Merlin (Moesin-ezrin-radixin-like protein, also known as schwannomin) is a tumor suppressor protein encoded by the neurofibromatosis type 2 gene NF2. Loss of function mutations or deletions in NF2 cause neurofibromatosis type 2 (NF2), a multiple tumor forming disease of the nervous system. NF2 is characterized by the development of bilateral vestibular schwannomas. Patients with NF2 can also develop schwannomas on other cranial and peripheral nerves, as well as meningiomas and ependymomas. The only potential treatment is surgery/radiosurgery, which often results in loss of function of the involved nerve. There is an urgent need for chemotherapies that slow or eliminate tumors and prevent their formation in NF2 patients. Interestingly NF2 mutations and merlin inactivation also occur in spontaneous schwannomas and meningiomas, as well as other types of cancer including mesothelioma, glioma multiforme, breast, colorectal, skin, clear cell renal cell carcinoma, hepatic and prostate cancer. Except for malignant mesotheliomas, the role of NF2 mutation or inactivation has not received much attention in cancer, and NF2 might be relevant for prognosis and future chemotherapeutic approaches. This review discusses the influence of merlin loss of function in NF2-related tumors and common human cancers. We also discuss the NF2 gene status and merlin signaling pathways affected in the different tumor types and the molecular mechanisms that lead to tumorigenesis, progression and pharmacological resistance.

Neurofibromatosis type 2 (NF2) is caused by mutations in the NF2 gene that encodes a tumor-suppressor protein called merlin. NF2 is characterized by formation of multiple schwannomas, meningiomas and ependymomas. Merlin loss-of-function is associated with increased activity of Rac and p21-activated kinases (PAKs) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrate for Cdc42/Rac-PAK and modulate actin dynamics by phosphorylating cofilin at serine-3. This modification inactivates the actin severing and depolymerizing activity of cofilin. LIMKs also translocate into the nucleus and regulate cell cycle progression. Significantly, LIMKs are overexpressed in several tumor types, including skin, breast, lung, liver and prostate. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2^ΔEx2) exhibited increased levels of LIMK1, LIMK2 and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared with wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared with normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2^ΔEx2 MSC reduced LIMK1 and LIMK2 levels. We show that pharmacological inhibition of LIMK with BMS-5 decreased the viability of Nf2^ΔEx2 MSCs in a dose-dependent manner, but did not affect viability of control MSCs. Similarly, LIMK knockdown decreased viability of Nf2^ΔEx2 MSCs. The decreased viability of Nf2^ΔEx2 MSCs was not due to caspase-dependent or -independent apoptosis, but rather due to inhibition of cell cycle progression as evidenced by accumulation of cells in G₂/M phase. Inhibition of LIMKs arrests cells in early mitosis by decreasing aurora A activation. Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.

Myelination is a complex process requiring coordination of directional motility and an increase in glial cell size to generate a multilamellar myelin sheath. Regulation of actin dynamics during myelination is poorly understood. However, it is known that myelin thickness is related to the abundance of neuregulin-1 (NRG1) expressed on the axon surface. Here we identify cofilin1, an actin depolymerizing and severing protein, as a downstream target of NRG1 signaling in rat Schwann cells (SCs). In isolated SCs, NRG1 promotes dephosphorylation of cofilin1 and its upstream regulators, LIM kinase (LIMK) and Slingshot-1 phosphatase (SSH1), leading to cofilin1 activation and recruitment to the leading edge of the plasma membrane. These changes are associated with rapid membrane expansion yielding a 35–50% increase in SC size within 30 min. Cofilin1-deficient SCs increase phosphorylation of ErbB2, ERK, focal adhesion kinase, and paxillin in response to NRG1, but fail to increase in size possibly due to stabilization of unusually long focal adhesions. Cofilin1-deficient SCs cocultured with sensory neurons do not myelinate. Ultrastructural analysis reveals that they unsuccessfully segregate or engage axons and form only patchy basal lamina. After 48 h of coculturing with neurons, cofilin1-deficient SCs do not align or elongate on axons and often form adhesions with the underlying substrate. This study identifies cofilin1 and its upstream regulators, LIMK and SSH1, as end targets of a NRG1 signaling pathway and demonstrates that cofilin1 is necessary for dynamic changes in the cytoskeleton needed for axon engagement and myelination by SCs.

