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Expression of the tumor suppressor gene NR4A3 is silenced in the blasts of acute myeloid leukemia (AML), irrespective of the karyotype. Although the transcriptional reactivation of NR4A3 is considered to have a broad-spectrum anti-leukemic effect, the therapeutic modalities targeting this gene have been hindered by our minimal understanding of the transcriptional mechanisms regulating its expression, particularly in human AML. Here we show the role of intragenic DNA hypermethylation in reducing the expression of NR4A3 in AML. Bisulfite sequencing analysis revealed that CpG sites at the intragenic region encompassing exon 3 of NR4A3, but not the promoter region, are hypermethylated in AML cell lines and primary AML cells. A DNA methyltransferase inhibitor restored the expression of NR4A3 following a reduction in DNA methylation levels at intragenic CpG sites. The in silico data revealed an enrichment of H3K4me1 and H2A.Z at exon 3 of NR4A3 in human non-malignant cells but that was excluded specifically in leukemia cells with CpG hypermethylation. This suggests that exon 3 represents a functional regulatory element involved in the transcriptional regulation of NR4A3. Our findings improve the current understanding of the mechanism underlying NR4A3 silencing and facilitate the development of NR4A3-targeted therapy.

Although autologous stem cell transplantation can achieve excellent responses in patients with POEMS syndrome, the optimal regimen for peripheral blood stem cell (PBSC) collection is still controversial. We retrospectively investigated the safety and efficacy of 41 PBSC collecting procedures in 37 patients with POEMS syndrome. PBSC mobilization was performed using cyclophosphamide + granulocyte colony–stimulating factor (G-CSF) (CG, n = 14) or G-CSF alone (G, n = 27). Twelve (85.7%) patients in the CG group and all (100%) patients in the G group received induction chemotherapy before PBSC collection. The proportions of good mobilizers (≥2.0 × 106 CD34+ cells/kg) were comparable between the 2 groups (CG versus G: 78.6% versus 70.4%, P = .71). Two (14.3%) patients in the CG group developed severe capillary leak symptoms during the PBSC mobilization period, whereas no patient in the G group experienced severe adverse events. Appropriate induction therapies followed by the G-CSF monotherapy compose an optimal strategy for PBSC collection.

A 34-year-old man who had been referred to our hospital was diagnosed with acute promyelocytic leukemia (APL). All-trans retinoic acid (ATRA), oral administration, was initiated. On day 25, he developed fever and respiratory distress with bilateral pulmonary infiltrates, suggesting differentiation syndrome (DS) caused by ATRA. These symptoms showed amelioration after discontinuing ATRA and initiating methylprednisolone. ATRA was re-started on day 29 at half the original dose because of residual APL blasts. The patient subsequently developed fever, severe stomatitis, and oropharyngeal ulcers, which persisted even after discontinuing ATRA. On day 48, he suddenly developed severe abdominal pain with free air, observable on an abdominal X-ray, and underwent emergency ileocecal resection. Pathological examination of the resected ileocecal intestines revealed multiple ulcers and perforations. No leukemic cell infiltration was observed. In this case, only ATRA was administered for APL treatment. These findings suggest that ileocecal ulcerations and perforations, as well as oropharyngeal ulcers, might have been caused by DS or ATRA. Furthermore, DNA typing of the HLA-B locus revealed that the patient had HLA-B51 associated with Behçet's disease. Therefore, hypercytokinemia with DS might have induced Behçet's disease-like symptoms, including stomatitis and ileocecal perforation, complications that are particularly observed in patients with HLA-B51.

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 (Tet2KD/KDEzh2Δ/Δ). 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 Tet2KD/KDEzh2Δ/Δ 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.

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.

