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.

ABT-737, an Inhibitor of Bcl-2/Bcl-XL Proteins, Is Cytotoxic in Multiple Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1589-1589
Author(s):  
Michael Kline ◽  
Terry Kimlinger ◽  
Michael Timm ◽  
Jessica Haug ◽  
John A. Lust ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Tumor Microenvironment ◽  
Cell Lines ◽  
Plasma Cells ◽  
Annexin V ◽  
Significant Activity ◽  
Rpmi 8226 ◽  
Dose Dependent

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that is incurable with the currently available therapeutics. New therapies based on better understanding of the disease biology are urgently needed. MM is characterized by accumulation of malignant plasma cells predominantly in the bone marrow. These plasma cells exhibit a relatively low proliferative rate as well as a low rate of apoptosis. Elevated expression of the anti-apoptotic Bcl-2 family members has been reported in MM cell lines as well as in primary patient samples and may be correlated with disease stage as well as resistance to therapy. ABT-737 (Abbott Laboratories, Abbott Park, IL) is a small-molecule inhibitor designed to specifically inhibit anti-apoptotic proteins of the Bcl-2 family and binds with high affinity to Bcl-XL, Bcl-2, and Bcl-w. ABT-737 exhibits toxicity in human tumor cell lines, malignant primary cells, and mouse tumor models. We have examined the in vitro activity of this compound in the context of MM to develop a rationale for future clinical evaluation. 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 of ABT-737 was measured using the MTT viability assay. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Flow cytometry was also used to measure BAX: Bcl-2 ratios after ABT-737 treatment and cell permeabilization with FIX & PERM (Caltag Laboratories, Burlingame, CA) Results: ABT-737 exhibited cytotoxicity in several MM cell lines including RPMI 8226, KAS-6/1, OPM-1, OPM-2, and U266 with an LC50 of 5-10μM. The drug also had significant activity against MM cell lines resistant to conventional agents such as melphalan (LR5) and dexamethasone (MM1.R) with similar LC50 (5-10 μM), as well as against doxorubicin resistant cells (Dox40), albeit at higher doses. Furthermore, ABT-737 retained activity in culture conditions reflective of the permissive tumor microenvironment, namely in the presence of VEGF, IL-6, or in co-culture with marrow-derived stromal cells. ABT-737 was also cytotoxic to freshly isolated primary patient MM cells. Time and dose dependent induction of apoptosis was confirmed using Annexin V/PI staining of the MM cell line RPMI 8226. Flow cytometry analysis of cells treated with ABT-737 demonstrated a time and dose dependent increase in pro-apoptotic BAX protein expression without significant change in the Bcl-XL or Bcl-2 expression. Ongoing studies are examining the parameters and mechanisms of ABT-737 cytotoxicity to MM cells in more detail. Conclusion: ABT-737 has significant activity against MM cell lines and patient derived primary MM cells in vitro. It is able to overcome resistance to conventional anti-myeloma agents suggesting a different mechanism of toxicity that may replace or supplement these therapies. Additionally, it appears to be able to overcome resistance offered by elements of the tumor microenvironment. The results of these studies will form the framework for future clinical evaluation of this agent in the clinical setting.

Hedgehog Pathway as a Potential Therapeutic Target in Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 672-672
Author(s):  
Simona Blotta ◽  
Joeseph Negri ◽  
Purushothama Nanjappa ◽  
Anne-Sophie Moreau ◽  
Rao Prabhala ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Viability ◽  
Cell Lines ◽  
Small Molecule ◽  
Plasma Cells ◽  
Annexin V ◽  
Inhibition Of Proliferation ◽  
Ic50 Values ◽  
Gli 1

