ALBERT

Description: Abstract 4231 Introduction: Fifty percent of Diamond–Blackfan anemia (DBA) patients possess mutations in ribosomal protein genes. Although several ribosomal protein genes, RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26, have been reported to be mutated in some DBA patients, including point mutations, nonsense mutations, deletions, splice site mutations, and translocations, other DBA patients appear to have intact ribosomal protein genes. To identify new mutations in ribosomal protein genes from a different aspect, we focused on extensive deletions in these genes, such as mutations involving loss of a whole allele. In this study, we applied quantitative genomic PCR, and successfully developed a convenient method for detecting extensive deletions designated the “DBA gene copy number assay”. Methods: DBA patients should have an intact allele and a mutated allele for the responsible ribosomal protein gene, meaning that they will have an abnormal karyotype (gene copy number of N) if they have an extensive deletion. We attempted to clarify the copy numbers of ribosomal protein genes by the difference in a 1-cycle delay of threshold in a quantitative PCR (q-PCR) assay. To detect extensive deletions, at least 2 sets of gene-specific primers for each DBA responsible gene (RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26) were prepared. Appropriate primers to fit the setting that the threshold cycle (Ct) of the q-PCR should occur within 1 cycle of the Ct scores of other primer sets were selected. After validation, we identified 6, 3, 4, 3, 3, 6, 9, 3, and 2 specific primer sets for RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26, respectively. By simply looking at the q-PCR amplification curves by eye, we were easily able to judge the copy numbers of 2N (normal) or N (abnormal) for the ribosomal protein genes. Results: We performed the DBA gene copy number assay for 14 randomly selected undiagnosed patients from the Japanese DBA genomic resource at the University of Hirosaki, who had no mutations by genomic sequencing analyses. For each case, all the DBA responsible genes were confirmed using the diagnostic primers. The results of the DBA gene copy number assays revealed that 5 of the 14 probands (36%) had an extensive deletion in one of the DBA responsible genes. As an interesting case among the 5 positive cases, we confirmed an extensive deletion in the RPS19 gene. The Ct scores for 4 of the 9 primer sets for RPS19 demonstrated a 1-cycle delay, while the scores for the other 5 primer sets were normal. By genomic PCR amplification analyses, we identified a deletion from nt. -1400 to +5757 (7157 nucleotides) in the RPS19 gene. The deleted region included the promoter region, and exons 1, 2, and 3 of the RPS19 gene. The remaining 4 cases were 1 proband with an RPL5 deletion, 1 with an RPL35A deletion and 2 with RPS17 deletions. In particular, the extensive deletions in the RPL5 and RPS17 alleles are the first such cases reported. Discussion: Since it has been difficult to address the loss of a whole allele in DBA, such mutations have not been precisely examined within the DBA responsible genes. Our data suggest that extensive deletions in ribosomal protein genes comprise a significant proportion of DBA cases in Japan. Our novel method could become a useful tool for screening the gene copy numbers of ribosomal protein genes, and for identifying new pathological mutations. Disclosures: No relevant conflicts of interest to declare.

Description: Adult T-cell leukemia/lymphoma (ATL) is a malignant lymphoproliferative disorder caused by HTLV-I infection. In ATL, chemotherapeutic responses are generally poor, which has suggested the existence of chemotherapy-resistant cancer stem cells (CSCs). To identify CSC candidates in ATL, we have focused on a Tax transgenic mouse (Tax-Tg) model, which reproduces ATL-like disease both in Tax-Tg animals and also after transfer of Tax-Tg splenic lymphomatous cells (SLCs) to nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Using a limiting dilution transplantation, it was estimated that one CSC existed per 104 SLCs (0.01%). In agreement with this, we have successfully identified candidate CSCs in a side population (0.06%), which overlapped with a minor population of CD38−/CD71−/CD117+ cells (0.03%). Whereas lymphoma did not develop after transplantation of 102 SLCs, 102 CSCs could consistently regenerate the original lymphoma. In addition, lymphoma and CSCs could also be demonstrated in the bone marrow and CD117+ CSCs were observed in both osteoblastic and vascular niches. In the CSCs, Tax, Notch1, and Bmi1 expression was down-regulated, suggesting that the CSCs were derived from Pro-T cells or early hematopoietic progenitor cells. Taken together, our data demonstrate that CSCs certainly exist and have the potential to regenerate lymphoma in our mouse model.

