Patient samples
This study was approved by the internal review board of the Korea University Anam Hospital (IRB No. 2015AN0267). Bone marrow (BM) blood from CML patients (n = 38) and healthy donors (n = 10) was collected after obtaining written informed consent, in accordance with the Declaration of Helsinki. BM mononuclear cells (BMMNCs) were separated, as described previously (30). CD34+ BMMNCs were sorted using a Super MACS instrument (Miltenyi Biotech Inc., Auburn, CA, USA). CML CD34+ cells were cultured in serum-free expansion medium (Sigma-Aldrich, St. Luis, MO, USA) containing a recombinant human cytokine cocktail (StemCell Technologies, Vancouver, Canada). The remaining CD34– cells were used to produce CML-derived BM stromal cells (BMSCs), which were cultured in mesenchymal stem cell growth medium (Lonza, Walkersville, MD, USA).
Reagents and cell lines
The CXCR2 antagonists, SB225002 and SB265610, were obtained from Calbiochem (San Diego, CA, USA) and Tocris Bioscience (Bristol, United Kingdom), respectively. Imatinib and nilotinib were purchased from Selleck Chemicals (Munich, Germany). Drug compounds were dissolved in Dimethyl Sulfoxide (DMSO) at a concentration of 10 mM. K562, KU812, and CD34+ cells were purchased from the American Type Culture Collection (Manassas, VA, USA). CML cells were maintained in RPMI 1640 medium (Corning, NY, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco Life Technologies, Grand Island, NY, USA) at 37 °C in a humidified atmosphere containing 5% CO2.
Development of TKI-resistant cell lines
To establish imatinib- and nilotinib-resistant CML cell lines, K562 and KU812 cell lines were gradually exposed to imatinib (from 0.01 µmol/L to 10 µmol/L) or to nilotinib (from 0.1 nmol/L to 100 nmol/L). The resulting imatinib- and nilotinib-resistant cells were maintained with 1 µmol/L imatinib and 20 nmol/L nilotinib, respectively. BCR-ABL gene mutations generated during the acquisition of TKI resistance were identified using next-generation sequencing. Library preparation and data analysis are described in Additional File 1, Table S1.
Cytokine array analysis
A cytokine array analysis was performed on K562, K562 imatinib-resistant cells (K562/IR), and K562 nilotinib-resistant cells (K562/NR) cultured in cell culture-conditioned medium for 48 h and BM blood serum samples from CML patients. Serum samples were prepared by centrifugation for 10 min at 1000 × g. The human cytokine antibody array (Ray Biotech, Norcross, GA, USA) was performed according to the manufacturer’s instructions (see in Additional File 1: Table S2), and dot blot quantitation was performed using Image Lab 5.0 software (Bio-Rad, Hercules, CA, USA).
ELISA
Human Interleukin-8 (IL-8) and Growth-Regulated Oncogene (GRO) alpha concentrations in culture supernatants and BM blood plasma were determined using ELISA kits (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. The levels of ligands were measured using a standard curve provided with the kit. All samples were measured in duplicate.
Colony formation assay
CML cells were cultured for 72 h after treatment with SB225002 or vehicle. Treated cells were harvested, resuspended in Iscove’s Modified Dulbecco’s Medium (IMDM; Corning, NY, USA), and mixed with semisolid culture medium (methylcellulose H4034; Stem Cell Technologies, Grenoble, France). Cells were seeded onto 35 mm dishes (5 × 103 cells/dish). Each assay was performed in duplicate, and the cells were counted after 12–14 culture days.
RT-qPCR
Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. RT-qPCR was performed as previously described (30). SYBR Green PCR Master Mix was used with a CFX96 RT-PCR machine (Bio-Rad, Hercules, CA, USA) as follows: 95 °C for 60 s, followed by 95 °C for 10 s and 60 °C for 60 s for 45 cycles. The primer sequences are listed in Table S3, Additional File 1. To evaluate relative mRNA expression, the comparative ΔΔCt method was used, with GAPDH as a reference gene.
Cell proliferation assay
CML cells were cultured in 96-well plates at 5 × 103 cells/well. After 24 h, cells were treated with SB225002 at different concentrations for 48 h. Cell proliferation was evaluated by adding 10 µL Cell Counting Kit-8 (CCK-8) reagent (Dojindo Laboratories; Japan) to each well and incubating it for 2 h. Absorbance at 450 nm was measured using a microplate spectrophotometer. The half maximal inhibitory concentration (IC50) values were calculated using GraphPad Prism 6.0.
Cell cycle analysis
For DNA content analysis, cells were harvested and fixed with 80% ethanol overnight at –20 °C. The cells were washed twice with PBS and incubated overnight at 37 °C in 0.5 mL PBS containing 100 µg/mL RNase A. Fixed cells were stained with 50 µg/mL propidium iodide (PI) solution and analyzed using FACS Canto Ⅱ (BD, Franklin Lakes, NJ, USA). Cell cycle analysis was performed using FlowJo software (FlowJo LLC, Ashland, OR, USA).
