Effects of dual inhibitor-induced NF-κB activation on EGR1 expression and BIM upregulation. (A) Jurkat cells treated with diluent (lanes 1 and 2) or 10 (lanes 3 and 4) or 20 μM OSI-027 (lanes 5 and 6) were fractionated into nuclei (N) and cytoplasm (C) for immunoblotting. (B,C,E) Jurkat cells transfected with empty vector or the IκB SS/AA super-repressor were treated with diluent or 10 μM OSI-027 and harvested for (B,E) qRT-PCR or (C) immunoblotting. Results of drug-treated samples in B and E were normalized to corresponding diluent-treated controls, which were set to 1. In panel B, *P = .008 and **P = .04 relative to empty vector control. In panel E, *P = .02 and **P = .04, respectively. (D) Jurkat cells transfected with 40 µg empty vector or IκB SS/AA along with 10 µg 800-bp BIM promoter luciferase reporter and 5 µg pTK-Renilla were treated with diluent or 10 µM OSI-027 and assayed for luciferase activities. In panel D, *P < .02. (F-G) Aliquots of a (F) clinical ALL sample and (G) CML-biphenotypic blast crisis sample incubated for 48 hours with diluent (lane 1), 5 µM OSI-027 (lane 2), 10 µM OSI-027 (lane 3), 20 µM OSI-027 (lane 4), or 1 µM MLN0128 (lane 5) were harvested for immunoblotting. Patient (Pt) numbers refer to supplemental Table 1. (H) Proposed model of mTOR dual inhibitor-induced BH3-only protein upregulation. As discussed in the text, various lymphoid cell lines and ALL specimens show activation of 1 or both pathways after mTOR dual inhibitor treatment. Reagents shown in gray inhibit downstream steps.