bluuebunny/crossref_metadata_2025_split huggingface dataset:
Domain Subclass Conversion Improved Activity of Anti-Mpl Agonist Antibodies in the Form of Whole IgG.
Authors: Kai Masayuki , Motoki Kazuhiro , Yoshida Hideaki , Tsuruhata Kumi , Endo Chisato , Muto Mari , Emuta Chie , Chisaka Yukiko , Shimabe Munetake , Miyazaki Hiroshi , Kataoka Shiro 
Published: April 2020

Abstract
Since the molecular cloning of thrombopoietin (TPO), two types of recombinant human TPO, full-length recombinant human TPO (rhTPO) and pegylated recombinant human megakaryocyte growth and development factor (PEG-rhMGDF), have been tested in a variety of clinical trials. The agents showed clinical efficacy in some cases. However, neutralizing antibodies against endogenous TPO were generated in some healthy volunteers who received PEG-rhMGDF, resulting in thrombocytopenia. Recently, several TPO mimetics are being developed, such as peptide-based molecules, nonpeptidyl small compounds and agonist antibodies. Here we report generation of novel fully human anti-human Mpl agonist antibodies. Two lead monoclonal antibodies MA01 and MA02 were cloned from human antibody-producing mice (KM mice™) immunized with human Mpl-expressing cells. Both MA01 and MA02 were of immunoglobulin gamma 1 (IgG1). These IgG1 agonists promoted proliferation of UT7/TPO cells in vitro, but were &gt;100-fold less effective than TPO. We, therefore, genetically modified MA01 and MA02 in order to enhance the activity and to minimize the effector function. First, constant heavy (CH) region of MA01 was converted to IgG4 (MA01G4) to reduce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). This conversion affected neither binding affinity nor agonistic activity of MA01. Next, hinge region of MA01G4 was changed to human IgG3 hinge (MA01G4344). MA01G4344 showed no change in binding affinity, but showed 10-fold higher activity than original MA01. In the case of MA02, the CH region was converted from G1 to G3344 to generate MA02G3344, in which CH1-hinge region was of IgG3 and CH2-CH3 region was of IgG4. The conversion drastically improved agonistic activity. Although MA02 (G1) activated Jak2 and Erk1/2 but not STAT5 and Akt, MA02G3344 activated all of them. These data suggest that conversion of CH1-hinge region to IgG3 type is essential for agonistic activity of anti-Mpl antibodies. The effective concentration 50 (EC50) values of MA01G4344 and MA02G3344 in UT7/TPO assay were in the range of 0.01–0.1nM. These antibodies stimulated human cord blood-derived CD34+ cells to give rise to CFU-MK colonies without any other supportive cytokines. A single injection of MA01G4344 increased platelet count of human Mpl transgenic mice for over one month. These domain subclass-converted antibodies are expected to provide a new medical option for treatment of chronic thrombocytopenia without losing good properties of natural antibody such as long serum half-life and low immunogenicity.
Figure Figure
Endolyn (CD164) Interacts with CXCR4 and Modulates the CXCL12-Mediated Homing and Migration of Cord Blood CD133+ Hematopoietic Precusor Cells.
Authors: Watt Suzanne M. , Forde Sinead , Tye Brit Jorgensen , Newey Sarah , Roubelakis Maria 
Published: April 2020

Abstract
The sialomucin, endolyn or CD164, has been shown to act as an important regulator in the adhesion of human haemopoietic stem/precursor cells (HPC) to stromal niche cells, while also controlling the entry of primitive human CD34+CD38lo HPC into cycle. Here, we define a novel function for endolyn, by identifying its ability to modulate CD133+ cord blood HPC migration on fibronectin towards CXCL12 in vitro. Interestingly, CD133+ cell migration on fibronectin to CXCL12 was reduced 1) by engaging the functional class II glycosylation-dependent epitope on endolyn with the 103B2/9E10 class II but not N6B6 class III antibody; and 2) by RNAi knockdown of endolyn in both CD133+ HPC and Jurkat cells. The inhibition of migration was more pronounced in the more primitive CD34+CD38lo/− HPC subset than in the CD38+ subset. We show a direct and temporal association of endolyn with the CXCR4 receptor, at the leading edge of CD133+ HPC. When CXCL12 is presented on fibronectin, we first see an upregulation in the association of CXCR4 with the a-4 and a-5 integrins that is closely followed by recruitment of endolyn to this complex. This was confirmed by co-immunoprecipitation studies. Knock-down of endolyn using siRNAs revealed that signaling through CXCR4 via PKC-zeta and Akt pathways was significantly dampened, while leaving MAPK phosphorylation unaffected. Our current studies are aimed at examining the in vivo importance of endolyn in HPC homing to the bone marrow of NOD/SCID mice. Our findings support a novel association between three distinct families of cell surface receptors that regulate both cell migratory and proliferative responses and identify endolyn as a key regulator of CXCR4/CXCL12 function.
Metaphase Cytogenetics Adds to Gene Expression Profiling in the Identification of High Risk Multiple Myeloma.
Authors: Tricot Guido , Zhan Fenghuang , Barlogie Bart , Huang Yongsheng , Sawyer Jeffrey , Shaughnessy John 
Published: April 2020

