Multiple Cyclin-dependent Kinases Research Articles

The Malaria Parasite Cyclin H Homolog PfCyc1 Is Required for Efficient Cytokinesis in Blood-Stage Plasmodium falciparum.

ABSTRACTAll well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs), and these protein kinase complexes are viable drug targets. The regulatory control of the Plasmodium falciparum cell division cycle remains poorly understood, and the roles of the various CDKs and cyclins remain unclear. The P. falciparum genome contains multiple CDKs, but surprisingly, it does not contain any sequence-identifiable G1-, S-, or M-phase cyclins. We demonstrate that P. falciparum Cyc1 (PfCyc1) complements a G1 cyclin-depleted Saccharomyces cerevisiae strain and confirm that other identified malaria parasite cyclins do not complement this strain. PfCyc1, which has the highest sequence similarity to the conserved cyclin H, cannot complement a temperature-sensitive yeast cyclin H mutant. Coimmunoprecipitation of PfCyc1 from P. falciparum parasites identifies PfMAT1 and PfMRK as specific interaction partners and does not identify PfPK5 or other CDKs. We then generate an endogenous conditional allele of PfCyc1 in blood-stage P. falciparum using a destabilization domain (DD) approach and find that PfCyc1 is essential for blood-stage proliferation. PfCyc1 knockdown does not impede nuclear division, but it prevents proper cytokinesis. Thus, we demonstrate that PfCyc1 has a functional divergence from bioinformatic predictions, suggesting that the malaria parasite cell division cycle has evolved to use evolutionarily conserved proteins in functionally novel ways.

Multiple CDK inhibitor dinaciclib suppresses neuroblastoma growth via inhibiting CDK2 and CDK9 activity.

Neuroblastoma (NB), the most common extracranial solid tumor of childhood, is responsible for approximately 15% of cancer-related mortality in children. Aberrant activation of cyclin-dependent kinases (CDKs) has been shown to contribute to tumor cell progression in many cancers including NB. Therefore, small molecule inhibitors of CDKs comprise a strategic option in cancer therapy. Here we show that a novel multiple-CDK inhibitor, dinaciclib (SCH727965, MK-7965), exhibits potent anti-proliferative effects on a panel of NB cell lines by blocking the activity of CDK2 and CDK9. Dinaciclib also significantly sensitized NB cell lines to the treatment of chemotherapeutic agents such as doxorubicin (Dox) and etoposide (VP-16). Furthermore, dinaciclib revealed in vivo antitumor efficacy in an orthotopic xenograft mouse model of two NB cell lines and blocked tumor development in the TH-MYCN transgenic NB mouse model. Taken together, this study suggests that CDK2 and CDK9 are potential therapeutic targets in NB and that abrogating CDK2 and CDK9 activity by small molecules like dinaciclib is a promising strategy and a treatment option for NB patients.

Opinion Paper: Targeting Multiple Cyclin-Dependent Kinases (CDKs): A New Strategy for Molecular Docking Studies.

Opinion Paper: Targeting Multiple Cyclin-Dependent Kinases (CDKs): A New Strategy for Molecular Docking Studies.

Down-regulation of multiple CDK inhibitor ICK/KRP genes promotes cell proliferation, callus induction and plant regeneration in Arabidopsis.

The ICK/KRP cyclin-dependent kinase (CDK) inhibitors are important plant cell cycle regulators sharing only limited similarity with the metazoan CIP/KIP family of CDK inhibitors. Information is still limited regarding the specific functions of different ICK/KRP genes in planta. We have shown previously that down-regulation of multiple CDK inhibitor ICK/KRP genes up-regulates the E2F pathway and increases cell proliferation, and organ and seed sizes in Arabidopsis. In this study, we observed that the quintuple ick1/2/5/6/7 mutant had more cells in the cortical layer of the root apical meristem (RAM) than the wild type (Wt) while its RAM length was similar to that of the Wt, suggesting a faster cell cycle rate in the quintuple mutant. We further investigated the effects of down-regulating ICK genes on tissue culture responses. The cotyledon explants of ick1/2/5/6/7 could form callus efficiently in the absence of cytokinin and also required a lower concentration of 2,4-D for callus induction compared to the Wt plants, suggesting increased competence for callus induction in the mutant. In addition, the quintuple ick mutant showed enhanced abilities to regenerate shoots and roots, suggesting that increased competence to enter the cell cycle in the quintuple mutant might make it possible for more cells to become proliferative and be utilized to form shoots or roots. These findings indicate that CDK activity is a major factor underlying callus induction and increased cell proliferation can enhance in vitro organogenesis.

