Competitor Cells Research Articles

Mathematical Modeling of Oncogenesis Control in Mature T-Cell Populations

T-cell receptor (TCR) polyclonal mature T cells are surprisingly resistant to oncogenic transformation after retroviral insertion of T-cell oncogenes. In a mouse model, it has been shown that mature T-cell lymphoma/leukemia (MTCLL) is not induced upon transplantation of mature, TCR polyclonal wild-type (WT) T cells, transduced with gammaretroviral vectors encoding potent T-cell oncogenes, into RAG1-deficient recipients. However, further studies demonstrated that quasi-monoclonal T cells treated with the same protocol readily induced MTCLL in the recipient mice. It has been hypothesized that in the TCR polyclonal situation, outgrowth of preleukemic cells and subsequent conversion to overt malignancy is suppressed through regulation of clonal abundances on a per-clone basis due to interactions between TCRs and self-peptide-MHC-complexes (spMHCs), while these mechanisms fail in the quasi-monoclonal situation. To quantitatively study this hypothesis, we applied a mathematical modeling approach. In particular, we developed a novel ordinary differential equation model of T-cell homeostasis, in which T-cell fate depends on spMHC-TCR-interaction-triggered stimulatory signals from antigen-presenting cells (APCs). Based on our mathematical modeling approach, we identified parameter configurations of our model, which consistently explain the observed phenomena. Our results suggest that the preleukemic cells are less competent than healthy competitor cells in acquiring survival stimuli from APCs, but that proliferation of these preleukemic cells is less dependent on survival stimuli from APCs. These predictions now call for experimental validation.

Biological Analysis of SRSF2 Mutations in Leukemogenesis

Abstract 1282Emerging evidence is establishing a connection between MDS and spliceosome mutations. Spliceosome including SF3b1, U2AF1 and SRSF2 are frequently and exclusively mutated in myelodysplastic syndromes (MDS) and related myeloid neoplasms. Spliceosome mutations occur at varying frequencies in different disease subtypes. SF3B1 was shown to be highly associated with MDS characterized by increased ring sideroblasts and SRSF2 mutations are more prevalent in chronic myelomonocytic leukemia. In spite of the fact that the recent discovery constitutes a novel class of genomic lesions and defines an entirely new pathogenic pathway of leukaemogenesis, the pathogenesis of spliceosome mutation is not largely understood. To understanding the biological consequences of spliceosomal mutations, we previously reported mutant U2AF1 cause altered RNA splicing, and overexpressed mutant U2AF1 decrease in cell proliferarion. However, currently, no functional analysis of SRSF2 mutation has been published.SRSF2 belongs to the serine/arginine-rich (SR) protein family. SR proteins are a family of RNA binding proteins characterized by one or two RNA recognition motifs (RRMs) and a signature RS domain enriched with arginine and serine repeats (RS domain).Growing body of evidence suggests that SR protein may be directly involved in the process of carcinogenesis. Gene knockout experiment indicated SRSF2 is involved with specific pathways in regulating cell proliferation and genomic stability during mammalian organogenesis. In neck and head tumor, SRSF2 is frequently overexpressed. And upregulated SRSF2 increases missplicing and downregulates E-cadherin expression, which is an important tumor suppressor gene. Therefore SRSF2 potential function in tumorigenesis is suggested in epithelial cancers. SRSF2 mutations with MDS exclusively occur at P95 within an intervening sequence between RRM and RS domains, indicating a gain-of-function nature of these mutations.So, to clarify the biological role of SRSF2 mutations in leukemogenesis, we evaluated the oncogenic role of SRSF mutations by expressing a mutant SRSF2 allele in Jurkat cells. The cells transduced with a tumor-derived SRSF2 allele showed reduced cell proliferation and increased apoptosis compared to the mock and wild type SRSF2-transduced cells. Next we performed in vitro colony assay using a highly purified hematopoietic stem cell population (CD34-c-Kit+ScaI+ Lin-(CD34-KSL) cells) collected from C57BL/6 (B6)-Ly5.1 mouse that was retrovirally transduced with mock, mutant or wild-type SRSF2 construct. The mutant SRSF2-transduced cells showed reduced cell proliferation compared with mock- or wild-type SRSF2 transduced cells. Subsequently, we conducted bone marrow transplantaion assay. We collected CD34-KSL cells from B6-Ly5.1 mouse, and retrovirally transduce mock, mutant or wild-type SRSF2 construct, each harbouring the EGFP marker gene. And these cells were sorted by EGFP marker, and transplanted with competitor cells (B6-Ly5.1/5.2 F1 mice origin) into lethally irradiated B6-Ly5.2 mice. The wild-type SRSF2-transduced cells showed a lower reconstitution capacity than the mock-transduced cells. On the other hand, the recipients of the cells transduced with the mutant SRSF2 showed lower EGFP-positive cell chimaerism than those of the mock- or the wild-type SRSF2-transduced. Therefore, the mutant SRSF2 was indicated to have a negative effect on cellular proliferation capacity in vitro and in vivo, and a gain-of-function nature of these mutations is suggested. These results are similar to the effect of U2AF1 mutant, which we reported mutant U2AF1 transduced TF-1 and HeLa cells present with a decrease in cell proliferation and hematopoietic stem cells expressing mutant U2AF1 also displayed lower reconstitution capacity by competitive reconstitution assay in mice. So far, the mechanism responsible for the growth advantage of mutant cells in patient is unclear. We furthermore observe hematopoietic phenotype of the bone marrow transplanted model mouse. SRSF2 mutations can coexist with mutations in TET2, ASXL1 and RUNX1. Therefore we performed additionally bone marrow transplantation assay, utilizing hematopoietic cells derived from TET2 knockdown mice, as a model of multistep carcinogenesis. We will present the results of our biological assay on the SRSF2 mutations and discuss the pathogenesis of MDS. Disclosures:No relevant conflicts of interest to declare.

Levels of the c-Myb Protein Indicate Repopulating Capacity in Long-Term Hematopoietic Stem Cells

Abstract 1196c-Myb is a transcription factor essential for the proliferation of hematopoietic cells: conventional c-myb deficient mice died around E14 when their hematopoietic progenitors/stem cells fail to proliferate in the fetal livers. Recently, c-myb has also been reported to be crucial for the differentiation of hematopoietic progenitors. We have previously reported that the differentiation into erythrocytes, megakaryocytes and B-lymphocytes is regulated by c-myb levels utilizing ES cell in vitro differentiation combined with a tetracycline-inducible gene expression system. The gene-altered c-myb mice, such as knockdown or conditional knockout mice in the hematopoietic cell lineages, showed that c-myb controlled hematopoietic stem cells (HSCs). In order to examine the levels of the c-Myb protein in HSCs, we established c-Myb reporter mice in which the EGFP cDNA was linked to the coding sequence of the c-myb gene (c-MybEGFP). Homozygous c-MybEGFP mice, showing normal hematopoiesis, expressed EGFP in hematopoietic progenitors. EGFP+ cells were observed in most long-term (LT) HSCs (90–95%), which were defined as CD34− Lin− Sca-1+c-Kithigh cells (34LSKs), CD150+CD48−LSKs and side-population LSKs. To evaluate c-Myb function in LT-HSCs, we transplanted 100 cells of EGFPlow and EGFPhigh of 34LSKs into irradiated mice along with competitor cells (0×106 cells). Both LT-HSC populations presented multilineage repopulating capacity over 20 weeks. In addition, the EGFPlow cells indicated higher chimerism in the total peripheral blood than the EGFPhigh cells at any given time point. The contribution of the EGFPlow-derived cells in the peripheral blood of the recipient mice increased over time whereas EGFPhigh progeny gradually decreased over time. Under a stringent transplantation condition (30 donor cells with 0.4×106 competitor cells), 83.3% of the recipients that received the EGFPlow34LSK showed donor-derived progeny while the EGFPhigh were lower (20.0%) 8 weeks after transplantation. At Week 12, all the recipients with the EGFPlow34LSKs demonstrated donor-derived progeny; however, EGFPhigh 34LSKs-derived cells disappeared totally in all the transplants. These results suggest that the EGFPlow and the EGFPhigh cells in LT-HSCs possess distinct repopulating capacity: the EGFPlow cells are high and the EGFPhigh cells are low. To investigate the relationship between the EGFPlow and the EGFPhigh LT-HSC, we examined EGFP expression levels in the recipient mice grafted EGFPlow34KSL at least 24 weeks after transplantation. EGFPlow34LSK generated EGFPhigh cells in the donor-derived 34LSK population in the recipient mice, suggesting the possibility that the EGFPlow LT-HSCs support the production of the EGFPhigh LT-HSCs. In conclusion, we found that the expression levels of c-Myb protein subdivide LT-HSC population in correspondence with their respective multilineage repopulating capacities. Disclosures:No relevant conflicts of interest to declare.

