Single-cell profiling of alveolar rhabdomyosarcoma reveals RAS pathway inhibitors as cell-fate hijackers with therapeutic relevance

Abstract

Rhabdomyosarcoma (RMS) is a group of pediatric cancers with features of developing skeletal muscle. The cellular hierarchy and mechanisms leading to developmental arrest remain elusive. Here, we combined single-cell RNA sequencing, mass cytometry, and high-content imaging to resolve intratumoral heterogeneity of patient-derived primary RMS cultures. We show that the aggressive alveolar RMS (aRMS) subtype contains plastic muscle stem-like cells and cycling progenitors that drive tumor growth, and a subpopulation of differentiated cells that lost its proliferative potential and correlates with better outcomes. While chemotherapy eliminates cycling progenitors, it enriches aRMS for muscle stem-like cells. We screened for drugs hijacking aRMS toward clinically favorable subpopulations and identified a combination of RAF and MEK inhibitors that potently induces myogenic differentiation and inhibits tumor growth. Overall, our work provides insights into the developmental states underlying aRMS aggressiveness, chemoresistance, and progression and identifies the RAS pathway as a promising therapeutic target.

Single cell transcriptomic profiling identifies tumor-acquired and therapy-resistant cell states in pediatric rhabdomyosarcoma

“…Despite tumors retaining the tripartite muscle lineage programs across models (Figure S3C), neuronal subpopulations were detected in larger numbers in patient tumors and PDXs, but rarely or not at all in primary cultures or commercial cell lines [Figure S4; Table S7]. The absence of neuronal cells within cell lines may explain why this rare subpopulation was not identified previously by scRNA sequencing analysis by Danielli et al [23]…”

“…In contrast, Wei et al examined 9 PDX and 4 patient tumors to identify four RMS-specific cell subpopulations that they called mesenchymal, proliferative, differentiated, and ground cells [22]. Finally, Danielli et al studied 14 PDCs and 3 conventional cell lines to group RMS cells into muscle stem-cell-like cells, cycling progenitors, and differentiated cells [23].…”

“…The 10X Genomics scRNAseq/snRNAseq data was collected from previously published datasets [21][22][23]25]. All datasets are available at the NCBI Gene Expression Omnibus (GEO) database under the following accession numbers : GEO: GSE218974…”

“…For samples derived from Wei et al and Danielli et al, filtered Seurat objects were downloaded from GEO repositories. Those objects were generated as previously described [22,23] For samples derived from Patel et al, we generated single-cell Seurat objects following the original pipeline [21]. In short, raw sequencing FASTQ files available from GEO were aligned to the human hg19 (for patient samples) or to the combined human hg19 and mouse mm10 references (for PDX samples) using the 10X Genomics Cell Ranger pipeline (version 3.0.0).…”

“…Here, we present a consensus evaluation of intratumoral heterogeneity in human RMS by combining datasets encompassing 72 samples from patient tumors, patientderived xenograft (PDX) models, PDX-derived primary cell cultures (PDCs), and commercial cell lines (CLs) [21][22][23]25]. By uniformly processing and integrating these datasets, we generated a comprehensive and unified annotation of RMS-specific cell subpopulations.…”