A High-Content Screening of Anticancer Compounds Suggests

Published OnlineFirst April 25, 2018; DOI: 10.1158/1535-7163.MCT-17-0841

Small Molecule Therapeutics Molecular Cancer Therapeutics A High-Content Screening of Anticancer Compounds Suggests the Multiple Tyrosine Kinase Inhibitor Ponatinib for Repurposing in Neuroblastoma Therapy Viktoryia 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- NB treatment or enrolled in NB clinical trials. Hence, 20 drugs ma (NB), paving the way for more effective treatments. However, were further tested for their efﬁcacy in inhibiting NB cell viability, children with high-risk NB still show high mortality rates prompt- both in two-dimensional and 3D models. Dose-response curves ing for a search of novel therapeutic options. Here, we aimed at were then supplemented with the data on side effects, therapeutic repurposing FDA-approved drugs for NB treatment by performing index, and molecular targets, suggesting two multiple tyrosine a high-content screening of a 349 anticancer compounds library. kinase inhibitors, ponatinib and axitinib, as promising candidates In the primary screening, we employed three NB cell lines, grown for repositioning in NB. Indeed, both drugs showed induction of as three-dimensional (3D) multicellular spheroids, which were cell-cycle block and apoptosis, as well as inhibition of colony treated with 10 mmol/L of the library compounds for 72 hours. formation. However, only ponatinib consistently affected migra- The viability of 3D spheroids was evaluated using a high-content tion and inhibited invasion of NB cells. Finally, ponatinib also imaging approach, resulting in a primary hit list of 193 com- proved effective inhibition of tumor growth in orthotopic NB pounds. We selected 60 FDA-approved molecules and prioritized mice, providing the rationale for its repurposing in NB therapy. drugs with multi-target activity, discarding those already in use for Mol Cancer Ther; 17(7); 1405–15. 2018 AACR.

Introduction of patients' risk, which has permitted reducing or withholding cytotoxic therapies without affecting the outcome for low- In the last decade, several studies have shed light on the biology intermediate risk patients. However, for children with high-risk of neuroblastoma (NB), a pediatric cancer of the sympathetic NB, the rate of mortality is still high (40%–50%), and few nervous system (1–3), allowing for a more accurate stratification survival improvements have been recorded in the last years (3). On the contrary, for this group of patients, the intensification of therapeutic aggressiveness has increased treatment morbidity. 1 2 Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy. UOC Therefore, the development of novel therapeutic options is Bioterapie, Ospedale Policlinico San Martino, Genova, Italy. 3Istituto di Ricerca 4 urgently needed. Pediatrica (IRP), Citta della Speranza, Padova, Italy. High Throughput Screening fi Core Facility, CIBIO, University of Trento, Trento, Italy. 5University of Naples The repositioning of FDA-approved drugs, that is the identi - Federico II, Napoli, Italy. 6CEINGE Biotecnologie Avanzate, Napoli, Italy. 7Animal cation and development of new applications for existing or Facility, Ospedale Policlinico San Martino, Genova, Italy. 8University of Genova, abandoned pharmacotherapies, is a strategy that has already been Genova, Italy. 9Ospedale Policlinico San Martino, Genova, Italy. 10IRCCS SDN, successfully used to repurpose approved compounds with studied Istituto di Ricerca Diagnostica e Nucleare, Napoli, Italy. bioavailability and safety profiles, known formulation and Note: Supplementary data for this article are available at Molecular Cancer manufacturing routes, and well-characterized pharmacology Therapeutics Online (http://mct.aacrjournals.org/). (4). This approach can significantly reduce the risks associated Current address for L. Pasini: Laboratory of Biosciences, Istituto Scientifico with drug development and potentially facilitate repositioned Romagnolo per lo Studio e la Cura dei Tumori (IRST) Srl - IRCCS, Meldola, Italy. drugs to enter clinical phases more rapidly and at a lower cost than Corresponding Authors: Luca Longo, Ospedale Policlinico San Martino, L.go R. novel compounds. As a matter of fact, several drugs have already Benzi, 10, 16132 Genova, Italy. Phone: 39-010-555-8430; Fax: 39-010-555-8487; been proposed for repurposing for one or more conditions of E-mail: [email protected]; and Viktoryia Sidarovich, Laboratory of Transla- interest to pediatric hematology oncology (4). More specifically, tional Genomics, Centre for Integrative Biology (CIBIO), University of Trento, Via two drugs currently used for other pathologies have been pro- Sommarive, 9, 38123 Trento, Italy. Phone: 39-0461-283096; Fax: 39-0461- posed as potentially useful in NB (5, 6). One of these drugs is the 283937; E-mail: [email protected] antianginal agent perhexiline, which was demonstrated to act in doi: 10.1158/1535-7163.MCT-17-0841 NB by increasing the expression of the noncoding RNA NDM29, 2018 American Association for Cancer Research. conferring susceptibility to the effects of cisplatin (5). More

