Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK7 inhibition augments response to multidrug chemotherapy in pancreatic cancer

doi: 10.1186/s13046-022-02443-w

Figure Lengend Snippet: Targeted inhibition of CDK7 reverses chemoresistance in pancreatic cancer. A Western blotting of TB32047 and MIA PaCa-2 gemcitabine- and paclitaxel-resistant cells (TBGPR, MIAR) and parental cells (TBWT, MIAWT). B The CDK7 inhibitor THZ1 synergistically enhances the antiproliferative effects of GEM and PTX in TBGPR and MIAGPR cells. The proliferation was determined after 72 h of treatment with THZ1 and GEM/PTX alone or in combination. Below is a plot of the fraction impacted versus the combination index. CI < 1.0, synergism. C Diagrammatic representation of the research outcome. Gemcitabine and paclitaxel combined with CDK7 inhibition induces DNA damage, G2-M arrest, and subsequent apoptosis

Article Snippet: The small-molecule inhibitor targeting CDK7 (THZ1, Cat. #HY-80013) was purchased from MedChemExpress.

Techniques: Inhibition, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK7 inhibition augments response to multidrug chemotherapy in pancreatic cancer

doi: 10.1186/s13046-022-02443-w

Figure Lengend Snippet: CRISPR-Cas9 knockout screens to identify chemosensitivity targets in pancreatic cancer cells treated with gemcitabine and paclitaxel. A Schematic diagram of the timeline and experimental procedure for CRISPR-Cas9 screening using protein kinase library. B, C Volcano plots showing log 2 -transformed sgRNA normalized fold change and -log 10 -transformed p -value of gemcitabine ( B ) and paclitaxel ( C ) versus vehicle-treated TB32047 cells, with significant genes in positive and negative selection highlighted in red and blue, respectively. D Venn diagram showing the shared genes of the top 20 candidate genes for gemcitabine and paclitaxel negative screening, which are Cdk7, Wee1, Fgfrl1, and Havcr2. E Scatterplots showing log 2 -transformed sgRNA normalized read counts of gemcitabine (left) and paclitaxel (right) versus vehicle-treated TB32047 cells, with sgRNAs targeting Cdk7 highlighted. F Data from TCGA-PDAC were applied for survival analysis. Kaplan–Meier survival analysis shows that higher CDK7 mRNA expression is associated with a tendency toward poor overall survival. G The analysis of CDK7 expression in PDAC tissues and normal pancreatic tissues was conducted using the TCGA (PDAC) and GTEx (Pancreas) databases. Taken together, the results indicate that CDK7 expression levels were significantly higher in pancreatic cancer than in the corresponding normal pancreatic tissue. H Western blot analysis revealed a variation in CDK7 expression between pancreatic cancer subtypes. * p < 0.05; *** p < 0.001, and **** p < 0.0001 were calculated using the unpaired Student’s t test

Article Snippet: The small-molecule inhibitor targeting CDK7 (THZ1, Cat. #HY-80013) was purchased from MedChemExpress.

Techniques: CRISPR, Knock-Out, Transformation Assay, Selection, Expressing, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK7 inhibition augments response to multidrug chemotherapy in pancreatic cancer

doi: 10.1186/s13046-022-02443-w

Figure Lengend Snippet: CDK7 knockout in combination with gemcitabine and paclitaxel treatment inhibits proliferation and increases apoptosis in pancreatic cancer cells. A Western blotting of TB32047 and MIA PaCa-2 cells transfected with CDK7 sgRNAs or non-targeting control. B The dose–response curves for GEM and PTX in TB32047 and MIA PaCa-2 CDK7 KO or NC cells. Seventy-two hours following treatment, cell survival was measured and normalized. Data are presented as the mean of three independent experiments ( n = 3). C–F CDK7-WT (NC sgRNA) and CDK7-knockout TB32047 ( C , D ) cells, as well as MIA PaCa-2 ( E , F ) cells, were treated with vehicle or chemotherapy for 24 h and then analyzed for apoptosis using flow cytometry. Data are presented as the mean of three independent experiments ( n = 3). ** p < 0.01; *** p < 0.001, and **** p < 0.0001 by two-way ANOVA with a Tukey multiple comparison test. G and H TB32047 ( G ) and MIA PaCa-2 ( H ) cells expressing CDK7 or control sgRNAs were treated with GEM and PTX; caspase 3/7 activity was measured 24 h later. Data are presented as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by two-way ANOVA with a Tukey multiple comparison test. I . Western blot analysis of TB32047 (left) and MIA PaCa-2 (right) cells transfected with non-targeting control (NC sgRNA) or CDK7-targeting sgRNAs (CDK7-sg1, CDK7-sg2, CDK7-sg3, CDK7-sg4) and treated for 24 h with vehicle or GEM plus PTX

Article Snippet: The small-molecule inhibitor targeting CDK7 (THZ1, Cat. #HY-80013) was purchased from MedChemExpress.

