TSC2 loss in neural progenitor cells suppresses translation of ASD/NDD-associated transcripts in an mTORC1- and MNK1/2-reversible fashion.

Tuberous sclerosis complex (TSC), an inherited neurodevelopmental (ND) disorder with frequent manifestations of epilepsy and autism spectrum disorder (ASD). TSC is caused by mutations in TSC1 or TSC2 tumor suppressor genes, with encoded proteins hamartin/TSC1 and tuberin/TSC2 forming a functional complex inhibiting mechanistic target of rapamycin complex-1 (mTORC1) signaling, leading to FDA-approved allosteric mTORC1-selective rapamycin analogs for TSC tumors. Rapalogs are effective for TSC-associated hamartomas, however, they are not effective for treating ND manifestations. mTORC1 signaling plays an essential role in protein synthesis through mTORC1-eIF4F and MNK-eIF4E-mediated mRNA translation. Further, the effects on mRNA translation by specific mTORC1 and MNK inhibitors such as RMC-6272 and eFT-508 in TSC have never been explored. Here, employing CRISPR-modified, isogenic TSC2 patient-derived neural progenitor cells (NPCs), we have examined mRNA translation upon loss of TSC2 . Our results reveal dysregulated translation in TSC2 -Null NPCs, which significantly overlap with the translatome from TSC1 -Null NPCs, which we reported recently. Most notably, numerous non-monogenic ASD-NDD- and epilepsy-associated genes identified in patients harboring putative loss-of-function mutations, including protein truncating, or damaging missense variants, were translationally suppressed in TSC2 -Null NPCs, and their translation were reversed upon RMC-6272 or eFT-508 treatment. Our study here establishes the importance of mTORC1-eIF4F and MNK-eIF4E-mediated mRNA translation in TSC, ASD and other neurodevelopmental disorders and lay the groundwork for evaluating drugs in clinical development that target these pathways as a treatment strategy for TSC as well as ASD/NDD.

Evolving concepts in meningioma management in the era of genomics.

Meningioma is the most common type of primary brain tumor. Surgical resection followed by surveillance is the first-line treatment for the majority of symptomatic meningiomas; however, recent advances in molecular sequencing, DNA methylation, proteomics, and single-cell sequencing provide insights into further characterizing this heterogeneous group of tumors with a wide range of prognoses. A subset of these tumors are highly aggressive and cause severe morbidity and mortality. Therefore, identifying those individuals with a poor prognosis and intervening are critical. This review aims to help readers interpret the molecular profiling of meningiomas to identify patients with worse prognoses and guide the management and strategy for surveillance.

Erratum: Gene replacement therapy in a schwannoma mouse model of neurofibromatosis type 2.

[This corrects the article DOI: 10.1016/j.omtm.2022.06.012.].

Preclinical evaluation of the third-generation, bi-steric mechanistic target of rapamycin complex 1-selective inhibitor RMC-6272 in NF2-deficient models.

Background: NF2-associated meningiomas are progressive, highly morbid, and nonresponsive to chemotherapies, highlighting the need for improved treatments. We have established aberrant activation of the mechanistic target of rapamycin (mTOR) signaling in NF2-deficient tumors, leading to clinical trials with first- and second-generation mTOR inhibitors. However, results have been mixed, showing stabilized tumor growth without shrinkage offset by adverse side effects. To address these limitations, here we explored the potential of third-generation, bi-steric mTOR complex 1 (mTORC1) inhibitors using the preclinical tool compound RMC-6272. Methods: Employing human NF2-deficient meningioma lines, we compared mTOR inhibitors rapamycin (first-generation), INK128 (second-generation), and RMC-6272 (third-generation) using in vitro dose-response testing, cell-cycle analysis, and immunoblotting. Furthermore, the efficacy of RMC-6272 was assessed in NF2-null 3D-spheroid meningioma models, and its in vivo potential was evaluated in 2 orthotopic meningioma mouse models. Results: Treatment of meningioma cells revealed that, unlike rapamycin, RMC-6272 demonstrated superior growth inhibitory effects, cell-cycle arrest, and complete inhibition of phosphorylated 4E-BP1 (mTORC1 readout). Moreover, RMC-6272 had a longer retention time than INK128 and inhibited the expression of several eIF4E-sensitive targets on the protein level. RMC-6272 treatment of NF2 spheroids showed significant shrinkage in size as well as reduced proliferation. Furthermore, in vivo studies in mice revealed effective blockage of meningioma growth by RMC-6272, compared with vehicle controls. Conclusions: Our study in preclinical models of NF2 supports possible future clinical evaluation of third-generation, investigational mTORC1 inhibitors, such as RMC-5552, as a potential treatment strategy for NF2.

