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

Lu H, Xue Y, Xue Y, Yu GK, Arias C, Lin J, Fong S, Faure M, Weisburd B, Ji X, Mercier A, Sutton J, Luo K, Gao Z, Zhou Q - Elife (2015)

Bottom Line: Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing.While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment.Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9's activity and resistance to inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

ABSTRACT
CDK9 is the kinase subunit of positive transcription elongation factor b (P-TEFb) that enables RNA polymerase (Pol) II's transition from promoter-proximal pausing to productive elongation. Although considerable interest exists in CDK9 as a therapeutic target, little progress has been made due to lack of highly selective inhibitors. Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing. While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment. Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9's activity and resistance to inhibition. Because the i-CDK9-induced MYC expression and binding to P-TEFb compensate for P-TEFb's loss of activity, only simultaneously inhibiting CDK9 and MYC/BRD4 can efficiently induce growth arrest and apoptosis of cancer cells, suggesting the potential of a combinatorial treatment strategy.

No MeSH data available.


Related in: MedlinePlus

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 cells treated with the indicated drugs and their concentrations were analyzed by immunoblotting, with quantification of the pSer2 signals shown at the bottom. (D and E) Nuclear extracts (NE) of HeLa-based cells expressing MYC-F and untreated (−) or treated with i-CDK9 or DMSO were subjected to anti-Flag immunoprecipitation. The immunoprecipitates (IP) were examined by immunoblotting for the indicated proteins. (F, G, H, and I) HeLa (F and H) and H1792 (G and I) cells were incubated with JQ1 or i-CDK9 alone or together at various concentrations. The concentrations of each drug (IC50), either used as a single agent or in combination, that caused 50% of cells to show growth inhibition in Celltiter-Glo assay (F and G) or produce Caspase 3/7 (H and I) were plotted using the isobologram method. The dotted lines denote the IC50 values of i-CDK9 and JQ1 had the effects of the two compounds been simply additive. (J and K) HeLa cells were treated with the indicated concentrations of i-CDK9 plus JQ1 (J) or i-CDK9 plus 10,058-F4 (K) and measured by flow cytometry for propidium iodide (PI)-stained sub-G1 population. The error bars represent mean ± SD from three independent measurements.DOI:http://dx.doi.org/10.7554/eLife.06535.021
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fig7: 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 cells treated with the indicated drugs and their concentrations were analyzed by immunoblotting, with quantification of the pSer2 signals shown at the bottom. (D and E) Nuclear extracts (NE) of HeLa-based cells expressing MYC-F and untreated (−) or treated with i-CDK9 or DMSO were subjected to anti-Flag immunoprecipitation. The immunoprecipitates (IP) were examined by immunoblotting for the indicated proteins. (F, G, H, and I) HeLa (F and H) and H1792 (G and I) cells were incubated with JQ1 or i-CDK9 alone or together at various concentrations. The concentrations of each drug (IC50), either used as a single agent or in combination, that caused 50% of cells to show growth inhibition in Celltiter-Glo assay (F and G) or produce Caspase 3/7 (H and I) were plotted using the isobologram method. The dotted lines denote the IC50 values of i-CDK9 and JQ1 had the effects of the two compounds been simply additive. (J and K) HeLa cells were treated with the indicated concentrations of i-CDK9 plus JQ1 (J) or i-CDK9 plus 10,058-F4 (K) and measured by flow cytometry for propidium iodide (PI)-stained sub-G1 population. The error bars represent mean ± SD from three independent measurements.DOI:http://dx.doi.org/10.7554/eLife.06535.021

Mentions: Having identified the mechanism of MYC induction by i-CDK9, we also wanted to determine the biological significance of this phenomenon. Given that MYC induction was triggered by inhibition of cellular CDK9 kinase, we hypothesized that MYC may normally facilitate CDK9's function and that the elevated MYC expression in i-CDK9-treated cells is therefore a cellular attempt to compensate for the loss of CDK9. To test this hypothesis, we first determined whether the shRNA-mediated MYC KD would affect the global pSer2 levels before and after the treatment with i-CDK9. Indeed, even though the KD was incomplete, it markedly reduced pSer2 levels in both untreated cells as well as in cells exposed to a range of i-CDK9 concentrations (Figure 7A), revealing a requirement of MYC for CDK9 to phosphorylate the Pol II CTD under both normal and inhibitory conditions.10.7554/eLife.06535.021Figure 7.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.


