Regulation Of Myeloma Research Articles

Integrating Gene and Mir Expression Profiles and Regulatory Network Structures to Define Aberrent Feed Forward Loops with Functional and Clinical Implications in Myeloma.

AbstractAbstract 2386Multiple myeloma (MM) is a clonal plasma cell malignancy with a heterogeneous genetic background. Extensive gene expression profile analysis have provided interesting insight into the disease biology and its correlation with clinical outcome; however, we have begun to realize the significant limitations of expression profile data alone. Therefore there is a growing understanding that additional genomic correlates need to be incorporated to develop an integrated oncogenomic analysis. We have here developed and defined multi-gene transcriptional and post-transcriptional feed-forward loop (FFL) These conceptual FFLs consist of a master TF which regulates a miR and together with it controls a set of specific common gene/s. These recurrent and important network motifs form functional nodes in the larger regulatory network, and are considered linchpins of disease causing genomic alterations in cancer and MM in particular. We have developed a comprehensive novel integrative analysis method, dChip-GemiNI (Gene and miRNA Network-based Integration), which combines gene and miR expression profiles, and also incorporates regulatory network structure in the form of computationally identified TF–miRNA FFLs. The dChip-GemiNI method statistically ranks computationally predicted FFLs by their explanatory power to account for differential gene and miRNA expression. We have next applied dChip-GemiNi to a training dataset of 60 MM patients and 5 normal plasma cells (NPCs) with both gene expression (GE) and miR profiles (dataset GSE16558) in order to identify FFLs containing TF-miR-gene networks with loss of negative feedback regulation in MM, supporting the uncontrolled growth, anti-apoptosis and/or other oncogenic effects. We have identified 20 FFLs significantly aberrant between NPC and MM cells. Prominent FFLs involve known MM dysregulated TFs such as MYC, TP53 and Sp1. In addition, we have utilized 3 available myeloma datasets with both miR and GE profiles (GSE16558, GSE17306, GSE17498), and classified MM samples into hyperdiploid MM (HMM) and non-hyperdiploid MM (NHMM) subtype groups by GE profiles at an accuracy >85%. These two groups have different survival outcomes (p-value < 0.01). We have identified 55 FFLs altered between these two MM subtypes. In particular we have observed that the FFL involving CREB1- miR-20a and target genes RRAGD, PIP4K2A, RHOC and CCND2 is altered between HMM and NHMM and is common between the 3 datasets. We have now begun to statistically ranks computationally predicted FFLs and develop a motif score to develop an integrated risk stratification model. In conclusion, FFLs form critical regulatory loops driving the functional behavior of MM cells. Analyzing the molecular impact of FFLs as a unit combining the aggregate impact of TF-miR and the target gene/s in MM will be instrumental in understanding the biology of the disease, developing clinically relevant integrated risk models, and translating basic research into targeted therapy. The ultimate goal is to develop strategies to regulate homeostatic control of these loops and overcome their oncogenic effects that drive the malignant phenotype. Disclosures:Munshi:Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.

Anti-Myeloma Activity of Modified Galectin-9 through JNK and p38 MAPK Pathways.

