Imatinib mesylate directly impairs class switch recombination through down-regulation of AID: its potential efficacy as an AID suppressor

Activation-induced cytidine deaminase (AID) is essential for class switch recombination (CSR) and somatic hypermutation.¹  Deregulated expression of AID acts as a genomic mutator and can contribute to tumorigenesis through genomic recombination and aberrant somatic hypermutation.^2-4  E2A, which harbors 2 binding sites in the AID promoter, is the crucial transcription factor for induction of AID.⁵  Imatinib mesylate (IM) has diverse immunomodulatory effects,^6,7  including reduction of T-cell proliferation and inhibition of T-cell effector functions.^8,9  Previously, we reported that serum titers of IgG and IgA, but not IgM, were significantly lower in chronic myeloid leukemia patients treated with IM versus those treated with IFN-α,¹⁰  suggesting that IM impairs CSR. In the present study, we investigated the effects of IM on CSR both in vitro and in vivo. Here, we present evidence that IM inhibits CSR through down-regulation of AID expression in splenic B cells.

Eight-week-old mice were immunized as previously reported,¹  with or without 50 mg/kg imatinib mesylate. The experiments were approved by the Committee of Animal Care at the Institute of Medical Science, University of Tokyo.

Immunostaining for AID was performed on frozen sections following the manufacturer's instructions using an AID antibody (H-80; Santa Cruz Biotechnology).

Primer sequences, reagents, and more detailed methods are shown in supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article).

CSR is induced in splenic B cells by stimulation with IL-4 and lipopolysaccharide (LPS).¹¹  After stimulation with IL-4 and LPS for 72 hours, IM decreased the proportion of IgG1-positive B cells dose-dependently. The proportion of B cells expressing surface IgG1 was approximately 16% without IM but was significantly reduced to approximately 3% with 10μM IM (Figure 1A). In the present culture system, only B cells can survive and proliferate,¹  suggesting that IM may act directly on B cells and inhibit their CSR.

Next, we examined expression of the germline transcript directed by the I promoter of IgG1 and AID, both of which are essential for CSR after B-cell stimulation.¹²  Expression of AID was suppressed by IM dose-dependently (Figure 1B), whereas the IgG1 germline transcripts were not decreased by IM (Figure 1C). Likewise, IgA CSR in CH12F3-2A cells was impaired by IM in a dose-dependent manner (Figure 1D). These results showed that AID, but not the germline transcript, was responsible for inhibition of CSR by IM. BrdU, CFSE, and annexin V analysis revealed that IM affected proliferation but not apoptosis (Figure 1E). Importantly, 1μM of IM did not decrease proliferation but down-regulates AID (Figure 1B,E). In addition, 5-fluorouracil decreased proliferation but did not down-regulate AID (supplemental Figure 1), suggesting that proliferation is not necessarily coupled with expression of AID. Therefore, it is possible to differentiate the effect of IM on proliferation and AID expression.

To further confirm that CSR is impaired by IM through down-regulation of AID in vivo, immunohistochemical analysis was performed on splenic tissues from nonimmunized and sheep red blood cells–immunized C57BL/6 mice with or without IM treatment (Figure 1F). The individual germinal centers from SRBC-immunized IM (+) mice were significantly smaller than those from SRBC-immunized IM (−) mice and comparable with those from nonimmunized mice (Figure 1F). As expected from these findings, AID expression, which is induced in germinal center-activated B cells, was barely detectable in the spleens of IM-treated mice but was strongly positive in those of nontreated mice. In addition, IM significantly suppressed AID expression, even in the residual germinal centers. Expression of AID was confirmed by real-time RT-PCR analysis. The results of IgG1 expression did not conflict with these results (Figure 1G). Compatible with the results obtained by in vitro stimulation of spleen cells, IM down-regulated expression of IgG1 as well as AID. Although enlargement of germinal center formation has been reported in AID knockout mice,¹  it is assumed that the immunomodulatory effects of IM on B cells, T cells, and dendritic cells^6,7  resulted in impairment of germinal center formation in our system.