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recently, the carbonic anhydrase inhibitor acetazolamide, which the magnitude of the difference between negative controls and is routinely administered for the treatment of high altitude response of cells under drug exposure. For samples without sickness and glaucoma (7), has emerged as a possible beneficial replicates, we used robust SSMD based on uniformly minimal novel therapeutic approach in NB, when used in combination variance unbiased estimate: n YieY G ð N 1Þ with a histone deacetylase inhibitor (6). pffiffiffiffiffiffiffiffiffiffiffiffiffiffiN 2 2 SSMD ¼ K ¼ 2 ð n Þ where nN is the 2ðn Þ G ð N 2Þ Here, we aimed at identifying candidate drugs for repositioning k N 1 MADN 2 ~ in high-risk NB. Therefore, we performed a high-content screening number of wells for a negative reference in a plate; YN and of a library of anticancer compounds in NB cell lines. The MADN are the median and median of absolute deviation of screening of this library, consisting of 349 small molecules either measured values in a negative reference; and G (*) is a gamma in vitro FDA-approved or under clinical trials, and subsequent and function. For samples with replicates, we used unpaired SSMD in vivo X X experiments indicated the multiple tyrosine kinase inhib- piffiffiffiffiffiffiffiffiffiffiN 2 under unequal variance: SSMD ¼ ,whereXi and si are s2þs2 itor ponatinib as a promising drug for repurposing in NB therapy. i N the sample mean and variance of measured values in all Materials and Methods 2 replicates of each compound, respectively. XN and sN are the Cell lines and reagents sample mean and variance of the negative controls in all plates, IMR-32, SK-N-AS, CHP-212 (purchased from the ATCC in June respectively. A compound was classified as a primary hit when 2014), and CHP-134, SK-N-BE(2) (purchased from ECACC in it demonstrated SSMD –3 in at least two cell lines. July 2008) NB cell lines were thawed upon arrival, expanded, and stored in a cell bank. All experiments were performed within 5 to 7 Hits validation passages from the thawing. We did not perform any additional Following manual curation of the primary hit list, the selected cell line authentication. Cells were cultured according to suppli- 20 compounds were validated in CHP-134 cells grown as 3D and ers' instructions. Mycoplasma testing was routinely performed two-dimensional (2D) cultures. For 3D spheroids, CHP-134 cells once per month using a PlasmoTest Mycoplasma detection kit were plated in ultra-low attachment 96-well plates at 1,000 cells/ (InvivoGen). The anticancer compound library, consisting of 349 well density 3 days before treatment, whereas for 2D experiments, small molecules, as well as single compounds ponatinib and 7,000 cells/well were plated in white flat-bottom 96-well plates axitinib, was purchased from Selleckchem and stored following (Nunc) a day before drug addition. Compounds were added at the manufacturer's instruction. a 0.625–80 mmol/L range for 72 hours. Because DMSO itself had no effect on spheroid viability at concentration up to 0.8 % High-content screening (v/v; Supplementary Fig. S1A), all controls were treated with a For spheroids generation, cells were seeded in transparent single DMSO concentration (0.1 % v/v), corresponding to DMSO round-bottom ultra-low attachment 96-well plates (Corning) in content in 10 mmol/L treatment. Cell viability was determined by 100 mL, using 1,000 (CHP-134) or 2,000 (SK-N-BE(2) and using either high-content imaging approach described above (3D IMR-32) cells per well. All dispensing steps were performed cultures) or CellTiter-Glo Luminescent Cell Viability assay (3D by the Tecan EVO 200 robot. Optimal three-dimensional and 2D cultures). (3D) structures were achieved after 3 days of incubation. Spher- oids were treated with the Selleckchem library compounds at Cell-cycle and apoptosis analyses 10 mmol/L by adding 100 mL/well of culture media containing 2 NB cell lines were treated with either ponatinib or axitinib the final concentration of the library compounds. Control spher- (0.4–1–3 mmol/L) for 48 hours. For cell-cycle analysis, cells oids were treated with DMSO (negative control) or 10 mmol/L were harvested, fixed with 70% ethanol, and left at –20Cfor vincristine and 5-fluorouracil (positive controls). Following a 2 hours. Subsequently, cells were stained with propidium 72-hour exposure to compounds, 3D spheroids viability was iodide (PI)/RNase A for 30 minutes in the dark and cell-cycle evaluated using a high-content imaging approach. All wells were profiles checked by flow cytometry (FACS; Becton-Dickinson). m supplemented with 1 g/mL calcein-AM dye that once metabo- The percentage of cells in G1,S,andG2–M phase of cell cycle lized by viable cells becomes fluorescent. After 30 minutes, bright- was calculated according to the obtained DNA content using field and fluorescent spheroid photographs were automatically CellQuest software. Apoptotic cells were determined by FACS captured by Operetta imaging system (PerkinElmer) using a 10 after double staining with FITC–Annexin-V and PI. PARP was objective. Images were processed in Harmony software detected by Western blot analysis. Total proteins were extracted (PerkinElmer), which quantitated the spheroids area and inten- following a standard protocol and separated in Mini-PROTEAN sity of calcein staining in the rim and the center of each spheroid. TGX Precast 4%–20% Gels (Bio-Rad). Transfer was performed The final high-content output value for each spheroid was calcu- by Trans-Blot Turbo transfer system (Bio-Rad). After incubation lated using the following equation: (Asph/Actr) (FIR/FIcore) 0 with primary and secondary antibodies (Supplementary Mate- (if FIsph FIbgr), where Asph is the area of a spheroid of interest, Actr rials and Methods), blots were developed with ECL Prime (GE is the mean area of DMSO-treated control spheroids, FIR is the Healthcare) and images acquired by a chemiluminescence fluorescent intensity in the spheroid's ring region, FIcore is the detection system (Uvitec Cambridge). Quantification of blot fluorescent intensity in the core region, FIsph is the mean fluores- bands was performed by ImageJ. cent intensity of entire spheroid, and FIbgr is the background fluorescence intensity. Colony formation assays NB cell lines were seeded with Methocult H4100, previously Strictly standardized mean difference for hit identification prepared by adding 40 mL of Methocult in 60 mL of culture 3D viability data were analyzed employing the Strictly Stan- medium, in 6-well plates at a concentration of 7,000 cells/well, dardized Mean Difference (SSMD) metric (8, 9), which measures 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 