phospho cdk7  (Cell Signaling Technology Inc)

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

Journal: mBio

Article Title: Functional insight into cyclin-dependent kinase (CDK)7 via chemical inhibition of the priority fungal pathogen Cryptococcus neoformans

doi: 10.1128/mbio.02898-25

Figure Lengend Snippet: Phylogenetic/guide trees obtained following multiple amino acid sequence alignment of the CDK7, cyclin H, and Mat1 homologs from C. neoformans ( Cn ), Homo sapiens ( Hs ), S. cerevisiae ( Sc ), and S. pombe ( Sp ). The trees show that Cn CDK7 and Cn Mat1 proteins cluster with the human homologs. Cn CDK7 (CNAG_06445 and XP_012053336.1 ), Hs CDK7 ( NP_001790.1 ), Sc CDK7/ Sc Kin28 ( NP_010175.1 ), Sp CDK7/ Sp Mcs6 ( NP_596349.1 ), Cn Cyclin H (CNAG_04405 and XP_012051791.1 ), Hs CycH ( NP_001230.1 ), Sc CycH/ Sc Ccl1 ( NP_015350.1 ), Sp CycH/ Sp Mcs2 ( NP_595776.1 ), Cn Mat1 (CNAG_05877 and XP_012050601.1 ), Hs Mat1 ( NP_002422.1 ), Sc Mat1/ Sc Tfb3 ( NP_010748.3 ), and Sp Mat1/ Sp Pmh1 ( NP_596334.1 ). Numbers represent sequence distances.

Article Snippet: ChromoTek mNeonGreen-Trap Agarose beads (cat. no. nta) and V5-Trap agarose beads (cat. no. v5ta) were used to pull down mNeonGreen-tagged CDK7 from the cleared lysates, with KN99 serving as a control for untagged CDK7.

Techniques: Sequencing

Journal: mBio

Article Title: Functional insight into cyclin-dependent kinase (CDK)7 via chemical inhibition of the priority fungal pathogen Cryptococcus neoformans

doi: 10.1128/mbio.02898-25

Figure Lengend Snippet: Cn CDK7 forms a CAK complex with Mat1 and cyclin H that localizes to the nucleus. ( A ) Schematic depicting the addition of the mNeonGreen (mNG), V5 epitope (V5), and 6× His (His) tags on CDK7, CycH, and Mat1, respectively, in the CDK7 triple-tagged strain as described in the Supplemental Method. ( B ) Immunoprecipitation (IP) and Western blotting with antibodies to each tag demonstrates formation of a CAK complex: in the left panel, Cn CDK7 was immunoprecipitated from the CDK7 triple-tagged strain with anti-mNG trap and subjected to SDS-PAGE and Western blotting with anti-mNG, anti-6× His, and anti-V5, detecting CDK7, CycH, and Mat1, respectively (lane 2). In the right panel, CycH was immunoprecipitated from the CDK7 triple-tagged strain with V5-Trap, followed by Western blotting with the various antitag antibodies (lane 2). In both panels, the non-tagged parent KN99 WT strain (WT) was taken through the same procedure as a negative control (lane 1). The total lysates used for each IP were probed with anti-PSTAIR to demonstrate the presence of Cdc2 in all IP samples. Epifluorescence microscopy validated the fluorescence of Cn CDK7 in the CDK7 triple-tagged strain ( C ) and the nuclear localization of the Cn CAK complex ( D ). DIC, Differential interference contrast.

Article Snippet: ChromoTek mNeonGreen-Trap Agarose beads (cat. no. nta) and V5-Trap agarose beads (cat. no. v5ta) were used to pull down mNeonGreen-tagged CDK7 from the cleared lysates, with KN99 serving as a control for untagged CDK7.

Techniques: Immunoprecipitation, Western Blot, SDS Page, Negative Control, Epifluorescence Microscopy, Fluorescence

Journal: mBio

Article Title: Functional insight into cyclin-dependent kinase (CDK)7 via chemical inhibition of the priority fungal pathogen Cryptococcus neoformans

doi: 10.1128/mbio.02898-25

Figure Lengend Snippet: Chemical structures of the human CDK7 inhibitors tested in this study. Inhibitors are arranged based on their molecular scaffold (in blue): pyrimidine (THZ1, SY-1365, and CDK7-IN-3), pyrrolidinopyrazole from p21-activated kinase (PAK4) (CDK7-IN-1, YKL-5-124, and IV-361) and pyrazolopyrimidine (samuraciclib). Red regions indicate the covalent warhead. THZ1 served as a molecular starting point for the creation of all compounds (indicated by the arrows), except Samuraciclib.

