RESUMEN
The rapid evolution of mass spectrometry-based single-cell proteomics now enables the cataloging of several thousand proteins from single cells. We investigated whether we could discover cellular heterogeneity beyond proteome, encompassing post-translational modifications (PTM), protein-protein interaction, and variants. By optimizing the mass spectrometry data interpretation strategy to enable the detection of PTMs and variants, we have generated a high-definition dataset of single-cell and nuclear proteomic-states. The data demonstrate the heterogeneity of cell-states and signaling dependencies at the single-cell level and reveal epigenetic drug-induced changes in single nuclei. This approach enables the exploration of previously uncharted single-cell and organellar proteomes revealing molecular characteristics that are inaccessible through RNA profiling.
Asunto(s)
Espectrometría de Masas , Procesamiento Proteico-Postraduccional , Proteómica , Transducción de Señal , Análisis de la Célula Individual , Análisis de la Célula Individual/métodos , Humanos , Espectrometría de Masas/métodos , Proteómica/métodos , Orgánulos/metabolismo , Proteoma/metabolismoRESUMEN
Nitrotyrosine, or 3-nitrotyrosine, is an oxidative post-translational modification induced by reactive nitrogen species. Although nitrotyrosine is considered a marker of oxidative stress and has been associated with inflammation, neurodegeneration, cardiovascular disease, and cancer, identification of nitrotyrosine-modified proteins remains challenging owing to its low stoichiometric levels in biological samples. To facilitate a comprehensive analysis of proteins and peptides containing nitrotyrosine, we optimized an immunoprecipitation-based enrichment workflow using a cell line model. The identification of proteins and peptides containing nitrotyrosine residues was carried out after peroxynitrite treatment of cell lysates, which generated modified nitrotyrosine residues on susceptible sites on proteins. We evaluated the efficacy of enriching nitrotyrosine-modified proteins and peptides by employing four different commercially available monoclonal antibodies directed against nitrotyrosine. LC-MS/MS analysis resulted in the identification of 1377 and 1624 nitrotyrosine-containing peptides from protein- and peptide-based enrichment experiments, respectively. Although the yield of nitrotyrosine-containing peptides was higher in experiments where peptides rather than proteins were enriched, we found a substantial proportion (37-65%) of identified nitrotyrosine-containing peptides contained nitrotyrosine at the N-terminus. However, in protein-based immunoprecipitation <9% of nitrotyrosine-containing peptides had nitrotyrosine modification at the N-terminus of the peptide. Overall, our study resulted in the identification of 2603 nitrotyrosine-containing peptides of which >2000 have not previously been reported. We synthesized 101 novel nitrotyrosine-containing peptides identified in our analysis and analyzed them by LC-MS/MS to validate our findings. We have confirmed the validity of 70% of these peptides, as they demonstrated a similarity score exceeding 0.7 when compared to peptides identified through experimental methods. Finally, we also validated the presence of nitrotyrosine modification on PKM and EF2 proteins in peroxynitrite-treated samples by immunoblot analysis. The large catalog presented in this study along with the workflow should facilitate the investigation of nitrotyrosine as an oxidative modification in a variety of settings in greater detail.
