scholarly journals Casein Kinase 1D Encodes a Novel Drug Target in Hedgehog—GLI-Driven Cancers and Tumor-Initiating Cells Resistant to SMO Inhibition

Cancers ◽  
2021 ◽  
Vol 13 (16) ◽  
pp. 4227
Author(s):  
Elisabeth Peer ◽  
Sophie Karoline Aichberger ◽  
Filip Vilotic ◽  
Wolfgang Gruber ◽  
Thomas Parigger ◽  
...  
Keyword(s):  
Critical Role ◽  
Treatment Strategies ◽  
Casein Kinase ◽  
Tumor Initiating Cells ◽  
Casein Kinase 1 ◽  
Hh Signaling ◽  
Novel Drug ◽  
Smo Inhibitor

(1) Background: Aberrant activation of the hedgehog (HH)—GLI pathway in stem-like tumor-initiating cells (TIC) is a frequent oncogenic driver signal in various human malignancies. Remarkable efficacy of anti-HH therapeutics led to the approval of HH inhibitors targeting the key pathway effector smoothened (SMO) in basal cell carcinoma and acute myeloid leukemia. However, frequent development of drug resistance and severe adverse effects of SMO inhibitors pose major challenges that require alternative treatment strategies targeting HH—GLI in TIC downstream of SMO. We therefore investigated members of the casein kinase 1 (CSNK1) family as novel drug targets in HH—GLI-driven malignancies. (2) Methods: We genetically and pharmacologically inhibited CSNK1D in HH-dependent cancer cells displaying either sensitivity or resistance to SMO inhibitors. To address the role of CSNK1D in oncogenic HH signaling and tumor growth and initiation, we quantitatively analyzed HH target gene expression, performed genetic and chemical perturbations of CSNK1D activity, and monitored the oncogenic transformation of TIC in vitro and in vivo using 3D clonogenic tumor spheroid assays and xenograft models. (3) Results: We show that CSNK1D plays a critical role in controlling oncogenic GLI activity downstream of SMO. We provide evidence that inhibition of CSNK1D interferes with oncogenic HH signaling in both SMO inhibitor-sensitive and -resistant tumor settings. Furthermore, genetic and pharmacologic perturbation of CSNK1D decreases the clonogenic growth of GLI-dependent TIC in vitro and in vivo. (4) Conclusions: Pharmacologic targeting of CSNK1D represents a novel therapeutic approach for the treatment of both SMO inhibitor-sensitive and -resistant tumors.

scholarly journals Casein kinase 1D encodes a novel drug target in Hedgehog-GLI driven cancers and tumor-initiating cells resistant to SMO inhibition

2021 ◽  
Author(s):  
Elisabeth Peer ◽  
Sophie K Aichberger ◽  
Filip Vilotic ◽  
Gruber Wolfgang ◽  
Thomas Parigger ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Critical Role ◽  
Casein Kinase ◽  
Tumor Initiating Cells ◽  
Casein Kinase 1 ◽  
Hh Signaling ◽  
Novel Drug ◽  
Smo Inhibitor

Abstract: (1) Background: Aberrant activation of the Hedgehog (HH)/GLI pathway in stem-like tumor initiating cells (TIC) is a frequent oncogenic driver signal in various human malignancies. Remarkable efficacy of anti-HH therapeutics led to the approval of HH inhibitors targeting the key pathway effector Smoothened (SMO) in basal cell carcinoma and acute myeloid leukemia. However, frequent development of drug resistance and severe adverse effects of SMO inhibitors pose major challenges that require alternative treatment strategies targeting HH/GLI in TIC downstream of SMO. We therefore investigated members of the casein kinase 1 (CSNK1) family as novel drug targets in HH/GLI driven malignancies. (2) Methods: We genetically and pharmacologically inhibited CSNK1D in HH-dependent cancer cells displaying either sensitivity or resistance to SMO inhibitors. To address the role of CSNK1D in oncogenic HH signaling and tumor growth and initiation, we quantitatively analyzed HH target gene expression, performed genetic and chemical perturbations of CSNK1D activity and monitored oncogenic transformation of TIC in vitro and in vivo using 3D clonogenic tumor spheroid assays and xenograft models. (3) Results: We show that CSNK1D plays a critical role in controlling oncogenic GLI activity downstream of SMO. We provide evidence that inhibition of CSNK1D interferes with oncogenic HH signaling in both SMO-inhibitor sensitive and resistant tumor settings. Furthermore, genetic and pharmacologic perturbation of CSNK1D decreases the clonogenic growth of GLI-dependent tumor-initiating cancer cells in vitro and in vivo. (4) Conclusions: Pharmacologic targeting of CSNK1D represents a novel therapeutic approach for the treatment of both SMO inhibitor sensitive and resistant tumors.

