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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 1 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 2 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, NFB p65, phospho- NFB p65 (Ser536), IBphospho- IB (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 NFB 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. 3 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 4 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 5 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, 11 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 6 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). References 1. Hideshima T, Catley L, Yasui H, Ishitsuka K, Raje N, Mitsiades C, et al. Perifosine, an oral bioactive novel alkylphospholipid, inhibits Akt and induces in vitro and in vivo cytotoxicity in human multiple myeloma cells. Blood 2006; 107: 4053-4062. 2. Liu S, Hsieh D, Yang YL, Xu Z, Peto C, Jablons DM, et al. Coumestrol from the national cancer Institute's natural product library is a novel inhibitor of protein kinase CK2. BMC Pharmacol Toxicol 2013; 14: 36. 3. Ikeda H, Hideshima T, Fulciniti M, Lutz RJ, Yasui H, Okawa Y, et al. The monoclonal antibody nBT062 conjugated to cytotoxic Maytansinoids has selective cytotoxicity against CD138-positive multiple myeloma cells in vitro and in vivo. Clin Cancer Res 2009; 15: 4028-4037. 4. Cirstea D, Hideshima T, Rodig S, Santo L, Pozzi S, Vallet S, et al. Dual inhibition of akt/mammalian target of rapamycin pathway by nanoparticle albumin-bound-rapamycin 7 and perifosine induces antitumor activity in multiple myeloma. Mol Cancer Ther 2010; 9: 963-975. 5. Hideshima T, Neri P, Tassone P, Yasui H, Ishitsuka K, Raje N, et al. MLN120B, a novel IkappaB kinase beta inhibitor, blocks multiple myeloma cell growth in vitro and in vivo. Clin Cancer Res 2006; 12: 5887-5894. 6. Hideshima T, Bergsagel PL, Kuehl WM, Anderson KC. Advances in biology of multiple myeloma: clinical applications. Blood 2004; 104: 607-618. 7. Okawa Y, Hideshima T, Steed P, Vallet S, Hall S, Huang K, et al. SNX-2112, a selective Hsp90 inhibitor, potently inhibits tumor cell growth, angiogenesis, and osteoclastogenesis in multiple myeloma and other hematologic tumors by abrogating signaling via Akt and ERK. Blood 2009; 113: 846-855. 8. Mitsiades CS, Mitsiades NS, McMullan CJ, Poulaki V, Kung AL, Davies FE, et al. Antimyeloma activity of heat shock protein-90 inhibition. Blood 2006; 107: 1092-1100. 9. Hideshima T, Mitsiades C, Tonon G, Richardson PG, Anderson KC. Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets. Nat Rev Cancer 2007; 7: 585-598. 10. Chauhan D, Uchiyama H, Akbarali Y, Urashima M, Yamamoto K, Libermann TA, et al. Multiple myeloma cell adhesion-induced interleukin-6 expression in bone marrow stromal cells involves activation of NF-kappa B. Blood 1996; 87: 1104-1112. 8 11. Mitsiades CS, Mitsiades NS, McMullan CJ, Poulaki V, Shringarpure R, Akiyama M, et al. Inhibition of the insulin-like growth factor receptor-1 tyrosine kinase activity as a therapeutic strategy for multiple myeloma, other hematologic malignancies, and solid tumors. Cancer Cell 2004; 5: 221-230. 9