidence indicates that LSC evasion of TKI-induced cell death is BCR–ABL1-independent [24,25], and likely requires aberrant activation of additional cell-intrinsic pathways regulating cell survival, self-renewal, as well as cell-extrinsic pathways that promote or interfere with interactions with supportive hematopoietic microenvironments [26,27]. Elucidating the full array of molecular alterations that drive LSC generation and maintenance will facilitate the development of novel therapeutic strategies and distinguish strong candidates for curative therapies in leukemic disorders. Deregulation of critical cellular processes such as survival and self-renewal can contribute to the persistence of disease-propagating cells that are practically immortal, allowing endless regeneration of the disease. Thus, through acquired activation of pro-survival and self-renewal pathways, LSC drive disease relapse and resistance to standard therapies. This highlights the

5.1. Modulating LSC survival through apoptotic factors Previous studies have reported that cell lines expressing BCR– ABL are resistant to apoptosis [31–33]. However this effect may be dependent on BCR–ABL expression levels [34], which is known to be increased in the blast crisis stage of disease [13]. Additional works have demonstrated that dormant BCR–ABL+, CD34+ CML progenitors are resistant to apoptosis induced by imatinib [35] or dasatinib [36]. This suggests that cellular anti-apoptosis pathways may be aberrantly activated in CML LSC, and it should be possible to target these activated survival pathways to promote programmed cell death of LSC. Intrinsic apoptosis occurs via Mitochondrial Outer Membrane Permeabilization (MOMP), which is a direct result of activation of BAX or BAK. Oligomerization of these proteins results in pore formation in the outer membrane, thus allowing leakage of cytochrome c from the mitochondrion which in turn activates caspases in the cytoplasm. Pro-survival proteins such as members of the B-cell lymphoma/leukemia-2 (Bcl-2) family interface with Bcl-2 associated X protein (BAX) and Bcl-2 homologous antagonist killer (BAK) at the mitochondrial membrane and prevent pore formation. They also sequester pro-apoptosis family members such as BCL2-like 11 (BIM) and BH3 interacting domain death agonist (BID). Up-regulation of BIM is required for TKIs to induce apoptosis in kinase-driven cancers, and a recent study identified a common intronic deletion polymorphism in the gene encoding BIM [37]. Notably, the polymorphism was sufficient to confer intrinsic TKI resistance in CML cell lines, and individuals with CML harboring the polymorphism experienced significantly inferior responses to TKIs than did individuals without the polymorphism [37]. Thus, Bcl-2 family proteins and interacting factors represent novel potential therapeutic targets for antagonizing aberrant survival signals in CML LSC. A number of inhibitors of the Bcl-2 family are in preclinical and clinical development [38] in addition to agents that impact both the intrinsic and extrinsic apoptosis pathways [39]. Recent studies in CML cell lines and primary CML patients samples demonstrated that a pharmacological inhibitor of two Bcl-2 family members (Bcl2 and Bcl-XL), ABT-737, modified Bcl-2 protein interactions toward a pro-apoptotic phenotype and promoted apoptosis in quiescent CD34(+) progenitor cells [40,41]. Its combination with TKI resulted in a synergistic effect on apoptosis in CML cell lines [41]. However, as ABT-737 only inhibits two of the six anti-apoptotic Bcl-2 family members, and does not target Mcl-1 which has been implicated in CML pathogenesis [42], the therapeutic indices between normal HSC and LSC will need to be carefully established in vitro and

Please cite this article in press as: L.A. Crews, C.H.M. Jamieson, Selective elimination of leukemia stem cells: Hitting a moving target, Cancer Lett. (2012), http://dx.doi.org/10.1016/j.canlet.2012.08.006

