Spindle Checkpoint Activation Research Articles

Eg5 UFMylation promotes spindle organization during mitosis

UFMylation is a highly conserved ubiquitin-like post-translational modification that catalyzes the covalent linkage of UFM1 to its target proteins. This modification plays a critical role in the maintenance of endoplasmic reticulum proteostasis, DNA damage response, autophagy, and transcriptional regulation. Mutations in UFM1, as well as in its specific E1 enzyme UBA5 and E2 enzyme UFC1, have been genetically linked to microcephaly. Our previous research unveiled the important role of UFMylation in regulating mitosis. However, the underlying mechanisms have remained unclear due to the limited identification of substrates. In this study, we identified Eg5, a motor protein crucial for mitotic spindle assembly and maintenance, as a novel substrate for UFMylation and identified Lys564 as the crucial UFMylation site. UFMylation did not alter its transcriptional level, phosphorylation level, or protein stability, but affected the mono-ubiquitination of Eg5. During mitosis, Eg5 and UFM1 co-localize at the centrosome and spindle apparatus, and defective UFMylation leads to diminished spindle localization of Eg5. Notably, the UFMylation-defective Eg5 mutant (K564R) exhibited shorter spindles, metaphase arrest, spindle checkpoint activation, and a failure of cell division in HeLa cells. Overall, Eg5 UFMylation is essential for proper spindle organization, mitotic progression, and cell proliferation.

Chromosome segregation: Mps1 and Dam1 battle to bind a shared interaction site at the kinetochore

The attachment of kinetochores to spindle microtubules is highly regulated to ensure proper chromosome segregation. Three new studies identify an interaction hub at the kinetochore that integrates kinetochore attachment state with spindle checkpoint activity and kinetochore assembly.

Spindle checkpoint activation by fungal orthologs of the S. cerevisiae Mps1 kinase.

There is an ongoing need for antifungal agents to treat humans. Identification of new antifungal agents can be based on screening compounds using whole cell assays. Screening compounds that target a particular molecule is possible in budding yeast wherein sophisticated strain engineering allows for controlled expression of endogenous or heterologous genes. We have considered the yeast Mps1 protein kinase as a reasonable target for antifungal agents because mutant or druggable forms of the protein, upon inactivation, cause rapid loss of cell viability. Furthermore, extensive analysis of the Mps1 in budding yeast has offered potential tactics for identifying inhibitors of its enzymatic activity. One such tactic is based on the finding that overexpression of Mps1 leads to cell cycle arrest via activation of the spindle assembly checkpoint. We have endeavored to adapt this assay to be based on the overexpression of Mps1 orthologs from pathogenic yeast in hopes of having a whole-cell assay system to test the activity of these orthologs. Mps1 orthologous genes from seven pathogenic yeast or other pathogenic fungal species were isolated and expressed in budding yeast. Two orthologs clearly produced phenotypes similar to those produced by the overexpression of budding yeast Mps1, indicating that this system for heterologous Mps1 expression has potential as a platform for identifying prospective antifungal agents.

Meiotic cells escape prolonged spindle checkpoint activity through kinetochore silencing and slippage.

To prevent chromosome mis-segregation, a surveillance mechanism known as the spindle checkpoint delays the cell cycle if kinetochores are not attached to spindle microtubules, allowing the cell additional time to correct improper attachments. During spindle checkpoint activation, checkpoint proteins bind the unattached kinetochore and send a diffusible signal to inhibit the anaphase promoting complex/cyclosome (APC/C). Previous work has shown that mitotic cells with depolymerized microtubules can escape prolonged spindle checkpoint activation in a process called mitotic slippage. During slippage, spindle checkpoint proteins bind unattached kinetochores, but the cells cannot maintain the checkpoint arrest. We asked if meiotic cells had as robust of a spindle checkpoint response as mitotic cells and whether they also undergo slippage after prolonged spindle checkpoint activity. We performed a direct comparison between mitotic and meiotic budding yeast cells that signal the spindle checkpoint through two different assays. We find that the spindle checkpoint delay is shorter in meiosis I or meiosis II compared to mitosis, overcoming a checkpoint arrest approximately 150 minutes earlier in meiosis than in mitosis. In addition, cells in meiosis I escape spindle checkpoint signaling using two mechanisms, silencing the checkpoint at the kinetochore and through slippage. We propose that meiotic cells undertake developmentally-regulated mechanisms to prevent persistent spindle checkpoint activity to ensure the production of gametes.

