VX-984 is a selective inhibitor of non-homologous end joining, with possible preferential activity in transformed cells

Abstract

// Atif J. Khan 1, 2 , Sarah M. Misenko 3 , Aditya Thandoni 2 , Devora Schiff 2 , Sachin R. Jhawar 2 , Samuel F. Bunting 3 and Bruce G. Haffty 2 1 Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY 10011, USA 2 Department of Radiation Oncology, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA 3 Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA Correspondence to: Atif J. Khan, email: khana7@mskcc.org Keywords: DNA-PK; non-homologous end joining; DNA repair; double-strand break repair; radiation therapy Received: December 05, 2017      Accepted: April 25, 2018      Published: May 25, 2018 ABSTRACT Purpose: DNA double-strand breaks (DSBs) can be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). We demonstrate the selectivity of VX-984, a DNA-PK inhibitor, using assays not previously reported. Experimental Design: The class switch recombination assay (CSR) in primary B cells was used to measure efficiency of NHEJ. A cellular reporter assay (U2OS EJ-DR) was used to assess the efficiency of HR and NHEJ in cells treated with VX-984. Immunofluorescence assays (IF) evaluated γ-H2AX foci for DSB repair kinetics in human astrocytes and T98G glioma cells. Western blotting was used to evaluate phosphorylation of DNA-PKcs substrates. Results: We found a dose-dependent reduction in CSR efficiency with VX-984, and through the EJ-DR assay, dramatic dose-dependent increases in HR and mNHEJ. Immunofluorescence assays showed an inability of malignant cells to resolve γ-H2AX foci in the presence of VX-984. Radiation-induced phosphorylation of DNA-PK substrates was further reduced by treatment with VX-984. Conclusions: VX-984 efficiently inhibits NHEJ, resulting in compensatory increases in alternative repair pathways, increases DSBs, and appears to affect transformed cells preferentially.