Background: BAL0891 is a dual inhibitor of threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1). These kinases collaborate in activating the mitotic spindle assembly checkpoint (SAC) at the kinetochore (KT) to regulate chromosome alignment and segregation prior to mitotic exit. In this work, kinase inhibition by BAL0891 was linked to effects on SAC integrity and aberrant mitotic progression in tumor cells. Comparison with a TTK-specific inhibitor (CFI-402257, CFI) allowed further evaluation of the contribution of dual TTK/PLK1 inhibition to anti-cancer activity, associated with a promising anti-proliferative profile across diverse tumor cell lines.

Methods: Kinase assays used a radiometric assay. Target residency was measured using surface plasmon resonance with recombinant kinase. Anti-proliferative activity was assessed with crystal violet or YO-PRO assay (5 days incubation), in-cell target inhibition by immunoblotting for phospho-TTK following drug wash-out. Effects on SAC integrity were followed by immunoprecipitation (IP) mitotic progression by flow cytometry/mitotic marker expression. Cells were blocked in mitosis using the microtubule-targeting agent nocodazole or the PLK1 inhibitor onvansertib. SAC KT accumulation was evaluated by immunofluorescence (IF) for co-localization of BubR1 with CENPC. Comparative studies with CFI used anti-proliferative IC50 concentrations.

Results: In vitro kinase profiling showed that BAL0891 has low nM IC50s against TTK and PLK1, with prolonged TTK (>12 h) and transient PLK1 (4 min) target residency. Prolonged TTK inhibition (≥38 h) was also observed in HT29 tumor cells. Consistent with a dominant TTK-targeting activity, BAL0891 treatment of HT29 cells blocked in mitosis with nocodazole or the PLK1 inhibitor onvansertib led to aberrant mitotic release and accumulation of polyploid cells. This was preceded by SAC disruption as visualized by IP assays. Effects on the SAC and mitotic exit were evaluated in comparative studies with CFI; BAL0891 exhibited faster kinetics for both parameters suggesting a contribution of PLK1 inhibition. This was confirmed by directly evaluating acute effects on SAC integrity at the KT by IF. Specifically, 1 h BAL0891 treatment of mitotic HT29 cells resulted in a highly reproducible and significant reduction in KT-associated SAC (p<0.0001) which was not observed with CFI in the same conditions. An extensive in vitro BAL0891 anti-proliferative screen indicated a broad anti-cancer potential, with low nM GI50s observed for most tumor lines and minimal activity on non-immortalized cells (GI50s >5 uM).

Conclusion: BAL0891 is a novel dual TTK/PLK1 mitotic checkpoint inhibitor. In tumor cells, prolonged effects on TTK and transient effects on PLK1 contribute to rapid SAC disruption and aberrant mitotic exit. This is associated with potent anti-proliferative activity in diverse tumor lines.

Citation Format: Elisa Zanini, Nicole Forster-Gross, Felix Bachmann, Nicole Willemsen-Seegers, Jos de Man, Guido J. Zaman, Rogier C. Buijsman, Anna Groner, Mila Roceri, Karin Burger, Paul McSheehy, Laurenz Kellenberger, Heidi A. Lane. BAL0891: A novel, small molecule, dual TTK/PLK1 mitotic checkpoint inhibitor (MCI) that drives aberrant tumor cell division [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5646.