Chromosomal Passenger Protein Complex Research Articles

Dynamic localization of the chromosomal passenger complex in trypanosomes is controlled by the orphan kinesins KIN-A and KIN-B.

The chromosomal passenger complex (CPC) is an important regulator of cell division, which shows dynamic subcellular localization throughout mitosis, including kinetochores and the spindle midzone. In traditional model eukaryotes such as yeasts and humans, the CPC consists of the catalytic subunit Aurora B kinase, its activator INCENP, and the localization module proteins Borealin and Survivin. Intriguingly, Aurora B and INCENP as well as their localization pattern are conserved in kinetoplastids, an evolutionarily divergent group of eukaryotes that possess unique kinetochore proteins and lack homologs of Borealin or Survivin. It is not understood how the kinetoplastid CPC assembles nor how it is targeted to its subcellular destinations during the cell cycle. Here, we identify two orphan kinesins, KIN-A and KIN-B, as bona fide CPC proteins in Trypanosoma brucei, the kinetoplastid parasite that causes African sleeping sickness. KIN-A and KIN-B form a scaffold for the assembly of the remaining CPC subunits. We show that the C-terminal unstructured tail of KIN-A interacts with the KKT8 complex at kinetochores, while its N-terminal motor domain promotes CPC translocation to spindle microtubules. Thus, the KIN-A:KIN-B complex constitutes a unique 'two-in-one' CPC localization module, which directs the CPC to kinetochores from S phase until metaphase and to the central spindle in anaphase. Our findings highlight the evolutionary diversity of CPC proteins and raise the possibility that kinesins may have served as the original transport vehicles for Aurora kinases in early eukaryotes.

Dynamic localization of the chromosomal passenger complex in trypanosomes is controlled by the orphan kinesins KIN-A and KIN-B

The chromosomal passenger complex (CPC) is an important regulator of cell division, which shows dynamic subcellular localization throughout mitosis, including kinetochores and the spindle midzone. In traditional model eukaryotes such as yeasts and humans, the CPC consists of the catalytic subunit Aurora B kinase, its activator INCENP, and the localization module proteins Borealin and Survivin. Intriguingly, Aurora B and INCENP as well as their localization pattern are conserved in kinetoplastids, an evolutionarily divergent group of eukaryotes that possess unique kinetochore proteins and lack homologs of Borealin or Survivin. It is not understood how the kinetoplastid CPC assembles nor how it is targeted to its subcellular destinations during the cell cycle. Here, we identify two orphan kinesins, KIN-A and KIN-B, as bona fide CPC proteins in Trypanosoma brucei, the kinetoplastid parasite that causes African sleeping sickness. KIN-A and KIN-B form a scaffold for the assembly of the remaining CPC subunits. We show that the C-terminal unstructured tail of KIN-A interacts with the KKT8 complex at kinetochores, while its N-terminal motor domain promotes CPC translocation to spindle microtubules. Thus, the KIN-A:KIN-B complex constitutes a unique ‘two-in-one’ CPC localization module, which directs the CPC to kinetochores from S phase until metaphase and to the central spindle in anaphase. Our findings highlight the evolutionary diversity of CPC proteins and raise the possibility that kinesins may have served as the original transport vehicles for Aurora kinases in early eukaryotes.

AURKB promotes bladder cancer progression by deregulating the p53 DNA damage response pathway via MAD2L2

BackgroundBladder cancer (BC) is the most common urinary tract malignancy. Aurora kinase B (AURKB), a component of the chromosomal passenger protein complex, affects chromosomal segregation during cell division. Mitotic arrest-deficient 2-like protein 2 (MAD2L2) interacts with various proteins and contributes to genomic integrity. Both AURKB and MAD2L2 are overexpressed in various human cancers and have synergistic oncogenic effects; therefore, they are regarded as emerging therapeutic targets for cancer. However, the relationship between these factors and the mechanisms underlying their oncogenic activity in BC remains largely unknown. The present study aimed to explore the interactions between AURKB and MAD2L2 and how they affect BC progression via the DNA damage response (DDR) pathway.MethodsBioinformatics was used to analyze the expression, prognostic value, and pro-tumoral function of AURKB in patients with BC. CCK-8 assay, colony-forming assay, flow cytometry, SA-β-gal staining, wound healing assay, and transwell chamber experiments were performed to test the viability, cell cycle progression, senescence, and migration and invasion abilities of BC cells in vitro. A nude mouse xenograft assay was performed to test the tumorigenesis ability of BC cells in vivo. The expression and interaction of proteins and the occurrence of the senescence-associated secretory phenotype were detected using western blot analysis, co-immunoprecipitation assay, and RT-qPCR.ResultsAURKB was highly expressed and associated with prognosis in patients with BC. AURKB expression was positively correlated with MAD2L2 expression. We confirmed that AURKB interacts with, and modulates the expression of, MAD2L2 in BC cells. AURKB knockdown suppressed the proliferation, migration, and invasion abilities of, and cell cycle progression in, BC cells, inducing senescence in these cells. The effects of AURKB knockdown were rescued by MAD2L2 overexpression in vitro and in vivo. The effects of MAD2L2 knockdown were similar to those of AURKB knockdown. Furthermore, p53 ablation rescued the MAD2L2 knockdown-induced suppression of BC cell proliferation and cell cycle arrest and senescence in BC cells.ConclusionsAURKB activates MAD2L2 expression to downregulate the p53 DDR pathway, thereby promoting BC progression. Thus, AURKB may serve as a potential molecular marker and a novel anticancer therapeutic target for BC.

