Abstract
Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma, with poor outcomes in high-risk and relapsed patients. Here, we identify de novo cholesterol biosynthesis as a critical metabolic vulnerability in RMS. The transcription factor PROX1, previously implicated in RMS growth, acts as an upstream regulator of cholesterol biosynthesis, promoting expression of key pathway genes. Inhibition of cholesterol biosynthesis, either genetically or pharmacologically, impaired RMS cell proliferation, caused a broad halt of cell cycle progression, and activated ER stress-mediated apoptosis through the PERK-ATF4-CHOP axis. Notably, RMS cells could not be rescued by exogenous LDL cholesterol, indicating a unique reliance on endogenous cholesterol production, whereas normal cells, including myoblasts and astrocytes, largely relied on extracellular cholesterol uptake. Clinical and single-cell RNA-seq analyses further revealed that high expression of cholesterol biosynthesis genes correlate with poor survival and enrichment of cell cycle-related gene signatures across RMS subtypes. Together, these findings mechanistically link cholesterol biosynthesis to proliferative signaling and ER stress response in RMS and highlight this pathway as a promising, non-redundant therapeutic target.