bluuebunny/crossref_metadata_2025_split huggingface dataset:
KIR-LIGAND Mismatched Natural Killer Cells Effectively Kill Primary Myeloma and Myeloma Cell Lines.
Authors: van Rhee Frits , Szmania Susann M. , Shi JuMei , Cottler-Fox Michele , Dyniewski Justin W. , Tricot Guido J. 
Published: April 2020

Abstract
Recent observations in haplo-identical, T-cell-depleted allogeneic transplantation have focused attention on the rapid and remarkable anti-tumor effect mediated by Killer Inhibitory Receptor (KIR) ligand mismatched natural killer (NK) cells. Most data are confined to the role of KIR-ligand mismatched NK cells for acute leukemia after allogeneic transplantation. In this study, we report both the ability of KIR mismatched NK cells to kill primary myeloma cells, and the effects of interleukin (IL)2 and IL15 on NK cells in vitro. NK cells were prepared from healthy donor PBMCs by depletion of T-lymphocytes using immunomagnetic beads conjugated to anti-CD3 followed by depletion of monocytes using a simple adherence step. After overnight incubation in cytokines (IL2/IL15 as indicated) standard chromium release assays determined the cytotoxicity of NK cells against target cells and 3H-thymidine incorporation assessed NK cell proliferation. KIR-ligand mismatched NK cells incubated overnight in 300 IU/ml IL2 killed primary MM cells from four individuals (76, 64,60,48% lysis) as well as MM lines U266, NCI H929, and the NK sensitive line K562 (95, 86, 76% lysis, respectively) at an E:T ratio of 20:1. NK cells were not cytotoxic towards autologous phytohemagglutinin (PHA) or allogeneic PHA-induced T-cell blasts. Increasing the IL2 concentration during overnight NK cell incubation from 300 to 1000 IU/ml did not significantly enhance the cytotoxicity against U266 myeloma cells or control K562 cells. IL15 was more potent than IL2 in inducing NK cell proliferation. The optimal IL15 concentration was 300 IU/ml (range tested: 0–3000 IU/ml). The use of both cytokines in concert was less effective than the use of IL15 alone, probably due to homology of the IL2 and IL15 b and g receptors. We conclude that T cell and monocyte depletion of PBMC results in a preparation significantly enriched for NK cells (±50%) that effectively kills KIR-ligand mismatched primary myeloma cells and myeloma cell lines. IL15 is the superior cytokine for enhancing NK cell proliferation. The exciting anti-leukemic effects mediated by KIR-ligand mismatched NK cells have thus far only been reported in the haplo-identical transplant setting. In view of our positive in vitro data in MM, we will evaluate in a phase II trial wheter the repeated transfusion of KIR-ligand-mismatched, T-cell depleted NK cells from haplo-identical donors after immunosuppressive treatment with fludarabine and dexamethasone (to avoid rejection of donor NK cells) and tumor reduction with melphalan followed by a delayed auto-transplant can improve outcome in patients who have high risk MM or who have relapsed after a previous auto-transplant. This will be the first clinical application of KIR-ligand-mismatched NK cells in autologous transplantation and the first such trial in myeloma.
Figure Figure
Caveolin-1 Is Required for VEGF- Triggered Multiple Myeloma Cell Migration and Is Targeted by Bortezomib (Velcade®).
Authors: Podar Klaus , Shringarpure Reshma , Tai Yu-Tzu , Simoncini Melissa , Sattler Martin , Ishitsuka Kenji , Richardson Paul G. , Hideshima Teru , Chauhan Dharminder , Kenneth Anderson C. 
