Dovitinib induces mitotic defects and activates the G2 DNA damage checkpoint.

Dovitinib (TKI258; formerly CHIR-258) is an orally bioavailable inhibitor of multiple receptor tyrosine kinases. Interestingly, Dovitinib triggered a G2 /M arrest in cancer cell lines from diverse origins including HeLa, nasopharyngeal carcinoma, and hepatocellular carcinoma. Single-cell analysis revealed that Dovitinib promoted a delay in mitotic exit in a subset of cells, causing the cells to undergo mitotic slippage. Higher concentrations of Dovitinib induced a G2 arrest similar to the G2 DNA damage checkpoint. In support of this, DNA damage was triggered by Dovitinib as revealed by γ-H2AX and comet assays. The mitotic kinase CDK1 was found to be inactivated by phosphorylation in the presence of Dovitinib. Furthermore, the G2 arrest could be overcome by abrogation of the G2 DNA damage checkpoint using small molecule inhibitors of CHK1 and WEE1. Finally, Dovitinib-mediated G2 cell cycle arrest and subsequent cell death could be promoted after DNA damage repair was disrupted by inhibitors of poly(ADP-ribose) polymerases. These results are consistent with the recent finding that Dovitinib can also target topoisomerases. Collectively, these results suggest additional directions for use of Dovitinib, in particular with agents that target the DNA damage checkpoint.

Pharmacological inactivation of CHK1 and WEE1 induces mitotic catastrophe in nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is a rare but highly invasive cancer. As radiotherapy is the primary treatment for NPC, this offers a rationale to investigate if uncoupling the DNA damage responses can sensitize this cancer type. The G2 DNA damage checkpoint is controlled by a cascade of protein kinases: ATM/ATR, which phosphorylates CHK1/CHK2, which in turn phosphorylates WEE1. A number of small molecule inhibitors have been developed against these kinases as potential therapeutic agents. Here we demonstrated that compare to that in immortalized nasopharyngeal epithelial cells, ATR, CHK1, and WEE1 were overexpressed in NPC cell lines. Inhibitors of these kinases were unable to promote extensive mitotic catastrophe in ionizing radiation-treated NPC cells, indicating that they are not very effective radiosensitizer for this cancer. In the absence of prior irradiation, however, mitotic catastrophe could be induced with inhibitors against CHK1 (AZD7762) or WEE1 (MK-1775). NPC cells were more sensitive to WEE1 inactivation than nasopharyngeal epithelial cells. Targeting CHK1 and WEE1 together induced more extensive mitotic catastrophe than the individual components alone. Taken together, our results show that NPC cells depend on CHK1 and WEE1 activity for growth and that inhibitors of these kinases may serve as potential therapeutics for NPC.

Co-inhibition of polo-like kinase 1 and Aurora kinases promotes mitotic catastrophe.

Mitosis is choreographed by a number of protein kinases including polo-like kinases and Aurora kinases. As these kinases are frequently dysregulated in cancers, small-molecule inhibitors have been developed for targeted anticancer therapies. Given that PLK1 and Aurora kinases possess both unique functions as well as co-regulate multiple mitotic events, whether pharmacological inhibition of these kinases together can enhance mitotic catastrophe remains an outstanding issue to be determined. Using concentrations of inhibitors that did not induce severe mitotic defects on their own, we found that both the metaphase arrest and mitotic slippage induced by inhibitors targeting Aurora A and Aurora B (MK-5108 and Barasertib respectively) were enhanced by a PLK1 inhibitor (BI 2536). We found that PLK1 is overexpressed in cells from nasopharyngeal carcinoma, a highly invasive cancer with poor prognosis, in comparison to normal nasopharyngeal epithelial cells. Nasopharyngeal carcinoma cells were more sensitive to BI 2536 as a single agent and co-inhibition with Aurora kinases than normal cells. These observations underscore the mechanism and potential benefits of targeting PLK1 and Aurora kinases to induce mitotic catastrophe in cancer cells.

Pharmacological targeting the ATR-CHK1-WEE1 axis involves balancing cell growth stimulation and apoptosis.

