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Background In patients with myelodysplastic syndrome (MDS), bone marrow cells have an increased predisposition to apoptosis, yet MDS cells outcompete normal bone marrow (BM)-- suggesting that factors regulating growth potential may be important in MDS. We previously identified v-Erb A related-2 (EAR-2, NR2F6) as a gene involved in control of growth ability. Methods Bone marrow obtained from C57BL/6 mice was transfected with a retrovirus containing EAR-2-IRES-GFP. Ex vivo transduced cells were flow sorted. In some experiments cells were cultured in vitro, in other experiments cells were injected into lethally irradiated recipients, along with non-transduced bone marrow cells. Short-hairpin RNA silencing EAR-2 was also introduced into bone marrow cells cultured ex vivo. Results Here, we show that EAR-2 inhibits maturation of normal BM in vitro and in vivo and that EAR-2 transplant chimeras demonstrate key features of MDS. Competitive repopulation of lethally irradiated murine hosts with EAR-2-transduced BM cells resulted in increased engraftment and increased colony formation in serial replating experiments. Recipients of EAR-2-transduced grafts had hypercellular BM, erythroid dysplasia, abnormal localization of immature precursors and increased blasts; secondary transplantation resulted in acute leukemia. Animals were cytopenic, having reduced numbers of erythrocytes, monocytes and granulocytes. Suspension culture confirmed that EAR-2 inhibits granulocytic and monocytic differentiation, while knockdown induced granulocytic differentiation. We observed a reduction in the number of BFU-E and CFU-GM colonies and the size of erythroid and myeloid colonies. Serial replating of transduced hematopoietic colonies revealed extended replating potential in EAR-2-overexpressing BM, while knockdown reduced re-plating ability. EAR-2 functions by recruitment of histone deacetylases, and inhibition of differentiation in 32D cells is dependent on the DNA binding domain. Conclusions This data suggest that NR2F6 inhibits maturation of normal BM in vitro and in vivo and that the NR2F6 transplant chimera system demonstrates key features of MDS, and could provide a mouse model for MDS. Electronic supplementary material The online version of this article (10.1186/s40364-018-0149-4) contains supplementary material, which is available to authorized users.

We have studied, as part of a group of international multicenter phase II clinical trials, the toxicity and effectiveness of CAMPATH1H administered intravenously three times a week in an outpatient setting to patients with recurrent or progressive low grade lymphoma. We report here on the toxicity and therapeutic results of the first seven patients treated before the study was closed prematurely because of unacceptable toxicity. Classical complete or partial responses of treatment were seen in three of seven patients. One complete response lasted 8.5 months and the other complete response is ongoing at 1 year. Responses occurred in nodal sites as well as in skin and peripheral blood. The first three or four antibody infusions in each patient was associated with grade 1 or 2 side-effects including rigor, fever, facial flushing, nausea, vomiting, hives, wheezes, hypotension, and/or diarrhea but these subsequently decreased or disappeared. The most significant toxicity was profound lymphopenia and associated infection, usually viral. Six of seven patients had culture or serologically documented infections and four patients had two or more such episodes. All infections responded to temporary discontinuation of antibody therapy and appropriate antiviral or antibiotic agents. We conclude that CAMPATH1H monoclonal antibody has therapeutic activity against low grade non-Hodgkin's lymphoma but that this activity is limited by marked lymphopenia and an unacceptably high frequency of serious infection at the dose and schedule used in this trial.

We report a patient in whom a cell line of 47,XY,+X,t(1;14)(q21;q32) constitution was found in a lymph node excised from the neck. Histologic examination and immunophenotyping both in situ and by flow cytometry failed to confirm a diagnosis of B-cell lymphoma, but Southern analysis indicated the presence of B-cell clonal expansion. These observations support the concept that primary chromosomal abnormalities occur in clonal expansions in the very earliest stages of tumorigenesis.

