Identification of Oncogenic EZH2 Mutations In Myelodysplastic Syndromes and Related Myeloid Malignancies

Abstract 607

Using single nucleotide polymorphism arrays (SNP-A) as a karyotyping tool led to the recognition that segmental somatic uniparental disomy (UPD) is a common and important defect in myelodysplastic syndromes (MDS) and related conditions. Homozygous mutations associated with UPD have been found. Additionally, SNP-A-detected submicroscopic deletions have proven helpful in narrowing the search for mutant genes to shared regions of loss of heterozygosity (LOH). Mapping of such microdeletions was instrumental in the identification of TET2 and CBL mutations, frequently found in a homozygous constellation within UPD of the corresponding chromosomal regions. UPD7q is one of the most frequent areas of UPD present in MDS, MDS/MPN and sAML. We hypothesized that this region may be associated with the presence of pathogenic mutations in a homozygous configuration. We utilized two strategies: i) because of the large size of the area commonly affected by UPD7, we applied next generation sequencing (NGS) for the identification of affected genes, and ii) we systematically sequenced genes contained in the overlapping deleted regions.

In total, 15 patients with UPD7q were identified. Exome libraries were generated from 2 cases with UPD7q and subjected to sequencing. In one, a missense C to T mutation was found at nt148137376 of EZH2 (100% of sequences), resulting in R690H. The somatic origin of this homozygous mutation was confirmed by sequencing of DNA from a skin biopsy. Simultaneously, we identified 2 patients with overlapping microdeletion involving 7q36.1. The minimally affected area involved 2 genes: CUL1 and EZH2. Sequenced all exons of both genes revealed no mutations in CUL1. However, a mutation in EZH2 exon 19 was identified in proximity to the mutation detected through NGS. The mutation involved position Ile715 producing a frame shift. We detected EZH2 mutations in 10/351 patients (7% of MDS/MPN, 2% of MDS and 0.9% of AML). We found 8 different EZH2 mutations present in 4/15 (27%) of cases with UPD7q, 2/30 (7%) patients with deletion 7q and 4/306 (1%) without LOH. Of note is that both hemizygous mutations were found in patients with microdeletions and none of the patients with large del(7q) or monosomy 7 harbored EZH2 mutation. There were 8 missense and 2 frame shift mutations, located in the SET domain of the EZH2 gene. Diagnoses included 3 cases of refractory cytopenia with multilineage dysplasia, while 5 had myelomonocytoid malignancies, and one each with MDS/MPN unclassifiable and atypical chronic myeloid leukemia (aCML). Overall, EZH2 mutations were detected in 4/53 (8%) cases of CMML, consistent with the high prevalence of somatic UPD7 in this disease. Interestingly, no EZH2 mutant case had a chromosome 7 abnormality by metaphase cytogenetics, but SNP-A karyotyping detected cryptic LOH7q in 6/10 patients with EZH2 mutations. EZH2 is a polycomb associated gene encoding a methyltransferase targeting H3K27, thereby producing a repressive mark. Loss of function or hypomorphic mutations of EZH2 would thus be predicted to result in abrogation of inhibitory chromatin marks and chromatin decompaction, conducive to expression of oncogenes. Immunofluorescence and western blot showed that EZH2 mutations in myeloid malignancies lead to decreased methylation of H3K27 and thereby are functionally relevant. Expression analysis of EZH2 did not show significantly decreased expression in MDS or haploinsuffciency in patients with del7/7q. In a patient with aCML and UPD7q, homozygous R690H EZH2 mutation was detected and trimethylated H3K27 was abrogated while H3K9 methylation appeared normal, consistent with functional impairment of the SET domain. Interestingly, an EZH2 mutation was associated with heterozygous TET2 S733fsX21 and heterozygous ASXL1 W538X mutations, suggesting multiple genes associated with epigenetic regulation may be mutated and act synergistically in malignant evolution. These triple mutant cells ultimately formed lethal tumors when injected to NOD SCID gamma mice.

In summary, our investigations demonstrate EZH2 mutations in patients with myeloid malignancies frequently associated with UPD7q, or 7q36.1 microdeletion, but not monosomy 7 and del(7q). These findings suggest that an increasing possibility that mutations in the polycomb gene family represent a new class of molecular lesions conveying a clonal epigenetic instability phenotype and can constitute leukemogenic events.