MKC-3946 inhibits XBP1 splicing in a model of ER stress in vivo, associated with significant growth inhibition of MM cells, alone or with bortezomib. (A) SCID mice were treated with Tm (1 mg/kg intraperitoneally) for 4 or 6 hours. Two hours after Tm was administered, mice were treated with MKC-3946 50 mg/kg intraperitoneally. After 4-hour exposure to MKC-3946, mice were killed. Livers were harvested, and total RNA was prepared. RT-PCR was performed using murine-specific XBP1 primers. Each lane represents a single mouse. The vertical line indicates a repositioned gel lane. (B-D) SCID mice were injected subcutaneously with 1 × 107 RPMI 8226 cells on day 0 and treated with 100 mg/kg MKC-3946 intraperitoneally daily (MKC-3946, n = 8), 0.15 mg/kg bortezomib intravenously twice a week (bortezomib, n = 8), or 100 mg/kg MKC-3946 intraperitoneally daily and 0.15 mg/kg bortezomib intravenously twice a week (combination, n = 8), for 21 days starting on day 1. A vehicle control group received intraperitoneal injections of vehicle and intravenous injection of saline (vehicle, n = 8). (B) Tumor volume was calculated from caliper measurements every 3 to 4 days, and data represent mean ± SE. (C) Survival in the plasmacytoma model was evaluated from the first day of treatment using Kaplan-Meier curves. (D) Total RNA was prepared from subcutaneous plasmacytoma harvested from each group of mice after 3 weeks of treatment. XBP1 and β-actin mRNA were examined using RT-PCR. The graph represents fold changes of XBP1s density relative to β-actin. (E) Growth of INA6 cells engrafted in human bone chips in SCID mice was monitored by serial serum measurements of shuIL-6R in the graph. Mice were treated with MKC-3946 100 mg/kg (n = 3) or control vehicle (n = 3), and shuIL-6R levels were determined weekly by ELISA. Error bars represent ± SE. Total RNA was prepared from tumor harvested from the MKC-3946– and control-treated SCID-hu mice at 3 weeks. XBP1 and β-actin mRNA were examined using RT-PCR in the right panel.
