Centrosome Maturation Research Articles

A phase 1a/1b study of aurora kinase A inhibitor VIC-1911 as monotherapy and in combination with sotorasib for the treatment of KRAS G12C-mutant non–small-cell lung cancer.

TPS9140 Background: Activity of direct covalent KRASG12C inhibitors in NSCLC is limited by mutational and adaptive resistance. AURKA has roles in centrosome maturation and cytokinesis. AURKA amplification/overexpression is reported in multiple tumors, including NSCLC, and AURKA signaling may mediate adaptive resistance to KRAS inhibition. VIC-1911 is a highly selective, orally active small molecule inhibitor of AURKA. VIC-1911 demonstrated monotherapy activity in vitro in KRASG12C-mutant human NSCLC cells with intrinsic and acquired resistance to the KRASG12C inhibitor sotorasib, and combination VIC-1911 and sotorasib showed synergy in the same cell lines. Combination VIC-1911 and sotorasib delayed the emergence of adaptive resistance in vitro. These findings suggest that 1) AURKA activation is present in both intrinsic and acquired resistance to sotorasib in KRASG12C-mutant NSCLC and 2) the combination of VIC-1911 and sotorasib is a potential therapeutic approach for KRASG12C-mutant NSCLC with intrinsic or acquired resistance to sotorasib monotherapy. In addition, in vivo data suggest both sotorasib and adagrasib are synergistic in combination with VIC-1911 in human KRASG12C-mutant NSCLC cell line xenograft models. Methods: A non-randomized, open-label Phase 1a/1b study of VIC-1911, an aurora kinase A inhibitor, administered as monotherapy and in combination with sotorasib for the treatment of advanced KRASG12C-mutant NSCLC is in progress. Participants ≥18 years, with locally advanced or metastatic KRASG12C-mutant NSCLC, prior treatment with at least 1 line of PD-1/PD-L1 inhibitor therapy with or without platinum-based chemotherapy and have adequate organ function are eligible. Both KRASG12C inhibitor naïve and experienced patients are eligible. VIC-1911 monotherapy is given at oral doses of 25 – 90 mg twice daily in dose escalation and at the VIC-1911 maximum tolerated dose (MTD) in the expansion cohort in patients who are refractory/relapsed on prior KRASG12C inhibitor therapy. The combination regimen includes dose escalation with VIC-1911 at oral doses of 75, 150 or 200 mg twice daily on days 1-4, 8-11 and 15-18 every 28-day cycle plus sotorasib 960 mg daily in patients who are refractory/relapsed or naïve to prior KRASG12C inhibitor therapy. In dose escalation, a 3+3 schema is followed with dose limiting toxicity (DLT) criteria for toxicity and a sample size ≤ 36. For expansion cohorts, the sample size is based on Simon 2-stage optimal design, with a sample size ≤ 104. VIC-1911 and sotorasib dose modification criteria are provided. Supportive medications are allowed and early stopping rules for toxicity will be followed. VIC-1911 pharmacokinetics will be assessed. Pharmacodynamics will be determined from ctDNA and tumor biomarker analysis. Clinical trial information: NCT05374538 .

Centrosome fragmentation and malposition is common in the first cell division of equine ICSI embryos

In vitro production of embryos via Intra-Cytoplasmic-Sperm-Injection (ICSI) has become popular in the horse industry. Although improving, the blastocyst yield in clinical practice remains sub-optimal (∼20%: Stout. JEVS 2020; 89:103011). The high incidence of developmental arrest of ICSI embryos is suggested a result of mitotic errors during the first zygotic cell divisions. A better understanding of the cause of these errors could help develop strategies to prevent mitotic errors in equine in vitro produced (IVP) embryos. In somatic cells, the centrosomes, composed of two centrioles surrounded by pericentriolar material are the major microtubule-organizing centers and play essential roles in spindle assembly and chromosome segregation. Mammalian oocytes lack centrosomes and although two centrioles are re-introduced by the spermatozoon at fertilization, centrosomes make only a minor contribution to zygotic spindle assembly (Schneider et al., J.Cell. Biol. 2021; 220(11):e202010106). Although not essential for spindle assembly, the role of the centrosomes in ensuring fidelity of chromosome segregation during zygotic cell division is unclear. The aim of this study was to evaluate the position and number of centrosomes in horse ICSI zygotes during the first mitotic division. Cumulus-Oocyte-Complexes (COCs) were obtained from Warmblood mares by Ovum-Pick-Up, maintained in H-SOF at room temperature overnight and then matured in vitro, fertilized by ICSI and cultured as described previously (Ducheyne et al. Reprod. Fertil. Dev. 2019; 31(12):1830). After 24h, injected oocytes were fixedand immunostained for alpha-tubulin, acetylated tubulin, (peri-)centrosome material (NEDD1) and chromatin (DAPI). For each zygote, confocal microscopy was performed using a Leica SPE II microscope and a 0.17µm interval z stack generated. Of 326 COCs collected, 177 reached MII and were injected and cultured further. Of the 177, 136 zygotes were fixed and imaged, 13 were at the two pronucleus, 11 at prophase/prometaphase, 14 at metaphase, 11 at anaphase or telophase, 54 at the two cell stage and 33 were either degenerated or poorly stained. During the two pronucleus and prophase/prometaphase stages, centrosomes mainly localized at the interface between the two pronuclei. In 56% (n=14/25) of zygotes in pro-metaphase and metaphase, independent spindles formed around each of the two parental pronuclei. The majority of dual spindles were closely adjacent and synchronous. Moreover, in 64.2% (n=9/14) of the metaphase zygotes, the (peri-)centrosomes were either malpositioned (e.g. not at the poles of the spindle) or fragmented. Fragmented or multiple centrosomes are known to predispose to abnormal chromosome-microtubule attachment and aberrant chromosome segregation. We therefore hypothesize that malposition and fragmentation of the centrosomes contributes to mitotic errors and developmental arrest of equine ICSI embryos. Acknowledgements to CAPES, Brazil, for financial support (Code 88882.433332/2019-01 and 88887.467895/2019-00)

