The therapeutic potential of pharmacologically targeting kinases involved in regulating the DNA damage response (DDR) has been investigated for over two decades. Inhibitors of ATM, ATR, CHK1, CHK2 and WEE1 have been developed with the aim of subverting cell cycle checkpoint function in cancer cells, promoting cell death. The DNA repair pathway non-homologous end-joining can also be targeted through DNA-PK inhibition. However, despite extensive preclinical and clinical studies, none of the many candidate inhibitors have yet made it through to clinical approval. Emerging evidence for tumour biomarkers associated with enhanced sensitivity to DDR kinase inhibition may provide a way through this impasse. Clinical testing in appropriately stratified cohorts is now becoming increasingly common, with some promising results. Building on results obtained with small-molecule inhibitors, targeted protein degradation (TPD) utilising proteolysis-targeting chimaeras (PROTACs) or molecular glues for degradation of DDR kinases is a rapidly developing strategy. This review discusses the current ATM, ATR, DNA-PK, CHK1, CHK2 and WEE1 inhibitors that show the most promise as monotherapies and combination treatments in solid tumours, as well as the potential benefits of using TPD technology over small-molecule inhibitors. Established and emerging biomarkers that can be applied to patient selection are also discussed.

Cancer cells often display centrosome amplification, requiring the kinesin KIFC1/HSET for centrosome clustering to prevent multipolar spindles and cell death. In parallel siRNA screens of deubiquitinase enzymes, we identify OTUD6B as a positive regulator of KIFC1 expression that is required for centrosome clustering in triple-negative breast cancer (TNBC) cells. OTUD6B can localise to centrosomes and the mitotic spindle and interacts with KIFC1. In OTUD6B-deficient cells, we see increased KIFC1 polyubiquitination and premature KIFC1 degradation during mitosis. Depletion of OTUD6B increases multipolar spindles without inducing centrosome amplification. Phenotypic rescue is dependent on OTUD6B catalytic activity and evident upon KIFC1 overexpression. OTUD6B is commonly overexpressed in breast cancer, correlating with KIFC1 protein expression and worse patient survival. TNBC cells with centrosome amplification, but not normal breast epithelial cells, depend on OTUD6B to proliferate. Indeed CRISPR-Cas9 editing results in only OTUD6B-/+ TNBC cells which fail to divide and die. As a deubiquitinase that supports KIFC1 expression, allowing pseudo-bipolar cell division and survival of cancer cells with centrosome amplification, OTUD6B has potential as a novel target for cancer-specific therapies.

Uveal melanoma (UM) is the most common intraocular cancer in adults. Whilst treatment of primary UM (PUM) is often successful, around 50% of patients develop metastatic disease with poor outcomes, linked to chromosome 3 loss (monosomy 3, M3). Advances in understanding UM cell biology may indicate new therapeutic options. We report that UM exhibits centrosome abnormalities, which in other cancers are associated with increased invasiveness and worse prognosis, but also represent a potential Achilles' heel for cancer-specific therapeutics. Analysis of 75 PUM patient samples revealed both higher centrosome numbers and an increase in centrosomes with enlarged pericentriolar matrix (PCM) compared to surrounding normal tissue, both indicative of centrosome amplification. The PCM phenotype was significantly associated with M3 (t-test, p < 0.01). Centrosomes naturally enlarge as cells approach mitosis; however, whilst UM with higher mitotic scores had enlarged PCM regardless of genetic status, the PCM phenotype remained significantly associated with M3 in UM with low mitotic scores (ANOVA, p = 0.021) suggesting that this is independent of proliferation. Phenotypic analysis of patient-derived cultures and established UM lines revealed comparable levels of centrosome amplification in PUM cells to archetypal triple-negative breast cancer cell lines, whilst metastatic UM (MUM) cell lines had even higher levels. Importantly, many UM cells also exhibit centrosome clustering, a common strategy employed by other cancer cells with centrosome amplification to survive cell division. As UM samples with M3 display centrosome abnormalities indicative of amplification, this phenotype may contribute to the development of MUM, suggesting that centrosome de-clustering drugs may provide a novel therapeutic approach.