Introduction: Polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome is a rare paraneoplastic disease due to an underlying monoclonal plasma cell (PC) dyscrasia. Despite of dynamic symptoms associated with highly elevated VEGF, monoclonal PCs are thought to be quite small, and pathogenic significance of these PCs remains undetermined. In this study, we performed whole exome sequencing (WES), target sequencing, and RNA sequencing of PCs in patients with POEMS syndrome in order to define its genetic profiles. Methods: Patients diagnosed with POEMS syndrome at Chiba University Hospital from July 2014 to June 2016 were enrolled. DNA was extracted from either PCs which were isolated from patients' bone marrow (BM) using CD138 MACS (Miltenyi) or buccal cells as controls. WES and target sequencing were performed using HiSeq2500 (Illumina) and MiSeq (Illumina), respectively. The data of WES and target sequencing were analyzed by Empirical Bayesian mutation Calling (EBCall). Copy number was analyzed using the data of WES. RNA sequencing of PCs isolated by MACS and FACS sorting was conducted using HiSeq 1500 (Illumina). PCs from some patients diagnosed with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) were also collected as controls for RNA sequencing. Results: Twenty POEMS patients (M:F 12:8, mean age 42.6, range 16-78; 15 newly diagnosed, 5 refractory or relapsed cases) were included in this study. Regarding the types of M protein, 55% (11/20) were IgA-λ, 25% (5/20) were IgG-λ, and each individual case of the following; IgA-λ+IgG-λ, BJP-λ, IgG-κ, and Castleman's variant with no M protein. The mean serum VEGF was 6,471 pg/ml (range 1,190-13,800), and the mean PCs percentage in the BM was 4.4% (range 0.8-10.5). WES was performed in 15 cases; a total of 359 somatic mutations in 334 genes were revealed in 93.3% of cases (14/15) with a mean number of 23.9 (range 0-119) in each. All these mutated genes were significantly enriched in several pathways related to cell adhesion. Importantly, frequently mutated genes in MM such as NRAS, KRAS, and TP53 were not identified. Among all mutations, 1.7% were frameshift insertions, 2.0% were frameshift deletions, 4.2% were stop gains, 0.8% were non-frameshift deletions, 60.2% were other non-synonymous single nucleotide variants (SNVs), 29.5% were synonymous SNVs, and 1.7% were splicing mutations which were within 2-bp of a splicing junction. Copy-number variations were detected in 33.3% of cases (5/10) including -13 (2 cases), +1q (2 cases), and hyperdiploidy (2 cases). To carry out target sequencing in all 20 cases, we defined 51 target genes which included recurrently mutated genes from our WES data, frequently mutated genes in hematopoietic and lymphoid tissues according to the database (COSMIC), and 15 frequently mutated genes in MM (NRAS, KRAS, TP53, BRAF, CDKN2C, FGFR3, BIRC3, DIS3, CYLD, KDM6A, LRP1B, FAM46C, COL6A3, DNAH5, and KRT6A). A total of 60 somatic mutations were revealed in 65% of cases (13/20), and 9 new somatic mutations were found in the cases in which WES was also performed. Ten recurrently mutated genes were identified; KLHL6 in 20% of cases (4/20), each of LTB, RYR1 in 15% of cases (3/20), and each of EHD1, EML4, HEPHL1, HIPK1, PCDH10, USH2A, and ZNF645 in 10% of cases (2/20). Among frequently mutated genes in MM, only 3 genes (FAM46C, LRP1B, and DNAH5) were mutated, each in a single case. We finally conducted RNA sequencing of the FACS-sorted PCs in 5 POEMS patients compared to 5 MGUS and 4 MM patients. Upregulated genes were significantly enriched in some gene sets, gene ontology terms, and pathways related to immune response and cell adhesion, whereas downregulated genes were related to tumorigenesis. Of note, VEGF was not significantly upregulated in POEMS patients. Principal component analysis distinguished the 3 disease groups of patients with marginal overlaps between POEMS and MGUS, and also MGUS and MM. Conclusions: Our data clearly demonstrate that the genetic profiles of PCs in POEMS syndrome are distinct from those in MM and MGUS. Notably, PCs may not be the main source of extremely elevated VEGF in POMES syndrome. On-going further investigation will help clarify the molecular pathogenesis of POEMS syndrome. Disclosures Ogawa: Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

