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Purpose: Rhabdomyosarcomas are a major cause of cancer death in children, described with MYCN amplification and, in the alveolar subtype, transcription driven by the PAX3-FOXO1 fusion protein. Our aim was to determine the prevalence of N-Myc protein expression and the potential therapeutic effects of reducing expression in rhabdomyosarcomas, including use of an antigene strategy that inhibits transcription.Experimental Design: Protein expression was assessed by immunohistochemistry. MYCN expression was reduced in representative cell lines by RNA interference and an antigene peptide nucleic acid (PNA) oligonucleotide conjugated to a nuclear localization signal peptide. Associated gene expression changes, cell viability, and apoptosis were analyzed in vitro. As a paradigm for antigene therapy, the effects of systemic treatment of mice with rhabdomyosarcoma cell line xenografts were determined.Results: High N-Myc levels were significantly associated with genomic amplification, presence of the PAX3/7-FOXO1 fusion genes, and proliferative capacity. Sustained reduction of N-Myc levels in all rhabdomyosarcoma cell lines that express the protein decreased cell proliferation and increased apoptosis. Positive feedback was shown to regulate PAX3-FOXO1 and N-Myc levels in the alveolar subtype that critically decrease PAX3-FOXO1 levels on reducing N-Myc. Pharmacologic systemic administration of the antigene PNA can eliminate alveolar rhabdomyosarcoma xenografts in mice, without relapse or toxicity.Conclusion: N-Myc, with its restricted expression in non-fetal tissues, is a therapeutic target to treat rhabdomyosarcomas, and blocking gene transcription using antigene oligonucleotide strategies has therapeutic potential in the treatment of cancer and other diseases that has not been previously realized in vivo. Clin Cancer Res; 18(3); 796–807. ©2011 AACR.

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CCR Translation for This Article from Antitumor Activity of Sustained N-Myc Reduction in Rhabdomyosarcomas and Transcriptional Block by Antigene Therapy

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Purpose: Rhabdomyosarcomas are a major cause of cancer death in children, described with MYCN amplification and, in the alveolar subtype, transcription driven by the PAX3-FOXO1 fusion protein. Our aim was to determine the prevalence of N-Myc protein expression and the potential therapeutic effects of reducing expression in rhabdomyosarcomas, including use of an antigene strategy that inhibits transcription. Experimental Design: Protein expression was assessed by immunohistochemistry. MYCN expression was reduced in representative cell lines by RNA interference and an antigene peptide nucleic acid (PNA) oligonucleotide conjugated to a nuclear localization signal peptide. Associated gene expression changes, cell viability, and apoptosis were analyzed in vitro. As a paradigm for antigene therapy, the effects of systemic treatment of mice with rhabdomyosarcoma cell line xenografts were determined. Results: High N-Myc levels were significantly associated with genomic amplification, presence of the PAX3/7-FOXO1 fusion genes, and proliferative capacity. Sustained reduction of N-Myc levels in all rhabdomyosarcoma cell lines that express the protein decreased cell proliferation and increased apoptosis. Positive feedback was shown to regulate PAX3-FOXO1 and N-Myc levels in the alveolar subtype that critically decrease PAX3-FOXO1 levels on reducing N-Myc. Pharmacologic systemic administration of the antigene PNA can eliminate alveolar rhabdomyosarcoma xenografts in mice, without relapse or toxicity. Conclusion: N-Myc, with its restricted expression in non-fetal tissues, is a therapeutic target to treat rhabdomyosarcomas, and blocking gene transcription using antigene oligonucleotide strategies has therapeutic potential in the treatment of cancer and other diseases that has not been previously realized in vivo. Translational Relevance Rhabdomyosarcomas are the most common pediatric soft tissue sarcoma. They are a leading cause of cancer death in children with few therapeutic options in highrisk categories. N-Myc is a transcription factor belonging to the MYC protein family that has a highly restricted expression pattern in non-fetal tissues. N-Myc protein expression in patient samples of rhabdomyosarcoma and an apoptotic response to sustained reduction of expression in rhabdomyosarcoma cell lines imply that N-Myc represents a tumor-specific therapeutic target in these tumors. Systemic treatment of mice with rhabdomyosarcoma cell line xenografts using an antigene peptide nucleic acid (PNA) oligonucleotide conjugated to a nuclear localization signal peptide that inhibits transcription of MYCN shows promise by eliminating tumors. This direct antigene approach may have therapeutic potential in the treatment of other cancers and diseases.

Background: The determination of cellular β-galactocerebrosidase activity is an established procedure to diagnose Krabbe disease and monitor the efficacy of gene/stem cell-based therapeutic approaches aimed at restoring defective enzymatic activity in patients or disease models. Current biochemical assays for β-galactocerebrosidase show high specificity but generally require large protein amounts from scanty sources such as hematopoietic or neural stem cells. We developed a novel assay based on the hypothesis that specific measurements of β-galactocerebrosidase activity can be performed following complete inhibition of β-galactosidase activity. Methods: We performed the assay using 2–7.5 μg of sample proteins with the artificial fluorogenic substrate 4-methylumbelliferone-β-galactopyranoside (1.5 mmol/L) resuspended in 0.1/0.2 mol/L citrate/phosphate buffer, pH 4.0, and AgNO3. Reactions were incubated for 30 min at 37 °C. Fluorescence of liberated 4-methylumbelliferone was measured on a spectrofluorometer (λex 360 nm, λem 446 nm). Results: AgNO3 was a competitive inhibitor of β-galactosidase [inhibition constant (Ki) = 0.12 μmol/L] and completely inhibited β-galactosidase activity when used at a concentration of 11 μmol/L. Under this condition, the β-galactocerebrosidase activity was preserved and could be specifically and accurately measured. The assay can detect β-galactocerebrosidase activity in as little as 2 μg cell protein extract or 7.5 μg tissue. Assay validation was performed using (a) brain tissues from wild-type and twitcher mice and (b) murine GALC−/− hematopoietic stem cells and neural precursor cells transduced by GALC-lentiviral vectors. Conclusions: The procedure is straightforward, rapid, and reproducible. Within a clinical context, our method unequivocally discriminated cells from healthy subjects and Krabbe patients and is therefore suitable for diagnostic applications.

The interaction with DNA of a peptide nucleic acid (PNA) oligomer (16nt) conjugated with a nuclear localization signal (NLS) peptide, which was previously found to be able to inhibit tumor cell proliferation through block of transcription of the MYCN oncogene, was studied by UV and CD spectroscopy. While data obtained by UV were not conclusive, the use of circular dichroism gave clear-cut evidence of the formation of a PNA:DNA duplex of exceptionally high stability (Tm ≥ 90°C). Using the same approach, the effect of mutations on DNA:PNA stability was evaluated, and was found in accordance with that expected for a Watson–Crick interaction. The role of the NLS peptide was evaluated by using a PNA lacking of this part, which gave rise to less stable PNA:DNA duplexes. Finally, a competition experiment carried out with a 26mer dsDNA, containing the target 16mer sequence in its middle region, in the presence of PNA-NLS gave evidence for the formation of a ternary complex at 25°, while at higher temperature, the PNA:DNA duplex and the displaced homologous DNA strand were detected. The present results support the possibility of an analogous mechanism of action of this antitumor PNA in vivo. Chirality, 2008. © 2007 Wiley-Liss, Inc.