scholarly journals Functional Characterization of Brain Tumor-Initiating Cells: Implications for Preclinical Models and Drug Development

Neurosurgery ◽  
2019 ◽  
Vol 66 (Supplement_1) ◽  
Author(s):  
Cesar A Garcia ◽  
Adip Guruprasad Bhargav ◽  
Sujan K Mondal ◽  
Karim ReFaey ◽  
Natanael Zarco ◽  
...  
Keyword(s):  
Brain Tumor ◽  
Critical Role ◽  
Functional Characterization ◽  
Tumor Initiating Cells ◽  
Retroviral Transduction ◽  
Significant Difference ◽  
Brain Tumor Initiating Cells ◽  
Functional Features

Abstract INTRODUCTION Glioblastoma (GBM) is the deadliest and most common primary brain cancer in adults. Brain tumor-initiating cells (BTICs) are a heterogeneous subset of stem-like, invasive cells that play a critical role in treatment failure and recurrence. METHODS Here, we propose a system to functionally characterize patient-derived BTICs to identify features that will guide assessment of therapeutics in a BTIC subpopulation-specific manner. We established and evaluated 5 BTIC populations based on (1) proliferation, (2) stemness, (3) migration, (4) tumorigenesis, (5) clinical characteristics, and (6) therapeutic sensitivity. RESULTS Overall, in Vitro growth trends reflected in Vivo growth rates. However, a significant difference was found between tumor growth in male versus female mice in 3 BTIC lines (QNS108 P = .0011; QNS120 P < .0001; QNS 140 P < .0001). Differences in survival were observed, where BTICs derived from male and female patients produced faster morbidity in mice of the opposite sex (male derived QNS108 male vs female P = .0039; female derived QNS203 male vs female P = .029). QNS203, which was isolated from a tumor in contact with the anterior subventricular zone, decreased survival at a faster rate compared to other cell lines (n = 10 per line, 5 males/5 females, P < .0001). Stem-like properties of BTICs were assessed via differentiation marker expression, sphere-forming capacity, and detection of canonical marker CD133. Higher CD133 expression correlated with faster in Vitro doubling time and greater tumor burden. Histology reflected similar patient tumor features such as migration across the corpus callosum and cystic formation. BTICs revealed varying responses to therapies (TMZ, Radiation, TRAIL, BMP4) and varied competence to retroviral transduction. CONCLUSION By studying the functional features of BTICs within our model of GBM heterogeneity, it was shown that several factors influenced tumorigenesis and survival. These included original tumor location, stemness, variation in therapeutic sensitivity, and a critical finding for the role of sex, an unexplored area for creating next-generation, sex-specific, and BTIC-specific therapeutics.

scholarly journals Generation of strong casein kinase 1 inhibitor of Arabidopsis thaliana

2019 ◽  
Author(s):  
Ami N Saito ◽  
Hiromi Matsuo ◽  
Keiko Kuwata ◽  
Azusa Ono ◽  
Toshinori Kinoshita ◽  
...  
Keyword(s):  
Bromine Atom ◽  
Casein Kinase ◽  
In Planta ◽  
Vitro Assay ◽  
Casein Kinase 1 ◽  
Yeast Fungi ◽  
Genes Encoding ◽  
Period Lengthening

