Abstract P58: A Novel ALK-R-Spondin-Wnt Signaling Axis in Neuroblastoma: Insights from In Silico And In Vitro Analysis

Abstract

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase implicated in the pathogenesis of several cancers, including anaplastic large cell lymphoma (ALCL), non-small cell lung cancer (NSCLC), and neuroblastoma. While ALK activation typically occurs through its ligands ALKAL1 and ALKAL2, in cancer it is often activated in a ligand-independent manner through oncogenic fusion proteins that retain the kinase domain. Activated ALK engages multiple downstream signaling pathways, including Ras/Raf/MEK/ERK, JAK/STAT3, and PI3K/AKT. Recent studies also suggest ALK may influence Wnt signaling, although its mechanism is not fully understood. To investigate ALK’s involvement in Wnt signaling, we performed network-based gene association analysis using GeneMANIA and identified R-spondins (Rspo1–4), known Wnt enhancers, as potential novel interacting partners of ALK. Protein-protein interaction (PPI) analysis indicated that R-spondins bind the TNF-like and EGF-like domains of ALK-regions critical for ALKAL2 binding. Molecular docking and dynamics simulations validated the structural stability and favorable binding of the ALK-Rspo complex. These computational findings were corroborated by in vitro experiments using neuroblastoma cell lines IMR32 and SH-SY5Y. Co-treatment with Wnt3a and Rspo2 significantly enhanced Wnt signaling activity in these cell lines, which was markedly diminished upon treatment with the ALK inhibitor crizotinib. This suggests a functional role for ALK in modulating Wnt signaling via its interaction with R-spondins. Together, our findings reveal a novel ALK-Rspo axis that contributes to the regulation of Wnt signaling in neuroblastoma.