An inhibitor of the EGF receptor family blocks myeloma cell growth factor activity of HB-EGF and potentiates dexamethasone or anti–IL-6 antibody-induced apoptosis

Abstract We previously found that some myeloma cell lines express the heparin-binding epidermal growth factor–like growth factor (HB-EGF) gene. As the proteoglycan syndecan-1 is an HB-EGF coreceptor as well as a hallmark of plasma cell differentiation and a marker of myeloma cells, we studied the role of HB-EGF on myeloma cell growth. The HB-EGF gene was expressed by bone marrow mononuclear cells in 8 of 8 patients with myeloma, particularly by monocytes and stromal cells, but not by purified primary myeloma cells. Six of 9 myeloma cell lines and 9 of 9 purified primary myeloma cells expressed ErbB1 or ErbB4 genes coding for HB-EGF receptor. In the presence of a low interleukin-6 (IL-6) concentration, HB-EGF stimulated the proliferation of the 6 ErbB1+ or ErbB4+ cell lines, through the phosphatidylinositol 3-kinase/AKT (PI-3K/AKT) pathway. A pan-ErbB inhibitor blocked the myeloma cell growth factor activity and the signaling induced by HB-EGF. This inhibitor induced apoptosis of patients'myeloma cells cultured with their tumor environment. It also increased patients' myeloma cell apoptosis induced by an anti–IL-6 antibody or dexamethasone. The ErbB inhibitor had no effect on the interaction between multiple myeloma cells and stromal cells. It was not toxic for nonmyeloma cells present in patients' bone marrow cultures or for the growth of hematopoietic progenitors. Altogether, these data identify ErbB receptors as putative therapeutic targets in multiple myeloma.

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Sphingolipids As a Novel Target For The Treatment Of Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.3163.3163 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3163-3163 ◽

Cited By ~ 1

Author(s):

