
==== Front
J Cancer Res Clin Oncol
J Cancer Res Clin Oncol
Journal of Cancer Research and Clinical Oncology
0171-5216
1432-1335
Springer Berlin Heidelberg Berlin/Heidelberg

39299959
5939
10.1007/s00432-024-05939-4
Research
Imatinib mesylate reduces c-MYC expression in double-hit lymphoma cells by suppressing inducible cytidine deaminase
Zhang JingCheng 1
Zhou Sheng 2
Jiang SiSi 3
He Fang 1
Tu Yan 1
Hu HuiXian huhuixian@zju.edu.cn

1
1 https://ror.org/04dzvks42 grid.412987.1 0000 0004 0630 1330 Department of Hematology, Affiliated Jinhua Hospital, Zhejiang University School of Medicine, Jinhua, 321000 China
2 https://ror.org/05b2ycy47 grid.459702.d Department of Hematology, Lanxi People’s Hospital, Lanxi, 321100 China
3 https://ror.org/05kqdk687 grid.495271.c Department of Internal Medicine, Yongkang Traditional Chinese Medicine Hospital, Yongkang, 321000 China
20 9 2024
20 9 2024
2024
150 9 42613 8 2024
3 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Background

Double-hit lymphoma (DHL) with c-MYC gene translocation is highly aggressive and has a poor prognosis. In DHL cells, activation-induced cytidine deaminase (AID) promotes antibody class switch recombination (CSR), ultimately leading to c-MYC gene translocation caused by Myc/IgH DNA double-strand breaks. However, currently there is still no method to suppress the expression of AID.

Methods

In this study, we compared the clinical significance of AID expression in DHL, Additionally, two human double-hit lymphoma cell lines were used to analyze the effect of imatinib mesylate on c-MYC in vitro, and the therapeutic effect was also evaluated in xenograft mouse models.

Results

Imatinib mesylate downregulated the AID and c-MYC proteins in patients with chronic myelogenous leukemia associated with DHL. In addition, imatinib mesylate reduced AID and c-MYC expression in SU-DHL-4 and OCI-Ly18 DHL cells. Imatinib mesylate exerted significant inhibitory effects on the proliferation and metastasis of SU-DHL-4 and OCI-Ly18 cells. Finally, imatinib mesylate reduced not only tumor burden in DHL mouse models, but also AID and c-MYC expression in vivo.

Conclusion

These findings reveal that imatinib mesylate effectively reduces the carcinogenic function of c-MYC in DHL, providing novel strategies for developing therapies targeting c-MYC-driven DHL.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s00432-024-05939-4.

Keywords

Activation-induced cytidine deaminase
Imatinib mesylate
C-Myc
Diffuse large B-cell lymphoma
Double-hit lymphoma
issue-copyright-statement© Springer-Verlag GmbH Germany, part of Springer Nature 2024
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pmcIntroduction

Diffuse large B-cell lymphoma (DLBCL) is the commonest type of non-Hodgkin lymphoma, and 6–14% of DLBCL cases have c-MYC gene translocations, often accompanied by BCL-2 or BCL-6 translocations, which is referred to as double-hit lymphoma (DHL) (Zhuang et al. 2022). The outcome of conventional R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy for DHL patients is poor, and intensive chemotherapy regimens or CD19 chimeric antigen receptor T cells (CART), combined with autologous stem cell transplantation (ASCT) consolidation, are often utilized (Friedberg 2017; González Barca 2022). However, a multi-center comparative study reported that DHL cases do not benefit from the above treatment (Clémentine et al. 2015).

The most important factor affecting DHL occurrence in DLBCL is c-MYC gene translocation (Nguyen et al. 2020). The c-MYC gene is upregulated in almost all cancers, controlling the expression of many carcinogenic tumor genes, including tumor cell proliferation, metabolism, differentiation, sensitivity to apoptosis stimulation, and genetic instability, which are closely related to tumor initiation and progression (Zhao et al. 2021; Meyer and Penn 2009). Due to its central role in tumorigenesis, c-MYC has become a promising independent molecular therapeutic target for tumors with c-MYC oncogenes. c-MYC was discovered more than 30 years ago, and anti-c-MYC drugs have been developed, such as antisense oligonucleotides, small interfering RNAs (siRNAs) or phospho-diamine morpholino oligomers (PMO), which induce tumor cell growth arrest, differentiation, or apoptosis (Dhanasekaran and Deutzmann 2022; Fatma et al. 2022). However, the development of drugs directly targeting the c-MYC gene is very limited, so novel treatment methods are needed to block the excessive activity of c-MYC more effectively in cancer cells.

