Pharmacological inhibitors of autophagy as novel cancer therapeutic agents

Accepted Manuscript Title: Pharmacological inhibitors of autophagy as novel cancer therapeutic agents Author: Cheng Wang Qidong Hu Han-Ming Shen PII: DOI: Reference:

S1043-6618(16)30062-7 http://dx.doi.org/doi:10.1016/j.phrs.2016.01.028 YPHRS 3054

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Pharmacological Research

Received date: Accepted date:

22-1-2016 22-1-2016

Please cite this article as: Wang Cheng, Hu Qidong, Shen Han-Ming.Pharmacological inhibitors of autophagy as novel cancer therapeutic agents.Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2016.01.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Pharmacological inhibitors of autophagy as novel cancer therapeutic agents Cheng Wang1, Qidong Hu1, Han-Ming Shen2* 1

Department of Anatomy, 2 Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Republic of Singapore

*Corresponding author: Dr. Han-Ming Shen, Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597. Email: [email protected]; Tel: 6565164998 Graphical abstract:

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Abstract Autophagy is an evolutionarily conserved cellular degradative process in which intracellular components (cellular proteins and organelles) are engulfed in autophagosomes which then fuse with lysosomes to form autolysosome for degradation. Autophagy is closely implicated in various physio-pathological processes and human diseases. Among them, the roles of autophagy in cancer have been extensively studied. Increasing evidence has demonstrated that inhibiting autophagy is a novel and promising approach in cancer therapy, based on the notion that autophagy is a pro-survival mechanism in cancer cells under therapeutic stress, and induction of autophagy is associated with chemoresistance of cancer cells to chemotherapeutic agents. Thus, suppression of autophagy would sensitize resistance tumor cells to cancer therapeutic agents, thereby supporting the clinical application of autophagy inhibitors. In recent years, significant progress has been achieved in developing autophagy inhibitors and testing their therapeutical potential, either as standalone or as adjuvant therapeutic agents, in cell and animal models, and more importantly in clinical trials. In this review, we will discuss some of these recent advances in development of novel small molecules autophagy inhibitors and their mechanisms of action, together with their applications in clinical trials.

1. Introduction Autophagy is an evolutionarily conserved process in which cellular proteins and organelles are engulfed in autophagosomes which then fuse with lysosomes to form autolysosome for degradation [1]. There are three types of autophagy in mammalian cells: macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). Among them, both micro- and macro-autophagy are competent to engulf large cellular structures through distinctive mechanisms, whereas chaperone-mediated autophagy (CMA) selectively degrades soluble proteins but without involvement of vesicular structures [2]. Macroautophagy (referred as autophagy hereafter in this review) is the most widely studied form of autophagy, which is believed to be a non-selective degradation system for proteins, and organelles, via the formation of double-membraned vesicles termed as autophagosomes. In general, the process of cellular self-digestion is considered to be beneficial for adapting cells or organisms to stress conditions such as starvation [3].In the past decade, remarkable progress has been made in understanding the molecular mechanisms of autophagy regulation, the biological functions of autophagy, the implication in human diseases and the potential application by targeting autophagy in control of human diseases. 1.1 The Autophagic Process At present, it has been well-established that the whole autophagy process is controlled by a group of proteins encoded by the autophagy-related-genes (Atgs). Since the first Atg was identified by Ohsumi’s group in yeast [4], so far more than 30 Atgs have been discovered in yeast, and many of them have the mammalian orthologues that control the various processes of autophagy, as briefly described below (partly illustrated in Figure 1).

2

(i)

Induction or initiation: The initiation of autophagy starts with emergence of phagophore or preautophagosomal structure (PAS), and is regulated by the ULK1 (unc51 like autophagy activating kinase 1, the mammalian orthologue of Atg1), consisting of ULK1, Atg13, FIP200 and Atg101 complex, downstream of mammalian target of rapamycin complex 1 (mTORC1). The mTORC1 plays a role in the regulation of cell growth and protein synthesis by the phosphorylation of two key translational regulators, eukaryote initiation factor 4E-binding protein (4EBP1) and S6 kinase (S6K) [5,6]. Activated mTORC1 is able to phosphorylates ULK1 and suppress its kinase activity. Thus, mTOR inhibitors such as rapamycin are well known to induce autophagy.

(ii)

Nucleation/Expansion/Elongation: This process is first mediated by the Beclin1 and hVPS34/class III phosphatidylinositol 3-kinases (PI3K) complex (Choi et al., 2013; Wong et al., 2011). Beclin1 is the mammalian orthologue of yeast Atg6 and the membrane binding domain of proved to be important in the nucleation process (Huang et al., 2012). Subsequently, the completion of autophagosome formation is controlled by two conjugation systems: Atg12-Atg5-Atg16 and LC3-PE (phosphatidylethanolamine).

