disruption of autophagic degradation with roc-325 ... · acridine orange ﬂuorescence. red...

Cancer Therapy: Preclinical

Disruption of Autophagic Degradation withROC-325 Antagonizes Renal Cell CarcinomaPathogenesisJennifer S. Carew1,2, Claudia M. Espitia3,William Zhao3,Yingchun Han2,Valeria Visconte2,James Phillips2, and Steffan T. Nawrocki1

Abstract

Purpose: Although autophagy plays important roles in malig-nant pathogenesis and drug resistance, there are few clinicalagents that disrupt this pathway, and the potential therapeuticbenefit of autophagy inhibition remains undetermined. We usedmedicinal chemistry approaches to generate a series of novelagents that inhibit autophagic degradation.

Experimental Design: ROC-325 was selected as a lead com-pound for further evaluation. Comprehensive in vitro and in vivostudieswere conducted to evaluate the selectivity, tolerability, andefficacy of ROC-325 in preclinical models of renal cell carcinoma(RCC) with HCQ serving as a comparator. Markers of autophagyinhibition and cell death were evaluated in tumor specimens.

Results: ROC-325 exhibited superior in vitro anticancer effectscompared with the existing autophagy inhibitor hydroxychlor-oquine (HCQ) in 12 different cancer cell lineswith diverse geneticbackgrounds. Focused studies of the mechanism of action and

efficacy of ROC-325 in RCC cells showed that drug treatmentinduced hallmark characteristics of autophagy inhibition, includ-ing accumulation of autophagosomes with undegraded cargo,lysosomal deacidification, p62 stabilization, and disruption ofautophagic flux. Subsequent experiments showed that ROC-325antagonized RCC growth and survival in an ATG5/7-dependentmanner, induced apoptosis, and exhibited favorable selectivity.Oral administration of ROC-325 to mice bearing 786-0 RCCxenografts was well tolerated, was significantly more effective atinhibiting tumor progression than HCQ, and inhibited auto-phagy in vivo.

Conclusions: Our findings demonstrate that ROC-325 hassuperior preclinical anticancer activity compared with HCQ andsupport the clinical investigation of its safety and preliminaryefficacy in patients with RCC and other autophagy-dependentmalignancies. Clin Cancer Res; 23(11); 2869–79. �2016 AACR.

IntroductionAutophagy is an evolutionarily conserved mechanism of lyso-

somal proteolysis that is used for the turnover of organelles andproteins with long half-lives and also functions to generatealternative sources of metabolic fuel via nutrient recycling understress conditions (1, 2). Although multiple key studies havedemonstrated that autophagy functions as amechanism of tumorsuppression via the elimination of defective premalignant cells,overwhelming evidence supports a major role for autophagicdegradation in the maintenance of bioenergetic homeostasisunder stress conditions, including hypoxia and nutrient depriva-tion (3). Additionally, autophagy has emerged as an importantmechanism of resistance to radiation, classical chemotherapy,and targeted anticancer agents due to its ability to augment the

survival capacity of malignant cells (4–8). The data supportingroles for autophagy as a mediator of drug resistance and malig-nant progression provided a logical foundation to devise strate-gies to impair this process for therapeutic benefit.

Chloroquine (CQ) and hydroxychloroquine (HCQ) have beenused for decades to treat malaria, rheumatoid arthritis, and lupusand represent two of a very small group of FDA-approved drugsthat disrupt lysosomal function and consequently inhibit autop-hagy (6). These specific properties of CQ/HCQ spurred numerouspreclinical investigations focused on establishing the safety andtherapeutic benefit of inhibiting autophagy to increase the efficacyof a diverse range of anticancer agents (9, 10). Based on thepositive impact of HCQ in this scenario, we and others initiateda series of phase I and phase I/II trials to investigate the safety andpreliminary efficacy of the addition of HCQ to existing anticancerregimens (11–16). Although the addition of HCQ was generallysafe and preliminary efficacy was observed in a minority ofpatients treated with HCQ-based regimens, it was unclear if themaximum tolerated dose (MTD) of HCQ in these studies resultedin complete autophagic inhibition. The results of these initialclinical studies ofHCQ-based combination regimensunderscoredthe need for better agents to antagonize lysosomal proteolysis.However, no comprehensive structure activity relationship (SAR)analyses on CQ/HCQ had been conducted to date, and it wastherefore unclear which specific structural modifications to theseagents may result in increased antiautophagic potency or efficacy.

Lys05 is a novel dimeric form of CQ connected with the spacerN,N-bis(2-aminoethyl)methylamine that was designed to take

1Department of Medicine, Division of Translational and Regenerative Medicine,University of Arizona Cancer Center, Tucson, Arizona. 2Taussig Cancer Institute,Cleveland Clinic, Cleveland, Ohio. 3Division of Hematology/Oncology, CancerTherapy and Research Center at The University of Texas Health Science Centerat San Antonio, San Antonio, Texas.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

CorrespondingAuthor: Jennifer S. Carew, University of Arizona, Arizona CancerCenter, Room 3985C, 1515 North Campbell Avenue, Tucson, AZ 85724. Phone:520-626-2272; Fax: 520-626-7395; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-16-1742

�2016 American Association for Cancer Research.

