Supplementary Information
Materials and Methods
Antibodies and Chemicals
Rabbit polyclonal antibodies were purchased from the following sources: caspase-3, caspase-9, phospho-Erk, Bad, and XIAP from Cell Signaling Technology (Beverly, MA); Bim from CalBiochem Co. (San Diego, CA); survivin from R&D Systems (Minneapolis, MN); Bax, cytochrome c, and Bcl-XL from BD Biosciences (San Jose, CA); and Bak, phospho-MEK1/2 from Upstate USA, Inc. (Charlottesville, VA). Mouse monoclonal antibodies were purchased from the following sources: caspase-8 from Cell Signaling Technology; Mcl-1 from Pharmingen (San Diego, CA); Erk2 from Santa Cruz Biotechnology (Santa Cruz, CA); and Bcl-2 from DAKO Cytomation (Carpinteria, CA). All other chemicals and solvents used were of the highest commercially available grade.
Cell lines and primary patient samples
The human AML cell lines U937, HL60, and KG-1 were obtained from the American Type Culture Collection (Rockville, MD). OCI/AML3 cells were kindly provided by Dr. M. Minden (Princess Margaret Hospital, Toronto, Canada). Lymphoblastic leukemic Jurkat and I2.1 cells (a Jurkat clone with a caspase-8 mutation) were purchased from the American Type Culture Collection (Manassas, VA). Bone marrow and peripheral bloodprimary samples were obtained from patients with newly diagnosed or recurrent AML or from patients with advanced, relapsed, or refractory AML or MDS, who were enrolled in the Phase I clinical trial with sorafenib. Informed consent was obtained following institutional guidelines.
Immunoprecipitation analyses
For the immunoprecipitation studies, cells lysates (0.4 mg of protein for each sample) were incubated overnight with anti-tubulin or Bcl-XL primary antibody at 4°C. The indicated proteins were pulled down by protein A/G PLUS-agarose (Santa Cruz Biotechnology), and then were resolved by SDS-PAGE, immunoblotted with Bim antibody. The amount of tubulin and Bcl-XL immunoprecipitated from each sample was determined by immunoblot with anti- tubulin and anti-Bcl-XL antibodies.
Lentiviral transduction
The oligonucleotide sequence of Bim-targeting shRNA (1) and of irrelevant shRNA of identical GC content (2) were as follows: 5’-CGCGTCCCCCTACCTCCCTACAGACA
GATTCAAGAGATCTGTCTGTAGGGAGGTAGTTTTTGGAAAT-3’(BIM) and 5’-CGCGTCCCCCGTACGCGGAATACTTCGATTCAAGAGATCGAAGTATTCCGCGTACGTTTTTGGAAAT-3’ (SCR). The lentiviral vectors were generated by inserting the above sequence between the Mlu I and Cla I sites of pLVTHM, a bicistronic lentiviral transfer vector that also expresses green fluorescent protein (a kind gift from Dr. Didier Trono (School of Life Sciences, Swiss Federal Institute of Technology, Lausanne, Switzerland)). Lentivirus was generated using 293T. At 48 hours post-transfection, virus-containing cell supernatants were collected and titered by the method of Dr. Trono.
Exponentially growing U937 cells were infected at a total multiplicity of infection of 0.4 to 0.5 TU per cell by spinoculation. After 48 hours of post infection, GFP positive U937 cells were collected by flow cytometry sorting. The cells were treated with sorafenib for 48 hours. The proportion of transduced cells undergoing apoptosis were stained with APC-conjugated-Annexin V and measured by flow cytometry.
Immunofluorescence staining and confocal analyses
U937 cells treated with 2.5 µM sorafenib for 3 hours were either stained live cells with Mitotracker (CMXRos) for 1 hour at 37°C in RPMI medium or remained unstained. The cells were then harvested, washed with PBS, and subsequently fixed in 100% cold methanol for 10 minutes at -20°C, after which they were exposed for an additional 1 minute to cold acetone to allow permeabilization. After washing and blocking in 5% goat serum-PBS, the cells were incubated with the first primary antibody overnight at 4°C and then with the first secondary biotinylated antibody for 30 minutes. The cells were next incubated with fluorescein avidin D for another 30 minutes, after which the cells were blocked using an avidin/biotin blocking kit. The cells were further incubated with second primary antibody and the biotinylated secondary antibody, followed by Texas Red Avidin D staining. The cells were then counterstained with DAPI mounting medium and observed under a confocal laser scanning microscope (Olympus FV500 ver.4.3 confocal system; Olympus, NY). Images were digitalized to obtain pseudocolored green for FITC, red for Texas Red (or CMXRos), and blue for DAPI. A yellow color results from the overlapping of green and red. Each image represents a single section, for which the confocal system was adjusted to allow a field depth of about 0.8 μm. As a control, U937 cells were incubated with pre-immune serum instead of primary antibodies.
AML blast colony and soft agar assays
For the colony formation assay, primary bone marrow blasts or normal bone marrow cells were obtained for in vitro studies from patients with newly diagnosed or recurrent AML or from normal bone marrow donors after informed consent had been obtained according to institutional guidelines. A previously described method was used to determine AML blast colony formation (3)
For AML cell lines, OCI/AML3 and U937 cells were seeded in 24-well plates in culture medium containing 0.35% low-melting agarose over a 0.7% agarose base layer in the presence of different concentrations of sorafenib or vehicle and incubated for 14 days at 37°C in a humidified 95% O2-5% CO2 atmosphere in RPMI supplemented with 10% FCS and L-glutamine. Colonies were then stained with p-iodonitrotetrazolium violet for 16 hours. Colonies larger than 100 mm in diameter were counted under a microscope in triplicate wells.
Phase I patient sample analyses
Whole-blood samples acquired from patients of sorafenib Phase I clinical trial according to institutional guidelines were collected under informed consent at indicated time points after the administration of sorafenib during the first course of therapy (Sorafenib: 600mg q.d. x 14d/21d for case 1, and 600mg q.d. x 5d/wk for case 2). The whole blood was subjected to hypotonic RBC lysis and held at room temperature for 5 to 10 minutes. After centrifugation, granulocytes were resuspended in PBS buffer and washed once. For the evaluation of apoptosis, the cells were stained with annexin V as described above (was this gated o CD34+?-pls check with Teresa). For the phospho-ERK analyses, the cells were fixed in 2% paraformaldehyde/PBS for 10 minutes at room temperature, permeabilized in 90% cold methanol for 20 minutes, and then were stained with APC-conjugated CD34 and PE-conjugated phospho-ERK antibodies, and the phospho-ERK levels were determined by flow cytometry. For Western blot analyses, the mononuclear cells were lysed in cell lysis buffer and protein levels were determined using corresponding antibodies, as described above.