Cyclin Partners Research Articles

Abstract 2489: Mre11-CDK2 interaction during the DNA double-strand break response

Abstract The MRN (Mre11/Rad50/NBS1) complex is a group of highly conserved proteins central to the detection and repair of DNA double-strand breaks (DSBs). Inherited deficiencies in DNA DSB signaling result in a spectrum of disorders featuring cancer predisposition, neurodegeneration, and immunodeficiency. MRN directly binds DNA DSB ends and facilitates activation of ataxia-telangiectasia mutated (ATM) kinase, which initiates cellular responses including DNA repair, cell cycle checkpoints, and in some circumstances, apoptosis. Cellular responses to DNA DSBs require rapid communication between DNA repair complexes and the cell cycle machinery, but much about this relationship remains unclear. Our lab recently discovered an interaction between Mre11 and Cyclin-dependent kinase 2 (CDK2), a key component of the cell cycle machinery (Buis et al., NSMB, 2012). CDK2, when bound to its regulatory partner Cyclin A, promotes cell cycle progression through S phase. We demonstrated that the Mre11-CDK2/Cyclin A interaction is important in regulating the capacity for DNA repair by homologous recombination in normally dividing cells. Current work is focused on understanding the role of this interaction during DNA damage responses. We hypothesize that Mre11 interaction with CDK2 is a component of a molecular switch between the normal cell cycle and the DNA damage response. To test our hypothesis, we take advantage of murine alleles of Mre11 previously generated in the lab. We performed immunoprecipitation studies to investigate the protein-protein interaction in the presence of DNA damage. We also utilized kinase inhibitors to investigate the role of apical kinases in regulating the interaction. Finally, we are currently optimizing in vitro kinase assays to further understand a relationship between Mre11 and CDK2 enzymatic activity.Unpublished findings reveal that the Mre11-CDK2/Cyclin A interaction is altered upon DNA damage, a process which appears to be important for cell cycle checkpoint regulation. In conclusion, we demonstrate that the Mre11-CDK2 interaction plays important roles both in normally dividing cells and in the DNA damage response. Taken together, our data suggests that Mre11 may act to bridge the gap between DNA repair complexes and the cell cycle machinery. Citation Format: Mary J. Morgan, Todd A. Festerling, Jeffrey Buis, David O. Ferguson. Mre11-CDK2 interaction during the DNA double-strand break response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2489. doi:10.1158/1538-7445.AM2017-2489

Cyclin C influences the timing of mitosis in fission yeast.

The multiprotein Mediator complex is required for the regulated transcription of nearly all RNA polymerase II-dependent genes. Mediator contains the Cdk8 regulatory subcomplex, which directs periodic transcription and influences cell cycle progression in fission yeast. Here we investigate the role of CycC, the cognate cyclin partner of Cdk8, in cell cycle control. Previous reports suggested that CycC interacts with other cellular Cdks, but a fusion of CycC to Cdk8 reported here did not cause any obvious cell cycle phenotypes. We find that Cdk8 and CycC interactions are stabilized within the Mediator complex and the activity of Cdk8-CycC is regulated by other Mediator components. Analysis of a mutant yeast strain reveals that CycC, together with Cdk8, primarily affects M-phase progression but mutations that release Cdk8 from CycC control also affect timing of entry into S phase.

Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16.

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has a unique consensus substrate phosphorylation motif compared with conventional CDKs. To elucidate the structure and inhibitor-binding properties of this atypical CDK, we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multitargeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity, suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts.

Inhibition of CDK8 mediator kinase suppresses estrogen dependent transcription and the growth of estrogen receptor positive breast cancer.