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advantageof the conceptofpolyvalencyandwas reported toexhibitsuperior autophagic inhibition and anticancer activity comparedwith CQ (17). Polyvalent molecules have the potential to yieldnonlinear, multifold potency against their respective targets com-pared with their corresponding monomers (18, 19). With the goalof generating new autophagy inhibitors with better potency, tol-erability, and anticancer efficacy thanHCQ, we used logicalmedic-inal chemistry approaches to generate a series of new dimericcompounds containing modified core elements of HCQ, CQ, andthe antischistosomal drug lucanthone (Miracil D; ref. 20). Here, wereport that ROC-325, a novel compound with structural motifs ofboth HCQ and lucanthone, inhibits autophagy at significantlylower doses and exhibits significantly superior single agent anti-cancer activity against a broad range of tumor types compared withHCQ. Focused studies in models of renal cell carcinoma (RCC)demonstrated that ROC-325 treatment led to the deacidification oflysosomes, accumulation of autophagosomes, and disruptedautophagic flux. Targeted knockdown of the essential autophagygenes ATG5 and ATG7 severely blunted the anticancer effects ofROC-325, thus indicating that its autophagy inhibitory propertieswere a critical component of its mechanism of action. Oral admin-istration of ROC-325 to mice bearing RCC xenografts was welltolerated and yielded dose-dependent inhibition of tumor growththat was significantly more efficacious than a higher dose of HCQ.Analysis of tumor specimens from mice treated with ROC-325demonstrated in vivo autophagy inhibition, reduced tumor cellproliferation, and apoptosis. Our collective findings establish thefoundation for further investigation of ROC-325 as a novel agentfor autophagy-dependent malignancies and other disorders wherelysosomal activity contributes to disease pathogenesis.

Materials and MethodsSynthesis of ROC-325, other ROC series compounds, and Lys05

The synthesis of ROC-325 was performed following scheme I.

Scheme I: (a) Pd(II)acetate, BINAP, K3PO4, dioxane, 100�C, 17hours; (b) 4N HCl in dioxane; (c) Pd(II)acetate, BINAP, K3PO4,dioxane, 100�C, 15 hours; (d) Methanol, 4N HCl in dioxane.

The procedures for the synthesis of ROC-325 are representativeof the preparation of the derivatives shown in Fig. 1A. Compoundnumbers detailed here correspond to the compound numbers inbold text in the above reaction scheme. A suspension of 1-Bromo-4-methylthiaxanthone (Compound1, 3.23 g, 10.62mmol/L), {2-[(2-Amino-ethyl)-methyl-amine]-ethyl}-carbamic acid tert-butylester amine (Compound 2, 1.53 g, 7.08mmol/L), Binap (2,20-bis(diphenylphosphino)-1,10-binaphthyl) (0.88 g, 1.4 mmol/L),and K3PO4 (7.5 g, 35.0 mmol/L) in 70mL of dioxane was purgedwith N2 for 25 minutes. The reaction was kept under N2 and Pd(OAc)2 (0.159 g, 0.708 mmol/L) added. The reaction was heatedat 100�C for 17 hours. The reaction was cooled, dichloromethane(100mL) added, and the crudemixture filtered and concentrated.Purification by flash silica chromatography eluting with ethylacetate/hexanes (4:6 to 6:4) provided 1.5 g of a red solid (Com-pound 3). Thirtymilliliters of 4NHCl was added to Compound 3(0.53 g). The suspension was stirred at room temperature for 1.5hours, and the solid was collected by filtration, washed withdichloromethane (3 � 15 mL), and dried at 50�C to give 150mg of a red solid (Compound 4). Next, amine Compound 4(0.250 g, 0.733 mmol/L), 4,7-dichloroquinoline Compound 5(0.329 g, 1.66 mmol/L), BINAP (0.091 g, 0.146 mmol/L), andK3PO4 (0.778g, 3.66mmol/L)were suspended indrydioxane (25mL). The mixture was purged with N2 for 25 minutes and thenkept underN2. Pd(OAc)2 (0.016 g, 0.073mmol/L)was added andthe reaction heated for 16 hours at 95�C. The reaction was cooledand filtered with the aid of dichloromethane (50mL). The filtratewas concentrated and the crude purified by flash chromatographyusing ethyl acetate/dichloromethane (1:9) andgave 205mgof redfoam Compound 6. The tri–HCl salt of Compound 6 was pre-pared by suspending it in 70mLofmethanol, adding 10mLof 4NHCl in dioxane, and stirring for 15 hours at room temperature.The precipitate was collected by filtration, washedwithmethanol,and then ethyl acetate (15/15), and dried at 50�C under vacuumfor 12 hours. The structure of ROC-325 was confirmed by 1Hnuclear magnetic resonance (NMR) and high-resolution massspectrometry (HRMS) analyses, and these data are included inSupplementary Figs. S1 and S2.

Cells and cell cultureA498, 786-0, Achn, and Caki-2 cells were obtained fromATCC.

Cells were cultured with medium supplemented with 10% FBS at37�C with 5% CO2 as previously described (21). Human normalrenal proximal tubule epithelial cells (RPTEC) were purchasedfrom Clonetics and cultured in REGM media (REGM BulletKit,Clonetics). Cell lines were authenticated by the source banksusing short tandem repeat (STR) DNA profiling techniques.

