Human Myeloma-derived Cell Lines Research Articles

Robust anti-myeloma effect of TAS0612, an RSK/AKT/S6K inhibitor, with venetoclax regardless of cytogenetic abnormalities.

Multiple myeloma (MM) remains a difficult-to-treat disease even with the latest therapeutic advances due to the complex, overlapping, and heterogeneous cytogenetic, genetic, and molecular abnormalities. To address this challenging problem, we previously identified the universal and critical roles of RSK2 and AKT, the effector signaling molecules downstream of PDPK1, regardless of cytogenetic and genetic profiles. Based on this, in this study, we investigated the anti-myeloma potency of TAS0612, a triple inhibitor against RSK, including RSK2, AKT, and S6K. Treatment with TAS0612 exerted the anti-proliferative effect via cell cycle blockade and the induction of apoptosis in human myeloma-derived cell lines (HMCLs) with diverse cytogenetic and genetic profiles. Ex vivo treatment with TAS0612 also significantly reduced the viability of patient-derived primary myeloma cells with diverse cytogenetic profiles. TAS0612 simultaneously caused the upregulation of several tumor suppressor genes, modulated prognostic genes according to the MMRF CoMMpass data, and downregulated a series of Myc- and mTOR-related genes. Moreover, the combination of TAS0612 with venetoclax (VEN) showed the synergy in inducing apoptosis in HMCLs irrespective of the t(11;14) translocation status. TAS0612 alone and combined with VEN are new potent candidate therapeutic strategies for MM, regardless of cytogenetic/genetic profiles, facilitating its future clinical development.

TAS0612, a Triple Inhibitor for RSK, AKT, and S6K, Is a Promising Anti-Myeloma Agent Regardless of Types of Cytogenetic/Genetic Abnormality

Introduction: Despite advancements in therapeutic approaches, treatment outcome for multiple myeloma (MM) remains unsatisfactory. One critical factor of the poor outcomes is the complex inter-patient and intra-clonal heterogeneity of cytogenetic, genetic, and molecular abnormalities. To overcome this, the exploration of therapeutic target molecules that play a universal and essential role in MM regardless of the types of molecular profile is crucial even in the post-genomic era. We have previously demonstrated that RSK2 Ser227 at the N-terminal kinase domain (NTKD), as the downstream signaling hub of the RAS/ERK pathway, plays essential roles in the proliferation and survival of myeloma cells (Shimura Y, Mol Cancer Ther 2012). We also identified that RSK2-NTKD is constitutively activated by constitutive active PDPK1, the master serine-threonine kinase of >20 AGC kinases, in myeloma cells of approximately 90% of MM patients regardless of cytogenetic abnormalities (Chinen Y, Cancer Res 2014, Tatekawa S, Br J Haematol 2017). Moreover, the combinatory blockade of RSK2 and AKT, one of the other important molecules regulated by PDPK1, exhibited synergistic anti-myeloma efficacy through the modulation of various critical gene sets involved in myeloma pathophysiology, such as MYC (Isa R, Int J Mol Sci 2022). Based upon these, this study investigates the anti-myeloma potency of TAS0612 (Taiho Oncology Inc., Princeton, NJ) that targets RSK, including RSK2, AKT, and S6K, and inhibits the bindings to their respective substrate molecules (Shibata T, Mol Cancer Ther 2020). Methods and Results: Firstly, we examined the activation status of RSK, AKT, and S6K in eight human myeloma-derived cell lines (HMCLs) with diverse genetic and molecular profiles at the protein level. RSK2 Ser227 was found to be commonly activated through phosphorylation in all HMCLs examined. In contrast, the activation patterns of p-AKT Ser473 and p-S6K Thr389 were inconsistent among eight HMCLs, and depending on cell types. Despite these cell type-dependent effects, the exposure to TAS0612 resulted in the accumulation of p-total RSK Ser380, p-AKT Ser473, and p-S6K Thr389 in the majority of HMCLs examined, and this led to the subsequent inactivation of their respective target substrates, such as p-YB1 Ser102, p-PRAS Thr246, and p-S6 Ser235/236, through dephosphorylation. In accompany with these molecular effects, the modified MTT assay showed the dose-dependent growth inhibitory effects of TAS0612 in all cell lines with various types of cytogenetic/genetic abnormalities with IC50 values mostly below 0.5 μM (48 hours). In addition, flow cytometry analyses for the cell cycle analysis and the apoptosis assay by the counterstaining of propidium iodide and Annexin-V showed that TAS0612 induced G1/S cell cycle arrest and apoptotic cell death. Furthermore, patient-derived primary myeloma cells were labeled with anti-CD138 MicroBeads, and were positively isolated from bone marrow mononuclear cells from patients with newly diagnosed MM (n=7) and relapsed/refractory MM (n=11), and, then, were treated by TAS0612. As the results, the ex vivo treatment of 2.5-5.0 μM TAS0612 for 72 hours showed robust cell reductive effects on primary-myeloma cells from all 18 patients examined irrespective of cytogenetic profile. To clarify the precise molecular effects of TAS0612 treatment in myeloma cells, we conducted a comprehensive molecular analysis with TAS0612 treatment by gene expression profiling. The results showed that TAS0612 treatment significantly modulated the gene sets involved in Myc signaling, mTOR signaling, and the core serum response (CSR). Additionally, TAS0612 caused the concomitant upregulation of several bona-fide tumor suppressor genes, including TP53INP, HBP1, FBOX32, and YPEL3, while the downregulation of oncogenes LYAR and HSPA1. Intriguingly, according to MMRF CoMMpass Study, 8 of 18 upregulated genes by TAS0612 in HMCLs were reported to be associated with favorable survival outcomes when highly expressed in myeloma cells. Conclusion: Our findings strongly support the potential of TAS0612 as an effective anti-myeloma agent. Further preclinical investigations are necessary to substantiate these results and facilitate its future clinical development.