We report a case of acute monocytic leukemia (FAB-5a) with a very aggressive clinical course and multiple chromosomal abnormalities. There were several sublines, each with trisomy 8 and a translocation involving 3q13.3 as a common breakpoint region. This region is an uncommon site of chromosomal breakage in malignancies and has not hitherto been reported as a breakpoint site in "jumping" translocations.

Effective spleen-derived humoral responses are essential for protecting hosts from blood-borne pathogens. Increased susceptibility to infection is therefore a risk post-splenectomy but is also associated with smoking, chemotherapy and aging. In fact, decreased vaccine efficacy in the elderly may be due to splenic hypofunction. While investigating the role of sensory nerves in hematopoiesis, we discovered a lymphocytosis phenotype involving the splenic marginal zone (MZ). Dividing red and white pulp in the spleen, the MZ is a specialized micro-anatomic structure facilitating innate and adaptive crosstalk to incoming antigen. Sensory nerves were found to synapse at this site and in mice deficient in the major sensory neurotransmitter substance P (Tac-1-/-), MZs were expanded histologically and confirmed by flow cytometry as an increase involving the IgMhiIgDloCD21hiCD23lo MZ B cell population. Tac-1-/- mice also exhibited splenomegaly with overall Ki-67 proliferation indices of 40% vs. 5% in controls. In the peripheral blood, Tac-1-/- mice had persistent polyclonal pan-B cell lymphocytosis with an absolute increase of 200% relative to wildtype. Aberrant B cell homeostasis had functional consequences as demonstrated by significantly decreased total basal plasma IgG (P Disclosures No relevant conflicts of interest to declare.

The leukemia stem cell model suggests that elucidation of the genes that regulate growth ability within the leukemia cell hierarchy will have important clinical relevance. We showed that the expression of NR2F6 (EAR-2), is greater in clonogenic leukemia single cells than in leukemia cells that do not divide, and that this gene is over-expressed in patients with acute myeloid leukemia and myelodysplastic syndrome. In vivo, overexpression of EAR-2 using a retroviral vector in a chimeric mouse model leads to a condition that resembles myelodysplastic syndrome with hypercellular bone marrow, increased blasts, abnormal localization of immature progenitors, morphological dysplasia of the erythroid lineage and a competitive advantage over wild-type cells, that eventually leads to AML in a subset of the mice, or after secondary-transplantation. Interestingly, animals transplanted with bone marrow that over-expresses EAR-2 develop leukemia that is preceded by expansion of the stem cell compartment in the transplanted mice—suggesting that EAR-2 is an important regulator of hematopoietic stem cell differentiation. Here we report that over-expression of EAR-2 also has a profound effect on the differentiation of erythroid progenitor cells both in vitro and in vivo. Studies of the roles of EAR-2 in normal primary bone marrow cells in vitro showed that overexpression of EAR-2 profoundly impaired differentiation along the erythroid lineage. EAR-2 over-expressing bone marrow cells formed 40% fewer BFU-E colonies, but had greatly extended replating capacity in colony assays. While knockdown of EAR-2 increased the number of cells produced per BFU-E colony 300%. Normal mice transplanted with grafts of purified bone marrow cells that over-expressed EAR-2 developed a rapidly fatal leukemia characterized by pancytopenia, enlargement of the spleen, and infiltration of blasts into the spleen, liver and peripheral blood. Sick animals had profound reduction of peripheral blood cell counts, particularly anemia with a 55% reduction in hemoglobin levels. Anemia was evident even on gross inspection of the blood and the liver in EAR-2 overexpressing animals. Analysis of the leukemic cells revealed an erythroblastic morphology, with the immunophenotype lineageneg, CD71high, TER119med. Hence, we wondered weather EAR-2 caused leukemia by arresting erythroid progenitor cell differentiation. Examination of the bone marrow of pre-leukemic animals showed a four-fold increase in cells with a pro-erythroblastic immunophenotype (CD71highTER119med , region I), and a four-fold