ABSTRACT
Targeted protein degradation is a groundbreaking modality in drug discovery; however, the regulatory mechanisms are still not fully understood. Here, we identify cellular signaling pathways that modulate the targeted degradation of the anticancer target BRD4 and related neosubstrates BRD2/3 and CDK9 induced by CRL2VHL- or CRL4CRBN -based PROTACs. The chemicals identified as degradation enhancers include inhibitors of cellular signaling pathways such as poly-ADP ribosylation (PARG inhibitor PDD00017273), unfolded protein response (PERK inhibitor GSK2606414), and protein stabilization (HSP90 inhibitor luminespib). Mechanistically, PARG inhibition promotes TRIP12-mediated K29/K48-linked branched ubiquitylation of BRD4 by facilitating chromatin dissociation of BRD4 and formation of the BRD4-PROTAC-CRL2VHL ternary complex; by contrast, HSP90 inhibition promotes BRD4 degradation after the ubiquitylation step. Consequently, these signal inhibitors sensitize cells to the PROTAC-induced apoptosis. These results suggest that various cell-intrinsic signaling pathways spontaneously counteract chemically induced target degradation at multiple steps, which could be liberated by specific inhibitors.
Subject(s)
Cell Cycle Proteins , Proteolysis , Signal Transduction , Transcription Factors , Ubiquitination , Humans , Signal Transduction/drug effects , Proteolysis/drug effects , Transcription Factors/metabolism , Transcription Factors/genetics , Cell Cycle Proteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Cell Line, Tumor , Apoptosis/drug effects , Cyclin-Dependent Kinase 9/metabolism , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Bromodomain Containing ProteinsABSTRACT
RNA polymerase II (RNAPII) transcription initiates bidirectionally at many human protein-coding genes. Sense transcription usually dominates and leads to messenger RNA production, whereas antisense transcription rapidly terminates. The basis for this directionality is not fully understood. Here, we show that sense transcriptional initiation is more efficient than in the antisense direction, which establishes initial promoter directionality. After transcription begins, the opposing functions of the endonucleolytic subunit of Integrator, INTS11, and cyclin-dependent kinase 9 (CDK9) maintain directionality. Specifically, INTS11 terminates antisense transcription, whereas sense transcription is protected from INTS11-dependent attenuation by CDK9 activity. Strikingly, INTS11 attenuates transcription in both directions upon CDK9 inhibition, and the engineered recruitment of CDK9 desensitises transcription to INTS11. Therefore, the preferential initiation of sense transcription and the opposing activities of CDK9 and INTS11 explain mammalian promoter directionality.
Subject(s)
Cyclin-Dependent Kinase 9 , Promoter Regions, Genetic , Transcription Initiation, Genetic , Humans , Cyclin-Dependent Kinase 9/metabolism , Cyclin-Dependent Kinase 9/genetics , Gene Expression Regulation , Nuclear Proteins , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , Transcription, Genetic , Transcriptional Elongation FactorsABSTRACT
CONTEXT: Cyclin-dependent kinase 9 (CDK9) plays a significant role in gene regulation and RNA polymerase II transcription under basal and stimulated conditions. The upregulation of transcriptional homeostasis by CDK9 leads to various malignant tumors and therefore acts as a valuable drug target in addressing cancer incidences. Ongoing drug development endeavors targeting CDK9 have yielded numerous clinical candidate molecules currently undergoing investigation as potential CDK9 modulators, though none have yet received Food and Drug Administration (FDA) approval. METHODS: In this study, we employ in silico approaches including the molecular docking and molecular dynamics simulations for the virtual screening over the natural compounds library to identify novel promising selective CDK9 inhibitors. The compounds derived from the initial virtual screening were subsequently employed for molecular dynamics simulations and binding free energy calculations to study the compound's stability under virtual physiological conditions. The first-generation CDK inhibitor Flavopiridol was used as a reference to compare with our novel hit compound as a CDK9 antagonist. The 500-ns molecular dynamics simulation and binding free energy calculation showed that two natural compounds showed better binding affinity and interaction mode with CDK9 receptors over the reference Flavopiridol. They also showed reasonable figures in the predicted absorption, distribution, metabolism, excretion, and toxicity (ADMET) calculations as well as in computational cytotoxicity predictions. Therefore, we anticipate that the proposed scaffolds could contribute to developing potential and selective CDK9 inhibitors subjected to further validations.
