Cancer cell death by mitotic failure upon combined kinase inhibition
von Antonia Kleeberg
Datum der mündl. Prüfung:2022-06-15
Betreuer:Prof. Dr. Matthias Dobbelstein
Gutachter:Prof. Dr. Matthias Dobbelstein
Gutachter:Prof. Dr. Elisabeth Heßmann
EnglischCancer cells are prone to replicative stress to a greater extent than healthy cells are. Thus, replicative stress might be a way to target cancer cells in a more specific way than conventional chemotherapeutics do. According to previous results, one way to exert replicative stress on cells is the inhibition of the centrosomal protein Polo-like kinase 4 (PLK4). The small compound CFI-400945 is a PLK4 inhibitor. CFI-400945 is currently being tested in phase I and II clinical trials, still its mechanisms of action partly remain elusive. Therefore, we examined the effect of CFI-400945 on DNA replication and cell proliferation in H1299 cells, with methods including cell microscopy, DNA fiber assays, western blot analyses, flow cytometry and immunofluorescence. As a result, we found that CFI-400945 manages to reduce the proliferation of cells by two means: Firstly by the exertion of replicative stress through the p38-MK2 signaling pathway and secondly by the induction of mitotic failure and mitotic catastrophe. The number of centrosomes in our experiments was affected in an unexpected way: In response to CFI-400945 treatment, we observed amplification of centrosomes – an effect that cannot be recapitulated by PLK4 knockdown. This strongly indicates an off-target effect executed by CFI-400945. As it is mechanistically conceivable, supported by literature and suggested by further experiments, we propose that it is Aurora kinase B inhibition that contributes to the effects of CFI-400945. The additional Aurora kinase B inhibition by CFI-400945 would also explain the synergistic cell lethality that we observed with combined CFI-400945 and MK2 inhibitor treatment. Although replication fork progression was rescued partly by the additional MK2 inhibitor treatment, the cell proliferation was reduced even more than with single CFI-400945 treatment. We propose that this can be explained by the combination of enhanced DNA replication and ablation of cell cycle checkpoints. Combined targeting of checkpoints governing DNA replication and mitosis might prove to represent a viable strategy of eliminating cancer cells.
Keywords: replicative stress; mitotic failure; CFI-400945; PLK4; Polo-like kinase 4; MK2; AURKB; mitotic catastrophe; targeted therapy; cancer; MAPKAP2