miRNA Expression in Multiple Myeloma as Predictive Model of Response to Bortezomib.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4918-4918 ◽  
Author(s):  
Paola Neri ◽  
Kathy Gratton ◽  
Li Ren ◽  
Adnan Mansoor ◽  
Peter Duggan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Mirna Expression ◽  
Response To Therapy ◽  
Differentially Expressed ◽  
Mirna Signature ◽  
Prediction Of Response ◽  
Mirna Profiling

Abstract Abstract 4918 Background Bortezomib therapeutic efficacy is well established in multiple myeloma (MM) however response to this therapy remains difficult to predict with resistant disease observed in nearly 20% of MM patients. Through DNA microarrays, predictive models of response to stem cell transplant and Bortezomib were reported correlating mRNA expression data with disease outcomes and response to therapy. MicroRNAs (miRNAs) are a key class of small, non-coding RNA molecules that modulate post-transcriptional regulation of gene expression and were recently described to be involved in deregulation of gene expression in many cancers including MM. Little evidence however is available concerning the role of miRNA expression in the prediction of response to Bortezomib in MM. We aimed to assess the expression of miRNAs in a panel of Bortezomib highly sensitive and relatively resistant MM cell lines as well as primary MM cells and identify miRNA expression patterns that are associated with response to Bortezomib. Methods We have used miRNA microarrays (Affymetrix miRNA GeneChip) as well as liquid phase Luminex microbead miRNA profiling (Flexmir, Luminex) to profile miRNA expression in MM cell lines (MM1S, KMS11, INA6, U266) and sorted CD138+ bone marrow PCs from MM patients prior to treatment with Bortezomib (n=5; 3 sensitive and 2 resistant) and PCs from a healthy normal donor (n=1). The MM cell lines included in this analysis were classified as sensitive (S) or resistant (R) based on their Bortezomib IC50 at 48 hours (IC50 for MM1S and KMS11 ∼ 5 nM versus INA6 and U266 ∼ 20nM). For the microarray studies the hybridization signal values for the multiple probes for each miRNA were obtained and normalized with the use of miRNA QC tool (Affymetrix) and analyzed using Partek Genomics Suite software. Thereafter, filters were applied to identify the miRNA probes whose normalized signal were at least 2 folds differentially expressed between sensitive (MM1S) and resistant (INA6) cell lines with a P value < 0.05 (ANOVA) and a FDR of 0.05. Bortezomib sensitive (n=3) and resistant (n=2) primary MM samples were subjected to the same miRNA array analysis and filtering. Liquid phase Luminex microbead miRNA profiling (FlexmiR) was used for the confirmation (MM1S and INA6) and validation of the array results in other MM cell lines KMS11 (IC50 5nM) and U266 (IC50 20nM). Results Using Affymetrix miRNA GeneChip we identified 22 differentially expressed miRNA with overexpression of miR-155, miR-342-3p, miR-181a and b, miR-128, miR-20b and downregulation miR-let-7b, miR-let-7i, miR-let-7d, miR-let-7c, miR-222, miR-221, miR-23a, miR-27a and miR-29a in bortezomib relatively resistant (INA6) versus sensitive (MM1S) cell line. These results were confirmed in INA6 and MM1S cells with the use of Luminex microbead miRNA profiling and validated to be similarly differentially expressed between KMS11 (sensitive) and relatively U266 (resistant) cell lines. Furthermore, TargetScan algorithms and Ingenuity Pathway Analysis software were used to identify predicted miRNAs-targeted mRNAs or potentially regulated networks and included genes involved in cell cycle regulation, cell growth, apoptosis and ubiquitin-conjugation pathways. Lastly to further investigate the clinical relevance of miRNAs in MM in terms of prediction of response and outcome to Bortezomib, we correlated miRNA expression profile of sorted CD138+ bone marrow PCs from Bortezomib sensitive (n=3) and resistant (n=2) MM patients with their response to therapy. Unsupervised analysis of the data revealed that the Bortezomib sensitive MM patients clustered with MM1S cell line while resistant patients segregated into the INA6 cluster. Conclusion In summary, we have described a MM miRNA signature, which includes miRNAs that modulate the expression of proteins critical to myeloma pathogenesis and is predictive of response to Bortezomib. Further validation of this miRNA signature in a larger cohort of Bortezomib-treated MM patients is ongoing. Disclosures Stewart: Glaxo-Smith-Kline: Research Funding.

scholarly journals Mir-485-3p and Mir-654-3p Expression in Bone Marrow Mesenchymal Stromal Cells in Patients with Monoclonal Gammopathies Is Related to the Status of the Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3155-3155
Author(s):  
Carlos Fernandez de Larrea ◽  
Tania Diaz ◽  
Alfons Navarro ◽  
Ester Lozano ◽  
Mari-Pau Mena ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Mirna Expression ◽  
Stromal Cells ◽  
Plasma Cells ◽  
Differentially Expressed ◽  
Monoclonal Gammopathies ◽  
The Status

