Oncogene (2015), 1–11 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc

REVIEW

Emerging strategies to effectively target autophagy in cancer VW Rebecca and RK Amaravadi Autophagy serves a dichotomous role in cancer and recent advances have helped delineate the appropriate settings where inhibiting or promoting autophagy may confer therapeutic efficacy in patients. Our evolving understanding of the molecular machinery responsible for the tightly controlled regulation of this homeostatic mechanism has begun to bear fruit in the way of autophagy-oriented clinical trials and promising lead compounds to modulate autophagy for therapeutic benefit. In this manuscript we review the recent preclinical and clinical therapeutic strategies that involve autophagy modulation in cancer. Oncogene advance online publication, 20 April 2015; doi:10.1038/onc.2015.99

INTRODUCTION The impact autophagy has on human health and disease are far and wide, with reports demonstrating important functions in bacterial1 and viral infections,2 suppression of inflammation,3 adaptive immune responses4 and immunosurveillance,5 neurodegeneration,6 heart disease7 and cancer.8 Aberrant autophagic activity is an emerging hallmark of cancer,9 serving a critical function in the pathogenesis, survival and response to therapy in a growing number of cancers. In general, autophagy provides the means by which cells mitigate metabolic and therapeutic stresses, remove waste and manage toxic byproducts of anabolism and catabolism, such as reactive oxygen species.10 The role autophagy serves specifically in cancer has been controversial, with some reports indicating autophagy suppresses tumor development, whereas other reports providing evidence that autophagy promotes the growth of established tumors.11 The overarching question is whether or not autophagy can be effectively modulated to impair cancer initiation or progression. Recent advances in the fundamental understanding of the contextdependent consequences of autophagy defects in the setting of activated oncogenes will likely pave the way for new strategies to either induce or impair autophagy therapeutically. Meanwhile, the first deliberate attempt to modulate autophagy therapeutically has been accomplished through the publication of the first seven clinical trials involving hydroxychloroquine (HCQ) in cancer patients.12–18 Lessons learned from these clinical trials have raised new questions that can be answered in the laboratory. Finally, a deeper understanding of how autophagy is regulated at the genetic, epigenetic and posttranslational level, and how autophagy can regulate itself and be regulated by drugs, extracellular components and metabolites, may point to new therapeutic targets that can directly or indirectly modulate autophagy. Here we discuss the latest developments in the field’s understanding of autophagy in cancer and novel strategies to effectively modulate autophagic activity. AUTOPHAGY FORM AND FUNCTION The dissection of the autophagy pathway was first described in yeast19 where it clearly serves as an intracellular, self-preservation

mechanism providing internal nutrients to cells in times of stress.20 Although autophagy is evolutionarily conserved across organisms, its role in multicellular organisms is more nuanced than it is in yeast. Recent evidence indicates autophagic flux is not only dependent on the expression of the canonical autophagy machinery, but through genetic, epigenetic, metabolic, posttranslational and extracellular regulation of this machinery. This complex regulation of autophagy may enable its multiple roles in cancer. Autophagic flux occurs at a basal rate in all eukaryotic cells to maintain equilibrium through the recycling of nonessential components within the cell.8 Under challenging conditions such as nutrient deprivation,21 hypoxia22 or targeted therapy,23 autophagic flux can be increased via multiple stimuli to elicit homeostatic regulation over critical metabolic building blocks including amino acids, nucleic acids and monosaccharides necessary for cell survival (Figure 1). Multiple forms of autophagy exist in mammalian cells, each with well-characterized mechanisms that differ in the way material destined for degradation is sequestered and transported to the lysosome (micro, chaperone mediated and macroautophagy).24 Macroautophagy represents the most multifunctional and best-described form of autophagy, comprising a complex, tightly regulated process where doublemembrane autophagic vesicles (termed autophagosomes) are generated. Autophagosomes function by sequestering damaged or misfolded proteins, engulfing mitochondria (termed mitophagy) and internalizing endoplasmic reticulum (ER; amongst other cytoplasmic components) through the aid of cargo adaptor proteins before ultimately fusing to the lysosome for degradation and recycling of internal contents to sustain cellular viability.25,26 Autophagy can be characterized as canonical or non-canonical, depending upon the molecular machinery involved in the biogenesis of autophagosomes. Canonical autophagy is regulated by a number of autophagy-related (ATG) proteins and non-ATG proteins (such as class III phosphatidylinositol 3-kinase (PI3KIII), p150 and (activating molecule in Beclin-1-regulated autophagy) Ambra1) that choreograph the initiation, elongation, maturation and fusion stages of the pathway.27 Non-canonical autophagy is not as well understood, where autophagosomes can be created independently of Atg5 or Atg7.28 Recently, ferritin clusters have been reported to accumulate at the site of autophagosome

The Department of Medicine and Abramson Cancer Center; University of Pennsylvania School of Medicine, Philadelphia, PA USA. Correspondence: Dr RK Amaravadi, The Department of Medicine and Abramson Cancer Center, University of Pennsylvania School of Medicine, 16 Penn Tower, 3400 Spruce Street, Philadelphia, PA 19063, USA. E-mail: [email protected] Received 31 December 2014; revised 18 February 2015; accepted 18 February 2015

Autophagy inhibitor therapy VW Rebecca and RK Amaravadi

2 extracellular intracellular

Acetylation

p150

ULK1/2 ATG101

Autophagy

mATG13 FIP200

Autophagy genes Foxk proteins TFEB

Me Ac Tf

p53 ATF4

LC3 ATG4 ATG5 ATG7 ATG12 ATG16

P62 Nix NBr

e Amino Acids Ammonia Glucose Lipids

Recycling

d • • • •

Histone Methylation Demethylation Acetylation Deacetylation

Epigenetic

c

Transcriptional

Initiation

Ubiquitination

Vps34 ATG14L

Cargo Adaptors

Phosphorylation

Beclin 1

Lysosomal degradation

Nucleation

a

Metabolic

PostTranslational

b

Maturation

Nutrient /Energy Sensors AMPK mTOR

XBP1

Nucleus

Potentially druggable targets

Figure 1. Autophagy regulators and points of intervention. (a) Autophagy occurs through a multistep process that includes four control points: initiation, nucleation, maturation, and lysosomal fusion and degradation of autophagosome contents. Successful autophagy results in the recycling of nutrients into the cytoplasm. (b–e) Autophagy is regulated on multiple levels with four major classes of regulation including posttranslational, transcriptional, epigenetic and metabolic regulation. Potential druggable targets are depicted (red star) with a promise to better modulate autophagy than strategies currently being implored.

