Cytotoxic effect of R-etodolac (SDX-101) in combination with purine analogs or monoclonal antibodies on ex vivo B-cell chronic lymphocytic leukemia cells

R-etodolac (SDX-101) is an isoform of the non-steroidal anti-inflammatory drug, etodolac, and is currently being tested in phase II clinical trials for the treatment of refractory B-cell chronic lymphocytic leukemia (B-CLL). The aim of this study was to evaluate the cytotoxicity of SDX-101 combined with agents proven to be effective as first-line treatment of B-CLL: the purine nucleoside analogs, fludarabine (FA) and cladribine (2-CdA), and the monoclonal antibodies, anti-CD52 (alemtuzumab; ALT) and anti-CD20 (rituximab; RIT). The cytotoxicity and specific pro-apoptotic effects of the study drugs on B-CLL cells were assessed in vitro in samples from overall 37 untreated patients. The combinations of SDX-101 with 2-CdA, FA or RIT exerted additive effects in B-CLL cells, with the following combination indices (CI): 0.89 for SDX-101 + 2-CdA, 0.95 for SDX-101 + RIT, and 1.17 for SDX-101 + FA. The main mechanism of these interactions was caspase-mediated apoptosis. The SDX-101 plus ALT combination resulted in only sub-additive cytotoxicity (CI = 1.25). In conclusion, these data obtained in vitro indicate that addition of 2-CdA, FA or RIT to SDX-101 significantly enhance cytotoxicity in B-CLL cells.     

R-etodolac (SDX-101) and the related indole–pyran analogues SDX-308 and SDX-309 potentiate the antileukemic activity of standard cytotoxic agents in primary chronic lymphocytic leukaemia cells

Objective SDX-101 is the non-cyclooxygenase 2-inhibiting R-enantiomer of the non-steroid anti-inflammatory drug etodolac, and has anti-tumour activity in chronic lymphocytic leukaemia (CLL). SDX-308 and SDX-309 are more potent, structurally related indole–pyran analogues of SDX-101. The current study was performed to investigate and quantify the cytotoxic potentiating effects resulting from a combination of either SDX-101, SDX-308 or SDX-309 with standard cytotoxic agents used in the CLL treatment today. Methods The lymphoma cell line U937-gtb was used, together with primary tumour cells isolated from seven CLL patients. Combinations between chlorambucil and each one of the agents etodolac, SDX-101, SDX-308 and SDX-309 were studied. In addition, SDX-309 was combined with fludarabine, doxorubicin or vincristine. Both simultaneous and sequential exposures were explored using the median-effect method. Results Most combinations were additive, which could be of clinical benefit since SDX-101 has been shown to be well tolerated. At the 70% effect level, synergy was observed between SDX-308 and chlorambucil in U937-gtb cells and in two-third of the CLL samples. Since chlorambucil is the most important drug in CLL therapy today and SDX-308 is presently targeted as the lead clinical candidate, this combination would be interesting for further studies. Vincristine and SDX-309 were synergistic in two-fourth of CLL samples. Conclusions To conclude, the non-COX-inhibiting etodolac-derivatives SDX-101, SDX-308 and SDX-309 are potential candidates for combination treatment of CLL. Especially, SDX-308 in combination with chlorambucil warrants further evaluation.     

Immunomodulatory drug lenalidomide (CC-5013, IMiD3) augments anti-CD40 SGN-40-induced cytotoxicity in human multiple myeloma: clinical implications

SGN-40, a humanized immoglobulin G1 (IgG1) anti-CD40 monoclonal antibody, mediates cytotoxicity against human multiple myeloma (MM) cells via suppression of interleukin (IL)-6-induced proliferative and antiapoptotic effects as well as antibody-dependent cell-mediated cytotoxicity (ADCC). Here, we studied the clinical significance of an immunomodulatory drug lenalidomide on SGN-40-induced cytotoxicity against CD138(+)CD40(+) MM lines and patient MM cells. Pretreatment with lenalidomide sensitized MM cells to SGN-40-induced cell death. Combined lenalidomide and SGN-40 significantly induced MM apoptosis, evidenced by enhanced cleavage of caspase-3/8/poly(ADP-ribose)polymerase and increased sub-G(0) cells, compared with either single agent at the same doses. Pretreatment of effector cells with lenalidomide augmented SGN-40-induced MM cell lysis, associated with an increased number of CD56(+)CD3(-) natural killer (NK) cells expressing CD16 and LFA-1. Importantly, pretreatment with lenalidomide or lenalidomide and SGN-40 markedly enhanced NK-cell-mediated lysis of autologous patient MM cells triggered by SGN-40. Lenalidomide also up-regulated CD40L on CD56(+)CD3(-) NK cells, facilitating IL-2-mediated activation of NK cells. In addition, lenalidomide induced the CD56(dim) NK subset, which are more potent mediators of ADCC against target MM cells than the CD56(bright) NK subset. Finally, pretreatment of both effector and target MM cells with lenalidomide markedly enhanced SGN-40-mediated ADCC against CD40-expressing MM cells. These studies, therefore, show that the addition of lenalidomide to SGN-40 enhances cytotoxicity against MM cells, providing the framework for combined lenalidomide and SGN-40 in a new treatment paradigm to both target MM cells directly and induce immune effectors against MM.     

