Growth inhibition (GI) and gene expression profiles across the 60 cell lines of the NCI-60 screen were analyzed to elucidate mechanistic differences between a nucleolus-targeting platinum-acridine (PA) hybrid anticancer agent (compound 1) and the standard-of-care chemotherapies cisplatin, oxaliplatin, mitomycin c, doxorubicin, and topotecan. The study utilized Pearson correlation and functional enrichment analysis tools (including gene set enrichment analysis, GSEA) in combination with the gene ontology (GO) resource to identify cellular processes that might contribute to the unique spectrum of activity and high potency of compound 1. The GSEA results are consistent with DNA being a major target of compound 1 based on the negative correlation observed between its potency and expression levels of genes implicated in DNA double-strand break (DSB) repair. GO terms related to RNA processing, including ribosome biogenesis and RNA splicing, are also negatively enriched in GSEA for compound 1, suggesting a mechanism by which these processes render cells more resistant to the hybrid agent. The opposite trend is observed for the other DNA-targeted drugs. Significant functional interactions (STRING) exist between genes/gene products involved in ribosome biogenesis and DSB repair in rDNA, including BRCA1, BABAM1, RPA2, MDM2, RAD21, RAD54L, RPL5, and PAK1IP1. The results suggest that high levels of ribosome biogenesis may buffer compound 1’s cytotoxicity by triggering the ribosomal protein (RPL5)-MDM2-p53 surveillance pathway to facilitate downstream repair of DSBs in rDNA as a nucleolar stress response.