Tumor-Specific Epha2 Receptor Tyrosine Kinase Inhibits Antitumor Immunity By Recruiting Suppressive Myeloid Populations In Non-Small Cell Lung Cancer

Background: Given the success of both targeted and immunotherapies against cancer, there is increasing utility for identifying targeted agents that also promote anti-tumor immunity. EphA2 is a receptor tyrosine kinase that contributes to tumor growth and metastasis in various cancer types and has been identified as a viable target for non-small cell lung cancer (NSCLC). Here, we examine how tumor-specific EphA2 affects the infiltration of different immune populations, the activation or inhibition of those populations, and the cytokine and chemokine milieu in mouse models of NSCLC. Methods: We generated two murine NSCLC cell lines, one derived from Lewis Lung Carcinoma and the other derived from a primary lung tumor harboring a KRAS mutation, as well as p53 and LKB1 loss of function alterations created using CRISPR-Cas9 gene editing. Effects of EphA2 overexpression in these cells were evaluated in both in vitro assays and in vivo models via subcutaneous tumor implantation and tail vein-injected orthotopic tumor formation in immunocompetent, syngeneic mice. Tumor immune infiltrate was assessed by flow cytometry, and cytokine and chemokine RNA expression levels were evaluated using Nanostring’s PanCancer Immune Profiling Panel. Results: Although EphA2 overexpression in these cell lines did not display significant proliferative advantage in vitro in MTT and colony formation assays, it did confer a growth advantage in vivo. Analysis of lung tumor immune infiltrate revealed decreased NK and T cells in the EphA2-overexpressing tumors, as well as increased myeloid populations, such as macrophages and monocytes. Furthermore, T cell activation markers, such as CD25, CD44, and CD69, in both CD4 and CD8 T cells were decreased in the EphA2-overexpressing tumors, while the percentage of regulatory T cells was increased. These changes in T cell activity were accompanied by increased presence of monocyte-derived CD11b+ Ly6C+ Ly6G- cells and Gr1- F4/80+ MHCII+ tumor-associated macrophages (TAMs). Higher infiltration of these myeloid populations coincided with increased monocyte-attracting chemokine and receptor expression, including CCL2, CCL7, CCL8, CCL12, and CCR2 and increased expression of immunosuppressive proteins, including TGFβ and arginase 1. Conclusion: Our studies suggest tumor-specific EphA2 inhibits infiltration and activation of key lymphocytic populations, especially T cells, while recruiting monocytes and likely promoting their transformation into myeloid-derived suppressor cells (MDSCs) and TAMs. Further functional studies are needed to validate the role of these myeloid cells. Elucidation of EphA2’s role in regulating immune recruitment and function will advance our understanding of tumor immune evasion and the potentially beneficial consequences of targeting EphA2 in cancer on the tumor microenvironment. Citation Format: Eileen Shiuan, Wenqiang Song, Shan Wang, Mark Boothby, Jin Chen. Tumor-specific EphA2 receptor tyrosine kinase inhibits anti-tumor immunity by recruiting suppressive myeloid populations in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1174.