ABSTRACT
The identification of genes involved in replicative stress is key to understanding cancer evolution and to identify therapeutic targets. Here, we show that CDK12 prevents transcription-replication conflicts (TRCs) and the activation of cytotoxic replicative stress upon deregulation of the MYC oncogene. CDK12 was recruited at damaged genes by PARP-dependent DDR-signaling and elongation-competent RNAPII, to repress transcription. Either loss or chemical inhibition of CDK12 led to DDR-resistant transcription of damaged genes. Loss of CDK12 exacerbated TRCs in MYC-overexpressing cells and led to the accumulation of double-strand DNA breaks, occurring between co-directional early-replicating regions and transcribed genes. Overall, our data demonstrate that CDK12 protects genome integrity by repressing transcription of damaged genes, which is required for proper resolution of DSBs at oncogene-induced TRCs. This provides a rationale that explains both how CDK12 deficiency can promote tandem duplications of early-replicated regions during tumor evolution, and how CDK12 targeting can exacerbate replicative-stress in tumors.
Subject(s)
Cyclin-Dependent Kinases , DNA Replication , Transcription, Genetic , Humans , Cyclin-Dependent Kinases/metabolism , Cyclin-Dependent Kinases/genetics , DNA Breaks, Double-Stranded , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins c-myc/genetics , Cell Line, Tumor , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , DNA DamageABSTRACT
Protein-protein interactions (PPIs) stabilization with molecular glues plays a crucial role in drug discovery, albeit with significant challenges. In this study, we propose a dual-site approach, targeting the PPI region and its dynamic surroundings. We conduct molecular dynamics simulations to identify critical sites on the PPI that stabilize the cyclin-dependent kinase 12 - DNA damage-binding protein 1 (CDK12-DDB1) complex, resulting in further cyclin K degradation. This exploration leads to the creation of LL-K12-18, a dual-site molecular glue, which enhances the glue properties to augment degradation kinetics and efficiency. Notably, LL-K12-18 demonstrates strong inhibition of gene transcription and anti-proliferative effects in tumor cells, showing significant potency improvements in MDA-MB-231 (88-fold) and MDA-MB-468 cells (307-fold) when compared to its precursor compound SR-4835. These findings underscore the potential of dual-site approaches in disrupting CDK12 function and offer a structural insight-based framework for the design of cyclin K molecular glues.
Subject(s)
Cyclin-Dependent Kinases , Protein Binding , Humans , Cell Line, Tumor , Cell Proliferation , Cyclin-Dependent Kinases/metabolism , Cyclins , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/chemistry , Molecular Dynamics SimulationABSTRACT
Budding yeast, Saccharomyces cerevisiae, is widely used as a model organism to study the genetics underlying eukaryotic cellular processes and growth critical to cancer development, such as cell division and cell cycle progression. The budding yeast cell cycle is also one of the best-studied dynamical systems owing to its thoroughly resolved genetics. However, the dynamics underlying the crucial cell cycle decision point called the START transition, at which the cell commits to a new round of DNA replication and cell division, are under-studied. The START machinery involves a central cyclin-dependent kinase; cyclins responsible for starting the transition, bud formation, and initiating DNA synthesis; and their transcriptional regulators. However, evidence has shown that the mechanism is more complicated than a simple irreversible transition switch. Activating a key transcription regulator SBF requires the phosphorylation of its inhibitor, Whi5, or an SBF/MBF monomeric component, Swi6, but not necessarily both. Also, the timing and mechanism of the inhibitor Whi5's nuclear export, while important, are not critical for the timing and execution of START. Therefore, there is a need for a consolidated model for the budding yeast START transition, reconciling regulatory and spatial dynamics. We built a detailed mathematical model (START-BYCC) for the START transition in the budding yeast cell cycle based on established molecular interactions and experimental phenotypes. START-BYCC recapitulates the underlying dynamics and correctly emulates key phenotypic traits of ~150 known START mutants, including regulation of size control, localization of inhibitor/transcription factor complexes, and the nutritional effects on size control. Such a detailed mechanistic understanding of the underlying dynamics gets us closer towards deconvoluting the aberrant cellular development in cancer.