and Institutional guidelines for the care and use of animals. The allowed to grow for about 2 weeks, and colonies were counted procedures and methodologies performed on animals were after a 4-hours MTT staining. approved by the Review Board of the IRCCS AOU San Martino-IST, Genoa, and the Italian Ministry of Health (Head Migration and invasion assays Dr. Luca Longo—Ministerial authorization n 680/2016-PR, CHP-134, IMR-32, and SK-N-BE(2) cells were plated in onto an issued on July 11, 2016). Oris 96 TC plate with stoppers in media containing 2% FBS, thus limiting cell proliferation over a 24-hour period of treatment. The next day, the stoppers were removed, and premigration images Results were captured by Operetta (PerkinElmer). Then, cells were treated 3D high-content screening suggests a list of FDA-approved with either ponatinib or axitinib (0.4–1–3 mmol/L) for 24 hours compounds active on NB followed by an acquisition of postmigration sample images. The To identify the candidate molecules for repositioning in NB, we brightfield photographs were analyzed by the Harmony software, selected the focused anticancer compound library from Selleck- calculating the cell-covered and cell-free area. Percentage of chem, which comprises 349 small molecules. The scheme of the scratch closure was calculated as the cell-free area at time zero screening and follow-up experiments is outlined in Fig. 1A. We ran minus the cell-free area at the end point expressed as the percent- the primary screening in three NB cell lines (CHP-134, IMR-32, age of the cell-free area at time zero. and SK-N-BE(2)), which were grown as 3D multicellular spher- For invasion assay, spheroids were generated as described oids (MCS). Compounds were added at 10 mmol/L concentration, above. Formed CHP-134 and IMR-32 spheroids were embedded and spheroids were imaged using the high-content imaging in 500 mg/mL CellMatrix basement membrane gel (ATCC) in system Operetta, after 72 hours of incubation. Before imaging, medium containing 0.5% and 2% FBS, respectively. Three days cells were supplemented with calcein-AM dye that once metab- later, spheroids were treated with 0.4 mmol/L ponatinib or axi- olized by viable cells becomes fluorescent. Examples of DMSO- tinib for 72 hours. The images were acquired after 1-hour incu- treated spheroids for all three NB cell lines are shown in Fig. 1B. bation with Calcein-AM using Zeiss Observer Z1 microscope, Images were then analyzed using the Harmony software, and imported, and analyzed using Columbus image data storage and spheroid viability was evaluated by an in-house developed algo- analysis system (PerkinElmer). The image analysis algorithm rithm, which quantitated the spheroids area and intensity of quantitated area of a spheroid body and an entire spheroid with calcein staining in the rim and core of each spheroid (Fig. 1C; extending into extracellular matrix protrusions (Supplementary Supplementary Table S1). Compact spheroids showed a partic- Materials and Methods). The change in the spheroid body area ular pattern, where the edge was brighter than the center because could be associated with growth/regression of spheroid itself, calcein-AM could not freely diffuse into the inner part but was whereas an increase in the area of the entire spheroid with predominantly metabolized by the surface cell layer (10). Upon protrusions reflected invasion process. drug treatment, when spheroid compaction was lost, calcein-AM penetrated into a spheroid center, resulting in a brighter/irregular Generation of SK-N-BE(2) cells carrying luciferase reporter signal in the core region. The lack of fluorescence emission SK-N-BE(2) cells expressing luciferase were obtained by infec- indicated that all cells were dead. Combination of the two para- tion with lentiviral particles carrying luciferase reporter from meters, spheroid area together with the pattern of fluorescent pLenti CMV Puro LUC plasmid (Addgene #17477). Lentiviral signal, delineated the difference in mechanisms of compound- particles were produced by cotransfecting the reporter plasmid mediated cellular toxicity, i.e., cytotoxic or cytostatic (Fig. 1D). We with the packaging vector psPAX2 (Addgene #12260) and the next analyzed the data by employing the SSMD metric. We envelope plasmid pMD2.G (Addgene #12259) into HEK-293T selected candidate hits using a SSMD cutoff < –3, i.e., classified cells (ICLC) in Opti-MEM culture medium (Gibco) with as the compounds with very and extremely strong effect. A total of 0.5 mg/mL polyethylenimine (Sigma-Aldrich). The cells stably 193 compounds were classified as primary hits in at least two of expressing luciferase were selected with 1 mg/mL puromycin. three cell lines and thus considered as authentic hits (Fig. 1E; Supplementary Table S1). Finally, we applied a filter, focusing In vivo mouse model only on FDA-approved compounds. This resulted in a list of 60 About 2 106 and 1.5 106 luciferase-expressing SK-N-BE(2) FDA-approved hits: 50 identified in all three NB cell lines, and 10 and IMR-32 cells, respectively, were orthotopically injected in the in at least two of them. Noteworthy, this list of selected com- left adrenal gland of 5-week-old females, CD1 nude mice pounds was enriched in tyrosine kinase, DNA/RNA synthesis, and (Charles River). Animals were anaesthetized with a mixture of topoisomerases inhibitors. xylazine (10 mg/kg) and ketamine (90 mg/kg; Imalgene 1000) and subjected to laparotomy. Each treatment and control group Validation screening identifies two leading compounds for NB initially consisted of 8 mice. Tumors were allowed to grow for repurposing about 2 (SK-N-BE(2)) or 4 (IMR-32) weeks. Health conditions of The list of 60 FDA-approved hits was manually curated. In animals were checked up daily. Both ponatinib and axitinib were particular, we discarded compounds already in use for NB treat- administered at 30 mg/kg by gavage, once a day for 16 consecutive ment or enrolled in NB clinical trials. Besides, compounds with days (17 days for ponatinib used in IMR-32–derived xenografts) multi-target activities were prioritized. Eventually, we selected 20 as a solution of 0.5% methylcellulose in PBS. Control mice were compounds for confirmation (Supplementary Table S1) that were administered with the vehicle alone (DMSO). Tumor growth was tested at the 0.625–80 mmol/L range in CHP-134 cells, grown monitored every 3 to 4 days by the Caliper IVIS Imaging Sys- either as 3D or as 2D cultures. As a result, we obtained three tem 100. All experiments involving animals were performed dose-response curves for each drug: one from cells grown as following the 3 Rs guiding principles and National and monolayer, and two from CHP-134 cultured as 3D MCS (one

