RESUMEN
Small-molecule tools have enabled mechanistic investigations and therapeutic targeting of the protein kinase-like (PKL) superfamily. However, such tools are still lacking for many PKL members, including the highly conserved and disease-related UbiB family. Here, we sought to develop and characterize an inhibitor for the archetypal UbiB member COQ8, whose function is essential for coenzyme Q (CoQ) biosynthesis. Guided by crystallography, activity assays and cellular CoQ measurements, we repurposed the 4-anilinoquinoline scaffold to selectively inhibit human COQ8A in cells. Our chemical tool promises to lend mechanistic insights into the activities of these widespread and understudied proteins and to offer potential therapeutic strategies for human diseases connected to their dysfunction.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/metabolismo , Ubiquinona/farmacología , Ubiquinona/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Pharmacological inhibition of protein kinases induces adaptive reprogramming of tumor cell regulatory networks by altering expression of genes that regulate signaling, including protein kinases. Adaptive responses are dependent on transcriptional changes resulting from remodeling of enhancer and promoter landscapes. Enhancer and promoter remodeling in response to targeted kinase inhibition is controlled by changes in open chromatin state and by activity of specific transcription factors, such as c-MYC. This review focuses on the dynamic plasticity of protein kinase expression of the tumor cell kinome and the resulting adaptive resistance to targeted kinase inhibition. Plasticity of the functional kinome has been shown in patient window trials where triple-negative and human epidermal growth factor receptor 2-positive breast cancer patient tumors were characterized by RNAseq after biopsies before and after 1 week of therapy. The expressed kinome changed dramatically during drug treatment, and these changes in kinase expression were shown in cell lines and xenografts in mice to be correlated with adaptive tumor cell drug resistance. The dynamic transcriptional nature of the kinome also differs for inhibitors targeting different kinase signaling pathways (e.g., BRAF-MEK-ERK versus PI3K-AKT) that are commonly activated in cancers. Heterogeneity arising from differences in gene regulation and mutations represents a challenge to therapeutic durability and prevention of clinical drug resistance with drug-tolerant tumor cell populations developing and persisting through treatment. We conclude that understanding the heterogeneity of kinase expression at baseline and in response to therapy is imperative for development of combinations and timing intervals of therapies making interventions durable.
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Neoplasias de la Mama , Ensamble y Desensamble de Cromatina , Inhibidores de Proteínas Quinasas , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Humanos , Ratones , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas QuinasasRESUMEN
The maternal embryonic leucine zipper kinase (MELK) has been implicated in the regulation of cancer cell proliferation. RNAi-mediated MELK depletion impairs growth and causes G2/M arrest in numerous cancers, but the mechanisms underlying these effects are poorly understood. Furthermore, the MELK inhibitor OTSSP167 has recently been shown to have poor selectivity for MELK, complicating the use of this inhibitor as a tool compound to investigate MELK function. Here, using a cell-based proteomics technique called multiplexed kinase inhibitor beads/mass spectrometry (MIB/MS), we profiled the selectivity of two additional MELK inhibitors, NVS-MELK8a (8a) and HTH-01-091. Our results revealed that 8a is a highly selective MELK inhibitor, which we further used for functional studies. Resazurin and crystal violet assays indicated that 8a decreases triple-negative breast cancer cell viability, and immunoblotting revealed that impaired growth is due to perturbation of cell cycle progression rather than induction of apoptosis. Using double-thymidine synchronization and immunoblotting, we observed that MELK inhibition delays mitotic entry, which was associated with delayed activation of Aurora A, Aurora B, and cyclin-dependent kinase 1 (CDK1). Following this delay, cells entered and completed mitosis. Using live-cell microscopy of cells harboring fluorescent proliferating cell nuclear antigen, we confirmed that 8a significantly and dose-dependently lengthens G2 phase. Collectively, our results provide a rationale for using 8a as a tool compound for functional studies of MELK and indicate that MELK inhibition delays mitotic entry, likely via transient G2/M checkpoint activation.
