ALBERT

Description: Junctional adhesion molecule-A (JAM-A/JAM-1/F11R) is a cell adhesion molecule expressed in epithelial cells, endothelial cells and also hematopoietic cells such as leukocytes, platelets and erythrocytes. However, the expression of JAM-A in hematopoietic stem cell (HSC) had not been known. Here we show that JAM-A is expressed at a high level in the enriched HSC fraction, i.e. CD34+ c-Kit+ cells in embryonic day 11.5 (E11.5) aorta-gonod-mesonephros (AGM) and E11.5 fetal liver (FL) as well as c-Kit+ Sca-1+ Lineage- (KSL) cells in E14.5 FL, E18.5FL and adult bone marrow (BM). While the percentage of JAM-A+ cells in those tissues decreases during the development, the expression in HSC fraction is maintained throughout life. In fact, c-Kit+Sca-1+ cells are enriched approximately 200-fold from whole BM cells by anti-JAM-A antibody alone, i.e. the c-Kit+Sca-1+ cells in whole BM was about 0.15% and that in JAM-A+ cells was about 30%. Colony forming assays reveal that multi-lineage colony forming activity (CFU-mix) in JAM-A+ cells is higher than that in JAM-A- cells in the enriched HSC fraction in all those tissues. HSC transplantation assay revealed that long-term repopulating HSC (LTR-HSC) activity is present in the mice received 100 JAM-A+ KSL (7/9) cells and 300 KSL cells (5/6). Since the ratio of JAM-A+ cells to JAM-A- cells in the KSL fraction is about 6.5:3.5, 100 JAM-A- KSL cells are equivalent to 285 total KSL cells. In contrast to JAM-A+ cells, the mice received 1000 JAM-A- KSL cells, which are equivalent to more than 2850 total KSL cells, failed to engraft for long-term (0/6). These data revealed that long-term repopulating HSC (LTR-HSC) activity is present exclusively in the JAM-A+ cells, but not in JAM-A- cells. Moreover only 100 JAM-A+ cells isolated from whole BM cells by anti-JAM-A antibody alone reconstituted the hematopoietic system for long-term (4/7). Together these results indicate that JAM-A is expressed on hematopoietic precursors in various hematopoietic tissues and is an excellent and convenient marker to enrich LTR-HSC from BM cells.

Description: Additional sex combs-like 1 (ASXL1), an epigenetic modulator, is frequently mutated in myeloid neoplasms. Recent analyses of mutant ASXL1 conditional knock-in (ASXL1-MT-KI) mice suggested that ASXL1-MT alone is insufficient for myeloid transformation. In our previous study, we utilized retrovirus-mediated insertional mutagenesis, which exhibited susceptibility of ASXL1-MT-KI hematopoietic cells to transform into myeloid leukemia cells. In this screening, we identified Hematopoietically expressed homeobox (HHEX) gene as one of the common retrovirus integration sites. In this study, we investigated the potential cooperation between ASXL1-MT and HHEX in myeloid leukemogenesis. Expression of HHEX enhanced proliferation of ASXL1-MT expressing HSPCs by inhibiting apoptosis and blocking differentiation, whereas it showed only modest effect in normal HSPCs. Moreover, ASXL1-MT and HHEX accelerated the development of RUNX1-ETO9a and FLT3-ITD leukemia. Conversely, HHEX depletion profoundly attenuated the colony-forming activity and leukemogenicity of ASXL1-MT-expressing leukemia cells. Mechanistically, we identified MYB and ETV5 as downstream targets for ASXL1-MT and HHEX by using transcriptome and ChIP-seq analyses. Moreover, we found that expression of ASXL1-MT enhanced the binding of HHEX to the promoter loci of MYB or ETV5 via reducing H2AK119ub. Depletion of MYB or ETV5 induced apoptosis or differentiation in ASXL1-MT-expressing leukemia cells, respectively. In addition, ectopic expression of MYB or ETV5 reversed the reduced colony-forming activity of HHEX-depleted ASXL1-MT-expressing leukemia cells. These findings indicated that the HHEX-MYB/ETV5 axis promotes myeloid transformation in ASXL1-mutated preleukemia cells.

