bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ex-Vivo Expansion of Human Cord Blood CD34+ Cells by Overexpression of Bmi-1.
Authors: Rizo Aleksandra , Vellenga Edo , de Haan Gerald , Schuringa Jan Jacob 
Published: April 2020

Abstract
Hematopoietic stem cells (HSCs) are able to self-renew and differentiate into cells of all hematopoietic lineages. Because of this unique property, they are used for HSC transplantations and could serve as a potential source of cells for future gene therapy. However, the difficulty to expand or even maintain HSCs ex vivo has been a major limitation for their clinical applications. Here, we report that overexpression of the Polycomb group gene Bmi-1 in human cord blood-derived HSCs can potentially overcome this limitation as stem/progenitor cells could be maintained in liquid culture conditions for over 16 weeks. In mouse studies, it has been reported that increased expression of Bmi-1 promotes HSC self-renewal, while loss-of-function analysis revealed that Bmi-1 is implicated in maintenance of the hematopoietic stem cells (HSC).
In a clinically more relevant model, using human cord blood CD34+ cells, we have established a long-term ex-vivo expansion method by stable overexpression of the Bmi-1 gene. Bmi-1-transduced cells proliferated in liquid cultures supplemented with 20% serum, SCF, TPO, Flt3 ligand, IL3 and IL6 for more than 4 months, with a cumulative cell expansion of more then 2×105-fold. The cells remained cytokine-dependent, while about 4% continued to express CD34 for over 20 weeks of culture. The cultured cells retained their progenitor activity throughout the long-term expansion protocol. The colony-forming units (CFUs) were present at a frequency of ~ 30 colonies per 10 000 cells 16 weeks after culture and consisted of CFU-GM, BFU-E and high numbers of CFU-GEMM type progenitors.
After plating the transduced cells in co-cultures with the stromal cell line MS5, Bmi-1 cells showed a proliferative advantage as compared to control cells, with a cumulative cell expansion of 44,9 fold. The non-adherent cells from the co-cultures gave rise to higher numbers of colonies of all types (~70 colonies/10.000 cells) after 4 weeks of co-culture. The LTC-IC frequencies were 5-fold higher in the Bmi-1-transduced cells compared to control cells (1/361 v.s. 1/2077, respectively). Further studies will be focused on in-vivo transplantation of the long-term cultured cells in NOD/SCID mice to test their repopulating capacity. In conclusion, our data implicate Bmi-1 as an important modulator of human HSC self-renewal and suggest that it can be a potential target for therapeutic manipulation of human HSCs.
The Hematopoietic Activity of the Homeobox Gene HOXB4 Depends on a N-Terminal Proline-Rich Region In Vitro and In Vivo.
Authors: Cusan Monica , Deshpande Aniruddha , Hiddemann Wolfgang , Humphries R. Keith , Buske Christian , Feuring-Buske Michaela 
Published: April 2020

Abstract
HOXB4 belongs to the family of homeobox transcription factors, which play a key role in hematopoietic development. The expression of HOXB4 induces a significant increase of long-term repopulating stem cells (SC) in human and mouse models, without perturbing hematopoietic differentiation. So far the underlying mechanisms of the SC amplificatory impact of HOXB4 are poorly understood. In an attempt to understand the unique characteristics of HOXB4, we performed a mutational study by deleting its proline - rich N-terminus, which has been described to act as a transcriptional activation domain in many other proteins, like non-homeobox genes (e.g. p53, AP2) and other homeobox genes (e.g. HOXD4 and HOXA13). We performed in vitro and in vivo experiments transducing murine 5-FU enriched HSCs with the pMSCV-IRES-GFP based retroviral vector harbouring the HOXB4 wild-type (wt) and several mutants, including a Δproline HOXB4 mutant (ΔP), where the proline-rich sequence between the aminoacidic positions 71–123 is deleted.