Abstract LB_C10: An orally available small molecule inhibitor for synthetic lethal targeting of MYC expressing tumors

Abstract The potential for synthetic lethality occurs when the same oncogenic events that promote carcinogenesis, also create vulnerabilities for cancer cells. Targeting cancers with these vulnerabilities, but not resident normal tissues, is the promise of synthetic lethal therapies. The compound LC30 is a potent and orally bioavailable compound that is synthetic lethal with deregulated MYC. No off-target liabilities have been demonstrated by kinome and safety profiling and long-term treatment is well tolerated by rodents and canines. LC30 is active against a wide spectrum of cell line xenografts derived from both liquid and solid cancers, including B and T cell malignancies, lung, colon, gastric, mammary and liver cancer cell lines, with efficacy that correlates with levels of MYC. Experiments aimed at unlocking the key to LC30’s synthetic lethality have revealed that LC30 is active in both resting cells and during cell division, so it is not simply an anti-mitotic compound. In resting cells, LC30 disrupts golgi structure, a phenotype that possibly relates to MYC control of centrosomal gene expression. Anti-angiogenic effects are observed in xenografts and have been confirmed using in vitro assays of endothelial cell migration and tubule formation. We speculate that the golgi phenotype and resulting faulty trafficking of secretory vesicles containing pro-angiogenic factors may underly the vascular phenotype. In dividing cells, LC30 potently deregulates the positioning of the chromosomal passenger protein complex (CPPC), a mitotic complex comprised of a catalytic subunit, Aurora B kinase (AURKB), the scaffolding protein inner centromere protein (INCENP) as well as survivin and borealin. The CPPC is dynamically relocated during mitotic progression so that AURKB activity can be localized to specific locations where activity is required. The phenotypic effects of LC30 treatment are consistent with this compound functioning as an inhibitor of the localization, but not catalytic activity of the CPPC. This includes the induction of multipolar spindles, micronuclei and polyploidy, leading to mitotic arrest and both mitotic and post-mitotic apoptosis. This unique mode-of-action, to disrupt CPPC passage in the mitotic cell, is a key component of the MYC synthetic lethality as cells without MYC overexpression do not develop multipolar spindles, lethal polyploidy and apoptose with treatment. LC30 represents a new class of anticancer compound that is a non-kinase, synthetic lethal inhibitor that unlocks MYC-induced vulnerabilities in cancer cells. Citation Format: Qiong Shi, Ting Zhang, Julia Kalashova, Jinhua Li, Chenglu Yang, Hongmei Li, Xiaohu Zhou, Yan Long, Yidan Xia Abuliezi, Gang Lv, Duo Yu, Shenqiu Zhang, Jing Zhang, Thaddeus D Allen, Hong Liu, Dun Yang. An orally available small molecule inhibitor for synthetic lethal targeting of MYC expressing tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C10.

Synthetic Antibodies Detect Distinct Cellular States of Chromosome Passenger Complex Proteins

High performance affinity reagents are essential tools to enable biologists to profile the cellular location and composition of macromolecular complexes undergoing dynamic reorganization. To support further development of such tools, we have assembled a high-throughput phage display pipeline to generate Fab-based affinity reagents that target different dynamic forms of a large macromolecular complex, using the Chromosomal Passenger Complex (CPC), as an example. The CPC is critical for the maintenance of chromosomal and cytoskeleton processes during cell division. The complex contains 4 protein components: Aurora B kinase, survivin, borealin and INCENP. The CPC acts as a node to dynamically organize other partnering subcomplexes to build multiple functional structures during mitotic progression. Using phage display mutagenesis, a cohort of synthetic antibodies (sABs) were generated against different domains of survivin, borealin and INCENP. Immunofluorescence established that a set of these sABs can discriminate between the form of the CPC complex in the midbody versus the spindle. Others localize to targets, which appear to be less organized, in the nucleus or cytoplasm. This differentiation suggests that different CPC epitopes have dynamic accessibility depending upon the mitotic state of the cell. An Immunoprecipitation/Mass Spectrometry analysis was performed using sABs that bound specifically to the CPC in either the midbody or MT spindle macromolecular assemblies. Thus, sABs can be exploited as high performance reagents to profile the accessibility of different components of the CPC within macromolecular assemblies during different stages of mitosis suggesting this high throughput approach will be applicable to other complex macromolecular systems.

