Effects of the inhibition of SK-1 and S1P2 on nilotinib-induced apoptosis in drug resistant 32D cells, expressing the T315I-Bcr-Abl1 in situ and in severe combined immunodeficiency mice in vivo. (A) 32D/T315I-Bcr-Abl1 cells were treated with increasing concentrations of nilotinib (0.05, 0.1, and 0.5μM for 48 hours) in the absence/presence of SKI-II or JTE-031 (alone or in combination), and then their effects on cell survival were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide; 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (left panel). Effects of nilotinib, SKI-II, JTE-013, or SKI-II/JTE-013 treatments on the levels of P-T315I-Bcr-Abl1, normalized to actin levels, were determined by Western blotting using the antibody that recognizes P-Bcr-Abl1 at Y177 compared with untreated cells. Relative expression levels of P-Bcr-Abl1 (normalized to actin levels) detected in Western blots are shown (right panel). (B) Role of SK-1 in the regulation of nilotinib-mediated growth inhibition (at 0.05, 0.1, and 0.5μM for 48 hours) was examined using trypan blue exclusion in the absence/presence of Scr or SK-1 siRNAs in 32D/T315I-Bcr-Abl1 cells. The data represent at least 2 independent trials performed in duplicates, and error bars represent SD. (C) Effects of the inhibition of SK-1 and S1P2 using SKI-II and JTE-013 in the absence/presence of nilotinib on the growth of 32D/T315I-Bcr-Abl1–derived tumors (n = 8) were determined in severe combined immunodeficiency/Balb/c mice. Mice containing 32D/wt-Bcr-Abl1– or 32D/T315I-Bcr-Abl1–derived allografts were treated with nilotinib at 10 mg/kg (every 3 days for 18 days), and untreated mice were used as a control (left panel). Mice containing the 32D/T315I-Bcr-Abl1–derived allografts were treated with 10 mg/kg nilotinib in the absence/presence of SKI-II and/or JTE-013 at 15, or 0.5 mg/kg,^34,37  respectively (right panel). Treatments were performed at days 1, 8, 15, and tumor growth was measured at days 1, 6, 11, 14, and 18 after treatments. Error bars represent SD, and *P < .05 was considered significant. (D) The proposed mechanism by which SK-1/S1P/S1P2 signaling regulates drug resistance. Regulation of drug resistance by SK-1/S1P involves the modulation of PP2A-dependent and SHP1-mediated degradation of Bcr-Abl1 (wt and mutants) selectively via S1P2 signaling. In addition, targeting SK-1/S1P2 signaling using pharmacologic (SKI-II and/or JTE-013) or molecular (siRNAs against SK-1 or S1P2) tools induces PP2A activity, leading to degradation of Bcr-Abl1, and attenuate resistance to TKIs (imatinib or nilotinib).