Gastrointestinal stromal cells (including GIST T1 and GIST 430) carrying KIT mutations were a gift from Professor Haibo Qiu (Sun Yat-Sen University Cancer Center, Guangdong, China) and cultured as described in previous literatures [21, 22]. Neither cell line matched with any reference cell line in the cell bank database (ATCC, DSMZ, JCRB and RIKEN), as indicated by less than 80% of match in STR profiles (data not shown). Their KIT mutations were validated by PCR and sequencing (data not shown) and were shown in the Table 2. GIST T1 cell line was cultured in RPMI 1640 medium (GIBCO, Grand Island, NY, USA) containing 300 mg/L l-glutamine and 2.0 g/L sodium bicarbonate, which was supplemented with 15% fetal bovine serum, 1% Penicillin/Streptomycin, 1% l-glutamine and 2 μg/mL Gentamycin. GIST 430 cell line was cultured in a 50:50 (v/v) mixture of DMEM/Ham’s F-12 supplemented with 15% fetal bovine serum, 1% Penicillin/Streptomycin, 1% l-glutamine and 2 μg/mL Gentamycin. In addition, 200 nM imatinib was added into GIST 430 cells to maintain drug resistance. These two cell lines were maintained at 37 °C in a humidified atmosphere of 5% CO2. All the reagents used for cell culture were from Shanghai Basalmedia Technologies Co., Ltd. (Shanghai, China), unless specified elsewhere.
HQP1351 was provided by Ascentage Pharma Group Corp., Ltd. (Jiangsu, China). Imatinib was purchased from Aikang Biopharmaceutical Co., Ltd. (Jiangsu, China). Ponatinib was purchased from Ailikaide Chemical Engineering Co., Ltd. (Jiangsu, China). Regorafenib and sunitinib were purchased from Selleck (Shanghai, China).
Kinase binding assays
The binding activities of HQP1351 at the concentration of 10 and 100 nM with native or mutant KIT were analyzed by KINOMEscan™ kinase assay system. Briefly, when the competition reaction was completed, the amount of kinase captured by the immobilized ligand was quantified with quantitative PCR. Inhibition rate (%) of HQP1351 against native or mutant KIT was then calculated to evaluate the inhibition potency. Data was represented as a percentage of control (% Ctrl) where 100% indicated that the test compound did not inhibit kinase activity and lower numbers indicated stronger hits. Negative control was DMSO control (100% Ctrl), and positive control was control compound (0% Ctrl). Inhibition rate (%) was calculated by subtracting % Ctrl from 100.
In vitro antiproliferative assays
The anti-proliferative activity was determined by a water-soluble tetrazolium (WST)-based assay using Cell Counting Kit-8 (CCK-8) (Tianjin Biolite Biotech Co., Ltd., Tianjin, China). Briefly, GIST cells were treated with a series of concentrations of test articles for 72 h. Each treatment was tested in 3 replicates. At the end of the treatments, CCK-8 reagent (Tianjin Biolite Biotech Co., Ltd., Tianjin, China) was added for another 2–4 h of incubation. A microplate reader was used to determine the OD450 value of each sample. The cell viability was calculated using the mean OD value of replicated wells using the following equation: (OD sample − OD blank)/(OD cell control − OD blank) × 100. GraphPad Prism software (version 6.0, Golden software, CA, USA) was employed to calculate IC50 values of each drug. The experiment was repeated twice, and IC50 was expressed as mean ± standard deviation (SD).
Colony formation assay
GIST 430 cells were incubated with 0.3 μM imatinib, ponatinib or HQP1351 for 14 days at the density of 1000 cells/well. After the removal of drug-containing medium, the cells were fixed, followed by the staining with 0.5% crystal violet. Finally, the colonies were photographed, and the colony numbers were counted using AlphaImager HP system (Proteinsimple, CA, USA).
Cell migration assay
GIST 430 cells were seeded at the density of 106 cells/well. A P-1000 pipette was used to create a slash in the near-confluent cell cultures, which was then further incubated with 0.3 μM imatinib, ponatinib or HQP1351. The average extent of wound closure was evaluated on day 0, 3, and 5 under an inverted microscope (Leica Microsystems, Germany). Each treatment was tested in 3 replicates.
Cell invasion assay
Transwell invasion assay was conducted using 24-well Transwell chamber with an 8 μm pore size polycarbonate filter membrane (Corning, NY, USA). Approximately 5 × 104 GIST 430 cells in 200 μL FBS-free culture media were seeded onto the upper chamber, and 600 μL culture media with 10% FBS were added in the lower chamber. After further incubation with 0.3 μM imatinib, ponatinib or HQP1351 for 24 h, the cells on the upper side of the wells were softly scraped off. Cells that migrated to the lower side of the wells were fixed by methanol and stained with 0.1% crystal violet for 30 min, then photographed by microscope. Each treatment was tested in 3 replicates.
