A High-Content Screening of Anticancer Compounds Suggests ... · two drugs currently used for other...

Small Molecule Therapeutics

A High-Content Screening of AnticancerCompounds Suggests the Multiple TyrosineKinase Inhibitor Ponatinib for Repurposing inNeuroblastoma TherapyViktoryia Sidarovich1, Marilena De Mariano2, Sanja Aveic3, Michael Pancher4,Valentina Adami4, Pamela Gatto4, Silvia Pizzini1, Luigi Pasini1, Michela Croce2,Federica Parodi2, Flora Cimmino5,6, Marianna Avitabile5,6, Laura Emionite7,Michele Cilli7, Silvano Ferrini2, Aldo Pagano8,9, Mario Capasso5,6,10,Alessandro Quattrone1, Gian Paolo Tonini3, and Luca Longo2

Abstract

Novel druggable targets have been discovered in neuroblasto-ma (NB), paving the way for more effective treatments. However,childrenwith high-risk NB still showhighmortality rates prompt-ing for a search of novel therapeutic options. Here, we aimed atrepurposing FDA-approved drugs forNB treatment byperforminga high-content screening of a 349 anticancer compounds library.In the primary screening, we employed three NB cell lines, grownas three-dimensional (3D) multicellular spheroids, which weretreated with 10 mmol/L of the library compounds for 72 hours.The viability of 3D spheroids was evaluated using a high-contentimaging approach, resulting in a primary hit list of 193 com-pounds. We selected 60 FDA-approved molecules and prioritizeddrugswithmulti-target activity, discarding those already in use for

NB treatment or enrolled in NB clinical trials. Hence, 20 drugswere further tested for their efficacy in inhibiting NB cell viability,both in two-dimensional and 3D models. Dose-response curveswere then supplemented with the data on side effects, therapeuticindex, and molecular targets, suggesting two multiple tyrosinekinase inhibitors, ponatinib andaxitinib, as promising candidatesfor repositioning in NB. Indeed, both drugs showed induction ofcell-cycle block and apoptosis, as well as inhibition of colonyformation. However, only ponatinib consistently affected migra-tion and inhibited invasion of NB cells. Finally, ponatinib alsoproved effective inhibition of tumor growth in orthotopic NBmice, providing the rationale for its repurposing in NB therapy.Mol Cancer Ther; 17(7); 1–11. �2018 AACR.

IntroductionIn the last decade, several studies have shed light on the biology

of neuroblastoma (NB), a pediatric cancer of the sympatheticnervous system (1–3), allowing for a more accurate stratification

of patients' risk, which has permitted reducing or withholdingcytotoxic therapies without affecting the outcome for low-intermediate risk patients. However, for children with high-riskNB, the rate of mortality is still high (�40%–50%), and fewsurvival improvements have been recorded in the last years (3).On the contrary, for this group of patients, the intensification oftherapeutic aggressiveness has increased treatment morbidity.Therefore, the development of novel therapeutic options isurgently needed.

The repositioning of FDA-approved drugs, that is the identifi-cation and development of new applications for existing orabandoned pharmacotherapies, is a strategy that has already beensuccessfully used to repurpose approved compoundswith studiedbioavailability and safety profiles, known formulation andmanufacturing routes, and well-characterized pharmacology(4). This approach can significantly reduce the risks associatedwith drug development and potentially facilitate repositioneddrugs to enter clinical phasesmore rapidly and at a lower cost thannovel compounds. As a matter of fact, several drugs have alreadybeen proposed for repurposing for one or more conditions ofinterest to pediatric hematology oncology (4). More specifically,two drugs currently used for other pathologies have been pro-posed as potentially useful in NB (5, 6). One of these drugs is theantianginal agent perhexiline, which was demonstrated to act inNB by increasing the expression of the noncoding RNA NDM29,conferring susceptibility to the effects of cisplatin (5). More

1Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy. 2UOCBioterapie, Ospedale Policlinico San Martino, Genova, Italy. 3Istituto di RicercaPediatrica (IRP), Citt�adella Speranza, Padova, Italy. 4HighThroughput ScreeningCore Facility, CIBIO, University of Trento, Trento, Italy. 5University of NaplesFederico II, Napoli, Italy. 6CEINGE Biotecnologie Avanzate, Napoli, Italy. 7AnimalFacility, Ospedale Policlinico San Martino, Genova, Italy. 8University of Genova,Genova, Italy. 9Ospedale Policlinico San Martino, Genova, Italy. 10IRCCS SDN,Istituto di Ricerca Diagnostica e Nucleare, Napoli, Italy.

