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Introduction

According to gene expression profiling, diffuse large B-cell lymphoma (DLBCL) can be classified into three main subtypes: i) Activated B-cell-like (ABC); ii) germinal center B-cell-like; and iii) primary mediastinal B-cell lymphoma (1-3). Standard chemoimmunotherapy drugs, such as rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP), as well as novel drugs combined with chemotherapy (R-CHOP+X), have resulted in a notable therapeutic effect in ~40% of patients with DLBCL (4,5). However, the ABC-DLBCL subtype has been found to be more resistant to the aforementioned standard treatments (6,7). One contributing factor to this resistance is a mutation in CD79B, which encodes a subunit of the B-cell receptor (BCR) (8). Several studies have demonstrated that BCR signaling remains active in ABC-DLBCL (2,8,9). Another possible explanation for the resistance of ABC-DLBCL is the inability to undergo class switch recombination (CSR) due to the deletion of the Sµ region in immunoglobulin (Ig) genes (10).

Following antigen stimulation, the BCR triggers a continuous proliferation of B cells. As a result, a significant number of large B cells accumulate in the germinal center (GC), but they fail to differentiate into plasma cells and memory B cells, ultimately leading to the development of DLBCL (2,9). During B cell differentiation, activation-induced cytidine deaminase (AID) is upregulated by TLR signaling through BCR signaling. AID converts cytosine (C) to uracil (U) to generate the U:G mismatch, and the U:G mismatch is subsequently repaired through DNA replication, base excision repair or mismatch repair (11,12). However, incomplete repair leads to mutations or deletions, which are essential in inducing somatic hypermutation (SHM) in the variable region of Ig genes and CSR in the constant region of Ig genes (11,12). Accurate and effective SHM and CSR could facilitate antibody affinity maturation (11,12). ABC-DLBCL is a type of lymphoma that arises from the abnormal process of large B cells that possess high levels of AID, but fail to complete antibody affinity maturation (13-15). In approximately half of patients with ABC-DLBCL, the Sµ region in Ig genes, which is a key target of AID in SHM and CSR, is deleted (10). Generally, it is considered that Sµ deletion or translocation are the direct result of AID-mediated abnormal CSR (10). At present, the majority of studies focus on the role of AID in DLBCL, including deamination induced mutations and chromosome translocations (1,2). It has been widely reported that the overexpression of AID of is associated with B-cell derived malignancies including ABC-DLBCL (13), and AID expression contributes to poor prognosis of patients with DLBCL treated with CHOP-based chemotherapy (16). This indicates a negative function of AID in the clinical treatment of DLBCL. However, previous research has shown that AID has a positive effect in inhibiting DLBCL progression (14). Therefore, restoring and enhancing the ability of AID to mediate antibody affinity maturation may be a viable option for developing effective therapy for ABC-DLBCL.

Proteasome inhibitors, such as MG132, is well-established for their ability to inhibit the ubiquitin-proteasome system, which regulates the levels of numerous cancer-related molecules in cells, such as cyclins, cyclin-dependent kinases, tumor suppressors and nuclear factor-κB (17-20). A previous study demonstrated the potent anti-lymphoma effect of MG132 in DLBCL (13). The present study aims to elucidate the mechanism of the anti-lymphoma effect of MG132 on ABC-DLBCL, which has been shown to be more refractory in clinical settings (1-3). It has been reported that the proteasome pathway plays a role in the post-transcriptional regulation of AID (21). Therefore, the present study aims to explore the ability of MG132 to inhibit AID degradation through the proteasome pathway, and develop the possibility of proteasome inhibitors in the treatment for ABC-DLBCL.

Materials and methods

In vivo tumor cell engraftment and treatment of mice

The present study utilized 10 female NOD/SCID mice (age, 6-8 weeks; Henan Skobes Biotechnology Co., Ltd.) raised in an independent ventilation system with a constant temperature of 22˚C and a light/dark cycle of 12/12 h. These mice (weight, 18-22 g) were maintained in specific pathogen-free conditions at Xinxiang Medical University (Xinxiang, China). A mouse model of human DLBCL was established by subcutaneously injecting 2x107 OCI-LY10 cells into the right flank of the NOD/SCID mice (14). Tumor volume was quantified using the formula: Volume=(length x width2)/2. Once the average tumor volume reached 80-100 mm3, treatment with MG132 commenced. The NOD/SCID mice bearing OCI-LY10 tumors were then allocated into a control group (n=5) or a treatment group (n=5). Mice in the treatment group received intraperitoneal injections of MG132 at a concentration of 50 mg/kg (200 µl), while the control group was administered an equal volume of the solvent (4% DMSO + 30% PEG300 + 20% propylene glycol + ddH2O) via the same route (14). Tumor growth was monitored every two days. All mice were sacrificed 45 days following the initiation of MG132 therapy. Euthanasia was performed using an overdose of sodium pentobarbital (100 mg/kg), administered intravenously to ensure rapid and painless loss of consciousness. The tumors in the control and treatment groups were excised and weighed. The present study was approved by The Ethics Committee of Xinxiang Medical University (Xinxiang, China; approval no. XYLL-2022001253), and all the animal procedures were performed in accordance with the ‘Guide for the Care and Use of Laboratory Animals’ published by the National Institutes of Health (22).

