Journal: bioRxiv

Article Title: Sensitizing tumor response to topoisomerase I antibody drug conjugate by selective CDK7 inhibition

doi: 10.1101/2025.11.23.690049

Figure Lengend Snippet: ( A ) Chemical structure of Q901. ( B ) Assessment of the covalent binding site was conducted via mass spectrometry analysis of the recombinant CDK7-cyclin H-MAT1 (CAK) trimetric complex. The recombinant CAK complex was incubated with Q901 or DMSO for 1 h, digested for 18 h, and analyzed by mass spectrometry. ( C ) KinMap image illustrating the kinase inhibition profile of Q901 against a panel of 410 kinases (397 protein kinase assays and 13 lipid kinase assays). The inhibition profile was determined by measuring the residual activity at 1 μM for 1 h using the PanQinase® Activity Assay. ATP concentration was set at the apparent ATP-Km value for each kinase. A red dot indicates 99% inhibition of CDK7 by Q901 at this concentration. ( D ) Efficacy and selectivity of Q901 against other CDKs at ATP concentrations corresponding to the apparent ATP-Km value for each kinase. Residual activity (%) was measured after a 1 h incubation with Q901 at the indicated concentrations. ( E ) CDK7 target occupancy assay using Bio-QS, a biotinylated analog of Q901. Cell lysates were prepared from A2780 cells treated with Q901 or DMSO for 4 h at the indicated concentrations and subjected to immunoprecipitation (IP) using Bio-QS and streptavidin agarose beads (SA). IP samples and whole-cell lysates were immunoblotted with an anti-CDK7 antibody. ( F ) Washout-based target occupancy assay to measure the duration of CDK7 inhibition. A2780 cells treated with 6 nM Q901 for 4 h were washed with fresh medium and incubated for the indicated times. Cells were lysed, treated with Bio-QS, and immunoprecipitated with streptavidin agarose beads (SA). The percentage of free CDK7 was calculated by normalizing CDK7 levels in IP samples from Q901 treatment to those in IP samples from the DMSO-treated group (n = 3; two-way ANOVA with Tukey’s multiple comparisons test, data represent mean ± SD).

Article Snippet: The following antibodies were used for immunoblotting: total CDK7 (Cell Signaling Technology, Danvers, MA, USA, 2090S, RRID: AB_2077140), MYC (Cell Signaling Technology, 13987, RRID: AB_2631168), E2F1 (Cell Signaling Technology, 3742, RRID: AB_2096936), MYC S62P (Cell Signaling Technology, 13748, RRID: AB_2687518), MYC T58P (Cell Signaling Technology, 46650), Rb S795P (Cell Signaling Technology, 9301, RRID: AB_330013), Rb S780P (Cell Signaling Technology, 9307, RRID: AB_330015), MAX (Cell Signaling Technology, 4739, RRID: AB_2281777), T-H2AX (Santa Cruz, Dallas, TX, USA, sc-517336, RRID: AB_3675923), P-H2AX (Cell Signaling Technology, 2577, RRID: AB_2118010), Ubiquitin (Santa Cruz, sc-8017, RRID: AB_628423), TOP1 (BD Biosciences, San Jose, CA, USA, 556597, RRID: AB_396474), β-actin (Santa Cruz, sc-47778, RRID: AB_626632), Affinity Purified Goat Anti-Rabbit IgG (H+L)-HRP (Bio-Rad, Hercules, CA, USA, 1706515, RRID: AB_11125142), and Affinity Purified Goat Anti-Mouse IgG (H+L)-HRP (Bio-Rad, 1706516, RRID: AB_11125547).

Techniques: Binding Assay, Mass Spectrometry, Recombinant, Incubation, Inhibition, Activity Assay, Concentration Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: Sensitizing tumor response to topoisomerase I antibody drug conjugate by selective CDK7 inhibition

doi: 10.1101/2025.11.23.690049

Figure Lengend Snippet: A ) 1 H NMR spectrum of Q901 was acquired using variable temperature (VT) NMR in DMSO-d□. ( B ) Targeted proteomics analysis to determine the Q901 binding sites on CDK7. The recombinant CAK trimeric complex was incubated with Q901 or DMSO, followed by protease digestion and peptide mapping via LC-MS/MS. Chromatograms show peptide fragments generated by ArgC (Clostripain) digestion (right) and ArgC/Trypsin digestion (left). The expanded boxes highlight the peak of C312 containing peptides, which are reduced following Q901 treatment, indicating covalent modification at this site. ( C ) Representative Western blot images from the pulse-chase assay described in . A2780 cells were treated with 6 nM Q901 for 4 h and then divided into two groups. One group (- wash out) remained in the Q901-containing medium for continuous incubation, while the other group (+ wash out) underwent a drug washout, where the medium was completely removed and replaced with fresh drug-free medium before further incubation for the indicated times. Bio-QS-labeled CDK7 was immunoprecipitated using streptavidin agarose beads (SA), and the levels of free CDK7 were analyzed by immunoblotting. These images in this figure were quantified in .

