Myeloma Exosomes Prime the Microenvironment to Support Survival and Growth of Myeloma Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2067-2067 ◽  
Author(s):  
Bangzheng Chen ◽  
Sharmin Khan ◽  
Bozena M Laska ◽  
Gareth J Morgan ◽  
Shmuel Yaccoby ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Healthy Donor ◽  
Plasma Cells ◽  
Bone Marrow Cells ◽  
Bone Marrow Microenvironment ◽  
Control Group ◽  
Myeloma Cells ◽  
Expression Of Genes ◽  
Survival And Growth

Abstract Introduction Myeloma bone marrow serum primes healthy donor (HD) bone marrow cells to support the survival and growth of myeloma plasma cells (MMPC) in co-culture. Following interaction with the MMPC, the phenotype of the HD bone marrow stroma changes and the expression of immune regulatory factors is increased while expression of genes associated with osteoblastogenesis and WNT signaling is suppressed. The aim of this study was to determine whether exosomes in the myeloma bone marrow serum have a role in priming the microenvironment. Methods Serum was separated from clotted bone marrow aspirates from myeloma patients obtained during scheduled clinic visits, and from healthy donors of similar ages. Exosomes were prepared from 1-2 ml of serum using Invitrogen's Total Exosome Isolation kit according to manufacturer's instruction. Exosomes were 30-130 nm in diameter as determined by Dynamic Light Scatter on a Brookhaven Instruments Corp. ZetaPlus Particle Sizing Software, and visualized by scanning electron microscopy. Small RNA (≤200 nucleotides) was isolated using Qiagen's miRNeasy kits, quality and quantity assessed with Qubit 2. 96 microRNAs (miRs) were analyzed by qRT-PCR on Fluidigm's BioMark platform using 96x96 multiplex chips and normalized to spiked in C. elegance miR mimetic. Primary CD138-expressing cells were isolated from EDTA-anticoagulated bone marrow aspirates. Luciferase-expressing stroma-dependent myeloma cells were described previously (Bam, BMC Cancer 2015:864). Healthy donor mesenchymal stem cells (MSC) were prepared from bone particles obtained during orthopedic surgery, and cultured in a-MEM supplemented with 10% FBS. Primary myeloma cells or luciferase-expressing stroma dependent myeloma cells were added to wells containing monolayers of MSC in α-MEM supplemented with 10% FBS in 96 well culture plates. For each experiment 3 conditions were used: A control group, a group to which 10% myeloma bone marrow serum was added, and a third group to which exosomes isolated from a matching bone marrow serum aliquot were added. The number of primary MMPC dictated that these experiments were carried out in duplicates, while the stroma dependent cells were plated in 5 replicates. Viable primary cells counts or luciferase bioluminescence were evaluated after 4 days of co-culture. Results Exosomes supported survival and growth of primary MMPC in co-culture with HD MSC, but had no effect on MMPC cultured alone, indicating that the myeloma bone marrow serum exosomes prime the MSC to support the MMPC. Exosomes from 5 of the 7 patients supported growth of stroma-dependent myeloma cells co-cultured with HD MSC. In a representative experiment, the bioluminescence of stroma-dependent myeloma cells was 46% higher in co-cultures supplemented with bone marrow serum (p=7.3E-6) and 23% higher (p=2.0E-6) when exosomes were used. The effect was reproduced when primary MMPC were used: in a representative experiment, the number of primary MPC recovered from the co-cultures supplemented with bone marrow serum was 41% higher, and when supplemented with exosomes 28% higher than in controls. Primary MMPC did not survive in the absence of MSCs and bone marrow serum or exosomes did not enhance their survival. Conclusions Exosome from bone marrow serum of myeloma patients prime the bone marrow microenvironment to support survival and growth of primary MMPC. Bone marrow serum is more effective, indicating that other factors in the serum are also important. The differences in microRNA contents between healthy donor and myeloma exosomes may provide an insight of the regulatory mechanisms molding the myeloma bone marrow microenvironment. Disclosures Morgan: Univ of AR for Medical Sciences: Employment; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria; Janssen: Research Funding.