Lentiviral shRNA gene silencingTargeted knockdown of ATG5, ATG7, and CTSD was achieved

using lentiviral shRNA using commercially available lentiviralshRNAs (Santa Cruz Biotechnology; Cat# sc-41445-V for ATG5,Cat# sc-41447-V for ATG7, Cat# sc-29239-V for CTSD) accordingto the manufacturer's instructions.

Chemicals and reagentsReagents were obtained from the following sources: giemsa

stain, acridine orange, bafilomycin A1, propidium iodide, 3-(4,5-

Translational Relevance

Autophagy plays an important role in cancer progressionand drug resistance, yet clinically available agents to target thispathway currently are limited. We used logical medicinalchemistry approaches to developROC-325, an orally availablenovel inhibitor of autophagic degradation. Comprehensive invitro and in vivo studies in preclinical models of renal cellcarcinoma (RCC) demonstrated that ROC-325 induced all ofthe hallmark features of autophagy inhibition, was well tol-erated, andwas significantlymore effective thanhydroxychlor-oquine. Our findings provide a strong rationale for clinicalinvestigation of the safety and efficacy of ROC-325 for RCCand other autophagy-dependent malignancies.

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Clin Cancer Res; 23(11) June 1, 2017 Clinical Cancer Research2870




dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),HCQ, and anti-b tubulin antibody (Sigma), anti-active caspase-3(Cell Signaling Technology), anti-LC3B, anti-p62, ant-ATG5, andanti-ATG7 (Abcam), anti-cathepsin D (Santa Cruz Biotech), goatanti-rabbit horseradish peroxidase (HRP)-conjugated secondaryantibody (The Jackson Laboratory), sheep anti-mouse-HRP anddonkey anti-rabbit-HRP (Amersham). Additional details regard-ing these reagents were previously described (14).

Measurement of cell proliferationCells infected with nontargeted control and ATG5 or ATG7-

targeted shRNA were plated in triplicate. Cell numbers were

counted daily for 5 consecutive days via automated trypan blueexclusion with the assistance of a Vi-Cell XR system (Beckman-Coulter).

Transmission electron microscopyTransmission electron microscopy of cells was performed as

previously described (22). RCC cells were treated with ROC-325for 24 hours and harvested for imaging. Briefly, sections were cutin an LKBUltracut microtome (Leica), stained with uranyl acetateand lead citrate, and examined in a JEM 1230 transmissionelectronmicroscope (JEOL). Images were captured using the AMTImaging System (Advanced Microscopy Techniques Corp).

Figure 1.

ROC-325 induced vacuolization andlysosome membrane permeability. A,Chemical structures of ROC-325 andrelated compounds. B, ROC-325induces vacuolization. 786-O and A498cells were treated for 24 hours with 5mmol/L ROC-325. Cell morphology andvacuolization were visualized byGiemsa staining. Scale bars, 20 mm.C, Electron microscopy demonstratesvacuolization and electron-denseparticle accumulation. Cells weretreated with 5 mmol/L ROC-325 for 24hours. Cells were fixed and prepared forelectron microscopy. Arrows indicatevacuoles with undegraded cargo in thecytosol of imaged cells. Scale bars forimages with 8,000� magnificationindicate 2 mm, and scale bars for imageswith 20,000� magnification indicate500 nm. D, Measurement of lysosomemembrane permeability by loss ofacridine orange fluorescence. Redacridine orange staining was measuredin 786-O and A498 cells byimmunofluorescence and quantifiedusing ImageJ software. Mean � SD,n ¼ 5. � , statistically significantdifference from the controls; P < 0.05.

Targeting Autophagy with ROC-325 Impairs RCC Pathogenesis

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Giemsa stainingCells were plated in chamber slides as previously described and

treated with ROC-325 for 24 hours (20). Following drug treat-ment, cells were washed with PBS and fixed in methanol for 5minutes. Cells were then incubated for 1 hour in Giemsa staindiluted 1:20 with deionized water. Cells were rinsed with waterand imaged using an Olympus fluorescent microscope with aDP71 camera. Image-Pro Plus software Version 6.2.1 (MediaCybernetics) was used for image acquisition.

Acridine orange stainingAcidic lysosomes were visualized by acridine orange staining as

previously described (20). After treatment with ROC-325 for 24hours, cells were stained with 1 mmol/L acridine orange for 15minutes at 37�C. Cells were washed with PBS, and images werecaptured using an Olympus fluorescent microscope. Acidic lyso-somes appear as orange fluorescent cytoplasmic vesicles. Quan-tification of 5 random fields of acridine orange intensity andimage acquisition were performed using Image-Pro Plus softwareVersion 6.2.1.

ImmunocytochemistryCells were plated on chamber slides and allowed to attach

overnight. Cells were then treated for 24 hours with ROC-325.Following drug treatment, cells were fixed with 4% paraformal-dehyde, permeabilized using 0.2% triton-X-100, and incubatedovernight with anti-LC3B antibody. Alexa Fluor 594–conjugatedfluorescent secondary antibody was used to visualize proteinlocalization. DAPI was utilized to stain the nucleus. Images werecaptured using an Olympus fluorescent microscope with a DP71camera and a 60� objective. Image-Pro Plus software Version6.2.1 (MediaCybernetics) was used for image acquisition (20).

