Promoting Osteoblastogenesis Using a Novel Dkk-1 Neutralizing Antibody in the Treatment of Multiple Myeloma Related Bone Disease

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2739-2739 ◽  
Author(s):  
Samantha Pozzi ◽  
Hua Yan ◽  
Sonia Vallet ◽  
Nileshwari Vaghela ◽  
Mariateresa Fulciniti ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Bone Disease ◽  
Human Monoclonal Antibody ◽  
Calcium Deposition ◽  
Chimeric Antibody ◽  
Dependent Manner ◽  
Alizarin Red ◽  
Pit Formation

Abstract Treatment of bone disease in multiple myeloma (MM) has largely focussed on the osteoclast axis while the osteoblast axis has been underexploited. Dkk1, an inhibitor of the wingless int (wnt) pathway, is important in osteoblastogenesis. Increased expression of Dkk1 in a subset of MM patients and its association with lytic bone disease opens up the potential of targeting the osteoblast axis. The aim of this study was to test the effect of a Dkk-1 neutralizing chimeric antibody (Mab B3) on osteoblasts (OB), osteoclasts (OC) and MM cells in the context of the bone microenvironment. First, we tested the expression of Dkk1 in plasma and bone marrow of 16 MM patients and 10 MM cell lines. Dkk1 levels were >18 ng/mL in 2 out of 16 patients; levels were comparable in blood and bone marrow plasma. In contrast, very little Dkk1 (2–9 ng/ml) was produced by bone marrow stromal cells (BMSC). One out of 10 MM cell lines (INA-6) expressed low concentrations of Dkk1 in the supernatant. Next, we tested the effect of Mab B3 on MM cell lines, in the presence or absence of BMSC, and on OB and OC from MM patient derived bone marrow. The effects on OC were evaluated by TRAP staining and pit formation. Effects on OB were assayed by alkaline phosphatase staining and alizarin red assays for calcium deposition. Mab B3 treatment did not demonstrate direct cytotoxic effects on MM cell lines negative for Dkk1. Mab B3, however, enhanced OB differentiation and calcium deposition in a dose dependent manner and inhibited OC differentiation and function, as evidenced by a decrease in number of multinucleated TRAP+ cells and a decrease in pit formation. Ongoing studies are addressing the effect of Mab B3 on MM cells in the context of OC and OB. Mab B3 is also undergoing in vivo testing in a SCID-hu model bearing INA-6 MM cells. These studies and the underlying mechanism of action of Mab B3 will be presented. Our preliminary data suggests that Mab B3 has anabolic bone effects; a corresponding human monoclonal antibody may be useful for the treatment of MM related bone disease. Future studies will evaluate Mab B3 in combination with catabolic agents such as bisphosphonates with the goal of restoring normal bone homeostatsis.

Multiple Myeloma Cell-Osteoblast Interaction Results in Impaired Bone Formation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4764-4764
Author(s):  
Sonia Vallet ◽  
Teru Hideshima ◽  
Samantha Pozzi ◽  
Nileshwari Vaghela ◽  
Gaurav Gharti-Chhetri ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Formation ◽  
Cell Lines ◽  
Bone Matrix ◽  
Type I ◽  
Dependent Manner ◽  
Alizarin Red ◽  
Matrix Mineralization ◽  
Osteolytic Bone Disease

