bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibitors of mToR Mediate Anti-Myeloma Activity by Apoptosis through the Extrinsic Pathway.
Authors: Günther Andreas , Tiemann Markus , Bakker Frank , Baum Wolfgang , Brocke-Heidrich Katja , Horn Friedemann , Burger Renate , Gramatzki Martin 
Published: October 2019

Abstract
The mammalian target of rapamycin (mToR) plays a crucial role in cell growth due to its role as nutrient dependent regulator of important cytokine signalling pathways. In multiple myeloma (MM) mToR is involved in the AKT pathway which can be activated by the loss of the tumor suppressor PTEN or stimulation with growth and survival factors such as interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1). mToR can be blocked by rapamycin or everolimus. When various human plasmacytoma lines were tested, rapamycin and everolimus induced a dose-dependent growth inhibition. The addition of bone marrow stromal cells, IL-6 or IGF-1 was unable to prevent the inhibitory activity. Growth inhibition was mediated not only by G1 cell cycle arrest but also by an induction of apoptosis as measured by propidium iodide staining. Further analysis was performed to elucidate the mechanism of apoptosis in the particular sensitive plasma cell line INA-6. mToR inhibition induced exclusively cleavage of pro-caspase 8 (extrinsic pathway) but not of pro-caspase 9 (intrinsic pathway). To confirm this observation, INA-6 cells were transfected with BCL-XL, an inhibitor of the intrinsic pathway, and treated with rapamycin. The transfectants showed similar sensitivity to rapamycin as the paternal cell line. Rapamycin was also tested in the INA-6 SCID mouse xenograft model.
Rapamycin was given orally for two weeks starting 24 h after tumor inoculation. A significant survival benefit of the rapamycin treated group compared to control animals (p= 0.0004) was observed. Six out of 14 treated mice did not develop plasmacytomas during the observation period of 149 days. In addition, even short term treatment of plasmacytoma bearing mice led to a significant shrinkage of the tumor mass. Histological examination revealed an increase of apoptotic plasma cells according to morphology after 72 h of treatment compared to controls. Induction of apoptosis in tumor cells was confirmed by immunohistological staining using an antibody specific for the human cleaved form of poly (ADP-ribose) polymerase. Since drugs inhibiting mToR are inducing apoptosis in human malignant plasma cells not only in vitro but also in vivo, the inhibition of mToR may represent an attractive new concept for multiple myeloma therapy.
On the Molecular Mechanism of DKK1 Inhibition of Osteoblast Differentiation in Multiple Myeloma.
Authors: Qiang Ya-Wei , Chen Yu , Stephens Owen , Rubin Jeff , Rudikoff Stuart , Barlogie Bart , Shaughnessy John D. 
Published: October 2019

Abstract
Lytic bone destruction is a severe complication of multiple myeloma growth in the bone. We have demonstrated that Dkk1, an antagonist of Wnt signaling which is essential for osteoblast growth and differentiation, is expressed by myeloma plasma cells leading to increased levels of this protein in serum. Myeloma serum can block osteoblast differentiation in-vitro and a neutralizing anti-DKK1 antibody can inhibit this. These data suggest that MM cells disrupt Wnt signaling and that this promotes uncoupled bone turnover. However, the molecular mechanism responsible for this processes remains to be elucidated. Here we investigated Wnt signaling in osteoblast differentiation and identified the molecular mechanism regulating this process. Using qRT-PCR and sequence confirmation we showed that multiple Frizzled (Fz) mRNAs and LRP5/6 co-receptors were expressed in C2C12 cells and three human osteoblast cell lines, hFOB1.19, MG63 and Saos-2. Furthermore, these cells had high levels of total beta-catenin an indicator of active Wnt signaling. Wnt3a conditioned medium (CM) and recombinant Wnt3a protein increased uncomplexed beta-catenin level as assessed by GST-E-cadherin binding assay. Blocking autocrine Wnt signaling with Dkk1 blocked Wnt-3a-induced increase in beta-catenin levels in dose-dependent fashion. Co-culturing of OPM-2 MM cells ectopically expressing Dkk1 inhibited the Wnt-3a induced beta-catenin stabilization in C2C12 cells. Transfection of C2C12 cells with a dominant negative beta-catenin (DNBC) completely blocked endogenous and Wnt-3a-induced TCF/LEF transcriptional activity as evidenced by TOPflash luciferase activity. Furthermore, BMP-2 induced increase in alkaline phosphatase (ALP) activity was completed abrogated in C2C12 cells expressing DNCB. Silencing LRP5/6 mRNA with small inference RNAs blocked BMP-2 induced increase in ALP expression, suggesting that canonical Wnt pathway contributes to BPM-2 induced OB differentiation. BMP-2 induced phosphorylation of Smad 1, 5 and 8 whereas Wnt-3a had no effect on phosphorylation of these Smads. Moreover, Dkk1 did not block BMP-2 induced phosphorylation of Smad-1, −5 and −8 and did not alter transcriptional activity of Cbfa1, an essential transcriptional factor for OB differentiation. Taken together, these data suggest that Wnt induction of OB cell differentiation through canonical Wnt-beta-catenin pathway is independent of the activation of the Smad pathway. Our results suggested that canonical Wnt signaling pathway plays an important role in osteoblastogenesis and that Dkk1 inhibits OB cell differentiation in a way that may contribute to MM bone lesions. New therapies targeting DKK1 in myeloma may represent a novel treatment strategy.