Techniques: Knock-Out, Western Blot, Transfection, Control, Flow Cytometry, Comparison, Expressing, Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK7 inhibition augments response to multidrug chemotherapy in pancreatic cancer

doi: 10.1186/s13046-022-02443-w

Figure Lengend Snippet: Targeted inhibition of CDK7 enhanced gemcitabine and paclitaxel chemotherapy response in pancreatic cancer in vitro. A Dose–response curves of TB32047 and MIA PaCa-2 wild-type cells to THZ1. Cell proliferation was evaluated 72 h after drug treatment. Data are presented as the mean of three independent experiments (n = 3). B and C The CDK7 inhibitor THZ1 enhances the antiproliferative impact of GEM and PTX in pancreatic cancer cells synergistically. Proliferation analysis was performed on TB32047 ( B ) and MIA PaCa-2 ( C ) cells treated for 72 h with THZ1 and GEM/PTX alone or in combination. The plot of fraction affected versus combination index is shown below the x-axis. CI < 1.0, synergism. D and F Clonogenic assay of TB32047 ( D ) and MIA PaCa-2 ( F ) cells treated with vehicle or drugs for 72 h and cultured without drugs for an additional 7–10 days. The cells remaining after the treatment were fixed and stained with crystal violet. Representative images (left) and quantifications (right) of three independent experiments ( n = 3) are shown. * p < 0.05 and ** p < 0.01 by one-way ANOVA with a Tukey multiple comparison test. ns: not significant. E and G . Caspase 3/7 activity was evaluated in TB32047 ( E ) and MIA PaCa-2 ( G ) cells treated for 72 h with vehicle or drugs. Data are presented as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 by two-way ANOVA with a Tukey multiple comparison test. H and I Flow cytometry-based apoptotic assay of TB32047 and MIA PaCa-2 cells treated for 72 h with drugs or drug combination for 72 h. Representative images ( H ) and quantification ( I ) of three independent experiments ( n = 3) are shown. ** p < 0.005 by two-way ANOVA with a Tukey multiple comparison test

Article Snippet: The small-molecule inhibitor targeting CDK7 (THZ1, Cat. #HY-80013) was purchased from MedChemExpress.

Techniques: Inhibition, In Vitro, Clonogenic Assay, Cell Culture, Staining, Comparison, Activity Assay, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK7 inhibition augments response to multidrug chemotherapy in pancreatic cancer

doi: 10.1186/s13046-022-02443-w

Figure Lengend Snippet: Combined CDK7 inhibition with standard chemotherapy suppresses PDAC tumor growth in vivo. A Images of TB32047 orthotopic mouse model of pancreatic cancer after treatment for 16 days. B and C Relative tumor weights ( B ) and volumes ( C ) of TB32047 orthotopic pancreatic cancer mouse model after treatment. *** p < 0.001 and **** p < 0.0001 by one-way ANOVA with a Tukey multiple comparison test. ns: not significant. D Western blot analysis of tissue samples from mice with pancreatic cancer that had been treated with vehicle, GEM/nab-PTX, or a combination. E Hematoxylin and eosin (H&E) staining demonstrated that the combination of THZ1 and GEM with nab-PTX decreased tumor cell content compared to the group receiving single components and the control group. Scale bar (black), 50 μm

Article Snippet: The small-molecule inhibitor targeting CDK7 (THZ1, Cat. #HY-80013) was purchased from MedChemExpress.

Techniques: Inhibition, In Vivo, Comparison, Western Blot, Staining, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: CDK7 inhibition augments response to multidrug chemotherapy in pancreatic cancer

doi: 10.1186/s13046-022-02443-w

Figure Lengend Snippet: CDK7 inhibition induces cell cycle arrest, apoptotic cell death and DNA damage through the STAT3-MCL1-CHK1 axis. A Gene set enrichment analysis (GSEA) on the RNA-sequencing data of BXPC-3, Mia PaCa-2, and PANC-1 cells treated with THZ1, a selective CDK7 inhibitor. Transcriptomic gene expression data are based on the GEO dataset GSE121273. B Western blotting of TB32047 and MIA PaCa-2 cells treated with vehicle (DMSO), THZ1, and GEM plus PTX, alone or in combination, for 24 h. C Immunofluorescence of TB32047 cells treated with vehicle (DMSO), THZ1, and GEM/PTX, alone or in combination, for 24 h. Cells were subsequently stained with antibodies to γH2AX (green) and DAPI (blue; nuclei). Scale bar (white), 20 μm

Article Snippet: The small-molecule inhibitor targeting CDK7 (THZ1, Cat. #HY-80013) was purchased from MedChemExpress.

Techniques: Inhibition, RNA Sequencing, Gene Expression, Western Blot, Immunofluorescence, Staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of CDK7-dependent transcriptional addiction is a potential therapeutic target in synovial sarcoma.

doi: 10.1016/j.biopha.2022.112888

Figure Lengend Snippet: Fig. 1. CDK7 expression and clinicopathological features in synovial sarcoma. Expression of CDK7 in human synovial sarcoma tissue microarrays were evaluated by immunohistochemistry, and the associations between CDK7 expression levels and clinicopathological features of synovial sarcoma patients were analyzed. (A) Representative images of different immunohistochemical staining intensities of CDK7 (×200; scale bar:100 µm). On the basis of the percentage of cells with positive nuclear staining, CDK7 staining patterns were categorized into 6 groups: 0, 0% nuclear staining; 1+, 1–10% nuclear staining; 2+, 11–25% nuclear staining; 3+, 26–50% nuclear staining; 4+, 51–75% nuclear staining; 5+, 76–100% nuclear staining. Samples with a score of 0–2 were recognized as low CDK7 expression group and 3–5 were recognized as high CDK7 expression group. (B) Distribution of CDK7 staining scores among clinical stage ≥IIB and clinical stage

Article Snippet: The selective CDK7 inhibitor BS-181 (#S1572, Selleck Chemicals, Houston, TX) was used to suppress CDK7 in synovial sarcoma cells.