Co-Expression Of The MUC1 Oncoprotein and CD34 On Primary Myeloma Bone Marrow Cells Identifies a Population With Myeloma Initiating Potential

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 127-127
Author(s):  
Katarina Luptakova ◽  
Jacalyn Rosenblatt ◽  
Dina Stroopinsky ◽  
Heidi Mills ◽  
Jana Jakubikova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Plasma Cells ◽  
Bone Marrow Cells ◽  
Newly Diagnosed ◽  
Aldh Activity ◽  
Myeloma Cells ◽  
Hoechst Dye ◽  
Immunocompromised Mice ◽  
Facs Sorting ◽  
Marrow Cells

Abstract Introduction A major challenge in the development of effective myeloma (MM) therapy is addressing tumor heterogeneity, with the presence of sub-clones that exhibit resistance to standard therapy. An ongoing area of investigation focuses on identification of myeloma initiating cells that demonstrate greater capacity for self-renewal and serve as a potential reservoir for disease recurrence. It has been postulated that MM cells arise from a primitive B cell precursor population distinct from the more differentiated malignant plasma cell population. The critical feature of these myeloma-propagating cells is thought to be the ability to efficiently recapitulate MM in immunocompromised mice. MUC1 is an oncoprotein aberrantly expressed in malignant cells, including multiple myeloma, that interacts with multiple transcription factors, such as NF-κB and the β-catenin/TCF4 complex, that regulate cell survival and proliferation critical for malignant transformation. We have previously demonstrated that MUC1 is expressed by AML leukemic blasts, as compared to normal hematopoietic stem cells, and blockade of MUC1 signaling prevents establishment of leukemia in immunocompromised animals. In the present project, we identify a unique population of CD34+/MUC1+/CD138+/CD20+ cells in primary MM bone marrow samples that exhibit features of myeloma initiating cells, as manifested by high levels of enzymatic ALDH activity, the ability to efflux Hoechst dye represented as “side population” (SP), and the ability to establish disease in immunocompromised mice. Of note, MM engraftment of unselected primary myeloma cells in a xenograft model has a low success rate, and typically requires the introduction of an artificial stromal support network. Methods and results Bone marrow aspirates were obtained from newly diagnosed MM patients using an established protocol approved by the IRB. Expression of MUC1, myeloid and lymphoid markers was assessed using multicolor flow cytometric analysis. While MUC1 shows only a minimal expression (<5%, n=8) in normal CD34+ hematopoietic progenitors, we have demonstrated that on average 54% of CD34+ cells isolated from bone marrow samples of MM patients expressed MUC1 (n=7, p<0.05), in addition to other MM and lymphoid markers. MM derived CD34+MUC1+ cells segregated with SP by the ability to efflux Hoechst dye and expressed high levels of ALDH as assessed by the Aldefluor assay (11% of CD34+MUC+ cells had high ALDH activity as opposed to less than 1% in bulk MM marrow cells, n=3). CD34+MUC+ cells co-expressed CD138+, CD20+, and were CD38 dim (n=7), consistent with the phenotypic markers that have been previously described in association with myeloma propagating cells. In order to study the capacity of CD34+MUC1+ cells to recapitulate MM in a murine model, a bone marrow sample was obtained from a patient with newly diagnosed MM with a cytogenetic abnormality characterized by the rearrangement of the CCND1/IGH loci. Primary bone marrow cells were fluorescently labeled and CD34-MUC+ (consistent with mature CD138+CD38hi plasma cells) and CD34+MUC+ populations of cells were isolated using FACS sorting. Cells from each population were injected into an irradiated NOD/SCID mouse (0.5x106cells/mouse). After 13 weeks, no human engraftment was detected in the 4/4 mice injected with CD34-MUC+ population of mature plasma cells. In contrast, 2/2 mice injected with CD34+MUC+ cells demonstrated human engraftment. Engrafted cells were isolated by FACS sorting and transferred onto glass slides for cytogenetic analysis by FISH. Notably, the engrafted cells harbored rearrangement at CCND1/IGH loci consistent with the originating MM clone. In another experiment, 2/2 NOD/SCID mice inoculated with CD34+MUC1+ primary MM cells demonstrated MM engraftment after 12 weeks, characterized by the presence of CD138+CD45- human plasma cells in the murine bone marrow. Conclusions We have identified a subpopulation of primitive myeloma cells that coexpress CD34 and the MUC1 oncoprotein. CD34+MUC1+ cells express CD20 and CD138, and express high levels of ALDH. CD34+MUC1+ cells demonstrate the capacity to engraft human MM cells in immunocompromised mice, even without an artificial stromal framework. Inhibition of MUC1 signaling thus may offer new avenues to target critical myeloma subclones. Disclosures: No relevant conflicts of interest to declare.

