The BAF complex blocks Myogenesis in rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a highly malignant pediatric cancer of skeletal muscle lineage. The aggressive alveolar subtype is commonly characterized by t(2;13) or t(1;13) translocations with the expression of PAX3- or PAX7-FOXO1 fusion transcription factors respectively and is known as fusion positive RMS (FP-RMS). FP-RMS tumors rely on the fusion gene to maintain their proliferative state while avoiding terminal differentiation program. Since disruptions of normal development are likely governed by epigenetic changes in these low-mutational-burden tumors, we investigated the contributions of chromatin regulatory complexes to RMS tumor maintenance. We demonstrate the essentiality of the mSWI/SNF ATPase BRG1 for FP-RMS proliferation and discover that RMS significantly overexpress SMARCA4 (encoding BRG1) compared to skeletal muscle. We define the mSWI/SNF subunit repertoire in FP-RMS and provide evidence for the assembly of multiple mSWI/SNF complexes including canonical BAF, PBAF and non-canonical (nc)BAF in FP-RMS. Proteomic studies suggest proximity between PAX3-FOXO1 and BAF complexes, which was further supported by genome-wide binding profiles revealing enhancer colocalization of BAF with FP-RMS core regulatory transcription factors. We report that mSWI/SNF complexes act as sensors of chromatin state, which are recruited to sites of de novo histone acetylation. Phenotypically, interference with mSWI/SNF complex function induces transcriptional activation of the skeletal muscle differentiation program associated with MYCN enhancer invasion at myogenic target genes. Finally, BRG1 targeting compounds reproduce the effects of genetic ablation. We conclude that inhibition of BRG1 overcomes the differentiation blockade of FP-RMS cells and may provide a therapeutic strategy for treating this lethal childhood tumor. Biological triplicate samples of Cas9 expressing RH4 transduced with indicated sgRNAs were collected 7 days after transduction for RNA isolation. Total RNAs were extracted by RNeasy Plus Mini Kit (Qiagen). Paired-end mRNA libraries were prepared using Truseq Stranded Total RNA Library Prep Kit (Illumina) and sequenced on a Novaseq system as 2x150 base reads by Atlas Biolab GmbH (Berlin, Germany).