The phytochemical, corynoline, diminishes Aurora kinase B activity to induce mitotic defect and polyploidy

Plants are a rich source for bioactive compounds. However, plant extracts can harbor a mixture of bioactive molecules that promote divergent phenotypes and potentially have confounding effects in bioassays. Even with further purification and identification, target deconvolution can be challenging. Corynoline and acetylcorynoline, are phytochemicals that were previously isolated through a screen for compounds able to induce mitotic arrest and polyploidy in oncogene expressing retinal pigment epithelial (RPE) cells. Here, we shed light on the mechanism by which these phytochemicals can attack human cancer cells. Mitotic arrest was coincident to the induction of centrosome amplification and declustering, causing multi-polar spindle formation. Corynoline was demonstrated to have true centrosome declustering activity in a model where A549 cells were chemically induced to have more than a regular complement of centrosomes. Corynoline could inhibit the centrosome clustering required for pseudo-bipolar spindle formation in these cells. The activity of AURKB, but not AURKA or polo-like kinase 4, was diminished by corynoline. It only partially inhibited AURKB, so it may be a partial antagonist or corynoline may work upstream on an unknown regulator of AURKB activity or localization. Nonetheless, corynoline and acetylcorynoline inhibited the viability of a variety of human cancer derived cell lines. These phytochemicals could serve as prototypes for a next-generation analog with improved potency, selectivity or in vivo bioavailability. Such an analog could be useful as a non-toxic component of combination therapies where inhibiting the chromosomal passenger protein complex is desired.

Synthetic Antibody Binders Detect Distinct Cellular States of Chromosome Passenger Complex Proteins

Synthetic Antibody Binders Detect Distinct Cellular States of Chromosome Passenger Complex Proteins

The Phytochemical Scoulerine Inhibits Aurora Kinase Activity to Induce Mitotic and Cytokinetic Defects.

To identify novel bioactive compounds, an image-based, cell culture screening of natural product extracts was conducted. Specifically, our screen was designed to identify phytochemicals that might phenocopy inhibition of the chromosomal passenger protein complex in eliciting mitotic and cytokinetic defects. A known alkaloid, scoulerine, was identified from the rhizomes of the plant Corydalis decumbens as being able to elicit a transient mitotic arrest followed by either apoptosis induction or polyploidy. In examining the mitotic abnormality further, we observed that scoulerine could elicit supernumerary centrosomes during mitosis, but not earlier in the cell cycle. The localization of NUMA1 at spindle poles was also inhibited, suggesting diminished potential for microtubule recruitment and spindle-pole focusing. Polyploid cells emerged subsequent to cytokinetic failure. The concentration required for scoulerine to elicit all its cell division phenotypes was similar, and an examination of related compounds highlighted the requirement for proper positioning of a hydroxyl and a methoxy group about an aromatic ring for activity. Mechanistically, scoulerine inhibited AURKB activity at concentrations that elicited supernumerary centrosomes and polyploidy. AURKA was only inhibited at higher concentrations, so AURKB inhibition is the likely mechanism by which scoulerine elicited division defects. AURKB inhibition was never complete, so scoulerine may be a suboptimal AURK inhibitor or work upstream of the chromosomal passenger protein complex to reduce AURKB activity. Scoulerine inhibited the viability of a variety of human cancer cell lines. Collectively, these findings uncover a previously unknown activity of scoulerine that could facilitate targeting human cancers. Scoulerine, or a next-generation analogue, may be useful as a nontoxic component of combination therapies where inhibiting the chromosomal passenger protein complex is desired.

Aurora Kinase B Inhibition: A Potential Therapeutic Strategy for Cancer.

Aurora kinase B (AURKB) is a mitotic serine/threonine protein kinase that belongs to the aurora kinase family along with aurora kinase A (AURKA) and aurora kinase C (AURKC). AURKB is a member of the chromosomal passenger protein complex and plays a role in cell cycle progression. Deregulation of AURKB is observed in several tumors and its overexpression is frequently linked to tumor cell invasion, metastasis and drug resistance. AURKB has emerged as an attractive drug target leading to the development of small molecule inhibitors. This review summarizes recent findings pertaining to the role of AURKB in tumor development, therapy related drug resistance, and its inhibition as a potential therapeutic strategy for cancer. We discuss AURKB inhibitors that are in preclinical and clinical development and combination studies of AURKB inhibition with other therapeutic strategies.

