2.1 Reagents

All reagents used in this study were of analytical grade. Linsitinib, a selective inhibitor of the IGF-1R, was purchased from Selleck Chemicals (Houston, TX, USA). Linsitinib stock solutions were prepared in dimethyl sulfoxide and stored at − 20 °C. Imatinib, a specific inhibitor of BCR::ABL1, was provided by Novartis Pharma AG (Basel, Switzerland). Additional reagents were acquired from Merck KGaA (Darmstadt, Germany).

2.2 Cell culture

The chronic myeloid leukemia cell line K562 was acquired from the American Type Culture Collection (Manassas, VA, USA). Additionally, T315I-mutant Ba/F3 cells and wild-type BCR::ABL-transfected Ba/F3 cells were obtained, as described previously. [13]. Imatinib-resistant K562K562 IR cells were established through culture with a low imatinib concentration [14]. Ponatinib-resistant K562 (K562 PR) cells were also characterized [15]. K562 PR cells were established through N-ethyl-N-nitrosourea (ENU) mutagenesis screening and were cultured with ponatinib [15]. Ba/F3 cells were maintained in a standard RPMI 1640 medium supplemented with 10% (vol/vol) fetal calf serum and 10% (vol/vol) conditioned medium from the WEHI-3B cell line (as a source of IL-3). All cell lines were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640) medium, supplemented with 10% fetal calf serum (FCS), 1% penicillin–streptomycin, and 1% glutamine, at 37 °C in a humidified atmosphere containing 5% carbon dioxide (CO2).

2.3 Data collection and processing

To identify differentially expressed and spliced transcripts in CML, messenger ribonucleic acid (RNA) profiles of peripheral blood mononuclear cells from five patients with chronic-phase chronic myeloid leukemia (CP-CML) and five healthy volunteers were generated through deep sequencing using the NextSeq 500 system (GSE100026 dataset; Illumina, San Diego, CA, USA) [16]. In patients with CP-CML who were undergoing frontline imatinib treatment, an inability to achieve an early molecular response (EMR), defined as BCR-ABL1 > 10% (International Scale [IS]) at 3 months, was indicative of a poor prognosis. This factor was evaluated using the Gene Expression Omnibus (GEO) dataset GSE130404 in which patients with EMR failure (n = 13) were compared with patients with EMR success (n = 83) [17]. Data were obtained from the GEO. GEO2R uses GEOquery and limma to perform differential expression analysis using original submitter-supplied processed data tables as input. The edgeR is an R/Bioconductor software package for differential analyses of sequencing data in the form of read counts for genes or genomic features used, according to user guide instructions. The RNA-Seq data analysis software for academic research was procured from BxINFO LLC (Shinagawa-ku, Tokyo, Japan). Additionally, RNA-Seq data visualization was conducted using the open bioinformatics web tool RaNA-Seq.eu) [18].

2.4 Cell viability assay

The cells were seeded in 96-well plates at a density of 4 × 103 cells or 8 × 103 cells per well. Cells were treated with imatinib and/or linsitinib for 72 h, after which cell viability was assessed using the Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) or determined using the Trypan Blue Staining assay (Bio-Rad, Hercules, CA, USA) following the manufacturer’s instructions. After 72 h of culture, samples were examined using the EnSpire Multimode Plate Reader (PerkinElmer, Waltham, MA, USA) or the TC10 Automated Cell Counter (Bio-Rad). Proliferation rates were reported as percentages relative to that of the control and untreated cells.

2.5 Determination of caspase 3/7 activities

Caspase activity in CML cells was evaluated using the Caspase-Glo 3/7 Assay Kit (Promega Corporation, Madison, WI, USA), based on the manufacturer’s protocol. CML cells were treated with imatinib, linsitinib, or a combination of these at various doses. After 48 h of incubation, the Caspase-Glo 3/7 reagent was dispensed into each well in equal amounts. Luminescence was then measured using the EnSpire Multimode Plate Reader (PerkinElmer) to determine the activity of caspase 3/7.

2.6 Cytotoxicity assays

The CML cells were treated with imatinib and/or linsitinib at various concentrations. Cytotoxicity was assessed by measuring the release of lactate dehydrogenase (LDH) from damaged cells using a cytotoxicity LDH assay kit with water-soluble tetrazolium salt (Dojindo Laboratories). After 48 h of treatment, LDH levels were quantified using the EnSpire Multimode Plate Reader (PerkinElmer), following the manufacturer's protocols.

2.7 Reverse transcription-quantitative polymerase chain reaction analysis (RT-qPCR)

Total RNA was extracted from CML cells with the RNAqueous-4PCR Kit (Life Technologies Japan Ltd., Minato-ku, Tokyo, Japan) and reverse transcribed using the First-Strand cDNA Synthesis Kit (OriGene Technologies, Rockville, MD, USA). Real-time polymerase chain reaction (PCR) was conducted using the Roche LightCycler 2.0 system (Roche Diagnostic GmbH, Minato-ku, Tokyo, Japan). Human IGF-1R, IGFBP2, IGF-1 and β-actin expression was quantified using the SYBR Green PCR Kit (Roche), following the manufacturer's instructions. Specific PCR primers were purchased from Takara Bio Inc. (Otsu, Shiga, Japan).

2.8 Small interfering RNA and small interfering RNA transfection

Small interfering RNA (siRNA) targeting the IGF-1R was acquired from Merck KGaA (Darmstadt, Germany). K562 PR cells were transfected with the IGF-1R siRNA via electroporation. In brief, cells were electroporated in 400 μL of culture medium at a density of 2 × 107/mL in electroporation cuvettes, combined with 1 nmol of siRNA. Electroporation was performed via the Gene Pulser II System (Bio-Rad) set to 0.2 kV and 950 μF capacitance. Fifteen minutes postelectroporation, cells were diluted in culture medium and incubated at 37 °C with 5% CO2 and 92% humidity. Gene expression in the transfected cells was assessed via RT-PCR. After 48 h, the cells were treated with imatinib and analyzed.

2.9 Mitochondrial membrane potential assay

The mitochondrial membrane potential (MMP) was evaluated using a mitochondrial staining kit (Merck KGaA), following the manufacturer's protocol. The cells were thereafter treated for 48 h with or without imatinib and/or linsitinib. JC-1 monomers and aggregates were measured using the EnSpire Multimode Plate Reader (PerkinElmer).

2.10 Statistical analyses

All data were analyzed using Prism 10 (GraphPad Software, Inc., San Diego, CA, USA). All data are presented as the mean ± standard deviation. Each experiment was performed three times. The paired Student’s t-test was used to calculate the significance of the differences between two groups. When one of the groups in this study was considered the control group, data were analyzed using Dunnett’s test as a post hoc test, followed by an analysis of variance with an alpha of 0.05 (n ≥ 3). Significance was expressed as *p < 0.05, **p < 0.01, ***p < 0.001, or ****p < 0.0001.