Abstract Osteolytic bone disease is a common complication of multiple myeloma (MM) resulting from uncoupled bone remodeling due to enhanced bone resorption and reduced bone formation. Bone formation is a complex process requiring functionally mature osteoblasts (OB). Mesenchymal stem cells differentiate into mature OB and following an active period of bone matrix synthesis lasting 1–2 weeks, they finally differentiate into inactive bone-lining cells or osteocytes. Although several studies have demonstrated that MM cells inhibit osteoblastogenesis via secretion of DKK1, a Wnt-pathway antagonist, the functional sequelae of interaction of mature OB with MM cells remains to be elucidated. Here, we studied the morphological and functional consequences induced by MM cells interacting with mature OB. Mature OB were generated from MM patients’ bone marrow mononuclear cells by cultivation in differentiation media consisting of αMEM with 20% fetal bovine serum, β-glycerol phosphate (2.16 mg/ml), ascorbic acid (0.05 mg/ml) and dexamethasone (10 nM). These mature OBs were alkaline phosphatase (ALP) positive and secreted and mineralized bone matrix, as demonstrated by Alizarin Red staining. MM cell lines INA6 and MM1.S were co-cultured with mature OB at a 5:1 ratio for 2, 4 and 7 days in OB differentiation media and bone marrow stromal cells (BMSC) were used as negative controls. After 4 days of co-culture, we observed phenotypic changes featured by acquisition of a spindle-like shape with reduced ALP staining in OB. In contrast, OB alone were intensely ALP-positive and cuboidal-shaped cells. Co-culture with INA-6 MM cells induced a reduction in ALP enzymatic activity in a time-dependent manner by 28% (± 10%) at day 2 and 72% (± 5%) at day 4 (p<0.05), respectively, whereas co-culture with MM1.S induced a 38% (± 5%) reduction after 4 days. Other MM cell lines induced similar effects. We then verified OB activity by assessing osteocalcin release and matrix mineralization. Importantly, osteocalcin secretion was completely abrogated in the presence of INA6, while MM1.S reduced it by 50% as early as day 2 (p<0.05). Moreover, Alizarin red staining demonstrated an impairment of matrix mineralization after 7 days of co-culture. Reduced OB function in the presence of MM cells was further confirmed by downregulation of Type-I collagen expression in OB. These effects were associated with only modest (10%) OB apoptosis as demonstrated by APO2.7 staining after 4 days of co-culture compared to OB alone. These phenotypic and functional sequelae on OB were not induced by co-culture supernatants, suggesting the requirement for direct MM cell/OB contact. These results therefore suggest that MM cell/mature OB interactions result in inhibition of bone formation by inactivation of mature OB. Ongoing studies are characterizing the mechanism by which MM cells induce OB inactivation and whether these changes affect the OC compartment. These studies of MM cell-OB interactions will form the basis for evaluation of novel agents with anabolic effects on the bone in the future.

Inhibition of ERK1/2 Activity by the MEK1/2 Inhibitor AZD6244 (ARRY-142886) Induces Human Multiple Myeloma Cell Apoptosis in the Bone Marrow Microenvironment: A New Therapeutic Strategy for MM.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3460-3460 ◽  
Author(s):  
Yu-Tzu Tai ◽  
Xian-Feng Li ◽  
Iris Breitkreutz ◽  
Weihua Song ◽  
Peter Burger ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Cell Lines ◽  
Caspase 3 ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Growth And Survival ◽  
Cyclin E1 ◽  
Human Multiple Myeloma

Abstract Activation of the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) signaling pathway mediates tumor cell growth in many cancers, including human multiple myeloma (MM). Specifically, this pathway mediates MM cell growth and survival induced by cytokines/growth factors (i.e. IL-6, IGF-1, CD40, BAFF) and adhesion to bone marrow stromal cells (BMSCs), thereby conferring resistance to apoptosis in the bone marrow (BM) milieu. In this study, we therefore examined the effect of the MEK1/2 inhibitor AZD6244 (ARRY-142886), on human MM cell lines, freshly isolated patient MM cells and MM cells adhered to BMSCs. AZD6244, inhibits constitutive and cytokine (IL-6, IGF-1, CD40)-stimulated ERK1/2, but not AKT phosphorylation. Importantly, AZD6244 inhibits the proliferation and survival of human MM cell lines, regardless of sensitivity to conventional chemotherapy, as well as freshly isolated patient MM cells. AZD6244 induces apoptosis in patient MM cells even in the presence of BMSCs, as evidenced by caspase 3 activity and PARP cleavage at concentrations as low as 20 nM. AZD6244 overcomes resistance to apoptosis in MM cells conferred by IL-6 and BMSCs, and inhibits IL-6 secretion induced by MM adhesion to BMSCs. AZD6244 suppresses MM cell survival/growth signaling pathways (i.e., STAT3, Bcl-2, cyclin E1, CDK1, CDK3, CDK7, p21/Cdc42/Rac1-activated kinase 1, casein kinase 1e, IRS1, c-maf) and up-regulates proapoptotic cascades (i.e., BAX, BINP3, BIM, BAG1, caspase 3, 8, 6). AZD6244 also upregulates proteins triggering cell cycle arrest (i.e. p16INK4A, p18INK4C, p21/WAF1 [Cdkn1a], p27 [kip1], p57). In addition, AZD6244 inhibits adhesion molecule expression in MM cells (i.e. integrin a4 [VLA-4], integrin b7, ICAM-1, ICAM-2, ICAM-3, catenin a1, c-maf) associated with decreased MM adhesion to BMSCs. These pleiotropic proapoptotic, anti-survival, anti-adhesion and -cytokine secretion effects of AZD6244 abrogate BMSC-derived protection of MM cells, thereby sensitizing them to both conventional (dexamethasone) and novel (perifosine, lenalidomide, and bortezomib) therapies. In contrast, AZD6244 has minimal cytotoxicity in BMSCs and does not inhibit DNA synthesis in CD40 ligand-stimulated CD19 expressing B-cells derived from normal donors at concentrations toxic to MM cells (between 0.02–2 mM). Furthermore, AZD6244 inhibits the expression/secretion of osteoclast (OC)-activating factors (i.e., macrophage inflammatory protein (MIP)-1a, MIP-1b, IL-1b, VEGF) from MM cells. It also downregulates MM growth and survival factors (IL-6, BAFF, APRIL) in OC cultures derived from MM patient peripheral blood mononuclear cells (PBMCs). Significantly, AZD6244 inhibits OC differentiation from MM PBMCs (n=10) in a dose-dependent manner. Together these results provide the preclinical basis for clinical trials with AZD6244 (ARRY-142886) in MM.

