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Head and neck cancer (HNC), the sixth most common cancer worldwide, is increasing in incidence, with oral squamous cell carcinoma (OSCC) as the predominant subtype. OSCC mainly affects middle-aged to elderly males, often occurring on the posterior lateral border of the tongue, leading to significant disfigurement and functional impairments, such as swallowing and speech difficulties. Despite advancements in understanding OSCC's genetic and epigenetic variations, survival rates for advanced stages remain low, highlighting the need for new treatment options. Primary treatment includes surgery, often combined with radiotherapy (RT) and chemotherapy (CT). Cetuximab-based chemotherapy, targeting the overexpressed epidermal growth factor receptor (EGFR) in 80–90% of HNCs, is commonly used but correlates with poor prognosis. Additionally, monopolar spindle 1 (MPS1), a spindle assembly checkpoint (SAC) component, is a significant target due to its role in genomic fidelity during mitosis and its overexpression in several cancers. This review explores EGFR and MPS1 as therapeutic targets in HNC, analyzing their molecular mechanisms and the effects of their inhibition on cancer cells. It also highlights the promise of combinatorial approaches, such as microtubule-targeting agents (MTAs) and antimitotic agents, in improving HNC therapies, patient outcomes, and survival rates. [ABSTRACT FROM AUTHOR]

The spindle assembly checkpoint (SAC) proteins are conserved among eukaryotes safeguarding chromosome segregation fidelity during mitosis. However, their biological functions in plant‐pathogenic fungi remain largely unknown. In this study, we found that the SAC protein MoMad1 in rice blast fungus (Magnaporthe oryzae) localizes on the nuclear envelope and is dispensable for M. oryzae vegetative growth and tolerance to microtubule depolymerizing agent treatment. MoMad1 plays an important role in M. oryzae infection‐related development and pathogenicity. The monopolar spindle 1 homologue in M. oryzae (MoMps1) interacts with MoMad1 through its N‐terminal domain and phosphorylates MoMad1 at Ser‐18, which is conserved within the extended N termini of Mad1s from fungal plant pathogens. This phosphorylation is required for maintaining MoMad1 protein abundance and M. oryzae full virulence. Similar to the deletion of MoMad1, treatment with Mps1‐IN‐1 (an Mps1 inhibitor) caused compromised appressorium formation and decreased M. oryzae virulence, and these defects were dependent on its attenuating MoMad1 Ser‐18 phosphorylation. Therefore, our study indicates the function of Mad1 in rice blast fungal pathogenicity and sheds light on the potential of blocking Mad1 phosphorylation by Mps1 to control crop fungal diseases. [ABSTRACT FROM AUTHOR]

Head and neck cancer (HNC), the sixth most common cancer worldwide, is increasing in incidence, with oral squamous cell carcinoma (OSCC) as the predominant subtype. OSCC mainly affects middle-aged to elderly males, often occurring on the posterior lateral border of the tongue, leading to significant disfigurement and functional impairments, such as swallowing and speech difficulties. Despite advancements in understanding OSCC’s genetic and epigenetic variations, survival rates for advanced stages remain low, highlighting the need for new treatment options. Primary treatment includes surgery, often combined with radiotherapy (RT) and chemotherapy (CT). Cetuximab-based chemotherapy, targeting the overexpressed epidermal growth factor receptor (EGFR) in 80–90% of HNCs, is commonly used but correlates with poor prognosis. Additionally, monopolar spindle 1 (MPS1), a spindle assembly checkpoint (SAC) component, is a significant target due to its role in genomic fidelity during mitosis and its overexpression in several cancers. This review explores EGFR and MPS1 as therapeutic targets in HNC, analyzing their molecular mechanisms and the effects of their inhibition on cancer cells. It also highlights the promise of combinatorial approaches, such as microtubule-targeting agents (MTAs) and antimitotic agents, in improving HNC therapies, patient outcomes, and survival rates.

The spindle assembly checkpoint (SAC) is a cellular surveillance mechanism that functions to ensure accurate chromosome segregation during mitosis. Macromolecular complexes known as kinetochores, act as the interface of sister chromatid attachment to spindle microtubules. In response to unattached kinetochores, the SAC activates its effector, the mitotic checkpoint complex (MCC), which delays mitotic exit until all sister chromatid pairs have achieved successful attachment to the bipolar mitotic spindle. Formation of the MCC (composed of Mad2, BubR1, Bub3 and Cdc20) is regulated by an Mps1 kinase‐dependent phosphorylation signaling cascade which assembles and repositions components of the MCC onto a catalytic scaffold. This scaffold functions to catalyze the conversion of the HORMA‐domain protein Mad2 from an "inactive" open‐state (O‐Mad2) into an "active" closed‐Mad2 (C‐Mad2), and simultaneous Cdc20 binding. Here, our current understanding of the molecular mechanisms underlying the kinetic barrier to C‐Mad2:Cdc20 formation will be reviewed. Recent progress in elucidating the precise molecular choreography orchestrated by the catalytic scaffold to rapidly assemble the MCC will be examined, and unresolved questions will be highlighted. Ultimately, understanding how the SAC rapidly activates the checkpoint not only provides insights into how cells maintain genomic integrity during mitosis, but also provides a paradigm for how cells can utilize molecular switches, including other HORMA domain‐containing proteins, to make rapid changes to a cell's physiological state. [ABSTRACT FROM AUTHOR]

The protein kinase Mps1 (monopolar spindle 1) is an important regulator of the Spindle Assembly Checkpoint (SAC), the evolutionary conserved checkpoint system of higher organisms that monitors the proper bipolar attachment of all chromosomes to the mitotic spindle during cell division. Defects in the catalytic activity and the transcription regulation of Mps1 are associated with genome instability, aneuploidy, and cancer. Moreover, multiple Mps1 missense and frameshift mutations have been reported in a wide range of types of cancer of different tissue origin. Due to these features, Mps1 arises as one promising drug target for cancer therapy. In this contribution, we developed a computational biology approach to study the dynamics of human Mps1 kinase interaction with isoflavones, a class of natural flavonoids, and compared their predicted mode of binding with that observed in the crystal structure of Mps1 in complex with reversine, a small-sized inhibitor of Mps1 and Aurora B kinases. We concluded that isoflavones define a chemical scaffold that can be used to develop new Mps1 inhibitors for the treatment of cancer associated with Mps1 amplification and aberrant chromosome segregation. In a broader context, the present report illustrates how modern chemoinformatics approaches can accelerate drug development in oncology. [ABSTRACT FROM AUTHOR]

Abstract Background It has been reported that the oncoprotein E7 from human papillomavirus type 16 (HPV16-E7) can induce the excessive synthesis of centrosomes through the increase in the expression of PLK4, which is a transcriptional target of E2F1. On the other hand, it has been reported that increasing MPS1 protein stability can also generate an excessive synthesis of centrosomes. In this work, we analyzed the possible role of MPS1 in the amplification of centrosomes mediated by HPV16-E7. Results Employing qRT-PCR, Western Blot, and Immunofluorescence techniques, we found that E7 induces an increase in the MPS1 transcript and protein levels in the U2OS cell line, as well as protein stabilization. Besides, we observed that inhibiting the expression of MPS1 in E7 protein-expressing cells leads to a significant reduction in the number of centrosomes. Conclusions These results indicate that the presence of the MPS1 protein is necessary for E7 protein to increase the number of centrosomes, and possible implications are discussed.

