An integrative analysis of colon cancer identifies an essential function for PRPF6 in tumor growth.

The spliceosome machinery is composed of multimeric protein complexes that generate a diverse repertoire of mRNA through coordinated splicing of heteronuclear RNAs. While somatic mutations in spliceosome components have been discovered in several cancer types, the molecular bases and consequences of spliceosome aberrations in cancer are poorly understood. Here we report for the first time that PRPF6, a member of the tri-snRNP (small ribonucleoprotein) spliceosome complex, drives cancer proliferation by preferential splicing of genes associated with growth regulation. Inhibition of PRPF6 and other tri-snRNP complex proteins, but not other snRNP spliceosome complexes, selectively abrogated growth in cancer cells with high tri-snRNP levels. High-resolution transcriptome analyses revealed that reduced PRPF6 alters the constitutive and alternative splicing of a discrete number of genes, including an oncogenic isoform of the ZAK kinase. These findings implicate an essential role for PRPF6 in cancer via splicing of distinct growth-related gene products.

Cdk8 deletion in the Apc(Min) murine tumour model represses EZH2 activity and accelerates tumourigenesis.

CDK8 is a dissociable kinase module of the Mediator complex and has been shown to play an important role in transcriptional regulation in organisms as diverse as yeast and humans. Recent studies suggest that CDK8 functions as an oncoprotein in melanoma and colon cancer. Importantly, these studies were conducted using in vitro cell line models and the role of CDK8 in tumourigenesis in vivo has not been explored. We have generated a mouse with a Cdk8 conditional knockout allele and examined the consequences of Cdk8 loss on normal tissue homeostasis and tumour development in vivo. Cdk8 deletion in the young adult mouse did not induce any gross or histopathological abnormalities, implying that Cdk8 is largely dispensable for somatic cellular homeostasis. In contrast, Cdk8 deletion in the Apc(Min) intestinal tumour model shortened the animals' survival and increased tumour burden. Although Cdk8 deletion did not affect tumour initiation, intestinal tumour size and growth rate were significantly increased in Cdk8-null animals. Transcriptome analysis performed on Cdk8-null intestinal cells revealed up-regulation of genes that are governed by the Polycomb group (PcG) complex. In support of these findings, Cdk8-null intestinal cells and tumours displayed a reduction in histone H3K27 trimethylation, both globally and at the promoters of a number of PcG-regulated genes involved in oncogenic signalling. Together, our findings uncover a tumour suppressor function for CDK8 in vivo and suggest that the role of CDK8 activity in driving oncogenesis is context-specific. Sequencing data were deposited at GEO (Accession No. GSE71385).

RNAi of mitotic cyclins in Drosophila uncouples the nuclear and centrosome cycle.

BACKGROUND: Successful cell duplication requires orderly progression through a succession of dramatic cell-cycle events. Disruption of this precise coupling can compromise genomic integrity. The coordination of cell-cycle events is thought to arise from control by a single master regulator, cyclin:Cdk, whose activity oscillates. However, we still know very little of how individual cell-cycle events are coupled to this oscillator and how the timing of each event is controlled. RESULTS: We developed an approach with RNA interference (RNAi) and real-time imaging to study cyclin contributions to the rapid syncytial divisions of Drosophila embryos. Simultaneous knockdown of all three mitotic cyclins blocked nuclei from entering mitosis. Despite nuclear arrest, centrosomes and associated myosin cages continued to divide until the midblastula transition. Centrosome division was synchronous throughout the embryo and the period of the uncoupled duplication cycle increased over successive divisions. In contrast to its normal actions, injection of a competitive inhibitor of the anaphase-promoting complex/cyclosome (APC/C) after knockdown of the mitotic cyclins did not interfere with the centrosome-duplication cycles. Finally, we examined how cyclin knockdown affects the onset of cellularization at the midblastula transition and found that nuclear cell-cycle arrest did not advance or delay onset of cellularization. CONCLUSIONS: We show that knockdown of mitotic cyclins allows centrosomes to duplicate in a cycle that is uncoupled from other cell-cycle events. We suggest that high mitotic cyclin normally ensures that the centrosome cycle remains entrained to the nuclear cycle.