Abstract Background: Crosstalk between malignant plasma cells and surrounding cells in the bone marrow (BM), such as mesenchymal stromal cells (MSCs), endothelial cells and immune cells, is crucial for pathogenesis of multiple myeloma (MM) and in asymptomatic monoclonal gammopathies. In these diseases, microRNAs (miRNAs) could be useful as biomarkers for diagnosis, prognosis and evaluation of treatment response. miRNAs can be released to the serum and transferred among MM cells and BM-MSCs as cell-cell communication. Previously, we have showed a serum 14-miRNA signature associated with complete remission (CR) after autologous stem-cell transplantation (ASCT). In this sense, patients in CR with partial recovery of two normal serum miRNA levels, similar to those with monoclonal gammopathy of undetermined significance (MGUS), was associated with better prognosis. The aim of this study was to analyze the miRNAs profile in mesenchymal stromal cells derived from bone marrow of patients with multiple myeloma in different status of the disease, comparing with MGUS controls. Methods: We analyzed samples from 95 patients with MGUS (N=23), MM at diagnosis (N=14), relapsed/refractory MM (N=14), MM in partial response (PR) or very good partial response (VGPR) (N=15), MM in CR (N=24) and healthy donors (N=5). Mononuclear cells from BM samples were cultured in DMEM containing 10% FBS. After a week, non-adherent cells were removed, whereas BM-MSCs were selected by their adherence to the plastic and their phenotype was confirmed by multiparametric flow cytometry. In a first screening phase, we analyzed 670 microRNAs in 20 primary BM-MSC from patients with MGUS (N=4), symptomatic MM (N=8) and MM in CR (N=8). miRNAs differentially expressed were identified according to a supervised analysis using significance analysis of microarrays (SAM) and Student's t-test based on multivariate permutation (with random variance model). miRNAs differentially expressed between groups of patients were validated in the whole cohort of BM-MSC from patients. Paired malignant plasma cells (CD38+) miRNA expression from patients with symptomatic MM as well as miRNA in serum samples paired with BM-MSC samples were also compared. RmiR package was used to identify miRNA targets, cross-correlating the miRNA expression data from the present study with our findings on the gene expression signature (Affymetrix Human Genome U219 array) in 12 BM-MSCs from patients (4 MGUS, 4 symptomatic MM and 4 in CR), based on the predicted targets from TargetScan and miRBase databases. Results: In the screening phase, we identified a miRNA profile of 10 miRNAs (miR-663b, miR-654-3p, miR-206, miR-411*, miR-885-5p, miR-668, miR-638, miR-485-3p, miR-744* and miR-199a) differentially expressed between patients with symptomatic MM and MM in CR (adjusted p-value <0.0001). In the validation phase, miR-485-3p and miR-654-3p resulted differentially expressed in the three groups of patients: MGUS, symptomatic MM and patients in CR (ANOVA test: p=0.0101 and p=0.0228, respectively). The levels of these miRNAs were significantly decreased in patients with MM than in those with MGUS, and these levels seemed to recover when patients achieved CR. These two miRNAs (miR-485-3p and miR-654-3p) were also correlated with all degrees of response in MM and with asymptomatic gammopathies (ANOVA test: p=0.0154 and p=0.0487, respectively). Moreover, paired cross-correlation among these two miRNAs expression with our results in mRNA gene expression profile data showed 324 for miR-485-3p and 265 for miR-654-3p genes (correlation index < -0.8) (Figure 1A and 1B). miR-485-3p and miR-654-3p showed a higher expression in BM-MSC than in MM CD38+ cells, suggesting MSC as cell of origin for these miRNAs. Serum expression of these two miRNAs was concordant with the observed in BM-MSC, with higher in patients in CR and MGUS than in those with symptomatic MM (Figure 1C and 1D). miRNA expression in BM-MSC supernatant as well as the identification of the biological role and validation of the miRNA targets are ongoing. Conclusion: miR-485-3p and miR-654-3p expression in mesenchymal stromal cells from bone marrow in patients with multiple myeloma and asymptomatic monoclonal gammopathies is related to the status of the disease and the response to treatment. These miRNAs are also expressed in serum, resulting in potential biomarkers for disease activity and risk of progression. Disclosures Rosinol: Janssen, Celgene, Amgen, Takeda: Honoraria. Bladé:Janssen: Honoraria.

C-met Receptor as a Potential Target for the Treatment of Patients with Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3395-3395
Author(s):  
Marcin Majka ◽  
Artur Jurczyszyn ◽  
Anna Zebzda ◽  
Wojciech Czogala ◽  
Ewa Lesko ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Protein Level ◽  
Bone Marrow Cells ◽  
Response To Therapy ◽  
Poor Responders ◽  
Met Receptor ◽  
Potential Target ◽  
Marrow Cells

