Materials. Imatinib, danusertib, volasertib and AZD1775 were purchased from Selleck Chemicals. For Western blotting, 10% acrylamide gels, running buffer (MOPS), transfer buffer and polyvinylidene difluoride (PVDF) transfer membrane were bought from Thermo Scientific. For immunofluorescence, the FITC-conjugated anti-mouse IgG, the anti-rabbit conjugated with Alexa Fluor 568 antibodies and DAPI (6-diamidino-2-phenylindole ) were purchased from Sigma-Aldrich.
The anti-caspase 3, anti-caspase 9, anti-Bax, anti-CHK1, anti-phospho-CHK1 (S317), anti-CHK2, anti-phospho-CHK2 (T68), anti-cyclin B1, anti-phospho-cyclin B1 (S133), anti CDC25C, anti-phospho-CDC25C (S198), anti-WEE1, anti-phospho-WEE1 (S642), anti-CDK1, anti-phospho-CDK1 (Y15), anti-phospho-H2AX (S139), anti-Aurora kinase A, anti-phospho-Aurora kinase A (T288), anti-Plk1, anti-phospho-Plk1 (T210) and anti-PARP antibodies were purchased from Cell Signaling Technology. The anti-β-actin antibody used as loading control was purchased from Santa Cruz Biotechnology.
Cell lines, patients and drug treatments. The parental K562 BCR-ABL1 + cell line (K562-S) was maintained in RPMI 1640 medium (Lonza) supplemented with 10% fetal calf serum (FCS, Gibco), 1% L-Glutamine and antibiotics in 5% CO2 and fully humidified atmosphere at 37°C. K562-R cells were generated by progressive increase of imatinib concentration (from 0.05 µM to 10 µM) in the culture medium. Drug resistance was confirmed by clonogenic assays and BCR-ABL1 dephosphorylation (indicating the survival in the presence of imatinib was sustained by a BCR-ABL1-independent mechanism of TKI resistance) was confirmed by western blotting [15].
Four CML patients in BC showing resistance to multiple lines of TKI therapy were included in our study; samples were collected with informed consent according to institutional guidelines. This study was conducted according to the principles of the Declaration of Helsinki and was approved by the Independent Ethics Committee of the “S. Orsola-Malpighi” University Hospital of Bologna (protocol 112/2014/U/Tess). The mononuclear cell fraction (MCF) from bone marrow or peripheral blood samples of patients and peripheral blood apheresis products of 8 healthy donors (HD) were obtained by Ficoll-Hypaque gradient centrifugation. CD34 + cells were isolated by immunomagnetic separation (mini-MACS, Miltenyi Biotec). Briefly, the MCF was incubated at 4° C for 30’ with magnetic microbeads coated with anti-CD34 antibody (Miltenyi Biotec). CD34 + cells were flown through a separation column in a magnetic field. Cell purity was confirmed using flow cytometric analysis of anti-CD34-FITC antibody (BD Biosciences); it was > 84% in all cases (data not shown).
Imatinib (1 µM), danusertib (1 µM) and volasertib (1 µM) were used to inhibit BCR-ABL1, Aurora kinase A and Plk1 respectively. The WEE1 inhibitor AZD1775 (1 µM) was used alone or in combination with danusertib (500 nM) and volasertib (500 nM).
Analysis of cell cycle distribution and apoptosis. Cell cycle distribution was performed on 5 × 105 cells fixed overnight in 70% ethanol and treated with 1 µg/µL propidium iodide (PI) and RNase (both from Sigma) at 37°C for 30 min. Apoptotic cells were recognized by cytofluorimetric analysis of fluorescinated Annexin V and PI uptake (Roche). Cell fluorescence and PI uptake were measured by mean of a FACScan flow cytometer (set at 488 nm excitation and 530 nm bandpass filter wave length for fluorescin detection or > 580 nm for PI detection) and two different dedicated softwares were used to analyze results obtained (Modfit and Diva software from Beckton Dickinson) [33].
Protein analyses. Whole cell lysates were used for protein analyses (Western blot) and evaluation of protein post-translational modifications. Briefly, 10x106 cells before and after treatments, were resuspended in 200µl of lysis solution (20 mM Tris-HCl, pH 7.5) and maintained in constant agitation for 30 min at 4°C. Lysates were then centrifuged in a microcentrifuge at 4°C for 30 min at 12.000 rpm. The supernatant was aspirated and placed in a fresh tube kept on ice, and the pellet discarded. 40µg of the whole cell lysates obtained were resolved by SDS PAGE. Gels were removed from the electrophoresis apparatus and transferred on a PVDF transfer membrane. Signal intensities in single blots obtained in three separate experiments were revealed by means of ChemiDoc XRS + Gel Imaging System (BioRad) equipped with a dedicated software (Image Lab, BioRad).
Clonogenic assays. Drug cytotoxicity was evaluated in cell lines (K562-S and K562-R) and CD34 + progenitor cells from BC patients or HD by clonogenic assays. The reduction of colony number (generated in 0.9% methylcellulose supplemented with 30% fetal calf serum) in the presence of increasing doses of imatinib (0.025-0.1 µM), danusertib (0.025-0.1 µM) and volasertib (0.025-0.1 µM) was assessed after 14 days of incubation at 37°C in a fully humidified atmosphere and 5% CO2. Nonlinear regression analyses (GraphPad Prism; GraphPad Software Inc.) were used to calculate the lethal dose (LD50) of the different drugs and combinations.
Immunofluorescence (IF) analysis. Cells set on poly-L-lysine-coated glass slides were fixed with 3% paraformaldehyde for 10’ at 37°C, washed with 0.1M glycine in phosphate-buffered saline (PBS), permeabilized in 70% ice-cold ethanol for 2’ at -20° C and incubated overnight at 4°C with primary anti-ɤH2AX and anti-RAD51 antibodies. Slides were then stained with a secondary anti-rabbit antibody conjugated with Alexa Fluor 568 for 2 h at room temperature and a subsequent anti-mouse antibody fluoresceinated. 6-diamidino-2-phenylindole (DAPI) was used to stain the nuclear compartment. IF analyses were performed using an Axiovert 40 CFL microscope (Zeiss). Images were acquired with a 100X objective and analyzed with the AxioVision software.
Statistics. Data are presented as mean values ± SD and were analyzed for statistical significance by Student t-test (GraphPad Prism Software). A P value of < 0.05 was considered as statistically significant.