Phenotypic Characterization of the CD45+ and CD45− Plasma Cell Compartments in Monoclonal Gammopathies.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3505-3505
Author(s):  
Teresa K. Kimlimger ◽  
Michael M. Timm ◽  
S. Vincent Rajkumar ◽  
Jessica L. Haug ◽  
Michael P. Kline ◽  
...  
Keyword(s):  
Plasma Cells ◽  
Cellular Adhesion ◽  
Receptor Expression ◽  
Phenotypic Characterization ◽  
Vegf Receptor ◽  
Myeloma Cells ◽  
Monoclonal Gammopathies ◽  
Normal Plasma ◽  
Significant Difference ◽  
Two Populations

Abstract Background: Multiple myeloma cells are heterogenous in terms of the expression of CD45 and previous studies have suggested important clinical and biological implications of the CD45 expression. It has been shown that expression of CD45 on myeloma cells has prognostic value and that the CD45 positive cells contain the proliferative fraction of the myeloma cells. We have also demonstrated that the CD45+ population has a higher apoptotic rate compared to CD45− cells. Given the hypothesis that CD45+ cells represent myeloma cells earlier in their development and are more proliferative and more dependent on the marrow microenvironment we studied differences between the two populations in patients with different stages of monoclonal gammopathies and in normal plasma cells. Methods: Fresh bone marrow aspirates were subjected to ACK lysis for removal of RBCs subjected to multicolor flow cytometry to study the expression of different surface antigens on the plasma cells. Plasma cells were identified based on their characteristic CD38/45 expression. The surface expression of CD19 (B cell marker), CD56 (NCAM), CD87, (involved in cellular adhesion, cell motility and angiogenesis), CD126 (IL-6 receptor alpha chain), sVEGF (surface VEGF; surrogate measure of VEGF receptor expression), and CD71 (proliferation marker) were studied and compared between the CD45 + and CD45− cells by gating separately on these populations. Results: Patients with active myeloma (76), smoldering myeloma (32), MGUS/amyloid (44), and normal patients (23) were studied. Not all markers were performed on all patients. Among the patient samples, the expression of CD19, CD87, CD71, CD126, and sVEGF were higher among the CD45+ cells compared to CD45− cells (Table: % of cells with expression). There was no significant difference between the two in terms of the expression of CD56. Among the normal plasma cells, the expression of CD19, CD87, CD126 and sVEGF was greater on the CD45+ cells compared to CD45− plasma cells and CD56 was not different. When the patient cells were compared to normal cells, the expression of CD19 was higher among the normal CD45+ plasma cells and CD56, CD87 and CD126 were less. The expression sVEGF was comparable Conclusion: In this study we demonstrate differences between CD45+ and CD45− plasma cells in patients with monoclonal gammopathies. These findings are important for understanding the disease biology and provide an insight into the functional differences seen between these two populations. The increased expression of CD19 in the CD45+ cells supports the notion that these cells are likely to be at an earlier developmental stage. The comparable CD56 expression is not in accordance with previous studies and needs to be examined in a larger group of patients. Increased expression of sVEGF and CD126 as well as the adhesion molecules is in accordance with increased responsiveness and dependence of the CD45+ cells to VEGF and IL-6 and the increased proliferative rates. These findings indirectly support the hypothesis that CD45 negative cells are less dependent on the marrow microenvironment and the cytokine milieu that it provides.

A Potential Biomarker Panel of Multiple Myeloma Identified Using Label-Free Mass Spectrometry-Based Quantitative Proteomics

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2890-2890
Author(s):  
Attaya Suvannasankha ◽  
Colin D. Crean ◽  
Heather M. Sahm ◽  
Rafat Abonour ◽  
Sherif Farag ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Multiple Myeloma ◽  
Protein Expression ◽  
Plasma Cells ◽  
Expression Profiles ◽  
Peptide Identification ◽  
Clinical Samples ◽  
Normal Plasma ◽  
Potential Biomarker ◽  
Significant Difference

