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Supplementary Information
Materials and Methods:
Reagents
HSP90 inhibitor TAS-116 was provided by TAIHO Oncology (Tsukuba, Ibaraki, Japan). This
compound is an ATP-competitive HSP90 inhibitor. Its unique HSP90 binding mode leads to
selective inhibition of cytosolic HSP90/. Bortezomib (BTZ), PF-04928473 (SNX-2112),
PF-04929113 (SNX-5422) were obtained from Selleck Chemicals (Houston, TX, USA);
17-allylamino-17-demethoxygeldanamycin (17-AAG) from Sigma-Aldrich (St Louis, MO, USA);
and recombinant human IL-6 from R&D Systems (Minneapolis, MN, USA).
Human 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, USA). RPMI-8226, U266, and NCI-H929
human MM cell lines, as well as ARPE-19 human retinal pigment epithelial cell line, were
obtained from ATCC (Manassas, VA, USA). OPM1 and OPM2 plasma cell leukemia cell lines
were kindly provided by Dr Edward Thompson (University of Texas Medical Branch, Galveston,
TX, USA). IL-6-dependent INA6 human cell line was provided by Dr Renate Burger (University
of Kiel, Kiel, Germany). NALM-6 B-cell leukemia cell line was kindly provided by Dr James
Griffin (Dana-Farber Cancer Institute, Boston, MA, USA). All MM and NALM-6 cell lines were
cultured in RPMI 1640 containing 10% FBS (Sigma-Aldrich), 2 M L-glutamine, 100 U/mL
penicillin, and 100 g/mL streptomycin (Invitrogen, Carlsbad, CA, USA), with 2.5 ng/mL of IL-6
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only in INA6 cells. ARPE-19 cell line was cultured in DMEM:F12 containing 10% FBS
(Sigma-Aldrich), 100 U/mL penicillin, and 100 g/mL streptomycin (Invitrogen).
Primary cells
Blood samples collected from healthy volunteers were processed by Ficoll-Hypaque (GE
Healthcare, Pittsburgh, PA, USA) gradient to obtain peripheral blood mononuclear cells. Patient
MM cells and bone marrow stromal cells (BMSCs) were obtained from BM samples after
informed consent was obtained, in accordance with the Declaration of Helsinki and approval by
the Institutional Review Board of the Dana-Farber Cancer Institute. Mononuclear cells were
separated using Ficoll-Hypaque density sedimentation, and plasma cells were purified (> 95%
CD138+) by positive selection with anti-CD138 magnetic-activated cell separation microbeads
(Miltenyi Biotec, San Diego, CA, USA). Tumor cells were also purified from the BM of MM
patients using the RosetteSep negative selection system (StemCell Technologies, Vancouver,
BC, Canada). BMSCs were generated by culturing BM mononuclear cells for 4 to 6 weeks in
DMEM medium supplemented with 15% FBS, 100 U/mL penicillin, and 100 g/mL streptomycin.
Growth Inhibition assay
The growth inhibitory effect of TAS-116 or other HSP90 inhibitors in cell lines, peripheral blood
mononuclear cells (PBMNCs), and BMSCs was assessed by measuring
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT; Sigma-Aldrich) dye
absorbance or CellTiter-Glo® assay (Promega, Madison, WI, USA), as previously described.1, 2
To measure proliferation of MM cells with or without BMSCs, the rate of DNA synthesis was
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measured by [3H]-thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, USA)
uptake, as previously described.3
Detection of apoptosis by annexin V/propidium iodide staining
Detection of apoptotic cells was done with the annexin V/propidium iodide (PI) detection kit
(Immunotech/Beckman Coulter, Indianapolis, IN, USA), as previously described 4. Apoptotic
cells were analyzed on a BD FACSCanto II (BD Biosciences, San Jose, CA, USA) using
FACSDiva (BD Biosciences). Cells that were annexin V positive and PI negative were
considered early apoptotic cells, whereas positivity for both annexin V and PI was associated
with late apoptosis or necrosis.
