A cfDNA methylation-based tissue-of-origin classifier for cancers of unknown primary.

Cancers of Unknown Primary (CUP) remains a diagnostic and therapeutic challenge due to biological heterogeneity and poor responses to standard chemotherapy. Predicting tissue-of-origin (TOO) molecularly could help refine this diagnosis, with tissue acquisition barriers mitigated via liquid biopsies. However, TOO liquid biopsies are unexplored in CUP cohorts. Here we describe CUPiD, a machine learning classifier for accurate TOO predictions across 29 tumour classes using circulating cell-free DNA (cfDNA) methylation patterns. We tested CUPiD on 143 cfDNA samples from patients with 13 cancer types alongside 27 non-cancer controls, with overall sensitivity of 84.6% and TOO accuracy of 96.8%. In an additional cohort of 41 patients with CUP CUPiD predictions were made in 32/41 (78.0%) cases, with 88.5% of the predictions clinically consistent with a subsequent or suspected primary tumour diagnosis, when available (23/26 patients). Combining CUPiD with cfDNA mutation data demonstrated potential diagnosis re-classification and/or treatment change in this hard-to-treat cancer group.

Functional Characterisation of the ATOH1 Molecular Subtype Indicates a Pro-Metastatic Role in Small Cell Lung Cancer.

Molecular subtypes of Small Cell Lung Cancer (SCLC) have been described based on differential expression of transcription factors (TFs) ASCL1, NEUROD1, POU2F3 and immune-related genes. We previously reported an additional subtype based on expression of the neurogenic TF ATOH1 within our SCLC Circulating tumour cell-Derived eXplant (CDX) model biobank. Here we show that ATOH1 protein was detected in 7/81 preclinical models and 16/102 clinical samples of SCLC. In CDX models, ATOH1 directly regulated neurogenesis and differentiation programs consistent with roles in normal tissues. In ex vivo cultures of ATOH1-positive CDX, ATOH1 was required for cell survival. In vivo, ATOH1 depletion slowed tumour growth and suppressed liver metastasis. Our data validate ATOH1 as a bona fide oncogenic driver of SCLC with tumour cell survival and pro-metastatic functions. Further investigation to explore ATOH1 driven vulnerabilities for targeted treatment with predictive biomarkers is warranted.

Lineage Plasticity in SCLC Generates Non-Neuroendocrine Cells Primed for Vasculogenic Mimicry.

INTRODUCTION: Vasculogenic mimicry (VM), the process of tumor cell transdifferentiation to endow endothelial-like characteristics supporting de novo vessel formation, is associated with poor prognosis in several tumor types, including SCLC. In genetically engineered mouse models (GEMMs) of SCLC, NOTCH, and MYC co-operate to drive a neuroendocrine (NE) to non-NE phenotypic switch, and co-operation between NE and non-NE cells is required for metastasis. Here, we define the phenotype of VM-competent cells and molecular mechanisms underpinning SCLC VM using circulating tumor cell-derived explant (CDX) models and GEMMs. METHODS: We analyzed perfusion within VM vessels and their association with NE and non-NE phenotypes using multiplex immunohistochemistry in CDX, GEMMs, and patient biopsies. We evaluated their three-dimensional structure and defined collagen-integrin interactions. RESULTS: We found that VM vessels are present in 23/25 CDX models, 2 GEMMs, and in 20 patient biopsies of SCLC. Perfused VM vessels support tumor growth and only NOTCH-active non-NE cells are VM-competent in vivo and ex vivo, expressing pseudohypoxia, blood vessel development, and extracellular matrix organization signatures. On Matrigel, VM-primed non-NE cells remodel extracellular matrix into hollow tubules in an integrin ß1-dependent process. CONCLUSIONS: We identified VM as an exemplar of functional heterogeneity and plasticity in SCLC and these findings take considerable steps toward understanding the molecular events that enable VM. These results support therapeutic co-targeting of both NE and non-NE cells to curtail SCLC progression and to improve the outcomes of patients with SCLC in the future.

