Centromere-associated Protein E Research Articles

Abstract 4945: Computational modeling-based discovery of novel anticancer drug candidates targeting centromere-associated protein E

Abstract Purpose: Centromere-associated protein E (CENP-E), a mitotic motor protein, is crucial for cell division. Inhibition of CENP-E can lead to chromosome misalignment and aneuploidy. Our recent studies revealed that the CENP-E inhibitor (CENP-Ei), Compound A, induces aneuploid-mediated cGAS-STING pathway activation in cancer cell lines, which is expected to trigger innate immune response and induce immunological conversion of the tumor microenvironment from cold to hot. However, known CENP-Eis exhibited limited efficacy, and none have gone beyond phase I trials since 2012. The aim of this study is to develop a workflow integrating structure and artificial intelligence-based modeling approaches to accelerate the discovery of novel CENP-Eis with improved efficacy and overcome related challenges. Method: The inhibitor-bound CENP-E structure was modeled using chimeric homology modeling followed by induced fit docking with known CENP-Eis. Structure-based virtual screening (VS) was performed on large compound libraries in Enamine REAL, Synthetically Accessible Virtual Inventory, and MolPort databases. Consensus scoring methods were applied by averaging ranks of individual molecules obtained from VS against different CENP-E conformations. An active learning-enhanced VS pipeline was developed using ATOM Modeling PipeLine (AMPL) to find the best-ranked ligands more efficiently. CENP-E ligand candidates were further filtered based on their safety properties predicted by machine learning models trained with curated databases of adverse drug reactions using AMPL. The success of this study was measured by its ability to reveal novel chemotypes that could modulate CENP-E in kinesin ATPase assays. Result: Conformations of CENP-E’s ligand binding site were predicted for VS using the homology model. Structure-based VS was performed to select high-ranked compounds based on consensus scoring. The active learning-enhanced VS pipeline achieved 75% accuracy in ranking the top 20% of hit compounds. The graph convolutional network model with a ROCAUC, 0.76, was created using AMPL to filter ligand candidates based on their predicted safety properties. Seventy-three potential CENP-E ligands computationally prioritized from the Molport database were experimentally tested using kinesin ATPase assays from which 4 potential inhibitors and 8 potential activators were identified indicating overlapping binding sites of inhibitors and activators. Conclusion: Our integrated workflow is effective in discovering novel CENP-E modulators. The discovery of CENP-E activators provides an opportunity to identify novel therapeutics in hepatocellular carcinoma malignancies known to experience decreased CENP-E activity leading to aneuploidy. The predictions generated by computational models combined with experimental validations could further accelerate anticancer drug discovery. Citation Format: Pinyi Lu, Ryo Kamata, Michael R. Weil, Naomi Ohashi, Akihiro Ohashi, Eric A. Stahlberg. Computational modeling-based discovery of novel anticancer drug candidates targeting centromere-associated protein E [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 4945.

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

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.

Targeting CENP-E augments immunotherapy in non-small cell lung cancer via stabilizing PD-L1

Centromere-associated protein E (CENP-E) plays a critical role in mitosis and chromosome misalignment, which may represent a potential therapeutic target in tumors. CENP-E is frequently overexpressed in lung cancer and act as a driver gene. However, it remains unclear whether CENP-E regulates the immune microenvironment in non-small cell lung cancer (NSCLC). Our study revealed that CENP-E is highly expressed and predicts a worse survival in NSCLC patients; inhibition of CENP-E leads to an upregulation of PD-L1 expression, consequently impacting the immune microenvironment of NSCLC by modulating the balance between CD8+ T cells and regulatory T cells (Tregs). Mechanistically, we demonstrated that downregulation of CENP-E could stabilize PD-L1 mRNA through the targeting of its 3′UTR by TTP. The genetic knockdown or pharmacological inhibition of CENP-E, in combination with PD-L1 antibody, could enhance the antitumor effect in NSCLC. Thus, our findings have revealed a role of CENP-E in immunotherapy and suggest that combination of CENP-E inhibitor with PD-L1 antibody could be an effective treatment option for NSCLC.

