Cyclin-dependent Kinase Family Research Articles

Abstract 2823: Identification of highly selective inhibitors of cyclin-dependent kinase 12

Abstract CDK12 and CDK13 belong to the cyclin-dependent kinase (CDK) family, which includes at least 20 different human CDKs and CDK-like enzymes. One subclass of CDKs (e.g. CDK7, CDK8, CDK9, CDK12 and CDK13) preferentially regulates transcription by phosphorylation of the C-terminal domain of RNA polymerase II (RNAPII), whereas another subgroup of CDKs plays a pivotal role in controlling cell cycle progression (e.g. CDK1, CDK2, CDK4, CDK6 and CDK7). Both groups - cell cycle as well as transcriptional CDKs - have been reported to be involved in cancer development and progression. Recent reports indicated CDK12 to be involved in DNA damage repair mechanisms and linked its mutation to high-grade serous ovarian carcinoma rendering CDK12 a promising target for drug development. However, the pivotal role of individual CDKs and their complex regulation requests for highly selective inhibitors to avoid unwanted adverse effects. Non-specific first-generation CDK-inhibitors cause toxicity in vivo issues. They showed little to no therapeutic window in clinical trials and thus shifted the focus to the optimization of selective small molecule CDK inhibitors. To identify selective small molecular weight inhibitors of CDK12 and CDK13 we employed a rationale drug discovery approach based on a focused kinase library screen. Screening of slightly over 16.000 compounds identified several hit compound classes. Medicinal chemistry based optimization yielded nanomolar CDK12 inhibitors. The selectivity of these improved CDK12 inhibitors is excellent, when tested against 12 CDK family members. Such selective CDK12 inhibitors have also shown promising physicochemical properties, indicating lead- and drug-likeness. Although, potency on the target still needs to be improved, the frontrunners from our selective CDK12 inhibitor family have shown cellular inhibition of CDK12 substrate phosphorylation such as those of RNA polymerase II. Currently, potency of these CDK12 inhibitors is being improved by using structure-guided medicinal chemistry-based optimization. The resulting lead candidates will then be tested in in vivo cancer models. Citation Format: Axel Choidas, Carsten Schultz-Fademrecht, Carsten Degeenhardt, Peter Habenberger, Uwe Koch, Jan E. Eickhoff, Ann Kathrin Greifenberg, Peter Nussbaumer, Matthias Geyer, Bert M. Klebl. Identification of highly selective inhibitors of cyclin-dependent kinase 12. [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 2823.

Abstract 2824: G1T38, a novel, oral, potent and selective CDK4/6 inhibitor for the treatment of Rb competent tumors

Abstract The development of therapeutically effective inhibitors of the cyclin dependent kinase (CDK) family has been challenging due to a poor understanding of target and structural biology leading to the development of drugs that are toxic with limited efficacy. Recently, the first highly selective CDK4/6 inhibitor, palbociclib, was approved by the FDA for use in combination with letrozole as a first line treatment in patients with ER+/ HER2- metastatic breast cancer. While this first approved CDK4/6 inhibitor is highly efficacious, it causes severe myelosuppression during daily treatment resulting in at least a 7 day treatment holiday in every 28 day cycle to allow recovery of neutrophil counts. This leads to an increased risk of febrile neutropenia, potential tumor growth during the treatment holiday, and emergence of drug resistance. CDK4/6-induced myelosuppression is the result of on-target inhibition of hematopoietic stem and progenitor cell proliferation causing a narrow therapeutic window between tumor efficacy and neutropenia. Thus, a next generation CDK4/6 inhibitor will need to produce a potent, selective G1 arrest in Rb competent tumors while minimizing the effect on the bone marrow. With a new understanding of CDK4/6 biology, we have generated compounds with unique properties that maintain tumor efficacy while minimizing inhibition of bone marrow proliferation. Here we describe G1T38, a novel, oral, potent, and selective CDK4/6 inhibitor. Biochemical profiling demonstrates G1T38 is a competitive, nanomolar inhibitor of CDK4/6 with highly selectivity for CDK4-cyclin D1 and CDK6-cyclin D3. Kinome profiling exhibits on-target selectivity across 468 independent kinases at 100 nM, with no significant activity against other cell cycle or mitotic kinases. G1T38 elicits a precise G1 arrest profile along with loss of Rb phosphorylation in Rb competent cells with no impact in Rb deficient cells up to three orders of magnitude above the biochemical IC50. In cell proliferation assays, G1T38 exhibits a low EC50 (<100 nM) in Rb competent cell lines compared to >3 μM in Rb null cells. In vivo, daily oral treatment with G1T38 causes significant, durable growth inhibition of tumors in a HER2/neu GEMM and in MCF7 xenograft breast cancer models. G1T38 is cleared from the plasma but significantly accumulates in tumors. The level of G1T38 in the tumor correlates with significant reductions in Rb phosphorylation and tumor cell proliferation. Additionally, daily oral dosing of G1T38 in mice, rats and dogs for up to 28 days has shown a dose dependent decrease in neutrophils without severe neutropenia. These data demonstrate that the unique pharmacokinetic and pharmacodynamic properties of G1T38 allow it to be highly efficacious against tumors while having a mild effect on the bone marrow, thus making it optimal for use as a daily oral antineoplastic agent. Citation Format: Jessica A. Sorrentino, John E. Bisi, Patrick J. Roberts, Jay C. Strum. G1T38, a novel, oral, potent and selective CDK4/6 inhibitor for the treatment of Rb competent tumors. [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 2824.