Measuring the stoichiometry and physical interactions between components elucidates the architecture of the vertebrate kinetochore.

Vertebrate kinetochores contain over 50 different proteins organized into three distinct regions: the inner plate, outer plate, and fibrous corona. The present study characterizes numerous precursors of kinetochore assembly in a system free of centromeric chromatin, Xenopus extracts. Hydrodynamic analysis suggests there are a minimum of two monomeric proteins and six pre-assembled complexes that accumulate on centromeres to form the kinetochore. The inner and outer kinetochore assemble from at least two distinct kinetochore complexes containing the proteins Mis12, Zwint, and Ndc80, all of which interact by immunoprecipitation. There is also a network of interactions between the fibrous corona proteins that is dissociated by microtubules. We quantify the number of molecules of specific proteins assembled into a single kinetochore. There are between 800 and 1200 molecules of the measured inner and outer kinetochore proteins, demonstrating that the components in these regions are in similar stoichiometry. In contrast, the measured fibrous corona proteins are present at 250-300 molecules per kinetochore. Zwint, but not Mis12, requires the Ndc80 complex for assembly into the kinetochore. Further, Ndc80 requires Zwint for assembly, indicating a co-dependency for these two proteins. Our data provide a model for the structural architecture and assembly pathway of the vertebrate kinetochore.

Inhibition of aurora B kinase blocks chromosome segregation, overrides the spindle checkpoint, and perturbs microtubule dynamics in mitosis.

How kinetochores correct improper microtubule attachments and regulate the spindle checkpoint signal is unclear. In budding yeast, kinetochores harboring mutations in the mitotic kinase Ipl1 fail to bind chromosomes in a bipolar fashion. In C. elegans and Drosophila, inhibition of the Ipl1 homolog, Aurora B kinase, induces aberrant anaphase and cytokinesis. To study Aurora B kinase in vertebrates, we microinjected mitotic XTC cells with inhibitory antibody and found several related effects. After injection of the antibody, some chromosomes failed to congress to the metaphase plate, consistent with a conserved role for Aurora B in bipolar attachment of chromosomes. Injected cells exited mitosis with no evidence of anaphase or cytokinesis. Injection of anti-Xaurora B antibody also altered the microtubule network in mitotic cells with an extension of the astral microtubules and a reduction of kinetochore microtubules. Finally, inhibition of Aurora B in cultured cells and in cycling Xenopus egg extracts caused escape from the spindle checkpoint arrest induced by microtubule drugs. Our findings implicate Aurora B as a critical coordinator relating changes in microtubule dynamics in mitosis, chromosome movement in prometaphase and anaphase, signaling of the spindle checkpoint, and cytokinesis.

The vertebrate Ndc80 complex contains Spc24 and Spc25 homologs, which are required to establish and maintain kinetochore-microtubule attachment.

How kinetochores bind to microtubules and move on the mitotic spindle remain unanswered questions. Multiple systems have implicated the Ndc80/Hec1 (Ndc80) kinetochore complex in kinetochore-microtubule interaction and spindle checkpoint activity. In budding yeast, Ndc80 copurifies with three additional interacting proteins: Nuf2, Spc24, and Spc25. Although functional vertebrate homologs of Ndc80 and Nuf2 exist, extensive sequence similarity searches have not uncovered homologs of Spc24 and Spc25. We have purified the xNdc80 complex to homogeneity from Xenopus egg extracts and identified two novel interacting proteins. Although the sequences have greatly diverged, we have concluded that these are the functional homologs of the yeast Spc24 and Spc25 proteins based on limited sequence similarity, common coiled-coil domains, kinetochore localization, similar phenotypes, and copurification with xNdc80 and xNuf2. Using both RNAi and antibody injection experiments, we have extended previous characterization of the complex and found that Spc24 and Spc25 are required not only to establish, but also to maintain kinetochore-microtubule attachments and metaphase alignment. In addition, we show that Spc24 and Spc25 are required for chromosomal movement to the spindle poles in anaphase.

Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae.

