key: cord-0261306-wq6g5box authors: Salehi, Pezhman; Zallocchi, Marisa; Vijayakumar, Sarath; Urbanek, Madeleine; Giffen, Kimberlee P.; Li, Yuju; Hati, Santanu; Zuo, Jian title: In silico transcriptomics identifies FDA-approved drugs and biological pathways for protection against cisplatin-induced hearing loss date: 2022-01-28 journal: bioRxiv DOI: 10.1101/2022.01.26.477836 sha: 2c0bdc8cd95b43f781e3fab86cb3ad44a42177a0 doc_id: 261306 cord_uid: wq6g5box Acquired hearing loss is a major health problem that affects 5-10% of the world population. However, there are no FDA-approved drugs for the treatment or prevention of hearing loss. Employing the Connectivity Map (CMap) that contains >54,000 compounds, we performed an unbiased in silico screen using the transcriptomic profiles of cisplatin-resistant and -sensitive cancer cell lines. Pathway enrichment analysis identified gene-drug targets for which 30 candidate drugs were selected with potential to confer protection against cisplatin-induced ototoxicity. In parallel, transcriptomic analysis of a cisplatin-treated cochlear-derived cell line identified common enriched pathway targets. We subsequently tested these top 30 candidate compounds, 15 (50%) of which are FDA-approved for other indications, and 26 (87%) of which were validated for their protective effects in either a cochlear-derived cell line or zebrafish lateral line neuromasts, thus confirming our in silico transcriptomic approach. Among these top compounds, niclosamide, a salicyanilide drug approved by the FDA for treating tapeworm infections for decades, protected from cisplatin- and noise-induced hearing loss in mice. Finally, niclosamide and ezetimibe (an Nrf2 agonist) exerted synergistic protection against cisplatin-ototoxicity in zebrafish, validating the Nrf2 pathway as part of niclosamide’s mechanism of action. Taken together, employing the CMap, we identified multiple pathways and drugs against cisplatin ototoxicity and confirmed that niclosamide can effectively be repurposed as an otoprotectant for future clinical trials against cisplatin- and noise-induced hearing loss. Significant Statement Employing the Connectivity Map as our in silico transcriptomic screening strategy we identified FDA-approved drugs and biological pathways for protection against cisplatin-induced hearing loss. Abstract: 23 Acquired hearing loss is a major health problem that affects 5-10% of the world 24 population. However, there are no FDA-approved drugs for the treatment or prevention been treated with pharmacological agents or genetic manipulations (e.g., CRISPR 67 genomic editing). We reasoned that transcriptomic profiles favoring cisplatin resistance 68 in the cancer cell lines should link to many drugs in the broad chemical space that are 69 likely to induce transcriptional profiles that mimic the cisplatin-resistant phenotype, thus 70 identifying drugs that may have novel therapeutic use for the treatment of cisplatin toxicity 71 within the cochlea, as long as these repurposed drugs do not interfere with cisplatin's 72 cancer killing ability. In addition to drug identification, these transcriptomic in silico 73 screens explore diverse biological pathways associated with cisplatin resistance in an 74 unbiased manner. There are several successful studies using the CMap, including a 75 recent study in the hearing field focusing on heat shock protein activators to treat 76 aminoglycoside ototoxicity (9) and others repurposing existing drugs for SARS-CoV2 77 treatment (9, 15, 16) . 78 In this study, we utilized transcriptomic profiles of cisplatin-resistant cancer cell lines to 88 Furthermore, we demonstrated that niclosamide also had protective effects against noise- Given that HCs in the inner ear are highly sensitive to cisplatin toxicity (1-8), our in silico 101 approach aimed to identify small molecules capable of inducing a transcriptomic profile 102 that could confer resistance to CIHL. For this purpose, we used publicly available RNA-103 seq datasets from the GEO and identified nine RNA-seq studies investigating cisplatin 104 resistance in several cancer cell lines. In each of these studies, the transcriptomic profiles 