Abstract
The International Staging System (ISS), based on B2-microglobulin and albumin levels at the time of diagnosis, has now generally been adopted as a new prognostic classification system for multiple myeloma (MM). While readily and widely applicable, ISS does not account for genetic disease features, such as metaphase (CA) and interphase fluorescence in situ hybridization (FISH) cytogenetic abnormalities, which when examined in the context of standard prognostic variables, confer higher hazards of relapse and disease-related death. Recently, gene expression profiling (GEP) uncovered the major prognostic significance for outcome of high expression of CKS1B, a gene mapping to an amplicon at chromosome 1q21. We have performed a comprehensive study of CA, FISH, GEP and ISS staging in 351 newly diagnosed MM patients, treated uniformly on Total Therapy 2. We have analyzed outcome based on a combination of high CKS1B by GEP and CA. GEP-based t(11;14) was prognostically favorable, irrespective of expression of CKS1B and, therefore, was removed from the group of patients with high CKS1B expression. After this adjustment, with the combination of both CA and high CKS1B (approximately 10% of all patients) conferred a very poor outcome with only 24% and 40% of such patients being event-free and/surviving at 3 years, compared with 72% and 84% for the others (p values : &lt;.0001). Such patients fared poorly, irrespective of their ISS stage. Similar prognostic information could be gained by combining CA with FISH-defined amplification of 1q21 and t(11;14). Because of their major prognostic impact, all newly diagnosed patients should be tested for these genetic markers. Novel treatment modalities are justified in the small subgroup of such poor prognosis patients, since they derive only a minor benefit from advances in MM therapy.
CKS1B Q4 + CA (with no CCND1) vs. Others CKS1B Q4 + CA (with no CCND1) vs. Others
GATA-1, Transcriptionally, and Thrombopoietin-Mediated Akt Activation, Post-Translationally, Regulate Continuous Expression of Bcl-xL Protein during Megakaryopoiesis.
Authors: Kozuma Yukinori , Kojima Hiroshi , Yuki Satoshi , Suzuki Hidenori , Nagasawa Toshiro 
Published: April 2020

Abstract
Thrombopoietin (TPO) plays a relevant role for megakaryocyte differentiation from stem cells. One of the important biological activities of TPO is to prevent the apoptosis of megakaryocytic cells. As an anti-apoptotic protein Bcl-xL, which has been proved to be indispensable for erythroid differentiation, is also abundantly expressed in megakaryocytes, it is assumed that Bcl-xL plays an important role for megakaryopoiesis. We thus investigated the expression of Bcl-xL during megakaryopoiesis and the underlying regulatory mechanism. In stem cell-derived megakaryocytes, expression of Bcl-xL increased in the early- and mid-stages of the differentiation. Both in vitro in stem cell-derived megakaryocyteic cell culture and in vivo in an animal model injected with anti-platelet antibody, expression of Bcl-xL protein was maintained until platelet-producing stage of the megakaryopoiesis. TPO-depletion caused significant decrease in Bcl-xL protein level without affecting its mRNA in both stem cell-derived megakaryocytes and TPO-dependent megakaryocytic UT7/TPO cells. As a 12-kD fragment of Bcl-xL appeared by the withdrawal of TPO, we considered that Bcl-xL was cleaved upon TPO-depletion. This cleavage was blocked by a caspase-3-specific inhibitor, suggesting that caspase cleaves Bcl-xL in TPO-depleted megakaryocytes. Furthermore, pretreatment of UT7/TPO cells with a phosphatidylinositol 3-kinase (PI3K) inhibitor resulted in the cleavage of Bcl-xL even in the presence of TPO. We thus hypothesized that PI3K or its downstream signaling molecule inhibits the activation of caspase-3 and consequent cleavage of Bcl-xL. To prove this possibility, we prepared UT7/TPO cells transfected with constitutively active Akt-1. When TPO was depleted, the transfectant was significantly less liable to caspase-3 activation and Bcl-xL cleavage. Concerning transcriptional regulation of Bcl-xL, suppression of GATA-1 in UT7/TPO using siRNA caused decreased expression of both c-Mpl and Bcl-xL. Taken together, we conclude that GATA-1 regulates the expression of both c-Mpl and Bcl-xL, and once Bcl-xL is expressed, its protein level is maintained by the TPO-mediated Akt activation.
A Putative Role for VEGFR-1 (FLT-1) in B Cell Commitment and Differentiation.
Authors: Fragoso Rita , Igreja Catia , Appleton Claudia , Henriques Alexandra , Clode Nuno , Wu Yan , Zhu Zhenping , Dias Sergio 
Published: April 2020

