Direct Targeting of MYCN Gene Ampliﬁcation by Site-Speciﬁc ... · Neuroblastoma cells were cultured on glass coverslips and treated with compounds for 24 hours before experiment

Translational Science

Direct Targeting of MYCN Gene Amplification bySite-Specific DNA Alkylation in NeuroblastomaHiroyuki Yoda1,2,3, Takahiro Inoue1,3, Yoshinao Shinozaki1, Jason Lin1,Takayoshi Watanabe1, Nobuko Koshikawa1, Atsushi Takatori2, and Hiroki Nagase1,3

Abstract

Amplification of MYCN plays a pivotal role in multipletypes of tumors and correlateswithpoor prognosis in high-riskneuroblastoma. Despite recent advances in the treatment ofneuroblastoma, no approaches directly target themaster onco-gene MYCN. Difficulties in targeting the MYCN proteininspired us to develop a new gene-level–inhibitory strategyusing a sequence-specific gene regulator. Here, we generated aMYCN-targeting pyrrole-imidazole (PI) polyamide, MYCN-A3, which directly binds to and alkylates DNA at homingmotifs within the MYCN transcript. Pharmacologic suppres-sion of MYCN inhibited the proliferation of cancer cellsharboring MYCN amplification compared with MYCN non-amplified cancer cells. In neuroblastoma xenograft mousemodels, MYCN-A3 specifically downregulated MYCN expres-

sion and suppressed tumor progression with no detectableadverse effects and resulted in prolonged overall survival.Moreover, treatment with MYCN-A3, but not MYCN non-targeting PI polyamide, precipitated a copy number reduc-tion of MYCN in neuroblastoma cells with MYCN amplifi-cation. These findings suggest that directly targeting MYCNwith MYCN-A3 is a novel therapeutic approach to reducecopy number of the MYCN gene for MYCN-amplifiedneuroblastoma.

Significance: This study presents a novel approach to drug-ging an amplified oncogene by showing that targeting geneamplification of MYCN suppresses MYCN expression andneuroblastoma growth.

IntroductionNeuroblastoma is the most common extracranial solid tumor

of childhood and accounts for 15%of deaths in pediatric patientswith cancer. Although recent advances in neuroblastoma therapyhave improved the clinical cure rate, children with high-riskfeatures have a poor prognosis despite multimodal treatment(1, 2). Notably, amplification ofMYCN is found in 25% of high-risk cases, correlates with tumor aggressiveness, and is a predictorfor clinical outcome of patients with neuroblastoma (3, 4).Silencing of MYCN expression by antisense oligonucleotides orRNA interference causes apoptosis, differentiation, and suppres-sion of tumor growth in neuroblastoma (5, 6). Therefore, inhi-bition of MYCN is a promising therapeutic approach for MYCN-driven neuroblastoma. However, the development of inhibitorsdirectly targeting MYCN has been technically and physically

challenging, as MYCN lacks enzymatic activity and globularfunctional domains (7). Besides, MYCN proteins are composedof two extended a-helical conformations with no obvious sur-faces (8), implying that development of a MYCN-targeted drug atthe protein level could be prohibited. Although several preclinicalstudies did show promising results against MYCN-driven neuro-blastoma (9–15), so far no drug linked to MYCN pathwayinhibition including the direct targeting of MYCN has yet devel-oped for clinical use.

Recently developed genome-editing technologies, such as theCRISPR/Cas9 system, have applied to various human diseasetherapies (16). CRISPR/Cas9 nucleases can efficiently inducesite-specific DNA damage within the essential copy number gainregion, that is, MYC and HER2, resulting in cancer cell death(17, 18). Such an approach is, however, currently difficult owingto technical limitations on clinical use for cancer therapy (19). Inthis study, we explored an alternative DNA-damaging strategyemploying a class of sequence-specific, double-stranded DNAminor groove binders: pyrrole-imidazole (PI) polyamides.PI polyamides are mainly composed of N-methylpyrrole andN-methylimidazole, which can bind to the gene of interestaccording to a binding rule based on the difference in the numberof hydrogen bonds in the nucleic acid bases (20, 21). To date,numerous reports have independently shown that PI polyamidesexert antitumor effects against various cancer cells (22–24).Remarkably, PI polyamides, when conjugated with DNA-alkylat-ing agents, induce sequence-specific DNA alkylation to suppresstarget gene expression by impairing the transcriptional elongationmachinery (25, 26). We also reported that PI polyamide targetingthemutantKRAS gene suppressed tumor progression in colorectalcancer (27).

1Division of Cancer Genetics, Chiba Cancer Center Research Institute, Chiba,Japan. 2Division of Innovative Cancer Therapeutics, Chiba Cancer CenterResearch Institute, Chiba, Japan. 3Department of Molecular Biology and Oncol-ogy, Graduate School of Medical and Pharmaceutical Sciences, Chiba University,Chiba, Japan.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Atsushi Takatori, Chiba Cancer Center Research Insti-tute, 666-2 Nitona, Chuo-ku, Chiba 260-8717, Japan. Phone: 81-43-264-5431;Fax: 81-43-265-4459; E-mail: [email protected]; and Hiroki Nagase,[email protected]

doi: 10.1158/0008-5472.CAN-18-1198

�2018 American Association for Cancer Research.