The main reason for c-MYC translocation in B cells is the excessive antibody class switch recombination (CSR). The immunoglobulin (Ig) gene undergoes two DNA alterations to enhance antibody specificity and functionality: somatic hypermutation (SHM) and class switch recombination (CSR) (Durandy 2003; Kumar et al. 2018). To date, activation-induced deaminase (AID) is the only enzyme reported to induce SHM and CSR (Jason et al. 2014; Bednarski and Sleckman 2019). The double-strand breaks (DSBs) triggered by AID replace the constant region of IgM’s heavy chain with other isotypes to achieve CSR, ultimately causing c-MYC/IgH gene double-strand breaks and carcinogenic chromosomal translocations (Matthews et al. 2013; Ramiro 2006). AID’s off-target mutations and subsequent DSBs as well as chromosomal translocations often promote tumorigenesis, especially in multiple B-cell lymphoma types (Pasqualucci 2008; Xu et al. 2010). Current data have shown that imatinib mesylate can be utilized as an AID inhibitor in B-cell lymphoma to reduce CSR in vivo (Kawamata et al. 2012). Imatinib mesylate has been clinically used for more than a decade, with significant success in the treatment of BCR/ABL-positive chronic myelogenous leukemia (CML) and gastrointestinal stromal tumors characterized by activated c-kit, becoming a first-line treatment option (O'Brien et al. 2003; Paul 2015). If imatinib mesylate can inhibit the expression of AID in B-cell lymphoma, it may reduce c-MYC in vivo. However, there is currently no relevant research.

To further characterize the molecular effects of imatinib mesylate, this study found that AID and c-MYC were downregulated in chronic myelogenous leukemia patients with DHL treated with imatinib mesylate. Additionally, two human double-hit lymphoma cell lines were used to analyze the effect of imatinib mesylate on c-MYC in vitro, and the therapeutic effect was also evaluated in xenograft mouse models. In summary, our findings reveal a new mechanism of imatinib mesylate in DHL cells. Due to the close associations among the invasive characteristics of c-MYC in DHL cells, imatinib mesylate may become a novel agent to regulate c-MYC-dependent carcinogenic signaling and be used to develop effective treatment strategies for DHL.

Materials and methods

Patients and cell culture

A total of 44 patients with DLBCL, treated in the Department of Hematology, Jinhua Hospital affiliated to Zhejiang University School of Medicine from January 2019 to December 2021. Among them, 22 patients harboring c-MYC (8q24) and BCL-2 (18q21) or/and BCL-6 (3q27) translocated DLBCL were defined as DHL, while the other 22 patients without c-MYC (8q24) and BCL-2 (18q21) or/and BCL-6 (3q27) translocated DLBCL were defined as Non-DHL. One patient with chronic myelogenous leukemia combined with DHL was also included in this study. These tumor tissue specimens were collected with the consent of the patients or their families, and those tissue collected as part of routine for the purpose of diagnosis. All patients received R-CHOP like chemotherapy on the first-line. Ethical approval was received by the institutional review boards of participating study sites, and all patients provided written informed consent. AID positive cells greater than 30% are considered AIDhigh, while those less than 30% are considered AIDlow. Ki-67 positive cells greater than 70% are considered Ki-67high, while those less than 70% are considered Ki-67low.The study was conducted in accordance with the Declaration of Helsinki. SU-DHL-4 and OCI-Ly18 cells in this study were purchased from Shanghai SLAE. SU-DHL-4 and OCI-Ly18, both DLBCL cells lines, are negative for EBV and carry c-MYC gene translocation (Bittremieux et al. 2019; Johnson-Farley et al. 2015). SU-DHL-4 and OCI-Ly18 cells were cultured in RPMI 1640 medium (Gibco BRL, Rockville, MD, USA) containing 10% fetal calf serum (FCS), L-glutamine (746 μg/ml), sodium bicarbonate (0.2%), streptomycin (90 μg/ml) and penicillin G (90 μg/ml). All cells were cultured at 37 °C in a humid 5% CO2 environment, with the medium replaced every 2 days.