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

Lu H, Xue Y, Xue Y, Yu GK, Arias C, Lin J, Fong S, Faure M, Weisburd B, Ji X, Mercier A, Sutton J, Luo K, Gao Z, Zhou Q - Elife (2015)

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 cells treated with the indicated drugs and their concentrations were analyzed by immunoblotting, with quantification of the pSer2 signals shown at the bottom. (D and E) Nuclear extracts (NE) of HeLa-based cells expressing MYC-F and untreated (−) or treated with i-CDK9 or DMSO were subjected to anti-Flag immunoprecipitation. The immunoprecipitates (IP) were examined by immunoblotting for the indicated proteins. (F, G, H, and I) HeLa (F and H) and H1792 (G and I) cells were incubated with JQ1 or i-CDK9 alone or together at various concentrations. The concentrations of each drug (IC50), either used as a single agent or in combination, that caused 50% of cells to show growth inhibition in Celltiter-Glo assay (F and G) or produce Caspase 3/7 (H and I) were plotted using the isobologram method. The dotted lines denote the IC50 values of i-CDK9 and JQ1 had the effects of the two compounds been simply additive. (J and K) HeLa cells were treated with the indicated concentrations of i-CDK9 plus JQ1 (J) or i-CDK9 plus 10,058-F4 (K) and measured by flow cytometry for propidium iodide (PI)-stained sub-G1 population. The error bars represent mean ± SD from three independent measurements.DOI:http://dx.doi.org/10.7554/eLife.06535.021
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Related In: Results  -  Collection

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fig7: 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 cells treated with the indicated drugs and their concentrations were analyzed by immunoblotting, with quantification of the pSer2 signals shown at the bottom. (D and E) Nuclear extracts (NE) of HeLa-based cells expressing MYC-F and untreated (−) or treated with i-CDK9 or DMSO were subjected to anti-Flag immunoprecipitation. The immunoprecipitates (IP) were examined by immunoblotting for the indicated proteins. (F, G, H, and I) HeLa (F and H) and H1792 (G and I) cells were incubated with JQ1 or i-CDK9 alone or together at various concentrations. The concentrations of each drug (IC50), either used as a single agent or in combination, that caused 50% of cells to show growth inhibition in Celltiter-Glo assay (F and G) or produce Caspase 3/7 (H and I) were plotted using the isobologram method. The dotted lines denote the IC50 values of i-CDK9 and JQ1 had the effects of the two compounds been simply additive. (J and K) HeLa cells were treated with the indicated concentrations of i-CDK9 plus JQ1 (J) or i-CDK9 plus 10,058-F4 (K) and measured by flow cytometry for propidium iodide (PI)-stained sub-G1 population. The error bars represent mean ± SD from three independent measurements.DOI:http://dx.doi.org/10.7554/eLife.06535.021
Mentions: Having identified the mechanism of MYC induction by i-CDK9, we also wanted to determine the biological significance of this phenomenon. Given that MYC induction was triggered by inhibition of cellular CDK9 kinase, we hypothesized that MYC may normally facilitate CDK9's function and that the elevated MYC expression in i-CDK9-treated cells is therefore a cellular attempt to compensate for the loss of CDK9. To test this hypothesis, we first determined whether the shRNA-mediated MYC KD would affect the global pSer2 levels before and after the treatment with i-CDK9. Indeed, even though the KD was incomplete, it markedly reduced pSer2 levels in both untreated cells as well as in cells exposed to a range of i-CDK9 concentrations (Figure 7A), revealing a requirement of MYC for CDK9 to phosphorylate the Pol II CTD under both normal and inhibitory conditions.10.7554/eLife.06535.021Figure 7.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.

Bottom Line: Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing.While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment.Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9's activity and resistance to inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

ABSTRACT
CDK9 is the kinase subunit of positive transcription elongation factor b (P-TEFb) that enables RNA polymerase (Pol) II's transition from promoter-proximal pausing to productive elongation. Although considerable interest exists in CDK9 as a therapeutic target, little progress has been made due to lack of highly selective inhibitors. Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing. While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment. Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9's activity and resistance to inhibition. Because the i-CDK9-induced MYC expression and binding to P-TEFb compensate for P-TEFb's loss of activity, only simultaneously inhibiting CDK9 and MYC/BRD4 can efficiently induce growth arrest and apoptosis of cancer cells, suggesting the potential of a combinatorial treatment strategy.

No MeSH data available.


Related in: MedlinePlus