Abstract 5483: High-throughput transcriptomics dose response profiling: identification of functional molecular signatures reflecting efficacy and safety of mTOR inhibitors

Abstract Background and Purpose: Recent publications demonstrate the utility of the TempO-Seq® high-throughput transcriptomics (HTTr) assay in generating in vitro dose response data for 1751 compounds in MCF-1 cells [Harrill et al, Tox Sci 2024, 202(1): 103-122] and 1201 compounds in U-2OS cells [Bundy et al, Tox & Applied Pharmacol 491 (2024) 117073]. These studies identified biological pathway altering concentrations (BPACs) for functional molecular signature patterns by evaluating 11, 037 functional gene signatures for each compound and dose. We applied this same approach to identify novel differences in the modulation of molecular/functional signatures by mTOR rapalogs and ATP-competitive inhibitors. Methods: To cover a broad range of functional molecular signatures we profiled the selected compounds, at 8 concentrations each, in MCF-7, U-2OS, and HepaRG cells using the commercial TempO-Seq whole transcriptome v2.1 assay. We identified differentially expressed genes between treated and untreated cells using DESeq2, calculated BPACs as previously described [Harrill et. al. Tox Sci 2021, 18(1): 68-89], then identified which of the 11, 037 functional molecular signatures were modulated (as published by Harrill and Bundy). Results: Signature patterns with different BPACs were identified for each compound. Each cell line generated a set of compound modulated molecular signatures that overlapped with one or both of the other cell lines, as well as unique molecular signatures to that cell line. From these data, we identified a set of functional molecular signatures associated with the class of mTOR inhibitors. We also identified molecular signatures that differentiated the rapalogs, which bind to FKBP12 and the mTORC1 complex to inhibit its kinase activity, from the ATP-competitive inhibitors, which inhibit the kinase activity of both mTORC1 and mTORC2. Finally, we identified compound-specific molecular signatures. These data will be presented. Conclusions: The results demonstrate the utility of HTTr dose response compound profiling in differentiating compound activity using a database of 11, 037 functionally annotated molecular signatures. The TempO-Seq assay effectively distinguished these activities for each compound and provided data that reflected potential efficacy and safety. Each cell line contributed unique modulated signatures that were absent in others, offering a more complete view of the activity of each compound. These findings demonstrate the value to therapeutic target focused drug discovery programs of the TempO-Seq HTTr assay in generating functional mechanistic dose response data, and underscore the potential of HTTr TempO-Seq assays to differentiate chemical series not only based on compound structure, but also on functional dose response activity that may impact both efficacy and safety. Citation Format: Bruce Seligmann, Megan Opichka, Monica Hernandez, Zhoutao Chen, Joanne M. Yeakley, Salvatore Camiolo. High-throughput transcriptomics dose response profiling: identification of functional molecular signatures reflecting efficacy and safety of mTOR inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5483.