The Rag1 mutant mouse pro-B (D345) cell line was established by infection of bone marrow cells from BCL-2 transgenic mice with the v-Abl retrovirus, which was was kindly provided by Dr David Schatz (Yale University, New Haven, USA) and stored in our laboratory (35). The D345 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), non-essential amino acids and 1% penicillin-streptomycin (all from Hyclone; Cytiva) and β-mercaptoethanol (50 µM) at 37°C with 5% CO₂. The 293T cells were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM (Hyclone; Cytiva) supplemented with 10% FBS, non-essential amino acids and penicillin-streptomycin (1%) at 37°C with 5% CO₂.

MSCV vector co-expressing human BCR-ABL1 (p210) and hCD4 (MSCV-BCR-ABL1-IRES-hCD4) and MSCV vector expressing hCD4 have been previously described (36). 293T cells were transfected with MSCV vectors and PKAT2 package vector using X-tremeGENE HP DNA transfection reagent (Roche Diagnostics) at 37°C. After 48 h of transfection, viral supernatants were collected, filtered, and stored at −80°C.

BCR-ABL1-transformed pro-B cells and the control cells were obtained by transduction of D345 cells with pMSCV-BCR-ABL1-hCD4 or pMSCV-EV-hCD4 viral supernatants with 1 µg/ml polybrene; then the six-well dishes were spun at 1,000 × g for 1.5 h at room temperature. The medium was changed after 4 h and incubated at 37°C. After 72 h, the cells were incubated with APC-conjugated anti-human CD4 antibody (cat. no. 561840; BD Biosciences) at a 1:10 dilution for 20 min at 4°C, and sorted by BD FACSAria II (BD Biosciences). Flow cytometric data were analyzed with Kaluza Analysis Software 2.1 (Beckman Coulter, Inc.).

A Cell Counting Kit-8 (CCK-8) assay was used to detect cell growth according to the manufacturer's instructions. The cells were suspended with FBS-deficient RPMI-1640 medium supplemented with non-essential amino acids, penicillin-streptomycin, and β-mercaptoethanol (50 µM). Six replicates of 2×10⁴ cells were seeded in a 96-well plate with 100 µl/well. Cells were cultured for 24, 48 and 72 h, then incubated with 10 µl CCK-8 solution (product no. FXP132-500; Beijing 4A Biotech Co., Ltd.) for 2 h. The absorbance at 450 nm was measured by a microplate reader.

The CCK-8 assay was also used to measure cell viability. Cells (2×10⁵) were seeded in a 96-well plate with 100 µl/well in six replicates. Cells were treated with vehicle (PBS) or imatinib (0, 3, 6 and 9 µM) for 24 h. Subsequently, the cells were incubated with 10 µl CCK-8 solution for 2 h. The absorbance values at 450 nm were measured by a microplate reader.

The pL-CRISPR.EFS.PAC was a gift from Dr Junjie Zhang of the University of Southern California (Los Angeles, USA). The AID, BLNK and FOXO1 single guide RNA (sgRNA) were designed at the CRISPR design website from Zhang laboratory (http://crispr.mit.edu/) and sequenced (Sunny Biotech Co., Ltd.), sgRNA that does not target the genome was used as a control, which was acquired from the Mouse GeCKOv2 Library (37,38). The gRNA sequences are listed in Table SI. The 293T cells were then independently transfected with the pCas9-AID, pCas9-BLNK and pCas9-FOXO1, pCas9-non-targeting guides along with ΔR9 and pVSVG helper plasmids using the X-tremeGENE HP DNA transfection reagent (Roche Diagnostics) at 37°C. The viral supernatants were collected 48 h after transfection.

BCR-ABL1-transformed pro-B cells were each infected with control, Aid-, Blnk-, or Foxo1-knockout viral supernatants in the presence of 1 µg/ml polybrene. Then the six-well dishes were spun at 1,000 × g for 1.5 h at room temperature. The medium was changed after 4 h and incubated at 37°C. After 72 h, cells were selected with puromycin (0.6 µg/ml) to enrich the transduced cells.

