Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases
- The Institute of Cancer Research, United Kingdom
- Merck KGaA, Germany
- Cardiff University, United Kingdom
Figures
Figure 1 with 2 supplements
Optimised compounds for exploring CDK8 and CDK19 function.
(A) Chemical structure and activity of compounds 2, 3 and 4 (n > 2, mean ± s.d.). (B) Overlay of 3 (grey; ocd 5HBJ) and 4 (pink; Pdb code: 5IDN) bound to CDK8/CCNC. Key interactions (yellow) and …
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Figure 1—source data 1
Properties of CDK8/19 ligands and their effects on reporter expression and cell proliferation in a human colorectal cancer cell line panel.
- https://doi.org/10.7554/eLife.20722.004
Figure 1—figure supplement 1
Effect of CDK8 and CDK19 shRNA and siRNA treatment in CDK8-amplified human colorectal cancer cell lines.
(A) WNT pathway reporter activity, cell viability and CDK8/19 transcript levels in COLO205-cl4 TCF/LEF reporter cells expressing either an inducible CDK8 shRNA, a constitutive CDK19 shRNA, an …
Figure 1—figure supplement 2
Comparison of CDK8 and CDK19 gene copy number or protein expression with sensitivity to treatment with compound.
Data from Figure 1—source data 1 for CDK8 gene copy number or protein expression, or CDK19 protein expression, were compared with the effects of 1, 3 and 4 on 14 d colony growth assay. Pearson r2 …
Figure 2 with 4 supplements
Target inhibition and antitumor activity of CDK8/19 ligands 3 and 4 in established human colorectal cancer cell line xenografts.
(A) Level of p-STAT1SER727 in SW620 human colorectal cancer xenografts following a single dose of 4, relative to the p-STAT1SER727 level in vehicle-treated mice. Significance was determined by …
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Figure 2—source data 1
Details of human colorectal cancer cell line xenograft studies.
- https://doi.org/10.7554/eLife.20722.008
Figure 2—figure supplement 1
Differential antitumor activity in human LS513 colorectal cancer xenografts treated with CDK8/19 ligand 1.
Antitumor activity in (A) HCT116 and (B) LS513 colorectal cancer xenografts treated with 1. (C) target engagement in LS513 colorectal cancer xenografts treated with 1. STAT1 and p-STAT1SER727 levels …
Figure 2—figure supplement 2
Pharmacodynamic profiling of CDK8/19 ligand 3 in human colorectal cancer xenografts.
(A, B) Level of p-STAT1SER727 in HCT116 colorectal cancer xenografts in mice treated with single doses of 3. Significance was determined by Kruskal-Wallis test and Dunn’s post-test (*p=<0.001, **p=<0…
Figure 2—figure supplement 3
Antitumor activity and target engagement in human colorectal cancer xenografts treated with CDK8/19 ligands 3 and 4.
(A, B) Antitumor activity (A) and target engagement (B) in SW620 colorectal cancer xenografts treated with 3. (C, D) Antitumor activity (C) and target engagement (D) in LS1034 colorectal cancer …
Figure 2—figure supplement 4
LS1034 colony assay.
(A) Growth of LS1034 cell colonies treated for 14 d with 350 nM test compounds 1, 3 and 5 (n = 3, mean ± s.d.).
Figure 3 with 2 supplements
In vitro and in vivo activity of CDK8/19 ligands in patient-derived tumour xenograft models.
(A) GI50 values for 2 in PDX soft agar colony cultures. (B) Exemplar dose-response profiles for selected colorectal cancer clonogenic assays treated with 2. (C) Volume of human colorectal cancer CXF …
Figure 3—figure supplement 1
In vivo activity of CDK8/19 ligand 3 in PDXs.
Volume of PDXs treated with vehicle, 3 and/or irinotecan/oxaliplatin, 0 (n = 10, mean ± s.e.m). Xenografts tested: A, CFX 883 (KRASMUT, CTNNB1MUT, PTENMUT); C, CFX 1729 (p53MUT); E, CFX 280 (KRASMUT,…
Figure 3—figure supplement 2
Pharmacodynamic and antitumor activity of 3 and 4 in AML models.
(A) Percentage of AML Nomo-1 cells detected in the peripheral blood of NOD/Shi-SCID/IL-2Rγnull mice treated with 10 mg/kg po bid 3 or a vehicle control (p=<0.001, Kruskal-Wallis and Dunn’s post …
Figure 4 with 1 supplement
Microarray gene expression profiling following in vivo treatment of human colorectal cancer xenografts with CDK8/19 ligands.
Mice were treated with 70 mg/kg po 1 (SW620 and COLO205), 20 mg/kg po 3 (COLO205). (A) Venn plots of transcription factor-associated genesets or those encoding or regulating pathways enriched in …
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Figure 4—source data 1
Geneset expression analysis of microarray data following in vivo treatment of SW620 or COLO205 cells.
- https://doi.org/10.7554/eLife.20722.017
Figure 4—figure supplement 1
Microarray gene expression profiling following in vivo treatment of colorectal cancer cell line xenografts.
