Brigatinib causes tumor shrinkage in both NF2-deficient meningioma and schwannoma through inhibition of multiple tyrosine kinases but not ALK.

Neurofibromatosis Type 2 (NF2) is an autosomal dominant genetic syndrome caused by mutations in the NF2 tumor suppressor gene resulting in multiple schwannomas and meningiomas. There are no FDA approved therapies for these tumors and their relentless progression results in high rates of morbidity and mortality. Through a combination of high throughput screens, preclinical in vivo modeling, and evaluation of the kinome en masse, we identified actionable drug targets and efficacious experimental therapeutics for the treatment of NF2 related schwannomas and meningiomas. These efforts identified brigatinib (ALUNBRIG®), an FDA-approved inhibitor of multiple tyrosine kinases including ALK, to be a potent inhibitor of tumor growth in established NF2 deficient xenograft meningiomas and a genetically engineered murine model of spontaneous NF2 schwannomas. Surprisingly, neither meningioma nor schwannoma cells express ALK. Instead, we demonstrate that brigatinib inhibited multiple tyrosine kinases, including EphA2, Fer and focal adhesion kinase 1 (FAK1). These data demonstrate the power of the de novo unbiased approach for drug discovery and represents a major step forward in the advancement of therapeutics for the treatment of NF2 related malignancies.

Fluorescence in situ hybridization detection of chromosome 22 monosomy in pleural effusion cytology for the diagnosis of mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is characterized by mutations in several genes, including cyclin-dependent kinase-inhibitor 2A/p16 in the 9p21 locus, BRCA1-associated protein 1 (BAP1), and neurofibromatosis type 2 (NF2) in the 22q12 locus. Recent studies indicate that fluorescence in situ hybridization (FISH) detects hemizygous loss of NF2 in tissue specimens of MPM. The authors investigated whether NF2 FISH, either alone or in combination with other diagnostic assays (9p21 FISH, methylthioadenosine phosphorylase [MTAP] immunohistochemistry [IHC], and BAP1 IHC), effectively distinguishes MPM cells from reactive mesothelial cells (RMCs) in cell blocks prepared from pleural effusions. METHODS: FISH assays were used to examine the deletion status of NF2 and 9p21, and IHC was used to determine the expression of MTAP and BAP1 in cell blocks from 54 cases with MPM and 18 cases with RMCs. RESULTS: Hemizygous NF2 loss (chromosome 22 monosomy or hemizygous deletion) showed 51.9% sensitivity (48.1% for chromosome 22 monosomy and 3.7% for hemizygous deletion) and 100% specificity in differentiating MPM cells from RMCs. Combinations of NF2 FISH, 9p21 FISH, and BAP1 IHC assays yielded greater sensitivity (98.1%) than any assay alone (9p21 FISH, 61.1%; MTAP IHC, 52.8%; or BAP1 IHC, 60.4%). The level of hemizygous NF2 loss in cell blocks positively correlated with that in corresponding tissues. Furthermore, to overcome cytologic specimen-specific challenges, FISH combined with cytokeratin AE1/AE3 immunofluorescence was necessary in 25.9% of MPM cases for FISH assessment of predominantly scattered MPM cells. CONCLUSIONS: NF2 FISH alone or in combination with other diagnostic assays effectively differentiates MPM cells from RMCs in cell blocks prepared from pleural effusions.

mTOR kinase inhibition disrupts neuregulin 1-ERBB3 autocrine signaling and sensitizes NF2-deficient meningioma cellular models to IGF1R inhibition.