S81694 is an inhibitor of monopolar spindle 1 kinase, a target expressed in proliferating cells. CL1-81694-001 was the first-in-human study aiming at identifying a safe dosing schedule in solid tumour patients. This trial was based on inter-individual dose-escalation of single agent S81694 in cohorts of ≥3 patients to assess the safety and tolerability and determine dose-limiting toxicities (DLTs), maximum tolerated dose (MTD) and recommended phase II dose (RP2D), with S81694 given on days 1,8,15 of a 28-day cycle as 1-h infusion. 38 patients were treated at doses ranging from 4 to 135 mg/m2/week; 144 cycles were administered (median 2/patient; range 1–32 cycles). Patients discontinued treatment for disease progression (78.9%), adverse events (AE; 18.4%) or withdrawal of consent (2.6%). Treatment modifications occurred in 22 patients (57.9%; 49 cycles). Common treatment-emergent AEs were fatigue (22 patients;57.9%), anaemia (17;44.7%) and nausea (12;31.6%). Haematological toxicity was mild, with Grade 3 anaemia observed in three patients and neutropenia mainly seen at the 135 mg/m2 dose level. Three first cycle DLTs included G3 anaemia (4 mg/m2 dose), G4 hypertension (20 mg/m2), G3 fatigue (135 mg/m2). MTD was not reached due to premature discontinuation of enrolment based on a sponsor decision. Among 35 patients evaluable for response, one (renal cell carcinoma) had a complete response, one (hepatocellular carcinoma) had a transient decrease of target lesions and 13 had stable disease. Seven patients remained on study for ≥6 cycles, two at the 135 mg/m2 dose. S81694 can be administered safely as a single agent in adults with solid tumours on days 1,8,15 of a 28-day cycle up to a dose of 135 mg/m2/week without reaching MTD. The RP2D was not defined due to the prioritization of the use of S81694 in combination with cytotoxic agents, based on emerging preclinical data. EudraCT number: 2014-002023-10; ISRCTN registry ISRCTN35641359. • First trial with monopolar spindle 1 kinase inhibitor in solid tumour patients. • MTD not reached when exploring doses up to 135 mg/m2/week. • Well tolerated and early signs of anti-tumour activity. • Further development warranted, ideally in combination with other drugs. [ABSTRACT FROM AUTHOR]

For correct chromosome segregation in mitosis, eukaryotic cells must establish chromosome biorientation where sister kinetochores attach to microtubules extending from opposite spindle poles. To establish biorientation, any aberrant kinetochore–microtubule interactions must be resolved in the process called error correction. For resolution of the aberrant interactions in error correction, kinetochore–microtubule interactions must be exchanged until biorientation is formed (the SWAP process). At initiation of biorientation, the state of weak kinetochore–microtubule interactions should be converted to the state of stable interactions (the SWITCH process)—the conundrum of this conversion is called the initiation problem of biorientation. Once biorientation is established, tension is applied on kinetochore–microtubule interactions, which stabilizes the interactions (the STABILIZE process). Aurora B kinase plays central roles in promoting error correction, and Mps1 kinase and Stu2 microtubule polymerase also play important roles. In this article, we review mechanisms of error correction by considering the SWAP, SWITCH, and STABILIZE processes. We mainly focus on mechanisms found in budding yeast, where only one microtubule attaches to a single kinetochore at biorientation, making the error correction mechanisms relatively simpler. [ABSTRACT FROM AUTHOR]

The Mps1 and Aurora B kinases regulate and monitor kinetochore attachment to spindle microtubules during cell division, ultimately ensuring accurate chromosome segregation. In yeast, the critical spindle attachment components are the Ndc80 and Dam1 complexes (Ndc80c and DASH/Dam1c, respectively). Ndc80c is a 600-Å-long heterotetramer that binds microtubules through a globular "head" at one end and centromere-proximal kinetochore components through a globular knob at the other end. Dam1c is a heterodecamer that forms a ring of 16–17 protomers around the shaft of the single kinetochore microtubule in point-centromere yeast. The ring coordinates the approximately eight Ndc80c rods per kinetochore. In published work, we showed that a site on the globular "head" of Ndc80c, including residues from both Ndc80 and Nuf2, binds a bipartite segment in the long C-terminal extension of Dam1. Results reported here show, both by in vitro binding experiments and by crystal structure determination, that the same site binds a conserved segment in the long N-terminal extension of Mps1. It also binds, less tightly, a conserved segment in the N-terminal extension of Ipl1 (yeast Aurora B). Together with results from experiments in yeast cells and from biochemical assays reported in two accompanying papers, the structures and graded affinities identify a communication hub for ensuring uniform bipolar attachment and for signaling anaphase onset. • The kinase Mps1 binds the Ndc80 complex through a conserved motif • X-ray crystallography shows a common Ndc80c binding site for Mps1 and Dam1 • The Ipl1 N-terminal extension recognizes the Ndc80c site bound by Mps1 and Dam1 • Alphafold predictions suggest broad conservation of these shared interactions Zahm and Harrison combine binding studies and structure determinations to identify a common interaction site on the Ndc80 complex of yeast kinetochores for kinases Mps1 and Ipl1 and for the DASH subunit, Dam1. Sequences and alphafold predictions suggest a widely conserved regulatory hub on Ndc80c, consistent with data in accompanying articles. [ABSTRACT FROM AUTHOR]

