Serum Of Myeloma Patients Research Articles

Glycosylationof Immunoglobulins Determine Bone Loss in Multiple Myeloma

About 80% of patients with multiple myeloma develop a severe osteolytic bone disease causing pain and fractures. The myeloma cells secrete immunoglobulins and the presence of monoclonal immunoglobulins in serum is a hallmark of the disease. Immunoglobulins play a role in bone loss, but have not been linked with the bone disease in multiple myeloma. In this work, we isolated immunoglobulins from serum of myeloma patients using protein G coupled magnetic beads and columns. We found that immunoglobulins from patients with bone disease (n=16) promoted osteoclast differentiation when added to human monocyte-derived pre-osteoclasts (p=0.006). We next fractionated the immunoglobulin samples by size-exclusion chromatography and found that the "osteoclast promoting activity" was in the high-molecular weight fractions, suggesting that they are in complexes. Since extent of complex formation may be determined by glycosylation, we examined whether there is a difference in immunoglobulin glycosylation between healthy controls and patients, and whether it changes during disease progression. To this end we analysed IgG glycosylation in serum samples from patients (n=72) and age and sex matched controls (n=51). These analyses showed that patient IgG was less galactosylated (p=0.02) and less sialylated (p=0.04) compared with control IgG. Moreover, patients with bone disease (n=43) had significantly less galactose on IgG compared with patients without bone disease (p=0.02, n=33). Supporting this data, we found that galactosidase treatment of immunoglobulins from patients without bone disease induced osteoclastogenesis (p=0.03), whereas addition of galactose to immunoglobulins of patients with bone disease removed their pro-osteoclastogenic effect (p=0.01). Further, the glycosyltransferases ST6GAL1 and B4GALT11, which add sialic acid and galactose to the sugar chain, respectively, are less expressed in plasma cells obtained from patients with bone disease (n=137) compared with those without (n=36, p<0.002, p<0.001, GSE755). Importantly, we observed a significant reduction of IgG glycosylation (p=0.02, n=8) in serum samples obtained from individual patients before and after the onset of bone disease. Taken together, our data support that immunoglobulins promote bone loss in multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Correlations of DKK1 with pathogenesis and prognosis of human multiple myeloma.

Human multiple myeloma (MM) is a kind of common tumor in middle-aged and elderly people, in which the osteolytic lesion is formed mainly through inhibiting osteoblast (OB) differentiation and promoting osteoclast (OC) differentiation. Dickkopf-1 (DKK1) is a soluble Wnt inhibitor, which has an important correlation with the pathogenesis of human MM. Therefore, the correlations of DKK1 with pathogenesis and prognosis of human MM were investigated in this study. The DKK1 expression in tissues and serum of myeloma patients was detected via immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). Correlation between DKK1 expression and survival time of patients was analyzed via Kaplan-Meier analysis. To further study the mechanism of DKK1 expression in pathogenesis and prognosis of human MM, MM cells were treated with DKK1 neutralizing antibody (BHQ880) or transfected with DKK1-small-interfering ribonucleic acid (siRNA) to study its effects on OB differentiation, osteocalcin level, β-catenin and interleukin-6 (IL-6) secretion. Moreover, the effect of DKK1-siRNA transfection on the activity of U266 cells was detected via methyl thiazolyl tetrazolium (MTT) assay. The DKK1 expression in tissues and serum of myeloma patients was significantly higher than that in control group (p< 0.01). In terms of survival time, the median survival time (45months) in patients with low DKK1 expression was significantly longer than that in patients with high DKK1 expression (only 22months). The DKK1 neutralizing antibody (BHQ880) and DKK1-siRNA significantly reduced the DKK1 level in MM cells, promoted the OB differentiation, increased the osteocalcin deposition, promoted the β-catenin expression and decreased the IL-6 expression and β-catenin phosphorylation. DKK1-siRNA could also reduce the proliferative activity of MM cells. DKK1 is closely related to the pathogenesis and prognosis of human MM, which might be a potential biomarker for the diagnosis of MM.

Chondroitin sulfate proteoglycan serglycin influences protein cargo loading and functions of tumor-derived exosomes.