Abstract 3470: Transforming growth factor-beta (TGFβ)-mediated growth inhibition of human myeloid leukemia cells is CAK-independent but links to cdc25c-CDK1 pathway

Abstract The cell cycle progression is controlled by cyclin-dependent kinases (CDKs). CDK-Activating Kinase (CAK) is an enzyme complex that is capable of phosphorylating (activating) all CDKs and is essential for G1 and G2 CDK activities. CAK is composed of CDK7, cyclin H, and Mat 1. CDK (CDK1 and CDK2) activity is also controlled by two inhibitory phosphorylation sites (tyrosine 15 and threonine 14) on the CDK. Dephosphorylation of these two sites by cdc25c activates CDK activity. We have previously reported that TGFβ inhibits growth of human myeloid leukemia cells via downregulation of multiple CDKs and cyclins. In this study, we investigated the effect of TGFβ on activity and expression of CAK and cdc25, the two upstream molecules of cell cycle regulatory molecules in human myeloid leukemia cells. TGF-β inhibited the proliferation of TF-1 and MV4-11 accompanied by an increase in p27 level and decrease in CDK1, CDK2, CDK4, cyclin A, cyclin D3, and cylcin B. However, TGF-β had no effect on the expression of CDK7, CDK 9, CDK11, cyclin H, Mat1, and their complexes (CDK7-cyclin H and CDK7-Mat1) for the time period tested up to 72 hours, detected by Western blot and immunoprecipitation, respectively. There were no detectable changes in the phosphorylation status of CDK7, CDK9, CDK11 in the cells treated with TGF-β as compared with the cells without TGFβ. In contrast, these cells showed marked decrease in the levels of MAT1 and CDK7 in response to retinoic acid and phorbol ester (PMA) stimulation. On the other hand, TGFβ significantly downregulated expression of cdc25c starting from 3h and with a maximum inhibition being observed at 72h. We also observed a transient dephosphorylation (activation) of cdc25c followed by significant decrease in the amount of dephophorylated 25c at 48 and 72h. Taken together, our data suggest that TGFβ-induced growth inhibition of human myeloid leukemia cells is CAK-independent but is linked to inhibition of early entry into mitosis of the cells by downregulating cdc25c-CDK1 pathway. Most likely, the downregulation of multiple CDKs and cyclins by TGFβ is regulated at translational or posttranslational but not transcriptional levels, because TGFβ had no any effect on the expression and phosphorylation of the CDKs that are involved in regulation of transcription factor TFII (CDK7 and CDK11) and TAK/P-TEFb (CDK9), respectively. (Supported by Faculty Incentive Grant and NIH-NIGMS MBRS RISE: R25 GM059244-13, Barry University). Citation Format: Alejandra Toro, Daria Vasilyeva, Talia Guardia, Tamara Guardia, Jazmine Duran, Xiaotang Hu. Transforming growth factor-beta (TGFβ)-mediated growth inhibition of human myeloid leukemia cells is CAK-independent but links to cdc25c-CDK1 pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3470. doi:10.1158/1538-7445.AM2014-3470

MEN1 tumorigenesis in the pituitary and pancreatic islet requires Cdk4 but not Cdk2.