Furthermore, we investigated whether ectopic expression of AID could rescue inhibition of CSR by IM. IgG1 expression in spleen cells decreased with IM treatment, whereas ectopic expression of AID completely rescued impairment of CSR under the condition that cell proliferation was suppressed by IM (Figure 2A-B). The results indicated that impairment of CSR by IM was at least in part the result of down-regulation of AID.

Finally, we examined the mechanism of down-regulation of AID by IM. Recently, Tran et al reported that Aicda regulation involved derepression by several layers of positive regulatory elements in addition to the 5′-promoter region.^5,13  Promoter region 2 in the first intron contains the functional binding elements for the ubiquitous silencers c-Myb and E2f and for the B cell–specific activators Pax5 and E2A (Figure 2C).^5,13  Surprisingly, all of these transcription factors were down-regulated by IM. Among them, expression of E2A was most markedly reduced (to 1 of 500) by IM (Figure 2D). We further found that levels of E2A protein as well as E-box binding activity were markedly reduced by IM (Figure 2E), suggesting that down-regulation of E2A by IM causes significant suppression of AID.

For the first time, our findings elucidate a mechanism of hypogammaglobulinemia caused by IM, which has been observed frequently in IM-treated chronic myeloid leukemia patients.^8,9  Its adverse effects as well as the immunomodulatory functions of each drug and their underlying mechanisms must be examined in more extensive studies.

AID was previously reported to be induced by BCR-ABL1 in Ph1⁺ pre B-ALL cell lines and inhibited by IM through ID2 up-regulation. Interestingly, neither PAX5 nor E2A showed changes in expression.¹⁴  In the present study using normal mature B cells, PAX5 and E2A levels were significantly decreased by IM, whereas ID2 was not increased (data not shown). PDGFR¹⁵  and c-kit,¹⁶  kinases that are also inhibited by IM, were not expressed in mature B cells. Together, these results could be induced by the off-target multikinase inhibitory effects of IM. The results of microarray analysis (supplemental Table 1; supplemental Figures 2-3) are consistent with this hypothesis. All microarray data are available for viewing on the Gene Expression Omnibus under accession number GSE35559.

Inappropriate expression of AID affects many diseases, such as malignancy and autoimmune diseases.^17,18  It probably also affects allergic disorders because AID is also essential to CSR from IgM to IgE, deregulation of which is an important causative factor of allergic disorders. The results of the present study suggest that IM, which has been used safely for several decades in clinical settings, can be used for various diseases involving AID. Indeed, dramatic resolution by IM has been reported in several cases of rheumatoid arthritis or asthma complicated with chronic myeloid leukemia.^19,20 

In conclusion, suppression of AID by IM is responsible for CSR impairment, leading to the frequent adverse effects of IM. IM may also be clinically useful as an AID suppressor.

The authors thank Dr Tasuku Honjo, Dr Toru Nakano, Mrs Bidisha Chanda, Mr Haruo Onoda, and Mr Keisuke Takahashi for critical comments or technical assistance. The array analysis was performed by Mr Masayuki Tanaka and Hideki Hayashi (Education and research Support Center, Toaki University).

This work was supported by the Japan Society for the Promotion of Science, Takeda Science Foundation, Sagawa Cancer Foundation, The Foundation for Cancer Research, Kobayashi Cancer Research Foundation, Mochida Science Foundation, Sankyo Science Foundation, and Shiseido Science Grant for the female researcher (to A.K.).

Contribution: T.K., J.L., T.S., A.K., and M.T. designed, performed, and analyzed the experiments and wrote the manuscript; T.T., H.N., Y.A., K.Y., N.O., and N.N. contributed vital reagents; K.A. collected the clinical samples; and A.T. supervised the research.

Conflict-of-interest disclosure: A.T. has received honoraria and research funding from Novartis. The remaining authors declare no competing financial interests.

Correspondence: Arinobu Tojo, Department of Hematology-Oncology, Research Hospital, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; e-mail: a-tojo@ims.u-tokyo.ac.jp; and Ai Kotani, Tokai University Institute of Innovative Science and Technology, Medical Science Division, Shimokasuya 143, Isehara City, Kanagawa Prefecture 259-1193, Japan; e-mail: ka102009@tsc.tokai-u.ac.jp.