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Chemoproteomic Discovery of a Ritanserin-Targeted Kinase Network Mediating Apoptotic Cell Death of Lung Tumor Cells S

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Chemoproteomic Discovery of a Ritanserin-Targeted Kinase Network Mediating Apoptotic Cell Death of Lung Tumor Cells S Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2018/08/29/mol.118.113001.DC1 1521-0111/94/5/1246–1255$35.00 https://doi.org/10.1124/mol.118.113001 MOLECULAR PHARMACOLOGY Mol Pharmacol 94:1246–1255, November 2018 Copyright ª 2018 by The American Society for Pharmacology and Experimental Therapeutics Chemoproteomic Discovery of a Ritanserin-Targeted Kinase Network Mediating Apoptotic Cell Death of Lung Tumor Cells s Sean T. Campbell,1 Caroline E. Franks,1 Adam L. Borne,1 Myungsun Shin, Liuzhi Zhang, and Ku-Lung Hsu Departments of Chemistry (S.T.C., C.E.F., M.S., L.Z., K.-L.H.), Pathology (S.T.C.), and Pharmacology (A.L.B., K.-L.H.), University of Virginia Cancer Center (K.-L.H.), University of Virginia, Charlottesville, Virginia Received May 15, 2018; accepted August 23, 2018 Downloaded from ABSTRACT Ritanserin was tested in the clinic as a serotonin receptor types. Whereas ritanserin exhibited polypharmacology in inverse agonist but recently emerged as a novel kinase NSCLC proteomes, this compound showed unexpected inhibitor with potential applications in cancer. Here, we specificity for c-RAF in the SCLC subtype, with negligible discovered that ritanserin induced apoptotic cell death of activity against other kinases mediating mitogen-activated non–small cell lung cancer (NSCLC) and small cell lung protein kinase signaling. Here we show that ritanserin blocks molpharm.aspetjournals.org cancer (SCLC) cells via a serotonin-independent mechanism. c-RAF but not B-RAF activation of established oncogenic We used quantitative chemical proteomics to reveal a signaling pathways in live cells, providing evidence in support ritanserin-dependent kinase network that includes key me- of c-RAF as a key target mediating its anticancer activity. diators of lipid [diacylglycerol kinase a, phosphatidylinositol Giventheroleofc-RAFactivationinRAS-mutatedcancers 4-kinase b] and protein [feline encephalitis virus-related resistant to clinical B-RAF inhibitors, our findings may have kinase, rapidly accelerated fibrosarcoma (RAF)] signaling, implications in overcoming resistance mechanisms associated metabolism [eukaryotic elongation factor 2 kinase, eukaryotic with c-RAF biology. The unique target landscape combined translation initiation factor 2-a kinase 4], and DNA damage with acceptable safety profiles in humans provides new response [tousled-like kinase 2] to broadly kill lung tumor cell opportunities for repositioning ritanserin in cancer. at ASPET Journals on September 28, 2021 Introduction similarities that led to its discovery as an inhibitor of diacylglycerol kinase-a (DGKa) (Purow, 2015; Boroda et al., Ritanserin is a serotonin (5-hydroxytryptamine) receptor 2017) (Fig. 1A). We recently used quantitative chemical (5-HTR) inverse agonist with specificity for the serotonin proteomics to discover ritanserin as an active-site inhibitor receptor type 2 (5-HT ) subtype (Peng et al., 2018). As a drug 2 of DGKa and the nonreceptor tyrosine protein kinase feline candidate, ritanserin was tested for treatment of several encephalitis virus-related kinase (FER) (Franks et al., 2017; neuropsychiatric disorders but never received approval for McCloud et al., 2018). Although they are distinct in substrate clinical use (Barone et al., 1986). Its oral bioavailability and preference, DGKa (Sakane et al., 2007) and FER (Greer, 2002) lack of adverse side effects in humans have since prompted studies to explore ritanserin for clinical applications beyond are kinases related by their role in coupling receptor activa- serotonin signaling (Purow, 2015). Comparison of ritanserin tion with intracellular signaling important for cell survival with existing lipid kinase inhibitors revealed structural and proliferation. Thus, ritanserin is capable of perturbing cellular signaling through serotonin-independent mecha- This work was supported by the University of Virginia [Startup Funds (to nisms. We and others have proposed that ritanserin may K.-L.H.)], the National Institutes of Health National Institute on Drug Abuse have potential applications in oncology by disrupting regu- [Grants DA035864 and DA043571 (to K.-L.H.)], the National Institutes of Health National Institute of General Medical Sciences [Grant T32-GM007055 latory pathways through its largely unexplored action (to C.E.F.)], the National Institutes of Health National Cancer Institute [Grant against the kinase superfamily. T32-CA009109 (to A.L.B. and S.T.C.)], the Schiff Foundation [Brain Tumor In this study, we set out to define the target spectrum of Research Award (to K.