Generation of Centromere-Associated Protein-E <em>CENP-E<sup>-/-</sup></em> Knockout Cell Lines using the CRISPR/Cas9 System

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing in a variety of organisms. Centromere-associated protein-E (CENP-E) is a plus-end-directed kinesin required for kinetochore-microtubule capture, chromosome alignment, and spindle assembly checkpoint. Although cellular functions of the CENP-E proteins have been well studied, it has been difficult to study the direct functions of CENP-E proteins using traditional protocols because CENP-E ablation usually leads to spindle assembly checkpoint activation, cell cycle arrest, and cell death. In this study, we have completely knocked out the CENP-E gene in human HeLa cells and successfully generated the CENP-E-/- HeLa cells using the CRISPR/Cas9 system. Three optimized phenotype-based screening strategies were established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, which effectively improve the screening efficiency and experimental success rate of the CENP-E knockout cells. Importantly, CENP-E deletion results in chromosome misalignment, the abnormal location of the BUB1 mitotic checkpoint serine/threonine kinase B (BubR1) proteins, and mitotic defects. Furthermore, we have utilized the CENP-E knockout HeLa cell model to develop an identification method for CENP-E-specific inhibitors. In this study, a useful approach to validate the specificity and toxicity of CENP-E inhibitors has been established. Moreover, this paper presents the protocols of CENP-E gene editing using the CRISPR/Cas9 system, which could be a powerful tool to investigate the mechanisms of CENP-E in cell division. Moreover, the CENP-E knockout cell line would contribute to the discovery and validation of CENP-E inhibitors, which have important implications for antitumor drug development, studies of cell division mechanisms in cell biology, and clinical applications.

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

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.

Comprehensive pan-cancer analysis identifies centromere-associated protein E as a novel prognostic and immunological biomarker in human tumors

Centromere-associated protein E (CENP-E), a core component of the kinetochore, mediates chromosome congression and spindle microtubule capture during mitosis. Partial experimental evidence has illustrated the carcinogenic effects of CENPE in tumors, but the corresponding pan-cancer analysis of CENPE still lacking. Based on public databases, including the Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), and Human Protein Atlas (HPA), we take an array of bioinformatics methods to investigate the potential oncogenic roles of CENPE. Then, we validated CENPE, cell cycle-related proteins, and immune checkpoint molecule findings expression in clinical colon cancer samples by western blot. Our results showed that CENPE was up-regulated in almost all tumors, and the expression level of CENPE was associated with worse overall survival (OS) and disease-specific survival (DSS) in patients. The strong relationship between CENPE with gene mutation and MMR has also been validated. Moreover, CENPE gene expression was positively correlated with immune checkpoint molecular, and reversely correlated with infiltration levels of most immune cells. In the human colon cancer tissues, the expression of CENPE, cell cycle-related proteins, and immune checkpoint molecules were significantly higher than in the adjacent normal tissues. Our results indicated that CENPE can function as an oncogene in various cancers, and may be regarded as a promising prognostic and diagnostic biomarker in cancer treatment.

Crystal structure of the motor domain of centromere-associated protein E in complex with a non-hydrolysable ATP analogue.

Centromere-associated protein E (CENP-E) is a kinesin motor protein essential for mitosis and a new target for anticancer agents with less side effects. To rationally design anticancer drug candidates based on structure, it is important to determine the three-dimensional structure of the CENP-E motor domain bound to its inhibitor. Here, we report the first crystal structure of the CENP-E motor domain in complex with a non-hydrolysable ATP analogue, adenylyl-imidodiphosphate (AMPPNP). Furthermore, the structure is compared with the ADP-bound form of the CENP-E motor domain as well as the AMPPNP-bound forms of other kinesins. This study indicates that helix α4 of CENP-E participates in the slow binding of CENP-E to microtubules. These results will contribute to the development of anticancer drugs targeting CENP-E.