Nfix Is Required for Hematopoietic Stem- and Progenitor Cell in Vivo Repopulating Potential.

Abstract 2320Hematopoietic stem cells (HSCs) are both necessary and sufficient to sustain the complete blood system of vertebrates. Specified at several locations during fetal development, they ultimately congregate in the fetal liver for rapid expansion. Around birth HSCs relocate to the bone marrow (BM) and enter a state of cellular quiescence, cycling intermittently to supply progenitor cells, which differentiate into the distinct blood lineages. Due to their regenerative potential, HSCs are heavily utilized in the clinic for bone marrow transplants (BMTs) to treat a variety of diseases. However, a lack of suitable donors and inefficiency in recipient engraftment currently limit this life saving therapy. To improve BMT regimens, a better understanding of regulators of HSC BM engraftment is required.Recently, we examined the gene expression patterns of HSCs as they emerge throughout murine ontogeny (McKinney-Freeman et al., Cell Stem Cell, in press). We observed that the transcription factor Nfix, a member of the nuclear factor I (NFI) family of transcription factors never before linked to HSC biology, was highly expressed by both fetal liver and BM HSCs. These data suggest that Nfix may play a novel role in regulating HSC function in the BM and/or fetal liver. To test this hypothesis, HSCs were enriched from E14.5 fetal liver or adult BM (Lineage-, c-kit+, Sca-1+ (LSK) cells) and then transduced with lentiviruses carrying shRNAs targeting Nfix. Twenty-four hours post-transduction, cells were injected into lethally irradiated mice along with untransduced BM LSK competitor cells congenic at the CD45 allele. The peripheral blood of recipient mice was then analyzed periodically over 16 weeks for engraftment of the Nfix-depleted cells. Depletion of Nfix by two independent shRNA (confirmed by Western blot analysis to deplete NFIX protein levels to <20% of baseline) resulted in a significant decrease in the repopulating activity of BM LSK cells relative to LSK cells transduced with either of two independent control shRNAs. As early as two weeks post-transplant, a 22% +/− 5% (p=0.03) reduction in repopulating activity was observed. By 16 weeks post-transplant, this reduction in repopulating potential had gradually increased to 55% +/−8% (p<0.0001) in four independent experiments. Depletion of Nfix in fetal liver-derived LSK cells resulted in a similar loss of repopulating potential. Critically, in vitro analysis of BM LSK expansion in liquid culture and differentiative potential, as analyzed by the methylcellulose based colony forming assay, revealed no differences in the activity of LSK cells transduced with Nfix-specific shRNAs compared to controls. Thus, it is unlikely that the observed decrease in BM repopulating activity is due to general cytotoxicity resulting from Nfix depletion or a block in differentiation. Concordantly, lineage analysis of peripheral blood of recipients showed no significant differences in the percentage of the major blood lineages derived from LSK cells transduced with Nfix-specific shRNAs compared to controls. Thus, the observed decrease in repopulating activity likely occurs at the level of HSCs and multipotent progenitors. In agreement with this conclusion, when BM of recipients transplanted with Nfix-depleted LSK cells are examined 4 and 16 weeks post transplant, a loss of phenotypic HSCs (LSK/CD150+/CD48-) relative to controls is evident.The loss of repopulating potential by Nfix-depleted cells as early as two weeks post-transplant suggests that Nfix may be involved in either the homing/lodgment of HSCs in the BM or their ability to expand soon after incorporation into the stem cell niche. We are presently teasing out the molecular mechanism behind this phenomenon. Furthermore, to examine a role for Nfix during HSC homeostasis, we are currently analyzing mice in which Nfix has been conditionally ablated in the hematopoietic compartment. Finally, functional analysis of two other members of the NFI gene family shown by our array analysis to be expressed by BM HSC, Nfia and Nfic, will further assess a role for this gene family in the regulation of HSCs.In summary, we have for the first time established a role for a member of the NFI gene family, Nfix, in HSC biology, as evident by a decrease in BM repopulating activity in Nfix-depleted HSCs. By dissecting the precise role of Nfix in HSC biology, we will glean insights that could improve our understanding of graft failure in clinical BMTs. Disclosures:No relevant conflicts of interest to declare.

Modeling and analysis of abnormality detection in biomolecular nano-networks

A scheme for detection of abnormality in molecular nano-networks is proposed. This is motivated by the fact that early diagnosis, classification and detection of diseases such as cancer play a crucial role in their successful treatment. The proposed nano-abnormality detection scheme (NADS) comprises of a two-tier network of sensor nano-machines (SNMs) in the first tier and a data gathering node (DGN) at the sink. The SNMs detect the presence of competitor cells as abnormality that is captured by variations in parameters of a nano-communications channel. In the second step, the SNMs transmit micro-scale messages over a noisy micro communications channel (MCC) to the DGN, where a decision is made upon fusing the received signals. The detection performance of each SNM is analyzed by setting up a Neyman-Pearson test. Next, taking into account the effect of the MCC, the overall performance of the proposed NADS is quantified in terms of probabilities of misdetection and false alarm. A design problem is formulated, when the optimized concentration of SNMs in a sample is obtained for a high probability of detection and a limited probability of false alarm.

Regulation of hematopoietic stem cells using protein transduction domain–fused Polycomb

The Polycomb-group complex is a chromatin regulatory factor that is classified into two different complexes: Polycomb repressive complex 1 and 2. Components of Polycomb repressive complex 1 are involved in the self-renewal of hematopoietic stem cells. Bmi1, one of these components, maintains the immaturity of neural and cancer stem cells as well as that of hematopoietic stem cells. We constructed recombinant protein transduction domain (PTD)-Polycomb proteins and transduced them into murine bone marrow (BM) cells. We designed and fused the PTD-protein transduction domain to three proteins (i.e., green fluorescent protein, Bmi1, and Mel18). Murine BM cells were incubated for 48 h and each PTD-Polycomb protein was added. Then, we analyzed the function of hematopoiesis using the colony assay and transplantation. BM cells exposed to PTD-Bmi1 showed an increased number of colonies. In contrast, BM cells exposed to PTD-Mel18 or to both proteins showed a decreased number of colonies. Hematopoietic cells derived from PTD-Bmi1-transduced BM cells were significantly increased in the peripheral blood at 6 weeks after transplantation. Moreover, 80% of mice transplanted with PTD-Bmi1-transduced BM cells died at 8 to 24 weeks after transplantation. However, only a few early deaths were observed in the mice transplanted with BM cells exposed to both PTD-Bmi1 and PTD-Mel18. We expect that hematopoietic stem cells could proliferate after transduction with PTD-Bmi1, but this may generate undesirable effects, e.g., tumorigenesis. Thus, Bmi1 and Mel18 have opposing functions and are present in distinct complexes.