Due to cell-cycle dysregulation, many cancer cells contain more than the normal compliment of centrosomes, a state referred to as centrosome amplification (CA). CA can drive oncogenic phenotypes and indeed can cause cancer in flies and mammals. However, cells have to actively manage CA, often by centrosome clustering, in order to divide. Thus, CA is also an Achilles' Heel of cancer cells. In recent years, there have been many important studies identifying proteins required for the management of CA and it has been demonstrated that disruption of some of these proteins can cause cancer-specific inhibition of cell growth. For certain targets therapeutically relevant interventions are being investigated, for example, small molecule inhibitors, although none are yet in clinical trials. As the field is now poised to move towards clinically relevant interventions, it is opportune to summarise the key work in targeting CA thus far, with particular emphasis on recent developments where small molecule or other strategies have been proposed. We also highlight the relatively unexplored paradigm of reversing CA, and thus its oncogenic effects, for therapeutic gain.

Ubiquitin-specific protease 15 (USP15) is a widely expressed deubiquitylase that has been implicated in diverse cellular processes in cancer. Here we identify topoisomerase II (TOP2A) as a novel protein that is regulated by USP15. TOP2A accumulates during G2 and functions to decatenate intertwined sister chromatids at prophase, ensuring the replicated genome can be accurately divided into daughter cells at anaphase. We show that USP15 is required for TOP2A accumulation, and that USP15 depletion leads to the formation of anaphase chromosome bridges. These bridges fail to decatenate, and at mitotic exit form micronuclei that are indicative of genome instability. We also describe the cell cycle-dependent behaviour for two major isoforms of USP15, which differ by a short serine-rich insertion that is retained in isoform-1 but not in isoform-2. Although USP15 is predominantly cytoplasmic in interphase, we show that both isoforms move into the nucleus at prophase, but that isoform-1 is phosphorylated on its unique S229 residue at mitotic entry. The micronuclei phenotype we observe on USP15 depletion can be rescued by either USP15 isoform and requires USP15 catalytic activity. Importantly, however, an S229D phospho-mimetic mutant of USP15 isoform-1 cannot rescue either the micronuclei phenotype, or accumulation of TOP2A. Thus, S229 phosphorylation selectively abrogates this role of USP15 in maintaining genome integrity in an isoform-specific manner. Finally, we show that USP15 isoform-1 is preferentially upregulated in a panel of non-small cell lung cancer cell lines, and propose that isoform imbalance may contribute to genome instability in cancer. Our data provide the first example of isoform-specific deubiquitylase phospho-regulation and reveal a novel role for USP15 in guarding genome integrity.

Post-translational modification of proteins by ubiquitylation is increasingly recognised as a highly complex code that contributes to the regulation of diverse cellular processes. In humans, a family of almost 100 deubiquitylase enzymes (DUBs) are assigned to six subfamilies and many of these DUBs can remove ubiquitin from proteins to reverse signals. Roles for individual DUBs have been delineated within specific cellular processes, including many that are dysregulated in diseases, particularly cancer. As potentially druggable enzymes, disease-associated DUBs are of increasing interest as pharmaceutical targets. The biology, structure and regulation of DUBs have been extensively reviewed elsewhere, so here we focus specifically on roles of DUBs in regulating cell cycle processes in mammalian cells. Over a quarter of all DUBs, representing four different families, have been shown to play roles either in the unidirectional progression of the cell cycle through specific checkpoints, or in the DNA damage response and repair pathways. We catalogue these roles and discuss specific examples. Centrosomes are the major microtubule nucleating centres within a cell and play a key role in forming the bipolar mitotic spindle required to accurately divide genetic material between daughter cells during cell division. To enable this mitotic role, centrosomes undergo a complex replication cycle that is intimately linked to the cell division cycle. Here, we also catalogue and discuss DUBs that have been linked to centrosome replication or function, including centrosome clustering, a mitotic survival strategy unique to cancer cells with supernumerary centrosomes.

The study of clathrin-coated vesicles (CCVs) isolated from various organs has revealed the identities and important features of many of the factors involved in membrane trafficking. The development of isolation methods using cultured cell lines has made it possible to manipulate the source material before isolation to ask important questions about the roles of these factors and the pathways in which they are involved. We discuss here the advantages and limitations of the use of cultured cell lines for the isolation of CCVs.

