Specific Inhibitor Of Cyclin-dependent Kinases Research Articles

Silencing of p53 and CDKN1A establishes sustainable immortalized megakaryocyte progenitor cells from human iPSCs.

SummaryPlatelet transfusions are critical for severe thrombocytopenia but depend on blood donors. The shortage of donors and the potential of universal HLA-null platelet products have stimulated research on the ex vivo differentiation of human pluripotent stem cells (hPSCs) to platelets. We recently established expandable immortalized megakaryocyte cell lines (imMKCLs) from hPSCs by transducing MYC, BMI1, and BCL-XL (MBX). imMKCLs can act as cryopreservable master cells to supply platelet concentrates. However, the proliferation rates of the imMKCLs vary with the starting hPSC clone. In this study, we reveal from the gene expression profiles of several MKCL clones that the proliferation arrest is correlated with the expression levels of specific cyclin-dependent kinase inhibitors. Silencing CDKN1A and p53 with the overexpression of MBX was effective at stably inducing imMKCLs that generate functional platelets irrespective of the hPSC clone. Collectively, this improvement in generating imMKCLs should contribute to platelet industrialization and platelet biology.

Cyclin-Dependent Kinase Inhibitors in Hematological Malignancies-Current Understanding, (Pre-)Clinical Application and Promising Approaches.

Simple SummaryCyclin-dependent kinases are involved in the regulation of cancer-initiating processes like cell cycle progression, transcription, and DNA repair. In hematological neoplasms, these enzymes are often overexpressed, resulting in increased cell proliferation and cancer progression. Early (pre-)clinical data using cyclin-dependent kinase inhibitors are promising but identifying the right drug for each subgroup and patient is challenging. Certain chromosomal abnormalities and signaling molecule activities are considered as potential biomarkers. We therefore summarized relevant studies investigating cyclin-dependent kinase inhibitors in hematological malignancies and further discuss molecular mechanisms of resistance and other open questions.Genetically altered stem or progenitor cells feature gross chromosomal abnormalities, inducing modified ability of self-renewal and abnormal hematopoiesis. Cyclin-dependent kinases (CDK) regulate cell cycle progression, transcription, DNA repair and are aberrantly expressed in hematopoietic malignancies. Incorporation of CDK inhibitors (CDKIs) into the existing therapeutic regimens therefore constitutes a promising strategy. However, the complex molecular heterogeneity and different clinical presentation is challenging for selecting the right target and defining the ideal combination to mediate long-term disease control. Preclinical and early clinical data suggest that specific CDKIs have activity in selected patients, dependent on the existing rearrangements and mutations, potentially acting as biomarkers. Indeed, CDK6, expressed in hematopoietic cells, is a direct target of MLL fusion proteins often observed in acute leukemia and thus contributes to leukemogenesis. The high frequency of aberrancies in the retinoblastoma pathway additionally warrants application of CDKIs in hematopoietic neoplasms. In this review, we describe the preclinical and clinical advances recently made in the use of CDKIs. These include the FDA-approved CDK4/6 inhibitors, traditional and novel pan-CDKIs, as well as dual kinase inhibitors. We additionally provide an overview on molecular mechanisms of response vs. resistance and discuss open questions.

Cyclin-dependent Kinases as Emerging Targets for Developing Novel Antiviral Therapeutics.

Besides its prominent role in cell proliferation, cyclin-dependent kinases (CDKs) are key players in viral infections as both DNA and RNA viruses modify CDK function to favor viral replication. Recently, a number of specific pharmacological CDK inhibitors have been developed and approved for cancer treatment. The repurposing of these specific CDK inhibitors for the treatment of viral infections may represent a novel effective therapeutic strategy to combat old and emergent viruses. In this review, we describe the role, mechanisms of action, and potential of CDKs as antiviral drug targets. We also discuss the current clinical state of novel specific CDK inhibitors, focusing on their putative use as antivirals, especially against new emerging viruses.

S-adenosylmethionine administration inhibits levodopa-induced vascular endothelial growth factor-A expression.

