bluuebunny/crossref_metadata_2025_split huggingface dataset:
SALL4, a Novel Oncogene Induces Myelodysplastic Syndrome and Acute Myeloid Leukemia Via Wnt/β-Catenin Pathway.
Authors: Chai Li , Cui Wei , Chang Jianchang , Di Chunhui , Amin Hesham , Ma Yupo 
Published: April 2020

Abstract
SALL4, a gene homologous to the Drosophila homeotic spalt, is a zinc finger transcriptional factor essential for human development. In Drosophila, spalt is regulated by the Wnt signaling pathway, a pathway critical for hematopoietic self-renewal of stem cells. We cloned SALL4 and its isoforms (SALL4A and SALL4B). We used immunohistochemistry, to demonstrate that SALL4 was constitutively expressed in primary acute myeloid leukemia cells (French American British, FAB: M1 to M5, N=81). The SALL4 in RNA was quantitative in bone marrow cells derived from acute myeloid leukemia (AML) and compared to non-neoplastic hematopietic cells from a purified CD34 progenitor pool, normal bone marrow and peripheral blood by RT-PCR. SALL4 expression was present in the purified normal CD34+ cells, AML blasts, but absent in mature myeloid cells. We tested the leukemogenic potential of constitutive overexpression of SALL4 in a murine hematopoietic model. All transgenic mice overexpressing SALL4B using the CMV promoter developed hematopoietic disorders, including myelodysplastic (MDS)-like symptoms and an AML transformation that disseminated to peripheral tissues including the spleen, liver, kidney and lymph nodes. The myelodysplastic features in these transgenic mice were pathologically similar to human MDSs and manifested as ineffective myelopoiesis. Dysplatic features such as Pseudo-Pelger-Huet like atypical white cells, and increased immature cells were detectable in the transgenic mouse peripheral blood. A high level of apoptosis and increased immature cells were also evident in transgenic mouse marrows and day-7 colony-forming unit assays in vitro. Both SALL4A and SALL4B were able to bind to β-catenin in vitro and synergistically enhanced the Wnt/β-catenin signaling pathway in a reporter gene assay. Our data suggests that the constitutive expression of SALL4 is causal to the leukemic phenotype. Our model should provide a useful platform to analyze the interaction of SALL4 with Wnt/β-catenin pathway in leukemia stem cells.
Hematopoietic Stem Cells Actively Extrude Imatinib-Mesylate Due to High ABCG2-Transporter Activity.
Authors: Scharenberg Christian , Gessner Michaela , Hoffmann Joerg , Robey Robert , Neubauer Andreas , Brendel Cornelia 
Published: April 2020

Abstract
The tyrosine kinase inhibitor imatinib mesylate (STI571) is a potent therapeutic agent for treatment of chronic myeloid leukemia (CML) due to its specific inhibition of bcr-abl kinase. However, patients with CML always relapse after withdrawal of imatinib therapy. With CML being the paradigm for a stem cell disease, we sought to investigate the influence of imatinib on the most primitive stem cell compartment.
Imatinib has recently been reported to be a high affinity substrate for the ABC-transporters ABCG2/BCRP (breast cancer resistance protein) and MDR1 (P-Glycoprotein). Given the high expression of these ABC-transporters in Hematopoietic Stem Cells (HSC), we examined the influence of imatinib on ABCG2- and MDR1-activity in human and murine HSC by using Hoechst- and Rhodamine-efflux assays. MDR1-mediated Rhodamine-efflux was only mildly influenced by Imatinib. However, addition of imatinib at therapeutic dosages completely abrogated the SP phenotype of total bone marrow after Hoechst 33342 staining. This effect was even more pronounced in cells of the HSC-phenotype, i.e., lineage-negative/AC133+ or c-kit+/Sca-1+/lin− (KSL) of human and murine marrow, respectively. In order to determine the effect of imatinib on stem cells in vivo, we isolated SP cells from 13 CML patients at various stages of disease using FACS. We employed quantitative RT-PCR-analysis for bcr-abl and demonstrated that the majority of CML patients had bcr-abl-negative SP cells, while peripheral blood mononuclear cells were mostly bcr-abl-positive. However, two patients in complete cytogenetic and molecular-genetic remission proved to be bcr-abl-positive within the SP cell population. This supports the notion that continuous imatinib-therapy may not eradicate the malignant stem cell pool and the leukemic clone may be able to expand even during permanent imatinib-therapy.
