Recurrent somatic mutations in POLR2A define a distinct subset of meningiomas

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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 genes2,3, including WNT6 and ZIC1/ZIC4. In addition to mutations in POLR2A, NF2, SMARCB1, TRAF7, KLF4, AKT1, PIK3CA, and SMO4,5,6,7,8, 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.

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Figure 1: POLR2A mutations define a distinct subset of benign meningiomas.
Figure 2: Meningioma driver genes in five major pathways account for the formation of >80% of benign meningiomas.
Figure 3: Meningioma subgroups cluster according to their genome-wide transcription profile and show significant differences in the expression of critical developmental regulators.
Figure 4: Super-enhancer-driven genes classify meningioma subgroups.

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Acknowledgements

We are grateful to the patients and their families who have contributed to this study. This work was supported by the Gregory M. Kiez and Mehmet Kutman Foundation, the Yale School of Medicine, the NIH (Medical Scientist Training Program grant T32GM00720 to V.E.C. and M.W.Y.; NIH/NCI NRSA F30CA183530-03 to V.E.C.), and the Austrian Science Fund (FWF; Erwin Schrödinger Fellowship J3490 to D.H.). Partial funding for sequencing of the tumor samples was provided through a research agreement between Gilead Sciences, Inc., and Yale University. We gratefully acknowledge T. Boggon for constructive comments regarding the structure of RNA polymerase II and N. Ivanova for providing expertise on the maintenance of mouse embryonic stem cells.

Author information

V.E.C. performed whole-exome sequencing analysis, molecular inversion probe (MIP) sequencing and analysis, amplicon sequencing and analysis, chr22 copy-number assessment via qPCR, structural analysis of mutations, H3K27ac ChIP-seq of POLR2A mutant tumors, TRAF7 splice mutation characterization, clinical correlations, and sample selection. V.E.C., M.W.Y., J.F.B., and D.D. performed Sanger confirmation of detected mutations for the 775-meningioma cohort and sample preparation. A.S.H. performed expression microarray analysis, CNV analysis, and RNA-seq variant calling. B.J.A. called super-enhancers on H3K27ac meningioma ChIP-seq samples. A.S.H., H.B., and V.E.C. performed differential super-enhancer binding analysis. H.B. performed predication analysis of microarray on meningioma samples. V.E.C. and M.W.Y. performed RNA-seq analysis. K.Y. created the bioinformatics pipelines used for DNA-seq experiments (whole-exome, MIPs, and amplicon). O.H. performed KLF4 luciferase experiments. T.I.L., A.G.E.-S., A.S.W., D.H., M.S., B.K., E.Z.E.-O., G.C.-G., K.M.-G., J.E.G., J.S., R.G., J.M.P., A.O.V., J.M.G., K.B., R.A.Y., and M.G. assisted with sample preparation and/or experimental design. V.E.C., M.W.Y., and M.G. wrote the manuscript, and A.S.H., H.B., T.I.L. and R.A.Y. edited the manuscript. M.G. designed and oversaw the project.

Correspondence to Murat Günel.

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Competing interests

Partial funding for sequencing of the tumor samples was provided through a research agreement between Gilead Sciences, Inc., and Yale University.

Integrated supplementary information

Supplementary Figure 1 POLR2A-mutant meningiomas do not have recurrent interchromosomal translocations.

Circos plots generated from somatic whole-genome sequencing data of three POLR2A mutant tumor-blood pairs: (a) MN-60355; (b) MN-63345; (c) MN-63670. Only structural events with a supporting read count >25 were plotted. For breakpoint call coordinates, see Supplementary Table 2.

Supplementary Figure 2 POLR2A-mutant meningiomas do not have increased numbers of somatic DNA mutations or transcriptional errors compared with other subgroups.

(a) Barplots of the number of somatic deleterious coding mutations, somatic silent mutations, and somatic non-coding mutations from 45 meningioma exomes normalized per megabase of sequencing (n=11 POLR2A mutant, orange; n=16 NF2/Chr22 loss, blue; n=18 non-NF2 mutant, green). The number of coding and non-coding somatic mutations from whole-exome sequencing data is not statistically different between POLR2A mutant and other meningioma samples (p=0.08 for deleterious coding; p=0.4 for noncoding; p=0.82 for all mutations). (b, c) Variants were called from RNAseq data for 25 meningiomas (n=8 POLR2A mutant, orange; n=9 NF2/Chr22 loss, blue; n=8 non-NF2 mutant, green). The number of coding (b) or non-coding (c) transcript mutations is not statistically different between POLR2A mutant and other samples (p=0.16 for deleterious coding; p=0.49 for noncoding). Student’s t-test was used to calculate significance.

