MerlinS13 phosphorylation regulates meningioma Wnt signaling and magnetic resonance imaging features.

Meningiomas are associated with inactivation of NF2/Merlin, but approximately one-third of meningiomas with favorable clinical outcomes retain Merlin expression. Biochemical mechanisms underlying Merlin-intact meningioma growth are incompletely understood, and non-invasive biomarkers that may be used to guide treatment de-escalation or imaging surveillance are lacking. Here, we use single-cell RNA sequencing, proximity-labeling proteomic mass spectrometry, mechanistic and functional approaches, and magnetic resonance imaging (MRI) across meningioma xenografts and patients to define biochemical mechanisms and an imaging biomarker that underlie Merlin-intact meningiomas. We find Merlin serine 13 (S13) dephosphorylation drives meningioma Wnt signaling and tumor growth by attenuating inhibitory interactions with ß-catenin and activating the Wnt pathway. MRI analyses show Merlin-intact meningiomas with S13 phosphorylation and favorable clinical outcomes are associated with high apparent diffusion coefficient (ADC). These results define mechanisms underlying a potential imaging biomarker that could be used to guide treatment de-escalation or imaging surveillance for patients with Merlin-intact meningiomas.

RNA splicing as a biomarker and phenotypic driver of meningioma DNA methylation groups.

BACKGROUND: Advances in our understanding of the molecular biology of meningiomas have led to significant gains in the ability to predict patient prognosis and tumor recurrence and to identify novel targets for therapeutic design. Specifically, classification of meningiomas based on DNA methylation has greatly improved our ability to risk stratify patients, however new questions have arisen in terms of the underlying impact these DNA methylation signatures have on meningioma biology. METHODS: This study utilizes RNA-seq data from 486 meningioma samples corresponding to three meningioma DNA methylation groups (Merlin-intact, Immune-enriched, and Hypermitotic), followed by in vitro experiments utilizing human meningioma cell lines. RESULTS: We identify alterations in RNA splicing between meningioma DNA methylation groups including individual splicing events that correlate with Hypermitotic meningiomas and predict tumor recurrence and overall patient prognosis and compile a set of splicing events that can accurately predict DNA methylation classification based on RNA-seq data. Furthermore, we validate these events using RT-PCR in patient samples and meningioma cell lines. Additionally, we identify alterations in RNA binding proteins and splicing factors that lie upstream of RNA splicing events, including upregulation of SRSF1 in Hypermitotic meningiomas which we show drives alternative RNA splicing changes. Finally, we design splice switching antisense oligonucleotides to target RNA splicing changes in NASP and MFF observed in Hypermitotic meningiomas, providing a rationale for RNA-based therapeutic design. CONCLUSIONS: RNA splicing is an important driver of meningioma phenotypes that can be useful in prognosticating patients and as a potential exploit for therapeutic vulnerabilities.

Glioblastoma Neurovascular Progenitor Orchestrates Tumor Cell Type Diversity.

Glioblastoma (GBM) is the deadliest form of primary brain tumor with limited treatment options. Recent studies have profiled GBM tumor heterogeneity, revealing numerous axes of variation that explain the molecular and spatial features of the tumor. Here, we seek to bridge descriptive characterization of GBM cell type heterogeneity with the functional role of individual populations within the tumor. Our lens leverages a gene program-centric meta-atlas of published transcriptomic studies to identify commonalities between diverse tumors and cell types in order to decipher the mechanisms that drive them. This approach led to the discovery of a tumor-derived stem cell population with mixed vascular and neural stem cell features, termed a neurovascular progenitor (NVP). Following in situ validation and molecular characterization of NVP cells in GBM patient samples, we characterized their function in vivo. Genetic depletion of NVP cells resulted in altered tumor cell composition, fewer cycling cells, and extended survival, underscoring their critical functional role. Clonal analysis of primary patient tumors in a human organoid tumor transplantation system demonstrated that the NVP has dual potency, generating both neuronal and vascular tumor cells. Although NVP cells comprise a small fraction of the tumor, these clonal analyses demonstrated that they strongly contribute to the total number of cycling cells in the tumor and generate a defined subset of the whole tumor. This study represents a paradigm by which cell type-specific interrogation of tumor populations can be used to study functional heterogeneity and therapeutically targetable vulnerabilities of GBM.