Description: In normal hematopoiesis, hematopoietic stem cells (HSCs) in bone marrow mainly produce various types of blood cells. HSCs have a function of self-renewal and are located in the osteoblastic and vascular niche, which regulates the stemness function as a stem cell microenvironment. Recent studies clearly show that several factors (N-Cadherin, b-Catenin, SDF-1a and Spp1) are involved in the maintenance of the stemness function in HSCs. However, it has also been shown that embryonic HSCs can proliferate and differentiate in extrameduller tissues like liver, spleen and placenta. In adults, extrameduller hematopoiesis (EMH) can be induced by a hematological malignancy and some infectious diseases. However, the mechanism of HSCs regulation and niche cells at the EMH has not been well defined. To reveal the mechanism of HSCs in the EMH, we utilized the c-fos knockout mouse (c-fos −/−) as an EMH model mouse. In c-fos −/−, hematopoiesis in bone marrow was absent as a result of marrow spaces being occupied by increasing the number of bone forming osteoblasts; EMH then began in the spleen. First, to identify the main site of the HSC-niche in the EMH spleen, we performed HSC localization analysis using in situ hybridization of various HSC markers. Surprisingly, some CD34, Sca-1, c-kit, SCL/tal-1, and Tie-2 expressing cells were located near megakaryocyte like cells (MLCs). These MLCs were mainly located in the red pulp region in the spleen, and 3–5 cells form a syncytium. Interestingly, these MLCs express various types of osteoblastic niche related molecules, (N-Cadherin, b-Catenin, Spp1 and SDF-1a) in addition to megakaryocytic markers (CD41, CD61, and b3-integrin), suggesting that MLC is a HSC niche candidate in the EMH. To confirm our hypothesis, we next performed CFU-S (colony forming unit-spleen) assay as a naturally induced EMH model. Total 1×105 bone marrow mononuclear cells isolated from Ly5.2 or GFP-transgenic mouse were transplanted into lethally irradiated Ly5.2 mouse. In this system, MLCs were first seen in the spleen at day 1. At day 8, the number of MLCs had increased and units of 10–15 MLCs aggregated and formed syncytium. About 80% of MLCs were derived from donor (Ly5.2) cells. More interestingly, and contrary to our expectations, these aggregated MLCs with Sca-1+ or c-kit+ hematopoietic progenitor cells (HPCs) were mainly located, not inside the colony, but in the interstitial region between the developing colonies. They also expressed osteoblastic niche molecules. It has been suggested that each colonies in spleen are derived from HPCs and that HPCs exist inside the colony. Our data indicate the possibility that HPCs were transiently- located outside the colony. To undertake a detailed characterization of MLCs, we sorted donor derived MLCs as a Lin-/CD41+ cells and performed Q-PCR analysis. In agreement with our histological analysis, the sorted MLCs expressed various types of niche molecules and cytokines, compared to megakaryocytes. As well, when we co-cultured HSCs with isolated MLCs, the numbers of HSC were significantly increased compare to with a liquid culture system. Taking these data together, we suggested that MLCs have the potential to support HSC proliferation. In this study, we first identified a candidate for EMH-niche cells and postulated a developmental mechanism in the spleen. Our findings provide a new functional insight into HSCs outside the bone marrow, and extend a new tool that supports ex vivo expansion of HSCs.

