Interleukin-6-dependent growth in a newly established plasmablastic lymphoma cell line and its therapeutic targets

Plasmablastic lymphoma (PBL) is a rare, highly aggressive subtype of non-Hodgkin lymphoma with plasma-cell differentiation occurring typically in immune-suppressed patients such as those with AIDS. This study reports the establishment and characterization of a new cell line, PBL-1, derived from a patient with AIDS-associated PBL. Morphological assessment of PBL-1 indicated plasma-cell differentiation with a CD20(−) CD38(+) CD138(+) immunophenotype and IgH/c-myc translocation. The cell line harbours Epstein-Barr virus, but a 52.7-kbp length defect was identified in its genome, resulting in no expression of viral microRNAs encoded in the BamHI-A Rightward Transcript region. Importantly, supplementation of culture medium with >5 ng/mL of interleukin-6 (IL-6) was required for PBL-1 growth. Starvation of IL-6 or addition of tocilizumab, an inhibitory antibody for the IL-6 receptor, induced apoptosis of PBL-1. Transduction of IL-6 into PBL-1 by lentivirus vector induced autologous growth without IL-6 supplementation of culture medium. These data indicate the IL-6 dependency of PBL-1 for proliferation and survival. mTOR inhibitors induced cell death effectively, suggesting mTOR in the IL-6 signalling pathway is a potential therapeutic target for PBL. This established PBL cell line will be a useful tool to further understand the pathophysiology of PBL and aid the future development of PBL treatment.

Immortalized cell lines are useful tools to investigate disease pathogenesis and to evaluate the effects of therapeutic agents. To the best of our knowledge, there has been no report describing a well-characterized PBL cell line established from typical PBL cases. In this study, we established a PBL cell line from a case of AIDS-associated PBL, and demonstrated its interleukin 6 (IL-6)-dependent growth. In addition, the treatment effect of therapeutic agents was investigated.

Results
Establishment of a PBL cell line. At the beginning of the primary culture of tumour cells from the patient ascites, cells grew in RPMI 1640 culture medium supplemented with 10% patient ascites, 10% foetal bovine serum (FBS), 10 ng/mL insulin, and 10 ng/mL of transferrin at 37 °C with 5% CO 2 . After several passages, autologous growth was observed. However, when the ascites was removed from the culture medium, the cells did not grow and became apoptotic. Therefore, we speculated that certain growth factor(s) were required for cell growth. Cytokines in the patient's ascites and the supplemented cultured medium were analysed by Luminex. A high amount of IL-6 was detected in the ascites (Supplementary Figure 1). Supplementation of RPMI 1640 containing 10% FBS with 5 ng/mL of IL-6 induced cell proliferation, and the percentage of live cells increased to >90%. Single cell cloning was performed successfully in medium containing IL-6 using a limiting dilution method. Five single clones with similar morphological phenotypes were obtained, and one of single cell-cloned cells was designated as PBL-1 and analysed.
Chromosomal analysis revealed a complex karyotype -the majority of the cells were diploid with modal 82 chromosomes (Supplementary Figure 4). Southern blotting analysis indicated an IgH gene rearrangement, and BioMed2 analysis revealed an immunoglobulin kappa gene rearrangement, indicating a B cell linage (Supplementary Figure 4). Fluorescence in situ hybridization (FISH) with a triple colour-dual fusion probe of IgH and c-myc revealed that PBL-1 had an IgH/c-myc rearrangement (Supplementary Figure 4d), whereas there were no bcl-2 or bcl-6 gene splits (data not shown). PCR and sequence analysis identified a break point in the IgH/c-myc rearrangement that was upstream of exon 1 of c-myc and downstream of the IgH regulatory region (Supplementary Figure 4e and f). Immunofixation electrophoresis of supernatants from the PBL-1 culture medium revealed that PBL-1 secreted no immunoglobulin heavy or light chains (data not shown). Sequence analysis of c-myc, p53, and myd88 revealed these genes had no recurring point mutations in PBL-1 compared with reference sequences (data not shown).

EBV infection in PBL-1.
