Interspecies differences in sensitivity to anticoagulant compounds remain an important issue in veterinary toxicology and wildlife risk assessment. In the present study, we developed an integrated pharmacokinetic-pharmacodynamic (PK/PD) model to quantitatively compare anticoagulant responses between rats and bats, linking hepatic exposure to vitamin K epoxide reductase (VKOR) inhibition and subsequent changes in prothrombin time-international normalized ratio (PT-INR). Species-specific pharmacokinetic parameters were estimated from experimental concentration-time data and incorporated into a mechanistic turnover model describing VKOR activity and clotting factor synthesis. The model reproduced dose-dependent PT-INR prolongation in rats and successfully described the delayed and attenuated anticoagulant responses observed in bats. Sensitivity analyses identified hepatic clearance (CLh), hepatic distribution volume (Vh), and the half-maximal inhibitory concentration for VKOR (IC50) as key determinants of PT-INR responses, with a smaller contribution from baseline coagulation turnover. In addition, structure-based binding affinity estimates were explored as surrogate indicators of inhibitory potency. Although computational affinity scores qualitatively reflected relative anticoagulant strength, substituting these scores for experimentally derived inhibitory parameters reduced predictive accuracy in some resistant species, highlighting the current limitations of structure-only approaches. These findings demonstrate that mechanistic PK/PD integration provides a quantitative framework for evaluating species differences in anticoagulant toxicity and may support veterinary risk assessment and wildlife protection strategies.

The gastric cancer (GC) tumor microenvironment (TME) constitutes a complex and dynamic ecosystem in which immune, stromal, and malignant cells (MCs) collectively shape therapeutic responses. The dynamics of GC TME remodeling in different treatment contexts remain unclear. We performed single-cell RNA sequencing (scRNA-seq) of GC biopsy specimens from treatment-naïve patients (TN) and patients treated with chemotherapy (C), chemotherapy plus nivolumab (CN), or chemotherapy plus trastuzumab (CT), and found that CN treatment enhanced natural killer (NK)/T cell-associated immunogenicity in GC TMEs. In CN TMEs, MCs upregulate MHC-I- and inflammation-related genes. Conventional type 1 dendritic cells (cDC1), one subcluster of dendritic cells (DCs), concurrently exhibited activation of antigen-presentation and T cell-stimulatory programs, indicating intensified communication between NK/CD8+ T cells and cDC1 in the CN TMEs. The cell-cell interaction analyses uncovered an intensified X-C Motif Chemokine Ligand 1 (XCL1) - X-C motif chemokine receptor 1 (XCR1) chemotactic axis linking NK/CD8+ T cells and cDC1, highlighting the formation of "the MCs-cDC1-NK/CD8+ T cells circuit" to reinforce antitumor immunity in the CN TMEs. Furthermore, a reduced cytotoxic NK sub-cluster, which showed high XCL1 expression, was enriched among CN responders, and its transcriptional signature was significantly correlated with favorable survival in the Cancer Genome Atlas GC cohort. Our findings delineate an immunostimulatory circuit driven by NK/CD8+ T-DCs-MCs interactions to orchestrate the immune cycle under CN therapy in GC TMEs.

Second-generation anticoagulant rodenticides (SGARs) were developed to overcome warfarin resistance in rodent populations; however, their prolonged hepatic retention has raised concerns regarding secondary poisoning of non-target wildlife. All major SGARs exist as cis-trans isomeric pairs, and differences in biological half-life between isomers have been reported, yet the molecular basis for such isomer-dependent pharmacokinetic behavior remains poorly understood. In this study, we conducted an integrated evaluation of cis and trans isomers of SGARs using in vivo, in vitro, and in silico approaches, with vitamin K epoxide reductase (VKOR) serving as the molecular target. The individual compounds exhibited distinct isomer-dependent profiles in hepatic retention, inhibitory potency (IC50), and VKOR interaction-related properties. Molecular dynamics simulations further revealed isomer-dependent differences in torsional flexibility around specific rotatable bonds and in ligand-VKOR interaction fractions. For flocoumafen and bromadiolone, the presence of an ether oxygen was associated with increased torsional and orientational flexibility and enhanced hydrogen-bonding potential, which may facilitate metabolic processing and contribute to the relatively faster elimination of cis isomers. Collectively, these results suggest that isomer-specific VKOR interaction patterns may contribute, in a compound-dependent manner, to isomer-dependent pharmacokinetic behavior, offering structural perspectives for the design of rodenticides with reduced ecological risk.