Expression microarraysRCC cells were treated with ROC-325 for 24 hours. Total

RNAs were isolated using the RNeasy Plus Mini Kit (Qiagen)and treated with the TURBO DNA-free Kit (Applied Biosys-tems). Total RNA (300 ng) per sample was amplified andhybridized to GeneChip Human Gene 1.0 ST arrays (Affyme-trix) according to the manufacturer's instructions. AffymetrixCEL files were imported into Partek Genomics Suite 6.4 (PartekInc.) using the default Partek normalization parameters and therobust multi-array average (RMA) analysis adjusted for probesequence and GC content (GC-RMA). Data normalization wasperformed across all arrays using quantile normalization (20).Gene ontology enrichments (GO) analyses were performed,and heat maps were also generated using Partek software.Microarray data were deposited with NCBI GEO and can beviewed under accession number GSE89766.

Quantitative real-time polymerase chain reactioncDNA from ROC-325 treated cells were used for relative

quantification by RT-PCR analyses. First-strand cDNA synthesiswas performed from 1 mg RNA in a 20-mL reaction mixtureusing the high-capacity cDNA Reverse Transcription Kit(Applied Biosystems). Cathepsin D (CTSD) and GAPDH tran-scripts were amplified using commercially available TaqManGene expression assays (Applied Biosystems). Relative geneexpression was calculated with the 2�DDCt method usingGAPDH as a housekeeping gene (23).

Quantification of drug-induced cytotoxicityCell viability was estimated by the MTT assay, which detects

the conversion of MTT to formazan by the mitochondria ofliving cells. Cells were seeded into 96-well microcultureplatesat 10,000 cells per well and allowed to attach for 24 hours.Cells were then treated with ROC-325 or HCQ for 72 hours.Following drug treatment, MTT was added and formazanabsorbance was quantified using a Molecular Devices micro-plate reader. The estimated cell viability under each experi-mental condition was calculated by normalizing the respectiveformazan optical density to the density of control cells. Proa-poptotic effects following in vitro drug exposure were quanti-fied by propidium iodide (PI) staining and fluorescence-acti-vated cell sorting (FACS) analysis of sub-G0/G1 DNA contentas previously described (24) and by measurement of activecaspase-3 by flow cytometry using a commercial kit (BDBiosciences).

ImmunoblottingRenal cancer cells were incubated with ROC-325 for 24

hours. Cells were harvested and were then lysed as previouslydescribed (25). Approximately 50 mg of total cellular proteinfrom each sample were subjected to SDS-PAGE, proteins weretransferred to nitrocellulose membranes, and the membraneswere blocked with 5% nonfat milk in a Tris-buffered salinesolution containing 0.1% Tween-20 for 1 hour. The blots werethen probed overnight at 4�C with primary antibodies,washed, and probed with species-specific secondary antibodiescoupled to horseradish peroxidase. Immunoreactive materialwas detected by enhanced chemiluminescence (West Pico,Pierce, Inc.).

In vivo evaluation of ROC-325 and HCQ786-O renal cancer cells (5 � 106) were suspended in a

mixture of HBSS and Matrigel and subcutaneously implantedinto female nude mice (BALB/c background; ref. 26). Tumor-bearing animals from each cell line xenograft were randomizedinto treatment groups. Mice were treated with vehicle (water),ROC-325 (25, 40, and 50 mg/kg PO), or HCQ (60 mg/kg IP)QD�5 for 6 weeks. Mice were monitored daily and tumorvolumes were measured twice weekly. At study completion,tumors from representative animals were excised from eachgroup, formalin-fixed, and paraffin-embedded for immunohis-tochemical analysis.

ImmunohistochemistryParaffin-embedded tumor sections were deparaffinized in

xylene, exposed to a graded series of alcohol, and rehydratedinPBS (pH 7.5). Heat-induced epitope retrieval on paraffin-embedded sections and probing with specific antibodies wereconducted as previously described (14). Positive reactionswere visualized using 3,30-diaminobenzidine (Dako). Imageswere captured using an Olympus fluorescent microscope with aDP71 camera and a 20� objective. Image-Pro Plus softwareVersion 6.2.1 (MediaCybernetics) was used for image acquisition.ImageJ software was used for quantification of LC3B and p62levels by densitometric analysis of five random fields containingviable tumor cells as previously described (14). Quantification ofcleaved caspase-3 was conducted by counting the number ofpositive cells in five random fields as previously described (27).

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Statistical analysesStatistical significance of differences observed between paired

samples was determined using the Student t test. Two-wayANOVA with the Tukey post hoc analysis was used to determinethe significance of differences between experimental conditions inmultiparameter assays, including the in vivo assessment of theeffects of ROC-325 and HCQ on RCC xenograft tumors. Differ-ences were considered significant in all experiments at P < 0.05with two-sided comparisons.

