Approximately half of high-risk neuroblastoma is characterized by MYCN amplification. N-Myc promotes tumor progression by inducing cell growth and inhibiting differentiation. MYCN has also been shown to play an active role in mitochondrial metabolism, but this relationship is not well understood. Although N-Myc is a known driver of the disease, it remains a target for which no therapeutic drug exists. Here, we evaluated a novel MYCN-specific antigene PNA oligonucleotide (BGA002) in MYCN-amplified (MNA) or MYCNexpressing neuroblastoma and investigated the mechanism of its antitumor activity. MYCN mRNA and cell viability were reduced in a broad set of neuroblastoma cell lines following BGA002 treatment. Furthermore, BGA002 decreased N-Myc protein levels and apoptosis in MNA neuroblastoma. Analysis of gene expression data from patients with neuroblastoma revealed that MYCN was associated with increased reactive oxygen species (ROS), downregulated mitophagy, and poor prognosis. Inhibition of MYCN caused profound mitochon-drial damage in MNA neuroblastoma cells through downregulation of the mitochondrial molecular chaperone TRAP1, which subsequently increased ROS. Correspondingly, inhibition of MYCN reactivated mitophagy. Systemic administration of BGA002 downregulated N-Myc and TRAP1, with a concomitant decrease in MNA neuroblastoma xenograft tumor weight. In conclusion, this study highlights the role of N-Myc in blocking mitophagy in neuroblastoma and in conferring protection to ROS in mitochondria through upregulation of TRAP1. BGA002 is a potently improved MYCN-specific antigene oligonucleotide that reverts N-Myc-dysregulated mitochondrial pathways, leading to loss of the protective effect of N-Myc against mitochondrial ROS.Significance: A second generation antigene peptide oligonucleotide targeting MYCN induces mitochondrial damage and inhibits growth of MYCN-amplified neuroblastoma cells.
Background Neuroblastoma is a deadly childhood cancer, and MYCN-amplified neuroblastoma (MNA-NB) patients have the worst prognoses and are therapy-resistant. While retinoic acid (RA) is beneficial for some neuroblastoma patients, the cause of RA resistance is unknown. Thus, there remains a need for new therapies to treat neuroblastoma. Here we explored the possibility of combining a MYCN-specific antigene oligonucleotide BGA002 and RA as therapeutic approach to restore sensitivity to RA in NB. Methods By molecular and cellular biology techniques, we assessed the combined effect of the two compounds in NB cell lines and in a xenograft mouse model MNA-NB. Results We found that MYCN-specific inhibition by BGA002 in combination with RA (BGA002-RA) act synergistically and overcame resistance in NB cell lines. BGA002-RA also reactivated neuron differentiation (or led to apoptosis) and inhibited invasiveness capacity in MNA-NB. Moreover, we found that neuroblastoma had the highest level of mRNA expression of mTOR pathway genes, and that BGA002 led to mTOR pathway inhibition followed by autophagy reactivation in MNA-NB cells, which was strengthened by BGA002-RA. BGA002-RA in vivo treatment also eliminated tumor vascularization in a MNA-NB mouse model and significantly increased survival. Conclusion Taken together, MYCN modulation mediates the therapeutic efficacy of RA and the development of RA resistance in MNA-NB. Furthermore, by targeting MYCN, a cancer-specific mTOR pathway inhibition occurs only in MNA-NB, thus avoiding the side effects of targeting mTOR in normal cells. These findings warrant clinical testing of BGA002-RA as a strategy for overcoming RA resistance in MNA-NB.
Purpose TH-MYCN transgenic mice represent a valuable preclinical model of neuroblastoma. Current methods to study tumor progression in these mice are inaccurate or invasive, limiting the potential of this murine model. The aim of our study was to assess the potential of small animal positron emission tomography (SA-PET) to study neuroblastoma progression in TH-MYCN mice. Procedure Serial SA-PET scans using the tracer 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG) have been performed in TH-MYCN mice. Image analysis of tumor progression has been compared with ex vivo evaluation of tumor volumes and histological features. Results [18F]FDG-SA-PET allowed to detect early staged tumors in almost 100 % of TH-MYCN mice positive for disease. Image analysis of tumor evolution reflected the modifications of the tumor volume, histological features, and malignancy during disease progression. Image analysis of TH-MYCN mice undergoing chemotherapy treatment against neuroblastoma provided information on drug-induced alterations in tumor metabolic activity. Conclusions These data show for the first time that [18F]FDG-SA-PET is a useful tool to study neuroblastoma presence and progression in TH-MYCN transgenic mice.
Small-cell lung cancer (SCLC) is the most aggressive lung cancer type, and is associated with smoking, low survival rate due to high vascularization, metastasis and drug resistance. Alterations in MYC family members are biomarkers of poor prognosis for a large number of SCLC. In particular, MYCN alterations define SCLC cases with immunotherapy failure. MYCN has a highly restricted pattern of expression in normal cells and is an ideal target for cancer therapy but is undruggable by traditional approaches. We propose an innovative approach to MYCN inhibition by an MYCN-specific antigene—PNA oligonucleotide (BGA002)—as a new precision medicine for MYCN-related SCLC. We found that BGA002 profoundly and specifically inhibited MYCN expression in SCLC cells, leading to cell-growth inhibition and apoptosis, while also overcoming multidrug resistance. These effects are driven by mTOR pathway block in concomitance with autophagy reactivation, thus avoiding the side effects of targeting mTOR in healthy cells. Moreover, we identified an MYCN-related SCLC gene signature comprehending CNTFR, DLX5 and TNFAIP3, that was reverted by BGA002. Finally, systemic treatment with BGA002 significantly increased survival in MYCN-amplified SCLC mouse models, including in a multidrug-resistant model in which tumor vascularization was also eliminated. These findings warrant the clinical testing of BGA002 in MYCN-related SCLC.
