Abstract A05: ARF6 inhibition enhances T cell-mediated cytotoxicity in triple negative breast cancer


 Triple negative breast cancer (TNBC) has the poorest clinical outcome owing to the lack of effective treatments out of all the breast cancer subtypes. Immune checkpoint blockade (ICB) therapies have shown promise in these patients but only a small proportion of patients respond to ICB therapy partly due to immune evasion. A primary mechanism by which tumor cells evade immune surveillance is downregulation of antigen presentation by decreasing the levels of antigen-MHC-I (major histocompatibility complex-I) complexes on the cancer cell surface. However, there are no known targets that would boost antigen presentation in the tumor cells and increase T cell functionality simultaneously to increase the efficacy of ICB therapy. The small GTPase ARF6 has been implicated in the internalization of MHC-I molecules into the cell. Additionally, ARF6 also plays a role in CD8+ T cell activation by reorganization of the actin cytoskeleton and inhibits the formation of the immunological sypnase. We hypothesize that by inhibiting ARF6 in both tumor cells and CD8+ T cells, T cell-mediated cytotoxicity would be increased. In cancer cells, ARF6 inhibition will inhibit endocytosis of MHC-I complexes and enhance antigen presentation, while in CD8+ T cells, Arf6 inhibition will promote the formation of the immunological synapse. We tested the dose-dependent effect of an ARF6 inhibitor NAV-2729 on the GTPase activity of ARF6 in tumor cells as it specifically inhibits ARF6 by blocking its GTP-binding domain. We observed a dose-dependent decrease in ARF6-GTP levels with NAV-2729 treatment. To this end, we inhibited ARF6 via the small molecule inhibitor NAV-2729 or knockdown by shRNA in murine and human TNBC cells and found that antigen presentation is enhanced when MHC-I mean fluorescence intensity (MFI) was measured by flow cytometry. We also inhibited ARF6 in CD8+ T cells by NAV-2729 pretreatment, activated them in vitro and measured their functionality by flow cytometry. Interestingly, inhibitor treatment led to higher IFNg, TNFa and IL2 production. In a T cell cytotoxicity assay, we observed increased T cell cytotoxicity in the coculture of T cells and tumor cells when either the tumor cells or CD8+ T cells were treated with inhibitor. Collectively, we show here that ARF6 inhibition in tumor cells and CD8+ T cells will enhance tumor cell killing due to enhanced antigen presentation and T cell functionality respectively. These results give us more insight into the use of Arf6 inhibitors in combination with ICB therapy, thus enhancing treatment efficacy.
 Citation Format: Ishara Moulana, Xiongbin Lu. ARF6 inhibition enhances T cell-mediated cytotoxicity in triple negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A05.