E3 ubiquitin ligase ASB8 promotes selinexor-induced proteasomal degradation of XPO1.

Selinexor (KPT-330), a small-molecule inhibitor of exportin-1 (XPO1, CRM1) with potent anticancer activity, has recently been granted FDA approval for treatment of relapsed/refractory multiple myeloma and diffuse large B-cell lymphoma (DLBCL), with a number of additional indications currently under clinical investigation. Since selinexor has often demonstrated synergy when used in combination with other drugs, notably bortezomib and dexamethasone, a more comprehensive approach to uncover new beneficial interactions would be of great value. Moreover, stratifying patients, personalizing therapeutics and improving clinical outcomes requires a better understanding of the genetic vulnerabilities and resistance mechanisms underlying drug response. Here, we used CRISPR-Cas9 loss-of-function chemogenetic screening to identify drug-gene interactions with selinexor in chronic myeloid leukemia, multiple myeloma and DLBCL cell lines. We identified the TGFß-SMAD4 pathway as an important mediator of resistance to selinexor in multiple myeloma cells. Moreover, higher activity of this pathway correlated with prolonged progression-free survival in multiple myeloma patients treated with selinexor, indicating that the TGFß-SMAD4 pathway is a potential biomarker predictive of therapeutic outcome. In addition, we identified ASB8 (ankyrin repeat and SOCS box containing 8) as a shared modulator of selinexor sensitivity across all tested cancer types, with both ASB8 knockout and overexpression resulting in selinexor hypersensitivity. Mechanistically, we showed that ASB8 promotes selinexor-induced proteasomal degradation of XPO1. This study provides insight into the genetic factors that influence response to selinexor treatment and could support both the development of predictive biomarkers as well as new drug combinations.

Target Identification of Small Molecules Using Large-Scale CRISPR-Cas Mutagenesis Scanning of Essential Genes.

Target deconvolution of new bioactive agents identified from phenotypic screens remains a challenging task. The discovery of mutations that confer resistance to such agents is regarded as the gold standard proof of target identification. Here, we describe a method that exploits the error-prone repair of CRISPR-induced DNA double-strand breaks to enhance mutagenesis and increase the incidence of drug resistance mutations in essential genes. As each DNA double-strand break is introduced at a targeted genomic site predefined by the presence of a protospacer adjacent motif (PAM) and a particular CRISPR single guide RNA (sgRNA), the genetic location of drug resistance mutations can be easily uncovered through targeted sequencing of CRISPR sgRNAs. Moreover, the method allows for the identification of not only the drug target gene, but also the drug-binding domain within the target gene.

Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes.

Unraveling the mechanism of action and molecular target of small molecules remains a major challenge in drug discovery. While many cancer drugs target genetic vulnerabilities, loss-of-function screens fail to identify essential genes in drug mechanism of action. Here, we report CRISPRres, a CRISPR-Cas-based genetic screening approach to rapidly derive and identify drug resistance mutations in essential genes. It exploits the local genetic variation created by CRISPR-Cas-induced non-homologous end-joining (NHEJ) repair to generate a wide variety of functional in-frame mutations. Using large sgRNA tiling libraries and known drug-target pairs, we validate it as a target identification approach. We apply CRISPRres to the anticancer agent KPT-9274 and identify nicotinamide phosphoribosyltransferase (NAMPT) as its main target. These results present a powerful and simple genetic approach to create many protein variants that, in combination with positive selection, can be applied to reveal the cellular target of small-molecule inhibitors.