Cyclin B1-Cdk1 Research Articles

Chloroform extract and acetyl-11-keto-beta-boswellic acid from Boswellia dalzielii stem bark induce apoptosis and cell cycle blockage in AW8507 cells

BackgroundGlobally, head and neck cancer is the sixth most common cancer. Despite the advancement in treatment, drug resistance remains a major cause for setback. In an earlier work, the authors reported that Boswellia dalzielii (Hutch) stem bark exhibited dose-dependent cytotoxicity in head and neck cancer cells, AW8507. Therefore, the cell death induction effect of Boswellia dalzielii stem bark chloroform extract in head and neck cancer cell line, AW8507, and its derived constituent on cell cycle and apoptosis proteins was further investigated.MethodsThe cell death induction activity of the Boswellia dalzielii stem bark chloroform fraction (CLBD) in AW8507 was determined using Annexin V-FITC/PI staining in flow cytometry. High-performance liquid chromatography-mass spectrometry was employed for compounds analysis of the CLBD, and reverse virtual screening was used to identify the mechanism of action of the compound, acetyl-11-keto-beta-boswellic acid, that was elucidated in the Boswellia dalzielii chloroform fraction.ResultsThe data obtained showed that Boswellia dalzielii stem bark Chloroform extract increased the percentage of cells presenting for early apoptosis from 4.14 to 10.10% in AW8507 cells. High-performance liquid chromatography-mass spectrometry analysis of the chloroform fraction identified acetyl-11-keto-beta-boswellic acid. Reverse virtual screening on selected proteins showed that acetyl-11-keto-beta-boswellic acid is a multi-protein target compound. It binds preferably to phosphorylated-cyclin dependent kinase 1 (p-CDK1) (binding score = − 9.2 kcal/mol), blocking the activation of cyclin B-CDK1 needed for cell cycle progression at G2/M phase of the cell cycle. Acetyl-11-keto-beta-boswellic acid also binds more tightly with αβ tubulin (binding score = 8.9 kcal/mol) than with the standard drug, docetaxel (binding score = 8.3 kcal/mol).ConclusionsThe results obtained confirmed the culpability of Boswellia dalzielii-derived acetyl-11-keto-beta-boswellic acid in the obstruction of the cell cycle progression in head and neck cancer cell line, AW8507; and the induction of apoptosis earlier reported for Boswellia dalzielii (Hutch) stem bark. Additional in vitro and/or in vivo studies would be required to validate in silico observations.

Structural basis of human separase regulation by securin and CDK1-cyclin B1.

In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex1. Separation of chromosomes during anaphase is triggered by separase-a large cysteine endopeptidase that cleaves the cohesin subunit SCC1 (also known as RAD212-4). Separase is activated by degradation of its inhibitors, securin5 and cyclin B6, but the molecular mechanisms of separase regulation are not clear. Here we used cryogenic electron microscopy to determine the structures of human separase in complex with either securin or CDK1-cyclin B1-CKS1. In both complexes, separase is inhibited by pseudosubstrate motifs that block substrate binding at the catalyticsite and at nearby docking sites. As in Caenorhabditis elegans7 and yeast8, human securin contains its own pseudosubstrate motifs. By contrast, CDK1-cyclin B1 inhibits separase by deploying pseudosubstrate motifs from intrinsically disordered loops in separase itself. One autoinhibitory loop is oriented by CDK1-cyclin B1 to block the catalyticsites of both separase and CDK19,10. Another autoinhibitory loop blocks substrate docking in a cleft adjacent to the separase catalytic site. A third separase loop contains a phosphoserine6 that promotes complex assembly by binding to a conserved phosphate-binding pocket in cyclin B1. Our study reveals the diverse array of mechanisms by which securin and CDK1-cyclin B1 bind and inhibit separase, providing the molecular basis for the robust control of chromosome segregation.

Coupling of Cdc20 inhibition and activation by BubR1.

Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1-Cdk1 independently inhibits APC/C-Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.

AMPKα2 activation by an energy-independent signal ensures chromosomal stability during mitosis

SummaryAMP-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.

