ALBERT

Description: The molecular basis for regulation of dendritic cell (DC) development and homeostasis remains unclear. Signal regulatory protein α (SIRPα), an immunoglobulin superfamily protein that is predominantly expressed in DCs, mediates cell-cell signaling by interacting with CD47, another immunoglobulin superfamily protein. We now show that the number of CD11chigh DCs (conventional DCs, or cDCs), in particular, that of CD8−CD4+ (CD4+) cDCs, is selectively reduced in secondary lymphoid tissues of mice expressing a mutant form of SIRPα that lacks the cytoplasmic region. We also found that SIRPα is required intrinsically within cDCs or DC precursors for the homeostasis of splenic CD4+ cDCs. Differentiation of bone marrow cells from SIRPα mutant mice into DCs induced by either macrophage-granulocyte colony-stimulating factor or Flt3 ligand in vitro was not impaired. Although the accumulation of the immediate precursors of cDCs in the spleen was also not impaired, the half-life of newly generated splenic CD4+ cDCs was markedly reduced in SIRPα mutant mice. Both hematopoietic and nonhematopoietic CD47 was found to be required for the homeostasis of CD4+ cDCs and CD8−CD4−(double negative) cDCs in the spleen. SIRPα as well as its ligand, CD47, are thus important for the homeostasis of CD4+ cDCs or double negative cDCs in lymphoid tissues.

Description: Key Points Naturally occurring oncogenic GATA1 mutants with internal deletions contribute to transient abnormal myelopoiesis in Down syndrome.

Description: Abstract 490 T lymphocytes play central roles in cellular immunity, exerting their proliferative and effector activities when they recognize antigens, in HLA-restricted and antigen-specific manner, via T-cell receptors (TCRs). Successful treatment of leukemias/cancers with T-lymphocytes infusions is a direct proof that human immunity has the potential to eradicate cancers. However, continuous exposure to tumor/self antigens drives T lymphocytes into a highly exhausted state, with loss of potentials for long-term survival, proliferation, and effector functions that can end up with deletion of antigen-responding T-lymphocyte pools. Several workers have endeavored to develop clinical protocols for expanding antigen-responding T cells from the few naïve T-cell pools remaining in the patient. However, highly expanded T cells in such protocols have not proved fully effective so far, because functional losses like those in the patient occur during ex vivo manipulation. To overcome this obstacle to T-lymphocyte based immunotherapy, we endeavored to induce antigen-specific TCR-expressing T lymphocytes from induced pluripotent stem (iPS) cells, which were derived from antigen-reactive single T lymphocytes. iPS cells have a capacity for unlimited self-renewal while maintaining pluripotency. These features enabled us to induce an unlimited number of T lymphocytes, especially naïve T lymphocytes, showing reactivity to specific antigens. If they retain properties of naïve T lymphocytes, they may proliferate for a longer period and achieve better therapeutic effects than their peripheral blood counterparts expanded in vitro. Peripheral T lymphocytes were isolated from healthy volunteers. Then three reprogramming factors (OCT4, SOX2, and KLF4) and additional factors (c-MYC and/or NANOG) were transduced into fresh or frozen/thawed T lymphocytes using a retrovirus. The virus-infected T lymphocytes were then transferred onto mouse embryonic fibroblasts (MEFs) in the presence of cytokines and chemicals favorable for T-lymphocyte survival/proliferation. iPS-like colonies were observed within 3 weeks after infections. Single T lymphocyte-derived colonies were isolated and clonally expanded. They exhibited standard ES-like morphology, cell surface markers and alkaline phosphatase activity, as well as differentiation potential into various