decrease in orthochromatophilic erythroblasts (CD71lowTER119high , region IV). We observed no change in the numbers of basophilic erythroblasts (CD71highTER119high , region II) or late basophilic and polychromatophilic erythroblasts (CD71medTER119high, region III). These data suggests that over-expression of EAR-2 blocks erythroid cell differentiation at the pro-erythroblastic stage. Since EAR-2 over-expressing recipients died within 4 week, we wanted to definitively test whether animals had compromised radioprotection. We showed that decreasing the size of the bone marrow graft, reduced survival of the EAR-2 over-expressing cohort by a week, but had no effect on control animals proving that EAR-2 over-expression has a profound effect on erythropoietic reconstitution in vivo. Mechanistically, we show that DNA binding is necessary for EAR-2 function, and that EAR-2 functions in an HDAC-dependent manner, regulating expression of several genes. Pre-leukemic pro-erythroblastic cells (CD71highTER119med) that over-expressed EAR-2 had lower expression of genes involved in erythroid differentiation such as GATA1, EBF1, inhibitor of NFKB (NFKBia), ETV6, CEBP/a, LMO2, and Nfe2, and increased expression of GATA2, GLI1, ID1 and PU.1 than GFP control pro-erythroblasts. These data establish that EAR-2 is a novel oncogene whose cellular function is to regulate terminal differentiation of erythroid cells at the proerythroblastic (CD71highTER119med) stage by deregulating gene expression necessary for erythroid differentiation. Disclosures Ichim: Entest BioMedical: Employment, Equity Ownership, Patents & Royalties, Research Funding. Koos:Entest BioMedical: Employment, Equity Ownership, Patents & Royalties, Research Funding.

A permanent cell line, HSC-M1, was established from a child with advanced CD30 (Ki-1)+ anaplastic large-cell lymphoma (ALCL). Clinical features included irritability, fever, weight loss, tender lymphadenopathy, pneumonitis, neutrophilia, and bone marrow erythrophagocytosis. While HSC-M1 cells exhibited an immunophenotype characteristic of ALCL of T-cell lineage, the cell line also demonstrated features of monocyte-macrophage lineage. Cytogenetic and polymerase chain reaction (PCR) analysis of the HSC-M1 cell line and involved bone marrow demonstrated the characteristic non-random chromosomal translocation t(2:5)(p23:q35). Reverse transcriptase PCR for mRNA expression of cytokines and cytokine receptors showed that HSC-M1 cells expressed the message for multiple cytokines and their receptors. Measurement of cytokine levels in serum samples using enzyme-linked immunosorbent assays showed increased concentrations of several cytokines. The increased levels of some cytokines correlated with disease activity and clinical symptoms. Although spontaneous production by HSC-M1 cells of some of these cytokines was demonstrated, the production of others was only detectable after stimulation with exogenous CD30 ligand. With few exceptions, there was good correlation between serum cytokine levels and cytokines produced by HSC-M1 cells. These findings indicate that cytokine production is a feature of ALCL cells and that some of the clinical manifestations in ALCL may result from cytokines produced by either the malignant or accessory cells.

OBJECTIVE: The objective of this study was to determine the changes in activated protein C resistance that occur during normal pregnancy. STUDY DESIGN: In this cross-sectional study activated protein C was measured in 128 women with normal pregnancies in the first, second, and third trimesters and in nonpregnant control subjects with 24 to 39 women in each group. In addition, factor V, factor VIII, free protein S, and functional protein C were measured and correlated with activated protein C levels. Polymerase chain reaction for factor V Leiden mutation was performed. RESULTS: There was a significant fall in the activity of activated protein C in the second and third trimesters of pregnancy ( p p = 0.002, R 2 = 0.20). The prevalence of the factor V Leiden mutation was 7.3%. CONCLUSION: Resistance to activated protein C is increased in the second and third trimesters of pregnancy. This is related to the alterations in other coagulation proteins, a phenomenon normally occurring during pregnancy. (Am J Obstet Gynecol 1997;177:162-9.)