Compartmentalized Control of Cdk1 Drives Mitotic Spindle Assembly

During cell division, dramatic microtubular rearrangements driven by cyclin B-cdk1 (Cdk1) kinase activity mark mitosis onset leading to interphase cytoskeleton dissolution and mitotic spindle assembly. Once activated by Cdc25, that reverses inhibitory phosphorylation operated by Wee1/Myt1, Cdk1 clears the cytoplasm from microtubules by inhibiting microtubule associated proteins (MAPs) with microtubule growth-promoting properties. Nevertheless, some of these MAPs are required for spindle assembly, creating quite a conundrum. We show here that a Cdk1 fraction bound to spindle structures escaped Cdc25 action and remained inhibited by phosphorylation (i-Cdk1) in mitotic human cells. Loss or restoration of i-Cdk1 inhibited or promoted spindle assembly, respectively. Furthermore, polymerizing spindle microtubules fostered i-Cdk1 by aggregating with Wee1 and excluding Cdc25. Our data reveal that spindle assembly relies on compartmentalized control of Cdk1 activity.

Different p53 genotypes regulating different phosphorylation sites and subcellular location of CDC25C associated with the formation of polyploid giant cancer cells

BackgroundOur previous studies have confirmed that cobalt chloride (CoCl2) can induce the formation of polyploid giant cancer cells (PGCCs), which is the key to the heterogeneity of solid tumors. PGCC formation is closely related to the abnormal expression of cell cycle-related proteins and cell fusion. In this study, we investigated the molecular mechanism of PGCCs formation by detecting the expression of cell cycle-related proteins in mutant and wild-type p53 cancer cell lines.MethodsHEY, BT-549, SKOv3 and MDA-MB-231 cells were treated with CoCl2 and the cell cycle was detected by flow cytometry. The expression and subcellular localization of cell cycle-related proteins, kinases, and P53 were compared before and after CoCl2 treatment. Immunoprecipitation was used to analyze the interacting proteins of pCDC25C-Ser216 and pCDC25C-Ser198. The clinicopathologic significances of these cell cycle-related proteins and protein kinases expression were studied.ResultsCoCl2 induced the formation of PGCCs and G2/M arrest. CDC25C, cyclin B1, and CDK1 expressions after CoCl2 treatment were lower than that in control cells. Cytoplasmic CDC25C was degraded by ubiquitin-dependent proteasome. The expression of P53 and phosphokinases including CHK1, CHK2, PLK1, and Aurora A increased after CoCl2 treatment. The expression of pCDC25C-Ser216 and pCDC25C-Ser198 depended upon the genotype of p53. The expressions of cell cycle-related proteins and kinases gradually increased with the development of ovarian cancer and breast cancer.ConclusionCHK1, CHK2–pCDC25C-Ser216–cyclin B1–CDK1, and Aurora A–PLK1–pCDC25C-Ser198–cyclin B1–CDK1 signaling pathways may participate in the formation of PGCCs and different phosphorylation sites of CDC25C may be associated with the genotype of p53.

Kinase inhibition profiles as a tool to identify kinases for specific phosphorylation sites

There are thousands of known cellular phosphorylation sites, but the paucity of ways to identify kinases for particular phosphorylation events remains a major roadblock for understanding kinase signaling. To address this, we here develop a generally applicable method that exploits the large number of kinase inhibitors that have been profiled on near-kinome-wide panels of protein kinases. The inhibition profile for each kinase provides a fingerprint that allows identification of unknown kinases acting on target phosphosites in cell extracts. We validate the method on diverse known kinase-phosphosite pairs, including histone kinases, EGFR autophosphorylation, and Integrin β1 phosphorylation by Src-family kinases. We also use our approach to identify the previously unknown kinases responsible for phosphorylation of INCENP at a site within a commonly phosphorylated motif in mitosis (a non-canonical target of Cyclin B-Cdk1), and of BCL9L at S915 (PKA). We show that the method has clear advantages over in silico and genetic screening.

Cyclin B1-Cdk1 facilitates MAD1 release from the nuclear pore to ensure a robust spindle checkpoint.

