ABSTRACT
Aberrant transcription in cancer cells involves the silencing of tumor suppressor genes (TSGs) and activation of oncogenes. Transcriptomic changes are associated with epigenomic alterations such as DNA-hypermethylation, histone deacetylation, and chromatin condensation in promoter regions of silenced TSGs. To discover novel drugs that trigger TSG reactivation in cancer cells, we used a GFP-reporter system whose expression is silenced by promoter DNA hypermethylation and histone deacetylation. After screening a natural product drug library, we identified that toyocamycin, an adenosine-analog, induces potent GFP reactivation and loss of clonogenicity in human colon cancer cells. Connectivity-mapping analysis revealed that toyocamycin produces a pharmacological signature mimicking cyclin-dependent kinase (CDK) inhibitors. RNA-sequencing revealed that the toyocamycin transcriptomic signature resembles that of a specific CDK9 inhibitor (HH1). Specific inhibition of RNA Pol II phosphorylation level and kinase assays confirmed that toyocamycin specifically inhibits CDK9 (IC50 = 79 nM) with a greater efficacy than other CDKs (IC50 values between 0.67 and 15 µM). Molecular docking showed that toyocamycin efficiently binds the CDK9 catalytic site in a conformation that differs from other CDKs, explained by the binding contribution of specific amino acids within the catalytic pocket and protein backbone. Altogether, we demonstrated that toyocamycin exhibits specific CDK9 inhibition in cancer cells, highlighting its potential for cancer chemotherapy.
ABSTRACT
PURPOSE: Embryonal rhabdomyosarcoma (eRMS) is the most common type of rhabdomyosarcoma in children. eRMS is characterized by malignant skeletal muscle cells driven by hyperactivation of several oncogenic pathways including the MYC pathway. Targeting MYC in cancer has been extremely challenging. Recently, we have demonstrated that the heart failure drug, proscillaridin A, produced anticancer effects with specificity toward MYC expressing leukemia cells. We also reported that decitabine, a hypomethylating drug, synergizes with proscillaridin A in colon cancer cells. Here, we investigated whether proscillaridin A exhibits epigenetic and anticancer activity against eRMS RD cells, overexpressing MYC oncogene, and its combination with decitabine. METHODS: We investigated the anticancer effects of proscillaridin A in eRMS RD cells in vitro. In response to drug treatment, we measured growth inhibition, cell cycle arrest, loss of clonogenicity and self-renewal capacity. We further evaluated the impact of proscillaridin A on MYC expression and its downstream transcriptomic effects by RNA sequencing. Then, we measured protein expression of epigenetic regulators and their associated chromatin post-translational modifications in response to drug treatment. Chromatin immunoprecipitation sequencing data sets were coupled with transcriptomic results to pinpoint the impact of proscillaridin A on gene pathways associated with specific chromatin modifications. Lastly, we evaluated the effect of the combination of proscillaridin A and the DNA demethylating drug decitabine on eRMS RD cell growth and clonogenic potential. RESULTS: Clinically relevant concentration of proscillaridin A (5 nM) produced growth inhibition, cell cycle arrest and loss of clonogenicity in eRMS RD cells. Proscillaridin A produced a significant downregulation of MYC protein expression and inhibition of oncogenic transcriptional programs controlled by MYC, involved in cell replication. Interestingly, significant reduction in total histone 3 acetylation and on specific lysine residues (lysine 9, 14, 18, and 27 on histone 3) was associated with significant protein downregulation of a series of lysine acetyltransferases (KAT3A, KAT3B, KAT2A, KAT2B, and KAT5). In addition, proscillaridin A produced synergistic growth inhibition and loss of clonogenicity when combined with the approved DNA demethylating drug decitabine. CONCLUSION: Proscillaridin A produces anticancer and epigenetic effects in the low nanomolar range and its combination with decitabine warrants further investigation for the treatment of eRMS.