A non-catalytic N-terminus domain of WRN prevents mitotic telomere deprotection

Telomeric ends form a loop structure (T-loop) necessary for the repression of ATM kinase activation throughout the normal cell cycle. However, cells undergoing a prolonged mitotic arrest are prone to lose the T-loop, resulting in Aurora B kinase-dependent mitotic telomere deprotection, which was proposed as an anti-tumor mechanism that eliminates precancerous cells from the population. The mechanism of mitotic telomere deprotection has not been elucidated. Here, we show that WRN, a RECQ helicase family member, can suppress mitotic telomere deprotection independently of its exonuclease and helicase activities. Truncation of WRN revealed that N-terminus amino acids 168–333, a region that contains a coiled-coil motif, is sufficient to suppress mitotic telomere deprotection without affecting both mitotic Aurora B-dependent spindle checkpoint and ATM kinase activity. The suppressive activity of the WRN168–333 fragment is diminished in cells partially depleted of TRF2, while WRN is required for complete suppression of mitotic telomere deprotection by TRF2 overexpression. Finally, we found that phosphomimetic but not alanine mutations of putative Aurora B target sites in the WRN168–333 fragment abolished its suppressive effect. Our findings reveal a non-enzymatic function of WRN, which may be regulated by phosphorylation in cells undergoing mitotic arrest. We propose that WRN enhances the protective function of TRF2 to counteract the hypothetical pathway that resolves the mitotic T-loop.

Aurora A phosphorylates Ndel1 to reduce the levels of Mad1 and NuMA at spindle poles.

Dynein inactivates the spindle assembly checkpoint (SAC) by transporting checkpoint proteins away from kinetochores toward spindle poles in a process known as "stripping." We find that inhibition of Aurora A kinase, which is localized to spindle poles, enables the accumulation of the spindle checkpoint activator Mad1 at poles where it is normally absent. Aurora kinases phosphorylate the dynein activator NudE neurodevelopment protein 1 like 1 (Ndel1) on Ser285 and Mad1 accumulates at poles when Ndel1 is replaced by a nonphosphorylatable mutant in human cells. The pole focusing protein NuMA, transported to poles by dynein, also accumulates at poles in cells harboring a mutant Ndel1. Phosphorylation of Ndel1 on Ser285 is required for robust spindle checkpoint activity and regulates the poles of asters inXenopusextracts. Our data suggest that dynein/SAC complexes that are generated at kinetochores and then transported directionally toward poles on microtubules are inhibited by Aurora A before they reach spindle poles. These data suggest that Aurora A generates a spatial signal at spindle poles that controls dynein transport and spindle function.

Analysis of the potential role of fission yeast PP2A in spindle assembly checkpoint inactivation.

As a surveillance mechanism, the activated spindle assembly checkpoint (SAC) potently inhibits the E3 ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) to ensure accurate chromosome segregation. Although the protein phosphatase 2A (PP2A) has been proposed to be both, directly and indirectly, involved in spindle assembly checkpoint inactivation in mammalian cells, whether it is similarly operating in the fission yeast Schizosaccharomycer pombe has never been demonstrated. Here, we investigated whether fission yeast PP2A is involved in SAC silencing by following the rate of cyclin B (Cdc13) destruction at SPBs during the recovery phase in nda3-KM311 cells released from the inhibition of APC/C by the activated spindle checkpoint. The timing of the SAC inactivation is only slightly delayed when two B56 regulatory subunits (Par1 and Par2) of fission yeast PP2A are absent. Overproduction of individual PP2A subunits either globally in the nda3-KM311 arrest-and-release system or locally in the synthetic spindle checkpoint activation system only slightly suppresses the SAC silencing defects in PP1 deletion (dis2Δ) cells. Our study thus demonstrates that the fission yeast PP2A is not a key regulator actively involved in SAC inactivation.

Ubc1 turnover contributes to the spindle assembly checkpoint in Saccharomyces cerevisiae.