Abstract Despite progress in the treatment of Multiple Myeloma (MM), it is still an incurable disease with average survival of 3–4 years. Because MM is often resistant to conventional therapies, new treatment strategies are necessary. The presence of elevated HGF (Hepatocytic Grow Factor) expression has been well documented in multiple myeloma. The c-met oncogene has been shown to be present in MM cell lines at the mRNA and protein level. Some data suggested that this axis could be responsible for proliferation and inhibition of apoptosis in MM cells. In this study we have analyzed c-met expression in 15 patients with (MM) before and after treatment. Seven of these pts responded well and eight pts responded poorly to the employed therapy. All 15 pts were c-met positive before therapy. Bone marrow cellularity of patients who responded well was 76% before (range: 10% – 100%) and 46% after treatment (range: 40% – 60%). In this group plasmocyte infiltration of bone marrow consisted of 59% before (range: 10% – 80%) and 9% after chemotherapy (range: 0% – 20%). Five of them had undetectable c-met positive cells among bone marrow cells after treatment. In the group of poor responders cellularity of bone marrow was 40% (range: 20% – 70%) before treatment and 46% (range: 20% – 70%) after therapy. Plasmocytes consisted of 20% (range: 10% – 50%) of bone marrow cells before and 44% (range: 10% – 90%) after treatment. All patients in this group had cells positive for c-met receptor after therapeutic regiment. This results suggested that c-met-HGF axis might be a good target for alternative therapy in MM. We looked for potential therapeutics that interferes with this axis and we found that geldanamycin (GA) has been shown to decrease expression of c-met at the protein level in several different cell types. Using inhibitors that belongs to geldanamycin family (GA, 17AAG and 17DMAG) we treated MM cell lines and primary sample. We found that these molecules strongly inhibited expression of c-met in both MM cell lines and patients sample as assessed by western blot analysis. We also tested the influence of these inhibitors on proliferation of MM cells. We found that 100nM dose of GA and 17DMAG inhibited growth of MM cell lines by 80% and 100nM dose of 17AAG inhibited growth of these cells by 20%. Primary cells were more resistant to treatment but we still obtained 30% inhibition with GA and 17DMAG. 17AAG was ineffective and proliferation decreased by less than 10%. Grow inhibition was probably not only due to c-met-HGF axis blockade because these molecules also inhibit other proteins (AKT, RAF). In our experiments we have shown that the level of c-met expression correlates with response to therapy. Patients who respond well had substantially decreased number of c-met positive plasmocytes after chemotherapy in comparison to poor responders. We have also showed that drugs that block c-met-HGF axis could be used in treatment of MM. These drugs could potentially inhibit cells proliferation, increase apoptosis and disrupt MM cells interaction with bone marrow environment. Based on these data we postulate that the c-met receptor is a potential target for MM therapy especially in patients who do not respond to the first line of treatment.

Junctional Adhesion Molecule-A/ F11 Receptor (JAM-A/ F11R) Expression in Multiple Myeloma (MM): a Candidate Biomarker of Aggressive Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2830-2830
Author(s):  
Bani M Azari ◽  
Marc J. Braunstein ◽  
H. Uwe Kluppelberg ◽  
Sadeaqua S Scott ◽  
Eric LP Smith ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Tumor Cells ◽  
Cell Lines ◽  
Adhesion Molecule ◽  
Mrna Levels

Abstract Abstract 2830 Poster Board II-806 Background: Multiple myeloma (MM) is an incurable disease of clonal plasma cells that accumulate in the bone marrow (BM), causing monoclonal IG production, bone marrow failure, osteolytic lesions and kidney disease. Although initially treatable, MM ultimately becomes refractory to treatment, and is invariably fatal, when tumor cells that harbor genetic mutations expand without regulation. Therefore novel treatment targets need to be identified. A key mechanism in MM pathogenesis is regulation of tumor growth by the bone marrow (BM) microenvironment, particularly by bone marrow neo-vascularization and adhesion of tumor cells to the marrow stroma. Aberrantly expressed genes that regulate angiogenesis by MM cells enhance MM progression and constitute targets in its treatment. JAM-A/F11R is an endothelial cell (EC) adhesion molecule of the immunoglobulin superfamily which is a multifunctional cell membrane protein that mediates intracellular signaling events that alter EC migration and paracellular permeability. For example, in breast cancer, attenuation of JAM-A increases tumor invasion and metastasis through a decrease in tumor adhesion (Ulas Naik Cell Adh Migr. 2008 Oct;2(4):249-51.). In this study we explored the JAM-A/F11R expression in MM tumor cells and in patients to determine the potential role of this molecule in the pathogenesis and progression of MM. Methods: The MM cell lines examined were RPMI-8266, U266, and NCI-H929. Human umbilical vein endothelial cells (HUVECs) served as controls. Informed consent was obtained from patients and control subjects. Primary BM tumor cells were enriched to > 95% CD138+ cells by positive selection using anti-CD138 MACS MicroBeads. The CD138-negative fraction was used for outgrowth of confluent EPCs (> 98% vWF/CD133/KDR+). JAM-A mRNA expression was assessed using an microarray gene expression profile, JAM-A probe based real-time PCR, and JAM-A levels in each sample were measure using a standard curve and normalized to GADPH. JAM-A protein levels in MM cell lines and primary tumor cells were measured by flow cytometry and immunofluorescence. For serum studies, peripheral blood was obtained from 25 newly diagnosed MM patients and 8 healthy, age- and sex-matched controls, and JAM-A levels were measured using an ELISA. Statistical analysis was performed using Student's t-test, two-tailed, with P ' .05 considered significant. Results: JAM-A mRNA levels were significantly increased in MM cell lines RPMI-8266, U266, and NCI-H929 compared to HUVECs (U266, P = 3×10-5; RPM1-8266, P = 1×10-6; NC1-H929, P= 5×10-4). The JAM-A mRNA levels were significantly greater in RPMI-8226; P < .04 compared to TNFα-activated HUVECs for 24 hours which is a proangiogenic switch for HUVEC gene expression. The elevated mRNA expression of the JAM-A in MM cell lines was confirmed by immunofluorescence and flow cytometry which showed the presence of both membrane and cytoplasmic JAM-A protein. Microarray analysis of gene expression profiles from 20 patients' corresponding tumor cells and microenvironmental EPCs showed that JAM-A had a higher level of expression in tumor cells versus MM EPC by 12.62 fold, (P=.0000642). Furthermore, JAM-A had a higher level of expression in MM EPC versus normal control EPC by 2.41 fold, (P=.00113) reflecting a complex regulatory role of F11 signaling in MM, similar to breast cancer (Naik, U. et al 2008). JAM-A was also found to be 12.6 fold greater in tumor cells compared to EPCS (P=.0000642). In addition, circulating levels of soluble JAM-A were found to be significantly greater in the serum of MM patients compared to controls (P < .005), with an average 2-fold increase. Serum levels of JAM-A in MM patients also decreased 71% with treatment n=5, P<.05. Conclusion: We show for the first time that JAM-A expression is highly elevated in MM tumor cells and its levels respond to treatment. In addition, MM patients have higher circulating JAM-A levels compared to healthy individuals and circulating JAM-A levels were reduced following treatment, suggesting that JAM-A may serve as a novel biomarker in MM. Current studies in the lab are aimed at correlating these levels with clinical parameters to determine whether JAM-A levels reflect disease severity and response to treatment. Results of these analyses, as well as results of ongoing experiments using JAM-A siRNA and antibody-inhibition approaches to target JAM-A in myeloma tumor and ECs will be presented. Disclosures: No relevant conflicts of interest to declare.