Constructs and cells

The pCas9-AID constructs were successfully generated through the ligation of gRNA targeting AID into the pL-CRISPR.EFS.PAC plasmids (cat. no. 57828; Addgene, Inc.) (12,13). The specific target sequences are detailed in Table SI (NCBI accession no. NM_020661.4). These gRNAs were designed using online software E-CRISPR (http://www.e-crisp.org/) to generate AID knock out. Additionally, the primer sequences utilized for amplifying the AID cDNA to produce the pWPI-AID-GFP lentivirus constructs are provided in Table SII.

The OCI-LY10 ABC-DLBCL cell line was acquired from the BeNa Culture Collection (Beijing Beina Chunglian Institute of Biotechnology; cat. no. BNCC337742). The 293T cells were maintained in the cell bank of Xinxiang Key Laboratory of Tumor Vaccine and Immunotherapy (Xinxiang, China). The AIDKO OCI-LY10 ABC-DLBCL cell line was generated by the AIDKO lentivirus, and the lentivirus was obtained from the supernatant of 293T cells co-transfected by pCas9-AID constructs (or pL-CRISPR.EFS.PAC plasmids as control) and virus generation plasmids including ΔR9 and pVSVG. Both cell lines were cultured in a humidified incubator at 37˚C and 5% CO2, using either Iscove's modified Dulbecco's medium or Dulbecco's modified Eagle's medium (Hyclone; Cytiva) as the base medium, supplemented with 10% fetal bovine serum (MilliporeSigma), non-essential amino acids and a 1% penicillin-streptomycin mixture.

To detect AID ubiquitination, pWPI-AID-GFP and Ub-HA constructs were co-transfected into 293T cells and cultured for 24, 48 and 72 h at 37˚C, respectively. Following these time points, cells were harvested for total protein extraction and subsequent immunoprecipitation analysis.

To assess MG132-induced cell death, cells were incubated with MG132 (10 µM; Selleck Chemicals; cat. no. S2619) for 8 h at 37˚C. The MG132 powder was prepared using DMSO as a solvent. As a control, an equivalent volume of DMSO was used in the treatment of the control cells.

RNA extraction and RT-qPCR

Total RNA was extracted from OCI-LY10 ABC-DLBCL cell pellets using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. 15596026) in accordance with the manufacturer's protocol. Subsequently, cDNA was synthesized utilizing the PrimeScript™ RT Reagent Kit (Takara Bio, Inc.; cat. no. RR037A) using reverse transcription for 15 min at 37˚C and terminated for 5 sec at 85˚C. qPCR was conducted on the QuantStudio™ 5 System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 15 sec at 95˚C, 30 sec at 60˚C and 30 sec at 72˚C, for a total of 40 cycles. The relative mRNA levels were determined using the 2-ΔΔCq method (23) calculated using Microsoft Office 2016, with the expression of β-actin taken as the internal control. The primer sequences employed for the quantification of AID and β-actin transcription are provided in Table SIII.

Flow cytometry and antibodies

To assess the apoptosis rate of OCI-LY10 ABC-DLBCL cells induced by MG132, the treated cells were harvested and twice washed with 1X PBS at 4˚C. The cells were then incubated with anti-PI and anti-Annexin V (BD Biosciences; cat. no. 556547) for 15 min at room temperature. Following incubation, the cells were resuspended in flow cytometry buffer and subjected to analysis via flow cytometry. All data were acquired and processed using a CytoFLEX Flow Cytometer (with BECKMAN CytoExpert version 2.4) (Beckman Coulter, Inc.).

