Abstract
Tumour oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state1, but direct pharmacological inhibition of transcription factors has so far proven difficult2. However, the transcriptional machinery contains various enzymatic cofactors that can be targeted for the development of new therapeutic candidates3, including cyclin-dependent kinases (CDKs)4. Here we present the discovery and characterization of a covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell-line profiling indicates that a subset of cancer cell lines, including human T-cell acute lymphoblastic leukaemia (T-ALL), have exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL cells shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and the key role of RUNX1 in the core transcriptional regulatory circuitry of these tumour cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumour types that are dependent on transcription for maintenance of the oncogenic state.
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Acknowledgements
We thank members of the Gray and Young laboratories for helpful discussions; D. Orlando, L. Lawton and L. Anders for advice; and C. Thoreen and D. Sabatini, as well as S. Cheng and G. Morin, for reagents. We thank K. Jones and N. Kohl for performing mouse studies and K. Jones and C. Christensen for prepping mouse tissues. We thank S. Riddle for performing LanthaScreen Eu Kinase Assays. This work was supported by the National Institutes of Health (R01 CA130876-04 and U54 HG006097-02 to N.S.G.; CA178860-01 and P01 NS047572-10 to J.A.M.; HG002668 and CA109901 to R.A.Y.), and the American Cancer Society Postdoctoral Fellowship 120272-PF-11-042-01-DMC (P.B.R.). J.R.B. is a Scholar in Clinical Research of the Leukemia Lymphoma Society and is supported by the Translational Research Program of the Leukemia Lymphoma Society and by the American Cancer Society.
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Authors and Affiliations
Contributions
N.S.G., N.K. and T.Z. conceived the project. N.S.G. and T.Z. conceived and directed the chemical synthesis of THZ1 and its analogues with input from T.Si. T.Z. performed chemical synthesis and small-molecule structure determination. R.A.Y., N.S.G., P.B.R. and N.K. conceived genomics studies. N.K., P.B.R., J.R.B., C.H.B., N.S.G. and R.A.Y. designed biological experimental research with input from J.R., B.J.A., D.M., T.Sa., T.L., A.P.W. and C.S.M. N.K., P.B.R., J.R., A.D., B.T., C.E.J. and N.M.H performed experimental biological research. S.B.F. designed and performed protein mass spectrometry on THZ1/CDK7 adducts with input from J.A.M. S.R. and A.A. performed elastic net regression analysis. N.D.K. performed molecular modelling studies with input from T.Sa. B.J.A. designed and performed genomics data analyses. N.K., N.S.G. and R.A.Y. co-wrote the paper. All authors edited the manuscript.
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N.S.G., T.Z. and N.K. are inventors on a patent application covering THZ1, which is licensed to a company co-founded by N.S.G. and R.A.Y.
Extended data figures and tables
Extended Data Figure 1 THZ1 demonstrates time-dependent inhibition of CDK7 in vitro and covalent binding of intracellular CDK7.
a, THZ1 but not THZ1-R shows time-dependent inhibition. LanthaScreen Eu Kinase Binding Assay was conducted at Life Technologies in a time-dependent manner (20, 60 and 180 min), demonstrating that THZ1 but not THZ1-R shows time-dependent inhibition of CDK7. b, c, Pre-incubation of THZ1 increases CDK7 inhibitory activity in vitro. Recombinant CAK complex was incubated with THZ1 (b) or THZ1-R (c) in a dose–response format with or without pre-incubation before ATP (25 μM) addition. The kinase reaction was then allowed to proceed for 45 min at 30 °C. d, Workflow of bio-THZ1 pull-down competition experiment. e, Bio-THZ1 pulls down CDK7 from cellular lysates. Loucy cellular lysates were incubated with bio-THZ1 (1 μM) with or without THZ1 (10 μM) and streptavidin-precipitated proteins were probed for CDK7. IB, immunoblot. f, Free intracellular THZ1 competes in a dose-dependent manner for bio-THZ1 binding to CDK7. Loucy cells were treated with increasing concentrations of THZ1 or with 10 μM THZ1-R for 4 h. Cellular lysates were incubated with bio-THZ1 and processed as indicated in a. g, Bio-THZ1 labels CDK7 in lysates. Loucy cellular lysates were incubated with bio-THZ1 at 4 °C for 12 h followed by immunoprecipitation of CDK7 at 4 °C for 3 h. Precipitated proteins were washed and probed with horseradish peroxidase (HRP)-conjugated streptavidin.
