Journal: International Journal of Molecular Sciences

Article Title: The Interactive Complex between Cytomegalovirus Kinase vCDK/pUL97 and Host Factors CDK7–Cyclin H Determines Individual Patterns of Transcription in Infected Cells

doi: 10.3390/ijms242417421

Figure Lengend Snippet: Schematic overview of the analyzed samples for the RNA-seq analysis. Individual steps of the procedure are indicated in the upper line: (1) HFFs were either treated under wild-type (WT) conditions or cyclin H knockout (KO) via lentiviral gene transduction. HFFs transduced with the CRISPR/Cas9 system without sgRNAs served as no KO controls. (2) Cells were infected with HCMV AD169 (MOI 0.1) or remained uninfected. (3) Inhibitors or solvent DMSO were added. (4) Cells were harvested and assayed in the RNA-seq procedure. Bold letters indicate the six analyzed treatment conditions, i.e., either uninfected control (mock-inf.), with (KO) or without cyclin H KO (no KO ctrl), or inhibitor treatment (DMSO, LDC4297, MBV).

Article Snippet: The CDK7-specific inhibitor LDC4297 was obtained from Lead Discovery Center GmbH, Dortmund, Germany. pUL97-specific inhibitor MBV was obtained from MedChemExpress, Monmouth Junction, NJ, USA.

Techniques: RNA Sequencing Assay, Knock-Out, Transduction, CRISPR, Infection, Solvent

Journal: International Journal of Molecular Sciences

Article Title: The Interactive Complex between Cytomegalovirus Kinase vCDK/pUL97 and Host Factors CDK7–Cyclin H Determines Individual Patterns of Transcription in Infected Cells

doi: 10.3390/ijms242417421

Figure Lengend Snippet: Principal component analysis (PCA): the individual biological triplicates are closely clustered, indicating consistency in their gene expression profiles. Each point on the scatter plot represents a sample, and its coordinates are determined by gene expression levels. Samples with similar gene expression profiles are positioned close together. The PCA plot displays the most significant patterns of variance within the high-dimensional dataset. Principal component 1 accounted for 20.4% of the total variance, capturing the primary pattern in the data. Principal component 2 represents the second most significant direction of variance and accounted for 15.5% of the total variance. Black indicates uninfected/mock control samples (non-bold) or DMSO-treated samples of HCMV infection (bold); green indicates samples of inhibitor treatment (MBV, LDC4297) or cyclin H knockout (KO).

Article Snippet: The CDK7-specific inhibitor LDC4297 was obtained from Lead Discovery Center GmbH, Dortmund, Germany. pUL97-specific inhibitor MBV was obtained from MedChemExpress, Monmouth Junction, NJ, USA.

Techniques: Expressing, Infection, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: The Interactive Complex between Cytomegalovirus Kinase vCDK/pUL97 and Host Factors CDK7–Cyclin H Determines Individual Patterns of Transcription in Infected Cells

doi: 10.3390/ijms242417421

Figure Lengend Snippet: Differential expression of biological pathways across various conditions in HCMV-infected HFFs # .

Article Snippet: The CDK7-specific inhibitor LDC4297 was obtained from Lead Discovery Center GmbH, Dortmund, Germany. pUL97-specific inhibitor MBV was obtained from MedChemExpress, Monmouth Junction, NJ, USA.

Techniques: Expressing, Transduction, Cell Differentiation

Journal: International Journal of Molecular Sciences

Article Title: The Interactive Complex between Cytomegalovirus Kinase vCDK/pUL97 and Host Factors CDK7–Cyclin H Determines Individual Patterns of Transcription in Infected Cells

doi: 10.3390/ijms242417421

Figure Lengend Snippet: Venn-diagram-based data evaluation of differential gene expression in HCMV-infected HFFs under various conditions. Gene expression in DMSO-treated samples served as a reference and was compared with mock-infected, cyclin H KO, MBV-treated, and LDC4297-treated samples. The number of significantly differentially expressed (DE) genes ( p ≤ 0.05) with at least a 1.5-fold change are listed. DE genes were categorized into cellular transcripts ( A ), viral transcripts ( B ), and a combination of both cellular and viral transcripts ( C ). Bold numbers indicate DE genes in MBV and cyclin H KO; underlined numbers indicate DE genes in MBV and LDC4297; asterisks indicate DE genes in MBV and cyclin H KO, but not in LDC4297.

