Abstract A011: Targeting the FES tyrosine kinase in antigen presenting cells to enhance anti-tumor cytotoxic lymphocyte responses
Authors: Dmytryk Natasha , Laight Brian , Greer Peter 
Published: December 2023

Abstract
For cytotoxic lymphocytes (CTLs) to effectively kill cancer cells, inflammatory signals from antigen presenting cells (APCs) are required. Targeting the signaling pathways that regulate production of these inflammatory cytokines represents an exciting strategy to bolster CTL activation in the context of cancer immunotherapy. Achieving optimal levels of CTL activation is critical to the success of immunotherapy treatments. Compelling advancements in CTL therapies including CAR-T cells and immune checkpoint inhibitors are showing great promise for improving patient outcomes. However, enhancing the innate immune system’s ability to strengthen anti-tumor CTL immune responses has been explored to a lesser extent. We intend to fill this gap by studying the APC-intrinsic role of the non-receptor tyrosine kinase FES in regulating the production of inflammatory cytokines. The first evidence of FES’s potential immune regulating role came from observations of increased lipopolysaccharide (LPS) sensitivity in fes-null mice. A transgenic mouse model of breast cancer expressing activated HER2/Neu in the mammary glands showed delayed tumor onset in mice targeted with a fes mutation that catalytically inactivated FES; and this delay correlated with increased immune infiltration and inflammation in pre-malignant mammary tissue. Previous studies have implicated FES in the activation of the SHP-2 phosphatase in macrophages, which can suppress toll-like receptor (TLR) pathways. TLR pathways govern strong innate inflammatory responses, including the expression of the so-called signal 3 cytokines required for full CTL activation (e.g. IL-12, IFNα/β). We therefore hypothesized that disrupting FES in APCs will relieve suppression of inflammatory cytokine production pathways, leading to increased CTL activation and cancer cell cytotoxicity. To investigate the impact of FES disruption on inflammatory signaling cascades, immunoblotting analysis of proteins in these signaling pathways was conducted on LPS-stimulated fes−/− and WT mouse bone marrow derived macrophages or dendritic cells. qRT-PCR was used to measure inflammatory cytokine transcript levels, including IFNα/β, IL-12, IL-1β and TNFα. To compare the ability of WT and fes−/− macrophages to present antigen and activate CTLs, OT-1 CTLs were co-cultured with OVA-presenting WT or fes−/− macrophages; and then evaluated by flow cytometry for IFNγ production. Evidence of increased activation of downstream mediators of TLR signaling including NFκB and TBK1 were seen in fes−/− macrophages and dendritic cells post LPS stimulation. fes−/− macrophages displayed increased inflammatory cytokine mRNA production in response to LPS stimulation. Finally, CTLs showed increased IFNγ expression when co-cultured with LPS-stimulated fes−/− macrophages compared to WT macrophages, indicating a higher degree of activation by fes−/− macrophages. The immunosuppressive function of FES makes it an actionable target for improving the efficacy of cancer immunotherapies and anti-cancer adaptive immune responses.
Citation Format: Natasha Dmytryk, Brian Laight, Peter Greer. Targeting the FES tyrosine kinase in antigen presenting cells to enhance anti-tumor cytotoxic lymphocyte responses [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A011.