Polyamine Depletion by D,L-α-Difluoromethylornithine Inhibits Ewing Sarcoma Metastasis by Inducing Ferroptosis

Purpose: Despite decades of clinical trials, no progress has been made in improving the survival of patients with Ewing sarcoma who either present with metastatic disease or suffer a metastatic relapse. In our preclinical models, we found differential levels of polyamines in tumors that metastasize compared with tumors that do not, leading us to investigate the potential for D,L-α-difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis, to prevent Ewing sarcoma metastasis. Experimental design: The effect of DFMO on Ewing sarcoma cell lines in vitro was studied by measuring proliferation, sphere formation, and clonogenic growth in soft agar. The effect in vivo was investigated using our orthotopic implantation/amputation model of metastasis. Transcriptomic changes were evaluated by RNA sequencing. Results: DFMO causes a cell cycle arrest and inhibits both sarcosphere formation and clonogenic growth in soft agar. In vivo, DFMO slows primary tumor growth and inhibits metastasis. RNA sequencing demonstrated gene expression patterns consistent with induction of ferroptosis caused by polyamine depletion, which was validated in vitro by demonstrating that DFMO treatment induces lipid peroxidation, and ferrostatin-1 and liproxstatin-1 allow sphere formation even in the presence of DFMO. Conclusions: DFMO slows the growth of Ewing sarcoma cells in vitro, with a profound impact on sphere formation and clonogenic growth, and affects all aspects of Ewing sarcoma tumorigenesis, including tumor initiation, tumor growth, and metastasis, probably through induction of ferroptosis mediated by polyamine depletion. Our results provide preclinical justification to test the ability of DFMO to prevent metastatic recurrence in patients with Ewing sarcoma.