Tumor cells produce and utilize exosomes to promote tumor growth and metastasis. Tumor-cell-derived exosomes deliver cargos that partially mimic the contents of the parent cell to nearby or distant normal or abnormal cells, thereby reprogramming the recipient cells to support tumor progression. Mechanisms by which tumor-derived exosomes subserve the tumor are under intense investigation. Here we demonstrate a critical role of the chondroitin sulfate proteoglycan serglycin in regulating the protein cargo and functions of myeloma cell-derived exosomes. Previous studies have shown that serglycin, the only known intracellular proteoglycan, functions mainly in the storage of basically charged components within the intracellular granules/vesicles via serglycin’s densely clustered, negatively charged glycosaminoglycan chains. Here we demonstrate that serglycin plays a critical role in the protein cargo loading of tumor-derived exosomes. Serglycin was detected in exosomes derived from cell culture supernatants of human myeloma cell lines and serum of myeloma patients. Mass spectrometry analysis of exosomal proteins identified significantly fewer protein components within exosomes derived from serglycin-knockdown myeloma cells than within exosomes from control cells. On gene ontology analysis, exosomes derived from serglycin-knockdown cells, but not from control cells, lacked many proteins that are required for mediating different cellular processes. In functional assays, exosomes from serglycin-knockdown cells failed to induce an invasive phenotype in myeloma cells and failed to promote migration of macrophages. These findings reveal that serglycin plays an important role in maintaining the protein cargo in tumor-derived exosomes and suggest that targeting serglycin may temper the influence of these exosomes on cancer progression.

Semaphorin 4D to suppress bone formation in multiple myeloma.

8039 Background: Myeloma bone disease is characterized by osteoclast activation and long-term osteoblast suppression. We investigated if Semaphorin 4D (Sema4D; CD100) plays a role in these processes. Sema4D has been shown to be a potent osteoblast inhibitor (Negishi-Koga T et al, Nat Med. 2011). A study recently identified that the breast cancer cell line MDA-MB-231 utilizes Sema4D to create osteolysis (Yang Y et al, PLOS One 2016). There have been previous data that Sema4D is increased in the serum of myeloma patients (Terpos et al, Blood 2012) and that co-culturing myeloma cell lines with osteocytes increases the expression of Sema4D mRNA in both (Suvannasankha et al, Blood 2016). We sought to investigate if myeloma cells are using Sema4D to suppress bone formation and if they affect the levels of Sema4D produced by osteoclasts. Methods: We used lentivirus carrying shRNA for Sema4D or control (Scr) to knock down the level of the protein in the 5TGM1 murine myeloma cell line. Knockdown was verified by qPCR and Western Blot. We subsequently co-cultured the 5TGM1 cells with the MC3T3-subclone M4 (MC4) murine stromal cell line for 2 days, removed the myeloma cells and then differentiated the MC4 cells using ascorbic acid and β-glycerolphosphate. At day 5, we analyzed the cells for Runx2 (a critical gene for the differentiation of stromal cells into osteoblasts) expression utilizing qPCR. Also, we performed qPCR in primary osteoclast (OCL) mouse cells differentiating into OCL with RANKL with or without pre-treatment with myeloma-conditioned media for 3 days before the addition of RANKL. Results: When 5TGM1-Scr were co-cultured with MC4 cells the expression of Runx2 on day 5 was decreased (p=0.02). Strikingly, the 5TGM1-shSema4D cells when co-cultured with MC4s did not have the same effect and allowed the upregulation of Runx2 expression on day 5 (p=0.01). Myeloma-conditioned media increased Sema4D expression by OCL throughout the 5 days of differentiation 2 to 3-fold (p=0.01 for day 5). Conclusions: The myeloma cells seem to be utilizing Sema4D both directly and indirectly to inhibit bone formation. Targeted therapy against Sema4D may improve outcomes and fracture-free survival for multiple myeloma patients.

Resistin Induces Multidrug Resistance in Myeloma By Inhibiting Cell Death and Upregulating ABC Transporter Expression

