Abstract A19: KPT0127, a novel, potent, and selective inhibitor of Crm1-mediated nuclear export shows selective cytotoxicity for neoplastic over normal cells and is well tolerated in animals
Authors: Shacham Sharon , Nir Raphael , Daelemans Dirk , Shechter Sharon , Vancurova Ivana , Juvekar Ashish , Lau Winnie , Higgins Guy , Draetta Giulio , Kauffman Michael G. 
Published: June 2012

Abstract
CRM1 (Xpo1) is the major export factor for proteins from the nucleus to the cytoplasm, including tumor suppressors (TSPs) and other modulators of proliferative responses such as p53, FOXO, c-Abl, pRB and IκB. Leptomycin B (LMB), a non-drug like natural product is a potent inhibitor of CRM1-mediated nucleocytoplasmic transport. LMB and related analogs trap TSPs and other proteins in the nucleus, forcing neoplastic cells into apoptosis while normal cells undergo reversible cell cycle arrest. However, LMB has limited efficacy in vivo due to its severe gastrointestinal toxicity. Here, we describe our lead compound KPT-0127 a novel small molecule, water soluble, drug-like, selective, irreversible CRM1 antagonist. Like LMB, KPT-0127 forms a covalent bond with Cys528 in the CRM1 cargo-binding pocket, abrogating most, but not all, export functions of CRM1. KPT-0127 exerts a potent (EC50 300-400nM) and prolonged inhibition of CRM1- mediated HIV-1 Rev, forkhead (FOXO), and p53 nuclear export in a variety of normal and transformed cell lines. In cytotoxicity assays, KPT-0127 showed high potency in most hematologic cancer cell lines (EC50 &lt;500nM, with leukemia and lymphoma lines often &lt;100nM) and variable activity in solid tumor cell lines (EC50 90-2000nM), with colon and melanoma cells being the most sensitive (EC50&lt; 100 nM). By contrast, normal cells were largely unaffected by treatment with KPT-0127 (EC50 &gt;5-10µM). Studies in the HCT-116 colon cancer cell line suggested that KPT-0127 dose dependently increases the nuclear levels of p53 and appears to induce cell cycle arrest at both the G1/S and G2/M checkpoints, prior to inducing apoptosis. In normal peripheral blood mononuclear cells (PBMCs) and in Hut78 leukemia cells, KPT-0127 potently increased the nuclear levels of IκB. However, KPT-0127 induced cell death of Hut78 cells with no effect on normal PBMCs. In drug combination studies, KPT-0127 showed additive or synergistic cytotoxicity activity with either 5-FU, carboplatin, or doxorubicin. Mechanism of action studies demonstrated that the Cys528 residue in the cargo-binding pocket of CRM1 is essential for the inhibitory effect of KPT-0127. Mutagenesis of Cys528 to a Ser completely abrogated KPT-0127 inhibition. KPT-0128, the transisomer of KPT-0127, shows little effect on both HIV-Rev nuclear export and in cytotoxicity assays (EC50s &gt; 10μM), supporting the specificity of KPT-0127 for CRM1. Moreover, the selectivity of KTP-0127 was demonstrated across a panel of 37 proteins including several cysteine proteases.
In single dose mouse toxicology studies, KPT-0127 was generally well tolerated (oral or SC) up to 560 mg/kg, and no deaths were observed. SC dosing daily for 5 days up to 100mg/kg (the highest dose tested) showed no behavioral, clinical chemistry, or hematogical effects in mice; Pharmacokinetics of KPT-0127 is adequate and in vivo efficacy studies are currently being performed; results will be presented. All together, these data demonstrate that KPT-0127 represents a novel, tumor selective, and well-tolerated irreversible Crm1 inhibitor which may be suitable for clinical development both as a single agent and in combination with standard therapies.
Citation Information: Clin Cancer Res 2010;16(14 Suppl):A19.