Background: Studies have demonstrated that S-adenosylmethionine could effectively affect the clinical wearing-off phenomena of levodopa, an antiparkinsonian agent; however, the detailed mechanisms for this effect need to be further clarified.Results: S-adenosylmethionine and levodopa had opposite effects on the protein stability of vascular endothelial growth factor-A. The analysis of tube formation and cell viability also showed the nonconforming functions of S-adenosylmethionine and levodopa on cell angiogenesis and proliferation. Meanwhile, S-adenosylmethionine could significantly abolish the increased angiogenesis and cell viability induced by levodopa. S-adenosylmethionine resulted in G1/S phase arrest, with decreased cyclin dependent kinase 4/6 and increased p16, a specific cyclin dependent kinase inhibitor. Mechanically, the different effects of levodopa and S-adenosylmethionine were dependent on the phosphorylation and activation of extracellular signal-regulated kinase. S-adenosylmethionine could be fitted into the predicted docking pocket in the crystal structure of vascular endothelial growth factor-A, enhancing its acetylation level and reducing half-life.Conclusions: These observations suggested that methyl donor S-adenosylmethionine could act as a potential agent against vascular endothelial growth factor-A-related diseases induced by levodopa treatment.Methods: We performed in vitro cytological analyses to assess whether S-adenosylmethionine intake could influence levodopa-induced vascular endothelial growth factor-A expression in human umbilical vein endothelial cells.

CDKN2C-Null Leiomyosarcoma: A Novel, Genomically Distinct Class of TP53/RB1–Wild-Type Tumor With Frequent CIC Genomic Alterations and 1p/19q-Codeletion

PURPOSELeiomyosarcoma (LMS) harbors frequent mutations in TP53 and RB1 but few actionable genomic alterations. Here, we searched for recurrent actionable genomic alterations in LMS that occur in the absence of common untreatable oncogenic drivers.METHODSTissues from 276,645 unique advanced cancers, including 2,570 uterine and soft tissue LMS, were sequenced by hybrid-capture–based next-generation DNA and RNA sequencing/comprehensive genomic profiling of up to 406 genes. We characterized clinicopathologic features of relevant patient cases.RESULTSOverall, 77 LMS exhibited homozygous copy loss of CDKN2C at chromosome 1p32.3 (3.0% of LMS). Genomic alterations (GAs) in TP53, RB1, and ATRX were rare compared with the remainder of the LMS cohort (11.7% v 73.4%, 0% v 54.5%, 2.6% v 24.5%, respectively; all P < .0001). CDKN2C-null LMS patient cases were significantly enriched for GAs in CIC (40.3% v 1.4%) at 19q13.2, CDKN2A (46.8% v 7.0%), and RAD51B (16.9% v 1.7%; all P < .0001). Chromosome arm-level aneuploidy analysis of available LMS patient cases (n = 1,284) found that 81% (58 of 72) of CDKN2C-null LMS exhibited 1p/19q-codeletion, a significant enrichment compared with 5.1% in the remainder of the LMS cohort (P < .0001). In total, 99% of CDKN2C-null LMS were in women; the median age was 61 years at surgery (range, 36-81 years). Fifty-five patient cases were uterine primary, four were nonuterine, and the remaining 18 were of uncertain primary site. Sixty percent of cases showed at least focal epithelioid variant histology. Most patients had advanced-stage disease, with 62% of confirmed uterine primary LMS at International Federation of Gynecology and Obstetrics stage IVB. We further validated our findings in two publicly available datasets: The Cancer Genome Atlas and the Project GENIE initiative.CONCLUSIONCDKN2C-null LMS defines a genomically distinct tumor that may have prognostic and/or therapeutic clinical implications, including possible use of specific cyclin-dependent kinase inhibitors.

Abstract PD2-04: ERα phosphorylation at pS294: A biomarker of ligand or mutational (Y537S, D538G) activation, and a receptor target for CDK2 inhibition

