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PRC2 is recurrently inactivated through EED or SUZ12 loss in malignant peripheral nerve sheath tumors

Nature Genetics volume 46, pages 1227–1232 (2014)Cite this article

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Abstract

Malignant peripheral nerve sheath tumors (MPNSTs) represent a group of highly aggressive soft-tissue sarcomas that may occur sporadically, in association with neurofibromatosis type I (NF1 associated) or after radiotherapy1,2,3. Using comprehensive genomic approaches, we identified loss-of-function somatic alterations of the Polycomb repressive complex 2 (PRC2) components (EED or SUZ12) in 92% of sporadic, 70% of NF1-associated and 90% of radiotherapy-associated MPNSTs. MPNSTs with PRC2 loss showed complete loss of trimethylation at lysine 27 of histone H3 (H3K27me3) and aberrant transcriptional activation of multiple PRC2-repressed homeobox master regulators and their regulated developmental pathways. Introduction of the lost PRC2 component in a PRC2-deficient MPNST cell line restored H3K27me3 levels and decreased cell growth. Additionally, we identified frequent somatic alterations of CDKN2A (81% of all MPNSTs) and NF1 (72% of non-NF1-associated MPNSTs), both of which significantly co-occur with PRC2 alterations. The highly recurrent and specific inactivation of PRC2 components, NF1 and CDKN2A highlights their critical and potentially cooperative roles in MPNST pathogenesis.

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Figure 1: The most frequent genetic alterations in MPNST (NF1 associated, sporadic, radiotherapy associated and epithelioid) and neurofibroma.
Figure 2: MPNSTs with PRC2 loss exhibit distinct gene expression pattern from MPNSTs with wild-type PRC2, signifying activation of developmentally suppressed pathways.
Figure 3: H3K27me3 immunohistochemistry correlates with PRC2 genetic status, and H3K27me3 loss characterizes progression from neurofibroma to MPNST.
Figure 4: PRC2 loss promotes cell proliferation and growth in MPNST with PRC2 loss.

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Acknowledgements

Next-generation sequencing and SNP6.0 analysis were performed at the Memorial Sloan Kettering Cancer Center (MSKCC) Genomics Core Facility, Center for Molecular Oncology (CMO). The authors thank members of the cBioPortal team (J. Gao, B. Gross, N. Schultz and C. Sander) for assistance with data analysis and visualization. The authors also thank T. Chan (MSKCC) and HOPP informatics for assistance with data analysis. This work was supported in part by a grant from the US National Institutes of Health (NIH) to W.L. (NCI U24-CA143840), the Harry J. Lloyd Trust–Translational Research grant to T.W., the Charles H. Revson Senior Fellowship to T.W., Jubilaeumsfonds of the Oesterreichische Nationalbank to T.W., grants from the US NIH to P.C. (P50CA140146, Career Development Award and Developmental Research Project.; DP2CA174499; K08CA151660), Y.C. (K08CA140946), L.-X.Q. (P50CA140146), C.R.A. (P50CA140146) and S.S. (P50CA140146), an award from the Sidney Kimmel Foundation to P.C. (Kimmel Scholar Award) and funding from the Cycle for Survival Fund to P.C.

Author information

Author notes

  1. William Lee, Sewit Teckie, Thomas Wiesner and Leili Ran: These authors contributed equally to this work.

Authors and Affiliations

  1. Computational Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    William Lee

  2. Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Sewit Teckie, Thomas Wiesner, Leili Ran, Sinan Zhu, Zhen Cao, Yupu Liang, Michael F Berger, Yu Chen & Ping Chi

  3. Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    William Lee & Sewit Teckie

  4. Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Carlos N Prieto Granada, Michael F Berger & Cristina R Antonescu

  5. Department of Genetics, Albert Einstein College of Medicine, Bronx, New York, USA

    Mingyan Lin & Deyou Zheng

  6. Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, New York, USA

    Andrea Sboner

  7. Institute for Computational Biomedicine, Weill Cornell Medical College, New York, New York, USA

    Andrea Sboner

  8. Institute for Precision Medicine, Weill Cornell Medical College, New York, New York, USA

    Andrea Sboner

  9. Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    William D Tap, Yu Chen & Ping Chi

  10. Department of Medicine, Weill Cornell Medical College, New York, New York, USA

    William D Tap, Yu Chen & Ping Chi

  11. Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts, USA

    Jonathan A Fletcher

  12. Genomics Core Laboratory, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Kety H Huberman & Agnes Viale

  13. Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Li-Xuan Qin

  14. Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Samuel Singer

  15. Department of Neurology, Albert Einstein College of Medicine, Bronx, New York, USA

    Deyou Zheng

  16. Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York, USA

    Deyou Zheng

Authors
  1. William Lee
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Contributions

