TY - JOUR A1 - Lagler, Charlotte A1 - El-Mesery, Mohamed A1 - Kübler, Alexander Christian A1 - Müller-Richter, Urs Dietmar Achim A1 - Stühmer, Thorsten A1 - Nickel, Joachim A1 - Müller, Thomas Dieter A1 - Wajant, Harald A1 - Seher, Axel T1 - The anti-myeloma activity of bone morphogenetic protein 2 predominantly relies on the induction of growth arrest and is apoptosis-independent JF - PLoS ONE N2 - Multiple myeloma (MM), a malignancy of the bone marrow, is characterized by a pathological increase in antibody-producing plasma cells and an increase in immunoglobulins (plasmacytosis). In recent years, bone morphogenetic proteins (BMPs) have been reported to be activators of apoptotic cell death in neoplastic B cells in MM. Here, we use bone morphogenetic protein 2 (BMP2) to show that the "apoptotic" effect of BMPs on human neoplastic B cells is dominated by anti-proliferative activities and cell cycle arrest and is apoptosis-independent. The anti-proliferative effect of BMP2 was analysed in the human cell lines KMS12-BM and L363 using WST-1 and a Coulter counter and was confirmed using CytoTox assays with established inhibitors of programmed cell death (zVAD-fmk and necrostatin-1). Furthermore, apoptotic activity was compared in both cell lines employing western blot analysis for caspase 3 and 8 in cells treated with BMP2 and FasL. Additionally, expression profiles of marker genes of different cell death pathways were analysed in both cell lines after stimulation with BMP2 for 48h using an RT-PCR-based array. In our experiments we observed that there was rather no reduction in absolute cell number, but cells stopped proliferating following treatment with BMP2 instead. The time frame (48–72 h) after BMP2 treatment at which a reduction in cell number is detectable is too long to indicate a directly BMP2-triggered apoptosis. Moreover, in comparison to robust apoptosis induced by the approved apoptotic factor FasL, BMP2 only marginally induced cell death. Consistently, neither the known inhibitor of apoptotic cell death zVAD-fmk nor the necroptosis inhibitor necrostatin-1 was able to rescue myeloma cell growth in the presence of BMP2. KW - apoptosis KW - gene expression KW - necrotic cell death KW - multiple myeloma KW - cell metabolism KW - cell cycle and cell division KW - B cells Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158993 VL - 12 IS - 10 ER - TY - JOUR A1 - Seher, Axel A1 - Lagler, Charlotte A1 - Stühmer, Thorsten A1 - Müller-Richter, Urs Dietmar Achim A1 - Kübler, Alexander Christian A1 - Sebald, Walter A1 - Müller, Thomas Dieter A1 - Nickel, Joachim T1 - Utilizing BMP-2 muteins for treatment of multiple myeloma JF - PLoS ONE N2 - Multiple myeloma (MM) represents a haematological cancer characterized by the pathological hyper proliferation of antibody-producing B-lymphocytes. Patients typically suffer from kidney malfunction and skeletal disorders. In the context of MM, the transforming growth factor β (TGFβ) member Activin A was recently identified as a promoter of both accompanying symptoms. Because studies have shown that bone morphogenetic protein (BMP)-2-mediated activities are counteracted by Activin A, we analysed whether BMP2, which also binds to the Activin A receptors ActRII and ActRIIB but activates the alternative SMAD-1/5/8 pathway, can be used to antagonize Activin A activities, such as in the context of MM. Therefore three BMP2 derivatives were generated with modified binding activities for the type II (ActRIIB) and/or type I receptor (BMPRIA) showing either increased or decreased BMP2 activity. In the context of MM these BMP2 muteins show two functionalities since they act as a) an anti-proliferative/apoptotic agent against neoplastic B-cells, b) as a bone-formation promoting growth factor. The molecular basis of both activities was shown in two different cellular models to clearly rely on the properties of the investigated BMP2 muteins to compete for the binding of Activin A to the Activin type II receptors. The experimental outcome suggests new therapeutic strategies using BMP2 variants in the treatment of MM-related pathologies. KW - multiple myeloma KW - signaling KW - cell proliferation KW - cell binding KW - membrane receptor signaling KW - BMP KW - gene expression KW - B cell receptors KW - B cells Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158144 VL - 12 IS - 5 ER - TY - THES A1 - Lagler, Charlotte T1 - Analyse von BMP2 und BMP2-Derivaten der TGF-β-Familie als potentielles Therapeutikum im Multiplen Myelom T1 - Analysis of BMP2 and BMP2 derivatives of the TGF-β-family as a potential therapeutic agent in multiple myeloma N2 - Diese Dissertation analysiert BMP2 und BMP2-Derivate als neue therapeutische Strategien für die Behandlung des Multiplen Myeloms (MM). Das MM ist eine maligne neoplastische Erkrankung des Knochenmarks mit Plasmazellvermehrung und erhöhten Leveln an Aktivin A im Blutserum, wobei eines der Hauptsymptome das Auftreten von schmerzvollen Osteolysen ist. In den letzten Jahren rückte Aktivin-A als interessantes Target