Recent studies suggest that physiological and tumorigenic proliferation of mammalian cells is controlled by multiple cyclin-dependent kinases (CDKs) largely in tissue-specific manners. We and others previously demonstrated that adult mice deficient for the D-cyclin-dependent kinase CDK4 (Cdk4−/− mice) exhibit hypoplasia in the pituitary and pancreatic islet due to primary postnatal defects in proliferation. Intriguingly, those neuroendocrine tissues affected in Cdk4−/− mice are the primary targets of tumorigenesis in the syndrome of multiple endocrine neoplasia type-1 (MEN1). Mice with heterozygous disruption of the tumor suppressor Men1 gene (Men1+/−) develop tumors in the pituitary, pancreatic islets and other neuroendocrine tissues, which is analogous to humans with MEN1 mutations. To explore the genetic interactions between loss of Men1 and activation of CDKs, we examined the impact of Cdk4 or Cdk2 disruption on tumorigenesis in Men1+/− mice. A majority of Men1+/− mice with wild-type CDKs developed pituitary and islet tumors by 15 months of age. Strikingly, Men1+/−; Cdk4−/− mice did not develop any tumors, and their islets and pituitaries remained hypoplastic with decreased proliferation. In contrast, Men1+/−; Cdk2−/− mice showed pituitary and islet tumorigenesis comparable to those in Men1+/− mice. Pituitaries of Men1+/−; Cdk4−/− mice showed no signs of loss of heterozygosity (LOH) in the Men1 locus, while tumors in Men1+/− mice and Men1+/−; Cdk2−/− mice exhibited LOH. Consistently, CDK4 knockdown in INS-1 insulinoma cells inhibited glucose-stimulated cell cycle progression with a significant decrease in phosphorylation of retinoblastoma protein (RB) at specific sites including Ser780. CDK2 knockdown had minimum effects on RB phosphorylation and cell cycle progression. These data suggest that CDK4 is a critical downstream target of MEN1-dependent tumor suppression and is required for tumorigenic proliferation in the pituitary and pancreatic islet, whereas CDK2 is dispensable for tumorigenesis in these neuroendocrine cell types.

Induction of β-cell replication by a synthetic HNF4α antagonist

Increasing the number of β cells is critical to a definitive therapy for diabetes. Previously, we discovered potent synthetic small molecule antagonists of the nuclear receptor transcription factor HNF4α. The natural ligands of HNF4α are thought to be fatty acids. Because obesity, in which there are high circulating levels of free fatty acids, is one of the few conditions leading to β-cell hyperplasia, we tested the hypothesis that a potent HNF4α antagonist might stimulate β-cell replication. A bioavailable HNF4α antagonist was injected into normal mice and rabbits and β-cell ablated mice and the effect on β-cell replication was measured. In normal mice and rabbits, the compound induced β-cell replication and repressed the expression of multiple cyclin-dependent kinase inhibitors, including p16 that plays a critical role in suppressing β-cell replication. Interestingly, in β-cell ablated mice, the compound induced α- and δ-cell, in addition to β-cell replication, and β-cell number was substantially increased. Overall, the data presented here are consistent with a model in which the well-known effects of obesity and high fat diet on β-cell replication occur by inhibition of HNF4α. The availability of a potent synthetic HNF4α antagonist raises the possibility that this effect might be a viable route to promote significant increases in β-cell replication in diseases with reduced β-cell mass, including type I and type II diabetes.

Abstract 1749: Cell cycle requirements shape ovarian cancer progression.

Abstract Acquired resistance to chemotherapeutic drugs and subsequent recurrence of drug-resistant cancer are serious challenges associated with epithelial ovarian cancer. Most high-grade tumors initially respond well to the combination of platinum and taxane-based agents but recur as platinum-resistant disease, which is incurable. In addition, a subset of primary tumors, including CCNE1-amplified tumors, are inherently less susceptible to standard chemotherapy. Studies by The Cancer Genome Atlas have revealed that CCNE1 (cyclin E) amplification is mutually exclusive with mutations in BRCA1 and BRCA2, which render ovarian cancer cells sensitive to DNA-damaging agents and PARP inhibitors, as a result of synthetic lethality. In order to develop a similar therapeutic concept for CCNE1-amplified cancers, we have studied responses and resistance mechanisms of human ovarian cancer cells to several pharmacological inhibitors of cyclin-dependent kinases (CDK). Functional and biochemical studies have established cyclin E as an activator of CDK, which phosphorylate the RB tumor suppressor. However, despite the functional importance of the RB pathway in cancer, CDK inhibitors (CDKi) have yet to make a significant impact on the management of solid tumors. Here, we describe oncogenic pathways that are activated in CDKi-resistant cells and play critical roles in tumor progression and drug resistance. Targeting individual CDK or subsets of multiple CDK in vitro and in vivo, we found that cyclin E dependence was the main driving force that shaped tumor progression in different model systems of CCNE1-amplified and recurrent ovarian cancer. We identified robust and specific compensatory mechanisms that sustain cyclin E-associated activity in CDKi-resistant cells via activation of oncogenic signaling upstream of the cyclin E-RB-E2F axis. Specific targeting of cooperating oncogenes increased CDKi efficacy and prevented the outgrowth of CDKi-resistant cancer cells. Collectively, our results suggest that CDKi can be highly effective elements of combination therapy. Citation Format: Barbie Taylor-Harding, Hasmik Agadjanian, Sandra Orsulic, Christine Walsh, Beth Y. Karlan, Wolf-Ruprecht Wiedemeyer. Cell cycle requirements shape ovarian cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1749. doi:10.1158/1538-7445.AM2013-1749

Abstract 576: Transforming growth factor-beta (TGF-β) inhibits growth of human myeloid leukemia cells and downregulates the expression of CDK inhibitor, p18INK4C.

