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High level of concomitant Bim and Bcl-2 expression correlates with sensitivity to ABBV-075-induced apoptosis

Apoptosis induction by MS417 across cancer cell lines

Restoration of Myc expression under BET inhibitor treatment failed to rescue cells from apoptosis or cell cycle arrest

ABBV-075 is a potent and selective BET family bromodomain inhibitor that recently entered phase I clinical trials. Comprehensive preclinical characterization of ABBV-075 demonstrated broad activity across cell lines and tumor models, representing a variety of hematologic malignancies and solid tumor indications. In most cancer cell lines derived from solid tumors, ABBV-075 triggers prominent G1 cell-cycle arrest without extensive apoptosis. In this study, we show that ABBV-075 efficiently triggers apoptosis in acute myeloid leukemia (AML), non-Hodgkin lymphoma, and multiple myeloma cells. Apoptosis induced by ABBV-075 was mediated in part by modulation of the intrinsic apoptotic pathway, exhibiting synergy with the BCL-2 inhibitor venetoclax in preclinical models of AML. In germinal center diffuse large B-cell lymphoma, BCL-2 levels or venetoclax sensitivity predicted the apoptotic response to ABBV-075 treatment. In vivo combination studies uncovered surprising benefits of low doses of ABBV-075 coupled with bortezomib and azacitidine treatment, despite the lack of in vitro synergy between ABBV-075 and these agents. The in vitro/in vivo activities of ABBV-075 described here may serve as a useful reference to guide the development of ABBV-075 and other BET family inhibitors for cancer therapy. Cancer Res; 77(11); 2976–89. ©2017 AACR.

ABBV-075 inhibits Bcl-XL transcription and displace BRD4 from Bcl-XL regulatory regions

Apoptosis induction by ABBV-075 across cancer cell lines

Induction of apoptosis across cancer cell lines and CD34 normal cells

Anti-proliferative activity of ABBV-075 across cancer cell lines

BET inhibitor causes cell cycle arrest and triggers senescence in solid tumor cells

supplemental figure legends

Expression of exogenous Bcl-XL rescues ABBV-075 induced apoptosis but not cell cycle arrest

Down regulation of Bcl-XL by MS417 and ABBV-075

ABBV-075 inhibits genes that are important for cell cycle

Context Pueraria lobata (Willd.) Ohwi (Fabaceae) root extract can lower blood glucose levels; however, whether Pueraria lobata root polysaccharide (PLP) possesses these effects is still unknown. Objective This study evaluates the therapeutic effect of PLP on diabetic metabolic syndrome. Materials and methods The db/m mice were assigned to normal control group (NC), db/db mice were divided into four groups randomly (n = 8). The db/db mice received rosiglitazone (10 mg/kg BW) or PLP (100 or 200 mg/kg BW) via oral gavage for 6 weeks. Afterward, blood glucose, insulin, and glycogen content were assayed, and insulin tolerance test (ITT), oral glucose tolerance test (OGTT) were performed. Glucose and lipid metabolism-related parameters and gene expression levels were assayed by ELISA and RT-PCR, respectively. Results After treatment with HPLP, the values of body weight, epididymal fat, subcutaneous fat, fasting blood glucose, insulin, and HOMA-IR decreased to 45.89 ± 1.66 g, 1.65 ± 0.14 g, 1.97 ± 0.16 g, 14.84 ± 1.52 mM, 9.35 ± 0.98 mU/L, and 5.56 ± 1.26, respectively; the levels of TG, TC, LDL-C, and FFA decreased to 1.67 ± 0.11 mmol/L, 6.23 ± 0.76 mmol/L, 1.29 ± 0.07 mmol/L, and 1.71 ± 0.16 mmol/L, respectively. HPLP down-regulated PEPCK, G6PC, FOXO1, SREBP-1, and ACC mRNA expression (p

The development of bromodomain and extraterminal domain (BET) bromodomain inhibitors and their examination in clinical studies, particularly in oncology settings, has garnered substantial recent interest. An effort to generate novel BET bromodomain inhibitors with excellent potency and drug metabolism and pharmacokinetics (DMPK) properties was initiated based upon elaboration of a simple pyridone core. Efforts to develop a bidentate interaction with a critical asparagine residue resulted in the incorporation of a pyrrolopyridone core, which improved potency by 9-19-fold. Additional structure-activity relationship (SAR) efforts aimed both at increasing potency and improving pharmacokinetic properties led to the discovery of the clinical candidate 63 (ABBV-075/mivebresib), which demonstrates excellent potency in biochemical and cellular assays, advantageous exposures and half-life both in animal models and in humans, and in vivo efficacy in mouse models of cancer progression and inflammation.

