Cyclin-dependent Kinases Targets Research Articles

CDK phosphorylation of Sfr1 downregulates Rad51 function in late-meiotic homolog invasions.

Meiosis is the developmental program that generates gametes. To produce healthy gametes, meiotic recombination creates reciprocal exchanges between each pair of homologous chromosomes that facilitate faithful chromosome segregation. Using fission yeast and biochemical, genetic, and cytological approaches, we have studied the role of CDK (cyclin-dependent kinase) in the control of Swi5-Sfr1, a Rad51-recombinase auxiliary factor involved in homolog invasion during recombination. We show that Sfr1 is a CDK target, and its phosphorylation downregulates Swi5-Sfr1 function in the meiotic prophase. Expression of a phospho-mimetic sfr1-7D mutant inhibits Rad51 binding, its robust chromosome loading, and subsequently decreases interhomolog recombination. On the other hand, the non-phosphorylatable sfr1-7A mutant alters Rad51 dynamics at late prophase, and exacerbates chromatin segregation defects and Rad51 retention observed in dbl2 deletion mutants when combined with them. We propose Sfr1 phospho-inhibition as a novel cell-cycle-dependent mechanism, which ensures timely resolution of recombination intermediates and successful chromosome distribution into the gametes. Furthermore, the N-terminal disordered part of Sfr1, an evolutionarily conserved feature, serves as a regulatory platform coordinating this phospho-regulation, protein localization and stability, with several CDK sites and regulatory sequences being conserved.

Efficacy of fadraciclib (CYC065), a novel dual CDK2/9 inhibitor, on patient-derived models of colorectal cancer.

3596 Background: Colorectal cancer (CRC) is the third most common type of cancer among adults in the United States. Given the heterogeneous nature of the disease and the limited number of treatments available, there is an unmet need to identify new therapeutic vulnerabilities for advanced CRC. One promising approach is targeting of cyclin dependent kinases (CDKs). In our current study, we sought to understand the potential efficacy of fadraciclib (CYC065), a novel dual CDK 2/9 inhibitor, as a novel treatment for metastatic CRC. Methods: Eighteen CRC patient-derived organoids (PDOs) were generated from patients undergoing biopsy or resection for their primary or metastatic CRC under an IRB approved protocol at Duke University. IHC and H&E staining were first performed on the established PDOs to confirm the diagnosis of CRC. PDOs were treated with standard of care chemotherapy (oxaliplatin, irinotecan (SN38) and 5-Flurouracil), CDK4/6 inhibitor palbociclib, and fadraciclib (CYC065). All drugs were used in doses ranging from 6.4nM to 100µM, and drug sensitivities were determined using Cell-Titer Glo (CTG) 72 hours after treatment. Subsequently, three matching PDXs were generated for in vivo validation and treated with fadraciclib (CYC065) via oral gavage at a dose of 25 mg/kg BID five days a week for two weeks. Target validation for CDK2/9 inhibition was performed via western blotting, cell cycle arrest was determined via flow cytometry, and killing by anaphase catastrophe was determined by immunofluorescence staining. Results: CRC PDOs were more sensitive to fadraciclib (CYC065) than chemotherapy and palbociclib, with IC50 values lower than chemotherapy or palbociclib (2.65±3.92µM for fadraciclib (CYC065) vs. 29.92±41.98µM for chemotherapy (P<0.05) or palbociclib (12.33±9.87µM)). Subsequently, we validated our findings in vivo using 3 matched PDX showing significant tumor growth inhibition (TGI) with fadraciclib (CYC065) compared to control (p<0.05) without serious adverse effects. We showed that fadraciclib (CYC065) induces G2/M cell cycle arrest, resulting in anaphase catastrophe as evident by the presence of multipolar mitosis in fadraciclib (CYC065)-treated cells. CDK2/9 inhibition leads to downregulation of c-Myc levels and upregulation of apoptosis induction as a second mechanism of action for this drug. Conclusions: Our data using both in vitro and in vivo patient-derived models support further exploration of fadraciclib (CYC065) has a potential therapy for advanced CRC. CDK2/9 inhibition impacts multiple critical pathways involved in transcription, mitosis and apoptosis. Further studies will be needed to reveal molecular biomarkers of sensitivity and resistance to this novel agent.

