RESUMEN
Casein kinase-2 (CK2) are serine/threonine kinases with dual co-factor (ATP and GTP) specificity, that are involved in the regulation of a wide variety of cellular functions. Small molecules targeting CK2 have been described in the literature targeting different binding pockets of the kinase with a focus on type I inhibitors such as the recently published chemical probe SGC-CK2-1. In this study, we investigated whether known allosteric inhibitors binding to a pocket adjacent to helix αD could be combined with ATP mimetic moieties defining a novel class of ATP competitive compounds with a unique binding mode. Linking both binding sites requires a chemical linking moiety that would introduce a 90-degree angle between the ATP mimetic ring system and the αD targeting moiety, which was realized using a sulfonamide. The synthesized inhibitors were highly selective for CK2 with binding constants in the nM range and low micromolar activity. While these inhibitors need to be further improved, the present work provides a structure-based design strategy for highly selective CK2 inhibitors.
Asunto(s)
Quinasa de la Caseína II , Inhibidores de Proteínas Quinasas , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Humanos , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo , Relación Estructura-Actividad , Estructura Molecular , Relación Dosis-Respuesta a Droga , Modelos Moleculares , Adenosina Trifosfato/metabolismo , Sitios de UniónRESUMEN
The pyrazolo[1,5-a]pyrimidine scaffold is a promising scaffold to develop potent and selective CSNK2 inhibitors with antiviral activity against ß-coronaviruses. Herein, we describe the discovery of a 1,2,4-triazole group to substitute a key amide group for CSNK2 binding present in many potent pyrazolo[1,5-a]pyrimidine inhibitors. Crystallographic evidence demonstrates that the 1,2,4-triazole replaces the amide in forming key hydrogen bonds with Lys68 and a water molecule buried in the ATP-binding pocket. This isosteric replacement improves potency and metabolic stability at a cost of solubility. Optimization for potency, solubility, and metabolic stability led to the discovery of the potent and selective CSNK2 inhibitor 53. Despite excellent in vitro metabolic stability, rapid decline in plasma concentration of 53 in vivo was observed and may be attributed to lung accumulation, although in vivo pharmacological effect was not observed. Further optimization of this novel chemotype may validate CSNK2 as an antiviral target in vivo.
Asunto(s)
Antivirales , Quinasa de la Caseína II , Pirimidinas , Triazoles , Replicación Viral , Triazoles/farmacología , Triazoles/química , Triazoles/síntesis química , Pirimidinas/farmacología , Pirimidinas/química , Pirimidinas/síntesis química , Antivirales/farmacología , Antivirales/química , Antivirales/síntesis química , Animales , Humanos , Replicación Viral/efectos de los fármacos , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo , Pirazoles/farmacología , Pirazoles/química , Pirazoles/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/síntesis química , Amidas/química , Amidas/farmacología , Amidas/síntesis química , Relación Estructura-Actividad , Ratones , Ratas , SARS-CoV-2/efectos de los fármacos , Descubrimiento de Drogas , MasculinoRESUMEN
Sodium and potassium channels, especially Nav1.5 and Kir2.1, play key roles in the formation of action potentials in cardiomyocytes. These channels interact with, and are regulated by, synapse-associated protein 97 (SAP97). However, the regulatory role of SAP97 in myocyte remains incompletely understood. Here, we investigate the function of SAP97 phosphorylation in the regulation of Nav1.5 and Kir2.1 channel complexes and the upstream regulation of SAP97. We found that SAP97 is phosphorylated by casein kinase II (CK2) in vitro. In addition, transfection of casein kinase 2 interacting protein-1 (CKIP-1) into cardiomyocytes to drive CK2 from the nucleus to the cytoplasm, increased SAP97 phosphorylation and Nav1.5 and Kir2.1 current activity. These findings demonstrated that CKIP-1 modulates the subcellular translocation of CK2, which regulates Nav1.5 and Kir2.1 channel complex formation and activity in cardiomyocytes.