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Ácido Peroxinitroso , Espectrometría de Masas en Tándem , Tirosina/análogos & derivados , Cromatografía Liquida/métodos , Proteínas/química , Péptidos/química , Tirosina/metabolismo , AnticuerposRESUMEN
BACKGROUND: Cell surface proteins perform critical functions related to immune response, signal transduction, cell-cell interactions, and cell migration. Expression of specific cell surface proteins can determine cell-type identity, and can be altered in diseases including infections, cancer and genetic disorders. Identification of the cell surface proteome remains a challenge despite several enrichment methods exploiting their biochemical and biophysical properties. METHODS: Here, we report a novel method for enrichment of proteins localized to cell surface. We developed this new approach designated surface Biotinylation Site Identification Technology (sBioSITe) by adapting our previously published method for direct identification of biotinylated peptides. In this strategy, the primary amine groups of lysines on proteins on the surface of live cells are first labeled with biotin, and subsequently, biotinylated peptides are enriched by anti-biotin antibodies and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: By direct detection of biotinylated lysines from PC-3, a prostate cancer cell line, using sBioSITe, we identified 5851 peptides biotinylated on the cell surface that were derived from 1409 proteins. Of these proteins, 533 were previously shown or predicted to be localized to the cell surface or secreted extracellularly. Several of the identified cell surface markers have known associations with prostate cancer and metastasis including CD59, 4F2 cell-surface antigen heavy chain (SLC3A2) and adhesion G protein-coupled receptor E5 (CD97). Importantly, we identified several biotinylated peptides derived from plectin and nucleolin, both of which are not annotated in surface proteome databases but have been shown to have aberrant surface localization in certain cancers highlighting the utility of this method. CONCLUSIONS: Detection of biotinylation sites on cell surface proteins using sBioSITe provides a reliable method for identifying cell surface proteins. This strategy complements existing methods for detection of cell surface expressed proteins especially in discovery-based proteomics approaches.
RESUMEN
Although tandem mass tag (TMT)-based isobaric labeling has become a powerful approach for multiplexed protein quantitation, automating the workflow for this technique has not been easy to achieve for widespread adoption. This is because preparation of TMT-labeled peptide samples involves multiple steps ranging from protein extraction, denaturation, reduction, and alkylation to tryptic digestion, desalting, labeling, and cleanup, all of which require a high level of proficiency. The variability resulting from multiple processing steps is inherently problematic, especially with large-scale clinical studies that involve hundreds of samples where reproducibility is critical for quantitation. Here, we sought to compare the performance of a recently introduced platform, AccelerOme, for an automated proteomic workflow employing TMT labeling with the manual processing of samples. Cell pellets were prepared and subjected to a 16-plex experiment using an automated platform and a conventional manual protocol. Single-shot liquid chromatography with tandem mass spectrometry analysis revealed a higher number of proteins and peptides identified using the automated platform. Efficiency of tryptic digestion, alkylation, and TMT labeling were similar in both manual and automated processes. In addition, comparison of quantitation accuracy and precision showed similar performance in an automated workflow compared to manual sample preparation by an expert. Overall, we demonstrated that the automated platform performs at a level similar to a manual process performed by an expert for TMT-based proteomics. We anticipate that this automated workflow will increasingly replace manual pipelines and has the potential to be applied to large-scale TMT-based studies, providing robust results and high sample throughput.
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Proteínas , Proteómica , Proteómica/métodos , Flujo de Trabajo , Reproducibilidad de los Resultados , Proteínas/química , Péptidos , Proteoma/análisisRESUMEN
Although single cell RNA-seq has had a tremendous impact on biological research, a corresponding technology for unbiased mass spectrometric analysis of single cells has only recently become available. Significant technological breakthroughs including miniaturized sample handling have enabled proteome profiling of single cells. Furthermore, trapped ion mobility spectrometry (TIMS) in combination with parallel accumulation-serial fragmentation operated in data-dependent acquisition mode (DDA-PASEF) allowed improved proteome coverage from low-input samples. It has been demonstrated that modulating the ion flux in TIMS affects the overall performance of proteome profiling. However, the effect of TIMS settings on the analysis of low-input samples has been less investigated. Thus, we sought to optimize the conditions of TIMS with regard to ion accumulation/ramp times and ion mobility range for low-input samples. We observed that an ion accumulation time of 180 ms and monitoring a narrower ion mobility range from 0.7 to 1.3 V s cm-2 resulted in a substantial gain in the depth of proteome coverage and in detecting proteins with low abundance. We used these optimized conditions for proteome profiling of sorted human primary T cells, which yielded an average of 365, 804, 1116, and 1651 proteins from single, five, ten, and forty T cells, respectively. Notably, we demonstrated that the depth of proteome coverage from a low number of cells was sufficient to delineate several essential metabolic pathways and the T cell receptor signaling pathway. Finally, we showed the feasibility of detecting post-translational modifications including phosphorylation and acetylation from single cells. We believe that such an approach could be applied to label-free analysis of single cells obtained from clinically relevant samples.
