(P 225) DETERMINING THE CONTRIBUTIONS OF STAT1 AND THE Y701 PHOSPHORYLATION TO THE UPS ANTI-TUMOR MICROENVIRONMENT

Objective: Sarcoma is a heterogeneous group of cancers with over 70 subtypes and survival rates have remained relatively stagnant in recent decades. We previously found that a subset of Undifferentiated Pleiomorphic Sarcoma (UPS) cases harboured tumor-infiltrating lymphocytes (TILs), expressed high levels of PD-1 ligand (PD-L1) RNA and had better clinical outcomes than those with low PD-L1 expression. Through RNA-sequencing, a list of genes differentially expressed but unique to the UPS cases with high PD-L1 expression was determined. Genes in the Th1 pathway were significantly overexpressed within this list. We hypothesize that tumor-related genes from the Th1 pathway like STAT1, a key transcription factor in the IFN-γ pathway, may be related to an anti-tumor microenvironment. Our aims are to identify the potential roles of STAT1 in malignant cell behaviours with the use of gene editing technologies. First, we characterized patient-derived PD-L1 high and low cell lines prior to creating STAT1 knockout lines. Knockout lines were characterized and then transfected to overexpress wildtype or phosphomutant STAT1; the Y701 phosphorylation is theorized to be a molecular switch between pro and anti-tumour functions of STAT1. Transcriptome, cytokine profile, proliferation, migration, and protein expression analyses of wildtype and gene-edited cell lines were employed to investigate the role of STAT1 and the Y701 phosphorylation in UPS cell lines.

Methods: Sarcoma specimens were used to derive primary cell lines and a subset were assessed for STAT1 protein expression, growth, and migration rate using Western blots, DNA-content cell enumeration, and scratch/wound healing analyses. Cell lines were treated with recombinant IFN-γ and characterized as described above to determine if IFN-γ pathway activation shaped cell behaviours differently across four primary UPS lines. STAT1 expression was disrupted with CRISPR-Cas9 within two cell lines and the isogenic lines were assessed for changes in proliferation, migration, cytokine secretion and in the transcriptome in the presence of absence of IFN-γ. Lentiviral constructs that overexpress wildtype or Y701-phosphomutant STAT1 were transfected into the knockout backgrounds and cell behaviour was assessed as outlined above to determine if a phospho-STAT1-dependent role exists in UPS cell behaviour.

Results: Four cell lines from PD-L1 high and low cases show distinct doubling times however, three of four cases show longer doubling times in the presence of IFN-γ. Migration rates were unique for each cell line assessed and were not impacted by IFN-γ treatment. Transcriptomes of cell lines were all impacted by IFN-γ where gene expression of anti-tumour cytokines is significantly upregulated. Two UPS cell lines have been successfully gene-edited to be deficient in STAT1 protein production, and phenotypic characterizations demonstrated that the loss of STAT1 increased cell growth in one cell line. Overexpression of both wildtype and Y701-phosphomutant STAT1 decreased migration in that same cell line. Experiments are underway to characterize the other impacts of wildtype and phosphomutant overexpression in the second STAT1 knockout cell line.

Conclusion: Cell lines derived from PD-L1 high and low UPS cell lines demonstrate unique rates of proliferation and migration, which could be related to tumour behaviour and the microenvironment. While some cell lines show IFN-γ-dependent migratory and proliferative behaviour, upregulation of anti-tumour cytokines are driven by IFN-γ treatment in all lines. One upregulated STAT1 phosphomutant line showed increased migratory behaviour relative to the wildtype upregulated cell line, suggesting the pY701 may impact cell migration. By assessing the functional consequences of IFN-y pathway activation, STAT1-deficiency, and the Y701 phosphorylation, we aim to clarify how STAT1-directed cellular behaviour may relate the improved anti-tumour microenvironment of UPS PD-L1 high cases.