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

Current address for L. Pasini: Laboratory of Biosciences, Istituto ScientificoRomagnolo per lo Studio e la Cura dei Tumori (IRST) Srl - IRCCS, Meldola, Italy.

Corresponding Authors: Luca Longo, Ospedale Policlinico San Martino, L.go R.Benzi, 10, 16132 Genova, Italy. Phone: 39-010-555-8430; Fax: 39-010-555-8487;E-mail: [email protected]; and Viktoryia Sidarovich, Laboratory of Transla-tional Genomics, Centre for Integrative Biology (CIBIO), University of Trento, ViaSommarive, 9, 38123 Trento, Italy. Phone: 39-0461-283096; Fax: 39-0461-283937; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-17-0841

�2018 American Association for Cancer Research.

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recently, the carbonic anhydrase inhibitor acetazolamide, whichis routinely administered for the treatment of high altitudesickness and glaucoma (7), has emerged as a possible beneficialnovel therapeutic approach in NB, when used in combinationwith a histone deacetylase inhibitor (6).

Here, we aimed at identifying candidate drugs for repositioninginhigh-riskNB. Therefore, we performed ahigh-content screeningof a library of anticancer compounds in NB cell lines. Thescreening of this library, consisting of 349 small molecules eitherFDA-approved or under clinical trials, and subsequent in vitro andin vivo experiments indicated the multiple tyrosine kinase inhib-itor ponatinib as a promising drug for repurposing in NB therapy.

Materials and MethodsCell lines and reagents

IMR-32, SK-N-AS, CHP-212 (purchased from the ATCC in June2014), and CHP-134, SK-N-BE(2) (purchased from ECACC inJuly 2008) NB cell lines were thawed upon arrival, expanded, andstored in a cell bank. All experimentswere performedwithin 5 to 7passages from the thawing. We did not perform any additionalcell line authentication. Cells were cultured according to suppli-ers' instructions. Mycoplasma testing was routinely performedonce per month using a PlasmoTest Mycoplasma detection kit(InvivoGen). The anticancer compound library, consisting of 349small molecules, as well as single compounds ponatinib andaxitinib, was purchased from Selleckchem and stored followingthe manufacturer's instruction.

High-content screeningFor spheroids generation, cells were seeded in transparent

round-bottom ultra-low attachment 96-well plates (Corning) in100 mL, using 1,000 (CHP-134) or 2,000 (SK-N-BE(2) andIMR-32) cells per well. All dispensing steps were performedby the Tecan EVO 200 robot. Optimal three-dimensional(3D) structures were achieved after 3 days of incubation. Spher-oids were treated with the Selleckchem library compounds at10mmol/L by adding 100 mL/well of culturemedia containing 2�the final concentration of the library compounds. Control spher-oids were treated with DMSO (negative control) or 10 mmol/Lvincristine and 5-fluorouracil (positive controls). Following a72-hour exposure to compounds, 3D spheroids viability wasevaluated using a high-content imaging approach. All wells weresupplemented with 1 mg/mL calcein-AM dye that once metabo-lized by viable cells becomes fluorescent. After 30minutes, bright-field and fluorescent spheroid photographs were automaticallycaptured by Operetta imaging system (PerkinElmer) using a 10�objective. Images were processed in Harmony software(PerkinElmer), which quantitated the spheroids area and inten-sity of calcein staining in the rim and the center of each spheroid.The final high-content output value for each spheroid was calcu-lated using the following equation: (Asph/Actr) � (FIR/FIcore) � 0(if FIsph� FIbgr), where Asph is the area of a spheroid of interest, Actr

is the mean area of DMSO-treated control spheroids, FIR is thefluorescent intensity in the spheroid's ring region, FIcore is thefluorescent intensity in the core region, FIsph is the mean fluores-cent intensity of entire spheroid, and FIbgr is the backgroundfluorescence intensity.

Strictly standardized mean difference for hit identification3D viability data were analyzed employing the Strictly Stan-

dardized Mean Difference (SSMD) metric (8, 9), which measures

the magnitude of the difference between negative controls andresponse of cells under drug exposure. For samples withoutreplicates, we used robust SSMD� based on uniformly minimalvariance unbiased estimate:

SSMD� ¼ Yi�eYNffiffiffiffiffiffiffiffiffiffiffiffiffiffi2kðnN�1Þ

p�MADN

K ¼ 2 � ðG ðnN�12 Þ

G ðnN�22 Þ

Þ2 where nN is the

number of wells for a negative reference in a plate; ~YN andMADN are the median and median of absolute deviation ofmeasured values in a negative reference; and G (*) is a gammafunction. For samples with replicates, we used unpaired SSMD

under unequal variance: SSMD ¼ Xi�XNffiffiffiffiffiffiffiffiffiffis2i þs2N

p , where Xi and s2i are

the sample mean and variance of measured values in allreplicates of each compound, respectively. XN and s2N are thesample mean and variance of the negative controls in all plates,respectively. A compound was classified as a primary hit whenit demonstrated SSMD � –3 in at least two cell lines.