105 of sensitive parental cell lines were compared to those of resistant counterparts ( Figure 106 1-table supplement 1). These nine studies were analyzed using the NCBI's GEO2R tool 107 (https://www.ncbi.nlm.nih.gov/geo/geo2r/) from which we obtained differentially 108 expressed gene (DEG) lists. To identify compounds that will mimic the cisplatin-resistant 109 transcriptomic profiles, we subsequently uploaded the DEGs from each study into the 110 LINCS and GDA databases. Combined, these two CMap databases contain more than 111 50,000 compounds with their corresponding transcript perturbation profiles from various 112 cancer cell lines. Our CMap database search identified more than 500 unique small 113 molecules associated with the cisplatin-resistant phenotype. Figure 1 (blue-shaded box) 114 summarizes our transcriptomic-based in silico approach. In parallel to our drug screening approach, we also aimed to identify enriched signaling 116 pathways associated with cisplatin resistance in the nine RNA-seq datasets. Each DEG drug-induced genetic perturbations to identify drugs and pathways to protect from CIHL. 159 neuromasts in vivo 160 To provide direct experimental evidence for our transcriptomic in silico screening, we 161 used HEI-OC1 cells to validate our top 30 identified candidate drugs in an assay similar 162 to our previous drug screening (5). Caspase activity was measured using Caspase-Glo The ABR wave-1 amplitude represents the summed activity of the cochlear nerve, and 214 therefore, an informative measure of auditory synapse function. We measured mean These ABR wave-1 amplitude results provide further evidence of niclosamide's 223 otoprotection in vivo. 224 We further quantified the number of outer HCs (OHCs) at the mid-basal region, the most 225 protected frequency region shown by ABR threshold and wave I amplitude 226 measurements. Representative images of cochlear HCs are displayed in Figure 4C . 227 Quantitative data for HC count at the mid-basal region are displayed in Figure 4D . The 228 one-way ANOVA revealed a significant group effect (P <0.05). The post-hoc test revealed 229 that while inner HC survival was not affected, the cisplatin-niclosamide group had more 230 OHC survival than the cisplatin alone group in the mid-basal region ( Figure 4D ). These 231 ABR and HC count data together, confirmed that niclosamide protects OHCs against 232 cisplatin damage. Niclosamide protects NMDA-induced HC loss in zebrafish in vivo 234 Since CIHL and NIHL share mechanistic commonalities (23, 24), we examined whether 235 niclosamide had any protective effects in a zebrafish model for HC excitotoxicity (25). As 236 previously described, neuromast HC numbers were reduced after exposure to 300 μM 237 NMDA (22). Conversely, post-treatment of the zebrafish exposed to 300 µM NMDA with 242 We further investigated niclosamide's therapeutic effects against NIHL in FVB/NJ mice. 243 We first injected the mice with 10 mg/kg niclosamide via IP once per day for four 244 consecutive days, starting one day before noise exposure, the day of the noise exposure, 245 and two more days after noise exposure. Control animals received vehicle injections on 246 the same schedule. Noise exposure was administered at 8-16 kHz at 100 dB SPL for 2 247 hrs. Noise-induced ABR threshold shifts were obtained by subtraction of the pre-exposure 248 from the post-exposure thresholds. Two-way ANOVA followed by Sidak's multiple 249 comparison test revealed that the niclosamide-noise exposed group had lower threshold 250 shifts than noise-exposed group across most of the tested frequencies (16 kHz, 32 kHz 251 and 63 kHz) at day 14 ( Figure 5B and Figure 4-figure supplement 1) . These results 252 demonstrate that niclosamide also protects against NIHL in mice and suggest that its 253 action is independent of cisplatin inactivation. To determine whether niclosamide prevents NIHL by protecting OHCs, we measured the 255 DPOAE amplitudes at the different f2 frequencies with L2 levels ranging from 10 to 70 dB 256 SPL ( Figure 5C ). In the noise-niclosamide group, DPOAE amplitudes were not 257 significantly higher than the noise-saline group at day 15 post-noise