Journal: Science immunology

Article Title: Conversion of antigen-specific effector/memory T cells into Foxp3-expressing T reg cells by inhibition of CDK8/19.

doi: 10.1126/sciimmunol.aaw2707

Figure Lengend Snippet: Fig. 3. Interaction of CDK8/19 and STAT5 in inducing Foxp3 expression. (A) Mouse CD4+ T cells were mock-infected or infected with retrovirus harboring the WT CDK8 and STAT5b genes, stimulated with anti-CD3/CD28 and IL-2, and subjected to immunoprecipitation and immunoblotting for CDK8, STAT5b, and MED12. Data are representative of two independent experiments. (B) STAT5 serine phosphorylation by CDK8. Recombinant GST-STAT5b incubated with recombinant WT or KD CDK8 in the presence or absence of 1.0 M AS with 100 M ATP and 10 mM MgCl2 was subjected to immunoblotting for phosphoserine (pS) of STAT5b. Data are representative of two independent experiments. (C) Control of serine and tyrosine phos- phorylation by AS in activated T cells. Mouse CD4+ T cells were stimulated with anti-CD3/CD28 in the presence or absence of TGF- for 22 hours and in the absence (DMSO) or presence of 100 nM AS, lysed, and subjected to immunoblot analysis for STAT5b, pS-STAT5b, or pY-STAT5. Signal intensity was quantified and normalized by GAPDH (n = 3 or 4). **P < 0.01 (Student’s t test). AU, arbitrary units. (D and E) Mouse naїve CD4+ T cells were incubated in the presence or absence of anti-CD3/28 for 22 hours, and PLA was performed to assess interaction between CDK8 and STAT5. Images were obtained using an LSM710 confocal microscope. Data were presented as maximum intensity projection (n = 3). Each red spot represents a single interaction, and DNA was stained with DAPI. (F) Mouse naїve CD4+ T cells were incubated in the presence or absence of anti-CD3/CD28 for 22 hours and examined for expression of CDK8 and STAT5. DNA was stained with Hoechst33342. Images were obtained using an LSM710 confocal microscope. Data are representative of two independent experiments. (G) Mouse CD4+ T cells infected with retrovirus encoding WT or S730A mutant STAT5b were stimulated with anti-CD3/CD28 and IL-2, without TGF-, and subjected to immunoblotting for STAT5b (left), or assessed for the percentage of Foxp3+ cells among live virus–infected (i.e., GFP+) CD4+ T cells by flow cytometry (n = 7) (right). ***P < 0.001 (Student’s t test).

Article Snippet: A fragment encoding mouse CDK8, CDK19, or STAT5b cDNA was obtained from pCMV6-CDK8 (OriGene, MR218219), pCMV6-CDK19 (OriGene, MR215712), or pCMV6-STAT5b (OriGene, MR210649) and subcloned into a retroviral plasmid pMCs-IRES-GFP (Cell Biolabs).

Techniques: Expressing, Infection, Immunoprecipitation, Western Blot, Phospho-proteomics, Recombinant, Incubation, Control, Microscopy, Staining, Mutagenesis, Virus, Flow Cytometry

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: Superposition of docked Sorafenib (cyan carbon atoms) onto its corresponding crystallographic structure. Sorafenib from 3RGF is depicted with light gray carbon atoms. As no structure of CDK8 with ATP is available, the ATP (dark gray carbon atoms) of the superposed structure of the human CDK2/Cyclin A complex (PDB ID: 4EOQ) is shown to highlight its potential binding site in CDK8.

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: Compounds were tested in duplicate at 10 µM in kinase assays with recombinant CDK8/Cyc C complex, in the presence of 15 µM [γ- 33 P] ATP. The graph represents the residual CDK8 activity (%) for each compound tested (blue dots); background controls (red dots) and negative control (DMSO 1%, green dots). 36 compounds inhibit CDK8 to below 50%.