Mir-106a-5p and Mir-146a-5p Delivered By Tumor-Derived Exosomes Promote the Induction of Myeloid-Derived Suppressor Cells from Normal Peripheral Blood Mononuclear Cells in Multiple Myeloma

Despite the great advance in treatment, including immunochemotherapy using monoclonal antibodies and immunomodulatory drugs, and cell therapies, such as chimeric-antigen receptor T cell therapies, multiple myeloma (MM) remains to be mostly incurable. One of the major mechanisms for the resistance to various immune-mediated treatment approaches for MM is the collapse of the tumor-immune surveillance system which is induced by the predominance of immunosuppressive cellular components over the surveillance system. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid cells which suppress T-cell-mediated immune response, and its increase has been associated with disease progression and treatment resistance in MM. We previously reported that the co-culture with some human-myeloma-derived cell lines (HMCLs), KMS-12-BM cells, and KMS-28-PE cells, but not others, such as AMO-1 cells and RPMI8226 cells, induced monocytic-MDSCs (M-MDSCs) from normal peripheral blood mononuclear cells (PBMCs), and showed that the combinatory secretion of CCL5, MIF, and MIP-1a, by HMCLs partly contributed to the induction of M-MDSCs in MM (Kuwahara-Ota S, Br J Haematol 2020). In addition to soluble factors, exosomes play a crucial role in cell-to-cell communication by transferring various molecules, including proteins, mRNAs, and microRNAs (miRNAs), and we here investigated the role of tumor-derived exosomes (TEXs) in the induction of M-MDSCs in MM. First, we isolated TEXs from the culture supernatant of various HMCLs. The presence of TEXs was confirmed by nanoparticle tracking analysis and flow cytometry. To examine whether TEXs promote the induction of M-MDSC, we treated PBMCs with TEXs from HMCLs. As the result, the exposure of TEXs from MDSC-inducible HMCLs, but not from MDSC-non-inducible HMCLs, induced M-MDSCs from PBMCs in a dose-dependent manner, indicating the involvement of TEXs in M-MDSC induction in MM. Next, we comparatively performed the comprehensive miRNA profiling by microarray analysis among TEXs from MDSC-inducible and non-inducible HMCLs and identified that miRNAs, including miR-16-5p, miR-17-5p, miR-20a-5p, miR-106a-5p, miR-107, and miR-146a-5p, were specifically upregulated in TEXs from MDSC-inducible HMCLs compared with MDSC-non-inducible HMCLs, which was validated by the quantitative RT-PCR. Interestingly, the expression levels of these 6 miRNAs were mostly equivalent in MDSC-inducible HMCLs and MDSC-non-inducible HMCLs, suggesting the crucial role of TEX-mediated delivery of those miRNAs. By the transfection experiments of these 6 miRNAs into PBMCs, we identified that PBMCs transfected by miR-106a-5p and miR-146a-5p exhibited differentiation into M-MDSCs. In addition, the combination of miRNA, i.e., miR-106a-5p or miR-146a-5p, and chemokine, namely, CCL5, MIP-1a, or MIF, further enhanced the induction of M-MDSC from PBMCs. Finally, to clarify the functional roles of miR-106-5p and miR-146a-5p in the TEX-mediated induction of M-MDSCs, we analyzed the comprehensive gene expression changes by the introduction of miR-106-5p or miR-146a-5p in PBMCs using gene expression microarray. As the result, the gene introduction of miR-106a-5p or miR-146a-5p was found to modulate various gene sets, including those associated with IFN-α signaling, IFN-γ signaling, inflammatory response, TNF-a signaling via NF-kB, or interleukin-6-JAK-STAT3 signaling, in CD33-positive myeloid cells, and these findings mirror the gene expression modulation induced by the co-culture with MDSC-inducible HMCLs in CD33-positive myeloid cells. In conclusion, this study for the first time identified the multi-layer functional involvement of TEX-mediated miR-106a-5p and miR-146a-5p transfer in the induction of M-MDSCs in MM.

