Abstract 727: NF-κB Mediates Resistance to Rapamycin in Neuroblastoma

Abstract

Abstract Neuroblastoma is the most common extracranial pediatric solid tumor and is the most prevalent malignancy of infancy. We were interested in the potential use of rapamycin for the treatment of neuroblastoma. However, 7/8 of neuroblastoma cell lines were found to be rapamycin resistant in vitro, independently of MYCN status. The sensitive cell line (CHLA90) and 2 of the most resistant cell lines (SKNAS [non-MYCN amplified] and NB1691 [MYCN amplified]) were further analyzed to characterize their response to rapamycin. Electromobility shift assay analysis showed that treatment of resistant cells with rapamycin activated NF-κB, while NF-κB activation was reduced in sensitive cells. Immunoblotting with specific NF-κB antibodies showed that rapamycin induced p50 but not p65 nuclear translocation in resistant cells, suggesting that the NF-κB complex activated by rapamycin represents p50 homodimers. Furthermore, immunocytochemical analysis showed nuclear translocation the p50 protein following treatment with rapamycin. To attempt to overcome rapamycin resistance, we tested the combination of two different NF-κB inhibitors (Velcade, a proteasome inhibitor and BMS-345541, a highly selective IκB kinase inhibitor) with rapamycin. Sub-lethal concentrations that resulted in NF-κB inhibition of both agents were identified and these doses were used in combination with rapamycin treatment. A synergy index (SI) was calculated and in both cell lines significant synergy (SI<0 and 95% confidence interval<0) between rapamycin and the two NF-κB inhibitory agents tested, was observed. Orthotopic neuroblastoma mouse models were established in the retroperitoneal space of SCID mice. Tumors were sized matched into cohorts of 5-10 mice each. Treatment groups included vehicle only, single agent (Velcade: 0.5mg/kg, 3x/w; rapamycin: 5mg/kg, 3x/w) and combination groups. Mice were sacrificed after 2 weeks of treatment and final tumor volumes calculated. In SKNAS, while Velcade and rapamycin individually had only modest effects on tumor volume (Control 6.6±3.2cm3; Velcade 4.5±1.8cm3; Rapamycin 2.6±0.95cm3), combination treatment was significantly more efficacious than either monotherapy (0.99±0.24cm3) (p<0.0002). In NB1691, Velcade has no effect on tumor size (Control 2.68±0.53cm3; Velcade 2.67±0.58cm3) while rapamycin had a modest effect on tumor volume (1.28±0.34cm3). The combination treatment was significantly more efficacious than either monotherapy (0.71±0.15cm3) (p<0.008). A synergy index (SI) was calculated and in both models significant synergy (SI<0 and 95% confidence interval<0) between rapamycin and Velcade was observed. In conclusion, the efficacy of rapamycin treatment of neuroblastoma may be limited by cellular resistance to the cytostatic and cytotoxic effects of rapamycin. However resistance can be overcome by NF-κB inhibitors, which results in significantly synergistic inhibition of tumor growth in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 727. doi:10.1158/1538-7445.AM2011-727