phospho cdk7  (Cell Signaling Technology Inc)

Journal: Nucleic Acids Research

Article Title: RNA polymerase II degradation triggered by DNA repair occurs in trans and independently of how the lesion is recognized

doi: 10.1093/nar/gkaf1416

Figure Lengend Snippet: Transcriptionally inactive RPB1 molecules can be targeted for degradation. ( A ) G1-arrested WT human keratinocytes were incubated for 3 h with the kinase inhibitor DRB (100 µM) and then irradiated with the indicated doses of UV light. Cells were harvested 10 h after irradiation. Relative RPB1 abundance was assessed by western blot using antibodies against the NTD of RPB1 and Vinculin as a loading control. IIo and IIa indicate the phosphorylated and unphosphorylated forms of RPB1. Images of a representative experiment and mean ± SEM of RPB1 abundance relative to 0 J/m 2 condition are shown ( n = 3 independent biological replicates). Two-way ANOVA.** P < .01; *** P < .001; **** P < .0001. ( B ) G1-arrested WT human keratinocytes were irradiated with 40 J/m 2 of UV light. Immediately after irradiation, the CDK7 inhibitor THZ1 (100 nM) or the CDK9 inhibitor BAY 1251152 (400 nM) was added, and cells were harvested 12 h after. Relative RPB1 abundance was assessed as before. IIo and IIa indicate the phosphorylated and unphosphorylated forms of RPB1. Images of a representative experiment and mean ± SEM of RPB1 abundance relative to 0 J/m 2 condition are shown ( n = 3 independent biological replicates). Two-way ANOVA. *** P < .001; **** P < .0001. ( C ) HEK293T human cells were transfected with a plasmid encoding an HA-tagged version of RPB1 that cannot be phosphorylated at serines 2 and 5 because of alanine replacement (A2A5-RPB1). Forty-eight hours after transfection, cells were irradiated with the indicated doses of UV light and harvested after 6 or 12 h. Relative levels of HA-tagged A2A5-RPB1 were assessed by western blot using antibodies against the HA tag and Vinculin as a loading control. Images of a representative experiment and mean ± SEM of HA-tagged A2A5-RPB1 abundance relative to 0 J/m 2 condition are shown ( n = 4 independent biological replicates). Two-way ANOVA.** P < .01; *** P < .001.

Article Snippet: When indicated, cells were incubated with the following drugs, which were kept in culture medium until harvesting: IlludinS (MedChemExpress #HY-125098); MG132 (Selleckchem #S2619); Spironolactone (Selleckchem #S4054); α-amanitin (Sigma #A2263); Aphidicolin (MedChemExpress #HY-N6733); ATR inhibitor VE822 (Selleckchem # S7102); ATM inhibitor KU60019 (Selleckchem #S1570); DNA-PK inhibitor NU7441 (Selleckchem #S2638); GSK3 inhibitor CHIR-99021 (Sigma); 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DRB) (Sigma #D1916); CDK7 inhibitor THZ1 (Selleckchem #S7449); CDK9 inhibitor BAY1251152 (Selleckchem #S8730); and MLN4924 (Selleckchem #S7109).

Techniques: Incubation, Irradiation, Western Blot, Control, Transfection, Plasmid Preparation

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: Gene expression and protein analysis of human FLC samples (A) mRNA expression of various known super-enhancer-driven genes in FLC were assessed in human tumor samples ( n = 35) and paired normal liver samples ( n = 10) ( t test, ∗∗ p < 0.0001). (B) Individual patient tumor samples with matched normal liver (FCF 82, 83, and 106) were evaluated for phosphorylated RPB-1 (Ser 2, 5, and 7) and CDK7. Fibrolamellar cancer was confirmed by demonstrating presence (tumor) and absence (normal) of the DNAJ-PKAc oncoprotein. (C) A phosphorylation index was calculated for each sample and phosphorylation level is presented as Phosphorylation index (Tumor)/Phosphorylation index (Adjacent) for RPB-1 (Ser 2, 5, and 7) and CDK7. Seven to nine sets of samples are shown from FLC tumor and adjacent normal tissue samples (including samples from ). (D) The data suggest that DNAJ-PKAc is correlated with heightened CDK7 activity (dotted green line). CDK7 forms a trimeric complex with cyclin H and MAT1 to phosphorylate serine-5 (preferentially) and serine-7 residues of a 52 heptad repeat in RNA polymerase II. Similarly, CDK9 dimerizes with cyclin T to preferentially phosphorylate serine-2 residues. SY-5609 and YKL-5-124 inhibit CDK7 activity; similarly VIP-152 and NVP-2 inhibit CDK9 activity. [Image created with Biorender.com ].

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Gene Expression, Expressing, Phospho-proteomics, Activity Assay

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 regulation of RNA Polymerase II phosphorylation and super-enhancer gene expression (A) Protein analysis for phosphorylated RNA polymerase II (serine-2, serine-5, and serine-7), CDK2 and CDK7 was performed and phosphorylation levels were quantified using ImageJ. Shown are two separate biological replicates for each line. Statistically significant differences between HepG2 and H33 are shown ( t test, ∗ p < 0.05). (B) RNA sequencing of HepG2 cells and H33 cells treated at varying doses of SY-5609 for 24 h ( n = 6 biological replicates per group) were evaluated for prominent super-enhancer-associated genes, including SLC16A14 and LINC00473 . Findings from the H33 clone were confirmed in a separate DNAJB1-PRKACA -expressing clone (H12) ( t test, ∗∗ p < 0.01). (C) DNAJB1-PRKACA -expressing H33 cells were treated with a selective CDK7 inhibitor, SY5609 (100 nM, 1 μM, and 10 μM), or DMSO control (0) for 24 h ( n = 3 biological replicates per group, two representative images shown per group for western blot). Known substrate targets of CDK7 were assessed including RNA Pol II CTD (Ser 2, 5, and 7), Thr160 phosphorylated CDK2 (pCDK2) and Thr170 phosphorylated CDK7 (pCDK7) ( t test, ∗ p < 0.05). (D and E) To assess for CDK7-dependent expression of FLC-specific genes, H33 cells were treated with SY-5609 (1–300 nM) for 24 h and levels of mRNA expression (RT-qPCR) versus DMSO control were evaluated, including SLC16A14 and LINC00473 . This was repeated with a separate covalent-binding selective and specific CDK7 inhibitor (YKL-5-124). Shown are three biological replicates per drug dose per mRNA with statistical significance denoted as compared to DMSO control ( t test, ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Phospho-proteomics, Gene Expression, RNA Sequencing, Expressing, Control, Western Blot, Quantitative RT-PCR, Binding Assay