Extended Data Figure 2 THZ1 covalently binds CDK7 C312.
a, b, Total ion chromatograms (TIC) and extracted ion chromatograms (XIC) for CDK7 peptides recorded during analysis of CAK complexes treated with DMSO (a) or THZ1 (b). c, Efficiency of labelling was estimated to be approximately 85%, as gauged by the reduction in signal of triply and quadruply charged YFSNRPGPTPGCQLPRPNCPVETLK ions (residues 294–318). The peptides VPFLPGDSDLDQLTR (residues 180–194) and LDFLGEGQFATVYK (residues 15–28) were used for normalization. d, Orbitrap HCD tandem mass spectrometry (MS/MS) spectrum of a quadruply charged CDK7-derived peptide (residues 294–318) labelled by THZ1 at C312. Fragment ions containing the peptide C terminus (y-type) or N terminus (b-type), along with the associated mass errors are shown in red and blue, respectively. Fragment ions marked by an asterisk contain the inhibitor and have the expected heavy isotope contribution from chlorine. The site of labelling was determined to be C312 (as opposed to C305) on the basis of fragment ions observed in additional MS/MS spectra (for example, y113+ observed with <3 p.p.m. mass error by fragmentation of the +6 charged precursor; see inset mass spectrum). e, C312S mutation eliminates THZ1 covalent binding. Cellular lysates from HCT116 cells expressing either Flag–CDK7 wild type or C312S were incubated with bio-THZ1 for 12 h at 4 °C and then at room temperature for 3 h to facilitate covalent binding. Precipitated proteins were then probed for the presence of Flag-tagged CDK7.
Extended Data Figure 3 THZ1 inhibits CDK12 but at higher concentrations compared with CDK7.
a, Protein sequence alignment of the C-terminal regions of all human (hs) CDKs and mouse (m) CDK7 using Uniprot default settings. Note that the canonical cell-cycle CDKs 1, 2 and 4, as well as 5, do not have C-terminal domains that extent to the equivalent position of CDK7 C312 and therefore do not show aligned sequence in this region. b, Bio-THZ1 covalently pulls down CDK7 from cellular lysates. Jurkat cellular lysates were incubated with bio-THZ1 (1 μM) at 4 °C for 12 h and for 2 h at room temperature. Precipitated proteins were washed with or without urea (4 M), here used as a denaturing agent, and probed for the indicated CDKs. c, Bio-THZ1 pulls down Flag–CDK12 from lysates. Lysates from 293A cells stably expressing Flag-tagged wild-type CDK12 were incubated with bio-THZ1 (1 μM) at 4 °C for 12 h and for 2 h at room temperature. Immunoprecipitated proteins were probed with Flag antibody to recognize CDK12 or with CDK7 antibody. d, Bio-THZ1 pulls down cyclin K from cellular lysates. Jurkat cellular lysates were incubated with bio-THZ1 (1 μM) at 4 °C for 12 h and for 2 h at room temperature. Precipitated proteins were probed for the indicated proteins. e, THZ1 inhibits CDK12 in an in vitro kinase assay. 293A cells stably expressing Flag-tagged wild-type CDK12 were treated with THZ1 or THZ1-R for 4 h. Exogenous CDK12 was immunoprecipitated from cellular lysates using Flag antibody. Precipitated proteins were washed and subjected to in vitro kinase assays at 30 °C for 30 min using the large subunit of RNAPII (RPB1) as substrate and 25 μM ATP. CS, Coomassie stain. f, Quantification of in vitro kinase assay conducted in d.
