Amplification of MYCN plays a pivotal role in multiple types of tumors and correlates with poor prognosis in high-risk neuroblastoma. Despite recent advances in the treatment of neuroblastoma, no approaches directly target the master oncogene MYCN. Difficulties in targeting the MYCN protein inspired us to develop a new gene-level–inhibitory strategy using a sequence-specific gene regulator. Here, we generated a MYCN-targeting pyrrole-imidazole (PI) polyamide, MYCN-A3, which directly binds to and alkylates DNA at homing motifs within the MYCN transcript. Pharmacologic suppression of MYCN inhibited the proliferation of cancer cells harboring MYCN amplification compared with MYCN nonamplified cancer cells. In neuroblastoma xenograft mouse models, MYCN-A3 specifically downregulated MYCN expression and suppressed tumor progression with no detectable adverse effects and resulted in prolonged overall survival. Moreover, treatment with MYCN-A3, but not MYCN nontargeting PI polyamide, precipitated a copy number reduction of MYCN in neuroblastoma cells with MYCN amplification. These findings suggest that directly targeting MYCN with MYCN-A3 is a novel therapeutic approach to reduce copy number of the MYCN gene for MYCN-amplified neuroblastoma.

Significance:

This study presents a novel approach to drugging an amplified oncogene by showing that targeting gene amplification of MYCN suppresses MYCN expression and neuroblastoma growth.

Neuroblastoma is the most common extracranial solid tumor of childhood and accounts for 15% of deaths in pediatric patients with cancer. Although recent advances in neuroblastoma therapy have improved the clinical cure rate, children with high-risk features have a poor prognosis despite multimodal treatment (1, 2). Notably, amplification of MYCN is found in 25% of high-risk cases, correlates with tumor aggressiveness, and is a predictor for clinical outcome of patients with neuroblastoma (3, 4). Silencing of MYCN expression by antisense oligonucleotides or RNA interference causes apoptosis, differentiation, and suppression of tumor growth in neuroblastoma (5, 6). Therefore, inhibition of MYCN is a promising therapeutic approach for MYCN-driven neuroblastoma. However, the development of inhibitors directly targeting MYCN has been technically and physically challenging, as MYCN lacks enzymatic activity and globular functional domains (7). Besides, MYCN proteins are composed of two extended α-helical conformations with no obvious surfaces (8), implying that development of a MYCN-targeted drug at the protein level could be prohibited. Although several preclinical studies did show promising results against MYCN-driven neuroblastoma (9–15), so far no drug linked to MYCN pathway inhibition including the direct targeting of MYCN has yet developed for clinical use.

Recently developed genome-editing technologies, such as the CRISPR/Cas9 system, have applied to various human disease therapies (16). CRISPR/Cas9 nucleases can efficiently induce site-specific DNA damage within the essential copy number gain region, that is, MYC and HER2, resulting in cancer cell death (17, 18). Such an approach is, however, currently difficult owing to technical limitations on clinical use for cancer therapy (19). In this study, we explored an alternative DNA-damaging strategy employing a class of sequence-specific, double-stranded DNA minor groove binders: pyrrole-imidazole (PI) polyamides. PI polyamides are mainly composed of N-methylpyrrole and N-methylimidazole, which can bind to the gene of interest according to a binding rule based on the difference in the number of hydrogen bonds in the nucleic acid bases (20, 21). To date, numerous reports have independently shown that PI polyamides exert antitumor effects against various cancer cells (22–24). Remarkably, PI polyamides, when conjugated with DNA-alkylating agents, induce sequence-specific DNA alkylation to suppress target gene expression by impairing the transcriptional elongation machinery (25, 26). We also reported that PI polyamide targeting the mutant KRAS gene suppressed tumor progression in colorectal cancer (27).

In this study, we have developed a novel DNA-alkylating PI polyamide directly regulating MYCN gene transcription (MYCN-A3), and validate the biological effects of MYCN-A3 in in vitro and in vivo systems. To our knowledge, this is the first study that demonstrates the promising potential of gene-level inhibition as a therapeutic strategy targeting MYCN amplification.

Compound synthesis

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

Cell lines and cell culture

Human neuroblastoma CHP-134, Kelly, and NB69 cells were maintained in RPMI1640; IMR-32 cells were maintained in DMEM. SK-N-BE(2) cells were maintained in DMEM/F12, and SK-N-AS cells were maintained in MEM/F12. All cell lines were supplemented with 10% FBS (Thermo Fisher Scientific) and penicillin/streptomycin (Thermo Fisher Scientific) and cultured in a humidified atmosphere at 37°C with 5% CO2. CHP-134, Kelly, SK-N-BE(2), and SK-N-AS cells were obtained from the European Collection of Authenticated Cell Cultures, NB69 cells from the RIKEN Cell Bank, and IMR-32 cells from the JCRB Cell Bank. Mycoplasma contamination was tested by Mycoplasma Detection Set (Takara) and short tandem repeat analysis was performed for cell authentication (Promega). All cells were used within 10 passages after thawing. Detailed information can be found in the Supplementary Information.

