Abstract PR002: Domain-specific oncogenic functions of VGLL2::NCOA2 in infantile rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children and has molecular features of immature skeletal muscle development. Spindle-cell RMS subtype in infant patients has been associated with the presence of chromosomal translocation event in the form of fusion-oncogenes. The most common fusion-oncogene in these patients is VGLL2::NCOA2, the fusion between vestigial-like family member 2 (VGLL2) and nuclear receptor coactivator 2 (NCOA2). Conventional chemotherapeutic approaches do not target the fusion-oncogene itself, are broadly toxic, and often impose lifelong adverse side effects that decrease the patient’s quality of life. Previous work from our lab has shown that the VGLL2::NCOA2 fusion is oncogenic in zebrafish and mouse allograft models, with tumors forming without requiring secondary mutations. Importantly, these tumors histologically and transcriptionally recapitulated the human disease. Understanding fusion-oncogene biology is critical to identifying novel molecular therapeutic targets to improve patient outcomes. Here, we implemented a structure-function-based strategy to define the requirements of each functional domain of VGLL2::NCOA2 in fusion-driven tumorigenesis. We hypothesize that the protein domains retained in the fusion-oncogene influence tumorigenic mechanisms. We generated deletions of the functional protein domain vestigial/Tondu (TDU) of VGLL2 and activating domains (AD1 and AD2) of NCOA2, both of which are retained in the fusion. We transfected C2C12 mouse myoblast cells with either the full-length VGLL2::NCOA2 fusion construct or the mutant domain deletion constructs. Using immunofluorescence staining and western blotting, we found that the VGLL2::NCOA2 fusion localized in the nucleus, whereas the single deletions of the TDU and AD1 domains resulted in decreased nuclear localization. C2C12 cells transfected with the full-length fusion readily formed colonies in soft agar; however, removing the TDU and AD1 domains suppressed colony formation. We complemented our in vitro studies with a mouse allograft approach. Mice injected with the C2C12 cells stably expressing VGLL2::NCOA2 with deletions of TDU and/or AD1 had a delayed tumor onset with more differentiated tumors compared to mice injected with the full-length fusion. Furthermore, bulk RNA-sequencing transcriptional analysis of transfected cells demonstrated that deletion of the TDU and AD1 domains resulted in an upregulation of skeletal muscle myogenesis gene sets, suggesting promotion of muscle differentiation compared to cells expressing the full-length fusion. Overall, our results indicate that VGLL2::NCOA2 is dependent on the TDU and AD1 domains for supporting tumorigenic activities and that targeting these domains or their interacting protein partners represents a potential therapeutic opportunity. Citation Format: Chinmay S. Sankhe, Delia Calderon, Cenny Taslim, Melissa Sammons, Lindsay Ryan, Amy C. Gross, Joanna Cyrta, Olivier Delattre, Ryan D. Roberts, Genevieve C. Kendall. Domain-specific oncogenic functions of VGLL2::NCOA2 in infantile rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr PR002.