Role of B-cell-activating factor in adhesion and growth of human multiple myeloma cells in the bone marrow microenvironment

Recent studies have underscored the role of B-cell-activating factor (BAFF), a member of the tumor necrosis factor superfamily, in promoting the survival of malignant B cells, including human multiple myeloma. We here characterized the functional significance of BAFF in the interaction between multiple myeloma and bone marrow stromal cells (BMSC) and further defined the molecular mechanisms regulating these processes. BAFF is detected on BMSCs derived from multiple myeloma patients as evidenced by flow cytometry. BAFF secretion is 3- to 10-fold higher in BMSCs than in multiple myeloma cells, and tumor cell adhesion to BMSCs augments BAFF secretion by 2- to 5-fold, confirmed by both ELISA and immunoblotting. Adhesion of MM1S and MCCAR multiple myeloma cell lines to KM104 BMSC line transfected with a luciferase reporter vector carrying the BAFF gene promoter (BAFF-LUC) significantly enhanced luciferase activity, suggesting that nuclear factor-kappaB (NF-kappaB) activation induced by multiple myeloma adhesion to BMSCs mediates BAFF up-regulation. Moreover, BAFF (0-100 ng/mL) increases adhesion of multiple myeloma lines to BMSCs in a dose-dependent manner; conversely, transmembrane activator and calcium modulator and cyclophylin ligand interactor-Ig or B-cell maturation antigen/Fc blocked BAFF stimulation. Using adenoviruses expressing dominant-negative and constitutively expressed AKT as well as NF-kappaB inhibitors, we further showed that BAFF-induced multiple myeloma cell adhesion is primarily mediated via activation of AKT and NF-kappaB signaling. Importantly, BAFF similarly increased adhesion of CD138-expressing patient multiple myeloma cells to BMSCs. These studies establish a role for BAFF in localization and survival of multiple myeloma cells in the bone marrow microenvironment and strongly support novel therapeutics, targeting the interaction between BAFF and its receptors in human multiple myeloma.     

Human anti-CD40 antagonist antibody triggers significant antitumor activity against human multiple myeloma

Monoclonal antibodies (mAb) directed against lineage-specific B-cell antigens have provided clinical benefit for patients with hematologic malignancies, but to date no antibody-mediated immunotherapy is available for multiple myeloma. In the present study, we assessed the efficacy of a fully human anti-CD40 mAb CHIR-12.12 against human multiple myeloma cells. CHIR-12.12, generated in XenoMouse mice, binds to CD138-expressing multiple myeloma lines and freshly purified CD138-expressing cells from >80% multiple myeloma patients, as assessed by flow cytometry. Importantly, CHIR-12.12 abrogates CD40L-induced growth and survival of CD40-expressing patient multiple myeloma cells in the presence or absence of bone marrow stromal cells (BMSC), without altering constitutive multiple myeloma cell proliferation. Immunoblotting analysis specifically showed that PI3-K/AKT, nuclear factor-kappaB (NF-kappaB), and extracellular signal-regulated kinase activation induced by CD40L (5 mug/mL) was inhibited by CHIR-12.12 (5 mug/mL). Because CD40 activation induces multiple myeloma cell adhesion to both fibronectin and BMSCs, we next determined whether CHIR-12.12 inhibits this process. CHIR-12.12 decreased CD40L-induced multiple myeloma cell adhesion to fibronectin and BMSCs, whereas control human IgG1 did not. Adhesion of multiple myeloma cells to BMSCs induces interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) secretion, and treatment of multiple myeloma cells with CD40L further enhanced adhesion-induced cytokine secretion; conversely, CHIR-12.12 blocks CD40L-enhanced IL-6 and VEGF secretion in cocultures of multiple myeloma cells with BMSCs. Finally, CHIR-12.12 triggered lysis of multiple myeloma cells via antibody-dependent cellular cytotoxicity (ADCC) but did not induce ADCC against CD40-negative multiple myeloma cells, confirming specificity against CD40-expressing multiple myeloma cells. These results provide the preclinical rationale for clinical trials of CHIR-12.12 to improve patient outcome in multiple myeloma.     

