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

“…Several groups have investigated the transcriptional heterogeneity of rhabdomyosarcoma (RMS) using single-cell RNA (scRNAseq) and/or single-nucleus RNA sequencing (snRNAseq). These studies identified RMS cell states using different bioinformatic methods, leading to divergent and often confusing nomenclature [21][22][23].…”

“…In previous reports, each group identified clusters of cells/nuclei with transcriptomic similarity to muscle-lineage cells [21][22][23]. Despite these similarities, each used different sample types, computational methods to define gene expression signatures, and differing nomenclature for each subpopulation.…”

“…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].…”

“…Thus, it is critical to understand the non-genetic heterogeneity within RMS, and the role that specific cell subpopulations play in driving the clinical behavior of RMS. Indeed, multiple groups have applied single-cell transcriptomics to discover distinct RMS cell subpopulations [21][22][23][24]. These studies consistently identified malignant cells with expression patterns similar to developing skeletal muscle, yet each study introduced different nomenclature and classification strategies due to limited sample numbers, differences in bioinformatic approaches, and mapping shared developmental cell states across mouse and/or human muscle.…”

“…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.…”