145 - CDKN2A Expression Is Associated with Chemoresistance in Ewing Sarcoma

Publication Number: 2139.145

Phani krishna Parcha, University of Florida, Gainesville, FL, United States; Nathan D. Seligson, Nemours Children's Health, Jacksonville, FL, United States

Background: Ewing sarcoma (EwS) is a rare, aggressive malignancy primarily affecting children and adolescents. Despite multimodal therapy, approximately 40% of patients experience relapse, which is associated with a 5-year survival rate below 30%. While disease recurrence reflects chemotherapy failure, mechanisms of resistance remain poorly understood. Currently, no validated molecular biomarkers exist to predict chemotherapy response in EwS. Retrospective analyses suggest a correlation between elevated CDKN2A expression and poor survival outcomes in EwS, raising the possibility of a role in chemoresistance. However, its functional role in chemotherapy sensitivity has not been directly tested.

Objective: To explore the relationship between CDKN2A expression and chemotherapy sensitivity in EwS using preclinical models.

Design/Methods: EwS cell lines were obtained from the Childhood Cancer Repository and the American Type Culture Collection. CDKN2A overexpression was achieved using a pLenti-C-Myc-DDK-IRES-Neo Lentiviral Gene Expression Vector. Protein expression was measured by western blot and flow cytometry. Cell viability was measured by CellTiter-Glo. Data were analyzed in Graphpad Prism 10. P values < 0.05 were considered significant.

Results: To assess the effect of CDKN2A expression on chemosensitivity in Ewing sarcoma, we transduced a lentiviral gene expression vector of CDKN2A in four cell lines with homozygous deletion of the CDKN2A locus. CDKN2A-transduced cells showed robust p16 expression (p < 0.0001). CDKN2A overexpression was associated with increased β-galactosidase activity (p < 0.0001) and G0/G1 and S phase accumulation. When treated with doxorubicin, etoposide, or cyclophosphamide, CDKN2A-transduced cells exhibited significantly higher IC₅₀ values compared to parental lines, indicating reduced chemosensitivity. CDKN2A-expressing cells exhibited a 2.6-fold increase in IC₅₀ across all drugs tested (standard deviation: 1.6). These findings indicate that CDKN2A expression contributes to a chemoresistant phenotype in EwS.

Conclusion(s): CDKN2A is present in approximately 85% of EwS tumors. These findings suggest that CDKN2A expression may contribute to a chemoresistant phenotype in EwS and support its potential as a predictive biomarker and therapeutic target.