High-Throughput Screening Approach for Identifying Compounds That Inhibit Nonhomologous End Joining

Andrea L. Bredemeyer, Bruce S. Edwards, Mark K. Haynes, Abigail J. Morales, Yinan Wang, Oleg Ursu, Anna Waller, Larry A. Sklar, Barry P. Sleckman

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

DNA double-strand breaks (DSBs) are repaired primarily by homologous recombination (HR) or nonhomologous end joining (NHEJ). Compounds that modulate HR have shown promise as cancer therapeutics. The V(D)J recombination reaction, which assembles antigen receptor genes in lymphocytes, is initiated by the introduction of DNA DSBs at two recombining gene segments by the RAG endonuclease, followed by the NHEJ-mediated repair of these DSBs. Here, using HyperCyt automated flow cytometry, we develop a robust high-throughput screening (HTS) assay for NHEJ that utilizes engineered pre-B-cell lines where the V(D)J recombination reaction can be induced and monitored at a single-cell level. This approach, novel in processing four 384-well plates at a time in parallel, was used to screen the National Cancer Institute NeXT library to identify compounds that inhibit V(D)J recombination and NHEJ. Assessment of cell light scattering characteristics at the primary HTS stage (83,536 compounds) enabled elimination of 60% of apparent hits as false positives. Although all the active compounds that we identified had an inhibitory effect on RAG cleavage, we have established this as an approach that could identify compounds that inhibit RAG cleavage or NHEJ using new chemical libraries.

Original languageEnglish
Pages (from-to)624-633
Number of pages10
JournalSLAS Discovery
Volume23
Issue number7
DOIs
StatePublished - Aug 1 2018

Keywords

  • cancer and cancer drugs
  • cell-based assays
  • immune system diseases
  • oncology

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