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in vivo. It will also be important to test the efficacy of broader inhibitors of the Bcl-2 family with activity against Mcl-1 as well as other members of the intrinsic apoptosis pathway in the eradication of CML LSC. Together, therapeutic strategies targeting prosurvival factors such as the Bcl-2 family may represent a key strategy towards preventing therapeutic resistance and may be more broadly applicable to CSC in other malignancies. 5.2. Targeting LSC self-renewal A common theme among candidate pathways that are de-regulated in LSC include canonical developmental pathways, such as the Wnt/b-catenin and Hedgehog signaling pathways, which are evolutionarily conserved and direct essential developmental processes including embryonic patterning by dictating cell fate decisions and cell migration. While these pathways are tightly controlled and generally inactive in adult tissues, they play an important role in normal adult stem cells. Increasingly, it has become clear that these and other stem cell pathways are aberrantly activated in a wide range of human cancers. The Wnt family of secreted proteins is composed of at least 19 isoforms that are activating ligands for the two distinct Wnt signaling pathways. The non-canonical Wnt signaling pathway involves a b-catenin-independent mechanism that influences cell polarity, asymmetric cell division and cell migration during development [43], while the canonical Wnt signaling pathway functions through b-catenin to affect pattern formation during development. The Wnt/b-catenin signaling pathway has been extensively studied in tumorigenesis and cancer progression [44]. Seminal gene microarray studies indicated that deregulation of the Wnt/b-catenin pathway occurs during the progression of CML from chronic to accelerated and finally blast crisis phases [45]. A key characteristic of normal hematopoietic stem cells (HSCs) is the ability to self-renew. During normal development this self-renewal potential is dependent on b-catenin function [46], as genetic deletion of b-catenin during fetal HSC development leads to impairment of self-renewal while b-catenin is dispensable in fully developed adult HSCs. During the progression of CML to the blast crisis stage, activation of b-catenin in CML granulocyte–macrophage progenitors enhances the self-renewal activity and leukemic potential of these cells [13]. This effect may be driven directly by BCR–ABL kinase activity, as enforced BCR–ABL expression drives increased b-catenin levels [47], and BCR–ABL controls b-catenin protein stabilization by affecting its phosphorylation status in CML cells [48]. Furthermore, silencing of b-catenin inhibited proliferation and clonogenicity of BCR–ABL(+) CML cells [48]. This suggests that b-catenin might be a useful therapeutic target for CML LSC harboring enhanced self-renewal potential due to aberrant activation of the canonical Wnt signaling pathway. Interestingly, deregulation of this pathway appears to be a common event in leukemic transformation, as similar activation of b-catenin has been observed in AML [15], suggesting that therapeutic targeting of this pathway could have broader application to multiple hematologic malignancies. Selective Wnt/b-catenin inhibitors have shown promise as novel therapeutic agents for the treatment of CML [49,50]. Thus, inhibiting b-catenin by pharmacologic modulation may be an effective combination therapy with imatinib to specifically target CML stem cells. However, it should be noted that genetic deletion of b-catenin after CML initiation did not lead to a significant increase in survival of mice [50], suggesting that other factors may contribute to therapeutic resistance. Another important signaling molecule that interacts with the canonical Wnt/b-catenin signaling pathway is GSK3b, a serine/ threonine kinase that functions as a negative regulator of b-catenin. Our previous gene targeted Wnt pathway sequencing research revealed that novel splice acceptor sites in GSK3b resulted in gen-