Article Snippet: The CDK7-specific inhibitor LDC4297 was obtained from Lead Discovery Center GmbH, Dortmund, Germany. pUL97-specific inhibitor MBV was obtained from MedChemExpress, Monmouth Junction, NJ, USA.

Techniques: Expressing, Infection

Journal: International Journal of Molecular Sciences

Article Title: The Interactive Complex between Cytomegalovirus Kinase vCDK/pUL97 and Host Factors CDK7–Cyclin H Determines Individual Patterns of Transcription in Infected Cells

doi: 10.3390/ijms242417421

Figure Lengend Snippet: Differentially expressed genes that were exclusively affected by cyclin H KO and MBV but not by LDC4297 treatment # .

Article Snippet: The CDK7-specific inhibitor LDC4297 was obtained from Lead Discovery Center GmbH, Dortmund, Germany. pUL97-specific inhibitor MBV was obtained from MedChemExpress, Monmouth Junction, NJ, USA.

Techniques: Virus, Membrane, Sublimation, Modification, Transduction, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: The Interactive Complex between Cytomegalovirus Kinase vCDK/pUL97 and Host Factors CDK7–Cyclin H Determines Individual Patterns of Transcription in Infected Cells

doi: 10.3390/ijms242417421

Figure Lengend Snippet: Predicted location of pUL97 in the preinitiation complex (PIC) of RNAP II-mediated eukaryotic transcription. ( A ) Structure of the RNAP II-specific PIC in white-space-filled presentation with the bound template DNA in yellow. The CDK7-MAT1-cyclin H unit, located at the periphery of the PIC, in association with RNAP II (1–1487) are shown in colors. The region in the dashed box is shown as an enlargement in panel ( B ). ( B ) The putative pUL97 binding site in the PIC (ribbon presentation of the PIC proteins). Note that the main cyclin-binding interface (IF2) of pUL97, comprising amino acids 231–280, is shown in the purple-space-filled presentation and the pUL97 (329–634) kinase domain is schematically outlined as a purple oval. The C-terminal domain (CTD) of RNAP II, which is not resolved in the experimental PIC structure, is shown as an orange dashed line. Filled orange spots indicate CTD phosphorylation sites located in spatial proximity of CDK7 and pUL97.

Article Snippet: The CDK7-specific inhibitor LDC4297 was obtained from Lead Discovery Center GmbH, Dortmund, Germany. pUL97-specific inhibitor MBV was obtained from MedChemExpress, Monmouth Junction, NJ, USA.

Techniques: Binding Assay

Journal: EMBO Molecular Medicine

Article Title: CDK7/12/13 inhibition targets an oscillating leukemia stem cell network and synergizes with venetoclax in acute myeloid leukemia

doi: 10.15252/emmm.202114990

Figure Lengend Snippet: Gene expression of GPR56 , SMO (left), and TGFb targets SRC, ZEB1, and ZEB2 (right) normalized to GAPDH in AML‐491 cells determined by q‐RT‐PCR 4 h after treatment with THZ1 0.5 µM or LDC4297 2 µM. Unpaired t ‐test. Mean, SD, and individual values from three individual treatments. *** P < 0.0005, ** P < 0.005, * P < 0.05. Setup of in vivo drug treatment. NSG mice were injected with 10 5 AML 04H112 cells. Four weeks post injection, bone marrow (BM) was analyzed for human leukemic engraftment by BM aspiration. Treatment with either THZ1 or vehicle was started in the following week as indicated. BM was analyzed again after the end of the 4‐week treatment period. Overall percentage of human (huCD45 + ) leukemic cells in mice before and at the end of the 4‐week treatment period. Individual mice and mean engraftment are shown. Unpaired t ‐test. *** P < 0.0005. N = 10 mice for each group. Left : Representative FACS plots showing the typical CD34 low GPR56 high profile of sample 04H112 before injection and after the 4‐week treatment with THZ1. Right : both LSC compartments, the CD34 ‐ GPR56 + and the CD34 + GPR56 + fractions were significantly reduced in vivo in the THZ1 treatment group. Individual mice and mean engraftment are shown. Unpaired t ‐test. *** P < 0.0005, ** P < 0.005, * P < 0.05. N = 10 mice for each group. Left : Overall percentage of human (huCD45 + ) leukemic cells and the fraction of CD34 + GPR56 + among human cells (mean, individual mice) two weeks after drug withdrawal. Right : FACS plot visualizing that the CD34 + GPR56 + is re‐established upon drug removal. Unpaired t ‐test, *** P < 0.0005, ** P < 0.005, * P < 0.05. Correlation plot showing an anti‐correlation between the percentage of CD34 + GPR56 + cells in AML samples and the corresponding IC50s for THZ1 determined in the respective samples. Pearson correlation. Each dot represents one sample. See Dataset for sample characteristics. FACS histogram plot (left) and summary bar graph from three replicate wells (right) showing reduction of GPR56 surface expression on the GPR56‐positive AML cell line HEL only in conditions that contain the more specific CDK7 inhibitor YKL‐5‐124, but not those that contain only the specific CDK12/13 inhibitor THZ531. Unpaired t ‐test, bars, and error bars represent mean and SD of three individual treatments, *** P < 0.0005, ** P < 0.005, * P < 0.05. SRF reporter assay showing dose‐dependent suppression of the GPR56‐CTF‐induced SRF signal by the CDK7 inhibitor YKL‐5‐124, but not by the CDK12/13 inhibitor THZ531. Four technical replicates. One of the four individual experiments. Unpaired t ‐test, bars and error bars represent mean and SD, *** P < 0.0005, ** P < 0.005, * P < 0.05.