P21 and p27 Immunoexpression in Upper Urinary Tract Urothelial Carcinomas.

p21 and p27 are members of cyclin-dependent kinase family, which function as tumor suppressors and they are involved in development and progression of several malignancies. We investigated their expression in upper urinary tract urothelial carcinoma (UUTUC). Radical nephroureterectomy materials of 34 patients were assessed by immunohistochemistry to evaluate expression of p21 and p27 in UUTUC. Results were correlated with various clinicopathological variables as age, gender, tumor grade and stage, tumor architecture, multifocality, subsequent bladder carcinoma development and clinical outcome.p21 and p27 expression was observed in 52.9% (n=18) and 88.2% (n=30), respectively. A total of 21 tumors (61.7%) showed either total loss of p21 expression (n=16, 47%) or lower expression (n=5, 14.7%). No correlation was found between p21 expression and clinicopathologic variables. Cases showing total loss or lower p27 expression (11.7% and <25.6%, respectively) (n=19, 55.8%) constituted 67.6% (n=23) of the cases totally. This loss or lower p27 expression correlated with a shorter overall survival in both univariate and multivariate analysis (p=0.039 and p=0.037, respectively). None of the noninvasive tumors (papillary and nodular tumors) showed loss of p27 (p=0.016) while 33.3% of invasive ones showed p27 loss. Noninvasive tumor architecture also correlated with subsequent bladder carcinoma development (p=0.032) while invasive tumor architecture correlated with advanced stage (T3 and T4) (p=0.003). p27 is widely expressed in UUTUC, while p21 expression is observed in half of the cases. Loss of p27 expression correlated with tumor architecture and overall survival in UUTUC. However, further research is needed to assess their role in UUTUC.

The emerging roles and therapeutic potential of cyclin-dependent kinase 11 (CDK11) in human cancer.

Overexpression and/or hyperactivation of cyclin-dependent kinases (CDKs) are common features of most cancer types. CDKs have been shown to play important roles in tumor cell proliferation and growth by controlling cell cycle, transcription, and RNA splicing. CDK4/6 inhibitor palbociclib has been recently approved by the FDA for the treatment of breast cancer. CDK11 is a serine/threonine protein kinase in the CDK family and recent studies have shown that CDK11 also plays critical roles in cancer cell growth and proliferation. A variety of genetic and epigenetic events may cause universal overexpression of CDK11 in human cancers. Inhibition of CDK11 has been shown to lead to cancer cell death and apoptosis. Significant evidence has suggested that CDK11 may be a novel and promising therapeutic target for the treatment of cancers. This review will focus on the emerging roles of CDK11 in human cancers, and provide a proof-of-principle for continued efforts toward targeting CDK11 for effective cancer treatment.

A Tale of the Good and Bad: Remodeling of the Microtubule Network in the Brain by Cdk5.

Cdk5, a cyclin-dependent kinase family member, is a global orchestrator of neuronal cytoskeletal dynamics. During embryogenesis, Cdk5 is indispensable for brain development. In adults, it is essential for numerous neuronal processes, including higher cognitive functions such as learning and memory formation, drug addiction, pain signaling, and long-term behavior changes through long-term potentiation and long-term depression, all of which rely on rapid alterations in the cytoskeleton. Cdk5 activity becomes deregulated in various brain disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, attention-deficit hyperactivity disorder, epilepsy, schizophrenia, and ischemic stroke; these all result in profound remodeling of the neuronal cytoskeleton. This Commentary specifically focuses on the pleiotropic contribution of Cdk5 in regulating neuronal microtubule remodeling. Because the vast majority of the physiological substrates of Cdk5 are associated with the neuronal cytoskeleton, our emphasis is on the Cdk5 substrates, such as CRMP2, stathmin, drebrin, dixdc1, axin,MAP2, MAP1B, doublecortin, kinesin-5, and tau, that have allowed to unravel the molecular mechanisms through which Cdk5 exerts its divergent roles in regulating neuronal microtubule dynamics, both in healthy and disease states.