Hits validationFollowing manual curation of the primary hit list, the selected

20 compounds were validated in CHP-134 cells grown as 3D andtwo-dimensional (2D) cultures. For 3D spheroids, CHP-134 cellswere plated in ultra-low attachment 96-well plates at 1,000 cells/well density 3 days before treatment, whereas for 2D experiments,7,000 cells/well were plated in white flat-bottom 96-well plates(Nunc) a day before drug addition. Compounds were added ata 0.625–80 mmol/L range for 72 hours. Because DMSO itselfhad no effect on spheroid viability at concentration up to 0.8 %(v/v; Supplementary Fig. S1A), all controls were treated with asingle DMSO concentration (0.1% v/v), corresponding toDMSOcontent in 10 mmol/L treatment. Cell viability was determined byusing either high-content imaging approach described above (3Dcultures) or CellTiter-Glo Luminescent Cell Viability assay (3Dand 2D cultures).

Cell-cycle and apoptosis analysesNB cell lines were treated with either ponatinib or axitinib

(0.4–1–3 mmol/L) for 48 hours. For cell-cycle analysis, cellswere harvested, fixed with 70% ethanol, and left at –20�C for2 hours. Subsequently, cells were stained with propidiumiodide (PI)/RNase A for 30 minutes in the dark and cell-cycleprofiles checked by flow cytometry (FACS; Becton-Dickinson).The percentage of cells in G1, S, and G2–M phase of cell cyclewas calculated according to the obtained DNA content usingCellQuest software. Apoptotic cells were determined by FACSafter double staining with FITC–Annexin-V and PI. PARP wasdetected by Western blot analysis. Total proteins were extractedfollowing a standard protocol and separated in Mini-PROTEANTGX Precast 4%–20% Gels (Bio-Rad). Transfer was performedby Trans-Blot Turbo transfer system (Bio-Rad). After incubationwith primary and secondary antibodies (Supplementary Mate-rials and Methods), blots were developed with ECL Prime (GEHealthcare) and images acquired by a chemiluminescencedetection system (Uvitec Cambridge). Quantification of blotbands was performed by ImageJ.

Colony formation assaysNB cell lines were seeded with Methocult H4100, previously

prepared by adding 40 mL of Methocult in 60 mL of culturemedium, in 6-well plates at a concentration of 7,000 cells/well,with a ratio cells/Methocult of 1:10. Cells were incubated with

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either ponatinib or axitinib (1 and 3 mmol/L) or DMSO andallowed to grow for about 2 weeks, and colonies were countedafter a 4-hours MTT staining.

Migration and invasion assaysCHP-134, IMR-32, and SK-N-BE(2) cells were plated in onto an

Oris 96 TC plate with stoppers in media containing 2% FBS, thuslimiting cell proliferation over a 24-hour period of treatment. Thenext day, the stoppers were removed, and premigration imageswere captured byOperetta (PerkinElmer). Then, cells were treatedwith either ponatinib or axitinib (0.4–1–3 mmol/L) for 24 hoursfollowed by an acquisition of postmigration sample images. Thebrightfield photographs were analyzed by the Harmony software,calculating the cell-covered and cell-free area. Percentage ofscratch closure was calculated as the cell-free area at time zerominus the cell-free area at the end point expressed as the percent-age of the cell-free area at time zero.

For invasion assay, spheroids were generated as describedabove. Formed CHP-134 and IMR-32 spheroids were embeddedin 500 mg/mL CellMatrix basement membrane gel (ATCC) inmedium containing 0.5% and 2% FBS, respectively. Three dayslater, spheroids were treated with 0.4 mmol/L ponatinib or axi-tinib for 72 hours. The images were acquired after 1-hour incu-bation with Calcein-AM using Zeiss Observer Z1 microscope,imported, and analyzed using Columbus image data storage andanalysis system (PerkinElmer). The image analysis algorithmquantitated area of a spheroid body and an entire spheroid withextending into extracellular matrix protrusions (SupplementaryMaterials and Methods). The change in the spheroid body areacould be associated with growth/regression of spheroid itself,whereas an increase in the area of the entire spheroid withprotrusions reflected invasion process.