Subject(s)
Cell Cycle , Models, Biological , Saccharomyces cerevisiae , Cell Cycle/physiology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , DNA Replication , Computational Biology , Saccharomycetales/genetics , Saccharomycetales/metabolism , Saccharomycetales/physiology , Cyclin-Dependent Kinases/metabolism , Cyclin-Dependent Kinases/genetics , Phosphorylation , Repressor ProteinsABSTRACT
Cyclin-dependent kinase 7 (Cdk7) is required in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of transcription factor TFIIH. Cdk7 forms active complexes by associating with Cyclin H and Mat1, and is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates basic residues conserved in other CDKs, pS164 nucleates an arginine network unique to the ternary Cdk7 complex, involving all three subunits. We identify differential dependencies of kinase activity and substrate recognition on the individual phosphorylations. CAK function is unaffected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by T170 phosphorylation. Moreover, dual T-loop phosphorylation stimulates multisite phosphorylation of the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7 activation is a two-step process wherein S164 phosphorylation precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing processivity, while pT170 enhances activity towards key transcriptional substrates.
Subject(s)
Cyclin-Dependent Kinase-Activating Kinase , Cyclin-Dependent Kinases , Phosphorylation , Humans , Cyclin-Dependent Kinases/metabolism , Cyclin-Dependent Kinases/chemistry , Cyclin-Dependent Kinases/genetics , Cyclin H/metabolism , Cyclin H/chemistry , Cyclin H/genetics , Crystallography, X-Ray , RNA Polymerase II/metabolism , RNA Polymerase II/chemistry , Transcription Factor TFIIH/metabolism , Transcription Factor TFIIH/chemistry , Transcription Factor TFIIH/genetics , Models, Molecular , Transcription Factors/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics , Protein Domains , Cell Cycle ProteinsABSTRACT
In cells, signal transduction heavily relies on the intricate regulation of protein kinases, which provide the fundamental framework for modulating most signaling pathways. Dysregulation of kinase activity has been implicated in numerous pathological conditions, particularly in cancer. The druggable nature of most kinases positions them into a focal point during the process of drug development. However, a significant challenge persists, as the role and biological function of nearly one third of human kinases remains largely unknown.Within this diverse landscape, cyclin-dependent kinases (CDKs) emerge as an intriguing molecular subgroup. In human, this kinase family encompasses 21 members, involved in several key biological processes. Remarkably, 13 of these CDKs belong to the category of understudied kinases, and only 5 having undergone broad investigation to date. This knowledge gap underscores the pressing need to delve into the study of these kinases, starting with a comprehensive review of the less-explored ones.Here, we will focus on the PCTAIRE subfamily of CDKs, which includes CDK16, CDK17, and CDK18, arguably among the most understudied CDKs members. To contextualize PCTAIREs within the spectrum of human pathophysiology, we conducted an exhaustive review of the existing literature and examined available databases. This approach resulted in an articulate depiction of these PCTAIREs, encompassing their expression patterns, 3D configurations, mechanisms of activation, and potential functions in normal tissues and in cancer.We propose that this effort offers the possibility of identifying promising areas of future research that extend from basic research to potential clinical and therapeutic applications.
Subject(s)
Cyclin-Dependent Kinases , Humans , Cyclin-Dependent Kinases/metabolism , Animals , Neoplasms/metabolism , Neoplasms/pathology , Neoplasms/genetics , Signal Transduction , Structure-Activity Relationship , Protein ConformationABSTRACT
Cyclin-dependent kinases (CDKs) play essential roles in regulating the cell cycle and are among the most critical targets for cancer therapy and drug discovery. The primary objective of this research is to derive general structure-activity relationship (SAR) patterns for modeling the selectivity and activity levels of CDK inhibitors using machine learning methods. To accomplish this, 8592 small molecules with different binding affinities to CDK1, CDK2, CDK4, CDK5, and CDK9 were collected from Binding DB, and a diverse set of descriptors was calculated for each molecule. The supervised Kohonen networks (SKN) and counter propagation artificial neural networks (CPANN) models were trained to predict the activity levels and therapeutic targets of the molecules. The validity of models was confirmed through tenfold cross-validation and external test sets. Using selected sets of molecular descriptors (e.g. hydrophilicity and total polar surface area) we derived activity and selectivity maps to elucidate local regions in chemical space for active and selective CDK inhibitors. The SKN models exhibited prediction accuracies ranging from 0.75 to 0.94 for the external test sets. The developed multivariate classifiers were used for ligand-based virtual screening of 2 million random molecules of the PubChem database, yielding areas under the receiver operating characteristic curves ranging from 0.72 to 1.00 for the SKN model. Considering the persistent challenge of achieving CDK selectivity, this research significantly contributes to addressing the issue and underscores the paramount importance of developing drugs with minimized side effects.