In vitro the ΔP-HOXB4 mutant led to a 3.2fold decrease in the number of hematopoietic cells harvested after 1 week of liquid expansion compared to the HOXB4 wt (n=3; p&lt;0.05). Furthermore, deletion of the proline - rich region resulted in a 7.5fold decrease in the total number of CFCs per 500 cells plated (n=3; p&lt;0.03), compared to the HOXB4 wt. The ΔP-HOXB4 mutant also generated less secondary colonies (18.5 versus 103 per 500 cells initially plated; n=2; p&lt;0.0001) compared to the HOXB4-wt. Of note and in contrast to the ΔP-HOXB4 mutant, the deletion of the PBX interacting domain (Pbx-HOXB4, aa W→A) or deletion of the C-terminal stretch (aa 221 −251; cDel-HOXB4) did not reduce the formation of primary colonies compared to HOXB4 wt (n=3; 178 and 124 versus 226 colonies per 500 cells initially plated, respectively). On the level of the short-term repopulating stem cells constitutive expression of HOXB4 wt (n=3) induced on average a 1,565fold increase in the frequency of ΔCFU-S in comparison to the GFP control (n=11) (10,645 versus 6.8 colonies/45,000 input cells; p&lt;0.0000001). The deletion on the N-terminal proline-rich region of HOXB4 (n=14) led to a significant decrease in the Δ-CFU-S frequency in comparison to the HOXB4-wt (mean 42fold; p&lt;0.000001), while it still generated significantly more Δ-CFU-S compared to the GFP control (252 versus 6.8/45,000 input cells; p&lt;0.001).
Taken together, these results characterize the N-terminal proline - rich stretch between aminoacidic positions 71–123 as a domain, which is highly relevant for the hematopoietic activity of HOXB4.
Differential Signaling of G Protein-Coupled Chemotactic Receptors (CXCR4, cysLT1 and S1P1) Expressed in Hematopoietic Progenitor Cells.
Authors: Xue Xingkui , Boehmler Andreas , Kanz Lothar , Seitz Gabriele , Mohle Robert 
Published: April 2020

Abstract
We have previously demonstrated consistent and high expression of the G protein-coupled receptors (GPCR) CXCR4, cysLT1 and S1P1 in human hematopoetic progenitor and stem cells (HPC). All of these receptors induced chemotactic responses in mobilized CD34+ HPC. As their ligands are present in the hematopoietic microenvironment, these GPCR may be involved in HPC homing and/or proliferation. Ligands for CXCR4 (SDF-1) and cysLT1 (cys-leukotrienes) are both produced by stromal cells, while the S1P1 ligand (sphingosine 1-phosphate, S1P) is released by megakaryocytes and platelets. As GPCR may activate multiple signaling pathways resulting in different functional effects, we compared the signaling capacities of CXCR4, cysLT1 and S1P1 in CD34+ HPC and CD34+ progenitor cell lines. At optimal ligand concentrations, cysLT1 mediated the strongest intracellular calcium fluxes, in contrast to CXCR4 (intermediate response) and S1P1 (almost absent calcium fluxes). Pertussis toxin (PTX) only partially inhibited cysLT1-mediated calcium signaling, indicating coupling of the receptor to both Gi and Gq proteins, while CXCR4-mediated responses were exclusively PTX-sensitive and therefore only Gi-dependent. Both cysLT1 and CXCR4 induced phosphorylation of the proliferation-related p44/42 (Erk) MAP kinase. Again, much stronger responses were mediated by cysLT1, which again were only partially PTX-sensitive. No Erk/MAP kinase activation was mediated by S1P1. We could demonstrate that pyk2, a kinase that links calcium signaling with Erk/MAP kinase activation, was involved in Gq-mediated signaling of cysLT1, as its phosphorylation was not inhibited by PTX. The fact that cysLT1 employs both Gi and Gq-mediated signaling pathways, while CXCR4 signaling was exclusively Gi-dependent, may explain why enhanced proliferation of CD34+ hematopoietic progenitor cells in cytokine-supplemented serum-free liquid cultures was only seen in response to ligands of cysLT1, but not in response to the CXCR4 ligand SDF-1. Activation of CXCR4 resulted in phosphorylation of Akt, which was not observed in response to ligands of cysLT1, while both GPCR induced translocation of NFkappaB to the nucleus. We conclude that in CD34+ HPC, Gi protein-mediated signaling of CXCR4 is mainly involved in cell migration, while Gi and Gq-mediated signaling of cysLT1 also affects cell proliferation predominantly by combined and profound activation of the Erk/MAP kinase pathway. The signaling capacity of S1P1 however was limited compared to the other GPCRs.
The GEF Lsc Regulates Hematopoietic Stem Cell Motility, Mobilization and Recruitment.
