CX-5461 and BMH-21 were obtained from Selleck Chemicals. Imatinib mesylate was from Cayman Chemical Company. The compounds were initially dissolved in DMSO and a series of 200X stock solutions were prepared according to proposed final concentrations. Drug treatment was performed by adding 5 µl of the stock solution in each ml of culture medium. DMSO was dissolved 1:200 in the culture medium as vehicle control. Primary antibodies used were: Anti-Kif1b (cat. no. A6638; ABclonal Biotech Co., Ltd. ) and anti-Rbfox2 (cat. no. A05389-1; Wuhan Boster Biological Technology, Ltd. ). The dilution ratios of anti-Kif1b and anti-Rbfox2 used in western blotting experiments were 1:500 and 1:1,000 respectively. The secondary antibody used was horseradish peroxidase-conjugated goat anti-rabbit IgG (cat. no. SA00001-2; Wuhan Sanying Biotechnology; 1:5,000).

K562 (p53^-/Ph⁺) and NALM-6 (p53⁺/Ph^-) cells were originally obtained from American Type Culture Collection. The THP-1 cell line (p53^-/Ph^-) was obtained from National Collection of Authenticated Cell Cultures. All of the cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics (all from Thermo Fisher Scientific, Inc.), in a humidified environment with 5% CO₂ at 37˚C. Untreated cells were maintained at a density of below 1x10⁶ cells/ml. Medium renewal was performed every 2 to 3 days.

The number of viable cells was assessed using a colorimetric Enhanced Cell Counting Kit-8 (cat. no. C0042; Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Briefly, 100 µl aliquots of cell suspension were transferred to a 96-well plate, and 10 µl of the CCK-8 solution was added to each well. After mixing, the cells were placed back to the cell incubator and further incubated for 2 h at 37˚C. Cell number was assessed by measuring absorbance at 450 nm using a microplate reader (EMax Plus; Molecular Devices, LLC).

Cell apoptosis was assessed using a FITC-Annexin V Apoptosis Detection kit (BD Biosciences). Cells were washed with cold PBS and resuspended in 1X Binding Buffer at a concentration of 1x10⁶ cells/ml. Then 100 µl aliquot of the cell suspension was transferred to a 5-ml test tube, and working solutions (5 µl each) of Annexin V and propidium iodide (PI), prepared according to the manufacturer's protocols, were added. After 15 min of incubation at room temperature in the dark, 400 µl of the Binding Buffer was added to each tube. Flow cytometry analysis was performed using a BD Accuri C6 Plus machine (BD Biosciences). The following controls were used to set up the quadrants: Unstained cells, cells stained with Annexin only, and cells stained only with PI. Cells in both early stage (Annexin⁺/PI⁻) and late stage (Annexin⁺/PI⁺) apoptosis were equally counted as apoptotic cells. Data were analyzed with WinMDI (version 2.9; The Scripps Institute; http://www.cyto.purdue.edu/flowcyt/software/Winmdi.htm) and FlowJo (version 10; FlowJo LLC) software.

Cells were divided into three treatment groups (with 3 independent biological replications in each group): control, imatinib alone, and imatinib + CX-5461. Total RNA was extracted using TRIzol^® (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. RNA integrity was confirmed with an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc.). Qualified total RNA was further purified by RNAClean XP Kit (Beckman Coulter, Inc.) and RNase-Free DNase Set (Qiagen GmbH). RNA concentration was determined using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Inc.). Library construction was performed using VAHTS Stranded mRNA-seq Library Prep Kit for Illumina (Vazyme Biotech Co., Ltd.). RNA sequencing was performed using a NextSeq Illumina550 platform (Illumina, Inc.) in a paired-end manner. RNA processing, library construction and sequencing services were provided by Shanghai Biotechnology Corporation. The complete dataset of the raw count values and fragments per kilo base per million mapped reads (FPKM) values are provided in Table SI. Differentially expressed genes were defined by the false discovery rate (q value) <0.05 and fold change >2. The reproducibility of the sequencing assay was confirmed by the degree of correlation between biological duplicate samples (see ENCODE Guidelines and Best Practices for RNA-Seq; https://www.encodeproject.org/about/experiment-guidelines) (27).

