Myeloma Bone Marrow Stromal Cells Research Articles

A Hyaluronan and Proteoglycan Link Protein 1 Matrikine: Role of Matrix Metalloproteinase 2 in Multiple Myeloma NF-κB Activation and Drug Resistance.

HAPLN1 and MMP2 produced by BMSCs obtained from patients with multiple myeloma promote NF-κB activity and resistance to bortezomib toxicity in multiple myeloma cells, uncovering their potential as biomarkers or therapeutic targets to address bortezomib resistance in patients with multiple myeloma.

Altered Genomic and Epigenetic Profiling of Myeloma Bone Marrow Stromal Cells Identifies Targets for Current and Future Immunotherapeutic Approaches

Background: Novel drug discoveries have shifted the treatment paradigms of most hematological malignancies including multiple myeloma (MM), but minimal residual disease and drug resistance underlie relapses in MM. Although many genetic and epigenetic alterations regulate MM progression, MM cells are not autonomous. Dynamic interactions between MM cells and cells of the bone marrow (BM) microenvironment have been reported by our group and others. MM plasma cells (PCs) depend on interactions with bone marrow stromal cells (BMSCs) for their survival and growth, but little is known about the specific genetic events taking place in the MM BM microenvironment. Methods: Here we report a detailed analysis of the genetic and epigenetic events that are characteristic of MM BMSC as compared to HD-BMSC interacting with BM PCs. To evaluate genetic and epigenetic landscapes, RNA was extracted from bulk sorted populations of 16 MM-BMSC, 3 HD-BMSC, and 10 autologous MM cells. We prepared libraries for 32 samples using the NEBNext Ultra II Stranded Poly A kit, and then sequenced on the NextSeq 500, PE150. Sequencing data were analyzed using a custom computational and statistical pipeline at the Department of Biostatistics, School of Public Health and Partek software. Results: Unsupervised clustering showed that MM-BMSC samples clustered as a distinct and completely separate cluster from HD-BMSC and autologous MM cells. Gene level analyses of these three groups identified 990 genes differentially expressed (upregulated or downregulated, P< 0.005). Sequential filtration analyses of the differentially expressed genes in MM-BMSC identified significant deregulation of : transcripts in the Jak-STAT signaling pathway (JAK3, PIM1, IL6, CSF2R, AKT1/2, BCL2L1, CDKN1A and range of IL transcripts); genes encoding extracellular matrix interacting proteins (CD36, CD49, LAMA3, CD44, CD47); and various plasma membrane proteins that define different subpopulations of hematopoietic cells. These genes were deregulated in >24% of MM-BMSC samples analyzed as compared to HD-BMSC samples. These transcripts were downregulated in autologous tumor cells. Next, we interrogated the epigenomic landscape and identified the splicing signature of MM-BMSC as compared to HD-BMSC, and autologous MM cells. Comparison of the splicing patterns (exon skipping, intron retention, novel splice acceptor and/or donor activation) of these three distinct groups showed that a total of 2,100 genes were differentially expressed and 566 were alternatively spliced among the three groups (P < 0.01). These analyses identified a limited number of the transcripts with ~3% significantly spliced in MM-BMSC compared to HD-BMSC. However, comparing MM-BMSC splicing events to MM cells splicing events, we identified >30% of genes which were alternatively spliced in MM cells but not in MM-BMSC. Further, gene enrichment and pathway analyses identified a selective set of transcripts that were alternatively and differentially spliced in MM-BMSC including genes involved in MAPK and Ras signaling pathways, homologous recombination, mismatch repair, and adherens junction. Conclusions: Taken together, our studies identified marked differences between important stromal elements in MM- and HD-BM. We identified genes that were specifically upregulated/suppressed in MM-BMSC compared to MM-cells and HD-BMSC. Within MM BMSCs, we identified several splicing events on genes of signaling pathways implicated in development and progression of MM. Furthermore, altered splicing events identified on these transcripts represent potential new immunotherapeutic targets. Disclosures Chu: Gilead: Honoraria; Celgene: Honoraria; Teva: Consultancy; Amgen Inc.: Honoraria; AstraZeneca: Honoraria. Anderson:C4 Therapeutics: Other: Scientific founder ; Gilead Sciences: Other: Advisory Board; OncoPep: Other: Scientific founder ; Sanofi-Aventis: Other: Advisory Board; Janssen: Other: Advisory Board.

