Bortezomib increases MLN4924-induced cell cytotoxicity. (A) MM.1S, MM.1R, and U266 were treated with MLN4924 (2.5 or 5 µM) separately or concurrently with MEL (5 or 10 µM) or BTZ (2 or 4 nM). MTT assay was used to evaluate growth inhibition. (B) MM.1R cells or primary human MM cells (D) were treated with 2 nM bortezomib, 2.5 µM MLN4924, or the combination for 24 hours with increased inhibition of the PI3K/mTOR pathway and increased PARP and caspase cleavage. (C) MM.1R cells were treated with serial concentrations of MLN4924 and BTZ. MTT assays were performed to assess the growth inhibition effects. Combination index values are shown in Table 1 using the Calcusyn software program. (E) MM.1R cells were treated for 24 hours with 2 nM bortezomib, 2.5 µM MLN4924 alone or in combination in the presence or absence of IGF-1 (25 ng/mL) or IL-6 (10 ng/mL). Cell death was assessed using annexin V/PI staining. BTZ, bortezomib; MEL, melphalan.