Cells (1×10⁶) were treated with imatinib (0, 3, 6 and 9 µM), and after 24 h, the cells were collected and washed twice with cold PBS, and incubated for 20 min at room temperature in 1:20 diluted PE-conjugated Annexin V and anti-7-aminoactinomycin D (7-AAD) (cat. no. 559763; BD Biosciences), according to the manufacturer's instructions. Then, the cells were analyzed by flow cytometer (Beckman Coulter, Inc.). The Aid KO and Aid WT BCR-ABL1-transformed pro-B cells were starved in FBS-free medium for 24 h, and measured for apoptosis by flow cytometry. Flow cytometric data were analyzed with Kaluza Analysis Software 2.1 (Beckman Coulter, Inc.).

Proliferation assays were performed using the BrdU kit (cat. no. 552598; BD Biosciences) according to the manufacturer's instructions. Briefly, a population of 1×10⁶ cells were cultured in 6-well plates and labeled with BrdU labeling reagent for 45 min before harvesting. The cells were centrifuged at 300 × g for 5 min at 4°C, then fixed and permeabilized with Cytofix/Cytoperm buffer and Cytoperm permeabilization buffer plus. Next, cells were treated with DNase for 1 h at 37°C and further incubated with APC-conjugated anti-BrdU for 20 min at room temperature. BrdU incorporation was measured using a flow cytometer (Beckman Coulter, Inc.). Flow cytometric data were analyzed with Kaluza Analysis Software 2.1 (Beckman Coulter, Inc.).

The total RNA of cell pellets was isolated by TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Before cDNA synthesis, 1 µg of total RNA was digested with 1 U/µl RNA-free recombination DNAse I. The cDNA was then transcribed with the PrimeScript™ RT reagent Kit (TaKaRa Bio, Inc.) according to the manufacturer's instructions. Quantitative PCR experiments were performed in triplicate with SYBR-Green dye (TaKaRa Bio, Inc.) on an Mx3000P qPCR system (Agilent Technologies, Inc.). All data were analyzed using the 2-ΔΔCq (relative quantification) method (39). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 10 min; 40 cycles at 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec; and a final step at 95°C for 1 min, 55°C for 30 sec and 95°C for 30 sec. Gene expression was normalized to GAPDH. The primer sequences used for quantitative PCR are listed in Table SII. The gene expression levels of B-cell differentiation components were normalized to the GAPDH level in the corresponding cell line and represented as a heatmap using GraphPad Prism 8 (GraphPad Software, Inc.).

The cell pellets were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris pH 7.4, 1% Triton X-100, 0.5% NaDoc, 10% glycerol, and 2.5% sodium deoxycholate) with protease and phosphatase inhibitors. Protein concentration was determined using a BCA kit (cat. no. P0010; Beyotime Institute of Biotechnology) and Multiskan™ FC Microplate Reader (Thermo Fisher Scientific Inc.), and analyzed using Skanit software version 3.1 (Thermo Fisher Scientific Inc.). A total of 60 µg of proteins were loaded onto 12% sodium dodecyl sulfide-polyacrylamide gel electrophoresis (SDS-PAGE) for electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; cat. no. IPVH00010; 0.45 µm; EMD Millipore). Membranes were blocked in 5% milk solution for 2 h at room temperature, and then incubated overnight at 4°C with indicated primary monoclonal antibodies with their respective dilutions (Table SIII). The membranes were then incubated with goat anti-rabbit IgG (H+L)-HRP (1:5,000; cat. no. 31466; Thermo Fisher Scientific Inc.), goat anti-rat IgG (H+L)-HRP (1:5,000; cat. no. 31470; Thermo Fisher Scientific Inc.) and goat anti-mouse IgG (H+L)-HRP (1:5,000; cat. no. 31431; Thermo Fisher Scientific Inc.) for 1 h at room temperature, and blots were visualized with Western Lighting Pro ECL (Fusion FX5; Vilber).