ABBV-075 is a potent and selective BET family bromodomain inhibitor that recently entered phase I clinical trials. Comprehensive preclinical characterization of ABBV-075 demonstrated broad activity across cell lines and tumor models, representing a variety of hematologic malignancies and solid tumor indications. In most cancer cell lines derived from solid tumors, ABBV-075 triggers prominent G1 cell-cycle arrest without extensive apoptosis. In this study, we show that ABBV-075 efficiently triggers apoptosis in acute myeloid leukemia (AML), non-Hodgkin lymphoma, and multiple myeloma cells. Apoptosis induced by ABBV-075 was mediated in part by modulation of the intrinsic apoptotic pathway, exhibiting synergy with the BCL-2 inhibitor venetoclax in preclinical models of AML. In germinal center diffuse large B-cell lymphoma, BCL-2 levels or venetoclax sensitivity predicted the apoptotic response to ABBV-075 treatment. In vivo combination studies uncovered surprising benefits of low doses of ABBV-075 coupled with bortezomib and azacitidine treatment, despite the lack of in vitro synergy between ABBV-075 and these agents. The in vitro/in vivo activities of ABBV-075 described here may serve as a useful reference to guide the development of ABBV-075 and other BET family inhibitors for cancer therapy. Cancer Res; 77(11); 2976–89. ©2017 AACR.

Faithful segregation of mitotic chromosomes requires bi-orientation of sister chromatids, which relies on the sensing of correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying PLK1 activation have been extensively studied, the regulatory mechanisms that couple PLK1 activity to accurate chromosome segregation are not well understood. In particular, PLK1 is implicated in stabilizing kinetochore–microtubule attachments, but how kinetochore PLK1 activity is regulated to avoid hyperstabilized kinetochore–microtubules in mitosis remains elusive. Here, we show that kinetochore PLK1 kinase activity is modulated by SET7/9 via lysine methylation during early mitosis. The SET7/9-elicited dimethylation occurs at the Lys191 of PLK1, which tunes down its activity by limiting ATP utilization. Overexpression of the non-methylatable PLK1 mutant or chemical inhibition of SET7/9 methyltransferase activity resulted in mitotic arrest due to destabilized kinetochore–microtubule attachments. These data suggest that kinetochore PLK1 is essential for stable kinetochore–microtubule attachments and methylation by SET7/9 promotes dynamic kinetochore–microtubule attachments for accurate error correction. Our findings define a novel homeostatic regulation at the kinetochore that integrates protein phosphorylation and methylation with accurate chromosome segregation for maintenance of genomic stability.

Summary AMP-activated protein kinase (AMPK) senses energy status and impacts energy-consuming events by initiating metabolism regulatory signals in cells. Accumulating evidences suggest a role of AMPK in mitosis regulation, but the mechanism of mitotic AMPK activation and function remains elusive. Here we report that AMPKα2, but not AMPKα1, is sequentially phosphorylated and activated by CDK1 and PLK1, which enables AMPKα2 to accurately guide chromosome segregation in mitosis. Phosphorylation at Thr485 by activated CDK1-Cyclin B1 brings the ST-stretch of AMPKα2 to the Polo box domain of PLK1 for subsequent Thr172 phosphorylation by PLK1. Inserting of the AMPKα2 ST-stretch into AMPKα1, which lacks the ST-stretch, can correct mitotic chromosome segregation defects in AMPKα2-depleted cells. These findings uncovered a specific signaling cascade integrating sequential phosphorylation by CDK1 and PLK1 of AMPKα2 with mitosis to maintain genomic stability, thus defining an isoform-specific AMPKα2 function, which will facilitate future research on energy sensing in mitosis., Graphical abstract, Highlights • AMPKα2 is selectively activated during mitosis by CDK1 and PLK1 • A conserved motif in AMPKα2 determines its interaction with and activation by PLK1 • Mitotic AMPK activation contributes to maintain genomic stability in normal mitosis, Biological Sciences ; Cell Biology ; Cell