Interrogating the precancerous evolution of pathway dysfunction in lung squamous cell carcinoma using XTABLE.

Lung squamous cell carcinoma (LUSC) is a type of lung cancer with a dismal prognosis that lacks adequate therapies and actionable targets. This disease is characterized by a sequence of low- and high-grade preinvasive stages with increasing probability of malignant progression. Increasing our knowledge about the biology of these premalignant lesions (PMLs) is necessary to design new methods of early detection and prevention, and to identify the molecular processes that are key for malignant progression. To facilitate this research, we have designed XTABLE (Exploring Transcriptomes of Bronchial Lesions), an open-source application that integrates the most extensive transcriptomic databases of PMLs published so far. With this tool, users can stratify samples using multiple parameters and interrogate PML biology in multiple manners, such as two- and multiple-group comparisons, interrogation of genes of interests, and transcriptional signatures. Using XTABLE, we have carried out a comparative study of the potential role of chromosomal instability scores as biomarkers of PML progression and mapped the onset of the most relevant LUSC pathways to the sequence of LUSC developmental stages. XTABLE will critically facilitate new research for the identification of early detection biomarkers and acquire a better understanding of the LUSC precancerous stages.

Lung squamous cell carcinoma is the second most common lung cancer. However, very little is known about how normal tissues in the lung develop in to these tumours. Like many cancers, this transformation comprises of an intermediate phase where healthy cells begin to form lesions that may (or may not) progress in to tumours. Understanding the biology of these lesions in lung squamous cell carcinoma may help clinicians detect them before they become cancerous. Knowing which genes are switched on and off during this intermediary phase can provide clues as to how these lesions form. There are already some publicly available transcriptional datasets showing the activity of tens of thousands of genes in pre-cancerous lesions extracted from patients with lung squamous cell carcinoma. But not every laboratory has the bioinformatic tools and skills required to interrogate these extensive databases. To address this, Roberts et al. built an open-source platform called XTABLE (short for Exploring Transcriptomes of Bronchial Lesions) which can analyse transcriptional datasets in multiple ways depending on the needs of the user. For instance, the tool can stratify the data into groups based on different parameters, such as the lesions potential to progress in to cancer, to see how the genes of the groups compare. It can also analyse the activity of individual genes and sets of genes involved in the same biological processes. Using XTABLE, Roberts et al. showed that a biological process linked to lung squamous cell carcinoma is also involved in the formation of pre-cancerous lesions. This suggests that molecules and genes associated with this process could potentially help scientists design prevention strategies. XTABLE will help researchers to better understand the biology of pre-cancerous lesions and how they develop in to tumours. Moreover, it will make it easier for scientists to validate their hypotheses using data collected from patients. The tool could also be useful for scientists interested in other types of lung cancers that share a similar biology.

cfDNA methylome profiling for detection and subtyping of small cell lung cancers.

Small cell lung cancer (SCLC) is characterized by morphologic, epigenetic and transcriptomic heterogeneity. Subtypes based upon predominant transcription factor expression have been defined that, in mouse models and cell lines, exhibit potential differential therapeutic vulnerabilities, with epigenetically distinct SCLC subtypes also described. The clinical relevance of these subtypes is unclear, due in part to challenges in obtaining tumor biopsies for reliable profiling. Here we describe a robust workflow for genome-wide DNA methylation profiling applied to both patient-derived models and to patients' circulating cell-free DNA (cfDNA). Tumor-specific methylation patterns were readily detected in cfDNA samples from patients with SCLC and were correlated with survival outcomes. cfDNA methylation also discriminated between the transcription factor SCLC subtypes, a precedent for a liquid biopsy cfDNA-methylation approach to molecularly subtype SCLC. Our data reveal the potential clinical utility of cfDNA methylation profiling as a universally applicable liquid biopsy approach for the sensitive detection, monitoring and molecular subtyping of patients with SCLC.