Subject(s)
Cyclin-Dependent Kinase 9 , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Kinase Inhibitors , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Cyclin-Dependent Kinase 9/metabolism , Cyclin-Dependent Kinase 9/chemistry , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Humans , Protein Binding , Biological Products/chemistry , Biological Products/pharmacology , Flavonoids/chemistry , Flavonoids/pharmacology , PiperidinesABSTRACT
Breast cancer is one of the most prevalent malignancies affecting women worldwide, underscoring the urgent need for more effective and specific treatments. Proteolysis-targeting chimeras (PROTACs) have emerged as a promising strategy to develop new lead compounds by selectively targeting oncoproteins for degradation. In this study, we designed, synthesized and evaluated a CRBN-based PROTAC, L055, which targets CDK9. Our findings demonstrate that L055 effectively inhibits the proliferation, induces cell cycle arrest, and decreases the survival of ERα-positive breast cancer cells in vitro. L055 specifically binds to CDK9, facilitating its degradation via the CRBN-dependent proteasomal pathway. Additionally, L055 suppressed the growth of organoids and tumors derived from T47D and MCF7 cells in nude mice. Thus, L055 represents a potential novel therapeutic agent for ERα-positive breast cancer and potentially other malignancies.
Subject(s)
Breast Neoplasms , Cell Proliferation , Cyclin-Dependent Kinase 9 , Estrogen Receptor alpha , Mice, Nude , Proteolysis , Humans , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Female , Animals , Estrogen Receptor alpha/metabolism , Proteolysis/drug effects , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Cyclin-Dependent Kinase 9/metabolism , Cell Proliferation/drug effects , Cell Line, Tumor , MCF-7 Cells , Xenograft Model Antitumor Assays , Mice, Inbred BALB C , Mice , Antineoplastic Agents/pharmacology , Cell Cycle Checkpoints/drug effects , Ubiquitin-Protein Ligases/metabolism , Adaptor Proteins, Signal Transducing/metabolismABSTRACT
Triple-negative breast cancer (TNBC) represents a highly aggressive and heterogeneous malignancy. Currently, effective therapies for TNBC are very limited and remain a significant unmet clinical need. Targeting the transcription-regulating cyclin-dependent kinase 9 (CDK9) has emerged as a promising avenue for therapeutic treatment of TNBC. Herein, we report the design, synthesis, optimization, and evaluation of a new series of aminopyrazolotriazine compounds as orally bioavailable, potent, and CDK9/2 selectivity-improved inhibitors, enabling efficacious inhibition of TNBC cell growth, as well as notable antitumor effect in TNBC models. The compound C35 demonstrated low-nanomolar potency with substantially improved CDK9/2 selectivity, downregulated the CDK9-downstream targets (e.g., MCL-1), and induced apoptosis in TNBC cell lines. Moreover, with the desired oral bioavailability, oral administration of C35 could significantly suppress the tumor progression in two TNBC mouse models. This study demonstrates that target transcriptional regulation is an effective strategy and holds promising potential as a targeted therapy for the treatment of TNBC.
Subject(s)
Antineoplastic Agents , Cyclin-Dependent Kinase 9 , Protein Kinase Inhibitors , Triple Negative Breast Neoplasms , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Cyclin-Dependent Kinase 9/metabolism , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/pathology , Humans , Animals , Female , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Administration, Oral , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/chemical synthesis , Mice , Cell Line, Tumor , Structure-Activity Relationship , Biological Availability , Cell Proliferation/drug effects , Apoptosis/drug effects , Drug Discovery , Transcription, Genetic/drug effects , Mice, Nude , Xenograft Model Antitumor AssaysABSTRACT
The Myb proto-oncogene encodes the transcription factor c-MYB, which is critical for hematopoiesis. Distant enhancers of Myb form a hub of interactions with the Myb promoter. We identified a long non-coding RNA (Myrlin) originating from the -81-kb murine Myb enhancer. Myrlin and Myb are coordinately regulated during erythroid differentiation. Myrlin TSS deletion using CRISPR-Cas9 reduced Myrlin and Myb expression and LDB1 complex occupancy at the Myb enhancers, compromising enhancer contacts and reducing RNA Pol II occupancy in the locus. In contrast, CRISPRi silencing of Myrlin left LDB1 and the Myb enhancer hub unperturbed, although Myrlin and Myb expressions were downregulated, decoupling transcription and chromatin looping. Myrlin interacts with the KMT2A/MLL1 complex. Myrlin CRISPRi compromised KMT2A occupancy in the Myb locus, decreasing CDK9 and RNA Pol II binding and resulting in Pol II pausing in the Myb first exon/intron. Thus, Myrlin directly participates in activating Myb transcription by recruiting KMT2A.