Characterization of Mesenchymal Stem Cells Abnormalities in Multiple Myeloma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 511-511 ◽  
Author(s):  
Philippe Bourin ◽  
Jill Corre ◽  
Karène Mahtouk ◽  
Mélanie Gadelorge ◽  
Patrick Laharrague ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Plasma Cells ◽  
Osteoblastic Differentiation ◽  
Differentially Expressed ◽  
Matrix Mineralization ◽  
Protein Levels

Abstract Introduction: The bone marrow microenvironnement (BMMe) play a significant role in the physiopathology of the multiple myeloma (MM). However, its abnormality still remains controversial. To address this question, we studied bone marrow mesenchymal stem cells (MSCs), the only long-lived cells of the BMMe. We compared, at a genomic and functional level, the MSCs isolated from patients with MM, to MSCs isolated from healthy subjects and those with monoclonal gammopathy of unknown significance (MGUS). Material and methods : Bone marrow samples from 26 MM patients, 7 MGUS patients and 11 healthy individuals were compared. The MSCs were selected by their adherence on plastic and were cultured in alpha-MEM medium + 10% SVF and antibiotics during 2 passages (primo-culture = P0 and first passage = P1). The gene expression profiling was carried out by Affymetrix GeneChip microarrays (U133 plus 2.0). The expression of interesting differentially expressed genes was validated by ELISA or qRT-PCR. The phenotype was studied by flow cytometry (CD45, CD90, CD73, CD13, CD14). The CFU-F frequencies in BM samples and in cell suspensions after P0 and P1 were studied as well as the cell productions after P0 and P1. The osteoblastic differentiation was evaluated both by alkaline phosphatase dosing and matrix mineralization quantification. We also carried out co-cultures of the MSCs with CD34+ cells to quantify their hematopoietic supportive potential. Finally XG1 and Molp-6, respectively stroma independent and stroma dependent cell lines, were co-cultured with MSCs to check the capacity of the MSCs to support malignant plasma cell growth. Results: Gene expression profile independently classified the MSCs in a normal and in a MM group. MGUS MSCs were interspersed between those 2 groups. 145 distinct genes were differentially expressed in MM and normal MSCs. Among them, 46% could be involved in tumor-microenvironment cross-talk. Known soluble factors involved in MM physiopathologic features, such as IL-6, IL-1ß, DKK1 and amphiregulin, were identified and new ones found. In particular growth and differentiation factor-15 (GDF-15), already described as a accurate biomarker of numerous tumours, was significantly overexpressed (p&lt;0.001) in MM MSCs both at mRNA and protein levels (183.5 ± 64.9 vs 749 ± 90.9 for mRNA, 1 10−4 pg/cell ± 1.9 10−5 pg/cell vs 4.3 10−4 pg/cell ± 1.4 10−4 pg/cell for protein respectively for normal and MM MSCs). It was also able to induce dose-dependant growth of Molp-6, in the absence of a supportive stroma. The phenotype and the CFU-F frequencies and the cell productions were similar in the 3 groups of MSCs and their hematopoietic supportive capacity was maintained. The MM MSCs complete differentiation towards the osteoblastic lineage, evaluated quantitatively, was faded. And very importantly, MM MSCs constituted a better supportive feeder layer for the Molp-6 cell line as compared to normal MSCs (cell expansion after 7 days : 2.1 ± 0.3 vs 3.3 ± 0.4, p = 0.04, respectively for normal and MM MSCs). On the other hand, the growth of XG1 was not influence by the subject origin of the MSCs. Conclusion: Our results show that the MSCs, like the malignant plasma cells, are abnormal in MM. This confirms the place of microenvironnement in the physiopathology of the MM and makes it possible to identify new potential therapeutic targets.

The Location of Multiple Myeloma Adhesion Variants in Diverse Niches within the Bone Marrow Affects Plasma Cells Enumeration.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5063-5063
Author(s):  
Liat Nadav ◽  
Ben-Zion Katz ◽  
Shoshana Baron ◽  
Lydia Lydia ◽  
Aaron Polliack ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Gene Expression Profile ◽  
Plasma Cell ◽  
Expression Profile ◽  
Plasma Cells ◽  
Response To Therapy ◽  
Morphological Evaluation