Asunto(s)
Quinasa de la Caseína II , Miocitos Cardíacos , Canal de Sodio Activado por Voltaje NAV1.5 , Canales de Potasio de Rectificación Interna , Miocitos Cardíacos/metabolismo , Quinasa de la Caseína II/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio de Rectificación Interna/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/genética , Animales , Ratas , Fosforilación , Transporte de Proteínas , Humanos , Proteínas Portadoras/metabolismo , Ratas Sprague-DawleyRESUMEN
Most biological processes are regulated by signaling modules that bind to short linear motifs. For protein kinases, substrates may have full or only partial matches to the kinase recognition motif, a property known as "substrate quality." However, it is not clear whether differences in substrate quality represent neutral variation or if they have functional consequences. We examine this question for the kinase CK2, which has many fundamental functions. We show that optimal CK2 sites are phosphorylated at maximal stoichiometries and found in many conditions, whereas minimal substrates are more weakly phosphorylated and have regulatory functions. Optimal CK2 sites tend to be more conserved, and substrate quality is often tuned by selection. For intermediate sites, increases or decreases in substrate quality may be deleterious, as we demonstrate for a CK2 substrate at the kinetochore. The results together suggest a strong role for substrate quality in phosphosite function and evolution. A record of this paper's transparent peer review process is included in the supplemental information.
Asunto(s)
Quinasa de la Caseína II , Quinasa de la Caseína II/metabolismo , Fosforilación , Humanos , Especificidad por Sustrato , Cinetocoros/metabolismo , Evolución Molecular , Sitios de UniónRESUMEN
Transcription is extremely important for cellular processes but can be hindered by RNA polymerase II (RNAPII) pausing and stalling. Cockayne syndrome protein B (CSB) promotes the progression of paused RNAPII or initiates transcription-coupled nucleotide excision repair (TC-NER) to remove stalled RNAPII. However, the specific mechanism by which CSB initiates TC-NER upon damage remains unclear. In this study, we identified the indispensable role of the ARK2N-CK2 complex in the CSB-mediated initiation of TC-NER. The ARK2N-CK2 complex is recruited to damage sites through CSB and then phosphorylates CSB. Phosphorylation of CSB enhances its binding to stalled RNAPII, prolonging the association of CSB with chromatin and promoting CSA-mediated ubiquitination of stalled RNAPII. Consistent with this finding, Ark2n-/- mice exhibit a phenotype resembling Cockayne syndrome. These findings shed light on the pivotal role of the ARK2N-CK2 complex in governing the fate of RNAPII through CSB, bridging a critical gap necessary for initiating TC-NER.
Asunto(s)
Síndrome de Cockayne , ADN Helicasas , Enzimas Reparadoras del ADN , Reparación del ADN , Proteínas de Unión a Poli-ADP-Ribosa , ARN Polimerasa II , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Humanos , Animales , Ratones , ADN Helicasas/metabolismo , ADN Helicasas/genética , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Transcripción Genética , Fosforilación , Quinasa de la Caseína II/metabolismo , Quinasa de la Caseína II/genética , Ratones Noqueados , Daño del ADN , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , Cromatina/metabolismo , Ubiquitinación , Reparación por EscisiónRESUMEN
The RNA cap methyltransferase CMTR1 methylates the first transcribed nucleotide of RNA polymerase II transcripts, impacting gene expression mechanisms, including during innate immune responses. Using mass spectrometry, we identify a multiply phosphorylated region of CMTR1 (phospho-patch [P-Patch]), which is a substrate for the kinase CK2 (casein kinase II). CMTR1 phosphorylation alters intramolecular interactions, increases recruitment to RNA polymerase II, and promotes RNA cap methylation. P-Patch phosphorylation occurs during the G1 phase of the cell cycle, recruiting CMTR1 to RNA polymerase II during a period of rapid transcription and RNA cap formation. CMTR1 phosphorylation is required for the expression of specific RNAs, including ribosomal protein gene transcripts, and promotes cell proliferation. CMTR1 phosphorylation is also required for interferon-stimulated gene expression. The cap-snatching virus, influenza A, utilizes host CMTR1 phosphorylation to produce the caps required for virus production and infection. We present an RNA cap methylation control mechanism whereby CK2 controls CMTR1, enhancing co-transcriptional capping.