Protein phase separation: new insights into cell division.

As the foundation for the development of multicellular organisms and the self-renewal of single cells, cell division is a highly organized event which segregates cellular components into two daughter cells equally or unequally, thus producing daughters with identical or distinct fates. Liquid-liquid phase separation (LLPS), an emerging biophysical concept, provides a new perspective for us to understand the mechanisms of a wide range of cellular events, including the organization of membrane-less organelles. Recent studies have shown that several key organelles in the cell division process are assembled into membrane-free structures via LLPS of specific proteins. Here, we summarize the regulatory functions of protein phase separation in centrosome maturation, spindle assembly and polarity establishment during cell division.

Multiple pathways for reestablishing PAR polarity in C. elegans embryo

Asymmetric cell divisions, where cells divide with respect to a polarized axis and give rise to daughter cells with different fates, are critically important for development. In many such divisions, the conserved PAR polarity proteins accumulate in distinct cortical domains in response to a symmetry breaking cue. The one-cell C. elegans embryo is a paradigm for understanding mechanisms of PAR polarization, but much less is known about polarity in subsequent divisions. Here, we investigate the polarization of the P1 cell of the two-cell embryo. A posterior PAR-2 domain forms in the first 4 ​min, and polarization becomes stronger over time. Initial polarization depends on the PAR-1 and PKC-3 kinases, and the downstream polarity regulators MEX-5 and PLK-1. However, par-1 and plk-1 mutants exhibit delayed polarization. This late polarization correlates with the time of centrosome maturation and actomyosin flow, and loss of centrosome maturation or myosin in par-1 mutant embryos causes an even stronger polarity phenotype. Based on these and other results, we propose that PAR polarity in the P1 cell is generated by at least two redundant mechanisms: There is a novel early pathway dependent on PAR-1, PKC-3 and cytoplasmic polarity, and a late pathway that resembles symmetry breaking in the one-cell embryo and requires PKC-3, centrosome associated AIR-1 and myosin flow.

Abstract 5811: Epigenetic regulation of chromosomal instability in triple-negative breast cancer

Abstract Metastasis is the leading cause of cancer-related death among women with breast cancer. Chromosomal instability (CIN) has emerged as a hallmark of triple-negative breast cancer (TNBC) as it has recently been shown to promote metastasis. However, the underlying molecular mechanisms by which CIN drives metastasis are not completely understood. We have identified a discrete population of highly metastatic SOX2/OCT4+ cells expressing elevated levels of epigenetic regulator EZH2 associated with increased CIN in both human and mouse TNBC. EZH2 histone methyl transferase (HMT) is the catalytic subunit of the Polycomb repressive complex 2 (PRC2), represses target genes through trimethylation of Histone 3 at lysine 27 (H3K27me3). Importantly, genetic and pharmacologic inhibition of EZH2 lead to reduction of CIN and impaired metastasis. These findings led to the hypothesis that EZH2-mediated CIN constitutes a novel mechanism of metastasis regulation. To directly demonstrate epigenetic regulation of CIN, we used genome wide-Cleavage Under Targets and Release Using Nuclease (CUT&RUN) in parallel with RNA-seq. Gene set enrichment of EZH2-repressed target genes directly implicated the spindle formation pathway network. The central core of this network comprised of tankyrase (TNKS), a multifunctional poly (ADP-ribose) polymerase (PARP) previously implicated in DNA repair, telomere function and centrosome maturation. ChIP-PCR confirmed that EZH2 directly binds to the TNKS promoter, and CRISPR knockout of TNKS abrogated the ability of EZH2 inhibition in suppressing CIN, consistent with pharmacological inhibition of TNKS. More specifically, dysregulation of TNKS by EZH2 in OCT4/SOX2+ cells lead to increased numbers of centrosomes and multipolar mitosis. To directly demonstrate the role of aberrant H3K27me3 in regulating chromosomal segregation during mitosis, we used dCas9-EZH2 or dCas9-EZH2 catalytically dead mutant together with chromosome-specific CRISPR guides to ectopically enhance H3K27 trimethylation at the pericentromeric region of specific chromosomes. Ectopic deposition of EZH2 at pericentromeric regions lead to increased CIN. Conceptually, our work provides an unappreciated link between epigenetic regulation and CIN which have been hitherto studied in isolation. From a clinical perspective, demonstrating epigenetic regulation of CIN has opened the possibility for the development of first CIN suppressive therapeutic strategies targeting TNBC metastasis. Citation Format: Yang Bai, Albert Agustinus, Cem Meydan, Dylan R. McNally, Shira Yomtoubian, Liron Yoffe, Ari M. Melnick, Samuel Bakhoum, Vivek Mittal. Epigenetic regulation of chromosomal instability in triple-negative breast cancer [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 5811.