A cyclin-dependent kinase-mediated phosphorylation switch of disordered protein condensation

Cell cycle transitions result from global changes in protein phosphorylation states triggered by cyclin-dependent kinases (CDKs). To understand how this complexity produces an ordered and rapid cellular reorganisation, we generated a high-resolution map of changing phosphosites throughout unperturbed early cell cycles in single Xenopus embryos, derived the emergent principles through systems biology analysis, and tested them by biophysical modelling and biochemical experiments. We found that most dynamic phosphosites share two key characteristics: they occur on highly disordered proteins that localise to membraneless organelles, and are CDK targets. Furthermore, CDK-mediated multisite phosphorylation can switch homotypic interactions of such proteins between favourable and inhibitory modes for biomolecular condensate formation. These results provide insight into the molecular mechanisms and kinetics of mitotic cellular reorganisation.

Imidazole-4-N-acetamide Derivatives as a Novel Scaffold for Selective Targeting of Cyclin Dependent Kinases.

The rational design of cyclin-dependent protein kinase (CDK) inhibitors presumes the development of approaches for accurate prediction of selectivity and the activity of small molecular weight anticancer drug candidates. Aiming at attenuation of general toxicity of low selectivity compounds, we herein explored the new chemotype of imidazole-4-N-acetamide substituted derivatives of the pan-CDK inhibitor PHA-793887. Newly synthesized compounds 1-4 containing an aliphatic methyl group or aromatic radicals at the periphery of the scaffold were analyzed for the prediction of relative free energies of binding to CDK1, -2, -5, and -9 using a protocol based on non-equilibrium (NEQ) thermodynamics. This methodology allows for the demonstration of a good correlation between the calculated parameters of interaction of 1-4 with individual targets and the values of inhibitory potencies in in vitro kinase assays. We provide evidence in support of NEQ thermodynamics as a time sparing, precise, and productive approach for generating chemical inhibitors of clinically relevant anticancer targets.

A synthetic biology approach reveals diverse and dynamic CDK response profiles via multisite phosphorylation of NLS-NES modules.

The complexity of multisite phosphorylation mechanisms in regulating nuclear localization signals (NLSs) and nuclear export signals (NESs) is not understood, and its potential has not been used in synthetic biology. The nucleocytoplasmic shuttling of many proteins is regulated by cyclin-dependent kinases (CDKs) that rely on multisite phosphorylation patterns and short linear motifs (SLiMs) to dynamically control proteins in the cell cycle. We studied the role of motif patterns in nucleocytoplasmic shuttling using sensors based on the CDK targets Dna2, Psy4, and Mcm2/3 of Saccharomyces cerevisiae. We designed multisite phosphorylation modules by rearranging phosphorylation sites, cyclin-specific SLiMs, phospho-priming, phosphatase specificity, and NLS/NES phospho-regulation and obtained very different substrate localization dynamics. These included ultrasensitive responses with and without a delay, graded responses, and different homeostatic plateaus. Thus, CDK can do much more than trigger sequential switches during the cell cycle as it can drive complex patterns of protein localization and activity by using multisite phosphorylation networks.

Targeting of cyclin-dependent kinases in atypical teratoid rhabdoid tumors with multikinase inhibitor TG02.