Article Snippet: The following antibodies were used for immunoblotting: total CDK7 (Cell Signaling Technology, Danvers, MA, USA, 2090S, RRID: AB_2077140), MYC (Cell Signaling Technology, 13987, RRID: AB_2631168), E2F1 (Cell Signaling Technology, 3742, RRID: AB_2096936), MYC S62P (Cell Signaling Technology, 13748, RRID: AB_2687518), MYC T58P (Cell Signaling Technology, 46650), Rb S795P (Cell Signaling Technology, 9301, RRID: AB_330013), Rb S780P (Cell Signaling Technology, 9307, RRID: AB_330015), MAX (Cell Signaling Technology, 4739, RRID: AB_2281777), T-H2AX (Santa Cruz, Dallas, TX, USA, sc-517336, RRID: AB_3675923), P-H2AX (Cell Signaling Technology, 2577, RRID: AB_2118010), Ubiquitin (Santa Cruz, sc-8017, RRID: AB_628423), TOP1 (BD Biosciences, San Jose, CA, USA, 556597, RRID: AB_396474), β-actin (Santa Cruz, sc-47778, RRID: AB_626632), Affinity Purified Goat Anti-Rabbit IgG (H+L)-HRP (Bio-Rad, Hercules, CA, USA, 1706515, RRID: AB_11125142), and Affinity Purified Goat Anti-Mouse IgG (H+L)-HRP (Bio-Rad, 1706516, RRID: AB_11125547).

Techniques: Targeted Proteomics, Binding Assay, Recombinant, Incubation, Liquid Chromatography with Mass Spectroscopy, Generated, Modification, Western Blot, Pulse Chase, Labeling, Immunoprecipitation

Journal: bioRxiv

Article Title: Sensitizing tumor response to topoisomerase I antibody drug conjugate by selective CDK7 inhibition

doi: 10.1101/2025.11.23.690049

Figure Lengend Snippet: ( A ) MCF-7 cells were treated with Q901 at the indicated concentrations for 72, 96, or 120 h. Cell viability was measured using the ATP Lite™ system. Inhibition (%) was plotted against the log-transformed Q901 concentration (µM) (n = 3 to 4). Data represent mean ± SD. ( B and C ) RNAPII ChIP-seq was performed following treatment with 100 nM Q901 for the indicated duration. (B) Volcano plot of pan RNAPII ChIP-seq signals after Q901 treatment (n = 2; blue dot indicates p-value ≤ 0.05 and log2FC ≤ -0.58; red dot indicates p-value ≤ 0.05 and log2FC ≥ 0.58). (C) Average ChIP-seq signal plots of various RNAPII forms for genes downregulated by Q901. ( D ) Average CDK7 ChIP-seq signal plots of downregulated and upregulated genes at the TSS. CDK7 ChIP-seq was performed with 100 nM Q901 for the indicated durations.

Article Snippet: The following antibodies were used for immunoblotting: total CDK7 (Cell Signaling Technology, Danvers, MA, USA, 2090S, RRID: AB_2077140), MYC (Cell Signaling Technology, 13987, RRID: AB_2631168), E2F1 (Cell Signaling Technology, 3742, RRID: AB_2096936), MYC S62P (Cell Signaling Technology, 13748, RRID: AB_2687518), MYC T58P (Cell Signaling Technology, 46650), Rb S795P (Cell Signaling Technology, 9301, RRID: AB_330013), Rb S780P (Cell Signaling Technology, 9307, RRID: AB_330015), MAX (Cell Signaling Technology, 4739, RRID: AB_2281777), T-H2AX (Santa Cruz, Dallas, TX, USA, sc-517336, RRID: AB_3675923), P-H2AX (Cell Signaling Technology, 2577, RRID: AB_2118010), Ubiquitin (Santa Cruz, sc-8017, RRID: AB_628423), TOP1 (BD Biosciences, San Jose, CA, USA, 556597, RRID: AB_396474), β-actin (Santa Cruz, sc-47778, RRID: AB_626632), Affinity Purified Goat Anti-Rabbit IgG (H+L)-HRP (Bio-Rad, Hercules, CA, USA, 1706515, RRID: AB_11125142), and Affinity Purified Goat Anti-Mouse IgG (H+L)-HRP (Bio-Rad, 1706516, RRID: AB_11125547).

Techniques: Inhibition, Transformation Assay, Concentration Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: Sensitizing tumor response to topoisomerase I antibody drug conjugate by selective CDK7 inhibition

doi: 10.1101/2025.11.23.690049

Figure Lengend Snippet: ( A ) The results of SE calling using the ROSE program with H3K27ac ChIP-seq (GSE62229). ( B ) Expression levels of enhancer target genes (pan RNAPII ChIP-seq; n = 2, Q901 1h treatment condition, data represent mean ± SEM). ( C ) Average fastGRO signals of four enhancer groups. ( D ) GO analysis results of target genes regulated by CDK7-bound SE and CDK7-bound TE. ( E ) Track images showing ChIP-seq signals for H3K27ac, CDK7, pan RNAPII, MYC, and E2F1, along with fastGRO, at a representative CDK7-bound SE region (highlighted in yellow) and it associated target genes.

