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Cell Death and Survival

Spindle Assembly Disruption and Cancer CellApoptosis with a CLTC-Binding CompoundMichael J. Bond1,2, Marina Bleiler1, Lauren E. Harrison1, Eric W. Scocchera3,Masako Nakanishi4, Narendran G-Dayanan3, Santosh Keshipeddy3,Daniel W. Rosenberg4, Dennis L.Wright3, and Charles Giardina1

Abstract

AK3 compounds are mitotic arrest agents that induce highlevels of gH2AX during mitosis and apoptosis followingrelease from arrest. We synthesized a potent AK3 derivative,AK306, that induced arrest and apoptosis of the HCT116colon cancer cell line with an EC50 of approximately 50nmol/L. AK306 was active on a broad spectrum of cancercell lines with total growth inhibition values ranging fromapproximately 25 nmol/L to 25 mmol/L. Using biotin andBODIPY-linked derivatives of AK306, binding to clathrinheavy chain (CLTC/CHC) was observed, a protein with rolesin endocytosis and mitosis. AK306 inhibited mitosis andendocytosis, while disrupting CHC cellular localization.Cells arrested in mitosis by AK306 showed the formationof multiple microtubule-organizing centers consisting ofpericentrin, g-tubulin, and Aurora A foci, without apparent

centrosome amplification. Cells released from AK306 arrestwere unable to form bipolar spindles, unlike nocodazole-released cells that reformed spindles and completed divi-sion. Like AK306, CHC siRNA knockdown disrupted spindleformation and activated p53. A short-term (3-day) treat-ment of tumor-bearing APC-mutant mice with AK306increased apoptosis in tumors, but not normal mucosa.These findings indicate that targeting the mitotic CHCcomplex can selectively induce apoptosis and may havetherapeutic value.

Implication: Disruption of clathrin with a small-moleculeinhibitor, AK306, selectively induces apoptosis in cancercells by disrupting bipolar spindle formation. Mol Cancer Res;16(9); 1361–72. �2018 AACR.

IntroductionMitotic disruption can trigger a number of cell responses,

ranging from cell-cycle arrest and repair, to senescence, mitoticcatastrophe, and apoptosis (1, 2). How the cell makes thisdecision is not entirely clear, but it appears to involve the natureof the damage and a range of cell-specific variables. One pathwaythat can link aberrant mitosis with cell death is the p53 pathway(3–7). Cells arrested in mitosis can activate the ATM–p53 path-way, which can promote apoptosis directly, or after an aberrantcell division. Cells stalled in mitosis can also become moresensitive to death ligands, which may provide an additionalmeans of eliminating cells at risk of undergoing an aberrantdivision (8, 9). A better understanding of howmitotic disruption

can lead to apoptosis could provide insight into the mechanismsprotecting cells from aneuploidy.

Cancer cells often carry mutations and other defects thatdebilitate the mitotic checkpoint. Mutations in genes for BUB1,BUBR1,MAD1, andMAD2 can directly affect the ability of cells tosense the spindle assembly checkpoint, whereas mutations inATM, ATR, and p53 can interfere with a cell's ability to translatethis checkpoint into apoptosis (10–17). In contrast with the lossof checkpoint proteins, cancer cells frequently overexpressmitotickinases such as the Aurora and Polo-like kinases (18–21). Theoverexpression of these kinasesmay serve to drivemitosis, even inthe presence of cellular damage (22). Aberrant mitoses can resultdirectly in the generation of aneuploid cells. In addition, failedcytokinesis can result in the generation of tetraploid cells, whichare at an elevated risk of erroneous chromosome segregation (23–25). Although the mitotic defects in cancer cells can lead tochromosomal instability, these defects may also increase cancercell sensitivity to agents that interfere with mitosis (26–28).

We have been studying a class of compounds selected for theirability to arrest cells in amitotic state that transitions to apoptosiswith high efficiency. These "AK3" compounds induce a mitoticarrest state with elevated levels of gH2Ax that resolves into a p53-facilitated cell death following release from arrest (8, 9, 29). Thesecompounds can also enhance ligand-induced cell death in p53-mutant cells through amechanism that involves a tighter couplingbetween the death receptors and caspase-8. Although the AK3compounds have an affinity for tubulin, their apoptotic potencycannot be easily explained by targeting tubulin alone (8); tradi-tional spindle poisons do not induce gH2Ax signaling as robustlyas the AK3 compounds and are less apoptotic (29). Because

1Department of Molecular and Cell Biology, University of Connecticut, Storrs,Connecticut. 2Department of Pharmacology, Yale University, New Haven,Connecticut. 3Department of Medicinal Chemistry, University of Connecticut,Storrs, Connecticut. 4Center forMolecularOncology, UConnHealth, Farmington,Connecticut.

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

C. Giardina is the lead author of this article.

Corresponding Author: Charles Giardina, University of Connecticut, 91 NorthEagleville Road, Storrs, CT 06269. Phone: 860-486-0089; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-18-0178

�2018 American Association for Cancer Research.

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gH2AX levels are increased specifically inmitotically arrested cells,we have proposed that the AK3 compounds cause DNA shearingthroughaberrant spindlepulling (aspreviouslydescribed; ref. 30),although we cannot rule out other mechanisms such as DNA/chromatin binding or DNA repair enzyme inhibition.

To better understand how this class of molecules works, wegenerated a series of AK3 derivatives for structure–activityrelationship (SAR) studies. Based on these finding, we con-structed affinity and fluorescent probe molecules, and foundselective binding to clathrin heavy chain (CHC), a proteininvolved in endocytosis and mitosis. Our findings support acentral role of CHC in centrosome complex integrity and reveala close linkage between CHC-directed centrosome complexmaintenance and the regulation of cell death signaling. Giventhe range of centrosome defects found in cancer cells, our dataindicate that CHC-directed centrosome assembly pathways area potentially fruitful target for novel therapeutic agent devel-opment (26, 31, 32).