Activated Leukocyte Cell Adhesion Molecule (ALCAM) Regulate Myeloma Cancer Stem Cell Mediated Myelomagenesis and Chemoresistance

IntroductionMultiple myeloma (MM) is an incurable plasma cell cancer. People introduced the concept of cancer stem cells (CSCs) in MM, known as MM-CSCs, to characterize a small population of malignant cells with distinguishable activities of self-renewal and differentiation into regular MM cells. In this study, we showed that activated leukocyte cell adhesion molecule (ALCAM) interacted with epidermal growth factor receptor (EGFR). ALCAM-EGFR axis regulated MM-CSC mediated myelomagenesis and chemoresistance.MethodsMM-CSCs in MM cell lines and primary MM cells were identified by Hoechst staining or ALDEFLUOR dye, followed by flow cytometry analyses. MM-CSC clonalization activity was examined by soft agar colony formation assay. The protein-protein interaction was identified by co-immunoprecipitation, proximity ligation assay, and proteins' structure simulation. MM-CSC mediated myelomagenesis in vivo was examined in human MM xenograft mouse model and murine adoptive MM model. MM-CSC mediated chemoresistance in vivo was examined using 5T MM model. The clinical significance of ALCAM expression in MM was analyzed using patients' gene expression profiles.ResultsBoth MM cell lines and primary MM cells expressed ALCAM. When cultured with bone marrow stromal cells (BMSCs) or in BMSC-conditioned medium, ALCAM knocked down (AL-KD) MM cells exhibited increased colony formation activity. Hoechst staining showed that under such culture conditions, AL-KD MM cells had increased stemness side population (SP), which was MM-CSC. Depletion of MM-CSC reduced MM colony formation activity. In human MM xenograft model, subcutaneous co-injection of AL-KD MM cells and BMSCs had large amount of MM-CSC and fast tumorigenesis. In murine adoptive MM model, which had tumor derived from viral transformed plasma cells with mandatory expression of MYC, KRAS genes and a shRNA sequence, ALCAM shRNA expression resulted significant more rapid tumor development than non-target shRNA.Mechanistic studies showed that ALCAM and EGFR co-immunoprecipitated in MM cells. Proximity ligation assay showed interaction of ALCAM and EGFR in primary MM cells. Immunofluorescent staining showed that ALCAM colocalized with unphosphorylated EGFR in MM. Epidermal growth factor (EGF), the canonical ligand of EGFR, stimulated EGFR phosphorylation and abolished ALCAM-EGFR colocalization. Protein structure simulation suggested that EGF and ALCAM mutual exclusively bound to the same region of EGFR. EGF addition stimulated MM-CSC in cultured MM. Inhibitors of EGFR, ERK, or hedgehog pathway repressed MM-CSC. EGFR activation led to hedgehog pathway activation via RAS/MEK/ERK cascade, while ALCAM inhibited such pro-MM-CSC cell signaling by binding to EGFR.Chemotherapeutics, such as bortezomib and melphalan, induced MM apoptosis, but also stimulated MM-CSCs, which were resistant to chemotherapeutics. Although CTR-KD and AL-KD MM had similar death rate after the drug treatment, AL-KD MM cells had more MM-CSC generated. MM-CSCs derived in AL-KD MM cells had more active cell proliferation even in the presence of drugs. Further study showed that chemotherapy stimulated EGFR expression and repressed ALCAM expression, therefore switched the balance of ALCAM-EGFR axis to pro-MM-CSC direction. EGFR inhibitors, such as gefitinib, did not have anti-MM activity, but repressed MM-CSCs. In 5T MM model, Gefitinib/melphalan combination therapy resulted prolonged remission period, and significant better mouse survival.We identified MM-CSC in patients BM. ALCAM expression correlated with MM overall survival (OS): ALCAMhigh patients had better OS. In ALCAM-EGFR axis, only ALCAM expression, but not EGFR expression, correlated with MM OS. ALCAM expression varied in different patients, while EGFR expression was more even. Therefore we believe in ALCAM-EGFR axis, it was ALCAM, but not the EGFR, that played the regulatory role. Furthermore, pathway enrichment analysis showed that MM-CSC, identified by Hoechst staining, and MM minimum residual disease (MRD) shared similarities in signaling pathways, as well as pattern of ALCAM expressions.Conclusion and DiscussionALCAM-EGFR axis regulates MM-CSC mediated myelomagenesis and chemoresistance. EGFR inhibitors repress chemoresistant MM-CSC. Combination therapy with conventional chemotherapeutics and an EGFR inhibitor might be promising in MM treatment. DisclosuresNo relevant conflicts of interest to declare.

Modeling multiple myeloma-bone marrow interactions and response to drugs in a 3D surrogate microenvironment.

Multiple myeloma develops primarily inside the bone marrow microenvironment, that confers pro-survival signals and drug resistance. 3D cultures that reproduce multiple myeloma-bone marrow interactions are needed to fully investigate multiple myeloma pathogenesis and response to drugs. To this purpose, we exploited the 3D Rotary Cell Culture System bioreactor technology for myeloma-bone marrow co-cultures in gelatin scaffolds. The model was validated with myeloma cell lines that, as assessed by histochemical and electron-microscopic analyses, engaged contacts with stromal cells and endothelial cells. Consistently, pro-survival signaling and also cell adhesion-mediated drug resistance were significantly higher in 3D than in 2D parallel co-cultures. The contribution of the VLA-4/VCAM1 pathway to resistance to bortezomib was modeled by the use of VCAM1 transfectants. Soluble factor-mediated drug resistance could be also demonstrated in both 2D and 3D co-cultures. The system was then successfully applied to co-cultures of primary myeloma cells-primary myeloma bone marrow stromal cells from patients and endothelial cells, allowing the development of functional myeloma-stroma interactions and MM cell long-term survival. Significantly, genomic analysis performed in a high-risk myeloma patient demonstrated that culture in bioreactor paralleled the expansion of the clone that ultimately dominated in vivo. Finally, the impact of bortezomib on myeloma cells and on specialized functions of the microenvironment could be evaluated. Our findings indicate that 3D dynamic culture of reconstructed human multiple myeloma microenvironments in bioreactor may represent a useful platform for drug testing and for studying tumor-stroma molecular interactions.

