The primary mechanism of cytotoxicity of the chemotherapeutic agent CX-5461 is topoisomerase II poisoning

Significance Small molecules can directly and indirectly interfere with multiple cellular processes simultaneously. However, there is a difference between what a molecule can do and what mechanisms cause cellular cytotoxicity. Genetic logic using CRISPR works well when discriminating between essential and nonessential target hypotheses. However, discriminating between competing hypotheses across multiple essential pathways is more challenging. Here we combine three complementary data-driven approaches to create multimodal measurements of the mechanism of cytotoxicity. Our multimodal data mining approach coupled to genetic, cell biologic, and biochemical experiments allowed us to detangle essential processes and reannotate CX-5461 as a topoisomerase II poison. Correct annotation is critical for charting a clinical development path and effectively using CX-5461 as a chemical genetic probe.