Abstract P164: Identification of the p53 negative feedback loop as a target for enhancing selinexor activity in neuroblastoma
Authors: Nguyen Rosa , Wang Hong , Sun Ming , Lee Dong Geun , Peng Junmin , Thiele Carol J. 
Published: December 2021

Neuroblastoma (NB) is the most common extra-cranial pediatric solid tumor and accounts for 15% of cancer-related deaths in children, highlighting an unmet need for novel therapies. Selinexor is a small molecule inhibitor of XPO1. XPO1 shuffles cargo proteins with a nuclear export sequence from the nucleus to the cytosol, many of which are essential for cancer growth and cell maintenance. XPO1 also has a prognostic significance in patients with NB since those with high XPO1 levels have an inferior event-free (P = 1.2e-5, Bonferroni P = 5.4e-3) and overall survival (P = 4.5e-7, Bonferroni P = 2.1e-4) compared to patients with low XPO1 expression. To characterize the global transcriptomic consequences of XPO1 inhibition, we performed RNA-sequencing in the NB cell line IMR-5 where we performed a knockdown of XPO1 or treated cells with selinexor. Conducting a gene set enrichment analysis, we found genes encoding MYCN targets were significantly downregulated by selinexor (normalized enrichment score [NES] = -2.7; nominal P = &lt;0.001) or XPO1 knockdown (NES = 2.2; nominal P = &lt;0.001). Genes associated with the p53 pathway were significantly enriched (NES = 2.7; nominal P = &lt;0.001 [selinexor treatment]; NES = 2.2; nominal P = &lt;0.001 [XPO1knockdown]). Given the large number of XPO1-targets, we applied an advanced tandem mass tag (TMT)-liquid chromatography (LC)/LC-MS/MS based pipeline for deep whole-cell proteomic and phospho-proteomic analyses to interrogate unknown mechanisms of selinexor’s action. We found that selinexor induced its cytotoxic effects in NB through the nuclear accumulation of p53. Drug sensitivity assays with selinexor against seven human and one murine NB cell lines yielded good (median EC50=28.7nM) and intermediate responses (median EC50=133.5nM) with all intermediate responders harboring a TP53 mutation while good responders had TP53 wild type status. Further, phosphoproteomic analysis showed that nuclear accumulation of p53 led to an increase in p53 phosphorylation at site S315 marking p53 for MDM2-mediated ubiquitination. Since this phosphorylation step is undertaken predominantly by aurora kinase A (AURKA), we used a clinically available AURKA inhibitor, alisertib, in combination with selinexor and found synergistic killing activity in vitro and enhanced growth inhibition in two orthotopic xenograft mouse models. These findings suggest a potential therapeutic benefit using selinexor and alisertib to synergistically increase p53-mediated cytotoxicity of high-risk NB. Given the known toxicity profiles and potential therapeutic benefits, these findings support a clinical trial of selinexor and alisertib in children with high-risk NB.

Citation Format: Rosa Nguyen, Hong Wang, Ming Sun, Dong Geun Lee, Junmin Peng, Carol J. Thiele. Identification of the p53 negative feedback loop as a target for enhancing selinexor activity in neuroblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P164.