Abstract The cell cycle progression controlled by cyclin-dependent kinases (CDKs) is counterbalanced by CDK inhibitors (CKIs). The CKIs have two families: CIP/KIP and INK. The CIP/KIP members are composed of p21WAF1/CIP1, p27KIP1 and p57KIP2, which bind to and inhibit both cyclin D-CDK4/6 kinases and cyclin-E/A-CDK2. The INK members consist of p15INK4B, p16INK4A, p18INK4C and p19INK4D. The members in this family only bind to and inhibit G1CDKs (CDK4 and CDK6). We have previously reported that p27KIP1 plays both a positive and a negative role in human myeloid leukemia cells. In this study, we investigated whether p18INK4C plays any role in TGF-β-mediated cell cycle control of human myeloid leukemia cells. TGF-β significantly inhibited proliferation of MV4-11 and TF-1 cells, with upregulation of p27KIP1and downregulation of multiple CDKs and cyclins. Surprisingly, TGF-β significantly inhibited the expression of p18INK4C, a traditional G1 CDK inhibitor. The inhibitory effect of TGFβ on p18INK4C was time dependent. The time course showed that early inhibition of p18INK4C occurred at 3h after initiating TGFβ treatment in the cells with a maximal inhibition of 70% being observed at 72h incubation with TGF-β. The control cells treated with the solvent for TGFβ did not show any significant changes in the levels of p18INK4C. As a sample loading control, the cells treated with or without TGF-β expressed about the same levels of actin.TGFβ-induced p18INK4C inhibition is also dose dependent with a maximal inhibition being detected by Western blot when 30ng/ml of TGFβ was added to the culture. Low concentrations (5ng/ml or less) did not markedly affect the expression of p18INK4C. The association of p18INK4C with CDK4 was also decreased in response to TGF-β treatment detected by immunoprecipitation. Our data also showed that the two cell lines tested barely expresses p15INK4B, p16INK4A, p19INK4D, and p21WAF1/CIP1 and these inhibitors do not appear to play an important role, either positively or negatively, in the cell cycle control of the leukemia cells. Our data suggest that p18INK4C may be required for cell cycle progression in human myeloid leukemia cells tested. Whether such a positive role of p18INK4C is caused by a possible gene mutation(s) and thereby contributes to leoplasia of the leukemia cells is currently under investigation (supported by NIH-NIGMS MBRS RISE: R25 GM059244, Barry University). . Citation Format: Talia Guardia, Reshma Badaloo, Valentina Serrano, Alejandra Toro, Xiaotang Hu. Transforming growth factor-beta (TGF-β) inhibits growth of human myeloid leukemia cells and downregulates the expression of CDK inhibitor, p18INK4C. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 576. doi:10.1158/1538-7445.AM2013-576

Synthesis and in vitro biological evaluation of 2,6,9-trisubstituted purines targeting multiple cyclin-dependent kinases

Several inhibitors of cyclin-dependent kinases (CDKs), including the 2,6,9-trisubstituted purine derivative roscovitine, are currently being evaluated in clinical trials as potential anticancer drugs. Here, we describe a new series of roscovitine derivatives that show increased potency in vitro. The series was tested for cytotoxicity against six cancer cell lines and for inhibition of CDKs. For series bearing 2-(hydroxyalkylamino) moiety, cytotoxic potency strongly correlated with anti-CDK2 activity. Importantly, structural changes that increase biochemical and anticancer activities of these compounds also increase elimination half-life. The most potent compounds were investigated further to assess their ability to influence cell cycle progression, p53-regulated transcription and apoptosis. All the observed biological effects were consistent with inhibition of CDKs involved in the regulation of cell cycle and transcription.