exposure. A two-258 factor ANOVA (group x frequency) was used to compare pre-exposure amplitudes to day 259 15 amplitudes. The ANOVA revealed no significant two-way group x frequency interaction 260 indicating that the OHC function is similar between all groups and suggesting that 261 niclosamide's protective effect against noise could be due to prevention of synaptopathy 262 between inner HCs and cochlear nerves. To test our hypothesis, mean ABR wave-I 263 amplitudes at 8, 16, 32, and 40 kHz were measured at day 15 post-noise exposure. Amplitudes from the 10 to 90 dB SPL stimulus intensity were compared between groups 265 in the pre-noise test using a two-factor ANOVA (group x stimulus level), and no group 266 differences were detected (data not shown). At day 15, only the 50-90 dB SPL stimulus 267 levels were used because many of the subjects had no responses below 50 dB SPL. Results from these experiments showed that the wave I amplitudes from the niclosamide-269 noise group were increased at all the noise stimulus tested, with 80 and 90 dB SPL noise group had higher amplitudes at 80 and 90 dB SPL compared to the noise-exposed 273 group ( Figure 5D ). The ABR wave-I amplitude results showed that cochlear nerve activity 274 in the noise-niclosamide group was comparable to the aged-matched controls, with no 275 statistically significant difference between these groups, thus providing evidence of 276 niclosamide's protection from synaptopathy. To assess the protection of the ribbon synapses, the cochlear samples were 278 immunostained with CtBP2. Representative images of the mouse ribbon synapses at 16 279 kHz are displayed in Figure 5E . Quantitative data for ribbon synapses at 16 kHz are 280 displayed in Figure 5F . T-test statistical analyses revealed that the niclosamide-noise In summary, our work highlights that 1) by using the CMap it is possible to identify hearing loss, which affect much larger populations than CIHL, the CMap can therefore be 367 fruitful in these important unmet health arenas. 368 Although CMap has been a popular resource for data-driven drug repositioning using a HEI-OC1 cells were exposed to cisplatin and various concentrations of the corresponding compounds. Caspase-3/7 activity was measured and plotted as a function of log10 compound concentration (µM). Caspase activity for all the treatments was normalized to cells treated only with cisplatin. Whenever possible, ICsos were calculated using GraphPad Prism software. Mean± standard error (n=3 per group). 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The-Scientist Malaviya AN. Landmark papers on the discovery of methotrexate for the treatment 756 of rheumatoid arthritis and other systemic inflammatory rheumatic diseases: a 757 fascinating story A phase I study of niclosamide in 759 combination with enzalutamide in men with castration-resistant prostate cancer Phase II trial to 762 investigate the safety and efficacy of orally applied niclosamide in patients with 763 metachronous or sychronous metastases of a colorectal cancer progressing after 764 therapy: the NIKOLO trial Lovastatin protects against cisplatin-induced hearing loss in 767 mice 3D model of 769 frequency representation in the cochlear nucleus of the CBA/J mouse 3D model of 772 frequency representation in the cochlear nucleus of the CBA/J mouse Age-related cochlear 775 synaptopathy: an early-onset contributor to auditory functional decline Characterization of 778 quinoxaline derivatives for protection against iatrogenically induced hearing loss. 779 JCI Insights Italy for their initial assistance in the use of the GDA 788 database ONR-N00014-18-1-2507, and LB692/Creighton to JZ, by DoD-790 RH190050 and the Bellucci Foundation Award to MZ, by NIH-R43 DC018762 to PS (and 791 subcontract to JZ), and by NIH1P20GM139762 782 We thank Emma Malloy and Tal Teitz for their help on cancer cell lines, Zhuo Li and Kan 783 Lin for cell culture, Molly Kubesh for pathway analysis, and Mrs. Xianghong Liu for 793 JZ, PS, SV, and MZ are inventors on a provisional/PCT patent application filed for the 794 use of niclosamide in hearing protection. JZ is the co-founder of Ting Therapeutics LLC.