Subject(s)
Cyclin-Dependent Kinases , Machine Learning , Neural Networks, Computer , Protein Kinase Inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Structure-Activity Relationship , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Cyclin-Dependent Kinases/chemistry , Humans , Drug Discovery/methodsABSTRACT
Cyclin-dependent kinase 7, along with cyclin H and MAT1, forms the CDK-activating complex (CAK), which directs cell cycle progression via T-loop phosphorylation of cell cycle CDKs. Pharmacological inhibition of CDK7 leads to selective anti-cancer effects in cellular and in vivo models, motivating several ongoing clinical investigations of this target. Current CDK7 inhibitors are either reversible or covalent inhibitors of its catalytic activity. We hypothesized that small molecule targeted protein degradation (TPD) might result in differentiated pharmacology due to the loss of scaffolding functions. Here, we report the design and characterization of a potent CDK7 degrader that is comprised of an ATP-competitive CDK7 binder linked to a CRL2VHL recruiter. JWZ-5-13 effectively degrades CDK7 in multiple cancer cells and leads to a potent inhibition of cell proliferation. Additionally, compound JWZ-5-13 displayed bioavailability in a pharmacokinetic study conducted in mice. Therefore, JWZ-5-13 is a useful chemical probe to investigate the pharmacological consequences of CDK7 degradation.
Subject(s)
Cell Proliferation , Cyclin-Dependent Kinases , Protein Kinase Inhibitors , Humans , Animals , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/chemical synthesis , Cell Proliferation/drug effects , Mice , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Structure-Activity Relationship , Molecular Structure , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Small Molecule Libraries/chemical synthesis , Drug Discovery , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Dose-Response Relationship, Drug , Cyclin-Dependent Kinase-Activating Kinase , Proteolysis/drug effects , Cell Line, Tumor , Drug Screening Assays, AntitumorABSTRACT
Macrophages polarize into alternatively activated M2 macrophages through interleukin (IL)-4, and they express high levels of arginase-1, which promotes anti-inflammatory responses. Several studies have confirmed the anti-inflammatory effects of cyclin-dependent kinase (CDK) 8/19 inhibition, and hence, numerous CDK8/19 inhibitors, such as BRD6989, have been developed. However, the effects of CDK8/19 inhibitors on arginase-1 expression in macrophages have not yet been elucidated. This study investigated the effects of CDK8/19 inhibitor on arginase-1 expression in IL-4-activated macrophages. The results showed that BRD6989 increased arginase-1 expression transcriptionally in murine peritoneal macrophages and the murine macrophage cell line RAW264.7 in an IL-4-dependent manner. In addition, the results indicated that BRD6989 enhances signal transducer and activator of transcription (STAT) 6 phosphorylation. Meanwhile, BRD6989 exhibited the capability to activate p38 mitogen-activated protein kinase (MAPKï¼ even in the absence of IL-4 stimulation. Moreover, we observed that a p38 MAPK inhibitor suppressed the BRD6989-induced increase in arginase-1 expression. Besides, BRD6989 increased the surface expression of CD206, an M2 macrophage marker. Thus, this study demonstrated for the first time that CDK8/19 inhibition increases arginase-1 expression, suggesting that this mechanism involves the activation of STAT6 and p38 MAPK. This finding implies that CDK8/19 inhibition may facilitate the production of anti-inflammatory M2 macrophages.
