Mps1 Kinase Research Articles

Mps1 dimerization and multisite interactions with Ndc80 complex enable responsive spindle assembly checkpoint signaling.

Error-free mitosis depends on accurate chromosome attachment to spindle microtubules, which is monitored by the spindle assembly checkpoint (SAC) signaling. As an upstream factor of SAC, the precise and dynamic kinetochore localization of Mps1 kinase is critical for initiating and silencing SAC signaling. However, the underlying molecular mechanism remains elusive. Here, we demonstrated that the multisite interactions between Mps1 and Ndc80 complex (Ndc80C) govern Mps1 kinetochore targeting. Importantly, we identified direct interaction between Mps1 tetratricopeptide repeat domain and Ndc80C. We further identified that Mps1 C-terminal fragment, which contains the protein kinase domain and C-tail, enhances Mps1 kinetochore localization. Mechanistically, Mps1 C-terminal fragment mediates its dimerization. Perturbation of C-tail attenuates the kinetochore targeting and activity of Mps1, leading to aberrant mitosis due to compromised SAC function. Taken together, our study highlights the importance of Mps1 dimerization and multisite interactions with Ndc80C in enabling responsive SAC signaling.

Cyclin B1 scaffolds MAD1 at the kinetochore corona to activate the mitotic checkpoint

Cyclin B:CDK1 is the master kinase regulator of mitosis. We show here that, in addition to its kinase functions, mammalian Cyclin B also scaffolds a localised signalling pathway to help preserve genome stability. Cyclin B1 localises to an expanded region of the outer kinetochore, known as the corona, where it scaffolds the spindle assembly checkpoint (SAC) machinery by binding directly to MAD1. In vitro reconstitutions map the key binding interface to a few acidic residues in the N‐terminal region of MAD1, and point mutations in this sequence abolish MAD1 corona localisation and weaken the SAC. Therefore, Cyclin B1 is the long‐sought‐after scaffold that links MAD1 to the corona, and this specific pool of MAD1 is needed to generate a robust SAC response. Robustness arises because Cyclin B1:MAD1 localisation loses dependence on MPS1 kinase after the corona has been established, ensuring that corona‐localised MAD1 can still be phosphorylated when MPS1 activity is low. Therefore, this study explains how corona‐MAD1 generates a robust SAC signal, and it reveals a scaffolding role for the key mitotic kinase, Cyclin B1:CDK1, which ultimately helps to inhibit its own degradation.

Kinetochore Stretching-Mediated Rapid Silencing of Mitotic Checkpoint Required for Failsafe Chromosome Segregation

The spindle-assembly checkpoint facilitates mitotic fidelity by delaying anaphase onset in response to microtubule vacancy at kinetochores. Following microtubule attachment, kinetochores receive microtubule-derived forces, which causes kinetochores to undergo repetitive cycles of deformations, the motion referred to as kinetochore stretching. The nature of forces and the relevance relating this motion are not well understood. Here we show that kinetochore stretching occurs within a framework of single end-on attached kinetochores, irrespectively of microtubule poleward pulling-force. An experimental setup to conditionally interfere with the stretching allowed us to define that kinetochore stretching comprises an essential process of checkpoint silencing by promoting PP1 phosphatase recruitment, after establishing of end-on attachments and removing the majority of checkpoint-activating kinase Mps1 from kinetochores. Remarkably, we found that the lower frequency of kinetochore stretching correlates with prolonged metaphase and also with the degree of chromosomal instability in cancer cell lines. Perturbation of kinetochore stretching and checkpoint silencing in chromosomally stable cells produced anaphase bridges which can be alleviated by reducing chromosome-loaded cohesin. These observations indicate that kinetochore stretching-mediated checkpoint silencing provides an unanticipated etiology underlying chromosomal instability, and underscore the significance of a rapid metaphase-to-anaphase transition in sustaining mitotic fidelity.

High Proliferation Rate and a Compromised Spindle Assembly Checkpoint Confers Sensitivity to the MPS1 Inhibitor BOS172722 in Triple-Negative Breast Cancers.

