Recurrent somatic mutations in POLR2A define a distinct subset of meningiomas

Abstract

RNA polymerase II mediates the transcription of all protein-coding genes in eukaryotic cells, a process that is fundamental to life. Genomic mutations altering this enzyme have not previously been linked to any pathology in humans, which is a testament to its indispensable role in cell biology. On the basis of a combination of next-generation genomic analyses of 775 meningiomas, we report that recurrent somatic p.Gln403Lys or p.Leu438_His439del mutations in POLR2A, which encodes the catalytic subunit of RNA polymerase II (ref. 1), hijack this essential enzyme and drive neoplasia. POLR2A mutant tumors show dysregulation of key meningeal identity genes, including WNT6 and ZIC1/ZIC4. In addition to mutations in POLR2A, NF2, SMARCB1, TRAF7, KLF4, AKT1, PIK3CA, and SMO, we also report somatic mutations in AKT3, PIK3R1, PRKAR1A, and SUFU in meningiomas. Our results identify a role for essential transcriptional machinery in driving tumorigenesis and define mutually exclusive meningioma subgroups with distinct clinical and pathological features.

Figure 1. POLR2A mutations define a distinct subset of benign meningiomas

(a) The recurrent somatic POLR2A mutations localize to the highly conserved dock domain, where interactions with TFIIB mediate the formation of the pre-initiation complex. Alterations found in human meningioma were mapped to the Saccharomyces cerevisiae RPB1 structure (PDB ID 1R5U). Green, RPB1 p.Gln403Lys equivalent residue; teal, RPB1 p.Leu438_His439del equivalent residues; yellow, zinc ion. (b) Alignment of the protein sequences of the large Pol II subunit for seven species shows the dock domain, including the two residues altered by recurrent meningioma mutations, to be highly conserved. (c.d) Representative Sanger chromatograms of recurrent POLR2A mutations in tumor and matching blood show that the mutations are somatic. Mutant alleles are confirmed by RNA-seq reads. gDNA, genomic DNA. (e) A representative Circos plot of a whole-genome-sequenced POLR2A meningioma does not show any large-scale chromosomal rearrangements. (f) The percentage of the genome altered by copy-number variations between meningioma subgroups. The low proportion of the genome altered by copy-number variation was similar in POLR2A mutant tumors and other non-NF2 mutant meningiomas. (g) The number of somatic mutations (normalized per megabase pair of sequencing data) from whole-exome sequencing data that are protein-altering and predicted to be damaging. In f and g, lines indicate median values, box edges indicate 25th (bottom) and 75th (top) percentiles, and whiskers and points show the outliers. (h) POLR2A meningiomas are enriched along the skull base, near the tuberculum sellae region (highlighted in pink).

Figure 2. Meningioma driver genes in five major pathways account for the formation of >80% of benign meningiomas

(a) The distribution of the 182 TRAF7 mutations showed highly recurrent missense mutations that clustered in the WD40 repeat domains or mutations affecting the splice junction immediately upstream of the WD40 repeat domain (n = 12), but not frameshift or nonsense mutations. Del, deletion; ins, insertion; aa, ammo acid. (b) Alignment of TRAF7 WD40 repeats. Left: the previously reported R1–2, R1, and D1 WD40 mutation hot spots. Boxes A, B. C, and D identify recurrent structural motifs shared by WD40 repeat domains. Blue-shaded amino acids mark mutations detected in meningioma, whereas red-shaded amino acids represent highly recurrent meningioma mutations (seen in more than ten tumors). Right: typical WD40 repeat (here, FBW7 WD40 repeat 3, PDB ID 2QVQ) with color-coded structural motifs and hot-spot mutations. (c) Recurrent TRAF7 splice mutations promote intron inclusion, resulting in a 28-amino-acid in-frame insertion plus a Ser-to-Cys missense alteration. (d) Distribution of PIK3CA mutants showing hot spots at p.Glu545 and p.His1047, which have been previously reported to result in constitutive PI3K signaling^, . (e) Distribution of the driver genes detected in the benign (WHO grade I) meningioma cohort. Each bar represents a meningioma sample. Only genes screened via targeted next-generation sequencing and/or Sanger sequencing are plotted. Our analysis identified somatic mutations in POLR2A, PIK3R1, SUFU, and PRKAR1A that were mutually exclusive with the previously identified meningioma genes. (f) Benign meningiomas form mutually exclusive subgroups based on mutational background.

Figure 3. Meningioma subgroups cluster according to their genome-wide transcription profile and show significant differences in the expression of critical developmental regulators

(a) Unsupervised hierarchical clustering of gene expression in meningioma tumor samples. The width of each clustering silhouette is a measure of the tumor’s match within the indicated subgroup. Each point represents a single tumor. (b) The number of clusters used for analysis was chosen to maximize the mean width of the clustering silhouette. Clustering with five subgroups gave optimal results. (c) Subgroup-specific genes were selected for heat-map generation in a supervised approach via a classification algorithm. For each subgroup, representative genes and the pathways they belong to are presented on the right. Upregulated genes are shown in red, and downregulated ones in blue. Pathway enrichment was performed using a hypergeometric test: *P < 0.05, **q <0.1. (d) Samples clustered according to underlying driver mutation, q-values were calculated via two-sided Fisher’s exact test.

Figure 4. Super-enhancer-driven genes classify meningioma subgroups

(a) Unsupervised hierarchical clustering based on super-enhancer binding classifies meningioma subgroups. Super-enhancer binding scores were derived from affinity based on H3K27ac ChIP-seq read counts. (b–d) Concordant differential gene expression and differentially bound super-enhancers between meningioma subgroups. Red points represent a differentially high-binding super-enhancer (FDR < 0.05) with concordant upregulation of its nearby target gene (FDR < 0.05). Blue points represent a differentially low-binding super-enhancer that correlates with downregulation of its target gene. (e,f) Differential super-enhancer binding and gene expression was detected at the WNT6/WNT10A (e) and ZIC1/ZIC4 (f) loci in POLR2A mutant tumors compared with other meningioma subgroups and control dura (‘normal’). Background shading represents super-enhancers present in two or more samples per subtype. Expression is log₂ transformed, rpkm, reads per kilobase of transcript per million mapped reads. (g) Schematic outlining the roles of WNT6 and ZIC1 in controlling neural crest development and meningeal cell differentiation. During embryonic development, WNT6 is expressed and secreted by the non-neural ectoderm (purple) to trigger induction of neural crest cells (light blue), whereas ZIC1 is expressed by neural crest cells and is a marker for meningeal cells. (h) Compared to the wild-type KLF4 consensus binding sequence, the two 3′-most nucleotides of the KLF4 p.Lys409Gln mutant consensus DNA binding sequence were altered to T and G, respectively. (i) Using luciferase assays, we confirmed that KLF4 p.Lys409Gln binds to this novel mutant motif in vitro, activating higher downstream gene expression than either the control or wild-type KLF4. Error bars represent s.d., calculated over three replicates.