ta suggest that Pim kinases are targets in myeloma. SGI-1776 is an imidazo pyridazine small molecule. SGI-1776 was found to be a potent ATP competitive inhibitor of Pim-1, Pim-2 and Pim-3 kinases with an IC50 of 7, 363, and 69 nM, respectively23. Since all family members have high R-7128 homology at the amino acid level, the small molecule was expected to inhibit the three Pim kinases to a similar extent24. In addition, SGI-1776 was also found to inhibit FLT3 and haspin at similar low nanomolar concentrations23,25. Even though clinical trials in prostate cancer, non-Hodgkin’s lymphoma, and leukemia were suspended due to cardiac toxicity, SGI-1776 still represents a proof-of-principle compound due to its potent inhibitory activity on all three Pim kinases. Based on this we hypothesized that the Pim kinase inhibitor would result in MM cytotoxicity due to the specific targeting of the overexpressed Pim-2 kinase. Evaluation of our data indicated that SGI-1776 treatment in myeloma cells as well as bone marrow aspirates from MM patients elicits its deleterious effects through inhibition of translation and induction of autophagy rather than apoptosis. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Cell lines Materials Materials and Methods MM.1S cell line was obtained from Drs. Nancy Krett and Steve Rosen. U266 cell line was obtained from Dr. William S. Dalton. Both cell lines were maintained in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum in the presence of 5% CO2 at 37C. Cells were routinely tested for Mycoplasma infection. Approximate doubling times for MM.1S and U266 are 48 and 36 h, respectively. Primary bone marrow aspirates from MM patients Bone marrow samples from 14 MM patients were obtained with informed consent in accordance with the Declaration of Helsinki to participate in the laboratory protocol, which was approved by the Institutional Review Board of the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19844694 University of Texas M. D. Anderson Cancer Center. Samples were processed using a ficoll gradient. CD138+ cells were separated using a MACS magnetic separation technique, leaving the remaining cells to be were treated as CD138- cells. CD138+ and CD138- cells were suspended in RPMI-1640 medium supplemented with 10% human AB serum in the presence of 5% CO2 at 37C. SGI-1776 was provided by SuperGen as a powder dissolved in dimethylsulfoxide at a concentration of 10 mM and stored at -20C. Rapamycin and bafilomycin A1 were purchased from Sigma. Clin Lymphoma Myeloma Leuk. Author manuscript; available in PMC 2014 September 01. Cervantes-Gomez et al. Page 4 Radioactive thymidine Incorporation DNA synthesis was measured using Thymidine incorporation. MM cells were treated in a dose-response dependent manner for 24, 48, and 72 h. Thirty minutes before the end of incubation, the cells were labeled with Thymidine at 37C. The labeled samples were harvested, washed with 10 mL of cold PBS, and transferred to glass fiber filters using a Millipore vacuum manifold. The filters were then washed twice with 5 mL of cold 0.4 N perchloric acid and rinsed with 70% ethanol. The filters were dried overnight and transferred to scintillation vials containing 7 mL of scintillation fluid. The radioactivity on the filters was quantified by a liquid scintillation counter. Data were expressed as a percentage of untreated control. Protein extraction and immunoblot assays After treatment, cells were harvested, washed twice with PBS, and lysed using one tablet of Complet