bluuebunny/crossref_metadata_2025_split huggingface dataset:
The JAK Inhibitor INCB20 Induces Antiproliferative and Apoptotic Effects in Human Myeloma Cells In Vitro and In Vivo.
Authors: Burger Renate , Legouill Steven , Tai Yu-Tzu , Shringarpure Reshma , Podar Klaus , Catley Laurence , Tassone Pierfrancesco , Neri Paola , Hideshima Teru , Chauhan Dharminder , Caulder Eian , Vaddi Kris , Neilan Claire L. , Gramatzki Martin , Fridman Jordan S. , Anderson Kenneth C. 
Published: April 2020

Abstract
In multiple myeloma (MM), IL-6 plays an important role for tumor cell growth, survival, and drug resistance. Janus kinases (JAKs) are protein tyrosine kinases and constitutively associated with the gp130 chain of the IL-6 receptor complex. Their activation is one of the first steps in cytokine receptor-mediated signaling and critical for virtually all subsequent downstream signaling cascades. INCB20 is a small-molecule synthetic compound which, in biochemical assays, potently inhibited all four JAKs with IC50 values between 0.3 nM and 1.2 nM (for comparison, IC50 of AG490, another JAK inhibitor, was &gt;50 μM). Consistent with the central role of JAKs in gp130-mediated signaling, INCB20 inhibited IL-6 induced phosphorylation of SHP-2, STAT1, STAT3, ERK1/2, and AKT in MM1.S cells. In contrast, AKT phosphorylation induced by IGF-1 remained unchanged. Evaluation of the cellular efficacy of INCB20 was performed using the IL-6 dependent INA -6 cell line. Growth of INA-6 cells was inhibited in a dose-dependent manner with an IC50 of approx. 0.5 μM, as measured by [3H]-thymidine uptake and an MTS-based assay (for comparison, the cellular IC50 of AG490 was 15–20 μM). This correlated with an increase in the percentage of apoptotic cells, as evaluated by Apo2.7 staining after 48 hours. Importantly, INA-6 growth was inhibited in the presence of bone marrow stromal cells accompanied by a decrease in phospho-STAT3 levels. Furthermore, in a subcutaneous INA-6-SCID model, INCB20 inhibited tumor growth (and phosphorylated STAT3) in a dose-dependent manner. Our studies provide the conceptual basis for the use of JAK inhibitors as a therapeutic approach in MM.
Nucleoside Analogues of Cladribine Produce Enhanced Responses in Cell Lines.
Authors: Walsby Elisabeth , Congiatu Constantino , Schwappach Anna , Walsh Val , Burnett Alan K. , McGuigan Christopher , Mills Kenneth I. 
Published: April 2020

Abstract
Nucleoside analogues are antimetabolites that can be used to treat AML. These compounds mimic the effects of physiological nucleotides in terms of uptake and metabolism and become incorporated into nascent DNA resulting in synthesis inhibition and chain termination. Nucleoside drugs often suffer from poor metabolism to the active triphosphate forms, deactivation and clinical toxicities. Phosphate prodrugs (protides) have modified monophosphate groups on the drug which potentially enables them to enter cells and subsequently become metabolised into the monophosphate form. Production of the monophosphate form of the drug is the rate limiting step in the production of the active triphosphate form.
Cladribine (2-chlorodeoxyadenosine) has been used as a parent drug to produce a series of protides with altered monophosphate groups. The parent drug and the protides were used at concentrations between 10μM to 0.002μM on a panel of six myeloid cell lines (HL-60, NB4, NB4R2, U937, KG1 and K562) for 72 hours. The effects of the protides were measured by proliferative assays (MTS) and IC50 values calculated. Two protides, CC122 and CC124, were assessed in combination with cytarabine (Ara-C) at ratios of 1:1 and 1:10 in the same manner as previously described.