Nicotinamide, a SIRT1 inhibitor, inhibits differentiation and facilitates expansion of hematopoietic progenitor cells with enhanced bone marrow homing and engraftment

Strategies that increase homing to the bone marrow and engraftment efficacy of exvivo expended CD34(+) cells are expected to enhance their clinical utility. Here we report that nicotinamide (NAM), a form of vitamin B-3, delayed differentiation and increased engraftment efficacy of cord blood-derived human CD34(+) cells cultured with cytokines. In the presence of NAM, the fraction of CD34(+)CD38(-) cells increased and the fraction of differentiated cells (CD14(+), CD11b(+), and CD11c(+)) decreased. CD34(+) cells cultured with NAM displayed increased migration toward stromal cell derived factor-1 and homed to the bone marrow with higher efficacy, thus contributing to their increased engraftment efficacy, which was maintained in competitive transplants with noncultured competitor cells. NAM is a known potent inhibitor of several classes of ribosylase enzymes that require NAD for their activity, as well as sirtuin (SIRT1), class III NAD(+)-dependent-histone-deacetylase. We demonstrated that EX-527, a specific inhibitor of SIRT1 catalytic activity, inhibited differentiation of CD34(+) cells similar to NAM, while specific inhibitors of NAD-ribosylase enzymes did notinhibit differentiation, suggesting that the NAM effect is SIRT1-specific. Our findings suggesta critical function of SIRT1 in the regulation of hematopoietic stem cell activity and imply theclinical utility of NAM for exvivo expansion of functional CD34(+) cells.

FLVCR Deletion Does Not Impact Normal Hematopoietic Stem Cell Function but Is Necessary for Erythroid and T-Cell Maturation and Enhances Platelet Formation

Abstract 2363Hematopoietic stem cells (HSCs) are mainly in a quiescent state, but activate and proliferate in response to stress. To maintain quiescence, as well as respond to activation signals, HSCs must coordinately regulate cell cycle status versus metabolic demand. Mismatch in these pathways can lead to HSC exhaustion, failure or malignant transformation. One metabolic pathway that is essential to all cells is heme biosynthesis. Heme can act directly as a signaling molecule by modulating the function of transcriptional (Bach1) or translational (eIFa2 kinase) regulators. In addition, heme can act indirectly through the action of hemoproteins (hemoglobin, oxidases, peroxidases, cytochromes). However, excess free intracellular heme is toxic, therefore organisms have developed “safety valve” pathways to regulate intracellular heme concentrations. The most well studied mechanism is free heme degradation by the heme-oxygenase pathway. We have recently described a second pathway to prevent intracellular heme excess, namely heme export by the cytoplasmic transporter FLVCR. Previously we have shown that FLVCR is essential for erythropoiesis, as FLVCR−/− mice die of anemia (Keel et. al., Science 319: 825, 2008). FLVCR is expressed in many other tissues and blood cells, including HSCs (Lucas et. al., Blood 106:51, 2005). This suggests that FLVCR may be important in HSC function or the development of other lineages, besides red cells. We used a competitive transplantation model to determine the effect of FLVCR deletion on HSC function and lineage maturation. FLVCR−/− or wild type (WT) donor cells and equal numbers of competitor cells were transplanted into lethally irradiated, syngeneic recipients. FLVCR−/− marrow cells reconstitute the granulocyte compartment equally as well as WT cells in serial transplantation (up to tertiary transplants). Equal contributions also remain after myelotoxic stress (radiation or arsenic trioxide). Therefore, FLVCR may be dispensable for normal HSC function under steady-state and stress conditions. As anticipated, FLVCR is required for proerythroblast survival and maturation. Eight weeks after competitive transplantation with FLVCR−/− donor cells, 100% of red cells are derived from competitor cells. This is also seen when the ratio of FLVCR−/−:competitor cells is increased to 95:5 from 50:50. FLVCR−/− cells fail to mature past the proerythroblast stage (CD71pos lin-Ter119neg or lo) and undergo significantly more apoptosis than WT donor cells (88% vs. 36%, p<0.001). FLVCR deletion also blocks T-cell development. Due to biological variability in our experimental model, T-cell numbers were normalized to granulocytes in each determination, in each mouse and expressed as a percentage of the expected value. At 5 weeks post-transplant, the percentage of FLVCR−/− derived T-cells was 65.8% of the percentage of FLVCR−/− derived granulocytes. The percentage of WT derived T-cells was 93.4% of the percentage of WT derived granulocytes (p<0.02). Over time, FLVCR−/− derived T-cells steadily decreased, dropping to 22% of expected values by week 24, and remaining low, whereas T-cells derived from control, WT donor cells remained stable (74–98% of expected values). FLVCR−/− T-cells are arrested at the double positive (CD4+CD8+) stage (Philip et. al., Blood (ASH Abstracts) 114:913, 2009). Surprisingly, loss of FLVCR improves platelet production. FLVCR−/− donor cells make up ∼60% of all platelets (avg. 1.71 x expected, when normalized to granulocytes). In the separate control cohort competitively transplanted with WT donor cells, ∼40% of the platelet population was derived from WT cells (avg. 0.91 x expected, when normalized to granulocytes). Our studies suggest that the increased platelet production likely results from increased megakaryocyte ploidy (e.g. avg. 14.2N in FLVCR−/− vs 5.2N in control) and not from increased megakaryocyte progenitors. Of note, FLVCR−/− deleted mice have thrombocytosis (2221/μL vs 1008/μL, p<0.001 in littermate controls), a finding we initially dismissed as a stress response to severe anemia. Together, these findings reveal stage specific roles for FLVCR in discrete hematopoietic lineages and support the broader hypothesis that heme excess might impair cell division, leading to decreased red and T-cell production, but improved endomitosis and increased platelet production. Disclosures:No relevant conflicts of interest to declare.

Megakaryocyte Lineage Commitment in Hematopoietic Stem Cells

Abstract 909Hematopoietic stem cells (HSCs) are able to self-renew and differentiate into all blood lineages. Lineage commitment can take place at any stage from daughter cells of HSCs to multipotent progenitors. We have previously suggested that lineage commitment takes place at the level of HSCs (Takano et al. 2004). According to our asymmetric model, one HSC gives rise to two daughter cells consisting of one HSC and one lineage-committed progenitor. To verify this model, we performed a series of single-cell transplantation, followed by paired daughter cell experiments. The Ly5 congenic system has been commonly used to distinguish test donor cells from recipient cells and competitor cells. Since the Ly5 antigen is only expressed in leukocytes, platelets and erythrocytes have not been examined for evaluation of multi-lineage reconstitution. To assess lineage contribution more precisely, we have recently generated a transgenic mouse line in which Kusabira Orange (KuO) is expressed in all blood lineages including platelets and erythrocytes. CD150+CD41−CD34−/lowc-Kit+Sca-1+Lin− (CD150+CD41−CD34−KSL) cells, in which HSCs are highly enriched (Kiel et al. 2005, Morita et al. 2010), and CD150−CD41−CD34−KSL cells were individually isolated from KuO transgenic mice (B6-Ly5.1), and were transplanted into lethally irradiated mice (B6-Ly5.2), along with 2×105 bone marrow cells (B6-Ly5.1/Ly5.2). For secondary transplantation, 1×107 bone marrow cells from recipient mice were transplanted to lethally irradiated mice. Peripheral blood cells of primary and secondary recipient mice were periodically analyzed after transplantation. In some mice, full-lineage reconstitution was detected after transplantation with single CD150+CD41−CD34−KSL cells. In others, only common myeloid progenitor-type reconstitution was detected whereas common lymphoid progenitor-type reconstitution was not detected at all. Interestingly, megakaryocyte lineage (Meg)-restricted reconstitution and Meg/erythrocyte lineage-restricted (ME) reconstitution were observed after transplantation with single CD150+CD41−CD34−KSL cells, but not after transplantation with single CD150−CD41−CD34−KSL cells. These results suggest that Meg and ME progenitors are closely related to HSCs in their development. Furthermore, we performed paired daughter cell assays combined with single cell transplantation. Following division of CD150+CD41−CD34−KSL cells in culture, individual two daughter cells were separated by micromanipulation techniques and were transplanted into lethally irradiated mice with 2 × 105 competitor cells. Preliminary data showed in effect that the recipient mice of each single daughter cell derived from one CD150+CD41−CD34−KSL cell exhibited asymmetric differentiation; one with full-lineage reconstitution and the other with Meg-restricted reconstitution. Our findings offer a new perspective of HSC differentiation pathways. Disclosures:No relevant conflicts of interest to declare.