(iii) Maturation/degradation: This is the final step of autophagy, in which the outer membrane of the autophagosome fuses with a lysosome to form an autolysosome where the inner membrane and luminal contents are degraded via acidic lysosomal hydrolases. Lysosomes is the cellular organelles which contain hydrolase enzymes to play crucial roles in cellular clearance [7,8]. The acidic internal pH in lysosomes is generated by the action of V-ATPase, a proton-pumping membrane protein and maintained by the balance of counterion channels [9]. A recent study from our laboratory has demonstrated the functional activation of lysosome in the course of autophagy, a process depending on both mTORC1 inhibition and autophagosomelysosome fusion [10]. Moreover, it has been established that the fusion process required membrane hemifusion driven by SNAREs (soluble N-ethylmaleimidesensitive factor attachment protein receptors) [11-15]. 1.2 Biological Functions of Autophagy Understanding the biological function of autophagy is critically important when establishing its role in human diseases. The fundamental function of autophagy is to maintain the metabolic balance in the cell [16], through which it plays important roles in cellular processes including development, inflammation, metabolism and aging [17-20]. Basically, autophagy is able to produce essential nutrients like amino acids under various stress conditions such as starvation. This supports protein synthesis in addition to energy homeostasis through tricarboxylic acid (TCA) cycle. Another key purpose of autophagy is to eliminate the intracellular protein aggregates and damaged organelles, such as mitochondria [21]. Furthermore, autophagy has been linked to the immune system, based on the facts that autophagy is involved in antigen presentation via endosome transportation, as well as in elimination of invaded microorganisms [1,22]. Moreover, autophagy serves as an important source of neonate development as LC3, an autophagosomal marker, has been identified to increase in many tissues in neonatal starvation period [23]. 1.3 Autophagy in Cell Survival and Cell Death Among the biological functions of autophagy as described above, the implication of autophagy in cell survival and death remains rather controversial amongst all its biological 3

functions. At present, there is ample evidence supporting the notion that autophagy supports cell survival via suppression of cell death, including both apoptosis and necrosis or other forms of non-apoptotic cell death [24-26]. However, autophagy is also implicated in programmed cell death. Studies in Drosophila have also revealed that dying organs during late larval stage such as midgut and salivary gland display a high level of Atgs, and autophagic cell death is required for the tissue removal instead of apoptosis [27]. Likewise, autophagy and apoptosis work independently in mammalian cells to promote cell death. For instance, mouse embryonic fibroblasts that are deficient of key apoptotic molecules such as Bak-/- and Bax-/- double knockout cells still underwent autophagic cell death mediated by Beclin1 and ATG5, which could be rescued by autophagy inhibitors [28]. Additionally, autophagy selectively eliminates damaged organelles, for example, it tends to digest damaged mitochondria and peroxisomes whereas it preserves functional mitochondria even under nutrient depletion [29]. This implies that autophagy is precisely controlled to evade unwanted self-digestion in normal cells. Studies on nematodes and fruitflies indicate unregulated autophagy caused by overactive Atgs triggers cell death, and it can be rescued when autophagy is blocked [30,31]. Therefore, that autophagy has paradoxical roles in cell survival and death modulation result in detrimental effects on cells [25]. The paradoxical role of autophagy in cell survival/cell death is also the molecular basis for the close implication of autophagy in some important human diseases, including cancer, neurodegeneration, and autoimmune disorders [3,32-35]. 2. Autophagy in Cancer Based on the understanding of the biological function of autophagy as discussed above, especially the dual roles of autophagy in cell survival and cell death, autophagy has been widely recognized to act as a double-edged sword in cancer: autophagy serves as an important anti-cancer mechanisms to prevent cancer initiation, while autophagy is capable of promoting cancer development at the late stage [3,18,36,37]. 2.1 Suppressive Role of Autophagy in Tumorigenesis At present, the anti-cancer function of autophagy has been extensively studied. It is notable that decreased autophagy leads to higher chances of spontaneous tumors, which has been validated in both in vivo and in vitro models. For instance, deletion of Beclin1 and ATG5 displayed increased tumorigeneric potential associated with DNA damage and chronic inflammation in mouse [38]. Similarly, inhibition of autophagy promotes the tumor progression via activation of inflammatory responses including TNFRSF11A (Tumor Necrosis Factor Receptor Superfamily, Member 11a) and NF-κB [39], as well as accumulation of pro-angiogenic transcriptional factor HIF-2a (Hypoxia Inducible Factor-2 alpha) [40]. Multiple mechanisms have been implicated in the anti-cancer function of autophagy. First, it serves to limit the growth of precancerous cells as well as it mitigates cellular oxidative stress, probably through disposing the damaged organelles, especially the mitochondria [41]. Second, autophagy triggers programmed cell death when massive amounts of vesicles accumulate, and maintains the genome integrity both of which repress the tumorigenesis [3,32]. Third, activated autophagy is able to promote p62 protein degradation, thus to prevent the pro-tumorigenic activity of p62 [42,43], and allelic loss of BECN1 increased the incidence of carcinogenesis in vivo through accumulation of p62 [41,44]. The reduced autophagy by Beclin1 inhibition also promotes tumor progression and chemoresistance [45]. By and large, defective autophagy is correlated with metabolic stress, 4

