Abstract A049 A Wnt5a dependent feedback loop contributes to Ewing sarcoma cell motility

Abstract Introduction: Ewing sarcoma (ES) is the second most common bone tumor in pediatrics, primarily affecting the adolescent and young adult population. The most significant prognostic factor is the presence or absence of metastatic disease. Despite improvement in outcomes for children with localized ES, patients who present with metastatic disease face a grim prognosis with a 5-year event-free survival of approximately 25%. Therefore, the most pressing need for ES patients is prevention and treatment of metastatic disease. We have identified a novel signaling pathway in Wnt signaling that allows ES cells to migrate, specifically via the non-canonical Wnt family member Wnt5a. Our results demonstrate a direct interaction between Wnt5a and pro-motility signals within ES cells that have previously been identified in genetic screens in ES, but not reported as a pathway. Methods: We used a combination of in vitro work with ES cell lines, including CRISPR-Cas9 editing, and in vivo work in a zebrafish model of ES, to study the role of non-canonical Wnt signaling and how chemical perturbation of proteins in this pathway affect ES cell migration. Results: Inhibition of post-translation palmitoylation of Wnt5a, thereby disrupting its ability to signal at the cell surface, resulted in an increase in endogenous Wnt5a transcription and translation, both in cell lines and patient-derived xenografts, suggesting a feedback dependence. In addition, both chemical inhibition of palmitoylation as well as Crispr-Cas9 editing of Wnt5a in ES cell lines resulted in decreased cell migration with decreased formation of cell protrusions, which are needed for forward motility. Phosphorylation of FAK at Tyr397 and binding of ALCAM to vinculin were both found to be critical steps in ES motility, all of which were affected by Wnt5a. Chemical perturbations of several of these proteins in a zebrafish model of ES affect axis development and angiogenesis. Conclusion: Our data support a novel role for non-canonical Wnt signaling in ES. Wnt5a is an active signaling molecule in this aggressive cancer to which there is a cell-autonomous feedback mechanism. Our data further demonstrates that Wnt5a drives a signaling pathway that engages multiple components identified as important in the migratory and invasive phenotypes of other cancers. In particular, we show that activation of this pathway involves phosphorylation of both FAK and Src, modification of the actin cytoskeleton, and induction of a physical interaction between the actin binding protein vinculin and the cell adhesion molecular, ALCAM. Inhibition of Wnt signaling, FAK phosphorylation, or ALCAM palmitoylation can each diminish ES cell migration. Thus, our work strongly supports a more nuanced model of ES metastasis, involving both canonical and noncanonical intracellular pathways which can modify both the ECM and the way cells respond to their environment, and would also support the development of clinical trials focused on disrupting these pathways as a means to prevent metastatic relapses and improve patient outcomes. Citation Format: Alissa C. Baker. A Wnt5a dependent feedback loop contributes to Ewing sarcoma cell motility [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr A049.