Abstract
Exportin-1 (CRM1/XPO1) is a crucial nuclear export protein that transports a wide variety of proteins from the nucleus to the cytoplasm. These cargo proteins include tumor suppressors and growth-regulatory factors and as such XPO1 is considered a potential anti-cancer target. From this perspective, inhibition of the XPO1-mediated nuclear export by selective inhibitor of nuclear export (SINE) compounds has shown broad-spectrum anti-cancer activity. Furthermore, the clinical candidate SINE, selinexor, is currently in multiple phase I/II/IIb trials for treatment of cancer. Resistance against selinexor has not yet been observed in the clinic, but in vitro selection of resistance did not reveal any mutations in the target protein, XPO1. However, introduction of a homozygous mutation at the drug's target site, the cysteine 528 residue inside the XPO1 cargo-binding pocket, by genetic engineering, confers resistance to selinexor. Here we investigated whether this resistance to selinexor is recessive or dominant. For this purpose we have engineered multiple leukemia cell lines containing heterozygous or homozygous C528S substitutions using CRISPR/Cas9-mediated genome editing. Our findings show that heterozygous mutation confers similar resistance against selinexor as homozygous substitution, demonstrating that SINE resistance can be obtained by a single and dominant mutation of the cysteine528 residue in XPO1.
Highlights
Human exportin 1 (XPO1), known as chromosome region maintenance 1 protein (CRM1), is a ubiquitously expressed member of the importin-β family of nucleocytoplasmic transport proteins
Exportin-1 (XPO1, CRM1) is a crucial protein involved in the nuclear export of many cellular proteins [13], including various tumor suppressive and growth regulatory proteins
The drug has been shown to and selectively interact with the cysteine528 residue located in the hydrophobic cargo-binding pocket of XPO1 [12]
Summary
Human exportin 1 (XPO1), known as chromosome region maintenance 1 protein (CRM1), is a ubiquitously expressed member of the importin-β family of nucleocytoplasmic transport proteins ( known as karyopherins). Resistant cells displayed an altered expression www.impactjournals.com/oncotarget pattern in a number of key signaling pathways, including upregulation of anti-apoptotic and downregulation of pro-apoptotic genes These cells were >100 fold less sensitive to SINEs and even though they showed nuclear accumulation of XPO1 cargo-proteins in resistant cells after treatment as compared to the parental cells, they did not incorporate any mutations in the cargo-binding pocket of XPO1. In an effort to validate the SINE-XPO1 drug-target interaction, we were able to successfully engineer the genome of Jurkat leukemic T-cells to substitute cysteine528, the anchor point of selinexor inside the cargo-binding pocket of XPO1, with a serine These homozygous genome-edited XPO1C528S Jurkat cells were viable and showed a >250 fold resistance to SINE compounds, demonstrating that the anti-cancer activity of SINE compounds effectively results from XPO1 inhibition [12]. We investigate the effect of heterozygous substitutions of the XPO1 cysteine 528 residue on SINE activity in cellulo
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