ALBERT

Description: Abstract 1274 Poster Board I-296 Introduction RCAN1 (also called DSCR1, calcipressin 1, MCIP1, Adapt78) is known to be a regulator of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, and has also been shown to regulate the activity of certain transcription factors, including NF-AT and NF-kB. RCAN1 expression is widely found in various tissues, including the brain, kidney, liver and heart, but the expression level of RCAN1 is extremely low in normal hematopoiesis-related organs (bone marrow and spleen). Although RCAN1 has been shown to act as a negative regulator of FceRI-induced mast cell activation and NF-AT signaling in megakaryocytes, the cellular function of RCAN1 in myeloid cells remains unknown. Recently, we found that RCAN1 was highly expressed in acute phase of chronic myeloid leukemia (CML) by DNA microarray analysis. In this study, to explore the role of RCAN1 in leukemogenesis, we compared RCAN1 expression in leukemia cells and that in normal hematopoietic cells. We also analyzed regulatory mechanisms of RCAN1 gene promoter function and searched for novel RCAN1-interacting molecules in myeloid leukemia cells. Methods Expression of RCAN1 was examined by quantitative PCR using normal human bone marrow cells, bone marrow cells from patients with acute myeloid leukemia (AML) and patients with CML, and various human hematological tumor cell lines. Promoter activity of the human RCAN1 gene was evaluated by a luciferase assay using various deletion mutants in KCL22 cells. To identify novel RCAN1-interacting proteins, a yeast two-hybrid assay was performed using a cDNA library from K562 leukemia cells. Results Fractions of CD34+CD38-, CD34+CD38+, CD33+CD14-CD16-, CD33+CD14+ and CD33+CD16+ cells from normal human bone marrow showed extremely low levels of RCAN1, indicating that RCAN1 is scarcely expressed in normal hematopoietic cells, including stem cells and progenitor cells. In contrast, bone marrow mononuclear cells (BMMC) from 15 of 16 patients with AML and all four patients with CML in acute phase showed substantial levels of RCAN1. Furthermore, RCAN1 was expressed in all human cell lines examined in this study that were derived from AML (THP-1, U937, HL60, KY821), CML in acute phase (KCL22,K562, KU812), acute lymphoblastic leukemia (Jarkat, Raji) and multiple myeloma (RPMI8226, U266). Interestingly, expression levels of RCAN1 were significantly decreased in BMMC from three of four AML patients when the patients were in complete remission. These results suggest that RCAN1 is extraordinarily expressed in a wide range of hematological tumor cells. To reveal the underlying mechanisms of ectopic expression of RCAN1, we next examined the promoter function of RCAN1 gene, which consists of seven exons. Although RCAN1 expresses several alternative isoforms that selectively include each of exons 1-4 and use distinct promoters, we found that only exon 1 isoform was expressed in these leukemia cells. Analysis of the function of human RCAN1 gene promoter upstream of exon 1 demonstrated that NKX2-5 binding site is important for basal activity of RCAN1 gene promoter in KCL22 cells. NKX2-5 is a homeobox-containing transcription factor and is involved in leukemogenesis of T-cell acute lymphoblastic leukemia. Our results raised the possibility that NKX2-5 plays a role in malignant transformation partly through expression of RCAN1 in myeloid leukemia cells. Finally, we searched for RCAN1-interacting proteins in leukemia cells. By screening a cDNA library from K562 cells using yeast two-hybrid assay, we found that RCAN1 interacts with HINT1, HINT2 and RINT1. HINT1 has been reported to be a tumor suppressor against hepatocellular carcinoma, and HINT2 and RINT1 have also been found to function as tumor suppressors. It would be interesting to clarify whether RCAN1 affects the activity of these molecules, and it is important to reveal the involvement of these molecules in leukemogenesis. Conclusion RCAN1 is extraordinarily expressed in a wide range of hematological malignant cells, and NKX2-5 might be important for its ectopic expression. It is possible that RCAN1 is biologically significant in leukemogenesis through affecting certain tumor suppressors. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1150 Poster Board I-172 Introduction ABO-incompatibility is not generally considered to be a risk factor of hematopoietic stem cell transplantation (HSCT). However, in our single-institute study of 568 patients, the incidence of acute GVHD was higher in minor-ABO incompatible HSCT than others (45.3% vs 35.2%, p=0.019). We made a hypothesis that there is a potential association between donor-derived isohemagglutinin (IH) and the outcomes of minor-ABO incompatible HSCT. Patients and method There were 1052 patients undergone HSCT between 1988 and 2009 at Komagome hospital. We analyzed 130 of 1052 patients (12.4%), who had received minor- ABO incompatible HSCT. Blood type and anti-A/B antibody titers were evaluated at least weekly in first one month after HSCT, and were repeated monthly until blood type had completely changed to the donor type. Our analysis was especially