MM cells resistant to Akt inhibition can also be unresponsive to blockade of other survival pathways. (A) Western analysis of signaling pathway components in AMO-1 cells after knockdown of STAT3 and additional inhibition of the Ras/MAPK pathway with PD98059 (50 μM) and of Akt with Akti-1/2 (10 μM) (pSUPER = control vector, siSTAT3 = siRNA expression construct26 ). Transfected cells were purified 1 day after electroporation, cultured together with BMSCs, and treated with PD98059, Akti-1/2, or both. The next day, cells were harvested for Western blotting, and the same material was used to run 2 different gels to permit unequivocal staining of phosphorylated versus total protein. Only 1 of 2 identical tubulin controls is shown for clarity. (B) Viability of pSUPER-transfected AMO-1 cells after treatment with 10 μM Akti-1/2 and of siSTAT3-transfected cells additionally treated with 10 μM Akti-1/2 and 50 μM PD98059. Viability was assayed at day 5 of drug treatment; survival is calculated as the percentage of live cells compared with the pSUPER control sample. Cells were cocultured with BMSCs, and the graph displays the results from 3 independent experiments. Error bars mark standard deviations. (C) Signaling analysis of primary MM cells after treatment with Sant7 and PD98059. A total of 5 × 104 primary MM cells were kept in medium alone or in coculture with BMSCs for 24 hours. They were then treated overnight with Sant7 (50 μg/mL) or with an equivalent amount of buffer and changes in their phospho-STAT3 level were measured. Primary MM cells stimulated with 25 ng/mL IL-6 for 15 minutes served as positive control. Similarly, phospho-ERK1/2 signals were blocked with PD98059 (50 μM) and induced with PMA. (D) Viability of Akti-1/2–resistant/resilient primary MM cells (n = 7; individually coded) after additional blockade of the IL-6R/STAT3 and Ras/MAPK pathways. Freshly isolated MM cells were cocultured with BMSCs and incubated with Akti-1/2 (10 μM) or, in addition, with PD98059 (50 μM) and Sant7 (50 μg/mL). Viability was measured on day 5. Cells treated with DMSO served as control.
