Abstract 89: Role of CDK8 and CDK19 in STAT1 phosphorylation at serine 727

Transcription factor STAT1 is part of the JAK/STAT signaling cascade; it plays an essential role in mediating responses to interferons (IFN). STAT1 modulates many cellular processes and has been implicated in both tumor-suppressing and tumor-promoting activities. IFNγ treatment induces STAT1 phosphorylation at tyrosine 701, enabling the translocation of STAT1 from cytoplasm to the nucleus. STAT1 is also phosphorylated at serine 727; S727 phosphorylation modulates the transcriptional activity of STAT1. Transcription-regulating kinase CDK8 was shown to be primarily responsible for IFNγ-induced STAT1 S727 phosphorylation, and serine-phosphorylated form of STAT1 was proposed as a potential pharmacodynamic (PD) marker for the activity of CDK8, an emerging cancer drug target. However, the role of CDK8 in STAT1 S727 phosphorylation in the absence of IFNγ treatment is controversial, as is the role of CDK19, a closely related paralog of CDK8, as well as the relative contributions of kinase-dependent and kinase-independent activities of CDK8 and CDK19. We have found that Senexin B, a selective CDK8/19 inhibitor, decreased both IFNγ-induced and basal STAT1 S727 phosphorylation in all the tested types of tumor cells but some phosphorylation was maintained even at the highest concentrations of Senexin B. We then generated a series of derivatives of human 293 cells with CRISPR-mediated knockout of CDK8, CDK19 and both CDK8 and CDK19 (double knockout, dKO), with subsequent reconstitution of dKO with wild-type (WT) CDK8 or CDK19 or with kinase-dead (KD) mutants of these proteins. The knockout of CDK8 or CDK19 individually had only a moderate effect on IFNγ-induced and basal STAT1 S727 phosphorylation, which remained susceptible to Senexin B. In contrast, such phosphorylation was strongly decreased in dKO cells, where it was unaffected by Senexin B. Senexin B-sensitive STAT1 S727 phosphorylation was restored by re-introduction of WT CDK8 or CDK19 but not of their KD mutants. Hence, both CDK8 and CDK19 mediate STAT1 S727 phosphorylation in kinase-dependent manner. We also tested different cytokines and stress-inducing agents for the ability to induce STAT1 S727 phosphorylation in WT and dKO cells. Several treatments, including serum stimulation, EGF, TNFα, etoposide and taxol, increased this phosphorylation in dKO cells, indicating that STAT1 serine phosphorylation induced by many treatments was CDK8/19-independent. Hence, great caution needs to be utilized in using STAT1 S727 phosphorylation as a PD marker for CDK8 activity. Citation Format: Jing Li, Jiaxin Liang, Lili Wang, Eugenia Broude, Igor Roninson, Mengqian Chen. Role of CDK8 and CDK19 in STAT1 phosphorylation at serine 727 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 89.