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 is a novel therapeutic target in DNAJB1-PRKACA-expressing cells (A) To assess for CDK7 effect on cell viability, HepG2 cells and H33 cells underwent 48-h drug treatment with either SY5609 (1 pM–5 μM) or YKL-5-124 (100 pM–10 μM). Percent viability was determined by normalizing to control (DMSO treated). To confirm the findings in the DNAJB1-PRKACA -expressing H33 cells, a separate clone (H12) was tested with the same drugs over the same dose range. In each figure, the LC 50 (IC 50 ) is represented by the straight line. (B) HepG2 cells and H33 cells were synchronized and treated with either DMSO or SY5609 (1 μm) for 24 h. Percent of cells in G0/G1, S, and G2/M were determined by flow cytometry. Shown are four biological replicates per cell line per treatment ( t test, ∗ p = 0.001, ∗∗ p < 0.0001). (C) HepG2 cells and H33 cells were treated with DMSO (control) or increasing doses of SY5609 for 24 h and caspase 3/7 activity measured. To confirm the increased apoptotic activity in the H33 cells, a separate clone (H12) was utilized. ( t test ∗ p < 0.0001, for H33 vs. HepG2 and H12 vs. HepG2). (D) To validate the results, PARP and cleaved-PARP (marker for apoptosis) protein were evaluated in HepG2 and H33 cells, using two separate antibodies that either recognize both PARP and cleaved-PARP (top bands) or only cleaved-PARP alone (bottom band). (E) Primary human hepatocytes (PHHs) isolated fresh from human donor liver transplant specimens and H33 cells were treated with DMSO, SY5609 100 nM, or SY5609 1 μM for 24 and 48 h. The percent of viable cells was determined by normalizing to DMSO control. There were four biological replicates per group per time/treatment dose ( t test ∗ p = 0.02, ∗∗ p = 0.01, ∗∗∗ p < 0.0001). (F) PHHs and H33 cells were treated with SY5609 (100 pM–5 μM) for 48, 72, or 120 h and percent viability assessed, normalized to DMSO control. The LC 50 (IC 50 ) is demarcated by the solid line. There were four biological replicates for each drug dose at each time point for each line (PHH and H33).

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Expressing, Control, Flow Cytometry, Activity Assay, Marker, Isolation

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 inhibition is lethal in human FLC (A) An FLC cell line derived from human FLC (FLC-H) was grown for six days (control) or treated with DMSO (Control DMSO) versus SY5609 at two separate doses (500 nM and 1 μM). Viable cells were quantified at day zero and day six. Additionally, the day 6 percent survival compared to controls was calculated. Shown are two separate experiments with three biological replicates per experiment ( t test ∗ p < 0.01, ∗∗ p < 0.001 versus control and control DMSO). (B) In a separate experiment, an FLC cell line was derived from a patient FLC liver tumor (FLC1025), and another discrete cell line was derived from patient metastatic FLC tumor implants (FLCMet). Each line was treated with SY5609 (10 nM–10 μM). Percent survival was determined normalized to DMSO control, with three biological replicates per dose. Shown is the dose response with curve (and 95% CI) and LC 50 (IC 50 ) demonstrated by the straight line (LC 50 FLC1025 ∼300 nM, LC 50 FLCMet ∼20 nM). (C) Human tissue slices derived from a patient with FLC (FLC217) were treated with DMSO or SY5609 (500 nM), and percent viability determined compared to DMSO control (∗∗ p = 0.003). (D) In a separate experiment, tissue slices derived from human FLC grown in a patient derived xenograft (PDX) model were treated with DMSO, SY5609 (100 nM, 500 nM, and 1 μM) and staurosporine (STS) 500 nM (positive control). There were 3–4 biological replicates per group ( t test ∗ p < 0.01, ∗∗ p < 0.001).

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Inhibition, Derivative Assay, Control, Positive Control

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: Synergistic combination between CDK7 and CDK9 inhibition in vitro (A) DNAJB1-PRKACA expressing H33 cells were treated with SY-5609 alone, VIP-152 alone, or in combination for 24 h. Protein was isolated and western blot performed for measurement and quantification of phosphorylated RPB-1 (Ser 2, 5, and 7) ( n = 3 biological replicates per group, two representative western blots are shown, t test, ∗ p < 0.05). (B) Expression of SLC16A14 and LINC00473 was determined in H33 cells treated with SY-5609 alone (blue), VIP-152 alone (purple), or in combination (green). Statistical significance is denoted as compared to control ( t test, ∗∗ p < 0.01, ∗ p < 0.05). (C and D) Synergistic response to combination therapy of SY-5609 and VIP-152 in H33 cells was determined with potent reduction in percent survival (normalized to DMSO control). Depicted is a dose-response curve which demonstrates shift of the curve to the left for SY-5609 with increasing concentration of VIP-152 up to a dose of 30 nM VIP-152. The drug combination showed strong synergy using all metrics including HSA (mean 18.73, p = 6.31e-5), Bliss (mean 14.55, p = 3.46e-4), Loewe (mean 15.48, p = 1.98e-4), and ZIP (mean 13.54, p = 6.04e-4). The strongest synergistic doses occurred at 30 nM SY-5609 + 30 nM VIP152 (synergy score ∼38) and 10 nM SY-5609 + 30 nM VIP152 (synergy score ∼37).

