Supplementary Materials and Methods Human MM cell lines Dex

Supplementary Materials and Methods
Human MM cell lines
Dex-sensitive MM.1S and resistant MM.1R human MM cell lines were kindly
provided by Dr. Steven Rosen (Northwestern University, Chicago, IL). NCI-H929
(H929), U266, and RPMI8226 human MM cells were obtained from American Type
Culture Collection, and KMS11 from Japanese Collection of Research Bioresources.
Doxorubicin-resistant RPMI-DOX40 (DOX40) was kindly provided by Dr. William
Dalton (Lee Moffitt Cancer Center, Tampa, FL). OPM1 and OPM2 plasma cell
leukemia cell lines were kindly provided by Dr. Edward Thompson (University of
Texas Medical Branch, Galveston, TX). IL-6 dependent INA6 cell line was provided
by Dr. Renate Burger (University of Kiel, Germany). ANBL6 cell line was kindly
provided by Dr. Robert Orlowski (M.D. Anderson Cancer Center, Houston, TX). All
cell lines were cultured in RPMI-1640 containing 10% fetal bovine serum (FBS), 2
μM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (Life Technologies),
with 2.5 ng/ml of IL-6 in INA6 cells and 5 ng/ml of IL-6 in ANBL6 cell cultures.
Primary cells
Blood samples collected from healthy volunteers were processed by Ficoll-Paque
(GE Healthcare) gradient to obtain peripheral blood mononuclear cells. Patient MM
cells and bone marrow stromal cells (BMSCs) were obtained from bone marrow
samples after informed consent was obtained, in accordance with the Declaration
of Helsinki. The study was approved by the Institutional Review Board of the
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Dana-Farber Cancer Institute. Mononuclear cells were separated using
Ficoll-Paque density sedimentation, and plasma cells were purified (>95% CD138+)
by positive selection with anti-CD138 magnetic activated cell separation micro
beads (Miltenyi Biotec). Tumor cells were also purified from the bone marrow of MM
patients using the RosetteSep negative selection system (Stem Cell Technologies).
BMSCs were generated by culturing bone marrow mononuclear cells for 4-6 weeks
in DMEM medium supplemented with 15% FBS, 100 U/mL penicillin, and 100
μg/mL streptomycin. To generate conditioned medium derived from BMSCs,
RPMI-1640 with 10% FBS was used for culture of BMSCs overnight, and the
medium was then harvested by centrifugation.
Akt kinase assays
MT-tagged human full length Akt1, 2 and FLAG-tagged human full length Akt3
protein were expressed using baculovirus expression system and purified with an
affinity column, followed by incubation with purified PDK1 for activation. To
determine IC50 values of TAS-117, activated Akt enzymes were preincubated with
TAS-117 at various concentrations in reaction buffer (15 mM Tris [pH 7.5], 0.01%
Tween20 and 2 mM DTT) at 25 °C for 120 min. After the preincubation, ATP and
peptide substrate were added to start the enzymatic reaction, and then incubated at
25 °C for 60 min. The final reaction mixture contains an appropriate amount of Akt
enzyme, 150 μM ATP, 500 nM peptide substrate (biotin-KGSGSGRPRTSSFAEG)
and 10 mM MgCl2 in the reaction buffer. The reactions were terminated by adding
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EDTA, followed by the addition of detection mixture containing SureLightTM
Allophycocyanin-Streptavidin (PerkinElmer) and Eu-W1024-labeled
anti-phospho-Crosstide Antibody (PerkinElmer) in detection buffer (30 mM Tris [pH
7.5] and 0.2% Tween 20). After incubation at room temperature for 120 min, the
samples were excited at 337 nm, and emissions at 620 nm and 665 nm were read
using PHERAstar (BMG LABTECH). Emission ratio of 665 nm/620 nm of each
sample was calculated and plotted against the concentration of TAS-117. IC50
values were obtained by analyzing data using XLfit software (IDBS) model 205.
In vitro Akt IP-kinase assay was performed according to manufacturer's
protocol (Akt Kinase Assay Kit, Cell Signaling Technology) with modifications.
Briefly, MM.1S cell lysates were immunoprecipitated with immobilized-p-Akt
(Ser473) Ab, washed twice with lysis buffer and once with kinase buffer.
Immunoprecipitates were resuspended in kinase buffer and incubated with DMSO
control or TAS-117 for 10 min at 30°C. The samples were then incubated in the
presence of ATP and substrate (GSK3 fusion protein) for 30 min at 30°C. The
reaction was terminated by adding SDS-sample buffer, and subjected to
SDS-PAGE and immunoblotting using Abs shown in Supplementary Fig.S1D.
mTOR kinase assay
The inhibitory activity of TAS-117 against FRAP1 (mTOR) was determined by
SelectScreen® Biochemical Screening Service (Invitrogen) using Z’-LYTE® Assay,
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with 10 point titrations and3-fold serial dilutions conducted from 10 μM. The ATP
concentration was at Km of mTOR.
Kinase selectivity profiling study
To investigate the selectivity for Akts over other kinases, the inhibitory activity of
TAS-117 at 1 µM was determined by QuickScoutTM Selectivity Profiling Service
(Carna Biosciences) against 302 protein and lipid kinases using IMAPTM (Molecular
Devices) assay or Mobility Shift Assay. The ATP concentration in each assay was
approximately at Km of each kinase, except for assays in which the ATP
concentration was fixed at 1 mM.
