Allison Blair

Expression of CD133 on leukemia-initiating cells in childhood ALL

Optimization of therapy for childhood acute lymphoblastic leukemia (ALL) requires a greater understanding of the cells that proliferate to maintain this malignancy because a significant number of cases relapse, resulting from failure to eradicate the disease. Putative ALL stem cells may be resistant to therapy and subsequent relapses may arise from these cells. We investigated expression of CD133, CD19, and CD38 in pediatric B-ALL. Cytogenetic and molecular analyses demonstrated that karyotypically aberrant cells were present in both CD133(+)/CD19(+) and CD133(+)/CD19(-) subfractions, as were most of the antigen receptor gene rearrangements. However, ALL cells capable of long-term proliferation in vitro and in vivo were derived from the CD133(+)/CD19(-) subfraction. Moreover, these CD133(+)/CD19(-) cells could self-renew to engraft serial nonobese diabetic-severe combined immunodeficient recipients and differentiate in vivo to produce leukemias with similar immunophenotypes and karyotypes to the diagnostic samples. Furthermore, these CD133(+)/CD19(-) ALL cells were more resistant to treatment with dexamethasone and vincristine, key components in childhood ALL therapy, than the bulk leukemia population. Similar results were obtained using cells sorted for CD133 and CD38, with only the CD133(+)/CD38(-) subfraction demonstrating xenograft repopulating capacity. These data suggest that leukemia-initiating cells in childhood B-ALL have a primitive CD133(+)/CD19(-) and CD38(-) phenotype.

An immortalized adult human erythroid line facilitates sustainable and scalable generation of functional red cells

With increasing worldwide demand for safe blood, there is much interest in generating red blood cells in vitro as an alternative clinical product. However, available methods for in vitro generation of red cells from adult and cord blood progenitors do not yet provide a sustainable supply, and current systems using pluripotent stem cells as progenitors do not generate viable red cells. We have taken an alternative approach, immortalizing early adult erythroblasts generating a stable line, which provides a continuous supply of red cells. The immortalized cells differentiate efficiently into mature, functional reticulocytes that can be isolated by filtration. Extensive characterization has not revealed any differences between these reticulocytes and in vitro-cultured adult reticulocytes functionally or at the molecular level, and importantly no aberrant protein expression. We demonstrate a feasible approach to the manufacture of red cells for clinical use from in vitro culture.

Comparison of childhood leukemia initiating cell populations in NOD/SCID and NSG mice

Leukemia initiating cells (LICs) are defined by their ability to generate leukemias in animal models and to self-renew by repopulating serial recipients. Recent findings suggest that multiple sub-populations of acute leukemia cells are capable of engrafting immune deficient mice raise important questions about the biology of these diseases.1, 2, 3 If several cell populations have LIC properties, what are the relationships of these stem cell populations to each other and which populations are most important to target with therapy? To address these questions, it is crucial to utilize assays that are capable of identifying any leukemia cells that possess stem cell properties. The recently developed NOD/LtSz-scid IL-2Rγc null (NSG) mouse is now considered a more permissive host for the growth and development of normal and acute myeloid leukemia cells than the nonobese diabetic severe combined immune deficient (NOD/SCID) strain.4, 5 In acute lymphoblastic leukemia (ALL) engraftment of high-risk cases has been reported to be slower in NOD/SCID than NSG but otherwise leukemias developed in both strains.6 However, another study reported that engraftment of T-ALL cells was improved using NSG mice.7 Observations that different populations of cells can engraft different mouse strains raise questions as to how the reports that used only NOD/SCID mice should be interpreted. NSG and NOD/SCID strains have not previously been directly and quantitatively compared for engraftment potential of pediatric LIC sub-populations using the same source of cells. Such studies should provide clear information as to the permissiveness of each strain and which sub-populations have LIC potential.

Interleukin-15 enhances cellular proliferation and upregulates CNS homing molecules in pre-B acute lymphoblastic leukemia

Genome-wide association studies have consistently implicated the interleukin-15 (IL-15) gene in acute lymphoblastic leukemia (ALL) biology, including associations with disease susceptibility, and increased risk of central nervous system (CNS) involvement. However, whether pre-B ALL blasts directly respond to IL-15 is unknown. Here, we show that most pre-B ALL primary samples and cell lines express IL-15 and components of its receptor and that primary pre-B ALL cells show increased growth in culture in response to IL-15. Investigation of mechanisms of action using IL-15-responsive SD-1 cells shows this growth advantage is maximal under low-serum conditions, mimicking those found in cerebrospinal fluid. IL-15 also upregulates PSGL-1 and CXCR3, molecules associated with CNS trafficking. Investigation of downstream signaling pathways indicates that IL-15 induces signal transducer and activator of transcription 5 (STAT5), extracellular signal-regulated kinase (ERK) 1/2, and to a lesser extent phosphatidylinositol 3-kinase (PI3K) and nuclear factor κB (NF-κB) phosphorylation. The IL-15-mediated growth advantage is abolished by mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK), PI3K, and NF-κB inhibitors but preserved in the presence of STAT5 inhibition. Together, these observations provide a mechanistic link between increased levels of IL-15 expression and leukemogenesis, high-risk disease, and CNS relapse and suggest potential therapeutic targets.