RT-PCR revealed that PBL-1 was positive for EBER-1 and EBNA-1 mRNA but negative for EBNA-2, latent membrane protein 1 (LMP-1), LMP-2A, and LMP-2B mRNA, indicating that the EBV status in PBL-1 was Latency I (Supplementary Figure 3c). RT-PCR also revealed Qp promotor usage and that the Cp and Wp promoters were not active in PBL-1 (Supplementary Figure 3c). EBV genotype-specific PCR analysis revealed that EBV in PBL-1 was genotype 1 (Supplementary Figure 3b). Reactivation of EBV was investigated by the addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) or Ig in the culture supernatant of PBL-1. Real-time PCR analysis showed that the EBV copy number was stable at 1.26 copies/cell in PBL-1 regardless of stimulation by TPA or Ig. To investigate the expression of EBV-encoded miRNA in PBL-1, next generation sequencing (NGS) was performed on small RNA extracted from PBL-1. Surprisingly, NGS detected only 2 reads of miR-BHRF1, but no reads of miR-BART in 2,112,810 reads, while human miRNA were detected abundantly (Supplementary Table 2). NGS also detected only 1 read of miR-BHRF1 in 1,426,476 reads from an RNA sample of the original tumour. PCR with primers designed for the region of miR-BART clusters in the EBV genome demonstrated a defect in the region (Fig. 2). Genome walking and PCR analysis revealed a deletion from 110,920 to 163,702 in the EBV genome of PBL-1 (numbering is based on strain M81, GenBank accession no. KF373730), indicating a complete deletion of miR-BART clusters (Fig. 2). Southern blot analysis with the 5′ site of the EBV genome detected a band of unexpected size for the circular form of EBV (Fig. 3a). FISH using an EBV-specific probe detected 2-4 dots of positive signals per cell in chromosomes 1q and 5q in the metaphase or interface (Fig. 3b). In addition, digital PCR analysis showed that EBV copy number in PBL-1 was stable at 1.67-2.7 copies per 1 PBL-1 is dependent on IL-6 for proliferation and survival. PBL-1 showed constant growth in medium supplemented with 5 ng/mL of IL-6. The XTT assay showed the IL-6-dose-dependent viability of PBL-1 (EC 50 = 0.237 ng/mL) (Fig. 4a). The XTT assay and cell viability counting indicated that other cytokines including IL-8, IL-4, IL-11, Monocyte Chemotactic Protein-1 (MCP-1), and oncostatin M had no effect on PBL-1 viability (Fig. 4b). Western blot analysis showed that starvation of IL-6 led to PBL-1 apoptosis and the cleavage of caspases-2, -3, -9, and PARP (Fig. 4c). Flow cytometry revealed that annexin V expression was induced at 6 hours after IL-6 starvation in PBL-1 (Fig. 4d), and BrdU assay showed a decrease of DNA synthesis in PBL-1 at 24 hours (Supplementary Figure 5a). IL-6 binds to soluble or membranous IL-6 receptor (or gp80, IL-6Ra), and this IL-6/ IL-6Ra complex binds to two molecules of gp130, a ubiquitously expressed membranous protein 25 . The IL-6/ IL-6R/gp130 complex induces activation of the Jak/Signal Transducers and Activator of Transcription (STAT) 3 pathway [26][27][28] . Flow cytometry analysis revealed the expression of gp80 and gp130 in PBL-1 (Supplementary Figure 2). In addition, enzyme-linked immunosorbent assay demonstrated that PBL-1 secreted a high concentration of soluble IL-6Ra (4,482 pg/mL in the supernatant of PBL-1, Fig. 4e). Tocilizumab, an anti-IL-6 receptor antibody, inhibited mitochondrial metabolic activity and DNA synthesis in PBL-1 in a dose-dependent manner (IC 50 = 6.11 μg/mL,  Figure 5c). Lentiviral transduction of PBL-1 with human IL-6, but not a control-gene or vector-transduced cells, without supplementation of IL-6 induced significant autologous proliferation and survival of the transduced cells (p < 0.01) (Fig. 4g). These data demonstrated a dependency on IL-6 for PBL-1 growth.

IL-6/Jak/STAT3 and PI-3K/Akt/mTOR pathways in PBL-1.
