Methods Materials Amuvatinib was obtained from Astex Pharma ceuticals, Inc. and was dissolved in DMSO. Because of stability constraints, amuvatinib solution was prepared fresh for each experiment. Imatinib was purchased from Novartis and was dissolved in DMSO and stored in aliquots at 20 C. thymidine was obtained from Moravek Biochemical Inc. Cell culture and growth analysis The myeloma cell lines U266 and RPMI 8226/S were obtained from Dr. William Dalton at H. Lee Moffitt Cancer Center. NK tert human bone marrow stromal cells were obtained from Dr. Jan Burger at UT MD Anderson Cancer Center. The cell lines were maintained as described and rou tinely tested for Mycoplasma infection and authenticated by short tandem repeat analysis by UT MD Anderson Cancer Centers Characterized Cell Line Core facility.

Myeloma cell stromal co cultures were performed using U266 cells and NK tert cells at a ratio of 20 to 1. Stromal cells were plated at a concentration of 2 102 cells/mm2 surface area 5 hours before adding U266 cells at a 20 fold higher concentration. The cells were co cultured for 2 h prior to treatment with or without amuvatinib or bortezo mib for 48 h. At the end of incubation, the U266 cells, which are free floating in these cultures, were carefully removed for analysis, leaving the adherent stromal layer undisturbed. Additionally, the stromal cells were also har vested by trypsinization and similarly assessed. The effect of amuvatinib treatment on cell growth inhibition was measured in exponentially growing U266 cells. Cells were counted using a Coulter counter.

DNA synthesis was measured using thymidine incorporation as de scribed. Gene expression array analyses Expression data from 162 CD138 bone marrow plasma cell samples from healthy individuals as well as patients with MGUS, SMM, MM N, and MM R, which were mea sured by using Affymetrix U133A microarrays, were down loaded from GEO. Robust Multichip Average algorithm was used for normalization/ quantification of the data. The maximal values for the re spective probe sets of MET and HGF were used for gene expression profiling. The Kruskal Wallis test was applied to assess whether expression of MET and HGF was associ ated with defined clinical groups, and results are presented as box plots.

Isolation of CD138 AND CD138 cells from primary bone marrow aspirates from MM patients Primary samples were obtained from both male and female myeloma patients being treated at MD Anderson Cancer Center. Patient samples were obtained using an MD Anderson Cancer Center Institutional Review Board approved protocol. All patients signed an informed consent form to provide peripheral Anacetrapib blood and bone marrow samples. After collection of bone marrow samples, CD138 cells were isolated as described, suspended in RPMI 1640 with 10% human AB serum and used immediately for experiments.