How the cell rapidly and completely reorganizes its architecture when it divides is a problem that has fascinated researchers for almost 150 yr. We now know that the core regulatory machinery is highly conserved in eukaryotes, but how these multiple protein kinases, protein phosphatases, and ubiquitin ligases are coordinated in space and time to remodel the cell in a matter of minutes remains a major question. Cyclin B1-Cdk is the primary kinase that drives mitotic remodeling; here we show that it is targeted to the nuclear pore complex (NPC) by binding an acidic face of the kinetochore checkpoint protein, MAD1, where it coordinates NPC disassembly with kinetochore assembly. Localized cyclin B1-Cdk1 is needed for the proper release of MAD1 from the embrace of TPR at the nuclear pore so that it can be recruited to kinetochores before nuclear envelope breakdown to maintain genomic stability.

The Severe Fever with Thrombocytopenia Syndrome Virus NSs Protein Interacts with CDK1 To Induce G2 Cell Cycle Arrest and Positively Regulate Viral Replication.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly identified phlebovirus associated with severe hemorrhagic fever in humans. While many viruses subvert the host cell cycle to promote viral growth, it is unknown whether this is a strategy employed by SFTSV. In this study, we investigated how SFTSV manipulates the cell cycle and the effect of the host cell cycle on SFTSV replication. Our results suggest that cells arrest at the G2/M transition following infection with SFTSV. The accumulation of cells at the G2/M transition did not affect virus adsorption and entry but did facilitate viral replication. In addition, we found that SFTSV NSs, a nonstructural protein that forms viroplasm-like structures in the cytoplasm of infected cells and promotes virulence by modulating the interferon response, induces a large number of cells to arrest at the G2/M transition by interacting with CDK1. The interaction between NSs and CDK1, which is inclusion body dependent, inhibits formation and nuclear import of the cyclin B1-CDK1 complex, thereby leading to cell cycle arrest. Expression of a CDK1 loss-of-function mutant reversed the inhibitive effect of NSs on the cell cycle, suggesting that this protein is a potential antiviral target. Our study provides new insight into the role of a specific viral protein in SFTSV replication, indicating that NSs induces G2/M arrest of SFTSV-infected cells, which promotes viral replication.IMPORTANCE Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne pathogen that causes severe hemorrhagic fever. Although SFTSV poses a serious threat to public health and was recently isolated, its pathogenesis remains unclear. In particular, the relationship between SFTSV infection and the host cell cycle has not been described. Here, we show for the first time that both asynchronized and synchronized SFTSV-susceptible cells arrest at the G2/M checkpoint following SFTSV infection and that the accumulation of cells at this checkpoint facilitates viral replication. We also identify a key mechanism underlying SFTSV-induced G2/M arrest, in which SFTSV NSs interacts with CDK1 to inhibit formation and nuclear import of the cyclin B1-CDK1 complex, thus preventing it from regulating cell cycle progression. Our study highlights the key role that NSs plays in SFTSV-induced G2/M arrest.

Recent advances in understanding the role of Cdk1 in the Spindle Assembly Checkpoint.

The goal of mitosis is to form two daughter cells each containing one copy of each mother cell chromosome, replicated in the previous S phase. To achieve this, sister chromatids held together back-to-back at their primary constriction, the centromere, have to interact with microtubules of the mitotic spindle so that each chromatid takes connections with microtubules emanating from opposite spindle poles (we will refer to this condition as bipolar attachment). Only once all replicated chromosomes have reached bipolar attachments can sister chromatids lose cohesion with each other, at the onset of anaphase, and move toward opposite spindle poles, being segregated into what will soon become the daughter cell nucleus. Prevention of errors in chromosome segregation is granted by a safeguard mechanism called Spindle Assembly Checkpoint (SAC). Until all chromosomes are bipolarly oriented at the equator of the mitotic spindle, the SAC prevents loss of sister chromatid cohesion, thus anaphase onset, and maintains the mitotic state by inhibiting inactivation of the major M phase promoting kinase, the cyclin B-cdk1 complex (Cdk1). Here, we review recent mechanistic insights about the circuitry that links Cdk1 to the SAC to ensure correct achievement of the goal of mitosis.

CDC6 regulates both G2/M transition and metaphase-to-anaphase transition during the first meiosis of mouse oocytes.