Article Snippet: ChromoTek mNeonGreen-Trap Agarose beads (cat. no. nta) and V5-Trap agarose beads (cat. no. v5ta) were used to pull down mNeonGreen-tagged CDK7 from the cleared lysates, with KN99 serving as a control for untagged CDK7.

Techniques:

Journal: mBio

Article Title: Functional insight into cyclin-dependent kinase (CDK)7 via chemical inhibition of the priority fungal pathogen Cryptococcus neoformans

doi: 10.1128/mbio.02898-25

Figure Lengend Snippet: Cn CDK7 is inhibited by human CDK7 inhibitors. Kinase assays were performed over a 300 min time course in the absence (DMSO) and presence of the indicated concentrations of each CDK7 inhibitor, using pulled-down CAK, CDK7 peptide substrate, and Kinase-Glo reagent. The latter allowed ATP consumption (due to the phosphorylation of the peptide substrate by CDK7) to be measured as a relative luminescence unit. The full dose–response time curves are shown in . Only CDK7 enzyme activity at 300 min is plotted and is expressed as “relative activity (%)” after normalization as described in Materials and Methods. The CDK1/CDK2/CDK4/CDK5 inhibitor, Purvalanol A, is included as a negative control. The results represent the mean relative activity ± SEM ( n = 2–3 independent experiments). Statistical analysis was performed using ordinary one-way analysis of variance with Dunnett’s multiple comparison test, comparing each concentration to the DMSO control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns, not significant.

Article Snippet: ChromoTek mNeonGreen-Trap Agarose beads (cat. no. nta) and V5-Trap agarose beads (cat. no. v5ta) were used to pull down mNeonGreen-tagged CDK7 from the cleared lysates, with KN99 serving as a control for untagged CDK7.

Techniques: Phospho-proteomics, Activity Assay, Negative Control, Comparison, Concentration Assay, Control

Journal: mBio

Article Title: Functional insight into cyclin-dependent kinase (CDK)7 via chemical inhibition of the priority fungal pathogen Cryptococcus neoformans

doi: 10.1128/mbio.02898-25

Figure Lengend Snippet: Phosphoproteomic analysis of SY-1356-treated Cn strain H99. ( A ) K-means clustering using the STRING database was performed on the 122 proteins that had reduced phosphorylation following CDK7 inhibition with SY-1365 (see the Supplemental Method and for details). This resulted in four functional clusters (PPI enrichment P value <5 × 10⁻⁴). Proteins in pink are discussed in the main text. ( B ) Representative Western blot of n = 3 independent experiments (see scatter dot plot), validating reduced phosphorylation of the MAPK, Hog1 (Hog1p) following treatment with SY-1365, as compared to total Hog1 (Hog1t). In the scatter dot plot, the reduction in Hog1p levels after 1 h treatment with SY-1365 is statistically significant ( *P = 0.013, unpaired t -test; bars represent mean intensity ± SD).

Article Snippet: ChromoTek mNeonGreen-Trap Agarose beads (cat. no. nta) and V5-Trap agarose beads (cat. no. v5ta) were used to pull down mNeonGreen-tagged CDK7 from the cleared lysates, with KN99 serving as a control for untagged CDK7.

Techniques: Phospho-proteomics, Inhibition, Functional Assay, Western Blot

Journal: mBio

Article Title: Functional insight into cyclin-dependent kinase (CDK)7 via chemical inhibition of the priority fungal pathogen Cryptococcus neoformans

doi: 10.1128/mbio.02898-25

Figure Lengend Snippet: Cn CDK7 phosphorylates Ser5 and Ser2 in the CTD of the Rpb1 subunit of RNAPII in vivo ( A ) Representative Western blot of three independent experiments (see adjacent scatter dot plot) showing that SY-1365 inhibits phosphorylation of Rpb1 on Ser5 (Rpb1-Ser5p). In the adjacent scatter dot plot, the reduction in Rpb1-Ser5p after 1 h treatment with SY-1365 when normalized to DMSO treatment is statistically significant (* P = 0.013, unpaired t -test; bars indicate mean intensity ± SD). ( B ) Representative Western blot of three independent experiments (see adjacent scatter dot plot) showing that SY-1365 inhibits phosphorylation of Rpb1 on Ser2 (Rpb1-Ser2p). In the adjacent scatter dot plot, the reduction in Rpb1-Ser2p after 1 h treatment with SY-1365 when normalized to DMSO treatment is statistically significant (* P = 0.0145, unpaired t -test; bars indicate mean intensity ± SD). In panels A and B , the levels of Rpb1-Ser5p and Rpb1-Ser2p in the SY-1365- and DMSO-treated samples were normalized to histone H3, which was used as a loading control.