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In this study, we have developed a novel DNA-alkylating PIpolyamide directly regulating MYCN gene transcription (MYCN-A3), and validate the biological effects ofMYCN-A3 in in vitro andin vivo systems. To our knowledge, this is the first study thatdemonstrates thepromising potential of gene-level inhibition as atherapeutic strategy targeting MYCN amplification.

Materials and MethodsCompound synthesis

PI polyamides conjugated with a DNA-alkylating agent,MYCN-A3, MYCN-A4, and mismatch polyamide (27) weredesigned and synthesized by step-wise solid-phase synthesis ona peptide synthesizer PSSM-8 (Shimadzu). Synthetic procedurescan be found in the Supplementary Information.

Cell lines and cell cultureHuman neuroblastoma CHP-134, Kelly, and NB69 cells were

maintained in RPMI1640; IMR-32 cells were maintained inDMEM. SK-N-BE(2) cells were maintained in DMEM/F12, andSK-N-AS cells were maintained in MEM/F12. All cell lines weresupplemented with 10% FBS (Thermo Fisher Scientific) andpenicillin/streptomycin (Thermo Fisher Scientific) and culturedin a humidified atmosphere at 37�C with 5% CO2. CHP-134,Kelly, SK-N-BE(2), and SK-N-AS cells were obtained from theEuropean Collection of Authenticated Cell Cultures, NB69 cellsfrom the RIKEN Cell Bank, and IMR-32 cells from the JCRB CellBank. Mycoplasma contamination was tested by MycoplasmaDetection Set (Takara) and short tandem repeat analysis wasperformed for cell authentication (Promega). All cells were usedwithin 10 passages after thawing. Detailed information can befound in the Supplementary Information.

Cell proliferation assay (WST assay)Neuroblastoma cell lines and other types of cancer cell lines

were exposed to compounds. After 72hours, cell proliferationwasdeterminedbyWSTassay using theCell CountingKit-8 (Dojindo)following the manufacturer's instructions and quantified on aMTP-310 Microplate Reader (Corona Electric).

Bio-MYCN-A3 precipitation assayCHP-134 cells were exposed to Bio-MYCN-A3 for 12 hours.

After phenol extraction and ethanol precipitation, genomic DNAwas fragmented by sonication using the M220 DNA ShearingSystem (Covaris) and subjected to PCR, as described in theSupplementary Information.

PCR analysisNeuroblastoma cells were exposed to compounds for RT-PCR

(qPCR). After 24 hours, RNA was extracted using the RNeasy PlusMini Kit (Qiagen) following the manufacturer's instructions.Purified RNA (1 mg)was used for cDNA synthesis with SuperscriptVILO Master Mix (Thermo Fisher Scientific), before reaction andmeasurements on a Veriti Thermal Cycler or StepOne Plus Real-Time PCR (Thermo Fisher Scientific). PCR primer sequences canbe found in the Supplementary Information.

Western blotting analysisWestern blotting was performed as described previously (27).

Detailed information can be found in the SupplementaryInformation.

Annexin V stainingNeuroblastoma cells were exposed to compounds for 24 hours

before the experiment. Annexin V staining was performed usingMEBCYTO Apoptosis Kit (MBL) according to the manufacturer'sinstructions. Cells were analyzed by flow cytometer BD FACSCa-libur and FlowJo Software (Becton Dickinson).

TUNEL assay and immunofluorescence stainingNeuroblastoma cells were cultured on glass coverslips and

treated with compounds for 24 hours before experiment.TUNEL assays were performed using the In situ Cell DeathDetection Kit and TMR Red (Roche) according to the manu-facturer's instructions. Detailed information on immuno-fluorescence staining can be found in the SupplementaryInformation.

In vivo studyHuman neuroblastoma cells were mixed 1:1 with Matrigel

(Corning) and injected subcutaneously into the flank ofBALB/c nu/nu mice purchased from Charles River Laboratories.Intravenous administration of 0.1 or 0.3 mg/kg compoundsbegan when the average tumor size reached 150 mm3. DMSOwas administered as a control (n ¼ 7 or 8 mice in each group).Tumor volume (W�W� L/2) and body weights were measuredevery 2 or 3 days. The mice were sacrificed when tumor volumereached 2,000 mm3. Survival analysis was performed usingKaplan–Meier curves.

Hematoxylin and eosin and IHC stainingCompounds (0.3 mg/kg) were injected intravenously into

BALB/c nu/nu mice harboring human neuroblastoma xeno-grafts when the tumor volume surpassed 200 mm3. After 48hours, the tumor was resected and fixed in 4% paraformalde-hyde. Sections (4 mm) were deparaffinized by immersing inxylene and rehydrated, followed by staining with hematoxylinand eosin (H&E) according to standard procedures, and fol-lowed by immunostaining using antibodies. Detailed informa-tion on antibodies can be found in the Supplementary Infor-mation. The number of cleaved caspase-3–positive cells wascounted in three high-power fields of each tumor at �400magnification.