tissues related to all three germ layers. Human TCRs are encoded in four genes (TCRA, TCRB, TCRG, TCRD), which should be genetically assembled in an irreversible manner during T-lymphocyte development. This feature allowed us to retrospectively confirm the iPS cells were generated from T lymphocyte. The TCR genes rearrangement encoded in an iPS colony was single in all iPS lines, indicating that the iPS colony was derived from single T lymphocyte. Sequence analyses of TCR genes revealed whether the rearrangements were productive, and the productivity might promise the conservation of TCR genes rearrangement during the reprogramming process. Next, we tried to re-differentiate T-lymphocyte derived-iPS (T-iPS) cells into T-lineage cells by co-culturing them with murine stromal cell layers (OP9 and OP9-DL1). These T-lineage committed cells were expressed TCRab heterodimer and T-cell surface markers such as CD3. They could activate via TCR stimulation, and produce IL-2 and IFN-g as maturing T lymphocytes. The re-differentiation efficiency of T-iPS cells was higher than those of embryonic stem cells, fibroblasts derived-iPS cells, or cord blood derived-iPS cells. mRNA sequence of TCRs transcribed in re-differentiated T-lineage cells was identical to that engraved in the pre-differentiated T-iPS cells genome. The invariance of the sequence, especially antigen-recognition site sequence, indicated that the antigen-specificity in original T lymphocyte was conserved during re-differentiation process. Here we show that the conservation of the antigen-specificity encoded in TCR genes throughout induction of T-iPS cells and re-differentiation into T-lineage cells. These data suggest that further optimization of these processes for clinical application could open the door to the development of novel T-lymphocyte therapy, repeatedly supplying patient-compatible and disease-specific naïve T lymphocytes. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 703 Patient-specific, induced pluripotent stem cells (iPSCs) enable us to study disease mechanisms and drug screening. To clarify the phenotypic alterations caused by the loss of c-MPL, the thrombopoietin (TPO) receptor, we established iPSCs derived from skin fibroblasts of a patient who received curative bone marrow transplantation for congenital amegakarycytic thrombocytopenia (CAMT) caused by the loss of the TPO receptor gene, MPL. The resultant CAMT-iPSCs exhibited mutations corresponding to the original donor skin. Then using an in vitro culture system yielding hematopoietic progenitor cells (HPCs), we evaluated the role of MPL on the early and late phases of human hematopoiesis. Although CAMT-iPSCs generated CD34+ HPCs, per se, their colony formation capability was impaired, as compared to control CD34+ HPCs. Intriguingly, both Glycophorin A (GPA)+ erythrocyte development and CD41+ megakaryocyte yields from CAMT-iPSCs were also impaired, suggesting that MPL is indispensable for MEP (megakaryocyte erythrocyte progenitors) development. Prospective analysis along with the hematopoietic hierarchy revealed that, in CAMT-iPSCs but not control iPSCs expressing MPL, mRNA expression and phosphorylation of putative signaling molecules downstream of MPL are severely impaired, as is the transition from CD34+CD43+CD41-GPA- MPP (multipotent progenitors) to CD41+GPA+ MEP. Additional analysis also indicated that c-MPL is required for maintenance of a consistent supply of megakaryocytes and erythrocytes from MEPs. Conversely, complimentary transduction of MPL into CAMT-iPSCs using a retroviral vector restored the defective erythropoiesis and megakaryopoiesis; however, excessive MPL signaling appears to promote aberrant megakaryopoiesis with CD42b (GPIba)-null platelet generation and impaired erythrocyte production. Taken together, our findings demonstrate the usefulness of CAMT-iPSCs for validation of functionality in the human hematopoiesis system. For example, it appears that MPL is not indispensable for the emergence of HPCs, but is indispensible for their maintenance, and for subsequent MEP development. Our results also strongly indicate that an appropriate expression level of an administered gene is necessary to achieve curative gene correction / therapy using patient-derived iPSCs. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4585 Malignant infantile osteopetrosis is an autosomal recessive disease characterized by a lack of osteoclastic function with incidence of 1: 200,000 to 1: 300,000. In consequence of disturbed bone building and remodeling, affected patients have osteosclerosis, dense fragile bone, and a marked reduction in the bone marrow cavity. Clinical features, such as anemia, thrombocytopenia, hepatosplenomegaly, bone fractures, bone deformity, and cranial nerve entrapment, appear soon after birth or within the first years of life. In the natural course of the disease, only 30% of children can survive to more than six years old. Because osteoclasts are of hematopoietic origin, allogeneic stem cell transplantation is the only curable therapy. Recently, successful HLA-haploidentical hematopoietic stem cell transplantation (HSCT) has been reported for patients without HLA- matched donors. We also present an infant with malignant infantile osteopetrosis who underwent HLA-haploidentical HSCT. A four-month-old boy was referred to our hospital for splenomegaly and pancytopenia. A physical examination revealed failure to thrive, distention of anterior fontanel, and hepatosplenomegaly. Laboratory findings indicated thrombocytopenia and anemia, and elevated alkaline phosphatase. Osteoclasts were hyperplastic in bone marrow. Radiography showed homogeneous and sclerotic bones with absence of corticomedullary junction, and bone marrow scintigraphy showed no accumulation of bone marrow. Subsequently, TCIRG1 mutations are identified. All these findings are compatible with malignant infantile osteopetrosis and the diagnosis was made. We conducted HSCT to revive his osteoclastic function. However, he didn't have HLA-matched donors not only in relatives but also in the bone marrow bank and the cord blood bank of Japan. We attempted HLA-haploidentical bone marrow transplantation (BMT) from his father with written informed consent. The patient was prepared for BMT at 11 months of age by conditioning with busulfan (6 mg/kg × 4/day × 4 days), cyclophosphamide (360mg/m2 × 2 days) and antithymocyte globulin (2 mg/kg/day × 3 days). Prophylaxis for graft-versus-host disease (GVHD) included tacrolimus and short-term methotrexate. Bone marrow nuclear cells (1×109/kg) were collected from his father and transplanted without any manipulations. The engraftment of neutrophils was confirmed on day 9 after BMT. Acute GVHD was limited to the skin (grade I). At 2 months after BMT, neutrophil counts, platelet counts, and hemoglobin level were all within normal limits. Bone marrow scintigraphy revealed a significant uptake in bone marrow. There have been no chronic GVHD up to six months after BMT.HSCT for patients with osteopetrosis is challenging as it is reported that HLA-haploidentical HSCT has the high rate of acute and chronic GVHD. Fortunately, HLA-haploidentical BMT was successful in our patient. This might be due to our regimen for prophylaxis of rejection and GVHD. We used ATG in conjunction with tacrolimus: this regimen may be prophylactic not only for rejection but also for GVHD as previously reported (Schulz et al. Blood 2002; 99: 3458–3460). In conclusion, HLA-haploidentical family donors should be considered as the alternative hematopoietic stem cell source for patients with malignant infantile osteopetrosis in condition without HLA-matched donors. Disclosures: No relevant conflicts of interest to declare.