[Backgrounds] POEMS syndrome is a rare plasma cell dyscrasia and its pathogenesis including the significance of monoclonal plasma cells in disease progression is poorly understood. Monoclonal plasma cells only produce λ immunoglobulins, and genes encoding immunoglobulin λ light chain (IGL) V regions are derived from IGLV1-44 or IGLV1-40 germline sequences. Here we analyzed the clonality in IGLV gene rearrangements using next generation sequencing (NGS) to evaluate the significance of monoclonal plasma cell clone size and follow its changes in clinical course to understand the pathogenesis of POEMS syndrome. [Methods] Patients who were diagnosed with POEMS syndrome between November 2006 and October 2015 at Chiba University Hospital were included in the study. As positive controls, 3 multiple myeloma (MM) patients with λ-type monoclonal light chain, and 9 negative control patients were also analyzed. NGS libraries were constructed from genomic DNA samples, extracted from bone marrow mononuclear cells. The IGLV1 and IGLV2 genes were amplified by polymerase chain reaction (PCR) using a 5' primer for the IGLV1/2 framework 3 (FR3) region and 3ʹ consensus primers for the IGLJ1/2/3 joining regions. Multiple samples were pooled, and paired-end 2 × 250 base pair sequencing reactions were carried out using an Illumina MiSeq sequencer. The closest matched germline sequences were determined using the ImMunoGeneTics database. Subsequently, frequencies of each clonotype that were characterized by a unique V-J rearrangement, conserved complementarity determining region 3 (CDR3) anchors and a unique CDR3 amino acid sequence, were calculated. [Results] Twenty-eight patients with POEMS syndrome were enrolled. All the patients had λ-type M protein. The median follow up time of the patients was 24.4 months (range, 3.7 - 113.9). Firstly we analyzed the usage of IGLV germline genes in each case. In 8 cases, the POEMS syndrome-specific germline sequences, IGLV1-40 or IGLV1-44, were dominant; accounting for more than 40% of all germline sequence usage. However, other samples showed minimal or no differences from controls, indicating that the clonal expansion of monoclonal plasma cells is generally low in patients with POEMS syndrome. Analyzing frequencies of the most dominant rearrangement in each germline, the clonal IGLV gene rearrangements of POEMS syndrome-specific germline sequences were significantly increased in 10 POEMS patients (35.7%; IGLV1-44: n = 8, IGLV1-40: n = 2). Significant increase of clone sizes were not directly linked to the initial disease status (vascular endothelial growth factor [VEGF] level and percentage of plasma cells in the bone marrow), overall survival and progression-free survival of POEMS patients. In 12 patients that we were able to follow their clinical courses, the clone size of IGLVgene rearrangements correlated with disease course assessed with serum VEGF level in most cases (n = 8), as they decreased with serum VEGF levels in disease remission and increased with re-elevation of serum VEGF in relapse cases. Clone sizes without significant increase at diagnosis were constantly flat even after achieving disease remission (n = 3). In one case, clone size with significant increase at diagnosis was unchanged even after serum VEGF level decreased (n = 1). Further observation is needed in this case. [Discussion] Considering the cases with significant increase of IGLV rearrangement clones, it was confirmed that clonal light chain gene expression is restricted to the IGLV1-44 and IGLV1-40 germline sequences, as previously reported. IGLV gene rearrangement clone was not detected as significant increase in some cases. Therefore, we speculated that there are certain numbers of patients with POEMS syndrome with extremely low frequency of clone cells. On the other hand, significant increase of clone size did not reflect disease status, suggesting that disease status is not regulated by tumor burden alone. By contrast, in cases with significantly increased clones, clone sizes changed dependiing on the disease status. These data do demonstrate that monoclonal plasma cells are related to the pathogenesis of POEMS syndrome. [Conclusions] Our analysis of IGLV gene rearrangements has demonstrated the association between the size of IGLV gene rearrangement clones and the clinical courses in POEMS syndrome. Disclosures No relevant conflicts of interest to declare.