Extended Data Figure 4 THZ1 irreversibly inhibits RNAPII CTD and CAK phosphorylation.
a, THZ1 exhibits time-dependent inactivation of intracellular CDK7. Loucy cells were treated with THZ1 or THZ1-R for 0–4 h. At each time point, cells were harvested, lysed and the cellular lysates were probed with antibodies against the specified proteins. b, THZ1 inhibits RNAPII CTD phosphorylation. Loucy cells were treated with THZ1 or THZ1-R for 4 h. Cellular lysates were then probed with antibodies recognizing the Ser 2, Ser 5 and Ser 7 CTD RNAPII phospho-epitopes. c, Loucy cells were treated with THZ1 or THZ1-R for 4 h followed by washout of inhibitor-containing medium. Cells were allowed to grow in medium without inhibitor for 0–6 h. At each time point cells were lysed and the cellular lysates were probed with antibodies against the specified proteins. ‘N’ indicates cells for which medium was never washed out. d, Apoptotic signalling is maintained despite washout of THZ1. Loucy cells were treated with THZ1 or THZ1-R for 4 h followed by washout of inhibitor-containing medium, at which point cells were allowed to grow in medium with or without inhibitor for 0–48 h. At each time point, cells were lysed and the cellular lysates were probed with antibodies against the specified proteins. e, Antiproliferative effects of THZ1 are impervious to inhibitor washout. Loucy cells were treated with THZ1 or THZ1-R in a dose–response format for 72 h. Antiproliferative effects were determined using CellTiter-Glo analysis. f, THZ1 reduces the T-loop phosphorylation status of CDK1 and CDK2 in Jurkat cells over a 3 h exposure. Asynchronous cells were treated with increasing concentrations of THZ1 or THZ1-R for 3 h. Cellular lysates were then probed with antibodies against the indicated proteins or phosphoproteins. g, THZ1, but not THZ1-R, completely inhibits T-loop phosphorylation of CDK1 and CDK2 after treatment over one cell cycle. Loucy cells were treated with THZ1, THZ1-R, Flavopiridol or DMSO vehicle at the indicated concentrations for 24 and 14 h, respectively (roughly one cell cycle). Cell lysates were harvested and probed with antibodies against the specified proteins or phosphoproteins. h, HeLa S3 cells stably expressing Flag-tagged wild-type CDK7 were treated with THZ1 (1 µM) or DMSO vehicle for 5 h with or without the presence of doxycycline. Proteins were immunoprecipitated using Flag antibody. Precipitated proteins were probed using the indicated antibodies. Asterisk indicates heavy chain from IgG antibody.
Extended Data Figure 5 Mutation of CDK7 C312 to serine rescues Ser 5/7 and partially rescues Ser 2 RNAPII CTD phosphorylation.
a, Expression of C312S rescues Ser 5/7 and partially rescues Ser 2 RNAPII CTD phosphorylation. HeLa S3 cells stably carrying a doxycycline-inducible Flag–CDK7 C312S construct were treated with THZ1 or DMSO for 5 h with or without the presence of doxycycline. Cellular lysates were then probed for the indicated proteins. b, Phenotypic rescue is specific to the C312S mutation, as rescue is not achieved with overexpression of Flag–CDK7 wild type (WT). HeLa S3 cells stably carrying doxycycline-inducible Flag–CDK7 wild-type and C312S constructs (or empty vector) were treated with THZ1 or DMSO for 5 h in the presence of doxycycline. c, Expression of C312S largely restores CDK1/2 T-loop phosphorylation. HeLa S3 cells stably carrying a doxycycline-inducible Flag–CDK7 C312S construct were treated with THZ1 or DMSO for 5 h with or without the presence of doxycycline. Cellular lysates were then probed for the indicated proteins or phosphoproteins. d, Overexpression of Flag–CDK7 C312 rescues the expression of a subset of transcripts in HeLa S3 cells. Log2 fold change in gene expression in HeLa S3 cells expressing Flag–CDK7 wild type (x-axis) and Flag–CDK7 C312S (y-axis) after a 4 h treatment with 500 nM THZ1. e, GO molecular function analysis of transcripts increased by 1 log2 order or more after expression of Flag–CDK7 C312S compared with Flag–CDK7 wild type in the presence of 500 nM THZ1.