AbstractCasein kinase 1 (CK1) is an evolutionarily conserved protein kinase among eukaryotes. Studies on yeast, fungi, and animals have revealed that CK1 plays roles in divergent biological processes. By contrast, the collective knowledge regarding the biological roles of plant CK1 lags was behind those of animal CK1. One of reasons for this is that plants have more multiple genes encoding CK1 than animals. To accelerate the research for plant CK1, a strong CK1 inhibitor that efficiently inhibits multiple members of CK1 proteins in vivo (in planta) is required. Here, we report a novel strong CK1 inhibitor of Arabidopsis (AMI-331). Using a circadian period-lengthening activity as estimation of the CK1 inhibitor effect in vivo, we performed a structure-activity relationship (SAR) study of PHA767491 (1,5,6,7-tetrahydro-2-(4-pyridinyl)-4H-pyrrolo[3,2-c]pyridin-4-one hydrochloride), a potent CK1 inhibitor of Arabidopsis, and found that PHA767491 analogues bearing a propargyl group at the pyrrole nitrogen atom (AMI-212) or a bromine atom at the pyrrole C3 position (AMI-23) enhance the period-lengthening activity. The period lengthening activity of a hybrid molecule of AMI-212 and AMI-23 (AMI-331) is about 100-fold stronger than that of PHA767491. An in vitro assay indicated a strong inhibitory activity of CK1 kinase by AMI-331. Also, affinity proteomics using an AMI-331 probe showed that targets of AMI-331 are mostly CK1 proteins. As such, AMI-331 is a strong potent CK1 inhibitor that shows promise in the research of CK1 in plants.

scholarly journals Altered synaptic adaptation and gain in sensory circuits of the casein kinase 1 delta (CK1dT44A) mouse model of migraine

2019 ◽  
Author(s):  
Pratyush Suryavanshi ◽  
Punam Sawant Pokam ◽  
KC Brennan
Keyword(s):  
Steady State ◽  
Cortical Neurons ◽  
Casein Kinase ◽  
Local Network ◽  
Casein Kinase 1 ◽  
Thermal Pain ◽  
Readily Releasable Pool ◽  
Short Stimulus

AbstractMigraine is a very common and disabling neurological disorder that remains poorly understood at the cellular and circuit level. Transgenic mice harboring a mutation in casein kinase 1 delta (CK1dT44A) represent the first animal model of non-hemiplegic migraine. These mice have decreased sensory thresholds to mechanical and thermal pain after treatment with the migraine trigger nitroglycerin; and an increased susceptibility to cortical spreading depression (CSD), which models the migraine aura. In this study, we investigated cellular and synaptic mechanisms within sensory cortical circuits that might underlie the migraine relevant phenotypes of CK1dT44A mice, using in vitro and in vivo whole cell electrophysiology. Surprisingly we found that at resting state, CK1dT44A neurons exhibited hyperpolarized membrane potentials, due to increased tonic inhibition. Despite this reduction in baseline excitability, CK1dT44A neurons fired action potentials more frequently in response to current injection. And despite similar synaptic and dendritic characteristics to wild type neurons, excitatory but not inhibitory CK1dT44A synapses failed to adapt to high frequency short-stimulus trains, resulting in elevated steady state excitatory currents. The increased steady state currents were attributable to an increased replenishment rate of the readily releasable pool, providing a presynaptic mechanism for the CK1dT44A phenotype. Finally, during in vivo experiments, CK1dT44A animals showed increased duration and membrane potential variance at ‘cortical up states’, showing that the intrinsic and synaptic changes we observed have excitatory consequences at the local network level. In conclusion excitatory sensory cortical neurons and networks in CK1dT44A animals appear to exhibit decreased adaptation and increased gain that may inform the migraine phenotype.

scholarly journals Identification of Domains Responsible for Ubiquitin-Dependent Degradation of dMyc by Glycogen Synthase Kinase 3β and Casein Kinase 1 Kinases

2009 ◽  
Vol 29 (12) ◽  
pp. 3424-3434 ◽  
Author(s):  
Margherita Galletti ◽  
Sara Riccardo ◽  
Federica Parisi ◽  
Carlina Lora ◽  
Mahesh Kumar Saqcena ◽  
...  
Keyword(s):  
Compound Eye ◽  
Glycogen Synthase ◽  
Physiological Role ◽  
Casein Kinase ◽  
Glycogen Synthase Kinase ◽  
Glycogen Synthase Kinase 3Β ◽  
Casein Kinase 1 ◽  
The One

