Abstract Focal high-copy number oncogene and resistance gene amplification via extrachromosomal DNA (ecDNA) is associated with high-risk disease and therapeutic failure across many cancer types. New therapies that target ecDNA function are needed to address this significant unmet need. We identified a novel kinesin motor protein, Kinesin, as a key regulator of proper mitotic ecDNA segregation and ecDNA maintenance in cancer cells. Genetic inactivation of Kinesin resulted in ecDNA mis-segregation during anaphase, reduced ecDNA levels, and a corresponding loss of tumor cell proliferation and survival. Pharmacological degradation of Kinesin using the highly selective heterobifunctional degrader KNSNd-1 recapitulated these cellular phenotypes. Furthermore, the antiproliferative effects of KNSNd-1 coincided with induction of pHH3 and γH2AX, indicating mitotic arrest and genotoxic stress as a putative mechanism of action underlying sensitivity. KNSNd-1 demonstrated potent degradation in vitro (∼1 nM DC₅₀ in HEK293T Kinesin-HiBiT cells), with exquisite selectivity. Global proteomics studies in CAL51 breast cancer cells treated with KNSNd-1 revealed Kinesin as the only protein significantly degraded (log₂ fold change 1; −log₁₀ p-value 4). In vitro sensitivity to KNSNd-1 correlated with ecDNA status, as determined by metaphase analysis (p = 0. 01, Mann-Whitney test), across a panel of breast cancer cell lines. Oral administration of KNSNd-1 monotherapy resulted in significant tumor growth inhibition (TGI), including tumor regressions, and this activity correlated with ecDNA status across multiple models and indications. Moreover, combination treatment with KNSNd-1 and targeted therapies in ecDNA-positive breast and gastric cancer models resulted in significantly greater TGI compared with targeted therapy alone. Together, these findings support the clinical development of Kinesin degraders for the treatment of ecDNA-associated high-risk cancers, including breast cancer. Citation Format: Deepti Wilkinson, Frederick A. Derheimer, Ardalan Adreshi, Joan Chen, Yuan Chien, Cory DuPai, Rachel Elsdon, Ryan J. Hansen, Kimberly Johnson, Shailaja Kasibhatla, Laura Layman, Zhengke Li, Ronghua Li, Debbie Liao, Taylor Loe, Jacques Mauger, Jafar Moininazeri, Ben Norman, Anne Marie Pferdekamper, Joshua Plum, Casey Sarkissian, Dave Solis, Auzon Steffy, Huikai Sun, Yen Truong, Julie Wiese, Anthony B. Pinkerton, Christian A. Hassig. Selective degradation of a novel kinesin as a potential therapeutic strategy addressing high-risk extrachromosomal DNA (ecDNA) positive cancers, including breast cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts) ; 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (8Suppl): Abstract nr LB361.
Wilkinson et al. (Fri,) studied this question.