Abstract BACKGROUND Meningioma is the most common primary intracranial tumor. Although ~80% are benign some WHO grade I are clinically aggressive. Chemotherapies are ineffective and biomarkers for clinical management are lacking. Approximately 60% sporadic meningiomas harbor mutations in the NF2 gene andutations in TRAF7, KLF4, AKT1, SMO and PIK3CA have been identified in the majority NF2-positive tumors esp lower grade. However, the molecular mechanisms behind meningioma tumourigenesis is still unclear. We aim to identify novel biomarkers and therapeutic targets of meningioma by characterizing the proteomic landscape. MATERIAL AND METHODS We analysed grade I, II and III frozen meningioma specimens and three different mutational groups: AKT1/TRAF7, KLF4/TRAF7 and NF2 -/- using LC-MS/MS to analyse global proteins, enriched phosphoproteins and phosphopeptides. Differential expression and functional annotation of proteins was completed using Perseus, IPA® and DAVID. For mutational subtypes quantitative phosphoproteomics was performed using TMT 10plex labeling approach followed by motif analysis using motif-X algorithm. We validated differential expression of proteins and phosphoproteins by Western blot and immunohistochemistry. RESULTS We quantified 3888 proteins and 3074 phosphoproteins across all meningioma grades. Bioinformatics analysis revealed commonly upregulated (phospho)proteins to be enriched in Gene Ontology terms associated with RNA metabolism. Validation confirmed significant overexpression of proteins such as EGFR, CKAP4, the nuclear proto-oncogene SET, the splicing factor SF2/ASF as well as total and activated phosphorylated form of the NIMA-related kinase, NEK9, involved in mitotic progression. Hexokinase 2 was overexpressed in higher grades. For the mutation subtypes we have quantified 4162 proteins across all mutational meningioma subgroups. Analysis showed distinct proteomic profiles of mutational subgroups. Comparative analysis showed 10 proteins were commonly significantly upregulated among all mutational subtypes vs. normal meninges. 257 proteins were commonly significantly downregulated and enriched with molecular functions including aldehyde dehydrogenase and oxido-reductase. Mutational subtype-specific analysis identified 162 proteins significantly upregulated in AKT1/TRAF7 vs. remaining sample groups to be enriched in the oxidative phosphorylation pathway. 14 and 7 proteins were commonly significantly upregulated in KLF4/TRAF7 and NF2 -/- mutant meningioma subtypes respectively. Several of these up-regulated proteins including ANNEXIN-3, CRABP2, CLIC3 and Endoglin were verified via WB. Lastly, analyses of 6600 phosphosites predicted regulatory kinases CONCLUSION We show extensive proteomic and phospophoproteomics analysis of meningioma and suggest new therapeutic and biomarker candidates.

Abstract BACKGROUND Meningiomas are the most frequent primary brain tumours of the central nervous system. The standard of treatment is (radio)surgery, but the lack of knowledge of their tumorigenesis contributes to delay the development of additional therapeutic options. MATERIAL AND METHODS We used a variety of differen model systems and cell biology techniques RESULTS We found STAT1 widely overexpressed in meningioma tumours and in patient-derived meningioma cells but not in the corresponding healthy controls. The protein showed a constitutive phosphorylation on both phosphosites (Y701 and S727), which was not dependent on the JAK/STAT pathway. STAT1 knocked down resulted in a significant reduction of cellular proliferation, showed as a decrease in Ki67-positive cells and Cyclin D1, and deactivation of AKT and ERK 1/2. By studying STAT1 binding partners we isolated a complex composed by STAT1, STAT2, PRMT5 and MEP50. As PRMT5 is known to interact with EGFR, we tested the tyrosine kinase and found that EGFR was constitutively active in meningioma and was responsible for the aberrant phosphorylation of STAT1 on both phosphosites. We tested different drugs inhibiting of EGFR phosphorylation, Canertinib was most effective. It caused a significant reduction in meningioma cells proliferation and a reduction of overall levels of Cyclin D1, phospho- AKT and phospho-ERK 1/2. Hence, STAT1 constitutive phosphorylation, initiated by EGFR activation, is responsible for inducing a positive feedback loop causing its own overexpression and consequently an increased proliferation of the tumour cells. CONCLUSION These findings underline a pivotal role of the EGFR and STAT1 axis in meningioma and provides the rationale for further studies aiming to identify novel and effective therapeutic options.

Meningioma is the most frequent primary intracranial tumour. Surgical resection remains the main therapeutic option as pharmacological intervention is still hampered by the poor knowledge of the molecular signature of these tumours In order to elucidate the proteomic profiling of meningiomas and identify proteins involved in their pathogenesis, we completed a comparative mass spectrometry analysis of meningioma tissue of all WHO grades, analysing global proteins, phosphoproteins and phosphopeptides. We performed differential expression analyses and functional annotation studies to identify commonly upregulated proteins and phosphoprotein in all grades of meningioma compared to meningeal tissue as well as grade-specific candidates relevant for tumour progression. Top candidates werevalidated by Western blotting and immunohistochemistry in an additional sample set We confirmed significant overexpression of proteins including EGFR, STAT2 and CKAP4 across all grades, as well as the aberrant activation of the downstream PI3K/AKT pathway, which seems differential between grades. Further, we validated upregulation in all grades of the total and activated phosphorylated form of the NIMArelated kinase, NEK9, involved in mitotic progression. Novel proteins identified in meningioma and validated as commonly overexpressed in all grades were the nuclear proto-oncogene SET and the splicing factor SF2/ASF, while another newly identified protein that was specific for higher grades was hexokinase-2, involved in cellular metabolism Overall, we generated a proteomic thesaurus of meningiomas in order to decipher aberrantly expressed proteins and activated pathways; this body of knowledge will eventually lead to the identification of relevant biomarkers and therapeutic targets.