Abstract 2770: Phenotypic profiling of Aurora inhibitors using the OncoPanelTMplatform

Abstract Aurora kinases (Aur) are a family of serine/threonine kinases essential for genetic stability and cell division during mitosis. In humans, activity of the three paralogs (AurA, AurB, and AurC) is regulated throughout the cell cycle, with peak activity during mitosis. Each paralog shares a conserved C-terminal catalytic domain, but differs in substrate specificity, sub-cellular localization, and function. AurA mainly controls centrosome maturation and bipolar spindle assembly; AurB phosphorylates histone H3 (Ser10), which aids in chromatin condensation and separation; AurC, whose expression is restricted to germ cells of both genders, shares similar characteristics to AurB including substrate localization, specificity, and function during mitosis. Aberrant Aur expression leads to genomic instability or aneuploidy, and overexpression of the three paralogs is found in several cancer types, including lung, colon, and gliomas. Aur inhibition induces growth arrest and apoptosis in a variety of cancer cell types. Thus, design and development of Aur inhibitors as anti-tumor agents has been widely explored in recent years, and many are under preclinical and clinical investigation. We used the Eurofins Discovery OncoPanel™ cell-based profiling platform to investigate the specific effects of AurA, AurB, and AurC inhibitors against a select panel of human cancer cell lines. To determine the mechanistic effect of Aur inhibition in cells, we measured phospho-histone H3 (Ser10) by high-content analysis. OncoPanel human tumor cell lines arrested with nocodazole were treated with Aur inhibitors for 2 hours. Phospho-histone H3 (Ser10) levels were measured and the percentage of the DMSO vehicle-treated control cell signal was calculated for each test point. Ten-point dose-response curves were fitted using a 4-parameter log-logistic model with custom curve-fitting software to determine IC50 values. The inhibitors were also evaluated for their effect on tumor cell proliferation, apoptosis, and cell cycle arrest over the same dose-response range, after a 10-day incubation. Following exposure, the cells were fixed, stained with DAPI, anti-cleaved caspase-3, and anti-phospho-histone H3 (Ser28), and imaged. Cell proliferation dose-response curves were also fitted using the same model as above. Correlations were then made between observed mechanistic and functional activity. Activity of the AurA-selective inhibitor, MLN8054, was also evaluated in the Eurofins Discovery BioMAP® Diversity PLUS human primary cell phenotypic profiling service to give a broader view of its biological activity. These studies demonstrate the combined value of using the OncoPanel and BioMAP platforms to determine mechanistic, functional, and broader biological activity of therapeutic candidates in relevant cellular models for a better translational understanding of potential therapeutic capabilities and accelerate decision-making. Citation Format: Luciano Galdieri, Justin Lipner, Steven M. Garner, Jillian Krings, Emily Hoehn, Brendan Lahm, Kaleb Collver, Kaitlyne Powers, Alastair J. King. Phenotypic profiling of Aurora inhibitors using the OncoPanelTMplatform [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 2770.