Healthy Donor Whole Bone Marrow Cells Preconditioned With Myeloma Patient Serum Support Long-Term Survival Of Primary Myeloma and Reveal Altered Microenvironmental Pathways

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3118-3118
Author(s):  
Rakesh Bam ◽  
Sathisha Upparahalli Venkateshaiah ◽  
Xin Li ◽  
Sharmin Khan ◽  
Wen Ling ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Healthy Donor ◽  
Plasma Cells ◽  
Healthy Adult ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Statistical Significance ◽  
Term Survival ◽  
Long Term Survival

Abstract Primary human myeloma (MM) cells do not survive in culture while current in vitro and in vivo systems for growing these cells are limited to coculture with specific bone marrow (BM) cell type or growth in immunodeficient animal model. The aim of the study was to determine long-term survival and interaction of primary MM plasma cells with a healthy adult human BM that include immune cells capable of functional activation. This system is different from the autologous BM culture that is already affected by the disease. Whole BM cells from healthy donors were cultured in αMEM medium supplemented with 10% FBS and 10% serum pooled from MM patients. Following 7-9 days the cultures were composed of adherent and nonadherent cellular compartments. The nonadherent compartment contained typical BM hematopoietic cells such as monocytes, B and T lymphocytes and NK and normal plasma cells as assessed by flow cytometry, while the adherent compartment contained cells that morphologically resemble macrophages, osteoclasts, megakaryocytes and fibroblast-like cells. At this culture stage, CD138-selected MM cells from 20 patients were added to the BM cultures (4:1 BM:MM cell ratio) and survival and growth of MM cells were determined after 7 days by assessing proportion of CD45low/intermediate/CD38high MM plasma cells among total number of cells. MM and BM cell viability was constantly high (>90%) in cocultures. Subsets of primary MM plasma cells, regardless of molecular risk or subtype, were survived and detected in all cases while in 14 of 20 experiments, number of MM plasma cells was increased by 58±12% (p<0.0005, n=14). MM cell proliferation following long-term coculture was evident by the loss of cell membrane PKH26 dye or by BudR uptake in dividing cocultured MM cells. Growth of primary MM was superior in cocultures supplemented with patient serum compared to healthy donor serum. In additional study, we stably infected IL6- or stroma-dependent MM lines, or two primary MM cell cases capable of passaging in SCID-hu mice with EGFP/luciferase construct and demonstrated increased MM cell growth in all experiments in coculture using bioluminescence analysis (statistical significance range: p<0.04 to p<0.0003). Growth of OPM2 MM line was also enhanced in coculture compared to culture alone. The coculture conditions protected OPM2 cells from dexamethasone but not bortezomib while proportion of MM cell killing by lenalidomide was enhanced compared to culture of OPM2 cells alone. To assess the effect of MM cells on BM cells in coculture, global gene expression profile was performed on BM cells cultured alone or plasma cell-depleted BM after coculture with MM cells from 4 patients. Among the top underexpressed genes we identified immunoglobulin genes related to polyclonal plasma cells, extracellular factors associated with osteoblastogenesis (e.g. MGP, IGFBP2), WNT signaling (e.g. SOX4, LRP1, LRP6) and TGFb bioavailability (e.g. FBN1, LTBP1). Top upregulated genes include immuneregulatory factors such as PROK2, LRG1, OLFM4 and IL16, and cellular markers (e.g. ARG1 expressed by MDSCs). This culture system demonstrates the ability of primary MM cells to interact with and to survive in coculture with healthy adult BM that was first cultivated by patients' serum and is appropriate for studying MM-microenvironment interaction, characterization of MM cell subpopulations capable of long term survival and targeted therapy. Disclosures: No relevant conflicts of interest to declare.

Inhibition of SUV39H Methyltransferase As a Potent Therapeutic Target in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1771-1771 ◽  
Author(s):  
Julie Devin ◽  
Elena Viziteu ◽  
Laurie Herviou ◽  
Anja Seckinger ◽  
Grandmougin Camille ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
Normal Bone ◽  
Significant Toxicity ◽  
Wide Range ◽  
Travel Grant

