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Autophagy in Chronic Myeloid Leukaemia: Stem Cell Survival and Implication in Therapy Gudmundur V. Helgason1,*, Arunima Mukhopadhyay1, Maria Karvela1, Paolo Salomoni2, Bruno Calabretta3 and Tessa L. Holyoake1 1

Paul O’Gorman Leukaemia Research Centre, Institute of Cancer Sciences, College of Medical, Veterinary & Life Sciences, Institute of Cancer Sciences, University of Glasgow, Glasgow, G12 0ZD, UK; 2Samantha Dickson Brain Cancer Unit, UCL Cancer Institute, Paul O'Gorman Building, London, WC1E 6BT, UK; 3Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA Abstract: The insensitivity of Chronic Myeloid Leukaemia (CML) stem cells to Tyrosine Kinase Inhibitor (TKI) treatment is now believed to be the main reason for disease persistence experienced in patients. It has been shown that autophagy, an evolutionarily conserved catabolic process that involves degradation of unnecessary or harmful cellular components via lysosomes, is induced following TKI treatment in CML cells. Of clinical importance, autophagy inhibition, using the anti-malarial drug hydroxychloroquine (HCQ), sensitised CML cells, including primitive CML stem cells, to TKI treatment. In this review we discuss the role of autophagy in the maintenance and survival of stem cells in more detail, with a focus on its role in survival of CML stem cells and the possibility to inhibit this pathway as a way to eliminate persistent CML stem cells in vitro and in patients.

Keywords: Autophagy, Bcr-Abl, CML, clinical trials, glioblastoma, haemopoietic stem cells, HCQ, ROS. INTRODUCTION Peter C. Nowell and David Hungerford first provided a genetic link to cancer in 1960 when they described a minute abnormal chromosome in leukocytes from Chronic Myeloid Leukaemia (CML) patients [1]. This chromosome, that is now known to be the hallmark of CML, was called the Philadelphia (Ph) chromosome after the city in which it was discovered. 13 years later, Janet Rowley using chromosomal banding techniques, demonstrated that the Ph chromosome resulted from a reciprocal translocation between the long arms of chromosomes 9 and 22, t(9:22)(q34;q11) [2], that was later shown to lead to generation of the fusion oncogene Bcr-Abl [3], that is sufficient to induce leukaemia as a sole oncogenic event [4, 5]. Around the same time that the Ph chromosome was first described, another scientist, Christian de Duve, introduced the term autophagy, that is derived from the Greek words “auto” (self) and “phagy” (eating), to describe intracellular vesicles that contained degraded cytoplasmic material [6]. Since then, autophagy has been characterised as a cell survival pathway that functions to degrade and recycle cellular components, such as aged proteins and organelles, that can be re-used to generate ATP and essential building blocks during nutrient and/or oxygen deprivation [7] to maintain homeostasis. Autophagy was first linked to disease in 1999 when BECLIN1, an essential autophagy gene, was shown to have

*Address correspondence to this author at the Paul O’Gorman Leukaemia Research Centre, 21 Shelley Road, Glasgow, G12 0ZD, UK; Tel: 0044 1413017884; Fax: 0044 1413017898; E-mail: [email protected]

1873-5576/13 $58.00+.00

tumour suppressive function and to be expressed at decreased levels in human breast carcinoma [8]. Although the precise mechanism by which autophagy mediates tumour suppression is not clear, it has been suggested that the ability of autophagy to remove damaged organelles, especially mitochondria, that are the source of reactive oxygen species (ROS) generation, is crucial for its suppressive function [9]. However, the role of autophagy in tumour suppression is complex and many tumour suppressor genes, such as PTEN and Tuberous Sclerosis Complex component 1 (TSC1) and TSC2, may stimulate autophagy. In addition, p53, the most commonly mutated tumour suppressor gene, has been shown to have both positive and negative effects on autophagy, depending on its localisation [10, 11]. However, the current view is that autophagy functions as both a tumour suppressor pathway by preventing tumour initiation and as a pro-survival pathway that helps tumour cells tolerate metabolic stress and resist death triggered by anti-cancer agents [12]. In line with this, allelic disruption of BECLIN renders epithelial cancer cells susceptible to metabolic stress and cells die due to inhibition of autophagy [13]. Furthermore, while there is still controversy as to whether autophagy represents a mechanism of cell death during chemotherapy, in recent years it has become clear that autophagy induced following anti-cancer therapy plays a protective role and can enhance the survival of drug-treated cells [14, 15]. This indicates that autophagy inhibition in combination with therapy may increase the anticancer effect of currently used drugs [16] and has been the rationale for initiation of many clinical trials where autophagy inhibition is being tested [17, 18]. We have recently shown that following Bcr-Abl inhibition by c-Abl specific tyrosine kinase inhibitors (TKIs), autophagy © 2013 Bentham Science Publishers

