Pharmacological Inhibitors Of Cyclin-dependent Kinases Research Articles

Combined Treatment with Host-Directed and Anticytomegaloviral Kinase Inhibitors: Mechanisms, Synergisms and Drug Resistance Barriers.

Despite the availability of currently approved antiviral drugs, infections with human cytomegalovirus (HCMV) still cause clinically challenging, sometimes life-threatening situations. There is an urgent need for enhanced anti-HCMV drugs that offer improved efficacy, reduced dosages and options for long-term treatment without risk of the development of viral drug resistance. Recently, we reported the pronounced anti-HCMV efficacy of pharmacological inhibitors of cyclin-dependent kinases (CDKs), in particular, the potential of utilizing drug synergies upon combination treatment with inhibitors of host CDKs and the viral CDK-like kinase pUL97 (vCDK/pUL97). Here, we expand this finding by further assessing the in vitro synergistic antiviral interaction between vCDK and CDK inhibitors towards HCMV as well as non-human cytomegaloviruses. An extension of this synergy approach was achieved in vivo by using the recombinant MCMV-UL97/mouse model, confirming the high potential of combination treatment with the clinically approved vCDK inhibitor maribavir (MBV) and the developmental CDK7 inhibitor LDC4297. Moreover, mechanistic aspects of this synergistic drug combination were illustrated on the levels of intracellular viral protein transport and viral genome replication. The analysis of viral drug resistance did not reveal resistance formation in the case of MBV + LDC4297 combination treatment. Spanning various investigational levels, these new results strongly support our concept, employing the great potential of anti-HCMV synergistic drug treatment.

CDK9 inhibition induces epigenetic reprogramming revealing strategies to circumvent resistance in lymphoma

Diffuse large B-cell lymphoma (DLBCL) exhibits significant genetic heterogeneity which contributes to drug resistance, necessitating development of novel therapeutic approaches. Pharmacological inhibitors of cyclin-dependent kinases (CDK) demonstrated pre-clinical activity in DLBCL, however many stalled in clinical development. Here we show that AZD4573, a selective inhibitor of CDK9, restricted growth of DLBCL cells. CDK9 inhibition (CDK9i) resulted in rapid changes in the transcriptome and proteome, with downmodulation of multiple oncoproteins (eg, MYC, Mcl-1, JunB, PIM3) and deregulation of phosphoinotiside-3 kinase (PI3K) and senescence pathways. Following initial transcriptional repression due to RNAPII pausing, we observed transcriptional recovery of several oncogenes, including MYC and PIM3. ATAC-Seq and ChIP-Seq experiments revealed that CDK9i induced epigenetic remodeling with bi-directional changes in chromatin accessibility, suppressed promoter activation and led to sustained reprograming of the super-enhancer landscape. A CRISPR library screen suggested that SE-associated genes in the Mediator complex, as well as AKT1, confer resistance to CDK9i. Consistent with this, sgRNA-mediated knockout of MED12 sensitized cells to CDK9i. Informed by our mechanistic findings, we combined AZD4573 with either PIM kinase or PI3K inhibitors. Both combinations decreased proliferation and induced apoptosis in DLBCL and primary lymphoma cells in vitro as well as resulted in delayed tumor progression and extended survival of mice xenografted with DLBCL in vivo. Thus, CDK9i induces reprogramming of the epigenetic landscape, and super-enhancer driven recovery of select oncogenes may contribute to resistance to CDK9i. PIM and PI3K represent potential targets to circumvent resistance to CDK9i in the heterogeneous landscape of DLBCL.

Pan Cyclin-Dependent Kinase Inhibitors for the Treatment of Breast Cancer

Background: Despite extensive attempts for the treatment of breast cancer (BC), it is the most prevalent cancer type among women, and its treatment remains elusive, particularly in patients with advanced disease. Although there are several therapeutic options, none of them is effective for complete relief, especially in metastatic patients. Cancer cells exhibit a high proliferation rate, which is usually associated with the dysregulation of cell cycle progress. Various proteins such as cyclin-dependent kinases (CDKs) and cyclins are involved in cell cycle modulation. Methods: Databases including PubMed, Scopus, WebofScience and Google Scholar were used to extract information. Articles published in English until 2022 were used. Results: Regarding the dysregulation of various CDKs in several cancer types, the pharmacologicalinhibitors of CDKs have extensively been evaluated to treat several cancer types such as BC. The blockade of CDKs strongly suppresses tumor growth through cell cycle arrest. Moreover, the combination of CDK inhibitors and other anti-cancer therapeutics has demonstrated potent synergistic effects on the treatment of various cancers. Conclusion: Therefore, various CDK inhibitors have been designed and evaluated as antiproliferative therapeutics to suppress the proliferation of cancer cells. Pan CDK inhibitors, including flavopiridol, dinaciclib, purvalanol A, SNS-032, and roscovitine, are the most effective CDK inhibitors investigated in several studies. They inhibit various CDKs such as the CDK1, 2, 4, 5, 6, 7, and 9. In this review, it is attempted to discuss the efficacy of Pan CDK inhibitors as an anti-cancer therapy in BC.