Abstract Background: Certain ERα phosphorylation (p) sites are essential for ERα transcriptional activity; and with development of ERα p-specific antibodies, some of these sites predict endocrine responsiveness. Unlike other ERα p-sites, pS294 has been shown to be induced by ligand activation and not by cross-talking growth factor signals. With development of a new rabbit monoclonal, pS294 induction was found to be dependent on a cyclin-dependent kinase (CDK). This study aimed to identify the specific CDK mediating induction of pS294, determine if ligand-independent ERα activating mutations (Y537S, D538G) also induce pS294, and learn if specific CDK inhibitors might enhance endocrine therapeutic efficacy by suppressing pS294. Methods: MCF7 cells, untreated (stripped media) or stimulated by estradiol (E2, 10nM) or growth factor (EGF, 5nM), were treated with either CDK-specific knockdown siRNAs or small molecule CDK inhibitors (with indicated specificities): Roscovitine (pan-CDKs); Dinaciclib (CDK1, CDK2, CDK5, CDK9); Palbociclib (CDK4, CDK6); JNJ7706621 (CDK1, CDK2); BMS265246 (CDK1, CDK2); and SNS032 (CDK2, CDK7, CDK9). Whole cell, nuclear or cytosolic lysates were either Western blotted (for ERα or specific CDKs) or first immunoprecipitated (total ERα, pS294-ERα) and then immunoblotted. RT-PCR of cellular RNA quantified pS294-ERα induced transcripts (EGF3, AREG, CXCL12 vs. GAPDH) potentially inhibited by CDK inhibitors. MCF7 overexpressing ERα activating mutations (Y537S, D538G) were produced by either transient transfection or knock-in; knock-in clones were innoculated into immunocompromised mice to assess ligand-independent xenograft tumor growth in vivo, while transfected cells and tumors were assessed for ligand-independent ERα phosphorylation. Results: CDK2 was determined to be the primary kinase mediating ligand-dependent induction of pS294-ERα, with co-precipitation of cyclins A/E confirming the expected mechanism of CDK2 recruitment to chromatin-bound pS294-ERα. Knock-in MCF7 cells expressing either Y537S or D538G ERα rapidly formed tumors in vivo without E2 supplementation; tumors and transiently transfected cells overexpressing mutated ERα showed pS294 &amp;gt;&amp;gt; pS118 expression, with constitutive pS294 suppressed by Dinaciclib but not by Palbociclib. CDK1/2 inhibitors (Dinaciclib, BMS265246) but not a CDK4/6 inhibitor (Palbociclib) cooperated with tamoxifen (4-HT) to induce apoptosis in wildtype MCF7. Conclusion: CDK2 is the primary mediator of pS294 induced by either ligand stimulation or ligand-independent mutational activation of ERα. While the CDK4/6 inhibitor Palbociclib is a recently approved adjunct to endocrine therapy, it enhances cytostatic growth arrest without affecting ERα phosphorylation or receptor induced gene expression. In contrast, CDK2 inhibitors like Dinaciclib should be explored for their ability to enhance ER-positive breast cancer cell death in combination with antiestrogens and for their ability to prevent the emergence of constitutively active ERα mutations by suppressing pS294 induction, essential for ERα mediated gene transactivation and breast tumor growth. Citation Format: Benz CC, Scott GK, Chu D, Kaur R, Muthurajah M, Rothschild D, Frazier K, Park BH. ERα phosphorylation at pS294: A biomarker of ligand or mutational (Y537S, D538G) activation, and a receptor target for CDK2 inhibition. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD2-04.

Protein-Protein Interaction for the De Novo Design of Cyclin-Dependent Kinase Peptide Inhibitors.

The homology of the inhibitor binding site regions on the surface of cyclin-dependent kinases (CDKs) makes actual CDK inhibitors unable to bind specifically to their molecular targets. Most of them are ATP competitive inhibitors with low specificity that also affect the phosphorylation mechanisms of other nontarget kinases giving rise to harmful side effects. So, the search of specific and potent inhibitors able to bind to the desired CDK target is still a pending issue. Structure based drug design minimized the erroneous binding and increased the affinity of the inhibitor interaction. In the case of CDKs their activation and regulation mechanisms mainly depend on protein-protein interactions (PPIs). The design of drugs targeting these PPIs makes feasible and promising towards the discovery of new and specific CDK inhibitors. Development of peptide inhibitors for a target protein is an emerging approach in computer aided drug designing. This chapter describes in detail methodology for use of the VitAL-Viterbi algorithm for de novo peptide design of CDK2 inhibitors.

Abstract 4555: Inhibition of CDK1/2 but not CDK4/6 cooperates with tamoxifen to induce apoptosis

