The objective of this study was to identify amino acid substitutions in human Topo-IIαα that reduce sensitivity to topoisomerase II inhibitors and to evaluate their implications for therapeutic response. The human HTOP2A gene which encodes Human Topoisomerase IIα was subjected to random mutagenesis and expressed in a yeast model system, followed by selection under increasing concentrations of Etoposide to isolate resistant variants. Resistant clones were characterized through sequencing and quantitative survivorship analyses to define resistance phenotypes.

Multiple mutations associated with decreased sensitivity to Etoposide were identified, demonstrating that single amino acid substitutions can significantly impair drug efficacy. The 14 most potent mutations were selected for further examination, revealing clusters in the 448-492 base pair region and near the catalytic Tyr805. The discovered variant R487I possessed the highest resistance of all mutations, displaying greater than 50% resistance even at high concentrations of Etoposide.

These findings provide mechanistic insight into topoisomerase II inhibitor resistance and support the utility of directed evolution approaches in predicting therapeutic response. Furthermore, integrating resistance profiles with survivorship outcomes offers a framework for evaluating the relative effectiveness of Etoposide in comparison to other topoisomerase-targeting agents and informing more personalized, cost-effective treatment strategies.