From the Lab to the Clinic: Exploring DFMO’s Potential to Prevent Metastatic Disease in Children With Osteosarcoma and Ewing Sarcoma

Osteosarcoma and Ewing sarcoma are the 2 most common primary malignant bone tumors in children. The most significant prognostic factor is the presence or absence of metastatic disease.1 Despite advances in the 1980s, we have been unable to cure more children with metastatic disease now than we did 30 years ago, with a plateau in survival rate of approximately 25% for this patient population.2 Approximately 30% to 35% of patients diagnosed with osteosarcoma or Ewing sarcoma will have a metastatic relapse, which carries a poor prognosis.

Although there have been some responses to second-line chemotherapy regimens, these have come without improvement in long-term survival. Many targeted therapies are currently being studied, but developing targeted therapies focused on and measured by local tumor response fails to address the most pressing need for these patients: the prevention and treatment of metastatic disease. D,L-α-difluoromethylornithine (DFMO) inhibits polyamine synthesis, a metabolic pathway strongly implicated in tumor cell proliferation.3 DFMO has been studied in several different cancers as either a therapeutic or a chemopreventive agent and is now FDA approved to reduce the risk of relapse in patients with newly diagnosed high-risk neuroblastoma.4,5

A Novel and Clinically Relevant Mouse Model

The Loeb lab at Montefiore Einstein Comprehensive Cancer Center and the Children’s Hospital at Montefiore in the Bronx, New York, developed a novel, clinically relevant mouse model of Ewing sarcoma and osteosarcoma metastasis that allows the study of spontaneous distant sarcoma metastasis. Fragments of patient-derived xenografts are implanted into the pretibial space, and affected limbs are amputated after tumor growth. In contrast to subcutaneous flank tumors, spontaneous distant spread of the tumor is consistently detected using this model. At the time of amputation, disseminated cancer cells can be detected in the lungs and/or bone marrow of mice; however, overt metastases are not evident.

Using this model, we tested DFMO given after tumor amputation and in combination with neoadjuvant chemotherapy. In control mice, metastatic tumors were found within the lungs and in various abdominal organs. We noted a statistically significant decrease in metastatic burden in mice with Ewing sarcoma treated with adjuvant DFMO (P = .045). Furthermore, we demonstrated that neoadjuvant ifosfamide administered daily for 3 days every 3 weeks in combination with adjuvant 2% DFMO decreases the metastatic burden in mice with Ewing sarcoma compared with both control mice and those that received ifosfamide alone (P = .0005).

Mice with osteosarcoma experienced similar results. In mice treated with adjuvant DFMO, we similarly saw a decrease in both local and distant metastases (P = .02). Moreover, mice treated with neoadjuvant ifosfamide in combination with adjuvant DFMO developed far fewer metastases compared with control mice or mice treated with ifosfamide alone (P = .0005).

With these promising results, we investigated the mechanism by which DFMO can prevent metastasis in both Ewing sarcoma and osteosarcoma. There is increasing recognition that the iron-dependent mode of programmed cell death known as ferroptosis is an important restraint on metastasis.6-8 Furthermore, it was recently reported that p53-mediated induction of ferroptosis requires expression of spermidine/spermine N1-acetyltransferase 1 (SAT1), the rate-limiting catabolic enzyme present in polyamine excretion from cells.9

Induction of SAT1 is correlated with increased expression of arachidonate 15-lipoxygenase (ALOX15), a key mediator of the lipid peroxidation that characterizes ferroptosis.9,10 We found that DFMO induces a statistically significant increased expression of ALOX15 in all Ewing sarcoma lines tested. This increase was abolished in the presence of exogenous polyamines, proving that polyamine depletion drives the increased expression of this enzyme.

The ability to form spherical colonies under nonadherent conditions (sarcospheres) is a well-established surrogate marker of a cancer stem cell phenotype. Ferroptosis can be inhibited by cell-cell and cell-matrix contact. We found that although DFMO is only cytostatic to cells growing on tissue culture dishes, it prevents the formation of sarcospheres in nonadherent culture conditions.

To validate that ferroptosis is the mechanism of cell death that prevents sphere formation, cells were plated at clonogenic density on ultralow adherence plates and then treated with varying concentrations of multiple cell death inhibitors in the presence of DFMO. After 7 days of growth, we observed well-established sarcospheres in all cell lines that were treated with DFMO and either ferrostatin-1 or liproxstatin—both inhibitors of ferroptosis, or n-acetylcysteine—a nonspecific inhibitor of reactive oxygen species and a key mediator of ferroptotic cell death. In contrast, Q-VD-OPh, a potent inhibitor of enzymatic activity of caspases and thus an inhibitor of cell death, was unable to rescue the inhibition of sphere formation by DFMO. Finally, RNA sequencing performed on the primary tumors treated with or without DFMO demonstrated gene expression patterns consistent with induction of ferroptosis caused by polyamine depletion.

