CDK9 activity is critical for maintaining MDM4 overexpression in tumor cells

Abstract

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.

Fig. 1. Pan-specific CDK inhibitors roscovitine and flavopiridol inhibit the growth of melanoma cell lines expressing high MDM4 levels.

a Colony formation assay: A375 and MEL-JUSO cells were treated with 20 µM roscovitine or 500 nM flavopiridol for 24 h, washed, and further cultivated in a drug-free growth medium. Cells were fixed and stained with 6.0 % glutaraldehyde + 0.5 % crystal violet solution. b Activation of p53 by roscovitine and flavopiridol: A375 and MEL-JUSO cells stably transfected with a p53 transcriptional activity luciferase reporter construct were treated with the indicated concentrations of roscovitine and flavopiridol for 24 h, lysed, and luciferase activity in lysates was determined. Relative p53 transcriptional activity was calculated. The values represent the mean ± SD; N = 3; *P < 0.05. c Roscovitine and flavopiridol downregulate MDM4 at lower concentrations than MDM2, concurrent MDM4 and MDM2 downregulation leads to stabilization of p53. Western blot analysis of protein lysates of A375 and MEL-JUSO cells after 24-h treatment with CDK inhibitors. The levels of PCNA protein served as a loading control.

Fig. 2. Pan-CDK inhibitors predominantly target MDM4 expression in melanoma and breast carcinoma cells.

Palbociclib, lacking activity toward CDK9, affects neither MDM4 nor MDM2 levels. Western blot analysis of cancer cell lysates after 24-h treatment with the indicated concentrations of CDK inhibitors in A375 (a) and MCF7 cells (b).

Fig. 3. CDK9 inhibition diminishes MDM4 expression and activates p53 in melanoma cells.

a Activation of p53-dependent transcription by CDK9 inhibitor atuveciclib and CDK1 inhibitor RO3306. A375 cells expressing luciferase under the control of a p53-responsive promoter were treated with specific CDK inhibitors for 24 h. Relative p53 activity in cell lysates was determined. The values represent the mean ± SD; N = 3; *P < 0.05; **P < 0.01. b Inhibitors of transcriptional CDKs THZ1, THZ531, and atuveciclib inhibit MDM4 expression and stabilize p53, with minimal impact on MDM2 levels. Western blot analysis of A375 cell lysates after 24-h treatment with the indicated concentrations of specific CDK inhibitors. c Time-course experiment with CDK9 inhibitor atuveciclib. A375 cells were treated with atuveciclib for 1, 3, 6, and 16 h. Proteins of interest were visualized by western blotting. d CDK9 knockdown downregulates MDM4 levels. Western blot analysis of A375 response to siRNA-mediated CDK9 knockdown. Commercially available non-targeting siRNAs (control siRNA 1) and GFP-targeting siRNAs (control siRNA 2 and 3) were used as controls. e CDK9 PROTAC THAL-SNS-032. The dual affinity molecule binds E3 ubiquitin ligase CRBN and induces selective degradation of the CDK9 protein. f Selective knockdown of CDK9 by THAL-SNS-032 induces a decrease in MDM4 levels. Western blot analysis of A375 cell lysates after 1, 6, and 24-h treatment with the indicated concentrations of THAL-SNS-032.

Fig. 4. Inhibition of P-TEFb-mediated transcription contributes to MDM4 depletion.

a MDM4 gene expression analysis by qRT-PCR. Six-hour treatment with dinaciclib and atuveciclib was used to inhibit CDK9-dependent transcription in A375 cells. Total RNA was isolated using RNA Blue reagent, reverse transcribed, and real-time quantitative PCR was performed in triplicates. The values represent the mean ± SD; N = 4; *P < 0.05. b mNET-Seq analysis of RNA polymerase II position along the MDM4 gene. Raji B cells harboring an analog sensitive CDK9 mutation were treated with adenine analog 1-NA-PP1 (CDK9as inhibited) or DMSO (control), and RNA polymerase II occupancy within the MDM4 gene region between control and CDK9-inhibited cells was compared. c Western blot analysis of MDM4, MDM2, p53, and p21 protein levels in A375 cell lysates after 24-hour treatment with the indicated concentrations of CDK9 inhibitor atuveciclib. PCNA served as a loading control. d Expression analysis of p53 target genes p21 and PUMA by qRT-PCR. A375 cells were treated with CDK9 inhibitor atuveciclib for 14 h before harvesting. Total RNA was isolated using RNA Blue reagent, reverse transcribed, and real-time quantitative PCR was performed in triplicates. The values represent the mean ± SD; N = 3; *P < 0.05; **P < 0.01.

Fig. 5. Atuveciclib acts synergistically with nutlin-3a in melanoma.

a Activation of p53 by atuveciclib combined with nutlin-3a treatment. To avoid a complete transcriptional block and thus interference with the luciferase reporter system, A375 cells were pretreated with atuveciclib for 16 h, washed, and treated with nutlin-3a for 10 h. DMSO pretreatment and treatment was used for control = DMSO + DMSO; ctrl = untreated control. The values are relative to the DMSO + nutlin-3a sample and represent the mean ± SD; N = 3; *P < 0.05; ***P < 0.001. b Western blot analysis of cell lysates after 24-h co-treatment with atuveciclib and nutlin-3a. c Effect of the drug combination on melanoma cell viability. Propidium iodide viability assay was performed after a 48-h co-treatment with atuveciclib and nutlin-3a in A375 and MEL-JUSO cells. The values represent the mean ± SD; N = 3; **P < 0.01; ***P < 0.001.

Fig. 6. CDK9 controls MDM4 levels in human pluripotent stem cells.

a CDK9 inhibition leads to MDM4 downregulation in hESCs. Western blot analysis of CCTL14 and CCTL12 cell lysates after 24-h treatment with selected CDK inhibitors. b Selective knockdown of CDK9 by THAL-SNS-032 PROTAC promotes MDM4 depletion in hESCs. Western blot analysis of CCTL14 lysates after 16-h THAL-SNS-032 treatment. c MDM4/MDM2 knockdown does not affect p53 levels in hESCs. Western blot and p53 transcriptional activity analysis in hESCs upon siRNA-mediated knockdown of MDM2 and MDM4. DNA-damaging agent doxorubicin served as a positive control. The p53 activity values are relative to control and error bars represent the SD; N = 3; double asterisk denotes P < 0.01.