Abstract 3946: VGLL2::NCOA2 structure-function analysis reveals domain requirements for fusion-driven infantile rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue sarcoma and is associated with impaired skeletal muscle differentiation. RMS has significant diagnostic and therapeutic challenges, and the presence of fusion oncogenes contributes to the aggressiveness of the disease. There exists lack of mechanistic understanding of fusion-oncogene driven tumorigenesis. As a result, more efforts need to be directed towards defining fusion-oncogene biology to identify novel therapeutic strategies and improve clinical outcomes in patients. A new family of fusion-oncogenes, including VGLL2::NCOA2, has been identified in the infantile RMS subtype. Normally, vestigial-like family member 2 (VGLL2) and nuclear receptor coactivator 2 (NCOA2) independently regulate skeletal muscle differentiation as transcriptional co-activators. We found the VGLL2::NCOA2 fusion is sufficient for tumorigenesis in multiple systems. Here, we elucidate whether protein domains contribute to this activity. VGLL2::NCOA2 retains the vestigial (TDU) domain of VGLL2 and activating domains (AD1 and AD2) of NCOA2. We hypothesize that TDU, AD1 and AD2 domains of VGLL2::NCOA2 contribute to fusion-driven tumorigenesis. We implemented our C2C12 mouse myoblast cell culture model and a structure-function analysis of VGLL2::NCOA2 to define domain requirements for transformation. We transfected mouse myoblast cells with the full-length human VGLL2::NCOA2 fusion construct and truncated mutant constructs containing deletions of TDU, AD1, and AD2 domains. Using biochemical approaches, we found higher nuclear localization of VGLL2::NCOA2 in cells transfected with the full-length fusion and AD2 domain deleted mutant fusion; however, deletion of either the TDU and AD1 domain resulted in decreased nuclear localization. Furthermore, in testing the oncogenic potential of transfected cells via soft agar colony formation assay, we found higher number of colonies in cells transfected with the full-length fusion and the AD2 domain deleted construct. In the mutants with the TDU and AD1 domains deleted, there is a significant reduction in colony formation. We complemented our cell culture strategies with a mouse allograft approach. Compared to the full-length VGLL2::NCOA2 fusion, mice allografted with myoblast cells containing the VGLL2::NCOA2 deleted TDU and AD1 domains form significantly delayed tumors, with onset beginning 40 days after the full-length fusion. Overall, our results suggest that TDU and AD1 domains are required to drive oncogenic mechanisms. Targeting these domains will facilitate identification of novel molecular targets that can suppress VGLL2::NCOA2 activity, and potentially be used as therapeutic opportunities. Citation Format: Chinmay S. Sankhe, Delia Calderon, Cenny Taslim, Melissa Sammons, Lindsay Ryan, Amy C. Gross, Olivier Delattre, Ryan D. Roberts, Genevieve C. Kendall. VGLL2::NCOA2 structure-function analysis reveals domain requirements for fusion-driven infantile rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3946.