<p>Figure S1: BGA002 induces apoptosis Figure S2: BGA002 inhibits MYCN expression in a broad neuroblastoma cell-line panel Figure S3: BGA002 is a specific MYCN inhibitor Figure S4: Mitochondria structural alteration are MYCN specific inhibition. Figure S5: BGA002 leads to MYCN-specific structural alteration in mitochondria. Figure S6: BGA001 does not lead to significant MYCN-specific structural alteration in mitochondria. Figure S7: MYCN inhibition leads to mitochondrial damage Figure S8: Alterations in mitochondrial pathways identify Neuroblastoma patients with poor prognosis. Figure S9: MYCN amplified patient are enriched for the mitochondrial gene signature. Figure S10: MYCN blocking leads to MYCN specific gene expression signature inhibition in NB cells and to mitophagy reactivation. Figure S11: OPTN controls mitophagy induction after MYCN silencing Figure S12: BGA002 reverts the MYCN control of ROS generation by TRAP1 down-regulation in MNA-NB cells. Figure S13: BGA002 causes elimination of MYCN-NB in mice</p>
<p>supplementary tables Supplementary Table 1: List of cell lines used in this study. Supplementary Table 2: List of the primers used in this study for quantitative Real Time PCR. Supplementary Table 3: Table showing the EC50 effect for BGA001 and BGA002 (MYCN mRNA, decrease in cell viability, N-myc protein and apoptosis induction in Kelly and SK-N-BE(2)c cell lines). Supplementary Table 4: List of genes present in the Mitochondrial-related signature in alphabetical order. Supplementary Table 5: Table summarizing principal patient features and their cluster attribution. More extensive information can be seen at http://www.ebi.ac.uk/arrayexpress; accession: E-MTAB-1781. Supplementary Table 6: Table summarizing the mitoscore and its components for each gene of the mitochondrial signature; genes are order based on decreasing mitoscore. Supplementary Table 7: List of the top 200 differentially expressed genes between Cluster 1 (left) and Cluster 2 (right) Supplementary Table 8: List of Cluster 1 and Cluster 2 associated genes used for the pathway network analysis. Supplementary Table 9: List of the ClueGo pathways obtained from the pathway network analysis.</p>
<div>Abstract<p>Approximately half of high-risk neuroblastoma is characterized by MYCN amplification. N-Myc promotes tumor progression by inducing cell growth and inhibiting differentiation. MYCN has also been shown to play an active role in mitochondrial metabolism, but this relationship is not well understood. Although N-Myc is a known driver of the disease, it remains a target for which no therapeutic drug exists. Here, we evaluated a novel MYCN-specific antigene PNA oligonucleotide (BGA002) in MYCN-amplified (MNA) or MYCN-expressing neuroblastoma and investigated the mechanism of its antitumor activity. MYCN mRNA and cell viability were reduced in a broad set of neuroblastoma cell lines following BGA002 treatment. Furthermore, BGA002 decreased N-Myc protein levels and apoptosis in MNA neuroblastoma. Analysis of gene expression data from patients with neuroblastoma revealed that MYCN was associated with increased reactive oxygen species (ROS), downregulated mitophagy, and poor prognosis. Inhibition of MYCN caused profound mitochondrial damage in MNA neuroblastoma cells through downregulation of the mitochondrial molecular chaperone TRAP1, which subsequently increased ROS. Correspondingly, inhibition of MYCN reactivated mitophagy. Systemic administration of BGA002 downregulated N-Myc and TRAP1, with a concomitant decrease in MNA neuroblastoma xenograft tumor weight. In conclusion, this study highlights the role of N-Myc in blocking mitophagy in neuroblastoma and in conferring protection to ROS in mitochondria through upregulation of TRAP1. BGA002 is a potently improved MYCN-specific antigene oligonucleotide that reverts N-Myc–dysregulated mitochondrial pathways, leading to loss of the protective effect of N-Myc against mitochondrial ROS.</p>Significance:<p>A second generation antigene peptide oligonucleotide targeting MYCN induces mitochondrial damage and inhibits growth of MYCN-amplified neuroblastoma cells.</p></div>
<p>supplementary tables Supplementary Table 1: List of cell lines used in this study. Supplementary Table 2: List of the primers used in this study for quantitative Real Time PCR. Supplementary Table 3: Table showing the EC50 effect for BGA001 and BGA002 (MYCN mRNA, decrease in cell viability, N-myc protein and apoptosis induction in Kelly and SK-N-BE(2)c cell lines). Supplementary Table 4: List of genes present in the Mitochondrial-related signature in alphabetical order. Supplementary Table 5: Table summarizing principal patient features and their cluster attribution. More extensive information can be seen at http://www.ebi.ac.uk/arrayexpress; accession: E-MTAB-1781. Supplementary Table 6: Table summarizing the mitoscore and its components for each gene of the mitochondrial signature; genes are order based on decreasing mitoscore. Supplementary Table 7: List of the top 200 differentially expressed genes between Cluster 1 (left) and Cluster 2 (right) Supplementary Table 8: List of Cluster 1 and Cluster 2 associated genes used for the pathway network analysis. Supplementary Table 9: List of the ClueGo pathways obtained from the pathway network analysis.</p>
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