The identification of the essential role of cyclin-dependent kinases (CDKs) in the control of cell division has prompted the development of small-molecule CDK inhibitors as anticancer drugs. For many of these compounds, the precise mechanism of action in individual tumor types remains unclear as they simultaneously target different classes of CDKs - enzymes controlling the cell cycle progression as well as CDKs involved in the regulation of transcription. CDK inhibitors are also capable of activating p53 tumor suppressor in tumor cells retaining wild-type p53 gene by modulating MDM2 levels and activity. In the current study, we link, for the first time, CDK activity to the overexpression of the MDM4 (MDMX) oncogene in cancer cells. Small-molecule drugs targeting the CDK9 kinase, dinaciclib, flavopiridol, roscovitine, AT-7519, SNS-032, and DRB, diminished MDM4 levels and activated p53 in A375 melanoma and MCF7 breast carcinoma cells with only a limited effect on MDM2. These results suggest that MDM4, rather than MDM2, could be the primary transcriptional target of pharmacological CDK inhibitors in the p53 pathway. CDK9 inhibitor atuveciclib downregulated MDM4 and enhanced p53 activity induced by nutlin-3a, an inhibitor of p53-MDM2 interaction, and synergized with nutlin-3a in killing A375 melanoma cells. Furthermore, we found that human pluripotent stem cell lines express significant levels of MDM4, which are also maintained by CDK9 activity. In summary, we show that CDK9 activity is essential for the maintenance of high levels of MDM4 in human cells, and drugs targeting CDK9 might restore p53 tumor suppressor function in malignancies overexpressing MDM4.

• MeSH
• Cyclin-Dependent Kinase 9 antagonists & inhibitors   metabolism   MeSH
• Transcription, Genetic MeSH
• Imidazoles pharmacology   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Humans MeSH
• Melanoma genetics   metabolism   pathology   MeSH
• MCF-7 Cells MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Breast Neoplasms genetics   metabolism   pathology   MeSH
• Piperazines pharmacology   MeSH
• Pluripotent Stem Cells metabolism   MeSH
• Cell Cycle Proteins biosynthesis   genetics   metabolism   MeSH
• Proto-Oncogene Proteins c-mdm2 biosynthesis   genetics   metabolism   MeSH
• Proto-Oncogene Proteins biosynthesis   genetics   metabolism   MeSH
• Roscovitine pharmacology   MeSH
• Sulfonamides pharmacology   MeSH
• Drug Synergism MeSH
• Transfection MeSH
• Triazines pharmacology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• atuveciclib MeSH Browser
• CDK9 protein, human MeSH Browser
• Cdk9 protein, mouse MeSH Browser
• Cyclin-Dependent Kinase 9 MeSH
• Imidazoles MeSH
• Protein Kinase Inhibitors MeSH
• MDM2 protein, human MeSH Browser
• Mdm2 protein, mouse MeSH Browser
• MDM4 protein, human MeSH Browser
• Mdm4 protein, mouse MeSH Browser
• nutlin 3 MeSH Browser
• Piperazines MeSH
• Cell Cycle Proteins MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH
• Proto-Oncogene Proteins MeSH
• Roscovitine MeSH
• Sulfonamides MeSH
• Triazines MeSH

Tumor suppressor p53 is mutated in about 50% of cancers. Most malignant melanomas carry wild-type p53, but p53 activity is often inhibited due to overexpression of its negative regulators Mdm2 or MdmX. We performed high throughput screening of 2448 compounds on A375 cells carrying p53 activity luciferase reporter construct to reveal compounds that promote p53 activity in melanoma. Albendazole and fenbendazole, two approved and commonly used benzimidazole anthelmintics, stimulated p53 activity and were selected for further studies. The protein levels of p53 and p21 increased upon the treatment with albendazole and fenbendazole, indicating activation of the p53-p21 pathway, while the levels of Mdm2 and MdmX decreased in melanoma and breast cancer cells overexpressing these proteins. We also observed a reduction of cell viability and changes of cellular morphology corresponding to mitotic catastrophe, i.e., G2/M cell cycle arrest of large multinucleated cells with disrupted microtubules. In summary, we established a new tool for testing the impact of small molecule compounds on the activity of p53 and used it to identify the action of benzimidazoles in melanoma cells. The drugs promoted the stability and transcriptional activity of wild-type p53 via downregulation of its negative regulators Mdm2 and MdmX in cells overexpressing these proteins. The results indicate the potential for repurposing the benzimidazole anthelmintics for the treatment of cancers overexpressing p53 negative regulators.

