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Rhabdomyosarcoma (RMS) is a highly aggressive pediatric soft tissue cancer, associated with the skeletal muscle lineage. Despite undoubted expression of key myogenic regulatory factors, RMS cells are blocked in a proliferative state and do not complete terminal differentiation. The extent to which the skeletal muscle lineage is represented in RMS tumors, as well as the mechanisms leading to developmental arrest, remain elusive. We therefore aimed to identify RMS subpopulations, understand why RMS cells are stalled in their differentiation path, and determine mechanisms to pharmacologically restore myogenic differentiation. Using single-cell RNA sequencing and mass cytometry analysis, we profiled cells from 14 PDX-derived primary cultures and three cell lines of alveolar (aRMS) and embryonal RMS (eRMS) subtypes. We discovered that both RMS subtypes contain different cell subpopulations distributed along the myogenic lineage. aRMS tumors recapitulate a yet unrecognized branched myogenic trajectory, where progenitor cells either commit to differentiation or give rise to actively cycling myoblasts. Following in vitro exposure to chemotherapy, aRMS cells show compositional shifts towards undifferentiated states, consistent with a model where treatment rewires cellular trajectories. To quantify aRMS cellular states, we developed an automated image-based single-cell approach and applied it to identify pro-differentiating agents among a library of >240 FDA-approved drugs. We identified the RAS pathway as an important mediator of myogenic differentiation in several aRMS cultures, demonstrating the potential for differentiation therapy. Current studies are ongoing to validate these results in vivo and to uncover drug combinations that completely overcome the differentiation block. Taken together, this study reveals possible cellular origins for RMS and identifies a potential novel treatment strategy for aRMS that targets cellular differentiation. Citation Format: Sara G. Danielli, Ermelinda Porpiglia, Andrea J. De Micheli, Ingrid Bechtold, Joana G. Marques, Stephanie Kasper, Helen M. Blau, Marco Wachtel, Beat W. Schäfer. Single-cell profiling reveals a conserved myogenic hierarchy in pediatric rhabdomyosarcomas amenable to differentiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1679. |
