Abstract C044: Analysis of 1000 secreted proteins in functional genomics and compound screens reveals cytotoxic and immunomodulatory targets

Abstract The identification of novel cancer immunotherapies and the characterization of their immunomodulatory effects is complicated by the plasticity of immune cells. However, the high cost of capturing this diversity at scale limits typical drug discovery efforts to simple readouts. We previously described the Nomic platform, a proteomics tool capable of quantifying 1000 proteins at high-throughput and low cost. Here, we leverage the Nomic platform in functional genomics and compound screens to characterize their immunomodulatory properties and assess potential toxicities.To compare genomic and chemical perturbations, we collected supernatants for secretome analysis from the CPJUMP1 dataset, in which U2OS osteosarcoma cells were perturbed with matched ORF, CRISPR KO, and compound libraries targeting 161 genes, and quantified 191 cytokines using a Nomic Flex panel. Despite conceptually opposite approaches of the ORF and CRISPR libraries, overexpression and KO of the same gene did not result in opposite phenotypes. Rather, we observed overexpression of kinases, especially those upstream of multiple signaling pathways, induced an inflammatory phenotype, though with distinct inflammatory mediators, consistent with established oncogene-induced inflammation. To further characterize on-target and off-target immunomodulatory effects of compounds, we collected supernatants from hepatocytes, cardiomyocytes, and microglia treated with 510 compounds at 3 concentrations. We used Nomic’s Omni 1000 to measure 1,000 proteins across 20,000 samples, generating 20 million data points. Each cell type presented unique secretory features in response to diverse sets of compounds; though, certain phenotypes were shared. Cytotoxic compounds resulted in leaked intracellular proteins, detectable in the supernatant. TLR agonists (resiquimod) induced the expression of cytokines, which were inhibited by anti-inflammatory compounds (corticosteroids). Clinically-relevant signs of immune modulation were captured: methylprednisolone treatment of hepatocytes resulted in paradoxical increases in SAA and IL-6, consistent with immunotoxic effects in patients receiving high dose systemic corticosteroids. We also captured both the cytotoxic and inflammatory effects of actinomycin D, consistent with the immune contribution to this compound’s anti-cancer properties. In the case of cycloheximide, we capture 3 distinct, dose dependent activities: low dose inhibition of protein synthesis, superinduction of IL-6 at intermediate doses, and high dose cytotoxicity. Finally, we demonstrated the capacity of secretome analysis to probe pathways regulating cytokine expression. For example, comparison of a set of 11 mTOR inhibitors identified on-target immune modulation and off target effects at increasing doses, enabling characterization of compound potency and specificity.Our results demonstrate the value of high-throughput proteomics to identify new immunomodulatory targets and compounds, evaluate potential toxicities, and characterize potency and specificity. Citation Format: Alyssa Rosenbloom, Nathaniel Robichaud`, Kiran Edwardson, Srinivas N. Chandrasekaran, Shantanu Singh, Amy R. Johnson, Jens Eberlein, Alistaire Sherman, Nicole Paul, Erroll Rueckert, Anne Carpenter, Eric Miller, Narges Rashidi, Milad Dagher. Analysis of 1000 secreted proteins in functional genomics and compound screens reveals cytotoxic and immunomodulatory targets abstract. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C044.