Impaired condensin complex and Aurora B kinase underlie mitotic and chromosomal defects in hyperdiploid B-cell ALL

AbstractB-cell acute lymphoblastic leukemia (ALL; B-ALL) is the most common pediatric cancer, and high hyperdiploidy (HyperD) identifies the most common subtype of pediatric B-ALL. Despite HyperD being an initiating oncogenic event affiliated with childhood B-ALL, the mitotic and chromosomal defects associated with HyperD B-ALL (HyperD-ALL) remain poorly characterized. Here, we have used 54 primary pediatric B-ALL samples to characterize the cellular-molecular mechanisms underlying the mitotic/chromosome defects predicated to be early pathogenic contributors in HyperD-ALL. We report that HyperD-ALL blasts are low proliferative and show a delay in early mitosis at prometaphase, associated with chromosome-alignment defects at the metaphase plate leading to robust chromosome-segregation defects and nonmodal karyotypes. Mechanistically, biochemical, functional, and mass-spectrometry assays revealed that condensin complex is impaired in HyperD-ALL cells, leading to chromosome hypocondensation, loss of centromere stiffness, and mislocalization of the chromosome passenger complex proteins Aurora B kinase (AURKB) and Survivin in early mitosis. HyperD-ALL cells show chromatid cohesion defects and an impaired spindle assembly checkpoint (SAC), thus undergoing mitotic slippage due to defective AURKB and impaired SAC activity, downstream of condensin complex defects. Chromosome structure/condensation defects and hyperdiploidy were reproduced in healthy CD34+ stem/progenitor cells upon inhibition of AURKB and/or SAC. Collectively, hyperdiploid B-ALL is associated with a defective condensin complex, AURKB, and SAC.

Kinetochore proteins suppress neuronal microtubule dynamics and promote dendrite regeneration.

Kinetochores connect centromeric chromatin to spindle microtubules during mitosis. Neurons are postmitotic, so it was surprising to identify transcripts of structural kinetochore (KT) proteins and regulatory chromosome passenger complex (CPC) and spindle assembly checkpoint (SAC) proteins in Drosophila neurons after dendrite injury. To test whether these proteins function during dendrite regeneration, postmitotic RNA interference (RNAi) was performed and dendrites or axons were removed using laser microsurgery. Reduction of KT, CPC, and SAC proteins decreased dendrite regeneration without affecting axon regeneration. To understand whether neuronal functions of these proteins rely on microtubules, we analyzed microtubule behavior in uninjured neurons. The number of growing plus, but not minus, ends increased in dendrites with reduced KT, CPC, and SAC proteins, while axonal microtubules were unaffected. Increased dendritic microtubule dynamics was independent of dual leucine zipper kinase (DLK)-mediated stress but was rescued by concurrent reduction of γ-tubulin, the core microtubule nucleation protein. Reduction of γ-tubulin also rescued dendrite regeneration in backgrounds containing kinetochore RNAi transgenes. We conclude that kinetochore proteins function postmitotically in neurons to suppress dendritic microtubule dynamics by inhibiting nucleation.

Direct Phosphorylation and Stabilization of MYC By Aurora B Kinase Promotes T Cell Leukemogenesis

