Follicular lymphoma (FL) and the GCB subtype of diffuse large B-cell lymphoma (DLBCL) derive from germinal center B-cells1. Targeted re-sequencing studies have revealed mutations in various genes in the NFkB 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 oncogene6 EZH2, which encodes a histone methyltransferase responsible for tri-methylating H3K27. After the recent discovery of mutations in the histone H3K27me3 demethylase UTX in several cancer types5, EZH2 is the second histone methyltransferase affected by mutations in cancer. These mutations, which replace a single tyrosine in the SET domain of the EZH2 protein (Y641), occur in 21.7% of GCB DLBCLs, 7.2% of FLs and are absent from ABC DLBCLs. Our data are consistent with the notion that EZH2 proteins with mutated Y641 have reduced enzymatic activity in vitro.
Advances in DNA sequencing technology have recently enabled the characterization of genomes and transcriptomes at sufficient resolution for identification of somatic point mutations7–9. To develop new insight into novel mutations potentially contributing to B-cell Non Hodgkin lymphomas (NHL), we used Illumina technology to sequence genomic DNA and RNA purified from a malignant lymph node biopsy obtained from a FL patient (“FL Sample A”)(Methods). FL patient A qualified as a grade 1 FL that co-expressed CD10, BCL2 and BCL6 by immuno-histochemistry. This sample was chosen for sequence analysis because it exhibited an unusually simple karyotype (Supplementary Figure 1), lacking t(14;18)(q32;q21), and other large-scale alterations (Supplementary Figures 2–5; Supplementary Tables 1 and 2). We analyzed the exon sequences of this tumor for mutations in both the genome (WGSS) and transcriptome (WTSS)(Table 1 and Methods). Matched constitutional DNA from the FL patient was sequenced to reveal “germline” sequence variants (Methods). We produced 25.6 aligned gigabases (Gb) from the tumor genomic library, yielding 9.47-fold redundant base coverage on average, and an additional 2.2 Gb of aligned sequence from the WTSS library, yielding 18.86-fold redundant base coverage on average within exons (Table 1; Methods). We focused our analysis on novel changes predicted to affect protein-coding sequence (Methods). Among these variants (Supplementary Table 3), we discovered a mutation affecting exon 15 of the EZH2 gene, which encodes a portion of the EZH2 SET domain. EZH2 is the catalytic component of the PRC2 complex, which is responsible for adding methyl groups to lysine 27 of histone 3 (H3K27)10, thereby repressing transcription at loci associated with histones bearing tri-methylated H3K27. We established that this mutation, which is predicted to replace Y641 with a histidine, was somatic in nature by confirming its presence in tumor DNA, and absence in constitutional non-tumor DNA (Methods). We also confirmed that the mutation was heterozygous in this patient sample.
To determine EZH2 mutational status in DLBCL samples, we next used WTSS to sequence the transcriptomes of 31 DLBCL patient samples and seven DLBCL cell lines. Based on cell of origin (COO) expression classification11, the primary lymphoma samples were classified as belonging to either the ABC (n=12), GCB (n=15) or unclassifiable subtypes (n=2) (Methods; Supplementary Table 4). Coverage of EZH2 in the WTSS libraries was consistently high, ranging in the patient samples from 5.3-fold to 187-fold redundant base-pair coverage (median 48.7-fold). Coverage of codon 641 ranged from 5-fold to 295-fold (median 46.5-fold; Supplementary Table 4). We identified Y641 mutations in four of these 31 patient samples and four of the cell lines (Table 2). No other mutations in EZH2 were detected and all mutations appeared to be heterozygous. The striking recurrence of these mutations suggested that mutation of Y641 in EZH2 was a common feature of lymphoma. Notably, despite a median base coverage depth of 11.4-fold in the UTX locus, we found no evidence for UTX mutations in these libraries.
We determined the prevalence of Y641 mutations in both FL and DLBCL tumors by Sanger sequencing the exon containing this codon in 251 FL samples, of which 30 had matched DLBCL samples taken at histological transformation and 320 primary DLBCL samples (including the original 31 patient samples)(Supplementary Table 5). This revealed a total of 18 FL and 35 DLBCL samples with heterozygous Y641 mutations. Of note, all Y641 mutations detected by WTSS demonstrated clear evidence for expression of both alleles (Supplementary Table 4). To search for additional sites mutated in this gene, we also sequenced all exons of EZH2 in 24 FL patients (in addition to FL patient A) and found only one example of an EZH2 mutation not affecting Y641 (Figure 1; Supplementary Table 5). This mutation, affecting N635, was found in conjunction with a Y641 mutation and we confirmed that both mutations were in a cis orientation. We confirmed that these mutations were somatic in the 7 FL (including “Sample A”) and 2 DLBCL patients for which germline DNA was available.