Entering Phase 2 Research

Our clinically relevant preclinical model of spontaneous distant osteosarcoma and Ewing sarcoma metastasis support the concept that DFMO, administered at a point where animals are radiographically free of disease but at high risk of developing overt metastasis due to dissemination of tumor cells, can considerably decrease metastatic recurrence. Furthermore, the combination of chemotherapy and DFMO dramatically decreases metastatic burden and provides an appropriate rationale for the design of a clinical trial. Based on these insights, a phase 2 single-institution clinical trial (NCT06892678) is ongoing at our Children’s Hospital at Montefiore and Montefiore Einstein Comprehensive Cancer Center.11

The primary objective is to determine the feasibility of administering DFMO to patients with relapsed Ewing sarcoma and osteosarcoma who have completed all planned therapy and have no evidence of disease. Secondary aims will explore the event-free survival (EFS) of patients who receive therapy with DFMO after relapsed osteosarcoma or Ewing sarcoma without evidence of disease at the completion of all planned therapy.

Looking ahead, we anticipate the opening of a multi-institutional clinical trial with 4 cohorts in the fall of 2025 through the Beat Childhood Cancer Research Consortium in collaboration with US WorldMeds, the manufacturer of DFMO. This will include the integration of DFMO as maintenance into both up-front osteosarcoma and Ewing sarcoma therapy, as well as in the relapsed and refractory setting.

Cohort 1 will determine whether relapse-free survival in patients with relapsed or refractory Ewing sarcoma treated with multiagent chemotherapy is improved with the addition of DFMO compared with historical outcomes of patients treated with the same multiagent chemotherapy without DFMO. Cohort 2 will determine whether EFS in patients with newly diagnosed metastatic Ewing sarcoma treated with multiagent chemotherapy is improved with the addition of DFMO compared with historical outcomes of patients treated with the same multiagent chemotherapy without DFMO. Cohort 3 will determine the 12-month disease control rate in patients with completely resected recurrent osteosarcoma treated with DFMO compared with the historical Children’s Oncology Group experience. Cohorts 4A and 4B will examine whether the addition of DFMO to postoperative chemotherapy with standard-of-care therapy improves the EFS for patients with resectable osteosarcoma either having localized disease with a poor histological response to 10 weeks of preoperative chemotherapy (cohort 4A) or metastatic disease at diagnosis (cohort 4B).

When the trial opens, it will be available at all participating Beat Childhood Cancer Research Consortium sites, including the Children’s Hospital at Montefiore. For accrual interest, please contact the principal investigators of the trial at rzylber@montefiore.org or lfabish@montefiore. org and authors of this piece or Abigail Moore, program manager at Beat Childhood Cancer, at BCCEnroll@pennstatehealth.psu.edu.

References

1. Goldstein SD, Hayashi M, Albert CM, Jackson KW, Loeb DM. An orthotopic xenograft model with survival hindlimb amputation allows investigation of the effect of tumor microenvironment on sarcoma metastasis. Clin Exp Metastasis. 2015;32(7):703-715. doi:10.1007/s10585-015-9738-x
2. Lagmay JP, Krailo MD, Dang H, et al. Outcome of patients
   with recurrent osteosarcoma enrolled in seven phase II trials through Children’s Cancer Group, Pediatric Oncology Group, and Children’s Oncology Group: learning from the past to move forward. J Clin Oncol. 2016;34(25):3031-303. doi:10.1200/ JCO.2015.65.5381
3. Smith MK, Trempus CS, Gilmour SK. Co-operation between follicular ornithine decarboxylase and v-Ha-ras induces spontaneous papillomas and malignant conversion in transgenic skin. Carcinogenesis. 1998;19(8):1409-1415. doi:10.1093/ carcin/19.8.1409
4. Gerner EW, Meyskens FL, Jr. Polyamines and cancer: old molecules, new understanding. Nat Rev Cancer. 2004;4(10):781- 792. doi:10.1038/nrc1454
5. FDA approves eflornithine for adult and pediatric patients with high- risk neuroblastoma. FDA. December 13, 2023. Updated December 14, 2023. Accessed August 6, 2025. https://www.fda.gov/drugs/resources-information-approved-drugs/fda-approves-eflornithine-adult-and-pediatric-patients-high-risk-neuroblastoma
6. Wu M, Zhang X, Zhang W, et al. Cancer stem cell regulated phenotypic plasticity protects metastasized cancer cells from ferroptosis. Nat Commun. 2022;13(1):1371. doi:10.1038/ s41467-022-29018-9
7. Sun X, Ou Z, Xie M, et al. HSPB1 as a novel regulator of ferroptotic cancer cell death. Oncogene. 2015;34(45):5617– 5625. doi:10.1038/onc.2015.32
8. Tan Q, Fang Y, Gu Q. Mechanisms of modulation of ferroptosis and its role in central nervous system diseases. Front Pharmacol. 2021;4(12):657033. doi:10.3389/ fphar.2021.657033
9. Ou Y, Wang SJ, Li D, Chu B, Gu W. Activation of SAT1 engages polyamine metabolism with p53-mediated ferroptotic responses. Proc Natl Acad Sci U S A. 2016;113(44):E6806– E6812. doi:10.1073/pnas.1607152113
10. Vucetic M, Daher B, Cassim S, Meira W, Pouyssegur J. Together we stand, apart we fall: how cell-to-cell contact/interplay provides resistance to ferroptosis. Cell Death Dis. 2020;11(9):789. doi:10.1038/s41419-020-02994-w
11. DFMO maintenance for patients with relapsed/refractory Ewing sarcoma or osteosarcoma (DFMO). ClinicalTrials.gov. Updated May 11, 2025. Accessed August 6, 2025. https://clinicaltrials. gov/study/NCT06892678