Cell culture
The human CML cell line K562S was obtained from the Korean Cell Line Bank. KCL22S and KCL22R were obtained from the American Type Culture Collection. The K562R cell line was generated from K562S by treating cells with gradually increasing concentrations of TKIs. All cell lines were maintained at 37 °C and 5% CO2 in a humidified cell culture incubator and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (HyClone) supplemented with 10% fetal bovine serum (HyClone), 100 U/ml penicillin, and 100 μg/ml streptomycin. To maintain TKI resistance, K562R and KCL22R cells were cultured in medium supplemented with 1 μM imatinib.
Primary human samples
Peripheral blood or bone marrow samples were obtained from 58 CML patients (clinicopathological characteristics listed in Additional file 1: Table S1) at Hwasun Chonnam National University Hospital. Samples were obtained at the time of diagnosis and prior to any treatment with TKIs (diagnosis), and again at follow-up after treatment with TKIs (follow-up). Mononuclear cells isolated from the patients were frozen and stored in liquid nitrogen until further use. Informed consent was obtained in accordance with the Declaration of Helsinki, and all procedures were approved by the Institutional Review Board of Seoul National University (E2103/003–007) and the Gwangju Institute of Science and Technology (20180629-BR-36-01-02). TKI-responder and TKI-resistant patients were classified clinically according to the European Leukemia Net guidelines [29]. The mutational status of BCR-ABL in CML samples was confirmed by sequencing analysis.
Plasmids, antibodies, and reagents
DSG1 and HA-tagged S100A4, CYP11A1, ACSS1, LY6G6D, NR5A1, AIF1L, and FAM167A were cloned into the MigR1 vector. pClneo-Myc-Erbin was obtained from Addgene. pFLAG-NIK-DN was described previously [30]. Anti-FAM167A (sc-393999), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sc-166574), anti-p100/p52 (sc-7386 X), anti-DSG1 (sc-59904), and anti-ubiquitin (sc-8017) antibodies were purchased from Santa Cruz Biotechnology; anti-NIK (4994), anti-p100/p52 (4882), anti-Myc (2276), anti-TRAF3 (4729), anti-CHIP (2080), and anti-c-cbl (2747) antibodies were purchased from Cell Signaling Technology; the anti-DSG1 antibody (32–6000) was purchased from Invitrogen; the anti-Erbin antibody (NBP2–13968) was purchased from Novus Biologicals; and the control IgG (H9658) was purchased from Sigma-Aldrich. Imatinib (sc-202180). DSG1-specific short hairpin RNA (shRNA) lentiviral particles (sc-35224-V) and control shRNA lentiviral particles (sc-108080) were obtained from Santa Cruz Biotechnology. Nilotinib (1750) was obtained from BioVision, recombinant FAM167A (MBS1363522) was obtained from MyBioSource, SN50 (481480) and MG132 (474790) were obtained from Sigma-Aldrich, and the Pierce Silver Stain Kit (24612) was obtained from Thermo Fisher Scientific.
Microarray and RNA-seq analyses
Total RNA was extracted with TRI Reagent (Molecular Research Center). For the microarray, synthesis of cDNA and biotinylated cRNA was performed using the Illumina TotalPrep RNA Amplification Kit (Ambion). Labeled cRNAs were hybridized to the Human HT-12 v4 Expression BeadChip (Illumina), and arrays were scanned with a Bead Array Reader confocal scanner (Illumina). For RNA-seq, libraries were prepared using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina) in accordance with the Illumina TruSeq protocol. Paired-end sequencing was performed using a NovaSeq 6000 system (Illumina). Genes showing a fold-change ≥2 were defined as differentially expressed and displayed in a heatmap generated by Multiple Experiment Viewer. For in silico analyses of activated transcription factors, information about transcription factors associated with their respective binding sites in differentially expressed or randomly selected gene promoters was obtained from the GeneCards database (https://www.genecards.org/), and plots were generated using Cytoscape. The enrichment score of a transcription factor was calculated using the following equation: enrichment score = (proportion of the transcription factor associated with the differentially expressed genes)/(proportion of the transcription factor associated with randomly selected genes) × (number of differentially expressed genes associated with the transcription factor).
Luciferase reporter assay
K562S and K562R cells were co-transfected with a Renilla luciferase vector [31] and an NF-κB-dependent reporter construct (pBIIx-luc) [31], or an AP-1-dependent reporter construct (AP-1-luc) [32] and other plasmids, using Lipofectamine 2000 (Invitrogen), and then treated as indicated. Twenty-four hours after transfection, luciferase activity was measured using the Dual-luciferase Reporter Assay Kit (Promega) and normalized to Renilla luciferase activity.
