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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

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 detectable Pol II signals in HeLa cells as revealed by ChIP-seq analysis. (B and C) GSEA results for the 27 BDPR genes at 2 hr (B) and 8 hr (C) post CDK9 inhibition. NES: Normalized Enrichment Score; FDR: False Discovery Rate. (D) Distribution of Pol II-bound genes with a given TR as determined by ChIP-seq. The genes are grouped by the indicated gene types and treatment conditions. The top panel compares the 23 BDPR genes to the remaining Pol II-bound genes in the genome, and the bottom compares the BDPR genes to 23 randomly selected genes. (E, F, G) Occupancy of Pol II across three representative BDPR genes as revealed by ChIP-seq. The read coverage is shown for the entire gene plus a margin on either side equal to 7% of the gene length.DOI:http://dx.doi.org/10.7554/eLife.06535.020
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fig6: 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 detectable Pol II signals in HeLa cells as revealed by ChIP-seq analysis. (B and C) GSEA results for the 27 BDPR genes at 2 hr (B) and 8 hr (C) post CDK9 inhibition. NES: Normalized Enrichment Score; FDR: False Discovery Rate. (D) Distribution of Pol II-bound genes with a given TR as determined by ChIP-seq. The genes are grouped by the indicated gene types and treatment conditions. The top panel compares the 23 BDPR genes to the remaining Pol II-bound genes in the genome, and the bottom compares the BDPR genes to 23 randomly selected genes. (E, F, G) Occupancy of Pol II across three representative BDPR genes as revealed by ChIP-seq. The read coverage is shown for the entire gene plus a margin on either side equal to 7% of the gene length.DOI:http://dx.doi.org/10.7554/eLife.06535.020

Mentions: MYC is a classic example of the so-called PRGs, which are a set of genes that can be induced in response to both extracellular and intracellular signals without the requirement for de novo protein synthesis (Fowler et al., 2011). Given the above demonstrations that BRD4 and its interaction with P-TEFb played an essential role in i-CDK9-induced MYC expression, we next investigated whether other BRD4-dependent primary response (BDPR) genes such as those identified in bone marrow-derived macrophages (Figure 6A; [Hargreaves et al., 2009]) might behave similarly as MYC in their response to i-CDK9. To this end, we performed gene set enrichment analysis (GSEA) of expression microarray data obtained from HeLa cells treated with either DMSO or i-CDK9 for 2 and 8 hr (Figure 6B,C). At the 2-hr time point, GSEA reveals a marked i-CDK9-induced reduction of expression (normalized enrichment score or NES = −2.28) for essentially all the 27 genes within the gene set (Figure 6A,B). However, at the 8-hr time point, the trend was completely reversed with virtually all the genes displaying significant up-regulation by i-CDK9 (Figure 6C; NES = +2.26). Thus, the biphasic transcriptional signature of the curated BDPR genes is the same as that of MYC in their response to i-CDK9.10.7554/eLife.06535.020Figure 6.i-CDK9 affects the expression of other BRD4-dependent primary response genes similarly as it does to MYC.


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)

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 detectable Pol II signals in HeLa cells as revealed by ChIP-seq analysis. (B and C) GSEA results for the 27 BDPR genes at 2 hr (B) and 8 hr (C) post CDK9 inhibition. NES: Normalized Enrichment Score; FDR: False Discovery Rate. (D) Distribution of Pol II-bound genes with a given TR as determined by ChIP-seq. The genes are grouped by the indicated gene types and treatment conditions. The top panel compares the 23 BDPR genes to the remaining Pol II-bound genes in the genome, and the bottom compares the BDPR genes to 23 randomly selected genes. (E, F, G) Occupancy of Pol II across three representative BDPR genes as revealed by ChIP-seq. The read coverage is shown for the entire gene plus a margin on either side equal to 7% of the gene length.DOI:http://dx.doi.org/10.7554/eLife.06535.020
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4490784&req=5

fig6: 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 detectable Pol II signals in HeLa cells as revealed by ChIP-seq analysis. (B and C) GSEA results for the 27 BDPR genes at 2 hr (B) and 8 hr (C) post CDK9 inhibition. NES: Normalized Enrichment Score; FDR: False Discovery Rate. (D) Distribution of Pol II-bound genes with a given TR as determined by ChIP-seq. The genes are grouped by the indicated gene types and treatment conditions. The top panel compares the 23 BDPR genes to the remaining Pol II-bound genes in the genome, and the bottom compares the BDPR genes to 23 randomly selected genes. (E, F, G) Occupancy of Pol II across three representative BDPR genes as revealed by ChIP-seq. The read coverage is shown for the entire gene plus a margin on either side equal to 7% of the gene length.DOI:http://dx.doi.org/10.7554/eLife.06535.020
Mentions: MYC is a classic example of the so-called PRGs, which are a set of genes that can be induced in response to both extracellular and intracellular signals without the requirement for de novo protein synthesis (Fowler et al., 2011). Given the above demonstrations that BRD4 and its interaction with P-TEFb played an essential role in i-CDK9-induced MYC expression, we next investigated whether other BRD4-dependent primary response (BDPR) genes such as those identified in bone marrow-derived macrophages (Figure 6A; [Hargreaves et al., 2009]) might behave similarly as MYC in their response to i-CDK9. To this end, we performed gene set enrichment analysis (GSEA) of expression microarray data obtained from HeLa cells treated with either DMSO or i-CDK9 for 2 and 8 hr (Figure 6B,C). At the 2-hr time point, GSEA reveals a marked i-CDK9-induced reduction of expression (normalized enrichment score or NES = −2.28) for essentially all the 27 genes within the gene set (Figure 6A,B). However, at the 8-hr time point, the trend was completely reversed with virtually all the genes displaying significant up-regulation by i-CDK9 (Figure 6C; NES = +2.26). Thus, the biphasic transcriptional signature of the curated BDPR genes is the same as that of MYC in their response to i-CDK9.10.7554/eLife.06535.020Figure 6.i-CDK9 affects the expression of other BRD4-dependent primary response genes similarly as it does to MYC.

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