Immunoblot analysis

For protein extraction, the OCI-LY10 cell pellet was lysed in RIPA buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) for 30 min. The RIPA preparation included 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate and 0.1% SDS. The lysates were subsequently centrifuged at 13,000 x g for 20 min at 4˚C, and the protein-containing supernatant was collected. The quality and concentration of the total protein were determined by BCA protein assay Kit (Beyotime Institute of Biotechnology). Protein samples (20-100 µg per lane) were then loaded onto 10% (w/v) Tris-HCl SDS-PAGE gels for electrophoresis, transferred to a PVDF membrane (MilliporeSigma), and subjected to blotting. The membrane was blocked with 5% (w/v) skimmed milk, then probed with an anti-AID antibody (1:1,000; cat. no. 4959; CST Biological Reagents Co., Ltd.) and anti-GAPDH (1:5,000; cat. no. ab9485; Abcam) served as the loading control. The primary antibody incubation was performed at 4˚C overnight. The signal was detected using a goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (1:1,000; cat. no. 31460; Thermo Fisher Scientific, Inc.). The secondary anti-rabbit antibody was incubated with the membrane for 1 h at room temperature. The band signals were captured using the FUSION FX7 system (Vilber Lourmat).

Immunoprecipitation

For protein extraction, the OCI-LY10 cell pellet was lysed in RIPA buffer (Beyotime Institute of Biotechnology) for 30 min. The lysates were subsequently centrifuged at 13,000 x g for 20 min at 4˚C, and the protein-containing supernatant was collected. The quality and concentration of the total protein were determined by BCA protein assay Kit (Beyotime Institute of Biotechnology). Following pre-clearance of the chromatin using Dynabeads Protein G beads (2X; 25 µl for each IP reaction; cat. no. 10003D; Invitrogen; Thermo Fisher Scientific, Inc.), a portion of the aliquot was set aside as the input sample. Subsequently, proteins from 5x106 cells were incubated with 5 µg of a specific antibody or normal goat IgG (Santa Cruz Biotechnology, Inc.; cat. no. sc2346) overnight at 4˚C. Anti-GFP (Abcam; cat. no. ab13970) immune complexes were then captured by incubating with Dynabeads Protein G beads (2X; 25 µl for each IP reaction) (2X; Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. 10003D) for 3 h. The beads were washed at 4˚C using RIPA buffer containing varying concentrations of NaCl. The proteins from the pull down were denatured at 100˚C and loaded onto SDS-PAGE gels for subsequent immunoblotting.

Detection of CSR

The AID-/- mice were a gift from Professor Ji of Xi'an Jiaotong University (Xi'an, China) (14,15), and were bred in the Laboratory Animal Centre of Xinxiang Medical University (Xinxiang, China). The WT and AID-/- C57 mice were confirmed to carry the homozygous disruption of the AID gene according to the generation of AID-/- mice reported by Honjo T (24). C57 and AID-/- mice were administered a tail vein injection of MG132 (10 µg/kg/day) for 24 h (25). Splenic resting B cells were purified by negative selection using anti-CD43-conjugated microbeads (Miltenyi Biotec GmbH) (26). A total of ~3-5x107 splenic resting B cells were obtained from one mouse spleen. Purified B cells were cultured at 5x105 cells/ml in RPMI medium supplemented with 15% fetal bovine serum (MilliporeSigma), non-essential amino acids, penicillin-streptomycin (1%) and β-mercaptoethanol (50 µM). Cells were cultured in a humidified incubator at 37˚C and 5% CO2. Cells were cultured in the presence of LPS and IL-4 for 5 days: i) LPS (50 µg/ml); and ii) LPS (3 µg/ml) plus IL-4 (50 ng/ml), to induce class switching to IgG1, IgG3 and IgE. MG132 (5 µm) treatment was sustained throughout the stimulation process. The antibody class switch was detected using RT-qPCR. The relative mRNA levels were determined using the 2-ΔΔCq method (23) calculated using Microsoft Office 2016, with the expression of β-actin taken as the internal control. The primers for RT-qPCR performed as aforementioned are listed in Table SIV (NCBI accession no. MF430613.1, V00818.1 and KU613484.1).

Bioinformatics

The expression levels of AID in clinical specimens were accomplished through interrogating the online databases GENT2 (http://gent2.appex.kr/gent2/) by searching tissue, blood disease and gene symbols. The significance cut-off level was defined as P<0.05 and |fold-change|>2.