Chemoresistance is a major hurdle in multiple myeloma. Most patients are prone to develop resistance to a wide spectrum of anticancer agents, significantly hampers the patients' long term outcome. Many studies point to bone marrow microenvironment as an important player in myeloma chemoresistance, in which marrow stromal cells and stromal-secreted soluble factors are shown to promote myeloma cell growth and survival. Our previous study has demonstrated that marrow-derived adipocytes protect myeloma cells against chemotherapy-induced apoptosis through adipocyte-secreted adipokines, one of such is leptin. However, the level of leptin expression in myeloma patients is not significantly changed, indicating the involvement of additional adipokines in this process. Interestingly, in a clinical study, an elevation of the adipokine resistin in the serum of myeloma patients after thalidomide treatment were observed as compared with that in patients before treatment, suggesting a potential role of this adipokine in response to chemotherapy. As a 12.5-kDa hormone that is mainly secreted by adipocytes and also secreted by other cells, resistin has a function in production of inflammatory cytokines that are important for cancer development. We thus hypothesized that resistin protects myeloma cells against chemotherapy. In our experiments, human myeloma cell lines and primary myeloma cells isolated from patient bone marrow aspirates were cultured in medium with addition of the recombinant human resistin and chemotherapy drugs melphalan or bortezomib for 24 hours. Cells without resistin served as a control. After cultures, an annexin-V binding assay for assessing apoptosis, western blot analysis for assessing cleavage of caspases and phosphorylation of signaling kinases, and the eFluxx-ID Gold uptake assay for examining ABC transporters activity were performed. In the animal study, myeloma-bearing SCID mice were treated with or without resistin and/or melphalan. Our results showed that resistin treatment reduced melphalan- or bortezomib-induced apoptosis both in vitro and in vivo. This protective effect has been further confirmed by the reduced cleavage of caspase-9, caspase-3, and poly (ADP-ribose) polymerase in myeloma cells. Mechanistic studies showed that culturing myeloma cells with resistin upregulated expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL and downregulated expression of the pro-apoptotic protein Bax via the NF-kB and the PI3K/Akt signaling pathways. Addition of resistin also reduced the intracellular accumulation of eFluxx-ID gold fluorescence in myeloma cells ARP-1 and MM.1S, when compared to that in cells without resistin. In addition, resistin significantly increased the mRNA and protein expression of ATP-binding cassette (ABC) transporters in myeloma cells by downregulating the expression of DNA methyltransferase 1 and 3a, and CpG methylation in the promoters of ABC transporters. Thus, our study demonstrates that resistin is a novel factor contributing to myeloma chemoresistance, and also implicates that disruption of its protective effect can be a potential strategy to improve current chemotherapy in patients and prolong survival. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1710: Bortezomib and lenalidomide resistant myeloma cells overexpress the hepatocyte growth factor/MET signaling axis and respond to MET kinase inhibitors

Abstract Multiple Myeloma (MM) is a plasma cell neoplasm that responds well to therapeutic agents such as bortezomib, a proteasome inhibitor, and lenalidomide, an immunomodulatory drug. However, myeloma patients acquire resistance to these agents and disease relapses. Previously, it has been established that levels of hepatocyte growth factor (HGF) are high in serum of myeloma patients. In concert, our studies in myeloma indicated that HGF gene expression is also high in CD138+ myeloma plasma cells. High HGF levels in serum and in myeloma cells are correlated with poor prognosis and advanced disease. HGF is the ligand for the MET receptor tyrosine kinase that controls proliferation, survival and migration. Our earlier studies demonstrated that MET acts as a survival factor in myeloma cells. We hypothesized that MET/HGF axis may serve as a resistant mechanism in myeloma. To test this, we examined the HGF/MET signaling pathway in bortezomib and lenalidomide resistant myeloma cell lines. These lines were developed by continuous exposure of cells to increasing concentrations of the respective drug. We used three paired cell lines; KAS-6/1 wild type (WT) and bortezomib resistant (KAS-6/V10R) as well as lenalidomide resistant (KAS-6/R10R); WT MM1.S and its lenalidomide resistant counterpart (MM1.R10R), and WT ANBL-6 and its bortezomib resistant (ANBL-6/V10R) pair. Compared to WT cells (KAS-6/1, MM.1S), resistant cell lines overexpress MET at both the protein and mRNA levels. MET is four-fold higher in KAS-6/V10R and KAS-6/R10R compared to KAS-6/1 at both the protein and mRNA level while it is three-fold higher in MM.1/R10R compared to MM.1S at the protein level. On the other hand, compared to WT ANBL-6 cells, the bortezomib resistant cell line ANBL-6/V10R does not overexpress MET but secretes higher (2.6x) levels of HGF into the medium. The overexpression of MET in the resistant cell lines is associated with increased signaling of the HGF/MET pathway at both basal conditions and upon HGF-stimulated activation of the HGF/MET pathway as we observed higher levels of phospho-AKT(S473), phospho-GSK3β(S9) and phospho-ERK1/2 in lenalidomide resistant cell lines compared to WT controls. To test utility of MET kinase inhibitors in bortezomib and lenalidomide resistant cell lines, we used crizotinib, SU11274, ARQ 197 and SGX523, all small-molecule MET kinase inhibitors. Our studies demonstrate that targeted MET kinase inhibition induced apoptosis with concomitant inhibition of growth in these resistant myeloma cell lines. Depending on the potency of the compound, we observed 1 - 90% apoptosis and 15 - 70 % growth inhibition with these inhibitors at 3 μM level. Taken together, out study implies that bortezomib and lenalidomide resistant myeloma may have active HGF/MET axis and MET inhibitors may have therapeutic efficacy for these patients. Citation Format: Shadia Zaman, Christine M. Stellrecht, Robert Z. Orlowski, Varsha Gandhi. Bortezomib and lenalidomide resistant myeloma cells overexpress the hepatocyte growth factor/MET signaling axis and respond to MET kinase inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1710. doi:10.1158/1538-7445.AM2014-1710

