bluuebunny/crossref_metadata_2025_split huggingface dataset:
Human CD34+ Myeloid Leukemic Progenitor Cells Are Susceptible to Lysis by Minor Histocompatibility Antigen LRH-1-Specific Cytotoxic T Lymphocytes.
Authors: Dolstra Harry , Overes Ingrid , Jedema Inge , Vos Annemieke , van Horssen-Zoetbrood Agnes , Norde Wieger , van der Voort Robbert , Falkenburg J.H. Frederik , Schattenberg Anton V. , de Witte T.M. 
Published: April 2020

Abstract
Allogeneic stem cell transplantation (SCT) is a specialized form of immunotherapy for treating patients with hematological malignancies. The curative potential is attributed to the graft-versus-tumor (GVT) response during which donor-derived cytotoxic T lymphocytes (CTL) eliminate malignant cells of the recipient. Minor histocompatibility antigens (MiHA) are the major targets of the GVT response, and expansion of MiHA-specific CTL has been shown to coincide with tumor remission following SCT. Recently, we identified a novel hematopoietic cell-restricted MiHA, designated LRH-1, which is presented by HLA-B7 and encoded by the P2X5 purinergic receptor gene (J. Clin. Invest.2005:115:3506–3516). Interestingly, tetramer analysis showed a direct association between in vivo expansion of LRH-1-specific CD8+ T cells and the disappearance of Bcr-Abl positive tumor cells in the CML patient from whom LRH-1-specific CTL was originally isolated. In addition, we detected in vivo expansion of LRH-1-specific CTL in an AML patient who was in clinical remission without GVHD. Furthermore, we demonstrated that P2X5 mRNA is significantly expressed in leukemic CD34+ progenitor cells from most CML as well as AML patients. These findings indicate a role for LRH-1 in inducing GVT immunity against myeloid leukemic progenitor cells. Here, we investigated the ex vivo responsiveness of myeloid leukemic CD34+ progenitor cells to LRH-1-specific CTL. First, we addressed this question using the CD34+ KG1 cell line which is positive for LRH-1. By using a CFSE-based survival assay we demonstrated that KG1 cells stably transfected with HLA-B7 could be efficiently lysed by LRH-1-specific CTL. Next, we determined responsiveness of purified CD34+ progenitor cells from HLA-B7+ CML patients to LRH-1 CTL-mediated killing. In the CFSE-based survival assay as well as a hematopoietic progenitor cell inhibition assay we showed that LRH-1-specific CTL efficiently recognize and kill CD34+ progenitor cells from LRH-1+ CML patients. In contrast, LRH-1-specific CTL did not inhibit the proliferation of CD34+ progenitor cells from LRH-1- CML patients. These findings illustrate that the P2X5-encoded LRH-1 antigen is an attractive target for adequate eradication of myeloid leukemic progenitor cells after allogeneic SCT.
MicroRNA hsa-mir-16 Contributes to Regulation of Myeloid Differentiation of Human CD34+ Cells.
Authors: Hildreth Richard , Georgantas Robert W. , Patel Roshan , Morisot Sebastien , Alder Jonathan , Civin Curt I. 