Atypical teratoid rhabdoid tumors (ATRTs) are aggressive pediatric brain tumors with no current standard of care and an estimated median patient survival of 12 to 18 months. Previous genetic analyses have implicated cyclin D1 and enhancer of zeste homolog 2 (EZH2), a histone methyltransferase that is implicated in many cancers, as key drivers of tumorigenicity in ATRTs. Since the effects of EZH2 and cyclin D1 are facilitated by a host of cyclin-dependent kinases (CDKs), the authors sought to investigate the potential therapeutic effects of targeting CDKs in ATRTs with the multi-CDK inhibitor, TG02. Human ATRT cell lines BT12, BT37, CHLA05, and CHLA06 were selected for investigation. The effects of TG02 on cell viability, proliferation, clonogenicity, and apoptosis were assessed via Cell Counting Kit-8 assays, cell counting, clonogenic assays, and flow cytometry, respectively. Similar methods were used to determine the effects of TG02 combined with radiation therapy (RT) or cisplatin. Synergism indices for TG02-cisplatin combination therapy were calculated using CompuSyn software. TG02 was observed to significantly impair ATRT cell growth in vitro by limiting cell proliferation and clonogenicity, and by inducing apoptosis. TG02 inhibited ATRT cell proliferation and decreased cell viability in a dose-dependent manner with nanomolar half maximal effective concentration (EC50) values (BT12, 207.0 nM; BT37, 127.8 nM; CHLA05, 29.7 nM; CHLA06, 18.7 nM). TG02 (150 nM) dramatically increased the proportion of apoptotic ATRT cells 72 hours posttreatment (TG02 8.50% vs control 1.52% apoptotic cells in BT12, p < 0.0001; TG02 70.07% vs control 15.36%, p < 0.0001). Combination therapy studies revealed that TG02 acted as a potent radiosensitizer in ATRT cells (BT12 surviving fraction, RT 51.2% vs RT + TG02 21.7%). Finally, CompuSyn analysis demonstrated that TG02 acted synergistically with cisplatin against ATRT cells at virtually all therapeutic doses. These findings were consistent in cell lines that cover all three molecular subgroups of ATRTs. The results of this investigation have established that TG02 is an effective therapeutic against ATRTs in vitro. Given the lack of standard therapy for ATRTs, these findings help fill an unmet need and support further study of TG02 as a potential therapeutic option for patients with this deadly disease.

Inhibitor of Multi-cyclin-dependent Kinases (AT7519) Reduced Survival of U937 Leukemic Cells and Enhanced Anti-leukemic Effect of Vincristine: A Highlight to CDK Inhibition Efficacy in Acute Leukemia

Background: The conservative character of the cell cycle outlined that any dysregulation in the regulatory components of this process in normal cells opens a gate toward neoplastic transformation. Objectives: Given the critical role of cyclin-dependent kinases (CDKs) in cancer pathogenesis and based on their frequent aberrancy in human leukemia, the present study aimed at evaluating the suppressive effect of a multi-CDK inhibitor AT7519 on acute myeloid leukemia-derived U937 cells. Methods: To assess the anti-leukemic effects of the inhibitor on acute myeloid leukemia (AML) cells, we used MTT and trypan blue assays. Flow cytometric analysis and q-RT-PCR were also applied to evaluate the impact of AT7519 on cell cycle and apoptosis. Results: The results suggested that suppression of CDK in U937 cells hampered the proliferation of leukemic cells through a G2/M arrest mediated by p21 gene. Additionally, the anti-survival impact of AT7519 on these cells was shown to be along with the apoptosis initiation not only through the increment of pro-apoptotic gene expression but also through diminishing the mRNA levels of both Pin1 and Survivin. Notably, the potent anti-leukemic property of this agent has become more prominent when we found that the blockage of CDKs in AML cells could synergize with the cytotoxic effect of vincristine (VCR). To the best of our knowledge, little is known about the molecular mechanisms of resistance to AT7519 and we proposed that the effectiveness of this agent was partially attenuated through either c-Myc or autophagy activation in U937 cells. Conclusions: This study suggests that the pharmacological targeting of CDKs could probably unwind the complexity of therapeutic obstacles on the way of acute leukemia, either in the context of mono- or combined-modal strategy.

A new linear cyclin docking motif that mediates exclusively S-phase CDK-specific signaling.

Cyclin‐dependent kinases (CDKs), the master regulators of cell division, are activated by different cyclins at different cell cycle stages. In addition to being activators of CDKs, cyclins recognize various linear motifs to target CDK activity to specific proteins. We uncovered a cyclin docking motif, NLxxxL, that contributes to phosphorylation‐dependent degradation of the CDK inhibitor Far1 at the G1/S stage in the yeast Saccharomyces cerevisiae. This motif is recognized exclusively by S‐phase CDK (S‐CDK) Clb5/6‐Cdc28 and is considerably more potent than the conventional RxL docking motif. The NLxxxL and RxL motifs were found to overlap in some target proteins, suggesting that cyclin docking motifs can evolve to switch from one to another for fine‐tuning of cell cycle events. Using time‐lapse fluorescence microscopy, we show how different docking connections temporally control phosphorylation‐driven target degradation. This also revealed a differential function of the phosphoadaptor protein Cks1, as Cks1 docking potentiated degron phosphorylation of RxL‐containing but not of NLxxxL‐containing substrates. The NLxxxL motif was found to govern S‐cyclin‐specificity in multiple yeast CDK targets including Fin1, Lif1, and Slx4, suggesting its wider importance.

