Abstract 301: CENP-E inhibitors activate cGAS-STING pathway by inducing chromosome misalignment and micronucleation after mitotic slippage

Abstract

Abstract Background: cGAS-STING, which monitors cytosolic DNA and triggers innate immune response, has recently attracted attention as a molecular target to sensitize cancer immune therapies. Since DNAs enclosed in micronucleus frequently leak into cytoplasm, micronucleus generated by centromere-associated protein-E (CENP-E) inhibition is expected to activate cGAS-STING pathways. We validated that small-molecule CENP-E inhibitors profoundly generate micronucleus through misaligned chromosomes. The aim of this study is to elucidate the possibility that micronucleus formation by CENP-E inhibitors activates the cGAS-STING pathway in cancer cells and induces immunological conversion of the tumor microenvironment from cold to hot. Material and Methods: GSK923295 and Cmpd-A were used as CENP-E inhibitors. HeLa and A549 cells were treated with the CENP-E inhibitors at the indicated concentrations. RNA sequencing data were subjected to gene set enrichment analysis (GSEA) to determine the signaling pathways modulated by Cmpd-A treatment compared to DMSO (control). Reporter assays for IRF and NF-kB were performed in A549 dual-reporter cells. High-throughput screening on IRF3 reporter activity was performed in A549 dual reporter cells in combination with -1,400 kinase inhibitor library and GSK923295. Results: The CENP-E inhibitors, GSK-923295 and Cmpd-A, induced chromosome misalignment, resulting in micronucleus formation after mitotic slippage. Phosphorylations of TBK1 and IRF3, cGAS-STING pathway markers, were remarkably upregulated in Cmpd-A-treated cells concordantly with micronucleus formation. The reporter assays also demonstrated that both IRF and NF-kB pathways were significantly activated in Cmpd-A-treated cells in a time-dependent manner. Transcriptome analysis of GSEA demonstrated that the gene ontologies (GO) for innate immune pathways, such as cytosolic DNA sensing pathway and cytokine-cytokine receptor interaction pathway, were significantly enriched in Cmpd-A-treated cells. The kinase library screening revealed that a series of inhibitors targeting PI3K-AKT-mTOR signaling pathways intensively suppressed CENP-E inhibitor-induced IRF activation, which occupied ~40% of hit compounds. Conclusions: The CENP-E inhibitors generated micronucleus activating the cGAS-STING pathway in multiple cancer cell lines. PI3K-AKT-mTOR signaling pathway may play important roles in the CENP-E inhibitor-induced cGAS-STING pathway activation. We are also initiating AI-based drug discovery, aiming to generate novel CENP-E inhibitors by performing virtual screening and generative molecular design. Citation Format: Ryo Kamata, Hitoshi Saito, Yumi Hakozaki, Yukie Kashima, Tomoko Yamamori. Morita, Pinyi Lu, Akihiro Ohashi. CENP-E inhibitors activate cGAS-STING pathway by inducing chromosome misalignment and micronucleation after mitotic slippage [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 301.