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Polyamine Depletion by D,L-α-Difluoromethylornithine Inhibits Ewing Sarcoma Metastasis by Inducing Ferroptosis
Rachel Offenbacher1,2, Kyle W Jackson3,4, Masanori Hayashi3,5
1Department of Pediatrics, Albert Einstein College of Medicine, Bronx, New York.
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.
Insights
D, L-alpha-difluoromethylornithine (DFMO) inhibits Ewing sarcoma growth and metastasis by depleting polyamines, likely inducing ferroptosis. This preclinical finding supports testing DFMO to prevent metastatic recurrence in patients.
Area of Science:
- Oncology
- Cancer Biology
- Translational Medicine
Background:
- Ewing sarcoma metastasis remains a significant challenge, with no improvements in patient survival despite extensive clinical trials.
- Preclinical models revealed differential polyamine levels in metastatic versus non-metastatic tumors.
- Polyamines are crucial for cancer cell proliferation and survival.
Purpose of the Study:
- To investigate the potential of D, L-alpha-difluoromethylornithine (DFMO), a polyamine synthesis inhibitor, to prevent Ewing sarcoma metastasis.
- To evaluate the efficacy of DFMO in preclinical models of Ewing sarcoma.
Main Methods:
- In vitro studies assessed DFMO's effects on Ewing sarcoma cell proliferation, sphere formation, and clonogenic growth.
- In vivo efficacy was evaluated using an orthotopic implantation/amputation metastasis model.
- RNA sequencing was employed to analyze transcriptomic changes induced by DFMO.
Main Results:
- DFMO induced cell cycle arrest, inhibited sphere formation, and reduced clonogenic growth in vitro.
- In vivo, DFMO treatment slowed primary tumor growth and significantly inhibited metastasis.
- RNA sequencing revealed gene expression patterns indicative of ferroptosis induction due to polyamine depletion, further validated by in vitro lipid peroxidation assays.
Conclusions:
- DFMO demonstrates significant preclinical efficacy against Ewing sarcoma, impacting tumor initiation, growth, and metastasis.
- The mechanism likely involves polyamine depletion-mediated induction of ferroptosis.
- These findings provide a strong rationale for clinical trials evaluating DFMO's ability to prevent metastatic recurrence in Ewing sarcoma patients.
