TRAF7 WD40-DOMAIN MUTATIONS DRIVE MENINGIOMA TRANSDIFFERENTIATION AND INTRATUMOR HYPEROSTOSIS

Abstract AIMS Meningiomas are the only primary tumors that undergo hyperostosis in the tumor microenvironment. Clinical observations suggest TRAF7short somatic variants (SSVs) are enriched in skull-base meningiomas, and that SSVs targeting the TRAF7 WD40-domain may be associated with meningioma-induced hyperostosis (MIH). Here, we integrate single-cell RNA sequencing and lineage tracing data with mechanistic and functional studies across re- gionally distinct primary meningioma cell cultures. We find that TRAF7 WD40-domain mutations drive menin- gioma cell transdifferentiation via PRRX1 into an osteoblast-like state that is associated with mineralization in vitro and MIH in vivo. METHODS Single-cell RNA sequencing,mitochondrial alteration enrichment from single-cell transcriptomes to establish re- latedness (MAESTER) lineage tracing, RNA velocity and pseudotime analyses were used to define cell types, cell states, and evolutionary trajectories from 8 human samples. Regionally distinct primary meningioma cell cul- tures (n=6) from 3 meningiomas with MIH were derived for mechanistic and functional studies. Targeted next- generation DNA sequencing, QPCR, immunofluorescence, cell proliferation, immunoprecipitation with western blot, and mineralization assays with alizarin red stains were used to define how TRAF7 WD40-domain mutations influence protein function and cell state. RESULTS Single-cell transcriptomics and lineage tracing identified a differentiation event from meningioma origin clus- ter to an osteoblast-like meningioma cell cluster that was enriched for bone-associated genes, driven by genes associated with PRRX1. Primary meningioma cell cultures were validated using DNA methylation profiling and SSTR2A expression. Only cell cultures from intraosseous regions expressed RUNX2, SP7, or ALPL, and under- went mineralization in vitro. Suppression of mutant TRAF7 using siRNAs blocked in vitro mineralization, and media from meningioma cell cultures from intraosseous regions induced mineralization of TRAF7 wildtype meningioma cell cultures in vitro. CONCLUSION These data demonstrate that TRAF7 WD40-domain mutations drive meningioma cell differentiation and intra- tumor hyperostosis via expression of PRRX1, and shed light on fundamental mechanisms underlying lamellar bone formation.