Article Snippet: The following antibodies were used for immunoblotting: total CDK7 (Cell Signaling Technology, Danvers, MA, USA, 2090S, RRID: AB_2077140), MYC (Cell Signaling Technology, 13987, RRID: AB_2631168), E2F1 (Cell Signaling Technology, 3742, RRID: AB_2096936), MYC S62P (Cell Signaling Technology, 13748, RRID: AB_2687518), MYC T58P (Cell Signaling Technology, 46650), Rb S795P (Cell Signaling Technology, 9301, RRID: AB_330013), Rb S780P (Cell Signaling Technology, 9307, RRID: AB_330015), MAX (Cell Signaling Technology, 4739, RRID: AB_2281777), T-H2AX (Santa Cruz, Dallas, TX, USA, sc-517336, RRID: AB_3675923), P-H2AX (Cell Signaling Technology, 2577, RRID: AB_2118010), Ubiquitin (Santa Cruz, sc-8017, RRID: AB_628423), TOP1 (BD Biosciences, San Jose, CA, USA, 556597, RRID: AB_396474), β-actin (Santa Cruz, sc-47778, RRID: AB_626632), Affinity Purified Goat Anti-Rabbit IgG (H+L)-HRP (Bio-Rad, Hercules, CA, USA, 1706515, RRID: AB_11125142), and Affinity Purified Goat Anti-Mouse IgG (H+L)-HRP (Bio-Rad, 1706516, RRID: AB_11125547).

Techniques: ChIP-sequencing, Expressing

Journal: bioRxiv

Article Title: Sensitizing tumor response to topoisomerase I antibody drug conjugate by selective CDK7 inhibition

doi: 10.1101/2025.11.23.690049

Figure Lengend Snippet: ( A ) Average ChIP-seq signal plots of CDK7 and pan RNAPII ChIP-seq across four enhancer groups. ( B ) Average ChIP-seq signal plots for CDK7 and pan RNAPII for protein-coding genes regulated by four enhancer groups. ( C ) Bar graph shows the proportion of downregulated genes in each enhancer groups (Pan RNAPII ChIP-seq; n = 2; p-value ≤ 0.05 and log2FC ≤ -0.58). ( D ) Scatter plot shows the expression levels and log2FC of target genes regulated by CDK7-bound SE and CDK7-bound TE (pan RNAPII ChIP-seq; n = 2; p-value ≤ 0.05).

Article Snippet: The following antibodies were used for immunoblotting: total CDK7 (Cell Signaling Technology, Danvers, MA, USA, 2090S, RRID: AB_2077140), MYC (Cell Signaling Technology, 13987, RRID: AB_2631168), E2F1 (Cell Signaling Technology, 3742, RRID: AB_2096936), MYC S62P (Cell Signaling Technology, 13748, RRID: AB_2687518), MYC T58P (Cell Signaling Technology, 46650), Rb S795P (Cell Signaling Technology, 9301, RRID: AB_330013), Rb S780P (Cell Signaling Technology, 9307, RRID: AB_330015), MAX (Cell Signaling Technology, 4739, RRID: AB_2281777), T-H2AX (Santa Cruz, Dallas, TX, USA, sc-517336, RRID: AB_3675923), P-H2AX (Cell Signaling Technology, 2577, RRID: AB_2118010), Ubiquitin (Santa Cruz, sc-8017, RRID: AB_628423), TOP1 (BD Biosciences, San Jose, CA, USA, 556597, RRID: AB_396474), β-actin (Santa Cruz, sc-47778, RRID: AB_626632), Affinity Purified Goat Anti-Rabbit IgG (H+L)-HRP (Bio-Rad, Hercules, CA, USA, 1706515, RRID: AB_11125142), and Affinity Purified Goat Anti-Mouse IgG (H+L)-HRP (Bio-Rad, 1706516, RRID: AB_11125547).

Techniques: ChIP-sequencing, Expressing

Journal: bioRxiv

Article Title: Sensitizing tumor response to topoisomerase I antibody drug conjugate by selective CDK7 inhibition

doi: 10.1101/2025.11.23.690049

Figure Lengend Snippet: ( A and B ) Average ChIP-seq signal plots of CDK7 (A) and pan RNAPII (B) for gene sets related to DNA repair, MYC targets V1, and E2F targets. ( C ) Track image of SRSF6 gene, a representative gene from the DNA repair pathway. ( D ) Track image of RPLP0 gene, a representative gene from the MYC targets V1 pathway. ( E ) Track image of EZH2 gene, a representative gene from the E2F targets pathway. ( F ) Heatmap showing log2FC values of DNA damage/repair genes expression from pan RNAPII ChIP-seq and mRNA-seq data (right; n = 3, FDR ≤ 0.1).