Perspective of Cyclin-dependent kinase 9 (CDK9) as a Drug Target

Deregulation of cyclin-dependent kinases (CDKs) has been associated with many cancer types and has evoked an interest in chemical inhibitors with possible therapeutic benefit. While most known inhibitors display broad selectivity towards multiple CDKs, recent work highlights CDK9 as the critical target responsible for the anticancer activity of clinically evaluated drugs. In this review, we discuss recent findings provided by structural biologists that may allow further development of highly specific inhibitors of CDK9 towards applications in cancer therapy. We also highlight the role of CDK9 in inflammatory processes and diseases.

Electrostatics and Intrinsic Disorder: A Single-Molecule Study of the Sic1 Protein

Intrinsically disordered proteins (IDPs) play critical yet often poorly understood roles in a variety of cellular processes. Despite an apparent antagonism between structural disorder and protein recognition, the large number of IDPs involved in protein regulation suggests that they could in fact provide advantages in recognition over well-folded proteins. Sic1 is an IDP inhibitor of a cyclin dependent kinase (CDK) in yeast, which interacts with a single site on its acceptor Cdc4 only upon phosphorylation of its multiple dispersed CDK sites. The multiple phosphorylation events can in principle be the basis for ultrasensitivity in protein-protein binding, however its molecular basis remains elusive1.We performed a systematic study of the Sic1's fluctuating conformations in different salt and denaturant concentrations using single-molecule Forster energy transfer (smFRET) and fluorescence correlation spectroscopy (FCS). smFRET data suggests that Sic1 protein is comprised of a continuum of conformers with varying end-end distances. Denaturant titration measurements suggest that these conformers are characterized by non-cooperative interactions. From FCS experiments, the exchange between Sic1 conformational states was found to occur on both ultrafast (10-100 ns timescale) and slow (10-100 ms) timescale. Burst smFRET experiments show that Sic1’s end-end distances do not vary significantly upon addition of salt, which suggests that charge-shielding by salt may only affect the structure locally, around charged amino acids. Our single-molecule data resolves conformational heterogeneity and dynamics in a model IDP protein and represent the first step towards the validation of the polyelectrostatic model of the Sic1-Cdc4 interaction2.(1) Nash, P., Tang, X., Orlicky, S., Chen, Q., Gertler, F. B., Mendenhall, M. D., Sicheri, F., Pawson, T., Tyers, M. Nature 2001, 414, 514.(2) Borg, M., Mittag, T., Pawson, T., Tyers, M., Forman-Kay, J. D., Chan, H. S. Proc. Natl. Acad. Sci. USA 2007, 104, 9650.

A phase I pharmacokinetic and pharmacodynamic study of AT7519, a cyclin-dependent kinase inhibitor in patients with refractory solid tumors

BackgroundAT7519 is an inhibitor of multiple cyclin-dependent kinases (CDKs). Based on potent antitumor activity in preclinical models, a first-in-human clinical trial in refractory solid tumors investigated its safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD). Patients and methodsAT7519 was administered in a ‘3 + 3’ dose- escalation scheme on 5 consecutive days every 3 weeks to patients with advanced, refractory solid tumors. Samples to monitor AT7519 PK and PD were obtained. ResultsTwenty-eight patients were treated at seven dose levels (1.8–40 mg/m2/day). At 40 mg/m2/day, one patient developed hypotension and ST segment elevation. At 34 mg/m2/day, dose-limiting toxic effects (DLTs) were QTc prolongation with one death (grade 5), fatigue (grade 4) and mucositis (grade 3). Electrocardiogram review suggested a dose-dependent increase in QTc and recruitment was discontinued without establishing a maximum tolerated dose. Four patients exhibited stable disease for >6 months and one had a prolonged partial response. PK profile revealed modest interpatient variation with linear exposure at increasing doses. Inhibition of markers of CDK activity was observed across the dose range and manifested in antiproliferative activity at a dose of 28 mg/m2. ConclusionAT7519 elicited clinical and PD activity resulting from CDK inhibition at doses below the appearance of DLT of QTc prolongation.