Interleukin-32 Induce Production of IL-6 in Multiple Myeloma Bone Marrow Stromal Cells

Background: Multiple myeloma (MM) is closely associated with inflammation. Patients with auto-immune disease history of infection and other inflammatory disease have higher incidence of MM. IL-6 is the most important inflammatory factor in MM which plays a key role in the proliferation and progression. We previously demonstrated that MM cells were modified by bone marrow stromal cells (BMSCs) that formulate a inflammatory microenvironment in bone marrow (BM) and secret IL-6. How the inflammation makes BMSCs secret IL-6, however, remained undocumented. Our subsequent study compared the differential secretion of peripheral blood (PB) between MM patients and normal people by cytokine array, and showed that interleukin 32(IL-32) is highly expressed in MM patients. IL-32, also named natural killer 4(NK-4), is a newly found inflammatory factor. It was reported in solid tumors IL-32 is a pro-inflammatory factor which triggers a massive amplification of inflammatory process resulting in the change of other inflammatory factor including IL-6,IL-10,TNF-α. In this study, we examined BMSCs cytokines in MM BM and found that IL-32 was a functional factor in the process of inflammation in MM BM microenvironment.Results: First, to test our previous study, we detected IL-32 in BM supernatant and PB supernatant in both MM patients (n=45) and healthy controls (n=13) by ELISA. Result showed that in both BM and PB, MM patients have higher expression of IL-32 compared to healthy controls (P<0.05). Next, total BM cells(both CD138+ and CD138- cells) from MM patients were assayed by qRT-PCR for gene expression analysis.IL-32 were highly expressed in MM BM cells and the CD138+ cells (P<0.05). We also detected IL-32 in MM cell lines (RPMI 8226,OPM-2) and BMSCs isolated from MM patients by qRT-PCR, Western blot, and ELISA, and found that IL-32 was highly expressed in MM cell lines than BMSCs. In contrast, proteinase 3(PR3, receptor of IL-32) was highly expressed in BMSCs compared to MM cell lines.Second, we stimulated the MM BMSCs with recombinant IL-32α, and found that the secretion of IL-6,CCL3 (MIP1-α), CCL4(MIP-1β) were significantly increased and CCL-5(RANTES)and IL-10 were decreased (P<0.05). Further, Western blot was applied to detect the inflammation molecular pathway in BMSCs. JAK-STAT pathway and NF-κB pathway were activated, and the phosphorylation of STAT3 was increased. After we knock down the PR3 in BMSCs, these changes were reduced. We repeated these experiments in BMSCs isolated from 15 different MM patients, the phenomenon mentioned above showed in 11 patients. The recombinant IL-32α was also used to stimulate 8226 and OPM-2 cells, but these two kinds of MM cells didn’t secret IL-6, and no significant change in cell proliferation or cell apoptosis.Finally, our group co-cultured the MM BMSCs with 8226 and OPM-2 cells. The secretion of IL-6 and the phosphorylation of JAK-STAT pathway in BMSCs were also increased. Knockdown of IL-32 in 8226 and OPM-2 cells weakened these changes.MM Cell proliferation and cell cycles after co-culture with MM BMSCs are under investigation.Conclusion: our findings suggest that IL-32 is mainly secreted by MM cells. It may not directly promote the MM cells to grow. However, IL-32 promote the MM BMSCs to secret more cytokines including IL-6,CCL3,CCL4 by activating the JAK-STAT3 pathway, which lead to a amplification of inflammation in BM environment, resulting in the cell proliferation . DisclosuresNo relevant conflicts of interest to declare.