zur Behandlung des Multiplen Myeloms in den Vordergrund. Die Reduzierung der Aktivin-A Level durch decoy-Rezeptoren führte zu einer signifikanten Verbesserung der Osteolysen und einem reduzierten Proliferationsverhalten der neoplastischen B-Zellen, sowohl im Tierexperiment als auch in Studien der klinischen Phase II. Die Aktivin-A-Antagonisierung ist somit ein neuer und vielversprechender Ansatz in der Therapie des Multiplen Myeloms. Das Bone Morphogenetic Protein 2 ist aufgrund seiner molekularen und biologischen Eigenschaften ein interessantes Target für die Therapie des Multiplen Myeloms. Es ist auf molekularer Ebene ein Aktivin-A-Antagonist, besitzt aber auch osteoinduktives Potential und apoptotische bzw. anti-proliferative Eigenschaften auf neoplastische B-Zellen. Da die in der Literatur bereits beschriebenen, durch Mitglieder der TGF-β-Familie induzierten Apoptosemechanismen, noch nicht genauer untersucht waren, wurde in dieser Arbeit die BMP2-induzierte Apoptose in 10 unterschiedlichen humanen MM-Zellen analysiert. Erstens konnte dabei nachgewiesen werden, dass 7 von 10 Zelllinien nicht BMP2-responsiv waren. Eine genauere Untersuchung ergab, dass neben der Expression spezifischer BMP-Rezeptoren auch die Expression von inhibitorischen Smad-Proteinen über die BMP2-Responsivität entscheidet. Zweitens zeigte die genauere Analyse der Apoptosemechanismen, dass entgegen der in der Literatur publizierten Ergebnisse, BMP2 keine apoptotische Wirkung auf die von uns untersuchten Zelllinien hat. Mehrere verschieden durchgeführte Experimente, u.a. die Verwendung von spezifischen Inhibitoren des programmierten Zelltodes, unterstützen dieses Ergebnis und klassifizieren BMP2 als einen rein anti-proliferativen Faktor. Der letzte Teil der Arbeit befasst sich mit der Analyse von potentiellen Aktivin-A-Antagonisten in Form verschiedener BMP2- und GDF5-Derivate und inwiefern sie sich zum Einsatz in der Therapie des Multiplen Myeloms eignen. Die unterschiedlichen Eigenschaften der einzelnen Mutanten wurden in verschiedenen Zellsystemen getestet. So konnte aufgezeigt werden, dass neben einer erhöhten biologischen Aktivität in Form eines gesteigerten osteoinduktiven und anti-proliferativen Potentials auf neoplastische B-Zellen (Superagonisten), sich die verschiedenen Derivate als Super-Antagonisten zu Aktivin A eignen und damit unterschiedlichen Ansprüchen der adjuvanten Therapie im Multiplen Myelom gerecht werden. N2 - This dissertation analyses BMP2 and BMP2 derivatives as new therapeutic agents in multiple myeloma (MM). MM is a malignant neoplastic disease of bone marrow with plasmatic cell proliferation and increased Activin-A-level in blood serum. A particular symptom of this disease is the occurrence of painful osteolysis. In the last few years Activin A has become an important target for the treatment of multiple myeloma. Reducing Activin A levels by decoy receptors led to a significant improvement in osteolysis and reduced proliferation behavior of neoplastic B cells, both in animal experiments and clinical phase II trials. Activin A antagonization is thus a new and promising approach in the treatment of multiple myeloma. Bone Morphogenetic Protein 2 is a promising prospect for the treatment of multiple myeloma due to its molecular and biological properties. It is an Activin-A-antagonist at the molecular level, but also has osteoinductive potential and apoptotic or anti-proliferative properties on neoplastic B cells as already described in literature. Since the apoptotic mechanisms, which members of the TGF-β family induced in MM-cells, have not yet been investigated in detail, the BMP2-induced apoptosis was analyzed in 10 different human MM cells. Firstly it was shown that 7 out of 10 cell lines were not responsive to BMP2. A more detailed analysis revealed that, besides the expression of specific BMP receptors, the expression of inhibitory Smad proteins determines BMP responsiveness. Secondly the more precise analysis of the apoptotic mechanisms revealed that, contrary to the results published in the literature, BMP2 has no apoptotic effect on the cell lines we have examined. Several different experiments, e.g. the use of specific inhibitors of programmed cell death, support this result and classify BMP2 as a purely anti-proliferative factor. The last part of this research deals with the analysis of potential Activin-A-antagonists in form of different BMP2 and GDF5 derivatives and how they are suitable for use in the therapy of multiple myeloma. The different properties of the individual mutants were tested in diverse cell systems. The results demonstrate that in addition to increased biological activity in form of increased osteoinductive and anti-proliferative potential on neoplastic B cells (superagonist), the various derivatives are suitable as super-antagonist to Activin A and cope with different requirements of adjuvant therapy in context of multiple myeloma. KW - BMP KW - Plasmozytom KW - BMP2 KW - Multiples Myelom Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-155553 ER -