Xenografts were treated with 70 mg/kg po 1 (SW620 and COLO205) and 20 mg/kg po 3 (COLO205). Scatterplots of false discovery rate (FDR-q) versus normalized enrichment score (NES) for indicated gene …
Figure 5 with 1 supplement
Treatment with CDK8/19 ligand 1 reduces the hyperplastic crypt stem cell population.
Gene expression, measured by RT-PCR, in the intestinal epithelial stem and TA cells isolated from mice expressing a Dox-inducible activated β-catenin transgene. (A) Transcript abundance, relative to …
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Figure 5—source data 1
Antibodies and PCR primers used for analysis of mouse intestinal epithelial cells.
- https://doi.org/10.7554/eLife.20722.020
Figure 5—figure supplement 1
Analysis of stem and TA cells isolated from the hyperplastic crypts of mice expressing a Dox-inducible activated β-catenin transgene.
(A) Experimental design. (B) FACS approach used to isolate the stem and TA cells. (C) Abundance of beta-actin control transcripts following treatment.
Figure 6 with 1 supplement
Effect of CDK8/19 chemical ligands on bone development and the immune response in model systems.
Mouse KS483 osteoprogenitor cells were treated with LGK974 (red) or compound 3 (black) for 13 d and bone matrix formation determined by measuring (A) N-terminal propeptide of type I procollagen …
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Figure 6—source data 1
Culture conditions and data from CDK8/19 ligand profiling in the culture/co-culture cell model panel.
- https://doi.org/10.7554/eLife.20722.023
Figure 6—figure supplement 1
Effect of CDK8/19 ligands 1-4 on 12 culture/coculture cell models.
(A) Log2 ratio of cell model biomarkers plotted relative to levels under control conditions. The gray area indicates the control envelope (>95% confidence). Points outside this area were considered …
Figure 7 with 1 supplement
Examples of degenerative and proliferative lesions in rats treated with CDK8/19 ligands 3 or 4.
(A) Intact proliferative zone in the bone growth plate of a control rat. (B) Dysplastic proliferative zone, showing disturbance of regular endochondrial ossification, from a rat treated with 20 …
Figure 7—figure supplement 1
Pharmacodynamic response in Wistar rats treated with CDK8/19 ligand 3.
(A) Immunoblot showing p-STAT1SER727 and STAT1 levels in lysates made from rat spleens harvested 2, 6 or 24 hr after the final dose of 3. (B) Ratio of p-STAT1SER727 to STAT1 in data from A.
Tables
Table 1
CDK8/19 ligands 3 and 4 adversely affect multiple organs in rats and dogs.
Wistar rats (5 male and 5 female per cohort) or Beagle dogs (2 male and 2 female per cohort for 3 and 1 male and 1 female for 4) received a daily oral dose of 3 or 4 for 14 days. In the rat study of 4, all animals were prematurely culled at 60 mg/kg and one male and female at 20 mg/kg, as a result of compound toxicity. In the dog studies, all animals were prematurely culled in the study of 3 and one female following exposure to 4 as a result of toxicity. The most severely affected organs are indicated in bold. The fold efficacious dose was calculated from a plasma PK measurement of compound exposure in satellite animals run in parallel to the tolerability study and compared to exposures at efficacious doses in human tumour xenograft models in mice (m – male and f – female).
| Rat | Dog | |||
|---|---|---|---|---|
| Low dose | Mid dose | High dose | Low dose | |
| CMPD 3 (mg/kg) | 5 | 10 | 20 | 5 |
| Target organs | Bone, bone, marrow, heart, liver, lung, lymph nodes, pancreas, reproductive tract (m), spleen, thymus. | Bone, bone marrow, heart, liver, lung, lymph nodes, pancreas, reproductive tract (m and f), spleen, thymus. | Bone, bone marrow, heart, liver, lung, lymph nodes, pancreas, reproductive tract (m and f), spleen, thymus. | Bone marrow, gastrointestinal mucosa, heart, lymphatic system |
| Fold of efficacious dose; 10 mg/kg | ~0.3 (m) – 1.3 (f) | ~0.5 (m) – 2 (f) | ~1 (m) – 5 (f) | ~0.3 (m) – 0.3 (f) |
| CMPD 4 (mg/kg) | 10 | 20 | 60 | 20 |
| Target organs | Bone, bone marrow, intestines, liver, lung, lymph nodes, mammary gland, pancreas, reproductive tract (m and f), skin, spleen, stomach, thymus. | Bone, bone marrow, heart, intestines, liver, lung, lymph nodes, mammary gland, pancreas, reproductive tract (m and f), skin, spleen, stomach, thymus | Bone, bone marrow, brain, heart, intestines, liver, lung, lymph nodes, mammary gland, pancreas, reproductive tract (m and f), skin, spleen, stomach, thymus | Bone marrow, heart, Intestines, lymphatic system |
| Fold of efficacious dose; 10 mg/kg 30 mg/kg | ~0.9 (m) – 2.4 (f) ~0.3 (m) – 0.8 (f) | ~3.9 (m) – 5.7 (f) ~1.3 (m) – 1.9 (f) | ~10.8 (m) – 23.1 (f) ~3.6 (m) – 7.7 (f) | ~22 (m) – 46 (f) ~7 (m) – 15 (f) |
