High-Throughput Screening and Proteomic Characterization of Compounds Targeting Myeloid-Derived Suppressor Cells

摘要

•(Phospho-)proteome profiling of differentiated myeloid-derived suppressor cells.•Phenotypic screen of 21,000 small molecules to identify novel MDSC modulators.•Reduction of reactive oxygen species identified as potential mode of action.•Chemical proteomics reveals Acyp1 and Cd74 as potential target proteins. Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population of incompletely differentiated immune cells. They are known to suppress T cell activity and are implicated in multiple chronic diseases, which make them an attractive cell population for drug discovery. Here, we characterized the baseline proteomes and phospho-proteomes of mouse MDSC differentiated from a progenitor cell line to a depth of 7000 proteins and phosphorylation sites. We also validated the cellular system for drug discovery by recapitulating and identifying known and novel molecular responses to the well-studied MDSC drugs entinostat and mocetinostat. We established a high-throughput drug screening platform using a MDSC/T cell coculture system and assessed the effects of ∼21,000 small molecule compounds on T cell proliferation and IFN-γ secretion to identify novel MDSC modulator. The most promising candidates were validated in a human MDSC system, and subsequent proteomic experiments showed significant upregulation of several proteins associated with the reduction of reactive oxygen species (ROS). Proteome-wide solvent-induced protein stability assays identified Acyp1 and Cd74 as potential targets, and the ROS-reducing drug phenotype was validated by measuring ROS levels in cells in response to compound, suggesting a potential mode of action. We anticipate that the data and chemical tools developed in this study will be valuable for further research on MDSC and related drug discovery. Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population of incompletely differentiated immune cells. They are known to suppress T cell activity and are implicated in multiple chronic diseases, which make them an attractive cell population for drug discovery. Here, we characterized the baseline proteomes and phospho-proteomes of mouse MDSC differentiated from a progenitor cell line to a depth of 7000 proteins and phosphorylation sites. We also validated the cellular system for drug discovery by recapitulating and identifying known and novel molecular responses to the well-studied MDSC drugs entinostat and mocetinostat. We established a high-throughput drug screening platform using a MDSC/T cell coculture system and assessed the effects of ∼21,000 small molecule compounds on T cell proliferation and IFN-γ secretion to identify novel MDSC modulator. The most promising candidates were validated in a human MDSC system, and subsequent proteomic experiments showed significant upregulation of several proteins associated with the reduction of reactive oxygen species (ROS). Proteome-wide solvent-induced protein stability assays identified Acyp1 and Cd74 as potential targets, and the ROS-reducing drug phenotype was validated by measuring ROS levels in cells in response to compound, suggesting a potential mode of action. We anticipate that the data and chemical tools developed in this study will be valuable for further research on MDSC and related drug discovery. Treatment with immune checkpoint inhibitors, targeting cytotoxic T lymphocyte–associated protein 4 (CTLA4), programmed cell death protein-1 (PD-1) or its ligand (PD-L1) has provided substantial benefits to some patients with different tumor entities. This indicates that successful clinical responses can be obtained by blocking inhibitory mechanisms which suppress T cell functions in the tumor niche (1Hodi F.S. O’Day S.J. McDermott D.F. Weber R.W. Sosman J.A. Haanen J.B. et al.Improved survival with ipilimumab in patients with metastatic melanoma.N. Engl. J. Med. 2010; 363: 711-723Google Scholar, 2Brahmer J.R. Tykodi S.S. Chow L.Q. Hwu W.J. Topalian S.L. Hwu P. et al.Safety and activity of