eration of a kinase-deficient GSK3b splice isoform that promoted malignant reprogramming of progenitors into LSC contributing to CML progression [8,51]. GSK3b is a central signaling molecule involved in multiple cellular activities. Interestingly, in addition to its function in canonical Wnt/b-catenin signaling, it also regulates other stem cell pathways such as Hedgehog (Hh) signaling and phosphorylates downstream effectors of this pathway, namely GLI2 [52]. The Hh signaling pathway is another crucial regulator of embryogenesis that has activity in fetal development as well as in adult stem cell populations. These molecules primarily function as a morphogens during development, however Hh signaling is critical for the maintenance of adult hematopoietic [53–55], neural [56], and skin and hair follicle [57] stem cells. Previous studies have established a role for aberrant activation of Hh signaling in a number of solid tumors as well as hematologic malignancies [45,58,59]. In CML Hh signaling is vital for maintenance of LSC through SMO-mediated GLI1 activation but in humans GLI2 increases with blastic transformation [45]. Microarray studies have revealed that overexpression of Hh pathway components correlates with CD34 expression in CML. This suggests that the Hh pathway is stimulated in CML LSC, and is a role for Hh signaling in CML is further supported by evidence that Hh activation appears to increase as CML progresses from chronic phase to blast crisis [45]. In mouse models of CML, the Shh pathway can be activated in CML progenitors by over expression of SMO resulting in CML progression [44]. Levels of downstream mediators of Shh signaling were upregulated in human CML samples [58]. Intriguingly, loss of SMO expression by knockout or pharmacological inhibition significantly reduced the percentage of CML LSC and delayed the development of CML [58,59]. While Hh signaling can become activated at several points in the pathway, most drugs targeting this signal transduction cascade are directed against the SMO transmembrane protein. The first identified inhibitor of the Hh pathway was cyclopamine [60,61], an alkaloid teratogen derived from the corn lily. Exposure to this compound during development causes cyclopenia, phenocopying the embryonic defects observed in Shh mutant embryos [62–64]. Cyclopamine directly binds SMO to efficiently block the Hh signaling pathway and decrease CSC numbers in preclinical models of several tumors. Inhibition with cyclopamine in mouse cells reduced LSC in vivo and increased time to relapse after the end of treatment, an effect that was augmented by BCR–ABL inhibition [58]. Clinical use of cyclopamine has been limited by low oral bioavailability and poor pharmacokinetics [65], but these initial studies have paved the way for development of novel Hh pathway modulators and SMO antagonists with improved pharmacokinetic profiles. The first clinically approved targeted Hh pathway antagonist is vismodegib (GDC-0049), a small molecule inhibitor of SMO [66,67], which was recently approved by the FDA for the treatment of basal cell carcinoma following positive Phase II trial results. Several alternative SMO-targeted small molecules are being clinically evaluated including PF-04449913, BMS-833923 (XL139), LDE-225, LEQ506, and TAK-441 (www.clinicaltrials.gov). Among these, PF04449913 is being tested in a current Phase I trial for patients with hematologic malignancies, and a new Phase I trial with this same SMO antagonist has recently been initiated and will be recruiting patients to evaluate the use of this inhibitor in AML and high-risk Myelodysplastic Syndrome in combination with standard chemotherapeutic agents used to treat these diseases (www. clinicaltrials.gov). The Hh signaling pathway can also be targeted via its upstream ligands or its downstream effectors – the GLI family of transcriptional regulators. It is important to bear in mind that it may be necessary to target multiple points in the Hh signaling pathway and

Please cite this article in press as: L.A. Crews, C.H.M. Jamieson, Selective elimination of leukemia stem cells: Hitting a moving target, Cancer Lett. (2012), http://dx.doi.org/10.1016/j.canlet.2012.08.006

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effectively counter potential development of resistance to SMO antagonists [68]. Future research efforts will undoubtedly shed more light on the precise molecular regulators of Hh signaling that should be targeted in order to best induce selective eradication of LSC while sparing normal hematopoietic progenitors. 5.3. Targeting LSC through modulation of epigenetic, metabolic and molecular chaperone pathways The proliferation, differentiation, cell cycle status and survival of CML LSC can also be regulated by a number of other molecular pathways, such as through the activity of epigenetic, metabolic and molecular chaperone factors. For example, histone deacetylase (HDAC) enzymes, which regulate DNA expression by modifying histone proteins in chromatin, have been identified as therapeutic targets in several cancers [69,70]. Notably, HDAC inhibitors have been shown to induce apoptosis in non-proliferating cancer cell lines [71] – an observation that has important implications for targeting dormant TKI-resistant LSC. Notably, inhibitors of HDAC activity have shown efficacy at inducing apoptosis alone and in combination with TKIs in CML cell lines and in both BC CML and CP CML primary cells [72–76]. In these studies, treatment with HDAC inhibitors combined with imatinib induced apoptosis in quiescent CML progenitors that were resistant to imatinib alone, and eliminated CML stem cells with in vivo engraftment capacity [76]. While pan-HDAC inhibitor treatment represents an effective strategy to target LSCs in CML patients receiving TKIs, there are some concerns about toxicity of this strategy to normal HSC populations. Recent studies have shown that inhibition of the NADdependent histone deacetylase Sirtuin 1 (SIRT1) enhanced sensitivity of CML progenitors to imatinib-induced apoptosis but did not affect survival of normal progenitors [77]. Another potential LSC-specific target in CML is the arachidonate 5-lipoxygenase (5-LO) gene (Alox5), which has been identified as a critical regulator of LSC in BCR–ABL-induced CML [78]. In a mouse model of CML, recipients of BCR–ABL transduced bone marrow cells from Alox5 / donor mice failed to develop CML. Alox5 deficiency did not appear to have a significant effect on the functions of normal hematopoietic stem cells, making 5-LO an attractive target for pharmacological intervention. In this context, a specific 5LO inhibitor effectively impaired the function of LSCs in the same CML mouse model in a similar manner to genetic deficiency [78]. Other combination treatment strategies aimed at coupling TKIs with inhibitors targeting molecular chaperones have shown promise as potential CML therapies. Molecular chaperones such as heat shock proteins (Hsp) play a key role in directing the conformational maturation of oncogenic signaling proteins including BCR– ABL. For example, a small molecule inhibitor of heat shock protein 90 (Hsp90) demonstrated a desirable therapeutic index that reduced the in vivo growth of mutant forms of BCR–ABL-expressing cells in a mouse model of leukemia [79]. Future studies will be vital to fully elucidate whether these promising therapeutic strategies will have similar efficacy in human leukemia cells. 6. Targeting LSC through cell-extrinsic pathways In leukemic disorders, LSC can hibernate in supportive hematopoietic microenvironments such as the bone marrow niche [26,80]. Poor disease outcomes are likely fueled by resurgence of leukemic cells derived from these resistant populations of LSC that reside in supportive niches, harbor enhanced survival and self-renewal capacity, and persist in a quiescent state [81]. It has been shown that a small population of residual leukemic CD34+ progenitor cells can survive in the bone marrow microenvironment of CML patients after years of TKI treatment. LSC residing in the bone marrow end-