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.

CDK9 activity is critical for maintaining MDM4 overexpression in tumor cells

The identification of the essential role of cyclin-dependent kinases (CDKs) in the control of cell division has prompted the development of small-molecule CDK inhibitors as anticancer drugs. For many of these compounds, the precise mechanism of action in individual tumor types remains unclear as they simultaneously target different classes of CDKs – enzymes controlling the cell cycle progression as well as CDKs involved in the regulation of transcription. CDK inhibitors are also capable of activating p53 tumor suppressor in tumor cells retaining wild-type p53 gene by modulating MDM2 levels and activity. In the current study, we link, for the first time, CDK activity to the overexpression of the MDM4 (MDMX) oncogene in cancer cells. Small-molecule drugs targeting the CDK9 kinase, dinaciclib, flavopiridol, roscovitine, AT-7519, SNS-032, and DRB, diminished MDM4 levels and activated p53 in A375 melanoma and MCF7 breast carcinoma cells with only a limited effect on MDM2. These results suggest that MDM4, rather than MDM2, could be the primary transcriptional target of pharmacological CDK inhibitors in the p53 pathway. CDK9 inhibitor atuveciclib downregulated MDM4 and enhanced p53 activity induced by nutlin-3a, an inhibitor of p53-MDM2 interaction, and synergized with nutlin-3a in killing A375 melanoma cells. Furthermore, we found that human pluripotent stem cell lines express significant levels of MDM4, which are also maintained by CDK9 activity. In summary, we show that CDK9 activity is essential for the maintenance of high levels of MDM4 in human cells, and drugs targeting CDK9 might restore p53 tumor suppressor function in malignancies overexpressing MDM4.

Abstract TS3-2: Modulation of anti-tumor immunity using CDK4/6 inhibitors

Abstract Several pharmacological inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) have now been approved for the treatment of metastatic estrogen receptor-positive breast cancer. Although CDK4/6 inhibitors were initially developed to prevent cancer cell proliferation, preclinical studies have now demonstrated that they can also exert other biological effects in solid tumors including augmentation of anti-tumor immunity. The detailed mechanisms underlying this are currently under investigation, and likely include enhanced antigen presentation by tumor cells as well as an increase in the ratio of effector to regulatory T lymphocytes. In this presentation, I will discuss our current understanding of how CDK4/6 inhibitors might stimulate anti-tumor immune responses, focusing on breast cancer. I will also discuss if and how this unexpected phenomenon might be leveraged for therapeutic benefit. Citation Format: Goel S. Modulation of anti-tumor immunity using CDK4/6 inhibitors [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr TS3-2.

Pharmacological cyclin dependent kinase inhibitors: Implications for colorectal cancer.

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

Metabolomic Applications to the Characterization of the Mode-of-Action of CDK Inhibitors.

Cyclin-dependent kinases (CDKs) regulate cell cycle progression, and some of them are also involved in the control of cellular transcription. Dysregulation of these critical cellular processes, due to the aberrant expression of some of these proteins, is common in many neoplastic malignancies. Consequently, the development of chemical compounds capable of inhibiting the biological activity of CDKs represents an attractive strategy in the anticancer area. CDK inhibition can trigger apoptosis and could be particularly useful in hematological malignancies, which are more sensitive to inhibition of cell cycle and apoptosis induction. Over the last few years, a number of pharmacological inhibitors of CDKs (CDKIs) belonging to different chemical families have been developed, and some of them have been tested in clinical trials. Given the complexity of the role of CDKs in cell functioning, it would be desirable to develop new tools that could facilitate a better understanding of the new insights into CDK functions and the mode-of-actions of CDKIs. In this context, this chapter describes an experimental approach to evaluate the metabolic consequences of CDKIs at the cellular level based on metabolomics by NMR. More specifically, a description of a strategy to characterize the biochemical effects of CDKIs acting on mammalian cells is provided, including protocols for the extraction of hydrophilic and lipophilic metabolites, the acquisition of 1D and 2D metabolomic Nuclear Magnetic Resonance (NMR) experiments, the identification and quantification of metabolites, and the annotation of the results in the context of biochemical pathways.