Protein kinases are coded by more than 2,000 genes and thus constitute the largest single enzyme family in the human genome. Most cellular processes are in fact regulated by the reversible phosphorylation of proteins on serine, threonine, and tyrosine residues. At least 30% of all proteins are thought to contain covalently bound phosphate. Despite the importance and widespread occurrence of this modification, identification of sites of protein phosphorylation is still a challenge, even when performed on highly purified protein. Reported here is methodology that should make it possible to characterize most, if not all, phosphoproteins from a whole-cell lysate in a single experiment. Proteins are digested with trypsin and the resulting peptides are then converted to methyl esters, enriched for phosphopeptides by immobilized metal-affinity chromatography (IMAC), and analyzed by nanoflow HPLC/electrospray ionization mass spectrometry. More than 1,000 phosphopeptides were detected when the methodology was applied to the analysis of a whole-cell lysate from Saccharomyces cerevisiae. A total of 216 peptide sequences defining 383 sites of phosphorylation were determined. Of these, 60 were singly phosphorylated, 145 doubly phosphorylated, and 11 triply phosphorylated. Comparison with the literature revealed that 18 of these sites were previously identified, including the doubly phosphorylated motif pTXpY derived from the activation loop of two mitogen-activated protein (MAP) kinases. We note that the methodology can easily be extended to display and quantify differential expression of phosphoproteins in two different cell systems, and therefore demonstrates an approach for "phosphoprofiling" as a measure of cellular states.

Aurora B kinase exists in a complex with survivin and INCENP and its kinase activity is stimulated by survivin binding and phosphorylation.

Aurora B regulates chromosome segregation and cytokinesis and is the first protein to be implicated as a regulator of bipolar attachment of spindle microtubules to kinetochores. Evidence from several systems suggests that Aurora B is physically associated with inner centromere protein (INCENP) in mitosis and has genetic interactions with Survivin. It is unclear whether the Aurora B and INCENP interaction is cell cycle regulated and if Survivin physically interacts in this complex. In this study, we cloned the Xenopus Survivin gene, examined its association with Aurora B and INCENP, and determined the effect of its binding on Aurora B kinase activity. We demonstrate that in the Xenopus early embryo, all of the detectable Survivin is in a complex with both Aurora B and INCENP throughout the cell cycle. Survivin and Aurora B bind different domains on INCENP. Aurora B activity is stimulated >10-fold in mitotic extracts; this activation is phosphatase sensitive, and the binding of Survivin is required for full Aurora B activity. We also find the hydrodynamic properties of the Aurora B/Survivin/INCENP complex are cell cycle regulated. Our data indicate that Aurora B kinase activity is regulated by both Survivin binding and cell cycle-dependent phosphorylation.

CCAT1 is an enhancer-templated RNA that predicts BET sensitivity in colorectal cancer.

Colon tumors arise in a stepwise fashion from either discrete genetic perturbations or epigenetic dysregulation. To uncover the key epigenetic regulators that drive colon cancer growth, we used a CRISPR loss-of-function screen and identified a number of essential genes, including the bromodomain and extraterminal (BET) protein BRD4. We found that BRD4 is critical for colon cancer proliferation, and its knockdown led to differentiation effects in vivo. JQ1, a BET inhibitor, preferentially reduced growth in a subset of epigenetically dysregulated colon cancers characterized by the CpG island methylator phenotype (CIMP). Integrated transcriptomic and genomic analyses defined a distinct superenhancer in CIMP+ colon cancers that regulates cMYC transcription. We found that the long noncoding RNA colon cancer-associated transcript 1 (CCAT1) is transcribed from this superenhancer and is exquisitely sensitive to BET inhibition. Concordantly, cMYC transcription and cell growth were tightly correlated with the presence of CCAT1 RNA in a variety of tumor types. Taken together, we propose that CCAT1 is a clinically tractable biomarker for identifying patients who are likely to benefit from BET inhibitors.

Development of a Potent, Specific CDK8 Kinase Inhibitor Which Phenocopies CDK8/19 Knockout Cells.