Project planning and experimental design: P.C., C.R.A., Y.C., T.W., W.L., S.T. and L.R. Sample collection, clinical database and cell lines: S.T., S.S., C.R.A., C.N.P.G., J.A.F. and W.D.T. Pathology review: C.R.A. and C.N.P.G. Preparation of DNA, RNA and next-generation sequencing libraries: T.W., K.H.H., S.T. and A.V. Sequence data analysis: W.L., Y.C., S.T., P.C., M.F.B., M.L., D.Z., Y.L. and A.S. Immunohistochemistry: T.W. Protein blots, immunofluorescence, growth curves and all cellular assays: L.R. and S.Z. Generation of the expression vectors: Z.C. and L.R. Biostatistics: L.-X.Q. Manuscript writing: P.C., Y.C., W.L., T.W. and L.R. The final manuscript was reviewed by all authors.

Corresponding author

Correspondence to Ping Chi.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Summary of copy number variation by SNP6.0 array of MPNSTs.

(a) Whole-genome view of copy number variations (CNVs) using an integrative genome browser (IGV) in the 15 MPNST samples. The clinical subtype of MPNST is shown on the left: NF1 associated (green), sporadic (yellow), RT associated (red) and epithelioid (blue). (b) Zoomed-in view of 1-Mb windows around CDKN2A, EED, TP53 and NF1-SUZ12. Somatic single-nucleotide variations (SNVs) and indels are annotated by red asterisks. Transcript alterations seen on RNA-seq likely due to DNA structural variations are annotated by red plus signs.

Supplementary Figure 2 EED alterations in MPNST.

(a) Example of an EED splice-site mutation in sample 14T. DNA sequencing shows a donor splice-site mutation of exon 8 of the remaining intact allele. (The green line indicates an SNV relative to the reference genome, and all other SNVs represent SNPs found in both germline and tumor samples.) RNA-seq analysis shows that the EED transcript aberrantly splices from exon 7 into exon 10, skipping exons 8 and 9. The normal RNA splicing pattern of a reference EED-intact tumor (sample 21T) is shown as a reference. (b) Scatter plot of the allelic fraction of SNPs from the SNP6.0 array analysis of chromosome 11 for tumors 15T and 16T. The data show that the entire chromosome 11 in both tumors has undergone copy-neutral LOH.

Supplementary Figure 3 SUZ12 structural variants in MPNST.

(a) Copy number view of SUZ12 in seven MPNST samples that have either copy number loss of copy-neutral LOH. Samples 2T, 7T and 13T have structural variants of the remaining allele. Samples 8T and 10T have homozygous deletion. Samples 12T and 9T have intact wild-type RNA. (b) Sashimi plot showing that sample 2T has SUZ12 transcript truncation after exon 6 (sample 21T with intact transcript is shown as a reference). (c) RNA-seq view of sample 7T showing loss of the transcript in the middle of exon 10. DNA sequencing shows that there is a translocation at this position (21T is shown as a reference). (d) Sashimi plot showing that sample 13T has SUZ12 transcript truncation after exon 4 with reads mapping to intron 4 (21T is shown as a reference).

Supplementary Figure 4 Coexistent subpopulations as calculated by EXPANDS analysis of sample 16T exome sequencing data.

Eight subpopulations (SPs) were detected within the tumor, with the most prevalent being present in 84% of the tumor and containing the NF1 D1237_splice mutation. Subpopulation 3 is present in 57% of the sample and contains the EED mutation encoding p.Glu249fs, suggesting that the NF1 mutation occurred first in the progression of this sporadic MPNST. For each of 526 somatic mutations (x axis), the squares designate the SP to which the mutation has been assigned, the circles represent the copy number of the genomic locus of the mutation, and the asterisks represent the allele frequency of the mutation. Colors represent the chromosome where the mutation is located. SPs are sorted from most prevalent to least prevalent.

Supplementary Figure 5 RNA-seq profiles of MPNST cell lines ST88-14 and MPNST724 at the SUZ12 locus showing the absence of SUZ12 transcript in ST88-14 cells.

Supplementary Figure 6 Western blot of NF1, SUZ12 and various chromatin marks with introduction of Flag-HA-tagged SUZ12 (FH-SUZ12) and control vector (Flag-HA-tagged GUS) in PRC2-wild-type MPNST724 and SUZ12-deficient ST88-14 human MPNST cells.

*, exogenous Flag-HA-tagged SUZ12; **, endogenous SUZ12.

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Lee, W., Teckie, S., Wiesner, T. et al. PRC2 is recurrently inactivated through EED or SUZ12 loss in malignant peripheral nerve sheath tumors. Nat Genet 46, 1227–1232 (2014). https://doi.org/10.1038/ng.3095

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