Targeting Akt and heat shock protein 90 produces synergistic multiple myeloma cell cytotoxicity in the bone marrow microenvironment.

PURPOSE: We hypothesized that targeting both Akt and heat shock protein (HSP) 90 would induce cytotoxic activity against multiple myeloma (MM) cells and target the bone marrow (BM) microenvironment to inhibit angiogenesis, osteoclast formation, as well as migration and adhesion of MM cells. EXPERIMENTAL DESIGN: MM cell lines were incubated with perifosine (5 and 10 micromol/L) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG; 50 and 100 nmol/L) alone and in combination. RESULTS: The combination of Akt inhibitor perifosine and HSP90 inhibitor 17-DMAG was synergistic in inducing MM cell cytotoxicity, evidenced by inhibition of DNA synthesis and induction of apoptosis. In addition, perifosine and 17-DMAG almost completely inhibited osteoclast formation: perifosine interfered with both early and late stages of osteoclast progenitor development, whereas 17-DMAG targeted only early stages. We next showed that combined therapy overcomes tumor growth and resistance induced by BM stromal cells and endothelial cells as well as the proliferative effect of exogenous interleukin-6, insulin-like growth factor-I, and vascular endothelial growth factor. Moreover, the combination also induced apoptosis and growth inhibition in endothelial cells and inhibited angiogenesis. Finally, we showed that the two agents prevented migration of MM cells toward stromal-derived factor-1 and vascular endothelial growth factor, which are present in the BM milieu, and also prevented adhesion of MM cells to fibronectin. CONCLUSIONS: This study provides the preclinical framework for treatment protocols targeting both the Akt and HSP pathways in MM.     

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.     

Role of NKG2D in cytokine-induced killer cells against multiple myeloma cells

The cytokine-induced killer cells (CIK) have been reported to have potent cytotoxicity against a variety of tumor cells including multiple myleoma (MM) cells. The mechanisms that CIK cell recognizing MM cells remain unknown. Recent studies indicated that the interaction between NKG2D receptor and NKG2D ligands plays an important role in inducing cytotoxicity against various target cells by natural killer cells (NK). We suspect whether NKG2D receptor and NKG2D ligands interaction is also responsible for the killing of MM cells by CIK as the same way did NK cells. We expanded CIK cells from healthy controls with interferon (IFN)-γ, CD3 monoclonal antibodies (mAb) and interleukin-2 (IL-2), and checked expression of NK cell receptors on CIK cells by flow cytometry. About 86% bulk CIK cells expressed NKG2D receptor but not other NK receptors, such as CD158a, CD158b and NCRs. We analyzed NKG2D ligands expression in MM patients by flow cytometry, primary plasma cells from 8 out of 13 (62%) MM patients expressed different levels of ULBPs or MICA/B on the cell surface. Interestingly, when stimulated with MM cell line U266 that expressed some levels of MICA/B, only NKG2D expressing CIK cells released IFN-γ. CIK cells showed cytotoxicity against NKG2D ligands expressing U266 and primary MM cells, and the cytotoxicity was partially blocked by treating CIK with anti-NKG2D antibody. We conclude that NKG2D-NKG2D ligand interaction may be one of the mechanisms by which CIK cells kill MM cells.     

Identification and validation of novel therapeutic targets for multiple myeloma.

