Inhibition of GATE-16 attenuates ATRA-induced neutrophil differentiation of APL cells and interferes with autophagosome formation

Autophagy is an intracellular bulk degradation process involved in cell survival upon stress induction, but also with a newly identified function in myeloid differentiation. The autophagy-related (ATG)8 protein family, including the GABARAP and LC3 subfamilies, is crucial for autophagosome biogenesis. In order to evaluate the significance of the GABARAPs in the pathogenesis of acute myeloid leukemia (AML), we compared their expression in primary AML patient samples, CD34⁺ progenitor cells and in granulocytes from healthy donors. GABARAPL1 and GABARAPL2/GATE-16, but not GABARAP, were significantly downregulated in particular AML subtypes compared to normal granulocytes. Moreover, the expression of GABARAPL1 and GATE-16 was significantly induced during ATRA-induced neutrophil differentiation of acute promyelocytic leukemia cells (APL). Lastly, knocking down GABARAPL2/GATE-16 in APL cells attenuatedneutrophil differentiation and decreased autophagic flux. In conclusion, low GABARAPL2/GATE-16 expression is associated with an immature myeloid leukemic phenotype and these proteins are necessary for neutrophil differentiation of APL cells.     

Proteinase-Activated Receptor 1 (PAR1) Regulates Leukemic Stem Cell Functions

External signals that are mediated by specific receptors determine stem cell fate. The thrombin receptor PAR1 plays an important role in haemostasis, thrombosis and vascular biology, but also in tumor biology and angiogenesis. Its expression and function in hematopoietic stem cells is largely unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. AML patients'' blast cells expressed much lower levels of PAR1 mRNA and protein than CD34⁺ progenitor cells. Constitutive Par1-deficiency in adult mice did not affect engraftment or stem cell potential of hematopoietic cells. To model an AML with Par1-deficiency, we retrovirally introduced the oncogene MLL-AF9 in wild type and Par1^−/− hematopoietic progenitor cells. Par1-deficiency did not alter initial leukemia development. However, the loss of Par1 enhanced leukemic stem cell function in vitro and in vivo. Re-expression of PAR1 in Par1^−/− leukemic stem cells delayed leukemogenesis in vivo. These data indicate that Par1 contributes to leukemic stem cell maintenance.     

Targeting Aberrant Glutathione Metabolism to Eradicate Human Acute Myelogenous Leukemia Cells

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34⁺) leukemic versus normal specimens. Our data indicate that CD34⁺ AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34⁺ AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34⁺ cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34⁺ AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34⁺ cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells.     

Characteristics of Myeloid Differentiation and Maturation Pathway Derived from Human Hematopoietic Stem Cells Exposed to Different Linear Energy Transfer Radiation Types

Exposure of hematopoietic stem/progenitor cells (HSPCs) to ionizing radiation causes a marked suppression of mature functional blood cell production in a linear energy transfer (LET)- and/or dose-dependent manner. However, little information about LET effects on the proliferation and differentiation of HSPCs has been reported. With the aim of characterizing the effects of different types of LET radiations on human myeloid hematopoiesis, in vitro hematopoiesis in Human CD34⁺ cells exposed to carbon-ion beams or X-rays was compared. Highly purified CD34⁺ cells exposed to each form of radiation were plated onto semi-solid culture for a myeloid progenitor assay. The surviving fractions of total myeloid progenitors, colony-forming cells (CFC), exposed to carbon-ion beams were significantly lower than of those exposed to X-rays, indicating that CFCs are more sensitive to carbon-ion beams (D ₀ = 0.65) than to X-rays (D ₀ = 1.07). Similar sensitivities were observed in granulocyte-macrophage and erythroid progenitors, respectively. However, the sensitivities of mixed-type progenitors to both radiation types were similar.In liquid culture for 14 days, no significant difference in total numbers of mononuclear cells was observed between non-irradiated control culture and cells exposed to 0.5 Gy X-rays, whereas 0.5 Gy carbon-ion beams suppressed cell proliferation to 4.9% of the control, a level similar to that for cells exposed to 1.5 Gy X-rays. Cell surface antigens associated with terminal maturation, such as CD13, CD14, and CD15, on harvest from the culture of X-ray-exposed cells were almost the same as those from the non-irradiated control culture. X-rays increased the CD235a⁺ erythroid-related fraction, whereas carbon-ion beams increased the CD34⁺CD38⁻ primitive cell fraction and the CD13⁺CD14^+/−CD15⁻ fraction. These results suggest that carbon-ion beams inflict severe damage on the clonal growth of myeloid HSPCs, although the intensity of cell surface antigen expression by mature myeloid cells derived from HSPCs exposed to each type of radiation was similar to that by controls.     