Generation of AID knockout stable cell lines

To generate cell lines with stable AID knockout, the stock solution of the siRNA duplex was prepared according to the manufacturer’s instructions (stock concentration, 100 µM) (Bioneer). The siRNA duplex was transfected into SU-DHL-4 cells using Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) according to the manufacturer’s instructions. A nontarget siRNA duplex (Bioneer) was used as a negative control. The target or nontarget siRNA duplexes with the diluted LipofectamineTM RNAiMAX transfection reagent was added to each cell. After 3 h, the expression levels of AID were measured in SU-DHL-4 cells transfected with target or nontarget siRNA duplexes. After 3 passages in the presence of puromycin, the cultured cells were used for experiments without further colony selection.

Immunofluorescence

For immunofluorescence, SU-DHL-4 cells in the logarithmic phase were centrifuged at 1500 rpm for 5 min, and an appropriate amount of cells were added to each well of a 6-well plate to achieve a density of about 50–80% on the second day. After washing with PBS, the cells were fixed with 4% paraformaldehyde at room temperature. Next, 0.1% Triton X-100 was used to rupture the cell membrane with at room temperature. Then, primary c-MYC antibody (Abcam, Inc. Cambridge, UK) and Alexa Fluor secondary antibody were added, followed by counterstaining with 10 ng/ml DAPI (1:100 ~ 1:500) at 4 °C in the dark and microscopy. We used ImageJ software (Image J version 1.44 software, National Institute of Health, Bethesda, MD, USA) to quantitatively analyze the immunofluorescence.

Quantitative real-time PCR

For real-time PCR, total RNA was extracted with TRIzol reagent (Invitrogen), and cDNA was synthesized with the GoScript Reverse Transcription System and Oligo(dT)15 primer (Promega, Madison, WI). qRT-PCR was performed with the SsoFast EvaGreen Supermix kit (Bio-Rad) and the relative levels of mRNAs for various myeloid differentiation markers were normalized to GAPDH mRNA expression. Primers for c-MYC were forward 5′-GAAAGTCACGCTGGAGACC G-3′ and reverse 5′-TCTCATGCCGTCGCTTGG-3′, and primers for GAPDH were forward 5′-TGAAGCAGGCATCTGAGGG-3′ and reverse 5′-CGAAGGTGGAAGAGTGGGAG-3′. Primers for AID were forward 5′-TGACCTTCAAAGAAAACCACGA-3′ and reverse 5′-CTGGAAGGTGGACAGCGAGG-3′, and primers for GAPDH were forward 5′-TGACGTGGACATCCGCAAAG-3′ and reverse 5′-CTGGAAGGTGGACAGCGAGG-3′. The experiments were performed in triplicate and repeated at least once.

Western blot

SU-DHL-4 cells were lysed with RIPA buffer, and protein concentrations were determined with the BCA Protein Assay Kit. A total of 30 μg of soluble protein was subjected to SDS-PAGE and electro-transferred onto polyvinylidene difluoride (PVDF) membranes. Blots were blocked with 5% fat-free milk for 1 h before incubation with primary antibodies at 4 °C overnight. Appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies were added for 1 h at ambient. Protein bands were detected with SuperSignal Chemiluminescent Substrate (Bio-Rad) and visualized on a Chemi DocTM Touch Imaging System (Bio-Rad). Densitometric analysis of protein abundance was performed with the ImageJ software (Image J version 1.44 software, National Institute of Health, Bethesda, MD, USA).

Cell proliferation assay

The effect of imatinib mesylate on cell proliferation was determined by the MTS assay. Specifically, SU-DHL-4 and OCI-Ly18 cells were co-cultured with imatinib for 48 h after CSR induction as described above. A total volume of 100 μl of each cell suspension was added to each well of a flat 96-well plate, followed by addition of 20 μl of MTS solution (CellTiter 96 Aqueous, Promega, Madison, WI, USA) and incubation at 37 °C for 1 h. Finally, a microplate reader (Benchmark, Bio-Rad) was used for absorbance reading at 490 nm. For the soft agar colony formation assay, 15,000 SU-DHL-4 and OCI-Ly18 cells were seeded in 0.5 ml of 0.3% soft agar on a 2-ml base layer of 0.6% agar. The cells were allowed to settle, and 2 ml of fresh RPMI medium (with or without imatinib at concentrations of 5 μM, 10 μM, and 20 μM) was added to cover the wells. The plate was incubated at 37 °C in a CO2 incubator for up to three weeks. Cell growth medium containing the inhibitor was replaced every four days. At the end of the incubation, the cells underwent 0.25% crystal violet staining. Then, a Leica microscope was used for imaging at × 150.