Article Snippet: The following antibodies were used for immunoblotting: total CDK7 (Cell Signaling Technology, Danvers, MA, USA, 2090S, RRID: AB_2077140), MYC (Cell Signaling Technology, 13987, RRID: AB_2631168), E2F1 (Cell Signaling Technology, 3742, RRID: AB_2096936), MYC S62P (Cell Signaling Technology, 13748, RRID: AB_2687518), MYC T58P (Cell Signaling Technology, 46650), Rb S795P (Cell Signaling Technology, 9301, RRID: AB_330013), Rb S780P (Cell Signaling Technology, 9307, RRID: AB_330015), MAX (Cell Signaling Technology, 4739, RRID: AB_2281777), T-H2AX (Santa Cruz, Dallas, TX, USA, sc-517336, RRID: AB_3675923), P-H2AX (Cell Signaling Technology, 2577, RRID: AB_2118010), Ubiquitin (Santa Cruz, sc-8017, RRID: AB_628423), TOP1 (BD Biosciences, San Jose, CA, USA, 556597, RRID: AB_396474), β-actin (Santa Cruz, sc-47778, RRID: AB_626632), Affinity Purified Goat Anti-Rabbit IgG (H+L)-HRP (Bio-Rad, Hercules, CA, USA, 1706515, RRID: AB_11125142), and Affinity Purified Goat Anti-Mouse IgG (H+L)-HRP (Bio-Rad, 1706516, RRID: AB_11125547).

Techniques: ChIP-sequencing, Expressing

Journal: bioRxiv

Article Title: Sensitizing tumor response to topoisomerase I antibody drug conjugate by selective CDK7 inhibition

doi: 10.1101/2025.11.23.690049

Figure Lengend Snippet: ( A ) A model illustrating how Q901 enhances the activity of TOP1i and TOP1i-ADCs. (Left) Q901 promotes CDK7 accumulation at the TSS while reducing RNAPII binding. This also decreases MYC and E2F1 binding at the TSS, leading to downregulation of genes involved in the DNA damage response pathway. (Middle) The dual inhibition of CDK7 (by Q901) and TOP1 (by TOP1i) blocks the repair of TOP1i-induced DNA damage, ultimately leading to cell death. (Right) The combination of Q901 and a TOP1i-ADC shows potent enhanced anticancer activity, effectively inducing cancer cell death in vitro and significantly reducing tumor growth in vivo. ( B and C ) HCT116, HER2 ultra low/negative human colon cancer cell line, was treated with Q901, T-DXd (10 μg/ml), or their combination at the indicated concentrations for 72 h (B). Dose-response curves were plotted as a function of log-transformed concentration relative to IC□□ values. Cell viability was measured using the ATP Lite™ system (n = 2, data represent mean ± SD). (C) For in vivo efficacy study, HCT116 cells mixed with Matrigel (1:1) were subcutaneously implanted into BALB/c nude mice. When tumors reached an average size of 117 mm3, mice were randomized into groups (n = 8 per group) and treated with Q901 (10 mg/kg, intraperitoneally once daily), T-DXd alone (10 mg/kg, intravenous single injection on day 0) or the combination. ( D and F ) H292, TROP2 positive human lung cancer cell line, was treated with Q901 in combination with SG at the indicated concentrations for 72 h (D). Dose-response curves were generated using log-transformed concentrations normalized to IC□□ values. Cell viability was assessed using the ATP Lite™ system (n = 2, data represent mean ± SD). ( E and F ) For in vivo efficacy study, H292 cells were mixed with Matrigel (1:1) and implanted subcutaneously into BALB/c nude mice. When tumors reached an average size of 140 mm3, mice were randomized into groups (n = 8 per group) and treated with Q901 (10 or 3 mg/kg, intraperitoneally once daily), SG alone (3 or 10 mg/kg, intravenous single injection on day 1 and 8) or the combination of both. The graph shows the mean tumor volume ± SEM. Statistical significance was calculated using GraphPad Prism software (*: p < 0.01, ****: p < 0.0001 by two-way ANOVA followed by Tukey’s multiple comparison test).

Article Snippet: The following antibodies were used for immunoblotting: total CDK7 (Cell Signaling Technology, Danvers, MA, USA, 2090S, RRID: AB_2077140), MYC (Cell Signaling Technology, 13987, RRID: AB_2631168), E2F1 (Cell Signaling Technology, 3742, RRID: AB_2096936), MYC S62P (Cell Signaling Technology, 13748, RRID: AB_2687518), MYC T58P (Cell Signaling Technology, 46650), Rb S795P (Cell Signaling Technology, 9301, RRID: AB_330013), Rb S780P (Cell Signaling Technology, 9307, RRID: AB_330015), MAX (Cell Signaling Technology, 4739, RRID: AB_2281777), T-H2AX (Santa Cruz, Dallas, TX, USA, sc-517336, RRID: AB_3675923), P-H2AX (Cell Signaling Technology, 2577, RRID: AB_2118010), Ubiquitin (Santa Cruz, sc-8017, RRID: AB_628423), TOP1 (BD Biosciences, San Jose, CA, USA, 556597, RRID: AB_396474), β-actin (Santa Cruz, sc-47778, RRID: AB_626632), Affinity Purified Goat Anti-Rabbit IgG (H+L)-HRP (Bio-Rad, Hercules, CA, USA, 1706515, RRID: AB_11125142), and Affinity Purified Goat Anti-Mouse IgG (H+L)-HRP (Bio-Rad, 1706516, RRID: AB_11125547).