Dasatinib Promotes Osteoprogenitor Differentiation and Inhibition of Osteoclastogenesis: Rationale for Treatment of Myeloma Bone Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3834-3834
Author(s):  
Antonio Garcia-Gomez ◽  
Mercedes Garayoa ◽  
Enrique M. Ocio ◽  
Edvan Crusoe ◽  
Diego Fernández ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Bone Disease ◽  
Plasma Cells ◽  
Annexin V ◽  
Conditioned Media ◽  
Tartrate Resistant Acid Phosphatase ◽  
Alizarin Red ◽  
Alp Activity ◽  
And Function

Abstract Abstract 3834 Poster Board III-770 Introduction Multiple myeloma (MM), an hematological malignancy of terminally differentiated plasma cells, is characterized by the presence of bone disease, caused by increased osteoclast (OC) activity and differentiation as well as a reduction in osteoblast (OB) number and function. Dasatinib (BMS-354825) is an oral multitargeted tyrosin-kinase inhibitor approved for chronic myeloid leukemia which is also under clinical investigation in several other types of tumors. Preclinical data suggests that dasatinib can also be of value in MM based on its effects on myelomatous plasma cells and angiogenesis. In this study, we have further investigated the effects of dasatinib on in vitro OB differentiation and function, as well as on OC formation and resorption activity. Materials and methods For studies on OB differentiation and function, human mensenchymal stem cells (hMSC) from bone marrow samples of healthy donors and MM patients were used. Alternatively, the mesenchymal hMSC-TERT, the osteoblast-like (MG-63) and multiple myeloma (MM.1S) cell lines were employed. Dasatinib mechanism of action was investigated by Western blotting, PKH67/Annexin V/7 aminoactinomycin D staining, real time RT-PCR, alkaline phosphatase (ALP) activity and quantitative mineralization assays. Receptor activator of nuclear factor κ B ligand (RANKL) and osteoprotegerin (OPG) levels in conditioned media were measured by ELISA. OCs were generated by culture of peripheral blood mononuclear cells from healthy volunteers in medium containing macrophage colony stimulating factor and RANKL. The effect of dasatinib on osteoclastogenesis was assessed by enumeration of multinucleated (≥3) tartrate resistant acid phosphatase-positive cells, whereas bone resorption was calculated by the resorbed area on calcium-coated slides. Results We found that dasatinib inhibited platelet derived growth factor (PDGF)-stimulated activation of PDGF receptor β (PDGFRβ) and c-Src in hMSC-TERT and MG-63 cell lines, both tyrosin kinases implicated in OB remodelation processes. Inhibition of PDGFRβ and c-Src signalling correlated with diminished proliferation of the same cell lines without affecting cell viavility as assessed by PKH67/Annexin V/7 aminoactinomycin D staining. Also, treatment of human osteoprogenitor cells with low dasatinib concentrations (2 - 5 nM) promoted OB differentiation since ALP activity at day 7 and gene expression levels of bone formation markers (Runx2/Cbfa1, ALP, and COLIA1) at day 7 and 14 in the osteoblastic differentiation process, were elevated; besides, dasatinib also increased mineral nodular formation as per quantification of alizarin red staining. Finally, treatment with dasatinib decreased the RANKL/OPG ratio in conditioned media from co-cultures of MG-63 and MM.1S cell lines. Similar range of dasatinib concentrations markedly inhibited OC formation, both at the initial and late stages of differentiation from hemopoietic progenitor cells. Finally, a significant reduction of OC resorptive activity of a calcium-coated substrate was observed. Conclusion Our results indicate that dasatinib favours both OB differentiation and activity and markedly impairs osteoclastogenesis and OC resorption, thus providing rationale for its use to improve bone lesions in MM patients and other bone pathologies. This work was supported by grants from Ministerio de Ciencia e Innovación - ISCIII (PI081825); Fundación de Investigación Médica Mutua Madrileña AP27262008, and Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León 07-09, Consejería Sanidad JCyL-ISCIII. Disclosures: Garzon: Bristol-Myers Squibb Company: Employment.