Abstract Epigenetics is characterized by a wide range of changes that are reversible and orchestrate gene expression. Recent studies have shown that epigenetic modifications play a role in multiple myeloma (MM) by silencing various cancer-related genes. We investigated the epigenetic genes differentially expressed between normal bone marrow plasma cells (BMPC ; N=5) and MM plasma cells from patients (N=206). Using SAM (Significance Analysis of Microarrays) analysis, only 12 genes significantly differentially expressed between BMPC and MM cells (ratio > 2 and FDR (false discovery rate) < 5%) were identified, including the SUV39H1 histone methyltransferase. SUV39H1 and SUV39H2 are regulators of chromatin organization. SUV39H1-dependent trimethylation of H3K9 is essential for maintenance of both pericentromeric and telomeric heterochromatin. SUV39H1 deficiency reduced cell viability severely and is associated to heterochromatin decompaction, loss of silencing, genome instability, and a wide range of defects in cell cycle, cell growth, and meiosis. SUV39H1-mediated H3K9me has been linked to gene silencing of the tumor suppressor genes, such as p15INK4B and E-cadherin, in acute myeloid leukemia (AML). Therefore, it is highly possible that the default function of SUV39H1 is to maintain genome stability by limiting the acute activation of oncogenes while its dysregulation could cause tumor formation. We reported that high SUV39H1 expression, in MM cells, is associated with a poor prognosis in two independent cohorts of patients (Heidelberg-Montpellier cohort - N=206 and UAMS-TT2 cohort - N=345). SUV39H1 expression was downregulated by conditional shRNA expression through lentiviral delivery. SUV39H1 knock down significantly inhibits H3K9me3, growth of myeloma cells, induces apoptosis, cell cycle deregulation, reactive oxygen species production and spontaneous accumulation of DNA double strand breaks. According to these results, SUV39H1 depletion sensitizes myeloma cells to melphalan. Chaetocin is a selective inhibitor of SUV39H1. We identified that chaetocin has anti-myeloma effects at low nanomolar doses (range: 4 to 17 nM), on 11 different human myeloma cell lines, that are representative of the molecular heterogeneity of the patients, in association with H3K9 trimethylation inhibition. Furthermore, this significant toxicity of chaetocin in MM was confirmed on primary myeloma cells of 5 patients cocultured with their bone marrow microenvironment without significant toxicity on normal bone marrow cells and hematopoietic stem cells. Interestingly, the IC50 doses of chaetocin in MM were 50 fold lower compared to results published in AML, suggesting H3K9 histone methyltransferases could be a potent therapeutic target in MM. Disclosures Seckinger: EngMab AG: Research Funding; Takeda: Other: Travel grant. Goldschmidt:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millenium: Honoraria, Research Funding, Speakers Bureau; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Chugai: Honoraria, Research Funding, Speakers Bureau. Hose:EngMab AG: Research Funding; Takeda: Other: Travel grant.

Targeting EZH2 in Multiple Myeloma Could be Promising for a Subgroup of MM Patients in Combination with IMiDs

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 311-311 ◽  
Author(s):  
Laurie Herviou ◽  
Alboukadel Kassambara ◽  
Stephanie Boireau ◽  
Nicolas Robert ◽  
Guilhem Requirand ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Newly Diagnosed ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
Ezh2 Expression ◽  
Bone Marrow Plasma