ResultsROC-325 induces lysosomal deacidification andautophagosome formation

We first created a series of novel compounds that containedmotifs ofHCQand lucanthone (LUC)with the goal of developingnew agentswith both improved autophagic inhibition and single-agent anticancer activity (Fig. 1A; refs.6, 20). We evaluated theability of each of these compounds to reduce the viability ofmalignant cells and increase the expression of p62, a protein thatis known to be specifically turned over by autophagy (28). Basedon these initial assays, we identified ROC-325 as a lead agent (Fig.1A; Supplementary Figs. S1 and S2). Direct comparison of the invitro anticancer effects of ROC-325 and HCQ in 12 differenthuman cancer cell lines with diverse genetic backgroundsdemonstrated that ROC-325 possessed significantly lower IC50s(approximately 10-fold) thanHCQ in allmodels tested (Table 1).We selected RCC as a specific tumor type for further investigationof the pharmacologic properties of ROC-325 based on the sen-sitivity of A498 cells in these preliminary screens, the clinicalefficacy we observed in a patient with RCC treated with a com-bination of HCQ and vorinostat after failing seven lines of priortherapy in our recent investigator-initiated clinical trial, and a newreport suggesting that alterations in the autophagy pathwayin patients with renal cancer may be relevant for overall survival(14, 29). Giemsa staining of A498 and 786-0 cells demonstratedthat ROC-325 induced cytosolic vacuolization (Fig. 1B). Trans-mission electron microscopy analyses showed that it promotedthe accumulation of autophagosomes with undegraded cargo(Fig. 1C). Treatment with ROC-325 also resulted in a significantloss of acridine orange fluorescence, which is consistent with anincrease in lysosomal membrane permeability (LMP) and theconsequential deacidification of lysosomes (Fig. 1D). These col-lectivefindings demonstrated that the chemical refinements of theLUC andHCQ coremotifs that we designed to produce ROC-325resulted in significantly greater anticancer activity while retaininglysosomal disrupting properties.

ROC-325-triggered effects on cathepsin D promote apoptosis,but are not required for autophagy inhibition

We previously showed that CQ/HCQ and LUC induceincreased expression of the lysosomal protease cathepsin D(CTSD; refs. 20, 22, 30). The LMP-related effects of HCQ andLUC also promote its subcellular relocalization from lysosomalcompartments into the cytosol. Notably, in direct contrast to themajority of lysosomal proteases that require a strict acidic pHenvironment for their activity, CTSD retains proteolytic functionunder cytosolic conditions and has been shown to play an activerole in apoptosis when relocalized to the cytosol (31–33). Indeed,our earlier work showed that the proapoptotic effects of CQ andLUC in malignant cells were significantly impaired when CTSDwas targeted using RNAi approaches. We first conducted micro-array analyses to assess the global effects of ROC-325 on the geneexpression profiles of RCC cells. 786-0 andA498 cells were treatedwith 5 mmol/L ROC-325 for 24 hours and subjected to Affymetrixmicroarray quantification of pharmacodynamic changes in geneexpression. Heat map analyses identified significant upregulationof genes involved with proteolysis in each cell line (Supplemen-tary Fig. S3A). Similar to the autophagy inhibitors CQ/HCQ andLUC, ROC-325 triggered a highly significant increase in CTSDlevels, which was confirmed by qRT-PCR (Supplementary Fig.S3B). In addition to this specific effect, ROC-325 treatment alsostimulated the expression of other genes with important roles inthe control of protein turnover and ER stress-induced apoptosis,including UBA7, TNFAIP3, DDIT3, PMAIP1, and CASP4, indicat-ing a link between the induction of apoptosis and the disruptionof protein homeostasis.

In order to determine if drug-induced expression of CTSD isrequired for the autophagy inhibitory effects of ROC-325,weusedlentiviral shRNA to target CTSD in 786-0 RCC cells (Supplemen-tary Fig. S4A). These assays showed that antagonizing ROC-325'sability to trigger increasedCTSD levels did not interfere with drug-mediated stabilization of p62, an established marker of autop-hagy inhibition. However, CTSD knockdown did reduce theproapoptotic effects of ROC-325 (Supplementary Fig. S4B). Thesecollectivefindings indicate thatCTSD is not essential for ROC-325to inhibit autophagy, and that its increased expression is likely asecondary/downstream pharmacodynamic event that promotesapoptosis.

ROC-325 induces hallmark features of autophagy inhibitionand antagonizes autophagic flux

Our morphologic and gene expression analyses in RCC cellsindicated that ROC-325 treatment yielded pharmacodynamiceffects that were consistent with inhibition of autophagy. In orderto further investigate the specific effects of ROC-325 on autop-hagy, we first utilized fluorescent confocalmicroscopy to quantifythe impact of drug treatment on LC3B distribution. Treatmentwith 5mmol/L ROC-325 for 24hours led to the formationof LC3Bpunctae and a robust increase in LC3B levels in both A498 and786-0 RCC cells (Fig. 2A). Immunoblotting analyses conducted inbothA498and786-0 cells demonstrated that ROC-325promoteda dose-dependent increase in LC3B expression in a manner thatcorrelated with a corresponding increase in the levels of p62 andcathepsin D (Fig. 2B). Washout experiments where cells weretreated with ROC-325, the drug was removed, and the effects onp62 expression were followed over time showed that ROC-325achieved sustained autophagy inhibition for more than 24 hoursafter drug exposure ceased (Supplementary Fig. S5). We next used

Table 1. IC50 values from 72-hour MTT assays

Cell line Tumor typeROC-325 IC50

(mmol/L)HCQ IC50

(mmol/L)