Published: April 2020

Abstract
We recently demonstrated that caveolae, vesicular flask-shaped invaginations of the plasma membrane represent novel therapeutic targets in multiple myeloma (MM). In the present study, we demonstrate that vascular endothelial growth factor (VEGF) triggers Src-dependent phosphorylation of caveolin-1, which is required for p130Cas phosphorylation and MM cell migration. Conversely, depletion of caveolin-1 by antisense methodology abrogates p130Cas phosphorylation and VEGF-triggered MM cell migration. The proteasome inhibitor bortezomib, both inhibited VEGF-triggered caveolin-1 phosphorylation and markedly decreased caveolin-1 expression. Consequently, bortezomib inhibited VEGF- induced MM cell migration. Bortezomib also decreased VEGF secretion in the bone marrow microenvironment and inhibited VEGF-triggered tyrosine phosphorylation of caveolin-1, migration, and survival in HUVECs.
Taken together, these studies demonstrate the requirement of caveolae for VEGF-triggered MM cell migration and identify caveolin-1 in MM cells and HUVECs as a molecular target of bortezomib.
NY-ESO-1 Specific Antibodies Are Frequently Detected in Hight-Risk Myeloma.
Authors: van Rhee Frits , Szmania Susann M. , Pomtree Mindy , Zhan Frank , Moreno Amberly , Rosen Nancy , Fox Michele , Barlogie Bart , Shaughnessy John , Batchu Ramesh , Tricot Guido J. 
Published: April 2020

Abstract
We have previously reported that NY-ESO-1 is especially present in high-risk multiple myeloma (MM) as defined by abnormal metaphase cytogenetics. The presence of NY-ESO1 specific antibody (ab) response is an excellent surrogate marker for the presence of a CD4+ NY-ESO-1 specific T-cell response, which is essential to priming, expanding and sustaining CD8+ NY-ESO-1 specific T-cell responses. We therefore studied the sera of 56 MM patients enrolled in our Total Therapy II protocol for the presence of specific NY-ESO-1 ab by sandwich ELISA using highly purified recombinant NY-ESO-1 protein. NY-ESO-1 RNA expression of purified MM cells was determined by gene expression profiling. NY-ESO-1 specific ab were detected in 13/39 (33%) of patients with NY-ESO-1 positive MM. In contrast, patients with NY-ESO-1 negative MM (n=27) all tested negative for NY-ESO-1 ab by ELISA, as did normal donors (n=20). Antibodies to NY-ESO-1 were present in 9/19 (47.3%) of patients who were studied with frank, NY-ESO-1 positive MM (at diagnosis, progressive disease or at relapse). All patients with progressive/relapsed MM had previously received intensive chemotherapy and/or auto-transplantation. In NY-ESO-1 positive MM patients who had a very good partial or partial response to therapy NY-ESO-1 specific ab were detected in only 4 patients of 13 tested (30.7%). In contrast, no NY-ESO-1 specific ab were detected in 7 NY-ESO-1 positive MM patients who had achieved (near) complete remission. Nine of the 13 (70%) NY-ESO-1 ab positive patients had high-risk MM as defined by abnormal metaphase cytogenetics. We conclude that the presence of NY-ESO-1 positive MM, and thus antigenic stimulation, is the principal determinant of the ability to mount a humoral response to NY-ESO-1 protein despite the presence of an immune system compromised by the immunosuppressive effects of MM or (high dose) chemotherapy. Further, NY-ESO-1 specific ab are principally found in patients with high-risk MM. The presence of spontaneous humoral immune responses to NY-ESO-1 is important since it suggests that there is a reservoir of NY-ESO-1 specific CD4+ T-cells that could be expanded and utilized for the immunotherapy of high-risk MM by vaccinating with NY-ESO-1 peptides, proteins or DNA based vaccines.
A Novel Inosine Monophosphate Dehydrogenase Inhibitor VX-944 Overcomes Conventional Drug-Resistance in Multiple Mueloma Cells in the Bone Marrow Microenvironment.
Authors: Ishitsuka Kenji , Hideshima Teru , Hamasaki Makoto , Noopur Raje , Shaji Kumar , Yasui Hiroshi , Roccaro Aldo M. , Chauhan Dharminder , Podar Klaus , Tai Yu-Zu , Jain Jugnu , Anderson Kenneth C. 