Abstract Background - The diagnosis of multiple myeloma (MM) is based on clinical and laboratory criteria combined with bone marrow (BM) plasmocytosis, estimated by inspection of bone marrow aspirates. Recent advances in flow-cytometry (FCM) have provided an additional tool for the diagnosis of MM and for monitoring response to therapy. However, significant discrepancy has been reported regarding the enumeration of plasma cells in marrow samples of MM patients using these two methods. Aims - In this study we compared the bone marrow plasmocytosis by microscopic examination of BM aspirates, to the flow cytometry results in samples obtained form MM patients. We tested whether the noted discrepancy between these two methods applies only to MM, or represents a trend in other hematopoietic malignancies as well. We defined this discrepancy and explained it. Methods - The number of plasma cells or blasts from BM aspirates of 41 MM or seven acute myeloid leukemia (AML) patients respectively were analyzed simultaneously by morphological evaluation and by FCM. Each sample was assessed independently by two qualified laboratory specialists and/or hemato-pathologist. In MM we found plasma cell fractions that were characterized by FCM and gene expression profile. Results - In MM it was evident that FCM under-estimated the number of BM plasma cells samples by an average of 60%, compared with conventional morphological evaluation. On the other hand in AML there was a good correlation between the morphological and FCM assessments of the blast cell population, indicating that the discrepancy observed in the MM BM samples may be related to unique characteristics of the malignant plasma cells. Since flow cytometry is performed on the bone marrow fluid which is depleted of fat tissue-adhesive plasma cells, we disrupted spicules from MM BM samples (by repeated passages through 21g needle) and found a 40% increase in plasma cell compared with the fluid of the same BM samples. In order to determine the FCM profile of the cells in these two fractions, we isolated BM derived spicules from aspirates of MM patients and treated them with extracellular matrix (ECM) degrading enzymes followed by mechanical shearing. This combination released the highly adhesive plasma cells from the spicules. The released myeloma cells displayed a different FCM profile and in particular had a higher level of CD138 expression. Gene expression profile, which was performed on similar adhesion variants of cultured MM cells, demonstrated distinct oncogenic and transcriptional programs. Summary - We have shown a major discrepancy between the percentage of MM cells obtained by routine BM morphology and flow cytometry counts. It is possible that this discrepancy is partially attributable to the two distinct microenvironmental components occupied by MM cells in the BM sample - the lipid spicules, and the fluid phase. MM cells located in different niches of the BM also differ in their FCM and gene expression profile. This study indicates that multiple myeloma patients contain heterogeneous populations of malignant plasma cells. These sub-populations may play distinct roles in the biological and clinical manifestations of the disease and differ in their response to anti-myeloma therapy.

Survivin in Multiple Myeloma: Prognostic and Therapeutic Implications

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 137-137
Author(s):  
Verena Wagner ◽  
Dirk Hose ◽  
Anja Seckinger ◽  
Ludmila Weitz ◽  
Tobias Meißner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Stress Response ◽  
Cell Lines ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Survivin Expression ◽  
Marrow Stromal Cells ◽  
Er Stress Response

Abstract Abstract 137 The IAP (inhibitor-of-apoptosis) family member, survivin, is one of the most significantly over-expressed genes in malignant cells. Survivin has been reported to inhibit apoptosis and regulate mitosis as well as cytokinesis. We therefore first determined the expression of survivin in CD138-purified multiple myeloma (MM) cells from previously untreated patients at our centers' trial group (TG) (n=246) and the UAMS Arkansas (n=345) validation group (VG). Using the PANP algorithm, survivin is aberrantly expressed in 27% (TG) of MM cell samples. It is not expressed in normal bone marrow plasma cell samples (n=7) while expression increases significantly from MM stage I-III (P<.001). Survivin expression correlates with proliferation as assessed by gene expression- (r=.8, P<.001) or propidium iodide (r=.7, P<.001). Presence of survivin expression correlates with inferior event-free and overall survival in patients undergoing high-dose chemotherapy in the TG (22.6 vs. 35.4 months, P<.001, 52.9 vs. n.r., P=.002) as well as in the VG (12.3 vs. 54.1 months, P<.001, and 17.4 vs. n.r., respectively). These results support the further evaluation of survivin as a therapeutic target in MM. We next assessed the effects of siRNA-mediated knock-down of survivin in vitro. Suppression of survivin by siRNA induced cell cycle arrest and apoptosis in MM cell lines. The small molecule suppressant of survivin, YM155, is currently in clinical development for the treatment of solid tumors. Here, we investigated YM155 for its anti-MM activity. YM155 abrogated proliferation and induced apoptosis in a panel of 10 human MM cell lines and MM cells isolated from multidrug-resistant patients at an IC50 of 4–50nM while the IC50 was not reached in primary bone marrow stromal cells up to 500nM. YM155 was also able to overcome the protective effect of IL-6, IGF-1 and the presence of bone marrow stromal cells, respectively. The induction of apoptosis by YM155 closely correlated with down-regulation of intracellular survivin protein expression within 24 to 36h of treatment with 50–100nM of YM155. However, inhibition of cell proliferation is already detectable at 12h at 5–10nM, suggesting two different dose- and time-dependent mechanisms of action. We therefore performed gene expression and protein profiling on YM155-treated MM cells. Strikingly, these data revealed early up-regulation of the ER stress response (PERK, phospho-eIF2a, ATF4, ATF3) followed by increased CHOP expression and a profound abrogation of proliferation. This appeared to be independent of cellular survivin levels, indicating that the early proliferation arrest at very low nanomolar concentrations is mediated primarily by the ER stress response. Moreover, gene signatures regulated by the IL-6/STAT pathway (CCND1, BCL2L1, MCL1, BIRC5A) were markedly altered upon YM155 treatment. Importantly, IL-6 profoundly sensitized IL-6 responsive MM cell lines to treatment with YM155. We therefore hypothesized that YM155 might abrogate upstream regulatory signaling pathways of survivin expression. Indeed, YM155 abrogated constitutive as well as IL-6 induced phosphorylation of STAT3, an important transcription factor for survivin expression in MM cells. In contrast, phosphorylation of ERK1/2 and AKT remained unchanged. Dephosphorylation of STAT3 closely correlated with the loss of intracellular survivin. In conclusion, we have demonstrated the prognostic significance of survivin expression and a potential therapeutic role for the small molecule suppressant of survivin YM155 in MM. Disclosures: No relevant conflicts of interest to declare.