Antibodies for western blotting
Immunoblotting was performed using antibodies: anti-phospho (p)-Akt (Ser473),
p-Akt (Thr308), Akt, p-PDK1 (Ser241), p-FKHR (Thr24)/FKHRL1 (Thr32), GAPDH,
PARP, LC3A/B (LC3-I and II), p-mTOR (Ser2481 and Ser2448), mTOR, p-p70 S6
kinase (Thr389), p70 S6 kinase, α-tubulin, p-p65 (Ser276), p65, IRE1α, BiP/GRP78,
p-eIF2α (Ser51), eIF2α, CHOP, p-STAT3 (Tyr705), STAT3, p-ERK1/2
(Thr202/Tyr204), ERK, p-GSK3α/β (Ser21/9), GSK3, caspase-3, and caspase-8
(Cell Signaling); as well as anti-p-IRE1α (Ser724) (Thermo Scientific) Ab.
Side population analysis
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To detect the side population (SP) in MM, the cells were washed in pre-warmed
RPMI with 2% FBS and 10 mM Hepes buffer (Life Technologies) and resuspended
at a concentration 1 × 106 cells/mL in RPMI with 2% FBS and 10 mM Hepes buffer
containing 5 μg/mL of Hoechst 33342 dye; cells were incubated for 90 min at 37°C
with intermittent shaking. As a negative gating control, SP cells were preincubated
with 50 μM reserpine, an ABC transporter inhibitor. At the end of the incubation,
cells were washed with ice-cold PBS containing 2% FBS and 10 mM Hepes buffer.
To gate only viable cells, 7-actinomicin D (7-AAD; Molecular probes, final
concentration = 1 μg/mL) in PBS with 2% FBS and 10 mM Hepes buffer was added
to the cells. The analyses and sorting (>98% purities) were done on a FACS Aria
Special Sorter UV laser (Becton Dickinson).
Murine xenograft models of human MM
Male CB17 SCID mice (48-54 days old) were purchased from Charles River
Laboratories. Animal studies using the MM.1S xenograft model were conducted
according to protocols approved by the Animal Ethics Committee of the
Dana-Farber Cancer Institute. In this model, mice were irradiated (200cGy) and
then inoculated subcutaneously in the right flank with 5 × 106 MM.1S cells in 100 μl
RPMI-1640. After detection of tumor, mice were treated for 21 days with 12 mg/kg
oral TAS-117 daily for 5 days a week (n=10); 16 mg/kg oral TAS-117 daily for 5 days
a week (n=10); 0.5 mg/kg subcutaneous bortezomib in the left flank twice a week
(n=10); or 12 mg/kg oral TAS-117 daily for 5 days a week and 0.5 mg/kg
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subcutaneous bortezomib twice a week (n=10). A vehicle control group received
oral vehicle (0.5% hydroxypropyl methylcellulose) alone and subcutaneous saline
(n=9). Tumor volume was calculated from caliper measurements every 3-4 days
until day of first death in each group; mice were sacrificed when tumors reached 2
cm in length or were ulcerated. Survival was evaluated from the first day of
treatment until death. Tumors excised from mice treated with 16 mg/kg TAS-117 or
vehicle were evaluated by immunohistochemical analysis using p-Akt (Ser473) and
TdT-mediated d-UTP nick end labeling (TUNEL) assay. Images were obtained
using Axio Imager.Z1 microscope (Car Zeiss) with 40×/1.3 objective lens, and
acquired through AxioVision software.
For the H929 xenograft model, 9 week old female CB17 SCID mice
(Charles River Laboratories) were subcutaneously injected with 1 × 107 H929 cells
mixed with Matrigel (BD Biosciences). After detection of tumors, mice were treated
for 14 days with 8 mg/kg oral TAS-117-HCl daily (n=10); 12 mg/kg oral TAS-117-HCl
daily (n=10); or vehicle alone as a control (n=10). Tumor volume was calculated
from caliper measurements every 3-5 days until day 16. This study was conducted
by Piedmont Research Center, where the animal care and use program is
accredited by the Association for Assessment and Accreditation of Laboratory
Animal Care International (AAALAC).
Immunocytochemical detection of LC3
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MM.1S cells were cultured with or without TAS-117 (1 μM) for 6h and cytospins
were prepared. Cells were fixed in ice-cold methanol and acetone (1:1), washed,
and blocked with PBS with 5% BSA for 1 h. After washing again, cells were
permeabilized and stained for 4 h with a solution of PBS with 0.05% Triton X-100
and 0.5% BSA containing LC3 Ab (Cell Signaling) at a ratio of 1:100. Cells were
washed and incubated for 1 h with the secondary-fluorescent Ab (Alexa Fluor 488;
Cell Signaling). After washes, the nuclear content was stained with DAPI reagent
(Life Technologies) for 5 minutes and washed. The entire procedure was performed
at room temperature. The slides were then mounted with ProLong Gold Antifade
Reagent (Life Technologies), and images were taken using a Zeiss microscope
(Carl Zeiss) equipped with Hamamatsu ORCA-ER camera (Hamamatsu Photonics)
using 100× objective lens and analyzed with ImageJ software.
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