formation along with p62 in cells lacking Atg5, possibly shedding insight regarding non-canonical autophagosome biogenesis dynamics.29 The classical and perhaps best-characterized environmental-mediated regulation of canonical autophagy occurs via the growth factor/receptor tyrosine kinase/ phosphoinositide 3-kinase (PI3K)/ protein kinase B (also known as AKT)/ mechanistic target of rapamycin complex 1 (mTORC1) signaling axis, which directly controls autophagic activity through the phosphorylation and inhibition of Unc-51-like kinase 1 (ULK1), part of the first protein complex involved in autophagic vesicle formation.30 Under conditions in which growth factors and nutrients such as amino acids are rich in the extracellular space, the PI3K/AKT/mTORC1 pathway is highly active and mTORC1 inhibits ULK1 through the phosphorylation at its serine-757 residue.31 However, when growth factors become limited, mTORC1 becomes inactive and can no longer repress the complex consisting of ULK1, focal adhesion kinase family-interacting protein 200 kDa, ATG13 and ATG101, which favors the initiation of autophagy (the first phase of autophagy).32 AMP-activated protein kinase, in response to either glucose starvation or amino-acid deprivation, can also regulate ULK1 activity via fine-tuning of the phosphorylation status of ULK1.33 Once activated, ULK1 forms a complex with Beclin-1 via assistance from TRIM5α, acting as a protein platform, leading to the phosphorylation and activation of Beclin-1.34 Once active, Beclin-1 activates the class III PI3K vacuolar sorting protein 34 (Vps34), a component necessary both for endocytic sorting and in the ability of cells to respond to fluctuations in nutrients such as amino acids and insulin. Vps34 activity has also been demonstrated to not be inhibited by the TORC1 inhibitor rapamycin, suggesting that Vps34 can also function upstream of mTOR, serving as a vehicle for mTOR to monitor the levels of a wider net of critical nutrients for cell survival.35 Following Vps34 activation, autophagy cytoplasmic machinery is recruited onto the phospholipid membranes derived from various sources including the Oncogene (2015) 1 – 11

endoplasmic reticulum,36 plasma membrane,37 mitochondria38 and Golgi apparatus.39 The second phase of autophagy (nucleation) marks the beginning of autophagosome formation with the nucleation of membranes by Beclin-Vps34 and either ATG14L, Rubicon, Ambra, among other proteins. The third phase (elongation and maturation) allows for the maturation of autophagosomes and requires a ubiquitin ligase-like ATG5-ATG12-ATG16L complex (formed with the aid of ATG7 and ATG10).40 ATG4 can also contribute to the elongation phase, and has recently been implicated as a biomarker and potential therapeutic target for chronic myeloid leukemia stem/progenitor cells (Figure 1).41 The ubiquitin-like protein LC3/Atg8 is subsequently conjugated to the lipid phosphatidylethanolamine on the surface of autophagosome membranes. Once integrated in the lipid bilayer, LC3 interacts with adaptor proteins (autophagy receptors) such as p62, Nbr1, TRIM5α and NIX, which recruit cargo from the cytoplasm and promote autophagosome closure.34,42 Proteomic network analysis in cells undergoing autophagy reveal high connectivity between LC3/Atg8 and upstream autophagy components such as ULK1, Vps34 and ATG2A, suggesting that LC3/Atg8 may serve a more significant role in regulating autophagosome formation than was previously appreciated.43 Once autophagosomes have engulfed cargo and closed, they are ultimately trafficked and fused to lysosomes forming autophagolysosomes. This fusion allows for the pH-dependent degradation of cytosolic cargo via hydrolases located within the acidic environment of the autophagolysosome.44 Lysosomal permeases such as spinster permit the release of degradation products ranging from sugars, amino acids and nucleic acids into the cytosol for reuse by the cell45 (Figure 1). Our growing understanding of how autophagy is regulated has shed light on the potential novel druggable components for autophagy inhibition, which will be discussed later (see Figure 1 and below). © 2015 Macmillan Publishers Limited

Autophagy inhibitor therapy VW Rebecca and RK Amaravadi

MOUSE MODELS ADDRESS THE ROLE OF AUTOPHAGY IN TUMOR INITIATION AND MAINTENANCE A major breakthrough in understanding the role of autophagy in tumorigenesis was made when spontaneous lung and liver tumors were found to arise in Beclin-1 +/ − mice.46 Monoallelic deletion of the human homolog of Beclin-1 (BECN1) was initially reported to occur in 40–75% of cases of human sporadic ovarian, breast and prostate cancer.47 Taken together these results established BECN1 as the first autophagy-associated tumor suppressor gene.47,48 However, the proximity of BECN1 to the ovarian and breast tumor suppressor gene BRCA1 on chromosome 17q21 has decreased the certainty of Beclin-1’s role as a bona fide tumor suppressor gene. A recent report demonstrated Beclin-1 allele loss to be a rare event, assessed in human prostate, breast and ovarian tumor sequencing data from The Cancer Genome Atlas and other databases, except in the setting of loss of neighboring gene BRCA1.49 Further, a larger panel of cancers was analyzed with no evidence for BECN1 mutation or loss, leaving the function of BECN1 as a tumor suppressor in human cancer unclear. Adding more complexity to the role Beclin-1 serves in malignancy is a report showing Beclin-1 to share regulation with p53 at the level of proteasomal degradation in an ubiquitin-dependent manner; therefore suggesting that the spontaneous malignancy in Beclin-1 +/ − experimental systems may be due to lower p53 levels.50 Along a similar vein, Beclin-1 and the antiapoptotic Bcl-2 family member myeloid cell leukemia (Mcl-1) protein are both stabilized by binding to the deubiquitinase USP9X (ubiquitin-specific peptidase 9 X-linked), and negatively modulate the expression of each other through competitive displacement of USP9X.51 Beclin-1 expression levels were discovered to decrease in patient-derived melanoma tissues as Mcl-1 levels increased in a significant interdependent manner, independent of autophagy.51 Though Beclin-1 has recently been demonstrated to have a role in the response of lung cancer to epidermal growth factor receptor inhibition,52 further experimental validation is needed to determine the practical consequences of BECN1 heterozygosity in human tumors and to delineate whether the observations involving Beclin-1 are indeed dependent on the role autophagy serves in each of these experimental systems, or rather due to the confounding implications BRCA1, p53 and Mcl-1 each provide on cancer cell viability and disease progression. Beyond Beclin-1, mouse models with mosaic deletion of Atg5 and liver-specific deletion of Atg7 also resulted in a greater incidence of spontaneous liver adenomas; however, the tumors were benign suggesting autophagy may be necessary for the progression beyond the benign state.53 Deletion of Fip200 also prevented the development of breast cancer.54,55 Numerous mouse models have demonstrated autophagy to serve a critical capacity in disease progression in established oncogene-driven tumors, where inhibition of autophagy results in a reduction in

