title: A rapid screening system evaluates novel inhibitors of DNA methylation and suggests F-box proteins as potential therapeutic targets for high-risk neuroblastoma.
abstract: After extensive research on radiochemotherapy, 5-year survival rates of children with high risk neuroblastoma still do not exceed 50%, owing to adverse side-effects exemplified by doxorubicin-induced cardiomyopathy. A promising new approach is the combination of conventional therapies with specific modulation of cell signaling pathways promoting therapeutic resistance, such as inhibition of aberrant kinase activity or re-expression of silenced tumor suppressor genes by means of chromatin remodeling. In this regard, we established a system that allows to identify potential drug targets as well as to validate respective candidate inhibitors in high-risk neuroblastoma model cell lines. Cell culture, drug exposure, shRNA-mediated knockdown and phenotype analysis are integrated into an efficient and versatile single well-based protocol. By utilizing this system, we assessed RG108, SGI-1027 and nanaomycin A, three novel DNA methyltransferase inhibitors that have not been tested in neuroblastoma cell lines so far, for their potential of synergistic anti-tumor activity in combination with doxorubicin. We found that, similarly to azacytidine, SGI-1027 and nanaomycin A mediate synergistic growth inhibition with doxorubicin independently of N-Myc status. However, they display high cytotoxicity but lack global DNA demethylation activity. Secondly, we conducted a lentiviral shRNA screen of F-box proteins, key regulators of protein stability, and identified Fbxw11/β-TrCP2 as well as Fbxo5/Emi1 as potential therapeutic targets in neuroblastoma. These results complement existing studies and underline the reliability and versatility of our single well-based protocol.
authors.affiliation: Labor für Pädiatrische Molekularbiologie, Charité - Universitätsmedizin Berlin, 10117, Berlin, Germany. livius.penter@charite.de.
authors.forename: Livius
authors.initials: L
authors.lastname: Penter
authors.affiliation: Institut für Medizinische Immunologie, AG Chronobiologie, Charité - Universitätsmedizin Berlin, 10115, Berlin, Germany.
authors.forename: Bert
authors.initials: B
authors.lastname: Maier
authors.affiliation: Labor für Pädiatrische Molekularbiologie, Charité - Universitätsmedizin Berlin, 10117, Berlin, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Frede
authors.affiliation: CIPSM, Fakultät für Chemie und Pharmazie, Ludwig-Maximilians-Universität, 81377, München, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Hackner
authors.affiliation: CIPSM, Fakultät für Chemie und Pharmazie, Ludwig-Maximilians-Universität, 81377, München, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Carell
authors.affiliation: Labor für Pädiatrische Molekularbiologie, Charité - Universitätsmedizin Berlin, 10117, Berlin, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Hagemeier
authors.affiliation: Labor für Pädiatrische Molekularbiologie, Charité - Universitätsmedizin Berlin, 10117, Berlin, Germany. matthias.truss@charite.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Truss
mesh_terms: D000634:Aminoquinolines / Q000008:administration & dosage / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019175:DNA Methylation / Q000187:drug effects*; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors*; D004317:Doxorubicin / Q000008:administration & dosage / Q000494:pharmacology; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D044783:F-Box Proteins / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D058990:Molecular Targeted Therapy; D009285:Naphthoquinones / Q000008:administration & dosage / Q000494:pharmacology; D009447:Neuroblastoma / Q000188:drug therapy / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D010797:Phthalimides / Q000008:administration & dosage / Q000494:pharmacology; D011743:Pyrimidines / Q000008:administration & dosage / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics; D014364:Tryptophan / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology; D000095622:Benzoisochromanequinones
chemical_list: D000634:Aminoquinolines; D015254:DNA Modification Methylases; D004317:Doxorubicin; D004791:Enzyme Inhibitors; D044783:F-Box Proteins; D009285:Naphthoquinones; D010797:Phthalimides; D011743:Pyrimidines; D034741:RNA, Small Interfering; D014364:Tryptophan; C503639:RG108; C581203:SGI-1027; C008638:nanaomycin A; D000095622:Benzoisochromanequinones
keywords: DNA methyltransferase inhibitors; Doxorubicin; F-box proteins; Neuroblastoma; RNA-interference; Screening system