Asunto(s)
Espectrometría de Masas , Mitosis , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Histonas/metabolismo , Humanos , Mitosis/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Neoplasias de la Mama Triple Negativas/enzimología , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Parallel reaction monitoring (PRM) has emerged as a popular approach for targeted protein quantification. With high ion utilization efficiency and first-in-class acquisition speed, the timsTOF Pro provides a powerful platform for PRM analysis. However, sporadic chromatographic drift in peptide retention time represents a fundamental limitation for the reproducible multiplexing of targets across PRM acquisitions. Here, we present PRM-LIVE, an extensible, Python-based acquisition engine for the timsTOF Pro, which dynamically adjusts detection windows for reproducible target scheduling. In this initial implementation, we used iRT peptides as retention time standards and demonstrated reproducible detection and quantification of 1857 tryptic peptides from the cell lysate in a 60 min PRM-LIVE acquisition. As an application in functional proteomics, we use PRM-LIVE in an activity-based protein profiling platform to assess binding selectivity of small-molecule inhibitors against 220 endogenous human kinases.
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Espectrometría de Movilidad Iónica , Proteómica , Humanos , Espectrometría de Masas , Péptidos , ProteínasRESUMEN
A focused series of substituted 4H-1,2,6-thiadiazin-4-ones was designed and synthesized to probe the anti-cancer properties of this scaffold. Insights from previous kinase inhibitor programs were used to carefully select several different substitution patterns. Compounds were tested on bladder, prostate, pancreatic, breast, chordoma, and lung cancer cell lines with an additional skin fibroblast cell line as a toxicity control. This resulted in the identification of several low single digit micro molar compounds with promising therapeutic windows, particularly for bladder and prostate cancer. A number of key structural features of the 4H-1,2,6-thiadiazin-4-one scaffold are discussed that show promising scope for future improvement.
Asunto(s)
Antineoplásicos/farmacología , Diseño de Fármacos , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Tiadiazinas/química , Proliferación Celular , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Human cytomegalovirus (HCMV) is a significant cause of disease in immune-compromised adults and immune naïve newborns. No vaccine exists to prevent HCMV infection, and current antiviral therapies have toxic side effects that limit the duration and intensity of their use. There is thus an urgent need for new strategies to treat HCMV infection. Repurposing existing drugs as antivirals is an attractive approach to limit the time and cost of new antiviral drug development. Virus-induced changes in infected cells are often driven by changes in cellular kinase activity, which led us to hypothesize that defining the complement of kinases (the kinome), whose abundance or expression is altered during infection would identify existing kinase inhibitors that could be repurposed as new antivirals. To this end, we applied a kinase capture technique, multiplexed kinase inhibitor bead-mass spectrometry (MIB-MS) kinome, to quantitatively measure perturbations in >240 cellular kinases simultaneously in cells infected with a laboratory-adapted (AD169) or clinical (TB40E) HCMV strain. MIB-MS profiling identified time-dependent increases and decreases in MIB binding of multiple kinases including cell cycle kinases, receptor tyrosine kinases, and mitotic kinases. Based on the kinome data, we tested the antiviral effects of kinase inhibitors and other compounds, several of which are in clinical use or development. Using a novel flow cytometry-based assay and a fluorescent reporter virus we identified three compounds that inhibited HCMV replication with IC50 values of <1 µm, and at doses that were not toxic to uninfected cells. The most potent inhibitor of HCMV replication was OTSSP167 (IC50 <1.2 nm), a MELK inhibitor, blocked HCMV early gene expression and viral DNA accumulation, resulting in a >3 log decrease in virus replication. These results show the utility of MIB-MS kinome profiling for identifying existing kinase inhibitors that can potentially be repurposed as novel antiviral drugs.
Asunto(s)
Antivirales/farmacología , Citomegalovirus/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Células Cultivadas , Citomegalovirus/metabolismo , Reposicionamiento de Medicamentos , Humanos , Espectrometría de Masas/métodos , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
The kinase enzymes within a cell, known collectively as the kinome, play crucial roles in many signaling pathways, including survival, motility, differentiation, stress response, and many more. Aberrant signaling through kinase pathways is often linked to cancer, among other diseases. A major area of scientific research involves understanding the relationships between kinases, their targets, and how the kinome adapts to perturbations of the cellular system. This review will discuss many of the current and developing methods for studying kinase activity, and evaluate their applications, advantages, and disadvantages. J. Cell. Biochem. 118: 3595-3606, 2017. © 2017 Wiley Periodicals, Inc.