Gene Expression Changes Associated With Recurrence After Gross Total Resection of Newly Diagnosed World Health Organization Grade 1 Meningioma.

BACKGROUND AND OBJECTIVE: Patients who undergo gross total resection (GTR) of Central Nervous System World Health Organization (WHO) grade 1 meningioma constitute a "low-risk" group, but some low-risk meningiomas can recur despite reassuring clinical and histological features. In this study, gene expression values in newly diagnosed WHO grade 1 meningiomas that had undergone GTR were evaluated for their association with recurrence. METHODS: This was a retrospective, international, multicenter cohort study that included WHO grade 1 meningiomas that underwent GTR, as first treatment, based on postoperative magnetic resonance imaging. Normalized gene expression values from a previously validated 34-gene panel were evaluated for their association with recurrence. Kaplan-Meier, multivariable Cox proportional hazard analyses, and K-means clustering were performed to assess the association of genes of interest with recurrence and identify molecular subgroups among clinically and histologically low-risk meningiomas. RESULTS: In total, 442 patients with WHO grade 1 meningiomas that underwent GTR and had available gene expression profiling data were included in the study. The median follow-up was 5.0 years (interquartile range 2.6-7.7 years), local recurrence occurred in 36 patients (8.1%), 5-year local freedom from recurrence was 90.5%, and median time to recurrence was 2.9 years (range 0.5-10.7 years). Eleven genes were associated with local recurrence, including lower expression of ARID1B, ESR1, LINC02593, PGR, and TMEM30B and higher expression of CDK6, CDKN2C, CKS2, KIF20A, PGK1, and TAGLN. Of these genes, PGK1 had the largest effect size. K-means clustering based on these 11 genes distinguished 2 molecular groups of clinically and histologically low-risk meningiomas with significant differences in local freedom from recurrence (hazard ratio 2.5, 95% CI 1.2-5.1, P = .016). CONCLUSION: Gene expression profiling may help to identify newly diagnosed WHO grade 1 meningiomas that have an elevated risk of recurrence despite GTR.

cIMPACT-NOW Update 8: Clarifications on molecular risk parameters and recommendations for WHO grading of meningiomas.

Meningiomas are the most frequent primary intracranial tumors. Hence, they constitute a major share of diagnostic specimens in neuropathology practice. The 2021 WHO Classification of Central Nervous System Tumors ("CNS5") has introduced the first molecular grading parameters for meningioma with oncogenic variants in the TERT promoter and homozygous deletion of CDKN2A/B as markers for CNS WHO grade 3. However, after publication of the new classification volume, clarifications were requested, not only on novel but also on long-standing questions in meningioma grading that were beyond the scope of the WHO "blue book". In addition, more recent research into possible new molecular grading parameters could not yet be implemented in the 2021 classification but constitute a compelling body of literature. Hence, the cIMPACT-NOW Steering Committee convened a working group to provide such clarification and assess the evidence of possible novel molecular criteria. As a result, this cIMPACT-NOW update provides guidance for more standardized morphological evaluation and interpretation, most prominently pertaining to brain invasion, identifies scenarios in which advanced molecular testing is recommended, proposes to assign CNS WHO grade 2 for cases with CNS WHO grade 1 morphology but chromosomal arm 1p deletion in combination with 22q deletion and/or NF2 oncogenic variants, and discusses areas in which the current evidence is not yet sufficient to result in new recommendations.

Meningeal solitary fibrous tumor cell states phenocopy cerebral vascular development and homeostasis.