Statistical analysis

Data analysis were performed using GraphPad Prism 8.0 software (Dotmatics). Unpaired t-tests were conducted when comparing datasets of two groups. One-way ANOVA and Dunnett's multiple comparisons test were used to perform the comparison of multiple groups. Data shown are representative of 3 independent experiments and represented as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

Results

Upregulation of AID in DLBCL and the anti-lymphoma effect of MG132 on ABC-DLBCL

The association between the expression of AID and several lymphoma subtypes including Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) was examined using the GENT2 database, which showed that AID expression was significantly higher in the NHL subtype compared with the HL subtype (Fig. 1A). The association between the expression of AID and the subtypes of NHL including follicular lymphoma, mantle cell lymphoma, DLBCL and chronic lymphocytic leukemia were evaluated using the GENT2 database. AID showed a significantly higher expression in DLBCL in comparison with the other NHL subtypes (Fig. 1B). These data indicated that the upregulation of AID is associated with DLBCL progression.

Figure 1 MG132 manifests anti-lymphoma effects on ABC-DLBCL. (A) AID mRNA expression levels in HL and NHL, and (B) the subtypes of NHL (FL, MCL, DLBCL and CLL) were analyzed using an online database (http://gent2.appex.kr/gent2/). (C) Tumor volume of OCI-LY10 tumor bearing mice after MG132 treatment (n=5). All groups compared with the DLBCL group by pairwise comparison. (D) Averaged weights of tumors collected from with or without MG132 treatment groups on day 45 (n=5). Data are represented as the mean ± SD. **P<0.01 and ****P<0.0001. HL, Hodgkin lymphoma; NHL, non-Hodgkin lymphoma; ABC-DLBCL, activated B-cell-like diffuse large B-cell lymphoma; AID, activation-induced cytidine deaminase; FL, follicular lymphoma; MCL, mantle cell lymphoma; CLL, chronic lymphocytic leukemia.

To investigate therapeutic strategies to treat the refractory ABC-DLBCL subtype, MG132 was introduced on the basis of previous studies (14,15). ABC-DLBCL cell xenograft tumor-bearing mice were established to explore the MG132 treatment effect in vivo. Tumor growth was monitored for 45 days, while MG132 treatment was initiated on day 14, when tumor volume had reached 80-100 mm3. The results showed that tumor volume was decreased in the MG132 treated tumor-bearing mice in comparison with the mice in the DMSO-treated group (Fig. 1C; Fig. S1), and the tumor weight showed the same trend (Fig. 1D). These results indicate the inhibitory effect of MG132 on tumor growth in ABC-DLBCL xenograft tumors. Collectively, these results imply an association between MG132 treatment and AID in ABC-DLBCL.

AID suppresses ABC-DLBCL progression

The post-transcription regulation of AID is achieved through the proteasome pathway (21). Considering the significant effect of AID in DLBCL (Fig. 1) and the inhibition of MG132 to ubiquitination through the proteasome pathway (19-21), AID was taken as a key factor to link the mechanism of MG132 induced anti-lymphoma to ABC-DLBCL. The OCI-LY10 ABC-DLBCL cells were transduced using CRISPR/Cas9 with three gRNAs for AID to generate the AID knockout OCI-LY10 (10AIDKO) cell lines. The levels of mRNA and protein for AID were significantly depleted in 10AIDKO cells compared with their wild-type (WT) counterparts and the negative control (EV; Fig. 2A and B). Specifically, two OCI-LY10 cell lines (10AIDKO2 and 10AIDKO3) with efficient AID deficiency were identified.

Figure 2 AID suppresses ABC-DLBCL progression. AID depletion in OCI-LY10 cells was confirmed by (A) immunoblots and (B) RT-qPCR. A total of targeting gRNAs were designed to reduce AID gene transcription. GAPDH protein was used as an internal control for immunoblots. EV indicates OCI-LY10 transduced by the empty vector pL-CRISPR.EFS.PAC. (C) Flow cytometry was performed for the cell death makers Annexin V and PI and stained WT and AIDKO2 cells after DMSO and MG132 treatment (10 µm). Histograms indicate the percentage of dead cells. Data shown are representative of 3 independent experiments. Data are represented as the mean ± SD. **P<0.01. WT, wild type; EV, empty vector; AIDKO, AID knockout OCI-LY10; AID, activation-induced cytidine deaminase; Rel., relative.

To determine whether AID loss had an impact on cellular function, the apoptosis rate of 10AIDKO2 cells was examined using Annexin V and PI staining in presence of DMSO. The AIDKO2 cells presented significantly less Annexin V+PI+ populations compared with the WT cells (Fig. 2C), indicating reduced apoptosis rate of ABC-DLBCL cells after AID deficiency. By contrast, these results showed an anti-lymphoma effect of AID to ABC-DLBCL cells.