Translatome analysis of tuberous sclerosis complex 1 patient-derived neural progenitor cells reveals rapamycin-dependent and independent alterations.

BACKGROUND: Tuberous sclerosis complex (TSC) is an inherited neurocutaneous disorder caused by mutations in the TSC1 or TSC2 genes, with patients often exhibiting neurodevelopmental (ND) manifestations termed TSC-associated neuropsychiatric disorders (TAND) including autism spectrum disorder (ASD) and intellectual disability. Hamartin (TSC1) and tuberin (TSC2) proteins form a complex inhibiting mechanistic target of rapamycin complex 1 (mTORC1) signaling. Loss of TSC1 or TSC2 activates mTORC1 that, among several targets, controls protein synthesis by inhibiting translational repressor eIF4E-binding proteins. Using TSC1 patient-derived neural progenitor cells (NPCs), we recently reported early ND phenotypic changes, including increased cell proliferation and altered neurite outgrowth in TSC1-null NPCs, which were unaffected by the mTORC1 inhibitor rapamycin. METHODS: Here, we used polysome profiling, which quantifies changes in mRNA abundance and translational efficiencies at a transcriptome-wide level, to compare CRISPR-edited TSC1-null with CRISPR-corrected TSC1-WT NPCs generated from one TSC donor (one clone/genotype). To assess the relevance of identified gene expression alterations, we performed polysome profiling in postmortem brains from ASD donors and age-matched controls. We further compared effects on translation of a subset of transcripts and rescue of early ND phenotypes in NPCs following inhibition of mTORC1 using the allosteric inhibitor rapamycin versus a third-generation bi-steric, mTORC1-selective inhibitor RMC-6272. RESULTS: Polysome profiling of NPCs revealed numerous TSC1-associated alterations in mRNA translation that were largely recapitulated in human ASD brains. Moreover, although rapamycin treatment partially reversed the TSC1-associated alterations in mRNA translation, most genes related to neural activity/synaptic regulation or ASD were rapamycin-insensitive. In contrast, treatment with RMC-6272 inhibited rapamycin-insensitive translation and reversed TSC1-associated early ND phenotypes including proliferation and neurite outgrowth that were unaffected by rapamycin. CONCLUSIONS: Our work reveals ample mRNA translation alterations in TSC1 patient-derived NPCs that recapitulate mRNA translation in ASD brain samples. Further, suppression of TSC1-associated but rapamycin-insensitive translation and ND phenotypes by RMC-6272 unveils potential implications for more efficient targeting of mTORC1 as a superior treatment strategy for TAND.

Prospective phase II trial of the dual mTORC1/2 inhibitor vistusertib for progressive or symptomatic meningiomas in persons with neurofibromatosis 2.

Background: Meningiomas occur in 80% of persons with neurofibromatosis 2 (NF2) and cause significant mortality and morbidity, yet there are no effective medical treatments. NF2-deficient tumors have constitutive activation of mammalian/mechanistic target of rapamycin (mTOR), and treatment with mTORC1 inhibitors results in growth arrest in a minority of tumors, with paradoxical activation of the mTORC2/AKT pathway. We studied the effect of vistusertib, a dual mTORC1/mTORC2 inhibitor, in NF2 patients with progressive or symptomatic meningiomas. Methods: Vistusertib was administered orally at 125 mg twice daily for 2 consecutive days each week. The primary endpoint was the imaging response in the target meningioma, defined as a volume decrease of 20% compared with the baseline. Secondary endpoints included toxicity, imaging response of nontarget tumors, quality of life, and genetic biomarkers. Results: Eighteen participants (13 female), median age of 41 (range, 18-61) years, were enrolled. In target meningiomas, the best response was partial response (PR) in 1/18 tumors (6%) and stable disease (SD) in 17/18 tumors (94%). For all measured intracranial meningiomas and vestibular schwannomas, the best imaging response was PR in 6/59 tumors (10%) and SD in 53 (90%). Treatment-related grade 3/4 adverse events occurred in 14 (78%) participants, and 9 participants discontinued treatment due to side effects. Conclusions: Although the study did not meet the primary endpoint, vistusertib treatment was associated with high rates of SD in progressive NF2-related tumors. However, this dosing regimen for vistusertib was poorly tolerated. Future studies of dual mTORC inhibitors for NF2 should focus on optimizing tolerability and evaluating the relevance of tumor stability in participants.

