Abstract 1030: CENP-E inhibition generates micronucleus formation activating the cGAS-STING pathway in cancer cells

Abstract

Abstract Background: Micronuclear formation has been reported to activate innate immune response in tumor through the cGAS-STING pathway, which monitors cytosolic DNA primarily to detect bacteria or virus infection. We have developed a novel small molecule inhibitor targeting CENP-E, termed Cmpd-A, which intensively generate micronucleus formation in a broad range of cancer cells, causing a potent antiproliferation by aneuploid-mediated cellular stresses. In this study, using Cmpd-A as a tool compound, we aim to determine that micronucleus formation induced by CENP-E inhibitor could activate the cGAS-STING pathway in cancer cells, which may potentially induce an immunological conversion from cold to hot in cancer cells. Method: We generated micronuclear formation in cancer cells by Cmpd-A treatment. Phosphorylations of TBK1 and IRF3, indexes of the cGAS-STING pathway activation, were determined by immunoblotting. Gene set enrichment analysis of RNA-seq data was performed, comparing DMSO (control) and Cmpd-A treatment. Reporter assays for IRF and NF-kB were also performed. Results: Immunoblotting revealed that phosphorylations of TBK1 and IRF3 were remarkably upregulated in Cmpd-A-treated cells concordantly with micronuclear formation. Transcriptome analysis revealed that the down-stream genes in the cGAS-STING pathway (innate immune pathways) are elevated: IL6, IP10, CXCL1, IL8, TNF, IFNA families, and IFNB1. The Gene Set Enrichment Analysis (GSEA) also demonstrated that innate immune-associated pathways were significantly enriched in Cmpd-A-treated cells: cytosolic DNA sensing pathway, cytokine-cytokine receptor interaction pathway, and Toll-like receptor signaling pathway. Furthermore, the IRF-Lucia and NF-kB-SEAP reporter activity assays revealed that both IRF and NF-kB were significantly activated in Cmpd-A-treated cells in a time-dependent manner. Conclusions: The CENP-E inhibitor generates micronucleus formation to activate the cGAS-STING pathway in cancer cells. These findings suggest the CENP-E inhibitor could potentially induce the immunological conversion from cold to hot in cancer cells. Citation Format: Yumi Hakozaki, Yukie Kashima, Tomoko Y. Morita, Kosuke Tanaka, Susumu S. Kobayashi, Akihiro Ohashi. CENP-E inhibition generates micronucleus formation activating the cGAS-STING pathway in cancer cells [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 1030.