Jagadish Kummetha Venkata ◽

Robert K Stuart ◽

Luciano J Costa ◽

Ningfei An ◽

Houjian Cai ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Cell Growth ◽

Cell Lines ◽

Stromal Cells ◽

Myeloma Cell ◽

Sphingolipid Metabolism ◽

Myeloma Cells ◽

S1p Receptors

Abstract Introduction Multiple Myeloma (MM) is the second most common hematological malignancy in the United States and accounts for ∼10,600 deaths annually. MM remains an incurable disease and almost all patients will eventually relapse and become refractory to currently available therapeutic agents. There is an unmet need for better understanding of the disease’s molecular pathways and identifying novel therapeutic targets. Sphingolipid metabolism is being increasingly recognized as a key pathway in cancer biology. In particular, sphingosine kinases (SK1 and SK2) provide a potential site for manipulation of the ceramide / sphingosine 1-phosphate (S1P) rheostat that regulates the balance between tumor cell proliferation and apoptosis, as well as tumor sensitivity to drugs. Currently, very little is known about sphingolipid metabolism in MM. We herein for the first time provide a detailed analysis of sphingolipid metabolism in MM and demonstrate the potential of targeting SK2 for the treatment of MM. Methods We first quantified sphingolipid metabolites and sphingolipid metabolizing genes in myeloma cell lines, in freshly isolated human primary CD138+ myeloma cells, and in a publically available gene expression dataset from MM patients. We then tested the anti-myeloma activity of SK2-specific shRNA and determined the efficacy of a selective SK2 inhibitor (ABC294640) in killing myeloma cell lines and primary human myeloma cells in vitro. The mechanistic pathway of apoptosis was analyzed by immunoblotting and flowcytometry. MM cell lines stably expressing luciferase and eGFP were generated for xenograft experiments and for in vitro co-cultures with stromal cells. Results From the publically available GSE6477 microarray data set, we found that one third of the genes involved in sphingolipid metabolism were significantly different in CD138+ MM cells from newly diagnosed MM patients compared to normal individuals, including SK2 and S1P receptors. In 5 MM cell lines compared to immortalized B cells (IBC), 19 key sphingolipid metabolites were measured, and we found that ceramides were significantly reduced whereas S1P was significantly increased. mRNA analyses of 11 sphingolipid metabolizing genes including S1P receptors in 7 MMs showed that SK1, SK2, and alkaline ceramidases were significantly increased compared to IBC. Furthermore, we isolated CD138+ myeloma cells from 21 MM patients and found that 13 of the patients had higher SK2 expression in CD138+ MM cells compared to CD138-cells. These data demonstrated abnormal sphingolipid metabolism and dys-regulated SK2 in myeloma cells. We generated SK2-specific shRNA and found that SK2 shRNA down-regulated SK2 mRNA, inhibited proliferation, and induced death in myeloma cells, suggesting that SK2 is important in myeloma cell survival. We then tested the efficacy of ABC294640 (the most-advanced, non-lipid SK2 inhibitor) in 6 MM cell lines. ABC294640 inhibited myeloma cell growth with an IC50s of ∼30 μM, including steroid-resistant and doxorubicin-resistant myeloma cells. ABC294640 inhibited MM cell growth as early as 6 hours after exposure and induced apoptotic cell death as demonstrated by Annexin V staining, PARP cleavage and caspase 9 activation. ABC294640 inhibited primary human CD138+MM cells with the same efficacy as with MM cell lines, demonstrating the potential of ABC294640 for the treatment of MM. Additionally, we found that blocking S1P receptors with FTY720 (a S1PR agonist with receptor degradation) induced apoptosis in MM cells. We performed extensive mechanistic and signaling pathway analyses and found that ABC294640 inhibited Mcl-1 and C-Myc expression, but had no effects on Bcl2. Furthermore, ABC294640 induced cell death by directing Mcl-1 to proteosomal degradation. MM is dependent on the bone marrow niche microenvironment for survival and progression. We found that ABC294640 was effective in inducing apoptosis in MM cells even in the presence of stromal cells. Finally, we are currently testing the in vivo effect of ABC294640 alone and in combination with bortezomib, thalidomide and dexamethasone in MM xenograft model transplanted with MM cells stably expressing luciferase. Our early preliminary results were encouraging. Conclusion Our data demonstrate that sphingolipid metabolism is abnormal and provides an attractive target in the treatment of refractory/relapsed MM. Disclosures: Costa: Otsuka: Research Funding.

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Expression of functional interleukin-15 receptor and autocrine production of interleukin-15 as mechanisms of tumor propagation in multiple myeloma

Blood ◽

10.1182/blood.v95.2.610 ◽

2000 ◽

Vol 95 (2) ◽

pp. 610-618 ◽

Cited By ~ 87

Author(s):

Inge Tinhofer ◽

Ingrid Marschitz ◽

Traudl Henn ◽

Alexander Egle ◽

Richard Greil

Keyword(s):

Multiple Myeloma ◽

Cell Survival ◽

Cell Lines ◽

Plasma Cells ◽

Constitutive Expression ◽

Myeloma Cell ◽

Myeloma Cells ◽

Cytotoxic Treatment ◽

Interleukin 15 ◽

Induced Apoptosis

Interleukin-15 (IL-15) induces proliferation and promotes cell survival of human T and B lymphocytes, natural killer cells, and neutrophils. Here we report the constitutive expression of a functional IL-15 receptor (IL-15R) in 6 of 6 myeloma cell lines and in CD38high/CD45low plasma cells belonging to 14 of 14 patients with multiple myeloma. Furthermore, we detected IL-15 transcripts in all 6 myeloma cell lines, and IL-15 protein in 4/6 cell lines and also in the primary plasma cells of 8/14 multiple myeloma patients. Our observations confirm the existence of an autocrine IL-15 loop and point to the potential paracrine stimulation of myeloma cells by IL-15 released from the cellular microenvironment. Blocking autocrine IL-15 in cell lines increased the rate of spontaneous apoptosis, and the degree of this effect was comparable to the pro-apoptotic effect of depleting autocrine IL-6 by antibody targeting. IL-15 was also capable of substituting for autocrine IL-6 in order to promote cell survival and vice versa. In short-term cultures of primary myeloma cells, the addition of IL-15 reduced the percentage of tumor cells spontaneously undergoing apoptosis. Furthermore, IL-15 lowered the responsiveness to Fas-induced apoptosis and to cytotoxic treatment with vincristine and doxorubicin but not with dexamethasone. These data add IL-15 to the list of important factors promoting survival of multiple myeloma cells and demonstrate that it can be produced and be functionally active in an autocrine manner.