Curcumin in combination with bortezomib synergistically induced apoptosis in human multiple myeloma U266 cells

Growth of multiple myeloma cells is controlled by various factors derived from host bone marrow microenvironments. Interaction between multiple myeloma cells and bone marrow stromal cells (BMSCs) plays an important role in the expression of adhesive molecules and secretion of growth factors involved in multiple myeloma (MM) cell growth, survival, and resistance to anticancer drugs. Recently, the possibility of developing novel anti-cancer therapeutic strategies targeting both MM cells and MM cell–BMSC interactions has been discussed. Here we present data showing that curcumin, a major constituent of turmeric compounds extracted from the rhizomes of the plant Curcuma longa, effectively reduced the growth of MM cells and BMSCs. Upon treatment with curcumin, IL-6/sIL-6R-induced STAT3 and Erk phosphorylation was dramatically reduced in the co-cultured cells. In addition, curcumin inhibited the production of pro-inflammatory cytokines and VEGF, factors that are associated with the progression of multiple myeloma, from both MM cells and BMSCs. In a combination treatment with curcumin and bortezomib, IL-6/sIL-6R-induced STAT3 and Erk phosphorylation was effectively inhibited. Moreover, this combination treatment synergistically inhibited the growth of MM cells co-cultured with BMSCs as compared to controls. Taken together, these results indicate that curcumin potentiates the therapeutic efficacy of bortezomib in MM suggesting this combination therapy to be of value in the clinical management of MM.     

Adherence of multiple myeloma cells to bone marrow stromal cells upregulates vascular endothelial growth factor secretion: therapeutic applications.

Increased angiogenesis has recently been recognized in active multiple myeloma (MM). Since vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are two key mediators of angiogenesis, we characterized the production of VEGF, b-FGF and interleukin-6 (IL-6) (a MM growth and survival factor) in MM cell lines and Epstein-Barr virus (EBV) transformed B cell lines from MM patients, patient MM cells, as well as bone marrow stromal cells (BMSCs) from normal healthy donors and MM patients. We detected secretion of VEGF, but no bFGF and IL-6, in MM cell lines (MM.1S, RPMI 8226 and U266); EBV transformed B cell lines from MM patients (IM-9, HS-Sultan and ARH77); MM cell lines resistant to doxorubicin (RPMI-DOX40), mitoxantrone (RPMI-MR20), melphalan (RPMI-LR5) and dexamethasone (MM.1R); and patient MM cells (MM1 and MM2). BMSCs from MM patients and normal donors secreted VEGF, b-FGF and IL-6. Importantly, when MM cells were adhered to BMSCs, there was a significant increase in VEGF (1.5- to 3.1-fold) and IL-6 (1.9- to 56-fold) secretion. In contrast, the bFGF decreased in co-cultures of BMSCs and MM cells. Paraformaldehyde fixation of BMSCs or MM cells prior to adhesion revealed that VEGF was produced both from BMSCs and MM cells, though it may come primarily from BMSCs in some cultures. IL-6 was produced exclusively in BMSCs, rather than MM cells. Moreover, when MM cells were placed in Transwell insert chambers to allow their juxtaposition to BMSCs without cell to cell contact, induction of VEGF and IL-6 secretion persisted, suggesting the importance of humoral factors. Addition of exogenous IL-6 (10 ng/ml) increased VEGF secretion by BMSCs. Conversely, VEGF (100 ng/ml) significantly increased IL-6 secretion by BMSCs. Moreover, anti-human VEGF (1 microg/ml) and anti-human IL-6 (10 microg/ml) neutralizing antibodies reduced IL-6 and VEGF secretion, respectively, in cultures of BMSCs alone and co-cultures of BMSCs and MM cells. Finally, thalidomide (100 microM) and its immunomodulatory analog IMiD1-CC4047 (1 microM) decreased the upregulation of IL-6 and VEGF secretion in cultures of BMSCs, MM cells and co-cultures of BMSCs with MM cells. These data demonstrate the importance of stromal-MM cell interactions in regulating VEGF and IL-6 secretion, and suggest additional mechanisms whereby thalidomide and IMiD1-CC4047 act against MM cells in the BM millieu.     