EWSR1 overexpression is a pro-oncogenic event in multiple myeloma.

Multiple myeloma (MM) is cytogenetically, genetically and molecularly heterogenous even among subclones in one patient, therefore, it is essential to identify both frequent and patient-specific drivers of molecular abnormality. Following previous molecular investigations, we in this study investigated the expression patterns and function of the Ewing sarcoma breakpoint region 1 (EWSR1) gene in MM. The EWSR1 transcriptional level in CD138-positive myeloma cells was higher in 36.4% of monoclonal gammopathy of undetermined significance, in 67.4% of MM patients compared with normal plasma cells, and significantly higher in ten human myeloma-derived cell lines (HMCLs) examined. EWSR1 gene knockdown caused growth inhibition with an increase of apoptotic cells in NCI-H929 and KMS-12-BM cells. Gene expression profiling using microarray analysis suggested EWSR1 gene knockdown caused transcriptional modulation of several genes associated with processes such as cell proliferation, cell motility, cell metabolism, and gene expression. Of particular, EWSR1 gene knockdown caused upregulation of let-7c and downregulation of its known targets K-RAS and AKT. Finally, our analysis using community database suggested that high EWSR1 expression positively associates with poor prognosis and advanced disease stage in MM. These findings suggest that EWSR1 overexpression is a pro-oncogenic molecular abnormality that may participate in MM progression.

Aberrant BUB1 Overexpression Promotes Mitotic Segregation Errors and Chromosomal Instability in Multiple Myeloma.

Chromosome instability (CIN), the hallmarks of cancer, reflects ongoing chromosomal changes caused by chromosome segregation errors and results in whole chromosomal or segmental aneuploidy. In multiple myeloma (MM), CIN contributes to the acquisition of tumor heterogeneity, and thereby, to disease progression, drug resistance, and eventual treatment failure; however, the underlying mechanism of CIN in MM remains unclear. Faithful chromosomal segregation is tightly regulated by a series of mitotic checkpoint proteins, such as budding uninhibited by benzimidazoles 1 (BUB1). In this study, we found that BUB1 was overexpressed in patient-derived myeloma cells, and BUB1 expression was significantly higher in patients in an advanced stage compared to those in an early stage. This suggested the involvement of aberrant BUB1 overexpression in disease progression. In human myeloma-derived cell lines (HMCLs), BUB1 knockdown reduced the frequency of chromosome segregation errors in mitotic cells. In line with this, partial knockdown of BUB1 showed reduced variations in chromosome number compared to parent cells in HMCLs. Finally, BUB1 overexpression was found to promote the clonogenic potency of HMCLs. Collectively, these results suggested that enhanced BUB1 expression caused an increase in mitotic segregation errors and the resultant emergence of subclones with altered chromosome numbers and, thus, was involved in CIN in MM.

Lenalidomide and pomalidomide potently interfere with induction of myeloid-derived suppressor cells in multiple myeloma.

An increase in immunosuppressive myeloid-derived suppressor cells (MDSCs) is associated with disease progression and treatment resistance in multiple myeloma (MM). We investigated the mechanisms underlying MDSC induction, and sought to discover a strategy for prevention of MDSC induction in MM. Using a transwell co-culture system, four of nine examined human myeloma-derived cell lines (HMCLs) were potent in inducing monocytic (M)-MDSCs from normal peripheral blood mononuclear cells (PBMCs). As the results, we identified that secretion of C-C motif chemokine ligand 5 (CCL5) and macrophage migration inhibitory factor (MIF) by myeloma cells is a prerequisite for induction of MDSCs in MM. The immunomodulatory drug (IMiD) compounds, such as lenalidomide (LEN) and pomalidomide (POM), were identified as potent inhibitors of MDSC induction through bidirectional molecular effects of cereblon (CRBN)-dependent and -independent downregulation of CCL5 and MIF in myeloma cells; and downregulation of C-C motif chemokine receptor 5, a receptor for CCL5, and induction of interferon regulatory factor 8, a critical transcription factor for monocytic differentiation, in PBMCs. In the present study of the molecular mechanisms underlying MDSC induction, we identified a novel effect of LEN and POM of inhibiting MDSC induction via overlapping regulatory effects in myeloma cells and normal PBMCs.

