A small molecule drug screening identifies colistin sulfate as an enhancer of Natural Killer cell cytotoxicity

Because of their crucial role in tumor immunity, NK cells have quickly become a prime target for immunotherapies, with adoptive transfer of NK cells and the use of NK cell engagers quickly moving to clinical stage. On the other hand, only few studies have focused on small molecule drugs capable of unleashing NK cell against cancer. In this context, repurposing small molecule is an attractive strategy to identify new immunotherapies from already approved drugs. Here, we screened 1,200 FDA-approved drugs from the Prestwick Chemical Library, to identify compounds that increase NK cell cytotoxic potential. Using a high-throughput luciferase-release cytotoxicity assay, we found that the antibiotic colistin sulfate increased cytotoxicity of human NK cells towards cancer cells. The effect of colistin was short lived and was not observed when NK cells were pretreated with the drug, showing how NK cell activity was potentiated only when the compound was present at the time of recognition of cancer cells. Further studies are needed to uncover the mechanism of action and the pre-clinical efficacy of colistin sulfate in mouse cancer models.


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Seminal studies from the 1990s and early 2000s highlighted the importance of the immune system 36 in tumor biology through processes such as immunosurveillance and immunoediting 1 , which in 37 turn led to the most recent advances in cancer therapeutics: immunotherapies. Cancer 38 immunotherapy is an encompassing term referring to therapeutic strategies that target components 39 of the immune system to enhance clearance of the malignant cells. Various categories of 40 immunotherapies exist 2 and, encouragingly, some became first-line treatments in some cancer 41 types 3,4 . As the field of immunotherapy continues to develop, we have gained a better co-culture of NK92+K562-NL cells at a E:T ratio of 1 were treated with 10 μM of each drug for 148 5-hours at 37˚C. Each compound was evaluated in singlet over 2 independent experiments.

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The Prestwick Chemical Library's 15 stock plates were stored at -20˚C in 10% DMSO at a 151 concentration of 100 μM in deep well plates (Axygen, Tamaulipas, Mexico), with compounds only 152 in columns 2-11. On the day of the screen, the stock plates were thawed, and the Bravo Automated  After the incubation, 50 μL of supernatant from each assay plate was transferred to round-bottom 169 black 96-well plates (Corning, ME) using the Bravo Automated Liquid Handling Platform. Biotek 170 plate reader was used to dispense 25 μL of CTZ to each well and measure luminescence.  To identify compounds capable of enhancing NK92 cytotoxicity, the luminescent fold-change over 182 DMSO control was calculated for all compounds in the E:T=1 condition and K562-NL alone 183 condition. Compounds that had a fold-change ≥1.3 were considered drug hits. Drugs were 184 excluded if the fold-change was ≥1.3 in the K562-NL alone treated with drugs condition. 185 Fold-change for each plate was calculated by using the controls on individual plates.    224 Traditional methods to assess NK cell cytotoxicity such as chromium-release or flow 225 cytometry-based assays are difficult to scale up for a high-throughput use. Luciferase release-based 226 killing assays have proven useful to perform drug screenings 24 , and a luciferase released-based 227 screen was employed in a previous NK cell drug screening 18 . To generate target cells suitable for 228 a luciferase release-based NK cell killing assay, we transduced the myeloid leukemia cell line 229 K562 with a lentiviral plasmid encoding nano luciferase (NL). Expression of NL was assessed on 230 12 transduced K562 cells by exposing cellular lysates to the substrate: no signal was observed from 231 the lysate of control cells, whereas a robust signal was detected from the lysate of transduced K562 232 cells (Sup. Fig. 2A). Once we verified NL expression on K562 cells, we employed K562-NL as 233 targets in a luciferase release-based killing assays using the NK cell line NK92 as effectors.

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Consistent with what expected in cytotoxicity assays, the presence of effector cells increased the 235 luminescence signal in a dose-dependent manner, indicating that the target cells were effectively 236 killed, whereas the luminescence signal observed with target cells alone was similar to that of the 237 media only (Fig. 1A).

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Next, we sought to obtain a polyclonal population of K562 cells expressing NL from the 240 transduced population, which likely contained cells which were not transduced. Therefore, we 241 sorted single cells into five 96-well plates and tested wells where cell growth was observed for 242 luciferase expression. K562 clones expressing NL were then tested in cytotoxicity assay vis-à-vis 243 with the unsorted K562-NL population (Sup Fig. 2B). Clones that were killed by NK92 cells 244 similarly to the K562 bulk population were selected and mixed at an equal ratio to make a 245 polyclonal population of K562-NL cells, which was then used in all subsequent experiments. 248 For these first experiments, to test NL activity, we used furimazine (FMZ), the optimized substrate 249 for NL 25 . However, using furimazine in a high-throughput setting is not feasible due to the high 250 cost of the substrate. Therefore, we explored if the less expensive substrate coelenterazine (CTZ), 251 widely used for Renilla and Gaussia luciferase, could be used as an alternative. After conducting 252 a luciferase release-based cytotoxicity assay, we used either FMZ or CTZ to assess NL activity 253 13 side-by-side. Luminescent signal was detected with both substrates, although the magnitude of 254 luminescence was higher using FMZ (Fig. 1B). However, the dynamic range between targets only 255 and the E:T ratio of 1 was comparable between the two substrates, and CTZ maintained the same 256 dose-response observed using FMZ, indicating that CTZ could effectively replace FMZ as a 257 substrate for these experiments.