The Jak/STAT3 pathway is a major signal pathway downstream of IL-6/IL-6R/gp130 26 . To verify the effect of IL-6 on STAT3 in PBL-1, we performed western blotting in the presence or absence of IL-6. While p-STAT3 was abundant in total cell lysates of PBL-1 in the presence of IL-6 (5 ng/mL), the dephosphorylation of p-STAT3 was induced within 1 h after the starvation of IL-6 ( Fig. 5a). Moreover, the expression of total STAT3 decreased gradually after the starvation of IL-6. The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signalling pathway is another major pathway of IL-6 signalling downstream of IL-6R/gp130/Jak 29,30 . Western blotting also revealed that IL-6 starvation induced the phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) and p70S6K and the dephosphorylation of 4E-BP-1, which are factors downstream of the mTOR pathway (Fig. 5a). Phosphoinositide-dependent protein kinase-1 (PDK1), Akt, and p70S6K were phosphorylated at 1 to 6 hours after IL-6 starvation, but they were dephosphorylated immediately after phosphorylation. To compare the efficiency of drugs that affect the Jak/Stat3 and PI-3K/Akt/mTOR pathways with other pathways, we screened compound libraries containing more than 100 inhibitors for various oncogenic pathways. Inhibitors for Jak/STAT and PI3K/Akt/mTOR pathways induced cell death similar to general tyrosine kinase or cyclin dependent kinase inhibitors (Supplementary Table 3). The XTT assay revealed that the mTOR inhibitors, everolimus and rapamycin, also inhibited the mitochondrial metabolic activity of PBL-1 in a dose-dependent manner, and that the IC 50 of each drug for PBL-1 was 187 pM and 9.74 nM, respectively ( Fig. 5b and c). Everolimus was effective at a very low dose compared with the other compounds. These data suggested that the mTOR pathway has a significant effect on the growth of PBL-1.
Therapeutic effects of drugs. In addition to Jak, PI3K, Akt, and mTOR inhibitors, we tested other drugs that have an effect on malignant lymphoma or multiple myeloma in vitro and in vivo including proteasome inhibitors, melphalan, pomalidomide, and histone deacetylase (HDAC) inhibitors. The XTT assay showed that these drugs also inhibited the mitochondrial metabolic activity of PBL-1 in a dose-dependent manner (Fig. 6a, Supplementary Table 4). However, the IC 50 of these drugs was higher than for the mTOR inhibitors. The combined therapeutic effect of the drugs was assessed using the Chou-Talalay Combination index (CI) method. The combination of bortezomib and tocilizumab (CI = 0.83, Fig. 6b), everolimus and tocilizumab (0.95), and panobinostat and tocilizumab (0.55) exhibited a synergistic effect (CI < 1), whereas antagonistic effects (CI > 1) were observed for three combinations (Supplementary Table 5).

Discussion
The cell origin of PBL is thought to be the plasmablast 1, 31 , which is an activated B-cell in the germinal centre that has undergone somatic hypermutation and class switching recombination. The immunophenotype of plasmablasts is CD38 (+) MUM-1 (+) and CD20 (−), as reflected in PBL. The immunophenotype of PBL is similar to that of multiple myeloma except for those with EBV infection 32 . The established cell line, PBL-1, had an immunophenotype typical of plasmablasts, with morphological features that resembled plasmacytic tumours. To the best of our knowledge, there have been two previous reports of the establishment and characterization of cell lines from this distinct tumour 33,34 , and one report of a xenograft model of EBV-negative-PBL 35 . A study of two  34 . IL-6-dependency in the culture medium was not described for these reported cell lines, although IL-6 might be associated with the establishment of these cell lines in a xenograft model 34,35 .
The established PBL cell line in this report was IL-6 dependent. The dependency of cell activation by IL-6 through the gp130/Jak/STAT signalling pathway has been established in multiple myeloma 26,27 . Starvation of IL-6 in PBL-1 led to the dephosphorylation of p-STAT3 and the phosphorylation of Akt, PDK1, and p70 within 1 h; however, p-Akt, p-PDK1, and p-p70 were dephosphorylated within 24 h (Fig. 5a). Although the factors upstream of the mTOR pathway were temporarily phosphorylated, they were dephosphorylated within a few hours. This phenomenon might be explained by negative feedback downstream of mTOR 36 . Inhibitors to factors upstream of mTOR did not dephosphorylate p-p70, but rapamycin and everolimus effectively dephosphorylated p-p70 37,38 . mTOR is considered the bottleneck of the pathway. Tocilizumab is an antibody to soluble and membranous IL-6R, and inhibits IL-6 signal transduction 28 . Tocilizumab inhibited mitochondrial metabolic activity in PBL-1 in a dose-dependent manner (Fig. 4f), suggesting the inhibition of IL-6 signalling might be an effective treatment for PBL. In vivo, PBL cells require IL-6 secreted from surrounding cells such as macrophages. PBL can also secrete cytokines that act on surrounding cells; indeed, IL-8 expression was observed in the supernatant of PBL-1 (Supplementary Figure 1). Furthermore, an autocrine IL-6 mechanism might be involved in the growth of PBL. In primary effusion lymphoma, which is also considered to be derived from plasmablasts, KSHV-encoded viral IL-6 is secreted from lymphoma cells and induces the proliferation of the tumour by an autocrine mechanism 39-41 . The dependency of PBL on IL-6 secretion and the PBL morphology suggests PBL are biologically related to primary effusion lymphoma.
