Abstract 1529: STAT3 mediates epigenetic regulation of MDSCs in tumors

Abstract

Abstract This project aims to test the hypothesis that the epigenetic modulatory drug (EMD), entinostat, is capable of inhibiting the immunosuppressive capabilities of MDSCs by altering STAT3 activity. Our previous work has shown that the HDACi entinostat is capable of sensitizing the inflammatory environment in both pancreatic ductal adenocarcinoma (PDAC) and HER2+ breast cancer mouse models to the immune checkpoint inhibitors anti-PD1 and anti-CTLA4 by inhibiting the immunosuppressive function of intratumoral MDSCs. These MDSCs not only produced significantly less arginase-1 protein, but also demonstrated reduced suppression of T cell proliferation when co-cultured with activated CD8+ T cells. The MDSC dysfunction induced by these combinatorial therapies resulted in significantly improved survival in both models as well as significantly improved infiltration of cytotoxic effector CD8+ T cells. Western blot analysis of isolated intratumoral G-MDSCs from treated animals showed a decrease in levels of phospho-STAT3 (pSTAT3), an important regulator of MDSC immunosuppressive genes. We hypothesize that the reduced levels of pSTAT3 alters the promoter binding of STAT3, thus preventing the translation of genes key to the immunosuppressive activity of MDSCs. To test this hypothesis, we are conducting mechanistic studies in ex vivo and in vitro MDSC models. Our ex vivo system involves isolating intratumoral MDSCs and subsequently culturing the MDSCs using tumor conditioned media. Our in vitro model uses the previously described “MDSC-like” J774M cell line, which was derived by sorting CD11b+ Gr1+ cells from the ATCC cell line J774A. We have shown that this cell line expresses the surface markers Ly6G and Ly6C in a similar manner to what is observed in intratumoral MDSCs. We have also performed immunosuppression assays and determined this cell line is capable of inhibiting T cell proliferation in a dose dependent manner. In addition to establishing and characterizing these models, our work has demonstrated that entinostat treatment results in a significant decrease in the production of functional arginase-1 protein in both ex vivo G-MDSCs and J774M cells. Western blot analysis of the J774M cells showed that this decrease in arginase-1 production correlated with a decrease in pSTAT3. Our current studies aim to confirm a decrease in pSTAT3 in ex vivo MDSCs as well as determine the effect of entinostat on the ability of J774M cells to suppress T cell proliferation. Additionally, we are performing ChIP-Seq on entinostat treated and untreated ex vivo MDSCs and J774M cells to determine how entinostat alters the binding of STAT3 to the promoters of key immunosuppressive genes. These results will not only help elucidate the molecular mechanism of action involved in entinostat’s reprogramming of MDSCs, but it will also help identify new, more specific targets to manipulate MDSC function, and improve efficacy of checkpoint inhibition in non-immunogenic cancers. Citation Format: Brian J. Christmas, Christine I. Rafie, Elizabeth M. Jaffee, Evanthia T. Roussos Torres. STAT3 mediates epigenetic regulation of MDSCs in tumors [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 1529.