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques: Recombinant, Activity Assay, Negative Control

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: Thirty six selected compounds and Senexin A (SNX A) were tested in dose-response on CDK8/Cyc C in kinase assays. IC 50 is expressed in µM and calculated from dose-response curves (each curve was performed in duplicate). The graph represents the distribution of IC 50 obtained, with two threshold lines: at 100 nM (red line) and 1 µM (green line).

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques:

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: A. Left, DepMap analysis of dependecy of cancer cell lines on CDK8 and CDK1 (an example of an essential CDK), from genome-wide CRISPR screens. Score 0 indicates a non essential gene; score of -1 corresponds to a median of all common essential genes. Right, number of cancer cell lines dependent on the indicated gene according to the DepMap CRISPR screen database. All CDKs and kinases used to screen for CDK8/19 inhibitor specificity are shown. B. Expression levels (from RNAseq data, TPM-normalised and represented as log2(TPM+1)) and relative copy number (based on whole exome sequencing) in cancer cell lines. C. cBioPortal mutation analysis of the indicated genes in pancancer TCGA data.

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques: Genome Wide, CRISPR, Expressing, Sequencing, Mutagenesis

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: Induced fit docking experiments were performed using either CDK8 (PDB: 3RGF) or CDK2 (PDB: 4GCJ) structures as protein target. Upper panels: ID and structural formula of the seven selected compounds. Lower panels: 3D arrangement of the best docking pose (white carbon atoms) into CDK8 or CDK2 active site with its corresponding docking score. Amino acids from the CDK8 DMG loop are represented in ball and stick; corresponding residues (DFG) in CDK2 structure are also highlighted.

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques:

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: A. MEFs seeded at 10 6 in 10 cm plates were grown for 5 days at 10 μM of drugs and imaged. B. Colony formation assay (CFA) with WT SV-40-immortalised MEFs, seeded at 15 000 in 10cm plates, treated with inhibitors at 10 μM for 8 days. N=2. C. CFA with immortalised MEFs, WT, CDK8 -/- , CDK19 -/- , or double knock-out, treated with inhibitors at 10μM for 6d. N = 2. D. 3T3-immortalised MEFs were treated for 2h with the indicated inhibitors, after which γIFN (at 100 ng/ml) was added for 1h, without changing the media. Cells were collected and samples prepared for WB analysis. CDK8/19 double knockout MEF cells were used as a positive control (for the absence of STAT1-S727ph signal). N=2. E. HCT116 cells grown in spheroids were pre-incubated with γIFN (at 20 ng/ml) for 3h and treated with selected inhibitors at 10 μM or DMSO at 0.1% for 3h. Spheroids were lysed and analysed by Western blotting for the indicated proteins. Normalised STAT1-S727ph/STAT1 signal ratio are shown. N=2.

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques: Colony Assay, Knock-Out, Double Knockout, Positive Control, Incubation, Western Blot

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: SV-40-immortalised MEF cell lysates (A, in vitro N=4) or MEF cells (B, in vivo , N=6) were treated with the indicated CDK8/19 inhibitors, or DMSO as a control, followed by heat treatment at the indicated temperatures and Western Blot analysis with anti-CDK8 antibody.

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques: In Vitro, In Vivo, Control, Western Blot

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: A. CDK8 was immunoprecipitated from interphase egg extract and the beads were blotted for the indicated proteins. B. Interphase egg extract was fractionated by sucrose gradient and fractions were blotted for the indicated proteins. C. Left, chromosomal DNA replication quantified by 32 P-dCTP incorporation assay, in control conditions, or after addition of recombinant Geminin, to block replication licencing. Right, chromatin loading of pre-replication complex (ORC2, MCM5 and CDC6) and pre-initiation complex (PCNA) replication factors, CDK8 and Cyclin C during DNA replication time course. N=4. D. Left, immunodepletion of CDK8 (but not using Mock beads) removes majority of the kinase from the interphase egg extract; GADPH was used as loading control. Right, chromatin fractions from CDK8- or Mock-immunodepleted egg extract, or CDK8-depleted extract supplemented with recombinant CDK8-Cyclin C complex were analysed by WB with the indidated proteins. Slower migrating recombinant GST-CDK8 and His-Cyclin C are indidated. E. Chromosomal DNA replication in immunodepleted extracts, as in D, was quantified. N=3. Error bars show standard deviation. F,G. Chromosomal DNA replication quantified by 32 P-dCTP incorporation assay, in control conditions (DMSO), or in the presense of the indicated inhibitors.