Impaired phosphocreatine metabolism in white adipocytes promotes inflammation.

The mechanisms promoting disturbed white adipocyte function in obesity remain largely unclear. Herein, we integrate white adipose tissue (WAT) metabolomic and transcriptomic data from clinical cohorts and find that the WAT phosphocreatine/creatine ratio is increased and creatine kinase-B expression and activity is decreased in the obese state. In human in vitro and murine in vivo models, we demonstrate that decreased phosphocreatine metabolism in white adipocytes alters adenosine monophosphate-activated protein kinase activity via effects on adenosine triphosphate/adenosine diphosphate levels, independently of WAT beigeing. This disturbance promotes a pro-inflammatory profile characterized, in part, by increased chemokine (C-C motif) ligand 2 (CCL2) production. These data suggest that the phosphocreatine/creatine system links cellular energy shuttling with pro-inflammatory responses in human and murine white adipocytes. Our findings provide unexpected perspectives on the mechanisms driving WAT inflammation in obesity and may present avenues to target adipocyte dysfunction.

TIAM1-RAC1 promote small-cell lung cancer cell survival through antagonizing Nur77-induced BCL2 conformational change.

Small-cell lung cancer (SCLC), an aggressive neuroendocrine malignancy, has limited treatment options beyond platinum-based chemotherapy, whereafter acquired resistance is rapid and common. By analyzing expression data from SCLC tumors, patient-derived models, and established cell lines, we show that the expression of TIAM1, an activator of the small GTPase RAC1, is associated with a neuroendocrine gene program. TIAM1 depletion or RAC1 inhibition reduces viability and tumorigenicity of SCLC cells by increasing apoptosis associated with conversion of BCL2 from its pro-survival to pro-apoptotic function via BH3 domain exposure. This conversion is dependent upon cytoplasmic translocation of Nur77, an orphan nuclear receptor. TIAM1 interacts with and sequesters Nur77 in SCLC cell nuclei and TIAM1 depletion or RAC1 inhibition promotes Nur77 translocation to the cytoplasm. Mutant TIAM1 with reduced Nur77 binding fails to suppress apoptosis triggered by TIAM1 depletion. In conclusion, TIAM1-RAC1 signaling promotes SCLC cell survival via Nur77 nuclear sequestration.

Soluble guanylate cyclase signalling mediates etoposide resistance in progressing small cell lung cancer.

Small cell lung cancer (SCLC) has a 5-year survival rate of <7%. Rapid emergence of acquired resistance to standard platinum-etoposide chemotherapy is common and improved therapies are required for this recalcitrant tumour. We exploit six paired pre-treatment and post-chemotherapy circulating tumour cell patient-derived explant (CDX) models from donors with extensive stage SCLC to investigate changes at disease progression after chemotherapy. Soluble guanylate cyclase (sGC) is recurrently upregulated in post-chemotherapy progression CDX models, which correlates with acquired chemoresistance. Expression and activation of sGC is regulated by Notch and nitric oxide (NO) signalling with downstream activation of protein kinase G. Genetic targeting of sGC or pharmacological inhibition of NO synthase re-sensitizes a chemoresistant CDX progression model in vivo, revealing this pathway as a mediator of chemoresistance and potential vulnerability of relapsed SCLC.

The Rare YAP1 Subtype of SCLC Revisited in a Biobank of 39 Circulating Tumor Cell Patient Derived Explant Models: A Brief Report.