Asunto(s)
Quinasa de la Caseína II , Metiltransferasas , Caperuzas de ARN , Animales , Humanos , Quinasa de la Caseína II/metabolismo , Quinasa de la Caseína II/genética , Células HEK293 , Virus de la Influenza A , Gripe Humana/virología , Gripe Humana/metabolismo , Gripe Humana/genética , Metilación , Metiltransferasas/metabolismo , Fosforilación , Caperuzas de ARN/metabolismo , ARN Polimerasa II/metabolismoRESUMEN
Ischemic stroke (IS) is a serious threat to human health. The naturally derived small molecule (E)-5-(2-(quinolin-4-yl) ethenyl) benzene-1,3-diol (RV01) is a quinolinyl analog of resveratrol with great potential in the treatment of IS. The aim of this study was to investigate the potential mechanisms and targets for the protective effect of the RV01 on IS. The mouse middle cerebral artery occlusion and reperfusion (MCAO/R) and oxygen-glucose deprivation and reperfusion (OGD/R) models were employed to evaluate the effects of RV01 on ischemic injury and neuroprotection. RV01 was found to significantly increase the survival of SH-SY5Y cells and prevent OGD/R-induced apoptosis in SH-SY5Y cells. Furthermore, RV01 reduced oxidative stress and mitochondrial damage by promoting mitophagy in OGD/R-exposed SH-SY5Y cells. Knockdown of CK2α' abolished the RV01-mediated promotion on mitophagy and alleviation on mitochondrial damage as well as neuronal injury after OGD/R. These results were further confirmed by molecular docking, drug affinity responsive target stability and cellular thermal shift assay analysis. Importantly, in vivo study showed that treatment with the CK2α' inhibitor CX-4945 abolished the RV01-mediated alleviation of cerebral infarct volume, brain edema, cerebral blood flow and neurological deficit in MCAO/R mice. These data suggest that RV01 effectively reduces damage caused by acute ischemic stroke by promoting mitophagy through its interaction with CK2α'. These findings offer valuable insights into the underlying mechanisms through which RV01 exerts its therapeutic effects on IS.
Asunto(s)
Quinasa de la Caseína II , Infarto de la Arteria Cerebral Media , Accidente Cerebrovascular Isquémico , Ratones Endogámicos C57BL , Mitofagia , Fármacos Neuroprotectores , Resveratrol , Animales , Mitofagia/efectos de los fármacos , Humanos , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Quinasa de la Caseína II/metabolismo , Quinasa de la Caseína II/antagonistas & inhibidores , Masculino , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Accidente Cerebrovascular Isquémico/metabolismo , Resveratrol/farmacología , Resveratrol/uso terapéutico , Ratones , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/patología , Línea Celular Tumoral , Apoptosis/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Modelos Animales de Enfermedad , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Simulación del Acoplamiento Molecular , Quinolinas/farmacología , Quinolinas/uso terapéutico , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Naftiridinas , FenazinasRESUMEN
Calcineurin inhibitors, such as cyclosporine and tacrolimus (FK506), are commonly used immunosuppressants for preserving transplanted organs and tissues. However, these drugs can cause severe and persistent pain. GluA2-lacking, calcium-permeable AMPA receptors (CP-AMPARs) are implicated in various neurological disorders, including neuropathic pain. It is unclear whether and how constitutive calcineurin, a Ca2+/calmodulin protein phosphatase, controls synaptic CP-AMPARs. In this study, we found that blocking CP-AMPARs with IEM-1460 markedly reduced the amplitude of AMPAR-EPSCs in excitatory neurons expressing vesicular glutamate transporter-2 (VGluT2), but not in inhibitory neurons expressing vesicular GABA transporter, in the spinal cord of FK506-treated male and female mice. FK506 treatment also caused an inward rectification in the current-voltage relationship of AMPAR-EPSCs specifically in VGluT2 neurons. Intrathecal injection of IEM-1460 rapidly alleviated pain hypersensitivity in FK506-treated mice. Furthermore, FK506 treatment substantially increased physical interaction of α2δ-1 with GluA1 and GluA2 in the spinal cord and reduced GluA1/GluA2 heteromers in endoplasmic reticulum-enriched fractions of spinal cords. Correspondingly, inhibiting α2δ-1 with pregabalin, Cacna2d1 genetic knock-out, or disrupting α2δ-1-AMPAR interactions with an α2δ-1 C terminus peptide reversed inward rectification of AMPAR-EPSCs in spinal VGluT2 neurons caused by FK506 treatment. In addition, CK2 inhibition reversed FK506 treatment-induced pain hypersensitivity, α2δ-1 interactions with GluA1 and GluA2, and inward rectification of AMPAR-EPSCs in spinal VGluT2 neurons. Thus, the increased prevalence of synaptic CP-AMPARs in spinal excitatory neurons plays a major role in calcineurin inhibitor-induced pain hypersensitivity. Calcineurin and CK2 antagonistically regulate postsynaptic CP-AMPARs through α2δ-1-mediated GluA1/GluA2 heteromeric assembly in the spinal dorsal horn.