Article Snippet: ChromoTek mNeonGreen-Trap Agarose beads (cat. no. nta) and V5-Trap agarose beads (cat. no. v5ta) were used to pull down mNeonGreen-tagged CDK7 from the cleared lysates, with KN99 serving as a control for untagged CDK7.

Techniques: In Vivo, Western Blot, Phospho-proteomics, Control

Journal: mBio

Article Title: Functional insight into cyclin-dependent kinase (CDK)7 via chemical inhibition of the priority fungal pathogen Cryptococcus neoformans

doi: 10.1128/mbio.02898-25

Figure Lengend Snippet: RNA-seq data analysis of SY-1365-treated Cn identifies CDK7 functions including splicing regulation. RNA-seq was performed on untreated and SY-1365-treated Cn ( n = 3) as described in Materials and Methods. ( A ) A principal component analysis plot shows that replicates correlate well, with each treatment group having a distinct cluster profile. Gene set enrichment analysis (GSEA) was performed using the clusterProfiler R package to identify Gene Ontology categories ( B ) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways ( C ) that were significantly enriched following SY-1365 treatment. Diamond plots display the enriched pathways, where size represents the percentage of enriched genes and color indicates the statistical significance of enrichment. ( D ) Table presenting alternative splicing events (ASEs) determined upon SY-1365 treatment. Inhibition of Cn CDK7 leads to more intron retention events over intron exclusion. Normalized coverage plots of representative ASE with ( E ) intron retention and ( F ) intron exclusion upon SY-1365 treatment.

Article Snippet: ChromoTek mNeonGreen-Trap Agarose beads (cat. no. nta) and V5-Trap agarose beads (cat. no. v5ta) were used to pull down mNeonGreen-tagged CDK7 from the cleared lysates, with KN99 serving as a control for untagged CDK7.

Techniques: RNA Sequencing, Alternative Splicing, Inhibition

Journal: mBio

Article Title: Functional insight into cyclin-dependent kinase (CDK)7 via chemical inhibition of the priority fungal pathogen Cryptococcus neoformans

doi: 10.1128/mbio.02898-25

Figure Lengend Snippet: Model depicting the role of Cn CDK7 in regulating the cell cycle, transcriptional processes, and translation. Cell cycle regulation: in the nucleus, Cn CDK7 in the CAK complex activates Cdk1 to promote cell cycle progression through G 2 /M. Activated Cdk1 also phosphorylates Cdc24 to promote actin polymerization. Cn CDK7 either directly phosphorylates components of the Hog1 signaling pathway or acts through an intermediary protein affected by Cn CDK7-dependent phosphorylation. Transcription: also in the nucleus, TFIIH-associated CAK phosphorylates Ser5 on the CTD of the Rpb1 subunit of RNAPII (see ), allowing RNAPII release from the promoter to initiate transcription. Ser5 phosphorylated-RNAPII pauses ~30 bp post-initiation and interacts with the capping enzymes, Ceg1p (RNA guanylyltransferase) and Cet1p (RNA triphosphatase), to form a capping complex. This complex allows the co-transcriptional formation of the 7-methylguanosine (7 mG) cap (red circle) on the 5′ end of newly synthesized (nascent) mRNA, ensuring mRNA stability, nuclear export, and translation. Cbc1 (mammalian Cbp80 homolog) and Cbc2 (mammalian Cbp20 homolog) then form a heterodimeric cap binding complex (CBC) that binds to the 7mG cap to stimulate formation of the pre-initiation complex (PIC) via the transcription regulator, Mot1. Post-release of the RNAPII transcriptional pause, Cn CDK7 is essential for Ser2 phosphorylation of Rpb1 to allow transcription elongation (see ). This occurs via Cn CDK7-mediated activation of CDK9. The CBC also links capping to splicing by promoting the recruitment of U1 snRNP to the 5′ splice site to initiate spliceosome assembly. The spliceosome, comprising U1–U6 snRNPs, Sf3b1, Msl5 (binds to the branch point sequences of the intron), and Cwf19 (facilitates spliceosome disassembly and mRNA release), mediates intron removal. As in humans, Cn CDK7 may also enhance spliceosome maturation by activating Cn CDK11 to phosphorylate Sf3b1 . Translation: in the cytoplasm, Pab1 binds the 3′ poly(A) tail of mature mRNAs, protecting them from degradation. Two major deadenylation complexes then act sequentially to regulate mRNA stability and turnover: the Pan2/Pan3 complex trims the poly(A) tail, while the Ccr4-NOT complex trims it further, leading to mRNA decay. The CBC at the 5′ end is replaced by eIF4E, which, along with eIF4A and eIF4G, initiates recruitment of additional eIFs to assemble ribosomal subunits on the mRNA, ultimately leading to the formation of a functional ribosome and the initiation of translation. Proteins with solid lines were identified in the CDK7 phosphoproteome (see ), while proteins, interactions, and functions delineated by broken lines are based on studies in higher eukaryotes and the presence of the homologous protein in Cn .