Published: April 2020

Abstract
In a large microRNA-array and bioinformatics study, we determined all of the microRNAs (miRs) expressed by human CD34+ hematopoietic stem-progenitor cells (HSPCs) from bone marrow and G-CSF mobilized blood. When we combined miR expression data, mRNA expression data fro a previous study (Georgantas et al, Cancer Research 64:4434), and data from various published mir-target prediction algorithms, we were able to bioinformaticly predict the actions of miRs within the hematopoietic system. MicroRNA hsa-mir-16 was highly expressed in CD34+ HSPCs, and was predicted to target several HSPC-expressed mRNAs (CXCR4, HoxB7, Runx-1, ETS-1, and Myb) that encode proteins known to be critically involved specifically in myelopoiesis within the hematopoietic system. We first confirmed that protein expression from each of these putative target mRNAs was in fact regulated by mir-16. The 3′UTR sequence from each of these mRNAs was cloned behind a luciferase reporter. Each reporter construct was transfected into K562 cells, which strongly express mir-16. In all cases, protein expression from the predicted target mRNA was greatly reduced in K562 cells, as compared to controls. As a first determination of mir-16’s function in hematopoietic cells, HL60 and K562 cells were transduced with hsa-mir-16 lentivirus, then treated with various chemical differentiation inducers. As was predicted by bioinformatics, hsa-mir-16 halted myeloid differentiation of HL60 cells, but did not affect megakaryocytic differentiation or erythroid differentiation of K562 cells. These initial findings suggest that mir-16 is a specific negative regulator of myelopoiesis. We are currently evaluating the effects of mir-16 on normal human CD34+ cells by in vitro CFC and suspension culture assays, as well as in vivo by transplantation of hsa-mir-16 lentivirus transduced cells in immunodeficient mice.
Lenalidomide (CC-5013; Revlimid®) Promotes Erythropoiesis in Myelodysplastic Syndromes (MDS) by CD45 Protein Tyrosine Phosphatase (PTP) Inhibition.
Authors: List Alan F. , Estes Myka , Williams Ann , Sekharam Madhavi , Ozawa Ukiyasu , Gao Gui , Wu Jerry , Gao Gui , Sokol Lubomir 
Published: April 2020

Abstract
Lenalidomide displays erythroid and cytogenetic remitting activity in MDS that is karyotype-dependent, yielding a high response rate in patients with chromosome 5q deletion (del5q; 76%) whereas cytogenetic and pathologic improvement in non-del5q patients is less common despite transfusion response in a significant proportion of patients [NEJM2005;352:549–57]. We postulated that lenalidomide targets regulatory proteins that repress the erythropoietin (EPO) signal in non-del5q MDS while inhibiting a critical del5q-associated target to account for karyotype-specific clonal suppression. To evaluate the mechanism by which lenalidomide enhances erythropoiesis in non-del5q MDS, we investigated its effects in MDS [N=9] and normal [N=7] marrow specimens, and in an EPO-responsive UT7 cell line model. Pre-incubation exposure to lenalidomide augmented EPO-induced BFU-E recovery up to 2.5-fold in non-del5q MDS specimens [N=7] over a concentration range of 0.001–1 mM, whereas change in BFU-E recovery from normals was nominal. In UT7 cells, lenalidomide [1mM] enhanced clonogenic response to EPO in a supra-additive fashion. To characterize effects on receptor signal response, we investigated the interaction between lenalidomide and EPO on STAT5 activation, and corresponding EPO-receptor (R) associated kinases. Lenalidomide enhanced both EPO-induced STAT5 phosphorylation and DNA binding in UT7 and MDS CD71Hi MDS erythroid precursors with corresponding Bcl-XL up-regulation. The magnitude of lenalidomide enhancement of EPO/STAT5 phosphorylation was significantly higher in MDS CD71Hi erythroid precursors compared to normal donors (median log10 geometric MFI pSTAT5 EPO+CC5013:EPO, 0.989 [s.d.,0.25] MDS vs. 0.0195 [0.07] Normal; P=0.0005). Lenalidomide enhancement of the EPO-R/STAT5 signal was associated with increased Janus kinase-2 (Jak-2) and Lyn kinase auto-phosphorylation and was abolished only by dual Jak and Src kinase inhibition, implicating targeted inhibition of a common Jak/Lyn kinase regulatory molecule. Indeed, lenalidomide potentiation of the EPO/STAT5 axis was replicated by inhibition of the dual Jak-2/Lyn PTP, CD45, with the selective antagonist RWJ-60475. In a cell free assay employing rhu-CD45 and a pp60 c-Src reporter substrate, lenalidomide inhibited CD45 PTP activity in a time-dependent fashion with a 50% inhibitory concentration [IC50] of 10nM, whereas thalidomide was largely ineffective. PTP inhibition was CD45 selective, demonstrating no significant inhibition of rhu-SHP-2 PTP activity. Our data suggest that lenalidomide is a PTP inhibitor that enhances EPO receptor signaling in non-del5q erythroid precursors by relieving CD45 repression of ligand-dependent Jak2 and Lyn kinase mediated STAT5 activation. Moreover, these findings suggest that CD45 over-activity may contribute to impaired EPO-R signaling in MDS erythroid precursors.