BACKGROUND: Meningeal solitary fibrous tumors (SFTs) are rare mesenchymal neoplasms that are associated with local recurrence and hematogenous metastasis. The cell states and spatial transcriptomic architecture underlying the unique clinical behavior of meningeal SFTs are unknown. METHODS: Single-cell (n=4), spatial (n=8), and bulk RNA sequencing (n=22) was used to define the cell states and spatial transcriptomic architecture of meningeal SFTs across histological grades and in patient-matched pairs of primary/recurrent or intracranial/metastatic samples. Immunofluorescence, immunohistochemistry, and comparison of single-cell types to meningiomas, or to cerebral vascular development or homeostasis, were used for validation. RESULTS: Here we show meningeal SFTs are comprised of regionally distinct gene expression programs that resemble cerebral vascular development or homeostasis. Single-cell trajectory analysis and pseudotemporal ordering of single-cells suggest that meningeal SFT cell fate decisions are dynamic and interchangeable. Cell-cell communication analyses demonstrate receptor-ligand interactions throughout the meningeal SFT microenvironment, particularly between SFT cells, endothelia, and immature neurons. Direct comparison of single-cell transcriptomes from meningeal SFTs versus meningiomas shows that SFT cells are enriched in expression of endothelial markers while meningiomas cells are enriched in expression of mural cells markers. Meningeal SFT spatial transcriptomes show regionally distinct intratumor heterogeneity in cell states, gene expression programs, and cell-cell interactions across WHO histological grades and in patient-matched pairs of primary/recurrent or intracranial/metastatic samples. CONCLUSIONS: These results shed light on pathways underlying meningeal SFT biology in comparison to other central nervous system tumors and provide a framework for integrating single-cell, spatial, and bulk RNA sequencing data across human cancers and normal tissues.

Cilia-enriched oxysterol 7ß,27-DHC is required for polycystin ion channel activation.

Polycystin-1 (PC-1) and PC-2 form a heteromeric ion channel complex that is abundantly expressed in primary cilia of renal epithelial cells. This complex functions as a non-selective cation channel, and mutations within the polycystin complex cause autosomal dominant polycystic kidney disease (ADPKD). The spatial and temporal regulation of the polycystin complex within the ciliary membrane remains poorly understood. Using both whole-cell and ciliary patch-clamp recordings, we identify a cilia-enriched oxysterol, 7ß,27-dihydroxycholesterol (DHC), that serves as a necessary activator of the polycystin complex. We further identify an oxysterol-binding pocket within PC-2 and showed that mutations within this binding pocket disrupt 7ß,27-DHC-dependent polycystin activation. Pharmacologic and genetic inhibition of oxysterol synthesis reduces channel activity in primary cilia. In summary, our findings reveal a regulator of the polycystin complex. This oxysterol-binding pocket in PC-2 may provide a specific target for potential ADPKD therapeutics.

Elevated meningioma risk among individuals who are Non-Hispanic Black is strongest for grade 2-3 tumors and synergistically modified by male sex.

Background: Meningioma risk factors include older age, female sex, and African-American race. There are limited data exploring how meningioma risk in African-Americans varies across the lifespan, interacts with sex, and differs by tumor grade. Methods: The Central Brain Tumor Registry of the United States (CBTRUS) is a population-based registry covering the entire U.S. population. Meningioma diagnoses from 2004-2019 were used to calculate incidence rate ratios (IRRs) for non-Hispanic Black individuals (NHB) compared to non-Hispanic white individuals (NHW) across 10-year age intervals, and stratified by sex and by WHO tumor grade. Results: 53,890 NHB individuals and 322,373 NHW individuals with an intracranial meningioma diagnosis were included in analyses. Beginning in young adulthood, the NHB-to-NHW IRR was elevated for both grade 1 and grade 2/3 tumors. The IRR peaked in the seventh decade of life regardless of grade, and was higher for grade 2/3 tumors (IRR=1.57; 95% CI: 1.46-1.69) than grade 1 tumors (IRR=1.27; 95% CI: 1.25-1.30) in this age group. The NHB-to-NHW IRR was elevated in females (IRR=1.17; 95% CI: 1.16-1.18) and further elevated in males (IRR=1.28; 95% CI: 1.26-1.30), revealing synergistic interaction between NHB race/ethnicity and male sex (P Interaction =0.001). Conclusions: Relative to NHW individuals, NHB individuals are at elevated risk of meningioma from young adulthood through old age. NHB race/ethnicity conferred higher risk of meningioma among men than women, and higher risk of developing WHO grade 2/3 tumors. Results identify meningioma as a significant source of racial disparities in neuro-oncology and may help to improve preoperative predictions of meningioma grade.

Calcified Meningiomas Demonstrate Equivocal Grade, Proliferation, and Immediate Surgical Outcomes.