chromosome instability, constitutive inflammation and angiogenesis, all of which may lead to tumorigenesis. 2.2 Pro-survival Role of Autophagy in Established Tumors Despite the strong evidence indicating the anti-cancer function of autophagy as described above, autophagy has also been implicated in the tumorigenesis process as a pro-cancer mechanism. The pro-cancer of autophagy has been well established in several animal models in which suppression of autophagy is able to mitigate tumor progression in pancreatic, lung and liver cancers [46-48]. Several key mechanisms have been implicated in the pro-cancer function of autophagy. First, autophagy plays an important pro-survival role in cancer cells under various stress conditions, including oxidative stress, DNA damage, hypoxia and starvation [25,49]. Unrestrained tumor cells are generally adapted to rely on glycolysis (Warburg effect) more than Krebs cycles that are more efficient in energy production. As a result, cancer cells are more addicted on autophagy than untransformed cells since it neutralizes this metabolic stress caused by high energy demand, thereby meaning the activated autophagy will provide a survival edge for the cancer cells. Second, induction of autophagy augments its cytoprotective roles against the cytotoxic effect of cancer therapeutics. It has been extensively studied that stress-induced autophagy interferes with the efficacy of chemotherapy [50]. Recent studies reported increased chemoresistance in bladder and gastric cancer cells with concurrent upregulation of autophagy related genes [51,52]. Third, autophagy increases cell motility and migration and promotes metastasis and invasion. [53,54]. Lastly, recent studies have demonstrated the possible role of, autophagy in supporting cancer stem cells (CSCs). ATG5 and ATG12 were reported to highly express in dormant breast cancer stem cells [55]. Besides, overexpression of either Beclin1 or ATG5 is believed to be essential for maintenance of CSCs in breast cancers and brain tumors [56,57]. Similarly, bladder cancer cells that overexpress autophagy related genes had concomitant high expression of stemness genes [58]. A very recent study reported the molecular mechanisms underlying the autophagydependent stemness maintenance, which prevents cell senescence [59]. In their study, it was found that basal autophagy is essential to maintain the stem-cell quiescent state in mice and failure of autophagy leads to senescence by impaired proteostasis, mitochondrial dysfunction and oxidative stress [59]. Based on the understanding of the double-edged role of autophagy in cancer as described above, it is conceivable that different approaches should be used for cancer prevention and cancer therapy: promotion of autophagy for the purpose of cancer prevention and suppression of autophagy for the aim of cancer therapy. In fact, it has been reported that suppression of Atgs restored the efficacy of chemotherapy in lymphoma, lung and breast cancers in preclinical outcomes [60-62]. Moreover, autophagy inhibition reduced the resistance of cancer cells to Bcl2-inhibitors, Tyrosine kinase inhibitor, Bortezomib and conventional radiotherapy [63-66]. In recent years, we have observed significant progresses in development of pharmacological inhibitors of autophagy, with great potential for clinical application in the past several years, to be discussed in details below. 3. Pharmacological Inhibitors of Autophagy

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In recent years, we have observed significant progresses in development of novel and specific autophagy inhibitors, in addition to the classical autophagy inhibitors, such as PI3K inhibitors 3-methyladenine (3-MA), Wortmannin, and LY294002, which work on the early stage to suppress autophagy, as well as chloroquine (CQ)/hydroxyl chloroquine (HCQ) and Bafilomycin A1, which mainly targets the late stage of autophagy via suppression of lysosomal function [67,68]. However, there are two important issues associated these two groups of inhibitors. First, all these inhibitors lack specificity as their molecular targets have broad spectrum of function beyond autophagy. Second, most of these inhibitors, except CQ/HCQ, are not clinically viable, most probably due to high toxicity. Therefore, development of specific autophagy inhibitors with potential clinical application becomes an important mission for the autophagy community. Table 1 summarizes the structure and target sites of various autophagy inhibitors to be discussed in this review. Their proposed sites of action are also illustrated in Figure 1. 3.1 Non-selective PI3K Inhibitors PI3K can be grouped to three categories according to their structural and catalytic variance: Class I, Class II and Class III PI3K (also known as VPS34/PIK3C3). It has been wellestablished that these three classes of PI3Ks play distinct roles in autophagy [69]. Class I PI3K was demonstrated to reduce autophagy whereas Class III PI3K induced autophagy [70]. This opposite observation is attributed to the divergent catalytic roles in autophagy signaling of these two classes. Class I PI3K recruits Akt/PKB in the presence of growth signals through generating PtdIns(3,4,5)P3. Akt serves to activate mTORC1 via tuberous sclerosis complex (TSC), whose inactivation retains RHEB(Ras homolog enriched in brain) in GTP bound state [71]. Conversely, VPS34/PIK3C3, known as the only Class III PI3K in eukaryotic cells, positively regulates autophagy via bridging Beclin1, UVRAG and ATG14 in spite of its capacity of activating mTORC1, as summarized in a recent review [69]. Due to the significant implications of PI3K in autophagy regulation, Class III PI3K comes into focus as a potent drug target of autophagy inhibition. Generally, the PI3K inhibitors can be sorted as pan-PI3K inhibitors, PI3K-mTOR dual inhibitors, Class I PI3K specific and Class III PI3K specific inhibitors. We will limit our discussion to those inhibitors targeting VPS34/PIK3C3 including Wortmannin (PX866), 3-methyladenine (3-MA) and LY294002 as well as some newly developed specific VPS34 inhibitors. Wortmannin is derived from fungal metabolite and covalently inhibits different classes of PI3K, and displays anti-tumor effect when treating alone [72]. 3-MA was first purified from the screening conducted in rat hepatocytes after isolation [73]. Both of them have been extensively studied as autophagy inhibitors. One interesting study from our laboratory elucidated their temporal effects on autophagy modulation. It described that the transient binding to Class III PI3K of 3-MA along with its constitutive effect on different classes of PI3K: 3-MA transiently inhibits class III PI3K and persistently blocks class I PI3K; while Wortmannin has the opposite pattern on these two classes of PI3K [74]. As a result, 3-MA has a dual regulatory effect on autophagy: while 3-MA is capable of inhibiting autophagy under certain context, prolonged treatment of 3-MA under nutrient rich conditions may promote autophagy via suppression of the class I PI3K-AKT-mTORC1 pathway [74] In addition to the lack of specificity of pan-PI3K inhibitors upon different classes of PI3K, both 3-MA and Wortmannin have inhibitory effect on other types of protein kinases such as mitogen-activated protein kinases (MAPK), mTOR and DNA-dependent protein kinase (DNA-PK), and such off-target effects further limit their potential clinical uses [75,76]. Moreover, some of these pan-PI3K inhibitors work in low potency, for instance, the IC50 of 3-MA is ~ 10mM, and that of LY294002 is > 1 µM, indicating that activity of irrelevant 6