Abstract 4997: PLK1 promotes the metastasis and drug resistance in melanoma

Abstract Melanoma is one of the most frequently diagnosed cancers in the caucasian population of both genders. The survival rate of advanced melanoma patients will be exceedingly poor, as low as 27.3%. BRAF-V600E is the most common mutation found clinically, resulting in the constitutive activation of the MAPK signaling pathway. Vemurafenib, a specific inhibitor of mutant BRAF, has shown an impressive response in phase 3 clinical trials and was approved by FDA in 2011. However, due to the rapid development of resistance, the duration of response under a single treatment is frequently short, highlighting the urgent requirement for novel therapy in melanoma treatment. Polo-like kinase 1 (PLK1), a crucial cell cycle regulator, participates in multiple mitotic processes, including centrosome maturation, mitotic entry, spindle assembly, sister chromatid segregation, mitotic exit, and cytokinesis. Compared to normal tissues, the expression level of PLK1 significantly upregulates in multiple cancers. More importantly, the expression level of PLK1 negatively correlates with the melanoma patients’ survival rate based on the TCGA database. Besides, PLK1 is identified as an oncogene to promote proliferation, motility, and resistance to various drugs. In the present study, we found that overexpression of PLK1 promotes tumor growth, metastasis, metabolic reprogramming, and vemurafenib resistance in the BrafCA/Ptenloxp mouse melanoma model. Through phosphorylation, overexpressed PLK1 stabilizes BACH1, a critical transcriptional factor of multiple metastatic and metabolic genes, and protects it from degradation. Subsequently, a higher level of BACH1 leads to increasing metastasis, upregulation of glycolysis, and suppression of OXPHOs. Besides, we have validated the strong synergy between PLK1 inhibitor volasertib and vemurafenib in treating melanoma carrying BRAF-V600E. Our in vitro and in vivo experiments have shown an improved efficacy of this combined therapy on inhibition of cell proliferation, induction of cell death, and suppression of cell metastasis compared to mono-treatment. In short, our data has indicated PLK1 as a potent and promising target in cancer treatment. Citation Format: Fengyi Mao, Yifan Kong, Chaohao Li, Yanquan Zhang, Xiongjian Rao, Qiongsi Zhang, Xinyi Wang, Zhiguo Li, Xiaoqi Liu. PLK1 promotes the metastasis and drug resistance in melanoma. [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 4997.

Abstract 1565: Targeting of KIF11 and AURKA to improve outcomes in Ewing sarcoma

Abstract Ewing Sarcoma (EWS) is a rare and aggressive pediatric malignancy of the bone and soft tissues. Despite intensive systemic combination chemotherapy and surgery/radiotherapy for local control, patients with metastatic disease have dismal prognosis (<30% overall survival rate). To identify better therapies for patients with refractory, recurrent and metastatic disease, we previously conducted in silico and drug re-purposing screens (PMID: 27863422; 31025088) that identified proteins essential for mitotic spindle formation and cell cycle progression such as Aurora Kinase A (AURKA), Kinesin Family Member 11 (KIF11), Kinesin Family Member 15 (KIF15) and its binding partner; targeting protein for Xklp2 (TPX2) that are upregulated by EWS-FLI1, the primary oncogenic driver of EWS, indicating their potential as therapeutic targets. KIF11 and KIF15 are mitotic motor kinesins essential for bipolar spindle formation, whereas AURKA is a mitotic kinase essential for centrosome maturation and mitotic spindle assembly. Though KIF11 and AURKA inhibitors have been extensively used in clinical trials before but have not been tested together in combination for any type of cancer. We hypothesized that targeting these two key proteins essential for mitotic progression provides a therapeutic vulnerability for Ewing Sarcoma by inducing G2/M arrest and mitotic catastrophe. Specific KIF11 (SB-743921) and AURKA (VIC-1911, VITRAC Therapeutics) inhibitors were used for targeting these proteins. We evaluated the in vitro activity of VIC-1911 and SB-743921 and synergy was measured by percentage inhibition of growth via SynergyFinder, that employs reference models (Bliss) to score synergy (scores >10 is synergistic). EWS showed strong synergistic interaction at physiologically relevant doses compared to control cell lines (e.g., mesenchymal stem cells and non-EWS tumor cells). This drug combination was effective in refractory EWS cells established post-chemotherapy (synergy score >24) and at much lower doses in cells established from pre-treatment tumor tissue (synergy score >27) indicating effectiveness at various clinical stages of EWS disease. Importantly, this therapy appeared preferential to EWS cells with an EWS-ETS fusion as compared to non-EWS cells. We observed zero colony formation in vitro with the combination treatment and increased percentage accumulation of cells in G2/M phase upon cell-cycle analysis indicating mitotic arrest. In vivo studies in EWS xenograft mouse models showed sustained tumor regression (NED at 30 days after ending therapy). The combination arm showed significant survival versus control (p≤ 0.001, HR=0.42, 95% CI=0.14-1.25), VIC-1911 (p≤ 0.001, HR=0.48, 95% CI=0.16-1.42) or SB-743921 (p ≤ 0.01, p≤ HR=0.71, 95% CI=0.21-2.34) alone. Efforts are underway to translate these preclinical studies into an early phase clinical trial of patients with histologically proven EWS who have failed standard of care therapy. Citation Format: Soumya M. Turaga, Vikalp Vishwakarma, Stacey Hembruff, Rajni Puri, Priya Sabu, Glenson Samuel, Andrew K. Godwin. Targeting of KIF11 and AURKA to improve outcomes in Ewing sarcoma [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 1565.

Inhibition of Polo-like kinase 1 (PLK1) triggers cell apoptosis via ROS-caused mitochondrial dysfunction in colorectal carcinoma.