Loss of RanGAP1 drives chromosome instability and rapid tumorigenesis of osteosarcoma.

Chromothripsis is a catastrophic event of chromosomal instability that involves intensive fragmentation and rearrangements within localized chromosomal regions. However, its cause remains unclear. Here, we show that reduction and inactivation of Ran GTPase-activating protein 1 (RanGAP1) commonly occur in human osteosarcoma, which is associated with a high rate of chromothripsis. In rapidly expanding mouse osteoprogenitors, RanGAP1 deficiency causes chromothripsis in chr1q, instant inactivation of Rb1 and degradation of p53, consequent failure in DNA damage repair, and ultrafast osteosarcoma tumorigenesis. During mitosis, RanGAP1 anchors to the kinetochore, where it recruits PP1-γ to counteract the activity of the spindle-assembly checkpoint (SAC) and prevents TOP2A degradation, thus safeguarding chromatid decatenation. Loss of RanGAP1 causes SAC hyperactivation and chromatid decatenation failure. These findings demonstrate that RanGAP1 maintains mitotic chromosome integrity and that RanGAP1 loss drives tumorigenesis through its direct effects on SAC and decatenation and secondary effects on DNA damage surveillance.

132 (PB122) - CENP-E inhibitor potently activates cGAS-STING pathway through misaligned chromosome-mediated micronucleation after mitotic slippage

Background: cGAS-STING, which monitors cytosolic DNA and triggers innate immune response, has recently attracted attention as a molecular target to sensitize cancer to immune therapies. Micronuclei generated by aberrant mitosis are known to profoundly activate the cGAS-STING pathway in cancer cells when they leak into the cytoplasm. We have validated that small-molecule inhibitors targeting centromere-associated protein-E (CENP-E) generate micronuclei through chromosome misalignment and mitotic slippage.

Photopharmacological Tools for Mitotic Cell Division

Genetic information is replicated and transmitted from a parent cell to two identical daughter cells through mitotic cell division. To accomplish this dynamic process with high accuracy and precision, various motor proteins work in a concerted manner. Especially in the metaphase, mitotic chromosomes are delivered by the motor protein of centromere-associated protein E (CENP-E) to the cell equatorial plane (metaphase plate) along mitotic spindles. However, the critical functional failure of CENP-E can activate the spindle assembly checkpoint through the misalignment of chromosomes at the metaphase plate. In this symposium review, the reversibly photoswitchable CENP-E inhibitor PCEI-HU (5) is reported. Compound 5 exhibited almost quantitative trans-cis photoisomerization of the arylazopyrazole photoswitch by illuminating light at 365 nm and 510 nm. Depending on the photoisomerization, CENP-E activity was regulated not only in vitro but also in cells. We successfully established a novel technique using 5 to dynamically photocontrol the CENP-E-dependent chromosome movement and mitotic progression in a living cell.

Identification of potential druggable targets of cell cycle with small-molecule inhibitors in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors worldwide and there are few crucial regulators and druggable targets for early diagnosis. Therefore, the identification of biomarkers for the early diagnosis and druggable targets of OSCC is imminent. In this study, we integrated gene set enrichment analysis, differential gene expression analysis based on the negative binomial distribution, weighted correlation network analysis, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes into analyzing the OSCC cohort downloaded from The Cancer Genome Atlas, and found that cell cycle and related biologic processes are significantly enriched. Then, we constructed the core gene network of OSCC, which showed the connection of encode human Cyclin-A2 protein, encode RAD51-associated protein 1, encode human centromere-associated protein E (CENPE), encode humans centromere protein I (CENPI) and encode polo-like kinase 1 (PLK1) to several cell cycle-related genes. Survival analysis further showed that low expression of these genes was associated with a better prognosis. Furthermore, we utilized a high-throughput virtual screening to find new CENPE and PLK1 inhibitors, and one of the CENPE inhibitor DB04517 suppressed the proliferation of OSCC cells by cell cycle arrest of cell cycle. Taken together, these candidate regulators could serve as the candidate diagnostic and prognostic biomarkers for OSCC, and specific suppression of these genes may be a potential approach to prevent and treat OSCC with the candidate inhibitors.

