Abstract A027: Structure-function analysis of the infantile rhabdomyosarcoma VGLL2::NCOA2 fusion-oncogene reveals domain requirements for tumorigenesis

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue sarcoma and is associated with arrested skeletal muscle differentiation. RMS poses considerable challenges in both diagnosis and treatment, and the presence of fusion oncogenes is predictive of more aggressive forms of the disease. There is a lack of mechanistic understanding of fusion-oncogene-driven tumorigenesis. Thus, efforts need to be directed toward 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 will evaluate what 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 immunofluorescence staining and 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 a 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. Structure-function analysis of the infantile rhabdomyosarcoma VGLL2::NCOA2 fusion-oncogene reveals domain requirements for tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr A027.