Description: Background We have previously identified very primitive human cord blood (CB)-derived CD34-negative (CD34-) severe combined immunodeficiency (SCID)- repopulating cells (SRCs) using the intra-bone marrow injection (IBMI) method (Blood 2003:101;2924). A series of our studies suggests that the identified CD34- SRCs are a distinct class of primitive hematopoietic stem cell (HSC) and that they are at the apex of human HSC hierarchy. Recently, we developed a high-resolution purification method for primitive CD34- SRCs using 18 lineage (Lin)-specific antibodies, which can enrich CD34- SRC at 1/1,000 level (Exp Hematol 2011: 39:203). In the present study, we tried to identify the positive marker of CD34- SRCs in order to further purify and characterize the CD34- SRCs (HSCs). Materials and Methods First, we extensively analyzed candidate positive markers, including known HSC markers and various adhesion molecules by FACS using highly purified CB-derived 18Lin-CD34+/- cells. Finally, we identified CD133 as a positive marker of human CB-derived CD34- SRCs. Then, CB-derived 18Lin- CD34+/-CD133+/- cells were sorted by FACS, and hematopoietic stem/progenitor cell (HSPC) capacities of these four fractions of cells were extensively investigated. HSPC capacities were evaluated using (1) colony-forming cell (CFC) assays, (2) measurement of maintenance/production of CD34+ cell capacities in co-cultures with human bone marrow-derived mesenchymal stromal cells (BM-MSCs) (Blood 2010:24:162), (3) SRC activities using NOG mice, (4) limiting dilution analyses (LDA) to determine the SRC frequency in the 18Lin-CD34-CD133+ fractions, and (5) comparison of gene expression profiles between 18Lin-CD34+/-CD133+/- cells by real-time RT-PCR. Results Seventy-five percent of 18Lin-CD34+ and 13.5% of 18Lin-CD34- cells highly expressed CD133. In the CFC assays, the plating efficiencies of 18Lin-CD34+CD133+, CD34+CD133-, CD34-CD133+ and CD34-CD133- cells were 57%, 65%, 39% and 19%, respectively. Interestingly, most of 18Lin-CD34-CD133+/- cells formed erythroid-bursts (71% and 73%) and erythro/megakaryocytes-containing mixed colonies (25% and 27%). On the contrary, they formed few granulocyte/macrophage colonies (4.2% and 0%). Then, we co-cultured these four fractions of cells with human BM-MSCs. One thousand of 18Lin-CD34+/-CD133+/- cells were seeded into each well and cells were co-cultured for 7 days in the presence of SCF+TPO+FL+IL-3+IL-6 +G-CSF. Both the 18Lin-CD34-CD133+/- cells produced CD34+ cells. However, the percentage and absolute number of CD34+ cells produced from 18Lin-CD34-CD133+ cells (31.7 % and 3.2 x 104 cells) were greater than those of 18Lin-CD34-CD133- cells (13.2 % and 0.4 x 104 cells). In addition, both the 18Lin-CD34- CD133+/- cells generated higher percentages (13.5 % and 11.5%) of CD41+ cells compared to those of the 18Lin- CD34+CD133+/- (1.8% and 4.2%) cells. Collectively, 18Lin-CD34+/-CD133+/- cells showed different in vitro lineage differentiation potentials. Then, these four fractions of cells were transplanted into NOG mice by IBMI. We performed primary and secondary transplantations for up to 36 weeks. In the results, all of the mice received 18Lin-CD34+CD133+ cells (n = 5) or 18Lin-CD34-CD133+ cells (n = 9) showed primary and secondary human CD45+ cell repopulations. However, neither 18Lin-CD34+CD133- cells nor 18Lin-CD34-CD133- cells showed human cell repopulations (n = 6 in each group). These results clearly demonstrated that the CD133 expression clearly segregated SRC activities in the 18Lin-CD34+/- cells. Moreover, LDA demonstrated that the frequency of SRCs in the 18Lin-CD34-CD133+ fraction was 1/142. Interestingly, HSC self-renewal maintenance genes, such as Notch1, HoxB4, HoxA9, and Bmi-1, were highly expressed in both 18Lin-CD34+/-CD133+ cells. Conclusion These results clearly demonstrated that CD133 is a positive marker of human CB-derived CD34- SRCs (HSCs). Furthermore, CD133 segregated SRC activities of 18Lin-CD34- as well as 18Lin-CD34+ cells in its positive fractions. More importantly, these findings suggest that number of CD133+ cells in cord blood units is a more appropriate marker to detect/predict HSC potentials in cord blood stem cell transplantation in comparison to currently used CD34+ cell numbers. Disclosures: No relevant conflicts of interest to declare.