INTRODUCTION: Recent consensus defines four SCLC subtypes on the basis of transcription factor expression: ASCL1, NEUROD1, POU2F3, and YAP1. The rare YAP1 subtype is associated with "neuroendocrine (NE)-low" cells among SCLC cell lines and patient samples. We evaluated YAP1 in 39 patients with phenotypically diverse circulating tumor cell-derived explant (CDX) models and revisited YAP1 in terms of prevalence, cell phenotype, and intertumor and intratumor heterogeneity. METHODS: YAP1 transcript and protein expression were assessed by RNA sequencing and immunohistochemistry or multiplexed immunofluorescence of NE and non-NE CDX subpopulations. Physically separated NE and non-NE CDX ex vivo culture lysates were Western blotted for YAP1, NE marker SYP, and AXL. RESULTS: RNA sequencing normalized for the four subtype transcription factors identified YAP1 expression in 14 of 39 CDX. A total of 10 CDX expressed YAP1 protein, and eight had strong YAP1 expression confined to rare non-NE cell clusters. This was confirmed in ex vivo CDX cultures in which adherent non-NE cells lacking SYP expression expressed YAP1. However, in two CDX, weaker cellular YAP1 expression was observed, widely dispersed in SYP-positive NE cells. CONCLUSIONS: YAP1 was predominantly expressed in non-NE cell clusters in SCLC CDX, but two of 39 CDX expressed YAP1 in NE cells. CDX22P, with relatively high YAP1 expression, is an ASCL1 NE subtype with a low NE score and an outlier within this subtype in our CDX biobank. These descriptive data reveal subtly different YAP1 expression profiles, paving the way for functional studies to compare YAP1 signaling in non-NE and low NE cell contexts for potentially personalized therapeutic approaches.

A biobank of small cell lung cancer CDX models elucidates inter- and intratumoral phenotypic heterogeneity.

Although small cell lung cancer (SCLC) is treated as a homogeneous disease, biopsies and preclinical models reveal heterogeneity in transcriptomes and morphology. SCLC subtypes were recently defined by neuroendocrine transcription factor (NETF) expression. Circulating-tumor-cell-derived explant models (CDX) recapitulate donor patients' tumor morphology, diagnostic NE marker expression and chemotherapy responses. We describe a biobank of 38 CDX models, including six CDX pairs generated pretreatment and at disease progression revealing complex intra- and intertumoral heterogeneity. Transcriptomic analysis confirmed three of four previously described subtypes based on ASCL1, NEUROD1 and POU2F3 expression and identified a previously unreported subtype based on another NETF, ATOH1. We document evolution during disease progression exemplified by altered MYC and NOTCH gene expression, increased 'variant' cell morphology, and metastasis without strong evidence of epithelial to mesenchymal transition. This CDX biobank provides a research resource to facilitate SCLC personalized medicine.

A rapid computational approach identifies SPICE1 as an Aurora kinase substrate.

Aurora kinases play a major role in mitosis by regulating diverse substrates. Defining their critical downstream targets is important in understanding Aurora kinase function. Here we have developed an unbiased computational approach to identify new Aurora kinase substrates based on phosphorylation site clustering, protein localization, protein structure, and species conservation. We validate the microtubule-associated proteins Clasp2, Elys, tubulin tyrosine ligase-like polyglutamylase residues 330-624 and spindle and centriole associated protein 1, residues 549-855 (SPICE1), as Aurora A and B kinases substrates in vitro. We also demonstrate that SPICE1 localization is regulated by Aurora kinases during mitosis. In the absence of Aurora kinase activity, SPICE1 remains at centrioles but does not target to the spindle. Similarly, a nonphosphorylatable SPICE1 mutant no longer localizes to the spindle. Finally, we show that misregulating SPICE1 phosphorylation results in abnormal centriole number, spindle multipolarity, and chromosome alignment defects. Overall, our work indicates that temporal and spatial Aurora kinase-mediated regulation of SPICE1 is important for correct chromosome segregation. In addition, our work provides a database-search tool that enables rapid identification of Aurora kinase substrates.

Telomere elongation through hTERT immortalization leads to chromosome repositioning in control cells and genomic instability in Hutchinson-Gilford progeria syndrome fibroblasts, expressing a novel SUN1 isoform.

