Abstract 6132: The Fes kinase plays an innate immune checkpoint role to suppress CD8+ T cell mediated anti-tumor immunity

Abstract Introduction: Immunotherapy is a powerful pillar of cancer therapy that still faces barriers due to cancer-mediated immunosuppression. Immunogenic cell death recruits and activates antigen-presenting cells (APCs) through engagement of pattern recognition receptors (PRRs), leading to pro-inflammatory Signal 3 cytokine production required for full activation of adaptive immune cells, including cytotoxic T lymphocytes (CTLs). The Fes tyrosine kinase suppresses innate immune responses in APCs by inhibiting PRR signaling and regulating cytokine trafficking. In non-cancer contexts, Fes may guard against consequences of overactive innate immunity, such as endotoxic shock or autoimmunity. However, this inhibitory effect on APCs may serve as a checkpoint to suppress successful anti-cancer immunotherapy by obstructing efficient priming of cancer specific CTLs. We hypothesize that Fes inhibition in APCs will improve Signal 3 cytokine signaling, resulting in greater adaptive immunity and tumor control. Methodology: Using bone marrow derived macrophages (BMDMs) from wildtype (WT) or Fes knockout (fes-/-) mice, we assessed LPS-induced PRR signalling by immunoblot analysis, production of key Signal 3 cytokines by multiplex ELISA, and the level of cell-associated cytokines by flow cytometry. The ability of BMDMs to prime CTLs was assessed in antigen cross-presentation co-cultures with OT-1 T cells. Using E0771 and B16F10 syngeneic orthotopic mouse engraftment models, we compared the efficacy of doxorubicin and anti-PD-1 to control tumor growth and prolong survival in WT and fes-/- mice. Tumors and spleen immune profiles were analyzed by flow cytometry. WT or fes-/- BMDMs were adoptively transferred into E0771 and B16F10 tumours to directly assess their relative ability to control tumour growth. Results: fes-/- BMDMs displayed improved PRR signaling, increased IFNβ, IL-12, and TNFα secretion, and greater levels of internal and cell-surface IL-12 following LPS stimulation. In antigen cross-presentation assays, LPS stimulated fes-/- BMDMs had improved CTL priming. Tumor control and survival were greater in fes-/- mice in both the E0771 and B16F10 engraftment models. Doxorubicin and the combination of doxorubicin and anti-PD-1 were more effective in fes-/-. This was especially striking in the B16F10 model, which is known to be resistant to PD-1 therapy. Immune profiling of tumors showed increased CTL and NK cell activation and PD-1 positivity, and a shift from predominately M2- to M1-polarized tumor associated macrophages in fes-/- mice. Intratumorally injected M1-polarized fes-/- BMDMs were better able to control tumor growth in both E0771 and B16F10 models. Conclusion: Fes acts as an innate immune checkpoint whose inhibition may enhance anti-cancer immunotherapy. We believe this effect is due to an increase in inflammatory signalling, and improved signal 3 cytokine signalling in fes-/- APCs. Citation Format: Brian J. Laight, Danielle Harper, Natasha Dmytryk, Connie S. Zhang, Changnian Shi, Sameh Basta, Madhuri Koti, Peter A. Greer. The Fes kinase plays an innate immune checkpoint role to suppress CD8+ T cell mediated anti-tumor immunity [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 6132.