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Interleukin-6 is a potent myeloma-cell growth factor in patients with aggressive multiple myeloma

Blood ◽

10.1182/blood.v74.1.11.11 ◽

1989 ◽

Vol 74 (1) ◽

pp. 11-13 ◽

Cited By ~ 182

Author(s):

XG Zhang ◽

B Klein ◽

R Bataille

Keyword(s):

Multiple Myeloma ◽

Growth Factor ◽

Cell Growth ◽

Interleukin 6 ◽

Myeloma Cell ◽

S Phase ◽

Myeloma Cells ◽

Severity Of The Disease

Abstract It has recently been demonstrated that interleukin-6 (IL-6) is a potent myeloma-cell growth factor in the majority of patients with multiple myeloma (MM). Using an anti-bromodeoxyuridine monoclonal antibody (MoAb) to specifically count myeloma cells in the S-phase (ie, labeling index, LI), we demonstrate that the IL-6 responsiveness of myeloma cells in vitro is directly correlated with their LI in vivo. Myeloma cells from all 13 patients with high LIs in vivo (greater than or equal to 1%) responded in vitro to IL-6, the strongest response occurring in cells from five patients with plasma-cell leukemia. In contrast, the cells of only two of eight patients with low myeloma-cell LIs in vivo (less than 1%) responded to IL-6 in vitro. After seven days of culturing with 1,000 U/mL recombinant IL-6 (rIL-6), the median LI value in the first group of patients (in vivo LI greater than or equal to 1%) was 11%, ie 11 times higher (P less than .01) than the median LI value (1%) in the second group of patients (in vivo LI less than 1%). Thus, the in vitro IL-6 responsiveness of myeloma cells is directly related to their in vivo proliferative status, and hence to the severity of the disease.

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PU.1 Is Downregulated in a Subset of Multiple Myeloma by the Methylation of Its -17 kb Upstream Regulatory Region and the Promoter.

Blood ◽

10.1182/blood.v108.11.3417.3417 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3417-3417

Author(s):

Yutaka Okuno ◽

Hiro Tatetsu ◽

Shikiko Ueno ◽

Hiroyuki Hata ◽

Yasuhiro Yamada ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Cell Lines ◽

Promoter Region ◽

Growth Arrest ◽

Myeloma Cell ◽

Upstream Region ◽

Cell Growth Arrest ◽

Myeloma Cells ◽

Expression Levels

Abstract It has been reported that disruption of transcription factors critical for hematopoiesis, such as C/EBPa and AML1, is involved in leukemogenesis. PU.1 is a transcription factor important for both myeloid and lymphoid development. We reported that mice in which the levels of PU.1 were 20% of that of wild-type developed acute myeloid leukemia, T cell lymphoma, and a CLL-like disease. These findings strongly suggest that PU.1 has tumor suppressive activity in multiple hematopoietic lineages. Last year, we reported that PU.1 is downregulated in a majority of multiple myeloma cell lines and and freshly isolated CD138 positive myeloma cells from certain number of myeloma patients, and that tet-off inducible exogenous expression of PU.1 in PU.1 negative myeloma cell lines induced cell growth arrest and apoptosis. Based on their PU.1 expression levels, we divided the myeloma patients into two groups, namely PU.1 high and PU.1 low-to-negative, (cutoff index of 25th percentile of the PU.1 expression level distribution among all patients). The PU.1 low-to-negative patients had a significantly poorer prognosis than the PU.1 high patients. To elucidate the mechanisms of downregulation of PU.1, we performed sequence and epigenetic analysis of the promoter region and the -17 kb upstream region that is conserved among mammalians and important for proper expression of PU.1. There are no mutations in these regions of all five myeloma cell lines. In contrast, the -17 kb upstream region was highly methylated in 3 of 4 PU.1 negative myeloma cell lines, while the promoter region was also methylated to various levels in all five myeloma cell lines including one PU.1 positive cell line. These data suggested that the downregulation of PU.1 in myeloma cell lines might be dependent on the methylation of both regulatory regions of PU.1 gene, especially the -17 kb upstream region. We also evaluated the mechanisms of cell growth arrest and apoptosis of myeloma cell lines induced by PU.1. Among apoptosis-related genes, we identified that TRAIL was upregulated after PU.1 induction. To evaluate the effect of upregulation of TRAIL, we stably introduced siRNA for TRAIL into myeloma cell lines expressing PU.1, and we found that apoptosis of these cells was partially suppressed by siRNA for TRAIL, suggesting that apoptosis of myeloma cells induced by PU.1 might be at least partially due to TRAIL upregulation. We are currently performing DNA microarray analysis to compare the expression levels of genes between before and after PU.1 induction, in order to further elucidate the mechanisms of cell growth arrest and apoptosis.