Reducing TGF‐β1 cooperated with StemRegenin 1 promoted the expansion ex vivo of cord blood CD34+ cells by inhibiting AhR signalling

ObjectiveAs an inhibitor of the AhR signalling pathway, StemRegenin 1 (SR1) not only promotes the expansion of CD34⁺ cells but also increases CD34⁻ cell numbers. These CD34⁻ cells influenced the ex vivo expansion of CD34⁺ cells. In this work, the effects of periodically removing CD34⁻ cells combined with SR1 addition on the ex vivo expansion and biological functions of HSCs were investigated.Materials and methodsCD34⁻ cells were removed periodically with SR1 addition to investigate cell subpopulations, cell expansion, biological functions, expanded cell division mode and supernatant TGF‐β1 contents.ResultsAfter 10‐day culture, the expansion of CD34⁺ cells in the CD34⁻ cell removal plus SR1 group was significantly higher than that in the control group and the SR1 group. Moreover, periodically removing CD34⁻ cells with SR1 addition improved the biological function of expanded CD34⁺ cells and significantly increased the percentage of self‐renewal symmetric division of CD34⁺ cells. In addition, the concentration of total TGF‐β1 and activated TGF‐β1 in the supernatant was significantly lower than those in the control group and the SR1 group. RT‐qPCR results showed that the periodic removal of CD34⁻ cells with cooperation from SR1 further reduced the expression of AhR‐related genes.ConclusionsPeriodic removal of CD34⁻ cells plus cooperation with SR1 improved the expansion of CD34⁺ cells, maintained better biological function of expanded CD34⁺ cells and reduced the TGF‐β1 contents by downregulating AhR signalling.

SR1 increased self‐renewal symmetric division of cord blood CD34⁺ cells. Removal of CD34⁻ cells cooperated with SR1 increased ex vivo expansion of cord blood CD34⁺ cells. Removal of CD34⁻ cells cooperated with SR1 further downregulated AhR signalling     

Mobilization of CD34~+CD38~- hematopoietic stem cells after priming in acute myeloid leukemia

AIM: To evaluate quantitatively and qualitatively the different CD34+cell subsets after priming by chemotherapy granulocyte colony-stimulating factor(± G-CSF)in patients with acute myeloid leukemia.METHODS: Peripheral blood and bone marrow sampleswere harvested in 8 acute myeloid leukemia patients during and after induction chemotherapy. The CD34/CD38 cell profile was analyzed by multi-parameter flow cytometry. Adhesion profile was made using CXC chemokine receptor 4(CXCR4)(CD184), VLA-4(CD49d/CD29) and CD47.RESULTS: Chemotherapy ± G-CSF mobilized immature cells(CD34+CD38 population), while the more mature cells(CD34+CD38lowand CD34+CD38+populations) decreased progressively after treatment. Circulating CD34+cells tended to be more sensitive to chemotherapy after priming with G-CSF. CD34+cell mobilization was correlated with a gradual increase in CXCR4 and CD47expression, suggesting a role in cell protection and the capacity of homing back to the marrow.CONCLUSION: Chemotherapy ± G-CSF mobilizes into the circulation CD34+bone marrow cells, of which, the immature CD34+CD38-cell population. Further manipulations of these interactions may be a means with which to control the trafficking of leukemia stem cells to improve patients’ outcomes.     

High Log-Scale Expansion of Functional Human Natural Killer Cells from Umbilical Cord Blood CD34-Positive Cells for Adoptive Cancer Immunotherapy

Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56⁺CD3⁻ NK cell products could be routinely generated from freshly selected CD34⁺ UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34⁺ UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56⁺ NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34⁺ cells for cancer immunotherapy.     

