DEPTOR maintains plasma cell differentiation and favorably affects prognosis in multiple myeloma

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: B lymphocyte differentiation; DEPTOR; Multiple myeloma; Plasma cell development; miRNAs.

Figures

Fig. 1

DEPTOR expression. aDEPTOR expression in normal B lymphocytes (NBL), normal PC (NPC), and myeloma cells obtained from gene expression arrays (GSE6691 at GEO repository). bDEPTOR expression detected by qRT-PCR in immature B cells, naive B cells, memory B cells, and NPC. (*p ˂ 0.05, **p ˂ 0.01, ***p ˂ 0.001)

Fig. 2

DEPTOR knockdown modulates transcription factors and markers related to B cell maturation. a mRNA levels of the indicated genes in H929 and MM1S determined by qRT-PCR 48 h after DEPTOR knockdown. b Western blot of DEPTOR, IRF4, and kappa and lambda light chain in H929 and MM1S. c Flow cytometry analysis of CD38, CD138, and cytoplasmic kappa light chain in H929 after DEPTOR knockdown (siDEPTOR) compared with non-targeting control (siNT); left panel: representative histograms; right panel: mean fluorescence intensity values (MFI). All results are presented as the means ± SD of three different experiments. (*p ˂ 0.05, **p ˂ 0.01, ***p ˂ 0.001)

Fig. 3

DEPTOR knockdown induces dedifferentiation of myeloma cells. a Giemsa stain of H929 and MM1S after DEPTOR knockdown. b Immunofluorescence of H929 and MM1S cells stained with ER-Tracker™ Red dye. c Average maximum diameter of H929 and MM1S transfected with non-targeting (siNT) and DEPTOR siRNA (siDEPTOR) measured from two independent experiments. At least 100 cells per experiment were counted. d Cell cycle analysis 48 h after DEPTOR knockdown. The percentages of cells in different phases of the cell cycle are indicated. e Cell size of H929 48 h after DEPTOR knockdown. Representative dot plots showing forward-scatter (FSC) versus side-scatter (SSC). All results are presented as the means ± SD of three different experiments. (*p ˂ 0.05, **p ˂ 0.01, ***p ˂ 0.001)

Fig. 4

Dedifferentiation of myeloma cells after DEPTOR silencing is independent of mTOR signaling. a Levels of mTORC1 and mTORC2 substrates after DEPTOR knockdown in H929 and MM1S. All experiments were carried out 48 h post-transfection. b Western blot of H929 transfected with siNT or siDEPTOR and cultured with control medium and rapamycin at 10 nM

Fig. 5

DEPTOR expression is controlled by miR135b and miR642a in MM. aDEPTOR mRNA levels measured by qRT-PCR (upper panel). Levels of DEPTOR in MM cell lines detected by Western blot (lower panel). b miR135b and miR642a expression in NPC and MM patients measured by qRT-PCR. microRNA expression was normalized to RNU43 (2−Δct). Two-tailed Welch’s t test p values for miR135b and miR642a were calculated (0.0081 and 0.0001, respectively). c Schematic diagram of the miR642a and miR135b predicted site on the DEPTOR 3′UTR, dark squares on DEPTOR 3′UTR WT, and DEPTOR 3′UTR MUT represents fragments cloned in pmiRGlo plasmid. d Luciferase activity in HEK293 cells cotransfected with pre-miR-NC or pre-miR135b/ miR642a and plasmid pmiR-Glo with the putative miR135b/miR642a binding site of DEPTOR cloned downstream of the luciferase reporter gene. Luciferase activity was normalized using Renilla. All results are presented as the means ± SD of three different experiments. (*p ˂ 0.05, **p ˂ 0.01, ***p ˂ 0.001)

Fig. 6

miR642a and miR135b regulate DEPTOR expression in MM. a miR-135b and miR-642a expression in MM cell lines measured by qRT-PCR. microRNA expression was normalized with respect to RNU43 (2−Δct). b Western blot of DEPTOR in H929 cells 72 h post-transfection with NC or miR-135b/miR-642a. c Western blot of DEPTOR in U266 and JJN3 transfected with NC or miR135b/miR642a inhibitors. (*p ˂ 0.05, **p ˂ 0.01, ***p ˂ 0.001)

Fig. 7

Upregulation of miR135b and miR642a results in myeloma cell dedifferentiation through negative regulation of DEPTOR. a Western blot of DEPTOR, IRF4, and kappa light chain in H929 cells 72 h post-transfection with NC or miR-135b/miR-642a. b Giemsa stain of H929 cells transfected with NC or miR-135b/miR-642a. c Immunofluorescence of H929 cells stained with ER tracker

Fig. 8

DEPTOR is differentially expressed in MM and its overexpression is associated with longer survival. a Levels of DEPTOR in MM patients assessed using capillary electrophoresis immunoassay. b Kaplan–Meier curves for progression-free survival (PFS) in 24 MM patients treated according to GEM2010