Loss of Notch Receptor-Ligand Engagement Leads to Increased Hematopoietic Stem and Progenitor Cell Egress and Mobilization

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 652-652
Author(s):  
Lan Zhou ◽  
Weihuan Wang ◽  
Grant Zimmerman ◽  
Jay Myers ◽  
Yuanshuai Huang ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Neutralizing Antibodies ◽  
Notch Receptor ◽  
Employment Equity ◽  
Receptor Ligand ◽  
Hematopoietic Stem ◽  
Notch Ligand ◽  
Stem And Progenitor Cells ◽  
Equity Ownership ◽  
Signaling Activation

Abstract BACKGROUND: Notch has long been recognized as an important molecule that regulates stem cell self-renewal and differentiation. Despite that Notch is required for the embryonic hematopoietic stem cell (HSC) generation, and that Notch is expressed in the adult HSC, the role of Notch pathway in adult HSC remains unclear. Recently it has been shown that Notch activation and the ability of interacting with Notch ligand Jagged1 is a signature of human primitive HSC and supports HSC regenerative potential. Here we study the physiological significance of Notch in the adult HSC population focusing on how Notch-ligand engagement regulates HSC quiescence and niche retention. METHODS: To better understand the role of Notch in adult HSC homeostasis, we examined HSC frequency, quiescence maintenance, niche occupancy, and HSC mobilization in mice with either conditional lack of RBP-JK, which mediates the canonical Notch signaling activity, or in mice with conditional lack of Pofut1 that catalyzes O-fucosylation of Notch EGF-like repeats and the generation of O-fucose glycans important for the binding of Notch ligands. We also tested Notch ligand neutralizing antibodies and Notch1 and Notch2 inhibitory antibodies to examine the effect of blocking Notch receptor-ligand engagement or the block of Notch signaling activation on HSC homeostasis. RESULTS: We report here that Pofut1-deficient hematopoietic stem and progenitor cells (HSPCs) display enhanced cell cycling and proliferation cell autonomously. These changes are accompanied by G-CSF-independent increased HSPC egress from the marrow to the periphery and other hematopoietic organs, and their enhanced sensitivity to mobilizing stimuli of G-CSF plus the CXCR4 antagonist, AMD3100. This phenotype is caused by reduced adhesion of Pofut1-deficient HSPC to Notch ligand-expressing stromal cells and the osteoblastic lineage cells. Adhesion to these cells by wild type but not Pofut1-deficient HSPCs can be blocked by recombinant Notch ligand Dll1 or Dll4. In addition, adhesion to these cells inhibits wild type but not Pofut1-deficient HSPC cycling, independent of RBP-Jk-mediated Notch signaling. Further, Pofut1-deficient HSPCs exhibit normal expression of key adhesion molecules and normal SDF-1-mediated chemotaxis, but show scattered and distal occupancy in the endosteal or osteoblastic niche. In support of roles for Notch-ligand engagement in facilitating HSPC niche retention, we show that mice receiving 4 doses of neutralizing antibodies to the Notch ligand Dll4 or Jagged1, but not Dll1, display ~2-3-fold increased HSC and progenitor egress when compared to mice receiving isotype control antibody, and further display 60% increased HSC mobilization when compared to mice receiving control antibody and treated similarly with G-CSF and AMD3100. Dll4 blockade also increases the sinusoidal endothelial cell population and HSPC cell cycling. In comparison, only a mild HSPC proliferation and egress is found in RBP-JK-deficient mice, or in mice receiving Notch2 inhibitory antibody. However, Notch2 blockade but not Notch1 blockade induces unique features of HSC and myeloid progenitor mobilization responding to G-CSF plus AMD3100. CONCLUSIONS: Based on these findings, we conclude that HSPC quiescence and retention in the marrow niche is facilitated by the interaction between Notch-expressing HSPCs and Jagged1- or Dll4-expressing niche cells, and is likely also contributed by Notch signaling activation. In addition, Notch receptor-ligand engagement in this process is strengthened by O-fucose modification of Notch receptors. Finally, the observations from our studies may provide a therapeutic indication. Inadequate mobilization in HSPC transplantation remains a clinical problem. Our findings that targeting Notch receptor-ligand interaction and/or inhibiting Notch2 activation increase HSPC emigration suggests a novel approach for enhancing mobilization of stem and progenitor cells for those patients who respond poorly to current mobilizing regimes. Disclosures Shim: Genentech: Employment, Equity Ownership. Yan:Genentech: Employment, Equity Ownership. Lowe:Genentech: Employment, Equity Ownership. Siebel:Genentech: Employment, Equity Ownership.