Description: Adult T cell leukemia (ATL) is a lymphoproliferative disorder caused by infection with HTLV-I. Although various chemotherapies have shown significant complete remission rates, most of the treated patients relapse. These data indicate the existence of leukemic stem cells (LSCs) and a specific niche that regulates stemness and protects LSCs from various chemotherapies. We have reported in previous studies that the ATL-LSCs isolated from a Tax-transgenic (Tax-Tg) mouse are enriched in the CD117+/CD38–/CD71– fraction of the lymphoma, and LSCs have the potential to reproduce the original tumor when transplanted into a NOD/SCID mouse (Yamazaki et al., Blood, 2009). However, the niche of ATL-LSCs is still unclear. To identify the ATL-LSC niche in vivo, we performed a homing assay. Splenic lymphoma cells isolated from a Tax-Tg mouse were GFP transduced by a lentivirus, and then sorted GFP+ cells were transplanted intra-peritoneally into a non-irradiated NOD/SCID mouse. The homing of GFP+ cells to tissues was assessed by flow cytometry (FCM) at 16 hours and 3, 7, 14 and 21 days after transplantation. As a result, GFP+ lymphoma cells were first detected in the spleen and BM at 16 hours after transplantation. No GFP+ lymphoma cells were detected in the thymus and LN. Interestingly, more than 60% of first colonized cells in the spleen and BM at 16 hours after transplantation were AT-LSCs (GFP+/CD117+ cells). From day 3 to 7, more than 40% of colonizing cells in the BM and spleen were ATL-LSCs. To identify the specific niche of ATL-LSCs in the BM, we performed imaging analysis of ATL-LSCs. ATL-LSCs (GFP+/CD117+ and CD38–/CD71–/CD117+ cells) were mainly localized near the endosteal region of trabecular bone in the BM. We found that ATL-LSCs were also attached to the reticular cells in the trabecular bone. In addition, we found the number of osteoclast was significantly increased at the trabecular region. Increasing number of osteoclasts correlates the increased the serum calcium concentration and decreased the mass of trabecular bone. FCM analysis and in vitro differentiation assay confirmed that the number of osteoclast precursors was increased in the ATL BM. To clarify the role of osteoclast in the ATL BM, we treated osteoclast inhibitor Zoledronic acid (ZOL) to the ATL mouse model. As a result, ZOL itself significantly reduced the number of GFP+ ATL cells in the BM. When we treated ZOL with anti cancer drug, GFP+ ATL cells were dramatically reduced in the BM and extend the mouse survival rate significantly despite anti cancer drug does not reduced the number of ATL cells itself. In addition, abnormal trabecular bone morphology was completely recovered in the treated mouse. These data suggest that osteoclast may have a function to support leukemic stem cell niche. To clarify the key signals to induce osteoclast in ATL BM, we checked the expression of RANKL and PTHrP. We found that RANKL was up-regulated both in the lymphoma cell and stromal cells in the bone marrow. 　　In this study, we found that ATL-LSC niche is located at the trabecular bone region in the BM and osteoclasts have a role to support ATL cell and develop LSCs niche in a mouse model of ATL. We conclude that osteoclast have a potential therapeutic target in the mouse model of ATL. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 984 Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome, characterized by red blood cell aplasia, macrocytic anemia, and increased risk of malignancy. Approximately 90% of patients present during the first year of life or in early childhood. About 40–50% of DBA cases are familial with autosomal dominant, while the remainder is sporadic cases whose mode of inheritance is largely unknown. Although anemia is the most prominent feature of DBA, up to 40% of patients also accompany other symptoms including growth retardation and/or a variety of congenital