Targeting MM at the Nexus between Cell Cycle and Transcriptional Regulation Via CDK7 Inhibition

Deregulated transcription and cell cycle control are hallmarks of cancer that are especially frequent in multiple myeloma (MM). Largely non-overlapping sets of cyclin-dependent kinases (CDKs) regulate cell division and RNA polymerase II (Pol II)-dependent transcription; and targeting of cell cycle CDKs has been long pursued as an attractive therapeutic strategy. Among CDKs, CDK7 presents a unique therapeutic opportunity as it functions as a CDK activating kinase (CAK), licensing the activity of cell cycle CDKs, and also serves as a core component of the general transcription factor TFIIH. Here we elucidated the biological role of CDK7 and its transcriptional regulatory landscape in MM, using genetic as well chemical approaches, including tools for CDK7 rapid protein degradation (dTAG) and the selective covalent inhibitor YKL-5-124 that targets a cysteine residue (C312) located outside of the kinase domain. We have observed that CDK7 inhibition via YKL-5-124 robustly inhibited the phosphorylation of the CDK1, 2 and 4 activation loops in a representative panel of MM cell lines at concentrations as low as 50 nM. This reduction was not observed in MM cells expressing a resistant mutation in the reactive cysteine (C312S). Consistent with decrease of CAK activity, we observed G1 arrest and S phase loss after CDK7 inhibition, which was also associated with a rapid and transient loss of Ser2 and Ser5 phosphorylation of the RNA Pol2 C-terminal domain. To understand the effect of CDK7 inhibition on MM cell growth and viability, we evaluated activity of YKL-5-124 across a large panel of 25 MM cell lines and observed a significant inhibition of MM cell proliferation, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs), suggesting a specific sensitivity of MM cells to CDK7 inhibition. Longer exposure to YKL-5-124 caused apoptotic cell death in MM cells; however treatment with an inactive analog or in cells expressing the C312S mutation failed to inhibit MM cell proliferation, confirming that the antiproliferative potency of YKL-5-124 resides in its unique characteristic to covalently bind to C312 domain. Importantly, CDK7 inhibition impaired primary MM cells proliferation alone and when cultured in the presence of BM microenvironment. Selective pharmacological degradation of endogenously tagged CDK7 confirmed impact of CDK7 inhibition on MM cell proliferation via inhibition of CDK7 transcriptional and cell cycle activities. To complement the pharmacological studies, we have established MM cells to express inducible CRISPR/Cas9 constructs encoding 4 independent small guide RNAs targeting CDK7, resulting in the reduction of the abundance of CDK7 protein by 20-60% which was sufficient to inhibit MM cell viability over time, phenocopying pharmacologic inhibition of CDK7. These results support the view that CDK7 is a pharmacologically relevant target for MM. Gene expression analysis after CDK7 inhibition in MM1S and H929 cells revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells confirmed using E2F-driven luciferase reporter. These data suggest significant role for CDK7 in the CDK-pRB-E2F pathway in MM, which was strengthened by the observation of a positive correlation between expression of CDK7 and expression of E2F target genes in primary MM cells (n=409). Finally, we have evaluated the in vivo effect of CDK7 inhibition in several murine models of human MM. In the localized subcutaneous model, and the disseminated MM model where treatment with YKL-5-124 decreased tumor burden and improved survival. The effect of CDK7 inhibition explored in an aggressive, genetically engineered model of Myc-dependent MM, revealed evidence of response by decline in measurement of monotypic serum immunoglobulins. In conclusion, our study demonstrates that CDK7 contributes to the 'transcriptional addiction' and the cell cycle deregulation frequently observed in MM and represents an attractive molecular vulnerability to be exploited therapeutically. Disclosures Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Celgene: Membership on an entity's Board of Directors or advisory committees. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties; BMS: Consultancy. Fulciniti:NIH: Research Funding.

The multi-targets mechanism of hydroxychloroquine in the treatment of systemic lupus erythematosus based on network pharmacology.

