Abstract 411: PI3K-AKT-mTOR signaling pathways play important roles in chromosomal instability-induced innate immune response in cancer cells

Abstract

Abstract Background: Chromosomal instability (CIN) frequently exerts innate immune response, being expected to sensitize immunotherapy. cGAS-STING pathway is a hub to regulate CIN-mediated innate immune activation. We previously demonstrated that small-molecule inhibitors targeting Centromere-associated Protein-E (CENP-E) profoundly accumulated CIN in cancer cells, leading to activation of cGAS-STING and its related innate immune pathways. In this study, aiming to deeply understand molecular mechanisms of CENP-E inhibitor (CENP-Ei)-induced CIN and innate immune activation, we conducted large-scale chemical screening with ~1,400 kinase inhibitors in CENP-Ei-treated reporter cells for NF-κB and IRF. We also experimentally validated the effects of hit compounds on CENP-Ei-induced innate immune activation. Material and Methods: GSK923295 and Cmpd-A were used as CENP-E inhibitors. HeLa, A549, A549 dual reporter cells were treated with the CENP-Ei at the indicated concentrations, subjected to immunofluorescent, transcriptome, FACS, reporter activity, or gene expression analyses. High-throughput screening (HTS) with ~1,400 kinase library was conducted in A549 dual reporter cells in combinational treatment with GSK923295. Combination effects of the representative hit compounds were confirmed in matrix-combination studies with the CENP-Ei. Results: Treatment with the CENP-Ei, Cmpd-A and GSK923295, induced CIN (e.g., multinucleation) in both A549 and A549 dual reporter cells, which elevated NFkB and IRF reporter activities as well as gene expressions of inflammatory cytokines and chemokines in innate immune pathways. Transcriptome analyses also confirmed that gene ontologies (GO) for innate immune response, such as cytosolic DNA sensing pathway and cytokine-cytokine receptor interaction pathway, were significantly enriched in CENP-Ei-treated CIN cells. Next, we conducted HTS of IRF-reporter activity with ~1,400 kinase library in combination with GSK923295 in A549 dual reporter cells. The HTS revealed that a series of inhibitors targeting PI3K-AKT-mTOR signaling pathways intensively suppressed CENP-Ei-induced IRF activation, which occupied ~40% of hit compounds. The matrix-combination studies confirmed that sapanisertib (TORC1/2 inhibitor) significantly attenuated CENP-Ei-induced IRF activity. Conclusions: CENP-Ei-induced CIN potently activates innate immune response pathways in cancer cells, in which PI3K-AKT-mTOR signaling pathway appears to be involved. We are initiating AI-based drug discovery to develop novel CENP-E inhibitors. Citation Format: Ryo Kamata, Hitoshi Saito, Yumi Hakozaki, Yukie Kashima, Gaku Yamamoto, Tomoko Yamamori Morita, Pinyi Lu, Akihiro Ohashi. PI3K-AKT-mTOR signaling pathways play important roles in chromosomal instability-induced innate immune response in cancer cells [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 411.