malformations. Recent studies have shown that the disease could be associated with heterozygous mutations in ribosomal protein (RP) genes, including six small subunit RP genes RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26 as well as four large subunit RP genes RPL5, RPL11, RPL26, and RPL35A, which collectively account for about 50% of patients with DBA. In addition, germline mutations in the GATA1 gene encoding a hematopoietic transcription factor, have been also reported in two DBA families. However, it is clear that the molecular etiology of many DBA cases remains to be covered. To identify new mutations that are responsible for DBA, we performed whole-exome sequencing on 40 DBA patients with no documented mutations/deletions involving known DBA genes. After excluding all variants registered in the 1000 Genomes Project, or dbSNP131, or found in our inhouse SNP database, we searched for non-synonymous mutations involving RP genes as possible candidate for novel DBA genes. In this study, we identified probable pathogenic mutations in two novel RP genes, RPS27 and RPL27 in two patients. The first case was a 1-year-old girl who harbored a single nucleotide substitution at the splice acceptor site in intron 1 of RPL27 (c.-2–1G〉A), which results in splicing error. She had atrial septal defect and pulmonary stenosis, and responded to steroid treatment. The second case was a 2-year-old girl carrying a frameshift deletion of RPS27 (c.90delC, p.Tyr31ThrfsX5), leading to a premature truncation. This patient had no abnormalities and responded to steroid treatment. An additional five missense SNVs affecting single cases was identified in five genes, including RPL3L, RPL8, RPL13, RPL18A, and RPL31, together with two in-frame deletions of RPL6 and RPL14 in two patients, which cause deletion of a single amino-acid. However, the pathological significance in these 7 cases is uncertain. In the remaining 31 patients, no mutations were detected in RP genes. In conclusion, we identified novel germline mutations of RP genes that could be responsible for DBA, further confirming the concept that the RP genes are common targets of germline mutations in DBA patients and also suggested the presence of non-RP gene targets for DNA. Disclosures: No relevant conflicts of interest to declare.

Description: Fifty percent of Diamond-Blackfan anemia (DBA) patients possess mutations in genes coding for ribosomal proteins (RPs). To identify new mutations, we investigated large deletions in the RP genes RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26. We developed an easy method based on quantitative-PCR in which the threshold cycle correlates to gene copy number. Using this approach, we were able to diagnose 7 of 27 Japanese patients (25.9%) possessing mutations that were not detected by sequencing. Among these large deletions, similar results were obtained with 6 of 7 patients screened with a single nucleotide polymorphism array. We found an extensive intragenic deletion in RPS19, including exons 1-3. We also found 1 proband with an RPL5 deletion, 1 patient with an RPL35A deletion, 3 with RPS17 deletions, and 1 with an RPS19 deletion. In particular, the large deletions in the RPL5 and RPS17 alleles are novel. All patients with a large deletion had a growth retardation phenotype. Our data suggest that large deletions in RP genes comprise a sizable fraction of DBA patients in Japan. In addition, our novel approach may become a useful tool for screening gene copy numbers of known DBA genes.