Combination of Gene Expression Data from Myeloid Cell Lines with In Vitro Response to Molecularly Targeted Therapies.
Authors: Mills Ken I. , Gilkes Amanda F. , Walsby Beth J. , Woodward Eleanor L. , Austin Steve J. , Jenkins Chris , Knapper Steven , Burnett Alan K. 
Published: April 2020

Abstract
AML is currently classified using a combination of morphology and major cytogenetic abnormalities. There is considerable heterogeneity with respect to secondary cytogenetics, molecular mutations, response to therapy, remission rate, and overall survival. New molecular therapies are being developed constantly and are tested in vitro using patient samples in order to balance the drug-patient relationship. Use of the same drug produces patient specific responses, whereas different molecular drugs give drug specific responses.
In order to rationalise and enhance the drug-patient relationship we have, using MTS assays, tested a range of molecular inhibitors against FLT3, PKC, HSP90, aminopeptidases and NFκB as well as modified nucleoside analogues on a panel of myeloid cell lines. These cell lines have also been profiled for gene expression. The combination of drug response and gene expression levels, were analysed by statistical algorithms including SAM, ANOVA and T-test to identify genes that differentiated between molecular responses or the overall therapeutic index (TI). Forty one genes distinguished between the molecular responses of FLT3, PKC, HSP90, and NFkB inhibitors. Around 100 genes were needed for the Ara-C response. The gene expression profiles of 187 diagnostic AML samples were also available with full clinical and molecular analysis. Some of these samples have been tested for in vitro responses to the molecular therapies, such as those targeted to FLT3 or HSP90. The TI gene list was used to analyse AML sub-groups including elderly patients (&gt;60 yrs old); younger patients (&lt;60 yrs old); cytogenetic risk groups, and those patients with FLT3 mutations (either ITD or TKD mutations) in an attempt to identify patients who may respond to specific molecularly targeted therapy. The Ara-C genes were used to predict whether a CR response was achieved. In patients less than 60 yrs old this correlation was not significant due to the great majority of patients achieving a complete remission. However, this was significant (p&lt;0.031) in patients &gt;60 yrs. Around 12% of patients were predicted to be responsive to HSP90 inhibitors whilst 18% might respond to FLT3 inhibitors. Several patients were in both groups, so 17% of patients were sensitive to a least one of the drugs. Whilst the potential added value of identifying patients sensitive to one of these drugs was only an additional 1–3%, this study shows the potential of using expression data to identify patients for specific molecular therapies and increasing the possibility of achieving a remission.
Dasatinib (BMS354825) Inhibits IgE-Dependent Activation and Histamine Release in Human Blood Basophils.
Authors: Kneidinger Michael , Valent Peter , Gleixner Karoline V. , Florian Stefan , Boehm Alexandra , Sonneck Karoline , Weghofer Margit , Lupinek Christian , Vrtala Susanne , Vales Anja , Valenta Rudolf , Superti-Furga Giulio , Lee Francis Y. 
