Mitotic inhibitors such as the PLK-1 inhibitor volasertib are promising candidates for cancer therapy. Failure of cell cycle regulation in tumors often follows the loss of RB-E2F and DREAM, two central cell cycle transcriptional repressors. Their repressive function depends on the two proteins RB and LIN37, respectively. LIN37 is part of DREAM and required for its repressor function. Without RB and LIN37/DREAM, cells express cell cycle genes largely independent of the cell cycle phase.

HCT116 WT, HCT116 RB-/-, HCT116 LIN37-/- and HCT116 RB-/- LIN37-/- cells were treated with 1 µM volasertib over a time course of 48 h. Samples were taken every 2 h (until 28 h) and every 4 h (until 48 h). The cells were analyzed for their cell cycle phases via flow cytometry. To assess drug resistance, cells were cultivated with 1 µM volasertib for 14 days. Cells were manually counted from microscopic images taken during the treatment period. Phosphorylation of H3Ser28 was utilized as a marker for mitosis to distinguish G2 and M phases in volasertib-treated cells by flow cytometry.

Flow cytometric cell cycle phase analyses revealed that HCT116 WT cells entered a sustained G2/M arrest after exposure to 1 µM volasertib. Loss of LIN37/DREAM promoted the escape from volasertib-induced arrest and allowed cells to enter endoreplication. Loss of RB alone showed no substantial effect on the volasertib-induced G2/M arrest, however, in combination with DREAM deficiency, it further promoted the escape and endoreplication. Since endoreplication is a known mechanism for drug resistance in cancer, we next analyzed cell survival over a 14-day treatment period. Concordant to the enhanced endoreplication in DREAM-deficient cells, these cells showed an increased rate of survival. The additional loss of RB further supported survival. Resistance to mitotic inhibitors and subsequent endoreplication can emerge from two processes: mitotic slippage or mitotic skipping. To characterize whether LIN37/DREAM deficiency promotes resistance via mitotic slippage or mitotic skipping, we tested volasertib-treated cells for histone H3 phosphorylation at Ser28, which served as a marker for mitosis. After 20 h of volasertib exposure, a substantial population of sensitive HCT116 WT cells were in mitosis. In contrast, resistant HCT116 RB-/- LIN37-/- cells largely remained in G2, suggesting that the resistance relies on the process of mitotic skipping.

Our work reveals that loss of LIN37/DREAM promotes resistance to mitotic inhibition by allowing endoreplication. Loss of RB alone does not confer resistance but can improve it in LIN37/DREAM-deficient cells. Further, we show that resistance arises from mitotic skipping, a process that prevents cells from entering mitosis and allows them to progress to the next cell cycle round, ultimately resulting in polyploidy.