Published: April 2020

Abstract
Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme required for the de novo synthesis of guanine nucleotides from IMP. VX-944 (Vertex Pharmaceuticals, Cambridge, MA) is a small molecule, selective, uncompetitive novel inhibitor directed against human IMPDH enzyme. IMPDH inhibitors have been demonstrated to induce growth arrest, and extensively investigated as immunosuppressants. Here we show that VX-944 inhibits growth of human multiple myeloma (MM) cell lines, including those resistant to conventional agents, via induction of apoptosis and S phase arrest in vitro. Interleukin-6, insulin-like growth factor-1, or co-culture with bone marrow stromal cells (BMSCs), do not protect against VX-944-induced MM cell growth inhibition. We next delineated the molecular mechanism of VX-944-induced MM cell death in the MM.1S human MM cell line. VX-944 induced apoptosis in MM.1S cells, confirmed by PARP cleavage as well as flow cytometric detection of the mitochondrial membrane protein 7A6 and TdT-mediated dUTP nick-end labelling (TUNEL) positive cells, without significant cleavage of caspases 3, 8 and 9. While the pan-caspase inhibitor z-VAD-fmk did not inhibit the VX-944-induced apoptosis and cell death suggesting that VX-944 triggers apoptosis in MM1.S cells primarily via caspase-independent pathway. Importantly, VX-944 augments the cytotoxicity of doxorubicin, melphalan and bortezomib, all of which activate caspases in MM cells and induce apoptosis, even in the presence of BMSCs. Taken together, our data demonstrate non-caspase-dependent apoptotic pathway triggered by VX-944 thereby providing a rationale to enhance MM cell cytotoxicity by combining this agent with conventional and/or novel agents which trigger caspase activation. Our ongoing studies are delineating the mechanisms whereby VX-944 induces MM cell apoptosis.
Safety and Efficacy Results from a Phase I Trial of Single-Agent Lumiliximab (Anti-CD23 Antibody) for Chronic Lymphocytic Leukemia.
Authors: Byrd John C. , O’Brien Susan , Flinn Ian , Kipps Thomas J. , Weiss Mark A. , Rai Kanti , Lin Thomas S. , Reid Jennifer , Wynne Dee , Leigh Bryan R. 
Published: April 2020

Abstract
The CD23 antigen is expressed at high density on the cell surface of certain B-cell malignancies, including chronic lymphocytic leukemia (CLL). Lumiliximab (L-mab), a macaque-human chimeric anti-CD23 monoclonal antibody, has been reported to have antitumor activity against CLL in preclinical studies. In this Phase I multicenter study, the safety, efficacy, and pharmacokinetics of single-agent L-mab were evaluated in 46 patients with relapsed or refractory CLL. Therapy consisted of intravenous L-mab given as 6 regimens: (1) 125 mg/m2/wk for 4 weeks; (2) 250 mg/m2/wk for 4 weeks; (3) 375 mg/m2/wk for 4 weeks; (4) 500 mg/m2/wk for 4 weeks; (5) 500 mg/m2 for 3 doses during Week 1, then 500 mg/m2/wk during Weeks 2 to 4; and (6) 500 mg/m2 three times a week for 4 weeks. Patient characteristics were as follows: median age of 62 years (range 47 to 80 years), 93% Caucasian, 72% male, 54% fludarabine-refractory, 48% Rai stage III/IV, and 78% WHO Performance Status 1. At study entry, patients had progressive CLL after 1 to 13 prior treatment regimens (median = 4 prior regimens). Antibody infusions, administered over 2 hours in an outpatient setting, were well tolerated. Study-related adverse events (probable, possible, or unknown relationship to study treatment) were reported in 40 of 46 patients (87%). The majority of events were Grade 1 or 2; the most common were headache, constipation, nausea, and cough. Grade 3 or 4 study-related adverse events were reported in 7 of 46 patients (15%) and included neutropenia and dyspnea. Evidence of clinical activity consisted of reductions in absolute lymphocyte counts (ALC) and lymphadenopathy. Decreases in ALC were observed in 42 of 46 (91%) patients, and decreases ≥ 50% were observed in 11 of 40 (28%) patients enrolled at 375 mg/m2/week or higher. Of 37 patients evaluated for change in lymphadenopathy, reductions were observed in 22 (59%). Flow cytometry revealed that L-mab saturated CD23 sites on CLL cells at doses above 375 mg/m2/week without down regulating CD23 expression. These results suggest that single-agent L-mab can be administered safely with evidence of clinical activity in patients with heavily pretreated CLL. Ongoing clinical studies are assessing the potential of L-mab in combination with rituximab and fludarabine-based chemotherapy.