Description: Chronic myeloid leukemia (CML) is effectively treated with imatinib mesylate (IM), a small molecule inhibitor of the BCR-ABL tyrosine kinase that is expressed in the entire hematopoietic compartment including stem cells (HSC) and progenitors in CML patients. While IM induces disease remission, it does not appear to eradicate BCR-ABL-positive stem cells. We analyzed the HSC/Progenitors profiles using fluorescence-activated cell sorting (FACS) and investigated the minimal residual disease (MRD) in HSC and myeloid progenitors from patients with CML chronic phase (CP) after IM-therapy. HSC identified as CD34+CD38–Lin–, were separated to Thy-1+ (HSC/Thy-1+) and Thy-1– (HSC/Thy-1–) cells. HSC/Thy-1+ (CD34+CD38–Thy-1+Lin–), HSC/Thy-1– (CD34+CD38–Thy-1–Lin–), common myeloid progenitors (CMP: CD34+CD38+IL-3Rα+CD45RA–Lin–), granulocyte–macrophage progenitors (GMP: CD34+CD38+IL-3Rα+CD45RA+Lin–), and megakaryocyte–erythroid progenitors (MEP: CD34+CD38+IL-3Rα–CD45RA– Lin–) were isolated by cell sorting and MRD was quantified with real-time polymerase chain reaction detecting BCR-ABL transcripts. FACS analysis revealed higher levels of the HSC/Thy-1– cells and progenitors (CD34+CD38+Lin– cells) in bone marrow from patients with CML CP than in normal bone marrow although the level of long-term HSC in CML CP was similar with normal bone marrow. After the IM-therapy the proportion of progenitor pools (CD34+Lin– cells) within Lin– were remarkably reduced, especially that of HSC/Thy-1– cells and progenitor cells. The proportion of MEP was increased and that of GMP was decreased in bone marrow from patients with CML CP as compared with their normal counterparts. BCR-ABL positive progenitors in bone marrow were eradicated within 12 month in 5 out of 9 patients. BCR-ABL positive cells, however, remained in the stem cell population. They were positive even after achieving undetectable levels of BCR-ABL transcript in total RNA isolated from the bone marrow. The ratio of BCR-ABL to BCR was significantly decreased with the continuation of imatinib, however the retention of BCR-ABL-positive cells was observed in HSC/Thy-1– or HSC/Thy-1+ populations except one out of 9 cases. These results indicated retention but significant reduction of BCR-ABL positive stem cells in CML during IM-therapy. They also implicate that the sorted and purified stem cells are useful for more sensitive quantification of BCR-ABLpositive MRD.

Description: Abstract 1977 Poster Board I-1000 Anemia is a common complication of chronic kidney disease (CKD), mainly due to the inability of the kidneys to secrete enough erythropoietin to adequately stimulate hematopoiesis. Further, given that the lifespan of red blood cells (RBCs) has been reported to be reduced in CKD patients, this reduction in RBC lifespan is believed to be a part of the etiology of renal anemia. In the present study, we focused on RBC survival and measured the lifespan of RBCs in rats with nephrogenic anemia. We also examined the effects of erythropoietin on RBC lifespan in this anemia model. Nephrogenic anemia was induced by oral administration of adenine (600 mg/kg/day for 10 days) to male Wistar rats. Progressive, serious anemia associated with increased levels of plasma creatinine was observed in the rats. On Day 40, the number of RBCs and hemoglobin (HGB) levels were lower in the adenine-treated rats than in normal, control rats (normal: 930×104/μL, anemic: 677×104/μL for RBC and normal: 17.2 g/dL, anemic: 13.4 g/dL for HGB). However, the number of reticulocytes did not change in the anemic rats (normal: 299×103/μL, anemic: 329×103/μL, P = 0.102). The percentage of annexin V-binding erythrocytes was increased in anemic rats (normal: 0.77%, anemic 1.76%) and inversely correlated with RBC count and HGB levels, suggesting that apoptosis of RBCs increased as anemia progressed. Taking these findings into account, we measured the lifespan of RBCs in rats with nephrogenic anemia. We transfused 5-chloromethylfluorescein diacetate (CMFDA)-labeled RBCs from normal donor rats into either normal or anemic recipients and determined the number of labeled RBCs present in the peripheral blood at various time points thereafter. The time course of the reduction in the percentage of labeled RBCs in peripheral blood revealed that the half-life (t1/2) of RBCs in anemic rats was shorter than in normal rats (normal: 22.5 d, anemic: 13.3 d). This reduction in RBC lifespan was also observed in a rat model of cisplatin-induced renal anemia. Injection of anemic rats with recombinant human erythropoietin (rhEPO) restored the number of RBCs and HGB concentration to normal levels. However, the t1/2of RBCs in these rats was not changed. The clearance of RBCs in anemic rats does not appear to be influenced by rhEPO injection. In conclusion, the survival of RBCs was reduced in rats with nephrogenic anemia, an observation consistent with the shortened survival time of RBCs in renal failure patients. This finding suggests that this model is suitable for investigating drugs which may be used in the treatment of renal anemia. Further, because EPO therapy did not affect the lifespan of RBCs, agents which improve the shortened RBC survival inherent in CKD patients may be useful in treating renal anemia. Disclosures: Iwatsuki: Astellas Pharma Inc.: Employment. Kitamura:Astellas Research Institute of America LLC: Employment. Suzuki:Astellas Pharma Inc.: Employment.