Abstract We have previously demonstrated that a consistent feature of malignant plasma cells of multiple myeloma (MM) is the aberrant expression of genes important in patterning and development, such as members of Hedghehog (Hh) pathway (FE Davies et al, Blood 2003). These findings suggest that overexpression of genes of this pathway, already involved in many solid tumors and recently implicated in maintaining a proposed MM stem cell compartment (CD Peacock et al, PNAS 2007), might be one of the mechanism through which Hh-signaling contributes to tumorigenesis in MM. Therefore, several small molecule modulators of Hh-pathway, which work as agonists and antagonists, are currently under development. We evaluated, by microarray analysis, the expression of Hh pathway genes in MM cell lines and primary MM cells vs. plasma cells from healthy donors. We found that primary MM cells overexpress Sonic (Shh), Smoothened (Smo), Patched (Ptc), Gli-1 and Gli-3 (relative expression ratios ranging from +1.8 to +5.0). Overexpression of Patched was also observed in most of the MM cell lines analyzed (+5.0 ratio in 5 of 6 MM cell lines). Additionally, we confirmed the expression of Shh and of Gli-1, by flow cytometry and western blotting respectively, in a large panel of MM cell lines. These data suggest an activation of the Hh-pathway in MM that, in some cell lines, is Shh-dependent. Therefore, we investigated the therapeutic potential of Hh-inhibitors in MM. We assayed the cell viability and proliferation, by MTT and Thymidine uptake respectively, in 8 MM cell lines after 72 hours of treatment with the small molecule Smo-inhibitor CUR-0199691 (Genentech). We observed a reduction in MM cell viability, with IC50 values ranging between 4.5–9.5 μM in these 8 cell lines and an inhibition of MM cell proliferation with IC50 values ranging between 0.5 and 2.5μM in the same cell lines. MM cell sensitivity to this compound appears to be related to the level of expression of Gli-1, since the cell lines with lower level of expression of Gli-1 were more sensitive. The treatment of these MM cell lines with Cyclopamine, another Hh-inhibitor, showed an IC50 between 7.5μM and 10μM after at least 96 hours of treatment in 4 of the MM cell lines tested. CUR-0199691 is also active in primary MM cells, triggering inhibition of proliferation by 50% at 5μM after only 24h of treatment, while cyclopamine reduces MM cell proliferation (normalized to the effect of tomatidine, its inactive analog) by 30% at 20μM after a 48 hour treatment. Annexin V-PI staining of Hh inhibitor-treated KMS11 cells, which are one of the most sensitive MM cell lines, showed induction of apoptosis, evidenced by detection of 12 and 15% of MM cells being Annexin V+/PI- after 48h and 72h respectively with 5μM of CUR-0199691. These results, taken together, show that the Hh-pathway is fuctionally active in MM and that the novel Hh pathway inhibitor CUR-0199691 is 4–5 times more effective than cyclopamine in both MM cell lines and primary MM cells. These studies provide the framework for further preclinical evaluation of CUR-0199691 in MM models towards possible future clinical trials.

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.

Distribution of Bim Determines MCL-1 Dependence or Co-Dependence on BCL-xL/BCL-2 In Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4054-4054
Author(s):  
Lawrence H. Boise ◽  
Alejo A Morales ◽  
Metin Kurtoglu ◽  
Shannon M Matulis ◽  
Rebecca Markovitz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Cellular Localization ◽  
Plasma Cells ◽  
Annexin V ◽  
Initial Distribution ◽  
Normal Plasma ◽  
Significant Difference ◽  
Dependent Cell ◽  
Smoldering Myeloma

Abstract Abstract 4054 The inappropriate activation of oncogenes can result in the up-regulation of pro-apoptotic signals often in the form of BH3-only proteins such as Bim, Noxa or Puma. This renders cells dependent on anti-apoptotic proteins including BCL-2, BCL-xL and MCL-1. Thus cancer cells would be predicted to be more susceptible to inhibition of BCL-2 family proteins, prompting the development and testing of several small molecule inhibitors of this class of proteins. Multiple myeloma is a plasma cell malignancy of the bone marrow and like normal plasma cells, myeloma plasma cells express MCL-1. A search of gene expression profile data from normal plasma cells (n=22), MGUS plasma cells (n=12), plasma cells from patients with asymptomatic (smoldering) myeloma (n=44) or newly diagnosed multiple myeloma (n=538) revealed no significant difference in MCL-1 mRNA expression associated with progression of disease. We then determined MCL-1 dependence through the introduction of siRNA in 4 MM cell lines and consistent with previous findings using anti-sense oligonucleotides in additional lines, we demonstrated that all cell lines tested were MCL-1 dependent. However using the BCL-2/BCL-xL/BCL-w-selective inhibitor ABT-737 we found that 3 of the 6 MCL1-dependent cell lines tested were sensitive (IC50 for Annexin V-FITC positive at 24 hrs of 300 – 600 nM) and therefore also dependent on BCL-xL/BCL-2. Taken together this is the first formal demonstration that cells can be co-dependent on multiple Bcl-2 family members. We have previously reported that ABT-737 sensitivity, and what we now refer to as co-dependence on MCL-1 and BCL-xL/BCL-2, is determined by the distribution of BIM on the anti-apoptotic BCL-2 proteins in these cells. We have now expanded these findings to patient samples that displayed sensitivity to ABT-737 that is similar to what we have observed in the co-dependent cell line MM.1s. Consistent with these findings co-immunoprecipation revealed BIM binding predominantly to BCL-xL. Additionally we have now demonstrated that BIM binding is not simply controlled by the expression levels of BCL-xL or MCL-1 as enforced over-expression of each protein could alter the sensitivity of co-dependent cell lines to ABT-737 but did not alter the initial distribution of BIM amongst these proteins. These data suggest that additional factors regulate the association of BIM with anti-apoptotic BCL-2 proteins. These factors could include differences in cellular localization of these proteins as well as differences in post-translational modifications of either pro- or anti-apoptotic BCL-2 family proteins. Disclosures: Boise: University of Chicago: Patents & Royalties.

