Primary cells collection
Mononuclear cells were isolated from the bone marrow or peripheral blood of patients with ALL with de novo diagnosed (ND) and relapsed/refractory (R/R) status, or bone marrow from complete remission (CR), or peripheral blood from healthy donors in the Department of Hematology, which was approved by the Ethics Committee and the Institutional Review Board of Fujian Medical University Union Hospital (2014058). All patients provided informed consent by the Declaration of Helsinki. ALL was diagnosed based on guidelines of the Chinese Medical Association and National Comprehensive Cancer Network (NCCN)14. Subject received ≥ 1 course of chemotherapy under protocol CALLG2008 of the Chinese Acute Lymphoblastic Leukemia Cooperative Group15. ND or R/R patient specimens with over 80% leukemia cells after isolation were included in the analysis. Blood and bone marrow samples’ mononuclear cells were purified by Ficoll-Paque® (GE Healthcare, Chicago, IL, USA) density-gradient centrifugation and proteins isolated as described 16.
Human T-ALL cell lines Jurkat and Molt-4, B-ALL cell line Nalm-6, BALL-1 and B-lymphoblast cells Hmy2.CIR with short tandem repeat (STR) certification and checking for negative mycoplasma contamination were preserved in the Fujian Institute of Hematology and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C with 5% CO2. Molt-4 and Nalm-6 cells were exposed to stepwise increasing concentrations of adriamycin to develop resistant cell lines. Single-cell clones were developed by limiting dilution17. Adriamycin-resistant Molt-4 and Nalm-6 cell clones were designated Molt-4/ADR(MAR) and Nalm-6/ADR(NAR). Adriamycin (ADR), 1 µg/ml, was used to maintain resistance16.
Full-length human NCL cDNA (2,174 bp) was synthesized by the polymerase chain reaction (PCR) inserted into a lentiviral-GV320-Cherry expression vector and designated LV-NCL-OE for over-expression of NCL. The empty vector for the negative control was designated LV-NC. A plasmid expressing short hairpin RNA (shRNA) targeting NCL was constructed by inserting double-stranded oligonucleotides into a lentiviral pGCL-tet-puro-EGFP vector and designated LV-shNCL. The plasmid carrying the non-targeting scramble sequence was designated LV-shNT. Lentiviral vectors were produced in HEK293T cells by transient transfection of the relevant plasmids. Primer sequences are displayed in Supplement Table S1.
Developing stable cell lines
Jurkat, Molt-4, and Nalm-6 cells were transfected with lentiviral particles LV-NCL-OE carrying the NCL cDNA or the empty vector LV-NC to produce NCL-overexpressing cells designated over-expression (OE) or negative control (NC). Jurkat, Molt-4, Nalm-6, and Molt-4/ADR cells were transfected with LV-shNCL, or LV-shNT (as negative control) to produce shRNA-expressing cells designated knockdown (KD) or Scramble (Scr). Transfected cells were selected in the presence of puromycin, 0.3 μg/ml, and expression of shRNA induced by adding 2 µg/ml of doxycycline for 24 h followed by maintenance in a complete medium containing puromycin, 0.2 µg/ml, and doxycycline, 2 μg/ml.
The half-maximal inhibitory concentration (IC50) assay
4×104 cells were cultured in the presence of adriamycin, dexamethasone, vincristine, L-asparaginase, and methotrexate at various concentrations for 48 h. Cells were added to 10 μl of a solution containing 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT, Sigma, MO, USA) and incubated for 4 h. 100 μl dimethyl sulfoxide (DMSO, Sigma) was added to dissolve formazan crystalline, and absorbance was measured using Elx808 Absorbance Microplate Spectrophotometer (BioTek, Winooski, VT, USA) at reference wavelengths of 490 and 630 nm22 and optical density (OD) values determined. Inhibitory rates were calculated according to the following formulae: (1– OD drug-treated cells/ OD untreated cells) ×100%. Change in drug sensitivity was expressed by taking the ratio of IC50 values of the NCL over-expressing or knock-down cells versus control.
Intra-cellular adriamycin accumulation assay
Intra-cellular adriamycin accumulation was determined using a BD FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA). Cells were cultured at a density of 5.0×105 cells/ml in the presence of 0, 0.5, or 1 µg/ml adriamycin for 12 h, collected and washed twice for multi-parameter flow cytometric (MPFC) analyses. DAPI buffer was used to stain DNA. Adriamycin mean fluorescence intensity (MFI) was analyzed using the FlowJo software 7.6.1. (Tomy Digital Biology, Tokyo, Japan).