Hormone therapy targeting estrogen receptor (ER) is the principal treatment for ER-positive breast cancers. However, many cancers develop resistance to hormone therapy while retaining ER expression. Identifying new druggable mediators of ER function can help to increase the efficacy of ER-targeting drugs. Cyclin-dependent kinase 8 (CDK8) is a Mediator complex-associated transcriptional regulator with oncogenic activities. Expression of CDK8, its paralog CDK19 and their binding partner Cyclin C are negative prognostic markers in breast cancer. Meta-analysis of transcriptome databases revealed an inverse correlation between CDK8 and ERα expression, suggesting that CDK8 could be functionally associated with ER. We have found that CDK8 inhibition by CDK8/19-selective small-molecule kinase inhibitors, by shRNA knockdown or by CRISPR/CAS9 knockout suppresses estrogen-induced transcription in ER-positive breast cancer cells; this effect was exerted downstream of ER. Estrogen addition stimulated the binding of CDK8 to the ER-responsive GREB1 gene promoter and CDK8/19 inhibition reduced estrogen-stimulated association of an elongation-competent phosphorylated form of RNA Polymerase II with GREB1. CDK8/19 inhibitors abrogated the mitogenic effect of estrogen on ER-positive cells and potentiated the growth-inhibitory effects of ER antagonist fulvestrant. Treatment of estrogen-deprived ER-positive breast cancer cells with CDK8/19 inhibitors strongly impeded the development of estrogen independence. In vivo treatment with a CDK8/19 inhibitor Senexin B suppressed tumor growth and augmented the effects of fulvestrant in ER-positive breast cancer xenografts. These results identify CDK8 as a novel downstream mediator of ER and suggest the utility of CDK8 inhibitors for ER-positive breast cancer therapy.

There will be blood: hematopoiesis control by mediator subunit MED12.

Hematopoiesis is sustained by the presence of hematopoietic stem cells (HSCs) that balance self-renewal and differentiation through controlled expression of enhancer-dependent transcriptional programs enforced by lineage-specific master transcription factors. The regulatory potential of these cardinal transcription factors is often realized with the assistance of Mediator, a conserved multiprotein interface between distal, enhancer-bound regulators and the RNA polymerase II machinery assembled on core promoters. Within Mediator, CDK8 and its partner Cyclin C (CycC) combine with MED12 and MED13 to form a 4-subunit kinase module that variably associates with a 26-subunit Mediator core (1). MED12, which anchors and activates CycC-CDK8 in Mediator, has previously been implicated in hematopoiesis through forward genetic and mutational screening in zebrafish embryos and human cancers, respectively, revealing links to both myelopoiesis and leukemia (2,3). However, the molecular basis for MED12 in these biological and pathological processes is unclear. Against this backdrop, the Aifantis laboratory recently undertook to more rigorously investigate the role of MED12 in the hematopoietic system (4).

Progress with palbociclib in breast cancer: latest evidence and clinical considerations.

Deregulation of the cell cycle is a hallmark of cancer, and research on cell cycle control has allowed identification of potential targets for anticancer treatment. Palbociclib is a selective inhibitor of the cyclin-dependent kinases 4 and 6 (CDK4/6), which are involved, with their coregulatory partners cyclin D, in the G1-S transition. Inhibition of this step halts cell cycle progression in cells in which the involved pathway, including the retinoblastoma protein (Rb) and the E2F family of transcription factors, is functioning, although having been deregulated. Among breast cancers, those with functioning cyclin D-CDK4/6-Rb-E2F are mainly hormone-receptor (HR) positive, with some HER2-positive and rare triple-negative cases. Deregulation results from genetic or otherwise occurring hyperactivation of molecules subtending cell cycle progression, or inactivation of cell cycle inhibitors. Based on results of randomized clinical trials, palbociclib was granted accelerated approval by the US Food and Drug Administration (FDA) for use in combination with letrozole as initial endocrine-based therapy for metastatic disease in postmenopausal women with HR-positive, HER2-negative breast cancer, and was approved for use in combination with fulvestrant in women with HR-positive, HER2-negative advanced breast cancer with disease progression following endocrine therapy. This review provides an update of the available knowledge on the cell cycle and its regulation, on the alterations in cyclin D-CDK4/6-Rb-E2F axis in breast cancer and their roles in endocrine resistance, on the preclinical activity of CDK4/6 inhibitors in breast cancer, both as monotherapy and as partners of combinatorial synergic treatments, and on the clinical development of palbociclib in breast cancer.

Distinct and Overlapping Requirements for Cyclins A, B, and B3 in Drosophila Female Meiosis.