The spindle assembly checkpoint protects the integrity of the genome by ensuring that chromosomes are properly attached to the mitotic spindle before they are segregated during anaphase. Activation of the spindle checkpoint results in inhibition of the Anaphase-Promoting Complex (APC), an E3 ubiquitin ligase that triggers the metaphase–anaphase transition. Here, we show that levels of Ubc1, an E2 enzyme that functions in complex with the APC, modulate the response to spindle checkpoint activation in Saccharomyces cerevisiae. Overexpression of Ubc1 increased resistance to microtubule poisons, whereas Ubc1 shut-off sensitized cells. We also found that Ubc1 levels are regulated by the spindle checkpoint. Checkpoint activation or direct APC inhibition led to a decrease in Ubc1 levels, charging, and half-life. Additionally, stabilization of Ubc1 prevented its down-regulation by the spindle checkpoint and increased resistance to checkpoint-activating drugs. These results suggest that down-regulation of Ubc1 in response to spindle checkpoint signaling is necessary for a robust cell cycle arrest.

INVESTIGATION OF PATHOGENIC VARIANTS OF BUB1 AND BUBR1 GENES IN THE ABORTED ANEUPLOID FETUSES

Chromosome aberrations certainly aneuploidies are the cause of the majority of spontaneous abortions in humans. Although the incidence of aneuploidies rises significantly with maternal age, it is frequent in young women. BUB1 (budding uninhibited by benzimidazole 1) and BUBR1(budding uninhibited by benzimidazole 1-related 1)are two key proteins mediating spindle-checkpoint activation that play a role in the inhibition of the anaphase-promoting complex/cyclosome (APC/C), delaying the onset of anaphase and ensuring proper chromosome segregation.

Transcriptomic analysis of nonylphenol effect on Saccharomyces cerevisiae.

Nonylphenol (NP) is a bioaccumulative environmental estrogen that is widely used as a nonionic surfactant. We have previously examined short-term effects of NP on yeast cells using microarray technology. In the present study, we investigated the adaptive response of Saccharomyces cerevisiae BY4742 cells to NP exposure by analyzing genome-wide transcriptional profiles using RNA-sequencing. We used 2 mg/L NP concentration for 40 days of exposure. Gene expression analysis showed that a total of 948 genes were differentially expressed. Of these, 834 genes were downregulated, while 114 genes were significantly upregulated. GO enrichment analysis revealed that 369 GO terms were significantly affected by NP exposure. Further analysis showed that many of the differentially expressed genes were associated with oxidative phosphorylation, iron and copper acquisition, autophagy, pleiotropic drug resistance and cell cycle progression related processes such as DNA and mismatch repair, chromosome segregation, spindle checkpoint activity, and kinetochore organization. Overall, these results provide considerable information and a comprehensive understanding of the adaptive response to NP exposure at the gene expression level.

Tissue-Specific Transcriptome Analysis Reveals Candidate Transcripts Associated with the Process of Programmed B Chromosome Elimination in Aegilops speltoides.

Some eukaryotes exhibit dramatic genome size differences between cells of different organs, resulting from programmed elimination of chromosomes. Here, we present the first transcriptome analysis of programmed chromosome elimination using laser capture microdissection (LCM)-based isolation of the central meristematic region of Aegilops speltoides embryos where B chromosome (B) elimination occurs. The comparative RNA-seq analysis of meristematic cells of embryos with (Bplus) and without Bs (B0) allowed the identification of 14,578 transcript isoforms (35% out of 41,615 analyzed transcript isoforms) that are differentially expressed during the elimination of Bs. A total of 2908 annotated unigenes were found to be up-regulated in Bplus condition. These genes are either associated with the process of B chromosome elimination or with the presence of B chromosomes themselves. GO enrichment analysis categorized up-regulated transcript isoforms into 27 overrepresented terms related to the biological process, nine terms of the molecular function aspect and three terms of the cellular component category. A total of 2726 annotated unigenes were down-regulated in Bplus condition. Based on strict filtering criteria, 341 B-unique transcript isoforms could be identified in central meristematic cells, of which 70 were functionally annotated. Beside others, genes associated with chromosome segregation, kinetochore function and spindle checkpoint activity were retrieved as promising candidates involved in the process of B chromosome elimination.