Translatome analysis of Tuberous Sclerosis Complex-1 patient-derived neural progenitor cells reveal rapamycin-dependent and independent alterations.

Tuberous sclerosis complex (TSC) is an inherited neurocutaneous disorder caused by mutations in TSC1 or TSC2 genes, with patients often exhibiting neurodevelopmental (ND) manifestations termed TSC-associated neuropsychiatric disorders (TAND) including autism spectrum disorder (ASD). The hamartin-tuberin (TSC1-TSC2) protein complex inactivates mechanistic target of rapamycin complex 1 (mTORC1) signaling, leading to increased protein synthesis via inactivation of translational repressor eIF4E-binding proteins (4E-BPs). In TSC1-null neural progenitor cells (NPCs), we previously reported early ND phenotypic changes, including increased proliferation/altered neurite outgrowth, which were unaffected by mTORC1-inhibitor rapamycin. Here, using polysome-profiling to quantify translational efficiencies at a transcriptome-wide level, we observed numerous TSC1-dependent alterations in NPCs, largely recapitulated in post-mortem brains from ASD donors. Although rapamycin partially reversed TSC1-associated alterations, most neural activity/synaptic- or ASD-related genes remained insensitive but were inhibited by third-generation bi-steric, mTORC1-selective inhibitor RMC-6272, which also reversed altered ND phenotypes. Together these data reveal potential implications for treatment of TAND.

Proteasomal pathway inhibition as a potential therapy for NF2-associated meningioma and schwannoma.

BACKGROUND: Neurofibromatosis 2 (NF2) is an inherited disorder caused by bi-allelic inactivation of the NF2 tumor suppressor gene. NF2-associated tumors, including schwannoma and meningioma, are resistant to chemotherapy, often recurring despite surgery and/or radiation, and have generally shown cytostatic response to signal transduction pathway inhibitors, highlighting the need for improved cytotoxic therapies. METHODS: Leveraging data from our previous high-throughput drug screening in NF2 preclinical models, we identified a class of compounds targeting the ubiquitin-proteasome pathway (UPP), and undertook studies using candidate UPP inhibitors, ixazomib/MLN9708, pevonedistat/MLN4924, and TAK-243/MLN7243. Employing human primary and immortalized meningioma (MN) cell lines, CRISPR-modified Schwann cells (SCs), and mouse Nf2-/- SCs, we performed dose response testing, flow cytometry-based Annexin V and cell cycle analyses, and RNA-sequencing to identify potential underlying mechanisms of apoptosis. In vivo efficacy was also assessed in orthotopic NF2-deficient meningioma and schwannoma tumor models. RESULTS: Testing of three UPP inhibitors demonstrated potent reduction in cell viability and induction of apoptosis for ixazomib or TAK-243, but not pevonedistat. In vitro analyses revealed that ixazomib or TAK-243 downregulates expression of c-KIT and PDGFRα, as well as the E3 ubiquitin ligase SKP2 while upregulating genes associated with endoplasmic reticulum stress-mediated activation of the unfolded protein response (UPR). In vivo treatment of mouse models revealed delayed tumor growth, suggesting a therapeutic potential. CONCLUSIONS: This study demonstrates the efficacy of proteasomal pathway inhibitors in meningioma and schwannoma preclinical models and lays the groundwork for use of these drugs as a promising novel treatment strategy for NF2 patients.

Gene replacement therapy in a schwannoma mouse model of neurofibromatosis type 2.

Loss of function of the neurofibromatosis type 2 (NF2) tumor suppressor gene leads to the formation of schwannomas, meningiomas, and ependymomas, comprising ∼50% of all sporadic cases of primary nervous system tumors. NF2 syndrome is an autosomal dominant condition, with bi-allelic inactivation of germline and somatic alleles resulting in loss of function of the encoded protein merlin and activation of mammalian target of rapamycin (mTOR) pathway signaling in NF2-deficient cells. Here we describe a gene replacement approach through direct intratumoral injection of an adeno-associated virus vector expressing merlin in a novel human schwannoma model in nude mice. In culture, the introduction of an AAV1 vector encoding merlin into CRISPR-modified human NF2-null arachnoidal cells (ACs) or Schwann cells (SCs) was associated with decreased size and mTORC1 pathway activation consistent with restored merlin activity. In vivo, a single injection of AAV1-merlin directly into human NF2-null SC-derived tumors growing in the sciatic nerve of nude mice led to regression of tumors over a 10-week period, associated with a decrease in dividing cells and an increase in apoptosis, in comparison with vehicle. These studies establish that merlin re-expression via gene replacement in NF2-null schwannomas is sufficient to cause tumor regression, thereby potentially providing an effective treatment for NF2.