Why (multi)targeting of cyclin-dependent kinases is a promising therapeutic option for hormone-positive breast cancer and beyond.

Estrogens, via induction of their specific receptors (e.g., ER-α), regulate cell proliferation, differentiation and morphogenesis in mammary epithelium. Cell-cycle progression is driven by activation of complexes consisting of cyclin-dependent kinases (CDKs) and cyclins, which also modulate the activity of ER-α. Loss of control over the cell-cycle results in accelerated cell division and malignant transformation. Thus, a reciprocal relation exists between estrogen signaling and cell proliferation. Based on these findings, a new concept was developed to reduce ER-α activity and bring the cell cycle in transformed cells to heel. Prevention of ER-α activation and control over the deregulated cell cycle was achieved by supplementation with pharmacological CDK inhibitors alone or in combination with selective antiestrogens.

Inhibition of Post-Transcriptional RNA Processing by CDK Inhibitors and Its Implication in Anti-Viral Therapy

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and RNA polymerase II mediated transcription. Several pharmacological CDK inhibitors are currently in clinical trials as potential cancer therapeutics and some of them also exhibit antiviral effects. Olomoucine II and roscovitine, purine-based inhibitors of CDKs, were described as effective antiviral agents that inhibit replication of a broad range of wild type human viruses. Olomoucine II and roscovitine show high selectivity for CDK7 and CDK9, with important functions in the regulation of RNA polymerase II transcription. RNA polymerase II is necessary for viral transcription and following replication in cells. We analyzed the effect of inhibition of CDKs by olomoucine II on gene expression from viral promoters and compared its effect to widely-used roscovitine. We found that both roscovitine and olomoucine II blocked the phosphorylation of RNA polymerase II C-terminal domain. However the repression of genes regulated by viral promoters was strongly dependent on gene localization. Both roscovitine and olomoucine II inhibited expression only when the viral promoter was not integrated into chromosomal DNA. In contrast, treatment of cells with genome-integrated viral promoters increased their expression even though there was decreased phosphorylation of the C-terminal domain of RNA polymerase II. To define the mechanism responsible for decreased gene expression after pharmacological CDK inhibitor treatment, the level of mRNA transcription from extrachromosomal DNA was determined. Interestingly, our results showed that inhibition of RNA polymerase II C-terminal domain phosphorylation increased the number of transcribed mRNAs. However, some of these mRNAs were truncated and lacked polyadenylation, which resulted in decreased translation. These results suggest that phosphorylation of RNA polymerase II C-terminal domain is critical for linking transcription and posttrancriptional processing of mRNA expressed from extrachromosomal DNA.

CDK/CK1 inhibitors roscovitine and CR8 downregulate amplified MYCN in neuroblastoma cells

To understand the mechanisms of action of (R)-roscovitine and (S)-CR8, two related pharmacological inhibitors of cyclin-dependent kinases (CDKs), we applied a variety of '-omics' techniques to the human neuroblastoma SH-SY5Y and IMR32 cell lines: (1) kinase interaction assays, (2) affinity competition on immobilized broad-spectrum kinase inhibitors, (3) affinity chromatography on immobilized (R)-roscovitine and (S)-CR8, (4) whole genome transcriptomics analysis and specific quantitative PCR studies, (5) global quantitative proteomics approach and western blot analysis of selected proteins. Altogether, the results show that the major direct targets of these two molecules belong to the CDKs (1,2,5,7,9,12), DYRKs, CLKs and CK1s families. By inhibiting CDK7, CDK9 and CDK12, these inhibitors transiently reduce RNA polymerase 2 activity, which results in downregulation of a large set of genes. Global transcriptomics and proteomics analysis converge to a central role of MYC transcription factors downregulation. Indeed, CDK inhibitors trigger rapid and massive downregulation of MYCN expression in MYCN-amplified neuroblastoma cells as well as in nude mice xenografted IMR32 cells. Inhibition of casein kinase 1 may also contribute to the antitumoral activity of (R)-roscovitine and (S)-CR8. This dual mechanism of action may be crucial in the use of these kinase inhibitors for the treatment of MYC-dependent cancers, in particular neuroblastoma where MYCN amplification is a strong predictor factor for high-risk disease.