ZIO-101, a Novel Organic Arsenic, Inhibits Human Myeloma Cell Growth in a SCID-hu Model.
Authors: Campbell Richard A. , Sanchez Eric , Chen Haiming , Turker Lauren , Li Mingjie , Trac Olivia , Shalitin Dror , Gordon Melinda S. , Pang Shen , Bonavida Benjamin , Said Jonathan , Wallner Barbara , Gale Robert P. , Berenson James R. 
Published: October 2019

Abstract
Inorganic arsenics like arsenic trioxide (ATO) are novel anti-cancer drugs active in acute promyelocytic leukemia (APL) and multiple myeloma (MM). ATO induces apoptosis of plasma cells by several mechanisms including down-regulation of BCL-2 expression and inhibition of DNA-binding by NF-κB. The amount of ATO that can be safely given is low because of QTc-prolongation. ZIO-101, a new organic arsenic, is in phase-1/-2 trials. ZIO-101 can be safely given at much higher doses than ATO. We evaluated the in vivo anti-myeloma activity of ZIO-101 in a SCID-hu mouse model of human myeloma. LAGλ-1 was developed from a person with melphalan-resistant and LAGλ-1B from a person with bortezomib-resistant myeloma. Each severe combined immunodeficient (SCID) mouse was implanted with a 2–4 mm3 fragment of LAGλ-1B or LAGλ-1 into the left superficial gluteal muscle. Fragments were allowed to grow for 14 d when human IgG was first detectable. ZIO-101 was given IV 1 or 2 times/d using three different schedules: one day/w, 3 days/w or 5 days/w at doses of 50– 200 mg/kg/d. Tumor volume and human IgG were assessed weekly. Doses up to 200 mg/kg were well-tolerated. Anti-myeloma effects were observed in both models at doses of 100 – 200 mg/kg on all 3 schedules. Mice receiving 100 mg/kg twice daily thrice weekly and those receiving 200 mg/kg once weekly showed marked anti-myeloma activity (100 mg/kg, P = 0.03; 200 mg/kg, P = 0.001) and reduced human IgG levels (100 mg/kg, P < 0.001; 200 mg/kg, P = 0.01) compared to controls. We are evaluating ZIO-101 in other SCID-hu models of human myeloma and exploring different doses and schedules of ZIO-101 alone or combined with other anti-myeloma agents. In summary, these data show activity of ZIO-101 in human myeloma in vivo. These studies provide the bases for future clinical trials.
Inhibition of Multiple Myeloma Cells Growth by AS602868, a Pharmacological Inhibitor of IKK2.