PS1354 MYELOMA‐INDUCED MYELOID‐DERIVED SUPPRESSOR CELLS ARE TARGETED BY IMMUNOMODULATORY DRUGS THAT REGULATE THE CCL5/CCR5 AXIS AND MIF AND IRF8 EXPRESSION

Background:The recent advent of immunochemotherapy using immunomodulatory drugs (IMiDs), such as lenalidomide (LEN) and pomalidomide (POM), has greatly improved treatment outcomes of multiple myeloma (MM); however, generation of immunosuppression in the tumor microenvironment hampers the therapeutic potency of immunochemotherapy. Myeloid‐derived suppressor cells (MDSCs), which are a heterogeneous group of immunosuppressive myeloid polymorphonuclear (PMN‐MDSCs) and monocytic (M‐MDSCs) cells, play critical roles in impairing cancer immunosurveillance, and an increase of MDSCs has been associated with treatment failure, disease progression, and poor prognosis in various cancers, including MM.Aims:In this study, we examined the molecular mechanisms underlying induction of MDSCs in MM, and showed that the inhibitory effects of IMiDs on myeloma‐induced MDSCs occur through novel mechanisms of action.Methods:Six human myeloma‐derived cell lines (HMCLs) (KMS‐12‐BM, KMS‐28‐PE, KMS‐34, AMO‐1, RPMI‐8226, and LP‐1) were utilized. HMCLs were co‐cultured with peripheral blood mononuclear cells (PBMCs) from a healthy donor using a transwell system. After 5–7 days, induction of MDSCs from PBMCs was evaluated by flow cytometry. CD14+/CD33+/HLA‐DR‐ cells and CD14‐/CD15+ cells were regarded as M‐MDSCs and PMN‐MDSCs, respectively. Soluble factors in the conditioned medium (CM) of each HMCL and co‐culture system were measured using a Human Cytokine Array Kit (R&D Systems, Minneapolis, U.S.A) for 36 cytokines/chemokines and ELISA kits. LEN and POM were provided by Celgene Corporation.Results:Two of the six cell lines (KMS‐12‐BM, KMS‐28‐PE) induced M‐MDSCs that strongly expressed indoleamine‐2, 3‐dioxygenase (IDO) and interleukin‐8, but not PMN‐MDSCs, from PBMCs in the co‐culture experiments using a transwell system. The increase of M‐MDSCs was accompanied by an increase of regulatory T cells in our system. Analyses of soluble factors in CM derived from HMCLs that did and did not induce MDSCs showed that CCL5 was secreted only by MDSC‐inducible HMCLs, while MIF was secreted by all HMCLs examined. Moreover, addition of an anti‐CCL5 antibody or a MIF inhibitor partly blocked induction of MDSCs, and the combination of an anti‐CCL5 antibody and a MIF inhibitor showed an additive effect in blocking MDSC induction. Supplementation of CCL5 enabled induction of MDSCs in several HMCLs that were otherwise unable to induce MDSCs, although the effect was minor. Collectively, combined secretion of CCL5 and MIF by MM cells was identified as the prerequisite for induction of M‐MDSCs. Next, among currently available drugs for MM, we found that LEN and POM had marked suppressive effects on induction of MDSCs by co‐existing myeloma cells. Both agents significantly reduced CCL5 and MIF at the transcriptional level in MM cells and at the protein level in CM from MM cells. Moreover, IMiDs reduced expression of CCR5, a receptor of CCL5, and increased transcription of IRF8, which is a negative regulator of MDSC differentiation in PBMCs.Summary/Conclusion:This study identified CCL5 and MIF secreted by tumor cells as proteins required for induction of MDSCs in MM, and showed that IMiDs have suppressive effects on MDSC induction through overlapping molecular mechanisms in myeloma cells and PBMCs. These results provide new insights into monitoring of the effects of IMiD‐containing immunochemotherapy and may permit development of a novel therapeutic approach

Transcriptional dysregulation of the deleted in colorectal carcinoma gene in multiple myeloma and monoclonal gammopathy of undetermined significance.