scholarly journals Genome Editing of the Bcl11A Erythroid Specific Enhancer in Bone Marrow Derived Hematopoietic Stem and Progenitor Cells for the Treatment of Sickle Cell Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 203-203 ◽  
Author(s):  
Siyuan Tan ◽  
Kai-Hsin Chang ◽  
Sarah Smith ◽  
Kai Chen ◽  
Timonthy Sullivan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Genome Editing ◽  
Cell Lineage ◽  
Cell Disease ◽  
Employment Equity ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Equity Ownership ◽  
Patent Applications

Abstract Ablation of Bcl11A could be a viable approach for the treatment of β-hemoglobinopathies such as β-thalassemia and sickle cell disease (SCD), since patients with Bcl11A haploinsufficiency have persistently high levels of fetal hemoglobin (HbF) (up to 30%), which are associated with development of minimal to no disease symptoms. Genome editing by engineered zinc-finger nucleases that target either the exon 2 (exon ZFN) or the GATA motif of the erythroid specific enhancer (enhancer ZFN) of Bcl11A has been shown to increase HbF level in erythroid progeny from mobilized peripheral hematopoietic stem and progenitor cells (PB-CD34+ HSPCs). However, peripheral mobilization of CD34+ cells is associated with high risk and currently is not an option for SCD patients. Therefore, we investigated the efficacy of genome editing of Bcl11A in bone marrow derived CD34+ cells (BM-CD34+ HSPCs). We first established a clinically compatible large-scale process to isolate CD34+ HSPCs from human bone marrow aspirates and to transiently express the ZFN protein by mRNA electroporation. The CD34+ isolation process resulted in ~ 95% pure CD34+ cells with greater than 90% viability. Both the exon and the enhancer ZFN drove 50-60% Bcl11A gene editing, resulting in a robust elevation of HbF in the erythroid progeny. Notably, the BM-CD34+ HSPCs were found to contain a small population (10 to 25%) of CD34+CD19+ pro-B cells that were refractory to ZFN transfection under our current electroporation condition. Since CD34+CD19+ pro-B cells are not expected to contribute to reconstituting the hematopoietic system other than B-cell lineage, the Bcl11A editing efficiency in the multipotent BM-CD34+ HSPC could be even higher. The engraftment abilities of Bcl11A edited BM-CD34+ cells were then investigated in an immunodeficient NOD/scid/gamma (NSG) mouse model. At a dose of 1 million cells per mouse, treatment with either the exon ZFN or the enhancer ZFN did not detectably impact engraftment or multi-lineage reconstitution compared with untreated cells. However, Bcl11A marking in engrafted human cells was found to be markedly higher in the mice treated by the enhancer ZFN than that by the exon ZFN. The exon ZFN resulted in a strong bias towards in-frame mutations across multi-lineages with the strongest effect observed in the B-cell lineage, suggesting that a threshold level of Bcl11A is required for efficient hematopoietic reconstitution and that cells fully lacking it due to disruption of the coding sequence are at a disadvantage. In contrast, the enhancer ZFN resulted in comparable Bcl11A marking across all lineages with no apparent selection for cells with a functional GATA sequence. Collectively, these data indicate that genome editing of the erythroid specific enhancer of Bcl11A in BM-CD34+ promotes HbF reactivation in the erythroid progeny while maintaining the engraftment and multi-lineage repopulating activities of edited BM-CD34+ HSPCs, which supports further clinical development of this approach for the treatment of SCD. Disclosures Tan: Biogen: Employment, Equity Ownership. Chang:Biogen: Employment, Equity Ownership. Smith:Biogen: Employment, Equity Ownership. Chen:Biogen: Employment, Equity Ownership. Sullivan:Biogen: Employment, Equity Ownership. Zhou:Biogen: Employment, Equity Ownership. Reik:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Urnov:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Rebar:Sangamo BioSciences: Employment. Danos:Biogen: Employment, Equity Ownership. Jiang:Biogen: Employment, Equity Ownership.

scholarly journals Changing the Natural History of Fanconi Anemia Complementation Group-A with Gene Therapy: Early Results of U.S. Phase I Study of Lentiviral-Mediated Ex-VivoFANCA Gene Insertion in Human Stem and Progenitor Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3350-3350
Author(s):  
Agnieszka Czechowicz ◽  
Maria Grazia Roncarolo ◽  
Brian C Beard ◽  
Ken Law ◽  
Eileen Nicoletti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Phase I ◽  
Peripheral Blood ◽  
Research Funding ◽  
Phase I Study ◽  
Employment Equity ◽  
Hematopoietic Stem ◽  
Early Evidence ◽  
Stem And Progenitor Cells ◽  
Equity Ownership

