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.
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.
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.
While drug resistance mutations provide the gold standard proof for drug target engagement, target deconvolution of inhibitors identified from a phenotypic screen remains challenging. Genetic ...screening for functional in-frame drug resistance mutations by tiling CRISPR-Cas nucleases across protein coding sequences is a method for identifying a drug’s target and binding site. However, the applicability of this approach is constrained by the availability of nuclease target sites across genetic regions that mediate drug resistance upon mutation. In this study, we show that an enhanced AsCas12a variant (enAsCas12a), which harbors an expanded targeting range, facilitates screening for drug resistance mutations with increased activity and resolution in regions that are not accessible to other CRISPR nucleases, including the prototypical SpCas9. Utilizing enAsCas12a, we uncover new drug resistance mutations against inhibitors of NAMPT and KIF11. These findings demonstrate that enAsCas12a is a promising new addition to the CRISPR screening toolbox and allows targeting sites not readily accessible to SpCas9.
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CRISPR-Cas mutagenesis screening across protein coding sequences allows for drug target deconvolution. However, the applicability of this approach can be severely restricted by the absence of CRISPR target sites. In this study, we show that the enhanced AsCas12a (enAsCas12a) CRISPR endonuclease, which harbors an expanded targeting range, facilitates efficient screening for drug resistance mutations.
The common participation of oncogenic KRAS proteins in many of the most lethal human cancers, together with the ease of detecting somatic KRAS mutant alleles in patient samples, has spurred ...persistent and intensive efforts to develop drugs that inhibit KRAS activity. However, advances have been hindered by the pervasive inter- and intra-lineage diversity in the targetable mechanisms that underlie KRAS-driven cancers, limited pharmacological accessibility of many candidate synthetic-lethal interactions and the swift emergence of unanticipated resistance mechanisms to otherwise effective targeted therapies. Here we demonstrate the acute and specific cell-autonomous addiction of KRAS-mutant non-small-cell lung cancer cells to receptor-dependent nuclear export. A multi-genomic, data-driven approach, utilizing 106 human non-small-cell lung cancer cell lines, was used to interrogate 4,725 biological processes with 39,760 short interfering RNA pools for those selectively required for the survival of KRAS-mutant cells that harbour a broad spectrum of phenotypic variation. Nuclear transport machinery was the sole process-level discriminator of statistical significance. Chemical perturbation of the nuclear export receptor XPO1 (also known as CRM1), with a clinically available drug, revealed a robust synthetic-lethal interaction with native or engineered oncogenic KRAS both in vitro and in vivo. The primary mechanism underpinning XPO1 inhibitor sensitivity was intolerance to the accumulation of nuclear IκBα (also known as NFKBIA), with consequent inhibition of NFκB transcription factor activity. Intrinsic resistance associated with concurrent FSTL5 mutations was detected and determined to be a consequence of YAP1 activation via a previously unappreciated FSTL5-Hippo pathway regulatory axis. This occurs in approximately 17% of KRAS-mutant lung cancers, and can be overcome with the co-administration of a YAP1-TEAD inhibitor. These findings indicate that clinically available XPO1 inhibitors are a promising therapeutic strategy for a considerable cohort of patients with lung cancer when coupled to genomics-guided patient selection and observation.
Abstract
Human exportin-1 (XPO1) is the key nuclear-cytoplasmic transport protein that exports a wide variety of different cargo proteins including tumor suppressors out of the cell's nucleus. ...Inhibition of XPO1 function consequently restores nuclear localization of these proteins and is a promising therapeutic strategy for cancer. Selective inhibitor of nuclear export (SINE) compounds are inhibitors of the XPO1-mediated nuclear export with potent anti-cancer activity. The oral clinical candidate SINE selinexor (KPT-330) is currently in Phase-II/IIb clinical trials and demonstrated remission in patients as a single agent or in combination therapy in trials for pre-treated, relapsed and refractory hematological and solid tumor malignancies. The drug is generally well tolerated when dosed every other day 1-3 times a week. However, XPO1 inhibitors with improved tolerability allowing more frequent dosing can be expected to have a substantial clinical benefit.
Here we present the anti-leukemic activity of a second-generation XPO1 inhibitor KPT-8602 with improved tolerability allowing for a daily dosing regimen. First, its anti-XPO1 activity was assessed; KPT-8602 potently inhibited the XPO1-mediated nuclear protein export at nanomolar concentrations and it blocked the interaction of XPO1 with cargo protein. KPT-8602 also induced potent cytotoxicity on a panel of T-ALL and B-ALL cell lines. Cytotoxicity correlated with the induction of caspase-dependent apoptosis and the nuclear accumulation of p53 as well as the subsequent induction of p53 response. To further investigate the mechanism of action of KPT-8602 we applied CRISPR/Cas9 to introduce a Cys528Ser mutation in the XPO1 gene of four different leukemia cell lines. Mutant cells were over 100 times resistant to KPT-8602. In addition, drug-target interaction was confirmed by pull-down of wild-type XPO1 protein out of cells using biotinylated KPT-8602 while it was unable to pull down mutated XPO1C528S out of mutant cells. These results illustrate the highly specific interaction of the drug for its target and prove that the anti-leukemic activity of KPT-8602 is caused by inhibition of XPO1.
To examine the anti-leukemic activity of KPT-8062 in vivo, mice engrafted with patient-derived T-cell acute lymphoblastic leukemia were treated with KPT-8602 or placebo. Mice were daily treated by oral gavage for 3 weeks. Treatment with KPT-8602 led to a significant reduction of leukemia cell numbers in blood as measured by weekly blood counts, without affecting normal erythropoiesis. Animals treated with KPT-8602 had prolonged survival compared to placebo treated animals. KPT-8602 also showed potent anti-leukemia activity in a mouse T-ALL leukemia model.
In conclusion, KPT-8062 is a second-generation XPO1 inhibitor with high specificity for its target and with potent anti-ALL activity. It displays better tolerability as compared to the first-generation SINE selinexor allowing it for daily dosing resulting in effective anti-ALL activity in in vivo PDX models warranting further evaluation of this new drug in patients. As such, in January 2016 a phase I/II study with KPT-8602 has been initiated.
Citation Format: Dirk Daelemans, Jasper Edgar Neggers, Jolien De Bie, Maarten Jacquemyn, Astrid D’Hoore, Els Vanstreels, Erkan Baloglu, Yosef Landesman, Sharon Shacham, William Senapedis, Antonis Dagklis, Thomas Vercruysse, Jan Cools. KPT-8602 is a second-generation XPO1 inhibitor with improved in vivo tolerability that demonstrates potent acute lymphoblastic leukemia activity. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-210.
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