Novel inosine monophosphate dehydrogenase inhibitor VX-944 induces apoptosis in multiple myeloma cells primarily via caspase-independent AIF/Endo G pathway

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme required for the de novo synthesis of guanine nucleotides from IMP. VX-944 (Vertex Pharmaceuticals, Cambridge, MA, USA) is a small-molecule, selective, noncompetitive inhibitor directed against human IMPDH. In this report, we show that VX-944 inhibits in vitro growth of human multiple myeloma (MM) cell lines via induction of apoptosis. Interleukin-6, insulin-like growth factor-1, or co-culture with bone marrow stromal cells (BMSCs) do not protect against VX-944-induced MM cell growth inhibition. VX-944 induced apoptosis in MM cell lines with only modest activation of caspases 3, 8, and 9. Furthermore, the pan-caspase inhibitor z-VAD-fmk did not inhibit VX-944-induced apoptosis and cell death. During VX-944-induced apoptosis, expressions of Bax and Bak were enhanced, and both apoptosis-inducing factor (AIF) and endonuclease G (Endo G) were released from the mitochondria to cytosol, suggesting that VX-944 triggers apoptosis in MM cells primarily via a caspase-independent, Bax/AIF/Endo G pathway. Importantly, VX-944 augments the cytotoxicity of doxorubicin and melphalan even in the presence of BMSCs. Taken together, our data demonstrate a primarily non-caspase-dependent apoptotic pathway triggered by VX-944, thereby providing a rationale to enhance MM cell cytotoxicity by combining this agent with conventional agents which trigger caspase activation.     

Biologic sequelae of c-Jun NH(2)-terminal kinase (JNK) activation in multiple myeloma cell lines

Although c-Jun NH(2)-terminal kinase (JNK) is activated by treatment with therapeutic agents, the biologic sequelae of inhibiting constitutive activation of JNK has not yet been clarified. In this study, we examine the biologic effect of JNK inhibition in multiple myeloma (MM) cell lines. JNK-specific inhibitor SP600125 induces growth inhibition via induction of G1 or G2/M arrest in U266 and MM.1S multiple myeloma cell lines, respectively. Neither exogenous IL-6 nor insulin-like growth factor-1 (IGF-1) overcome SP600125-induced growth inhibition, and IL-6 enhances SP600125-induced G2/M phase in MM.1S cells. Induction of growth arrest is mediated by upregulation of p27(Kip1), without alteration of p53 and JNK protein expression. Importantly, SP600125 inhibits growth of MM cells adherent to bone marrow stromal cells (BMSCs). SP600125 induces NF-kappaB activation in a dose-dependent fashion, associated with phosphorylation of IkappaB kinase alpha (IKKalpha) and degradation of IkappaBalpha. In contrast, SP600125 does not affect phosphorylation of STAT3, Akt, and/or ERK. IKK-specific inhibitor PS-1145 inhibits SP600125-induced NF-kappaB activation and blocks the protective effect of SP600125 against apoptosis. Our data therefore demonstrate for the first time that inhibiting JNK activity induces growth arrest and activates NF-kappaB in MM cells.     

