CFI-400945 enhances radiosensitivity in colorectal cancer models by promoting mitotic dysregulation and DNA damage-associated apoptosis

Radioresistance remains a major barrier to improving radiotherapy (RT) efficacy in colorectal cancer (CRC). Since mitotic fidelity and centrosome homeostasis influence cell fate under genotoxic stress, we investigated whether CFI-400945 enhances the radiation response in CRC models and whether this effect is associated with polo-like kinase 4 (PLK4)-related mitotic dysregulation. HCT116 and HT29 CRC cells were treated with CFI-400945 and/or irradiation. Radiation response was evaluated using MTT viability and clonogenic survival assays, alongside analyses of cell-cycle distribution, centrosome-associated abnormalities, nuclear phenotypes, DNA damage-associated signaling, and apoptotic markers. PLK4 involvement was evaluated using siRNA-mediated PLK4 depletion, and Aurora kinase B (AURKB) phosphorylation was assessed to examine potential Aurora B-related effects. Combination treatment was also tested in non-malignant CCD 841 CoN colon-derived control cells and patient-derived CRC organoids. Combined CFI-400945 and RT treatment reduced short-term viability and long-term clonogenic survival in HCT116 and HT29 cells compared with either monotherapy. In CCD 841 CoN cells, CFI-400945 did not significantly enhance radiation-associated loss of clonogenic survival versus RT alone. Combination treatment increased G2/M accumulation, >4 N and sub-G1 fractions, γ-tubulin-positive foci, multinucleation, and micronucleation, consistent with centrosome-associated mitotic abnormalities and reduced survival. DNA damage-associated signaling was enhanced, with increased DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and phosphorylated histone H2AX (γH2AX) levels and increased phosphorylation of ataxia telangiectasia mutated (ATM) and CHK2. Apoptotic marker induction also increased, including robust cleavage of caspase-3 and PARP1. CFI-400945 did not markedly suppress AURKB phosphorylation under the experimental conditions. Importantly, siRNA-mediated PLK4 depletion reduced clonogenic survival after irradiation and increased cleaved PARP1 and cleaved caspase-3, supporting PLK4 involvement in the radiation response. In patient-derived CRC organoids, combination treatment reduced viability and increased DNA damage-associated and apoptotic markers compared with single treatments. CFI-400945 enhanced radiation response in CRC cell lines and patient-derived organoids, accompanied by centrosome-associated mitotic abnormalities, increased DNA damage-associated signaling, and apoptotic marker induction. siRNA-mediated PLK4 depletion partially recapitulated these effects, supporting a role for PLK4-associated mitotic regulation. However, because CFI-400945 may also target non-PLK4 mitotic kinases, these support an association --between radiosensitization and PLK4-related mitotic dysregulation rather than an exclusively PLK4-dependent mechanism.