Description: To understand myelodysplastic syndromes (MDS) at the molecular level, we used a mouse bone marrow transplant (BMT) model. Several leukemogenic fusion proteins were demonstrated to induce leukemia in mouse BMT models. However, analysis of molecular basis of MDS has been hampered by the lack of mouse MDS models. Mutations of a transcription factor AML-1/Runx-1 were identified in 15–40% of MDS-RAEB and MDS/AML and 5–10% of de novo AML. To test transforming abilities of the mutant AML-1, we transduced AML-1 with different mutations (#5 and #27) into mouse bone marrow cells using our highly efficient retrovirus-mediated gene transfer, transplanted the transduced cells to irradiated mice, and found that most mice developed leukemia within 4–13 months after the transplant (table1 and figure1). The leukemic mice exhibited marked hepato-splenomegaly, and morphological abnormalities of myeloid and erythroid lineages were frequently observed. Some mice developed to leukemia after a long latency with MDS-like symptoms, either with pancytopenia or leukocytosis with anemia, thus mimicking the human disease. To clarify the differences between the leukemic mice with early onsets and those that developed leukemia after a long period of MDS-like symptoms in the mutant AML-1-induced mouse diseases, the integration sites of the transduced retroviruses were investigated using a polymerase chain reaction (PCR)-based technique bubble PCR. Our preliminary results suggested that the integration sites of the retroviruses harboring the mutant AML-1 contribute to the early onset of leukemia. In conclusion, we have developed a useful mouse in vivo model of MDS/AML that should help understand the molecular basis of MDS and progression of MDS to leukemia. penetrance and latency mutation number position form penetrance latency #5 D171N point mutation in RHD 69.6 % 4–13 months #27 S291fsX300 truncation type 100 % 4–9 months Figure Figure

Description: Chronic graft-versus-host disease (cGVHD) is a limiting factor in allogeneic hematopoietic stem cell transplantation (alloHSCT) for the treatment of leukemia and other malignancies. Relative to the process that initiates and promotes cGVHD, the regulation is poorly understood. In this study, we examined the role of naturally occurring regulatory dendritic cells (DCregs) in murine major histocompatibility complex (MHC)-compatible and multiple minor histocompatibility antigen (miHAg)–incompatible model of cGVHD in alloHSCT. DCregs generated from bone marrow in vitro (BM-DCregs) exclusively expressed CD200 receptor 3 (CD200R3), which exerted a suppressive function in the Ag-specific CD4+ T-cell response. CD49+CD200R3+ cells showed similarities in phenotype and function to BM-DCregs, which formally distinguishes them from other leukocytes, suggesting that they are the natural counterpart of BM-DCregs. Treatment of the recipient mice after alloHSCT with the recipient-type CD49+CD200R3+ cells as well as BM-DCregs protected against cGVHD, and the protection was associated with the generation of Ag-specific anergic CD4+ T cells as well as CD4+CD25+Foxp3+ regulatory T cells (Tregs) from donor-derived alloreactive CD4+CD25−Foxp3− T cells. In addition, the depletion of CD49+CD200R3+ cells before alloHSCT enhanced the progression of cGVHD. In conclusion, CD49+CD200R3+ cells act as naturally occurring DCregs to regulate the pathogenesis of cGVHD in alloHSCT mediated through the control of the transplanted alloreactive CD4+ T cells.

Description: Natural killer (NK) cells and NKT cells play critical early roles in host defense. Here we show that MIST, an adaptor protein belonging to the SLP-76 family, functions negatively in NK cells but positively in CD4+NKT cells. NK-cell receptor-mediated IFN-γ production was enhanced in NK cells, whereas TCR- or NK-cell receptor-mediated cytokine production was reduced in CD4+NKT cells from MIST-deficient mice. These opposite effects of MIST paralleled the exclusive expression of the Src family kinase, Fgr, in NK cells between the 2 cell populations. We further demonstrated that interaction of MIST with Fgr, mediated by the C-terminal proline-rich region of MIST and the SH3 domain of Fgr, was required for the suppression of NK-cell receptor-induced IFN-γ production. This functional interdependence of signaling molecules demonstrates a new mechanism by which adaptor proteins can act as molecular switches to control diverse responses in different cell populations.