Fourteen protides of cladribine were analysed in addition to cladribine itself and Ara-C. The average IC50 value for each protide, across all the cell lines, tested were reduced by 2 to 11 fold compared to cladribine and Ara-C. One protide (CPF193) had no effect on any of the cell lines, whilst CPF194 was found only to be active in the KG1 cell line with a 2.7 fold reduction in the concentration required kill 50% of the cells treated compared to cladribine. In general, K562 cells did not respond to treatment with Ara-C, cladribine or 6 of the protides although they did respond to the remaining protides with IC50 values slightly higher than the other cell lines.
The IC50 values for each protide in each cell line individually was also calculated and some cell lines, particularly NB4, showed dramatic reductions in IC50. In particular protide CC124 showed over 40-fold reduction from 9.4μM for cladribine to 0.23μM for CC124. This was also lower than the IC50 for Ara-C in this cell line (0.53μM).
Ara-C and cladribine combinations, at a ratio of 1:1, gave an average combination index (CI) at the IC50 of 0.9857 across all the cell lines, excluding K562, indicating an additive effect only. However, protide CC124 and Ara-C in combination, at the same ratio, produced an average CI of 0.6334 indicating synergistic activity.
This study has shown that altered monophosphate groups on the nucleoside inhibitor cladribine reduces the IC50, particularly when used in combination with Ara-C. The reduced doses would be beneficial in a clinical environment.
Inhibition of Bcl-2 Signaling by Small Molecule BH3 Inhibitor GX15-070 as a Novel Therapeutic Strategy in AML.
Authors: Contractor Rooha , Konopleva Marina , Samudio Ismael , Kantarjian Hagop , Andreeff Michael 
Published: April 2020

Abstract
GX15-070 is a novel cycloprodigiosin derived small molecule BH3 inhibitor that binds with moderate affinity to all antiapoptotic Bcl-2 family members, including Mcl-1, and is currently undergoing Phase I clinical trials in CLL. In this study, we investigated anti-leukemic activity of GX15-070 in acute myeloid leukemia (AML) cell lines and primary AML samples. GX15-070 inhibited cell growth of HL-60, U937, OCI-AML3 and KG-1 at IC50’s of 0.1, 0.5, 0.5 and 2.5μM, respectively as determined by trypan blue exclusion method at 72 hours. Overexpression of Bcl-2 or Bcl-XL did not confer resistance to GX15-070 (IC50’s of HL-60/neo, HL-60/Bcl-2 and HL-60/Bcl-XL cells 0.25μM). MDR-overexpressing HL-60/Dox cells were similarly sensitive compared to their non-MDR counterpart, and specific MDR inhibitor (PSC 833) did not enhance apoptosis caused by GX15-070. GX15-070 (250nM) inhibited Mcl-1/Bak heterodimerization in HL-60 cells starting at 3 hours as evidenced by IP/Western blot analysis, however it did not affect Bax/Bcl-2 heterodimerization. This was associated with cytosolic release of cytochrome c followed by an increase in annexin positivity (21±2.5% annexin + cells with 250nM) and a decrease in mitochondrial inner membrane potential with 64±4.8% of cells losing membrane potential at 72 hours. GX15-070 did not affect cell cycle distribution of HL-60 cells. In 6/7 primary AML samples, GX15-070 induced apoptosis in CD34+ progenitor cells at an average IC50 of 3.6±1.2μM at 24 hours. In conclusion, the small molecule BH3 inhibitor GX15-070 potently induces apoptosis in myeloid leukemia cells via disruption of Mcl-1/Bak dimerization and activation of the intrinsic apoptotic cascade. Our findings provide rationale for the further development of this BH3 inhibitor as a novel therapeutic strategy for AML.
Fully Human CD38 Antibodies Efficiently Trigger ADCC of Multiple Myeloma Cell Lines and Primary Tumor Cells.
Authors: Peipp Matthias , de Weers Michel , Beyer Thomas , Repp Roland , Parren Paul , Valerius Thomas , van de Winkel Jan G.J. 