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protects CML stem cells, indicating that autophagy might contribute to disease persistence experienced by CML patients [19]. Here, we discuss the potential role of autophagy in normal and leukaemic stem cell maintenance and survival and the possibility of inhibiting autophagy as a way to provide cure for CML patients.

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initiation of the phagophores [26]. The phosphorylation status of the ULK1 complex changes depending on cellular nutrient conditions. In mammals mTORC1 (mTOR complex 1), that is activated under growing (nutrient rich) conditions, interacts with and phosphorylates ULK1 and ATG13 proteins of the initiation complex to inhibit membrane targeting of the complex, and therefore acts as a negative regulator of autophagy [27]. During starvation, mTORC1 dissociates from the ULK1 complex and AMPK (AMPactivated protein kinase) can phosphorylate ULK1 directly to initiate autophagy [28], allowing the complex to associate with membranes resulting in the initiation of phagophore formation [29]. The class III PI3K (PI3K-III), VPS34, is critical for expansion of the phagophores to autophagosomes, a double-membraned vesicle carrying cargo for lysosomal delivery [30]. VPS34 forms a complex with VPS15, BECLIN1 (yeast Atg6) and the anti-apoptotic protein BCL-2, that can inhibit autophagy by binding to the BH3 domain in BECLIN1 [31]. The BECLIN1/BCL-2 interaction is reduced following starvation, freeing BECLIN1 to engage in autophagy activation [32]. BECLIN1 is also associated with AMBRA1 and phosphorylation of AMBRA1 by ULK1 upon autophagy induction frees BECLIN1 for membrane association [33]. Other BECLIN1 binding partners like UVRAG, Atg14L and Rubicon can interact with BECLIN1 and positively regulate autophagy [34, 35]. The expansion of the phagophores to autophagosomes leading to autophagosome maturation, also depends on two ubiquitin-like conjugation systems forming complexes essential for autophagy [36]: 1) The ATG12/ ATG5/ATG16 and 2) ATG8-PE (phosphatidylethanolamine) (ATG8 is also known as microtubule-associated protein 1 light chain 3, hereafter called LC3). The ATG12/ATG5/

MOLECULAR REGULATION OF AUTOPHAGY An understanding of the molecular mechanism of macroautophagy (hereafter referred to as autophagy) has only been gained in the last 15 years and many regulators of autophagy, or autophagy-related (ATG) genes, have now been identified. Most of these genes were initially identified in yeast and several of them have now been shown to have functional orthologues in mammalian cells [20]. The evolutionarily conserved mechanism of autophagy has been thoroughly investigated and many detailed reviews have been published about the protein complexes that contribute to each step [21, 22]. In short, the autophagy process starts with the initiation complex, a serine/threonine kinase complex containing ULK1 and ULK2 (yeast Atg1), ATG13, FIP200 (a mammalian functional homologue of Atg17) and ATG101 (sometimes this complex is called the ULK1 complex [23] (Fig. 1). These proteins are predominately localised in the cytosol and associated with the phagophore (also called the isolation membrane) upon autophagy induction. Ever since the discovery of autophagy by de Duve about 50 years ago [6], the source of the membrane has been investigated and debated, and it is now supposed that endoplasmic reticulum (ER), golgi, mitochondria [24] and the plasma membrane [25] may all provide a membrane source for