Article Snippet: The CDK12/13 inhibitor THZ531 (Selleckchem #S6595), the CDK7/12/13 inhibitor THZ1 (Selleckchem #S7549), and the CDK7 inhibitors YKL‐5‐124 (Selleckchem #S8863) and LDC4297 (Selleckchem #S7992) were dissolved in DMSO at 50 mM stock concentration and diluted in media so that final DMSO concentration was 0.1% in all conditions.

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, In Vivo, Injection, Expressing, Reporter Assay

Journal: EMBO Molecular Medicine

Article Title: CDK7/12/13 inhibition targets an oscillating leukemia stem cell network and synergizes with venetoclax in acute myeloid leukemia

doi: 10.15252/emmm.202114990

Figure Lengend Snippet: Gene expression of GPR56 , SMO (left), and TGFb targets SRC, ZEB1, and ZEB2 (right) normalized to GAPDH in AML‐491 cells determined by q‐RT‐PCR 4 h after treatment with THZ1 0.5 µM or LDC4297 2 µM. Unpaired t ‐test. Mean, SD, and individual values from three individual treatments. *** P < 0.0005, ** P < 0.005, * P < 0.05. Setup of in vivo drug treatment. NSG mice were injected with 10 5 AML 04H112 cells. Four weeks post injection, bone marrow (BM) was analyzed for human leukemic engraftment by BM aspiration. Treatment with either THZ1 or vehicle was started in the following week as indicated. BM was analyzed again after the end of the 4‐week treatment period. Overall percentage of human (huCD45 + ) leukemic cells in mice before and at the end of the 4‐week treatment period. Individual mice and mean engraftment are shown. Unpaired t ‐test. *** P < 0.0005. N = 10 mice for each group. Left : Representative FACS plots showing the typical CD34 low GPR56 high profile of sample 04H112 before injection and after the 4‐week treatment with THZ1. Right : both LSC compartments, the CD34 ‐ GPR56 + and the CD34 + GPR56 + fractions were significantly reduced in vivo in the THZ1 treatment group. Individual mice and mean engraftment are shown. Unpaired t ‐test. *** P < 0.0005, ** P < 0.005, * P < 0.05. N = 10 mice for each group. Left : Overall percentage of human (huCD45 + ) leukemic cells and the fraction of CD34 + GPR56 + among human cells (mean, individual mice) two weeks after drug withdrawal. Right : FACS plot visualizing that the CD34 + GPR56 + is re‐established upon drug removal. Unpaired t ‐test, *** P < 0.0005, ** P < 0.005, * P < 0.05. Correlation plot showing an anti‐correlation between the percentage of CD34 + GPR56 + cells in AML samples and the corresponding IC50s for THZ1 determined in the respective samples. Pearson correlation. Each dot represents one sample. See Dataset for sample characteristics. FACS histogram plot (left) and summary bar graph from three replicate wells (right) showing reduction of GPR56 surface expression on the GPR56‐positive AML cell line HEL only in conditions that contain the more specific CDK7 inhibitor YKL‐5‐124, but not those that contain only the specific CDK12/13 inhibitor THZ531. Unpaired t ‐test, bars, and error bars represent mean and SD of three individual treatments, *** P < 0.0005, ** P < 0.005, * P < 0.05. SRF reporter assay showing dose‐dependent suppression of the GPR56‐CTF‐induced SRF signal by the CDK7 inhibitor YKL‐5‐124, but not by the CDK12/13 inhibitor THZ531. Four technical replicates. One of the four individual experiments. Unpaired t ‐test, bars and error bars represent mean and SD, *** P < 0.0005, ** P < 0.005, * P < 0.05.

