(P 036) STAG2 LOSS ENCHANCES SENSITIVITY TO DNA DAMAGE YET REDUCES DNA DAMAGE-INDUCED INFLAMMATION IN EWING SARCOMA

Objective: Ewing sarcoma is a primary bone cancer that most commonly occurs in adolescents. ~20% of Ewing tumors demonstrate loss of expression of STAG2, a member of the cohesin complex. Emerging data suggests that loss of STAG2 expression is associated with inferior outcomes in patients with Ewing sarcoma. Thus, it is imperative to better understand how Ewing tumors with loss of STAG2 differ in response to therapy as compared to Ewing tumors with intact STAG2 expression. Mutations in cohesin complex proteins have been linked with impaired DNA damage repair. In AML, it has been shown that loss of STAG2 enhances sensitivity to Poly (ADP-ribose) polymerase (PARP) inhibitors. Significant cross-talk occurs between DNA damage and inflammatory pathways in cancer, and thus, STAG2 loss has the potential to impact inflammatory signaling pathways. For this study, we chose to study the impact of STAG2 loss on radiation and radiation-induced inflammation in Ewing sarcoma, as patients with high risk Ewing sarcoma often receive radiation as part of their treatment plan. We hypothesize that Ewing cells with STAG2 loss will demonstrate enhanced sensitivity to radiation. Further, we hypothesize that despite enhanced sensitivity to DNA damage, Ewing tumors with STAG2 loss will demonstrate a more immunosuppressive tumor microenvironment during radiation therapy, thus promoting immune evasion.

Methods: In vitro assays were conducted using multiple, established Ewing sarcoma cell lines and primary Ewing tumor cells. STAG2 siRNA (or control) and CRISPR STAG2 KO cells (or NT control cells) were treated -/+ radiation using a MultiRad350 Precision irradiator and were subject to live cell imaging for monitoring of confluence and apoptosis over time. For in vivo studies, as no syngeneic or transgenic murine models of Ewing sarcoma exist, we established Ewing tumors using Ewing CRISPR STAG2 KO cells (or CRISPR control cells expressing STAG2) in humanized mice in order to study immune cell populations in the Ewing microenvironment. Blood from humanized mice was analyzed by flow cytometry to confirm human immune cell reconstitution. Ewing tumors in humanized mice were radiated using a MultiRad350 Precision irradiator and lead shields to direct radiation exposure to the tumor only. Ewing tumors were collected and analyzed using bulk RNA-sequencing and multiplex immunofluorescence to analyze immune populations.

Results: We demonstrate that Ewing cells with STAG2 loss are significantly more sensitive to radiation across doses as compared to cells with intact STAG2 expression. Initial RNAseq analysis of Ewing tumor specimens -/+ radiation from humanized mice reveal enhanced inflammatory signatures present in specimens receiving radiation. However, this radiation induced inflammation is greatly reduced in Ewing tumors lacking STAG2 expression. Additional analyses of tumor infiltrating leukocytes and mechanism regulating this finding are ongoing.

Conclusion: Personalized treatment approaches are needed in order to better treat patients with metastatic and relapsed Ewing sarcoma. We have demonstrated that Ewing tumors with loss of STAG2 expression are more sensitive to radiation-induced apoptosis yet demonstrate less inflammation following radiation. Ongoing in vitro and in vivo studies are aimed at determining therapeutic combinations to reverse the immunosuppressive phenotype of Ewing tumors with STAG2 loss.