In vitro FISH analysisNeuroblastoma cells were exposed to compounds. After

72 hours, cells were collected, mounted on the slide glass, andfixed with Carnoy fixative. VYSIS LSI MYCN (2p24) spectrumgreen/VYSIS CEP2 spectrum orange probe was obtained fromAbbott. SureFISH for MYCN, PAX3, and MYC probes wereobtained from Agilent. The experiments were performed fol-lowing the manufacturers' instructions. Cell nuclei were stainedwith ProLong Gold Antifade Mountant with DAPI (ThermoFisher Scientific). The fluorescence images were captured byconfocal microscope TCS SP8 (Leica) or fluorescence micro-scope BZ-X700 (Keyence), and the signal intensities werequantified in five randomly selected fields using WinROOFsoftware (Mitani).

CRISPR/Cas9–mediated DNA cleavage of MYCN geneCRISPR/Cas9–mediated targeting of the MYCN gene was per-

formed using Alt-R S.p. Cas9 Nuclease 3NLS, Alt-R CRISPR/Cas9crRNAs targeting MYCN (MYCNcr-a and MYCNcr-b), Alt-R

Targeting MYCN Amplification by MYCN-Specific Polyamide

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CRISPR/Cas9 Human Negative Control crRNA, and Alt-RCRISPR/Cas9 tracrRNA-ATTO550 (Integrated DNA Technolo-gies), as described in the Supplementary Information.

Southern blotting analysisGenomic DNA was extracted from CHP-134 cells exposed to

compounds for 72hours anddigestedwithEcoRI. After separationin 0.8%agarose gel electrophoresis, DNAwas transferred to nylonmembranes (Hybond Nþ, GE Healthcare). Digoxigenin-labeledprobes were synthesized by a PCR DIG Probe Synthesis Kit(Roche) according to manufacturer's instructions using the fol-lowing primers: MYCN, 50-TGTGTTTGAGCTGTCGGAGAG-30

and 50-CCCCCTTTGTAAAAATGCAACC-30; PAX3, 50-TTTATTC-CATGGGGCTAGGAG-30 and 50-GATGCCGTGTTCTCTTTTCC-30.

In vivo FISH analysisFormalin-fixed paraffin-embedded (FFPE) tumor blocks were

sliced (4mm)anddeparaffinized by immersing in xylene and thenwere rehydrated. FISH analyses were performed using aHistologyFISH Accessory Kit (Dako) according to the manufacturer'sinstructions. The fluorescence images were captured by confocalmicroscope TCSSP8orfluorescencemicroscope BZ-X700, and thesignal intensities were quantified in five randomly selected fieldsusing WinROOF software.

Statistical analysisStatistical analysis was performed using GraphPad Prism 6.0

(GraphPad Software). Detailed information can be found in theSupplementary Information.

Ethical statementThis study was performed in strict accordance with the

recommendations in the Guide for the Care and Use of Lab-oratory Animals of the Ministry of Education, Culture, Sports,Science and Technology of Japan. The protocol was approvedby the Committee on the Ethics of Animal Experiments ofChiba Cancer Center. All efforts were made to minimizesuffering.

ResultsThe amplified MYCN gene is a potential target inneuroblastoma

We first investigated whether MYCN inhibition would inducegrowth inhibition in MYCN-amplified neuroblastoma cells. Thedepletion of MYCN using siRNAs targeting MYCN showed theinhibition of cell proliferation in MYCN-amplified neuroblasto-ma cells (Supplementary Fig. S1A and S1B), supporting theprevious reports (5, 6).

Recently, it has proposed that cancer cells are vulnerable toCRISPR/Cas9–induced site-specific DNA breaks within ampli-fied regions of the genome (17). To test this hypothesis inMYCN-amplified neuroblastoma cells, we employed theCRISPR/Cas9 system targeting MYCN as a site-specific DNAdamage agent. As expected, crRNAs targeting MYCN signifi-cantly increased the number of apoptotic cells compared withcontrol crRNA in MYCN-amplified CHP-134 and Kelly cells,whereas no induction of apoptosis was observed in MYCNnonamplified SK-N-AS cells (Supplementary Fig. S1C andS1D). Under these conditions, we observed that crRNAs target-

ing MYCN decreased MYCN expression in CHP-134 and Kellycells (Supplementary Fig. S1E). According to these results,direct targeting of the MYCN gene is an attractive therapeuticapproach for MYCN-amplified neuroblastoma, even thoughcurrent CRISPR/Cas9 or interfering RNA molecules may notbe clinically applicable owing to a lack of appropriate deliverymethods. Thus, we took an approach of chemical biology withPI polyamides capable of penetrating into the nuclei of cellsand binding to the specific sequence of target DNA.