EBV infected PBL-1 show Latency I. The integration of EBV in host cells is often observed in tumours and cell lines 12 . Generally, integration by a virus occurs randomly at fragile sites in the host genome 42 . Although the integration site of EBV has not been identified in PBL-1, Southern blot analysis using an EBV-Bam-L probe (Fig. 3a) and dot-like signals of EBV observed in chromosomes by FISH (Fig. 3b), suggested that EBV did integrate into the host genome of PBL-1. Real-time PCR and droplet digital PCR analysis revealed that chemical and immunological stimulations did not induce the reactivation of EBV in PBL-1. In addition, a miRNA cluster in the BamHI-A Rightward Transcript (BART) region of the EBV genome was completely defective in PBL-1. Thus, miR-BARTs are not essential for maintaining this cell line. B95.8 also has a large deletion in BART DNA; however, no report has described deletion of the whole BART genome in EBV among any cases of lymphoma or EBV strains isolated from various diseases or healthy donors 43 . miR-BARTs were reported to have anti-apoptotic or oncogenic functions 44,45 . A large deletion causing the complete lack of miR-BARTs might be associated with an abortive state. Only EBNA-1 and Qp promoters were expressed in PBL-1, whereas other EBV-encoded genes were not expressed. Recently, it was reported that EBNA-1 enhanced the expression of IL-6R 46,47 or had linkage with STAT regulation 48 , suggesting it might play a role in the survival of PBL-1.
In this study, bortezomib, pomalidomide, tocilizumab, and mTOR inhibitors (rapamycin, everolimus) were effective at inducing apoptosis in PBL-1. Everolimus, an mTOR inhibitor, is an immunosuppressive drug used for the treatment of breast cancer, neuroendocrine tumours, and kidney cancer; however, there has been no report on the therapeutic use of everolimus for PBL. Inhibitors of the mTOR pathway such as everolimus or rapamycin were more effective on PBL-1 than inhibitors to other factors downstream of IL-6 signalling such as the Jak/Stat pathway. This suggests that mTOR-associated factors play an important role in the IL-6 signalling pathway in PBL-1, and that IL-6 signalling pathways, especially mTOR, are a potential therapeutic target for PBL. Furthermore, in this study, bortezomib, a proteasome inhibitor used for the therapy of multiple myeloma, also exhibited a cytotoxic effect on PBL-1. This finding may indicate that PBL-1 has similar pharmacological responses to multiple myeloma. A combination of HDAC and proteasome inhibitors enhanced apoptosis in some lymphomas, multiple myeloma, EBV-associated tumours and cell lines [49][50][51][52][53] . These drugs might be potential therapeutic candidates for the treatment of PBL.

Patient.
A 60-year-old Japanese male patient with HIV-1 infection was admitted with fever, weight loss, and ascites. His blood CD4 (+) cell counts were 316/μL. Flow cytometry analysis indicated the presence of CD19 (−), CD20 (−), CD38 (+) cells in the ascites. Positron emission transfer imaging revealed a large tumour in the transversus colon, and its biopsied specimen showed the infiltration of tumour cells similar to those in the ascites. Cell culture. To establish a new cell line, tumour cells were collected from the patient ascites and cultured in RPMI 1640 culture medium supplemented with 10% patient ascites, 10% FBS, 10 ng/mL of insulin (Sigma-Aldrich, St. Louis, MO), and 10 ng/mL of transferrin (Sigma-Aldrich) at 37 °C with 5% CO 2 . Supplementation of insulin and transferrin was gradually reduced during passages. Finally, cells were grown in RPMI 1640 with 5 ng/mL of IL-6 (PeproTech, Rocky Hill, NJ) and 10% FBS. Single cell cloning was performed using a limiting dilution method in IL-6 containing medium. For the IL-6 starvation study, cells were centrifuged, washed twice with phosphate buffered saline (PBS), and cultured in RPMI 1640 supplemented with 10% FBS without IL-6. EBV-positive human cell lines (Raji and LCL), B95.8, an EBV-positive marmoset cell line, and EBV-negative cell lines (Bjab, BCBL-1, TY-1, PCM6, and Ramos) were cultured in RPMI 1640 medium with 10% FBS. Cells were stained with trypan blue and cell viability was measured using a TC10 automated cell counter (Bio-Rad, New York, NY).