Lethal Myelofibrosis Induced by Bmi1-Deficient Hematopoietic Cells Unveils a Tumor Suppressor Function of the Polycomb Group Genes

Abstract 390Polycomb-group (PcG) proteins form polycomb repressive complex (PRC) 1 and PRC2, and function as transcriptional repressors through histone modifications. They maintain proliferative capacity of hematopoietic stem and progenitor cells by repressing transcription of tumor suppressor genes, thus have been characterized as oncogenes. However, identification of inactivating mutations of a PcG gene, EZH2, unveiled its tumor suppressor function in myeloid malignancies including primary myelofibrosis (PMF). Here, we show that loss of another PcG gene, Bmi1, causes pathological hematopoiesis reminiscent of PMF in mice. We previously reported that deletion of both Ink4a and Arf in Bmi1-deficient mice substantially restores the defective self-renewal capacity of HSCs. To evaluate the repopulating capacity of Bmi1−/−Ink4a-Arf−/− BM cells precisely, we transplanted wild-type, Ink4a-Arf−/−, and Bmi1−/−Ink4a-Arf−/− BM cells into lethally irradiated mice without competitor cells. The recipients repopulated with Bmi1−/−Ink4a-Arf−/− donor cells had 2-fold more megakaryocyte/erythroid progenitors (MEPs) and extramedullary hematopoiesis as evident from a significant increase in the number of LSK HSCs/MPPs in spleen. All the control recipient mice repopulated with Ink4a-Arf−/− BM cells eventually developed sarcoma or lymphoma and succumbed to die by 11 months post-transplant as previously reported with the Ink4a-Arf−/− mice. On the other hand, the recipient mice repopulated with Bmi1−/−Ink4a-Arf−/− BM cells died much earlier than the Ink4a-Arf−/− controls, displayed more progressive pancytopenia, and showed marked hepatosplenomegaly and hypoplastic BM with massive fibrosis at their terminal stage. Although abnormal megakaryocytosis was not obvious in BM and spleen because of severe fibrosis at the terminal stage of the disease, the mice at earlier time point after transplantation showed marked megakaryocytosis in both BM and spleen, implicating pathological megakaryocytosis in the development of lethal myelofibrosis. Together, lethal myelofibrosis induced by Bmi1−/−Ink4a-Arf−/− hematopoietic cells follows the natural course of human PMF.To identify the responsible genes for PMF-like disease in the absence of Bmi1, we compared gene expression profiles of LSK HSCs/MPPs and common myeloid progenitors (CMPs) from wild-type, Ink4a-Arf −/−, and Bmi1−/−Ink4a-Arf −/− BM cells. Absence of Bmi1 caused de-repression of a cohort of genes. We then compared the list of de-repressed genes with that of PMF-associated genes identified by a gene expression profiling of CD34+ cells from human PMF patients (Guglielmelli et al., Stem Cells 25:165–173, 2007). Hmga2, a well-known oncogene, appeared to be commonly upregulated in Bmi1−/−Ink4a-Arf −/− CMPs and PMF CD34+ cells. Chromatin immunoprecipitation assays demonstrated that Bmi1 directly represses the expression of Hmga2 by marking its promoter with a repressive histone mark. To test contribution of Hmga2 to the development of PMF-like disease in mice, we examined the effects of Hmga2 overexpression on hematopoiesis. Forced expression of Hmga2 in HSCs promoted expansion of progenitor cells and significantly facilitated megakaryocytopoiesis in vitro. Hmga2-overexpressing HSCs also induced a mild myeloproliferative state with enhanced megakaryocytopoiesis in recipient mice, although fibrosis was not obviously induced. These results implicated Hmga2 in the development of pathological hematopoiesis in the absence of Bmi1.Collectively, our findings unveiled the tumor suppressor function of Bmi1. Corresponding to our findings, mice with hypomorphic mutations of Eed and Suz12 reportedly showed enhanced hematopoiesis. All these findings might be in line with tumor suppressor function of EZH2 observed in MDS and MPN patients, and suggestive of a broad range of target genes of the PcG proteins, which include both oncogenes and tumor suppressor genes. Although the tumor suppressor genes have been stresses as PcG targets, our findings shed a light on the role of PcG proteins in gene silencing of oncogenes. Thus, the PcG proteins fine-tune the growth of hematopoietic cells in both a positive and a negative manner to maintain hematopoietic homeostasis. Disclosures:No relevant conflicts of interest to declare.

A Functional In Vivo RNAi screen Involving Jumonji C Domain Containing Candidates Unravels Kdm5b As a Negative Modulator of Hematopoietic Stem Cell Self-Renewal

Abstract 385Epigenetic modifications influence chromatin accessibility, impacting on cell fate decisions, such as stem cell self-renewal and differentiation, in both normal and leukemic stem cells (LSC). To investigate the putative role of histone demethylases (HDM) in modulating primary hematopoietic stem cell (HSC) fate, an in vivo functional screen was performed, using an RNAi based strategy, involving 25 members of the Jumonji (JmjC) domain protein family.As a first step, expression profile studies of these gene candidates were undertaken. Transcripts of all these enzymes were detected in isolated HSC populations (frequency 1:2) from fetal liver (n=1) and bone marrow (n=2), except for Hairless. As compared to unsorted bone marrow (BM), stem cells harboured higher expression of Jarid1b (relative-fold enrichment (RQ) of 3.9±1.7), Jmjd2d (RQ3.8±1.9), and Jhdm1b (3.1±1.7). Next, 5shRNA were designed against each of the 25 JmjC containing proteins, and cloned into a retroviral LMP vector encoding GFP to permit tracking of transduced cells in vivo. HSC-enriched CD150+CD48−Lin−cells (∼60 LT-HSC) were infected over 5 days by co-culture with retroviral producer cells in an arrayed 96-well format, with one shRNA per well. Directly after infection, the in vivo reconstituting potential of ¼ of each well was evaluated through duplicate competitive repopulation assays involving the co-transplantation of 1.5 × 105 congenic BM competitor cells into irradiated recipients. The remaining cell fraction served to asses gene transfer by GFP epifluorescence measurements, and RNA isolated from sorted GFP+ cells was used to evaluate gene knockdown levels by Q-RT-PCR analysis. Blood reconstitution was evaluated at an early (4wks) and late time point (16–20wks), tracking the contribution of the donor CD45.1+ transduced (GFP+) cells to recipient hematopoiesis over time. As baseline references, sh-RNA to Luciferase (no effect) and the histone acetyl transferase Myst3 (stem cell loss) were used, as well as Hoxb4 over-expression (stem cell expansion). The primary screen, followed by validation experiments, unravelled one positive (Jhdm1f/Phf8) and two negative (Jarid1b, Hif1an) regulators of HSC activity. The strongest impact was seen with Jarid1b knockdown, and the resulting gain in HSC activity. As a confirmation step, cells were kept in culture for one week, to better contrast an increase in HSC activity, compared to control HSC. After 7 days in vitro, 1/8 equivalents of single well cultures were transplanted into 3 mice, and blood reconstitution levels serially assessed. Cells transduced with sh-RNA against Jarid1b contributed more significantly to host hematopoiesis than sh-RNA Luciferase transduced cells (58±16% vs 26±3% GFP), or Hoxb4 over-expressing cells (37±2% GFP), at comparable gene transfer rates, at the 16 week time point and beyond (3 independent experiments). Long-term HSC frequencies were evaluated from these cultures, and found to be 6–10 fold increased in shJari1d1b-cell cultures. In long-term recipients, differentiation potential of these cells was preserved, as evidenced by CD4+CD8+ thymic cells, B220+ splenic cells and CD11b+ bone marrow cells in the GFP positive contingent. Clonality studies on DNA isolated from these sorted populations confirmed oligoclonality of the stem cell expansion, and HSC pluripotency. There were no cases of leukemic transformation in all of the transplant recipients (n>30). As assessed by Q-RT-PCR, levels of HoxA5, HoxA9, HoxA10 and CxCl5 were increased in day7 sh3Jarib1b-cells (vs ctl), while the levels of the tumor suppressors Cav1, Sash1 and Egr1 were decreased. A more detailed assessment of the HoxA cluster revealed predominant expression of 5’ cluster genes in expanding shJarib1b-cells, from HoxA5 to HoxA11, with a concomitant increase in the level of H3K4 tri-methylation, as assessed by ChIP-CHIP.In conclusion, HDM of the JmjC family can modulate HSC activity, both positively and negatively. These data suggest that the H3K4 demethylase Jarid1b (KDM5b) restrains stem cell self-renewal, acting as a co-repressor, possibly via epigenetic regulation of the HoxA gene cluster, among other target genes. This observation could be further exploited as an HSC expansion strategy. Disclosures:No relevant conflicts of interest to declare.