Beginning with promiscuous COT inhibitors, which were found to inhibit CDK8, a series of 6-aza-benzothiophene containing compounds were developed into potent, selective CDK8 inhibitors. When cocrystallized with CDK8 and cyclin C, these compounds exhibit an unusual binding mode, making a single hydrogen bond to the hinge residue A100, a second to K252, and a key cation-π interaction with R356. Structure-based drug design resulted in tool compounds 13 and 32, which are highly potent, kinase selective, permeable compounds with a free fraction >2% and no measurable efflux. Despite these attractive properties, these compounds exhibit weak antiproliferative activity in the HCT-116 colon cancer cell line. Further examination of the activity of 32 in this cell line revealed that the compound reduced phosphorylation of the known CDK8 substrate STAT1 in a manner identical to a CDK8 knockout clone, illustrating the complex effects of inhibition of CDK8 kinase activity in proliferation in these cells.

Design and Development of a Series of Potent and Selective Type II Inhibitors of CDK8.

Using Sorafenib as a starting point, a series of potent and selective inhibitors of CDK8 was developed. When cocrystallized with CDK8 and cyclin C, these compounds exhibit a Type-II (DMG-out) binding mode.

Purification of the Ndc80 kinetochore subcomplex from Xenopus eggs.

The identification of protein binding partners often facilitates understanding of protein complex function. However, identifying binding partners has proven difficult because proteins are often bound to insoluble structures or are only present during certain stages of the cell cycle. Fortunately, Xenopus eggs stockpile many proteins, which are typically insoluble, as soluble subcomplexes to facilitate rapid early embryonic divisions. We exploited this by developing a purification scheme using Xenopus egg extracts to isolate Coomassie-stainable amounts of the xNdc80 kinetochore complex. In this scheme Xenopus eggs are directly made into a mitotic high-speed supernatant and then flowed over three chromatographic columns: heparin, Mono-Q, and Superose 6 gel filtration columns. A final immunoprecipitation is then performed from the peak Superose 6 column to yield Ndc80 complex purified to homogeneity. With minor modification and manipulation of Xenopus egg extracts, this protocol can easily be adapted for purification of other protein complexes.

Lactate dehydrogenase B is required for the growth of KRAS-dependent lung adenocarcinomas.

PURPOSE: This study is aimed to identify genes within the KRAS genomic amplicon that are both coupregulated and essential for cell proliferation when KRAS is amplified in lung cancer. EXPERIMENTAL DESIGN: We used an integrated genomic approach to identify genes that are coamplified with KRAS in lung adenocarcinomas and subsequently preformed an RNA interference (RNAi) screen to uncover functionally relevant genes. The role of lactate dehydrogenase B (LDHB) was subsequently investigated both in vitro and in vivo by siRNA and short hairpin RNA (shRNA)-mediated knockdown in a panel of lung adenocarcinoma cells lines. LDHB expression was also investigated in patient tumors using microarray and immunohistochemistry analyses. RESULTS: RNAi-mediated depletion of LDHB abrogated cell proliferation both in vitro and in xenografted tumors in vivo. We find that LDHB expression correlates to both KRAS genomic copy number gain and KRAS mutation in lung cancer cell lines and adenocarcinomas. This correlation between LDHB expression and KRAS status is specific for lung cancers and not other tumor types that harbor KRAS mutations. Consistent with a role for LDHB in glycolysis and tumor metabolism, KRAS-mutant lung tumors exhibit elevated expression of a glycolysis gene signature and are more dependent on glycolysis for proliferation compared with KRAS wild-type lung tumors. Finally, high LDHB expression was a significant predictor of shorter survival in patients with lung adenocarcinomas. CONCLUSION: This study identifies LDHB as a regulator of cell proliferation in a subset of lung adenocarcinoma and may provide a novel therapeutic approach for treating lung cancer.

CDK8 maintains tumor dedifferentiation and embryonic stem cell pluripotency.