Article Snippet: The CDK12/13 inhibitor THZ531 (Selleckchem #S6595), the CDK7/12/13 inhibitor THZ1 (Selleckchem #S7549), and the CDK7 inhibitors YKL‐5‐124 (Selleckchem #S8863) and LDC4297 (Selleckchem #S7992) were dissolved in DMSO at 50 mM stock concentration and diluted in media so that final DMSO concentration was 0.1% in all conditions.

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, In Vivo, Injection, Expressing, Reporter Assay

Journal: EMBO Molecular Medicine

Article Title: CDK7/12/13 inhibition targets an oscillating leukemia stem cell network and synergizes with venetoclax in acute myeloid leukemia

doi: 10.15252/emmm.202114990

Figure Lengend Snippet: Gene expression of GPR56 , SMO (left), and TGFb targets SRC, ZEB1, and ZEB2 (right) normalized to GAPDH in AML‐491 cells determined by q‐RT‐PCR 4 h after treatment with THZ1 0.5 µM or LDC4297 2 µM. Unpaired t ‐test. Mean, SD, and individual values from three individual treatments. *** P < 0.0005, ** P < 0.005, * P < 0.05. Setup of in vivo drug treatment. NSG mice were injected with 10 5 AML 04H112 cells. Four weeks post injection, bone marrow (BM) was analyzed for human leukemic engraftment by BM aspiration. Treatment with either THZ1 or vehicle was started in the following week as indicated. BM was analyzed again after the end of the 4‐week treatment period. Overall percentage of human (huCD45 + ) leukemic cells in mice before and at the end of the 4‐week treatment period. Individual mice and mean engraftment are shown. Unpaired t ‐test. *** P < 0.0005. N = 10 mice for each group. Left : Representative FACS plots showing the typical CD34 low GPR56 high profile of sample 04H112 before injection and after the 4‐week treatment with THZ1. Right : both LSC compartments, the CD34 ‐ GPR56 + and the CD34 + GPR56 + fractions were significantly reduced in vivo in the THZ1 treatment group. Individual mice and mean engraftment are shown. Unpaired t ‐test. *** P < 0.0005, ** P < 0.005, * P < 0.05. N = 10 mice for each group. Left : Overall percentage of human (huCD45 + ) leukemic cells and the fraction of CD34 + GPR56 + among human cells (mean, individual mice) two weeks after drug withdrawal. Right : FACS plot visualizing that the CD34 + GPR56 + is re‐established upon drug removal. Unpaired t ‐test, *** P < 0.0005, ** P < 0.005, * P < 0.05. Correlation plot showing an anti‐correlation between the percentage of CD34 + GPR56 + cells in AML samples and the corresponding IC50s for THZ1 determined in the respective samples. Pearson correlation. Each dot represents one sample. See Dataset for sample characteristics. FACS histogram plot (left) and summary bar graph from three replicate wells (right) showing reduction of GPR56 surface expression on the GPR56‐positive AML cell line HEL only in conditions that contain the more specific CDK7 inhibitor YKL‐5‐124, but not those that contain only the specific CDK12/13 inhibitor THZ531. Unpaired t ‐test, bars, and error bars represent mean and SD of three individual treatments, *** P < 0.0005, ** P < 0.005, * P < 0.05. SRF reporter assay showing dose‐dependent suppression of the GPR56‐CTF‐induced SRF signal by the CDK7 inhibitor YKL‐5‐124, but not by the CDK12/13 inhibitor THZ531. Four technical replicates. One of the four individual experiments. Unpaired t ‐test, bars and error bars represent mean and SD, *** P < 0.0005, ** P < 0.005, * P < 0.05.

Article Snippet: The CDK12/13 inhibitor THZ531 (Selleckchem #S6595), the CDK7/12/13 inhibitor THZ1 (Selleckchem #S7549), and the CDK7 inhibitors YKL‐5‐124 (Selleckchem #S8863) and LDC4297 (Selleckchem #S7992) were dissolved in DMSO at 50 mM stock concentration and diluted in media so that final DMSO concentration was 0.1% in all conditions.

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, In Vivo, Injection, Expressing, Reporter Assay