Abstract Abstract 2890 Background: Multiple myeloma is an incurable and fatal hematologic malignancy. Recent gene microarray studies showed distinct gene expression profiles defining MM subgroups and their association with cytogenetic abnormalities and treatment outcome. However, aside from transcriptional control, a variety of post-transcriptional/post-translational modifications likely play an important role in regulating protein expression and function, and ultimately may prove informative for predicting tumor behavior. Objectives: We hypothesize that the protein profile in MM cells is different than normal plasma cells. Methodology: Normal plasma cells and myeloma cells were isolated using CD138 immune magnetic beads from bone marrow aspirates from healthy volunteers or patients with newly diagnosed MM, respectively. CD138+ cells were frozen and subsequently analyzed in one batch. Proteins were digested by trypsin. Tryptic peptides were injected onto an HPLC system and analyzed on a Thermo-Fisher LTQ mass spectrometer. Peptide identification and quantification were carried out using proprietary algorithms. Identified proteins were categorized into priority groups based on the quality of the peptide identification by tandem mass spectrometry. Proteins with significant changes in expression level were further analyzed by bioinformatics tools for the determination of the biological significance. Results: In the discovery phase of this study, 433 proteins were identified and their expression levels were quantitatively compared. 169 of these proteins demonstrated a significant difference between normal plasma cells and MM cells. Among the significantly changed proteins, 18 were identified and quantified with high confidence, and were therefore chosen for further validation. The identified proteins are known to be involved in the glycolysis/gluconeogenesis pathway, the oxidative phosphorylation pathway, cysteine metabolism and the pentose phosphate pathway. None of these proteins are known to be of prognostic value or being currently targeted for therapy in MM. A high-throughput LC/MS-based multiple-reaction-monitoring (MRM) assay for quantitative validation of these candidates with clinical samples is ongoing. To date, using the MRM assay, we were able to detect MRM peptides for 13 of the 18 targeted proteins in clinical samples. The quantification of these peptides will be further confirmed using a separate set of clinical samples. Conclusion: Significant differences in protein expression were observed between MM and normal plasma cells. The study presents an important step toward using proteomics as a tool to develop diagnostic and/or prognostic biomarkers in the clinical setting. However, both follow-up analytical and clinical validations are required before they can serve as disease-specific biomarkers. Disclosures: Abonour: Celgene: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Anti-VEGF Drugs in the Treatment of Multiple Myeloma Patients

2020 ◽  
Vol 9 (6) ◽  
pp. 1765 ◽  
Author(s):  
Roberto Ria ◽  
Assunta Melaccio ◽  
Vito Racanelli ◽  
Angelo Vacca
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Plasma Cells ◽  
Vascular Endothelial Cells ◽  
Vasculogenic Mimicry ◽  
Inflammatory Cells ◽  
Receptor Expression ◽  
Antiangiogenic Agents ◽  
Vegf Receptor ◽  
Vascular Endothelial

The interaction between the bone marrow microenvironment and plasma cells plays an essential role in multiple myeloma progression and drug resistance. The vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) pathway in vascular endothelial cells activates and promotes angiogenesis. Moreover, VEGF activates and promotes vasculogenesis and vasculogenic mimicry when it interacts with VEGF receptors expressed in precursor cells and inflammatory cells, respectively. In myeloma bone marrow, VEGF and VEGF receptor expression are upregulated and hyperactive in the stromal and tumor cells. It has been demonstrated that several antiangiogenic agents can effectively target VEGF-related pathways in the preclinical phase. However, they are not successful in treating multiple myeloma, probably due to the vicarious action of other cytokines and signaling pathways. Thus, the simultaneous blocking of multiple cytokine pathways, including the VEGF/VEGFR pathway, may represent a valid strategy to treat multiple myeloma. This review aims to summarize recent advances in understanding the role of the VEGF/VEGFR pathway in multiple myeloma, and mainly focuses on the transcription pathway and on strategies that target this pathway.

