Histone deacetylase 3 (HDAC3) is one of four members of the human class I HDACs that regulates gene expression by deacetylation of histones and nonhistone proteins. Early studies have suggested that HDAC3 activity is regulated by association with the corepressors N-CoR and SMRT. Here we demonstrate that, in addition to protein-protein interactions with NCoR/SMRT, the activity of HDAC3 is regulated by both phosphorylation and dephosphorylation. A protein kinase CK2 phosphoacceptor site in the HDAC3 protein was identified at position Ser 424 , which is a nonconserved residue among the class I HDACs. Mutation of this residue was found to reduce deacetylase activity. Interestingly, unlike other class I HDACs, HDAC3 uniquely copurifies with the catalytic and regulatory subunits of the protein serine/threonine phosphatase 4 complex (PP4 c /PP4 R1 ). Furthermore, HDAC3 complexes displayed protein phosphatase activity and a series of subsequent mutational analyses revealed that the N terminus of HDAC3 (residues 1-122) was both necessary and sufficient for HDAC3-PP4 c interactions. Significantly, both overexpression and siRNA knock-down approaches, and analysis of cells devoid of PP4 c , unequivocally show that HDAC3 activity is inversely proportional to the cellular abundance of PP4 c . These findings therefore further highlight the importance of protein-protein interactions and extend the significance of dephosphorylation in the regulation of HDAC activity, as well as present a novel alternative pathway by which HDAC3 activity is regulated.

To clarify the cooperative roles of recurrently identified mutations and to establish a more precise risk classification system in acute myeloid leukemia (AML), we comprehensively analyzed mutations in 51 genes, as well as cytogenetics and 11 chimeric transcripts, in 197 adult patients with de novo AML who were registered in the Japan Adult Leukemia Study Group AML201 study. We identified a total of 505 mutations in 44 genes, while only five genes, FLT3, NPM1, CEBPA, DNMT3A and KIT, were mutated in more than 10% of the patients. Although several cooperative and exclusive mutation patterns were observed, the accumulated mutation number was higher in cytogenetically normal AML and lower in AML with RUNX1-RUNX1T1 and CBFB-MYH11, indicating a strong potential of these translocations for the initiation of AML. Furthermore, we evaluated the prognostic impacts of each sole mutation and the combinations of mutations and/or cytogenetics, and demonstrated that AML patients could be clearly stratified into five risk groups for overall survival by including the mutation status of DNMT3A, MLL-PTD and TP53 genes in the risk classification system of the European LeukemiaNet. These results indicate that the prognosis of AML could be stratified by the major mutation status in combination with cytogenetics.

The zinc finger transcription factor GATA-2 plays a critical role in the survival and proliferation of hematopoietic stem cells. This study examined the interaction of GATA-2 with histone deacetylases (HDACs) to define the involvement of HDACs in the regulation of GATA-2 function. GATA-2 directly associates with HDAC3 but not with HDAC1. Consistent with this, HDAC3 suppressed the tran-scriptional potential of GATA-2, whereas HDAC1 did not affect GATA-2-dependent transcription. Results further demonstrated that GATA-2 and HDAC3 colocal-ized in the nucleus. These results identify GATA-2 as a nuclear target for HDAC3-mediated repression. Furthermore, GATA-2 also directly associated with HDAC5 but not with other class II HDACs examined, that is, HDAC4 and HDAC6. This is the first demonstration that a tissue-specific transcription factor directly and selectively interacts with HDAC3 and HDAC5 among HDAC family members. (Blood. 2001;98:2116-2123)

Epstein–Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) of the elderly (EBV[+]DLBCL-E) is classified as a subtype of DLBCL. Until now, its molecular pathogenesis has remained unknown. To identify pathways characteristic of EBV(+)DLBCL-E, gene expression profiling of five EBV(+)DLBCL-E and seven EBV-negative DLBCL (EBV[−]DLBCL) cases was undertaken using human oligonucleotide microarray analysis. Gene set enrichment analysis and gene ontology analysis showed that gene sets of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) and nuclear factor kappa B (NF-κB) pathways were enriched in EBV(+)DLBCL-E cases. To confirm the results of the expression profiles, in vitro analysis was performed. Expression profiling analysis showed that high activation of the JAK-STAT and NF-κB pathways was induced by EBV infection into DLBCL cell lines. Activation of the NF-κB pathway was confirmed in EBV-infected cell lines using an electrophoretic mobility shift assay. Western blot analysis revealed an increased protein expression level of phosphorylated signal transducer and activator of transcription 3 (STAT3) in an EBV-infected cell line. Protein expression of phosphorylated STAT3 was frequently observed in lymphoma cells of EBV(+)DLBCL-E clinical samples using immunohistochemistry (EBV[+]DLBCL-E: 80.0% [n = 20/25] versus EBV[−]DLBCL: 38.9% [n = 14/36]; P = 0.001). The results of the present study suggest that activation of the JAK-STAT and NF-κB pathways was characteristic of EBV(+)DLBCL-E, which may reflect the nature of EBV-positive tumor cells. Targeting these pathways as therapies might improve clinical outcomes of EBV(+)DLBCL-E.

