Selective Kinase Inhibition Shows That Bur1 (Cdk9) Phosphorylates the Rpb1 Linker In Vivo.
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ABSTRACT:
SUBMITTER: Chun Y
PROVIDER: S-EPMC6639251 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Aberrant transcription in cancer cells involves the silencing of tumor suppressor genes (TSGs) and activation of oncogenes. Transcriptomic changes are associated with epigenomic alterations such as DNA-hypermethylation, histone deacetylation, and chromatin condensation in promoter regions of silenced TSGs. To discover novel drugs that trigger TSG reactivation in cancer cells, we used a GFP-reporter system whose expression is silenced by promoter DNA hypermethylation and histone deacetylation. After screening a natural product drug library, we identified that toyocamycin, an adenosine-analog, induces potent GFP reactivation and loss of clonogenicity in human colon cancer cells. Connectivity-mapping analysis revealed that toyocamycin produces a pharmacological signature mimicking cyclin-dependent kinase (CDK) inhibitors. RNA-sequencing revealed that the toyocamycin transcriptomic signature resembles that of a specific CDK9 inhibitor (HH1). Specific inhibition of RNA Pol II phosphorylation level and kinase assays confirmed that toyocamycin specifically inhibits CDK9 (IC50 = 79 nM) with a greater efficacy than other CDKs (IC50 values between 0.67 and 15 µM). Molecular docking showed that toyocamycin efficiently binds the CDK9 catalytic site in a conformation that differs from other CDKs, explained by the binding contribution of specific amino acids within the catalytic pocket and protein backbone. Altogether, we demonstrated that toyocamycin exhibits specific CDK9 inhibition in cancer cells, highlighting its potential for cancer chemotherapy.
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Project description:BUR1 and BUR2 were previously identified by a genetic selection for mutations that increase transcription from basal promoters in vivo. BUR1 encoded a putative protein kinase with greatest similarity to members of the cyclin-dependent kinase (CDK) family, although that similarity was not sufficient to classify it as a CDK. It was also not known whether Bur1 activity was cyclin dependent and, if so, which cyclins stimulated Bur1. The molecular cloning and characterization of BUR2 presented here sheds light on these issues. Genetic analysis indicates that BUR2 function is intimately related to that of BUR1: bur1 and bur2 mutations cause nearly identical spectra of mutant phenotypes, and overexpression of BUR1 suppresses a bur2 null allele. Biochemical analysis has provided a molecular basis for these genetic observations. We find that BUR2 encodes a cyclin for the Bur1 protein kinase, based on the following evidence. First, the BUR2 amino acid sequence reveals similarity to the cyclins; second, Bur1 and Bur2 coimmunoprecipitate from crude extracts and interact in the two-hybrid system; and third, BUR2 is required for Bur1 kinase activity in vitro. Our combined genetic and biochemical results therefore indicate that Bur1 and Bur2 comprise a divergent CDK-cyclin complex that has an important functional role during transcription in vivo.
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Project description:The EphA2 receptor tyrosine kinase signals through two distinct mechanisms, one regulated by tyrosine phosphorylation and the other by serine/threonine phosphorylation. Serine 892 (S892) is one of the major serine/threonine phosphorylation sites in EphA2, but little is known about its regulation and function. S892 is located in the linker connecting the EphA2 kinase and SAM domains, and is part of a cluster of five phosphorylated residues that includes the well characterized S897. EphA2 can be phosphorylated on S897 by the RSK, AKT and PKA kinases to promote a non-canonical form of signaling that plays an important role in cancer malignancy. Here we show that the Protein Kinase C (PKC) family phosphorylates the EphA2 S892 motif in vitro and in cells. By using a newly developed phosphospecific antibody, we detected EphA2 S892 phosphorylation in a variety of cell lines. As expected for a PKC target site, the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) increases S892 phosphorylation whereas the broad-spectrum PKC inhibitor Go 6983 inhibits both basal and TPA-induced S892 phosphorylation. Besides phosphorylating S892, PKC can also increase EphA2 phosphorylation on S897 through the MEK kinase, which regulates the ERK-RSK signaling axis. We also found that S892 and S897 phosphorylation induced by PKC activation can be downregulated by ephrin ligand-induced EphA2 canonical signaling. Our data reveal that the PKC family contributes to the phosphorylation cluster in the EphA2 kinase-SAM linker, which regulates EphA2 non-canonical signaling and cancer malignancy.