Published: April 2020

Abstract
Although new treatment modalities have recently been added to the standard regimens for multiple myeloma, the clinical outcome for patients with advanced disease is often limited. Monoclonal antibodies are increasingly used for tumor therapy, and may also represent interesting options for multiple myeloma patients. CD38 is one of the most promising target antigens on malignant plasma cells, which are evaluated in preclinical and early clinical studies as targets for antibody therapy. CD38 is a type II transmembrane protein with ectoenzymatic activity, which is involved in calcium mobilization. Human CD38 is predominantly expressed by bone marrow precursor cells and by terminally differentiated plasma cells. Multiple myeloma cells show moderate to high expression levels - making CD38 a potential candidate as target for immunotherapy.
A panel of 42 fully human CD38 antibodies was generated by immunizing human Ig transgenic mice. Immunofluorescence studies with CD38 transfected cells demonstrated antigen-specific, high affinity binding, and cross-blocking experiments revealed four distinct epitope groups. Seven antibodies, representing each of the four groups, were selected for further analyses. ADCC and CDC activity against CD38-positive myeloma cell lines (AMO-1 and JK6), and against freshly-isolated primary multiple myeloma cells was investigated. Human whole blood served as effector source, which was then fractionated into plasma (containing human complement), mononuclear (MNC) or granulocytic (PMN) effector cells. All antibodies mediated concentration-dependent killing of both multiple myeloma cell lines - using human mononuclear cells as effector source. Also complement-dependent killing of freshly isolated myeloma cells was observed. However, none of the antibodies recruited PMN for tumor cell lysis. Importantly, CD38 antibodies also killed freshly isolated tumor cells from a rare patient with a CD38/138- positive plasma cell leukemia, which was chemotherapy- refractory at the time of analysis. Furthermore, CD38 antibodies effectively prevented outgrowth of CD38-positive tumor cells in SCID mouse xenograft models. Antibody 005 was significantly more effective in these assays compared to the remaining panel of CD38 antibodies.
In conclusion, CD38 antibodies efficiently mediated killing of multiple myeloma cell lines as well as freshly isolated tumor cells and prevented tumor outgrowth in xenografted SCID mice. Antibody 005 was superior in mediating CDC and ADCC via MNC - particularly at low antibody concentrations.
The Y-box Binding Protein YB-1 Is Associated with Progressive Disease and Mediates Survival and Drug Resistance in Multiple Myeloma.
Authors: Chatterjee Manik , Ransco Christoph , Stühmer Thorsten , Eckstein Niels , Royer Hans-Dieter , Bargou Ralf 
Published: April 2020

Abstract
Introduction: The Y-box binding protein YB-1 is a member of the cold shock domain protein superfamily and represents one of the most evolutionary conserved nucleic-acid binding proteins. Yb-1 is involved in a wide variety of cellular functions, such as regulation of transcription and translation, but also in DNA-repair and stress response to extracellular signals. Furthermore, recent reports from our group could show that overexpressed YB-1 plays a role in drug resistance of breast cancer cells and might act as an oncogene. The goal of this study was to investigate a potential pathogenetic role of YB-1 in MM.
Material and Methods: For the detection of YB-1 expression in vivo, bone marrow biopsies of MM patients were analyzed by immunohistochemistry. The expression of YB-1 protein in a number of MM cell lines was analyzed by Western Blotting. The regulation of YB-1 expression through major signaling pathways, e.g. IL-6R/STAT3, Ras/MAPK and PI3K/AKT, was analyzed using specific inhibitors of these pathways (Sant7, PD98059 and Ly294002). To determine the role of YB-1 for survival and proliferation, siRNA technology was exploited to transiently knockdown the expression of YB-1 in the MM cell lines INA-6 and MM.1s. In addition, these YB-1 knockdown cells were exposed to doxorubicin and melphalan to evaluate the influence of YB-1 on drug resistance.