Abstract Multiple Myeloma is a B cell neoplasia characterized by the accumulation of clonal plasma cells within the bone marrow.Epigenetics is characterized by a wide range of changes that are reversible and orchestrate gene expression. Recent studies have shown that epigenetic modifications play a role in multiple myeloma (MM) by silencing various cancer-related genes. We investigated the epigenetic genes differentially expressed between normal bone marrow plasma cells (BMPC ; N=5) and MM plasma cells from patients (N=206). Using SAM (Significance Analysis of Microarrays) analysis, only 12 genes significantly differentially expressed between BMPC and MM cells (ratio > 2 and FDR (false discovery rate) < 5%) were identified, including the EZH2 histone methyltransferase. EZH2, the enzymatic subunit of Polycomb Repressive Complex 2, is a histone methyltransferases able to repress gene expression by catalyzing H3K27me3 histone mark. EZH2 overexpression has been associated with numerous hematological malignancies, including MM. We thus studied EZH2 role in MM physiopathology and drug resistance. EZH2 expression was analyzed in normal bone marrow plasma cells (BMPCs; N=5), primary myeloma cells from newly diagnosed patients (MMCs; N=206) and human myeloma cell lines (HMCLs; N=40) using Affymetrix microarrays. EZH2 gene is significantly overexpressed in MMCs of patients (median 574, range 105 - 4562) compared to normal BMPCs (median = 432; range: 314 - 563) (P < 0.01). The expression is even higher in HMCLs (median 4481, range 581 - 8455) compared to primary MMCs or BMPCs (P < 0.001). High EZH2 expression is associated with a poor prognosis in 3 independent cohorts of newly diagnosed patients (Heidelberg-Montpellier cohort - N=206, UAMS-TT2 cohort - N=345 and UAMS-TT3 cohort - N =158). Furthermore, GSEA analysis of patients with high EZH2 expression highlighted a significant enrichment of genes involved in cell cycle, downregulated in mature plasma cells vs plasmablasts, and EZH2 targets. Specific EZH2 inhibition by EPZ-6438 EZH2 inhibitor induced a significant decrease of global H3K27me3 in all the HMCLs tested (P < 0.01) and inhibited MM cell growth in 5 out of the 6 HMCLs tested. The inhibitory effect of EZH2 inhibitor on MM cell growth appeared at day 6 suggesting that it is mediated by epigenetic reprogramming. To confirm that EZH2 is also required for the survival of primary MMCs from patients, primary MM cells (n = 17 patients) co-cultured with their bone marrow microenvironment and recombinant IL-6 were treated with EPZ-6438. As identified in HMCLs, EZH2 inhibition significantly reduced the median number of viable myeloma cells by 35% (P = 0.004) from a subset of patients (n=9) while the other group (n=8) was resistant. Of interest, EPZ-6438 induced a significant global H3K27me3 decrease in both groups of patient. RNA sequencing of 6 HMCLs treated with EPZ-6438 combined with H3K27me3 ChIP analyses allowed us to create an EZ GEP-based score able to predict HMCLs and primary MM cells sensitivity to EZH2 inhibitors. We also observed a synergy between EPZ-6438 and Lenalidomide, a conventional drug used for MM treatment. More interestingly, pretreatment of myeloma cells with EPZ-6438 significantly re-sensitize drug-resistant MM cells to Lenalidomide. Investigating the effect of EPZ-6438/Lenalidomide combination in MMC, we identified that IKZF1, IRF4 and MYC protein levels were significantly more inhibited by the combination treatment (65.5%, 63.9% and 14.8% respectively) compared with Lenalidomide (51.5%, 43% and 2.2%) or EPZ-6438 (45.2%, 38.7% and 6.2%) alone. Clinical trials are ongoing with EZH2 inhibitors in lymphoma and could be promising for a subgroup of MM patients in combination with IMiDs. Furthermore, the EZ score enables identification of MM patients with an adverse prognosis and who could benefit from treatment with EZH2 inhibitors. Disclosures Goldschmidt: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Hose:EngMab: Research Funding; Takeda: Other: Travel grant; Sanofi: Research Funding.

The Proliferative Potential of Myeloma Plasma Cells Manifest in the SCID-hu Host

Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3576-3582 ◽  
Author(s):  
Shmuel Yaccoby ◽  
Joshua Epstein
Keyword(s):  
Bone Marrow ◽  
Plasma Cell ◽  
Blood Cells ◽  
Plasma Cells ◽  
Bone Marrow Cells ◽  
Severe Combined Immunodeficiency ◽  
Proliferative Potential ◽  
Myeloma Cells ◽  
Host System ◽  
Human Bones

The low proliferative activity of myeloma plasma cells prompted the notion that the clonotypic B cells that exist in the blood and bone marrow of all myeloma patients contain the proliferative myeloma cells (stem cell). We have exploited our severe combined immunodeficiency (SCID)-hu host system for primary myeloma to investigate whether myeloma plasma cells are capable of sustained proliferation. Purified CD38++CD45− plasma cells consistently grew and produced myeloma and its manifestations in SCID-hu hosts (8 of 9 experiments). In contrast, the plasma cell-depleted bone marrow cells from 6 patients did not grow or produce myeloma in SCID-hu hosts. Similarly, whereas plasma-cell containing blood cells from 4 patients grew and produced myeloma in hosts, neither the PC-depleted blood cells from 3 of the patients nor a blood specimen that did not contain plasma cells grew in SCID-hu hosts, regardless of their CD19-expressing cell contents. Also, in hosts injected with blood cells, although the myeloma cells were able to disseminate through the murine host system, they were only able to grow in the human bones within a human microenvironment and were not detectable in the murine blood or other organs. Interestingly, the circulating plasma cells appear to grow more avidly in the SCID-hu hosts than their bone marrow counterparts, suggesting that they represent a subpopulation of the plasma cells in the bone marrow. Although our studies clearly demonstrate the proliferative potential of myeloma plasma cells, they are suggestive, not conclusive, as to the existence of a preplasmacytic myeloma progenitor cell.