ABSTRACT In the present study, we report that ubiquitin-mediated degradation of dMyc, the Drosophila homologue of the human c-myc proto-oncogene, is regulated in vitro and in vivo by members of the casein kinase 1 (CK1) family and by glycogen synthase kinase 3β (GSK3β). Using Drosophila S2 cells, we demonstrate that CK1α promotes dMyc ubiquitination and degradation with a mechanism similar to the one mediated by GSK3β in vertebrates. Mutation of ck1α or -ε or sgg/gsk3β in Drosophila wing imaginal discs results in the accumulation of dMyc protein, suggesting a physiological role for these kinases in vivo. Analysis of the dMyc amino acid sequence reveals the presence of conserved domains containing potential phosphorylation sites for mitogen kinases, GSK3β, and members of the CK1 family. We demonstrate that mutations of specific residues within these phosphorylation domains regulate dMyc protein stability and confer resistance to degradation by CK1α and GSK3β kinases. Expression of the dMyc mutants in the compound eye of the adult fly results in a visible defect that is attributed to the effect of dMyc on growth, cell death, and inhibition of ommatidial differentiation.

scholarly journals Casein kinase-1γ1 and 3 stimulate tumor necrosis factor-induced necroptosis through RIPK3

2019 ◽  
Vol 10 (12) ◽  
Author(s):  
Song-Yi Lee ◽  
Hyunjoo Kim ◽  
Cathena Meiling Li ◽  
Jaemin Kang ◽  
Ayaz Najafov ◽  
...  
Keyword(s):  
Ectopic Expression ◽  
Specific Inhibitor ◽  
Casein Kinase ◽  
In Vitro Assays ◽  
Serine Threonine Kinase ◽  
Casein Kinase 1 ◽  
Threonine Kinase ◽  
Necrosis Factor

AbstractUpon necroptosis activation, receptor interacting serine/threonine kinase (RIPK)1 and RIPK3 form a necrosome complex with pseudokinase mixed lineage kinase-like (MLKL). Although protein phosphorylation is a key event for RIPK1 and RIPK3 activation in response to a necroptosis signal, relatively little is known about other factors that might regulate the activity of these kinases or necrosome formation. Through a gain-of-function screen with 546 kinases and 127 phosphatases, we identified casein kinase 1 gamma (CK1γ) as a candidate necroptosis-promoting factor. Here, we show that the decreased activity or amounts of CK1γ1 and CK1γ3, either by treatment with a chemical inhibitor or knockdown in cells, reduced TNFα-induced necroptosis. Conversely, ectopic expression of CK1γ1 or CK1γ3 exacerbated necroptosis, but not apoptosis. Similar to RIPK1 and RIPK3, CK1γ1 was also cleaved at Asp343 by caspase-8 during apoptosis. CK1γ1 and CK1γ3 formed a protein complex and were recruited to the necrosome harboring RIPK1, RIPK3 and MLKL. In particular, an autophosphorylated form of CK1γ3 at Ser344/345 was detected in the necrosome and was required to mediate the necroptosis. In addition, in vitro assays with purified proteins showed that CK1γ phosphorylated RIPK3, affecting its activity, and in vivo assays showed that the CK1γ-specific inhibitor Gi prevented abrupt death in mice with hypothermia in a model of TNFα-induced systemic inflammatory response syndrome. Collectively, these data suggest that CK1γ1 and CK1γ3 are required for TNFα-induced necroptosis likely by regulating RIPK3.

scholarly journals miR-5000-3p confers oxaliplatin resistance by targeting ubiquitin-specific peptidase 49 in colorectal cancer

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Yan-yan Zhuang ◽  
Wa Zhong ◽  
Zhong-sheng Xia ◽  
Shu-zhen Lin ◽  
Man chung Chan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Colorectal Cancer ◽  
Drug Resistance ◽  
Critical Role ◽  
Treatment Strategies ◽  
Luciferase Reporter ◽  
Multi Drug Resistance ◽  
Gastrointestinal Malignancies