Parthenolide eliminates leukemia initiating cell populations and improves survival in xenografts of childhood acute lymphoblastic leukemia

Approximately 20% of children with acute lymphoblastic leukemia (ALL) relapse because of failure to eradicate the disease. Current drug efficacy studies focus on reducing leukemia cell burden. However, if drugs have limited effects on leukemia-initiating cells (LICs), then these cells may expand and eventually cause relapse. Parthenolide (PTL) has been shown to cause apoptosis of LIC in acute myeloid leukemia. In the present study, we assessed the effects of PTL on LIC populations in childhood ALL. Apoptosis assays demonstrated that PTL was effective against bulk B- and T-ALL cells, whereas the CD34(+)/CD19(-), CD34(+)/CD7(-), and CD34(-) subpopulations were more resistant. However, functional analyses revealed that PTL treatment prevented engraftment of multiple LIC populations in NOD/LtSz-scid IL-2Rγ(c)-null mice. PTL treatment of mice with established leukemias from low- and high-risk patients resulted in survival and restoration of normal murine hemopoiesis. In only 3 cases, disease progression was significantly slowed in mice engrafted with CD34(+)/CD19(-) or CD34(+)/CD7(-) and CD34(-) cells, but was not prevented, demonstrating that individual LIC populations within patients have different responses to therapy. These observations indicate that PTL may have therapeutic potential in childhood ALL and provide a basis for developing effective therapies that eradicate all LIC populations to prevent disease progression and reduce relapse.

Ex vivo expansion of megakaryocyte progenitor cells from normal bone marrow and peripheral blood and from patients with haematological malignancies

Summary. A number of haematological and non‐haematological malignancies can be successfully treated using high‐dose chemotherapy ± irradiation followed by haematopoietic progenitor cell transplantation. Post transplant, thrombocytopenia and neutropenia always occur and patients require platelet transfusions. It may be possible to reduce the period of thrombocytopenia by re‐infusion of ex vivo expanded megakaryocyte progenitors (MP), derived from the progenitor cell graft. We have investigated the expansion of MP from CD34+ enriched cells from normal bone marrow (NBM) and peripheral blood (PB) and remission BM or PB samples from patients with haematological malignancies. CD34+ cells were cultured in serum‐free medium supplemented with thrombopoietin (TPO), interleukin 1 (IL‐1), IL‐6 and stem cell factor (SCF) for 7 d, then cell proliferation was assessed by flow cytometry using lineage‐specific markers. It was possible to significantly expand the number of MP cells from all sources. There were no major differences in yields of MP from normal BM or PB, or BM from multiple myeloma and non‐Hodgkins lymphoma patients. However, expansion of MP in acute myeloid leukaemia samples was lower than all other samples and the number of megakaryocyte colony‐forming units was reduced. Several cytokine combinations were evaluated to optimize MP expansion from NBM. Equivalent yields of MP were obtained using TPO and one of IL‐1, IL‐3, granulocyte–macrophage colony‐stimulating factor or SCF, suggesting that large cytokine combinations are not necessary for this procedure. It should be possible to scale up the culture conditions described to produce effective MP doses for clinical transplantation.

An optimised biphasic culture system for the generation of functional dendritic cells from patients with acute lymphoblastic leukaemia at presentation and in clinical remission

We have tested the hypothesis that functional dendritic cells (DC) may be generated from patients with acute lymphoblastic leukaemia (ALL). We evaluated the production of DC from blast cells taken at presentation from nine children with ALL. Blast cells were expanded in serum-free medium supplemented with Flt3L, G-CSF, GM-CSF, IL-3, IL-6 and SCF for 7 days and subsequently stimulated with Flt3L, GM-CSF and TGF-β for a further 14 days, with the addition of TNF-α for the final 48 h of culture. Cultured cells had the morphological appearance of DC and expressed the DC-associated antigens CD1A (range 2–87%) and CD83 (15–44%). Expression of the co-stimulatory molecules CD80 and CD86 was increased and the majority of these cells retained their expression of CD34 (73 ± 4%) and HLA-DR (79 ± 5%). Seven of the nine ALL had a leukaemia-specific abnormality and DC generated from five of these seven cases were derived from the leukaemic clone. Leukaemic DC derived from four HLA-A*02-positive ALL pulsed with CMV-associated peptides could induce significant proliferation of peptide-specific CD8+ T cells. This specificity was verified using tetrameric complexes of HLA class I/antigenic peptide. DC could also be generated from cells taken at times of complete remission of ALL and from normal controls using these culture conditions. These findings show that functional DC can be generated both from ALL blasts and from patients in remission; these might be utilised in future for immunotherapeutic strategies in the treatment of ALL.