Phagophore

Lysosome ULK2

ULK1

Atg13 Atg101

1) Initiation complex

PE LC3-II

p

Vps34 BECLIN1

4) Completion

Vps15 UVRAG Rubicon

AMBRA1

2) Phagophore formation

5) Degradation Atg16 Atg12

Atg5

Atg3 Atg7

Atg4

3) Autophagosome maturation

Fig. (1). Molecular regulation of autophagy. Autophagy starts with activation of the initiation complex that contains ULK1, ULK2, ATG13, FIP200 and ATG101. This complex then becomes active after AMPK activation and mTORC1 inhibition, for example, during starvation or drug treatment. The PI3K-III VPS34 is critical for phagophore formation. VPS34 forms a complex with VPS15, UVRAG, AMBRA1, Rubicon and BECLIN1. This complex can be inhibited by the anti-apoptotic protein BCL-2, which can interact with BECLIN1 through the BH3 domain in BECLIN1. Autophagosome maturation is mediated by the ATG12/ATG5/ATG16 and ATG8 (LC3)-PE conjugation systems. These systems perform the lipid modification of LC3-I, leading to LC3-II-PE binding to the autophagosomal membrane. Completed autophagosomes contain materials such as proteins and organelles that are degraded after autophagosome fusion with lysosomes that have low pH and an abundance of pH-sensitive enzymes that break down the waste materials.

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ATG16 complex formation starts with conjugation of ATG5 with ATG12, an ubiquitin-like modifier, by E1-like enzyme ATG7 and E2-like enzyme ATG10, whereas E1-ubiquitin enzymes ATG4 and ATG7 and E2-ubiquitin enzyme ATG3 are the key players in mediating the lipid modification of LC3. ATG12-ATG5 complex non-covalently interacts with multimeric ATG16 and this multimer functions as an E3-like enzyme and regulates the binding of lipidated LC3 (LC3-II or ATG8-PE) to the autophagosomal membrane [37]. This alteration is most commonly used to monitor autophagy by various assays. However, correct interpretation of LC3-I/II conversion is very important and guidelines for monitoring autophagy have therefore recently been updated by Klionsky et al., [38]. The completion process of autophagy includes the fusion of the outer membrane of autophagosomes with lysosomes (that contain pH-sensitive degradative enzymes) to form autolysosomes. Within autolysosomes, the inner single membrane of the autophagosome and its cargo is lysed by lysosomal hydrolases, especially cathepsins, and the content degraded [39]. The detailed mechanism of the autophagosome fusion with the lysosomes in order to form the autolysosomes is not very well understood, although it has been shown to require the lysosomal protein LAMP-2, the small GTPase RAB7 and UVRAG [40-42]. This multistep autophagic pathway is tightly controlled by several signaling mechanisms as discussed later in this review, such as the PI3K/Akt/mTORC1 pathway, that is also crucial for normal cell survival and tumourigenesis. THE ROLE OF THE PI3K/AKT/MTORC1 PATHWAY IN NORMAL HAEMOPOIETIC STEM CELL (HSC) MAINTENANCE HSCs live in the bone marrow, sit on top of a hierarchical system and give rise to all the short-lived cells within the blood that are needed over the course of our lifetime [43]. HSCs can be found within specific niches or microenvironment within the bone marrow [44, 45] and are mostly in a quiescent state [46]. HSCs can be stimulated to selfrenew and generate two HSCs (symmetric division) or to undergo asymmetric cell division, to generate one HSC and a progenitor cell that can then repopulate differentiated haemopoietic cell lineages [43]. HSC’s quiescence needs to be tightly controlled, as deregulation in cell cycle entry or differentiation can lead to exhaustion of the HSC pool, that is needed for blood cell replenishment [46, 47]. For example, homozygous deletion of the cyclin-dependent kinase p21 leads to increased proliferation of HSCs, followed by a loss of stem cells and increased susceptibility to stress-induced exhaustion of the stem cell pool [48]. In addition to cyclindependent kinase inhibition, the PI3K/Akt/mTORC1 pathway also plays an important role in regulation of HSC’s quiescence and deregulation of this pathway can lead to leukaemia [49-53]. Studies using mouse models have shown that 1) conditional deletion of the Pten tumour suppressor gene (a phosphatase that counters PI3K phosphorylation) in HSCs led to their short-term expansion and long-term depletion and to impaired ability to sustain haemopoietic reconstitution and eventually to the development of a myeloproliferative disorder (MPD) [51] (Fig. 2). These events were mostly mediated by mTORC1 as rapamycin rescued normal HSC function after Pten deletion [50]. 2)