Angiogenesis in Multiple Myeloma (MM): Angiogenic Switch or Reflexion of Plasma Cell Number? A Gene Expression Based Survey in Primary Myeloma Cells and the Bone Marrow Microenvironment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3405-3405
Author(s):  
Dirk Hose ◽  
John DeVos ◽  
Christiane Heiß ◽  
Jean-Francois Rossi ◽  
Angela Rösen-Wolff ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Plasma Cell ◽  
Empirical Bayes ◽  
Plasma Cells ◽  
De Novo ◽  
Cell Number ◽  
Bone Marrow Microenvironment ◽  
Myeloma Cells ◽  
Angiogenic Switch

Abstract BACKGROUND. Angiogenesis is a hallmark of active multiple myeloma. However, two etiologic hypotheses have been proposed: an angiogenic switch (i.e. differential or de novo expression of pro/antiangiogenic genes in MM), and, alternatively an effect of increased plasma cell number. AIM of this study was to investigate the angiogenic signature of multiple myeloma cells (MMC), normal bone marrow plasma cells (BMPC), the bone marrow microenvironment (BMME) and cellular subfractions therein. PATIENTS AND METHODS. 128 newly diagnosed MM-patients (65 training (TG) / 63 independent validation group (VG)) and 14 normal donors (ND) were included. Bone marrow aspirates were CD138-purified by activated magnetic cell sorting. Whole bone marrow (n=49) and FACSAria sorted subfractions thereof (n=5) were investigated. RNA was in-vitro transcribed and hybridised to Affymetrix HG U133 A+B GeneChip (TG) and HG U133 2.0 plus arrays (VG). Expression data were gcrma-normalised and the empirical Bayes algorithm used. p-Values were adjusted using the Benjamini-Hochberg method (Bioconductor). iFISH was performed on purified MM-cells using probesets for chromosomes 1q21, 9q34, 11q23, 11q13, 13q14, 15q22, 17p13, 19q13, 22q11 and the translocations t(4;14) and t(11;14). HGF expression was verified by real time RT-PCR and western blotting. Based on Medline review, we established a list of 89 pro- and 56 antiangiogenic genes and investigated their expression according to the stage of disease: BMPC vs. MGUS, SD stage I (asymptomatic myeloma) vs. SD stage II/III (symptomatic myeloma requiring therapy). RESULTS. BMPC express pro- (e.g. VEGFA) and antiangiogenic genes (e.g. TIMP2). Only one pro-angiogenic gene (hepatocyte growth factor, HGF) is significantly overexpressed in MMC compared to BMPC. HGF has previously been linked with myeloma progression and induction of angiogenesis. Six antiangiogenic genes (TIMP2, SERPINF1, COL18A1, PF4, THBS1, CXCL14) are downregulated in MMC compared with BMPC. Compared to healthy donors, the BMME of MM shows a significant downregulation of PLAU (urokinase, antiangiogenic) and upregulation of TNF(proangiogenic). CONCLUSION. Upregulation of HGF-expression, downregulation of TIMP2, SERPINF1, COLA18A1, PF4, THBS1 and CXCL14 expression in MMC as well as downregulation of PLAU and upregulation of TNFα in the BMME seem to indicate an “angiogenic switch”. However, given the relatively low number of differentially expressed genes (7/145) and the expression of angiogenic genes by BMPC, an effect caused by an increasing number of plasma cells might be evenly important.

High-Throughput Microrna Profiling in Patients with Waldenstrom’s Macroglobulinemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1704-1704
Author(s):  
Sophia Adamia ◽  
Samir B Amin ◽  
Cheng Li ◽  
Christopher J Patterson ◽  
Herve AvetLoiseau ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Healthy Donor ◽  
Plasma Cells ◽  
Bone Marrow Cells ◽  
Critical Role ◽  
Protein Coding ◽  
Growth And Survival ◽  
Small Noncoding Rnas ◽  
Marrow Cells