Superior survival of ex vivo cultured human reticulocytes following transfusion into mice

The generation of cultured red blood cells from stem cell sources may fill an unmet clinical need for transfusion-dependent patients, particularly in countries that lack a sufficient and safe blood supply. Cultured red blood cells were generated from human CD34+ cells from adult peripheral blood or cord blood by ex vivo expansion, and a comprehensive in vivo survival comparison with standard red cell concentrates was undertaken. Significant amplification (>105-fold) was achieved using CD34+ cells from both cord blood and peripheral blood, generating high yields of enucleated cultured red blood cells. Following transfusion, higher levels of cultured red cells could be detected in the murine circulation compared to standard adult red cells. The proportions of cultured blood cells from cord or peripheral blood sources remained high 24 hours post-transfusion (82±5% and 78±9%, respectively), while standard adult blood cells declined rapidly to only 49±9% by this time. In addition, the survival time of cultured blood cells in mice was longer than that of standard adult red cells. A paired comparison of cultured blood cells and standard adult red blood cells from the same donor confirmed the enhanced in vivo survival capacity of the cultured cells. The study herein represents the first demonstration that ex vivo generated cultured red blood cells survive longer than donor red cells using an in vivo model that more closely mimics clinical transfusion. Cultured red blood cells may offer advantages for transfusion-dependent patients by reducing the number of transfusions required.

Junctional Adhesion Molecule-A Is Highly Expressed on Human Hematopoietic Repopulating Cells and Associates with the Key Hematopoietic Chemokine Receptor CXCR4.

Hematopoietic stem/progenitor cells (HSPCs) reside in specialized bone marrow microenvironmental niches, with vascular elements (endothelial/mesenchymal stromal cells) and CXCR4‐CXCL12 interactions playing particularly important roles for HSPC entry, retention, and maintenance. The functional effects of CXCL12 are dependent on its local concentration and rely on complex HSPC‐niche interactions. Two Junctional Adhesion Molecule family proteins, Junctional Adhesion Molecule‐B (JAM)‐B and JAM‐C, are reported to mediate HSPC‐stromal cell interactions, which in turn regulate CXCL12 production by mesenchymal stromal cells (MSCs). Here, we demonstrate that another JAM family member, JAM‐A, is most highly expressed on human hematopoietic stem cells with in vivo repopulating activity (p < .01 for JAM‐Ahigh compared to JAM‐AInt or Low cord blood CD34+ cells). JAM‐A blockade, silencing, and overexpression show that JAM‐A contributes significantly (p < .05) to the adhesion of human HSPCs to IL‐1β activated human bone marrow sinusoidal endothelium. Further studies highlight a novel association of JAM‐A with CXCR4, with these molecules moving to the leading edge of the cell upon presentation with CXCL12 (p < .05 compared to no CXCL12). Therefore, we hypothesize that JAM family members differentially regulate CXCR4 function and CXCL12 secretion in the bone marrow niche. Stem Cells 2016;34:1664–1678

Functionalized Triblock Copolymer Vectors for the Treatment of Acute Lymphoblastic Leukemia

The chemotherapeutic Parthenolide is an exciting new candidate for the treatment of acute lymphoblastic leukemia, but like many other small-molecule drugs, it has low aqueous solubility. As a consequence, Parthenolide can only be administered clinically in the presence of harmful cosolvents. Accordingly, we describe the synthesis, characterization, and testing of a range of biocompatible triblock copolymer micelles as particle-based delivery vectors for the hydrophobic drug Parthenolide. The drug-loaded particles are produced via an emulsion-to-micelle transition method, and the effects of introducing anionic and cationic surface charges on stability, drug sequestration, biocompatibility, and efficacy are investigated. Significantly, we demonstrate high levels of efficacy in the organic solvent-free systems against human mesenchymal stem cells and primary T-acute lymphoblastic leukemia patient cells, highlighting the effectiveness of the delivery vectors for the treatment of acute lymphoblastic leukemia.