Cells (1×10⁴) were affixed to frosted X slides and fixed with 4% paraformaldehyde solution for 15 min at room temperature, followed by permeabilization with 0.2% Triton X-100 for 10 min at room temperature. The slides were blocked with 5% BSA and 10% horse serum in PBS at room temperature for 1 h and were incubated with anti-γH2AX (1:200; product no. 9718; Cell Signaling Technology, Inc.) at 4°C overnight. After three rinses with PBS, the cells were incubated with anti-rabbit IgG (H+L) Alexa Fluor^® 555 Conjugate (1:500; product no. 4413; Cell Signaling Technology, Inc.) for 1 h. Then, the cells were washed twice and stained with 1 µg/ml DAPI for 10 min at room temperature. Images were acquired with a confocal microscope (Leica TCS SP8 STED 3X; Leica Microsystems, Inc.) at a magnification of ×40 and processed with ImageJ v.1.8.0 software (National Institutes of Health).

Genomic DNA was extracted from 5×10⁶ cells using the DNA Extraction Kit (cat. no. D824A; TaKaRa Bio, Inc.) according to the manufacturer's instructions. The ABL1 kinase portion of the BCR-ABL1 gene was amplified with two rounds of PCR. In the first round, the BCR-ABL1 fragments were amplified using BCR- (exon 13) and ABL1- (exon 9) specific primers to prevent co-amplification of normal ABL1. Primers for the second round of amplification focused on the ABL1 kinase domain (exons 3-8). The genomic fragments were amplified using high fidelity DNA polymerase KOD-Plus-Neo (code no. KOD-401; Toyobo Life Science). PCR products were purified with the Gel Extraction Kit (SKU no. D2500-01; Omega Bio-Tek, Inc.) and cloned into the pMD^® 18-T vector (cat. no. 6011; TaKaRa Bio, Inc.). Clones were sequenced commercially (Sunny Biotech Co., Ltd.). PCR primers used for amplification are listed in Table SIV.

Unpaired t-test was performed for comparison of two groups and one-way ANOVA followed by Bonferroni's post hoc tests were performed for multiple comparisons using the SPSS 18.0 statistical software package (SPSS, Inc.). Results were presented as the mean ± SEM. P<0.05 was considered to indicate a statistically significant difference.

To demonstrate that Bcr-Abl kinase was sufficient to induce pro-B cell transformation, the retroviral vector pMSCV-BCR-ABL1-IRES-hCD4 and empty vector pMSCV–IRES-hCD4 were individually introduced into the D345 cell line. The D345 cell line harbors the Rag1 catalytic mutant (D708A), which retains the native ability to interact with Rag2 and bind to DNA but lacks catalytic activity, thus failing to induce the rearrangement of immunoglobulin genes, ultimately leading to arrested B-cell development at the pro-B stage (35). After flow cytometric sorting, over 90% of infected cells were positive for hCD4 expression (Fig. S1A and B). A large amount of Bcr-Abl expression was detected in pro-B cells infected with retroviral vectors carrying BCR-ABL1 (Fig. 1A). Faster growth of BCR-ABL1-transformed pro-B cells than the control cells in the absence of nutrition for 72 h was observed (Fig. 1B). After treatment with the tyrosine kinase inhibitor, imatinib, at the indicated concentrations for 24 h, the cell viability of BCR-ABL1-transformed pro-B cells significantly decreased compared to that of the control cells (Fig. 1C). The percentage of apoptotic cells was quantified by 7-AAD and Annexin V staining, and BCR-ABL1-transformed pro-B cells exhibited higher apoptotic cell populations than the control cells (Fig. 1D). Moreover, phosphorylated Crkl (p-Crkl) is the main target of Bcr-Abl tyrosine kinase and serves as the connector for downstream effector molecules (40). The p-Crkl levels as indicators of Bcr-Abl kinase activity were therefore determined, and it was revealed that p-Crkl was upregulated following BCR-ABL1 overexpression, while its levels were significantly reduced after imatinib treatment (Fig. 1E). The data revealed that Bcr-Abl kinase activity was required to promote pro-B cell growth in vitro.

Considering the possibility that Bcr-Abl kinase enhances cell survival by activating the PI3K/Akt pathway to induce Foxo1 phosphorylation in BCR-ABL1-transformed pro-B cells (29), western blotting was performed to examine the phosphorylation of Akt and Foxo1 in BCR-ABL1-transformed pro-B cells. The data indicated that the levels of p-Akt and p-Foxo1 were increased in BCR-ABL1-transformed pro-B cells compared with that in control cells but were significantly reduced in the presence of 6 µM imatinib (Fig. 1E). Furthermore, the expression of Foxo1 target genes BIM, TRAIL, CDKN1B, and GADD45α, which are critical apoptosis and cell-cycle mediators (30), were upregulated after imatinib treatment in BCR-ABL1-transformed pro-B cells (Fig. 1F). Collectively, these results are consistent with previous studies revealing that Bcr-Abl kinase stimulates the PI3K/Akt pathway to inactivate Foxo1, leading to aberrant proliferation of pro-B cells (8,41,42).