Subject(s)
Enhancer Elements, Genetic , Histone-Lysine N-Methyltransferase , Myeloid-Lymphoid Leukemia Protein , Proto-Oncogene Proteins c-myb , Transcription, Genetic , Proto-Oncogene Proteins c-myb/metabolism , Proto-Oncogene Proteins c-myb/genetics , Animals , Myeloid-Lymphoid Leukemia Protein/metabolism , Myeloid-Lymphoid Leukemia Protein/genetics , Mice , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Enhancer Elements, Genetic/genetics , RNA Polymerase II/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Humans , Cyclin-Dependent Kinase 9/metabolism , Cyclin-Dependent Kinase 9/genetics , Proto-Oncogene Mas , Protein Binding , Cell Differentiation/genetics , Enhancer RNAsABSTRACT
Acute myeloid leukemia (AML) accounts for greater than twenty thousand new cases of leukemia annually in the United States. The average five-year survival rate is approximately 30%, pointing to the need for developing novel model systems for drug discovery. In particular, patients with chromosomal rearrangements in the mixed lineage leukemia (MLL) gene have higher relapse rates with poor outcomes. In this study we investigated the expression of human MLL-ENL and MLL-AF9 in the myeloid lineage of zebrafish embryos. We observed an expansion of MLL positive cells and determined these cells colocalized with the myeloid markers spi1b, mpx, and mpeg. In addition, expression of MLL-ENL and MLL-AF9 induced the expression of endogenous bcl2 and cdk9, genes that are often dysregulated in MLL-r-AML. Co-treatment of lyz: MLL-ENL or lyz:MLL-AF9 expressing embryos with the BCL2 inhibitor, Venetoclax, and the CDK9 inhibitor, Flavopiridol, significantly reduced the number of MLL positive cells compared to embryos treated with vehicle or either drug alone. In addition, cotreatment with Venetoclax and Flavopiridol significantly reduced the expression of endogenous mcl1a compared to vehicle, consistent with AML. This new model of MLL-r-AML provides a novel tool to understand the molecular mechanisms underlying disease progression and a platform for drug discovery.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic , Cyclin-Dependent Kinase 9 , Leukemia, Myeloid, Acute , Myeloid-Lymphoid Leukemia Protein , Oncogene Proteins, Fusion , Proto-Oncogene Proteins c-bcl-2 , Zebrafish , Zebrafish/genetics , Zebrafish/embryology , Animals , Cyclin-Dependent Kinase 9/genetics , Cyclin-Dependent Kinase 9/metabolism , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Myeloid-Lymphoid Leukemia Protein/genetics , Myeloid-Lymphoid Leukemia Protein/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Humans , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/metabolism , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Sulfonamides/pharmacology , Piperidines/pharmacology , Embryo, Nonmammalian , Flavonoids/pharmacology , Myeloid Cells/metabolism , Myeloid Cells/drug effects , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
The intestinal environment facilitates HIV-1 infection via mechanisms involving the gut-homing vitamin A-derived retinoic acid (RA), which transcriptionally reprograms CD4+ T cells for increased HIV-1 replication/outgrowth. Consistently, colon-infiltrating CD4+ T cells carry replication-competent viral reservoirs in people with HIV-1 (PWH) receiving antiretroviral therapy (ART). Intriguingly, integrative infection in colon macrophages, a pool replenished by monocytes, represents a rare event in ART-treated PWH, thus questioning the effect of RA on macrophages. Here, we demonstrate that RA enhances R5 but not X4 HIV-1 replication in monocyte-derived macrophages (MDMs). RNA sequencing, gene set variation analysis, and HIV interactor NCBI database interrogation reveal RA-mediated transcriptional reprogramming associated with metabolic/inflammatory processes and HIV-1 resistance/dependency factors. Functional validations uncover post-entry mechanisms of RA action including SAMHD1-modulated reverse transcription and CDK9/RNA polymerase II (RNAPII)-dependent transcription under the control of mammalian target of rapamycin (mTOR). These results support a model in which macrophages residing in the intestine of ART-untreated PWH contribute to viral replication/dissemination in an mTOR-sensitive manner.
