Abstract
Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma with 5-year survival below 30% in high-risk/metastatic cases, was investigated through integrated bioinformatics analysis (identifying 269 conserved differentially expressed genes in GEO datasets GSE28511/GSE141690) and experimentally validated thiostrepton (TST), a ribosomal-targeting antibiotic, as a potent therapeutic candidate via Connectivity Map analysis (p < 0.05, score ≈ -1). In-vitro studies demonstrated TST's dose-/time-dependent suppression of RMS proliferation (IC50 4.986-9.764 μmol/L), migration and invasion, G0/G1 cell cycle arrest, and apoptosis induction. In vivo, TST (3.4 mg/mL, 4 weeks) significantly inhibited tumor growth (p < 0.05 vs. phosphate buffered saline [PBS]) without organ toxicity. RNA sequencing identified the phosphatidylinositol 3-kinase/protein kinase B (PI3K-AKT) pathway as the primary suppressed pathway (False Discovery Rate [FDR] < 0.05), with concurrent downregulation of downstream regulators (AKT, JAK, CDKs). This was confirmed by PI3K activator 740 Y-P rescue experiments, which partially reversed the effects of TST (p < 0.05). These findings establish TST as a multi-mechanism PI3K-AKT inhibitor for refractory RMS while validating Connectivity Map (Cmap)-driven drug repurposing for pediatric oncology.