Background: Fanconi anemia (FA) is a rare genetic disorder characterized by defective cellular deoxyribonucleic acid (DNA) repair, associated with developmental abnormalities, progressive bone marrow failure (BMF), and a predisposition to hematologic malignancies and solid tumors. 80% of FA patients develop BMF. Although allogeneic hematopoietic stem cell transplant (allo-HSCT) is a curative treatment for BMF, its utilization and efficacy is limited by availability of suitable human leukocyte antigen (HLA)-matched donors, risk of graft-versus-host disease (GVHD) and transplant-related toxicities. Ex-vivo insertion of a functional FANCA gene into autologous FA-A CD34+ enriched hematopoietic stem and progenitor cells (HSPCs) has been shown in preclinical studies to provide a survival advantage to the gene-modified stem cells, leading to correction of BMF. Feasibility of this approach was established in the FANCOLEN-1 clinical trial (Spain), although cell doses and transduction levels varied considerably. Modifications to the collection and manufacturing processes were made in the clinical studies to enhance the dose of transduced HSPCs, with the goal of preventing progression of BMF to obviate the need of an allo-HSCT. Design and Methods: RP-L102-0418 (clinicaltrials.gov # NCT03814408) is a U.S. Phase I clinical trial evaluating the feasibility and safety of autologous CD34+ cells transduced with a lentiviral vector (LV) carrying the FANCA gene (PGK-FANCA-WPRE) in two pediatric patients with FA-A. Patients <12 years of age, with early evidence of cytopenias, but with bone marrow (BM) CD34+ count >30/µL were eligible for treatment. Peripheral blood mononuclear cells were collected via leucocytapheresis on two consecutive days after mobilization with granulocyte-colony stimulating factor (G-CSF) and Plerixafor (Mozobil). CD34+ HSPCs were enriched, placed in culture with cytokines, and transduced with PGK-FANCA-WPRE LV. The investigational drug product (DP) (RP-L102) was infused fresh into patients within 4 hours of release, without any prior conditioning regimen. Patients are being followed for 3 years post-infusion for safety assessments (replication competent lentivirus (RCL), insertion site analysis (ISA)) and to ascertain early evidence of efficacy (increasing peripheral blood vector copy number (VCN) and BM mitomycin-C (MMC) resistance), along with stabilization/correction of cytopenias. Results: Two FA-A patients (aged 5 and 6 years) were consented and enrolled on the study at Stanford University. Mobilization and apheresis procedures were performed successfully without any serious adverse events. DP was successfully manufactured using "Process B" optimization including transduction enhancers, commercial-grade vector, and modified cell processing. Because of higher transduced CD34+ and colony forming cell (CFC) doses, we anticipate early development of BM MMC resistance in the current study patients. Safety and efficacy data 4 to 6 months post-treatment, including peripheral blood VCN, blood counts and bone marrow MMC resistance, will be available at the time of presentation. Conclusions: DP has been successfully manufactured in the Phase I study (N=2) to meet the required specifications.Patients are being monitored for early efficacy assessments; 6+ months of follow-up may be required to observe the proliferative advantage of transduced HSPCs.Plans for Phase II portion of the study are in progress. Disclosures Czechowicz: Rocket Pharmaceuticals, Inc.: Research Funding. Beard:Rocket Pharmaceuticals: Employment, Equity Ownership. Law:Rocket Pharmaceuticals: Employment, Equity Ownership. Nicoletti:Rocket Pharmaceuticals, Inc.: Employment, Equity Ownership. Río:Rocket Pharmaceuticals: Equity Ownership, Patents & Royalties, Research Funding. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding. Schwartz:Rocket Pharmaceuticals: Employment, Equity Ownership.

Crispr/Cas9- Mediated Genome Editing of Human CD34+ Cells Upregulate Fetal Hemoglobin to Clinically Relevant Levels in Single Cell-Derived Erythroid Colonies

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3623-3623 ◽  
Author(s):  
Bibhu Mishra ◽  
Song Chou ◽  
Michelle I Lin ◽  
Elizabeth J Paik ◽  
Ying Zhang ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Genetic Variants ◽  
Fetal Hemoglobin ◽  
Regulatory Sequences ◽  
Employment Equity ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Naturally Occurring ◽  
Stem And Progenitor Cells ◽  
Equity Ownership