The Bone Marrow Hematopoietic Microenvironment Is Functionally Impaired in Iron-Overloaded Mice,

Abstract 3173 Background and Purpose:Deferasirox (DFX) is an oral iron chelator that enables effective chelation by once daily administration.Since the introduction of DFX, iron chelation therapy (ICT) for transfusional iron overload has attracted increased attention. It is known that excess iron increases oxidative stress and affects various organs, such as the liver, heart and endocrine glands, negatively. Sufficient ICT can remove excess iron and improve organ dysfunction in iron-overloaded patients, and accumulating data has indicated that efficient ICT improves the survival of transfusion-dependent patients with myelodysplastic syndromes (MDS). Recently, we experienced a case of MDS with transfusional iron overload in which the hematopoietic data improved unexpectedly after administration of DFX without any other specific treatments (Okabe H et al. Rinsho Ketsueki, 2009). An increasing number of similar cases has been reported. This clinical observation indicates that iron overload could also affect the hematopoietic system unfavorably, via, as yet, unknown mechanisms. Methods and Results:We generated iron-overloaded mice to investigate how iron overload affects hematopoiesis in vivo. C57BL6 mice were injected with a total of 200 mg of iron dextran, intraperitoneally over 4 weeks. The iron-overloaded mice showed pigmented skin and hepatosplenomegaly, and histological examination showed excess iron deposition in the bone marrow, liver, spleen and heart. The serum and organ iron concentrations in these mice markedly increased. However, the iron-overloaded mice did not show any significant changes in peripheral blood counts or the proportion of immature hematopoietic cells in the bone marrow. To further examine the effects of excess iron on the biological functions of hematopoietic stem and progenitor cells (HSPCs), we performed bone marrow transplantation (BMT) assays. First, to assess the hematopoietic reconstitutional capacity of the HSPCs of iron-overloaded mice, we transplanted bone marrow cells (1×106 cells) from iron-overloaded mice or normal mice into lethally irradiated normal recipient mice along with the same number of normal competitor cells. We found no significant difference in hematopoietic reconstitution between the iron-overloaded donor cells and the normal donor cells, suggesting that the hematopoietic reconstitutional capacity of HSPCs in iron-overloaded mice is not significantly affected by iron. In contrast, when we transplanted bone marrow cells from normal mice (2×106 cells) into iron-overloaded recipients, hematopoietic recovery was significantly delayed, in particular platelet counts (at 2 weeks after BMT, normal recipients vs. iron-overloaded recipients, 63.4±9.4 vs. 18.7±4.7×104/μl, respectively, p<0.001). This indicates that excess iron disturbs the function of the bone marrow microenvironment and delays hematopoietic reconstitution. Microarray and quantitative RT-PCR analysis of non-hematopoietic bone marrow cells (CD45-/Ter119-) from the iron-overloaded mice demonstrated significant reductions in CXCL12, VCAM-1, Kit-ligand and IGF-1, which are important regulators of hematopoiesis. In addition, in the iron-overloaded mice, the serum concentration of erythropoietin and the expression level of thrombopoietin in the liver were also significantly reduced. Furthermore, increased oxidative stress levels were observed in the iron-overloaded liver and bone marrow. Conclusion:We did not observe any direct effects of excessive iron on hematopoietic cells, but found significant impairment of the hematopoietic microenvironment in the bone marrow of iron-overloaded mice. These results suggest that oxidative stress induced by excess iron could disturb the hematopoiesis-supporting capacity of the bone marrow microenvironment by reducing the expression of many essential molecules. Disclosures:No relevant conflicts of interest to declare.

Homeostasis of hematopoietic stem cells regulated by the myeloproliferative disease associated-gene product Lnk/Sh2b3 via Bcl-xL

Hematopoietic stem cells (HSCs) are maintained at a very low frequency in adult bone marrow under steady-state conditions. However, it is not fully understood how homeostasis of bone marrow HSCs is maintained. We attempted to identify a key molecule involved in the regulation of HSC numbers, a factor that, in the absence of Lnk, leads to HSC expansion. Here, we demonstrate that upon stimulation with thrombopoietin, expression of Bcl-xL, an antiapoptotic protein, was highly enhanced in Lnk-deficient HSCs compared to normal HSCs. As a result, Lnk-deficient HSCs underwent reduced apoptosis following exposure to lethal radiation. Downregulation of Bcl-xL expression in Lnk-deficient HSCs by short-hairpin RNA resulted in a great reduction of their capacity for reconstitution. These findings suggest that Lnk/Sh2b3 constrains the expression of Bcl-xL and that the loss of Lnk/Sh2b3 function enhances survival of HSCs by inhibiting apoptosis. Furthermore, our observations indicate that HSCs in patients with an Lnk/Sh2b3 mutation might become resistant to apoptosis due to thrombopoietin-mediated enhanced expression of Bcl-xL. Consequently, reduced apoptosis could facilitate accumulation of HSCs with oncogenic mutations leading to development of myeloproliferative disorders.

JAK2-V617F Expressing Stem Cells Display a Competitive Advantage At Low Limiting Dilution and Are Capable of Initiating MPN Phenotype

Abstract 615JAK2-V617F is the most common genetic alteration in myeloproliferative neoplasms (MPN). Forced expression of JAK2-V617F in animal models induced a MPN phenotype. However, MPN in these models was initiated by polyclonal expansion of multiple hematopoietic progenitor and stem cells. In contrast, patients with MPN display clonal hematopoiesis. Frequently, additional somatic mutations precede the acquisition of JAK2-V617F, suggesting that a pre-JAK2 mutation could be required for clonal expansion. Compatible with this hypothesis, in one JAK2-V617F knockin model the MPN phenotype was not transplantable and the stem cells showed signs of exhaustion. To address the question of whether JAK2-V617F alone is sufficient to initiate MPN we performed competitive repopulation assays and serial transplantations in a transgenic JAK2-V617F mouse model.Our transgenic mice express the human JAK2-V617F under the endogenous JAK2 promoter and display a polycythemia vera (PV) or essential thrombocythemia (ET) phenotype. The transgene can be activated by Cre recombinase and the donors were induced 5–6 weeks prior to transplantation. To allow monitoring of the reconstitution, a GFP expressing transgenic strain was used as the source of competitor cells. Bone marrow transplantations were performed into lethally irradiated mice in the inbred BL/6 background. A total of 2×106 bone marrow cells per recipient were transplanted with ratios of mutant (V617F) to wild type (WT) set as 1:1, 1:10 or 1:100. Results:At 1:1 and 1:10 ratios, the mice displayed a PV phenotype beginning at 4 weeks post transplantation (Hb ∼200-220 g/L and neutrophil count of 5–20×109 /L) that remained stable for ∼26 weeks. An increase in platelet counts (4-6×1012/L) followed at later time points. V617F cells rapidly outcompeted WT cells reaching >95% chimerisms in peripheral blood. The MPN phenotype was transplantable into secondary and tertiary recipients and no signs of exhaustion were observed. An increase in the frequencies of lineage-negative, Sca-1+ c-kit+ (LSK) cell numbers in bone marrow was noted (4.1% in V617F vs 1.2% for WT, p<0.05). To assess their cycling rate, LSK cells were sorted, labeled with carboxyfluorescein succinimidyl ester and transplanted into non-conditioned recipients. After 8 weeks, the number of LSK cells was 62-fold increased and V617F LSK cells outcompeted host cells (52% vs. 3% for WT, p<0.01). The number of slow-dividing (0-2 divisions) LSK cells was substantially reduced in the V617F transplanted mice (0.095% vs. 7.54% for WT, p<0.01). Thus, V617F increases the cycling of LSK cells and provides a competitive advantage.To allow detecting low numbers of donor cells in 1:100 transplantations, the GFP transgene was bred into the V617F background and a mixture of 2×104 V617F/GFP bone marrow cells plus 1.98×106 WT competitors was used. Based on the expected frequency of 3 long term hematopoietic stem cells (HSC) per 105 bone marrow cells, the recipients should receive on average 0.6 GFP-positive long term HSC. In mice transplanted with V617F/GFP and WT competitors at 1:100, we detected reconstitution defined as >0.1% GFP positive cells in 12 of 40 recipients (28%) that persisted beyond 20 weeks, whereas in mice transplanted with WT/GFP and WT competitors, only 5/40 (12%) showed reconstitution. The relative contribution in the WT group ranged from 0.1 – 3%, whereas in the V617F group we observed chimerism between 5 –100%. Four of the 12 mice that reconstituted with V617F/GFP cells developed a MPN phenotype (3PV, 1ET). Terminal workup of 3 mice (2PV and 1ET) at 29 weeks revealed that mice with erythrocytosis had enlarged spleens (∼150mg vs. 65mg for WT) and increased erythropoiesis in spleen histology. In contrast the mouse with ET phenotype showed only a slight increase in spleen size (90mg vs. 65mg for WT) and no increase in spleen erythropoiesis. Conclusions:Our results show that JAK2-V617F positive cells were able to rapidly outcompete WT cells. The MPN phenotype was transplantable into secondary and tertiary recipients. At low limiting dilution (1:100), V617F cells displayed > 2-fold higher engraftment rate (28%) than WT cells (12%) and a higher contribution to hematopoiesis (5-100% chimerism vs. 0.1–3%). Since 4/12 mice (30%) reconstituted with V617F cells also developed a MPN phenotype, our data demonstrate than JAK2-V617F as the sole genetic alteration can initiate MPN under conditions resembling monoclonal disease. Disclosures:No relevant conflicts of interest to declare.

Leukemogenesis and ageing: ‘fit for transformation’ ?

There is longstanding recognition of the striking association between ageing in humans and the incidence rates of many common types of cancer. The biological basis of this link is likely to be multi-factorial including both age-associated decline in anti-oncogenic functions, such as repair of oxidative damage to DNA, timing of etiologic exposures and/or protracted time required for accumulation of a full set of oncogenic mutations [1,2]. Acute leukemias have an unusual age-associated incidence with a peak at a young age (2-5 years) followed by a later increasing incidence with age in adults. In this instance, however, the biological and genetic subtypes of leukemia are distinct at different ages [3]. BCR-ABL1 gene fusion is the result of the ‘Philadelphia’ chromosome - the first specific genetic abnormality described in cancer and which is a translocation between chromosomes 9 and 22 [4]. Two versions of the fusion exist involving different exons of BCR and generating two different sized proteins: p210 BCR-ABL1 predominantly associated with chronic myeloid leukemia and p190 BCR-ABL1 linked to B cell precursor acute lymphoblastic leukemia (ALL). Both sub-types of leukemia have increased incidence with age. So what might be the biological basis of the age-associated incidence of B-ALL with p190 BCR-ABL1? Vicente-Duenas et al [5] designed a novel experiment to address this question which conveniently bypassed uncertainties over required exposures for the natural disease and age-associated alterations in the host tissue micro-environment. Using an inducible p190 BCR-ABL transgene, they found that ‘old’ (= 20 months) cells develop leukemias at a much faster rate (~2x) than young' (= 4 months) cells when transplanted into recipients of the same age (4 weeks). The key to the experiment is that BCR-ABL1 expression is not activated until after the transplant. Although all mice eventually developed ALL, this result is compatible with the notion that ‘old’ B progenitors have some competitive advantage when expressing BCR-ABL1. What might this be? A plausible explanation is offered in another paper recently published by Henry et al [6]. This group have previously suggested that age-related fitness decline in stem and progenitor cells might increase selective pressure for oncogenic mutations [7]. In their new study [6], they also evaluated the impact of age on the transformability of hematopoietic cells by p190 BCR-ABL1. In their case, this was performed by transfecting ‘young’ (2 months) versus ‘old’ (22-24 months) bone marrow cells with a p190 BCR-ABL retrovirus and transplanting these into recipients which were selected to provide either ‘young’ (2 months) or ‘old’ (22-24 months) competitor normal cells. They observed higher rates of leukemia (up to 200 days post-transplant) with transfer of ‘older’ cells. More rapid onset of leukemia in aged p190 BCR-ABL1 cells or more efficient leukemogenesis probably reflects more ‘target’ cells being transformed which, in turn, would increase the probability of the acquisition of secondary genetic abnormalities such as IKZF1 deletion which, at least for leukemia in patients with BCR-ABL1+ ALL, appear to be critical for malignant progression [8]. This interpretation accords with the observation of Henry et al [6] that ‘older’ mouse cells transfected with BCR-ABL1 undergo more rapid polyclonal expansion early after transplantation. Additionally, Henry et al [6] observed that ‘young’ competitor cells in the recipient had a restraining impact on the leukemogenic efficiency of ‘old’ transformed cells, indicating that the age-associated leukemogenesis involves both cell intrinsic (or autonomous) and extrinsic factors perhaps related to competitive occupancy of critical bone marrow niches. Henry et al argue that BCR-ABL1 actually has more potent selective advantage in older cells because it restores defective kinase signalling in ageing cells. They provide supportive evidence for this by showing increased expansion of young BCR-ABL1+ B cell progenitors under conditions of reduced kinase signalling via the regulatory molecule IL7 [6]. These data are compelling but one alternative explanation might also be considered. B cell lymphopoiesis declines dramatically with age in both mice and humans and this inevitably produces substantial changes in the cellular architecture of bone marrow [9]. Transforming functions of oncogenes, including leukemic fusion genes, in very cell context dependent [10]. It is possible that it is not only some qualitative feature of the target cell that changes with age but the number of the appropriate cells available for transformation. With ionizing radiation-induced ALL, children [11] or young mice [12] are more vulnerable, as are young post-adolescent females with respect to irradiation-induced breast cancer [13]. This is most easily interpreted as reflecting age-associated changes in the number and proliferative activity of the particular stem and progenitor cells that provide the vulnerable ‘targets’ for transformation. Whatever the mechanisms, or combination of mechanisms, involved, these two new papers add a new dimension to our understanding of the complexity of age-associated leukemogenesis and, perhaps, cancer in general.

RhoA GTPase Is Dispensable for Hematopoietic Stem Cell Maintenance but Essential for Multipotent Progenitor and Lower Hierarchical Hematopoietic Differentiation.

AbstractAbstract 2618Rho family small GTPases are critical regulators of multiple functions of blood cells, including adhesion, migration, proliferation, survival and gene expression. Elucidation of their roles in hematopoiesis could be fundamental for understanding the mechanisms of various blood diseases and improving therapeutic outcomes for hematopoietic abnormalities. The Rho GTPases Rac1, Rac2 and Cdc42 have been found essential in maintaining hematopoietic stem cell (HSC) niche residency and regulating myelopoiesis and lymphopoiesis in previous mouse gene targeting studies. Using a dominant-negative mutant overexpression approach, an earlier study showed that suppression of RhoA activity enhanced HSC proliferation and engraftment potential; however, the bone fide role of RhoA in blood development remains unknown given limitations of such a mutant overexpression approach in specificity, dosage effect, and physiological relevance. Here, we stringently define the function of RhoA in HSC maintenance and hematopoiesis using an interferon inducible RhoA conditional knockout mouse model (Mx-cre+; RhoAloxp/loxp). Systematic deletion of RhoA caused lethality of the mice 7 days post polyIC induction due to hematopoietic failure that was accompanied by drastically decreased bone marrow (BM) cellularity (to ∼1/3 of wild type controls), a loss of splenocytes, and a significant reduction of cell counts of most cell lineages in peripheral blood, suggesting RhoA is required for multiple blood cell lineage differentiation and production. Syngenic transplant experiments yielded similar results, demonstrating that these effects are intrinsic to the hematopoietic compartment. The observed cytopenia resulting from RhoA loss was associated with the exhaustion of BM phenotypic HSPC (Lin−Sca1+c-kit+, LSK) and the hierarchical progenitor cells (Lin−c-kit+, LK) in number and frequency, a complete loss of colony forming activities, and a total engraftment failure. In addition, BrdU chase labeling and Annexin V/7-AAD staining revealed that RhoA deletion caused a transient increase of proliferation (1.7 fold increase in S phase) and reduction of survival (16.8 fold reduction in Annexin V− 7-AAD−) of remaining LSK in the BM. These results indicate that RhoA plays an indispensible, cell autonomous role in HSPC maintenance and hematopoiesis. In a competitive transplantation model where Mx-cre+;RhoAloxp/loxp or Mx-cre−;RhoAloxp/loxp CD45.2+ BM cells and WT CD45.1+ competitor cells were transplanted at 1:1 ratio into syngenic CD45.1+ recipients prior to polyIC induction, RhoA deletion also caused a complete extinction of donor derived (CD45.2+) Mac1+Gr1+ myeloid cells, B220+ B cells, as well as CD3+ T cells, in the peripheral. Interestingly, distinct from these more differentiated lineages, BM CD45.2+ LSK population was only marginally affected (88.9 % of pre-polyIC induction level) while the CD45.2+ LK cells and later hierarchical lineages were rapidly eliminated after RhoA deletion in this competitive transplant model. This was associated with increased apoptosis in CD45.2+ RhoA−/− LK, but not LSK, cells, suggesting a specific requirement of RhoA in the myeloid progenitor cell survival. Further, the CD45.2+ RhoA−/− LSK and LSKCD150+ cell populations, not differentiated donor-derived progenitors, from the primary competitive transplant BM, were able to be maintained in secondary transplant recipients 5 months post-transplantation, indicating that RhoA serves as a key regulator at an early progenitor differentiation step. Interestingly, RhoA deletion did not affect lin− cell p-MLC and p-cofilin contents and in vitro expansion in response to SCF stimulation, suggesting that RhoA is not required for actomyosin signaling nor SCF induced proliferation. Taken together, our results implicate RhoA as a unique and essential regulator of multipotent progenitor differentiation and survival that controls multi-lineage hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Chronic RPS19 Deficiency Leads to Bone Marrow Failure In a Mouse Model for Diamond-Blackfan Anemia

AbstractAbstract 193Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical malformations and predisposition to cancer. Of the many different DBA disease genes known, all encode for ribosomal proteins, suggesting that DBA is a disorder relating to ribosomal biogenesis or function. Among these genes, ribosomal protein S19 (RPS19) is the most frequently mutated (25 % of the patients). The generation of animal models for DBA is pivotal in order to understand the disease mechanisms and to evaluate novel therapies. We have generated two mouse models for RPS19-deficient DBA by taking advantage of RNA interference (Jaako et al, 2009 ASH meeting abstract). These models contain RPS19-targeting shRNAs expressed by a doxycycline-responsive promoter downstream of the Collagen A1 locus allowing an inducible and dose-dependent regulation of shRNA. As we have previously reported, the induction of RPS19 deficiency results in a reduction in the number of erythrocytes, platelets and white blood cells, and flow cytometric analysis of bone marrow after a short-term induction reveals increased frequencies of hematopoietic stem and progenitor cells reflecting the onset of stress hematopoiesis. In the current study we have analyzed the long-term effect of RPS19 deficiency in bone marrow. In contrast to a short-term induction, flow cytometric analysis of bone marrow after 51 days revealed decreased frequencies of hematopoietic stem and progenitor cells that correlate with a severe peripheral blood phenotype. In addition, we observed a 3–6 fold increase in apoptosis in RPS19-deficient bone marrow compared to controls based on TUNEL assay. Furthermore, transplantation of whole bone marrow cells from transgenic donors into wild type lethally irradiated recipients confirms that the observed phenotype is autonomous to the blood system. To study whether long-term RPS19 deficiency functionally impairs hematopoietic stem cells, we pre-induced mice for 30 days followed by 15 days without doxycycline to restore the RPS19 expression. Mice were sacrificed and total bone marrow cells were transplanted together with wild-type competitor cells (1:1) into wild type lethally irradiated recipients without doxycycline. This experimental setting allows us to assess the functionality of pre-induced hematopoietic stem cells in absence of ribosomal stress. Flow cytometric analysis of peripheral blood one month after transplantation clearly demonstrates decreased reconstitution from pre-induced donors compared to the wild-type competitor. While this time point reflects mainly the function of transplanted progenitors, long-term analysis of hematopoietic stem cell function in these recipients is ongoing. To study the molecular mechanisms underlying the hematopoietic defect we performed comparative microarray analysis. We chose to analyze preCFU-E/CFU-E erythroid progenitors since we have previously located the erythroid defect at the CFU-E – proerythroblast transition based on flow cytometry and clonogenic proliferation cultures of prospectively isolated erythroid progenitors. Microarray analysis of preCFU-E/CFU-E progenitors reveals deregulation of several genetic pathways, including a robust upregulation of p53 pathway genes, and these targets have been confirmed by real-time PCR. Furthermore, many of p53 target genes are also upregulated in the Lineage− Sca-1+ c-Kit+ (LSK) population that contains immature hematopoietic progenitors and stem cells suggesting that the activation of p53 is not restricted to the erythroid lineage. To ask whether increased activity of p53 can solely explain the hematopoietic phenotype, we have crossed our mouse model into a p53-null background. In summary, our data suggest that RPS19-deficient mice fail to uphold stress hematopoiesis for extended periods of time, with chronic RPS19 deficiency causing bone marrow failure. Disclosures:No relevant conflicts of interest to declare.

Histone Demethylases as Modulators of Hematopoietic Cell Fate.

Abstract 393Epigenetic modifications influence cell fate decisions, such as stem cell self-renewal and differentiation, both in normal and leukemic stem cells (LSC). On this premise, an expression profile study of the emerging class of histone demethylating enzymes (HDE) in stem cell enriched populations was designed. Transcripts of all these enzymes (n=27) were detected in isolated HSC populations (frequency 1:2) from fetal liver and bone marrow. Similar results were obtained in HoxA9-Meis1 induced leukemia, with an inverse correlation between LSC content and the HDE Jarid1b expression level. As a functional follow-up, systematic knockdown of known or presumed HDE was undertaken in primary HSC using a RNA interference (RNAi) based approach. Five sh-RNA were designed and generated against every HDE and cloned into a retroviral vector, containing a GFP reporter gene. The screen was set as a loss-of-function assay, where sorted CD150+CD48-Lin- cells were co-cultured on retroviral producers for 5 days, in a 96-well setting, and a fraction of each well was then transplanted into 2 mice (day 0), along with competitor cells. The remaining cell fraction served to asses gene transfer by GFP epifluorescence measurements, and RNA isolated from sorted GFP+ cells was used to evaluate gene knockdown levels by Q-PCR analysis. Blood reconstitution was evaluated at regular intervals, tracking the contribution of the donor CD45.1+ transduced (GFP+) cells to recipient hematopoiesis over time. As baseline references, sh-RNA to luciferase (no effect) and the histone acetyl transferase myst3 (stem cell loss) were used, as well as hoxb4 over-expression (stem cell expansion). In the primary screen, RNAi against jarid1a, lsd1 and an arginine demethylating enzyme, resulted in a progressive decline in blood reconstitution levels from the transduced cells, ranging from a 50% to 80% reduction of the GFP content within the graft. On the other hand, knockdown of Jarid1b conferred an in vivo competitive advantage to the transduced cells. As a validation procedure, a 7 day culture step was introduced to the assay, hence increasing the signal to noise ratio, to better detect an increase in HSC activity, as recently reported. After a week in vitro (day 7), 1/8 equivalents of single well cultures were transplanted into 3 independent mice, and blood reconstitution levels serially assessed. Cells transduced with sh-RNA against jarid1b (3 constructs out of 6) contributed more significantly to host hematopoiesis than sh-RNA luciferase transduced cells (58±16% vs 26±3% GFP), or hoxb4 over-expressing cells (37±2% GFP), at comparable gene transfer rates, at the 14 week time point and beyond (3 independent experiments). In long-term recipients, differentiation potential of these cells was preserved, as evidenced by CD4+CD8+ thymic cells, B220+ splenic cells and CD11b+ bone marrow cells in the GFP positive contingent. Clonality studies on DNA isolated from these sorted populations are ongoing. There were no cases of leukemic transformation in all of the transplant recipients (n>30).In conclusion, this RNAi based study performed on primary hematopoietic cells links histone demethylases to modulations in cell fate. An opposite role seems to emerge among the H3K4me3/me2 HDE JARID1a and JARID1b regarding HSC activity, yet these proteins are highly homologous. Sequence alignments pinpoint to differences within the C-terminal portion, containing a PHD domain, and interestingly, dysregulation of this chromatin-binding PHD finger (NUP98-JARID1a) has been recently shown to play a direct role in leukemogenesis Disclosures:No relevant conflicts of interest to declare.

Competition Triggers Plasmid-Mediated Enhancement of Substrate Utilisation in Pseudomonas putida

Competition between species plays a central role in the activity and structure of communities. Stable co-existence of diverse organisms in communities is thought to be fostered by individual tradeoffs and optimization of competitive strategies along resource gradients. Outside the laboratory, microbes exist as multispecies consortia, continuously interacting with one another and the environment. Survival and proliferation of a particular species is governed by its competitive fitness. Therefore, bacteria must be able to continuously sense their immediate environs for presence of competitors and prevailing conditions. Here we present results of our investigations on a novel competition sensing mechanism in the rhizosphere-inhabiting Pseudomonas putida KT2440, harbouring gfpmut3b-modified KanR TOL plasmid. We monitored benzyl alcohol (BA) degradation rate, along with GFP expression profiling in mono species and dual species cultures. Interestingly, enhanced plasmid expression (monitored using GFP expression) and consequent BA degradation were observed in dual species consortia, irrespective of whether the competitor was a BA degrader (Pseudomonas aeruginosa) or a non-degrader (E. coli). Attempts at elucidation of the mechanistic aspects of induction indicated the role of physical interaction, but not of any diffusible compounds emanating from the competitors. This contention is supported by the observation that greater induction took place in presence of increasing number of competitors. Inert microspheres mimicking competitor cell size and concentration did not elicit any significant induction, further suggesting the role of physical cell-cell interaction. Furthermore, it was also established that cell wall compromised competitor had minimal induction capability. We conclude that P. putida harbouring pWW0 experience a competitive stress when grown as dual-species consortium, irrespective of the counterpart being BA degrader or not. The immediate effect of this stress is a marked increase in expression of TOL, leading to rapid utilization of the available carbon source and massive increase in its population density. The plausible mechanisms behind the phenomenon are hypothesised and practical implications are indicated and discussed.

DNMT3b's Role in Hematopoietic Stem Cell Self-Renewal.

DNA methylation is an epigenetic means of gene regulation and is carried out by a family of methyltransferases of which DNMT1 acts to maintain methylation marks following DNA replication and DNMT3a and DNMT3b methylate DNA de novo. DNMT3b has been shown to be essential for mammalian development and necessary for differentiation of germline and neural progenitor cells. Mutations of DNMT3b in humans lead to a rare autosomal recessive disorder characterized by immunodeficiency, centromeric instability, and facial abnormalities. We have shown by real-time, RT-PCR that DNMT3b mRNA is uniquely over-expressed by approximately 30-fold in immunophenotypically-defined longterm repopulating hematopoietic stem cells (HSCs) that are CD34−lineage−c-kit+Sca-1+ as compared to progenitor and differentiated cell types within the bone marrow and with respect to the other members of the DNMT family, namely DNMT1 and DNMT3a. To determine DNMT3b's function in HSCs competitive bone marrow transplantation was undertaken. Isolated lineage− enriched bone marrow cells were transduced with a retroviral backbone based on the Murine Stem Cell Virus (MSCV) carrying either GFP and a short, hairpin RNA (shRNA) targeting DNMT3b or GFP alone. Following transduction 1×105 GFP+ cells along with 1×105 competitor cells were transplanted into 9.5 Gray irradiated congenic recipients. Two months following transplantation mice receiving bone marrow cells transduced with DNMT3b shRNA showed a significantly lower engraftment of donor cells as a percentage of total competitor cell engraftment in the peripheral blood as compared to those receiving cells transduced with GFP alone (24.8 vs 3.7, p<0.05) which persisted at 3 months (22.8 vs 1.5, p<0.05). Similarly, within the donor derviced cells in the peripheral blood there was a lower percentage of myeloid (CD11b+) cells at 2 and 3 months in the recipients of DNMT3b shRNA transduced cells as compared to controls. However there was no observed difference in the percentage of peripheral B (CD45R+) or T (CD3+) cells within the donor-derived cells. To determine the mechanism behind the observed engraftment defect with DNMT3b knockdown we cultured GFP+ transduced bone marrow cells in vitro with minimal cytokine support. As a control for our targeting methodology we also transduced bone marrow cells from mice harboring two floxed DNMT3b alleles with a MSCV carrying Cre recombinase and GFP. While lineage− bone marrow cells transduced with GFP alone increased 10-fold in number over two weeks of culture, cells in which DNMT3b was down regulated by shRNA or Cre-mediated recombination only doubled. Culture of lineage− bone marrow cells in methylcellulose medium by the colony-forming cell (CFC) assay revealed increases in the granulocytic and total number of colonies with DNMT3b knockdown or Cre-mediated recombination of DNMT3b similar to the increased myeloid engraftment of DNMT3b shRNA transduced cells observed 1 month following competitive bone marrow transplantation. However when 5,000 of these cells from the first CFC assay were sub-cultured there was a significant loss of colony forming ability within all lineages when DNMT3b was targeted by shRNA or Cre-mediated recombination. Taken together with the decreased engraftment of DNMT3b shRNA cells following competitive bone marrow transplantation, the observed limited proliferation in liquid culture and loss of colony forming ability during serial CFC assays is suggestive of a self-renewal defect of HSCs in the absence of DNMT3b, that was previously only reported in the absence of both DNMT3a and DNMT3b. Further elucidation of this proposed self-renewal defect is being undertaken and results of ongoing studies including long-term culture initiating cell (LTC-IC) assays and identification of genomic sites of DNA methylation within different hematopoietic subsets will also be presented.