Materials and MethodsCell culture

HCT116 colon cancer cell linewas obtained from the AmericanType Culture Collection. The p53-mutant HCT116 cell line wasthe kind gift of Dr. Bert Vogelstein. The p53-null status of thesecells was confirmed by Western blotting. Cells were culturedinMcCoy's 5Amedium, with 10% (v/v) FBS, nonessential aminoacids, and antibiotic/antimycotic (Gibco, Thermo Fisher).The immortalized primary colon cell line, Young Adult MouseColonocytes (YAMC), was a gift from Dr. R Whitehead (Vander-bilt University, Nashville, TN). YAMCs were cultured in RPMImedium containing 5% FBS, nonessential amino acids, antibi-otic/antimycotic, insulin–transferrin–selenium, and 5 units ofmurine gamma interferons (Gibco), and were grown at 33�C.Cell lines were expanded and frozen upon receipt with freshcultures started every 3 to 4 months. Cells are examined formycoplasma contamination at least monthly by DAPI staining.

Chemicals and reagentsCompounds were synthesized as described below. Nocodazole

was purchased from Sigma. Transferrin-Alexa Fluor 488 waspurchased from Molecular Probes Thermo Fisher. All treatmentswith chemicals were performed approximately 24 hours afterpassage unless otherwise indicated. AK306 was used at a concen-tration of 100 to 200 nmol/L, unless otherwise indicated.

Chemical synthesisAll reactions, unless specified, were conducted under an atmo-

sphere ofArgon in glassware that hadbeenflamedried.Methylenechloride (CH2Cl2) was bought from Baker Cycle-Tainers. Anhy-drous toluene, triethylamine, and dimethylformamide were pur-chased from Sigma-Aldrich. N-Boc-piperazine, 2-ethoxy benzoicacid, 2-propoxy benzoic acid, and 1,4-benzodioxan-5-carboxylicacidwere purchased fromAK Scientific. 2-ethoxybenzoyl chlorideand 1-bromo-3(trifluoromethoxy)benzene were bought fromAlfa Aesar. 1-bromo-3,5-dichlorobenze was bought from AcrosOrganics. 2-ethoxy-4-nitro benzoic acid was purchased fromSigma-Aldrich. Boc-5-aminovaleric acid was purchased fromChem-Impex Int'L INC. NHS-Biotin was purchased from APEx-BIO. BDP FL NHS ester was bought from Lumiprobe Life ScienceSolutions. Where appropriate, control of temperature was

achieved with a Neslab Cryocool CC-100 II immersion cooler,ice bath, or a heated oil bath. Flash chromatography was per-formed on Silica Gel, 40 microns, 32-63 flash silica, and/or -NH2capped spherical silica gel. High-performance liquid chromatog-raphy was performed on Phenomenex C18 silica gel column,5 microns 250 � 4.60 mm, and monitored using the ShimadzuSIL-20AC equipped with a UV detector. Thin layer chromatogra-phy was performed on silica gel (Silica Gel 60 F254) glass plates,and the compounds were visualized by UV and/or potassiumpermanganate stain. Details on specific compounds are providedas Supplementary Information File S2.

Flow cytometryCells were analyzed for DNA content by ethanol fixation and

staining with propidium iodide as previously described (33).The stained cells were filtered prior to analysis on FACSCaliburflow cytometer (BD Biosciences) using Cell Quest software (BDBiosciences). The data were analyzed using FlowJo (TreeStar Inc.).

Affinity chromatography and BODIPY probingPierce Avidin Agarose (Thermo Scientific) slurry was added to

two tubes, washed with 10 column volumes of PBS to removepreservative sodium azide, and suspended in 1 column volume ofWash Buffer (PBS, 2 mmol/L MgCl2, 0.1% protease inhibitorcocktail, 0.1% Triton X-100, 1 mmol/L DTT). Molar excess ofbiotinylated AK328 (relative to the avidin bead capacity)was added to the first tube. As a control, an equimolar amountof D-(þ)-biotin (Sigma-Aldrich) was added to the second tube.Themixtures were then incubated for 1 hour at room temperatureto allow binding of biotin to avidin and washed twice with 10column volumes of PBS to remove unboundmaterial. Whole-cellextract of HCT116 cells prepared withM-PER buffer was added tothe tubes, and the solutions were incubated at 4�C overnightunder mild agitation to allow the compound to bind to cellularproteins (�15 mg of protein per column). The slurries were thenpacked into two Gravity Flow Poly-Prep Chromatography col-umns (Bio-Rad), and the flow through was collected. Columnswerewashedwith 10 columnvolumes ofWashBufferwith the lastwash fraction collected to check for efficiency of the washing step.The proteinswere elutedwith 5 sequential column volumes of theElution Buffer (PBS, 2 mmol/L MgCl2, 1X protease inhibitorcocktail, 0.1% Triton X-100, 1 mmol/L DTT). For SDS elution,the slurry was heated to 90�C for 5 minutes in the presence of 1%SDS prior to elution. The proteins in each elution fraction wereconcentrated using PierceTM 3K MWCO PES Protein Concentra-tors (Thermo Scientific). Before loading in the concentratorchamber, fractions were adjusted to 1% SDS to prevent proteinbinding and clogging of themembrane. Concentrated eluates, cellextract, flow through, and wash fractions from each column werethen analyzed using SDS-PAGE. Proteins were separated on SDSpolyacrylamide gels (Bio-Rad) and stained with SYPRORuby RedGel stain (Bio-Rad). Bands were excised from the gel and sentto the Yale/Keck MS and Proteomics Resource facility formass spectrometry. To confirm specific CHC binding to thecolumn, the column was eluted with 1 mmol/L AK306 (atroom temperature) with the resulting fractions analyzed byWestern blotting using a CHC primary antibody (P1663; CellSignaling Technology) and a IRDye 680RD Goat anti-Rabbitsecondary antibody for detection (LI-COR Biosciences). Imageswere acquired and analyzed using the LiCor Odyssey CLxImaging System.

Bond et al.

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To determine the affinity of purified clathrin for the affinitycolumn, clathrin was purified from mouse liver by differentialcentrifugation methods as previously described (34). Purifiedprotein (�70 mg) was loaded on to the affinity and controlcolumns as described above, and eluted sequentially with AK306and 1% SDS. The resulting fractions were analyzed by SDS PAGEand SYPRO gel staining.

