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Supplementary Figure 1: U266 cells were transiently transfected with 500 nM of small interfering RNA (siRNA) SMARTpool for IGF1R or nonspecific control duplexes (pool of four; Thermo Scientific Dharmacon) using the Cell Line Nucleofector Kit V Solution (Amaxa Biosystems, Cologne, Germany), program X-005. After collection, cells were plated in a 96 well plate (20000 cells/well) and 72 h MTT viability assay was performed. Western blot analysis 72 hr after transfection shows a decrease in IGF1R expression in the U266 cell line ( lower panel). For the MTT assay, control cells (transfected with non specific control) were treated with and without AZD8055 100 nM, 66% decrease in viability was observed in presence of AZD8055. Transfected cells (IGF1R knock out cells) were cultured with and without AZD8055 100 nM. IGF1R knockout cells show 39% decrease in viability compared to scramble control and the combination of IGF1R knock out and AZD8055 100 nM show a 78% decrease in viability compared to scramble control. This finding suggests that IGF1R knockout sensitizes to AZD8055 inhibition similar to IGF1R antibody.

Despite promising preclinical results with mTOR kinase inhibitors in multiple myeloma, resistance to these drugs may arise via feedback activation loops. This concern is especially true for insulin-like growth factor 1 receptor (IGF1R), because IGF1R signaling is downregulated by multiple AKT and mTOR feedback mechanisms. We have tested this hypothesis in multiple myeloma using the novel selective mTOR kinase inhibitor AZD8055. We evaluated p-mTOR S2481 as the readout for mTORC2/Akt activity in multiple myeloma cells in the context of mTOR inhibition via AZD8055 or rapamycin. We next validated AZD8055 inhibition of mTORC1 and mTORC2 functions in multiple myeloma cells alone or in culture with bone marrow stroma cells and growth factors. Unlike rapamycin, AZD8055 resulted in apoptosis of multiple myeloma cells. AZD8055 treatment, however, induced upregulation of IGF1R phosphorylation in p-Akt S473–expressing multiple myeloma cell lines. Furthermore, exposure of AZD8055-treated cells to IGF1 induced p-Akt S473 and rescued multiple myeloma cells from apoptosis despite mTOR kinase inhibition and TORC2/Akt blockage. The addition of blocking IGF1R antibody resulted in reversing this effect and increased AZD8055-induced apoptosis. Our study suggests that combination treatment with AZD8055 and IGF1R-blocking agents is a promising strategy in multiple myeloma with potential IGF1R/Akt signaling–mediated survival. Mol Cancer Ther; 13(11); 2489–500. ©2014 AACR.