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Inhibition, In Vitro, Expressing, Isolation, Western Blot, Control, Concentration Assay

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 and CDK9 inhibition in an organoid model (A) Tissue from a patient with FLC (FLC4-PDX) was propagated and developed into a patient-derived cancer organoid model, FLC4 (organoid). Western blot shows the presence of DNAJ-PKAc oncoprotein and phosphorylated RPB-1 (Ser 2, 5, and 7) in the organoid compared to immortalized human hepatocytes (IHH, n = 4) which displays only native PKAc and low levels of phosphorylated RPB-1. Phosphorylated RPB-1 (Ser 2, 5, and 7) was measured and quantified. Statistical significance was calculated ( t test, ∗∗∗ p < 0.001, ∗∗ p < 0.01). (B) RNA was isolated from organoids treated with SY-5609 (1 nM, 3 nM, 10 nM, and 30 nM) and VIP-152 (30 nM, 100 nM, 300 nM, and 500 nM) and SLC16A14 and LINC00473 expression was determined by qPCR relative to DMSO control. Due to the limited availability of organoid tissue available only one replicate is represented per dose.

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Inhibition, Derivative Assay, Western Blot, Isolation, Expressing, Control

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: Synergistic combination of CDK7 and CDK9 inhibition in an organoid model (A) FLC organoids were treated with SY-5609 alone, VIP-152 alone or in combination and western blot was performed showing phosphorylated RPB-1 (Ser 2, 5, and 7). Protein levels were measured and quantified. (B) FLC organoids were treated with SY-509 alone and in combination with VIP-152 at various doses which showed a dose-dependent decrease in cell survival as compared to DMSO. (C) RNA was isolated from FLC organoids treated with combination SY-5609 and VIP-152 for 96 h (1nM/10 nM, 10nM/10 nM, and 100nM/100 nM) and expression of SLC16A14 and LINC00473 was determined. Due to the limited availability of organoid tissue available only one replicate is represented per dose.

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Inhibition, Western Blot, Isolation, Expressing

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: Gene expression and protein analysis of human FLC samples (A) mRNA expression of various known super-enhancer-driven genes in FLC were assessed in human tumor samples ( n = 35) and paired normal liver samples ( n = 10) ( t test, ∗∗ p < 0.0001). (B) Individual patient tumor samples with matched normal liver (FCF 82, 83, and 106) were evaluated for phosphorylated RPB-1 (Ser 2, 5, and 7) and CDK7. Fibrolamellar cancer was confirmed by demonstrating presence (tumor) and absence (normal) of the DNAJ-PKAc oncoprotein. (C) A phosphorylation index was calculated for each sample and phosphorylation level is presented as Phosphorylation index (Tumor)/Phosphorylation index (Adjacent) for RPB-1 (Ser 2, 5, and 7) and CDK7. Seven to nine sets of samples are shown from FLC tumor and adjacent normal tissue samples (including samples from ). (D) The data suggest that DNAJ-PKAc is correlated with heightened CDK7 activity (dotted green line). CDK7 forms a trimeric complex with cyclin H and MAT1 to phosphorylate serine-5 (preferentially) and serine-7 residues of a 52 heptad repeat in RNA polymerase II. Similarly, CDK9 dimerizes with cyclin T to preferentially phosphorylate serine-2 residues. SY-5609 and YKL-5-124 inhibit CDK7 activity; similarly VIP-152 and NVP-2 inhibit CDK9 activity. [Image created with Biorender.com ].

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

Techniques: Gene Expression, Expressing, Phospho-proteomics, Activity Assay

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 regulation of RNA Polymerase II phosphorylation and super-enhancer gene expression (A) Protein analysis for phosphorylated RNA polymerase II (serine-2, serine-5, and serine-7), CDK2 and CDK7 was performed and phosphorylation levels were quantified using ImageJ. Shown are two separate biological replicates for each line. Statistically significant differences between HepG2 and H33 are shown ( t test, ∗ p < 0.05). (B) RNA sequencing of HepG2 cells and H33 cells treated at varying doses of SY-5609 for 24 h ( n = 6 biological replicates per group) were evaluated for prominent super-enhancer-associated genes, including SLC16A14 and LINC00473 . Findings from the H33 clone were confirmed in a separate DNAJB1-PRKACA -expressing clone (H12) ( t test, ∗∗ p < 0.01). (C) DNAJB1-PRKACA -expressing H33 cells were treated with a selective CDK7 inhibitor, SY5609 (100 nM, 1 μM, and 10 μM), or DMSO control (0) for 24 h ( n = 3 biological replicates per group, two representative images shown per group for western blot). Known substrate targets of CDK7 were assessed including RNA Pol II CTD (Ser 2, 5, and 7), Thr160 phosphorylated CDK2 (pCDK2) and Thr170 phosphorylated CDK7 (pCDK7) ( t test, ∗ p < 0.05). (D and E) To assess for CDK7-dependent expression of FLC-specific genes, H33 cells were treated with SY-5609 (1–300 nM) for 24 h and levels of mRNA expression (RT-qPCR) versus DMSO control were evaluated, including SLC16A14 and LINC00473 . This was repeated with a separate covalent-binding selective and specific CDK7 inhibitor (YKL-5-124). Shown are three biological replicates per drug dose per mRNA with statistical significance denoted as compared to DMSO control ( t test, ∗ p < 0.05, ∗∗ p < 0.01).