Techniques: Expressing, Immunohistochemistry, Immunohistochemical staining, Staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of CDK7-dependent transcriptional addiction is a potential therapeutic target in synovial sarcoma.

doi: 10.1016/j.biopha.2022.112888

Figure Lengend Snippet: Fig. 2. Expression of CDK7 correlated with clinical prognosis in various sarcomas. Expression of CDK7 in various human sarcoma tissue microarrays were evaluated by immunohistochemistry, and the correlations between CDK7 expression levels and clinicopathological prognosis of various sarcomas patients were analyzed. (A) Representative images of different immunohistochemical staining intensities of CDK7 (×200; scale bar:100 µm). Based on the percentage of cells with positive nuclear staining, CDK7 staining patterns were categorized into 6 groups: 0, 0% nuclear staining; 1+, 1–10% nuclear staining; 2+, 11–25% nuclear staining; 3+, 26–50% nuclear staining; 4+, 51–75% nuclear staining; 5+, 76–100% nuclear staining. Samples with a score of 0–2 were recognized as the low CDK7 expression group and 3–5 were recognized as the high CDK7 expression group. (B) Distribution of CDK7 staining scores in various sarcoma tissue samples from survival and non- survival patients. (C) Kaplan–Meier survival curve of sarcoma patients with CDK7 high staining (≥3) or low staining (<3).

Article Snippet: The selective CDK7 inhibitor BS-181 (#S1572, Selleck Chemicals, Houston, TX) was used to suppress CDK7 in synovial sarcoma cells.

Techniques: Expressing, Immunohistochemistry, Immunohistochemical staining, Staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of CDK7-dependent transcriptional addiction is a potential therapeutic target in synovial sarcoma.

doi: 10.1016/j.biopha.2022.112888

Figure Lengend Snippet: Fig. 3. CDK7 nuclear localization and expression in human synovial sarcoma cell lines. Representative images of the CDK7 localization determined by immuno fluorescence in (A) Fuji and (B) SYO-1 cells. Cells were visualized by fluorescence microscopy after. incubation with Alexa Fluor 488 goat anti-rabbit IgG (CDK7; green) or Alexa Fluor 594 goat anti-mouse IgG (β-Actin; red) or Hoechst 33342 (blue) nuclear stain. The images were merged to show the subcellular co-localization. (C) Expression levels of CDK7, RNAP II, and phosphorylation of RNAP II (Ser2 and Ser5) in four synovial sarcoma cell lines SYO-1, Fuji, Yamato, and Aska were detected using Western blotting. (D) Relative expressions of CDK7 and RNAP II Ser5 compared with α-Tubulin in different synovial sarcoma cell lines. Scale bar is 50 µm. The data are mean±SD of the experiment carried out in triplicate.

Article Snippet: The selective CDK7 inhibitor BS-181 (#S1572, Selleck Chemicals, Houston, TX) was used to suppress CDK7 in synovial sarcoma cells.

Techniques: Expressing, Fluorescence, Microscopy, Incubation, Staining, Phospho-proteomics, Western Blot

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of CDK7-dependent transcriptional addiction is a potential therapeutic target in synovial sarcoma.

doi: 10.1016/j.biopha.2022.112888

Figure Lengend Snippet: Fig. 4. Knockdown of CDK7 with siRNA decreases cell growth and proliferation in synovial sarcoma cells. MTT assays demonstrate a dose-dependent reduction of cell proliferation after CDK7 siRNA transfection in (A) Fuji and (B) SYO-1 cell lines. **P < 0.01 compared with the cell only (no treatment) group. (C) Representative images of the morphologic changes observed in synovial sarcoma cells by microscopy after CDK7 siRNA treatment. Scale bar: 50 µm. The expression of CDK7 and the transcription-related proteins RNAP II, and phosphorylation of RNAP II (Ser2 and Ser5) were evaluated by Western blotting in (D) Fuji and (E) SYO-1 synovial sarcoma cells after 48 h of siRNA transfection. The data are mean ± SD of the experiment carried out in triplicate.

Article Snippet: The selective CDK7 inhibitor BS-181 (#S1572, Selleck Chemicals, Houston, TX) was used to suppress CDK7 in synovial sarcoma cells.

Techniques: Knockdown, Transfection, Microscopy, Expressing, Phospho-proteomics, Western Blot

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of CDK7-dependent transcriptional addiction is a potential therapeutic target in synovial sarcoma.

doi: 10.1016/j.biopha.2022.112888

Figure Lengend Snippet: Fig. 5. Inhibiting CDK7 activity with BS-181 blocks synovial sarcoma cell proliferation and induces cell apoptosis. Cell viability was determined by MTT assays after BS-181 treatment for 5 days in (A) Fuji and (B) SYO-1 synovial sarcoma cells. (C) Representative images of Fuji and SYO-1 cells after increasing concentrations of BS- 181. Morphologic changes were observed by microscopy. Scale bar: 50 µm. Western blotting after 24 h of BS-181 treatment, showing the expression of CDK7, RNAP II, phosphorylation of RNAP II (Ser2 and Ser5) in (D) Fuji and (E) SYO-1 synovial sarcoma cells. The data are mean ± SD of the experiment carried out in triplicate.