The expression of NR4A3, which is a member of the gene encoding NR4A orphan nuclear receptor subfamily, has been reported to be commonly silenced in blasts of patients with acute myeloid leukemia (AML), irrespective of karyotype. In line with this finding, Nr4a1-/-/Nr4a3-/- mice rapidly develop AML within one month following birth (Mullican et al., 2007). In addition, Nr4a1+/-/Nr4a3-/- and Nr4a1-/-/Nr4a3+/- mice show myelodysplastic/myeloproliferative neoplasms (Ramirez-Herrick et al., 2011), suggesting that NR4A3 functions as a tumor suppressor gene in myeloid malignancies. The extremely short latency of AML development in Nr4a1-/-/Nr4a3+/- mice indicates that silencing these tumor suppressors is sufficient to induce AML and that NR4A3 has a crucial role in the pathogenesis of AML. Thus, unveiling the molecular mechanism that regulates NR4A3 expression in AML would facilitate the development of novel therapies, including transcriptional reactivation of the gene. However, the therapeutic modalities targeting NR4A3 have been hindered by our minimal understanding of the mechanism underlying reduced NR4A3 expression, particularly in human AML cells. Abnormal epigenetic regulation is a common mechanism in the pathogenesis of several types of cancers. For instance, the expression of several tumor suppressor genes, such as p16 and MLH1, is repressed due to DNA hypermethylation at their promoter regions. Given that loss-of-function mutations in NR4A3 have not been reported in AML to date, we hypothesized that DNA hypermethylation contributes to a reduction in NR4A3 expression in AML. To test our hypothesis, we analyzed DNA methylation status of NR4A3 in human AML cells. We first compared the level of NR4A3 expression in eight human AML cell lines and two human primary AML samples, with that in CD34+ mononuclear bone marrow (BM) cells from healthy human controls. As expected, the expression of NR4A3 was markedly reduced in all of the AML cell lines and primary AML cells compared with that in the cells of the healthy controls. To evaluate the function of NR4A3 in human AML cells, we ectopically overexpressed NR4A3 in a human AML cell line (NB4 cells). The growth of NR4A3 -overexpressing NB4 cells was remarkably compromised compared with that of the controls, suggesting a tumor suppressive function of NR4A3 in both human AML and murine cells. To investigate the DNA methylation status of NR4A3, we performed bisulfite sequencing assays using eight human AML cell lines (HL60, NB4, Kasumi, TF1, U937, K562, MOLM13, and THP1) as well as CD34+ BM cells from healthy controls. Unexpectedly, a hypermethylated CpG site in the promoter region was not detected in any of the cell lines. However, the drastically or mildly methylated region including twenty eight CpGs was identified approximately 3 kb downstream of the transcription start site in six AML cell lines (97.5%, 78.3%, 77.1%, 89.9%, 95.2%, and 86.9% in HL60, NB4, Kasumi, TF1, U937, and K562, respectively) and two mixed lineage leukemia-related cell lines (31.0% and 53.6% in MOLM13 and THP1, respectively), whereas this site was hypomethylated in the controls (n = 2; mean, 12.7%; range, 7.1%-18.2%). To evaluate the contribution of this hypermethylated region to reduced NR4A3 expression, the six AML cell lines with heavily hypermethylated CpGs at NR4A3 and two human primary AML cell samples were treated with a DNA methyltransferase inhibitor (decitabine; DAC) for three or five days. DAC exposure inhibited cell growth and restored the expression of NR4A3 in all AML cell lines and primary cells in a dose- and time-dependent manner. Next, we examined the status of DNA methylation at the CpG site following DAC treatment with bisulfite sequencing assays. The frequencies of methylated CpG in HL60, NB4, and K562 cells was reduced from 97.5% to 53.6%, 78.3% to 68.5%, and 86.9% to 67.5% after DAC treatment, respectively. In contrast, the methylation status in Kasumi, TF1, and U937 cells did not significantly changed after DAC treatment. Our findings in the present study suggest that DNA hypermethylation may partially account for the transcriptional inactivation of NR4A3 in AML. However, the mechanism of reduced NR4A3 expression is complex and variable depending on the genetic background. We are currently working on a more detailed analysis of DNA methylation using human primary cells, by extending the regions for investigation, such as enhancer regions. Disclosures Nakaseko: Novartis: Honoraria, Research Funding, Speakers Bureau; Otsuka: Honoraria, Research Funding; BMS: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau.