BOS172722 (CCT289346) is a highly potent, selective, and orally bioavailable inhibitor of spindle assembly checkpoint kinase MPS1. BOS172722 treatment alone induces significant sensitization to death, particularly in highly proliferative triple-negative breast cancer (TNBC) cell lines with compromised spindle assembly checkpoint activity. BOS172722 synergizes with paclitaxel to induce gross chromosomal segregation defects caused by MPS1 inhibitor-mediated abrogation of the mitotic delay induced by paclitaxel treatment. In in vivo pharmacodynamic experiments, BOS172722 potently inhibits the spindle assembly checkpoint induced by paclitaxel in human tumor xenograft models of TNBC, as measured by inhibition of the phosphorylation of histone H3 and the phosphorylation of the MPS1 substrate, KNL1. This mechanistic synergy results in significant in vivo efficacy, with robust tumor regressions observed for the combination of BOS172722 and paclitaxel versus either agent alone in long-term efficacy studies in multiple human tumor xenograft TNBC models, including a patient-derived xenograft and a systemic metastasis model. The current target indication for BOS172722 is TNBC, based on their high sensitivity to MPS1 inhibition, the well-defined clinical patient population with high unmet need, and the synergy observed with paclitaxel.

Autophosphorylation is sufficient to release Mps1 kinase from native kinetochores

Accurate mitosis depends on a surveillance system called the spindle assembly checkpoint. This checkpoint acts at kinetochores, which attach chromosomes to the dynamic tips of spindle microtubules. When a kinetochore is unattached or improperly attached, the protein kinase Mps1 phosphorylates kinetochore components, catalyzing the generation of a diffusible "wait" signal that delays anaphase and gives the cell time to correct the error. When a kinetochore becomes properly attached, its checkpoint signal is silenced to allow progression into anaphase. Recently, microtubules were found to compete directly against recombinant human Mps1 fragments for binding to the major microtubule-binding kinetochore element Ndc80c, suggesting a direct competition model for silencing the checkpoint signal at properly attached kinetochores. Here, by developing single-particle fluorescence-based assays, we tested whether such direct competition occurs in the context of native kinetochores isolated from yeast. Mps1 levels were not reduced on kinetochore particles bound laterally to the sides of microtubules or on particles tracking processively with disassembling tips. Instead, we found that Mps1 kinase activity was sufficient to promote its release from the isolated kinetochores. Mps1 autophosphorylation, rather than phosphorylation of other kinetochore components, was responsible for this dissociation. Our findings suggest that checkpoint silencing in yeast does not arise from a direct competition between Mps1 and microtubules, and that phosphoregulation of Mps1 may be a critical aspect of the silencing mechanism.

Abstract 2211: Discovery of orally available and potent MPS1(TTK) kinase inhibitors for anti-cancer drugs

Abstract There are many cell cycle regulating kinases such as CDKs, Polo, Aurora, Bub, NEK/NimA, etc. Dual specificity protein kinase, MPS1(TTK) is one of them. The role of MPS1 is to regulate spindle assembly checkpoint(SAC) during cell mitosis. The inhibition of MPS1 activity causes cell death by premature mitotic exit. Some of MPS1 inhibitors (NMS-P153, BOS-172722, CFI-402257, BAY-1217389, and BAY-1161909) are in clinical trials. These compounds are treated for solid tumors with or without Paclitaxel. We discovered new orally available and potent inhibitors of MPS1 kinase (MPS1 enzyme IC50 < 10nM / MDA-MB-231 GI50 < 50nM, MDA-MB-468 GI50 < 50nM / HCT116 GI50 < 50nM, F% > 30%). The compounds selectively inhibit MPS1 based on kinase profiling and decrease phosphorylation of MPS1 and Phospho-HH3 signaling, effectively. Some of compounds were selected for further preclinical studies. Citation Format: Younho Lee, Juhee Kang, Sein Kang, Hyunkyung Kim, Kyung-ah Seo, Jieun Choi, Jaeyoung Ahn, Jung Beom Son, Nam Doo Kim. Discovery of orally available and potent MPS1(TTK) kinase inhibitors for anti-cancer drugs [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 2211.