Abstract Fetal hemoglobin (HbF) is abundant in the late stage fetus and newborns, but is progressively lost over the first 6 months of life as the genes encoding the g-globin subunit of HbF are repressed. HbF expression can provide clinical benefit to patients with deficient or defective b-globin, for example in β-thalassemia and Sickle Cell Disease (SCD), respectively. CRISPR-Cas9 technology offers a unique treatment modality that can be used to ex vivo edit regulatory DNA sequences in patient CD34+ hematopoietic stem and progenitor cells (HSPC) containing hematopoietic stem and progenitor cells in order to upregulate HbF. Reinfusion of edited CD34+ HSPC would be expected to lead to the production of erythrocytes expressing high levels of HbF, and amelioration of disease. There are a number of reports of naturally occurring genetic variants that are associated with Hereditary Persistence of Fetal Hemoglobin (HPFH), presumably through the loss of regulatory sequences that would otherwise bind regulatory proteins that developmentally down-regulate the expression of HbF. Thus, by using the CRISPR-Cas9 system to re-create the DNA sequence variations associated with HPFH in CD34+ HSPC, we aim to relieve transcriptional inhibition of ɣ-globin, resulting in upregulation of HbF. Here, we transfected healthy primary human CD34+ HSPC with Cas9 and guide RNAs in order to re-create naturally occurring HPFH genetic variants. Transfected CD34+ HSPC were sorted as single cells post-editing and expanded as erythroid colonies. These colonies were then genotyped to confirm editing and further assessed for globin transcript and protein. Clonal analysis demonstrated Non-Homologous End Joining (NHEJ) mediated editing in greater than 90% of the colonies. These editing events re-created the intended HPFH genetic variants in up to 75% of the colonies. For some, but not all of the HPFH genetic variants, both mono-and bi-allelic genetic modifications led to significant upregulation of HbF. These results provide us with an editing strategy that support a viable therapeutic approach for the treatment of β-thalassemia and SCD. Disclosures Mishra: CRISPR Therapeutics: Employment. Chou:CRISPR Therapeutics: Employment. Lin:CRISPR Therapeutics: Employment. Paik:CRISPR Therapeutics: Employment. Zhang:CRISPR Therapeutics: Employment, Equity Ownership. Liang:CRISPR Therapeutics: Employment. Tomkinson:CRISPR Therapeutics: Employment. Pettiglio:CRISPR Therapeutics: Employment. Sanginario:CRISPR Therapeutics: Employment. Allen:CRISPR Therapeutics: Employment. Cradick:CRISPR Therapeutics: Employment. John:CRISPR Therapeutics: Employment. Chakraborty:CRISPR Therapeutics: Employment. Cowan:CRISPR Therapeutics: Employment, Equity Ownership. Novak:CRISPR Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Lundberg:CRISPR Therapeutics: Employment, Equity Ownership.

scholarly journals Ex vivo expansion of human hematopoietic stem and progenitor cells

Blood ◽  
2011 ◽  
Vol 117 (23) ◽  
pp. 6083-6090 ◽  
Author(s):  
Ann Dahlberg ◽  
Colleen Delaney ◽  
Irwin D. Bernstein
Keyword(s):  
Stem Cell ◽  
Growth Factors ◽  
Notch Signaling ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Growth Factors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

AbstractDespite progress in our understanding of the growth factors that support the progressive maturation of the various cell lineages of the hematopoietic system, less is known about factors that govern the self-renewal of hematopoietic stem and progenitor cells (HSPCs), and our ability to expand human HSPC numbers ex vivo remains limited. Interest in stem cell expansion has been heightened by the increasing importance of HSCs in the treatment of both malignant and nonmalignant diseases, as well as their use in gene therapy. To date, most attempts to ex vivo expand HSPCs have used hematopoietic growth factors but have not achieved clinically relevant effects. More recent approaches, including our studies in which activation of the Notch signaling pathway has enabled a clinically relevant ex vivo expansion of HSPCs, have led to renewed interest in this arena. Here we briefly review early attempts at ex vivo expansion by cytokine stimulation followed by an examination of our studies investigating the role of Notch signaling in HSPC self-renewal. We will also review other recently developed approaches for ex vivo expansion, primarily focused on the more extensively studied cord blood–derived stem cell. Finally, we discuss some of the challenges still facing this field.

scholarly journals POS0042 NOTCH 1 INHIBITION INCREASES OSTEOCLAST PROGENITOR ACTIVITY IN THE MOUSE MODEL OF RHEUMATOID ARTHRITIS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 226.3-226
Author(s):  
M. Filipović ◽  
A. Šućur ◽  
D. Flegar ◽  
Z. Jajić ◽  
M. Ikić Matijašević ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Notch Signaling ◽  
Neutralizing Antibodies ◽  
Osteoclast Differentiation ◽  
Notch Receptor ◽  
Notch 1 ◽  
Collagen Induced Arthritis ◽  
Notch Receptors