Tetraspanin Family Member,CD82, Regulates Expression of EZH2 via Inactivation of p38 MAPK Signaling in Leukemia Cells

PurposeWe recently found that the tetraspanin family member, CD82, which is aberrantly expressed in chemotherapy-resistant CD34⁺/CD38⁻ acute myelogenous leukemia (AML) cells, negatively regulates matrix metalloproteinase 9, and plays an important role in enabling CD34⁺/CD38⁻ AML cells to adhere to the bone marrow microenvironment. This study explored novel functions of CD82 that contribute to AML progression.ResultsMicroarray analysis revealed that levels of EZH2 decreased after shRNA-mediated depletion of CD82 in CD34⁺/CD38⁻ AML cells. Moreover, the antibody-mediated blockade of CD82 in leukemia cells lowered EZH2 expression via activation of p38 MAPK signaling, decreased the amount of EZH2 bound to the promoter regions of the tumor suppressor genes, and inhibited histone H3 lysine 27 trimethylation in these promoter regions, resulting in upregulation of the tumor suppressors at both the mRNA and protein levels.     

Intrahepatic transplantation of CD34+ cord blood stem cells into newborn and adult NOD/SCID mice induce differential organ engraftment

In vivo studies concerning the function of human hematopoietic stem cells (HSC) are limited by relatively low levels of engraftment and the failure of the engrafted HSC preparations to differentiate into functional immune cells after systemic application. In the present paper we describe the effect of intrahepatically transplanted CD34⁺ cells from cord blood into the liver of newborn or adult NOD/SCID mice on organ engraftment and differentiation.Analyzing the short and long term time dependency of human cell recruitment into mouse organs after cell transplantation in the liver of newborn and adult NOD/SCID mice by RT-PCR and FACS analysis, a significantly high engraftment was found after transplantation into liver of newborn NOD/SCID mice compared to adult mice, with the highest level of 35% human cells in bone marrow and 4.9% human cells in spleen at day 70. These human cells showed CD19 B-cell, CD34 and CD38 hematopoietic and CD33 myeloid cell differentiation, but lacked any T-cell differentiation. HSC transplantation into liver of adult NOD/SCID mice resulted in minor recruitment of human cells from mouse liver to other mouse organs. The results indicate the usefulness of the intrahepatic application route into the liver of newborn NOD/SCID mice for the investigation of hematopoietic differentiation potential of CD34⁺ cord blood stem cell preparations.     

Human Herpesvirus 6A Infection and Immunopathogenesis in Humanized Rag2?/?γc?/? Mice

Although serious human diseases have been correlated with human herpesvirus 6A (HHV-6A) and HHV-6B, the lack of animal models has prevented studies which would more definitively link these viral infections to disease. HHV-6A and HHV-6B have recently been classified as two distinct viruses, and in this study we focused specifically on developing an in vivo model for HHV-6A. Here we show that Rag2^−/−γc^−/− mice humanized with cord blood-derived human hematopoietic stem cells produce human T cells that express the major HHV-6A receptor, CD46. Both cell-associated and cell-free viral transmission of HHV-6A into the peritoneal cavity resulted in detectable viral DNA in at least one of the samples (blood, bone marrow, etc.) analyzed from nearly all engrafted mice. Organs and cells positive for HHV-6A DNA were the plasma and cellular blood fractions, bone marrow, lymph node, and thymic samples; control mice had undetectable viral DNA. We also noted viral pathogenic effects on certain T cell populations. Specific thymocyte populations, including CD3⁻ CD4⁺ CD8⁻ and CD3⁺ CD4⁻ cells, were significantly modified in humanized mice infected by cell-associated transmission. In addition, we detected significantly increased proportions of CD4⁺ CD8⁺ cells in the blood of animals infected by cell-free transmission. These findings provide additional evidence that HHV-6A may play a role in human immunodeficiencies. These results indicate that humanized mice can be used to study HHV-6A in vivo infection and replication as well as aspects of viral pathogenesis.     