In vitro and in vivo models have been developed that have allowed for delineation of mechanisms of multiple myeloma (MM) cell homing to bone marrow (BM); tumor cell adhesion to extracellular matrix proteins and BM stromal cells; and cytokine-mediated growth, survival, drug resistance, and migration within the BM milieu. Delineation of the signaling cascades mediating these sequelae has identified multiple novel therapeutic targets in the tumor cell and its BM microenvironment. Importantly, novel therapies targeting the tumor cell and the BM, as well as those targeting the tumor cell or BM alone, can overcome the growth, survival, conventional drug resistance, and migration of MM cells bound to BM using both in vitro and in vivo severe combined immunodeficiency mouse models of human MM. These studies have translated rapidly from the bench to the bedside in derived clinical trials, and have already led to the United States Food and Drug Administration approval of the novel proteasome inhibitor bortezomib for treatment of relapsed/refractory MM. Novel agents will need to be combined to enhance cytotoxicity, avoid development of drug resistance, and allow for use of lower doses in combination therapies. Genomics, proteomics, and cell signaling studies have helped to identify in vivo mechanisms of sensitivity versus resistance to novel therapies, as well as aiding in the rational application of combination therapies. These studies have therefore provided the framework for a new treatment paradigm targeting the MM cell in its BM milieu to overcome drug resistance and improve patient outcome in MM.     

Etodolac induces apoptosis and inhibits cell adhesion to bone marrow stromal cells in human myeloma cells

OBJECTIVES: Cyclooxygenase-2 (COX-2) is reported to regulate apoptosis and to be an important cellular target for therapy. METHODS: We examined whether etodolac, meloxicam, and thalidomide inhibited proliferation and induced apoptosis in myeloma cell lines (RPMI 8226 and MC/CAR cells). RESULTS: Etodolac induced apoptosis more strongly compared with thalidomide or meloxicam. Etodolac induced down-regulation of Bcl-2 protein and mRNA, activation of Caspase-9, -7 and -3, cIAP-1 and Survivin, and loss of mitochondrial membrane potential in a dose-dependent manner. In addition, when myeloma cells were coincubated with 50 microM etodolac on bone marrow stromal cells (BMSCs), myeloma cell adhesion to BMSCs was significantly inhibited compared with thalidomide or meloxicam coincubation, and the adhesion molecules VLA-4, LFA-1 (CD11a), CXCX4, and CD44 were suppressed on myeloma cells treated with etodolac. Moreover, 50-100 microM racemate of etodolac significantly inhibited the proliferation of myeloma cells compared to 100 microM R-etodolac or S-etodolac. CONCLUSIONS: Etodolac induced loss of mitochondrial membrane potential and apoptosis via a COX-2-independent pathway, suppressed the expression of adhesion molecules, and inhibited myeloma cell adhesion to BMSCs compared with thalidomide or meloxicam. The activities of etodolac potentially extend to the treatment of patients with myeloma resistant to standard chemotherapy, including thalidomide.     

Combination of bendamustine and entinostat synergistically inhibits proliferation of multiple myeloma cells via induction of apoptosis and DNA damage response

Bendamustine, a hybrid molecule of purine analog and alkylator, induces cell death by activation of apoptosis, DNA damage response, and mitotic catastrophe. Entinostat, a selective class I inhibitor of histone deacetylase (HDAC), exerts anti-tumor activity in various cancer types, including multiple myeloma (MM). We sought to determine the combinatorial effects of bendamustine and entinostat on MM cells. Cell growth assays showed that bendamustine or entinostat inhibited proliferation in a dose-dependent manner, and their combinations synergistically induced growth inhibition in all MM cells tested. An apoptotic-ELISA and western blot assays on PARP cleavage and caspase-8 and caspase-3 revealed that bendamustine in combination with entinostat exhibited a much more potent activity than either agent alone to promote the MM cells undergoing apoptosis in a dose-dependent manner. Flow cytometric analysis found that entinostat exhibited distinct effects on cell cycle progression in different lines and bendamustine mainly arrested the cells at S phase, whereas their combinations dramatically blocked the S cells entering G2/M phase. Furthermore, studies on DNA damage response indicated that phospho-histone H2A.X (P-H2A.X), a hall marker of DNA double strand break, along with phosphorylated CHK2 (P-CHK2) was significantly enhanced by the combinations of bendamustine and entinostat as compared to either agent alone. These molecular changes were correlated with the increases in mitotic catastrophe. Collectively, our data demonstrate that bendamustine in combination with entinostat exhibit potent anti-proliferative/anti-survival activity in MM cells via induction of apoptosis and DNA damage response. Regimens consisting of bendamustine and/or entinostat may represent novel therapeutic strategies against MM.     

The hepatocyte growth factor/Met pathway controls proliferation and apoptosis in multiple myeloma.