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Project description:Sustained neutrophilic inflammation is detrimental for cardiac repair and associated with adverse outcomes following myocardial infarction (MI). An attractive therapeutic strategy to treat MI is to reduce or remove infiltrating neutrophils to promote downstream reparative mechanisms. CDK9 inhibitor compounds enhance the resolution of neutrophilic inflammation; however, their effects on cardiac repair/regeneration are unknown. We have devised a cardiac injury model to investigate inflammatory and regenerative responses in larval zebrafish using heartbeat-synchronised light-sheet fluorescence microscopy. We used this model to test two clinically approved CDK9 inhibitors, AT7519 and flavopiridol, examining their effects on neutrophils, macrophages and cardiomyocyte regeneration. We found that AT7519 and flavopiridol resolve neutrophil infiltration by inducing reverse migration from the cardiac lesion. Although continuous exposure to AT7519 or flavopiridol caused adverse phenotypes, transient treatment accelerated neutrophil resolution while avoiding these effects. Transient treatment with AT7519, but not flavopiridol, augmented wound-associated macrophage polarisation, which enhanced macrophage-dependent cardiomyocyte number expansion and the rate of myocardial wound closure. Using cdk9-/- knockout mutants, we showed that AT7519 is a selective CDK9 inhibitor, revealing the potential of such treatments to promote cardiac repair/regeneration.
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Project description:Targeted treatment for triple-negative breast cancer (TNBC) remains an elusive clinical challenge. Cyclin-dependent kinase 9 (CDK9) is a transcriptional regulator shown to promote growth of multiple cancers, including TNBC, and represents a potential new therapeutic target. CDK9 increases RNA Polymerase II (Pol II) activity, which facilitates sustained expression of short-lived oncogenic and anti-apoptotic proteins. However, pre-clinical evaluation of CDK9 as a therapeutic target has been hampered by the poor selectivity of existing CDK9 inhibitors (CDK9i). Here, we report a novel CDK9i; D11-8, which exhibits high potency against CDK9 (Ki = 8 nM) and displays remarkable selectivity over other CDKs and human kinases. D11-8 inhibited TNBC cell line proliferation,, induced G2/M cell cycle arrest, and increased apoptosis. Mechanistically, D11-8 inhibited CDK9; reducing Pol II phosphorylation, down-regulating expression of proto-oncogene MYC and anti-apoptotic marker MCL1, and dramatically inducing Pol II promoter-proximal pausing at MYC, G2/M checkpoint, and E2F2 target genes. D11-8 was further validated for TNBC in vivo, and inhibited TNBC cell line tumour growth without toxicity. To further elucidate the cancer cell specificity of D11-8, normal breast tissue was collected from women undergoing reduction mammoplasty surgery and treated with D11-8 ex vivo as patient-derived explants. High doses of D11-8 (2.7 µM) had no effect on the proliferative capacity of normal breast epithelial cells (Ki67 positivity), and tissues appeared histologically normal. Collectively, these data demonstrate that D11-8 effectively inhibits CDK9 in TNBC cell lines, resulting in growth inhibition without short-term toxicity.
