Abstract 98: SETDB1 promotes tumorigenesis in endometrial cancer through suppressing anti-tumor immune response and increasing proliferation

Abstract

Abstract Background: An H3K9 methyltransferase and cancer driver gene, SETDB1, promotes tumorigenesis in many cancer types, such as melanoma, and breast cancer. SETDB1 is often overexpressed and correlates with worse survival. Multiple mechanisms have been reported for its oncogenesis from promoting tumorigenesis and regulating immune evasion. The roles of SETDB1 in driving endometrial cancer (EC) progression is not well-studied. Methods: EC cell lines were used for knockout SETDB1 using CRISPR-Cas9 system, RNA-seq was conducted to discover SETDB1 regulated genes. ChIP-qPCR was performed to identify SETDB1 direct targets. Immunocompromised NSG and immunocompetent C57BL/6 mice were utilized to investigate SETDB1 promoted tumor growth, immune evasion, and mice survival. IHC and flow cytometry was conducted to evaluate macrophage infiltration. Cytokines were screened by qPCR for potential macrophage chemoattractant. Results: In vitro, knockout SETDB1 decreased cell proliferation and enhanced G1/S cell cycle arrest. RNA-seq data revealed that in SETDB1−/− clones, many important oncogenes such as POLR2A, MUC5AC, and MKI67 were downregulated, while tumor suppressors such as PGR, RERG, and ZNF582 were upregulated. ChIP assay discovered that SETDB1 directly binds and represses ZNF582, ZNF266, ZNF841 expression via depositing H3K9me3 at promoter region. In vivo, SETDB1−/- significantly reduced tumor growth and prolonged NSG mice survival up to 100 days. SETDB1 promoted tumor growth via 1. Intrinsic: increasing mitotic figure, pH3-S10 and Ki67 level, 2. Extrinsic: blocking macrophage infiltration and chemokine secretion. Our in vitro functionality assay confirmed that bone-marrow derived M1-polarized macrophages promoted greater killing for SETDB1−/− cells. We surveyed 17 cytokines and found CCL5, CXCL9, and IL6 mRNA expression were enhanced in SETDB1−/− tumors suggesting active immune response. Consistent with the elevated CCL5 mRNA expression, CCL5 protein level was also upregulated in macrophages and cancer cells in SETDB1−/- tumors. To prove SETDB1 directly regulates CCL5 expression, we re-expressed SETDB1 in SETDB1−/− cells, and indeed, the SETDB1 re-expression did suppress CCL5 expression. Using immunocompetent C57BL/6 mice, we further confirmed that SETDB1 promotes tumor growth of MSH2-369 mice cells and blocks macrophage infiltration. To understand SETDB1 regulated pathways in EC patients’ samples, using EC-TCGA data, we discovered that high SETDB1 expression correlated with higher level of active transcription genes and reduced immune response genes. Our findings provide the mechanisms behind these observations. Conclusion: SETDB1 drives EC tumorigenesis by both intrinsically upregulating cell proliferation genes and promoting immune escape extrinsically. Our novel findings suggest SETDB1 is a promising anticancer target for EC patients. Citation Format: Kiarash Salari, Jiaqing Hao, Matthew Wells, Eleanor Johnson, Ryan Jilek, Jack Gilbert, Ryan Mclerran, David Meyerholz, Melinda Yates, Bing Li, Shujie Yang. SETDB1 promotes tumorigenesis in endometrial cancer through suppressing anti-tumor immune response and increasing proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 98.