Protein Self-Organization Patterns in Dried Serum Reveal Changes in B-Cell Disorders

Detection of serum monoclonal proteins is a common laboratory analysis used in the evaluation of patients with B-cell disorders. Since many individuals with elevated immunoglobulin have no symptoms, it is important to have simple methods for initial screening of patients with suspected B-cell disorders. Samples of serum from healthy donors and from patients with elevated immunoglobulin levels were tested using a technology named Droplet MicroChromatography (DMC). DMC was developed at Artann Laboratories (West Trenton, New Jersey, USA) for the rapid assessment of changes in the composition of serum. DMC is based on the dynamics of the sediment pattern formation during drying of a fluid microdroplet. Results of this pilot study confirm the hypothesis that the pattern formation created by drying droplets of serum would differ between normal samples and those containing monoclonal proteins. Reproducible differences in the patterns formed by the two types of specimens are shown. Strong correlation between abnormally elevated levels of immunoglobulins in the serum of myeloma patients and the patterns formed by drying droplets of serum indicates that the DMC technique may be suitable for semi-quantitative analysis of serum samples. We also demonstrate that computer identification of the drying droplet structure and dynamics is a tractable issue. DMC has significant diagnostic potential and can serve as a basis for development of a simple, rapid, and inexpensive method for initial screening of patients suspected of having multiple myeloma and other pathologies of lymphoid origin that are associated with the overproduction of monoclonal immunoglobulins. The DMC test requires only approximately 1 microL of serum and could therefore be performed in any facility where it is safe to work with serum.

Significance of macrophage inflammatory protein-1 alpha (MIP-1α) in multiple myeloma

Macrophage inflammatory protein-1 alpha (MIP-1α) is a member of the CC chemokine family and is primarily associated with cell adhesion and migration. It is produced by myeloma (MM) cells and directly stimulates osteoclast formation and differentiation in a dose dependent way. MIP-1α protein levels were elevated in the bone marrow plasma of MM patients and correlated with disease stage and activity. MIP-1α was also elevated in the serum of myeloma patients with severe bone disease and correlated positively with bone resorption markers providing evidence for a causal role of MIP-1α in the development of lytic bone lesions in MM. MIP-1α has also been found to stimulate proliferation, migration and survival of plasma cells. Mice, which were inoculated with myeloma cells and treated with a monoclonal rat anti-mouse MIP-1α antibody, showed a reduction of both paraprotein and lytic lesions. In addition, MIP-1α enhanced adhesive interactions between myeloma and marrow stromal cells, increasing the expression of RANKL and IL-6, which further increased bone destruction and tumor burden. Myeloma patients with high MIP-1α serum levels have poor prognosis. The positive correlation between MIP-1α and β2-microglobulin that has been observed in MM patients at diagnosis further supports the notion that MIP-1α is not only a chemokine with osteoclast activity function but is also implicated in myeloma growth and survival. Therefore, MIP-1α pathway may serve as a target for the development of novel anti-myeloma therapies.

Pleitrophin Is Highly Expressed by Myeloma Cells, Elevated in the Serum of Myeloma Patients, and Is a New Autocrine Growth Factor for This B Cell Malignancy.