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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 on the left. Numbers represent the considered candidate compounds after each phase. B, Examples of brightfield and fluorescent images of DMSO-treated MCS obtained from three NB cell lines. C, Illustration of image analysis with an in-house–developed algorithm evaluating spheroids viability. Values in black correspond 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 results of the primary screening in CHP-134. Each dot represents one compound, and the white color indicates primary hits. E, Venn diagram showing the number of primary hits (n ¼ 193), among which 60 are FDA-approved, emerged as potentially effective in at least two NB cell lines.

after high-content image analysis, and the second after CellTiter- CHP-212, IMR-32, SK-N-AS, and SK-N-BE(2)) with increasing Glo 3D luminescent assay; Supplementary Fig. S1B). Despite a concentrations of ponatinib and axitinib for 48 hours. All five high degree of overlap between 2D and 3D cellular response to the NB cell lines showed a similar response to treatment, with CHP- treatment, we observed that cells cultured in 3D were more 212 cells being slightly more sensitive. The average IC50 values resistant to certain compounds as compared with cell monolayers, were 1.5 mmol/L (range, 0.35–2.315 mmol/L) for ponatinib in accordance with published literature (11). Noteworthy, com- and 2.2 mmol/L (range, 0.5–8.3 mmol/L) for axitinib (Fig. 2B). parison of the dose-response curves obtained by image analysis Of note, although ponatinib resulted in complete inhibition of and commercial CellTiter-Glo 3D luminescent assay further cell viability at higher concentrations, axitinib decreased cell confirmed the validity of high-content algorithm for spheroid viability to a certain cell line–specific threshold (Fig. 2B). viability evaluation. Finally, we surveyed the validated 20 compounds in terms of severe side effects, therapeutic index, and Ponatinib and axitinib induce block of cell-cycle progression molecular targets, and prioritized the multiple tyrosine kinase and apoptosis inhibitors ponatinib and axitinib to follow up (Fig. 2A). Hence, We next evaluated whether the effect of ponatinib and axitinib we treated five NB cell lines grown as monolayer (CHP-134, on cell viability could be attributed to inhibition of proliferation