Asunto(s)
Calcineurina , Quinasa de la Caseína II , Receptores AMPA , Médula Espinal , Tacrolimus , Animales , Receptores AMPA/metabolismo , Ratones , Calcineurina/metabolismo , Masculino , Femenino , Tacrolimus/farmacología , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Quinasa de la Caseína II/metabolismo , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Ratones Endogámicos C57BL , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Sinapsis/fisiología , Inhibidores de la Calcineurina/farmacología , Fenotipo , Canales de CalcioRESUMEN
Protein kinase CK2 is a holoenzyme composed of two regulatory subunits (CK2ß) and two catalytic subunits (CK2α and CK2α'). CK2 controls several cellular processes, including proliferation, inflammation, and cell death. However, CK2α and CK2α' possess different expression patterns and substrates and therefore impact each of these processes differently. Elevated CK2α participates in the development of cancer, while increased CK2α' has been associated with neurodegeneration, especially Huntington's disease (HD). HD is a fatal disease for which no effective therapies are available. Genetic deletion of CK2α' in HD mouse models has ameliorated neurodegeneration. Therefore, pharmacological inhibition of CK2α' presents a promising therapeutic strategy for treating HD. However, current CK2 inhibitors are unable to discriminate between CK2α and CK2α' due to their high structural homology, especially in the targeted ATP-binding site. Using computational analyses, we found a potential type IV ("D" pocket) allosteric site that contained different residues between CK2α and CK2α' and was distal from the ATP-binding pocket featured in both kinases. We decided to look for allosteric modulators that might interact in a biased fashion with the type IV pocket on both CK2α and CK2α'. We screened a commercial library containing â¼29,000 allosteric-kinase-inhibitor-like compounds using a CK2α' activity-dependent ADP-Glo Kinase assay. Obtained hits were counter-screened against CK2α using the ADP-Glo Kinase assay, revealing two CK2α'-biased compounds. These two compounds might serve as the basis for further medicinal chemistry optimization for the potential treatment of HD.
Asunto(s)
Adenosina Trifosfato , Quinasa de la Caseína II , Ensayos Analíticos de Alto Rendimiento , Inhibidores de Proteínas Quinasas , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo , Adenosina Trifosfato/metabolismo , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Animales , Descubrimiento de Drogas/métodos , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Introduction: In classical Hodgkin lymphoma (cHL), the survival of neoplastic cells is mediated by the activation of NF-κB, JAK/STAT and PI3K/Akt signaling pathways. CK2 is a highly conserved serine/threonine kinase, consisting of two catalytic (α) and two regulatory (ß) subunits, which is involved in several cellular processes and both subunits were found overexpressed in solid tumors and hematologic malignancies. Methods and results: Biochemical analyses and in vitro assays showed an impaired expression of CK2 subunits in cHL, with CK2α being overexpressed and a decreased expression of CK2ß compared to normal B lymphocytes. Mechanistically, CK2ß was found to be ubiquitinated in all HL cell lines and consequently degraded by the proteasome pathway. Furthermore, at basal condition STAT3, NF-kB and AKT are phosphorylated in CK2-related targets, resulting in constitutive pathways activation. The inhibition of CK2 with CX-4945/silmitasertib triggered the de-phosphorylation of NF-κB-S529, STAT3-S727, AKT-S129 and -S473, leading to cHL cell lines apoptosis. Moreover, CX-4945/silmitasertib was able to decrease the expression of the immuno-checkpoint CD274/PD-L1 but not of CD30, and to synergize with monomethyl auristatin E (MMAE), the microtubule inhibitor of brentuximab vedotin. Conclusions: Our data point out a pivotal role of CK2 in the survival and the activation of key signaling pathways in cHL. The skewed expression between CK2α and CK2ß has never been reported in other lymphomas and might be specific for cHL. The effects of CK2 inhibition on PD-L1 expression and the synergistic combination of CX-4945/silmitasertib with MMAE pinpoints CK2 as a high-impact target for the development of new therapies for cHL.
