(P 55) THE EFFECT OF STAT1 Y701 PHOSPHORYLATION ON GENE EXPRESSION IN UPS

Objective: Investigating the signal transduction pathways related to immunotherapy could improve the survival rates for some sarcoma types. We previously found that a subset of Undifferentiated Pleomorphic Sarcoma (UPS) cases had tumor-infiltrating lymphocytes, high PD-L1 RNA expression, and improved clinical outcomes in comparison to those with low PD-L1 expression. Genes differentially expressed but unique to the PD-L1 high UPS cohort were significantly enriched in the Th1 pathway. We hypothesized that the expression of Th1-related genes such as STAT1, a transcription factor activated by IFN-γ, may be associated with an anti-tumor microenvironment. STAT1 can exist in a tyrosine 701-phosphorylated (pSTAT1) or unphosphorylated (uSTAT1) state. pSTAT1 is associated with anti-tumor and anti-proliferative functions, whereas uSTAT1 has been linked to increased DNA resistance gene expression programs. We aimed to investigate how STAT1 phosphorylation influenced gene expression in UPS.

Methods: STAT1 knockouts (STAT1KO) were generated from primary UPS cell lines. Lentiviral vectors encoding wildtype STAT1 or a Y701F phosphomutant were transfected into two UPS STAT1KO cell lines. RNA was extracted from cells overexpressing wildtype STAT1 (pSTAT1) or Y701F-mutant STAT1 (uSTAT1), along with empty vector controls (EV), and subjected to transcriptomic analysis. Differentially expressed genes (DEGs) associated with STAT1 Y701 phosphorylation were identified and subjected to pathway analyses.

Results: DEGs based on STAT1 Y701 phosphorylation status were identified. In pathway analyses from one case, all pathways were negatively enriched when comparing DEGs between the uSTAT1 and pSTAT1 cell lines. Further, when comparing uSTAT1 cells to the EV, all pathways—including type I IFN signalling—were negatively enriched. This may indicate an inhibitory role for uSTAT1 on other STATs or downstream effectors within these pathways.

Conclusion: In UPS, uSTAT1 was associated with a negative enrichment in multiple pathways, suggesting the Y701 residue is critical in IFN-γ signal transduction. The negative enrichment of pathways in uSTAT1 compared to the EV suggests that uSTAT1 may negatively regulate transcriptional programs mediated by other STAT family members. Together, these findings support that STAT1 phosphorylation status impacts expression programs and may influence UPS cell properties as well as the tumor microenvironment.