CENPE contributes to pulmonary vascular remodeling in pulmonary hypertension

Hypoxic pulmonary vascular remodeling is a pathological feature of pulmonary hypertension (PH). Our results showed that centromere-associated protein E (CENPE) expression in PH patients and hypoxia-induced PH rats was significantly higher than that in normal controls. In addition, CENPE deficiency significantly inhibited the development of pulmonary vascular remodeling and right ventricular hypertrophy. Moreover, knocking out CENPE effectively inhibited the proliferation and induced the apoptosis of primary pulmonary artery smooth muscle cells (PASMCs) in vivo. Furthermore, CENPE silencing by small interference significantly inhibited abnormal proliferation, apoptosis resistance, migration, and cell cycle arrest in hypoxia-induced PASMCs. Interestingly, we found that CENPE might exert its biological effect by targeting the transcription of CDK1 proteins.

A non-canonical function for Centromere-associated protein-E controls centrosome integrity and orientation of cell division

Centromere-associated protein-E (CENP-E) is a kinesin motor localizing at kinetochores. Although its mitotic functions have been well studied, it has been challenging to investigate direct consequences of CENP-E removal using conventional methods because CENP-E depletion resulted in mitotic arrest. In this study, we harnessed an auxin-inducible degron system to achieve acute degradation of CENP-E. We revealed a kinetochore-independent role for CENP-E that removes pericentriolar material 1 (PCM1) from centrosomes in late S/early G2 phase. After acute loss of CENP-E, centrosomal Polo-like kinase 1 (Plk1) localization is abrogated through accumulation of PCM1, resulting in aberrant phosphorylation and destabilization of centrosomes, which triggers shortened astral microtubules and oblique cell divisions. Furthermore, we also observed centrosome and cell division defects in cells from a microcephaly patient with mutations in CENPE. Orientation of cell division is deregulated in some microcephalic patients, and our unanticipated findings provide additional insights into how microcephaly can result from centrosomal defects.

CENPE Inhibition Leads to Mitotic Catastrophe and DNA Damage in Medulloblastoma Cells.

Medulloblastoma (MB) is the most frequent brain tumor in children. The standard treatment consists in surgery, followed by radiotherapy and chemotherapy. These therapies are only partially effective since many patients still die and those who survive suffer from neurological and endocrine disorders. Therefore, more effective therapies are needed. Primary microcephaly (MCPH) is a rare disorder caused by mutations in 25 different genes. Centromere-associated protein E (CENPE) heterozygous mutations cause the MCPH13 syndrome. As for other MCPH genes, CENPE is required for normal proliferation and survival of neural progenitors. Since there is evidence that MB shares many molecular features with neural progenitors, we hypothesized that CENPE could be an effective target for MB treatment. In ONS-76 and DAOY cells, CENPE knockdown induced mitotic defects and apoptosis. Moreover, CENPE depletion induced endogenous DNA damage accumulation, activating TP53 or TP73 as well as cell death signaling pathways. To consolidate CENPE as a target for MB treatment, we tested GSK923295, an allosteric inhibitor already in clinical trial for other cancer types. GSK923295, induced effects similar to CENPE depletion with higher penetrance, at low nM levels, suggesting that CENPE's inhibition could be a therapeutic strategy for MB treatment.

Rational design and development of a lit-active photoswitchable inhibitor targeting CENP-E.