Abstract B48: Targeting chemo-resistance in CCNE1-amplified ovarian cancer

Abstract The putative oncogene CCNE1 (cyclin E1) is frequently amplified in human high-grade serous ovarian cancer. CCNE1-amplified cancers tend to be wildtype for BRCA1 and BRCA2 and are associated with shorter survival and resistance to platinum-based chemotherapy. Cyclin E1 is an activating cofactor for cyclin-dependent kinases (CDK), which stimulate cell cycle progression through phosphorylation of the retinoblastoma (RB) protein and subsequent induction of E2F transcriptional activity (cyclin E1-RB-E2F signaling). Since BRCA1 and BRCA2 are known target genes of E2F transcription factors, we hypothesize that cyclin E1 signaling actively contributes to high DNA repair capacity and chemo-resistance in a BRCA1/2-dependent manner. We predict CCNE1-amplified ovarian cancers to exhibit an “anti-BRCAness” phenotype. In two independent gene expression datasets, generated by TCGA and The Women's Cancer Program at Cedars-Sinai, we found that CCNE1-amplified ovarian cancers maintain higher levels of BRCA1 than CCNE1-wildtype tumors. Moreover, we have employed genetic and pharmacological targeting approaches in order to assess if inhibition of cyclin E1-dependent signaling can induce BRCAness and restore chemo-sensitivity. Here, we show that pharmacological CDK inhibitors (CDKi) effectively shut down E2F-mediated transcription, resulting in downregulation of BRCA1 and BRCA2 in CDKi-treated cells. Long-term exposure of ovarian cancer cell lines to CDKi selected for cells with reduced dependency on cyclin E1, and genomic profiling revealed de novo DNA copy number changes which compensate for loss of cyclin E1 function. Importantly, CDKi-resistant subclones retained lower levels of BRCA1 and were significantly more sensitive to cisplatin than parental cell lines. Collectively, our results suggest that cyclin E1 signaling is required for BRCA1 expression and chemo-resistance. Currently available CDKi such as Dinaciclib, which is in a phase 3 clinical trial, may be useful to specifically sensitize CCNE1-amplified ovarian cancers to cisplatin. Citation Format: Barbie Taylor-Harding, Hasmik Agadjanian, Paul Joseph Aspuria, Takako Mizuno, Dong-Joo Cheon, Sandra Orsulic, Beth Karlan, Christine Walsh, Wolf Ruprecht Wiedemeyer. Targeting chemo-resistance in CCNE1-amplified ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B48.

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

The Impact Of CDK Inhibition In Human Malignancies Associated With Pronounced Defects In Apoptosis: Advantages Of Multi-Targeting Small Molecules

Malignant cells in chronic lymphocytic leukemia (CLL) and related diseases are heterogeneous and consist primarily of long-lived resting cells in the periphery and a minor subset of dividing cells in proliferating centers. Both cell populations have different molecular signatures that play a major role in determining their sensitivity to therapy. Contemporary approaches to treating CLL are heavily reliant on cytotoxic chemotherapeutics. However, none of the current treatment regimens can be considered curative. Pharmacological CDK inhibitors have extended the repertoire of potential drugs for CLL. Multi-targeted CDK inhibitors affect CDKs involved in regulating both cell cycle progression and transcription. Their interference with transcriptional elongation represses anti-apoptotic proteins and, thus, promotes the induction of apoptosis. Importantly, there is evidence that treatment with CDK inhibitors can overcome resistance to therapy. The pharmacological CDK inhibitors have great potential for use in combination with other therapeutics and represent promising tools for the development of new curative treatments for CLL.

Promotion of Apoptosis in Cancer Cells by Selective Purine-Derived Pharmacological CDK Inhibitors: One Outcome, Many Mechanisms