Techniques: Activity Assay, Binding Assay, Inhibition, In Vitro, In Vivo, Transformation Assay, Concentration Assay, Injection, Generated, Software, Comparison

Journal: Molecular Cancer Therapeutics

Article Title: Triptolide Induces Cell Killing in Multidrug-Resistant Tumor Cells via CDK7/RPB1 Rather than XPB or p44

doi: 10.1158/1535-7163.mct-15-0753

Figure Lengend Snippet: Figure 3. Triptolide induces RPB1 degradation in both parental and MDR cell lines and RPB1 phosphorylation at Ser1878. A, MES/SA, MES-SA/MX5, KB, and KB/VCR cells were treated as shown in Materials and Methods and immunoblotted for RPB1. B, SK-OV-3, KB, and KB/VCR cells were pretreated with a CDK7- specific inhibitor and then assayed for RPB1. C, SK-OV-3 cells were treated as indicated and immunoblotted for phosphorylated RPB1 at Ser5 and phosphorylated CDK7 at Thr170. D, KB and KB/VCR cells were treated as indicated and immunoblotted for phosphorylated RPB1 at Ser5 and phosphorylated CDK7 at Thr170. E, cells were cultured as depicted in Materials and Methods, and IC50 values were measured using a Cell Counting Kit-8 (CCK-8) assay. F, KB and KB/VCR cells were cultured as indicated and immunoblotted for RPB1. G, SK-OV-3 cells were treated as indicated and immunoprecipitated RPB1 and separated with SDS-PAGE. Gel bands were excised, digested, and assessed using LC/MS-MS to identify the phosphorylation site of RPB1. m/z, mass-to-charge ratio.

Article Snippet: All antibodies were commercially available: RPB1 [carboxy-terminal domain (CTD) repeat], p-S5-RPB1, XPB, OCT-4, SOX-2, NANOG, ubiquitin, and p-Thr170-CDK7were fromAbcam,GAPDHand IgGwere from Beyotime Institute of Biotechnology (Haimen, China), CDK7 and p44 were from Santa Cruz Biotechnology, and c-MYC was from BD Biosciences.

Techniques: Phospho-proteomics, Cell Culture, Cell Counting, CCK-8 Assay, Immunoprecipitation, SDS Page, Liquid Chromatography with Mass Spectroscopy

Journal: Virus research

Article Title: Cytomegalovirus cyclin-dependent kinase ortholog vCDK/pUL97 undergoes regulatory interaction with human cyclin H and CDK7 to codetermine viral replication efficiency.

doi: 10.1016/j.virusres.2023.199200

Figure Lengend Snippet: Fig. 1. Structural model of the interaction between pUL97 and cyclin H. (A) Predicted binding site of pUL97(231-280) (orange) superimposed with the experimental cyclin H–CDK7–MAT1 complex structure (gray, cyan, and dark blue). This model suggests that pUL97(231-280) uses the same binding pocket as MAT1 for targeting the cyclin H–CDK7 complex. (B) Model of a ternary pUL97–cyclin H–CDK7 complex, in which pUL97 is attached to cyclin H exclusively through IF2 formed by the 231-280 sequence stretch. The pUL97 kinase domain (residues 329-634, marked in red) is connected to the complex by a nonstructured, flexible linker (residues 281-328, indicated as dark orange connecting line). (C) Model of a pUL97–cyclin H complex, in which pUL97 interacts with cyclin H both through IF2, pUL97(231-280) (orange), and the globular kinase domain IF1, pUL97(329-634) (red), thereby displacing CDK7.

Article Snippet: The following antibodies were used in this study: mAb-UL97.01, mAb-UL53, mAb-UL50, and mAb-UL56 (kindly provided by T. Lenac and S. Jonick, University of Rijeka, Croatia), mAb-IE1 (kindly provided by William Britt, Birmingham, AL, USA), mAb-UL44 and mAb-MCP (kindly provided by Bodo Plachter, University of Mainz, Mainz, Germany), mAb-pp65 and mAb-UL69 (kindly provided by T. Stamminger, Ulm, Germany), mAb-β-actin (A5441, Sigma Aldrich), pAb-cyclin H (LSC331195, LS Bio), pAb-CDK7 pSer164 (PA5-105583, Sigma-Aldrich), pAb-CDK7 pThr170 (ab155976, Abcam), pAb-CDK9 pThr186 (2549, Cell signaling), mAb-CDK7 (sc-7344, Santa Cruz Biotechnology), pAbRNAPII CTD pSer2 (NB100-1805, Novus Biologicals), mAb-Flag (F1804, Sigma-Aldrich).