A miRNA Risk Score for the Prediction of Response to Lenalidomide in Multiple Myeloma (MM) Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 987-987 ◽  
Author(s):  
Paola Neri ◽  
Andrew R Belch ◽  
Jordan Johnson ◽  
Kathy J Gratton ◽  
Li Ren ◽  
...  
Keyword(s):  
Risk Score ◽  
Mirna Expression ◽  
Research Funding ◽  
Plasma Cells ◽  
Expression Profiles ◽  
Response To Therapy ◽  
Differentially Expressed ◽  
P Value ◽  
Prediction Of Response ◽  
Mirnas Expression

Abstract Abstract 987 Background: Lenalidomide has demonstrated clinical activity in patients with newly diagnosed or relapsed MM, however nearly a third of relapsed patients fail to respond to it. While preclinical studies have reported variable biomarkers and pathways (Wnt-GSK3b-beta catenin; eIF4E-C/EBPb-IRF4; Cul4A-DDB1-Cereblon) as mediating the anti-MM effects of IMiDs, a comprehensive risk score for prediction of response to Lenalidomide is lacking. MiRNA are highly preserved non-coding RNAs that act post-transcriptionally to regulate gene expression by binding to the 3'UTR of mRNAs. To date studies have reported selective miRNAs expression in plasma cells at different stages of the clonal disease progression (MGUS to MM), correlated miRNA expression with distinct MM molecular subgroups and demonstrated a genome-wide elevated expression of miRNAs in high-risk MM. Herein, we have conducted a comprehensive profiling of miRNA and mRNA expression in Lenalidomide treated MM patients and established a miRNA-based risk score that is predictive of response to therapy. Methods and results: We have postulated that a miRNA signature in MM is predictive of response to Lenalidomide based therapy. To test this hypothesis, we analyzed in a testing cohort (n=20) the miRNA and mRNA signatures of Lenalidomide sensitive “S” and resistant “R” MM patients. In order to account for the role of the bone marrow environment in this disease, RNAs were extracted from 1mm punched biopsies of non-sorted and plasma cells enriched areas of the bone marrows collected immediately prior to initiating Lenalidomide. MiRNAs were hybridized to the miRNA Affymetrix gene-chip and raw miRNA expression values were log2 transformed and normalized (miRNA-QC tool, Affymetrix). Comparison of normalized miRNAs expression in “S” versus “R” patients (Anova testing) identified 29 differentially expressed miRNAs (Fold change < −2 or > 2 with a p value and FDR <0.01) between these two groups. In order to establish a risk score (RS) for prediction of response to Lenalidomide, we performed a stepwise canonical discriminant analysis with response to Lenalidomide as grouping variable and the 29 differentially expressed miRNA as independent variables. The discriminant analysis identified a RS based on the log2-scale expression of 4 miRNAs using the following equation: RS= ((1.068*hsa-miR-21) + (1.367*hsa-miR-26b) +(1.761* has-miR-3147)+(3.523*has-miR-34a) – 36.692). This univariate summary (ie, RS) of the miRNA expression profiles for each patient enabled accurate (100%) prediction of response to Lenalidomide. All patients with a RS < 0 were sensitive to Lenalidomide with a mPFS and mOS of 21.4 and 39.5 months respectively. In contrast, all patients with a RS > 0 were resistant to Lenalidomide with a mPFS and mOS of 4.1 and 24 months respectively. Validation of this RS was also performed in an independent cohort (n=20) of MM patients treated with lenalidomide. In this validation cohort the miRNA RS accurately predicted response to therapy in 90% of the cases. The mPFS and mOS were 32.0 and 43.1 months in patients with a RS < 0 (predicted as responders) as opposed to a mPFS and mOS of 6.6 and 7.6 months in patients with a RS > 0 (predicted as non-responders) (Figure below). mRNA profiling (U133A Plus2 array chip) was also performed on the plasma cells enriched bone marrow sections with 554 genes identified as differentially expressed (Fold change < −2 or > 2 with a p value and FDR <0.005) between Lenalidomide S and R patients. Using the TargetScan miRNA target mRNA prediction tool, combinatory analysis of miRNA and mRNA expression profiles of these MM patients identified positive and negative correlations (p<0.05) between differentially expressed miRNA and mRNAs. Lastly in a multivariate Cox regression analysis that included ISS stage, FISH cytogenetics ((del17p and t(4;14)) and the 4 miRNA RS, these variables were independent predictors of survival post Lenalidomide based therapy. Conclusion: We believe that this miRNA Risk Score provides a robust method of predicting sensitivity or resistance to Lenalidomide in MM patients and warrants further validation in a larger prospective study. The biological functions of these 4 miRNAs and their regulation of MM cells sensitivity to Lenalidomide is currently being investigated in vitro in a library of MM cell lines. Disclosures: Neri: Celgene: Honoraria, Research Funding. Belch:Celgene: Research Funding; Onyx: Research Funding. Bahlis:Celgene: Honoraria, Speakers Bureau.

