ARTICLE ABSTRACTPurpose: Radiation therapy continues to be an important therapeutic strategy for providing definitive local/regional control of human cancer. However, oncogenes that harbor driver mutations and/or amplifications can compromise therapeutic efficacy. Thus, there is a need for novel approaches that enhance the DNA damage produced by ionizing radiation.Experimental Design: A forward chemical genetic approach coupled with cell-based phenotypic screening of several tumor cell lines was used to identify a novel chemical entity (NCE) that functioned as a radiation sensitizer. Proteomics, comet assays, confocal microscopy, and immunoblotting were used to identify the biological target.Results: The screening process identified a 5-((N-benzyl-1H-indol-3-yl)-methylene)pyrimidine-2,4,6(1H,3H,5H)trione as an NCE that radiosensitized cancer cells expressing amplified and/or mutated RAS, ErbB, PIK3CA, and/or BRAF oncogenes. Affinity-based solid-phase resin capture followed by liquid chromatography/tandem mass spectrometry identified the chaperone nucleophosmin (NPM) as the NCE target. SiRNA suppression of NPM abrogated radiosensitization by the NCE. Confocal microscopy showed that the NCE inhibited NPM shuttling to radiation-induced DNA damage repair foci, and the analysis of comet assays indicated a diminished rate of DNA double-strand break repair.Conclusion: These data support the hypothesis that inhibition of DNA repair due to inhibition of NPM shuttling increases the efficacy of DNA-damaging therapeutic strategies. Clin Cancer Res; 17(20); 6490–9. ©2011 AACR.