The deregulation of apoptosis and the cell cycle are important steps in the onset of cancer, giving cells unlimited reproductive potential and increasing their likelihood of survival. The cell cycle is an essential and tightly regulated four-stage process that effects the accurate duplication and transmission of genetic content to cells' progeny. Cyclin-dependent kinases (CDKs) are key elements of the mammalian cell cycle machinery. Their activity is normally regulated via cyclin binding, phosphorylation events, and interactions with endogenous CDK inhibitors. Malfunctions in the control of the cell cycle can be specifically countered using pharmacological CDK inhibitors. Importantly, CDK inhibitors are very effective against both rapidly dividing and quiescent cancer cells; this is particularly relevant in the treatment of malignancies such as chronic lymphatic leukemia (CLL) and multiple myeloma (MM) that exhibit both a low mitotic index and apoptotic defects. The high efficacy of pharmacological CDK inhibitors against CLL and MM is attributable to their ability to eliminate leukemic cells by apoptosis. Indeed, not only do pharmacological CDK inhibitors block cell cycle progression; they also promote apoptosis and thereby destroy irrevocably malignant cells. This article focuses on the impact of inhibiting individual cellular CDKs on apoptosis. We discuss in detail the molecular mechanisms by which CDK inhibitors are able to bypass chemoresistance in tumor cells and trigger apoptosis. Remarkably, recent findings suggest that the pharmacological utility of CDK inhibitors may not be restricted to the treatment of cancer: some may be efficacious in the treatment of patients with neurodegenerative and cardiovascular diseases.

Reconstitution of human MCF‐7 breast cancer cells with caspase‐3 does not sensitize them to action of CDK inhibitors

Human MCF-7 breast cancer cells are resistant to pro-apoptotic stimuli due to caspase-3 inactivation. On the other hand, they should be sensitive to agents like selective pharmacological inhibitors of cyclin-dependent kinases (CDKs) that (re)activate p53 tumor suppressor protein because they harbor intact p53 pathways. In this study we examined whether reconstitution of caspase-3 in MCF-7 cells sensitizes them to inhibitors of CDKs, by analyzing the effects of roscovitine (ROSC) and olomoucine (OLO), two closely related selective pharmacological CDK inhibitors, on both mother MCF-7 cells and a secondary mutant line, MCF-7.3.28 that stably expresses human caspase-3. The results show that ROSC is, as expected, much more potent than OLO. Surprisingly; however, ROSC and OLO reduced proliferation of parental MCF-7 cells more strongly than caspase-3-proficient counterparts. Both inhibitors arrest human breast cancer cells at the G(2)-phase of the cell cycle. Analysis of cell-cycle regulators by immunoblotting revealed that ROSC strongly induces p53 protein activity by inducing its phosphorylation at Ser46 in the MCF-7 cells lacking caspase-3, but not in caspase-3-proficient cells. Furthermore, reconstitution of caspase-3 in MCF-7 cells neither elevates the mitochondrial apoptosis rate nor significantly increases caspases activity upon ROSC treatment. However, the stabilization of p53 in response to DNA damaging agents is the same in both caspase negative and positive MCF-7 cells. Cytotoxic agents induce caspase-3-dependent apoptosis in caspase-3-proficient cells. These results indicate that reconstitution of MCF-7 cancer cells with caspase-3 sensitize them to the action of DNA damaging agents but not to ATP-like pharmacological inhibitors of CDKs.

Delayed Treatment with Systemic (S)-Roscovitine Provides Neuroprotection and Inhibits In Vivo CDK5 Activity Increase in Animal Stroke Models

BackgroundAlthough quite challenging, neuroprotective therapies in ischemic stroke remain an interesting strategy to counter mechanisms of ischemic injury and reduce brain tissue damage. Among potential neuroprotective drug, cyclin-dependent kinases (CDK) inhibitors represent interesting therapeutic candidates. Increasing evidence indisputably links cell cycle CDKs and CDK5 to the pathogenesis of stroke. Although recent studies have demonstrated promising neuroprotective efficacies of pharmacological CDK inhibitors in related animal models, none of them were however clinically relevant to human treatment.Methodology/Principal FindingsIn the present study, we report that systemic delivery of (S)-roscovitine, a well known inhibitor of mitotic CDKs and CDK5, was neuroprotective in a dose-dependent manner in two models of focal ischemia, as recommended by STAIR guidelines. We show that (S)-roscovitine was able to cross the blood brain barrier. (S)-roscovitine significant in vivo positive effect remained when the compound was systemically administered 2 hrs after the insult. Moreover, we validate one of (S)-roscovitine in vivo target after ischemia. Cerebral increase of CDK5/p25 activity was observed 3 hrs after the insult and prevented by systemic (S)-roscovitine administration. Our results show therefore that roscovitine protects in vivo neurons possibly through CDK5 dependent mechanisms.Conclusions/SignificanceAltogether, our data bring new evidences for the further development of pharmacological CDK inhibitors in stroke therapy.

