The CBFA2T3-GLIS2 (C/G) fusion is a product of a cryptic translocation primarily seen in infants and early childhood and is associated with dismal outcome. Here, we demonstrate that the expression of the C/G oncogenic fusion protein promotes the transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) in an endothelial cell coculture system that recapitulates the transcriptome, morphology, and immunophenotype of C/G acute myeloid leukemia (AML) and induces highly aggressive leukemia in xenograft models. Interrogating the transcriptome of C/G-CB cells and primary C/G AML identified a library of C/G-fusion-specific genes that are potential targets for therapy.
View Article and Find Full Text PDFTo establish novel and effective treatment combinations for chronic myelomonocytic leukemia (CMML) preclinically, we hypothesized that supplementation of CMML cells with the human oncogene Meningioma 1 (MN1) promotes expansion and serial transplantability in mice, while maintaining the functional dependencies of these cells on their original genetic profile. Using lentiviral expression of MN1 for oncogenic supplementation and transplanting transduced primary mononuclear CMML cells into immunocompromised mice, we established three serially transplantable CMML-PDX models with disease-related gene mutations that recapitulate the disease in vivo. Ectopic MN1 expression was confirmed to enhance the proliferation of CMML cells, which otherwise did not engraft upon secondary transplantation.
View Article and Find Full Text PDFPurpose: A cryptic inv(16)(p13.3q24.3) encoding the fusion is associated with poor outcome in infants with acute megakaryocytic leukemia.
View Article and Find Full Text PDFThe ability to expand hematopoietic stem and progenitor cells (HSPCs) ex vivo is critical to fully realize the potential of HSPC-based therapies. In particular, the application of clinically effective therapies, such as cord blood transplantation, has been impeded because of limited HSPC availability. Here, using 3D culture of human HSPCs in a degradable zwitterionic hydrogel, we achieved substantial expansion of phenotypically primitive CD34 cord blood and bone-marrow-derived HSPCs.
View Article and Find Full Text PDF, and are the most frequently mutated genes in cytogenetically normal acute myeloid leukemia (AML), but little is known about how these mutations synergize upon cooccurrence. Here we show that triple-mutated AML is characterized by high leukemia stem cell (LSC) frequency, an aberrant leukemia-specific CD34 immunophenotype, and synergistic upregulation of Hepatic Leukemia Factor (). Cell sorting based on the LSC marker GPR56 allowed isolation of triple-mutated from double-mutated subclones.
View Article and Find Full Text PDFMol Ther Methods Clin Dev
September 2018
Retroviral integration site analysis and barcoding have been instrumental for multiplex clonal fate mapping, although their use imposes an inherent delay between sample acquisition and data analysis. Monitoring of multiple cell populations in real time would be advantageous, but multiplex assays compatible with flow cytometric tracking of competitive growth behavior are currently limited. We here describe the development and initial validation of three generations of lentiviral fluorescent genetic barcoding (FGB) systems that allow the creation of 26, 14, or 6 unique labels.
View Article and Find Full Text PDFThe hematopoietic syndrome of acute radiation syndrome (h-ARS) is characterized by severe bone marrow aplasia, resulting in a significant risk for bleeding, infections, and death. To date, clinical management of h-ARS is limited to supportive care dictated by the level of radiation exposure, with a high incidence of mortality in those exposed to high radiation doses. The ideal therapeutic agent would be an immediately available, easily distributable single-agent therapy capable of rapid in vivo hematopoietic reconstitution until recovery of autologous hematopoiesis occurs.
View Article and Find Full Text PDFCord blood transplantation (CBT) recipients are at increased risk for delayed engraftment and primary graft failure, complications that are often indistinguishable early post-transplantation. Current assays fail to accurately identify recipients with slow hematopoietic recovery and distinguish them from those with pending graft failure. To address this, we prospectively examined the kinetics of immune cell subset recovery in the peripheral blood of 39 patients on days +7 and +14 after double-unit CBT (dCBT) by multiparametric flow cytometry analysis, which we term real-time immunophenotyping (RTIP).
View Article and Find Full Text PDFHerein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light-inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells.
View Article and Find Full Text PDFAcute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy, which is initiated and driven by a rare fraction of leukemia stem cells (LSCs). Despite the difficulties of identifying a common LSC phenotype, there is increasing evidence that high expression of stem cell gene signatures is associated with poor clinical outcome. Identification of functionally distinct subpopulations in this disease is therefore crucial to dissecting the molecular machinery underlying LSC self-renewal.
View Article and Find Full Text PDFLeukemic transformation of human cells is a complex process. Here we show that forced expression of MN1 in primitive human cord blood cells maintained on stromal cells in vitro induces a transient, but not serially transplantable, myeloproliferation in engrafted mice. However, cotransduction of an activated HOX gene (NUP98HOXD13) with MN1 induces a serially transplantable acute myeloid leukemia (AML).