Switching Cdk2 On or Off with Small Molecules to Reveal Requirements in Human Cell Proliferation

Multiple cyclin-dependent kinases (CDKs) control eukaryotic cell division, but assigning specific functions to individual CDKs remains a challenge. During the mammalian cell cycle, Cdk2 forms active complexes before Cdk1, but lack of Cdk2 protein does not block cell-cycle progression. To detect requirements and define functions for Cdk2 activity in human cells when normal expression levels are preserved, and nonphysiologic compensation by other CDKs is prevented, we replaced the wild-type kinase with a version sensitized to specific inhibition by bulky adenine analogs. The sensitizing mutation also impaired a noncatalytic function of Cdk2 in restricting assembly of cyclin A with Cdk1, but this defect could be corrected by both inhibitory and noninhibitory analogs. This allowed either chemical rescue or selective antagonism of Cdk2 activity in vivo, to uncover a requirement in cell proliferation, and nonredundant, rate-limiting roles in restriction point passage and S phase entry.

A first in man, phase I dose-escalation study of PHA-793887, an inhibitor of multiple cyclin-dependent kinases (CDK2, 1 and 4) reveals unexpected hepatotoxicity in patients with solid tumors

Background: PHA-793887 is an inhibitor of multiple cyclin dependent kinases (CDK) with activity against CDK2, CDK1 and CDK4. The primary objectives of this first in man study were to determine the dose limiting toxicities (DLTs), maximum tolerated dose (MTD) and recommended phase II dose of PHA-793887. Results: Although toxicity was acceptable at initial dose levels, PHA-793887 was poorly tolerated at doses ≥44 mg/m2. The most frequent events across all dose levels were gastrointestinal or nervous system events. DLTs were experienced by two of three patients at the dose level of 66 mg/m2, and by three of nine patients at the dose level of 44 mg/m2. In all but one patient the DLT was hepatotoxicity; fatal hepatorenal failure was seen in one patient treated at the 44 mg/ m2 dose level. There were no objective responses, but disease stabilization was observed in five patients. Over the dose range investigated, pharmacokinetic studies showed that systemic exposure to PHA-793887 increased with the dose and was time-independent. The study terminated after the enrolment of 19 patients due to the severe hepatic toxicity. Patients and Methods: Cohorts of three to six patients were treated at doses of 11, 22, 44 and 66 mg/m2 of PHA-793887 administered as 1-hour intravenous infusion on days 1, 8 and 15 in a 4-week cycle. Safety and pharmacokinetics were investigated. Conclusion: PHA-793887 induces severe, dose-related hepatic toxicity, which was not predicted by pre-clinical models and currently precludes its further clinical development.

Whether to target single or multiple CDKs for therapy? That is the question

Complexes consisting of cyclin-dependent kinases (CDKs) and their regulatory subunits (the cyclins) control the progression of normal mammalian cells through the cell cycle. However, during malignant transformation this regulatory apparatus malfunctions, allowing cells to undergo unchecked proliferation. In many cases, the high mitotic potential of malignant cells is due to the constitutive activation of CDK-cyclin complexes, facilitated by the inactivation of cellular CDK inhibitors, such as p16(INK4A) or p27(Kip1), and the loss of functional tumor suppressors, such as the p53 and pRb proteins. It has recently been suggested that pharmacological intervention based on remedying the deficiency or loss of activity of these negative regulators of the cell cycle could be a very effective therapeutic option in the treatment of cancer. Multiple CDK inhibitors have been synthesized over the last two decades, spanning at least five classes of compounds. While these inhibitors can be classified on the basis of their chemical structure, it may be more interesting to categorize them according to their pharmacological nature, as broad-spectrum unspecific, pan-specific, or very selective antagonists. This review offers a critical assessment of the advantages and disadvantages of both pan-specific and highly selective CDK inhibitors in therapy.

Cyclin-dependent kinases (cdks) and the DNA damage response: rationale for cdk inhibitor–chemotherapy combinations as an anticancer strategy for solid tumors

Importance of the field: The eukaryotic cell division cycle is a tightly regulated series of events coordinated by the periodic activation of multiple cyclin-dependent kinases (cdks). Small-molecule cdk-inhibitory compounds have demonstrated preclinical synergism with DNA-damaging agents in solid tumor models. An improved understanding of how cdks regulate the DNA damage response now provides an opportunity for optimization of combinations of cdk inhibitors and DNA damaging chemotherapy agents that can be translated to clinical settings.Areas covered in this review: Here, we discuss novel work uncovering multiple roles for cdks in the DNA-damage-response network. First, they activate DNA damage checkpoint and repair pathways. Later their activity is turned off, resulting in cell cycle arrest, allowing time for DNA repair to occur. Recent clinical data on cdk inhibitor–DNA-damaging agent combinations are also discussed.What the reader will gain: Readers will learn about novel areas of cdk biology, the complexity of DNA damage signaling networks and clinical implications.Take home message: New data demonstrate that cdks are ‘master’ regulators of DNA damage checkpoint and repair pathways. Cdk inhibition may therefore provide a means of potentiating the clinical activity of DNA-damaging chemotherapeutic agents for the treatment of cancer.