Description: ABL1 gene encoding a non-receptor tyrosine kinase has been involved as ABL1 chimeric genes in hematological malignancies. Their partner genes include BCR in chronic myeloid leukemia (CML), B-acute lymphoblastic leukemia (B-ALL), T-acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML), ETV6 in B-ALL, T-ALL, AML and myeloproliferative neoplasms (MPNs), RCSD1, SFPQ, ZMIZ1, FOXP1, SNX2, GAG in B-ALL, NUP214 and EML1 in T-ALL. The resultant chimeric gene products have been shown to associate with a cellular proliferation through constitutive activation of ABL1 signaling. In addition, they are the therapeutic targets for tyrosine kinase inhibitors (TKIs). We identified a novel chimeric fusion gene SEPT9 (Septin9)-ABL1 in a case of T-prolymphocytic leukemia (T-PLL) using 5’- RACE PCR. It is generated by an in-frame fusion between SEPT9 exon 4 and ABL1 exon 2. The case was treated with imatinib and dasatinib, but the tumor burden did not reduce at all. The purpose of this study was to analyze the biological functions of the SEPT9-ABL1 gene product including TKIs sensitivity. The expression analysis in the case using RT-PCR and Western blot revealed that there were at least 4 isoforms applying SEPT9a, SEPT9d, SEPT9e and SEPT9f as the fusion partners. SEPT9a-ABL1 was expressed most strongly among the SEPT9-ABL1 isoforms. The SEPT9-ABL1 isoforms were retrovirally transduced into 32D cells, a murine interleukin3 (IL-3) dependent hematopoietic cell line. All of these isoforms caused 32D cells an IL-3 independent proliferation. Their proliferation rates were less than 32D cells with BCR-ABL1, and that of 32D cells with SEPT9d-ABL1 was subtle (day0, 1×105 cells/ ml; day2, the densities of 32D cells with BCR-ABL1, SEPT9a-ABL1, SEPT9d-ABL1 SEPT9e-ABL1 and SEPT9f-ABL1 were 17.8×105, 6.7×105, 2.7×105, 6.0×105 and 6.1×105 cells /ml, respectively). In consistent with the results of cellular proliferation, the immunocytological analysis revealed SEPT9a-ABL1, SEPT9e-ABL1 and SEPT9f-ABL1 proteins localized mainly in the cytoplasm. When these cells were transplanted into C3H/HeJ mice (5×106/ mouse). Mice with SEPT9e-ABL1- and SEPT9f-ABL1-infected cells died with the infiltration of transplanted cells into bone marrow, spleen and liver within 50 days. It was later than mice with BCR-ABL1 infected cells. Half maximal inhibitory concentrations (IC50) of imatinb in 32D cells infected with BCR-ABL1, SEPT9a-ABL1, SEPT9e-ABL1 and SEPT9f-ABL1 were 2.52μM, 0.96μM, and 2.08μM, respectively. They were higher than IC50 with BCR-ABL1 (0.28μM)( SEPT9a-ABL1, 9.0 times, p

Description: Background: A proto-oncogene BCL6 is a known transcriptional repressor and a master regulator of germinal center B cell program. It represses expression of DNA damage response genessuch as p53, ATR, CHEK1 and p21, which helps B cells to survive and expand during antigen receptor-diversification reactions. It also plays a pivotal role in the formation of germinal centers and lymphomagenesis. BCL6 is down-regulated in normal plasma cells while it is aberrantly expressed in bone marrow residing myeloma cells. Although the role of BCL6 in B cell lymphomas has been intensively studied, its role in plasma cell neoplasms remains to be elucidated. In this study we asked whether BCL6 plays a role in DNA damage response of myeloma cells. Methods: Bone marrow samples obtained from 36 of primary plasma cell dyscrasia patients (5 cases of MGUS, 30 of multiple myeloma (MM) and 1 plasma cell leukemia (PCL)) were subjected to the study after informed consent. This study was approved by IRBs of Gunma University Hospital and Nishigunma National Hospital. CD138-positive bone marrow plasma cells were isolated as a purity of 〉95% using magnetic beads and RNAs were extracted. Expression levels of BCL6, p53, ATR, CHEK1 and p21 were quantified by real time PCR using Taqman-probes. For retroviral infection, BCL6 was cloned into pMY-IRES-GFP vector and transfected to PlatA cells using lipofection reagents. 