SIX1 Reprograms Myogenic Transcription Factors to Maintain the Rhabdomyosarcoma undifferentiated state

Rhabdomyosarcoma (RMS) is a pediatric skeletal muscle sarcoma characterized by the expression of the myogenic-lineage transcription factors (TF) MYOD1 and MYOG. Despite high expression of these TFs, RMS cells fail to terminally differentiate, suggesting the presence of factors that alter their myogenic function. Here, we demonstrate that the developmental TF, SIX1, is highly expressed in RMS and is critical to maintain a muscle progenitor-like state. In contrast to its traditional role in development promoting differentiation, SIX1 loss induces terminal differentiation of RMS cells into myotube-like cells and dramatically impedes tumor growth in vivo. We show that SIX1 maintains the RMS undifferentiated state by controlling enhancer activity and MYOD1 occupancy at stem/progenitor loci over terminal muscle differentiation loci, and that loss of SIX1 reprograms MYOD1 to induce terminal differentiation in RMS. Finally, we demonstrate that a gene signature derived from SIX1 loss correlates with differentiation status in RMS and predicts RMS progression in human disease. Our findings demonstrate differential functions for SIX1 in development and RMS where SIX1 serves as a master regulator to repress RMS differentiation via genome-wide alterations in MYOD1-mediated transcription.