(841) Elucidating Molecular Mechanisms in Vestibular Schwannoma

Introduction: Vestibular schwannomas (VS) arise from NF2 mutations that inactivate Merlin, a tumor suppressor regulating Schwann cell proliferation. Merlin has been shown to interact with E3 ubiquitin ligase Arkadia to promote degradation of transcriptional corepressor SKOR2, facilitating TGFβ/SMAD signaling. Loss of Merlin is associated with pathway disruption, SKOR2 accumulation, and increased Schwann cell proliferation. The role of Arkadia in VS remains incompletely defined.

Methods: CRISPR-engineered NF2 mutant and wild-type human induced pluripotent stem cells (iPSCs) were differentiated into Schwann cells and validated by immunohistochemistry. Proliferation was assessed by cell counting using brightfield imaging across maturation stages (15-60 days post-differentiation). Lentiviral shRNA constructs targeting Arkadia were optimized using real-time PCR and puromycin selection. Western blot analysis of Merlin, SKOR2, Arkadia, SMAD2/3, and Ki-67 was performed for VS tumors and greater auricular nerve controls.

Results: Both NF2 mutant and wild-type iPSCs were differentiated into Schwann cells. Time-course analyses showed differences in proliferation patterns between NF2 mutant and wild-type cells across developmental stages. Screening of Arkadia-targeting shRNA constructs identified a construct that reproducibly reduced Arkadia mRNA expression while preserving viability. Western blot analyses demonstrated expression of Merlin–Arkadia–SKOR2 pathway components in VS tumors and control nerve tissue.

Conclusions: We established a human iPSC-derived Schwann cell and VS platform to support investigation of Arkadia-associated signaling in VS. This system provides a foundation for future studies examining how Arkadia modulation may influence Schwann cell proliferation in VS.