Autologous CMV-Specific T Cells as Effector Cells in Immunotherapy of B Cell Chronic Lymphocytic Leukemia.
Authors: Kater Arnon P. , Remmerswaal Esther E.B.M , Nolte Martijn A. , Eldering Eric , van Lier Rene A.W , van Oers Marinus H.J 
Published: April 2020

Abstract
Introduction. B cell Chronic Lymphocytic Leukemia (B-CLL) is prototypic for human cancers that escape immune surveillance. These cells not only lack immunogenic tumor-specific epitopes but also express low levels of surface molecules that are necessary for optimal interaction with naive T cells. Different strategies have been tried to generate B-CLL specific T cells that can be used for adoptive immunotherapy. Yet it is uncertain whether these T cell specific responses are strong enough to eliminate the neoplastic clone. Previously, we have demonstrated that CMV seropositive patients with B-CLL have significantly increased relative and absolute numbers of CD8+ T cells exhibiting a CD45RA+CD27− cytotoxic CMV specific phenotype. In the present study we investigated the possibility to direct these in vivo generated CMV specific effector cells to the poorly immunogenic B-CLL cells.
Results. A considerable fraction (6%; range 1.0 to 16.4) of the circulating CD8+ T cell pool of the four B-CLL patients studied consisted of CMV-specific T cells, as visualized with either tetrameric HLA-A2.1/NLVPMVATV complexes or HLA-B7.2/TPRVTGGGAM complexes. Activation of these cells by CMV-peptide in the presence of IL-2 at day 0, and restimulation at day 8 with either CMV peptide loaded EBV transformed cell-lines or autologous peptide-loaded B-CLL cells, resulted in a near 100-fold absolute expansion of tetramer positive CD8+ T cells (on average 1.9 x 106 at day 0, and 2.2 x 108 at week 3). These in vitro restimulated CMV-specific T cells were non-cytotoxic for non-peptide loaded B-CLL cells, but showed a very efficient killing of CMV peptide loaded B-CLL cells, with a 50% specific lysis at an effector:target ratio as low as 4:1. Thus, despite their general anti-apoptotic profile, B-CLL cells are excellent targets and undergo apoptosis after CTL attack. This CTL specific killing was found to be completely dependent on the granzyme and perforin pathway. Finally we showed that the CMV specific effector cells in B-CLL patients do not need restimulation to effectuate a cytotoxic response towards CMV peptide loaded B-CLL cells.
Conclusion. Directing virus-specific T cells to B-CLL tumors may overcome the inadequate immunostimulatory capacity of these cells and could be exploited for T-cell mediated immunotherapy.
Novel Anti-CD37 Small Modular Immunopharmaceutical (SMIP) Induces B-Cell-Specific, Caspase-Independent Apoptosis in Human CLL Cells.
Authors: Zhao Xiaobin B. , Biswas Sabyasachi , Mone Andrew , Ledbetter Jeffrey , Ledbetter Martha Hayden , Grosmaire Laura , Espling Erik , Cheney Carolyn , Young Donn , Lee Robert J. , Muthusamy Natarajan , Byrd John C. 
