NF2-related schwannomatosis (NF2) is a genetic syndrome characterized by the growth of benign tumors in the nervous system, particularly bilateral vestibular schwannomas, meningiomas, and ependymomas. This review consolidates the current knowledge on NF2 syndrome, emphasizing the molecular pathology associated with the mutations in the gene of the same name, the NF2 gene, and the subsequent dysfunction of its product, the Merlin protein. Merlin, a tumor suppressor, integrates multiple signaling pathways that regulate cell contact, proliferation, and motility, thereby influencing tumor growth. The loss of Merlin disrupts these pathways, leading to tumorigenesis. We discuss the roles of another two proteins potentially associated with NF2 deficiency as well as Merlin: Yes-associated protein 1 (YAP), which may promote tumor growth, and Raf kinase inhibitory protein (RKIP), which appears to suppress tumor development. Additionally, this review discusses the efficacy of various treatments, such as molecular therapies that target specific pathways or inhibit neomorphic protein-protein interaction caused by NF2 deficiency. This overview not only expands on the fundamental understanding of NF2 pathophysiology but also explores the potential of novel therapeutic targets that affect the clinical approach to NF2 syndrome.

Neurofibromatosis type 2 (NF2)-related schwannomatosis is a rare autosomal dominant monogenic disorder caused by mutations in the NF2 gene. The hallmarks of NF2-related schwannomatosis are bilateral vestibular schwannomas (VS). The current treatment options for NF2-related schwannomatosis, such as observation with serial imaging, surgery, radiotherapy, and pharmacotherapies, have shown limited effectiveness and serious complications. Therefore, there is a critical demand for novel effective treatments. Gene therapy, which has made significant advancements in treating genetic diseases, holds promise for the treatment of this disease. This review covers the genetic pathogenesis of NF2-related schwannomatosis, the latest progress in gene therapy strategies, current challenges, and future directions of gene therapy for NF2-related schwannomatosis.

Solid tumours can universally evade contact inhibition of proliferation (CIP), a mechanism halting cell proliferation when cell-cell contact occurs. Merlin, an ERM-like protein, crucially regulates CIP and is frequently deactivated in various cancers, indicating its significance as a tumour suppressor in cancer biology. Despite extensive investigations into Merlin\'s role in cancer, its lack of intrinsic catalytic activity and frequent conformation changes have made it notoriously challenging to study. To address this challenge, we harnessed innovative luciferase technologies to create and validate a NanoBiT split-luciferase biosensor system in which Merlin is cloned between two split components (LgBiT and SmBiT) of NanoLuc luciferase. This system enables precise quantification of Merlin\'s conformation and activity both in vitro and within living cells. This biosensor significantly enhances the study of Merlin\'s molecular functions, serving as a potent tool for exploring its contributions to CIP and tumorigenesis.

Traumatic brain injury (TBI) is a common disease worldwide with high mortality and disability rates. Besides the primary mechanical injury, the secondary injury associated with TBI can also induce numerous pathological changes, such as brain edema, nerve apoptosis, and neuroinflammation, which further aggravates neurological dysfunction and even causes the death due to the primary injury. Among them, neuronal apoptosis is a key link in the injury. Melanocortin-1 receptor (MC1R) is a G protein coupled receptor, belonging to the melanocortin receptor family. Studies have shown that activation of MC1R inhibits oxidative stress and apoptosis, and confers neuroprotective effects against various neurological diseases. Merlin is a protein product of the NF2 gene, which is widely expressed in the central nervous system (CNS) of mice, rats, and humans. Studies have indicated that Merlin is associated with MC1R. In this study, we explored the anti-apoptotic effects and potential mechanisms of MC1R. A rat model of TBI was established through controlled cortical impact. The MC1R-specific agonist Nle4-D-Phe7-α-Melanocyte (NDP-MSH) and the inhibitor MSG-606 were employed to explore the effects of MC1R and Merlin following TBI and investigated the associated mechanisms. The results showed that the expression levels of MC1R and Merlin were upregulated after TBI, and activation of MC1R promoted Merlin expression. Further, we found that MC1R activation significantly improved neurological dysfunction and reduced brain edema and neuronal apoptosis induced by TBI in rats. Mechanistically, its neuroprotective function and anti-apoptotic were partly associated with MC1R activation. In conclusion, we demonstrated that MC1R activation after TBI may inhibit apoptosis and confer neuroprotection by upregulating the expression of Merlin.

