Introduction: Anaplastic thyroid cancer (ATC) is among the most aggressive of all human cancers with a median survival of 4.3 months. Currently, there is no effective treatment for most ATC patients - surgery, radiation, and chemotherapy all fail to significantly prolong ATC patient survival. YM155, first identified as a survivin inhibitor, was highlighted in a high-throughput screen performed by the National Cancer Institute, killing anaplastic thyroid cancer cells in vitro and in vivo. However, there was no association between survivin expression and response to YM155 in clinical trials (not including ATC), and YM155 has been mostly abandoned for development despite favorable pharmacokinetic and toxicity profiles. A number of additional mechanisms have been proposed for YM155. The purpose of this study was to investigate the mechanisms underlying YM155-mediated ATC cell death. Methods: ATC cell line THJ16T was used as the model for this study. AlamarBlue was used to measure cell viability. Immunofluorescent detection of phosphorylated histone H2AX (γ-H2AX, p-Ser139) foci was used as a surrogate marker for double-strand DNA breaks. siRNA was used to knockdown topoisomerase 2α (Top2α), and Lipofectamine-3000 was used to overexpress Top2α plasmids. In-vivo complex of enzyme assay was used to measure the association between Top2α and DNA. Modeling software Molecular Operating Environment and the crystal structure of human Top2α bound to AMP-PNP (Protein Data Bank # 1ZXM) were used to estimate binding affinities for ATP and YM155 in the ATP binding site of Top2α. Site-directed mutagenesis was used to introduce mutation Ser148 → Ala into a recombinant human Top2α. Results: Previously, we presented gene expression data from ATC patients showing significant overexpression of Top2α compared to benign thyroid samples. ATC cell lines that overexpress Top2α are more sensitive to YM155 and exhibit larger increases in DNA damage with YM155 treatment. In this study, ATC cells grown to be resistant to YM155 show decreased expression of Top2α, and overexpression of Top2α re-sensitizes resistant cells to YM155. Molecular modeling shows favorable binding for YM155 in the Top2α ATP binding site and identifies key amino acids for YM155-Top2α interaction. An engineered Top2α mutant abrogates the effect of YM155 as measured by cell viability and DNA damage assays, confirming the contribution of Top2α to YM155 mechanism of action. Conclusions: Our results strongly suggest that Top2α is an important target mediating YM155’s anticancer activity in ATC. We propose that Top2α inhibition by YM155 leads to DNA damage and eventual cell death. Given YM1559s potent anti-proliferative effects in ATC cells with sparing of benign thyroid cells, along with a plausible mechanism of action, we suggest further evaluation and optimization of YM155 for future clinical application in ATC. In conclusion, our study lays the foundation for targeting the enzymatic activity of Top2α as a novel therapeutic strategy for the treatment of ATC, with YM155 serving as the predicate drug. Citation Format: Ryan Mackay, Paul Weinberger, Elahe Mahdavian, Qinqin Xu. YM155 induces DNA damage and cell death in anaplastic thyroid cancer cells by inhibiting DNA topoisomerase IIα at the ATP binding site [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA033.