Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin

Journal name:
Nature Genetics
Volume:
42,
Pages:
181–185
Year published:
DOI:
doi:10.1038/ng.518
Received
Accepted
Published online

Follicular lymphoma (FL) and the GCB subtype of diffuse large B-cell lymphoma (DLBCL) derive from germinal center B cells1. Targeted resequencing studies have revealed mutations in various genes encoding proteins in the NF-κB pathway2, 3 that contribute to the activated B-cell (ABC) DLBCL subtype, but thus far few GCB-specific mutations have been identified4. Here we report recurrent somatic mutations affecting the polycomb-group oncogene5 EZH2, which encodes a histone methyltransferase responsible for trimethylating Lys27 of histone H3 (H3K27). After the recent discovery of mutations in KDM6A (UTX), which encodes the histone H3K27me3 demethylase UTX, in several cancer types6, EZH2 is the second histone methyltransferase gene found to be mutated in cancer. These mutations, which result in the replacement of a single tyrosine in the SET domain of the EZH2 protein (Tyr641), occur in 21.7% of GCB DLBCLs and 7.2% of FLs and are absent from ABC DLBCLs. Our data are consistent with the notion that EZH2 proteins with mutant Tyr641 have reduced enzymatic activity in vitro.

At a glance

Figures

  1. Recurrent mutations of Tyr641 in EZH2.
    Figure 1: Recurrent mutations of Tyr641 in EZH2.

    (a) Genomic organization of the EZH2 locus, alternative exons and protein domain structure. The location of the mutation affecting Tyr641 in exon 15 of the EZH2 gene and protein is indicated with a red asterisk in each case. (b) Illustration of sequencing results. Three of the five distinct mutations and amino acid replacements in codon 641 from different lymphoma samples as detected by capillary sequencing (left) or Illumina WTSS (right). (c) A multiple alignment of EZH2, EZH1 (its paralog), the Drosophila ortholog E(Z) and six other human SET-domain proteins demonstrates the intra- and interspecies sequence conservation of SET domains. Conservation codes reported by ClustalX are shown above24. The predominant mutation in EZH2 affects a key tyrosine in the catalytic site of the SET domain (orange) conserved in the Drosophila ortholog E(Z). With one exception, all EZH2 mutations found in FL and DLBCL alter this amino acid. The exception was a double mutant (FL) with a second somatic mutation affecting Asn635 (blue). The mutants identified comprise five of the eight possible nonsynonymous variants of this codon (lower right, in red). Notably, the five observed amino acid changes were not found at equal frequencies. We detected a slight enrichment for Y641F (49%), with lower frequencies for Y641S (21%), Y641N (15%) and Y641H (13%), and only a single example of Y641C (2%) (Supplementary Table 5). Of the unobserved variants (blue), two would result in a truncated protein and the third would introduce an aspartate residue. The pattern and nature of these changes (A→G, A→T, T→G, T→A) indicated to us that these mutations are not likely to arise from activation-induced cytidine deaminase (AID)-induced somatic hypermutation at this locus25.

  2. In vitro assembly and functional analysis of PRC2 with mutant and wild-type EZH2.
    Figure 2: In vitro assembly and functional analysis of PRC2 with mutant and wild-type EZH2.

