Abstract 2998: Inhibition of a small GTPase in tumor cells enhances antigen presentation and tumor cell killing in breast cancer

Abstract

Abstract Breast cancer is the leading cause of cancer in women and the second leading cause of death in female patients. Immune checkpoint blockade (ICB) therapies have shown promise in treating breast cancer patients but only a small proportion of patients respond to ICB therapy partly due to lack of immune cell infiltration into the tumor. Reduced immune infiltration is a result of downregulation of antigen presentation in the tumor cell by decreasing the levels of antigen-MHC-I (major histocompatibility complex-I) molecules on the tumor cell surface. To investigate pathways upregulated in breast cancer, we performed a paradigm tool analysis (Broad Institute) of RNA expression data using TCGA breast invasive carcinoma datasets and found that signaling events of a small GTPase (SGX) ranked 8th among upregulated pathways. SGX has been implicated in the internalization of MHC-I molecules into the cell. To investigate the effects of SGX on antigen presentation, we generated SGX knockdown EO771 and 4T1 murine and MDA-MB-231 human breast tumor cell lines and measured their antigen presentation by flow cytometry. We observed a significant increase in MHC-I levels upon SGX knockdown. We then performed an antibody internalization assay on SGX knockdown cells where fluorophore-labeled MHC-I antibodies were allowed to internalize for 10 min followed by early endosome isolation. There was a significant reduction of MHC-I antibody-containing early endosomes in SGX knockdown cells. Using a commercially available SGX inhibitor, we tested the pharmacological inhibition of SGX and observed a dose-dependent increase in MHC-I MFI. If a SGX inhibitor was to be administered in vivo, we would need to test the impact of inhibitor treatment on CD8+ T cells. To this end, we isolated CD8+ T cells from a C57BL/6 mouse and pretreated them with the inhibitor, activated them in vitro and measured their functionality. Interestingly, inhibitor treatment led to higher IFNg, TNFa and IL-2 production. To explore the possibility of dual SGX inhibition in tumor and T cells, we performed a T cell cytotoxicity assay using EO771 tumor cells and autologous CD8+ T cells and observed increased tumor cell killing when either the tumor cells or T cells were treated with inhibitor. Collectively, we show here that dual SGX 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 SGX inhibitors in combination with ICB, thus enhancing treatment efficacy. Citation Format: Ishara Moulana, Samantha Sharma, Xinna Zhang, Xiongbin Lu. Inhibition of a small GTPase in tumor cells enhances antigen presentation and tumor cell killing in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2998.