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A rapid screening system evaluates novel inhibitors of DNA methylation and suggests F-box proteins as potential therapeutic targets for high-risk neuroblastoma

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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.

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Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Labor für Pädiatrische Molekularbiologie, Charité - Universitätsmedizin Berlin, 10117, Berlin, Germany

    Livius Penter, Ute Frede, Christian Hagemeier & Matthias Truss

  2. Institut für Medizinische Immunologie, AG Chronobiologie, Charité - Universitätsmedizin Berlin, 10115, Berlin, Germany

    Bert Maier

  3. CIPSM, Fakultät für Chemie und Pharmazie, Ludwig-Maximilians-Universität, 81377, München, Germany

    Benjamin Hackner & Thomas Carell

Authors
  1. Livius Penter
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  2. Bert Maier
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  3. Ute Frede
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  4. Benjamin Hackner
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  5. Thomas Carell
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  6. Christian Hagemeier
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  7. Matthias Truss
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Corresponding authors

Correspondence to Livius Penter or Matthias Truss.

Electronic supplementary material

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Online Resource 1

Effects of azacytidine, RG108, SGI-1027 and nanaomycin A +/-doxorubicin on cell cycle distribution. Cell cycle distribution measured by flow cytometry using propidium iodide fluorescence labeling: DNA content of individual cells is represented by pulse area (PE-A). Pulse width (PE-W) was used to exclude cell doublets. a) SK-N-AS and SH-EP cells (N-Myc single copy).b) LAN-1 and SK-N-BE(2) cells (N-Myc amplified). (PDF 78 kb)

Online Resource 2

Effects of azacytidine, RG108, SGI-1027 and nanaomycin A +/-doxorubicin on apoptosis quantified with flow cytometry using Annexin V (AV) and propidium iodide (PI) fluorescence labeling. Viable and dead cells are labeled AV-/PI- and AV+/PI+, while early and late apoptosis are characterized as AV+/PI- and AV+/PI+. a) SK-N-AS and SH-EP cells (N-Myc single copy). b) LAN-1 and SK-N-BE(2) cells (N-Myc amplified). (PDF 101 kb)

Online Resource 3

Overview of shRNAs against F-box proteins employed in the paper (PDF 14 kb)

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Penter, L., Maier, B., Frede, U. et al. A rapid screening system evaluates novel inhibitors of DNA methylation and suggests F-box proteins as potential therapeutic targets for high-risk neuroblastoma. Targ Oncol 10, 523–533 (2015). https://doi.org/10.1007/s11523-014-0354-5

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  • DOI: https://doi.org/10.1007/s11523-014-0354-5

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