Advances in research of monopolar spindle protein kinase 1 in tumor therapy

The Spindle assembly checkpoint (SAC) is an extremely important monitoring step in the mitosis process, ensuring that all sister chromatids are placed on the medium-term plate before the mid-mitotic transformation. Recent studies have shown that this checkpoint abnormality has a higher correlation with tumorigenesis, development and metastasis mechanisms. Monopolar spindle 1 (Mps1) is a conserved bispecific protein kinase whose primary function is centrosome replication and activation of SAC, both of which help prevent mitotic errors and prevent tumorigenesis. The latest study found that the expression level of Mps1 is related to the degree of malignancy of breast cancer. More notably, inhibition of Mps1 kinase activity or knockdown of Mps1 by siRNA results in a barrier to tumor cell division, leading to tumor cell apoptosis without affecting normal cell activity. The dysregulation of the cell cycle is also closely related to the invasive biological behavior of malignant tumors, and becomes a basic marker of tumor progression. The cell cycle is mainly regulated by a number of checkpoint regulatory pathways, which are specifically dysregulated in tumor cells and Has the potential to become a target for the design of selective treatment of tumor cells. This article reviews how Mps1 regulates SAC and its recent advances in cancer therapy Key words: Spindle assembly checkpoint; Monopolar spindle 1; Gene targeting therapy; Osteosarcoma

Mps1 regulates spindle morphology through MCRS1 to promote chromosome alignment.

Accurate partitioning of chromosomes during mitosis is essential for genetic stability and requires the assembly of the dynamic mitotic spindle and proper kinetochore–microtubule attachment. The spindle assembly checkpoint (SAC) monitors the incompleteness and errors in kinetochore–microtubule attachment and delays anaphase. The SAC kinase Mps1 regulates the recruitment of downstream effectors to unattached kinetochores. Mps1 also actively promotes chromosome alignment during metaphase, but the underlying mechanism is not completely understood. Here, we show that Mps1 regulates chromosome alignment through MCRS1, a spindle assembly factor that controls the dynamics of the minus end of kinetochore microtubules. Mps1 binds and phosphorylates MCRS1. This mechanism enables KIF2A localization to the minus end of spindle microtubules. Thus, our study reveals a novel role of Mps1 in regulating the dynamics of the minus end of microtubules and expands the functions of Mps1 in genome maintenance.

Interactions between N-terminal Modules in MPS1 Enable Spindle Checkpoint Silencing

Faithful chromosome segregation relies on the ability of the spindle assembly checkpoint (SAC) to delay anaphase onset until chromosomes are attached to the mitotic spindle via their kinetochores. MPS1 kinase is recruited to kinetochores to initiate SAC signaling and is removed from kinetochores once stable microtubule attachments have been formed to allow normal mitotic progression. Here, we show that a helical fragment within the kinetochore-targeting N-terminal extension (NTE) module of MPS1 is required for interactions with kinetochores and forms intramolecular interactions with its adjacent tetratricopeptide repeat (TPR) domain. Bypassing this NTE-TPR interaction results in high MPS1 levels at kinetochores due to loss of regulatory input into MPS1 localization, inefficient MPS1 delocalization upon microtubule attachment, and SAC silencing defects. These results show that SAC responsiveness to attachments relies on regulated intramolecular interactions in MPS1 and highlight the sensitivity of mitosis to perturbations in the dynamics of the MPS1-NDC80-C interactions.

MAD1-dependent recruitment of CDK1-CCNB1 to kinetochores promotes spindle checkpoint signaling.

Cyclin B-dependent kinase (CDK1-CCNB1) promotes entry into mitosis. Additionally, it inhibits mitotic exit by activating the spindle checkpoint. This latter role is mediated through phosphorylation of the checkpoint kinase MPS1 and other spindle checkpoint proteins. We find that CDK1-CCNB1 localizes to unattached kinetochores and like MPS1 is lost from these structures upon microtubule attachment. This suggests that CDK1-CCNB1 is an integral component and not only an upstream regulator of the spindle checkpoint pathway. Complementary proteomic and cell biological analysis demonstrate that the spindle checkpoint protein MAD1 is one of the major components of CCNB1 complexes, and that CCNB1 is recruited to unattached kinetochores in an MPS1-dependent fashion through interaction with the first 100 amino acids of MAD1. This MPS1 and MAD1-dependent pool of CDK1-CCNB1 creates a positive feedback loop necessary for timely recruitment of MPS1 to kinetochores during mitotic entry and for sustained spindle checkpoint arrest. CDK1-CCNB1 is therefore an integral component of the spindle checkpoint, ensuring the fidelity of mitosis.

