Tyrosine 641 of the EZH2 Oncogene Is Frequently Mutated in Follicular and Diffuse Large B-Cell Lymphomas of Germinal Center Origin.

Diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) constitute 70% of all non-Hodgkin lymphomas (NHL). Both malignancies derive from germinal center (GC) B-cells and are characterized by clinical and genomic heterogeneity. Chromosomal alterations that deregulate oncogenes as well as mutations in genes involved in cell proliferation and apoptosis have been described in DLBCL. This disease can also be divided into distinct molecular subtypes by gene expression profiling. Differences observed between the activated B-cell (ABC) and germinal center B cell (GCB) subtypes result in part from distinct genomic alterations (Lenz, PNAS 2008) . For example, recent targeted re-sequencing efforts have revealed mutations in various genes in the NFkB signaling pathway, which likely contribute mainly to the ABC subtype (Compagno, Nature 2009). Thus far, few GCB-specific mutations other than t(14;18) have been identified.

Targeted re-sequencing studies are only able to reveal mutations in pre-selected candidate genes and so much of the genome has not yet been investigated in lymphomas. To obtain a more global view of the mutational landscape of NHL, we applied Illumina massively parallel second-generation sequencing to sequence the genomic DNA of a FL tumor sample. In parallel, we sequenced the transcriptomes (i.e. mRNA) of 31 DLBCL tumor samples using the same technology.

Genomic sequencing revealed a mutation altering tyrosine 641 (Y641) in the polycomb group oncogene Enhancer of Zeste Homolog 2 (EZH2), a gene responsible for trimethylating lysine 27 on histone H3 (H3K27). Mutations affecting the same codon were also observed in 4 DLBCL samples. These mutations were confirmed to be somatic in nature by Sanger sequencing exon 15 in tumor DNA and germline DNA derived from peripheral blood in these 5 patients. Sanger sequencing all coding exons in EZH2 from an additional 25 FL samples revealed that mutations were restricted to codon 641. These mutations could change the tyrosine residue to Asparagine, Serine, Phenylalanine or Histidine. The frequency of EZH2 Y641 mutations in GCB-derived lymphomas was determined by Sanger sequencing exon 15 in a total of 479 lymphoma samples. EZH2 (Y641) mutations occurred predominantly in lymphomas of the GCB type: 18/80 (22%) GCB-type de novo DLBCL, 6/52 (11.5)% of “transformed” DLBCL derived from FL and 16/225 (7.1%) of pre-treatment FL samples. No EZH2 mutations were found in 42 DLBCL of the ABC subype, 25 mantle cell lymphomas, 30 small lymphocytic lymphomas or 25 T cell lymphomas or 23 reactive tonsils. Introducing each of the four Y641 mutations into wild-type EZH2 resulted in 15-fold reduction in the ability to trimethylate lysine 27 on H3 peptides in-vitro.

Over-expression of EZH2 is thought to drive malignancy in a variety of solid tumors but mutations in this gene have never been described. The trimethylated H3K27 epigenetic mark is used to silence the transcription of genes involved in differentiation and hematopoiesis. In Drosophila, the phenotypic consequence of mutating the orthologous tyrosine residue in the E(z)(“Enhancer of Zeste”) gene indicates an apparent gain-of-function (Jones, Genetics 1990). Taken together, our mutation frequency and biochemical data are compatible with the notion that alteration of the ability of germinal center B-cells to trimethylate H3K27 may promote the development of FL and the GCB subtype of DLBCL.

Zhu:BPS Biosciences: Employment. Kimbara:BPS Biosciences: Employment. Shashkin:BPS Biosciences: Employment. Charlot:BPS Biosciences: Employment. Tcherpakov:BPS Biosciences: Employment.