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Proteoma , Proteómica , Humanos , Proteoma/análisis , Proteómica/métodos , Espectrometría de Movilidad Iónica/métodos , Espectrometría de Masas/métodos , Procesamiento Proteico-PostraduccionalRESUMEN
PARP inhibitors (PARPi) have activity in homologous recombination (HR) repair-deficient, high-grade serous ovarian cancers (HGSOC). However, even responsive tumors develop PARPi resistance, highlighting the need to delay or prevent the appearance of PARPi resistance. Here, we showed that the ALK kinase inhibitor ceritinib synergizes with PARPis by inhibiting complex I of the mitochondrial electron transport chain, which increases production of reactive oxygen species (ROS) and subsequent induction of oxidative DNA damage that is repaired in a PARP-dependent manner. In addition, combined treatment with ceritinib and PARPi synergized in HGSOC cell lines irrespective of HR status, and a combination of ceritinib with the PARPi olaparib induced tumor regression more effectively than olaparib alone in HGSOC patient-derived xenograft (PDX) models. Notably, the ceritinib and olaparib combination was most effective in PDX models with preexisting PARPi sensitivity and was well tolerated. These findings unveil suppression of mitochondrial respiration, accumulation of ROS, and subsequent induction of DNA damage as novel effects of ceritinib. They also suggest that the ceritinib and PARPi combination warrants further investigation as a means to enhance PARPi activity in HGSOC, particularly in tumors with preexisting HR defects. SIGNIFICANCE: The kinase inhibitor ceritinib synergizes with PARPi to induce tumor regression in ovarian cancer models, suggesting that ceritinib combined with PARPi may be an effective strategy for treating ovarian cancer.
Asunto(s)
Antineoplásicos/administración & dosificación , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Carcinoma Epitelial de Ovario/metabolismo , Daño del ADN/efectos de los fármacos , Reposicionamiento de Medicamentos/métodos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Ftalazinas/administración & dosificación , Piperazinas/administración & dosificación , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Pirimidinas/administración & dosificación , Sulfonas/administración & dosificación , Animales , Carcinoma Epitelial de Ovario/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Humanos , Ratones , Ratones SCID , Neoplasias Ováricas/patología , Células PC-3 , Reparación del ADN por Recombinación/efectos de los fármacos , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Although inhibitors of the kinases CHK1, ATR, and WEE1 are undergoing clinical testing, it remains unclear how these three classes of agents kill susceptible cells and whether they utilize the same cytotoxic mechanism. Here we observed that CHK1 inhibition induces apoptosis in a subset of acute leukemia cell lines in vitro, including TP53-null acute myeloid leukemia (AML) and BCR/ABL-positive acute lymphoid leukemia (ALL), and inhibits leukemic colony formation in clinical AML samples ex vivo. In further studies, downregulation or inhibition of CHK1 triggered signaling in sensitive human acute leukemia cell lines that involved CDK2 activation followed by AP1-dependent TNF transactivation, TNFα production, and engagement of a TNFR1- and BID-dependent apoptotic pathway. AML lines that were intrinsically resistant to CHK1 inhibition exhibited high CHK1 expression and were sensitized by CHK1 downregulation. Signaling through this same CDK2-AP1-TNF cytotoxic pathway was also initiated by ATR or WEE1 inhibitors in vitro and during CHK1 inhibitor treatment of AML xenografts in vivo. Collectively, these observations not only identify new contributors to the antileukemic cell action of CHK1, ATR, and WEE1 inhibitors, but also delineate a previously undescribed pathway leading from aberrant CDK2 activation to death ligand-induced killing that can potentially be exploited for acute leukemia treatment. SIGNIFICANCE: This study demonstrates that replication checkpoint inhibitors can kill AML cells through a pathway involving AP1-mediated TNF gene activation and subsequent TP53-independent, TNFα-induced apoptosis, which can potentially be exploited clinically.