Somatic loss-of-function mutations of epigenetic regulators are frequently found in MDS patients. Although DNA hypomethylating agents have been shown to improve the clinical outcomes in patients with MDS, it remains unknown how altered DNA methylation promotes the development of MDS. We have recently shown that concurrent loss of Tet2 and Ezh2 promotes the development of MDS in mice compared with the individual loss by utilizing hypomorphic Tet2 mice and Ezh2 conditional knockout mice. In this study, we transplanted Cre-ERT (WT), Tet2KD/KD, Cre-ERT;Ezh2fl/fl, and Tet2KD/KD;Cre-ERT;Ezh2fl/fl fetal liver cells into lethally irradiated recipient mice, and deleted Ezh2 at 4 weeks post-transplantation. To understand how DNA hypermethylation contributed to the development of MDS, we performed Reduced Representation Bisulfite Sequence (RRBS) in Lin- Sca1+ Kit+ (LSK) stem/progenitor cells isolated from WT, Tet2KD/KD, Ezh2Δ/Δ, and Tet2KD/KD Ezh2Δ/Δ mice. Both Tet2 loss and Ezh2 loss caused hypermethylation in CpG islands (CGIs) and promoter regions. However, the impact of Tet2 loss was much evident in the enhancer elements and ~20 % of them underwent hypermethylation upon Tet2 loss. Combined loss of Tet2 and Ezh2 caused hypermethylation in the transcriptional regulatory regions including both the promoter regions and enhancer elements. Importantly, the majority of hyper-differentially methylated regions (hyper-DMRs) in Tet2KD/KD Ezh2Δ/Δ -MDS LSK cells showed a distribution that was distinct from those of single mutant LSK cells and only the hyper-DMRs in the enhancer elements in Tet2KD/KD Ezh2Δ/Δ -MDS LSK cells correlated with a significant reduction in gene expression levels. Hyper-DMRs in MDS LSK cells were significantly enriched in genes involved in transcriptional regulation and cell differentiation by GO analysis. These findings suggest that combined loss of Tet2 and Ezh2 cooperatively remodeled DNA methylation to an extent not observed in either mutant allele alone and contributed to the pathogenesis of MDS. Notably, we found that 375 out of 780 hyper-DMRs in Tet2KD/KD Ezh2Δ/Δ MDS HSPCs were overlapped with DMRs in CD34+ HSPCs in patients with MDS (Maegawa S, et al. Genome Research 2014). We next focused on the 131 genes with hyper-DMRs that showed reduced expression levels in Tet2KD/KD Ezh2Δ/Δ -MDS LSK cells. Those included Gata2, Gata3, Evi1, and Nr4a2. Given that Nr4a2/Nurr1, a nuclear receptor transcription factor, restricts the proliferation of hematopoietic stem cells (HSCs), we transduced WT and Tet2KD/KD Ezh2Δ/Δ HSCs with an Nr4a2 retrovirus in vitro. We found that exogenous Nr4a2 severelyimpairs proliferative capacity of Tet2KD/KD Ezh2Δ/Δ HSCs but moderately that of WT HSCs, implying that MDS HSCs are more susceptible to activation of Nr4a2 compared to WT HSCs. Next, we sought to determine whether DMRs in CGIs were associated with altered histone modifications upon the deletion of Ezh2, we performed chromatin immunoprecipitation (ChIP) sequencing by using H3K27me3 and H3K4me3 antibodies. We defined canonical polycomb repressive complexes 2 (PRC2) targets