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

Techniques: Phospho-proteomics, Gene Expression, RNA Sequencing, Expressing, Control, Western Blot, Quantitative RT-PCR, Binding Assay

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 is a novel therapeutic target in DNAJB1-PRKACA-expressing cells (A) To assess for CDK7 effect on cell viability, HepG2 cells and H33 cells underwent 48-h drug treatment with either SY5609 (1 pM–5 μM) or YKL-5-124 (100 pM–10 μM). Percent viability was determined by normalizing to control (DMSO treated). To confirm the findings in the DNAJB1-PRKACA -expressing H33 cells, a separate clone (H12) was tested with the same drugs over the same dose range. In each figure, the LC 50 (IC 50 ) is represented by the straight line. (B) HepG2 cells and H33 cells were synchronized and treated with either DMSO or SY5609 (1 μm) for 24 h. Percent of cells in G0/G1, S, and G2/M were determined by flow cytometry. Shown are four biological replicates per cell line per treatment ( t test, ∗ p = 0.001, ∗∗ p < 0.0001). (C) HepG2 cells and H33 cells were treated with DMSO (control) or increasing doses of SY5609 for 24 h and caspase 3/7 activity measured. To confirm the increased apoptotic activity in the H33 cells, a separate clone (H12) was utilized. ( t test ∗ p < 0.0001, for H33 vs. HepG2 and H12 vs. HepG2). (D) To validate the results, PARP and cleaved-PARP (marker for apoptosis) protein were evaluated in HepG2 and H33 cells, using two separate antibodies that either recognize both PARP and cleaved-PARP (top bands) or only cleaved-PARP alone (bottom band). (E) Primary human hepatocytes (PHHs) isolated fresh from human donor liver transplant specimens and H33 cells were treated with DMSO, SY5609 100 nM, or SY5609 1 μM for 24 and 48 h. The percent of viable cells was determined by normalizing to DMSO control. There were four biological replicates per group per time/treatment dose ( t test ∗ p = 0.02, ∗∗ p = 0.01, ∗∗∗ p < 0.0001). (F) PHHs and H33 cells were treated with SY5609 (100 pM–5 μM) for 48, 72, or 120 h and percent viability assessed, normalized to DMSO control. The LC 50 (IC 50 ) is demarcated by the solid line. There were four biological replicates for each drug dose at each time point for each line (PHH and H33).

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

Techniques: Expressing, Control, Flow Cytometry, Activity Assay, Marker, Isolation

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 inhibition is lethal in human FLC (A) An FLC cell line derived from human FLC (FLC-H) was grown for six days (control) or treated with DMSO (Control DMSO) versus SY5609 at two separate doses (500 nM and 1 μM). Viable cells were quantified at day zero and day six. Additionally, the day 6 percent survival compared to controls was calculated. Shown are two separate experiments with three biological replicates per experiment ( t test ∗ p < 0.01, ∗∗ p < 0.001 versus control and control DMSO). (B) In a separate experiment, an FLC cell line was derived from a patient FLC liver tumor (FLC1025), and another discrete cell line was derived from patient metastatic FLC tumor implants (FLCMet). Each line was treated with SY5609 (10 nM–10 μM). Percent survival was determined normalized to DMSO control, with three biological replicates per dose. Shown is the dose response with curve (and 95% CI) and LC 50 (IC 50 ) demonstrated by the straight line (LC 50 FLC1025 ∼300 nM, LC 50 FLCMet ∼20 nM). (C) Human tissue slices derived from a patient with FLC (FLC217) were treated with DMSO or SY5609 (500 nM), and percent viability determined compared to DMSO control (∗∗ p = 0.003). (D) In a separate experiment, tissue slices derived from human FLC grown in a patient derived xenograft (PDX) model were treated with DMSO, SY5609 (100 nM, 500 nM, and 1 μM) and staurosporine (STS) 500 nM (positive control). There were 3–4 biological replicates per group ( t test ∗ p < 0.01, ∗∗ p < 0.001).

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

Techniques: Inhibition, Derivative Assay, Control, Positive Control

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: Synergistic combination between CDK7 and CDK9 inhibition in vitro (A) DNAJB1-PRKACA expressing H33 cells were treated with SY-5609 alone, VIP-152 alone, or in combination for 24 h. Protein was isolated and western blot performed for measurement and quantification of phosphorylated RPB-1 (Ser 2, 5, and 7) ( n = 3 biological replicates per group, two representative western blots are shown, t test, ∗ p < 0.05). (B) Expression of SLC16A14 and LINC00473 was determined in H33 cells treated with SY-5609 alone (blue), VIP-152 alone (purple), or in combination (green). Statistical significance is denoted as compared to control ( t test, ∗∗ p < 0.01, ∗ p < 0.05). (C and D) Synergistic response to combination therapy of SY-5609 and VIP-152 in H33 cells was determined with potent reduction in percent survival (normalized to DMSO control). Depicted is a dose-response curve which demonstrates shift of the curve to the left for SY-5609 with increasing concentration of VIP-152 up to a dose of 30 nM VIP-152. The drug combination showed strong synergy using all metrics including HSA (mean 18.73, p = 6.31e-5), Bliss (mean 14.55, p = 3.46e-4), Loewe (mean 15.48, p = 1.98e-4), and ZIP (mean 13.54, p = 6.04e-4). The strongest synergistic doses occurred at 30 nM SY-5609 + 30 nM VIP152 (synergy score ∼38) and 10 nM SY-5609 + 30 nM VIP152 (synergy score ∼37).