MG132 induces AID accumulation by inhibiting ubiquitination

To assess the effect of MG132 on AID degradation through proteasomes, the anti-lymphoma effect of MG132 on ABC-DLBCL was confirmed through inhibiting AID degradation. The ubiquitination of AID after MG132 treatment was detected, with the anti-GFP purified proteins indicating the ubiquitination of AID with MG132 treatment. The results showed that AID underwent degradation without MG132 administration (Fig. S2; Fig. 3A, lanes 1 and 2; Fig. 3B), while MG132 administration resulted in almost a complete disappearance of ubiquitination and a single AID band in the immunoblots (Fig. 3A, lane 3 and lane 4; Fig. 3B), suggesting that MG132 resulted in the inhibition of proteasome degradation of AID. Therefore, the results indicate that MG132 induces AID accumulation by inhibiting ubiquitination.

Figure 3 MG132 induces AID accumulation by inhibiting ubiquitination. Immunoprecipitation of 293T cells cotransfected pWPI-AID-GFP and Ub-HA. (A) The anti-GFP and normal IgG purified proteins were indicated. (B) The anti-GFP purified proteins were indicated. AID, activation-induced cytidine deaminase; Ub, ubiquitin; HA, hemagglutinin.

MG132 modulates AID upregulation and elevates CSR

To further explore the mechanism of MG132 treating ABC-DLBCL, the inhibitory effects of MG132 on AID and the important role of AID in CSR was assessed. Therefore, class switch in WT and AID-/- mice treated with or without MG132 in vivo was detected (Fig. 4A). AID accumulation was observed after MG132 treatment (WT + MG132 group; Fig. 4B), suggesting that MG132 induced AID accumulation, which may be associated with the anti-lymphoma effect of MG132 on ABC-DLBCL.

Figure 4 MG132 inhibited AID ubiquitination. (A) C57 mice and AID-/- mice were administered MG132 through tail intravenous injection. The mice in four treatment groups: i) WT + PBS; ii) WT + MG132; iii) AID-/- + PBS; and iv) AID-/- + MG132 were sacrificed 24 h after injection. The CD43- B cells were further divided into three groups and were stimulated by LPS (50 µg/ml), LPS (3 µg/ml) plus IL-4 (500 U/ml) for 5 days. (B) The AID and GAPDH of four treatment groups was detected by immunoblotting. (C) A scheme depicting the primers used to detect the CSR of IgM to IgG1, IgG3 and IgE assessed by RT-qPCR. CSR occurs between a Sµ switch region and a S region located upstream of another C region, such as Sγ1, which will result in the switching from IgM to IgG1. The combinations of cytokines and LPS that lead to the switching of Ig isotypes are indicated on the top. Ovals indicate switch regions, arrows mark the promoters and rectangles are constant region exons (Cµ, Cγ3, Cγ1 and Cε). Class-switching can result in the formation of a switch circle, from which a hybrid transcript is expressed. These transcripts can be reverse-transcribed for cDNA, and using specific primers (such as arrows marked with C and A for detecting the switching to Cγ1 isotype, with B and A for detecting the switching to Cγ3 isotype or D and A for detecting the switching to Cε), quantitate switch circle formation by qPCR. (D) The class switch of IgM to IgG1, IgG3 and IgE in the resting B cells was identified by RT-qPCR. All groups were compared with the WT LPS + IL4 group by pairwise comparison. Data shown are representative of three independent experiments. Data are represented as the mean ± SD. *P<0.05 and **P<0.01. WT, wild type; AID, activation-induced cytidine deaminase; CSR, class switch recombination; VDJ, variable diversity joining.

After confirmation of MG132 inhibition of AID ubiquitination, the AID-induced class switch in the presence or absence of MG132 was also assessed in vivo. By stimulating with LPS or/and LPS plus IL-4, the purified spleen resting B cells underwent class switch to IgG1, IgG3 and IgE (Fig. 4C). Through stimulation of LPS (3 µg/ml) plus IL-4 (50 ng/ml), a notable elevated class switch to IgG1 was detected in mice with complete AID expression, while no class switch to IgG1 was observed in spleen resting B cells from AID-/- mice treated with MG132 (Fig. 4D). Similar trends were also observed in the detection of class switch to IgG3 and IgE with stimulation by LPS (50 µg/ml) only and LPS (3 µg/ml) plus IL-4 (50 ng/ml) for 5 days (Fig. 4D). As hypothesized, notable class switching to IgE and IgG3 was detected by RT-qPCR in spleen resting B cells of WT mice, while AID-/- mice had no class switching to IgE and IgG3 due to AID deficiency (Fig. 4D). Collectively, MG132 inhibits AID ubiquitination to elevate AID levels, which thus induces apparent CSR.