CDK1-CCNB1 creates a spindle checkpoint-permissive state by enabling MPS1 kinetochore localization.

Spindle checkpoint signaling is initiated by recruitment of the kinase MPS1 to unattached kinetochores during mitosis. We show that CDK1-CCNB1 and a counteracting phosphatase PP2A-B55 regulate the engagement of human MPS1 with unattached kinetochores by controlling the phosphorylation status of S281 in the kinetochore-binding domain. This regulation is essential for checkpoint signaling, since MPS1S281A is not recruited to unattached kinetochores and fails to support the recruitment of other checkpoint proteins. Directly tethering MPS1S281A to the kinetochore protein Mis12 bypasses this regulation and hence the requirement for S281 phosphorylation in checkpoint signaling. At the metaphase-anaphase transition, MPS1 S281 dephosphorylation is delayed because PP2A-B55 is negatively regulated by CDK1-CCNB1 and only becomes fully active once CCNB1 concentration falls below a characteristic threshold. This mechanism prolongs the checkpoint-responsive period when MPS1 can localize to kinetochores and enables a response to late-stage spindle defects. By acting together, CDK1-CCNB1 and PP2A-B55 thus create a spindle checkpoint-permissive state and ensure the fidelity of mitosis.

A crystal structure of the human protein kinase Mps1 reveals an ordered conformation of the activation loop.

Monopolar spindle 1 (Mps1) is a dual‐specificity protein kinase, orchestrating faithful chromosome segregation during mitosis. All reported structures of the Mps1 kinase adopt the hallmarks of an inactive conformation, which includes a mostly disordered activation loop. Here, we present a 2.4 Å resolution crystal structure of an “extended” version of the Mps1 kinase domain, which shows an ordered activation loop. However, the other structural characteristics of an active kinase are not present. Our structure shows that the Mps1 activation loop can fit to the ATP binding pocket and interferes with ATP, but less so with inhibitors binding, partly explain the potency of various Mps1 inhibitors.

Cytotoxic and Apoptotic Effects of Novel Pyrrolo[2,3-d]Pyrimidine Derivatives Containing Urea Moieties on Cancer Cell Lines.

Pyrrolo[2,3-d]pyrimidines have been recently reported to have anticancer activities through inhibition of different targets such as, Epidermal Growth Factor Receptor (EGFR) tyrosine kinase, Janus Kinase (JAK), mitotic checkpoint protein kinase (Mps1), carbonic anhydrase, MDM-2. On the other hand, aryl urea moieties which are found in some tyrosine kinase inhibitors such as Sorafenib and Linifanib have aroused recent attention as responsible for anticancer activities. The aims of this paper are to synthesize pyrrolo[ 2,3-d]pyrimidine derivatives containing urea moiety and evaluate their anti-cancer activity against human lung cancer cell line (A549), prostate cancer cell line (PC3), human colon cancer cell line (SW480) and human breast cancer cell line (MCF-7). A series of new pyrrolo[2,3-d]pyrimidines containing urea moieties have been synthesized as Scheme 1. In vitro cytotoxicity of target compounds were evaluated against, SW480, PC3, A549 and MCF-7 human cancer cell lines using a MTT assay. In order to evaluate the mechanism of cytotoxic activity of compounds 9e, 10a and 10b, having the best cytotoxic activity, Annexin V binding assay, cell cycle analysis and western blot analysis were performed. Among the target compounds, 10a (IC50 = 0.19 µM) was found to be the most potent derivative against PC3 cells. Compound 10b and 9e showed the strong cytotoxic activity against MCF-7 and A549 cells with IC50 value of 1.66 µM and 4.55 µM, respectively. Flow cytometry data suggest that the cytotoxic activity of the compounds on cancer cells might be mediated by apoptosis revealing a significant increase in the percentage of late apoptotic cells and causing a cell cycle arrest at different stages. Western blot analysis of apoptosis marker demonstrated that these compounds induce apoptosis through the intrinsic pathway. Compound 9e displayed the strongest cytotoxicity against A549 cancer cell line, and induced late apoptosis in A549, as confirmed by cell cycle arrest in G0/G1 phase. In addition, compound 9e reduced expression of the anti-apoptotic protein Bcl-2 and enhanced expression of the pro-apoptotic protein Bax, besides increased caspase-9 and caspase-3, as well as cleavage of PARP levels. These results suggest that compound 9e showed a cytotoxic effect in A549 cells through activation of the mitochondrial apoptotic pathway. Further studies will be undertaken in our laboratory to improve cytotoxic activity of compound 9e and to identify the biological targets of 9e which are responsible for anticancer activity.

