Abstract B58: The novel MDM2 inhibitor RG7388 is highly effective against neuroblastoma in vitro and in vivo

Abstract

Abstract Introduction: Novel less toxic therapeutic approaches to neuroblastoma therapy are urgently needed to improve survival and reduce treatment related side effects. As neuroblastoma is nearly uniformly p53 wild-type, research efforts have focused on activating innate p53-mediated apoptotic mechanisms, specifically through the inhibition of MDM2, the primary inhibitor of p53. The compound RG7388 has proven effective against adult tumors and is under consideration for clinical trials in pediatrics. Here we demonstrate the efficacy of this new class of MDM2 inhibitors against p53 wild-type neuroblastoma in vitro and in vivo orthotopic models of neuroblastoma. Methods: The human neuroblastoma cell lines, NGP, SH-SY5Y, LAN5 and LAN5 si-p53 (p53 silenced by shRNA), were utilized for in vitro and in vivo studies. Using graded concentrations of RG7388 (0 nM - 1500 nM), cell viability was measured by MTS assay. Apoptosis with RG7388 (0 nM, 100 nM, 500 nM) was measured using a flow cytometric annexin assay. In vivo effect of RG7388 was assessed in an orthotopic mouse model of neuroblastoma, in which neuroblastoma cells were intrarenally implanted into 80 female NCr nude mice (NGP n=22, SH-SY5Y n=18, LAN5 n=20, LAN5 si-p53 n=20). The mice were randomly divided into a control group which received vehicle (hydroxypropylcellulose/Tween 80) and a treatment group which received RG7388. RG7388 was initiated three weeks post-implantation by intraperitoneal injection once daily for 14 days at a concentration of 25 mg/kg for mice implanted with NGP and SH-SY5Y, and 35 mg/kg for mice implanted with LAN5 and LAN5 si-p53. At five weeks post-implantation, all mice were sacrificed, xenograft tumors resected and weighed. Xenograft tumor specimens underwent immunohistochemistry for apoptosis. Results: Using MTS assay and annexin apoptosis assay to measure cell viability and apoptosis in several cell lines of neuroblastoma (NGP, SH-SY5Y, LAN5) treated with RG7388, we found robust decrease in cell proliferation and increase in apoptosis compared to vehicle control in all p53 wild-type cell lines. The p53 silenced cell line, LAN5 si-p53, was found to be resistant to RG7388 with no decrease in cell proliferation and no increase in apoptosis. In vivo, all mice tolerated RG7388 well without morbidity. RG7388 significantly inhibited tumor growth by 59% in NGP (p=0.003), 67% in SH-SY5Y (p=0.006), and 75% in LAN5 (p=0.0019) xenograft tumors. However, RG7388 had no inhibitory effect on LAN5 si-p53 xenograft tumors compared to vehicle treatment (p=0.57). Tumor apoptosis was analyzed by cleaved caspase-3 staining, which showed xenografts treated with RG7388 had significantly increased apoptotic cells per high power field in NGP xenografts (p<0.0001) and SH-SY5Y xenografts (p=0.0014). Conclusion: The MDM2 small molecule inhibitor RG7388 significantly inhibits p53 wild-type neuroblastoma tumor growth but has no effect on p53-silenced neuroblastoma xenografts in an orthotopic mouse model. Our studies suggest that RG7388 inhibits tumor growth by p53-mediated apoptosis. Ongoing studies include assessment of the effect of RG7388 on tumor angiogenesis and metastasis. In vitro studies are ongoing to verify the molecular mechanism of RG7388 and confirm increase in p53 levels as well as downstream p53 pathways. Further studies are also being performed in several other p53 mutant neuroblastoma cell lines to elucidate the role of p53 for RG7388 treatment. Citation Format: Anna Lakoma, Eveline Barbieri, Matthew C. McVay, Zaowen Chen, Charitra Adhikari, Jason M. Shohet, Eugene S. Kim. The novel MDM2 inhibitor RG7388 is highly effective against neuroblastoma in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B58.