Meiosis, like mitosis, depends on the activity of the cyclin dependent kinase Cdk1 and its cyclin partners. Here, we examine the specific requirements for the three mitotic cyclins, A, B, and B3 in meiosis of Drosophila melanogaster. We find that all three cyclins contribute redundantly to nuclear envelope breakdown, though cyclin A appears to make the most important individual contribution. Cyclin A is also required for biorientation of homologs in meiosis I. Cyclin B3, as previously reported, is required for anaphase progression in meiosis I and in meiosis II. We find that it also plays a redundant role, with cyclin A, in preventing DNA replication during meiosis. Cyclin B is required for maintenance of the metaphase I arrest in mature oocytes, for spindle organization, and for timely progression through the second meiotic division. It is also essential for polar body formation at the completion of meiosis. With the exception of its redundant role in meiotic maturation, cyclin B appears to function independently of cyclins A and B3 through most of meiosis. We conclude that the three mitotic cyclin-Cdk complexes have distinct and overlapping functions in Drosophila female meiosis.

Cdk7 mediates RPB1-driven mRNA synthesis in Toxoplasma gondii.

Cyclin-dependent kinase 7 in conjunction with CyclinH and Mat1 activates cell cycle CDKs and is a part of the general transcription factor TFIIH. Role of Cdk7 is well characterized in model eukaryotes however its relevance in protozoan parasites has not been investigated. This important regulator of key processes warrants closer examination particularly in this parasite given its unique cell cycle progression and flexible mode of replication. We report functional characterization of TgCdk7 and its partners TgCyclinH and TgMat1. Recombinant Cdk7 displays kinase activity upon binding its cyclin partner and this activity is further enhanced in presence of Mat1. The activated kinase phosphorylates C-terminal domain of TgRPB1 suggesting its role in parasite transcription. Therefore, the function of Cdk7 in CTD phosphorylation and RPB1 mediated transcription was investigated using Cdk7 inhibitor. Unphosphorylated CTD binds promoter DNA while phosphorylation by Cdk7 triggers its dissociation from DNA with implications for transcription initiation. Inhibition of Cdk7 in the parasite led to strong reduction in Serine 5 phosphorylation of TgRPB1-CTD at the promoters of constitutively expressed actin1 and sag1 genes with concomitant reduction of both nascent RNA synthesis and 5′-capped transcripts. Therefore, we provide compelling evidence for crucial role of TgCdk7 kinase activity in mRNA synthesis.

Abstract 4337: Novel potent and selective orally available CDK8/19 kinase inhibitors

Abstract CDK8 and its paralog CDK19 are cyclin-dependent kinases and together with CDK7 and CDK9 belongs to the group of C-terminal domain (CTD) kinases that phosphorylate the CTD of RNA polymerase II, thus regulating transcription. CDK8 together with its partner Cyclin C, MED12 and MED13 are components of multi-protein Mediator complex which couples action of transcription factors with the molecular machinery that carries out transcription, e.g. CDK8 couple basal transcriptional machinery to sequence-specific transcription factors such as Notch, p53, β-catenin, and also repress the transcription of other genes. As Mediator independent roles, CDK8 has been shown to act as part of a separate complex behaving as a histone kinase. Moreover, CDK8 has been identified as a major kinase in the response to IFN signaling mediated STAT1-S727 phosphorylation. Several studies indicated that high overexpression and activity of CDK8 could be a driver of malignant progression in colorectal cancer being a marker of poor prognosis. Moreover, in gastric cancer CDK8 expression and the delocalization of β-catenin expression showed a significant positive correlation with carcinogenesis and tumor progression, especially lymph node metastasis. Recently, gene amplification of CDK8, CDK19, CCNC and MED13 in breast cancers has been related with poor response to adjuvant therapy. These results suggest that CDK8 inhibitors may become a unique class of anticancer drugs that could increase the efficacy of cancer therapy by blocking chemotherapy-induced production of tumor-promoting secreted factors. We have carried out a medium throughput screening campaign which led to the discovery of several low nanomolar hits belonging to 3 different chemical series. Later on, we have performed a Hit Generation phase, designing novel inhibitors within a patentable chemical space. Here, we have taken into account information from the chemical structures of the screening hits, X-ray structure of CDK8 protein co-crystalized with sorafenib, docking studies, chemical feasibility and intellectual property. We have identified a novel chemical series of CDK8/19 inhibitors. After a HtL exploration we have reached lead compounds with potency in the picomolar range and high selectivity versus a panel of 468 protein kinases. The leads display cellular activity by blocking β−catenin reporter activity and STAT1 phoshorylation in the low nanomolar range. Lead compounds have been screened in a panel of tumoral cell lines, showing a defined profile in terms of sensitivity with GI50s which range from nanomolar to low micromolar values. These inhibitors induce cell death in a dose response manner. The novel chemical series show a drug-like profile in terms of solubility, permeability, CYP450 inhibition and hERG. The identified lead compounds are orally bioavailable, with good clearance, volume of distribution and exposure levels. A selected lead compound has been used in in vivo PK-PD studies showing positive results. Citation Format: Carmen Blanco Aparicio, Oliver Renner, Elena Gomez-Casero, Maria Isabel Albarran, Antonio Cebria, Borja Barrera, Enara Aguirre, Maria del Carmen Rodriguez de Miguel, Manuel Urbano, Ana Isabel Hernandez, Cristina Gomez de la Oliva, Virginia Rivero, Rosario Concepcion Riesco, Sonia Martinez Gonzalez, Joaquin Pastor. Novel potent and selective orally available CDK8/19 kinase inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4337.