The transcription factor GATA-1 plays a significant role in erythroid differentiation and association with CBP stimulates its activity by acetylation. It is possible that histone deacetylases (HDACs) repress the activity of GATA-1. In the present study, we investigated whether class I and class II HDACs interact with GATA-1 to regulate its function and indeed, GATA-1 is directly associated with HDAC3, HDAC4 and HDAC5. The expression profiling and our previous observation that GATA-2 interacts with members of the HDAC family prompted us to investigate further the biological relevance of the interaction between GATA-1 and HDAC5. Coexpression of HDAC5 suppressed the transcriptional potential of GATA-1. Our results demonstrated that GATA-1 and HDAC5 colocalized to the nucleus of murine erythroleukemia (MEL) cells. Furthermore, a portion of HDAC5 moved to the cytoplasm concomitant with MEL cell erythroid differentiation, which was induced by treatment with N,N 0 -hexamethylenebisacetamide. These observations support the suggestion that control of the HDAC5 nucleocytoplasmic distribution might be associated with MEL cell differentiation, possibly through regulated GATA-1 transactivation.

We retrospectively reviewed 104 biopsy specimens of previously untreated skin acute graft-versus-host disease (GVHD) within 100 days after allogeneic stem cell transplantation, and analyzed the relationship between types of infiltrating cells and clinical outcomes. Counting the total number of CD8 ؉ T cells, CD163 ؉ macrophages, and CD1a ؉ dendritic cells in 4 fields under original magnification IntroductionMacrophages are phagocytic cells with various abilities, such as phagocytosis, antigen-presenting, and secretion of cytokines. 1,2 Recently, it was revealed in human sequential biopsy data that recipient macrophages contributed to acute graft-versus-host disease (GVHD) by antigen-presenting and secreting cytokines, causing the activation and proliferation of CD8 ϩ T cells. 3 We focused on macrophage involvement in acute GVHD, especially on the relationship between the macrophage infiltration of skin lesions and refractory GVHD. MethodsBetween January 1997 and October 2007 at the Japanese Red Cross Nagoya First Hospital, we used skin biopsy specimens within 100 days after allogeneic stem cell transplantation (allo-SCT) of skin lesions clinically considered acute GVHD without any GVHD treatment from 104 patients who underwent allo-SCTs. We analyzed the relationship between types of infiltrating cells and clinical outcomes by counting the total number of CD8 ϩ T cells, CD163 ϩ macrophages, and CD1a ϩ dendritic cells in 4 fields of a skin biopsy specimen under original magnification ϫ200. Immunohistochemical analysis using paraffin sections was performed using monoclonal antibodies against CD8, CD163, and CD1a (Novocastra). CD163 is a member of the scavenger receptor cystein-rich superfamily and is an exclusive marker for macrophages, playing a major role in the scavenging components of damaged cells. [4][5][6][7] The endpoints of this study were the outcomes of acute GVHD and overall survival (OS). Acute GVHD was diagnosed and graded according to the consensus criteria. 8 We defined refractory GVHD as that exhibited by patients who had persistent lesions after primary steroid treatments. To establish parameters, we analyzed the numbers of infiltrating , disease risk (low vs high), human leukocyte antigen (HLA) disparity (match vs mismatch), donor source (related vs unrelated), graft source (bone marrow vs peripheral blood), age at allo-SCT (Յ 50 years vs Ͼ 50 years), conditioning regimen (conventional regimens vs reduced intensity regimens), and skin GVHD stage at biopsy (stages 1-2 vs stages 3-4). A significance level of P Ͻ .05 was used for all analyses, which were based on all data available as of August 31, 2008. Protocols were approved by the Japanese Red Cross Nagoya First Hospital's Institutional Review Board, and all patients provided informed consent in accordance with the Declaration of Helsinki. Table 1 summarizes the characteristics of patients and information gathered about GVHD. We divided patients into 4 groups according to the amount of infiltrating cells (FM and FT, 60.6%; MT and FM, 18.2%; MT...

Key Points• HDCA plus CY/TBI improved overall survival relative to CY/TBI in CBT for myeloid malignancy.• HDCA suppressed relapse but did not increase the incidence of severe adverse events or nonrelapse mortality.Cord blood transplantation (CBT) is an effective therapeutic option for adults with acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS) after the conventional cyclophosphamide and total body irradiation (CY/TBI) regimen, but posttransplant relapse is still of high importance. High-dose cytarabine (HDCA) can be added to CY/TBI for an intensified regimen; however, its additional effects have not yet been completely elucidated. Therefore, we conducted a cohort study to compare the prognosis of HDCA/ CY/TBI (n 5 617) and CY/TBI (n 5 312) in CBT for AML/MDS, using a Japanese transplant registry database. The median age was 40 years, and 86.2% of the patients had AML; highrisk disease was observed in 56.2% of the patients. The median follow-up period after CBT was approximately 3.5 years. Overall survival was significantly superior in the HDCA/CY/ TBI group (adjusted hazard ratio [HR], 0.56; 95% confidence interval [CI], 0.45-0.69; P < .01), and tumor-related mortality was lower (HR, 0.50; P < .01). The incidence of grade II to IV acute graft-vs-host disease (aGVHD) and chronic GVHD was significantly higher in the HDCA/CY/TBI group (HR, 1.33 and 2.30, respectively), but not grade III to IV aGVHD. Incidence of infectious episodes showed no significant difference. Nonrelapse mortality was not increased by the addition of HDCA. Higher-dose CA (12 rather than 8 g/m 2 ) was more effective, particularly in patients at high-risk for disease. This study is the first to show the superiority of HDCA/CY/TBI to CY/TBI in CBT for AML/MDS. A large-scale prospective study is warranted to establish new conditioning regimens including HDCA administration. (Blood. 2015;126(3):415-422)

Key Points The machine learning algorithms produced clinically reasonable and robust risk stratification scores for aGVHD. Predicting scores for aGVHD also demonstrated the link between risk of development of aGVHD and overall survival after HSCT.