Assessment of in vivo antitumor activity

The experimental protocol was approved by the Animal Experimental Ethics Committee of Jinhua Hospital affiliated to Zhejiang University School of Medicine (Ethics approval number 2019-147-001). SPF-grade animals were purchased at 4 weeks of age from Shanghai SLAC Company SCXK (Hu) 2017-0005 (certificate number 20170005054151). A total of 2 × 106 SU-DHL4 cells was injected subcutaneously. When the tumor volume reached approximately 100 mm3, SU-DHL-4 lymphoma-bearing nude mice were randomly divided into the PBS and imatinib mesylate groups. According to literature reports imatinib mesylate was administered at a dose of 50 mg/kg/day for 30 days (Kawamata et al. 2012). The suspension of imatinib mesylate was directly administered into the animal stomach by gavage. Tumors in all nude mice were measured with a digital caliper, and their volumes were derived according to the following formula: V tumor = (tumor length) × (tumor width)2/2. Tumor size and body weight were monitored every 5 days. After 30 days of treatment, all nude mice were sacrificed with carbon dioxide, and lymphoma samples and major organs were extracted and fixed with formalin (10%).

Immunohistochemistry

Tumor tissues were fixed in 4% buffered formaldehyde, embedded in paraffin, with a paraffin section thickness of 4–6 μm. sectioned and stained by H&E or immunohistochemistry. Then, the sections were treated with the antigen retrieval buffer in a microwave for 1 min. After washing, the sections were blocked with 10% horse serum at ambient for 1 h. The sections were incubated with primary antibodies against AID (Affymetrix eBioscience, San Diego, CA, USA) and c-MYC (Clone EP1176Y, Abcam; 1:100) overnight at 4 °C. After washing, the secondary antibody was added, and slides were incubated with DAB (Vector Laboratories, Burlingame, CA) for 30 s and counterstained with hematoxylin (Harris) for 30 s. The Vectastain Elite Impression kit was used for blocking, antibody treatment, and color development. A Nikon digital camera mounted on a Leica microscope was used for imaging at × 600.

Fluorescence in situ hybridization

For FISH, based on H&E staining, the tumor cell-rich area was selected as the hybridization area. Probes (including c-MYC dual-color separation probe; QP-30-191096) and DAPI were purchased from Vysis, Inc. (USA). Streptavidin Alexa594 (Molecular Probes, Eugene, OR, USA) and fluorescein isothiocyanate-anti-digoxin (Roche) were used to detect the labeled probes in the red and green spectra, respectively.

Statistical analysis

The sample size (n) indicates the number of independent biological samples in each experiment. Sample sizes and experimental repeats are indicated in Figures and their legends. Generally, all experiments were performed with n ≥ 3, with * indicating p < 0.05 and ** reflecting p < 0.01. Analyses were performed with the GraphPad Prism 9.0 software.

Results

AID promotes the occurrence of double-hit lymphoma

To assess the role of AID in the development of double-hit lymphoma, the expression of the AID protein was examined in double-hit lymphoma (DHL) and ordinary diffuse large B-cell lymphoma (DLBCL) cells. In immunohistochemical staining, AID expression was higher in DHL compared with ordinary DLBCL (Fig. 1A, B). Moreover, DHL expressed higher Ki-67 levels, indicating enhanced DHL tumor proliferation (Fig. 1A and C). Based on AID expression in tissue specimens from DHL patients, a survival curve was generated (Fig. 1D). Patients with high AID expression had significantly reduced survival compared with those with low AID expression (Fig. 1D). In addition, c-MYC expression was associated with patient survival, and individuals with high c-MYC expression had significantly lower survival (Fig. 1H). Next, 18F-FDG PET-CT was used to evaluate the standard uptake value (SUV) of patients with high AID expression in DHL and low AID expression in DLBCL (Fig. 1E), and SUVmax in patients with high AID expression in DHL was significantly higher than that of DLBCL cases (Fig. 1F). Finally, LDH levels were assessed in the peripheral blood of DHL and DLBCL patients, The results showed that LDH was significantly higher in the peripheral blood of patients with high AID expression in DHL (Fig. 1G). In summary, the retrospective analysis showed that AID plays an important role in the development of DHL, and high AID expression in DHL may be considered a marker of poor prognosis.Fig. 1 AID is a marker of poor prognosis in double hit lymphoma. A Immunohistochemical detection of AID and Ki-67 expression in DLBCL and DHL; B The percentage of AID positive cells is in DLBCL and DHL (n = 44); C The percentage of Ki-67 positive cells is in DLBCL and DHL (n = 44); D The survival curves of AIDlow and AIDHigh in double hit lymphoma; E 8F-FDG PET-CT imaging in DLBCL and DHL patients; F The 18F-FDG PET-CT SUVmax in DLBCL and DHL (n = 10); G Serum lactate dehydrogenase (LDH) levels in DLBCL and DHL(n = 20); H The survival curves of c-MYClow and c-MYCHigh in double hit lymphoma. Results are expressed as the means ± SD. *p < 0.05; **p < 0.01