The aberrant activation of oncogene MYC (also termed c-MYC) is one of the most common features in human cancer. The unleashed MYC oncogene frequently produces abundant MYC protein, which mediates a transcriptional response involved in a variety of biological processes, contributing to almost every aspect of tumorigenesis. The significance of MYC deregulation has been recognized in T-cell acute lymphoblastic leukemia (T-ALL), a life-threatening hematological malignancy with dismal outcome due to disease relapse and drug resistance. Therapeutic efforts aimed at inhibiting MYC expression or activity should have an important clinical relevance. However, attempts to directly disrupt MYC function have met with limited success, in part due to its "undruggable" protein structure. Aurora kinases, a multi-genic family of serine/threonine kinases, consist Aurora A (AURKA), Aurora B (AURKB) and Aurora C (AURKC). They are known to play an integral role in the regulation of cell division and chromosomal segregation. Amplification or overexpression of Aurora kinases is frequently found in human cancers with clear evidence of oncogenic potential, implicating Aurora kinases as rational anti-tumor targets. AURKB is the catalytic subunit of the chromosomal passenger protein complex (CPPC) which regulates multiple facets of cell division. Overexpression of AURKB has been reported in a variety of cancers and predicts poor overall survival. AZD1152 is a highly potent and selective inhibitor of Aurora B; preclinical evidence of anti-tumor efficacy with AZD1152 has extended to the clinical setting with tolerable toxicity. Despite considerable study, it remains largely unclear how expression of AURKB is elevated and, in particular, how elevated levels of AURKB reprogram cells to promote the cancer progression. We identified AURKB as a novel MYC binding partner using liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. To assess the potential role of AURKB in regulating MYC, we inhibited AURKB in multiple T-ALL cell lines and found that MYC protein expression was diminished significantly. Notably, depletion of AURKB failed to downregulate MYC mRNA, suggesting a post-transcriptional regulation of the MYC protein. We then assessed MYC protein decay in the presence or absence of AURKB expression. Time course experiments revealed that knockdown of AURKB significantly shortened the half-life of endogenous MYC in T-ALL cells. Consistently, AURKB depletion resulted in significant downregulation of MYC-induced gene expression programs, corroborating an essential role of AURKB in sustaining MYC transcriptional activities. Regulation of MYC degradation by ubiquitin-proteasome system is dependent on MYC phosphorylation. Initiation of MYC turnover requires GSK3b-mediated phosphorylation of threonine 58 (p-T58). Time-course analysis showed that treatment with AZD1152 in T-ALL cells resulted in a notable increase in MYC p-T58. We performed in vitro kinase assays and found that MYC is phosphorylated directly by AURKB. This phosphorylation counteracted GSK3b-directed Thr58 phosphorylation and subsequent FBW7-mediated proteasomal degradation and enhanced the MYC protein stability. The AURKB mRNA and protein are generally more abundant in human T-ALL than normal thymocytes. We demonstrated that MYC, in concert with TAL1, directly binds to the AURKB promoter and activates its transcription, thus constituting a reciprocal regulation between MYC and AURKB. In order to clarify the biological significance of the AURKB-MYC circuit, we co-injected the one-cell-stage zebrafish embryos with the rag2:EGFP-Myc construct alone or together with rag2:AURKB followed by fluorescent microscopy analysis. Our results demonstrate that AURKB phosphorylation of MYC is functionally important for T cell leukemogenesis in vivo. Moreover, inhibition of AURKB elicits dramatic MYC degradation in association with robust apoptosis in wild type-FBW7 T-ALL cells, suggesting that FBW7 mutational status may serve as a genetic predictor of sensitivity to AURKB inhibitors. In this study, we unravel a non-mitotic role of AURKB in promoting MYC stabilization via direct MYC phosphorylation at an unconventional site. Our findings decipher a previously unsuspected mechanism involved in MYC deregulation and highlight disruption of the AURKB-MYC signaling circuit as a promising T-ALL therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

Functional SNPs of INCENP Affect Semen Quality by Alternative Splicing Mode and Binding Affinity with the Target Bta-miR-378 in Chinese Holstein Bulls

Inner centromere protein (INCENP) plays an important role in mitosis and meiosis as the main member of chromosomal passenger protein complex (CPC). To investigate the functional markers of the INCENP gene associated with semen quality, the single nucleotide polymorphisms (SNPs) g.19970 A>G and g.34078 T>G were identified and analyzed. The new splice variant INCENP-TV is characterized by the deletion of exon 12. The g.19970 A>G in the exonic splicing enhancer (ESE) motif region results in an aberrant splice variant by constructing two minigene expression vectors using the pSPL3 exon capturing vector and transfecting vectors into MLTC-1 cells. INCENP-TV was more highly expressed than INCENP-reference in adult bull testes. The g.34078 T>G located in the binding region of bta-miR-378 could affect the expression of INCENP, which was verified by luciferase assay. To analyze comprehensively the correlation of SNPs with sperm quality, haplotype combinations constructed by g.19970 A>G and g.34078 T>G, as well as g.-692 C>T and g.-556 G>T reported in our previous studies, were analyzed. The bulls with H1H12 and H2H2 exhibited a higher ejaculate volume than those with H2H10 and H9H12, respectively (P < 0.05). Bulls with H11H11 and H2H10 exhibited higher initial sperm motility than those with H2H2 (P < 0.05). The expression levels of INCENP in bulls with H1H12 and H11H11 were significantly higher than those in bulls with H9H12 (P < 0.05), as determined by qRT-PCR. Findings suggest that g.19970 A>G and g.34078 T>G in INCENP both of which appear to change the molecular and biological characteristics of the mRNA transcribed from the locus may serve as a biomarkers of male bovine fertility by affecting alternative splicing mode and binding affinity with the target bta-miR-378.