Dual Regulation by Pairs of Cyclin-Dependent Protein Kinases and Histone Deacetylases Controls G1 Transcription in Budding Yeast

Author SummaryEukaryotic cells grow and divide by progressing through carefully orchestrated stages of the cell cycle characterized by stage-specific patterns of gene expression, DNA replication, and scission. How stage-specific gene expression is coordinated with cell cycle progression is only partially understood. The phase known as G1 marks the initiation of the cell cycle (called START in yeast) and involves the coordinated expression of more than 200 genes regulated by two transcription factors, SBF and MBF. The activity of SBF and MBF is restrained by binding of the repressor protein Whi5 to the two transcription factors early in G1 phase. Phosphorylation of Whi5 by G1-specific forms of the cyclin-dependent kinase (CDK) Cdc28 promotes dissociation of Whi5 from SBF and its export from the nucleus; this, in turn, releases SBF to activate G1-specific transcription. This G1 transcriptional circuit is analogous to that defined in mammals by the E2F family of transcription factors and the retinoblastoma (Rb) tumor suppressor protein. Rb further contributes to the repression of G1-specific transcription in mammals by recruiting histone deacetylases (HDACs), which are chromatin remodeling complexes that regulate promoter accessibility. Here, we show that regulation of G1-specific transcription in yeast also involves repressor-mediated recruitment of HDACs. We demonstrate that repression by Whi5 is modulated by both Cln-Cdc28 and a second G1-specific CDK, Pcl-Pho85, and further show that both kinases regulate the interaction of Whi5 with HDACs. We propose that regulation of the repressor by more than one G1-specific CDK ensures definitive inactivation of Whi5, a critical event for appropriate cell cycle initiation.

Factors Affecting the Diversity of DNA Replication Licensing Control in Eukaryotes

Replication of eukaryotic genomes is limited to once per cell cycle, by a two-step mechanism. DNA replication origins are first "licensed" during G1 phase by loading of an inactive DNA helicase (Mcm2-7) into pre-replicative complexes (pre-RCs). Initiation then occurs during S phase, triggered by cyclin-dependent kinases (CDKs), which promote recruitment of proteins required for helicase activation and replisome assembly. CDKs and the anaphase promoting complex/cyclosome (APC/C) restrict licensing to G1 phase by directly and indirectly regulating pre-RC components, including ORC, Cdc6, Cdt1, and Mcm2-7. Despite the fundamental importance of licensing regulation, the mechanisms by which pre-RC components are regulated differ widely across Eukarya. Here we show that even within the genus Saccharomyces, Cdc6 is regulated differently in different species. We propose that two factors contribute to the rapid evolution of licensing regulation. The first is redundancy: eliminating any single pre-RC-regulatory mechanism has very little affect on viability. The second is interchangeability: we show that regulatory mechanisms can be swapped between pre-RC components without compromising the block to re-replication. These experiments provide a framework for understanding the diversity of licensing regulation in eukaryotes and provide new tools for manipulating the chromosome-replication cycle.

Dynamic equilibrium engagement of a polyvalent ligand with a single-site receptor

Intrinsically disordered proteins play critical but often poorly understood roles in mediating protein interactions. The interactions of disordered proteins studied to date typically entail structural stabilization, whether as a global disorder-to-order transition or minimal ordering of short linear motifs. The disordered cyclin-dependent kinase (CDK) inhibitor Sic1 interacts with a single site on its receptor Cdc4 only upon phosphorylation of its multiple dispersed CDK sites. The molecular basis for this multisite-dependent interaction with a single receptor site is not known. By NMR analysis, we show that multiple phosphorylated sites on Sic1 interact with Cdc4 in dynamic equilibrium with only local ordering around each site. Regardless of phosphorylation status, Sic1 exists in an intrinsically disordered state but is surprisingly compact with transient structure. The observation of this unusual binding mode between Sic1 and Cdc4 extends the understanding of protein interactions from predominantly static complexes to include dynamic ensembles of intrinsically disordered states.