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Tumor necrosis factor (TNF)-α and its receptors (TNFR) have been identified in the sera of CLL patients in increased concentrations, and high TNF-α levels are indicative for an aggressive disease, thus suggesting a role in CLL progression.13-16 TNF-α was shown to act as an autocrine growth factor in CLL.17,18 The inhibition of TNFR signaling by etanercept, a recombinant TNFR-2 derivative, in combination with the anti-CD20 antibody rituximab, caused durable remissions in refractory patients without 17p deletion.19 However, the detailed pathomechanism of TNFR signaling in CLL development and progression remains largely unknown. TNF-α is a pro-inflammatory cytokine that exerts its pleiotropic effects via two recep- tors, TNFR-1 (P55) and TNFR-2 (P75).20 Only TNFR-1 is endowed with an intracellular death domain, and can thereby induce either caspase-mediated apoptosis or pro- survival signals via NFκB activation.21 TNF-α-induced NFκB activation was shown to be blocked by wogonin, a naturally occurring flavonoid, resulting in a shift of TNFR- 1 signaling towards apoptosis induction.22 In a multitude of in vitro and in vivo studies, wogonin has been demon- strated to exert anti-oxidant, anti-inflammatory and anti- tumor activities.23
To elucidate the oncogenic role of TNFR-1 in CLL and to test its potential as a therapeutic target, we analyzed TNFR-1 expression and function in primary CLL cell co- cultures and Em-T-cell leukemia 1 (TCL1) mice in the presence and absence of wogonin, and used these plat- forms for pre-clinical evaluation of TNFR-1 as a drug tar- get in CLL.
Methods
Samples
PB of CLL patients (Online Supplementary Table S1) and healthy donors (HD) was obtained after informed consent and in accor- dance with the Declaration of Helsinki. The study was approved by the Institutional Review Board.
Quantification of Soluble TNFR-1
Sera from 247 CLL patients from the German CLL8 study (Online Supplementary Table S2) and from 50 age- and sex-matched healthy controls were used to quantify soluble TNFR-1 by cyto- metric bead arrays (BD Biosciences, Heidelberg, Germany), according to the manufacturer's protocol. Capture beads were synthesized by coupling anti-TNFR-1 antibody (Duoset, R&D Systems, Minneapolis, MN, USA) to functionalized beads. A biotinylated detection antibody (Duoset, R&D Systems) and a streptavidin conjugate were used for visualization. Data was acquired on a FACSCanto II flow cytometer and analyzed with FCAP software (BD Biosciences).
Sera from final stage leukemic Em-TCL1 mice24 presenting with splenomegaly and more than 90% leukemic cells in the blood, and matched wild-type (WT) controls were screened for 144 inflam- matory factors using Mouse Cytokine Array G2000 (RayBiotech, Norcross, GA, USA), according to the manufacturer’s protocol.
haematologica | 2018; 103(4)
TNF receptor signaling as therapeutic target in CLL
stantially contributes to the observed high efficacy of these inhibitors.10-11 Albeit, despite their clinical success, CLL remains an incurable disease due to clonal evolution of malignant cells under treatment, followed by drug resistance and relapse.12 The current challenge is to devel- op new strategies by targeting not only CLL cells, but also the microenvironment, with the goal being that of eradi- cating the malignant cells.
Glass slides were scanned on an Agilent microarray scanner (Agilent Technologies, Santa Clara, CA, USA) and data was ana- lyzed using GenePix Pro software (Molecular Devices, San José, CA, USA).
Serum soluble (s)TNFR-1 was quantified in mice after adoptive transfer (AT) of CLL using mouse sTNFR-1 enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems).
Statistical analysis
Details of statistical analysis are provided in the Online SupplementaryMethods.
Gene expression analysis
CLL cells and CD19-sorted B cells of HD were cultured for one day in high cell density (1x107 cells in 4 mL per well in 6-well plates) and total ribonucleic acid (RNA) was isolated before (day 0 [d0]) and after culture (d1). Microarray-based transcriptome analy- sis and quantitative reverse transcription- polymerase chain reac- tion (RT-PCR) was performed as previously described.3
Tissue microarrays and immunohistochemistry
Tissue microarrays (TMAs) including BM trephines (n=20 CLL patients; n=16 HD), LN sections (n=58 CLL patients; n=14 coinci- dental LN taken for non-malignant pathologies with no evidence of germinal center formation), and reactive LNs (n=28) were stained with hematoxylin and eosin (H&E) and antibodies against CD20 (Dako, clone L26, Agilent), TNFR-1 (polyclonal, Abcam, Cambridge, UK), CD3 (Labvision, clone SP7, ThermoFisher), and CD68 (Dako, clone KP1, Agilent), as detailed in Online Supplementary Methods.
Animal models and treatments
AT of TCL1 splenocytes was performed as previously described.25 Briefly, 6-8-week-old C57BL/6 WT females (Charles River Laboratories, UK) were transplanted with 4x107 splenocytes pooled from leukemic TCL1 mice24 (>95% CD19+CD5+ cells) via tail vein injection. In early treatment studies, animals were ran- domized to daily treatment with 40 mg/kg wogonin in H2O con- taining arginine as an adjuvant to improve the solubility of wogo- nin, administered by intraperitoneal (i.p.) injection from 48 hours after AT for three weeks. For late treatment studies, PB tumor load was determined 21 days post-AT and animals were randomized to treatment with phosphate buffered saline (PBS) or 40 mg/kg wogonin (BIOTREND Chemicals AG, Wangen, Switzerland) solved in dimethyl sulfoxide (DMSO) for 21 days using daily oral gavage. At the endpoints, serum, peritoneal exudate, PB and sin- gle-cell suspensions of lymphoid organs were prepared as described previously.26
Results
sTNFR-1 serum level predicts overall survival and tumor-associated deaths in CLL
sTNFR-1 was quantified in serum from 247 CLL patients (Online Supplementary Table S2) and 50 age- and sex-matched controls by cytometric bead arrays. The analysis revealed a significantly higher median serum con- centration of sTNFR-1 in CLL patients (2.30 ng/mL, range: 0.50-7.31) compared to controls (1.35 ng/mL, range: 0.76- 1.74; P<0.0001; Figure 1A). Further, sTNFR-1 serum con- centrations moderately but significantly correlated with β2-microglobulin (R=0.582; P<0.001; Figure 1B) and thymidine kinase (R=0.263; P<0.001; Figure 1C) serum levels, suggesting the malignant cells as the source of
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