by >2-fold enrichment of H3K27me3 and bivalent genes by enrichment of both H3K27me3 and H3K4me3 in WT LSK cells. We found that approximately half of the hyper-DMRs in CGIs were enriched in canonical PRC2 targets and bivalent genes in LSK cells of all mutant genotypes. Notably, these hyper-DMRs in PRC2 targets, including bivalent genes, were significantly associated with reduced expression levels only in Tet2KD/KD Ezh2Δ/Δ -MDS LSK cells but not in either single mutant alone. We also observed that these PRC2 targets with hyper-DMRs still retained reduced but higher levels of H3K27me3 compared to the others despite the absence of Ezh2, implying that both DNA hypermethylation and residual H3K27me3 by Ezh1 cooperate to repress the transcription of these genes critical for hematopoiesis. Finally, we examined the impact of decitabine, a demethylating agent in vivo. Although the treatment of decitabine did not improve the survival of Tet2KD/KD Ezh2Δ/Δ -MDS mice, it partly resolved hyper-DMRs at critical genes including Gata2, Gata3, and Nr4a2, followed by a significant elevation in platelets counts in vivo. Taken together, our study unveils the cooperative biological function of aberrant DNA methylation and histone modifications in the pathogenesis of MDS. Disclosures Nakaseko: BMS: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Otsuka: Honoraria, Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau.

Precise understanding of radiation effects is critical to develop new modalities for the prevention and treatment of radiation-induced damage. We previously reported that non-lethal doses of X-ray irradiation induce DNA damage in human hematopoietic stem and progenitor cells (HSPCs) reconstituted in NOD/Shi-scid IL2rγnull (NOG) immunodeficient mice and severely compromise their repopulating capacity. In this study, we analyzed in detail the functional changes in human HSPCs in NOG mice following non-lethal radiation. We transplanted cord blood CD34+ HSPCs into NOG mice. At 12 weeks post-transplantation, the recipients were irradiated with 0, 0.5, or 1.0 Gy. At 2 weeks post-irradiation, human CD34+ HSPCs recovered from the primary recipient mice were transplanted into secondary recipients. CD34+ HSPCs from irradiated mice showed severely impaired reconstitution capacity in the secondary recipient mice. Of interest, non-lethal radiation compromised contribution of HSPCs to the peripheral blood cells, particularly to CD19+ B lymphocytes, which resulted in myeloid-biased repopulation. Co-culture of limiting numbers of CD34+ HSPCs with stromal cells revealed that the frequency of B cell-producing CD34+ HSPCs at 2 weeks post-irradiation was reduced more than 10-fold. Furthermore, the key B-cell regulator genes such as IL-7R and EBF1 were downregulated in HSPCs upon 0.5 Gy irradiation. Given that compromised repopulating capacity and myeloid-biased differentiation are representative phenotypes of aged HSCs, our findings indicate that non-lethal ionizing radiation is one of the critical external stresses that promote aging of human HSPCs in the bone marrow niche.