scholarly journals The Purine Metabolic Enzyme AMPD1 Is a Novel Therapeutic Target for Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5614-5614
Author(s):  
Yawara Kawano ◽  
Yuki Inada ◽  
Takayuki Sasano ◽  
Nao Nishimura ◽  
Hiroyuki Hata ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Plasma Cells ◽  
Hypoxic Condition ◽  
Myeloma Cell ◽  
Novel Agents ◽  
Metabolic Enzyme ◽  
Myeloma Cells

Abstract Introduction. Novel agents have improved the prognosis of multiple myeloma. However, side effects of novel agents have been a huge issue for especially elderly and frail patients. Additionally, despite the high remission rate by novel agents, multiple myeloma is still an incurable disease. In order to improve those issues, it is necessary to develop a new therapeutic strategy which is highly specific to myeloma cells and which targets a different pathway from the present anti-myeloma agents used in the clinic. In the present study, we attempt to identify a specific molecule which is specifically expressed in plasma cells and myeloma cells, and to examine whether it can be a novel target for multiple myeloma therapy. Materials and methods. A public available gene expression website GenomicScape (http://www.genomicscape.com/) and Genevestigator (https://genevestigator.com/gv/index.jsp) were utilized in order to study genes specifically expressed in human plasma cells and myeloma cells. To examine gene expression in myeloma cell lines, we utilized the gene expression data set of multiple cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE: http://www.broadinstitute.org/ccle). AMPD1 (AMP deaminase 1) gene expression in normal leukocytes and hematological malignancies were analyzed by RT-PCR. AMPD1 protein expression was analyzed by westernblot and immunohistochemistry. Genes co-expressed with AMPD1 in human myeloma cells were identified using public available gene expression datasets (GSE 4581, GSE 9782). Molecular pathways associated with genes co-expressed with AMPD1 were analyzed using Molecular Signatures Database (http://software.broadinstitute.org/gsea/msigdb/index.jsp). Cell viability of myeloma cell lines and peripheral blood mononuclear cells (PBMCs) treated by AMPD inhibitors (compound #3, #4) (Admyre T et al. Chemistry & Biology. 2014; 21: 1486-1496.) were analyzed using 7AAD dye and flow-cytometry. Results. Public gene expression screening analysis identified several genes specifically expressed in human plasma cells and myeloma cells. Among the identified genes, we focused on AMPD1, which has not been previously studied in multiple myeloma. CCLE analysis and RT-PCR analysis showed that AMPD1 is specifically expressed in bone marrow plasma cells, myeloma cell lines and patient derived myeloma cells. AMPD1 protein expression was limited to myeloma cell lines, human bone marrow myeloma cells and extramedullary plasmacytomas. Genes co-expressed with AMPD1 in human myeloma cells were associated with hypoxic pathways. Myeloma cell lines cultured under hypoxic condition had significantly higher AMPD1 expression compared to cell lines cultured under normoxic condition. AMPD inhibitors induced cell death in myeloma cell lines from around 50 uM, while the effect against PBMCs were minimal. AMPD inhibitors were more effective against myeloma cell lines under hypoxic condition compared to normoxic condition, reflecting the higher AMPD1 expression under hypoxia. Conclusions. AMPD1 is a purine metabolic enzyme that converts adenosine monophosphate (AMP) to inosine monophosphate (IMP), freeing ammonia during the process. AMPD1 has been previously reported that its expression is limited to skeletal muscles. This is the first report so far that among leukocytes and hematological malignancies, AMPD1 is specifically expressed in bone marrow plasma cells and myeloma cells. We also showed that AMPD1 expression in myeloma cells are increased under hypoxia. This indicates that AMPD1 plays a significant role in myeloma cells surviving under hypoxic conditions such as the bone marrow microenvironment. AMPD inhibitors showed cytotoxicity on myeloma cell lines in vitro, while PBMCs were not affected. Additionally, AMPD inhibitors were more effective under hypoxic condition, suggesting that AMPD1 inhibition works more specifically in the bone marrow microenvironment. Our report raise the possibility that AMPD1 inhibition can be a novel therapeutic strategy for multiple myeloma. Detailed analysis of myeloma cell death by AMPD1 inhibition is now undergoing. Disclosures Matsuoka: Bristol Myers Squibb: Research Funding.

Inhibition of Multiple Myeloma Cells Growth by AS602868, a Pharmacological Inhibitor of IKK2.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3472-3472 ◽  
Author(s):  
Michel Jourdan ◽  
Jerome Moreaux ◽  
Dirk Hose ◽  
Jean-Francois Rossi ◽  
Karène Mahtouk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Mononuclear Cells ◽  
Bone Marrow Mononuclear Cells ◽  
Hematopoietic Stem ◽  
Myeloma Cells