Cell proliferation assay (WST assay)

Neuroblastoma cell lines and other types of cancer cell lines were exposed to compounds. After 72 hours, cell proliferation was determined by WST assay using the Cell Counting Kit-8 (Dojindo) following the manufacturer's instructions and quantified on a MTP-310 Microplate Reader (Corona Electric).

Bio-MYCN-A3 precipitation assay

CHP-134 cells were exposed to Bio-MYCN-A3 for 12 hours. After phenol extraction and ethanol precipitation, genomic DNA was fragmented by sonication using the M220 DNA Shearing System (Covaris) and subjected to PCR, as described in the Supplementary Information.

PCR analysis

Neuroblastoma cells were exposed to compounds for RT-PCR (qPCR). After 24 hours, RNA was extracted using the RNeasy Plus Mini Kit (Qiagen) following the manufacturer's instructions. Purified RNA (1 μg) was used for cDNA synthesis with Superscript VILO Master Mix (Thermo Fisher Scientific), before reaction and measurements on a Veriti Thermal Cycler or StepOne Plus Real-Time PCR (Thermo Fisher Scientific). PCR primer sequences can be found in the Supplementary Information.

Western blotting analysis

Western blotting was performed as described previously (27). Detailed information can be found in the Supplementary Information.

Annexin V staining

Neuroblastoma cells were exposed to compounds for 24 hours before the experiment. Annexin V staining was performed using MEBCYTO Apoptosis Kit (MBL) according to the manufacturer's instructions. Cells were analyzed by flow cytometer BD FACSCalibur and FlowJo Software (Becton Dickinson).

TUNEL assay and immunofluorescence staining

Neuroblastoma cells were cultured on glass coverslips and treated with compounds for 24 hours before experiment. TUNEL assays were performed using the In situ Cell Death Detection Kit and TMR Red (Roche) according to the manufacturer's instructions. Detailed information on immunofluorescence staining can be found in the Supplementary Information.

In vivo study

Human neuroblastoma cells were mixed 1:1 with Matrigel (Corning) and injected subcutaneously into the flank of BALB/c nu/nu mice purchased from Charles River Laboratories. Intravenous administration of 0.1 or 0.3 mg/kg compounds began when the average tumor size reached 150 mm3. DMSO was administered as a control (n = 7 or 8 mice in each group). Tumor volume (W × W × L/2) and body weights were measured every 2 or 3 days. The mice were sacrificed when tumor volume reached 2,000 mm3. Survival analysis was performed using Kaplan–Meier curves.

Hematoxylin and eosin and IHC staining

Compounds (0.3 mg/kg) were injected intravenously into BALB/c nu/nu mice harboring human neuroblastoma xenografts when the tumor volume surpassed 200 mm3. After 48 hours, the tumor was resected and fixed in 4% paraformaldehyde. Sections (4 μm) were deparaffinized by immersing in xylene and rehydrated, followed by staining with hematoxylin and eosin (H&E) according to standard procedures, and followed by immunostaining using antibodies. Detailed information on antibodies can be found in the Supplementary Information. The number of cleaved caspase-3–positive cells was counted in three high-power fields of each tumor at ×400 magnification.

In vitro FISH analysis

Neuroblastoma cells were exposed to compounds. After 72 hours, cells were collected, mounted on the slide glass, and fixed with Carnoy fixative. VYSIS LSI MYCN (2p24) spectrum green/VYSIS CEP2 spectrum orange probe was obtained from Abbott. SureFISH for MYCN, PAX3, and MYC probes were obtained from Agilent. The experiments were performed following the manufacturers’ instructions. Cell nuclei were stained with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). The fluorescence images were captured by confocal microscope TCS SP8 (Leica) or fluorescence microscope BZ-X700 (Keyence), and the signal intensities were quantified in five randomly selected fields using WinROOF software (Mitani).

CRISPR/Cas9–mediated DNA cleavage of MYCN gene

CRISPR/Cas9–mediated targeting of the MYCN gene was performed using Alt-R S.p. Cas9 Nuclease 3NLS, Alt-R CRISPR/Cas9 crRNAs targeting MYCN (MYCNcr-a and MYCNcr-b), Alt-R CRISPR/Cas9 Human Negative Control crRNA, and Alt-R CRISPR/Cas9 tracrRNA-ATTO550 (Integrated DNA Technologies), as described in the Supplementary Information.

Southern blotting analysis

Genomic DNA was extracted from CHP-134 cells exposed to compounds for 72 hours and digested with EcoRI. After separation in 0.8% agarose gel electrophoresis, DNA was transferred to nylon membranes (Hybond N+, GE Healthcare). Digoxigenin-labeled probes were synthesized by a PCR DIG Probe Synthesis Kit (Roche) according to manufacturer's instructions using the following primers: MYCN, 5′-TGTGTTTGAGCTGTCGGAGAG-3′ and 5′-CCCCCTTTGTAAAAATGCAACC-3′; PAX3, 5′-TTTATTCCATGGGGCTAGGAG-3′ and 5′-GATGCCGTGTTCTCTTTTCC-3′.