SMARCE1 deficiency generates a targetable mSWI/SNF dependency in clear cell meningioma.

Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes establish and maintain chromatin accessibility and gene expression, and are frequently perturbed in cancer. Clear cell meningioma (CCM), an aggressive tumor of the central nervous system, is uniformly driven by loss of SMARCE1, an integral subunit of the mSWI/SNF core. Here, we identify a structural role for SMARCE1 in selectively stabilizing the canonical BAF (cBAF) complex core-ATPase module interaction. In CCM, cBAF complexes fail to stabilize on chromatin, reducing enhancer accessibility, and residual core module components increase the formation of BRD9-containing non-canonical BAF (ncBAF) complexes. Combined attenuation of cBAF function and increased ncBAF complex activity generates the CCM-specific gene expression signature, which is distinct from that of NF2-mutated meningiomas. Importantly, SMARCE1-deficient cells exhibit heightened sensitivity to small-molecule inhibition of ncBAF complexes. These data inform the function of a previously elusive SWI/SNF subunit and suggest potential therapeutic approaches for intractable SMARCE1-deficient CCM tumors.

High-content image-based analysis and proteomic profiling identifies Tau phosphorylation inhibitors in a human iPSC-derived glutamatergic neuronal model of tauopathy.

Mutations in MAPT (microtubule-associated protein tau) cause frontotemporal dementia (FTD). MAPT mutations are associated with abnormal tau phosphorylation levels and accumulation of misfolded tau protein that can propagate between neurons ultimately leading to cell death (tauopathy). Recently, a p.A152T tau variant was identified as a risk factor for FTD, Alzheimer's disease, and synucleinopathies. Here we used induced pluripotent stem cells (iPSC) from a patient carrying this p.A152T variant to create a robust, functional cellular assay system for probing pathophysiological tau accumulation and phosphorylation. Using stably transduced iPSC-derived neural progenitor cells engineered to enable inducible expression of the pro-neural transcription factor Neurogenin 2 (Ngn2), we generated disease-relevant, cortical-like glutamatergic neurons in a scalable, high-throughput screening compatible format. Utilizing automated confocal microscopy, and an advanced image-processing pipeline optimized for analysis of morphologically complex human neuronal cultures, we report quantitative, subcellular localization-specific effects of multiple kinase inhibitors on tau, including ones under clinical investigation not previously reported to affect tau phosphorylation. These results demonstrate the potential for using patient iPSC-derived ex vivo models of tauopathy as genetically accurate, disease-relevant systems to probe tau biochemistry and support the discovery of novel therapeutics for tauopathies.

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.

Gene therapy for tuberous sclerosis complex type 2 in a mouse model by delivery of AAV9 encoding a condensed form of tuberin.

Tuberous sclerosis complex (TSC) results from loss of a tumor suppressor gene - TSC1 or TSC2, encoding hamartin and tuberin, respectively. These proteins formed a complex to inhibit mTORC1-mediated cell growth and proliferation. Loss of either protein leads to overgrowth lesions in many vital organs. Gene therapy was evaluated in a mouse model of TSC2 using an adeno-associated virus (AAV) vector carrying the complementary for a "condensed" form of human tuberin (cTuberin). Functionality of cTuberin was verified in culture. A mouse model of TSC2 was generated by AAV-Cre recombinase disruption of Tsc2-floxed alleles at birth, leading to a shortened lifespan (mean 58 days) and brain pathology consistent with TSC. When these mice were injected intravenously on day 21 with AAV9-cTuberin, the mean survival was extended to 462 days with reduction in brain pathology. This demonstrates the potential of treating life-threatening TSC2 lesions with a single intravenous injection of AAV9-cTuberin.

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.

Combination therapy with mTOR kinase inhibitor and dasatinib as a novel therapeutic strategy for vestibular schwannoma.