In contrast to adult bone marrow (BM) hematopoiesis, fetal liver (FL) hematopoiesis involves mainly erythropoiesis and myelopoiesis, with limited lymphopoiesis. Fetal hematopoietic stem cells (HSCs) are known to have greater repopulating capacity as compared with adult HSCs. Polycomb group (PcG) protein Ezh2 participates in gene silencing by catalyzing the trimethylation on H3K27 (H3K27me3). We previously reported that in transplantation assays, Ezh2-deficient FL hematopoietic cells have greater reconstitution capacity and establish significantly higher ratios of myeloid to lymphoid reconstitution relative to wild-type (WT). From these results, we hypothesized that adult BM hematopoietic cells repopulated with Ezh2-deficient FL cells partially retain the features of fetal hematopoietic cells as compared with WT. To address this hypothesis, we transplanted Cre-ERT;Ezh2fl/fl or WT FL hematopoietic cells and deleted Ezh2 (Ezh2 KO) via intraperitoneal administration of tamoxifen at 4 weeks after transplantation. At 3 months post-deletion of Ezh2, donor-derived lin-Sca-1+c-Kit+ (LSK) HSC/MPP fraction were recovered and subjected to microarray analysis, together with LSK cells from WT E15.5 FL and adult BM. Within genes which showed higher expression in Ezh2 KO BM LSK cells compared with WT (453 genes: Ezh2 KO-gene), and higher in FL compared with adult BM LSK cells (1139 genes: FL-gene), 102 genes (23% of Ezh2 KO-gene and 9% of FL-specific gene) were overlapped (p-value < 1.0x10-16). Recently, Copley et al. (Nat Cell Biol, 2013) revealed that Lin28b is exclusively expressed in FL HSCs and the Lin28b/let7/Hmga2 axis plays a critical role in controlling developmental changes in HSC properties. As reported, we found that Lin28b was detected only in FL LSK cells, but of note, remarkably higher expression was observed in Ezh2 KO relative to WT LSK cells in adult BM. Lin28b is known to block the maturation of let-7 microRNAs, thereby up-regulates expression of let-7 target genes. Within 1648 putative let-7 miRNA target genes, 136 genes were highly expressed in FL LSK cells (FL-LSK let-7 target). Gene set enrichment analysis (GSEA) showed that FL-LSK let-7 target gene set was ectopically enriched in Ezh2 KO LSK and also GMP cells but not in WT cells in adult BM. We next performed ChIP-seq analysis to evaluate the direct target of Ezh2 in adult BM, Lin28b promoter region appeared to be marked with PcG histone marks H3K27me3 and H2AK119Ub1 but not in FL. The levels of PcG histone marks were significantly low in Ezh2-deficient BM LSK cells and GMPs relative to WT. These findings indicate that Lin28 is directly regulated by PcG histone modifications in adult BM. Interestingly, Within FL-LSK let-7 target, 16 genes underwent H3K27me3 histone modifications in adult BM and several of them escaped gene silencing in the absence of Ezh2, including Igf2bp3, Hmga2, Jam3, Dcbld1 and Zfp354a., in the same way as Lin28b. These results demonstrated that a part of let-7 target genes are negatively regulated in BM not only by let-7 but also by PcG histone modifications in adult BM. Recent analyses have revealed that inactivating somatic mutations in epigenetic regulator genes such as EZH2 or TET2 occur frequently in patients with myelodysplastic disorders. We previously reported that Ezh2 is largely dispensable for adult HSCs in contrast to fetal hematopoiesis but after a long latency, deletion of Ezh2 in adult hematopoietic cells induces myelodysplastic/myeloproliferative neoplasm (MDS/MPN)-like disorders characterized by myelodysplasia with higher repopulating capacity of HSCs. Furthermore, concurrent depletion of Ezh2 and Tet2 markedly accelerated the development of myelodysplastic syndrome (MDS) and MDS/MPN. Lin28b and its targets Igf2bp1 and Igf2bp3 are characterized as ‘oncofetal’ genes, which is highly expressed not only in early embryogenesis but also in various tumors. Corresponding to these findings, FL-LSK let-7 target genes were highly and more significantly enriched in Tet2KD/KDEzh2Δ/Δ(DKO) LSK and GMPs from MDS or MDS/MPN mice relative to Ezh2 KO in adult BM. Taken together, Ezh2 is indispensable for the developmental stage-specific regulation of the Lin28b-let7 pathway, and Ezh2 deletion results in insufficient suppression of a subset of the oncofetal genes, which may contribute to the FL-like characteristics and the development of hematopoietic disorders. Disclosures No relevant conflicts of interest to declare.