Abstract A024: Cell state regulation of metastasis in Fusion-negative Rhabdomyosarcoma

Abstract

Fusion-negative Rhabdomyosarcoma (FN-RMS) is a common childhood soft tissue sarcoma with hundreds of cases diagnosed annually in the United States. Despite having good treatment outcomes in the primary tumor setting, metastatic RMS has an abysmal prognosis with less than 20% of patients surviving the disease. Thus, there is a critical need to uncover the molecular pathways that drive metastasis. Recently, we used single-cell RNA sequencing (scRNA-seq) and functional assays to identify molecularly distinct FN-RMS cell states, including i) progenitor/CSCs that are largely quiescent but divide after chemotherapy or radiation, ii) mid-differentiated proliferative cells that drive growth under non-stress conditions, and iii) differentiated cells that express mature skeletal muscle-lineage factors. Using cell surface markers that define specific cell states, FACS, and mouse xenografts, we made the unexpected finding that CSCs are less metastatic than other cell states. In fact, it is the most differentiated TSPAN33+/CD44- cell state that is the most efficient at driving metastatic seeding. Consistent with this, FN-RMS cells differentiated with the MEK-inhibitor trametinib have increased metastatic tumor formation in xenografted mice and have enhanced seeding in the caudal hematopoietic tissue (CHT), the primary metastatic niche in zebrafish. Mechanistically, we found that the transcription factor myogenin is induced following differentiation of FN-RMS cells concurrent with elevated expression of the integrin subunits ITGA4 and ITGB6. Given the established role of integrin signaling in extravasation and metastatic colonization, this upregulation may underlie the enhanced metastatic seeding observed upon differentiation and suggests a role for myogenin in regulating the process. Together, these findings delineate the cellular and molecular mechanisms of FN-RMS metastasis and identify new predictive biomarkers and therapeutic targets for preventing relapse, progression, and metastasis.