This study was conducted using the rituximab-sensitive human activated B-cell-like-DLBCL cell line, NU-DUL-1. The NU-DUL-1 cell line was purchased from American Type Culture Collection (Manassas, VA, USA). The cells were maintained in the Roswell Park Memorial Institute Medium 1,640 supplemented with 10% fetal bovine serum and penicillin-streptomycin (100 U/mL) at 37°C and 5% CO₂ in a humidified incubator. Human serum, a source of complement, was purchased from Sigma-Aldrich (St. Luis, MO, USA).

To develop resistant cell lines, the naïve cell line was gradually exposed to an escalating dose of rituximab (0.1–128 μg/mL). After 24 h of incubation with a specific dose of rituximab and human serum, cells were centrifuged and maintained in fresh growth medium for several days. Once the log phase of growth was reached, the same process was repeated with an escalating dose of rituximab 10 times [9].

AntiCD 20 monoclonal antibody, rituximab (Mabthera), was provided by Roche (Basel, Switzerland). Deferasirox and SB 203580 were purchased from Sigma-Aldrich. BIRB 796 was purchased from MedChemExpress (Princeton, NJ, USA). Primary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA; antibodies against Erk1/2, phosphoErk1/2, SAPK/JNK, phosphoSAPK/JNK, p38 mitogen-activated protein kinase [MAPK], phosphop38 MAPK, phosphoNF-κB, antipoly [ADP-ribose] polymerase [PARP], and Bcl-2) and BD Biosciences (San Jose, CA, USA; antibody against β-actin). The secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA; anti-mouse immunoglobulin (Ig)G and anti-rabbit IgG).

Cell viability was evaluated using the EZ-cytox Cell Viability Assay Kit (Water water-soluble tetrazolium salt, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate) (Daeillab service, Seoul, Korea). Cells were seeded in 96-well plates (2.0 × 10⁴ cells per well), and various concentrations of rituximab (0–80 μg/mL), deferasirox (0–25 μM), or rituximab and deferasirox were administered with 20% human serum. After 48 hours of incubation, 10 μL of the kit reagent was added to each well, and the cells were further incubated for 4 hours. Absorbance was measured at 450 nm using a SpectraMax 190 Microplate Reader (Molecular Devices, San Jose, CA, USA).

Rituximab-resistant cells were treated with the same concentration of rituximab (10 μg/mL) and human serum. Treated cells were stained with a FITC-conjugated Annexin V apoptosis detection kit (BD Biosciences) and analyzed through flow cytometry using a Navios EX Flow Cytometer (Beckman Coulter, Pasadena, CA, USA).

Total RNA was extracted from previously collected rituximab-sensitive cell line (RSCL) and RRCL using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The library was constructed using the TruSeq Stranded Total RNA LT Single Prep Kit (Gold) and mapped to the reference genome (GRCh37) using HISAT version 1.10 and Bowtie2 aligner 2.3.4.1. Transcript assembly was performed using StringTie, version 2.1.3b. The fragment counts were measured in units of reads per kilobase of transcript per million mapped reads (RPKM). The values of log₂(RPKM+1) were calculated for differentially expressed gene (DEG) analysis, and DEG was analyzed using DESeq2.

Whole cells were solubilized in mammalian protein extraction reagent (ThermoFicher Scientific, BRIMS, Cambdridge, MA, USA) supplemented with protease inhibitor cocktail tablets (Roche^®, Indianapolis, IN, USA). The supernatant was collected after centrifugation (15,000 × g, 4°C, 15 min), and protein concentrations were measured using a BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Equal quantities of protein were electrophoresed on a Novex WedgeWell Trisglycine gel (ThermoFicher Scientific, BRIMS) and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Richmond, CA, USA). The membranes were blocked with phosphate-buffered saline containing 0.2% Tween 20 and 5% nonfat dry milk and incubated overnight at 4°C with primary antibodies against the following proteins: ERK1/2, phosphoERK1/2, SAPK/JNK, phosphoSAPK/JNK, p38 MAPK, phosphop38 MAPK, phosphoNF-κB, PARP, Bcl-2, and β-actin. The membranes were then incubated with the following horseradish peroxidase-conjugated secondary antibodies: antirabbit IgG and antimouse IgG. The immunoblots were visualized using enhanced chemiluminescence solution (DoGenBio, Seoul, Korea) and exposed to X-ray film.