Article Snippet: The selective CDK7 inhibitor BS-181 (#S1572, Selleck Chemicals, Houston, TX) was used to suppress CDK7 in synovial sarcoma cells.

Techniques: Activity Assay, Microscopy, Western Blot, Expressing, Phospho-proteomics

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of CDK7-dependent transcriptional addiction is a potential therapeutic target in synovial sarcoma.

doi: 10.1016/j.biopha.2022.112888

Figure Lengend Snippet: Fig. 6. Inhibition of CDK7 suppresses spheroid and colony formation in human synovial sarcomas. Representative images of cell colony formation after treatment with BS-181 were shown in (A) Fuji and (C) SYO-1 cells. Quantification of clonogenic assays reveals a reduced rate of cell colony formation after with BS-181 in (B) Fuji and (D) SYO-1 cells. **P < 0.01 compared with the cell only group. Representative images of (E) Fuji and (G) SYO-1 cell spheroids after 14 days in 3D culture with or without BS-181 treatment. Scale bar: 100 µm. The relative diameters of spheroids compared with untreated groups were analyzed in (F) Fuji and (H) SYO-1 synovial sarcoma cells. **P < 0.01 compared with the cell only group. The data are mean ± SD of the experiment carried out in triplicate.

Article Snippet: The selective CDK7 inhibitor BS-181 (#S1572, Selleck Chemicals, Houston, TX) was used to suppress CDK7 in synovial sarcoma cells.

Techniques: Inhibition

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of CDK7-dependent transcriptional addiction is a potential therapeutic target in synovial sarcoma.

doi: 10.1016/j.biopha.2022.112888

Figure Lengend Snippet: Fig. 7. Inhibition of CDK7 promotes cell apoptosis and reduces human synovial sarcoma cell migration. Annexin V/PI staining assays were used to determine the apoptosis rate of synovial sarcoma cells (A) Fuji and (B) SYO-1 cells treated with vehicle or BS-181 for 24 h. The proportion of apoptotic cells was calculated. **P < 0.05 compared with the control group. Data represents mean ± SD. Relative migration distance of (C) Fuji and (D) SYO-1 synovial sarcoma cells at different time points (0, 24, 48, and 72 h) after exposure to BS-181. Scale bar: 100 µm. **P < 0.01 compared with the cell only group. The data are mean ± SD of the experiment carried out in triplicate. Quantification of the cell migration distance of (E) Fuji and (F) SYO-1 cells following BS-181 treatment.

Article Snippet: The selective CDK7 inhibitor BS-181 (#S1572, Selleck Chemicals, Houston, TX) was used to suppress CDK7 in synovial sarcoma cells.

Techniques: Inhibition, Migration, Staining, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of CDK7-dependent transcriptional addiction is a potential therapeutic target in synovial sarcoma.

doi: 10.1016/j.biopha.2022.112888

Figure Lengend Snippet: Fig. 8. CDK7-dependent transcriptional addiction in tumor cell survival of synovial sarcoma. (A) CDK7 plays a role in the TFIIH complex that regulates transcription and mRNA synthesis. During transcription initiation, the activity of CDK7 is required for the activation of the CTD phosphorylation segment of RNAP II. CDK7 also participates in transcriptional elongation by promoting phosphorylation of the conserved threonine residue within the T-loop region to fully activate CDK9 and others. Through affecting stability of preinitiation complexes, CDK7 controls mRNA synthesis and leads to altered gene expression, cell cycle progression, and survival of tumor cells, which together promote cell proliferation and facilitate metastasis. (B) Targeted inhibition of CDK7 blocks transcriptional progression and induces cell apoptosis, offering a potentially effective tumor therapy in synovial sarcoma.

Article Snippet: The selective CDK7 inhibitor BS-181 (#S1572, Selleck Chemicals, Houston, TX) was used to suppress CDK7 in synovial sarcoma cells.

Techniques: Activity Assay, Activation Assay, Phospho-proteomics, Residue, Gene Expression, Inhibition

Journal: Cancer cell

Article Title: BCL2 Amplicon Loss and Transcriptional Remodeling Drives ABT-199 Resistance in B Cell Lymphoma Models

doi: 10.1016/j.ccell.2019.04.005

Figure Lengend Snippet: (A) Image-based cell-viability assays (upper panel) and western blots (lower panel) of control (GFP) or CDK7 knockdown derivatives of parental (left), I10 (middle), and L20 (right) cells treated with THZ1 (220 nM).

Article Snippet: CDK7 , Cell Signaling Technologies , Cat# 2916; RRID: AB_2077142.

