Polyamine Depletion Inhibits Ewing Sarcoma Metastasis by Inducing Ferroptosis

Polyamine metabolism and signaling play important roles in multiple cancers including neuroblastoma and breast cancer but have not previously been studied in Ewing sarcoma. Prior work in our lab suggested that tumor microenvironment affects arginase expression -- orthotopically implanted hindlimb tumors with enhanced metastatic potential demonstrate diminished arginase expression compared to subcutaneously implanted tumors that do not metastasize. As arginase catalyzes the conversion of arginine to ornithine, the precursor for polyamine synthesis, this suggested a potential role for polyamine synthesis in Ewing sarcoma metastasis and a target for therapeutic intervention. Here, we show that blocking polyamine synthesis with D, L-alpha-difluoromethylornithine (DFMO) causes a G1 cell cycle arrest, dose-dependent decreases in sarcosphere formation from Ewing sarcoma cell lines growing in non-adherent conditions and a decrease in clonogenic growth in soft agar. Further, we utilized our orthotopic implantation/amputation model of Ewing sarcoma metastasis to investigate the impact of disrupting polyamine synthesis on tumor growth and metastasis. When mice were treated with low dose DFMO, there was no effect on primary tumor growth or metastatic potential; however, at higher concentrations, systemic DFMO slowed primary tumor growth in addition to limiting metastasis. RNA sequencing demonstrated gene expression patterns consistent with induction of ferroptosis caused by polyamine depletion, and this was confirmed experimentally. Collectively, these results reveal a novel mechanism by which DFMO prevents metastasis – induction of ferroptosis due to polyamine depletion, and our results suggest that DFMO may be a readily translatable and well-tolerated treatment to prevent metastatic recurrence in patients with high-risk Ewing sarcoma.