focused on the impact of IH on the outcome of minor-ABO incompatible HSCT. Result Minor ABO-incompatible HSCT were 130 cases that included AML (n=40), ALL (n=28), CML (n=27), MDS (n=15), SAA (n=9), NHL (n=7), MM (n=2) and ATL (n=2). Median age was 37 (range: 16-67) years old. Seventy-seven patients were male, 53 were female. Stem cell sources were bone marrow (n=96), peripheral blood (n=16) and umbilical cord blood (n=18). Eleven cases were HLA-matched and 9 were HLA-mismatched transplants. Anti-host IgG and IgM IH had been detected in 20 of 130 transplants undergoing minor ABO-incompatible HSCT (15.4%). Median time to IH detection was 13 days after HSCT (12-39days). There was a higher incidence of IH production in the HLA-mismatched group than in the HLA-matched group (p=0.007).There was also a higher incidence of IH production in the unrelated transplants group than in the related transplants group. (p=0.021). None of 18 patients receiving umbilical cord blood transplantation showed evidence of IH production. The incidence of grade II-IV acute GVHD was significantly higher (90% vs 60%, p

Description: Abstract 3648 Poster Board III-584 GATA1 is a transcription factor essential for the differentiation of erythroid cells and megakaryocytes. Since GATA1 regulates genes related to the survival, proliferation and differentiation of hematopoietic cells, regulation of the Gata1 gene expression is critically important for the understanding of hematopoiesis. The Gata1 locus contains multiple untranslated first exons plus five common coding exons. Of these first exons, erythroid first exon (IE exon) is important for the Gata1 gene expression in the hematopoietic lineages. However, due to the embryonic lethality of this IE exon knockdown mice, less is understood about the contribution of the IE exon to adult hematopoiesis. Here, we achieved specific deletion of the IE exon in adulthood by crossing the IE-floxed mice with the interferon-inducible Mx1-Cre transgenic mice. This conditional IE-deletion mouse (ΔIE mouse) showed severe thrombocytopenia with increased premature megakaryocytes similarly to the phenotypes reported in the conditional Gata1 knockout mice in which the entire Gata1 gene was deleted in adulthood. In addition, the ΔIE mice showed severe anemia with skewed erythroid maturation, and importantly this erythroid phenotypes substantially differed from those observed in the conditional Gata1 knockout mice. Further analyses revealed that the Gata1 mRNA level in the megakaryocytic lineage was significantly downregulated. By contrast, in the erythroid lineage, Gata1 mRNA was retained at a comparable level to that in control mice utilizing two alternative first exons; one was the IEb/c, which was previously reported as a first exon rarely used in hematopoietic cells, and the other was newly identified IEd exon located within the second intron. Surprisingly, in the ΔIE mice these transcripts failed to produce full-length GATA1 protein, but instead inefficiently yielded GATA1 lacking the N-terminal 83 amino acids. This form of GATA1 is often observed in Down syndrome-associated transient myeloproliferative disorder and acute megakaryoblastic leukemia. Of note, the transcript derived from exon IEb/c preserved the first translation initiation codon in exon 2 but lost the potential to select the first translation initiation codon or failed to produce full-length GATA1. The present study demonstrates that the IE exon is instrumental to adult erythropoiesis by regulating the proper level of transcription and by selecting the correct translation start site for production of adequate full-length GATA1 protein. Disclosures: No relevant conflicts of interest to declare.

Description: Clinical trials with antiangiogenic agents have not been able to validate plasma or serum levels of angiogenesis regulators as reliable markers of cancer presence or therapeutic response. We recently reported that platelets contain numerous proteins that regulate angiogenesis. We now show that accumulation of angiogenesis regulators in platelets of animals bearing malignant tumors exceeds significantly their concentration in plasma or serum, as well as their levels in platelets from non–tumor-bearing animals. This process is selective, as platelets do not take up a proportional amount of other plasma proteins (eg, albumin), even though these may be present at higher concentrations. We also find that VEGF-enriched Matrigel pellets implanted subcutaneously into mice or the minute quantities of VEGF secreted by microscopic subcutaneous tumors (0.5-1 mm3) result in an elevation of VEGF levels in platelets, without any changes in its plasma levels. The profile of other angiogenesis regulatory proteins (eg, platelet-derived growth factor, basic fibroblast growth factor) sequestered by platelets also reflects the presence of tumors in vivo before they can be macroscopically evident. The ability of platelets to selectively take up angiogenesis regulators in cancer-bearing hosts may have implications for the diagnosis and management of many angiogenesis-related diseases and provide a guide for antiangiogenic therapies.