Faithful chromosome segregation in mammalian cells requires the bi-orientation of sister chromatids which relies on sensing correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying cyclin-dependent kinase 1 (CDK1) activation that triggers mitotic entry is extensively studied, the regulatory mechanisms that couple CDK1-cyclin B activity to chromosome stability are not well understood. Here, we identified a signaling axis in which Aurora B activity is modulated by CDK1-cyclin B via acetyltransferase TIP60 (Tat-interactive protein 60 kDa) in human cell division. CDK1-cyclin B phosphorylated Ser90 of TIP60, which elicited TIP60-dependent acetylation of Aurora B and promoted accurate chromosome segregation in mitosis. Mechanistically, TIP60 acetylation of Aurora B at Lys215 protected the phosphorylation of its activation loop from PP2A reactivation-elicited dephosphorylation to ensure a robust, error-free metaphase-anaphase transition. These findings delineated a conserved signaling cascade that integrates protein phosphorylation and acetylation to cell cycle progression for maintenance of genomic stability.

Although loss of Sirt1 leads to chromosome aneuploidy, which accounts for higher tumor susceptibility, the molecular mechanisms remain unclear. Herein, we demonstrate that Sirt1 directly regulates Plk1, of which activity is critical for mitotic progression and spindle dynamics. Depletion or inhibition of Sirt1 significantly perturbs the formation of the mitotic spindle, leading to defective chromosome segregation. Elevated depolymerization of the mitotic spindle following loss of Sirt1 was associated with the deregulation of Plk1 activity. Thus, we conclude that Sirt1 may contribute to a mitotic regulator that controls spindle dynamics through Plk1 activity, resulting in fine-tuning of Plk1 dependent microtubule dynamics.

Protein-protein interactions (PPIs) are critical regulatory events in physiology and pathology, and they represent an important target space for pharmacological intervention. However, targeting PPIs with small molecules is challenging owing to the large surface area involved in protein-protein binding and the lack of obvious small-molecule-binding pockets at many protein-protein interfaces. Nonetheless, successful examples of small-molecule modulators of PPIs have been growing in recent years. This article reviews some of the recent advances in the discovery of small-molecule regulators of PPIs that involve key oncogenic proteins. Our discussion focuses on the three key modes of action for these small-molecule modulators: orthosteric inhibition, allosteric regulation, and interfacial binding/stabilization. Understanding the opportunities and challenges of these diverse mechanisms will help guide future efforts in developing small-molecule modulators against PPIs.

Long-term nonprogressive human immunodeficiency virus type 1 (HIV-1) infection and its transition to progressive infection presents an opportunity to identify the molecular determinants of HIV-1 attenuation and pathogenesis. We studied an individual who underwent a transition from long-term nonprogressive to rapidly progressive infection. Because HIV-1 RNA genomes in plasma represent replicating virus, we developed a technique to clone full-length HIV-1 RNA genomes from plasma and used this technique to obtain clones from this individual before and during the transition. Most clones assayed were infectious, demonstrating that the RNA genomes encoded viable virus. Analysis of 20 complete HIV-1 RNA genomic sequences revealed one major difference between sequences found during the two phases of infection. During the nonprogressive phase, the predominant sequences had a large deletion in an Sp1-binding site and adjacent promoter in the U3 part of the long terminal repeat (LTR); when the infection became progressive, all viruses had intact Sp1 and promoter sequences and were derived from a minor species present earlier. Analysis of 184 clones of the LTR region obtained at five time points spanning a 7-year period confirmed this switch. In an in vitro assay, the deletion downregulated LTR-driven transcription of a reporter gene. In addition, analysis of cytotoxic T lymphocyte (CTL) epitopes predicted from the complete viral RNA genomes revealed multiple potential escape mutants that accumulated by the time of progression. These studies suggest that during the nonprogressive phase, the Sp1 enhancer-promoter deletion is likely to have played a role in decreasing replication, thereby attenuating HIV-1. The accumulation of CTL escape mutants suggests that a breakdown in immunologic surveillance may have allowed proliferation of intact virus, thus leading to rapid disease progression. These data reveal the viral and immune interactions characterizing a transition from long-term nonprogressive to rapidly progressive infection.

The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras. High-resolution cocrystal structures delineated a unique ligand-binding pocket on the Ras protein that is adjacent to the switch I/II regions and can be expanded upon compound binding. Structure analysis predicts that compound-binding interferes with the Ras/SOS interactions. Indeed, selected compounds inhibit SOS-mediated nucleotide exchange and prevent Ras activation by blocking the formation of intermediates of the exchange reaction. The discovery of a small-molecule binding pocket on Ras with functional significance provides a new direction in the search of therapeutically effective inhibitors of the Ras oncoprotein.