Bone marrow stromal cells create a permissive microenvironment for myeloma development: a new stromal role for Wnt inhibitor Dkk1

The rapid progression of multiple myeloma is dependent upon cellular interactions within the bone marrow microenvironment. In vitro studies suggest that bone marrow stromal cells (BMSC) can promote myeloma growth and survival and osteolytic bone disease. However, it is not possible to recreate all cellular aspects of the bone marrow microenvironment in an in vitro system, and the contributions of BMSCs to myeloma pathogenesis in an intact, immune competent, in vivo system are unknown. To investigate this, we used a murine myeloma model that replicates many features of the human disease. Coinoculation of myeloma cells and a BMSC line, isolated from myeloma-permissive mice, into otherwise nonpermissive mice resulted in myeloma development, associated with tumor growth within bone marrow and osteolytic bone disease. In contrast, inoculation of myeloma cells alone did not result in myeloma. BMSCs inoculated alone induced osteoblast suppression, associated with an increase in serum concentrations of the Wnt signaling inhibitor, Dkk1. Dkk1 was highly expressed in BMSCs and in myeloma-permissive bone marrow. Knockdown of Dkk1 expression in BMSCs decreased their ability to promote myeloma and the associated bone disease in mice. Collectively, our results show novel roles of BMSCs and BMSC-derived Dkk1 in the pathogenesis of multiple myeloma in vivo.     

Growth inhibition of multiple myeloma cells by a novel IkappaB kinase inhibitor.

Involvement of nuclear factor-kappaB (NF-kappaB) in cell survival and proliferation of multiple myeloma has been well established. In this study we observed that NF-kappaB is constitutively activated in all human myeloma cell lines, thus confirming the previous studies. In addition, we found the phosphorylation of p65 subunit of NF-kappaB in addition to the phosphorylation of IkappaBalpha and the activation of NF-kappaB DNA binding and that various target genes of NF-kappaB including bcl-x(L), XIAP, c-IAP1, cyclin D1, and IL-6 are up-regulated. We then examined the effect of a novel IkappaB kinase inhibitor, 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile (ACHP). When myeloma cells were treated with ACHP, the cell growth was efficiently inhibited with IC(50) values ranging from 18 to 35 mumol/L concomitantly with inhibition of the phosphorylation of IkappaBalpha/p65 and NF-kappaB DNA-binding, down-regulation of the NF-kappaB target genes, and induction of apoptosis. In addition, we observed the treatment of ACHP augmented the cytotoxic effects of vincristine and melphalan (l-phenylalanine mustard), conventional antimyeloma drugs. These findings indicate that IkappaB kinase inhibitors such as ACHP can sensitize myeloma cells to the cytotoxic effects of chemotherapeutic agents by blocking the antiapoptotic nature of myeloma cells endowed by the constitutive activation of NF-kappaB.     

Novel therapies targeting the myeloma cell and its bone marrow microenvironment.

Novel therapies in multiple myeloma (MM) target not only the tumor cell but also the bone marrow (BM) microenvironment. Thalidomide (Thal), as well as derivative immunomodulatory drugs (IMiDs), directly induce apoptosis or G1 growth arrest in MM cell lines and patient's MM cells which are resistant to melphalan (Mel), doxorubicin (Dox), and dexamethasone (Dex). Although Thal and IMiDs do not alter adhesion of MM cells to bone marrow stromal cells (BMSCs), they inhibit the upregulation of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) secretion triggered by the binding of MM cells to BMSCs. Proteasome inhibitors represent another potential anticancer therapy targeting the MM cell and the BM microenvironment. The proteasome inhibitor PS-341 directly inhibits proliferation and induces apoptosis in both human MM cell lines and freshly isolated patient's MM cells which are resistant to Mel, Dox, and Dex. PS-341 inhibits p44/42 mitogen-activated protein kinase (MAPK) growth signaling triggered by IL-6 and induces apoptosis, despite induction of p21 and p27, in p53 wild-type and p53 mutant MM cells. PS-341 adds to the anti-MM activity of dexamethasone and overcomes IL-6-mediated protection against dexamethasone-induced apoptosis. PS-341 blocks the paracrine growth of human MM cells by decreasing their adherence to BMSCs and related NF-kappaB-dependent induction of IL-6 secretion in BMSCs. Moreover, proliferation and MAPK growth signaling of those residual adherent MM cells is also inhibited. Tumor necrosis factor-alpha (TNF-alpha), which is produced by some MM cells, induces only low-level MM proliferation and MAPK activation in MM cells, but markedly upregulates IL-6 secretion from BMSCs and upregulates expression of adhesion molecules (VLA-4 and LFA-1) on MM cells and their receptors (VCAM-1 and ICAM-1) on BMSCs, with resultant increased binding of MM cells to BMSCs. Inhibition of TNF-alpha-induced NF-kappaB activation with PS-341 inhibits both the upregulation of these molecules on MM cells and BMSCs and the resultant increased adhesion. Therefore, inhibiting TNF-alpha and its sequelae may be useful treatment strategies in MM. Our data show that VEGF causes proliferation and enhances migration of MM as well as plasma cell leukemia (PCL) cells. VEGF induced twofold activation of cell migration in MM cells and more than 100-fold activation of cell migration in PCL cells, suggesting an important role of VEGF in the progression of MM to PCL. These data indicate that VEGF plays a pivotal role not only in neoangiogenesis in MM BM but also in proliferation and migration of tumor cells.     