Generation of SK-N-BE(2) cells carrying luciferase reporterSK-N-BE(2) cells expressing luciferase were obtained by infec-

tion with lentiviral particles carrying luciferase reporter frompLenti CMV Puro LUC plasmid (Addgene #17477). Lentiviralparticles were produced by cotransfecting the reporter plasmidwith the packaging vector psPAX2 (Addgene #12260) and theenvelope plasmid pMD2.G (Addgene #12259) into HEK-293Tcells (ICLC) in Opti-MEM culture medium (Gibco) with0.5 mg/mL polyethylenimine (Sigma-Aldrich). The cells stablyexpressing luciferase were selected with 1 mg/mL puromycin.

In vivo mouse modelAbout 2� 106 and 1.5� 106 luciferase-expressing SK-N-BE(2)

and IMR-32 cells, respectively, were orthotopically injected in theleft adrenal gland of 5-week-old females, CD1� nude mice(Charles River). Animals were anaesthetized with a mixture ofxylazine (10 mg/kg) and ketamine (90 mg/kg; Imalgene 1000)and subjected to laparotomy. Each treatment and control groupinitially consisted of 8 mice. Tumors were allowed to grow forabout 2 (SK-N-BE(2)) or 4 (IMR-32) weeks. Health conditions ofanimals were checked up daily. Both ponatinib and axitinib wereadministered at 30mg/kg by gavage, once a day for 16 consecutivedays (17 days for ponatinib used in IMR-32–derived xenografts)as a solution of 0.5% methylcellulose in PBS. Control mice wereadministered with the vehicle alone (DMSO). Tumor growth wasmonitored every 3 to 4 days by the Caliper IVIS Imaging Sys-tem 100. All experiments involving animals were performedfollowing the 3 Rs guiding principles and National and

Institutional guidelines for the care and use of animals. Theprocedures and methodologies performed on animals wereapproved by the Review Board of the IRCCS AOU SanMartino-IST, Genoa, and the Italian Ministry of Health (HeadDr. Luca Longo—Ministerial authorization n� 680/2016-PR,issued on July 11, 2016).

Results3D high-content screening suggests a list of FDA-approvedcompounds active on NB

To identify the candidatemolecules for repositioning inNB, weselected the focused anticancer compound library from Selleck-chem, which comprises 349 small molecules. The scheme of thescreening and follow-up experiments is outlined in Fig. 1A.We ranthe primary screening in three NB cell lines (CHP-134, IMR-32,and SK-N-BE(2)), which were grown as 3D multicellular spher-oids (MCS).Compoundswere added at 10mmol/L concentration,and spheroids were imaged using the high-content imagingsystem Operetta, after 72 hours of incubation. Before imaging,cells were supplemented with calcein-AM dye that once metab-olized by viable cells becomes fluorescent. Examples of DMSO-treated spheroids for all three NB cell lines are shown in Fig. 1B.Images were then analyzed using the Harmony software, andspheroid viability was evaluated by an in-house developed algo-rithm, which quantitated the spheroids area and intensity ofcalcein staining in the rim and core of each spheroid (Fig. 1C;Supplementary Table S1). Compact spheroids showed a partic-ular pattern, where the edge was brighter than the center becausecalcein-AM could not freely diffuse into the inner part but waspredominantly metabolized by the surface cell layer (10). Upondrug treatment, when spheroid compaction was lost, calcein-AMpenetrated into a spheroid center, resulting in a brighter/irregularsignal in the core region. The lack of fluorescence emissionindicated that all cells were dead. Combination of the two para-meters, spheroid area together with the pattern of fluorescentsignal, delineated the difference in mechanisms of compound-mediated cellular toxicity, i.e., cytotoxic or cytostatic (Fig. 1D).Wenext analyzed the data by employing the SSMD metric. Weselected candidate hits using a SSMD cutoff < –3, i.e., classifiedas the compounds with very and extremely strong effect. A total of193 compounds were classified as primary hits in at least two ofthree cell lines and thus considered as authentic hits (Fig. 1E;Supplementary Table S1). Finally, we applied a filter, focusingonly on FDA-approved compounds. This resulted in a list of 60FDA-approved hits: 50 identified in all three NB cell lines, and 10in at least two of them. Noteworthy, this list of selected com-poundswas enriched in tyrosine kinase, DNA/RNA synthesis, andtopoisomerases inhibitors.