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Triggering of Toll-Like Receptor-4 In Human Multiple Myeloma Cells Promotes Proliferation and Alters Cell Responses to Immune and Chemotherapy Drug Attack

Blood ◽

10.1182/blood.v116.21.1905.1905 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1905-1905

Author(s):

Zhen Cai ◽

Hanying Bao ◽

Peilin Lu ◽

Lijuan Wang ◽

Donghua He ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Proliferation ◽

T Cell ◽

Tumor Cells ◽

Cell Lines ◽

Myeloma Cell ◽

Myeloma Cells ◽

Response To Chemotherapy ◽

Tlr4 Ligand ◽

Induced Apoptosis

Abstract Abstract 1905 Multiple myeloma (MM) is a fatal plasma cell malignancy mainly localized in the bone marrow. The clonal expansion of tumor cells is associated with the disappearance of normal plasma cells and with a marked depression in the production of normal immunoglobulin (Ig). This makes MM patients highly vulnerable to bacterial, fungal and viral infections and recurrent infections remain to be a major cause of death in MM patients. It has been shown that most primary myeloma cells and cell lines express multiple Toll-like receptors (TLRs). Among them, TLR4 is most frequently expressed. To investigate TLR-initiated responses in MM cells including proliferation, anti-apoptosis and immune escape, we first screened four commonly used human myeloma cell line (HMCL) for the expression of major TLRs by RT-PCR. Surprisingly, all the HMCL expressed multiple TLRs. We also examined primary myeloma cells from 4 patients with MM and our results showed that TLR4 was expressed by all the tumor cells. We incubated myeloma cells with LPS, the natural ligand for TLR4, and found that cell proliferation increased significantly. Targeting TLRs on malignant B cells can induce resistance to chemotherapeutic agents but can also be exploited for combined therapeutic approaches. As mechanisms involved in the resistance to apoptosis play a major role in MM escape to therapies, we sought to determine the capacity of TLR4 ligand to promote the survival of HMCL cells. Myeloma cells were pretreated for four hours with LPS before being induced apoptosis by adriamycin. Results showed that LPS pretreatment partially protected the cells from adriamycin-induced apoptosis. The TLR signaling pathway activates several signaling elements, including NF-kB and ERK/JNK/p38 MAPKs, which regulate many immunologically relevant proteins. Time-dependent MAPK phosphorylation was measured to assess the activation of these kinases upon treatment with LPS in cell lines. ERK1/2, p38, and JNK phosphorylation and NF-kB were significantly up-regulated following LPS treatment. Moreover, our findings demonstrated that LPS-induced cell proliferation was dependent on JNK, ERK and p38 signaling. IL-18, a recently described member of the IL-1 cytokine superfamily, is now recognized as an important regulator of innate and acquired immune responses. In this study, we found that LPS induced IL-18 secretion and activated MAPK and NF-kB signaling simultaneously. Therefore, our results suggest that activation of the MAPK signaling and secretion of IL-18 are interconnected. Tumors evade immune surveillance by multiple mechanisms, including the production of factors such as TGF-β and VEGF, which inhibit and impair tumor-specific T cell immunity. Our study also showed that T cell proliferation induced by allostimulatory cells decreased when the HMCL were pre-treated with LPS. Moreover, immunoregulatory molecules on HMCL, such as B7-H1, B7-H2 and CD40, were upregulated after treatment with LPS, suggesting that TLR4 ligand LPS facilitates tumor cell evasion of the immune system. Our results show that TLRs are functional on myeloma tumor cells, and the ligands to these TLRs have a functional role in affecting myeloma cell proliferation, survival, and response to chemotherapy and immune attacks. Disclosures: No relevant conflicts of interest to declare.