Summary Cell cycle progression requires the E3 ubiquitin ligase anaphase-promoting complex (APC/C), which uses the substrate adaptors CDC20 and CDH1 to target proteins for proteasomal degradation. The APC CDH1 substrate cyclin A is critical for the G1/S transition and, paradoxically, accumulates even when APC CDH1 is active. We show that the deubiquitinase USP37 binds CDH1 and removes degradative polyubiquitin from cyclin A. USP37 was induced by E2F transcription factors in G1, peaked at G1/S, and was degraded in late mitosis. Phosphorylation of USP37 by CDK2 stimulated its full activity. USP37 overexpression caused premature cyclin A accumulation in G1 and accelerated S phase entry, whereas USP37 knockdown delayed these events. USP37 was inactive in mitosis because it was no longer phosphorylated by CDK2. Indeed, it switched from an antagonist to a substrate of APC CDH1 and was modified with degradative K11-linked polyubiquitin.

Mitotic kinases orchestrate cell cycle processes by phosphorylation of cell cycle regulators. DDA3, a spindle-associated phosphor-protein, is a substrate of mitotic kinases that control chromosome movement and spindle microtubule (MT) dynamics. Through a mass spectrometry analysis, we identified phosphorylation sites on the endogenous mitotic DDA3, which include Ser22, Ser65, Ser70, and Ser223. Phosphorylation of these residues converts interphase form of DDA3 to mitotic form by changing its biochemical activity, as unphosphorylated DDA3 processed both the MT polymerizing and bundling activities, whereas phosphor-mimic mutants lost both activities, only retaining the MT-binding activity. We found that mitotic kinases, such as Cdk1, Aurora A, and Plk1, phosphorylate DDA3 in vitro. Whereas Cdk1 and Aurora A negatively regulate MT-polymerizing and MT-bundling activities, Plk1 does not affect these activities. Interestingly, the phosphorylation of DDA3 by Aurora A and Plk1 inhibits the phosphorylation by other kinases, indicating that sequential phosphorylation is important for the regulation of DDA3 function. We conclude that kinases control the function of DDA3 in the cell cycle by regulating its MT-polymerizing/bundling activities through sequential phosphorylation.

DDA3 is a microtubule-associated protein that controls chromosome congression and segregation by regulating the mitotic spindle. Depletion of DDA3 alters spindle structure, generates unaligned chromosomes at metaphase, and delays the mitotic progression. Through a mass spectrometry analysis, we found that DDA3 is phosphorylated on Ser225 during mitosis. Phosphorylation of this residue is important for the mitotic function of DDA3, as the phospho-mimicking DDA3-S225D variant, but not the nonphosphorable DDA3-S225A mutant, rescues the DDA3-knockdown phenotype. We conclude that the mitotic function of DDA3 is regulated by phosphorylation on the Ser225 residue.

DDA3 is a microtubule-associated protein that controls chromosome congression and segregation by regulating the dynamics of the mitotic spindle. Depletion of DDA3 alters spindle structure, generates unaligned chromosomes at metaphase, and delays the mitotic progression. DDA3 interacts with the microtubule depolymerase Kif2a and controls the association of Kif2a to the mitotic spindle and the dynamic turnover of microtubules in the spindle. To understand the function and regulation of DDA3, we analyzed its domain structure and found that the C-terminal domain of DDA3 directly binds to microtubules in vitro and associates with the mitotic spindle in vivo. The N-terminal domain of DDA3 does not interact with microtubules, but acts dominant negatively over the wild-type protein. Ectopic expression of this domain prevents the endogenous DDA3 from association with the spindle and results in a high frequency of unaligned chromosomes in metaphase cells, a phenotype similar to that in metaphase cells depleted of DDA3. Mechanistically, expression of N-terminal DDA3 reduces the amount of spindle-associated Kif2a and increases the spindle microtubule density, pheno-copying those in DDA3-depleted cells. We conclude that DDA3 has a distinct domain structure. The C-terminal domain confers its ability to associate with the mitotic spindle, while the regulatory N-terminal domain controls the microtubule-binding by the C-terminal domain and determines the cellular activity of the DDA3 protein.