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Next, we set to determine the ideal E:T ratio to use for the drug screening. We conducted several 260 luciferase release-based cytotoxicity assays that included a range of E:T ratios and chose to use a 261 E:T ratio of 1 as this ratio shows minimal killing but still has detectable luminescence above 262 K562-NL target cells alone and there is large dynamic range between the 1 and 81 ratios, an E:T 263 ratio that shows saturation in killing (Fig. 1C).

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As a positive control for the screen, we decided to use the mild detergent digitonin, as we found it 266 able to effectively lyse targets cells without compromising NL activity (Fig. 1D).

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For these set-up experiments, the supernatant from the luciferase-release cytotoxicity assay was 269 collected and transferred to a new plate after a centrifugation step, which would be hardly feasible 270 in high-throughput conditions. Our concern was that by skipping the centrifugation step prior to 271 collecting the supernatant, we would capture live target cells that would lyse after addition of the 272 substrate, resulting in similar luminescence detection between target cells alone and 273 target+effector cells. Therefore, to determine if this step was required, we tested the difference 274 between directly collecting the assay's supernatant at the end of the cytotoxicity assay with or 275 without a centrifugation step. To our advantage, the difference between the target cells alone and 276 14 target+effector cells condition was still retained without the centrifugation step (Fig. 1E). Based 277 on these results, we deemed that a centrifugation step prior to supernatant collection was 278 unnecessary and decided to proceed with directly collecting the assay supernatant for the drug 279 screening.

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Finally, to optimize the high-throughput drug screening workflow, we needed to estimate if leaving 282 the effector or target cells at room temperature for an extended amount of time before they were 283 seeded would affect the results as, logistically, we could not keep cells in their cell culture 284 conditions (humidified incubator, 37˚C, 5% CO2) when seeding the drug screening assay plates. 285 We simulated drug screen plating conditions by incubating NK92 and K562-NL cells separately 286 at room temperature for 0, 60, 120, 180, and 240 minutes before the cells were seeded into assay 287 plates. We observed that leaving the cells at room temperature more than 120 minutes before being 288 seeded into assay plates gradually but substantially decreased NK92 cytotoxicity (Fig. 1F). We 289 also observed a slight increase in spontaneous lysis in the K562-NL alone condition as time 290 progressed, shown by the increase in luminescence detection at the last two time points (Fig. 1F).

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Based on these results, we concluded that cells had to be seeded within 1-hour to maintain the 292 dynamic range between the experimental and control conditions.  295 To identify compounds that enhanced NK cell cytotoxicity, we employed the Prestwick Chemical 296 Library. K562-NL cells alone or NK92+K562-NL cells mixed at a E:T ratio of 1 were treated with 297 10 μM of each drug for 5 hours at 37˚C ( Fig. 2A). Each compound was evaluated in singlet over 298 2 biological replicates. To identify compounds that increased NK92 cytotoxicity, the luminescent 299 values of all wells containing drugs were compared to the DMSO control wells from the same 300 plate and this difference was quantified as fold-change over DMSO control (Fig. 2B) Alexidine dihydrochloride proved to be toxic for target cells even in absence of effectors, and was 308 therefore not further considered. 14 compounds from the total drugs identified had a fold-change 309 ≥ 1.3 on both screening days (Table 1) To evaluate the overall screening assay stability, Z' factor was calculated for each assay plate from 318 the screening of the Prestwick Chemical Library. The screening assay had an average Z' factor of 319 0.72 for K562-NL alone plates treated with drugs and 0.44 for the NK92 + K562-NL (E:T of 1) 320 plates treated with drugs. A Z' factor close to 0.5 is considered fair and Z' factor 0.5-1 is considered 321 16 good 26 . Z'-factor for each individual plate can be found in Supplementary Table 3. Z'-factor 322 analysis suggests that the overall quality of the drug screening was fair.  (Fig. 3A), whereas the other 7 drugs did not change, or even reduced, the ability of 334 NK cells to kill target cells ( Fig. 3B-H).

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NK92 cells were co-cultured with K562-NL cells at the indicated E:T ratios for 5-hrs at 37˚C.

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After incubation, the supernatant was collected, the indicated substrate added, and luminescence 563 read by Biotek Synergy microplate reader. A. Luciferase-release cytotoxicity assay using 564 transduced K562-NL cells (bulk population, unsorted). 20,000 K562-NL targets per well. After 565 the incubation, luciferase activity was measured using Promega Nano-glo luciferase assay system.

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Mean +/-SD of three technical replicates. B. After the incubation, luciferase activity was measured 567 after addition of either Promega Nano-glo luciferase assay system (FMZ) or CTZ substrate. 10,000