Abstract Body: CX-5461 is a chemotherapeutic drug that is in clinical trials for the treatment of solid tumors enriched for DNA-repair deficiencies. While CX-5461 was initially described as an RNA pol I inhibitor, it has recently been shown to be a topoisomerase II poison that primarily targets TOP2B. Anti-cancer drugs such as Doxorubicin (DOX) also target TOP2B which can result in cardiotoxicity. It has therefore been suggested that CX-5461 may also induce cardiotoxicity. We therefore designed a study to investigate the effects of CX-5461 on iPSC-derived cardiomyocytes from six individuals. We compared the cellular and molecular response to CX-5461 and DOX at 0.1 and 0.5 uM concentrations at three, 24 and 48 hours post-treatment. CX-5461 is less cardiotoxic than DOX based on cell viability measurements 24 hours following treatment (LD50 DOX = 0.65 uM, CX-5461 = 9.98 uM). Similarly, the proportion of cells expressing the DNA damage marker γ-H2AX 24 hours post sub-micromolar drug treatment is higher in DOX-treated cells (DOX = 90%, CX-5461 = 6%). Consistent with the strong cellular effects of DOX, transcriptomic analysis between DOX and a vehicle control identified thousands of gene expression changes. In contrast CX-5461 induced 441 gene expression changes across drug concentrations and time. Comparison of gene expression drug response trajectories over time reveals response signatures that are shared between CX-5461 and DOX, and specific to DOX. There are no CX-5461 unique signatures. Shared response genes correspond to pathways related to the cell cycle and DNA replication. A total of 33 genes in loci associated with DOX-induced cardiotoxicity show gene expression changes in response to DOX and only one gene (GPSM2) in response to CX-5461. Our results indicate that CX-5461 treatment of cardiomyocytes induces gene expression changes that mirror some of those induced by DOX, but these effects do not coincide with the cardiotoxicity observed with DOX treatment.