The spindle assembly checkpoint (SAC) controls the anaphase onset by preventing premature chromosome segregation until bipolar microtubule (MT) attachment and inter-kinetochore tension are fully established for every kinetochore pair. Once the SAC is off, activation of the Anaphase-Promoting Complex/Cyclosome, a ubiquitin ligase, leads to the degradation of securin and cyclin B, the activation of separase and the initiation of anaphase. We report here the identification and characterization of a G(2)-induced gene, SKAP, as a regulator for the anaphase onset. SKAP localizes to spindle MTs and kinetochores in mitosis. Depletion of SKAP does not activate the SAC, but substantially increases the duration of metaphase, delays the activation of separase, and decreases the fidelity of chromosome segregation. Our study identifies SKAP as a novel regulator of the metaphase-to-anaphase transition and demonstrates that misregulation of the separase activation results in a reduced fidelity of chromosome segregation and a reduced genomic stability independent of the SAC.

HURP is a spindle-associated protein that mediates Ran-GTP-dependent assembly of the bipolar spindle and promotes chromosome congression and interkinetochore tension during mitosis. We report here a biochemical mechanism of HURP regulation by Aurora A, a key mitotic kinase that controls the assembly and function of the spindle. We found that HURP binds to microtubules through its N-terminal domain that hyperstabilizes spindle microtubules. Ectopic expression of this domain generates defects in spindle morphology and function that reduce the level of tension across sister kinetochores and activate the spindle checkpoint. Interestingly, the microtubule binding activity of this N-terminal domain is regulated by the C-terminal region of HURP: in its hypophosphorylated state, C-terminal HURP associates with the microtubule-binding domain, abrogating its affinity for microtubules. However, when the C-terminal domain is phosphorylated by Aurora A, it no longer binds to N-terminal HURP, thereby releasing the inhibition on its microtubule binding and stabilizing activity. In fact, ectopic expression of this C-terminal domain depletes endogenous HURP from the mitotic spindle in HeLa cells in trans, suggesting the physiological importance for this mode of regulation. We concluded that phosphorylation of HURP by Aurora A provides a regulatory mechanism for the control of spindle assembly and function.

Through a convergence of functional genomic and proteomic studies, we identify Bora as a previously unknown cell cycle protein that interacts with the Plk1 kinase and the SCF–β-TrCP ubiquitin ligase. We show that the Bora protein peaks in G2 and is degraded by proteasomes in mitosis. Proteolysis of Bora requires the Plk1 kinase activity and is mediated by SCF–β-TrCP. Plk1 phosphorylates a conserved DSGxxT degron in Bora and promotes its interaction with β-TrCP. Mutations in this degron stabilize Bora. Expression of a nondegradable Bora variant prolongs the metaphase and delays anaphase onset, indicating a physiological requirement of Bora degradation. Interestingly, the activity of Bora is also required for normal mitotic progression, as knockdown of Bora activates the spindle checkpoint and delays sister chromatid segregation. Mechanistically, Bora regulates spindle stability and microtubule polymerization and promotes tension across sister kinetochores during mitosis. We conclude that tight regulation of the Bora protein by its synthesis and degradation is critical for cell cycle progression.

Dynamic turnover of the spindle is a driving force for chromosome congression and segregation in mitosis. Through a functional genomic analysis, we identify DDA3 as a previously unknown regulator of spindle dynamics that is essential for mitotic progression. DDA3 depletion results in a high frequency of unaligned chromosomes, a substantial reduction in tension across sister kinetochores at metaphase, and a decrease in the velocity of chromosome segregation at anaphase. DDA3 associates with the mitotic spindle and controls microtubule (MT) dynamics. Mechanistically, DDA3 interacts with the MT depolymerase Kif2a in an MT-dependent manner and recruits Kif2a to the mitotic spindle and spindle poles. Depletion of DDA3 increases the steady-state levels of spindle MTs by reducing the turnover rate of the mitotic spindle and by increasing the rate of MT polymerization, which phenocopies the effects of partial knockdown of Kif2a. Thus, DDA3 represents a new class of MT-destabilizing protein that controls spindle dynamics and mitotic progression by regulating MT depolymerases.

Accurate chromosome segregation is controlled by the spindle checkpoint, which senses kinetochore– microtubule attachments and tension across sister kinetochores. An important step in the tension-signaling pathway involves the phosphorylation of an unknown protein by polo-like kinase 1/Xenopus laevis polo-like kinase 1 (Plx1) on kinetochores lacking tension to generate the 3F3/2 phosphoepitope. We report here that the checkpoint protein BubR1 interacts with Plx1 and that phosphorylation of BubR1 by Plx1 generates the 3F3/2 epitope. Formation of the BubR1 3F3/2 epitope by Plx1 requires a prior phosphorylation of BubR1 on Thr 605 by cyclin-dependant kinase 1 (Cdk1). This priming phosphorylation of BubR1 by Cdk1 is required for checkpoint-mediated mitotic arrest and for recruitment of Plx1 and the checkpoint protein Mad2 to unattached kinetochores. Biochemically, formation of the 3F3/2 phosphoepitope by Cdk1 and Plx1 greatly enhances the kinase activity of BubR1. Thus, Cdk1-mediated phosphorylation of BubR1 controls checkpoint arrest and promotes the formation of the kinetochore 3F3/2 epitope.