Published: April 2020

Abstract
Dasatinib (BMS354825) is a small molecule-type inhibitory drug that blocks several tyrosine kinases including PDGFR, KIT, BCR/ABL, and several src kinases. Dasatinib has recently been introduced as a new effective antileukemic drug in patients with imatinib-resistant chronic myeloid leukemia. However, because of the many targets of dasatinib and the broad range of potential target-cells in physiologic and pathologic tissues, dasatinib has recently been examined for its immunosuppressive and anti-inflammatory effects. We here report that dasatinib at 1 μM completely blocks the anti-IgE-induced release of histamine in human blood basophils in healthy subjects. The effects of dasatinib on histamine secretion in basophils were dose-dependent (IC50: 50–100 nM). Dasatinib was also found to block recombinant allergen-induced release of histamine from human basophils in sensitized individuals. In addition, as assessed by flow cytometry, dasatinib (1 μM) was found to inhibit the IgE-dependent upregulation of several activation-linked antigens, including aminopeptidase N (CD13), LAMP-3 (CD63), endolyn (CD164), and the basophil-specific ectoenzyme E-NPP3 (CD203c) on the cell surface of human basophils. The effects of dasatinib on upregulation of basophil differentiation antigens were found to be dose-dependent and were seen in normal subjects as well as in allergic individuals. Together, these data show that dasatinib blocks IgE-mediated activation and histamine release in human blood basophils. These anti-allergic effects of dasatinib may have clinical implications and may point to additional indications for this multifunctional drug.
Analysis of Aurora Kinase Expressions and Cell Cycle Regulation by Aurora-C in Leukemia Cells.
Authors: Kobayashi Miki , Nakamura Satoki , Ono Takaaki , Sugimoto Yuya , Sahara Naohi , Shinjo Kaori , Shigeno Kazuyuki , Ohnishi Kazunori 
Published: April 2020

Abstract
Background: The conserved Aurora family kinases, a family of mitotic serine/threonine kinases, have three members (Aurora-A, -B and -C) in mammalian cells. The Aurora kinases are involved in the regulation of cell cycle progression, and alterations in their expression have been shown to associate with cell malignant transformation. Aurora A localizes to the centrosomes during anaphase, and it is required for mitotic entry. Aurora B regulates the formation of a stable bipolar spindle-kinetochore attachment in mitosis. The function of Aurora-C in mammalian cells has not been studied extensively. In this study, we investigated that human leukemia cells expressed all 3 Aurora kinases at both protein and mRNA level, and the mechanisms of cell cycle regulation by knock down of Aurora C in leukemia cells.
Methods: In this study, we used the 7 human leukemia cell lines, K562, NB4, HL60, U937, CEM, MOLT4, SUP-B15 cells. The expression levels of mRNA and proteins of Aurora kinases were evaluated by RT-PCR and western blot. The analysis of proliferation and cell cycle were performed by MTT assay and FCM, respectively.
Results: The mRNA of Aurora-A and Aurora-B are highly expressed in human leukemia cell lines (K562, NB4, HL60, U937, CEM, MOLT4, SUP-B15 cells), while the mRNA of Aurora C is not only expressed highly in all cells. In contrast, an increase in the protein level of the 3 kinases was found in all cell lines. These observations suggested posttranscriptional mechanisms, which modulate the expression of Aurora C. In cell cycle analysis by flow cytometory, the knock down of Aurora C by siRNA induced G0/G1 arrest and apoptosis in leukemia cells, and increased the protein levels of p27Kip1 and decreased Skp2 by western blot. In MTT assay, it was revealed that the growth inhibition of leukemia cells transfected with siRNA Aurora C compared with leukemia cells untransfected with siRNA Aurora C. Moreover, We showed that Aurora C was associated with Survivin and directly bound to Survivin by immunoprecipitation and western blot.
Conclusion: We found that human leukemia cells expressed all 3 members of the Aurora kinase family. These results suggest that the Aurora kinases may play a relevant role in leukemia cells. Among these Aurora kinases, Aurora C interacted with Survivin and prevented apoptosis of leukemia cells, and induced cell cycle progression. Our results showed that Aurora-C may serve as a key regulator in cell division and survival. These results suggest that the Aurora C kinase may play an important role in leukemia cells, and may represent a target for leukemia therapy.
Inhibition of T315I Bcr-Abl and Other Imatinib-Resistant Bcr-Abl Mutants by the Selective Abl Kinase Inhibitor SGX70393.