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

Techniques: Inhibition, In Vitro, Expressing, Isolation, Western Blot, Control, Concentration Assay

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 and CDK9 inhibition in an organoid model (A) Tissue from a patient with FLC (FLC4-PDX) was propagated and developed into a patient-derived cancer organoid model, FLC4 (organoid). Western blot shows the presence of DNAJ-PKAc oncoprotein and phosphorylated RPB-1 (Ser 2, 5, and 7) in the organoid compared to immortalized human hepatocytes (IHH, n = 4) which displays only native PKAc and low levels of phosphorylated RPB-1. Phosphorylated RPB-1 (Ser 2, 5, and 7) was measured and quantified. Statistical significance was calculated ( t test, ∗∗∗ p < 0.001, ∗∗ p < 0.01). (B) RNA was isolated from organoids treated with SY-5609 (1 nM, 3 nM, 10 nM, and 30 nM) and VIP-152 (30 nM, 100 nM, 300 nM, and 500 nM) and SLC16A14 and LINC00473 expression was determined by qPCR relative to DMSO control. Due to the limited availability of organoid tissue available only one replicate is represented per dose.

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

Techniques: Inhibition, Derivative Assay, Western Blot, Isolation, Expressing, Control

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: Synergistic combination of CDK7 and CDK9 inhibition in an organoid model (A) FLC organoids were treated with SY-5609 alone, VIP-152 alone or in combination and western blot was performed showing phosphorylated RPB-1 (Ser 2, 5, and 7). Protein levels were measured and quantified. (B) FLC organoids were treated with SY-509 alone and in combination with VIP-152 at various doses which showed a dose-dependent decrease in cell survival as compared to DMSO. (C) RNA was isolated from FLC organoids treated with combination SY-5609 and VIP-152 for 96 h (1nM/10 nM, 10nM/10 nM, and 100nM/100 nM) and expression of SLC16A14 and LINC00473 was determined. Due to the limited availability of organoid tissue available only one replicate is represented per dose.

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

Techniques: Inhibition, Western Blot, Isolation, Expressing

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: Gene expression and protein analysis of human FLC samples (A) mRNA expression of various known super-enhancer-driven genes in FLC were assessed in human tumor samples ( n = 35) and paired normal liver samples ( n = 10) ( t test, ∗∗ p < 0.0001). (B) Individual patient tumor samples with matched normal liver (FCF 82, 83, and 106) were evaluated for phosphorylated RPB-1 (Ser 2, 5, and 7) and CDK7. Fibrolamellar cancer was confirmed by demonstrating presence (tumor) and absence (normal) of the DNAJ-PKAc oncoprotein. (C) A phosphorylation index was calculated for each sample and phosphorylation level is presented as Phosphorylation index (Tumor)/Phosphorylation index (Adjacent) for RPB-1 (Ser 2, 5, and 7) and CDK7. Seven to nine sets of samples are shown from FLC tumor and adjacent normal tissue samples (including samples from ). (D) The data suggest that DNAJ-PKAc is correlated with heightened CDK7 activity (dotted green line). CDK7 forms a trimeric complex with cyclin H and MAT1 to phosphorylate serine-5 (preferentially) and serine-7 residues of a 52 heptad repeat in RNA polymerase II. Similarly, CDK9 dimerizes with cyclin T to preferentially phosphorylate serine-2 residues. SY-5609 and YKL-5-124 inhibit CDK7 activity; similarly VIP-152 and NVP-2 inhibit CDK9 activity. [Image created with Biorender.com ].

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Gene Expression, Expressing, Phospho-proteomics, Activity Assay

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 regulation of RNA Polymerase II phosphorylation and super-enhancer gene expression (A) Protein analysis for phosphorylated RNA polymerase II (serine-2, serine-5, and serine-7), CDK2 and CDK7 was performed and phosphorylation levels were quantified using ImageJ. Shown are two separate biological replicates for each line. Statistically significant differences between HepG2 and H33 are shown ( t test, ∗ p < 0.05). (B) RNA sequencing of HepG2 cells and H33 cells treated at varying doses of SY-5609 for 24 h ( n = 6 biological replicates per group) were evaluated for prominent super-enhancer-associated genes, including SLC16A14 and LINC00473 . Findings from the H33 clone were confirmed in a separate DNAJB1-PRKACA -expressing clone (H12) ( t test, ∗∗ p < 0.01). (C) DNAJB1-PRKACA -expressing H33 cells were treated with a selective CDK7 inhibitor, SY5609 (100 nM, 1 μM, and 10 μM), or DMSO control (0) for 24 h ( n = 3 biological replicates per group, two representative images shown per group for western blot). Known substrate targets of CDK7 were assessed including RNA Pol II CTD (Ser 2, 5, and 7), Thr160 phosphorylated CDK2 (pCDK2) and Thr170 phosphorylated CDK7 (pCDK7) ( t test, ∗ p < 0.05). (D and E) To assess for CDK7-dependent expression of FLC-specific genes, H33 cells were treated with SY-5609 (1–300 nM) for 24 h and levels of mRNA expression (RT-qPCR) versus DMSO control were evaluated, including SLC16A14 and LINC00473 . This was repeated with a separate covalent-binding selective and specific CDK7 inhibitor (YKL-5-124). Shown are three biological replicates per drug dose per mRNA with statistical significance denoted as compared to DMSO control ( t test, ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Phospho-proteomics, Gene Expression, RNA Sequencing, Expressing, Control, Western Blot, Quantitative RT-PCR, Binding Assay