Furthermore, these data imply that the detoxifying function of ABC transporters on stem cells may be altered under imatinib treatment. Using HEK-293 cells transfected with ABCG2, we demonstrate that imatinib reverses mitoxantrone-resistance - potentially due to high affinity substrate inhibition.
In summary, our data suggest the novel resistance mechanism of imatinib being actively extruded from primitive leukemic stem cells, and that addition of chemotherapeutic agents to imatinib therapy may facilitate the eradication of bcr-abl-positive stem cells in chronic myeloid leukemia.
The Role of RUNX1 DNA-Binding Mutations in Acute Myeloid Leukemia.
Authors: Cammenga Jorg , Putz Gabriela , Niebuhr Birte , Horn Stefan , Bergholz Ulla , Buchholz Frank , Stocking Carol 
Published: April 2020

Abstract
The RUNX1 gene encodes an alpha subunit of the core-binding factor (CBF), an important heterodimeric transcription factor in hematopoietic ontogeny and development, and is one of the most frequently disrupted genes in acute leukemia. In addition to its involvement in several translocations, the RUNX1 gene is often subject to deletions or point mutations in acute myelogenous leukemia (AML). Interestingly, in addition to complete loss-of-function mutations, many of the alterations involve missense point mutations within the Runt domain that disrupt DNA binding activity (DB-mutants). In vitro assays have suggested that these DB mutants have a dominant-negative (DN) activity, presumably due to their ability to bind and sequester CBF beta but inability to bind DNA. A strict correlation between the type of mutation and its monoallelic or biallelic incidence is not apparent even though a DN mutant should only affect one allele while a loss of function mutation should affect both alleles. It has been hypothesized that loss of one allele (haploinsufficiency) is sufficient for loss of tumor suppressor activity but the relative high incidence of specific DB mutations suggests a more complex scenario. We thus sought to determine if expression of DB mutants in murine bone marrow (BM) resulted in a similar phenotype as the loss of Runx1, or if these mutations are associated with a gain-of-function. Two RUNX1-DB mutants were thus evaluated using the established retroviral transduction/transplantation mouse model. Between 3 and 6 months after transplantation, peripheral blood, spleen and BM cells were analyzed. Long-term repopulating cells expressing RUNX1 DB-mutants were able to contribute normally to both myeloid and lymphoid compartments, although a disproportionate increase in the B-cell compartment was observed in 3 out of 10 mice. Surprisingly, and inconsistent with a DN activity, disruption of normal T-cell or megakaryocytic development was not observed in the mice, in contrast to Runx1−/+ mice. Significantly, however, replating assays in vitro demonstrated that RUNX1-DB mutants lead to a significant increase in self-renewal activity, in contrast to BM cells of floxed Runx1 mice expressing the Cre recombinase, which showed a less dramatic effect on self-renewal. Colonies derived from CFU-Cs expressing RUNX1-DB mutants were composed of dysplastic granulocytic and monocytic cells, with an increasing number of immature blasts after multiple replatings (&gt;7), whereas residual colonies from Runx1fl/− BM receiving CRE showed a different morphology with more mature cells. Thus our data suggest that RUNX1-DB mutants do not act in a dominant negative fashion to inhibit normal RUNX1 function, but impart a gain-of-function that results in impaired myeloid differentiation and increased self-renewal potential, consistent with its association with AML.