Abstract 1855 Introduction: Solid tumors commonly recruit and condition immature myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMΦs), which impair immune-surveillance and facilitate growth. Whether they play a pathogenic role in hematological malignancies is not known. Our previous studies in an MDS/CMML model (SHIP-/- mice) revealed MΦs that are skewed to an alternatively activated (M2) phenotype (reminiscent of TAMΦs), characterized by arginase 1 (Arg1)-mediated redirection of L-arginine metabolism away from cytotoxic NO and towards the production of healing intermediates, as well as over-expression of the Th2-skewing and anti-apoptotic chitinase-like protein, Ym1. Moreover, MDSCs are also increased in SHIP-/- mice, leading to T-cell suppression. We therefore hypothesized that MDSCs/M2-MΦs may be expanded in the BM of MDS and CMML patients and/or their properties hijacked by the malignant clones. Methods: With informed consent, BM mononuclear cells (MNCs) and plasma were obtained from 40 specimens in the Sunnybrook MDS Biobank (6 normals, 11 CMML-1, 1 CMML-2, 1 RARS, 2 del(5q), 6 RCMD, 2 RCMD-RS, 8 RAEB-1, and 3 RAEB-2) (20 at presentation; 20 at follow-up). BM plasma was subjected to YKL-40 ELISA and Quantibody (RayBiotech) cytokine array (bFGF, BMP6, IL-1α, IL-1β, IL-6, PDGF-BB, TGFβ1, TNFα, VEGF, and VEGF-R2). BM-MNC protein lysates were prepared and subjected to arginase enzymatic assay, and Arg1/YKL-40 Westerns. Micro-vessel density (MVD) was recorded as the concordant counts of two observers per mm2 of H&E BM biopsies. Surrogate MVD analysis was performed by automated CD31 immunohistochemistry (IHC) analysis (Vias, Ventana). Linear regression and unpaired, two-tailed t tests were performed with Prism (GraphPad) software. Results: Mean BM-MNC arginase activity (μg urea/h/μg protein, recorded as % of control mean) and BM plasma YKL-40 levels were significantly higher in CMML patients, as compared to controls (350 ± 69 % vs 100 ± 5 %, p = 0.022, and 300 ± 32 μg/L vs 150 ± 22 μg/L, p = 0.0033, respectively). Westerns confirmed intracellular Arg1/YKL-40 over-expression in CMML BM-MNCs. Heterogeneous Arg1/YKL-40 over-expression was observed in MDS patients, and was strongly associated with the lowest risk IPSS and WPSS MDS scoring categories (Figure 1) (CMML not included). Moreover, high Arg1, but not YKL-40, was able to further discriminate IPSS Low/Int1-risk patients with poor prognosis using the low-risk MD Anderson score (Figure 1). This low-risk MDS/high-Arg1/YKL-40 profile was confirmed by negative correlations with (poor-prognostic) MVD (Arg1: p = 0.049, YKL-40: p = 0.089) (similar trends with CD31), albeit in a seemingly paradoxical pro-angiogenic BM plasma milieu (trends to increased bFGF, PDGF-BB, VEGF, and VEGF-R2). These results were unexpected, given that Arg1/YKL-40 are normally thought to be pro-angiogenic, and were reminiscent of recent studies suggesting VEGF-mediated recruitment of suppressive myeloid cells may inhibit angiogenesis and tumor progression in some situations (Stockmann et al., Nature 456:814-8). Accordingly, MDS BM arginase activity was associated with a larger BM myeloid compartment (using > 60% as a cutoff; p = 0.042) and a striking linear correlation with peripheral blood (PB) neutrophil counts on the day of BM collection (p = 0.0003), likely reflecting BM and systemic expansion of suppressive myeloid populations. MDS BM-MNC Arg1/YKL-40 Westerns, stratified by PB neutrophil counts, confirmed these associations. Conclusions: 1) Arg1/YKL-40, effectors of MDSCs/M2-MΦs, are over-expressed in BM MNCs and plasma in most CMML patients, as predicted from SHIP-/- mouse studies. Perturbations in SHIP and associated pathways may warrant investigation in CMML. 2) Arg1/YKL-40 are over-expressed in low-risk MDS patients, as assessed using 3 prognostic scoring systems. Their immune-suppressive nature and pro-proliferative/anti-apoptotic effects may create a permissive environment for low-risk MDS clones. 