Subject(s)
Arginase , Cyclin-Dependent Kinase 8 , Cyclin-Dependent Kinases , Interleukin-4 , STAT6 Transcription Factor , p38 Mitogen-Activated Protein Kinases , Animals , Arginase/metabolism , Arginase/antagonists & inhibitors , STAT6 Transcription Factor/metabolism , Mice , p38 Mitogen-Activated Protein Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , RAW 264.7 Cells , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Interleukin-4/metabolism , Cyclin-Dependent Kinase 8/antagonists & inhibitors , Cyclin-Dependent Kinase 8/metabolism , Protein Kinase Inhibitors/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Phosphorylation/drug effects , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/metabolism , Enzyme Activation/drug effects , Flavonoids , Piperidines , Cyclin-Dependent Kinase 9ABSTRACT
The regulation of the cancer cell cycle heavily relies on cyclin-dependent kinases (CDKs). Targeting CDKs has been identified as a promising approach for effective cancer therapy. In recent years, there has been significant attention paid towards developing small-molecule CDK inhibitors in the field of drug discovery. Notably, five such inhibitors have already received regulatory approval for the treatment of different cancers, including breast tumors, lung malignancies, and hematological malignancies. This review provides an overview of the synthetic routes used to produce 17 representative small-molecule CDK inhibitors that have obtained regulatory approval or are currently being evaluated through clinical trials. It also discusses their clinical applications for treating CDK-related diseases and explores the challenges and limitations associated with their use in a clinical setting, which will stimulate the further development of novel CDK inhibitors. By integrating therapeutic applications, synthetic methodologies, and mechanisms of action observed in various clinical trials involving these CDK inhibitors, this review facilitates a comprehensive understanding of the versatile roles and therapeutic potential offered by interventions targeting CDKs.
Subject(s)
Antineoplastic Agents , Cyclin-Dependent Kinases , Neoplasms , Protein Kinase Inhibitors , Small Molecule Libraries , Humans , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/chemical synthesis , Neoplasms/drug therapy , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Small Molecule Libraries/pharmacology , Small Molecule Libraries/chemistry , Small Molecule Libraries/therapeutic use , Small Molecule Libraries/chemical synthesis , Animals , Drug Discovery , Clinical Trials as TopicABSTRACT
Cell cycle regulation is largely abnormal in cancers. Molecular understanding and therapeutic targeting of the aberrant cell cycle are essential. Here, we identified that an underappreciated serine/threonine kinase, cyclin-dependent kinase-like 3 (CDKL3), crucially drives rapid cell cycle progression and cell growth in cancers. With regard to mechanism, CDKL3 localizes in the nucleus and associates with specific cyclin to directly phosphorylate retinoblastoma (Rb) for quiescence exit. In parallel, CDKL3 prevents the ubiquitin-proteasomal degradation of cyclin-dependent kinase 4 (CDK4) by direct phosphorylation on T172 to sustain G1 phase advancement. The crucial function of CDKL3 in cancers was demonstrated both in vitro and in vivo. We also designed, synthesized, and characterized a first-in-class CDKL3-specific inhibitor, HZ1. HZ1 exhibits greater potency than CDK4/6 inhibitor in pan-cancer treatment by causing cell cycle arrest and overcomes acquired resistance to CDK4/6 inhibitor. In particular, CDKL3 has significant clinical relevance in colon cancer, and the effectiveness of HZ1 was demonstrated by murine and patient-derived cancer models. Collectively, this work presents an integrated paradigm of cancer cell cycle regulation and suggests CDKL3 targeting as a feasible approach in cancer treatment.
