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.