Table 1.

3 tumor volume in established tumors. In a mouse xenograft model utilizing immortalized baby mouse kidney epithelial cell lines engineered to express constitutive activity of RAS (H-rasV12) while also possessing defects in apoptotic machinery (Bax/Bak-deficient), autophagy was found to support survival of cancer cells undergoing metabolic stress and was localized to the poorly vascularized, hypoxic cores of tumors.56 Further, cell lines engineered with constitutive activity of AKT (myr-AKT) along with apoptotic defects displayed high levels of necrosis, mechanistically due to the coordinate inhibition of apoptosis (via Bax/Bak deficiency) and autophagy (inhibited by AKT activity). Although these data were critical, what were sorely needed were genetically engineered mouse models of oncogene-driven cancers with and without defects in autophagy genes. These models have emerged recently (Table 1) and reveal a theme where the majority of mice with defects in key autophagy machinery display accelerated the development of benign tumors, however, autophagy appears to be essential for the progression of benign tumors to a more malignant state. Once a tumor is established, autophagy has been clearly demonstrated to also have a role in promoting the survival of existing tumor cells within the tumor microenvironment.57 Two models of spontaneous Kras-driven lung cancer, one with tumor cell deletion of Atg758 and one with tumor cell deletion of Atg5,59 explored the importance of autophagy in the context of Ras oncogenes (Table 1). In the KrasG12D/Atg7fl/fl model, the deletion of Atg7 resulted in a significant reduction in tumor burden and an increase in tumor lipid accumulation; however, no difference in the overall survival could be noted due to an increase in death by inflammation in mice with Atg7-deficient tumors.58 In the KrasG12D/Atg5fl/fl model, the deletion of Atg5 resulted in increased tumor initiation; however, tumor cells exhibited decreased mitochondrial bioenergetics, and the deletion of Atg5 also enhanced survival of mice.59 Each of these mouse models revealed autophagy to be necessary for cancer cell proliferation and progression of lung tumors from adenomas to carcinomas. These findings strengthen the concept that Ras-driven cancers rely on autophagy for sustained metabolism and growth. A mouse model with Cre-activatable BRAF (BrafV600E) driven lung cancer, with and without the conditional knockout of Atg7 was generated to determine the role of autophagy in BRAF-driven lung cancers. Autophagy was required for the growth of established BrafV600Edriven lung cancers via the preservation of mitochondrial function and the supply of metabolic substrates critical for sustained tumorigenesis.60 Atg7-deficient mice experienced increased early tumorigenesis in an oxidative stress-dependent manner compared with mice with intact Atg7; however, as in the Kras-driven lung cancer model, Atg7 deletion converted BrafV600E-driven adenomas to tumors that had the histological appearance of benign oncocytomas rather than carcinomas.60

Mouse models testing the effects of tumor-specific autophagy deficiency in cancer

Genotype

Cancer type

Atg knockout

MMTV-PyMT Breast FIP200 lox-stop-lox-KrasG12D; Tp53flox/flox Lung Atg7 lox-stop-lox-KrasG12D Lung Atg5 V600E flox/flox Braf ; Tp53 Lung Atg7 G12D frt/frt frt-stop-frt-Kras ; Tp53 Lung Atg7 lox-stop-lox-KrasG12D; Tp53flox/flox; Pdx-cre Pancreas Atg5 or Atg7 lox-stop-lox-KrasG12D; Pdx-cre Pancreas Atg5 or Atg7 lox-stop-lox-KrasG12D; Tp53flox/+; Pdx-cre Pancreas Atg5

Tumor initiationa

Tumor progressiona

Decreased ND Increased Increased Increased Increased Increased Increased

Decreased Decreased Decreased Decreased Decreased Increased Decreased Decreased

Mouse survival following References post-tumor autophagy inhibition Increased No difference Increased Increased ND Decreased Decreased Increased

Wei et al.55 Guo et al.58 Rao et al.59 Strohecker et al.60 Karsli-Uzunbas et al.63 Rosenfeldt et al.64 Rosenfeldt et al.64 Iacobuzio-Donhue et al.68

