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.…”

Epigenetic determinants of fusion-driven sarcomas: paradigms and challenges

“…It is of high interest that within the spectrum of fusion-driven sarcomas, there are varying degrees of mechanistic interrelatedness of the fusion oncoprotein with ATP-dependent remodeling complexes. A great set of examples is that mammalian SWI/SNF (BAF) complexes 1) directly incorporate the SS fusion oncoproteins (Kadoch and Crabtree, 2013), while 2) the ES fusions interact with SWI/SNF in the absence of chromatin but can participate in DNA binding site selection (Boulay et al, 2017), and 3) in RMS canonical BAF complexes interact with fusion oncoproteins supported through chromatin but not in the absence of chromatin (Laubscher et al, 2021). Each of these contexts provide a framework through which to examine fusion oncoproteinremodeler interactions, which we hypothesize occur through intrinsically disordered domains (IDRs) of fusions across a constellation of childhood sarcomas.…”

“…In FP-RMS, cBAF interacts with PAX3-FOXO1 through a DNA/ chromatin interface (Laubscher et al, 2021). There is evidence of a hierarchical program in which CR TFs establish the enhancer landscape leading to cBAF recruitment at acetylated sites, thus fine-tuning CR TFs transcriptional activity.…”

“…This stabilized RMS enhancer network interferes with the myogenic differentiation process locking RMS cells in a myoblastic-like state. Excitingly, the genetic depletion of SWI/SNF, or strategies for targeted protein degradation/inhibition lead a cell cycle arrest and the induction of myogenic enhancers (Laubscher et al, 2021). As an interesting parallel, in SS, SS18-SSX1 fusion protein has been described as a BAF subtype-specific subunit (Middeljans et al, 2012).…”