Abstract As essential drivers of cell cycle progression and transcription, cyclin-dependent kinases (CDKs) often become dysregulated during tumorigenesis, thus motivating extensive efforts towards development of CDK inhibitors as a new class of promising anticancer agents with clear CDK specificity including those targeting mainly CDK4 and CDK6 (e.g. PD0332991, LY2835219) vs. those primarily targeting CDK1 and CDK2 (e.g. BMS265246, Dinaciclib). Given there different cyclin dependencies and mitotic phase mechanisms, including the relative cell cycle independence of some CDKs, specific CDK inhibitors are expected to exhibit different anticancer activities, side effects, and interactions with other anticancer agents, and require different clinical biomarkers. With regard to breast cancers in which at least 75% overexpress estrogen receptor (ER, alpha isoform), it is particularly important to establish the type of CDK inhibitor most effective in combination with anti-estrogenic agents like tamoxifen or aromatase inhibitors. To preclinically determine the potential added efficacy of specific CDK inhibition in combination with anti-estrogen therapy, the ER-positive human breast cancer cell line MCF7 was used to compare the relative efficacy of CDK1/2 inhibition vs. CDK4/6 inhibition in combination with 4-hydroxytamoxifen (Tam) in promoting breast cancer cell death (apoptosis). These studies were also designed based on our recent evidence that CDK1/2 inhibition, but not CDK4/6 inhibition, suppressed transcriptional upregulation of ER inducible genes by impairing ER hinge domain phosphorylation at Ser294, suggesting that CDK1/2 inhibition would preferentially cooperate with Tam to impair ER-positive breast cancer viability by their convergent suppression of ligand-dependent ER transcription. We confirmed this hypothesis by demonstrating that CDK1/2 inhibitors, but not CDK4/6 inhibitors, suppressed ligand induced ER phosphorylation at Ser294, without altering Ser118 phosphorylation which remained constant despite either CDK1/2 or CDK4/6 inhibition. Consistent with its ability to suppress ER phosphorylation at Ser294, CDK1/2 inhibition, in combination with Tam, enhanced MCF7 apoptosis, while CDK4/6 inhibition had no such cooperative effect. These ER-positive breast cancer cell line and molecular studies should guide further preclinical and clinical efforts in evaluating the optimal choice of CDK inhibitor for use in combination with endocrine agents. These findings also suggest that clinical emphasis should be placed on the evaluation of CDK1/2 inhibitors in combination with anti-estrogens like tamoxifen, where loss of ER phosphorylation at Ser294 can also be used as an early biomarker of response to monitor this therapeutic combination. Citation Format: Gary K. Scott, Daniel Rothschild, Ravneet Kaur, Christopher Benz. Inhibition of CDK1/2 but not CDK4/6 cooperates with tamoxifen to induce apoptosis. [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 4555. doi:10.1158/1538-7445.AM2014-4555

Cyclin-dependent Kinase 9 Links RNA Polymerase II Transcription to Processing of Ribosomal RNA

Ribosome biogenesis is a process required for cellular growth and proliferation. Processing of ribosomal RNA (rRNA) is highly sensitive to flavopiridol, a specific inhibitor of cyclin-dependent kinase 9 (Cdk9). Cdk9 has been characterized as the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Here we studied the connection between RNAPII transcription and rRNA processing. We show that inhibition of RNAPII activity by α-amanitin specifically blocks processing of rRNA. The block is characterized by accumulation of 3' extended unprocessed 47 S rRNAs and the entire inhibition of other 47 S rRNA-specific processing steps. The transcription rate of rRNA is moderately reduced after inhibition of Cdk9, suggesting that defective 3' processing of rRNA negatively feeds back on RNAPI transcription. Knockdown of Cdk9 caused a strong reduction of the levels of RNAPII-transcribed U8 small nucleolar RNA, which is essential for 3' rRNA processing in mammalian cells. Our data demonstrate a pivotal role of Cdk9 activity for coupling of RNAPII transcription with small nucleolar RNA production and rRNA processing.

A novel CyclinE/CyclinA-CDK Inhibitor targets p27Kip1 degradation, cell cycle progression and cell survival: Implications in cancer therapy

p27Kip1 (p27) binds and inhibits the cyclin E- or cyclin A-associated cyclin-dependent kinases (CDKs)2 and other CDKs, and negatively regulates G1–G2 cell cycle progression. To develop specific CDK inhibitors, we have modeled the interaction between p27 and cyclin A-CDK2, and designed a novel compound that mimics p27 binding to cyclin A-CDK2. The chemically synthesized inhibitor exhibited high potency and selective inhibition towards cyclin E/cyclin A-CDK2 kinase in vitro but not other kinases. To facilitate permeability of the inhibitor, a cell penetrating peptide (CPP) was conjugated to the inhibitor to examine its effect in several cancer cell lines. The CPP-conjugated inhibitor significantly inhibited the proliferation of cancer cells. The treatment of the inhibitor resulted in the increased accumulation of p27 and p21Cip1/Waf1 (p21) and hypo-phosphorylation of retinoblastoma protein (Rb). The degradation of p27, mediated through the phosphorylation of threonine-187 in p27, was also inhibited. Consequently, exposure of cells to the inhibitor caused cell cycle arrest and apoptosis. We conclude that specific cyclinE/cyclin A-CDK2 inhibitors can be developed based on the interaction between p27 and cyclin/CDK to block cell cycle progression to prevent tumor growth and survival.

