Drugs
MG132 were purchased from Selleck Chemicals(Houston, Texas, USA). ST1326 and ABT199 was purchased from Sigma-Aldrich (St Louis, MO, USA)
Cell culture
HL-60, THP-1, OCI-AML2 and OCI-AML3 cell lines were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences. KASUMI-1 cell line was gifted by Professor Chen Saijuan (Shanghai Institute of Hematology, Shanghai, China). These cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum(FBS)(Gibco) at 37°C in a humidified incubator containing 5% CO2. MV4-11 and MOLM-13 cell lines were a kind gift from Professor Ravi Bhatia (City of Hope National Medical Center, Duarte, CA, USA). These two cell lines were cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% (FBS).
Diagnostic AML patient samples were purified by standard Ficoll-Hypaque (Sigma-Aldrich) density centrifugation, then cultured in RPMI 1640 with 10% FBS.
Clinical samples
Clinical data were collected from Zhejiang Institute of Hematology, China. Informed consent was provided from all patients according to institutional guidelines. This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University. From July 2010 to July 2016, 325 patients were included in this study with detailed diagnostic and treatment information. Cytogenetically normal acute myeloid leukemia (CN-AML) was defined as AML with the karyotype 46 XY [20] or 46 XX [20] in all 20 metaphase cells analyzed. Gene mutations were analyzed by whole gene sequencing. Use descriptive statistics to summarize patient characteristics, including frequency counts, medians, and ranges.
Cell viability assay
AML Cells were seeded in 96-well plates at 1–2×104 (AML cell lines) or 1×105 (primary AML cells) per well. Then cells were treated with variable concentrations of ST1326 and/or ABT199 for 48 hours. Next,10 µl MTS solution (Promega, Madison, WI) was added to each well. Cells were incubated for 4 hoursat 37°C in a humidified incubator containing 5% CO2.Finally, cells at plates were assessed at a wavelength of 490 nm. For the AML cell lines, experiments were performed 3 independent times in triplicate, while primary patient sample experiments were performed once in triplicate due to limited sample.
RNA knockdown in human leukemia cell lines
To knock down CPT1a in human leukemia cell lines, short hairpin RNAs (shRNAs) were designed and cloned into amodified psi-LVRU6GP-shRNA plasmid. The sequences of sh1 was GCTCTTAGACAAATCTATCTC. The sequence of sh2 was GCCTTTGGTAAAGGAATCATC. The sequences of NC shRNA were ACAGAAGCGATTGTTGATC. These vectors were then packaged with human embryonic kidney 293T cells for the infection of leukemic cells. Then virus supernatant was collected at 48h and 72h. AML cell lines were transfected with the shRNA or control lentiviruses and incubated for 72 h. Next, cells were continuously cultured in the medium containing 1.0 µg/mL puromycin.
Growth curve assay
Cells were seeded in 96-well plates (1.0 × 104 cells per well), blank medium as a control, 10 µL of MTS solution (Promega CellTitre96) (5mg/mL) were added to each well at 0h, 24h, 48h and 72h, and the cells were incubated for an additional 4h at 37℃, the absorbance was measured at 490 nm.
Annexin V/propidium iodide staining and flow cytometry analysis
AML cells were treated with ST1326 or ABT199, alone or in combination for 48hours.Then cells were co-stainedwith Annexin VFITC and Propidium Iodide (PI) for 15 min using an apoptosis detection kit (Beckman Coulter, Brea, CA, USA) in the dark. Apoptotic cells were analyzed by flow cytometry using FACScan™ flow cytometer (Becton Dickinson, San Diego, CA, USA). Results are expressed as percent annexin V + cells.
Western blot analysis
Cells were lysed in radioimmunoprecipitation (RIPA) buffer (Cell Signaling Technology) on ice for 30 min. After centrifugation of the cell lysate at 12000 ×g for 15 min at 4°C, Protein concentration of the cellular supernatant was determined using BCA reagent(BBI life science, Shanghai, China). Cell lysates were then loaded onto 10% SDS-PAGE (Life Technologies, Carlsbad, CA, USA). After electrophoresis, proteins were transferred to PVDF membrane (Millipore, Billerica, MA, USA). Then, the membranes were blocked with 5% non-fat milk for 1 h and incubated withprimary antibodies overnight at 4°C. Membranes were incubated with secondary antibodies (Cell Signaling Technology) for 1 h at room after washing three times with TBST buffer temperature. The target proteins were visualized using an ECL detection kit (Amersham, Little Chalfont, UK) and analyzed using Image Lab™ software (Bio-Rad Laboratories, Hercules, CA, USA).