kinases are largely disrupted with such concentration [77]. Therefore, development of potent selective PI3K inhibitors holds the limelight in autophagy research. 3.2 Selective VPS34 Inhibitors VPS34 is the Class III PI3K that plays a vital role in autophagy via production of phosphatidylinositol 3-phosphate (PI3P) which is required for autophagosome biogenesis [69]. In recent years, great effort has been paid for identifying specific inhibitors for VPS34 that can be potentially applied to autophagy-targeted cancer therapy. Via high-throughput compounds screening of a series of tetrahydropyrimidopyrimidinone analogs, which were synthesized to selectively suppress VPS34, one compound (Compound 31) has been tested to constitute optimized VPS34 inhibition both in vivo and in vitro [78]. SAR405 is another highly selective VPS34 antagonist which reduces autophagy via preventing vesicle trafficking from late endosomes to lysosomes without disturbing the function of early endosomes [79]. It is exceedingly specific to VPS34 since the IC50 of Class I PI3K was shown to be more than 10000 fold of that of VPS34 (1.2nM), and thus effectively prevents autophagy in a dose-dependent pattern [79]. Two Bisaminopyrimidines molecules (VPS34In1 and PIKIII, Novartis) have been developed as highly selective antagonists for VPS34 through structure-based high-throughput screening [80,81]. PI3P level at endosomes rapidly decreases after administration of VPS34-IN at nanomolar level without compromising activity of other kinases such as Akt and SGK2 in cells [80]. PIKIII is another selective VPS34 inhibitor that is developed by Norvartis with the capacity of binding to a featured hydrophobic pocket in the ATP binding site of VPS34 that is not found in Class I PI3K. The pocket in VPS34 is rather proximal toward a hinge structure that is bridged by a phenylalanine compared with that in Class I PI3K [81]. As a result, this compound displayed suppression of VPS34 with IC50 of ~50nM, that is up to 100 fold higher than that against Class I PI3K [81]. With the availability of the above-mendtioned specific VPS34 inhibitors, it remains to be further studied how these Vps34 inhibitors could be applied to autophagy-related human diseases such as cancer. Much more work is required to explore their anti-tumor effects via animal models and clinical trials. 3.3. Specific ULK1 inhibitors As discussed earlier, ULK1 is able to form a complex with adaptor proteins FIP200, Atg13 and Atg101, and functions as a key autophagy regulator, downstream of mTORC1 in control of autophagy initiation [82]. mTORC1 suppresses ULK1 by direct phosphorylation [82]. Moreover, nutrient deprivation signals activates AMPK which then directly activates ULK1 via phosphorylation [83,84]. Therefore, inhibition of ULK1 becomes a logic strategy in suppression of autophagy. Multiple strategies have been applied to suppress autophagy by targeting ULK1. First, ubiquitinaiton of ULK1 influences autophagic flux. Treatment using small molecule deubiquitinase inhibitor WP1130 reduces autophagy via enhanced ubiquitination of ULK1 followed by its translocation into aggresomes [85]. Similarly, a recent report demonstrated that ULK1 and VPS34 are subject to ubiquitination and proteolysis in the course of autophagy, as a self-termination process [86]. Second, two small molecules, MRT68921 and MRT67307, with capacity of competitive ATP binding, have been reported to specifically reduce ULK1/2 kinase activity, leading to blockage of basal autophagy as indicated by increased LC3-II level [87]. Notably, both of these two compounds require the key methionine 92 in the ATP binding pocket of ULK1, and probably disrupt the interaction between ULK1 and the scaffold proteins [87]. Interestingly, MRT68921 exhibits a more potent role in autophagy inhibition than MRT67307 for it works at a lower concentration as well as has higher selectivity to ULK1 [87]. Third, SBI-0206965 is a recently reported small molecule that are selective for ULK1/2 up to ~700 nM, which 7