Combination therapy with interleukin-6 receptor superantagonist Sant7 and dexamethasone induces antitumor effects in a novel SCID-hu In vivo model of human multiple myeloma.

Interleukin-6 (IL-6) protects multiple myeloma cells against apoptosis induced by glucocorticoids. Here, we investigated whether inhibition of the IL-6 signaling pathway by the IL-6 receptor superantagonist Sant7 enhances the in vivo antitumor effects of dexamethasone on the IL-6-dependent multiple myeloma cell line INA-6. For this purpose, we used a novel murine model of human multiple myeloma in which IL-6-dependent INA-6 multiple myeloma cells were directly injected into human bone marrow implants in severe combined immunodeficient (SCID) mice (SCID-hu). The effect of in vivo drug treatments on multiple myeloma cell growth was monitored by serial determinations of serum levels of soluble IL-6 receptor (shuIL-6R), which is released by INA-6 cells and served as a marker of tumor growth. In SCID-hu mice engrafted with INA-6 cells, treatment with either Sant7 or dexamethasone alone did not induce significant reduction in serum shuIL-6R levels. In contrast, the combination of Sant7 with dexamethasone resulted in a synergistic reduction in serum shuIL-6R levels after 6 consecutive days of treatment. Gene expression profiling of INA-6 cells showed down-regulation of proliferation/maintenance and cell cycle control genes, as well as up-regulation of apoptotic genes in multiple myeloma cells triggered by Sant7 and dexamethasone combination. In vitro colony assays showed inhibition of myeloid and erythroid colonies from normal human CD34(+) progenitors in response to dexamethasone, whereas Sant7 neither inhibited colony growth nor potentiated the inhibitory effect of dexamethasone. Taken together, these results indicate that inhibition of IL-6 signaling by Sant7 significantly potentiates the therapeutic action of dexamethasone against multiple myeloma cells, providing the preclinical rationale for clinical trials of Sant7 in combination with dexamethasone to improve patient outcome in multiple myeloma.     

