Erlotinib

Compounds used in the CIVO and systemic studies were purchased from Selleck Chemicals (Everolimus, Sunitinib, 5-Fluorouracil, Rapamycin, Gemcitabine, ABT-199, ABT-263, Erlotinib), Chemietek (ABT-263, ABT-199) and Medkoo Biosciences (Mitomycin C). Abraxane^® was manufactured by Celgene Corporation (San Diego, CA).

A number of anti-neoplastic drugs were tested for their ability to inhibit growth of MM cells: the tyrosine kinase inhibitors (TKI) bosutinib, dasatinib, imatinib, sorafenib, sunitinib, and nilotinib, the ErbB-receptor inhibitors lapatinib, erlotinib, and gefitinib, the Aurora-kinase inhibitor VX-680, the HSP90 inhibitor 17AAG, the PLK-1 inhibitor BI2536, the pan-BCL-2 antagonist obatoclax, and the HDAC-inhibitor vorinostat were purchased from Chemietek (Indianapolis, IN, USA). The PI3 kinase/mTOR inhibitor BEZ235 was obtained from Selleck Chemicals (Houston, TX, USA). Stock solutions of drugs were prepared by dissolving in dimethylsulfoxide, DMSO (Merck, Darmstadt, Germany). RPMI 1640 medium and fetal calf serum (FCS) were purchased from PAA Laboratories (Pasching, Austria), and ³H-thymidine from PerkinElmer (Waltham, MA, USA). FITC-labeled CD34 monoclonal antibody (mAb) 581, PE-labeled CD34 mAb 581, FITC-labeled CD138 mAb MI15, PE-labeled CD138 mAb DL-101, PerCP-labeled CD45 mAb 2D1, APC-labeled CD38 mAb HIT2, PE-labeled and Alexa Fluor® 647-labeled active caspase-3 mAb C92-605 were purchased from BD Biosciences (San Jose, CA, USA). The PerCP-labeled CD20 mAb 2H7 and the APC-labeled CD27 mAb O323 were obtained from Biolegend (San Diego, CA, USA), and an Annexin V/FITC kit from eBioscience (San Diego, CA, USA).

We purchased the drugs from the commercial sources: paclitaxel, doxorubicin/adriamycin, 5-fluorouracil, epirubicin, cyclophosphamide, crizotinib, salinomycin, thioridazine, phenethyl isothiocyanate (PEITC), sodium valproate, sodium butyrate, 5-azacytidine, and 6-mercaptopurine (Sigma-Aldrich, St. Louis, MO), NVP-BEZ235 (Cayman Chemical, Ann Arbor, MI), and itraconazole (Selleckchem, Houston, TX). Naoto Ueno kindly provided AZD6244 (AstraZeneca, Wilmington, DE) and erlotinib (ChemieTek, Indianapolis, IN). We dissolved BEZ235, PEITC, itraconazole, crizotinib, salinomycin, doxorubicin, paclitaxel, AZD6244, and erlotinib in dimethyl sulfoxide (DMSO), thioridazine, sodium valproate, and sodium butyrate in water, 6-mercaptopurine in 0.1 M NaOH, and 5-azacytidine in dulbecco’s phosphate buffered saline. To prepare FAC (5-fluorouracil, adriamycin, cyclophosphamide) and FEC (5-fluorouracil, epirubicin, cyclophosphamide) chemotherapy combinations, drugs were measured individually and dissolved into DMSO and combined. Final concentrations of drugs were 250 nM 5-fluorouracil, 25 nM adriamycin, and 250 nM cyclophosphamide for FAC and 250 nM 5-fluorouracil, 50 nM epirubicin, and 250 nM cyclophosphamide for FEC. We added equal volume of the solvent in all dishes including the control dishes without drugs. DMSO volume was ≤0.04% of the volume of the culture medium.