3) The unexpected association of high Arg1/YKL-40 with a pro-angiogenic BM milieu and paradoxically low MVD, may be in keeping with the ability of MDSCs/M2-MΦs to inhibit angiogenesis under certain conditions. Understanding what regulates the “immune/angiogenic switch” to low suppressive myeloid/high MVD/high-risk MDS may have important future treatment implications. Disclosures: Krystal: Aquinox Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Abstract 438 Type II Abl kinase inhibitors such as imatinib achieve high selectivity by specifically targeting the inactive Abl/c-Kit conformation. However, these inhibitors are prone to inactivation by resistance mutations that cause conformational changes. A better understanding of mutant Abl conformations would provide information to design inhibitors that recognize multiple states of Abl with lower rates of resistance. To more easily examine Abl kinase conformations, conformer specific reagents are needed that are (i) easily generated against a given Abl conformation, (ii) compatible with genetic selections, in vivo and in vitro assays, and structural studies, and (iii) amenable to chemical synthesis. Recently, we developed a novel genetic screen for isolating “lariat” peptide inhibitors of protein function. Lariats are lactone-cyclized peptides that possess the above-mentioned characteristics and can be used to evaluate the function and therapeutic potential of proteins. Previously, we isolated specific lariat inhibitors against the inactive conformation of the bacterial repressor LexA. To obtain conformer specific lariats against Abl, we screened a combinatorial seven amino acid lariat library for interactions with the Abl SH1 domain using the yeast two-hybrid (Y2H) assay. We obtained two lariat peptides named A1 (SGWQRLPFEY) and A2 (SGWHRLSEEY) that interacted with the Abl SH1 domain. In vitro studies with purified Abl kinase demonstrated that the A1 lariat competitively inhibited ATP binding with an inhibitory constant of 5.95 μM. By performing site-saturation mutagenesis, we defined acceptable and tolerable substitutions at each position of the A1 lariat. To obtain tighter binding variants of the A1 lariat, we rationally designed mutations and constructed a second-generation lariat (GWQTLDWNY) with | 10 times higher affinity for Abl. We compared the affinity of the lariats for imatinib resistant Abl kinase mutants using the lariat Y2H binding assay. Mutations that (i) promote conformational dynamics of the kinase T315I), (ii) destabilize the Abl/c-Kit like inactive state (H396R), (iii) disrupt the flexibility or hydrophobicity of the lariat-binding pocket (E355G), and (iv) distort the ATP phosphate binding loop (Y253F), have a marked decrease in lariat affinity. Together, these results show that the lariat preferentially bound to the Abl/c-Kit like inactive conformation and act similarly to type II kinase inhibitors. A specificity screen against a panel of related and distant kinases also demonstrated the conformer specific nature of the lariats. We used lariats that recognized the Abl/c-Kit like inactive state to probe various conformations of Abl His396 mutants. His396 is located in the activation loop and does not alter the Abl/c-Kit like inactive form. His396Pro and His396Arg substitutions are clinically relevant imatinib resistant mutations that alter the conformation of Abl. Previous structural studies showed that the His396Pro mutation causes Abl to adopt the active conformation. It is hypothesized that the His396Arg mutation favors the inactive Src/Cdk like state. Using inactive conformation specific Abl lariats as probes, we showed that these lariats interact weakly with the His396Arg relative to His396, suggesting that this mutation destabilized the Abl/c-Kit like inactive state. We also showed that the lariats interacted stronger with a previously unreported His396Ile mutation than His396, indicating that His396Ile shifts Abl to a highly inactive conformation. Preliminary studies suggest that this residue plays a key role in determining the flexibility of the activation loop and hence the conformational state of Abl. These results provide information on the structure and sequence requirements for maintaining an auto-inhibited activation loop conformation. In summary, we isolated lariat peptide inhibitors against the Abl SH1 domain, characterized their mechanism of action, and improved their affinity for wild type and mutant Abl SH1domains. Additionally, we demonstrated that lariat peptides can be used as affinity reagents to probe conformational states of Abl. Disclosures: No relevant conflicts of interest to declare.