Meningiomas (MNs), arising from the arachnoid/meningeal layer, are nonresponsive to chemotherapies, with ∼50% showing loss of the Neurofibromatosis 2 (NF2) tumor suppressor gene. Previously, we established NF2 loss activates mechanistic target of rapamycin complex 1 (mTORC1) and mechanistic target of rapamycin complex 2 (mTORC2) signaling, leading to clinical trials for NF2 and MN. Recently our omics studies identified activated ephrin (EPH) receptor and Src family kinases upon NF2 loss. Here, we report increased expression of several ligands in NF2-null human arachnoidal cells (ACs) and the MN cell line Ben-Men-1, particularly neuregulin-1/heregulin (NRG1), and confirm increased NRG1 secretion and activation of V-ERB-B avian erythroblastic leukemia viral oncogene homolog 3 (ERBB3) receptor kinase. Conditioned-medium from NF2-null ACs or exogenous NRG1 stimulated ERBB3, EPHA2, and mTORC1/2 signaling, suggesting pathway crosstalk. NF2-null cells treated with an ERBB3-neutralizing antibody partially downregulated mTOR pathway activation but showed no effect on viability. mTORC1/2 inhibitor treatment decreased NRG1 expression and downregulated ERBB3 while re-activating pAkt T308, suggesting a mechanism independent of NRG1-ERBB3 but likely involving activation of another upstream receptor kinase. Transcriptomics after mTORC1/2 inhibition confirmed decreased ERBB3/ERBB4 while revealing increased expression of insulin-like growth factor receptor 1 (IGF1R). Drug treatment co-targeting mTORC1/2 and IGF1R/insulin receptor attenuated pAkt T308 and showed synergistic effects on viability. Our findings indicate potential autocrine signaling where NF2 loss leads to secretion/activation of NRG1-ERBB3 signaling. mTORC1/2 inhibition downregulates NRG1-ERBB3, while upregulating pAkt T308 through an adaptive response involving IGF1R/insulin receptor and co-targeting these pathways may prove effective for treatment of NF2-deficient MN.

NF2 deficiency accelerates neointima hyperplasia following vascular injury via promoting YAP-TEAD1 interaction in vascular smooth muscle cells.

Neurofibromin 2 (NF2), a potent tumor suppressor, is reported to inhibit proliferation in several cell types. The role of NF2 in neointima hyperplasia after vascular injury is unknown. We explored the role of NF2 in proliferation, migration of vascular smooth muscle cell (VSMC) and neointima hyperplasia after vascular injury. NF2 phosphorylation was elevated in VSMC subjected to platelet-derived growth factor (PDGF)-BB and in artery subjected to vascular injury. Mice deficient for Nf2 in VSMC showed enhanced neointima hyperplasia after injury, increased proliferation and migration of VSMC after PDGF-BB treatment. Mechanistically, we observed increased nuclear p-NF2, declined p-Yes-Associated Protein (YAP), nuclear translocation of YAP after PDGF-BB treatment or injury. NF2 knockdown or YAP overexpression showed similar phenotype in VSMC proliferation, migration and neointima hyperplasia. YAP inhibition abolished the above effects mediated by NF2 knockdown. Finally, NF2 knockdown further promoted YAP-TEA Domain Transcription Factor 1 (TEAD1) interaction after PDGF-BB treatment. Inhibition of TEAD1 blocked PDGF-BB-induced VSMC proliferation and migration, which were not reversed by either NF2 knockdown or YAP overexpression. In conclusion, NF2 knockdown promotes VSMC proliferation, migration and neointima hyperplasia after vascular injury via inducing YAP-TEAD1 interaction.

Merlin regulates signaling events at the nexus of development and cancer.

BACKGROUND: In this review, we describe how the cytoskeletal protein Merlin, encoded by the Neurofibromin 2 (NF2) gene, orchestrates developmental signaling to ensure normal ontogeny, and we discuss how Merlin deficiency leads to aberrant activation of developmental pathways that enable tumor development and malignant progression. MAIN BODY: Parallels between embryonic development and cancer have underscored the activation of developmental signaling pathways. Hippo, WNT/ß-catenin, TGF-ß, receptor tyrosine kinase (RTK), Notch, and Hedgehog pathways are key players in normal developmental biology. Unrestrained activity or loss of activity of these pathways causes adverse effects in developing tissues manifesting as developmental syndromes. Interestingly, these detrimental events also impact differentiated and functional tissues. By promoting cell proliferation, migration, and stem-cell like phenotypes, deregulated activity of these pathways promotes carcinogenesis and cancer progression. The NF2 gene product, Merlin, is a tumor suppressor classically known for its ability to induce contact-dependent growth inhibition. Merlin plays a role in different stages of an organism development, ranging from embryonic to mature states. While homozygous deletion of Nf2 in murine embryos causes embryonic lethality, Merlin loss in adult tissue is implicated in Neurofibromatosis type 2 disorder and cancer. These manifestations, cumulatively, are reminiscent of dysregulated developmental signaling. CONCLUSION: Understanding the molecular and cellular repercussions of Merlin loss provides fundamental insights into the etiology of developmental disorders and cancer and has the potential, in the long term, to identify new therapeutic strategies. Video Abstract.