Authors: Petit Isabelle , Kaul Prashant , Lerner Daniel J. , Rafii Shahin 
Published: April 2020

Abstract
Lsc is a Rho GTPase guanine nucleotide exchange factor (RhoGEF) that physically and functionally links G-protein coupled receptors (GPCR) to the monomeric GTPase RhoA in mature hematopoietic and other cells. Lsc−/− (LscKO) mice have a peripheral leukocytosis, abnormal neutrophil and B cell motility, and immune response deficiencies. Although Lsc is required for neutrophil homeostasis, its role in hematopoietic stem and progenitor cells is unknown. In this study, we have used LscKO mice to determine if Lsc is required for normal stem cell motility and mobilization. Initially, we used immunofluorescence labeling to demonstrate that hematopoietic stem and progenitor cells express Lsc. This suggested that Lsc may be required for normal hematopoietic stem and progenitor cell migration. Stromal-cell derived factor-1 (SDF-1) is a potent chemokine for hematopoietic stem cells and activates the CXCR4 GPCR. It has been reported that Lsc is not required for SDF-1-stimulated migration of mature murine T and B cells. However, using a bare-filter transwell assay, we found that while LscKO Sca-1+ cells and Sca-1+Lin- cells have normal spontaneous migration, they have significantly increased SDF-1-stimulated migration compared to their wild-type (WT) counterparts, 1.4 and 2.3 fold, respectively. We then demonstrated that adhesion of LscKO Sca-1+ cells to bone marrow (BM) stromal MS-5 cells was normal, indicating that impaired adhesion was not responsible for the abnormal SDF-1-stimulated migration. Using colony assay, we demonstrated that LscKO mice have a normal number of circulating peripheral stem and progenitor cells. Strikingly, after 5 days of G-CSF administration, LscKO mice have 1.6 fold and 2.3 fold the number of peripheral mature WBC and stem and progenitor cells (colony forming units), respectively, compared to WT mice. Recruitment of BM CXCR4+ pro-angiogenic stem and progenitor cells has been linked to enhanced tumor angiogenesis. Because LscKO BM cells had abnormal SDF-1-stimulated migration and mobilization, we hypothesized that Lsc might regulate tumor angiogenesis as well. To this end, we assessed tumor growth in LscKO mice by injecting congenic Lewis lung carcinoma cells subcutaneously into LscKO mice and WT controls. Preliminary experiments revealed that tumors were 3.3 times larger in the LscKO mice as compared to WT mice. Quantification of the tumor vessels with anti-CD31 staining demonstrated that the tumors in LscKO mice were 1.4 fold more vascularized than controls. In summary, our results demonstrate that the Rho GEF Lsc is essential for normal hematopoietic stem cell migration and mobilization. In addition, we propose that absence of Lsc facilitates tumor growth by promoting BM stem and progenitor cell recruitment to the neo-angiogenic vessels, possibly augmenting tumor vascularization.
A NUP98-HOX Fusion Gene Containing the Homeodomain of HOXA10 Promotes Significant Expansion of Primitive Human Hematopoietic Cells in Extended Cultures.
Authors: Imren Suzan , Sauvageau Guy , Eaves Connie J. , Humphries R. Keith 
Published: April 2020

Abstract
Expanding human hematopoietic stem cells (HSCs) in vitro is a major goal in clinical hematology but remains a major challenge due to the potent differentiating activity of known cytokines. We recently demonstrated that a NUP98-HOX fusion gene containing only the homeodomain (hd) of HOXA10 (NUP98-HOXA10hd) is a powerful stimulator of murine HSC expansion in vitro - causing &gt;1000-fold net HSC increases in 10 days (Sekulovic et al, ISEH 2005). To investigate the proliferative effect of NUP98-HOXA10hd on primitive human hematopoietic cells, highly enriched CD34+ cord blood cells were prestimulated overnight and exposed to self-inactivating MNDUSNUP98-HOXA10hd pgkGFP or control pgkGFP lentiviruses for 6h. The gene transfer efficiency into CD34+ cells determined 4 days after infection was 56 ± 5% for NUP98-HOXA10hd and 66 ± 5% for the GFP control. GFP+ cells were sorted on day 5 and then maintained for another 5 days in serum-free cultures containing Flt3-ligand, Steel factor, IL-3, IL-6 and G-CSF. An aliquot of each was then plated into “primary” colony-forming cell (CFC) assay cultures. No difference was detected in either the numbers or the types of colonies generated in these primary CFC assays of the 10-day cultured cells from the NUP98-HOXA10hd and control arms. However, when these primary CFC assays were replated into secondary CFC assays, the number of colonies obtained from the NUP98-HOXA10hd-transduced cells was 5-fold higher as compared to the GFP-control transduced cells and, upon replating into tertiary CFC assays, this difference increased to over a 100-fold. To determine the effect of NUP98-HOXA10hd on more primitive hematopoietic cells, 104 day-10 GFP+ cells were co-cultured on mouse fibroblast feeders engineered to produce human SF, IL-3 and G-SCF. At the end of 6 weeks, 13-fold more cells were recovered from the cultures initiated with NUP98-HOXA10hd-transduced cells than from the control cultures (474,000 ± 190,000 vs 37,000 ± 16,000, 3 experiments). CFC outputs were also greatly enhanced (21-fold more CFC than in the controls cultures, range=20–80, 3 experiments). Moreover, the proportion of progenitors in the assays of the cultures initiated with NUP98-HOXA10hd cells that were multi-lineage (CFU-GEMM) was &gt;10-fold higher as compared to the CFCs obtained from the control cultures (8 ± 3% vs 0.7 ± 0.7%). When this experiment was repeated using limiting dilutions of initial day-10 cells, the frequency of NUP98-HOXA10hd-transduced cells able to generate CFCs another 6 weeks later was 10-fold higher as compared to the day-10 GFP control-transduced cells. These findings document an unprecedented potency of NUP98-HOXA10hd for stimulating the ex-vivo expansion of very primitive pluripotent human hematopoietic cells.