Supplementary Figure 3 Recurrent TRAF7 splice mutations promote intron inclusion, resulting in a 28-amino-acid in-frame insertion plus an S/C missense.

(a) Location of the recurrent canonical (green) and non-canonical (crimson) TRAF7 splice mutations. The number above the diamond indicates the number of meningiomas mutant at that position. (b) RNAseq read alignments of a TRAF7 splice mutant (top) and a TRAF7 splice wildtype (bottom) reveal that TRAF7 splice mutant meningiomas have reads spanning intron 11 (grey shading above reads). This is not observed in non-splice mutant TRAF7 meningiomas. (c) An enhanced view of the RNAseq reads reveals the reads spanning the intron contain the non-canonical splice mutation (G/A change 5 basepairs intronic). (d) Sanger sequencing of cDNA libraries created from TRAF7 splice wildtype meningiomas reveal that intron 10 is spliced. Top: alignment of the Sanger bases to the human mRNA reference sequence; Bottom: Sanger chromatograms. (e) Sanger sequencing of cDNA libraries created from TRAF7 splice mutant meningiomas reveals mRNA reads spanning intron 10. Top: alignment of the Sanger bases to the human genomic reference sequence; Bottom: Sanger chromatograms.

Supplementary Figure 4 Schematic of Sonic hedgehog (SHH) signaling.

Mutually exclusive somatic mutations in SMO, PRKAR1A and SUFU that activate SHH signaling were identified in the 775 meningioma cohort.

Supplementary Figure 5 Spike-in normalized RNA-seq data from POLR2A-mutant v6.5 mouse ESCs do not reveal evidence for global transcriptional amplification in this model.

(a) The FPKM (fragments per kilobase of exon per million mapped reads) was computed for spike-in control normalized RNAseq data from knock-in POLR2A mutant mES cells (two independent Q403K clones, A7 and B10) and compared to wildtype v6.5 mouse ES cells. Transcripts with an FPKM value of 1 or greater in wildtype cells were selected, and the density of transcripts with a given FPKM was plotted. The FPKM distribution is not significantly different between the samples, consistent with a lack of evidence for global transcriptional amplification. (b) In contrast, identical analysis of previously published spike-in normalized RNAseq data with high- and low- expression of c-Myc reveals a shift in the distribution of normalized FPKM in the cells with high expression of c-Myc, consistent with global transcriptional amplification.

Supplementary Figure 6 Gene expression clustering of benign meningiomas using non-PAM methods.

Clustering using (a) consensus, (b) NMF, (c) hierarchical, or (d) PCA methods resulted in 5 discrete subgroups that were separated by the underlying driver gene mutations. Grade I, genomically unstable NF2 samples were excluded from analysis.

Supplementary Figure 7 Super-enhancers with concordant changes in gene expression that are more active in meningiomas compared to dura.

(a-c) Shading represents high confidence super-enhancers that were detected in at least 2 samples per subgroup. Chromosomal coordinates are hg19. (d-e) Concordant differential gene expression and differentially-bound super enhancers between meningioma subgroups. Red points represent a differentially highly bound super-enhancer (FDR < 0.05) with concordant up-regulation of its nearby target gene (FDR < 0.05). Blue points represent a differentially lower bound super-enhancer that correlates with down-regulation of its target gene.

Supplementary Figure 8 Super-enhancers with concordant changes in gene expression that are more active in specific meningioma subgroups.

