Comprehensive Molecular Profiling Identifies FOXM1 as a Key Transcription Factor for Meningioma Proliferation

Abstract

Meningioma is the most common primary intracranial tumor, but the molecular drivers of aggressive meningioma are incompletely understood. Using 280 human meningioma samples and RNA sequencing, immunohistochemistry, whole-exome sequencing, DNA methylation arrays, and targeted gene expression profiling, we comprehensively define the molecular profile of aggressive meningioma. Transcriptomic analyses identify FOXM1 as a key transcription factor for meningioma proliferation and a marker of poor clinical outcomes. Consistently, we discover genomic and epigenomic factors associated with FOXM1 activation in aggressive meningiomas. Finally, we define a FOXM1/Wnt signaling axis in meningioma that is associated with a mitotic gene expression program, poor clinical outcomes, and proliferation of primary meningioma cells. In summary, we find that multiple molecular mechanisms converge on a FOXM1/Wnt signaling axis in aggressive meningioma.

Keywords: DNA methylation; FOXM1; NF2; RNA sequencing; Wnt; epigenome; genome; meningioma; transcriptome; whole-exome sequencing.

Figure 1.. Study Design

Comprehensive molecular profiling strategy of meningioma (IHC^β-catenin, β-catenin IHC; IHC^FOXM, FOXM1 IHC; IHC, immunohistochemistry; DM, 850K DNA methylation profiling; NS, NanoString targeted gene expression profiling; RNA-seq, RNA sequencing; WES, whole-exome sequencing).

Figure 2.. RNA Sequencing Identifies Distinct Transcriptomic Clusters of Meningiomas and a Mitotic Signature Associated with FOXM1 Activity in Aggressive Tumors

(A) Unsupervised hierarchical clustering on the basis of the top 2,000 most variable genes segregates meningiomas into two transcriptomic clusters (n = 42). (B) Differential expression analysis of RNA-seq data on the basis of meningioma grade reveals a total of 2,012 significant genes (q < 0.1) with the greatest differences between WHO grade I and grade III meningiomas. (C) GO analysis for biological processes shows enrichment for cell division genes in WHO grade III meningiomas compared with WHO grade I meningiomas. (D) ChEA identifies FOXM1 as a putative regulator of the transcriptomic signature in WHO grade III meningiomas. (E) High-grade meningiomas are enriched in FOXM1 mRNA by RNA-seq (fragments per kilobase of transcript per million reads, FKPM). (F and G) Meningiomas with FOXM1 FKPM ≥ 1 are associated with poor LRFS (F) and OS (G) relative to meningiomas with FOXM1 FKPM < 1.

Figure 3.. FOXM1 Protein Expression Is Associated with Meningioma Proliferation and Poor Clinical Outcomes

(A) Hematoxylin staining with IHC for FOXM1 from two meningiomas reveals significant intra- and inter-tumor heterogeneity of FOXM1 expression. Scale bars, 100 μm. (B) Co-IHC for FOXM1 and the meningioma cell marker SSTR2A demonstrates that FOXM1 expression is restricted to a subpopulation of meningioma cells. Scale bar, 100 μm. (C) Co-IHC for FOXM1 and the cell proliferation marker Ki-67, denoted by the MIB1 clone, shows that proliferating meningioma cells are characterized by nuclear FOXM1 which decorates mitotic spindles (arrowhead). Scale bar, 25 μm. (D) Meningioma proliferation, denoted by MIB1 labeling index of the cell proliferation marker Ki-67, is associated with FOXM1 expression (p < 0.0001, n = 38 meningiomas). Dashed lines denote thresholds for subsequent outcomes analyses according to MIB1 labeling index and FOXM1 expression levels. (E and F) Elevated meningioma MIB1 labeling index is associated with poor LRFS (E) and OS (F) relative to meningiomas with low MIB1 labeling index (n = 40). (G and H) Elevated meningioma FOXM1 protein expression is associated with poor LRFS (G) and OS (H) relative to meningiomas with low FOXM1 protein expression (N = 52).

Figure 4.. Whole-Exome Sequencing Reveals High Somatic Mutation Burden in Aggressive Meningiomas

(A) Analysis of recurrent somatic mutations occurring in at least three tumors confirms that NF2 is the most commonly mutated gene in aggressive meningioma (n = 24). (B) The number of somatic mutations per meningioma ranges from 17 to 98, with 4–23 synonymous mutations and 13–75 nonsynonymous mutations. (C) The number of large-scale chromosomal alterations per meningioma, defined as comprising greater than one-third of a chromosomal arm, ranges from 0 to 10, with 0–3 amplifications and 0–10 deletions. (D) The number of somatic mutations per meningioma is associated with patient age for all meningioma grades (p < 0.0001). Dashed line denotes median somatic mutation count used as a cutoff for subsequent outcomes analyses according to mutation burden. (E) High meningioma somatic mutation burden is associated with poor DSS relative to low meningioma somatic mutation burden.