The deleted in colorectal carcinoma (DCC) gene at 18q21 encodes a netrin-1 receptor, a tumor suppressor that prevents cell growth. While allele loss or decreased expression of DCC has been associated with the progression of solid tumors and hematologic malignancies, including leukemias and malignant lymphomas, its involvement has not been evaluated in multiple myeloma (MM), a plasma cell malignancy characterized by complex and heterogenous molecular abnormalities. We here show that 10 of 11 human myeloma-derived cell lines (HMCLs) expressed non-translated aberrant DCC transcriptional variants, in which exon 2 fuses with intron 1 instead of exon 1 (mt.DCC). Among them, two co-expressed wild type transcripts (wt.DCC), while eight co-expressed the splicing variant (sv.DCC) lacking exon 1. The remaining HMCL expressed only sv.DCC. In addition, analyses revealed that there were two types of mt.DCC that differed in their fusion of intron 1 with exon 2. In patient-derived samples from 30 MM and 8 monoclonal gammopathy of undetermined significance (MGUS) patients, wt.DCC was expressed in 53% of MM, but not in MGUS, while 23% of MM and 75% of MGUS expressed only sv.DCC. Considering that 25% of MGUS, 57% of MM, and 91% HMCLs expressed mt.DCC, our results suggest that the acquisition of mt.DCC might be a secondary genetic change in plasma cell dyscrasia.

RSK2Ser227 Is a Therapeutic Target of Myeloma Cells Regardless of Upstream Signalings.

AbstractAbstract 2945 Background:Despite recent advances of therapeutic strategies, multiple myeloma (MM) still remains mostly incurable due to its drug resistance by both various cell intrinsic molecular abnormalities and the supports of myeloma microenvironment factors. The search for new therapeutic target molecule upon which various MM-promoting signalings converge is essential for the development of more effective therapy for MM.RSK2 is a member of the 90 kDa ribosomal S6 kinases (RSKs) family of serine (Ser) /threonine (Thr) kinases and lies downstream of RAS/ERK-1/2 signaling cascade. In MM, it has been shown that RSK2 mediates signaling initiated by FGFR3 activation for cell survival, but the functional role and the value as the therapeutic target of RSK2 have not been clearly defined. In this study, we precisely studied the association with RSK2 activation and chromosomal status, especially focusing on RSK2Ser227, which is responsible for downstream signaling phosphorylation, and the value of RSK2 as the therapeutic target in MM. Methods:Twelve human myeloma-derived cell lines and primary MM cells were utilized in this study. The use of human samples was approved by the ethical review board of our institute. Cell killing assay was performed using flow cytometry. Growth inhibitory assay was performed with modified MTT assay. Protein levels were examined by Western blotting. BI-D1870, a RSK2 inhibitor, was purchased from Symansis Limited (Auckland, New Zealand). RNA interference for RSK2 was performed by transfection of small interfering RNA (siRNA) into NCI-H929 cells by means of CLB-Transfection Kit (Lonza, Swiss) with protocol 9. Drug combination assays were evaluated with CalcuSyn software (Biosoft, Cambridge, UK). Results:RSK2Ser227 was shown to be phosphorylated in all 12 MM cell lines and 6 of 9 primary MM cells. t(4;14) involving IgH/FGFR3 was detected only in 7 cell lines and high FGFR3 expression was identified only in 6 cell lines. RSK2Ser227 was phosphorylated even in the absence of ERK1/2 or RSK2Tyr529 phosphorylation in several MM cell lines, indicating that their activation was not mandatory to RSK2Ser227 phosphorylation. Our study also did not show any positive relationship between RSK2Ser227 phosphorylation and other so-called “high-risk” cytogenetic abnormalities, such as 13q-, t(11;14), t(8;14), t(14;16), 1q21+, or 17p-.Treatment with BI-D1870 (RSK2 inhibitor) resulted in complete dephosphorylation of RSK2Ser227, but not RSK2Tyr529. The phosphorylation status of ERK1/2, or related signaling kinases, such as AKT, p38 MAPK, or JNK was also not affected. BI-D1870 showed dose-dependent growth inhibition effect in 6 MM-derived cell lines by inducing apoptosis. At molecular level, Mcl-1, p21WAF1/CIP1, cyclin D2 and c-Myc was significantly down-regulated and Bim was up-regulated by BI-D1870 intervention in NCI-H929 cell. BI-D1870 didn't modulate expression level of IRF-4. These change were also observed in other MM cell lines (KMS-34, AMO-1).To exclude the possibility that above BI-D1870 induced changes in protein expression and cell death are due to its off-target effects, we performed gene knockdown experiments of RSK2. Transient gene knockdown of RSK2 by means of RNAi caused downregulation of cyclin D2, p21WAF1/CIP1, c-Myc, while it induced cell death in NCI-H929 cells. In contrast, Bim expression was not altered by RSK2 knockdown.Lenalidomide (LEN) is the most powerful currently available IMiD for MM treatment. While cell death induction by LEN (100μM) was partly prevented by IL-6 (50ng/ml) in NCI-H929 cells, co-administration of BI-D1870 (0.2 μM, at the degree of low concentration not to affect cell viability when used solely) overcame the resistance to LEN induced by IL-6. KMS-11/BTZ were highly resistant to bortezomib (BTZ)-induced cell death, by contrast, it showed the similar sensitivities to BI-D1870-induced cell death when compared with their parental cells. And more, BI-D1870 showed synergistic or additive effects with upcoming new anti-MM agents, such as RAD001, MS-275 and ABT-263 in MM cell lines. Conclusions:RSK2, especially RSK2Ser227, may be a universal therapeutic target for MMs with diverse molecular signatures. Disclosures:Iida:Janssen Pharmaceutical K.K.: Honoraria.