The Senescence-Associated Secretory Phenotype In Multiple Myeloma

IntroductionThe incidence of multiple myeloma (MM) increases with age, yet some cytogenetic changes are actually more common in younger patients with MM (Avet-Loiseau J Clin Oncol 2013). This suggests that a mechanism other than chromosomal changes underlies the increased incidence with age. Senescent cells secrete a number of proinflammatory cytokines, chemokines, growth factors and proteases resulting in the senescence-associated secretory phenotype (SASP), which can promote tumor growth. Preclinical data suggests that myeloma bone marrow stromal cells express the SASP (Andre PLOS ONE 2013). We hypothesized that SASP factors correlate with age in patients with MM. MethodsPeripheral blood serum and matched bone marrow aspirate plasma from patients with multiple myeloma were evaluated for selected factors associated with the SASP using quantitative multiplex immunoassay (Rules Based Medicine, Austin TX USA). SASP factors with a known role in MM [interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-15 (IL-15), granulocyte-macrophage colony-stimulating factor (GMCSF), intercellular adhesion molecule 1(ICAM1), osteoprotegerin (OPG), hepatocyte growth factor (HGF), insulin-like growth factor-binding protein(IGFBP-1), interleukin-1 beta (IL1b), monocyte chemotactic protein 1(MCP-1), macrophage inflammatory protein-1 alpha(MIP-1a), angiogenin, leptin, vascular endothelial growth factor receptor 1(VEGFR1) and stem cell factor(SCF)] were selected. The relationship between age and SASP factors were analyzed using Kendall tau rank correlation coefficient. ResultsSamples from 25 patients (each with peripheral blood serum and matched bone marrow aspirate plasma) were analyzed. The median age was 62 (range 47 - 74). Disease states were as follows: 36% newly diagnosed/untreated, 24% pretransplant and 40% relapsed. ISS stage included 40% stage I, 28% stage II and 32% stage III. Three of the selected SASP factors in the peripheral blood correlated with age: IL-8 (Kendall Tau 0.334, p=0.027), OPG (Kendall Tau 0.289, p=0.046) and MCP-1 (Kendall Tau 0.332, p=0.022). No SASP factors tested in the bone marrow plasma were significantly correlated with age. ConclusionsWe demonstrated age-associated differences in the SASP factors IL-8, OPG and MCP-1 in the peripheral blood of myeloma patients. Future research will examine differences between patients with myeloma and age-matched controls without cancer. Disclosures:Vij:Celgene : Honoraria, Research Funding, Speakers Bureau; Millennium: Honoraria, Speakers Bureau; Onyx: Honoraria, Research Funding, Speakers Bureau. Stockerl-Goldstein:Celgene : Speakers Bureau; Celgene : Speakers Bureau; Millennium: Speakers Bureau; Millennium: Speakers Bureau.

Abstract 4111: SDF1/CXCR4 regulates stiffness of bone marrow stromal cell through RhoA and Rac1 GTPases pathway to support myeloma cell proliferation in multiple myeloma.

Abstract Abstract As the second most prevalent hematologic malignancy, multiple myeloma (MM) remains high incurable and relapse owing to intrinsic or acquired drug resistance. Therefore, new therapeutic strategies that are active in drug resistance and target molecular processes of MM progress are attractive. MM is characterized by bone marrow infiltration of malignant plasma cells. The bone marrow microenvironment is a complex structure of various extracellular components and many cell types including haematopoietic stem cells, bone marrow stromal cells (BMSCs), immune cells, bone marrow endothelial cells, as well as osteoclasts and osteoblasts. Recently study showed the physical interaction of multiple myeloma cells with BMSCs from patients of multiple myeloma in the bone marrow milieu, provokes a positive feedback loop and results in multiple myeloma pathogenesis. We observed the myeloma BMSCs are much stiffer and more contractile than normal BMSCs. Stiffer hydrogels support colony formation and adhension of MM better than softer hydrogels. These suggested that myeloma BMSCs provide myeloma cell-friendly microenvironments for myeloma cells via exerting biomechanical forces. In order to elucidate the molecular mechanisms leading to the change of myeloma BMSCs biochemical phenotype, we analyzed myeloma BMSCs and normal BMSCs gene expression profiles. Microarray data analysis demonstrated stromal cell derived factor-1(SDF1) was over expressed in MM BMSCs. In vitro, 100ng/ml SDF1 significantly induced actin polymerization and polarization in MM BMSCs, but not in normal BMSCs. In order to understand how SDF1 regulate BMSCs’ contractile, we conducted reverse phase protein array (RPPA) experiment. Based on RPPA data, we determined RhoA and Rac1 GTPases played important roles in SDF1 induced stiffer of BMSCs. Western blot results also approved SDF1 increased phosphate RhoA and phosphate Rac1 expression level. In conclusion, we characterized the pivotal role of RhoA and Rac1 GTPases in regulating BMSCs stiffness and BMSCs-MM interaction. Targeting biomechanics of BMSCs in myeloma patients, restoring to their normal state would be essential for the long term cure of myeloma, provoking RhoA and Rac1 pathway as novel MM therapy. Citation Format: Dan Wu, Jing Su, Qianqian Song, Xiaobo Zhou. SDF1/CXCR4 regulates stiffness of bone marrow stromal cell through RhoA and Rac1 GTPases pathway to support myeloma cell proliferation in multiple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4111. doi:10.1158/1538-7445.AM2013-4111

In Vivo Targeting of Stromal-Derived Factor-1 As a Strategy to Prevent Myeloma Cell Dissemination to Distant Bone Marrow Niches