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Figure 2. Effect of ponatinib and axitinib on NB cell viability. A, CHP-134 cells growing in 3D and 2D were treated for 72 hours with ponatinib and axitinib (0.625–80 mmol/L). Images represent merge of brightfield and fluorescent photos of CHP-134 spheroids. Viability was analyzed by using high-content approach (3D) or CellTiter-Glo luminescent assay (3D and 2D). B, Dose-response curves obtained in five NB cell lines treated in 2D for 48 hours with ponatinib and axitinib at 0.05–20 mmol/L concentration range. Data represent the mean percentage of viable cells SD, n ¼ 3, of one from two biological replicates.

and/or induction of apoptosis. Five NB cell lines were treated with specific, causing either G1 accumulation in CHP-212 and SK-N-BE either ponatinib or axitinib (0.4–1–3 mmol/L) for 48 hours and (2) cells or G2–M accumulation in CHP-134 cells (Fig. 3; analyzed to determine cell-cycle distribution and apoptosis by Supplementary Fig. S2A). Characterization of apoptotic cell flow cytometry. Data showed an accumulation of G2–M phase for populations by FACS indicated a high degree of variability among all cell lines treated with axitinib in a dose-dependent manner the cell lines tested (Fig. 4A). Upon treatment with axitinib, we (Fig. 3; Supplementary Fig. S2A). Ponatinib effects were cell type observed a statistically significant increase of the combined

