Opm 2

RT112, OPM2, CAL51, HCC366 and HCC78 cells were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). EBC-1, MKN45, MKN1 and MKN28 cells were purchased from JCRB Cell Bank (Tokyo, Japan). UMUC14 cells were obtained from the European Collection of Cell Cultures (Salisbury, UK). SUM52PE cells were obtained from Asterand Company. MGC-823 and MDA-MB-453 cells were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). SGC-7901 cells were obtained from the Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine (Shanghai, China). Human umbilical vein endothelial cells (HUVECs) were obtained from ScienCell Research Laboratories (Shanghai, China). Other cell lines used in the present study were purchased from American Type Culture Collection (Manassas, United States). Some background information of cell lines used in the study was listed in Additional file 1: Table S1. All cell lines were obtained between 2000 and 2017 and cultured according to the provided instructions. The cells were confirmed to be free of mycoplasma and were passaged no more than 25–30 times after thawing. The cell lines were characterized using short tandem repeat markers by Genesky Biopharma Technology (most recent test in 2017).

This study used 6 HMCLs, NCI-H929, OPM-2, AMO-1 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Neddersassen, Germany), RPMI8226 (American Type Culture Collection, Manassas, VA, USA), KMS-12-BM and KMS-28-PE (kind gifts from Dr. Ohtsuki T (Kawasaki Medical School, Kurashiki, Okayama, Japan)), harboring various types of chromosomal abnormalities and gene mutations (Supplementary Information). HMCLs were grown in RPMI-1640 with 10% fetal calf serum, 2 mM L-glutamate, and penicillin/streptomycin at 37 °C in a fully humidified atmosphere of 5% CO₂ in the air. Patient-derived CD138-positive myeloma cells were isolated using MACSprep^TM Multiple Myeloma CD138 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Nordrhein-Westfalen, Germany) [5 (link),29 (link)], and were grown in RPMI-1640 with 10% fetal calf serum, 2 mM L-glutamate, penicillin/streptomycin, and CellXVivo Human B Cell Expansion Kit (R&D systems, Minneapolis, MN, USA). The agents used were BI-D1870, which selectively inhibits the phosphorylation of RSK2^Ser227 at NTKD in an ATP-competitive manner [64 (link)] (Cayman Chemical Company, Ann Arbor, MI, USA), and ipatasertib, which binds to AKT in an ATP-competitive manner and prevents substrate phosphorylation [65 (link)] (Selleck Biotech Ltd., Tokyo, Japan).

The HMCL MM1S was acquired from American Type Culture Collection, and JJN3, RPMI-8226, U266, and OPM2 were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen. LINF903, an Epstein–Barr virus-transformed B-cell line established from a healthy individual, was obtained from the National DNA Bank of the University of Salamanca, Spain. MM and LINF903 cell lines were cultured in RPMI 1640–l-glutamine medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% of fetal bovine serum (Sigma-Aldrich) and antibiotics (Gibco Life Technologies, Grand Island, NY, USA). All cells were incubated at 37°C in a 5% CO₂ atmosphere. The presence of mycoplasma was routinely checked with a MycoAlert kit (Lonza, Basel, Switzerland) and only mycoplasma-free cells were used in the experiments.

Human multiple myeloma cell lines (HMCLs) U266, KMS-12-BM, OPM-2, NCI-H929, SK-MM-1 and RPMI8226 were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). MM.1S and MM.1R cells were kindly provided by Dr. Steven Rosen (Northwestern University, Chicago, IL). A human bone marrow mesenchymal stromal cell line immortalized by enforced expression of telomerase (BMSC TERT⁺) was kindly provided by Dr. Dario Campana (St. Jude Children’s Research Hospital, Memphis, TN). All cell lines were cultivated in RPMI-1640 medium supplemented with 10 % heat-inactivated fetal bovine serum, 2 mM L-glutamine and 100 U/ml penicillin/streptomycin (Gibco). BMSC TERT⁺ cells were supplemented with 1 μM hydrocortisone (Sigma-Aldrich). For co-culture experiments, 1 × 10⁵ BMSC TERT⁺ cells were seeded in 24-well plates and cultured overnight before 2 × 10⁵ MM cells were added per well for 72 h.

Human myeloma cell lines AMO-1, MOLP-8, RPMI-8226, KMS-12-BM, EJM, IM-9, U-266, OPM-2, and LP-1, chronic myeloid leukemia cell line K562, and human embryonic kidney cell line 293 were newly obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Two additional human myeloma cell lines, Brown and SK-007, were provided by the New York branch of the Ludwig Institute for Cancer Research (LICR). Upon arrival in our laboratory, the authenticity of the cell lines was verified using cytology and flow cytometry. All cell lines were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) with penicillin–streptomycin (Invitrogen) and 10% fetal calf serum (Lonza, Basel, Switzerland).