Poly (ADP-ribose) polymerase (PARP) inhibitors have improved the prognosis of homologous recombination deficient (HRD) ovarian cancer (OC), while effective therapeutic strategies for HR-proficient (HRP) OC still need to be established. This study investigates senescence-mediated inflammation as a novel mechanism of action for PARP inhibitors in HRP cancers. Transcriptome analyses were performed in olaparib-treated HeLa cells as a HRP model. Interferon regulatory factor-Lucia luciferase (IRF-Luc) reporter activity was assessed. The effects of PARP inhibitors on senescence-like phenotypes were assessed in seven HRP cancer cell lines, based on morphological changes, senescence-associated β-galactosidase (SA-β-GAL) activity, cellular granularity, and senescence-associated secretory phenotype (SASP)-related gene expression. Peripheral blood mononuclear cell (PBMC) migration assays were also performed with the conditioned medium in treatment with the PARP inhibitor. Transcriptome analyses revealed numbers of inflammatory cytokine- and chemokine-related pathways were significantly upregulated in olaparib-treated HeLa cells, which were confirmed by IRF-Luc reporter assays. The PARP inhibitors induced senescent phenotypes in HRP cancer cell lines: flattened and enlarged morphology, increased SA-β-GAL activity, elevated cellular granularity, and upregulated expressions of SASP-related genes (e.g., IL1B, IL6, and CXCL10). Furthermore, in vitro migration assays revealed that PARP inhibitor-treated HRP cancer cells attracted PBMCs more abundantly, suggesting the potential for recruiting immune cells to HRP cancer cells through senescence-mediated immunological activation. Our findings suggest that PARP inhibitors recruit immune cells to HRP cancer cells, potentially activating immune responses in the tumor microenvironment, providing new insights into the clinical benefits of PARP inhibitors in immunotherapy for patients with HRP OC.

Control of rodents remains a critical global challenge due to their role in spreading zoonotic diseases and damaging ecosystems, but current rodenticides, such as anticoagulant rodenticides, have significant limitations, including the development of resistance in rodents and bioaccumulation leading to secondary toxicity in nontarget species. Dcha-20, a lithocholic acid derivative and vitamin D receptor agonist, was synthesized to potentially address these issues. This study investigates its rodenticidal efficacy, safety profile, and mechanism of action. The oral LD50 estimate of Dcha-20 was 4.9 mg/kg in male and female rats, ranking it among the most potent of currently available rodenticides. Pharmacokinetic analysis revealed a short half-life and low bioavailability (4.5% in males and 12.8% in females), suggesting minimal risk of secondary toxicity. Unlike vitamin D3, Dcha-20 impeded plasma calcium elevation observed with vitamin D3, minimizing the risk of hypercalcemia in nontarget animals. Transcriptome analysis highlighted upregulation of pathways associated with vitamin D metabolism and inflammatory responses, indicating acute renal failure as the primary toxic mechanism. Histopathological and biochemical analyses confirmed renal damage, including elevated blood urea nitrogen and creatinine levels. Dcha-20 is stable to light and heat, unlike vitamin D3 derivatives. These findings suggest that Dcha-20 is a promising candidate for safe, effective rodent control, with potential for large-scale use.

KRASG12C inhibitors sotorasib and adagrasib have been approved for the treatment of KRASG12C-mutant non-small cell lung cancer (NSCLC). However, the efficacy of single-agent treatments is limited, presumably due to multiple resistance mechanisms. To overcome these therapeutic limitations, combination strategies that potentiate the antitumor efficacy of KRASG12C inhibitors must be developed. Through unbiased high-throughput screening of 1,395 kinase inhibitors, we identified adavosertib, a WEE1 inhibitor, as a promising combination partner of sotorasib. The combination of sotorasib and adavosertib exhibited synergistic antiproliferative activities both in vitro and in vivo, irrespective of TP53, STK11, and KEAP1 co-mutation profiles. WEE1 inhibition potentiated MCL-1-mediated apoptosis in sotorasib-treated cancer cells. Mechanistically, the combination downregulated MCL-1 protein levels by attenuating de novo translation and enhancing its degradation. WEE1 overexpression conferred resistance against sotorasib via MCL-1 upregulation. Moreover, cells that acquired sotorasib resistance profoundly upregulated both WEE1 and MCL-1 proteins, highlighting WEE1 as a crucial driver of sotorasib resistance. Importantly, WEE1 inhibition re-sensitized resistant cells to sotorasib treatment. The current findings demonstrate that combined inhibition of KRASG12C and WEE1 not only exhibits synergistic antitumor efficacy but also overcomes resistance to KRASG12C inhibitors, thus representing a novel therapeutic strategy for KRASG12C-mutant NSCLC.