Chromosome segregation regulation in human zygotes: altered mitotic histone phosphorylation dynamics underlying centromeric targeting of the chromosomal passenger complex.

Are the kinase feedback loops that regulate activation and centromeric targeting of the chromosomal passenger complex (CPC), functional during mitosis in human embryos? Investigation of the regulatory kinase pathways involved in centromeric CPC targeting revealed normal phosphorylation dynamics of histone H2A at T120 (H2ApT120) by Bub1 kinase and subsequent recruitment of Shugoshin, but phosphorylation of histone H3 at threonine 3 (H3pT3) by Haspin failed to show the expected centromeric enrichment on metaphase chromosomes in the zygote. Human cleavage stage embryos show high levels of chromosomal instability. What causes this high error rate is unknown, as mechanisms used to ensure proper chromosome segregation in mammalian embryos are poorly described. In this study, we investigated the pathways regulating CPC targeting to the inner centromere in human embryos. We characterized the distribution of the CPC in relation to activity of its two main centromeric targeting pathways: the Bub1-H2ApT120-Sgo-CPC and Haspin-H3pT3-CPC pathways. The study was conducted between May 2012 and March 2014 on human surplus embryos resulting from in vitro fertilization treatment and donated for research. In zygotes, nuclear envelope breakdown was monitored by time-lapse imaging to allow timed incubations with specific inhibitors to arrest at prometaphase and metaphase, and to interfere with Haspin and Aurora B/C kinase activity. Functionality of the targeting pathways was assessed through characterization of histone phosphorylation dynamics by immunofluorescent analysis, combined with gene expression by RT-qPCR and immunofluorescent localization of key pathway proteins. Immunofluorescent analysis of the CPC subunit Inner Centromere Protein revealed the pool of stably bound CPC proteins was not strictly confined to the inner centromere of prometaphase chromosomes in human zygotes, as observed in later stages of preimplantation development and somatic cells. Investigation of the regulatory kinase pathways involved in centromeric CPC targeting revealed normal phosphorylation dynamics of histone H2A at T120 (H2ApT120) by Bub1 kinase and subsequent recruitment of Shugoshin. However, phosphorylation of histone H3 at threonine 3 (H3pT3) by Haspin kinase failed to show the expected centromeric enrichment on metaphase chromosomes in the zygote, but not at later stages. Inhibition of Haspin revealed this activity to be essential for proper mitotic checkpoint complex activation in human zygotes, thus demonstrating an active mitotic checkpoint under normal conditions. Abolishment of H3pT3 during zygotic prometaphase further shows that centromeric H2ApT120 alone is not sufficient for proper shugoshin and CPC localization. As the removal of H3pT3 from the chromosome arms during prometaphase normally contributes to further centromeric enrichment of the CPC in somatic cells, CPC targeting may be less accurate in human zygotes. Owing to ethical limitations, tripronuclear zygotes were used in functional experiments. Although these represent the best available models, it is unknown if they are completely representative for dipronuclear zygotes. In addition, further research is needed to determine to what extent the differences we observed in H3T3 phosphorylation dynamics and CPC localization affect chromosome attachment. In the zygote, paternal and maternal chromosomes coming from two separate pronuclei, and with contrasting epigenetic signatures, need to be aligned on a single metaphase plate. Our results suggest that adaptations in mechanisms regulating CPC targeting exist in the human zygote, to ensure symmetric recruitment despite the epigenetic asymmetry between maternal and paternal chromosomes. This adaptation may come at a price regarding chromosome segregation fidelity. This study was funded by the Portuguese Fundação para a Ciência e Tecnologia and the Netherlands Organization for Scientific Research. The authors have no conflicts of interest to declare.

Reed-Sternberg cells form by abscission failure in the presence of functional Aurora B kinase.

Large multinucleated Reed-Sternberg cells (RS) and large mononucleated Hodgkin cells (H) are traditionally considered to be the neoplastic population in classical Hodgkin lymphoma, (cHL) and postulated to promote the disease. However, the contribution of these larger cells to the progression of cHL remains debatable. We used established cHL cell lines and cHL cellular fractions composed of small mononucleated cells only or enriched in large RS/H cells to investigate RS/H cell origin and to characterize the cells which they derive from. We confirm that the small mononucleated cells give rise to RS/H cells, and we show that the latter proliferate significantly more slowly than the small cells. By using live-cell imaging, we demonstrate that binucleated RS cells are generated by failure of abscission when a few small cells attempt to divide. Finally, our results reveal that the small mononucleated cells are chromosomally unstable, but this is unlikely to be related to a malfunctioning chromosomal passenger protein complex. We propose that the small mononucleated cells, rather than the RS/H cells, are the main drivers of cHL.