Abstract A32: Selective inhibition of AuroraA by LY3295668 erbumine in neuroblastoma models induces apoptosis through a combination of S-phase DNA damage and mitotic arrest

Abstract Aurora kinase A (AuroraA) is a serine/threonine protein kinase critical for centrosome maturation, mitotic spindle formation, and spindle checkpoint activation. Aberrant expression of AuroraA promotes tumor initiation and disease progression, making it a promising therapeutic target in several cancer types. However, recent clinical trials of purported selective AuroraA inhibitors failed due to lack of efficacy and/or hematologic toxicity, likely due to concurrent inhibition of Aurora kinase B (AuroraB). Selective AuroraA inhibition leads to mitotic arrest and apoptosis, while AuroraB inhibition causes persistent DNA endoreduplication and polyploidy leading to genomic instability. LY3295668 erbumine (LY3295668) is a selective AuroraA inhibitor with preclinical activity in multiple models of cancer including neuroblastoma (NBL). High-risk NBL, a devastating pediatric cancer with few effective treatment options, is mainly stratified by MYCN amplification status. In NBL, AuroraA stabilizes N-MYC post-transcriptionally; moreover, AuroraA also controls N-MYC-dependent transcription during S-phase. Here, we evaluated LY3295668 in multiple NBL cell lines and in vivo mouse models where treatment resulted in apoptosis and tumor regressions, respectively. We compared LY3295668 to alisertib (a dual AuroraA/B inhibitor) in multiple cell-based assays that differentiate AuroraA activity from that of AuroraB and showed that LY3295668 has a distinct pharmacologic profile from alisertib. In addition, we determined the mechanism of action for LY3295668 includes DNA damage prior to mitotic block. Our results further support that AuroraA controls MYC-dependent transcription in S-phase and selective inhibition of AuroraA with LY3295668 promotes transcription-replication conflicts leading to DNA damage. In addition to AuroraA-mediated mitotic arrest, this DNA damage may contribute to the hypersensitivity of MYC-driven tumors such as NBL. In summary, LY3295668 is a selective AuroraA inhibitor with a dual mechanism of action in NBL involving DNA damage in S-phase and a cell cycle block in mitosis. These combined activities contribute to sensitivity of preclinical NBL models. Citation Format: Michele Dowless, Jill Kremer, Caitlin Lowery, Xueqian Gong, Joseph Klahn, Jennifer Stephens, Yu-Hua Hui, Wayne Blosser, Ricardo Martinez, Jian Du, Louis Stancato. Selective inhibition of AuroraA by LY3295668 erbumine in neuroblastoma models induces apoptosis through a combination of S-phase DNA damage and mitotic arrest [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A32.

Deregulation of Neuro-Developmental Genes and Primary Cilium Cytoskeleton Anomalies in iPSC Retinal Sheets from Human Syndromic Ciliopathies.

SummaryCiliopathies are heterogeneous genetic diseases affecting primary cilium structure and function. Meckel-Gruber (MKS) and Bardet-Biedl (BBS) syndromes are severe ciliopathies characterized by skeletal and neurodevelopment anomalies, including polydactyly, cognitive impairment, and retinal degeneration. We describe the generation and molecular characterization of human induced pluripotent stem cell (iPSC)-derived retinal sheets (RSs) from controls, and MKS (TMEM67) and BBS (BBS10) cases. MKS and BBS RSs displayed significant common alterations in the expression of hundreds of developmental genes and members of the WNT and BMP pathways. Induction of crystallin molecular chaperones was prominent in MKS and BBS RSs suggesting a stress response to misfolded proteins. Unique to MKS photoreceptors was the presence of supernumerary centrioles and cilia, and aggregation of ciliary proteins. Unique to BBS photoreceptors was the accumulation of DNA damage and activation of the mitotic spindle checkpoint. This study reveals how combining cell reprogramming, organogenesis, and next-generation sequencing enables the elucidation of mechanisms involved in human ciliopathies.

38P - Bub3 is phosphorylated by the ataxia-telangiectasia mutated kinase in mitosis and required for activation of the mitotic spindle checkpoint in breast cancer

