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DEPTOR Maintains Plasma Cell Differentiation and Favorably Affects Prognosis in Multiple Myeloma Dalia Quwaider1,3, Luis A

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DEPTOR Maintains Plasma Cell Differentiation and Favorably Affects Prognosis in Multiple Myeloma Dalia Quwaider1,3, Luis A Quwaider et al. Journal of Hematology & Oncology (2017) 10:92 DOI 10.1186/s13045-017-0461-8 RESEARCH Open Access DEPTOR maintains plasma cell differentiation and favorably affects prognosis in multiple myeloma Dalia Quwaider1,3, Luis A. Corchete1,3, Irena Misiewicz-Krzeminska1,3,4, María E. Sarasquete1,3, José J. Pérez2, Patryk Krzeminski1,3, Noemí Puig1,2,3, María Victoria Mateos1,2,3, Ramón García-Sanz1,2,3, Ana B. Herrero1,3† and Norma C. Gutiérrez1,2,3*† Abstract Background: The B cell maturation process involves multiple steps, which are controlled by relevant pathways and transcription factors. The understanding of the final stages of plasma cell (PC) differentiation could provide new insights for therapeutic strategies in multiple myeloma (MM). Here, we explore the role of DEPTOR, an mTOR inhibitor, in the terminal differentiation of myeloma cells, and its potential impact on patient survival. Methods: The expression level of DEPTOR in MM cell lines and B cell populations was measured by real-time RT-PCR, and/or Western blot analysis. DEPTOR protein level in MM patients was quantified by capillary electrophoresis immunoassay. RNA interference was used to downregulate DEPTOR in MM cell lines. Results: DEPTOR knockdown in H929 and MM1S cell lines induced dedifferentiation of myeloma cells, as demonstrated by the upregulation of PAX5 and BCL6, the downregulation of IRF4, and a clear reduction in cell size and endoplasmic reticulum mass. This effect seemed to be independent of mTOR signaling, since mTOR substrates were not affected by DEPTOR knockdown. Additionally, the potential for DEPTOR to be deregulated in MM by particular miRNAs was investigated. The ectopic expression of miR-135b and miR-642a in myeloma cell lines substantially diminished DEPTOR protein levels, and caused dedifferentiation of myeloma cells. Interestingly, the level of expression of DEPTOR protein in myeloma patients was highly variable, the highest levels being associated with longer progression-free survival. Conclusions: Our results demonstrate for the first time that DEPTOR expression is required to maintain myeloma cell differentiation and that high level of its expression are associated with better outcome. Primary samples used in this study correspond to patients entered into GEM2010 trial (registered at www. clinicaltrials.gov as #NCT01237249, 4 November 2010). Keywords: DEPTOR, Multiple myeloma, Plasma cell development, B lymphocyte differentiation, miRNAs Background monoclonal immunoglobulin. Although several therapeutic Multiple myeloma (MM) is a clonal disorder of B cells agents are available, MM remains incurable. Knowledge of (BCs) in the final stage of differentiation that accounts for all the factors involved in PC differentiation could provide approximately 10% of all hematological cancers [1]. MM is new insights of relevance to therapeutic strategies for MM. characterized by clonal accumulation of malignant plasma In fact, in some hematological neoplasms, the malignant cells (PCs) in the bone marrow, which secrete a transformation of BC has been associated with the disrup- tion of the B cell differentiation process, such as mutations * Correspondence: [email protected] of certain key BC maturation factors [2–4]. The transition †Equal contributors 1Cancer Research Center-IBMCC (USAL-CSIC), Salamanca, Spain from B lymphoid precursors to antibody-secreting PCs in- 2Department of Hematology, University Hospital of Salamanca, Paseo San volves several molecular and cellular modifications includ- Vicente, 58-182, Salamanca 37007, Spain ing transcriptional changes, expansion of the cytoplasm Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Quwaider et al. Journal of Hematology & Oncology (2017) 10:92 Page 2 of 12 and the secretory organelles to accommodate high-rate syn- were obtained from the Deutsche Sammlung von Mik- thesis of immunoglobulins, unfolded protein response roorganismen and Zellkulturen (DSMZ). Cell line identity (UPR) activation, and changes in cell surface antigen was confirmed periodically by STR analysis with the expression [5–8]. It has been demonstrated that the tran- PowerPlex 16 HS system kit (www.promega.com) and on- scriptomes of BC and PC are maintained by two groups of line STR matching analysis (www.dsmz.de/fp/cgi-bin/ transcriptional factors: thosethatpromotetheBcell str.html). Cell lines were cultured in RPMI 1640 medium program, such as PAX5, BCL6, and BACH2, and those that supplemented with 10% fetal bovine serum and antibiotics favor and facilitate PC differentiation, notably IRF4, (Gibco Life Technologies, Grand Island, NY, USA). Bone BLIMP1, and XBP1 [9]. Interestingly, many of these tran- marrow (BM) samples from ten healthy donors were scription factors repress others required for the alternative sorted by a FACSAria equipment into four BC popula- developmental state, thereby establishing mutually exclu- tions: immature B cells (CD34−, CD19 +, CD10+, CD38+ sive gene expression programs [9–12]. Besides transcrip- +), naive B cells (CD19+, CD27−,CD10−), memory B cells tional factors, other types of proteins and biochemical (CD19+, CD138−, CD27+, CD38+), and plasma cells pathways could be involved in the transformation of BC (CD38+++, CD138+, CD45low). Monoclonal antibodies into mature PC. were purchased as follows: anti-CD45-FITC (clone D3/9) Using microarray expression data, we found that the and anti-CD19-PECy7 (clone A3-B1) from Immunostep mRNA-encoding DEPTOR, an inhibitor of mTORC1 (Salamanca, Spain); anti-CD38-PerCP-Cy™5.5 (clone and mTORC2 kinases activities [13], was overexpressed HIT2), anti-CD34-APC (clone 8G12), and anti-CD27- in normal PCs (NPCs) and myeloma cells compared BV421 (clone M-T271) from BD Biosciences (San Jose, with normal B lymphocytes (NBLs) [14], which raised CA, USA); anti- CD138-Pacific OrangeTM (clone B-A38) the possibility that this protein contributes to PC differ- from Exbio Praha (Vestec, Czech Republic); and anti- entiation. The complete role of DEPTOR within the cells CD10-PE (clone ALB1) from Beckman Coulter (Pasadena, has not yet been fully elucidated, although the involve- CA, USA). CD138+ plasma cells were isolated from BM ment of DEPTOR in several biological processes, such samples of 24 patients with newly diagnosed MM as cell growth, apoptosis, and autophagy, has been included in the GEM2010 Spanish trial (bortezomib, reported [15]. A potential role of DEPTOR as a tumor melphalan, and prednisone plus lenalidomide and dexa- suppressor or as an oncogene, depending on cell methasone), using an autoMACS separation system context, has also been described. It is considered a (Miltenyi-Biotec, Auburn, CA, USA). tumor suppressor, functioning by the inhibition of mTOR, whose activity is frequently hyperactivated in RNA extraction and quantitative real-time PCR analysis many human tumors. Indeed, DEPTOR has been found RNA was extracted from the cell lines using an RNeasy to be downregulated in many types of human cancers. mini kit (Qiagen, Valencia, USA) according to the stand- However, it is also overexpressed in many other tumor ard protocol. RNA integrity was assessed using an types, including chronic myeloid leukemia, and MM [13, Agilent 2100 Bioanalyzer (Agilent Technologies, Santa 16]. The overexpression of DEPTOR in MM has been Clara, CA, USA). Total RNA (1 μg) was reverse- associated with translocations involving MAF transcrip- transcribed to complementary DNA (cDNA) using tion factors and CCND1 and CCND3 genes [13]. Fur- High-Capacity cDNA Reverse Transcription Kits (Ap- thermore, DEPTOR seems to be overexpressed in MM plied Biosystems, Foster City, CA, USA). Expression of with copy number gains of 8q24 where DEPTOR is lo- target genes was assessed using TaqMan qRT-PCR cated [17]. assays (Applied Biosystems). Relative gene expression −Δ Here, we report for the first time that DEPTOR main- was calculated by the 2 Ct method using GAPDH as tains the terminal differentiation of MM cells. Knock- the endogenous control for normalization. down of DEPTOR reverts the transcriptional program of To detect mature miR135b and miR642a expression the PC to that characteristic of a BC. In addition, we levels, TaqMan quantitative real-time polymerase chain found that microRNA deregulation in MM, specifically reaction (qRT-PCR) micro RNA (miRNA) assay (Applied miR642a and miR135b downregulation, may also under- Biosystems) was performed. The relative levels of ex- pin the overexpression of DEPTOR. pression of mature miR135b and miR642a normalized with respect to the RNU43 endogenous control were −Δ Methods determined by the 2 Ct method. Each measurement Cell lines and primary samples was performed in triplicate. The human multiple myeloma cell lines (MMCL), NCI- H929, MM1S, and U266 were acquired from the ATCC Transfections (American Type Culture Collection), and the JJN3, RPMI- Cell lines were transfected using the nucleofector II sys- 8226, OPM-2, KMS12BM,
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