The evolution of multiple myeloma (MM) depends on complex signals from the bone marrow (BM) microenvironment, supporting the proliferation and survival of malignant plasma cells. An interesting candidate signal is hepatocyte growth factor/scatter factor (HGF), since its receptor Met is expressed on MM cells, while HGF is produced by BM stromal cells and by some MM cell lines, enabling para- or autocrine interaction. To explore this hypothesis, we studied the biological effects of HGF stimulation on MM cell lines and on primary MMs. We observed that Met is expressed by the majority of MM cell lines and by approximately half of the primary plasma cell neoplasms tested. Stimulation of MM cells with HGF led to the activation of the RAS/mitogen-activated protein kinase and phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) pathways, signaling routes that have been implicated in the regulation of cell proliferation and survival. Indeed, functional studies demonstrated that HGF has strong proliferative and anti-apoptotic effects on both MM cell lines and primary MM cells. Furthermore, by applying specific signal-transduction inhibitors, we demonstrated that MEK is required for HGF-induced proliferation, whereas activation of PI3K is required for both HGF-induced proliferation and for rescue of MM cells from apoptosis. Taken together, our data indicate that HGF is a potent myeloma growth and survival factor and suggest that the HGF/Met pathway is a potential therapeutic target in MM.     

R-etodolac induces E-cadherin and suppresses colitis-related mouse colon tumorigenesis

Colorectal cancer is one of the most serious complications of ulcerative colitis (UC), and the risk of UC-associated neoplasia increases as the region and duration of the disease increase. Selective cyclooxygenase (COX)-2 inhibitors effectively diminish carcinogenesis in a murine UC model. However, this may exacerbate colitis. The selective COX-2 inhibitor etodolac is marketed as a racemic mixture of the R- and S-enantiomers. The biochemical and pharmacological effects of etodolac are caused by the S-enantiomer, while the R-enantiomer lacks COX-inhibitory activity. In this study, we evaluated the effect of R-etodolac on colitis-related mouse colon tumorigenesis. The mice received 1,2-dimethlhydrazine (DMH), and then chronic colitis was induced by administration of two cycles of DSS (each cycle: 3% DSS for 7 days followed by distilled water for 14 days). The mice were sacrificed 28 days after the completion of both cycles. Mice were divided into the following groups: group A served as a disease control; group B received a low (2-mg/kg) dose of R-etodolac every 3 days during the entire period; group C received a high (10-mg/kg) dose of R-etodolac on the same schedule as group B; and group D served as a normal control. Administration of R-etodolac decreased the disease activity index during the DSS administration cycle. The mean number of tumors was 17.8, 15.2, 6.0, and 0 in groups A-D, respectively. In group C, R-etodolac significantly suppressed the occurrence of neoplasia (p<0.05). Although R-etodolac treatment did not affect COX-2 expression, it significantly enhanced expression of E-cadherin in both neoplastic lesions and background mucosa (i.e., lesion-free colon). Thus, administration of R-etodolac exerts a suppressive effect on the development of neoplasia in a murine model of DSS-induced colitis without exacerbation of the colitis. These results suggest that R-etodolac could be useful in the prevention of UC-associated neoplasia.     

Mechanisms by which SGN-40, a humanized anti-CD40 antibody, induces cytotoxicity in human multiple myeloma cells: clinical implications

CD40 is expressed on B-cell malignancies, including human multiple myeloma (MM) and a variety of carcinomas. We examined the potential therapeutic utility of SGN-40, the humanized anti-CD40 monoclonal antibody, for treating human MM using MM cell lines and patient MM cells (CD138(++), CD40(+)). SGN-40 (0.01-100 micro g/ml) induces modest cytotoxicity in MM cell lines and patient MM cells. In the presence of de novo protein synthesis inhibitor cycloheximide, SGN-40 significantly induced apoptosis in Dexamethasone (Dex)-sensitive MM.1S and Dex-resistant MM.1R cells and in patient MM cells. SGN-40-mediated cytotoxicity is associated with up-regulation of cytotoxic ligands of the tumor necrosis factor family (Fas/FasL, tumor necrosis factor-related apoptosis-inducing ligand, and tumor necrosis factor alpha). SGN-40 treatment also induces a down-regulation of CD40 dependent on an endocytic pathway. Consequently, pretreatment of MM cells with SGN-40 blocked sCD40L-mediated phosphatidylinositol 3'-kinase/AKT and nuclear factor kappaB activation. Importantly, pretreatment of MM.1S and MM.1R cells with SGN-40 inhibited proliferation triggered by interleukin 6 (IL-6) but not by insulin-like growth factor-I. In addition, SGN-40 pretreatment of MM.1S cells blocked the ability of IL-6 to protect against Dex-induced inhibition of DNA synthesis. This was associated with a 2-4-fold reduction of IL-6 receptor at protein and mRNA levels in SGN-40-treated MM.1S cells and patient MM cells. Taken together, these results provide the preclinical rationale for the evaluation of SGN-40 as a potential new therapy to improve patient outcome in MM.     