Network pharmacology is used with bioinformatic tools to broaden the understanding of drugs' potential targets and the intersections with key genes of particular disease. Here we applied network pharmacology to collect testable hypotheses about the multi-targets mechanism of hydroxychloroquine (HCQ) against systemic lupus erythematosus (SLE). Firstly, we predicted the potential targets of HCQ. Secondly, we got the related genes of SLE returned from databases. Thirdly, the intersections of the potential targets (HCQ) and related genes (SLE) were analyzed with gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Finally, we validated our predictions of the potential targets by performing docking studies with HCQ. The results suggest that the efficacy of HCQ against SLE is mainly associated with the targets of cyclin-dependent kinase 2 (CDK2), estrogen receptor alpha (ESR1) and CDK1, which regulate PI3K/Akt/GSK3β as well as interferon (IFN) signaling pathway. Biological process of the network associated with the three targets is concentrated in the inhibition of immune response, negative regulation of gene expression and regulation of immune system process. Molecular docking analysis proves that hydrogen bonding and π-π stacking are the main forms of interaction. Our research provides protein targets affected by HCQ in the treatment of SLE. Three key targets (CDK2, ESR1 and CDK1) involving 1766 proteins become the multi-targets mechanism of HCQ in the treatment of SLE. As well, the research also provides a new idea for introducing network pharmacology into the evaluation of the drugs with multi-targets in dermatology.

CDK1 in Breast Cancer: Implications for Theranostic Potential.

Breast cancer has been identified as one of the main cancer-related deaths among women during some last decades. Recent advances in the introduction of novel potent anti-cancer therapeutics in association with early detection methods led to a decrease in the mortality rate of breast cancer. However, the scenario of breast cancer is yet going on and further improvements in the current anti-cancer therapeutic approaches are needed. Several factors are present in the tumor microenvironment which help to cancer progression and suppression of anti-tumor responses. Targeting these cancer-promoting factors in the tumor microenvironment has been suggested as a potent immunotherapeutic approach for cancer therapy. Among the various tumorsupporting factors, Cyclin-Dependent Kinases (CDKs) are proposed as a novel promising target for cancer therapy. These factors in association with cyclins play a key role in cell cycle progression. Dysregulation of CDKs which leads to increased cell proliferation has been identified in various cancers, such as breast cancer. Accordingly, the development and use of CDK-inhibitors have been associated with encouraging results in the treatment of breast cancer. However, it is unknown that the inhibition of which CDK is the most effective strategy for breast cancer therapy. Since the selective blockage of CDK1 alone or in combination with other therapeutics has been associated with potent anti-cancer outcomes, it is suggested that CDK1 may be considered as the best CDK target for breast cancer therapy. In this review, we will discuss the role of CDK1 in breast cancer progression and treatment.

Theileria annulata surface protein (TaSP) is a target of cyclin-dependent kinase 1 phosphorylation in Theileria annulata-infected cells.

Leucoproliferative Theileria parasites possess the unique capability to transform their bovine host cell, resulting in tumour-like characteristics like uncontrolled proliferation. The molecular mechanisms underlying this parasite-dependent process are only poorly understood. In the current study, bioinformatic analysis of the Theileria annulata surface protein (TaSP) from different T.annulata isolates identified a conserved CDK1 phosphorylation motif T131 PTK within the extracellular, polymorphic domain of TaSP. Phosphorylation assays with radioactively labelled ATP as well as ELISA-based experiments using a phospho-threonine-proline (pThr-Pro) antibody revealed, that CDK1-cyclin B specifically phosphorylates T131 , identifying TaSP as a substrate in vitro. Confocal microscopy and proximity ligation assays suggest an interaction between CDK1 and TaSP in T.annulata-infected cells. Further studies demonstrated a nearly complete co-localization of the pThr-Pro signal and TaSP only in cells in interphase, pointing towards a cell cycle-dependent event. Immunostainings of isolated, non-permeabilized schizonts confirmed the presence of the pThr-Pro epitope on the schizont's surface. Lambda phosphatase treatment abolished the pThr-Pro signal of the schizont, which was reconstituted by the addition of CDK1-cyclin B. Treatment of T.annulata-infected cells with the CDK1 inhibitor purvalanol A resulted in morphological changes characterized by tubulin-rich cell protrusions and an extension of the schizont, and a dose-dependent reduction of BrdU incorporation and Ki67 staining of T.annulata-infected cells, demonstrating a clear impact on the Theileria-dependent proliferation of the bovine host cell. Our data reveal the parasite surface protein TaSP as a target for the host cell kinase CDK1, a major player during cell division. Targeting the uncontrolled proliferation of Theileria-infected cells is a novel and reasonable approach to limit parasite load in order to facilitate a successful cellular immune response against the parasite.

