Abstract 3040: Inhibition of Exportin-1 as part of combinatorial treatment for patients with high-risk neuroblastoma

Abstract

Abstract Background: In many adult cancers, upregulation of the nuclear-to-cytoplasmic transport protein Exportin-1 (XPO1) correlates with poor outcome and responsiveness to selinexor, an FDA-approved XPO1 inhibitor. Similar data are emerging in childhood cancers, for which selinexor is being evaluated in early phase clinical studies. Our aims are to identify patients most likely to benefit from selinexor combination therapy and define the cellular mechanism responsible this effect. We hypothesize that selinexor and the proteasome inhibitor, bortezomib, act synergistically to decrease NF-kB-mediated proliferation by halting excessive exportation and proteasome degradation of IkB, an XPO1 cargo protein. Methods: We performed ion intensity-based label-free semiquantitative proteomic profiling on primary tumor material from fifty patients with uniformly treated high-risk NB, half of whom were long term survivors and half of whom died <18 months from diagnosis from disease progression. We cross-referenced this dataset with gene expression profiling data available through the R2 database. A panel of neuroblastoma cell lines was screened for XPO1 expression using Western blotting, sensitivity to selinexor and bortezomib as single agents, and in combination using cellular viability assays. Localization and quantification of IkB after vehicle or selinexor treatment was done by confocal imaging, using immunofluorescent labeling and Volocity software. Flow cytometry was used to define treatment effect on cell cycle arrest and apoptosis using BrdU and Annexin-V labeling, respectively. siRNA-mediated knockdown of IkB was used to investigate the relationship between presence of IkB and drug effectiveness. NF-kB activity was measured with a luciferase assay. Results: High tumor protein and gene expression of XPO1 correlates with poor outcome. Neuroblastoma cell lines demonstrate variably high XPO1 expression compared to normal tissue, as well as sensitivity to selinexor (IC50 range 4-300nM). IkB is retained in the nuclear compartment after treatment with selinexor. When selinexor is used in combination with bortezomib the effect on cell proliferation is synergistic. Flow cytometry demonstrates that selinexor alone contributes to G1-0 arrest while combination treatment results in significantly enhanced apoptosis. Measurement of NF-kB transcriptional activity shows decrease in activity with single agents and the least amount of activity in combination and siRNA against IkB diminishes the effect of single and combination treatment. Conclusions: This work demonstrates that XPO1 expression is a biomarker of highly aggressive neuroblastoma and may define patients most likely to be responsive to treatment with selinexor-based combinatorial therapies. Synergistic treatment effect with selinexor and bortezomib is mediated, in part, through the presence of IkB to regulate NF-kB signaling. Citation Format: Basia Galinski, Yossef Landesman, Daniel Weiser. Inhibition of Exportin-1 as part of combinatorial treatment for patients with high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3040.