Extended Data Figure 6 THZ1 potently disrupts T-ALL proliferation.
a, THZ1, but not THZ1-R, exhibits strong antiproliferative effects against T-ALL cell lines. Cells were treated with THZ1, THZ1-R or DMSO vehicle for 72 h and assessed for antiproliferative effect by CellTiter Glo analysis. Error bars show ± s.d. b, THZ1 causes cell-cycle arrest. Jurkat (top) and Loucy (bottom) T-ALL cells were treated with THZ1 for the indicated time periods. Cell-cycle progression was assessed using FACS cell-cycle analysis. 2N = G1, 4N = G2. c, Treatment with THZ1 decreases CDK1/2 T-loop phosphorylation. Jurkat cells were incubated with THZ1 for the indicated duration of time and lysates were probed for the specified proteins.
Extended Data Figure 7 Treatment with THZ1 induces apoptosis in T-ALL cells.
a, Representative annexin V and propidium iodide stainings for Jurkat cells incubated with THZ1 for the indicated amount of time and harvested to determine the percentage of apoptotic and/or dead cells by annexin V and propidium iodide staining, respectively. The percentage of cells in each cell population is shown in the four quadrants. b, Treatment with THZ1 induces apoptosis. Quantification of annexin V and propidium iodide staining data from a. Experiments were performed in biological triplicates. Error bars show ± s.d. c, Representative annexin V and propidium iodide stainings for Loucy cells incubated with THZ1 for the indicated amount of time and harvested to determine the percentage of apoptotic and/or dead cells by annexin V and propidium iodide staining, respectively. The percentage of cells in each cell population is shown in the four quadrants. d, Treatment with THZ1 induces apoptosis. Quantification of annexin V and propidium iodide staining data from c. Experiments were performed in biological triplicates. Error bars show ± s.d. e, f, Sustained treatment with THZ1 induces apoptosis coincident with loss of RNAPII CTD phosphorylation and a reduction in anti-apoptotic proteins. Jurkat (e) and Loucy (f) cells were incubated with THZ1 for the indicated duration of time and lysates were probed for the specified proteins. Apoptosis was monitored by PARP cleavage.
Extended Data Figure 8 THZ1 demonstrates potent killing of primary chronic lymphocytic leukaemia cells and antiproliferative activity against primary T-ALL cells and in vivo against a human T-ALL xenograft.
a, Patient-derived chronic lymphocytic leukaemia (CLL) samples were obtained and cultured in vitro for 24 hours in the presence of escalating doses of the specified compounds (n = 10 samples, 1 technical replicate per condition, per sample). Cell death upon compound exposure was evaluated using FITC Annexin V Apoptosis Kit I (BD Biosciences) and 1x104 events were collected and analysed using a BD FACSCanto II flow cytometer. Results shown are mean normalized percentage death based on Annexin V and propidium iodide single- and double-positive cells (± s.d.) normalized to baseline death in the vehicle (DMSO) control condition of each sample. Compounds tested were THZ1, THZ1-R and Flavopiridol (THZ1 versus THZ1-R P = 1.5 × 10−38; THZ1 versus Flavopiridol P = 0.05). P values were generated using an ANOVA model. b, Patient-derived xenografts (patient IDs 3255-1, M18-1-5 and D135-1-5; n = 3) were treated with THZ1 for 3 h followed by compound washout. An aliquot of input cells was then counted by flow cytometry using a known quantity of flow cytometry calibration beads (data not shown; Molecular Probes). The remaining cells were plated onto MS5-DL1 feeder cells in the presence of serum-free media (supplemented with 0.75 µM SR1, 10 ng ml−1 interleukin (IL)-7, 10 ng ml−1 IL-2). Seventy-two hours later, cultures were harvested by vigorous pipetting with Trypsin, filtered through nylon mesh to deplete feeders, and counted by flow cytometry using a known quantity of flow cytometry calibration beads and with gating to discriminate between T-ALL cells and carryover feeders. The final cell number was normalized to the input cell number to calculate fold expansion. This experiment was performed once per patient-derived sample. c, Bioluminescent images of two representative mice treated with either vehicle control, 10 mg kg−1 THZ1 once daily (qD), or 10 mg kg−1 THZ1 twice daily (BID) for the indicated number of days. d, Spleen tissue from mice treated with THZ1 shows decreased RNAPII CTD phosphorylation. Mice were treated with THZ1 10 mg kg−1 once daily or twice daily or vehicle control. The animals were killed and spleen tissues were isolated. Lysates prepared from homogenized spleen tissue were probed for RNAPII CTD phosphoepitopes. e, THZ1 binds directly to CDK7 in mouse tissues. Mice were treated with THZ1 10 mg kg−1 once daily or twice daily or vehicle control. The animals were killed and spleen tissues were isolated. Lysates prepared from homogenized spleen tissue were incubated with bio-THZ1 for 12 h at 4 °C and 2 h at room temperature to induce covalent bond formation. Proteins pulled down were then probed for the presence of CDK7. f, Body weights of mice treated with either vehicle control, 10 mg kg−1 THZ1 once daily, or 10 mg kg−1 THZ1 twice daily over the duration of the drug treatment.