Asunto(s)
Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Pirazinas/farmacología , Pirazoles/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apoptosis , Proliferación Celular , Femenino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CPX-351 is a liposomally encapsulated 5:1 molar ratio of cytarabine and daunorubicin that recently received regulatory approval for the treatment of therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes based on improved overall survival compared to standard cytarabine/daunorubicin therapy. Checkpoint kinase 1 (CHK1), which is activated by DNA damage and replication stress, diminishes sensitivity to cytarabine and anthracyclines as single agents, suggesting that CHK1 inhibitors might increase the effectiveness of CPX-351. The present studies show that CPX-351 activates CHK1 as well as the S and G2/M cell cycle checkpoints. Conversely, CHK1 inhibition diminishes the cell cycle effects of CPX-351. Moreover, CHK1 knockdown or addition of a CHK1 inhibitor such as MK-8776, rabusertib or prexasertib enhances CPX-351-induced apoptosis in multiple TP53-null and TP53-wildtype AML cell lines. Likewise, CHK1 inhibition increases the antiproliferative effect of CPX-351 on primary AML specimens ex vivo, offering the possibility that CPX-351 may be well suited to combine with CHK1-targeted agents.
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Apoptosis , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Citarabina/farmacología , Daunorrubicina/farmacología , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Leucemia Mieloide Aguda/patología , Leucocitos Mononucleares/patología , Inhibidores de Proteínas Quinasas/farmacología , Proliferación Celular , Humanos , Técnicas In Vitro , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/enzimología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/enzimología , Células Tumorales CultivadasRESUMEN
The BCL2 family of proteins regulates cellular life and death decisions. Among BCL2 family members, BH3-only proteins have critical roles by neutralizing antiapoptotic family members, as well as directly activating BAX and BAK. Despite widespread occurrence of BH3-only protein upregulation in response to various stresses, this process is rarely quantified. Moreover, it is unclear whether all BH3-only proteins are equipotent at inducing cell death. Here we show that BH3-only proteins increase as much as 15- to 20-fold after various treatments and define a parameter, termed BH3-only tolerance, which measures how many copies of a particular BH3-only protein can be expressed before the majority of cells in a population undergo apoptosis. We not only assess the relative contributions of anti- and proapoptotic BCL2 family members to BH3-only tolerance, but also illustrate how the study of this parameter can be used to understand cellular sensitivity to anticancer drugs and new combinations. These observations provide a new quantitative framework for assessing apoptotic susceptibility under various conditions.
Asunto(s)
Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/análisis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Células HEK293 , HumanosRESUMEN
Senescent cells accumulate in fat with aging. We previously found genetic clearance of senescent cells from progeroid INK-ATTAC mice prevents lipodystrophy. Here we show that primary human senescent fat progenitors secrete activin A and directly inhibit adipogenesis in non-senescent progenitors. Blocking activin A partially restored lipid accumulation and expression of key adipogenic markers in differentiating progenitors exposed to senescent cells. Mouse fat tissue activin A increased with aging. Clearing senescent cells from 18-month-old naturally-aged INK-ATTAC mice reduced circulating activin A, blunted fat loss, and enhanced adipogenic transcription factor expression within 3 weeks. JAK inhibitor suppressed senescent cell activin A production and blunted senescent cell-mediated inhibition of adipogenesis. Eight weeks-treatment with ruxolitinib, an FDA-approved JAK1/2 inhibitor, reduced circulating activin A, preserved fat mass, reduced lipotoxicity, and increased insulin sensitivity in 22-month-old mice. Our study indicates targeting senescent cells or their products may alleviate age-related dysfunction of progenitors, adipose tissue, and metabolism.