AbstractAbstract 440 Background.Multiple myeloma (MM) patients present with multiple lytic lesions at diagnosis, indicating the presence of continuous dissemination of MM cells from the primary site of tumor development to multiple distant bone marrow (BM) niches. We hypothesized that stromal-derived factor-1 (SDF-1) may represent a target for preventing transition from MGUS (micrometastatic stage) to active-MM (macrometastatic stage); thus resulting in inhibition of MM progression. We therefore evaluated SDF-1 expression in the BM of patients with MGUS, MM, compared to healthy individuals; and tested NOX-A12, a high affinity l-oligonucleotide (Spiegelmer) binder to SDF-1 in MM, looking at its ability to modulate MM cell tumor growth and MM cell homing to the BM in vitro and in vivo . Methods.SDF-1 levels were evaluated by immunohistochemistry on BM specimens obtained from patients with MGUS, active-MM, or healthy individuals; and confirmed by ELISA, using conditioned-medium of BM-mesenchymal stromal cells obtained from MGUS, active-MM and healthy individuals. BM metastatic lesions from primary epithelial tumors were also considered. Co-localization of MM tumor cells (MM.1S-GFP+) with SDF-1 was tested in vivo by in vivo confocal microscopy, using both AlexaFluor633-conjugated-anti-SDF-1 monoclonal antibody and AlexaFluor647-conjugated-NOX-A12 oligonucleotide. Effect of NOX-A12 on modulating MM cell dissemination was tested in vivo, by using in vivo confocal microscopy. In vivo homing and in vivo tumor growth of MM cells (MM.1S-GFP+/luc+) were assessed by using in vivo confocal microscopy and in vivo bioluminescence, in SCID mice treated with 1) vehicle; 2) NOX-A12; 3) bortezomib; 4) NOX-A12+bortezomib. Detection of mobilized MM-GFP+ cells ex vivo was performed by flow cytometry. Effects of drug combination on dissemination of MM cells to distant BM niches was evaluated ex vivo by immunofluorescence on femurs obtained from each cohort of mice. DNA synthesis and adhesion of MM cells in the context of NOX-A12 (50–100nM) treated primary MM BM stromal cells (BMSCs) in presence or absence of bortezomib (2.5–5nM) were tested by thymidine uptake and adhesion in vitro assay, respectively. Synergism was calculated by using CalcuSyn software. NOX-A12-dependent-modulation of signaling was evaluated by western blot on MM cells exposed or not to primary BM-MSCs. Results.Patients with active-MM present with higher BM SDF-1 expression vs. MGUS patients and healthy individuals. Similarly, BM presenting with metastasis from epithelial primary malignancies had higher SDF-1 levels compared to healthy subjects, thus suggesting the importance of SDF-1 in favoring tumor cell metastasis to BM niches. SDF-1 co-localized at BM level with MM tumor cells in vivo. In vitro, NOX-A12 induced a dose-dependent de-adhesion of MM cells from the BMSCs supported by inhibition of BM-MSC-mediated phosphorylation of ERK1/2 and cofilin. These findings were corroborated and validated in vivo: NOX-A12 induced MM cell mobilization from the BM to the peripheral blood as shown ex vivo, by reduced percentage of MM cells in the BM and increased number of MM cells within the peripheral blood of mice treated with NOX-A12 vs. control (BM: 57% vs. 45%; PB: 2.7% vs. 15%). This was supported by inhibited homing of MM cells to the BM of those mice pre-treated with NOX-A12. We next showed that NOX-A12-dependent de-adhesion of MM cells from BMSCs lead to enhanced MM cell sensitivity to bortezomib, as shown in vitro, where a synergistic effect between NOX-A12 and bortezomib was observed (C.I.: .57-.76). These findings were validated in vivo: tumor burden was similar between NOX-A12- and control mice whereas bortezomib-treated mice showed significant reduction in tumor progression compared to the control (P<.05); importantly, significant reduction of tumor burden in those mice treated with sequential administration of NOX-A12 and bortezomib was observed, compared to bortezomib alone-treated mice (P <.05). Similarly, NOX-A12+bortezomib combination induced significant inhibition of MM cell homing, as shown by in vivo confocal microscopy. Conclusion.SDF-1 represents a valid target for inhibiting MM cell dissemination to distant BM niches, thus providing the evidence for using the SDF-1 inhibiting Spiegelmer NOX-A12 to target MM cells at the stage of micrometastasis (MGUS), thus preventing development of symptomatic macrometastatic MM. Disclosures:Zboralski:NOXXON Pharma AG, Berlin, Germany: Employment. Kruschinski:NOXXON Pharma AG, Berlin, Germany: Employment. Ghobrial:Novartis: Advisory Board Other; Onyx: Advisory Board, Advisory Board Other; Millennium: Advisory Board, Advisory Board Other; Bristol Myers Squibb: Advisory Board, Advisory Board Other.