In vivo FISH analysis

Formalin-fixed paraffin-embedded (FFPE) tumor blocks were sliced (4 μm) and deparaffinized by immersing in xylene and then were rehydrated. FISH analyses were performed using a Histology FISH Accessory Kit (Dako) according to the manufacturer's instructions. The fluorescence images were captured by confocal microscope TCS SP8 or fluorescence microscope BZ-X700, and the signal intensities were quantified in five randomly selected fields using WinROOF software.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software). Detailed information can be found in the Supplementary Information.

Ethical statement

This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Education, Culture, Sports, Science and Technology of Japan. The protocol was approved by the Committee on the Ethics of Animal Experiments of Chiba Cancer Center. All efforts were made to minimize suffering.

The amplified MYCN gene is a potential target in neuroblastoma

We first investigated whether MYCN inhibition would induce growth inhibition in MYCN-amplified neuroblastoma cells. The depletion of MYCN using siRNAs targeting MYCN showed the inhibition of cell proliferation in MYCN-amplified neuroblastoma cells (Supplementary Fig. S1A and S1B), supporting the previous reports (5, 6).

Recently, it has proposed that cancer cells are vulnerable to CRISPR/Cas9–induced site-specific DNA breaks within amplified regions of the genome (17). To test this hypothesis in MYCN-amplified neuroblastoma cells, we employed the CRISPR/Cas9 system targeting MYCN as a site-specific DNA damage agent. As expected, crRNAs targeting MYCN significantly increased the number of apoptotic cells compared with control crRNA in MYCN-amplified CHP-134 and Kelly cells, whereas no induction of apoptosis was observed in MYCN nonamplified SK-N-AS cells (Supplementary Fig. S1C and S1D). Under these conditions, we observed that crRNAs targeting MYCN decreased MYCN expression in CHP-134 and Kelly cells (Supplementary Fig. S1E). According to these results, direct targeting of the MYCN gene is an attractive therapeutic approach for MYCN-amplified neuroblastoma, even though current CRISPR/Cas9 or interfering RNA molecules may not be clinically applicable owing to a lack of appropriate delivery methods. Thus, we took an approach of chemical biology with PI polyamides capable of penetrating into the nuclei of cells and binding to the specific sequence of target DNA.

MYCN-amplified neuroblastoma cells are highly sensitive to MYCN-A3 targeting the 3′UTR of the MYCN gene transcript region

To induce DNA damage directly in the amplified region of the MYCN gene, we designed and synthesized two sequence-specific DNA-alkylating PI polyamides, MYCN-A3 and MYCN-A4, which bind to and alkylate MYCN transcript regions (MYCN-A3, 5′-TGGGWGCCW-3′; MYCN-A4, 5′-TGGGWCGGW-3′; W, A, or T; Fig. 1A; Supplementary Fig. S2A).

Figure 1.

Amplification of the MYCN gene is a potential therapeutic vulnerability. A, Chemical structures of MYCN-A3 and biotinylated MYCN-A3 (Bio-MYCN-A3), sequence-specific DNA-alkylating agents directly targeting the MYCN gene. MYCN-A3 was designed to bind to 3′UTR within the MYCN gene (5′-TGGGWGCCW-3′; W, A, or T). The site of alkylation is shown in red. B, IC50 values were determined in 12 MYCN-amplified (Amp) and 15 MYCN nonamplified (non-Amp) cancer cell lines treated with MYCN-A3 for 72 hours by WST assay. Each dot represents an individual cell line. Horizontal lines indicate the median values (Amp, 4.5 nmol/L; non-Amp, 50.4 nmol/L; IC50 values are specified in Supplementary Table S1). P values were determined by nonparametric Mann–Whitney U tests. C, Distribution of Bio-MYCN-A3 in MYCN-amplified CHP-134 and Kelly, MYCN nonamplified SK-N-AS, and NB69 neuroblastoma cells. Blue, DAPI; red, Bio-MYCN-A3. DMSO was used as a control. Scale bars, 5 μm. D, Bio-MYCN-A3 precipitation assay after biotin–streptavidin interactions to test the direct binding of MYCN-A3 to the MYCN gene in CHP-134 cells. The schematic represents the positions of PCR primer sets 1, 2, and 3. The region of primer set 3 includes the binding site of MYCN-A3. DMSO was used as a control.

Figure 1.