Phenotypic difference of normal plasma cells from mature myeloma cells

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2658-2663 ◽  
Author(s):  
H Harada ◽  
MM Kawano ◽  
N Huang ◽  
Y Harada ◽  
K Iwato ◽  
...  
Keyword(s):  
Plasma Cells ◽  
Fluorescein Isothiocyanate ◽  
Color Analysis ◽  
Phenotypic Difference ◽  
Phenotypic Analysis ◽  
Color Flow ◽  
Myeloma Cells ◽  
Normal Plasma ◽  
Healthy Donors ◽  
Chronic Lymphadenitis

We have recently shown that two-color analysis with fluorescein isothiocyanate (FITC)-anti-CD38 antibody could clearly distinguish myeloma cells (plasma cells) from other hematopoietic cells in the bone marrow. Myeloma cells (plasma cells) alone were located at CD38strong positive (++) fractions. To further distinguish normal plasma cells from mature myeloma cells phenotypically, we examined immunophenotypes of normal plasma cells and myeloma cells by two-color flow cytometry with FITC-anti-CD38 antibody and phycoerythrin staining with antibody to VLA-4, MPC-1, CD44, CD56, CD19, CD20, CD24, or CD10. Normal plasma cells were all VLA-4+VLA-5+MPC-1+CD44+ CD19+CD56- in the bone marrows from seven healthy donors, tonsils from four patients with chronic tonsillitis, a spleen from one patient with idiopathic thrombocytopenic purpura, and lymph nodes from two patients with chronic lymphadenitis, respectively. On the other hand, mature myeloma cells (12 of 20 cases), VLA-4+VLA-5+MPC-1+, were all CD19- and most of them CD56+, and there were no myeloma cells with the CD19+CD56- phenotype in the 20 cases of myelomas we tested. Thus, as for the expression of CD19 and CD56, normal plasma cells from various tissues are all CD19+CD56-, whereas no myeloma cells have the CD19+CD56- phenotype. According to this finding, we investigated the expression of CD19 and CD56 on plasma cells (CD38++ fractions) in monoclonal gammopathy of undetermined significance (MGUS). Both CD19+CD56- and CD19-DC56+ plasma cells were found in all five cases of MGUS we tested, suggesting that MGUS consists of phenotypically normal plasma cells and myeloma cells. Therefore, it is reasoned that phenotypic analysis of plasma cells with anti-CD19 and anti-CD56 antibodies can distinguish normal plasma cells from malignant plasma cells (myeloma cells), and can detect malignant plasma cells even in MGUS or premyeloma states.

Phenotypic characterization of the human myeloma cell growth fraction

Blood ◽  
2005 ◽  
Vol 105 (12) ◽  
pp. 4845-4848 ◽  
Author(s):  
Nelly Robillard ◽  
Catherine Pellat-Deceunynck ◽  
Régis Bataille
Keyword(s):  
Plasma Cells ◽  
Ex Vivo ◽  
Inverse Correlation ◽  
B Cell Lymphoma ◽  
Myeloma Cell ◽  
Phenotypic Characterization ◽  
Growth Fraction ◽  
Myeloma Cells ◽  
Brdu Labeling

Abstract In this study we quantified the proliferation rate of normal and malignant plasma cells (PCs) by ex vivo incorporation of 5-bromo-2′-deoxyuridine (BrdU; labeling index, LI) using flow cytometry. We show that all bone marrow PCs, either normal or malignant, include a subset of proliferating PCs present within the CD45bright fraction. Indeed, medullary normal and malignant PCs were always heterogeneous for CD45 expression, and proliferation was always restricted primarily to the CD45bright compartment. Moreover, an inverse correlation was found between LI or CD45 and B-cell lymphoma 2 (Bcl-2) in both malignant and normal PCs, the most proliferating CD45bright PCs have the lowest Bcl-2 expression. We investigated expression of molecules of interest in multiple myeloma (MM)—that is, CD138, CD19, CD20, CD27, CD28, CD56, and CD11a—to further characterize the CD45bright fraction. Among all of these molecules, only CD11a was exclusively expressed by CD45bright proliferating myeloma cells. In conclusion, proliferating myeloma cells are characterized by the specific CD45bright CD11apos Bcl-2low phenotype. (Blood. 2005;105:4845-4848)

