ARTICLE ABSTRACTSW044248, identified through a screen for chemicals that are selectively toxic for non–small cell lung cancer (NSCLC) cell lines, was found to rapidly inhibit macromolecular synthesis in sensitive, but not in insensitive, cells. SW044248 killed approximately 15% of a panel of 74 NSCLC cell lines and was nontoxic to immortalized human bronchial cell lines. The acute transcriptional response to SW044248 in sensitive HCC4017 cells correlated significantly with inhibitors of topoisomerases and SW044248 inhibited topoisomerase 1 (Top1) but not topoisomerase 2. SW044248 inhibited Top1 differently from camptothecin and camptothecin did not show the same selective toxicity as SW044248. Elimination of Top1 by siRNA partially protected cells from SW044248, although removing Top1 was itself eventually toxic. Cells resistant to SW044248 responded to the compound by upregulating CDKN1A and siRNA to CDKN1A sensitized those cells to SW044248. Thus, at least part of the differential sensitivity of NSCLC cells to SW044248 is the ability to upregulate CDKN1A. Mol Cancer Ther; 15(1); 23–36. ©2015 AACR.