Dual Inhibition of the STAT3 and Src Pathways by NS-018, a JAK2 and Src-Family Kinase Inhibitor, Reduces Myeloma Cell Proliferation and Adhesion and Suppresses Osteoclast Formation

Abstract 2900Multiple myeloma (MM) is a clonal B-cell malignancy characterized by the accumulation of malignant plasma cells in the bone marrow, and it finally leads to osteolytic bone destruction and impaired hematopoiesis. The pathophysiology of MM is closely linked with the bone marrow microenvironment, which consists of various cell components including bone marrow stromal cells (BMSCs) and bone marrow endothelial cells as well as osteoclasts and osteoblasts. Interaction between MM cells and BMSCs through cell-adhesion molecules confers drug resistance to myeloma cells and stimulates the release of cytokines such as interleukin-6 (IL-6) and receptor activator of NF-kappaB ligand (RANKL) from BMSCs. IL-6 is a major cytokine which enhances cell proliferation and promotes the survival of MM cells by downstream signaling through Janus kinase (JAK) and signal transducer and activator of transcription (STAT). RANKL triggers osteoclast differentiation and activation leading to bone resorption, lytic bone lesion, and osteopenia. The Src family kinases (SFKs), c-Src and Fyn, mediate signaling by cell-adhesion molecules and the RANKL receptor, and they play important roles in cell adhesion and osteoclast differentiation.NS-018 is a potent and selective dual JAK2/SFKs inhibitor which is under phase1/2 clinical development for the treatment of myelofibrosis. A previous study has shown that NS-018 inhibits JAK2, c-Src and Fyn kinases with IC50 values of 0.72, 6.0 and 7.3 nmol/L, respectively. NS-018 also inhibits JAK2 and SFKs in cells as evidenced by its antiproliferative effect against Ba/F3 cells expressing constitutively activated JAK2 and NIH3T3 cells transformed by v-Src.In the present study, the ability of NS-018 to inhibit JAK2/STAT3 signaling was examined in IL-6-responsive human MM cell lines such as U266, RPMI 8226, and PCM6. NS-018 suppressed IL-6-induced phosphorylation of STAT3 in a dose-dependent manner at concentrations greater than 100 nmol/L. In addition, NS-018 inhibited the IL-6-enhanced the proliferation of PCM6 cells at concentrations similar to those required to inhibit STAT3 phosphorylation.To assess whether SFKs inhibition by NS-018 could contribute to an improvement in MM pathology, we next investigated the effect of NS-018 on the adhesion of myeloma cells to cell-adhesion molecules and on osteoclast formation. NS-018 (100 nmol/L) inhibited the adhesion of RPMI 8226 cells to collagen type 1 and VCAM-1 by about 40%. NS-018 also suppressed RANKL-induced differentiation of human osteoclast precursor cells to mature osteoclasts. The numbers of TRAP-positive multinucleated osteoclasts were reduced to about one-half at 100 nmol/L NS-018 and none were observed at 1000 nmol/L. NS-018 similarly suppressed the differentiation of murine RAW264.7 cells to mature osteoclasts. The suppression of cell adhesion and osteoclast formation by NS-018 could both be mediated by c-Src and/or Fyn inhibition, because both inhibitory effects were observed with a typical SFKs inhibitor but not with a typical JAK2 inhibitor.In conclusion, NS-018 reduced IL-6-enhanced myeloma cell proliferation through inhibition of the JAK2/STAT3 signaling pathway and suppressed cell adhesion and osteoclast formation through inhibition of c-Src and/or Fyn. These results suggest that NS-018 has a dual mechanism of action in MM by simultaneously blocking the JAK2/STAT3 and SFKs pathways. Treatment with NS-018 is a potential new therapeutic option to improve the complex pathological condition of patients with MM. Disclosures:No relevant conflicts of interest to declare.

Bone Marrow Stromal Cells From Myeloma Patients Support the Growth of Myeloma Stem Cells

It has been well documented that bone marrow stromal cells (BMSCs) of multiple myeloma patients play a pivotal role in supporting the growth of mature myeloma cells. With evolving concepts concerning the presence of myeloma stem (initiating) cells, we aimed this investigation to specifically address the supportive role of BMSCs for myeloma stem cell growth in vitro and in vivo. BMSC lines were derived from myeloma or control patients (myeloma or control BMSCs). Myeloma stem cells of the RPMI 8226 myeloma cell line were recognized through the identification of "side populations" (SP) with Hoechst dye staining. SP cells formed more colonies when grown on myeloma BMSC than on control BMSC. Additionally, higher percentages of SP cells were observed when grown on myeloma BMSCs than on control BMSCs. In the mouse model, SP cells inoculated with myeloma BMSCs grew faster than those inoculated with control BMSCs. Of note, SP cells demonstrated an increased expression of CD184 (CXCR4) compared with non-SP cells. The expression of CD184 in SP cells was further increased when they were cultured with myeloma BMSCs. CD184(+) SP cells formed more colonies than CD184(-) SP cells. Treatment with AMD 3100, an inhibitor of CD184, reduced colony formation by CD184(+) SP cells when co-cultured with myeloma BMSCs. This was associated with the decreased activation of ERK, a downstream target of activated CD184, in myeloma cells. These findings indicate that the myeloma BMSCs create a microenvironment supportive of myeloma stem cells via, at least partially, the CXCR4 signaling pathway.