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Adipogénesis , Senescencia Celular , Células Madre/fisiología , Activinas/metabolismo , Animales , Humanos , RatonesRESUMEN
Chronic, low grade, sterile inflammation frequently accompanies aging and age-related diseases. Cellular senescence is associated with the production of proinflammatory chemokines, cytokines, and extracellular matrix (ECM) remodeling proteases, which comprise the senescence-associated secretory phenotype (SASP). We found a higher burden of senescent cells in adipose tissue with aging. Senescent human primary preadipocytes as well as human umbilical vein endothelial cells (HUVECs) developed a SASP that could be suppressed by targeting the JAK pathway using RNAi or JAK inhibitors. Conditioned medium (CM) from senescent human preadipocytes induced macrophage migration in vitro and inflammation in healthy adipose tissue and preadipocytes. When the senescent cells from which CM was derived had been treated with JAK inhibitors, the resulting CM was much less proinflammatory. The administration of JAK inhibitor to aged mice for 10 wk alleviated both adipose tissue and systemic inflammation and enhanced physical function. Our findings are consistent with a possible contribution of senescent cells and the SASP to age-related inflammation and frailty. We speculate that SASP inhibition by JAK inhibitors may contribute to alleviating frailty. Targeting the JAK pathway holds promise for treating age-related dysfunction.
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Adipocitos/enzimología , Senescencia Celular/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/enzimología , Quinasas Janus/antagonistas & inhibidores , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , Adipocitos/citología , Tejido Adiposo/citología , Tejido Adiposo/enzimología , Animales , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Senescencia Celular/genética , Matriz Extracelular/metabolismo , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Quinasas Janus/genética , Quinasas Janus/metabolismo , Macrófagos/citología , Macrófagos/enzimología , Ratones , ARN Interferente Pequeño/genética , Transducción de Señal/genéticaRESUMEN
Mitochondrial outer membrane permeabilization (MOMP), a key step in the intrinsic apoptotic pathway, is incompletely understood. Current models emphasize the role of BH3-only BCL2 family members in BAX and BAK activation. Here we demonstrate concentration-dependent BAK autoactivation under cell-free conditions and provide evidence that this autoactivation plays a key role in regulating the intrinsic apoptotic pathway in intact cells. In particular, we show that up to 80% of BAK (but not BAX) in lymphohematopoietic cell lines is oligomerized and bound to anti-apoptotic BCL2 family members in the absence of exogenous death stimuli. The extent of this constitutive BAK oligomerization is diminished by BAK knockdown and unaffected by BIM or PUMA down-regulation. Further analysis indicates that sensitivity of cells to BH3 mimetics reflects the identity of the anti-apoptotic proteins to which BAK is constitutively bound, with extensive BCLXLâ¢BAK complexes predicting navitoclax sensitivity, and extensive MCL1â¢BAK complexes predicting A1210477 sensitivity. Moreover, high BAK expression correlates with sensitivity of clinical acute myelogenous leukemia to chemotherapy, whereas low BAK levels correlate with resistance and relapse. Collectively, these results inform current understanding of MOMP and provide new insight into the ability of BH3 mimetics to induce apoptosis without directly activating BAX or BAK.
Asunto(s)
Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Activación Transcripcional/genética , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células Jurkat , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/fisiopatología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfonamidas/farmacología , Proteína bcl-X/metabolismoRESUMEN
Bcl-2, the founding member of a family of apoptotic regulators, was initially identified as the protein product of a gene that is translocated and overexpressed in greater than 85% of follicular lymphomas (FLs). Thirty years later we now understand that anti-apoptotic Bcl-2 family members modulate the intrinsic apoptotic pathway by binding and neutralizing the mitochondrial permeabilizers Bax and Bak as well as a variety of pro-apoptotic proteins, including the cellular stress sensors Bim, Bid, Puma, Bad, Bmf and Noxa. Despite extensive investigation of all of these proteins, important questions remain. For example, how Bax and Bak breach the outer mitochondrial membrane remains poorly understood. Likewise, how the functions of anti-apoptotic Bcl-2 family members such as eponymous Bcl-2 are affected by phosphorylation or cancer-associated mutations has been incompletely defined. Finally, whether Bcl-2 family members can be successfully targeted for therapeutic advantage is only now being investigated in the clinic. Here we review recent advances in understanding Bcl-2 family biology and biochemistry that begin to address these questions.
