(P 66) EWING TUMORS WITH STAG2 LOSS DEMOSNTRATE ALTERED INTERFERON SIGNATURES AND IMPAIRED CXCL10 EXPRESSION

Objective: Ewing sarcoma is a fusion oncoprotein-driven bone sarcoma diagnosed in adolescents and young adults. Studies of Ewing molecular biomarkers associated with inferior patient survival are emerging. Approximately ~15-20% of Ewing tumors demonstrate loss of STAG2 expression and tumors with STAG2 loss are more aggressive both in vitro and in vivo and are associated with inferior outcomes. Understanding factors driving aggressiveness and treatment resistance in Ewing tumors with STAG2 loss is a priority. Tumor intrinsic and extrinsic response to inflammatory signaling can alter tumor immune cell infiltration, anti-tumor immune response, and importantly, tumor cell apoptosis. Here, we investigate the response of Ewing tumors with STAG2 loss to type 1 and type 2 interferon both at baseline and following DNA damage. We hypothesize that Ewing tumors with STAG2 loss will demonstrate impaired interferon responses, including after DNA damage. These data enhance the biologic understanding of Ewing tumors with STAG2 loss and pinpoint inflammatory pathway proteins that can be targeted to potentially improve tumor control.

Methods: Sequenced human Ewing patient tumors and cell lines were bioinformatically analyzed, including assessment of STAG2 status. Ewing cell lines with deficient STAG2, and Ewing cells with STAG2 modified by CRISPR/Cas-9 technology were utilized. Ewing cells were treated with small weekly fractions of radiation for months to generate control and STAG2 KO Ewing cells with sub-lethal accumulation of DNA damage (xGy cells and ctGy no treatment controls). Tumor cell apoptosis after interferon stimulation was monitored via IncuCyte caspase 3/7 activity assays. Ewing tumors with STAG2 loss were examined in vivo utilizing both immunodeficient and immunocompetent, humanized mouse models to determine immune cell contributions to interferon signatures. RNAseq analysis of type 1 and type 2 interferon-treated control xGy and STAG2 KO xGy Ewing cells and controls was conducted. LEGENDplex cytokine assays were utilized.

Results: Bioinformatic analyses reveal that human Ewing tumors and cells with STAG2 loss demonstrate reduced type 1 and type 2 interferon signatures at baseline as compared to STAG2 intact Ewing tumors. We found inflammatory signaling and inflammation-induced apoptosis response heterogeneity in Ewing sarcoma; some Ewing tumors are more interferon-responsive and alternatively, some tumors demonstrate predominant TNF-alpha responses. STAG2 loss does not alter interferon-gamma receptor expression or localization. RNAseq analysis of interferon treated STAG2 KO Ewing cells reveals altered and delayed upregulation of interferon-regulated genes, including CXCL10, a cytokine that regulates immune cell trafficking and tumor cell proliferation. Cytokine (protein) analyses confirm impaired CXCL10 production in Ewing cells with STAG2 loss. Analyses from Ewing tumors developed in immunodeficient versus immunocompetent mice pinpoint the contribution of immune cells on this effect. Interferon response and apoptosis analyses of control and STAG2 KO xGy cells (cells with DNA damage accumulation) are ongoing.

Conclusion: These data provide clues to dysregulated tumor intrinsic and extrinsic interferon responses in Ewing tumors with loss of STAG2 expression at baseline and after DNA damage. The heterogeneity of these responses, even in the context of STAG2 loss, is detailed. Next steps include therapeutically manipulating these pathways with the goal of improving tumor control.