Lenalidomide

Human myeloma cell lines (HMCL) were obtained from ATCC (MM1.S [CRL‐9068], U266B1 [TIB‐196], NCI H929 [CRL‐9068], RPMI 8226 [CCL‐155]) or JCRB (KMS11 [1179]) and were confirmed mycoplasma‐negative and validated by 9‐marker Short Tandem Repeat profiling (IDEXX BioResearch). Other HMCLs were obtained from DSMZ (JJN3, LP‐1), ATCC (MM1.R), or RG Hawley (8226Dox40, KMS34, KMS34CFZ; George Washington University).25 Lysates from kidney cancer cell lines Caki‐2 and RCW were provided by Y. Nakamura. Cells were cultured in RPMI 1640 (Life Technologies, #72400) with 10% fetal calf serum and penicillin/streptomycin under 5% CO₂. OTS514 and OTS964 were provided by OncoTherapy Science, Inc. Lenalidomide and verapamil were purchased from Cayman Chemical. For viability assays, 2 × 10⁴ cells/well were cultured in 96‐well plates with increasing concentrations of test drug for 72 hours (minimum 6 wells per dose per cell line). MTT (thiazolyl blue tetrazolium bromide, Acros Organics) was then added and incubated at 37°C for up to 1.5 hours. Cells were lysed overnight, absorbance at 570 nm was read, and IC₅₀ was calculated using the four‐parameter log (inhibition) vs response fit in Prism 7 (GraphPad Software). Multi‐agent curves were evaluated for pharmacological synergy by the Chou‐Talalay method with CalcuSyn Software (ComboSyn, Inc).26

B-lymphoma cell line DB and SU-DHL-4 were available from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in RPMI-1640 medium with 10% heat-inactivated fetal bovine serum (FBS) in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C. Specific TGF-β/NODAL/Smad inhibitor SB431542 was from Sigma (St. Louis, MO, USA). Lenalidomide was from Cayman Chemical (Ann Arbor, MI, USA). Nucleic acid synthesis inhibitor Actinomycin was from Abcam (Cambridge, UK).

FPFT-2216 and pomalidomide were synthesized by Fujimoto Pharmaceutical Group. Avadomide, iberdomide, siremadlin, idasanutlin, navtemadlin, milademetan, and MG-132 were procured from MedChemExpress; Z-VRPR-FMK and phorbol 12-myristate 13-acetate (PMA) were procured from Adipogen Life Sciences. Lenalidomide and ionomycin were obtained from Cayman Chemical, BAY 11-7082 from FUJIFILM Wako Pure Chemical Corporation, and safimaltib from Selleck Chemicals. Rituxan Intravenous Infusion (rituximab) was obtained from Zenyaku Kogyo. Z-VRPR-FMK was dissolved in PBS, and other reagents were dissolved in DMSO. The final DMSO concentration used for in vitro experiments did not exceed 0.25%.