Characterization and targeting the CXCR2-STAT-1/3 axis in metastatic Ewing sarcoma

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Project Summary Ewing Sarcoma (EwS) is the second most common bone tumor in children. At diagnosis, approximately 25-30% of patients with EwS have metastatic disease. For localized disease, systemic chemotherapy regimen along with surgery and/or radiotherapy has significantly increased the survival rate of patients to approximately 70%. Unfortunately, the prognosis for metastatic EwS has remained dismal, with a 5-year survival rate of 20–30%. This prognosis has not changed in last several decades. Thus, it is imperative to focus on the mechanisms of EwS metastasis and identify specific biological features of the metastatic tumor. In this regard, it is important to study the tumor biology not in isolation, but in the context of the tumor stroma, to better understand the interaction between the sarcoma cells and the tumor associated stromal cells that facilitates EwS metastatic growth. Given the limitation in representative vertebrate models for EwS, our approach of integrating tumor intrinsic and stromal factors that lead to metastatic phenotypes is closer to the physiological context than studying the tumor in isolation. Using serial transplantation approaches, we have generated novel lung metastatic EwS cell lines. Cytokine analysis from co-culture of these cells with lung fibroblast cells, revealed upregulation of several cytokines, including CXCL-1/2, CXCL-5, and CXCL-8, which are ligands to the receptor CXCR2. This observation was confirmed from transcriptomic analysis using Nanostring technology from paired primary with lung metastatic patient samples. Further, integrated analysis of proteomics from multiple metastatic models, converged on STAT1 as a significantly differentially expressed protein in metastatic samples. Thus, through the integration of multi-omic approaches on relevant innovative in vivo metastatic models and human patient specimens, we have identified a novel molecular mechanism of metastasis in EwS that we can readily assess using our unique resources. Our overarching hypothesis is that the CXCR2-STAT1 axis is a critical pathway in metastatic EwS and an attractive therapeutic target. To address this hypothesis, and to bridge the current gaps in knowledge and potential therapeutic opportunities, we propose two innovative, translation and exploratory aims. Aim1 will characterize the tumorigenic effects of the CXCR2-STAT1 pathway in primary and metastatic tumor by identifying STAT1 regulated genes induced by CXCR2 and assessing genetic and pharmacological inhibition of this pathway as a novel therapeutic intervention. Aim2 will investigate the role of extra cellular vesicles, as an intercellular mediator of tumor-stroma communication that facilitates CXCR2-STAT1 signaling by micro RNA (miRNA) regulation. The treatment of aggressive and metastatic Ewing sarcomas remains a challenge therapeutically. Successful completion of our aims will provide novel molecular, and valuable pre- clinical therapeutic insights for metastatic Ewing sarcoma.