Abstract 204 Treatment of chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, and dasatinib, results in a dramatic reduction in proliferating BCR-ABL expressing leukemia cells. However, these agents do not eliminate the CML stem cell population, indicating that inhibiting BCR-ABL kinase activity alone is not sufficient to eradicate the disease. In vitro studies of human CML cell lines and CD34+ cells isolated from CML patients, have shown that bone marrow stromal cell factor (BMSF) conditioned media can maintain important pro-survival and self-renewal activities downstream of BCR-ABL in the presence of TKIs, suggesting a role for secreted BMSFs in innate resistance to BCR-ABL kinase inhibition. However, the ability of BMSFs to maintain the leukemic potential of CML stem cells upon exposure to TKIs has not been reported. We used a standard murine retroviral transduction system to model CML blast crisis (BC-CML) and obtain cells highly enriched for leukemia initiating potential. Purified LIN-, Sca-1+, CD117+ cells (LSKs) were isolated from the bone marrow of C57BL6/J mice and retrovirally-transduced with BCR-ABL-GFP and Nup98/HoxA9-YFP then injected intravenously into recipient C57BL6/J mice. All animals developed leukemia within 21 days characterized by leukocytosis and extensive infiltration of bone marrow and spleen with leukemic blasts. LSKs expressing both BCR-ABL-GFP and Nup98/HoxA9-YFP (GFP+/YFP+ LSKs) were purified from the spleens or bone marrows of leukemic mice and cultured for 72 hrs in BMSF conditioned media across a range of concentrations (0% - 50%) in the presence and absence of imatinib (0 - 1000 nM). BMSF conditioned media reduced the cytotoxic effects of imatinib on GFP+/YFP+ LSKs as assessed by cell counts, trypan blue viability assays, and Annexin V expression by flow cytometry. Furthermore, BMSF conditioned media reduced the inhibitory effects of imatinib on GFP+/YFP+ LSK colony formation in methylcellulose, and beta-catenin expression as assessed by flow cytometry. These observations strongly suggest that signaling by stromal cell-derived soluble factors protects BC-CML stem cells from imatinib therapy by re-activating pro-survival and self-renewal pathways. The ability of BMSFs to reduce the inhibitory effect of imatinib on BC-CML stem cell self-renewal in vivo was assessed by performing secondary transplantation assays. GFP+/YFP+ LSKs were purified from primary CML mice and transplanted into secondary recipients following in vitro exposure to BMSF conditioned media in the presence and absence of 1000 nM imatinib. Survival after transplantation was compared in cohorts of 5 mice per experimental condition: Group 1 (0% BMSF, 0 nM imatinib), Group 2 (50% BMSF, 0 nM imatinib), Group 3 (50% BMSF, 1000 nM imatinib) and Group 4 (0% BMSF, 1000 nM imatinib). Survival was significantly prolonged in Group 4 mice treated with 1000 nM imatinib and this effect was abrogated by treatment with 50% BMSF conditioned media, indicating that cell-derived soluble factors contribute to maintaining BC-CML stem cell function in the presence of imatinib. Our findings strongly suggest that signaling by soluble BMSFs plays an important role in the innate imatinib resistance of CML stem cells, implicating these factors in disease relapse. Genetically defined murine models of CML provide a powerful in vivo system to identify and target soluble factors that contribute to stromal-mediated cytoprotection of CML stem cells from TKIs. Disclosures: No relevant conflicts of interest to declare.