Techniques: Binding Assay, Sequencing

Journal: Virus research

Article Title: Cytomegalovirus cyclin-dependent kinase ortholog vCDK/pUL97 undergoes regulatory interaction with human cyclin H and CDK7 to codetermine viral replication efficiency.

doi: 10.1016/j.virusres.2023.199200

Figure Lengend Snippet: Fig. 3. Viral protein substrate phosphorylation by CDK7. HFFs were infected with HCMV AD169-GFP at MOI of 1 and lysed 3 d.p.i. (A) Viral proteins were coimmunoprecipitated with cellular kinase CDK7 (lanes 3-10) and exposed to the non-radioactive IVKA reaction. Specific phosphorylation signals are marked to indicate single (◦) or continuous (arrows in respective colors) bands. Autophosphorylation signals of viral kinase pUL97 served as a positive control (lane 2). Du plicates of each immunoprecipitate were treated with 5 µM of CDK7-inhibitor LDC4297 shortly before the IVKA reaction (lanes 4, 6, 8, 10). (B) An IVKA reaction conducted in parallel without ATPγS served as a negative control. Successful IP (C) and reliable expression levels (D) of all proteins of interest were demonstrated by Wb analysis. (E) Phosphorylation signals from IVKA reactions shown in (A) were quantitated three times with varying background parameters, resulting in triplicate determinations of each sample, and mean values ± SD were then normalized to IP control.

Article Snippet: The following antibodies were used in this study: mAb-UL97.01, mAb-UL53, mAb-UL50, and mAb-UL56 (kindly provided by T. Lenac and S. Jonick, University of Rijeka, Croatia), mAb-IE1 (kindly provided by William Britt, Birmingham, AL, USA), mAb-UL44 and mAb-MCP (kindly provided by Bodo Plachter, University of Mainz, Mainz, Germany), mAb-pp65 and mAb-UL69 (kindly provided by T. Stamminger, Ulm, Germany), mAb-β-actin (A5441, Sigma Aldrich), pAb-cyclin H (LSC331195, LS Bio), pAb-CDK7 pSer164 (PA5-105583, Sigma-Aldrich), pAb-CDK7 pThr170 (ab155976, Abcam), pAb-CDK9 pThr186 (2549, Cell signaling), mAb-CDK7 (sc-7344, Santa Cruz Biotechnology), pAbRNAPII CTD pSer2 (NB100-1805, Novus Biologicals), mAb-Flag (F1804, Sigma-Aldrich).

Techniques: Phospho-proteomics, Infection, Positive Control, Negative Control, Expressing, Control

Journal: Virus research

Article Title: Cytomegalovirus cyclin-dependent kinase ortholog vCDK/pUL97 undergoes regulatory interaction with human cyclin H and CDK7 to codetermine viral replication efficiency.

doi: 10.1016/j.virusres.2023.199200

Figure Lengend Snippet: Fig. 6. Human CDK7 does not increase pUL97 in vitro kinase activity. (A) 293T cells were transiently transfected with plasmids encoding pUL97-Flag. Cells were lysed 2 d post-transfection, and pUL97-Flag and CDK7 were immu noprecipitated using the indicated antibodies; a mouse Fc fragment served as a negative control. Dynabeads-bound proteins were eluted in 150 µl enzyme buffer. 25 µl of eluted Dynabeads were denatured in 25 µl 2x loading buffer and subjected to SDS-PAGE and Wb to confirm successful precipitation of pUL97- Flag and CDK7. (B) Samples derived from immunoprecipitation of CDK7 plus pUL97-Flag, as well as Fc control samples, were subjected to a qSox-IVKA (using the pUL97-specific sensor peptide AQT0258). Optionally, 0.01 µM of CDK7-specific inhibitor LDC4297 (corresponds to the mean antiviral EC50 value) or equal amounts of DMSO were added to the reactions. Background activity determined with the Fc control was subtracted from values of combined pUL97-Flag plus CDK7 kinase activity. Mean values ± SD derived from one representative experiment are given, as derived from measurements in tripli cates. Statistical analysis was performed using an ordinary two-way ANOVA: n. s., not significant.

Article Snippet: The following antibodies were used in this study: mAb-UL97.01, mAb-UL53, mAb-UL50, and mAb-UL56 (kindly provided by T. Lenac and S. Jonick, University of Rijeka, Croatia), mAb-IE1 (kindly provided by William Britt, Birmingham, AL, USA), mAb-UL44 and mAb-MCP (kindly provided by Bodo Plachter, University of Mainz, Mainz, Germany), mAb-pp65 and mAb-UL69 (kindly provided by T. Stamminger, Ulm, Germany), mAb-β-actin (A5441, Sigma Aldrich), pAb-cyclin H (LSC331195, LS Bio), pAb-CDK7 pSer164 (PA5-105583, Sigma-Aldrich), pAb-CDK7 pThr170 (ab155976, Abcam), pAb-CDK9 pThr186 (2549, Cell signaling), mAb-CDK7 (sc-7344, Santa Cruz Biotechnology), pAbRNAPII CTD pSer2 (NB100-1805, Novus Biologicals), mAb-Flag (F1804, Sigma-Aldrich).