BACKGROUND/OBJECTIVE: Meningioma calcification is thought to predict reduced growth potential and aggression. However, historical studies have primarily focused on correlating calcification in small meningiomas (diameter less than 2.5 cm) rather than analyzing characteristics of calcified meningiomas across all sizes. In this study, we investigate the pathologic and clinical implications of meningioma calcification. METHODS: We utilized a historical database of 342 consecutive newly diagnosed intracranial meningiomas with preoperative computed tomography and magnetic resonance imaging scans treated at a single institution from 2005 to 2019. We correlated the presence of calcification with patient demographics, grade, Mindbomb Homolog-1 index, location, volume, Simpson grade, and recurrence using both univariate and multivariate generalized linear models. RESULTS: On univariate analysis, no single variable correlated with tumor calcification. Notably, neither tumor 2021 World Health Organization grade (P = 0.91) nor Mindbomb Homolog-1 index (P = 0.62) predicted calcification. After accounting for demographic characteristics and tumor volume and location, there was no significant association between 2021 World Health Organization grade (P = 0.52) and Mindbomb Homolog-1 index (P = 0.54) and calcification. Calcification had no influence on resection grade (P = 0.59) or recurrence (P = 0.80). CONCLUSIONS: In this series, calcified meningiomas exhibited similar 2021 World Health Organization tumor grading distribution, proliferation indexes, and immediate surgical outcomes compared to their noncalcified counterparts. These findings question the historical role of using meningioma calcification as an independent guide to their management.

Perivascular NOTCH3+ stem cells drive meningioma tumorigenesis and resistance to radiotherapy.

Meningiomas are the most common primary intracranial tumors. Treatments for patients with meningiomas are limited to surgery and radiotherapy, and systemic therapies remain ineffective or experimental. Resistance to radiotherapy is common in high-grade meningiomas and the cell types and signaling mechanisms that drive meningioma tumorigenesis and resistance to radiotherapy are incompletely understood. Here we report NOTCH3 drives meningioma tumorigenesis and resistance to radiotherapy and find that perivascular NOTCH3+ stem cells are conserved across meningiomas from humans, dogs, and mice. Integrating single-cell transcriptomics with lineage tracing and imaging approaches in genetically engineered mouse models and xenografts, we show NOTCH3 drives tumor initiating capacity, cell proliferation, angiogenesis, and resistance to radiotherapy to increase meningioma growth and reduce survival. To translate these findings to patients, we show that an antibody stabilizing the extracellular negative regulatory region of NOTCH3 blocks meningioma tumorigenesis and sensitizes meningiomas to radiotherapy, reducing tumor growth and improving survival.

Meningioma transcriptomic landscape demonstrates novel subtypes with regional associated biology and patient outcome.

Meningiomas, although mostly benign, can be recurrent and fatal. World Health Organization (WHO) grading of the tumor does not always identify high-risk meningioma, and better characterizations of their aggressive biology are needed. To approach this problem, we combined 13 bulk RNA sequencing (RNA-seq) datasets to create a dimension-reduced reference landscape of 1,298 meningiomas. The clinical and genomic metadata effectively correlated with landscape regions, which led to the identification of meningioma subtypes with specific biological signatures. The time to recurrence also correlated with the map location. Further, we developed an algorithm that maps new patients onto this landscape, where the nearest neighbors predict outcome. This study highlights the utility of combining bulk transcriptomic datasets to visualize the complexity of tumor populations. Further, we provide an interactive tool for understanding the disease and predicting patient outcomes. This resource is accessible via the online tool Oncoscape, where the scientific community can explore the meningioma landscape.

Leveraging single-cell sequencing to classify and characterize tumor subgroups in bulk RNA-sequencing data.

PURPOSE: Accurate classification of cancer subgroups is essential for precision medicine, tailoring treatments to individual patients based on their cancer subtypes. In recent years, advances in high-throughput sequencing technologies have enabled the generation of large-scale transcriptomic data from cancer samples. These data have provided opportunities for developing computational methods that can improve cancer subtyping and enable better personalized treatment strategies. METHODS: Here in this study, we evaluated different feature selection schemes in the context of meningioma classification. To integrate interpretable features from the bulk (n = 77 samples) and single-cell profiling (∼ 10 K cells), we developed an algorithm named CLIPPR which combines the top-performing single-cell models, RNA-inferred copy number variation (CNV) signals, and the initial bulk model to create a meta-model. RESULTS: While the scheme relying solely on bulk transcriptomic data showed good classification accuracy, it exhibited confusion between malignant and benign molecular classes in approximately ∼ 8% of meningioma samples. In contrast, models trained on features learned from meningioma single-cell data accurately resolved the sub-groups confused by bulk-transcriptomic data but showed limited overall accuracy. CLIPPR showed superior overall accuracy and resolved benign-malignant confusion as validated on n = 789 bulk meningioma samples gathered from multiple institutions. Finally, we showed the generalizability of our algorithm using our in-house single-cell (∼ 200 K cells) and bulk TCGA glioma data (n = 711 samples). CONCLUSION: Overall, our algorithm CLIPPR synergizes the resolution of single-cell data with the depth of bulk sequencing and enables improved cancer sub-group diagnoses and insights into their biology.