Abstract
VEGF and its receptors are expressed in the hematopoietic system. A role for FLT-1 in particular was described in monocyte-macrophage migration and lineage differentiation (Sawano A et al, 2001), megakaryocytes maturation (Casella I et al, 2003) and dendritic cell differentiation (Dikov M et al, 2005). Given that the expression of this receptor in the lymphoid lineage is not known, we to studied FLT-1 expression and a putative function in normal lymphoid progenitors. To address this question we induced in vitro CD34+ cells differentiation into the B cell lineage using a well established assay (on S17 stromal cells). With this approach, we observed that FLT-1 is expressed throughout B cell differentiation increasing along the differentiation process, and reaching its highest at the “immature B cell” stage. We also neutralized FLT-1 during B cell differentiation in vitro. Surprisingly, in the presence of the FLT-1 neutralizing antibody (6.12 monoclonal Ab, from ImClone systems), at the end of the assays (4 different experiments) a significantly higher number of CD19+ cells (mainly immature B cells) were detected. Analyzing some of the transcription factors known to be involved in the commitment and differentiation of lymphoid B cells, we observed that the expression of PU.1, Pax5 and E47 was up-regulated by FLT-1 neutralization. Next, given that FLT-1 function was mainly associated with cell migration, and since it is expressed in B cells that are ready to exit the bone marrow into secondary lymphoid organs, we reasoned that FLT-1 might have a role in B cells exit from the bone marrow. For this purpose, we treated mice with the FLT-1 neutralizing Ab for 3 days and analyzed B cells levels in bone marrow and peripheral blood. FLT-1 neutralization led to a significant decrease (p&lt;0.05) in B cells in the bone marrow and peripheral blood. Taken together, our data supports a clear role for FLT-1 in B cell commitment. To understand if VEGF/PlGF signalling through FLT-1 promotes myeloid differentiation, suppresses B cell differentiation or simply regulates the quiescent state of hematopoietic stem cells, we differentiated in vitro CD34+/FLT-1− cells and CD34+/FLT-1+ cells (10% of CD34+ cells) using the assay described above. Interestingly, CD34+/FLT-1− differentiation in vitro largely promoted B cell differentiation, while CD34+/FLT-1+ cells originated mostly myeloid cell differentiation. We are currently exploiting the molecular basis whereby FLT-1 signalling may impair B cells commitment and possibly promotes myeloid differentiation.
PRV-1 Interacts with the Cytoplasmic Domain of Mpl and Interferes with TPO-Induced Signaling Cascades.
Authors: Johnson Jan , Ghandi Manish , Minear Jennifer N. , Lannutti Brian J. 
Published: April 2020