Ectopic Activation of the Spindle Assembly Checkpoint Signaling Cascade Reveals Its Biochemical Design

Switch-like activation of the spindle assembly checkpoint (SAC) is critical for accurate chromosome segregation and for cell division in a timely manner. To determine the mechanisms that achieve this, we engineered an ectopic, kinetochore-independent SAC activator: the "eSAC." The eSAC stimulates SAC signaling by artificially dimerizing Mps1 kinase domain and a cytosolic KNL1 phosphodomain, the kinetochore signaling scaffold. By exploiting variable eSAC expression in a cell population, we defined the dependence of the eSAC-induced mitotic delay on eSAC concentration in a cell to reveal the dose-response behavior of the core signaling cascade of the SAC. These quantitative analyses and subsequent mathematical modeling of the dose-response data uncover two crucial properties of the core SAC signaling cascade: (1) a cellular limit on the maximum anaphase-inhibitory signal that the cascade can generate due to the limited supply of SAC proteins and (2) the ability of the KNL1 phosphodomain to produce the anaphase-inhibitory signal synergistically, when it recruits multiple SAC proteins simultaneously. We propose that these properties together achieve inverse, non-linear scaling between the signal output per kinetochore and the number of signaling kinetochores. When the number of kinetochores is low, synergistic signaling by KNL1 enables each kinetochore to produce a disproportionately strong signal output. However, when many kinetochores signal concurrently, they compete for a limited supply of SAC proteins. This frustrates synergistic signaling and lowers their signal output. Thus, the signaling activity of unattached kinetochores will adapt to the changing number of signaling kinetochores to enable the SAC to approximate switch-like behavior.

Pign Suppresses CIN and Leukemia Progression Via Regulation of the Spindle Assembly Checkpoint