A498 Renal 4.9 52A549 Lung 11 >75CFPAC-1 Pancreas 4.6 >75COLO-205 Colon 5.4 51DLD-1 Colon 7.4 >75IGROV-1 Ovarian 11 >75MCF-7 Breast 8.2 >75MiaPaCa-2 Pancreas 5.8 >75NCI-H69 Lung 5.0 54PC-3 Prostate 11 58RL NHL 8.4 35UACC-62 Melanoma 6.0 >75






the autophagy inhibitor bafilomycin A1 as a tool to evaluate theeffects of ROC-325 on autophagic flux (20). 786-0 cells weretreated with ROC-325 alone and in combination with bafilomy-cin A1 and the levels of LC3B and p62 were quantified byimmunoblotting. No significant differences in the levels of eitherLC3B or p62 were detected when ROC-325 was combined withbafilomycin A1, thus indicating that ROC-325 effectively inhib-ited autophagic flux (Fig. 2C). Furthermore, the addition ofbafilomycin A1 did not significantly augment the ability ofROC-325 to induce apoptosis (Fig. 2D). Our results collectivelydemonstrate that ROC-325 disrupts autophagic degradation.

The anticancer effects of ROC-325 are severely diminished incells with genetically impaired autophagy

All anticancer drugs have multiple effects. To assess whetherautophagic inhibition is a major mechanism of action that con-tributes to the anticancer properties of ROC-325, we geneticallyimpaired two different genes that have been established to beessential for functional autophagy, ATG5 (34) and ATG7 (35),using lentiviral shRNA approaches in 786-0 RCC cells (Fig. 3A).Cells with targeted knockdown of either gene proliferated moreslowly than cells infected with nontargeted control shRNA, indi-cating that functional autophagy promotes RCC cell proliferation

Figure 2.

ROC-325 inhibits autophagy. A,ROC-325 induces LC3B accumulation.RCC cells were treated with 5 mmol/LROC-325 for 24 hours. LC3Baccumulation was visualized byimmunocytochemistry. B, ROC-325increases LC3B, p62, and cathepsin Dexpression. 786-O and A498 cells weretreated with the indicatedconcentrations of ROC-325 for 24 hours.Protein levels were determined byimmunoblotting. The lower band on allLC3 blots in the manuscript depicts thelipidated (LC3-II) form of LC3B, which isan established autophagosome marker.C and D, Bafilomycin A1 does notaugment ROC-325–mediated LC3B orp62 accumulation or apoptosis. 786-Ocells were treated with 5 mmol/L ROC-325, 100 nmol/L bafilomycin A1, or bothagents for 48 hours. Protein expressionwas determined by immunoblotting andapoptosis by PI-FACS analysis. Mean �SD, n ¼ 3. � , statistically significantdifference from control; P < 0.05. Thereis no significant difference amongbafilomycin A1, ROC-325, and ROC-325þ bafilomycin A1 treatments.

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Figure 3.

ROC-325 has selective anticancer effects that are dependent upon autophagy function.A,The essential autophagy genesATG5 andATG7were knockeddown in 786-0 RCC cells using lentiviral shRNA. Knockdown efficiency was assessed by immunoblotting. Tubulin documented equal protein loading. B, Cells infected withnontargeted control, ATG5- or ATG7-directed lentiviral shRNAs were treated with the indicated concentrations of ROC-325 for 72 hours. The effects of drugtreatment on cell viability were quantified for each experimental condition by theMTT assay. Mean� SD, n¼ 3. � , P <0.05.C,ROC-325 selectively decreases RCC cellline viabilitymore effectively than HCQ. RCC cell lines and normal RPTEC cellswere treatedwith varying concentrations of ROC-325 or HCQ for 72 hours. Cell viabilitywasmeasured using theMTT assay.Mean� SD, n¼ 3.D,ROC-325 stimulates apoptosis. RCC cell lineswere treatedwith the indicated concentrations of ROC-325 for48 hours. Active caspase-3 (left) was measured using a FITC-labeled active caspase-3 antibody followed by flow cytometric analysis. Mean � SD, n ¼ 3. DNAfragmentation (right) was measured by PI-FACS analysis. Mean � SD, n ¼ 3.






and thereby affirming that autophagy inhibition may be aneffective approach to antagonize RCC pathogenesis (Supplemen-tary Fig. S6A).Genetic impairment of eitherATG5orATG7 yieldedsimilar results with respect to the anticancer effects of ROC-325 inthat the genetic disruption of autophagy rendered it significantlyless effective (Fig. 3B). Supporting experiments with HCQ alsoshowed that this agent was less effective at reducing RCC cellviability when autophagy was genetically impaired (Supplemen-tary Fig. S6B). This indicates that autophagy inhibition is a keyanticancermechanismof actionof ROC-325 at the concentrationsused in this study.