Erratum to: Controlling the response to DNA damage by the APC/C-Cdh1.

Reproduction depends on successful cell division, which is coordinated in the cell cycle. Especially in the context of multicellular organisms, proper control of cell cycle initiation and completion is essential for successful development and homeostasis. Research in the past decades has revealed how cell cycle progression is coordinated by various pathways, which show extensive feedback loops and display multiple levels of cross-talk. Despite this complexity, the core of the cell cycle machinery is constituted by a highly evolutionary conserved molecular ‘engine’, called the cyclin/cyclin-dependent kinase (CDK) complex [1]. Unicellular eukaryotic organisms, such as the budding yeast S. cerevisiae, only have limited genes encoding cyclins and CDKs. In contrast, multicellular organisms, including mammals, express multiple different CDKs, which are often able to bind more than one cyclin, giving rise to a range of distinct cyclin-CDK complexes, each of their activities characterizing discrete phases of the cell cycle. The activity of CDKs is regulated on multiple transcriptional and post-translational levels. CDK activity is, for instance, extensively controlled through activating (e.g. by the CDK-activating kinase (CAK) [2, 3]) and inhibitory phosphorylation (e.g. by the Wee1 and Myt1 kinases [4–6]. However, probably the most important regulatory layer of oscillating CDK activity relates to the controlled production and down-regulation of their cyclin partners, as CDKs are typically only active when bound to a cyclin. The controlled production and down-regulation of cyclins also holds the key as to how the cell cycle can only progress in a unidirectional fashion, and how S-phase and mitosis are limited to once per cell cycle [7]. The timely destruction of cyclin proteins is accounted for by ubiquitin ligation and ensuing degradation by the 26S proteasome [8]. Ubiquitination of mitotic A- and B-type cyclins is accounted for by the anaphase-promoting complex/cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase. During S and G2 phase of the cell cycle, the APC/C remains inactive, which allows the gradual accumulation of mitotic cyclins [9, 10]. Once cells have entered mitosis and have properly aligned their chromosomes, the APC/C is activated and ubiquitinates—among other substrates—mitotic cyclins, and thereby constitutes an important part of the mitotic exit machinery, allowing cells to complete cell division. Through this mechanism, the APC/C forms an integral part of the machinery that ensures periodicity of the cell cycle [8]. Recent evidence has shown that the APC/C also performs additional functions, for instance in response to DNA damage. In this review, we provide a short background on the APC/C and the cellular response to DNA damage. Subsequently, we summarize the current literature on APC/C-Cdh1 activation after DNA damage, and how this affects DNA repair, checkpoint duration and cell fate.