Around 5x10⁵ cells were homogenized in TRIzol^® (Thermo Fisher Scientific, Inc.) and total RNA was isolated according to the manufacturer's instructions. The RNA concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). cDNA was synthesized from 50 ng of total RNA using the PrimeScript RT-PCR Kit (cat. no. RR037A; Takara Bio, Inc.) according to the manufacturer's instructions. Reverse transcription was carried out using 100 µM of random 6mers, under a thermocycler condition of 37˚C for 15 min followed by 85˚C for 5 sec. The real-time PCR reaction was carried out using the UltraSYBR Mixture kit (cat. no. CW0957M; CWBio) according to the manufacturer's instructions in a LightCycler 96 Instrument (Roche Diagnostics). The primer sequences used in the study are listed in Table SII. The reaction was carried out using 5 ng of cDNA in a 50-µl reaction volume, containing 0.2 µM of primers. The following thermocycler settings were applied: 95˚C for 10 min; 40 cycles of 95˚C for 16 sec and 60˚C for 1 min. Human GAPDH was used as the housekeeping gene. Fold changes were determined using the 2^-ΔΔCq method, where ΔCq=Cq target gene-Cq housekeeping gene; ΔΔCq=ΔCq test sample-ΔCq calibrator (control) sample (see ‘Real-time PCR handbook’ at https://www.thermofisher.com/content/dam/LifeTech/global/Forms/PDF/real-time-pcr-handbook.pdf) (28).

siRNA constructs targeting RBFOX2 (sense sequence CCGGAGUUAUAUGCAGCAUTT; anti-sense AUGCUGCAUAUAACUCCGGTT) and KIF1B-β (sense GCCAUCCUCUCCCUAAAUATT; anti-sense UAUUUAGGGAGAGGAUGGCTT) were obtained from Shanghai GenePharma Co., Ltd. The RBFOX2 siRNA was designed using RBFOX2 transcript variant 1 mRNA (Reference Sequence accession: NM_001031695.4) as the template, targeting the 54-nucleotide exon which was conserved in transcript variants 1-18. The KIF1B-β siRNA was designed using KIF1B transcript variant 1 mRNA (Reference Sequence accession: NM_015074.3) as the template, targeting the nucleotides 4108 to 4126. A universal non-targeting siRNA was used as control (sense UUCUCCGAACGUGUCACGUTT; anti-sense ACGUGACACGUUCGGAGAATT). K562 cells were re-suspended in antibiotic-free Opti-MEM I Reduced Serum medium (cat. no. 31985-062 from Thermo Fisher Scientific, Inc.) at 1x10⁶ cells per ml, and transfected using Lipofectamine^® RNAiMAX (cat. no. 13778-075; Thermo Fisher Scientific, Inc.) with a final siRNA concentration of 200 nM at 37˚C. Fresh medium was replenished after 6 h of incubation with siRNA, and the cells were used for experimentation at 48 h after transfection.

Total proteins were extracted using RIPA lysis buffer (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) containing Protein Phosphatase Inhibitors cocktail (cat. no. P1260; Beijing Solarbio Science & Technology Co., Ltd.). The protein concentration was determined using a BCA protein assay kit (cat. no. P0010; Beyotime Institute of Biotechnology). Samples were boiled in SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol and 0.02% bromophenol blue). Protein samples (25 µg per lane) were loaded onto precast 4-20% gradient Bis-Tris PAGE gel (Dalian Meilun Biotechnology Co., Ltd.), separated by electrophoresis, and transferred to Immobilon-NC nitrocellulose membrane (Merck KGaA). The membrane was blocked with 5% non-fat milk for 2 h at room temperature, and then incubated with primary antibodies overnight at 4˚C. After washing, the membrane was probed with horseradish peroxidase-conjugated secondary antibody (1:5,000) for 1-2 h at room temperature. The bands were developed by enhanced chemiluminescence using SuperKine West Femto Maximum Sensitivity Substrate (cat. no. BMU102-CN; Abbkine Scientific Co., Ltd.), and visualized with a Tanon 3500 Gel Imaging System (Tanon Science and Technology Co., Ltd.). Band densitometry analysis was performed using ImageJ software (version 1.46r) (National Institutes of Health).