LATS suppresses mTORC1 activity to directly coordinate Hippo and mTORC1 pathways in growth control.

The Hippo and mammalian target of rapamycin complex 1 (mTORC1) pathways are the two predominant growth-control pathways that dictate proper organ development. We therefore explored potential crosstalk between these two functionally relevant pathways to coordinate their growth-control functions. We found that the LATS1 and LATS2 kinases, the core components of the Hippo pathway, phosphorylate S606 of Raptor, an essential component of mTORC1, to attenuate mTORC1 activation by impairing the interaction of Raptor with Rheb. The phosphomimetic Raptor-S606D knock-in mutant led to a reduction in cell size and proliferation. Compared with Raptor+/+ mice, RaptorD/D knock-in mice exhibited smaller livers and hearts, and a significant inhibition of elevation in mTORC1 signalling induced by Nf2 or Lats1 and Lats2 loss. Thus, our study reveals a direct link between the Hippo and mTORC1 pathways to fine-tune organ growth.

Loss of PTPRJ/DEP-1 enhances NF2/Merlin-dependent meningioma development.

INTRODUCTION: Meningiomas are common tumors in adults, which develop from the meningeal coverings of the brain and spinal cord. Loss-of-function mutations or deletion of the NF2 gene, resulting in loss of the encoded Merlin protein, lead to Neurofibromatosis type 2 (NF2), but also cause the formation of sporadic meningiomas. It was shown that inactivation of Nf2 in mice caused meningioma formation. Another meningioma tumor-suppressor candidate is the receptor-like density-enhanced phosphatase-1 (DEP-1), encoded by PTPRJ. Loss of DEP-1 enhances meningioma cell motility in vitro and invasive growth in an orthotopic xenograft model. Ptprj-deficient mice develop normally and do not show spontaneous tumorigenesis. Another genetic lesion may be required to interact with DEP-1 loss in meningioma genesis. METHODS: In the present study we investigated in vitro and in vivo whether the losses of DEP-1 and Merlin/NF2 may have a combined effect. RESULTS: Human meningioma cells deficient for DEP-1, Merlin/NF2 or both showed no statistically significant changes in cell proliferation, while DEP-1 or DEP1/NF2 deficiency led to moderately increased colony size in clonogenicity assays. In addition, the loss of any of the two genes was sufficient to induce a significant reduction of cell size (p < .05) and profound morphological changes. Most important, in Ptprj knockout mice Cre/lox mediated meningeal Nf2 knockout elicited a four-fold increased rate of meningioma formation within one year compared with mice with Ptprj wild type alleles (25% vs 6% tumor incidence). CONCLUSIONS: Our data suggest that loss of DEP-1 and Merlin/NF2 synergize during meningioma genesis.

YAP/TAZ Inhibition Induces Metabolic and Signaling Rewiring Resulting in Targetable Vulnerabilities in NF2-Deficient Tumor Cells.

Merlin/NF2 is a bona fide tumor suppressor whose mutations underlie inherited tumor syndrome neurofibromatosis type 2 (NF2), as well as various sporadic cancers including kidney cancer. Multiple Merlin/NF2 effector pathways including the Hippo-YAP/TAZ pathway have been identified. However, the molecular mechanisms underpinning the growth and survival of NF2-mutant tumors remain poorly understood. Using an inducible orthotopic kidney tumor model, we demonstrate that YAP/TAZ silencing is sufficient to induce regression of pre-established NF2-deficient tumors. Mechanistically, YAP/TAZ depletion diminishes glycolysis-dependent growth and increases mitochondrial respiration and reactive oxygen species (ROS) buildup, resulting in oxidative-stress-induced cell death when challenged by nutrient stress. Furthermore, we identify lysosome-mediated cAMP-PKA/EPAC-dependent activation of RAF-MEK-ERK signaling as a resistance mechanism to YAP/TAZ inhibition. Finally, unbiased analysis of TCGA primary kidney tumor transcriptomes confirms a positive correlation of a YAP/TAZ signature with glycolysis and inverse correlations with oxidative phosphorylation and lysosomal gene expression, supporting the clinical relevance of our findings.