Cyclin-Dependent Kinases: Bridging Their Structure and Function Through Computations

Cyclin-dependent kinases (CDKs) are one of the most promising target families for drug discovery for several diseases, such as cancer and neurodegenerative disorders. Over the years, structural insights on CDKs have demonstrated high protein plasticity, with several cases where two or more structures of the same protein adopt different conformations. This has generated a great deal of interest in understanding the relationship between CDK structure and function. Here, we highlight how computer simulations have recently contributed in characterizing some key rare and transient events in CDKs, such as the reaction transition state and activation loop movement. Although not yet fully defined, we can now portray the enzymatic mechanism and plasticity of CDKs at high spatial and temporal resolution. These theoretical studies bridge with experiments and highlight structural determinants that could help in designing specific CDK inhibitors.

Quantitative analysis of PD 0332991 in mouse plasma using automated micro-sample processing and microbore liquid chromatography coupled with tandem mass spectrometry

In the oncology therapeutic area, the mouse is the primary animal model used for efficacy studies. Often with mouse pharmacokinetic (PK) and pharmacokinetic/pharmacodynamic (PK/PD) studies, less than 20 μL of total plasma sample volume is available for bioanalysis due to the small size of the animal and the need to split samples for other measurements such as biomarker analyses. The need to conduct automated “small volume” sample processing for quantitative bioanalysis has therefore increased. An automated fit for purpose protein precipitation (PPT) method using a Hamilton MicroLab Star (Reno, NV, USA) to support mouse PK and PK/PD studies for an oncology drug candidate PD 0332991, (a specific inhibitor of cyclin-dependent kinase 4 (CDK-4) currently in development) for processing “small volumes” was developed. The automated PPT method was achieved by extracting and processing 10 μL out of a minimum sample volume of 15 μL plasma utilizing the Hamilton MicroLab Star. A 96-conical shallow well plate by Agilent Technologies, Inc (Wilmington, DE, USA) was the labware of choice used in the automated Hamilton “small volume” method platform. Analyses of a 10 μL plasma aliquot from 15 μL of plasma study samples were conducted by both automated and manual PPT method. All plasma samples were quantitated using a Sciex API 4000 triple quadrupole mass spectrometer coupled with an Eksigent Express HT Ultra HPLC system. The chromatography was achieved using an Agilent microbore C 18 Extend, 1.0 × 50 mm, 3.5 μm column at a flow rate of 0.150 mL/min with a total run time of 1.8 min. Accuracy and precision of standard and QC concentration levels were within 90–107% and <14%, respectively. Calibration curves were linear over the dynamic range of 1.0–1000 ng/mL. PK studies for PD 0332991 were conducted in female C3H mice following intravenous administration at 1 mg/kg and oral administration at 2 mg/kg. PK values such as area under curve (AUC), volume of distribution (Vd), clearance (Cl), half life ( T 1/2) and bioavailability ( F%) demonstrated less than 11% difference between the automated Hamilton and manual PPT methods. The results demonstrate that the automated Hamilton PPT method can accurately and precisely aliquot 10 μL of plasma from 15 μL or larger volume plasma samples. The fit for purpose Hamilton PPT method is suitable for routine analyses of plasma samples from micro-sampling PK and PK/PD samples to support discovery studies.

High Throughput Screens Yield Small Molecule Inhibitors of Leishmania CRK3:CYC6 Cyclin-Dependent Kinase

Background Leishmania species are parasitic protozoa that have a tightly controlled cell cycle, regulated by cyclin-dependent kinases (CDKs). Cdc2-related kinase 3 (CRK3), an essential CDK in Leishmania and functional orthologue of human CDK1, can form an active protein kinase complex with Leishmania cyclins CYCA and CYC6. Here we describe the identification and synthesis of specific small molecule inhibitors of bacterially expressed Leishmania CRK3:CYC6 using a high throughput screening assay and iterative chemistry. We also describe the biological activity of the molecules against Leishmania parasites.Methodology/Principal FindingsIn order to obtain an active Leishmania CRK3:CYC6 protein kinase complex, we developed a co-expression and co-purification system for Leishmania CRK3 and CYC6 proteins. This active enzyme was used in a high throughput screening (HTS) platform, utilising an IMAP fluorescence polarisation assay. We carried out two chemical library screens and identified specific inhibitors of CRK3:CYC6 that were inactive against the human cyclin-dependent kinase CDK2:CycA. Subsequently, the best inhibitors were tested against 11 other mammalian protein kinases. Twelve of the most potent hits had an azapurine core with structure activity relationship (SAR) analysis identifying the functional groups on the 2 and 9 positions as essential for CRK3:CYC6 inhibition and specificity against CDK2:CycA. Iterative chemistry allowed synthesis of a number of azapurine derivatives with one, compound 17, demonstrating anti-parasitic activity against both promastigote and amastigote forms of L. major. Following the second HTS, 11 compounds with a thiazole core (active towards CRK3:CYC6 and inactive against CDK2:CycA) were tested. Ten of these hits demonstrated anti-parasitic activity against promastigote L. major.Conclusions/SignificanceThe pharmacophores identified from the high throughput screens, and the derivatives synthesised, selectively target the parasite enzyme and represent compounds for future hit-to-lead synthesis programs to develop therapeutics against Leishmania species. Challenges remain in identifying specific CDK inhibitors with both target selectivity and potency against the parasite.

