Abstract 3947: Defining VGLL2::NCOA2 and ARF6 cooperation in infantile rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is most common pediatric soft tissue sarcoma, is aggressive, and has characteristics of arrested skeletal muscle differentiation. Infantile RMS, a subtype of RMS, is genetically driven by a family of fusion-oncogenes involving key proteins essential for skeletal muscle development. One example is the fusion between transcriptional coactivators, VGLL2 and NCOA2. Recent VGLL2::NCOA2-driven RMS cases have shown that this fusion-oncogene can be lethal; however, there is a lack of significant knowledge surrounding the biological mechanisms of the fusion and an unmet need of effective targeted therapies. To assess VGLL2::NCOA2 tumorigenic mechanisms and therapeutic vulnerabilities, we generated two new VGLL2::NCOA2 driven tumor models, a C2C12 mouse allograft and transgenic zebrafish model. Both of these models histologically and transcriptionally recapitulate the human disease. Shared transcriptional features between patient data, mouse allograft, and transgenic zebrafish tumors revealed suppression of skeletal muscle specific genes and either reactivation or inappropriate developmental persistence of the small GTPase, ADP ribosylation factor 6 (ARF6). ARF6 is important for myogenic development and was significantly overexpressed across all cross-species tumors and is being pursued as a therapeutic opportunity in other cancer contexts. Knockout of Arf6 in our C2C12-VGLL2::NCOA2 cell culture model suppressed VGLL2::NCOA2 colony formation and restored the expression levels of critical muscle differentiation specific genes. To define the role of ARF6 in vivo, we used CRISPR/Cas9 to generate a zebrafish arf6b germline knockout. Although arf6 is duplicated in the fish, the Arf6b homozygous null fish have a significant reduction in Arf6 protein levels and are viable as adults and fertile. We then injected wildtype and arf6b knockout fish with mosaic human VGLL2::NCOA2 to determine genetic cooperation in the disease. We found arf6b knockout fish still developed VGLL2::NCOA2 tumors; however, transcriptional analysis showed that these tumors downregulated VGLL2::NCOA2 expression and were more differentiated with a concomitant upregulation of skeletal muscle specific pathways. Given that infantile RMS displays features of arrested skeletal muscle differentiation, these results highlight the contribution of ARF6 with VGLL2::NCOA2 for oncogenesis. Our findings are consistent with our data that C2C12-VGLL2::NCOA2 cell models are unable to terminally differentiate, but that removing Arf6 ameliorates that suppression. We are also performing mouse allograft studies, to continue to delineate the in vivo oncogenic mechanisms of ARF6 cooperation in infantile RMS. We will assess tumor morphology and genetic features, and tumor incidence. Overall, our findings underscore the cooperative role of ARF6 in VGLL2::NCOA2 driven tumor highlighting its potential as a therapeutic target. Citation Format: Delia Calderon, Chinmay Sankhe, Cenny Taslim, Olivier Delattre, Genevieve Kendall. Defining VGLL2::NCOA2 and ARF6 cooperation in 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 3947.