Pleitrophin (PTN) was originally described as a development regulatory cytokine. This protein regulates the growth of neuroectodermal and mesodermal cell lineages during early embryogenesis but becomes down-regulated during late phase of embryogenesis and shows a very restricted expression pattern in adult neural system. This protein has been recently shown to both induce angiogenesis and bind to syndecan. Thus, we determined whether this protein may be found in tumor cells from myeloma patients and its potential to influence their growth. First, we measured serum levels of PTN in myeloma (n=115) and age-matched controls (n=50) using an ELISA-based technique. PTN levels in the serum of myeloma patients were markedly elevated compared to the normal control group (P<0.02). The serum concentration of PTN in MM patients averaged 28 pg (range, 8 to 110 pg). In contrast, the control serum levels averaged only 12 pg (range, 0 to 20 pg. Interestingly, the PTN concentration in two patients with plasma leukemia was much higher than other multiple myeloma patients. Next, we analyzed the expression of PTN using RT-PCR on RNA from myeloma cell lines (RPMI8226, U266), and multiple myeloma patients' and normal control bone marrow aspirates. Results showed that the PTN mRNA was strongly expressed in multiple myeloma cell lines, myeloma bone marrow samples but not in the normal control bone marrow specimens.In order to determine whether PTN stimulates multiple myeloma growth, we further cloned a whole PTN sense or anti-sense sequencing DNA into the multiple myeloma cell lines RPMI8226 and U266. Cells transduced with sense PTN showed markedly increased proliferation compared to cells transduced with antisense or vector alone. The role of PTN in hematological malignancies has not been previously defined. Due to the restricted expression pattern of PTN in adults, PTN is suggested as a potential new target for treatment of multiple myeloma. Further investigations are defining the prognostic value of PTN serum levels and the mechanism by which this cytokine drives myeloma growth.

Hepatocyte Growth Factor and Its Receptor c-Met in Multiple Myeloma

We have examined whether the hepatocyte growth factor (HGF)/c-met receptor-ligand pair is expressed in freshly isolated and highly purified myeloma cells and whether HGF can be found in the sera of myeloma patients. Myeloma cells were purified with an immunomagnetic method using the syndecan 1-specific antibody B-B4. HGF and c-met mRNA in these cells were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). HGF and c-met proteins were detected by enzyme-linked immunosorbent assay (ELISA) and Western blot, respectively. Serum from 13 myeloma patients was obtained at diagnosis and the levels of HGF were determined by ELISA. HGF and c-met mRNA were expressed in all examined samples (n = 7). HGF was detected in the supernatants of 17 of 20 primary cultures of myeloma cells, whereas bone marrow mononuclear cells from normal controls did not produce detectable amounts of HGF (n = 3). The mean HGF level in serum of myeloma patients at diagnosis was more than fourfold higher than the mean level in normal controls. Possible implications of HGF/c-met expression for the pathophysiology of multiple myeloma are discussed.

Myeloma neuropathy: passive transfer from man to mouse.

Mice were injected daily, for up to 10 weeks, with purified monoclonal immunoglobulin G from patients with myelomatous polyneuropathy or benign gammopathy. The animals developed a demyelinating polyneuropathy with slowed nerve conduction velocities. The putative antinerve factor may be an antibody since injection of Fab fragments from the monoclonal immunoglobulin G produced a similar demyelination. This provides evidence of a circulating factor in the serum of myeloma patients with polyneuropathy that reproduces typical features of the human disease on passive transfer. This disorder is thus distinguished from other neuropathies that occur as remote effects of malignant disease but have no identified pathogenic factors associated with them.

An assay for osteoclast-activating factor (OAF) in biological fluids: Detection of OAF in the serum of myeloma patients

A procedure is described for the partial purification and biological assay of a lymphokine, osteoclast-activating factor, in samples of serum. This procedure involves two steps of ultra-filtration of samples, followed by polyacrylamide gel electrophoresis and bioassay of fractions eluted from the gels. The procedure effectively discriminates between osteoclast-activating factor and other agents with similar biological activities (parathyroid hormone, prostaglandin E 2, and 1, 25-dihydroxyvitamin D 3), when each agent is added to normal human serum. Using this procedure as a specific assay for osteoclast-activating factor, serum from normal individuals and myeloma patients was examined. The activity was detected in 11 of 23 myeloma sera but in none of 12 normal sera.