RNAseq of mouse RAW264.7 macrophages with CRISPR deletion of SGK1, SGK3, or SGK1/3 or treated with SGKi Sanofi-14h with or without DMXAA stimulation.
The STING signaling pathway is essential for the innate immune response to DNA viruses and bacteria and is important in tumor immunity. STING binding to cGAMP or to synthetic agonists leads to the activation of the kinase TBK1 which phosphorylates the transcription factor IRF3 which promotes expression of type 1 interferons such as IFNb to block viral activity. Aberrant type 1 IFN expression is associated with human diseases including autoimmunity, HIV, and cancer. Here we identify N-[4-(1H-pyrazolo[3,4-b] pyrazin-6-yl)-phenyl]-sulfonamide (Sanofi-14h), a compound with preference for inhibition of the AGC family kinase SGK3, as an inhibitor of IFNb gene expression in response to STING stimulation of macrophages. Sanofi-14h abrogated SGK activity and also impaired activation of the critical TBK1/IRF3 pathway downstream of STING activation, notably blocking the ligand-induced interaction of STING with TBK1. Deletion of SGK1 and SGK3 in macrophages suppressed activation of IFNb transcription but did not block TBK1/IRF3 activation downstream of STING. Gene and protein expression analysis revealed that deletion of SGK1/3 in a macrophage cell line decreases basal expression of critical transcription factors required for the innate immune response, such as IRF7 and STAT1.  Additional studies reveal that other AGC kinase inhibitors block TBK1 and IRF3 activation suggesting common action on a critical regulatory node in the STING pathway. Thus, studies with Sanofi-14h have revealed both SGK-dependent and SGK-independent effects in the STING pathway and suggest a mechanism to alter type 1 IFN transcription through small molecule therapy Overall design: Murine RAW264.7 macrophages were CRISPR edited with sgRNAs against SGK1 or SGK3 or control sgRNA. Dual SGK1/3 KO cells were also constructed. RNA was collected from cells under resting (unstimulated) conditions or after 2 hours of stimulation with the murine STING agonist DMXAA (10µg/mL). In addition, a set of WT cells were pretreated with the SGK inhibitor Sanofi-14h (500nM) or vehicle (DMSO) for 2 hours prior to stimulation.