Background:Osteoclasts mediate periarticular and systemic bone loss in rheumatoid arthritis (RA). Osteoclast progenitor cells (OCPs) derived from the myeloid lineage are susceptible to regulation through Notch signaling. Murine bone marrow and splenic OCPs, identified as CD45+Ly6G-CD3-B220-NK1.1-CD11blo/+CD115+CCR2+ cells, are specifically increased in arthritis. We previously identified an increased frequency of OCPs expressing Notch receptors in arthritic mice.Objectives:Several studies suggested that Notch signaling modulation affects the course of experimental arthritis. We aimed to determine the effects of Notch receptor signaling inhibition on OCP activity and arthritis severity in murine collagen-induced arthritis (CIA).Methods:Male C57/Bl6 and DBA mice were immunized with chicken type II collagen and treated with i.p. injections of anti-Notch 1 neutralizing antibodies (1mg/kg). Notch receptor 1 through 4 expression on OCPs was analyzed by flow cytometry in periarticular bone marrow (PBM) and spleen (SPL). Gene expression of Notch receptors, ligands and transcription targets as well as osteoclast differentiation genes RANK, cFos and cFms was determined by qPCR from tissues and sorted OCPs. FACS sorted OCPs were stimulated by osteoclastogenic factors (M-CSF and RANKL), in control, IgG, Jagged (Jag)1 or Delta-like (DLL)1 coated wells, with or without anti-Notch 1 antibodies. Research was approved by the Ethics Committee.Results:We confirmed the expression of Notch receptors on OCPs by flow cytometry with Notch 1 and 2 being most abundantly expressed (around 25% and 40% positive OCPs in PBM and 35% and 20% in SPL respectively), with a significant increase of Notch 2 expression in arthritis. Seeding OCPs on DLL1 coated wells significantly increased while seeding on Jag1 coated wells significantly decreased osteoclastogenesis as reflected on the number of TRAP+ osteoclasts and expression of osteoclast differentiation genes. The addition of anti-Notch 1 antibodies to ligand-stimulated OCPs resulted in an increased number of TRAP+ osteoclasts, partially reversing Jag1 inhibition. In vivo treatment with anti-Notch 1 antibodies did not affect total OCP frequency, but increased expression of Notch 4 both in PBM and SPL as seen by flow cytometry and qPCR. Additionally, anti-Notch 1 treatment stimulated Notch transcription factors HES and HEY. Both PBM and SPL cultured OCPs from anti-Notch 1 treated mice produced a higher number of large TRAP+ osteoclasts, doubling the area covered with osteoclasts in the latter compared to untreated mice. Increased osteoclastogenesis in vitro was further confirmed by an increased expression of osteoclast differentiation genes in the treated group.Conclusion:Our results confirm that Notch signaling may represent an important therapeutic target for the regulation of osteoclast activity in arthritis. Both in vitro and in vivo anti-Notch 1 neutralizing antibodies enhanced osteoclastogenesis in CIA model, implying an inhibitory role of Notch 1 signaling in osteoclast differentiation. As Notch 2 expression is increased on OCPs of arthritic mice, we next plan to determine the effects of Notch 2 neutralization on osteoclast activity and arthritis severity.References:[1]Ikić Matijašević M, Flegar D, Kovačić N, Katavić V, Kelava T, Šućur A, et al. Increased chemotaxis and activity of circulatory myeloid progenitor cells may contribute to enhanced osteoclastogenesis and bone loss in the C57BL/6 mouse model of collagen-induced arthritis. Clin Exp Immunol. 2016;186(3):321–35.[2]Šućur A, Filipović M, Flegar D, Kelava T, Šisl D, Lukač N, et al. Notch receptors and ligands in inflammatory arthritis – a systematic review. Immunology Letters 2020 Vol. 223, p. 106–14.Acknowledgements:The work has been supported by Croatian Science Foundation projects IP-2018-01-2414, UIP-2017-05-1965 and DOK-2018-09-4276.Disclosure of Interests:None declared.

scholarly journals The retromer complex safeguards against neural progenitor-derived tumorigenesis by regulating Notch receptor trafficking

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Bo Li ◽  
Chouin Wong ◽  
Shihong Max Gao ◽  
Rulan Zhang ◽  
Rongbo Sun ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Molecular Mechanisms ◽  
Neural Progenitor ◽  
Tumor Formation ◽  
Neural Progenitors ◽  
Notch Receptor ◽  
Dependent Manner ◽  
Cell Fate Decisions ◽  
Retromer Complex ◽  
Signaling Activation

The correct establishment and maintenance of unidirectional Notch signaling are critical for the homeostasis of various stem cell lineages. However, the molecular mechanisms that prevent cell-autonomous ectopic Notch signaling activation and deleterious cell fate decisions remain unclear. Here we show that the retromer complex directly and specifically regulates Notch receptor retrograde trafficking in Drosophila neuroblast lineages to ensure the unidirectional Notch signaling from neural progenitors to neuroblasts. Notch polyubiquitination mediated by E3 ubiquitin ligase Itch/Su(dx) is inherently inefficient within neural progenitors, relying on retromer-mediated trafficking to avoid aberrant endosomal accumulation of Notch and cell-autonomous signaling activation. Upon retromer dysfunction, hypo-ubiquitinated Notch accumulates in Rab7+ enlarged endosomes, where it is ectopically processed and activated in a ligand-dependent manner, causing progenitor-originated tumorigenesis. Our results therefore unveil a safeguard mechanism whereby retromer retrieves potentially harmful Notch receptors in a timely manner to prevent aberrant Notch activation-induced neural progenitor dedifferentiation and brain tumor formation.

