Characterization of molecular and cellular functions of the cyclin-dependent kinase CDK9 using a novel specific inhibitor

Abstract

Background and purpose: The cyclin-dependent kinase CDK9 is an important therapeutic target but currently available inhibitors exhibit low specificity and/or narrow therapeutic windows. Here we have used a new highly specific CDK9 inhibitor, LDC000067 to interrogate gene control mechanisms mediated by CDK9.

Experimental approach: The selectivity of LDC000067 was established in functional kinase assays. Functions of CDK9 in gene expression were assessed with in vitro transcription experiments, single gene analyses and genome-wide expression profiling. Cultures of mouse embryonic stem cells, HeLa cells, several cancer cell lines, along with cells from patients with acute myelogenous leukaemia were also used to investigate cellular responses to LDC000067.

Key results: The selectivity of LDC000067 for CDK9 over other CDKs exceeded that of the known inhibitors flavopiridol and DRB. LDC000067 inhibited in vitro transcription in an ATP-competitive and dose-dependent manner. Gene expression profiling of cells treated with LDC000067 demonstrated a selective reduction of short-lived mRNAs, including important regulators of proliferation and apoptosis. Analysis of de novo RNA synthesis suggested a wide ranging positive role of CDK9. At the molecular and cellular level, LDC000067 reproduced effects characteristic of CDK9 inhibition such as enhanced pausing of RNA polymerase II on genes and, most importantly, induction of apoptosis in cancer cells.

Conclusions and implications: Our study provides a framework for the mechanistic understanding of cellular responses to CDK9 inhibition. LDC000067 represents a promising lead for the development of clinically useful, highly specific CDK9 inhibitors.

Keywords: CDK9; RNA polymerase II; cyclin-dependent kinase; gene expression; transcription.

Figure 1

Inhibition of in vitro transcription by LDC067. (A) Molecular structure of LDC000067. (B) Inhibition of kinase catalytic activity by 10 μM LDC067. A radiometric in vitro kinase assay was used to determine residual kinase activity (expressed as percentage of remaining substrate phosphorylation compared to the DMSO control reaction). Each kinase was measured in duplicate and data shown are means and range of the two measurements. The stippled line indicates residual P-TEFb activity (6.2%) in this assay. (C and D) Influence of inhibitors on in vitro transcription using HEK293T nuclear extract. Transcripts originate from a 380 bp G-free cassette in pGal-ML. Transcript (Tx) levels were determined by phosphorimaging. (E) Comparison of the effects of LDC067 in different nuclear extracts of the indicated cell lines under low (60 μM) and high (500 μM) ATP concentrations. Final inhibitor concentration was 10 μM. (F) Influence of magnesium on transcription inhibition by LDC067 (10 μM) with low ATP (60 μM) in HEK293T nuclear extracts.

Figure 2

Cellular effects of CDK9 inhibition by LDC067. (A) Western blot analysis of global Ser2-P levels in mESCs treated for 90 or 180 min with DMSO, 10 μM LDC067 or 1 μM flavopiridol (FP). Ser2-P signals were quantified using ImageJ (http://rsbweb.nih.gov/ij/) and normalized to RNAPII signals. (B) Effects of CDK9 inhibitor treatment (60 min) in HeLa cells. THAP1 served as loading control. Quantification of CTD modifications was carried out as in (A). (C) Kinase assay using GST-CTD as substrate in HeLa nuclear extract treated with 10 μM LDC067 or 1 μM flavopiridol for 30 min. GST-CTD Ser2-P levels were quantified using ImageJ and Tubulin signals for normalization. (D) MCF7 cells were treated for 4 h with the indicated concentrations of CDK9 inhibitors and analysed by Western blot with the indicated antibodies. TBP served as loading control. E – H. Apoptosis induction in the indicated cell lines and patient-derived leukaemia (AML) blasts after 24 hours treatment. Apoptosis was assayed using flow cytometry of annexin V-propidium iodide double-stained cells. Signals are represented by the means ± SD (error bars) of three biological replicates, except for AML blasts from a single patient, where three independent technical replicates were analysed. *P < 0.05, significantly different from DMSO or untreated values;Student's t-tests.

Figure 3

RT-qPCR analysis of de novo transcription. (A and B) Increasing concentrations of LDC067 (1/2/4/6/10 μM in A; 2.5/5/10/20/40 μM in B) were applied for 90 min and the indicated genes were analysed in A549 cells (A) or mESCs (B) using exon-intron primers (e-i) and, for the latter, also exon-exon (e-e) primers. Half-maximal inhibition is indicated by the dotted line. (C) RT-qPCR analysis of luciferase reporter gene transcripts in stably transfected HeLa cells treated with 10 μM LDC067 or DMSO for 30 min followed by 1 μg·mL⁻¹ doxycycline (Dox) for 60 min. Scheme of the luciferase (LUC) reporter gene and primer locations are shown at the top. (D) Luciferase assay of Dox-induced HeLa reporter cells treated with increasing amounts of LDC067 for the indicated times.

Figure 4

Microarray analysis of THP1 cells treated with LDC067. (A) Statistics of regulated genes. (B) Gene ontology (GO) analysis of down-regulated mRNAs. (C) RT-qPCR analysis of unspliced de novo transcripts covering exon-intron sequences of the indicated genes in control or LDC067-treated THP1 cells. Genes to the left showed unchanged steady-state mRNA levels, while genes to the right (FOS, MYC, PLK2) were significantly down-regulated in the microarray analysis.

Figure 5

Increase of RNAPII pausing at MYC in the presence of LDC067. (A) Scheme of the human MYC gene with qPCR amplicons used for ChIP analysis indicated underneath. Distribution of RNAPII (B), Ser2-P (C), Ser5-P (D) and Ser7-P (E) was determined by ChIP of HeLa cells treated with 10 μM LDC067 or DMSO for 1 h.