Rhodamine-123 efflux assay
A rhodamine-123 efflux assay was used to study the function of ABC transporters. Cells were incubated at 5×105 cells/ml with 1 μmol/L rhodamine-123 (Sigma-Aldrich, St. Louis, MO, USA) in RPMI-1640 medium at 37°C for 30 min, washed and incubated in the rhodamine-123-free medium at 37°C for 60 min, and analyzed in MPFC. Unexposed cells were negative controls. Results were analyzed using the FlowJo software.
Quantitative Real-time PCR (qRT-PCR)
Total cell RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. cDNA was synthesized from 1μg RNA using the GoScriptTM reverse transcription system (Promega, Madison, WI, USA). cDNA was mixed with 2 × SYBR Green PCR Master Mix (Applied Biosystems, Thousand Oaks, CA, USA) with gene-specific primers. qRT-PCR was run using AB 7500 Real-Time PCR system (Applied Biosystems). Samples were run in triplicate and normalized using 18S rRNA. Primer specificity was verified by melting curve analyses. Relative gene expression was determined using the comparative threshold cycle (Ct) and 2-∆∆Ct method. Primer sequences are displayed in Supplementary Table S1.
Western blotting was performed as described21. β-actin, GADPH, NCL, and BCRP antibodies were from Santa Cruz Technology (Dallas, TX, USA), α-Tublin, Histone H3, phospho-ERK1/2, ERK1/2, phospho-Raf, Raf, phospho-MEK1, and MEK1 from Cell Signaling Technology (Danvers, MA, USA), and Ras, P-gp, MRP1, and LRP from Abcam Inc. (Cambridge, UK). Anti-mouse and anti-rabbit secondary antibodies were used following the manufacturer’s instructions (Abcam Inc.). Immune reactivity was detected by chemiluminescence reaction using an enhanced chemiluminescence (ECL) kit (Pierce, Rockford, IL, USA).
Pathway suppression assay
Cells were cultured at a density of 1×106 cells/ml in media containing 10 nM SCH772984 ERK1/2 inhibitor, 10nM GSK1120212 MEK1/2 inhibitor, 100nM PLX-4720 B-RafV600E inhibitor (Selleck Chemicals, TX, USA) or mock medium control in triple 6-well plates. 1 µg/ml ADR was added to one plate and cells were cultured for 12 h and analyzed for intra-cellular adriamycin accumulation (described above). Cells of the other two plates were cultured for 48 h and analyzed for RNA and protein concentrations by qRT-PCR and western blotting after inhibiting the ERK pathway.
Immune precipitation analyses
Protein co-immune precipitation analyses to detect the interactions between nucleolin and other proteins were done by Crosslink Magnetic IP/Co- IP Kit (Pierce, Rockford, IL, USA) according to manufacturer’s protocol. Briefly, monoclonal antibodies (5 μg) were coupled to pre-washed protein A/G magnetic beads for 15 minutes and cross-linked with disuccinimidyl suberate (DSS) for 30 minutes. Antibody-crosslinked beads (25 μl) were incubated with 500 μl cell lysate from 10 million cells overnight at 4°C on a rotator. The beads were washed, and the bound proteins were eluted from the beads in elution buffer for 5 minutes at room temperature and then neutralized with buffer. Eluates were used for western blot analyses.
Immune fluorescence staining
2×105 Jurkat, Molt-4, Nalm-6, and Hmy2.CIR cells were fixed in 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.2% Triton X-100 in PBS for 10 min. Non-specific binding of the antibody was blocked with 1% bovine serum albumin and 5% goat serum in PBS for 1 h at room temperature. Dishes were incubated overnight at 4°C with primary anti-NCL antibody (1:100 dilution in blocking buffer), washed in PBS, and incubated with secondary PE-conjugated goat anti-mouse IgG (diluted 1:500 in blocking buffer) for 1 h at room temperature. DAPI buffer (10 μg/mL) was used to stain DNA. Cells were washed thrice in PBS and studied using a fluorescence microscope. Images were overlaid.
Plasm& membrane (PM) and Nuclear (N) protein extraction
Intracellular expression of NCL protein was done by Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, CN) according to manufacturer’s protocol. Briefly, PM and N proteins were extracted by cytoplasm lysis buffer and nuclear lysis buffer respectively with 5×106 Jurkat, Molt-4, Nalm-6, BALL-1, Hmy2.CIR cells and PBMC from healthy donors or patients with ALL. The extractions were used for western blot analyses with 20μg/test. α-tubulin protein was used as loading control of cytoplasm protein(diluted 1:400 in blocking buffer). Histone H3 protein was used as loading control of nuclear protein (diluted 1:1000 in blocking buffer).