Oncoprotein 18 (Op18) : A Differentially Expressed Gene Is a Novel Therapeutic Target in Multiple Myeloma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2741-2741
Author(s):  
Dheeraj Pelluru ◽  
Jingyi Wang ◽  
Rao Prabhala ◽  
Mariateresa Fulciniti ◽  
Masood A. Shammas ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Plasma Cells ◽  
Differentially Expressed Gene ◽  
Myeloma Cell ◽  
Differentially Expressed ◽  
Myeloma Cells ◽  
Normal Plasma ◽  
Novel Therapeutic

Abstract Stathmin(Op18) is a ubiquitous cytosolic 18Kda protein regulating microtubule (MT) dynamics via binding and stabilizing activities. The activity of Op18 is down regulated during cell cycle by phosphorylation at four Ser residues. The phospho-Op18 is unable to bind to tubulin allowing the progression of the cell cycle. Two additional Op18-binding proteins, KIS prevents Op18 dephosphorylation and activation while iASPP (inhibitor of apoptosis stimulatory protein phosphatase) binds to WT-p53 and inhibits p53-related apoptosis. We have identidfied Op18 as a differentially expressed gene by suppression subtractive hybridization in myeloma. We have also confirmed differential expression of KIS in myeloma cells compared to normal plasma cells. Overexpression of Op18 was confirmed at both RNA and at protein level by RT –PCR and Western blotting in human myeloma cell lines as well as primary samples compared to normal plasma cells and normal human fibroblasts. In contrast, none of the tonsillar CD138+ plasma cells and normal bone marrow mononuclear cells showed Op18 overexpression. To establish the role of Op18 overexpression in MM cell transformation, the human MM cell lines were treated with antisense Op18 oligodeoxynucleotides (ODN). The growth rate of the ODN treated human myeloma cells was significantly reduced compared to the control cells along with cell cycle arrest in G2/M phase and increase in apoptosis as measured by immunohistochemical staining. Additionally, we have silenced Op18 by transfecting Op18-specific siRNA in MM cells and observed that KIS is translocated to cytoplasm, from nucleus as well as MM cells are arrested in G2/M. Importantly, Op18 silencing increased sensitivity of MM cells to microtubule drugs suggesting possible combination approach for therapeutic application. Its role in networking cellular signal transduction pathways in myeloma is under investigation. Recent publication identifying Op18 as one of the 15 most relevant genes determining outcome in myeloma (Avet-loiseau et al, JCO 2008) adds to the validity of Op18 as a molecule playing important role in myeloma cell growth and survival and warrant investigation as a novel therapeutic target.

Distribution of Bim Determines MCL-1 Dependence or Co-Dependence on BCL-xL/BCL-2 In Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4054-4054
Author(s):  
Lawrence H. Boise ◽  
Alejo A Morales ◽  
Metin Kurtoglu ◽  
Shannon M Matulis ◽  
Rebecca Markovitz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Cellular Localization ◽  
Plasma Cells ◽  
Annexin V ◽  
Initial Distribution ◽  
Normal Plasma ◽  
Significant Difference ◽  
Dependent Cell ◽  
Smoldering Myeloma