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Proteínas de Neoplasias/análisis , Neoplasias/enzimología , Proteínas Quinasas/análisis , Animales , HumanosRESUMEN
The ARF family of regulatory GTPases, within the RAS superfamily, is composed of ~30 members in mammals, including up to six ARF and at least 18 ARF-like (ARL) proteins. They exhibit significant structural and biochemical conservation and regulate a variety of essential cellular processes, including membrane traffic, cell division, and energy metabolism; each with links to human diseases. We previously identified members of the ELMOD family as GTPase-activating proteins (GAPs) for ARL2 that displayed crossover activity for ARFs as well. To further characterize the GAP activities of the three human ELMODs as GAPs we developed new preparations of each after overexpression in human embryonic kidney (HEK293T) cells. This allowed much higher specific activities and enhanced stability and solubility of the purified proteins. The specificities of ELMOD1-3 as GAPs for six different members of the ARF family were determined and found to display wide variations, which we believe will reveal differences in cellular functions of family members. The non-opioid sigma-1 receptor (S1R) was identified as a novel effector of GAP activity of ELMOD1-3 proteins as its direct binding to either ELMOD1 or ELMOD2 resulted in loss of GAP activity. These findings are critical to understand the roles of ELMOD proteins in cell signaling in general and in the inner ear specifically, and open the door to exploration of the regulation of their GAP activities via agonists or antagonists of the S1R.
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Factores de Ribosilacion-ADP/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Receptores sigma/metabolismo , Factores de Ribosilacion-ADP/genética , Proteínas del Citoesqueleto/genética , Proteínas de Unión al GTP/genética , Proteínas Activadoras de GTPasa/genética , Células HEK293 , Humanos , Receptores sigma/genética , Receptor Sigma-1RESUMEN
Protein kinases are a primary focus in targeted therapy development for cancer, owing to their role as regulators in nearly all areas of cell life. Recent strategies targeting the kinome with combination therapies have shown promise, such as trametinib and dabrafenib in advanced melanoma, but empirical design for less characterized pathways remains a challenge. Computational combination screening is an attractive alternative, allowing in-silico filtering prior to experimental testing of drastically fewer leads, increasing efficiency and effectiveness of drug development pipelines. In this work, we generated combined kinome inhibition states of 40,000 kinase inhibitor combinations from kinobeads-based kinome profiling across 64 doses. We then integrated these with transcriptomics from CCLE to build machine learning models with elastic-net feature selection to predict cell line sensitivity across nine cancer types, with accuracy R2 â¼ 0.75-0.9. We then validated the model by using a PDX-derived TNBC cell line and saw good global accuracy (R2 â¼ 0.7) as well as high accuracy in predicting synergy using four popular metrics (R2 â¼ 0.9). Additionally, the model was able to predict a highly synergistic combination of trametinib and omipalisib for TNBC treatment, which incidentally was recently in phase I clinical trials. Our choice of tree-based models for greater interpretability allowed interrogation of highly predictive kinases in each cancer type, such as the MAPK, CDK, and STK kinases. Overall, these results suggest that kinome inhibition states of kinase inhibitor combinations are strongly predictive of cell line responses and have great potential for integration into computational drug screening pipelines. This approach may facilitate the identification of effective kinase inhibitor combinations and accelerate the development of novel cancer therapies, ultimately improving patient outcomes.
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Antineoplásicos , Melanoma , Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Biología Computacional/métodos , Antineoplásicos/uso terapéutico , Melanoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Línea Celular TumoralRESUMEN
The oxindole scaffold has been the center of several kinase drug discovery programs, some of which have led to approved medicines. A series of two oxindole matched pairs from the literature were identified where TLK2 was potently inhibited as an off-target kinase. The oxindole has long been considered a promiscuous kinase inhibitor template, but across these four specific literature oxindoles TLK2 activity was consistent, while the kinome profile was radically different ranging from narrow to broad spectrum kinome coverage. We synthesized a large series of analogues, utilizing quantitative structure-activity relationship (QSAR) analysis, water mapping of the kinase ATP binding sites, kinome profiling, and small-molecule x-ray structural analysis to optimize TLK2 inhibition and kinome selectivity. This resulted in the identification of several narrow spectrum, sub-family selective, chemical tool compounds including 128 (UNC-CA2-103) that could enable elucidation of TLK2 biology.