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Normal Stem Cell Alterations that affect normal HSCs:

PTEN

PI3K Akt TSC1/2

Deletion of Pten Deletion of p85/ subunits of PI3K Constitutive activation of Akt

Deletion of TSC1

mTORC1

Autophagy

Deletion of FIP200 Deletion of Atg7

ROS

“Stemness” Fig. (2). Regulation of “stemness”. The PI3K/Akt/mTORC1 pathway plays an important role in regulation of HSC’s quiescence and deregulation of this pathway, such as Bcr-Abl expression, can lead to leukaemia. Deregulated stimulation of this pathway may also lead to inhibition of autophagy, resulting in increased ROS levels that can affect stem cell maintenance. Examples of gene/protein alterations that affect normal HSCs are shown and include: Deletion of Pten in HSCs leading to reduction of HSC numbers, impaired haemopoiesis and development of MPD. Deletion of p85/ subunits of PI3K leading to reduction in progenitor cells and decreased repopulation potential of HSCs. Constitutive activation of Akt leading to increased cycling of HSCs, impaired engraftment and development of MPD. Deletion of TSC1 leading to increased cycling and depletion of HSCs, defective repopulation potential and defective haemopoietic development. Conditional deletion of FIP200 leading to loss of HSC maintenance/function, increased HSC proliferation, aberrant myelopoiesis and increased mitochondrial mass/ROS. Conditional deletion of Atg7 leading to loss of HSC maintenance/function, failure to reconstitute the haemopoietic system, increased proliferation of HSC/progenitor cells, and accumulation of mitochondria/ROS/DNA damage.

Similar to the phenotype of the mice with Pten deletion, constitutive activation of Akt (using myristoylated Akt) resulted in increased cycling of HSCs, impaired engraftment and development of MPD, T cell lymphoma or Acute Myeloid Leukaemia (AML) [52]. Rapamycin increased survival of the mice indicating that most of the effects were mTORC1 dependent. 3) Deletion of TSC1 resulted in increased mTORC1 activity, increased HSC cell cycling resulting in depletion, mobilisation, impairment of long-term repopulation and defective haemopoietic lineage development [53-55]. The effects of TSC1 deletion on repopulation potential and haemopoietic lineage development were shown to be mediated through mTORC1 [53]. 4) Genetic loss of the promyelocytic leukaemia gene (PML), that may negatively

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regulate the PI3K/Akt/mTORC1 pathway at multiple levels [56], resulted in increased mTORC1 activity and increased cycling of HSCs, leading to their exhaustion [57]. Rapamycin treatment restored normal HSC function. 5) Conversely, genetic deletion of p85alpha and p85beta regulatory subunits of PI3K resulted in reduction in absolute number of haemopoietic progenitor cells and decreased multilineage repopulating the ability of HSCs [49]. Therefore, several different types of modulation of the PI3K/Akt/mTORC1 signalling pathway suggest that this axis is fundamental for the switch of commitment of HSCs from quiescent to proliferating and mTORC1 plays a critical role in this control. But how can increased mTORC1 activity lead to decreased HSCs maintenance and/or survival? One possibility is that increased mTORC1 activity results in a higher metabolic state and increase in ROS. As mentioned earlier, HSCs sit in a special niche, which has been shown to be a low-oxygen part of the bone marrow, far away from circulating oxygenated blood [58]. This hypoxic microenvironment might be important for maintaining HSCs in an undifferentiated state and migration to parts with normoxia might promote their metabolic activity and drive differentiation [47, 59]. In fact, early HSC can be functionally isolated by taking advantage of their relatively low intracellular ROS activity [60]. Interestingly, the ROSlow population has been shown to be enriched for cells in G0, with higher selfrenewal potential and expressing higher levels of cyclinkinase inhibitors, whereas HSC exhaustion was observed following serial transplantation of the ROShigh population. Interestingly, mTORC1 activity was higher in the ROShigh population compared to the ROSlow population and rapamycin was able to restore HSC function in the ROShigh population by inhibiting mTORC1 [60]. This is in line with the fact that TSC1 inactivation in mice resulted in higher ROS levels, as well as increased mitochondrial mass and mitochondrial DNA copy number [54, 55, 61]. The importance of low ROS levels in quiescent HSCs is further supported by experiments with the FOXO (Forkhead transcription factors of the class O), transcription factors that are controlled by Akt, and function as key regulators of cell cycle, apoptosis and oxidative stress response [62]. FoxO3a is phosphorylated by Akt in proliferating cells leading to its cytoplasmic localisation and inactivation. However, in some quiescent HSCs, Akt is not activated and FoxO3a, which is important for suppressing ROS levels [63], is localised to the nucleus. Upon activation of Akt by cytokine stimulation and lipid raft clustering, Foxo3a is exported from the nucleus to the cytoplasm, followed by HSCs re-entry into the cell cycle [64]. In line with this, conditional deletion of FoxO1, FoxO3 and FoxO4 in the adult haemopoietic system resulted in myeloid lineage expansion, lymphoid developmental abnormalities, and a marked decrease of HSCs [65]. And interestingly, there was a marked increase in the ROS levels in FoxO deficient HSCs compared to normal HSCs, indicating that functional interplay between PI3K/Akt/ mTORC1 pathway and FoxO transcription factors is essential for maintenance of normal quiescent HSC.