Selective CDK7 inhibition with BS-181 suppresses cell proliferation and induces cell cycle arrest and apoptosis in gastric cancer.

Cyclin-dependent kinase (CDK) family members have been considered as attractive therapeutic targets for cancer. In this study, we aim to investigate the anticancer effects of a selective CDK7 inhibitor, BS-181, in gastric cancer (GC) cell line. Human GC cells (BGC823) were cultured with or without BS-181 at different concentrations for 24–72 hours. BS-181 significantly reduced the activity of CDK7 with downregulation of cyclin D1 and XIAP in GC cells. Treatment with BS-181 induced cell cycle arrest and apoptosis. The expression of Bax and caspase-3 was significantly increased, while Bcl-2 expression was decreased in cells treated with BS-181. In addition, the inhibition of CDK7 with BS-181 resulted in reduced rates of proliferation, migration, and invasion of gastric cells. Those results demonstrated the anticancer activities of selective CDK7 inhibitor BS-181 in BGC823 cells, suggesting that CDK7 may serve as a novel therapeutic target or the treatment of GC.

Neuroprotective Mechanisms Mediated by CDK5 Inhibition.

Cyclin-dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase belonging to the family of cyclin-dependent kinases. In addition to maintaining the neuronal architecture, CDK5 plays an important role in the regulation of synaptic plasticity, neurotransmitter release, neuron migration and neurite outgrowth. Although various reports have shown links between neurodegeneration and deregulation of cyclin-dependent kinases, the specific role of CDK5 inhibition in causing neuroprotection in cases of neuronal insult or in neurodegenerative diseases is not wellunderstood. This article discusses current evidence for the involvement of CDK5 deregulation in neurodegenerative disorders and neurodegeneration associated with stroke through various mechanisms. These include upregulation of cyclin D1 and overactivation of CDK5 mediated neuronal cell death pathways, aberrant hyperphosphorylation of human tau proteins and/or neurofilament proteins, formation of neurofibrillary lesions, excitotoxicity, cytoskeletal disruption, motor neuron death (due to abnormally high levels of CDK5/p25) and colchicine- induced apoptosis in cerebellar granule neurons. A better understanding of the role of CDK5 inhibition in neuroprotective mechanisms will help scientists and researchers to develop selective, safe and efficacious pharmacological inhibitors of CDK5 for therapeutic use against human neurodegenerative disorders, such as Alzheimer's disease, amyotrophic lateral sclerosis and neuronal loss associated with stroke.

Abstract A02: CDK6 controls platinum sensitivity via the regulation of FOXO3a/ATR: A new actionable pathway for ovarian cancer patients.

