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

Abstract

Follicular lymphoma (FL) and the GCB subtype of diffuse large B-cell lymphoma (DLBCL) derive from germinal center B cells. Targeted resequencing studies have revealed mutations in various genes encoding proteins in the NF-kappaB pathway that contribute to the activated B-cell (ABC) DLBCL subtype, but thus far few GCB-specific mutations have been identified. Here we report recurrent somatic mutations affecting the polycomb-group oncogene 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 types, 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.

Figure 1. Recurrent mutations of Y641 in EZH2

(A) Genomic organization of the EZH2 locus, alternative exons and protein domain structure. The location of the mutation affecting Y641 in exon 15 of the EZH2 gene and protein is indicated with a red asterisk. (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 inter-species sequence conservation of SET domains. Conservation codes reported by ClustalX are shown above. 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 in FL and DLBCL alter this amino acid. The exception was a double mutant (FL), with a second somatic mutation affecting N635 (blue). All mutants comprise 5 of the 8 possible non-synonymous 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%) followed by Y641S (21%), Y641N (15%) and Y641H (13%) and only a single example of Y641C (2%)(Supplementary Table 5). Of the unobserved variants (D, 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 do not likely arise from AID-induced somatic hypermutation at this locus.

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 Y641 mutants were co-expressed along with wild-type AEPB2, EED, SUZ12 and RbAp48 in SF9 cells using a baculovirus expression system (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 Western blot. (C) The purified complex was then assayed using biotinylated histone H3 (21-44) peptide along with S-adenosylmethionine (in the assay buffer) to detect enzyme activity. Methylated histone H3 was measured using a highly specific antibody, which recognizes only the tri-methylated K27 residue of histone H3 (Methods). The secondary antibody, which is labeled with Europium, was detected using time-resolved fluorescence (620nm). PRC2 methylase activity of each mutant (and wild-type EZH2) was tested at varying purified PRC2 amounts (between 0 and 200ng). The specific activity for the four mutants was calculated to be 0.001, 0.0012, 0.0011 and 0.0009 pmol/min/ug for the H, N, S and F 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 standard deviation of triplicate measurements.