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Figure 3. Cell-cycle distribution after ponatinib and axitinib treatment. NB cell lines were treated with increasing concentration (0.4–1–3 mmol/L) of ponatinib or axitinib for 48 hours. The data represent the mean SD of at least three independent measurements. A two-tailed t test was applied to calculate statistical signiﬁcance; P < 0.05 is indicated with an asterisk ().

fractions of early and late apoptotic cells at 3 mmol/L concentra- all cell lines in a dose-dependent manner for both drugs (Fig. 4B). tion in SK-N-BE(2) cells, and at all concentrations in SK-N-AS Only in CHP-134 cells, we had to increase the concentration of cells. Similarly, 3 mmol/L ponatinib induced a signiﬁcant enrich- ponatinib to 5 mmol/L to detect a 49% cleavage of PARP (Sup- ment in early and late apoptotic cells in CHP-134, CHP-212, and plementary Fig. S2B). SK-N-AS cells, with the latter being also responsive to 1 mmol/L We then investigated the survival and proliferation of NB cells ponatinib. To allow for a more in-depth analysis of apoptosis by clonogenic assays. Both drugs generally showed a complete induction, we additionally investigated the cleavage of PARP, inhibition of colony formation in all tested NB cell lines at 3 another hallmark of the process. Indeed, upon exposure of NB mmol/L and an effective inhibition at 1 mmol/L in 3 of 4 NB cell cells to either ponatinib or axitinib, we detected PARP cleavage in lines (Fig. 4C; Supplementary Fig. S2C).

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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 as relative 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 calculated as 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 number represents the mean SD of two replicates. IMR-32 cells did not form colonies.

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Ponatinib shows efﬁcacy in 3D invasion and migration assays for 3 days with 0.4 mmol/L ponatinib or axitinib. Ponatinib and inhibits receptor tyrosine kinase downstream effector inhibited invadopodia development, whereas spheroid area pathways remained unchanged as compared with vehicle-treated controls We next asked whether ponatinib and axitinib could affect (Fig. 5B). Axitinib showed no effect on invasion into the 3D migration of NB cells. We observed that ponatinib drastically extracellular matrix. Finally, we examined phosphorylation status affected the ability of cells to migrate at all concentrations, of several downstream effectors commonly shared by receptor whereas axitinib revealed its migration-inhibitory activity only tyrosine kinases (RTK), targeted by ponatinib. As shown in Fig. 5C at 3 mmol/L (Fig. 5A). We then investigated the ability of these ponatinib effectively inhibited the phosphorylation of AKT, drugs to inhibit cell invasion. Spheroids of CHP-134 and IMR-32 mTOR, Stat3, and S6 ribosomal protein in a time-dependent cells were embedded in the extracellular matrix and incubated manner 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 SD of 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 spheroids embedded 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 the entire 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. Mean SD, n ¼ 4, 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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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 lighter in the ponatinib-treated group. D, Inhibition of tumor growth by ponatinib in IMR-32–derived xenografts. E, Photos of IMR-32–derived tumors treated with ponatinib. F, Tumor weight of IMR-32–derived xenografts was significantly lighter after ponatinib treatment. A two-tailed t test was applied to calculate statistical significance.