Inhibition Of PI3K Classia Kinases Using GDC0941 Overcomes Protection Of Multiple Myeloma Cells In The Bone Marrow Microenvironment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3169-3169
Author(s):  
Hugh Kikuchi ◽  
Amofa Eunice ◽  
Maeve McEnery ◽  
Farzin Farzaneh ◽  
Stephen A Schey ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Bone Resorption ◽  
Cell Lines ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Pi3k Pathway ◽  
Dependent Manner ◽  
Dose Dependent

Abstract Despite of newly developed and more efficacious therapies, multiple myeloma (MM) remains incurable as most patient will eventually relapse and become refractory. The bone marrow (BM) microenvironment provides niches that are advantageous for drug resistance. Effective therapies against MM should ideally target the various protective BM niches that promote MM cell survival and relapse. In addition to stromal mesenchymal/myofibroblastic cells, osteoclasts play a key supportive role in MM cell viability. Additionally, 80% of patients develop osteolytic lesions, which is a major cause of morbidity. Increased osteoclast activity is characteristic in these patients and targeting osteoclast function is desirable to improve therapies against MM. Osteoclasts need to form an F-actin containing ring along the cell margin that defines a resorbing compartment where protons and degradative enzymes are secreted for dissolution of bone mineral. Remodelling of F-actin and vesicle secretion are regulated by the class IA PI3K pathway during osteoclastic bone resorption. Additionally, it has recently been shown that inhibition of the class IA PI3K pathway in MM cells with GDC0941 induces apoptosis-mediated killing. We hypothesised that GDC0941 could be used as a therapeutic agent to overcome MM-induced osteoclast activation. GDC0941 inhibited maturation of osteoclasts derived from BM aspirates from MM patients in a dose dependent manner. This correlated with decreased bone resorption of osteoclasts cultured on dentine discs. Exposure of mature osteoclasts to GC0941 resulted in abnormal organisation of larger F-actin rings, suggesting a negative effect on the dynamics of the actin cytoskeleton required for bone resorption. We also found that GDC-0941 can prevent protection of the MM cell lines MM1.S and MM1.R by osteoclasts against killing. GDC-0941 alone blocked MM cell proliferation independently of the presence of BM stromal cells and synergised with other therapeutic agents including Lenalidomide, Pomalidomide, Bortezomid and Dexamethasone. We also found that in the presence of MM cells, Dexamethasone (a drug commonly used alone or in combination with new drugs against MM) induced the proliferation of BM stromal cells and adhesion of MM cells on this protective stroma in a dose dependent manner. Dexamethasone is highly effective at MM cell killing when cells are cultured alone. However, we found that at low doses (below 1 uM) and in the presence of BM stromal cells, Dexamethasone could induce MM cell proliferation. GDC0941 enhanced Dexamethasone killing even in the presence of BM stromal cells by blocking Dexamethasone-induced stromal cell proliferation and adhesion of MM cells on the stroma. Targeting individual the PI3K Class IA isoforms alpha, beta, delta or gamma proved to be a less efficient strategy to enhance Dexamethasone killing. Previous work has shown that efficacy of targeting individual PI3K Class I A isoforms would be low for activation of caspases in MM cells as it would be dependent on relative amounts of isoforms expressed by the MM patient. GDC-0941 also inhibited the proliferation of MM1.R and RPMI8266 MM cell lines, which are less sensitive to treatment to Dexamethasone. Co-culture of MM cells with BM stromal cells induced the secretion of IL-10, IL-6, IL-8, MCP-1 and MIP1-alpha. The dose-dependant increased proliferation of Dexamethasone-treated MM cells in the presence of the BM stroma correlated with the pattern of secretion of IL-10 (a cytokine that can induce B-cell proliferation) and this was blocked by the combination of Dexamethasone with GDC0941. GDC-0941 alone or in combination with Dexamethasone was more efficacious at inducing MM cell apoptosis in the presence of the BM stroma cells vs treatment of MM cells alone. These are very encouraging results as they suggest that GDC-0941 in combination with Dexamethasone would be potentially highly efficacious for targeting MM cells in the BM microenvironment. We are currently performing in vivo data using C57BL/KaLwRij mice injected with 5T33-eGFP MM cells that will be discussed at the meeting. We propose that MM patients with active bony disease may benefit from treatment with GDC0941 alone or in combination with currently used therapeutic drugs against MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals TBK1/Ikkε Inhibitor Amlx Blocks Multiple Myeloma Cell Growth in Vitro and In Vivo