Structural and Functional Analysis of Cyclin D1 Reveals p27 and Substrate Inhibitor Binding Requirements

An alternative strategy for inhibition of the cyclin dependent kinases (CDKs) in antitumor drug discovery is afforded through the substrate recruitment site on the cyclin positive regulatory subunit. Critical CDK substrates such as the Rb and E2F families must undergo cyclin groove binding before phosphorylation, and hence inhibitors of this interaction also block substrate specific kinase activity. This approach offers the potential to generate highly selective and cell cycle specific CDK inhibitors and to reduce the inhibition of transcription mediated through CDK7 and 9, commonly observed with ATP competitive compounds. While highly potent peptide and small molecule inhibitors of CDK2/cyclin A, E substrate recruitment have been reported, little information has been generated on the determinants of inhibitor binding to the cyclin groove of the CDK4/cyclin D1 complex. CDK4/cyclin D is a validated anticancer drug target and continues to be widely pursued in the development of new therapeutics based on cell cycle blockade. We have therefore investigated the structural basis for peptide binding to its cyclin groove and have examined the features contributing to potency and selectivity of inhibitors. Peptidic inhibitors of CDK4/cyclin D of pRb phosphorylation have been synthesized, and their complexes with CDK4/cyclin D1 crystal structures have been generated. Based on available structural information, comparisons of the cyclin grooves of cyclin A2 and D1 are presented and provide insights into the determinants for peptide binding and the basis for differential binding and inhibition. In addition, a complex structure has been generated in order to model the interactions of the CDKI, p27(KIP)¹, with cyclin D1. This information has been used to shed light onto the endogenous inhibition of CDK4 and also to identify unique aspects of cyclin D1 that can be exploited in the design of cyclin groove based CDK inhibitors. Peptidic and nonpeptidic compounds have been synthesized in order to explore structure-activity relationship for binding to the cyclin D1 groove, which to date has not been carried out in a systematic fashion. Collectively, the data presented provide new insights into how compounds can be developed that function as chemical biology probes to determine the cellular and antitumor effects of CDK inhibition. Furthermore, such compounds will serve as templates for structure-guided efforts to develop potential therapeutics based on selective inhibition of CDK4/cyclin D activity.

Quantitative analysis of PD 0332991 in xenograft mouse tumor tissue by a 96-well supported liquid extraction format and liquid chromatography/mass spectrometry

Phase II attrition of clinical candidates in the drug development cycle is currently a major issue facing the pharmaceutical industry. To decrease phase II attrition, there is an increased emphasis on validation of mechanism of action, development of efficacy models and measurement of drug levels at the site of action. PD 0332991, a highly specific inhibitor of cyclin-dependent kinase 4 (CDK-4) is currently in clinical development for the treatment of solid tumor. A clinical presurgical study will be required to better understand how PD 0332991 affects signaling pathways and how the intratumoral concentration of PD 0332991 correlates with plasma PK parameters and molecular alterations in breast cancer tissues after PD 0332991 treatment. Before conducting such a clinical study, it is important to evaluate PD 0332991 levels in tumor tissue samples from a xenograft mouse model for the determination of drug exposure at the site of action. Therefore, the objectives of this study were (1) to develop and validate a sensitive LC–MS/MS method to quantify PD 0332991 in mouse tumor tissues from MDA-MB-231-Luc human breast tumor xenografts in SCID-beige mice; (2) to quantify PD 0332991 levels in mouse tumor tissues after oral administration of PD 0332991 at 10 and 100 mg/kg using the validated LC–MS/MS method. Both liquid–liquid extraction (LLE) and supported liquid extraction (SLE) in a 96-well format were developed and evaluated to achieve optimal extraction recovery with minimal matrix effects. The newly developed SLE method is more efficient (speed and ease) and demonstrates comparable recovery (93.1–100% at three different concentrations) compared to the traditional LLE method. The validated LC–MS/MS for PD 032291 in mouse tumor tissue homogenate method exhibited a linear dynamic range of 0.1–100 ng/mL with inter-day accuracy and precision within 15%. The validated method was successfully applied to measure PD 0332991 levels in tumor tissues in MDA-MB-231-Luc human breast tumor xenografts in SCID beige mice. The mean tumor concentrations at 6 h post-oral PD 0332991 administration at 10 and 100 mg/kg were 1793 (±1008) and 25,163 (±3959) ng/g, respectively.