The spindle assembly checkpoint complex (SAC) is responsible for proper chromosomal segregation during mitosis. The SAC stalls mitotic exit until proper attachment of mitotic spindles to the chromosomes and bi-orientation of the chromosomes on the spindles are achieved. Dysregulation of the SAC may result in chromosomal instability (CIN) which is known to drive leukemia progression. We previously assessed the impact of phosphatidylinositol glycan anchor biosynthesis class N (PIGN) expression aberrations on leukemia progression and showed that PIGN expression aberrations were linked with CIN and leukemia transformation in high-risk myelodysplastic syndrome (MDS) patients. An in-depth understanding of the mechanistic basis of PIGN involvement in CIN and leukemic progression would have boundless therapeutic and diagnostic implications for patients. Thus, we investigated the mechanistic link between PIGN, CIN and the SAC.PIGN downregulation via RNAi and CRISPR/Cas9 as well as ectopic overexpression studies, co-immunoprecipitation, and confocal microscopy were employed to decipher the relationship between PIGN, CIN, and SAC signaling. Additionally, we tested whether the depletion of PIGN results in aberrant cell cycle signaling and defective chromosomal segregation using flow cytometry and mitotic index assays. We initially performed cell cycle synchronization experiments using myeloid and lymphoblastoid cell lines and examined PIGN expression at different stages of the cell cycle via Western blot analyses and RT-qPCR.Our results indicated that PIGN expression was cell cycle-regulated and PIGN loss significantly impacted the expression of SAC-related proteins. CRISPR/Cas9 mediated knockout of PIGN in CD34+ mononuclear cells derived from a healthy individual resulted in the suppression of MAD1 and MAD2. A similar observation was made in HEK293 PIGN CRISPR/Cas9 knockout cells. PIGN loss in the HEK293 cells resulted in MAD1, MAD2, and MPS1 suppression but led to BUBR1 upregulation. PIGN downregulation resulted in impaired mitotic checkpoint activation and consequently impacted mitotic exit. PIGN downregulation results in defective mitotic checkpoint signaling and mitotic exit with an accumulation of missegregation errors. Interestingly, ectopic overexpression of PIGN restored the MAD1 and MAD2 expression. Co-immunoprecipitation experiments and confocal analyses in cell cycle synchronized cells respectively revealed direct interactions and co-localization between PIGN and the SAC proteins MAD1, MAD2, as well as the mitotic kinase MPS1 thus unveiling a novel spatiotemporal regulatory mechanism. PIGN physically interacts with and regulates the SAC via MAD1, MAD2, MPS1 and BUBR1 during mitotic cell cycle progression. The co-purification of PIGN with some of these mitotic checkpoint proteins showed the direct role that PIGN may play in the regulation of mitotic checkpoint signaling. Thus, PIGN as a CIN suppressor may be crucial in the regulation of mitotic integrity via the SAC as part of maintaining genome stability.Despite the ubiquity of CIN in leukemia progression, there is still limited knowledge about the mechanism(s) involved. Also, since its discovery as a CIN suppressor, the molecular mechanism by which the loss of PIGN leads to CIN has until now remained elusive. However, this study revealed a novel mechanism in which PIGN may maintain genome stability via SAC regulation. Our findings open the possibility to study PIGN as a tumor suppressor because its loss significantly altered the expression of SAC-related proteins. Ultimately, PIGN modulation could be adopted as a therapeutic approach in leukemia treatment, more specifically in the averting leukemia progression in high-risk MDS patients. DisclosuresNo relevant conflicts of interest to declare.

Distinct Roles of RZZ and Bub1-KNL1 in Mitotic Checkpoint Signaling and Kinetochore Expansion

The Mad1-Mad2 heterodimer is the catalytic hub of the spindle assembly checkpoint (SAC), which controls M phase progression through a multi-subunit anaphase inhibitor, the mitotic checkpoint complex (MCC) [1, 2]. During interphase, Mad1-Mad2 generates MCC at nuclear pores [3]. After nuclear envelope breakdown (NEBD), kinetochore-associated Mad1-Mad2 catalyzes MCC assembly until all chromosomes achieve bipolar attachment [1, 2]. Mad1-Mad2 and other factors are also incorporated into the fibrous corona, a phospho-dependent expansion of the outer kinetochore that precedes microtubule attachment [4-6]. The factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes remain controversial [7-12], and the specific phosphorylation event(s) that trigger corona formation remain elusive [5, 13]. We used genome editing to eliminate Bub1, KNL1, and the Rod-Zw10-Zwilch (RZZ) complex in human cells. We show that RZZ's sole role in SAC activation is to tether Mad1-Mad2 to kinetochores. Separately, Mps1 kinase triggers fibrous corona formation by phosphorylating two N-terminal sites on Rod. In contrast, Bub1 and KNL1 activate kinetochore-bound Mad1-Mad2 to produce a "wait anaphase" signal but are not required for corona formation. We also show that clonal lines isolated after BUB1 disruption recover Bub1 expression and SAC function through nonsense-associated alternative splicing (NAS). Our study reveals a fundamental division of labor in the mammalian SAC and highlights a transcriptional response tononsense mutations that can reduce or eliminate penetrance in genome editing experiments.

422P - First-in-human study of the monopolar spindle 1 (Mps1) kinase inhibitor BAY 1161909 in combination with paclitaxel in subjects with advanced malignancies

422P - First-in-human study of the monopolar spindle 1 (Mps1) kinase inhibitor BAY 1161909 in combination with paclitaxel in subjects with advanced malignancies

Leader of the SAC: molecular mechanisms of Mps1/TTK regulation in mitosis.

