Ezh2-Y641f Mutation Interacts With Stat3 To Regulate The Melanoma Tumor Immune Response Via Disruption Of Ifn Signaling.

Abstract Cancer genetic sequencing studies have identified systematic alterations of epigenetic and chromatin modifiers in most cancers, consistent with the fact that chromatin modifiers, such as the Polycomb Repressive Complex 2 (PRC2), regulate the expression of tumor suppressors. In melanoma, 10% of patients exhibit amplifications or point mutations in EZH2, the enzymatic subunit of PRC2; however, the oncogenic mechanisms of EZH2 mutations in melanoma remain elusive. Previously, we observed that expression of Ezh2-Y641F in melanoma cells resulted in upregulation of IFN-related genes. We thus sought to better understand this mutation and the consequences of IFN signaling changes on tumor growth and the tumor immune response. We discovered that Ezh2-Y641F directly interacts with and methylates Stat3, a known regulator of interferon signaling. To understand the effects of this interaction, we performed ChIP-seq for Ezh2 and Stat3 in melanoma cells and found that the binding profiles of Stat3 and Ezh2 are greatly changed in Ezh2-Y641F cells. Furthermore, Ezh2 and Stat3 binding sites co-localized at several genomic loci in Ezh2-Y641F cells, including a region that regulates Ezh2. Knockdown of Stat3 in melanoma cells decreased Ezh2 expression and partially reduced expression of the IFN-related genes that were initially upregulated by Ezh2-Y641F. Analysis of the tumor-infiltrating immune cells by flow cytometry revealed increased CD8+ T cells and decreased myeloid cells in Ezh2-Y641F melanomas, consistent with a more favorable immune response, although tumor growth was unchanged. Genetic inhibition of Stat3 expression via shRNA rescued this phenotype. Our data suggest a novel interaction between the PRC2 complex and Stat3 in melanoma, which has significant physiological consequences on the tumor immune response. Molecular and genomic analysis suggest that Ezh2 and Stat3 together regulate gene expression, including Ezh2 itself. Overall, these data highlight the significant role of chromatin regulators on the tumor immune response. Citation Format: Sarah M. Zimmerman, Samantha Nixon, George P. Souroullas. Ezh2-Y641F mutation interacts with Stat3 to regulate the melanoma tumor immune response via disruption of IFN signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2132.