Structure-Function Analysis of an Infantile Rhabdomyosarcoma Fusion-Oncogene Reveals Domain Requirements for Tumorigenesis

Rhabdomyosarcoma is a pediatric soft-tissue sarcoma that reflects impaired skeletal muscle differentiation. Spindle cell/sclerosing rhabdomyosarcoma is a histological subtype found in children and adults. In children under two, spindle cell rhabdomyosarcoma is genetically driven by fusion-oncogenes of transcription factors or co-activators important for skeletal muscle lineage commitment and development. The most common fusion-oncogene in this group is VGLL2::NCOA2, a fusion between vestigial-like family member 2 (VGLL2) and nuclear receptor coactivator 2 (NCOA2). Children with fusion-driven rhabdomyosarcoma are treated with general chemotherapy, surgery, and radiation, which are not specific to the molecular underpinnings, and increase risk of secondary malignancies and premature death. Previously, we showed that VGLL2::NCOA2 is transforming in mouse myoblasts and transgenic zebrafish tumor models. Here, we build on this work by detailing the structure-function relationship of the retained VGLL2::NCOA2 protein domains to define domain requirements and their molecular interactions for tumorigenesis. We created deletion mutants of the VGLL2 vestigial/Tondu (TDU) domain and the NCOA2 activating domains 1 and 2 (AD1 and AD2), and a double mutant lacking the TDU+AD1 domains and assessed their function in a mouse myoblast model. We find that the TDU and AD1 domains support nuclear fusion-oncogene localization, transformation in vitro, and tumorigenesis in a mouse allograft model. Furthermore, transcriptomic analysis demonstrates that the TDU or AD1 mutant VGLL2::NCOA2 drives an upregulation of myogenesis gene sets and supports terminal differentiation. Overall, our results indicate that VGLL2::NCOA2 requires the TDU and AD1 domains for optimal transformation, and that targeting these domains, or their interacting protein partners, represents a therapeutic opportunity.