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osteal niche are predominantly quiescent [82]. Since non-proliferating CD34+ CML progenitors are resistant to apoptosis induced by imatinib [35], and moreover BCR–ABL(+) stem cells persist in CML patients despite prolonged treatment with imatinib [83], this suggests that the quiescent cell population is uniquely resistant to TKI treatment [84]. Cellular quiescence induced by niche localization may also protect cells from radiation or chemotherapy [84]. Interactions with the stromal microenvironment can alter the expression patterns of survival pathways implicated in LSC generation and maintenance. For example, co-culture with stromal cells increased expression of the pro-survival proteins, BCL2 and BCL-XL in CD34+ AML LSC, thus impeding apoptosis after chemotherapy exposure [85]. Enhanced cytokine signaling in medullary and extra-medullary niches [86] can result in activation of pathways such as JAK/STAT signaling that promote LSC survival and self-renewal [87]. Bone marrow (BM) stroma has been implicated in the longterm survival of leukemic cells, and contributes to the expansion and proliferation of both transformed and normal hematopoietic cells. Moreover, recent studies have revealed that imatinib therapy may induce acquisition of quiescence and stroma-mediated chemoresistance of CML progenitor cells in the bone marrow [88]. Together these results indicate that the bone marrow microenvironment provides a privileged sanctuary for LSC that protects them from TKI intervention and facilitates the development of therapeutically resistant LSC. These conditions present a clear obstacle for any leukemia treatment strategy, and thus the LSC niche must be considered in any new therapeutic approaches – both as a therapeutic target itself as well as in evaluating the efficacy of novel treatments targeting LSC in the context of various supportive microenvironments. Targeting the adhesion molecules as well as signals that induce quiescence or recruit and retain LSC to the niche could be implemented to impair LSC settling and survival in the protective bone marrow microenvironment. 6.1. Targeting the hematopoietic microenvironment in CML HSC in the bone marrow respond to numerous extracellular signals that modulate their activity and interaction within this unique microenvironment. A broad array of soluble factors has been implicated in regulating the response of tumor cells to chemotherapy. Signaling molecules such as cytokines (GM-CSF, GMSF, IL-4, IL-6, IL-8), angiogenic factors (VEGF, nitric oxide) and TGF-b present in the niche environment can influence response to chemotherapy [89–92]. It is possible to target some of these factors to interfere with LSC survival in the niche. For example, G-CSF treatment induced LSC in the bone marrow to reenter the cell cycle and increased their sensitivity to chemotherapy [93]. Other molecules that influence LSC interactions with the niche microenvironment include cell surface markers and signaling receptors. Preclinical studies identified the importance of a putative CSC marker, CD44 – a b-catenin target gene – in regulating the interaction of the CSC with the bone marrow niche. Notably, monoclonal antibody-mediated inhibition of CD44 resulted in a reduction of the human AML LSC burden in xenograft models [94] or LSC burden in murine recipients of BCR–ABL1-transduced progenitors from CD44-mutant donors [95]. Another critical cell surface receptor implicated in the process of HSC homing to the bone marrow and acquisition of quiescence is C-X-C chemokine receptor type 4 (CXCR4) and its ligand, CXCL12 (SDF-1) [96,97]. Recent studies focused on elucidating the mechanisms of LSC migration into and out of the bone marrow niche demonstrated that CML cells expressed CXCR4 [98], and that upregulation of CXCR4 by imatinib promotes migration of CML cells to the bone marrow, inducing cell cycle arrest and thus ensuring the survival of quiescent CML progenitor cells [88]. Inhibition of the interactions between CXCR4 and its activating ligand using