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 is a novel therapeutic target in DNAJB1-PRKACA-expressing cells (A) To assess for CDK7 effect on cell viability, HepG2 cells and H33 cells underwent 48-h drug treatment with either SY5609 (1 pM–5 μM) or YKL-5-124 (100 pM–10 μM). Percent viability was determined by normalizing to control (DMSO treated). To confirm the findings in the DNAJB1-PRKACA -expressing H33 cells, a separate clone (H12) was tested with the same drugs over the same dose range. In each figure, the LC 50 (IC 50 ) is represented by the straight line. (B) HepG2 cells and H33 cells were synchronized and treated with either DMSO or SY5609 (1 μm) for 24 h. Percent of cells in G0/G1, S, and G2/M were determined by flow cytometry. Shown are four biological replicates per cell line per treatment ( t test, ∗ p = 0.001, ∗∗ p < 0.0001). (C) HepG2 cells and H33 cells were treated with DMSO (control) or increasing doses of SY5609 for 24 h and caspase 3/7 activity measured. To confirm the increased apoptotic activity in the H33 cells, a separate clone (H12) was utilized. ( t test ∗ p < 0.0001, for H33 vs. HepG2 and H12 vs. HepG2). (D) To validate the results, PARP and cleaved-PARP (marker for apoptosis) protein were evaluated in HepG2 and H33 cells, using two separate antibodies that either recognize both PARP and cleaved-PARP (top bands) or only cleaved-PARP alone (bottom band). (E) Primary human hepatocytes (PHHs) isolated fresh from human donor liver transplant specimens and H33 cells were treated with DMSO, SY5609 100 nM, or SY5609 1 μM for 24 and 48 h. The percent of viable cells was determined by normalizing to DMSO control. There were four biological replicates per group per time/treatment dose ( t test ∗ p = 0.02, ∗∗ p = 0.01, ∗∗∗ p < 0.0001). (F) PHHs and H33 cells were treated with SY5609 (100 pM–5 μM) for 48, 72, or 120 h and percent viability assessed, normalized to DMSO control. The LC 50 (IC 50 ) is demarcated by the solid line. There were four biological replicates for each drug dose at each time point for each line (PHH and H33).

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Expressing, Control, Flow Cytometry, Activity Assay, Marker, Isolation

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 inhibition is lethal in human FLC (A) An FLC cell line derived from human FLC (FLC-H) was grown for six days (control) or treated with DMSO (Control DMSO) versus SY5609 at two separate doses (500 nM and 1 μM). Viable cells were quantified at day zero and day six. Additionally, the day 6 percent survival compared to controls was calculated. Shown are two separate experiments with three biological replicates per experiment ( t test ∗ p < 0.01, ∗∗ p < 0.001 versus control and control DMSO). (B) In a separate experiment, an FLC cell line was derived from a patient FLC liver tumor (FLC1025), and another discrete cell line was derived from patient metastatic FLC tumor implants (FLCMet). Each line was treated with SY5609 (10 nM–10 μM). Percent survival was determined normalized to DMSO control, with three biological replicates per dose. Shown is the dose response with curve (and 95% CI) and LC 50 (IC 50 ) demonstrated by the straight line (LC 50 FLC1025 ∼300 nM, LC 50 FLCMet ∼20 nM). (C) Human tissue slices derived from a patient with FLC (FLC217) were treated with DMSO or SY5609 (500 nM), and percent viability determined compared to DMSO control (∗∗ p = 0.003). (D) In a separate experiment, tissue slices derived from human FLC grown in a patient derived xenograft (PDX) model were treated with DMSO, SY5609 (100 nM, 500 nM, and 1 μM) and staurosporine (STS) 500 nM (positive control). There were 3–4 biological replicates per group ( t test ∗ p < 0.01, ∗∗ p < 0.001).

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Inhibition, Derivative Assay, Control, Positive Control

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: Synergistic combination between CDK7 and CDK9 inhibition in vitro (A) DNAJB1-PRKACA expressing H33 cells were treated with SY-5609 alone, VIP-152 alone, or in combination for 24 h. Protein was isolated and western blot performed for measurement and quantification of phosphorylated RPB-1 (Ser 2, 5, and 7) ( n = 3 biological replicates per group, two representative western blots are shown, t test, ∗ p < 0.05). (B) Expression of SLC16A14 and LINC00473 was determined in H33 cells treated with SY-5609 alone (blue), VIP-152 alone (purple), or in combination (green). Statistical significance is denoted as compared to control ( t test, ∗∗ p < 0.01, ∗ p < 0.05). (C and D) Synergistic response to combination therapy of SY-5609 and VIP-152 in H33 cells was determined with potent reduction in percent survival (normalized to DMSO control). Depicted is a dose-response curve which demonstrates shift of the curve to the left for SY-5609 with increasing concentration of VIP-152 up to a dose of 30 nM VIP-152. The drug combination showed strong synergy using all metrics including HSA (mean 18.73, p = 6.31e-5), Bliss (mean 14.55, p = 3.46e-4), Loewe (mean 15.48, p = 1.98e-4), and ZIP (mean 13.54, p = 6.04e-4). The strongest synergistic doses occurred at 30 nM SY-5609 + 30 nM VIP152 (synergy score ∼38) and 10 nM SY-5609 + 30 nM VIP152 (synergy score ∼37).

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Inhibition, In Vitro, Expressing, Isolation, Western Blot, Control, Concentration Assay

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 and CDK9 inhibition in an organoid model (A) Tissue from a patient with FLC (FLC4-PDX) was propagated and developed into a patient-derived cancer organoid model, FLC4 (organoid). Western blot shows the presence of DNAJ-PKAc oncoprotein and phosphorylated RPB-1 (Ser 2, 5, and 7) in the organoid compared to immortalized human hepatocytes (IHH, n = 4) which displays only native PKAc and low levels of phosphorylated RPB-1. Phosphorylated RPB-1 (Ser 2, 5, and 7) was measured and quantified. Statistical significance was calculated ( t test, ∗∗∗ p < 0.001, ∗∗ p < 0.01). (B) RNA was isolated from organoids treated with SY-5609 (1 nM, 3 nM, 10 nM, and 30 nM) and VIP-152 (30 nM, 100 nM, 300 nM, and 500 nM) and SLC16A14 and LINC00473 expression was determined by qPCR relative to DMSO control. Due to the limited availability of organoid tissue available only one replicate is represented per dose.

