Class I HDAC and PIM inhibition cooperatively suppresses MM cell growth in vitro and in vivo. (A) MM.1S or RPMI 8226 cells were transduced with shLuc or shPIM2 (#1). Transduced cells were treated with or without MS-275 (0.5 μM) in the presence or absence of IL-6 (10 ng/mL) for 48 hours, and cell viability was then assessed by the CCK-8 assay. Lysates extracted from transduced cells after puromycin selection were subjected to immunoblotting using the indicated antibodies. β-Actin served as a loading control. Relative expression levels of each target, which are normalized to its loading control, are shown below for each immunoblotting image. Error bars show the SD of triplicates. ∗∗P < .01, ∗∗∗P < .001 significantly different from the shLuc or shPIM2 condition with the MS-275 treatment; the Tukey-Kramer multiple comparison test. (B-C) MM.1S, RPMI 8226 (B), and primary CD138-positive cells (C) were treated with or without MS-275 (0.5 μM), SMI-16a (50 μM), or their combination in the presence or absence of IL-6 (10 ng/mL) for 48 hours. Cell viability was assessed by the CCK-8 assay. Error bars show the SD of triplicates. ∗∗P < .01, ∗∗∗P < .001; the Tukey-Kramer multiple comparison test. (D) After the development of measurable tumors (>50 mm3), cohorts were treated for 3 weeks with the vehicle control (n = 8; blue line), 3.5 mg/kg MS-275 3 days a week (n = 8; red line), 20 mg/kg SMI-16a 5 days a week (n = 8; green line), or 3.5 mg/kg MS-275 3 days a week with 20 mg/kg SMI-16a 5 days a week (n = 9; yellow line). Tumor growth was monitored with caliper measurements every 3 days. Error bars show the SEM of tumor volumes in each group. ∗P < .05 (control vs MS-275, SMI-16a vs MS-275 plus SMI-16a), ∗∗P < .01 (control vs MS-275 plus SMI-16a) on day 34; the Tukey-Kramer multiple comparison test. (E) Images show representative in vivo images, ordered from left to right: vehicle control, MS-275, SMI-16a, and the combination group of MS-275 with SMI-16a at the time of treatment on days 1 and 28.