Techniques: Western Blot, Control, Knockdown

Journal: Cancer cell

Article Title: BCL2 Amplicon Loss and Transcriptional Remodeling Drives ABT-199 Resistance in B Cell Lymphoma Models

doi: 10.1016/j.ccell.2019.04.005

Figure Lengend Snippet: Model for the evolution of ABT-199 persistence/resistance in 18q21-amplified mantle cell lymphoma and double-hit lymphoma models, which occurs through the selection for rare clones having loss or reductions in copy number of 18q21 amplicons that harbor BCL2, and via adaptive super-enhancer (SE)-driven transcriptional reprogramming. The latter mechanism confers vulnerability to transcriptional inhibition by targeting CDK7.

Article Snippet: CDK7 , Cell Signaling Technologies , Cat# 2916; RRID: AB_2077142.

Techniques: Amplification, Selection, Clone Assay, Inhibition

Journal: Cancer cell

Article Title: BCL2 Amplicon Loss and Transcriptional Remodeling Drives ABT-199 Resistance in B Cell Lymphoma Models

doi: 10.1016/j.ccell.2019.04.005

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CDK7 , Cell Signaling Technologies , Cat# 2916; RRID: AB_2077142.

Techniques: Virus, Recombinant, Protease Inhibitor, Immunoprecipitation, Library Quantification, Membrane, cDNA Synthesis, SYBR Green Assay, Gene Expression, Software

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. In theory, acute inhibition of transcriptional CDKs, particularly those involved in phosphorylating RPB1 CTD Ser2 phosphorylation, is expected to globally suppress gene expression, predominantly affecting mRNAs with short half-lives. B. Volcano plots of gene expression derived from 4-6 hours treatment of CDK7 inhibitor THZ1 (250 nM) in ovarian cancer cell line Kuramochi , CDK9 inhibitor HH1 (10 µM) in a cell line (YB5) derived from the SW48 colon cancer cell line , or CDK12 inhibitor SR-4835 (90 nM) in triple-negative breast cancer line MDA-MB-231 . RNA-seq data were downloaded from the Gene Expression Omnibus (GEO) and analyzed. C. A volcano plot of nascent RNA expression from neuroblastoma cells (IMR32) treated with 400 nM THZ531 for 2 hours . Note that the sequencing involved 4-thiouridine-pulse labeling and included RNA spike-in control. D. (Top) selection of TCGA ovarian serous adenocarcinoma samples with low or high expression of the indicated CDK genes (the top and bottom 5% in terms of CDK7, CDK9 or CDK12 mRNA expression in ovarian cancer samples with expression data; n = 17 each group). (Bottom) volcano plots of differential gene expression in tumors with low expressing CDKs compared to those with high expressing CDKs. For all volcano plots, genes significantly upregulated or downregulated (absolute log 2 fold change (FC) ≥ 1, p < 0.1) are colored in red and blue, respectively.

Article Snippet: The following primary antibodies were purchased and used for fluorescence immunoblotting: RNA polymerase II subunit B1 (phospho CTD Ser-2) Antibody, clone 3E10 (EMD Millipore, #04-1571); Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (Cell Signaling Technology, #13499); Phospho RNA Polymerase II (S2) Antibody, (Bethyl Laboratories, A300-654A); RNA polymerase II subunit B1 (phospho-CTD Ser-5) Antibody, clone 3E8 (EMD Millipore, #04-1572); Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (Cell Signaling Technology, #13523) RNA polymerase II subunit B1 (phospho-CTD Ser-7) Antibody, clone 4E12 (EMD Millipore, #04-1570); RNA Polymerase II Antibody (Bethyl Laboratories, A300-653A); RNA Polymerase II RPB1, clone 8WG16 (BioLegend, #664906); Rpb1 NTD (D8L4Y) Rabbit mAb (Cell Signaling Technology, #14958); c-Myc (clone Y69) Rabbit mAb (Abcam, #ab32072); c-Myc (D84C12) Rabbit mAb (Cell Signaling Technology, #5605); anti-CDK12 (Cell Signaling Technology, #11973); anti-CDK12 (proteintech, #26816-1-AP); anti-CDK12, clone 45F7-H2 (BIO-RAD, #VMA00874); anti-CDK13, clone 46B7-G7 (BIO-RAD, #VMA00875); CDK7 Recombinant Monoclonal Antibody (BL-80-5D4) (Bethyl, #A700-006); CDK7 (MO1) Mouse mAb (Cell Signaling Technology, #2916); CDK8 (D6M3J) Rabbit mAb (Cell Signaling Technology, #17395); CDK9 (C12F7) Rabbit mAb (Cell Signaling Technology, #2316); Anti-CDK19 (Sigma-Aldrich, #HPA007053); JunB Rabbit mAb, clone ARC0268 (ABclonal, #A4848); c-Jun (60A8) Rabbit mAb (Cell Signaling Technology, #9165); c-Fos (9F6) Rabbit mAb (Cell Signaling Technology, #2250); Anti-c-ErbB2/c-Neu (Ab-3) Mouse mAb (3B5) (EMD Millipore, #OP15); anti-PARP (Cell Signaling Technology, #9542); anti-β-Actin, clone AC-15 (Sigma, A5441); anti-Vinculin (Sigma, V9131); anti-β-Tubulin (BioLegend, # 903401).