The novel JAK inhibitor AZD1480 blocks STAT3 and FGFR3 signaling, resulting in suppression of human myeloma cell growth and survival

IL-6 and downstream JAK-dependent signaling pathways have critical roles in the pathophysiology of multiple myeloma (MM). We investigated the effects of a novel small-molecule JAK inhibitor (AZD1480) on IL-6/JAK signal transduction and its biological consequences on the human myeloma-derived cell lines U266 and Kms.11. At low micromolar concentrations, AZD1480 blocks cell proliferation and induces apoptosis of myeloma cell lines. These biological responses to AZD1480 are associated with concomitant inhibition of phosphorylation of JAK2, STAT3 and MAPK signaling proteins. In addition, there is inhibition of expression of STAT3 target genes, particularly Cyclin D2. Examination of a wider variety of myeloma cells (RPMI 8226, OPM-2, NCI-H929, Kms.18, MM1.S and IM-9), as well as primary myeloma cells, showed that AZD1480 has broad efficacy. In contrast, viability of normal peripheral blood (PB) mononuclear cells and CD138(+) cells derived from healthy controls was not significantly inhibited. Importantly, AZD1480 induces cell death of Kms.11 cells grown in the presence of HS-5 bone marrow (BM)-derived stromal cells and inhibits tumor growth in a Kms.11 xenograft mouse model, accompanied with inhibition of phospho-FGFR3, phospho-JAK2, phospho-STAT3 and Cyclin D2 levels. In sum, AZD1480 blocks proliferation, survival, FGFR3 and JAK/STAT3 signaling in myeloma cells cultured alone or cocultured with BM stromal cells, and in vivo. Thus, AZD1480 represents a potential new therapeutic agent for patients with MM.

Adhesive interactions of human multiple myeloma cell lines with different extracellular matrix molecules.

Multiple myeloma represents a human B cell malignancy which is characterized by a predominant localization of the malignant cell clone within the bone marrow. With the exception of the terminal stage of the disease the myeloma tumor cells do not circulate in the peripheral blood. The bone marrow microenvironment is believed to play an important role in homing, proliferation and terminal differentiation of myeloma cells. Here we have studied the expression of several extracellular matrix (ECM) molecules in the bone marrow of multiple myeloma patients and analyzed their adhesive capacities with four different human myeloma-derived cell lines. All ECM molecules analyzed (tenascin, laminin, fibronectin, collagen types I, III, V and VI) could be detected in bone marrow cryostat sections of multiple myeloma patients. Adhesion assays showed that only laminin, the microfibrillar collagen type VI and fibronectin were strong adhesive components for the myeloma cell lines U266, IM-9, OPM-2 and NCI-H929. Tenascin and collagen type I were only weak adhesive substrates for these myeloma cells. Adhesion to laminin and fibronectin was beta 1-integrin-mediated since addition of anti-beta 1-integrin antibodies could inhibit the binding of the four different cell types to both matrix molecules. In contrast, integrins do not seem to be involved in binding of the myeloma cells to collagen type VI. Instead, inhibition of binding by heparin suggested that membrane-bound heparan sulfate proteoglycans are responsible ligands for binding to collagen type VI. Adhesion assays with several B-cell lines resembling earlier differentiation stages revealed only weak interactions with tenascin and no interactions with collagen type VI, laminin or fibronectin. In summary, the interactions of human myeloma cells with the extracellular matrix may explain the specific retention of the plasma cells within the bone marrow.