Article Snippet: ChromoTek mNeonGreen-Trap Agarose beads (cat. no. nta) and V5-Trap agarose beads (cat. no. v5ta) were used to pull down mNeonGreen-tagged CDK7 from the cleared lysates, with KN99 serving as a control for untagged CDK7.

Techniques: Phospho-proteomics, Synthesized, Binding Assay, Activation Assay, Functional Assay

Journal: bioRxiv

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

doi: 10.1101/2025.11.23.690049

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

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

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

Journal: bioRxiv

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

doi: 10.1101/2025.11.23.690049

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

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

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

Journal: bioRxiv

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

doi: 10.1101/2025.11.23.690049

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

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

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

Journal: bioRxiv

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

doi: 10.1101/2025.11.23.690049

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

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

Techniques: ChIP-sequencing, Expressing

Journal: bioRxiv

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

doi: 10.1101/2025.11.23.690049

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

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

Techniques: ChIP-sequencing, Expressing

Journal: bioRxiv

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

doi: 10.1101/2025.11.23.690049

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

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

Techniques: ChIP-sequencing, Expressing

Journal: bioRxiv

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

doi: 10.1101/2025.11.23.690049

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

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

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

Journal: bioRxiv

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

doi: 10.1101/2025.11.23.690049

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

Article Snippet: Proteins were transferred to PVDF membranes, blocked with 5% BSA, and immunoblotted with a CDK7 primary antibody (Cell Signaling Technology, 2090S) followed by a secondary HRP-conjugated antibody.

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

Journal: bioRxiv

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

doi: 10.1101/2025.11.23.690049

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

Article Snippet: Proteins were transferred to PVDF membranes, blocked with 5% BSA, and immunoblotted with a CDK7 primary antibody (Cell Signaling Technology, 2090S) followed by a secondary HRP-conjugated antibody.

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

Journal: bioRxiv

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

doi: 10.1101/2025.11.23.690049

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

Article Snippet: Proteins were transferred to PVDF membranes, blocked with 5% BSA, and immunoblotted with a CDK7 primary antibody (Cell Signaling Technology, 2090S) followed by a secondary HRP-conjugated antibody.

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

Journal: bioRxiv

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

doi: 10.1101/2025.11.23.690049

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

Article Snippet: Proteins were transferred to PVDF membranes, blocked with 5% BSA, and immunoblotted with a CDK7 primary antibody (Cell Signaling Technology, 2090S) followed by a secondary HRP-conjugated antibody.

Techniques: ChIP-sequencing, Expressing

Journal: bioRxiv

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

doi: 10.1101/2025.11.23.690049

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

Article Snippet: Proteins were transferred to PVDF membranes, blocked with 5% BSA, and immunoblotted with a CDK7 primary antibody (Cell Signaling Technology, 2090S) followed by a secondary HRP-conjugated antibody.

Techniques: ChIP-sequencing, Expressing

Journal: bioRxiv

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

doi: 10.1101/2025.11.23.690049

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

Article Snippet: Proteins were transferred to PVDF membranes, blocked with 5% BSA, and immunoblotted with a CDK7 primary antibody (Cell Signaling Technology, 2090S) followed by a secondary HRP-conjugated antibody.

Techniques: ChIP-sequencing, Expressing

Journal: bioRxiv

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

doi: 10.1101/2025.11.23.690049

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

Article Snippet: Proteins were transferred to PVDF membranes, blocked with 5% BSA, and immunoblotted with a CDK7 primary antibody (Cell Signaling Technology, 2090S) followed by a secondary HRP-conjugated antibody.

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