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Progresión de la Enfermedad , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Terapia Molecular Dirigida , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
The healthspan of mice is enhanced by killing senescent cells using a transgenic suicide gene. Achieving the same using small molecules would have a tremendous impact on quality of life and the burden of age-related chronic diseases. Here, we describe the rationale for identification and validation of a new class of drugs termed senolytics, which selectively kill senescent cells. By transcript analysis, we discovered increased expression of pro-survival networks in senescent cells, consistent with their established resistance to apoptosis. Using siRNA to silence expression of key nodes of this network, including ephrins (EFNB1 or 3), PI3Kδ, p21, BCL-xL, or plasminogen-activated inhibitor-2, killed senescent cells, but not proliferating or quiescent, differentiated cells. Drugs targeting these same factors selectively killed senescent cells. Dasatinib eliminated senescent human fat cell progenitors, while quercetin was more effective against senescent human endothelial cells and mouse BM-MSCs. The combination of dasatinib and quercetin was effective in eliminating senescent MEFs. In vivo, this combination reduced senescent cell burden in chronologically aged, radiation-exposed, and progeroid Ercc1(-/Δ) mice. In old mice, cardiac function and carotid vascular reactivity were improved 5 days after a single dose. Following irradiation of one limb in mice, a single dose led to improved exercise capacity for at least 7 months following drug treatment. Periodic drug administration extended healthspan in Ercc1(-/∆) mice, delaying age-related symptoms and pathology, osteoporosis, and loss of intervertebral disk proteoglycans. These results demonstrate the feasibility of selectively ablating senescent cells and the efficacy of senolytics for alleviating symptoms of frailty and extending healthspan.
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Envejecimiento/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Dasatinib/farmacología , Osteoporosis/prevención & control , Quercetina/farmacología , Transcriptoma , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Adipocitos/patología , Envejecimiento/genética , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/patología , Senescencia Celular/genética , Fosfatidilinositol 3-Quinasa Clase I , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Combinación de Medicamentos , Endonucleasas/genética , Endonucleasas/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Efrinas/genética , Efrinas/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Perfilación de la Expresión Génica , Corazón/efectos de los fármacos , Corazón/fisiopatología , Disco Intervertebral/química , Disco Intervertebral/efectos de los fármacos , Disco Intervertebral/patología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Ratones , Ratones Noqueados , Osteoporosis/genética , Osteoporosis/metabolismo , Osteoporosis/patología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidor 2 de Activador Plasminogénico/genética , Inhibidor 2 de Activador Plasminogénico/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Follicular lymphoma (FL), an indolent neoplasm caused by a t(14;18) chromosomal translocation that juxtaposes the BCL2 gene and immunoglobulin locus, has a variable clinical course and frequently undergoes transformation to an aggressive lymphoma. Although BCL2 mutations have been previously described, their relationship to FL progression remains unclear. In this study, we evaluated the frequency and nature of BCL2 mutations in 2 independent cohorts of grade 1 and 2 FLs, along with the correlation between BCL2 mutations, transformation risk, and survival. The prevalence of BCL2 coding sequence mutations was 12% in FL at diagnosis and 53% at transformation (P < .0001). The presence of these BCL2 mutations at diagnosis correlated with an increased risk of transformation (hazard ratio 3.6; 95% CI, 2.0-6.2; P < .0001) and increased risk of death due to lymphoma (median survival of 9.5 years with BCL2 mutations vs 20.4 years without; P = .012). In a multivariate analysis, BCL2 mutations and high FL international prognostic index were independent risk factors for transformation and death due to lymphoma. Some mutant Bcl-2 proteins exhibited enhanced antiapoptotic capacity in vitro. Accordingly, BCL2 mutations can affect antiapoptotic Bcl-2 function, are associated with increased activation-induced cytidine deaminase expression, and correlate with increased risk of transformation and death due to lymphoma.