We report here an efficient functional genomic analysis by combining information on the gene expression profiling, cellular localization, and loss-of-function studies. Through this analysis, we identified Cep55 as a regulator required for the completion of cytokinesis. We found that Cep55 localizes to the mitotic spindle during prometaphase and metaphase and to the spindle midzone and the midbody during anaphase and cytokinesis. At the terminal stage of cytokinesis, Cep55 is required for the midbody structure and for the completion of cytokinesis. In Cep55-knockdown cells, the Flemming body is absent, and the structural and regulatory components of the midbody are either absent or mislocalized. Cep55 also facilitates the membrane fusion at the terminal stage of cytokinesis by controlling the localization of endobrevin, a v-SNARE required for cell abscission. Biochemically, Cep55 is a microtubule-associated protein that efficiently bundles microtubules. Cep55 directly binds to MKLP1 in vitro and associates with the MKLP1-MgcRacGAP centralspindlin complex in vivo. Cep55 is under the control of centralspindlin, as knockdown of centralspindlin abolished the localization of Cep55 to the spindle midzone. Our study defines a cellular mechanism that links centralspindlin to Cep55, which, in turn, controls the midbody structure and membrane fusion at the terminal stage of cytokinesis.

Through a functional genomic screen for mitotic regulators, we identified hepatoma up-regulated protein (HURP) as a protein that is required for chromosome congression and alignment. In HURP-depleted cells, the persistence of unaligned chromosomes and the reduction of tension across sister kinetochores on aligned chromosomes resulted in the activation of the spindle checkpoint. Although these defects transiently delayed mitotic progression, HeLa cells initiated anaphase without resolution of these deficiencies. This bypass of the checkpoint arrest provides a tumor-specific mechanism for chromosome missegregation and genomic instability. Mechanistically, HURP colocalized with the mitotic spindle in a concentration gradient increasing toward the chromosomes. HURP binds directly to microtubules in vitro and enhances their polymerization. In vivo, HURP stabilizes mitotic microtubules, promotes microtubule polymerization and bipolar spindle formation, and decreases the turnover rate of the mitotic spindle. Thus, HURP controls spindle stability and dynamics to achieve efficient kinetochore capture at prometaphase, timely chromosome congression to the metaphase plate, and proper interkinetochore tension for anaphase initiation.

TPX2, a microtubule-associated protein, is required downstream of Ran-GTP to induce spindle assembly. TPX2 activity appears to be tightly regulated during the cell cycle, and we report here one molecular mechanism for this regulation. We found that TPX2 protein levels are cell cycle regulated, peaking in mitosis and declining sharply during mitotic exit. TPX2 is degraded in mitotic extracts, as well as in HeLa cells exiting from mitosis. This instability depends, both in vitro and in vivo, on the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls mitotic progression. In a reconstituted system, TPX2 is efficiently ubiquitinated by APC/C that has been activated by Cdh1. Two discrete elements in TPX2 are required for recognition by APC/C(Cdh1): a KEN box and a novel element in amino acids 1 to 86. Interestingly, the latter element, which has no known APC/C recognition motifs, is required for the ubiquitination of TPX2 by APC/C(Cdh1) in vitro and for its degradation in vivo. We conclude that APC/C(Cdh1) controls the stability of TPX2, thereby ensuring accurate regulation of the spindle assembly in the cell cycle.

