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.