ROC-325 exhibits therapeutic selectivity and has significantlygreater anticancer activity than HCQ

We next treated four different RCC cell lines (786-0, A498,Achn, andCaki-2)with a range of concentrations of ROC-325 andHCQ for 72 hours. The effects of each agent on cell viability weredetermined by the MTT assay. A direct comparison of the activityof ROC-325 andHCQ revealed that ROC-325 was approximately10-fold more potent than HCQ based on IC50 analyses andexhibited favorable therapeutic selectivity for malignant versusnormal renal proximal tubule epithelial cells (RPTEC) treatedwith ROC-325 (Fig. 3C). Because the reduction in cell viability weobserved in RCC cells treated with ROC-325 could have resultedfrom inhibition of proliferation, cell death, or both, we nextquantified the effects of ROC-325 on apoptosis by measuringdrug-inducedDNA fragmentation and caspase-3 activation. Treat-ment of RCC cells with ROC-325 for 48 hours led to dose-dependent increases in the percentages of cells with active cas-pase-3 expression and fragmentedDNA(Fig. 3D) in amanner thatcorrelated with the reduction in cell viability. Additionally, treat-ment of 786-0 cells with ROC-325 in combination with the FDA-approved drugs vorinostat, temsirolimus, or sorafenib yieldedsignificantly greater levels of apoptosis than any single agent(Supplementary Fig. S7). This suggests that the ability of ROC-325 to trigger apoptosis accounts for a significant portion of itsanticancer effects and that it has potential applications as atherapeutic partner to existing anticancer agents.

Oral administration of ROC-325 is well tolerated andantagonizesRCC tumorprogressionmore effectively thanHCQ

The in vivo anticancer activity of ROC-325 was evaluated byadministering vehicle control (water), ROC-325 (25, 40, or 50mg/kgQD), orHCQ(60mg/kgQD) tonudemice implantedwith786-0 RCC xenografts. ROC-325 treatment led to significant,dose-dependent inhibition of disease progression in a mannerthat was superior to HCQ (Fig. 4A). ROC-325 was well toleratedand no notable toxicities were observed other than a verymodest,nonsignificant reduction inmean body weight at the highest dose(Fig. 4B). Immunohistochemical analysis of specimens collectedfrom animals treated with ROC-325, HCQ, or vehicle controldemonstrated significant, dose-dependent increases in the autop-hagic markers LC3B (Fig. 5A) and p62 (Fig. 5B) and increasedapoptosis (cleaved caspase-3; Fig. 5C). Our collective data dem-onstrate that ROC-325 is orally bioavailable, significantly moreefficacious as a monotherapy than HCQ, exhibits favorable tol-erability, and inhibits autophagy in vivo. These findings supportfurther investigation of the safety and efficacy of ROC-325 as anovel agent for the treatment of autophagy-driven tumors andother disorders where lysosomal activity contributes to diseasepathogenesis.

DiscussionThe rational ability of autophagic degradation to help fulfill the

fundamental need of tumors to maintain energy metabolism todrive their expansion, metastasis, and sustain their survival undertherapy- and microenvironment-induced stress gave rise to a newfield focused on determining the contributions of autophagy tomalignant pathogenesis. More than a decade later, autophagy hasbeendefined as amechanism that fuelsmalignant bioenergetics ina manner that contributes to both disease progression and drugresistance in a diverse range of solid and hematologic cancers(1, 2). Accordingly, a tremendous number of preclinical investiga-tions showed that genetic or pharmacological inhibition of autop-hagy diminished drug resistance and augmented the efficacy of aplethora of cytotoxic and targeted anticancer agents and radiationtherapy (5, 6). The findings of several of these studies directlyestablished the foundation for multiple early-phase clinical trialsthat investigated the safety and preliminary efficacy of the anti-malarial autophagy inhibitor HCQ in combination with several

Figure 4.

ROC-325 reduces tumor burden more effectively than HCQ in RCC xenografts.A, 786-O cells were injected into the flanks of nude mice. Mice were pair-matched and randomized into groups when mean tumor burden reachedapproximately 100 mm3. Mice were treated with 25, 40, or 50 mg/kg ROC-325PO and 60 mg/kg HCQ IP QD�5 throughout the course of the study. Tumorvolumes were measured twice weekly. Mean � SEM, n ¼ 5. � , statisticallysignificant difference in tumor burden compared with vehicle control based ontwo-way ANOVA analyses; P < 0.05. B, ROC-325 is well-tolerated in mice. Bodyweight was determined at the end of the study (day 46) to quantify drug-induced weight loss. Mean � SD, n ¼ 5.

Carew et al.





other FDA-approved anticancer agents and radiotherapy (11–16).The initial series ofHCQ combination clinical trials demonstratedthat the addition of HCQ to these specific regimens yielded anacceptable safety profile. Preliminary efficacy was observed in asmall subset of patients (such as the RCC patient on our HCQ þ

vorinostat phase I trial) treated in these clinical studies, but thequestion of whether the MTD of HCQ in these studies resulted incomplete autophagic inhibition remained unanswered.

Themodest efficacy that was observed in this first series of trialsseeking to deliberately inhibit autophagy as a novel therapeutic

Figure 5.