Although esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy, there is little knowledge about its tumor heterogeneity affecting treatment response. To decipher transcriptomic heterogeneity of tumor microenvironment (TME) from a therapeutic perspective, we conducted CITE-seq and immunostaining by Hyperion in ESCC with or without chemotherapeutic stimuli. Tumor biopsies of ESCC were sequentially collected by endoscopy from 3 patients during chemotherapy treatments. CITE-seq samples were processed with Chromium Next GEM Single Cell 5′ Reagent Kit v2 (10 x Genomics) and TotalSeq Universal Cocktails (BioLegend) as per the manufacturer′s recommendations, respectively. Cell-to-cell interaction analyses in CITE-seq and neighborhood analyses in Hyperion were performed. Clinical responses of 3 patients for ESCC tumor biopsies were PD, SD, and PR, respectively. Cell annotation analyses in TME components revealed that populations of myeloid cells exhibited an overall decrease during the treatment across three patients, while the decrease in myeloid cell populations was more pronounced in the PR patient. The cluster analyses in epithelial cells exhibited 6 unique subclusters, which were transcriptionally characterized in proliferation, EMT, inflammation, and metabolic pathways. In spatial analyses, the epithelial cells in the PD patient adhered tightly to each other, while the epithelial cells in the SD or PR patient clustered relatively mixed with immune and stromal cells. In SD or PR patients, furthermore, varieties of T cell populations were more accumulated in response to the chemotherapy treatments compared to the PD patient. Neighborhood analysis and cell-cell interaction analysis together identified cell type pairs that showed consistent changes in physical and molecular perspectives. In PD patient, dendritic cell (DC)-macrophage pair was enriched in the proximity of each other, and also increased molecular interactions after chemotherapy. In contrast, activated T-Treg pair was depleted and decreased molecular interactions. Interestingly not just activated T-Treg interaction decreased, but proliferating T cells were also depleted from the tumor. We conducted integrative analyses with cutting-edge technologies to determine temporal dynamics of TME landscape in ESCC during chemotherapy treatment. Our observations may indicate that chemotherapy increased DC-macrophage interaction to suppress antigen presentation by DC, which suppressed T cell activation and proliferation. The mechanism might contribute to the chemotherapy resistance. These findings will shed light on the molecular insights to deeply understand complexity of the TME networks for chemotherapy resistance in ECSS. Hiroki Yamashita, Chiaki Mashima, Maiko Narahara, Ryo Kamata, Kenji Watanabe, Ryuichi Nakamura, Sumie Muramatsu, Ayumi Tezuka, Eri Otsuka, Chisa Wada, Yoko Ishimoto, Daisuke Kotani, Takashi Kojima, Naoya Wada, Hiroyuki Sumi, Tomonori Yano, Gensuke Takayama, Akihiro Ohashi. Temporal dynamics of transcriptional and spatial landscape in tumor microenvironment of esophageal squamous cell carcinoma during chemotherapy treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5303.

Identifying the molecular targets of toxic compounds remains a major challenge in toxicology, particularly when adverse effects occur in off-target organs and the mechanism of action is unknown. To address this issue, a comprehensive computational pipeline was developed to perform high-throughput molecular docking across the entire AlphaFold2-predicted structural proteome of representative organisms such as human and mouse, followed by enrichment analysis to estimate biological processes potentially affected by ligand binding. The pipeline was first evaluated using six known drug-target pairs. In several cases, the known targets were ranked between the top 2 and 250 proteins (top 0.009-1.15%) among more than 21,000 proteins, and displayed docking poses consistent with experimentally observed binding conformations. However, performance was limited for certain targets, such as carbonic anhydrase II with acetazolamide, where the binding pocket was broad, leading to inaccurate docking results. The pipeline was subsequently applied to puberulic acid, a compound suspected of causing severe nephrotoxicity. Screening identified sodium/myo-inositol cotransporter 2 (SLC5A11) as a high-affinity target in both human and mouse, suggesting a mechanism involving disruption of renal osmoregulation. Although docking scores represent only theoretical binding estimates and do not directly imply physiological effects, their distribution was independent of protein length and AlphaFold2 confidence scores (pLDDT), supporting the methodological robustness. This in silico framework enables hypothesis-driven identification of potential target proteins for toxicants or therapeutics and offers a useful tool for predictive toxicology, particularly when experimental data are limited. The pipeline is available at: https://github.com/toxtoxcat/reAlldock.