48 hours later, supernatants were collected and centrifuged for 16 hours and the pellets were used for infection. GFP positive cells were collected and used for following experiments. For γH2AX foci formation analysis, the cells were given ionized irradiation at doses of 0, 3, 5 and 10Gy, and used after an hour incubation. Cells were also exposed to different concentration of etoposide (0, 1, 5, 10, 50, 100μM) for 30minute, then washed with fresh media and incubated for an hour. Cells were stained with a FITC-labeled anti-γH2AX antibody as reported (Muslimovic et al, Nat. Protoc. 2008). For cell cycle analysis by flow cytometry, EdU uptake and 7AAD DNA staining were performed according to a manufacture’s protocol (Life Technologies). Correlation of expression levels between each of genes was assessed using Spearman’s non-parametric correlation analysis. Results: Median of BCL6 expression levels of MM and PCL cells (Qty median=1.47, range 0.09-17.2) was not significantly different from that of MGUS cells (Qty median=1.84, range 0.8-4.2, p=0.51). In marked contrast to germinal center B cells, expression levels of BCL6 and p53 were positively correlated in MM, PCL and MGUS (r=0.457, p=0.007). The correlation between expression of BCL6 and ATR did not reach statistical significance (r=0.323, p=0.062). ATR and p53 were also positively correlated (r=0.549, p=0.001). The expression level of p21, a well-known target of p53, was positively correlated to that of p53 (r=0.681, p=0.03), which supports our data qualification. We also examined mRNA expression levels of BCL6 in MM cell lines, RPMI8226, KMS11, KMS12PE, KMS12BM, KMS18, KMS26, ARH 77 and U266. U266, KMS12PE, KMS26 expressed little amount of BCL6 compared to the patient samples. The other five cell lines did not express BCL6. In order to study BCL6 functions in MM cells, we retrovirally expressed BCL6 in the KMS12BM cell line. The expression level of BCL6 was comparable to the patient samples after the retroviral expression (QTy 6.70). Since BCL6 is known to be a transcriptional repressor and supposed to directly repress p53, ATR, CHEK1 and p21 in B cells, we analyzed expression levels of these genes. Intriguingly, p53, ATR, CHEK1 and p21 are not repressed by overexpression of BCL6 in KMS12BM. These results suggest that alternative regulatory mechanisms of transcriptional regulation by BCL6 are present in MM cells. For further evaluation of the DNA damage response by BCL6 expression, we irradiated or treated these cells with etoposide and analyzed for γH2AX foci formation, a hallmark of DNA double strand break. No differences in the foci formation between mock-infected and BCL6-infected-KSM12BM were detected either with irradiation or etoposide exposure. We also studied cell cycle progressionin these cells. Flow cytometry analysis showed no significant difference between these cells. Conclusion: Unlike B cells in germinal centers, BCL6 expression in myeloma cells did not repress DNA damage response gene expression. Disclosures No relevant conflicts of interest to declare.