AbstractColorectal cancer (CRC) is the most common form of gastrointestinal malignancies. A growing number of reports focusing on oxaliplatin (OXA) resistance in CRC treatment have revealed that drug resistance is an urgent issue in clinical applications, especially for finding effective therapeutic targets. Recently, microRNAs (miRNAs) are reported to play a critical role in tumor progressions and multi-drug resistance. The main aim of this study is to establish whether miR-5000-3p is an oncogene that is resistant to OXA and further confirm its underlying regulatory role in CRC. The OXA-associated gene expression dataset in CRC cells was downloaded from Gene Expression Omnibus (GEO) database. Statistical software R was used for significance analysis of differentially expressed genes (DEGs) between OXA-resistant (OR)-CRC cells and CRC cells, and results indicated ubiquitin-specific peptidase 49 (USP49) was upregulated in OR-CRC cells. Luciferase reporter assay showed that USP49 was verified to act as a downstream target gene of miR-5000-3p. From the results of TCGA database, miR-5000-3p expression was upregulated and USP49 was downregulated in patients with CRC. The function of miR-5000-3p was detected using MTT assay, wound healing, Transwell, and flow cytometry assays. Moreover, through in vitro and in vivo experiments, miR-5000-3p expression was confirmed to be upregulated in CRC cells or OR-CRC cells comparing to normal cell lines. Molecular mechanism assays revealed that USP49 binds to the miR-5000-3p promoter to increase the expression of miR-5000-3p, resulting in cancer cells sensitized to OXA. To sum up, these results suggest that miR-5000-3p may be a novel biomarker involved in drug-resistance progression of CRC. Moreover, the drug-resistance mechanism of miR-5000-3p/USP49 axis provides new treatment strategies for CRC in clinical trials.

TAMI-51. HORIZONTAL MITOCHONDRIAL TRANSFER FROM THE TUMOR MICROENVIRONMENT TO GLIOBLASTOMA INCREASES TUMORIGENICITY

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi208-vi209
Author(s):  
Dionysios Watson ◽  
Defne Bayik ◽  
Justin Lathia
Keyword(s):  
Bone Marrow ◽  
Tumor Microenvironment ◽  
Tumor Cells ◽  
Bone Marrow Cells ◽  
Treatment Strategies ◽  
Tumor Initiating Cells ◽  
Mitochondrial Transfer ◽  
Mitochondria Transfer

Abstract Communication between glioblastoma (GBM) and its microenvironment facilitates tumor growth and therapeutic resistance, and is facilitated through a variety of mechanisms. Organelle transfer between cells was recently observed, including mitochondria transfer from astrocytes to neurons after ischemic stroke. Given the dependence of GBM on microenvironmental interactions, we hypothesized that mitochondria transfer from tumor microenvironment to GBM cells could occur and affect metabolism and tumorigenicity. We interrogated this in vivo by establishing intracranial GBM tumors in mito::mKate2 mice (with trackable fluorescent mitochondria) using syngeneic GFP-expressing tumor cells (SB28 and GL261 models). We also cultured stromal cell types from mito::mKate2 mice with tumor cells, enabling sorting of tumor cells with and without exogenous mitochondria. Confocal microscopy revealed horizontal transfer of mKate2+ mitochondria from mouse cells to implanted GBM cells in vivo and was confirmed by flow cytometry where 20-40% of GBM cells acquired exogenous mitochondria. Transfer was negligible in wildtype mice transplanted with mito::mKate2 bone marrow cells, suggesting that brain-resident cells were the main donors. In vitro, astrocytes and microglia exhibited 5 to 10-fold higher mitochondrial transfer rate than bone-marrow derived macrophages. Seahorse metabolic profiling revealed that GBM cells with mKate2+ mitochondria had 40% lower respiratory reserve compared to cells without exogenous mitochondria. Median survival of mice implanted with SB28 that acquired mitochondria was significantly shorter and in vivo limiting dilution confirmed the frequency of tumor-initiating cells was 3-fold higher in SB28 cells with exogenous mitochondria. Our data indicate that horizontal mitochondrial transfer from brain-resident glia to mouse GBM tumors alters tumor cell metabolism and increases their tumorigenicity. Ongoing studies are assessing gene expression in GBM cells acquiring exogenous mitochondria; validating findings in human specimens; and screening for transfer inhibitor drugs. Horizontal mitochondrial transfer represents a foundational tumor microenvironment interaction contributing to glioblastoma plasticity, and is likely to inform next-generation treatment strategies.