The Immunotoxin HM1.24-ETA′ Is a Potent Inducer of Apoptosis in Malignant Plasma Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3658-3658
Author(s):  
Matthias Staudinger ◽  
Anja Muskulus ◽  
Renate Burger ◽  
Andreas Guenther ◽  
Roland Repp ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Annexin V ◽  
Myeloma Cell ◽  
Parp Cleavage ◽  
Single Chain ◽  
Potent Inducer ◽  
Promising Target

Abstract Despite new treatment modalities, the clinical outcome of at least a subgroup of patients with multiple myeloma (MM) still needs improvement. Antibody-based targeted therapies are increasingly used for tumor therapy, and may represent interesting options for MM patients. HM1.24 is a surface molecule that is over expressed on malignant plasma cells and efficiently internalized from the cell surface. It may represent a promising target for the development of myeloma-directed immunoconstructs. Here, the development and characterization of a novel single-chain immunotoxin, HM1.24-ETA′, is described. HM1.24-ETA′ was generated by genetic fusion of an HM1.24-specific single-chain Fv (scFv) antibody and a truncated variant of Pseudomonas aeruginosa exotoxin A (ETA′). The immunotoxin was expressed in E. coli and purified to homogeneity by affinity chromatography. Specific binding to HM1.24 was demonstrated by immunofluorescence staining and flow cytometry using antigen positive and negative cells. HM1.24-ETA′ efficiently inhibited growth of IL-6 dependent and IL-6 independent myeloma cell lines (INA-6, RPMI8226, U266). Half maximal growth inhibition was observed at low nanomolar concentrations. Further analyses demonstrated that target cell killing occurred via induction of apoptosis, as evidenced by Annexin V/propidium iodide staining and detection of PARP cleavage. The cytotoxic effect was completely blocked by adding excess of unconjugated parental antibody, demonstrating that the effect was antigen-specific and not mediated by non-specific uptake of the immunotoxin. Importantly, HM1.24-ETA′ efficiently triggered apoptosis (>80% Annexin V positive cells) of freshly isolated plasma cell leukemia cells within 48h. In conclusion, HM1.24-ETA′ efficiently triggered apoptosis of multiple myeloma cell lines as well as freshly isolated tumor cells. These results indicate that HM1.24 may represent a promising target structure for efficient antigen-specific delivery of cytotoxic compounds to plasma cell tumors.

The Sequence-Selective DNA Cross-Linking Agent SJG-136 (SG2000, BN2629) Is Highly Potent in Multiple Myeloma Cells and Is Synergistic with Bortezimib

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5166-5166
Author(s):  
Elisabeth J Walsby ◽  
Alan Burnett ◽  
Steven Knapper ◽  
Chris Fegan ◽  
Chris Pepper
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
Cell Lines ◽  
Plasma Cells ◽  
Single Agent ◽  
Annexin V ◽  
Cross Linking ◽  
Incurable Disease ◽  
High Level

Abstract Despite recent advances in the treatment of multiple myeloma (MM), including the introduction of bortezimib, it remains an incurable disease with a short median survival. The development of new agents with efficacy in MM therefore remains both important and urgent. SJG-136 (SG2000, BN2629) is a novel DNA cross-linking agent that binds in a sequence-selective manner in the minor groove of the DNA helix. It is structurally novel compared with other clinically used DNA cross-linking agents and has exhibited a unique pattern of activity in the NCI 60-cell line screen. We have previously shown that this agent is highly effective against primary chronic lymphocytic leukaemia cells. In this study we evaluated its potential as a therapy for MM in cell lines and plasma cells derived from patients. The MM cell lines H929, U266 and JJN3 were shown to have a mean LD50 of 5.1nM (±3.4) following in vitro culture with SJG-136 for 48 hours. This was accompanied by a dose-dependent increase in the percentage of apoptotic cells as evidenced by Annexin V positivity and caspase-3 activation. In comparison to the standard therapies for MM, SJG-136 demonstrated significantly lower LD50 values than doxorubicin (P=0.004), Melphalan (P=0.004) and bortezimib (P=0.03). We also assessed the ability of SJG-136 to kill plasma cells from primary myeloma samples (identified by CD38 and CD138 positivity). These cells were sensitive to SJG-136 with a mean LD50 of 2.3nM (± 0.96) following 48 hour exposures to SJG-136. In contrast, normal bone marrow was significantly less affected by SJG-136 than the myeloma samples (P<0.0001). Finally, we assessed the potential for synergy between SJG-136 and these standard treatments using the H929 cell line as a model. SJG-136 was combined with doxorubicin (1:125), melphalan (1:4000) and bortezimib (1:8) and the combination index (CI) was calculated to assess synergy. A CI of less than 1 was considered synergistic. We found no evidence for synergy between SJG-136 and doxorubicin or melphalan (CI=1.29 and 1.75 respectively). However, SJG-136 was synergistic with bortezimib (CI=0.47) possibly reflecting their different mechanisms of action. In conclusion, SJG-136 is a potentially valuable addition to the battery of drugs available for treatment of MM. Not only was SJG-136 highly potent as a single agent in MM cell lines and primary plasma cells but it also showed a high level of synergy with the proteasome inhibitor bortezimib.

