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Selective CDK8 inhibitor SEL120-34A alters expression of interferon-related DNA damage resistance signature genes in colorectal cancer

ABSTRACT: CDK8 (cyclin-dependent kinase 8) is a kinase component of a multi - protein Mediator complex, involved in transcription control. Several studies indicated that high overexpression and activity of CDK8 could be a driver of malignant progression in colorectal cancer (CRC). Herewith we present molecular insights into mechanism of action of SEL120-34A - a selective small molecule inhibitor of CDK8 kinase. Biochemical and binding studies indicated that SEL120-34A selectively binds and inhibits enzymatic activity of CDK8 in the low nM range. Recently CDK8 has been described as a regulator of STAT1 activity in NK cells where by phosphorylating STAT1 serine 727 (Ser727) influences a possible immunoescape mechanism in various cancers. Consistently, SEL120-34A and other recently reported selective CDK8 inhibitors could repress phosphorylation of STAT1 at a Ser727 at low nM concentrations in cancer cells without any significant changes on tyrosine sites directly regulated by JAK kinases. SEL120-34A inhibited expression of several STAT1 dependent genes in CRC cell lines, stimulated by various cytokines and growth factors. These results were further corroborated with specific CDK8 siRNA knockdown experiments and chromatin immunoprecipitation studies showing CDK8 occupancy on promoters of SEL120-34A regulated genes. In order to better characterize in vivo mechanism of action, mice bearing HCT116 and Colo205 xenograft tumors were treated with SEL120-34A and gene expression changes were measured with microarrays in excised tumors. In animals treated with the CDK8 inhibitor a dose dependent repression of STAT1 Ser727 was observed. The functional analyses of significantly (adj. p. value < 0.05) altered genes with Gene Ontology revealed that those with reduced expression belong to interferon I pathway and type I interferon-mediated signaling pathway terms. This subset of STAT regulated genes was further characterized as an interferon-related DNA damage resistance signature (IRDS) - a prosurvival pathway which correlated strongly with resistance to radiation and chemotherapy in various tumors. Consistently, SEL120-34A has shown very potent cytotoxic synergy with standard of care drugs in CRC, particularly in cells stimulated with interferons. Taken together, for the first time we have shown that selective CDK8 inhibitors are potent regulators of STAT related - IRDS signaling pathway in vitro and in vivo. In addition to previously reported stand-alone efficacy of CDK8 inhibitors in vivo, we provide also a combination treatment rationale for CRC.

ORGANISM(S): Homo sapiens

PROVIDER: GSE77987 | GEO | 2016/03/01

SECONDARY ACCESSION(S): PRJNA312303

REPOSITORIES: GEO

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Project description:CDK8 (cyclin-dependent kinase 8) is a kinase component of a multi - protein Mediator complex, involved in transcription control. Several studies indicated that high overexpression and activity of CDK8 could be a driver of malignant progression in colorectal cancer (CRC). Herewith we present molecular insights into mechanism of action of SEL120-34A - a selective small molecule inhibitor of CDK8 kinase. Biochemical and binding studies indicated that SEL120-34A selectively binds and inhibits enzymatic activity of CDK8 in the low nM range. Recently CDK8 has been described as a regulator of STAT1 activity in NK cells where by phosphorylating STAT1 serine 727 (Ser727) influences a possible immunoescape mechanism in various cancers. Consistently, SEL120-34A and other recently reported selective CDK8 inhibitors could repress phosphorylation of STAT1 at a Ser727 at low nM concentrations in cancer cells without any significant changes on tyrosine sites directly regulated by JAK kinases. SEL120-34A inhibited expression of several STAT1 dependent genes in CRC cell lines, stimulated by various cytokines and growth factors. These results were further corroborated with specific CDK8 siRNA knockdown experiments and chromatin immunoprecipitation studies showing CDK8 occupancy on promoters of SEL120-34A regulated genes. In order to better characterize in vivo mechanism of action, mice bearing HCT116 and Colo205 xenograft tumors were treated with SEL120-34A and gene expression changes were measured with microarrays in excised tumors. In animals treated with the CDK8 inhibitor a dose dependent repression of STAT1 Ser727 was observed. The functional analyses of significantly (adj. p. value < 0.05) altered genes with Gene Ontology revealed that those with reduced expression belong to interferon I pathway and type I interferon-mediated signaling pathway terms. This subset of STAT regulated genes was further characterized as an interferon-related DNA damage resistance signature (IRDS) - a prosurvival pathway which correlated strongly with resistance to radiation and chemotherapy in various tumors. Consistently, SEL120-34A has shown very potent cytotoxic synergy with standard of care drugs in CRC, particularly in cells stimulated with interferons. Taken together, for the first time we have shown that selective CDK8 inhibitors are potent regulators of STAT related - IRDS signaling pathway in vitro and in vivo. In addition to previously reported stand-alone efficacy of CDK8 inhibitors in vivo, we provide also a combination treatment rationale for CRC. Female mice, SCID beige , 7-8 weeks old, were maintained according to the standards of the specific pathogen free conditions. Colo205 or HCT116 cells were centrifuged at 1000 rpm for 5 min and counted using Trypan blue method. A 5M cells were suspended in PBS and MatrigelÂ® (BD Biosciences) (3:1, v:v) and were injected subcutaneously (SC), on the right hind limb. As the tumor was maintained until it reached ~100-150 mm3. Mice were randomized into uniform groups and subjected to the compound administrations. Prior to use, compound was dissolved freshly in water and administered 30mg/kg BIDx3 per os using cannula in a volume of 10 ul per 1 g of body weight. Tumor samples were collected 4 and 16 hours after last compound dose and preserved in RNAlater.