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and maintains low ROS levels in HSCs? It is tempting to speculate that autophagy, that is known to be negatively regulated by mTORC1 [66], plays a part in maintenance of mitochondrial biogenesis and ROS homeostasis within HSCs (Fig. 2). Interestingly, in recent years it has become clear that autophagy plays a critical role for HSC maintenance. The first indication came from Liu et al., who in 2010 showed that conditional deletion of FIP200 (focal adhesion kinase family interacting protein of 200 kD), a newly identified essential autophagy gene and a component of the initiation complex, resulted in severe anaemia and perinatal lethality [67]. FIP200 was required for the maintenance and function of fetal HSCs and FIP200 deficient HSCs were unable to reconstitute lethally irradiated recipients. FIP200 ablation did result in increased rate of HSC proliferation and aberrant myelopoiesis evidenced by the presence of a nearly 10-fold increase in the number of myeloid cells in the peripheral blood of FIP200 knockout mice, without an increase in apoptosis of HSCs. FIP200-null fetal HSCs exhibited increased mitochondrial mass and high ROS levels, indicating a role for autophagy in the maintenance of fetal haemopoiesis and HSCs. In another study by Mortensen et al., the essential autophagy gene Atg7 was conditionally deleted in the haemopoietic system [68]. This also resulted in loss of normal HSC function and autophagy deficient HSCs failed to reconstitute the haemopoietic system of lethally irradiated mice. Moreover, in addition to increased proliferation, the HSC and the progenitor cell compartment displayed an accumulation of mitochondria, ROS and DNA damage, further supporting a role for autophagy in regulating mitochondrial quantity and quality in HSCs. The production of both lymphoid and myeloid progenitors was impaired in the absence of Atg7 and the mice developed MPD and died within weeks, indicating that autophagy may protect against leukaemogenesis. However, autophagy plays additional roles beyond regulation of mitochondria in HSCs. In mammalian red blood cells, the removal of the nucleus followed by the elimination of other organelles, such as mitochondria, are necessary steps that enable differentiation to proceed. It has also recently been shown that mitophagy (mitochondrial sequestration by autophagosomes, followed by lysosomal degradation), a known mechanism of mitochondrial turnover, is essential during erythropoiesis and a necessary developmental step in erythroid cells [69]. Using mice lacking Atg7 in the haemopoietic system, Mortensen et al., showed that loss of mitochondria is mediated by autophagy, as Atg7 knockout erythroblasts and reticulocytes accumulate damaged mitochondria, increased ROS and undergo apoptosis [70]. Taken together, these data strongly suggest that the mTORC1mitochondria-ROS axis, crucial for HSC “stemness”, also links to autophagy which in turn plays a role in quiescence, differentiation and pluripotency of HSCs. Therefore, autophagy can act as a tumour suppressor by maintaining a healthy state of stem cells through regulation of mitochondrial mass/number and by minimising ROS levels and DNA damage. THE ROLE OF AUTOPHAGY IN CML STEM CELLS