Techniques: In Vitro, Activity Assay, Transfection, Negative Control, SDS Page, Derivative Assay, Immunoprecipitation, Control

Journal: Virus research

Article Title: Cytomegalovirus cyclin-dependent kinase ortholog vCDK/pUL97 undergoes regulatory interaction with human cyclin H and CDK7 to codetermine viral replication efficiency.

doi: 10.1016/j.virusres.2023.199200

Figure Lengend Snippet: Fig. 7. Phosphorylation patterns of CDK7 depend on the state of HCMV infection and pUL97 kinase activity. (A) HFFs were seeded in T75 cell culture flasks and infected 1 d later with HCMV AD169, or pUL97 kinase-deficient mutant ORF-UL97 K355Δ at MOI of 0.5, or remained-mock infected. 24 h after infection HCMV AD169 infected cells were treated with 0.35 µM (i.e. EC50) pUL97-specific inhibitor maribavir (MBV), or with equal amounts of DMSO. Cells were harvested 3 d p.i. and samples were subjected to standard SDS-PAGE and Wb analysis. Proteins of interest and site-specific phosphorylation were stained using the indicated antibodies. (B) Mean values ± SD of phosphorylation intensity of CDK7 at sites Ser164 and Thr170, as well as CDK9 Thr186, was determined by quadruplicate densitometric quantitation of those Wbs depicted in (A) together with a second biological replicate (shown in Fig. S5). Statistical analysis was performed using an ordinary one-way ANOVA and post-hoc Tukey correction: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n.s., not significant.

Article Snippet: The following antibodies were used in this study: mAb-UL97.01, mAb-UL53, mAb-UL50, and mAb-UL56 (kindly provided by T. Lenac and S. Jonick, University of Rijeka, Croatia), mAb-IE1 (kindly provided by William Britt, Birmingham, AL, USA), mAb-UL44 and mAb-MCP (kindly provided by Bodo Plachter, University of Mainz, Mainz, Germany), mAb-pp65 and mAb-UL69 (kindly provided by T. Stamminger, Ulm, Germany), mAb-β-actin (A5441, Sigma Aldrich), pAb-cyclin H (LSC331195, LS Bio), pAb-CDK7 pSer164 (PA5-105583, Sigma-Aldrich), pAb-CDK7 pThr170 (ab155976, Abcam), pAb-CDK9 pThr186 (2549, Cell signaling), mAb-CDK7 (sc-7344, Santa Cruz Biotechnology), pAbRNAPII CTD pSer2 (NB100-1805, Novus Biologicals), mAb-Flag (F1804, Sigma-Aldrich).

Techniques: Phospho-proteomics, Infection, Activity Assay, Cell Culture, Mutagenesis, SDS Page, Staining, Quantitation Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Novel covalent CDK7 inhibitor potently induces apoptosis in acute myeloid leukemia and synergizes with Venetoclax.

doi: 10.1186/s13046-023-02750-w

Figure Lengend Snippet: Fig. 1 Efficacy of XL102 and its effects on downstream targets of CDK7. A Kinase activity of CDK7 in the presence of XL102 was measured using the LANCE TR-FRET in vitro kinase assay by incubating both CDK7 and XL102 followed by addition of ATP and U-light-MBP peptide substrate. Time-resolved fluorescence (excitation, 320 nm; emission donor, 615 nm; emission acceptor, 665 nm) was monitored by using 2030 multilabel reader Victor5 (PerkinElmer). The IC50 values were derived by fitting a sigmoidal dose–response curve to a plot of assay readout over inhibitor concentration, computed with the Graph Pad Prism. B The pull-down assay using bio-THZ1 show a dose dependent target engagement in AML cells harvested after 3 h of XL102 treatment followed by washing to remove any unbound XL102 (time point-0 h). C XL102 inhibited CTD phosphorylation of conserved residues of RPII in AML cells in dose dependent manner at 6 h and 24 h along with quantification of Western blot data. Results are the mean ± SD of three independent experiments

Article Snippet: Antibodies used against various proteins were CDK7 (Bethyl Laboratories, Massachusetts, US) and Beta actin (Santa Cruz Biotechnology, Texas, US).