2023-11-25 | GSE184335 | GEO
Project description:Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in many cancer cells without causing toxicity in vivo. However, to date, TRAIL-receptor agonists have only shown limited therapeutic benefit in clinical trials. This can, most likely, be attributed to the fact that 50% of all cancer cell lines and most primary human cancers are TRAIL resistant. Consequently, future TRAIL-based therapies will require the addition of sensitizing agents that remove crucial blocks in the TRAIL apoptosis pathway. Here, we identify PIK-75, a small molecule inhibitor of the p110α isoform of phosphoinositide-3 kinase (PI3K), as an exceptionally potent TRAIL apoptosis sensitizer. Surprisingly, PI3K inhibition was not responsible for this activity. A kinome-wide in vitro screen revealed that PIK-75 strongly inhibits a panel of 27 kinases in addition to p110α. Within this panel, we identified cyclin-dependent kinase 9 (CDK9) as responsible for TRAIL resistance of cancer cells. Combination of CDK9 inhibition with TRAIL effectively induced apoptosis even in highly TRAIL-resistant cancer cells. Mechanistically, CDK9 inhibition resulted in downregulation of cellular FLICE-like inhibitory protein (cFlip) and Mcl-1 at both the mRNA and protein levels. Concomitant cFlip and Mcl-1 downregulation was required and sufficient for TRAIL sensitization by CDK9 inhibition. When evaluating cancer selectivity of TRAIL combined with SNS-032, the most selective and clinically used inhibitor of CDK9, we found that a panel of mostly TRAIL-resistant non-small cell lung cancer cell lines was readily killed, even at low concentrations of TRAIL. Primary human hepatocytes did not succumb to the same treatment regime, defining a therapeutic window. Importantly, TRAIL in combination with SNS-032 eradicated established, orthotopic lung cancer xenografts in vivo. Based on the high potency of CDK9 inhibition as a cancer cell-selective TRAIL-sensitizing strategy, we envisage the development of new, highly effective cancer therapies.
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Project description:Aberrant transcription in cancer cells is characterized by silencing of tumor suppressor genes (TSG) and activation of oncogenes. Transcriptomic changes are associated with epigenomic alterations such as DNA hypermethylation, histone deacetylation, and chromatin condensation in promoter regions of silenced TSG. To discover novel drugs that trigger tumor suppressor genes reactivation in cancer cells, we used a GFP reporter system whose expression is silenced by promoter DNA hypermethyation and histone deacetylation. After screening a natural product drug library, we identified toyocamycin, an adenosine analog that induced potent GFP reactivation and loss of clonogenicity in human colon cancer cells. Toyocamycin induced a downregulation in gene pathways involved in RNA pol II transcription regulation. Connectivity mapping analysis revealed that toyocamycin produced a pharmacological signature mimicking cyclin-dependent kinase (CDK) inhibitors. Using RNA-sequencing, we showed that toyocamycin transcriptomic signature closely resembled the profile of a specific CDK9 inhibitor (HH1). Specific inhibition of RNA pol II phosphorylation level and enzymatic kinase assays confirmed that toyocamycin inhibits specifically CDK9 (IC50 = 79nM) with a greater efficacy than other CDKs (IC50 values between 0.67-15µM). Molecular docking showed that toyocamycin binds efficiently CDK9 catalytic site with a different pose than in other CDKs, which was explained by the binding contribution of specific amino acids within the catalytic pocket and the backbone of the protein. Altogether, we demonstrated that toyocamycin exhibits specific CDK9 inhibition in cancer cells, highlighting its potential for cancer chemotherapy.
2022-05-14 | GSE202744 | GEO
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2009-03-30 | E-MEXP-1746 | biostudies-arrayexpress
Project description:Telomere length regulation is essential for cell viability in eukaryotes. While many pathways that affect telomere length are known, we do not yet have a complete understanding of the mechanism of length regulation. To identify new pathways that might regulate telomere length, we carried out a genetic screen in yeast and identified the cyclin-dependent kinase complex Bur1/2 as a regulator of telomere length. Mutations in either BUR1 cyclin-dependent kinase or the associated BUR2 cyclin resulted in short telomeres. This regulation did not function through the known role of BUR1 in regulating histone modification as bur1∆ set2∆ and bur2∆ set2∆ double mutants rescued cell growth but did not rescue the telomere shortening effects. We found that both bur1∆ and bur2∆ set2∆ were also defective in de novo telomere addition, and deletion of SET2 did also not rescue this elongation defect. The Bur1/2 cyclin-dependent kinase regulates transcription of many genes. We found that TLC1 RNA levels were reduced in bur2∆ set2∆ mutants; however, overexpression of TLC1 restored the transcript levels but did not restore de novo telomere elongation or telomere length. These data suggest that the Bur1/2 kinase plays a role in telomere elongation separate from its role in transcription of telomerase components. Dissecting the role of the Bur1/2 kinase pathway at telomeres will help complete our understanding of the complex network of telomere length regulation.
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