To exclude the possibility that such mutations can also occur in non-malignant germinal center B-cells or other types of lymphoma, we sequenced this region of exon 15 in eight CD77+-enriched centroblast samples from reactive tonsils and 23 reactive lymph nodes (a source of normal B-cells) and 80 samples of other lymphoma types using both Sanger sequencing and targeted ultra-deep Illumina re-sequencing (Methods; Supplementary Figure 6; Supplementary Table 6). We also sequenced WTSS libraries generated from two additional normal centroblast samples (Supplementary Table 4). Consistent with our hypothesis that Y641 mutations are unique to malignant B-cells, none of these samples showed evidence for mutations at Y641 or elsewhere within the sequenced region (Table 3). Notably, all of the DLBCL samples with known COO and which were also positive for EZH2 mutations were of the GCB subtype and not the ABC subtype. This revealed a significant enrichment of Y641 mutations among the GCB subtype of DLBCLs (Table 3; n=18/83 GCB vs 0/42 ABC, P = 0.00168, two-tailed Fisher’s Exact Test).
We next assessed the effect various Y641 mutations would have on the structure, and potentially the function, of the EZH2 SET domain by generating a computational model (Supplementary Figure 7) using the crystal structure of the highly conserved MLL1 SET domain12 as the structural template (Methods). Our model indicates that Y641 interacts with the lysine 27 side chain of the H3 histone tail, as has been suggested in other SET domain proteins13. Though no EZH2 SET domain mutations have been reported in humans, detailed mutant phenotypes have been described in Drosophila. A mutation altering the tyrosine orthologous to EZH2 Y641 has been characterized in the Drosophila ortholog E(z) in an allele known as “E(z)1”. Drosophila E(z)1 mutant protein was found to be incapable of tri-methylating H3K27 in vitro14.
We sought to directly determine whether EZH2 with mutated Y641 impacts the catalytic activity of PRC2 in a cell-free methylation assay. Individual clones, each containing one of the four most frequently detected mutations (Figure 1), were first expressed along with the other components of PRC2. PRC2 complexes were purified and tested in vitro for H3K27 tri-methylation activity using ELISA and an antibody specific for H3K27me3 (Methods). The results (Figure 2) indicated that, compared to wild-type EZH2, all four Y641 mutants consistently demonstrated a marked reduction (~7-fold) in their ability to tri-methylate the H3K27 peptide. This biochemical result suggested that the four predominant Y641 variants observed in our sequencing study could confer reduced ability of PRC2 complexes to tri-methylate H3K27 in vivo.
Other reports have suggested that increased abundance of EZH2 mRNA correlates with cancer progression in tissues in which EZH2 expression is normally low or undetectable, such as breast and prostate6,15. However, EZH2 mRNA is known to be abundant in normal germinal center B-cells16, and a conditional knock-out of the mouse EZH2 ortholog indicated that the SET domain is required for early B-cell development, including rearrangement of the immunoglobulin heavy chain (IGH) locus17. Given the apparent requirement for EZH2 in germinal center B-cells, it is possible that the mechanism by which EZH2 contributes to lymphomagenesis is distinct from the apparently straightforward increases in EZH2 mRNA abundance observed in breast6 and prostate15 cancers. Expression of both EZH2 and BMI1 (the catalytic component of PRC1) have been linked to the degree of malignancy of B-cell NHL and perturbations in the balance of the quantities of these two proteins has been suggested as an early event in lymphomagenesis18. However, mutation of EZH2 has not, to date, been implicated in B-cell malignancies or any other cancer.
Though the biological mechanism is not known, our findings suggest that EZH2 Y641 mutations, and possibly a reduction in H3K27 tri-methylation, have a role in the pathogenesis of GCB lymphomas. The well-studied Phe/Tyr switch19 site is known to regulate the number of methyl groups a SET domain-containing protein can add without compromising its overall catalytic activity. Although the Y641 residue is distinct from the Phe/Tyr switch site, the result of our in vitro experiment does not rule out the possibility that these mutations may alter the product (or target) specificity of EZH2. Our finding is particularly timely in light of recent studies demonstrating enhanced DNA methylation at PRC2 targets in lymphoma as compared to normal B-cells20,21. H3K27 tri-methylation via PRC2 can be a precursor to DNA methylation and, in some cases, DNA methyltransferase may be physically coupled with PRC222. Hence, Y641 mutations may contribute to the differential DNA methylation that has been observed at polycomb targets in FL20 and DLBCL21.
In conclusion, we identified novel recurrent somatic mutations affecting the EZH2 SET domain and have associated these with FL and DLBCL cases of only the GCB subtype. Our data indicate that mutation of the EZH2 SET domain is among the most frequent genetic events observed in GCB malignancies after t(14;18)(q32;q21). The mutated tyrosine corresponds to a key residue in the active site of the EZH2 protein and, consistent with functional studies of a comparable mutation in the Drosophila E(z) ortholog, we show that PRC2 complexes containing mutated EZH2 protein exhibit reduced H3K27 tri-methylation activity in vitro. We have shown that a wild-type copy of EZH2 is present in all samples with Y641 mutations and have also detected expression of both alleles in the mutant samples profiled by transcriptome sequencing. This, along with the fact that all lymphomas with mutations in EZH2 appear to have a mutation affecting Y641, sets EZH2 apart from the pattern of mutational inactivation seen in the case of UTX, which appears to behave as a tumor suppressor gene5. Aside from the recurrence of inactivating mutations in UTX, EZH2 is the only protein affecting H3K27 methylation status to be identified as a target of somatic mutation in cancer, and the first in which recurrent mutations of the SET domain appear restricted to a specific lymphoma subtype.