A cerebellopontine angle mouse model for the investigation of tumor biology, hearing, and neurological function in NF2-related vestibular schwannoma.

Neurofibromatosis type II (NF2) is a disease that lacks effective therapies. NF2 is characterized by bilateral vestibular schwannomas (VSs) that cause progressive and debilitating hearing loss, leading to social isolation and increased rates of depression. A major limitation in NF2 basic and translational research is the lack of animal models that allow the full spectrum of research into the biology and molecular mechanisms of NF2 tumor progression, as well as the effects on neurological function. In this protocol, we describe how to inject schwannoma cells into the mouse brain cerebellopontine angle (CPA) region. We also describe how to apply state-of-the-art intravital imaging and hearing assessment techniques to study tumor growth and hearing loss. In addition, ataxia, angiogenesis, and tumor-stroma interaction assays can be applied, and the model can be used to test the efficacy of novel therapeutic approaches. By studying the disease from every angle, this model offers the potential to unravel the basic biological underpinnings of NF2 and to develop novel therapeutics to control this devastating disease. Our protocol can be adapted to study other diseases within the CPA, including meningiomas, lipomas, vascular malformations, hemangiomas, epidermoid cysts, cerebellar astrocytomas, and metastatic lesions. The entire surgical procedure takes ~45 min per mouse and allows for subsequent longitudinal imaging, as well as neurological and hearing assessment, for up to 2 months.

Loss of NF2 Induces TGFß Receptor 1-mediated Noncanonical and Oncogenic TGFß Signaling: Implication of the Therapeutic Effect of TGFß Receptor 1 Inhibitor on NF2 Syndrome.

Neurofibromatosis type 2 (NF2) syndrome is a very rare human genetic disease, and there has been no proper treatment for it until now. In our recent study, it has been reported that the loss of NF2 activates MAPK signaling through reduction of RKIP in a mesothelioma model. Here, we show that loss of NF2 induces reduction of the TGFß receptor 2 (TßR2) expression, and an overwhelming expression of TGFß receptor 1 (TßR1) is activated by physical stimuli such as pressure or heavy materials. Activated TßR1 induces the phosphorylation and degradation of RKIP. RKIP reduction consequently results in MAPK activation as well as Snail-mediated p53 suppression and occurrence of EMT in NF2-deficient cells by physical stimuli. Thus, TßR1 kinase inhibitors restore cell differentiation and induce growth suppression in NF2-deficient Schwannoma cell line and MEF. Moreover, TEW7197, a specific TßR1 kinase inhibitor, reduces tumor formation in the NF2-model mouse (Postn-Cre;NF2f/f). Gene expression profiling reveals that TEW7197 treatment induces the expression of lipid metabolism-related gene set, such as NF2-restored cells in HEI-193 (NF2-deficient Schwannoma). Our results indicate that reduction or deletion of TßR2 or NF2 induces the TßR1-mediated oncogenic pathway, and therefore inhibition of the unbalanced TGFß signaling is a putative strategy for NF2-related cancers (NF2 syndrome and mesothelioma) and TßR2-mutated advanced cancers. Mol Cancer Ther; 17(11); 2271-84. ©2018 AACR.

The ERM family member Merlin is required for endometrial gland morphogenesis.

Disruption of endometrial gland formation or function can cause female infertility. Formation of endometrial glands via tubulogenesis of luminal epithelial cells requires the establishment and maintenance of cell polarity and cell adhesion. The FERM domain-containing protein Merlin coordinates epithelial cell polarity and cell adhesion and is critical for epithelial tissue function in the skin and kidney. We now demonstrate a requirement for Merlin in endometrial gland development. Conditional deletion of Merlin in the endometrium results in female infertility caused by the absence of gland formation. Interestingly, we observed glandular epithelial markers within discrete groups of cells in the Merlin-deficient luminal epithelium. Wnt signaling, a pathway necessary for endometrial gland development is maintained in Merlin-deficient endometrium, suggesting the glandular fate program is active. Instead, we observe increased levels of apical actin and markers indicative of high membrane tension on the basal surface of the Merlin-deficient luminal epithelium. These findings suggest that the structural integrity of the luminal epithelium during gland formation is required for appropriate endometrial tubulogenesis and tissue function. Moreover, our work implicates Merlin-dependent regulation of mechanical tension in the proper formation of endometrial gland architecture and function.