Results: Immunohistochemical analyses of bone marrow biopsies revealed that YB-1 is strongly expressed only in MM cells of samples that show a highly proliferative phenotype. This subgroup of MM patients was characterized by an aggressive clinical course. In contrast, MM cells of samples with a slow proliferative status did not show YB-1 expression. Interestingly, tumor cells of patients that responded to chemotherapy did not express YB-1. Furthermore, neither normal bone marrow plasma cells nor premalignant plasma cells of MGUS patients showed YB-1 expression. YB-1 was detected in all of the evaluated MM cell lines. YB-1 expression was not regulated by the IL-6R/STAT3, Ras/MAPK and PI3K/AKT pathways. Knockdown of YB-1 in INA-6 and MM1.s cells by siRNA resulted in a slow proliferation phenotype with a higher apoptotic cell fraction. In addition, we observed that YB-1 knockdown remarkably sensitized cells towards drug-induced apoptosis.
Conclusions: YB-1 expression in MM appears to be correlated with a highly proliferative phenotype and with disease progression. YB-1 contributes to the malignant growth and drug resistance and might be therefore an attractive therapeutical target to circumvent aquired drug resistance in MM.
Extracellular Endosulfatases (Sulfs) Inhibit Myeloma Tumor Growth In Vivo.
Authors: Dai Yuemeng , Yang Yang , MacLeod Veronica , Yue Xinping , Rapraeger Alan C. , Shriver Zachary , Venkataraman Ganesh , Sasisekharan Ram , Sanderson Ralph D. 
Published: April 2020

Abstract
Human sulfatases Hsulf-1 and Hsulf-2 are extracellular endosulfatases that specifically remove 6-O sulfates from heparan sulfate chains. Published studies have shown that expression of Hsulf-1 in some tumor cell lines diminishes their response to heparin-binding growth factors (e.g., FGF-2, HGF) leading to speculation that these enzymes may regulate tumor growth. To examine this possibility in vivo, the cDNA for either Hsulf-1 or Hsulf-2 was expressed in the human myeloma cell line CAG. Disaccharide composition analysis of heparan sulfate chains isolated from the surface of the transfected cells reveals that 6-O sulfation of trisulfated disaccharides is diminished by 70% and 50% on cells expressing Hsulf-1 and Hsulf-2, respectively, as compared to control cells transfected with empty vector only. When stimulated with exogenous FGF-2, downstream signaling as measured by the level of phosphorylated ERK1/2, is reduced in HSulf-1 or Hsulf-2 transfectants as compared to controls. To determine if Hsulf-1 and Hsulf-2 regulate myeloma tumor cell growth in vivo, cells were injected subcutaneously into the left flank of SCID mice. Monitoring of whole animal tumor burden by analysis of human kappa light chain present in the serum revealed that tumors formed from cells expressing either Hsulf-1 or Hsulf-2 exhibited a dramatic inhibition of tumor growth as compared to tumors formed from control cells (p&lt;0.0004). Upon sacrifice, mean weights of primary tumors were 0.46 g (n=12), 0.18 g (n=17) and 2.44 g (n=16) for Hsulf-1, Hsulf-2 and control tumors, respectively (p&lt;0.0001). To confirm that the endosulfatase enzymes were active in vivo, assembly of the FGF-2 ternary signaling complex (FGF-2/heparan sulfate/FGF Receptor 1) was analyzed on frozen tissue sections of the tumors. Incorporation of the FGFR1 into the complex requires the presence of 6-O sulfate groups in the endogenous heparan sulfate chains. Experiments show that FGF-2 binds equally well to the heparan sulfate present in all tumors, but the FGFR1 is not incorporated into the complex on tumors expressing either Hsulf-1 or Hsulf-2. This indicates that the Sulfs are active in vivo and that this activity alters heparin-binding growth factor signaling of the tumor cells. Additionally, we find that the elevation of Sulf expression causes a marked increase in extracellular matrix (ECM) deposition within tumors which may, along with attenuated growth factor signaling, contribute to the reduction in tumor growth. These studies indicate that dynamic regulation of heparan sulfate structure by Sulfs present within the tumor microenvironment can have a dramatic impact on the growth and progression of myeloma tumors in vivo.