THE ROLE OF AUTOPHAGY IN HSCS But what is the biological process by which the PI3K/ Akt/mTORC1 pathway regulates mitochondrial biogenesis

As mentioned earlier, the hallmark of CML is the BcrAbl fusion oncoprotein that is caused by a reciprocal translocation between the long arms of chromosomes 9 and

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22 within the HSC [1-3]. Bcr-Abl kinase activity is both required and sufficient to induce CML in mice when expressed in HSCs [4, 71]. We and others have shown that Bcr-Abl regulates PI3K activity [72, 73], that leads to mTORC1 activation [74] and that PI3K/Akt/mTORC1 pathway activation is essential for Bcr-Abl mediated transformation [75]. Furthermore, Bcr-Abl expression has also been shown to induce differentiation and to decrease self-renewal capacity and number of mouse HCSs [76, 77], consistent with the effect of deregulation of the PI3K/Akt/ mTORC1 pathway in normal stem cells [49-55]. In 1999 we showed evidence for the existence of a population of leukaemic stem cells in patients with CML [78]. These cells are Bcr-Abl positive, able to remain quiescent (undivided) for up to 4 days in liquid culture and capable of engrafting in immunodeficient mice. In collaboration with Ravi Bhatia, we have further demonstrated that their insensitivity to all three generations of TKIs, imatinib [79, 80], dasatinib [81, 82], nilotinib [83, 84] and bosutinib [85] is the cause of disease persistence in CML patients. Importantly, most recently we and Corbin et al., demonstrated that CML stem cells can survive prolonged TKI treatment that fully inhibits Bcr-Abl kinase activity [86, 87], indicating that combination therapy is needed to provide a cure for CML patients. This has prompted us to search for additional survival mechanisms that are present or induced following treatment in the surviving TKI-resistant stem/progenitor cells. One such mechanism is autophagy. Autophagy, has been shown to be induced by various drugs in CML cells [16], including TKI treatment in both CML cell lines and primary stem/progenitor (CD34+38-) cells [19, 88]. We further showed that specific autophagy inhibition, either with ATG7 or ATG5 knockdown, or pharmacological inhibition using CQ, resulted in enhanced TKI-induced death in CML cell lines and primary CML stem cells. These promising results have led to the initiation of the CHOICES phase II clinical trial, where HCQ is being tested in combination with imatinib in imatinib-sensitive patients (see more in “Autophagy as a Therapeutic Target in CML and Glioblastoma Multiforme (GBM)” section) [89, 90]. So what is the effect of Bcr-Abl expression on autophagy and how does autophagy protect CML stem cells from TKI treatment? The fact that autophagy is induced following BcrAbl inhibition suggests that Bcr-Abl down-regulates autophagy. In support of this hypothesis, Altman et al., who in 2010 developed a growth factor-dependent haemopoietic cell culture model in which autophagy was acutely disrupted through conditional Cre-mediated excision of Atg3, [91] demonstrated that Bcr-Abl expressing cells exhibited low basal autophagy, but were highly dependent on it. They also showed that autophagy inhibition following Atg3 deletion in Bcr-Abl expressing cells led to increased p53 phosphorylation and accumulation, as well as increased expression of the p53 target gene, p21 and the pro-apoptotic Bcl-2 family protein Puma. Interestingly, H2A.X, a marker of DNA double strand breaks (that may be upstream of p53), was also increased following atg3 deletion. However, it was not clear if autophagy inhibition had any effect on mitochondrial number or function that could possibly lead to