ROC-325 significantly increases LC3Band p62 expression and inducesapoptosis in RCC xenografts. A,Tumors were harvested 6 hours afterthe last dose of each drugwas administered. LC3Bimmunohistochemistry. Tumors werestained with an anti-LC3B antibody,and the relative intensity ofexpression was quantified bydensitometry. Mean � SD, n ¼ 5.B, p62 immunohistochemistry.Tumors were stainedwith an anti-p62antibody, and the relative intensity ofexpression was quantified bydensitometry. Mean � SD, n ¼ 5. C,Apoptosis was determined by activecaspase-3 immunohistochemistry.Tumorswere stainedwith an antibodyto cleaved caspase-3. The percentageof positive stained cells wasdetermined manually under 20�magnification. Mean � SD, n ¼ 5.� , statistically significant differencefrom vehicle; P < 0.05.






strategy likely stemmed from two major issues. First, the lack ofvalidated predictive biomarkers that define genetic features oftumors that render them autophagy-dependent prevented therefinement of the eligibility criteria in a way that would facilitatethe targeted enrollment of specific patients that may be morelikely to benefit from treatment with autophagy inhibitors. It hasbeen reported in preclinical studies that mutations in RAS conferautophagy addiction and that RAS-driven cancers may be hyper-sensitive to autophagy inhibition (36, 37). However, this has notbeen clinically proven to date. It is possible that the importance ofRAS in this context may be tumor type specific and that otheroncogenes with mechanistic links to the control of autophagymay also be involved in determining autophagy dependence. Inorder to ultimately position autophagy inhibitors to effectivelybenefit patients, it is imperative to define a genetic, epigenetic,metabolic, and phenotypic tumor signature that predicts suscep-tibility to this precision therapeutic approach. Additionally, theanticancer agents whose efficacy is most significantly reduced bytherapy-triggered autophagy must be established to identify opti-mal and rational partners for combination regimens. Advances inboth of these areaswould dramatically improve the opportunitiesfor this class of drugs to successfully impact cancer outcomes.

A secondmajor underlying cause of the limited efficacy that weand our colleagues observed is likely due to the pharmacologicproperties of HCQ itself. HCQ has been used for the treatment ofmalaria, rheumatoid arthritis, and systemic lupus for many years.However, it was not designed to be an autophagy inhibitor.Rather, it has been attempted to be repurposed as such due tothe lack of rationally designed autophagy inhibitors available forclinical use and the overwhelming interest to quickly translate theexcitement of preclinical findings regarding autophagy inhibitioninto potential therapeutic benefit for patients with cancer. Thus,the limited clinical efficacy of HCQ in this patient populationcould be due, in part, to the insufficient inhibition of autophagy atdoses of HCQ that yield acceptable safety and tolerability.

We sought to develop new agents that exhibited both super-ior autophagic inhibition and anticancer activity than HCQ.Because no comprehensive SAR studies determining whichchemical motifs are essential to disrupt autophagy at thelysosomal level have been conducted to date, we analyzed thestructure of HCQ and other reported agents that inhibit autop-hagy and designed a series of new compounds with variousmodifications to key chemical motifs in HCQ and these otherdrugs. Our initial screening assays identified ROC-325 as a leadcompound. Notably, the structure of ROC-325 contains ele-ments of both HCQ and LUC, which we previously discoveredto inhibit autophagy, but is chemically distinct from Lys05,another new dimeric agent that has been demonstrated toinhibit autophagy (17, 20).

Comprehensive preclinical studies with ROC-325 demonstrat-ed that it induced all of the hallmark features of genetic andpharmacologic autophagy inhibition including the formation ofLC3Bpunctae, accumulation of autophagosomes, stabilization ofp62, and disruption of autophagic flux. ROC-325 also triggeredthe expression of the lysosomal protease CTSD, which we previ-ously showed plays a key role in mediating CQ/HCQ and LUC-induced apoptosis in addition to other genes with establishedroles in controlling protein degradation and endoplasmic retic-ulum (ER) stress-induced cell death (20, 22, 30). Preliminarystudies demonstrated that ROC-325 also appears to augment theanticancer effects of several FDA-approved drugs. Although all ofthese findings are scientifically important and collectively definethemechanism of action of this new drug, themost critical aspectof this study is the evidence demonstrating that ROC-325 is orallybioavailable and inhibits autophagy in vivo while yielding signif-icantly greater single-agent efficacy against RCC xenograft tumorsthan a higher dose of HCQ administered on the same schedule.Taken together, our findings establish ROC-325 as a novel autop-hagy inhibitor that warrants further investigation to better defineits safety and efficacy for the treatment of autophagy-dependentmalignancies and other lysosome-centric disorders.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J.S. Carew, J. Phillips, S.T. NawrockiDevelopment ofmethodology: J.S. Carew, C.M. Espitia, J. Phillips, S.T. NawrockiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.S. Carew, C.M. Espitia, W. Zhao, Y. Han, V. Visconte,J. Phillips, S.T. NawrockiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.S. Carew, C.M. Espitia,W. Zhao, Y.Han, V. Visconte,J. Phillips, S.T. NawrockiWriting, review, and/or revision of the manuscript: J.S. Carew, J. Phillips,S.T. NawrockiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C.M. Espitia, W. Zhao, Y. Han, J. Phillips,S.T. NawrockiStudy supervision: J.S. Carew, S.T. Nawrocki

Grant SupportThis work was supported by grants from the Scott Hamilton CARES Initiative

(J.S. Carew) and the National Cancer Institute (R01CA172443, J.S. Carew;R01CA190789, S.T. Nawrocki; and P30CA023074).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 8, 2016; revised November 10, 2016; accepted November 14,2016; published OnlineFirst November 23, 2016.

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2017;23:2869-2879. Published OnlineFirst November 23, 2016.Clin Cancer Res Jennifer S. Carew, Claudia M. Espitia, William Zhao, et al. Renal Cell Carcinoma PathogenesisDisruption of Autophagic Degradation with ROC-325 Antagonizes

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