TP-2Rho Is a Sensitive Solvatochromic Red-Shifted Probe for Monitoring the Interactions between CDK4 and Cyclin D.

Understanding the intricate steps of protein kinase regulation requires characterization of protein-protein interactions between the catalytic subunit, its regulatory partners and the substrate. Fluorescent probes are useful tools with which to study such interactions and to gain insight into their affinities and specificities. Solvatochromic probes, which display changes in their fluorescence emission in response to changes in the polarity of the medium, are particularly attractive. Here we describe conjugation of a switchable fluorescent dye, TP-2Rho, to peptide and protein derivatives of cyclin-dependent kinase 4 (CDK4) and its application to characterization of the interactions between the catalytic subunit of this kinase, its regulatory partner cyclin D1 and a peptide substrate. We demonstrate the sensitivity of TP-2Rho in relation to of those other dyes used for monitoring peptide-protein and protein-protein interactions. Moreover, we show that TP-Rho-labelled peptides can be introduced into living cells to probe endogenous CDK4/cyclin D.

Pharmacological cyclin dependent kinase inhibitors: Implications for colorectal cancer.

Colorectal cancer accounts for a significant proportion of cancer deaths worldwide. The need to develop more chemotherapeutic agents to combat this disease is critical. Cyclin dependent kinases (CDKs), along with its binding partner cyclins, serve to control the growth of cells through the cell cycle. A new class of drugs, termed CDK inhibitors, has been studied in preclinical and now clinical trials. These inhibitors are believed to act as an anti-cancer drug by blocking CDKs to block the uncontrolled cellular proliferation that is hallmark of cancers like colorectal cancer. CDK article provides overview of the emerging drug class of CDK inhibitors and provides a list of ones that are currently in clinical trials.

Cyclic Dependent Kinase (CDK): Role in Cancer Pathogenesis and as Drug Target in Cancer Therapeutics

The cell cycle is the process by which mammalian cells regulate proliferation and has S, M, G2 and G1 phase. Loss of these cell-cycle control and increased resistance to apoptosis (programmed cell death) represent major hallmarks of cancer. Cyclic dependent kinases (CDKs), a family of serine/threonine, can control the cell cycle progression and transcription. Besides, they are also involved in regulating mRNA processing, the differentiation of nerve cells and glucose homeostasis. Therefore, CDKs are multiple function proteins. Cellular proliferation, driven by CDKs and their cyclin partners, is decontrolled in cancer; therefore, cancer is considered as a proliferative disorder and targeting the cell cycle, therefore, seems to be a good strategy for new targeted anticancer agents. CDKs activity is closely associated with specific cyclin co-factors and at least 12 separate genetic loci are known to code for the CDKs. Therefore, cyclins are considered to be the gears that are changed to aid the transition between cycle phases. CDKs are generally classified into two major groups, based on whether they control cell cycle progression which includes CDK1 to CDK6 or regulate gene transcription by RNAPII that includes CDK 7, CDK8, CDK9 and CDK19. Increases in level of CDKs are observed in cancer. Inhibition of CDKs, which are the key regulators of the cell-cycle progression and RNA transcription, represents a good strategy for cancer drug discovery and development as well as therapy. This review was briefly described the above-mentioned possible roles of CDKs in the physiological and pathological mechanisms of cancer, further discussing recent advances and challenges in CDKs as a therapeutic target.

Abstract LB-B22: CCNY2 promotes lung cancer cell migration and invasion via regulating F-actin expression