Data analysis was performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). The data were analyzed using student’s t-test, and statistical significance was defined as a p value < 0.05.

We developed a RRCL by sequential exposure to rituximab in human serum. To determine whether rituximab resistance was obtained, cell viability was confirmed after administration of various concentrations of RSCL and RRCL. Rituximab caused a dose-dependent reduction in the viability of both cell lines. After treatment with rituximab 8 μg/mL, the viability of RRCL cells was reduced to 60%, whereas that of RSCL cells was reduced to 8.7%. Cell viability was significantly reduced in RSCL compared to RRCL at all concentrations (p < 0.0001) (Fig. 1). Analysis of apoptosis using annexin V/PI staining also showed similar results. Both cell lines were treated with rituximab (10 μg/mL) and human serum (20%) for 48 hours, and RRCL showed decreased early and late apoptosis compared with RSCL (Fig. 2). These results revealed that the cells that were gradually exposed to an escalating dose of rituximab acquired rituximab resistance.

To determine the differences in gene expression between RSCL and RRCL, we performed NGS. A total of 1,879 DEGs were identified in RRCL compared to RSCL (|fold change| ≥ 2 and p < 0.05) (Fig. 3). Subsequently, we applied ConsensusPathDB (CPDB) to the 1,879 genes to determine which functional signaling pathway was associated with rituximab resistance. In the over-representation analysis of CPDB, 20 pathways showed statistical significance (q-value < 0.1) (Table 1). As the MAPK signaling pathway is known to be associated with cell survival and tumorigenesis, we identified DEGs related to the MAPK signaling pathway (Table 2). MAPK13, which encodes the p38δ protein, was expressed more than four-fold in RRCL.

We then performed western blot analysis for the major proteins of the MAPK pathway and found that phosphop38 expression was increased in RRCL (Fig. 4). There were four isoforms of p38 MAPK (α, β, γ, and δ), which are known to be different MAPK subgroups, so we performed western blot analysis after administration of two kinds of p38 inhibitors: SB205380, an α and β selective p38 inhibitor and BIRB 796, a panp38 inhibitor. When SB205380 was administered, phosphop38 expression slightly decreased compared to that in controls, who were not administered any drugs. However, phosphop38 expression significantly decreased when BIRB 796 was administered (Fig. 5). Therefore, we elucidated that p38 δ expression was increased in RRCL, and that p38 MAPK activation was associated with rituximab resistance.

Since deferasirox, an orally administered iron chelator, is also known to suppress phosphorylation of the MAPK pathway and inhibit NF-κB activity, we hypothesized that deferasirox could be a candidate for overcoming rituximab resistance. Deferasirox caused a dose-dependent reduction in cell viability in both RSCL and RRCL, and there were no significant differences between the two cell lines (p = 0.4481) (Fig. 6). To determine the combined effects of deferasirox and rituximab, RRCL was treated with gradually increasing concentrations of rituximab (0–10 μg/mL) alone or in combination with 10 μM deferasirox. When RRCL was treated with 1.25 μg/mL rituximab only, 56.6% of the cells survived, but when treated with 1.25 μg/mL rituximab and 10 μM deferasirox, only 28.3% of the cells survived. Cell viability of RRCL was significantly reduced at all of the combination concentrations when rituximab and deferasirox were coadministered compared to when rituximab was administered alone (p < 0.0001) (Fig. 7).

To determine whether there was a synergistic effect of the two drugs, the Chou–Talalay combination index (CI) was performed using CompuSyn software. The CI for deferasirox and rituximab was < 1 (0.43–0.68) at all combination concentrations, indicating that the combination of deferasirox and rituximab had a synergistic effect.