Loss-of-function mutations of EZH2, a catalytic component of polycomb repressive complex 2 (PRC2), are observed in B10% of patients with myelodysplastic syndrome (MDS), but are rare in acute myeloid leukaemia (AML). Recent studies have shown that EZH2 mutations are often associated with RUNX1 mutations in MDS patients, although its pathological function remains to be addressed. Here we establish an MDS mouse model by transducing a RUNX1S291fs mutant into hematopoietic stem cells and subsequently deleting Ezh2. Ezh2 loss significantly promotes RUNX1S291fs-induced MDS. Despite their compromised proliferative capacity of RUNX1S291fs/Ezh2-null MDS cells, MDS bone marrow impairs normal hematopoietic cells via selectively activating inflammatory cytokine responses, thereby allowing propagation of MDS clones. In contrast, loss of Ezh2 prevents the transformation of AML via PRC1-mediated repression of Hoxa9. These findings provide a comprehensive picture of how Ezh2 loss collaborates with RUNX1 mutants in the pathogenesis of MDS in both cell autonomous and non-autonomous manners.

Deletion of Ezh2 results in transcriptional repression of developmental regulator genes, derepression of oncogenic polycomb targets, and induction of MDS/MPN-like disease in mice that is exacerbated by concurrent deletion of Tet2.

Key Points Ezh2 loss in hematopoietic stem cells predisposes mice to develop heterogeneous hematologic malignancies. Ezh1 is essential to maintain hematopoiesis in the setting of Ezh2 loss.

No abstract

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.

Fetal liver hematopoietic stem cells (HSCs) seed bone marrow (BM) and undergo reprograming into adult-type HSCs that are largely quiescent and restricted in their self-renewal activity. Here we report that in the absence of the polycomb-group gene Ezh2, a cohort of fetal-specific genes, including let-7 target genes, were activated in BM hematopoietic stem/progenitor cells (HSPCs), leading to acquisition of fetal phenotypes by BM HSPCs, such as enhanced self-renewal activity and production of fetal-type lymphocytes. The Lin28b/let-7 pathway determines developmentally timed changes in HSPC programs. Of note, many of the fetal-specific let-7 target genes, including Lin28, appear to be transcriptionally repressed by Ezh2-mediated H3K27me3 in BM HSPCs, and Ezh2 loss results in their ectopic expression, particularly in hematologic malignancies that develop in the absence of Ezh2. These findings suggest that Ezh2 cooperates with let-7 microRNAs in silencing the fetal gene signature in BM HSPCs and restricts their transformation.

Thrombopoietin-receptor agonists have been recently introduced for a second-line treatment of immune thrombocytopenia (ITP). Splenectomy has tended to be avoided because of its complications, but the response rate of splenectomy is 60-80% and it has still been considered for steroid-refractory ITP. We performed partial splenic embolization (PSE) as an alternative to splenectomy. Between 1988 and 2013, 91 patients with steroid-resistant ITP underwent PSE at our hospital, and we retrospectively analyzed the efficacy and long-term outcomes of PSE. The complete response rate (CR, platelets > 100 × 10/L) was 51% (n = 46), and the overall response rate (CR plus response (R), > 30 × 10/L) was 84% (n = 76). One year after PSE, 70% of patients remained CR and R. The group with peak platelet count after PSE ≥ 300 × 10/L (n = 29) exhibited a significantly higher platelet count than the group with platelet count < 300 × 10/L (n = 40) at any time point after PSE. The failure-free survival (FFS) rates at 1, 5, and 10 years were 78, 56, and 52%, respectively. Second PSE was performed in 20 patients who relapsed (n = 14) or had no response to the initial PSE (n = 6), and the overall response was achieved in 63% patients. There were no PSE-related deaths. These results indicate that PSE is a safe and effective alternative therapy to splenectomy for patients with steroid-resistant ITP as it generates long-term, durable responses.

Somatic inactivating mutations in epigenetic regulators are frequently found in combination in myelodysplastic syndrome (MDS). However, the mechanisms by which combinatory mutations in epigenetic regulators promote the development of MDS remain unknown. Here we performed epigenomic profiling of hematopoietic progenitors in MDS mice hypomorphic for Tet2 following the loss of the polycomb-group gene Ezh2 (Tet2Ezh2). Aberrant DNA methylation propagated in a sequential manner from a Tet2-insufficient state to advanced MDS with deletion of Ezh2. Hyper-differentially methylated regions (hyper-DMRs) in Tet2Ezh2 MDS hematopoietic stem/progenitor cells were largely distinct from those in each single mutant and correlated with transcriptional repression. Although Tet2 hypomorph was responsible for enhancer hypermethylation, the loss of Ezh2 induced hyper-DMRs that were enriched for CpG islands of polycomb targets. Notably, Ezh2 targets largely lost the H3K27me3 mark while acquiring a significantly higher level of DNA methylation than Ezh1 targets that retained the mark. These findings indicate that Ezh2 targets are the major targets of the epigenetic switch in MDS with Ezh2 insufficiency. Our results provide a detailed trail for the epigenetic drift in a well-defined MDS model and demonstrate that the combined dysfunction of epigenetic regulators cooperatively remodels the epigenome in the pathogenesis of MDS.