BackgroundATM is an important cell cycle checkpoint kinase. This protein is an controller of cell cycle checkpoint that is required for cell response to DNA damage and for genome stability. The spindle assembly checkpoint (SAC) controls accurate chromosome segregation to maintain genome stability. All kinetochores correctly attach to the spindle, which allow mitosis to proceed. Bub3 is the mainly component of the SAC, in which its kinase activity can stabilize the correct kinetochore–microtubule attachments. Genomic stability is a hallmarks of breast cancer, and the level of Bub3 in breast cancer are higher than that in normal tussue. Understanding how the ATM-Bub3 signal contributes to kinetochore-based signaling may help us to recognize the occurrence of breast cancer. MethodsStable Isotope Labeling with Amino Acid in Cell Culture (SILAC) to identify ATM interation protein in breast cancer. Bioinformatics analyze the phosphorylation site of Bub3. Biacore assay and in vitro kinase assay to validate the phosphorylation site. Flow cytometry assay to check the mitotic index. Mutation of the phosphorylation site of bub3 and Co-IP were proformed to identify the interation of Bub3 and Bub1 or ZNF207, and western blot to be used to test the activity of Bub1. ResultsThrough Stable Isotope Labeling with Amino Acid in Cell Culture (SILAC), we identified additional mitotic proteins that interact and can be phosphorylated by mitotically activated ATM kinase. We provide both in vitro and in vivo evidence that ATM phosphorylates Bub3 on Serine 135. This phosphorylation event promotes Bub1 activity in the mitotic spindle checkpoint. Further, we find that phosphorylation of Bub3 on Ser135 is required for formation of the Bub3-Bub1 complex, which is essential for Bub1 Ser314 phosphorylation. Mutation of Ser135 to alanine of Bub3 resulted in an enhanced interaction with BuGZ/ZNF207 and reduced interaction with Bub1, which lead to a spindle checkpoint defect. ConclusionsOur findings highlight the functional significance of ATM-mediated kinetochore protein phosphorylation and elucidate a detailed regulatory mechanism of the SAC in breast cancer. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

9PD - Aurora-B mediated snail phosphorylation is essential for mitotic spindle checkpoint and for preventing chromosomal instability in breast cancer

BackgroundSnail is a transcriptional repressor that promotes epithelial-to-mesenchymal transition (EMT). Snail is upregulated in breast cancer and it is associated with chromosomal instability (CIN) and metastasis. The mitotic spindle assembly checkpoint (SAC) is a critical mechanism to prevent CIN. Extensive studies have investigated signaling pathways that involve in protein-protein interactions and post translational modifications. However, it not known how mitosis might be impacted by transcriptional regulation. Due to the fact that Snail is a transcriptional regulator and that Snail is upregulated in breast cancer with CIN, it is critical to understand whether and how abnormally regulated Snail contributes to breast cancer CIN via transcriptional regulation. MethodsWe used siRNA to knockdown Snail in breast caner cell lines to look for a change in SAC. We developed a phosphor-specific antibody to study Aurora-B mediated phosphorylation. Site specific mutagenesis was used to generate phosphorylation site mutants, and exogenous proteins were expressed in cells to look for an effect in SAC and CIN. ResultsWe found that Snail is an essential element that is required for the SAC. Genetic depletion of Snail in breast cancer cells by siRNA led to a defect in activation of SAC (measured by accumulation of Histone Serine 10 phosphorylation in response to spindle damaging agents). Interestingly we also found that the Snail protein level was significantly enhanced in the presence of nocodazole. Further, we found that Snail upregulation was through phosphorylation in an Aurora-B dependent manner. We identified a serine site that can be phosphorylated by Aurora-B. Using a phosphor-specific antibody we have developed, we further proved that the phosphorylation event was initiated in mitosis. Functional studies reveal a critical role for Snail phosphorylation in activation of the SAC and prevention of CIN. Conclusions1. Snail is required for mitotic spindle checkpoint activation; 2. Snail is phosphorylated by Aurora B in mitosis; 3. Aurora-B medicated Snail phosphorylation is required for optinal activation of the spindle checkpoint and prevention of chromosomal instability in breast cancer. Legal entity responsible for the studyThe authors. FundingTianjin Medical University Cancer Institute and Hospital. DisclosureAll authors have declared no conflicts of interest.

Selective Inhibition of Spindle Microtubules by a Tubulin-Binding Quinazoline Derivative.