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increased DNA damage, as has been shown in normal HSCs [67, 68]. Although we showed that TKI-induced autophagy was associated with ER stress and was suppressed by depletion of intracellular Ca2+ [19], the exact mechanism by which Bcr-Abl suppresses autophagy is not entirely clear. However, two recent papers have shed some light on this. Firstly, Sheng et al., showed that Bcr-Abl, through the PI3K/Akt pathway, transcriptionally up-regulated activating transcription factor 5 (ATF5) in a FoxO4 dependent manner [92] and ATF5 in turn, stimulated mTORC1 transcription, required for autophagy inhibition. So this model suggests that BcrAbl not only activates mTORC1 kinase activity, but also leads to increased mTORC1 transcription. The authors further showed that imatinib-induced autophagy was dependent on inhibition of the PI3K/Akt/mTORC1 pathway, as ectopic expression of constitutively active PI3K (PI3KCAE545K) suppressed autophagy induced by imatinib. Secondly, Yu et al., showed that imatinib inhibited expression of microRNA-30a (mir-30a) in CML cells leading to autophagy induction and up-regulation of BECLIN1 and ATG5 expression [93]. Taken together this suggests that Bcr-Abl expression inhibits autophagy by at least two mechanisms 1) in an mTORC1 independent manner by inducing mir-30a expression and 2) in an mTORC1 dependent manner by inducing mTORC1 activity/expression. To support the involvement of active mTORC1 in autophagy inhibition in CML cells, OSI-027, an mTOR inhibitor (inhibits both mTORC1 and mTORC2 complexes) has been shown to induce protective autophagy in K562 cells and combination of OSI-027 and CQ-mediated autophagy inhibition resulted in increased apoptosis compared to OSI-027 alone [94]. Therefore, autophagy may be a key defensive mechanism that provides survival and/or limits the apoptotic responses following mTOR inhibition in CML cells and combined use of potent mTOR inhibitors with autophagy inhibitors may provide an approach to enhance the effect of single drug treatment. In reality, autophagy, a tumour suppressor in normal HSC can be exploited therapeutically in CML. AUTOPHAGY AS A THERAPEUTIC TARGET IN CML AND GLIOBLASTOMA MULTIFORME (GBM) There are currently no specific autophagy inhibitors available to be used in the clinic. However, autophagy can be inhibited using the anti-malarial drug HCQ, which has also been approved for treatment of autoimmune disorders, such as rheumatoid arthritis [16]. HCQ is a lysosomotropic weak base, meaning it preferentially accumulates within the acidic environment of lysosomes, where it raises the pH and impairs autophagic protein degradation. Over 40 clinical trials have been initiated in cancer patients using HCQ, alone or in combination with cytotoxic agents, as an autophagy inhibitor [14, 17, 18, 95]. Our promising in vitro data on the effect of TKI/CQ combination on survival of CML stem cells [19] have led to a phase II clinical trial (CHOICES; CHlOroquine and Imatinib Combination to Eliminate Stem cells - ClinicalTrials.gov: NCT01227135), the first clinical trial using autophagy inhibitions in CML. The CHOICES trial aims to test the combination of imatinib with HCQ in

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CML patients who have achieved major cytogenetic response (MCyR), but who continue to show evidence for residual disease detectable by quantitative PCR. Our more recent work on the role of autophagy in GBM stem cells (Salomoni’s laboratory), raises the question of whether autophagy is the Achilles heel in other cancer stem cells when hit with radio-, chemo- or targeted therapy. We have shown that a number of autophagy regulators, such as DRAM1 and p62, are highly expressed in GBM tumours and regulate invasive properties of GBM stem cells. Furthermore, DRAM1 expression is associated with shorter overall survival in GBM patients [96]. Notably, a trial is currently ongoing in which HCQ is being tested in combination with radiation in elderly patients with GBM (ClinicalTrials.gov: NCT00486603). In addition to playing a protective role in CML stem cell persistence, autophagy might also serve as a therapeutic target in CML cells that are resistant to TKI treatment. Results from Carew et al., showed that CQ enhanced the anti-cancer activity of the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) in imatinib-resistant primary cells [97], suggesting that autophagy might also play a role in CML cells when Bcr-Abl is fully active. Recently, some forms of macrolide antibiotics, such as azithromycin [98] and clarithromycin [99] and desmethylclomipramine, an active metabolite of clomipramine, that is used for the treatment of psychiatric disorders [100], have been shown to inhibit autophagy and interestingly, in one small study by Carella et al., clarithromycin strongly potentiated TKI treatment in four patients with advanced CML, who were resistant to TKI alone [101, 102]. However it still needs to be shown that concentrations of clarithromycin used in this study can inhibit autophagy in patients and that the beneficial effect was solely mediated through autophagy inhibition (experiments are underway in Carella’s laboratory to investigate this). In addition, Lys05, a CQ derivative that is 10 times more potent than HCQ [103], mefloquine, another lysosomotropic and anti-malarial agent [104] and spautin-1, a small molecule inhibitor of VPS34 [105], have all recently shown promising results in preclinical models and are under investigation in our laboratory. It is still early days for autophagy inhibition in the clinic and some questions remain unanswered. Although HCQ has been shown to block autophagy and enhance the effect of many anti-cancer agents in vitro in the 1-10M concentration range, it still remains to be shown that autophagy can be blocked in patients receiving 400-800 mg daily. Therefore development of more potent/specific autophagy inhibitors for the use in the clinic is the challenge for the pharmaceutical industry and academia. Using the best animal and in vitro models to verify potential future specific autophagy inhibitors will be crucial, as it is still possible that inhibition of the later stages of autophagy (inhibition of autolysosomes/ lysosome fusion) is toxic to cells and that similar effects will not be observed with drugs that inhibit the initiation or formation of autophagosomes. Another challenge is to find the best way to monitor autophagy, a very dynamic process, in patient samples, using methods that minimise handling to ensure consistency.