The ATR-CHK1-WEE1 kinase cascade's functions in the DNA damage checkpoints are well established. Moreover, its roles in the unperturbed cell cycle are also increasingly being recognized. In this connection, a number of small-molecule inhibitors of ATR, CHK1, and WEE1 are being evaluated in clinical trials. Understanding precisely how cells respond to different concentrations of inhibitors is therefore of paramount importance and has broad clinical implications. Here we present evidence that in the absence of DNA damage, pharmacological inactivation of ATR was less effective in inducing mitotic catastrophe than inhibition of WEE1 and CHK1. Small-molecule inhibitors of CHK1 (AZD7762) or WEE1 (MK-1775) induced mitotic catastrophe, as characterized by dephosphorylation of CDK1(Tyr15), phosphorylation of histone H39(Ser10), and apoptosis. Unexpectedly, partial inhibition of WEE1 and CHK1 had the opposite effect of accelerating the cell cycle without inducing apoptosis, thereby increasing the overall cell proliferation. This was also corroborated by the finding that cell proliferation was enhanced by kinase-inactive versions of WEE1. We demonstrated that these potential limitations of the inhibitors could be overcome by targeting more than one components of the ATR-CHK1-WEE1 simultaneously. These observations reveal insights into the complex responses to pharmacological inactivation of the ATR-CHK1-WEE1 axis.

PARP1 is overexpressed in nasopharyngeal carcinoma and its inhibition enhances radiotherapy.

Nasopharyngeal carcinoma is a rare but highly invasive cancer. As options of agents for effective combination chemoradiotherapy for advanced nasopharyngeal carcinoma are limited, novel therapeutic approaches are desperately needed. The ubiquitin ligase CHFR is known to target PARP1 for degradation and is epigenetically inactivated in nasopharyngeal carcinoma. We present evidence that PARP1 protein is indeed overexpressed in nasopharyngeal carcinoma cells in comparison with immortalized normal nasopharyngeal epithelial cells. Tissue microarray analysis also indicated that PARP1 protein is significantly elevated in primary nasopharyngeal carcinoma tissues, with strong correlation with all stages of nasopharyngeal carcinoma development. We found that the PARP inhibitor AZD2281 (olaparib) increased DNA damage, cell-cycle arrest, and apoptosis in nasopharyngeal carcinoma cells challenged with ionizing radiation or temozolomide. Isobologram analysis confirmed that the cytotoxicity triggered by AZD2281 and DNA-damaging agents was synergistic. Finally, AZD2281 also enhanced the tumor-inhibitory effects of ionizing radiation in animal xenograft models. These observations implicate that PARP1 overexpression is an early event in nasopharyngeal carcinoma development and provide a molecular basis of using PARP inhibitors to potentiate treatment of nasopharyngeal carcinoma with radio- and chemotherapy.

Novel functions of the phosphatase SHP2 in the DNA replication and damage checkpoints.

Replication stress- and DNA damage-induced cell cycle checkpoints are critical for maintaining genome stability. To identify protein phosphatases involved in the activation and maintenance of the checkpoints, we have carried out RNA interference-based screens with a human phosphatome shRNA library. Several phosphatases, including SHP2 (also called PTPN11) were found to be required for cell survival upon hydroxyurea-induced replicative stress in HeLa cells. More detailed studies revealed that SHP2 was also important for the maintenance of the checkpoint after DNA damage induced by cisplatin or ionizing radiation in HeLa cells. Furthermore, SHP2 was activated after replicative stress and DNA damage. Although depletion of SHP2 resulted in a delay in cyclin E accumulation and an extension of G(1) phase, these cell cycle impairments were not responsible for the increase in apoptosis after DNA damage. Depletion of SHP2 impaired CHK1 activation, checkpoint-mediated cell cycle arrest, and DNA repair. These effects could be rescued with a shRNA-resistant SHP2. These results underscore the importance of protein phosphatases in checkpoint control and revealed a novel link between SHP2 and cell cycle checkpoints.

Tetraploidization increases sensitivity to Aurora B kinase inhibition.

Aurora kinases are overexpressed in many cancers and are targets for anticancer drugs. The yeast homolog of Aurora B kinase, IPL1, was found to be a ploidy-specific lethality gene. Given that polyploidization is a common feature of many cancers, we hypothesized polyploidization also sensitizes mammalian cells to inhibition of Aurora kinases. Using two models of apparent diploid vs. tetraploid cell lines (one based on the hepatocellular carcinoma cell line Hep3B and another on untransformed mouse fibroblasts), we found that tetraploid cells were more sensitive to Aurora B inhibition than their diploid counterparts. Apoptosis could be induced in tetraploid cells by two different Aurora B inhibitors. Furthermore, tetraploid cells were sensitive to Aurora B inhibition but were not affected by Aurora A inhibition. Interestingly, the underlying mechanism was due to mitotic slippage and the subsequent excessive genome reduplication. In support of this, abolition of cytokinesis with dihydrocytochalasin B resulted in similar effects on tetraploid cells as Aurora B inhibition. These results indicate that inhibition of Aurora B or cytokinesis can promote apoptosis effectively in polyploid cancer cells.