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.

Impact of Natural Dietary Agents on Multiple Myeloma Prevention and Treatment: Molecular Insights and Potential for Clinical Translation

2020 ◽  
Vol 27 (2) ◽  
pp. 187-215 ◽  
Author(s):  
Lavinia Raimondi ◽  
Angela De Luca ◽  
Gianluca Giavaresi ◽  
Agnese Barone ◽  
Pierosandro Tagliaferri ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Multiple Myeloma ◽  
Natural Products ◽  
Bone Disease ◽  
Molecular Mechanisms ◽  
Plasma Cells ◽  
Hematologic Malignancy ◽  
Signal Transduction Pathways ◽  
Pharmacologically Active ◽  
Dietary Agents

: Chemoprevention is based on the use of non-toxic, pharmacologically active agents to prevent tumor progression. In this regard, natural dietary agents have been described by the most recent literature as promising tools for controlling onset and progression of malignancies. Extensive research has been so far performed to shed light on the effects of natural products on tumor growth and survival, disclosing the most relevant signal transduction pathways targeted by such compounds. Overall, anti-inflammatory, anti-oxidant and cytotoxic effects of dietary agents on tumor cells are supported either by results from epidemiological or animal studies and even by clinical trials. : Multiple myeloma is a hematologic malignancy characterized by abnormal proliferation of bone marrow plasma cells and subsequent hypercalcemia, renal dysfunction, anemia, or bone disease, which remains incurable despite novel emerging therapeutic strategies. Notably, increasing evidence supports the capability of dietary natural compounds to antagonize multiple myeloma growth in preclinical models of the disease, underscoring their potential as candidate anti-cancer agents. : In this review, we aim at summarizing findings on the anti-tumor activity of dietary natural products, focusing on their molecular mechanisms, which include inhibition of oncogenic signal transduction pathways and/or epigenetic modulating effects, along with their potential clinical applications against multiple myeloma and its related bone disease.

Ruxolitinib Regulates the Autophagy Machinery in Multiple Myeloma Cells

2020 ◽  
Vol 20 (18) ◽  
pp. 2316-2323 ◽  
Author(s):  
Alican Kusoglu ◽  
Bakiye G. Bagca ◽  
Neslihan P.O. Ay ◽  
Guray Saydam ◽  
Cigir B. Avci
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
B Lymphocyte ◽  
Plasma Cells ◽  
Annexin V ◽  
Myeloma Cell ◽  
Control Group ◽  
Multiple Myeloma Cell ◽  
Ic50 Values ◽  
Stat Pathway

Background: Ruxolitinib is a selective JAK1/2 inhibitor approved by the FDA for myelofibrosis in 2014 and nowadays, comprehensive investigations on the potential of the agent as a targeted therapy for haematological malignancies are on the rise. In multiple myeloma which is a cancer of plasma cells, the Interleukin- 6/JAK/STAT pathway is emerging as a therapeutic target since the overactivation of the pathway is associated with poor prognosis. Objective: In this study, our purpose was to discover the potential anticancer effects of ruxolitinib in ARH-77 multiple myeloma cell line compared to NCI-BL 2171 human healthy B lymphocyte cell line. Methods: Cytotoxic effects of ruxolitinib in ARH-77 and NCI-BL 2171 cells were determined via WST-1 assay. The autophagy mechanism induced by ruxolitinib measured by detecting autophagosome formation was investigated. Apoptotic effects of ruxolitinib were analyzed with Annexin V-FITC Detection Kit and flow cytometry. We performed RT-qPCR to demonstrate the expression changes of the genes in the IL-6/JAK/STAT pathway in ARH-77 and NCI-BL 2171 cells treated with ruxolitinib. Results: We identified the IC50 values of ruxolitinib for ARH-77 and NCI-BL 2171 as 20.03 and 33.9μM at the 72nd hour, respectively. We showed that ruxolitinib induced autophagosome accumulation by 3.45 and 1.70 folds in ARH-77 and NCI-BL 2171 cells compared to the control group, respectively. Treatment with ruxolitinib decreased the expressions of IL-6, IL-18, JAK2, TYK2, and AKT genes, which play significant roles in MM pathogenesis. Conclusion: All in all, ruxolitinib is a promising agent for the regulation of the IL-6/JAK/STAT pathway and interferes with the autophagy mechanism in MM.

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