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers. Polo-like kinase 1 (PLK1), a member of the serine/threonine kinase PLK family, is the most investigated and essential in the regulation of cell cycle progression, including chromosome segregation, centrosome maturation and cytokinesis. However, the nonmitotic role of PLK1 in CRC is poorly understood. In this study, we explored the tumorigenic effects of PLK1 and its potential as a therapeutic target in CRC. GEPIA database and immunohistochemistry analysis were performed to evaluate the abnormal expression of PLK1 in CRC patients. MTT assay, colony formation and transwell assay were performed to assess cell viability, colony formation ability and migration ability after inhibiting PLK1 by RNAi or the small molecule inhibitor BI6727. Cell apoptosis, mitochondrial membrane potential (MMP) and ROS levels were evaluated by flow cytometry. Bioluminescence imaging was performed to evaluate the impact of PLK1 on CRC cell survival in a preclinical model. Finally, xenograft tumor model was established to study the effect of PLK1 inhibition on tumor growth. First, immunohistochemistry analysis revealed the significant accumulation of PLK1 in patient-derived CRC tissues compared with adjacent healthy tissues. Furthermore, PLK1 inhibition genetically or pharmacologically significantly reduced cell viability, migration and colony formation, and triggered apoptosis of CRC cells. Additionally, we found that PLK1 inhibition elevated cellular reactive oxygen species (ROS) accumulation and decreased the Bcl2/Bax ratio, which led to mitochondrial dysfunction and the release of Cytochrome c, a key process in initiating cell apoptosis. These data provide new insights into the pathogenesis of CRC and support the potential value of PLK1 as an appealing target for CRC treatment. Overall, the underlying mechanism of inhibiting PLK1-induced apoptosis indicates that the PLK1 inhibitor BI6727 may be a novel potential therapeutic strategy in the treatment of CRC.

SH2D4A promotes centrosome maturation to support spindle microtubule formation and mitotic progression

Mitotic progression requires the precise formation of spindle microtubules based on mature centrosomes. During the G2/M transition, centrosome maturation progresses, and associated microtubules bundle to form mitotic spindle fibers and capture the chromosomes for alignment at the cell equator. Mitotic kinases-induced phosphorylation signaling is necessary for these processes. Here, we identified SH2 domain-containing protein 4A (SH2D4A/PPP1R38) as a new mitotic regulator. SH2D4A knockdown delays mitotic progression. The time-lapse imaging analysis showed that SH2D4A specifically contributes to the alignment of chromosomes. The cold treatment assay and microtubule regrowth assay indicated that SH2D4A promotes microtubule nucleation to support kinetochore–microtubule attachment. This may be due to the centrosome maturation by SH2D4A via centrosomal recruitment of pericentriolar material (PCM) such as cep192, γ-tubulin, and PLK1. SH2D4A was found to be a negative regulator of PP1 phosphatase. Consistently, treatment with a PP1 inhibitor rescues SH2D4A-knockdown-induced phenotypes, including the microtubule nucleation and centrosomal recruitment of active PLK1. These results suggest that SH2D4A is involved in PCM recruitment to centrosomes and centrosome maturation through attenuation of PP1 phosphatases, accelerating the spindle formation and supporting mitotic progression.

Transfer of Galectin-3-Binding Protein via Epididymal Extracellular Vesicles Promotes Sperm Fertilizing Ability and Developmental Potential in the Domestic Cat Model.

Key proteins transferred by epididymal extracellular vesicles (EVs) to the transiting sperm cells contribute to their centrosomal maturation and developmental potential. Although not reported in sperm cells yet, galectin-3-binding protein (LGALS3BP) is known to regulate centrosomal functions in somatic cells. Using the domestic cat model, the objectives of this study were to (1) detect the presence and characterize the transfer of LGALS3BP via EVs between the epididymis and the maturing sperm cells and (2) demonstrate the impact of LGALS3BP transfer on sperm fertilizing ability and developmental potential. Testicular tissues, epididymides, EVs, and spermatozoa were isolated from adult individuals. For the first time, this protein was detected in EVs secreted by the epididymal epithelium. The percentage of spermatozoa with LGALS3BP in the centrosome region increased as cells progressively incorporated EVs during the epididymal transit. When LGALS3BP was inhibited during in vitro fertilization with mature sperm cells, less fertilized oocytes and slower first cell cycles were observed. When the protein was inhibited in epididymal EVs prior to incubation with sperm cells, poor fertilization success further demonstrated the role of EVs in the transfer of LGALS3BP to the spermatozoa. The key roles of this protein could lead to new approaches to enhance or control fertility in clinical settings.

Multiple Roles of PLK1 in Mitosis and Meiosis.

Cells are equipped with a diverse network of signaling and regulatory proteins that function as cell cycle regulators and checkpoint proteins to ensure the proper progression of cell division. A key regulator of cell division is polo-like kinase 1 (PLK1), a member of the serine/threonine kinase family that plays an important role in regulating the mitotic and meiotic cell cycle. The phosphorylation of specific substrates mediated by PLK1 controls nuclear envelope breakdown (NEBD), centrosome maturation, proper spindle assembly, chromosome segregation, and cytokinesis. In mammalian oogenesis, PLK1 is essential for resuming meiosis before ovulation and for establishing the meiotic spindle. Among other potential roles, PLK1 regulates the localized translation of spindle-enriched mRNAs by phosphorylating and thereby inhibiting the translational repressor 4E-BP1, a downstream target of the mTOR (mammalian target of rapamycin) pathway. In this review, we summarize the functions of PLK1 in mitosis, meiosis, and cytokinesis and focus on the role of PLK1 in regulating mRNA translation. However, knowledge of the role of PLK1 in the regulation of meiosis remains limited.