Aurora B kinase, a subunit of the chromosomal passenger protein complex, plays essential roles in spindle assembly, chromosome bi-orientation, and cytokinesis. The kinase activity of Aurora B, which peaks in mitosis, is tightly controlled in the cell cycle. Modulation of Aurora B protein levels could partly account for the regulation of its kinase activity in the cell cycle. However, little is known on the molecular mechanism of regulation of Aurora B levels. Here, we examined Aurora B protein levels and confirmed that they fluctuate during the cell cycle, peaking in mitosis and dropping drastically in G1. This profile for Aurora B in the cell cycle is reminiscent of those for substrates of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase essential for mitotic progression. Indeed, Aurora B is a substrate of APC/C both in vitro and in vivo. Aurora B is efficiently ubiquitinated in an in vitro reconstituted system by APC/C that had been activated by Cdh1. The recognition of Aurora B by APC/C-Cdh1 is specific as it requires the presence of a conserved D-box at the COOH terminus of Aurora B. Furthermore, endogenous Aurora B and Cdh1 form a complex exclusively in mitotic cells. Degradation of Aurora B at the end of mitosis requires Cdh1 in vivo as a reduction of the Cdh1 level by RNA interference stabilizes the Aurora B protein. We conclude that, as a key mitotic regulator, Aurora B is regulated both by its activation during early mitosis and by its destruction by APC/C-Cdh1 in late mitosis and in G1.

Anillin, an actin-binding protein localized at the cleavage furrow, is required for cytokinesis. Through an in vitro expression screen, we identified anillin as a substrate of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls mitotic progression. We found that the levels of anillin fluctuate in the cell cycle, peaking in mitosis and dropping drastically during mitotic exit. Ubiquitination of anillin required a destruction-box and was mediated by Cdh1, an activator of APC/C. Overexpression of Cdh1 reduced the levels of anillin, whereas inactivation of APC/C(Cdh1) increased the half-life of anillin. Functionally, anillin was required for the completion of cytokinesis. In anillin knockdown cells, the cleavage furrow ingressed but failed to complete the ingression. At late cytokinesis, the cytosol and DNA in knockdown cells underwent rapid myosin-based oscillatory movement across the furrow. During this movement, RhoA and active myosin were absent from the cleavage furrow, and myosin was redistributed to cortical patches, which powers the random oscillatory movement. We concluded that anillin functions to maintain the localization of active myosin, thereby ensuring the spatial control of concerted contraction during cytokinesis.

Initiation of cytokinesis requires the establishment of the cleavage plane, the assembly of the contractile ring, and the ingression of the cleavage furrow. MgcRacGAP, a GTPase-activating protein for RhoA, is required for cytokinesis, but the mechanism of its action remains unknown. We report here that MgcRacGAP is required for the assembly of anillin and myosin into the contractile ring. In addition, MgcRacGAP is required for the localized activation of myosin through the RhoA-mediated phosphorylation of the myosin regulatory light chain. Cells with MgcRacGAP RNA interference (RNAi) failed cytokinesis without any ingression of the cleavage furrow. Paradoxically, MgcRacGAP, a GTPase-activating protein, associates during cytokinesis with ECT2, a guanine nucleotide exchange factor for RhoA, and the localization of ECT2 to both the central spindle and the contractile ring depends on MgcRacGAP. Knockdown of ECT2 phenocopies that of MgcRacGAP. We conclude that MgcRacGAP controls the initiation of cytokinesis by regulating ECT2, which in turn induces the assembly of the contractile ring and triggers the ingression of the cleavage furrow.

Tumorigenesis is a consequence of loss of tumor suppressors and activation of oncogenes. Expression of the mitotic checkpoint protein Chfr is lost in 20-50% of primary tumors and tumor cell lines. To explore whether downregulation of Chfr contributes directly to tumorigenesis, we generated Chfr knockout mice. Chfr-deficient mice are cancer-prone, develop spontaneous tumors and have increased skin tumor incidence after treatment with dimethylbenz(a)anthracene. Chfr deficiency leads to chromosomal instability in embryonic fibroblasts and regulates the mitotic kinase Aurora A, which is frequently upregulated in a variety of tumors. Chfr physically interacts with Aurora A and ubiquitinates Aurora A both in vitro and in vivo. Collectively, our data suggest that Chfr is a tumor suppressor and ensures chromosomal stability by controlling the expression levels of key mitotic proteins such as Aurora A.