    (a) Wild-type EZH2 and each of the four Tyr641 mutants were coexpressed along with wild-type AEPB2, EED, SUZ12 and RbAp48 in Sf9 cells using a baculovirus expression system (Online Methods). Together, these five proteins associate to form an enzymatically active PRC2 complex in vitro. The purified complex from the Sf9 cells showed strong expression of each of these proteins and confirmed their association and assembly into PRC2. (b) Expression of EZH2 protein from each of the four mutant constructs was confirmed by protein blotting. (c) The purified complex was then assayed using biotinylated histone H321–44 peptide along with S-adenosylmethionine (in the assay buffer) to detect enzyme activity. Methylated histone H3 was measured using a highly specific antibody that recognizes only the trimethylated Lys27 residue of histone H3 (Online Methods). Europium-labeled secondary antibody was detected by time-resolved fluorescence (620 nm). PRC2 methylase activity of each mutant (and of wild-type EZH2) was tested at varying amounts of purified PRC2 (between 0 and 200 ng). The specific activities for the four mutants were calculated to be 0.001, 0.0012, 0.0011 and 0.0009 pmol/min/μg for the Y641H, Y641N, Y641S and Y641F mutants, respectively (mean = 0.00105). The wild-type enzyme (blue) showed a specific activity of 0.0071 (~6.8-fold greater). Error bars reflect the s.d. of triplicate measurements.

Accession codes

Referenced accessions

GenBank/EMBL/DDBJ

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Author information

Affiliations

  1. Genome Sciences Centre, BC Cancer Agency, Vancouver, British Columbia, Canada.

    • Ryan D Morin,
    • Tesa M Severson,
    • Andrew J Mungall,
    • Jianghong An,
    • Rodrigo Goya,
    • Jessica E Paul,
    • Obi L Griffith,
    • Richard Corbett,
    • Angela Tam,
    • Richard Varhol,
    • Duane Smailus,
    • Michelle Moksa,
    • Yongjun Zhao,
    • Allen Delaney,
    • Hong Qian,
    • Inanc Birol,
    • Jacqueline Schein,
    • Richard Moore,
    • Robert Holt,
    • Steven Jones,
    • Martin Hirst &
    • Marco A Marra
  2. BC Cancer Agency, Vancouver, British Columbia, Canada.

    • Nathalie A Johnson,
    • Merrill Boyle,
    • Bruce W Woolcock,
    • Florian Kuchenbauer,
    • Damian Yap,
    • R Keith Humphries,
    • Sohrab Shah,
    • Joseph M Connors &
    • Samuel Aparicio
  3. BPS Biosciences, San Diego, California, USA.

    • Henry Zhu,
    • Michelle Kimbara,
    • Pavel Shashkin,
    • Jean F Charlot &
    • Marianna Tcherpakov
  4. Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada.

    • Doug E Horsman &
    • Randy D Gascoyne
  5. Division of Medical Oncology, University of British Columbia, Vancouver, British Columbia, Canada.

    • Joseph M Connors
  6. Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.

    • Marco A Marra

Contributions

M.A.M., R.D.G., D.E.H. and J.M.C. conceived of the study and led the design of the experiments. R.D.M. performed the WGSS and WTSS analysis, produced Figures 1 and 2 and, with M.A.M., wrote the manuscript. N.A.J. prepared the samples, performed sample sorting and COO analysis and contributed to the text. O.L.G. and R.D.M. analyzed gene expression data. T.M.S., A.J.M. and J.E.P. performed sequence validation experiments and visual inspection of capillary sequence data. D.S. and M.M. constructed multiplexed libraries for deep resequencing of EZH2. H.Z., M.K., P.S., J.F.C., D.Y. and M.T. conducted enzymatic assays. I.B. performed statistical analysis and contributed to the manuscript. J.A. and S.J. produced the model for EZH2 and contributed to the manuscript. M.B. and B.W.W. prepared samples and performed FACS. F.K. and R.K.H. validated expression findings in the RNA. A.D., H.Q., R.C. and S.S. performed copy number analysis. A.T., Y.Z., R.H., M.H. and R.M. produced the sequencing libraries and performed the sequencing. R.V. processed raw sequencing data. R.G. identified candidate mutations. J.S., M.H. and S.A. conceived of experiments and contributed to the text.

Competing financial interests

Authors H.Z., M.K., P.S., J.F.C. and M.T. are employees of BPS Bioscience. This company manufactures commercial EZH2 assay kits.

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Supplementary information

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    Supplementary Figures 1–7 and Supplementary Tables 1–7

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