This study presents a novel in silico screening method for identifying potential protein-ligand interactions within the complete structural proteome, as predicted by AlphaFold2, using puberulic acid as a test case. The methodology utilizes molecular docking simulations to predict interactions based on structural compatibility and binding affinity estimations. This approach highlights proteins, such as sodium/myo-inositol cotransporters, which exhibit theoretical high affinity for puberulic acid, suggesting possible competitive inhibition or mimetic interactions that could disrupt osmoregulation. It is important to clarify that these simulations provide a hypothetical framework for understanding potential interactions. The high docking scores do not directly translate to biological effects but serve as a basis for further experimental validation. This cautious interpretation acknowledges the inherent limitations of predictive modeling while maintaining confidence in the robustness of the computational process. Moreover, the exploratory nature of this study is supported by the fact that binding affinity predictions are not influenced by protein amino acid length or the predictive accuracy (pLDDT scores) provided by AlphaFold2. This independence from structural prediction metrics suggests that the docking results offer a reliable exploration of potential interactions across a wide array of proteins, making this approach a valuable asset in the fields of drug discovery and toxicological research. The ability to comprehensively identify candidate proteins with potential affinity to chemical compounds enhances the predictive capacity of toxicological assessments and supports the strategic development of safer pharmaceutical agents. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Gastric cancer (GC) in the fifth-leading types of cancer and the third-leading cause of death from cancer. Characterization of tumor microenvironment (TME) in GC is important not only to comprehensively understand tumor biology in GC but also to develop a novel cancer therapeutics targeting TME. In this study, we conducted multi-omics analyses of single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing (bulk RNA-seq), and whole exome sequencing (WES) using biopsied tumor samples through endoscopy from 23 GC patients: 3 treatment-naïve patients, 7 patients in treatment with chemotherapy (Chemo), 5 patients in combination treatment with chemotherapy and immune checkpoint blockade (Chemo+CPI), 3 patients in combination treatment with chemotherapy and Her2-antibody targeted therapy (Chemo+Her2i), and 5 patients in third-line or later treatment. We initially classified the GC samples into 4 molecular subtypes (CIN, GS, MSI, and EBV) using datasets of WES, bulk RNA-seq and scRNA-seq, showing that our samples were mainly composed of CIN (n=7) and GS (n=16) types. In scRNA-seq, we revealed that drug treatments profoundly modulated not only proportions of TME components but also their molecular characteristics. For instance, Chemo+CPI treated patients showed higher proportion of CD8+ cytotoxic T cells with activation and/or exhaustion markers. Combination treatments with Chemo+Her2i, in addition, resulted in higher Trail and Estrogen signaling pathways in epithelial cells of TME components. These findings suggest that interventions with certain cancer therapeutic drugs could substantially convert transcriptome landscape and reconstitute GC TME components. High-resolution of GC TME analyses should provide a clue for developing a next generation of cancer therapeutic drugs. Citation Format: Hiroki Yamashita, Jason Guoqiang Zhang, Chiaki Mashima, Ryo Kamata, Tomoko Yamamori, Izuma Nakayama, Akihito Kawazoe, Kohei Shitara, Tomonori Yano, Qunli Xu, Akihiro Ohashi, Takeshi Saito. Transcriptomic landscape of tumor microenvironment in gastric cancer in treatment with different types of cancer drugs [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 5075.

Per- and poly-fluoroalkyl substances (PFAS) exhibit high persistence in the environment and accumulate within the human body, warranting a thorough assessment of their toxicity. In this study, we exposed mice (male C57BL/6J mice aged 8 weeks) to a composite of nine PFAS, encompassing both long-chain PFAS (e.g., perfluorooctanoic acid and perfluorooctanesulfonic acid) and short-chain PFAS (e.g., perfluorobutanoic acid and perfluorobutanesulfonic acid). The exposure concentrations of PFAS were equivalent to the estimated daily human intake in the composition reported (1 µg/L (sum of the nine compounds), representing the maximum reported exposure concentration). Histological examination revealed hepatocyte vacuolization and irregular hepatocyte cord arrangement, indicating that exposure to low levels of the PFAS mixture causes morphological changes in liver tissues. Transcriptome analysis revealed that PFAS exposure mainly altered a group of genes related to metabolism and chemical carcinogenesis. Machine learning analysis of the liver metabolome showed a typical concentration-independent alteration upon PFAS exposure, with the annotation of substances such as glutathione and 5-aminovaleric acid. This study demonstrates that daily exposure to PFAS leads to morphological changes in liver tissues and alters the expression of metabolism- and cancer-related genes as well as phospholipid metabolism.