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Transformación Celular Neoplásica/genética , Linfoma Folicular/genética , Linfoma Folicular/mortalidad , Mutación , Proteínas Proto-Oncogénicas c-bcl-2/genética , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Cromosomas Humanos Par 14/genética , Cromosomas Humanos Par 14/metabolismo , Cromosomas Humanos Par 18/genética , Cromosomas Humanos Par 18/metabolismo , Estudios de Cohortes , Citidina Desaminasa/biosíntesis , Citidina Desaminasa/genética , Supervivencia sin Enfermedad , Femenino , Regulación Enzimológica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Linfoma Folicular/metabolismo , Masculino , Persona de Mediana Edad , Prevalencia , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factores de Riesgo , Tasa de SupervivenciaRESUMEN
Although farnesyltransferase inhibitors have shown promising activity in relapsed lymphoma and sporadic activity in acute myelogenous leukemia, their mechanism of cytotoxicity is incompletely understood, making development of predictive biomarkers difficult. In the present study, we examined the action of tipifarnib in human acute myelogenous leukemia cell lines and clinical samples. In contrast to the Ras/MEK/ERK pathway-mediated Bim upregulation that is responsible for tipifarnib-induced killing of malignant lymphoid cells, inhibition of Rheb-induced mTOR signaling followed by dose-dependent upregulation of Bax and Puma occurred in acute myelogenous leukemia cell lines undergoing tipifarnib-induced apoptosis. Similar Bax and Puma upregulation occurred in serial bone marrow samples harvested from a subset of acute myelogenous leukemia patients during tipifarnib treatment. Expression of FTI-resistant Rheb M184L, like knockdown of Bax or Puma, diminished tipifarnib-induced killing. Further analysis demonstrated that increased Bax and Puma levels reflect protein stabilization rather than increased gene expression. In U937 cells selected for tipifarnib resistance, neither inhibition of signaling downstream of Rheb nor Bax and Puma stabilization occurred. Collectively, these results not only identify a pathway downstream from Rheb that contributes to tipifarnib cytotoxicity in human acute myelogenous leukemia cells, but also demonstrate that FTI-induced killing of lymphoid versus myeloid cells reflects distinct biochemical mechanisms downstream of different farnesylated substrates. (ClinicalTrials.gov identifier NCT00602771).
Asunto(s)
Antineoplásicos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Leucemia Mieloide Aguda/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Neuropéptidos/metabolismo , Quinolonas/farmacología , Proteína X Asociada a bcl-2/metabolismo , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Farnesiltransferasa/metabolismo , Humanos , Prenilación/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteína Homóloga de Ras Enriquecida en el Cerebro , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Células U937RESUMEN
Bcl-2 is phosphorylated on Ser(70) after treatment of cells with spindle poisons. On the basis of effects observed in cells overexpressing Bcl-2 S70E or S70A mutants, various studies have concluded that Ser(70) phosphorylation either enhances or diminishes Bcl-2 function. In the present study, the ability of phosphorylated Bcl-2, as well as the S70E and S70A mutants, to bind and neutralize proapoptotic Bcl-2 family members under cell-free conditions and in intact cells was examined in an attempt to resolve this controversy. Surface plasmon resonance indicated that phosphorylated Bcl-2, Bcl-2 S70E, and Bcl-2 S70A exhibit enhanced binding to Bim and Bak compared with unmodified Bcl-2. This enhanced binding reflected a readily detectable conformation change in the loop domain of Bcl-2. Furthermore, Bcl-2 S70E and S70A bound more Bak and Bim than wild-type Bcl-2 in pull-downs and afforded greater protection against several chemotherapeutic agents. Importantly, binding of endogenous Bcl-2 to Bim also increased during mitosis, when Bcl-2 is endogenously phosphorylated, and disruption of this mitotic Bcl-2/Bim binding with navitoclax or ABT-199, like Bcl-2 downregulation, enhanced the cytotoxicity of paclitaxel. Collectively, these results provide not only a mechanistic basis for the enhanced antiapoptotic activity of phosphorylated Bcl-2, but also an explanation for the ability of BH3 mimetics to enhance taxane sensitivity.