Western blotting
MM cells were treated with or without novel or conventional agents; cells were then harvested,
washed, and lysed, as in prior studies.1, 5 Cell lysates were subjected to SDS-PAGE, transferred
to membranes, and immunoblotted with the following antibodies: anti-Akt, phospho-Akt
(Ser473), ERK, phospho-ERK (Thr202/Tyr204), C-Raf, phosphor-C-Raf (Ser338), MEK1/2,
phosphor-MEK1/2 (Ser217/221), XIAP, CDK4, caspase-3, caspase-8, PARP, NFB p65,
phospho- NFB p65 (Ser536), IBphospho- IB (Ser32/36), IKK, IKK,
phospho-IKK/Ser176/180), IRE1, PERK, BiP/GRP78, eIF2, phospho- eIF2 (Ser51),
CHOP, GAPDH, HSP27, HSP70, HSP90, and -Tubulin (Cell Signaling, Beverly, MA, USA);
phospho-IRE1 (Ser724; Thermo Scientific, West Palm Beach, FL, USA); and NFB p50
(Santa Cruz Biotechnology, Dallas, TX, USA). Protein expression was quantified using ImageJ
(National Institutes of Health, Bethesda, MD, USA). FL indicates full-length; CF, cleaved form.
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Murine xenograft models of human MM
CB17 SCID mice (48-54 days old) were purchased from Charles River Laboratories
(Wilmington, MA, USA). All animal studies were conducted according to protocols approved by
the Animal Ethics Committee of the Dana-Farber Cancer Institute. Mice were irradiated (200
cGy), injected subcutaneously with 5 × 106 MM.1S cells in the right flank on day 0, and then
received treatment for 28 days after detection of tumor. Mice were treated with 10 mg/kg oral
TAS-116 5 days a week (n = 10); 15 mg/kg oral TAS-116 5 days a week (n = 10); 0.5 mg/kg
subcutaneous BTZ twice a week (n = 8); or 0.5 mg/kg subcutaneous BTZ twice a week and 10
mg/kg oral TAS-116 5 days a week (n = 10) for 28 days. A vehicle control group received oral
vehicle only and subcutaneous saline (n = 9). Tumor size was measured every other day in 2
dimensions using calipers, and tumor volume was calculated with the formula: V = 0.5(a × b2)
where “a” is the long diameter of the tumor and “b” is the short diameter of the tumor. Mice were
sacrificed when the tumor reached 2 cm3 or mice appeared moribund, to prevent unnecessary
morbidity. Survival was evaluated from the first day of treatment until death. For analysis of
tumor tissues, mice in both control and treatment groups were sacrificed at day 3 after treatment
with vehicle or TAS-116. Tumors excised from mice were evaluated by TdT-mediated d-UTP
nick end labeling (TUNEL) assay and immunohistochemical analysis using cleaved caspase-3
staining. For evaluation of retinal tissue damage, TAS-116 (15 mg/kg; 5 days a week),
PF-04929113 (SNX-5422) (40 mg/kg; 3 times per week), or vehicle was administered orally in
SCID mice for two weeks. Photoreceptor cell death was evaluated by TUNEL staining.
Statistical analysis
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Statistical significance was determined by Student’s t-test. The minimal level of significance was
P < 0.05. Overall survival (OS) was assessed using Kaplan-Meier curves and log-rank analysis.
The combination index (CI) values were calculated by isobologram analysis using the
CompuSyn Version 1.0 software program (ComboSyn, Paramus, NJ, USA). CI values < 1.0
indicate synergism; CI = 1.0, additive effect; and CI > 1.0, antagonism.