Electron microscopy. PBL-1 cells were pelleted by centrifugation, fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 h at room temperature, post-fixed in 1% osmium tetroxide, and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (HT7700, Hitachi High Technologies, Tokyo, Japan) at 80 kV.

RT-PCR.
RT-PCR to detect EBV latency-associated transcripts was performed as described previously 54 .
XTT assay. The viability of PBL-1 cells was examined using a XTT (tetrazolium derivative) assay kit (Roche Molecular Biochemicals, Indianapolis, IN). Cells were cultured in 96-well plates in triplicate at a density of 2 × 10 5 cells/mL in RPMI 1640 medium containing 10% FBS in the presence or absence of 5 ng/mL IL-6 and/ or applied drugs. After 48 h of culture, the XTT labelling mixture was added according to the manufacturer's instructions. Colour development of formazan production was measured spectrophotometrically with Multiskan MS (LabSystems/Thermo Fisher Scientific) at 450 nm 55 . The cell proliferation ratio was calculated by the OD of a sample divided by the OD of the positive controls (IL-6-supplied PBL-1).
Western blot analysis. Protein extraction and immunoblotting were performed as described previously 56 . Briefly, 1 × 10 6 cells were lysed in 100 μL of M-PER lysis buffer containing Halt protease and phosphatase inhibitor cocktail (Pierce Biotechnology, Rockford, IL). Cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride microporous membrane (Immobilon-P Transfer Membrane, Millipore, Bedford, MA) using the NuPAGE system (Life Technologies, Carlsbad, CA). The membranes were blocked with Block Ace and probed with primary antibodies followed by horse radish peroxidase-conjugated anti-mouse or anti-rabbit antibodies (Promega, Madison, WI) with an immunoreaction enhancer solution (Can Get Signal, Toyobo, Osaka, Japan). Blots were visualized by Super-Signal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific) and images were captured with a C-Digit Blot Scanner (LI-COR Biosciences, Lincoln, NE).
Anti-IL6R antibody and drug screening. Anti-IL6R antibody, tocilizumab (Actemra), was purchased from Chugai Pharmaceutical, Tokyo, Japan, and used at 48 ng-100 μg/mL. To identify drugs that inhibited PBL-1 cell proliferation, PBL-1 cells were cultured in RPMI 1640 with 10% FBS, 5 ng/mL of IL-6, and drugs from the InhibitorSelect 96-well Protein Kinase Inhibitor Library I, II, III (Calbiochem, San Diego, CA) in a 96-well plate at two final concentrations (10 nM and 1 μM). We also added 7 HDAC inhibitors (HDAC inhibitor Set II: Sigma Aldrich) and other antitumour drugs to PBL-1 cells with 5 ng/mL of IL-6 and cultured them for 48 h.
Analysis for synergism and antagonism of drugs. PBL-1 cells were cultured in RPMI 1640 with 10% FBS, 5 ng/mL of IL-6 and two drugs (bortezomib, everolimus, tocilizumab and panobinostat) each added as a 2-fold dilution series from 6.4 μg/mL (tocilizumab), 32 nM (bortezomib and panobinostat) or 1.6 nM (everolimus) to determine the IC 50 . XTT assay was performed after 48 hr of culture. A constant combination ratio experiment for the 8 point assay was carried out, as the ratio of (IC 50 of drug 1)/(IC 50 of drug 2) was constant. The Chou-Talalay Combination index (CI) was calculated according to a study by Chou et al. using CompuSyn Version 1.0 software (http://www.combosyn.com/) 57 . A CI value of <1, = 1, or >1 indicates synergy, additivity, and antagonism, respectively. Statistical analysis. All results were expressed as the mean ± SD of data obtained in triplicate, from at least three individual experiments. All statistical analyses were performed with Excel software (Microsoft, Bellevue, WA), and GraphPad Prism Version 6.0 software (GraphPad Software, La Jolla, CA). P values < 0.05 were considered statistically significant.
Ethical statement. The study protocol was approved by the Institutional Review Board, National Institute of Infectious Diseases (Approval No. 512) and Tokyo Metropolitan Komagome Hospital (Approval No. 1552). Informed consent was obtained from the patient with PBL. All methods were performed in accordance with the relevant guidelines and regulations.
Further methods are provided in the supplemental Materials and Methods.
Data availability. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.