Abstract Epithelial Ovarian Cancers (EOCs) consist in a heterogeneous group of tumors. Late diagnosis and platinum-resistant recurrences make EOCs the most lethal gynecological malignancy. The central role played by CDKs (Cyclin-Dependent Kinase) in several cellular mechanisms, such as control of cell cycle progression, DNA repair and transcription and the association of their activity in different apoptotic pathways, make them attractive targets to overcome platinum-resistance in EOCs. We explored the possibility that the activity of one or more CDK was necessary to overcome the platinum-induced cell death in EOC cells. To this aim we used an unbiased functional genomic screening targeting, with specific shRNAs, all the 23 members of the CDKs family alone or in association with platinum. This screening identified CDK6 as the CDK most significantly involved in the regulation of platinum sensitivity in EOC cells since CDK6 silencing did not affect cell survival per se but strongly increased platinum-induced cell death. The effect of CDK6 silencing on the sensitivity to both carboplatinum and cisplatin was confirmed in a larger panel of EOC cell lines using multiple shRNAs. Next, the use of CDK6 dominant negative (CDK6D163N) and constitutively active (CDK6R31C) mutants demonstrated that CDK6 kinase activity was necessary to protect from platinum-induced death. Accordingly, an orally active CDK4/CDK6 inhibitor, PD0332991, was able to sensitize EOC cells to CBDCA or CDDP treatment both in vitro and in vivo, in a CDK6-dependent, CDK4-independent manner. To understand the molecular mechanism whereby CDK6 regulates platinum-induced cell death in EOC we focused on specific CDK6 phosphorylation targets and demonstrated that the transcription factor FOXO3a was a relevant downstream target of CDK6. Functional and biochemical analyses showed that upon platinum exposure, CDK6 binds and phosphorylates FOXO3a on a specific Serine residue. We identified the Cyclin D3/CDK6 complex as the one principally involved in FOXO3a phosphorylation and proved that FOXO3a phosphorylation by the Cyclin D3/CDK6 complex leaded to increased FOXO3a protein stability. Accordingly, FOXO3a downregulation sensitizes EOC cells to platinum while FOXO3a overexpression in CDK6 silenced cells was able to rescue the effect of CDK6 silencing on platinum-induced cell death. Mechanistically, we showed that the CDK6/FOXO3a axis was necessary to regulate the DNA damage response in EOC cells through the regulation of ATR/CHK1 pathway since, after platinum treatment, the knock-down of CDK6 or FOXO3a induced the downregulation of ATR and the decrease of CHK1 phosphorylation. Accordingly, we identified in the ATR promoter three putative FOXO3a binding sites and, using chromatin immunoprecipitation, we confirmed that ATR was a FOXO3a transcriptional target, in platinum treated EOC cells. Inhibition of CDK6, FOXO3a or ATR increased platinum dependent-cell death via the induction of the so-called Premature Chromosome Condensation (PCC), a lethal event that selectively kills cancer cells when the ATR/CHK1 pathway is inhibited in the presence of damaged DNA. Overall we demonstrated that in EOC platinum sensitivity could be increased by specific CDK6 inhibition also using a clinically available small molecule, the PD0332991. We are currently analyzing if in primary EOC the expression of CDK6, FOXO3a and/or Cyclin D3 could be used as biomarkers to select those patients that may benefit from a combination therapy based on platinum and CDK6 inhibition, further increasing the translational relevance of our work. Citation Format: Alessandra Dall'Acqua, Schiappacassi Monica, Gustavo Baldassarre. CDK6 controls platinum sensitivity via the regulation of FOXO3a/ATR: A new actionable pathway for ovarian cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A02.

TFP5/TP5 peptide provides neuroprotection in the MPTP model of Parkinson′s disease

Cyclin-dependent kinase 5 (Cdk5) is a member of the serine-threonine kinase family of cyclin-dependent kinases. Cdk5 is critical to normal mammalian nervous system development and plays important regulatory roles in multiple cellular functions. Recent evidence indicates that Cdk5 is inappropriately activated in several neurodegenerative conditions, including Parkinson's disease (PD). PD is a chronic neurodegenerative disorder characterized by the loss of dopamine neurons in the substantia nigra, decreased striatal dopamine levels, and consequent extrapyramidal motor dysfunction. During neurotoxicity, p35 is cleaved to form p25. Binding of p25 with Cdk5 leads deregulation of Cdk5 resulting in number of neurodegenerative pathologies. To date, strategies to specifically inhibit Cdk5 hyperactivity have not been successful without affecting normal Cdk5 activity. Here we show that inhibition of p25/Cdk5 hyperactivation through TFP5/TP5, truncated 24-aa peptide derived from the Cdk5 activator p35 rescues nigrostriatal dopaminergic neurodegeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP+) in a mouse model of PD. TP5 peptide treatment also blocked dopamine depletion in the striatum and improved gait dysfunction after MPTP administration. The neuroprotective effect of TFP5/TP5 peptide is also associated with marked reduction in neuroinflammation and apoptosis. Here we show inhibition of Cdk5/p25-hyperactivation by TFP5/TP5 peptide, which identifies Cdk5/p25 as a potential therapeutic target to reduce neurodegeneration in PD.

Abstract B63: CDK6 controls platinum sensitivity via the regulation of FOXO3a/ATR: a new actionable pathway for ovarian cancer patients