Accurate chromosome segregation relies on kinetochores carrying out multiple functions, including establishing and maintaining microtubule attachments, forming precise bi-oriented attachments between sister chromatids, and activating the spindle assembly checkpoint. Central to these processes is the highly conserved Ndc80 complex. This kinetochore subcomplex interacts directly with microtubules but also serves as a critical platform for recruiting kinetochore-associated factors and as a key substrate for error correction kinases. The precise manner in which these kinetochore factors interact and regulate each other's function remains unknown, considerably hindering our understanding of how Ndc80 complex-dependent processes function together to orchestrate accurate chromosome segregation. Here, we aimed to uncover the role of Nuf2's CH domain, a component of the Ndc80 complex, in ensuring these processes. Through extensive mutational analysis, we identified a conserved interaction domain composed of two segments in Nuf2's CH domain that form the binding site for Mps1 within the yeast Ndc80 complex. Interestingly, this site also associates with the Dam1 complex, suggesting Mps1 recruitment may be subject to regulation by competitive binding with other factors. Mutants disrupting this "interaction hub" exhibit defects in spindle assembly checkpoint function and severe chromosome segregation errors. Significantly, specifically restoring Mps1-Ndc80 complex association rescues these defects. Our findings shed light on the intricate regulation of Ndc80 complex-dependent functions and highlight the essential role of Mps1 in kinetochore bi-orientation and accurate chromosome segregation. [Display omitted] • Two segments of Nuf2's CH domain recruit the Mps1 kinase to kinetochores • Other factors, including the Dam1 complex, also bind this "interaction hub" • Kinetochore-bound Mps1 promotes recruitment of the Dam1 complex • Association of Mps1 with the hub is essential for bi-orientation and cell viability Parnell et al. identify an "interaction hub" in the Ndc80 complex via mutational analysis in yeast, which associates with both Mps1 and Dam1 complex. Mutants at this site show inviability due to severe defects in bi-orientation and chromosome segregation; however, restoring Mps1-Ndc80c association rescues viability, highlighting its critical role. [ABSTRACT FROM AUTHOR]

The CRISPRi system using dCas9 Sth1 from Streptococcus thermophilus developed for Mycobacterium tuberculosis and M. smegmatis was modified to allow gene knock-out in M. abscessus. Efficacy of the knock-out system was evaluated by applying deletions and insertions to the mps1 gene. A comparative genomic analysis of mutants and wild type validated the target specificity. • Streptococcus thermophilus CRISPR/Cas (Cas9 Sth1) used for gene knock-out in Mycobacterium abscessus. • A large variety of deletion size obtained with CRISPR/Cas9 Sth1 in M. abscessus. • High gene target specificity provided by CRISPR/ Cas9 Sth1 in M. abscessus. [ABSTRACT FROM AUTHOR]

Background: It has been reported that the oncoprotein E7 from human papillomavirus type 16 (HPV16-E7) can induce the excessive synthesis of centrosomes through the increase in the expression of PLK4, which is a transcriptional target of E2F1. On the other hand, it has been reported that increasing MPS1 protein stability can also generate an excessive synthesis of centrosomes. In this work, we analyzed the possible role of MPS1 in the amplification of centrosomes mediated by HPV16-E7. Results: Employing qRT-PCR, Western Blot, and Immunofluorescence techniques, we found that E7 induces an increase in the MPS1 transcript and protein levels in the U2OS cell line, as well as protein stabilization. Besides, we observed that inhibiting the expression of MPS1 in E7 protein-expressing cells leads to a significant reduction in the number of centrosomes. Conclusions: These results indicate that the presence of the MPS1 protein is necessary for E7 protein to increase the number of centrosomes, and possible implications are discussed. [ABSTRACT FROM AUTHOR]

The mucopolysaccharidoses (MPS), Pompe Disease (PD), and Krabbe disease (KD) are inherited conditions known as lysosomal storage disorders (LSDs) The resulting enzyme deficiencies give rise to progressive symptoms. The United States Department of Health and Human Services’ Recommended Uniform Screening Panel (RUSP) suggests LSDs for inclusion in state universal newborn screening (NBS) programs and has identified screening deficiencies in MPS I, KD, and PD NBS programs. MPS I NBS programs utilize newborn dried blood spots and assay alpha L-iduronidase (IDUA) enzyme to screen for potential cases. Glycosaminoglycans (GAGs) offer potential as a confirmatory test. KD NBS programs utilize galactocerebrosidase (GaLC) as an initial test, with psychosine (PSY) activity increasingly used as a confirmatory test for predicting onset of Krabbe disease, though with an excessive false positive rate. PD is marked by a deficiency in acid α-glucosidase (GAA), causing increased glycogen, creatine (CRE), and other biomarkers. Bivariate normal limit (BVNL) methods have been applied to GaLC and PSY activity to produce a NBS tool for KD, and more recently, to IDUA and GAG activity to develop a NBS tool for MPS I. A BVNL tool based on GAA and CRE is in development for infantile PD diagnosis. Early infantile KD, MPS I, and PD cases were pre-symptomatically identified by BVNL-based NBS tools. This article reviews these developments, discusses how they address screening deficiencies identified by the RUSP and may improve NBS more generally.

The protein kinase Mps1 (monopolar spindle 1) is an important regulator of the Spindle Assembly Checkpoint (SAC), the evolutionary conserved checkpoint system of higher organisms that monitors the proper bipolar attachment of all chromosomes to the mitotic spindle during cell division. Defects in the catalytic activity and the transcription regulation of Mps1 are associated with genome instability, aneuploidy, and cancer. Moreover, multiple Mps1 missense and frameshift mutations have been reported in a wide range of types of cancer of different tissue origin. Due to these features, Mps1 arises as one promising drug target for cancer therapy. In this contribution, we developed a computational biology approach to study the dynamics of human Mps1 kinase interaction with isoflavones, a class of natural flavonoids, and compared their predicted mode of binding with that observed in the crystal structure of Mps1 in complex with reversine, a small-sized inhibitor of Mps1 and Aurora B kinases. We concluded that isoflavones define a chemical scaffold that can be used to develop new Mps1 inhibitors for the treatment of cancer associated with Mps1 amplification and aberrant chromosome segregation. In a broader context, the present report illustrates how modern chemoinformatics approaches can accelerate drug development in oncology.

The goal of mitosis is to form two daughter cells each containing one copy of each mother cell chromosome, replicated in the previous S phase. To achieve this, sister chromatids held together back-to-back at their primary constriction, the centromere, have to interact with microtubules of the mitotic spindle so that each chromatid takes connections with microtubules emanating from opposite spindle poles (we will refer to this condition as bipolar attachment). Only once all replicated chromosomes have reached bipolar attachments can sister chromatids lose cohesion with each other, at the onset of anaphase, and move toward opposite spindle poles, being segregated into what will soon become the daughter cell nucleus. Prevention of errors in chromosome segregation is granted by a safeguard mechanism called Spindle Assembly Checkpoint (SAC). Until all chromosomes are bipolarly oriented at the equator of the mitotic spindle, the SAC prevents loss of sister chromatid cohesion, thus anaphase onset, and maintains the mitotic state by inhibiting inactivation of the major M phase promoting kinase, the cyclin B-cdk1 complex (Cdk1). Here, we review recent mechanistic insights about the circuitry that links Cdk1 to the SAC to ensure correct achievement of the goal of mitosis.