Clathrin-mediated endocytosis (CME) is the major internalisation route for many different receptor types in mammalian cells. CME is shut down during early mitosis, but the mechanism of this inhibition is unclear. In this study, we show that the mitotic shutdown is due to an unmet requirement for actin in CME. In mitotic cells, membrane tension is increased and this invokes a requirement for the actin cytoskeleton to assist the CME machinery to overcome the increased load. However, the actin cytoskeleton is engaged in the formation of a rigid cortex in mitotic cells and is therefore unavailable for deployment. We demonstrate that CME can be ‘restarted’ in mitotic cells despite high membrane tension, by allowing actin to engage in endocytosis. Mitotic phosphorylation of endocytic proteins is maintained in mitotic cells with restored CME, indicating that direct phosphorylation of the CME machinery does not account for shutdown.

The characterization of clathrin-coated vesicles (CCVs), including the effects of genetic or biochemical manipulations on their composition, can be studied by mass spectrometry analysis of HeLa cell fractions enriched for CCVs. This protocol describes the preparation of samples by in-solution proteolytic digest and subsequent peptide fractionation, followed by analysis in a Q Exactive mass spectrometer.

The characterization of clathrin-coated vesicles (CCVs), including the effects of genetic or biochemical manipulations on their composition, can be studied by mass spectrometry analysis of HeLa cell fractions enriched for CCVs. This protocol describes the preparation of samples by tryptic in-gel digest and peptide extraction followed by analysis in an Orbitrap mass spectrometer.

HeLa cell lines can be experimentally manipulated using drugs or gene-silencing techniques such as RNA interference. Fractions enriched for clathrin-coated vesicles (CCVs) can be isolated from these cell lines and used to study the effects of these manipulations on the composition of CCVs. This protocol, originally developed in the laboratory of Margaret Robinson (Cambridge, United Kingdom), describes the preparation of a HeLa cell fraction that is enriched for a mixed population of CCVs and is suitable for analysis by mass spectroscopy, western blotting, or electron microscopy.

Most normal cells have two centrosomes that form bipolar spindles during mitosis, while cancer cells often contain more than two, or "supernumerary" centrosomes. Such cancer cells achieve bipolar division by clustering their centrosomes into two functional poles, and inhibiting this process then leads to cancer-specific cell death. A major problem with clinically used anti-mitotic drugs, such as paclitaxel, is their toxicity in normal cells. To discover new compounds with greater specificity for cancer cells, we established a high-content screen for agents that block centrosome clustering in BT-549 cells, a breast cancer cell line that harbors supernumerary centrosomes. Using this screen, we identified 14 compounds that inhibit centrosome clustering and induce mitotic arrest. Some of these compounds were structurally similar, suggesting a common structural motif important for preventing centrosome clustering. We next compared the effects of these compounds on the growth of several breast and other cancer cell lines, an immortalized normal human mammary epithelial cell line, and progenitor-enriched primary normal human mammary epithelial cells. From these comparisons, we found some compounds that kill breast cancer cells, but not their normal epithelial counterparts, suggesting their potential for targeted therapy. One of these compounds, N2-(3-pyridylmethyl)-5-nitro-2-furamide (Centrosome Clustering Chemical Inhibitor-01, CCCI-01), that showed the greatest differential response in this screen was confirmed to have selective effects on cancer as compared to normal breast progenitors using more precise apoptosis induction and clonogenic growth endpoints. The concentration of CCCI-01 that killed cancer cells in the clonogenic assay spared normal human bone marrow hematopoietic progenitors in the colony-forming cell assay, indicating a potential therapeutic window for CCCI-01, whose selectivity might be further improved by optimizing the compound. Immunofluorescence analysis showed that treatment with CCCI-01 lead to multipolar spindles in BT-549, while maintaining bipolar spindles in the normal primary human mammary epithelial cells. Since centrosome clustering is a complex process involving multiple pathways, the 14 compounds identified in this study provide a potentially novel means to developing non-cross-resistant anti-cancer drugs that block centrosome clustering.