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques: Immunoprecipitation, Control, Recombinant, Blocking Assay, Immunodepletion, Standard Deviation

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: A. Alignment of the 20 CDK sequences. Only the regions containing the amino acids responsible for interactions with the compounds are shown. Interacting amino acids and their conservation are highlighted. B. View of the CDK8 amino acid residues involved in contact with compounds. The seven compounds are superposed and shown with gray balls. Interacting residues are depicted by colored balls and sticks according to their percentage of conservation (red: highly conserved residues (71-100%); pink: moderately conserved residues (35-70%); green: poorly conserved resides (10-34%)). C. Highlight of virtually mutated residues. Left panel: wild-type CDK8 showing the seven residues selected (ball and stick and dark grey carbon) to assess the effect of point mutations on compound binding. The induced fit pose of molecule #82 is shown (ball and stick and light grey carbon). Right panel: superposition of the poses obtained for each mutant following induced-fit docking. For clarity, only one of the two side chains is shown (ball and stick and dark grey carbon) in the case of the A100M/L and L158R/W mutants. In contrast, all the positions obtained for compound #82 are shown (ball and stick and light grey carbon).

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques: Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: Discovery and chemical biology of CDK8 inhibitors reveals insights for kinase inhibitor development

doi: 10.1101/2025.11.06.686927

Figure Lengend Snippet: Overlay of structures of selected inhibitors with the inactive (DMG-out, PDB 3RGF; A, cyan) and active (DMG-in) conformations, without (PDB 5XS2; A and B, blue) and with MED12 (PDB 8TQ2; B, cyan). Zoom-in on ATP-binding pocket of CDK8, with side chains of D173 and M174 shown. In red, inhibitor docked into 5XS2; in yellow, inhibitor docked into 3RGF, A, and 8TQ2, B. Induced fit docking scores for compared structures are indicated.

Article Snippet: CDK8 - Santa Cruz, sc-13155 CDK19 – Sigma, HPA007053 Cyclin C – Abcam, ab85927 STAT1 S727-ph - Cell Signaling, #9177 STAT1-Cell Signaling, #9172 Alpha-Tubulin (clone B-5-1-2), Sigma #T5168 XMcm3, XOrc2, XRPA, XCdc6 – gifts from M. Méchali MCM5 – Abcam, CRCT5.1 (A2.7A3), ab6154 PCNA – Abcam, ab18197

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: CDK8 Inhibition Increases E2F1 Transcriptional Activity and Promotes STAT3-Dependent Suppression of Mcl-1 in Triple-Negative Breast Cancer Cell Line MDA-MB-468

doi: 10.3390/ijms27020897

Figure Lengend Snippet: Effect of CDK8 inhibitor Q12 treatment of MDA-MB-468 cells on E2F1 protein and E2F1-dependent luciferase expression. ( A ) Effect of treatment with 10 µM Q12 (6 h and 24 h) on E2F1 ( left ) and pE2F1(Ser375) ( right ) expression in MDA-MB-468 cells compared to vehicle-treated control (DMSO) cells ( n = 3–6). ( B ) Relative luciferase expression in MDA-MB-468 cells transfected with an E2F1-responsive luciferase plasmid following treatment with Q12 (10 µM) or SEL120 (1µM) compared to vehicle-treated control (DMSO) cells ( n = 4). Unpaired Student’s t -test was used to determine significance. * ρ < 0.05, ** ρ < 0.01, **** ρ < 0.0001.

