Inactivating somatic mutations in polycomb-group (PcG) genes such as EZH2 and ASXL1occur frequently in patients with myelodysplastic syndromes (MDS), myeloproliferative neoplasm (MPN) and MDS/MPN overlap disorders. While these mutations suggest a tumor suppressor function of polycomb repressive complex 2 (PRC2)-related genes in these diseases, both the impact of each PcG mutation and its interplay with coinciding mutations remain largely unknown.
To understand the contribution of inactivating PcG mutations to the development of myeloid malignancies, genomic DNA from 119 patients with MDS and related neoplasms were analyzed for mutations in EZH2, ASXL1 and TET2 by high-throughput sequencing. Inactivating mutations in EZH2 and ASXL1 were detected in 8.4 and 16.8 % of patients, respectively. Moreover, 3.4 % of patients had deletion of EZH2 (located at 7q36) associated with -7 and 7q- chromosomal abnormalities. Notably, 57.1 % of these EZH2 mutations coexisted with TET2 mutations. Conversely, 34.8 % of patients with TET2 mutations had coexisting EZH2mutations.
In order to understand the impact of inactivating EZH2 mutations and concurrent EZH2 and TET2 mutations on hematopoiesis, we crossed Cre-ERT;Ezh2fl/fl mice and Tet2 gene trap mice (Tet2KD/KD). Due to the early time of death in Tet2KD/KD mice and a necessity to exclude the influence of the loss of Tet2 and Ezh2 in BM niche cells, we transplanted E14.5 fetal liver cells from Cre-ERT control (WT), Cre-ERT;Tet2KD/KD, Cre-ERT;Ezh2fl/fl and Cre-ERT;Tet2KD/KDEzh2fl/fl CD45.2 mice into lethally irradiated CD45.1 recipient mice and deleted Ezh2 by intraperitoneal injection of tamoxifen at 4 weeks post-transplantation. During a long observation period, we found that Ezh2Δ/Δ mice developed MDS/MPN and half of the mice died by 10 months post-transplantation. They showed myeloproliferative features characterized by extramedullary hematopoiesis in the spleen as evident from splenomegaly with a marked increase in LSK cells. They were anemic and showed increased apoptosis in Ter119highCD71high erythroblasts in the BM, suggesting ineffective erythropoiesis, a feature compatible with myelodysplastic disorders. Ezh2Δ/Δ mice also showed dysplasia of myeloid cells, including a pseudo Pelger-Huët anomaly. To our surprise, concurrent deletion of Tet2 and Ezh2 significantly shortened the latency of disease development of not only MDS/MPN but also MDS, and all of the compound mice died of pneumonia by 10 months. Tet2KD/KDEzh2Δ/Δ MDS/MPN mice showed myeloproliferative features, including monocytosis and/or splenomegaly with extramedullary hematopoiesis. In contrast, Tet2KD/KDEzh2Δ/Δ MDS mice did not show obvious myeloproliferative features, but showed a trend of pancytopenia. The proportion of Annexin V+ cells in CD71highTer119high erythroblasts was significantly higher in both MDS/MPN and MDS mice compared to their WT counterparts, implicating enhanced apoptosis as a cause of anemia. Furthermore, myeloid dysplasia was more pronounced in these mice compared to Ezh2Δ/Δmice.
Gene set enrichment analysis with microarray data showed that the Myc module was significantly enriched in Ezh2Δ/Δ LSK cells and became highly enriched in Tet2KD/KDEzh2Δ/Δ LSK cells during the development of MDS/MPN and MDS in Tet2KD/KDEzh2Δ/Δ mice. As expected, all of the PRC2 gene sets (Ezh2 targets and Ezh1 targets) showed a trend of positive enrichment in Ezh2Δ/Δ and Tet2KD/KDEzh2Δ/Δ LSK cells. Notably, however, Ezh1 targets became negatively enriched in Tet2KD/KDEzh2Δ/Δ LSK cells during the development of myelodysplastic disorders. ChIP-seq and microarray analysis data showed that upon deletion of Ezh2, a series of potential PcG related target oncogenes, such as Hmga2 and Pbx3, became derepressed in LSK cells. On the other hand, key developmental regulator genes, such as genes encoding homeobox, paired-box, T-box, forkhead and Gata family transcription factors and zinc finger DNA-binding proteins, were kept transcriptionally repressed by the compensatory action of Ezh1.
Our findings provide the first evidence of the tumor suppressor function of EZH2 and demonstrate the cooperative effect of concurrent gene mutations in the pathogenesis of myelodysplastic disorders. These two models represent novel, genetically accurate models of myelodysplastic disorders amenable to epigenomic as well as preclinical therapeutic studies.
Disclosures:
No relevant conflicts of interest to declare.
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