Integrin-linked kinase (ILK) localizes to both focal adhesions and centrosomes in distinct multiprotein complexes. Its dual function as a kinase and scaffolding protein has been well characterized at focal adhesions, where it regulates integrin-mediated cell adhesion, spreading, migration and signaling. At the centrosomes, ILK regulates mitotic spindle organization and centrosome clustering. Our previous study showed various spindle defects after ILK knockdown or inhibition that suggested alteration in microtubule dynamics. Since ILK expression is frequently elevated in many cancer types, we investigated the effects of ILK overexpression on microtubule dynamics. We show here that overexpressing ILK in HeLa cells was associated with a shorter duration of mitosis and decreased sensitivity to paclitaxel, a chemotherapeutic agent that suppresses microtubule dynamics. Measurement of interphase microtubule dynamics revealed that ILK overexpression favored microtubule depolymerization, suggesting that microtubule destabilization could be the mechanism behind the decreased sensitivity to paclitaxel, which is known to stabilize microtubules. Conversely, the use of a small molecule inhibitor selective against ILK, QLT-0267, resulted in suppressed microtubule dynamics, demonstrating a new mechanism of action for this compound. We further show that treatment of HeLa cells with QLT-0267 resulted in higher inter-centromere tension in aligned chromosomes during mitosis, slower microtubule regrowth after cold depolymerization and the presence of a more stable population of spindle microtubules. These results demonstrate that ILK regulates microtubule dynamics in both interphase and mitotic cells.

Endocytosis and mitosis are fundamental processes in a cell's life. Nearly 50 years of research suggest that these processes are linked and that endocytosis is shut down as cells undergo the early stages of mitosis. Precisely how this occurs at the molecular level is an open question. In this review, we summarize the early work characterizing the inhibition of clathrin-mediated endocytosis and discuss recent challenges to this established concept. We also set out four proposed mechanisms for the inhibition: mitotic phosphorylation of endocytic proteins, altered membrane tension, moonlighting of endocytic proteins, and a mitotic spindle-dependent mechanism. Finally, we speculate on the functional consequences of endocytic shutdown during mitosis and where an understanding of the mechanism of inhibition will lead us in the future.

Abstract Most normal cells have two centrosomes that form bipolar spindles during mitosis, while cancer cells often contain extra centrosomes. Such cancer cells then achieve bipolar division by clustering centrosomes into two functional poles. Our lab has previously shown that inhibition of Integrin-linked kinase, which is a signaling and scaffold protein in focal adhesion and is also a centrosomal protein, results in scattered centrosomes and multipolar spindles, which leads to cancer specific cell death (Fielding et al., 2011, 30:521-534, Oncogene). A major problem with clinically used anti-mitotic drugs, such as taxol, is their toxicity in normal cells. Our goal is to develop new drugs that act specifically on cancer cells through targeting extra centrosomes without affecting normal cells. To discover new drugs, we established a high-content screen that automatically detects cells with de-clustered centrosomes. BT-549, a breast cancer cell line, was used for the screen, since it is known to effectively cluster centrosomes. The Canadian Chemical Biology Network enabled us to test chemical libraries predicted to have drug-like properties. For screening, cells were grown in 96 well plates, treated with test compounds for 5 hours, and subjected to immunofluorescence to examine centrosome arrangement in mitotic cells. Images were taken and analyzed with an automated fluorescence imager, Cellomics Array Scan VTI. We have thus screened over 6000 compounds from which we identified 18 hits. Fifteen of these were confirmed to inhibit centrosome clustering in the cells cultured under standard conditions, and induce an arrest in mitosis. Three compounds are structurally similar, suggesting a common structural motif as preventing centrosome clustering. To compare the effects of these drugs on normal and cancer cells, the viability of several breast and other cancer cell lines, an immortalized model of normal human mammary epithelial cells (MCF10A), and freshly isolated primary normal human mammary epithelial cells were examined after two days of drug treatment using the MTT assay. These comparisons identified some compounds that selectively reduced the viability of cancer cells, but not normal mammary epithelial cells suggesting their potential for cancer-specific therapy. These small molecules are currently being tested in orthotopic and xenograft models, and will be studied for target identification and mechanism of action for further drug development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3887. doi:1538-7445.AM2012-3887