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A Critical Role for PIM2 Kinase in Multiple Myeloma Through NF-κB Activation

Blood ◽

10.1182/blood.v118.21.1839.1839 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1839-1839

Author(s):

Veerendra Munugalavadla ◽

Leanne Berry ◽

Jae Chang ◽

Geoffrey Del Rosario ◽

Jake Drummond ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Cyclin D1 ◽

Cell Lines ◽

Combination Treatment ◽

Single Agent ◽

Myeloma Cell ◽

Myeloma Cells ◽

Pim Kinases ◽

Pi3k Inhibition

Abstract Abstract 1839 The PIM kinases are a family of 3 growth factor- & cytokine-induced proteins hypothesized to have redundant survival and growth functions. Although PIM-1, -2 have been noted as highly expressed in multiple myeloma (MM) (Claudio JO et al., 2002), there are few data to support potential therapeutic utility of PIM inhibition in this indication. Here we show that the myeloma cell lines express all PIM protein isoforms to varying extents, and we describe the properties of a novel pan-PIM inhibitor GNE-652 with picomolar biochemical potency, an excellent selectivity profile, and favorable ADME properties. Myeloma cell lines and patient samples exhibit a striking prevalence of response to GNE-652 (23 of 25 lines with IC50 < 1 micromolar, median < 0.1 micromolar) and synergy in combination with the PI3K inhibitor GDC-0941 (mean combination index values ∼0.2 (n=25)). MM cells respond to this combination with cell cycle arrest and marked apoptosis in vitro. Conversely, a PIM-1, -3 selective inhibitor, GNE-568, failed to suppress MM cell growth and also failed to provide synergy in combination with PI3K inhibition, suggesting PIM-2 is a critical driver of MM cell growth & survival. Additional results suggest that PIM signaling converges on both TORC1 and AKT to generate differential synergies with PI3K/AKT/mTOR pathway inhibitors. PIM has been shown to potentially inactivate PRAS40, a negative regulator of TORC1 (Zhang et al., 2009). We demonstrate that PIM or PI3K inhibition caused a loss of phosphorylation on PRAS40 and resulted in a physical association of PRAS40 and TORC1 and a decrease in phosphorylated p70S6K and S6RP. These reductions were apparent in 7 of 7 cell lines assayed and enhanced by the combination of PI3K and PIM inhibition. Consistent with prior reports (Hammerman et al., 2005), we show that a second node of convergence between PIM and TORC1 is 4E-BP1. Both GDC-0941 and GNE-652 treatments reduced phosphorylation of 4E-BP1 in all the myeloma cell lines tested. Since dephosphorylated 4E-BP1 competes with eIF4G for the mRNA cap binding factor eIF4E, we assayed immunoprecipitates of eIF4E for the presence of eIF4G and 4E-BP1 and observed increased BP1 and decreased 4G. The combination treatment significantly enhanced the loss of 4G relative to either single agent, and importantly, even at 5 × IC50 concentrations for single agents, combination drug treatment achieved greater extent of effect than single agent treatment. It has been hypothesized that a subset of mRNAs are particularly sensitive to inhibition of cap-dependent translation, including a number of oncogenes such as cyclin D1. We noted across 7 different myeloma cell lines, strong decreases in levels of cyclin D1, and D3 that were further decreased by combination treatment of PIM and PI3K inhibition. In summary, we have identified several points at which PIM and PI3K/AKT/mTOR converge to provide synergy in multiple myeloma cell lines. As PIM isoforms are highly expressed in MM cells, we hypothesized that this could be due to proteosomal-mediated stability, and interestingly, MG132 and velcade each stabilized all PIM isoforms. It is commonly known that the JAK/STAT pathway regulates PIM transcription, but we show JAK inhibitors failed to abolish the expression of PIM in myeloma cells, suggesting a role for additional regulators. Recent genome sequencing studies from human myeloma samples (Chapman MA et al., 2011) confirmed the prevalence of NF-kB pathway activation, consistent with prior observations made in MM cell lines (Demchenko YN et al., 2010). The relationship of PIM and NF-kB is controversial in the literature (Hammerman PS et al., 2004 & Zhu N et al., 2002), with some groups placing PIM upstream of NF-kB and others the converse. Using an IκBα inhibitor, BMS-345541, we have examined the role for NF-kB in the regulation of PIM kinases. Here, we show that the BMS-345541 could preferentially suppress PIM2 expression in a dose dependent manner while PIM 1, 3 levels are modestly affected, suggesting that the high levels of PIM2 expression observed are partly driven by deregulation of the NF-kB pathway in MM. In conclusion, we provide pharmacological and biochemical evidence to suggest that PIM2 differentially regulate growth and survival of myeloma cells. Our results provide the rationale for further preclinical development of PIM inhibitors and the basis for a possible clinical development plan in multiple myeloma. Disclosures: Munugalavadla: Genentech: Employment. Berry:Genentech: Employment. Chang:Genentech: Employment. Rosario:Genentech: Employment. Drummond:Genentech: Employment. Du:Genentech: Employment. Fitzgerald:Genentech: Employment. Friedman:Genentech: Employment. Gould:Genentech: Employment. Maecker:Genentech: Employment. Moffat:Genentech: Employment. Slaga:Genentech: Employment. Xiaojing:Genentech: Employment. West:Genentech: Employment. Yu:Genentech: Employment. Ebens:Genentech: Employment.