Authors: O’Hare Thomas , Eide Christopher A. , Tyner Jeffrey W. , Wong Matthew J. , Smith Caitlyn A. , Corbin Amie S. , Buchanan Sean , Jessen Katayoun A. , Tang Crystal , Holme Kevin , Burley Stephen K. , Deininger Michael W.N. 
Published: April 2020

Abstract
Imatinib effectively inhibits the tyrosine kinase activity of Bcr-Abl, the molecular driver of CML. Emergence of imatinib resistance due to mutations within the Bcr-Abl kinase domain (KD) has prompted the development of new Abl kinase inhibitors. A particularly important target is Bcr-Abl(T315I), which accounts for 15–20% of patients with resistance. To address this unresolved need, we profiled the novel Abl kinase inhibitor SGX70393 against native and mutant Bcr-Abl. Methods: We assessed the efficacy of SGX70393 in cellular and biochemical assays against a panel of KD mutants. Cell proliferation assays and Bcr-Abl tyrosine phosphorylation immunoblot analyses were performed for parental Ba/F3 cells, Ba/F3 cells expressing unmutated Bcr-Abl, or Ba/F3 cells expressing a single Bcr-Abl KD mutation (M244V, G250E, Q252H, Y253F, Y253H, E255K, E255V, F311L, T315I, F317L, M351T, F359V, V379I, L387M, H396P, or H396R). The resistance profile of SGX70393 was also evaluated using a recently developed accelerated, cell-based mutagenesis assay (Bradeen, et al. Blood, June 2006; doi:10.1182). Results: SGX70393 inhibited growth of cells expressing Bcr-Abl(T315I) (IC50: 7.3 nM) or unmutated Bcr-Abl (IC50: 12 nM). Sensitivity of Bcr-Abl mutants to SGX70393 partitioned into three categories: high (IC50&lt;25 nM: M244V, T315I, F359V, V379I, L387M, H396P, and H396R), medium (IC50&lt;300 nM: Q252H, Y253H, E255K, and F311L), and low (IC50&gt;500 nM: G250E, Y253F, E255V, and F317L). A cell-based mutagenesis screen for Bcr-Abl kinase domain escape mutants emerging in the presence of SGX70393 revealed a concentration-dependent reduction in surviving clones, with five previously reported Bcr-Abl mutations (L248M; G250E; Y253F; E255V; F317V) accounting for almost all resistance. Conclusions: (a) SGX70393 is a potent inhibitor of native and T315I mutant Bcr-Abl. (b) SGX70393 coverage extends to most clinically relevant mutants except mutations of the p-loop and F317.
Use of RNA Electroporated DC for Activation of LRH-1 Specific Cytotoxic T Lymphocytes in the Treatment of Lymphoid Malignancies.