Bcr-Abl Inhibitors Induce Expression of HoxA10, Playing a Role as an Inhibitor of the Proliferation through PI3K/PKB Pathway in Chronic Myelogenous Leukemia Cells.
Authors: Sugimoto Yuya , Nakamura Satoki , Ono Takaaki , Kobayashi Miki , Sahara Naohi , Shinjo Kaori , Shigeno Kazuyuki , Ohnishi Kazunori 
Published: April 2020

Abstract
[Background] Homeobox (Hox) genes are grouped together in 4 clusters, A to D. Recent studies has shown that the Hox proteins are important in the control of differentiation and proliferation in hematopoietic cells. It is reported that HoxA10 has been expressed in all types acute myeloid leukemias (except for APL) but not in acute lymphoid leukemias, implicating an important role as a regulator of myeloid progenitors. Moreover, overexpression of HoxA10 induses the proliferation of early progenitor cells and leads to the development of myeloid leukemias. However, we found that Bcr-Abl inhibitors increased the expression of HoxA10 gene in CML cells. In this study, we investigated the mechanism controlling HoxA10 in CML cells treated with Bcr-Abl inhibitors.
[Methods] The cells used in this study were Ph1 positive CML cells, K562 and Meg01, and U937 cells for a Ph1 negative cell. We used AMN107 and BMS354825 for Bcr-Abl inhibitors, LY294002 for a PI3K inhibitor, PP2 for a Src kinase inhibitor, and SB203580 for a p38 MAP kinase inhibitor. For analysis of HoxA10 mRNA and protein, RT-PCR and western blot were performed in K562, Meg-01 and U937 cells, which untreated or treated with AMN107, BMS354825, LY294002, PP2, or SB203580 respectively. We then attempted to localize the intracellular locations of HoxA10 in K562 and Meg01, which untreated or treated with AMN107, BMS354825, or LY294002 by using conforcal fluorescence microscopy. Finally, for analysis of proliferation in K562, Meg-01 and U937 transfected with siRNA HoxA10, MTT assays were performed in untransfected or transfected K562, Meg-01 and U937 treated with or without AMN107, BMS354825, orLY294002.
[Results] Both K562 and Meg01 cells expressed HoxA10 mRNA and protein at lower level compared to U937 cells. Interestingly, treatment with AMN107, BMS354825, or LY294002 increased the expression of HoxA10 mRNA and protein in both K562 and Meg01 cells. In contrast, treatment with PP2 or SB203580 did not affect the expression of HoxA10 mRNA and protein in both K562 and Meg01 cells. The fluorescence of HoxA10 was more strongly observed in the area corresponding to the cell’s cytoplasm than nucleus, and the treatment with AMN107, BMS354825, or LY294002 increased the fluorescence in nucleus of K562 and Meg01 cells in a time-dependent manner. In K562 and Meg01 cells transfected with the siRNA HoxA10, treatment with AMN107 or BMS354825 slightly inhibited the proliferation compared to K562 and Meg01 transfected with control siRNA.
[Conclusions] In this study, we showed that Bcr-Abl inhibitors induced the expression of HoxA10, and HoxA10 was regulated by PI3K/P[Kappa[B pathway in CML cells. This finding indicates a new insight in the regulation of cell proliferation via the PI3K/PKB signal pathway in CML cells.
Generation of Specific Resistance Profiles in FLT3-ITD and FIP1L1-PDGFRalpha towards Specifically Acting Tyrosine Kinase Inhibitors.