Subject(s)
HIV-1 , Macrophages , TOR Serine-Threonine Kinases , Tretinoin , Virus Replication , Macrophages/metabolism , Macrophages/virology , Macrophages/drug effects , Humans , HIV-1/drug effects , TOR Serine-Threonine Kinases/metabolism , Tretinoin/pharmacology , Virus Replication/drug effects , Reverse Transcription/drug effects , SAM Domain and HD Domain-Containing Protein 1/metabolism , SAM Domain and HD Domain-Containing Protein 1/genetics , HIV Infections/virology , HIV Infections/drug therapy , HIV Infections/metabolism , Cyclin-Dependent Kinase 9/metabolism , RNA Polymerase II/metabolism , Transcription, Genetic/drug effectsABSTRACT
Sarcomas include various subtypes comprising two significant groups - soft tissue and bone sarcomas. Although the survival rate for some sarcoma subtypes has improved over time, the current methods of treatment remain efficaciously limited, as recurrent, and metastatic diseases remain a major obstacle. There is a need for better options and therapeutic strategies in treating sarcoma. Cyclin dependent kinase 9 (CDK9) is a transcriptional kinase and has emerged as a promising target for treating various cancers. The aberrant expression and activation of CDK9 have been observed in several sarcoma subtypes, including rhabdomyosarcoma, synovial sarcoma, osteosarcoma, Ewing sarcoma, and chordoma. Enhanced CDK9 expression has also been correlated with poorer prognosis in sarcoma patients. As a master regulator of transcription, CDK9 promotes transcription elongation by phosphorylation and releasing RNA polymerase II (RNAPII) from its promoter proximal pause. Release of RNAPII from this pause induces transcription of critical genes in the tumor cell. Overexpression and activation of CDK9 have been observed to lead to the expression of oncogenes, including MYC and MCL-1, that aid sarcoma development and progression. Inhibition of CDK9 in sarcoma has been proven to reduce these oncogenes' expression and decrease proliferation and growth in different sarcoma cells. Currently, there are several CDK9 inhibitors in preclinical and clinical investigations. This review aims to highlight the recent discovery and results on the transcriptional role and therapeutic potential of CDK9 in sarcoma.
Subject(s)
Cyclin-Dependent Kinase 9 , Sarcoma , Transcription, Genetic , Humans , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Cyclin-Dependent Kinase 9/metabolism , Cyclin-Dependent Kinase 9/genetics , Sarcoma/genetics , Sarcoma/drug therapy , Sarcoma/metabolism , Sarcoma/pathology , Sarcoma/therapy , Animals , Gene Expression Regulation, Neoplastic , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/pharmacologyABSTRACT
Cyclin-dependent kinase 9 (CDK9) plays a role in transcriptional regulation, which had become an attractive target for discovery of antitumor agent. In this work, beyond traditional CDK9 inhibitor with bidentate ligands in ATP binding domain, a series of novel CDK9 inhibitor with tridentate ligand were designed and synthesized. Surprisingly, this unique tridentate ligand structure endows better CDK9 inhibition selectivity compared to other CDK subtypes, and the lead candidate compound Z4-7a showed effective proliferation inhibition in HCT116 cells with acceptable pharmacokinetic properties. Research on the mechanism indicated that Z4-7a could induce apoptosis in the HCT116 cell line by inhibiting phosphorylation of RNA polymerase II at Ser2, which resulted in the inhibition of apoptosis-related genes and proteins expression. In brief, introduction of tridentate ligand might work as a promising strategy for the development of novel selective CDK9 inhibitor.
Subject(s)
Antineoplastic Agents , Apoptosis , Cell Proliferation , Cyclin-Dependent Kinase 9 , Dose-Response Relationship, Drug , Drug Design , Protein Kinase Inhibitors , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Cyclin-Dependent Kinase 9/metabolism , Humans , Ligands , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Cell Proliferation/drug effects , Structure-Activity Relationship , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Molecular Structure , Apoptosis/drug effects , Drug Screening Assays, Antitumor , Drug Discovery , Animals , HCT116 CellsABSTRACT
Treatment-naïve small cell lung cancer (SCLC) is typically susceptible to standard-of-care chemotherapy consisting of cisplatin and etoposide recently combined with PD-L1 inhibitors. Yet, in most cases, SCLC patients develop resistance to first-line therapy and alternative therapies are urgently required to overcome this resistance. In this study, we tested the efficacy of dinaciclib, an FDA-orphan drug and inhibitor of the cyclin-dependent kinase (CDK) 9, among other CDKs, in SCLC. Furthermore, we report on a newly developed, highly specific CDK9 inhibitor, VC-1, with tumour-killing activity in SCLC. CDK9 inhibition displayed high killing potential in a panel of mouse and human SCLC cell lines. Mechanistically, CDK9 inhibition led to a reduction in MCL-1 and cFLIP anti-apoptotic proteins and killed cells, almost exclusively, by intrinsic apoptosis. While CDK9 inhibition did not synergise with chemotherapy, it displayed high efficacy in chemotherapy-resistant cells. In vivo, CDK9 inhibition effectively reduced tumour growth and improved survival in both autochthonous and syngeneic SCLC models. Together, this study shows that CDK9 inhibition is a promising therapeutic agent against SCLC and could be applied to chemo-refractory or resistant SCLC.