Notch-Dependent Control of Blood Lineage Development is Modified by Fucosylation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2448-2448
Author(s):  
Lan Zhou ◽  
Quanjian Yan ◽  
David Yao ◽  
Lebing W Li ◽  
Stanton L. Gerson ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Notch Signaling ◽  
Es Cells ◽  
Notch Receptor ◽  
Wild Type ◽  
Notch Ligand ◽  
Hematopoietic Reconstitution ◽  
Myeloid Development

Abstract Notch receptors are conserved cell surface molecules essential for hematopoietic cell fate determination. Activated Notch enhances self-renewal of hematopoietic stem cells and promotes T lymphopoiesis. O-linked fucose moieties attached to the EGF domains of Notch receptors and its modification by Fringe can strongly modulate Notch signaling. Our recently published results indicate that Notch-dependent signaling controls myelopoiesis both in vitro and in vivo, and identify a requirement for Notch fucosylation in the expression of Notch ligand binding activity and Notch signaling efficiency in hematopoietic progenitor cells. In the current study, we tested the hypothesis that fucosylation controlled Notch signaling regulates hematopoietic lineage homeostasis. Genetically-modified mouse embryonic stem (ES) cells deficient in Notch1 receptor (NOTCH1−/−) or pofut1 (POFUT1−/−) that controls O-fucose modification of Notch receptor EGF repeats are studied in an in vitro co-culture assay with Notch ligand-expressing OP9 cells. Activation of Notch in wild type ES cells promotes T lymphopoiesis, while exposure of NOTCH1−/− or POFUT1−/− ES cells to Notch ligand failed to generate T lymphocytes but sustained granulocytic production. When probed with recombinant Notch ligand Dll1 or Dll4, hematopoietic cells derived from wild type ES line displayed robust Notch ligand binding, but cells from NOTCH1−/− or POFUT1−/− ES lines showed completely absent or reduced Notch ligand interaction, respectively. In comparison, ES cells deficient in pofut2 (POFUT2−/−) that controls O-fucose modification on thrombospondin repeats (TSR) displayed a wild type lineage development phenotype and normal Notch ligand binding ability. When examined for their in vivo hematopoietic reconstitution, blood cells derived from NOTCH1−/− or POFUT1−/− ES lines, but not POFUT2−/− ES line, showed enhanced granulocytic but suppressed T and B lymphoid lineage development. These results are consistent with our bone marrow transplantation findings that hematopoietic reconstitution by fucosylation-deficient marrow progenitor cells exhibited increased granulocytopoiesis while wild type or fucosylation-intact marrow cells have normal lineage distribution. Our observations indicate that Notch signaling maintains blood lineage homeostasis by promoting lymphoid lineage development and suppressing overt myeloid development. O-fucose modification of EGF repeats on Notch receptor is essential for this Notch-dependent control of blood lineage homeostasis as deficiency of fucose on Notch receptor results in enhanced myeloid development.

Use of Genome-Wide Expression Analysis to Optimize An Ex Vivo clinical Protocol for 16,16-Dimethyl Prostaglandin E2 Enhancement of Umbilical Cord Blood In Hematopoietic Stem Cell Transplantation.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1451-1451
Author(s):  
Caroline Desponts ◽  
David Robbins ◽  
Thuy Le ◽  
Annie Chi ◽  
Scott Thies ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Treatment Protocol ◽  
Employment Equity ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Biologic Activity ◽  
Genome Wide ◽  
Cd34 Cells ◽  
Equity Ownership ◽  
Genome Wide Expression

Abstract Abstract 1451 A systematic investigation was performed to optimize the treatment protocol for ex vivo incubation of human hematopoietic stem cells (HSCs) with 16,16-dimethyl prostaglandin E2 (FT1050) prior to transplantation. This protocol is part of an ongoing Phase Ib clinical trial of FT1050-enhanced double cord blood (CB) transplantation after reduced intensity conditioning. FT1050 has been previously shown in vertebrate models to improve the engraftment potential of HSCs from bone marrow (BM) and CB after a brief ex vivo treatment. In these models, treatment of BM or CB with FT1050 was performed for 1 to 2 hours at 4 °C, followed by a wash and subsequent cell infusion into the recipient (North et al. Nature 2007, Hoggatt et al. Blood 2009). Several groups have demonstrated that under these conditions, FT1050-treated cells have an engraftment advantage over vehicle treated cells. The objective of the current investigation was to identify a set of conditions that maximizes the biologic activity of FT1050. Genome-wide expression analysis and cAMP assays were used to optimize the ex vivo FT1050 treatment protocol with respect to concentration, time and temperature. Using this approach, hundreds of up- and down-regulated genes were identified in FT1050-treated CD34+ cells. These signature genes include upregulation of CXCR4, a known mediator of HSC homing via SDF-1a, and CREB, a key gene involved in cAMP signaling. Results from these experiments demonstrated that FT1050 concentrations above 10 μM did not result in increased levels of biologic activity. In terms of duration of incubation, cAMP activity reached maximal levels within 30 minutes of exposure while a 2 hour treatment period was necessary to maximize the changes in gene expression. Finally, the biologic activity of FT1050 was highly sensitive to temperature, with treatment of cells at 37 °C yielding larger changes in cAMP production and gene expression as compared to incubation of cells at 25 °C and 4 °C. The biological effects of FT1050 on subsets of CD34+ cells isolated from CB were also determined. Interestingly, the stem/progenitor subsets of CD34+ cells (Lin-CD34+CD38-CD90+CD45RA-, Lin-CD34+CD38-CD90-CD45RA- and Lin-CD34+) had a greater response to FT1050 relative to the lineage positive cells. The different conditions were also evaluated using CFU-C and 7-AAD assays. No evidence of adverse effects were observed. Based upon these findings, the ongoing clinical trial incorporates the optimized FT1050 ex vivo treatment protocol (10 μM for 120 minutes at 37 °C). Disclosures: Desponts: Fate Therapeutics, Inc.: Employment, Equity Ownership. Robbins:Fate Therapeutics, Inc.: Employment, Equity Ownership. Le:Fate Therapeutics, Inc.: Employment, Equity Ownership. Thies:Fate Therapeutics, Inc.: Employment, Equity Ownership. Mendlein:Fate Therapeutics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Grayson:Fate Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Multani:Fate Therapeutics, Inc.: Employment, Equity Ownership. Shoemaker:Fate Therapeutics: Employment, Equity Ownership.