MYCN-amplified neuroblastoma cells are highly sensitive toMYCN-A3 targeting the 30UTR of the MYCN gene transcriptregion

To induce DNA damage directly in the amplified region of theMYCN gene, we designed and synthesized two sequence-specificDNA-alkylating PI polyamides, MYCN-A3 and MYCN-A4, whichbind to and alkylate MYCN transcript regions (MYCN-A3, 50-TGGGWGCCW-30; MYCN-A4, 50-TGGGWCGGW-30; W, A, orT; Fig. 1A; Supplementary Fig. S2A).

The cytotoxicity of neuroblastoma cells with or withoutMYCNamplification showed that CHP-134 cells were highly sensitive toboth MYCN-A3 and MYCN-A4 when compared with SK-N-AScells, and MYCN-A3 exhibited stronger cytotoxicity than MYCN-A4 (Supplementary Fig. S2B), prompting us to proceed withMYCN-A3 as the lead candidate. We further assessed the cytotox-icity of MYCN-A3 in different neuroblastoma cell lines (Supple-mentary Fig. S2C) and other types of cancer cell lines with orwithoutMYCN amplification.MYCN-A3 displayed selective cyto-toxicity against MYCN-amplified cancer cells with the medianIC50 ¼ 4.5 nmol/L, nearly a tenth of the cytotoxicity in MYCNnonamplified cancer cells (IC50 ¼ 50.4 nmol/L; Fig. 1B; Supple-mentary Table S1).

To verify the binding site of MYCN-A3 in the region ofMYCNgene, we synthesized a biotinylated derivative of MYCN-A3(Bio-MYCN-A3; Fig. 1A). We initially confirmed the intranuc-lear localization of Bio-MYCN-A3 in CHP-134, Kelly, SK-N-AS,and NB69 cells by using Cy3-conjugated streptavidin (Fig. 1C).To examine a site-specific covalent alkylation of DNA by Bio-MYCN-A3 in CHP-134 cells, we designed three primer setswithin the MYCN gene (primer set 1 in exon-1; primer set 2in exon-3; primer set 3 in 30UTR that contains a MYCN-A3–binding motif; Fig. 1D). After extracting genomic DNA fol-lowed by biotin–streptavidin interactions, we observed theenrichment of the DNA fragments in the region of primer set3, as compared with upstream regions of the MYCN-A3 motif(primer sets 1 and 2), suggesting that MYCN-A3 induces a site-specific alkylation at the 30UTR of MYCN gene containing itstarget DNA sequence. Bio-MYCN-A3 demonstrated a compa-rable IC50 value to MYCN-A3 in CHP-134 cells (SupplementaryFig. S2D), indicating that the additional biotinylated moietydid not interfere with the pharmacologic characteristics ofMYCN-A3. To further confirm the direct alkylation of MYCNgene with MYCN-A3, we performed ligation-mediated PCR,which amplifies DNA fragments with blunt ends generated atalkylated sites (27). Genomic DNA collected from MYCN-A3–treated cells displayed a PCR band of the expected size, whilevehicle control and mismatch polyamide that has no bindingmotif for the MYCN gene failed to show the PCR product(Supplementary Fig. S3A and S3B). We next tested the bindingspecificity of MYCN-A3 by surface plasmon resonanceassay. MYCN-A3 showed 212-fold higher binding affinity to

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the full-match sequence compared with mismatch polyamide(Supplementary Fig. S3C and S3D). These data suggest thatMYCN-A3 specifically alkylates the targeted DNA sequence ofMYCN gene in MYCN-amplified neuroblastoma cells.

MYCN-A3 suppresses MYCN expression in MYCN-amplifiedneuroblastoma cells

To determine whether MYCN-A3 inhibits the transcriptionalelongation of the MYCN gene by specific alkylation at thetargeted DNA sequence, we performed qPCR and Westernblotting analyses. The treatment of MYCN-A3, but not Mis-match polyamide, demonstrated dose-dependent suppressionof MYCN expression at the mRNA and protein levels in MYCN-amplified neuroblastoma cells (Fig. 2A and B; SupplementaryFig. S4A and S4B). We also confirmed that MYCN-A4 decreasedlevels of MYCN expression and cell proliferation, whereasmismatch polyamide showed little, if any, influence onMYCN-amplified neuroblastoma cells (Supplementary Fig.S4C and S4D). Among direct transcriptional target genes ofMYCN, NLRR1, DKC1, TWIST1, and BMI1 are essential forMYCN-mediated neuroblastoma tumorigenesis (28–31) andfree of the MYCN-A3–binding motif. MYCN-A3 treatmentdecreased the expression levels of those genes in a similarmanner to the MYCN gene (Fig. 2C). These results suggest that

MYCN-A3 suppresses MYCN expression at the transcriptionallevel, resulting in the downregulation of MYCN target genes.