AURORA KINASE A and related downstream molecules: A potential network for cancer therapy.

Aurora-A kinase (AURKA) belongs to the serine/threonine kinase family specific to cell division. In normal cells, activation of the AURKA protein is essential for regulating chromosomal segregation and centrosome maturation. The physiological concentration of AURKA accumulation has utmost importance during cell division. AURKA starts accumulating during the S phase of the cell cycle, gets functionally activated during the G2/M phase, attaches to the microtubule, and gets degraded during mitotic exit. Overexpression of AURKA could lead to deregulated cell cycle division, which is intrinsic to numerous cancers. Moreover, dysregulated AURKA affects various downstream molecules that aid in cancer pathogenesis. AURKA phosphorylates its substrates, including oncoproteins, transcriptional factors, tumor suppressor proteins, or other kinases central to various oncogenic signaling pathways critical to cancer. Considering the central role of AURKA in cell proliferation and tumorigenesis, targeting AURKA can be a novel alternative to cancer management. Several AURKA inhibitors have shown promising responses against different cancers either as a single agent or combined with various therapies. This chapter briefly discusses the role of AURKA and its downstream molecules in cancer vis-à-vis the role of AURKA inhibitor in chemoprevention.

CCHCR1-astrin interaction promotes centriole duplication through recruitment of CEP72

BackgroundThe centrosome is one of the most important non-membranous organelles regulating microtubule organization and progression of cell mitosis. The coiled-coil alpha-helical rod protein 1 (CCHCR1, also known as HCR) gene is considered to be a psoriasis susceptibility gene, and the protein is suggested to be localized to the P-bodies and centrosomes in mammalian cells. However, the exact cellular function of HCR and its potential regulatory role in the centrosomes remain unexplored.ResultsWe found that HCR interacts directly with astrin, a key factor in centrosome maturation and mitosis. Immunoprecipitation assays showed that the coiled-coil region present in the C-terminus of HCR and astrin respectively mediated the interaction between them. Astrin not only recruits HCR to the centrosome, but also protects HCR from ubiquitin-proteasome-mediated degradation. In addition, depletion of either HCR or astrin significantly reduced centrosome localization of CEP72 and subsequent MCPH proteins, including CEP152, CDK5RAP2, and CEP63. The absence of HCR also caused centriole duplication defects and mitotic errors, resulting in multipolar spindle formation, genomic instability, and DNA damage.ConclusionWe conclude that HCR is localized and stabilized at the centrosome by directly binding to astrin. HCR are required for the centrosomal recruitment of MCPH proteins and centriolar duplication. Both HCR and astrin play key roles in keeping normal microtubule assembly and maintaining genomic stability.

Design and Synthesis of Aminopyrimidinyl Pyrazole Analogs as PLK1 Inhibitors Using Hybrid 3D-QSAR and Molecular Docking.

Cancer continues to be one of the world’s most severe public health issues. Polo-like kinase 1 (PLK1) is one of the most studied members of the polo-like kinase subfamily of serine/threonine protein kinases. PLK1 is a key mitotic regulator responsible for cell cycle processes, such as mitosis initiation, bipolar mitotic spindle formation, centrosome maturation, the metaphase to anaphase transition, and mitotic exit, whose overexpression is often associated with oncogenesis. Moreover, it is also involved in DNA damage response, autophagy, cytokine signaling, and apoptosis. Due to its fundamental role in cell cycle regulation, PLK1 has been linked to various types of cancer onset and progression, such as lung, colon, prostate, ovary, breast cancer, melanoma, and AML. Hence, PLK1 is recognized as a critical therapeutic target in the treatment of various proliferative diseases. PLK1 inhibitors developed in recent years have been researched and studied through clinical trials; however, most of them have failed because of their toxicity and poor therapeutic response. To design more potent and selective PLK1 inhibitors, we performed a receptor-based hybrid 3D-QSAR study of two datasets, possessing similar common scaffolds. The developed hybrid CoMFA (q2 = 0.628, r2 = 0.905) and CoMSIA (q2 = 0.580, r2 = 0.895) models showed admissible statistical results. Comprehensive, molecular docking of one of the most active compounds from the dataset and hybrid 3D-QSAR studies revealed important active site residues of PLK1 and requisite structural characteristics of ligand to design potent PLK1 inhibitors. Based on this information, we have proposed approximately 38 PLK1 inhibitors. The newly designed PLK1 inhibitors showed higher activity (predicted pIC50) than the most active compounds of all the derivatives selected for this study. We selected and synthesized two compounds, which were ultimately found to possess good IC50 values. Our design strategy provides insight into development of potent and selective PLK1 inhibitors.

