Identifying Drug-Target Selectivity of Small-Molecule CRM1/XPO1 Inhibitors by CRISPR/Cas9 Genome Editing

Abstract

Validation of drug-target interaction is essential in drug discovery and development. The ultimate proof for drug-target validation requires the introduction of mutations that confer resistance in cells, an approach that is not straightforward in mammalian cells. Using CRISPR/Cas9 genome editing, we show that a homozygous genomic C528S mutation in the XPO1 gene confers cells with resistance to selinexor (KPT-330). Selinexor is an orally bioavailable inhibitor of exportin-1 (CRM1/XPO1) with potent anticancer activity and is currently under evaluation in human clinical trials. Mutant cells were resistant to the induction of cytotoxicity, apoptosis, cell cycle arrest, and inhibition of XPO1 function, including direct binding of the drug to XPO1. These results validate XPO1 as the prime target of selinexor in cells and identify the selectivity of this drug toward the cysteine 528 residue of XPO1. Our findings demonstrate that CRISPR/Cas9 genome editing enables drug-target validation and drug-target selectivity studies in cancer cells.

Development and potential applications of CRISPR-Cas9 genome editing technology in sarcoma

“…To validate this drug-target interaction, CRISPR-Cas9 was adopted to introduce a single XPO1 C528S mutation in acute T cell leukemia Jurkat cells. As expected, this mutation prevented Selinexor-meditated functional inhibition of XPO1 by blocking XPO1-Selinexor binding [53, 77]. The study confirmed that XPO1 served as the prime target of Selinexor in cancer cells [53, 77].…”

“…Human exportin-1 (XPO1), also known as chromosome region maintenance 1 protein (CRM1), is considered an anticancer target [53]. In recent years, over expression of XPO1 has been observed in osteosarcoma and has been correlated with poor prognosis and resistance to therapy [83].…”

“…In recent years, over expression of XPO1 has been observed in osteosarcoma and has been correlated with poor prognosis and resistance to therapy [83]. Selinexor (KPT-330), an inhibitor of XPO1, is currently undergoing phase 2 clinical trials and has demonstrated high response rates as a therapeutic agent in phase 1 trials for heavily pretreated, relapsed, and refractory hematological and solid tumor malignancies in humans [16, 36, 53]. Selinexor could inhibit the function and formation of XPO1, possibly through binding to the cysteine 528 residue of XPO1, which leads to an accumulation of tumor suppressor proteins in the nucleus of treated cells, and thus cell cycle arrest and apoptosis [16, 36, 53].…”

Global profiling of lysine reactivity and ligandability in the human proteome

“…By combining features of recognition and reactivity, covalent ligands have the potential to target sites on proteins that are difficult to address by reversible binding interactions alone 3 . While original covalent probes often targeted essential catalytic residues within the active sites of enzymes, in particular, serine 4 and cysteine 5 residues of enhanced nucleophilicity, more recent successes in covalent ligand development include electrophilic small molecules that react with non-catalytic cysteines across diverse protein classes, including kinases 6,7 , GTPases 8 , and non-enzymatic proteins (e.g., nuclear export factors 9 ). These efforts have culminated in the approval of several covalent kinase inhibitors as drugs for treating diverse cancers 6,7 .…”

Therapeutic implication of concomitant chromosomal aberrations in patients with aggressive B-cell lymphomas

“…56 Preclinical data from our group revealed that inhibition of XPO1 results in nuclear entrapment of several key oncogenic transcripts such as MYC and BCL6 in DH/TH lymphoma cells ultimately resulting in protein reduction. 55 Selinexor is a small molecule covalent inhibitor of XPO1 57 that reduces the expression of MYC and BCL6 in DHL/THL cells and displays anti-lymphoma activity in vitro and in vivo toward this aggressive subgroup of DLBCL. 55 …”