Wednesday, January 15, 2025
1/15/2025
ABSTRACT Durable tumor regression by immune checkpoint blockade (ICB) has been observed in a small cohort of cancer patients, but ICB efficacy varies widely on an individual basis. ICB largely targets cytotoxic CD8+ T cells, but the mechanisms regulating CD8+ T cell responses to ICB are yet to be elucidated. CX3CR1+ CD8+ T cells remain highly cytotoxic and proliferative within the tumor microenvironment and single-cell RNA-sequencing of CX3CR1+ CD8+ T cells from ICB responders exhibited a consistent decrease in expression of Dual Specificity Phosphatase 2 (DUSP2), a nuclear phosphatase, compared to CX3CR1- CD8+ T cells. Peripheral human CD8+ T cells with DUPS2 knockdown (KD) increased tumor cytotoxicity in vitro, whereas overexpression (OE) trended towards decreased secretion of effector molecules, Granzyme B and Interferon gamma. Recent reports have additionally documented increased in vivo anti-tumor capacity in DUSP2 KO mice and have loosely made a connection between DUSP2 and CX3CR1 expression6, but the role of DUSP2 in CD8+ T cells and in ICB responses specifically, is a context not yet explored. Furthermore, the signaling pathways in human primary cells have yet to be revealed. Our preliminary data initiated a goal to uncover DUSP2’s mechanistic role in ICB response in hopes of pinpointing therapeutic targets that may improve ICB response rates. This proposal will investigate the hypothesis that DUSP2 expression in CD8+ T cells negatively regulates effector function in response to ICB. The proposed specific aims of this proposal are to 1) characterize the impact of DUSP2 expression in CD8+ T cells on ICB response and 2) determine the regulatory mechanisms of DUSP2 transcription in CD8+ T cells and the downstream pathways modulated by DUSP2. To determine the impact of DUSP2 on ICB response, tumor size and kinetics, as well as CD8+ T cell phenotype will be analyzed in CD8+ T cell Dusp2 conditional knockout (cKO) mice receiving tumor and ICB injection. The DUSP2 signaling network will be uncovered by promoter pull-down assay, single-cell profiling, and RNA-sequencing analyses of human CD8+ T cells with DUSP2 KD or OE. Identified pathways will be modulated (blocked or activated) in human CD8+ T cells by in vitro administration of pathway inhibitors and agonists. DUSP2 expression will be subsequently measured by quantitative reverse transcription PCR, western blot, and flow cytometry and CD8+ T cells will be phenotyped by flow cytometry and included in tumor co-culture cytotoxicity assays. The proposed in vivo experiments will expose DUSP2’s role in regulating ICB response. Uncovering the DUSP2 interactome in human CD8+ T cells can provide a novel and translationally significant opportunity to improve ICB response in oncology patients with limited therapeutic alternatives.
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