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Interleukin-6 is a potent myeloma-cell growth factor in patients with aggressive multiple myeloma

Blood ◽

10.1182/blood.v74.1.11.bloodjournal74111 ◽

1989 ◽

Vol 74 (1) ◽

pp. 11-13 ◽

Cited By ~ 3

Author(s):

XG Zhang ◽

B Klein ◽

R Bataille

Keyword(s):

Multiple Myeloma ◽

Growth Factor ◽

Cell Growth ◽

Interleukin 6 ◽

Myeloma Cell ◽

S Phase ◽

Myeloma Cells ◽

Severity Of The Disease

It has recently been demonstrated that interleukin-6 (IL-6) is a potent myeloma-cell growth factor in the majority of patients with multiple myeloma (MM). Using an anti-bromodeoxyuridine monoclonal antibody (MoAb) to specifically count myeloma cells in the S-phase (ie, labeling index, LI), we demonstrate that the IL-6 responsiveness of myeloma cells in vitro is directly correlated with their LI in vivo. Myeloma cells from all 13 patients with high LIs in vivo (greater than or equal to 1%) responded in vitro to IL-6, the strongest response occurring in cells from five patients with plasma-cell leukemia. In contrast, the cells of only two of eight patients with low myeloma-cell LIs in vivo (less than 1%) responded to IL-6 in vitro. After seven days of culturing with 1,000 U/mL recombinant IL-6 (rIL-6), the median LI value in the first group of patients (in vivo LI greater than or equal to 1%) was 11%, ie 11 times higher (P less than .01) than the median LI value (1%) in the second group of patients (in vivo LI less than 1%). Thus, the in vitro IL-6 responsiveness of myeloma cells is directly related to their in vivo proliferative status, and hence to the severity of the disease.

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Paracrine interactions of basic fibroblast growth factor and interleukin-6 in multiple myeloma

Blood ◽

10.1182/blood-2002-09-2907 ◽

2003 ◽

Vol 101 (7) ◽

pp. 2775-2783 ◽

Cited By ~ 85

Author(s):

Guido Bisping ◽

Regine Leo ◽

Doris Wenning ◽

Berno Dankbar ◽

Teresa Padró ◽

...

