ARTICLE ABSTRACTAPTO-253 is a small molecule with antiproliferative activity against cell lines derived from a wide range of human malignancies. We sought to determine the mechanisms of action and basis for resistance to APTO-253 so as to identify synthetic lethal interactions that can guide combination studies. The cellular pharmacology of APTO-253 was analyzed in Raji lymphoma cells and a subline selected for resistance (Raji/253R). Using LC/MS/ESI analysis, APTO-253 was found to convert intracellularly to a complex containing one molecule of iron and three molecules of APTO-253 [Fe(253)3]. The intracellular content of Fe(253)3 exceeded that of the native drug by approximately 18-fold, and Fe(253)3 appears to be the most active form. Treatment of cells with APTO-253 caused DNA damage, which led us to ask whether cells deficient in homologous recombination (i.e., loss of BRCA1/2 function) were hypersensitive to this drug. It was found that loss of either BRCA1 or BRCA2 function in multiple isogenic paired cell lines resulted in hypersensitivity to APTO-253 of a magnitude similar to the effects of PARP inhibitors, olaparib. Raji cells selected for 16-fold acquired resistance had 16-fold reduced accumulation of Fe(253)3. RNA-seq analysis revealed that overexpression of the ABCG2 drug efflux pump is a key mechanism of resistance. ABCG2-overexpressed HEK-293 cells were resistant to APTO-253, and inhibition of ABCG2 reversed resistance to APTO-253 in Raji/253R. APTO-253 joins the limited repertoire of drugs that can exploit defects in homologous recombination and is of particular interest because it does not produce myelosuppression. Mol Cancer Ther; 17(6); 1167–76. ©2018 AACR.