Critical Role of flt3 in Fetal and Adult T Lymphopoiesis.
Authors: Sitnicka Ewa , Buza-Vidas Natalija , Ahlenius Henrik , Cilio Corrado M. , Gekas Christos , Nygren Jens M. , Jensen Christina T. , Svensson Marcus , Agace William W. , Jacobsen Sten Eirik W. 
Published: April 2020

Abstract
We recently demonstrated that signalling through the cytokine tyrosine kinase receptor flt3 and interleukin-7 receptor α (IL-7Rα ) is indispensable for fetal and adult B cell commitment and development (Sitnicka et al., J. Exp. Med. 198: 1495, 2003). We have also shown that flt3 ligand (FL)-deficient mice have reduced levels of the common lymphoid progenitor (CLP) but normal levels of mature T cells (Sitnicka et al., Immunity, 17:463, 2002). However, previous studies failed to demonstrate a role of flt3 and its ligand in T lymphopoiesis. Herein, although having normal numbers of thymocytes, FL-deficient mice were shown to have distinct reductions in the earliest double negative (DN) progenitors in the fetal, postnatal and adult thymus. A critical role of flt3 in thymocyte development was most evident in the absence of Interleukin-7 receptor a (IL-7Rα ) signalling. Strikingly, in adult FL−/−IL-7Rα −/− (double deficient) mice thymic cellularity was reduced 400 fold as compared to FL−/− and wild type (WT) controls and 30 fold as compared to IL-7Rα −/− mice. In agreement with previous studies, IL-7Rα −/− thymocytes revealed a partial block at the progression from the DN2 (CD4−CD8−CD44+CD25+) to DN3 (CD4−CD8−CD44−CD25+) stage, while in FL−/−IL-7Rα −/− mice DN1 (CD4−CD8−CD44+CD25−), DN2 and DN3 thymic progenitors were undetectable. The DP CD4+CD8+ thymocytes were reduced more than 1000 fold as compared to IL-7Rα −/− mice and low levels of peripheral T cells remaining in FL−/−IL-7Rα −/− mice had exclusively an activated/memory phenotype. Furthermore, FL−/−IL-7Rα −/− bone marrow cells lacked T cell reconstituting potential after transplantation into lethally irradiated recipients. Taken together these data demonstrate indispensable and complementary roles of flt3 and IL-7Rα in T lymphopoiesis.
TRAILR Triggering by Human Antibodies Induces Apoptosis through an Early Cleavage of Mcl-1 and Caspase 3 in Myeloma Cells.
Authors: Pellat-Deceunynck Catherine , Gomez-Bougie Patricia , Daniels Sylvanne , Geffroy-Luseau Alexandrine , Bataille Regis , Amiot Martine 
Published: April 2020

Abstract
We investigated TRAILR expression and sensitivity of myeloma cells in vitro. This study was done using a panel of 20 myeloma cell lines that are representative of primary myeloma cells (14q chromosomal translocation, IL-6 dependency, phenotype, oncogenes mutation). TRAILR were stimulated with agonistic human antibodies directed against either TRAIL-R1/DR4 (HGS-ETR1, mapatumumab) or TRAIL-R2/DR5 (HGS-ETR2), provided by Human Genome Sciences, Rockville, MD. This approach allowed us to analyze the contribution of each receptor separately. We show that a wide majority of cell lines, 16 of 20 were killed upon either TRAIL-R1 or R2 stimulation in the presence or absence of IL-6. However, 4 cell lines were resistant to HGS-ETR1 and 6 to HGS-ETR2 and 3 to both. Activation of both caspase 8 and Bid has been extensively described as being associated with TRAIL response. Indeed, we observed an activation of both caspase 8 and Bid. Cleaved molecules were detected 6 to 18h after antibody addition but after detection of cellular apoptosis. However, we show that Mcl-1L, a key molecule for myeloma survival, was downregulated and cleaved as soon as 3h after Ab addition. The cleaved form of Mcl-1 has been shown to behave like a proapoptotic molecule. Since caspase 3 has been reported to cleave Mcl-1, we looked at caspase 3 activation. Indeed, we observed that caspase 3 cleavage occured early and concomitantly to the one of Mcl-1. Our data show that in a wide majority of myeloma cell lines (80%) TRAILR triggering induces massive apoptosis that was not prevented by IL-6, the major myeloma cell growth and survival factor. Moreover, apoptosis induced upon TRAILR triggering was fully correlated to an early cleavage of both caspase 3 and Mcl-1 and to a delayed one of both caspase 8 and Bid.