Neurofibromatosis type 2 (NF2) is a genetic tumor-predisposition disorder caused by NF2/merlin tumor suppressor gene inactivation. The hallmark of NF2 is formation of bilateral vestibular schwannomas (VS). Because merlin modulates activity of the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, we investigated repurposing drugs targeting MEK1 and/or MEK2 as a treatment for NF2-associated schwannomas. Mouse and human merlin-deficient Schwann cell lines (MD-MSC/HSC) were screened against 6 MEK1/2 inhibitors. Efficacious drugs were tested in orthotopic allograft and NF2 transgenic mouse models. Pathway and proteome analyses were conducted. Drug efficacy was examined in primary human VS cells with NF2 mutations and correlated with DNA methylation patterns. Trametinib, PD0325901, and cobimetinib were most effective in reducing MD-MSC/HSC viability. Each decreased phosphorylated pERK1/2 and cyclin D1, increased p27, and induced caspase-3 cleavage in MD-MSCs. Proteomic analysis confirmed cell cycle arrest and activation of pro-apoptotic pathways in trametinib-treated MD-MSCs. The 3 inhibitors slowed allograft growth; however, decreased pERK1/2, cyclin D1, and Ki-67 levels were observed only in PD0325901 and cobimetinib-treated grafts. Tumor burden and average tumor size were reduced in trametinib-treated NF2 transgenic mice; however, tumors did not exhibit reduced pERK1/2 levels. Trametinib and PD0325901 modestly reduced viability of several primary human VS cell cultures with NF2 mutations. DNA methylation analysis of PD0325901-resistant versus -susceptible VS identified genes that could contribute to drug resistance. MEK inhibitors exhibited differences in antitumor efficacy resistance in schwannoma models with possible emergence of trametinib resistance. The results support further investigation of MEK inhibitors in combination with other targeted drugs for NF2 schwannomas.

Neurofibromatosis type 2 (NF2) is a nervous system tumor disorder caused by inactivation of the merlin tumor suppressor encoded by the NF2 gene. Bilateral vestibular schwannomas are a diagnostic hallmark of NF2. Mainstream treatment options for NF2-associated tumors have been limited to surgery and radiotherapy; however, off-label uses of targeted molecular therapies are becoming increasingly common. Here, we investigated drugs targeting two kinases activated in NF2-associated schwannomas, c-Met and Src. We demonstrated that merlin-deficient mouse Schwann cells (MD-MSC) treated with the c-Met inhibitor, cabozantinib, or the Src kinase inhibitors, dasatinib and saracatinib, underwent a G1 cell-cycle arrest. However, when MD-MSCs were treated with a combination of cabozantinib and saracatinib, they exhibited caspase-dependent apoptosis. The combination therapy also significantly reduced growth of MD-MSCs in an orthotopic allograft mouse model by greater than 80% of vehicle. Moreover, human vestibular schwannoma cells with NF2 mutations had a 40% decrease in cell viability when treated with cabozantinib and saracatinib together compared with the vehicle control. This study demonstrates that simultaneous inhibition of c-Met and Src signaling in MD-MSCs triggers apoptosis and reveals vulnerable pathways that could be exploited to develop NF2 therapies. Mol Cancer Ther; 16(11); 2387–98. ©2017 AACR.

Ponatinib promotes a G 1 cell-cycle arrest of merlin/NF2-deficient human schwann cells Ponatinib promotes a G1 cell-cycle arrest of merlin/NF2-deficient human schwann cells