Abstract The NF-κB pathway is involved in the physiological regulation of cell proliferation in many cell types as well as in the resistance of several malignancies to cell death. The pathophysiologic basis for multiple myeloma (MM) has been attributed to the dysregulation of various paracrine or autocrine growth factor loops and to perturbations in several signal transduction pathways including IKK/NF-κB. The aim of the present study was to investigate the effect of a pharmaceutical IKK2 inhibitor (IKK2-I), the anilinopyrimidine derivative AS602868 (Serono International SA), on the in vitro growth of human MM cell lines (HMCL) and primary MM cells. We evaluated the effect of AS602868 on the proliferation and the survival of 12 IL-6-dependent HCML and 2 autonomously growing HCML as well as on the survival of total bone marrow mononuclear cells from patients with newly diagnosed MM (n = 6) or with relapsing MM (n = 7). Results show that using HMCL or primary MM cells, AS602868 induces a clear dose-dependent inhibition of MM cell growth (the 50% inhibitory concentration (IC50) ranging from 0.28 to 8.3 μM, mean IC50 = 2.6 μM on HCML). It was shown using HMCL that the growth inhibition induced by AS602868 is the result of a simultaneous induction of apoptosis and inhibition of the cell cycle progression. Importantly, AS602868 does not alter the survival of other bone marrow mononuclear cells (CD138−) co-cultured with primary MM (CD138+) cells except on CD34+ hematopoietic stem cells. Interestingly, using gene expression profiling with Affymetrix microarrays on 13 HMCL, we show that the resistance (high IC50) to AS602868 inhibitor is strongly correlated to APRIL gene expression (r =.7603, p <.01). Thus, an autocrine production of APRIL confers a resistance to the AS602868 IKK2-I. This can be explained since APRIL has been shown to activate the alternative NF-κB pathway which implicates an IKK complex composed of IKK1 homodimers instead of the IKK1/IKK2/NEMO complex involved in the canonical NF-κB pathway. The results demonstrate the important role of NF-κB in maintaining survival of MM cells and suggest that a pharmacological inhibition of the NF-κB pathway by an IKK2-I, AS602868, can efficiently kill myeloma cell lines or primary myeloma cells and might represent an innovative approach for treating MM patients.

scholarly journals Gene Expression Profile of Circulating Myeloma Cells Reveals CD44 and CD97 (ADGRE5) Overexpression

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5639-5639
Author(s):  
Tereza Sevcikova ◽  
Fedor Kryukov ◽  
Lucie Brozova ◽  
Jana Filipova ◽  
Zuzana Kufova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Research Funding ◽  
Plasma Cells ◽  
Flow Cytometric Analysis ◽  
Differentially Expressed ◽  
Paired Samples ◽  
Flow Cytometric

Abstract Introduction: Release of the aberrant plasma cells (PC) from the bone marrow (BM) and their presence in the peripheral blood (PB) is a maker of disease progression and worse survival in multiple myeloma (MM) (Nowakowski et al., 2005). Circulating plasma cells (cPCs) are able to survive without homing microenvironment, evade the original tumor and colonize other bone marrow niche. Detailed analysis of various surface proteins showed that cPCs display decreased levels of integrins, adhesion molecules N-CAM (CD56) and the stem cell factor receptor (Paiva et al., 2013). Comprehensive analysis of the genome-wide gene expression profiling that could provide deeper insight into the expression patterns of cPCs of MM is still lacking. Aims: To identify differentially expressed genes in paired samples of aberrant plasma cells from BM and PB and to describe potential biomarkers of cPCs in MM. Material and methods: Ten patients with multiple myeloma (seven new diagnoses and three relapses) have been included in the study after signing the informed consent form. Paired samples of aberrant plasma cells from bone marrow and peripheral blood were obtained from each patient. Aberrant plasma cells (aPCs) were sorted according to the immunophenotype as CD45dim/CD38+/CD19-/CD56-/+ cells. Gene expression profiling (GEP) was performed on paired samples using Affymetrix GeneChip Human Gene ST 1.0 array. RMA normalized data at gene level were analyzed using Wilcoxon paired test with Benjamini-Hochberg multiple testing correction. Results: The median infiltration of aberrant PC in the BM was 27.5% (range 1.1 - 93%) and 1.2% (range 0.19 - 2.8%) for cPCs in the PB. The median level of M-protein was 32.35 g/l (range 18.6 - 62.2 g/l). GEP analysis of paired BM and PB samples revealed 1001 significantly changed genes in cPCs (adjusted p-value<0.05). Gene ontology analysis did not reveal any significantly affected pathways. Nevertheless, two genes upregulated in cPCs, ADGRE5 and CD44, can be suggested as biologically relevant potential biomarkers of cPCs (Figure 1). Conclusion: The infiltration of aPCs in the bone marrow does not correlate with the amount of cPCs (p=0.16). Among differentially expressed genes, two surface markers upregulated in cPCs are of particular interest: CD44 and ADGRE5 (CD97). The CD44 antigen is a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration. Moreover, CD44 contribute to lenalidomide resistance in multiple myeloma (Bjorklund et al., 2014). CD97 is encoded by ADGRE5 gene and belongs to the EGF-TM7 subgroup of adhesion G-protein-coupled receptors. The expression of CD97 has been linked to invasive behavior in thyroid and colorectal cancer. Moreover, higher CD97 expression levels have been detected in 54% (208/385) of primary AML samples based on flow cytometric analysis (Wobus et al., 2015). Nevertheless, neither ADGRE5 nor CD97 expression were described in plasma cell dyscrasia previously. Thus, despite non-systemic changes of gene expression at the whole transcriptome level, cPCs in MM likely represent distinct biological entity with specific expression profile underlying advanced PC malignant transformation. To confirm the results, flow cytometric analysis on the bigger cohort will be performed. Acknowledgment: This study was supported by Institutional Development Plan of University of Ostrava (IRP201550) and The Ministry of Education, Youth and Sports (Specific university research of the Faculty of Medicine, University of Ostrava) project no. SGS03/LF/2015-2016, Ministry of Health Czech Republic RVO-FNOs/2014/17P and RVO-FNOs/2016/21. Figure 1 Genes of interest differentially expressed in the bone marrow (BM) versus peripheral blood (PB) aberrant plasma cells. Figure 1. Genes of interest differentially expressed in the bone marrow (BM) versus peripheral blood (PB) aberrant plasma cells. Disclosures Hajek: BMS: Honoraria; Onyx: Consultancy; Novartis: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding.

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