Abstract Waldenström’s macroglobulinemia (WM) is an incurable B cell malignancy characterized by the accumulation of IgM secreting clonally related bone marrow lymphoplasmacytic cells (LPC) including CD19+ B-cells and CD138+ plasma cells. Despite intense research efforts, the pathogenetic basis for this disease remains to be clarified. MicroRNAs are small noncoding RNAs that regulate the expression of protein-coding genes by inducing translational inhibition and through cleavage of targeted transcripts by partial or complete base pairing. We therefore evaluated the expression of 384 microRNAs in CD19+ and CD138+ sorted bone marrow lymphoplasmacytic from 13 untreated WM patients, and compared their expression profiling to analogous lymphoplasmacytic cells taken from the bone marrows of 13 healthy donors. Data obtained from microRNA arrays was analyzed using SDS, RQ manager, R and dChip softwares. Relative expression was calculated using the comparative Ct method through RQ manager and dChip softwares. Of the 384 microRNAs evaluated in CD19+ patient cells, miR-192, -125b, -21, -155 demonstrated significant upregulation, whereas miR-181c, -572, and -650 were significantly down regulated compared to healthy donor CD19+ bone marrow cells (p&lt;0.05). Analysis of bone marrow derived CD138+ cells from WM patients demonstrated significant upregulation in miR-192, -193b, -17-3p, -585, -148b, whereas miR-29c, -155, -126, -148a, -125a, -181d, -30a-3p, let-7b, let-7c were downregulated in comparison to healthy donor CD138+ bone marrow cells (p&lt;0.05). Importantly, characterization of the modulated microRNAs found in these studies demonstrated a critical role in growth and survival pathways through modulation of several genes including HOX, BCL-2 and c-myc. Taken together, these studies demonstrate significant differences in microRNA expression between comparable WM and healthy donor lymphoplasmacytic cell populations, and identify aberrancies in microRNAs with a pivotal role in the growth and survival of B-cells.

A Novel Role For Histone Deacetylase 11 (HDAC11) In Plasma Cell Differentation and Survival

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1907-1907
Author(s):  
Eva Sahakian ◽  
Jason B. Brayer ◽  
John Powers ◽  
Mark Meads ◽  
Allison Distler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cells ◽  
Plasma Cell ◽  
Research Funding ◽  
Plasma Cells ◽  
Myeloma Cells ◽  
Peripheral Blood Plasma

Abstract The role of HDACs in cellular biology, initially limited to their effects upon histones, is now appreciated to encompass more complex regulatory functions that are dependent on their tissue expression, cellular compartment distribution, and the stage of cellular differentiation. Recently, our group has demonstrated that the newest member of the HDAC family of enzymes, HDAC11, is an important regulator of IL-10 gene expression in myeloid cells (Villagra A Nat Immunol. 2009). The role of this specific HDAC in B-cell development and differentiation is however unknown. To answer this question, we have utilized a HDAC11 promoter-driven eGFP reporter transgenic mice (TgHDAC11-eGFP) which allows the monitoring of the dynamic changes in HDAC11 gene expression/promoter activity in B-cells at different maturation stages (Heinz, N Nat. Rev. Neuroscience 2001). First, common lymphoid progenitors are devoid of HDAC11 transcriptional activation as indicated by eGFP expression. In the bone marrow, expression of eGFP moderately increases in Pro-B-cells and transitions to the Pre- and Immature B-cells respectively. Expression of eGFP doubles in the B-1 stage of differentiation in the periphery. Of note, examination of both the bone marrow and peripheral blood plasma cell compartment demonstrated increased expression of eGFP/HDAC11 mRNA at the steady-state. These results were confirmed in plasma cells isolated from normal human subjects in which HDAC11 mRNA expression was demonstrated. Strikingly, analysis of primary human multiple myeloma cells demonstrated a significantly higher HDAC11 mRNA expression in malignant cells as compared to normal plasma cells. Similar results were observed in 4/5 myeloma cell lines suggesting that perhaps HDAC11 expression might provide survival advantage to malignant plasma cells. Support to this hypothesis was further provided by studies in HDAC11KO mice in which we observed a 50% decrease in plasma cells in both the bone marrow and peripheral blood plasma cell compartments relative to wild-type mice. Taken together, we have unveiled a previously unknown role for HDAC11 in plasma cell differentiation and survival. The additional demonstration that HDAC11 is overexpressed in primary human myeloma cells provide the framework for specifically targeting this HDAC in multiple myeloma. Disclosures: Alsina: Millennium: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Baz:Celgene Corporation: Research Funding; Millenium: Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding.