Cell surface expression and functional significance of adhesion molecules on human myeloma‐derived cell lines

Multiple myeloma is characterized by the presence of malignant plasma cells predominantly localized in bone marrow. Our prior studies have suggested that human myeloma derived-cell lines adhere specifically to fibronectin and to bone marrow stromal cells (BMSCs) via beta 1 and beta 2 integrins as well as RGD peptide, and that tumour cell to BMSC contact triggers interleukin-6 (IL-6) secretion from BMSCs. Since IL-6 is a growth factor for myeloma, adhesion may be important in paracrine IL-6 mediated tumour cell growth. We therefore examined phenotypic expression of adhesion molecules on the U266 and IM-9 human myeloma-derived cell lines using the panel of monoclonal antibodies (MoAbs) directed at adhesion molecules submitted to the Vth International Conference on Human Leukocyte Differentiation Antigens. U266 and IM-9 myeloma cell lines express mainly CD29, CD49d, VLA-1, CD18, CD54, ICAM-2 and ICAM-3. In contrast, CD49b, VLA-3, CD49f, CD11b, VCAM-1, selectins and selectin-ligands were not expressed on these cell lines. Specific adherence of IM-9 cells to BMSC line LP101 was demonstrated which could be partially blocked by pre-incubation and culture of tumour cells with anti-beta 1 integrin, anti-beta 2 integrin, anti-CD49d, anti-VLA-5, anti-CD11a, anti-CD44 and anti-CD54 MoAbs. The combination of these MoAbs (anti-CD29, CD18, CD11a, CD49d, VLA-5, CD44, CD54, ICAM-2, ICAM-3 MoAbs) decreased but did not completely abrogate binding of IM-9 to BMSCs. Moreover, increases in IL-6 secretion from BMSCs after adherence of IM-9 cells were also partially blocked by these MoAbs. These findings suggest that multiple adhesion pathways may mediate adherence of myeloma cell lines to BMSCs, localizing tumour cells in the marrow microenvironment and triggering IL-6 secretion by BMSCs which may augment tumour cell growth.

Adhesion of human myeloma-derived cell lines to bone marrow stromal cells stimulates interleukin-6 secretion

Previous studies show that human myeloma-derived cell lines specifically adhere to fibronectin (FN) through very late antigen-4 (VLA-4; alpha 4 beta 1 integrin complex) and RGD-peptide mechanisms, which may contribute to the localization of tumor cells in bone marrow (BM). In these studies, we characterized the adhesion of myeloma- derived cell lines to both normal and myeloma BM stromal cells (BMSCs) and the effect of adhesion on DNA synthesis. Because interleukin-6 (IL- 6) plays an important role in the pathogenesis of multiple myeloma, we also examined the effects of tumor cell adhesion on IL-6 secretion by BMSCs. In 51chromium binding assays, the U266, ARH-77, and IM-9 cell lines showed 52% +/- 12%, 55% +/- 6%, and 47% +/- 7% specific adherence, respectively, to normal BMSCs and 74% +/- 4%, 60% +/- 3%, and 61% +/- 6% specific adherence, respectively, to myeloma BMSCs. In contrast, only 12% to 13% specific binding of HS-Sultan cells to BMSCs was noted. The binding of myeloma cells to BMSCs was partially blocked with anti-beta 1 monoclonal antibody (MoAb), anti-beta 2 integrin MoAb, and excess RGD peptide, suggesting multiple mechanisms for the adhesion of myeloma cell lines to BMSCs. Binding of cell lines to FN or myeloma BMSCs did not affect cell line proliferation; however, adhesion of myeloma cell lines to normal BMSCs decreased DNA synthesis, ie, stimulation indices are 0.1 +/- 0.04, 0.2 +/- 0.1, 0.2 +/- 0.07, and 0.1 +/- 0.06 for the adherent non-IL-6-dependent U266, ARH-77, HS- Sultan, and IM-9 cells, respectively (n = 5, P < .01). In contrast, adherence of IL-6-dependent B9 cells increased their proliferation (stimulation index, 3.2 +/- 0.7). Significant (twofold to eightfold) increases in IL-6 secretion were evident in cell line-adherent (> or = 12 hours) normal and myeloma BMSC cultures. Paraformaldehyde fixation of BMSCs before adhesion completely abrogated IL-6 secretion, suggesting that IL-6 secretion was triggered in BMSCs rather than in cell lines. Partial blocking of cell line adhesion to BMSCs, using anti- beta 1 integrin and anti-beta 2 integrin MoAbs and RGD peptide, also partially blocked the triggering of IL-6 secretion by BMSCs. When cell lines were placed in Transwell inserts and then cultured with either normal or myeloma BMSCs, permitting juxtaposition without cell to cell contact between myeloma cell lines and BMSCs, no increase in IL-6 secretion was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Adhesion of Human Myeloma-Derived Cell Lines to Bone Marrow Stromal Cells Stimulates Interleukin-6 Secretion