MYCN-A3 shows the similar genome-wide effects of targetingMYCN CRISPR/Cas9 system

We performed expression microarrays to assess the genome-wide effect of MYCN-A3 in comparison with CRISPR/Cas9 silenc-ing of MYCN (MYCNcr-a; Supplementary Fig. S5A), and foundboth treatments to be generally similar in terms of overall expres-sion changes. Volcano plots revealed similar correlations betweenchanges in expression and significance level, especially for thosenear log2 FC � 0 (Supplementary Fig. S5B). We also found thatboth treatments tended to downregulate a large portion of com-mon elements (Supplementary Fig. S5C), suggesting that thereduction of MYCN expression by MYCN-A3 and MYCNcr-awould have a similar effect overall, despite that both treatmentsare mechanistically different. To further assess the biologicaleffects of the two treatments, we decided to explore the behavioraldifferences by comparing aggregated changes in expression on apathway level. To do so, we categorized, per pathway, geneexpressions between MYCN-A3 and MYCNcr-a, and used Fisherexact test to evaluate whether both treatments lead to similar fold-change outcomes. These pathway-level comparisons also sug-gested that only a small number of pathways (as annotated in

Figure 1.

Amplification of theMYCN gene is apotential therapeutic vulnerability.A, Chemical structures ofMYCN-A3 and biotinylatedMYCN-A3 (Bio-MYCN-A3),sequence-specific DNA-alkylatingagents directly targeting theMYCNgene. MYCN-A3 was designed tobind to 30UTR within theMYCNgene (50-TGGGWGCCW-30 ; W, A, orT). The site of alkylation is shown inred. B, IC50 values were determinedin 12MYCN-amplified (Amp) and 15MYCN nonamplified (non-Amp)cancer cell lines treated withMYCN-A3 for 72 hours byWSTassay. Each dot represents anindividual cell line. Horizontal linesindicate the median values (Amp,4.5 nmol/L; non-Amp, 50.4 nmol/L;IC50 values are specified inSupplementary Table S1). P valueswere determined by nonparametricMann–Whitney U tests. C,Distribution of Bio-MYCN-A3 inMYCN-amplified CHP-134 and Kelly,MYCN nonamplified SK-N-AS, andNB69 neuroblastoma cells. Blue,DAPI; red, Bio-MYCN-A3. DMSOwas used as a control. Scale bars,5 mm. D, Bio-MYCN-A3 precipitationassay after biotin–streptavidininteractions to test the directbinding of MYCN-A3 to theMYCNgene in CHP-134 cells. Theschematic represents the positionsof PCR primer sets 1, 2, and 3. Theregion of primer set 3 includes thebinding site of MYCN-A3. DMSOwas used as a control.






the Kyoto Encyclopedia of Genes andGenomes) were found to bedifferent statistically (Supplementary Fig. S5D, histogram of Pvalues). Furthermore, most of the statistically significant path-ways, for instance glycosylphosphatidylinositol (GPI)-anchorbiosynthesis (P ¼ 0.0119) appeared not to be intimately con-nected to the carcinogenesis of neuroblastoma or MYCN (Sup-plementary Table S2); in sharp contrast, the more relevant path-ways (e.g., MAPK, ErbB, Ras signaling pathways) appeared to berelatively invariant between the two treatments, as indicated bytheir high P values (P � 1).

MYCN-A3 induces apoptosis in MYCN-amplifiedneuroblastoma cells

Cell morphologic abnormalities such as cell shrinkage anddetachment were observed in CHP-134 and Kelly cells, but notSK-N-AS and NB69 cells, after MYCN-A3 treatment (Supple-mentary Fig. S6A). We next analyzed the cell surface Annexin Vbinding, which can detect apoptotic cell death, to investigatethe mechanism of cell death after MYCN-A3 treatment(Fig. 3A). At 24 hours, CHP-134 and Kelly cells showedmarkedly increased Annexin V binding. We then performedthe TUNEL assay to ensure that CHP-134 and Kelly cellsunderwent apoptosis after MYCN-A3 treatment (Fig. 3B). West-ern blotting analysis also demonstrated that the levels ofapoptotic markers, cleavage of caspase-3 and PARP, wereincreased in CHP-134 and Kelly cells (Fig. 3C). Notably,Western blotting analysis and immunofluorescence stainingexhibited elevated levels of gH2AX in CHP-134 and Kelly cellscompared with in SK-N-AS and NB69 cells (Fig. 3C and D;Supplementary Fig. S6B). MYCN-A3 treatment led to the ele-

vated levels of p53 phosphorylation at Ser-15 and the expres-sions of p53 and its family members in all tested cells (Sup-plementary Fig. S6C). These results suggest that MYCN-ampli-fied neuroblastoma cells accumulate DNA damage after MYCN-A3 treatment and undergo apoptosis.