Reversine is a selective inhibitor of mitotic kinase monopolar spindle 1 (MPS1) and has been reported as an anticancer agent in various cancers. The effects of reversine on bile duct cancer, cholangiocarcinoma (CCA), a lethal cancer in Northeastern Thailand, were investigated. This study reports that reversine inhibited cell proliferation of CCA cell lines in dose- and time-dependent manners but had less inhibitory effect on an immortalized cholangiocyte cell line. Reversine also triggered apoptotic cell death by decreasing anti-apoptotic proteins, Bcl-XL and Mcl-1, increasing Bax pro-apoptotic protein and activating caspase-3 activity. Moreover, reversine induced autophagic cell death by increasing LC3-II and Beclin 1 while decreasing p62. Reversine activated autophagy via the AKT signaling pathway. Additionally, this study demonstrated for the first time that reversine could diminish the expression of Hypoxia-Inducible Factor 1-alpha (HIF-1α) and glucose transporter 1 (GLUT1), resulting in a reduction of glucose uptake and energy production in CCA cell lines. These findings suggest that reversine could be a good candidate as an alternative or supplementary drug for CCA treatment. [ABSTRACT FROM AUTHOR]

During mitosis, the identical sister chromatids of each chromosome must attach through their kinetochores to microtubules emanating from opposite spindle poles. This process, referred to as chromosome biorientation, is essential for equal partitioning of the genetic information to the two daughter cells. Defects in chromosome biorientation can give rise to aneuploidy, a hallmark of cancer and genetic diseases. A conserved surveillance mechanism called spindle assembly checkpoint (SAC) prevents the onset of anaphase until biorientation is attained. Key to chromosome biorientation is an error correction mechanism that allows kinetochores to establish proper bipolar attachments by disengaging faulty kinetochore–microtubule connections. Error correction relies on the Aurora B and Mps1 kinases that also promote SAC signaling, raising the possibility that they are part of a single sensory device responding to improper attachments and concomitantly controlling both their disengagement and a temporary mitotic arrest. In budding yeast, Aurora B and Mps1 promote error correction independently from one another, but while the substrates of Aurora B in this process are at least partially known, the mechanism underlying the involvement of Mps1 in the error correction pathway is unknown. Through the characterization of a novel mps1 mutant and an unbiased genetic screen for extragenic suppressors, we recently gained evidence that a common mechanism based on Mps1-dependent phosphorylation of the Knl1/Spc105 kinetochore scaffold and subsequent recruitment of the Bub1 kinase is critical for the function of Mps1 in chromosome biorientation as well as for SAC activation (Benzi et al. EMBO Rep, 2020). [ABSTRACT FROM AUTHOR]

The relationship between kinetochores and nuclear pore complexes (NPCs) is intimate but poorly understood. Several NPC components and associated proteins are relocated to mitotic kinetochores to assist in different activities that ensure faithful chromosome segregation. Such is the case of the Mad1‐c‐Mad2 complex, the catalytic core of the spindle assembly checkpoint (SAC), a surveillance pathway that delays anaphase until all kinetochores are attached to spindle microtubules. Mad1‐c‐Mad2 is recruited to discrete domains of unattached kinetochores from where it promotes the rate‐limiting step in the assembly of anaphase‐inhibitory complexes. SAC proficiency further requires Mad1‐c‐Mad2 to be anchored at NPCs during interphase. However, the mechanistic relevance of this arrangement for SAC function remains ill‐defined. Recent studies uncover the molecular underpinnings that coordinate the release of Mad1‐c‐Mad2 from NPCs with its prompt recruitment to kinetochores. Here, current knowledge on Mad1‐c‐Mad2 function and spatiotemporal regulation is reviewed and the critical questions that remain unanswered are highlighted. [ABSTRACT FROM AUTHOR]

The Mps1 kinase corrects improper kinetochore–microtubule attachments, thereby ensuring chromosome biorientation. Yet, its critical phosphorylation targets in this process remain largely elusive. Mps1 also controls the spindle assembly checkpoint (SAC), which halts chromosome segregation until biorientation is attained. Its role in SAC activation is antagonised by the PP1 phosphatase and involves phosphorylation of the kinetochore scaffold Knl1/Spc105, which in turn recruits the Bub1 kinase to promote assembly of SAC effector complexes. A crucial question is whether error correction and SAC activation are part of a single or separable pathways. Here, we isolate and characterise a new yeast mutant, mps1‐3, that is severely defective in chromosome biorientation and SAC signalling. Through an unbiased screen for extragenic suppressors, we found that mutations lowering PP1 levels at Spc105 or forced association of Bub1 with Spc105 reinstate both chromosome biorientation and SAC signalling in mps1‐3 cells. Our data argue that a common mechanism based on Knl1/Spc105 phosphorylation is critical for Mps1 function in error correction and SAC signalling, thus supporting the idea that a single sensory apparatus simultaneously elicits both pathways. Synopsis: Through isolation and characterization of the new mps1‐3 mutant, this study proposes that Mps1‐dependent phosphorylation of Spc105 contributes to both proper chromosome segregation and the spindle assembly checkpoint in budding yeast. mps1‐3 temperature‐sensitive mutant fails to correct faulty kinetochore‐microtubule attachments and activate the spindle assembly checkpoint.Mps1‐3 is hyperactive and does not localize at kinetochores.Suppressing mutations reducing PP1 activity at kinetochores restore proper chromosome segregation and checkpoint signalling in mps1‐3 mutant cells.The mps1‐3 mutant is defective in Spc105 phosphorylation and Bub1 kinetochore recruitment.Artificial recruitment of Bub1 to Spc105 rescues the chromosome segregation and checkpoint defects of mps1‐3 cells. [ABSTRACT FROM AUTHOR]