AbstractPrevious studies show that human myeloma-derived cell lines specifically adhere to fibronectin (FN) through very late antigen-4 (VLA-4; α4β1 integrin complex) and RGD-peptide mechanisms, which may contribute to the localization of tumor cells in bone marrow (BM). In these studies, we characterized the adhesion of myeloma-derived cell lines to both normal and myeloma BM stromal cells (BMSCs) and the effect of adhesion on DNA synthesis. Because interleukin-6 (IL-6) plays an important role in the pathogenesis of multiple myeloma, we also examined the effects of tumor cell adhesion on IL-6 secretion by BMSCs. In 51chromium binding assays, the U266, ARH-77, and IM-9 cell lines showed 52% ± 12%, 55% ± 6%, and 47% ± 7% specific adherence, respectively, to normal BMSCs and 74% ± 4%, 60% ± 3%, and 61 % ± 6% specific adherence, respectively, to myeloma BMSCs. In contrast, only 12% to 13% specific binding of HS-Sultan cells to BMSCs was noted. The binding of myeloma cells to BMSCs was partially blocked with anti- β1 monoclonal antibody (MoAb), anti-β2 integrin MoAb. and excess RGD peptide, suggesting multiple mechanisms for the adhesion of myeloma cell lines to BMSCs. Binding of cell lines to FN or myeloma BMSCs did not affect cell line proliferation; however, adhesion of myeloma cell lines to normal BMSCs decreased DNA synthesis, ie, stimulation indices are 0.1 ± 0.04, 0.2 ± 0.1, 0.2 ± 0.07, and 0.1 ± 0.06 for the adherent non-IL-6-dependent U266, ARH-77, HS-Sultan, and IM-9 cells, respectively (n = 5, P < .01). In contrast, adherence of IL-6-dependent B9 cells increased their proliferation (stimulation index, 3.2 ± 0.7). Significant (twofold to eightfold) increases in IL-6 secretion were evident in cell line-adherent (µ12 hours) normal and myeloma BMSC cultures. Paraformaldehyde fixation of BMSCs before adhesion completely abrogated IL-6 secretion, suggesting that IL-6 secretion was triggered in BMSCs rather than in cell lines. Partial blocking of cell line adhesion to BMSCs, using anti-β1 integrin and anti-β2 integrin MoAbs and RGD peptide, also partially blocked the triggering of IL-6 secretion by BMSCs. When cell lines were placed in Transwell inserts and then cultured with either normal or myeloma BMSCs, permitting juxtaposition without cell to cell contact between myeloma cell lines and BMSCs, no increase in IL-6 secretion was observed. These studies suggest that adhesion to BMSCs may not only localize tumor cells in marrow but also permit cell-to-cell contact and facilitate stimulation of IL-6 secretion by BMSCs in a paracrine growth mechanism.

Lack of a role of interleukin 11 in the growth of multiple myeloma

Interleukin (IL)11 is a recently described lymphokine which, like IL-6, stimulates normal hematopoietic murine and human hematopoietic progenitor cells and therefore has potential value for either enhancing hematopoiesis in disease states or augmenting hematopoietic recovery after myeloablative theorapies. Since IL-6 is known to promote the growth of human myeloma, either in an autocrine or paracrine fashion, we examined the effect of IL-11 on the growth of a murine plasmacytoma cell line, human myeloma-derived cell lines, and freshly isolated human myeloma cells. Interleukin 11 does increase DNA synthesis by the murine plasmacytoma line T10 in the presence of neutralizing antibody to IL-6. However, neither human myeloma cells nor derived cell lines express IL-11 mRNA; secrete IL-11; express IL-11 cell surface receptors; or augment either DNA synthesis or Ig secretion in response to exogenous IL-11. These findings strongly suggest that IL-11 does support the growth of a murine plasmacytoma cell line but does not play a role in the growth of either freshly isolated human myeloma cells or derived cell lines.