Combination of AURKA inhibitor and HSP90 inhibitor to treat breast cancer with AURKA overexpression and TP53 mutations.

Breast cancer is the most common cancer among women worldwide. Researches show that Aurora kinase A (AURKA) is highly expressed in approximately 73% of breast cancer patients, which induces drug resistance in breast cancer patients and decreases the median survival time. AURKA regulates spindle assembly, centrosome maturation, and chromosome alignment. AURKA overexpression affects the occurrence and development of breast cancer. Besides AURKA overexpression, heat shock protein 90 (HSP90) maintains the survival and proliferation of tumor cells by stabilizing the structure of oncoproteins, including P53 mutants (mtP53). TP53 mutations accounted for approximately 13%, 40%, 80%, 33%, 71%, and 82% of luminal A, Luminal B, Luminal C, normal basal-like, HER2-amplified, and basal-like breast cancers, respectively. TP53 mutation can aggravate cell genome instability and enhance the invasion, migration, and resistance of cancer cell. This review describes the research status of AURKA and HSP90 in breast cancer, summarizes the structure, function, and the chaperone cycle of HSP90, elaborates the interrelation between HSP90, mtP53, P53, and AURKA, and proposes the combination of HSP90 inhibitor and AURKA inhibitor to treat breast cancer. Targeting AURKA and HSP90 to treat cancer with AURKA overexpression and TP53 mutations will help improve the specificity and efficiency of breast cancer treatment and solve the problem of drug resistance.

Design, synthesis and biological evaluation of acyl hydrazones-based derivatives as RXRα-targeted anti-mitotic agents

RXRα, a unique and important nuclear receptor, plays a vital role in various biological and pathological pathways, including growth, differentiation, and apoptosis. We recently reported a transcription-independent function of RXRα in cancer cells in which RXRα is phosphorylated by Cdk1 at the onset of mitosis, resulting in its translocation to the centrosome, where the phosphorylated RXRα (p-RXRα) interacts with polo-like kinase 1 (PLK1) to promote centrosome maturation and mitotic progression. Significantly, we also identified that a small molecule XS-060 binds to RXRα and selectively inhibits the p-RXRα/PLK1 interaction to induce mitotic arrest and catastrophe in cancer cells. Here, we report our design, synthesis, and biological evaluation of a series of XS-060 analogs as RXRα-targeted anti-mitotic agents. Our results identified B10 as an improved anti-mitotic agent. B10 bound to RXRα (Kd = 3.04 ± 0.58 μM) and inhibited the growth of cervical cancer cells (HeLa, IC50 = 1.46 ± 0.10 μM) and hepatoma cells (HepG2, IC50 = 3.89 ± 0.45 μM and SK-hep-1, IC50 = 5.74 ± 0.50 μM) with low cytotoxicity to nonmalignant cells(LO2, IC50 > 50 μM). Furthermore, our mechanistic studies confirmed that B10 acted as an anticancer agent by inhibiting the p-RXRα/PLK1 pathway. These results provide a basis for further investigation and optimization of RXRα-targeted anti-mitotic molecules for cancer therapy.

The ciliopathy protein CCDC66 controls mitotic progression and cytokinesis by promoting microtubule nucleation and organization.

Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division and signaling via the primary cilium in quiescent cells. Microtubule-associated proteins (MAPs) govern assembly, maintenance, and remodeling of diverse microtubule arrays. While a set of conserved MAPs are only active during cell division, an emerging group of MAPs acts as dual regulators in dividing and nondividing cells. Here, we elucidated the nonciliary functions and molecular mechanism of action of the ciliopathy-linked protein CCDC66, which we previously characterized as a regulator of ciliogenesis in quiescent cells. We showed that CCDC66 dynamically localizes to the centrosomes, the bipolar spindle, the spindle midzone, the central spindle, and the midbody in dividing cells and interacts with the core machinery of centrosome maturation and MAPs involved in cell division. Loss-of-function experiments revealed its functions during mitotic progression and cytokinesis. Specifically, CCDC66 depletion resulted in defective spindle assembly and orientation, kinetochore fiber stability, chromosome alignment in metaphase as well as central spindle and midbody assembly and organization in anaphase and cytokinesis. Notably, CCDC66 regulates mitotic microtubule nucleation via noncentrosomal and centrosomal pathways via recruitment of gamma-tubulin to the centrosomes and the spindle. Additionally, CCDC66 bundles microtubules in vitro and in cells by its C-terminal microtubule-binding domain. Phenotypic rescue experiments showed that the microtubule and centrosome-associated pools of CCDC66 individually or cooperatively mediate its mitotic and cytokinetic functions. Collectively, our findings identify CCDC66 as a multifaceted regulator of the nucleation and organization of the diverse mitotic and cytokinetic microtubule arrays and provide new insight into nonciliary defects that underlie ciliopathies.

