Abstract A profile of microRNA (miRNA) and mRNA expression patterns across the NCI-60 cell-line screen was analyzed to identify expression signatures that correlate with sensitivity to FdUMP[10], fluorouracil (5FU), floxuridine (FdU), topotecan, and irinotecan. Genome-wide profile analyses revealed FdUMP[10] resembles FdU most closely and shows dissimilarities with 5FU. FdUMP[10] had the largest dynamic range of any of these drugs across the NCI-60 indicative of cancer cell–specific activity. Genes involved in endocytosis, such as clathrin (CLTC), SNF8, annexin A6 (ANXA6), and amyloid protein-binding 2 (APPBP2) uniquely correlated with sensitivity to FdUMP[10], consistent with a protein-mediated cellular uptake of FdUMP[10]. Genes involved in nucleotide metabolism were enriched for the three fluoropyrimidine drugs, with the expression profile for 5FU correlated to an RNA-mediated cytotoxic mechanism, whereas expression of glycosyltransferases (XYLT2) that use UDP sugars as substrates and the nucleoside diphosphatase and metastasis suppressor NM23 (NME1) were associated with FdUMP[10] sensitivity. Topotecan and irinotecan had significant negative correlations with miR-24, a miRNA with a high aggregate PCT score for topoisomerase 1 (Top1). Our results reveal significant new correlations between FdUMP[10] and Top1 poisons, as well as new information on the unique cytotoxic mechanism and genomic signature of FdUMP[10]. Mol Cancer Ther; 9(12); 3105–14. ©2010 AACR.