Authors: von Bubnoff Nikolas , Thoene Silvia , Gorantla Sivahari P. , Saenger Jana , Peschel Christian , Duyster Justus 
Published: April 2020

Abstract
BCR-ABL kinase domain mutations constitute the major mechanism of resistance in patients with chronic myelogenous leukemia treated with the ABL kinase inhibitor imatinib. Mutations causing resistance to therapeutic kinase inhibition were also identified in other target kinases in various malignant diseases, such as FLT3-ITD in acute myelogenous leukemia, cKit in gastrointestinal stromal tumors, EGFR in patients with lung cancer, and FIP1L1-PDGFRalpha in hypereosinophilic syndrome. Thus, mutations in kinase domains seem to be a general mechanism of resistance to therapeutically applicated tyrosine kinase inhibitors. We recently developed a cell-based screening strategy that allows one to predict the pattern and relative abundance of BCR-ABL resistance mutations emerging in the presence of imatinib, and the novel ABL kinase inhibitor AMN107 (nilotinib). We therefore intended to determine, if this method would also allow the generation of resistant cell clones with other oncogeneic tyrosine kinases as targets in the presence of specifically acting kinase inhibitors. When FLT3-ITD and su5614 were used as drug/target combination in our cell-based method, the frequency of resistant clones in the presence of su5614 at 10 times the IC50 was 0.17 per million cells. In 40 per cent of resistant clones, point mutations were detected leading to amino acid exchanges within the FLT3-ITD split kinase domain. The yield of resistant clones was increased by the factor of 14 to 2.37 per million cells by adding ethyl-nitrosourea (ENU), a potent inducer of point mutations. Also, the proportion of mutant clones increased from 40 to 74 per cent. In 83 mutant clones that were examined so far, we detected eight exchanges affecting kinase domain two (TK2) of the split kinase domain within or shortly behind the FLT3-ITD activation loop (A-loop). We did not detect exchanges affecting TK1. We next examined whether resistant clones would also come up with FIP1L1-PDGFRalpha-transformed cells in the presence of imatinib. Again, the yield of resistant clones increased when cells were pretreated with ENU, and a proportion of resistant clones contained mutations in the FIP1L1-PDGFRalpha kinase domain, affecting the nucleotide-binding loop (P-loop) and A-loop. We conclude that cell-based resistance screening is a simple and powerful tool that allows prediction of resistance mutations towards kinase inhibitors in various relevant oncogeneic kinases.
Combination of Imatinib and Hypusination Inhibitors Represents a Novel Therapeutic Strategy in Bcr-Abl Positive Leukemias.
Authors: Balabanov Stefan , Gontarewicz Artur , Ziegler Patrick , Hartmann Ulrike , Copland Mhairi , Brassat Ute , Hauber Ilona , Kanz Lothar , Bokemeyer Carsten , Nordheim Alfred , Hauber Joachim , Holyoake Tessa L. , Brummendorf Tim H. 
Published: April 2020

Abstract
In order to identify new cellular Bcr-Abl downstream targets, we have recently detected differential regulation of eukaryotic initiation factor 5A (eIF-5A) in Bcr-Abl positive cells. EIF-5A represents the only known eukaryotic protein activated by post-translational hypusination. Hypusination inhibitors (HI) GC7 and ciclopirox alone exerted an anti-proliferative effect on Bcr-Abl positive (K562, BA/F3p210) and negative human leukemia cell lines (HL-60, BA/F3) in vitro. No direct effect of HI treatment on phosphorylation of Bcr-Abl downstream targets (global p-tyrosination, p-CRKL, p-STAT5) was observed. However, the synergistic dose-effect relationship for apoptosis induction, cytotoxicity and inhibition of proliferation found for the combination of Imatinib and HI was restricted to Bcr-Abl-positive cells. Furthermore, this synergistic effect was confirmed by cytotoxicity assays, cell cycle analysis and CFSE-labeling of primary CD34+ CML cells in comparison to CD34+ cells from healthy donors. In parallel to these findings, eIF-5A mRNA is expressed at clearly (if significant then say significant and add p-value, otherwise delete clearly) elevated levels in PBMCs from CML patients at different disease stages when compared with PBMCs from healthy donors. Specificity of this effect could be demonstrated by co-treatment of K562 cells with Imatinib and a stable lentiviral siRNA against eIF-5A. Based on this observation, we observed a strong antiprolifrative effect of HI as single agents on BA/F3p210 cells, including the Imatinib-resistant mutants T315I, M351T or E255K. Furthermore, a combination of Imatinib and HI had a synergistic effect in inducing cytotoxicity, apoptosis and inhibition of proliferation in leukemic cell lines with moderate resistance to imatinib (M351T). In contrast, in BA/F3p210T315I (T315I) cells, which are resistant to Imatinib as a single agent, the co-treatment of Imatinib and HI did not lead to additive or synergistic effects. In conclusion, we have identified inhibition of eIF-5A hypusination as a promising new approach for single agent or combination therapy in Bcr-Abl