Published: April 2020

Abstract
Tru 16.4 is a single chain protein with a modified CD37 binding Fv domain linked to a modified human IgG1 hinge, CH2 and CH3 domains. It is a member of a novel composition class called small modular immunopharmaceuticals (SMIP) construct. CD37 is a stable marker for B-cell chronic lymphocytic leukemia (BCLL) and non-Hodgkins lymphoma (NHL). Although radiolabeled murine anti-CD37 antibody MB-1 has been evaluated in NHL patients and shown to be able to target tumors, efficacy was not established. There has been no evaluation of an unconjugated anti-CD37 antibody in human clinical trials. Here we report that Tru 16.4, binds to CD37 on primary CLL cells surface, and induces caspase-independent apoptosis that is further enhanced by a secondary crosslinking with goat anti-human antibody. The percentage of dead cell population over control based upon Annexin V/PI staining of 12 BCLL patient samples (28.3%, SD 15.4%) is significantly greater than that of isotype control, trastuzumab (3.4%, SD 7.6%). The expression of CD37 on T cells is low, and the exposure of Tru 16.4 under the same conditions does not demonstrate significant binding or induce noticeable apoptosis on normal T cells at 24 hours (0.47%, SD 0.99%). This provides a selective advantage for BCLL antibody therapy. Results from a comparison study utilizing 6 additional patient samples demonstrated that Tru 16.4 induced apoptosis (31.8%, SD 12.2%) is significantly higher than that of the anti-CD20 antibody, rituximab (21.8%, SD 5.25%) and similar to that observed with the anti-CD52 antibody, alemtuzumab (36.6%, SD 19.5%). The cell death induced by Tru 16.4 is dependent upon dose and duration of treatment, indicating 5μg/mL as a saturation concentration and 24 hours as the optimal time for maximal in vitro apoptosis. The degree of CLL cell death was found to be directly proportionate to cell surface membrane expression of CD37, based on nonparametric Spearman rank correlation analysis of the mean fluoresecnce intensity (MFI) after PE-labelled anti-CD37 antibody immunostaining and percentages of Annexin V/PI positive cells, with a correlation factor rs = 0.80 ( P &lt; 0.01). Ongoing studies suggest Tru 16.4 induces intracellular signaling following CD37 antigen ligation and current efforts are focused on identifying the specific death pathways mediated by this treatment. Overall, these findings indicate that Tru 16.4 mediates B-cell specific apoptosis through a novel caspase independent mechanism and supports effort for further clinical investigation in CLL.
Modification of B-CLL Cells Via Infection with a Replication-Defective MVA Virus Encoding Three Costimulatory Molecules: A Potential Approach to Tumor Cell Immunotherapy of B-CLL.
Authors: Palena Claudia , McIntosh Loni , Schlom Jeffrey , Foon Kenneth , Panicali Dennis , Tsang Kwong Y. 