BACKGROUND: To test current models for how unattached and untense kinetochores prevent Cdc20 activation of the anaphase-promoting complex/cyclosome (APC/C) throughout the spindle and the cytoplasm, we used GFP fusions and live-cell imaging to quantify the abundance and dynamics of spindle checkpoint proteins Mad1, Mad2, Bub1, BubR1, Mps1, and Cdc20 at kinetochores during mitosis in living PtK2 cells. RESULTS: Unattached kinetochores in prometaphase bound on average only a small fraction (estimated at 500-5000 molecules) of the total cellular pool of each spindle checkpoint protein. Measurements of fluorescence recovery after photobleaching (FRAP) showed that GFP-Cdc20 and GFP-BubR1 exhibit biphasic exponential kinetics at unattached kinetochores, with approximately 50% displaying very fast kinetics (t1/2 of approximately 1-3 s) and approximately 50% displaying slower kinetics similar to the single exponential kinetics of GFP-Mad2 and GFP-Bub3 (t1/2 of 21-23 s). The slower phase of GFP-Cdc20 likely represents complex formation with Mad2 since it was tension insensitive and, unlike the fast phase, it was absent at metaphase kinetochores that lack Mad2 but retain Cdc20 and was absent at unattached prometaphase kinetochores for the Cdc20 derivative GFP-Cdc20delta1-167, which lacks the major Mad2 binding domain but retains kinetochore localization. GFP-Mps1 exhibited single exponential kinetics at unattached kinetochores with a t1/2 of approximately 10 s, whereas most GFP-Mad1 and GFP-Bub1 were much more stable components. CONCLUSIONS: Our data support catalytic models of checkpoint activation where Mad1 and Bub1 are mainly resident, Mad2 free of Mad1, BubR1 and Bub3 free of Bub1, Cdc20, and Mps1 dynamically exchange as part of the diffuse wait-anaphase signal; and Mad2 interacts with Cdc20 at unattached kinetochores.

Members of the Ndc80/Nuf2 complex have been shown in several systems to be important in formation of stable kinetochore-microtubule attachments and chromosome alignment in mitosis 1, 2, 3, 4, 5, 6, 7, 8 and 9. In HeLa cells, we have shown that depletion of Nuf2 by RNA interference (RNAi) results in a strong prometaphase block with an active spindle checkpoint, which correlates with low but detectable Mad2 at kinetochores that have no or few stable kinetochore microtubules [5]. Another RNAi study in HeLa cells reported that Hec1 (the human Ndc80 homolog) is required for Mad1 and Mad2 binding to kinetochores and that kinetochore bound Mad2 does not play a role in generating and maintaining the spindle assembly checkpoint [6]. Here, we show that depletion of either Nuf2 or Hec1 by RNAi in HeLa cells results in reduction of both proteins at kinetochores and in the cytoplasm. Mad1 and Mad2 concentrate at kinetochores in late prophase/early prometaphase but become depleted by 5-fold or more over the course of the prometaphase block, which is Mad2 dependent. The reduction of Mad1 and Mad2 is reversible upon spindle depolymerization. Our observations support a model in which Nuf2 and Hec1 function to prevent microtubule-dependent stripping of Mad1 and Mad2 from kinetochores that have not yet formed stable kinetochore-microtubule attachments.

The abnormal expression of breast cancer-specific gene 1 (BCSG1) in malignant mammary epithelial cells is highly associated with the development and progression of breast cancer. A series of in vitro and in vivo studies performed in our laboratory and others have demonstrated that BCSG1 expression significantly stimulates proliferation, invasion, and metastasis of breast cancer cells. However, currently little is known about how BCSG1 exerts its oncogenic functions. To elucidate the cellular mechanisms underlying the effects of BCSG1 in breast cancer cells, we used a yeast two-hybrid system to screen for proteins that could associate with BCSG1. Through this screening, we identified the mitotic checkpoint protein BubR1 as a novel binding partner of BCSG1. The specific association of BCSG1 with BubR1 in breast cancer cells was demonstrated by immunoprecipitation and GST pull-down assays. Intriguingly, experiments conducted in four different cell lines all showed that exogenous expressions of BCSG1 consistently reduce the cellular levels of the BubR1 protein without affecting BubR1 mRNA expression. The tendency of endogenous BCSG1 expression coinciding with lower BubR1 protein levels was also observed in seven out of eight breast cancer cell lines. We further showed that the reducing effect of BCSG1 on BubR1 protein expression could be prevented by treating BCSG1-transfected cells with MG-132, a selective 26S proteasome inhibitor, implying that the proteasome machinery may be involved in the BCSG1-induced reduction of the BubR1 protein. Accompanied with a reduction of BubR1 protein level, BCSG1 expression resulted in multinucleation of breast cancer cells upon treatment with spindle inhibitor nocodazole, indicating an impaired mitotic checkpoint. Taken together, our novel findings suggest that BCSG1 may accelerate the progression of breast cancer at least in part by compromising the mitotic checkpoint control through inactivation of BubR1.