Keyword(s):

Multiple Myeloma ◽

Growth Factor ◽

Fibroblast Growth Factor ◽

Cell Lines ◽

Stromal Cells ◽

Interleukin 6 ◽

Marrow Stromal Cells ◽

Fibroblast Growth ◽

Myeloma Cells ◽

Stimulation Of

Myeloma cells express basic fibroblast growth factor (bFGF), an angiogenic cytokine triggering marrow neovascularization in multiple myeloma (MM). In solid tumors and some lymphohematopoietic malignancies, angiogenic cytokines have also been shown to stimulate tumor growth via paracrine pathways. Since interleukin-6 (IL-6) is a potent growth and survival factor for myeloma cells, we have studied the effects of bFGF on IL-6 secretion by bone marrow stromal cells (BMSCs) and its potential reverse regulation in myeloma cells. Both myeloma-derived cell lines and myeloma cells isolated from the marrow of MM patients were shown to express and secrete bFGF. Cell-sorting studies identified myeloma cells as the predominant source of bFGF in MM marrow. BMSCs from MM patients and control subjects expressed high-affinity FGF receptors R1 through R4. Stimulation of BMSCs with bFGF induced a time- and dose-dependent increase in IL-6 secretion (median, 2-fold; P < .001), which was completely abrogated by anti-bFGF antibodies. Conversely, stimulation with IL-6 enhanced bFGF expression and secretion by myeloma cell lines (2-fold;P = .02) as well as MM patient cells (up to 3.6-fold; median, 1.5-fold; P = .002). This effect was inhibited by anti–IL-6 antibody. When myeloma cells were cocultured with BMSCs in a noncontact transwell system, both IL-6 and bFGF concentrations in coculture supernatants increased 2- to 3-fold over the sum of basal concentrations in the monoculture controls. The IL-6 increase was again partially, but significantly, inhibited by anti-bFGF. The data demonstrate a paracrine interaction between myeloma and marrow stromal cells triggered by mutual stimulation of bFGF and IL-6.

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Analysis of Intratumor Heterogeneity in Multiple Myeloma Using Methylcellulose Clonogenic Assays.

Blood ◽

10.1182/blood.v104.11.2346.2346 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2346-2346

Author(s):

Bonnie K. Arendt ◽

Raphael Fonseca ◽

Gregory Ahmann ◽

Diane F. Jelinek

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Cell Line ◽

Cell Lines ◽

Growth Regulation ◽

Myeloma Cell ◽

Intratumor Heterogeneity ◽

Parental Cell Line ◽

Myeloma Cells ◽

Igf I

Abstract Multiple myeloma (MM) is a fatal disease characterized by the accumulation of malignant plasma cells in the bone marrow. Although progress has been made in better understanding growth control of this disease, effective treatment of MM patients with this disease is likely complicated by the extensive patient to patient variability that exists, as well variability within the tumor population itself. Thus, there is abundant evidence that intraclonal or intratumor heterogeneity exists in myeloma as revealed by morphologic and phenotypic heterogeneity in primary myeloma cells isolated from a single patient. We have had a long-standing interest in the growth regulation of myeloma cells and have hypothesized, along with other investigators, that there may only be a subset of myeloma cells that exhibits extensive proliferative potential. Understanding how cellular compartments within the malignant clone, as defined by identical immunoglobulin variable region sequence, may vary in growth regulation properties in isolation or in the company of less proliferative tumor cell subsets is key to understanding disease progression and how to better target the putative proliferative subset in myeloma. In this study, we have used a methylcellulose clonogenic assay to study intraclonal heterogeneity in a panel of human MM cell lines. Each of these cell lines, DP-6, KAS-6/1, KP-6, and, exhibit a variable response to IL-6 and IGF-I, and our goal was to evaluate growth responsiveness of individual subclones from each of these cell lines. Myeloma cell lines were plated at a concentration of 200-1000 cells in 1 ml Methocult H4533 in 35 mm gridded dishes with or without various cytokines. Following 3 weeks of culture, colonies were scored and those consisting of >40 cells were isolated, expanded, and studied further. Of interest, subclones isolated from each of the cell lines displayed significant differences in growth response to various cytokines in addition to specific morphologic and phenotypic differences. In this regard, results emerging from the DP-6 cell line were particularly intriguing. We have previously shown that the DP-6 cell line displays a proliferative response to both IL-6 and IGF-I and expresses autocrine IL-6 at a low level. Analysis of the growth properties of individual DP-6 clones revealed the existence of DP-6 cells (clone 1-15) that proliferate at a rapid rate in the apparent absence of exogenous growth factors. Whereas a neutralizing antibody to IL-6 did not inhibit cell growth, addition of a blocking antibody to the IGF-IR, (αIR3), completely blocked growth factor independent proliferation. Phenotypic analysis also displayed variation between the parental cell line and its subclone. For instance, the parental DP-6 cells largely expressed CD45 at a high level, whereas the clone 1-15 did not. Finally, we have also further characterized MM cell line subclones by gene profiling and FISH (fluorescence in situ hybridization) analysis to link specific phenotype and genotypes with patterns of cell growth. These results provide additional evidence that intratumor heterogeneity exists in myeloma. These studies further demonstrate how growth regulation may vary considerably among cellular subsets of the malignant population. Understanding what factors regulate the balance of specific myeloma cell subpopulations is key to an understanding of tumor progression. In summary, these studies provide a necessary foundation for future studies of the growth potential of subsets found in primary MGUS, SMM and MM patient samples.