Abbreviation: ND, not determined. aFollowing genetic autophagy inhibition

© 2015 Macmillan Publishers Limited

Oncogene (2015) 1 – 11

Autophagy inhibitor therapy VW Rebecca and RK Amaravadi

4 In mouse models of pancreatic cancer, autophagy was discovered to be vital and essential for tumorigenic growth of pancreatic cancers de novo.61 Pancreatic ductal adenocarcinoma (PDAC) cell lines and primary tumor possess constitutively activated autophagy (as seen by GFP-LC3 puncta and cleaved LC3-A IHC (LC3-II)) and a unique dependence upon autophagy. Importantly, the genetic (suppression of ATG5 expression by shRNAs) or chemical inhibition (chloroquine) of autophagy leads to robust tumor regression and prolonged survival in pancreatic cancer xenografts and genetic mouse models.61 KRAS mutations are one of the known drivers in PDAC, and a recent report leveraging an inducible mouse model of mutated Kras (KrasG12D) in a p53Lox/WT background shed further light on the role autophagy serves in pancreatic cancer. In a temporal and pancreas-specific manner, the authors ablated KRAS activity, which resulted in pancreatic tumor regression within 2–3 weeks followed by relapse a few months thereafter. The cancer cells surviving KRAS ablation were studied with transcriptome analysis and gene set enrichment analysis revealing a significant enrichment of genes involved in lysosomal activity, mitochondrial electron transport chain and autophagy, among other cellular processes.62 Although the genetically engineered mouse models described above were incredibly useful in shedding light on the effects of autophagy defects on the tumorigenesis of oncogene-driven cancer, they did not effectively model the therapeutic ablation of autophagy. With cancer therapy, drugs will typically impact the pathway throughout the body and are often administered only after the tumor becomes apparent (stage IV) or in a high-risk (stage III) setting. Although tumor xenografts address this to some degree, those models are artificial because mice lack immune systems and the tumor is typically grown out of context in the flanks of the mice. To address all of these concerns, a genetically engineered mouse model of an inducible Kras-driven lung cancer was generated where Atg7 could be systemically deleted in a conditional manner. When systemic Atg7 deletion was engaged in adult mice, mice initially were asymptomatic, but eventually died of neurodegeneration at roughly 3 months.63 However, when Atg7 was systemically ablated in mice before the induction of Krasdriven lung cancer, the rate of lung nodules appeared to increase, but the nodules failed to progress to cancer before the mice succumbed to the effects of systemic Atg7 depletion. When Atg7 was systemically deleted in mice after Kras-driven tumors were allowed to form, massive tumor regression and apoptosis was observed before the toxicity of Atg7 depletion on normal tissue was evident. These observations are valuable as they reveal that chronic autophagy inhibition may yield toxicities, supporting the exploration of optimal treatment regimens that minimize exposure to autophagy inhibitors while still maximizing the antitumor benefit conferred from autophagy inhibition. In general, mouse models show that autophagy is critical in the transition from premalignant to malignant, however, autophagy promotes growth of established tumors. These recent results partially reconcile the dichotomy of autophagy in tumorigenesis, and support a role for the inhibition of autophagy as a therapeutic strategy in certain advanced cancers. There was an exception reported, where a model of pancreas-specific Kras-mutant, Trp53−/− tumors was treated with autophagy inhibition with either genetic ablation of Atg5 or Atg7, or chemically with HCQ, resulting in the promotion of tumorigenesis (Table 1).64 From both the strategies, autophagy inhibition was found to accelerate the formation of PDAC in mice due to enhanced glucose uptake and enrichment of anabolic pathways.65 A wrinkle in this model is its use of an embryonic pancreas-specific homozygous deletion of Trp53 in the context of Kras mutation, which results in advanced cancers in early development. In nature, p53 is most frequently found as missense mutations in Kras-mutant pancreatic cancers.66 The heterozygous expression of mutant Trp53 in the context of Oncogene (2015) 1 – 11

oncogenic Kras is postulated to give rise to precancerous lesions called pancreatic intraepithelial neoplasias, with the subsequent loss of heterozygosity of the wild-type TP53 allele driving the progression from pancreatic intraepithelial neoplasias to PDAC.65 Thus, the model64 utilizing homozygous deletion of Trp53 did not fully recapitulate the step-wise progression of pancreas cancer as is found in humans. To address this important issue, a pancreasspecific Kras-mutant Trp53+/ − mouse model was generated that experiences loss of heterozygosity of the wild-type Trp53 allele during PDAC progression, therefore mirroring the step-wise development of human pancreas cancer.67 Within this model with Trp53 (loss of heterozygosity), autophagy inhibition via ablation of Atg5 or with HCQ was found to increase the overall survival in a mouse preclinical trial leveraging cohorts of genetically characterized, patient-derived xenografts. Trp53 status was not found to correlate with the response in tumor cell lines or patient-derived xenograft models, and although autophagy inhibition in the pancreas lead to an increase in tumor initiation, few of these premalignant lesions could develop into invasive tumors and the mice treated with autophagy inhibition lived longer overall.67 These findings are of the upmost importance, as conclusions drawn from the Trp53 model that did not recapitulate human pancreas cancer development64 lead to premature recommendations that patients with Trp53 mutations should not receive treatment with HCQ.68 Due to the high profile of the Journal in which this opinion piece was published, it is possible that patients who may have benefited from clinical trials utilizing HCQ may have been directed to other therapies by their physicians. Insight from these studies will also help design therapy regimens, where exposure to autophagy inhibitors will be strategically timed to allow for optimal therapeutic benefit in the absence of potential hazards from the chronic inhibition of autophagy. It appears that in most cases autophagy defects lead to accelerated tumor initiation, but impaired tumor maintenance. It is for these reasons why much effort in developing therapeutics targeting autophagy is focused on advanced cancers where concerns about developing secondary benign tumors will be less problematic if the advanced cancer that is putting the patient’s life immediately at risk can be halted or regressed. A deeper understanding of how autophagy is regulated on multiple levels could unravel the switch that turns autophagy from a tumor suppressor to a tumor promoter. CANCER THERAPY CAN PRODUCE AUTOPHAGIC/ IMMUNOGENIC CELL DEATH: THE ARGUMENT TO INDUCE AUTOPHAGY Observations that therapy-induced autophagy can have a role in tumor cell cytotoxicity have been reported; however, they commonly depend upon pre-existing defective apoptotic machinery in order for the autophagic cell death to manifest. Bcl-2 homology 3 mimetics such as gossypol have been demonstrated to elicit autophagic cell death in apoptosis-deficient malignant glioma and prostate cancer, by way of disrupting physical interactions between Bcl-2 family members and Beclin-1.69 Autophagic cell death refers to cell death that is accompanied by extensive cytoplasmic vacuolization, often correlated to increased autophagic flux.70 The use of the term autophagic cell death is controversial, as since its conception the phrase is commonly misused to suggest that autophagy actively contributes to cell death. Although autophagy frequently occurs concurrently with regulated cell death, autophagy is directly responsible for the death of tumor cells in only a few cases.71 To date, there have been no deliberate attempts to induce autophagy specifically in a cancer model. Autophagy appears to be responsible for the death of some cancer cells with defective apoptotic machinery, such as inhibited caspase-8, in an ATG7 and © 2015 Macmillan Publishers Limited