沙利度胺对难治、复发多发性骨髓瘤患者骨髓微环境的影响

背景与目的:越来越多的研究表明,骨髓瘤细胞的生长、生存以及耐药的产生与骨髓微环境密切相关。沙利度胺(thalidomide,Thal)是作用于骨髓微环境的药物之一,我们通过观察沙利度胺对难治、复发多发性骨髓瘤(multiplemyeloma,MM)患者骨髓微环境的影响,进一步了解Thal的作用机制。方法:用流式细胞仪检测沙利度胺治疗前后难治、复发的MM患者骨髓基质细胞膜表面的细胞间粘附分子(ICAM-1)和血管细胞粘附分子(VCAM-1)的表达强度,半定量RT-PCR法检测骨髓基质细胞IL-1βmRNA、IL-6mRNA、TNF-αmRNA的表达,采用酶联免疫吸附法(ELISA)测定其血清VEGF、bFGF水平。结果:难治、复发MM患者骨髓基质细胞(BMSC)膜表面ICAM-1、VCAM-1平均荧光强度分别为13.28±4.26、10.35±2.47,用Thal治疗有效的难治、复发MM患者BMSC膜表面ICAM-1、VCAM-1平均荧光强度分别为4.29±0.98、3.54±0.62,明显受抑制(P<0.05)。难治、复发MM患者BMSCIL-1βmRNA、IL-6mRNA、TNF-αmRNA与β-actin的比值分别为1.83±0.64、24.52±11.46、3.42±1.83,用Thal后有效的难治、复发MM患者BMSCIL-1βmRNA、IL-6mRNA、TNF-αmRNA与β-actin的比值分别为0.58±0.11、13.47±14.31、1.25±0.76,明显受抑制(P<0.05)。而治疗无效的难治、复发MM患者BMSC膜表面IC     

The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells

Human multiple myeloma (MM) is a presently incurable hematological malignancy, and novel biologically based therapies are urgently needed. Proteasome inhibitors represent a novel potential anticancer therapy. In this study, we demonstrate that the proteasome inhibitor PS-341 directly inhibits proliferation and induces apoptosis of human MM cell lines and freshly isolated patient MM cells; inhibits mitogen-activated protein kinase growth signaling in MM cells; induces apoptosis despite induction of p21 and p27 in both p53 wild-type and p53 mutant MM cells; overcomes drug resistance; adds to the anti-MM activity of dexamethasone; and overcomes the resistance to apoptosis in MM cells conferred by interleukin-6. PS-341 also inhibits the paracrine growth of human MM cells by decreasing their adherence to bone marrow stromal cells (BMSCs) and related nuclear factor kappaB-dependent induction of interleukin-6 secretion in BMSCs, as well as inhibiting proliferation and growth signaling of residual adherent MM cells. These data, therefore, demonstrate that PS-341 both acts directly on MM cells and alters cellular interactions and cytokine secretion in the BM millieu to inhibit tumor cell growth, induce apoptosis, and overcome drug resistance. Given the acceptable animal and human toxicity profile of PS-341, these studies provide the framework for clinical evaluation of PS-341 to improve outcome for patients with this universally fatal hematological malignancy.     

The bone marrow stromal microenvironment influences myeloma therapeutic response in vitro.

The bone marrow microenvironment supports growth and differentiation of normal hematopoietic cells and can contribute to malignant growth. Since myeloma cells localize and accumulate in bone marrow, it is important to understand the influence of the bone marrow microenvironment not only on the growth of the malignant cells, but also on the therapeutic response of myeloma cells. Growth factors such as interleukin-6 (IL-6) produced by bone marrow stromal cells can protect myeloma cells from glucocorticoid-induced apoptosis. We examined the effect of myeloma cells-bone marrow stromal cells interaction in vitro on several therapeutic treatments. An interleukin-6-dependent myeloma cell line ANBL6 was used and treated with dexamethasone, doxorubicin, and melphalan in the presence of bone marrow stromal cells. Stromal cells were able to protect ANBL6 from dexamethasone, but significantly enhanced the effect of doxorubicin and melphalan. IL-6-induced bcl-XL and cyclin D2 expression in ANBL6 cells, but dexamethasone was able to suppress both bcl-XL and cyclin D2 expression in ANBL6. Doxorubicin and melphalan were able to suppress bcl-XL expression only in the presence of IL-6. We also looked at the effect of activating mutations of N-ras in myeloma cells interacting with stromal cells on therapeutic responses. Surprisingly, ANBL6 N-ras shows significant resistance to all drugs used. Notably, the presence of stromal cells did not alter ANBL6 Nras cells' drug resistance. These results suggest both the bone marrow microenvironment and genetic alterations of myeloma cells can independently impact on therapeutic responses.