Drugs targeting the spindle assembly checkpoint (SAC), such as inhibitors of Aurora kinase B (AURKB) and dual specific protein kinase TTK, are in different stages of clinical development. However, cell response to SAC abrogation is poorly understood and there are no markers for patient selection.
A panel of 53 tumor cell lines of different origins was used. The effects of drugs were analyzed by MTT and flow cytometry. Copy number status was determined by FISH and Q-PCR; mRNA expression by nCounter and RT-Q-PCR and protein expression by Western blotting. CRISPR-Cas9 technology was used for gene knock-out (KO) and a doxycycline-inducible pTRIPZ vector for ectopic expression. Finally, in vivo experiments were performed by implanting cultured cells or fragments of tumors into immunodeficient mice.
Tumor cells and patient-derived xenografts (PDXs) sensitive to AURKB and TTK inhibitors consistently showed high expression levels of BH3-interacting domain death agonist (BID), while cell lines and PDXs with low BID were uniformly resistant. Gene silencing rendered BID-overexpressing cells insensitive to SAC abrogation while ectopic BID expression in BID-low cells significantly increased sensitivity. SAC abrogation induced activation of CASP-2, leading to cleavage of CASP-3 and extensive cell death only in presence of high levels of BID. Finally, a prevalence study revealed high BID mRNA in 6% of human solid tumors.
The fate of tumor cells after SAC abrogation is driven by an AURKB/ CASP-2 signaling mechanism, regulated by BID levels. Our results pave the way to clinically explore SAC-targeting drugs in tumors with high BID expression.

Cells evoke the DNA damage checkpoint (DDC) to inhibit mitosis in the presence of DNA double-strand breaks (DSBs) to allow more time for DNA repair. In budding yeast, a single irreparable DSB is sufficient to activate the DDC and induce cell cycle arrest prior to anaphase for about 12 to 15 hours, after which cells \"adapt\" to the damage by extinguishing the DDC and resuming the cell cycle. While activation of the DNA damage-dependent cell cycle arrest is well-understood, how it is maintained remains unclear. To address this, we conditionally depleted key DDC proteins after the DDC was fully activated and monitored changes in the maintenance of cell cycle arrest. Degradation of Ddc2ATRIP, Rad9, Rad24, or Rad53CHK2 results in premature resumption of the cell cycle, indicating that these DDC factors are required both to establish and to maintain the arrest. Dun1 is required for establishment, but not maintenance of arrest, whereas Chk1 is required for prolonged maintenance but not for initial establishment of the mitotic arrest. When the cells are challenged with 2 persistent DSBs, they remain permanently arrested. This permanent arrest is initially dependent on the continuous presence of Ddc2 and Rad53; however, after 15 hours both proteins become dispensable. Instead, the continued mitotic arrest is sustained by spindle-assembly checkpoint (SAC) proteins Mad1, Mad2, and Bub2 but not by Bub2\'s binding partner Bfa1. These data suggest that prolonged cell cycle arrest in response to 2 DSBs is achieved by a handoff from the DDC to specific components of the SAC. Furthermore, the establishment and maintenance of DNA damage-induced cell cycle arrest requires overlapping but different sets of factors.

Aneuploidy is the gain or loss of entire chromosomes, chromosome arms or fragments. Over 100 years ago, aneuploidy was described to be a feature of cancer and is now known to be present in 68-90% of malignancies. Aneuploidy promotes cancer growth, reduces therapy response and frequently worsens prognosis. Chromosomal instability (CIN) is recognized as the main cause of aneuploidy. CIN itself is a dynamic but stochastic process consisting of different DNA content-altering events. These can include impaired replication fidelity and insufficient clearance of DNA damage as well as chromosomal mis-segregation, micronuclei formation, chromothripsis or cytokinesis failure. All these events can disembogue in segmental, structural and numerical chromosome alterations. While low levels of CIN can foster malignant disease, high levels frequently trigger cell death, which supports the \"aneuploidy paradox\" that refers to the intrinsically negative impact of a highly aberrant karyotype on cellular fitness. Here, we review how the cellular response to CIN and aneuploidy can drive the clearance of karyotypically unstable cells through the induction of apoptosis. Furthermore, we discuss the different modes of p53 activation triggered in response to mitotic perturbations that can potentially trigger CIN and/or aneuploidy.

The study of the size of cells and organelles has a long history, dating back to the 1600s when cells were defined. In particular, various methods have elucidated the size of the nucleus and the mitotic spindle in several species. However, little research has been conducted on oocyte size and organelles in mammals, and many questions remain to be answered. The appropriate size is essential to cell function properly. Oocytes have a very large cytoplasm, which is more than 100 times larger than that of general somatic cells in mammals. In this review, we discuss how oocytes acquire an enormous cytoplasmic size and the adverse effects of a large cytoplasmic size on cellular functions.

The kinetochore is the multiprotein complex of eukaryotic organisms that is assembled on mitotic or meiotic centromeres to connect centromeric DNA with microtubules. Its function involves the coordinated action of more than 100 different proteins. The kinetochore acts as an organiser hub that establishes physical connections with microtubules and centromere-associated proteins and recruits central protein components of the spindle assembly checkpoint (SAC), an evolutionarily conserved surveillance mechanism of eukaryotic organisms that detects unattached kinetochores and destabilises incorrect kinetochore-microtubule attachments. The molecular communication between the kinetochore and the SAC is highly dynamic and tightly regulated to ensure that cells can progress towards anaphase until each chromosome is properly bi-oriented on the mitotic spindle. This is achieved through an interplay of highly cooperative interactions and concerted phosphorylation/dephosphorylation events that are organised in time and space.This contribution discusses our current understanding of the function, structure and regulation of the kinetochore, in particular, how its communication with the SAC results in the amplification of specific signals to exquisitely control the eukaryotic cell cycle. This contribution also addresses recent advances in machine learning approaches, cell imaging and proteomics techniques that have enhanced our understanding of the molecular mechanisms that ensure the high fidelity and timely segregation of the genetic material every time a cell divides as well as the current challenges in the study of this fascinating molecular machine.