Description: Background: 5-methylation (5-mC) is the predominant epigenetic mark in mammalian genomic DNA. When promoter region of certain gene is hypermethylated, the gene becomes transcription silent. Promoter of tumor suppressor genes (TSG) usually exists in CpG islands, and silencing of TSGs in cancer cells is often associated with hypermethylation. p15, CDH1 are frequently methylated in myeloid malignancies such as acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Common Fragile Site (CFS) is a fragile site on the chromosomes easy to produce gap and break, and it contains putative TSGs. FHIT, WWOX and PARK2 are the CFS genes known to be frequently methylated in solid tumors, but their status of hematologic malignancies has not been fully elucidated yet. 5-hydroxymethylaiton (5-hmC) is a newly discovered epigenetic modification that is presumably generated by oxidation of 5-mC by the TET family of cytosine oxygenases. Techniques identifying 5-mC cannot distinguish between 5-mC and 5-hmC, therefore 5-hmC status of the genes have not fully elucidated yet too. Recently it has been demonstrated that mutation of epigenetic modifiers (DNMT3A, TET2, IDH1/2) play important role on AML pathogenesis. We tried to clarify 5-mC and 5-hmC status of TSG p15, CDH1 and CFS genes FHIT, WWOX and PARK2 by using new techniques and the relationships with expression levels of epigenetic modifiers in AML. Methods: BM samples obtained from 74 of AML patients are subjected to the study after informed consent. This study was approved by IRB of Gunma University Hospital. DNA, RNA were extracted from BM mononuclear cells. Methylation specific PCR (MSP) was carried out to assay 5-mC of p15, CDH1, WWOX, PARK2. Quantification of 5-mC and 5-hmC (except PARK2) was carried out by methylation sensitive restriction enzyme assay (MSRE) with glucosylation and Q-PCR. Total DNA 5-mC and 5-hmC were analyzed by ELISA. The mRNA expression levels of p15, CDH1, FHIT, WWOX, PARK2, DNMT1, 3A, TET2 were quantified by Q-PCR. Results: MSP revealed that p15, CDH1, WWOX and PARK2 were methylated in 43.1%, 94.3%, 35.7% and 36.9% of AML, respectively. PARK2 methylation was not found in t(15;17) APL, but in 32% of normal karyotype AML (NK-AML), in 67% of t(8;21) CBF-AML. In contrast, the p15 methylation was found in 83.3% of APL, 45.5% of NK-AML, 50% of CBF-AML. WWOX methylation was found in 42.9% of APL, in 16% of NK-AML and 66.7% of CBF-AML. Adverse karyotype AML (adv-AML) tended to show lower % of WWOX, PARK2 and p15 methylation with 15.8%, 21.1% and 18.8% compare to good risk karyotype. The frequency of the methylation of PARK2 and WWOX were varied among karyotypes and the methylation was mutually exclusive. ELISA demonstrated that mean % of total 5-mC DNA was 1.08% and ratio of 5-hmC in 5-mC was 0.95% in AML. Interestingly, 5-hmC was 0% in adv-AML although 5-mC existed (mean: 1.05%). Locus specific MSRE-QPCR demonstrated that mean % of 5-mC of p15, CDH1, WWOX and FHIT were 6.62%, 1.25%, 8.33%, 2.88%, respectively., In adv-AML, 5-hmC of CDH1, WWOX and FHIT were not detected, although 5-mC of these genes were detected (0.41%, 9.0%, 2.14%) in accordance with whole DNA analysis. In good and intermediate AML, 5-hmC of these genes was 3.44%, 1.07%, 2.69% ,respectively. RQ-PCR demonstrated that CDH1, p15, WWOX, PARK2 and epigenetic modifier DNMT1, DNMT3A and TET2 expression were not different among various karyotype risks, but only FHIT expression significantly higher in good risk group (p=0.047). The expression levels of the genes were not significantly different between mentylated and unmethylated. The ratio of 5-hmC/5-mC of the TSGs tended to be associated with the expression levels of the corresponding genes, but the association did not reach statistical significance. DNMT3A expression in AML with 5-mC PARK2 was higher than in other AML (p=0.016). Contrary to the intuition, DNMT3A expression was positively correlated with FHIT, PARK2 expression (r=0.776, p