Previous studies have reported that aberrant Aid expression depends on Bcr-Abl kinase activity in BCR-ABL1⁺ B-ALL (14,43). Consistent with these findings, the mRNA and protein expression levels of AID gene were induced in BCR-ABL1-transformed pro-B cells, while imatinib treatment suppressed Aid expression (Fig. 2A and B). When Aid was deleted using CRISPR/Cas9 gene-editing, the protein level of Aid was significantly reduced in Aid-knockout (KO) BCR-ABL1-transformed pro-B cells compared to BCR-ABL1-transformed pro-B cells infected with non-targeting control viral supernatants (hereinafter named WT cells) (Fig. S2). Cell growth and apoptosis assays were used to determine the role of Aid in the cell survival of BCR-ABL1-transformed pro-B cells. By using the CCK-8 assay, significant inhibition of cell growth was revealed in Aid KO BCR-ABL1-transformed pro-B cells in the absence of nutrition for 72 h (Fig. 2C). The proportion of apoptotic cells was significantly increased in Aid KO cells compared to WT cells (Fig. 2D). These results revealed that Aid can enhance BCR-ABL1-transformed pro-B cell survival.

The Rag mutant cell line was utilized to directly examine the contribution of dysregulated Aid on genomic instability. The γH2AX foci, a reliable marker of DNA double-strand breaks (DSB), was assessed through immunofluorescence to characterize the genomic instability (44). As anticipated, increased γH2AX foci per cell were observed in BCR-ABL1-transformed pro-B cells compared with control cells (Fig. 2E). However, the γH2AX foci per cell were less numerous in Aid-KO BCR-ABL1-transformed pro-B cells than WT cells (Fig. 2F), suggesting that Bcr-Abl kinase activity-mediated aberrant Aid expression contributed to genomic instability in BCR-ABL1-transformed pro-B cells. It was concluded that Bcr-Abl kinase activity initiated malignant transformation characterized by pro-B cell proliferation and genomic instability.

To investigate B-cell differentiation function of components in BCR-ABL1-transformed pro-B cells, the expression of eight key components associated with the pre-BCR differentiation pathway was analyzed. The results revealed that the mRNA and protein expression levels of BLNK and FOXO1 were upregulated in BCR-ABL1-transformed pro-B cells compared to control cells (Figs. 1E and 3A and B). To ascertain the exact role of Blnk and Foxo1 in BCR-ABL1-transformed pro-B cells, the Blnk- or Foxo1-KO BCR-ABL1-transformed pro-B cell lines were generated using CRISPR/Cas9-based gene-editing technology. The Blnk KO3 cells and the Foxo1 KO3 cells exhibited the greatest knockout efficiency (Fig. 3C-3F). After being treated with imatinib, the depletion of Blnk or Foxo1 in BCR-ABL1-transformed pro-B cells revealed the higher levels of viable cells and lower levels of apoptotic cells, indicating that either Blnk or Foxo1 deletion improved the anti-apoptosis ability (Fig. 4A and B). In addition, the cell growth ability of Blnk-KO3 or Foxo1-KO3 BCR-ABL1-transformed pro-B cells was assessed by the BrdU incorporation assay and the results revealed that depletion of Blnk or Foxo1 induced a significant increase in the S phase and a reduction in the G0/G1 phase (Fig. 4C). Collectively, these observations indicated the inhibitory effects of Blnk and Foxo1 on cell growth in BCR-ABL1-transformed pro-B cells.