Cell cycle analysis
GIST 430 cells were first incubated with imatinib, ponatinib or HQP1351 at 0.3 μM for 24 h. Then, the cells were washed with PBS, trypsinized and harvested by centrifugation. The obtained cell pellet was fixed with cold ethanol (70%) at 4 °C for 4 h. Finally, a FACS Calibur flow cytometer (BD Biosciences, CA, USA) was used to analyze the cell cycle distribution right after the nuclear staining with propidium iodide (PI).
GIST 430 cells were first treated with imatinib, ponatinib or HQP1351 at 0.3 μM for 48 h. Then, the cells were washed with PBS, trypsinized and harvested by centrifugation. Finally, the obtained cell pellet was stained with Annexin-V-fluorescein isothiocyanate (FITC)/PI, and subjected to a FACS flow cytometer (BD Biosciences, CA, USA). A minimum of 30,000 events were analyzed.
Western blot analysis
GIST T1 and GIST 430 cells were treated with the indicated concentrations of HQP1351, ponatinib, or imatinib for 24 and 72 h, and dissolved in 1 × SDS sample lysis buffer. The protein lysates after sonication and boiling were separated by electrophoresis using 10% SDS-PAGE and transferred to a PVDF membrane. After being blocked with 1 × TBS containing 0.1% Tween-20 and 5% nonfat milk, the membrane was then incubated with the primary antibodies followed by HRP-conjugated secondary antibody [Goat Anti-Rabbit IgG (H + L), MultiSciences (Lianke) Biotech Co., Ltd., Hangzhou, China]. Finally, bound secondary antibody was visualized using ECL Western Blotting Detection Kit (Yeasen Biotech Co., Ltd., Shanghai, China). The primary antibody include c-KIT (D13A2) XP® rabbit monoclonal antibody (mAb), phospho-c-KIT (Tyr703) (D12E12) rabbit mAb, SRC (36D10) rabbit mAb, phospho-SRC family (Tyr416) (D49G4) rabbit mAb, AKT (pan) (11E7) rabbit mAb, phospho-AKT (Ser473) (D9E) XP® rabbit mAb, p44/42 MAPK (ERK1/2) (137F5) rabbit mAb, phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (197G2) rabbit mAb, STAT3 (124H6) mouse mAb and phospho-STAT3 (Tyr705) antibody. All of the primary antibodies were purchased from Cell Signaling Technology (MA, USA). Anti-β-Actin antibody mouse mAb was from Sigma-Aldrich (St. Louis, MO, USA).
In vivo efficacy study
All animal experiments were approved by Shanghai Laboratory Animal Research Center (Shanghai, China) and Shanghai Ascentage Pharmaceutical Technology Co., Ltd. (Shanghai, China). Tumors were established by subcutaneous implantation of GIST T1 or GIST 430 cells (5–10 × 106/0.2 mL) into the right flank of each mouse (female, 6–8 weeks old). Balb/c nu/nu mice (Shanghai Laboratory Animal Research Center, Shanghai, China) and SCID Beige mice (Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were used for the implantation of GIST T1 cells and GIST 430 cells, respectively.
In efficacy studies, mice were randomly assigned to different groups when the average tumor volume reached 50–200 mm3. Mice were then treated by oral gavage with HQP1351, ponatinib, or vehicle following predefined dosing regimens. Both HQP1351 and ponatinib were administered as a suspension in 0.2% HPMC (Colorocon, Shanghai, China) in water. At a predefined time point, the tumor size was measured, and tumor volume was calculated (width2 × length/2). The body weight of mice was recorded as well. At the end of experiments, T/C% and RTV (relative tumor volume) were calculated to evaluate the antitumor activity of the test articles. T and C were the mean volume of the tumors from the treatment groups and control group respectively. The tumor volume at the end of experiment was expressed as RTV and calculated by dividing the absolute tumor volume at the end of experiment with the absolute tumor volume at day 1.
In vitro data were expressed as mean ± SD. Tumor volume and body weight data were expressed as mean ± SEM. Statistical analyses were performed using one-way ANOVA with post hoc Tukey’s HSD test. Statistical significance was accepted at the level of P < 0.05. All data in the study have been recorded at Sun Yat-sen University Cancer Center for future reference (number RDDB2018000420).