Supplemental information
Supplemental Methods
Assays for growth inhibition and apoptosis
Assays for growth inhibition and apoptosis were performed as described in the Supplementary information. Growth-inhibitory effect was analyzed by a modified MTT assay using the Cell Counting Kit-8 (Dohjindo Molecular Technologies, Kumamoto, Japan). For the examination of apoptosis, cells were counterstained with Annexin V-FITC and propidium iodide (PI) and subjected to flow cytometric analysis. Data obtained by flow cytometric analyses were analyzed using FLOWJO software Ver. X (Tomy Digital Biology, Tokyo, Japan).
Western blot analysis
Primary antibodies used in this study were against PDPK1, phospho (p)-PDPK1Ser241, ERK1/2, p-ERK1/2Thr202/Tyr204, RSK2, p-RSK2Ser227, p-RSK2Tyr529, AKT, p-AKTThr308, cleaved caspase-3 (Cell Signaling Technologies, Beverly, MA, USA), MYC, FGFR3, BAD, p-BADSer112, IRF4 (Santa Cruz Biotechnology), BCL2 (Upstate Biotechnology, Inc., Lake Placid, NY, USA), BIM (Enzo Life Sciences International Inc., Farmingdale, NY, USA), Cyclin D1 (CCND1), Cyclin D2 (CCND2) (Becton Dickinson, San Diego, CA, USA), DEPTOR (Millipore, Billerica, MA, USA), and β-Actin (Sigma, St Louis, MO, USA).
Primers used for the analyses of N-Ras, K-RAS and BRAF mutations
The following primers were used: N-RAS forward, GCT GTG GTC CTA AAT CTG TC; N-RAS reverse, TCG CCT GTC CTC ATG TAT TG; K-RAS forward, CGG CTC GGC CAG TAC TCC (for codons 12 and 13) and GAG AGG CCT GCT GAA AAT GA (for codon 61); and K-RAS reverse, TGC ACT GTA CTC CTC TTG AC (for codons 12 and 13) and CCT ACT AGG ACC ATA GGT AC (for codon 61); BRAF forward, ACA CGC CAA GTC AAT CAT CC; BRAF reverse, TCT GGT CCC TGT TGT TGA TG.
Probes utilized for fluorescence in situ hybridization (FISH)
Interphase FISH studies for the detection of immunoglobulin heavy chain (IGH) translocations were used for the analyses of t(4;14)(p16;q32) (LSI IGH/FGFR3), t(11;14) (LSI IGH/CCND1), t(8;14) (LSI IGH/MYC) and t(14;16)(q32;q23) (LSI IGH/MAF), all obtained from Vysis, Bergisch-Gladbach, Germany. The 13q deletion, 17p deletion and 1q21 abnormalities were evaluated using probes specific for 13q14.3 (LSI D13S319) (Vysis), 17p13.1 (LSI TP53) (Vysis) and 1q21 (Cytocell, Cambridge, UK), respectively.
Supplemental legend
Supplementary Table S1.
Clinical information of patients whose myeloma cells were subjected to Western blotting and in vitro treatment with BX-912. Abbreviations; Pt.: patient, M: male, F: female, Ig; immunoglobulin, BJP: Bence-Jones protein type, NA: not applicable, NE: not examined, None: no abnormal signals were identified by FISH. MP: melphalan plus prednisolone, VMP: bortezomib (BTZ) plus MP, MPT; MP plus thalidomide, BD: BTZ plus dexamethasone (DEX), CBD: cyclophosphamide plus BD plus, Rd: lenalidomide plus DEX, CP: cyclophosphamide plus prednisolone, ASCT: high-dose chemotherapy supported by autologous stem cell transplantation.
Supplementary Fig. S1
AMO-1 cells and NCI-H929 cells were seeded at 2 x 105 cells/ml on day 0, and were treated either by BX-912 or AR12 at their concentrations of 5, 10, or 20 mM for 24, 48, and 72 hours. Cells were directly stained by trypan blue, and viable cell numbers were counted under the inverted microscope. Results were mean±SD of triplicate analyses.
Supplementary Fig. S2
A, DNA content was examined by propidium iodide (PI)-staining of formalin-fixed cells using flow cytometric analysis. The X-axis represents DNA content, and the Y-axis represents the relative cell numbers. AR-12 (AR) treatment reduced G1 population (light green fraction) and increased the subG1 fraction, a hallmark of apoptosis induction (indicated by arrow), in the myeloma AMO-1 and NCI-H929 cell lines. AMO-1 and NCI-H929 cells were treated with AR at their IC80 concentrations for 12 and 24 hours. B, AMO-1 and NCI-H929 cells were treated with AR at their IC80 concentrations for 6 to 12 hours, and were subjected to double staining for Annexin-V (X-axis) and PI (Y-axis). AR treatment caused the increase of cells undergoing early apoptosis (Annexin-V-positive/PI-negative fraction) first, and, then, resulted in the increase of cells undergoing late apoptosis (double positive for Annexin-V and PI) in both AMO-1 cells and NCI-H929 cells.
Supplementary Fig. S3
AMO-1 cells and NCI-H929 cells were co-cultured with HS-5 cells for 48 hours, and then were treated by BX-912 at its IC50s for 3 hours. The activities of PDPK1Ser241, RSK2Ser227 and AKTThr308 were examined by Western blotting. Ctl. indicates cells cultured without the co-culture.
Supplementary Fig. S4.
A. BX-912 shows no cross-resistance with BTZ. BX-912 exerted its growth inhibitory effect on BTZ-resistant KMS-11/BTZ cells and OPM-2/BTZ cells (red lines) as effectively as on their respective parental cells (blue dotted lines). Cells were treated with the indicated concentrations of either BX-912 or BTZ for 48 hours. The viable cell number of untreated cells was considered to be 1.0. B. The combination of BX-912 and the NF-κB inhibitor, BAY11-7085, exhibited an additive growth inhibitory effect on BTZ-resistant KMS-11/BTZ cells and OPM-2/BTZ cells. Shaded areas represent the additive effects of the two agents.
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