Techniques: Inhibition, Phospho-proteomics, Gene Expression, Derivative Assay, RNA Sequencing, RNA Expression, Sequencing, Labeling, Control, Selection, Expressing

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. HCC1954 cells were treated with THZ531 or NVP-2 for 4 hours, after which whole cell lysates were harvested for fluorescent immunoblotting. The molecular weights of the fluorescent protein markers and clone identities for monoclonal antibodies are indicated. Merged images show signals from two primary antibodies raised in different species. B. Volcano plots of gene expression. HCC1954 cells were treated for 4 h with THZ531 (200 nM), or NVP-2 (200 nM), or vehicle control. Total RNA was extracted and subjected to library construction and deep sequencing. Dotted lines in the volcano plots (left) indicate the thresholds used for log-transformed fold change (1, -1). The Tukey box plots (right) illustrate the comparison between the magnitudes of change in significantly up- or down-regulated genes. **** p < 0.0001; ns, not significant (Mann-Whitney test). C. Analysis of gene size among groups of genes impacted by CDK12 or 9 inhibition. **** p < 0.0001; ns, not significant (Mann-Whitney test). D. Traces of RNA-seq coverage over the gene BLM. Note that CDK12 inhibition decreases coverage over exons, but concomitantly produces reads at positions known to harbor intronic polyadenylation. The boxed regions are also displayed with re-scaled y-axis (bottom). E. Metagene profile plots of genes that are commonly downregulated in HCC1954 cells treated with THZ531, NVP-2, or THZ1 and that have been segmented by gene size. Note that CDK12 inhibition is uniquely associated with a size-dependent elongation defect. F. Overrepresentation analysis of significantly downregulated genes in HCC1954 cells that were treated as indicated in the x-axis. Gene sets in the KEGG and Reactome pathway databases were evaluated. Dot color denotes the statistical significance of gene set enrichment within the group of downregulated genes (-log q) and dot size denotes the fraction of downregulated genes corresponding to that gene set. G. Summary plot of normalized enrichment scores (NES) for gene sets that are significantly downregulated in HCC1954 cells treated with CDK12 inhibitor (nominal p-value < 0.05). Blue dots denote gene sets implicated in the DNA damage response. H. Venn diagram for gene sets that are downregulated by CDK12, CDK9, and CDK7 inhibition. Note that gene sets involved in the DNA damage response are specific to CDK12 inhibition. The right bottom illustrates metagene profile plots of genes in the gene set Reactome_ Homologous DNA Pairing and Strand Exchange in HCC1954 cells treated with THZ531, NVP-2, or THZ1 and that have been segmented by gene size.

Article Snippet: The following primary antibodies were purchased and used for fluorescence immunoblotting: RNA polymerase II subunit B1 (phospho CTD Ser-2) Antibody, clone 3E10 (EMD Millipore, #04-1571); Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (Cell Signaling Technology, #13499); Phospho RNA Polymerase II (S2) Antibody, (Bethyl Laboratories, A300-654A); RNA polymerase II subunit B1 (phospho-CTD Ser-5) Antibody, clone 3E8 (EMD Millipore, #04-1572); Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (Cell Signaling Technology, #13523) RNA polymerase II subunit B1 (phospho-CTD Ser-7) Antibody, clone 4E12 (EMD Millipore, #04-1570); RNA Polymerase II Antibody (Bethyl Laboratories, A300-653A); RNA Polymerase II RPB1, clone 8WG16 (BioLegend, #664906); Rpb1 NTD (D8L4Y) Rabbit mAb (Cell Signaling Technology, #14958); c-Myc (clone Y69) Rabbit mAb (Abcam, #ab32072); c-Myc (D84C12) Rabbit mAb (Cell Signaling Technology, #5605); anti-CDK12 (Cell Signaling Technology, #11973); anti-CDK12 (proteintech, #26816-1-AP); anti-CDK12, clone 45F7-H2 (BIO-RAD, #VMA00874); anti-CDK13, clone 46B7-G7 (BIO-RAD, #VMA00875); CDK7 Recombinant Monoclonal Antibody (BL-80-5D4) (Bethyl, #A700-006); CDK7 (MO1) Mouse mAb (Cell Signaling Technology, #2916); CDK8 (D6M3J) Rabbit mAb (Cell Signaling Technology, #17395); CDK9 (C12F7) Rabbit mAb (Cell Signaling Technology, #2316); Anti-CDK19 (Sigma-Aldrich, #HPA007053); JunB Rabbit mAb, clone ARC0268 (ABclonal, #A4848); c-Jun (60A8) Rabbit mAb (Cell Signaling Technology, #9165); c-Fos (9F6) Rabbit mAb (Cell Signaling Technology, #2250); Anti-c-ErbB2/c-Neu (Ab-3) Mouse mAb (3B5) (EMD Millipore, #OP15); anti-PARP (Cell Signaling Technology, #9542); anti-β-Actin, clone AC-15 (Sigma, A5441); anti-Vinculin (Sigma, V9131); anti-β-Tubulin (BioLegend, # 903401).