In-gel staining using AK327-BODIPY was performed byrunning 2 mg of purified clathrin, BSA (Sigma), and tubulin(Cytoskeleton, Inc.) on duplicate SDS-PAGE gels. One gel wasstained with SYPRO. The other was washed in 25 mmol/L Tris,192 mmol/L glycine buffer to remove SDS, followed by incu-bation in HKM buffer (25 mmol/L Hepes, pH 7.4, 125 mmol/Lpotassium acetate, and 5 mmol/L magnesium acetate with1 mmol/L DTT) with 500 nmol/L AK327-BODIPY overnightat 4�C. BODIPY was then detected by illumination on a UVlight box.

Immunofluorescence microscopyCells were seeded on glass coverslips coated with 1% gelatin in

24-well plates at 1 � 105 cells per well. After 24 hours, cells werefixed with 100% ice-cold methanol for 5 minutes at –20�C or 4%paraformaldehyde for 15 minutes at room temperature followedby permeabilization with 0.5% Triton X-100 in PBS. Next, cellswere blocked in 5% serum in PBS and then incubated withprimary antibody for 1 hour at room temperature. The followingantibodies were used for these studies: b-tubulin (E7monoclonalantibody, Developmental Studies Hybridoma Bank), g-tubulin(ab11316, Abcam), Aurora A (630938, BD Transduction Labo-ratories), centrin-3 (PA5-66623, Invitrogen), CHC, s-20 (sc-6579,Santa Cruz Biotechnology), Pericentrin (PCNT; ab4448, Abcam),and Phospho p53, s-15 (9284, Cell Signaling Technology).Appropriate secondary antibodies (Life Technologies or JacksonImmunoResearch) were used for 45-minute incubation. Nucleiwere visualized using DAPI (5 mg/mL in PBS; DI306, Life Tech-nologies). Coverslips were mounted on slides using ProLongGold Antifade Reagent (Life Technologies). For experiments inwhich cells were stained with AK327-BODIPY, antibody stainingwas performed first, followed by incubation with 500 nmol/LAK327-BODIPY in PBS and direct imaging (without mounting).

Images were acquired using Nikon A1R confocal laser micro-scope and NIS-Elements Advanced Research Software (version4.4, Nikon Instruments Inc.). Image brightness and contrast weremodified when appropriate. Background subtraction was accom-plished by setting the minimum displayed brightness/contrastvalue to the value of a region of known background. Quantifica-tion of immunostaining was performed using ImageJ/FIJI imageprocessing software (http://fiji.sc/). The pixel value thresholdswere set manually at identical ranges. Menu command Analyse/Analyse particleswasused to generate the IntegratedDensity value(integrated fluorescence intensity) of the area of interest, whichwas then divided by the number of nuclei in the area to obtainIntegrated Density value per cell. Calculations were conducted inMS Excel.

Endocytosis assayHCT116 cells plated onto glass cover slips were serum starved

overnight in McCoy's media plus 0.5% BSA. Cells were thentreated with DMSOor varying concentrations of AK306 dissolvedinDMSO for 30minutes at 37�C. AK306 treatment was limited to30 minutes to limit the mitotic arrest action of the compound.

Media were then removed, and fresh media containing 0.5%Transferrin (Tfn) Alexa Fluor 488 Conjugate (Thermo Fischer)were added. Cells were then placed at 37�C for 30 minutes. Afterexposure to transferrin, media were removed, and cells werewashedoncewith PBS and thenfixedwith 4%PFA for 10minutes.Cells were then imaged as described above. Following back-ground subtraction and image stacking, both DAPI and fluores-cent Tfn channels were merged. Images were processed using FIJI.Cells were analyzed using the particle analysis plugin, standardwith ImageJ image analysis software (http://rsb.info.nih.gov/ij).Image brightness and contrast were modified with Adobe Photo-shop software CS6 (Adobe Systems).

Live cell imagingCells were plated on 8-well Nunc Lab-Tek II Chambered

Coverglass slides at 2 � 104 cells per well. To visualize micro-tubules, cells were incubated with CellLight Tubulin-GFP con-struct (Invitrogen) for 24 hours. Time-lapse imaging was per-formed in a humidified CO2 chamber at 37�C using the NikonA1R confocal microscope and NIS-Elements software describedabove. Images were acquired every 45 seconds as z-stacks withthe spacing between slices of 0.3 mm. Videos were projected andadjusted for background and brightness/contrast using ImageJ/FIJI software as described above.

siRNA transfectionON-TARGET plus SMART pool siRNA duplexes against CHC

and nontargeting siRNA were obtained from GE Dharmacon.Double reverse siRNA transfection was performed in a 24-wellplate using Lipofectamine RNAiMAX (Invitrogen). Briefly, cellswere lifted using trypsin-EDTA, diluted in OPTI-MEM medium(Life Technologies), pelleted, and resuspended in OPTI-MEMmedium. siRNA duplexes diluted in the samemedium to achievefinal concentration of 100 nmol/L and 4 mL of LipofectamineRNAiMAX were added to each well. Note that 500 mL of resus-pended cells were added on top of the siRNA–Lipofectaminemixture. Six hours later, the OPTI-MEM medium was replacedwith the regularMcCoy's 5A. After 48 hours, the same transfectionprocedure was repeated. The cells were then incubated for 36hours before proceeding with immunofluorescence (IF).

Mouse treatment and tissue analysisApcD14/þ mice were kindly provided by Dr. Christine Perret at

the Universite' Paris and maintained in the animal facility atUConn Health (35). All mice were maintained in a light-cycled,temperature-controlled room and allowed free access to drinkingwater and diet ad libitum. All animal experiments were conductedwith approval from the Center for Comparative Medicine atUConn Health. Genotyping for Apc was performed using tailbiopsies (35). Sixteen-week-old male ApcD14/þ mice were admin-istered five i.p. injections of AK306 or vehicle over the course of 3days: injections were performed at 10 am and 5 pm on days 1 and2, and at 10 amonday3. Animalswere sacrificed at 3 pmonday 3.Each dose of AK306 was 30 mg/kg in 200 mL. Control animalsreceived 200 mL of vehicle (saline with 20% DMSO cosolvent).Three animals were in the vehicle group and four were in thetreated group.