Autophagy inhibitor therapy VW Rebecca and RK Amaravadi

Beclin-1-dependent manner in vitro.72 Another study reported reexpression of (ARHI) aplasia Ras homolog I in human ovarian cancer cell lines resulted in autophagic cell death in vitro.73 However, in vivo autophagy enabled these cells to remain dormant in the context of ARHI re-expression, with chloroquine treatment markedly reducing the regrowth of xenografts. Similar results were also observed in vitro when cells were cultured with factors found in vivo such as IGF-I, M-CSF and IL-8, suggesting autophagy serves a protective role when experimental conditions recapitulate those found within the tumor microenvironment. A recent consensus statement on cell death nomenclature warned about the fact that regulated cell death mechanisms frequently interact with each other and it may be that in many cases persistent autophagy can activate other forms of cell death that are actually responsible for the death that ensues.71 There may exist multiple checkpoints that limit autophagic cell death from occurring in vivo, such as growth factor availability and functional apoptotic machinery. Interestingly, autophagy has also been reported to serve a role in the recruitment of immune system effectors. Chemotherapy in autophagy-competent cancers recruited dendritic cells and T lymphocytes to the tumor bed in an ATP-dependent fashion.74 Inhibiting autophagy suppressed the release of ATP and attenuated the recruitment of immune cells. Similar results were observed in melanoma where chemotherapy75 or radiotherapy76 each led to an increase in mannose-6-phosphate receptor on the tumor cell surface, making tumor cells more susceptible to lysis by cytotoxic T cells, in an autophagy-dependent manner. The implications these findings hold in regard to the clinical utilization of autophagy inhibitors moving forward remain to be determined. A potential combination of an immune checkpoint inhibitor, such as anti-PD-1 antibody,77 with an autophagy inhibitor can be envisioned to ensure potential secondary effects on the immune response to cancer cells do not blunt the antitumor effect of autophagy inhibition. CANCER THERAPY CAN PRODUCE CYTOPROTECTIVE AUTOPHAGY: THE ARGUMENT TO INHIBIT AUTOPHAGY Autophagy was convincingly shown as a key survival mechanism in apoptosis-defective transformed cells subjected to growth factor withdrawal. Cells that survived growth factor withdrawal or other modes of starvation could be killed when autophagy was inhibited with either 3-methyladenine or CQ, and the autophagic phenotype was reversible once growth factors were replenished.21 Utilizing a Myc-induced model of lymphoma, the role of autophagy in the survival of tumor cells in vivo was demonstrated where treatment with either CQ or ATG5 shRNAs enhanced the ability of alkylating drug therapy to induce tumor cell death.78 Since then, a multitude of papers have been published demonstrating utility in combining autophagy inhibitors with cancer therapy.11 In addition to autophagy serving a critical role in tumorigenesis, many cancer drugs have been reported to induce autophagy that can be cytoprotective. Traditional cytotoxic chemotherapeutics and targeted therapies induce autophagy through a number of signaling pathways including the DNA damage response, mTOR and AMP-activated protein kinase signaling, the ER stress response and others.11 Inhibition of autophagy with chloroquine in preclinical models improves the response of tumor cells to alkylating agents, suggesting that autophagy promotes survival.79 Another report observed cytoprotective autophagy to serve a critical resistance mechanism to BRAF inhibition in BRAF-mutant melanoma.23 This finding was of particular interest, as the role autophagy has in resistance to targeted therapies that target PI3K/AKT/mTOR signaling have been well studied;80,81 however, the function of autophagy in the context of MAPK pathway inhibition has not been well characterized. Mechanistically, BRAF inhibition leads to a physical © 2015 Macmillan Publishers Limited

5 interaction between mutant BRAF and GRP78, a master regulator of ER stress activity, which results in the downstream activation of the ER stress pathway effector PERK. PERK activation results in an induction of cytoprotective autophagy. BRAF inhibitor-induced autophagy was observed at a high rate in tumors obtained at the time of progression on BRAF inhibitor therapy.23 Targeting autophagy with HCQ concurrently with BRAF inhibitor therapy resulted in significant tumor regression in mouse xenografts studies. This finding was reproduced in in vitro and in vivo studies in pediatric gliomas that harbor BRAFV600E mutations, and the addition of HCQ to a BRAF inhibitor overcame the resistance to BRAF inhibition in a patient with pediatric glioma.82 Many other examples exist supporting the concept of combining chemotherapy or targeted therapy with a chloroquine derivative, providing rationale for launching cancer clinical trials involving HCQ. CLINICAL TRIALS OF HCQ, THE FIRST AUTOPHAGY INHIBITOR The seminal discoveries of these recent mouse models and preclinical investigations dovetail nicely with the publishing of the first set of HCQ clinical trials in patients with advanced cancers (Table 2). Six phase I/II trials were performed in human patients diagnosed with glioblastoma multiforme,16 relapsed/refractory myeloma17 and melanoma in addition to other advanced tumors.13–15 One additional clinical trial was published wherein pet dogs diagnosed with spontaneously occurring lymphoma were also treated with HCQ-based combination therapies.12 Each trial involved a combination therapy that had preclinical studies to justify clinical translation.78,83–86 The major finding from these trials is that, based on electron microscopy-based pharmacodynamic assays, autophagy can be modulated therapeutically with chloroquine derivatives. Remarkably, across all of the trials o 10% of patients had severe non-hematological toxicity. Specifically, there was no evidence of extensive metabolic toxicity, liver injury or neurologic impairment in these trials despite some evidence that chronic modulation of autophagy was achieved in patients, as seen by the accumulation of autophagic vesicles in peripheral blood mononuclear cells and tumor cells. When combined with radiation therapy and concurrent and adjuvant temozolomide, HCQ produced dose-limiting myelosuppression at doses above 600 mg HCQ. At these doses only a subset of patients had evidence of autophagy modulation detectable in their peripheral blood mononuclear cells, which may be one reason there was no significant improvement in the overall survival compared with the historical controls of temozolomide and radiation alone.16 Significant therapy-associated increases in AVs and LC3-II were observed in peripheral blood mononuclear cells in a concentration-dependent manner, demonstrating HCQ could modulate autophagy in vivo. Combined treatment with the proteasome inhibitor bortezomib and HCQ resulted in a greater perturbation of tumor cell autophagy compared with peripheral blood mononuclear cell autophagy, arguing that HCQ may selectively accumulate in tumor cells.17 Similar results were observed in the phase I trial of vorinostat and HCQ13 and in the canine lymphoma trial using doxorubicin with HCQ.12 Although these phase I studies were not powered to determine efficacy, response rates in unselected patient populations were generally low. However, there were a number of striking responses and prolonged stable disease observed in patients with melanoma, renal cell carcinoma, colon cancer and myeloma, that suggest that a specific subset of cancers may be susceptible to regimens containing chloroquine-based autophagy inhibitors. Critical to the future success of autophagy-oriented clinical trials are biomarkers that may aid in patient selection. Current biomarkers to assess autophagy modulation in clinical trials consist of monitoring the accumulation of autophagic vesicles in peripheral blood mononuclear cells and tumor cells by electron microscopy, as well as checking for changes in LC3 lipidation by western blotting and Oncogene (2015) 1 – 11