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4504-4504
Author(s):  
Quanhong Sun ◽  
Peng Zhang ◽  
Juraj Adamik ◽  
Konstantinos Lontos ◽  
Valentina Marchica ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Growth ◽  
Cell Lines ◽  
Bone Disease ◽  
Research Funding ◽  
Ex Vivo ◽  
Dominant Negative ◽  
Induction Of Apoptosis

Abstract Multiple myeloma (MM) is the most frequent cancer to involve the skeleton and remains incurable for most patients, thus novel therapies are needed. MM bone disease is characterized by osteolytic lesions that contribute significantly to patient morbidity and mortality. We showed that TBK1 signaling is a novel pathway that increases osteoclast (OCL) formation in Paget's disease, an inflammatory bone disease. Therefore, we hypothesized that TBK1 plays a similar role in MM induction of OCL. We found that MM conditioned media (MM-CM) dose-dependently increased bone marrow monocyte (BMM) expression of activated TBK1 protein and enhanced RANKL-driven OCL formation. TBK1 knockdown by shRNA transduction into BMM significantly attenuated the ability of MM-CM to increase OCL differentiation without altering OCL differentiation in control media. We found that the TBK1/IKKε inhibitor Amlexanox (Amlx) blocked normal and MM-enhanced OCL formation. Importantly, TBK1 mRNA expression in CD138+ plasma cells (PC) isolated from MM or PC leukemia patients is significantly higher as compared to PC from Monoclonal Gammopathy of Undetermined Significance (MGUS) patients. Therefore, we tested whether targeting the TBK1/ IKKε signaling pathways would also affect MM cells. We found that Amlx strongly decreased the viability of several MM cell lines and primary MM cells via induction of apoptosis. Amlx treatment of MM cell lines also induced a G1/S blockade, decreased activated ERK1/2, and increased translation of the dominant-negative C/EBPb-LIP isoform in several MM cell lines. The positive-acting C/EBPb-LAP isoform was previously shown to be a critical transcription factor for MM viability. Importantly, Amlx also enhanced the effectiveness of the proteasome inhibitors bortezomib and carfilzomib to kill MM cells in culture. Further, Amlx sensitized MM1.S cells to the induction of apoptosis by the autophagic inhibitor Bafilomycin A. Amlx dose-dependently inhibited tumor growth in a syngeneic MM mouse model in which 5TGM1 MM cells expressing secreted GLuc were injected subcutaneously into immunocompetent C57Bl/KaLwRij. Tumor growth was assessed by measuring tumor volumes and by the levels of secreted GLuc in the blood. Further, OCL formation ex vivo from bone marrow monocytes obtained from AMLX-treated mice versus controls was decreased. Amlx did not affect the viability of primary BMM, bone marrow stromal cells (BMSC), or splenocytes. Further, Amlx treatment of primary BMSC from MM patients or normal donors decreased expression of TNFα, IL-6 and RANKL, thereby decreasing BMSC support of MM survival and OCL differentiation. Amlx pretreatment of BMSC and murine pre-osteoblast MC4 cells also decreased VCAM1 expression and reduced MM cell adhesion, another mechanism for Amlx reduction of bone microenvironmental MM support. These data suggest that targeting TBK1/IKKε signaling may decrease MM bone disease by slowing MM growth, directly and indirectly, and preventing MM-induced osteolysis. Disclosures Giuliani: Janssen Pharmaceutica: Other: Avisory Board, Research Funding; Celgene Italy: Other: Avisory Board, Research Funding; Takeda Pharmaceutical Co: Research Funding. Roodman:Amgen Denosumab: Membership on an entity's Board of Directors or advisory committees.

Development of a Novel Phthalimide Derivative, Preclinical Effects on High-Risk Myeloma Cells and Osteoclasts

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5718-5718
Author(s):  
Yutaka Hattori ◽  
Maiko Matsushita ◽  
Noriko Tabata ◽  
Hirokazu Shiheido ◽  
Hiroshi Yanagawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Cell Lines ◽  
Pharmacokinetic Study ◽  
Bone Disease ◽  
Mouse Bone Marrow ◽  
Dependent Manner ◽  
Phthalimide Derivative