Cell division cycle protein, CDC6, is essential for the initiation of DNA replication. CDC6 was recently shown to inhibit the microtubule-organizing activity of the centrosome. Here, we show that CDC6 is localized to the spindle from pro-metaphase I (MI) to MII stages of oocytes, and it plays important roles at two critical steps of oocyte meiotic maturation. CDC6 depletion facilitated the G2/M transition (germinal vesicle breakdown [GVBD]) through regulation of Cdh1 and cyclin B1 expression and CDK1 (CDC2) phosphorylation in a GVBD-inhibiting culture system containing milrinone. Furthermore, GVBD was significantly decreased after knockdown of cyclin B1 in CDC6-depleted oocytes, indicating that the effect of CDC6 loss on GVBD stimulation was mediated, at least in part, by raising cyclin B1. Knockdown of CDC6 also caused abnormal localization of γ-tubulin, resulting in defective spindles, misaligned chromosomes, cyclin B1 accumulation, and spindle assembly checkpoint (SAC) activation, leading to significant pro-MI/MI arrest and PB1 extrusion failure. These phenotypes were also confirmed by time-lapse live cell imaging analysis. The results indicate that CDC6 is indispensable for maintaining G2 arrest of meiosis and functions in G2/M checkpoint regulation in mouse oocytes. Moreover, CDC6 is also a key player regulating meiotic spindle assembly and metaphase-to-anaphase transition in meiotic oocytes.

Identification of Differentially Expressed Genes (DEGs) Relevant to Prognosis of Ovarian Cancer by Use of Integrated Bioinformatics Analysis and Validation by Immunohistochemistry Assay.

BackgroundThe aim of this study was to investigate the differentially expressed genes (DEGs) relevant to prognosis of ovarian cancer by use of integrated bioinformatics analysis.Material/MethodsThe DEGs between normal ovariy tissue and ovarian cancer tissue were screened in GSE54388, GSE14407, and GSE18520 datasets and the overlapping DEGs were then indentified. GO and KEEG enrichment were performed to analyze the biological functions and pathways of the DEGs. A protein–protein interaction (PPI) network of the identified DEGs was constructed using the STRING database. Differences in prognosis between low and high expression of the hub DEGs were also evaluated using the Kaplan-Meier Plotter database. Protein expression of 2 hub genes – BUB1B and KIF201A – was assessed by immunohistochemistry assay and evaluated with the patient’s clinical pathology characteristics.ResultsWe identified 361 DEGs, mainly involving oncogene-induced cell senescence, cyclin B1-CDK1 complex, protein kinase A catalytic subunit binding, cell cycle, and p53 signaling pathway. Ten hub genes were identified from among the 361 DEGs. The overall survival (OS) and progression-free survival (PFS) of these 10 hub genes were evaluated in the Kaplan-Meier-plotter database. Three (BUB1B, KIF11, and KIF20A) of the 10 hub genes were found to be correlated with ovarian cancer OS and PFS. BUB1B expression level was correlated with ovarian FIGO stage (p<0.05) and tumor differentiation (p<0.05). For KIF20A, the expression level was correlated with FIGO stage (p<0.05) and intraperitoneal metastasis (p<0.05).ConclusionsDEGs can participate in ovarian cancer development and can be used as biomarkers for prognosis. Patients with upregulated BUB1B, KIF11, and KIF20A tend to have worse overall survival and disease-free survival compared with patients who have low expression.

Myc stimulates cell cycle progression through the activation of Cdk1 and phosphorylation of p27

Cell cycle stimulation is a major transforming mechanism of Myc oncoprotein. This is achieved through at least three concomitant mechanisms: upregulation of cyclins and Cdks, downregulation of the Cdk inhibitors p15 and p21 and the degradation of p27. The Myc-p27 antagonism has been shown to be relevant in human cancer. To be degraded, p27 must be phosphorylated at Thr-187 to be recognized by Skp2, a component of the ubiquitination complex. We previously described that Myc induces Skp2 expression. Here we show that not only Cdk2 but Cdk1 phosphorylates p27 at the Thr-187. Moreover, Myc induced p27 degradation in murine fibroblasts through Cdk1 activation, which was achieved by Myc-dependent cyclin A and B induction. In the absence of Cdk2, p27 phosphorylation at Thr-187 was mainly carried out by cyclin A2-Cdk1 and cyclin B1-Cdk1. We also show that Cdk1 inhibition was enough for the synthetic lethal interaction with Myc. This result is relevant because Cdk1 is the only Cdk strictly required for cell cycle and the reported synthetic lethal interaction between Cdk1 and Myc.