-L.H.)], and the U.S. Department of Defense [Grant W81XWH-17-1-0487 (to K.-L.H.)]. ritanserin to better understand its mode of action in tumor 1S.T.C., C.E.F., and A.L.B. contributed equally to this work. cells. Previous reports demonstrated that ritanserin is cyto- https://doi.org/10.1124/mol.118.113001. s This article has supplemental material available at molpharm. toxic against glioblastoma and melanoma through putative aspetjournals.org. downstream targets of DGKa, including mammalian target of ABBREVIATIONS: 5-HTR, 5-hydroxytryptamine receptor; 5-HT2, serotonin receptor type 2; ANOVA, analysis of variance; DGKa, diacylglycerol kinase a; DMSO, dimethylsulfoxide; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; FER, feline encephalitis virus-related kinase; HEK293T, human embryonic kidney 293 T; LC-MS, liquid chromatography-mass spectrometry; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; MS, mass spectrometry; NSCLC, non–small cell lung cancer; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; RAF, rapidly accelerated fibrosarcoma; SCLC, small cell lung cancer; SILAC, stable isotope labeling with amino acids in cell culture; SR,SILACratio. 1246 Target Landscape of Ritanserin in Lung Cancer Proteomes 1247 Fig. 1. Ritanserin shows cytotoxic activ- ity in lung tumor cells. (A) Ritanserin is a 5-HT2R inverse agonist with known activ- ity against lipid (DGKa) and protein (FER) kinases. Ketanserin is a 5-HT2R inverse agonist that lacks DGKa/FER in- hibitory activity and serves as a negative control. (B) Cell viability dose-response curves for NSCLC (A549, H1650) and SCLC (H82) tumor cells treated with ritanserin or ketanserin at the indicated concentrations for 2 days. (C) Time course of cell viability in tumor cells treated with 25 mM ritanserin, 25 mM ketanserin, or 1 mM staurosporine for 4 days. Stauro- sporine is a pan-kinase inhibitor and was included as a positive control of tumor cell death. All experiments were performed in triplicate and data are from two indepen- dent biologic replicates performed on Downloaded from separate days (n = 6). Statistical signifi- cance was calculated with respect to ketanserin treatment. Data are shown as means 6 S.E.M. *P # 0.05; **P # 0.01; ***P # 0.001; ****P # 0.0001. 5-HT2R, 5-HT2 receptor. molpharm.aspetjournals.org rapamycin (Dominguez et al., 2013), hypoxia-inducible factor and then returned to the incubator for 2 hours. Plates were again 1-a (Dominguez et al., 2013), and geranylgeranyl transferase I shaken, followed by measurement of absorbance at 450 nm. Data were (Olmez et al., 2018). We hypothesize that ritanserin’s cellular normalized to DMSO-treated wells and significance values were activity is mediated through blockade of kinase networks to determined with one-way analysis of variance (ANOVA). explain its broad action against diverse tumor cell types. An Cell Counts. Tumor cells were plated in 60-mm plates at a density of 100,000 cells/ml (A549, H1650) or 200,000 cells/ml (H82) and a advantage of multitargeted strategies is to minimize the volume of 3.5 ml/plate. Cells were treated with inhibitors at the potential for development of resistance mechanisms (Knight indicated concentrations (final DMSO concentration of 0.5%) for et al., 2010). We conducted cell viability assays to determine at ASPET Journals on September 28, 2021 48 hours at 37°C under 5% CO2. After incubation, adherent cells were the impact of ritanserin treatments on survival of different washed and detached with trypsin and all cells were collected and lung cancer subtypes. We used quantitative chemoproteomics concentrated by spinning at 400g for 3 minutes, followed by aspiration to determine the kinase targets of ritanserin in both of media. Cells were resuspended in 10 nM Trypan Blue and 10 mlof non–small cell lung cancer (NSCLC) and small cell lung cancer this solution was counted via a hemocytometer. Dead cells were (SCLC) proteomes. Our findings reveal that ritanserin shows excluded from all counts. Data were normalized to DMSO-treated novel activity against c-RAF (rapidly accelerated fibrosarcoma) wells and significance values were determined with one-way ANOVA. in SCLC proteomes. The lack of activity against other kinases CaspaseGlo Assays. Assays were performed as directed by the manufacturer (Promega). Briefly, tumor cells were plated in black involved in mitogen-activated protein kinase (MAPK) signaling tissue-culture treated transparent-bottom 96-well plates at a density suggests that ritanserin mediates its cellular activity in SCLC of 200,000 cells/ml (A549, H1650) or 400,000 cells/ml (H82) in a volume cells at least in part through blockade of c-RAF. of 50 ml/well. Cells were treated with inhibitors at the indicated concentrations (final DMSO concentration of 0.5%) for 24 hours at 37°C under 5% CO2.
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