In the emerging field of photopharmacology, synthetic photoswitches based on reversible photochemical reactions are fused to bioactive molecules. Azobenzene derivatives, which can undergo trans-cis photoisomerization, are typical photoswitches. Most azobenzene-based photochemical tools are active in the thermodynamically stable trans, but not cis, form. cis-Active photochemical tools would be ideal because they can be "initially inactive and active after light illumination" in a reversible mode only by light illumination. However, only a few rational strategies for constructing such "lit-active" photopharmacological tools has been developed. Herein, we report a rationally designed lit-active photoswitchable inhibitor targeting centromere-associated protein E (CENP-E). Using the lit-active inhibitor, we were able to photoregulate CENP-E-dependent mitotic chromosome location in cells. This study provides a framework to facilitate further progress in the development of photopharmacological tools.

P53 Is Not Required for High CIN to Induce Tumor Suppression.

Chromosomal instability (CIN) is a hallmark of cancer. While low levels of CIN can be tumor promoting, high levels of CIN cause cell death and tumor suppression. The widely used chemotherapeutic, paclitaxel (Taxol), exerts its anticancer effects by increasing CIN above a maximally tolerated threshold. One significant outstanding question is whether the p53 tumor suppressor is required for the cell death and tumor suppression caused by high CIN. Both p53 loss and reduction of the mitotic kinesin, centromere-associated protein-E, cause low CIN. Combining both genetic insults in the same cell leads to high CIN. Here, we test whether high CIN causes cell death and tumor suppression even in the absence p53. Despite a surprising sex-specific difference in tumor spectrum and latency in p53 heterozygous animals, these studies demonstrate that p53 is not required for high CIN to induce tumor suppression. Pharmacologic induction of high CIN results in equivalent levels of cell death due to loss of essential chromosomes in p53+/+ and p53-/- cells, further demonstrating that high CIN elicits cell death independently of p53 function. IMPLICATIONS: These results provide support for the efficacy of anticancer therapies that induce high CIN, even in tumors that lack functional p53.

Ruminal transcript abundance of the centromere-associated protein E gene may influence residual feed intake in beef steers.

A better understanding regarding the mechanisms by which the rumen processes feed may assist us in identifying animals with superior feed efficiency. Studies to evaluate the gene expression of rumen tissue have previously been performed to analyze their relationship with feed efficiency. Continuing this research is critical to determine whether the expression of the genes identified is associated with feed efficiency in additional populations of beef cattle to ensure that they are robust across breed and environment. A previous rumen-transcriptome study on Hereford×Angus steers identified 122 differentially expressed genes (PFDR <0.05) associated with residual feed intake (RFI), a measure of feed efficiency. The purpose of our study was to test the most divergent, up- and down-regulated genes in the rumen tissue of an unrelated population of Hereford×Angus steers that included two contemporary groups. A total of 13 genes were evaluated by quantitative real-time PCR. The centromere-associated protein E (CENPE) gene was expressed in lower concentrations in the rumen epithelium of steers in the more efficient (low RFI) group in both contemporary groups of animals, which was the same as the previous study. In addition, CENPE, a gene involved in chromosome alignment during mitosis, has also been associated with growth traits in cattle and pigs. There was no relationship between the expression of the other 12 genes tested with RFI in the population of steers in this study, which illustrates the importance of validating gene expression data in additional populations.

Reduced expression of CENP-E contributes to the development of hepatocellular carcinoma and is associated with adverse clinical features

Human kinesin centromere-associated protein E (CENP-E), one of spindle checkpoint proteins, has been identified as a tumor suppressor in several types of cancer, however, its role in hepatocarcinogenesis remains unknown. Here we investigated the role of CENP-E in human hepatocellular carcinoma (HCC) employing HCC cell lines (Hep3B, SMMC7721, and QGY7701), animal models, and patient’s clinical samples and data. We demonstrated that down-regulation of CENP-E by CENP-E-silencing shRNAs significantly promoted HCC proliferation/growth both in vitro and in vivo. Further studies found that CENP-E suppressed the proliferation of HCC cells by halting cell cycle progression at the G1-S phase and accelerating cell apoptosis. Analyses of HCC patient samples and clinical data revealed that CENP-E was significantly down-regulated in HCC tissues and low CENP-E expression was significantly associated with patient’s adverse clinicopathological features: poor prognosis, advanced TNM stage, metastasis, and larger tumor size. Multivariate analysis indicated that CENP-E was an independent prognostic factor predicting outcomes of advanced HCC patients. Our data suggest that loss of CENP-E contributes to HCC development and is strongly associated with adverse HCC clinical pathology. Thus, CENP-E could be a novel target for new treatments and a useful prognostic biomarker for HCC patients.

