Supplementary Figure S1: Tumor growth curves for individual mouse xenografts treated with 4-HPR/LXS and ABT-751; Supplementary Figure S2: Dose escalation study of n-acetylcysteine (NAC) and sodium thiosulfate (STS); Supplementary Figure S3: Effect of 4-HPR + ABT-751 on mitochondrial membrane depolarization, cytochrome c release, and caspase and poly (ADPribose) polymerase (PARP) cleavage; Supplementary Figure S4: Cytotoxicities of fenretinide(4-HPR) in combination with five microtubule inhibitors in three neuroblastoma cell lines (SMS-KCNR, CHLA-119, and CHLA-140); Supplementary Figure S5: Effect of the antioxidants vitamin C (Vit C) and vitamin E (Vit E) on the cytotoxiciity of fenretinide (4-HPR) in combination with five microtubule inhibitors in two neuroblastoma cell lines.
ARTICLE ABSTRACTABT-751 is a colchicine-binding site microtubule inhibitor. Fenretinide (4-HPR) is a synthetic retinoid. Both agents have shown activity against neuroblastoma in laboratory models and clinical trials. We investigated the antitumor activity of 4-HPR + the microtubule-targeting agents ABT-751, vincristine, paclitaxel, vinorelbine, or colchicine in laboratory models of recurrent neuroblastoma. Drug cytotoxicity was assessed in vitro by a fluorescence-based assay (DIMSCAN) and in subcutaneous xenografts in nu/nu mice. Reactive oxygen species levels (ROS), apoptosis, and mitochondrial depolarization were measured by flow cytometry; cytochrome c release and proapoptotic proteins were measured by immunoblotting. 4-HPR + ABT-751 showed modest additive or synergistic cytotoxicity, mitochondrial membrane depolarization, cytochrome c release, and caspase activation compared with single agents in vitro; synergism was inhibited by antioxidants (ascorbic acid, α-tocopherol). 4-HPR + ABT-751 was highly active against four xenograft models, achieving multiple maintained complete responses. The median event-free survival (days) for xenografts from 4 patients combined was control = 28, 4-HPR = 49, ABT-751 = 77, and 4-HPR + ABT-751 > 150 (P < 0.001). Apoptosis (terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling, TUNEL) was significantly higher in 4-HPR + ABT-751–treated tumors than with single agents (P < 0.01) and was inhibited by ascorbic acid and α-tocopherol (P < 0.01), indicating that ROS from 4-HPR enhanced the activity of ABT-751. 4-HPR also enhanced the activity against neuroblastoma xenografts of vincristine or paclitaxel, but the latter combinations were less active than 4-HPR + ABT-751. Our data support clinical evaluation of 4-HPR combined with ABT-751 in recurrent and refractory neuroblastoma. Mol Cancer Ther; 15(11); 2653–64. ©2016 AACR.