Abstract BACKGROUND: Despite recent advances in the use of newly developed drugs including immune-modulatory drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide and proteasome inhibitors such as bortezomib, carfilzomib, and MLN9708, MM is still an incurable disease. In particular, MM patients harboring 17p deletion, t(14;16), t(14;20), or t(4;14) are classified as a high-risk group and have shown significantly shorter survival. With the goal of helping prolong the survival of these high-risk MM patients, we screened 29 synthetic phthalimide derivatives and found a novel compound, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3-dione (TC11), which induced the apoptosis of KMS34 cells with t(4;14) and del17p13. PURPOSE:The purpose of this project is to clarify preclinical effects of the synthetic phthalimide derivative, TC11, on high-risk MM cell lines and osteoclasts. Namely, anti-myeloma and anti-osteoclastogenic activities and pharmacokinetic study in mice were shown. We also try to isolate directly binding molecules. Safety issues including hematological toxicities and teratogenicity were also discussed. METHODS AND RESULTS: TC11 significantly inhibited growth of MM cell lines (IC50 4-8μM) including KMS34 and KMS11 cells which have high-risk chromosomal abnormalities. TC11 also suppressed the proliferation of all of the bone marrow cells obtained from the MM patients, in a dose-dependent manner. TC11 increased annexin V-positive fraction and induced apoptosis. TC11 was injected intraperitonealy into myeloma (KMS34 and KMS11 cells)-bearing lcr/SCID mice, and anti-myeloma activity was evaluated in vivo. Twenty mg/kg of TC11 significantly inhibited growth of KMS34 or KMS11-derived plasmacytomas. Apoptosis of MM cells was observed by histopathological examination. In order to evaluate hematological toxicity of TC11, growth of colony-forming cells was examined. In the presence of 5μM of TC11, formation of CFCs was not significantly suppressed, suggesting low hematopoietic toxicity. In the pharmacokinetic analyses using lcr mice, the plasma concentrations of TC11 was examined; Cmaxwas 18.1μM at 1.5hr (Tmax), and T1/2 was 2.5hr, when 100mg/kg of TC11 was injected. If 20mg/kg was injected, Cmaxwas 2.1μM at 1.0hr (Tmax), and T1/2 was 1.2hr. Oral administration of TC11 to Icr mice was safely carried out, and results of pharmacokinetic study will be shown. Aiming at the therapeutic use of TC11 to bone disease, anti-osteoclastogenic activity was examined. Mouse bone marrow mononuclear cells were incubated in the presence of M-CSF and RANK-ligand. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts was reduced in number in the presence of 1μM of TC11. It was also found that 1μM of TC11 inhibited bone resorption by pit assay. We have identified nucleophosmin 1 (NPM1) and α-tubulin as TC11-binding molecules using our unique in vitro selection system using mRNA display, in vitro virus (IVV) method. However, cereblon (CRBN) was not detected as a TC11-binding protein by this method. The immunofluorescent analysis showed that TC11-treated cells exhibited elevated levels of α-tubulin fragmentation. Together with our previous observation of induction of centrosomal disruption of HeLa cells by NPM1-knock down, TC11 may cause anti-myeloma effects via mitotic catastrophe. CONCLUSION: We have demonstrated that TC11, a novel phthalimide derivative, has anti-tumor activity against MM cells with high-risk genetic abnormality including del 17p and t(4;14), in vitro and in vivo. This novel compound also down-regulates the differentiation and function of osteoclasts. Our data provide a strong preclinical rationale for TC11 as a safe and effective drug for the treatment of high-risk MM patients with bone disease. The actions of this drug relating to α-tubulin and NPM1 remain to be further investigated. TC11 exerts its anti-myeloma effect via molecular interactions which do not involve CRBN. In addition, TC11 does not form racemate and is expected to lack teratogenicity. The results of our present study suggest that new phthalimide derivatives other than thalidomide, lenalidomide and pomalidomide could be developed by drug designing for the treatment of MM. Disclosures No relevant conflicts of interest to declare.

CK2 Kinase Inhibitors Display Anti-Myeloma Effects and Antagonize Osteoclast Activity in Models of Multiple Myeloma Bone Marrow Microenvironment

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 444-444
Author(s):  
Sabrina Manni ◽  
Denise Toscani ◽  
Anna Colpo ◽  
Alessandra Brancalion ◽  
Fortunato Zaffino ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Death ◽  
Cell Growth ◽  
Cell Lines ◽  
Bone Disease ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Plasma Cells ◽  
Ck2 Inhibitors

