Selective and Potent Akt Inhibition Triggers Anti-Myeloma Activities and Enhances Fatal Endoplasmic Reticulum Stress Induced by Proteasome Inhibition

TAS-117 abrogates the cytoprotective effect of the BM microenvironment associated with Akt inhibition in both MM cells and BMSCs. A, MM.1S cells were cultured for 48 hours with increasing concentrations of TAS-117 (left) or perifosine (right) in the presence () or absence () of BMSCs. Cell proliferation was assessed by [³H]-thymidine uptake of quadruplicate cultures. Data, the mean ± SD [³H]-thymidine incorporation [counts per minute (CPM)]. B, MM.1S cells were treated with or without TAS-117 (0.5 μmol/L) for 6 hours with normal medium (control) or conditioned medium derived from culture supernatant of BMSCs (BMSC). Whole-cell lysates were subjected to Western blotting using p-Akt (Ser473), Akt, and GAPDH Abs. C, MM.1S cells were cultured for 48 hours with increasing concentrations of TAS-117 or perifosine in RPMI-1640 containing 10% or 25% FBS. Cell growth was assessed by CellTiter-Glo Luminescent Cell Viability Assay of quadruplicate cultures, expressed as the percentage of untreated control. Data, mean ± SD. D, BMSCs from two patients with MM were treated with or without TAS-117 (2 μmol/L) for 6 hours. Whole-cell lysates were subjected to Western blotting using p-p65, p65, p-Akt (Ser473), Akt, and GAPDH Abs. E, BMSCs derived from three patients with MM were cultured for 24 hours with control medium () or TAS-117 (2 μmol/L; ) in the presence or absence of TNFα (0.25 ng/mL). IL6 in culture supernatant was measured by ELISA of triplicate cultures. Data, mean ± SD; *, P < 0.01; **, P < 0.001.

TAS-117 inhibits tumor growth in murine xenograft models of human MM. A to D, SCID mice were injected s.c. with 5 × 10⁶ MM.1S cells and treated for 21 days with 12 mg/kg oral TAS-117 daily for 5 days a week (n = 10); 16 mg/kg oral TAS-117 daily for 5 days a week (n = 10); or vehicle as a control (n = 9). A, tumor volume was calculated from caliper measurements every 3 to 4 days, and data, mean ± SE. B, body weight of mice was expressed as the percentage of baseline. Data, mean ± SD. C, survival was evaluated from the first day of treatment using Kaplan–Meier curves. D, tumors harvested from TAS-117- (16 mg/kg) and vehicle control–treated mice after 5 days of treatment were subjected to immunohistochemical analysis using p-Akt (Ser473) and TUNEL assay. E, SCID mice were injected s.c. with 1 × 10⁷ H929 cells and treated daily for 14 consecutive days with 8 mg/kg oral TAS-117-HCl (n = 10); 12 mg/kg oral TAS-117-HCl (n = 10); or vehicle alone as a control (n = 10). Tumor volume was calculated from caliper measurements every 3 to 5 days, and data, mean ± SE.

TAS-117 triggers G₀–G₁ arrest followed by apoptosis, associated with induction of autophagy and ER stress response. A, MM.1S and H929 cells were treated with or without TAS-117 (0.5 μmol/L) for 24 hours and then subjected to PI staining for cell-cycle analysis by flow cytometry. B, MM.1S and H929 cells were treated with or without TAS-117 (1 μmol/L) for 48 hours. Apoptotic cells were analyzed by flow cytometry using Annexin V/PI staining. C, MM.1S and H929 cells were treated with TAS-117 (1 μmol/L) for the indicated times. Whole-cell lysates were then subjected to Western blotting using LC3A/B and GAPDH Abs. Note: the GAPDH blot in MM.1S cells is the same as that in Fig. 1B (right) because the same cell lysates were used in these experiments. D, MM.1S and H929 cells were treated with or without TAS-117 (1 μmol/L) for 6 hours. Whole-cell lysates were subjected to Western blotting using LC3A/B, p-mTOR (Ser2481 and Ser2448), mTOR, p-p70 S6 kinase, p70 S6 kinase, and α-tubulin Abs. E, MM.1S cells were treated with or without TAS-117 (1 μmol/L), and H929 cells were treated with or without TAS-117 (0.5 μmol/L), for the indicated times. Whole-cell lysates were subjected to Western blotting using p-Akt (Ser473), Akt, p-IRE1α, IRE1α, BiP/GRP78, p-eIF2α, eIF2α, CHOP, and GAPDH Abs.

TAS-117 enhances bortezomib-induced cytotoxicity in vitro and in vivo. A, MM.1S cells were cultured for 48 hours with bortezomib (0–2 nmol/L) in combination with TAS-117 0 μmol/L (), 0.25 μmol/L (), 0.5 μmol/L (), 1 μmol/L () in normal medium or conditioned medium derived from culture supernatant of BMSCs (BMSC medium). Cell growth was assessed by MTT assay of triplicate cultures, expressed as the percentage of untreated control. Data, mean ± SD. B, MM.1S cells were treated with or without TAS-117 (1 μmol/L), bortezomib (2 nmol/L), or the combination for 24 hours. Apoptotic cells were analyzed by flow cytometry using Annexin V/PI staining. C, MM.1S cells were treated with or without bortezomib (10 nmol/L) in the presence or absence of TAS-117 (0.5 μmol/L) for 8 hours. Whole-cell lysates were subjected to Western blotting using p-Akt (Ser473), Akt, CHOP, PARP, and GAPDH Abs. The graph, fold changes of CHOP density relative to GAPDH. D, RPMI-8226 cells were cultured for 48 hours with bortezomib (0–3 nmol/L) in combination with TAS-117 0 μmol/L (), 0.25 μmol/L (), 0.5 μmol/L (), 1 μmol/L (). Cell viability was assessed by MTT assay of triplicate cultures, expressed as the percentage of untreated control. Data, mean ± SD. E, RPMI-8226 cells were treated with or without bortezomib (10 nmol/L) in the presence or absence of TAS-117 (1 μmol/L) for 8 hours. Whole-cell lysates were subjected to Western blotting using p-Akt (Ser473), Akt, and GAPDH Abs. F and G, SCID mice were injected s.c. with 5 × 10⁶ MM.1S cells and treated for 21 days with 0.5 mg/kg s.c. bortezomib twice a week (n = 10); 12 mg/kg oral TAS-117 5 days a week (n = 10); 0.5 mg/kg s.c. bortezomib twice a week and 12 mg/kg oral TAS-117 5 days a week (n = 10); or vehicle alone as a control (n = 9). Note: we performed the in vivo studies using MM.1S cells at the same time for Fig. 3A–D, so the vehicle control and TAS-117 (12 mg/kg) groups are commonly used. F, tumor volume was calculated from caliper measurements every 3 to 4 days, and data, mean ± SE. G, survival was evaluated from the first day of treatment using Kaplan–Meier curves.