Radiotherapy (RT) is important for local control at primary and metastatic sites in pediatric rhabdomyosarcoma (RMS) even though loco-regional recurrence is frequently observed. Therefore, radio-sensitizers with high toxicity to cancer and low to normal tissues are urgently needed. The alveolar (A)RMS and the embryonal (E)RMS variant, despite genetic differences, are sustained by common molecular networks. Identifying and targeting the crucial nodes of the molecular network supporting RMSs radio-resistance could radio-sensitize RMSs. With this project we want to identify pathways regulating intrinsic and promoting acquired radio-resistance in RMS and select and validate in vitro pharmacological compounds targeting identified pathways. To this purpose, shRNA-based strategy will be used to identify genes sustaining intrinsic radio-resistance. Pathways involved in acquiring radio-resistant phenotype will be identified using transcriptomics, mass cytometry and phosphoproteomics. Then, pharmacological compounds selected versus candidate targets previously identified and prioritized will be screened. These will be validated, in vitro, for their ability to counteract intrinsic and acquired radio-resistance. The validated pathways will be studied in patients¿ samples to evaluate their relevance as clinical correlate to inform future treatments. Expected results are to unveil molecular mechanisms responsible for radio-resistance and to identify pharmacological strategies to counteract intrinsic and acquired radio-resistance. Data collected from this project will contribute to identify the mechanisms responsible of radio-resistance and could have a significant impact in planning new RT-based therapeutic strategies able to reduce the toxicity and improve the efficacy of RT.