Abstract 2899: Selinexor inhibits NF-κB activity by sequestering IkB-a in the nucleus and blocking IkB-a degradation

Abstract

Background: Selinexor is a small-molecule therapeutic that inhibits XPO1 mediated nuclear export, resulting in nuclear accumulation of tumor suppressor proteins (TSPs) and subsequently cancer cell death while sparing normal cells. It has been previously demonstrated that the inhibitor of NFκB, IκB-, is localized to the cytoplasm by XPO1 in several cancer cell lines and that treatment of cancer cells with selinexor reduces NF-κB transcriptional activity. The mechanism of NF-κB inhibition by selinexor, however, is not fully understood. We hypothesized that nuclear retention of IκB- and down-regulation of IκB- kinase (IKK) in response to selinexor treatment would inhibit NF-κB transcriptional activity. Methods: U2OS (Osteosarcoma) cells were treated with selinexor in the presence or absence of TNF stimulation and whole protein lysates were analyzed by Western blotting. IκB- localization was evaluated by immunofluorescence microscopy and NF-κB transcriptional activity by ELISA (Thermo Scientific). Results: TNFα induced the phosphorylation of NF-κB p65 subunit on serine 536 and IκB- on serine 32/36 through IKK. This resulted in the dissociation of IκB-α from NF-κB and led to IκB-α degradation via the 26S proteasome. Free NF-κB could now migrate into the nucleus and initiate transcriptional activation supporting tumorigenicity and inflammation. The IKK kinase is a complex made of two kinases (IKKα and IKKβ) and one regulatory subunit, NEMO/IKKγ. We found that selinexor treatment blocked IKK activity through the down regulation of the IKK (IKKγ) gamma subunit protein levels. This inhibition was dose dependent and prevented IκB- phosphorylation, thereby protecting IκB- from degradation. The protection of intact IκB- from degradation and its forced nuclear accumulation through XPO1 inhibition enabled inhibition of NF-κB transcriptional activity even in the presence of TNFα. Selinexor did not alter the protein levels of IKK or IKK. Conclusions: Selinexor blocks TNFα induced degradation of IκB- by reducing the levels of IKK. In addition, selinexor increased nuclear IκB- levels through the inhibition of nuclear export. This blocked NF-κB activity and enhanced cancer cell death. We are currently investigating the beneficial effects of combining selinexor with proteasome inhibitors, which are known to prevent IκB- degradation. Citation Format: Trinayan Kashyap, Christian Argueta, Boris Klebanov, TJ Unger, Benjamin Link, Maxwell Werman, Margaret Lee, Sharon Shacham, Yosef Landesman. Selinexor inhibits NF-κB activity by sequestering IkB-a in the nucleus and blocking IkB-a degradation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2899.