Aneuploid human eggs (oocytes) are a major cause of infertility, miscarriage, and chromosomal disorders. Such aneuploidies increase greatly as women age, with defective linkages between sister chromatids (cohesion) in meiosis as a common cause. We found that loss of a specific pool of the cohesin protector protein, shugoshin 2 (SGO2), may contribute to this phenomenon. Our data indicate that SGO2 preserves sister chromatid cohesion in meiosis by protecting a "cohesin bridge" between sister chromatids. In human oocytes, SGO2 localizes to both sub-centromere cups and the pericentromeric bridge, which spans the sister chromatid junction. SGO2 normally colocalizes with cohesin; however, in meiosis II oocytes from older women, SGO2 is frequently lost from the pericentromeric bridge and sister chromatid cohesion is weakened. MPS1 and BUB1 kinase activities maintain SGO2 at sub-centromeres and the pericentromeric bridge. Removal of SGO2 throughout meiosis I by MPS1 inhibition reduces cohesion protection, increasing the incidence of single chromatids at meiosis II. Therefore, SGO2 deficiency in human oocytes can exacerbate the effects of maternal age by rendering residual cohesin at pericentromeres vulnerable to loss in anaphase I. Our data show that impaired SGO2 localization weakens cohesion integrity and may contribute to the increased incidence of aneuploidy observed in human oocytes with advanced maternal age. [Display omitted] • SGO2 localizes to centromeric cups and the pericentromeric bridge in human oocytes • Human SGO2 prevents premature loss of cohesion and aneuploidy during meiosis • Bridge SGO2 is lost in human oocytes from women of advanced age • MPS1 and BUB1 kinases contribute to SGO2 localization in human oocytes Human fertility declines with maternal age due to aneuploidy affecting the oocyte. Mihalas et al. use super-resolution microscopy in human oocytes and provide evidence that age-dependent loss of the SGO2 cohesin protector protein could contribute to this phenomenon. [ABSTRACT FROM AUTHOR]

Bub1 is a conserved mitotic kinase involved in signaling of the spindle assembly checkpoint. Multiple phosphorylation sites on Bub1 have been characterized, yet it is challenging to understand the interplay between the multiple phosphorylation sites due to the limited availability of phosphospecific antibodies. In addition, phosphoregulation of Bub1 in Schizosaccharomyces pombe is poorly understood. Here we report the identification of a new Mph1/Mps1-mediated phosphorylation site, i.e., Ser532, of Bub1 in Schizosaccharomyces pombe. A phosphospecific antibody against phosphorylated Bub1-Ser532 was developed. Using the phosphospecific antibody, we demonstrated that phosphorylation of Bub1-Ser352 was mediated specifically by Mph1/Mps1 and took place during early mitosis. Moreover, live-cell microscopy showed that inhibition of the phosphorylation of Bub1 at Ser532 impaired the localization of Bub1, Mad1, and Mad2 to the kinetochore. In addition, inhibition of the phosphorylation of Bub1 at Ser532 caused anaphase B lagging chromosomes. Hence, our study constitutes a model in which Mph1/Mps1-mediated phosphorylation of fission yeast Bub1 promotes proper kinetochore localization of Bub1 and faithful chromosome segregation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Aneuploidy is a condition in which cells have an abnormal number of chromosomes that is not a multiple of the haploid complement. It is known that aneuploidy has detrimental consequences on cell physiology, such as genome instability, metabolic and proteotoxic stress and decreased cellular fitness. Importantly, aneuploidy is a hallmark of tumors and it is associated with resistance to chemotherapeutic agents and poor clinical outcome. To shed light into how aneuploidy contributes to chemoresistance, we induced chromosome mis-segregation in human cancer cell lines, then treated them with several chemotherapeutic agents and evaluated the emergence of chemoresistance. By doing so, we found that elevation of chromosome mis-segregation promotes resistance to chemotherapeutic agents through the expansion of aneuploid karyotypes and subsequent selection of specific aneuploidies essential for cellular viability under those stressful conditions. Here, we describe a method to generate aneuploid cell populations and to evaluate their resistance to anti-cancer agents. This protocol has been already successfully employed and can be further utilized to accelerate the exploration of the role of aneuploidy in chemoresistance., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

In mitosis, the spindle assembly checkpoint (SAC) monitors the formation of microtubule‐kinetochore attachments during capture of chromosomes by the mitotic spindle. Spindle assembly is complete once there are no longer any unattached kinetochores. Here, we will discuss the mechanism and key components of spindle checkpoint signalling. Unattached kinetochores bind the principal spindle checkpoint kinase monopolar spindle 1 (MPS1). MPS1 triggers the recruitment of other spindle checkpoint proteins and the formation of a soluble inhibitor of anaphase, thus preventing exit from mitosis. On microtubule attachment, kinetochores become checkpoint silent due to the actions of PP2A‐B56 and PP1. This SAC responsive period has to be coordinated with mitotic spindle formation to ensure timely mitotic exit and accurate chromosome segregation. We focus on the molecular mechanisms by which the SAC permissive state is created, describing a central role for CDK1‐cyclin B1 and its counteracting phosphatase PP2A‐B55. Furthermore, we discuss how CDK1‐cyclin B1, through its interaction with MAD1, acts as an integral component of the SAC, and actively orchestrates checkpoint signalling and thus contributes to the faithful execution of mitosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Chemically induced dimerisation (CID) uses small molecules to control specific protein-protein interactions. We employed CID dependent on the plant hormone abscisic acid (ABA) to reconstitute spindle checkpoint signalling in fission yeast. The spindle checkpoint signal usually originates at unattached or inappropriately attached kinetochores. These are complex, multiprotein structures with several important functions. To bypass kinetochore complexity, we took a reductionist approach to studying checkpoint signalling. We generated a synthetic checkpoint arrest ectopically by inducing heterodimerisation of the checkpoint proteins Mph1 (the fission yeast homologue of Mps1) and Spc7 (the fission yeast homologue of KNL1). These proteins were engineered such that they cannot localise to kinetochores, and only form a complex in the presence of ABA. Using this novel assay we were able to checkpoint arrest a synchronous population of cells within 30 min of ABA addition. This assay allows detailed genetic dissection of checkpoint activation and, importantly, also provides a valuable tool for studying checkpoint silencing. To analyse silencing of the checkpoint and the ensuing mitotic exit, we simply washed out the ABA from arrested fission yeast cells. We show here that silencing is critically dependent on protein phosphatase 1 (PP1) recruitment to Mph1-Spc7 signalling platforms. [ABSTRACT FROM AUTHOR]