Abstract 952Galectins constitute a family of animal lectins that show affinity for β-galactosides and share conserved amino acid sequences in their carbohydrate recognition domains (CRDs). Galectin-9 is a tandem-repeat type galectin with two CRDs and has been shown to be crucially involved in a variety cellular functions, such as cell adhesion, cell proliferation, apoptosis and mRNA splicing. Recent studies have indentified anti-cancer properties of galectin-9 against several cancerous diseases through the induction of apoptosis and the prevention of metastasis. In this study, we investigated for the first time the anti-multiple myeloma (MM) activity of a recombinant mutant form of human galectin-9 (hGal9) both in vitro and in vivo, and also examined the potential utility of hGal9 for treatment-resistant MM. hGal9 was synthesized by the Galpharma Research Institute (Kagawa, Japan). While the natural form of galectin-9 having a long linker peptide is a substrate for proteolysis, the deletion of the entire linker region renders hGal9 highly stable against proteolysis. In vitro, hGal9 inhibited the cell proliferation of four myeloma cell lines examined (KMS-12-BM, AMO-1, NCI-H929, IM9), including a bortezomib-low sensitive IM9 subcell line, with IC50 between 75.1 and 280.0 nM. This growth inhibitory effect was mediated by the induction of apoptosis as was demonstrated by the increased number of Annexin V-positive cells with the activation of caspase-8, -9, and -3 in hGal9-treated cells. β-galactoside binding activity is essential in the anti-myeloma activity of hGal9, as the addition of lactose completely abrogated hGal9-mediated growth inhibitory effect on myeloma cell lines and the degree of cell surface binding affinity of hGal9 tends to correlate with IC50 of hGal9 in specific cell lines. To determine the molecular pathway responsible for the anti-myeloma activity of hGal9, we used RNA microarray technology to analyze the changes in gene expression patterns by hGal9. Two MM cell lines, IM9 and KMS-12-BM, were analyzed, and the genes showing more than 2.0-fold changes in their expression level after 6h of hGal9 treatment in both cell lines were assessed. Sixteen genes were upregulated both in KMS-12-BM and IM9, and we focused on c-jun (2.64-fold in IM9 and 6.90-fold in KMS-12-BM) and junD (2.14-fold in IM9 and 3.03-fold in KMS-12-BM), major effector molecules downstream of JNK and p38 MAPK, which are known to be essential for cell survival and death regulation of MM. The upregulation of c-Jun and JunD proteins by hGal9 was also confirmed with Western blotting. We then focused on the relationship between the anti-MM effect of hGal9 and the cross-talk networks of JNK and p38 MAPK signaling pathways. JNK and p38 MPAK phosphorylation by hGal9 were evident in both cell lines, and hGal9 also induced H2AX phosphorylation in myeloma cell lines, suggesting that JNK-H2AX and/or p38-H2AX signaling pathways are involved in the apoptotic induction by hGal9 in myeloma cells. Importantly, the blockade of either the JNK by JNK inhibitor VIII or p38 MAPK pathway by SB203580 partially eliminates the cytotoxic effect of hGal9 on myeloma cells. Taken together, our findings suggest that the anti-myeloma activity of hGal9 at least partly mediates the activation of the JNK and p38 MAPK pathways. hGal9 also induced cell death in patient-derived CD138-positive myeloma cells from ten patients, some with poor risk factors, such as chromosomal deletion of 13q or translocation t(4;14)(p16;q32). Finally, hGal9 potently inhibited the growth of human myeloma cells xenografted in nude mice. Collectively, our study suggest that hGal9 is a potential candidate for a new therapeutic approach for MM that may overcome resistance to conventional chemotherapy. Disclosures:No relevant conflicts of interest to declare.

Correlation of differential expression of NALP7 and NALP11 with glucocorticoid sensitivity in multiple myeloma cell lines

19516 Background: Glucocorticoids (GC) are effective anti-myeloma therapy. However, GC-resistance almost invariably develops. To study the mechanism of GC-resistance in multiple myeloma, we compared the gene expression profiles of the clinically relevant myeloma clones derived from the same patient which are either sensitive (MM.1S) or resistant (MM.1R) to GC-induced apoptosis and growth inhibition. Methods: The MM.1 cell lines were previously described. cDNA microarray analysis was conducted using the GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix). Gene expression of individual genes were analyzed by quantitative real-time PCR using gene- specific primer-probe sets (Applied Biosystems), with relative gene expression level normalized to an internal control (RNaseP). Gene knock- down or overexpression was achieved by transfecting MM.1 cells with gene-specific siRNA (Invitrogen) or cDNA expression construct, respectively. Results: By cDNA microarray analysis, we identified NALP7 and NALP11 among the few genes that showed most significant differential levels of expression between the sensitive and resistant myeloma lines. Both genes belong to the NALP gene family implicated in innate immunity. We confirmed with qPCR that NALP7 expression was significantly decreased whereas NALP11 expression was markedly increased in GC-resistant MM.1 cells compared to the GC-sensitive MM.1 cells. We further showed that both genes are down-regulated by GC treatment. Next, we investigated the biological significance of the NALP genes by altering the NALP gene expression level using transfection. Suppression of NALP7 by siRNA-mediated gene knock-down or exogenous overexpression of NALP11, either alone or in combination, did not alter the GC-sensitivity of MM.1S cells, suggesting that altering NALP7 and NALP11 expression is not sufficient to induce GC resistance. Conclusions: Our studies demonstrated that the differential expression of two NALP family genes, NALP7 and NALP11, correlates with GC sensitivity in myeloma, and that both genes are regulated by glucocorticoid signaling. The biological significance of their differential regulation in myeloma remains to be determined. No significant financial relationships to disclose.