Dominant and Pharmacologically Sensitized ENU Mutagenesis Screens Uncover Novel Regulators of Hematopoiesis and Model Hematopoietic Disease.
Authors: Stanford William L. , Anderson Nicole M. , Milenkovic Zorana , Zitano Lia , Adamson Lee , Wang Chen , Cybulsky Myron , McNagny Kelly , Paulson Robert F. , Barber Dwayne L. , Minden Mark D. 
Published: April 2020

Abstract
To generate new mouse models of human hematopoietic disease and increase our knowledge of the genetic networks that control hematopoiesis, we are performing dominant (generation 1 or G1) and pharmacologically-sensitized forward ethylnitrososurea (ENU) mutagenesis screens. Mice are phenotyped by saphenous vein peripheral blood analysis using an automated hematological analyzer. ENU is ideally suited to generating models of human disease and annotating gene function because the spectrum of mutations (point mutations generating leading to subtle amino acid substitutions, splicing errors, or premature termination) are similar to those often found in human disease. Furthermore, null mutations often do not represent the full extent of a gene’s function, requiring multiple alleles to fully define gene function. While dominant mutations can unequivocally cause some human diseases, often mutations in multiple genes interact and contribute to disease progression. Thus, we have developed sensitized screens that induce transient cytopenias using various pharmacological agents (5-fluorouracil, phenylhydrazine, and hydroxyurea) and analyzing the recovery in peripheral blood levels of red blood cells, white blood cells and platelets. This strategy enables identification of hematopoietic mutants that do not present abnormal blood cell counts in a homeostatic state. The induced cytopenia recovery assay is also being used as a secondary phenotyping assay for some of our G1 dominant mutants. The combined dominant and sensitized screens have yielded 14 heritable dominant mutants to date plus four additional mutants in hereditary testing. The array of mutations that we are analyzing are models for the following diseases: polycythemia, thrombocythemia, leukocytosis, anemia, and thrombocytopenia. I will discuss the progress of the mutagenesis screen and several ENU mutants, including a novel mutation in the protein tyrosine kinase Jak2, leading to thrombocythemia. This point mutation in the protein kinase domain will help us to dissect the recently discovered role of Jak2 in Myeloproliferative Diseases including Essential Thrombocythemia.
Differentiation of Human Embryonic Stem Cells into Mesenchymal Stem Cells.
Authors: Olivier Emmanuel N. , Rybicki Anne C. , Bouhassira Eric E. 
Published: April 2020

Abstract
Mesenchymal stem cells (MSC) are multipotent progenitors that contribute to the formation of many connective tissues including fat, bone, cartilage and muscle. Because of this great versatility MSCs have a large therapeutic potential particularly in the areas of cell therapy and regenerative and reconstructive medicine. MSCs are rare cells that can be isolated from tissues such as bone marrow, cartilage and muscles. Since no unique marker characteristic of MSCs has been identified, investigators have relied on a series of functional and morphological criteria to identify them. These criteria include growth on plastic, resistance to trypsin, presence of specific cell surface antigens and potential to differentiate into adipocytes, chondrocytes and osteoblasts.We report here a method to reproducibly differentiate human embryonic stem cells (hESCs) into MSCs by pre-differentiating for 8 days hESCs growing on MEF, mechanically dissociating the differentiated colonies, replating the differentiated colonies in DMEM, 10% FBS and 7.5% CO2 for 4 to 8 weeks until a thick multi-layer epithelium-like sheet of cells develop; and dissociating these multi-layer structures with a combination of proteases. The cells obtained with this procedure are morphologically similar to MSCs, are contact-inhibited, can be grown in culture for about 20–25 passages and have a gene expression profile, as determined by cDNA micro-array, similar to published profiles of mesenchymal stem cells. Immuno-phenotyping of these hESC-derived MSCs revealed a immuno-profile similar to bone marrow MSCs (negative for CD34, CD45 and positive for CD13, CD44, CD71, CD73, CD105, CD166, HLA ABC, SSEA4 and TRA1-85). Functional differentiation experiments revealed that hESC-derived MSCs can be differentiated into osteocytes and adipocytes as demonstrated by staining with alizarin red and oil red O. Finally, we have also shown that hESC-derived MSCs can support the growth of undifferentiated hESCs cells and of CD34+ hematopoietic cells.The ability to produce MSCs from hESCs should prove useful to produce large amount of genetically identical and genetically modifiable MSCS that can be used to study the biology of MSCs and for therapeutic applications.