The increased expression of receptor activator of nuclear‐κB ligand (RANKL) of multiple myeloma bone marrow stromal cells is inhibited by the bisphosphonate ibandronate

The receptor activator of nuclear factor-kappaB ligand (RANKL) and interleukin-1beta are osteoclast activating factors which are abnormally expressed in bone marrow stromal cells and plasma cells of multiple myeloma patients. In this work we analyzed RANKL expression in human bone marrow mesenchymal stromal cells and the effect of the bisphosphonate ibandronate on RANKL expression after IL-1beta activation of ERK pathway. Mesenchymal stromal cells were obtained from bone marrow iliac aspirates from multiple myeloma patients at stages II/III and non-osteoporotics control donors; these cells were maintained under long-term culture conditions. Cells were cultured in the presence or the absence of 5 ng/ml IL-1beta and/or 5 microM ibandronate, during selected periods. mRNA for RANKL and protein levels were assayed by RT-PCR and Western blot, respectively. Human bone marrow stromal cell line HS-5 was used for assessing IL 1beta- and ibandronate-ERK phosphorylation responses. Multiple myeloma mesenchymal stromal cells differentiate from control cells by increased basal RANKL expression. IL-1beta up regulated RANKL expression showed dependent on activated MEK/ERK pathway. Finally, the bisphosphonate ibandronate, that hindered activation of the MEK/ERK pathway significantly inhibited both basal and IL-1beta dependent RANKL expression by cells. Results indicate that RANKL expression involves the MEK/ERK pathway in multiple myeloma mesenchymal stromal cells, and that early obstruction of this path, such as that achieved with ibandronate, significantly deters RANKL protein expression.

Normal Bone Marrow Stromal Cells Inhibit the Growth of Myeloma Cells While Myeloma Bone Marrow Stromal Cells Improve It.

Objective: Observe the effect of normal and myeloma bone marrow stromal cells (BMSC) to the growth of myeloma cells. Evaluate the function of adhesion molecules in the mutual action of BMSC and myeloma cells.Methods: Immunomagnetic beads and anti-CD138 antibody were used to separate myeloma cells from patients' bone marrow of multiple myeloma (MM). U266 and freshly purified myeloma cells respectively co-cultured with normal BMSC and myeloma BMSC. Single U266 or fresh myeloma cells were cultured as control group. Anti-CD11a, anti-CD29, anti-CD44 and anti-CD49d antibodies were respectively added in the co-cultured system and single myeloma cells. After culture for 72 hours, cell cycle of myeloma cells and the ratio of apoptotic cells were tested by flowcytometry. The level of interleukin 6 (IL-6) in the supernate was measured.Results: Normal BMSC inhibited the growth of myeloma cells while myeloma BMSC improved it. As to U266, S+G2% of myeloma cells in normal BMSC group and myeloma BMSC group were 55.5±4.0% and 65.4±4.5%(p<0.05)respectively. The ratio of apoptotic cells were 3.92±1.83% and 0.86±0.58%(p<0.05)respectively(figure1 from left to right: U266, U266+normal BMSC, U266+myeloma BMSC, red: apoptotic cells). The level of IL-6 in the supernate were 1236±146.2pg/ml and 1661±161.5pg/ml(p<0.05)respectively. After co-cultured with normal BMSC and myeloma BMSC, S+G2% of freshly separated myeloma cells were 5.2±4.1% and 15.9±5.5%(p<0.05)respectively. The ratio of apoptotic cells were 18.7±7.8% and 8±3.9%(p<0.05) respectively (figure2 from left to right: fresh myeloma cells, fresh myeloma cells+normal BMSC, fresh myeloma cells+myeloma BMSC, red: apoptotic cells). The level of IL-6 in the supernate were 1621.2±121.1pg/ml and 2151.1±170.4pg/ml(p<0.05)respectively. The function of four adhesion molecules was different in the co-culture system. Anti-CD11a hardly affected the growth of myeloma cells. But anti-CD29 and anti-CD44 showed their significant inhibition to the growth of myeloma cells, including the decrease of supernatant IL-6 level, percentage of phase S+G2 and the increase of apoptotic cells (fresh myeloma cells). Anti-CD49d indicated partial inhibition, but no significant change.Conclusion: We firstly proved that normal BMSC inhibited the growth of myeloma cells. Anti-CD29 and anti-CD44 can block the adhesion of myeloma cells and BMSC, resulting in the growth inhibition of myeloma cells. [Display omitted]

Zoledronic acid down‐regulates adhesion molecules of bone marrow stromal cells in multiple myeloma