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Oncogene (2015) 1 – 11

Abbreviations: HCQ, hydroxychloroquine; HDAC, histone deacetylase; MTD, maximal tolerable dose; mTOR, mechanistic target of rapamycin complex; NCI, the National Cancer Institute; PMBC, peripheral blood mononuclear cell; PET, positron emission tomography.

Dana–Farber Autophagy Phase II HCQ 7

Pancreatic

Colorado State DNA damage Dog lymphoma Phase I doxorubicin+ HCQ 6

mTOR HDAC Phase I temsirolimus+ HCQ Phase I vorinostat+ HCQ 4 5

Proteasome Myeloma DNA damage Solid tumors, melanoma Phase I bortezomib + HCQ Phase I temozolomide + HCQ 2 3

Solid tumors, melanoma Penn, Pfizer Solid tumors, renal, colon San Antonio, NCI

30 Comparison of purified tumor cells with PBMC 40 High-dose chemo and HCQ well tolerated; clinical activity None 35 Serial PET scans, tumor biopsies; clinical activity Grade 3 fatigue at 800 mg 24 Tumor biopsies; clinical activity 600 mg MTD Sepsis, death, 12.5 mg/kg 27 100% clinical benefit MTD Grade 3/4 lymphopenia 20 High-dose HCQ well tolerated

92 Survival outcome National trial

ABTC (15 centers), NCI Myelosuppression at 800 mg 600 mg MTD Penn, Millenium None Penn, Merck None Phase I/II temozolomide/radiation + HCQ DNA damage Glioma 1

Trial Agents

Table 2.

Clinical trials involving HCQ

Pathway

Disease

Sites/funding

DLT/MTD

N Unique features

6 total LC3 protein by immunohistochemistry. Interestingly, a recent study profiled the secreted factors unique to tumor cells with high levels of autophagy relative to those with low levels of autophagy, suggesting the measurement of these autophagy-associated secreted proteins in plasma may serve as surrogates for intratumoral autophagy levels.87 An additional phase II trial was recently published where patients with previously treated metastatic pancreatic cancer were administered HCQ as a single agent.18 Although HCQ monotherapy did not demonstrate significant therapeutic efficacy, highdose HCQ was well tolerated. HCQ has also been demonstrated to synergize with chemotherapeutics and targeted agents, which may explain the lack of efficacy as a single agent. There are numerous ongoing trials utilizing HCQ in combination therapies, a summary of which can be found in Table 3. More potent inhibitors of autophagy possessing greater in vivo activity relative to what is currently achievable by HCQ are urgently needed. Inhibitors such as Lys05 (see below) have been developed and are in the steps of optimization for clinical use, which should result in an increase in detectable autophagy inhibition in patients and an increase in clinical benefit. A definitive test of the role that autophagy serves in the setting of anticancer therapy for patients awaits randomized studies of HCQ and the new generation of autophagy inhibitors where autophagy can be more robustly inhibited in vivo. Insight gained from recent preclinical and clinical studies identify potential side-effects from autophagy inhibition in vivo including myelosuppression, lymphopenia and Paneth cell dysfunction, a characteristic resembling the intestinal phenotype of humans with genetic defects in ATG16L1.88 Ongoing trials utilizing HCQ in combination therapy will expand our knowledge regarding the proper context where autophagy inhibition may elicit the greatest clinical activity (Table 3). OTHER AGENTS BEING DEVELOPED AS AUTOPHAGY INHIBITORS FOR CLINICAL TRIALS Our understanding of the autophagic pathway and its importance in cancer has increased exponentially within the last decade, providing new promising molecular targets for cancer therapy. Druggable autophagy targets include Beclin-1, ULK1, ATG4, ATG7 and recently Vps34 (Figure 1). To date, no kinase inhibitors against ULK1 have entered clinical trials, however, a peptide has been described that may have utility in modulating autophagy. Highthroughput screening efforts to identify novel autophagy inhibitors resulted in the development of SAR405, a lowmolecular mass kinase inhibitor of Vps34. SAR405 was recently described to possess a unique binding mode and molecular interaction within the ATP-binding cleft of human Vps34.89 Inhibition of Vps34 with SAR405 led to significant impairment of lysosomal function and could prevent the autophagy induced by starvation conditions or the inhibition of mTOR with everolimus. This study revealed synergy between SAR405 and everolimus in renal cell carcinoma studies. Another study utilizing the selective Vps34 inhibitor PIK-III, demonstrated PIK-III potently inhibited the formation of mCherry-positive autolysosomes (in cells expressing the mCherry-GFP-LC3 reporter), and prevented the clearance of mitochondria in a carbonyl cyanide m-chlorophenylhydrazoneinduced mitophagy model.90 These findings reveal Vps34 to have a pivotal role in the initiation of autophagy and degradation of substrates, and encourage further studies to establish whether Vps34 inhibitors should be explored in future clinical trials. Although it is clear that HCQ exerts part of its effects through its action on autophagy, chloroquine derivatives likely harm cancer cells by engaging other targets. This observation is reverberated with a recent report demonstrating the efficacy of CQ in vivo relied upon its ability to normalize tumor vessel structure and increase perfusion, consequently reducing hypoxia, cancer cell invasion and metastasis, irrespective of autophagy inhibition.91 In addition, © 2015 Macmillan Publishers Limited

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7 Table 3.