Abstract
Polycythemia rubra vera-1 (PRV-1) is a member of the uPAR/CD59/Ly6 family of cell surface receptors. An increase in the expression level of PRV-1 mRNA has been shown in patients with polycythemia vera and essential thrombocythemia, however, the ligand and function of PRV-1 remains unclear. Here, we show for the first time that PRV-1 expression alters the TPO-mediated Mpl receptor-signaling pathway. We also report a novel interaction between PRV-1 and the cytoplasmic region of Mpl (121 amino acids) using a yeast two-hybrid screen. Further characterization of this interaction in yeast containing truncated versions of the Mpl receptor cytoplasmic region indicates that the binding of PRV-1 to Mpl is regulated at the distal end of the cytoplasmic region. Deletion analysis utilizing Mpl receptors truncated after 53 (T53) or 69 (T69) cytoplasmic amino acid residues failed to show an association with PRV-1. Further mapping of the cytoplasmic domain of Mpl showed that truncations equal or greater than 111 cytoplasmic residues (T111) restored the interaction with PRV-1. To examine the physiological relevance of this interaction, we co-expressed Mpl and PRV-1 in BaF3 cells. Western blot analysis with an anti-phosphotyrosine antibody on TPO-stimulated BaF3/Mpl/PRV-1 whole cell lysates demonstrated a noticeable change in intracellular phosphorylation when compared to BaF3/Mpl. We found no affect on Jak2 and Mpl tyrosine phosphorylation. In contrast, there were higher levels of tyrosine-phosphorylated STAT5 and STAT3 in BaF3/Mpl cells expressing PRV-1. We then further investigated the effect of PRV-1 expression on MAPK signaling. We found that TPO-stimulation of BaF3/Mpl/PRV-1 cells resulted in a reduction of MAPK phosphorylation compared with cells expressing Mpl alone. These results indicate a novel function for PRV-1 as a signaling molecule in cytokine signaling cascades and may lead to a greater understanding of the mechanism of overexpression of PRV-1 in myeloproliferative disorders.
Telintra™ (TLK199) Enhances Granulocyte Colony-Stimulating Factor Production.
Authors: Stofega Mary R. , Cheng Mingshan , Ma Huiping , Sambucetti Lidia , Keck James G. , Brown Gail L. , Cai Danying 
Published: April 2020

Abstract
TLK199 is a novel glutathione analog inhibitor of the enzyme GST P1-1. GST P1-1 has been shown to be a negative regulator of c-Jun N-terminal kinase, which has been implicated in the control of cellular growth and differentiation. Studies in rodents have shown that TLK199 treatment increases the levels of circulating white blood cells and stimulates the production of CFU-GM in bone marrow. In a rodent model of 5-FU-induced neutropenia, treatment with TLK199 accelerated the recovery of neutrophil levels at a rate similar to that observed with G-CSF. In a recently reported Phase 2 clinical trial in patients with refractory MDS involving multilineage blood cell dysfunction, treatment with TLK199 resulted in a 65% Hematological Improvement of neutrophils, erythrocytes and/or platelets, confirming the myelorestorative effect of TLK199 in humans. The present study was designed to determine if the myelostimulative effects of TLK199 on neutrophils and their precursors are due in part to increased levels of endogenous G-CSF. TLK199 was tested in normal and neutropenic rodents for its effects on G-CSF production. A single administration of TLK199 (p.o. or i.p.) to normal mice resulted in up to 500% increase in G-CSF serum levels. In a rat model of 5-FU-induced neutropenia, TLK199 administration accelerated the recovery of circulating neutrophils, which corresponded to maximal G-CSF serum levels being observed earlier than vehicle control. The ability of TLK199 to enhance G-CSF production in primary human bone marrow stromal cells was also examined. Treatment of these cells with TLK199 potentiated IL-1b-induced production of G-CSF, GM-CSF and IL-6 from 200 to 400%. These data suggest that the enhancement of G-CSF production may contribute to the accelerated recovery of neutrophils observed with TLK199 treatment under neutropenic conditions. Together with the results from the Phase 2 clinical trial in MDS, these data support further clinical assessment of TLK199 treatment for chemotherapy-induced cytopenia.
Polymorphisms in Human Homeobox HLX1 and DNA Repair RAD51 Genes Increase the Risk of Therapy-Related Acute Myeloid Leukemia.
Authors: Jawad Mays , Seedhouse Claire , Russell Nigel H. , Plumb Mark 
Published: April 2020