Validation screening identifies two leading compounds for NBrepurposing

The list of 60 FDA-approved hits was manually curated. Inparticular, we discarded compounds already in use for NB treat-ment or enrolled in NB clinical trials. Besides, compounds withmulti-target activities were prioritized. Eventually, we selected 20compounds for confirmation (Supplementary Table S1) thatweretested at the 0.625–80 mmol/L range in CHP-134 cells, growneither as 3D or as 2D cultures. As a result, we obtained threedose-response curves for each drug: one from cells grown asmonolayer, and two from CHP-134 cultured as 3D MCS (one

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after high-content image analysis, and the second after CellTiter-Glo 3D luminescent assay; Supplementary Fig. S1B). Despite ahighdegree of overlap between2Dand3D cellular response to thetreatment, we observed that cells cultured in 3D were moreresistant to certain compounds as comparedwith cellmonolayers,in accordance with published literature (11). Noteworthy, com-parison of the dose-response curves obtained by image analysisand commercial CellTiter-Glo 3D luminescent assay furtherconfirmed the validity of high-content algorithm for spheroidviability evaluation. Finally, we surveyed the validated 20compounds in terms of severe side effects, therapeutic index, andmolecular targets, and prioritized the multiple tyrosine kinaseinhibitors ponatinib and axitinib to follow up (Fig. 2A). Hence,we treated five NB cell lines grown as monolayer (CHP-134,

CHP-212, IMR-32, SK-N-AS, and SK-N-BE(2)) with increasingconcentrations of ponatinib and axitinib for 48 hours. All fiveNB cell lines showed a similar response to treatment, with CHP-212 cells being slightly more sensitive. The average IC50 valueswere 1.5 mmol/L (range, 0.35–2.315 mmol/L) for ponatiniband 2.2 mmol/L (range, 0.5–8.3 mmol/L) for axitinib (Fig. 2B).Of note, although ponatinib resulted in complete inhibition ofcell viability at higher concentrations, axitinib decreased cellviability to a certain cell line–specific threshold (Fig. 2B).

Ponatinib and axitinib induce block of cell-cycle progressionand apoptosis

We next evaluated whether the effect of ponatinib and axitinibon cell viability could be attributed to inhibition of proliferation

Figure 1.

High-content drug screening. A, Schematic of the screening process and hit selection. Phases of 3D screening and follow-up drugs testing are listed onthe left. Numbers represent the considered candidate compounds after each phase. B, Examples of brightfield and fluorescent images of DMSO-treated MCSobtained from three NB cell lines. C, Illustration of image analysis with an in-house–developed algorithm evaluating spheroids viability. Values in blackcorrespond to the spheroid area as compared with vehicle-treated controls, whereas the ratio of calcein fluorescence in the rim and center of each spheroid is in red.Zero multiplier (light green) is introduced when calcein staining of a spheroid is comparable with the background. D, Scatter plot demonstrating the resultsof the primary screening in CHP-134. Each dot represents one compound, and the white color indicates primary hits. E, Venn diagram showing the numberof primary hits (n ¼ 193), among which 60 are FDA-approved, emerged as potentially effective in at least two NB cell lines.

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and/or induction of apoptosis. FiveNB cell lines were treatedwitheither ponatinib or axitinib (0.4–1–3 mmol/L) for 48 hours andanalyzed to determine cell-cycle distribution and apoptosis byflow cytometry. Data showed an accumulation of G2–Mphase forall cell lines treated with axitinib in a dose-dependent manner(Fig. 3; Supplementary Fig. S2A). Ponatinib effects were cell type

specific, causing eitherG1 accumulation inCHP-212 and SK-N-BE(2) cells or G2–M accumulation in CHP-134 cells (Fig. 3;Supplementary Fig. S2A). Characterization of apoptotic cellpopulations by FACS indicated a high degree of variability amongthe cell lines tested (Fig. 4A). Upon treatment with axitinib, weobserved a statistically significant increase of the combined

Figure 2.

Effect of ponatinib and axitinib on NBcell viability. A, CHP-134 cells growingin 3D and 2Dwere treated for 72 hourswith ponatinib and axitinib(0.625–80 mmol/L). Images representmerge of brightfield and fluorescentphotos of CHP-134 spheroids. Viabilitywas analyzed by using high-contentapproach (3D) or CellTiter-Gloluminescent assay (3D and 2D).B, Dose-response curves obtainedin five NB cell lines treated in 2D for48 hours with ponatinib and axitinibat 0.05–20 mmol/L concentrationrange. Data represent the meanpercentage of viable cells SD, n¼ 3,of one from two biological replicates.