Abstract Epithelial Ovarian Cancers (EOC) consist in a heterogeneous group of tumors. Late diagnosis and platinum-resistant recurrences make EOC the most lethal gynecological malignancies. The central role played by CDKs (Cyclin-Dependent Kinase) in some cellular mechanisms, like control of cell cycle progression, DNA repair and transcription and their activity in apoptotic pathways, make them attractive targets to overcome platinum-resistance in EOC. We explored if the activity of any CDK was necessary to overcome the platinum-induced cell death in EOC cells. We used an unbiased functional genomic screening targeting, with specific shRNAs, all the 23 members of the CDKs family alone or in association with platinum. This screening identified CDK6 as the most significantly involved in the regulation of platinum sensitivity since CDK6 silencing did not affect cell survival per se but strongly increased platinum-induced cell death. The effect of CDK6 silencing on the sensitivity to platinum drugs was confirmed in a larger panel of EOC cell lines. The use of CDK6 dominant negative (CDK6D163N) and constitutively active (CDK6R31C) mutants showed that CDK6 kinase activity was necessary to protect from platinum-induced death. Thus, an orally active CDK4/CDK6 inhibitor, PD0332991, was able to sensitize EOC cells to platinum treatment both in vitro and in vivo, in a CDK6-dependent CDK4-independent manner. To unveil the mechanism whereby CDK6 regulates platinum-induced cell death in EOC we focused on specific CDK6 phosphorylation targets and demonstrated that the transcription factor FOXO3a was a relevant downstream target of CDK6. Functional and biochemical analyses showed that upon platinum exposure, CDK6 binds and phosphorylates FOXO3a on a specific Serine residue. We identified the Cyclin D3/CDK6 complex as the one mainly involved in FOXO3a phosphorylation and proved that FOXO3a phosphorylation by the Cyclin D3/CDK6 complex leaded to increased FOXO3a protein stability. Moreover, FOXO3a downregulation sensitizes EOC cells to platinum while FOXO3a overexpression in CDK6 silenced cells was able to rescue the effect of CDK6 silencing on platinum-induced cell death. We showed that the CDK6/FOXO3a axis was necessary to regulate the DNA damage response in EOC cells through the regulation of ATR/CHK1 pathway, since after platinum treatment the silencing of both CDK6 or FOXO3a induced the downregulation of ATR and the decrease in CHK1 phosphorylation. We identified in the ATR promoter three putative FOXO3a binding sites and, using chromatin immunoprecipitation, we confirmed that ATR is a FOXO3a transcriptional target. Hence, inhibition of CDK6, FOXO3a or ATR increased platinum dependent-cell death via the induction of the so-called Premature Chromosome Condensation (PCC), a lethal event that selectively kills cancer cells when the ATR/CHK1 pathway is inhibited in the presence of damaged DNA. We demonstrated that in EOC, platinum sensitivity could be increased by specific CDK6 inhibition also using a clinically available small molecule, the PD0332991. We are currently analyzing if in primary EOC the expression of CDK6, FOXO3a and/or Cyclin D3 could be used as biomarkers to select patients that may benefit from a combination therapy based on platinum and CDK6 inhibition further increasing the translational relevance of our work. Citation Format: MONICA B. SCHIAPPACASSI, Alessandra Dall'acqua, Gustavo Baldassarre. CDK6 controls platinum sensitivity via the regulation of FOXO3a/ATR: a new actionable pathway for ovarian cancer patients. [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 B63.

Studying Nestin and its Interrelationship with Cdk5.

Current research utilizes the specific expression pattern of intermediate filaments (IF) for identifying cellular state and origin, as well as for the purpose of disease diagnosis. Nestin is commonly utilized as a specific marker and driver for CNS progenitor cell types, but in addition, nestin can be found in several mesenchymal progenitor cells, and it is constitutively expressed in a few restricted locations, such as muscle neuromuscular junctions and kidney podocytes. Alike most other members of the IF protein family, nestin filaments are dynamic, constantly being remodeled through posttranslational modifications, which alter the solubility, protein levels, and signaling capacity of the nestin filaments. Through its interactions with kinases and other signaling executors, resulting in a complex and bidirectional regulation of cell signaling events, nestin has the potential to determine whether cells divide, differentiate, migrate, or stay in place. In this review, the broad and similar roles of IFs as dynamic signaling scaffolds, is exemplified by observations of nestin functions and its interaction with the cyclin- dependent kinase 5, the atypical kinase in the family of cyclin-dependent kinases.

MiR-16 induction after CDK4 knockdown is mediated by c-Myc suppression and inhibits cell growth as well as sensitizes nasopharyngeal carcinoma cells to chemotherapy.

Cyclin-dependent kinase 4 (CDK4) is a member of cyclin-dependent kinase family which regulates G1 to S cell cycle transition. CDK4 activity is increased in many tumor types. Here, we report a negative automodulatory feedback loop between CDK4 and miR-16 that regulates cell cycle progression in nasopharyngeal carcinoma (NPC). By miRNA array and real-time PCR, we identified upregulation of tumor suppressor miR-16a, which inhibited cell cycle progression and sensitized NPC cells to chemotherapy. CDK4 knockdown reduced the expression of c-Myc, the latter of which directly suppresses the miR-16 expression by directly binding to the miR-16 promoter. Moreover, we found that miR-16 upregulation could reduce CDK4 expression by repressing CCND1 and thus forms a feedback loop via the CDK4/c-Myc/miR-16/CCND1 pathway. Finally, miR-16 was negatively correlated with CDK4 expression in NPC biopsies. In summary, our results define a double-negative feedback loop involving CDK4 and miR-16 mediated by c-Myc that modulates NPC cell growth and chemotherapy sensitivity.