Extended Data Figure 9 THZ1 inhibits RNAPII CTD phosphorylation and causes cell-cycle arrest in non-transformed cell lines.
a, b, THZ1 inhibits RNAPII CTD phosphorylation. RPE-1 (a) and BJ fibroblasts (b) were treated with THZ1 or THZ1-R for 4 h. Cellular lysates were then probed with antibodies against the indicated proteins. c, d, THZ1 causes cell-cycle arrest in non-transformed cells. RPE-1 (c) and BJ fibroblast (d) cells were treated with THZ1, Flavopiridol, Staurosporine or DMSO vehicle for the indicated time periods. Cell-cycle progression was analysed after permeabilization and staining with propidium iodide. e, f, THZ1 inhibits proliferation of non-transformed cell lines. RPE-1 (e) and BJ fibroblast (f) cells were treated with THZ1, THZ1-R, Flavopiridol or Staurosporine for 72 h and antiproliferative effect were determined by CellTiter Glo. Error bars show ± s.d.
Extended Data Figure 10 High-dose THZ1 reduces global steady-state mRNA levels, but low-dose THZ1 preferentially downregulates components of the TAL1/RUNX1/GATA3 transcriptional circuit.
a, THZ1, but not THZ1-R, causes global downregulation of steady-state mRNA levels. Jurkat cells were treated with THZ1 (250 nM) or THZ1-R (250 nM) for 4 h. Total RNA was isolated and ERCC spike-in controls were added relative to cell number and analysed using Affymetrix PrimeView microarrays. Heatmaps displaying the log2 fold change in gene expression versus DMSO for 22,310 genes expressed in DMSO conditions at 6 h in THZ1 or THZ1-R. b, Total H3K27ac ChIP-seq signal (length × density) in enhancer regions for all stitched enhancers in Jurkat. Enhancers are ranked by increasing H3K27ac ChIP-seq signal. c, d, Gene tracks of H3K27ac (top), CDK7 (middle) and RNAPII (bottom) ChIP-seq occupancy at the TSS, gene body and enhancer regions of TAL1 (c) and MYB (d). e, THZ1 downregulates mRNA transcripts of the TAL1/RUNX1/GATA3 transcriptional circuitry. Quantitative polymerase chain reaction with reverse transcription (RT–qPCR) expression analysis in Jurkat cells of transcripts identified as downregulated after THZ1 treatment. qPCR was carried out using Taqman probes according to the manufacturer’s protocol. All experiments shown were performed in biological triplicate with each individual biological sample qPCR amplified in technical triplicate. Expression was normalized to ACTB, and fold change in expression was calculated relative to DMSO. Error bars show ± s.d. f, THZ1 treatment reduces the protein levels of TAL1/RUNX1/GATA3 transcriptional circuitry. Jurkat cells treated with THZ1 for the indicated time points were probed for the specified proteins.
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Kwiatkowski, N., Zhang, T., Rahl, P. et al. Targeting transcription regulation in cancer with a covalent CDK7 inhibitor. Nature 511, 616–620 (2014). https://doi.org/10.1038/nature13393
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DOI: https://doi.org/10.1038/nature13393
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