Dysfunctional CAF-I reveals its role in cell cycle progression and differential regulation of gene silencing

ABSTRACT Chromatin Assembly Factor I (CAF-I) plays a central role in the reassembly of H3/H4 histones during DNA replication. In S. cerevisiae CAF-I is not essential and its loss is associated with reduced gene silencing at telomeres and increased sensitivity to DNA damage. Two kinases, Cyclin Dependent Kinase (CDK) and Dbf4-Dependent Kinase (DDK), are known to phosphorylate the Cac1p subunit of CAF-I, but their role in the regulation of CAF-I activity is not well understood. In this study we systematically mutated the phosphorylation target sites of these kinases. We show that concomitant mutations of the CDK and DDK target sites of Cac1p lead to growth retardation and significant cell cycle defects, altered cell morphology and increased sensitivity to DNA damage. Surprisingly, some mutations also produced flocculation, a phenotype that is lost in most laboratory strains, and displayed elevated expression of FLO genes. None of these effects is observed upon the destruction of CAF-I. In contrast, the mutations that caused flocculation did not affect gene silencing at the mating type and subtelomeric loci. We conclude that dysfunctional CAF-I produces severe phenotypes, which reveal a possible role of CAF-I in the coordination of DNA replication, chromatin reassembly and cell cycle progression. Our study highlights the role of phosphorylation of Cac1p by CDK and a putative role for DDK in the transmission and re-assembly of chromatin during DNA replication.

Multisite phosphorylation code of CDK.

The quantitative model of cyclin-dependent kinase (CDK) function states that cyclins temporally order cell cycle events at different CDK activity levels, or thresholds. The model lacks a mechanistic explanation, as it is not understood how different thresholds are encoded into substrates. We show that a multisite phosphorylation code governs the phosphorylation of CDK targets and that phosphorylation clusters act as timing tags that trigger specific events at different CDK thresholds. Using phospho-degradable CDK threshold sensors with rationally encoded phosphorylation patterns, we were able to predictably program thresholds over the entire range of the Saccharomyces cerevisiae cell cycle. We defined three levels of CDK multisite phosphorylation encoding: (i) Ser-Thr swapping in phosphorylation sites, (ii) patterning of phosphorylation sites, and (iii) cyclin-specific docking combined with modulation of CDK activity. Thus, CDK can signal via hundreds of differentially encoded targets at precise times to provide a temporally ordered phosphorylation pattern required for cell division.

CDK activity provides temporal and quantitative cues for organizing genome duplication.

In eukaryotes, the spatial and temporal organization of genome duplication gives rise to distinctive profiles of replication origin usage along the chromosomes. While it has become increasingly clear that these programs are important for cellular physiology, the mechanisms by which they are determined and modulated remain elusive. Replication initiation requires the function of cyclin-dependent kinases (CDKs), which associate with various cyclin partners to drive cell proliferation. Surprisingly, although we possess detailed knowledge of the CDK regulators and targets that are crucial for origin activation, little is known about whether CDKs play a critical role in establishing the genome-wide pattern of origin selection. We have addressed this question in the fission yeast, taking advantage of a simplified cell cycle network in which cell proliferation is driven by a single cyclin-CDK module. This system allows us to precisely control CDK activity in vivo using chemical genetics. First, in contrast to previous reports, our results clearly show that distinct cyclin-CDK pairs are not essential for regulating specific subsets of origins and for establishing a normal replication program. Importantly, we then demonstrate that the timing at which CDK activity reaches the S phase threshold is critical for the organization of replication in distinct efficiency domains, while the level of CDK activity at the onset of S phase is a dose-dependent modulator of overall origin efficiencies. Our study therefore implicates these different aspects of CDK regulation as versatile mechanisms for shaping the architecture of DNA replication across the genome.