blocks Ser249 phosphorylation of VPS34 by ULK1 [88]. It also displays capability of inhibiting autophagy induced by mTOR inhibitor AZD8055 as it relocates the stress induced cytostatic responses into cytotoxic responses in tumor cells [88]. Though SBI-0206965 exhibited to be more effective than chloroquine when synergized with mTOR inhibitors in this study, the high off-target inhibitory effect on other kinases remains a knotty problem in cellular study [77,88]. At present, more work is needed to test the potential cancer therapeutic effects of those specific ULK1 inhibitors in animal models and clinical trials. 3.4 Specific Beclin1 inhibitors Beclin1, the mammalian orthologue of Atg6, is a key autophagy regulator, which forms a complex together with VPS15 and VPS34 (PI3KCIII), downstream of ULK1/2 [89]. It behaves to be both oncogenic and tumor suppressive considering its involvement in multiple cellular processes. Targeting Beclin1 either by antagonizing or agonizing has emerged as an effective tool in modulation of autophagy and as possible anti-tumor therapies. At present, several compounds have been reported to specifically target Beclin1. For instance, 3-MA and Wortmannin were able to inhibit Beclin1 function by dissociating the binding with VPS34 [90,91]. Recently, specific and potent autophagy inhibitor-1 (Spautin-1) was first discovered by an image based compound screening using LC3-GFP as indicator of autophagy [92,93]. Spautin-1, originally named as MBCQ, was found to inhibit a hydrolase of cGMP termed as phosphodiesterase type 5 (PDE5) in 2006 [94]. Subsequently, Liu and Xia et al. presented the novel function of spautin-1 (MBCQ) in selective degradation of Beclin1-VPS34 complex [93]. Briefly, spautin-1 targets two ubiquitin-specific peptidases (USP10 and USP13), both of which mediate deubiquitination of Beclin1 and p53, thus leading to their proteasomal degradation [93]. Nutrient deprived cells are far more sensitive to spautin-1 than normal cells. Moreover, several subsequent studies have examined the potential application of spautin-1 in adjuvant cancer therapy via suppression of autophagy. For instance, Shao et al. showed that Spautin-1 improved the efficacy of Imatinib by activating Akt/GSK3β mediated apoptosis in chronic myeloid lynphoma (CML) [95]. Similar enhanced effect by spautin-1 on mTOR and Akt inhibitors were reported correspondingly in osteosarcoma and epithelial ovarian cancer when they were coordinated with spautin-1 [96,97]. Also, spautin-1 serves to activate nucleotide excision repair (NER) which alleviates cytotoxicity stress-induced by DNA damage via suppression of Xeroderma pigmentosum, complementation group C (XPC) transcription [98]. This observation was validated through combined treatment using spautin-1 and rapamycin in established tumor [98]. In addition to small molecule inhibitors of Beclin1 as discussed above, preclinical studies also demonstrated that siRNA or shRNA-mediated knockdown of Beclin1 would suppress autophagy, augment the therapeutic efficacy of chemotherapy, and restrain invasion and metastasis in osteosarcoma and hepatocellular carcinoma [99,100]. 3.5 Lysosome inhibitors Chloroquine (CQ) was originally developed as an antimalarial drug in 1930s by Bayer Laboratory, but it is recently applied to suppress autophagy. Though the underlying mechanism remains to be fully elucidated, it is believed that CQ serves to reduce lysosomal acidification after its accumulation inside the acidic compartments such as lysosomes [101]. This disrupts the lysosomal functions, leading to cell death [102]. For the last decade, CQ and its derivatives, like HCQ, have been mainly implicated in anticancer therapies for their capacity of inhibiting autophagy which is concurrently induced by other anticancer drugs [103]. CQ/HCQ treatment enhanced radiation therapy as well as temozolomide in glioma 8