Description: Myelodysplastic syndrome (MDS) is a hematopoietic stem-cell disorder characterized by trilineage dysplasia and susceptibility to acute myelogenous leukemia (AML). Analysis of molecular basis of MDS has been hampered by the heterogeneity of the disease. Recently, mutations of the transcription factor AML1/RUNX1 have been identified in 15% to 40% of MDS–refractory anemia with excess of blasts (RAEB) and MDS/AML. We performed mouse bone marrow transplantation (BMT) using bone marrow cells transduced with the AML1 mutants. Most mice developed MDS and MDS/AML-like symptoms within 4 to 13 months after BMT. Interestingly, among integration sites identified, Evi1 seemed to collaborate with an AML1 mutant harboring a point mutation in the Runt homology domain (D171N) to induce MDS/AML with an identical phenotype characterized by marked hepatosplenomegaly, myeloid dysplasia, leukocytosis, and biphenotypic surface markers. Collaboration between AML1-D171N and Evi1 was confirmed by a BMT model where coexpression of AML1-D171N and Evi1 induced acute leukemia of the same phenotype with much shorter latencies. On the other hand, a C-terminal truncated AML1 mutant (S291fsX300) induced pancytopenia with erythroid dysplasia in transplanted mice, followed by progression to MDS-RAEB or MDS/AML. Thus, we have developed a useful mouse model of MDS/AML that should help in the understanding of the molecular basis of MDS and the progression of MDS to overt leukemia.

Description: Pre–B-cell leukemia spontaneously develops in BLNK-deficient mice, and pre–B-cell acute lymphoblastic leukemia cells in children often lack BLNK protein expression, demonstrating that BLNK functions as a tumor suppressor. However, the mechanism by which BLNK suppresses pre–B-cell leukemia, as well as the identification of other genetic alterations that collaborate with BLNK deficiency to cause leukemogenesis, are still unknown. Here, we demonstrate that the JAK3/STAT5 signaling pathway is constitutively activated in pre-B leukemia cells derived from BLNK−/− mice, mostly due to autocrine production of IL-7. Inhibition of IL-7R signaling or JAK3/STAT5 activity resulted in the induction of p27kip1 expression and cell-cycle arrest, accompanied by apoptosis in the leukemia cells. Transgene-derived constitutively active STAT5 (STAT5b-CA) strongly synergized with the loss of BLNK to initiate leukemia in vivo. In the leukemia cells, exogenously expressed BLNK inhibited autocrine JAK3/STAT5 signaling, resulting in p27kip1 induction, cell-cycle arrest, and apoptosis. BLNK-inhibition of JAK3 was dependent on the binding of BLNK to JAK3. These data indicate that BLNK normally regulates IL-7–dependent proliferation and survival of pre–B cells through direct inhibition of JAK3. Thus, somatic loss of BLNK and concomitant mutations leading to constitutive activation of Jak/STAT5 pathway result in the generation of pre–B-cell leukemia.

Description: Human embryonic stem cells provide a unique tool to study early events occurring in the development of human embryonic hematopoiesis, and their totipotent capability indicates a potent clinical application based on the cellular therapy and the evaluation of drug effects on hematopoietic and blood cells. To achieve efficient production of hematopoietic cells from human embryonic stem cells, we attempted to reproduce the circumstance surrounding embryonic hematopoietic cells in vitro. Since fetal liver is the predominant source of hematopoietic and blood cells in mammalian embryogenesis, we established stromal cells from mouse fetal liver at days 14 to 15 of gestation. In the co-culture of human embryonic stem cells with the established stromal cells, a number of hematopoietic progenitors were generated at around day 14 of co-culture, and this hematopoietic activity was highly enriched in the cobble stone-like cells under the stromal layer. Most of the cobble stone-like cells collected expressed CD34 and contained a variety of hematopoietic colony-forming cells, especially multilineage colony-forming cells, at a high frequency. The multipotential hematopoietic progenitors in the cobble stone-like cells produced all types of mature blood cells, including adult type hemoglobin-synthesizing erythrocytes and tryptase and chymase-bouble positive mast cells in the suspension cultiue with a cytokine cocktail. The developed co-culture system of human embryonic stem cells should offer a novel source for hematopoietic and blood cells applicable to cellular therapies and drug screening.