Identification of Key Genes and Prognostic Value Analysis in Hepatocellular Carcinoma by Integrated Bioinformatics Analysis.

Emerging evidence indicates that various functional genes with altered expression are involved in the tumor progression of human cancers. This study is aimed at identifying novel key genes that may be used for hepatocellular carcinoma (HCC) diagnosis, prognosis, and targeted therapy. This study included 3 expression profiles (GSE45267, GSE74656, and GSE84402), which were obtained from the Gene Expression Omnibus (GEO). GEO2R was used to analyze the differentially expressed genes (DEGs) between HCC and normal samples. The functional and pathway enrichment analysis was performed by the Database for Annotation, Visualization and Integrated Discovery. A protein-protein interaction (PPI) network of the identified DEGs was constructed using the Search Tool for the Retrieval of Interacting Gene, and hub genes were identified. ONCOMINE and CCLE databases were used to verify the expression of the hub genes in HCC tissues and cells. Kaplan-Meier plotter was used to assess the effects of the hub genes on the overall survival of HCC patients. A total of 99 DEGs were identified from the 3 expression profiles. These DEGs were enriched with functional processes and pathways related to HCC pathogenesis. From the PPI network, 5 hub genes were identified. The expression of the 5 hub genes was all upregulated in HCC tissues and cells compared with the control tissues and cells. Kaplan-Meier survival curves indicated that high expression of cyclin-dependent kinase (CDK1), cyclin B1 (CCNB1), cyclin B2 (CCNB2), MAD2 mitotic arrest deficient-like 1 (MAD2L1), and topoisomerase IIα (TOP2A) predicted poor overall survival in HCC patients (all log-rank P < 0.01). These results revealed that the DEGs may serve as candidate key genes during HCC pathogenesis. The 5 hub genes, including CDK1, CCNB1, CCNB2, MAD2L1, and TOP2A, may serve as promising prognostic biomarkers in HCC.

A squalamine derivative, NV669, as a novel PTP1B inhibitor: in vitro and in vivo effects on pancreatic and hepatic tumor growth.

NV669 is an aminosterol derived from squalamine found to possess strong anticancer effects. The aim of this study was to investigate NV669’s beneficial effects on human pancreatic and hepatic cancer models and to decipher the cellular and molecular mechanisms involved in tumor growth decrease upon treatment with NV669.Pancreatic (BxPC3, MiaPaCa-2) and hepatic (HepG2, Huh7) cancer cells were treated with NV669, and the effects recorded on proliferation, cell cycle and death. Results showed that NV669 inhibited the viability of cancer cells, induced cell cycle arrest and subsequently promoted apoptosis. This was accompanied by a decrease in the expression of cyclin B1 and phosphorylated Cdk1 and by a cleavage of pro-apoptotic caspase-8 and PARP-1. Taken together, our studies showed that NV669 inhibits the proliferation of pancreatic and hepatic cancer cells through the regulation of G2/M phase transition via the cyclin B1-Cdk1 complex. In vitro NV669 inhibits PTP1B activity and FAK expression. NV669 impacts on the expression of adhesion molecules CDH-1, -2 and -3 in BxPC3 and Huh7 lines that form cell monolayers. Consecutively NV669 induces cell detachment. This suggests that NV669 by inhibiting PTP1B induces cell detachment and apoptosis. Subsequently, our in vivo results showed that NV669 inhibited the growth of pancreatic and hepatic tumor xenografts with a significant cell cycle arrest in pre-mitotic phase and an increase of tumor cell apoptosis. Therefore, NV669 may serve as an alternative anticancer agent, used alone or in association with other medications, for the treatment of pancreatic adenocarcinoma and hepatocellular carcinoma.

Orchestration of the spindle assembly checkpoint by CDK1-cyclin B1.