Subject(s)
Cyclin-Dependent Kinase 9 , Indolizines , Lung Neoplasms , Pyridinium Compounds , Small Cell Lung Carcinoma , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Cyclin-Dependent Kinase 9/metabolism , Small Cell Lung Carcinoma/drug therapy , Small Cell Lung Carcinoma/pathology , Humans , Animals , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Cell Line, Tumor , Mice , Pyridinium Compounds/pharmacology , Pyridinium Compounds/therapeutic use , Indolizines/pharmacology , Cyclic N-Oxides/pharmacology , Apoptosis/drug effects , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic useABSTRACT
The development of distinct dendritic cell (DC) subsets, namely, plasmacytoid DCs (pDCs) and conventional DC subsets (cDC1s and cDC2s), is controlled by specific transcription factors. IRF8 is essential for the fate specification of cDC1s. However, how the expression of Irf8 is regulated is not fully understood. In this study, we identified TRIM33 as a critical regulator of DC differentiation and maintenance. TRIM33 deletion in Trim33fl/fl Cre-ERT2 mice significantly impaired DC differentiation from hematopoietic progenitors at different developmental stages. TRIM33 deficiency downregulated the expression of multiple genes associated with DC differentiation in these progenitors. TRIM33 promoted the transcription of Irf8 to facilitate the differentiation of cDC1s by maintaining adequate CDK9 and Ser2 phosphorylated RNA polymerase II (S2 Pol II) levels at Irf8 gene sites. Moreover, TRIM33 prevented the apoptosis of DCs and progenitors by directly suppressing the PU.1-mediated transcription of Bcl2l11, thereby maintaining DC homeostasis. Taken together, our findings identified TRIM33 as a novel and crucial regulator of DC differentiation and maintenance through the modulation of Irf8 and Bcl2l11 expression. The finding that TRIM33 functions as a critical regulator of both DC differentiation and survival provides potential benefits for devising DC-based immune interventions and therapies.
Subject(s)
Bcl-2-Like Protein 11 , Cell Differentiation , Dendritic Cells , Homeostasis , Interferon Regulatory Factors , Mice, Inbred C57BL , Transcription Factors , Animals , Interferon Regulatory Factors/metabolism , Interferon Regulatory Factors/genetics , Dendritic Cells/metabolism , Dendritic Cells/cytology , Mice , Transcription Factors/metabolism , Transcription Factors/genetics , Bcl-2-Like Protein 11/metabolism , Bcl-2-Like Protein 11/genetics , Transcription, Genetic , Apoptosis , RNA Polymerase II/metabolism , Cyclin-Dependent Kinase 9/metabolism , Trans-Activators/metabolism , Trans-Activators/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins/genetics , Mice, Knockout , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/cytologyABSTRACT
Reactivating p53 activity to restore its anticancer function is an attractive cancer treatment strategy. In this study, we designed and synthesized a series of novel PROTACs to reactivate p53 via the co-degradation of CK1α and CDK7/9 proteins. Bioactivity studies showed that the selected PROTAC 13i exhibited potency antiproliferative activity in MV4-11 (IC50 = 0.096 ± 0.012 µM) and MOLM-13 (IC50 = 0.072 ± 0.014 µM) cells, and induced apoptosis of MV4-11 cells. Western-blot analysis showed that PROTAC 13i triple CK1α and CDK7/9 protein degradation resulted in the significantly increased expression of p53. At the same time, the transcriptional repression due to the degradation significantly reduced downstream gene expression of MYC, MDM2, BCL-2 and MCL-1, and reduced the inflammatory cytokine levels of TNF-α, IL-1ß and IL-6 in PMBCs. These results indicate the beneficial impact of simultaneous CK1α and CDK7/9 degradation for acute myeloid leukemia therapy.