A long-standing paradigm in cell biology is the shutdown of endocytosis during mitosis. There is consensus that transferrin uptake is inhibited after entry into prophase and that it resumes in telophase. A recent study proposed that endocytosis is continuous throughout the cell cycle and that the observed inhibition of transferrin uptake is due to a decrease in available transferrin receptor at the cell surface, and not to a shutdown of endocytosis. This challenge to the established view is gradually becoming accepted. Because of this controversy, we revisited the question of endocytic activity during mitosis. Using an antibody uptake assay and controlling for potential changes in surface receptor density, we demonstrate the strong inhibition of endocytosis in mitosis of CD8 chimeras containing any of the three major internalization motifs for clathrin-mediated endocytosis (YXXΦ, [DE]XXXL[LI], or FXNPXY) or a CD8 protein with the cytoplasmic tail of the cation-independent mannose 6-phosphate receptor. The shutdown is not gradual: We describe a binary switch from endocytosis being “on” in interphase to “off” in mitosis as cells traverse the G 2 /M checkpoint. In addition, we show that the inhibition of transferrin uptake in mitosis occurs despite abundant transferrin receptor at the surface of HeLa cells. Our study finds no support for the recent idea that endocytosis continues during mitosis, and we conclude that endocytosis is temporarily shutdown during early mitosis.

Abstract Dysregulated mitosis is a hallmark of cancer. Many centrosomal proteins, including the integrin-linked kinase (ILK), that regulate microtubule organization and mitotic spindle assembly have been implicated in cancers. Inhibition or depletion of ILK result in a mitotic spindle defect that has not been well characterized and while ILK is overexpressed in many cancers, the effect of ILK overexpression on mitotic spindle function is unknown. In this study, we have characterized the defective spindle phenotype, and also looked at the effect of ILK overexpression on mitotic spindle function. HeLa cells stably over-expressing wild-type ILK were used for overexpression studies while parental HeLa cells were treated with QLT-0267, an ILK-specific inhibitor, to study the defective spindle phenotype. While ILK inhibition led to an abnormal mitotic spindle phenotype, the spindle contained bona fide kinetochore-microtubules that persisted after cold- or calcium-treatment. ILK inhibition, however, led to higher sister-centromere stretch in chromosomes aligned between two spindle poles, suggesting that kinetochores were under abnormally high tension, likely due to inhibition of microtubule polymerization. Microtubule regrowth in mitotic cells after cold depolymerization was significantly slower with ILK inhibition. In the context of ILK overexpression in cancers, we first measured the duration of mitosis and showed that ILK-overexpressing cells completed mitosis at a faster rate and also required shorter durations between metaphase and anaphase. ILK overexpression also resulted in increased resistance to paclitaxel-induced cell death. Collectively, our data suggest that ILK regulates microtubule dynamics, which is the focus of ongoing investigations in our laboratory, and ILK overexpression has clinical implications by inducing resistance to paclitaxel, possibly through increased microtubule dynamics. This work was supported by funding from the Canadian Institutes of Health Research. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2989. doi:10.1158/1538-7445.AM2011-2989

Many cancer cells contain more than two centrosomes, which imposes a potential for multipolar mitoses, leading to cell death. To circumvent this, cancer cells develop mechanisms to cluster supernumerary centrosomes to form bipolar spindles, enabling successful mitosis. Disruption of centrosome clustering thus provides a selective means of killing supernumerary centrosome-harboring cancer cells. Although the mechanisms of centrosome clustering are poorly understood, recent genetic analyses have identified requirements for both actin and tubulin regulating proteins. In this study, we demonstrate that the integrin-linked kinase (ILK), a protein critically involved in actin and mitotic microtubule organization, is required for centrosome clustering. Inhibition of ILK expression or activity inhibits centrosome clustering in several breast and prostate cancer cell lines that have centrosome amplification. Furthermore, cancer cells with supernumerary centrosomes are significantly more sensitive to ILK inhibition than cells with two centrosomes, demonstrating that inhibiting ILK offers a selective means of targeting cancer cells. Live cell analysis shows ILK perturbation leads cancer cells to undergo multipolar anaphases, mitotic arrest and cell death in mitosis. We also show that ILK performs its centrosome clustering activity in a focal adhesion-independent, but centrosome-dependent, manner through the microtubule regulating proteins TACC3 and ch-TOG. In addition, we identify a specific TACC3 phosphorylation site that is required for centrosome clustering and demonstrate that ILK regulates this phosphorylation in an Aurora-A-dependent manner.