In the research field of tubulin-binding agents for the development of anticancer agents, hidden targets are emerging as a problem in understanding the exact mechanisms of actions. The quinazoline derivative 1-(4-methoxyphenyl)-1-(quinazolin-4-yl)ethan-1-ol (PVHD121) has anti-cell proliferative activity and inhibits tubulin polymerization by binding to the colchicine site of tubulin. However, the molecular mechanism of action of PVHD121 in cells remains unclear. Here, we demonstrate that PVHD121 delays mitotic entry and efficiently causes mitotic arrest with spindle checkpoint activation, leading to subsequent cell death. The dominant phenotype induced by PVHD121 was aberrant spindles with robust microtubules and unseparated centrosomes. The microtubules were radially distributed, and their ends appeared to adhere to kinetochores, and not to centrosomes. Extensive inhibition by high concentrations of PVHD121 eliminated all microtubules from cells. PVHD277 [1-(4-methoxyphenyl)-1-(2-morpholinoquinazolin-4-yl)ethan-1-ol], a PVHD121 derivative with fluorescence, tended to localize close to the centrosomes when cells prepared to enter mitosis. Our results show that PVHD121 is an antimitotic agent that selectively disturbs microtubule formation at centrosomes during mitosis. This antimitotic activity can be attributed to the targeting of centrosome maturation in addition to the interference with microtubule dynamics. Due to its unique bioactivity, PVHD121 is a potential tool for studying the molecular biology of mitosis and a potential lead compound for the development of anticancer agents. SIGNIFICANCE STATEMENT: Many tubulin-binding agents have been developed as potential anticancer agents. The aim of this study was to understand the subcellular molecular actions of a quinazoline derivative tubulin-binding agent, 1-(4-methoxyphenyl)-1-(quinazolin-4-yl)ethan-1-ol (PVHD121). As expected from its binding activity to tubulin, PVHD121 caused aberrant spindles and inhibited mitotic progression. However, in addition to tubulin, PVHD121 also targeted an unexpected biomolecule involved in centrosome maturation. Due to targeting the biomolecule just before entering mitosis, PVHD121 preferentially inhibited centrosome-derived microtubules rather than chromosome-derived microtubules during spindle formation. This study not only revealed the molecular action of PVHD121 in cells but also emphasized the importance of considering possible tubulin-independent effects of tubulin-binding agents via hidden targeted biomolecules for future use.

Abstract CT019: Population pharmacokinetics of an aurora kinase A inhibitor, LY3295668 erbumine (AK-01), in patients with locally advanced or metastatic solid tumors

Abstract Background: The aurora kinase family plays a vital regulatory role in mitotic and meiotic events, with aurora kinase A (AurA) critical to centrosome maturation, mitotic spindle formation and checkpoint activation. LY3295668 erbumine (AK-01) is an inhibitor of AurA with 1000-fold selectivity for inhibition of AurA over aurora kinase B (AurB). Pharmacokinetic-pharmacodynamic (PK-PD) nonclinical models have shown that 90% inhibition of AurA phosphorylation (pAurA) over an extended period (≥ 16 hours per day) was required to yield efficacy in H446 (small cell lung cancer) xenograft models. Nonclinical plasma LY3295668 concentrations associated with 90% pAurA inhibition (IC90) were estimated previously using a direct sigmoidal relationship between PK and pAurA. In this first-in-man study, PK of LY3295668 in patients with advanced/metastatic cancer were evaluated. Human PK profiles were simulated subsequently and compared to pAurA IC90 to estimate a human efficacious dose range. Methods: LY3295668 erbumine was administered to 13 patients with locally advanced or metastatic solid tumors over a dose range of 25 to 75 mg given twice daily (BID). In the first cycle, serial plasma PK samples were collected up to 8 hours on Day 1 and Day 15. Additional trough samples were obtained prior to dosing on Day 2, on Day 8 and at discontinuation if applicable. PK parameter estimations were performed using non-linear mixed effects models in NONMEM v7.3. Results: The population PK model was developed using 146 plasma concentrations collected from 13 patients. A 2-compartment PK model with first order absorption best described the disposition of LY3295668. Following oral administration, LY3295668 was rapidly absorbed with peak concentrations occurring within 1 to 2 hours. The estimated elimination half-life was approximately 21 hours; therefore, steady state was attained within 4 to 5 days of dosing. Simulations of plasma concentration-time profiles showed that at the maximum tolerated dose (MTD) of 25 mg BID, 90% of patients are expected to achieve steady-state plasma concentrations greater than the pAurA IC90 for the entire day. Patients with dose limiting toxicities (DLTs) had the highest model-predicted exposures amongst the 13 patients. Conclusion: At the MTD of 25 mg BID, steady state LY3295668 plasma concentrations are maintained above the pAurA IC90 for the entire dosing interval, exceeding the minimum requirements for efficacy associated with non-clinical xenograft models. Trial Registration: NCT03092934 Citation Format: Eunice Yuen, Yu-Hua Hui, Amparo de la Peña, Sonya C. Tate, John R. Stille, Andrew Lithio, Patricia S. Smith, Quincy Chu, Gerald Batist, Nathaniel Bouganim, Caroline Fortier, Sara Zaknoen, Jill Kremer. Population pharmacokinetics of an aurora kinase A inhibitor, LY3295668 erbumine (AK-01), in patients with locally advanced or metastatic solid tumors [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 CT019.