Cyclin-Dependent Kinase 5 Phosphorylates Endothelial Nitric Oxide Synthase at Serine 116

Nitric oxide (NO) production in endothelial cells (EC) is regulated by multisite phosphorylation of specific serine and threonine residues in endothelial NO synthase (eNOS). Among these, eNOS-Ser116 is phosphorylated in the basal state, and its phosphorylation contributes to basal NO production. Here, we investigated the mechanism by which eNOS-Ser116 is phosphorylated during the basal state using bovine aortic EC. Although a previous study suggested that protein kinase C was involved in eNOS-Ser116 phosphorylation, overexpression of various protein kinase C isoforms did not affect eNOS-Ser116 phosphorylation. An in silico analysis using a motif scan revealed that the eNOS-Ser116 residue might be a substrate for proline-directed protein kinases. Roscovitine, a specific inhibitor of cyclin-dependent kinase (CDK), 1, 2, and 5, but not an inhibitor of mitogen-activated protein kinase kinase or glycogen synthase kinase 3beta, inhibited eNOS-Ser116 phosphorylation dose dependently. Furthermore, purified CDK1, 2, or 5 directly phosphorylated eNOS-Ser116 in vitro. Ectopic expression of the dominant-negative CDK5 but not dominant-negative CDK1 or dominant-negative CDK2 repressed eNOS-Ser116 phosphorylation and increased NO production. In addition, CDK5 activity was detected in bovine aortic EC, and coimmunoprecipitation and confocal microscopy studies revealed a colocalization of eNOS and CDK5. Cotransfection of CDK5 and p25, the specific CDK5 activator, increased eNOS-Ser116 phosphorylation and decreased NO production, but its parent molecule, p35, and p39, another activator, were not detected in bovine aortic EC, which suggests the existence of a novel CDK5 activator. Overall, this is the first study to find that CDK5 is a physiological kinase responsible for eNOS-Ser116 phosphorylation and regulation of NO production.

Expression of p16 protein in acral lentiginous melanoma

Acral lentiginous melanoma (ALM) is a clinicopathologic subtype of cutaneous malignant melanoma. ALM is the most common type of melanoma amongst Asians, Africans, and patients with mixed ancestry. In Brazil, ALM comprises 10% of cutaneous melanoma. ALM develops on palmar, plantar, and subungual skin, and its biology is different from that of other cutaneous melanomas, where sunlight is the major known environmental determinant. Alterations and inactivation of the p16INK4 gene that encodes a specific inhibitor of cyclin-dependent kinase have been related to melanoma genesis and progression. Few studies, however, have addressed p16 expression in ALM. In order to verify and compare p16 protein expression, 32 paraffin-embedded ALM specimens were subjected to a immunohistochemical technique using a monoclonal anti-p16 antibody. The tumors were classified according to thickness (up to 1.0 mm and thicker than 1.0 mm) and the presence of ulceration. Twenty-five (78%) ALMs displayed positive p16 protein expression: 21 of the 25 (84%) with a thickness of more than 1.0 mm, and four of the seven (57%) with a thickness of 1.0 mm or less. Thirteen of the 17 (76%) nonulcerated lesions and 12 of the 15 (80%) ulcerated lesions displayed positive p16 protein expression. The data obtained suggest that p16 protein expression per se may not represent a marker of retinoblastoma protein (pRb) pathway disturbance in ALM tumorigenesis.