EPH receptor signaling as a novel therapeutic target in NF2-deficient meningioma.

Background: Meningiomas are the most common primary brain tumor in adults, and somatic loss of the neurofibromatosis 2 (NF2) tumor suppressor gene is a frequent genetic event. There is no effective treatment for tumors that recur or continue to grow despite surgery and/or radiation. Therefore, targeted therapies that either delay tumor progression or cause tumor shrinkage are much needed. Our earlier work established mammalian target of rapamycin complex mTORC1/mTORC2 activation in NF2-deficient meningiomas. Methods: High-throughput kinome analyses were performed in NF2-null human arachnoidal and meningioma cell lines to identify functional kinome changes upon NF2 loss. Immunoblotting confirmed the activation of kinases and demonstrated effectiveness of drugs to block the activation. Drugs, singly and in combination, were screened in cells for their growth inhibitory activity. Antitumor drug efficacy was tested in an orthotopic meningioma model. Results: Erythropoietin-producing hepatocellular receptor tyrosine kinases (EPH RTKs), c-KIT, and Src family kinase (SFK) members, which are biological targets of dasatinib, were among the top candidates activated in NF2-null cells. Dasatinib significantly inhibited phospho-EPH receptor A2 (pEPHA2), pEPHB1, c-KIT, and Src/SFK in NF2-null cells, showing no cross-talk with mTORC1/2 signaling. Posttreatment kinome analyses showed minimal adaptive changes. While dasatinib treatment showed some activity, dual mTORC1/2 inhibitor and its combination with dasatinib elicited stronger growth inhibition in meningiomas. Conclusion: Co-targeting mTORC1/2 and EPH RTK/SFK pathways could be a novel effective treatment strategy for NF2-deficient meningiomas.

Deficiency of tumor suppressor Merlin facilitates metabolic adaptation by co-operative engagement of SMAD-Hippo signaling in breast cancer.

The NF2 gene encodes the tumor and metastasis suppressor protein Merlin. Merlin exerts its tumor suppressive role by inhibiting proliferation and inducing contact-growth inhibition and apoptosis. In the current investigation, we determined that loss of Merlin in breast cancer tissues is concordant with the loss of the inhibitory SMAD, SMAD7, of the TGF-ß pathway. This was reflected as dysregulated activation of TGF-ß signaling that co-operatively engaged with effectors of the Hippo pathway (YAP/TAZ/TEAD). As a consequence, the loss of Merlin in breast cancer resulted in a significant metabolic and bioenergetic adaptation of cells characterized by increased aerobic glycolysis and decreased oxygen consumption. Mechanistically, we determined that the co-operative activity of the Hippo and TGF-ß transcription effectors caused upregulation of the long non-coding RNA Urothelial Cancer-Associated 1 (UCA1) that disengaged Merlin's check on STAT3 activity. The consequent upregulation of Hexokinase 2 (HK2) enabled a metabolic shift towards aerobic glycolysis. In fact, Merlin deficiency engendered cellular dependence on this metabolic adaptation, endorsing a critical role for Merlin in regulating cellular metabolism. This is the first report of Merlin functioning as a molecular restraint on cellular metabolism. Thus, breast cancer patients whose tumors demonstrate concordant loss of Merlin and SMAD7 may benefit from an approach of incorporating STAT3 inhibitors.

A Xenograft Model of Vestibular Schwannoma and Hearing Loss.

HYPOTHESIS: Microsurgical implantation of mouse merlin-deficient Schwann cells (MD-SC) into the cerebellopontine angle of immunodeficient rats will initiate tumor formation, hearing loss, and vestibular dysfunction. BACKGROUND: The progress in identifying effective drug therapies for treatment of Neurofibromatosis type II (NF2) is limited by the availability of animal models of VS that develop hearing loss and imbalance. METHODS: A microsurgical technique for implanting MD-SCs onto the cochleovestibular nerve of rats was developed. Ten Rowett Nude rats were implanted with either ∼10 MD-SCs expressing luciferase (N = 5) or vehicle (N = 5). Rats received bioluminescence imaging, auditory brainstem response testing, and were observed for head tilt every 2 weeks after surgery, for a total of 6 weeks. Tumors were harvested and processed with hematoxylin & eosin staining and immunohistochemistry was performed for S100. RESULTS: Rats implanted with MD-SCs developed significantly higher tumor bioluminescence measurements and hearing threshold shifts at multiple frequencies by the 4th and 6th weeks post-implantation, compared with control rats. Rats implanted with MD-SCs also developed gross tumor. The tumor volume was significantly greater than nerve volumes obtained from rats in the control group. All rats with tumors developed a head tilt, while control rats had no signs of vestibular dysfunction. Tumors demonstrated histological features of schwannoma and express S100. CONCLUSION: Using this microsurgical technique, this xenograft rat model of VS develops tumors involving the cochleovestibular nerve, shifts in hearing thresholds, and vestibular dysfunction. This animal model can be used to investigate tumor-mediated hearing loss and perform preclinical drug studies for NF2.