Amplification of the MYCN gene is a potential therapeutic vulnerability. A, Chemical structures of MYCN-A3 and biotinylated MYCN-A3 (Bio-MYCN-A3), sequence-specific DNA-alkylating agents directly targeting the MYCN gene. MYCN-A3 was designed to bind to 3′UTR within the MYCN gene (5′-TGGGWGCCW-3′; W, A, or T). The site of alkylation is shown in red. B, IC50 values were determined in 12 MYCN-amplified (Amp) and 15 MYCN nonamplified (non-Amp) cancer cell lines treated with MYCN-A3 for 72 hours by WST assay. Each dot represents an individual cell line. Horizontal lines indicate the median values (Amp, 4.5 nmol/L; non-Amp, 50.4 nmol/L; IC50 values are specified in Supplementary Table S1). P values were determined by nonparametric Mann–Whitney U tests. C, Distribution of Bio-MYCN-A3 in MYCN-amplified CHP-134 and Kelly, MYCN nonamplified SK-N-AS, and NB69 neuroblastoma cells. Blue, DAPI; red, Bio-MYCN-A3. DMSO was used as a control. Scale bars, 5 μm. D, Bio-MYCN-A3 precipitation assay after biotin–streptavidin interactions to test the direct binding of MYCN-A3 to the MYCN gene in CHP-134 cells. The schematic represents the positions of PCR primer sets 1, 2, and 3. The region of primer set 3 includes the binding site of MYCN-A3. DMSO was used as a control.

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The cytotoxicity of neuroblastoma cells with or without MYCN amplification showed that CHP-134 cells were highly sensitive to both MYCN-A3 and MYCN-A4 when compared with SK-N-AS cells, and MYCN-A3 exhibited stronger cytotoxicity than MYCN-A4 (Supplementary Fig. S2B), prompting us to proceed with MYCN-A3 as the lead candidate. We further assessed the cytotoxicity of MYCN-A3 in different neuroblastoma cell lines (Supplementary Fig. S2C) and other types of cancer cell lines with or without MYCN amplification. MYCN-A3 displayed selective cytotoxicity against MYCN-amplified cancer cells with the median IC50 = 4.5 nmol/L, nearly a tenth of the cytotoxicity in MYCN nonamplified cancer cells (IC50 = 50.4 nmol/L; Fig. 1B; Supplementary Table S1).

To verify the binding site of MYCN-A3 in the region of MYCN gene, we synthesized a biotinylated derivative of MYCN-A3 (Bio-MYCN-A3; Fig. 1A). We initially confirmed the intranuclear 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 sets within the MYCN gene (primer set 1 in exon-1; primer set 2 in exon-3; primer set 3 in 3′UTR that contains a MYCN-A3–binding motif; Fig. 1D). After extracting genomic DNA followed by biotin–streptavidin interactions, we observed the enrichment of the DNA fragments in the region of primer set 3, 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 3′UTR of MYCN gene containing its target DNA sequence. Bio-MYCN-A3 demonstrated a comparable IC50 value to MYCN-A3 in CHP-134 cells (Supplementary Fig. S2D), indicating that the additional biotinylated moiety did not interfere with the pharmacologic characteristics of MYCN-A3. To further confirm the direct alkylation of MYCN gene with MYCN-A3, we performed ligation-mediated PCR, which amplifies DNA fragments with blunt ends generated at alkylated sites (27). Genomic DNA collected from MYCN-A3–treated cells displayed a PCR band of the expected size, while vehicle control and mismatch polyamide that has no binding motif for the MYCN gene failed to show the PCR product (Supplementary Fig. S3A and S3B). We next tested the binding specificity of MYCN-A3 by surface plasmon resonance assay. MYCN-A3 showed 212-fold higher binding affinity to the full-match sequence compared with mismatch polyamide (Supplementary Fig. S3C and S3D). These data suggest that MYCN-A3 specifically alkylates the targeted DNA sequence of MYCN gene in MYCN-amplified neuroblastoma cells.

MYCN-A3 suppresses MYCN expression in MYCN-amplified neuroblastoma cells

To determine whether MYCN-A3 inhibits the transcriptional elongation of the MYCN gene by specific alkylation at the targeted DNA sequence, we performed qPCR and Western blotting analyses. The treatment of MYCN-A3, but not Mismatch polyamide, demonstrated dose-dependent suppression of MYCN expression at the mRNA and protein levels in MYCN-amplified neuroblastoma cells (Fig. 2A and B; Supplementary Fig. S4A and S4B). We also confirmed that MYCN-A4 decreased levels of MYCN expression and cell proliferation, whereas mismatch polyamide showed little, if any, influence on MYCN-amplified neuroblastoma cells (Supplementary Fig. S4C and S4D). Among direct transcriptional target genes of MYCN, NLRR1, DKC1, TWIST1, and BMI1 are essential for MYCN-mediated neuroblastoma tumorigenesis (28–31) and free of the MYCN-A3–binding motif. MYCN-A3 treatment decreased the expression levels of those genes in a similar manner to the MYCN gene (Fig. 2C). These results suggest that MYCN-A3 suppresses MYCN expression at the transcriptional level, resulting in the downregulation of MYCN target genes.

Figure 2.