fractions of early and late apoptotic cells at 3 mmol/L concentra-tion in SK-N-BE(2) cells, and at all concentrations in SK-N-AScells. Similarly, 3 mmol/L ponatinib induced a significant enrich-ment in early and late apoptotic cells in CHP-134, CHP-212, andSK-N-AS cells, with the latter being also responsive to 1 mmol/Lponatinib. To allow for a more in-depth analysis of apoptosisinduction, we additionally investigated the cleavage of PARP,another hallmark of the process. Indeed, upon exposure of NBcells to either ponatinib or axitinib, we detected PARP cleavage in

all cell lines in a dose-dependentmanner for both drugs (Fig. 4B).Only in CHP-134 cells, we had to increase the concentration ofponatinib to 5 mmol/L to detect a 49% cleavage of PARP (Sup-plementary Fig. S2B).

We then investigated the survival and proliferation of NB cellsby clonogenic assays. Both drugs generally showed a completeinhibition of colony formation in all tested NB cell lines at 3mmol/L and an effective inhibition at 1 mmol/L in 3 of 4 NB celllines (Fig. 4C; Supplementary Fig. S2C).

Figure 3.

Cell-cycle distribution after ponatiniband axitinib treatment. NB cell lineswere treated with increasingconcentration (0.4–1–3 mmol/L) ofponatinib or axitinib for 48 hours.The data represent the mean SDof at least three independentmeasurements. A two-tailed t testwas applied to calculate statisticalsignificance; P < 0.05 is indicatedwith an asterisk (�).

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Figure 4.

Apoptosis analysis and clonogenic assays. A, The percentage of both early and late apoptotic cells was determined by FACS analysis, expressed asrelative to the vehicle-treated control, and then summed. A two-tailed t test was applied to calculate statistical significance; P < 0.05 is indicated with an asterisk (�).B, Detection of PARP activation at 48 hours. After normalizing the intensity of the bands to GAPDH signal, the percentage of cleaved PARP was calculatedas relative to the control and indicated by the number at the bottom of each lane. DMSO effect, if any, was subtracted and set as zero. C, Colony numberrepresents the mean SD of two replicates. IMR-32 cells did not form colonies.






Ponatinib shows efficacy in 3D invasion and migration assaysand inhibits receptor tyrosine kinase downstream effectorpathways

We next asked whether ponatinib and axitinib could affectmigration of NB cells. We observed that ponatinib drasticallyaffected the ability of cells to migrate at all concentrations,whereas axitinib revealed its migration-inhibitory activity onlyat 3 mmol/L (Fig. 5A). We then investigated the ability of thesedrugs to inhibit cell invasion. Spheroids of CHP-134 and IMR-32cells were embedded in the extracellular matrix and incubated

for 3 days with 0.4 mmol/L ponatinib or axitinib. Ponatinibinhibited invadopodia development, whereas spheroid arearemained unchanged as compared with vehicle-treated controls(Fig. 5B). Axitinib showed no effect on invasion into the 3Dextracellular matrix. Finally, we examined phosphorylation statusof several downstream effectors commonly shared by receptortyrosine kinases (RTK), targeted by ponatinib. As shown in Fig. 5Cponatinib effectively inhibited the phosphorylation of AKT,mTOR, Stat3, and S6 ribosomal protein in a time-dependentmanner in the two NB cell lines tested.

Figure 5.

Inhibition of migration and invasion capability of NB cells. A, CHP-134, IMR-32, and SK-N-BE(2) cells were treated with ponatinib and axitinib (0.4–1–3 mmol/L)for 24 hours. Postmigration sample images acquired in digital phase contrast channel are shown. PC and NC: positive and negative controls. Mean values SDof triplicates are provided. Asterisks indicate statistical significance in the two-tailed t test; �,P <0.05; �� ,P<0.01; and �� ,P<0.001.B,CHP-134 and IMR-32 spheroidsembedded in an extracellular matrix were treated with 0.4 mmol/L ponatinib or axitinib for 72 hours. The output values represent total area covered by theentire invading spheroid (left Y axis, bars) or spheroid body alone (right Y axis, dots), both normalized to the mean spheroid body area of DMSO-treated controls.MeanSD, n¼4, of a representative experiment from three biological replicates is provided. Representative fluorescent (calcein-AM) images are shown.C,SK-N-BE(2) and IMR-32 cells treated with 1 mmol/L ponatinib for up to 24 hours were subjected to SDS-PAGE and immunoblotted with indicated antibodies.