Published: April 2020

Abstract
Chronic lymphocytic leukemia of B-cell origin (B-CLL) is a lymphoproliferative disorder characterized by the accumulation of slowly dividing, apoptosis-defective, clonal CD5+ B cells that typically involve the bone marrow, lymph nodes, spleen and blood of affected individuals. New therapeutic approaches are necessary to be investigated for the treatment of B-CLL. Several characteristics of the neoplastic B cells make B-CLL a good candidate for immunotherapy; evidence exists that B-CLL cells can be recognized by autologous anti-tumor T cells in an MHC-restricted manner. B-CLL cells, however, are themselves ineffective antigen-presenting cells mainly because of their inadequate costimulatory capacity. We have investigated the ability of in vitro manipulated B-CLL cells that express a triad of costimulatory molecules, designated TRICOM, to stimulate effective anti-tumor T-cell responses and their potential as cancer vaccines. Delivery of transgenes to the B-CLL cells could be accomplished through the use of a modified vaccinia virus strain Ankara designated MVA. This is a highly attenuated, non-replicative virus that has been demonstrated to be safe and well tolerated in both animals and humans. Here we report the use of a recombinant MVA vector to deliver the simultaneous expression of three human costimulatory molecules (B7-1, ICAM-1, LFA-3) on the surface of B-CLL cells. Infection was conducted using various multiplicity-of-infection (MOI) of MVA-TRICOM and the efficiency of the vector to enhance the expression of the encoded transgenes was evaluated by FACS analysis. The infection of B-CLL cells with MVA-TRICOM markedly increased the expression of B7-1, ICAM-1 and LFA-3 on the cell surface. The ability of the MVA-TRICOM-infected B-CLL cells to activate specific T cells was subsequently analyzed in proliferation assays, cytotoxic, and cytokine production assays. Proliferation of both autologous and allogeneic T cells was observed when MVA-TRICOM-infected B-CLL cells were used as stimulators in mixed lymphocyte reactions. Autologous CTLs were then generated in vitro using MVA-TRICOM-infected B-CLL cells as APC; these CTLs were capable of lysing uninfected autologous as well as allogeneic B-CLL cells. From our results we conclude that MVA-TRICOM-infected B-CLL cells can be used to induce B-CLL-specific CTLs that, in turn, can mediate the lysis of unmodified B-CLL cells. Our findings have implications for a potential use of MVA-TRICOM-infected B-CLL cells as tumor vaccines in the immunotherapy of B-CLL.
Constitutive Activation of Akt and mTOR by Flt3 Internal Tandem Duplications Mediates Myeloid Leukemogenesis and Can Be Inhibited by Rapamycin.
Authors: Brandts Christian H. , Sargin Bulent , Rode Miriam , Biermann Christoph , Lindtner Beate , Schwable Joachim , Buerger Horst , Muller-Tidow Carsten , Choudhary Chunaram , McMahon Martin , Berdel Wolfgang E. , Serve Hubert L. 
Published: April 2020

Abstract
Up to 30% of patients with acute myeloid leukemia (AML) harbor internal tandem duplications (ITD) within the FLT3 gene, a tyrosine kinase receptor. The signalling pathways and downstream targets are only partly understood. We demonstrated that the presence of Flt3-ITD constitutively activated Akt (PKB), a key serine-threonine kinase within the PI3-kinase pathway. The constitutive activation of Akt induced phosphorylation and transcriptional inhibition of the transcription factor Foxo3a. Also, forced Foxo3a activity reversed Flt3-ITD-mediated growth properties. Using a novel Akt inhibitor, we showed that Flt3-ITD-mediated Foxo3a phosphorylation was dependent on Akt. Conditional Akt activation targeted to the cell membrane conferred cytokine-independent survival, proliferation and transformation of myeloid progenitor cells. Induction of Akt activity in vivo promoted the development of a myeloproliferative disease in a syngeneic mouse model. Importantly, phosphorylation of Akt was frequently found in primary myeloid blasts, supporting that this signalling pathway may be important in the development of AML. Furthermore, activation of Akt constitutively activated the downstream kinase mammalian target of rapamycin (mTOR). mTOR has several important biological functions, notably in nutrient sensing and protein translation. Treatment with rapamycin, a specific inhibitor of mTOR, inhibited mTOR kinase activity at 1nM in all examined cell systems. Rapamycin caused cell cycle arrest, apoptosis and completely inhibited transformation in FLT3-ITD-positive cells. These effects were most pronounced in the low nanomolar range, which are therapeutic concentrations of rapamycin-treated patients. The biological advantage in Akt-expressing cells was fully reversed by treatment with rapamycin. In all examined primary blasts from AML patients, mTOR was inhibited by rapamycin. In short term proliferation assays, twelve out of 18 (67%) examined showed marked sensitivity with an IC50 below 10nM rapamycin, while one third were found to be more resistant.
Taken together, increased survival, proliferation and leukemic transformation by Flt3-ITD is mediated by Akt and mTOR, and can be inhibited by rapamycin. We conclude that rapamycin may be an attractive substance for the treatment of patients with AML.