Abstract 2350: In vivo E2F reporting on efficacious dosing schedules of MEK plus CDK4/6 inhibition in melanoma

Abstract Pharmacological targeting of cyclin dependent kinases 4 and 6 (CDK4/6) could represent a viable therapeutic option in combination with BRAF and/or MEK inhibitors in different genetic subsets of melanoma. Indeed, continuous and concurrent dosing of MEK inhibitor (MEKi) plus a CDK4/6 inhibitor (CDK4/6i) leads to melanoma regressions in in vivo models and delays the onset of MEKi resistance. Current scheduling in breast cancer patients for the CDK4/6i, palbociclib, is intermittent due to adverse effects resulting in neutropenia. It is unclear what the most efficacious schedules are of palbociclib plus a MEKi. Utilizing an E2F reporter system, we sought to analyze the efficacy of different CDK4/6i plus MEKi schedules in a quantitative and temporal manner. Intermittent dosing (3 weeks on/1 week off) of both CDK4/6i and MEKi combination therapy resulted in tolerant tumors and rapid reactivation of E2F activity during drug holiday. Continuous MEKi with intermittent CDK4/6i led to more complete responses as compared to continuous CDK4/6i with intermittent MEKi. Weight loss of mice was also evident in the continuous CDK4/6i plus intermittent MEKi arm suggesting adverse events related to continuous CDK inhibition. Importantly, functional proteomic analysis revealed distinct mechanisms of acquired resistance/drug tolerance that arose from the three scheduling arms. Here, we report that upregulation of S6 phosphorylation is associated with acquired resistance to MEK plus CDK4/6 inhibition and can be overcome with an mTOR inhibitor. Taken together, in vivo reporting allows for quantitative measurement of pathway activity associated with inhibitor resistance and can be utilized to optimize combination schedules to improve the therapeutic index in patients. Citation Format: Jessica Teh, Neda Nikbakht, Timothy Purwin, Inna Chervoneva, Prem Patel, Michael Davies, Andrew Aplin. In vivo E2F reporting on efficacious dosing schedules of MEK plus CDK4/6 inhibition in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2350. doi:10.1158/1538-7445.AM2017-2350

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.

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.

Penetration Peg Formation and Invasive Hyphae Development Require Stage-Specific Activation of MoGTI1 in Magnaporthe oryzae.

The hemibiotrophic pathogen Magnaporthe oryzae causes one of the most destructive diseases in cultivated rice. Complex infection-related morphogenesis and production of various effectors are known to be important for successful colonization and disease development. In this study, we characterized the activation of the MoGTI1 transcription factor and its role in infection-related morphogenesis and effector gene expression. The Mogti1 mutant was nonpathogenic, although it was normal in appressorium formation and turgor generation. Close examination showed that Mogti1 was defective in penetration and growth of normal invasive hyphae. Deletion of MoGTI1 affected the expression of the majority of effector genes. The expression of MoGti1 appeared to be controlled by the Mps1 but not Pmk1 mitogen-activated protein kinase (MAPK), and the mps1 and Mogti1 mutants had similar phenotypes in plant infection and cell wall integrity defects. However, lack of MAPK phosphorylation sites and dispensability of the putative MAPK docking site suggested that MoGti1 is not a direct target of Mps1. Site-specific mutagenesis analyses showed that the putative protein kinase A phosphorylation site was not essential for localization of MoGti1 to the nucleus but important for its normal function. Although the cyclin-dependent kinase (CDK) phosphorylation site of MoGti1 is dispensable during vegetative growth and appressorium formation, the S77A mutation affected penetration and invasive growth. Localization of MoGti1(S77A)-green fluorescent protein to the nucleus in late stages of appressorium formation and during invasive growth was not observed, suggesting a stage-specific CDK phosphorylation of MoGti1. Overall, our data indicate that Mps1 may indirectly regulate the expression of MoGti1 in maintaining cell wall integrity, conidiation, and plant infection. MoGti1 is likely a stage-specific target of CDK and plays a crucial role in effector gene expression and morphogenesis related to the development of penetration pegs and invasive hyphae.

Evolutionary conservation of the CDK targets in eukaryotic DNA replication initiation

A fundamental requirement for all organisms is the faithful duplication and transmission of the genetic material. Failure to accurately copy and segregate the genome during cell division leads to loss of genetic information and chromosomal abnormalities. Such genome instability is the hallmark of the earliest stages of tumour formation. Cyclin-dependent kinase (CDK) plays a vital role in regulating the duplication of the genome within the eukaryotic cell cycle. Importantly, this kinase is deregulated in many cancer types and is an emerging target of chemotherapeutics. In this review, I will consider recent advances concerning the role of CDK in replication initiation across eukaryotes. The implications for strict CDK-dependent regulation of genome duplication in the context of the cell cycle will be discussed.