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A Small Molecule Drug Screening Identifies Colistin Sulfate As an Enhancer of Natural Killer

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A Small Molecule Drug Screening Identifies Colistin Sulfate As an Enhancer of Natural Killer bioRxiv preprint doi: https://doi.org/10.1101/2021.08.20.457155; this version posted August 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 A small molecule drug screening identifies colistin sulfate as an enhancer of Natural Killer 2 cell cytotoxicity. 3 Serena Cortés-Kaplan1,2,3, Mohammed S. Hasim1,2,3, Shelby Kaczmarek2,3, Zaid Taha1,2,3, Glib 4 Maznyi1,2,3, Scott McComb2,3,4, Seung-Hwan Lee2,3, Jean-Simon Diallo1,2,3, Michele Ardolino1,2,3 5 6 1: Cancer Therapeutics Program, Ottawa Hospital Research Institute, Ottawa, ON 7 2: CI3, University of Ottawa, Ottawa, ON 8 3: Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, 9 ON 10 4: Human Health Therapeutics Research Centre, National Research Council, Canada 11 12 * Correspondence: 13 Michele Ardolino 14 501 Smyth Road, Cancer Center, 3-328, Ottawa, ON, K1H 8M2 15 [email protected] 16 Tel: +1-613-737-8899 ext 77257 17 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.20.457155; this version posted August 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 18 Abstract: 19 Because of their crucial role in tumor immunity, NK cells have quickly become a prime target for 20 immunotherapies, with adoptive transfer of NK cells and the use of NK cell engagers quickly 21 moving to clinical stage. On the other hand, only few studies have focused on small molecule 22 drugs capable of unleashing NK cell against cancer. In this context, repurposing small molecule is 23 an attractive strategy to identify new immunotherapies from already approved drugs. Here, we 24 screened 1,200 FDA-approved drugs from the Prestwick Chemical Library, to identify compounds 25 that increase NK cell cytotoxic potential. Using a high-throughput luciferase-release cytotoxicity 26 assay, we found that the antibiotic colistin sulfate increased cytotoxicity of human NK cells 27 towards cancer cells. The effect of colistin was short lived and was not observed when NK cells 28 were pretreated with the drug, showing how NK cell activity was potentiated only when the 29 compound was present at the time of recognition of cancer cells. Further studies are needed to 30 uncover the mechanism of action and the pre-clinical efficacy of colistin sulfate in mouse cancer 31 models. 32 33 Keywords: 34 NK cells, immunotherapy, functional screen, drug repurposing 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.20.457155; this version posted August 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 35 Introduction 36 Seminal studies from the 1990s and early 2000s highlighted the importance of the immune system 37 in tumor biology through processes such as immunosurveillance and immunoediting1, which in 38 turn led to the most recent advances in cancer therapeutics: immunotherapies. Cancer 39 immunotherapy is an encompassing term referring to therapeutic strategies that target components 40 of the immune system to enhance clearance of the malignant cells. Various categories of 41 immunotherapies exist2 and, encouragingly, some became first-line treatments in some cancer 42 types3,4. As the field of immunotherapy continues to develop, we have gained a better 43 understanding of how immune cells contribute to immunotherapy efficacy, for instance Natural 44 Killer (NK) cells. NK cells are innate lymphoid cells that play a crucial role in tumor surveillance 45 and clearance5. The importance of NK cells in immunosurveillance is appreciated from the 46 observation that mice without functional NK cells show impaired tumor control6,7 and that patients 47 with defective or decreased frequency of NK cells are at greater risk of developing malignancies, 48 specifically virally induced cancers8,9. Recently, a thorough systematic review and meta-analysis 49 encompassing 15 solid cancer types found that NK cell infiltration in solid tumors was associated 50 with improved overall survival10, whereas lower low frequency of circulating or tumor-infiltrating 51 NK cells or NK cells that display impaired function are associated with worse prognosis in several 52 cancer types11-14. NK cells can either be administered for adoptive cell therapy or can be directly 53 targeted to enhance their anti-tumor activity5. Within