Ponatinib inhibits tumor growth in SK-N-BE(2) and IMR-32 appeared less vascularized. Conversely, tumors from animals orthotopic NB mouse models treatedwithaxitinibweremorevariableinsizeanddidnot To evaluate the in vivo efficacy of ponatinib and axitinib in show significant differences in weight (P ¼ 0.18) as compared inhibiting tumor growth, we orthotopically injected luciferase- with vehicle-treated mice (Fig. 6C). Results obtained for pona- expressing SK-N-BE(2) cells in immunodeficient nude mice and tinib were then confirmed in a second in vivo mouse model, allowed tumors to develop. On average, upon treatment, mice using IMR-32 cells. Again, ponatinib significantly inhibited NB showed no overt changes in weight and both drugs were well growth after 17 days of treatment (P ¼ 0.001), as compared tolerated with the exception of a mild-to-moderate skin peeling with controls (Fig. 6D; Supplementary Fig. S4). Tumors from and an increased water consumption observed with the use of ponatinib-treated mice were smaller (Fig. 6E), and tumor ponatinib. Although daily administration of axitinib did not weight significantly (P ¼ 0.01) decreased (Fig. 6F). exert significant impairment of NB growth (P ¼ 0.25), ponatinib resulted in a reduced tumor growth after 16 days of treatment (P ¼ 0.003), as compared with controls (Fig. 6A; Discussion Supplementary Fig. S3). Moreover, tumors from mice admin- Preclinical testing of potential anticancer agents involves in vitro istered with ponatinib were evidently smaller (Fig. 6B), and analyses in disease-relevant cell line models, which are typically tumor weight was indeed significantly (P ¼ 0.02) decreased grown as monolayers. However, the several shortcomings of (Fig. 6C). In addition, ponatinib-treated tumors visually monolayer cultures in mimicking real-life in vivo scenarios

