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.

PURPOSE Rhabdomyosarcomas (RMS) are rare neoplasms affecting children and young adults. Efforts to improve patient survival have been undermined by a lack of suitable disease markers. Plasma circulating tumor DNA (ctDNA) has shown promise as a potential minimally invasive biomarker and monitoring tool in other cancers; however, it remains underexplored in RMS. We aimed to determine the feasibility of identifying and quantifying ctDNA in plasma as a marker of disease burden and/or treatment response using blood samples from RMS mouse models and patients. METHODS We established mouse models of RMS and applied quantitative polymerase chain reaction (PCR) and droplet digital PCR (ddPCR) to detect ctDNA within the mouse plasma. Potential driver mutations, copy-number alterations, and DNA breakpoints associated with PAX3/ 7-FOXO1 gene fusions were identified in the RMS samples collected at diagnosis. Patient-matched plasma samples collected from 28 patients with RMS before, during, and after treatment were analyzed for the presence of ctDNA via ddPCR, panel sequencing, and/or whole-exome sequencing. RESULTS Human tumor-derived DNA was detectable in plasma samples from mouse models of RMS and correlated with tumor burden. In patients, ctDNA was detected in 14/18 pretreatment plasma samples with ddPCR and 7/7 cases assessed by sequencing. Levels of ctDNA at diagnosis were significantly higher in patients with unfavorable tumor sites, positive nodal status, and metastasis. In patients with serial plasma samples (n = 18), fluctuations in ctDNA levels corresponded to treatment response. CONCLUSION Comprehensive ctDNA analysis combining high sensitivity and throughput can identify key molecular drivers in RMS models and patients, suggesting potential as a minimally invasive biomarker. Preclinical assessment of treatments using mouse models and further patient testing through prospective clinical trials are now warranted.

No abstract

Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children and adolescents, represents an aberrant form of skeletal muscle differentiation. Both skeletal muscle development, as well as regeneration of adult skeletal muscle are governed by members of the myogenic family of regulatory transcription factors (MRFs), which are deployed in a highly controlled, multi-step, bidirectional process. Many aspects of this complex process are deregulated in RMS and contribute to tumorigenesis. Interconnected loops of super-enhancers, called core regulatory circuitries (CRCs), define aberrant muscle differentiation in RMS cells. The transcriptional regulation of MRF expression/activity takes a central role in the CRCs active in skeletal muscle and RMS. In PAX3::FOXO1 fusion-positive (PF+) RMS, CRCs maintain expression of the disease-driving fusion oncogene. Recent single-cell studies have revealed hierarchically organized subsets of cells within the RMS cell pool, which recapitulate developmental myogenesis and appear to drive malignancy. There is a large interest in exploiting the causes of aberrant muscle development in RMS to allow for terminal differentiation as a therapeutic strategy, for example, by interrupting MEK/ERK signaling or by interfering with the epigenetic machinery controlling CRCs. In this review, we provide an overview of the genetic and epigenetic framework of abnormal muscle differentiation in RMS, as it provides insights into fundamental mechanisms of RMS malignancy, its remarkable phenotypic diversity and, ultimately, opportunities for therapeutic intervention.

Rhabdomyosarcoma (RMS) is an aggressive human pediatric cancer. Despite robust expression of myogenic regulatory factors, RMS cells are blocked in a proliferative state and do not terminally differentiate. The extent to which the skeletal muscle lineage is represented in RMS tumors and the mechanisms leading to developmental arrest remain elusive. Here, we combined single-cell RNA sequencing (scRNAseq), mass cytometry (CyTOF) and high-content imaging to resolve RMS heterogeneity. ScRNAseq and CyTOF analysis of a total of 17 patient-derived primary cultures and three cell lines uncovered plastic myogenic subpopulations that delineate a branched trajectory. The less aggressive embryonal RMS (eRMS) harbor primarily muscle stem cell (MuSC)-like cells and exhibit sparse commitment to differentiation. The more aggressive alveolar RMS (aRMS) comprise primarily actively cycling committed progenitors with a paucity of differentiated cells. The oncogenic fusion protein PAX3:FOXO1 sustains aRMS cells in the cycling trajectory loop, which we show can re-wired towards differentiation upon its downregulation or by dual pharmacological RAF and MEK inhibition. Our findings provide insights into the developmental states and trajectories underlying RMS progression and identify the RAS pathway as a promising target of differentiation therapy for human aRMS.STATEMENT OF SIGNIFICANCEWe present the first comprehensive single-cell transcriptomic and proteomic atlas of pediatric rhabdomyosarcoma (RMS), in which we identify impaired myogenic trajectories with prognostic value. We demonstrate that RAS pathway inhibitors disrupt the oncogenic trajectory and induce terminal differentiation, revealing novel therapeutic targets for the aggressive alveolar RMS subtype.