Project Summary In response to stimulation by type I interferon (IFN-I), STAT2 is phosphorylated by JAK tyrosine kinases and then binds to phosphorylated STAT1 and IRF9 to form ISGF3, which drives the transcriptional response. This canonical function of STAT2 is indispensable for the biological responses to IFN-I, which are cytostatic and also lead to the activation of anti-tumor immunity in cancer cells. Consequently, STAT2 has been assumed to help suppress cancer progression and enhance therapy. However, this original view of STAT2 is challenged by several new lines of evidence that reveal a previously unknown pro-tumor impact of STAT2. A higher level of STAT2 expression is associated with a worse prognosis in lung cancer and an aggressive phenotype in breast cancer, and STAT2 deficiency protects mice from chemically induced skin and colon cancer. However, the molecular mechanisms underlying these pro-tumor activities of STAT2 are not yet well understood. Helping to bridge this gap, we have identified two novel non-canonical, pro-tumor activities of STAT2 that do not require tyrosine phosphorylation. First, STAT2 lacking phosphorylation of Y690 (U-STAT2) forms a complex with IRF-9, even in the absence of IFN-I stimulation. Intriguingly, unlike ISGF3, this constitutive U-STAT2:IRF9 complex increases the expression of a set of NFκB-dependent genes, including IL-6, facilitating the development of a mesenchymal/stem-like phenotype in lung cancer cells. Second, U-STAT2 binds to the cytosolic sensor STING, preventing STING from stimulating IFN-I synthesis in response to DNA or synthetic agonists of cGAS. This is the first example of a STAT2 function that is completely independent of any role in transcription. Notably, both these non-canonical STAT2 activities are regulated by a previously unknown phosphorylation of threonine 404 in STAT2 (T403 in mice), which promotes the formation of U-STAT2:IRF9 and U-STAT2:STING complexes. Consequently, elevated level of T404 phosphorylated STAT2 result in increased resistance to cisplatin in lung cancer cells and enhanced tumor growth in a xenograft model. Furthermore, while the level of P-T404 STAT2 in human lung adenocarcinoma specimens correlates with reduced immune cell infiltration, prevention of T403 phosphorylation by a T-to-A mutation in mice leads to better tumor control and enhanced anti-tumor immunity. We propose that the T404 phosphorylation of U-STAT2 promotes cancer progression and resistance to therapy. To test this hypothesis, we will determine the tumor-intrinsic roles of U-STAT2 in cancer progression and chemoresistance; and investigate the role of the T404-regulated U-STAT2/STING complex in resistance to immunotherapy.