Up-Regulation of NK Cell Activating Receptors Associated with Elevated IL-15 Levels Post-Transplant.
Authors: Boyiadzis Michael , Carson Jesse , Allen Kenton , Memon Sarfraz A. , Dean Robert A. , Odom Jeanne , Bishop Michael R. , Gress Ronald E. , Hakim Frances T. 
Published: April 2020

Abstract
NK cells express diverse activating and inhibitory receptors, which collectively determine the NK cytotoxic response. Because NK cells can be potent anti-tumor effectors in the post transplant period of minimal residual disease, we investigated the receptor expression on NK cells following non-myeloablative, HLA-matched, allogeneic hematopoietic stem cell transplant (HSCT). We focused on activating receptors, comparing the expression of natural cytotoxicity receptors (NCR) and C-type lectin receptors on circulating NK cells at one, three, six and twelve months following HSCT with that of the donor-derived mobilized stem cell apheresis. During the first post-transplant month, the absolute numbers of NK cells recovered to normal levels in the 14 patients studied, but the subpopulation of CD56++(bright) CD16− NK cells increased disproportionately compared to the more common CD56+(dull) CD16+ NK cells. By six months the proportions of the NK cell subsets normalized to pre-transplant levels. At one month post transplant, the median percentage of NK cells expressing the anti-tumor NCR NKp46 increased from 15 to 64% and that expressing NKp30 increased from 23 to 40% as compared to the donor’s NK cells; expression remained elevated during the first year. Expression of NKG2D, the homodimeric activating C-type lectin receptor, similarly increased post-transplant. CD94 was also upregulated on NK cells, but the activating heterodimeric partner of CD94, NKG2C, did not change significantly. To investigate these shifts in NK populations and receptor expression, we investigated the effect of cytokine milieu post transplant on these shifts in NK populations and receptor expression. We have determined that plasma levels of IL-15, a cytokine critical in NK differentiation and expansion, increase more than 50-fold in these HSCT patients from pretreatment to the day of transplant and decline in the first weeks post transplant, inversely proportional to NK recovery. When NK cells were cultured with rIL-15, we observed a disproportionate expansion of CD56++ NK cells and a rapid up-regulation of the NCR and NKG2D receptors, similar to the changes observed post-transplant. These data suggest that the cytokine milieu of transplant, in particular elevated levels of IL-15, may contribute to anti-tumor efficacy post transplant by affecting the recovery of NK subsets and modulating expression of activating receptors.
High Avidity Cyclin E-Derived Peptide-Specific CTL Contribute to Induction of Remission after Stem Cell Transplantation without Associated Graft-Versus-Host Disease.