Description: Abstract 3185 Human ALAS2 gene encodes erythroid-specific 5-aminolevulinate synthase, the rate-limiting enzyme of heme biosynthetic pathway in erythroid cells. The mutation of ALAS2 gene causes X-linked sideroblastic anemia (XLSA), and approximate 50 different mutations have been reported in the coding region of ALAS2 gene as disease causative mutations. Here, we report two novel mutations of ALAS2 gene identified in a pedigree with XLSA and one male patient with congenital sideroblastic anemia (CSA). Incidentally, both mutations were predicted to impair or abolish the function of the specific GATA transcription factor-binding motif (GATA element) located at the middle of the first intron of ALAS2 gene (referred as ALAS2int1GATA). In a proband of the pedigree of XLSA, the “GATA” sequence in ALAS2int1GATA, which is the core sequence of consensus for GATA element (WGATAR), was changed to “GGTA”, and the same mutation was identified in two male relatives, his mother's cousins, both of whom were diagnosed as sideroblastic anemia. Furthermore, a patient with CSA carries a deletion of 35 bps in the first intron of ALAS2 gene, the deleted region of which contains ALAS2int1GATA, although this deletion was not detected in his parents' ALAS2 gene. No other mutation was detected in the proximal promoter region, the known enhancer region present in eighth intron, and the coding region and exon-intron boundaries of ALAS2 gene. Moreover, no mutation was detected in the coding region and exon-intron boundaries of SLC25A38, ABCB7, GLRX5, PUS1 or SLC19A2 gene, each of which was reported as a responsible gene for CSA. It is of interest that ALAS2int1GATA is present within one of “GATA transcription factor-occupying regions in K562 erythroleukemia cells” identified by genome-wide analysis using chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) (Fujiwara et al., Mol Cell, vol. 36: p667–681, 2009), suggesting that ALAS2int1GATA may act as a cis-regulatory element for ALAS2 transcription in vivo. In fact, our ChIP-PCR analysis in K562 cells, which focused on the proximal promoter (−267 to +29) and the first intron of ALAS2 gene, confirmed that GATA-1 protein selectively bound to the GATA element in the proximal promoter region as well as ALAS2int1GATA out of 17 consensus GATA elements present in the first intron. Then, we examined whether the 467-bp region in the first intron of ALAS2 gene, defined by ChIP-seq analysis (referred as “ALAS2 ChIP-peak”), acts as an enhancer for ALAS2 expression using transient reporter assay, because ALAS2 ChIP-peak contains ALAS2int1GATA. For this assay, we used pGL3-AEpro, which contains the firefly luciferase gene under the control of the ALAS2 proximal promoter, as a parent reporter vector. The presence of ALAS2 ChIP-peak in the downstream region of the luciferase gene increased ALAS2 proximal promoter activity about ten-folds in K562 cells. However, this enhancing activity of ALAS2 ChIP-peak was not observed in non-erythroid HEK293 embryonic kidney cells. Importantly, each newly identified mutation at ALAS2int1GATA diminished the enhancer activity of ALAS2 ChIP-peak on ALAS2 proximal promoter in K562 cells. Moreover, electrophoretic mobility shift assay (EMSA) revealed that GATA-1 protein in nuclear extracts of K562 cells or HEK293 cells overexpressing GATA-1 could bind to a wild-type probe containing ALAS2int1GATA, but GATA-1 failed to bind to a mutant probe, which carries a single base change in ALAS2int1GATA or a deletion of ALAS2int1GATA that was identified in patients with XLSA or CSA, respectively. These results suggest that ALAS2int1GATA plays an essential role for enhancing ALAS2 expression as a core of the ALAS2 ChIP-peak, the function of which may depend on the erythroid-specific transcription factor, GATA-1. Thus, the mutation at or the deletion of ALAS2int1GATA impairs the recruitment of GATA transcription factor(s) to the ALAS2 ChIP-peak, which in turn decreases the transcription of ALAS2 gene, thereby causing XLSA or CSA. In conclusion, we provide the evidence for the existence of an enhancer element in the first intron of ALAS2 gene. Moreover, the loss-of-function mutation at the newly identified enhancer element of ALAS2 gene is associated with XLSA or CSA. Disclosures: No relevant conflicts of interest to declare.