In Vitro and in Vivo Quantification of Exportin-1 (XPO1) Occupancy By the Oral Selective Inhibitor of Nuclear Export (SINE) Selinexor in Multiple Myeloma, Acute Myeloid Leukemia, and Healthy PBMCs

Introduction: SINE are a family of small molecules that selectively inhibit nuclear export by forming a slowly reversible covalent bond with Cysteine 528 (Cys528) in the cargo binding pocket of Exportin 1 (XPO1/CRM1). SINE binding to XPO1 leads to forced nuclear retention and activation of major tumor suppressor proteins (TSPs) such as p53, FOXO, pRB and IkB, resulting in selective death of cancer cells. Selinexor is an orally bioavailable SINE compound currently in human phase I and II clinical trials for advanced hematological and solid cancers. Oral selinexor demonstrates maximal pharmacokinetic exposure at 1-2 hours in humans with associated increases in pharmacodynamic markers of XPO1 inhibition in 2-4 hours that last for up to 48 hours. The goal of this study was to develop a binding assay that would enable quantification of XPO1 occupancy in PBMCs from patients following oral administration of selinexor.

Methods: To measure the binding of SINE to XPO1, biotinylated leptomycin B (LMB) was utilized. Biotinylated LMB binds covalently and irreversibly to Cys528 in the cargo-binding site of free XPO1 with activity confirmed to be similar to that of unmodified LMB in cytotoxicity assays. To measure SINE binding to XPO1 in vitro, cancer cell lines and PBMCs from normal human donors were treated with SINE compounds prior to treatment with biotinylated LMB. Any XPO1 that did not bind SINE instead binds to biotinylated LMB and can be quantified. In in vivo studies, mice were treated with selinexor, followed by collection of PBMCs for treatment with biotinylated LMB. After incubation with biotinylated LMB, cells were harvested, lysed, and protein lysates were subjected to pull-down experiments with streptavidin-conjugated beads followed by immunoanalysis of XPO1.

Results: To evaluate selinexor-XPO1 binding kinetics in vitro, MM.1S, AML2, AML3, and HEL cells were treated with 0 - 10 µM of SINE compounds and unbound XPO1 was pulled down from cell lysates treated with biotinylated LMB. Immunoanalysis showed that 50% XPO1 occupancy with selinexor was achieved at 0.07 µM in MM.1S, 0.1 µM in AML2, 0.03 µM in AML3, and 0.12 µM in HEL cells. Selinexor-XPO1 occupancy experiments using human PBMCs isolated from donor whole blood showed 50% XPO1 occupancy at 0.05 µM. In mice, 50% XPO1 occupancy in PMBCs was achieved after 4 hours treatment with 1.2 mg/kg (3.6 mg/m²) selinexor, while 90% XPO1 occupancy was achieved at 8.1 mg/kg (24.3 mg/m²). Mice treated with a single dose of selinexor from 1.5 to 10 mg/kg for 4-96 hours revealed sustained, dose dependent XPO1 occupancy in PBMCs for up to 72 hours.

Conclusions: We have developed a sensitive and robust assay to measure selinexor binding to XPO1 that can be used to evaluate drug exposure following treatment with oral selinexor in preclinical and clinical studies. Studies are ongoing to determine whether there is a correlation between XPO1 occupancy (pharmacodynamics measurement) with disease response in patients with solid and hematological malignancies.