Authors: Ishiyama Ken , Kondo Yukio , Wieder Eric , Lu Sijie , Molldrem Jeffrey 
Published: April 2020

Abstract
Cyclin E1 (CCNE1) and cyclin E2 (CCNE2) are cell cycle genes that are overexpressed in AML, ALL, and CML, and in solid tumors such as breast cancer, lung cancer, and gastric cancer. We reported that two homologous nonameric peptides from CCNE1 (CCNE1144-152, ILLDWLMEV) and CCNE2 (CCNE2144-152, ILLDWLLEV), which differ by a single amino acid at position 7, have equal binding affinity for HLA-A2 and that CCNE1- and CCNE2- specific CTL elicited from healthy donors kill ALL and CML cells that overexpress CCNE1 and CCNE2. Interestingly, CCNE1/A2 and CCNE2/A2 tetramers bind to both T-cell receptors (TCR), but the longer tetramer dissociation t1/2 of CCNE1/A2 compared with CCNE2/A2 tetramer suggests CCNE1 is the more dominant epitope. To determine the clinical significance of CTL immunity to these self-epitopes, we studied peripheral blood mononuclear cells from 18 patients before and after allogeneic stem cell transplantation (SCT) using CCNE1/A2 and CCNE2/A2 tetramers. An increase in the absolute number of circulating CCNE1- or CCNE2-CTL after SCT was considered evidence of an immune response (IR). Of the18 patients, there were 10 with CML and 8 with ALL, and all were HLA-A2+ and had sufficient blood samples cryopreserved from various time points before SCT and 3–12 months after SCT for analysis. Ten patients, including all 8 ALL patients, were in complete remission (CR) prior to SCT and 5 patients achieved CR after SCT. An IR to CCNE1 was found in 12 of 18 (67%) patients and an IR to CCNE2 was found in 14 of 18 (78%) patients. All 12 patients who had an IR to CCNE1 also had an IR to CCNE2, reflecting possible cross-recognition of both peptides by the same CTL clones. By Chi-square analysis, IR to either CCNE1 or CCNE2 did not correlate with diagnosis (CML vs. ALL), source of the graft (matched related donors vs. mismatched donors), or disease status prior to SCT (remission vs. no remission). Six patients developed acute graft-versus-host disease (aGVHD) and 12 developed chronic graft-versus-host disease (cGVHD). IR to either CCNE1 or CCNE2 occurred more frequently in patients without aGVHD than in the patients with aGVHD (92% vs. 50%, p&lt;0.05), although no significant difference in the IR frequency was observed amongst those who developed cGVHD versus those that did not. Of 8 CML patients who were not in CR prior to SCT, IR to either CCNE1 or CCNE2 occurred more frequently in patients that achieved CR compared to those that did not achieve CR after SCT (100% vs. 33%, respectively; p&lt;0.04). These results provide clinical evidence that both CCNE1 and CCNE2 peptides are novel leukemia-associated antigens, and they justify future clinical trials to determine whether CTL immunity can be enhanced against these antigens in patients with ALL and CML.
Impact of Recovery of CD4 and CD8 Lymphocyte Subsets on Acute GVHD after Reduced Intensity Conditioning (RIC) Allogeneic Stem Cell Transplantation (allo-SCT).
Authors: Mohty Mohamad , Gaugler Beatrice , Faucher Catherine , Sainty Danielle , Arnoulet Christine , Gastaut Jean-Albert , Viens Patrice , Olive Daniel , Blaise Didier 
Published: April 2020

Abstract
RIC regimens for allo-CST are being explored with good results concerning feasibility and engraftment. However, little is known about the immune recovery pattern in these patients, especially the different CD4 and CD8 lymphoid T cell subsets. Here, we assessed at different time points after allo-SCT, the kinetic of recovery of naïve (CD45RA+/CD27+), central memory (CD45RA−/CD27+), and terminally differentiated (CD45RA+/CD27−) CD4+ and CD8+ T lymphocytes in 64 patients from a single center, receiving HLA-identical RIC allo-SCT. Patients and graft characteristics are: age 48 y (27–63), diagnoses: 22 myeloid malignancies (34%), 22 lymphoid malignancies (34%) and 20 metastatic solid tumors (31%). 