scholarly journals T. cruzi DNA polymerase beta (Tcpolβ) is phosphorylated in vitro by CK1, CK2 and TcAUK1 leading to the potentiation of its DNA synthesis activity

2021 ◽  
Vol 15 (7) ◽  
pp. e0009588
Author(s):  
Edio Maldonado ◽  
Diego A. Rojas ◽  
Fabiola Urbina ◽  
Aldo Solari
Keyword(s):  
Protein Kinases ◽  
Dna Polymerase ◽  
Excision Repair ◽  
Polymerase Activity ◽  
Casein Kinase ◽  
Phosphorylation Sites ◽  
Casein Kinase 1 ◽  
Synthesis Activity

The unicellular protozoan Trypanosoma cruzi is the causing agent of Chagas disease which affects several millions of people around the world. The components of the cell signaling pathways in this parasite have not been well studied yet, although its genome can encode several components able to transduce the signals, such as protein kinases and phosphatases. In a previous work we have found that DNA polymerase β (Tcpolβ) can be phosphorylated in vivo and this modification activates the synthesis activity of the enzyme. Tcpolβ is kinetoplast-located and is a key enzyme in the DNA base excision repair (BER) system. The polypeptide possesses several consensus phosphorylation sites for several protein kinases, however, a direct phosphorylation of those sites by specific kinases has not been reported yet. Tcpolβ has consensus phosphorylation sites for casein kinase 1 (CK1), casein kinase 2 (CK2) and aurora kinase (AUK). Genes encoding orthologues of those kinases exist in T. cruzi and we were able to identify the genes and to express them to investigate whether or no Tcpolβ could be a substrate for in vitro phosphorylation by those kinases. Both CK1 and TcAUK1 have auto-phosphorylation activities and they are able to phosphorylate Tcpolβ. CK2 cannot perform auto-phosphorylation of its subunits, however, it was able to phosphorylate Tcpolβ. Pharmacological inhibitors used to inhibit the homologous mammalian kinases can also inhibit the activity of T. cruzi kinases, although, at higher concentrations. The phosphorylation events carried out by those kinases can potentiate the DNA polymerase activity of Tcpolβ and it is discussed the role of the phosphorylation on the DNA polymerase and lyase activities of Tcpolβ. Taken altogether, indicates that CK1, CK2 and TcAUK1 can play an in vivo role regulating the function of Tcpolβ.

scholarly journals MiR-125b-2 knockout increases high-fat diet-induced fat accumulation and insulin resistance

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Li-Min Wei ◽  
Rui-Ping Sun ◽  
Tao Dong ◽  
Jie Liu ◽  
Ting Chen ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Critical Role ◽  
Treatment Strategies ◽  
Normal Diet ◽  
Healthy Weight ◽  
Fat Accumulation ◽  
Obesity And Diabetes ◽  
White Fat

AbstractObese individuals are more susceptible to comorbidities than individuals of healthy weight, including cardiovascular disease and metabolic disorders. MicroRNAs are a class of small and noncoding RNAs that are implicated in the regulation of chronic human diseases. We previously reported that miR-125b plays a critical role in adipogenesis in vitro. However, the involvement of miR-125b-2 in fat metabolism in vivo remains unknown. In the present study, miR-125b-2 knockout mice were generated using CRISPR/CAS9 technology, resulting in mice with a 7 bp deletion in the seed sequence of miR-125b-2. MiR-125b-2 knockout increased the weight of liver tissue, epididymal white fat and inguinal white fat. MiR-125b-2 knockout also increased adipocyte volume in HFD-induced obese mice, while there were no significant differences in body weight and feed intake versus mice fed a normal diet. Additionally, qRT-PCR and western blot analysis revealed that the expression of the miR-125b-2 target gene SCD-1 and fat synthesis-associated genes, such as PPARγ and C/EBPα, were significantly up-regulated in miR-125b-2KO mice (P < 0.05). Moreover, miR-125b-2KO altered HFD-induced changes in glucose tolerance and insulin resistance. In conclusion, we show that miR-125b-2 is a novel potential target for regulating fat accumulation, and also a candidate target to develop novel treatment strategies for obesity and diabetes.

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