Mitotic adaptations shape acquired resistance and vulnerabilities to KIF18A inhibition in cancer

KIF18A inhibition selectively kills cancer cells by inducing chromosome alignment defects that activate a spindle assembly checkpoint (SAC)-dependent mitotic arrest. The mechanisms by which initially sensitive cancer cells acquire resistance to KIF18A inhibitors (KIF18Ai), the vulnerabilities of resistant cells, and the initial genetic determinants of KIF18Ai sensitivity remain poorly understood. Using orthogonal CRISPR-Cas9 screening and long-term drug adaptation approaches, we identify two convergent resistance mechanisms. Resistant cells either partially override the SAC, permitting mitotic exit despite chromosome misalignment, or adapt spindle microtubule dynamics to restore chromosome alignment in the absence of KIF18A. Both strategies sustain mitotic progression without inducing KIF18Ai dependence or increasing sensitivity to other mitotic perturbations. In contrast, reduced activity of the mitotic exit regulators PP2A or APC/C enhances KIF18Ai sensitivity. Accordingly, Mps1 inhibitor-driven APC/C mutations enhance responsiveness to KIF18A inhibition. In conclusion, resistance to KIF18A inhibition emerges rapidly, yet distinct genetic contexts create exploitable vulnerabilities to KIF18Ai.