MYCN-A3 suppresses tumor progression in humanneuroblastoma xenograft mouse models

On the basis of these findings in vitro, we next evaluated theantitumor potential of MYCN-A3 in neuroblastoma xenograftsusing immunodeficient BALB/c nu mice. Single-dose administra-tions of MYCN-A3 significantly suppressed tumor growth inmiceharboring CHP-134 xenografts (Supplementary Fig. S7A). Threeweekly injections with MYCN-A3 significantly suppressed tumorgrowth compared with the treatment of CBI, a DNA-alkylatingagent without the core PI polyamide (Supplementary Fig. S7B).Antitumor activity of MYCN-A3 was also confirmed in Kellyxenografts bymultiple administrations ofMYCN-A3without lossof body weight (Fig. 4A–C). The survival rate of mice significantlyprolonged with multiple administration of MYCN-A3 in a dose-dependent manner (Fig. 4D). In contrast to MYCN-A3, mismatchpolyamide did not induce the inhibition of tumor growth(Fig. 4E–H).

We also performed histologic studies on the antitumor effectof MYCN-A3 using CHP-134 and Kelly xenograft–derivedtumor tissue sections at 48 hours after administration ofMYCN-A3 or mismatch polyamide. H&E staining revealedchromatin condensation and nuclear fragmentation in tumorstreated with MYCN-A3. Furthermore, IHC staining using anti-MYCN, 53BP1, and cleaved caspase-3 antibodies demonstrated

Figure 2.

MYCN-A3 inhibits MYCN expressioninMYCN-amplified neuroblastomacell lines. A and B,MYCN-amplifiedCHP-134, Kelly, SK-N-BE(2), andIMR-32 cells treated with 10 nmol/LMYCN-A3 or mismatch polyamidefor 24 hours. MYCNmRNA (A) andprotein (B) were analyzed by qPCRandWestern blotting analysis,respectively. P values weredetermined by one-way ANOVAfollowed by Dunnett posttest(�� , P < 0.01; ��, P < 0.001;�� , P < 0.0001). Data aremean� SD from three independentexperiments. C, CHP-134, Kelly,SK-N-BE(2), and IMR-32 cellstreated with 10 nmol/L MYCN-A3 for24 hours. mRNA of theMYCN-targeted genes, such as NLRR1,DKC1, TWIST1, and BMI1, wasanalyzed by qPCR. DMSOwas usedas a control. RPS18was used as aninternal control.

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Figure 3.

MYCN-A3 induces apoptotic cell death inMYCN-amplified neuroblastoma cell lines in vitro. A–D, CHP-134, Kelly, SK-N-AS, and NB69 cells treated with 10 nmol/LMYCN-A3 or mismatch polyamide for 24 hours. Representative images show that Annexin V–positive cells were detected by using flow cytometry (A); apoptoticcell death was detected by TUNEL assay (B); protein expression of cleaved caspase-3, PARP, and gH2AX was detected byWestern blotting analysis (C); andgH2AX was detected by immunofluorescence staining (D). Scale bars, 50 mm. DMSOwas used as a control.






Figure 4.

MYCN-A3 suppresses tumor growth in humanMYCN-amplified neuroblastoma xenograft models.A�H, Kelly cells were subcutaneously injected into the flankof female immune-deficient BALB/c nu/nu mice. Administration of MYCN-A3 or mismatch polyamide began when average tumor size reached 150mm3. Tumorvolume and body weight of mice were measured every 3 days. Red arrows, timepoint of administration of MYCN-A3 or mismatch polyamide. P values weredetermined by repeated measures ANOVA followed by a Bonferroni/Dunn posttest. Data are represented as mean� SD (A, B, E, and F); representative imagesof mice with Kelly xenografts administered 0.3 mg/kg MYCN-A3 or mismatch polyamide at day 12 after administration (C and G). Kaplan–Meier plots ofoverall survival in MYCN-A3- or mismatch polyamide–administered mice with Kelly xenograft tumors. P values were determined from two-sided log-rank tests(D and H). DMSOwas used as a control. ns, nonsignificant.

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the suppressed MYCN expression, and the elevated levels ofDNA damage and apoptotic markers by MYCN-A3 administra-tion (Fig. 5A and B; Supplementary Fig. S8A and S8B). Collec-tively, MYCN-A3 suppressed MYCN expression, increased DNAdamage, and induced apoptosis, resulting in the inhibition oftumor progression in MYCN-amplified neuroblastoma mousemodels.

MYCN-A3 disrupts conformation of the amplified MYCN geneloci in MYCN-amplified neuroblastoma cells

We further investigated whether site-specific DNA damage attheMYCN gene loci alters the amplification status of theMYCN

gene in neuroblastoma cells. FISH analysis using a probe forMYCN at 2p24.3 displayed a typical diffused pattern in thenuclei of control CHP-134 and Kelly cells, while a probe forCEP2 (2p11.1-q11.1) showed a normal disomic signal distri-bution (Fig. 6A, left). Intriguingly, CRISPR/Cas9 againstMYCN(MYCNcr-a) attenuated the probe signals in CHP-134 cells(Fig. 6A, right; Supplementary Fig. S9A). The reduced signalintensity of the MYCN probe was observed in CHP-134 andKelly cells treated with MYCN-A3, whereas the influence ofmismatch polyamide on FISH signals was marginal (Fig. 6B;Supplementary Fig. S9B). Probes for the PAX3 (2q36.1) andMYC (8q24.21) genes that possess no MYCN-A3–binding

Figure 5.