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Interleukin-10 is a growth factor for human myeloma cells by induction of an oncostatin M autocrine loop

Blood ◽

10.1182/blood.v88.10.3972.bloodjournal88103972 ◽

1996 ◽

Vol 88 (10) ◽

pp. 3972-3986 ◽

Cited By ~ 86

Author(s):

ZJ Gu ◽

V Costes ◽

ZY Lu ◽

XG Zhang ◽

V Pitard ◽

...

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Cell Growth ◽

Cell Lines ◽

Interleukin 10 ◽

Myeloma Cell ◽

Oncostatin M ◽

Autocrine Growth ◽

Myeloma Cells ◽

Autocrine Loop

We have a previously reported that interleukin-10 (IL-10) is a potent but IL-6-unrelated growth factor for freshly explanted myeloma cells (Lu et al, Blood 85:2521, 1995). We have also shown that exogenous IL-10 supported the growth of XG-1 and XG-2 human myeloma cell lines (HMCL) through an IL-6-independent mechanism. (Lu et al, Blood 85:2521, 1995). Because the IL-10 receptor does not involve the gp 130 IL-6 transducer, we have attempted to elucidate the mechanisms of IL-10 action on myeloma cells. Our results indicate that the myeloma cell growth factor activity of IL-10 was abrogated by an antibody to the gp 130 IL-6 transducer, indicating that it was mediated through one of the gp 130-activating cytokines. We found that myeloma cells from XG-1 and XG-2 HMCL and from 5 of 6 patients' tumoral samples produced oncostatin M (OM) constitutively but failed to produce IL-6, IL-11 and leukemia- inhibitory factor (LIF). The autocrine OM was inactive in the absence of IL-10 due to lack of a functional OM receptor on myeloma cells. IL- 10, by inducing the receptor for LIF (LIFR), produced a functional autocrine OM loop in XG-1 and XG-2 cells and in primary myeloma cells from 2 patients. We also found that some myeloma cell lines (XG-4, XG- 6, and XG-7) an fresh myeloma cells from 3 of 6 patients produced an autocrine IL-10 and that these cells constitutively expressed LIFR. One HMCL (XG-7) produced IL-10, OM, and IL-6 an expressed LIFR. The XG-7 cells used OM and IL-6 as autocrine growth factors. We have previously shown that IL-10 could induce IL-11 receptor in myeloma cells and confer on them sensitivity to IL-11 (Lu et al, FEBS Lett 377:515, 1995). Taken together, these results show that IL-10 is a key cytokine for inducing the expression of LIFR and IL-11R and possibly another uncharacterized OM coreceptor on myeloma cells and that OM and IL-10 might be produced by myeloma cells. They also emphasize that all myeloma cell growth factors reported to data involve an activation of the gp130 IL-6 transducer.

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