MYCN-A3 inhibits MYCN expression in MYCN-amplified neuroblastoma cell lines. A and B,MYCN-amplified CHP-134, Kelly, SK-N-BE(2), and IMR-32 cells treated with 10 nmol/L MYCN-A3 or mismatch polyamide for 24 hours. MYCN mRNA (A) and protein (B) were analyzed by qPCR and Western blotting analysis, respectively. P values were determined by one-way ANOVA followed by Dunnett posttest (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Data are mean ± SD from three independent experiments. C, CHP-134, Kelly, SK-N-BE(2), and IMR-32 cells treated with 10 nmol/L MYCN-A3 for 24 hours. mRNA of the MYCN-targeted genes, such as NLRR1, DKC1, TWIST1, and BMI1, was analyzed by qPCR. DMSO was used as a control. RPS18 was used as an internal control.

Figure 2.

MYCN-A3 inhibits MYCN expression in MYCN-amplified neuroblastoma cell lines. A and B,MYCN-amplified CHP-134, Kelly, SK-N-BE(2), and IMR-32 cells treated with 10 nmol/L MYCN-A3 or mismatch polyamide for 24 hours. MYCN mRNA (A) and protein (B) were analyzed by qPCR and Western blotting analysis, respectively. P values were determined by one-way ANOVA followed by Dunnett posttest (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Data are mean ± SD from three independent experiments. C, CHP-134, Kelly, SK-N-BE(2), and IMR-32 cells treated with 10 nmol/L MYCN-A3 for 24 hours. mRNA of the MYCN-targeted genes, such as NLRR1, DKC1, TWIST1, and BMI1, was analyzed by qPCR. DMSO was used as a control. RPS18 was used as an internal control.

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

We performed expression microarrays to assess the genome-wide effect of MYCN-A3 in comparison with CRISPR/Cas9 silencing of MYCN (MYCNcr-a; Supplementary Fig. S5A), and found both treatments to be generally similar in terms of overall expression changes. Volcano plots revealed similar correlations between changes in expression and significance level, especially for those near log2 FC ∼ 0 (Supplementary Fig. S5B). We also found that both treatments tended to downregulate a large portion of common elements (Supplementary Fig. S5C), suggesting that the reduction of MYCN expression by MYCN-A3 and MYCNcr-a would have a similar effect overall, despite that both treatments are mechanistically different. To further assess the biological effects of the two treatments, we decided to explore the behavioral differences by comparing aggregated changes in expression on a pathway level. To do so, we categorized, per pathway, gene expressions between MYCN-A3 and MYCNcr-a, and used Fisher exact test to evaluate whether both treatments lead to similar fold-change outcomes. These pathway-level comparisons also suggested that only a small number of pathways (as annotated in the Kyoto Encyclopedia of Genes and Genomes) were found to be different statistically (Supplementary Fig. S5D, histogram of P values). Furthermore, most of the statistically significant pathways, for instance glycosylphosphatidylinositol (GPI)-anchor biosynthesis (P = 0.0119) appeared not to be intimately connected to the carcinogenesis of neuroblastoma or MYCN (Supplementary Table S2); in sharp contrast, the more relevant pathways (e.g., MAPK, ErbB, Ras signaling pathways) appeared to be relatively invariant between the two treatments, as indicated by their high P values (P ∼ 1).

MYCN-A3 induces apoptosis in MYCN-amplified neuroblastoma cells

Cell morphologic abnormalities such as cell shrinkage and detachment were observed in CHP-134 and Kelly cells, but not SK-N-AS and NB69 cells, after MYCN-A3 treatment (Supplementary Fig. S6A). We next analyzed the cell surface Annexin V binding, which can detect apoptotic cell death, to investigate the mechanism of cell death after MYCN-A3 treatment (Fig. 3A). At 24 hours, CHP-134 and Kelly cells showed markedly increased Annexin V binding. We then performed the TUNEL assay to ensure that CHP-134 and Kelly cells underwent apoptosis after MYCN-A3 treatment (Fig. 3B). Western blotting analysis also demonstrated that the levels of apoptotic markers, cleavage of caspase-3 and PARP, were increased in CHP-134 and Kelly cells (Fig. 3C). Notably, Western blotting analysis and immunofluorescence staining exhibited elevated levels of γH2AX in CHP-134 and Kelly cells compared with in SK-N-AS and NB69 cells (Fig. 3C and D; Supplementary Fig. S6B). MYCN-A3 treatment led to the elevated levels of p53 phosphorylation at Ser-15 and the expressions of p53 and its family members in all tested cells (Supplementary Fig. S6C). These results suggest that MYCN-amplified neuroblastoma cells accumulate DNA damage after MYCN-A3 treatment and undergo apoptosis.

Figure 3.

MYCN-A3 induces apoptotic cell death in MYCN-amplified neuroblastoma cell lines in vitro. A–D, CHP-134, Kelly, SK-N-AS, and NB69 cells treated with 10 nmol/L MYCN-A3 or mismatch polyamide for 24 hours. Representative images show that Annexin V–positive cells were detected by using flow cytometry (A); apoptotic cell death was detected by TUNEL assay (B); protein expression of cleaved caspase-3, PARP, and γH2AX was detected by Western blotting analysis (C); and γH2AX was detected by immunofluorescence staining (D). Scale bars, 50 μm. DMSO was used as a control.