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highlighted the need for more representative models. Over the leukemia (Phþ ALL; ref. 20). Currently, there are 34 clinical past years, various types of 3D culture systems have received studies that aim to determine the efficacy of ponatinib in increased recognition as reliable preclinical models to recapitulate various malignancies, which include: acute myeloid leukemia, drug responses of solid tumors (11). Cells grown in a 3D spatial non–small cell lung cancer, head and neck cancer, glioblasto- layout re-establish metabolic and proliferative gradients, as ma, and patients whose advanced solid tumor has activating well as morphologic and functional properties of the correspond- mutations in FGFRs, RET and KIT genes (https://clinicaltrials. ing tissue in vivo, thus promising greater power to predict gov). Interestingly, the latter two genes have been reported as clinical efficacies. involved in NB (21–22) and may thus act as potential targets of The advantage of high-content approach over standard homo- ponatinib in a subset of patients. geneous assays for quantification of treatment-induced responses Although a remarkable clinical efficacy in patients with CML is the combination of quantitative and qualitative image data. was observed, ponatinib also showed an increased risk of Merging brightfield and fluorescent calcein-AM images allowed us cardiovascular events (23–24). The incidence of adverse events to develop the algorithm quantitating high-content readouts that was correlated with the dose-intensity of the drug. As a conse- can be attributed to spheroid viability. The final quantitative quence, the phase III clinical trial was terminated (25). Fol- metric demonstrated a high degree of reliability, as validated by lowing a careful assessment of the risk/benefitratio,theFDA comparison with the commercially available luminescent viabil- has then allowed the reintroduction of ponatinib with a boxed ity assay. The 3D high-content screening performed in CHP-134, warning calling attention on the risk of cardiovascular events IMR-32, and SK-N-BE(2) spheroids resulted in compound lists (23). Recently, it has been suggested that the use of reduced greatly overlapping among the cell lines tested. In addition, doses of ponatinib (down to 15 mg/day instead of the usual compounds that were already in use for NB treatment (i.e., dosage of 45 mg/day) might maintain therapeutic activity vincristine, carboplatin, doxorubicin, etoposide, teniposide, in most patients while decreasing the vascular adverse events topotecan) were present in the final hit list, indicating the overall (24, 26). Although clinicians will have to tackle the side effects reliability and robustness of the developed assay. Eventually, our observed upon ponatinib administration, the results achieved analysis indicated two multiple kinase inhibitors, namely pona- through our study, as well as others (17, 18), indicate that this tinib and axitinib, as candidates for repositioning in NB. drug has the potential to be employed in clinical trials aimed to Indeed, ponatinib was initially reported as a potent, orally evaluate benefits in high-risk NB patients. available multi-target kinase inhibitor active against BCR-ABL In conclusion, our survey of a comprehensive anticancer mutants and indicated for the treatment of chronic myeloid compound library provides rationale for further preclinical leukemia (CML; ref. 12). Moreover, ponatinib was reported to testing of ponatinib in NB, and eventually for setting up a inhibit Src and members of the VEGFR and PDGFR families of clinical trial involving children with NB, who have no alterna- RTKs (12). Besides, it impairs the in vitro kinase activity of all four tive therapeutic options. FGFRs (12, 13), as well as of RET kinase, showing promising preclinical activity in models of RET-driven medullary thyroid Disclosure of Potential Conflicts of Interest carcinoma (14). On the other hand, axitinib proved potent No potential conflicts of interest were disclosed. inhibition of multiple targets, such as VEGFRs, PDGFRb, and c-Kit (15). Authors' Contributions Our experiments showed that both drugs led to the accumu- Conception and design: V. Sidarovich, M. Capasso, A. Quattrone, G.P. Tonini, lation of either G1 or G2–M cell-cycle phases, as well as to the L. Longo activation of PARP, indicating that they can, indeed, impair Development of methodology: V. Sidarovich, S. Aveic, M. Pancher, V. Adami, proliferation and induce apoptosis in NB cells. Moreover, both P. Gatto, L. Pasini, F. Cimmino, M. Avitabile, M. Cilli Acquisition of data (provided animals, acquired and managed patients, compounds exerted striking efficacy in inhibiting the formation of provided facilities, etc.): V. Sidarovich, M. De Mariano, S. Aveic, M. Pancher, colonies, but only ponatinib resulted effective in inhibiting cell V. Adami, P. Gatto, M. Croce, L. Emionite, M. Cilli, L. Longo invasion and migration. Finally, in vivo experiments demonstrated Analysis and interpretation of data (e.g., statistical analysis, biostatistics, that ponatinib has the potential to inhibit tumor growth, making computational analysis): V. Sidarovich, M. De Mariano, S. Aveic, M. Pancher, this compound a suitable candidate for repositioning in NB V. Adami, P. Gatto, S. Pizzini, M. Croce, F. Parodi, S. Ferrini, A. Pagano, therapy. Although we could not prove a significant activity of M. Capasso, A. Quattrone, L. Longo Writing, review, and/or revision of the manuscript: V. Sidarovich, S. Aveic, axitinib in our in vivo model, one previous study indicated that M. Pancher, L. Pasini, L. Emionite, M. Cilli, S. Ferrini, A. Quattrone, G.P. Tonini, axitinib could partially impair the growth of human NB xeno- L. Longo grafts by inhibiting angiogenesis (16). Notably, independent Administrative, technical, or material support (i.e., reporting or organizing investigations have very recently proposed ponatinib itself as a data, constructing databases): V. Sidarovich, L. Longo drug displaying antitumor efficacy against NB (17, 18). Ponatinib Study supervision: V. Sidarovich, A. Quattrone, L. Longo proved to inhibit the FGFR1-activated signaling pathway and enhance the cytotoxic effects of doxorubicin on NB cells (18). Acknowledgments Moreover, ponatinib also interferes with the insulin-like growth This study was funded by the Italian Neuroblastoma Foundation Grant (L. Longo). factor-1 receptor signaling pathways and Src activity, inhibiting cell migration and inducing apoptosis in NB (19). In addition to The costs of publication of this article were defrayed in part by the payment of these results, we now add further evidence that supports a possible page charges. This article must therefore be hereby marked advertisement in clinical use of ponatinib in NB. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Ponatinib was developed by Ariad Pharmaceuticals, Inc., and initially approved at the end of 2012 for the treatment of CML Received September 5, 2017; revised January 12, 2018; accepted April 10, and Philadelphia-chromosome positive acute lymphoblastic 2018; published first April 25, 2018.

1414 Mol Cancer Ther; 17(7) July 2018 Molecular Cancer Therapeutics

Repurposing of Ponatinib in Neuroblastoma

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www.aacrjournals.org Mol Cancer Ther; 17(7) July 2018 1415

A High-Content Screening of Anticancer Compounds Suggests the Multiple Tyrosine Kinase Inhibitor Ponatinib for Repurposing in Neuroblastoma Therapy

Viktoryia Sidarovich, Marilena De Mariano, Sanja Aveic, et al.

Mol Cancer Ther 2018;17:1405-1415. Published OnlineFirst April 25, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-17-0841

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Cited articles This article cites 26 articles, 4 of which you can access for free at: http://mct.aacrjournals.org/content/17/7/1405.full#ref-list-1

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