Discovered in 1991 in a screen for genes involved in spindle pole body duplication, the monopolar spindle 1 (Mps1) kinase has since claimed a central role in processes that ensure error-free chromosome segregation. As a result, Mps1 kinase activity has become an attractive candidate for pharmaceutical companies in the search for compounds that target essential cellular processes to eliminate, for example, tumour cells or pathogens. Research in recent decades has offered many insights into the molecular function of Mps1 and its regulation. In this review, we integrate the latest knowledge regarding the regulation of Mps1 activity and its spatio-temporal distribution, highlight gaps in our understanding of these processes and propose future research avenues to address them.

Key phosphorylation events in Spc29 and Spc42 guide multiple steps of yeast centrosome duplication.

Phosphorylation modulates many cellular processes during cell cycle progression. The yeast centrosome (called the spindle pole body, SPB) is regulated by the protein kinases Mps1 and Cdc28/Cdk1 as it nucleates microtubules to separate chromosomes during mitosis. Previously we completed an SPB phosphoproteome, identifying 297 sites on 17 of the 18 SPB components. Here we describe mutagenic analysis of phosphorylation events on Spc29 and Spc42, two SPB core components that were shown in the phosphoproteome to be heavily phosphorylated. Mutagenesis at multiple sites in Spc29 and Spc42 suggests that much of the phosphorylation on these two proteins is not essential but enhances several steps of mitosis. Of the 65 sites examined on both proteins, phosphorylation of the Mps1 sites Spc29-T18 and Spc29-T240 was shown to be critical for function. Interestingly, these two sites primarily influence distinct successive steps; Spc29-T240 is important for the interaction of Spc29 with Spc42, likely during satellite formation, and Spc29-T18 facilitates insertion of the new SPB into the nuclear envelope and promotes anaphase spindle elongation. Phosphorylation sites within Cdk1 motifs affect function to varying degrees, but mutations only have significant effects in the presence of an MPS1 mutation, supporting a theme of coregulation by these two kinases.

Abstract 1849: Determining the molecular requirements for paclitaxel-induced spindle multipolarity

Abstract Paclitaxel (TaxolTM) and other microtubule poisons are standard chemotherapy agents used in the treatment of breast cancer. However, only a subset of patients experience positive therapeutic effects from these drugs, underscoring the importance of further mechanistic study. Recently we showed that paclitaxel does not accumulate in primary breast cancers at sufficient concentrations to cause mitotic arrest, as it does at typically used concentrations in cell culture. Instead, paclitaxel therapy induces multipolar spindles in breast cancers. Breast cancer cell lines treated with low nanomolar, clinically relevant doses of paclitaxel exhibit chromosome missegregation due to multipolar divisions. We have now extended this to demonstrate that low nanomolar concentrations of other clinically used microtubule poisons (including ixabepilone, vinorelbine, and eribulin) also induce multipolarity in breast cancer cells, suggesting that multipolar mitotic divisions may be a common mechanism of cytotoxicity for microtubule-targeted drugs. Importantly, supernumerary centrosomes are not required for microtubule poison-induced multipolar spindles. To gain mechanistic insight into the formation of paclitaxel-induced multipolar spindles, a variety of mitotic regulators were tested for their ability to affect spindle multipolarity in breast cancer cells treated with clinically relevant concentrations of paclitaxel. Our results demonstrate that the mitotic kinesin Eg5 and the kinase Plk1 promote both the formation and the maintenance of paclitaxel-induced spindle multipolarity, while the mitotic kinesin CENP-E and the kinase Mps1 do not. Interestingly, we found that decreasing the time to anaphase onset decreases the number of multipolar divisions in paclitaxel, resulting in reduced cytotoxicity. Previous studies have shown that Mps1 inhibition sensitizes cells to paclitaxel, and this combination therapy has entered clinical trials (NCT02138812 and NCT02366949). However, our evidence suggests that Mps1 inhibition reduces the efficacy of paclitaxel as a result of decreased time to anaphase onset and fewer multipolar divisions. Additionally, our results suggest that inhibitors of spindle pole clustering increase the number of paclitaxel-induced multipolar divisions and therefore have the potential to increase its efficacy. Identification of the cellular requirements for multipolarity induced by paclitaxel, and of agents that drive the metaphase-to-anaphase transition in the presence of multipolar spindles, may improve the clinical utility of paclitaxel and other microtubule poisons. Citation Format: Christina Scribano. Determining the molecular requirements for paclitaxel-induced spindle multipolarity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1849.