Spatial genomic, biochemical and cellular mechanisms underlying meningioma heterogeneity and evolution.

Intratumor heterogeneity underlies cancer evolution and treatment resistance, but targetable mechanisms driving intratumor heterogeneity are poorly understood. Meningiomas are the most common primary intracranial tumors and are resistant to all medical therapies, and high-grade meningiomas have significant intratumor heterogeneity. Here we use spatial approaches to identify genomic, biochemical and cellular mechanisms linking intratumor heterogeneity to the molecular, temporal and spatial evolution of high-grade meningiomas. We show that divergent intratumor gene and protein expression programs distinguish high-grade meningiomas that are otherwise grouped together by current classification systems. Analyses of matched pairs of primary and recurrent meningiomas reveal spatial expansion of subclonal copy number variants associated with treatment resistance. Multiplexed sequential immunofluorescence and deconvolution of meningioma spatial transcriptomes using cell types from single-cell RNA sequencing show decreased immune infiltration, decreased MAPK signaling, increased PI3K-AKT signaling and increased cell proliferation, which are associated with meningioma recurrence. To translate these findings to preclinical models, we use CRISPR interference and lineage tracing approaches to identify combination therapies that target intratumor heterogeneity in meningioma cell co-cultures.

Meningioma: International Consortium on Meningiomas (ICOM) consensus review on scientific advances & treatment paradigms for clinicians, researchers, and patients.

Meningiomas are the most common primary intracranial tumors in adults and are increasing in incidence due to the aging population and the rising availability of neuroimaging. While most exhibit non-malignant behaviour, a subset of meningiomas are biologically aggressive and lead to significant neurological morbidity and mortality. In recent years, meaningful advances in our understanding of the biology of these tumors have led to the incorporation of molecular biomarkers into their grading and prognostication. However, unlike other central nervous system tumors, a unified molecular taxonomy for meningiomas has not yet been established and remains an overarching goal of the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy-Not Official WHO (cIMPACT-NOW) working group. There also remains clinical equipoise on how specific meningioma cases and patient populations should be optimally managed. To address these existing gaps, members of the International Consortium on Meningiomas (ICOM) including field-leading experts, have prepared a comprehensive consensus narrative review directed towards clinicians, researchers, and patients. Included in this manuscript are detailed overviews of proposed molecular classifications, novel biomarkers, contemporary treatment strategies, trials on systemic therapies, health-related quality of life studies, and management strategies for unique meningioma patient populations. In each section we discuss the current state of knowledge as well as ongoing clinical and research challenges to road map future directions for further investigation.

Eed controls craniofacial osteoblast differentiation and mesenchymal proliferation from the neural crest.

The histone methyltransferase Polycomb repressive complex 2 (PRC2) is required for specification of the neural crest, and mis-regulation of neural crest development can cause severe congenital malformations. PRC2 is necessary for neural crest induction, but the embryonic, cellular, and molecular consequences of PRC2 activity after neural crest induction are incompletely understood. Here we show that Eed, a core subunit of PRC2, is required for craniofacial osteoblast differentiation and mesenchymal proliferation after induction of the neural crest. Integrating mouse genetics with single-cell RNA sequencing, our results reveal that conditional knockout of Eed after neural crest cell induction causes severe craniofacial hypoplasia, impaired craniofacial osteogenesis, and attenuated craniofacial mesenchymal cell proliferation that is first evident in post-migratory neural crest cell populations. We show that Eed drives mesenchymal differentiation and proliferation in vivo and in primary craniofacial cell cultures by regulating diverse transcription factor programs that are required for specification of post-migratory neural crest cells. These data enhance understanding of epigenetic mechanisms that underlie craniofacial development, and shed light on the embryonic, cellular, and molecular drivers of rare congenital syndromes in humans.