TACI and BCMA, Unlike BAFF-R, Are Expressed by Human Myeloma Cell Lines and TACI+ Cell Lines Have a Gene Signature Indicative of Mature Bone Marrow Plasma Cells and Bone Marrow Environment Dependence.
Authors: Moreaux Jerome , Cremer Friedrich W. , Reme Thierry , Mahtouk Karene , Pantesco Veronique , de Vos John , Moos Marion , Robert Nicolas , Goldschmidt Hartmut , Rossi Jean-François , Hose Dirk , Klein Bernard 
Published: April 2020

Abstract
B-cell activating factor (BAFF) and A Proliferation-Inducing Ligand (APRIL) are produced by the BM microenvironment of patients with multiple myeloma (MM) and are growth factors of multiple myeloma cells (MMC). BAFF has three receptors: BAFF-R, TACI and BCMA, and APRIL has 2 receptors: TACI and BCMA. Using real time RT-PCR and FACS analysis, we found that TACI and BCMA are expressed by MMC, unlike BAFF-R. Gene expression profiling (GEP) of purified MMC from newly-diagnosed patients has indicated that primary MMC with high TACI expression (TACI[suphigh] MMC) have a mature plasma cell gene signature linked to a dependence on the bone marrow environment. In contrast, primary MMC with a low TACI expression (TACI[suplow] MMC) have a gene signature of proliferating plasmablasts. TACI expression was either undetectable (11 HMCLs) or present (7 HMCLs) on a panel of 18 HMCLs, whereas BCMA expression was detected in all HMCLs with sensitive real-time RT-PCR and FACS analysis. GEP of the 18 HMCLs were determined with Affymetrix U133 DNA microarrays that interrogate 33000 genes or ESTs. 109 out of the 33000 genes/ESTs were differentially expressed between TACI[sup+] and TACI[sup-] HMCLs (ratio ≤ 0.5 and ≥ 2, P ≤ 0.05). TACI[sup+] HMCLs have a gene signature indicative of a BM environment dependence close to that of normal bone marrow plasma cells, including genes involved in intercellular communication and signal transduction genes. TACI[sup+] HMCLs with a BM environment dependence signature include the RPMI8226 and LP1 HMCLs that produce BAFF or APRIL as autocrine growth factors, a property of the bone marrow environment in patients with intramedullary MM. The microenvironment dependence signature was also found in 5 HMCLs (XG-2, XG-12, XG-13, XG-19 and XG-20) whose growth requires addition of exogenous IL-6 or BAFF/APRIL in vitro. These data indicate that a high TACI expression and the associated microenvironment dependence gene signature found in primary MMC is kept in HMCLs that have been maintained in culture for 10–30 years, and whose growth does not need anymore interaction with BM environment. One hypothesis is that this gene signature may be a characteristic of the MMC clone at the occurrence of the disease. The MM stem cell could be immortalized at different stages of normal plasma cell differentiation. Transformation at the plasmablastic stage would give rise to a MMC clone with a plasmablastic gene signature. Transformation at a later plasma cell differentiation stage would give rise to a MMC clone with a gene signature of mature BM plasma cells.