Highlights XPO1 is overabundant in high-risk neuroblastoma and correlates with poor survival. Neuroblastoma cells are sensitive to XPO1 inhibition with selinexor. Use of selinexor results in nuclear retention of IkB, diminishing NF-kB activity. Selinexor and bortezomib act synergistically through promotion of apoptosis. Synergy is mediated in part, through IkB regulation of NF-kB activity.
Background: Half of patients with high-risk neuroblastoma succumb to disease, yet these patients with inferior outcome cannot be identified at diagnosis despite contemporary risk stratification that integrates MYCN copy number status, tumor histology, and patient age. We have shown that overexpression of Exportin-1 (XPO1) is associated with poor survival in neuroblastoma, affirming what has been identified across a range of malignancies. Selinexor (KPT-330, Karyopharm Therapeutics), an XPO1 inhibitor in early phase clinical trials, inhibits the nuclear to cytoplasmic translocation of tumor suppressor and growth regulatory proteins implicated in oncogenesis. We hypothesized that protein expression level of XPO1 in neuroblastoma cell lines predict the effective response of treatment with selinexor. Methods: We used a panel of early passage patient derived neuroblastoma cell lines including IMR5, NLF, KELLY, NB-EBC1, and SKNSH that are representative of the spectrum and genetic diversity of human disease. Cell lines were treated with varying concentrations of selinexor. RNA and whole cell protein extract as well as nuclear and cytoplasmic fractionated protein was obtained from untreated and treated conditions. Treated and untreated cells were also pelleted, and, along with patient tumor samples, formalin-fixed and paraffin-embedded for microscopic and immunohistochemical (IHC) evaluation. Results: Compared to noncancerous cell lines, neuroblastoma cell lines have high protein and mRNA expression of XPO1; higher expression correlates with greater sensitivity to selinexor. Selinexor treatment reduced XPO1 protein and increased XPO1 mRNA expression, and it lead to nuclear retention of XPO1 cargo such as p53 and survivin. Comparison of microscopic features and protein staining intensity of patient samples with known XPO1 expression is ongoing. Conclusions: This study provides rationale for XPO1 protein abundance as a potential biomarker of therapeutic response to the drug selinexor. By using patient derived neuroblastoma cell lines, we are able to evaluate the response of selinexor and XPO1 expression in the context of this enigmatic disease. These data suggest that initial drug response to selinexor can be predicted early in the course of disease and more investigation of the high expression of XPO1 seen in patients with poor outcome will provide strategies for combinatorial treatment approaches. Citation Format: Basia Galinski, Marcus Luxemburg, Michelle Ewart, Yosef Landesman, Daniel Weiser. Exportin-1 (XPO1) is a novel therapeutic biomarker for patients with neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1938. doi:10.1158/1538-7445.AM2017-1938
Background: Neuroblastoma is an embryonic tumor of the peripheral nervous system. Comparative proteomics of high-risk neuroblastoma patients with poor prognosis outcomes demonstrated increased expression of Exportin-1 (XPO1), a nuclear transport protein with over 200 recognized cargo, in those who do not survive. Inhibition of nuclear export with the selective inhibitor of nuclear export, orally bioavailable drug molecule selinexor (Karyopharm Therapeutics) has shown anticancer activity. Treatment decreased tumor burden and increased survival in animal models. Advance-phase clinical trials using selinexor in hematologic and solid cancers are ongoing. In neuroblastoma cell lines, we have found correlation between decreased steady state protein levels of IkB, the inhibitor of NF-kB, and increased XPO1 protein expression. Likewise, proteasome inhibition with bortezomib leads to decreased NF-kB levels and decreased proliferation in neuroblastoma cells. We hypothesize that combination treatment of selinexor and bortezomib will have a greater than additive effect on the inhibition of neuroblastoma cellular proliferation. Such effect would be due in part to decreased NF-kB transcription activity through stabilization of IkB as a result of proteasome activity inhibition and forced localization of IkB into the nucleus where it may be protected from degradation. Methods: Neuroblastoma cell lines were used for all experiments. MTT assays were used to generate dose-response curves for single-agent selinexor and bortezomib, as well as sequential treatment combination (bortezomib then selinexor six hours later). Apoptosis and cell cycle regulation were assessed for all treatment conditions. NF-kB activity was measured by ELISA in cell lysates with decreased XPO1 expression through siRNA knockdown of XPO1. Western analysis and immunofluorescence were used to quantify NF-kB and IkB protein expression in treated and knockdown conditions. Results: Sequential combination treatment showed decreased cell viability compared to treatment with a single agent. Western analysis of NF-kB in treated single- and double-agent conditions saw decreased protein expression of NF-kB active and inactive forms. We expect flow cytometry experiments to demonstrate increased levels of apoptosis and cell cycle arrest. Future ELISA analysis of siRNA and combination treated cells will assess NF-kB transcription activity. Conclusions: Regulation of IkB protein expression through nuclear localization as well as decreased proteasome degradation can halt NF-kB-driven cellular proliferation. Combination therapy with selinexor and bortezomib has potential to be effective against highly aggressive neuroblastoma that is, in part, driven by NF-kB. Future work will define additional pathways altered with combination treatment and guide in vivo testing and clinical development of this therapeutic strategy. Citation Format: Basia Galinski, Marcus Luxemburg, Yosef Landesman, Daniel Weiser. Combination treatment with selinexor and bortezomib for management of highly aggressive neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3193.
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