In mitosis, the spindle assembly checkpoint (SAC) monitors the formation of microtubule-kinetochore attachments during capture of chromosomes by the mitotic spindle. Spindle assembly is complete once there are no longer any unattached kinetochores. Here, we will discuss the mechanism and key components of spindle checkpoint signalling. Unattached kinetochores bind the principal spindle checkpoint kinase monopolar spindle 1 (MPS1). MPS1 triggers the recruitment of other spindle checkpoint proteins and the formation of a soluble inhibitor of anaphase, thus preventing exit from mitosis. On microtubule attachment, kinetochores become checkpoint silent due to the actions of PP2A-B56 and PP1. This SAC responsive period has to be coordinated with mitotic spindle formation to ensure timely mitotic exit and accurate chromosome segregation. We focus on the molecular mechanisms by which the SAC permissive state is created, describing a central role for CDK1-cyclin B1 and its counteracting phosphatase PP2A-B55. Furthermore, we discuss how CDK1-cyclin B1, through its interaction with MAD1, acts as an integral component of the SAC, and actively orchestrates checkpoint signalling and thus contributes to the faithful execution of mitosis.

Spatiotemporal control of mitotic exit during anaphase by an aurora B-Cdk1 crosstalk.

According to the prevailing 'clock' model, chromosome decondensation and nuclear envelope reformation when cells exit mitosis are byproducts of Cdk1 inactivation at the metaphase-anaphase transition, controlled by the spindle assembly checkpoint. However, mitotic exit was recently shown to be a function of chromosome separation during anaphase, assisted by a midzone Aurora B phosphorylation gradient - the 'ruler' model. Here we found that Cdk1 remains active during anaphase due to ongoing APC/CCdc20- and APC/CCdh1-mediated degradation of B-type Cyclins in Drosophila and human cells. Failure to degrade B-type Cyclins during anaphase prevented mitotic exit in a Cdk1-dependent manner. Cyclin B1-Cdk1 localized at the spindle midzone in an Aurora B-dependent manner, with incompletely separated chromosomes showing the highest Cdk1 activity. Slowing down anaphase chromosome motion delayed Cyclin B1 degradation and mitotic exit in an Aurora B-dependent manner. Thus, a crosstalk between molecular 'rulers' and 'clocks' licenses mitotic exit only after proper chromosome separation.

Abstract 2539: Discovery of degradation pathway for maternal embryonic leucine zipper kinase (MELK): Implications for breast cancer therapy

Abstract Background: Despite previous studies suggesting that MELK is not involved in proliferation in cancer, many groups have shown that MELK is a critical mediator of radioresistance and may be an ideal target for radiosensitization. Furthermore, additional studies suggest MELK functions to regulate the cell-cycle and that its mRNA and protein levels are regulated during cell-cycle, although the mechanisms of this regulation are currently unknown. We sought to identify mediators of MELK protein regulation, determine whether it was ubiquitin-ligase mediated, and if so, which E3-ubiquitin ligase machinery degrades MELK during cell-cycle progression. Methods: Sequence alignments were carried out to identify degron motifs in MELK. Site directed mutagenesis were performed to mutate degrons in MELK. CDC20 and CDH1 were over-expressed to identify the co-activators of the E3-ubiquitin ligase complex. Cells were synchronized by double thymidine block or by nocodazole. CDC27/APC3 and CDC20 antibodies were used to pull-down the Anaphase Promoting Complex/Cyclosome (APC/C) and Mitotic Checkpoint Complex (MCC), respectively. Resulting pellets were probed with antibodies against BubR1/Mad3, Bub3, CDC20, CDC27, MELK, Cyclin B1, CDK1, BUB1, and Mad2. A role for MELK kinase activity on its half-life and formation of APC/C and MCC was evaluated using kinetically inactive (MELK-KD) mutant or OTSSP167. siRNA against MELK was used to confirm a requirement of MELK for formation of APC/C and MCC. The effect of MELK degron mutant on radiosensitization will be evaluated in MELK CRISPR TNBC cell lines. Results: In this study we provide evidence that MELK has two D-boxes and a KEN box which are largely conserved from C. elegance to H. sapiens. We identified that MELK is degraded by the E3-ubiquitin ligase APC/C with the coactivator being CDC20. Rescue with MG132 confirms that MELK is degraded by a proteosomal-mediated pathway. CO-IP studies show that MELK is required for APC/C and MCC formation and that MELK interacts with CDC20, CDK1, BUB1, and BUBR1. Cycloheximide chase studies show D-Box1 and KEN box mutants of MELK are more stable while D-BOX2 mutants are least stable. The KEN and D-Box mutant MELK show impairment in APC/C and MCC formation, altered Cyclin B1-CDK1 and Cyclin B1-CDC20 interactions. MELK KD and OTSSP prove that kinase activity of MELK plays a role in its stability. We anticipate that the least stable MELK mutant will result in higher radiosensitivity compared to MELK-WT. Conclusions: MELK is regulated by APC/CCDC20 through its D-Box1, KEN and D-Box2. Degron mutant and MELK KD show altered protein stability which leads to attenuated APC/C and MCC formation. These results provide proof that MELK is a cell-cycle checkpoint kinase and support the rationale for developing clinical strategies to degrade and inhibit MELK in combination with radiation therapy. Citation Format: Shyam Nyati, Benjamin Chandler, Anna Michmerhuizen, Andrea Pesch, Cassandra Ritter, Leah Moubadder, Meilan Liu, Meleah Cameron, Eric Olsson, Kari Wilder-Romans, Dipankar Ray, Theodore S. Lawrence, Felix Y. Feng, Lori J. Pierce, Corey Speers. Discovery of degradation pathway for maternal embryonic leucine zipper kinase (MELK): Implications for breast cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2539.