Figure 3.

MYCN-A3 induces apoptotic cell death in MYCN-amplified neuroblastoma cell lines in vitro. A–D, CHP-134, Kelly, SK-N-AS, and NB69 cells treated with 10 nmol/L MYCN-A3 or mismatch polyamide for 24 hours. Representative images show that Annexin V–positive cells were detected by using flow cytometry (A); apoptotic cell death was detected by TUNEL assay (B); protein expression of cleaved caspase-3, PARP, and γH2AX was detected by Western blotting analysis (C); and γH2AX was detected by immunofluorescence staining (D). Scale bars, 50 μm. DMSO was used as a control.

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MYCN-A3 suppresses tumor progression in human neuroblastoma xenograft mouse models

On the basis of these findings in vitro, we next evaluated the antitumor potential of MYCN-A3 in neuroblastoma xenografts using immunodeficient BALB/c nu mice. Single-dose administrations of MYCN-A3 significantly suppressed tumor growth in mice harboring CHP-134 xenografts (Supplementary Fig. S7A). Three weekly injections with MYCN-A3 significantly suppressed tumor growth compared with the treatment of CBI, a DNA-alkylating agent without the core PI polyamide (Supplementary Fig. S7B). Antitumor activity of MYCN-A3 was also confirmed in Kelly xenografts by multiple administrations of MYCN-A3 without loss of body weight (Fig. 4A–C). The survival rate of mice significantly prolonged with multiple administration of MYCN-A3 in a dose-dependent manner (Fig. 4D). In contrast to MYCN-A3, mismatch polyamide did not induce the inhibition of tumor growth (Fig. 4E–H).

Figure 4.

MYCN-A3 suppresses tumor growth in human MYCN-amplified neuroblastoma xenograft models. A−H, Kelly cells were subcutaneously injected into the flank of female immune-deficient BALB/c nu/nu mice. Administration of MYCN-A3 or mismatch polyamide began when average tumor size reached 150 mm3. Tumor volume and body weight of mice were measured every 3 days. Red arrows, timepoint of administration of MYCN-A3 or mismatch polyamide. P values were determined by repeated measures ANOVA followed by a Bonferroni/Dunn posttest. Data are represented as mean ± SD (A, B, E, and F); representative images of 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 of overall 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). DMSO was used as a control. ns, nonsignificant.

Figure 4.

MYCN-A3 suppresses tumor growth in human MYCN-amplified neuroblastoma xenograft models. A−H, Kelly cells were subcutaneously injected into the flank of female immune-deficient BALB/c nu/nu mice. Administration of MYCN-A3 or mismatch polyamide began when average tumor size reached 150 mm3. Tumor volume and body weight of mice were measured every 3 days. Red arrows, timepoint of administration of MYCN-A3 or mismatch polyamide. P values were determined by repeated measures ANOVA followed by a Bonferroni/Dunn posttest. Data are represented as mean ± SD (A, B, E, and F); representative images of 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 of overall 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). DMSO was used as a control. ns, nonsignificant.

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We also performed histologic studies on the antitumor effect of MYCN-A3 using CHP-134 and Kelly xenograft–derived tumor tissue sections at 48 hours after administration of MYCN-A3 or mismatch polyamide. H&E staining revealed chromatin condensation and nuclear fragmentation in tumors treated with MYCN-A3. Furthermore, IHC staining using anti-MYCN, 53BP1, and cleaved caspase-3 antibodies demonstrated the suppressed MYCN expression, and the elevated levels of DNA damage and apoptotic markers by MYCN-A3 administration (Fig. 5A and B; Supplementary Fig. S8A and S8B). Collectively, MYCN-A3 suppressed MYCN expression, increased DNA damage, and induced apoptosis, resulting in the inhibition of tumor progression in MYCN-amplified neuroblastoma mouse models.

Figure 5.

Pharmacologic effects of MYCN-A3 on human MYCN-amplified neuroblastoma xenograft models in vivo. A, BALB/c nu/nu mice with Kelly xenograft were administered 0.3 mg/kg MYCN-A3 or mismatch polyamide. At 48 hours after administration, the tumor tissues were collected and used for H&E and IHC with MYCN, 53BP1, and cleaved caspase-3. Scale bars, 50 μm. B, Quantification of the number of positive cells with cleaved caspase-3 in Kelly xenografts. P values were determined by one-way ANOVA followed by Dunnett posttest. Data are represented as mean ± SD. DMSO was used as a control.

Figure 5.

Pharmacologic effects of MYCN-A3 on human MYCN-amplified neuroblastoma xenograft models in vivo. A, BALB/c nu/nu mice with Kelly xenograft were administered 0.3 mg/kg MYCN-A3 or mismatch polyamide. At 48 hours after administration, the tumor tissues were collected and used for H&E and IHC with MYCN, 53BP1, and cleaved caspase-3. Scale bars, 50 μm. B, Quantification of the number of positive cells with cleaved caspase-3 in Kelly xenografts. P values were determined by one-way ANOVA followed by Dunnett posttest. Data are represented as mean ± SD. DMSO was used as a control.