Knockdown of PFTK1 Inhibits the Migration of Glioma Cells.

The prognosis of glioma patients is generally poor, so it is urgent to find out the underlying molecular mechanisms. PFTK1 is a member of cyclin-dependent kinases (Cdks) family and has been reported to contribute to tumor migration and invasion. In this study, we aimed to explore the expression and function in human glioma. Western blot and immunohistochemistry were used to evaluate the expression of PFTK1. PFTK1 expression was higher in glioma tissues compared with normal brain tissues, and its level was associated with the WHO grade in Western blot analysis. The suppression of PFTK1 expression by RNA interference was shown to inhibit the migration of glioma cells. Knockdown of PFTK1 increases E-cadherin expression and decreases vimentin expression. These data show that PFTK1 may participate in the pathogenic process of glioma, suggesting that PFTK1 can become a potential therapeutic strategy for gastric cancer.

Cyclin E Deregulation Promotes Loss of Specific Genomic Regions

Cell-cycle progression is regulated by the cyclin-dependent kinase (Cdk) family of protein kinases, so named because their activation depends on association with regulatory subunits known as cyclins. Cyclin E normally accumulates at the G1/S boundary, where it promotes S phase entry and progression by activating Cdk2. In normal cells, cyclin E/Cdk2 activity is associated with DNA replication-related functions. However, deregulation of cyclin E leads to inefficient assembly of pre-replication complexes, replication stress, and chromosome instability. In malignant cells, cyclin E is frequently overexpressed, correlating with decreased survival in breast cancer patients. Transgenic mice deregulated for cyclin E in the mammary epithelia develop carcinoma, confirming that cyclin E is an oncoprotein. However, it remains unknown how cyclin E-mediated replication stress promotes genomic instability during carcinogenesis. Here, we show that deregulation of cyclin E causes human mammary epithelial cells to enter into mitosis with short unreplicated genomic segments at a small number of specific loci, leading to anaphase anomalies and ultimately deletions. Incompletely replicated regions are preferentially located at late-replicating domains, fragile sites, and breakpoints, including the mixed-lineage leukemia breakpoint cluster region (MLL BCR). Furthermore, these regions are characterized by a paucity of replication origins or unusual DNA structures. Analysis of a large set of breast tumors shows a significant correlation between cyclin E amplification and deletions at a number of the genomic loci identified in our study. Our results demonstrate how oncogene-induced replication stress contributes to genomic instability in human cancer.

Heat Shock Inhibition of CDK5 Increases NOXA Levels through miR-23a Repression

Hyperthermia is a proteotoxic stress that is lethal when exposure is extreme but also cytoprotective in that sublethal exposure leads to the synthesis of heat shock proteins, including HSP70, which are able to inhibit stress-induced apoptosis. CDK5 is an atypical cyclin-dependent kinase family member that regulates many cellular functions including motility and survival. Here we show that exposure of a human lymphoid cell line to hyperthermia causes CDK5 insolubilization and loss of tyrosine-15 phosphorylation, both of which were prevented in cells overexpressing HSP70. Inhibition of CDK5 activity with roscovitine-sensitized cells to heat induced apoptosis indicating a protective role for CDK5 in inhibiting heat-induced apoptosis. Both roscovitine and heat shock treatment caused increased accumulation of NOXA a pro-apoptotic BH3-only member of the BCL2 family. The increased abundance of NOXA by CDK5 inhibition was not a result of changes in NOXA protein turnover. Instead, CDK5 inhibition increased NOXA mRNA and protein levels by decreasing the expression of miR-23a, whereas overexpressing the CDK5 activator p35 attenuated both of these effects on NOXA and miR-23a expression. Lastly, overexpression of miR-23a prevented apoptosis under conditions in which CDK5 activity was inhibited. These results demonstrate that CDK5 activity provides resistance to heat-induced apoptosis through the expression of miR-23a and subsequent suppression of NOXA synthesis. Additionally, they indicate that hyperthermia induces apoptosis through the insolubilization and inhibition of CDK5 activity.

Cell cycle control by a minimal Cdk network.

