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.

Introduction Selinexor (KPT-330) is a first-in-class selective inhibitor of XPO1-mediated nuclear export that has recently been approved by the US FDA for the treatment of relapsed/refractory multiple myeloma and relapsed diffuse large B-cell lymphoma. Additionally, the drug is being evaluated in ongoing clinical trials (phase I to III) on late-stage haematological and solid tumours. To improve outcomes and personalize therapeutics, specific biomarkers and underlying mechanisms of response to treatment need to be uncovered. Material and method We performed genome-wide CRISPR/Cas9-mediated loss-of-function genetic screening in two multiple myeloma (KMS-28-BM and SK-MM-1) and one chronic myeloid leukemia (HAP1) cell lines. After transduction with a knockout library, cells were divided and subsequently cultured into three parallel groups (control, IC20 and IC50) for at least 20 cell doublings. This setup allows monitoring of subtle relative changes between groups. After DNA extraction, sequencing of guide RNAs and statistical analysis, we identified genes that upon disruption confer either increased susceptibility or resistance to selinexor. Results and discussion For the multiple myeloma cell lines, the TGF-β/SMAD4 signalling pathway stood out as an important mediator of resistance to selinexor. Besides SMAD4 as a major hit in both cell lines, we identified five other genes from this pathway leading to resistance in KMS-28-BM cells, suggesting that downregulation of this pathway is a potential biomarker of response to SINE treatment in multiple myeloma. In addition, screens revealed significant variability across cell lines with hit genes involved in mRNA processing, proteasomal degradation and cell cycle regulation. Interestingly, we could simultaneously uncover resistance and sensitivity genes involved in the same pathway, suggesting that these have antagonistic effects. Notably, we identified knockout of the poorly characterized gene ASB8 as a strong, common sensitizer between the three cell lines. Conclusion Until now, most chemogenetic CRISPR/Cas9 knockout screens have focused on determining resistance genes, and few have employed the technique to screen for genes that enhance cell kill. We performed such screens with selinexor and found a number of general and cell type-specific hits. Genes identified to confer increased resistance are candidate biomarkers while genes inducing increased susceptibility represent new targets for drug combination therapies. Disclosures Kwanten: Karyopharm Therapeutics Inc.: Patents & Royalties: employees of KULeuven. KULeuven has a license agreement (royalties) with Karyopharm Therapeutics on XPO1 inhibitors (selinexor). Landesman:Karyopharm Therapeutics Inc: Current Employment, Current equity holder in publicly-traded company. Daelemans:Karyopharm Therapeutics Inc.: Patents & Royalties: employees of KULeuven. KULeuven has a license agreement (royalties) with Karyopharm Therapeutics on XPO1 inhibitors (selinexor).

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Identification and validation of the cellular target of bioactive hits identified in a phenotypic screen is crucial for their further development into a drug. The gold standard proof for identification of a small molecule's target is the discovery of mutations that confer resistance. However, selection of drug resistance and subsequent deconvolution of the relevant mutations that confer drug resistance remains time consuming. Therefore, a methodology that can accelerate the drug resistance selection process and that can simplify identification of relevant drug resistance mutations would benefit the drug discovery and development process greatly. Here we report a simple method to rapidly identify the mechanism of action of small molecules with anti-cancer activity. The method utilizes the positive selection of in-frame drug resistance mutations in the target protein of a small molecule derived from the localized genetic variation created by non-homologous end joining (NHEJ) repair of CRISPR/Cas9-induced double strand breaks. In brief, we introduced double strand breaks in the target proteins of selinexor, ispinesib and triptolide and treated these cells with the respective drug. Resistance was obtained rapidly and sequencing of resistant colonies revealed known as well as many new drug resistant protein variants. Next, we investigated whether this approach can be applied as a screening strategy on a subset of genes to identify the direct cellular target protein of a small molecule. We therefore, as proof of concept, designed a pooled sgRNA tiling library targeting all PAM sites of multiple genes and applied this to ispinesib. Drug resistant colonies formed rapidly and were enriched with sgRNAs targeting the ispinesib binding site in KIF11, ispinesib’s target protein. Further sequencing of the sgRNA target gene locus revealed drug resistance mutations in KIF11, validating the feasibility of the methodology to uncover the target of a small molecule from a small pool of candidate genes. To conclude, we provide a new method for identification of the cellular target protein and binding site of a small molecule based on positive selection of drug resistant protein variants generated by targeted CRISPR/Cas-induced NHEJ repair. Citation Format: Jasper E. Neggers, Bert Kwanten, Tim Dierckx, Dirk Daelemans. Identification of drug-target interactions through rapid selection of drug resistant protein variants generated by CRISPR/Cas9-induced NHEJ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5224. doi:10.1158/1538-7445.AM2017-5224

Exportin 1 (XPO1/CRM1) is a key nuclear transport receptor (karyopherin) responsible for the export of different cargo proteins out of the cell’s nucleus into the cytoplasm. Its correct function is essential for cellular homeostasis, however normal XPO1 function is often disrupted in malignant cells. XPO1 is overexpressed in solid and hematological tumors and higher XPO1 expression is often associated with poorer prognosis. XPO1-regulated cargo proteins include tumor suppressor proteins and cell cycle regulators that can be involved in tumorigenesis. For example, aberrant XPO1 function may lead to cytoplasmic mislocalization of tumor suppressor proteins which results in their functional inactivation and hence may cause tumorigenesis. Inhibition of XPO1 function will restore their proper subcellular localization and function and cause tumor regression. Although XPO1 has a central role in cellular homeostasis, it is a good target for cancer therapy, as illustrated by the clinical success of the selective inhibitor of nuclear export (SINE) selinexor. Selinexor is the first and currently the only XPO1 inhibitor clinically approved. It is applied for the treatment of patients with multiple myeloma after at least one prior treatment, and for the treatment of diffuse large B cell lymphoma. Selinexor covalently binds to XPO1. The second generation SINE eltanexor is also a covalent XPO1 inhibitor but has only minimal brain penetration and consequently lower toxicity in preclinical studies. Here, we describe a novel chemical class of reversible XPO1 inhibitors with high brain penetration but with good tolerability allowing frequent dosing in preclinical models. The lead compound, FR-027, potently inhibits XPO1 function (EC50 69 ± 10 nM) and shows potent cancer cell growth inhibition in vitro of both hematological and solid cancer cell lines (EC50 50-950 nM). FR-027 is a reversible inhibitor of XPO1 and unlike many other XPO1 inhibitors it does not induce XPO1 protein degradation; this may contribute to an increased tolerability. Indeed, while it shows high brain penetration, FR-027 allows frequent dosing in mice with good tolerability (body weight). It is orally bioavailable and demonstrates strong anti-leukemic efficacy in an aggressive MOLT-4 xenograft model. Importantly, it shows potent efficacy in both an orthotopic U87 MG brain tumor xenograft model and a metastatic syngeneic ID8-fLuc ovarian cancer model with significant survival benefit as monotherapy. Altogether, these results demonstrate that FR-027 is a novel, reversible XPO1 inhibitor with important molecular and pharmacological characteristics that warrant further clinical development. Citation Format: Janne Van Hauwenhuyse, Felien Reniers, Leentje Persoons, Sam Noppen, Eline Boel, Els Vanstreels, Ann Vankerckhoven, Bert Kwanten, An Coosemans, Guy Van den Mooter, Wim Dehaen, Dirk Daelemans. A novel reversible inhibitor of XPO1 with potent efficacy in multiple preclinical mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1652.