View Article and Find Full Text PDFThe small number of hematopoietic stem and progenitor cells in cord blood units limits their widespread use in human transplant protocols. We identified a family of chemically related small molecules that stimulates the expansion ex vivo of human cord blood cells capable of reconstituting human hematopoiesis for at least 6 months in immunocompromised mice. The potent activity of these newly identified compounds, UM171 being the prototype, is independent of suppression of the aryl hydrocarbon receptor, which targets cells with more-limited regenerative potential.
View Article and Find Full Text PDFAldehyde dehydrogenase (ALDH) activity is a widely used marker for human hematopoietic stem cells (HSCs), yet its relevance and role in murine HSCs remain unclear. We found that murine marrow cells with a high level of ALDH activity as measured by Aldefluor staining (ALDH(br) cells) do not contain known HSCs or progenitors. In contrast, highly enriched murine HSCs defined by the CD48(-)EPCR(+) and other phenotypes contain two subpopulations, one that stains dimly with Aldefluor (ALDH(dim)) and one that stains at intermediate levels (ALDH(int)).
View Article and Find Full Text PDFHigh levels of the aldehyde dehydrogenase isoform ALDH1A1 are expressed in hematopoietic stem cells (HSCs); however, its importance in these cells remains unclear. Consistent with an earlier report, we find that loss of ALDH1A1 does not affect HSCs. Intriguingly, however, we find that ALDH1A1 deficiency is associated with increased expression of the ALDH3A1 isoform, suggesting its potential to compensate for ALDH1A1.
View Article and Find Full Text PDFPre-B-cell leukemia homeobox interacting protein 1 or human PBX1 interacting protein (PBXIP1/HPIP) is a co-repressor of pre-B-cell leukemia homeobox 1 (PBX1) and is also known to regulate estrogen receptor functions by associating with the microtubule network. Despite its initial discovery in the context of hematopoietic cells, little is yet known about the role of HPIP in hematopoiesis. Here, we show that lentivirus-mediated overexpression of HPIP in human CD34(+) cells enhances hematopoietic colony formation in vitro, whereas HPIP knockdown leads to a reduction in the number of such colonies.
View Article and Find Full Text PDFObjective: Leukemia-initiating cells (LICs) have been the subject of considerable investigation because of their ability to self-renew and maintain leukemia. Thus, selective targeting and killing of LICs would provide highly efficient and novel therapeutic strategies. Here we explored whether we could use a canine leukemia cell line (G374) derived from a dog that received HOXB4-transduced repopulating cells to study leukemia in the murine xenograft model and the dog.
View Article and Find Full Text PDFObjective: Myeloid ectropic viral integration site 1 (MEIS1) is a Hox cofactor known for its role in development and is strongly linked to normal and leukemic hematopoiesis. Although previous studies have focused on identifying protein partners of MEIS1 and its transcriptionally regulated targets, little is known about the upstream transcriptional regulators of this tightly regulated gene. Understanding the regulation of MEIS1 is important to understanding normal hematopoiesis and leukemogenesis.
View Article and Find Full Text PDFCurr Protoc Stem Cell Biol
January 2008
Development of strategies to extensively expand hematopoietic stem cells (HSCs) in vitro will be a major factor in enhancing the success of a range of transplant-based therapies for malignant and genetic disorders. In addition to potential clinical applications, the ability to increase the number of HSCs in culture will facilitate investigations into the mechanisms underlying self-renewal. In this unit, we describe a robust strategy for consistently achieving over 1000-fold net expansion of HSCs in short-term in vitro culture by using novel engineered fusions of the N-terminal domain of nucleoporin 98 (NUP98) and the homeodomain of the hox transcription factor, HOXA10 (so called NUP98-HOXA10hd fusion).
View Article and Find Full Text PDFBackground And Objectives: Primitive human hematopoietic cells contain higher levels of aldehyde dehydrogenase (ALDH) activity than their terminally differentiating progeny but the particular stages when ALDH levels change have not been well defined. The objective of this study was to compare ALDH levels among the earliest stages of hematopoietic cell differentiation and to determine whether these could be exploited to obtain improved purity of human cord blood cells with long-term lympho-myeloid repopulating activity in vivo.
Design And Methods: ALDEFLUOR-stained human cord blood cells displaying different levels of ALDH activity were first analyzed for co-expression of various surface markers.
Transplantation of genetically corrected autologous hematopoietic stem cells is an attractive approach for the cure of sickle-cell disease and beta-thalassemia. Here, we infected human cord blood cells with a self-inactivating lentiviral vector encoding an anti-sickling betaA-T87Q-globin transgene and analyzed the transduced progeny produced over a 6-month period after transplantation of the infected cells directly into sublethally irradiated NOD/LtSz-scid/scid mice. Approximately half of the human erythroid and myeloid progenitors regenerated in the mice containing the transgene, and erythroid cells derived in vitro from these in vivo-regenerated cells produced high levels of betaA-T87Q-globin protein.
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