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Inhibition, Derivative Assay, Western Blot, Isolation, Expressing, Control

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: Synergistic combination of CDK7 and CDK9 inhibition in an organoid model (A) FLC organoids were treated with SY-5609 alone, VIP-152 alone or in combination and western blot was performed showing phosphorylated RPB-1 (Ser 2, 5, and 7). Protein levels were measured and quantified. (B) FLC organoids were treated with SY-509 alone and in combination with VIP-152 at various doses which showed a dose-dependent decrease in cell survival as compared to DMSO. (C) RNA was isolated from FLC organoids treated with combination SY-5609 and VIP-152 for 96 h (1nM/10 nM, 10nM/10 nM, and 100nM/100 nM) and expression of SLC16A14 and LINC00473 was determined. Due to the limited availability of organoid tissue available only one replicate is represented per dose.

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Inhibition, Western Blot, Isolation, Expressing

Journal: Nucleic Acids Research

Article Title: CDK7–CDK11 axis in spliceosome regulation and pre-mRNA splicing

doi: 10.1093/nar/gkaf1343

Figure Lengend Snippet: CDK7 transiently binds CDK11. ( A ) Schematic representation of proteomic BioID experiment for identification of proteins proximal to CDK11. The N-terminus of CDK11 fused to biotin ligase (BirA) biotinylates nearby proteins in cells. The biotinylated proteins are purified and identified by mass spectrometry. ( B ) Volcano plot of proteins identified in CDK11 BioID. Splicing factors are marked in orange, components of the CDK11 complex in blue, CDK7 in cyan and other proteins in green. ( C ) Immunoblot analysis of immunoprecipitations of endogenous CDK11 after 4 h treatment with 50 nM SY-351 in HCT116 cells. Detected proteins are indicated on the right. IgG = antibody control.

Article Snippet: Blocking was done with 3% fetal bovine serum (FBS) in PBS for 1 h. Subsequently, cells were incubated with primary antibodies (P-CDK11 Ab1, P-CDK11 Ab2, 1:100 dilution), SF3B1 (MBL, MB-D221-3, 1:300 dilution), CDK11 (Abcam, ab19393, 1:400 dilution), and CDK7 (Bethyl, A300-405A, 1:400 dilution) for 1 h and washed 3 × 5 min with PBS.

Techniques: Purification, Mass Spectrometry, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: CDK7–CDK11 axis in spliceosome regulation and pre-mRNA splicing

doi: 10.1093/nar/gkaf1343

Figure Lengend Snippet: CDK11 is phosphorylated on canonical activating Thr595 in cells. ( A ) Amino acid sequence of T-loop of CDK11B. Activating Thr595 is in red. Peptide sequences used for the production of three (Ab1, Ab2, and Ab3) phospho-specific Thr595 antibodies are indicated by black bars. ( B ) Multiple protein sequence alignment of T-loop of CDKs. Activating Thr residues are highlighted in red, Ser corresponding to Ser164 in CDK7 is in orange. xDFG and APE motifs are indicated. ( C ) Immunoblot analysis of CDK11 in HCT116 cells upon treatment with control or CDK11 siRNAs for 40 h. Bands corresponding to CDK11 110 and P-CDK11 220 (monitored by Ab3) are marked on the right with arrows. “Long” and “short” corresponds to long and short exposure of the film. Asterisk denotes nonspecific bands. ( D ) Immunoblot analysis of CDK11 in HCT116 cells upon treatment with 50 nM SY-351 or control DMSO for 4 h. See Fig. for the legend. ( E ) Immunofluorescence microscopy of HCT116 cells using DAPI stain and the P-CDK11 220 (Ab2) and SF3B1 antibodies upon 100 nM treatment with SY-351 or control DMSO for 4 h; scale bar = 10 µm. ( F ) Quantification of P-CDK11 intensity (monitored by Ab2) in Fig. . Box plots represent median and IQR, whiskers extend to the furthest value inside 1.5 × IQR, asterisk: P < 0.05.

Article Snippet: Blocking was done with 3% fetal bovine serum (FBS) in PBS for 1 h. Subsequently, cells were incubated with primary antibodies (P-CDK11 Ab1, P-CDK11 Ab2, 1:100 dilution), SF3B1 (MBL, MB-D221-3, 1:300 dilution), CDK11 (Abcam, ab19393, 1:400 dilution), and CDK7 (Bethyl, A300-405A, 1:400 dilution) for 1 h and washed 3 × 5 min with PBS.

Techniques: Sequencing, Western Blot, Control, Immunofluorescence, Microscopy, Staining