MG132-mediated AID upregulation rescues abnormal CSR in ABC-DLBCL cells

To evaluate the impact of MG132 on ABC-DLBCL cells, ABC-DLBCL OCI-LY10 cells were treated with MG132 (5 µM) in the presence or absence of AID. The ubiquitination of MG132 treatment at different times (24, 48 and 72 h) was detected by immunoblotting (Fig. 5A). The results showed that MG132-treated WT OCI-LY10 cells, specifically in the 24 h treatment group, showed increased AID accumulation compared with the untreated counterparts (Fig. 5A, lanes 2-4 compared with lane 1). In addition, as time progressed, AID accumulation reduced, which could be attributed to the declined drug efficiency along with increased time (Fig. 5A, lane 2-4).

Figure 5 MG132 induced-AID upregulation restores abnormal CSR in ABC-DLBCL cells. (A) 10WT and 10AIDKO2 cells were treated by MG132 (5 µM) for 24, 48 and 72 h, respectively. The ubiquitin, AID and GAPDH expression of different treatment groups was detected by immunoblotting. (B) Scheme depicting LPS and IL-4 stimulation. 10WT and 10AIDKO2 cells were stimulated by LPS (50 µg/ml) or LPS (3 µg/ml) plus IL-4 (50 ng/ml) for 3 days, respectively. (C) The class switch of IgM to IgG1, IgG3 and IgE in WT and AIDKO was detected by RT-qPCR. All groups were compared with the WT LPS + IL4 group by pairwise comparison. Data shown are representative of three independent experiments. Data are represented as the mean ± SD. **P<0.01, ***P<0.001 and ****P<0.001. WT, wild type; AIDKO, AID knockout OCI-LY10; AID, activation-induced cytidine deaminase; CSR, class switch recombination; Rel., relative.

In order to further detect MG132 induced CSR in ABC-DLBCL, 10WT and 10AIDKO cells were stimulated by LPS or/and LPS plus IL-4 (Fig. 5B). A significant increase in class switching to IgG1, IgG3 and IgE was detected in WT ABC-DLBCL cells stimulated by LPS (3 µg/ml) alone or LPS plus IL-4 (50 ng/ml), but not in AIDKO cells (Fig. 5C). Thus, the data indicate that MG132 inhibits AID ubiquitination to elevate AID levels, thus inducing apparent CSR in ABC-DLBCL cells.

Discussion

At present, the standard treatment strategy for DLBCL in clinical practice is R+CHOP therapy, which involves the combination of rituximab with cyclophosphamide, doxorubicin, vincristine and prednisone (1-3). This approach has led to significant improvements in the survival of patients with DLBCL, with a success rate of ~40% (1-5). However, the standard first-line R-CHOP is used to treat all subtypes of DLBCL instead of individual therapy targeting the subtypes of DLBCL (1-3). The majority of patients eventually suffer from refractory DLBCL, particularly those with the ABC-DLBCL subtype, following relapse in R-CHOP or R-CHOP-like chemotherapy (6,7). To address this issue, various strategies have been developed to improve the outcome of ABC-DLBCL, including the addition of new targets such as CD20 and CD47 to R-CHOP therapy (termed R-CHOP+X) (27,28), inhibitors targeting genetic aberrations involving oncogenic signaling pathways such as BCR, NOTCH, NF-κB and PI3K/AKT, and strategies targeting epigenetic modification factors (2,9,29,30). However, the efficacy of these therapeutic approaches in clinical practice remains unsatisfactory. In the present study, MG132 demonstrated a significant cell killing and tumor inhibition effect, indicating the feasibility of using proteasome inhibitors to treat ABC-DLBCL. However, MG132 is a peptide-like compound with poor protein kinase activity and is not the ideal proteasome inhibitor for in vivo use (18-20). The present study utilized the proteasome inhibitor MG132 as a tool to inhibit the ubiquitination of AID through the proteasome pathway, resulting in the accumulation of AID. These findings shed light on the anti-lymphoma effect of proteasome inhibitors on ABC-DLBCL. Notably, bortezomib, another proteasome inhibitor, is currently being investigated in 10 clinical trials listed on ClinicalTrials.gov (http://www.clinicaltrials.gov) (31-33), but the mechanisms of bortezomib in treating DLBCL have not been investigated. The present study elucidates the mechanism of MG132 treating DLBCL by inhibiting AID degradation. However, as a comprehensive proteasome instead of a 20S proteasome (such as bortezomib), the use of MG132 in clinical treatment may cause systematic targeting but not specific targeting (17-20). Therefore, the clinical translation of MG132 requires more research into the routes of drug administration and the dosage. The results of the present study demonstrate the mechanism of the proteasome inhibitor-mediated anti-lymphoma effect on ABC-DLBCL, showing that proteasome inhibitors elevate AID levels to rescue abnormal CSR in ABC-DLBCL. These findings enhance the potential clinical utility of proteasome inhibitors in treating refractory ABC-DLBCL in the future.