Figure 5.. DNA Methylation Profiling Identifies Distinct Epigenomic Clusters that Are Associated with Meningioma Somatic Mutation Burden and Clinical Outcomes

(A) Unsupervised hierarchical clustering on the basis of the top 2,000 most variable probes segregates meningiomas into three clusters according to high, low, and medium DNA methylation (n = 26). (B and C) High meningioma DNA methylation is associated with increased patient age (B) and elevated somatic mutation burden (C) relative to low and medium meningioma DNA methylation clusters (*p = 0.0123, **p < 0.0001). (D) High meningioma DNA methylation is associated with poor DSS relative to medium and low meningioma methylation clusters. (E) GREAT results for differentially methylated sites between high and low meningioma DNA methylation clusters identifies hypermethylation of H3K27me3 and PRC targets in the high-methylation cluster. (F and G) Meningiomas with increased FOXM1 mRNA expression by RNA-seq display decreased expression of PRC target genes BMP2 (F) and HOXD4 (G) (*p = 0.0010, **p = 0.0393).

Figure 6.. A FOXM1/Wnt Signaling Axis Promotes Cell Proliferation Gene Expression in Aggressive Meningioma

(A and B) Elevated meningioma FOXM1 mRNA expression by NanoString targeted gene expression profiling is associated with poor LRFS (A) and OS (B) relative to meningiomas with low FOXM1 mRNA expression (n = 96). (C and D) High-grade and recurrent meningiomas are enriched in FOXM1 mRNA by NanoString gene expression profiling. Red denotes FOMX1-high tumors, and blue denotes FOXM1-low tumors. (E) Meningioma β-catenin staining by IHC shows abundant cytoplasmic and occasional nuclear staining. Scale bar, 20 μm. (F) Meningioma FOXM1 and β-catenin nuclear staining and co-localization by immunofluorescence. Scale bar, 2 μm. (G) Meningioma nuclear β-catenin staining by IHC (n = 11) is associated with poor LRFS relative to meningiomas without nuclear β-catenin staining (n = 221). (H) Hypermethylation of the SFRP1 promoter by DNA methylation profiling is associated with poor LRFS relative to meningiomas without SFRP1 promoter hypomethylation (n = 24). (I) Meningiomas with elevated FOXM1 mRNA expression by RNA-seq display decreased SFRP1 expression (*p = 0.0378). (J) Unsupervised hierarchical clustering of meningiomas according to FOXM1 target gene expression (n = 69) by RNA-seq segregates tumors into two clusters (n = 42). (K and L) Elevated meningioma FOXM1 target gene expression is associated with poor LRFS (K) and OS (L) relative to meningiomas with low FOXM1 target gene expression. (M) A FOXM1 target protein-protein interaction (PPI) network constructed from genes enriched in high-grade meningiomas by RNA-seq highlights a cell proliferation program.

Figure 7.. FOXM1/Wnt Signaling Drives Primary Meningioma Cell Proliferation

(A–C) Immunofluorescence reveals an inverse relationship between SFRP1 and FOXM1 expression in M6 and M10 primary meningioma cells. Scale bar, 10 μm (*p < 0.0001, **p = 0.0008). (D) Immunofluorescence shows cytoplasmic and nuclear β-catenin staining in M10 primary meningioma cells. Scale bar, 5 μm. (E and F) Real-time qPCR and tetrazolium assays show a strong association between expression of FOXM1 (E), expression of FOXM1 target genes CCNA2 and CCNB2 (E), and proliferation of M3, M8, M12, BEN-MEN-1, and HBL-52 primary meningioma cells (F). (G) Overexpression of FOXM1-Myc induces HBL-52 primary meningioma cell proliferation as demonstrated by nuclear Ki-67 staining. Scale bar, 5 μm. (H and I) Real-time qPCR and tetrazolium assays demonstrate that overexpression of FOXM1 or CCNB1 induces expression of FOXM1 target genes CCNA2 and CCNB2 (H) and proliferation of BEN-MEN-1 primary meningioma cells (I) (*p < 0.002, **p < 0.02). (J and K) Tetrazolium assays in primary meningioma cells transduced with FOXM1 shRNAs, or treated with the FOXM1 antagonist FDI-6, show that FOXM1 knockdown (KD) and FOXM1 pharmacologic inhibition blocks primary meningioma cell proliferation (*p < 0.03). (L) An integrated molecular model of aggressive meningiomas, which are characterized by DNA hypermutation and hypermethylation. NF2 mutation stabilizes FOXM1 protein, and hypermethylation of H3K27me3 and PRC target genes facilitates a functional switch toward a FOXM1 transcriptional program. Hypermethylation of Wnt antagonists, such as SFRP1, activates β-catenin to cooperate with FOXM1 to drive expression of cell proliferation genes.