Generation and Use of Merlin-Deficient Human Schwann Cells for a High-Throughput Chemical Genomics Screening Assay.

Schwannomas are benign nerve tumors that occur sporadically in the general population and in those with neurofibromatosis type 2 (NF2), a tumor predisposition genetic disorder. NF2-associated schwannomas and most sporadic schwannomas are caused by inactivating mutations in Schwann cells in the neurofibromatosis type 2 gene (NF2) that encodes the merlin tumor suppressor. Despite their benign nature, schwannomas and especially vestibular schwannomas cause considerable morbidity. The primary available therapies are surgery or radiosurgery which usually lead to loss of function of the compromised nerve. Thus, there is a need for effective chemotherapies. We established an untransformed merlin-deficient human Schwann cell line for use in drug discovery studies for NF2-associated schwannomas. We describe the generation of human Schwann cells (HSCs) with depletion of merlin and their application in high-throughput screening of chemical libraries to identify compounds that decrease their viability. This NF2-HSC model is amenable for use in independent labs and high-throughput screening (HTS) facilities.

E-cadherin expression is correlated with focal adhesion kinase inhibitor resistance in Merlin-negative malignant mesothelioma cells.

Malignant mesothelioma (MM) is an aggressive tumor commonly caused by asbestos exposure after a long latency. Focal adhesion kinase (FAK) inhibitors inhibit the cell growth of Merlin-deficient MM cells; however, their clinical efficacy has not been clearly determined. The aim of this study was to evaluate the growth inhibitory effect of the FAK inhibitor VS-4718 on MM cell lines and identify biomarkers for its efficacy. Although most Merlin-deficient cell lines were sensitive to VS-4718 compared with control MeT-5A cells, a subset of these cell lines exhibited resistance to this drug. Microarray and qRT-PCR analyses using RNA isolated from Merlin-deficient MM cell lines revealed a significant correlation between E-cadherin mRNA levels and VS-4718 resistance. Merlin- and E-cadherin-negative Y-MESO-22 cells underwent apoptosis upon treatment with a low concentration of VS-4718, whereas Merlin-negative, E-cadherin-positive Y-MESO-9 cells did not undergo VS-4718-induced apoptosis. Furthermore, E-cadherin knockdown in Merlin-negative MM cells significantly sensitized cells to VS-4718 and induced apoptotic cell death upon VS-4718 treatment. Together, our results suggest that E-cadherin serves as a predictive biomarker for molecular target therapy with FAK inhibitors for patients with mesothelioma and that its expression endows MM cells with resistance to FAK inhibitors.

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

Neurofibromatosis type 2 (NF2) is a genetic syndrome that predisposes individuals to multiple benign tumors of the central and peripheral nervous systems, including vestibular schwannomas. Currently, there are no FDA approved drug therapies for NF2. Loss of function of merlin encoded by the NF2 tumor suppressor gene leads to activation of multiple mitogenic signaling cascades, including platelet-derived growth factor receptor (PDGFR) and SRC in Schwann cells. The goal of this study was to determine whether ponatinib, an FDA-approved ABL/SRC inhibitor, reduced proliferation and/or survival of merlin-deficient human Schwann cells (HSC). Merlin-deficient HSC had higher levels of phosphorylated PDGFRα/ß, and SRC than merlin-expressing HSC. A similar phosphorylation pattern was observed in phospho-protein arrays of human vestibular schwannoma samples compared to normal HSC. Ponatinib reduced merlin-deficient HSC viability in a dose-dependent manner by decreasing phosphorylation of PDGFRα/ß, AKT, p70S6K, MEK1/2, ERK1/2 and STAT3. These changes were associated with decreased cyclin D1 and increased p27Kip1levels, leading to a G1 cell-cycle arrest as assessed by Western blotting and flow cytometry. Ponatinib did not modulate ABL, SRC, focal adhesion kinase (FAK), or paxillin phosphorylation levels. These results suggest that ponatinib is a potential therapeutic agent for NF2-associated schwannomas and warrants further in vivo investigation.