In present-day eukaryotes, the cell division cycle is controlled by a complex network of interacting proteins, including members of the cyclin and cyclin-dependent protein kinase (Cdk) families, and the Anaphase Promoting Complex (APC). Successful progression through the cell cycle depends on precise, temporally ordered regulation of the functions of these proteins. In light of this complexity, it is surprising that in fission yeast, a minimal Cdk network consisting of a single cyclin-Cdk fusion protein can control DNA synthesis and mitosis in a manner that is indistinguishable from wild type. To improve our understanding of the cell cycle regulatory network, we built and analysed a mathematical model of the molecular interactions controlling the G1/S and G2/M transitions in these minimal cells. The model accounts for all observed properties of yeast strains operating with the fusion protein. Importantly, coupling the model’s predictions with experimental analysis of alternative minimal cells, we uncover an explanation for the unexpected fact that elimination of inhibitory phosphorylation of Cdk is benign in these strains while it strongly affects normal cells. Furthermore, in the strain without inhibitory phosphorylation of the fusion protein, the distribution of cell size at division is unusually broad, an observation that is accounted for by stochastic simulations of the model. Our approach provides novel insights into the organization and quantitative regulation of wild type cell cycle progression. In particular, it leads us to propose a new mechanistic model for the phenomenon of mitotic catastrophe, relying on a combination of unregulated, multi-cyclin-dependent Cdk activities.

Unraveling the role of Cdk1 in postnatal neurogenesis

Event Abstract Back to Event Unraveling the role of Cdk1 in postnatal neurogenesis Quentin Marlier1*, Sebastien Verteneuil1*, Renaud Vandenbosch1, Nicolas Caron2, Philipp Kaldis3, Laurent Nguyen1 and Brigitte Malgrange1 1 GIGA Neurosciences-University of Liege, Belgium 2 Affichem, France 3 Institute of Molecular and Cell Biology, Singapore Age-related neurological disorders, including stroke and neurodegenerative diseases are becoming a major health care problem in many countries as average life expectancy is increasing. One appealing therapeutic strategy to treat neurological disorders would consist of recruiting endogenous neural precursor cells (NPCs) to replace the lost neurons. NPCs are present in two specific areas of the postnatal brain, the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ) of the lateral ventricles. These NPCs give rise to neurons throughout life, a phenomenon termed postnatal neurogenesis. A prerequisite for the development of new therapeutic strategies involving postnatal NPCs is a better understanding of their molecular regulation. In this context, both extrinsic factors and intrinsic factors have been identified. For instance, the family of cell cycle regulators cyclin-dependent kinases (Cdks) are key regulators of postnatal neurogenesis since genetic invalidation of interphase Cdk2 or Cdk6 leads to a decrease of NPCs proliferation. Here, we investigated the role of Cdk1, an essential Cdk for M-phase in several tissues, in postnatal neurogenesis. Towards that purpose, we crossed mice bearing a conditional Cdk1 allele (Cdk1lox) with mice expressing a tamoxifen-inducible form of the Cre-recombinase under the control of the Sox2 promoter (Sox2CreER), allowing us to specifically delete Cdk1 in Sox2+ NPCs in the postnatal brain. Following Cdk1 loss, our preliminary results show a decrease in the percentage of proliferating Sox2+ cells as well as a decrease in the number of phospho-histone H3+ (ph-h3) in the DG, but there is no difference in the number of cells positive for the apoptotic marker activated-caspase 3. Interestingly, we also observed a long-term depletion of the pool of Sox2+ in the absence of Cdk1. Altogether, these data suggest a crucial role for Cdk1 in postnatal neurogenesis, but further investigations are required to identify its precise requirement in this process. Keywords: adult neurogenesis, Cell Cycle, Cdk1, Neural Stem Cells, proliferation Conference: 11th National Congress of the Belgian Society for Neuroscience, Mons, Belgium, 22 May - 22 May, 2015. Presentation Type: Poster presentation Topic: Neuroscience Citation: Marlier Q, Verteneuil S, Vandenbosch R, Caron N, Kaldis P, Nguyen L and Malgrange B (2015). Unraveling the role of Cdk1 in postnatal neurogenesis. Front. Neurosci. Conference Abstract: 11th National Congress of the Belgian Society for Neuroscience. doi: 10.3389/conf.fnins.2015.89.00069 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Apr 2015; Published Online: 05 May 2015. * Correspondence: Mr. Quentin Marlier, GIGA Neurosciences-University of Liege, Liège, 4000, Belgium, quentin.marlier@ulg.ac.be Mr. Sebastien Verteneuil, GIGA Neurosciences-University of Liege, Liège, 4000, Belgium, sebastien.verteneuil@hotmail.fr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Quentin Marlier Sebastien Verteneuil Renaud Vandenbosch Nicolas Caron Philipp Kaldis Laurent Nguyen Brigitte Malgrange Google Quentin Marlier Sebastien Verteneuil Renaud Vandenbosch Nicolas Caron Philipp Kaldis Laurent Nguyen Brigitte Malgrange Google Scholar Quentin Marlier Sebastien Verteneuil Renaud Vandenbosch Nicolas Caron Philipp Kaldis Laurent Nguyen Brigitte Malgrange PubMed Quentin Marlier Sebastien Verteneuil Renaud Vandenbosch Nicolas Caron Philipp Kaldis Laurent Nguyen Brigitte Malgrange Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Roscovitine-treated HeLa cells finalize autophagy later than apoptosis by downregulating Bcl‑2.