Article Snippet: Control siRNA-B (sc-44230), E2F-1 siRNA (h) (sc-29297), Cdk8 siRNA (h) (sc-29267), siRNA Transfection Medium (sc-36868) and siRNA Transfection Reagent (sc-29528), and mouse anti-Mcl-1 (22, sc-12756) (dil. 1:400) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Luciferase, Expressing, Control, Transfection, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: CDK8 Inhibition Increases E2F1 Transcriptional Activity and Promotes STAT3-Dependent Suppression of Mcl-1 in Triple-Negative Breast Cancer Cell Line MDA-MB-468

doi: 10.3390/ijms27020897

Figure Lengend Snippet: ( A ) p73 protein expression following treatment with 10 µM Q12 (1, 6, 12, 24 h) compared to vehicle-treated (DMSO) control ( n = 3). ( B ) CDK8 protein expression in MDA-MB-468 cell line following 24 h treatment with 10 µM Q12 ( n = 3). Images are representative of three independent experiments. Error bars represent mean ± SD. Unpaired Student’s t -test was used to determine significance. ** ρ < 0.01.

Article Snippet: Control siRNA-B (sc-44230), E2F-1 siRNA (h) (sc-29297), Cdk8 siRNA (h) (sc-29267), siRNA Transfection Medium (sc-36868) and siRNA Transfection Reagent (sc-29528), and mouse anti-Mcl-1 (22, sc-12756) (dil. 1:400) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: CDK8 Inhibition Increases E2F1 Transcriptional Activity and Promotes STAT3-Dependent Suppression of Mcl-1 in Triple-Negative Breast Cancer Cell Line MDA-MB-468

doi: 10.3390/ijms27020897

Figure Lengend Snippet: ( A ) Western blot assessment of siRNA-mediated CDK8 KD. ( B ) Western blot assessment of siRNA-mediated E2F1 KD. ( C ) Comparison of cell viability of wild-type, siRNA control-transfected, CDK8 siRNA-transfected, and E2F1 siRNA-transfected MDA-MB-468 cells treated with Q12 (20 µM) for 0.5, 1, 2, 6, and 24 h ( n = 3). Only significant differences are shown. * ρ ≤ 0.01.

Article Snippet: Control siRNA-B (sc-44230), E2F-1 siRNA (h) (sc-29297), Cdk8 siRNA (h) (sc-29267), siRNA Transfection Medium (sc-36868) and siRNA Transfection Reagent (sc-29528), and mouse anti-Mcl-1 (22, sc-12756) (dil. 1:400) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Western Blot, Comparison, Control, Transfection

Journal: International Journal of Molecular Sciences

Article Title: CDK8 Inhibition Increases E2F1 Transcriptional Activity and Promotes STAT3-Dependent Suppression of Mcl-1 in Triple-Negative Breast Cancer Cell Line MDA-MB-468

doi: 10.3390/ijms27020897

Figure Lengend Snippet: ( A ) Expression of Mcl-1 protein in MDA-MB-468 cells treated with Q12 (10 µM, 24 h), cryptotanshinone (CPT, 10 µM, 24 h), or co-treatment with Q12 (10 µM, 24 h) and CPT (10 µM, 24 h) compared to vehicle-treated (DMSO) control cells ( n = 3). ( B ) Proposed mechanism by which CDK8 inhibitor decreases viability of MDA-MB-468 triple-negative breast cancer cells. Images are representative of three independent experiments. Error bars represent mean ± SD. Unpaired Student’s t -test was used to determine significance. * ρ < 0.05, *** ρ < 0.001, ns = not significant.

Article Snippet: Control siRNA-B (sc-44230), E2F-1 siRNA (h) (sc-29297), Cdk8 siRNA (h) (sc-29267), siRNA Transfection Medium (sc-36868) and siRNA Transfection Reagent (sc-29528), and mouse anti-Mcl-1 (22, sc-12756) (dil. 1:400) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Control