Analysis of Mesenchymal Stromal Cells (MSC) and Their Interactions with CD34 Stem and Progenitor Cells in Patients with Myelodysplastic Syndromes (MDS)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2393-2393
Author(s):  
Stefanie Geyh ◽  
Ron Patrick Cadeddu ◽  
Julia Fröbel ◽  
Ingmar Bruns ◽  
Fabian Zohren ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Myelodysplastic Syndromes ◽  
Stromal Cells ◽  
Median Number ◽  
Lower Number ◽  
Differentiation Potential ◽  
Receptor Ligand ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Abstract 2393 Background: Myelodysplastic syndromes (MDS) represent a heterogeneous group of hematopoietic stem cell disorders and research in this field has mainly focused on hematopoietic stem and progenitor cells (HSPC). Still, recent data from mouse models indicate that the bone marrow (BM) microenvironment might be involved in the pathogenesis MDS (Raaijmakers et al., 2010). The role of mesenchymal stromal cells (MSC) in particular as a key component of the BM microenvironment remains elusive in human MDS and data so far are controversial. Design/Methods: We therefore investigated MSC and immunomagnetically enriched CD34+ HSPC from BM of 42 untreated patients (pts) with MDS (12 RCMD, 12 RAEB, 12 sAML, 3 del5q, 1 CMML-1, 1 MDS hypocellular, 1 MDS unclassifiable according to WHO) and age-matched healthy controls (HC, n=13). MSC were examined with regard to growth kinetics, morphology and differential potential after isolation and expansion according standard procedures in line with the international consensus criteria (Dominici et al., 2006). Furthermore corresponding receptor-ligand pairs on MSC and CD34+ cells (Kitlg/c-kit; CXCL12/CXCR4; Jagged1/Notch1; Angpt1-1/Tie-2; ICAM1/LFA-1) were investigated by RT-PCR. Results: In MDS, the colony forming activity (CFU-F) of MSC was significantly reduced in comparison to HC (median number of colonies per 1×107MNC in MDS: 8, range 2–74 vs. HC: 175, 10–646, p=0.003) and this was also true when looking at the different subtypes (RCMD median: 16, p=0.04; RAEB median: 8, p=0.31; sAML median: 26, p=0.02). According to this, MSC from pts with RCMD and del5q could only be maintained in culture for a lower number of passages (median number of passages: MDS 3 passages, range 1–15; HD 14 passages, range 8–15, p=0.01), had a lower number of cumulative population doublings (CPD) and needed a longer timer to reach equivalent CPD (MDS median: 18,16 CPD, HD median: 33,68 CPD, p=0,0059). All types of MDS-MSC showed an abnormal morphology, while an impaired osteogenic differentiation potential was exclusively observed in pts with RCMD. These findings of an altered morphology together with a diminished growth and differentiation potential prompted us to test, whether the interaction between MSC and CD34+ HSPC in BM of pts with MDS was also disturbed. On the MDS-MSC, we found a significant lower expression of Angpt1 in pts with RAEB (3.5-fold, p=0.01) and del5q (4.9-fold, p=0.009) compared to HD. The expression of CXCL12 (2.5-fold, p=0.057) and jagged1 was reduced in trend in MSC from pts with MDS, while no differences were observed with regard to the expression of kitlg and ICAM1. When looking on CD34+ cells, we found a significantly reduced expression of CXCR4 (RCMD 2.5-fold, p=0.02; RAEB 2.46-fold, p=0.02), notch1 (RCMD 6-fold, p=0.04) and Tie-2 (RAEB 5.91-fold, p=0.02) in pts with MDS, while LFA-1 was overexpressed in pts with RAEB (2.6-fold, p=0.036). Conclusion: Taken together, our data indicate that MSC from pts with MDS are structurally altered and that the crosstalk between CD34+ HSPC and MSC in the BM microenvironment of pts with MDS might be deregulated as a result of an abnormal expression of relevant receptor-ligand pairs. Ongoing research is required to corroborate these findings and to definitely address their functional relevance for the pathogenesis of MDS. Disclosures: No relevant conflicts of interest to declare.