Authors: Jourdan Michel , Moreaux Jerome , Hose Dirk , Rossi Jean-Francois , Mahtouk Karène , Aboulaze Mathieu , Robert Nicolas , Baudard Marion , Reme Thierry , Romanelli Angela , Goldschmidt Hartmut , Dreano Michel , Klein Bernard 
Published: October 2019

Abstract
The NF-κB pathway is involved in the physiological regulation of cell proliferation in many cell types as well as in the resistance of several malignancies to cell death. The pathophysiologic basis for multiple myeloma (MM) has been attributed to the dysregulation of various paracrine or autocrine growth factor loops and to perturbations in several signal transduction pathways including IKK/NF-κB. The aim of the present study was to investigate the effect of a pharmaceutical IKK2 inhibitor (IKK2-I), the anilinopyrimidine derivative AS602868 (Serono International SA), on the in vitro growth of human MM cell lines (HMCL) and primary MM cells. We evaluated the effect of AS602868 on the proliferation and the survival of 12 IL-6-dependent HCML and 2 autonomously growing HCML as well as on the survival of total bone marrow mononuclear cells from patients with newly diagnosed MM (n = 6) or with relapsing MM (n = 7). Results show that using HMCL or primary MM cells, AS602868 induces a clear dose-dependent inhibition of MM cell growth (the 50% inhibitory concentration (IC50) ranging from 0.28 to 8.3 μM, mean IC50 = 2.6 μM on HCML). It was shown using HMCL that the growth inhibition induced by AS602868 is the result of a simultaneous induction of apoptosis and inhibition of the cell cycle progression. Importantly, AS602868 does not alter the survival of other bone marrow mononuclear cells (CD138−) co-cultured with primary MM (CD138+) cells except on CD34+ hematopoietic stem cells. Interestingly, using gene expression profiling with Affymetrix microarrays on 13 HMCL, we show that the resistance (high IC50) to AS602868 inhibitor is strongly correlated to APRIL gene expression (r =.7603, p <.01). Thus, an autocrine production of APRIL confers a resistance to the AS602868 IKK2-I. This can be explained since APRIL has been shown to activate the alternative NF-κB pathway which implicates an IKK complex composed of IKK1 homodimers instead of the IKK1/IKK2/NEMO complex involved in the canonical NF-κB pathway. The results demonstrate the important role of NF-κB in maintaining survival of MM cells and suggest that a pharmacological inhibition of the NF-κB pathway by an IKK2-I, AS602868, can efficiently kill myeloma cell lines or primary myeloma cells and might represent an innovative approach for treating MM patients.
BSc2118, a Novel Proteasome Inhibitor, Shows Anti-Tumor Activity in Multiple Myeloma and Mantle Cell Lymphoma.
Authors: Sterz Jan , Jakob Christian , Kuckelkorn Ulrike , Braun Hannes A. , Kaiser Martin , Fleissner Claudia , Kleeberg Lorenz , Heider Ulrike , Possinger Kurt , Kloetzel Peter M. , Sezer Orhan , Zavrski Ivana 
Published: October 2019

Abstract
The ubiquitin-proteasome pathway was recently identified as a promising new therapeutic target in cancer treatment. An increased proteasome activity was described in certain cancer types, especially in multiple myeloma (MM) and also in mantle cell lymphoma (MCL). The proteasome inhibitor bortezomib has been approved for the treatment of refractory MM and has also shown reproducible activity in MCL. Recently we described a novel tripeptide compound, BSc2118, with inhibitory activity against all three proteolytic activities (post-glutamyl peptide hydrolase-like, trypsin-like and chymotrypsin-like activities) of the 20S proteasome (Cancer Res2006;66:7598–605). We investigated the in vitro effects of BSc2118 in MM and MCL cell lines by MTT cell viability and AnnexinV apoptosis-assays. Furthermore, the intracellular chymotrypsin-like proteasome activity and NF-κB activity were detected in MM and MCL cells by detection of proteasome-degraded peptides or NF-κB p65 subunit. In MM cell lines OPM-2 and U266 we could show a significant time and dose-dependent reduction of cell viability by BSc2118 with an IC50 at 48hrs of 52nM and 65nM, respectively, whereas the MM cell line RPMI-S was less sensitive with an IC50 of 287nM. Using AnnexinV assay, a dose-dependent induction of apoptosis by BSc2118 was shown after 48hrs incubation. Comparably, in MCL we also found a time and dose-dependent reduction of cell viability in the cell lines HBL-2, JeKo-1 and Granta-519 with an IC50 of 82nM, 130nM and 262nM, respectively. Furthermore, BSc2118 induced apoptosis in all three MCL cell lines. Additionally, we detected a significant dose-dependent inhibition of intracellular chymotrypsin-like proteasome activity in MM and MCL cells, and a dose-dependent inhibition of TNFα-induced NF-κB activation in the MM cell lines OPM-2 and RPMI-S. This is the first report of anti-tumor effects of the novel proteasome inhibitor BSc2118 in MM and MCL cells. The compound effectively reduces the cell survival and shows a high pro-apoptotic activity in the MM cell lines OPM-2 and U266 and a significant activity in MCL cell lines HBL-2 and JeKo-1. Mechanisms of action are the inhibition of proteasome and NF-κB activity. Since previous clinical trials have shown an activity of the proteasome inhibitor bortezomib in MM and MCL, and adverse effects of other proteasome inhibitors may differ, our preclinical data support the idea to consider BSc2118 as a promising new agent in anti-tumor drug development.