Neurofibromatosis Type 2 (NF2)-related schwannomatosis is a genetic disorder that causes development of multiple types of nervous system tumors. The primary and diagnostic tumor type is bilateral vestibular schwannoma. There is no cure or drug therapy for NF2. Recommended treatments include surgical resection and radiation, both of which can leave patients with severe neurological deficits or increase the risk of future malignant tumors. Results of our previous pilot high-throughput drug screen identified phosphoinositide 3-kinase (PI3K) inhibitors as strong candidates based on loss of viability of mouse merlin-deficient Schwann cells (MD-SCs). Here we used novel human schwannoma model cells to conduct combination drug screens. We identified a class I PI3K inhibitor, pictilisib and p21 activated kinase (PAK) inhibitor, PF-3758309 as the top combination due to high synergy in cell viability assays. Both single and combination therapies significantly reduced growth of mouse MD-SCs in an orthotopic allograft mouse model. The inhibitor combination promoted cell cycle arrest and apoptosis in mouse merlin-deficient Schwann (MD-SCs) cells and cell cycle arrest in human MD-SCs. This study identifies the PI3K and PAK pathways as potential targets for combination drug treatment of NF2-related schwannomatosis.

The type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family produces the critical lipid regulator phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the plasma membrane (PM). Here, we investigated the potential role of PIP5Kγ, a PIP5K isoform, in the Hippo pathway. The ectopic expression of PIP5Kγ87 or PIP5Kγ90, two major PIP5Kγ splice variants, activated large tumor suppressor kinase 1 (LATS1) and inhibited Yes-associated protein (YAP), whereas PIP5Kγ knockdown yielded opposite effects. The regulatory effects of PIP5Kγ were dependent on its catalytic activity and the presence of Merlin and LATS1. PIP5Kγ knockdown weakened the restoration of YAP phosphorylation upon stimulation with epidermal growth factor or lysophosphatidic acid. We further found that PIP5Kγ90 bound to the Merlin\'s band 4.1/ezrin/radixin/moesin (FERM) domain, forming a complex with PI(4,5)P2 and LATS1 at the PM. Notably, PIP5Kγ90, but not its kinase-deficient mutant, potentiated Merlin-LATS1 interaction and recruited LATS1 to the PM. Consistently, PIP5Kγ knockdown or inhibitor (UNC3230) enhanced colony formation in carcinoma cell lines YAP-dependently. In addition, PIP5Kγ90 interacted with heat shock cognate 71-kDa protein (Hsc70), which also contributed to Hippo pathway activation. Collectively, our results suggest that PIP5Kγ regulates the Hippo-YAP pathway by forming a functional complex with Merlin and LATS1 at the PI(4,5)P2-rich PM and via interplay with Hsc70.

Neurofibromatosis type 2 (NF2), a multiple neoplasia syndrome, is a manifestation of an impaired expression of the merlin protein, exerting inhibitory effects on cell proliferation signals due to abnormalities of the NF2 gene located on chromosome 22. About half of patients inherit a germline mutation from a parent, and nearly 60% of de novo NF2 patients are estimated to have somatic mosaicism. The development of technical methods to detect NF2 gene mutation, including targeted deep sequencing from multiple tissues, improved the diagnostic rate of mosaic NF2. With improved understanding of genetics and pathogenesis, the diagnostic criteria for NF2 were updated to assist in identifying and diagnosing NF2 at an earlier stage. The understanding of cell signaling pathways interacting with merlin has led to the development of molecular-targeted therapies. Currently, several translational studies are searching for possible therapeutic agents targeting VEGF or VEGF receptors. Bevacizumab, an anti-VEGF monoclonal antibody, is widely used in many clinical trials aiming for hearing improvement or tumor volume control. Currently, a randomized, double-masked trial to assess bevacizumab is underway. In this randomized control trial, 12 other Japanese institutions joined the principal investigators in the clinical trial originating at Fukushima Medical University. In this review, we will be discussing the latest research developments regarding NF2 pathophysiology, including molecular biology, diagnosis, and novel therapeutics.