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.     

A novel Bcl-2/Bcl-X(L)/Bcl-w inhibitor ABT-737 as therapy in multiple myeloma

Bcl-2 or Bcl-X(L) confers resistance to chemotherapy in multiple myeloma (MM). Here we characterized the effects of ABT-737, a potent small-molecule inhibitor of antiapoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w with markedly higher affinity than previously reported compounds, on human MM cells. ABT-737 induces apoptosis in MM cells, including those resistant to conventional therapy. Examination of purified patient MM cells demonstrated similar results, without significant toxicity against normal peripheral blood mononuclear cells and MM bone marrow stromal cells. Importantly, ABT-737 decreases the viability of bortezomib-, dexamethasone-(Dex) and thalidomide-refractory patient MM cells. Additionally, ABT-737 abrogates MM cell growth triggered by interleukin-6 or insulin-like growth factor-1. Mechanistic studies show that ABT-737-induced apoptosis is associated with activation of caspase-8, caspase-9 and caspase-3, followed by poly(ADP-ribose) polymerase cleavage. Combining ABT-737 with proteasome inhibitor bortezomib, melphalan or dexamethasone induces additive anti-MM activity. Taken together, our study provides the rationale for clinical protocols evaluating ABT-737, alone and together with botezomib, mephalan or dexamethasone, to enhance MM cell killing, overcome drug resistance conferred by Bcl-2 and improve patient outcome in MM.     

Rational combination treatment with histone deacetylase inhibitors and immunomodulatory drugs in multiple myeloma

Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide (Len) and pomalidomide trigger anti-tumor activities in multiple myeloma (MM) by targetting cereblon and thereby impacting IZF1/3, c-Myc and IRF4. Histone deacetylase inhibitors (HDACi) also downregulate c-Myc. We therefore determined whether IMiDs with HDACi trigger significant MM cell growth inhibition by inhibiting or downregulating c-Myc. Combination treatment of Len with non-selective HDACi suberoylanilide hydroxamic acid or class-I HDAC-selective inhibitor MS275 induces synergic cytotoxicity, associated with downregulation of c-Myc. Unexpectedly, we observed that decreased levels of cereblon (CRBN), a primary target protein of IMiDs, was triggered by these agents. Indeed, sequential treatment of MM cells with MS275 followed by Len shows less efficacy than simultaneous treatment with this combination. Importantly ACY1215, an HDAC6 inhibitor with minimal effects on class-I HDACs, together with Len induces synergistic MM cytotoxicity without alteration of CRBN expression. Our results showed that only modest class-I HDAC inhibition is able to induce synergistic MM cytotoxicity in combination with Len. These studies may provide the framework for utilizing HDACi in combination with Len to both avoid CRBN downregulation and enhance anti-MM activities.     

Chemokine receptor CCR2 is expressed by human multiple myeloma cells and mediates migration to bone marrow stromal cell-produced monocyte chemotactic proteins MCP-1, -2 and -3

The restricted bone marrow (BM) localisation of multiple myeloma (MM) cells most likely results from a specific homing influenced by chemotactic factors, combined with the proper signals for growth and survival provided by the BM microenvironment. In analogy to the migration and homing of normal lymphocytes, one can hypothesise that the BM homing of MM cells is mediated by a multistep process, involving the concerted action of adhesion molecules and chemokines. In this study, we report that primary MM cells and myeloma derived cell lines (Karpas, LP-1 and MM5.1) express the chemokine receptor CCR2. In addition, we found that the monocyte chemotactic proteins (MCPs) MCP-1, -2 and -3, three chemokines acting as prominent ligands for CCR2, are produced by stromal cells, cultured from normal and MM BM samples. Conditioned medium (CM) from BM stromal cells, as well as MCP-1, -2 and -3, act as chemoattractants for human MM cells. Moreover, a blocking antibody against CCR2, as well as a combination of neutralizing antibodies against MCP-1, -2 and -3, significantly reduced the migration of human MM cells to BM stromal cell CM. The results obtained in this study indicate the involvement of CCR2 and the MCPs in the BM homing of human MM cells.