After dosing, intestinal tissue was laid flat on filter paper, fixedin formalin overnight, and switched to 70% ethanol in themorning. Small intestines were rolled, embedded in paraffin, andsectioned onto glass slides (20 m). For immunostaining, tissue

Spindle Assembly Disruption and Apoptosis by AK306

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sampleswere deparaffinized and rehydratedwith antigen retrievalperformed in a pressure cooker using sodium citrate (pH 6.0).Tissues were blocked with 1% donkey serum in PBS, and primaryantibodies were applied in incubation buffer (1% BSA, 1%donkey serum, 0.3% Triton X-100, 0.01% sodium azide in PBS).Cy-3–conjugated secondary antibodies were used for detection,followed by DAPI staining and mounting. Samples were imagedusing a Leica SP8 confocal microscope. Z-stack images werecaptured and processed using ImageJ/FIJI software. Stacks werefirst separated by color and then averaged before beingmeasured.Each color channel was thresholded using the Yen algorithm.Menu command Analyze particle was used to count the numberof stained nuclei.

For terminal deoxynucleotidyl transferase–mediated dUTPnick end labeling (TUNEL) staining, sections were deparaffinized,rehydrated, and stained using the Trevigen TACS TdT Kit. Tissuesections were counterstained with DAPI and mounted. Sampleswere imaged using Nikon A1R confocal microscope, stacked, andprocessed with ImageJ/FIJI using the same parameter settings.Selective borders were manually drawn around tumor area, colorchannelswere separated, backgroundwas subtracted, imageswerefiltered and automatically thresholded (the Otsu algorithm wasused for green channel and the Percentile for DAPI channel),merged cells were separated using Watershed function, and cellswere counted using particle analyzing tool.

Statistical analysesA Student t test was used for comparing two treatment groups.

An ANOVA test was usedwhen comparingmore than two groups.A Tukey post hoc test was used to determine the significance ofdifferences between multiple groups. Significance was calculatedat an alpha of 0.05.

ResultsSAR study

We performed SAR studies on the AK301 compound with theintention of synthesizing molecular probes to identify possiblecellular targets.Our goalwas to createmorepotent compounds to:

(1) offset the loss in efficacy that typically accompanies theattachment of biotin or fluorescent molecules and (2) identifysites for attachment of these probe molecules. From previousstudies, we knew that the position of the chlorine on the phenylring and the position and length of the ether group on the benzoylring were important for maximal activity (8, 29). We thereforefocused on these two areas of the molecule. Figure 1 shows thenew derivatives generated from this study. A cell-cycle assay wasused to measure the percentage of cells arrested in G2–M. EC50

values were obtained from titration curves plotting the percentageof arrested HCT116 colon cancer cells (Fig. 1).

We first manipulated the phenyl ring of AK301. Because havingone chloro-group in the meta-position decreased EC50, we decid-ed to incorporate another chloro at the other meta-position.Because the chlorophenyl ring can rotate about its bond to thepiperazine, we reasoned that placement of another chlorine couldincrease the number of configurations that interact with thecellular target. Alternatively, the additional chlorine could resultin additional interactions within a hydrophobic space. Thishypothesis leads to the development of a dichlorobenzene deriv-ative named AK306. The EC50 was decreased by this modificationfrom approximately 150 to 75 nmol/L. Our next compoundaimed to examine the size of the hydrophobic pocket at thisposition. We developed a molecule with a trifluoromethyl ether(-OCF3) replacing the chlorine on AK301. OCF3 encompassesmore space than a chloro-group, but is similarly hydrophobic.Interestingly, the OCF3 derivative, called AK307, was significantlyless potent than AK301 with an EC50 near 800 nmol/L. Thisfinding implies that the hydrophobic pocket inwhich the chlorineis interacting is relatively small.

With the success of AK306, we decided to fix the dichlorophenyl group and continue SAR with the benzoyl ring. Frompreviouswork, we knew that the length of the ether contributed toefficacy, with an ethoxy group being more effective than a meth-oxy group (8, 29). To test whether increasing the length of thisgroup increased the activity further, a derivative was synthesizedwith a propoxy group. This derivative, AK311, did not havesignificantly higher potency than AK306. We therefore did notpursue a 4 or 5 carbon ether functionality. A second modified

Figure 1.

Compounds synthesized and tested in this study. The table indicates the EC50 concentration required to induce a G2–M arrest in 50% of HCT116 cells, following anovernight/16-hour exposure. The maximum percentage of cells arrested in G2–M was determined at approximately 4 times the EC50.

Bond et al.





benzoyl derivative was synthesized to determinewhether decreas-ing the flexibility of the ethoxy group improved activity. Wereplaced the ether moiety with 1,4 dioxane, a cyclic acetal. Thisderivative, named AK313, had a higher EC50 than AK306, indi-cating thatflexibility of the etherwas beneficial for activity.Wedidnot pursue further modifications on this site.

To find an attachment site for biotin and other probe mole-cules, we first attached an amino group, which can readily form apeptidebondwithNHSesters.Wedecided to introduce the aminogroup to the benzoyl ring because it appeared that the dichlorophenyl ring was important for activity. This aniline derivative,called AK327, was tested in HCT116 colon cancer and found tohave similar mitotic arresting activity to AK306. Once AK327 wassynthesized, we began attaching groups that could aid in thebiochemical identification of target proteins. AK328, which has abiotin attached to AK327, showed activity in HCT116 coloncancer cells and was therefore deemed useful for target identifi-cation purposes.

Finally, we tested the solvent accessibility of the benzoyl groupof AK327 at the amino group. The biotin group attached to thissite in AK328 includes a flexible carbon linker thatmay be solventaccessible. To determine how much space was available in thispart of themolecule, an isovaleric acid andbenzoic acid derivativewere synthesized (AK330 and AK332, respectively). Isovalericacid incorporates steric hindrance due to a branch point twocarbons from the acid carbonyl. Interestingly, this derivative was

inactive. Next, we tested AK332. Like with AK330, AK332was alsoinactive at concentrations of up to 4 mmol/L. However, if fourmethylenes were included before the branch point, activity of thecompound was improved (AK331). These data indicated that acarbon linker of at least four methylene groups was necessary forsolvent accessibility at this location.

