Abstract 363: Exploiting Rb-deficiency: Dual inhibition of TRIP13 and Aurora A as a path to mitotic catastrophe

Abstract Deregulation of the Rb pathway is found in approximately one-third of all solid tumors. Despite the high prevalence, there are still no clinically established therapies selectively targeting Rb-deficient cancers. Therefore, understanding how Rb function loss can be leveraged is crucial for developing novel, biomarker-driven, targeted therapeutic strategies. We and others have previously shown that Rb-deficient cancer cells depend on Aurora A kinases to bypass the primed spindle assembly checkpoint (SAC) and survive due to overexpression of the mitotic checkpoint protein Mad2. Thyroid hormone receptor interactor 13 (TRIP13) is a mitotic checkpoint modulatory protein that catalyzes the ATP-dependent conformational conversion of Mad2 to promote SAC silencing. Our previous research demonstrated that the combination of Aurora A inhibition using specific inhibitor alisertib and TRIP13 depletion induced synthetic lethality selectively in Rb-deficient cancer cells where the combination induced mitotic catastrophe. The combination also significantly increased the number of mitotic cells with an abnormal mitotic morphology by causing mitotic spindle defects and abnormalities in chromosomal alignment. Additionally, the combination led to prominent G2/M arrest in Rb-deficient cancer cells, while it only transiently delayed cell cycle progression in Rb-proficient cancer cells. Our live-cell imaging experiments using three Rb-deficient cell line models (HeLa, UMSCC47, ME180) showed that the combination induces marked prolonged arrest in mitosis and results in mitotic cell death. Furthermore, using auxin-inducible degron system and siRNA-mediated protein depletion, we showed that TRIP13 plays a modulatory role in mitotic exit as its depletion induced transient mitotic arrest and enhanced alisertib-induced lethality by further extending the mitotic arrest. Since mitotic catastrophe can cause DNA damage and this damage can be sensed by the inducers of immunogenic cell death (ICD), we also tested the effect of the combination on DNA damage and ICD. The combination caused marked DNA damage in mitotic Rb-deficient cancer cells, as measured by the increased γH2AX protein levels and tail-length using the neutral comet assay. Similarly, it increased the protein levels of ICD potentiators, cGAS and cleaved-gasdermin E. The mitotic entry was shown to be pivotal to the lethal effects of the combination, since the inhibition of mitotic entry with concurrent CDK1 inhibitor, RO-3306, prevented apoptosis and DNA damage, as determined by APO-DIRECT assay and immunoblotting. This is the first study to elucidate the mechanism behind the synthetic lethality of TRIP13 and Aurora A in Rb-deficient cancer cells. These findings may provide important insight into how to rationally design novel therapeutic strategies targeting mitotic regulators by harnessing the therapeutic vulnerability of Rb-deficient cancer cells. Citation Format: Lacin Yapindi, Soma Ghosh, Li Shen, Jing Wang, Faye M. Johnson. Exploiting Rb-deficiency: Dual inhibition of TRIP13 and Aurora A as a path to mitotic catastrophe [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 363.