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MYCN-A3 disrupts conformation of the amplified MYCN gene loci in MYCN-amplified neuroblastoma cells

We further investigated whether site-specific DNA damage at the MYCN gene loci alters the amplification status of the MYCN gene in neuroblastoma cells. FISH analysis using a probe for MYCN at 2p24.3 displayed a typical diffused pattern in the nuclei of control CHP-134 and Kelly cells, while a probe for CEP2 (2p11.1-q11.1) showed a normal disomic signal distribution (Fig. 6A, left). Intriguingly, CRISPR/Cas9 against MYCN (MYCNcr-a) attenuated the probe signals in CHP-134 cells (Fig. 6A, right; Supplementary Fig. S9A). The reduced signal intensity of the MYCN probe was observed in CHP-134 and Kelly cells treated with MYCN-A3, whereas the influence of mismatch polyamide on FISH signals was marginal (Fig. 6B; Supplementary Fig. S9B). Probes for the PAX3 (2q36.1) and MYC (8q24.21) genes that possess no MYCN-A3–binding motif indicated the limited influence of MYCN-A3 in a distant region of chromosome 2 and a different chromosome (Supplementary Fig. S9C).

Figure 6.

MYCN-A3 decreased the copy numbers of the MYCN gene in vitro and in vivo. A and B, Representative images of FISH analysis in CHP-134 cells at 48 hours after transfection with MYCNcr-a (A). Scale bars, 25 μm. B, CHP-134 and Kelly cells treated with MYCN-A3 or mismatch polyamide for 72 hours. Scale bars, 10 μm. Green, MYCN (2p24.3); red, CEP2 (2p11.1-q11.1; B). C, Southern blotting using MYCN and PAX3 probes in CHP-134 cells transfected with MYCNcr-a for 48 hours (top), treated with MYCN-A3 or mismatch polyamide for 72 hours (bottom). D, Representative images of the FISH analysis show that the mice with CHP-134 and Kelly xenografts administered with MYCN-A3 or mismatch polyamide. At 48 hours after administration, the tumor tissues were collected and used for FISH analysis. Scale bars, 25 μm. DMSO was used as a control.

Figure 6.

MYCN-A3 decreased the copy numbers of the MYCN gene in vitro and in vivo. A and B, Representative images of FISH analysis in CHP-134 cells at 48 hours after transfection with MYCNcr-a (A). Scale bars, 25 μm. B, CHP-134 and Kelly cells treated with MYCN-A3 or mismatch polyamide for 72 hours. Scale bars, 10 μm. Green, MYCN (2p24.3); red, CEP2 (2p11.1-q11.1; B). C, Southern blotting using MYCN and PAX3 probes in CHP-134 cells transfected with MYCNcr-a for 48 hours (top), treated with MYCN-A3 or mismatch polyamide for 72 hours (bottom). D, Representative images of the FISH analysis show that the mice with CHP-134 and Kelly xenografts administered with MYCN-A3 or mismatch polyamide. At 48 hours after administration, the tumor tissues were collected and used for FISH analysis. Scale bars, 25 μm. DMSO was used as a control.

Close modal

To further assess the change in the MYCN gene copy number, Southern blotting analysis was performed in CHP-134 cells. Notably, MYCNcr-a and MYCN-A3 treatment reduced the relative band intensity of the MYCN probe without affecting the band similarly detected by the PAX3 probe (Fig. 6C, top; Supplementary Fig. S9D), whereas mismatch polyamide did not affect either MYCN or PAX3 probes (Fig. 6C, bottom). Moreover, FISH analyses using FFPE samples revealed that FISH signal intensities of the MYCN probe were decreased by MYCN-A3 administration in the tissues of xenograft neuroblastoma tumors, whereas no influence was observed by treatment of mismatch polyamide (Fig. 6D; Supplementary Fig. S9E). Altogether, our findings suggest that site-specific DNA alkylation contributes to the selective destruction of the MYCN gene loci, resulting in the suppression of MYCN expression.

Recent research and development of new pharmaceutical candidates have improved our understanding of neuroblastoma biology and the overall survival of patients. However, the clinical prognosis remains poor for patients with MYCN-amplified neuroblastoma (32, 33). Therefore, those patients need MYCN-targeting agents in clinical practice.

Although MYCN-A3 preferentially affected MYCN-amplified tumor cells, the relationship between MYCN copy numbers and the sensitivity of current chemotherapeutic agents remains unclear. A recent study has reported that the CRISPR/Cas9 system targeting amplified genomic regions induces site-specific DNA damage accompanied by accumulation of γH2AX and cell death (34). In this data, γH2AX was elevated by MYCN-A3 treatment in MYCN-amplified neuroblastoma cells but not in MYCN nonamplified cells, suggesting that the higher copy number of the MYCN gene could lead to more events of sequence-dependent DNA alkylation. This novel strategy to target an amplified region of genomic DNA is attractive for developing new lead compounds against tumor types driven by oncogene amplification.