Neurofibromatosis type 2 (NF2) is an inherited disorder characterized by bilateral vestibular schwannomas (VS) that arise from neoplastic Schwann cells (SCs). NF2-associated VSs are often accompanied by meningioma (MN), and the majority of NF2 patients show loss of the NF2 tumor suppressor. mTORC1 and mTORC2-specific serum/glucocorticoid-regulated kinase 1 (SGK1) are constitutively activated in MN with loss of NF2. In a recent high-throughput kinome screen in NF2-null human arachnoidal and meningioma cells, we showed activation of EPH RTKs, c-KIT, and SFK members independent of mTORC1/2 activation. Subsequently, we demonstrated in vitro and in vivo efficacy of combination therapy with the dual mTORC1/2 inhibitor AZD2014 and the multi-kinase inhibitor dasatinib. For these reasons, we investigated activated mTORC1/2 and EPH receptor-mediated signaling in sporadic and NF2-associated VS. Using primary human VS cells and a mouse allograft model of schwannoma, we evaluated the dual mTORC1/2 inhibitor AZD2014 and the tyrosine kinase inhibitor dasatinib as monotherapies and in combination. Escalating dose-response experiments on primary VS cells grown from 15 human tumors show that combination therapy with AZD2014 and dasatinib is more effective at reducing metabolic activity than either drug alone and exhibits a therapeutic effect at a physiologically reasonable concentration (~0.1 µM). In vivo, while AZD2014 and dasatinib each inhibit tumor growth alone, the effect of combination therapy exceeds that of either drug. Co-targeting the mTOR and EPH receptor pathways with these or similar compounds may constitute a novel therapeutic strategy for VS, a condition for which there is no FDA-approved pharmacotherapy.

TSC patient-derived isogenic neural progenitor cells reveal altered early neurodevelopmental phenotypes and rapamycin-induced MNK-eIF4E signaling.

Background: Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with frequent occurrence of epilepsy, autism spectrum disorder (ASD), intellectual disability (ID), and tumors in multiple organs. The aberrant activation of mTORC1 in TSC has led to treatment with mTORC1 inhibitor rapamycin as a lifelong therapy for tumors, but TSC-associated neurocognitive manifestations remain unaffected by rapamycin. Methods: Here, we generated patient-specific, induced pluripotent stem cells (iPSCs) from a TSC patient with a heterozygous, germline, nonsense mutation in exon 15 of TSC1 and established an isogenic set of heterozygous (Het), null and corrected wildtype (Corr-WT) iPSCs using CRISPR/Cas9-mediated gene editing. We differentiated these iPSCs into neural progenitor cells (NPCs) and examined neurodevelopmental phenotypes, signaling and changes in gene expression by RNA-seq. Results: Differentiated NPCs revealed enlarged cell size in TSC1-Het and Null NPCs, consistent with mTORC1 activation. TSC1-Het and Null NPCs also revealed enhanced proliferation and altered neurite outgrowth in a genotype-dependent manner, which was not reversed by rapamycin. Transcriptome analyses of TSC1-NPCs revealed differentially expressed genes that display a genotype-dependent linear response, i.e., genes upregulated/downregulated in Het were further increased/decreased in Null. In particular, genes linked to ASD, epilepsy, and ID were significantly upregulated or downregulated warranting further investigation. In TSC1-Het and Null NPCs, we also observed basal activation of ERK1/2, which was further activated upon rapamycin treatment. Rapamycin also increased MNK1/2-eIF4E signaling in TSC1-deficient NPCs. Conclusion: MEK-ERK and MNK-eIF4E pathways regulate protein translation, and our results suggest that aberrant translation distinct in TSC1/2-deficient NPCs could play a role in neurodevelopmental defects. Our data showing upregulation of these signaling pathways by rapamycin support a strategy to combine a MEK or a MNK inhibitor with rapamycin that may be superior for TSC-associated CNS defects. Importantly, our generation of isogenic sets of NPCs from TSC patients provides a valuable platform for translatome and large-scale drug screening studies. Overall, our studies further support the notion that early developmental events such as NPC proliferation and initial process formation, such as neurite number and length that occur prior to neuronal differentiation, represent primary events in neurogenesis critical to disease pathogenesis of neurodevelopmental disorders such as ASD.

First International Conference on RASopathies and Neurofibromatoses in Asia: Identification and advances of new therapeutics.