cells. [104-106] Similarly, efficacy of mTOR inhibitor, everolimus, and tyrosine kinase inhibitor, gefitinib is enhanced with concurrent treatment of chloroquine compared with single drug administration [107-109]. CQ derivatives such as Lys-01 and Lys-05 also display anti-tumor effects in adapted cells [110,111]. On the other hand, there is evidence indicating autophagy-independent function of CQ. For instance, treating cancer cells with CQ alone displays reduced hypoxia, metastasis and invasion in an autophagy-independent manner [112]. Comparatively, it sensitizes breast cancer cells to chemotherapy, mTOR inhibitors and PI3K inhibitors that bypasses autophagy [113]. Moreover, hedgehog signaling could be another potential target of CQ, whose inhibition helps reducing cancer stem cells (CSCs) in stroma [114]. It is well acknowledged that CQ is a non-selective inhibitor which may affect lysosome-based activity and accumulation of autophagosomes [115]. Although CQ is considered as a safe drug, it should be taken into account that aside from impairing the kidney CQ possibly causes neurodegenerative and cardiovascular diseases if treated with high doses [116,117]. Despite the above-mentioned limitations, at present, CQ/HCQ are the only autophagy inhibitors that are under active clinical trials, either alone or as adjuvant cancer therapeutic agents in clinical trials (to be discussed in details below). In addition to CQ/HCQ, some other agents are developed to suppress autophagy activity by targeting lysosome at the late stage of autophagy. Namely, one bisbenzylisoquinoline alkaloid, Liensinine, is found to blocks the late stage autophagy via inhibiting the recruitment of RAB7A during autophagosome-lysosome fusion [118]. Similarly, antibiotics Elaiophylin and anti-schistome drug Lucanthone have been found to interfere autophagy via suppression of cathespin activity in lysosome [119,120]. Lysosomotropic agents ARN5187 is found to be more cytotoxic than CQ in autophagy inhibition, and is privileged for its additional inhibitory effect on nuclear receptor, REV-Erb beta (REV-ERBB), whose excessive signaling is closely related to tumorigenesis [121,122]. Besides, Oblongifolin C also interferes autophagosome-lysosome fusion via decreasing the expression of cathespins, which is purified from plants [123]. Research work from our laboratory also demonstrated that andrographolide, a diterpenoid lactone isolated from a medicinal herb, Andrographis paniculata , is capable of suppressing autophagy by blocking autophagosome-lysosome fusion and subsequently enhancing the killing effects of cisplatin in human cancer cells [124]. 4. Therapeutic Development and Clinical Trials with Autophagy Inhibitors in Cancer Therapy 4.1 Combinational Therapy with Other Established Cancer Therapeutic Agents Although autophagy inhibitors such as CQ/HCQ have been tested as standalone cancer therapeutics, a more practical and logical approach is to use combinational therapy by combining autophagy inhibitors with established cancer therapeutic agents [18,36,125]. There are several rationales supporting such approaches. First, cancer cells generally have higher level of autophagy which gets them adapted and survived unfavorable metabolic alterations such as hypoxia and acidity [126]. Second, many cancer therapeutic agents are known to induce autophagy via multiple mechanisms, such as oxidative stress and DNA damage, and activated autophagy confers competence of circumventing the attacks from chemodrugs upon tumor cells, thus results in chemoresistance [127]. Fourth, monotherapy using autophagy inhibitors alone performs poorly on limiting tumor progression, along with inconsistent outcomes from clinical results [128]. 9

At present, there is substantial evidence showing the improved therapeutic effects by using such combinational strategies. Wortmannin potentiates efficacy of an anti-mitotic genotoxic agent, docetaxel and cisplatin in multiple cancer cell lines such as lung, prostate, and glioma when corporately delivered in nanoparticles [129,130]. Identically, 3-MA improves the outcomes of various anticancer treatment in preclinical studies [131-134]. As mentioned earlier, spautin-1 dramatically increased the efficacy of Imatinib treatment for CML patients, in addition to apoptosis enhancement induced by mTORC suppression in osteosarcoma [95,97]. SAR405 slowed proliferation of renal carcinoma cells when synergized with everolimus [135]. Liensinine was implicated to enhance the efficacy of chemotherapies such as doxorubicin [118]. CQ/HCQ has been extensively used as in accessory cancer therapy owing to its relative low toxicity and side effects. There are reports describing combined treatment of CQ/HCQ with small molecules such as MEK, Akt or mTOR inhibitors, multikinases inhibitors, Histone deacetylase (HDAC) inhibitors, from which suppression of autophagy restores the sensitivity of cancer cells to these drugs [136140]. More specifically, CQ is able to overcome resistance to alkylating agents such as cisplatin in endometrial cancer cells and in murine lymphoma [141]. Evidence from murine models also indicates that HCQ-mediated autophagy inhibition enhances cytotoxicity of T cells/NK cells when synergized with IL-2, suggesting a promising synergy of targeting autophagy in immunotherapies [50], as well as that adjuvant CQ overcame acquired resistance to gefitinib, cyclophosphamide, vorinostat, and saracatinib by enhanced apoptosis [109,142,143]. Moreover, to overcome the potential side effects caused by combination of CQ administration and chemotherapy, substituting CQ/HCQ with verteporfin which treats occluded vessels has been reported to block autophagosome formation without worsening kidney injury that coordinately induced by alkylating agents and CQ/HCQ [144]. 4.2 Outcomes of Clinical Trials Using CQ/HCQ As early as in 2006, one clinical trial has been reported and demonstrated that chloroquine (CQ) increased overall survival for 24 months among glioblastoma patients, comparing to 11 months in patients synergized with placebo [145]. In Phase I trials, it has been warranted that combination of CQ with anticancer drugs such as mTOR inhibitor temsirolimus and alkylating agents temozolomide are well tolerated when dose of HCQ escalates up to 1200 mg/day [146]. CQ/HCQ also enhanced the efficacy of both drugs in treating advanced solid tumors and melanoma [146,147]. Besides, CQ/HCQ improved the outcomes of proteasome inhibitor Bortezomib in myeloma as well as that of HDAC inhibitor vorinostat in treating patients with renal carcinoma and colorectal cancer [136,148,149]. Despite CQ/HCQ may enhance efficacy of anticancer drugs in these published trial reports, some trials compromised that dose-limiting toxicity stays to the primary concern of HCQ adjuvant for radiation therapy, together with temozolomide in a Phase II study [105,128]. A Phase I trial concluded that HCQ as safe and promising in combined administration with anti-tumor drugs bevacizumab, followed by an ongoing Phase II trial in metastasis colorectal cancer treatment (NCT01206530) [150]. Conversely, administration of CQ alone had only negligible therapeutic efficacy [151]. Table 2 summarizes all the clinical trials included in this review. In general, outcomes from the clinical trials support that autophagy inhibition CQ/HCQ is effective in combinational therapy with other established cancer therapeutic agents. Moreover, active clinical trials are ongoing to test the clinical efficacy of autophagy inhibitors (mainly CQ/HCQ) as cancer therapeutics, as summarized in Table 3. Up to 17 clinical trials using CQ/HCQ as adjuvant in cancer therapy are ongoing currently (http://www.cancer.gov/about-cancer/treatment/clinical-trials) as summarized in Table 3. One single blind phase I trial (NCT01897116) meant to examine whether CQ/HCQ 10