The effects of Blnk or Foxo1 deletion on genomic instability were then further determined in BCR-ABL1-transformed pro-B cells. Increased levels of γH2AX were detected in Blnk- or Foxo1-deficient BCR-ABL1-transformed pro-B cells (Fig. 5A). Furthermore, higher AID transcript levels were accompanied by a corresponding change of protein in Blnk- or Foxo1-deficient BCR-ABL1-transformed pro-B cells (Fig. 5B-E). To analyze the somatic mutations of non-Ig genes dependent on Aid, genomic DNA was purified from WT, Blnk-KO3, and Foxo1-KO3 BCR-ABL1-transformed pro-B cells. An 852-bp segment located in the ABL1 kinase domain (exons 3-8), a known target of Aid, was amplified, sequenced, and analyzed for point mutations (Fig. S3A-C). The data revealed that the ABL1 region exhibited higher mutation frequencies in Blnk-KO3 cells (6.607×10⁻⁵ mutations per bp) and Foxo1-KO3 cells (8.533×10⁻⁵ mutations per bp) than that in WT counterparts (2.171×10⁻⁵ mutations per bp) (Fig. 5F; Table SV). Thus, the results indicated that Blnk or Foxo1 negatively regulated the genomic instability in BCR-ABL1-transformed pro-B cells.

As Blnk and Foxo1 decreased cell growth ability and genomic instability in BCR-ABL1-transformed pro-B cells, it was hypothesized that Blnk and Foxo1 regulated Bcr-Abl kinase during the pro-B cell stage. p-Crkl in Blnk-KO3 and Foxo1-KO3 BCR-ABL1-transformed pro-B cells were detected. Notably, following Blnk or Foxo1 deletion, the level of p-Crkl was significantly increased (Fig. 6A, lanes 1, 4, and 7). Notably, imatinib treatment attenuated p-Crkl in WT, Blnk-KO3, and Foxo1-KO3 BCR-ABL1-transformed pro-B cells (Fig. 6A, lanes 2, 3, 5, 6, 8 and 9), indicating that either Blnk- or Foxo1-deficient BCR-ABL1-transformed pro-B cells still responded to imatinib treatment. Thus, based on these results, it was argued that Blnk and Foxo1 served as tumor suppressors by inhibiting the Bcr-Abl kinase activity, hindering tumor initiation and progression in BCR-ABL1⁺ B-ALL.

To elucidate the interaction of Blnk and Foxo1 in BCR-ABL1-transformed pro-B cells, gene expression changes of Foxo1 target genes were assayed following Blnk or Foxo1 knockout. The results revealed that the expression levels of Foxo1 target genes BIM, TRAIL, CDKN1B and GADD45α were lower in Blnk- or Foxo1-deficient BCR-ABL1-transformed cells than in the WT cells (Fig. 6B). During B-cell development, Blnk inhibits Akt activity and activates Foxo proteins to initiate differentiation (22). Consistent with these findings, the present results revealed higher levels of p-Foxo1 and p-Akt (Fig. 6C, lanes 1 and 3) in Blnk-KO3 BCR-ABL1-transformed pro-B cells compared to the WT counterparts, and imatinib caused a decrease in p-Foxo1 and p-Akt levels (Fig. 6C, lanes 2 and 4), suggesting that Blnk restrains Akt activity-mediated reduction of Foxo1 phosphorylation in BCR-ABL1-transformed pro-B cells. The present findings indicated that, in BCR-ABL1-transformed pro-B cells, Blnk reduced Foxo1 phosphorylation and maintained Foxo1 activity via PI3K/Akt pathway inactivation, which ultimately was involved in regulation of the Bcr-Abl kinase.

Although overactive oncogenes induce apoptosis or senescence, the oncogenes could also suppress these ‘commits suicide’ processes to allow aberrant proliferation (45). It is worth mentioning that the mRNA and protein expression levels of BLNK and FOXO1 were significantly increased in the BCR-ABL1-transformed pro-B cells treated with imatinib (Figs. 1E and 7A and B). These results indicated the inhibitory effect of Bcr-Abl kinase on the expression of Blnk and Foxo1 in BCR-ABL1-transformed pro-B cells. Therefore, it was inferred that pro-B cells have an intrinsic antitumor ability, mediated by Blnk and Foxo1 upregulation in response to oncogenic stress. In contrast, Bcr-Abl kinase inhibited the expression of Blnk and Foxo1, thus affecting their tumor suppressor function and fueling malignant transformation of pro-B cells, marking the first step toward tumorigenesis.