Abstract Abstract 444 Background. Multiple myeloma (MM) plasma cell growth in the bone marrow (BM) microenvironment is fueled by survival signals delivered by the surrounding non-malignant cells (stromal and other types) and through contacts with the extracelllar matrix. Interactions of MM cells with osteoclasts and osteoblasts generate a milieu, in which bone resorption and bone loss occur more rapidly than bone deposition. Novel agents, such as bortezomib and lenalidomide, which target the MM BM microenvironment, have shown unprecedented anti-myeloma efficacy in part due to their ability to somewhat revert these microenvironmental alterations. However, often resistance occurs also to novel drugs and the disease progresses. We have described that targeting protein kinase CK2 with chemical inhibitors or RNA interference causes MM cell death, increases the sensitivity to chemotherapeutics and compromises the NF-κB and STAT3 activity (Piazza FA et al. 2006, Blood; 108: 1698). We also found that CK2 inhibitors synergize with Hsp90 inhibitors (Manni S et al. 2012, Clinical Cancer Res; 18: 1888) and bortezomib (Manni S et al., Blood (2011 ASH Annual Meeting Abstracts); 118; 1849) in inducing MM cell death. Moreover, a phase I clinical trial is ongoing in USA (ID: NCT01199718) testing the oral CK2 inhibitor CX4945 (Cylene Pharmaceuticals, CA, USA) in MM patients. Purpose. We investigated whether and how CK2 inhibition with ATP-competitive CX4945 and tTBB inhibitors could affect the growth of MM cells and of osteoprogenitors in models of BM microenvironemnt. The aim of the study was to provide further insights into the mechanism of action of CK2 inhibitors also in the MM microenvironment, in particular on the stromal cell-mediated MM cell survival and on the unbalanced bone metabolism. We ultimately aimed at generating original data useful for the design of novel rational combination therapies incorporating CK2 inhibitors in the therapy of MM and of MM-bone disease. Methods. MM plasma cells from patients and MM cell lines were cultured in the presence of BM stromal cells obtained from MM patients or BM stromal cell lines or in the presence of osteoclasts. ATP-competitive CK2 inhibitors were added to the co-cultures or to cultures of osteoblast cell lines or progenitors. Cell growth was evaluated with different means and signaling pathways were studied in MM plasma cells and in the stromal cells. NF-κB target gene expression and DNA binding was tested with microplate arrays. For osteoclast generation, CD14+ peripheral blood monocytes were stimulated in alpha-MEM medium with 10% FBS plus RANKL (60ng/ml) plus M-CSF (25ng/ml) for 28 days; early-osteoblasts colonies were obtained from BM cells stimulated under appropriate conditions. Results. CK2 inhibition with CX4945 or tTBB caused apoptosis of MM cells (either freshly isolated from patients or cell lines) cultured on patient-derived mesenchymal stromal cells (MSC) or on the BM stromal cell line HS-5. The inhibitors did not significantly affect MSC viability. A reduction of NF-κB activity evaluated in MM cells was found upon CK2 inhibition, with a parallel reduction of the production of NF-κB-dependent cytokines. When assayed on osteoprogenitors, CX4945 displayed an inhibitory effect on osteoclast formation from CD14+ monocytes even at low concentrations (1 μM up to 7 μM, comparable with the effects of zolendronate 1 μM), whereas it inhibited the formation of osteoblasts from BM colonies at day 14 at fairly higher concentrations (>5 μM). Moreover, CX4945 inhibited osteoblast proliferation at even higher concentration (>7.5 μM). The anti-myeloma effect of CK2 inhibitors was present also when MM cells (INA-6 cell line) were cultured in the presence of osteoclasts generated from CD14+ monocytes. Conclusions. Our study shows that inhibition of CK2 could profoundly affect the growth of MM cells in models of BM microenvironment while substantially sparing the normal cellular stromal counterparts and osteoblasts and suggests that CK2 inhibitors could be exploited to target the hyperactivity of osteoclast seen in MM bone disease. Disclosures: Giuliani: Celgene: Research Funding.

Bone disease as the first manifestation of systemic AL-amyloidosis

2020 ◽  
Vol 92 (7) ◽  
pp. 85-89
Author(s):  
L. P. Mendeleeva ◽  
I. G. Rekhtina ◽  
A. M. Kovrigina ◽  
I. E. Kostina ◽  
V. A. Khyshova ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Disease ◽  
Plasma Cells ◽  
Interventricular Septum ◽  
Bone Destruction ◽  
Amyloid Deposition ◽  
Bone Lesions ◽  
Al Amyloidosis ◽  
Linear Type