Subject(s)
Cyclin-Dependent Kinase 4 , Humans , Animals , Mice , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/genetics , Cell Line, Tumor , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Colonic Neoplasms/pathology , Colonic Neoplasms/metabolism , Colonic Neoplasms/genetics , Colonic Neoplasms/drug therapy , Cell CycleABSTRACT
The biology of the cyclin-dependent kinase-like (CDKL) kinase family remains enigmatic. Contrary to their nomenclature, CDKLs do not rely on cyclins for activation and are not involved in cell cycle regulation. Instead, they share structural similarities with mitogen-activated protein kinases and glycogen synthase kinase-3, although their specific functions and associated signaling pathways are still unknown. Previous studies have shown that the activation of CDKL5 kinase contributes to the development of acute kidney injury (AKI) by suppressing the protective SOX9-dependent transcriptional program in tubular epithelial cells. In the current study, we measured the functional activity of all five CDKL kinases and discovered that, in addition to CDKL5, CDKL1 is also activated in tubular epithelial cells during AKI. To explore the role of CDKL1, we generated a germline knockout mouse that exhibited no abnormalities under normal conditions. Notably, when these mice were challenged with bilateral ischemia-reperfusion and rhabdomyolysis, they were found to be protected from AKI. Further mechanistic investigations revealed that CDKL1 phosphorylates and destabilizes SOX11, contributing to tubular dysfunction. In summary, this study has unveiled a previously unknown CDKL1-SOX11 axis that drives tubular dysfunction during AKI.NEW & NOTEWORTHY Identifying and targeting pathogenic protein kinases holds potential for drug discovery in treating acute kidney injury. Our study, using novel germline knockout mice, revealed that Cdkl1 kinase deficiency does not affect mouse viability but provides protection against acute kidney injury. This underscores the importance of Cdkl1 kinase in kidney injury and supports the development of targeted small-molecule inhibitors as potential therapeutics.
Subject(s)
Acute Kidney Injury , Cyclin-Dependent Kinases , Mice, Knockout , SOXC Transcription Factors , Signal Transduction , Animals , Acute Kidney Injury/metabolism , Acute Kidney Injury/pathology , Acute Kidney Injury/genetics , Cyclin-Dependent Kinases/metabolism , Cyclin-Dependent Kinases/genetics , Phosphorylation , SOXC Transcription Factors/metabolism , SOXC Transcription Factors/genetics , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Rhabdomyolysis/metabolism , Mice, Inbred C57BL , Disease Models, Animal , Mice , Male , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Epithelial Cells/metabolismABSTRACT
Cyclin-dependent kinase-like 3 (CDKL3) has been identified as an oncogene in certain types of tumors. Nonetheless, its function in hepatocellular carcinoma (HCC) is poorly understood. In this study, we conducted a comprehensive analysis of CDKL3 based on data from the HCC cohort of The Cancer Genome Atlas (TCGA). Our analysis included gene expression, diagnosis, prognosis, functional enrichment, tumor microenvironment and metabolic characteristics, tumor burden, mRNA expression-based stemness, alternative splicing, and prediction of therapy response. Additionally, we performed a cell counting kit-8 assay, TdT-mediated dUTP nick-end Labeling staining, migration assay, wound healing assay, colony formation assay, and nude mouse experiments to confirm the functional relevance of CDKL3 in HCC. Our findings showed that CDKL3 was significantly upregulated in HCC patients compared to controls. Various bioinformatic analyses suggested that CDKL3 could serve as a potential marker for HCC diagnosis and prognosis. Furthermore, CDKL3 was found to be involved in various mechanisms linked to the development of HCC, including copy number variation, tumor burden, genomic heterogeneity, cancer stemness, and alternative splicing of CDKL3. Notably, CDKL3 was also closely correlated with tumor immune cell infiltration and the expression of immune checkpoint markers. Additionally, CDKL3 was shown to independently function as a risk predictor for overall survival in HCC patients by multivariate Cox regression analysis. Furthermore, the knockdown of CDKL3 significantly inhibited cell proliferation in vitro and in vivo, indicating its role as an oncogene in HCC. Taken together, our findings suggest that CDKL3 shows promise as a biomarker for the detection and treatment outcome prediction of HCC patients.