Description: Abstract 1877 Adult T cell leukemia (ATL) is a lymphoproliferative disorder caused by infection with HTLV-I. Although various chemotherapies have shown significant complete remission rates, most of the treated patients relapse. These data indicate the existence of leukemic stem cells (LSCs) and a specific niche that regulates stemness and protects these cells from chemotherapy. We have reported in previous studies that the ATL-LSCs isolated from a Tax-transgenic (Tax-Tg) mouse are enriched in the CD117+/CD38–/CD71– fraction of the lymphoma, and LSCs have the potential to reproduce the original tumor when transplanted into a NOD/SCID mouse (Yamazaki et al., Blood, 2009). However, the niche of ATL-LSCs in the spleen, bone marrow (BM), thymus and lymph node (LN) is still unclear. To identify the ATL-LSC niche in vivo, we performed a homing assay. Lymphoma cells isolated from a Tax-Tg mouse were GFP transduced by a lentivirus, and then sorted GFP+ cells (2×106) were transplanted intraperitoneally into a non-irradiated NOD/SCID mouse. The homing of GFP+ cells to tissues was traced by flow cytometry (FCM) at 16 hours and 3, 7, 14 and 21 days after transplantation. At 16 hours after transplantation, GFP+ lymphoma cells were detected in the spleen and BM. No GFP+ lymphoma cells were detected in the thymus and LN. Interestingly, more than 60% of first colonized cells in the spleen and BM at 16 hours were AT-LSCs (GFP+/CD117+ cells). From day 3 to 7, more than 40% of proliferating cells in the BM and spleen were ATL-LSCs. At day 3, only a few non-ATL-LSCs (GFP+/CD117–cells) were detected in the thymus, LN and peripheral blood. The number of GFP+ cells was drastically increased at day 14 in the spleen. These data indicate that ATL-LSCs prefer to colonize and proliferate in the spleen and BM. To identify the specific niche of ATL-LSCs in the spleen and BM, we performed imaging analysis of ATL-LSCs. ATL-LSCs (GFP+/CD117+ and CD38–/CD71–/CD117+cells) were mainly localized near the vascular region in the spleen and endosteal region of trabecular bone in the BM. We found that some ATL-LSCs were attached to reticular cells (RC) in the spleen. In the BM, ATL-LSCs cells were localized at the endosteal region of the trabecular bone. Interestingly, similar to the spleen, RCs were observed at the endosteal region and contacted ATL-LSCs in the BM. FCM analysis confirmed that the number of reticular cells and mesenchymal stem cells (MSCs), were increased in the ATL BM and spleen. These data suggest that RCs are a possible candidate for the ATL-LSC niche and may be a new target of therapy. Finally, to characterize the ATL-LSC niche, we isolated osteoblastic cells, blood endothelial cells, lymphatic endothelial cells and reticular cells from normal and ATL BM to compare the gene expression profiles of each niche cell type. Here, together with DNA microarray analysis of ATL-LSCs both in the BM and spleen, we have characterized ATL-LSC niche cells both in the spleen and BM. Disclosures: No relevant conflicts of interest to declare.

Description: Tie2 is a receptor-type tyrosine kinase expressed on hematopoietic stem cells and endothelial cells. We used cultured embryonic stem (ES) cells to determine the function of Tie2 during early vascular development and hematopoiesis. Upon differentiation, the ES cell–derived Tie2+Flk1+ fraction was enriched for hematopoietic and endothelial progenitor cells. To investigate lymphatic differentiation, we used a monoclonal antibody against LYVE-1 and found that LYVE-1+ cells derived from Tie2+Flk1+ cells possessed various characteristics of lymphatic endothelial cells. To determine whether Tie2 played a role in this process, we analyzed differentiation of Tie2-/- ES cells. Although the initial numbers of LYVE-1+ and PECAM-1+ cells derived from Tie2-/- cells did not vary significantly, the number of both decreased dramatically upon extended culturing. Such decreases were rescued by treatment with a caspase inhibitor, suggesting that reductions were due to apoptosis as a consequence of a lack of Tie2 signaling. Interestingly, Tie2-/- ES cells did not show measurable defects in development of the hematopoietic system, suggesting that Tie2 is not essential for hematopoietic cell development.