The Anaphase Promoting Complex (APC/C), a large cullin-RING E3-type ubiquitin ligase, constitutes the ultimate target of the Spindle Assembly Checkpoint (SAC), an intricate regulatory circuit that ensures the high fidelity of chromosome segregation in eukaryotic organisms by delaying the onset of anaphase until each chromosome is properly bi-oriented on the mitotic spindle. Cell-division cycle protein 20 homologue (CDC20) is a key regulator of APC/C function in mitosis. The formation of the APC/CCDC20 complex is required for the ubiquitination and degradation of select substrates, which is necessary to maintain the mitotic state. In contrast to the roles of CDC20 in animal species, little is known about CDC20 roles in the regulation of chromosome segregation in plants. Here we address this gap in knowledge and report the expression in insect cells; the biochemical and biophysical characterisation of Arabidopsis thaliana (AtCDC20) WD40 domain; and the nuclear and cytoplasmic distribution of full-length AtCDC20 when transiently expressed in tobacco plants. We also show that most AtCDC20 degrons share a high sequence similarity to other eukaryotes, arguing in favour of conserved degron functions in AtCDC20. However, important exceptions were noted such as the lack of a canonical MAD1 binding motif; a fully conserved RRY-box in all six AtCDC20 isoforms instead of a CRY-box motif, and low conservation of key residues known to be phosphorylated by BUB1 and PLK1 in other species to ensure a robust SAC response. Taken together, our studies provide insights into AtCDC20 structure and function and the evolution of SAC signalling in plants.

Pre-mRNA processing factor 4 kinase (PRP4K, also known as PRPF4B) is an essential kinase first identified in the fission yeast Schizosaccharomyces pombe that is evolutionarily conserved from amoebae to animals. During spliceosomal assembly, PRP4K interacts with and phosphorylates PRPF6 and PRPF31 to facilitate the formation of the spliceosome B complex. However, over the past decade additional evidence has emerged that PRP4K has many diverse cellular roles beyond splicing that contribute to tumour suppression and chemotherapeutic responses in mammals. For example, PRP4K appears to play roles in regulating transcription and the spindle assembly checkpoint (SAC), a key pathway in maintaining chromosomes stability and the response of cancer cells to taxane-based chemotherapy. In addition, PRP4K has been revealed to be a haploinsufficient tumour suppressor that promotes aggressive cancer phenotypes when partially depleted. PRP4K is regulated by both the HER2 and estrogen receptor, and its partial loss increases resistance to the taxanes in multiple malignancies including cervical, breast and ovarian cancer. Moreover, ovarian and triple negative breast cancer patients harboring tumours with low PRP4K expression exhibit worse overall survival. The depletion of PRP4K also enhances both Yap and epidermal growth factor receptor (EGFR) signaling, the latter promoting anoikis resistance in breast and ovarian cancer. Finally, PRP4K is negatively regulated during epithelial-to-mesenchymal transition (EMT), a process that promotes increased cell motility, drug resistance and cancer metastasis. Thus, as we discuss in this review, PRP4K likely plays evolutionarily conserved roles not only in splicing but in a number of cellular pathways that together contribute to tumour suppression.

Cellular function is highly dependent on genomic stability, which is mainly ensured by two cellular mechanisms: the DNA damage response (DDR) and the Spindle Assembly Checkpoint (SAC). The former provides the repair of damaged DNA, and the latter ensures correct chromosome segregation. This review focuses on recently emerging data indicating that the SAC and the DDR proteins function together throughout the cell cycle, suggesting crosstalk between both checkpoints to maintain genome stability.

Embryonic stem (ES) cells are pluripotent cells widely used in cell therapy and tissue engineering. However, the broader clinical applications of ES cells are limited by their genomic instability and karyotypic abnormalities. Thus, understanding the mechanisms underlying ES cell karyotypic abnormalities is critical to optimizing their clinical use. In this study, we focused on proliferating human and mouse ES cells undergoing multipolar divisions. Specifically, we analyzed the frequency and outcomes of such divisions using a combination of time-lapse microscopy and cell tracking. This revealed that cells resulting from multipolar divisions were not only viable, but they also frequently underwent subsequent cell divisions. Our novel data also showed that in human and mouse ES cells, multipolar spindles allowed more robust escape from chromosome segregation control mechanisms than bipolar spindles. Considering the frequency of multipolar divisions in proliferating ES cells, it is conceivable that cell division errors underlie ES cell karyotypic instability.

The Mad2 protein plays a key role in the spindle assembly checkpoint (SAC) function. The SAC pathway delays mitotic progression into anaphase until all kinetochores attach to the spindle during mitosis. The formation of the Mad2-p31comet complex correlates with the completion of spindle attachment and the entry into anaphase during mitosis. Herein, we showed that dynein intermediate chain 2c (DNCI2c)-a subunit of dynein motor protein-forms an immunocomplex with p31comet during mitosis. DNCI2c-knockdown resulted in prolonged mitotic arrest in a Mad2-dependent manner. Furthermore, DNCI2c-knockdown-induced mitotic arrest was not rescued by p31comet overexpression. However, the combination of p31comet overexpression with the mitotic drug treatment reversed the mitotic arrest in DNCI2c-knockdown. Together, these results indicate that the DNCI2c-p31comet complex plays an important role in exiting Mad2-dependent SAC.