Phenotypic small-molecule screening identifies JAK inhibitors as pharmacologic modulators of T cell exhaustion

Abstract Immune checkpoint blockade (ICB) therapy has emerged as viable first-line treatment for cancers such as melanoma. Despite the striking long-term clinical benefits that were previously unattainable, the effectiveness of ICB therapy remains limited in a majority of patients and cancer types. This is due in part to insufficient activation and/or restoration of anti-tumor T cell responses. Thus, understanding the signaling pathways that potentiate T cell exhaustion is essential for developing pharmacologic drugs that reactivate T cells and overcome the limitations of current ICB therapy. To this end, we developed a biologically relevant phenotypic screening system using the LCMV-CL13 infection model coupled with high-throughput flow cytometry to identify small-molecules that resurrect hypofunctional T cells. We discovered 19 hits following a screen of a manually curated collection of 12,000 repurposed drugs. Among the lead compounds identified were janus kinase (JAK) inhibitors including the anti-myelofibrosis drug ruxolitinib. Phenotypic rescue of exhausted T cells in vitro following ruxolitinib treatment resulted in a unique transcriptional signature that diverged from anti-PD-L1 treatment. Lineage-defining genes known to preserve precursor exhausted CD8+ T cells, including transcription factor 7 (Tcf7), were significantly upregulated following exposure to ruxolitinib. Mechanistically, ruxolitinib attenuated the cumulative pSTAT signaling signature within resurrected cells to enhance their survival. Collectively, these results demonstrate a disease-relevant framework for identifying small-molecule modulators of dysfunctional T cells and suggest that JAKs are viable targets for cancer immunotherapy.