Recent studies have further demonstrated that targeting MYCN transcription by the BET bromodomain inhibitor JQ1 or CDK7 inhibitor THZ1 effectively induces cell death in MYCN-amplified neuroblastoma cells (35, 36), indicating that transcriptional suppression of MYCN is a promising strategy in drug development. Previously, we also reported a PI polyamide designed to target a driver gene mutation of KRAS downregulating its transcription in colorectal cancer cells (27). Herein, MYCN-A3 targeted 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 is indispensable for MYCN-A3 to induce a massive neuroblastoma cell death, even though off-target genes with the full-match binding sequence may exist.

The pharmacologic mechanisms of action of DNA-alkylating PI polyamides to disrupt gene transcription remain to be determined. Upon MYCN-A3 treatment, the hybridization of FISH probes was repressed in the amplified genomic regions of MYCN, perhaps as a consequence of the covalent binding of PI polyamide. In contrast, other genomic DNA regions outside of the MYCN amplicon 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 at amplified MYCN gene loci. This amplified region also contains other transcripts including NCYM and lncUSMycN, a cis-antisense gene of MYCN and a long noncoding RNA gene, respectively (37, 38). NCYM and lncUSMycN are coamplified with MYCN and regulate MYCN expression and neuroblastoma tumorigenesis. FISH probes used in this study detected a >200 kb genomic region containing the MYCN gene and these two MYCN-associated genes. Because FISH analyses clearly showed reduced probe signals after MYCN-A3 treatment, it is likely that MYCN-A3 affects the flanking region of MYCN gene loci and thereby exerts high cytotoxicity by repression of neighbor genes in addition to transcriptional target genes of MYCN.

In human neuroblastoma xenograft mice, the weak signals of the MYCN probe in FFPE–FISH analyses strongly suggest that MYCN-A3 distributes in tumor tissues and cells in situ after intravenous administration. Although PI polyamides can be effluxed from the plasma after intravenous injection (39), they effectively accumulate in tumor tissue and are retained until at least 48 hours after administration (40, 41). These reports suggest that PI polyamides might have an enhanced permeability and retention effect (42), allowing for prolonged administration intervals to further reduce the potential side effects of the PI polyamides. Indeed, MYCN-A3 exhibits antitumor effects even at a low dose without severe side effects such as loss of body weight. Most current conventional chemotherapy regimens consist of various combinations of high-dose DNA-damaging agents (43, 44), and patients who survive high-risk neuroblastoma may suffer from significant subsequent effects such as growth failure and secondary malignancy (45).

Our current data suggest that MYCN-A3 offers a new therapeutic strategy with lesser side effects for patients with advanced neuroblastoma with MYCN amplification. In addition, loss of copy numbers of MYCN by MYCN-A3 or the CRISPR/Cas9 system might be a novel approach to accelerate drug development for clinical practice. To this end, we need further preclinical studies using patient-derived xenograft models and novel genetically engineered models with a transgene inducing multiple copy numbers of the entire MYCN including 3′UTR, because current MYCN-Tg mouse models do not have the 3′UTR of the human MYCN gene.

In conclusion, we have successfully developed a novel PI polyamide conjugated with a DNA-alkylating agent, MYCN-A3, which shows high cytotoxicity in MYCN-amplified tumor cells derived from neuroblastoma. Site-specific DNA damage induced by MYCN-A3 leads to suppression of the MYCN gene and potent antitumor activity against MYCN-amplified neuroblastoma. Targeting MYCN gene amplification using MYCN-A3 can be a novel approach for the development of next-generation cancer therapy for high-risk MYCN-amplified neuroblastoma.

No potential conflicts of interest were disclosed.

Conception and design: H. Yoda, A. Takatori, H. Nagase

Development of methodology: H. Yoda, A. Takatori

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H. Yoda, A. Takatori

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H. Yoda, J. Lin, A. Takatori

Writing, review, and/or revision of the manuscript: H. Yoda, J. Lin, A. Takatori, H. Nagase

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Inoue, J. Lin, T. Watanabe, N. Koshikawa

Study supervision: A. Takatori, H. Nagase

Others (organic synthesis and characterization of compounds): Y. Shinozaki, T. Watanabe

We are grateful to K. Hiraoka, Y. Suzuki, K. Sugimoto, Y. Kaihou, T. Koga, R. Igarashi, Y. Nakamura, and R. Murasugi for technical assistance. We also thank K. Shinohara, T. Bando, and H. Sugiyama for advice on chemical design. This work was supported in part by Practical Research for Innovative Cancer Control from the Japan Agency for Medical Research and Development (AMED, grant no. 15656919 to A. Takatori), MEXT KAKENHI (grant no. 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), and AMED (grant no. 18ae0101051 to H. Nagase).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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