Authors: Overes Ingrid , Fredrix Hanny , de Rijke Bjorn , van Horssen-Zoetbrood Agnes , van Krieken Han , Raymakers Reinier , van der Voort Robbert , de Witte T.M. , Dolstra Harry 
Published: April 2020

Abstract
Allogeneic hematopoietic stem cell transplantation (HSCT) is a potent treatment for patients with hematological malignancies. The therapeutic efficacy is attributed to the graft-versus-tumor (GVT) response during which donor-derived cytotoxic T lymphocytes (CTL) eliminate malignant cells of the recipient. Minor histocompatibility antigens (MiHA) are the major targets of the GVT response, and expansion of MiHA-specific CTL has been shown to coincide with tumor remission following SCT. Unfortunately, GVT response is often accompanied by graft-versus-host disease (GVHD) causing severe damage to skin, liver and gut. Therefore, it would be highly beneficial to direct GVT immunity to MiHA that are selectively expressed by “malignant” hematopoietic cells. Recently, we identified a novel lymphoid lineage-restricted MiHA, designated LRH-1, which is derived from a frame shift polymorphism in the P2X5 purinergic receptor protein (J. Clin. Invest.2005:115:3506–3516). Here, we examined by real-time quantitative RT-PCR mRNA expression of P2X5 in patient samples and cell lines from numerous lymphoid malignancies. We observed that P2X5 mRNA is highly expressed in tumor cells from all stages of lymphoid development. Furthermore, we demonstrated that LRH-1−specific CTL efficiently lyse LRH-1+ tumor cells and cell lines of lymphoid origin. These findings illustrate that LRH-1 is an attractive target for specific immunotherapy after allogeneic HSCT. A potentially efficient strategy is the application of MiHA-loaded dendritic cells (DC) to boost MiHA-specific T cell responses already primed in vivo after HSCT. To facilitate efficient presentation of LRH-1 by DC, we have optimized RNA electroporation of DC using in vitro-transcribed mRNA. We observed that RNA electroporation of mature DC resulted in a high yield of viable cells, high expression of co-stimulatory molecules, no loss of migratory capacity towards lymph node-specific chemokines, and high protein expression of the introduced antigens. Furthermore, we demonstrated that RNA-electroporated DC display long-lasting peptide presentation to LRH-1− specific CTL and induce proliferation of LRH-1− specific effector-memory CTL ex vivo. These data will be of importance in designing LRH-1− based immunotherapy in transplanted patients with lymphoid malignancies.
Bortezomib Inhibits Human Osteoclastogenesis.
Authors: Zavrski Ivana , Hecht Monica , Krebbel Holger , Fleissner Claudia , Mieth Maren , Kaiser Martin , Heider Ulrike , Jakob Christian , Sterz Jan , Possinger Kurt , Sezer Orhan 
Published: April 2020

Abstract
Enhanced osteoclastogenesis in cancer-induced bone disease may be caused by intercellular interactions between tumor cells and cells of the bone marrow microenvironment. In multiple myeloma, overexpression of RANKL in the bone marrow microenvironment may lead to the activation of TRAF-signaling and in consequence to increased NF-κB and AP-1 transcriptional activities in osteoclastic lineage cells. This results in enhanced osteoclast differentiation, activation and increased bone resorption. In this study, we have examined the effects of two NF-κB inhibitors towards their inhibitory potency on human osteoclastogenesis: proteasome inhibitor bortezomib and selective IKK inhibitor PS-1145. CD14+ osteoclast precursors from peripheral blood were stimulated with RANKL and M-CSF up to four weeks. Using MTT- and TUNEL-assays, cytotoxicity levels of each drug were determined on the differentiation stage day +1 (early osteoclast precursors), day +8 (preosteoclasts) and day +21 (osteoclasts). To evaluate the effects of both drugs considering osteoclast differentiation and -function, 2 sub-apoptotic doses of bortezomib, one sub-apoptotic and one low-apoptotic dose of PS-1145 were used. As revealed by the microscopic quantification of mature osteoclasts (TRAP-positive and multi-nucleated cells), the osteoclast differentiation was diminished by both drugs, whereas the effects were dose- and time-dependent. The microscopic quantification of resorption lacunae on dentine pits revealed that the resorptional activity was reduced by 65% for 0.1 nM bortezomib (p=0.007), by 79% for 1 nM bortezomib (p&lt;0.0005), by 60% for 1 μM PS-1145 (p=0.023) and by 91% for 10 μM PS-1145 (p&lt;0.0005). As shown by immunoblotting and by ELISA-based methods, the subcellular mechanisms of action involved in inhibition of early osteoclast differentiation were found to be related to the inhibition of p38 mitogen-activated protein kinase (MAPK) pathways, whereas the advanced differentiation and activation occurred in course of inhibition of AP-1 and NF-κB activation. The AP-1 blockade contributed to significant reduction of osteoclastic vascular endothelial growth factor (VEGF) production. In conclusion, our data demonstrate that proteasomal inhibition should be considered as a novel therapeutical principle of cancer-induced lytic bone disease.