CDK8 is a cyclin-dependent kinase that mediates transcriptional control of pathways linked to both cancer and stem cells. In this study, we show that CDK8 is required for both tumor growth and maintenance of tumor dedifferentiation in vivo and uncover a common role for CDK8 in controlling cancer and stem cell function. Acute CDK8 loss in vivo strongly inhibited tumor growth and promoted differentiation. Transcriptional profiling identified a set of embryonic stem cell-related genes that are activated by CDK8 in cancer. Consistent with this, we found that CDK8 expression correlated to the embryonic stem cell pluripotency state and loss of CDK8 caused embryonic stem cells to differentiate. This effect was, at least partially, mediated by the ability of CDK8 to regulate MYC protein and downstream MYC target gene expression. Similar regulation of MYC target genes by CDK8 was observed in colon tumor cells, and increased expression of a CDK8-regulated, embryonic stem cell MYC target gene signature was associated with loss of differentiation and poor outcome in primary human colon cancers. Together, these observations reveal that CDK8 acts, at least in part, through MYC to maintain both tumors and embryonic stem cells in an undifferentiated state. This raises the intriguing possibility that targeting CDK8 therapeutically may specifically inhibit the stem-like properties of cancer cells.

An integrated genomic screen identifies LDHB as an essential gene for triple-negative breast cancer.

Breast cancer has been redefined into three clinically relevant subclasses: (i) estrogen/progesterone receptor positive (ER+/PR+), (ii) HER2/ERRB2 positive, and (iii) those lacking expression of all three markers (triple negative or basal-like). While targeted therapies for ER+/PR+ and HER2+ tumors have revolutionized patient treatment and increased lifespan, an urgent need exists for identifying novel targets for triple-negative breast cancers. Here, we used integrative genomic analysis to identify candidate oncogenes in triple-negative breast tumors and assess their function through loss of function screening. Using this approach, we identify lactate dehydrogenase B (LDHB), a component of glycolytic metabolism, as an essential gene in triple-negative breast cancer. Loss of LDHB abrogated cell proliferation in vitro and arrested tumor growth in fully formed tumors in vivo. We find that LDHB and other related glycolysis genes are specifically upregulated in basal-like/triple-negative breast cancers as compared with other subtypes, suggesting that these tumors are distinctly glycolytic. Consistent with this, triple-negative breast cancer cell lines were more dependent on glycolysis for growth than luminal cell lines. Finally, we find that patients with breast cancer and high LDHB expression in their tumors had a poor clinical outcome. While previous studies have focused on the ubiquitous role of LDHA in tumor metabolism and growth, our data reveal that LDHB is upregulated and required only in certain cancer genotypes. These findings suggest that targeting LDHB or other components of lactate metabolism would be of clinical benefit in triple-negative breast cancer.

Developmental control of late replication and S phase length.

BACKGROUND: Fast, early embryonic cell cycles have correspondingly fast S phases. In early Drosophila embryos, forks starting from closely spaced origins replicate the whole genome in 3.4 min, ten times faster than in embryonic cycle 14 and a hundred times faster than in a wing disc. It is not known how S phase duration is regulated. Here we examined prolongation of embryonic S phases, its coupling to development, and its relationship to the appearance of heterochromatin. RESULTS: Imaging of fluorescent nucleotide incorporation and GFP-PCNA gave exquisite time resolution of S phase events. In the early S phases, satellite sequences replicated rapidly despite a compact chromatin structure. In S phases 11-13, a delay in satellite replication emerged in sync with modest and progressive prolongation of S phase. In S phase 14, major and distinct delays ordered the replication of satellites into a sequence that occupied much of S phase. This onset of late replication required transcription. Satellites only accumulated abundant heterochromatin protein 1 (HP1) after replicating in S phase 14. By cycle 15, satellites clustered in a compact HP1-positive mass, but replication occurred at decondensed foci at the surface of this mass. CONCLUSIONS: The slowing of S phase is an active process, not a titration of maternal replication machinery. Most sequences continue to replicate rapidly in successive cycles, but increasing delays in the replication of satellite sequences extend S phase. Although called constitutively heterochromatic, satellites acquire the distinctive features of heterochromatin, compaction, late replication, HP1 binding, and aggregation at the chromocenter, in successive steps coordinated with developmental progress.

Influence of cyclin type and dose on mitotic entry and progression in the early Drosophila embryo.