Abstract Background: While novel covalent inhibitors of KRAS G12C have been approved in non-small-cell lung cancer (NSCLC), their efficacies are known to be limited. To enhance the therapeutic potential of KRAS inhibitors, combination strategies need to be developed. In this study, we conducted a large-scale screening to identify novel combination partners that induced synergistic efficacy with KRAS inhibitors. Additionally, mechanistic analyses were conducted to clarify the synergistic effects in combination and their role in conferring resistance to KRAS inhibitors. Methods: To identify novel combination partners of KRAS inhibitors, we conducted high-throughput screening (HTS) using a chemical library composed of 1,400 kinase inhibitors. Sotorasib was used as a KRAS inhibitor. Anti-proliferative and apoptotic effects in vitro were assessed by ATP-based cell viability assay, and caspase 3/7 assay and/or Annexin V assay, respectively. Protein expression levels were assessed by immunoblotting. In vivo studies were performed using a H358 xenograft nude mouse model and a patient-derived xenograft model (PDX). PDX samples were provided by the National Cancer Center J-PDX library, Japan. Results: HTS identified the WEE1 inhibitor AZD1775, a G2/M checkpoint abrogator in clinical-stage development, as a promising combination partner of KRAS inhibitors in KRAS G12C mutant NSCLC. Synergistic effects of KRAS and WEE1 inhibitors were confirmed across multiple KRAS G12C mutant NSCLC cell lines, independently of their co-occurring oncogenic mutation profiles. The combination treatment enhanced apoptotic cell death, being upregulated pro-apoptotic protein BIM, while downregulated anti-apoptotic protein MCL1. In vivo efficacy studies in combination with sotorasib and AZD1775 also demonstrated remarkable tumor regression and durable response in both CDX and PDX models. We also generated sotorasib-resistant H23 (H23-SR) cells, confirming upregulation of both WEE1 and MCL1 in the resistant cells. Ectopic overexpression of WEE1 in H23, on the contrary, conferred resistance to sotorasib. Furthermore, WEE1 inhibition re-sensitized H23-SR cells to KRAS inhibitors. Conclusion: Our results suggest that the WEE1-MCL1 axis plays an important role in both the initial therapeutic efficacy and the acquired resistance to KRAS inhibitors. These findings could pave the way for a novel therapeutic strategy for KRAS G12C mutant NSCLC. Citation Format: Gaku Yamamoto, Kosuke Tanaka, Ryo Kamata, Takehiro Nakao, Shunta Mori, Jie Liu, Susumu S. Kobayashi, Akihiro Ohashi. WEE1 inhibition prevents and overcomes resistance to KRAS inhibitors in lung cancer by enhancing MCL1-mediated apoptosis [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 1938.

Aromatic sensitizers and related substances (SRCs), which are crucial in the paper industry for facilitating color‐forming and color‐developing chemical reactions, inadvertently contaminate effluents during paper recycling. Owing to their structural resemblance to endocrine‐disrupting aromatic organic compounds, concerns have arisen about potential adverse effects on aquatic organisms. We focused on SRC effects via the aryl hydrocarbon receptor (AHR), employing molecular docking simulations and zebrafish (Danio rerio) embryo exposure assessments. Molecular docking revealed heightened binding affinities between certain SRCs in the paper recycling effluents and zebrafish Ahr2 and human AHR, which are pivotal components in the SRC toxicity mechanism. Fertilized zebrafish eggs were exposed to SRCs for up to 96 h post fertilization; among these substances, benzyl 2‐naphthyl ether (BNE) caused morphological abnormalities, such as pericardial edema and shortened body length, at relatively low concentrations (1 μM) during embryogenesis. Gene expression of cytochrome P450 1A (cyp1a) and ahr2 was also significantly increased by BNE. Co‐exposure to the AHR antagonist CH‐223191 only partially mitigated BNE's phenotypic effects, despite the effects of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin being relatively well restored by CH‐223191, indicating BNE's AHR‐independent toxic mechanisms. Furthermore, some SRCs, including BNE, exhibited in silico binding affinity to the estrogen receptor and upregulation of cyp19a1b gene expression. Therefore, additional insights into the toxicity of SRCs and their mechanisms are essential. The present results provide important information on SRCs and other papermaking chemicals that could help minimize the environmental impact of the paper industry. Environ Toxicol Chem 2024;00:1–13. © 2024 SETAC

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.