Subject(s)
Biomarkers, Tumor , Carcinoma, Hepatocellular , Liver Neoplasms , Carcinoma, Hepatocellular/diagnosis , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/diagnosis , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Liver Neoplasms/metabolism , Humans , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Prognosis , Animals , Mice , Mice, Nude , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Male , Female , Cell Proliferation/geneticsABSTRACT
Cancer immunotherapies are ineffective in nonresponding patients due to absence of immune responses. Here, we identified that dihydroartemisinin (DHA) induced immunogenic cell death (ICD) in hepatocellular carcinoma (HCC), proved by release or surface expose of damage-associated molecular patterns and in vivo protective vaccine activity. Mechanistically, DHA can inhibit cyclin-dependent kinases (CDKs), leading to a buildup of intracellular reactive oxygen species (ROS), which induces immunogenic cell death. In both Hepa1-6 and H22 tumor bearing mice, DHA exerted anti-tumor activity through increasing tumor-infiltrating CD8+ T cells with expression of activation makers (CD25 and CD69), secretion of intracellular cytokines (IFN-γ and TNF-α) and activated dendritic cells expressing MHCâ ¡, CD80 and CD86. In hepa1-6 tumor bearing mice, DHA decreased immunosuppressive myeloid-derived suppressor cells. Furthermore, DHA enhanced the anti-PD-1 antibody and chimeric antigen receptor (CAR) T cell-mediated tumor suppression through recruitment and activation of endogenous CD8+ T cells. Overall, we demonstrated that by inhibiting CDKs, DHA can remodel tumor micro-environment to amplify anti-tumor immune responses in HCC. These findings provide a promising therapy option for HCC patients.
Subject(s)
Artemisinins , CD8-Positive T-Lymphocytes , Carcinoma, Hepatocellular , Cyclin-Dependent Kinases , Immunotherapy , Liver Neoplasms , Mice, Inbred C57BL , Tumor Microenvironment , Animals , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunology , Artemisinins/pharmacology , Artemisinins/therapeutic use , Mice , Carcinoma, Hepatocellular/therapy , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/drug therapy , Liver Neoplasms/immunology , Liver Neoplasms/drug therapy , Liver Neoplasms/therapy , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Cell Line, Tumor , Humans , Immunotherapy/methods , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , Reactive Oxygen Species/metabolism , Myeloid-Derived Suppressor Cells/drug effects , Myeloid-Derived Suppressor Cells/immunology , MaleABSTRACT
Fungi have historically been the source of numerous important medicinal compounds, but full exploitation of their genetic potential for drug development has been hampered in traditional discovery paradigms. Here we describe a radically different approach, top-down drug discovery (TD3), starting with a massive digital search through a database of over 100,000 fully genomicized fungi to identify loci encoding molecules with a predetermined human target. We exemplify TD3 by the selection of cyclin-dependent kinases (CDKs) as targets and the discovery of two molecules, 1 and 2, which inhibit therapeutically important human CDKs. 1 and 2 exhibit a remarkable mechanism, forming a site-selective covalent bond to the CDK active site Lys. We explored the structure-activity relationship via semi- and total synthesis, generating an analog, 43, with improved kinase selectivity, bioavailability, and efficacy. This work highlights the power of TD3 to identify mechanistically and structurally novel molecules for the development of new medicines.
Subject(s)
Cyclin-Dependent Kinases , Drug Discovery , Protein Kinase Inhibitors , Humans , Structure-Activity Relationship , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/chemical synthesis , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Animals , Genomics/methods , Models, MolecularABSTRACT
AIMS: Colorectal cancer (CRC) remains a major global health issue, with metastatic cases presenting poor prognosis despite advances in chemotherapy and targeted therapy. Irinotecan, a key drug for advanced CRC treatment, faces challenges owing to the development of resistance. This study aimed to understand the mechanisms underlying irinotecan resistance in colorectal cancer. MAIN METHODS: We created a cell line resistant to irinotecan using HT29 cells. These resistant cells were utilized to investigate the role of the CDK7-MDK axis. We employed bulk RNA sequencing, conducted in vivo experiments with mice, and analyzed patient tissues to examine the effects of the CDK7-MDK axis on the cellular response to irinotecan. KEY FINDINGS: Our findings revealed that HT29 cells resistant to irinotecan, a crucial colorectal cancer medication, exhibited significant phenotypic and molecular alterations compared to their parental counterparts, including elevated stem cell characteristics and increased levels of cytokines and drug resistance proteins. Notably, CDK7 expression was substantially higher in these resistant cells, and targeting CDK7 effectively decreased their survival and tumor growth, enhancing irinotecan sensitivity. RNA-seq analysis indicated that suppression of CDK7 in irinotecan-resistant HT29 cells significantly reduced Midkine (MDK) expression. Decreased CDK7 and MDK levels, achieved through siRNA and the CDK7 inhibitor THZ1, enhanced the sensitivity of resistant HT29 cells to irinotecan. SIGNIFICANCE: Our study sheds light on how CDK7 and MDK influence irinotecan resistance in colorectal and highlights the potential of MDK-targeted therapies. We hypothesized that irinotecan sensitivity and overall treatment efficacy would improve by inhibiting MDK. This finding encourages a careful yet proactive investigation of MDK as a therapeutic target to enhance outcomes in colorectal cancer patients.