The cell cycle is tightly regulated by the family of cyclin-dependent kinases (CDKs). CDKs act as regulatory factors on serine and threonine residues by phosphorylating their substrates and cyclins. CDK‑targeting drugs have previously demonstrated promising effects as cancer therapeutics both in vitro and in vivo. Roscovitine, a purine‑derivative and specific CDK inhibitor, has been demonstrated to arrest the cell cycle and induce apoptosis in a number of different cancer cell lines, including HeLa cervical cancer cells. In the present study, roscovitine was able to decrease both the cell viability and cell survival as well as induce apoptosis in a dose‑dependent manner in HeLa cells by modulating the mitochondrial membrane potential. The decrease of anti‑apoptotic B-cell lymphoma 2 (Bcl‑2) and Bcl-2 extra large protein expression was accompanied by the increase in pro‑apoptotic Bcl-2-associated X protein and P53-upregulated modulator of apoptosis expression. The marked decrease in Bcl‑2 following exposure to roscovitine (20 µM) for 48 h prompted us to determine the autophagic regulation. The outcome revealed that roscovitine triggered Beclin‑1 downregulation and microtubule-associated light chain 3 cleavage starting from 12 h of incubation. Another biomarker of autophagy, p62, a crucial protein for autophagic vacuole formation, was diminished following 48 h. In addition, monodansyl cadaverin staining of autophagosomes also confirmed the autophagic regulation by roscovitine treatment. The expression levels of different Bcl‑2 family members determined whether apoptosis or autophagy were induced following incubation with roscovitine for different time periods. Downregulation of pro‑apoptotic Bcl‑2 family members indicated induction of apoptosis, while the downregulation of anti‑apoptotic Bcl‑2 family members rapidly induced autophagosome formation in HeLa cells.

Polo kinase regulates the localization and activity of the chromosomal passenger complex in meiosis and mitosis in Drosophila melanogaster.

Cell cycle progression is regulated by members of the cyclin-dependent kinase (CDK), Polo and Aurora families of protein kinases. The levels of expression and localization of the key regulatory kinases are themselves subject to very tight control. There is increasing evidence that crosstalk between the mitotic kinases provides for an additional level of regulation. We have previously shown that Aurora B activates Polo kinase at the centromere in mitosis, and that the interaction between Polo and the chromosomal passenger complex (CPC) component INCENP is essential in this activation. In this report, we show that Polo kinase is required for the correct localization and activity of the CPC in meiosis and mitosis. Study of the phenotype of different polo allele combinations compared to the effect of chemical inhibition revealed significant differences in the localization and activity of the CPC in diploid tissues. Our results shed new light on the mechanisms that control the activity of Aurora B in meiosis and mitosis.

Proteomic Analysis of the Human Cyclin-dependent Kinase Family Reveals a Novel CDK5 Complex Involved in Cell Growth and Migration

Cyclin-dependent kinases (CDKs) are the catalytic subunits of a family of mammalian heterodimeric serine/threonine kinases that play critical roles in the control of cell-cycle progression, transcription, and neuronal functions. However, the functions, substrates, and regulation of many CDKs are poorly understood. To systematically investigate these features of CDKs, we conducted a proteomic analysis of the CDK family and identified their associated protein complexes in two different cell lines using a modified SAINT (Significance Analysis of INTeractome) method. The mass spectrometry data were deposited to ProteomeXchange with identifier PXD000593 and DOI 10.6019/PXD000593. We identified 753 high-confidence candidate interaction proteins (HCIPs) in HEK293T cells and 352 HCIPs in MCF10A cells. We subsequently focused on a neuron-specific CDK, CDK5, and uncovered two novel CDK5-binding partners, KIAA0528 and fibroblast growth factor (acidic) intracellular binding protein (FIBP), in non-neuronal cells. We showed that these three proteins form a stable complex, with KIAA0528 and FIBP being required for the assembly and stability of the complex. Furthermore, CDK5-, KIAA0528-, or FIBP-depleted breast cancer cells displayed impaired proliferation and decreased migration, suggesting that this complex is required for cell growth and migration in non-neural cells. Our study uncovers new aspects of CDK functions, which provide direction for further investigation of these critical protein kinases.