A chromosomal passenger complex protein signature model predicts poor prognosis for non-small-cell lung cancer.

AimThe chromosomal passenger complex (CPC) acts as a key modulator for mitosis and cell cytokinesis. High levels of CPC proteins are frequently observed in multiple cancers and are correlated with more progressive malignant behaviors. The aim of the study was to evaluate whether CPC components or their combinations could be used to assess the clinical risk of patients with non-small-cell lung cancer (NSCLC).MethodsThe expression levels of four CPC proteins – aurora B kinase (AURKB), borealin, inner centromere protein (INCENP), and survivin – were evaluated using immunohistochemistry in an independent cohort of NSCLC specimens. A molecular predictor model was developed based on the combination of the four CPC proteins.ResultsAll the CPC components were overexpressed in NSCLC tumors compared with their paired adjacent normal lung tissues. Survivin overexpression was significantly correlated with late tumor stage (P=0.0166). High expressions of AURKB, INCENP, and survivin, but not borealin, were associated with shorter survival in patients with NSCLC. The constructed 4-CPC-gene model divided the cohort into two different subgroups with significantly different prognoses (hazard ratio, HR =2.8915 [95% confidence interval, CI: 1.5187–5.5052]; P=0.0013) and was retained as an independent prognostic factor in multivariate analysis (HR =2.4398 [95% CI: 1.2631–4.7127], P=0.0082). Moreover, the 4-CPC-gene model demonstrated a higher predictive ability for overall survival than each individual CPC biomarker.ConclusionTaken together, our study suggests that a molecular prognostic model based on simultaneous detection of CPC components could serve as a complement to current clinical risk stratification approaches for patients with NSCLC.

Chromatin Protein HP1α Interacts with the Mitotic Regulator Borealin Protein and Specifies the Centromere Localization of the Chromosomal Passenger Complex

Accurate mitosis requires the chromosomal passenger protein complex (CPC) containing Aurora B kinase, borealin, INCENP, and survivin, which orchestrates chromosome dynamics. However, the chromatin factors that specify the CPC to the centromere remain elusive. Here we show that borealin interacts directly with heterochromatin protein 1 (HP1) and that this interaction is mediated by an evolutionarily conserved PXVXL motif in the C-terminal borealin with the chromo shadow domain of HP1. This borealin-HP1 interaction recruits the CPC to the centromere and governs an activation of Aurora B kinase judged by phosphorylation of Ser-7 in CENP-A, a substrate of Aurora B. Consistently, modulation of the motif PXVXL leads to defects in CPC centromere targeting and aberrant Aurora B activity. On the other hand, the localization of the CPC in the midzone is independent of the borealin-HP1 interaction, demonstrating the spatial requirement of HP1 in CPC localization to the centromere. These findings reveal a previously unrecognized but direct link between HP1 and CPC localization in the centromere and illustrate the critical role of borealin-HP1 interaction in orchestrating an accurate cell division.

Molecular Mechanisms of Contractility-Based Cellular Mechanosensing

Mechanosensing is fundamental to numerous cellular functions, and myosin II is a core component of this mechanosensory machinery. Given the importance of successful cell division for maintaining genomic integrity, we reasoned that myosin II-mediated mechanosensing would be a significant factor in ensuring successful cytokinesis. We found that dividing cells are exquisitely sensitive to mechanical stress inputs, relocating myosin II to sites of the externally imposed stress. This system has the hallmark of a soft mechanical check-point, slowing cytokinesis until the deformation is corrected. The myosin II-based mechanosensing is performed by a three-part sensor, including force amplification through the myosin II lever arm, myosin bipolar thick filament (BTF) assembly, and actin filament anchoring by cortexillin I. Multi-scale modeling demonstrates how myosin II force-sensing and cooperative actin-binding couples to BTF assembly and quantitatively accounts for the amounts and kinetics of myosin mechanosensitive accumulation. Furthermore, forces are shared between myosin and different actin crosslinkers with myosin having potentiating or inhibitory effects on certain crosslinkers. Additionally, different types of cell deformations elicit distinct responses: myosin and α-actinin respond to dilation while filamin mainly reacts to shear. This myosin II-based mechanosensory system is part of a larger control system that tunes myosin accumulation at the cleavage furrow under diverse mechanical constraints. In this context, cortexillin I not only anchors the actin filaments, but also links to signal transduction proteins. These signaling proteins, including IQGAPs and the chromosomal passenger complex proteins, reinforce 'normal' signals that emanate from the mitotic spindle to direct myosin II accumulation. Overall, this control system demonstrates how mechanical inputs can be converted to signaling outputs in a manner analogous to chemical signal transduction.