Therapies undergoing combinatorial testing with HCQ in cancer

Pathway

Target

Clinical agents

Company

Trial ID

PI3K PI3K PI3K MAPK MAPK MAPK Epigenetic Immune Other Other Other

AKT mTOR mTOR BRAF Dual MEK/BRAF Pan-Raf/Pan-RTK HDAC Interleukin-2 EGFR EGFR Nucleoside

MK2206 Sirolimus (rapamycin) Everolimus (RAD001) Vemurafenib (PLX4032) Trametinib (GSK1120212) dabrafenib (GSK2118436) Sorafenib Vorinostat Aldesleukin (IL-2) Gefitinib Erlotinib Gemcitabine

Merck Pfizer Novartis Roche/Plexxicon GlaxoSmithKline GlaxoSmithKline Bayer Merck Prometheus AstraZeneca Genentech/ Astellas Eli Lilly

NCT01480154 NCT01842594 NCT01510119 NCT01897116 NCT02257424 NCT01634893 NCT01023737 NCT01550367 NCT00809237 NCT01026844 NCT01506973

Abbreviations: EGFR, epidermal growth factor receptor; IL-2, interleukin 2; mTOR, mechanistic target of rapamycin complex; PI3K, phosphoinositide 3-kinase; RTK, receptor tyrosine kinase.

clinical trials indicate that high doses of HCQ produce only modest autophagy modulation in surrogate tissues. Efforts to identify more potent autophagy inhibitors have commenced. The existence of non-canonical autophagy brings up the possibility that any therapeutic strategy poised at modulating a canonical autophagy protein can be circumvented by an increase in the function of non-canonical autophagy; however, both canonical and non-canonical autophagy ultimately rely on the lysosome for final degradation, providing a potentially ideal target, which is currently being investigated. Lys05, a novel dimeric derivative of chloroquine was shown to have significant in vivo activity both as a single agent88 and in combination with a BRAF inhibitor.23 Efforts are underway to optimize Lys05 for clinical trials. VATG-027, a potent autophagy inhibitor identified through a highthroughput screen of anti-malarial compounds was found to have activity in melanoma cells.92 The interesting observation was made that the ability to inhibit autophagy was separate from the cytoxicity profiles of the compounds tested. CELL INTRINSIC REGULATION OF AUTOPHAGY POINTS TO NEW THERAPEUTIC TARGETS Recent work has increased our understanding of the cell intrinsic regulation of autophagy in cancer cells, and by doing so may point the way toward better therapeutic targets. Oncogene and tumor suppressor-dependent gene regulation has been investigated leveraging the mouse models mentioned above, which possess Kras mutations and p53 deletions to understand how each may regulate autophagy. These experiments may not recapitulate the human condition where oncogenes and tumor suppressor genes are mutated in the context of innumerable other genetic and epigenetic alterations in cancer that may convert a signal that suppresses autophagy into one that promotes it.93,94 Adding to the complexity of predicting autophagy regulation by studying recurrent somatic mutations associated with cancer, it is increasingly evident that besides genetic regulation of autophagy, transcriptional, epigenetic and posttranslational regulation of autophagy has a major impact on the eventual role of autophagy within a given cancer cell. Transcriptional regulation of autophagy has been demonstrated through Foxk proteins (Foxk1 and Foxk2) acting as transcriptional repressors of autophagy genes.95 Mechanistically, mTOR promotes the transcriptional activity of Foxk1 in nutrient-rich conditions, resulting in the co-localization of Foxk1 with Sin3A at the promoters of 79 known autophagy-associated genes. Interestingly, ablation of Foxk1 with siRNA resulted in the upregulation of critical components of the Ulk1 and Vps34 machinery, reinforcing the negative impact on autophagy served by Foxk1 transcriptional © 2015 Macmillan Publishers Limited

activity.95 Autophagy has been linked to lysosomal biogenesis through observations that starvation activates a transcriptional program largely coordinated by the transcription factor EB (TFEB), which results in the upregulation of autophagy and lysosomal genes to enable the cell to survive.96 TFEB, when overexpressed, significantly increases the number of autophagosomes in cells, and was found to be regulated through the phosphorylation of its serine 142 residue by ERK2, belonging to the MAPK pathway. P53 has also been shown to have a role in the transcription of autophagy genes, which compliment the mouse models described investigating the role mutant p53 may serve on the sensitivity to autophagy-based therapy. Global genomic profiling in mouse embryo fibroblasts revealed p53 to transcriptionally regulate a multitude of autophagy genes, where in response to DNA damage, an induction of autophagy relied on p53 transcriptional activity.97 It is worth noting that autophagy has been demonstrated to still occur in the absence of functional p53, suggesting that p53 does not solely regulate autophagy but rather has a part in the highly orchestrated symphony that is autophagy.63 ER stress also results in the upregulation of autophagy via activating transcription factor 4 increasing ULK1 mRNA and protein expression in cells undergoing severe ER stress,98 and has recently represented a significant resistance mechanism in melanoma cells treated with BRAF inhibitor therapy.23 Although transcription factors are not traditionally thought of as druggable targets, efforts are underway to develop strategies to activate or impair the transcriptional activity of p53, TFEB and FOXO proteins (Figure 1). Epigenetically, the acetylation status of histone H4 lysine 16 (H4K16) was found to regulate life or death decisions in autophagic cells, where an induction of autophagy results in a decrease of H4K16 acetylation (H4K16ac) and ultimately a decrease in the expression of ATG genes on a genome-wide level.99 Antagonizing the reduction in H4K16ac upon autophagy induction results in an increase in autophagic cell death.99 Another checkpoint is represented by the nutrients released from autophagic degradation such as amino acids, which stimulate the Ragulator complex, and result in the activation of mTORC1 and negative feedback on autophagic activity to maintain homeostasis.100 The metabolite acetyl-coenzyme A, recently reported to function as a suppressor of cytoprotective autophagy in aging cells, also occurs mechanistically through hyperacetylation of histone H3 leading to transcriptional downregulation of a number of autophagy genes.101 Methylation also has a role in autophagy regulation, with a genome-wide methylation analysis revealing hyper-methylation of the ULK2 gene, resulting in the inhibition of autophagy in glioblastoma cells.102 Epigenetic agents such as HDACs85 and demethylating agents103 have Oncogene (2015) 1 – 11