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Given the tumour suppressive function of autophagy, is there a potential risk associated with autophagy inhibition in humans? CQ and HCQ have been used extensively in the treatment of malaria and rheumatoid arthritis and are quite well tolerated. Since complete autophagy inhibition using mouse models has significant detrimental effects within the HSC compartment this level of tolerance suggests that the duration and/or magnitude of autophagy inhibition in current trials is well below maximal and infers that it is unlikely that severe toxicities or a higher incidence of secondary malignancies will be observed. Whether more proximal, specific and potent autophagy inhibitors will have similar safety profiles remains to be investigated. Interestingly, a recent study by Michaud et al., showed that autophagy may limit chemotherapy responses by preventing autophagydependent anti-cancer immune responses, raising the concern that acute autophagy inhibition may actually limit chemotherapy responses in certain cancers for which an immune reaction plays an important role in disease response [106]. It is hoped that current clinical trials of HCQ-mediated autophagy inhibition will provide critical information regarding many of these issues and act as proof of concept for the potential use of autophagy inhibition as a strategy to increase the efficacy of currently used anti-cancer treatment. CONCLUSION Although imatinib still represents the most successful example of targeted therapy in human cancer, the persistence of CML stem cells, inherently insensitive to Bcr-Abl inhibition, compels CML patients to life-long TKI treatment with the risk of toxicity, resistance, relapse and an enormous economic burden on the health system. However, autophagy, a process fundamental for maintenance and survival of normal HSCs and down-regulated in CML HSCs, is induced following Bcr-Abl inhibition in CML stem cells. This induction likely contributes to their insensitivity to TKI treatment and it is therefore hoped that autophagy inhibition may provide an enhanced response rate for CML patients. It is anticipated that current clinical trials in CML and other cancers, where the aim is to enhance the effect of anti-cancer drugs by combinatorial treatment with HCQ, will provide vital information about the role of autophagy in CML and other stem cell driven malignancies. CONFLICT OF INTEREST TLH has previously received research support from Bristol-Myers Squibb and Novartis. ACKNOWLEDGEMENTS G.V.H. was funded by the Kay Kendall Leukaemia Fund (KKL404) and currently is a KKLF Intermediate Research Fellow (KKL698)/Leadership Fellow. A.M. is funded by the Medical Research Council (G0900882, CHOICES, ISCRTN No. 61568166). G.V.H. and T.L.H. are supported by Scottish Universities Life Sciences Alliance (MSD23_G_HolyoakeChan). B.C. is supported in part by NCI CA95111 grant. PS is funded by the Brain Tumour Charity, CRUK, AICR and is supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre.

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ABBREVIATIONS

[15]

CML

=

Chronic Myeloid Leukaemia

[16]

TKI

=

Tyrosine Kinase Inhibitor

HCQ

=

hydroxychloroquine

Ph

=

Philadelphia

ROS

=

reactive oxygen species

PE

=

phosphatidylethanolamine

HSC

=

haemopoietic stem cell

GBM

=

Glioblastoma Multiforme

CHOICES

=

CHlOroquine and Imatinib Combination to Eliminate Stem cells

MCyR

=

major cytogenetic response

[20]

FOXO

=

Forkhead transcription factors of the class O

[21]

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Received: February 15, 2013

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Accepted: July 01, 2013

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