Asunto(s)
Antígeno B7-H1 , Quinasa de la Caseína II , Enfermedad de Hodgkin , Transducción de Señal , Humanos , Enfermedad de Hodgkin/metabolismo , Enfermedad de Hodgkin/tratamiento farmacológico , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/patología , Quinasa de la Caseína II/metabolismo , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/genética , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Línea Celular Tumoral , Fenazinas , Naftiridinas/farmacología , Apoptosis , Regulación Neoplásica de la Expresión Génica , FosforilaciónRESUMEN
The phosphorylation of eukaryotic translational initiation factors has been shown to play a significant role in controlling the synthesis of protein. Viral infection, environmental stress, and growth circumstances cause phosphorylation or dephosphorylation of plant initiation factors. Our findings indicate that casein kinase 2 can phosphorylate recombinant wheat eIFiso4E and eIFiso4G generated from E. coli in vitro. For wheat eIFiso4E, Ser-207 was found to be the in vitro phosphorylation site. eIFiso4E lacks an amino acid that can be phosphorylated at the position corresponding to Ser-209, the phosphorylation site in mammalian eIF4E, yet phosphorylation of eIFiso4E has effects on VPg binding affinity that are similar to those of phosphorylation of mammalian eIF4E. The addition of VPg and phosphorylated eIFiso4F to depleted wheat germ extract (WGE) leads to enhancement of translation of both uncapped and capped viral mRNA. The addition of PABP together with eIFiso4Fp and eIF4B to depleted WGE increases both uncapped and capped mRNA translation. However, it exhibits a translational advantage specifically for uncapped mRNA, implying that the phosphorylation of eIFiso4F hinders cap binding while promoting VPg binding, thereby facilitating uncapped translation. These findings indicate TEV virus mediates VPg-dependent translation by engaging a mechanism entailing phosphorylated eIFiso4Fp and PABP. To elucidate the molecular mechanisms underlying these observed effects, we studied the impact of PABP and/or eIF4B on the binding of VPg with eIFiso4Fp. The inclusion of PABP and eIF4B with eIFiso4Fp resulted in about 2-fold increase in affinity for VPg (Kd = 24 ± 1.7 nM), as compared to the affinity of eIFiso4Fp alone (Kd = 41.0 ± 3.1 nM). The interactions between VPg and eIFiso4Fp were determined to be both enthalpically and entropically favorable, with the enthalpic contribution accounting for 76-97% of the ΔG at 25°C, indicating a substantial role of hydrogen bonding in enhancing the stability of the complex. The binding of PABP to eIFiso4Fp·4B resulted in a conformational alteration, leading to a significant enhancement in the binding affinity to VPg. These observations suggest PABP enhances the affinity between eIFiso4Fp and VPg, leading to an overall conformational change that provides a stable platform for efficient viral translation.
Asunto(s)
Factores Eucarióticos de Iniciación , Proteínas de Unión a Poli(A) , Potyvirus , Unión Proteica , Biosíntesis de Proteínas , Triticum , Fosforilación , Potyvirus/metabolismo , Potyvirus/genética , Triticum/virología , Triticum/metabolismo , Triticum/genética , Factores Eucarióticos de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/genética , Proteínas de Unión a Poli(A)/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas Virales/metabolismo , Proteínas Virales/genética , Quinasa de la Caseína II/metabolismo , Quinasa de la Caseína II/genéticaRESUMEN
DNA replication is a vulnerable cellular process, and its deregulation leads to genomic instability. Here, we demonstrate that chromobox protein homolog 3 (CBX3) binds replication protein A 32-kDa subunit (RPA2) and regulates RPA2 retention at stalled replication forks. CBX3 is recruited to stalled replication forks by RPA2 and inhibits ring finger and WD repeat domain 3 (RFWD3)-facilitated replication restart. Phosphorylation of CBX3 at serine-95 by casein kinase 2 (CK2) kinase augments cadherin 1 (CDH1)-mediated CBX3 degradation and RPA2 dynamics at stalled replication forks, which permits replication fork restart. Increased expression of CBX3 due to gene amplification or CK2 inhibitor treatment sensitizes prostate cancer cells to poly(ADP-ribose) polymerase (PARP) inhibitors while inducing replication stress and DNA damage. Our work reveals CBX3 as a key regulator of RPA2 function and DNA replication, suggesting that CBX3 could serve as an indicator for targeted therapy of cancer using PARP inhibitors.
