Background: Meningiomas are the most common primary intracranial tumor, accounting for over 30% of all primary brain tumors. Although most are benign, up to 20% recur and can be considered aggressive. Based on RNA and whole-exome sequencing data, we identified three molecular subtypes of meningioma. Meningioma Group B and C (MenG B/C) are characterized by deletion of Neurofibromatosis Type 2 (NF2) gene. Simultaneously, the deactivation of the Hippo pathway has been linked to meningioma growth. NF2 inactivation has previously been linked to deregulating the Hippo signaling pathway; however, the mechanism of this relationship is unclear in meningioma. This study aims to investigate the impact of common meningiomal NF2 mutations on Hippo pathway activity and identify which specific NF2-Hippo signaling interactions contribute to these effects.

Methods: We conducted an extensive literature review to identify the most common NF2 mutations in meningioma. Six of these mutants were cloned and overexpressed in 293 cells with a N-terminal mono-GFP tag. Localization was assessed via fluorescence imaging and compared to wild-type (WT) NF2 localization. We then generated a split-luciferase Hippo pathway biosensor to quantify mutant NF2’s effects on downstream Hippo pathway activity. In this biosensor, one of two luciferase domains is attached to a small amino acid stretch of the YAP protein that harbors the phosphorylation site for degradation. The other part is attached to the phospho-dependent interactor 14-3-3. When Hippo is on, the YAP site gets phosphorylated, binds to 14-3-3 and completes a functional luciferase, which emits a luminescence signal upon substrate binding. WT and mutant NF2s were co-transfected with this biosensor and corresponding signals were quantified. In a second line of experiments WT and mutants were co-expressed with Hippo pathway kinases, MST1 and LATS1, then immunoprecipitated to investigate if these interact.

Results: We found 19 articles and one database describing 205 NF2 mutations in meningioma, most of which were nonsense mutations. We investigated six of the most common mutants along with the three most common WT NF2 variations (v1, v2, v7). NF2 var7 and NF2 mutants showed aberrant protein aggregation compared to var1 or var2 NF2 which correlated with severity of truncation. Luciferase assay revealed that var7 NF2 as well as mutant NF2 decreased YAP phosphorylation and subsequent Hippo pathway activity. Immunoprecipitation and Western Blot results found that although NF2 v1 and v2 show normal MST1 and LATS interaction, var7 lost interaction with MST1. Interestingly, the NF2 mutants lacked LATS1 interaction, but MST1 interacted even with the smallest truncation of NF2.

Conclusion: We propose that NF2 forms a tertiary complex with MST1 and LATS1 to activate the Hippo kinase cascade and prevent proliferation. NF2 nonsense mutations create aberrant non-functional proteins that disrupt this complex formation and attenuate Hippo signaling. The naturally occurring var7 NF2 loses MST1 interaction while the NF2 mutations lose LATS1 interaction. These findings have strong therapeutic implications in targeting the MST1-LATS interaction to suppress oncogenicity. Future directions suggest investigating the role of TRAF7 in Hippo signaling, enabling a complete picture of the Hippo pathway in meningioma.