Effect of compounds on G2–M arrest and apoptosisWe moved forward with an analysis of AK306, because it had

high potency, good solubility, and reproducible activity. Previouswork with this family of compounds indicated that they inducedmitotic arrest that resolved into apoptosis with relatively highefficiency following compound withdrawal (29). Cell-cycle dis-tribution analyzed by flow cytometry showed that both p53-nulland wild-type HCT116 cells arrested in G2–M at similar AK306concentrations (Fig. 2A). However, release of cells from arrest bycompound withdrawal leads to significantly more apoptosis ofp53-normal cells over a 24-hour period following release (Fig.2B). In addition to inducing apoptosis following release, titrationexperiments with AK306 showed that apoptosis could also beobserved at concentrations just below that required to induce amitotic arrest (Fig. 2C). This finding suggests that mitotic arrestand the apoptotic response are not strictly linked and that arrest inmitosis may preclude cells from entering apoptosis.

To assess the sensitivity of other cancer cell lines to AK306,the NCI-60 cancer cell line panel was analyzed (Supplementary

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The impact of p53 on AK306-induced G2–M arrest and apoptosis. A, A similar dose-dependent G2–M arrest in p53–wild-type and -mutant HCT116 cells.G2–M arrest was determined by flow cytometry. B, HCT116 cells were arrested with AK306, and then released from arrest by compound withdrawal (media change).The p53–wild-type cells showed higher levels of apoptosis between 2 and 24 hours, as quantified by cell fragmentation. C, A spike in p53-promotedapoptosis was observed a 50 nmol/L AK306. At higher concentrations, cells were arrested in G2–M. Asterisks indicate a significant increase in apoptosis inp53–wild-type-treated cells (P < 0.01). D, Schematic of affinity chromatography experiments to identify cellular targets. HCT116 cell extracts were passed over anavidin column bound with AK328-biotin or biotin (a negative control). Bound proteins were then eluted with SDS or AK306. E, Columnswere run as described in 2D.Shown are the final wash and the SDS elution from a biotin and an AK328-biotin column analyzed by SDS PAGE and SYPRO staining. The 200 kDa band enrichedin the AK328-biotin eluate was excised and identified as CHC by LC-MS. F, HCT116 cell extract was loaded on the AK328-biotin column and then eluted with1 mmol/L AK306. CHC was detected byWestern blotting in the load, flow through (FT), final wash, and AK306 eluate. G, CHC purified from mouse liver extract wasanalyzed by SYPRO staining (left plot). Purified CHC was loaded on an AK328-biotin or biotin control columns, eluted with sequential AK306 washes (1 mmol/L,E1 and E2), followed by an SDS elution. The resulting fractions were analyzed by SDS-PAGE followed by SYPRO staining (right plots).






Fig. S1). A wide range of sensitivities was observed. Of note,direct lethality was observed in some p53-mutant cell lines(at submicromolar concentrations), including HL-60 (leukemia),NCI-H522 (lung cancer), COLO205 and HT-29 (colon cancer),and MDA-MB-435 and M14 (melanoma). These data indicatethat cell sensitivity to AK306 is affected by a range of factors anddoes not always require p53 for cell killing.

Identification of CHC as a target of AK3 compoundsWe took advantage of our biotin-linked compound, AK328, to

identify potential cellular targets. Figure 2D shows the generalstrategy of our approach. Whole-cell extracts from HCT116 cellswere loaded on streptavidin columns coupled to biotin or thebiotinylated AK328 compound. Following extensive washing,elutionwas performedwith either SDSor AK306. Figure 2E showsthe results when elution was done with SDS and the resultingfractions analyzed on an SDS gel. A number of proteins werefound to elute from the columns, but a protein at approximately200 kDa appeared to be selectively retained on the AK328column. This 200 kDa protein was excised from the gel andanalyzed by LC-MS, which identified the192 kDa CHC. To con-firm the presence of CHC, and to obtain additional evidence forspecificity, cell extractwas again loaded on theAK328 column andelution was performed with AK306 (1 mmol/L). CHCwas presentin this elution, as determined by immunoblotting (Fig. 2F).Finally, to ensure the compounds were binding directly to CHC,CHCwas purified frommouse liver and tested for AK328 columnbinding. As shown in Fig. 2G, purified CHC bound to the AK328

column and could be eluted with AK306 (with residual bindingeluted with SDS), whereas CHC binding to the biotin controlcolumn was not detected.

We also took advantage of a BODIPY-linked compound,AK327-BODIPY. Although this compound was insufficientlypotent to induce G2–M arrest in the HCT116 cell line, it was ableto arrest the YAMC mouse colonocyte cell line at concentrationsabove 400 nmol/L (Supplementary Fig. S2). We therefore deter-mined the ability of AK327-BODIPY to bindmouseCHC in an in-gel staining experiment. Purified CHC (2 mg) was run on an SDSPAGE gel, along with comparable amounts of BSA and tubulin, asdetermined by SYPRO Ruby Gel Stain (Fig. 3A, top). A duplicategel was then soaked in Tris-glycine buffer to remove SDS andthen in HKM buffer to stabilize CHC. AK327-BODIPY was thenadded to the gel to determine protein binding (500 nmol/L,overnight). As shown in Fig. 3A, AK327-BODIPY was boundto CHC. There was also detectable binding to tubulin, but lessto BSA. This finding supports the ability of the AK327-BODIPY tobind CHC. We also determined the cell-staining capabilities ofAK327-BODIPY. YAMC cells were first fixed and immunostainedfor CHC, followed by treatment with 200 nmol/L AK327-BOD-IPY. As shown in Fig. 3B, considerable overlap between CHC andAK327-BODIPY is observed, consistent with AK327-BODIPYbinding CHC.