Techniques: Western Blot, Bioprocessing, Gene Expression, Control, Sequencing, Transformation Assay, Comparison, MANN-WHITNEY, Inhibition, RNA Sequencing

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. A volcano plot of gene expression in SKBR3 cells treated with THZ531 (200 nM, 4 h) compared with vehicle control (0.04% DMSO, v/v). Blue and red dots denote individual genes with significant change in expression (fold of change > 1, q < 0.1). B. (Left) A Tukey box plot indicates that, for genes significantly altered in expression by THZ531 treatment in SKBR3 cells, the magnitude of upregulation is even greater than gene downregulation (**p < 0.01, Mann-Whitney test). (Right) Comparison of gene size among groups of genes that differentially respond to CDK12 inhibition (****p < 0.0001, Mann-Whitney test). C. HCC1954 cells were treated with vehicle control, 200 nM THZ531 or THZ1 for 4 h, followed by lysis with 1x SDS sample buffer and fluorescent immunoblotting. Note that THZ1 treatment caused a complete electrophoretic mobility shift of total RBP1, indicating a substantial loss of CTD phosphorylation. Note that THZ1 also targets CDK12 and CDK13, and thus lacks a desired selectivity. THZ1 was chosen for the current study, instead of a more selective version of CDK7 inhibitor YKL-5-124, primarily because YKL-5-125 does not have any effect on CTD phosphorylation (Olson et al., 2019). D. A volcano plot of gene expression in HCC1954 cells treated with THZ1 (200 nM, 4 h) compared with vehicle control. Blue and red dots are genes with significant change in expression (fold of change > 1, q < 0.1). E. (Left) A Tukey box plot indicating that, for genes with expression significantly altered by THZ1 treatment in HCC1954 cells, the magnitude of downregulation is greater than gene upregulation (****p < 0.01). (Right) A comparison of gene size among groups of genes that differentially respond to CDK12 inhibition (n.s., not significant; Mann-Whitney test). F. Fluorescent immunoblotting of total cell lysates from HCC1954 cells treated with indicated doses of THZ531 for 4 or 24 hours. Note that cells treated with THZ531 for 24 hours demonstrate a reduced protein abundance of ATM, BRCA1, and EGFR, all of which are encoded by large genes (>100 kb). G. (Top) A schematic depicting mRNA with proximal or distal polyA. (Bottom) The percentage of mRNA in each treatment group (HCC1954) showing positive (proximal polyA) or negative (distal polyA) change of proximal polyadenylation site usage (△PPAU). H. Traces of RNA Seq coverage over the MAP3K9 gene. Note that CDK12 inhibition in HCC1954 cells abolishes reads corresponding to a distal polyA site (boxed) while exerting no effect on coverage over the exons.

Article Snippet: The following primary antibodies were purchased and used for fluorescence immunoblotting: RNA polymerase II subunit B1 (phospho CTD Ser-2) Antibody, clone 3E10 (EMD Millipore, #04-1571); Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (Cell Signaling Technology, #13499); Phospho RNA Polymerase II (S2) Antibody, (Bethyl Laboratories, A300-654A); RNA polymerase II subunit B1 (phospho-CTD Ser-5) Antibody, clone 3E8 (EMD Millipore, #04-1572); Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (Cell Signaling Technology, #13523) RNA polymerase II subunit B1 (phospho-CTD Ser-7) Antibody, clone 4E12 (EMD Millipore, #04-1570); RNA Polymerase II Antibody (Bethyl Laboratories, A300-653A); RNA Polymerase II RPB1, clone 8WG16 (BioLegend, #664906); Rpb1 NTD (D8L4Y) Rabbit mAb (Cell Signaling Technology, #14958); c-Myc (clone Y69) Rabbit mAb (Abcam, #ab32072); c-Myc (D84C12) Rabbit mAb (Cell Signaling Technology, #5605); anti-CDK12 (Cell Signaling Technology, #11973); anti-CDK12 (proteintech, #26816-1-AP); anti-CDK12, clone 45F7-H2 (BIO-RAD, #VMA00874); anti-CDK13, clone 46B7-G7 (BIO-RAD, #VMA00875); CDK7 Recombinant Monoclonal Antibody (BL-80-5D4) (Bethyl, #A700-006); CDK7 (MO1) Mouse mAb (Cell Signaling Technology, #2916); CDK8 (D6M3J) Rabbit mAb (Cell Signaling Technology, #17395); CDK9 (C12F7) Rabbit mAb (Cell Signaling Technology, #2316); Anti-CDK19 (Sigma-Aldrich, #HPA007053); JunB Rabbit mAb, clone ARC0268 (ABclonal, #A4848); c-Jun (60A8) Rabbit mAb (Cell Signaling Technology, #9165); c-Fos (9F6) Rabbit mAb (Cell Signaling Technology, #2250); Anti-c-ErbB2/c-Neu (Ab-3) Mouse mAb (3B5) (EMD Millipore, #OP15); anti-PARP (Cell Signaling Technology, #9542); anti-β-Actin, clone AC-15 (Sigma, A5441); anti-Vinculin (Sigma, V9131); anti-β-Tubulin (BioLegend, # 903401).