Quantitative reverse transcription PCR (qRT-PCR)
Total cellular RNA was isolated using the RNeasy Mini Kit (Qiagen) and cDNA was prepared with TOPscript RT Drymix (Enzynomics). qRT-PCR was performed using SYBR Green qPCR 2× Premix (Enzynomics) on a Stratagene Mx3000P (Agilent Technologies). The results were normalized to the level of GAPDH. The qRT-PCR primer sequences were as follows: FAM167A-F, 5′-GCACAGTGAACACAACTAACC-3′; FAM167A-R, 5′-CTTGGGGATGGCAGAGAGAT-3′; S100A4-F, 5′-TCTTGGGGAAAAGGACAGATG-3′; S100A4-R, 5′-CATTTCTTCCTGGGCTGCTTA-3′; CYP11A1-F, 5′-TTCCTGCCAAGACACTGGTG-3′; CYP11A1-R, 5′-GATCCGCCGTCCCAGACA-3′; ACSS1-F, 5′-TTGGAGGTCTGGATCCAGTC-3′; ACSS1-R, 5′-GACAAACTCTCC CTCCCCTA-3′; LY6G6D-F, 5′-TACCTGGAAACCCCCCAGT-3′; LY6G6D-R, 5′-TGCCTGCAGGGGCCACAT-3′; NR5A1-F, 5′-TTCAGCCTGGATTTGAAGTTC-3′; NR5A1-R, 5′-CTTGTGGTACAGGTACTCC-3′; AIF1L-F, 5′-AGGGTCTCAAGAGTTGTCCC-3′; AIF1L-R, 5′-ATACTTGGCAGTCCTCACGTT-3′; DSG1-F, 5′-TGCTGGAGTTGAAAGGCATTA-3′; DSG1-R, 5′-AGTGCAATGTGAAATGGGTCT-3′; Erbin-F, 5′-CAAGTCTCGGTGTTCCCTTT-3′; Erbin-R, 5′-AGATCCATTGTTCCGTGAGG-3′; GAPDH-F, 5′-GGAGCGAGATCCCTCCAAAAT-3′; and GAPDH-R, 5′-GGCTGTTGTCATACTTCTCATG-3′.
Cell fractionation
Cells were suspended in hypotonic buffer (10 mM HEPES, pH 7.9; 10 mM KCl; 1.5 mM MgCl2; 0.5 mM DTT; 0.5 mM PMSF) and incubated on ice for 15 min. Then, 0.05% Nonidet P-40 was added and the lysates were passed five times through a 25-gauge needle. After centrifugation (1000×g, 5 min, 4 °C) to sediment the nuclei, the supernatants were collected and separated by high-speed centrifugation (16,000×g, 5 min, 4 °C). The resulting supernatants were stored at − 80 °C as the cytosolic fraction. The pellets containing cell nuclei were washed with hypotonic buffer and suspended in hypertonic buffer (20 mM HEPES, pH 7.9; 420 mM NaCl; 1.5 mM MgCl2; 25% glycerol; 0.2 mM EDTA; 0.5 mM DTT; 0.5 mM PMSF; 1 μg/mL leupeptin; 1 μg/mL aprotinin; 1 μg/mL pepstatin A), followed by a 30-min incubation on ice with vortexing every 10 min. After centrifugation of the nuclear lysates (16,000×g, 5 min, 4 °C), the resulting supernatants were stored at − 80 °C as the nuclear fraction.
Immunoblot analysis
Proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on 8–15% gels and then transferred to polyvinylidene difluoride membranes. The membranes were probed with specific antibodies as indicated. Densitometry was performed using ImageJ software with GAPDH as an internal standard.
Immunoprecipitation
Cells were lysed for 30 min on ice in lysis buffer (20 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1% Triton X-100; 10% glycerol; 2 mM EDTA; 1 μg/ml leupeptin; 1 μg/ml aprotinin; 1 μg/ml pepstatin A; 0.1 mM PMSF). The lysates were then centrifuged for 15 min at 15,000×g and 4 °C and the supernatants were incubated at 4 °C under rotation with specific antibodies. Protein G Sepharose® beads (GE Healthcare) were added and incubated under rotation at 4 °C. After washing four times with lysis buffer, the immunoprecipitates were eluted by boiling in SDS sample buffer and then subjected to immunoblot analysis. Band intensities were analyzed by ImageJ software.