Abstract Background: Cyclin Y (CCNY) is a novel cyclin and is highly conserved in metazoan species. Cyclin Y mRNA has several transcripts and only ccny1 and ccny2 has been documented. A potential CDK partner of Cyclin Y is PFTAIRE kinase (PFTK1). In hepatocellular carcinoma, cell motility and invasion was enhanced by PFTK1 expression. But the function of CCNY1 and CCNY2 on cell migration and invasiveness has not been reported yet. Methods: Recombined plasmids carrying CCNY1 and CCNY2 were constructed and transfected to H1299 cells to obtain CCNY up-regulation cells. A lentivirus-based RNAi delivery system was used to inhibit CCNY mRNA expression. The role of CCNY in cell motility and invasion was investigated using wound healing and transwell assay. The protein levels in lung cancer cells were determined by western-blot, immunofluorescence technique and high-content cell analysis. Mouse xenograft experiments were carried out to study the metastasis ability of CCNY1 and CCNY2 in vivo. Immunohistochemistry was used to detect the CCNY protein level of lung cancer tissues. Results: cell motility and invasion activity were inhibited and MET (Mesenchymal - Epithelial Transition) was caused by down-regulating of CCNY in 95D and H1299 cells. CCNY2 could enhance cell migration and invasion activity in vivo and vitro. The F-actin level was regulated by CCNY2 expression. In non-small cell lung cancer tissues, CCNY2 was highly expressed and the CCNY2 expression was associated with histological grade. Conclusion: CCNY2 was firstly detected in lung cancer cells and non-small cell lung cancer tissues. Our findings demonstrated CCNY2 not CCNY1 promoted cell motility and invasion by regulating the expression of F-actin and modulating intracellular cytoskeletal components. Citation Format: xiaoting zhao, Mei Jiang, wentao Yue. CCNY2 promotes lung cancer cell migration and invasion via regulating F-actin expression. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B22.

Conformational Equilibrium of CDK/Cyclin Complexes by Molecular Dynamics with Excited Normal Modes

Cyclin-dependent kinases (CDKs) and their associated regulatory cyclins are central for timely regulation of cell-cycle progression. They constitute attractive pharmacological targets for development of anticancer therapeutics, since they are frequently deregulated in human cancers and contribute to sustained, uncontrolled tumor proliferation. Characterization of their structural/dynamic features is essential to gain in-depth insight into structure-activity relationships. In addition, the identification of druggable pockets or key intermediate conformations yields potential targets for the development of novel classes of inhibitors. Structural studies of CDK2/cyclin A have provided a wealth of information concerning monomeric/heterodimeric forms of this kinase. There is, however, much less structural information for other CDK/cyclin complexes, including CDK4/cyclin D1, which displays an alternative (open) position of the cyclin partner relative to CDK, contrasting with the closed CDK2/cyclin A conformation. In this study, we carried out normal-mode analysis and enhanced sampling simulations with our recently developed method, molecular dynamics with excited normal modes, to understand the conformational equilibrium on these complexes. Interestingly, the lowest-frequency normal mode computed for each complex described the transition between the open and closed conformations. Exploration of these motions with an explicit-solvent representation using molecular dynamics with excited normal modes confirmed that the closed conformation is the most stable for the CDK2/cyclin A complex, in agreement with their experimentally available structures. On the other hand, we clearly show that an open↔closed equilibrium may exist in CDK4/cyclin D1, with closed conformations resembling that captured for CDK2/cyclin A. Such conformational preferences may result from the distinct distributions of frustrated contacts in each complex. Using the same approach, the putative roles of the Thr160 phosphoryl group and the T-loop conformation were investigated. These results provide a dynamic view of CDKs revealing intermediate conformations not yet characterized for CDK members other than CDK2, which will be useful for the design of inhibitors targeting critical conformational transitions.

Abstract 2895: Phosphorylation of EZH2 at T416 by CDK2 promotes development of mammary tumors with basal-like phenotype