Wee1 Rather Than Plk1 Is Inhibited by AZD1775 at Therapeutically Relevant Concentrations.

Wee1 kinase is an inhibitor of cyclin-dependent kinase (cdk)s, crucial cell cycle progression drivers. By phosphorylating cdk1 at tyrosine 15, Wee1 inhibits activation of cyclin B-cdk1 (Cdk1), preventing cells from entering mitosis with incompletely replicated or damaged DNA. Thus, inhibiting Wee1, alone or in combination with DNA damaging agents, can kill cancer cells by mitotic catastrophe, a tumor suppressive response that follows mitosis onset in the presence of under-replicated or damaged DNA. AZD1775, an orally available Wee1 inhibitor, has entered clinical trials for cancer treatment following this strategy, with promising results. Recently, however, AZD1775 has been shown to inhibit also the polo-like kinase homolog Plk1 in vitro, casting doubts on its mechanism of action. Here we asked whether, in the clinically relevant concentration range, AZD1775 inhibited Wee1 or Plk1 in transformed and non-transformed human cells. We found that in the clinically relevant, nanomolar, concentration range AZD1775 inhibited Wee1 rather than Plk1. In addition, AZD1775 treatment accelerated mitosis onset overriding the DNA replication checkpoint and hastened Plk1-dependent phosphorylation. On the contrary selective Plk1 inhibition exerted opposite effects. Thus, at therapeutic concentrations, AZD1775 inhibited Wee1 rather than Plk1. This information will help to better interpret results obtained by using AZD1775 both in the clinical and experimental settings and provide a stronger rationale for combination therapies.

Mitotic CDK Promotes Replisome Disassembly, Fork Breakage, and Complex DNA Rearrangements

DNA replication errors generate complex chromosomal rearrangements and thereby contribute to tumorigenesis and other human diseases. One mechanism that triggers these errors is mitotic entry before the completion of DNA replication. To address how mitosis might affect DNA replication, we used Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive interphase egg extracts into a mitotic state, the replicative CMG (CDC45/MCM2-7/GINS) helicase undergoes ubiquitylation on its MCM7 subunit, dependent on the E3 ubiquitin ligase TRAIP. Whether replisomes have stalled or undergone termination, CMG ubiquitylation is followed by its extraction from chromatin by the CDC48/p97 ATPase. TRAIP-dependent CMG unloading during mitosis is also seen in C.elegans early embryos. At stalled forks, CMG removal results in fork breakage and end joining events involving deletions and templated insertions. Our results identify a mitotic pathway of global replisome disassembly that can trigger replication fork collapse and DNA rearrangements.