Cholangiocarcinoma (CCA) is a malignancy of the bile duct epithelium. The major problems of this cancer are late diagnosis and a high rate of metastasis. CCA patients in advanced stages have poor survival and cannot be cured with surgery. Therefore, targeting molecules involved in the metastatic process may be an effective CCA treatment. Monopolar spindle 1 (MPS1) is a kinase protein that controls the spindle assemble checkpoint in mitosis. It is overexpressed in proliferating cells and various cancers. The functional roles of MPS1 in CCA progression have not been investigated. The aims of this study were to examine the roles and molecular mechanisms of MPS1 in CCA progression. Immunohistochemistry results showed that MPS1 was up-regulated in carcinogenesis of CCA in a hamster model, and positive expression of MPS1 in human CCA tissues was correlated to short survival of CCA patients (n = 185). Small interfering RNA (siRNA)-induced knockdown of MPS1 expression reduced cell proliferation via G2/M arrest, colony formation, migration, and invasion. Moreover, MPS1 controlled epithelial to mesenchymal transition (EMT)-mediated migration via AKT and STAT3 signaling transductions. MPS1 was also involved in MMPs-dependent invasion of CCA cell lines. The current research highlights for the first time that MPS1 has an essential role in promoting the progression of CCA via AKT and STAT3 signaling pathways and could be an attractive target for metastatic CCA treatment.

c-Src tyrosine kinase plays roles in a wide range of signaling events and its increased activity is frequently observed in a variety of epithelial and non-epithelial cancers. v-Src, an oncogene first identified in the Rous sarcoma virus, is an oncogenic version of c-Src and has constitutively active tyrosine kinase activity. We previously showed that v-Src induces Aurora B delocalization, resulting in cytokinesis failure and binucleated cell formation. In the present study, we explored the mechanism underlying v-Src-induced Aurora B delocalization. Treatment with the Eg5 inhibitor (+)-S-trityl-L-cysteine (STLC) arrested cells in a prometaphase-like state with a monopolar spindle; upon further inhibition of cyclin-dependent kinase (CDK1) by RO-3306, cells underwent monopolar cytokinesis with bleb-like protrusions. Aurora B was localized to the protruding furrow region or the polarized plasma membrane 30 min after RO-3306 addition, whereas inducible v-Src expression caused Aurora B delocalization in cells undergoing monopolar cytokinesis. Delocalization was similarly observed in monopolar cytokinesis induced by inhibiting Mps1, instead of CDK1, in the STLC-arrested mitotic cells. Importantly, western blotting analysis and in vitro kinase assay revealed that v-Src decreased the levels of Aurora B autophosphorylation and its kinase activity. Furthermore, like v-Src, treatment with the Aurora B inhibitor ZM447439 also caused Aurora B delocalization at concentrations that partially inhibited Aurora B autophosphorylation. Given that phosphorylation of Aurora B by v-Src was not observed, these results suggest that v-Src causes Aurora B delocalization by indirectly suppressing Aurora B kinase activity. • CDK1 inhibition of STLC-arrested cells induces monopolar cytokinesis. • Aurora B localization can be analyzed in monopolar cytokinesis cells. • MPS1 inhibition instead of CDK1 inhibition causes monopolar cytokinesis in STLC-arrested cells. • v-Src reduces Aurora B kinase activity, causing the Aurora B delocalization. • LATS kinases inhibition causes the Aurora B delocalization. [ABSTRACT FROM AUTHOR]

Leaves play a key role in photosynthesis in rice plants. The premature senescence of such plants directly reduces the accumulation of photosynthetic products and also affects yield and grain quality significantly and negatively. A novel premature senescence mutant, mps1 (mid-late stage premature senescence 1), was identified from a mutant library consisting of ethyl methane sulfonate (EMS) induced descendants of Jinhui 10, an elite indica restorer line of rice. The mutant allele, mps1, caused no phenotypic differences from the wild type (WT), Jinhui 10, but drove the leaves to turn yellow when mutant plants grew to the tillering stage, and accelerated leaf senescence from the filling stage to final maturation. We characterized the agronomic traits, content of photosynthetic pigments and photosynthetic efficiency of mps1 and WT, and fine-mapped MPS1. The results showed that the MPS1-drove premature phenotype appeared initially on the leaf tips at the late tillering stage and extended to the middle of leaves during the maturing stage. Compared to the WT, significant differences were observed among traits of the number of grains per panicle (–31.7%) and effective number of grains per panicle (–38.5%) of mps1 individuals. Chlorophyll contents among the first leaf from the top (Top 1st), the second leaf from the top (Top 2nd) and the third leaf from the top (Top 3rd) of mps1 were significantly lower than those of WT (P

The spindle assembly checkpoint (SAC) is a key mechanism to regulate the timing of mitosis and ensure that chromosomes are correctly segregated to daughter cells. The recruitment of the Mad1 and Mad2 proteins to the kinetochore is normally necessary for SAC activation. This recruitment is coordinated by the SAC kinase Mps1, which phosphorylates residues at the kinetochore to facilitate binding of Bub1, Bub3, Mad1, and Mad2. There is evidence that the essential function of Mps1 is to direct recruitment of Mad1/2. To test this model, we have systematically recruited Mad1, Mad2, and Mps1 to most proteins in the yeast kinetochore, and find that, while Mps1 is sufficient for checkpoint activation, recruitment of either Mad1 or Mad2 is not. These data indicate an important role for Mps1 phosphorylation in SAC activation, beyond the direct recruitment of Mad1 and Mad2.

The molecular chaperone Hsp90 protects deregulated signaling proteins that are vital for tumor growth and survival. Tumors generally display sensitivity and selectivity toward Hsp90 inhibitors; however, the molecular mechanism underlying this phenotype remains undefined. We report that the mitotic checkpoint kinase Mps1 phosphorylates a conserved threonine residue in the amino-domain of Hsp90. This, in turn, regulates chaperone function by reducing Hsp90 ATPase activity while fostering Hsp90 association with kinase clients, including Mps1. Phosphorylation of Hsp90 is also essential for the mitotic checkpoint because it confers Mps1 stability and activity. We identified Cdc14 as the phosphatase that dephosphorylates Hsp90 and disrupts its interaction with Mps1. This causes Mps1 degradation, thus providing a mechanism for its inactivation. Finally, Hsp90 phosphorylation sensitizes cells to its inhibitors, and elevated Mps1 levels confer renal cell carcinoma selectivity to Hsp90 drugs. Mps1 expression level can potentially serve as a predictive indicator of tumor response to Hsp90 inhibitors.