Caspase 3 activity was measured with a colorimetric assay kit (cat. no. C1115; Beyotime Institute of Biotechnology), which detected the cleavage of substrate (Ac-DEVD-p-nitroanilide) to the yellow-colored product p-nitroaniline, according to the manufacturer's protocol. Briefly, 4x10⁵ cells were collected by centrifugation (600 x g for 5 min), washed with PBS, and incubated in 200 µl of the lysis buffer supplied on ice for 15 min with vortexing. The sample was then centrifuged at 16,000 x g 4˚C for 15 min. The supernatant was transferred to a test tube, mixed with 0.2 mM substrate, and incubated at 37˚C overnight. The absorbance at 405 nm was measured using a SUNRISE microplate reader (Tecan Group, Ltd.).

Data are expressed as the mean ± standard error of the mean. Data analysis was performed with unpaired t-test or one-way analysis of variance followed by post hoc Tukey's test as appropriate. All of the n values represented the number of independent biological replicates. P<0.05 was considered to indicate a statistically significant difference.

In order to determine the potential synergy between imatinib and CX-5461, the combination subthresholding approach was adopted, an effect-based method which detects additivity by showing that combination of non-effective doses of two drugs yields a significant effect (29). Following this principle, the synergy is determined by showing that neither [drug A] nor [drug B] has a significant effect individually, while the effect of [drug A]/[drug B] combination is significant. According to Picard et al (30), the effective plasma concentration of imatinib in humans is between 300-3,000 nM. Based on this information, 100 nM was selected as the reference concentration for imatinib in the following experiments. The rationale for using 100 nM of imatinib was that this concentration represented a non-effective one for single treatment, thereby allowing the present study to test drug synergy with the subthresholding approach. First, it was confirmed that at a concentration of 100 nM, neither imatinib nor CX-5461 individually had any significant effect on the viability of K562 cells after 48 h of treatment (Fig. 1). In comparison, under the same experimental conditions, combined treatment with CX-5461 and imatinib, both at 100 nM (for 48 h), significantly decreased the cell viability (Fig. 2A). To precisely define the nature of the synergistic effect of CX-5461 on imatinib-induced cell toxicity, concentration-response analysis was performed in the absence or presence of 100 nM CX-5461. As shown in Fig. 2B, CX-5461 increased the Emax (maximum effect) of imatinib (83.4±3.1 vs. 70.8±1.9%, P=0.026, unpaired t-test, n=3), but had no significant effect on the EC₅₀ value (concentration producing 50% of the maximum effect; 0.23±0.02 vs. 0.20±0.009 mM; P=0.215). Furthermore, treatment with imatinib/CX-5461 combination (for 48 h) increased the number of apoptotic cells as compared with CX-5461 or imatinib mono-treatment (Fig. 2C).

To clarify whether the synergistic action of CX-5461 with imatinib was due to inhibition of rDNA transcription, the cells were treated with another unrelated Pol I inhibitor BMH-21. It was found that treatment with BMH-21 at 1 µM for 48 h moderately reduced the cell viability (Fig. S1A). At this concentration, however, a synergistic effect of BMH-21 on imatinib-induced apoptosis was not observed (Fig. S1B). These data indicated that the synergistic effect of CX-5461 with imatinib was unlikely to be a direct result of the reduced rDNA transcription.

To clarify whether the synergistic effect of CX-5461 on imatinib-induced apoptosis was related to changed expressions of essential apoptosis-regulating factors, the mRNA levels of FAS, BAX, BIM, BCL-2 and BCL-XL genes were examined. As shown in Fig. 3, CX-5461 exhibited no significant effects on the expression of these genes either in the absence or presence of imatinib co-treatment.