Characterizing the Lymphopoietic Kinetics and Features of Hematopoietic Progenitors Contained in the Adult Murine Liver In Vivo

The appearance of donor-derived lymphocytes in liver transplant patients suggests that adult livers may contain cells capable of lymphopoiesis. However, only a few published studies have addressed the lymphopoietic capacity of adult liver cells, and its kinetics and features remain unclear. Herein, we investigated the lymphopoietic capacity of adult liver mononuclear cells (MNCs) and purified liver hematopoietic progenitor cells (HPCs) in vivo. Similar to bone-marrow transplantation (BMT), transplantation of liver MNCs alone was able to rescue survival of lethally irradiated mice. In terms of kinetics, liver MNC-derived myeloid lineage cells reconstituted more slowly than those from BMT. Liver MNC-derived lymphocyte lineage cells in the blood, spleen and BM also reconstituted more slowly than BMT, but lymphocytes in the liver recovered at a similar rate. Interestingly, liver MNCs predominantly gave rise to CD3⁺CD19⁻ T cells in both irradiated WT and non-irradiated lymphocyte-deficient Rag-1^−/−Il2rg^−/− recipients. To define the lymphopoietic potential of various cell populations within liver MNCs, we transplanted purified lineage-negative (Lin⁻) liver HPCs into recipient mice. Unlike total liver MNCs, liver HPCs reconstituted T and B cells in similar frequencies to BMT. We further determined that the predominance of T cells observed after transplanting total liver MNCs likely originated from mature T cells, as purified donor liver T cells proliferated in the recipients and gave rise to CD8⁺ T cells. Thus, the capacity of donor adult liver cells to reconstitute lymphocytes in recipients derives from both HPCs and mature T cells contained in the liver MNC population.     

Assessment of Human Multi-Potent Hematopoietic Stem/Progenitor Cell Potential Using a Single In Vitro Screening System

Hematopoietic stem cells are responsible for the generation of the entire blood system through life. This characteristic relies on their ability to self renew and on their multi-potentiality. Thus quantification of the number of hematopoietic stem cells in a given cell population requires to show both properties in the studied cell populations. Although xenografts models that support human hematopoietic stem cells have been described, such in vivo experimental systems remain restrictive for high throughput screening purposes for example. In this work we developed a conditional tetracycline inducible system controlling the expression of the human NOTCH ligand Delta-like 1 in the murine stromal MS5 cells. We cultured hematopoietic immature cells enriched in progenitor/stem cells in contact with MS5 cells that conditionally express Delta-like 1, in conditions designed to generate multipotential lineage differentiation. We show that upon induction or repression of DL1 expression during co-culture, human immature CD34⁺CD38^−/low(CD45RA⁻CD90⁺) cells can express their B, T, NK, granulo/monocytic and erythroid potentials in a single well, and at the single cell level. We also document the interference of low NOTCH activation with human B and myelo/erythroid lymphoid differentiation. This system represents a novel tool to precisely quantify human hematopoietic immature cells with both lymphoid and myeloid potentials.     

Leukemic Stem Cell Frequency: A Strong Biomarker for Clinical Outcome in Acute Myeloid Leukemia

Introduction

Treatment failure in acute myeloid leukemia is probably caused by the presence of leukemia initiating cells, also referred to as leukemic stem cells, at diagnosis and their persistence after therapy. Specific identification of leukemia stem cells and their discrimination from normal hematopoietic stem cells would greatly contribute to risk stratification and could predict possible relapses.

Results

For identification of leukemic stem cells, we developed flow cytometric methods using leukemic stem cell associated markers and newly-defined (light scatter) aberrancies. The nature of the putative leukemic stem cells and normal hematopoietic stem cells, present in the same patient''s bone marrow, was demonstrated in eight patients by the presence or absence of molecular aberrancies and/or leukemic engraftment in NOD-SCID IL-2Rγ-/- mice. At diagnosis (n = 88), the frequency of the thus defined neoplastic part of CD34+CD38- putative stem cell compartment had a strong prognostic impact, while the neoplastic parts of the CD34+CD38+ and CD34- putative stem cell compartments had no prognostic impact at all. After different courses of therapy, higher percentages of neoplastic CD34+CD38- cells in complete remission strongly correlated with shorter patient survival (n = 91). Moreover, combining neoplastic CD34+CD38- frequencies with frequencies of minimal residual disease cells (n = 91), which reflect the total neoplastic burden, revealed four patient groups with different survival.

Conclusion and Perspective

Discrimination between putative leukemia stem cells and normal hematopoietic stem cells in this large-scale study allowed to demonstrate the clinical importance of putative CD34+CD38- leukemia stem cells in AML. Moreover, it offers new opportunities for the development of therapies directed against leukemia stem cells, that would spare normal hematopoietic stem cells, and, moreover, enables in vivo and ex vivo screening for potential efficacy and toxicity of new therapies.