Immortalizing primary cells with human telomerase reverse transcriptase (hTERT) has been common practice to enable primary cells to be of extended use in the laboratory because they avoid replicative senescence. Studying exogenously expressed hTERT in cells also affords scientists models of early carcinogenesis and telomere behavior. Control and the premature ageing disease-Hutchinson-Gilford progeria syndrome (HGPS) primary dermal fibroblasts, with and without the classical G608G mutation have been immortalized with exogenous hTERT. However, hTERT immortalization surprisingly elicits genome reorganization not only in disease cells but also in the normal control cells, such that whole chromosome territories normally located at the nuclear periphery in proliferating fibroblasts become mislocalized in the nuclear interior. This includes chromosome 18 in the control fibroblasts and both chromosomes 18 and X in HGPS cells, which physically express an isoform of the LINC complex protein SUN1 that has previously only been theoretical. Additionally, this HGPS cell line has also become genomically unstable and has a tetraploid karyotype, which could be due to the novel SUN1 isoform. Long-term treatment with the hTERT inhibitor BIBR1532 enabled the reduction of telomere length in the immortalized cells and resulted that these mislocalized internal chromosomes to be located at the nuclear periphery, as assessed in actively proliferating cells. Taken together, these findings reveal that elongated telomeres lead to dramatic chromosome mislocalization, which can be restored with a drug treatment that results in telomere reshortening and that a novel SUN1 isoform combined with elongated telomeres leads to genomic instability. Thus, care should be taken when interpreting data from genomic studies in hTERT-immortalized cell lines.

Effect of irradiation and bone marrow transplantation on angiotensin II-induced aortic inflammation in ApoE knockout mice.

BACKGROUND AND AIMS: Angiotensin II (Ang II) infusion promotes the development of aortic aneurysms and accelerates atherosclerosis in ApoE-/- mice. In order to elucidate the role of hematopoietic cells in these pathologies, irradiation and bone marrow transplantation (BMT) are commonly utilized. The aim of this study was to investigate the effects of irradiation and BMT on abdominal and thoracic aortic aneurysm formation and acute leukocyte recruitment in the aortic root and descending aorta, in an experimental mouse model of aortic aneurysm formation. METHODS: ApoE-/- mice were either lethally irradiated and reconstituted with ApoE-/- bone marrow or non-irradiated. Following engraftment, mice were treated with Ang II to induce aortic inflammation and accelerate atherosclerosis. RESULTS: Ang II infusion (0.8 mg/kg/day) in BMT mice resulted in reduced aortic aneurysms and atherosclerosis with decreased leukocyte infiltration in the aorta compared to non-BMT mice, when receiving the same dose of Ang II. Furthermore, the reduced aortic infiltration in BMT mice was accompanied by increased levels of monocytes in the spleen and bone marrow. A dose of 3 mg/kg/day Ang II was required to achieve a similar incidence of aneurysm formation as achieved with 0.8 mg/kg/day in non-BMT mice. CONCLUSIONS: This study provides evidence that BMT can alter inflammatory cell recruitment in experimental mouse models of aortic aneurysm formation and atherosclerosis and suggests that irradiation and BMT have a considerably more complex effect on vascular inflammation, which should be evaluated.

A Genomic DNA Reporter Screen Identifies Squalene Synthase Inhibitors That Act Cooperatively with Statins to Upregulate the Low-Density Lipoprotein Receptor.