• Keywords
• Mdm2, MdmX, benzimidazoles, drug repurposing, melanoma, p53,
• MeSH
• Albendazole pharmacology   MeSH
• Benzimidazoles pharmacology   MeSH
• Down-Regulation MeSH
• Fenbendazole pharmacology   MeSH
• Nuclear Proteins metabolism   MeSH
• Humans MeSH
• Melanoma drug therapy   metabolism   MeSH
• MCF-7 Cells MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 metabolism   MeSH
• Drug Repositioning MeSH
• Cell Proliferation drug effects   MeSH
• Cell Cycle Proteins MeSH
• Proto-Oncogene Proteins c-mdm2 metabolism   MeSH
• Proto-Oncogene Proteins metabolism   MeSH
• Gene Expression Regulation, Neoplastic drug effects   MeSH
• High-Throughput Screening Assays MeSH
• Drug Screening Assays, Antitumor MeSH
• Cell Survival drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Albendazole MeSH
• Benzimidazoles MeSH
• Fenbendazole MeSH
• Nuclear Proteins MeSH
• MDM2 protein, human MeSH Browser
• MDM4 protein, human MeSH Browser
• Tumor Suppressor Protein p53 MeSH
• Cell Cycle Proteins MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH
• Proto-Oncogene Proteins MeSH
• TP53 protein, human MeSH Browser

MdmX overexpression contributes to the development of cancer by inhibiting tumor suppressor p53. A switch in the alternative splicing of MdmX transcript, leading to the inclusion of exon 6, has been identified as the primary mechanism responsible for increased MdmX protein levels in human cancers, including melanoma. However, there are no approved drugs, which could translate these new findings into clinical applications. We analyzed the anti-melanoma activity of enoxacin, a fluoroquinolone antibiotic inhibiting the growth of some human cancers in vitro and in vivo by promoting miRNA maturation. We found that enoxacin inhibited the growth and viability of human melanoma cell lines much stronger than a structurally related fluoroquinolone ofloxacin, which only weakly modulates miRNA processing. A microarray analysis identified a set of miRNAs significantly dysregulated in enoxacin-treated A375 melanoma cells. They had the potential to target multiple signaling pathways required for cancer cell growth, among them the RNA splicing. Recent studies showed that interfering with cellular splicing machinery can result in MdmX downregulation in cancer cells. We, therefore, hypothesized that enoxacin could, by modulating miRNAs targeting splicing machinery, activate p53 in melanoma cells overexpressing MdmX. We found that enoxacin and ciprofloxacin, a related fluoroquinolone capable of promoting microRNA processing, but not ofloxacin, strongly activated wild type p53-dependent transcription in A375 melanoma without causing significant DNA damage. On the molecular level, the drugs promoted MdmX exon 6 skipping, leading to a dose-dependent downregulation of MdmX. Not only in melanoma, but also in MCF7 breast carcinoma and A2780 ovarian carcinoma cells overexpressing MdmX. Together, our results suggest that some clinically approved fluoroquinolones could potentially be repurposed as activators of p53 tumor suppressor in cancers overexpressing MdmX oncoprotein and that p53 activation might contribute to the previously reported activity of enoxacin towards human cancer cells.

• MeSH
• Alternative Splicing drug effects   MeSH
• Apoptosis drug effects   MeSH
• Down-Regulation drug effects   MeSH
• Enoxacin pharmacology   MeSH
• Humans MeSH
• Melanoma genetics   metabolism   pathology   MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Breast Neoplasms genetics   metabolism   pathology   MeSH
• Ovarian Neoplasms genetics   metabolism   pathology   MeSH
• Ofloxacin pharmacology   MeSH
• DNA Damage drug effects   MeSH
• Cell Proliferation drug effects   MeSH
• Proto-Oncogene Proteins c-mdm2 genetics   metabolism   MeSH
• Signal Transduction drug effects   MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Enoxacin MeSH
• Tumor Suppressor Protein p53 MeSH
• Ofloxacin MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH