130. Design and Preclinical Characterization of SER-155, an Investigational Cultivated Microbiome Therapeutic to Restore Colonization Resistance and Prevent Infection in Patients Undergoing Hematopoietic Stem Cell Transplantation

Abstract Background During allogeneic hematopoietic stem cell transplant (HSCT), the diversity and stability of the GI microbiome is disrupted, increasing the risk of domination by pathogens associated with bacteremia, aGvHD, and mortality. SER-155 is an investigational, oral microbiome therapeutic composed of cultivated spores and vegetative bacterial strains rationally designed to reduce the risk of bacteremia and aGvHD in HSCT recipients by decolonizing potential pathogens and restoring GI colonization resistance. SER-155 was evaluated in vitro for key pharmacological properties associated with colonization resistance, and in vivo to assess its ability to restore colonization resistance by reducing Enterococcus and Enterobacteriaceae carriage. Methods The design of SER-155 leveraged genomic data from interventional and observational human datasets to include taxa associated with reduced risk of infection and aGvHD in HSCT. Strains of interest were phenotyped, and over 50 candidate consortia containing different combinations of over 150 species were designed and tested in vitro and in vivo. In vivo, candidate compositions were evaluated in mouse models of vancomycin-resistant Enterococcus faecium (VRE) and carbapenem-resistant Klebsiella pneumoniae (CRE) colonization. Results Oral administration of SER-155 led to a 2-3 Log10 reduction in VRE and CRE titers compared to untreated mice (Figure 1). In vitro, the carbon source utilization profile of VRE, CRE, and SER-155 strains were assessed using a panel of 85 carbon sources. All 56 carbon sources used by CRE or VRE for anaerobic growth were also utilized by SER-155 strains, supporting a model in which nutrient competition may contribute to reducing CRE and VRE carriage and restoring colonization resistance. Figure 1. SER-155 Efficacy in Mouse Models of VRE and CRE Colonization. The titers of VRE or CRE were quantified in fecal pellets by plating on selective agar at the indicated time-points. The median A) VRE and B) CRE CFU per gram of feces was calculated for each group and plotted on the line graph (n=6-10 per group). L.O.D., limit of detection. Data were analyzed using the Mann-Whitney t-test and significance was determined as a p-value of p< 0.05*, p<0.01**, p<0.001***, p<0.0001****. Conclusion SER-155 is an investigational cultivated microbiome therapeutic intended to reduce the risk of infection by engrafting human-commensal bacterial strains in adults undergoing allogeneic HSCT. Preclinical assessments in vitro and in vivo support the ability of SER-155 to reduce VRE and CRE carriage and restore colonization resistance in the gut. A Phase 1b study evaluating SER-155 in allogeneic HSCT patients is being planned. Disclosures Elizabeth Halvorsen, PhD, Seres Therapeutics (Employee, Shareholder) Marin Vulic, PhD, Seres Therapeutics (Employee) Edward J. O’Brien, PhD, Seres Therapeutics (Employee, Shareholder) Jessica Byrant, PhD, Seres Therapeutics (Employee, Shareholder) Mary-Jane Lombardo, PhD, Seres Therapeutics (Employee, Shareholder) Christopher Ford, PhD, Seres Therapeutics (Employee, Shareholder) Matt Henn, PhD, Seres Therapeutics (Employee, Shareholder)

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Combining Immunocytokine and Ex Vivo Activated NK Cells as a Platform for Enhancing Graft-Versus-Tumor Effects Against GD2+ Murine Neuroblastoma

Frontiers in Immunology ◽

10.3389/fimmu.2021.668307 ◽

2021 ◽

Vol 12 ◽

Author(s):

Paul D. Bates ◽

Alexander L. Rakhmilevich ◽

Monica M. Cho ◽

Myriam N. Bouchlaka ◽

Seema L. Rao ◽

...

Keyword(s):

Tumor Growth ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Allogeneic Hsct ◽

Tnf Α

Management for high-risk neuroblastoma (NBL) has included autologous hematopoietic stem cell transplant (HSCT) and anti-GD2 immunotherapy, but survival remains around 50%. The aim of this study was to determine if allogeneic HSCT could serve as a platform for inducing a graft-versus-tumor (GVT) effect against NBL with combination immunocytokine and NK cells in a murine model. Lethally irradiated C57BL/6 (B6) x A/J recipients were transplanted with B6 bone marrow on Day +0. On day +10, allogeneic HSCT recipients were challenged with NXS2, a GD2+ NBL. On days +14-16, mice were treated with the anti-GD2 immunocytokine hu14.18-IL2. In select groups, hu14.18-IL2 was combined with infusions of B6 NK cells activated with IL-15/IL-15Rα and CD137L ex vivo. Allogeneic HSCT alone was insufficient to control NXS2 tumor growth, but the addition of hu14.18-IL2 controlled tumor growth and improved survival. Adoptive transfer of ex vivo CD137L/IL-15/IL-15Rα activated NK cells with or without hu14.18-IL2 exacerbated lethality. CD137L/IL-15/IL-15Rα activated NK cells showed enhanced cytotoxicity and produced high levels of TNF-α in vitro, but induced cytokine release syndrome (CRS) in vivo. Infusing Perforin-/- CD137L/IL-15/IL-15Rα activated NK cells had no impact on GVT, whereas TNF-α-/- CD137L/IL-15/IL-15Rα activated NK cells improved GVT by decreasing peripheral effector cell subsets while preserving tumor-infiltrating lymphocytes. Depletion of Ly49H+ NK cells also improved GVT. Using allogeneic HSCT for NBL is a viable platform for immunocytokines and ex vivo activated NK cell infusions, but must be balanced with induction of CRS. Regulation of TNFα or activating NK subsets may be needed to improve GVT effects.

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Isolation and characterization of human CD34−Lin− and CD34+Lin− hematopoietic stem cells using cell surface markers AC133 and CD7

Blood ◽

10.1182/blood.v95.9.2813.009k20_2813_2820 ◽

2000 ◽

Vol 95 (9) ◽

pp. 2813-2820 ◽

Cited By ~ 215

Author(s):

Lisa Gallacher ◽

Barbara Murdoch ◽

Dongmei M. Wu ◽

Francis N. Karanu ◽

Mike Keeney ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Surface Markers ◽

Cell Surface Markers ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34−Lin−). A major barrier in the further characterization of human CD34− stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34−CD38−Lin− and CD34+CD38−Lin− cells derived from human cord blood. Although the majority of CD34−CD38−Lin− cells lack AC133 and express CD7, an extremely rare population of AC133+CD7− cells was identified at a frequency of 0.2%. Surprisingly, these AC133+CD7− cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34+ stem cells, and they were the only subset among the CD34−CD38−Lin− population capable of giving rise to CD34+ cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo–cultured AC133+CD7− cells isolated from the CD34−CD38−Lin− population, whereas 400-fold greater numbers of the AC133−CD7− subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34− cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34+ cells.

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Growth Suppressors IFI-204 and IFI-205 Inhibit Primitive Hematopoietic Cell Proliferation In Vitro and in Vivo.

Blood ◽

10.1182/blood.v104.11.1691.1691 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1691-1691

Author(s):

Kimberly Klarmann ◽

Daniel Gough ◽

Benyam Asefa ◽

Chris Clarke ◽

Katie Renn ◽

...

Keyword(s):

Stem Cell ◽

Cell Growth ◽

Cell Line ◽

Constitutive Expression ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Inhibit Cell Growth ◽

Stem And Progenitor Cells

Abstract Members of the interferon inducible-200 (IFI-200) family of proteins inhibit cell growth and may be important mediators of differentiation. We examined IFI-204 and IFI-205 mRNA expression in purified populations of hematopoietic stem and progenitor cells at different stages of maturation using quantitative RT-PCR and found that their expression markedly increased during myeloid maturation. To evaluate the effect of IFI-205 and IFI-204 on hematopoietic stem cell (HSC) growth, we transduced these genes into mouse bone marrow cells (BMC) using retroviral vectors. The presence IFI-204 or IFI-205 resulted in a decrease in cell growth in response to hematopoietic growth factors. Further analysis revealed the infected cells were 98% c-Kit+ Sca-1+, indicative of the stem cell surface phenotype, suggesting they may be blocked in a primitive stage of maturation. When transplanted, BMC transduced with IFI-204 or IFI-205 failed to engraft lymphoid, myeloid, or erythroid lineages in both short and long term reconstitution assays, suggesting that constitutive expression of IFI-204 and IFI-205 inhibited HSC development both in vitro and in vivo. However, based on the quantitative RT-PCR results, which show that IFI-205 increased during myeloid differentiation, we know its endogenous, regulated expression must permit the cells to mature. Therefore, to study of the effects of these genes on differentiation we transduced the mulitpotential EML (erythroid, myeloid, lymphoid) cell line with IFI-204 and IFI-205 to circumvent severe growth inhibition caused by expression of IFI-204 and Ifi-205 in normal cells. Single cell analysis of EMLs transduced with IFI-205 demonstrated that expression of IFI-205 in this cell line did not significantly inhibit cell growth. We have isolated EML clones from the transduced cells and verified IFI-205 expression. In addition, we generated transgenic mice that express IFI-205 under control of the Vav and MRP8 promoters, and we identified transgenic lines that express IFI-205 at higher levels compared to wild type controls. Analysis of hematopoiesis in these animals is currently in progress. Altogether, our data demonstrate 3 findings: 1) IFI-204 and IFI-205 expression increases during myeloid development based on quantitative RT-PCR analysis, 2) constitutive expression of IFI-204 and -205 results in potent inhibition of growth and maturation of normal hematopoietic stem and progenitor cells in vivo and in vitro and 3) these genes did not significantly inhibit the proliferation of the EML cell line, which provides us with a means to study the mechanism by which these molecules regulate myeloid maturation. Finally, the considerable inhibitory effects of these family members on normal hematopoietic cell growth suggest their potential as therapeutic modalities for treatment of leukemia.

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Involvement of the Integrin Alpha 6 Receptor in the Homing and Engraftment of Hematopoietic Stem and Progenitor Cells to Bone Marrow.

Blood ◽

10.1182/blood.v104.11.1293.1293 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1293-1293

Author(s):

Hong Qian ◽

Sten Eirik W. Jacobsen ◽

Marja Ekblom

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Progenitor Cell ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Functional Blocking

Abstract Within the bone marrow environment, adhesive interactions between stromal cells and extracellular matrix molecules are required for stem and progenitor cell survival, proliferation and differentiation as well as their transmigration between bone marrow (BM) and the circulation. This regulation is mediated by cell surface adhesion receptors. In experimental mouse stem cell transplantation models, several classes of cell adhesion receptors have been shown to be involved in the homing and engraftment of stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Using FACS analysis, the integrin a6 chain was now found to be ubiquitously (>95%) expressed in mouse hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, lin−Sca-1+c-Kit+CD34+) both in adult bone marrow and in fetal liver. In vitro, about 70% of mouse BM lin−Sca-1+c-Kit+ cells adhered to laminin-10/11 and 40% adhered to laminin-8. This adhesion was mediated by integrin a6b1 receptor, as shown by functional blocking monoclonal antibodies. We also used a functional blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of hematopoietic stem and progenitor cells. We found that the integrin a6 antibody inhibited the homing of bone marrow progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C was reduced by about 40% as compared to cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells (LTR), antibody treated bone marrow cells were first injected intravenously into lethally irradiated primary recipients. After three hours, bone marrow cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis 16 weeks after transplantation revealed an 80% reduction of stem cell activity of integrin a6 antibody treated cells as compared to cells treated with control antibody. These results suggest that integrin a6 plays an important role for hematopoietic stem and progenitor cell homing in vivo.

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Integrin alpha 6 Is Essential for Fetal Hematopoietic Progenitor, but Not Fetal Stem Cell Homing to Bone Marrow.

Blood ◽

10.1182/blood.v106.11.1387.1387 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1387-1387

Author(s):

Hong Qian ◽

Sten Eirik W. Jacobsen ◽

Marja Ekblom

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Progenitor Cell ◽

Fetal Liver ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Integrin Alpha 6

Abstract Homing of transplanted hematopoietic stem cells (HSC) in the bone marrow (BM) is a prerequisite for establishment of hematopoiesis following transplantation. However, although multiple adhesive interactions of HSCs with BM microenviroment are thought to critically influence their homing and subsequently their engraftment, the molecular pathways that control the homing of transplanted HSCs, in particular, of fetal HSCs are still not well understood. In experimental mouse stem cell transplantation models, several integrins have been shown to be involved in the homing and engraftment of both adult and fetal stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Furthermore, integrin a6 is required for adult mouse HSC homing to BM in vivo (Qian et al., Abstract American Society of Hematology, Blood 2004 ). We have now found that the integrin a6 chain like in adult HSC is ubiquitously (>99%) expressed also in fetal liver hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, LSK ). In vitro, fetal liver LSK cells adhere to laminin-10/11 and laminin-8 in an integrin a6b1 receptor-dependent manner, as shown by function blocking monoclonal antibodies. We have now used a function blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of fetal liver hematopoietic stem and progenitor cells to BM. The integrin a6 antibody inhibited homing of fetal liver progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C in BM was reduced by about 40% as compared to the cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells, BM cells were first incubated with anti-integrin alpha 6 or anti-integrin alpha 4 or control antibody, and then injected intravenously into lethally irradiated primary recipients. After three hours, BM cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis up to 16 weeks after transplantation showed that no reduction of stem cell reconstitution from integrin a6 antibody treated cells as compared to cells treated with control antibody. In accordance with this, fetal liver HSC from integrin a6 gene deleted embryos did not show any impairment of homing and engraftment in BM as compared to normal littermates. These results suggest that integrin a6 plays an important developmentally regulated role for homing of distinct hematopoietic stem and progenitor cell populations in vivo.

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The Activating KIR Genes 2DS1 and 2DS2 Regulate NK Alloreactivity In Vitro.

Blood ◽

10.1182/blood.v108.11.927.927 ◽

2006 ◽

Vol 108 (11) ◽

pp. 927-927

Author(s):

Joseph H. Chewning ◽

Charlotte N. Gudme ◽

Bo Dupont

Keyword(s):

Stem Cell ◽

Nk Cells ◽

Hematopoietic Stem Cell ◽

Hla Class I ◽

Class I ◽

Target Cells ◽

Ligand Specificity ◽

Hematopoietic Stem ◽

Allogeneic Hsct

Abstract The role of Natural Killer (NK) cells in host protection against viral infection and malignant transformation has been well described. NK cells may also lead to a reduction in post-transplant relapse and improved survival in hematopoietic stem cell transplantation (HSCT) for acute myelogenous leukemia (AML). It has been hypothesized that the genotype for the inhibiting killer immunoglobulin-like receptor (KIR) of the hematopoietic stem cell donor in combination with the HLA class I genotype of the recipient could control NK alloreactivity leading to a reduction in post-transplant complications. The KIR gene family encodes however both activating and inhibiting receptors. Here we test the hypothesis that activating KIRs with ligand specificity for HLA class I may contribute to alloreactivity, and potentially could be a genetic factor of significance in allogeneic HSCT. We tested this hypothesis in studies of two pairs of inhibiting and activating KIRs with highly homologous codon sequences in the extracellular domain, namely KIR2DL2/3-KIR2DS2 and KIR2DL1-KIR2DS1. Both the inhibitory 2DL1 and activating 2DS1 have ligand specificity for HLA-Cw group 2, and 2DL2 and 2DL3, have ligand specificity for HLA-Cw group 1, while the activating 2DS2 does not bind in vitro to C1 group. Using an EBV-transformed B-lymphoblastoid cell line (EBV-BLCL) target cell panel homozygous for HLA Class I alleles, we found that NK cells from donors with KIR haplotypes lacking KIR2DS1 or 2DS2 were not cytotoxic to allogeneic EBV-BLCL, independent of the target HLA class I genotype. Polyclonal NK cells obtained from KIR2DS1 positive and C1 group positive donors mediated NK cytotoxicity against C2 positive targets. In contrast, NK cells from KIR2DS1 positive, C2 group homozygous donors displayed minimal cytotoxicity against the C2 group targets (p<0.01). NK clones generated from 2DS1 positive, C2-group negative individuals were cytotoxic to C2-group target cells, while such NK clones could not be obtained from individuals positive for 2DS1 and cognate ligands. Similar findings were made for the relationship between 2DS2, 2DL2/3 and cognate ligand C1 group. Both polyclonal IL-2 propagated NK cells and NK clones from individuals positive for 2DS2 and homozygous for C2 group displayed specific cytotoxicity against C1 positive target cells. The cytotoxicity of 2DS2 positive, C1 group positive NK cells against the C1 positive BLCLs was minimal (p<0.01). These studies demonstrate that 2DS1 and 2DS2 are activating receptors that can induce an alloantigen response. We also present a model for combinations of KIR and HLA genotypes in which the allogeneic function of KIR2DS1 and 2DS2 is consistently seen in donor NK cells. Activating KIR may therefore play a role in allogeneic HSCT, and could contribute to the balance between activating and inhibiting signals for NK cells in HLA-Cw incompatible donor-recipient combinations. Activating KIR interactions with cognate ligand could potentially also play a role in the innate immune response. In the normal host, the increased affinity of the inhibiting KIR isoforms for HLA class I may prevent auto-reactivity, while the activating isoforms may only function in an HLA restricted pattern in context of specific pathogens or transformed cells. It is possible that the low affinity activating KIR may require additional co-stimulating signals that are up-regulated during cellular stress.

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The Role of Nucleophosmin In Hematopoietic Stem Cells and the Pathogenesis of Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v116.21.95.95 ◽

2010 ◽

Vol 116 (21) ◽

pp. 95-95 ◽

Cited By ~ 1

Author(s):

Keisuke Ito ◽

Paolo Sportoletti ◽

John G Clohessy ◽

Grisendi Silvia ◽

Pier Paolo Pandolfi

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Myelodysplastic Syndrome ◽

Cell Biology ◽

De Novo ◽

Stem Cell Biology ◽

Hematopoietic Stem

Abstract Abstract 95 Myelodysplastic syndrome (MDS) is an incurable stem cell disorder characterized by ineffective hematopoiesis and an increased risk of leukemia transformation. Nucleophosmin (NPM) is directly implicated in primitive hematopoiesis, the pathogenesis of hematopoietic malignancies and more recently of MDS. However, little is known regarding the molecular role and function of NPM in MDS pathogenesis and in stem cell biology. Here we present data demonstrating that NPM plays a critical role in the maintenance of hematopoietic stem cells (HSCs) and the transformation of MDS into leukemia. NPM is located on chromosome 5q and is frequently lost in therapy-related and de novo MDS. We have previously shown that Npm1 acts as a haploinsufficient tumor suppressor in the hematopoietic compartment and Npm1+/− mice develop a hematologic syndrome with features of human MDS, including increased susceptibility to leukemogenesis. As HSCs have been demonstrated to be the target of the primary neoplastic event in MDS, a functional analysis of the HSC compartment is essential to understand the molecular mechanisms in MDS pathogenesis. However, the role of NPM in adult hematopoiesis remains largely unknown as Npm1-deficiency leads to embryonic lethality. To investigate NPM function in adult hematopoiesis, we have generated conditional knockout mice of Npm1, using the Cre-loxP system. Analysis of Npm1 conditional mutants crossed with Mx1-Cre transgenic mice reveals that Npm1 plays a crucial role in adult hematopoiesis and ablation of Npm1 in adult HSCs leads to aberrant cycling and followed by apoptosis. Analysis of cell cycle status revealed that HSCs are impaired in their ability to maintain quiescence after Npm1-deletion and are rapidly depleted in vivo as well as in vitro. Competitive reconstitution assay revealed that Npm1 acts cell-autonomously to maintain HSCs. Conditional inactivation of Npm1 leads to an MDS phenotype including a profoundly impaired ability to differentiate into cells of the erythroid lineage, megakaryocyte dyspoiesis and centrosome amplification. Furthermore, Npm1 loss evokes a p53-dependent response and Npm1-deleted HSCs undergo apoptosis in vivo and in vitro. Strikingly, transfer of the Npm1 mutation into a p53-null background rescued the apoptosis of Npm1-ablated HSCs and resulted in accelerated transformation to an aggressive and lethal form of acute myeloid leukemia. Our findings highlight the crucial role of NPM in stem cell biology and identify a new mechanism by which MDS can progress to leukemia. This has important therapeutic implications for de novo MDS as well as therapy-related MDS, which is known to rapidly evolve to leukemia with frequent loss or mutation of TRP53. Disclosures: No relevant conflicts of interest to declare.

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Novel In Vivo Evidence That Not Only Androgens But Also Pituitary Gonadotropins and Prolactin Directly Stimulate Murine Bone Marrow Stem Cells – Implications For Potential Treatment Strategies In Aplastic Anemias

Blood ◽

10.1182/blood.v122.21.2476.2476 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2476-2476

Author(s):

Kasia Mierzejewska ◽

Ewa Suszynska ◽

Sylwia Borkowska ◽

Malwina Suszynska ◽

Maja Maj ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Sex Hormones ◽

Peripheral Blood ◽

Hematopoietic Stem ◽

Clonogenic Potential

Abstract Background Hematopoietic stem/progenitor cells (HSPCs) are exposed in vivo to several growth factors, cytokines, chemokines, and bioactive lipids in bone marrow (BM) in addition to various sex hormones circulating in peripheral blood (PB). It is known that androgen hormones (e.g., danazol) is employed in the clinic to treat aplastic anemia patients. However, the exact mechanism of action of sex hormones secreted by the pituitary gland or gonads is not well understood. Therefore, we performed a complex series of experiments to address the influence of pregnant mare serum gonadotropin (PMSG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), androgen (danazol) and prolactin (PRL) on murine hematopoiesis. In particular, from a mechanistic view we were interested in whether this effect depends on stimulation of BM-residing stem cells or is mediated through the BM microenvironment. Materials and Methods To address this issue, normal 2-month-old C57Bl6 mice were exposed or not to daily injections of PMSG (10 IU/mice/10 days), LH (5 IU/mice/10 days), FSH (5 IU/mice/10 days), danazol (4 mg/kg/10 days) and PRL (1 mg/day/5days). Subsequently, we evaluated changes in the BM number of Sca-1+Lin–CD45– that are precursors of long term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011;25:1278–1285) and bone forming mesenchymal stem cells (Stem Cell & Dev. 2013;22:622-30) and Sca-1+Lin–CD45+ hematopoietic stem/progenitor cells (HSPC) cells by FACS, the number of clonogenic progenitors from all hematopoietic lineages, and changes in peripheral blood (PB) counts. In some of the experiments, mice were exposed to bromodeoxyuridine (BrdU) to evaluate whether sex hormones affect stem cell cycling. By employing RT-PCR, we also evaluated the expression of cell-surface and intracellular receptors for hormones in purified populations of murine BM stem cells. In parallel, we studied whether stimulation by sex hormones activates major signaling pathways (MAPKp42/44 and AKT) in HSPCs and evaluated the effect of sex hormones on the clonogenic potential of murine CFU-Mix, BFU-E, CFU-GM, and CFU-Meg in vitro. We also sublethally irradiated mice and studied whether administration of sex hormones accelerates recovery of peripheral blood parameters. Finally, we determined the influence of sex hormones on the motility of stem cells in direct chemotaxis assays as well as in direct in vivo stem cell mobilization studies. Results We found that 10-day administration of each of the sex hormones evaluated in this study directly stimulated expansion of HSPCs in BM, as measured by an increase in the number of these cells in BM (∼2–3x), and enhanced BrdU incorporation (the percentage of quiescent BrdU+Sca-1+Lin–CD45– cells increased from ∼2% to ∼15–35% and the percentage of BrdU+Sca-1+Lin–CD45+ cells increased from 24% to 43–58%, Figure 1). These increases paralleled an increase in the number of clonogenic progenitors in BM (∼2–3x). We also observed that murine Sca-1+Lin–CD45– and Sca-1+Lin–CD45+ cells express sex hormone receptors and respond by phosphorylation of MAPKp42/44 and AKT in response to exposure to PSMG, LH, FSH, danazol and PRL. We also observed that administration of sex hormones accelerated the recovery of PB cell counts in sublethally irradiated mice and slightly mobilized HSPCs into PB. Finally, in direct in vitro clonogenic experiments on purified murine SKL cells, we observed a stimulatory effect of sex hormones on clonogenic potential in the order: CFU-Mix > BFU-E > CFU-Meg > CFU-GM. Conclusions Our data indicate for the first time that not only danazol but also several pituitary-secreted sex hormones directly stimulate the expansion of stem cells in BM. This effect seems to be direct, as precursors of LT-HSCs and HSPCs express all the receptors for these hormones and respond to stimulation by phosphorylation of intracellular pathways involved in cell proliferation. These hormones also directly stimulated in vitro proliferation of purified HSPCs. In conclusion, our studies support the possibility that not only danazol but also several other upstream pituitary sex hormones could be employed to treat aplastic disorders and irradiation syndromes. Further dose- and time-optimizing mouse studies and studies with human cells are in progress in our laboratories. Disclosures: No relevant conflicts of interest to declare.

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Stem Cell Factor Sall4 Regulates Normal and Malignant Hematopoiesis In an Isoform-Specific Manner

Blood ◽

10.1182/blood.v122.21.3728.3728 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3728-3728

Author(s):

Samuel Milanovich ◽

Jeremy Allred ◽

Jonathan Peterson ◽

Cary Stelloh ◽

Sridhar Rao

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem ◽

Empty Vector ◽

Bmi1 Expression ◽

Colony Forming Units ◽

Colony Forming Capacity

Abstract Stem cells play key roles in early normal development (e.g. embryonic stem cells (ESCs)), maintenance of adult organs (e.g. hematopoietic stem cells (HSCs)) and in some cancers (e.g. leukemia stem cells). To what degree these different types of stem cells rely upon shared versus distinct transcriptional programs remains controversial. Sall4 is a zinc finger transcription factor that exists in two distinct splice isoforms, Sall4a (long) and Sall4b (short). Sall4 has been implicated in embryonic, hematopoietic and malignant stem cell transcriptional regulation. Additionally, Sall4 has been proposed as a potential means of ex-vivo hematopoietic stem cell expansion prior to transplantation. Sall4 isoform-specific differences have been described in ESCs, with Sall4b shown to be critical for maintaining ESC “stemness”. Here we investigate the role of Sall4 isoforms in pediatric acute myeloid leukemia (AML) and murine hematopoiesis to unravel shared versus unique transcriptional programs across different stem cell types. Quantitative real time PCR shows that Sall4b is the predominant Sall4 isoform in murine HSCs and lin-, Sca1+, cKit+ (LSK) cells. Sall4b expression decreases in early lineage-committed progenitors, while Sall4a expression is minimal to absent across murine HSCs and progenitors. Next, we evaluated seven pediatric AML samples and found highly variable Sall4 expression across AML cases. All samples had measurable Sall4a and Sall4b; in 3/7 cases Sall4a and Sall4b expression was similar to that of ESCs, in the other 4 cases Sall4 expression was minimal (<3% of ESCs). To study overexpression of Sall4, we used a murine stem cell retrovirus system to express Sall4a or Sall4b. Bone marrow was harvested from C57/BL6 mice and lineage-committed cells were removed by magnetic column separation. Lineage-negative bone marrow was infected with either empty vector, Sall4a or Sall4b. Transduced bone marrow was then cultured in methylcellulose media to assess colony forming capacity and proliferation in vitro or transplanted in syngeneic mice to assess engraftment and hematopoietic reconstitution in vivo. Sall4a or Sall4b overexpression caused diminished colony forming capacity and cellular proliferation in vitro compared to bone marrow transduced with empty vector (Figure 1). In bone marrow transplant assays, all mice (4/4) transplanted with Sall4b-transduced bone marrow following lethal irradiation succumbed to bone marrow failure within 10 days of transplant. Transplantation of Sall4b-transduced bone marrow into sublethally irradiated mice failed to contribute to hematopoiesis as measured by peripheral blood leukocyte GFP expression (encoded by the viral vector). Together, this data shows that Sall4b-transduced hematopoietic cells fail to engraft and reconstitute hematopoiesis in vivo. We postulated that this phenotype might be mediated through the interaction of Sall4 with Bmi1. Bmi1 is a member of the polycomb complex necessary for normal hematopoiesis, and is known to be bound by Sall4. In preliminary experiments, we have found that overexpression of Sall4 leads to decreased Bmi1 expression at 48 hours post-infection compared to bone marrow infected with empty vector.Figure 1Lin- bone marrow expressing Sall4a, Sall4b or empty vector was cultured in methylcellulose; plates were flushed and replated out to three generations. Colony forming units were assessed (A) and viable cells were counted (B) after 7-10 days in culture.Figure 1. Lin- bone marrow expressing Sall4a, Sall4b or empty vector was cultured in methylcellulose; plates were flushed and replated out to three generations. Colony forming units were assessed (A) and viable cells were counted (B) after 7-10 days in culture. In conclusion, our data shows that Sall4b is expressed in murine hematopoietic stem cells and progenitors, suggesting that Sall4b but not Sall4a influences a hematopoietic cell fate. Additionally, Sall4 expression is variable in AML specimens, implicating a potential pathogenic role in some leukemias, while others are Sall4-independent. Lastly, Sall4 overexpression is associated with decreased expression of the critical hematopoietic gene Bmi1. Together this data suggests that hematopoiesis is dependent upon appropriately regulated Sall4 expression with alterations leading to impaired proliferation and self-renewal. These effects on hematopoiesis appear to be mediated at least in part through a dose-dependent effect on Bmi1 expression. Future studies will evaluate other genes targeted by Sall4 in hematopoiesis and leukemia to define Sall4-dependent gene signatures in normal versus malignant hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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Myeloablative Chemo-Conditioning for First Hematopoietic STEM CELL Transplantation in Children with ACUTE Lymphoblastic Leukemia in First or Second Remission

Blood ◽

10.1182/blood.v124.21.546.546 ◽

2014 ◽

Vol 124 (21) ◽

pp. 546-546 ◽

Cited By ~ 1

Author(s):

Christina Peters ◽

Jean-Hugues Dalle ◽

Stelios Graphakos ◽

Petr Sedlacek ◽

Antonio Campos ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Lymphoblastic Leukemia ◽

Peripheral Blood Stem Cells ◽

Hematopoietic Stem ◽

Allogeneic Hsct ◽

Donor Type ◽

Blood Stem Cells

Abstract Christina Peters, Petr Sedlacek, Jean Hugues Dalle, Stelios Graphakos, Antonio Campos, Akif Yesilipek, Jacek Wachowiak, Arjan Lankester, Andrea Pession, Amir Ali Hamidieh, Marianne Ifversen, Jochen Büchner, Gergely Krivan, Franca Fagioli, Arnaud Dalissier; Myriam Labopin; Peter Bader on behalf of the EBMT Pediatric Diseases Working Party Most children with acute lymphoblastic leukemia (ALL) with indication for allogeneic hematopoietic stem cell transplantation (HSCT) receive myeloablative conditioning with a total body irradiation (TBI)-containing regimen. To investigate the outcomes of patients (pts) who did not undergo TBI, we performed a retrospective registry based study on children below 18 years who received a myeloablative chemo-conditioning for a first allogeneic HSCT from different donors between 2000 and 2012. In this analysis, only chemotherapeutic regimens with more than 30 applications were included. In total, 732 pts were included: 313 pts who received bone marrow (BM) or peripheral blood stem cells (PBSC) in 1st CR, 247 pts with BM/PBSC transplantation in CR2, 85 pts and 52 pts who received umbilical cord blood (CB) in 1st or 2nd CR, respectively. The most commonly applied myeloablative chemo-combinations were: Busulfan (Bu)/Cyclophosphamide (Cy) (n=202), Bu/Cy/Etoposide (VP) (n=189), Bu/Cy/Melphalan (Mel) (n=93), Bu/AraC/Mel (n=80), Bu/Fludarabine (Flu)/Thiotepa (Thio) (n=62), Bu/Cy/Thio (n=53, Bu/Cy/Thio (n=53), and Bu/Flu (n=53). 313 pts received either BM or PBSC in CR1 with a median follow up of 26 months (1-156) and we compared Bu/Cy/VP vs the other chemo-conditioning regimens. The Bu/Cy/VP cohort had a longer follow up (med 37 vs. 20 months, p=0.002), pts were younger (med 3,6 vs. 6,5 years, p=0.003) and the median year of transplant was earlier (med 2009 vs. 2010, p=0.03). Donor type, CMV match, gender match, stem cell were comparable. In univariate analysis, conditioning with Bu/Cy/VP was better than all other combinations: relapse incidence (RI) 21% vs 32% (p=0.05), leukemia-free survival (LFS) 72 vs 54% (p=0.004), overall survival (OS) 79 vs 68% (p=0.03) and chronic GVHD (cGVHD) 9% vs 19% (p=0.014). Engraftment and incidence and severity of acute GVHD were similar and non- relapse mortality (NRM) was 7% vs 13% (p=0.10). Other significant influencing factors were: interval between diagnosis and transplantation below or beyond 208 days (NRM 6% vs 16%, p=0.015), donor sibling vs other (RI 35% vs 23%, p=0.01, NRM 5% vs 16%, p=0.001) and in vivo T cell depletion (TCD) vs no TCD (RI 35% vs. 19%, p=0.003; NRM 20% vs 4%, p=0.0001). In the cox model, conditioning type (Bu/CY/VP vs other), age, year of transplantation, interval from diagnosis to transplant, donor type, stem cell source and in vivo TCD were evaluated. For LFS only BU/CY/VP was associated with better outcome (p=0.004, HR .52), RI was lower after Bu/Cy/VP (HR .54, p=0.02), NRM was higher in pts older than 4,6 years (p=0.02, HR 2,48) and after TCD HSCT (p=0.01, HR 9,13) and OS was best after Bu/Cy/VP (p=0.03, HR 0.57). We conclude that omission of TBI is feasible for children who undergo first allogeneic HSCT in first or second complete remission. The combination of busulfan, cyclophosphamide and etoposide resulted in better LFS and OS with less NRM and RI for children who received bone marrow or peripheral blood stem cells in CR1. These observations should be the basis for prospective trials in homogenous patient groups. Disclosures No relevant conflicts of interest to declare.

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