Results:
TAS-116 induces apoptosis in MM cells
We investigated the mechanism of cytotoxicity triggered by TAS-116 using annexin V/PI staining
and immunoblotting in MM cells. The analysis showed a significant dose- and time-dependent
increase in annexin V-positive cells after treatment with TAS-116 in MM.1S cells
(Supplementary Figure S3A). Consistent with MM.1S cells, we also observed dose-dependent
increase in annexin V-positive cells in RPMI-8226 cells and patient MM cells (Supplementary
Figure S3B, S3C). In addition, TAS-116 markedly induced caspase-8, -3, and PARP cleavage in
MM cell lines (Supplementary Figure S3D). Importantly, the pan-caspase inhibitor zVAD-fmk
inhibited not only TAS-116-induced caspase and PARP cleavage in MM.1S cells
(Supplementary Figure S3E), but also blocked TAS-116-induced apoptosis in both MM.1S and
RPMI-8226 cells (P < 0.01, respectively; Supplementary Figure S3F). These results strongly
suggest that TAS-116 triggers caspase-dependent apoptosis in MM cells.
TAS-116 inhibits Akt and ERK pathway, and overcomes the growth stimulatory effects
triggered by cytokines and the bone marrow microenvironment
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We evaluated the potential mechanisms underlying the enhanced potency of TAS-116 in MM
cells. Because ERK and Akt signaling cascades mediate cell proliferation and drug resistance in
MM cells,6, 7 we examined whether TAS-116 suppresses these signaling cascades induced by
cytokines. TAS-116 significantly inhibited p-ERK and p-Akt in a time-dependent manner in
MM.1S cells (Supplementary Figure S5A left panel). Akt is a well-known HSP90 client protein
and degraded by HSP90 inhibitors,8 and TAS-116 significantly decreased phosphorylated Akt
relative to total Akt (Supplementary Figure S5A right panel).
BMSCs secrete cytokines such as IL-6 or IGF-1 which promote growth, survival, and drug
resistance in MM cells,9 and we next examined whether TAS-116 can suppress signaling
cascades induced by BMSCs or cytokines. Importantly, TAS-116 markedly inhibited IL-6-,
IGF-1-, and BMSC supernatant-induced p-ERK and p-Akt in MM.1S cells (Supplementary
Figure S4A left panel, S4B left panel, S4C left panel, S5B, S5C, S5D). Since we and others
have demonstrated that IL-6 and IGF-1 both induce growth and inhibit apoptosis in MM cells,10,
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we next determined whether TAS-116 can overcome the protective effects of exogenous IL-6
and IGF-1. Both IL-6 and IGF-1 trigger increased MM.1S cell growth, which was inhibited by
TAS-116 (P < 0.001; Supplementary Figure S4A right panel, S4B right right panel). We further
examined the inhibitory effect of TAS-116 on MM cell growth in the BM milieu: MM cell
adherence to BMSCs enhanced [3H]-thymidine uptake in MM.1S cells, which was inhibited by
TAS-116 (P < 0.001; Supplementary Figure S4C right panel). In addition, we tested the direct
cytotoxicity of TAS-116 on patient BMSCs using MTT assay: no significant growth inhibition in
BMSCs was triggered by TAS-116 (Supplementary Figure S5E). These data demonstrate that
TAS-116 potently inhibits BMSCs- and cytokine-induced phosphorylation of ERK and Akt in MM
cells, and also blocks the growth stimulatory effect of the BM microenvironment on MM cells.
TAS-116 triggers synergistic cytotoxicity with bortezomib in vitro
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We assessed the anti-MM effect of TAS-116 in combination with bortezomib using
[3H]-thymidine uptake and MTT assay. The combination of TAS-116 and bortezomib induced
synergistic cytotoxicity, with a combination index < 1.0, in MM cell lines (Supplementary Figure
S6A and Supplementary Table S1), as well as MM cells from 2 patients (Supplementary Figure
S6B and Supplementary Table S2). In addition, annexin V/PI staining showed that TAS-116
enhanced apoptosis induced by bortezomib in both MM cell lines (Supplementary Figure S6C),
and MM cells from 2 patients (Supplementary Figure S6D).
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