Journal: Nucleic Acids Research

Article Title: CDK7–CDK11 axis in spliceosome regulation and pre-mRNA splicing

doi: 10.1093/nar/gkaf1343

Figure Lengend Snippet: CDK7 is required for the formation of P-CDK11 220 and active spliceosomes. ( A ) Immunoblot analyses of indicated proteins in HCT116 cells treated with control DMSO or 50 nM SY-351 for 2 h and separated into cytoplasmic (cyto), nucleoplasm (nucl), and chromatin (chrom) fractions. Bands corresponding to CDK11 110 and P-CDK11 220 (monitored by Ab3) are marked on the right with arrows. CCNL1 is marked on the right with an arrow. “Long” and “short” corresponds to long and short exposure of the film. Asterisk denotes a nonspecific band. ( B ) Immunoblot analyses of indicated proteins in nucleoplasm and chromatin fractions (from Fig. ) separated by ultracentrifugation in 10%–40% glycerol gradient. Bands corresponding to CDK11 110 and P-CDK11 220 (monitored by Ab3) are marked on the right with arrows. Asterisks denote nonspecific bands. ( C ) Metagene analyses of P-CDK11 ChIP-Seq (using Ab2) on 8090 genes in cells treated with either control DMSO or 50 nM SY-351 for 2 h. Each transcript was divided into two parts with fixed length [transcription start site (TSS) −3 kb to +1.5 kb and transcription termination site (TTS) −1.5 kb to +20 kb] and a central part with variable length corresponding to the rest of gene body (shown in %). Each part was binned into a fixed number of bins (90/180/215), average coverage normalized to sequencing depth was calculated for each bin for each transcript in each sample and then averaged first across genes and second across samples. The color track at the bottom indicates the significance of paired Wilcoxon tests comparing the normalized transcript coverages for each bin between DMSO and SY-351 treatment. P ‐values are adjusted for multiple testing with the Bonferroni method within each subfigure; color code: red = adjusted P ‐value ≤ 10 −15 , orange = adjusted P ‐value ≤ 10 −10 , yellow = adjusted P ‐value ≤ 10 −3 . ( D ) IGV genome browser view of P-CDK11 220 ChIP-Seq on EZR gene in HCT116 cells treated with either control DMSO or 50 nM SY-351 for 2 h. SF3B1 and P-SF3B1 ChIP-seq on EZR gene in cells treated with control DMSO are shown below. noAb = control without antibody, R1, R2 = replicate 1, 2. T211 and T235 Ab = antibodies against P-Thr211 and P-Thr235 in SF3B1, respectively. Y -axis scale is denoted in square brackets.

Article Snippet: Blocking was done with 3% fetal bovine serum (FBS) in PBS for 1 h. Subsequently, cells were incubated with primary antibodies (P-CDK11 Ab1, P-CDK11 Ab2, 1:100 dilution), SF3B1 (MBL, MB-D221-3, 1:300 dilution), CDK11 (Abcam, ab19393, 1:400 dilution), and CDK7 (Bethyl, A300-405A, 1:400 dilution) for 1 h and washed 3 × 5 min with PBS.

Techniques: Western Blot, Control, ChIP-sequencing, Sequencing

Journal: Nucleic Acids Research

Article Title: CDK7–CDK11 axis in spliceosome regulation and pre-mRNA splicing

doi: 10.1093/nar/gkaf1343

Figure Lengend Snippet: Onset of splicing deficiency after CDK7 inhibition correlates with P-CDK11 220 dephosphorylation. ( A ) Immunoblot analyses of proteins after treatment of HCT116 cells with control DMSO or 50 nM SY-351 for the indicated times. Bands corresponding to CDK11 110 and P-CDK11 220 are marked on the right with arrows. ( B ) DNA gel-visualised RT-PCR analyses of splicing of ARRDC4, CCNL1 , and RIOK1 transcripts after treatment of HCT116 cells with 100 nM SY-351 for the indicated times. Schema of unspliced and spliced transcripts, including order of the tested exons in each transcript, are depicted on the right. DNA represents control RT-PCR product from genomic DNA. ( C ) Graph shows ratio of unspliced to spliced transcripts of six genes measured by RT-qPCR in HCT116 cells treated with 50 nM SY-351 for indicated times. mRNA levels were normalised to PPIA mRNA and expression in control DMSO condition was set as 1. Error bars = SD, n = 3.

Article Snippet: Blocking was done with 3% fetal bovine serum (FBS) in PBS for 1 h. Subsequently, cells were incubated with primary antibodies (P-CDK11 Ab1, P-CDK11 Ab2, 1:100 dilution), SF3B1 (MBL, MB-D221-3, 1:300 dilution), CDK11 (Abcam, ab19393, 1:400 dilution), and CDK7 (Bethyl, A300-405A, 1:400 dilution) for 1 h and washed 3 × 5 min with PBS.

Techniques: Inhibition, De-Phosphorylation Assay, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

Journal: Nucleic Acids Research

Article Title: CDK7–CDK11 axis in spliceosome regulation and pre-mRNA splicing

doi: 10.1093/nar/gkaf1343

Figure Lengend Snippet: Working Model. ( A ) CDK7 activity in normal cell. CDK7, as part of the TFIIH complex, phosphorylates the CTD of RNAPII to regulate promoter escape and normal transcription. CDK7, probably as part of the CAK complex, is also required for the onset of P-CDK11 220 , which is needed for the phosphorylation of SF3B1 (P-SF3B1) and activation of the spliceosome and normal splicing. ( B ) Consequences of short (<2 h) CDK7 inhibition with SY-351. Short inhibition blocks promoter escape, which results in a strong decrease in transcription, with some RNAPII slowly transcribing into the gene body. ( C ) Consequences of long (>2 h) CDK7 inhibition with SY-351. After a longer inhibition, the transcription is rescued by other transcriptional CDKs that phosphorylate the CTD of RNAPII . This results in faster elongation and synthesis of nascent RNA. The RNA is not spliced, because ongoing CDK7 inhibition blocks the formation of P-CDK11 220 and subsequent SF3B1 phosphorylation, which results in an inactive spliceosome and nonfunctional splicing.

Article Snippet: Blocking was done with 3% fetal bovine serum (FBS) in PBS for 1 h. Subsequently, cells were incubated with primary antibodies (P-CDK11 Ab1, P-CDK11 Ab2, 1:100 dilution), SF3B1 (MBL, MB-D221-3, 1:300 dilution), CDK11 (Abcam, ab19393, 1:400 dilution), and CDK7 (Bethyl, A300-405A, 1:400 dilution) for 1 h and washed 3 × 5 min with PBS.

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