Hypercholesterolemia remains one of the leading risk factors for the development of cardiovascular disease. Many large double-blind studies have demonstrated that lowering low-density lipoprotein (LDL) cholesterol using a statin can reduce the risk of having a cardiovascular event by approximately 30%. However, despite the success of statins, some patient populations are unable to lower their LDL cholesterol to meet the targeted lipid levels, due to compliance or potency issues. This is especially true for patients with heterozygous familial hypercholesterolemia who may require additional upregulation of the low-density lipoprotein receptor (LDLR) to reduce LDL cholesterol levels below those achievable with maximal dosing of statins. Here we identify a series of small molecules from a genomic DNA reporter screen that upregulate the LDLR in mouse and human liver cell lines at nanomolar potencies (EC50 = 39 nM). Structure-activity relationship studies carried out on the lead compound, OX03771 [(E)-N,N-dimethyl-3-(4-styrylphenoxy)propan-1-amine], led to the identification of compound OX03050 [(E)-3-(4-styrylphenoxy)propan-1-ol], which had similar potency (EC50 = 26 nM) but a much-improved pharmacokinetic profile and showed in vivo efficacy. Compounds OX03050 and OX03771 were found to inhibit squalene synthase, the first committed step in cholesterol biosynthesis. These squalene synthase inhibitors were shown to act cooperatively with statins to increase LDLR expression in vitro. Overall, we demonstrated here a novel series of small molecules with the potential to be further developed to treat patients either alone or in combination with statins.

Constrained release of lamina-associated enhancers and genes from the nuclear envelope during T-cell activation facilitates their association in chromosome compartments.

The 3D organization of the genome changes concomitantly with expression changes during hematopoiesis and immune activation. Studies have focused either on lamina-associated domains (LADs) or on topologically associated domains (TADs), defined by preferential local chromatin interactions, and chromosome compartments, defined as higher-order interactions between TADs sharing functionally similar states. However, few studies have investigated how these affect one another. To address this, we mapped LADs using Lamin B1-DamID during Jurkat T-cell activation, finding significant genome reorganization at the nuclear periphery dominated by release of loci frequently important for T-cell function. To assess how these changes at the nuclear periphery influence wider genome organization, our DamID data sets were contrasted with TADs and compartments. Features of specific repositioning events were then tested by fluorescence in situ hybridization during T-cell activation. First, considerable overlap between TADs and LADs was observed with the TAD repositioning as a unit. Second, A1 and A2 subcompartments are segregated in 3D space through differences in proximity to LADs along chromosomes. Third, genes and a putative enhancer in LADs that were released from the periphery during T-cell activation became preferentially associated with A2 subcompartments and were constrained to the relative proximity of the lamina. Thus, lamina associations influence internal nuclear organization, and changes in LADs during T-cell activation may provide an important additional mode of gene regulation.

Lin28a uses distinct mechanisms of binding to RNA and affects miRNA levels positively and negatively.

Lin28a inhibits the biogenesis of let-7 miRNAs by triggering the polyuridylation and degradation of their precursors by terminal uridylyltransferases TUT4/7 and 3'-5' exoribonuclease Dis3l2, respectively. Previously, we showed that Lin28a also controls the production of neuro-specific miRNA-9 via a polyuridylation-independent mechanism. Here we reveal that the sequences and structural characteristics of pre-let-7 and pre-miRNA-9 are eliciting two distinct modes of binding to Lin28a. We present evidence that Dis3l2 controls miRNA-9 production. Finally, we show that the constitutive expression of untagged Lin28a during neuronal differentiation in vitro positively and negatively affects numerous other miRNAs. Our findings shed light on the role of Lin28a in differentiating cells and on the ways in which one RNA-binding protein can perform multiple roles in the regulation of RNA processing.

The SNF2 family ATPase LSH promotes cell-autonomous de novo DNA methylation in somatic cells.

Methylation of DNA at carbon 5 of cytosine is essential for mammalian development and implicated in transcriptional repression of genes and transposons. New patterns of DNA methylation characteristic of lineage-committed cells are established at the exit from pluripotency by de novo DNA methyltransferases enzymes, DNMT3A and DNMT3B, which are regulated by developmental signaling and require access to chromatin-organized DNA. Whether or not the capacity for de novo DNA methylation of developmentally regulated loci is preserved in differentiated somatic cells and can occur in the absence of exogenous signals is currently unknown. Here, we demonstrate that fibroblasts derived from chromatin remodeling ATPase LSH (HELLS)-null mouse embryos, which lack DNA methylation from centromeric repeats, transposons and a number of gene promoters, are capable of reestablishing DNA methylation and silencing of misregulated genes upon re-expression of LSH. We also show that the ability of LSH to bind ATP and the cellular concentration of DNMT3B are critical for cell-autonomous de novo DNA methylation in somatic cells. These data suggest the existence of cellular memory that persists in differentiated cells through many cell generations and changes in transcriptional state.