Project description:Gene regulation by cytokine-activated STAT transcription factors requires serine phosphorylation within the transactivation domain (TAD). STAT1 and STAT3 TAD phosphorylation was reported to occur upon promoter binding by an unknown kinase. Here we show that the Mediator CDK8 module phosphorylates S727 of the STAT1 TAD in the interferon (IFN) signaling pathway as well as the TADs of other STATs. Microarray analysis reveals that CDK8-mediated STAT1 TAD phosphorylation positively or negatively regulates over 40% of IFN-gamma-responsive genes, and RNA polymerase II occupancy correlates with gene expression changes. This selective regulation occurs despite CDK8 occupancy and STAT1 S727 phosphorylation at both S727 phosphorylation-dependent and -independent IFN-gamma target genes. Independently of its role as STAT1 S727 kinase CDK8 acts as a positive regulator of IFN-gamma responses. These data reveal a dual input of CDK8 in STAT1-controlled transcription and propose a key role for CDK8 in TAD phosphorylation of other STATs during cytokine responses. STAT1 WT and STAT1 S727A mouse fibroblasts were treated with siRNA to CDK8 (siCdk8 smart pool, On Target Plus, Dharmacon) and control siRNA (siCtrl) and stimulated with IFN-gamma for 4 h or left untreated. Total RNA from three independent experiments for each treatment and each genotype was isolated from cells using Trizol reagent (Invitrogen) following the manufactures protocol and used for expression analysis using Agilent Whole Mouse Genome Microarrays, 8x60K. Standard protocols for labeling and hybridization were followed. In brief, fluorescent cRNA was generated using Low Input Quick Amp Labeling Kit (Agilent). The amplified cyanine 3-labeled cRNA samples were then purified using SV Total RNA Isolation System (Promega) and hybridized to microarray slides. Microarray slides were washed and scanned with an Agilent Scanner. Note: The outlier array #10 [SA.CDK.gamma.R3] was removed from subsequent analysis and its processed data was not provided. However, its raw data file has been linked as a supplementary file at the foot of the Series record.

Project description:There is an unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545 a WNT-pathway inhibitor discovered by cell-based screening is a potent and selective chemical probe for the Mediator complex-associated protein kinases CDK8 and CDK19. CCT251545 is an ATP competitive inhibitor with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand binding site. In contrast to Type II inhibitors of CDK8/19 CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogues not only alter WNT-pathway regulated gene expression but also other CDK8/19 targets including STAT1-regulated gene expression. Consistent with this we find that phospho-STAT1SER727 is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally we demonstrate in vivo activity of CCT251545 in WNT-dependent tumours. Compound 1,2, 6 and 9 are close analogues of a 3,4,5-trisubstituted pyridine series identified from a high-throughput cell-based reporter assay of WNT signalling. They were shown to be potent and selective inhibitors of the Mediator complex-associated protein kinases CDK8 and CDK19. They are ATP competitive inhibitors with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand-binding site. A 3D culture model of murine intestinal-derived organoids expressing a transgenic doxycycline-inducible mutant beta-catenin expression following removal of doxycycline results in reduced WNT signalling and was compared to treatment for 24 hours with coumpound 2, 6 and 9. Compound 2 (n=3), compound 6 (n=4), compound 9 (n=3), doxycycline removal (n=4), doxycycline treatment (n=7). Samples were hybridized agianst mouse reference. Compounds 2, 6 and 9 are close analogues of a 3,4,5-trisubstituted pyridine series identified from a high-throughput cell-based reporter assay of WNT signalling. They were shown to be potent and selective inhibitors of the Mediator complex-associated protein kinases CDK8 and CDK19. They are ATP competitive inhibitors with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand-binding site.

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Selective CDK8 inhibitor SEL120-34A alters expression of interferon-related DNA damage resistance signature genes in colorectal cancer

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