Abstract 1551: miR-625-3p enhances chemosensitivity in ovarian cancer cells through exosomal export of cisplatin and paclitaxel

Abstract Although a majority of women with high-grade serous ovarian cancer (HGSC) achieve a complete response to first-line platinum- and paclitaxel-based chemotherapy, around thirty percent of patients are identified as incomplete responders. Their diseases typically either does not respond or progresses during treatment (refractory) or recurs within 6 months of completing treatment (resistant), suggesting the inherit or intrinsic characteristics of the tumor. Currently, there are no biomarkers that can predict intrinsic resistance, which can avoid unnecessary treatment and toxicity. MicroRNAs (miRNAs) provide a novel master layer of regulation of gene expression. However, the molecular mechanisms by which miRNAs confer a chemoresistant phenotype in ovarian cancer have not been elucidated. To identify miRNAs that are associated with intrinsic chemoresistance in HGSC, Ion Torrent next-generation sequencing analysis of miRNAs was performed on microdissected chemosensitive and chemorefractory primary HGSC cases. Amongst all the miRNAs that were significantly down-regulated in chemorefractory cases, lower miR-625-3p expression was found to be associated with poorer overall and progression-free survivals. Further functional studies showed that overexpression of miR-625-3p significantly decreased cisplatin and paclitaxel resistance in ovarian cancer cells in vitro and in vivo. Online target prediction algorithms together with transcriptome profiling of HGSC cells transfected with miR-625-3p mimics or control mimics identified SSX2IP as a direct target of miR-625-3p, suggesting that SSX2IP may play a role in conferring cisplatin/paclitaxel resistance in HGSC by mediating the effect of miR-625-3p. SSX2IP is a microtubule anchoring and centriolar satellite protein, which plays a critical role in controlling microtubule length and orientation, and centrosome maturation. We demonstrated that HGSC cells transfected with SSX2IP had marked increases in microtubule masses, intracellular multivesicular body trafficking and exosomal cisplatin level; and a decrease in intracellular cisplatin level. In conclusion, down-regulation of miR-625-3p confers cisplatin/paclitaxel resistance in ovarian cancer cells via its novel direct target SSX2IP. SSX2IP alters the microtubule dynamics and subsequently mediates inter-organelle crosstalks between microtubules and multivesicular bodies, which facilitate exosomal export of cisplatin and paclitaxel of HGSC cells. This study is crucial for developing new predictive biomarkers for intrinsic chemoresistance, and new treatment strategies for HGSC based on upregulating miR-625-3p or downregulating SSX2IP expression in ovarian cancer cells, which will enhance cisplatin and paclitaxel sensitivity, and improve patient survival rates. Citation Format: Chi Lam Au Yeung, Tetsushi Tsuruga, Kay-Pong Yip, Sammy Ferri-Borgogno, Samuel Mok. miR-625-3p enhances chemosensitivity in ovarian cancer cells through exosomal export of cisplatin and paclitaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1551.

Abstract LB568: Protein phosphatase 4 deficiency protects mice against cerulein-induced acute pancreatitis

Abstract Background: Pancreatitis is an inflammatory disease of the pancreas that causes significant morbidity and mortality and is one of the major risk factors for pancreatic cancer. Protein phosphatase 4 (PP4) has been reported to be overexpressed in breast, lung and pancreatic cancers. PP4 plays an important role in the regulation of centrosome maturation, DNA repair, NF-κB, and c-jun-NH2-kinase (JNK) signaling pathways. However, the expression and functions of PP4 in pancreatitis have not been studied. Methods: To examine the functions of PP4 in cerulein-induced acute pancreatitis (AP), we crossed PP4flox/+ mice with pancreatic acinar cell specific cre, CreERT, which was driven by a full length pancreatic acinar specific elastase I promoter. PP4flox/flox, CreERT mice and control mice were treated with 8 hourly intraperitoneal injections of cerulein (50 µg/kg/h). The pancreata were harvested at different time points for histopathological examination and molecular analysis. Results: PP4 expression was increased at both protein and mRNA levels in mouse pancreas with AP. Compared to CreERT mice, PP4flox/flox, CreERT mice demonstrated significantly less edema, less inflammation, less necrosis or loss of pancreatic acinar cells in pancreas after cerulein treatment. The serum amylase and lipase levels were lower in cerulein-treated PP4flox/flox, CreERT mice compared with controls. The pancreata of PP4flox/flox, CreERT mice exhibited decreased STAT3 Tyr705 phosphorylation and decreased NF-κB activation cerulein-treated PP4flox/flox, CreERT mice than control mice. Conclusion: Our findings demonstrated that PP4 plays an important role in cerulein-induced AP through STAT3 and NF-κB signaling pathways. Inhibition of PP4 may help to reduce the pancreatic injuries and to prevent the development of chronic pancreatitis, which increases the risk of pancreatic cancer. Citation Format: Hua Wang, Reza Abbasgholizadeh, Rohan Moniruzzaman, Baoan Ji, Anirban Maitra, Huamin Wang. Protein phosphatase 4 deficiency protects mice against cerulein-induced acute pancreatitis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB568.