Knock out of AID leads to decrease in c-MYC expression

To assess if AID regulates c-MYC expression in DHL, the DHL cell line SU-DHL-4 with c-MYC translocation was infected with a lentivirus to knock out AID, and the transfection results were verified by qRT-PCR (Fig. 2A). Using immunofluorescence to analyze c-MYC expression in SU-DHL-4 cells after AID knockdown, SU-DHL-4 cells had significantly decreased fluorescence intensity for the c-MYC protein (Fig. 2B). Compared with the control group, without knockdown, knocking down AID significantly decreased the expression of the c-MYC protein (Fig. 2C). In addition, Western blot also demonstrated that AID knockout decreased c-MYC protein expression (Fig. 2D). After AID knockout, c-MYC mRNA levels were decreased (Fig. 2F). The above results indicate that knocking down AID in DHL cells can reduce c-MYC expression.Fig. 2 AID regulates c-MYC in double hit lymphoma cells. A qRT-PCR validation results after knocking down AID(n = 3); B The expression of c-MYC was detected by immunofluorescence assay; C ImageJ software to quantitatively analyze the immunofluorescence of c-MYC protein (n = 3); D Western blot analysis c-MYC protein expression, respectively (n = 3); E ImageJ software to quantitatively analyze the Western blot of c-MYC protein (n = 3). F qPCR analysis indicating c-MYC mRNA expression, respectively (n = 3). Results are expressed as the means ± SD. *p < 0.05; **p < 0.01 vs. NC group

Imatinib mesylate downregulates AID and c-MYC

In a patient with chronic myelogenous leukemia (CML) and DHL, imatinib mesylate at 400 mg was administered once daily for 2 months. Unexpectedly, cervical lymph node lesions in the patient were significantly decreased after treatment with imatinib mesylate. We used immunohistochemistry and fluorescence in situ hybridization (FISH) to detect the AID protein and c-MYC gene expression, respectively, in the tumor tissue specimen of this patient (Fig. 3A). To further confirm the potential of imatinib mesylate in reducing AID and c-MYC expression, SU-DHL-4 and OCI-Ly18 cells were co-cultured with imatinib mesylate. The results showed that imatinib mesylate inhibited AID mRNA expression in SU-DHL-4 and OCI-Ly18 cells (Fig. 3B), and also inhibited c-MYC mRNA expression (Fig. 3C). Using the MTS assay, the effects of imatinib mesylate at concentrations of 2, 5, 10, and 20 μM on the proliferation of SU-DHL-4 and OCI-Ly18 cells were examined. On day 3, imatinib mesylate exerted only a mild inhibitory effect on DLBCL cells at 10 μM, but showed a strong inhibitory effect on cell proliferation at 20 μM. On day 5, in SU-DHL-4 and OCI-Ly18 cells, the 50% inhibitory concentration (IC50) values of imatinib mesylate were 17.0 and 21.2 μM, respectively (Fig. 3D, E). In summary, these results suggest that imatinib mesylate can downregulate AID and c-MYC and reduce the proliferative ability of DHL cells in vitro.Fig. 3 Imatinib inhibits c-MYC expression in double hit lymphoma. A A patient with double hit lymphoma and chronic myeloid leukemia underwent 18F-FDG PET-CT imaging after 2 months of oral imatinib treatment, after oral administration of imatinib, both AID protein and c-MYC gene expression decreased; B, C Imatinib was co cultured with SU-DHL-4 and OCI-Ly18 cells, qPCR analysis indicating AID and c-MYC mRNA expression, respectively (n = 8); D, EIncubate DHL cells with a specified concentration of imatinib in a medium containing 2% FCS for 5 days. On the 5th day, from 10 to 20 μM imatinib has significant inhibitory effects on DHL-4 and OCI-Ly18 cells. The data is the mean ± SEM (n = 6). There was a significant difference (**P < 0.01) compared to the control group without imatinib