The neurofibromatoses, which include neurofibromatosis type I (NF1), neurofibromatosis type II (NF2), and schwannomatosis, are a group of syndromes characterized by tumor growth in the nervous system. The RASopathies are a group of syndromes caused by germline mutations in genes that encode components of the RAS/mitogen-activated protein kinase (MAPK) pathway. The RASopathies include NF1, Noonan syndrome, Noonan syndrome with multiple lentigines, Costello syndrome, cardio-facio-cutaneous syndrome, Legius syndrome, capillary malformation arterio-venous malformation syndrome, and SYNGAP1 autism. Due to their common underlying pathogenetic etiology, all these syndromes have significant phenotypic overlap of which one common feature include a predisposition to tumors, which may be benign or malignant. Together as a group, they represent one of the most common multiple congenital anomaly syndromes estimating to affect approximately one in 1000 individuals worldwide. The subcontinent of India represents one of the largest populations in the world, yet remains underserved from an aspect of clinical genetics services. In an effort to bridge this gap, the First International Conference on RASopathies and Neurofibromatoses in Asia: Identification and Advances of New Therapeutics was held in Kochi, Kerala, India. These proceedings chronicle this timely and topical international symposium directed at discussing the best practices and therapies for individuals with neurofibromatoses and RASopathies.

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.

Traditional and systems biology based drug discovery for the rare tumor syndrome neurofibromatosis type 2.

Neurofibromatosis 2 (NF2) is a rare tumor suppressor syndrome that manifests with multiple schwannomas and meningiomas. There are no effective drug therapies for these benign tumors and conventional therapies have limited efficacy. Various model systems have been created and several drug targets have been implicated in NF2-driven tumorigenesis based on known effects of the absence of merlin, the product of the NF2 gene. We tested priority compounds based on known biology with traditional dose-concentration studies in meningioma and schwann cell systems. Concurrently, we studied functional kinome and gene expression in these cells pre- and post-treatment to determine merlin deficient molecular phenotypes. Cell viability results showed that three agents (GSK2126458, Panobinostat, CUDC-907) had the greatest activity across schwannoma and meningioma cell systems, but merlin status did not significantly influence response. In vivo, drug effect was tumor specific with meningioma, but not schwannoma, showing response to GSK2126458 and Panobinostat. In culture, changes in both the transcriptome and kinome in response to treatment clustered predominantly based on tumor type. However, there were differences in both gene expression and functional kinome at baseline between meningioma and schwannoma cell systems that may form the basis for future selective therapies. This work has created an openly accessible resource (www.synapse.org/SynodosNF2) of fully characterized isogenic schwannoma and meningioma cell systems as well as a rich data source of kinome and transcriptome data from these assay systems before and after treatment that enables single and combination drug discovery based on molecular phenotype.

Pain correlates with germline mutation in schwannomatosis.

Schwannomatosis has been linked to germline mutations in the SMARCB1 and LZTR1 genes, and is frequently associated with pain.In a cohort study, we assessed the mutation status of 37 patients with clinically diagnosed schwannomatosis and compared to clinical data, whole body MRI (WBMRI), visual analog pain scale, and Short Form 36 (SF-36) bodily pain subscale.We identified a germline mutation in LZTR1 in 5 patients (13.5%) and SMARCB1 in 15 patients (40.5%), but found no germline mutation in 17 patients (45.9%). Peripheral schwannomas were detected in 3 LZTR1-mutant (60%) and 10 SMARCB1-mutant subjects (66.7%). Among those with peripheral tumors, the median tumor number was 4 in the LZTR1 group (median total body tumor volume 30 cc) and 10 in the SMARCB1 group (median volume 85cc), (P=.2915 for tumor number and P = .2289 for volume). mutation was associated with an increased prevalence of spinal schwannomas (100% vs 41%, P = .0197). The median pain score was 3.9/10 in the LZTR1 group and 0.5/10 in the SMARCB1 group (P = .0414), and SF-36 pain-associated quality of life was significantly worse in the LZTR1 group (P = .0106). Pain scores correlated with total body tumor volume (rho = 0.32471, P = .0499), but not with number of tumors (rho = 0.23065, P = .1696).We found no significant difference in quantitative tumor burden between mutational groups, but spinal schwannomas were more common in LZTR1-mutant patients. Pain was significantly higher in LZTR1-mutant than in SMARCB1-mutant patients, though spinal tumor location did not significantly correlate with pain. This suggests a possible genetic association with schwannomatosis-associated pain.