51 pts (80%) were considered as high risk. 49 pts (77%) received a fludarabine, busulfan and ATG-based RIC, while 15 pts (23%) received a low dose irradiation-based RIC. 91% of patients received a PBSC graft, with 42 (66%) receiving CSA alone for GVHD prophylaxis and 22 (34%) receiving CSA and MMF. 24 pts (38%) developed grade 2–4 acute GVHD at a median of 49 d (26–85). In this series, in contrast to CD4+ T cell subsets, CD8+ T cell subsets had a progressive and sustained recovery in the first 3 months after allo-SCT, with acquisition of functional markers such as 2B4 and perforin. Among the different subsets analyzed, the recovery of naïve CD4+ T cells, and central memory CD4+ and CD8+ T cells, measured at day 28 after allo-SCT and before onset of grade 2–4 acute GVHD, showed a significant correlation with the risk of grade 2–4 acute GVHD (P=0.001; P=0.002 and P=0.05 respectively). Patients developing grade 2–4 acute GVHD recovered a median of 47 naïve CD4+ T cells/μL prior to onset of GVHD as compared to 11 cells/μL in patients with grade 0–1 acute GVHD (Figure). Naïve CD4+ T cell levels significantly decreased after appropriate acute GVHD treatment. In a Cox multivariate analysis taking into account all relevant risk factors for acute GVHD, early recovery of naïve CD4+/CD45RA+/CD27+ T cells in the first month after allo-SCT was the strongest parameter significantly predictive of grade 2–4 acute GVHD development (P=0.006; RR=4.0; 95%CI, 1.5–11.0). Interestingly, there was a significant correlation between the total number of CD4+ T cells infused with the allogeneic graft and the early recovery of naïve CD4+ T cells (P=0.001), suggesting that graft manipulation might represent an attractive tool towards harnessing alloreactivity after RIC-allo-SCT.
Figure Figure
HIV-1 Proviral Integration Is Blocked by Agonists to the VpPAC2 Neuroendocrine Receptor.
Authors: Branch Donald R. , Bokaei Payman Baradar , Sakac Darinka , Ma Xue-Zhong 
Published: April 2020

Abstract
VPAC1 is a 7-transmembrane G-protein-coupled neuroendocrine receptor previously shown to transduce a facilitation signal for HIV-1 infection (Branch DR, et al., AIDS.2002;16:309–319). VPAC2, a related receptor, has been reported to have opposing function when compared to VPAC1 (Xia M, et al., J Immunol.1996;157:1132–1138; Tsutsumi M, et al., Diabetes.2002;51:1453–1460). We therefore examined whether stimulation of VPAC2, in contrast to VPAC1, may act to inhibit HIV-1 infection. Using three different and specific agonists of VPAC2, helodermin, RO 25-1553, and R3P55, daily treatment with low concentrations (10−9M) resulted in ~75% to 95% inhibition of either X4 or R5 HIV-1 productive infection in cell lines or primary peripheral blood mononuclear cells. The agonists VIP, PACAP, and secretin, that stimulate the two other VPAC receptor family members VPAC1 and PAC1, did not inhibit HIV-1 infection. Also, Hut78 cells, that lack VPAC2 and are infected by HIV-1, show no effect of VPAC2 agonists on HIV-1 infection. However, Hut78 cells transfected with human VPAC2 cDNA to overexpress this receptor become resistant to HIV-1 infection when treated with VPAC2 agonists.
VPAC2-mediated inhibition of productive HIV-1 infection was not due to effects on CD4 or chemokine co-receptor expression, cell growth, or apoptosis. Treatment with VPAC2 agonists also did not inhibit HIV-1 entry into the host cell. However, compared to untreated or cells treated with VPAC1-specific agonists, VPAC2 stimulation profoundly inhibits HIV-1 proviral DNA integration into the host genome. The block in integration was found to be due to the ability of agonists to VPAC2 to inhibit formation of 2-LTR circles, required for HIV-1 integration within the nucleus. We conclude that VPAC2-specific agonists are strongly inhibitory for productive HIV-1 infection. Furthermore, this inhibition is mediated by suppression of 2-LTR circle formation preventing proviral integration. Agonists of VPAC2 appear to be excellent candidates for future development as possible drugs for the amelioration of treatments aimed at the prevention of HIV/AIDS.