P01.14.B ROR1-WNT5A CROSSTALK MEDIATED BY M6A PROMOTES VASCULOGENIC MIMICRY IN GLIOMA STEM CELLS

Abstract BACKGROUND Glioma stem cells (GSCs) have the capacity to transdifferentiate into endothelial-like cells, contributing to glioblastoma (GBM) progression and therapy resistance. However, the mechanisms driving GSC-derived endothelial cell (GDEC) formation remain poorly understood. METHODS We employed single-cell RNA sequencing (scRNA-seq) and immunofluorescence to identify GSC subpopulations. Comparative RNA-seq, scRNA-seq, and scATAC-seq were used to identify regulators of endothelial differentiation. To investigate upstream control of ROR1 expression, we conducted MeRIP-seq, RIP-seq, and RNA stability assays. Additionally, scRNA-seq, spatial transcriptomics, Co-IP, and co-culture assays were used to explore immune-mediated regulation. In vivo effects of ROR1 targeting were evaluated via AAV-BR1 delivery, transmission electron microscopy (TEM), and TUNEL assays. RESULTS scRNA-seq identified a CD133⁺/CD31⁺ GSC subpopulation in GBM, validated by immunofluorescence. Comparative transcriptomic analyses and public datasets (CCGA, TCGA) revealed ROR1 as a key mediator of GSC-to-GDEC differentiation. Multi-omics integration (scRNA-seq, scATAC-seq, spatial RNA-seq) and in situ validation confirmed ROR1’s central role. ROR1 overexpression promoted, while its knockdown suppressed, GDEC formation and tumor growth in mouse models. Furthermore, Hypoxia induced WTAP upregulation and immunosuppressive microenvironment plays a vital role in this process. Mechanistically, hypoxia upregulated WTAP expression in GSCs, which, via m6A modification, stabilized ROR1 through HuR binding.ROR1 then activated WNT signaling by binding WNT5A, as shown by WNT5A stimulation and co-IP assays. Single-cell and spatial transcriptomics revealed that WNT5A is primarily secreted by glioma-associated macrophages (GAMs), with WNT5A⁺ TAMs found in proximity to ROR1⁺ GDECs. Finally,in vivo, AAV-BR1-mediated ROR1 inhibition reduced tumor growth, normalized vasculature, and enhanced temozolomide (TMZ) delivery and efficacy, as confirmed by TEM, HPLC, and TUNEL assays. CONCLUSION Our findings uncover a hypoxia-induced WTAP-ROR1 axis that drives GSC transdifferentiation into GDECs via m6A-dependent stabilization of ROR1. WNT5A from GAMs activates ROR1-mediated WNT signaling, further promoting this process. Targeting ROR1 via AAV-BR1 normalizes vasculature and enhances TMZ sensitivity, offering a promising therapeutic strategy for GBM.