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Ponatinib inhibits tumor growth in SK-N-BE(2) and IMR-32orthotopic NB mouse models

To evaluate the in vivo efficacy of ponatinib and axitinib ininhibiting tumor growth, we orthotopically injected luciferase-expressing SK-N-BE(2) cells in immunodeficient nude mice andallowed tumors to develop. On average, upon treatment, miceshowed no overt changes in weight and both drugs were welltolerated with the exception of a mild-to-moderate skin peelingand an increased water consumption observed with the use ofponatinib. Although daily administration of axitinib did notexert significant impairment of NB growth (P ¼ 0.25), pona-tinib resulted in a reduced tumor growth after 16 days oftreatment (P ¼ 0.003), as compared with controls (Fig. 6A;Supplementary Fig. S3). Moreover, tumors from mice admin-istered with ponatinib were evidently smaller (Fig. 6B), andtumor weight was indeed significantly (P ¼ 0.02) decreased(Fig. 6C). In addition, ponatinib-treated tumors visually

appeared less vascularized. Conversely, tumors from animalstreated with axitinib were more variable in size and did notshow significant differences in weight (P ¼ 0.18) as comparedwith vehicle-treated mice (Fig. 6C). Results obtained for pona-tinib were then confirmed in a second in vivo mouse model,using IMR-32 cells. Again, ponatinib significantly inhibited NBgrowth after 17 days of treatment (P ¼ 0.001), as comparedwith controls (Fig. 6D; Supplementary Fig. S4). Tumors fromponatinib-treated mice were smaller (Fig. 6E), and tumorweight significantly (P ¼ 0.01) decreased (Fig. 6F).

DiscussionPreclinical testing of potential anticancer agents involves in vitro

analyses in disease-relevant cell line models, which are typicallygrown as monolayers. However, the several shortcomings ofmonolayer cultures in mimicking real-life in vivo scenarios

Figure 6.

Effects of ponatinib and axitinib on NB tumor growth in vivo. A, Only ponatinib showed inhibition of tumor growth in SK-N-BE(2)–derived xenografts.B, Photos of SK-N-BE(2)–derived tumors treated with ponatinib and axitinib. C, Tumor weight of SK-N-BE(2)–derived xenografts was significantly lighterin the ponatinib-treated group. D, Inhibition of tumor growth by ponatinib in IMR-32–derived xenografts. E, Photos of IMR-32–derived tumors treatedwith ponatinib. F, Tumor weight of IMR-32–derived xenografts was significantly lighter after ponatinib treatment. A two-tailed t test was applied tocalculate statistical significance.






highlighted the need for more representative models. Over thepast years, various types of 3D culture systems have receivedincreased recognition as reliable preclinicalmodels to recapitulatedrug responses of solid tumors (11). Cells grown in a 3D spatiallayout re-establish metabolic and proliferative gradients, aswell as morphologic and functional properties of the correspond-ing tissue in vivo, thus promising greater power to predictclinical efficacies.

The advantage of high-content approach over standard homo-geneous assays for quantification of treatment-induced responsesis the combination of quantitative and qualitative image data.Merging brightfield andfluorescent calcein-AM images allowedusto develop the algorithm quantitating high-content readouts thatcan be attributed to spheroid viability. The final quantitativemetric demonstrated a high degree of reliability, as validated bycomparison with the commercially available luminescent viabil-ity assay. The 3D high-content screening performed in CHP-134,IMR-32, and SK-N-BE(2) spheroids resulted in compound listsgreatly overlapping among the cell lines tested. In addition,compounds that were already in use for NB treatment (i.e.,vincristine, carboplatin, doxorubicin, etoposide, teniposide,topotecan) were present in the final hit list, indicating the overallreliability and robustness of the developed assay. Eventually, ouranalysis indicated two multiple kinase inhibitors, namely pona-tinib and axitinib, as candidates for repositioning in NB.

Indeed, ponatinib was initially reported as a potent, orallyavailable multi-target kinase inhibitor active against BCR-ABLmutants and indicated for the treatment of chronic myeloidleukemia (CML; ref. 12). Moreover, ponatinib was reported toinhibit Src and members of the VEGFR and PDGFR families ofRTKs (12). Besides, it impairs the in vitro kinase activity of all fourFGFRs (12, 13), as well as of RET kinase, showing promisingpreclinical activity in models of RET-driven medullary thyroidcarcinoma (14). On the other hand, axitinib proved potentinhibition of multiple targets, such as VEGFRs, PDGFRb, andc-Kit (15).