the second category, small molecule drugs 54 have been surprisingly overlooked, despite evidence that small molecules can potentiate NK 55 anti-cancer functions15,16. In addition to enhancing cytotoxicity, small molecules can also be used 56 to promote proliferation and maturation in expansion protocols for NK cells that are used for 57 adoptive cell therapies15. Small molecule immunotherapies are advantageous as they are orally 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.20.457155; this version posted August 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 58 bioavailable, usually cost less than biological immunotherapies, can target both extracellular and 59 intracellular components and have a greater ability to penetrate through physiological barriers17. 60 However, from small molecule identification to development of a lead drug compound, the clinical 61 drug pipeline can take years if not decades before the drug sees use in the clinic. For this reason, 62 drug repurposing is an attractive alternative that identifies new indications for previously approved 63 drugs. Repurposed drugs already have a safety and efficacy profile associated with them which 64 makes this a favorable route. Several studies have conducted high-throughput drug screenings to 65 identify drugs that modulate NK cell activity. These studies utilized commercially available 66 libraries containing repurposed drugs18-20 or natural compound libraries21,22 and have identified 67 small molecules that were not previously known to modulate NK cell activity. Here, we screened 68 the Prestwick Chemical Library for compounds capable of enhancing cytotoxicity of human NK 69 cells towards leukemia target cells and identified the antibiotic colistin sulfate as an enhancer of 70 NK cell cytotoxicity. 71 72 Materials and Methods. 73 Cell culture 74 All cell lines were cultured in a humidified incubator at 37˚C and 5% CO2 in media supplemented 75 with 100 U/ml penicillin (Gibco, CA), 100 μg/ml streptomycin (Gibco), 10 g/ml gentamycin 76 sulfate (Gibco), 20 mM HEPES (Fisher, ON). NK92 cells were cultured in RMPI-1640 containing 77 10% fetal bovine serum (FBS) (Gibco). K562-NL cells were cultured in RMPI-1640 containing 78 5% FBS. A375-NL cells and 786O-NL cells were cultured in DMEM (Corning, VA) containing 79 10% FBS. 80 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.20.457155; this version posted August 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 81 Reagents and drugs 82 Preparation of coelenterazine substrate: 500 μg of coelenterazine substrate (CTZ) (Gold 83 Biotechnology, MO) was reconstituted in 610 μL of 100% ethanol and 6.2 μL of 12 N hydrochloric 84 acid. The reconstituted substrate was protected from light and stored at -80˚C until use. Prior to 85 measuring luciferase activity, the reconstituted CTZ was mixed with 1X salt buffer (45 mM 86 EDTA, 30 mM sodium pyrophosphate, 1.425 M NaCl) at a 1:200 dilution (5 μL CTZ per 1 mL 87 salt buffer). 88 89 The Prestwick Chemical Library (https://www.prestwickchemical.com) was kindly provided by 90 Dr. Diallo. 91 92 Preparation of candidate drugs: colistin sulfate salt (Sigma-Aldrich, MO), nicotinamide 93 (Sigma-Aldrich), monensin sodium salt (Sigma-Aldrich), zafirlukast (Sigma-Aldrich), tizanidine 94 hydrochloride (Sigma-Aldrich), closantel (Sigma-Aldrich), benazepril hydrochloride 95 (Sigma-Aldrich), and diflorasone diacetate (Sigma-Aldrich) were prepared at a master stock 96 concentration of 1 mM in 100% DMSO, with exception of colistin sulfate salt which was dissolved 97 in water. A working stock concentration was prepared for all candidate drugs of 100 μM in PBS 98 with a final DMSO concentration of 10%. All candidate drugs were stored at -20˚C until use. 99 Fluorochrome-conjugated antibodies, all from BD Biosciences, CA: AF647-CD3 (Clone 100 UCHT-1), APC-R700-CD4 (Clone RPA-T4), BV786-CD8 (Clone RPA-T8), PE-CD56 (Clone 101 B159), BV711-CD16 (Clone 3G8), BV650-CD19 (Clone SJ25-C1) and PerCP-Cy5.5-CD14 102 (Clone MφP9). 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.20.457155; this version posted August 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 103 Generation of cell lines 104 To generate K562, A375 and 78O cells expressing nanoluciferase, lentiviral particles were 105 produced by co-transfecting 293T cells with a lentiviral plasmid encoding nano luciferase plenti- 106 NL (a gift from Dr.
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