Introduction Meningiomas are the most common primary intracranial brain tumour arising from meningeal tissue. Despite the majority of them displaying benign features, they can cause mild to severe morbidity. The current main therapeutic approach is complete tumour resection commonly with adjunct radiation therapy. However, tumour location can hamper complete resection and chemotherapies are ineffective. In this study we aim to elucidate the pathogenic signature of these tumours and identify novel molecular targets by deciphering the global proteome and phosphoprotein profile of different grades of meningiomas. Material and methods Tumour lysates were collected from grade I, II and III frozen meningioma specimens and three normal healthy human meninges. Phosphoprotein purification was performed using Qiagen® PhosphoProtein Purification Kit. Proteins were separated by SDS-PAGE followed by in-gel tryptic digestion. Extracted peptides were purified and analysed by electrospray ionisation LC-MS/MS. Raw mass spectrometry files were analysed using MaxQuantTM. Expression data were validated by Western blot and immunohistochemistry. In silico functional annotation of expression data was completed using Perseus 1.5.0.31 software suite, Ingenuity Pathway Analysis (IPA®) and DAVID 6.8. Results and discussions We have quantified 3888 proteins and 3074 phosphoproteins across all grades of meningioma and normal meninges. Comparative analysis identified 181 proteins and 338 phosphoproteins to be commonly significantly upregulated (log2 fold-change ≥1.5; p Conclusion In summary, we performed a comprehensive quantitative proteomic analysis from meningioma tissue of all WHO grades compared to healthy meninges and have identified several potential candidates that may hold therapeutic potential for targeted treatment of these tumours.

Meningiomas are slow growing tumours of the meninges that affect brain and spinal cord. They account for a quarter of all primary brain tumours of the central nervous system. Accordingly to the WHO classification system, meningiomas are classified as grade I, atypical grade II and malignant grade III. Symptoms include headaches, focal neurological signs depending on localisation and seizures. The standard of treatment for these tumours is (radio)surgery. Nevertheless, it is estimated that one third of meningiomas cannot be operated or can be only partially resected, often leaving patients with significant morbidity. Current chemotherapies are not effective therefore it is a great medical need to find novel therapeutic options. We aimed to identify novel targets/biomarkers by analysing proteome and phosphoproteome of different grade meningiomas. Frozen tumour specimens as well as meningioma-derived primary tumour cells were analysed by mass spectrometry to decipher proteome and phosphoproteome. ­Phosphoproteins were isolated by an additional purification step. Overall we analysed 22 meningiomas (8 grade I, 8 grade II and 6 grade III) for the proteome and 14 for the phosphoproteome (5 grade I, 5 grade II and 4 grade III). Comparative studies were performed to identify aberrantly overexpressed proteins and dysregulated pathways compared to healthy human meninges (N=3). Among the proteins found significantly upregulated in meningioma vs. normal controls we identified STAT1, a member of the Jak/STAT signalling pathway. Validation studies performed on primary meningioma cells confirmed that the total amount of the protein was overexpressed about four times compared to normal human meningeal cells. Additionally, both phosphorylation sites (Y701 and S727) on STAT1 were aberrantly activated in meningioma cells but not in meningeal cells. Immunohistochemical analysis confirmed an upregulation of phosphorylated STAT1(Y701) especially on grade III meningiomas, in agreement with expression studies, although with high variability across samples. When the Jak/STAT signaling pathway gets activated in response to cytokines and growth factors, STAT1 is phosphorylated by activated JAKs and translocate into the nucleus to regulate gene expression. In primary meningioma cells we found that the amount of cytoplasmic pSTAT1(Y701) was markedly increased in vitro, without interferon stimulation and also in serum-free conditions, suggesting a cytokine-independent mechanism for pSTAT1 activation. Finally, STAT1 knocked down resulted in a significant reduction of cellular proliferation showed as a decrease in Ki67-positive cells and a decrease expression of Cyclin D1. These data altogether suggest STAT1 as a novel potential target in meningiomas.

Merlin has broad tumor-suppressor functions as its mutations have been identified in multiple benign tumors and malignant cancers. In all schwannomas, the majority of meningiomas and 1/3 of ependymomas Merlin loss is causative. In neurofibromatosis type 2, a dominantly inherited tumor disease because of the loss of Merlin, patients suffer from multiple nervous system tumors and die on average around age 40. Chemotherapy is not effective and tumor localization and multiplicity make surgery and radiosurgery challenging and morbidity is often considerable. Thus, a new therapeutic approach is needed for these tumors. Using a primary human in vitro model for Merlin-deficient tumors, we report that the Ras/Raf/mitogen-activated protein, extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) scaffold, kinase suppressor of Ras 1 (KSR1), has a vital role in promoting schwannomas development. We show that KSR1 overexpression is involved in many pathological phenotypes caused by Merlin loss, namely multipolar morphology, enhanced cell–matrix adhesion, focal adhesion and, most importantly, increased proliferation and survival. Our data demonstrate that KSR1 has a wider role than MEK1/2 in the development of schwannomas because adhesion is more dependent on KSR1 than MEK1/2. Immunoprecipitation analysis reveals that KSR1 is a novel binding partner of Merlin, which suppresses KSR1’s function by inhibiting the binding between KSR1 and c-Raf. Our proteomic analysis also demonstrates that KSR1 interacts with several Merlin downstream effectors, including E3 ubiquitin ligase CRL4DCAF1. Further functional studies suggests that KSR1 and DCAF1 may co-operate to regulate schwannomas formation. Taken together, these findings suggest that KSR1 serves as a potential therapeutic target for Merlin-deficient tumors.