Subject(s)
Antineoplastic Agents , Casein Kinase Ialpha , Cell Proliferation , Cyclin-Dependent Kinase 9 , Cyclin-Dependent Kinases , Drug Screening Assays, Antitumor , Leukemia, Myeloid, Acute , Tumor Suppressor Protein p53 , Humans , Tumor Suppressor Protein p53/metabolism , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Casein Kinase Ialpha/metabolism , Casein Kinase Ialpha/antagonists & inhibitors , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Cyclin-Dependent Kinase 9/metabolism , Structure-Activity Relationship , Molecular Structure , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Dose-Response Relationship, Drug , Apoptosis/drug effects , Drug Discovery , Cell Line, Tumor , Proteolysis/drug effects , Tumor Cells, Cultured , Proteolysis Targeting Chimera , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
Cancer cells frequently exhibit hyperactivation of transcription, which can lead to increased sensitivity to compounds targeting the transcriptional kinases, in particular CDK9. However, mechanistic details of CDK9 inhibition-induced cancer cell-selective anti-proliferative effects remain largely unknown. Here, we discover that CDK9 inhibition activates the innate immune response through viral mimicry in cancer cells. In MYC over-expressing prostate cancer cells, CDK9 inhibition leads to the gross accumulation of mis-spliced RNA. Double-stranded RNA (dsRNA)-activated kinase can recognize these mis-spliced RNAs, and we show that the activity of this kinase is required for the CDK9 inhibitor-induced anti-proliferative effects. Using time-resolved transcriptional profiling (SLAM-seq), targeted proteomics, and ChIP-seq, we show that, similar to viral infection, CDK9 inhibition significantly suppresses transcription of most genes but allows selective transcription and translation of cytokines related to the innate immune response. In particular, CDK9 inhibition activates NFκB-driven cytokine signaling at the transcriptional and secretome levels. The transcriptional signature induced by CDK9 inhibition identifies prostate cancers with a high level of genome instability. We propose that it is possible to induce similar effects in patients using CDK9 inhibition, which, we show, causes DNA damage in vitro. In the future, it is important to establish whether CDK9 inhibitors can potentiate the effects of immunotherapy against late-stage prostate cancer, a currently lethal disease.
Subject(s)
Cyclin-Dependent Kinase 9 , Immunity, Innate , Humans , Male , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 9/metabolism , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Prostatic Neoplasms/immunology , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Prostatic Neoplasms/metabolismABSTRACT
The scaffolds of two known CDK inhibitors (CAN508 and dinaciclib) were the starting point for synthesizing two series of pyarazolo[1,5-a]pyrimidines to obtain potent inhibitors with proper selectivity. The study presented four promising compounds; 10d, 10e, 16a, and 16c based on cytotoxic studies. Compound 16a revealed superior activity in the preliminary anticancer screening with GI % = 79.02-99.13 against 15 cancer cell lines at 10 µM from NCI full panel 60 cancer cell lines and was then selected for further investigation. Furthermore, the four compounds revealed good safety profile toward the normal cell lines WI-38. These four compounds were subjected to CDK inhibitory activity against four different isoforms. All of them showed potent inhibition against CDK5/P25 and CDK9/CYCLINT. Compound 10d revealed the best activity against CDK5/P25 (IC50 = 0.063 µM) with proper selectivity index against CDK1 and CDK2. Compound 16c exhibited the highest inhibitory activity against CDK9/CYCLINT (IC50 = 0.074 µM) with good selectivity index against other isoforms. Finally, docking simulations were performed for compounds 10e and 16c accompanied by molecular dynamic simulations to understand their behavior in the active site of the two CDKs with respect to both CAN508 and dinaciclib.