Multi-Parameter Flow Cytometry(FC) Can Detect Proliferating Myeloma Cells After Treatment: Implications for Targeting the Myeloma Proliferative Component

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4995-4995
Author(s):  
John Lust ◽  
Shaji Kumar ◽  
Michael Timm ◽  
Kathleen Donovan ◽  
Philip R. Greipp ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasma Cell ◽  
Research Funding ◽  
Plasma Cells ◽  
Bone Marrow Cells ◽  
Autologous Transplantation ◽  
Cell Labeling ◽  
Growth Fraction ◽  
Proliferating Cells ◽  
Myeloma Cells

Abstract Abstract 4995 Background: Multiple myeloma results from an accumulation of monoclonal nonproliferating plasma cells arising from a small subpopulation of proliferating myeloma cells. In an effort to optimize detection of the human myeloma cell growth fraction and obviate the need for slide review, a novel FC strategy was developed combining elements of light chain restriction, surface antigen expression, and ploidy analysis. Methods: Bone marrow cells from 41 patients with plasma cell proliferative disorders were lysed with ACK and resuspended in 3% BSA. Cells were stained using a 6-color assay with anti-CD45, anti-CD38, anti-CD138, and anti-CD19. Cells were washed and 100 ul Caltag solution A was added for 15 min. Cells were washed and 100 ul of Caltag solution B, anti-kappa and anti-lambda were added for 10 min. Cells were washed and 100 ul PBS and 3 ul RNAse are added for 15 min. Cells were washed and 400 ul of a 1:1000 solution of 3uM DAPI in Tris 0.1% NP-40 was added. Cells were incubated at 4°C for 45 minutes before running on a FACSCanto instrument for ploidy determination. All patients were analyzed both by flow cytometry and the slide based plasma cell labeling index. Results: Forty-one patients were studied; 34 demonstrated a proliferative fraction and 7 had too few plasma cells for analysis after therapy. Of the 34 patients, 6 had MGUS/SMM, 5 newly diagnosed MM, 7 amyloid, and 16 were treated MM. The mean percent proliferating cells were 1.1% (range 0 – 8.6%) with PCLI and 1.4% (range 0.1 – 12.7%) by flow. The correlation between PCLI and flow gave a RSquare value of 0.54. Twelve patients with a PCLI of 0% had a flow proliferation between 0.1 – 1.4% (mean 0.49%). Treated patients received lenalidomide, dexamethasone, bortezomib, and/or autologous transplantation. All 16 treated myeloma patients with adequate plasma cells had a flow proliferation between 0.2 – 12.7% (mean 2.2%). Conclusion: Flow cytometry offers a useful way to detect the proliferative myeloma component at diagnosis and after treatment. The continued presence of proliferating myeloma cells after treatment may explain why most patients relapse and offers another important marker to monitor and cell population to target in patients with active disease. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

scholarly journals Game of Bones: How Myeloma Manipulates Its Microenvironment

2021 ◽  
Vol 10 ◽  
Author(s):  
Tyler Moser-Katz ◽  
Nisha S. Joseph ◽  
Madhav V. Dhodapkar ◽  
Kelvin P. Lee ◽  
Lawrence H. Boise
Keyword(s):  
Bone Marrow ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Bone Marrow Microenvironment ◽  
Bone Marrow Niche ◽  
Hematological Cancer ◽  
Myeloma Cells ◽  
Cell Progression ◽  
Current Therapies ◽  
Advanced Stages

Multiple myeloma is a clonal disease of long-lived plasma cells and is the second most common hematological cancer behind Non-Hodgkin’s Lymphoma. Malignant transformation of plasma cells imparts the ability to proliferate, causing harmful lesions in patients. In advanced stages myeloma cells become independent of their bone marrow microenvironment and form extramedullary disease. Plasma cells depend on a rich array of signals from neighboring cells within the bone marrow for survival which myeloma cells exploit for growth and proliferation. Recent evidence suggests, however, that both the myeloma cells and the microenvironment have undergone alterations as early as during precursor stages of the disease. There are no current therapies routinely used for treating myeloma in early stages, and while recent therapeutic efforts have improved patients’ median survival, most will eventually relapse. This is due to mutations in myeloma cells that not only allow them to utilize its bone marrow niche but also facilitate autocrine pro-survival signaling loops for further progression. This review will discuss the stages of myeloma cell progression and how myeloma cells progress within and outside of the bone marrow microenvironment.

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