Merlin controls the repair capacity of Schwann cells after injury by regulating Hippo/YAP activity.

Loss of the Merlin tumor suppressor and activation of the Hippo signaling pathway play major roles in the control of cell proliferation and tumorigenesis. We have identified completely novel roles for Merlin and the Hippo pathway effector Yes-associated protein (YAP) in the control of Schwann cell (SC) plasticity and peripheral nerve repair after injury. Injury to the peripheral nervous system (PNS) causes a dramatic shift in SC molecular phenotype and the generation of repair-competent SCs, which direct functional repair. We find that loss of Merlin in these cells causes a catastrophic failure of axonal regeneration and remyelination in the PNS. This effect is mediated by activation of YAP expression in Merlin-null SCs, and loss of YAP restores axonal regrowth and functional repair. This work identifies new mechanisms that control the regenerative potential of SCs and gives new insight into understanding the correct control of functional nerve repair in the PNS.

Global Proteome and Phospho-proteome Analysis of Merlin-deficient Meningioma and Schwannoma Identifies PDLIM2 as a Novel Therapeutic Target.

Loss or mutation of the tumour suppressor Merlin predisposes individuals to develop multiple nervous system tumours, including schwannomas and meningiomas, sporadically or as part of the autosomal dominant inherited condition Neurofibromatosis 2 (NF2). These tumours display largely low grade features but their presence can lead to significant morbidity. Surgery and radiotherapy remain the only treatment options despite years of research, therefore an effective therapeutic is required. Unbiased omics studies have become pivotal in the identification of differentially expressed genes and proteins that may act as drug targets or biomarkers. Here we analysed the proteome and phospho-proteome of these genetically defined tumours using primary human tumour cells to identify upregulated/activated proteins and/or pathways. We identified over 2000 proteins in comparative experiments between Merlin-deficient schwannoma and meningioma compared to human Schwann and meningeal cells respectively. Using functional enrichment analysis we highlighted several dysregulated pathways and Gene Ontology terms. We identified several proteins and phospho-proteins that are more highly expressed in tumours compared to controls. Among proteins jointly dysregulated in both tumours we focused in particular on PDZ and LIM domain protein 2 (PDLIM2) and validated its overexpression in several tumour samples, while not detecting it in normal cells. We showed that shRNA mediated knockdown of PDLIM2 in both primary meningioma and schwannoma leads to significant reductions in cellular proliferation. To our knowledge, this is the first comprehensive assessment of the NF2-related meningioma and schwannoma proteome and phospho-proteome. Taken together, our data highlight several commonly deregulated factors, and indicate that PDLIM2 may represent a novel, common target for meningioma and schwannoma.

Generation of Noninvasive, Quantifiable, Orthotopic Animal Models for NF2-Associated Schwannoma and Meningioma.

Schwannomas and meningiomas are nervous system tumors that can occur sporadically or in patients with neurofibromatosis type 2 (NF2). Mutations of the Neurofibromatosis 2 (NF2) gene are frequently observed in these tumors. Schwannomas and meningiomas cause significant morbidities, and an FDA-approved medical therapy is currently not available. The development of preclinical animal models that accurately capture the clinical characteristics of these tumors will facilitate the evaluation of novel therapeutic agents for the treatment of these tumors, ultimately leading to more productive clinical trials. Here, we describe the generation of luciferase-expressing NF2-deficient schwannoma and meningioma cells and the use of these cells to establish orthotopic tumor models in immunodeficient mice. The growth of these tumors and their response to treatment can be measured effectively by bioluminescence imaging (BLI) and confirmed by small-animal magnetic resonance imaging (MRI). These and other animal models, such as genetically-engineered models, should substantially advance the investigation of promising therapies for schwannomas and meningiomas.