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8 already been shown to modulate autophagy, and these new findings could guide their development further as autophagy modulators. Posttranslationally, the autophagic machinery is regulated at multiple levels including phosphorylation, acetylation and ubiquitination. The phosphorylation status of multiple key players in the autophagy pathway has significant roles in the regulation of autophagy. When phosphorylated, mTORC1 is active and results in the inhibition of autophagy through the direct phosphorylation of ULK1 by mTORC1. LC3 can also be phosphorylated by PKA and PKC, resulting in the inability for LC3 to become lipidated, an essential step needed for LC3 incorporation within the autophagosome bilayer.104 Lysine acetylation has an inhibitory role, where under conditions of nutrient starvation, loss of acetylation results in an induction of autophagy.105 Silencing of acetyl-coenzyme A synthetase, leading to a decrease in the overall acetylation of cytoplasmic proteins, has also been reported to result in enhanced autophagy in Drosophila brains.106 Ubiquitination also helps regulate autophagy, with an emerging role for the E3 ubiquitin ligases Nedd4,107 Parkin108 and TRIM13109 in the initiation of autophagy, mitochondrial homeostasis and in substrate specificity for autophagic degradation.110 Metabolic regulation of autophagy occurs through the ability of upstream autophagy-regulating effectors to sense the intracellular levels of ammonia, amino acids, growth factors, glucose and lipids10 (Figure 1). Ammonia is created via amino-acid catabolism and induces autophagy by way of activating AMP-activated protein kinase and leading to the ER stress response.111 A drop in amino-acid levels is sensed by a few different mechanisms, which include (1) sensing of the resulting accumulation of uncharged tRNA species by GCN2,112 (2) lysosomal sensing that recruits mTORC1 to the lysosomal surface,113 (3) sensing of intracellular acetyl-CoA stores that are negatively impacted by low levels of various amino acids114 and (4) sensing the depletion of the metabolic intermediate α-ketoglutarate, another result of low amino-acid levels.115 All of these amino-acid sensing mechanisms result in an induction of autophagy to increase the intracellular degradation of nonessential components in an attempt to increase the pool of available amino acids to continue metabolism. Understanding these epigenetic, posttranslational and metabolic regulatory circuits may help define the autophagic switch that appears to occur in the transition from tumor suppressor to tumor promoter. The development of smallmolecule inhibitors that target cancer metabolism will certainly have an impact on autophagy, and perhaps in some cases these drugs can be repositioned or reconsidered as autophagy modulators if further research indicates that autophagy is responsible for the main changes observed with these inhibitors. NEW ROLES FOR THE FUNCTIONAL EFFECTS OF AUTOPHAGY Autophagy functionally protects cells by way of degrading intracellular components, which would have otherwise led to the loss of cellular fitness, while also simultaneously catering to the ever-changing metabolite demands of the cell with freshly digested building blocks for survival. Although it is clear that the degradation through autophagy of protein substrates has a role in cellular survival, the specificity of this process is unknown. Global proteome analysis comparing cells with intact-autophagy versus cells with defective autophagy (Atg5+/+ and Atg5−/−) revealed that autophagy preferentially degrades proteins that are toxic or nonessential for survival under stressful conditions, seeming to spare proteins involved in the maintenance of functional autophagy and stress survival.116 Interestingly, proteins found to increase in response to autophagy induction were involved in vesicle-mediated trafficking and lysosomal protein degradation, potentially providing a new suite of therapeutic targets that may augments strategies of inhibiting autophagy. Another report Oncogene (2015) 1 – 11

identified a specific protein turnover mechanism where autophagy was responsible for the degradation of the inhibitory p53 isoform Δ133p53α through interaction of the chaperoneassociated E3 ubiquitin ligase STUB1.117 Autophagy was also found to have a key role in the degradation of damaged nuclear DNA in cells deficient of Dnase2a.118 DNA accumulated in autophagy-deficient cells, which resulted in Sting-mediated inflammation. Autophagic activity can govern the secretory profile of cancer cells, where high autophagy is associated with melanoma metastasis, and serum from metastatic melanoma patients with high tumor autophagy levels contain a secretory signature found to correlate with cells displaying high autophagic activity.87 These findings are of immense importance as they provide a potential avenue to assess autophagic activity of tumors within patients from serum samples as well as the potential to provide a means to stratify potential responders in future autophagy-based therapy regimens. Autophagy also functionally inhibits apoptosis through indirect inhibition of p53-upregulated modulator of apoptosis, which demonstrates how autophagy can determine cell fate.119 In addition, autophagy has been demonstrated to directly impact proliferation by way of AMBRA promoting the dephosphorylation of c-Myc Ser62, resulting in the proteasomal degradation of c-Myc and a decrease in the rate of cell division.120 Finally, autophagy has a functional role in the immunogenic clearance of cancer cells. Immunogenic cell death (ICD) relies in part on the release of ATP from dying cells121 and autophagy has been found to be critical in the ICD-associated secretion of ATP.122 Mechanistically, ATP was found to release in a manner dependent upon the lysosomal protein LAMP1 and the opening of PANX1 (pannexin 1) channels. Implications on what effect utilization of autophagy inhibitors may confer upon ICD remains to be determined. However, future combination regimens can be envisioned where an autophagy inhibitor along with an immune-enhancing therapy can provide the best of both worlds of inhibiting cytoprotective autophagy while concurrently launching an effective immune response against the tumor cells. In summary, a first series of hurdles, including experiments in xenografts and genetically engineered mouse models, followed by the first series of HCQ trials have been overcome demonstrating the application of autophagy inhibitors in patients with advanced cancers could be done safely, and has resulted in encouraging antitumor results in selected patients. The stage is now set for the testing of more potent and specific inhibitors of the autophagic machinery. While this is being done in the clinic, translating knowledge about the regulation of autophagy and its full spectrum of functions in multicellular organisms will permit the development of new strategies for autophagy modulation in cancer. CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS This work was supported by R01 CA169134 (RKA) from the National Institutes of Health.

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