Compensatory induction of MYC expression by sustained CDK9 inhibition via a BRD4-dependent mechanism

  1. Huasong Lu
  2. Yuhua Xue
  3. Guoying K Yu
  4. Carolina Arias
  5. Julie Lin
  6. Susan Fong
  7. Michel Faure
  8. Ben Weisburd
  9. Xiaodan Ji
  10. Alexandre Mercier
  11. James Sutton
  12. Kunxin Luo
  13. Zhenhai Gao  Is a corresponding author
  14. Qiang Zhou  Is a corresponding author
  1. University of California, Berkeley, United States
  2. Xiamen University, China
  3. Novartis Institute for BioMedical Research, United States
9 figures

Figures

Figure 1 with 1 supplement
i-CDK9 is a potent and selective CDK9 inhibitor that elicits cellular responses indicative of P-TEFb inhibition.

(A and B) Structures and selectivity profiles of i-CDK9 (A) and falvopiridol (B). The numbers refer to the concentrations (µM) of the two compounds that resulted in 50% inhibition of the enzymatic …

https://doi.org/10.7554/eLife.06535.003
Figure 1—source data 1

Selectivity profile of i-CDK9.

https://doi.org/10.7554/eLife.06535.004
Figure 1—figure supplement 1
Recombinant CDK12-CycK is less sensitive to inhibition by i-CDK9.
https://doi.org/10.7554/eLife.06535.005
i-CDK9 causes widespread promoter-proximal pausing by Pol II and the biggest decrease in expression for genes involved in regulation of transcription and RNA metabolic process.

(A) Schematic diagram illustrating calculation of the Pol II traveling ratio (TR). (B) Distribution of Pol II-bound genes with a given TR as determined by ChIP-seq under the various conditions as …

https://doi.org/10.7554/eLife.06535.006
Figure 3 with 2 supplements
Induction of MYC mRNA production in response to sustained inhibition of CDK9 by i-CDK9 and the requirement of MYC's natural genomic structure in this process.

(A) The indicated tumor cell lines were treated with i-CDK9 (0.5 μM) for 2–16 hr and the MYC mRNA levels, which were divided by those in the DMSO-treated cells and averaged from three independent …

https://doi.org/10.7554/eLife.06535.007
Figure 3—figure supplement 1
Biphasic response of MYC mRNA production throughout the course of CDK9 inhibition by i-CDK9.
https://doi.org/10.7554/eLife.06535.008
Figure 3—Figure supplement 2
HEXIM1 expression is continuously suppressed throughout the entire course of i-CDK9 treatment of five different tumor cell lines.
https://doi.org/10.7554/eLife.06535.009
Figure 4 with 5 supplements
Activation of MYC transcription by i-CDK9 depends on induced transfer of kinase-active P-TEFb from 7SK snRNP to BRD4, binding of the BRD4-P-TEFb complex to acetylated MYC chromatin template, and BRD4-mediated increase in CDK9's catalytic activity and resistance to inhibition.

(A) The HeLa-based F1C2 cells stably expressing CDK9-F were incubated with the indicated concentrations of i-CDK9. Nuclear extracts (NE) and the anti-CDK9-F immunoprecipitates (IP) derived from NE …

https://doi.org/10.7554/eLife.06535.010
Figure 4—figure supplement 1
i-CDK9 (0.3 μM) induces disruption of 7SK snRNP at a time point much earlier than that required to cause about 50% reduction in global pSer2.
https://doi.org/10.7554/eLife.06535.011
Figure 4—figure supplement 2
JQ1 decreases associations of both BRD4 and CDK9 with the MYC locus.
https://doi.org/10.7554/eLife.06535.012
Figure 4—figure supplement 3
JQ1 blocks the i-CDK9-induced MYC expression in H1792 and A2058 cells.
https://doi.org/10.7554/eLife.06535.013
Figure 4—figure supplement 4
MYC induction by 0.3 μM i-CDK9 can be subsequently shut off by 2 μM of the drug.
https://doi.org/10.7554/eLife.06535.014
Figure 4—figure supplement 5
Examination of the purity and concentrations of WT and ∆PID BRD4 used in the CDK9 kinase assay.
https://doi.org/10.7554/eLife.06535.015
Figure 5 with 3 supplements
Treatment with i-CDK9 (0.3 μM for 8 hr) increases the levels of P-TEFb, BRD4, total Pol II, Pol II with pSer2 CTD and acetyl-H3/H4 at the MYC locus.

(A) Genomic structure of the MYC locus. Arrows indicate the positions and direction of the four MYC promoters P0 to P3. The small horizontal bars labeled with letters A to K mark the positions of 11 …

https://doi.org/10.7554/eLife.06535.016
Figure 5—figure supplement 1
The i-CDK9-induced increase in CDK9's binding to the MYC locus is mostly BRD4-dependent.
https://doi.org/10.7554/eLife.06535.017
Figure 5—figure supplement 2
Treatment with i-CDK9 (0.3 μM for 8 hr) decreases the levels of both total Pol II and Pol II with pSer2 CTD at the HEXIM1 locus.
https://doi.org/10.7554/eLife.06535.018
Figure 5—figure supplement 3
i-CDK9 does not affect the cellular levels of acetylated histones H3 and H4.
https://doi.org/10.7554/eLife.06535.019
i-CDK9 affects the expression of other BRD4-dependent primary response genes similarly as it does to MYC.

(A) The list of 27 curated BRD4-dependent primary response (BDPR) genes identified in bone marrow-derived macrophages is displayed in alphabetical order. The 23 genes in bold face type had …

https://doi.org/10.7554/eLife.06535.020
Simultaneous inhibition of CDK9 and MYC synergistically induces growth arrest and apoptosis of cancer cells due to the fact that MYC facilitates P-TEFb phosphorylation of Pol II CTD and increases binding to BRD4-P-TEFb upon CDK9 inhibition.

(A) Lysates of HeLa cells expressing the indicated shRNA and exposed to increasing concentrations of i-CDK9 were analyzed by immunoblotting for the indicated proteins. (B and C) Lysates of HeLa …

https://doi.org/10.7554/eLife.06535.021

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