Thymoglobulin (Polyclonal Rabbit Anti Thymocyte Globulin) Has In Vivo Activity in a Mouse Model of Myeloma.
Authors: Timm Michael , Wellik Linda , Kimlinger Teresa , Haug Jessica , Kline Michael , Rajkumar S. Vincent , Greipp Philip , Kumar Shaji 
Published: October 2019

Abstract
Background: Multiple myeloma remains incurable with current approaches and newer therapies are needed to improve the outcome of these patients. While monoclonal antibody based therapies have been successful in some of the hematological malignancies, such approaches have had limited efficacy in the setting of myeloma. Thymoglobulin (polyclonal rabbit antithymocyte globulin, Genzyme) (Thymo) has been extensively evaluated in the setting of allogeneic blood and marrow transplantation and solid organ transplants. Given the polyclonal nature of this product, with antibodies against different B cell antigens, we evaluated the in vitro and in vivo activity of Thymo in myeloma.
Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity following drug treatment was measured using the MTT viability assay. Apoptosis was measured by flow cytometry using Annexin V/PI in cell lines and Apo 2.7 in primary patient plasma cells. Shifts in expression of a variety of different B cell and plasma cell antigens were examined on several different myeloma cell lines following Thymo treatment in order to identify the potential antigenic targets. In vivo activity of thymo was evaluated in a SCID plasmacytoma model injected with RPMI myeloma cell lines.
Results: rATG was cytotoxic in vitro to several MM cell lines (RPMI 8226, U266, OPM1, OPM2) including the IL-6 dependent cell line Kas6/1 with LC50 of around 1 mg/mL. Additionally, thymo was cytotoxic MM cell lines resistant to conventional agents such as doxorubicin (Dox40), melphalan (LR5) and dexamethasone (MM1R). Thymo induced apoptosis in MM cell lines and in patient derived primary myeloma cells. When tested in combination with other anti-myeloma agents an additive effect was seen with doxorubicin, PS341 and melphalan. Using competitive flow cytometry, we identified CD138, CD38, Cd45, CD126, CD49d (VLA4), as well as CD20 as antigens likely to be targeted by Thymo. Tumor bearing mice injected with Thymo at two different doses (5 mg/kg and 10 mg/kg for five days) had significantly delayed tumor growth compared to non-injected mice, and this translated into a better survival for these mice. Mice receiving 10 mg/kg dose had a slower tumor growth compared to 5 mg/kg dose (Figure).
Conclusions: Thymoglobulin has promising in vitro and in vivo activity in the setting of myeloma. These studies will provide the rational for future clinical development of this agent in myeloma alone or in combination with other agents. Based on these results, we are in the process of initiating a clinical trial combining Thymo with Melphalan.
Figure Figure
Changes in Bone Marrow Plasma Cell Surface Markers Following CD138-Magnetic Bead Based Positive Selection.
Authors: Kimlinger Teresa K. , Rajkumar S. Vincent , Kline Michael P. , Haug Jessica L. , Timm Michael M. , Witzig Thomas E. , Kumar Shaji 
Published: October 2019

Abstract
Background: Isolation of malignant plasma cells from bone marrow of patients with monoclonal gammopathies is critical for studies into the disease biology. The isolation of plasma cells have generally been performed by positive selection using plasma cell markers such as CD138 or by negative selection by removing other marrow cells using a cocktail of antibodies. We have previously demonstrated differences in apoptotic rates in plasma cells with and without this enrichment step. Here we have examined the effect of CD138 magnetic bead selection on the surface phenotype of plasma cells by flow cytometry.
Methods: Bone marrow aspirates from patients with myeloma (n=12) were first washed, and then lysed with ACK to eliminate red cells. The samples were split with a portion of the cells further processed for CD138 selection. ACK lysed only whole bone marrow (WBM) and sorted cells were stained with CD38, CD45, CD56, activation markers CD71 and CD69, adhesion markers CD49d, CD11b, and CD66, B cell markers CD19 and CD20, and clonality (kappa and lambda). Gates were drawn around the plasma cells and plasma cell subsets on the basis of CD38/45 expression for both sorted and unsorted samples. In addition, an aliquot of the sorted preparation was examined by immunohistochemistry to calculate the purity of the sorted sample.