Abstract Basal-like breast cancer, also referred to as triple negative breast cancer (TNBC) due to the lack of expression of ErbB2, PR, and ER alpha receptors, is considered to be one of the most aggressive breast cancers, which display high rates of tumor cell proliferation, metastasis, and post-surgical re-occurrence. This subtype of breast cancer presents a major challenge for breast cancer oncologists as currently there is no effective therapy available. Here, we show that Cyclin E and EZH2, the methyltransferase component of the polycomb repressive complex 2 (PRC2) are exclusively co-expressed in TNBC tumor tissues. We further show that CDK2, the only known enzymatic partner of Cyclin E, can phosphorylate EZH2 at T416 in vivo. CDK2-mediated modification of EZH2 at T416 leads to gain-of-function, enhancing its ability to promote breast cancer cell migration/invasion, expansion and self-renewal of tumor sphere in vitro, and tumor growth in vivo. In addition, our immunohistochemistry-staining of 247 primary breast tumor tissues validates that high EZH2/T416 phosphorylation correlates with poor survival in TNBC but not in non-TNBC patients. To further attest the oncogenic role of EZH2/T416 phosphorylation by CDK2 in vivo, we generated transgenic mice expressing EZH2T416D mutant, a mimicking phosphorylated form of EZH2, in mammary glands. In contrast to previously reported EZH2WT transgenic mice, which did not develop mammary tumors, our EZH2T416D-expressing mice developed malignant mammary tumor with basal-like and highly metastatic potential phenotypes. These genetic evidence revealed that CDK2/Cyclin E mediated site-specific phosphorylation of EZH2 is capable of inducing the basal-like/TNBC phenotype and maintaining cancer stem cell population of breast cancers so that the T416 phosphorylation of EZH2 might serve as a biomarker for TNBC prognosis and effective therapy. Citation Format: Lei Nie, Yongkun Wei, Adam LaBaff, Cheng-Chieh Yang, Weiya Xia, Longfei Huo, Dongping Liu, Yi-Hsin Hsu, Gabriel N. Hortobagyi, Jun Yao, Celina G. Kleer, Mien-Chie Hung. Phosphorylation of EZH2 at T416 by CDK2 promotes development of mammary tumors with basal-like phenotype. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2895. doi:10.1158/1538-7445.AM2015-2895

CDK8-Cyclin C Mediates Nutritional Regulation of Developmental Transitions through the Ecdysone Receptor in Drosophila.

The steroid hormone ecdysone and its receptor (EcR) play critical roles in orchestrating developmental transitions in arthropods. However, the mechanism by which EcR integrates nutritional and developmental cues to correctly activate transcription remains poorly understood. Here, we show that EcR-dependent transcription, and thus, developmental timing in Drosophila, is regulated by CDK8 and its regulatory partner Cyclin C (CycC), and the level of CDK8 is affected by nutrient availability. We observed that cdk8 and cycC mutants resemble EcR mutants and EcR-target genes are systematically down-regulated in both mutants. Indeed, the ability of the EcR-Ultraspiracle (USP) heterodimer to bind to polytene chromosomes and the promoters of EcR target genes is also diminished. Mass spectrometry analysis of proteins that co-immunoprecipitate with EcR and USP identified multiple Mediator subunits, including CDK8 and CycC. Consistently, CDK8-CycC interacts with EcR-USP in vivo; in particular, CDK8 and Med14 can directly interact with the AF1 domain of EcR. These results suggest that CDK8-CycC may serve as transcriptional cofactors for EcR-dependent transcription. During the larval–pupal transition, the levels of CDK8 protein positively correlate with EcR and USP levels, but inversely correlate with the activity of sterol regulatory element binding protein (SREBP), the master regulator of intracellular lipid homeostasis. Likewise, starvation of early third instar larvae precociously increases the levels of CDK8, EcR and USP, yet down-regulates SREBP activity. Conversely, refeeding the starved larvae strongly reduces CDK8 levels but increases SREBP activity. Importantly, these changes correlate with the timing for the larval–pupal transition. Taken together, these results suggest that CDK8-CycC links nutrient intake to developmental transitions (EcR activity) and fat metabolism (SREBP activity) during the larval–pupal transition.

Two Distinct Cdc2 Pools Regulate Cell Cycle Progression and the DNA Damage Response in the Fission Yeast S.pombe