Techniques: Activity Assay, In Vitro, Kinase Assay, Fluorescence, Derivative Assay, Concentration Assay, Pull Down Assay, Drug discovery, Phospho-proteomics, Western Blot

Journal: International Journal of Oral Science

Article Title: Lysine-specific demethylase 1 controls key OSCC preneoplasia inducer STAT3 through CDK7 phosphorylation during oncogenic progression and immunosuppression

doi: 10.1038/s41368-025-00363-x

Figure Lengend Snippet: Proteomics analysis showing LSD1 inhibition impairs STAT3 protein and phospho-protein network: a Global proteomics of tongue tumor protein lysate from 4MOSC1 syngeneic mouse model showing SP2509 treatment reduces LSD1 and STAT3, whereas increased NFATc1 and IRF3. b Phosphoproteomics analysis of 4MOSC1 tumors treated with SP2509 reversed phosphorylated oncoproteins expression shown in the heat map, including phospho-CDK7 (Tyr170). c Kinase-substrate enrichment analysis (KSEA) in Phosphomatics tool reveals the kinase activity-based z-score (activation/deactivation) on the reduced activity for phosphorylated CDK7

Article Snippet: For genetic knockout, we used plasmids CDK7 sgRNA (BRDN0001162216) (Addgene #76077), EF.STAT3C.Ubc.GFP (Addgene #24983), KDM1A sgRNA CRISPR/Cas9 All-in-one Lentivector (abm #K2776607).

Techniques: Inhibition, Phospho-proteomics, Expressing, Activity Assay, Activation Assay

Journal: International Journal of Oral Science

Article Title: Lysine-specific demethylase 1 controls key OSCC preneoplasia inducer STAT3 through CDK7 phosphorylation during oncogenic progression and immunosuppression

doi: 10.1038/s41368-025-00363-x

Figure Lengend Snippet: Phosphoproteomics analysis showing LSD1 inhibition impairs CDK7 and EGFR-STAT3 network: a Kinase substrate interaction analysis in SP2509 treated groups shows inhibition of CDK7 phosphorylation, which has various substrates, including other CDKs and eukaryotic translation initiation factors. b IPA analysis generated by global proteomics data shows that SP2509 reduces the EGFR-STAT3 network, whereas upregulation of NFATc1 results in accumulation of inflammatory leukocyte network

Article Snippet: For genetic knockout, we used plasmids CDK7 sgRNA (BRDN0001162216) (Addgene #76077), EF.STAT3C.Ubc.GFP (Addgene #24983), KDM1A sgRNA CRISPR/Cas9 All-in-one Lentivector (abm #K2776607).

Techniques: Phospho-proteomics, Inhibition, Generated

Journal: International Journal of Oral Science

Article Title: Lysine-specific demethylase 1 controls key OSCC preneoplasia inducer STAT3 through CDK7 phosphorylation during oncogenic progression and immunosuppression

doi: 10.1038/s41368-025-00363-x

Figure Lengend Snippet: CDK7 is a key mediator of LSD1-induced STAT3 expression: RT-qPCR analysis to evaluate the effect of LSD1, STAT3, or CDK7 inhibitors on expression KDM1A, STAT3 , and CDK7 expression in; a HSC3 cells, and b CAL27 cells. RT-qPCR analysis to evaluate the effect of genetic knockout of KDM1A, STAT3 , or CDK7 on expression of KDM1A, STAT3 , and CDK7 in; c HSC3 cells, and d CAL27 cells. “ns” P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1

Article Snippet: For genetic knockout, we used plasmids CDK7 sgRNA (BRDN0001162216) (Addgene #76077), EF.STAT3C.Ubc.GFP (Addgene #24983), KDM1A sgRNA CRISPR/Cas9 All-in-one Lentivector (abm #K2776607).

Techniques: Expressing, Quantitative RT-PCR, Knock-Out

Journal: International Journal of Oral Science

Article Title: Lysine-specific demethylase 1 controls key OSCC preneoplasia inducer STAT3 through CDK7 phosphorylation during oncogenic progression and immunosuppression

doi: 10.1038/s41368-025-00363-x

Figure Lengend Snippet: Impact on phosphorylation of CDK7 and methylation of H3K4 and H3K9 after LSD1 inhibition: a Effect on phospho-CDK7 (T170) after LSD1, STAT3 and CDK7 inhibition. b Status of H3K4 and H3K9 methylation on STAT3 and CDK7. Statistical analysis was performed by t -test and one-way ANOVA. “ns” P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1

Article Snippet: For genetic knockout, we used plasmids CDK7 sgRNA (BRDN0001162216) (Addgene #76077), EF.STAT3C.Ubc.GFP (Addgene #24983), KDM1A sgRNA CRISPR/Cas9 All-in-one Lentivector (abm #K2776607).

Techniques: Phospho-proteomics, Methylation, Inhibition

Journal: International Journal of Oral Science

Article Title: Lysine-specific demethylase 1 controls key OSCC preneoplasia inducer STAT3 through CDK7 phosphorylation during oncogenic progression and immunosuppression

doi: 10.1038/s41368-025-00363-x

Figure Lengend Snippet: Graphical Abstract. The potential mechanism after blocking LSD1 inhibits novel CDK7 phospho-protein networks and STAT3 signaling ultimately promotes CD8+ T cell infiltration and activation by relieving CTLA4-mediated immunosuppression

Article Snippet: For genetic knockout, we used plasmids CDK7 sgRNA (BRDN0001162216) (Addgene #76077), EF.STAT3C.Ubc.GFP (Addgene #24983), KDM1A sgRNA CRISPR/Cas9 All-in-one Lentivector (abm #K2776607).

Techniques: Blocking Assay, Activation Assay