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Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas , Relación Estructura-Actividad Cuantitativa , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/síntesis química , Humanos , Estructura Molecular , Oxindoles/farmacología , Oxindoles/química , Oxindoles/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Relación Dosis-Respuesta a Droga , Modelos MolecularesRESUMEN
In search for broad-spectrum antivirals, we discovered a small molecule inhibitor, RMC-113, that potently suppresses the replication of multiple RNA viruses including SARS-CoV-2 in human lung organoids. We demonstrated selective dual inhibition of the lipid kinases PIP4K2C and PIKfyve by RMC-113 and target engagement by its clickable analog. Advanced lipidomics revealed alteration of SARS-CoV-2-induced phosphoinositide signature by RMC-113 and linked its antiviral effect with functional PIP4K2C and PIKfyve inhibition. We discovered PIP4K2C's roles in SARS-CoV-2 entry, RNA replication, and assembly/egress, validating it as a druggable antiviral target. Integrating proteomics, single-cell transcriptomics, and functional assays revealed that PIP4K2C binds SARS-CoV-2 nonstructural protein 6 and regulates virus-induced impairment of autophagic flux. Reversing this autophagic flux impairment is a mechanism of antiviral action of RMC-113. These findings reveal virus-induced autophagy regulation via PIP4K2C, an understudied kinase, and propose dual inhibition of PIP4K2C and PIKfyve as a candidate strategy to combat emerging viruses.
RESUMEN
Arf GAPs (ADP-ribosylation factor GTPase-activating proteins) are essential components of Arf (ADP-ribosylation factor) signaling pathways. Arf GAPs stimulate the hydrolysis of GTP to GDP to transition Arf from the active, GTP bound, state to the inactive, GDP bound, state. Based on this activity, Arf GAPs were initially proposed to function primarily or exclusively as terminators of Arf signaling. Further studies of Arf GAPs have revealed that they also function as effectors of Arf signaling in at least a few steps or processes in which Arfs are not directly involved. In this review we discuss the non-canonical functions of Arf GAPs and address several key questions in the field, including: whether (1) Arf GAPs are terminators or effectors of Arf signaling, (2) Arf GAPs positively or negatively regulate COPI assembly, (3) Arf GAPs are involved in vesicle fission, and (4) Arf GAPs regulate vesicle uncoating.
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Factores de Ribosilacion-ADP/metabolismo , Animales , Proteína Coat de Complejo I/metabolismo , Humanos , Unión Proteica , Transporte de Proteínas , Transducción de SeñalRESUMEN
The human family of ELMO domain-containing proteins (ELMODs) consists of six members and is defined by the presence of the ELMO domain. Within this family are two subclassifications of proteins, based on primary sequence conservation, protein size, and domain architecture, deemed ELMOD and ELMO. In this study, we used homology searching and phylogenetics to identify ELMOD family homologs in genomes from across eukaryotic diversity. This demonstrated not only that the protein family is ancient but also that ELMOs are potentially restricted to the supergroup Opisthokonta (Metazoa and Fungi), whereas proteins with the ELMOD organization are found in diverse eukaryotes and thus were likely the form present in the last eukaryotic common ancestor. The segregation of the ELMO clade from the larger ELMOD group is consistent with their contrasting functions as unconventional Rac1 guanine nucleotide exchange factors and the Arf family GTPase-activating proteins, respectively. We used unbiased, phylogenetic sorting and sequence alignments to identify the most highly conserved residues within the ELMO domain to identify a putative GAP domain within the ELMODs. Three independent but complementary assays were used to provide an initial characterization of this domain. We identified a highly conserved arginine residue critical for both the biochemical and cellular GAP activity of ELMODs. We also provide initial evidence of the function of human ELMOD1 as an Arf family GAP at the Golgi. These findings provide the basis for the future study of the ELMOD family of proteins and a new avenue for the study of Arf family GTPases.
Asunto(s)
Factores de Ribosilacion-ADP/genética , Proteínas del Citoesqueleto/genética , Evolución Molecular , Filogenia , Factores de Ribosilacion-ADP/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas Activadoras de GTPasa , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Estructura Terciaria de Proteína , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The oxindole scaffold has been the center of several kinase drug discovery programs, some of which have led to approved medicines. A series of two oxindole matched pairs from the literature were identified where TLK2 was a potent off-target kinase. The oxindole has long been considered a promiscuous inhibitor template, but across these 4 specific literature oxindoles TLK2 activity was consistent, while the kinome profile was radically different from narrow to broad spectrum coverage. We synthesized a large series of analogues and through quantitative structure-activity relationship (QSAR) analysis, water mapping of the kinase ATP binding sites, small-molecule x-ray structural analysis and kinome profiling, narrow spectrum, sub-family selective, chemical tool compounds were identified to enable elucidation of TLK2 biology.