The Chromosomal Passenger Protein Birc5b Organizes Microfilaments and Germ Plasm in the Zebrafish Embryo

Microtubule-microfilament interactions are important for cytokinesis and subcellular localization of proteins and mRNAs. In the early zebrafish embryo, astral microtubule-microfilament interactions also facilitate a stereotypic segregation pattern of germ plasm ribonucleoparticles (GP RNPs), which is critical for their eventual selective inheritance by germ cells. The precise mechanisms and molecular mediators for both cytoskeletal interactions and GP RNPs segregation are the focus of intense research. Here, we report the molecular identification of a zebrafish maternal-effect mutation motley as Birc5b, a homolog of the mammalian Chromosomal Passenger Complex (CPC) component Survivin. The meiosis and mitosis defects in motley/birc5b mutant embryos are consistent with failed CPC function, and additional defects in astral microtubule remodeling contribute to failures in the initiation of cytokinesis furrow ingression. Unexpectedly, the motley/birc5b mutation also disrupts cortical microfilaments and GP RNP aggregation during early cell divisions. Birc5b localizes to the tips of astral microtubules along with polymerizing cortical F-actin and the GP RNPs. Mutant Birc5b co-localizes with cortical F-actin and GP RNPs, but fails to associate with astral microtubule tips, leading to disorganized microfilaments and GP RNP aggregation defects. Thus, maternal Birc5b localizes to astral microtubule tips and associates with cortical F-actin and GP RNPs, potentially linking the two cytoskeletons to mediate microtubule-microfilament reorganization and GP RNP aggregation during early embryonic cell cycles in zebrafish. In addition to the known mitotic function of CPC components, our analyses reveal a non-canonical role for an evolutionarily conserved CPC protein in microfilament reorganization and germ plasm aggregation.

Abstract LB-287: TPX2 acts as a scaffold and co-activator protein for the oncogenic kinase Aurora B

Abstract Aurora B kinase is a master regulator of mitosis and is over-expressed in a variety of malignancies including astrocytomas, seminomas, ependymomas, prostate cancer and non small-cell lung cancer. Several Aurora B inhibitors are currently being evaluated for their efficacy in cancer therapy. Therefore, it is critical to understand the regulation of Aurora B activity by other proteins. Aurora B forms the enzymatic core of the Chromosomal Passenger Complex (CPC). The CPC consists of three other proteins besides Aurora B: INCENP, Survivin and Borealin. All these proteins have been known to promote Aurora B activation. The mitotic protein Targeting Protein for Xenopus kinesin-like protein 2 (TPX2) co-localizes with Aurora B at several stages of mitosis, hinting at the existence of a signaling cross-talk between Aurora B and TPX2. The main purpose of our study is to determine whether Aurora B and TPX2 can cross-talk with each other during mitosis, and if found so, to decode the mechanism by which this signaling cross-talk occurs. We hypothesize that TPX2 can transmit signals to regulate Aurora B activity during mitosis. To test our hypothesis, we have performed experiments utilizing M-phase Xenopus egg extracts (XEE) which possess an abundance of mitotic proteins, and human cell lines as model systems. By performing immunoprecipitation assays (IPs) in M-phase XEE, we detected an interaction between TPX2 and the CPC proteins - Aurora B, INCENP and Survivin. This validates the existence of a signaling cross-talk between TPX2 and the CPC proteins. Moreover, IPs and microtubule pelleting assays utilizing XEE revealed that, as is typical of scaffold proteins, either too much or too little TPX2 alters Aurora B activity. Significantly, immunodepletion of endogenous TPX2 from XEE decreases the association between Aurora B and its activators- Survivin and INCENP, leading to a consequent reduction in Aurora B activity. Additionally, in vitro binding assays demonstrate that, residues 138 to 328 of Xenopus TPX2 (TPX2 B) are sufficient to enhance the interaction between Aurora B and its activator, Survivin. Further, TPX2 B also increases Aurora B kinase activity in an in vitro kinase assay. These data suggest that under endogenous conditions, TPX2 functions as a co-activator of Aurora B by increasing Aurora B-Survivin and Aurora B-INCENP interactions. Importantly, IPs with Panc-1 and CFPAC-1 pancreatic cancer cell lines suggest that this mechanism of Aurora B activation by TPX2 is likely to be conserved in human cancer cell lines. Based upon our results, we conclude that TPX2 enhances Aurora B activity by serving as a scaffold protein for assembly of the CPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-287. doi:1538-7445.AM2012-LB-287