Abstract Abstract 4054 The inappropriate activation of oncogenes can result in the up-regulation of pro-apoptotic signals often in the form of BH3-only proteins such as Bim, Noxa or Puma. This renders cells dependent on anti-apoptotic proteins including BCL-2, BCL-xL and MCL-1. Thus cancer cells would be predicted to be more susceptible to inhibition of BCL-2 family proteins, prompting the development and testing of several small molecule inhibitors of this class of proteins. Multiple myeloma is a plasma cell malignancy of the bone marrow and like normal plasma cells, myeloma plasma cells express MCL-1. A search of gene expression profile data from normal plasma cells (n=22), MGUS plasma cells (n=12), plasma cells from patients with asymptomatic (smoldering) myeloma (n=44) or newly diagnosed multiple myeloma (n=538) revealed no significant difference in MCL-1 mRNA expression associated with progression of disease. We then determined MCL-1 dependence through the introduction of siRNA in 4 MM cell lines and consistent with previous findings using anti-sense oligonucleotides in additional lines, we demonstrated that all cell lines tested were MCL-1 dependent. However using the BCL-2/BCL-xL/BCL-w-selective inhibitor ABT-737 we found that 3 of the 6 MCL1-dependent cell lines tested were sensitive (IC50 for Annexin V-FITC positive at 24 hrs of 300 – 600 nM) and therefore also dependent on BCL-xL/BCL-2. Taken together this is the first formal demonstration that cells can be co-dependent on multiple Bcl-2 family members. We have previously reported that ABT-737 sensitivity, and what we now refer to as co-dependence on MCL-1 and BCL-xL/BCL-2, is determined by the distribution of BIM on the anti-apoptotic BCL-2 proteins in these cells. We have now expanded these findings to patient samples that displayed sensitivity to ABT-737 that is similar to what we have observed in the co-dependent cell line MM.1s. Consistent with these findings co-immunoprecipation revealed BIM binding predominantly to BCL-xL. Additionally we have now demonstrated that BIM binding is not simply controlled by the expression levels of BCL-xL or MCL-1 as enforced over-expression of each protein could alter the sensitivity of co-dependent cell lines to ABT-737 but did not alter the initial distribution of BIM amongst these proteins. These data suggest that additional factors regulate the association of BIM with anti-apoptotic BCL-2 proteins. These factors could include differences in cellular localization of these proteins as well as differences in post-translational modifications of either pro- or anti-apoptotic BCL-2 family proteins. Disclosures: Boise: University of Chicago: Patents & Royalties.

scholarly journals VS38 Identifies Myeloma Cells With Dim CD38 Expression and Plasma Cells Following Daratumumab Therapy, Which Interferes With CD38 Detection for 4 to 6 Months

2019 ◽  
Author(s):  
Elizabeth L Courville ◽  
Sophia Yohe ◽  
Paula Shivers ◽  
Michael A Linden
Keyword(s):  
Flow Cytometry ◽  
Plasma Cell ◽  
Fluorescence Intensity ◽  
Plasma Cells ◽  
Myeloma Cells ◽  
Flow Cytometry Data ◽  
Cd38 Expression ◽  
Cell Percentage ◽  
Significant Difference ◽  
Institutional Experience

Abstract Objectives We report our institutional experience using VS38 to evaluate plasma cells by flow cytometry. Methods Flow cytometry data were reanalyzed to compare plasma cell percentages between the standard panel and VS38 panel. Natural killer (NK) and plasma cell CD38 median fluorescence intensity (MFI) values were calculated. Results Our cohort included 63 specimens from 38 patients. Twenty-six had received daratumumab (monoclonal anti-CD38 therapy) between less than 1 month and 17 months prior. For NK and plasma cells, CD38 MFI values were suppressed for 0 to 4 months and started to increase 4 to 6 months after last exposure. There was no significant difference in clonal plasma cell percentage calculated by the VS38 and standard panels; however, identification and quantification using the VS38 panel were easier. Conclusions VS38 is a viable alternative to bright CD38 to identify plasma cells and particularly helpful in myeloma cases with dim CD38 and after daratumumab. Daratumumab interference with CD38 identification persists 4 to 6 months after the last exposure.

scholarly journals DNMTi/HDACi combined epigenetic targeted treatment induces reprogramming of myeloma cells in the direction of normal plasma cells

2018 ◽  
Vol 118 (8) ◽  
pp. 1062-1073 ◽  
Author(s):  
Angelique Bruyer ◽  
Ken Maes ◽  
Laurie Herviou ◽  
Alboukadel Kassambara ◽  
Anja Seckinger ◽  
...  
Keyword(s):  
Plasma Cells ◽  
Targeted Treatment ◽  
Myeloma Cells ◽  
Normal Plasma

