(P 266) TARGETING THE DNA DAMAGE RESPONSE TO ADVANCE PRECISION THERAPIES IN PAEDIATRIC SARCOMA

Objective: Outcomes for children with sarcoma remain poor, and standard treatments are associated with significant toxicity. Targeted therapies are not routinely available. Early evidence suggests that inhibitors of the DNA damage response (DDR) may offer therapeutic benefit in sarcoma. To identify which patients are most likely to benefit from DDR inhibitors by characterising the DDR landscape in paediatric sarcoma and evaluating its association with drug sensitivity.

Methods: Comprehensive genomic profiling of DDR aberrations was performed in 130 paediatric sarcoma patients using whole-genome sequencing. Based on these data, seven DDR-targeting agents were selected for in vitro testing, both as monotherapies and in combinations. Drug efficacy was assessed via cell viability assays. Cell cycle effects were evaluated by flow cytometry, molecular changes in DDR mediators by western blotting, and mitotic entry via immunofluorescence microscopy. Gene set enrichment analysis (GSEA) was used to explore the link between genomic profiles and drug responses. A genome-wide CRISPR-Cas9 screen is underway to uncover determinants of drug sensitivity.

Results: Frequent DDR gene alterations were observed across the sarcoma cohort. Inhibition of ATR, CHK1, or WEE1 led to potent growth inhibition in multiple paediatric sarcoma cell lines, with particularly strong responses in Ewing sarcoma (ES) and fusion-positive rhabdomyosarcoma (FP-RMS). Dual inhibition of the ATR pathway resulted in synergistic responses at doses well below clinically achievable concentrations. Mechanistically, ATR, CHK1, and WEE1 inhibitors induced S-phase and G2/M arrest, increased DNA damage, and triggered apoptosis. Immunofluorescence microscopy revealed elevated premature mitotic entry, consistent with checkpoint failure as a mechanism of cell death. GSEA revealed G2/M checkpoint upregulation in sensitive models, particularly in fusion-driven sarcomas, implicating fusion proteins in checkpoint control. Nonetheless, resistance was observed in a subset of ES models, suggesting additional pathways or biomarkers modulate response.

Conclusion: DDR pathway alterations are common in paediatric sarcomas. Inhibiting ATR, CHK1, or WEE1 is a promising therapeutic approach, particularly in fusion-driven subtypes such as ES and FP-RMS. These findings support further exploration of DDR-targeted therapy and highlight the need for predictive biomarkers to guide patient selection.