BAF complexes drive proliferation and block myogenic differentiation in fusion-positive rhabdomyosarcoma

Abstract

Rhabdomyosarcoma (RMS) is a pediatric malignancy of skeletal muscle lineage. The aggressive alveolar subtype is characterized by t(2;13) or t(1;13) translocations encoding for PAX3- or PAX7-FOXO1 chimeric transcription factors, respectively, and are referred to as fusion positive RMS (FP-RMS). The fusion gene alters the myogenic program and maintains the proliferative state wile blocking terminal differentiation. Here we investigated the contributions of chromatin regulatory complexes to FP-RMS tumor maintenance. We define, for the first time, the mSWI/SNF repertoire in FP-RMS. We find that SMARCA4 (encoding BRG1) is overexpressed in this malignancy compared to skeletal muscle and is essential for cell proliferation. Proteomic studies suggest proximity between PAX3-FOXO1 and BAF complexes, which is further supported by genome-wide binding profiles revealing enhancer colocalization of BAF with core regulatory transcription factors. Further, mSWI/SNF complexes act as sensors of chromatin state and 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 which is reproduced by BRG1 targeting compounds. We conclude that inhibition of BRG1 overcomes the differentiation blockade of FP-RMS cells and may provide a therapeutic strategy for this lethal childhood tumor.

Extracellular Vesicles from 3D Engineered Microtissues Harbor Disease‐Related Cargo Absent in EVs from 2D Cultures

“…[68] Spectra were searched against a Homo Sapiens canonical SwissProt proteome (version 2016-12-09), concatenated to its reversed decoyed fasta database and common protein contaminants. Protein fold changes were calculated according to the method described by Laubscher et al [69] Imaging Flow Cytometry: Sample preparation and data acquisition parameters were performed according to recommendations from Görgens et al [55] with slight modifications. To prepare the staining solution, fluorescent antibodies were diluted 1:10 in PBS and centrifuged at 17 000× g for 15 min at 10 °C.…”