Salt-inducible kinase 3 protects tumor cells from cytotoxic T cell attack by promoting TNF-induced NF-?B activation

Background: Cancer immunotherapeutic strategies showed unprecedented results in the clinic. However, many patients do not respond to immuno-oncological treatments due to the occurrence of a plethora of immunological obstacles, including tumor intrinsic mechanisms of resistance to cytotoxic T cell attack. Thus, a deeper understanding of these mechanisms is needed to develop successful immunotherapies. Methods: To identify novel genes that protect tumor cells from effective T cell-mediated cytotoxicity, we performed a genetic screening in pancreatic cancer cells challenged with TILs and antigen-specific T cells. Results: The screening revealed 108 potential genes that protected tumor cells from T cell attack. Among them, salt-inducible kinase 3 (SIK3) was one of the strongest hits identified in the screening. Both genetic and pharmacological inhibition of SIK3 in tumor cells dramatically increased T cell-mediated cytotoxicity in several in vitro co-culture models, using different sources of tumor and T cells. Consistently, adoptive T cell transfer of TILs led to tumor growth inhibition of SIK3 depleted cancer cells in vivo. Mechanistic analysis revealed that SIK3 rendered tumor cells susceptible to TNF secreted by tumor-activated T cells. SIK3 promoted NF-?B nuclear translocation and inhibited Caspase-8 and -9 after TNF stimulation. Chromatin accessibility and transcriptome analyses showed that SIK3 knockdown profoundly impaired the expression of pro-survival genes under the TNF-NF-?B axis. TNF stimulation led to SIK3-dependent phosphorylation of the NF-?B upstream regulators inhibitory-?B kinase (IKK) and NF-kappa-B inhibitor alpha (I?Ba) on the one side, and to inhibition of histone deacetylase 4 (HDAC4) on the other side, thus sustaining NF-?B activation and nuclear stabilization. A SIK3 dependent gene signature of TNF-mediated NF-?B activation was found in a majority of pancreatic cancers where it correlated with increased cytotoxic T cell activity and poor prognosis. Overall design: ATAC-seq of PANC1 cell line with shRNA mediated SIK3 knockdown was treated with human TNF or vehicle control