No abstract

WHO grade II and III as well as some WHO grade I meningioma are clinically aggressive. Approximately 60% sporadic meningiomas harbour mutations in the NF2 gene, others in genes including TRAF7, KLF4, AKT1, SMO and PIK3CA . However, the molecular mechanisms behind meningioma tumourigenesis are still unclear. We aim to identify novel biomarkers and therapeutic targets of meningioma by characterising the proteomic landscape. We performed (phosho)proteomic profiling of grade I, II and III meningiomas and three different mutational groups: AKT1 E17K /TRAF7, KLF4 K409Q /TRAF7 and NF2 -/-. We validated differential expression of proteins and phosphoproteins by Western blot on a meningioma validation set and by immunohistochemistry. Looking at all grades bioinformatics analysis revealed commonly upregulated proteins and phosphoproteins to be enriched in Gene Ontology terms associated with RNA metabolism. Validation studies confirmed significant overexpression of proteins such as EGFR and CKAP4 and upregulation and activation of the NIMA-related kinase, NEK9, involved in mitotic progression. Novel proteins described included the nuclear proto-oncogene SET, the splicing factor SF2/ASF and the higher-grade specific protein, Hexokinase 2. For the mutation subtypes we have quantified 4162 proteins across all mutational meningioma subgroups with proteomic profiles of mutational subgroups. Comparative analysis showed 10 proteins were commonly significantly upregulated among all mutational subtypes vs. normal meninges, indicating proteomic landscapes of mutational subtypes to be highly variable. 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 E17K /TRAF7 vs. remaining sample groups to be enriched in the oxidative phosphorylation pathway. Less proteins were commonly significantly upregulated in KLF4 K409Q /TRAF7 and NF2 -/- mutant meningioma subtypes respectively. Several of these up-regulated proteins including ANNEXIN-3, CRABP2, CLIC3 were verified. Analyses of 6600 phospho-sites predicted regulatory kinases. Further validation and functional verification of potential candidates is ongoing.

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.

Introduction Meningioma are the most common primary intracranial tumour. According to WHO, ~80% tumours are benign grade I. Although, some grade I tumour clinically show aggressive behaviour. Radio-surgery are the main therapeutic approaches, chemotherapies are ineffective. Accurate biomarkers for clinical management are lacking. The mutational profile of low-grade meningioma is well-defined, with non-NF2 mutated tumours harbouring recurrent mutations in genes including TRAF7, KLF4, AKT1 and SMO. Here, we aim to identify novel biomarkers and therapeutic targets of genetically stratified low-grade meningioma by characterising the proteomic landscape. Materials and methods Meningioma specimens were stratified according to mutational background: AKT1E17K/TRAF7, KLF4K409Q/TRAF7 and NF2-/-. Proteins were separated by SDS-PAGE followed by in-gel tryptic digestion and sample preparation for LC-MS/MS analysis. Raw mass spectrometry data files were processed by MaxQuant and Perseus software. Quantitative phospho-proteomics was performed using TMT-10plex labelling approach followed by motif analysis using motif-X algorithm. GO enrichment analyses were performed using DAVID against all human proteins. Results and Conclusions We have quantified 4162 proteins across all mutational meningioma subgroups and normal meninges (n=31). Hierarchical clustering analysis showed distinct proteomic profiles of mutational subgroups revealing clusters of differentially expressed proteins (DEPs). Comparative analysis showed 10 proteins were commonly significantly upregulated (log2 fold-change≥1; p<0.05) among all mutational subtypes vs. normal meninges, indicating proteomic landscapes of mutational subtypes to be highly variable. In contrast, 257 proteins were commonly significantly downregulated (log2 fold-change≤-1; p<0.05) and enriched with molecular functions including aldehyde dehydrogenase and oxido-reductase. Mutational subtype-specific analysis identified 162 proteins significantly upregulated in AKT1E17K/TRAF7 vs. remaining sample groups to be enriched in the oxidative phosphorylation pathway. Lastly, analyses of 6600 phospho-sites (n=8) predicted regulatory kinases including EGFR and PKCα. Several of these up-regulated proteins and kinases already verified via WB. Further validation and functional verification will allow us to identify potential drug targets/biomarkers for meningioma.