The Bone Marrow Microenvironment Influences the Differential Chemokine Receptor Expression of Normal and Neoplastic Plasma Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2353-2353
Author(s):  
Faith E. Davies ◽  
Mona H. Al Rayes ◽  
J. Anthony Child ◽  
Gareth J. Morgan ◽  
Andrew C. Rawstron
Keyword(s):  
Bone Marrow ◽  
Plasma Cell ◽  
Chemokine Receptor ◽  
Chemokine Receptors ◽  
Plasma Cells ◽  
Receptor Expression ◽  
Ccr2 Expression ◽  
Receptor Profile ◽  
Significant Difference ◽  
Neoplastic Process

Abstract The expression of cytokines and chemokines are under control of several factors, including the bone marrow (BM) microenvironment. The aim of this work was to study the chemokine receptor expression on normal and neoplastic PCs and to investigate the relationship between the BM microenvironment and plasma cell behaviour. The study included 20 patients with reactive disorders or normal BM, 20 individuals with MGUS (monoclonal gammopathy of undetermined significance) and 19 patients with multiple myeloma at presentation. A large panel of chemokine receptor-specific antibodies directed against CCR1, CCR2, CCR3, CCR5, CCR6, CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5 was characterized employing a four-colour flow cytometry approach. We demonstrate that normal and myeloma PCs have a specific chemokine receptor profile expressing 7/10 of the receptors studied. CCR2, CCR6, and CXCR1 showed decreased expression on myeloma PCs in comparison to normal PCs (average 3.3, 1.5 and 1.8-fold difference in expression level respectively). In contrast CXCR4 was upregulated on myeloma PCs on average 2.2-fold in comparison to normal PCs. There was no significant difference in expression between normal (CD19+) and neoplastic (CD19−) PCs from the same bone marrow environment in MGUS patients with respect to CCR6, CXCR1 and CXCR4. In contrast there was a significant difference in expression of CCR2 between CD19+ and CD19− PCs from the same BM (P=0.002). The level of expression on CD19− PCs was on average 1.6-fold lower than on their CD19+ counterparts (range 1.1 6.8-fold lower). In conclusion these data demonstrate that normal and myeloma PCs have a specific chemokine receptor profile. Myeloma PCs have a reduced level of some chemokine receptors compared to normal PCs which may account for their abnormal localization within the BM. Differences in expression of CXCR1, CXCR4, and CCR6 are not specific to the neoplastic process, as both normal and neoplastic plasma cells from the same marrow in MGUS patients show corresponding levels of expression. It is probable that feedback loops between neoplastic plasma cells and bone marrow stroma also influence normal plasma cell expression of these chemokine receptors. However, differences in CCR2 expression are not influenced by the marrow microenvironment, therefore downregulation of CCR2 expression is either a function of the neoplastic process or of the stage of differentiation of the originating neoplastic cell. Interfering with chemokines and their receptors which are related to the malignant transformation, particularly CCR2, may prove useful as adjunct to chemotherapy approaches.

The Epigenetic Repression of Mir-375 Is the Dominant Mechanism for the Constitutive Activation of PDPK1/RSK2 Signaling Axis in Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4182-4182
Author(s):  
Shotaro Tatekawa ◽  
Junya Kuroda ◽  
Yoshiaki Chinen ◽  
Yuji Shimura ◽  
Hisao Nagoshi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Copy Number ◽  
Plasma Cells ◽  
Expression Level ◽  
Chromosome 16 ◽  
Myeloma Cells ◽  
U6 Snrna ◽  
Normal Plasma ◽  
Signaling Axis