Asunto(s)
Quinasa de la Caseína II , Replicación del ADN , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Proteína de Replicación A , Humanos , Quinasa de la Caseína II/metabolismo , Quinasa de la Caseína II/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteína de Replicación A/metabolismo , Proteína de Replicación A/genética , Línea Celular Tumoral , Proteolisis , Daño del ADN , Fosforilación , Proteínas Cromosómicas no HistonaRESUMEN
Resistance to therapy commonly develops in patients with high-grade serous ovarian carcinoma (HGSC) and triple-negative breast cancer (TNBC), urging the search for improved therapeutic combinations and their predictive biomarkers. Starting from a CRISPR knockout screen, we identified that loss of RB1 in TNBC or HGSC cells generates a synthetic lethal dependency on casein kinase 2 (CK2) for surviving the treatment with replication-perturbing therapeutics such as carboplatin, gemcitabine, or PARP inhibitors. CK2 inhibition in RB1-deficient cells resulted in the degradation of another RB family cell cycle regulator, p130, which led to S phase accumulation, micronuclei formation, and accelerated PARP inhibition-induced aneuploidy and mitotic cell death. CK2 inhibition was also effective in primary patient-derived cells. It selectively prevented the regrowth of RB1-deficient patient HGSC organoids after treatment with carboplatin or niraparib. As about 25% of HGSCs and 40% of TNBCs have lost RB1 expression, CK2 inhibition is a promising approach to overcome resistance to standard therapeutics in large strata of patients.
Asunto(s)
Quinasa de la Caseína II , Proteínas de Unión a Retinoblastoma , Humanos , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo , Quinasa de la Caseína II/genética , Proteínas de Unión a Retinoblastoma/metabolismo , Proteínas de Unión a Retinoblastoma/genética , Femenino , Línea Celular Tumoral , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Carboplatino/farmacología , Mutaciones Letales Sintéticas , Replicación del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Antineoplásicos/farmacologíaRESUMEN
To identify novel susceptibility genes for hepatocellular carcinoma (HCC), we performed a rare-variant association study in Chinese populations consisting of 2,750 cases and 4,153 controls. We identified four HCC-associated genes, including NRDE2, RANBP17, RTEL1, and STEAP3. Using NRDE2 (index rs199890497 [p.N377I], p = 1.19 × 10-9) as an exemplary candidate, we demonstrated that it promotes homologous recombination (HR) repair and suppresses HCC. Mechanistically, NRDE2 binds to the subunits of casein kinase 2 (CK2) and facilitates the assembly and activity of the CK2 holoenzyme. This NRDE2-mediated enhancement of CK2 activity increases the phosphorylation of MDC1 and then facilitates the HR repair. These functions are eliminated almost completely by the NRDE2-p.N377I variant, which sensitizes the HCC cells to poly(ADP-ribose) polymerase (PARP) inhibitors, especially when combined with chemotherapy. Collectively, our findings highlight the relevance of the rare variants to genetic susceptibility to HCC, which would be helpful for the precise treatment of this malignancy.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Reparación del ADN por Recombinación , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Quinasa de la Caseína II/genética , Quinasa de la Caseína II/metabolismo , Línea Celular Tumoral , Predisposición Genética a la Enfermedad , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Reparación del ADN por Recombinación/efectos de los fármacos , Ratones Desnudos , Ratones Endogámicos BALB C , AdultoRESUMEN
AIMS: The aim of this study was to better understand how the chemotherapy drug doxorubicin contributes to the development of ß-cell dysfunction and to explore its relationship with mitochondrial aldehyde dehydrogenase-2 (ALDH2). MATERIALS AND METHODS: In order to investigate this hypothesis, doxorubicin was administered to INS-1 cells, a rat insulinoma cell line, either with or without several target protein activators and inhibitors. ALDH2 activity was detected with a commercial kit and protein levels were determined with western blot. Mitochondrial ROS, membrane potential, and lipid ROS were determined by commercial fluorescent probes. The cell viability was measured by CCK-assay. RESULTS: Exposure of INS-1 cells to doxorubicin decreased active insulin signaling resulting in elevated ALDH2 degradation, compared with control cells by the induction of acid sphingomyelinase mediated ceramide induction. Further, ceramide induction potentiated doxorubicin induced mitochondrial dysfunction. Treatment with the ALDH2 agonist, ALDA1, blocked doxorubicin-induced acid sphingomyelinase activation which significantly blocked ceramide induction and mitochondrial dysfunction mediated cell death. Treatment with the ALDH2 agonist, ALDA1, stimulated casein kinase-2 (CK2) mediated insulin signaling activation. CK2 silencing neutralized the function of ALDH2 in the doxorubicin treated INS-1 cells. CONCLUSIONS: Mitochondrial ALDH2 activation could inhibit the progression of doxorubicin induced pancreatic ß-cell dysfunction by inhibiting the acid sphingomyelinase induction of ceramide, by regulating the activation of CK2 signaling. Our research lays the foundation of ALDH2 activation as a therapeutic target for the precise treatment of chemotherapy drug induced ß-cell dysfunction.