Description: Mutations in ribosomal protein S19 (RPS19) gene is closely related to Diamond-Blackfan anemia (DBA). We have found point mutation, deletion and insertion in RPS19 gene in DBA patients (Blood, 100: 2724–31, 2002). In our analysis of DBA patients, gene expression of RPS19 from these mutated genomes were down regulated. Retroviral transduction of RPS19 gene to patient bone marrow cells restored the ability to differentiate into erythroid in vitro (Mol Ther., 7: 613–22, 2003). However, the mechanisms of how these mutations in RPS19 associate with anemia remains unknown. To analyze the mutated genome, we have made constructs of mutated RPS19 gene-expression vectors. From reported 56 different RPS19 mutations, we focused on the missense mutants and tried to characterize them. Flag-tagged twelve missense mutants (V15F, L18R, P47L, W52R, R56Q, S59F, A61Q, R62W, R101H, G120R, G127Q, and G131R) were exogenously expressed in several cell lines by retroviral vectors and were analysed by western blotting or FACS. When these 12 mutants were expressed in erythro-leukemic cell lines K562 and HEL, almost all the mutant proteins (except for G120R) were expressed at significantly low level compared to that of wild type. However, some of these mutants were expressed in human kidney epithelial cell line, 293T cells. These findings suggest that the expression level of RPS19 protein derived from mutated RPS19 genome is dependent on cell types, whether erythroid-lineage cells or others. In order to analyze the function of RPS19, doxycycline (Dox)-inducible expression of siRNA against RPS19 was induced in K562 cells and observed growth arrest at day 4 after induction of siRNA by Dox. Expression level of RPS19 decreased to one tenth of the normal level. Cell cycle analysis and quantitative-PCR analysis revealed that the growth arrest of K562 cells expressing siRNA against RPS19 was due to the induction of cell cycle arrest at G1 phase (G0/G1 54 % vs 36% ) coincide with the up-regulation of p21 and p57 mRNA level. From these results, we could possibly suggested that, in DBA patients, RPS19 protein expression is not stable and the decreased level of RPS19 protein prolongs G1 phase of cell cycle especially in the course of differentiation on erythroid cells, which may contribute to the retarded production of red blood cells. Our findings allow to understand the entire feature of DBA-associated RPS19 mutations especially on the erythorid differentiation and may help defining the therapeutic target to improve DBA.

Description: Hematopoietic stem cells (HSCs) have a potential to differentiate into variety types of blood cells, and have an ability of self-renewing in the bone marrow (BM). All these fates are regulated by intrinsic genetic pathways in the micro-enviroment (niche) where HSCs are located. Osteoblasts are thought to be the hematopoietic niche which regulates HSC function in the BM. To clarify the molecular basis of hematopoietic niche system, we compared the two somatic stem cells (Germ line stem cell (GS) and HSC) using cDNA suppression subtractive hybridization, and identified 12 stem cell-specific genes which are expressed both in the HSCs and GS. Among these genes, we analyzed Spp1, since it is a major glycoprotein produced by osteoblast and it has been reported to be a negative regulator of hematopoietic niche size in the BM. In situ Hybridization (ISH) with Spp1-specific probes demonstrated that Spp1-expressing osteobalsts were located at the endosteal region of trabecular bone (TB). In the TB of adult mouce, Spp1-expressing hematopoietic cells attached with Spp1-expressing osteobalsts. Double ISH analysis revealed that Spp1 and Tie2 were partially co-expressed in the TB. These data suggests that Spp1 is an important molecule of hematopoietic niche system which were produced and interacted both HSCs and niche cell. When we analyzed spp1 knock out mouse (Spp1−/−), adult Spp1−/− mice (8 weeks) appeared to be normal and fertile. However, the abnormal TB formation was observed in the younger Spp1−/− mice (2 weeks). A number of osteobalsts were increased and formed a multi-layer of osteoblasts at the endosteal region of TB. Along with the morphological change of the TB, distribution pattern of CD34, Tie2 and Bmi-1 expressing HSC was unusual. Tie2 expressing cells of Spp1−/− mouse were mainly located at vascular-enriched zone in the bone marrow, but few Tie2 expressing cells at osteoblastic zone. These data suggest that HSC and osteobalst do not form the correct niche in the Spp1−/− TB, and Spp1 is thought to be essential for the initial niche formation. To confirm the function of Spp1 at the initiation phase of niche formation, we performed the bone marrow transplantation. Mononuclear cells (1×105 cells) from Ly5.1 mice were transplanted into lethally irradiated Ly5.2 mice. At 12 days after transplantation, donor hematopoietic cells were observed in the recipient TB. At this day 12, the expression of Spp1 was up-regulated in the endosteal region of TB, however, Spp1 expression was decreased in the course of reconstitution. Thus, the findings regarding the hematopoietic niche formation in Spp1−/− mice and in the irradiated mice allow to understand the Spp1 function in the initial phase of niche formation in the bone marrow.