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A, AK3-BODIPY binding to CHC. Purified CHC, BSA, and tubulin were run on duplicate SDS gels. The top gel was stained with SYPRO, and the bottom gelwas incubatedwith AK327-BODIPY (500 nmol/L overnight in protein renaturation buffer). Both gels were viewed under UV/blue light illumination.B, Colocalizationof CHC and AK3-BODIPY staining. YAMC cells were fixed and stained for CHC using Cy3 (red fluorescence). AK3-BODIPY was applied approximately 30 minutesprior to cell imaging. The BODIPY signal was found to concentrate in areas where CHC was highest. The bar shown is 20 mm. C, Endocytosis assay. HCT116cellswere treatedwith AK306 for 30minutes, followed by addition of transferrin (TFN) conjugated toAlexa-488 (for 30minutes). Cellswere then fixed and analyzedby confocal microscopy. Bar indicates 20 mm. D, Dose-response comparing the inhibition of TFN-Alexa 488 internalization and G2–M arrest.

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endocytosis, we analyzed the impact of AK306 on fluorogenictransferrinuptake. As shown inFig. 3C,HCT116 cells takeupGFP-labeled transferrin and rapidly transport it to the perinuclearregion. AK306 prevented the trafficking of transferrin, leavingmuch of the signal at the plasma membrane (Fig. 3C). An AK306titration shows that endocytosis disruption and mitotic arrestoccur at similar concentrations (Fig. 3D). These data support thepossibility that both the mitotic and endocytic effects of AK306occur via the same target, potentially CHC, although indirectaffects through the interaction with other targets cannot yet beruled out.

We also determined the effect of AK306 on the cellularlocalization of CHC. Figure 4A shows the results of an immu-nostaining experiment performed on cells treated with AK306for 1 and 2 hours. These images reveal a rapid dispersal of theCHC away from the perinuclear region by 1 hour, and asignificant reduction in cell staining at 2 hours. These findingsindicate a rapid effect of AK306 on CHC cellular localizationconsistent with a direct binding of CHC. The decrease inimmunofluorescent staining was not mirrored by a decreasein CHC levels, as determined by Western blotting (Fig. 4B). Theeffect of AK306 on CHC IF is therefore more likely the result ofepitope masking. Figure 4C shows the quantification of CHC IFand Western blotting.

Mitotic effects of AK306CHC knockdown has been reported to disrupt mitosis

through a number of mechanisms, including the assembly andstability of the centrosome complex (38–41). We thereforeexamined the impact of AK306 on centrosome complex assem-bly by immunofluorescent staining for PCNT and g-tubulin.Treatment of HCT116 cells with AK306 resulted in the frag-mentation of PCNT and g-tubulin staining, consistent withcentrosome complex disruption (Fig. 4D and E). Whereascontrol cells displayed normal bipolar spindles with two PCNTfoci or two g-tubulin foci, AK306 cells typically displayedmultiple puncta. Quantification of PCNT staining indicated asignificant increase in the number of PCNT foci after AK306treatment (Fig. 4F).

The disrupted centrosome complex assembly was not repairedafter AK306 withdrawal. Figure 5A shows the formation of mul-tipolar centrosome assemblies following removal of AK306,based on PCNT staining. In addition, TACC3, a protein recruitedto the centrosome and mitotic spindle in a complex with CHC,was also mislocalized in released cells (Fig. 5A). Analysis of cellsexpressing GFP-tagged b-tubulin likewise showed multipolarspindle formation following AK306 withdrawal (Fig. 5B). Thisresponse can be compared with cells released from a reversiblearrest induced by themicrotubule disruptor nocodazole (Fig. 5C).

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A, Dispersion and reduction in CHC staining in response to AK306 treatment. HCT116 cells were treated with AK306 for 1 or 2 hours. Treated cells werefixed and stained with CHC antibody. Confocal images show disrupted CHC distribution and reduced CHC staining in AK306-treated cells. B,Western blot of CHCexpression after 1 or 2 hours of AK306 treatment. C, Quantification of CHC fluorescent staining and expression. The left plot quantifies the data from A andshows a reduction in CHC staining after 2 hours of treatment (P < 0.01). The right plot quantifies theWestern blot data from B. D, Effect of AK306 on PCNT. HCT 116cells treated with AK306 (for 2 hours) were immunostained for PCNT and analyzed confocal microscopy. Representative images show PCNT localized atcentrosomal foci in control cells, whereas AK306 caused the formation of multiple additional foci dispersed throughout the cells. Bar indicates 10 mm. E,Representative confocal images of cells stained for g-tubulin before and after treatment with AK306. Cells were treated as in D, with the exception that 50 nmol/LAK306 was used. F, The number of PCNT foci per cell was quantified using FIJI particle counter. Quantification of large PCNT foci (at least half the integratedfluorescence intensity of foci in control cells). The quantification indicated a significant increase in the number of foci after AK306 treatment (P < 0.01).






Whereas AK306-released cells displayedmultipolar spindles (yel-low arrows), cells released from nocodazole treatment displayednormal bipolar spindles (green arrows) andmidbodies indicativeof a successful division (red arrows). Thus, AK306-treated cellsappear to be unable to reform bipolar spindle assemblies evenafter the compound is withdrawn. This defect appears to be theresult of acentrosomal MTOC formation rather than centrioleover-replication, as centrin-3 staining showed that themajority ofcells exiting AK306-induced arrest with aberrant spindles showedonly two pairs of centrin-3 foci (Fig. 5D).

Effects of CHC knockdownTo further examine the relationship between clathrin and the

cellular effects of AK306, we utilized siRNA to knockdown CHCexpression in HCT116 cells. Figure 6A shows that CHC siRNAreduced CHC expression in HCT116 cells, relative to control,nontargeting siRNA. Cells were then analyzed by Aurora A stain-ing, a protein that normally associates with centrosomes and themitotic spindle (42). As shown in Fig. 6B and C, both CHC siRNAand AK306 disrupted Aurora A localization, leading to the for-mation of multiple foci. The number of Aurora A foci wassignificantly increased following CHC siRNA knockdown, com-pared with control siRNA (Fig. 6D).

Because AK306 induced a p53-dependent cell death, andcentrosome disruption has been shown to activate aDNAdamageresponse pathway, we analyzed the level of p53 phosphorylationat serine 15 (the ATM/ATR site) following AK306 treatment (Fig.6E, left plots) and after CHC knockdown (Fig. 6E, right plots;refs. 4, 5, 7, 29, 43, 44). Control cells had low levels of phospho-p53, whereas AK306 treatment resulted in dispersed nuclearstaining of phospho-p53. This staining concentrated into nuclearfoci following release from AK306. CHC knockdown likewiseincreased phospho-p53 staining, with dispersed nuclear stainingobserved. These data are consistent with AK306 and CHC siRNAactivating p53 though centrosome disruption.