RESUMEN
ELMODs are a family of three mammalian paralogues that display GTPase-activating protein (GAP) activity toward a uniquely broad array of ADP-ribosylation factor (ARF) family GTPases that includes ARF-like (ARL) proteins. ELMODs are ubiquitously expressed in mammalian tissues, highly conserved across eukaryotes, and ancient in origin, being present in the last eukaryotic common ancestor. We described functions of ELMOD2 in immortalized mouse embryonic fibroblasts (MEFs) in the regulation of cell division, microtubules, ciliogenesis, and mitochondrial fusion. Here, using similar strategies with the paralogues ELMOD1 and ELMOD3, we identify novel functions and locations of these cell regulators and compare them to those of ELMOD2, allowing the determination of functional redundancy among the family members. We found strong similarities in phenotypes resulting from deletion of either Elmod1 or Elmod3 and marked differences from those arising in Elmod2 deletion lines. Deletion of either Elmod1 or Elmod3 results in the decreased ability of cells to form primary cilia, loss of a subset of proteins from cilia, and accumulation of some ciliary proteins at the Golgi, predicted to result from compromised traffic from the Golgi to cilia. These phenotypes are reversed upon activating mutant expression of either ARL3 or ARL16, linking their roles to ELMOD1/3 actions.
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Proteínas Activadoras de GTPasa/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Animales , Cilios/metabolismo , Proteínas del Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Proteínas Activadoras de GTPasa/fisiología , Aparato de Golgi/metabolismo , Ratones , Microtúbulos/metabolismo , Dinámicas Mitocondriales , Transducción de Señal/genéticaRESUMEN
Deep annotation of a library of 4-anilinoquin(az)olines led to the identification of 7-iodo-N-(3,4,5-trimethoxyphenyl)quinolin-4-amine 16 as a potent inhibitor (IC50 =14â nM) of Protein Kinase Novel 3 (PKN3) with micromolar activity in cells. Compound 16 is a potential tool compound to study the cell biology of PKN3 and its role in pancreatic and prostate cancer and T-cell acute lymphoblastic leukemia. These 4-anilinoquin(az)olines may also be useful tools to uncover the therapeutic potential of PKN3 inhibition in a broad range of diseases.
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Neoplasias de la Próstata , Inhibidores de Proteínas Quinasas , Humanos , Masculino , Proteína Quinasa C , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Inhibition of the HER2/ERBB2 receptor is a keystone to treating HER2-positive malignancies, particularly breast cancer, but a significant fraction of HER2-positive (HER2+) breast cancers recur or fail to respond. Anti-HER2 monoclonal antibodies, like trastuzumab or pertuzumab, and ATP active site inhibitors like lapatinib, commonly lack durability because of adaptive changes in the tumor leading to resistance. HER2+ cell line responses to inhibition with lapatinib were analyzed by RNAseq and ChIPseq to characterize transcriptional and epigenetic changes. Motif analysis of lapatinib-responsive genomic regions implicated the pioneer transcription factor FOXA1 as a mediator of adaptive responses. Lapatinib in combination with FOXA1 depletion led to dysregulation of enhancers, impaired adaptive upregulation of HER3, and decreased proliferation. HER2-directed therapy using clinically relevant drugs (trastuzumab with or without lapatinib or pertuzumab) in a 7-day clinical trial designed to examine early pharmacodynamic response to antibody-based anti-HER2 therapy showed reduced FOXA1 expression was coincident with decreased HER2 and HER3 levels, decreased proliferation gene signatures, and increased immune gene signatures. This highlights the importance of the immune response to anti-HER2 antibodies and suggests that inhibiting FOXA1-mediated adaptive responses in combination with HER2 targeting is a potential therapeutic strategy.