Description: Abstract 3768 Poster Board III-704 Aberrant reactivation of Hedgehog (Hh) signaling has been described in a wide variety of human cancers and its role in maintenance of self-renewal of cancer stem cells. However, it is unclear whether Hh signaling contributes to the growth, survival and drug resistance of acute myeloid leukemic (AML) cells. Regarding normal CD34+hematopoietic cells, we previously showed that Indian Hh (Ihh) and its receptor molecules, Patched and Smoothened are expressed in cord blood (CB) CD34+ cells and Ihh regulate proliferation of short-term hematopoietic repopulating cells in vivo. In the present study, we assessed the possibility that Hh pathway activation contributes to survival and drug resistance of acute myeloid leukemic (AML) cells. Hh signaling in bone marrow (BM) CD34+ cells, leukemic cell lines and primary CD34+ leukemic cells were screened by RT-PCR and a Hh signaling reporter assay. We have found that Ihh were expressed in normal BM CD34+ cells, primary CD34+leukemic cells and most of human AML cell lines, HL60, U937, KG1, Kasumi-1, Kasumi-3 and TF-1. In contrast, Hh receptor components, Patched and Smoothened, were detected in BM CD34+ cells, primary CD34+leukemic cells (n=3) and cytokine responsible CD34+ AML cell lines such as Kasumi-1, Kasumi-3 and TF-1. Moreover, the downstream transcription factor GLI1 or GLI2 were expressed in BM CD34+ cells, primary CD34+leukemic cells and these CD34+ cell lines, indicating that Hh signaling was active in these cells. We further assessed whether Hh signaling transmit to GLI1 using GLI1-responsive luciferase assay. GLI-responsive reporter plasmid (TK-6GBS-Luc) was transduced into these cells in the presence or absence of anti-Hh neutralizing antibody 5E1. TK-6GBS-Luc-transduced Kasumi-1, Kasumi-3 and TF-1 cells showed high luciferase activity. Furthermore, the luciferase activity of these cells was significantly reduced in the presence of 10 μg/ml antibody 5E1. These results clearly indicated that Hh signaling could transmit into these CD34+ leukemic cell lines in autocrine manner. We next examined the effect of Hh inhibitors on these CD34+ leukemic cells. Inhibition of Hh signaling with the naturally derived chemical Smoothened antagonist Cyclopamine, endogenous Hh inhibitor Hedgehog-interacting protein or anti-Hedgehog neutralizing antibody induces apoptosis in these cells after 48 hr exposure although these CD34+ cell lines exhibit resistance to cytarabine (Ara-C) exposure. In contrast, cyclopamine did not affect growth and survival of the U937 or HL-60 cell line that lacks expression of Hh receptor components. Furthermore, combination of 10 μM cyclopamine significantly reduced the drug resistance against Ara-C in CD34+ cell lines as well as primary CD34+AML cells (Figure). These results suggest that Hh pathway activation is a feature of CD34+ myeloid leukemic cells and inhibition of Hh signaling pathway in combination with Ara-C etc. may be a new therapeutic strategy to eradicate CD34+ AML stem cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1439 Poster Board I-462 We have previously shown that cobblestone area (CA) could be observed underneath human stromal cells 2 weeks after coculture in serum free condition. However, it was difficult to evaluate 5 week CA-forming cells (CAFC) which is a surrogate indicator of hematopoietic stem cells because human stromal cells could not survive more than 4 weeks in a serum free media, X-VIVO10. In the present study, we optimized serum free media, combination of cytokines and chemical agents such as stem cell factor (SCF), thrombopoietin (TPO), flt3 ligand (FL) with or without delta like protein 4 (DLL4) and GSK inhibitor. In addition, we added the serum derived from aplastic anemia and MDS patients in this coculture system and evaluated the effect of serum on in vitro hematopoiesis. We found that certain serum free media could maintain the layer of human stromal cells more than 8 weeks. By using this serum free media, bone marrow (BM) CD34+ cells could be cocultured for 8 weeks without disruption of the human stromal layer. Five week CAFC could be observed in all condition of cytokine combination with or without chemical inhibitor. However, because cytoplasmic appearance of cells in some CAs is quite irregular without DLL4 or GSK inhibitor, we conducted immunohistochemical staining by using FITC-labeled anti human CD34, CD11b or CD33 antibodies. Surprisingly, we found that CD34+CAFCs were exclusively observed in the presence of DLL4 or GSK inhibitor. Otherwise, CAs was composed of CD11b+/CD45+ hematopoietic cells. Moreover, NOD/SCID repopulating cells were detected in the coculture containing CD34+CAFCs, suggesting that CD34+CAFCs were a valuable indicator of hematopoietic stem cells (HSCs). Using CD34+CAFCs as a surrogate maker of HSCs, we assessed whether the serum derived from patients of aplastic anemia and MDS could inhibit formation of CD34+CAFC. Notably, some of patient's serum could reduce the number of CD34+CAFCs. These results indicated the possibility that CD34+CAFCs underneath human stromal layer could be useful for screening certain soluble factors to inhibit hematopoiesis in aplastic anemia and MDS. Disclosures No relevant conflicts of interest to declare.