Our experiments showed that both drugs led to the accumu-lation of either G1 or G2–M cell-cycle phases, as well as to theactivation of PARP, indicating that they can, indeed, impairproliferation and induce apoptosis in NB cells. Moreover, bothcompounds exerted striking efficacy in inhibiting the formationofcolonies, but only ponatinib resulted effective in inhibiting cellinvasion andmigration. Finally, in vivo experiments demonstratedthat ponatinib has the potential to inhibit tumor growth, makingthis compound a suitable candidate for repositioning in NBtherapy. Although we could not prove a significant activity ofaxitinib in our in vivo model, one previous study indicated thataxitinib could partially impair the growth of human NB xeno-grafts by inhibiting angiogenesis (16). Notably, independentinvestigations have very recently proposed ponatinib itself as adrug displaying antitumor efficacy against NB (17, 18). Ponatinibproved to inhibit the FGFR1-activated signaling pathway andenhance the cytotoxic effects of doxorubicin on NB cells (18).Moreover, ponatinib also interferes with the insulin-like growthfactor-1 receptor signaling pathways and Src activity, inhibitingcell migration and inducing apoptosis in NB (19). In addition tothese results, wenowadd further evidence that supports a possibleclinical use of ponatinib in NB.

Ponatinib was developed by Ariad Pharmaceuticals, Inc., andinitially approved at the end of 2012 for the treatment of CMLand Philadelphia-chromosome positive acute lymphoblastic

leukemia (Phþ ALL; ref. 20). Currently, there are 34 clinicalstudies that aim to determine the efficacy of ponatinib invarious malignancies, which include: acute myeloid leukemia,non–small cell lung cancer, head and neck cancer, glioblasto-ma, and patients whose advanced solid tumor has activatingmutations in FGFRs, RET and KIT genes (https://clinicaltrials.gov). Interestingly, the latter two genes have been reported asinvolved in NB (21–22) and may thus act as potential targets ofponatinib in a subset of patients.

Although a remarkable clinical efficacy in patients with CMLwas observed, ponatinib also showed an increased risk ofcardiovascular events (23–24). The incidence of adverse eventswas correlated with the dose-intensity of the drug. As a conse-quence, the phase III clinical trial was terminated (25). Fol-lowing a careful assessment of the risk/benefit ratio, the FDAhas then allowed the reintroduction of ponatinib with a boxedwarning calling attention on the risk of cardiovascular events(23). Recently, it has been suggested that the use of reduceddoses of ponatinib (down to 15 mg/day instead of the usualdosage of 45 mg/day) might maintain therapeutic activityin most patients while decreasing the vascular adverse events(24, 26). Although clinicians will have to tackle the side effectsobserved upon ponatinib administration, the results achievedthrough our study, as well as others (17, 18), indicate that thisdrug has the potential to be employed in clinical trials aimed toevaluate benefits in high-risk NB patients.

In conclusion, our survey of a comprehensive anticancercompound library provides rationale for further preclinicaltesting of ponatinib in NB, and eventually for setting up aclinical trial involving children with NB, who have no alterna-tive therapeutic options.

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

Authors' ContributionsConception and design: V. Sidarovich, M. Capasso, A. Quattrone, G.P. Tonini,L. LongoDevelopment of methodology: V. Sidarovich, S. Aveic, M. Pancher, V. Adami,P. Gatto, L. Pasini, F. Cimmino, M. Avitabile, M. CilliAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): V. Sidarovich, M. De Mariano, S. Aveic, M. Pancher,V. Adami, P. Gatto, M. Croce, L. Emionite, M. Cilli, L. LongoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): V. Sidarovich, M. De Mariano, S. Aveic, M. Pancher,V. Adami, P. Gatto, S. Pizzini, M. Croce, F. Parodi, S. Ferrini, A. Pagano,M. Capasso, A. Quattrone, L. LongoWriting, review, and/or revision of the manuscript: V. Sidarovich, S. Aveic,M. Pancher, L. Pasini, L. Emionite, M. Cilli, S. Ferrini, A. Quattrone, G.P. Tonini,L. LongoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): V. Sidarovich, L. LongoStudy supervision: V. Sidarovich, A. Quattrone, L. Longo

AcknowledgmentsThis study was funded by the Italian Neuroblastoma Foundation Grant

(L. Longo).

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 September 5, 2017; revised January 12, 2018; accepted April 10,2018; published first April 25, 2018.

Sidarovich et al.




https://clinicaltrials.gov

https://clinicaltrials.gov


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Published OnlineFirst April 25, 2018.Mol Cancer Ther Viktoryia Sidarovich, Marilena De Mariano, Sanja Aveic, et al. Repurposing in Neuroblastoma Therapy

forSuggests the Multiple Tyrosine Kinase Inhibitor Ponatinib A High-Content Screening of Anticancer Compounds

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