Identification of benzo[d]pyrrolo[2,1-b]thiazole derivatives as CENP-E inhibitors

Kinesin centromere-associated protein E (CENP-E) has emerged as a potential target for the development of anticancer drugs due to its involvement in the mitotic progression of the cell cycle. Although several CENP-E inhibitors have been reported, more knowledge of chemical structures and inhibitory mechanisms is necessary for developing CENP-E inhibitors. Here, we describe the identification of new CENP-E inhibitors. Screening of a small-molecule chemical library identified benzo[d]pyrrolo[2,1-b]thiazole derivatives, including 1, as compounds with inhibitory activity against the microtubule-stimulated ATPase of the CENP-E motor domain. Among the mitotic kinesins examined, 1 selectively inhibited the kinesin ATPase activity of CENP-E. In a steady-state ATPase assay, 1 exhibited ATP-competitive behavior, which was different from the CENP-E inhibitor GSK923295. Compound 1 inhibited the proliferation of tumor-derived HeLa and HCT116 cells more efficiently than that of non-cancerous WI-38 cells. The inhibition of cell proliferation was attributed to the ability of 1 to induce apoptotic cell death. The compound showed antimitotic activity, which caused cell cycle arrest at mitosis via interference with proper chromosome alignment. We identified 1 and its derivatives as the lead compounds that target CENP-E, thus providing a new opportunity for the development of anticancer agents targeting kinesins.

Abstract 2008: CENP-E kinesin facilitates chemokine ligand CCL18 translocation to promote migration in triple negative breast cancer

Abstract Our research is focused on the molecular mechanisms responsible for enhanced cellular proliferation and migration as it relates to triple negative breast cancer (TNBC). Advances in visualization techniques and technologies work to provide an in-depth view of various cellular mechanisms, allowing us to capture molecular level protein-protein interactions previously unknown. There are more than 40 kinesin motor proteins found within the spindle assembly checkpoint, classified into 14 families, which each play essential roles in microtubule-dependent intracellular trafficking. This assembly is a critical component to the cell cycle control complex that acts during mitosis to prevent chromosome missegregation and aneuploidy. Centromere associated protein E (CENP-E) is a kinesin motor protein that shows aberrant overexpression in TNBC which facilitates exponential cell growth and eventual metastasis that occurs in TNBC. Growing awareness through scientific discoveries sheds light on the fact that it is a rare occurrence for biological functions to be attributed to a single mutated macromolecule. Most physiological processes arise from a complex orchestration of cellular events occurring between multiple molecules acting in concert. The nature of protein-protein transactions that drive gene activation appear to be dynamic in TNBC cells thus enabling uncontrolled proliferation. We believe that a pleiotropic network of small molecules may be working cooperatively with CENP-E to facilitate the exponential cell growth and eventual metastasis that occurs in TNBC cells. Currently, we are carrying out spectral imaging and biophysical force measurements to delineate CCL18-elicited cell mechanics and breast cancer metastasis. To address this, we have incorporated syntelin, a small inhibitory molecule targeted to CENP-E in an effort to analyze the resultant effect it has on this network of proteins we believe may be involved in the CENP-E signaling cascade. Taken together, these results indicate that syntelin may comprise a novel targeted chemotherapeutic intervention for TNBC. Citation Format: McKay M. Mullen. CENP-E kinesin facilitates chemokine ligand CCL18 translocation to promote migration in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2008.