Abstract B34: Characterizing the functional role of XPO1 overexpression in primary refractory neuroblastoma
Authors: Castellanos Raquel , Tauber David , Landesman Yosef , Weiser Daniel 
Published: May 2016

Abstract
Background: Neuroblastoma (NB) is a highly lethal childhood cancer of the sympathetic nervous system that accounts for a disproportionate number of childhood cancer deaths. It remains a difficult cancer to eradicate despite intensive treatment. Approximately 15% of patients show no response to initial chemotherapy and almost all die. Prior approaches to identify at diagnosis those patients who fare most poorly have been unsuccessful, driving the critical need to develop pre-treatment biomarkers that can guide precise and effective therapeutic strategies. Using comparative proteomic profiling to identify biological markers for primary refractory neuroblastoma (PRN) we identified Exportin-1 (XPO1/CRM1) to be significantly more abundant in patients that died from tumor progression. XPO1 is a nuclear transport receptor responsible for the movement of multiple tumor suppressor proteins into the cytoplasm. Overexpression of XPO1 has been implicated in tumor development and progression of multiple cancers including prostate cancer, melanoma, and glioblastoma. Treatment with SINE compounds, which target XPO1, have been shown to induce anti-tumor activity in tumors. In this study we look at the effects of KPT-330 treatment on neuroblastoma cell lines, specifically looking at nuclear exported proteins.
Methods: Whole protein cell lysates from neuroblastoma cell lines (CHLA15, Kelly, IMR5, NB-EBC1) and control RPE1 (non-NB) cells treated with KPT-330 (Selenixor) were extracted and analyzed by immunoblots. Cell lines were treated with a range of concentrations (250nM-1000nM) of KPT-330, lasting 24, 48, and 72 hours. Cytotoxic effects were measured by MTT proliferation assays at 72 hours of treatment with KPT-330 at doses ranging 100nM-1000nM. Expression of XPO1 and nuclear exported proteins was evaluated at treatment time-points mentioned above by immunoblots.
Results: Proliferation defects were observed in all NB cell lines when compared to vehicle (DMSO) treatment and RPE1 cell treatment. Expression of XPO1 was down-regulated in treated NB cells at 24, 48 and 72 hours time-points. BARD1 and Survivin, also showed down-regulated expression in the presence of KPT-330.
Conclusions: This study will provide a mechanistic explanation of how XPO1 overexpression contributes to tumor progression and validate it as a therapeutic biological marker for PRN. Treatment of neuroblastoma cell lines with KPT-330 leads to cell death and loss of proliferation, similar to what has been observed for other cancers. Inhibition of XPO1 function may have implications aside from nuclear export transport. Two nuclear exported proteins, BARD1 and Survivin were both downregulated in the presence of KPT-330. These preclinical studies will provide rationale for incorporation of an XPO1 inhibitor into clinical trials for children with refractory disease.
Citation Format: Raquel Castellanos, David Tauber, Yosef Landesman, Daniel Weiser. Characterizing the functional role of XPO1 overexpression in primary refractory neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B34.