mediated autophagy downregulation will extend the duration of responses to Vemurafenib among patients with advanced BRAF melanoma. Another phase II trial was launched to test the efficacy of combined therapy using HCQ, tubulin inhibitor paclitaxel, platinum derivatives carboplatin and angiogenesis inhibitor bevacizumab in treating Non-Small-Cell Lung Cancer (NSCLC). Nevertheless, none of these trials have reached Phase III, implying that inhibiting autophagy remains a rudimentary approach, and lots of work yet to be done in this field. As for synthesized novel selective autophagy inhibitors such as spautin-1, they are still mostly in the preclinical stage and much more efforts are needed for move these inhibitors into clinical trials as cancer therapeutic agents. 5. Conclusions and Future Directions As a highly conserved biological process, autophagy displays a dual function in cancer: suppression of cancer initiation at early stage and promotion of cancer progression at late stage. Accordingly, it is generally accepted inhibiting autophagy is a logic approach in cancer therapy, and autophagy inhibitors can be used as either standalone therapeutic agents or more practically as potential adjuvant therapy in combination with other established therapeutic agents. One key challenge is that conventional approaches to suppress autophagy are mostly non-selective, making it difficult to interpret the preclinical results from the complex autophagy network as it is intertwined with PI3K-Akt-mTOR, AMPK, and lysosome signaling. It necessitates the growing interest in developing specific inhibitors for autophagy. Up to date, we have observed significant progresses in developing such inhibitors by targeting specific autophagy regulators, such as ULK1, VPS34 and Beclin1, via high throughput chemical screening and specific structure biology-based selection [78,81,87,135]. Clearly, the remaining task is to test the clinical viability of these small molecule inhibitors via systematic preclinical investigations to support their potential applications in clinical trials of cancer therapy. At present, CQ and HCQ are the only autophagy inhibitors that currently undergo phase I and II clinical trials in which they are utilized together with a variety of other anti-cancer drugs. We expect that development of autophagy inhibitors arisen from the basic research of autophagy and understanding of the molecular mechanisms of the autophagic process will lead to real breakthrough in treatment of human diseases including cancer in the short years to come. Acknowledgements: Wang C is supported by a NUS research scholarship. The work in Shen HM’s lab is supported by research grants from Singapore National Medical Research Council (NMRC/CIRG/1346/2012 and NMRC/CIRG/1373/2013). The work in Hu Q’s lab is supported by research grants from NUS start-up fund, Singapore Ministry of Education (AcRF T1-2014 Apr-01) and Singapore National Medical Research Council (NMRC/BNIG/2028/2015).