Pharmacologic effects of MYCN-A3on humanMYCN-amplifiedneuroblastoma xenograft modelsin vivo. A, BALB/c nu/nu mice withKelly xenograft were administered0.3 mg/kg MYCN-A3 or mismatchpolyamide. At 48 hours afteradministration, the tumor tissueswere collected and used for H&Eand IHC with MYCN, 53BP1, andcleaved caspase-3. Scale bars, 50mm. B,Quantification of the numberof positive cells with cleavedcaspase-3 in Kelly xenografts.P values were determinedby one-way ANOVA followed byDunnett posttest. Data arerepresented as mean� SD. DMSOwas used as a control.






motif indicated the limited influence of MYCN-A3 in a distantregion of chromosome 2 and a different chromosome (Sup-plementary Fig. S9C).

To further assess the change in the MYCN gene copy number,Southern blotting analysis was performed in CHP-134 cells.Notably, MYCNcr-a andMYCN-A3 treatment reduced the relativeband intensity of the MYCN probe without affecting the bandsimilarly detectedby thePAX3probe (Fig. 6C, top; SupplementaryFig. S9D), whereas mismatch polyamide did not affect eitherMYCN or PAX3 probes (Fig. 6C, bottom). Moreover, FISH anal-yses using FFPE samples revealed that FISH signal intensities oftheMYCN probe were decreased by MYCN-A3 administration inthe tissues of xenograft neuroblastoma tumors, whereas no influ-ence was observed by treatment ofmismatch polyamide (Fig. 6D;Supplementary Fig. S9E). Altogether, our findings suggest thatsite-specific DNA alkylation contributes to the selective destruc-tion of theMYCN gene loci, resulting in the suppression ofMYCNexpression.

DiscussionRecent research and development of new pharmaceutical can-

didates have improved our understanding of neuroblastomabiology and the overall survival of patients. However, the clinicalprognosis remains poor for patients with MYCN-amplifiedneuroblastoma (32, 33). Therefore, those patients needMYCN-targeting agents in clinical practice.

Although MYCN-A3 preferentially affected MYCN-amplifiedtumor cells, the relationship betweenMYCN copy numbers andthe sensitivity of current chemotherapeutic agents remainsunclear. A recent study has reported that the CRISPR/Cas9 system targeting amplified genomic regions inducessite-specific DNA damage accompanied by accumulation ofgH2AX and cell death (34). In this data, gH2AX was elevatedby MYCN-A3 treatment in MYCN-amplified neuroblastomacells but not in MYCN nonamplified cells, suggesting that thehigher copy number of the MYCN gene could lead to more

Figure 6.

MYCN-A3 decreased the copynumbers of theMYCN gene in vitroand in vivo.A and B, Representativeimages of FISH analysis in CHP-134cells at 48 hours after transfectionwith MYCNcr-a (A). Scale bars,25 mm. B, CHP-134 and Kelly cellstreated with MYCN-A3 or mismatchpolyamide for 72 hours. Scale bars,10 mm. Green,MYCN (2p24.3); red,CEP2 (2p11.1-q11.1; B). C, Southernblotting usingMYCN and PAX3probes in CHP-134 cells transfectedwith MYCNcr-a for 48 hours (top),treated with MYCN-A3 or mismatchpolyamide for 72 hours (bottom).D, Representative images of theFISH analysis show that the micewith CHP-134 and Kelly xenograftsadministered with MYCN-A3 ormismatch polyamide. At 48 hoursafter administration, the tumortissues were collected and used forFISH analysis. Scale bars, 25 mm.DMSOwas used as a control.

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events of sequence-dependent DNA alkylation. This novelstrategy to target an amplified region of genomic DNA isattractive for developing new lead compounds against tumortypes driven by oncogene amplification.

Recent studies have further demonstrated that targetingMYCNtranscription by the BET bromodomain inhibitor JQ1 or CDK7inhibitor THZ1 effectively induces cell death inMYCN-amplifiedneuroblastoma cells (35, 36), indicating that transcriptionalsuppression of MYCN is a promising strategy in drug develop-ment. Previously, we also reported a PI polyamide designed totarget a driver gene mutation of KRAS downregulating its tran-scription in colorectal cancer cells (27). Herein, MYCN-A3 tar-geted the MYCN gene and had a similar impact on the genome-wide gene expression comparable with CRISPR/Cas9 system.These data suggest that primary targeting of the MYCN gene isindispensable for MYCN-A3 to induce a massive neuroblastomacell death, even though off-target genes with the full-matchbinding sequence may exist.