Subject(s)
Antineoplastic Agents , Bridged Bicyclo Compounds, Heterocyclic , Cyclic N-Oxides , Drug Design , Indolizines , Molecular Docking Simulation , Protein Kinase Inhibitors , Pyridinium Compounds , Humans , Pyridinium Compounds/pharmacology , Pyridinium Compounds/chemistry , Indolizines/pharmacology , Indolizines/chemistry , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Cyclic N-Oxides/pharmacology , Cyclic N-Oxides/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Cell Line, Tumor , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cyclin-Dependent Kinases/antagonists & inhibitors , Structure-Activity Relationship , Pyrimidines/pharmacology , Pyrimidines/chemistry , Drug Screening Assays, Antitumor , Cyclin-Dependent Kinase 5/antagonists & inhibitors , Cyclin-Dependent Kinase 5/metabolism , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Cyclin-Dependent Kinase 9/metabolismABSTRACT
The release of paused RNA polymerase II (RNAPII) from promoter-proximal regions is tightly controlled to ensure proper regulation of gene expression. The elongation factor PTEF-b is known to release paused RNAPII via phosphorylation of the RNAPII C-terminal domain by its cyclin-dependent kinase component, CDK9. However, the signal and stress-specific roles of the various RNAPII-associated macromolecular complexes containing PTEF-b/CDK9 are not yet clear. Here, we identify and characterize the CDK9 complex required for transcriptional response to hypoxia. Contrary to previous reports, our data indicate that a CDK9 complex containing BRD4 but not AFF1/4 is essential for this hypoxic stress response. We demonstrate that BRD4 bromodomains (BET) are dispensable for the release of paused RNAPII at hypoxia-activated genes and that BET inhibition by JQ1 is insufficient to impair hypoxic gene response. Mechanistically, we demonstrate that the C-terminal region of BRD4 is required for Polymerase-Associated Factor-1 Complex (PAF1C) recruitment to establish an elongation-competent RNAPII complex at hypoxia-responsive genes. PAF1C disruption using a small-molecule inhibitor (iPAF1C) impairs hypoxia-induced, BRD4-mediated RNAPII release. Together, our results provide insight into potentially targetable mechanisms that control the hypoxia-responsive transcriptional elongation.
Subject(s)
Nuclear Proteins , Transcription Factors , Humans , Transcription Factors/genetics , Transcription Factors/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Gene Expression Regulation , Cyclin-Dependent Kinases/metabolism , Cyclin-Dependent Kinase 9/genetics , Cyclin-Dependent Kinase 9/metabolism , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Phosphorylation , Hypoxia , Transcription, Genetic , Positive Transcriptional Elongation Factor B/genetics , Positive Transcriptional Elongation Factor B/metabolism , Bromodomain Containing Proteins , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolismABSTRACT
CDK9 is a cyclin-dependent kinase that plays pivotal roles in multiple cellular functions including gene transcription, cell cycle regulation, DNA damage repair, and cellular differentiation. Targeting CDK9 is considered an attractive strategy for antitumor therapy, especially for leukemia and lymphoma. Several potent small molecule inhibitors, exemplified by TG02 (4), have progressed to clinical trials. However, many of them face challenges such as low clinical efficacy and multiple adverse reactions and may necessitate the exploration of novel strategies to lead to success in the clinic. In this perspective, we present a comprehensive overview of the structural characteristics, biological functions, and preclinical status of CDK9 inhibitors. Our focus extends to various types of inhibitors, including pan-inhibitors, selective inhibitors, dual-target inhibitors, degraders, PPI inhibitors, and natural products. The discussion encompasses chemical structures, structure-activity relationships (SARs), biological activities, selectivity, and therapeutic potential, providing detailed insight into the diverse landscape of CDK9 inhibitors.
Subject(s)
Cyclin-Dependent Kinase 9 , Cyclin-Dependent Kinases , Cell Cycle Checkpoints , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein Kinase Inhibitors/chemistryABSTRACT
Cartilage defects in the knee are often associated with the progression of degenerative osteoarthritis (OA), and cartilage repair is a useful strategy for managing this disease. However, cartilage repair is challenging because of the unique environment within the tissue. Recently, stem cell-based therapies have shed new light on this issue. In this study, we prepared exosomes (EXOs) from cartilage stem/progenitor cells (CSPCs) and found that treatment with EXOs increased the viability, migration, and proliferation of cultured primary chondrocytes. In a subacute OA rat model, the application of EXOs facilitated cartilage regeneration as evidenced by histological staining. Exosomal protein analysis together with bioinformatics suggested that cyclin-dependent kinase 9 (CDK9) is a key factor for chondrocyte growth and migration. Functional studies confirmed this prediction, that is, inhibiting CDK9 reduced the beneficial effects induced by EXOs in primary chondrocytes; while overexpression of CDK9 recapitulated the EXOs-induced phenotypes. RNA-Seq data showed that a set of genes involved in cell growth and migration were up-regulated by EXOs in chondrocytes. These changes could be partially reproduced by CDK9 overexpression. Overall, our data suggest that EXOs derived from primary CSPCs hold great therapeutic potential for treating cartilage defect-associated disorders such as degenerative OA, and that CDK9 is a key factor in this process.