The activity of Cdc2 (CDK1) kinase, which coordinates cell cycle progression and DNA break repair, is blocked upon its phosphorylation at tyrosine 15 (Y15) by Wee1 kinase in the presence of DNA damage. How Cdc2 can support DNA repair whilst being inactivated by the DNA damage checkpoint remains to be explained. Human CDK1 is phosphorylated by Myt1 kinase at threonine 14 (T14) close to its ATP binding site before being modified at threonine 161 (T167Sp) in its T-loop by the CDK-activating kinase (CAK). While modification of T161 promotes association with the cyclin partner, phosphorylation of T14 inhibits the CDK1-cyclin complex. This inhibition is further enforced by the modification of Y15 by Wee1 in the presence of DNA lesions. In S.pombe, the dominant inhibition of Cdc2 is provided by the phosphorylation of Y15 and only a small amount of Cdc2 is modified at T14 when cells are in S phase. Unlike human cells, both inhibitory modifications are executed by Wee1. Using the novel IEFPT technology, which combines isoelectric focusing (IEF) with Phos-tag SDS electrophoresis (PT), we report here that S.pombe Cdc2 kinase exists in seven forms. While five forms are phosphorylated, two species are not. Four phospho-forms associate with cyclin B (Cdc13) of which only two are modified at Y15 by Wee1. Interestingly, only one Y15-modified species carries also the T14 modification. The fifth phospho-form has a low affinity for cyclin B and is neither Y15 nor T14 modified. The two unphosphorylated forms may contribute directly to the DNA damage response as only they associate with the DNA damage checkpoint kinase Chk1. Interestingly, cyclin B is also present in the unphosphorylated pool. We also show that the G146D mutation in Cdc2.1w, which renders Cdc2 insensitive to Wee1 inhibition, is aberrantly modified in a Wee1-dependent manner. In conclusion, our work adds support to the idea that two distinct Cdc2 pools regulate cell cycle progression and the response to DNA damage.

Characterization of human cyclin-dependent kinase 12 (CDK12) and CDK13 complexes in C-terminal domain phosphorylation, gene transcription, and RNA processing.

Cyclin-dependent kinase 9 (CDK9) and CDK12 have each been demonstrated to phosphorylate the RNA polymerase II C-terminal domain (CTD) at serine 2 of the heptad repeat, both in vitro and in vivo. CDK9, as part of P-TEFb and the super elongation complex (SEC), is by far the best characterized of CDK9, CDK12, and CDK13. We employed both in vitro and in vivo assays to further investigate the molecular properties of CDK12 and its paralog CDK13. We isolated Flag-tagged CDK12 and CDK13 and found that they associate with numerous RNA processing factors. Although knockdown of CDK12, CDK13, or their cyclin partner CCNK did not affect the bulk CTD phosphorylation levels in HCT116 cells, transcriptome sequencing (RNA-seq) analysis revealed that CDK12 and CDK13 losses in HCT116 cells preferentially affect expression of DNA damage response and snoRNA genes, respectively. CDK12 and CDK13 depletion also leads to a loss of expression of RNA processing factors and to defects in RNA processing. These findings suggest that in addition to implementing CTD phosphorylation, CDK12 and CDK13 may affect RNA processing through direct physical interactions with RNA processing factors and by regulating their expression.

Spatial reorganization of the endoplasmic reticulum during mitosis relies on mitotic kinase cyclin A in the early Drosophila embryo.

Mitotic cyclin-dependent kinase with their cyclin partners (cyclin:Cdks) are the master regulators of cell cycle progression responsible for regulating a host of activities during mitosis. Nuclear mitotic events, including chromosome condensation and segregation have been directly linked to Cdk activity. However, the regulation and timing of cytoplasmic mitotic events by cyclin:Cdks is poorly understood. In order to examine these mitotic cytoplasmic events, we looked at the dramatic changes in the endoplasmic reticulum (ER) during mitosis in the early Drosophila embryo. The dynamic changes of the ER can be arrested in an interphase state by inhibition of either DNA or protein synthesis. Here we show that this block can be alleviated by micro-injection of Cyclin A (CycA) in which defined mitotic ER clusters gathered at the spindle poles. Conversely, micro-injection of Cyclin B (CycB) did not affect spatial reorganization of the ER, suggesting CycA possesses the ability to initiate mitotic ER events in the cytoplasm. Additionally, RNAi-mediated simultaneous inhibition of all 3 mitotic cyclins (A, B and B3) blocked spatial reorganization of the ER. Our results suggest that mitotic ER reorganization events rely on CycA and that control and timing of nuclear and cytoplasmic events during mitosis may be defined by release of CycA from the nucleus as a consequence of breakdown of the nuclear envelope.