Results: Significant differences were observed in terms of the percentage of plasma cells expressing the different antigens when the cells were selected using CD138. This difference included a greater than 10% difference in expression between the two preparations as well as a change from positive to negative in several cases. There was a substantial loss in the expression of CD20, CD71 and CD11b on plasma cells following CD138 based sorting (Table). Among the other markers, CD49d remains unchanged and changes are variable for the other markers. In addition, in 8 of the 12 cases, there was a nearly complete loss of the CD45 positive subset with a loss of discrimination between CD45 negative and CD45 positive plasma cell subsets in the remaining CD138 sorted preparations (figure: unsorted left, sorted right).
Conclusion: In conclusion, the process of CD138 sorting of plasma cells appears to change important markers on the plasma cells and may even eliminate a key subset from further analysis. This should be kept in mind when isolating plasma cells using CD138 positive selection for analysis such as gene expression profiling. Consideration should be given to negative selection using antibodies against other cell types to deplete them.
Marker Unsorted % Sorted % CD56 74 67 CD69 11 2 CD71 23 8 CD49d 95 92 CD11b 33 4 CD66 5 1 CD20 16 6 Figure Figure
New Criteria To Identify Risk of Progression in Monoclonal Gammopathy of Uncertain Significance: Multiparametric Flow Cytometry Analysis of Bone Marrow Plasma Cells.
Authors: Perez-Persona Ernesto , Vidriales María-Belén , Mateo Gema , Sanz Ramón Garcia , Mateos Marivi , de Coca Alfonso Garcia , Galende Josefina , Martin-Nunez Guillermo , Alonso Jose M. , Heras Natalia , Hernandez Jose M. , Martin Alejandro , Lopez-Berges Consuelo , Orfao Alberto , Miguel J.F. San 
Published: October 2019

Abstract
Monoclonal Gammopathy of Uncertain Significance (MGUS) is a monoclonal disorder defined by the presence of a serum monoclonal protein <3g/dL, bone marrow plasma cells < 10% and absence of end-organ damage. The risk of progression to multiple myeloma (MM) is about 1% per year, and therefore these patients require long follow-up. Accordingly, the definition of new parameters that could be used for the identification of patients at risk of progression could be of great value. The aim of the present study is to evaluate the utility of multiparameter flow cytometry analysis of bone marrow (BM) plasma cells (PC) for predicting the risk of progression of MGUS patients. From January 1996 to September 2004, bone marrow aspirate samples from 350 patients, who fulfil the criteria of MGUS according to the International Myeloma Working Group criteria, were analysed by multiparametric flow cytometry. A specific gate on PC was performed based on CD138/CD38 expression and FSC/SSC characteristics and PC were immunophenotypically classified as normal (polyclonal) or aberrant (clonal) according to the expression of CD138, CD38, CD45, CD19 and CD56 antigens. Twenty seven patients (8 %) progressed to MM, with a median time to progression (TTP) of 46 months (range 9 to 109 months). Interestingly, the percentage of aberrant PC within the total BM PC compartment (aPC/BMPCc) clearly identify patients at different risk of progression. Thus, TTP in patients with ≥ 95% aPC/BMPCc was 85 months vs not reached cases with <95% aPC/BMPCc (p=0.0000). Other parameters with a significant influence on progression in the univariate analysis were: paraprotein level (higher vs lower of 2 mg/dl; p= 0.0004), the presence of immunoparesis (no paresis vs. decreased levels in one or two Ig. p= 0.0005), Bence-Jones proteinuria (p= 0.0003), PC BM infiltration assessed both by morphology and flow cytometry (p=0.0074; and p= 0.001, respectively), and DNA index assessed by flow cytometry (diploid vs aneuploid; p=0.0064). Moreover, the cut off level of 95% aPC/BMPCc, also allows the discrimination of two risk categories upon considering only patients at low risk of progression, based on a low paraprotein level or absence of inmunoparesis (p= 0.0000 and p= 0.0000, respectively). On multivariate analysis only the percentage of aPC/BMPCc (≥95%) (p=0.000), the DNA index (p=0.007), and the Bence-Jones proteinuria (p=0.000) showed independent prognostic value. In summary, our results show that multiparameter FC evaluation of BMPC at diagnosis is a simple, cost-effective and valuable tool for predicting the risk of progression of MGUS patients.