Dual Inhibition of TRIP13 and Aurora A Induces Mitotic DNA Damage and Concurrent Pyroptotic-apoptotic Cell Death in Rb-deficient Cancer Cells

PURPOSE: Cancers driven by a loss of tumor suppressor function lack actionable druggable targets. We investigated the cell cycle-specific mechanisms underlying the efficacy of co-targeting TRIP13 and Aurora A by defining their key functions in Rb-deficient cancers to develop effective treatment strategies. EXPERIMENTAL DESIGN: We used live-cell imaging to monitor individual cell fates and validated results using orthogonal measurements of apoptosis, pyroptosis, and cell cycle in vitro. In mouse xenografts, we used a clinically relevant Aurora A inhibitor and inducible TRIP13 protein degradation to elucidate this combination's effect in vivo. Human tumor mRNA expression was analyzed to establish clinical relevance. RESULTS: Co-targeting TRIP13 and Aurora A led to mitotic cell death by inducing prolonged mitotic arrest. We observed that TRIP13 contributes to this effect by further extending Aurora A inhibition-induced mitotic arrest, thereby enhancing its cytotoxicity. Orthogonal in vitro assays further revealed that dual targeting induces DNA damage and concurrent apoptotic and gasdermin E (GSDME)-mediated pyroptotic cell death in mitotically arrested Rb-deficient cancer cells. Additionally, this combination achieved marked antitumor efficacy in vivo accompanied by a measurable survival benefit in mice bearing Rb-deficient carcinoma. Rb-deficient human head and neck and lung squamous cell carcinoma tumors exhibited significantly higher CASP3 but lower GSDME expression, suggesting an adaptive mechanism to limit GSDME-mediated pyroptosis that is overcome by the combination. CONCLUSIONS: Combined inhibition of TRIP13 and Aurora A may have a high therapeutic index by inducing mitotic pyroptosis and apoptosis specifically in Rb-deficient cancer cells and potentially engaging anti-tumor immunity.