Downregulation of NOP53 Ribosome Biogenesis Factor Leads to Abnormal Nuclear Division and Chromosomal Instability in Human Cervical Cancer Cells.

NOP53 ribosome biogenesis factor (NOP53) is a nucleolar protein involved in oncogenesis/tumor suppression, cell cycle regulation, and cell death. Here, we investigated the role of NOP53 in the maintenance of normal nuclear shape and chromosomal stability. Depletion of NOP53 by shRNA caused abnormal nuclear morphology, including large nucleus, irregular nucleus, and multinucleated cells, and chromosomal instability resulting in micronucleus or nuclear bud formation. The abnormal nuclear shape and chromosomal instability were restored by re-expression of NOP53. We further showed that NOP53 was involved in chromosome congression in metaphase. Downregulation of NOP53 induced aberrant chromosome congression and spindle checkpoint activation, resulting in delayed mitosis and mitotic arrest. Thus, our findings demonstrated that the nucleolar protein NOP53 participated in mitotic progression and that dysregulated NOP53 expression caused chromosomal instability in cancer cells.

Mitotic Exit Dysfunction through the Deregulation of APC/C Characterizes Cisplatin-Resistant State in Epithelial Ovarian Cancer.

Purpose: Acquired resistance to cisplatin is a major barrier to success in treatment of various cancers, and understanding mitotic mechanisms unique to cisplatin-resistant cancer cells can provide the basis for developing novel mitotic targeted therapies aimed at eradicating these cells.Experimental Design: Using cisplatin-resistant models derived from primary patient epithelial ovarian cancer (EOC) cells, we have explored the status of mitotic exit mechanisms in cisplatin-resistant cells.Results: We have uncovered an unexpected role of long-term cisplatin treatment in inducing mitotic exit vulnerability characterized by increased spindle checkpoint activity and functional dependency on Polo-like kinase 1 (PLK1) for mitotic exit in the presence of anaphase promoting complex/cyclosome (APC/C) dysfunction in a cisplatin-resistant state. Accordingly, PLK1 inhibition decreased the survival of cisplatin-resistant cells in vitro and in vivo and exacerbated spindle checkpoint response in these cells. APC/CCDC20 inhibition increased sensitivity to pharmacologic PLK1 inhibition, further confirming the existence of APC/C dysfunction in cisplatin-resistant cells. In addition, we uncovered that resistance to volasertib, PLK1 inhibitor, is due to maintenance of cells with low PLK1 expression. Accordingly, stable PLK1 downregulation in cisplatin-resistant cells induced tolerance to volasertib.Conclusions: We provide the first evidence of APC/C dysfunction in cisplatin-resistant state, suggesting that understanding APC/C functions in cisplatin-resistant state could provide a basis for developing novel mitotic exit-based therapies to eradicate cisplatin-resistant cancer cells. Our results also show that PLK1 downregulation could underlie emergence of resistance to PLK1-targeted therapies in cancers. Clin Cancer Res; 24(18); 4588-601. ©2018 AACR.

Playing polo during mitosis: PLK1 takes the lead.

Polo-like kinase 1 (PLK1), the prototypical member of the polo-like family of serine/threonine kinases, is a pivotal regulator of mitosis and cytokinesis in eukaryotes. Many layers of regulation have evolved to target PLK1 to different subcellular structures and to its various mitotic substrates in line with its numerous functions during mitosis. Collective work is starting to illuminate an important set of substrates for PLK1: the mitotic kinases that together ensure the fidelity of the cell division process. Amongst these, recent developments argue that PLK1 regulates the activity of the histone kinases Aurora B and Haspin to define centromere identity, of MPS1 to initiate spindle checkpoint signaling, and of BUB1 and its pseudokinase paralog BUBR1 to coordinate spindle checkpoint activation and inactivation. Here, we review the recent work describing the regulation of these kinases by PLK1. We highlight common themes throughout and argue that a major mitotic function of PLK1 is as a master regulator of these key kinases.