Introduction: The biology of myelodysplastic syndrome (MDS) is poorly understood, and treatment options are limited. Thus, most MDS patients require chronic red blood cell transfusion, and many develop secondary iron overload. Although the pathophysiological consequences of iron overload to the heart, liver, and endocrine organs have been well characterized, its effects on haematopoiesis have not been studied. However, it has been observed that chelation therapy in iron-overloaded MDS patients may result in reduction of transfusion requirements, and recent studies have suggested a correlation between the use of iron chelation therapy and improvement in leukaemia-free survival in MDS. At the cellular level, iron toxicity is mediated in large part via the generation of reactive oxygen species (ROS). It has been shown in animal models that accumulation of ROS leads to senescence of haematopoietic stem cells, and that ROS cause DNA damage and promote the development of malignancy. These effects of ROS may be particularly important in MDS, in which haematopoiesis is already severely compromised and genetic instability is a striking feature. Hypothesis: We hypothesize that iron overload secondary to transfusion leads to increased levels of intracellular ROS in early haematopoeitic cells in MDS. The increase in intracellular ROS in MDS would be predicted to lead further impairment of haematopoiesis via stem cell exhaustion and while promoting accumulation of DNA damage by myelodysplastic stem cells and early progenitors, thus accelerating progression of MDS to acute leukaemia. Results: To test this hypothesis, we examined the relationship between transfusion-related iron overload and ROS content of CD34+ bone marrow cells in MDS. ROS content was measured in CD34+ cells by flow cytometry in bone marrow aspirates from 34 consecutive MDS patients (CMML=4, MDS/MPD=2, RA=4, RARS=3, RCMD=2, RAEB 1=6, RAEB 2=12, RAEB-t/AML=1). The patients represented a wide range of prior transfusion burden (0->300 units PRBC) and serum ferritin levels (11->10000 μg/L). ROS was strongly correlated with serum ferritin concentration for patients with iron overload (serum ferritin >1000 μg/L; n=14, R=0.733, p To assess the effect of iron overload on normal stem cell and progenitor function, we established a mouse model of subacute bone marrow iron overload. B6D2F1 mice were loaded with iron dextran by intraperitoneal injection (150mg total iron load over 21 days), and sacrificed three days after the end of iron loading. Iron staining of tissue sections confirmed iron deposition in the bone marrow, liver, and myocardium. The development of splenomegaly was noted in iron-loaded animals. Flow cytometric analysis revealed increased apoptosis of bone marrow cells in iron loaded mice based on annexin V+/7 AAD-staining (6.26±0.96% versus 3.54±0.99% for control mice, paired student’s t-Test p Conclusions: These results establish a relationship between CD34+ cell ROS content and serum ferritin concentration in MDS patients with iron overload, and indicate that iron chelation therapy in this patient population reverses this ROS accumulation. The physiological consequences of this relationship are currently being investigated in this patient set by haematopoietic colony assays and assessment of DNA damage in CD34+ cells. Nonethelesss, these data may have key implications for the deployment of iron chelation therapy in MDS patients, and may explain the association between the use of iron chelation and improved leukaemia-free survival in MDS.

A central conundrum in the biology of myelodysplastic syndrome (MDS) is the observation that haematopoietic cells derived from the MDS clone have an increased disposition to apoptosis, yet gain clonal dominance in the bone marrow (BM). One explanation for this apparent paradox could be that MDS stem cells attain clonal dominance by acquiring an augmented probability of self-renewal. We therefore sought to identify novel genes associated with self-renewal that are overexpressed in both MDS and AML. By analyzing the growth of individual clonal siblings derived from low passage cultures of OCI-AML4 we found the expression of the orphan nuclear receptor NR2F6 to be four-fold higher in leukaemia cells with high proliferative capacity, compared to those which spontaneously lose proliferative ability. NR2F6 mRNA transcripts were more abundant in patients (n=37) with MDS, CMML, and AML compared to normal BM (n=16; p