Radioprotection: smart games with death

The efficacy of cancer treatment by radiation and chemotherapeutic drugs is often limited by severe side effects that primarily affect the hematopoietic system and the epithelium of the gastrointestinal tract. Progress in understanding differences in the mechanisms involved in the responses of normal and tumor cells to genotoxic stress has led to the development of new rational approaches to selective protection of normal cells, such as suppression of apoptosis by pharmacological inhibition of p53 or activation of NF-kappaB. Another promising approach presented in this issue by Johnson et al. is based on the idea of using pharmacological inhibitors of cyclin-dependent kinases (CDKs) to convert normal cells into a radioresistant state by inducing reversible cell cycle arrest at the G1/S transition. The evidence indicates that this approach is likely to be specific for protection of normal cells and may, therefore, have clinical potential as an adjuvant in anticancer therapies.

Abstract 3490: Selective estrogen receptor modulators enhance efficacy of small molecule CDK inhibitors on human breast cancer cells differing in the expression of ER

Abstract Inactivation of cyclin-dependent kinases (CDKs) by pharmacological inhibitors is a very effective weapon against malignant cells. Roscovitine (ROSC), a selective CDK inhibitor primarily affecting CDK2, CDK1, and CDK7, reduces the number of human cancer cells in a concentration-dependent manner. At lower doses ROSC arrests cell cycle progression and at higher doses it induces apoptosis. ROSC inhibits efficiently proliferation of human ER+-ve MCF-7 breast cancer cells by induction of cell cycle arrest at the G2/M transition [1] and concomitantly initiates apoptosis by a p53-dependent pathway [2]. However, the effect of ROSC was much weaker in MCF-7 cells maintained in the presence of estrogen-mimicking compounds [3]. Therefore, we decided to examine the action of selective CDK inhibitors on other breast cancer cell lines differing in the status of p53 and ER and to prove the impact of selective estrogen receptor modulators (SERMs) on the efficacy of ROSC and olomoucine II (OLO II), a new pharmacological CDK inhibitor. ROSC and OLO II were effective on all tested breast cancer cell lines. They arrested MCF-7 and BT-20 cells at G2/M transition and SKBR3 cells in G1 phase and additionally induced apoptosis. The effect of the inhibition of CDKs on distinct cell cycle regulators and pro-survival factors was studied. Interestingly, SERM strongly affected all tested cell lines irrespective of their ER status. Its combination with CDK inhibitors had a different impact. It enhanced G1 or G2 arrest. Our results provide evidence that both CDK inhibitors exert a strong anti-proliferative and pro-apoptotic effect and that their mode of action depends on the cellular context. Moreover, our findings demonstrate that pharmacological CDK inhibitors can be combined with anti-estrogen therapy. The strong effect of anti-estrogens on ER-negative cancer cells indicates that SERMs crosstalk with other steroid hormone receptors. [1] Wojciechowski J. et al. 2003. Rapid onset of nucleolar disintegration preceding cell cycle arrest in roscovitine-induced apoptosis of human MCF-7 breast cancer cells. Int J Cancer 106: 486-95. [2] Wesierska-Gadek J. et al. 2005. Roscovitine-induced up-regulation of p53AIP1 protein precedes the onset of apoptosis in human breast cancer cells. Mol Cancer Ther 4: 113-124. [3] Wesierska-Gadek J. et al. 2006. Phenol red reduces ROSC mediated cell cycle arrest and apoptosis in human MCF-7 cells. J Cell Biochem. 98: 1369-1379. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3490.

Novel Potent Pharmacological Cyclin-Dependent Kinase Inhibitors

Progression of the cell cycle is controlled by various activating and inhibiting cellular factors. The subtle balance between these counteracting regulators in normal cells ensures proper cell cycle progression and facilitates cellular responses to a variety of stress stimuli. Key activators include cyclin-dependent kinases (CDKs) and, consequently, loss or inactivation of CDK inhibitors contributes to the escape of cancer cells from cell cycle control and hyperactivation of CDKs occurs in various neurodegenerative disorders. However, these adverse effects may be compensated by pharmacological counterparts. Inhibitors of CDKs representing various classes of compounds with diverse CDK inhibitory patterns have been developed, but inhibitors that have high selectivity and offer highly targeted activity against both cell cycle and transcriptional CDKs are of particular interest. This review focuses on pharmacological CDK inhibitors that have entered clinical trials and some compounds that have been evaluated preclinically. Recent discoveries in cell cycle regulation have provided rationales for clinical applications of CDK inhibitors in both monotherapeutic and combined therapeutic regimens.