Primary antibodies for immunoblotting were purchased from the following sources: caspase3, cleaved caspase3, PARP, Bad, Bax, BCL-2, MCL-1, BCL-xl, BIM, p-ERK, ERK, p-AKT, AKT, pGSK3β, GSK3β, p-Mcl1Thr163, GAPDH, β-tubulin and β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA,USA).
Real-time RT-PCR
Total RNA was isolated from the AML cells using TRIzol reagent according to the manufacturer's instructions. Reverse transcription was performed using the RNAPCR core kit (Life Technologies, Paisley, UK). Real-time quantitative PCR was performed on iQ5 System (Bio-Rad, Hercules, CA)using a SYBR Green qPCR master mix with GAPDH as an internal control. Primer sequences used are listed in following:
Mcl-1,5′-AAGAGGCTGGGATGGGTTTGTG-3′(forward),5′-TTGGTGGTGGTGGTGGTTGG-3′(reverse); CPT1a,5′-ATCAATCGGACTCTGGAAACGG-3′(forward), 5′-TCAGGGAGTAGCGCATGGT-3′(reverse); GAPDH,5′-GGAGCGAGATCCCTCCAAAAT-3′(forward) ,5′-GGCTGTTGTCATACTTCTCATGG-3′(reverse).
Sample Preparation and GC-TOFMS Analysis
12 bone marrow samples with aberrant CPT1a expression collected at disease diagnosis were stored frozen at − 80°C until use. Each 107 of bone marrow blasts was added into a 1.5 mL of tube followed by the addition of 400 µL of acetone for protein precipitation. The mixture was stirred by vortex for 30 s and centrifuged at 10000 rpm for 10 min. A 400-µL supernatant was transferred to a 500 µL of glass tube and dried under vacuum. The dried analytes were dissolved in 80 µL of methoxylamine hydrochloride (15 mg/mL, dissolved in pyridine) for 90 min at 30°C and then silylated with 80 µL N,O-bis-trimethylsilyl-trifluoroacetamideand Trimethylchlorosilane (in a ratio of 99:1) (Supelco) for 2 h at 70°C. Each 70-µL aliquot of hexane was added to the derivatization bottles. After the sample was stirred for 1 min and kept at room temperature for an hour, 1-µL aliquot of the solution was injected into a PerkinElmer gas chromatography coupled with a TurboMass-Autosystem XL mass spectrometer (PerkinElmer, Inc.) in the splitless mode. A DB-5MS capillary column coated with 5% Diphenyl cross-linked 95% dimethylpolysiloxane (30 m × 250 µm i.d., 0.25-µm film thickness; Agilent J&W Scientific, Folsom, CA) was used for separation. Both the injection temperature and the interface temperature were set to 260°C, and the ion source temperature was adjusted to 200°C. Initial GC oven temperature was set at 80°C for 2 min after injection, and was raised up to 285°C with 5°C/min and maintained at 285°C for 7 min. Helium at a flow rate of 1 mL/min was used as the carrier gas. The measurements were made with electron impact ionization (70 eV) in the full scan mode (m/z 30 − 550). A total of 71 metabolites were identified by the comparison with the internal library built with the standard reference compounds.
Statistical analysis
AML patient characteristics were summarized using descriptive statistics, which included frequency counts, median and interquartile range. Categorical variables were compared using Fisher’s exact test, and continuous variables were analyzed using a nonparameter T-test. OS was defined as time from the date of diagnosis until death due to any cause or the last follow-up. Univariate and multivariate analyses with a Cox proportional hazards models were performed to assess significant predictors. The proportional-hazards assumption was checked for each variable before fitting Cox models. Data were analyzed using GraphPad Prism 8.0 software. CalcuSyn software (Biosoft, Cambridge, UK) was used to calculate the combination index (CI).