Cancer-targeting by AK306To assess the potential anticancer effect of AK306, 16-week-old

ApcD14/þ mice received five doses of AK306 over the course of 3days (30 mg/kg per dose). On day 3, mice were euthanized, andintestinal tissuewas analyzed for apoptosis by TUNEL staining. Asshown in Fig. 7, the apoptosis rate in tumors was approximatelydoubled by AK306 administration, whereas no significant induc-tion of apoptosis was observed in normal tissue. No effect ofAK306 on proliferation or mitosis was observed (SupplementaryFig. S3). Although the apoptosis induction in the tumors was

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A, Analysis of mitotic structures following release from AK306 arrest. Release from AK306 arrest causes ectopic localization of centrosome-related proteins andabnormal mitosis. Confocal images of representative cells were collected after immunostaining for PCNT and TACC3. Mitotic abnormalities include multipolarspindles, an inability to line up chromosomesat themetaphaseplate, andmultipolar cell division. Thebar shown is 5mm.B,Time-lapse images showing tripolarmitosisafter removal of AK306, compared with normal bipolar mitosis. Cells were transduced with CellLightTubulin-GFP construct to label microtubules. Cells werethen treated with AK306 and released. The top row shows the formation of aberrant tripolar spindle and abnormal cytokinesis in an AK306 released cell. The bottomrow shows normal mitotic progression in a nontreated cell. The bar indicates 5 mm. C, Impeded cell division after AK306 withdrawal in contrast to nocodazolewithdrawal. Cells were treated with either AK306 or nocodazole and then released for 2 hours. Cells were fixed and stained with antibody to tubulin. Cells releasedfrom AK306 formed aberrant multipolar spindles (yellow arrows), whereas cells released from nocodazole were able to achieve metaphase (green arrow) andcomplete cytokinesis (as indicated by midbody formation; red arrows). The bar shown indicates 10 mm. D, Cells were released from AK306 arrest as in C, andthen stained for tubulin (red) and centrin-3 (green). Cell with aberrant spindleswere then scored for the number of centrin-3 foci. Representative images are shown inthe left plots and the quantified data in the graph. Most of the cells showed aberrant spindles in the absence of centrin over-replication.

Bond et al.





modest, these results support the idea that disrupting the CHCcomplexes may be a fruitful approach for targeting cancer cells.

DiscussionIn previous work, we described the effects of the AK301 com-

pound that arrests cells in a mitotic state characterized by theformation of multipolar spindles and elevated gH2AX expression(8, 29). Upon AK301 withdrawal, cells exit mitotic arrest with asignificant portion undergoing apoptosis (up to 40%). Classicspindle poisons vincristine and colchicine were found to be lessapoptotic in this treatment procedure (8, 29). Although AK301was found to moderately suppress microtubule polymerization,we reasoned that additional cellular targets were likely beingaffected to account for the strong apoptotic effect. We thereforeperformed a focused SAR study to increase compound potencyand to design probe compounds for target identification. Themost potent derivative synthesizedwas AK306,which has an EC50

of approximately 50nmol/L inHCT116 colon cancer cells. AK306was then used as a scaffold for biotinylated and BODIPY-labeledprobe molecules, with attachments made to a region of themolecule that did not interfere with its mitotic arrest activity.Affinity chromatography revealed that CHCwas a potential targetfor the AK3 compounds. In addition, a BODIPY-labeled AK3

compound bound to CHC in an in-gel binding assay. Thesefindings, together with the similar cellular effects caused byAK306 and CHC siRNA knockdown, support CHC as animportant cellular target for the AK3 family of compounds.Although other CHC-binding compounds have been described,the AK3 compounds are structurally distinct with nanomolar-level activity (45).

Clathrin plays a number of roles in mitosis. In relation to itsmembrane transport function, clathrin promotes cytokinesis andcell abscission (46, 47). Clathrin also forms a complex withTACC3 and chTOG, two nonmotor proteins involved in regulat-ingmitotic spindle dynamics (39, 48–51). CHC and TACC3 forma composite microtubule-binding domain that serves to stabilizeK-fibers attaching the centrosome to the kinetochore (52). In thiscomplex, the trimeric clathrin structure is envisioned to bundleand stabilize microtubules. Interestingly, CHC in conjunctionwith chTOG also plays a critical role in centrosome complexstability, with knockdown of either protein leading to the forma-tion of extra MTOCs in the cell (39). Our findings indicate thatAK306 binding to CHC may lead to a rapid and irreversiblefragmentation of the centrosome complex, resulting in the for-mation of acentrosomal MTOCs (which are observed within anhour of AK306 treatment). Even after AK306 is withdrawn,centrosome components (such as g-tubulin and PCNT) remain

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A, HCT116 cells were transfected with CHC siRNA or control (nontargeting) siRNA. After two rounds of transfection, immunostaining was performed withantibodies to CHC. Efficiency of CHC inhibition was assessed by IF visualization using confocal microscope. Left image shows expression of CHC in the control cells,and right image shows expression after knockdown with CHC siRNA. The bar shown indicates 20 mm. B, The effect of CHC knockdown on centrosomecomplexassemblywas investigatedby immunostaining for centrosome-associatedAurora kinaseA (AuKA). Comparedwith control cells (left plot), an increase in thenumber of mitotic cells with multiple AuKA foci appears after CHC knockdown (two right plots). The bar shown indicates 20 mm. C, Representative images ofAK306-treated cells (50 nmol/L) showing multiple Aurora A foci. The bar shown indicates 20 mm. D, Quantification of the increase in AuKA foci in CHCsiRNA–transfected cells. Cells with more than two AuKA foci were counted using the A1R confocal microscope with 60X objective. A significantly greater number ofcells with multiple AuKA foci appear after CHC siRNA transfection (Student t test; P < 0.01). E, CHC depletion leads to p53 phosphorylation at serine-15. Left plotshows phosphorylation of p53 in response to AK306 treatments. HCT116 cells were treated with AK306 overnight (16 hours) and then released for 3 hours, asindicated. Cells were then stained for phospho-p53 (ser-15). Nuclear staining is seen in treated and released cells, but not in control cells. Right plots similarly showphospho-p53 nuclear staining in CHC siRNA knockdown cells, which is absent in the nontargeting siRNA controls. The bar shown indicates 10 mm.






scattered throughout the cell. An important cellular outcome ofcentrosome complex disruption in this manner is p53 activationand apoptosis after release from arrest.