Supernumerary centrosomes promote the assembly of abnormal spindles in many human cancers. The observation that modest changes in the centrosomal levels of Mps1 kinase can cause centrosome overduplication in human cells suggests the existence of a regulatory system that may tightly control its centrosomal stability. Here, we show that Cdkn3, a Cdk-associated phosphatase, prevents Mps1-mediated centrosome overduplication. We identify Cdkn3 as a direct binding partner of Mps1. The interaction between Mps1 and Cdkn3 is required for Mps1 to recruit Cdkn3 to centrosomes. Subsequently, Mps1-bound Cdkn3 forms a regulatory system that controls the centrosomal levels of Mps1 through proteasome-mediated degradation and thereby prevents Mps1-mediated centrosome overduplication. Conversely, knockdown of Cdkn3 stabilizes Mps1 at centrosomes, which promotes centrosome overduplication. We suggest that Mps1 and Cdkn3 form a self-regulated feedback loop at centrosomes to tightly control the centrosomal levels of Mps1, which prevents centrosome overduplication in human cells.

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.

Cell division in mitosis and meiosis is governed by evolutionary highly conserved protein kinases and phosphatases, controlling the timely execution of key events such as nuclear envelope breakdown, spindle assembly, chromosome attachment to the spindle and chromosome segregation, and cell cycle exit. In mitosis, the spindle assembly checkpoint (SAC) controls the proper attachment to and alignment of chromosomes on the spindle. The SAC detects errors and induces a cell cycle arrest in metaphase, preventing chromatid separation. Once all chromosomes are properly attached, the SAC-dependent arrest is relieved and chromatids separate evenly into daughter cells. The signaling cascade leading to checkpoint arrest depends on several protein kinases that are conserved from yeast to man. In meiosis, haploid cells containing new genetic combinations are generated from a diploid cell through two specialized cell divisions. Though apparently less robust, SAC control also exists in meiosis. Recently, it has emerged that SAC kinases have additional roles in executing accurate chromosome segregation during the meiotic divisions. Here, we summarize the main differences between mitotic and meiotic cell divisions, and explain why meiotic divisions pose special challenges for correct chromosome segregation. The less-known meiotic roles of the SAC kinases are described, with a focus on two model systems: yeast and mouse oocytes. The meiotic roles of the canonical checkpoint kinases Bub1, Mps1, the pseudokinase BubR1 (Mad3), and Aurora B and C (Ipl1) will be discussed. Insights into the molecular signaling pathways that bring about the special chromosome segregation pattern during meiosis will help us understand why human oocytes are so frequently aneuploid.

In previous studies, the B‑Raf proto‑oncogene, serine/threonine kinase (BRAF)V600E mutation has been identified in multiple malignant tumors. BRAFV600E has been revealed to contribute to tumorigenesis by the activation of phospho‑mitogen‑activated protein kinases (MAPKs) and their downstream Monopolar spindle 1 (Mps1), leading to chromosome euploidy and tumor development. In the present study, the presence of phospho‑MAPK and Mps1 in 161 thyroid carcinoma cases with complete clinical parameters was analyzed by immunohistochemistry, and the BRAF mutation was detected by polymerase chain reaction‑direct sequencing. It was revealed that BRAFV600E was present in ~34% of thyroid cancer cases and was associated with age, clinical tumor stage and lymph node stage. However, the association of BRAFV600E with overall survival was not statistically significant. The expression of Mps1 was significantly increased in tumor tissues with BRAFV600E, however, this did not affect the expression of phospho‑MAPK in thyroid carcinomas. Collectively, the results of the present study suggested that BRAFV600E may regulate the expression of Mps1 in MAP kinase independent ways in thyroid carcinoma. Therefore, Mps1 expression is associated with BRAFV600E while the upstream signaling of phospho‑MAPK has no relevance. The specific mechanisms of BRAFV600E and the unknown pathway associated with Mps1 exhibit potential for further study, and provide a theoretical basis for the molecular treatment of thyroid carcinoma. [ABSTRACT FROM AUTHOR]

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit.

Drosophila Schneider 2 (S2) cells are a simple and powerful system commonly used in cell biology because they are well suited for high resolution microscopy and RNAi-mediated depletion. However, understanding dynamic processes, such as cell division, also requires methodology to interfere with protein function with high spatiotemporal control. In this research study, we report the adaptation of an optogenetic tool to Drosophila S2 cells. Light-activated reversible inhibition by assembled trap (LARIAT) relies on the rapid light-dependent heterodimerization between cryptochrome 2 (CRY2) and cryptochrome-interacting bHLH 1 (CIB1) to form large protein clusters. An anti-green fluorescent protein (GFP) nanobody fused with CRY2 allows this method to quickly trap any GFP-tagged protein in these light-induced protein clusters. We evaluated clustering kinetics in response to light for different LARIAT modules, and showed the ability of GFP-LARIAT to inactivate the mitotic protein Mps1 and to disrupt the membrane localization of the polarity regulator Lethal Giant Larvae (Lgl). Moreover, we validated light-induced co-clustering assays to assess protein-protein interactions in S2 cells. In conclusion, GFP-based LARIAT is a versatile tool to answer different biological questions, since it enables probing of dynamic processes and protein-protein interactions with high spatiotemporal resolution in Drosophila S2 cells.

Cells undergoing meiosis produce haploid gametes through one round of DNA replication followed by 2 rounds of chromosome segregation. This requires that cohesin complexes, which establish sister chromatid cohesion during S phase, are removed in a stepwise manner. At meiosis I, the separase protease triggers the segregation of homologous chromosomes by cleaving cohesin's Rec8 subunit on chromosome arms. Cohesin persists at centromeres because the PP2A phosphatase, recruited by the shugoshin protein, dephosphorylates Rec8 and thereby protects it from cleavage. While chromatids disjoin upon cleavage of centromeric Rec8 at meiosis II, it was unclear how and when centromeric Rec8 is liberated from its protector PP2A. One proposal is that bipolar spindle forces separate PP2A from Rec8 as cells enter metaphase II. We show here that sister centromere biorientation is not sufficient to “deprotect” Rec8 at meiosis II in yeast. Instead, our data suggest that the ubiquitin-ligase APC/CCdc20removes PP2A from centromeres by targeting for degradation the shugoshin Sgo1 and the kinase Mps1. This implies that Rec8 remains protected until entry into anaphase II when it is phosphorylated concurrently with the activation of separase. Here, we provide further support for this model and speculate on its relevance to mammalian oocytes. [ABSTRACT FROM PUBLISHER]