Asunto(s)
Aldehído Deshidrogenasa Mitocondrial , Apoptosis , Quinasa de la Caseína II , Supervivencia Celular , Doxorrubicina , Células Secretoras de Insulina , Mitocondrias , Transducción de Señal , Doxorrubicina/farmacología , Ratas , Animales , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Apoptosis/efectos de los fármacos , Aldehído Deshidrogenasa Mitocondrial/metabolismo , Aldehído Deshidrogenasa Mitocondrial/genética , Transducción de Señal/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Supervivencia Celular/efectos de los fármacos , Quinasa de la Caseína II/metabolismo , Quinasa de la Caseína II/antagonistas & inhibidores , Línea Celular Tumoral , Ceramidas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Antibióticos Antineoplásicos/farmacologíaRESUMEN
Homologous recombination (HR) plays a key role in maintaining genomic stability, and the efficiency of the HR system is closely associated with tumor response to chemotherapy. Our previous work reported that CK2 kinase phosphorylates HIV Tat-specific factor 1 (HTATSF1) Ser748 to facilitate HTATSF1 interaction with TOPBP1, which in turn, promotes RAD51 recruitment and HR repair. However, the clinical implication of the CK2-HTATSF1-TOPBP1 pathway in tumorigenesis and chemotherapeutic response remains to be elucidated. Here, we report that the CK2-HTATSF1-TOPBP1 axis is generally hyperactivated in multiple malignancies and renders breast tumors less responsive to chemotherapy. In contrast, deletion mutations of each gene in this axis, which also occur in breast and lung tumor samples, predict higher HR deficiency scores, and tumor cells bearing a loss-of-function mutation of HTATSF1 are vulnerable to poly(ADP-ribose) polymerase inhibitors or platinum drugs. Taken together, our study suggests that the integrity of the CK2-HTATSF1-TOPBP1 axis is closely linked to tumorigenesis and serves as an indicator of tumor HR status and modulates chemotherapy response.
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Proteínas Portadoras , Quinasa de la Caseína II , Proteínas de Unión al ADN , Transducción de Señal , Humanos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Transducción de Señal/efectos de los fármacos , Quinasa de la Caseína II/metabolismo , Quinasa de la Caseína II/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Animales , Femenino , Ratones , Línea Celular Tumoral , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patologíaRESUMEN
The molecular mechanisms governing the response of hematopoietic stem cells (HSCs) to stress insults remain poorly defined. Here, we investigated effects of conditional knock-out or overexpression of Hmga2 (High mobility group AT-hook 2), a transcriptional activator of stem cell genes in fetal HSCs. While Hmga2 overexpression did not affect adult hematopoiesis under homeostasis, it accelerated HSC expansion in response to injection with 5-fluorouracil (5-FU) or in vitro treatment with TNF-α. In contrast, HSC and megakaryocyte progenitor cell numbers were decreased in Hmga2 KO animals. Transcription of inflammatory genes was repressed in Hmga2-overexpressing mice injected with 5-FU, and Hmga2 bound to distinct regions and chromatin accessibility was decreased in HSCs upon stress. Mechanistically, we found that casein kinase 2 (CK2) phosphorylates the Hmga2 acidic domain, promoting its access and binding to chromatin, transcription of anti-inflammatory target genes, and the expansion of HSCs under stress conditions. Notably, the identified stress-regulated Hmga2 gene signature is activated in hematopoietic stem progenitor cells of human myelodysplastic syndrome patients. In sum, these results reveal a TNF-α/CK2/phospho-Hmga2 axis controlling adult stress hematopoiesis.