Imatinib mesylate inhibits the proliferation of tumor cells

To further demonstrate the inhibitory effect of imatinib mesylate on DHL cell proliferation, the soft agar colony formation assay was performed. The results showed that imatinib mesylate significantly affected the soft agar colony formation abilities of SU-DHL-4 and OCI-Ly18 cells. Inoculate SU-DHL-4 cells onto soft agar in RPMI medium, incubate for two weeks, stain cells with crystal violet, and observe under a microscope × 150 counts of growing colonies. Treatment with 5 μM imatinib mesylate significantly decreased the proliferation of SU-DHL-4 cells (Fig. 4A, B). Additionally, imatinib mesylate also reduced the proliferation of OCI-Ly18 cells (Fig. 4C, D). These results indicate that imatinib mesylate exerts a significant inhibitory effect on DHL cells.Fig. 4 Imatinib inhibits soft agar colony formation in DHL cells. A Icroscope × 150 counts of growing colonies in SU-DHL-4 cells (n = 3); B ImageJ software to quantitatively analyze the counts of growing colonies in SU-DHL-4 cells (n = 3); C Icroscope × 150 counts of growing colonies in OCI-Ly18 cells (n = 3); D ImageJ software to quantitatively analyze the counts of growing colonies in OCI-Ly18 cells (n = 3). Results are expressed as the means ± SD. *p < 0.05; **p < 0.01 vs. compare group

Imatinib mesylate inhibits the growth of tumor nodules in subcutaneous xenograft models

Considering the excellent targeting effect of imatinib mesylate on B-cell lymphoma cells in vitro, we next evaluated the potential anticancer activity of imatinib mesylate in an experimental B-cell lymphoma model. Based on in vitro data, SU-DHL-4 cells were selected to establish a DLBCL xenograft model. When the tumors grew to 100 mm3, imatinib mesylate was administered at a dose of 50 mg/kg/day for 30 days (Fig. 5A). Compared with the control group, daily imatinib (50 mg/kg/day) significantly inhibited tumor growth (Fig. 5B). After treatment with imatinib mesylate in tumor bearing mice, the tumor volume was significantly reduced compared to the control group. (Fig. 5C). To further examine cell proliferation, Ki-67 expression was determined. As shown in Fig. 5D, E, imatinib mesylate treatment decreased Ki-67 expression. Next, dual-color separation FISH probes were used to label the c-MYC gene, and imatinib mesylate treatment decreased c-MYC translocation (Fig. 5F, G). Finally, lactate dehydrogenase was detected in mice, and imatinib mesylate treatment decreased lactate dehydrogenase levels (Fig. 5H). Overall, imatinib mesylate successfully inhibited the proliferation of SU-DHL-4 cells in vivo, indicating strong efficacy in the treatment of malignant B-cell tumors. In this study, the excellent results in the subcutaneous lymphoma model support further investigation examining the safety and therapeutic efficacy of imatinib mesylate in a disseminated lymphoma model.Fig. 5 Effects of treatment of imatinib in DHL-tumor bearing mice. A Establish a xenograft model by injecting SU-DHL-4 cells into male SCID mice and administering imatinib orally for 30 days; B, C Tumor volume in the imatinib treatment group and control group(n = 10); D Immunohistochemical detection of Ki-67 protein expression in mouse tumor tissue(n = 10); E ImageJ software to quantitatively analyze the Ki-67; F FISH detection of c-MYC translocation in mouse tumor tissue(n = 10); G ImageJ software to quantitatively analyze the c-MYC translocation; H LDH expression levels in peripheral blood of mice(n = 10). Results are expressed as the means ± SD. *p < 0.05; **p < 0.01 vs. control group

Discussion

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma diagnosed clinically, and about half of the cases can be treated with the standard R-CHOP chemotherapy regimen (Schmitz et al. 2018). Retrospective studies have found that about one-third of DLBCL patients can survive for a long time, but patients with c-MYC gene translocations show particularly poor outcomes (Pederson et al. 2013).In up to 30% of DLBCL cases, increased c-MYC protein expression indicates that c-MYC changes may be an important secondary transformation event (Green et al. 2012).In B cells, the main reason for c-MYC translocation is that double-strand breaks (DSBs) initiated by AID replace the constant region of IgM heavy chain with other isotypes to achieve CSR (Peycheva and Neumann 2022), ultimately leading to c-MYC gene double-strand breaks and carcinogenic chromosomal translocations (Matthews et al. 2013; Ramiro 2006).

