Excessive activation?induced cytidine deaminase accumulated by proteasome inhibitors rescues abnormal class switch in activated B?cell?like diffuse large B?cell lymphoma

Abstract

Activation-induced cytidine deaminase (AID) is an enzyme that plays a crucial role in mediating somatic hypermutation and class-switch recombination (CSR). It has been found to be associated with aberrant immunoglobulin CSR in activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL). In the present study, MG132, a potent proteasome and calpain inhibitor, induced significant cell death in ABC-DLBCL cells and inhibited the growth of ABC-DLBCL cell xenograft tumors. The results also showed that MG132 induced AID accumulation by impairing proteasome degradation of AID. Excessive endogenous AID accumulation was observed in both AID-deficient and C57/BL6 wild-type mice treated with MG132, and apparent CSR of IgM to IgG1, IgG3 and IgE. Upon stimulation of cytokines such as LPS and/or IL-4, ABC-DLBCL cells also showed a noticeable increase in CSR of IgM to IgG1, IgG3 and IgE with decreased AID protein levels. The present study demonstrates that MG132 can induce AID accumulation, which in turn restores dysfunctional CSR in ABC-DLBCL. Using MG132 as a tool, the present study elucidates the anti-lymphoma effect of proteasome inhibitors on ABC-DLBCL by rescuing the abnormal AID-induced CSR.

Keywords: MG132; activated B-cell-like diffuse large B-cell lymphoma; activation-induced cytidine deaminase; class switch recombination; proteasome inhibitor; ubiquitination.

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.

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.

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.

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.

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.

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.