Membrane NCL staining assay by flow cytometry
1×106 Jurkat and primary ALL cells from relapsed-refractory ALL patients were incubated with 5μl anti-NCL-FITC (Biolegend, CA, US) or isotype control at 4°C for 15min. Cells were washed and detected using a BD FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA), and analyzed using the FlowJo software 7.6.1. (Tomy Digital Biology, Tokyo, Japan).
BALB/C nude mice (SLAC Laboratory Animal, Shanghai, China) were injected subcutaneously with 8 × 105 Nalm-6 or Nalm-6/ADR cells. Treatments were started after tumor volume reached about 100-300 mm3. Tumor volumes= shortest diameter2 × longest diameter/2. Nalm-6 nude mice (n=5) were received vehicle, adriamycin(1.5 mg/kg), aptamer control sequence (CCT-rich sequence, CRO, Sangon,15 μg per mouse), aptamer AS1411(15 μg per mouse) only, adriamycin with CRO or AS1411(Applied Adriamycin followed by AS1411 or CRO), respectively. For Nalm-6/ADR model, mice were divided into 7 cohorts each of 6 mice. Mice received AS1411, 75 μg per mouse and CRO, 75 μg per mouse. Adriamycin was injected intravenously via the tail vein once a day for 3 days. Aptamers were given by subcutaneous injection adjacent to the tumor daily for 7 days. Tumor suppression rate (%) = (1- mean tumor weight in cohort/mean tumor weight in controls) ×100%.
NCG mice (Nanjing Biomedical Research Institute, Nanjing, China) were injected intravenously with 1×104 Nalm-6 cells stably expressing firefly luciferase. One week later mice were injected with vehicle, adriamycin (1.5 mg/kg intravenously daily for 3 d), AS1411 (15 µg per mouse daily subcutaneously proximal to the tumor for 7 d) or the sequential combination of adriamycin and AS1411. Leukaemia development was monitored using the IVIS imaging system (PerkinElmer, Hopkinton, MA, USA). Human CD45+ (hCD45-positive) cells were detected in mouse bone marrow by MPFC as described. Moribund mice were euthanized and hCD45-positive leukaemia cells in spleen and bone marrow were analyzed by MPFC.
1×106 patients-derived xenograft (PDX) expanded B-ALL cells were injected via the tail vein into NOD-scid-IL2Rg-/(NSI) mice ages 8–10 weeks immediately after exposure to 1 Gy total body radiation. Two weeks later mice received adriamycin, 1.5 mg/kg intravenously daily for 3 d, AS1411 or CRO, 15 µg per mouse daily subcutaneously proximal to the tumor for 7 d or adriamycin, 1.5 mg/kg intravenously, daily for 3 d followed by AS1411, 15 µg per mouse daily subcutaneously proximal to the tumor daily for 7 d. Blood cells were sampled weekly to assess leukaemia.
All experiments were approved by the Ethics Committee of Institutional Animal Care and Use. BALB/C nude mice and NCG mice experiments complied with guidelines of Fujian Medical University. NSI mouse experiments complied with guidelines of the Laboratory Animal Center of the Guangzhou Institutes of Biomedicine and Health (GIBH).
Overall survival (OS) was defined as the interval from diagnosis to death or censored at transplant or last follow-up. Relapse-free survival (RFS) was defined as the interval from complete remission to relapse or death from any cause or transplant. Survival analysis of the patients was constructed using the R software, with the survival and survminer packages. Kaplan-Meier plots were created to illuminate the correlations between the genetic risk scores and the survival index of patients, including OS and RFS12. Univariate and multivariate COX regression analyses were used to test whether the genetic risk score model is an independent prognostic risk factor relative to the clinical characteristics of the total set. Statistical significance was tested using log-rank tests. The prognostic risk factors were analyzed using Cox Regression with the survival R packages. A p-value < 0.05 was considered to be statistically significantly different.
Experiments were done in triplicate. Concentrations were expressed as mean ± SEM (standard error mean) or median (interquartile range [IQR]). Students t-test, one-way analysis of variance (ANOVA), Kruskal–Wallis or chi-square tests were used as appropriate. Data were analyzed using SPSS statistics software 23.0 (IBM, Chicago, IL, USA) or GraphPad Prism Software 6.0 (GraphPad, San Diego, CA, USA).