The Hippo pathway plays a critical role in controlled cell proliferation. The tumor suppressor Merlin and large tumor suppressor kinase 1 (LATS1) mediate activation of Hippo pathway, consequently inhibiting the primary effectors, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Phosphatidylinositol 4,5-bisphosphate (PIP2), a lipid present in the plasma membrane (PM), binds to and activates Merlin. Phosphatidylinositol 4-phosphate 5-kinase α (PIP5Kα) is an enzyme responsible for PIP2 production. However, the functional role of PIP5Kα in regulation of Merlin and LATS1 under Hippo signaling conditions remains unclear.
PIP5Kα, Merlin, or LATS1 knockout or knockdown cells and transfected cells with them were used. LATS1, YAP, and TAZ activities were measured using biochemical methods and PIP2 levels were evaluated using cell imaging. Low/high cell density and serum starvation/stimulation conditions were tested. Colocalization of PIP5Kα and PIP2 with Merlin and LATS1, and their protein interactions were examined using transfection, confocal imaging, immunoprecipitation, western blotting, and/or pull-down experiments. Colony formation and adipocyte differentiation assays were performed.
We found that PIP5Kα induced LATS1 activation and YAP/TAZ inhibition in a kinase activity-dependent manner. Consistent with these findings, PIP5Kα suppressed cell proliferation and enhanced adipocyte differentiation of mesenchymal stem cells. Moreover, PIP5Kα protein stability and PIP2 levels were elevated at high cell density compared with those at low cell density, and both PIP2 and YAP phosphorylation levels initially declined, then recovered upon serum stimulation. Under these conditions, YAP/TAZ activity was aberrantly regulated by PIP5Kα deficiency. Mechanistically, either Merlin deficiency or LATS1 deficiency abrogated PIP5Kα-mediated YAP/TAZ inactivation. Additionally, the catalytic domain of PIP5Kα directly interacted with the band 4.1/ezrin/radixin/moesin domain of Merlin, and this interaction reinforced interaction of Merlin with LATS1. In accordance with these findings, PIP5Kα and PIP2 colocalized with Merlin and LATS1 in the PM. In PIP5Kα-deficient cells, Merlin colocalization with PIP2 was reduced, and LATS1 solubility increased.
Collectively, our results support that PIP5Kα serves as an activator of the Hippo pathway through interaction and colocalization with Merlin, which promotes PIP2-dependent Merlin activation and induces local recruitment of LATS1 to the PIP2-rich PM and its activation, thereby negatively regulating YAP/TAZ activity. Video Abstract.

Neurofibromatosis type 2 (NF2) is a genetic condition marked by the development of multiple benign tumors in the nervous system. The most common tumors associated with NF2 are bilateral vestibular schwannoma, meningioma, and ependymoma. The clinical manifestations of NF2 depend on the site of involvement. Vestibular schwannoma can present with hearing loss, dizziness, and tinnitus, while spinal tumor leads to debilitating pain, muscle weakness, or paresthesias. Clinical diagnosis of NF2 is based on the Manchester criteria, which have been updated in the last decade. NF2 is caused by loss-of-function mutations in the NF2 gene on chromosome 22, leading the merlin protein to malfunction. Over half of NF2 patients have de novo mutations, and half of this group are mosaic. NF2 can be managed by surgery, stereotactic radiosurgery, monoclonal antibody bevacizumab, and close observation. However, the nature of multiple tumors and the necessity of multiple surgeries over the lifetime, inoperable tumors like meningiomatosis with infiltration of the sinus or in the area of the lower cranial nerves, the complications caused by the operation, the malignancies induced by radiotherapy, and inefficiency of cytotoxic chemotherapy due to the benign nature of NF-related tumors have led a march toward exploring targeted therapies. Recent advances in genetics and molecular biology have allowed identifying and targeting of underlying pathways in the pathogenesis of NF2. In this review, we explain the clinicopathological characteristics of NF2, its genetic and molecular background, and the current knowledge and challenges of implementing genetics to develop efficient therapies.

Primary liver cancer is the third leading cause of cancer-related death worldwide. An increasing body of evidence suggests that the Hippo tumor suppressor pathway plays a critical role in restricting cell proliferation and determining cell fate during physiological and pathological processes in the liver. Merlin (Moesin-Ezrin-Radixin-like protein) encoded by the NF2 (neurofibromatosis type 2) gene is an upstream regulator of the Hippo signaling pathway. Targeting of Merlin to the plasma membrane seems to be crucial for its major tumor-suppressive functions; this is facilitated by interactions with membrane-associated proteins, including CD44 (cluster of differentiation 44). Mutations within the CD44-binding domain of Merlin have been reported in many human cancers. This study evaluated the relative contribution of CD44- and Merlin-dependent processes to the development and progression of liver tumors. To this end, mice with a liver-specific deletion of the Nf2 gene were crossed with Cd44-knockout mice and subjected to extensive histological, biochemical and molecular analyses. In addition, cells were isolated from mutant livers and analyzed by in vitro assays. Deletion of Nf2 in the liver led to substantial liver enlargement and generation of hepatocellular carcinomas (HCCs), intrahepatic cholangiocarcinomas (iCCAs), as well as mixed hepatocellular cholangiocarcinomas. Whilst deletion of Cd44 had no influence on liver size or primary liver tumor development, it significantly inhibited metastasis formation in Nf2-mutant mice. CD44 upregulates expression of integrin β2 and promotes transendothelial migration of liver cancer cells, which may facilitate metastatic spreading. Overall, our results suggest that CD44 may be a promising target for intervening with metastatic spreading of liver cancer.