Abstract [Introduction] Multiple myeloma (MM) is a cytogenetically/molecularly heterogeneous hematologic malignancy that remains mostly incurable, and the identification of a universal and relevant therapeutic target molecule is essential for the further development of therapeutic strategy. We have recently identified that 3-phosphoinositide-dependent protein kinase 1 (PDPK1), a serine threonine kinase, and its major downstream substrate RSK2, a member of the 90 kDa ribosomal S6 kinase family of serine threonine kinases, were universally active in eleven human MM-derived cell lines (HMCLs) examined regardless of the type of cytogenetic abnormality, the mutation state of RAS, RAF and FGFR3 genes and myeloma cells of approximately 90% of symptomatic patients at diagnosis. Our study also disclosed that PDPK1/RSK2 signaling axis played pivotal roles in myeloma pathophysiology by regulating series of downstream molecules, such as c-MYC, IRF4, D-type cyclins, or PLK1, while the inactivation of either PDPK1 or N-terminal domain of RSK2 resulted in the induction of apoptosis in myeloma cells which was accompanied by the activation of BH3-only proteins BIM and BAD (Shimura Y, Mol Caner Ther 2012; Chinen Y, Cancer Res 2014). Here we assessed the underlying mechanism for PDPK1 overexpression in MM. [Methods] The miR-375 expression level was analyzed by the quantitative RT-PCR in 11 HMCLs and 92 patient-derived myeloma cells isolated by CD138-positive cell sorting (normal plasma cells (N=10), MGUS (N=21), newly diagnosed MM (NDMM) (N=27), relapsed/refractory (RRMM) (N=34). The pre-miR-375 precursor molecule (miR-375 mimics), the siRNA targeted against PDPK1, or a negative control RNA-oligonucleotides was transfected into 8 cell lines by utilizing Hemagglutinating Virus of Japan (HVJ)-envelope vector. The copy number abnormality of PDPK1 gene was assessed by double-color FISH for PDPK1 gene and the centromere of chromosome 16. The methylation status of miR-375 promoter site was analyzed by methylation-specific PCR (MSP). This study was conducted in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Boards. Patient-derived samples were obtained with informed consent, and normal bone marrow plasma cells were obtained from volunteers who were not affected by hematologic disease. [Result] The level of miR-375 expression was calculated with 2-ΔCt methods. Human U6 snRNA was examined as the reference. The median log102-ΔCt ± SD of normal plasma cells, MGUS, NDMM, RRMM and HMCLs were -2.46 ± 0.67, -3.64 ± 0.68, -4.23 ± 0.95, -3.92 ± 1.24 and -3.69 ± 0.29 respectively. When compared to normal plasma cells, the miR-375 expression was significantly decreased in NDMM and RRMM (p<0.01, respectively), and tended to be decreased in MGUS (p=0.083) and HMCLs (p=0.097). As the causative of miR-375 repression, our study disclosed that the promoter sites of miR-375 gene were hypermethylated in 8/8 of HMCLs when examined by MSP. The interphase FISH for PDPK1 with centromere chromosome 16 indicated the copy number of PDPK1 gene was increased in 11/11 HMCLss, however, this was never the case with patient-derived myeloma cells (0/7). Importantly, the miR-375 gene transfection resulted in the reduction of PDPK1 expression in 7 of 8 HMCLs, and it simultaneously caused the reduction of the expression levels of IGF1 receptor and JAK2, the known targets of miR-375. Furthermore, when treated with 5-Azacitidine and/or Trichostatin A, miR-375 was markedly upregulated, suggesting that the overlapping epigenetic deregulations, such as DNA hypermethylation or histone deacetylation, are involved in the silencing of miR-375. [Conclusion and Discussion] PDPK1 is activated by autophosphorylation and, therefore, its expression level is the crucial determinant for its activity, Because our study revealed the miR-375 expression as the major regulator of PDPK1 expression, it is suggested that the abnormally repressed miR-375 is the major causative for the constitutive hyperactivation of the PDPK1/RSK2 signaling axis in MM. Moreover, since the decreased miR-375 expression was observed in plasma cells of MGUS and was more pronounced in MM, miR-375 repression by epigenetic deregulation may be involved in both disease development and progression of MM. Disclosures No relevant conflicts of interest to declare.

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