Please cite this article in press as: L.A. Crews, C.H.M. Jamieson, Selective elimination of leukemia stem cells: Hitting a moving target, Cancer Lett. (2012), http://dx.doi.org/10.1016/j.canlet.2012.08.006

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CXCR4 antagonizing agents in leukemia models impaired LSC adhesion to the extracellular matrix components of the bone marrow niche and increased the sensitivity of LSC to TKIs in vivo [98,99]. These results support the idea of using CXCR4 inhibition in conjunction with TKI therapeutics to override drug resistance in CML and suppress or eradicate residual disease. Cellular quiescence is yet another potential target that may facilitate selective targeting of resistant LSC within protective niches. While significant work remains to fully elucidate the mechanism of normal stem cell quiescence within particular microenvironments and the parallel mechanisms commandeered by LSC, preliminary evidence has suggested that mobilization of CSCs is possible and that re-awakened CSCs are more sensitive to TKIs [93,98]. As such, quiescence-targeted anti-LSC strategies may be more effective when combined with conventional therapies.

7. Conclusions In summary, it is clear that innovative therapeutic approaches are necessary to selectively target CSC that drive disease progression and resistance to therapy. While the majority of LSC targeted therapies are still in preclinical development at this time, a handful of compounds have progressed to clinical trials. LSC collected from patients with hematologic malignancies represent a unique opportunity to evaluate novel targeted prognostic and therapeutic strategies because it is possible to sequentially sample cells from these disorders and profile LSC before and after therapy. Combining LSC-targeted therapies with standard TKI therapy for leukemia may prove to be quite effective because these strategies could synergize by targeting proliferating and quiescent LSC populations, and thus may postpone disease relapse or development of LSC-mediated therapeutic resistance. Combination therapies provide a rational and effective strategy to treating complex disorders, as evidenced by other diseases where ‘‘cocktail’’ therapies are the norm, such as HAART therapies for HIV. A common result among anti-leukemia therapies is the acquired resistance to therapy, highlighting the importance of considering that the LSC pool is not a static population for which one therapy will be sufficient to cure disease. In fact, in the years since the introduction of imatinib, it has become clear that the LSC population evolves over time, acquiring survival and self-renewal advantage through upregulation of signaling pathways such as the Bcl-2 family, the Wnt/b-catenin, GSK3b and Hh pathways, and through interaction with protective niches (Figs. 1 and 2). Future strategies could include targeting other unique aspects of LSC biology, for example modulation of LSC dormancy through cell cycle manipulation. The shift towards strategies targeting multiple molecular pathways in a ‘‘two-pronged’’ attack on LSC may not only thwart development of drug resistance and disease relapse, but could even eradicate the LSC population and prevent the need for lifetime treatment of patients suffering from blood cancers. Development of curative strategies that eliminate LSC populations are the ultimate goal of the monumental research efforts that have illuminated these important LSC pathways in recent years. Novel therapies such as the strategies described here have the potential to dramatically improve the quality of life and lessen the financial burden on patients with LSC-driven leukemic disorders by shortening their course of treatment, reducing disease relapse, and providing more effective interventions for patients that are resistant to current therapies.

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Please cite this article in press as: L.A. Crews, C.H.M. Jamieson, Selective elimination of leukemia stem cells: Hitting a moving target, Cancer Lett. (2012), http://dx.doi.org/10.1016/j.canlet.2012.08.006