Techniques: Gene Expression, Control, Expressing, MANN-WHITNEY, Comparison, Inhibition, Lysis, Western Blot, Electrophoretic Mobility Shift Assay, Phospho-proteomics, Quantitative Proteomics, RNA Sequencing

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. (Left) Summary plot of normalized enrichment scores (NES) for gene sets that are significantly upregulated in HCC1954 cells treated with CDK12 inhibitor (nominal p-value < 0.05). Blue dots denote MYC signature. (Right) GSEA plot of Hallmark_MYC Targets V1 for genes altered by THZ531 (200 nM, 4h) in HCC1954 (top) and SKBR3 (bottom) cells. Normalized enrichment score (NES) and p values are indicated. B. Traces of RNA-seq coverage over MYC in cells that were treated as indicated. Enlarged views for traces of CDK9 and CDK7 inhibition are shown as inserts with the y-axis scaled down. C. HCC1954 cells were treated as indicated for 4 h followed by total RNA extraction and reverse transcription. Quantitative PCR was then performed for the indicated genes. Note that MYC expression is increased, while other select DDR genes demonstrated significant downregulation. *p<0.05, **p<0.01, ***p<0.001 (Student’s t tests). D. HER2+ breast cancer cells were treated with THZ531 (200 nM) for the indicated time points. Cell lysates were prepared in SDS sample buffer and subjected to fluorescent immunoblotting. E. HCC1954 cells were treated with increasing concentrations of THZ531 (0, 12.5, 25, 50,100, 200, 400, and 800 nM) for 4 hours. Cell lysates were prepared and analyzed as in (G). F. HCC1954 cells were treated with Actinomycin D at the indicated doses in the presence of vehicle control or THZ531 (400 nM). Four hours post-treatment, cells were lysed with 1x SDS sample buffer, and lysates were prepared for fluorescent immunoblotting. Merged images are shown for blots using antibodies raised in different species. The very left lane was loaded with protein markers that emit near-infrared fluorescence. G. THZ531-induced MYC expression relies on de novo protein synthesis. HCC1954 cells were treated with cycloheximide (6.25 μg/ml) and THZ531 (200 nM), either individually or in combination. Cells were harvested in 3 hours for immunoblotting. H. Tumors with low expression of CDK12 exhibit activation of a MYC signature. (Right) The consolidated MYC signature was developed by combining all 12 gene sets from MSigDB ( http://www.gsea-msigdb.org/ ) and identifying genes whose expression positively correlates with MYC across the 1305 cancer cell lines in the Cancer Cell Line Encyclopedia (CCLE, Pearson correlation r > 0.2). (Left) Expression of the 235 genes was then evaluated in CDK12, CDK7, CDK9-low ovarian tumors in comparison to their high expression counterparts. The top dot plot displays the differential expression of all 235 genes, while the bottom plot shows genes with significant differential expression (q < 0.1; n = 106, 60, 6 for the CDK12, CDK7, CDK9 groups, respectively). *p<0.05, ****p<0.0001 (Mann-Whitney test).

Article Snippet: The following primary antibodies were purchased and used for fluorescence immunoblotting: RNA polymerase II subunit B1 (phospho CTD Ser-2) Antibody, clone 3E10 (EMD Millipore, #04-1571); Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (Cell Signaling Technology, #13499); Phospho RNA Polymerase II (S2) Antibody, (Bethyl Laboratories, A300-654A); RNA polymerase II subunit B1 (phospho-CTD Ser-5) Antibody, clone 3E8 (EMD Millipore, #04-1572); Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (Cell Signaling Technology, #13523) RNA polymerase II subunit B1 (phospho-CTD Ser-7) Antibody, clone 4E12 (EMD Millipore, #04-1570); RNA Polymerase II Antibody (Bethyl Laboratories, A300-653A); RNA Polymerase II RPB1, clone 8WG16 (BioLegend, #664906); Rpb1 NTD (D8L4Y) Rabbit mAb (Cell Signaling Technology, #14958); c-Myc (clone Y69) Rabbit mAb (Abcam, #ab32072); c-Myc (D84C12) Rabbit mAb (Cell Signaling Technology, #5605); anti-CDK12 (Cell Signaling Technology, #11973); anti-CDK12 (proteintech, #26816-1-AP); anti-CDK12, clone 45F7-H2 (BIO-RAD, #VMA00874); anti-CDK13, clone 46B7-G7 (BIO-RAD, #VMA00875); CDK7 Recombinant Monoclonal Antibody (BL-80-5D4) (Bethyl, #A700-006); CDK7 (MO1) Mouse mAb (Cell Signaling Technology, #2916); CDK8 (D6M3J) Rabbit mAb (Cell Signaling Technology, #17395); CDK9 (C12F7) Rabbit mAb (Cell Signaling Technology, #2316); Anti-CDK19 (Sigma-Aldrich, #HPA007053); JunB Rabbit mAb, clone ARC0268 (ABclonal, #A4848); c-Jun (60A8) Rabbit mAb (Cell Signaling Technology, #9165); c-Fos (9F6) Rabbit mAb (Cell Signaling Technology, #2250); Anti-c-ErbB2/c-Neu (Ab-3) Mouse mAb (3B5) (EMD Millipore, #OP15); anti-PARP (Cell Signaling Technology, #9542); anti-β-Actin, clone AC-15 (Sigma, A5441); anti-Vinculin (Sigma, V9131); anti-β-Tubulin (BioLegend, # 903401).

Techniques: RNA Sequencing, Inhibition, RNA Extraction, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control, Fluorescence, Activation Assay, Comparison, Quantitative Proteomics, MANN-WHITNEY