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

Area of Science:

• Molecular Biology
• Genetics
• Immunology

Background:

• DNA double-strand breaks (DSBs) are critical DNA lesions repaired by homologous recombination (HR) or nonhomologous end joining (NHEJ).
• Modulators of HR show therapeutic potential in cancer treatment.
• The V(D)J recombination process in lymphocytes, essential for adaptive immunity, involves RAG endonuclease-initiated DSBs followed by NHEJ repair.

Purpose of the Study:

• To develop a robust high-throughput screening (HTS) assay for identifying inhibitors of nonhomologous end joining (NHEJ).
• To screen a large chemical library for compounds that modulate the V(D)J recombination pathway.

Main Methods:

• Utilized engineered pre-B-cell lines for single-cell level monitoring of V(D)J recombination.
• Implemented HyperCyt automated flow cytometry for parallel processing of 384-well plates.
• Incorporated cell light scattering analysis to filter false positives during HTS.

Main Results:

• Screened 83,536 compounds from the National Cancer Institute NeXT library.
• Eliminated 60% of apparent hits as false positives through light scattering assessment.
• Identified active compounds that inhibit RAG cleavage, a key step in V(D)J recombination.

Conclusions:

• Established a novel HTS approach for identifying inhibitors of NHEJ and RAG cleavage.
• The assay is adaptable for screening new chemical libraries to find modulators of DNA repair pathways.
• This method provides a platform for discovering novel therapeutic agents targeting DNA repair mechanisms.