Our case demonstrates severe bone disease in primary AL-amyloidosis without concomitant multiple myeloma. A 30-year-old man had spontaneous vertebral fracture Th8. A computed tomography scan suggested multiple foci of lesions in all the bones. In bone marrow and resected rib werent detected any tumor cells. After 15 years from the beginning of the disease, nephrotic syndrome developed. Based on the kidney biopsy, AL-amyloidosis was confirmed. Amyloid was also detected in the bowel and bone marrow. On the indirect signs (thickening of the interventricular septum 16 mm and increased NT-proBNP 2200 pg/ml), a cardial involvement was confirmed. In the bone marrow (from three sites) was found 2.85% clonal plasma cells with immunophenotype СD138+, СD38dim, СD19-, СD117+, СD81-, СD27-, СD56-. FISH method revealed polysomy 5,9,15 in 3% of the nuclei. Serum free light chain Kappa 575 mg/l (/44.9) was detected. Multiple foci of destruction with increased metabolic activity (SUVmax 3.6) were visualized on PET-CT, and an surgical intervention biopsy was performed from two foci. The number of plasma cells from the destruction foci was 2.5%, and massive amyloid deposition was detected. On CT scan foci of lesions differed from bone lesions at multiple myeloma. Bone fragments of point and linear type (button sequestration) were visualized in most of the destruction foci. The content of the lesion was low density. There was no extraossal spread from large zones of destruction. There was also spontaneous scarring of the some lesions (without therapy). Thus, the diagnosis of multiple myeloma was excluded on the basis based on x-ray signs, of the duration of osteodestructive syndrome (15 years), the absence of plasma infiltration in the bone marrow, including from foci of bone destruction by open biopsy. This observation proves the possibility of damage to the skeleton due to amyloid deposition and justifies the need to include AL-amyloidosis in the spectrum of differential diagnosis of diseases that occur with osteodestructive syndrome.

scholarly journals Diagnosis and Treatment of Bone Disease in Multiple Myeloma: Spotlight on Spinal Involvement

Scientifica ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Patrizia Tosi
Keyword(s):  
Spinal Cord ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Bone Resorption ◽  
Spinal Cord Compression ◽  
Bone Disease ◽  
Cord Compression ◽  
Diagnosis And Treatment ◽  
Conventional Chemotherapy ◽  
Spinal Involvement

Bone disease is observed in almost 80% of newly diagnosed symptomatic multiple myeloma patients, and spine is the bone site that is more frequently affected by myeloma-induced osteoporosis, osteolyses, or compression fractures. In almost 20% of the cases, spinal cord compression may occur; diagnosis and treatment must be carried out rapidly in order to avoid a permanent sensitive or motor defect. Although whole body skeletal X-ray is considered mandatory for multiple myeloma staging, magnetic resonance imaging is presently considered the most appropriate diagnostic technique for the evaluation of vertebral alterations, as it allows to detect not only the exact morphology of the lesions, but also the pattern of bone marrow infiltration by the disease. Multiple treatment modalities can be used to manage multiple myeloma-related vertebral lesions. Surgery or radiotherapy is mainly employed in case of spinal cord compression, impending fractures, or intractable pain. Percutaneous vertebroplasty or balloon kyphoplasty can reduce local pain in a significant fraction of treated patients, without interfering with subsequent therapeutic programs. Systemic antimyeloma therapy with conventional chemotherapy or, more appropriately, with combinations of conventional chemotherapy and compounds acting on both neoplastic plasma cells and bone marrow microenvironment must be soon initiated in order to reduce bone resorption and, possibly, promote bone formation. Bisphosphonates should also be used in combination with antimyeloma therapy as they reduce bone resorption and prolong patients survival. A multidisciplinary approach is thus needed in order to properly manage spinal involvement in multiple myeloma.

scholarly journals Establishment of Cell Lines from Both Myeloma Bone Marrow and Plasmacytoma: SNU_MM1393_BM and SNU_MM1393_SC from a Single Patient

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Youngil Koh ◽  
Woo-June Jung ◽  
Kwang-Sung Ahn ◽  
Sung-Soo Yoon
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Cultured Cells ◽  
Clonal Evolution ◽  
Myeloma Cell ◽  
Mouse Bone Marrow ◽  
Single Patient ◽  
U266 Cell

Purpose.We tried to establish clinically relevant human myeloma cell lines that can contribute to the understanding of multiple myeloma (MM).Materials and Methods.Mononuclear cells obtained from MM patient’s bone marrow were injected via tail vein in an NRG/SCID mouse. Fourteen weeks after the injection, tumor developed at subcutis of the mouse. The engraftment of MM cells into mouse bone marrow (BM) was also observed. We separated and cultured cells from subcutis and BM.Results.After the separation and culture of cells from subcutis and BM, we established two cell lines originating from a single patient (SNU_MM1393_BM and SNU_MM1393_SC). Karyotype of the two newly established MM cell lines showed tetraploidy which is different from the karyotype of the patient (diploidy) indicating clonal evolution. In contrast to SNU_MM1393_BM, cell proliferation of SNU_MM1393_SC was IL-6 independent. SNU_MM1393_BM and SNU_MM1393_SC showed high degree of resistance against bortezomib compared to U266 cell line. SNU_MM1393_BM had the greater lethality compared to SNU_MM1393_SC.Conclusion.Two cell lines harboring different site tropisms established from a single patient showed differences in cytokine response and lethality. Our newly established cell lines could be used as a tool to understand the biology of multiple myeloma.

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