The cellular events linking AK306 treatment and CHC siRNAknockdown to p53 phosphorylation/activation and apoptosis arepresently unclear. One possibility is that aberrant spindles gen-erated by centrosome complex fragmentation shear the DNA andactivate ATM and p53. However, ATM has also been shown tointeract directly with the centrosome and can be activated duringmitosis (4, 53). ATM activated during mitosis phosphorylatesp53, which then also localizes to the centrosome and can bereleased when the centrosome is heavily damaged (4, 5, 7). Ourdata suggest that AK306 (andCHCsiRNA) activates p53 throughacentrosome-based pathway and/or spindle-promoted DNAshearing, because DNA damage signaling is observed primarilyduring mitosis. However, we cannot rule out AK306 interactionswith chromatin DNA or the inhibition of cellular DNA repairenzymes.

Experiments with an APC-mutant model of intestinal cancershowed that cancers are more sensitive to the apoptotic effectsof AK306 than normal tissue. The mechanisms accounting forthis increased sensitivity are not entirely clear. Like many cancers,APC-mutant cancers have centrosomal defects; the normal APCprotein stabilizes the centrosome and facilitates microtubulegrowth, whereas the truncated APC proteins disrupt these activ-ities (26, 38, 39, 54, 55). Because CHC has functions at thecentrosome similar to APC, AK306 may provide a second cen-trosome hit in APC-mutant cancers. Other centrosomal defects incancer cells may likewise affect their sensitivity to AK306. TheHCT116 cells used in this study are APC-normal, but have an

activated ß-catenin oncogene that promotes the formation ofaberrant MT structures consisting of a subset of centrosomeproteins, including g-tubulin (56). The propensity of HCT116cells to form aberrant MTOC-nucleating centers may underlietheir sensitivity to AK306. Understanding the cellular defects thatsensitize cancer cells to AK306 could provide insight into howcentrosome defects might be utilized for therapeutic targeting.

Although our SAR study was limited, we were able to obtainimportant information regarding the potential binding pocket onCHC. The pocket surrounding the phenyl group is large enough toaccommodate chlorines on the ring's meta positions, but not thelarger -OCF3 substituent. The two chlorines of AK306may bind toseparate pockets or may provide two configurations with whichthe compound can bind CHC. AK306's second ring, the benzoylgroup, appears to lie in a portion of the protein that is partiallysolvent accessible. Specifically, the aniline derivative with an NH2

para to the benzoyl carbonyl allowed the attachment of straightcarbon chains, but not aryl or isovaleryl substituents, whilemaintaining nanomolar activity. This portion of the moleculemost likely sits at the edge of the protein. AK306 therefore appearsto be a good scaffold for the construction of novel molecularprobes to study the role ofCHC inmitosis and endocytosis. Futurestudies of the AK306-binding site on CHC may reveal adjacentpockets that could be exploited to increase the compound'seffectiveness or allow for the design of molecules that selectivelyinhibit clathrin's mitotic function.

Small molecules that bind CHC should prove useful for study-ing clathrin's cellular roles and to provide information onthe value of CHC as a therapeutic target. In addition to theirpotential anticancer activity, CHC inhibitorsmay also be useful as

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Tumor-bearing ApcD14/þ mice were treated with five injections of AK306 (30 mg/kg) over the course of 3 days. Animals were sacrificed and tissue analyzedfor apoptosis using TUNEL. A, Representive images of cancerous and normal tissue from animals treated with AK306 or vehicle (as indicated). Normal mucosa andcancerous tissue were demarcated for the analysis. B, The fraction of TUNEL-positive cells in the tissue was quantified using ratio imaging with DAPI stainingused to determine total cell number. Significant differences were observed between normal tissue and tumors, and in vehicle- and AK306-treated tumors(ANOVA, P < 0.001).

Bond et al.





therapeutics by affecting plasma membrane receptor trafficking.For example, AK306 can increase apoptotic signaling from deathreceptors by altering their trafficking, providing a potential meansfor enhancing apoptosis in TNF-rich microenvironments (8, 9).CHC-targeting drugs may also be useful for suppressing infectionby viruses that utilize clathrin-mediated endocytosis for cell entryor for limiting the uptake of some bacterial toxins. The workdescribed here focuses on the potential of cancer therapeutics, butother applications are also possible.

Disclosure of Potential Conflicts of InterestD.L Wright has ownership interest (including patents) in QMD LLC. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: M.J. Bond, E.W. Scocchera, D.L. Wright, C. GiardinaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.J. Bond, M. Bleiler, L.E. Harrison, E.W. Scocchera,M. Nakanishi, D.W. Rosenberg

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.J. Bond, M. Bleiler, L.E. Harrison, N. G-Dayanan,C. GiardinaWriting, review, and/or revision of the manuscript: M.J. Bond, M. Bleiler,D.W. Rosenberg, D.L. Wright, C. GiardinaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L.E. Harrison, S. Keshipeddy, D.W. Rosenberg

AcknowledgmentsThis work was funded by anNCI grant R21CA208638 (to C. Giardina) and a

SPARK Technology Commercialization Award (to C. Giardina, D.L.Wright, andD.W. Rosenberg).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 21, 2018; revised April 5, 2018; accepted April 25, 2018;published first May 16, 2018.

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Bond et al.




2018;16:1361-1372. Published OnlineFirst May 16, 2018.Mol Cancer Res Michael J. Bond, Marina Bleiler, Lauren E. Harrison, et al. CLTC-Binding CompoundSpindle Assembly Disruption and Cancer Cell Apoptosis with a

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