Asunto(s)
Quinasa de la Caseína II , Cromatina , Proteína HMGA2 , Células Madre Hematopoyéticas , Ratones Noqueados , Proteína HMGA2/metabolismo , Proteína HMGA2/genética , Animales , Células Madre Hematopoyéticas/metabolismo , Ratones , Humanos , Quinasa de la Caseína II/metabolismo , Quinasa de la Caseína II/genética , Cromatina/metabolismo , Cromatina/genética , Factor de Necrosis Tumoral alfa/metabolismo , Hematopoyesis , Estrés Fisiológico , Fluorouracilo/farmacología , Regeneración , Fosforilación , Síndromes Mielodisplásicos/patología , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Ratones Endogámicos C57BLRESUMEN
The autosomal dominant Okur-Chung neurodevelopmental syndrome (OCNDS: OMIM #617062) is a rare neurodevelopmental disorder first described in 2016. Features include developmental delay (DD), intellectual disability (ID), behavioral problems, hypotonia, language deficits, congenital heart abnormalities, and non-specific dysmorphic facial features. OCNDS is caused by heterozygous pathogenic variants in CSNK2A1 (OMIM *115440; NM_177559.3). To date, 160 patients have been diagnosed worldwide. The number will likely increase due to the growing use of exome sequencing (ES) and genome sequencing (GS). Here, we describe a novel OCNDS patient carrying a CSNK2A1 variant (NM_177559.3:c.140G>A; NP_808227.1:p.Arg47Gln). Phenotypically, he presented with DD, ID, generalized hypotonia, speech delay, short stature, microcephaly, and dysmorphic features such as low-set ears, hypertelorism, thin upper lip, and a round face. The patient showed several signs not yet described that may extend the phenotypic spectrum of OCNDS. These include prenatal bilateral clubfeet, exotropia, and peg lateral incisors. However, unlike the majority of descriptions, he did not present sleep disturbance, seizures or gait difficulties. A literature review shows phenotypic heterogeneity for OCNDS, whether these patients have the same variant or not. This case report is an opportunity to refine the phenotype of this syndrome and raise the question of the genotype-phenotype correlation.
Asunto(s)
Quinasa de la Caseína II , Trastornos del Neurodesarrollo , Niño , Humanos , Masculino , Quinasa de la Caseína II/genética , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/patología , Secuenciación del Exoma , Predisposición Genética a la Enfermedad , Heterocigoto , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Hipotonía Muscular/genética , Hipotonía Muscular/patología , Mutación , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/patología , Trastornos del Neurodesarrollo/diagnóstico , FenotipoRESUMEN
Senescent cell clearance is emerging as a promising strategy for treating age-related diseases. Senolytics are small molecules that promote the clearance of senescent cells; however, senolytics are uncommon and their underlying mechanisms remain largely unknown. Here, we investigated whether genomic instability is a potential target for senolytic. We screened small-molecule kinase inhibitors involved in the DNA damage response (DDR) in Zmpste24-/- mouse embryonic fibroblasts, a progeroid model characterized with impaired DDR and DNA repair. 4,5,6,7-tetrabromo-2-azabenzamidazole (TBB), which specifically inhibits casein kinase 2 (CK2), was selected and discovered to preferentially trigger apoptosis in Zmpste24-/- cells. Mechanistically, inhibition of CK2 abolished the phosphorylation of heterochromatin protein 1α (HP1α), which retarded the dynamic HP1α dissociation from repressive histone mark H3K9me3 and its relocalization with γH2AX to DNA damage sites, suggesting that disrupting heterochromatin remodeling in the initiation of DDR accelerates apoptosis in senescent cells. Furthermore, feeding Zmpste24-deficient mice with TBB alleviated progeroid features and extended their lifespan. Our study identified TBB as a new class senolytic compound that can reduce age-related symptoms and prolong lifespan in progeroid mice.