Transforming Growth Factor-β Requires Its Target Plasminogen Activator Inhibitor-1 for Cytostatic Activity

The cytokine transforming growth factor beta (TGFbeta) has strong antiproliferative activity in most normal cells but contributes to tumor progression in the later stages of oncogenesis. It is not fully understood which TGFbeta target genes are causally involved in mediating its cytostatic activity. We report here that suppression of the TGFbeta target gene encoding plasminogen activator inhibitor-1 (PAI-1) by RNA interference leads to escape from the cytostatic activity of TGFbeta both in human keratinocytes (HaCaTs) and primary mouse embryo fibroblasts. Consistent with this, PAI-1 knock-out mouse embryo fibroblasts are also resistant to TGFbeta growth arrest. Conversely, we show that ectopic expression of PAI-1 in proliferating HaCaT cells induces a growth arrest. PAI-1 knockdown does not interfere with canonical TGFbeta signaling as judged by SMAD phosphorylation and induction of bona fide TGFbeta target genes. Instead, knockdown of PAI-1 results in sustained activation of protein kinase B. Significantly, we find that constitutive protein kinase B activity leads to evasion of the growth-inhibitory action of TGFbeta. Our data are consistent with a model in which induction of PAI-1 by TGFbeta is critical for the induction of proliferation arrest.

Roscovitine Reduces Neuronal Loss, Glial Activation, and Neurologic Deficits after Brain Trauma

Traumatic brain injury (TBI) causes both direct and delayed tissue damage. The latter is associated with secondary biochemical changes such as cell cycle activation, which leads to neuronal death, inflammation, and glial scarring. Flavopiridol--a cyclin-dependent kinase (CDK) inhibitor that is neither specific nor selective--is neuroprotective. To examine the role of more specific CDK inhibitors as potential neuroprotective agents, we studied the effects of roscovitine in TBI. Central administration of roscovitine 30 mins after injury resulted in significantly decreased lesion volume, as well as improved motor and cognitive recovery. Roscovitine attenuated neuronal death and inhibited activation of cell cycle pathways in neurons after TBI, as indicated by attenuated cyclin G1 accumulation and phosphorylation of retinoblastoma protein. Treatment also decreased microglial activation after TBI, as reflected by reductions in ED1, galectin-3, p22(PHOX), and Iba-1 levels, and attenuated astrogliosis, as shown by decreased accumulation of glial fibrillary acidic protein. In primary cortical microglia and neuronal cultures, roscovitine and other selective CDK inhibitors attenuated neuronal cell death, as well as decreasing microglial activation and microglial-dependent neurotoxicity. These data support a multifactorial neuroprotective effect of cell cycle inhibition after TBI--likely related to inhibition of neuronal apoptosis, microglial-induced inflammation, and gliosis--and suggest that multiple CDKs are potentially involved in this process.

RETRACTION: Flavopiridol Suppresses Tumor Necrosis Factor-Induced Activation of Activator Protein-1, c-Jun N-Terminal Kinase, p38 Mitogen-Activated Protein Kinase (MAPK), p44/p42 MAPK, and Akt, Inhibits Expression of Antiapoptotic Gene Products, and Enhances Apoptosis through CytochromecRelease and Caspase Activation in Human Myeloid Cells

Although flavopiridol, a semisynthetic flavone, was initially thought to be a specific inhibitor of cyclin-dependent kinases, it has now been shown that flavopiridol mediates antitumor responses through mechanism(s) yet to be defined. We have shown previously that flavopiridol abrogates tumor necrosis factor (TNF)-induced nuclear factor-κB (NF-κB) activation. In this report, we examined whether this flavone affects other cellular responses activated by TNF. TNF is a potent inducer of activator protein-1 (AP-1), and flavopiridol abrogated this activation in a dose- and time-dependent manner. Flavopiridol also suppressed AP-1 activation induced by various carcinogens and inflammatory stimuli. When examined for its effect on other signaling pathways, flavopiridol inhibited TNF-induced activation of various mitogen-activated protein kinases, including c-Jun NH₂-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and p44/p42 MAPK. It is noteworthy that this flavone also suppressed TNF-induced activation of Akt, a cell survival kinase, and expression of various antiapoptotic proteins, such as IAP-1, IAP-2, XIAP, Bcl-2, Bcl-xL, and TRAF-1. Flavopiridol also inhibited the TNF-induced induction of intercellular adhesion molecule-1, c-Myc, and c-Fos, all known to mediate tumorigenesis. Moreover, TNF-induced apoptosis was enhanced by flavopiridol through activation of the bid-cytochrome-caspase-9-caspase-3 pathway. Overall, our results clearly suggest that flavopiridol interferes with the TNF cell-signaling pathway, leading to suppression of antiapoptotic mechanisms and enhancement of apoptosis.