Monopolar spindle1 (Mps1, also known as TTK) is the core component of the spindle assembly checkpoint, which functions to ensure proper distribution of chromosomes to daughter cells. Mps1 is hardly detectable in normal organs except the testis and placenta. However, high levels of Mps1 are found in many types of human malignancies, including glioblastoma, thyroid carcinoma, breast cancer, and other cancers. Several Mps1 inhibitors can inhibit the proliferation of cancer cells and exhibit demonstrable survival benefits. Mps1 can be utilized as a new immunogenic epitope, which is able to induce potent cytotoxic T lymphocyte activity against cancer cells while sparing normal cells. Some clinical trials have validated its safety, immunogenicity and clinical response. Thus, Mps1 may be a novel target for cancer therapy. Mps1 is differentially expressed between normal and malignant tissues, indicating its potential as a molecular biomarker for diagnosis. Meanwhile, the discovery that it clearly correlates with recurrence and survival time suggests it may serve as an independent prognostic biomarker as well. [ABSTRACT FROM PUBLISHER]

Summary The spindle checkpoint acts as a mitotic surveillance system, monitoring interactions between kinetochores and spindle microtubules and ensuring high-fidelity chromosome segregation [ 1–3 ]. The checkpoint is activated by unattached kinetochores, and Mps1 kinase phosphorylates KNL1 on conserved MELT motifs to generate a binding site for the Bub3-Bub1 complex [ 4–7 ]. This leads to dynamic kinetochore recruitment of Mad proteins [ 8, 9 ], a conformational change in Mad2 [ 10–12 ], and formation of the mitotic checkpoint complex (MCC: Cdc20-Mad3-Mad2 [ 13–15 ]). MCC formation inhibits the anaphase-promoting complex/cyclosome (Cdc20-APC/C), thereby preventing the proteolytic destruction of securin and cyclin and delaying anaphase onset. What happens at kinetochores after Mps1-dependent Bub3-Bub1 recruitment remains mechanistically unclear, and it is not known whether kinetochore proteins other than KNL1 have significant roles to play in checkpoint signaling and MCC generation. Here, we take a reductionist approach, avoiding the complexities of kinetochores, and demonstrate that co-recruitment of KNL1 Spc7 and Mps1 Mph1 is sufficient to generate a robust checkpoint signal and prolonged mitotic arrest. We demonstrate that a Mad1-Bub1 complex is formed during synthetic checkpoint signaling. Analysis of bub3 Δ mutants demonstrates that Bub3 acts to suppress premature checkpoint signaling. This synthetic system will enable detailed, mechanistic dissection of MCC generation and checkpoint silencing. After analyzing several mutants that affect localization of checkpoint complexes, we conclude that spindle checkpoint arrest can be independent of their kinetochore, spindle pole, and nuclear envelope localization. [ABSTRACT FROM AUTHOR]

The spindle- and kinetochore-associated (Ska) complex is essential for normal anaphase onset in mitosis. The C-terminal domain (CTD) of Ska1 binds microtubules and was proposed to facilitate kinetochore movement on depolymerizing spindle microtubules. Here, we show that Ska complex recruits protein phosphatase 1 (PP1) to kinetochores. This recruitment requires the Ska1 CTD, which binds PP1 in vitro and in human HeLa cells. Ska1 lacking its CTD fused to a PP1-binding peptide or fused directly to PP1 rescues mitotic defects caused by Ska1 depletion. Ska1 fusion to catalytically dead PP1 mutant does not rescue and shows dominant negative effects. Thus, the Ska complex, specifically the Ska1 CTD, recruits PP1 to kinetochores to oppose spindle checkpoint signaling kinases and promote anaphase onset. Microtubule binding by Ska, rather than acting in force production for chromosome movement, may instead serve to promote PP1 recruitment to kinetochores fully attached to spindle microtubules at metaphase.

Mono-polar spindle 1 (Mps1/TTK) represents a protein kinase reported to be vital for cell division processes and is generally regarded as an attractive target for the treatment of hepatocellular carcinoma, breast carcinoma, and colon cancer. However, the C604Y mutation has been linked to acquired resistance. Recently, three potential small-molecule inhibitors of Mps1 (i.e., reversine, NMS-P715, and its derivative Cpd-5) were reported for the C604Y mutation that exhibit significant resistance to NMS-P715 and Cpd-5, but retain affinity for reversine. In this study, classical molecular dynamic (MD) simulations, accelerated MD (aMD) simulations, and umbrella sampling (US) simulations were performed to illustrate the resistance mechanisms of inhibitors to Mps1. The classical MD simulations combined with free energy calculations revealed that reversine features similar binding affinity characteristics to both Mps1WT and Mps1C604Y, but both NMS-P715 and Cpd-5 feature much higher binding affinities to Mps1WT than to Mps1C604Y. The major variations were shown to be controlled by electrostatic energy and the conformational change of A-loop-induced entropy increased. The large conformational changes of Mps1C604Y bound to NMS-P715 and Cpd-5 were also observed in aMD simulations. The US simulation results further suggest that reversine and Cpd-5 both exhibit similar dissociation processes from both Mps1WT and Mps1C604Y, but Cpd-5 and NMS-P715 were found to dissociate more easily from Mps1C604Y than from Mps1WT, thus a reduced residence time was responsible for the inhibitors resistance to the C604Y mutation. The physical principles provided by the present study may provide important clues for the discovery and rational design of novel inhibitors to combat the C604Y mutation of Mps1.

Monopolar spindle kinase 1 (Mps1), a core component of the spindle assembly checkpoint (SAC), plays a crucial role in the transition of cells from mid-to late mitosis. As an attractive therapeutic target, inhibition of Mps1 induces cell cycle arrest and apoptosis in a variety of tumors, including breast cancer. However, early clinical development of Mps1 inhibitors remains unsatisfactory. Here, we designed and synthesized a new class of Mps1 inhibitors with 7 H -pyrrolo[2,3- d ]pyrimidine structure using a scaffold hopping approach. Structure–activity relationship (SAR) revealed that 12 is a potent Mps1 inhibitor (IC 50 = 29 nM), which inhibited phosphorylation of Mps1 in vitro and in vivo. Treatment with 12 not only impeded proliferation of breast cancer cell lines, but also induced cell cycle arrest and apoptosis of MCF-7 and 4T1 cells. 12 suppressed tumor growth in vivo , and no obvious toxicities were observed. These results demonstrated the potential of Mps1 inhibitor 12 for the treatment of breast cancer. [Display omitted] • A new class of Mps1 inhibitors were designed using scaffold hopping strategies. • The most potent compound 12 inhibited proliferation and colony formation of breast cancer cells. • 12 induced G2/M arrest and cell apoptosis of MCF-7 and 4T1 cells. • 12 effectively restrained tumor growth and was tolerated in vivo. • 12 inhibited phosphorylation of Mps1 both in vitro and in vivo. [ABSTRACT FROM AUTHOR]