Vincristine Sulfate Liposomes Injection (Marqibo®) Facilitates Durable Remissions and Potentially Curative Hematopoietic Stem Cell Transplantation in Adults with Advanced, Relapsed and/or Refractory Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4235-4235
Author(s):  
Gary J. Schiller ◽  
John Lister ◽  
Leonard T. Heffner ◽  
Stuart L. Goldberg ◽  
Lloyd E. Damon ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Salvage Therapy ◽  
Lymphoblastic Leukemia ◽  
Vincristine Sulfate ◽  
Employment Equity ◽  
Dose Intensification ◽  
Hematopoietic Stem ◽  
Equity Ownership

Abstract Abstract 4235 A durable response in advanced, relapsed and/or refractory adult acute lymphoblastic leukemia (ALL) may be defined as remission that results in a meaningful prolongation of life or response that facilitates “bridging” to a subsequent, potentially curative, hematopoietic stem cell transplantation (HSCT). Vincristine sulfate liposomes injection (VSLI, Marqibo®) is a sphingomyelin/cholesterol (SM/Chol) nanoparticle formulation of standard vincristine sulfate (VCR) designed to facilitate dose intensification, prolonged drug delivery and enhanced lymphoid malignancy penetration and concentration without increased toxicity. Recently, VSLI was evaluated in a multi-institutional, Phase 1/2 (VSLI-06; NCT00144963) clinical trial and a multi-national, Phase 2 (HBS407; NCT00495079) clinical trial in a combined 101 adults (median age 31 years [range 18 to 83 years]) with advanced, relapsed and/or refractory ALL. All but 1 patient had Philadelphia chromosome negative disease. Thirteen patients (13%) had extramedullary disease, 37 (37%) had undergone a prior HSCT, and 100% had received at least one prior line of therapy including standard VCR. Study VSLI-06 (N = 36) was a dose-ascending trial of weekly VSLI (1.5 to 2.4 mg/m2) combined with pulse dexamethasone. Study HBS407 was a single-arm trial of weekly single-agent VSLI at the maximum tolerated dose established in VSLI-06 of 2.25 mg/m2. Overall, 19 (19%) patients received VSLI as a first salvage therapy, 57 (56%) patients received VSLI as a second salvage therapy, and 25 (25%) patients received VSLI as a third or greater salvage therapy. All patients had to be deemed ineligible for immediate HSCT in order to enroll in VSLI-06 or HBS407. In the combined study population, the overall response rate (complete remission [CR], CR with incomplete hematologic recovery [CRi], partial remission [PR], and bone marrow blast response [BMB]) was 31% (95% CI: 22–41) with a 20% (95% CI: 13–29) rate of CR+CRi. Despite delivering intensified individual (2.8–5.5 mg) and cumulative (up to 70.1 mg) doses of VCR, VSLI had a similar safety profile to that reported for the approved dose of standard VCR. VSLI enabled bridging to a post-VSLI HSCT in 12 of 65 (18%) patients in HBS407 and 5 of 36 (14%) patients in VSLI06 for a total of 17 of 101 (17%). All 17 post-VSLI HSCT patients were under the age of 60 years. Three of 12 post-VSLI HSCT patients from HBS407 remain alive at greater than 28, 33, and 35 months following VSLI, respectively. All 12 patients lived for greater than 100 days after post-VSLI HSCT. Long-term survival (greater than 12 months) was achieved in 27% of those able to receive post-VSLI HSCT. These outcomes, that are important to patients, may reflect the effectiveness of the VCR dose intensification facilitated by VSLI. The neuropathy associated with the dose intensified VCR administered as VSLI was predictable, manageable, and comparable to that published for standard VCR. The lack of early, pre-day 100, mortality following post-VSLI HSCT suggests that the sphingomyelin-based liposomal formulation did not adversely affect subsequent transplantation procedures. In conclusion, VSLI produced both clinically important endpoints of prolonged survival and achievement of response allowing for a bridge to HSCT for advanced, relapsed and/or refractory ALL. Disclosures: Schiller: Talon Therapeutics: Research Funding. Silverman:Talon Therapeutics: Employment, Equity Ownership. Deitcher:Talon Therapeutics: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

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