Shading represents high confidence super-enhancers that were detected in at least 2 samples per subgroup. Chromosomal coordinates are hg19.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Tables 4 and 5. (PDF 2662 kb)

Supplementary Table 1

Genomic and clinical characterization of 775 meningiomas. (a) Per-sample summary of clinical data, detected mutations, method of sequencing, method of structural variation analysis, method of transcriptomic analysis, and method of ChIPseq analysis (if applicable). Anatomical location key: ASB: Anterior skull base, MSB_med: Middle skull basemedial, MSB_lat: Middle skull baselateral, PF_antmed: Anterior, medial posterior cranial fossa, PF_postlat: Posterior, lateral posterior cranial fossa, Spinal: Spinal, ST_ant: Anterior supratentorial, ST_post: Posterior supratentorial, Ventricular: Intraventricular, Multiple: Multiple. Coordinates are hg19. Exome = Whole-exome sequencing. MIPs = next-generation sequencing screening of the coding sequences of NF2, SMARCB1, TRAF7, PIK3CA, PIK3R1, PRKAR1A, SUFU, SMO, AKT1 p.Glu17Lys and KLF4 p.Lys409Gln. Amplicon = next-generation screening of the coding sequences of NF2, TRAF7, SMO, AKT1 p.Glu17Lys and KLF4 p.Lys409Gln. P = primary; R = Recurrent. M = Male; F = Female; U = Unknown; N/A = Not performed. (b) Cohort summary of genomic and epigenomic approaches for 775 meningiomas. (XLSX 198 kb)

Supplementary Table 2

Somatic breakpoints from three whole-genome-sequenced POLR2A-mutant meningiomas and matching blood pairs. Somatic breakpoints from three whole-genome sequenced POLR2A mutant meningiomas and matching blood pairs called by Breakdancer35 and passing filters as described in the methods. Chromosomal coordinates are hg19. (XLSX 65 kb)

Supplementary Table 3

Somatic variants from whole-exome-sequenced meningiomas. Somatic variants passing quality filters. Coordinates are hg19. (XLSX 456 kb)

Supplementary Table 6

Alternative splicing between meningioma subgroups. Transcripts were assembled from RNA-seq data using the Tuxedo suite, and alternative splicing and differential promoter usage was calculated between subgroups using CummeRbund37,38. There are no significant differences in alternative splicing between meningioma subtypes. (TXT 65341 kb)

Supplementary Table 7

Differential promoter usage between meningioma subgroups. Transcripts were assembled from RNAseq data using the Tuxedo suite, and differential promoter usage was calculated between subgroups using CummeRbund37,38. There are no significant differences in differential promoter usage between meningioma subtypes. (TXT 42857 kb)

Supplementary Table 8

Alternative splicing between v6.5 mESC knock-in models of POLR2AQ403K (clones A7 and B10) and wild-type cells. Transcripts were assembled from RNA-seq data using the Tuxedo suite, and alternative splicing and differential promoter usage was calculated between subgroups using CummeRbund37,38. There are no significant differences in alternative splicing between POLR2A mutant mES cells and wildtype. (TXT 19096 kb)

Supplementary Table 9

Differential promoter usage between v6.5 mESC knock-in models of POLR2AQ403K (clones A7 and B10) and wild-type cells. Transcripts were assembled from RNAseq data using the Tuxedo suite, and differential promoter usage was calculated between subgroups using CummeRbund37,38. There are no significant differences in differential promoter usage between meningioma subtypes. (TXT 10795 kb)

Supplementary Table 10

PAM clustering signature genes for meningioma subgroups. (XLSX 510 kb)

Supplementary Table 11

Concordant differentially bound super-enhancers and differential gene expression. Comparisons are between all meningiomas and dura. (XLSX 873 kb)

Supplementary Table 12

Concordant differentially bound super-enhancers and differential gene expression: comparisons between meningioma subgroups. (XLSX 174 kb)

Supplementary Table 13

Concordant differentially bound super-enhancers in at least two samples and differential gene expression: comparisons between meningioma subgroups. Super-enhancers assigned to a group (e.g. POLR2A) were present in at least two samples. Superenhancers present in dura were excluded from analysis. (XLSX 398 kb)

Supplementary Table 14

Whole-exome sequencing metrics. (a) Mean depth of sequencing coverage for tumor exomes was 205.08. (b) Mean depth of sequencing coverage for matching blood exomes was 115.19. (XLSX 69 kb)

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Clark, V., Harmancı, A., Bai, H. et al. Recurrent somatic mutations in POLR2A define a distinct subset of meningiomas. Nat Genet 48, 1253–1259 (2016) doi:10.1038/ng.3651

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