The BCR plays a crucial role in the activation and maturation of B cells. During the development of GC, BCR transduces the antigen stimulation signal, leading to an increase in BCL6 expression and the proliferation of B cells to form the dark zone. AID mediates CSR and SHM in the dark zone (34). As B cells differentiate into plasma cells and memory B cells in the bright zone of GC, the cells with high-affinity to antigens proliferate and leave the GC structure, while those with low-affinity to antigens undergo apoptosis (Fig. 6A) (34,35). Abnormalities in BCR are associated with an increasing number of B-cell malignancies (2,8,9). Mutations in signaling transducing components of the BCR pathway, such as CD79B and CARD11, have been identified in ABC-DLBCL (2,8,9). These mutations result in sustained and chronic active BCR signaling, leading to continuous activation of the NF-κB pathway (34). Sustained BCR signaling prevents large B cells from differentiating into plasma cells and memory B cells efficiently (36). Furthermore, ABC-DLBCL is deficient in CSR due to Sµ region deletions (10). ABC-DLBCL is characterized by the continuous proliferation and accumulation of large B cells, which prevents the cells from undergoing CSR (9). Therefore, increasing the level of AID could be a potential strategy to drive the differentiation of these accumulated large B cells. In the present study, it was demonstrated that using the proteasome inhibitor MG132 to inhibit AID degradation through the proteasome pathway can effectively up-regulate AID protein levels. This, in turn, promotes AID-induced CSR and facilitates the differentiation of GC B cells into mature B cells (Fig. 6B). The effect of MG132 on AID accumulation in ABC-DLBCL was investigated by detecting CSR to IgG1, IgG3 and IgE. The results demonstrate that MG132 mediates AID accumulation and apparent CSR using WT and AID-/- mice. It is hypothesized that AID may not induce CSR in vitro. However, notable CSR was observed in the detection of CSR in ABC-DLBCL cells with the stimulation of cytokines (LPS and/or IL4), indicating MG132 induced AID accumulation to mediate the restoration of abnormal CSR in ABC-DLBCL. This indicates that cytokines such as LPS and IL-4 may simulate the in vivo environment for CSR onset. It has been reported that half of patients with ABC-DLBCL have internal deletions in Sµ (10). Although apparent CSR was observed after LPS and/or LPS + IL4 stimulation, the effect of excessive AID on the Sµ region was not assessed in the present study. This may be due to various processes such as DNA repair, cell division and clone selection (37). Nonetheless, the restoration of CSR is a promising outcome induced by the elevated level of AID in ABC-DLBCL.

Figure 6 Proteasome inhibitor MG132 regulates AID-induced CSR by inhibiting AID ubiquitination in ABC-DLBCL. (A) After stimulating by antigens, BCR transduces signaling, which induces B cells to proliferate and form germinal centers. AID promotes SHM and CSR, which are essential to antibody affinity maturation. ABC-DLBCL is derived from the high level of AID but fails to promote CSR. (B) By treating ABC-DLBCL with the proteasome inhibitor MG132, AID accumulates in ABC-DLBCL, the process of CSR is restored and ABC-DLBCL cells undergo apoptosis. AID, activation-induced cytidine deaminase; BCR, B-cell receptor; CSR, class switch recombination; Ub, ubiquitin; ABC-DLBCL, activated B-cell-like diffuse large B-cell lymphoma; SHM, somatic hypermutation.