Myeloma plasma cells interact with the bone marrow microenvironment which, in turn, supports their growth and protects them from apoptosis. In vitro studies have demonstrated the antitumor potential of zoledronic acid (ZOL) on myeloma cell lines, but few data are available on its effects on bone marrow stromal cells (BMSCs). The aim of the current study was to evaluate the antiproliferative and apoptotic effect of ZOL on BMSCs, as well as its effect on the expression of adhesion molecules. BMSCs, obtained from bone marrow mononucleated cells of 8 patients with multiple myeloma, were treated with increasing concentrations of ZOL for 3 days. Cytotoxic effect was analyzed by 3-(4-5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide; thiazolyl blue (MTT) assay whereas the induction of apoptosis was evaluated by flow cytometric detection of fluorescein isothiocyanate-labeled annexin V, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and nuclear changes. Moreover, expression of CD106, CD56, CD50, CD49d, CD44, and CD40 was analyzed by flow cytometry. Data were evaluated by the Friedman test. After 3 days of exposure at concentrations of 10(-4) to 10(-5) M, ZOL induced a decrease in proliferation (P < 0.0001) and an increase in apoptosis (P < 0.002). Analysis of culture supernatants showed that myeloma BMSCs expressed interleukin (IL)-6, negligible levels of tumor necrosis factor-alpha, and no IL-1beta. In vitro exposure to the lowest concentrations of ZOL decreased IL-6 production by BMSCs. Among the adhesion molecules, CD106, CD54, CD49d, and CD40, which were strongly expressed at baseline, showed a statistically significant reduction compared with controls after exposure to ZOL. ZOL interfered with myeloma BMSCs by reducing proliferation, increasing apoptosis, and modifying the pattern of expression of adhesion molecules, especially those involved in plasma cell binding. These effects on BMSCs might explain the antitumor activity of ZOL.

Bone marrow stromal-derived soluble factors and direct cell contact contribute to de novo drug resistance of myeloma cells by distinct mechanisms.

The tumor microenvironment plays a critical role in determining the fate of tumor cells. We have previously reported that adhesion of human myeloma and leukemia cell lines to the extracellular matrix protein, fibronectin, confers a multidrug-resistant phenotype. Mechanisms associated with this cell adhesion-mediated drug resistance are drug-type specific. In the present study, we examined the influence of bone marrow stromal cells (BMSCs) on myeloma cell response to the topoisomerase II inhibitor, mitoxantrone. Apoptosis was inhibited by more than 50% when cells were adhered to BMSCs as compared to myeloma cells maintained in suspension. To investigate the mechanisms contributing to the resistance of myeloma cells in contact with BMSCs, we examined the protective effects of BMSCs under four separate conditions: (1) direct cell contact; (2) BMSCs conditioned medium; (3) medium conditioned by coculturing myeloma cells in direct contact with BMSCs; and (4) medium conditioned by coculturing myeloma cells and BMSCs without direct physical contact. Conditioned medium from BMSCs alone was not sufficient to protect myeloma cells from drug-induced apoptosis; however, soluble factors produced during the myeloma-BMSCs interaction decreased the sensitivity of myeloma cells to mitoxantrone, suggesting a dynamic interaction between myeloma cells and BMSCs. We also found that myeloma cells in direct contact with BMSCs underwent growth arrest, whereas soluble factors produced by myeloma cells-BMSCs coincubation stimulated the proliferation of myeloma cells. These data show that both cell-cell adhesion of BMSCs with myeloma cells and soluble factors induced by this cell-cell interaction are involved in the protection of myeloma cells from mitoxantrone-induced apoptosis; however, the mechanisms contributing to the drug resistance are different.

HHV-8 and multiple myeloma in the UK

Rettig and colleagues reported an association between HHV-8 and multiple myeloma. HHV-8 genomic sequences were detected by PCR in cultured bone-marrow stromal cells from 15 of 15 patients with multiple myeloma and from two of eight patients with monoclonal gammopathy of uncertain significance, but not from 26 controls. The association is biologically plausible since HHV-8 encodes an interleukin-6 (IL-6) homologue and IL-6 is known to be an important growth factor for myeloma cells; furthermore Rettig and colleagues showed expression of viral IL-6 in three of three myeloma bone-marrow stromal cells. The investigators therefore proposed that HHV-8 has a causative, albeit indirect, role in the pathogenesis of multiple myeloma. Unlike other human herpesviruses, with the exception of herpes simplex virus type 2, infection with HHV-8 is not widespread in the population. Serological assays seem to give the best indication of prevalence. On the basis of antibody reactivity to a latency associated nuclear antigen (LANA) and a recombinant capsid-related protein, encoded by open reading frame (orf) 65·2, HHV-8 seroprevalence in the UK is estimated at about 5%; in the USA it appears to be a little higher. In some Mediterranean countries HHV-8 seroprevalence is around 10–20%, and in Uganda around 50%. By an immunofluorescence assay for lytic HHV-8 antigens, an upper estimate for the HHV-8 prevalence in the USA is around 25%. All three assays suggest a marked geographical variation in prevalence. HHV-8 and multiple myeloma in France

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 &lt; .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 (&gt; 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.