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Resistencia a Antineoplásicos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Células Cultivadas , Evaluación Preclínica de Medicamentos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Humanos , Células Jurkat , Células K562 , Proteínas de la Membrana/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Interferente Pequeño/farmacologíaRESUMEN
A phase 2 study of the oral farnesyltransferase inhibitor tipifarnib was conducted in 93 adult patients with relapsed or refractory lymphoma. Patients received tipifarnib 300 mg twice daily on days 1-21 of each 28-day cycle. The median number of prior therapies was 5 (range, 1-17). For the aggressive B-cell, indolent B-cell, and T-cell and Hodgkin lymphoma (HL/T) groups, the response rates were 17% (7/42), 7% (1/15), and 31% (11/36), respectively. Of the 19 responders, 7 were diffuse large B-cell non-Hodgkin lymphoma (NHL), 7 T-cell NHL, 1 follicular grade 2, and 4 HL. The median response duration for the 19 responders was 7.2 months (mean, 15.8 months; range, 1.8-62), and 5 patients in the HL/T group are still receiving treatment at 29-64+ months. The grade 3/4 toxicities observed were fatigue and reversible myelosuppression. Correlative studies suggest that Bim and Bcl-2 should be examined as potential predictors of response in future studies. These results indicate that tipifarnib has activity in lymphoma, particularly in heavily pretreated HL/T types, with little activity in follicular NHL. In view of its excellent toxicity profile and novel mechanism of action, further studies in combination with other agents appear warranted. This trial is registered at www.clinicaltrials.gov as #NCT00082888.
Asunto(s)
Linfoma/tratamiento farmacológico , Quinolonas/uso terapéutico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/efectos adversos , Inhibidores Enzimáticos/uso terapéutico , Farnesiltransferasa/antagonistas & inhibidores , Femenino , Humanos , Linfoma/patología , Masculino , Persona de Mediana Edad , Quinolonas/administración & dosificación , Quinolonas/efectos adversos , Recurrencia , Resultado del Tratamiento , Adulto JovenRESUMEN
The mechanism of cytotoxicity of farnesyltransferase inhibitors is incompletely understood and seems to vary depending on the cell type. To identify potential determinants of sensitivity or resistance for study in the accompanying clinical trial (Witzig et al, page 4882), we examined the mechanism of cytotoxicity of tipifarnib in human lymphoid cell lines. Based on initial experiments showing that Jurkat variants lacking Fas-associated death domain or procaspase-8 undergo tipifarnib-induced apoptosis, whereas cells lacking caspase-9 or overexpressing Bcl-2 do not, we examined changes in Bcl-2 family members. Tipifarnib caused dose-dependent up-regulation of Bim in lymphoid cell lines (Jurkat, Molt3, H9, DoHH2, and RL) that undergo tipifarnib-induced apoptosis but not in lines (SKW6.4 and Hs445) that resist tipifarnib-induced apoptosis. Further analysis demonstrated that increased Bim levels reflect inhibition of signaling from c-Raf to MEK1/2 and ERK1/2. Additional experiments showed that down-regulation of the Ras guanine nucleotide exchange factor RasGRP1 diminished tipifarnib sensitivity, suggesting that H-Ras or N-Ras is a critical farnesylation target upstream of c-Raf in lymphoid cells. These results not only trace a pathway through c-Raf to Bim that contributes to tipifarnib cytotoxicity in human lymphoid cells but also identify potential determinants of sensitivity to this agent.