AID is an enzyme that induces SHM and CSR of Ig genes (11-13). However, the off-target effects of AID on non-Ig genes or the dysfunction of repairing AID-mediated double-strand breaks can cause point mutations or chromosome translocations (38). The off-target effects of AID is reported to be a leading cause of carcinogenesis, indicating that high levels of AID play a negative role in promoting the occurrence and progression of cancers (39). However, the present study indicates a positive role of excessive AID in treating ABC-DLBCL, providing a novel insight into re-evaluating the function of AID in cancer. Previous research has attempted to reveal the possible mechanism of excessive AID, showing that AID acts as a transcriptional factor to regulate complex gene networks (12,13). This provides a way to identify novel functions of AID in addition to its traditional role in carcinogenesis. In the present study, the positive role of AID in anti-lymphoma was verified, which was an alternative use of its traditional function, whereby excessive AID restores the dysfunction of CSR in ABC-DLBCL. However, a contradiction emerged between the beneficial role ascribed to AID in ABC-DLBCL and the previously reported harmful role of AID in DLBCL (13,16), as well as the detrimental impact of AID inferred from TCGA data. These previous studies and the TCGA data discussed the negative role of AID in DLBCL mainly through the results of the off-targets of AID (40), but omitting its mechanism in ABC-DLBCL with abnormal processing of antibody affinity maturation. The findings of the anti-lymphoma effects of AID in the present study may imply that the elevated AID expression in DLBCL could be a compensatory mechanism to counteract detrimental factors. Nonetheless, the robust expression of AID was insufficient to halt the progression of ABC-DLBCL. The research introduces a novel approach involving the artificial enhancement of AID levels through proteasome inhibition, aiming to counteract negative influences and exert an anti-tumor effect. Therefore, understanding the dual role of AID in carcinogenesis, elucidating the mechanism of AID in specific cancers and exploring the alternative function of AID (except for its mutational function) could lead to the development of future cancer treatments.

In conclusion, the findings of the present study have aided the understanding of the mechanisms underlying how proteasome inhibitors can effectively treat refractory ABC-DLBCL. The crucial role of MG132 in inducing AID accumulation through inhibition of AID degradation via the proteosome pathway was identified, and it was demonstrated that the elevation of AID levels rescues abnormal CSR. These results suggest the potential therapeutic value of AID in treating ABC-DLBCL, and the promising application of proteasome inhibitors in clinical therapy strategies for AID-associated DLBCL. However, there are limitations that warrant acknowledgment. Firstly, the findings on the mechanisms underlying the effect of MG132 on ABC-DLBCL was discussed from the aspect of AID-mediated antibody affinity maturation in ABC-DLBCL, and it would be beneficial to further investigate the systematic regulation mechanisms of MG132 in treating ABC-DLBCL. Secondly, for MG132 clinical translation, the potential side-effect and patient variability requires further investigation.

Supplementary Material

Subcutaneous tumors taken from OCI-LY10 ABC-DLBCL cell tumor bearing mice treated with DMSO or MG132. ABC-DLBCL, activated B-cell-like diffuse large B-cell lymphoma.

GFP fluorescence of 293T cells co-transfected by pWPI-AID-GFP and Ub-HA observed by fluorescence microscope. AID, activation-induced cytidine deaminase; Ub, ubiquitin; HA, hemagglutinin.

Sequences of the destroyed alleles in the AID gene.

Sequences of primers for amplifying AID cDNA.

Sequences of primers for quantitative AID transcription.

Reverse transcription-quantitative PCR primers for detecting antibody class switch.

Acknowledgements

The authors would like to thank Professor Yanhong Ji of Xi'an Jiaotong University (Xi'an, China) for providing the AID-/- mice used in the present study.

Funding

Funding: This research was funded by The Key Scientific Research Foundation of the Higher Education Institutions of Henan Province (grant no. 22A320041), Natural Science Foundation of Henan province (grant no. 232300421189) and The Undergraduate Innovation and Entrepreneurship Training Program of Henan province (grant no. 202310472044).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

JJ conceptualized the study; JJ and ZL performed the study methodology; ZL searched the online software; CX performed the formal analysis; ZL and ZW carried out the experiments; ZL, CX, ZW, ZLiu obtained the resources; CX and ZL curated the data and wrote the original draft; ZL reviewed and edited the manuscript, and supervised the study; JJ visualized the study, performed project administration and acquired the funding. All authors have read and approved the final version of the manuscript. ZL, CX, ZW, ZL and JJ confirm the authenticity of all the raw data.

Ethics approval and consent to participate

The present study was approved by The Ethics Committee of Xinxiang Medical University, China (Xinxiang, China; approval no. XYLL-2022001253). All the animal procedures were performed in accordance with The ‘Guide for the Care and Use of Laboratory Animals’ published by the National Institutes of Health (22).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References