Subject(s)
Cartilage, Articular , Cell Proliferation , Chondrocytes , Disease Models, Animal , Exosomes , Animals , Exosomes/metabolism , Rats , Chondrocytes/metabolism , Cartilage, Articular/metabolism , Cartilage, Articular/pathology , Stem Cells/metabolism , Stem Cells/cytology , Cell Movement , Rats, Sprague-Dawley , Cyclin-Dependent Kinase 9/metabolism , Cyclin-Dependent Kinase 9/genetics , Osteoarthritis, Knee/metabolism , Osteoarthritis, Knee/pathology , Osteoarthritis, Knee/therapy , Male , Cells, Cultured , Regeneration , Osteoarthritis/pathology , Osteoarthritis/metabolism , Osteoarthritis/therapyABSTRACT
Proteolysis targeting chimeras (PROTACs) have emerged as revolutionary anticancer therapeutics that degrade disease-causing proteins. However, the anticancer performance of PROTACs is often impaired by their insufficient bioavailability, unsatisfactory tumor specificity and ability to induce acquired drug resistance. Herein, we propose a polymer-conjugated PROTAC prodrug platform for the tumor-targeted delivery of the most prevalent von Hippel-Lindau (VHL)- and cereblon (CRBN)-based PROTACs, as well as for the precise codelivery of a degrader and conventional small-molecule drugs. The self-assembling PROTAC prodrug nanoparticles (NPs) can specifically target and be activated inside tumor cells to release the free PROTAC for precise protein degradation. The PROTAC prodrug NPs caused more efficient regression of MDA-MB-231 breast tumors in a mouse model by degrading bromodomain-containing protein 4 (BRD4) or cyclin-dependent kinase 9 (CDK9) with decreased systemic toxicity. In addition, we demonstrated that the PROTAC prodrug NPs can serve as a versatile platform for the codelivery of a PROTAC and chemotherapeutics for enhanced anticancer efficiency and combination benefits. This study paves the way for utilizing tumor-targeted protein degradation for precise anticancer therapy and the effective combination treatment of complex diseases.
Subject(s)
Nanoparticles , Prodrugs , Proteolysis , Von Hippel-Lindau Tumor Suppressor Protein , Prodrugs/chemistry , Prodrugs/pharmacology , Prodrugs/therapeutic use , Humans , Animals , Proteolysis/drug effects , Nanoparticles/chemistry , Cell Line, Tumor , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Female , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Cell Cycle Proteins/metabolism , Mice , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Cyclin-Dependent Kinase 9/metabolism , Transcription Factors/metabolism , Mice, Nude , Adaptor Proteins, Signal Transducing/metabolism , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Mice, Inbred BALB C , Drug Delivery Systems , Bromodomain Containing Proteins , Ubiquitin-Protein LigasesABSTRACT
Long non-coding RNAs (lncRNAs) play an essential role in vertebrate myogenesis and muscle diseases. However, the dynamic expression patterns, biological functions, and mechanisms of lncRNAs in skeletal muscle development and regeneration remain largely unknown. In this study, a novel lncRNA (named lncMGR) was differentially expressed during breast muscle development in fast- and slow-growing chickens. Functionally, lncMGR promoted myoblast differentiation, inhibited myoblast proliferation in vitro, and promoted myofiber hypertrophy and injury repair in vivo. Mechanistically, lncMGR increased the mRNA and protein expression of skeletal muscle myosin heavy chain 1â¯A (MYH1A) via both transcriptional and post-transcriptional regulation. Nuclear lncMGR recruited cyclin-dependent kinase 9 (CDK9) to the core transcriptional activation region of the MYH1A gene to activate MYH1A transcription. Cytoplasmic lncMGR served as a competitive endogenous RNA (ceRNA) to competitively absorb miR-2131-5p away from MYH1A and subsequently protected the MYH1A from miR-2131-5p-mediated degradation. Besides miR-2131-5p, cytoplasmic lncMGR could also sponge miR-143-3p to reconcile the antagonist between the miR-2131-5p/MYH1A-mediated inhibition effects and miR-143-3p-mediated promotion effects on myoblast proliferation, thereby inhibiting myoblast proliferation. Collectively, lncMGR could recruit CDK9 and sponge multiple miRNAs to regulate skeletal muscle development and regeneration, and could be a therapeutic target for muscle diseases.