NALP7 and NALP11 Differential Expression Correlates with Glucocorticoid Sensitivity in Multiple Myeloma Cell Lines.

Multiple myeloma is the second most common hematological malignancy and remains incurable. Glucocorticoids (GC) are among the most commonly used therapy for myeloma by inducing growth arrest and apoptosis in myeloma cells. While GC treatments are usually effective at first, all patients will eventually develop resistance. Understanding the mechanism of GC resistance is an important step towards developing new therapy to overcome this significant clinical problem. In order to study the mechanism underlying the GC resistance phenotype in myeloma, we compared the gene expression profiles of the clinically relevant myeloma clones derived from the same patient (MM.1) which are either sensitive or resistant to GC-induced apoptosis and growth inhibition. By cDNA microarray analysis, we identified NALP7 and NALP11 among the few genes that showed most significant differential levels of expression between the sensitive and resistant myeloma lines. Both genes belong to the NALP (NATCH-, LRR- and pyrin domain-containing proteins) family of proteins which have been implicated in the innate immune response. However, their function and regulation in myeloma has never been explored. Using a two-step quantitative real-time RT-PCR analysis, we have demonstrated that the NALP7 expression was significantly reduced whereas the NALP11 expression was markedly increased in GC-resistant MM.1 cells compared to the GC-sensitive MM.1 cells. We then investigated the biological significance of NALP gene differential expression in myeloma GC-resistance. To test whether suppressing NALP7 expression would lead to GC-resistance, we transfected the sensitive MM.1 cells with NALP-7 siRNA which effectively knocked down NALP7 mRNA level by over 80%. The knock-down of NALP-7, however, did not appear to affect the GC sensitivity of the transfected myeloma cells as dexamethasone (dex) treatment effectively caused growth arrest and apoptosis in transfected cells as in the control cells. We have also tested whether suppressing NALP11 expression would overcome the resistance to GC treatment by transfecting the GC-resistant MM.1 cells with NALP11 siRNA. While NALP11 expression was effectively knocked down to less than 20%, the transfected cells remain resistant to GC treatment as in the control cells, suggesting that NALP11 expression is not required to maintain the GC-resistant phenotype. We further investigated the upstream signaling cascades influencing NALP7 and NALP11 expression. Interestingly, dex treatment suppressed the expression of both NALP7 and NALP11 genes in the GC-sensitive MM.1 cells in a time- and dose-dependent manner. Co-treatment with glucocorticoid receptor (GR) antagonist RU486 blocked the down-regulation of NALP7 and NALP11 by dex, suggesting that a functional GR is required to mediate this action. In GC-resistant MM.1 cells which have much reduced levels of wild type GR, NALP7 and NALP11 mRNA levels were not affected by dex treatment, suggesting further that the suppression of NALP7 by GC requires sufficient levels of GR. Our studies demonstrated that the differential expression of two NALP family genes, NALP7 and NALP11, correlates with GC sensitivity in myeloma, and that both genes are regulated by glucocorticoid signaling. The biological significance of their differential regulation in myeloma remains to be determined.

Differential Expression of Type I MAGE in New and Relapsed Multiple Myeloma: Evidence for Association with Proliferation and Progression of Disease.