Cyclins are key cell cycle regulators, yet few analyses test their role in timing the events that they regulate. We used RNA interference and real-time visualization in embryos to define the events regulated by each of the three mitotic cyclins of Drosophila melanogaster, CycA, CycB, and CycB3. Each individual and pairwise knockdown results in distinct mitotic phenotypes. For example, mitosis without metaphase occurs upon knockdown of CycA and CycB. To separate the role of cyclin levels from the influences of cyclin type, we knocked down two cyclins and reduced the gene dose of the one remaining cyclin. This reduction did not prolong interphase but instead interrupted mitotic progression. Mitotic prophase chromosomes formed, centrosomes divided, and nuclei exited mitosis without executing later events. This prompt but curtailed mitosis shows that accumulation of cyclin function does not directly time mitotic entry in these early embryonic cycles and that cyclin function can be sufficient for some mitotic events although inadequate for others.

DNA replication times the cell cycle and contributes to the mid-blastula transition in Drosophila embryos.

We examined the contribution of S phase in timing cell cycle progression during Drosophila embryogenesis using an approach that deletes S phase rather than arresting its progress. Injection of Drosophila Geminin, an inhibitor of replication licensing, prevented subsequent replication so that the following mitosis occurred with uninemic chromosomes, which failed to align. The effect of S phase deletion on interphase length changed with development. During the maternally regulated syncytial blastoderm cycles, deleting S phase shortened interphase, and deletion of the last of blastoderm S phase (cycle 14) induced an extra synchronous division and temporarily deferred mid-blastula transition (MBT) events. In contrast, deleting S phase after the MBT in cycle 15 did not dramatically affect mitotic timing, which appears to retain its dependence on developmentally programmed zygotic transcription. We conclude that normal S phase and replication checkpoint activities are important timers of the undisturbed cell cycle before, but not after, the MBT.

The highly conserved Ndc80 complex is required for kinetochore assembly, chromosome congression, and spindle checkpoint activity.

We show that the Xenopus homologs of Ndc80/Tid3/HEC1 (xNdc80) and Nuf2/MPP1/Him-10 (xNuf2) proteins physically interact in a 190-kD complex that associates with the outer kinetochore from prometaphase through anaphase. Injecting function-blocking antibodies to either xNdc80 or xNuf2 into XTC cells caused premature exit from mitosis without detectable chromosome congression or anaphase movements. Injected cells did not arrest in response to microtubule drugs, showing that the complex is required for the spindle checkpoint. Kinetochores assembled in Xenopus extracts after immunodepletion of the complex did not contain xRod, xZw10, xP150 glued (Dynactin), xMad1, xMad2, xBub1, and xBub3, demonstrating that the xNdc80 complex is required for functional kinetochore assembly. In contrast, function-blocking antibodies did not affect the localization of other kinetochore proteins when added to extracts containing previously assembled kinetochores. These extracts with intact kinetochores were deficient in checkpoint signaling, suggesting that the Ndc80 complex participates in the spindle checkpoint. We also demonstrate that the spindle checkpoint can arrest budding yeast cells lacking Ndc80 or Nuf2, whereas yeast lacking both proteins fail to arrest in mitosis. Systematic deletion of yeast kinetochore genes suggests that the Ndc80 complex has a unique role in spindle checkpoint signaling. We propose that the Ndc80 complex has conserved roles in kinetochore assembly, chromosome congression, and spindle checkpoint signaling.

Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy.

Hemophilia B is an X-linked coagulopathy caused by absence of functional coagulation factor IX (FIX). Using adeno-associated virus (AAV)-mediated, liver-directed gene therapy, we achieved long-term (> 17 months) substantial correction of canine hemophilia B in 3 of 4 animals, including 2 dogs with an FIX null mutation. This was accomplished with a comparatively low dose of 1 x 10(12) vector genomes/kg. Canine FIX (cFIX) levels rose to 5% to 12% of normal, high enough to result in nearly complete phenotypic correction of the disease. Activated clotting times and whole blood clotting times were normalized, activated partial thromboplastin times were substantially reduced, and anti-cFIX was not detected. The fourth animal, also a null mutation dog, showed transient expression (4 weeks), but subsequently developed neutralizing anti-cFIX (inhibitor). Previous work in the canine null mutation model has invariably resulted in inhibitor formation following treatment by either gene or protein replacement therapies. This study demonstrates that hepatic AAV gene transfer can result in sustained therapeutic expression in a large animal model characterized by increased risk of a neutralizing anti-FIX response.