Subject(s)
Colorectal Neoplasms , Cyclin-Dependent Kinase-Activating Kinase , Cyclin-Dependent Kinases , Drug Resistance, Neoplasm , Irinotecan , Irinotecan/pharmacology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/genetics , Humans , Animals , Drug Resistance, Neoplasm/drug effects , Mice , HT29 Cells , Cyclin-Dependent Kinases/metabolism , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/genetics , Mice, Nude , Xenograft Model Antitumor Assays , Gene Expression Regulation, Neoplastic/drug effects , Mice, Inbred BALB C , Female , Cell Proliferation/drug effectsABSTRACT
Detection of disease biomarkers constitutes a major challenge for the development of personalized and predictive diagnostics as well as companion assays. Protein kinases (PKs) involved in the coordination of cell cycle progression and proliferation that are hyperactivated in human cancers constitute attractive pharmacological targets and relevant biomarkers. Although it is relatively straightforward to assess the relative abundance of PKs in a biological sample, there is not always a direct correlation with enzymatic activity, which is regulated by several posttranslational mechanisms. Studies of relative abundance therefore convey limited information, and the lack of selective, sensitive, and standardized tools together with the inherent complexity of biological samples makes it difficult to quantify PK activities in physio-pathological tissues. To address this challenge, we have developed a toolbox of fluorescent biosensors that report on CDK activities in a sensitive, selective, dose-dependent, and quantitative fashion, which we have implemented to profile CDK activity signatures in cancer cell lines and biopsies from human tumors. In this study, we report on a standardized and calibrated biosensing approach to quantify CDK1,2,4, and 6 activities simultaneously through a combination of four different biosensors in a panel of 40 lung adenocarcinoma and 40 follicular lymphoma samples. CDK activity profiling highlighted two major patterns which were further correlated with age, sex of patients, tumor size, grade, and genetic and immunohistochemical features of the biopsies. Multiplex CDKACT biosensing technology provides new and complementary information relative to current genetic and immunohistochemical characterization of tumor biopsies, which will be useful for diagnostic purposes, potentially guiding therapeutic decision. These fluorescent peptide biosensors offer promise for personalized diagnostics based on kinase activity profiling.
Subject(s)
Biosensing Techniques , Cyclin-Dependent Kinases , Humans , Biosensing Techniques/methods , Cyclin-Dependent Kinases/metabolism , Peptides/chemistry , Biopsy , Fluorescent Dyes/chemistry , Cell Line, Tumor , Lung Neoplasms/pathology , Lung Neoplasms/diagnosis , Lung Neoplasms/enzymologyABSTRACT
AML is an aggressive malignancy of immature myeloid progenitor cells. Discovering effective treatments for AML through cell differentiation and anti-proliferation remains a significant challenge. Building on previous studies on CDK2 PROTACs with differentiation-inducing properties, this research aims to enhance CDKs degradation through structural optimization to facilitate the differentiation and inhibit the proliferation of AML cells. Compound C3, featuring a 4-methylpiperidine ring linker, effectively degraded CDK2 with a DC50 value of 18.73 ± 10.78 nM, and stimulated 72.77 ± 3.51 % cell differentiation at 6.25 nM in HL-60 cells. Moreover, C3 exhibited potent anti-proliferative activity against various AML cell types. Degradation selectivity analysis indicated that C3 could be endowed with efficient degradation of CDK2/4/6/9 and FLT3, especially FLT3-ITD in MV4-11 cells. These findings propose that C3 combined targeting CDK2/4/6/9 and FLT3 with enhanced differentiation and proliferation inhibition, which holds promise as a potential treatment for AML.