To further explore the possible mechanism underlying the synergy between CX-5461 and imatinib, genome-wide RNA sequencing experiments were performed in cells which were untreated, treated with imatinib alone, and treated with imatinib/CX-5461 combination respectively. Technically, the reproducibility of the assay was confirmed by the high degree of correlation (coefficient >0.9) between biological duplicate samples (27). The specific correlation coefficient values between sample pairs are shown in Fig. 4A. Consistent with the sub-efficacious concentration of imatinib used in this experiment, it was shown that only a small number of gene expressions were altered by imatinib treatment alone (Fig. 4B). Specifically, nine genes were upregulated and 16 genes were downregulated significantly by imatinib. Similarly, it was demonstrated that in the presence of imatinib, only a minor group of genes were responsive to CX-5461 co-treatment (Fig. 4B and Table I). Specifically, nine genes were upregulated and five genes were downregulated significantly. As the transcription factor p53 has a central role in mediating the cellular effects of Pol I inhibition-induced NSR (18,19), in K562 cells (without functional p53) the CX-5461-responsive genes did not contain typical p53 targets (see Table I). Moreover, it was shown that under the present experimental settings, CX-5461-responsive genes in K562 cells were totally distinct from those responsive to imatinib (Fig. 4C). Among the differentially expressed genes responsive to CX-5461, special attention was paid to the RBFOX2 gene (downregulated), which encodes a RNA splicing factor (31-33). Rbfox2 mediates the splicing of KIF1B mRNA, while reduced Rbfox2 expression has been shown to facilitate the preferential expression of the KIF1B-β isoform (over KIF1B-α), which has a pro-apoptotic role in solid tumor cells (34-40).

The present study examined whether changed expression of KIF1B was involved in the pro-apoptotic effect of imatinib/CX-5461 combination. Using qPCR, it was found that CX-5461 treatment significantly upregulated the expression levels of both KIF1B-α and KIF1B-β in the presence of imatinib (Fig. 5A); however, CX-5461 alone had no significant effect on KIF1B expression in the absence of imatinib (Fig. S2). Similarly, imatinib alone did not significantly affect the expression of KIF1B (Fig. S2). To further confirm the finding that imatinib/CX-5461 could upregulate the expression of KIF1B, two additional PCR primers were designed targeting the N-terminal part of Kif1b, which was 100% identical for Kif1b-α and Kif1b-β. As shown in Fig. 5B, the mRNA level of total KIF1B was significantly increased in imatinib/CX-5461-treated cells. Moreover, it was confirmed that imatinib/CX-5461 significantly increased the protein level of total Kif1b as compared with the imatinib mono-treatment (Fig. 5B). Remarkably, the present study did not detect any significant change in the KIF1B-α to KIF1B-β ratio following imatinib/CX-5461 treatment (Fig. 5A), which seemed to be in contrast to the reported effect of Rbfox2(38). To clarify this question, gene silencing for RBFOX2 was performed (Fig. 5C), and it was demonstrated that knocking down the RBFOX2 expression in K562 cells did not change the expressions of KIF1B-α or KIF1B-β, nor did it affect the ratio between the two isoforms (Fig. 5D). These results suggest that there is no association between Rbfox2 and KIF1B mRNA splicing in K562 cells under the present experimental settings.

As KIF1B-α was not implicated in modulating cell apoptosis (34,40), it was then tested whether KIF1B-β was involved in mediating the pro-apoptotic action of imatinib/CX-5461 combination in K562 cells. For this purpose, K562 cells were transfected with KIF1B-β siRNA, of which the gene silencing effect was confirmed by western blotting (Fig. 6A). Flow cytometry assays, demonstrated that in cells transfected with the control siRNA, CX-5461 in the presence of imatinib increased the apoptotic response; by contrast, in cells transfected with KIF1B-β siRNA, the pro-apoptotic effect of CX-5461 was blunted (Fig. 6B). To further corroborate the above flow cytometry results, caspase 3 activity assay was performed in K562 cells. In cells transfected with the control siRNA, imatinib/CX-5461 increased the level of caspase 3 activation as compared with imatinib mono-treatment, whereas in cells transfected with KIF1B-β siRNA, the effect of CX-5461 was diminished (Fig. 6C).

The effects of imatinib/CX-5461 on KIF1B expression in NALM-6 (human acute lymphoblastic leukemia cell line; p53⁺/Ph^-) and THP-1 (human monocytic leukemia cell line; p53^-/Ph^-) cells was also tested. In NALM-6 cells, it was shown that only imatinib/CX-5461 combination significantly increased the expression of KIF1B-α and KIF1B-β (Fig. S3), whereas the effects of imatinib or CX-5461 alone did not reach a statistical significance, although the moderate increases induced by CX-5461 were significant using unpaired t-test. In THP-1 cells, however, imatinib and CX-5461, either alone or in combination, had no significant effects on the expression of KIF1B isoforms (Fig. S3). The partial effect of imatinib in NALM-6 cells (lacking Bcr-Abl) may be due to the off-target activities of the drug (41).