RESUMEN
ELMOD2 is a â¼32 kDa protein first purified by its GTPase-activating protein (GAP) activity toward ARL2 and later shown to have uniquely broad specificity toward ARF family GTPases in in vitro assays. To begin the task of defining its functions in cells, we deleted ELMOD2 in immortalized mouse embryonic fibroblasts and discovered a number of cellular defects, which are reversed upon expression of ELMOD2-myc. We show that these defects, resulting from the loss of ELMOD2, are linked to two different pathways and two different GTPases: with ARL2 and TBCD to support microtubule nucleation from centrosomes and with ARF6 in cytokinesis. These data highlight key aspects of signaling by ARF family GAPs that contribute to previously underappreciated sources of complexity, including GAPs acting from multiple sites in cells, working with multiple GTPases, and contributing to the spatial and temporal control of regulatory GTPases by serving as both GAPs and effectors.
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Citocinesis/fisiología , Proteínas del Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Animales , Técnicas de Cultivo de Célula , Centrosoma/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/fisiología , Fibroblastos/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Células Madre Embrionarias de Ratones/metabolismoRESUMEN
BACKGROUND: Imatinib mesylate (imatinib) is the first-line treatment for newly diagnosed chronic myeloid leukemia (CML) due to its remarkable hematologic and cytogenetic responses. We previously demonstrated that the imatinib-resistant CML cells (Myl-R) contained elevated Lyn activity and intracellular creatine pools compared to imatinib-sensitive Myl cells. METHODS: Stable isotope metabolic labeling, media creatine depletion, and Na+/K+-ATPase inhibitor experiments were performed to investigate the origin of creatine pools in Myl-R cells. Inhibition and shRNA knockdown were performed to investigate the specific role of Lyn in regulating the Na+/K+-ATPase and creatine uptake. RESULTS: Inhibition of the Na+/K+-ATPase pump (ouabain, digitoxin), depletion of extracellular creatine or inhibition of Lyn kinase (ponatinib, dasatinib), demonstrated that enhanced creatine accumulation in Myl-R cells was dependent on uptake from the growth media. Creatine uptake was independent of the Na+/creatine symporter (SLC6A8) expression or de novo synthesis. Western blot analyses showed that phosphorylation of the Na+/K+-ATPase on Tyr 10 (Y10), a known regulatory phosphorylation site, correlated with Lyn activity. Overexpression of Lyn in HEK293 cells increased Y10 phosphorylation (pY10) of the Na+/K+-ATPase, whereas Lyn inhibition or shRNA knockdown reduced Na+/K+-ATPase pY10 and decreased creatine accumulation in Myl-R cells. Consistent with enhanced uptake in Myl-R cells, cyclocreatine (Ccr), a cytotoxic creatine analog, caused significant loss of viability in Myl-R compared to Myl cells. CONCLUSIONS: These data suggest that Lyn can affect creatine uptake through Lyn-dependent phosphorylation and regulation of the Na+/K+-ATPase pump activity. GENERAL SIGNIFICANCE: These studies identify kinase regulation of the Na+/K+-ATPase as pivotal in regulating creatine uptake and energy metabolism in cells.
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Antineoplásicos/farmacología , Creatina/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Mesilato de Imatinib/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Familia-src Quinasas/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Células Tumorales CultivadasRESUMEN
Over 55,000 people in the United States are diagnosed with pancreatic ductal adenocarcinoma (PDAC) yearly, and fewer than 20% of these patients survive a year beyond diagnosis. Chemotherapies are considered or used in nearly every PDAC case, but there is limited understanding of the complex signaling responses underlying resistance to these common treatments. Here, we take an unbiased approach to study protein kinase network changes following chemotherapies in patient-derived xenograft (PDX) models of PDAC to facilitate design of rational drug combinations. Proteomics profiling following chemotherapy regimens reveals that activation of JNK-JUN signaling occurs after 5-fluorouracil plus leucovorin (5-FU + LEU) and FOLFOX (5-FU + LEU plus oxaliplatin [OX]), but not after OX alone or gemcitabine. Cell and tumor growth assays with the irreversible inhibitor JNK-IN-8 and genetic manipulations demonstrate that JNK and JUN each contribute to chemoresistance and cancer cell survival after FOLFOX. Active JNK1 and JUN are specifically implicated in these effects, and synergy with JNK-IN-8 is linked to FOLFOX-mediated JUN activation, cell cycle dysregulation, and DNA damage response. This study highlights the potential for JNK-IN-8 as a biological tool and potential combination therapy with FOLFOX in PDAC and reinforces the need to tailor treatment to functional characteristics of individual tumors.