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Figure legend: Figure 1: The autophagic process controlled under key ATG protein complexes and sites of action by various pharmacological autophagy inhibitors

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Table 1. Overview of Autophagy Inhibitors Name of Agents

Structure

Sites of Targets in Autophagy Signaling

Class I and Class III PI3K

3-methyladenine

Wortmannin

Class I and Class III PI3K

LY294002

Class I and Class III PI3K

MRT68921

ULK1 Complex

MRT67307

ULK1 Complex

SBI-0206965

ULK1 Complex

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Spautin-1

Beclin1-VPS34 Complex

SAR 405

VPS34

Vps34-In1

VPS34

PIK-III

VPS34

Compound 31

VPS34

Chloroquine

Lysosome

Hydrochloroquine

Lysosome

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V-ATPase, blocks lysosome fusion

Bafilomycin A

Lys05

Lysosome

Lucanthone

Attenuates lysosomal cathepsins activity

Elaiophylin

Attenuates lysosomal cathepsins activity

Liensinine

Blocks autophagosome formation

ARN5187

Blocks final maturation of autophagolysosome

Oblongifolin C

Raises lysosomal PH and attenuates lysosomal cathepsins activity

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Table 2. Summary of Reported Clinical Outcomes of CQ/HCQ Treatment in Cancer Therapy

Ref

Therapeutic Agents

Cancer Types

CQ (150 mg/d) combined with conventional [145] radiotherapy and chemotherapy for 12 months

Glioblastoma

Temsirolimus (25 [146] mg/week) and HCQ (1200 mg/d)

Advanced Solid Tumors

HCQ (200-800 mg/d) combined with radiotherapy [128] and temozolomide (50-100 mg/m2)

Glioblastoma

HCQ (200-1200 mg/d) and intense Advanced solid [147] temozolomide (150 tumors and 2 mg/m ) for 4 melanoma weeks Colorectal, HDAC inhibitor breast, nonvorinostat (400 [136] small-cell lung, mg/d) and CQ (600 ovarian and mg/d) renal cancer

Stages of Trials

N/A

Phase I

Phase I

Phase I

Relapsed/refract ory myeloma

HCQ (800-1200 [151] mg/d)

Metastatic pancreatic Phase II adenocarcinoma Metastic colorectal cancer

Outcomes

15 per set

Increased median survival by 13 months

26

3.5 months of median progression-free survival

15 for Phase Median Overall PhaseI/I I Survival of ~16 I 72 for Phase months II

Bortezomib (1.3 [149] mg/m2) and HCQ (1200 mg/d)

mFOLFOX6/bevac izumab (5 mg/kg) [150] and HCQ (1200 mg/d)

No. of Cases

Phase I

Phase I

25

37

The highest dose of 1200mg/d is well tolerated.

24

The highest dose of 800mg/d is well tolerated.

25

No dose-limiting toxicity was observed, and 1200mg/d of HCQ is well tolerated

20

Negligible therapeutic efficacy

23

The hightest dose of 1200mg/d is well tolerated

Table 3. Currently Approved Clinical Trials Using Adjuvant CQ/HCQ as Cancer Therapies Trial ID

Phase for Trial

Cancer Type

Dose & Purpose

Other Therapeutic Agents

Year of Start

NCT01206530

I, II

Colorectal cancer

HCQ (600800mg/day) Side effect and best dose to be given

Fluorouracil Leucovorin Calcium Oxaliplatin, Bevacizumab

2010

NCT01506973

I, II

Pancreatic cancer

HCQ (800 and 1200 Gemcitabine mg/day) Abraxane Toxicity and one year survival rate

NCT01510119

I, II

Kidney cancer

HCQ Rate of progression in 6 months and maximum tolerated dose

Rad001(everoli 2011 mus)

NCT01550367

I, II

Metastatic renal cell carcinoma

HCQ (600-1200 mg/day) Safety and toxicity

IL-2 (aldesleukin)

2012

NCT02013778

I, II

Liver cancer and Hepatocellular carcinoma

HCQ Maximum tolerated dose and complete response rate

Transarterial Chemoemboliz ation (TACE)

2013

NCT01006369

II

Metastatic Colorectal Cancer

HCQ (200mg/day) Partial response and overall survival

XELOXBevacizumab

2009

NCT01828476

II

Metastatic Castrate Refractory Prostate Cancer

Response with/without HCQ treatment

Navitoclax and Abiraterone acetate

2013

NCT01494155

II

Pancreatic Cancer

HCQ (400mg BID) Progression-free survival

Proton beam radiation therapy and Capecitabine

2011

NCT01649947

II

Recurrent Non-Small Cell Lung Cancer

CQ (200mg BID) Response to therapy

Bevacizumab, Carboplatin, Paclitaxel

2011

NCT01978184

II

Pancreatic Cancer

HCQ (1200mg/day) Antitumor Efficacy

Gemcitabine, Abraxane

2013

26

2011

NCT02316340

II

Refractory Metastatic Colorectal Cancer

HCQ (600mg/day) Vorinostat Clinical efficacy with progression free survival

2015

NCT01023737

I

Malignant Solid Tumour

HCQ (4001000mg/day) Maximum tolerated dose and safety

Vorinostat

2009

NCT01266057

I

Advanced Cancers

HCQ (200mg/day) Maximum tolerated dose and toxicity

Vorinostat, Sirolimus

2011

NCT01480154

I

Advanced HCQ Solid Tumors, Maximum tolerated Melanoma, dose Prostate or Kidney Cancer

Akt inhibitor MK2206

2011

NCT01897116

I

Stage IV Melanoma

Vemurafenib

2013

HCQ Maximum tolerated dose

＊Source: National Cancer Institute (USA) http://www.cancer.gov/clinicaltrials/

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