We have previously reported has shown that the expression of AID protein decreases in the serum of chronic myeloid leukemia treated with imatinib mesylate (Zhang et al. 2020). This is particularly important because AID protein can induce damage and mutation of c-MYC, and overexpression of AID is necessary in c-MYC translocation lesions (Robbiani et al. 2008). In Burkitt lymphoma with c-MYC translocation, a large number of studies have shown that AID causes c-MYC translocation (Davide et al. 2013). In addition, in several inert B cell malignancies including chronic lymphocytic leukemia and follicular lymphoma, AID expression increases CSR leading to higher-level transformation has been reported (Shikata et al. 2012; Reiniger et al. 2006). Our before study showed that imatinib mesylate inhibits AID and c-MYC protein expression in chronic myelogenous leukemia (Failed 2020). The possible mechanism is that the alteration of c-MYC by CSR requires the formation of paired DNA breaks, but DNA damage has already occurred in the malignant cells of chronic myelogenous leukemia mature cells (Richardson and Jasin 2000; Kuppers and Dalla-Favera 2001).

Previously, in vitro experiments have shown that imatinib mesylate can significantly inhibit AID expression (Kawamata et al. 2012). However, there has been no further study on the effect of imatinib mesylate on c-MYC. Our in vitro experiments showed that imatinib mesylate at a concentration of 10–20 μM can significantly inhibit AID protein and mRNA, as well as reduce c-MYC mRNA. In addition, we also demonstrated that imatinib mesylate can reduce c-MYC gene amplification in vitro. These findings open up the possibility of controlling the expression and function of c-MYC in DLBCL (and possibly other types) cancer cells through imatinib mesylate. c-MYC has been widely demonstrated to play a key role in the metastasis of various types of cancer cells (Baluapuri and Wolf 2020; Das et al. 2023). Therefore, we analyzed the effect of imatinib mesylate treatment on the invasion and soft agar colony formation ability of DHL cells. We observed that imatinib mesylate effectively blocked the clonogenicity in DHL cells. In addition, we observed that oral administration of imatinib mesylate reduced tumor growth in HDL tumor xenografts and decreased the levels of c-MYC gene and proteins in vivo. The c-MYC gene is overexpressed in most tumor cells, making c-MYC inhibitors promising for treating different types of cancer.

However, direct target therapy of c-MYC has not produced significant clinical benefits (Zhao et al. 2021; Prochownik and Vogt 2010). Due to the lack of evidence that c-MYC inhibitors have therapeutic effects, the recent enthusiasm for direct target therapy of c-MYC has decreased. In this context, our research results reveal the efficacy of imatinib mesylate in vitro and in vivo against c-MYC-translocated DHL cells. Our analysis of c-MYC expression and regulation in DHL not only reveals an unknown mechanism of action of imatinib mesylate, but also opens up a new utilization pathway. In summary, our work strongly supports the potential clinical application value of imatinib mesylate as a treatment for c-MYC gene amplification in DHL in the near future.

Supplementary Information

Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 292 KB)

Acknowledgements

This work was supported by Zhejiang Medical Association Science and Technology Project (No. 2022ZYC-Z3), and the Jinhua Science and Technology Research Program (No.2020-3-043, No.2023-04-067).

Author contributions

ZJC and HHX contributed to the conception and design of this study, had full access to all study data and take responsibility for the integrity of the data and the accuracy of the data analysis. YT contributed to manuscript writing. Zhou Sheng and JSS contributed to critical revision of the manuscript. HF and Zhu Ting-Jun contributed to statistical analysis. All authors contributed to data acquisition, analysis and interpretation and reviewed and approved the final version.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare no competing financial interest. No disclosures were reported by all the authors.

Ethics approval

All animal experiments were approved by the ethics committee of Affiliated Jinhua Hospital, Zhejiang University School of Medicine.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

JingCheng Zhang, Sheng Zhou and SiSi Jiang contributed equally to this work.
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