Abstract
It is generally accepted that genetic low penetrance susceptibility and resistance genes within the general population make a significant contribution to therapy-related acute myeloid leukaemia (t-AML) risk. The risk of malignant transformation is defined by the number of mutations required and the mutation rate, but one additional theoretical risk factor is the target cell frequency, as this defines the number of genomes at risk. Whilst the genetic control of target cell frequency is unknown in humans, a leading candidate gene is the HLX1 homeobox transcription factor gene. HLX1 is specifically expressed on CD34+ cells and plays a vital role in hemopoietic development. In addition the HLX1 gene lies within a proposed human t-AML susceptibility locus on chromosome 1, as defined by murine radiation-induced AML genetic studies. We have examined the distribution of a C/T-3′UTR polymorphism in HLX1 in 189 AML patients, including 42 cases of t-AML, and 169 control subjects. The presence of a variant HLX1-3′UTR T allele resulted in a significant increase in the risk of t-AML (OR= 3.36, 95% CI 1.65–6.84); to our knowledge, this is the first time that a gene which is implicated in target cell biology has been associated with an increased risk of malignant transformation. In addition we examined polymorphisms in genes involved in DNA repair (RAD51 and XRCC2), carcinogen detoxification (HYL1) and methionine metabolism (MTHFR and MS). There was no difference in the distribution of the XRCC2, HYL1, MTHFR or MS polymorphisms in the control and AML cohorts studied. The RAD51 homologous recombination DNA repair gene polymorphism (135G/C-5′UTR) has been shown to result in enhanced promoter activity with consequent elevated mRNA expression and this polymorphism has previously been demonstrated to increase t-AML risk (OR=2.66, 95% CI 1.17–6.02). When combined analysis was performed on RAD51 (135G/C- 5′ UTR) and HLX1 (C/T -3′UTR) a 9.5-fold increase in the risk of t-AML was associated with the presence of variant alleles of both genes (OR= 9.50, 95% CI 2.22–40.64). The synergistic genetic interaction between the HLX1-C/T (3′- UTR) and RAD51-135 G/C polymorphisms demonstrates that target cell biology together with an increased homologous recombination DNA repair capacity, significantly increases the risk of t-AML. We suggest that this is because the HLX1-C/T (3′- UTR) polymorphism results in a higher number of stem cells; hence during genotoxic therapy (for a primary malignancy) there is an increased target cell number for genotoxic damage and potential malignant transformation. An optimum repair system is necessary to ‘deal’ with this burden of repair, however, the presence of altered repair capacity due to the RAD51-135C/T polymorphism may result in mis-repair and together with lack of apoptosis (due to a over-efficient repair system) will encourage the perpetuation of the oncogenic transformation and AML susceptibility.
Gfi1 Protein Turnover Is Regulated by the Ubiquitin Ligase Triad1.
Authors: van der Meer Laurens T. , Marteijn Jurgen A.F. , de Witte Theo M. , Jansen Joop H. , van der Reijden Bert A. 
Published: April 2020

Abstract
The transcriptional repressor Growth factor independence-1 (Gfi1) plays an essential role during various stages of hematopoiesis. It is crucial for the self-renewal and long-term reconstituting potential of stem cells, essential for neutrophilic differentiation, and it plays an important role in T-cell and dendritic cell development. Gfi1 has also been implicated in malignant hematopoeisis because the Gfi1 gene is a common proviral integration site in murine leukemia models. We recently found that Gfi1 protein levels are mainly regulated by the ubiquitin-proteasome system. Although Gfi1 mRNA levels are low in primary human monocytes, the protein levels are high due to low proteasomal degradation. Conversely, in mature granulocytes Gfi1 mRNA levels are high but protein levels are low due to strong proteasome-mediated turnover. Because Gfi1 plays an important role in normal and malignant hematopoiesis it will be of great interest to identify the ubiquitin ligases that regulate its turnover.
Previously, we showed that the RING finger ubiquitin ligase Triad1 regulates myeloid cell proliferation. Using yeast-two-hybrid assays we found that Triad1 binds the zinc finger region of Gfi1. This interaction was confirmed in co-immunoprecipitation experiments. To study whether the turnover of Gfi1 is regulated by Triad1 we performed ubiquitination assays. To our suprise we found that instead of promoting ubiquitination, Triad1 inhibited Gfi1 protein ubiquitination, also in the presence of proteasome inhibitors. RNAi mediated down regulation of Triad1 protein levels stimulated Gfi1 ubiquitination. Importantly, expression of a Triad1 point mutant (H158A) that fails to bind the ubiquitin conjugating enzyme UbcH7 also inhibited Gfi1 ubiquitination. To study whether the observed diminished ubiquitination by Triad1 affected the turnover of Gfi1 we analyzed Gfi1 protein half-life using the protein synthesis inhibitor cycloheximide. This showed that Triad1 co-expression prolonged the half-life of Gfi1 significantly. We conclude that Triad1 inhibits Gfi1 ubiquitination, resulting in decreased turnover of the protein. As this inhibition also occurs in the presence of proteasome inhibitors and is independent of the ubiquitin ligase activity of Triad1, these data support a model in which Triad1 competes for Gfi1 binding with other ubiquitin ligases that do mark Gfi1 for proteasomal degradation. Currently, we are testing candidate ubiquitin ligases (RING finger and HECT proteins) that were found to associate with Gfi1 in yeast-two-hybrid assays to gain more insight in how the activity of this important transcription factor is regulated.