A method for quantification of exportin-1 (XPO1) occupancy by Selective Inhibitor of Nuclear Export (SINE) compounds

// Marsha L. Crochiere 1 , Erkan Baloglu 1 , Boris Klebanov 1 , Scott Donovan 1 , Diego del Alamo 1 , Margaret Lee 1 , Michael Kauffman 1 , Sharon Shacham 1 , Yosef Landesman 1 1 All authors are current or former employees of Karyopharm Therapeutics Inc., Newton, MA, 02459 U.S.A Correspondence to: Marsha L. Crochiere, e-mail: marsha@karyopharm.com Keywords: selinexor, export, occupancy, cancer, resistance Received: August 06, 2015      Accepted: November 18, 2015      Published: December 07, 2015 ABSTRACT Selective Inhibitor of Nuclear Export (SINE) compounds are a family of small-molecules that inhibit nuclear export through covalent binding to cysteine 528 (Cys528) in the cargo-binding pocket of Exportin 1 (XPO1/CRM1) and promote cancer cell death. Selinexor is the lead SINE compound currently in phase I and II clinical trials for advanced solid and hematological malignancies. In an effort to understand selinexor-XPO1 interaction and to establish whether cancer cell response is a function of drug-target engagement, we developed a quantitative XPO1 occupancy assay. Biotinylated leptomycin B (b-LMB) was utilized as a tool compound to measure SINE-free XPO1. Binding to XPO1 was quantitated from SINE compound treated adherent and suspension cells in vitro , dosed ex vivo human peripheral blood mononuclear cells (PBMCs), and PBMCs from mice dosed orally with drug in vivo . Evaluation of a panel of selinexor sensitive and resistant cell lines revealed that resistance was not attributed to XPO1 occupancy by selinexor. Administration of a single dose of selinexor bound XPO1 for minimally 72 hours both in vitro and in vivo . While XPO1 inhibition directly correlates with selinexor pharmacokinetics, the biological outcome of this inhibition depends on modulation of pathways downstream of XPO1, which ultimately determines cancer cell responsiveness.