Loss of Tet1-Associated 5-Hydroxymethylcytosine Is Concomitant with Aberrant Promoter Hypermethylation in Liver Cancer.

Aberrant hypermethylation of CpG islands (CGI) in human tumors occurs predominantly at repressed genes in the host tissue, but the preceding events driving this phenomenon are poorly understood. In this study, we temporally tracked epigenetic and transcriptomic perturbations that occur in a mouse model of liver carcinogenesis. Hypermethylated CGI events in the model were predicted by enrichment of the DNA modification 5-hydroxymethylcytosine (5hmC) and the histone H3 modification H3K27me3 at silenced promoters in the host tissue. During cancer progression, selected CGIs underwent hypo-hydroxymethylation prior to hypermethylation, while retaining H3K27me3. In livers from mice deficient in Tet1, a tumor suppressor involved in cytosine demethylation, we observed a similar loss of promoter core 5hmC, suggesting that reduced Tet1 activity at CGI may contribute to epigenetic dysregulation during hepatocarcinogenesis. Consistent with this possibility, mouse liver tumors exhibited reduced Tet1 protein levels. Similar to humans, DNA methylation changes at CGI in mice did not appear to be direct drivers of hepatocellular carcinoma progression, rather, dynamic changes in H3K27me3 promoter deposition correlated strongly with tumor-specific activation and repression of transcription. Overall, our results suggest that loss of promoter-associated 5hmC in liver tumors licenses reprograming of DNA methylation at silent CGI during progression. Cancer Res; 76(10); 3097-108. ©2016 AACR.

TD-60 links RalA GTPase function to the CPC in mitosis.

TD-60 (also known as RCC2) is a highly conserved protein that structurally resembles the Ran guanine exchange factor (GEF) RCC1, but has not previously been shown to have GEF activity. TD-60 has a typical chromosomal passenger complex (CPC) distribution in mitotic cells, but associates with integrin complexes and is involved in cell motility during interphase. Here we show that TD-60 exhibits GEF activity, in vitro and in cells, for the small GTPase RalA. TD-60 or RalA depletion causes spindle abnormalities in prometaphase associated with abnormal centromeric accumulation of CPC components. TD-60 and RalA apparently work together to contribute to the regulation of kinetochore-microtubule interactions in early mitosis. Importantly, several mitotic phenotypes caused by TD-60 depletion are reverted by the expression of a GTP-locked mutant, RalA (Q72L). The demonstration that a small GTPase participates in the regulation of the CPC reveals a level of mitotic regulation not suspected in previous studies.

Shugoshin biases chromosomes for biorientation through condensin recruitment to the pericentromere.

To protect against aneuploidy, chromosomes must attach to microtubules from opposite poles ('biorientation') prior to their segregation during mitosis. Biorientation relies on the correction of erroneous attachments by the aurora B kinase, which destabilizes kinetochore-microtubule attachments that lack tension. Incorrect attachments are also avoided because sister kinetochores are intrinsically biased towards capture by microtubules from opposite poles. Here, we show that shugoshin acts as a pericentromeric adaptor that plays dual roles in biorientation in budding yeast. Shugoshin maintains the aurora B kinase at kinetochores that lack tension, thereby engaging the error correction machinery. Shugoshin also recruits the chromosome-organizing complex, condensin, to the pericentromere. Pericentromeric condensin biases sister kinetochores towards capture by microtubules from opposite poles. Our findings uncover the molecular basis of the bias to sister kinetochore capture and expose shugoshin as a pericentromeric hub controlling chromosome biorientation. DOI: http://dx.doi.org/10.7554/eLife.01374.001.