Western blotting
Proteins were extracted using a protease inhibitor kit (Intron Biotechnology.; Seongnam, Korea). Immunoblotting was performed as previously described (29). Antibodies against the following proteins were used: CXCR2 and c-Myc (Abcam, Cambridge, UK); β-actin, cyclin B1, and p-CDC2 (Santa Cruz, CA, USA); and p-c-ABL, c-ABL, p-mTOR, mTOR, p-Akt, Akt, caspase-3, and cleaved PARP (Cell Signaling, Danvers, MA, USA).
Apoptosis assay
CML cells were seeded in six-well plates and treated with 1 µM SB225002 for 48 h. Cells were washed with PBS and resuspended in 0.5 mL binding buffer containing 1 µg/mL PI and 1 µg/mL FITC-labeled annexin V (Koma Biotechnology, Seoul, Korea). After incubating cells for 15 min at room temperature, apoptotic cells were quantified by measuring the percentage of annexin V+ cells using a BD FACS Canto II flow cytometer (BD, Franklin Lakes, NJ, USA). The data were analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA).
Transduction of lentivirus shRNA for CXCR2 silencing and activation
The pLKO.1-shRNA control and shRNA CXCR2 transduction particles were purchased from Sigma-Aldrich (St. Louis, MO, USA). For CXCR2 upregulation, control and CXCR2 lentiviral activation particles were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). K562 cells were resuspended in virus-containing media and mixed with 8 µg/mL polybrene. The cells were centrifuged at 800 × g for 2 h at 32 °C. Virus-containing medium was removed, and the cell pellet was resuspended in culture media. To select stably infected cells, the medium was replaced with fresh medium containing puromycin after 72 h.
Animal experiments
Animal experiments were approved by the Institutional Animal Care and Use Committee of the Korea University College of Medicine (IRB NO. KOREA-2018-0058) and performed according to institutional and national guidelines. Female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice (6–8-weeks-old) were purchased from Koatech (Pyeongtaek, South Korea). To establish the xenograft model, 5 × 106 K562, K562/imatinib resistance (IR), or K562/nilotinib resistance (NR) cells were suspended in culture media with Matrigel (356234; BD Bioscience, San Jose, CA, USA), and 100 µL suspended cells were implanted subcutaneously into the right flank. One week after inoculation, the mice were randomly divided into control and drug treatment groups. The control group was injected with DMSO, while the SB225002 (20,33), imatinib (34,35), and nilotinib (36) groups were intraperitoneally injected with 10 mg/kg of the respective drugs three times per week. The injections were continued for 14 days until tumors reached 20 mm, which is the maximum allowable size following animal ethics guidelines. Tumor size was ascertained three times a week, and the volume was calculated using the following formula: volume (mm3) = (length × height2)/2.
Immunohistochemistry
Formalin-fixed tissue was embedded in paraffin, and 3 µm-thick sections were cut. Slides were cleared in xylene and rehydrated with gradient ethanol to deparaffinize them. Samples were treated in citrate buffer for antigen retrieval. Then, samples were incubated overnight with CXCR2, c-Myc, and c-ABL (Abcam, Cambridge, UK), and mTOR (Invitrogen, Waltham, MA, USA) primary antibodies, followed by incubation with a biotinylated secondary anti-mouse antibody. Secondary antibody staining was visualized with DAB solution, and the samples were counterstained with hematoxylin. Images were analyzed using an Olympus BX 53 microscope (Tokyo, Japan).
Whole-genome sequencing
DNA was extracted from TKI-sensitive and -resistant cells (K562, K562/IR, K562/NR, KU812, KU812/IR, and KU812/NR). Libraries were prepared using TruSeq Nano DNA Sample Prep kits (Illumina, Inc., San Diego, CA, USA). DNA sequencing was performed using a NovaSeq 6000 system (Illumina, Inc.). The sequences were aligned to the reference genome (hg19) using the Isaac aligner v.iSAAC-03.16.12.05 (37). The Isaac aligner tool was used to remove adapter sequences and low-quality reads. Aligned reads were sorted according to location information. Single nucleotide variants and small indel mutations were confirmed to extract the variants calls using the Genome Variant Call Format File (gVCF) tool. The influence of mutations on the amino acid sequence was predicted using SnpEff v.4.2 (38) and SnpSift v.4.2 (39). Mutation annotations were added from the known variant databases dbSNP (40), COSMIC (41), ClinVar (42), and ExAC (43). Consensus sequences were obtained based on accumulated information in a specific region using Mpileup in Samtools v. 1.3.1 (44).
Statistical analysis
All results were obtained from at least three independent experiments. The data are represented as the mean ± standard deviation (SD). Statistical differences were calculated using Student’s t-test to compare two groups and one-way analysis of variance (ANOVA) with Tukey’s post hoc test for comparison of more than two groups. P < 0.05 was considered to be statistically significant. All statistical analyses were performed using GraphPad Prism V6.0 (GraphPad Software, San Diego, CA, USA).