Subject(s)
Antineoplastic Agents , Cyclin-Dependent Kinases , Drug Discovery , Leukemia, Myeloid, Acute , Protein Kinase Inhibitors , Proteolysis Targeting Chimera , Proteolysis , fms-Like Tyrosine Kinase 3 , Humans , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , fms-Like Tyrosine Kinase 3/metabolism , Leukemia, Myeloid, Acute/drug therapy , Molecular Structure , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/chemical synthesis , Structure-Activity Relationship , Proteolysis Targeting Chimera/chemistry , Proteolysis Targeting Chimera/pharmacology , Proteolysis Targeting Chimera/therapeutic useABSTRACT
The development of selective modulators of cyclin-dependent kinases (CDKs), a kinase family with numerous members and functional variations, is a significant preclinical challenge. Recent advancements in crystallography have revealed subtle differences in the highly conserved CDK pockets. Exploiting these differences has proven to be an effective strategy for achieving excellent drug selectivity. While previous reports briefly discussed the structural features that lead to selectivity in individual CDK members, attaining inhibitor selectivity requires consideration of not only the specific structures of the target CDK but also the features of off-target members. In this review, we summarize the structure-activity relationships (SARs) that influence selectivity in CDK drug development and analyze the pocket features that lead to selectivity using molecular-protein binding models. In addition, in recent years, novel CDK modulators have been developed, providing more avenues for achieving selectivity. These cases were also included. We hope that these efforts will assist in the development of novel CDK drugs.
Subject(s)
Cyclin-Dependent Kinases , Protein Kinase Inhibitors , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/chemical synthesis , Humans , Structure-Activity Relationship , Molecular Structure , Animals , Models, MolecularABSTRACT
BACKGROUND: ARID1A, a subunit of the SWI/SNF chromatin remodeling complex, is thought to play a significant role both in tumor suppression and tumor initiation, which is highly dependent upon context. Previous studies have suggested that ARID1A deficiency may contribute to cancer development. The specific mechanisms of whether ARID1A loss affects tumorigenesis by RNA editing remain unclear. RESULTS: Our findings indicate that the deficiency of ARID1A leads to an increase in RNA editing levels and alterations in RNA editing categories mediated by adenosine deaminases acting on RNA 1 (ADAR1). ADAR1 edits the CDK13 gene at two previously unidentified sites, namely Q113R and K117R. Given the crucial role of CDK13 as a cyclin-dependent kinase, we further observed that ADAR1 deficiency results in changes in the cell cycle. Importantly, the sensitivity of ARID1A-deficient tumor cells to SR-4835, a CDK12/CDK13 inhibitor, suggests a promising therapeutic approach for individuals with ARID1A-mutant tumors. Knockdown of ADAR1 restored the sensitivity of ARID1A deficient cells to SR-4835 treatment. CONCLUSIONS: ARID1A deficiency promotes RNA editing of CDK13 by regulating ADAR1.
Subject(s)
Adenosine Deaminase , Cyclin-Dependent Kinases , DNA-Binding Proteins , RNA Editing , RNA-Binding Proteins , Transcription Factors , Adenosine Deaminase/metabolism , Adenosine Deaminase/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Humans , Transcription Factors/metabolism , Transcription Factors/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Cyclin-Dependent Kinases/metabolism , Cyclin-Dependent Kinases/genetics , Cell Line, Tumor , CDC2 Protein KinaseABSTRACT
Lung cancer is one of the most significant malignancies, with both high morbidity and mortality. CDK10 is closely related to cancer progression and metastasis. However, its role in lung cancer radioresistance demands further clarification. In this study, we demonstrated that CDK10 was downregulated in lung cancer tissues, and CDK10 expression level was associated with the clinical prognosis in lung cancer patients. We also found that silencing CDK10 promoted lung cancer cell proliferation, migration, and radioresistance. We further verified that silencing CDK10 facilitated the activation of JNK/c-Jun signaling, and c-Jun depletion could reverse the effects of CDK10 knockdown in lung cancer cells. Our findings revealed that CDK10 plays an important role in cell growth and radioresistance by inhibiting JNK/c-Jun signaling pathway in lung cancer. Therefore, CDK10 might be a promising therapeutic target in lung cancer. [BMB Reports 2024; 57(7): 336-341].