Temporal Dynamics of SMARCA4 Loss Define Its Role in Myogenic Differentiation of Alveolar Rhabdomyosarcoma

Abstract

SMARCA4 (BRG1) is a critical ATPase subunit of canonical BAF complexes and a known long term dependency in alveolar rhabdomyosarcoma (ARMS). To define the short term consequences of SMARCA4 loss, we used a doxycycline inducible shRNA system in Rh4 cells. Doxycycline treatment produced strong SMARCA4 knockdown, yet acute knockdown did not alter proliferation or induce myogenic differentiation. Ki 67 positivity remained unchanged, and Myosin Heavy Chain (MHC) expression was absent, indicating preservation of the undifferentiated, proliferative ARMS phenotype. These findings contrast with CRISPR mediated SMARCA4 knockout studies, which report cell cycle exit and robust differentiation, suggesting that ARMS cells tolerate partial or early phase SMARCA4 loss without destabilizing BAF complex function or PAX3 FOXO1 driven regulatory circuitry. Together with prior long term knockdown studies, our results support a model in which SMARCA4 dependency in ARMS is a sustained phenomenon requiring deep, prolonged suppression to trigger differentiation and loss of tumor viability.