The type I Melanoma Antigen GEne (MAGE) proteins belong to the Cancer-Testis family of tumor-associated antigens and are found in a broad range of solid and hematologic malignancies. We previously showed that the type I MAGE proteins CT7 (MAGE-C1) and MAGE-A3 were commonly detected in primary myeloma by both RT-PCR and immunohistochemistry (IHC). Higher levels of MAGE protein expression had a positive correlation with abnormally elevated proliferation as measured by the Plasma Cell Proliferation Index (PCPI, percentage of Ki-67+ cells in the CD138+ myeloma cell compartment). These findings suggest that MAGE may play a role in abnormal cell cycle regulation in myeloma. We explored this hypothesis by examining type I MAGE gene expression and proliferation by IHC in 46 newly-diagnosed, untreated and 35 relapsed myeloma patients, based on the clinical observation that relapsed patients exhibit lower response rates to therapy and shorter time to progression, indicative of more aggressive disease. PCPI was significantly higher in relapsed patients (19.0 ± 3.5%) compared to newly diagnosed (6.9 ± 1.3%, p<0.0002). Expression of CT7 and CT10 (MAGE-C2), a type I MAGE not previously associated with myeloma, was stable between newly-diagnosed and relapsed patients (76.0% of new samples vs. 77.1% of relapsed for CT7, 48.5% vs. 50.0% for CT10). In contrast, MAGE-A3 was detected in a significantly greater percentage of relapsed patients (77.1%) compared to newly diagnosed (35.6%, p=0.0003). The link between MAGE expression and unrestricted proliferation was further supported by in vitro studies with human myeloma cell lines. Proliferating myeloma cells were metabolically labeled with the nucleotide analog bromodeoxyuridine (BrdU) followed by intracellular staining and flow cytometry. This assay demonstrated that proliferating myeloma cells that incorporated BrdU into their genomic DNA expressed higher levels of type I MAGE protein compared to non-proliferating cells. Arresting cells at the G1-S interface by double thymidine blockade lead to the accumulation of cells expressing high levels of MAGE, which rapidly entered S phase and incorporated BrdU upon release from block. These results strongly suggest that expression of CT7 and CT10 are relatively early and stable events in the pathogenesis of myeloma, whereas activation of MAGE-A3 expression is associated with disease progression. Furthermore, MAGE expression is correlated with abnormal proliferation in vitro and in vivo, suggesting a potential functional role in the dysregulation of the cell cycle that is a hallmark of this disease. These results support the further exploration of the role of type I MAGE expression in myeloma and the development of therapeutic agents targeting them, especially tumor vaccines.

The survival of IL-6-dependent myeloma cells critically relies on their capability to transit the G 1 to S phase interval of the cell cycle

Interleukin-6 (IL-6) has an essential role in the initial progression of myeloma cell tumours. IL-6 triggers proliferation of these cells via the Ras-mitogen-activated protein kinase (MAPK) cascade and is thought to promote their survival via signal transducer and activator of transcription (STAT) pathway-dependent regulation of Bcl-2 family antiapoptotic members. Using IL-6-dependent murine B9 hybridoma/plasmacytoma cells, we here report that exiting the cell cycle G1 phase is a crucial step contributing to maintain viability. We show that (1) drug-mediated reversible G 1 arrest triggered apoptosis despite the presence of IL-6; (2) a short IL-6 pulse to G 1-arrested cells was sufficient to induce S phase entry and prevent apoptosis; and (3) phorbol ester and related derivatives promoted S phase entry and survival of IL-6-starved cells without up-regulating bcl-X L expression. Furthermore, that the MAPK kinase (MEK) 1/2 inhibitor, U0126, blocked proliferation and induced death of B9 cells indicate that IL-6 may not exert its survival effect primarily through bcl-X L and emphasizes the key role of Ras-MAPK cascade elements in the regulation of myeloma growth/viability.

Interleukin-15 blocks apoptosis and induces proliferation of the human myeloma cell line OH-2 and freshly isolated myeloma cells.

The growth factor-dependent myeloma cell line OH-2, which has previously been shown to be responsive to interleukin (IL)-6, tumour necrosis factor (TNF)-alpha and lymphotoxin, was examined for response to other growth factors. Enhanced proliferation was found in the presence of IL-10, IL-15, IL-2 and insulin growth factor (IGF)-1. Proliferation was strongest in response to IL-6, intermediate and roughly equipotent in response to IL-15, IL-10 and TNF-alpha, and modest in response to IL-2 and IGF-1. IL-15 was synergistic with TNF-alpha, whereas combinations of IL-15 and the other cytokines were merely additive. IL-15-induced proliferation could not be blocked by neutralizing antibody against gp 130, the common transducer chain of IL-6 and related cytokines. IL-15 and IL-6 prevented apoptosis equally well, both better than TNF-alpha, IL-10, and IGF-1. In four out of six samples of purified primary cells, IL-15 and IL-6 induced proliferation. Furthermore, IL-15 mRNA was detected by RT-PCR in most myeloma cell lines and freshly isolated purified patient samples. IL-15 protein was detectable only in one out of about 20 tested cell supernatants from patients and myeloma cell lines. The OH-2 cell line is multi-responsive to cytokines and is a good system for the study of integration of cytokine signal transduction and growth control in myeloma. IL-15 represents a novel modality of growth regulation in myeloma.