EZH2 Is Either Mutated or Downregulated in Patients with Loss of Heterozygosity of Chromosome 7/7q and Leads to Epigenetic Dysregulation Via Histone H3K27.

Abstract 228

Chromosome 7 abnormalities occur either in isolation or in conjunction with other lesions (del(5q) or del/uniparental disomy (UPD)17p). To date the search for mutations affecting genes located on chromosome 7 has not been successful.

By combination of metaphase and single nucleotide polymorphism array based karyotyping, in a cohort of 929 patients with myeloid malignancies (myelodysplastic syndromes (MDS): n=366; myelodysplastic/myeloproliferative neoplasms (MDS/MPN): n=108; MPN: n=206; secondary acute myeloid leukemia (sAML): N=167; primary (p)AML: n=82), loss of heterozygosity (LOH) in chromosome 7q was observed in 11.8% of MDS, 11.3% of MDS/MPN, 8.1% of MPN, 7.6% of sAML and 12.6% of pAML. In addition to somatic interstitial deletions (del7q) and a total loss of chromosome 7 (del7), somatic UPD(7q) was found: UPD(7q) was most frequently observed in MDS/MPN (4.9%). Similar to del(7/7q), UPD(7q) also conveyed a poor prognosis in terms of overall survival (OS). We hypothesized that cases with LOH7q may harbor a mutated gene which would explain clonal malignant evolution. For identification of such a hypothetical gene, we used two strategies: 1) application of a next generation sequencing platform and 2) targeted sequencing of candidate genes in intervals delineated by the presence of somatic microdeletions in specific patients. Both approaches demonstrated that the EZH2 gene can be affected by somatic mutations. Subsequently, screening led to detection of a total of 16 cases with EZH2 mutations, which were present in the majority of UPD(7q) patients, but in only 10% of cases with del(7q). In 4 cases (without LOH7q), we also found heterozygous EZH2 mutations. In addition to mutations, expression of EZH2 was found to be significantly decreased in patients with del(7/7q), likely due to haploinsufficiency, and surprisingly decreased in the entire cohort of patients, including 119/542 cases of AML.

EZH2 mutations were observed in MDS/MPN (7.1%) and MPN (6.4%) but less frequently in AML. Further analyses of clinical phenotypes associated with the EZH2 mutation showed a significant negative impact on OS. In particular, the negative impact on OS was evident in the MDS/MPN group (p=0.0005) or in patients older than 60 years (p=0.0086). In the index case (sAML and UPD(7q)), 2 other mutations were detected: ASXL1 and TET2; all mutations were found at initial presentation prior to AML transformation. A concominant ASXL1 mutation was found in 3 other cases, while in 2 other patients, EZH2 mutations were found together with TET2 mutations.

We have also investigated the pathogenic mechanisms resulting form EZH2 mutations. Leukemic cells from a patient with a homozygous EZH2 mutation easily initiated xenografts in NSG mice, which within 4 weeks of injection developed massive infiltration of spleen and liver with leukemic blasts. As expected, WB, ELISA and immunohistochemistry performed on these blasts revealed decreased H3K27 trimethylation compared to EZH2 wild type CD34 positive cells. A similar effect was observed in mutant cell line SKM-1 when compared to cell lines with wild type EZH2. When we investigated the effects of EZH2 on the gene expression profile in EZH2 mutant and WT cell lines, we found a significant increase in expression of HOXA9, known to be regulated by EZH2, along with PU.1, CDKN2A, IRF4, RASSF1 and CEBPA. We also investigated the effect of EZH2 mutations on the structural state of chromatin using a PCR-based method using non-denatured chromatin. We set up primers prior to and/or at the beginning of the first exons of MYC, CDKN2A and PU.1 to asses the chromatin plasticity. The EZH2 mutation was associated with more efficient amplification of these genes, suggestive of a relaxed, transcriptionally active state of DNA on the corresponding sites.

In sum, mutations of EZH2 are present in patients with MDS/MPN and sAML. The mutations are mostly homozygous and are not commonly associated with del(7/7q). However, decreased expression of wild type EZH2 may be responsible for the pathogenesis associated with del(7/7q). Additionally, they convey a negative prognostic impact on OS. Mechanistically, decreased H3K27 trimethylation mediates downstream effects, such as persistent expression of various genes like HOXA9 and others. Mutations of EZH2 link the genomic instability to epigenetic dysregulation of gene expression.