Electrophoretic mobility shift assay (EMSA)
For the supershift analysis, nuclear fractions were incubated for 20 min at room temperature with an anti-p52 antibody or isotype control in binding buffer (5 mM Tris, pH 7.5; 25 mM KCl; 0.5 mM EDTA; 2.5% glycerol; 0.5 mM DTT; 0.1 μg/μl poly (dI/dC); 0.5 mg/ml BSA), followed by further incubation for 20 min at room temperature with a biotinylated double-stranded NF-κB probe (5′-AGTTGAGGGGACTTTCCCAGG-3′). The reaction samples were separated through 6% non-denaturing polyacrylamide gels and transferred to nylon membranes. The probes on the membranes were visualized using a LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific) and quantified by ImageJ.
Flow cytometry analysis and cell sorting
For surface DSG1 staining, cells were either incubated with Myc-tagged FAM167A (MyBioSource), followed by staining with an Alexa Fluor 488-conjugated anti-Myc antibody (9B11, Cell Signaling Technology), or stained with an extracellular domain-targeting anti-DSG1 antibody (129204, R&D Systems), followed by an Alexa Fluor 488-conjugated anti-mouse IgG antibody (Invitrogen). For primary cells, PE-conjugated anti-CD34 (4H11, eBioscience) and anti-DSG1 antibodies (129204, R&D Systems), conjugated to Alexa Fluor 488 using an Alexa Fluor 488 antibody labeling kit, (Invitrogen) were used to stain mononuclear cells. Flow cytometry analysis and cell sorting were performed using a Guava EasyCyte HT (Millipore), a FACSCanto II (BD Biosciences), or FACSAria III (BD Biosciences), and the data were analyzed with FlowJo software (TreeStar).
Immunofluorescence and immunohistochemical staining
For immunofluorescence staining, fixed and permeabilized cells were stained with an anti-DSG1 antibody (129204, R&D Systems), followed by an Alexa Fluor 488-conjugated anti-mouse IgG antibody (Invitrogen). Nuclei were visualized by Hoechst 33342 (Sigma-Aldrich) staining. An FV1000 confocal microscope (Olympus) and accompanying FV10-ASW software were used to acquire confocal images. For immunohistochemical staining, fixed and paraffin-embedded tumor sections were stained with hematoxylin and eosin (HE), anti-Ki-67 antibody (SP6, Abcam), and anti-NIK antibody (Abcam), using standard procedures. TUNEL staining was performed with the DeadEnd Colorimetric TUNEL System (Promega). Images were captured with an Eclipse Ti inverted microscope (Nikon) and accompanying NIS-Elements software. Quantification was performed using ImageJ software. The number of Ki-67+ or TUNEL+ cells in each field was counted. NIK expression in tumor cells was scored (intensity: 0 = negative, 1 = weak, 2 = moderate, 3 = strong), and the H score was calculated using the following equation: H score = 1 × (% of cells with intensity 1) + 2 × (% of cells with intensity 2) + 3 × (% of cells with intensity 3).
Cell viability and apoptosis assays
For the cell viability assays, K562S or K562R cells were seeded in 96-well plates and treated as indicated prior to transient transfection with expression constructs. After 72 h, cell viability was measured using WST reagent (DoGenBio). For the apoptosis assays, K562S or K562R cells were seeded in 6-well plates and treated as indicated. After 72 h, cells were stained with Annexin V-APC and propidium iodide using an Annexin V-APC apoptosis detection kit (Thermo Fisher Scientific) and analyzed on a Guava EasyCyte HT cytometer (Millipore).
Mouse xenograft model
K562R cells (1× 107) were suspended in 50% (v/v) serum-free Matrigel (Corning) and implanted subcutaneously into the right flanks of 6-week-old female BALB/c (nu/nu) mice. Tumor size was measured with calipers, and the tumor volume was calculated using a standard formula (width2 × length/2). When the tumor volume reached 100–200 mm3, the mice were randomly divided into groups and treated for 10 days with imatinib (10 mg/kg/day, intraperitoneally), an anti-FAM167A antibody or isotype control (2 mg/kg/3 days, intraperitoneally), or vehicle (saline, PBS). All animal experiments were performed according to protocols approved by Institutional Animal Care and Use Committees at Seoul National University (SNU-210315-6) and the Gwangju Institute of Science and Technology (GIST-2019-008).
Statistical analysis
The number of replicates for each experiment are indicated in the figure legends. Data are presented as the mean ± standard deviation (s.d.). Two-tailed unpaired Student’s t-tests were used to determine statistical significance. P values < 0.05 were considered significant (*P < 0.05, **P < 0.01, and ***P < 0.001).