The pharmacologicmechanisms of action ofDNA-alkylating PIpolyamides to disrupt gene transcription remain to be deter-mined. Upon MYCN-A3 treatment, the hybridization of FISHprobes was repressed in the amplified genomic regions ofMYCN,perhaps as a consequence of the covalent binding of PI polyam-ide. In contrast, other genomic DNA regions outside of theMYCNamplicon exhibited normal FISH probe binding in MYCN-A3–treated cells. Southern blotting analyses also supported the site-specific induction of DNA damage by MYCN-A3 treatment atamplified MYCN gene loci. This amplified region also containsother transcripts includingNCYM and lncUSMycN, a cis-antisensegene of MYCN and a long noncoding RNA gene, respectively(37, 38).NCYM and lncUSMycN are coamplified withMYCN andregulate MYCN expression and neuroblastoma tumorigenesis.FISH probes used in this study detected a >200 kb genomic regioncontaining the MYCN gene and these two MYCN-associatedgenes. Because FISH analyses clearly showed reduced probesignals afterMYCN-A3 treatment, it is likely thatMYCN-A3 affectsthe flanking region of MYCN gene loci and thereby exerts highcytotoxicity by repression of neighbor genes in addition to tran-scriptional target genes of MYCN.

In human neuroblastoma xenograft mice, the weak signals ofthe MYCN probe in FFPE–FISH analyses strongly suggest thatMYCN-A3 distributes in tumor tissues and cells in situ afterintravenous administration. Although PI polyamides can beeffluxed from the plasma after intravenous injection (39), theyeffectively accumulate in tumor tissue and are retained until atleast 48 hours after administration (40, 41). These reports suggestthat PI polyamides might have an enhanced permeability andretention effect (42), allowing for prolonged administrationintervals to further reduce the potential side effects of the PIpolyamides. Indeed, MYCN-A3 exhibits antitumor effects evenat a low dose without severe side effects such as loss of bodyweight. Most current conventional chemotherapy regimens con-sist of various combinations of high-dose DNA-damaging agents(43, 44), and patients who survive high-risk neuroblastoma maysuffer from significant subsequent effects such as growth failureand secondary malignancy (45).

Our current data suggest thatMYCN-A3offers a new therapeuticstrategy with lesser side effects for patients with advanced neuro-blastoma with MYCN amplification. In addition, loss of copynumbers of MYCN by MYCN-A3 or the CRISPR/Cas9 systemmight be a novel approach to accelerate drug development forclinical practice. To this end, we need further preclinical studiesusing patient-derived xenograft models and novel geneticallyengineered models with a transgene inducing multiple copynumbers of the entire MYCN including 30UTR, because currentMYCN-Tg mouse models do not have the 30UTR of the humanMYCN gene.

In conclusion, we have successfully developed a novel PIpolyamide conjugated with a DNA-alkylating agent, MYCN-A3,which shows high cytotoxicity in MYCN-amplified tumor cellsderived from neuroblastoma. Site-specific DNA damage inducedby MYCN-A3 leads to suppression of theMYCN gene and potentantitumor activity against MYCN-amplified neuroblastoma. Tar-getingMYCN gene amplification using MYCN-A3 can be a novelapproach for the development of next-generation cancer therapyfor high-risk MYCN-amplified neuroblastoma.

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

Authors' ContributionsConception and design: H. Yoda, A. Takatori, H. NagaseDevelopment of methodology: H. Yoda, A. TakatoriAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Yoda, A. TakatoriAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Yoda, J. Lin, A. TakatoriWriting, review, and/or revision of themanuscript:H. Yoda, J. Lin, A. Takatori,H. NagaseAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T. Inoue, J. Lin, T. Watanabe, N. KoshikawaStudy supervision: A. Takatori, H. NagaseOthers (organic synthesis and characterization of compounds): Y. Shinozaki,T. Watanabe

AcknowledgmentsWe are grateful to K. Hiraoka, Y. Suzuki, K. Sugimoto, Y. Kaihou, T. Koga,

R. Igarashi, Y. Nakamura, and R. Murasugi for technical assistance. We alsothank K. Shinohara, T. Bando, and H. Sugiyama for advice on chemicaldesign. This work was supported in part by Practical Research for InnovativeCancer Control from the Japan Agency for Medical Research and Develop-ment (AMED, grant no. 15656919 to A. Takatori), MEXT KAKENHI (grantno. 25830092 to A. Takatori), Takeda Science Foundation (to A. Takatori),Princess Takamatsu Cancer Research Fund (to H. Nagase), JSPS KAKENHI(grant nos. JP26290060, 17H03602, and JP16H01579 to H. Nagase), andAMED (grant no. 18ae0101051 to H. Nagase).

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 May 7, 2018; revised October 23, 2018; accepted December 17,2018; published first December 24, 2018.

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Yoda et al.




2019;79:830-840. Published OnlineFirst December 24, 2018.Cancer Res Hiroyuki Yoda, Takahiro Inoue, Yoshinao Shinozaki, et al. Alkylation in Neuroblastoma

Gene Amplification by Site-Specific DNAMYCNDirect Targeting of

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Direct Targeting of MYCN Gene Ampliﬁcation by Site-Speciﬁc ... · Neuroblastoma cells were cultured on glass coverslips and treated with compounds for 24 hours before experiment